Progress in Biological Chirality

Foreword of the Editors Progress in Biological Chirality is based on papers presented at the 3^^^ Interdisciplinary Sym...

Author: Gyula Pályi (Editor) | Claudia Zucchi (Editor) | Luciano Caglioti (Editor)

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Foreword of the Editors Progress in Biological Chirality is based on papers presented at the 3^^^ Interdisciplinary Symposium on Biological Chirality (April 30-May 4, 2003, Modena, Italy), it, however, contains a number of important contributions from Authors who could not participate at this Symposium. We believe, that this meeting and the book are presenting some very important advances after the first (Serramazzoni, Italy, 1998) and the second (Szeged, Hungary, 2000) meetings of this series. The phenomenon of the biological chirality (that is: the high enantiomeric excess of chiral substances used by all living organisms) represents a very attractive challenge to scientists since at least 150 years [1]. Corresponding research was often of speculative character [2], yielding highly intellectual approaches, which, however, got experimental verification only very recently [3]. The intellectual efforts resulted several excellent reviews (e.g. [4]) and even books (e.g. [5]) on this topic. We believe that the problems of biological chirality could be resolved by an unusually broad interdisciplinary effort. This, in a word of rapidly increasing specialisation, requires exceptional dedication from the scientists who are dealing with these problems. This was characteristic for the Authors of a previous book [5b] and also for the Authors of the present volume. We feel particularly honoured by being chosen for the coordination of these efforts, what - we are firmly convinced - represent in fact (not only in the title) a progress in biological chirality.

^ Tjyula Palyi ^ University of Modena and Reggio Emilia

Claudia Zucchi University of Modena and Reggio Emilia

ûciano Cagwoti University X a Sapienza" Roma [1] L. Pasteur, CK Acad. Set 16 (1848) 535-538; ^ m Chim. Phys. 24 (1848) 442-459. [2] (a) W.H. Mills, Chem. Ind. 51 (1932) 750-759. (b) F.C. Frank, Biochim. Biophys. Acta 11 (1953) 459-463. (c) V. Avetisov and V. Goldanskii, Proc. Natl. Acad ScL USA 93 (1996) 11435-11442. [3] (a) A Szabo-Nagy andL. Keszthelyi, Proc. Natl. Acad Sci. USA 96 (1999) 4252-4255. (b) K. Soai, I. Sato, S. Komiya, M. Hayashi, Y. Matsueda, H. Imamura, T. Hayase, H. Morioka, H. Tabira, J. Yamamoto and Y.Kowata, Tetrahedron Asymmetry 14 (2003) 185-188. [4] (a) L. Keszthelyi, Quart. Rev. Biophys. 28 (1995) 473-507. (b) L. Mark6, Diss Savariensis 24 (1998) 1-64. (c) B.L. Feringa and R. A van Delden, Angew. Chem., Int. Ed. 38 (1999) 3418-3438. [5] D.B. Cline (Ed.) Physical Origin of Homochirality in Life, AIP Press, Woodberg (NY, USA), 1996. (b) G. Palyi, C. Zucchi and L. CagUoti CEjds.)Advances in BioChirality Elsevier, Amsterdam, 1999. (c)H. Brunner, Rechts Oder Links, Wiley-VCH, Weinheim, 1999. (d) C. Mc Manus, Right Hand, Left Hand, Weidenfeld & Nicholson, London, 2002.

Preface This book. Progress in Biological Chirality, is dealing with an exciting area of Natural Sciences. The topic is interesting for (at least) two reasons. One reason is, that the biological chirality (some call it "homochirality"), is of enormous importance for the highly selective and very finely tuned, concerted chemical reactions, which make up life. According to some scientists, this phenomenon is one of the essential requisites of the origin of life on Earth. As a consequence of these considerations, the understanding of the origin, evolution and interrelations while in act of biological chirality is of fundamental importance for fields ranging from theoretical biology to planning the structure of new pharmaceuticals. Another reason is of emotional character. Biological chirality today represents one of the great unresolved secrets of Natural Sciences. It is something like, as the gravity was in Galileo's and Newton's age: you see everywhere the result of a principle, but it is not yet sufficiently documented how it came to existence. As a result of efforts described in the present book, we know today much more about these challenges than only a few years earlier. This progress is the result of several interdisciplinary efforts from theoretical physics, chemistry and biology, through palaeontology, paleobotanies to preparative efforts at modelling some aspects of biological chirality by the synthesis and structural characterisation of specially designed molecules. A broad spectrum of such research efforts is presented in this book, which will give a useful information source to specialists and a remarkable piece of reading to all who are interested in problems how life is operating and how it originated.

Prof Ferdinando Taddei President National Academy of Sciences, Letters and Arts (Modena)

Prof Gian Carlo Pellacani Rector Universit^f Modena and^Reggio Emilia I •/

iAt^C(ul2»^^^^>«'«^-^

Prof Raymond Daudel President European Academy of Sciences, Arts and Humanities (Paris)

,/jîy^^l Y

Board of the Referees Editors and Authors acknowledge thankfully the work of the following Referees: Vladik A. Avetisov N.N. Semenov Institute of Chemical Physics Russian Academy of Sciences Kossygina 4 117977 Moscow, Russian Federation [email protected] Krishnan Balasubramanian Department of Applied Science College of Engineering University of California, Davis Hertz Hall Bldg 661, PO Box 808, L-794, Livermore CA 94550 [email protected] Henri Brunner Institut fiir Anorganische Chemie Universitat Regensburg D-93040 Regensburg, Germany [email protected] Luciano Caglioti Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologiche Attive Universita "La Sapienza" P.le A. Moro 5 00185 ROMA, Italy caglioti @axrma.uniromal .it Janos Csapo Institute of Chemistry Department of Biochemistry and Food Chemistry University of Kaposvar GubaS.u. 40 H-7400 Kaposvar, Hungary [email protected] Jerzy Dzik Institute for Paleobiology PAN University of Warszawa Twarda 51/55 00818 Warszawa, Poland [email protected]

Arrigo Fomi Department of Chemistry University of Modena and Reggio Emilia viaCampi 183 1-41100 Modena, Italy [email protected] Noriko Fujii Research Reactor Institute Kyoto University Noda, Kumatori, Sennan Osaka 590-0494, Japan [email protected] Romeu Cardoso Guimaraes Departamento de Biologia Geral Inst. Ciencias Biologicas Universidade Federal de Minas Gerais 31270-901 Belo Horizonte MG, Brazil [email protected] Lajos Keszthelyi Institute of Biophysics Biological Research Centre Hungarian Academy of Sciences Temesvari krt. 62 P.O. Box 521 H-6701 Szeged, Hungary [email protected] Dilip Kondepudi Department of Chemistry Wake Forest University Winston-Salem NC 27109 USA [email protected] Andras Liptak Carbohydrate Research Group Hungarian Academy of Sciences Egyetem ter 1 H-4010 Debrecen Hungary [email protected]

Progress in Biological Chirality Laszlo Marko Department of Organic Chemistry University of Veszprem Veszprem, H-8201, Hungary [email protected] Koichiro Matsuno Department of Bioengineering Nagaoka University of Technology Nagaoka 940-2188, Japan [email protected] Paul G. Mezey Canada Research Chair in Scientific Modelling and Simulation Department of Chemistry and Department of Physics and Physical Oceanography Memorial University of Newfoundland Saint John's, NF, CANADA A1B 3X7 [email protected], [email protected] Lynn Mihichuk Department of Chemistry University of Regina Regina, Saskatchewan, CANADA S4S 0A2 [email protected] Maria Minunni Dipartimento di Chimica Universita di Firenze Via del la Lastruccia 3 50019 SestoF.no(FI), Italy [email protected] Gyula Palyi Department of Chemistry University of Modena and Reggio Emilia viaCampi 183 1-41100 Modena, Italy [email protected] Vince Pozsgay National Institutes of Health 31 Center Dr. Rm 2A25, MSC 2423 Bethesda, MD 20892-2423 [email protected] Livia Simon Sarkadi Dept. of Biochemistry and Food Technology Budapest Univ. of Technology and Economics Muegyetem rkp, 3

H-1111 Budapest, Hungary sarkadi @ mail, bme.hu Gyorgy Steinbrecher International Working Group FUSION-B.F.R. Association EURATOM-MEC Department of Theoretical Physics, Physics Faculty, University of Craiova, Str.A.I.Cuza 13, Craiova-1 100, Romania [email protected] Eors Szathmary Department of Plant Taxonomy and Ecology Eotvos University Ludovika ter 2 H-1083 Budapest Hungary [email protected] Ferdinando Taddei Department of Chemistry University of Modena and Reggio Emilia ViaCampi 183 1-4II00 Modena, Italy Taddei .ferdinando @unimo .it Giovanni Torre Department of Chemistry University of Modena and Reggio Emilia ViaCampi 183 1-41100 Modena, Italy Torre.giovanni @unimo.it Riccardo Zanasi Dipartimento di Chimica Universita di Salerno Via Salvador Allende 1-84081 Baronissi (SA), Italia [email protected] Geoffrey Zubay Columbia University New York City 10027 USA [email protected] Claudia Zucchi Department of Chemistry University of Modena and Reggio Emilia ViaCampi 183 1-41100 Modena, Italy [email protected]

Progress in Biological Chirality G. Palyi, C. Zucchi and L. Caglioti (Editors) © 2004 Elsevier Ltd. All rights reserved.

Chapter 1 Origin of Biomacromolecular Homochirality: in Search of Evolutional Dynamics Vladik Avetisov Semenov Institute of Chemical Physics of the Russian Academy of Sciences, Kossygina 4, 119991 Moscow, Russia avetisov@chph. ras. ru

1.

Introduction Biomacromolecular homochirality is one of the important attributes of the molecular background of life. It is well known that the nucleotide sequences (A, T(U), G, C) in DNA and RNA polymeric chains, as well as the amino acid sequences in enzymes, are "nearly random". In this respect, they are "slightly edited random copolymers" [1-4]. At the same time, these biopolymers have a universal and remarkable trait, namely, DNA and RNA consist of D nucleotides, while enzymes involve solely L amino acids. From the viewpoint of chirality, the primary structures of DNA, RNA, and enzymes are highly specific. They are homochiral. There is no exception to this rule as far as key biomacromolecules are concerned. Biomacromolecular homochirality is assumed to be necessary for reproduction of genetic information. Indeed, macromolecular chains of DNA and RNA are known to be templates (matrix) on which complementary copies are assembled, and any homochiral A, T(U), G, C sequence can serve as a template suitable for assembling of complementary replica. In opposite, as it was shown [5], pairing between a chirally defect unit in replica and the corresponding unit in homochiral template is impossible unless chemical bonds along the chains are broken. The reproduction of "chiral mutations" with a significantly high probability is impossible due to complete loss of the template profile around of chiral defect [6-8]. During the DNA, RNA, and enzymes biosynthesis, enantiomeric configuration of units is under precise control. It is less than one "chiral defecf per a biomacromolecular chain. The control is provided by biochemical functions of high enantioselectivity: They are realized, for it's turn, by particular homochiral macromolecules, enzymes. This "exclusive circle" is crucial from the origin-of-homochirality problem, because selection of macromolecular sequences through template directed replication requires homochirality and, what are important, enantiospecific functions capable of maintaining the assemblage of homochiral sequences. Let us consider macromolecular chain containing N units of L or D configurations. The number of all possible chains differing in L, D sequences is equal to 2^ and grows exponentially with N. The value of M becomes commensurate with the particle number fluctuations in "laboratory scale" systems (-10'^^) at N^40. Therefore, whenever A^ does not

4

Progress in Biological Chirality

exceed a couple dozens, it is possible to choose such conditions for the chain assembling that all conceivable sequences including homochiral ones may be realized. The origin of homochiral macromolecules of such length has no specific physical problem. Given an appropriate condition for polymerization, the probability for any predefined sequence to appear, in particular, a homochiral one, does not vanish even when the choice of L or D configuration is purely random. However, there are strong statistical limitations for assembling of long L,D-sequences. Indeed, comparison of M with the particle number fluctuations in a "cosmological scale" system consists, for instance, of 10^" particles (the number of carbon atoms in the Solar system) revels that M becomes of the same order of magnitude at A^«130. This means that each realized sequence of L, D-units containing more than a hundred units is certain to be "unique" for the overwhelming majority of sequences cannot be realized in principle, simply because even the whole Universe is too small for that. At such level of complexity, which is related to the simplest biomacromolecules, a relative part of realizable sequences is vanishingly small regardless of physical or chemical conditions. There is no looking over all possible sequences during the evolution. Hence "the memory of random choice" under such statistical limitations simply means that, in principle, any primary pool of sequences must be suitable for replication, mutation, and selection. Therefore, some explanation should be invoked to account for that whether homochiral sequences could be selected and kept under the replication-mutation processes at prebiotic stage of evolution. It is for this reason, the problem of the origin of rather complex homochiral macromolecules is of special interest in the theory of evolution.

2.

Evolution as DifTusion First, we discuss the origin of biomacromolecular homochirality in a fi"amework of the widely known model of evolution suggested by M. Eigen and coworkers [9, 10]. This model has also been reviewed in a number of publications [11-19]. Let us consider sequences /, (/=1,2. .2^, N»\) of L-, D-units. All possible sequences of length N are usefiilly to be described by a metric sequences space, in particular, by A^dimensional hypercube. Each point in the sequences space represents a particular sequence. Each of A^' neighbors of a given sequence differs from the last by chiral configuration of only one N units, i. e. each neighbour differs in a "point mutation". A distance between two sequences, /, and //, is measured by a minimal number of successive unit steps (point mutations) allowing transitionfi-om/, to /;. It is called the Hamming distance. Let us introduce the equations describing replication-mutation processes. In the quasispecies evolution model [10], the equations have the form; dx -± = ( 4 e „ -B^ ~cp)x^^Y.^,Q,^x, , a - 1,2...2" (1) where x. =c,0)/^c'.(r) is the relative part of sequences // among all sequences, c^(r) is the concentration of/;. The rate parameters A^ and B^ define replication and degeneration of the sequence /,, respectively. The value {A^ - B^) is referred to fitness o. of the sequence //. Parameter (p means the velocity of an outflow across the system. The total concentration of sequences being constant, the value (p is equal to mean fitness over the system.

Origin of Biomacromolecular Homochirality: in Search of Evolutional Dynamics

5

^ = 0 . This type of landscapes may be associated with the conditions, under which both short and long mutation jumps are realized with commensurable probabilities. Contrary to logarithmic landscapes, exponential landscape are characterized by an exponential suppression of the long mutation jumps, >o(|;c->'|^)~exp[-a|x->'|^J. Finally, on can consider an "intermediate" kind of landscapes - power landscapes with power incidence of the transition probability with respect to the mutation jump length,

Origin of Biomacromolecular Homochirality: in Search of Evolutional Dynamics

11

The solution of the equation (3) for the above three hierarchical landscapes have been found in [33]. A sketch of these results is as follow. The long-time behaviour of population degradation constrained by logarithmic landscapes is described by the Kohlrausch-Williams-Watts law, S{t) ~ exp[- 0 / T)"" J 0 < a < 1 . The long-time behaviour of population degradation constrained by linear landscapes is described by the power decay law, S{t)- (t/r)"' 0 < a . The long-time behaviour of population degradation constrained by exponential landscapes is described by the logarithmic decay law, S(t) ~ a[ln(r/ r)]"^ 1 < a. These results certainly give us an understanding how evolution is governed by long mutation jumps. However, a major intrigue lies in the questions what kind of error catastrophe is characteristic for the evolutional dynamics constrained by hierarchical fitness landscape. The answer may give some reasons to search hierarchically constrained dynamics capable to selection of homochiral macromolecules at the prebiotic stages of evolution.

6.

References

O.B. Ptitsin and M.V. Volkenstain, /. Biomol. Struct. Dynamics 4 (1986) 137-156. J. Monod, Chance and Necessity: an Essay on the Natural Philosophy of Modern Biology, Vintage Books, New York, 1971. [3 C.-K. Peng, S.V. Buldyrev, A.L. Goldberg, S. Havlin, F. Sciortino, M. Simons andH.E. Stanley, Nature 356 (1992) 168-170. 14 V.S. Pande, A.Y. Grosberg and T. Tanaka, Proa Nat. Acad Sci. USA 91 (1994) 12972-12975. [5 V.I. Goldanskii, V.A. Avetisov and VV. Kuz'min, FEES Lett. 101 (1986) 181-183. [6 G.F. Joyce, G.M. Visser, C. A. A. van Boeckel J.H. van Boom, L.E. Orgel and J. van Westrenen, Nature 310(1984)602-604. [7; G.F. Joyce, A.W. Schwartz, S.L. Miller and L.E. Orgel, Proc. Nat. Acad Sci. USA 84 (1987) 4398-4402. V.A. Avetisov, V.I. Goldanskii and VV. Kuz'min, Physics Today 44 (1991) 33-41. [9: M. Eigen, Naurwissenschaften 58 (1971) 465-526. [10 M. Eigen, J. McCaskii and P.J. Schuster, J. Phys. Chem. 92 (1988) 6881-6891. 111 B. Derida and L. Peliti, Bull. Math. Biol. 53 (1991) 355-363. 112 P. Tarazona, Phys. Rev. A 45 (1992) 6038-6050. [13 S. Franz, L. Peliti and M. Sellitto, J. Phys. A.: Math. Gen. 26 (1993) L1195-L1199. S. Franz and L. Peliti, J. Phys. A.: Math. Gen. 30 (1997) 4481-4487. [U [15 S. Galluccio, Phys. Rev. E. 56 (1997) 4526-4539. [16 F. Slanina and M. Kotrla, Phys. Rev. Lett. 83 (1999) 5587-5589. [17 F. Slanina and M. Kotrla, Phys. Rev. E 62 (2000) 6170-6179. [18 L. Peliti, Europhys. Lett. 57 (2002) 745-748. [19 V.A. Avetisov, in: Fundamentals of Life (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002, pp. 361-367. [20 V.A. Avetisov and V.I. Goldanskii, Physics-Uspekhi, 39 (1996) 819-835 [21 V.A. Avetisov and V.I. Goldanskii, Proc. Nat. Acad Sci. USA 93 (1996) 11435-11442. [22 V.I. Goldanskii and V.V Kuz'min, Physics-Uspekhi, 32 (1989) 1-49. [23 S. Kauffman, The Origin of Order, Oxford University Press, New York, 1993. [24 D. Sherrington, Physica D 107 (1997) 117-121. [25 M. Mezard, G. Parisi and M. Virasoro, Spin-Glass Theory and Beyond, World Scientific, Singapore, 1987. [26 H. Frauenfelder, Nature Struct. Biol. 2 (1995) 821-823. [27 D.T. Leeson and D.A. Wiersma, Nature Struct. Biol. 2 (1995) 849-852. [28 H. Frauenfelder and D.T. Leeson, Nature Struct. Biol. 5 (1998) 757-759. [29 D.J. Wales, M.A. Miller andT.R. Walsh, Nature 394 (1998) 758-760. [1 [2

12


[30] [31] [32] [33]

R. Raminal, G. Toulouse and M.A. Viiasoro, Rev. Mod. Phys. 58 (1986) 765-788. V.A. Avetisov, A.K. Bikulov and S.V. Kosyrev, J. Phys. A.:Math. Gen. 32 (1999) 8785-8791. V.A. Avetisov, A.K. Bikulov, S.V. Kosyrev, and V.A. Osipov, J. Phys. A.: Math. Gen. 35 (2002) 177-189. V.A. Avetisov, A.K. Bikulov and V.A. Osipov. J. Phys. A.: Math. Gen. 36 (2003) 4239-4246.


Chapter 2 Carbon Monoxide Clusters in the Formation of D-Sugars, LAmino Adds, Ribonucleotides and Deoxyribonucleic Acids in Prebiotic Molecular Evolution on Earth Nigel Aylward, Neville Bofinger* School of Natural Resource Sciences, Queensland University of Technology, George St., Brisbane, Queensland 4000, Australia n. aylward@student. qui. edu. au

1.

Introduction A property of most biological molecules is molecular asymmetry or chirality (Greek for hand) [1], such that the molecules are not identical to their mirror images, termed enantiomorphs. Most sugars found in nature are D-sugars, which are related to Dglyceraldehyde [2], whilst most amino acids found in proteins belong to the L-stereochemical series related to L-glyceraldehyde. Since the discovery of the configuration of glucose [3], absolute asymmetric synthesis has turned out to be a major challenge [4]. Recent successful studies have used photochemistry with circularly polarised light [5], chiral selection based on the electroweak interaction (although disputed) [6], and the combination of a magnetic field and non-polarized Ught [7], amongst others [8]. This paper proposes a prebiotic route to asymmetric sugars and amino acids based on adsorption of carbon monoxide on a cyclic porphin template. This seems possible as porphyrins have been shown to be present as geological deposits on Earth [9], form chargetransfer complexes with nitrogen containing compounds [10], and exhibit magneto-optical rotatory dispersion [11]. The synthesis is based on spontaneous reactions in a presumed primeval atmosphere. Although geological records of conditions in the atmosphere and hydrosphere of the early Earth are almost completely lacking [12], an atmosphere composed of hydrogen cyanide, formaldehyde, ammonia, hydrogen, carbon monoxide and water should give a large range of organic compounds [13]. For this work the assumption is made that carbon monoxide, and water were present and photolysis of water could have lead to a reducing medium [14]. An examination of the total energy of these molecules and their transformation products; sugars and amino acids; suggests that the latter could have arisen from reactions in a primeval atmosphere that may have existed on Earth [12], and would be expected to occur somewhere in the Universe provided that a suitable reaction mechanism could be envisaged. For the spontaneous chemical formation of these biologically important molecules it is

14

Progress in Biological Chiiality

satisfying if all are formed from the same initial milieu of reactants by established chemical reactions that are kinetically feasible and preferably thermodynamically favoured. Here, one such set of reactions is proposed based on the availability of the primary reactants, thermodynamic, and kinetic considerations.

2.

A Theory of Asymmetric Induction As this work is applied theoretical chemistry a simplified theory is given here to justify the suggested asymmetric induction. The following considerations apply to an adduct of carbon monoxide with the magnesium porphin complex of approximately C4V symmetry as designated in Fig. 1. Porphin and its derivatives [15], have been treated by the free electron model [16, 17], as an 18-membered benzenoidal ring where the secular equation becomes identical in form to the LCAO secular equation for the problem [15]. Porphin and tetrahydroporphin have been treated under the point group D4h using LCAOs to obtain more accurate wavefunctions and molecular orbital energies [18]. These are also available from present day ah initio calculations. Using the free-electronmodel or particle on a ring [19], the energy levels are given by. En^

n - 0 , 1,-1,2,-2,.

where the quantization is evident, and the wavefunctions by,

yn((t))=-V^""

n = 0, 1,-1, 2,-2,

Magnetic Field

Electric Field

Rl

MgCOporphin (2) Figure 1. The orientation and charges of the MgCOporphin charge transfer adduct indicating the in-plane electric vector and the perpendicular magnetic vector of theradiationto cause an allowed A2 -> E transition

Carbon Monoxide Clusters in the Formation of D-Sugars, L-Amino Acids, ...

15

The quantum numbers of the highest filled level are + 4 or -4, and of the lowest unfilled level, + 5 or -5, hence the transition is fourfold degenerate [15]. The electric transition moment for this transition is given by the integral, < \|/* I m I \|/> dx and for light polarized in the x-direction, the integral is [15]: rp/47i <e-'^* le^* + e " * | e ' ^ ^ > ^ 0 where rp is the radius of the circle. A consideration of the four transition moments shows that the allowed bands correspond to changes of the quantum number of+1 or -1. The angular momentum in this model is quantized and perpendicular to the plane of the ring with a value, Mz = m hJ2n

m = 0, 1, -1, 2, -2,...

where m is the magnetic quantum number - the same as the quantum number for the zcomponent of angular momentum. Thus, the interaction of the orbital motion of an electron with a magnetic field is quantized, only the values, E = - m pm | H |

m = 0, 1,-1, 2,-2,...

being possible, where the atomic unit of magnetic moment is given by [20], Pm= -eh/4^mec Using time-dependent perturbation theory [20], the electric moment induced by the magnetic field of the radiation perpendicular to the porphin plane is given by an expression proportional to the product of the electric and magnetic transition moments, where, mko =

and,

jdzok =

and H is the applied field. The presence of the radiation magnetic field H [21], perturbs the ground and excited wavefunctions requiring that these are described using a summation of higher eigenstates. The presence of these is sufficient for there to be an oscillating electric transition moment perpendicular to the ring which is finite. Thus, the presence of the magnetic field pointing upwards (i.e. arising from the direction of the incident radiation). Fig. 1, together with the allowed in-plane A2 -> E electronic transition from the electric transition moment, should result in an oscillating in-phase electric field perpendicular to the plane, and an angular momentum of the ring electrons in accordance with Lenz's law [22]. The electric transition imparts sufficient energy to the charge transfer adduct [10], (calculated as 0.2479 h from HOMO to LUMO) for the activation energy to be overcome. Fig. 2, and the dissociation energy to be exceeded, such that the carbon monoxide molecule moves to the periphery of the ring system where a transition state consisting of an aziridine-ring is ormed. The

16


Y DISTANCE(A)

X DISTANCE(A)

Figure 2. Total Energy (-1297 + X haitree) of the MgCO-porphin complex. Origin on the Mg"^ ion, x-axis through a pyrrole N atom, y-axis in-plane

carbon monoxide entity of the charge transfer complex is positively charged so that during half the cycle of the electric transition moment perpendicular to the ring this assists in increasing its height above the ring as shown in Fig. 3, and an increase in the energy of the system as shown in Fig 2.

R2 DISTANCE(A)

0.5

1 1.5 R1 DISTANCEfA)

Figure 3. Height of the caiixm monoxide entity above the ring as it traversesfromthe centre at coordinate (0,0,0) to near a pyrrole nitrogen atom. The coordinates of the caibon of the CO entity are at (2.3,0.6, 1.3). The ordinate A and abscissae are in miits of 10 '^ m (Angstrom)

Carbon Monoxide Clusters in the Formation of D-Sugars, L-Amino Acids,

17

-200

-400

R2 DISTANCE(A)

1

1

R1 DISTANCE(A)

Figure 4. The potential energy surface of the acetaldimine-carbon monoxide adduct on the surface of a porphin molecule. Magnesium ion at coordinate (0,0,0) with coordinated imine. Carbon monoxide entity at the transition state near a pyrrole nitrogen atom. The ordinate is the total energy (-1427.0 + X.10-3) (hartree)

This formation of a carbon monoxide adduct is postulated to be repeatable, with the subsequent formation of high energy reactive transition complexes. However, after the initial transition complex is formed the bonding in the porphin ring system is more fixed, with anticlockwise transition moments leading to reactions. On reduction these complexes should yield D-sugars. Moreover, the formation of charge-transfer complexes with imines predisposes them to form aziridine complexes hydrolysable to L-amino acids preferentially, as shown in Fig. 4.

3.

Methods of Calculation The computations tabulated in this paper used the SPARTAN (1994) and GAUSSIAN98(1998) packages, [23] using IBM RISC 6000 and SP2 computers. Equilibrium structures were calculated at the HF/6-31G* and MP2/6-31G* level. For the large porphins energies more accurate than MP2 were not available. For jobs run under GAUSSIAN98, geometry optimizations used the VTIGHT option. The basis set used in the MP2 geometry optimizations was the standard split-valence double zeta basis set, augmented with a d-type polarization function on the non-hydrogen atoms [24]. The lowest ab initio level of theory which provides an account of the dispersion forces and correlation correction to the dipole moment is the MP2 level (the second order MoUer-Plesset perturbation theory) [25]. This is a suitable level of theory for the systems considered in the present work. Since theoretical Hartree-Fock frequencies tend to exceed known experimental values, the calculated frequencies were scaled by a factor of 0.89, when used to derive zero point vibrational energies (ZPE) and thermochemical corrections to the total energies. This is

18


consistent with the recommendation of Pople et al. [26]. The zero-point energies were calculated at the HF level of accuracy. Calculations in this paper that use only HF energies without adding the zero-point energy are explicitly indicated with a subscript, e.g., A H(HF) The energy unit used for these calculations is the atomic unit for energy, the hartree. 1 hartree = 627.5095 kcal. mol"^ [23].

4.

Results

4.1 Total energies (hartrees) The total energies and zero point energies for the HF and MP2/6-31G* equilibrium geometries are given in Table 1. 4.2 The prebiotic synthesis ofD-sugars andL-amino acids The reactions proposed for the formation of D-glyceraldehyde and L-alanine involve the reactions of carbon monoxide either with itself (condensation) or with an imine as follows: Table 1. HF and MP2 /6-3IG* total energies (hartrees) for the respective equilibrium geometries Molecule (1) Mgporphin Mgporphin (triplet, 6-3IG**) (2) Mg(CO)porphin (3) MgporphinCO (4) Mgporphin(CO)3 (5) Mgporphin(CO-)3 (6) MgCOporphin(CO)2(CO-)2 (7) Mg(CO-)porphin(CO-), (8) Mg(C(OH)-)porphinCO(C(OH))2CH(OHr (9) Mg(C(OP=0-))-porphinCOC(OH)COC(OH)CH-0(10) Mg(C(OH)-)poiphinCOC=C(OH)CH(OH)^^ (11) Mg(C(OH)-)porphinCOUCHC(OH)CH(OH)^" (U-uridine) porphin Mg(CH3CH=NH)porphinCO Mg(CH3CH-NH)porphin \C0/ MgCOporphin(CO-CO)2 glyceraldehyde deoxyribose ribose CO H2 HO-P=0 0-P=0 OH" OH 2-methyl aziridine-3-one CH3-CH=NH uracil H2O

HF hartree

MP2 hartree

-1181.86208

-1185.12250 -1185.10392 -1298.13452 -1297.93784

-1294.60561 -1294.48601 -1519.80735 -1519.79776 -1745.17902 -1745.14346 -1747.89231 -2237.45751 -1672.19465 -2084.83363 -983.25693 -1427.59183 -1427.63029 -1745.17902 -341.64958 -494.58123 -569.43280 -112.73434 -1.12683 -491.05064 -490.49517 -75.32660 -75.38228 -245.77318 -133.07280 -412.47029 -76.01075

ZPE (HF) hartree

-1524.00686 -1749.95424 -1750.01626

-986.48501

0.31165

-1431.64414 -1749.97463 -342.56404 -495.93959 -570.97086 -113.02818 -1.14414

-246.48876 -133.49001 -413.63259 -76.19924

0.10256 0.17281 0.17816 0.00484 0.01033 0.01942 0.00711

0.08481 0.07392 0.02148

Carbon Monoxide Clusters in the Formation of D-Sugars, L-Amino Acids, 3C0

+

3H2 -

19

C3H6O3

glyceraldehyde AH(MP2/ZPE) = 0.00368 h

CH3-CH = NH + CO AH(MP2/ZPE) = 0.03481 h

CH3-CH - NH Rl \ / R2 CO

However, it is well known that these reactions do not proceed easily. A mechanism is needed whereby this could have occurred. The template proposed here is that of original porphin, and the mechanism is similar to present photosynthesis. However, the usual reliance on thermodynamic data or kinetic data will not justify the formation of the transition complex as photosynthesis as usually stated involves the input of electromagnetic energy without which it is less efficient. It is assumed here that the energy needed to form the complexes is provided by the radiation. 4.3 The formation of the magnesium-porphm'carbon monoxide complex In present day chlorophyll the porphin moiety often contains a magnesium ion. This structure has been taken as a basic template for the postulated sugar synthesis. It has been stated that there is not a distinction between the principal and subsidiary valences in the metallic complexes [18]. The reaction in Scheme 1 is proposed as the route by which carbon monoxide first complexes with magnesium chelated porphin.

Mg*porphin + CO

Mg-(CO)-porphin AH(HF) = -0.00919 h Scheme 1

(2)

If the Mg-porphin (triplet state) is in the triplet state, the enthalpy change is also favourable, as follows: A H(MP2/6-3iG**) ~ -0.10388 h

It is postulated that when the complex becomes excited from photolysis according to the equation, Mg-(CO)-porphin-

Mg-porphin-CO

20


the CO group is able to move through a transition state to the porphin ring, as shown. The energy of the product is almost identical to that of the excited reactant, but the excitation energy is more than the activation energy for the transformation.

Mg(CO)-porphin

(2)

Mg-porphinCO

(3)

AH(MP2) = 0.19668 h The potential energy surface for the magnesium-porphin-carbon monoxide comple is shown in Fig. 2. 4.4 The isomeric states of the magnesium-porphin complex The magnesium complex can be described classically by either of the two structures shown in Scheme 2.

Mg-porphin (la)

Mg-porphin (lb) Scheme 2

The actual structure may be resonant between them. However, when the CO group migrates to the ring it must pick a C=N bond from either structure (la) or (lb). The results of the following analysis are built on the assumptions of the model that the surface to react with the carbon monoxide is the same surface on which the radiation is incident. If the magnesium-porphin-carbon monoxide complex is considered approximately under the point group C4v, then the HOMO of symmetry A2 and LUMO of symmetry E, as are shown in Fig. 5. The A2 -> E transition between these states is allowed as previously illustrated [15]. The electric vector is expected to be in the plane of the porphin ring, with the magnetic vector perpendicular to the ring, as shown.

Carbon Monoxide Clusters in the Formation of D-Sugars, L-Amino Acids. ... (a)

^

21

(b)

Figure 5. (a) MgporphinCO HOMO and (b) MgporphinCO LUMO

This should result in a very small induced electric dipole moment perpendicular to the ring, whose direction should be in a direction to assist the carbon monoxide moiety to gain height above the ring whilst approaching the transition state, as shown in Fig. 3. With the magnetic vector perpendicular to the ring the electronic charge is expected to precess counterclockwise (when viewed from above) in accord with Lenz's law, to produce resonance form (la), whilst the induced electric dipole should also point upwards to assist the elevation of the carbon monoxide moiety, and result in the formation of a complex with form (la). Conversely, when the magnetic field of the radiation points downwards, the induced circulation of electron charge is expected to be clockwise, in accord with Lenz's law, but the induced electric dipole should point downwards thwarting the elevation of the carbon monoxide,moiety. The net result is that when reaction occurs it will be supposed to be with structure (la). If the magnesium ion subsequently takes other ligands then the CO group may preferentially react with those slightly counterclockwise to itself rather than those clockwise to itself Similarly, if clusters of CO groups are present on the ring, the transition moment is supposed to be more effective in linking them if moving counter-clockwise than clockwise i.e. it is supposed that the direction of the net electronic transition moment is important. The activation energy to bond the carbon monoxide entities is less than 80 kcal.mol" Wave functions for the LUMO suggest that it has a high TT* character as does the ground state of the magnesium-porphin-CO complex. This is here illustrated with the formation of a three-carbon bonded complex reducible to glyceraldehyde and glycerol.

Mg-porphin-(CO-)3 (5) radical AH(HF) = 0.00959 h

Mg-porphin-(CO)3 (4)

22


4.5 The formation of L-amino acids It is supposed that an imine residue such as acetaldimine coordinated to the magnesium ion can react with the high energy complexed CO group on the surface periphery of the porphin ring, but it can only approach for optimum reactivity in one orientation, as shown: Mg-porphin-CO + CH3CH=NH AH(HF) = -0.03303 h Mg.(CH3CH=NH)porphinCO AH(HF) = -0.03846 h

^ Mg-(CH3CH=NH)-porphin-CO

. Mg(CH3CH-NH)porphin

\ / CO

MgacetaldimineporphinCO complex The potential energy surface to form the acetaldimine - carbon monoxide complex is shown in Fig. 4. From this diagram the activation energy to form the 3-methyl aziridone is calculated as 0.08 h. A drawing of the transition state is given in Fig. 6.

Figure 6. Transition state of the MgacetaldimineporphinCO complex

Carbon Monoxide Clusters in the Formation of D-Sugars, L-Amino Acids,

23

4.6 The formation of carbohydrates from carbon monoxide and hydrogen The justification for the premises in this paper have a firm basis in the favourable energy changes involved in the conversion of carbon monoxide and hydrogen to sugars as exemplified by the following two equations involving the production of 2'-deoxyribose and ribose, respectively. 5 CO + 6H2

-^

C5H10O4 + H2O deoxyribose

The equation gives the enthalpy of reaction as, AH(MP2/zPE) = -0.03690 h This indicates that this is an exothermic reaction and may be spontaneous. Similarly, for the formation of ribose the equation is, 5 CO + 5H2

^ C5H10O5 ribose

The equation gives the enthalpy of reaction as, AH(MP2/zPE)= -0.01824 h Again, the enthalpy change suggests that the reaction could be spontaneous. However, the usual reliance on thermodynamic data or kinetic data will not justify the formation of the transition complex as photosynthesis as usually stated involves the input of electromagnetic energy without which it is less efficient. It is assumed here that the energy needed to form the complexes is provided by the radiation. 4.7 The formation of the pentoses The main interest is the formation of D-ribose and D-2'-deoxyribose from the bonding of 5 CO groups complexed to the porphin ring.

Mg(CO)porphin(CO)2(CO-)2 (6)

Mg-(CO-)-porphin-(CO-)4 (7) radical AH(MP2) = -0.06203 h

24


The pentacovalent complex is shown in Fig. 7. It is not unique as there are at least two other similar structures for the complex of entirely equivalent energy that might be expected to be reducible to D-arabinose and D-xylose. In the reduction of this complex it is assumed that the precessional movement of the electrons allows a proton to be bonded, with the increase of charge to +1, as shown.

+ 2H2 +

HT

Mg-(CO-)-porphin-(CO-)4 (7)

Mg(C(OH).)porphin-CO-(C(OH))2-CH(OH)'^ (8)

This reaction is energetically favoured. AH =-0.49519 h

Carbon Monoxide Clusters in the Formation of D-Sugars, L-Amino Acids, ... 4.8 The formation of ribose phosphate The supposition is that this just involves the addition of free radical electrons to the phosphorus atom shells of metaphosphite, whilst it is complexed to the Mg^^ ion, as shown. MgCOporphin(CO-CO)2 + HO-P=0 + H2

• Mg(COP02')porphin'(CO-(COH)2CHO-)

AH(HF) = -0.10102 h

*^

^

^

#

"-^^m' ^fe^

Mg-C(OPO-)-porphin-(CO-C(OH)-CO-C(OH)-CHO-) complex

W This may be compared to the following reaction involving charged species. Mg- COH)-porphin-(CO-(COH)2-CHOH)'^ -^ Mg-(COP02-)-porphin-(CO-(COH)2-CHO-) + 0-P=0' + H2 AH(HF) = -0.19686 h 4.9 The formation of a ribose ketene This reduced complex may lose hydroxyl ion to produce a ketene according to the energetically favoured reaction shown, HOC

+ H" Mg(C(OH)-)porphinCO(C(OH))2CH(OH)'^ (8)

+ H2O Mg(C(OH)-)porphinCOC=C(OH)CH(OH)^'^ (10)

AH =-0.31309 h

25

26


4.10 The formation of a D-2 '-deoxyhbose nucleoside This ketene is of high enough energy for the reaction with a nucleic acid base to be favourable, as shown,

+ U MgC(OH)-)porphinCOCC(OH)-CH(OH)^^ MgC(OH)-porphinCOUCHC(OH)CH(OH)'^ (10) (11) AH =-0.16868 h The structure of the nucleoside is shown in Fig. 8. Reduction of these complexes by the products of photolysis [14], should yield sugars, nucleosides or nucleotides. porphin-H20 + hv —• porphin^ H20-

+

H2O"

^ H- -f OH-

porphin^ + H2O —• porphin + H20^ H20^

•H^

+ OH

The reductions are always favourable as the molecules are of high energy resulting from the many photochemical excitations.

Figure 8. The structure of the nucleoside MgC(OH)-porphinCOUCHC(OH)CH(OH)^

Carbon Monoxide Clusters in the Formation of D-Sugars, L-Amino Acids, ...

27

eg. Mg(COH)porphin-(CO-(COH)2-CHOH)^ + BHi-^Mgporphin + HOCH2-(CH(OH))3-CHO + H ribose A H(HF) = -0.02209 h

5.

Discussion The exact sequence of reactions leading to ribose, deoxyribose, the nucleosides, and nucleotides, is open to conjecture, but it is postulated here to proceed via carbon monoxide oriented clusters excited by radiation. In the reduction and protonation of the Mg-porphin-(CO)5, it is expected that other acceptor molecules may be used apart from the proton. For example formaldehyde might lead to the hexoses. Similarly trimethyl sulphide cation may lead to the sugars rhamnose and fucose. The 1-CO group is similarly expected to be susceptible to attack by formaldehyde anion giving hexoses, whilst glyoxal anion might lead to heptoses. Finally, the ease of producing 2,3-unsaturated compounds leads to a strong indication of the very early formation of ascorbic acid in prebiotic molecular evolution.

6.

Conclusion From the postulates presented here it is clear that prebiotic paths to the biologically active sugars are conceivable. Further work at a higher level of accuracy may provide some more data to support this work and may change the enthalpy values tabulated in this paper.

7.

Acknowledgements Appreciation is expressed to Queensland University of Technology Centre for Instrumental and Developmental Chemistry for the equipment and facilities; to Mr. A. Lewis, S. Walsh, and Dr. J. Young and M. Barry, B. Savage, and A. Rasmussen of the Supercomputing Department, and Dr. A. Wiegand. N. Aylward is grateful for the award of a scholarship from QUT.

8.

References

[1] (a) G. Palyi, C. Zucchi and L. Caglioti, Eds., Fundamentals of Life, Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002. (b) G. Palyi, C. Zucchi and L. Caglioti, Eds., Advances in BioChirality, Elsevier, Amsterdam, 1999. (c) I. Tinoco, K. Sauer and J.C. Wang, Physical Chemistry, Prentice-Hall Inc., Englewood Cliffs, N.J., 1978. (d) R. Janoschek, Ed., Chirality from Weak Bosons to the Alpha Helix, Springer-Verlag, Berlin, 1991. (e) E.H. Rodd, Ed., Chemistry of Carbon Compounds, Elsevier, Amsterdam, 1951. [2] A.L. Lehninger, Biochemistry, Worth Pubhshers Inc., N.Y., 1975. [3] C.S. Hudson,^^. Carb. Chem. 3 (1948) 1-22. [4] B.L. Feringa and R.A. van Delden, Angew. Chem., Int. Ed 38 (1999) 3418-3438. [5] N.P.M. Huck, W.F.Jager, B. de Lange and B.L. Feringa, Science 111> (1996) 1686-1688. [6] R. Berger and M. Quack, J. Chem. Phys. 112 (2001) 3148-3158. [7] G.L.J.A. Rikken and E. Raupach, Nature 405 (2000) 932-935. [8] B.L. Feringa, Science 292 (2001) 2021-2022.

28 [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

[23]

[24] [25] [26]

Progress in Biological Chirality R. S. Czemuszewicz, J. Porphyrins Phthalocvanines 4 (2000) 426-431. D.G. Whitten, I.G. Lopp and JP.D. Wildes, J.Am. Chem. Soc. 90 (1968) 7196-7200. P.J. Stephens, W. Suetaak and P.N. Schatz, J. Chem. Phys. 44 (1966) 4592-4602. (a) E. Nisbct and N. Sleep. Nature 409 (2001) 1083-1093. (b) G. Palyi and C. Zucchi, Orig. Life Evol. Biosphere 30 (2000) 172. F. Raulin, Orig. Life Evol. Biosphere 30 (2000) 116-117. F.K. Fong, Light Reaction Path of Photosynthesis, Springer-Verlag, Berlin, 1982. W.T. Simpson, J. Chem. Phys. 17 (1949) 1218-1221. (a) N.S. Bayliss, J. Chem. Phys. 16 (1948) 287-292. (b) J.R. Piatt, J. Chem. Phys. 17 (1949) 484-495. W.T. Simpson, J. Chem. Phys. 16 (1948) 1124-1136. H.C. Longuet-Higgins, C.W. Rector and JR. Piatt, J. Chem. Phys. 18 (1950) 1174-1181. P.W. Atkins, Molecular Quantum Mechanics, Clarendon Press, Oxford, 1970. W. Kauzmann, Quantum Chemistry, Academic Press Inc., N.Y.. 1957. J.T. Hougen, J. Chem. Phys. 32 (1960) 1122-1125. (a) D.C. Giancoli, Physics, Prentice Hall Int. Inc., Englewood Cliffs, New Jersey 1985. (b) R.L. Weber, K.V. Manning, M.W. White and G.A. Weygand, College Physics, McGraw-Hill Book Company, N.Y., 1959. (a) SPARTAN (1994) Ver. 3.1, Wavefunction Inc., 18401 Von Karman Avenue, Irvine, Cahfomia 92715, U.S.A. (b) Gaussian98 (1998) Users Reference. Gaussian Inc., Carnegie Office Park, Bldg.6., Pittsburgh, PA 15106. USA. P C . Hariharan and J.A. Poplc, Theoret. Chim. Acta 28 (1973) 213-222. C. MoUcr and M.S. Plesset, Phys Rev. 46 (1934) 618-622. J.A. Pople, H.B. Schlegel, R. Krishnan, D.J. De Frees, J.S. Binkley, M.J. Frisch, R.A. Whiteside, R.J. Hout and W.J. Hehre. Int. J. Quantum Chem. Symp. S 15 (1981) 269-278.


Chapter 3 Molecular Clockworks as Potential Models for Biological Chirality Lajos Bencze,^'* Claudia Zucchi,^ Luciano Caglioti,'' and Gyula Palyi"*'* "^ Muller Laboratory, Department of Organic Chemistry, University of Veszprem, P.O. Box J58, Veszprem, H-8201, Hungary [email protected] Department of Chemistry, University ofModena and Reggio Emilia, Via Campi 183, Modena, 1-41100, Italy [email protected] ^ Department of Chemistry and Technology of Biologically Active Compounds, University "La Sapienza", P.le A. Moro 5,1-00185 Roma, Italy

1.

Introduction The origin of biological (homo)chirality is one of the last (?) classical great secrets of Natural Sciences [1]. Several elegant approaches have been postulated for the solution of this mystery [2]. These include the photochemical effect of circularly polarized sunlight in Nature [3], the influence of asymmetry of weak nuclear forces on molecular reactivity [4], autocatalytic amplification of mineral morphological (macroscopic) chirality [5] and other highly intellectual working hypotheses. We suspect that the interaction of coordinated ligands (prominently: chiral plus pro-chiral) may exert an, at least partial, influence on the origin of biological (or "pre-biological") enantioselection, on the maintenance of its level [6] or on the development of biological signal transduction systems [7]. Interestingly, the main focus of the research aimed at obtaining molecular level enantioselection is at the meeting point of two other great concerns of contemporaneous chemistry, namely (a) to achieve enantioselective syntheses by molecular catalysis [8] and (b) to miniaturize down to the molecular level [9]. Both are considered in the pioneering papers of Kelly et al. [10], reporting partially hindered molecular systems, where the reduction of rotational freedom produces brake/cogwheel like interactions. This principle, combined with application of photochemistry, is used also in the so-called molecular switches [11]. We have studied some intermediates of coordination catalysis from this standpoint. Here we summarize the results obtained with mononuclear complexes, alkylcobalt carbonyls, which are well known from C,C-bond making catalysis [12].

30

Progress in Biological Chiralin

RO

/^CH.

O

oc Co

CO

p^ -Ph Ph

Figure 1. Conformations of the of the ester group and of the triphenyl phosphine ligand (notation)

2.

Structural Studies X-Ray diffraction of crystalline phases of more than a dozen flexible ROC(0)CH2Co(CO)3PR'3 (1) type alkylcobalt carbonyl phosphine complexes showed [13, 14] that these complexes form enantiomeric conformations with achiral R and R'. Theseconformations are characterized by the opposed helical chirality of the PR'3 (mostly R' = phenyl) ligands [15] and each of these enantiomeric structures is accompanied by only one conformation of the ester fragment (Fig. 1). Essentially, the system exhibits 100% diastereomeric yield. This particular situation, which leads to a dramatic reduction in the number of the observed conformers, with respect to the number of statistically possible (energetically apparently equivalent) ones, prompted us to perform molecular mechanical and quantum chemical calculations on one of these compounds: [(ethoxycarbonyl)methyl]cobalt tricarbonyl triphenylphosphine, EtOC(0)CH2Co(CO)3PPh3. These studies enabled an attempt to be made at performing a preparative/structural evaluation of the theoretical considerations.

3.

Quantum Chemistry Equilibrium energy calculations [16, 17] showed that a//"statistically" possible, observed and non-observed stereoisomers are energetically equivalent in the ground state, within the limits of computational error. Thus, it should be concluded that the conformation preferences ("selection rules") observed for the crystalline phases are not (prevalently) of ''thermodynamic" origin. Consequently, the possible transformation routes ("racemization")

Molecular Clockworks as Potential Models for Biological Chirality

31

O—Et „ „ -^"^^r^

OC

ester fragment

^,vCO Co ^.^^

cobalt carbonyl fragment

/7

triphenyl phosphine ligand

Figure 2. Schematic representation of the ester, cobalt carbonyl and triphenyl phosphine fragments in the molecules

were analysed by quantum chemical methods [20]. These promised to afford greater insight into the forces operative in the intramolecular self-organization. Based on ground-state energy calculations, molecular graphical studies, rigid and relaxed rotor approach conformation analyses^ it is deduced that the mechanism of the inversion proceed with correlated motions of coaxial rotors, coupled bevel gear like rotors with different periodicity and the first example of a coupled conrotation ever observed in molecular systems. The most informative potential function is shown in Fig. 3. The results indicate that the rotation of the ester group (Fig. 2) to left or right proceeds with different energies because of steric crowding. As the ester group rotates, this induces a "concerted" rotation of the Co(CO)3 moiety in the equatorial plane of the complex. On the other hand, rotation of the Co(CO)3 moiety in the equatorial plane induces reorientation of the phenyl groups on the phosphine ligand through repulsive interactions with the ortho-H atoms of the Ph rings. Consequently, if the ester group spontaneously reorients from re position, then it will turn preferentially to left (towards the ester carbonyl group) causing clockwise (con)rotation of the Co(CO)3 moiety, which again generates M conformation of the PPhs ligand (Fig. 4). Thus, once rotated, the ester group will be in si position, accompanied by M-PPhs, while, starting from the ^/-ester, after rotation relY combination will develop, both being exactly what was observed experimentally in the crystalline phase. The steering effect of the ester group is also due to its rigidity, owing not only to the central sp^ carbon but also to the C-H O type H-bridge interaction [21], identifiable on the basis of the X-ray data, between one of the ethyl a-H-s and the ester carbonyl-0. It should be noted that this effect renders the two Et a-H atoms inequivalent, thus generating a centre of chirality on the ethyl-a-C atom. We analysed the correlation between the configuration of this carbon atom and the conformations developing in the crystalline phase, and found that the silM and re/P couplings always corresponded to the S a-C and R a-C configurations, respectively. ^ Racemization of the whole molecule involves the simultaneous rotation of 6 (formal) a-bonds, 16 dihedral angles and a total of 48 torsional interactions.

32

Progress in Biological Chirality 25

collision of the ester carbonyl group with the equatorial carbonyl ligands

collision of the ethoxy group with the equatorial carbonyl ligands

(0 0)

X

isomer _ re -50

175

-25

200

Dihedral Angle Co-C(H2)-Csp^=0 (©i), deg.

Figure 3. Conformational potential function of the ester group (torsion col) in EtOC(0)CH2Co(CO)3PPh3 The correspondence of the fragments of the EtOC(0)CH2Co(CO)3PPh3 molecule to the mechanism of parts of a classical (mechanical) clock [22] is shown in Fig. 4. These modelling studies revealed that the driving force of the high stereoselection is thermal energy, just as in the case of macromechanical devices, and the low-energy interconversion of enantiomers (when applicable) takes place through a clockwork mechanism, whereas the formation of the diastereomers corresponds to gear slippage, a malfunction also found in mechanical clocks. The ester fragment is always oriented quasi-parallel to the Co(CO)3 group (plane-to-plane angle 25°). Other possible conformations around the C{sp^)-Q{sp^) bond are not populated. Based on experimental data and density functional calculations it is concluded, that the

escapement

gear

balance

Figure 4. Mutual rotation of the fragments and analogy with mechanical clockwork in EtOC(0)CH2Co(CO)3PPh3


33

conformational chirality may be attributed mainly to the r|^ type coordination between the aester and the central cobalt atom [23]. This novel type of interaction (autosolvation) can open a new way for the consideration of esters as prochiral ligands invoking stereoselection amongst the incoming ligands and behaving as a source of chiral induction in catalysis, as well. Our X-ray studies [14, 24] on additional ROC(0)CH2Co(CO)3PR3 complexes showed different coupling of the si/re (ester) and MyP (phosphine) conformations, but always the same, or even larger reduction in the number of conformers. We attribute this to the influence of the R group on the potential curves. A greater reduction of the number of isomers was observed with chiral R or R' groups [25].

4.

Extension of the Clockwork-Principle We subjected the above structural/theoretical picture to a preparative/structural/spectroscopic analysis. First of all, it should be pointed out that the surprising early CD spectroscopic resuhs, obtained with chiral derivatives of complexes 1 [26], showing CD bands that could be attributed only to chiral perturbation of transitions involving the d electrons of the Co [23], are in excellent agreement with the novel structural and theoretical results [16, 23 d]. An interesting extension of the theoretical results obtained with an achiral complex 1 (R = Et) and structural observations with several additional achiral analogs was the introduction of centres of configurational chirality into the ester group. While at the achiral complexes the concerted development of chiral conformations resulted in the observation of only 50% of the statistically possible isomers, the introduction of, even racemic forms, of the 5Bu-group [25a] resulted in the development of only 25% of all possible isomers in the crystalline phase (Fig. 5). A solution spectroscopic (^H-NMR) study showed that this dramatic selection rule may be retained also in the liquid phase (Fig. 6). An additional possibility for a preparative/structural study can be deduced for other systems with C(5p^).CH2-Co-(CO)3-PPh3 sequences which should show comparable behaviour if our analysis is correct.

(a)

sQ\x (b)

(configuration)

S 1 R

S

12.5 % 1 si

Ester

25%

PPhs

50 %

R re

/////

S si

R

m

S

1R 1 re

Figure 5. Correlation of statistically possible isomers/conformers of ROC(0)CH2-Co(CO)3PPh3 complexes with those observed experimentally by X-ray diffraction. (a) R = achiral; (b) R = sBu; m observed combinations

34


Acetone - de

ppm

2.16

2.14

2.12

Figure 6. 'H-NMR spectrum of the a-CHj group of the 5BuOC(0)CH2Co(CO)3PPh3

A promising group of such compounds are the benzylcobalt carbonyl derivatives [27]. We have prepared and structurally characterized the following /rîw5-RCH2Co(CO)3PPh3 complexes: R = para-tBuC^- [28] (Fig. 7), meta-ClCeiU' [29] as well as two orthosubstituted phenylacetylcobah tricarbonyl triphenylphosphine derivatives (Fig. 8) [30]. These benzyl complexes show in the crystalline phase quasi-parallel position of the aromatic (benzyl) ring and the Co(CO)3fragment,as well as two helical isomers (M and P, in 1:1 ratio) of the PPha group. The symmetric para-tBu derivative shows two (crystallographically) independent molecules in the P f phase and their enantiomers (generated by an inversion centre), that is, a picture indicating the existence of all four isomers. Most interestingly the w^/a-Cl-derivative, which is asymmetric, but the Cl-substituent is farther from the carbonyl ligands (thus generating not overly strong crowding) shows the presence of all four isomers too, but in a

C13'

C12'

C24

C19'

Figure 7. ORTEP drawing of the two independent molecules (one enantiomer each) in the ciystalline phase of p-tBuC6H4CH2Co(CO)3PPh3 [28]


35

vC16

€34

IC33

C20/

C21

Figure 8. ORTEP drawing of the o-MeC6H4CH2C(0)Co(CO)3PPh3 and o-PhC6H4CH2-C(0)Co(CO)3PPh3 molecules (one enantiomer each) [30]

ratio of 12:38 % for each PPhs conformation (which however are in 50:50 % ratio). This latter effect perfectly reflects the "slipping" malfunction of the escapement/gear connection in mechanical clockworks [22]. These observations are in harmony with the theoretical analysis, and suggest directions of future preparative/structural and theoretical work. The striking similarity between this molecular system and the mechanical clock presents new synthetic and theoretical directions which are now under investigation in our laboratories. One of the most important of these directions is the connection of clockworktype steric hindrance to chiral induction, a phenomenon which is of utmost importance from the viewpoints of theoretical biology [1], of synthetic organic chemistry [8, 31] and perhaps even of the origin(s) of life on Earth [32].

5.

Acknowledgement The authors acknowledge financial support for this research to the [Hungarian] OTKA program (Grant Number T-016326, T-035221), to the [Italian] Ministry of University and Research (MURST) and [Italian] National Research Council (CNR) as well as Prof R.D. Adams (Columbus, SC, USA) for his comments on the manuscript.

6.

References

[1] G. P^yi, C. Zucchi and L. Caglioti, in: Advances in BioChirality (Eds. G. P^lyi, C. Zucchi and L. Caglioti) Elsevier, Amsterdam, 1999, pp. 3-12. [2] (a) L. Keszthelyi, Quart. Rev. Biophys. 28 (1995) 473-507. (b) L. Markd, Diss. Savariensis 24 (1998) 1-64. (c) G. P^yi, K. Micskei, L. Bencze and C. Zucchi, Magyar Kern. Lapja 58 (2003) 218-223.

36

Progress in Biological Chiralit>

[3] A. Vitkin, Opt. Photonics News 111 (1996) 30-33. [4] (a) A. Szabo-Nagy and L. Keszthelyi, Proc. Natl. Acad. Sci. USA 96 (1999) 4252-4255 and refs. therein. (b) W.A. Bonner, Chirality 12 (2000) 114-126. [5] (a) K. Soai, T. Shibata, H. Morioka and K. Choji, Nature 378 (1995) 767-768. (b) K. Soai and T. Shibata, in: Advances in BioChirality (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier, Amsterdam, 1999, pp. 125-136. (c) K. Soai, S. Osanai, K. Kadowaki, S. Yonekubo, T. Shibata and I. Sato, /. Am. Chem. Soc. ill (1999) 11235-11237. (d)K. Soai, T. Shibata and I. Sato,^cc. Chem. Res. 33 (2000) 382-390. (e) K. Soai, in: Fundamentals of Life (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scicnzc, Lettere ed Arti (Modena), Paris, 2002, pp. 427-435. [6J (a) G. Palyi, C. Zucchi, R. Boese, M. Szabo, R. Szilagyi and L. Bencze, 12"^ Internal Conf Origin Life (July 11-17, 1999, San Diego, CA, USA), Abstr. p. 42 (C.2.6). (b) G. Palyi, C. Zucchi and C. Hajdu, Atti Memorie, Accad Naz Sci Lett Arti (Modena) 316 [8/2J (2000) 389-415. [7] G. Varadi, M. Strobeck, S. Koch. L. Caghoti, C. Zucchi and G. Palyi, Critical Rev. Biochem. Mol. Biol. 34 (1999) 181-214. [8] (a) H. Brunner and W. Zetthneicr. Handbook of Enantioselective Catalysis, VCH, Weinheim, 1993, vol. 12. (b) J. Ojima, Ed., Catalytic Asymmetric Synthesis, VCH, New York. 1993. (c) C.C. Stinson, Chem. & Eng. News 76 (Sept. 21) (1998) 83-104; 77 (Oct. 11) (1999) 101-120; 78 (May 8) (2000) 59-70; 78 (July 10) (2000) 63-80; 78 (Oct. 23) (2000) 55-78; 79 (Oct. 1) (2001) 79-97; M. Jacoby, ibid. 80 (March 25) (2002) 43-46; A.M. Rouhi. ibid 80 (June 10) (2002) 43-50; 80 (June 10) (2002) 51-57; 81 (May 5) (2003) 45-55; 81 (May 5) (2003) 56-61. [9] (a) H.D. Gilbert Ed., Miniaturization, Reinhold, New York, 1961. (b) J. Rebek, Jr. Ace. Chem. Res. 17 (1984) 258-264. (c) K. Mislow, Chemtracts Org Chem. 2 (1989) 151-174. (d) J.-M. Lehn, Supramolecular Chemistry, VCH. Weinheim, 1995. (e) Special reports in: Nature 2000 (Aug. 20); Chem. & Eng. News 2000 (Oct. 16); Science 2000 (Nov. 24). [10] (a) T.R. Kelly, M.C. Bowyer, K. V. Bhaskar. D. Bebbington. A. Garcia, F. Lang, M.H. Kim and M.P. Jette, J. Am. Chem. Soc. 116 (1994) 3657-3658. (b) T.R. Kelly, 1. Tellitu and J.P. SesXclo, Angew. Chem., Int. Ed Engl. 36 (1997) 1866-1868. (c) A.P. Davis, ibid 37 (1998) 909-910. [11] B.L. Feringa, Ed., Molecular Switches, VCH-Wiley, Weinheim, 2001. [12] (a) V. Galamb and G. Palyi. Coord Chem. Rev. 59 (1984) 203-238. (b) I. Kovacs and F. Ungvary, ibid 161 (1997) 1-32. [13] G. Palyi, C. Zucchi, T. Bartik, T. Herbrich. C. Kriebel, R. Boese, A. Sorkau and G. Frater, Atti Accad. Sci. Bologna, Rend CL Sci. Fis. 281 [14/10] (1992/93) 159-167. [14] G. Palyi, K Alberts. T. Bartik, R. Boese, G. Frater, T. Herbrich. A. Herfurth, C. Kriebel, A. Sorkau, CM. Tschoemer and C. Zucchi, Organometallics 15 (1996) 3253-3255. [15] (a) P. Finocchiaro, D. Gust and K. Mislow, J. Am. Chem. Soc. 96 (1974) 3198-3205. (b) K. Mislow, D. Gust, P. Finocchiaro and R.J. Boettcher, Top Curr. Chem. 47 (1974) 1-28. (c) K. Mislow, Ace. Chem. Res. 9 (1976) 26-33. (d) H. Iwamura and K. Mislow, ibid 21 (1988) 175-182. (e) J. Polowin, S.C. Mackie and M.C. Baird, Organometallics 11 (1992) 3724-3730. (f) S.E. Gamer and A.G. Orpen, J. Chem. Soc, Dalton 7>fl«5. (1993)533-541. [16] (a) M.J. Szabo, L. Bencze, R.K. Szilagyi, G. Palyi and C. ZuQchi, Xlllth FECUEMConf Organomet. Chem. (Aug. 29 - Sept. 3, 1999, Lisboa, Vox^:)Abstr. (018). (b) L. Bencze, R.K. Szilagyi, M.J. Szabo, R. Boese, C. Zucchi and G. Palyi, in: Fundamentals of Life (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002, 451-471. [17] A systematic comparison of available semiempirical Hamiltonians [18] yielded reasonable equilibrium geometries, with the PM3^'^ method, these were used for obtaining equihbrium heats of formation by the Spartan programme [19]. Heat of formation (kcalmol^). X-ray geometry re/P -1570.05, si/M -1569.62; optimized geometry, all combinations -1812.16 ^ -1812.21. Thus the crystal field effect is cca. +240 kcal-mol'. [18] (a) J.J.P. Stewart,/ Comput. Chem. 10 (1989) 209-220. (b) J.J.P. Stewart, ibid 10 (1989) 221-264. (c) J. Yu, W.J. Here, personal communication to R.K. Szilagyi. [19] SPARTAN 4.0 Wavefunction Inc, 18401 von Karman, Suite 370, Irvine, CA, 92715 (USA) [20] PM3TN* (see refs. 18, 19) [21] (a) J. Rebek. Jr. Ace. Chem. Res. 23 (1990) 399-404. (b) G.R. Desiraju, ibid. 24 (1991) 290-296. (c) Idem, Angew. Chem., Int. Ed Engl. 34 (1995) 2311-2327.


37

[22] (a) F.J. Britten, The Watch and Clock Marker's Handbook, Dictionary and Guide, Antique Collector's Club Ltd, Woodbridge, Suftolk (UK), 1976, 11*^ ed. (b) F.W. Britten, Horological Hints and Helps, Antique Collector's Club Ltd, Woodbridge, Suftolk (UK) 1996, 4^^ ed. [23] Autosolvation: (a) G. Palyi and G. Varadi, J. Organomet. Chem. 86 (1975) 119-125 (b) G. Palyi, Transition Met. Chem. 2 (1977) 273-275. (c) G. Palyi, M. Kovacs-Toplak and G. Yai^ CI > O > N was used [7, 8]. Thus, from a rapidly interconverting mixture in solution the two diastereomers (R^,Sc) and (6'Rh,*Sc) crystallized in a 1:1 ratio in the same lattice. As expected, the bond lengths and bond angles of the two diastereomers (Rmx,Sc) and (*^Rh,-^c) are extremely similar [6]. In fact, the bond lengths from the Rh atom to its ligands deviate less than 1 % from each other. Figure 2, lower part, shows that the two diastereomers form an ''almost-racemate", connected by an "almost inversion center". In our analysis we place this inversion center exactly halfway between the two Rh atoms. The deviations of the four different substituents around the Rh atoms Cp(centroid), CI, O, N from inversion symmetry are marginal. Naturally, inconsistencies arise for the chiral nitrogen substituents which for both diastereomers have the same (S) configuration, excluding centrosymmetry. With 5.487 A the Rh-Rh distance in the "inversion pair" is extremely short. All the other Rh-Rh distances are much longer. Thus, in these "inversion pairs" two diastereomers assemble in the same way as two enantiomers do in a racemate related by an inversion center. In the middle the pairs of diastereomers {Rmi,Sc) and (^Rh,^c) are image/mirror image related, towards the outside, however, centrosymmetry is lost. The lattice is formed by translation of these inversion pairs.

3.

The Molecular Recognition Motif for the 1:1 Co-crystallization of the Diastereomers (/?RhA)- and (.S^RHA )-[(Cp)Rh(N-0)Cl] and Related Pairs of Diastereomers In the inversion pair (RRh,Sc)/(SKhySc) the two Rh atoms and the two Cp(centroids) define a "central plane" the dihedral angle Cpl-Rhl-Rh2-Cp2 being 174.9 ^ The angles Cpl-Rhl-Rh2 = 75.3 ° and Cp2-Rh2-Rhl = 78.5 ° show that the Rh-Cp centroids are slightly inclined towards Rhl-Rh2 forming a lying Z. This leaves the two Cp planes almost parallel to each other. Concerning the legs of the half-sandwich pianostools it is the Osai and CI substituents which point to the inside of the inversion pairs. This turns the N ligands, the chiral substituents of which disturb centrosymmetry, to the outside, the N atoms being close to the central plane. Molecular recognition occurs between two (Cp)Rh(Osai)Cl fragments with opposite metal configuration, which approach each other by inversion symmetry. With almost parallel Rh-Cp centroids in the central plane the fragments orient their Osai-Cl edges towards one another. There are 2x2 hydrogen bonds between the C-H groups of the Cp rings and the Osai and CI substituents of opposite molecules [distances (angles) C3-H-02 3.90 A (145.5 °), C29H-Ol 3.51 A (151.6 °), C2-H-.C12 3.65 A (153.1 °) and C28-H.-C11 3.51 A (136.9°)] giving rise to the motif of the inverted pianostools. The same molecular recognitionmotif is found in other half-sandwich complexes, for which 1:1 co-crystallization of two diastereomers in the same single crystal has been

Diastereomers Do, What They Should Not Do

43

Figure 3. Inversion pairs {RM,SC)' and (5'M,5'c)-[(Ar)MXY(NR*)] showing M-M distances of 5.6 - 5.8 A, the lying Z arrangement and hydrogen bonds C-H-X and C-H-Y

described in the literature [9, 10]. In these literature reports the phenomenon of 1:1 diastereomer co-crystallization has been stated, but not analyzed and understood. The inversion pairs of the compounds [(Cp)Ru(N-0)Cl] , N-0 = salicylaldimine anion derived from (5)-methyl valinate [11] (upper left of Figure 3), fS>l-phenylethylamine [12] (upper right), (i?)-l-hydroxybut-2-ylamine [13] (lower left), have distances and angles extremely close to iRmi,Sc)/(Smi,Sc) [6] (lower right). In the inverted pianostools of Figure 4 the diastereomers approach each other with their "racemic" sides. However, it should be kept in mind that it is not a "must" for such halfsandwich compounds to show 1:1 diastereomer co-crystallization. There are examples which crystallize as pure diastereomers (see discussion in refs. 9, 10). Thus, there is a delicate balance between the two situations 1:1 diastereomer co-crystallization and crystallization as a pure diastereomer. In solution the diastereomer ratios of such half-sandwich complexes vary depending on compound type and substitution pattern. In both crystallization aUernatives (diastereomer ratio 50:50 or 100:0) asymmetric transformations with respect to the metal configuration are involved [14-16], although they probably are not the reason for realizing one or the other.

4.

Analysis of the Molecular Recognition Motif in Compounds of the Type I(Ar)MXYZ], X and Y Being Electronegative Substituents The molecular recognition motif of the inverted pianostools is strictly obeyed in a dozen of compounds discussed in refs. 9 and 10, including the lying Z for the arrangement

44

Progress in Biological Chiralitv'

Ar(centroid)-M-M-Ar(centroid). Four of these pairs are shown in Figure 3 in a slight top view with respect to the 7i-bonded ligand. The M1-M2 distances are in the narrow range between 5.58 and 5.82 A. Similarly the X1-Y2 and Yl ••X2 distances are between 4.34 and 5.10 A, X being a halogen and Y the oxygen of the salicyl system. The distances Z1-Z2 range between 9.12 and 9.98 A indicating that the nitrogen substituents are far apart from each other pointing away from the center of the pairs. The C-H--X/Y hydrogen bonds within the pairs are all in the same range. The molecular recognition motif of Figures 2 and 3 explains the increased occurrence of 1:1 diastereomer co-crystallization of compounds [(Ar)MXY(NR*)] on the basis of a centrosymmetric arrangement of two mirror image (Ar)MXY fragments. A consequence should be that other compounds having the structural element (Ar)MXY with X and Y electronegative substituents should establish this molecular recognition motif as well, even if they have nothing to do with chirality. As the motif was increasingly found for (Cy)Ru compounds a search in the Cambridge File for [(Cy)RuLCl2] complexes was made, expecting pair formation as indicated in Figures 2 and 3 with the orientation of the ligands L to the outside of the pairs. There were 19 entries [10]. Ten nicely fitted the molecular recognition pattern. Four of them are shown in Figure 4 with ligands L = 4-cyanopyridine [17], nbutyl(diphenyl)phosphine [18], tribenzylphosphine [19] and tris(3-methylphenyl)phosphine [20]. Nine [(Cy)RuLCl2] systems did not fit this pattern. In these nine compounds the ligands L were similar to those which complied with the pattern. In these compounds there were no

Figure 4. The molecular recognition motif of the inverted pianostools for [(Cy)RuLCl2] compounds showing M-M distances of 5.6 - 5.8 A, the lying Z arrangement and hydrogen bonds C-H-X and C-H—Y


45

Figure 5. Racemate crystallization of [(Cy)Ru(N-0)Cl] derived from 8-hydroxyquinoline in the molecular recognition motif of the inverted pianostools

inversion pairs with Ru-Ru distances in the range 5.60-5.80 A. Thus, the situation corresponds to the diastereomers discussed above: The molecular recognition motif of the inverted pianostools is an attractive possibility, but it is not reinforced. There are always examples which could follow it, but fail to do so. The analysis given for the compounds [(Cy)RuLCl2] could easily be extended to related systems. A representative example is the compound [(Cy)Ru(N-0)Cl] [21] in which the unsymmetrical chelate ligand N-0 is the anion of 8-hydroxyquinoline (Figure 5). The chiral molecules form a racemate with a perfect inversion center between the two enantiomers. The Ru-Ru distance is 5.34 A and the hydrogen bonds C-H-0 3.279 A (angle 125.8 °) and C-H-Cl 3.674 A (angle 160.0 °) are fully conform with the molecular recognition pattern of the inverted pianostools. 5.

Relevance of the Inverted Pianostool Motif for Achiral Compounds, Enantiomers and Diastereomers As shown in Figure 4 for [(Cy)RuCl2L] derivatives, half-sandwich compounds of the type [(Ar)MLX2] tend to form the molecular recognition motif of the inverted pianostools in the solid state. Such a detail is overlooked as long as only a single molecule is considered as customary in today's publications. It is perceived only after the packing diagram has been taken into account. The left part of Figure 6 shows a representative example of an achiral [(Cy)RuLCl2] derivative with L = tribenzylphosphine. If enantiomers of the type [(Ar)MXYL] are crystallized, frequently the inverted pianostool motif is established resulting in a racemate, the usual situation in enantiomer crystallization as outlined in the introduction. As an example compound [(Cy)Ru(N-0)Cl] of Figure 5 is repeated in the middle of Figure 6.

46

Progress in Biological Chiralitv

^^^^^^Sî^^^^l^^,

interesting detail of packing diagram

Mmmtmmem

iliitf.

racennate crystallization

Figure 6. The molecular recognition motif of the inverted pianostools for achiral compounds (left), enantiomers (middle) and diastereomers (right) The most interesting case is the crystallization of diastereomers of the type [(Ar)MXY(NR*)]. Establishment of the inverted pianostool motif results in the interesting consequence that two diastereomers co-crystallize 1:1 in the same single crystal with the implications for the failure of isomer separation addressed in the introduction. Remarkably, the half-sandwich complexes showing 1:1 diastereomer co-crystallization not only comprise Rh compounds but also Ru, Os and Ir compounds, not only cyclopentadienyl

Z ^-••*' Figure 7. Generahzed representation of the inverted pianostool molecular recognition motif


47

derivatives but also benzene and cymene derivatives, not only chloro ligands but also iodo ligands and not only salicylaldiminates but also pyrrolylaldiminates. In addition, most of the compounds differ appreciably in the chiral nitrogen substituent [9, 10]. Thus, a generalized representation of the molecular recognition motif of the inverted pianostools with M - M distances of 5.6-5.8 A, the lying Z arrangement and hydrogen bonds between the C-H groups of the pianostool seat and electronegative substituents X and Y takes the form shown in Figure 7.

6.

References

[1] C.J. Welch, Chimlity 13 (2001) 425-427. [2] E.L. Eliel and S.H. Wilen, Stereochemistry of Organic Compounds, Wiley & Sons, New York, 1994. [3] G.B. Kaufiman, I. Bemal and H.-W. Schiitt, Enantiomer 4 (1999) 33-45. [4] B.S. Green, M. Lahav and D. Rabinovich, ylcc. Chem. Res. 12 (1979) 191-197. [5] L. Addadi, E. Gati, M. Lahav and L. Leiserowitz, Isr. J. Chem. 15 (1916-11) 116-123. [6] H. Brunner, A. Kolhiberger and M. Zabel, Polyhedron 22 (2003) 2639-2646. [7] C. Lecomte, Y. Dusausoy, J. Protas, J. Tirouflet and A. Dormond, J. Organomet. Chem. 73 (1974) 67-76. [8] H. Brunner, Enantiomer 2 {1991) 133-134. [9]H. Brunner, M. Weber and M. Zabel Angew. Chem. 115 (2003) 1903-1907: Angew. Chem. Int. Ed 42 (2003) 1859-1862. [10] H. Brunner, M. Weber and M. Zabel, Coord Chem. Rev. 242 (2003) 3-13. [11] H. Brunner, T. Zwack, M. Zabel, W. Beck and A. Bohm, Organometallics 11 (2003) 1741-1750. [12] S.K. Mandal and A.R. Chakravarty, J. Chem. Soc, Dalton Trans. (1992) 1627-1633. [13] H. Brunner, T. Neuhierl and B. Nuber, Eur. J. Inorg. Chem. (1998) 1877-1881. [14] H. BrumiQr,Adv. Organomet. Chem. 18 (1980) 151-206. [15] H. Brmmer,Angew. Chem. I l l (1999) 1149-1163;Angew. Chem. Int. Ed 38 (1999) 1194-1208. [16] H. Brunner, Eur. J. Inorg. Chem. (2001) 905-912. [17] D.K. Gupta, A.N. Sahay, D.S. Pandey, N.K. Jha, P. Sharma, G. Espinosa, A. Cabrera, M.C. Puerta and P. Valerga, J. Organomet. Chem. 568 (1998) 13-20. [18] G. Bruno, M. Panzalorto, F. Nicolo, C. G. Arena and P. Cardiano, Acta Crystallogr., Sect. C (Cr. Str. Comm.) 56 (2000) e429. [19] S. Serron, S.P. Nolan, Yu. A. Abramov, L. Brammer and J.L. Petersen, OrganometalUcs 17 (1998) 104110. [20] A. Hafner, A. Miihlebach and P.A. von der Schaaf, Angew. Chem. 109 (1997) 2213-2216; Angew. Chem. Int. Ed Engl 36 (1997) 1111-1114. [21] C. Gemel, R. John, C. Slugovc, K. Mereiter, R. Schmid and K. Kirchner, J. Chem. Soc, Dalton Trans. (2000) 2607-2612.

Progress in Biological Chirality G. Palyi, C. Zucchi and L. Caglioti (Editors) © 2004 Elsevier Ltd. Allrightsreserved.

Chapter 5 Molecular Origins of Life Homochirality as a Consequence of the Dynamic Co-Emergence and Co-Evolution of Peptides and Chemical Energetics Auguste Commeyras,'*'* Jacques Taillades,^ Helena Collet,^ Laurent Boiteau/ Odile Vandenabeele-Trambouze,^ Robert Pascal,^ Herve Cottet,^ Raphael Plasson,^ Jean-Philippe Biron,^ Eddy Souaid,^ Laurence Garrel/ Olivier Lagrille,^ Gregoire Danger,^ Jean-Christophe Rossi,^ Franck Selsis,^ Michel Dobrijevic" and Herve Martin"^ Ôrganisation Moleculaire, Evolution & Materiaux Fluores (UMR5073-CNRS), Departement de Chimie- CC017, Universite de Montpellier 2, F-34095 Montpellier Cedex 5, France [email protected] ^Centro de astrobiologia - INTA (CSIC), Ctra de Ajalvir km 4,E- 28850 Torrejon de Ardoz, Madrid, Spain ^Laboratoire d'Astrodynamique, d'Astrophysique et d'Aeronomie de Bordeaux (L3AB, UMR5804~CNRS), 2 rue de VObservatoire, BP89, F'33270 Floirac, France ^Laboratoire Magmas et Volcans, Universite Blaise Pascal-CNRS-OPGC, 5 rue Kessler, F-63038 Clermont-Ferrand cedex 1, France

1.

Introduction There are some obvious arguments that are hard to circumvent, such as; no dynamics without energy, no evolution without dynamics, no life without evolution, which result in postulating the existence of dynamic co-evolutionary processes at the origin of Ufe. The idea of dynamic co-evolution at the time of the origin of life requires the addition of the energy component to the old debate about the priority of emergence between peptides and RNAs This debate is far from being resolved and continues to evolve, since it is assumed that the emergence of the RNA world is hard to imagine [1] without the aid of peptides and their catalytic effects [2]. Putting into perspective the conditions of the emergence of the first peptides and their subsequent catalytic effects may have constituted one of the first steps in this extraordinary mechanism of the pathway from inert to "alive". To participate in this debate, we have carefully analysed the formation of a-amino acids discovered in meteorites, and shown that they are derived from the following system {H2O, HCN, R^COR^ NH3, R^NH2, CO2, NaHCOs, B(0H)4", NO, O2}, involving thirty five different carbonyl compounds ammonia and two amines [3]. By studying the evolution of these systems, we deduced an experimental scenario which can explain the emergence of

50


exogenous and endogenous a-amino acids as well as the emergence of peptides on the primitive Earth. The core feature of this scenario is the "molecular dynamics" which can be shown by the following course of events.

Experimental The experimental conditions are described in the quoted references.

3.

Results and Discussion

3. J a-Amino acids and related compounds from a multi-component reaction model We start, in Figure 1, with some of the essential molecules of prebiotic chemistry (water, hydrogen cyanide, carbonyl compounds, ammonia or amines, carbon dioxide and borates). 3.1.1 Reversible reactions The first reaction between these molecules is the formation of the a-hydroxynitriles (I). This reaction is very fast. It is followed by the formation of amino alcohols (II). These amino alcohols lose the OH" anion to give iminium ions (III). These iminium ions react with the cyanide anion to give a-amino nitriles (IV). These a-amino nitriles then react with the initial carbonyl derivatives to give new a-amino alcohols (V). As before, these new amino alcohols (V) in turn lose OH" and, together with the cyanide anion, give a-amino dinitriles (VII). But, in competition with this reaction, the amino alcohols (V) also lose H^, leading to amino alcoholates (VIII) [4-10]. Carbon dioxide, when present in the medium, specifically reacts with the a-amino nitriles (IV) to give carbamic acids (IX) and carbamates (X) [11, 12].

B.-%„">

HCN + RiR2C=0 + H2O+ NH3 + CO2 + B(OH)4

^

'NH2

(II)

I-

B(OH)4

.(IV)

R2

(III)

i (IV)

CO, I

CN ORis

R / ' ^ C ^ r (XI) OH

F^

H

R2

(V)

riT^ H

(IX)

HO

R2

(VIII) BIN/"

R^^ CN HO

(VI) OH

^R2

H

W"2 (Xd)

Figure 2. Five different and competitive irreversibie reactions (A), (B), (C), (D), (E) enable the reversible system to escape from reversibility-. To take into accomit the information from § 3.2, we have added the carbamoylation, reaction (F), to this figure

52


constant is very high compared with the previously mentioned reaction paths (A) and (B). When a small concentration of ammonia is used, the reaction (C) gives imino-diacids as proven by Laurence Garrel (personal communication). The discovery of these imino-diacids n Miller's type experiments [19], as in meteorites [20], confirms the relation between exogenous and endogenous a-amino acids, and gives the prebiotic status to the reaction (A) (B) and (C) [3]. The second reaction (D) starts from the carbamates (X). It leads to hydantoins (Xc) via the intermediary isocyanates (Xb). This reaction is also chemoselective [11, 21]. It is known as the Bucherer-Bergs reaction. Its rate constant is almost as high as that of the reaction (C). The third reaction (E) starts from the borates (XII), which are used as catalysts. This reaction is also chemoselective [13, 14]. It leads to the formation of a-hydroxy amides (XIIc). Its rate constant is lower than that of (C) and (D), but much higher than that of (A) and (B). All of the reactions (A) to (E) displace the species in equilibrium, preferentially giving aamino amides and hydantoins, and less effectively a-hydroxy amides. Thus the process is kinetically controlled, but only by the reactions (C), (D) and (E). Reactions (A) and (B) play a negligible role. The next step gives a-hydroxy acids (Xlld), a-amino acids (Vllld), and Ncarbamoylamino acids (Xd), all of which have the same rate. This means that the formation of these compounds is always kinetically controlled by the reactions (C), (D) and (E). The global process needs to be completed^ by the action of cyanate on a-amino acids (reaction F). This reaction is slow, so it does not interfere with the previous one, but it transforms quantitatively the free a-amino acids into N-carbamoyl amino acids [3, 22]. The consequence, for the primitive Earth, is that this whole set of reactions could have kinetically converged, not towards free a-amino acids, but rather towards N-carbamoyl amino acids. 3.2 Peptides from N-carbamoylamino acids through a primary pump At this point in the story we must ask why, N-carbamoyl amino acids were formed at all? The following step in this scenario is to go from N-carbamoyl amino acids to peptides. Our approach (Figure 3) is a molecular engine (also called a primary pump) fed with N-carbamoyl amino acids and frielled by a gaseous mixture of NO/O2 as the energy source [23, 24]. 3.2.1 Molecular engine: description The first step (1) is the formation of N-carbamoylamino acids in aqueous solution [22]. The second step (2) is the concentration of these N-carbamoyl amino acids. This could have taken place at low tide on the shores of the Hadean continent. In the dry phase, step (3), if NOx were present in the primitive atmosphere [24], it could nitrosate the N-carbamoylamino acids, and produce stoichiometric amounts of nitrous acid. These nitrosated intermediates are unstable and, through cyclization, lead to the Ncarboxyanhydrides of a-amino acids (NCAs). This reaction is quantitative at ambient temperature [25]. At the same time as producing NCAs, they form stoichiometric amounts of nitrogen and water. The nitrous acid stabilizes the NCAs for several hours. If the NCAs stay too long in an acid medium they are hydrolyzed into a-amino acids which are then recycled.

Molecular Origins of Life: Homochirality as a Consequence of the Dynamic... 53 H2N—C—C-OH I CH3 Non-racemic meteoritic a-methyl-aminoacids

ENERGY NO + 0 ,

^=-

NOx

N2 + H2O +CO2

l%<eeL fmatx. ^ s _

Figure 5. Algorithm showing the functioning scheme of the primary pump. The evolution of the enantiomeric excess during the beginning of its functioning shows the break of symmetry and its amplification

56


Either scenario could have lead the amplification of dissymmetry towards a unique enantiomeric form (L). Measurements are required to test the validity of these hypothesis. At this point, it must be made clear that the argument concerning homochirality, is not yet based on knowing all the kinetic constants. Only a complete kinetic modelling based on real kinetic constants can confirm such a scenario. We believe it is a price that must be paid in order to study such dynamic systems. Some journal referees need to be convinced that this is in fact the case, (c) Kinetic modeling While waiting for this ideal kinetic model, we have constructed one (see the algorithm) using estimated (or real cf § 3.4) kinetic constants, (ku. > kLo, kracem a-amino acids > kracem peptides, khydroiyse LD et DL > khydroiyse LL and DD). During its fianctioning, this model leads to a break of symmetry (L) or (D) and to its amplification (see the graphic in Figure 5). i. 3 Environmental requirements Before proceeding we have got information showing that the primitive Earth may have satisfied all the necessary requirements to make this primary pump realistic [24]. These results gave us greater motivation to proceed in detailing the primary pump, looking for mechanisms and kinetic constants for every step. 3.4 Primary pump step by step Only some details of step (1), (3) and (5) have been obtained. 3.4. J Step (J): carbamoylation Although the rate law is complex [22], V = d[NCO]' / dt = [NCO]' x (ko + k4 x[C03]' + ks x [NH3]' + k6 x[AA]') k6, the kinetic constant for the carbamoylation of amino acids at 50°C and pH 6.5 was obtained. 1300 lO-^mol'^Ls-^forVal, 1700 lO-^/mof^Ls-^ for Gly, 3530 10-^/mor'Ls"^forThr. These kinetic constants are very low, but they look well adapted to the dynamic of the system. Only kinetic modelling will prove or disprove this. 3.4.2 Step (3): nitrosation The gaseous mixture NO/O2 react with N-carbamoyl amino acids even in solid phase [25, 34]. The picture (a) in Figure 6 shows crystals of N-carbamoyl valine. In presence of a gaseous mixture of NO/O2. Picture (b) shows the surface of the crystal at the beginning of the reaction. The bubbles are N2. The liquid is water and nitrous acid. Picture (c) is the Valine NCA. This reaction is complete at 20 °C in half an hour. The kinetic constants for the nitrosation of these N-carbamoyl amino acids were obtained : - by HNO2 in water (kHN02 = 0.1 s'^), the constant is low, - by N2O4 in dry phase (kN204 = 1 7 s'^) the constant is 17 times bigger. - but by N2O3 in solid phase (kN203 = l.OxlO^s'^) the kinetic constant is 1 billion times bigger than with nitrous acid [35, 36].

Molecular Origins of Life: Homochirality as a Consequence of the Dynamic... 57

Figure 6. (a/ crystals of N-carbamoylvaline), (b/ action of NO/Oson a), (c/ valine NCA)

Note that N2O4 and N2O3 are present in equilibrium in the gaseous mixture NO/O2. Figure 7 summarizes the reversible reactions between NO, O2, N2O4 and N2O3 in the gaseous phase. 3.4.3 Step (5): peptides synthesis Our work gives a prebiotic status to these NC As. Except for those of Bartlett [37, 38] that have to be reconsidered, no kinetic measurements of the polycondensation of NCA can be found in the literature, in aqueous solvent.

I 1/2NO I

jl 1/2

Colourless

1/202

O-O-NO

t

ArH^9S = -57.2 KJ.mof ^0*^298 = -65.93 KJ.mol'

1/2NO

l/2 0]>ô/o-NO

I (A)

(B)

N-0-]>Q

-—^

NQz

• ;

NC^*^

Brown ^ NNO '

^^n-'^

I ^=?==^ 0=N-]>Q

(C)

: i

t; « - ^ ^ ^ ° (D)

o NiOi

II Colourless

ArH^298--57.12KJ.mo-f ArG°298 = -4.73 KJ.mo-f

Bl

Colourless

ArH°298 = -40.56 KJ.mol /VG^98=1.86KJ.mof

Figure 7. Equilibrium between NO and O2 in the gaseous phase

58


(a) Oligocondensation ofNCA and enantioselectivity In aqueous solution NCAs give two competitive reactions with different kinetic laws: - Vhyd = khyd [HO][NCA] for hydrolysis kcondens [-NH2][NCA] for oligocondensation. For example [39] for valine NCA : - the kinetic constant for hydrolysis is khydrolysis = 4.40xl0'^s"\ - and for the formation of the dipeptides H-L-Val-L-Val-OH and H-D-Val-L-Val-OH kLL =1.65x10-^ s-^Lmor\ kDL= 1.14x10"^ s'^Lmol"^ The first conclusion is that in water the kinetic constants for the divaline formation are one hundred times bigger than those of hydrolysis. This is a very promising result for peptide synthesis in aqueous solution. The second conclusion, when considering the ratio KLL/KDL = 152, is that a large enantioselectivity is observed already for the dipeptide formation. This ratio should be higher for the synthesis of larger peptides due to a probable micro phase separation around octapeptides, (see the discussion on this point by Kricheldorf p 180185 [40]) and possible assisted polycondensations of NCAs by these microphase like membranes [41]. In the near future these measurements will be, for example, extended to the synthesis of HL-VAL-L-isoVal-OH and H-D-VAL-L-isoVAL-OH from L-isoVAL on NCA-VAL, L and D and related compounds, to see one of the possible roles of the non-racemic exogenous amino acids in the emergence of the homochirality (see § 3.2.5b). Now it should be noted that in water, the formation rate of peptides is a function of their pKa (only the non protonated amino group is active) and, paradoxically the PKa's of peptides are not very well known. (b) FastpKa measurement of peptides We have developed a fast method for measuring the pKa using capillary electrophoresis. In the figure 8 we see that the electrophoretic mobility of peptides depends on the pH.

Figure 8. Capillary electrophoresis of polyglycines. Electrophoretic mobilities are pH dependent


25''C y = m1 +m2*exp(-m3*m0)-m4*m0 Error Value ml

7.9698

m2

14.82 2.2411 0.032703 0.0045027

m3 m4 1 Chisq

a •a

R^

0.041 3.5145 0.25004 0.0062972

0.99834

NA NA

JS 8-5

Figure 9. pKa of polyglycines from 1 to 9 residues

From these electrophoretic mobilities, we obtained the pKa's for various peptides such as the polyglycines (for details see PhD of Raphael Plasson 2003 University of Montpellier 2 to be published). The pKa of peptides decreases with their lengthening, (c) Lengthening From this information, we deduce that in aqueous solution, at equal concentrations, longer peptides (lower pKa) tend to lengthen faster than shorter peptides, by assuming that the kinetic lengthening constant is the same for all peptides. This also will be carefully controlled in the near future.

pH primitive ocean

Figure 10. In aqueous solution, at equal concentrations of peptides, the concentration of the non-protonated amino group of the longer peptides (lower pKa) is higher than the concentration of the non-protonated amino group of shorter peptides

60


L

ionic

W

*

VE VW I VVE VEV VEVE

current 8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

Elution time/min 26.00

28.00

30.00

32.00

34.00

36.00

Figure 11. Molecular engine running with 2 amino acids Val (V) and Glu (E) (HPLC/MS,Q-TOF analysis)

(d) Chemoselectivity This physico-chemical approach needs to be completed by synthesis techniques because, in water, other parameters are involved in the lengthening. Figure 11 shows the results obtained by running the primary pump with two amino acids: V (valine) and E (glutamic acid). Of the many possible peptides, many but not all are formed. The distribution of peptides formed in these experiments shows an effective chemoselectivity (Figure 12) during the lengthening process. Starting with 2 amino acids

4 /4 Dipeptides formed

4/8 Tripeptides formed

4/16 Tetrapeptides formed

5/32 pentapeptides formed

VVVV EVVV

..»•

VEVV

VEVW W E W

EEVV VVVE EWE VEVE

- ^

VEWE WEVE

EEVE VVEV EVEV VEEV EEEV WEE EVEE VEEE EEEE

Figure 12, Chemoselectivity during the formation of peptides from 2 amino acids as starting materials


(e) Selectivity due to hydrophobia With four amino acids (V, E, G, A) as the starting materials, MALDI-TOF analysis shows that the length of the peptides increases with the cycle number, we go from 800 Da for cycle 1, to 1000 Da for cycle 2, to 1300 Da for cycle 7... etc. But insoluble peptides are also formed, and are therefore missing in the soluble fraction. The insoluble fraction (separated after 7 cycles) is made up of peptides whose molecular weight is distributed around 800 Daltons, whereas the molecular weight of the soluble compounds is higher than 2000. So there is a selectivity process due to hydrophobia that is complementary to the others. The stimulating aspect of this point is that the existence of this stress (due to hydrophobia) eliminated the insoluble peptides. The only peptides capable of surviving (that is to say of evolving) were the soluble ones, in other words those, with good sequences, that were best adapted to their environment. Note that the inappropriately adapted species, the insoluble ones, are completely recycled after hydrolysis. We can therefore hypothesize that in aqueous solution, the selectivities described above could have considerably decreased the number of peptides formed and thus making realistic that the prebiotic synthesis of peptides occurred without the genetic code.

Cycle #2

'*'*i^,^ 1380

1920

2460

3000

Cycle #7 (soluble fraction)

Cycle #7 (insoluble fraction)

Figure 13. The molecular engine was run with 4 amino acids (V, E, G, A). MALDI-TOF analysis for the peptides formed is given for the cycle 1, 2, and 7 (soluble fraction and insoluble fraction)

62


3.5 Chemical energy Energy itself must have evolved. In the course of this research, we moved from radiation energy to NOx anhydrides. These NOx compounds then evolved towards a second group of anhydrides, by which we mean the NCAs. Why not try to find an affiliation between NCAs and ATP or equivalent? Energetically speaking this affiliation is correct, and nobody knows the origin of these fundamental molecules which must have been synthesized in a continuous flow. 3.6 Terrestrial energy In order to work, the primary pump requires an alternation of dry and aqueous periods for instance on tidal beaches. In addition to chemical energy, a terrestrial source of energy was also required in order to allow "primary pump efficiency"; this energy was not directly involved in the chemical reactions, but initially it created favourable conditions for their development. 3.7 Need of extension of kinetic modelling The global system already presented constitutes a primary pump, fed with a-amino acids, fuelled by a gaseous mixture NO/O2 which recycles the essentials of its material. With about fifty carbonyl compounds this molecular engine is capable of synthesizing all the a-amino acids of the primitive Earth. From these the homochiral peptides could have been built, using only twenty of them. To understand and appreciate the evolution of such systems, kinetic modelling is needed.

4.

Conclusion To summarize, we have presented a scenario for the dynamic co-evolution of peptides and energy on the primitive Earth. In this scenario homochirality and sequential peptides appear as a consequence of this dynamic co-evolution. This scenario is not necessarily the right one, but it is chemically original and has the virtue that it can be experimentally tested. During the course of these experiments, we hope to open up a gateway to the emergence of the catalytic activities of peptides. We have already begun to approach this aspect of the work [42].

5. Acknowledgements This work was supported by the European Community program COST D27 Prebiotic Chemistry and Early Evolution; I'Universite de Montpellier 2, le Ministere de VEducation Nationale et le Centre National de la Recherche Scientifique (Departement de Chimie et Institut National des Sciences de I 'Univers).

6.

References

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Molecular Origins of Life: Homochirality as a Consequence of the Dynamic... 63 [3] A. Commeyras. J. Taillades. H. Collet, L. Boiteau. R. Pascal O. Vandenabeele. A. Rousset. L. Garrel. J.C Rossi, H. Cottet, J.P. Biron, O. Lagrille, R. Plasson, E. Souaid, F. Selsis, M. Dobrijevic and H. Martin, Approche dynamique de la synthese des peptides et de leurs precurseurs sur la Terre primitive, in: Les traces du vivant. (Eds. M. Gargaud, D. Despois and J.P. Parisot) Presses Universitaires de Bordeaux, 2003, p. 115-163. [4] J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes: I-Etude de la decomposition en solution aqueuse des a-alcoyl-aminonitriles tertiaires. Mecanisme d'elimination du groupement nitrite. Tetrahedron 30 (1974) 127-132. [51 J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes: II-Mecanisme de formation en solution aqueuse des a-alcoylaminoisobutyronitrile a partir d'acetone, d'acide cyanhydrique et d'ammoniac, methyl ou dimethylamine. Tetrahedron 30 (1974) 2493-2501. [6] J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes: Ill-Etude en solution aqueuse de la stabilite et des conditions de synthese des a-aminonitriles tertiaires. Importance des protons portes par le groupement amine. Tetrahedron 30 (1974) 3407-3414. [7] M. Bejaud, L. Mion and A. Commeyras, Systemes de Strecker et Apparentes. VII- Etude stereochimique des a-aminodinitriles, produits secondaires de la synthese selon Strecker des a-aminonitriles. Tetrahedron Lett. 34 (1975) 2985-2986. [8] M. B6jaud, L. Mion, J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes. IV-Etude comparative de la reactivite des a-aminonitriles secondaires et tertiaires en solution aqueuse entre pH 10 et 14. Hydrolyse des a-aminonitriles secondaires et son importance dans la formation prebiotique des acides amines naturels. Tetrahedron 31 (1975) 403-410. [9] M. Bejaud, L. Mion and A. Conuneyras, Systemes de Strecker et Apparentes. VI- Stabilite des aminonitriles en fonction du pH. Bull Soc. Chim. France 1-2 (1976) 233-236. [10] M. Bejaud, L. Mion and A. Commeyras, Systemes de Strecker et Apparentes. VIII- Etude thermodynamique des systemes acetaldehyde-acide cyanhydrique-monomethylamine ou ammoniac en solution aqueuse. Stabihte en fonction du pH des cyanhydrines, a-aminonitriles et a-aminodinitriles formes. Bull. Soc. Chim. France 9-10 (1976) 1425-1430. [11] A. Rousset, M. Lasperas, J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes. XIFormation et stabihte de I'a-carboxyaminonitrile intermediaire essentiel dans la synthese des hydantoines selon Bucherer-Bergs. Tetrahedron 36 (1980) 2649-2661. [12] A. Rousset, M. Lasperas, J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes. XVComportement d'a-alcoylaminonitriles en presence de CS2 et de CO2. Bull Soc. Chim. France 5-6, II (1984)209-216. [13] J. Jammot, R. Pascal and A. Commeyras, Hydration of cyanohydrins in weakly alkaline solutions of boric acid salts. Tetrahedron Lett. 30 (1989) 563-564. [14] J. Jammot, R. Pascal and A. Commeyras, The influence of borate buffers on the hydration rate of cyanohydrins: evidence for an intramolecular mechanism. J. Chem. Soc, Perkin Trans. LI (1990) 157-162. [15] A. Strecker, y^ww. Chemie 75 (1850) 25-29. [16] R. Pascal, J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes. IX- L'acetone, catalyseur d'hydratation des a-aminonitriles tertiaires en solution aqueuse basique. Bull Soc Chim. France 3-4 (1978) 177-184. [17] R. Pascal, J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes. X- Decomposition et hydratation en milieu aqueux basique des a-aminonitriles secondaires. Processus dliydratation autocatalytique et catalyse par I'acetone. Tetrahedron 34 (1978) 2275-2281. [18] R. Pascal, J. Taillades and A. Commeyras, Systemes de Strecker et Apparentes. XII- Catalyse par les aldehydes de Thydratation intramoleculaire des a-aminonitriles. Tetrahedron 36 (1980) 2999-3008. [19] S.L. Miller, Production of some organic compounds under possible primitive Earth conditions. J. Am. Chem. Soc. 11 (1955) 2351-2361. [20] S. Pizzarello and G.W. Cooper, Molecular and chiral analyses of some protein amino acid derivatives in the Murchison and Murray meteorite. Meteoritics Planetary Science, 36 (2001) 897-909. [21] J. Taillades, J. Brugidou, R. Pascal, R. Sola, L. Mion and A. Commeyras, Nouvelles voies de synthese d'acides a-ammcs. Actualite Chimique (1986) 13-20. [22] J. Taillades, L. Boiteau, I. Beuzelin, O. Lagrille, J. Biron, W. Vayaboury, O. Vandenabeele-Trambouze, and A. Commeyras, A pH-dependent cyanate reactivity model: application to preparative Ncarbamoylation of amino acids. Perkin Trans. 2 (2001) 1247-1253.

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[23] A. Commeyras, J. Taillades. H. Collet. L. Mion. L. Boiteau, O. Trambouze-Vandenabeele, H. Cottet J.P. Biron, F. Schue, O. Giani, O. Lagrille, R. Plasson, W. Vayaboury, H. Martin, F. Selsis, M. Dobrijevic and M. Geffard. La Terre matrice de la vie: emergence avant-gardiste des peptides sur les plages de I'Hadeen., in: L'environnement de la Terre primitive, (Eds. M. Gargaud D. Despois and J.P. Parisot) Presses Universitaires de Bordeaux, 2001, p. 361-380. [24J A. Commeyras, H. Collet, L. Boiteau, J. Taillades, O. Vandenabeele-Trambouze, H. Cottet, J.-P. Biron, R. Plasson, L. Mion, O. Lagrille, H. Martin, F. Selsis and M. Dobrijevic, Prebiotic synthesis of sequential peptides on the hadean beach by a molecular engine working with nitrogen oxides as energy sources. Polymer Internat. 51 (2002)661-665. [25] H. Collet, C. Bied, L. Mion, J. Taillades and A. Commeyras, A new simple and quantitative synthesis of a-amino acid-N-carboxyanhydrides. Tetrahedron Lett. 37 (1996) 9043-9046. [26] S.J. Mojzsis, R. Krishnamurthy and G. Arrhenius, Before RNA and after: geophysical and geochemical constraints on molecular evolution, in: The RNA word second edition (Eds. R.F. Gesteland, T.R. Cech and J.F. Athins) Cold Spring Harbord, 1999 p. 1-47. [27] H. Collet, L. Boiteau, J. Taillades and A. Commeyras, Solid phase decarbamoylation of N-carbamoyl peptides and monoalkylureas using gaseous NOx: a new simple deprotection reaction with minimum waste. Tetrahedron Lett. 40 (1999) 3355-3358. [28] A. Radzicka and R. Wolfenden, Rates of uncatalysed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J. Am. Chem. Soc. 118 (1996) 6105-6109. [29] JR. Cronin and S. Pizzarello. Enantiomeric excesses in meteoritic amino acids. Science 275 (1997) 951955. [30] S. Pizzarello and J.R. Cronin, Non-racemic amino acids in the Murray and Murchison meteorites. Geochim. Cosmochim. Acta, 64 (2000) 329-338. [31] N.E. Blair and W. A. Bonner, A model for the enantiomeric enrichment of polypeptides on the primitive Earth. Origin Life Evol. Biosphere 11 (1981) 331-335. [32] K. Iwamoto and M. Seno, On a chemical system related to absolutely asymmetric synthesis. J. Chem. Phys. 76(1982)2347-2351. [33] D.K. Kondepudi and G.W. Nelson. Weak neutral currents and the origin of biomolecular chirahty. Nature 314(1985)438-441. [34] J. Taillades, H. Collet, L. Garrel. 1. Beuzelin, L. Boiteau, H. Choukroun and A. Commeyras, N-Carbamoyl amino acid solid-gas nitrosation by NO/NOx: A new route to oligopeptides via a-aminoacid Ncarboxyanhydride. Prebiotic imphcations. J. Mol. Evol. 48 (1999) 638-645. [35] O. Lagrille, Nitrosation de N-carbamoylamino acides solides par le melange gazeux NO/O2. Synthese de N-carboxyanhydrides (Anhydrides de Leuchs), in: Departement de chimie. Universite de Montpellier 2, Montpelher, Fr. 2001, p. 211. [36] O. Lagrille, J. Taillades, L. Boiteau and A. Commeyras, N-carbamoyl derivatives and their nitrosation by gazeous NOx-A new promissing tool in stepwise peptide synthesis. Eur. J. Org. Chem. (2002) 1026-1032. [37] P.D. Bartlett and D.C. Dittmer, A kinetic study of the Leuchs anhydrides in aqueous solution. II.,. J. Am. Chem. Soc. 79 (1957) 2159-2161. [38] P.D. Bartlett and R.H. Jones, A kinetic study of the Leuchs anhydrides in aqueous solution. I. /. Am. Chem. Soc. 79 (1957) 2153-2158. [39] R. Plasson, J.P. Biron, H. Cottet A. Commeyras and J. Taillades, Kinetic study of a-amino acids Ncarboxyanhydrides polymerisation in aqueous solution using capillary electrophoresis. J. Chrom. A., 952/1-2(2002)239-248. [40] H.R. Kricheldorf, a-Amino acid-N-carboxy-anhydrides and related heterocycles. Synthesis, properties, peptide Synthesis, polymerisation. Heterocycles. Springer-Verlag, Berhn, 1987, 213 pages. [41] PL. Luisi, P. Walde, M. Blocher and D. Liu, Research on the origin of life: membrane-assisted polycondensations of amino acids and peptides. Chimia, 54 (2000) 52-53. [42] R. Pascal, Catalysis by Induced Intramolecularity: What can be learned by mimicking enzymes with caibonyl compounds that covalently bind substrates?". Eur. J. Org. Chem. (2003) 1813-1824.


Chapter 6 Use of Amino Acids and Amino Acid Racemization for Age Determination in Archaeometry J. Csapo,^'* M. Collins,^ Z. Csapo-Kiss,^ E. Varga-Visi/ G. Pohn" and J. Csapo' Jr. ""University ofKaposvdr, Faculty ofAnimal Science, H'740l Kaposvdr. P.O.Box 16, Guba S. u, 40, Hungary csapo@mail atk. u-kaposvar. hu ^ Fossil Fuels and Environmental Geochemistry, NRG, Drummond Building, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NEl 7RU, United Kingdom ^Janus Pannonius University Faculty of Natural Sciences, Natural Geography Department, H-7624 Pecs, Ifjusdgu, 6, Hungary

1.

Introduction

/. / Age determination from fossil bones The subject of this paper is a survey on the possibilities of the determination of the age of archaeological samples (bones and teeth) containing proteins based on transformations of the amino acids therein. The first possibility is to estimate chronological age based upon the extent of racemization of the L-amino acids which are the constituents of native proteins. Amino acid contents in fossil shell, bone and tooth samples from were first reported by Abelson in 1954 [1]. In 1967, Hare and Abelson [2] reported that D-amino acids in fossils resulted from conversion of L-amino acids of protein. It was found that the older the fossil the higher the D/L ratio and, after a certain age, amino acids occurred in racemic form. The ratio of D-allo-isoleucine and L-isoleucine content in a fossilised shell sample was found to be 0.32 and the fossil was estimated to be 70 000 years old [3]. This is considered as the first application of amino acid racemization (AAR) - or more correctly epimerization (isoleucine has two chiral centres) - in geochronology. Subsequently, racemization of amino acids was used for age determination of various materials containing protein [4, 5, 6, 7, 8, 9, 10]. Isoleucine was given special attention because it could be reliably and consistently separated from the diastereoisomeric D-alloisoleucine by either ion exchange or reversed phase chromatography. Some errors of age determination based on AAR were reported [5]. Temperature, pH, soil composition and various contaminants would also impact upon the extent of racemization (and hence age estimation) in fossil bone samples. Indeed Marshall [11] in reporting the troubled history of racemization dating of bone highlighted the mismatch between early AAR estimates of Palaeoindina bones [7] and subsequent radiocarbon dating of the same amino

66


acids. Marshall [11] reports Milford Wolpoff s opinion that many people regarded AAR as "some kind of joke". Since various changes in temperature during the past the reaction temperature of racemization can only be estimated and not accurately determined. Therefore in this, as in earlier studies, DL ratios of independently dated samples have been used to 'calibrate' the rate. These data are then compared with data obtained from the analysis of amino acids in samples of unknown age. To make the comparison more accurate, the antecedents of samples of known age when analysed were the same as or similar to those of unknown age. All of the samples analysed in this study originated from the similar Hungarian environment. The samples were excavated from a depth of approximately 1.5-2.0 metres (the depth is very important because of the temperature; the yearly average temperature at this depth is ll^'C. The pH of the soil at this depth in most cases was very near to neutral and never exceeded 7.0 (the pH ranged between 5.5 and 6.8). Therefore, approx. 250 fossil bone samples previously analysed by the radiocarbon method were collected from various Hungarian museums, and their D- and L-amino acid contents were determined so far. The analyses were accomplished in the Radiocarbon Laboratory of the Nuclear Research Institute of Debrecen from collagen isolated from bone according to the generally accepted methods of radiocarbon laboratories. The DL ratio was plotted against time to produce a calibration curve. This curve can be used to estimate the age of samples of unknown age after their D- and L-amino acid contents have been determined. The DL ratio for 2-3 amino acids was determined for each sample and the mean value of ages estimated from calibration curves was used to derive the final age estimate. 1.2 Individual age determination from teeth The possible most accurate estimation of age at the time of death is a significant part of the study of historical populations. In everyday practice age means the time span between the birth and death of any individual human being. It is traditionally measured in years, months, weeks, etc. In other words it is calendar or chronological age. The usual age estimation methods of historical anthropology observe and analyse the changes occurring on the skeleton and on the teeth with the progress of age. Growth, maturation and aging are all processes that leave characteristic traces on human bones and teeth, therefore the estimation of the so called skeletal and dental ages can be estimated. Skeletal and dental ages come from the biological age of any given individual. If and when the person's biological age is close to his or her calendar age our estimate for his or her skeletal (or dental) age can fall very close to his or her calendar age [12]. This way we are able to produce an indirect estimation of calendar age via biological age determination. When estimating age the anthropologist analyses ontogenesis, the biological aging of the human organism. These processes are determined by a multitude of external (environmental) and internal (genetic) factors. Chronological age runs at a steady pace, but the passing of biological age can be and really is very varied between individuals and even within one human frame too, among its constituent parts. This is the reason for which we cannot achieve a truly accurate calibration of our age measuring methods compared to the passing of chronological time: this sort of calibration could be nothing else but an individual one. As biological and calendar ages often don't concur, chronological age in paleoanthropology may be estimated only within certain intervals (5 years at least). There can be no other realistic aim for us but to score within these limits with the greatest plausibility possible. Some

Use of Amino Acids and Amino Acid Racemization for Age Determination in Archaeometr>^ constituents of the human body, such as teeth, are easy to examine even centuries after the death of the individual, as their changes are not significantly affected by the environment. Their analysis could significantly improve the accuracy of age estimation. Helfman and Bada [13] were the first to declare that the aspartic acid racemization process within teeth could be utilised to estimate the age of living animals and of humans. Then they estabhshed the reaction rate coefficient of aspartic acid for human teeth at 8.29x10""* year"\ but they measured it at T-STxlO""* year"^ a year later [14]. Bada and Brown [15] produced a calibrating diagram by plotting In (H-D/L)/(l-D/L) against time. They found a satisfactory level of conformity between the actual age data and the data estimated on the basis of amino acid racemization. Gillard et al [16] analysed the D-Asp content of molars and premolars. They failed to establish significant differences between the D-Asp contents of the same teeth when taking samples fi-om several parts of their crowns and roots. Ohtani and Yamamoto [17] found significant differences between estimated and actual ages when comparing the D-Asp contents of dental enamel and dentin substance. They detected that racemization (kAsp: 5.75x10"^ year'^) is much faster in dentin than in enamel (kAsp: 4.47x10"'* year'^). They concluded that age could be more accurately estimated on the basis of dentin (±3 years) than on the basis of enamel (±2-11 years). They also calculated post-mortal reaction speed coefficients for an average temperature of 15 "^C (dentin: kAsp. 9.70x10"^ year'^ and enamel: 1.330x10"^ year-^). Ritz et al. [18] analysed the dentin substance of third molars' roots and they arrived at the conclusion that racemization of the root's dentin was somewhat different to that of the crown's dentin. They produced a special calibration diagram for those cases where age has to be determined but the crown substance was damaged or impaired. They established that the degree of Asp racemization was multiplied in acid solvent proteins compared to non-acid solvent ones. Ohtani [19] examined Asp racemization on central and lateral incisors, on first and second molars and he also treated the averages of these. He found a close correlation of actual ages and D/L Asp ratios. Ohtani concluded that racemization within deciduous teeth was a good indicator for the estimation of individual age, but racemization of permanent was far less useful for the same purpose. Ritz et al. [20] have reported the application of this method to biopsy samples of dentine. Thanks to the strictly regulated nature of the sampling process ages estimated on the basis of Asp racemization presented a very close conformity to actual ages. Ritz et al. established a margin of error not larger than ±year for 46.4% of the cases analysed and the error of age estimation never went beyond the ±5 years limit. Improvements in chromatographic methods have increased the number of chiral amino acids which can be separated in a single analysis. Nevertheless the ready separation, rapid racemization and high concentration of aspartic acid in teeth, means that it is still the most widely studied amino acid. We have previously estimated the racemization half-lives of different amino acids [21, 22]. It was concluded that D-enantiomers of amino acids with faster racemization than that of Asp promise to be just as good indicators of individual age as Asp, and amino acids falling of slower racemization rate could also provide useful information on age. We present in this paper two case studies: one regarding the age determination of fossil bone samples and the other concerning the estimation of individual age at the time of death. The development of the calibration curves used as well as the correlation of our results (based

67

68


on amino acid racemization) with standard paleoanthropological methods will be also described in this report. 2.

Materials and Methods

2. / Amino acid determination method The determination of the DL amino acid ratio in hydrolyzed proteins requires chromatographic methods which are suitable for the simultaneous separation and highly sensitive detection of several amino acids and their enantiomers. In order to be able to measure the changes of the amino acid composition and/or the enantiomeric ratio of the individual amino acids in very small archaeological samples the use of the highly selective and sensitive high-performance liquid chromatographic method (HPLC) is inevitable. Precolumn derivatization of a-amino acids using o-phthaldialdehyde (OPA) and various thiols as the reagents leads to fluorescent formed derivatives, while another reagent, 9-fluorenylmethyl chloroformate (FMOC-Cl) transforms a-amino and imino acids to fluorometrically active derivatives. Both types of derivatives have good chromatographic properties enabling several amino acids to be separated and measured within one chromatographic run at very low concentration levels. If the aim is the enantiomeric separation of amino acids, chiral derivatization agent should be used which transforms the amino acid enantiomers to pairs of diastereomeric derivatives separable on achiral HPLC columns. The chiral 1-(9-fluorenyl)-ethyl chloroformate (FLEC) reagent [23], and also the OPA/thiol reagent are suitable for this purpose if a chiral thiol is used in the latter case [24]. Both types of reagents were successfully used in our study. If the aim is only the separation of the isoleucine and D-allo-isoleucine, no precolumn derivatization is necessary since they are diastereoisomers, and the separation and quantitation can be made by amino acid analyser without chiral selectors. The prerequisite of a successful chromatographic analysis is the complete hydrolysis of the protein content of archaeological samples. All hydrolyses were carried out using 6M hydrochloric acid. If the aim is the determination of the enantiomeric ratio, it is very important to keep the extent of racemization at the lowest possible level. The effect of the reaction times and temperatures on the extent of the hydrolysis and racemization has been carefully investigated [25]. 2.2 Hydrolysis of proteins with reduced racemization The following materials were used for testing the racemization during hydrolysis: bovine ribonuclease, lysozyme, cytochrom C, fossil bone sample, and individual free amino acids as follows: L-aspartic acid, L-glutamic acid,, L-threonine„ L-alanine, L-valine, L-phenylalanine, L-histidine and L-tryptophan. The classical Moore and Stein [26] method using 6M hydrochloric acid medium for 24 h at 110 ""C was compared with hydrolysis in the same medium but at higher temperature (160-180 "^C) with shorter reaction times (15-60 min) using closed Pyrex tubes as reaction vessels. The use of microwave oven was also attempted. The method of Einarson et al. [23] was used to monitor the racemization (HPLC column: Kromasil C8 5 |am, 250 x 5.6 I.D.; eluent: gradient system A = 40% methanol in 9.5mM phosphate buffer at pH = 7.05, B = acetonitrile; pre-column derivatization reagent: ophthalaldehyde/2,3,4,6-tetra-0-acetyl-1 -thio-(3-D-glucopyranoside (OPA/TATG)).

Use of Amino Acids and Amino Acid Racemization for Age Determination in Archaeometry 3.

69

Results and Discussion

3. L Hydrolysis studies With the use of higher hydrolysis temperature e. g. 160 ""C, 170 ""C or 180 T , much shorter reaction time is adequate to achieve complete hydrolysis related to classic hydrolysis conditions when 24 hour heating is required for the same purpose at 110 ''C. Even splitting of the most stable bonds adjacent to Val, He and Leu is completely accomplished at 160 ''C for 60 min, at 170 ^C for 45 min and at 180 T for 30 min. The rate of the racemization in the course of the hydrochloride acid catalyzed protein hydrolysis decreases in the following order: aspartic acid, glutamic acid, threonine, phenylalanine, alanine, valine, histidine, as shown in Table 1 on the examples of lysozime, cytochrom and bone sample. Tryptophan was almost completely decomposed under the examined conditions [25]. The rate of the racemization of peptide bound amino acids in 6M hydrochloric acid between 110 and 170 ""C is 4-7 times higher than that of the free amino acids. Microwave-promoted hydrolysis is advantageous if racemization occuring due to this sort of treatment has no importance related to the subject of the study. If minimizing racemization is a prerequisite of the success of the research, this method is definitely disadvantageous. As seen in Table 1, the degree of racemization in case of high temperature - short reaction time methods is lower by 20-55% than at 110 "C for 24 h. Two optimal conditions were selected 160 ''C for 60 min or at 170 ""C for 45 min, and these were selected for the age estimation studies. Table 1. D- amino acid content of lysozyme (A), cytochrome (B) and fossil bone (C) hydrolysed by 6 M hydrochloric acid at different temperatures for different times Amino acid Asp Glu Thr Ala Val Phe His

A

110 "C for 24 h B

C

A

160 *C for 60 min. B

C

6.62 4.58 3.62 2.99 2.11 3.31 1.83

7.0i 4.61 3.74 3.21 2.24 3.42 1.89

7.89 5.93 4.38 4.02 2.53 3.64 2.38

3.27 2.79 2.29 1.69 1.69 3.19 1.64

3.42 2.84 2.31 1.65 1.84 3.37 1.67

4.15 3.61 3.14 2.13 2.33 3.57 2.01

A

170 T for 45 min. B

C

A

180 T for 30 min. B

C

3.29 2.81 2.11 1.72 1.71 2.89 1.52

3.57 2.89 2.23 1.77 1.82 3.11 1.60

4.42 3.74 3.04 2.11 2.27 3.60 1.99

3.84 3.51 2.87 2.81 2.54 2.97 1.79

3.99 3.63 3.14 2.89 2.57 2.83 1.93

4.67 3.92 3.42 3.04 2.82 3.20 2.11

Amino acid Asp Glu Thr Ala Val Phe His

The values refer to the percentage of racemization expressed as the ratio [D/(D+L)]xlOO. Each value is the mean of three determinations

70


3.2. Age determinations offossil bones 3.2.1. Epimerization of isoleucine. The bone samples were washed in running distilled water, dried in a vacuum drying oven and ground to produce powder material as fine as flour. Apolar contaminants were removed with petroleum ether in a Soxhlet extractor. The free amino acids were extracted by O.IM HCl solution for 16 hours. The nitrogen content of the residue was determined by Kjel-Foss nitrogen analyser. Sample size (200-2.000 mg residual material containing approx. 10-20 mg protein) was dependent on nitrogen content. Samples were weighed and hydrolysed with 6M HCl at 170 ""C for 30 min. The sample was then lyophylized, the residue was dissolved in water, and the precipitated silicate compounds were separated from the liquid containing free amino acids by centrifrigation. The pH of the solution was raised to pH=9 to precipitate metal hydroxides which were filtered off The hydrolysed solution was neutralised and evaporated to dryness by lyophylization. An aliquot of hydrolysed material was dissolved in a citrate buffer solution of pH=2.2, and isoleucine and D-allo-isoleucine were determined by LKB 4101 type amino acid analyser as described by Csapo et al. [27]. 3.2.2 Racemized amino acids determined with HPLC. The other D- and L-amino acids were separated by the method of Einarsson et al. [23]. The data obtained from the analyses on 24 fossil bone samples of known age from various Hungarian museums are summarised in Table 2 [10, 21]. Linear relationship was found between the D/L ratio and the age of the samples determined by the radiocarbon method. Seven amino acids (His= histidine, Phe= phenylalanine, Asx= aspartic acid / asparagine, Glx= glutamic acid / glutamJne, Ala= alanine, Ile= isoleucine, Val= valine) were investigated. These may be considered as being the most suitable for age determination because some of them show very fast racemization (His, Phe, Asp), while others show medium (Glu and Ala) and very slow racemization (He, Val); analytical data for other amino acids analysed are not presented in Table 2. None of the ratios lower than 0.1 or higher than 0.7 presented in Table 2 because, in these cases, the accuracy of age determination was doubtful. (The two acidic amino acids, aspartic acid and glutamic acid are exceptions, which mount up approx. 30% of the protein content of the bone and the tooth, so due to this the low rate of racemization resuhs in a well measurable D-enantiomers. The half life of amino acid racemization, i.e. the time required for the ratio of D- to L-amino acid to reach 0.333, was calculated by interpolation or extrapolation from the data of Table 2 and is presented in Table 3. The temperature of the samples during burying was approximately 1IX, while the pH of the soil in the surroundings of the bone was between 5.5 and 6.8. From the data of Table 2 and Table 3 it is evident, that D-His, D-Phe, D-Asp, D-Glu and D-Ala contents can be used for the age determination of samples which are 2-12.000, 320.000, 5-35.000, 8-70.000 and 10-80.000 years old, respectively. Age of samples older than 30.000 and 50.000 years can be determined on the basis of He and Val content, respectively. Data presented in Table 2 were corrected using the D-amino acid content of a fresh pig bone to correct for induced racemization. Concentrations of the D-form for all of the amino acids were negligible. However, all analyses were corrected for the small concentrations present in fresh pig bone. Studying the calibration curves, it can be concluded that in the case of D/L ratio lower than 0.1, the D-amino acid content is too low (with the exception of the aspartic acid and glutamic acid) and age determination is uncertain. It is obvious that the calibration curves can be used

Use of Amino Acids and Amino Acid Racemization for Age Determination in Archaeometry

71

Table 2. D/L ratios for various amino acids concerning ages of fossil samples determined by the radiocartwn method Age of samples determined by the ^'^C corrected method (year) 2200 2800 3110 3240 4630 5460 6850 11200 12400 15600 18600 20200 22600 25400 28600 30400 32500 36900 44600 46800 54300 62200 65000 72400

His

0.138 0.162 0.181 0.199 0.253 0.312 0.419 0.618 0.682

-

Phe

The D/L ratios for various amino acids Ala Glu Asp

_ 0.101 0.109 0.128 0.179 0.225 0.252 0.442 0.473 0.561 0.654 0.689

-

_ 0.109 0.128 0.171 0.271 0.289 0.378 0.432 0.491 0.543 0.580 0.621 0.643 0.702

-

_ 0.091 0.126 0.143 0.178 0.209 0.233 0.256 0.275 0.311 0.250 0.355 0.395 0.481 0.500 0.527 0.606 0.340

-

_ 0.112 0.131 0.158 0.192 0.209 0.228 0.246 0.289 0.321 0.343 0.381 0.465 0.483 0.510 0.586 0.613 0.652

lie

Val

-

-

0.099 0.118 0.134 0.142 0.169 0.188 0.199 0.221

Table 3. Half lives of racemization and epimerization of various amino acids found in Hungarian fossil bone samples* Amino acids Histidine Phenylalanine Tyrosine Aspartic acid Serine Threonine Glutamic acid Alanine Isoleucine Leucine Valine

Half life (year) 5500 8500 8600 13500 16500 17000 28500 32000 110000 140000 180000

The half life of amino acid racemization, i.e. the time required for the ratio of D- to L-amino acid to reach 0.333, was calculated from the data of Fig. 2. The temperature of the samples during burying was approximately 11 °C, while the pH of the soil in the surroundings of the bone was between 5.5 and 6.8.

0.100 0.115 0.119 0.136

72


for age determination most satisfactorily in the linear range (between 0.1 and 0.5 where Damino acids are present in well detectable amounts). The optimum D/L ratio for each sample can be found by analysing the amino acids best suited for age determination, e.g., for fossil bone samples of 11.200 years of age the D/L ratios for His, Phe, Asp and Ala are 0.682, 0.473, 0.271 and 0.112, respectively. In this case the D/L ratios of Phe and Asp are recommended for determining the age of samples; however, the D/L ratios of His and Ala can be used to confirm the estimate based on the ratios of Phe and Asp. The applicability of calibration curves is presented finally. As an example, one bone sample with unknown age and originated from the end of the Neolithic Age or from the beginning of the Copper Age according to the archaeologists was analysed for L- and Damino acids and the following results were obtained (the unknown sample was excavated from a depth of 1.85 metres and the pH of the soil was 6.50): L-His: 0.0697 mg, D-His: 0.0289 mg, D/Lnis= 0.428. Age calculated from calibration curve: 7100 years; SEM = 337. L-Phe: 0.0543 mg, D-Phe: 0.0138 mg, D/Lphe=0.254. Age calculated from calibration curve: 6950 years; SEM = 191. L-Asp: 0.1346 mg, D-Asp: 0.0245 mg, D/LASP=0.182. Age calculated from calibration curve: 6900 years; SEM = 465. The estimated age of the sample is the mean value of the above estimates, or 6980 years. This mean value has a standard error of 202 years and the 95% confidence interval would be 6554 to 7406 years. Of course the described method includes the analytical error of age estimation by the ^"^C method, but the effects on AAR of temperature, pH and the composition of soil have been eliminated. The D/L ratio for 2 to 3 amino acids should be determined for each sample, and the mean value of estimated ages based on calibration curves is considered the best estimate of age of the fossil sample. We have utilised this method very successfully for dating fossil bone samples from Hungary. The difference between the data from the calibration curve and those from ^"^C dating was generally negligible. From 1994 we have determined the age of several human bones and different species of animals (cattle, horse, red deer) to the greatest satisfaction of our archaeologist colleagues. However, our calibration curves should not be used in other environments, due to different conditions (temperature, pH). However, based on these results, other calibration curves can be formulated for each environment on the basis of the methods described here. The great advantage of this method compared to the other methods is that very low sample size is required. 2-10 mg sample size containing O.l-l.O mg protein is sufficient for age determination based on AAR. The radiocarbon age determination method can be used only for samples not older than 80.000 years. By means of AAR (especially by the means of He, Leu and Val) the age of the samples can be estimated up to 300-500 thousand years. 3.2.3. Age estimation of teeth based on the D-aspartic and D-glutamic acid contents. Our research project was carried out in collaboration with the Institute of Odontology and Earth Sciences Centre of Gothenburg University. We analysed two recent tooth sample series to establish the so called calibrating diagrams. In 1998-99 we gathered 22 teeth from the dental surgery of Pannon Agricultural University's Faculty of Animal Sciences in Kaposvar and we measured the D- and L-aspartic acid contents of them. When planning the sample we attempted to include individuals with in the largest possible age envelope (17-62 years), and


73

we also tried to select enough individuals from each age group to have a comprehensively representative sample. Table 4. contains the data produced by analysing the Kaposvar dental sample of 1998. We calculated ln(l+D/L)(l-D/L) correlations both for aspartic and glutamic acids besides D/L aspartic and D/L glutamic acid ratios. D/L ratios as well as the ln(l+D/L)(l-D/L) ftmction were presented as a function of age. We calculated correlations of known ages and the D/L ratios of the two amino acids by linear regression. We found a very close positive relation between D/L ratio and age in case of aspartic acid contents. The value of r was 0,91 for the D/L ratio as well as for the calculated function. When analysing glutamic acid we concluded that the values of r fell between 0,98-0,99 for the D/L ratio as well for the calculated function. Our examination of this dental sample of 22 teeth also led us to the conclusion that D-aspartic acid is a useful indicator for the estimation of individual age if treated to the analytical methods (protein hydrolysis, derivative production, separation and identification of D- and Lenantiomers) we applied. We also drew the conclusion that D-glutamic acid content is also suitable for accurate age estimation beside D-aspartic acid, though D-glutamic acid is present in teeth in a smaller concentration because of its different racemization half-period. That is the reason why it is more difficult to measure and its scoring is a more demanding job for researchers. Our first conclusions were based on a numerically small sample but we supported them by analysing 102 dental samples in 1999. At the same time we opened up our field of research from comparatively young age groups towards older ones. Our 1999 examinations produced r=0,93 positive correlations between D/L ratios and individual ages both in case of D-aspartic

Table 4. D-amino acid content of teeth of different age Age (year)

17 20 21 22 22 24 24 25 27 28 31 32 35 40 42 43 43 44 46 53 53 62

lii(l+D/L)/(l-D/L)

D/L ratio

Asp

Glu

Asp

Glu

0.034 0.035 0.036 0.037 0.038 0.039 0.038 0.041 0.042 0.043 0.044 0.044 0.047 0.050 0.052 0.053 0.053 0.053 0.055 0.059 0.060 0.065

0.017 0.017 0.019 0.019 0.020 0.021 0.019 0.021 0.021 0.021 0.022 0.022 0.024 0.026 0.026 0.027 0.028 0.027 0.028 0.031 0.030 0.033

0.068 0.070 0.072 0.074 0.076 0.078 0.076 0.082 0.084 0.086 0.088 0.088 0.094 0.100 0.104 0.106 0.106 0.106 0.110 0.118 0.120 0.130

0.034 0.034 0.038 0.038 0.040 0.042 0.038 0.042 0.042 0.042 0.044 0.044 0.048 0.052 0.052 0.054 0.056 0.054 0.056 0.062 0.060 0.066

74


and D-glutamic acids. The relation of an individual's age and the D-aspartic acid content of his or her tooth was presented in Figure 1, the correlation of age and D-glutamic acid was presented in Figure 2. These two correlations are eminently suitable to estimate the age of any individual in the age envelope of 40-86 years on the basis of the D-aspartic and D-glutamic content of his or her tooth. The results of our 1999 research work confirmed those arrived at in 1998, so we may state the existence of an extremely close link between any individual's age and the D-aspartic acid content of his or her tooth, and the D/L aspartic acid ratio on the basis of the analysis of a numerically large sample of teeth. Another one of our assumptions was also proven correct: it was not just D-aspartic acid content but also D-glutamic acid content that could be used to estimate the age of an individual if a sufficiently sensitive method of analysis was provided to measure the small concentration of D-glutamic acid present. In the third phase of our work we tried to apply the calibration diagrams produced by our amino acid racemization method on tooth samples originating in historical times. The age of individuals from Avar cemetery were estimated by the above mentioned anthropological methods in advance. The average of their age estimated by "traditional" anthropological methods, D/L aspartic and D/L glutamic ratios as well the age of these bone samples calculated on the basis of D/L aspartic acid and D/L glutamic acid was presented in Figure 3. In Figure 3 we also presented these ages by linear regression and this way it also presented the correlation of ages estimated by anthropological and by the two D-amino acid contents. It was evident from this figure that this correlation of ages estimated by the traditional and the new D-aspartic acid content methods was extremely close. When comparing the results of anthropological age estimation to those of the method based on amino acid racemization the value of "r" exceeded 0.9 with both amino acids.

50

60

70

80

Age of life (year)

Figure 1. Linear regression between the age of life of the individuals and the D/L aspartic acid ratio of their teeth

40

50

60

70

80

Age of life (year)

Figure 2. Linear regression between the age of life of the individuals and the D/L glutamic acid ratio of their teeth


75

E cp Q

^5

30

20

30

40

50

60

70

80

Age of life (year) estimated by anthropological methods

Figure 3. Linear regression between the age of life estimated by anthropological methods and D-amino acid content We investigated whether are there any differences in the D-aspartic acid and D-glutamic acid content of teeth from the same skull. For the sake of increasing the accuracy of the method we determined the concentration of the D-aspartic acid and D-glutamic acid and the D/L ratio on both two amino-acids of the incisors, eye teeth, premolar teeth and molar teeth taken from the skull of a female's skeleton estimated 40-45 years old by anthropological method. From our researches it seems that the incisors contain more glutamic acid than the others, and the aspartic acid content of the first incisor and the first molar tooth is also higher than the others (Figure 4). Examining the D-amino acids and the D/L ratio we ascertained that the D-aspartic acid and the D-glutamic acid content and the D/L ratio of the two incisors are the highest among all the teeth, followed by the eye teeth, the premolar teeth and the molar teeth (Figure 5). We received the least amount of D-amino acid content and D/L ratio at the third molar tooth. From our experiments it seems that the later growing teeth contain less Damino acids than the ones coming out earlier, which shall be taken into consideration at the age determination based on D-amino acid concentration of teeth. At the comparative experiments it is practical to use teeth from the same age, because the differences among the teeth of the individual can be bigger than the difference between individuals, which makes age determination uncertain. The significance of the anthropological utilisation of the amino acid racemization based age estimation method can be summarised as followings: A single tooth or even a fragment with some enamel is sufficient for amino acid based analysis. It could be an especially useful characteristic when treating poorly preserved remains. The amino acid racemization based age estimation method is built on an exact foundation of natural sciences. Its application was clear-cut, and therefore free of intra- and interpersonal errors. Amino acid racemization

76

Progress in Biological Chiralitv 1,4

Figure 4. L-aspartic acid and L-glutamic acid content of different teeth samplesfromthe same skull (Ii, I2 = incisor: C = canine; Pi, P2 = premolar; Mi, M2, M3 = molar)

0,06-

S 0,05o o 3

0,04-

I

•g

"o 05

o 0,03E 05

Q 0,02-

2 0,010,00-

I I D-aspartic acid I M I D/L Asp ratio I I D-glutamic acid • I D/L Glu ratio

mMz-+I1

in I2

Figure 5. Composition of the different teeth samplesfromthe same skull (Ii, l2= incisor C = canine; Pi, P2= premolar, Mi, M2, M3 = molar)

provides help where it is the most urgently needed: in the estimation of age within the most difficult adult group. The results produced by the amino acid racemization based method presented a fine correlation to those of other "classic" anthropological methods. When used in combination they could confirm each-other's results. For theoretical considerations this method was developed into a different entity compared to all routine age determination methods of anthropology. In contrast to all the other methods, it did not take into consideration the genetically programmed evolution of the organism, nor its responses to the environment or its physiological adaptation. It measured the structural

Use of Amino Acids and Amino Acid Racemization for Age Determination in Archaeometiy

77

alterations of amino acids, which are processes independent of circumstances of life and genetical facilities. In fact, amino acid racemization measured the passing of chronological time and not that of biological time, and therefore it gave a completely new meaning to the word age in historical anthropology. The advantage of the method elaborated by us compared to the previous ones can be summarized as follows: - we do not only determine the age of the individual by the aspartic acid, but by the D/L ratio of the aspartic acid and the glutamic acid, and the average of the two estimations are considered as the real age; - if different teeth are available from the different individuals then we can correct the age of the individual at the time of death by analysing the teeth of the same skull and the accuracy of the analysis can be improved if different teeth are available. 4.

Conclusion After reviewing previous attempts to use the extent of amino acid racemization for the determination of the age of archaeological samples containing proteins the authors describe their own approach. After an optimised protein hydrolysis with low racemization the D- and L-amino acid content in fossil bone samples of known age (radiocarbon method) was determined by HPLC after precolumn derivatization. Based on the obtained half-lives of racemization and plotting the D/L ratio as a function of time for various amino acids, calibration curves were obtained which can be used for the age determination of fossil bone samples in the range of 2000-500 000 years. Another method is presented for the determination of age at death based upon the racemization of aspartic acid in teeth. In addition to aspartic acid, D-glutamic acid was also found to be suitable for the estimation of age. Calibration curves based on these investigations were used for the age estimation of more than 200 skeletons of unknown age from the different historical periods. The correlation coefficient between our results and those obtained using standard paleoanthropological methods was over 0.9. From our experiments it seems that the later forming teeth racemized more slowly than those which erupt earlier, and this must be taken into consideration in age determination. Comparative experiments suggest that differences in extent of racemization of the same individual can be larger than the difference between individuals.

5.

References

[1] P.H. Abelson, Amino acids in fossils. Carnegie Inst. Washington Yearb. 53 (1954) 97-108. [2] P.E Hare and P.H. Abelson, Racemization of amino acids in fossil shells. Carnegie Inst. Washington Yearb. 66(1967)526-536. [3] P.E. Hare and R.M. Mitterer, Laboratory simulations of amino acid diagenezis in fossils. Carnegie Inst. Washington Yearb. 67 (1968) 205-212. [4] J.F. Wehmiller and P.E. Hare, Racemization of amino acids in marine sediments. Science 173 (1971) 907914. [5] K.M, Williams and G.G. Smith, A critical evaluation of the application of amino acid racemization to geochronology and geothermometiy. Orig. Life 8 (1977) 91-144. [6] G.H. Miller and P.E. Hare, Amino acid geochronology: Integrity of the carbonate matrix and potential of moUuscan fossils. In: Biogeochemistry of amino acids (Eds. P.E. Hare, T.C. Hoering and K. Jr. King) J. Wiley and Sons, NY, 1980, pp. 415-425.

78 [7 [8 [9 [10 [11 [12 [13 [14 [15 [16 [17; [18 [19 [20 [21 [22 [23

[24

[25

[26

[27

Progress in Biological Chirality J.L. Bada, Aspartic acid racemization ages of California Paleoindian skeletons. Am. Antiquity 50 (1985) 645-647. J.L. Bada, Amino acid racemization reactions andtiieirgeochemical implications. Ann. Rev. Earth Planet Sci. 13(1985)241. J. Csapo, I. Pap and L. Kolto, Archaeological age determination of fossil bone samples based on amino acid racenuzation and epimerization. Anthropologia Hungarica 1 (1988) 67-86. J. Csapo, Z. Csapo-Kiss, L. Kolto and I. Papp, Age determination of fossil bone samples based on the ratio of amino acid racemization. Archaeometry '90, Birkhauser Verlag, Basel 1990, pp. 627-635. E. Marshall, Racemization dating: Great expectations. Science 248 (1990) 799. D.H. Ubelaker, Human Skeletal Remains, Excavation, Analysis, Interpretation. Taraxacum, Washington, 1989, pp. 63-95. P.M. Helfman and J.L. Bada, Aspartic acidracemizationin tooth enamel from living hmnans. Prog. Natl. Acad. Sci. USA 72(1975)2891-2894. P.M. Helfman and J.L. Bada, Aspartic acidracemizationin dentine as a measure of ageing. Nature 262 (1976)279-281. J.L. Bada and S.E. Brown, Amino acidracemizationin living mammals: biochronological apphcations. Trends in Biochemical Sciences September 3-5, 1980. R.D. Gillard, A M Pollard, P. A. Sutton and D.K. Whittaker: An improved method for age at deatii determination from the measurement of D-aspartic acid in dental collagen. Archaeometry 32 (1990) 61-70. S. Ohtani and K. Yamamoto, Estimation of age from a tooth by means ofracemizationof an amino acid, especially aspartic acid - Comparison of enamel and dentin. J. Forensic Sciences 37 (1992) 1061-1067. S. Ritz, H.W. Schiitz and C. Peper, Postmortem estimation of age at death based on aspartic acid racemization in dentin. Its applicabihty for root dentin. Int. J. Legal Medicine 105 (1993) 289-293. S. Ohtani, Age estimation by aspartic acid racemization in dentin of deciduous teeth. Forensic Science International 68 (1994) 77-82. S. Ritz, R. Stock, H.W. Schutz and H.J. Kaatsch: Age estimation in biopsy specimens of dentin. Int. J. Legal Medicine 108(1995) 135-139. J. C s ^ , S. Nemethy, S. Folestad A. Tivesten, T.G. Martimand Z. Csapo-Kiss, Age determination based on amino acid racemization. A new possibility. Amino Acids 1 (1994) 317-325. J. Csapo, Z. Csapo-Kiss and J. Jr. Csapo, Use of the amino acids and amino acid racemization for age determination in archaeometry. Trends in Analytical Chemistry 17(1998) 140-148. S. Einarsson, S. Folestad and B. Josefsson, Separation of amino acid enantiomers using precolumn derivatization with o-phthalaldehyde and 2,3,4,6,-tetra-O-acetyl-l-thio-p-D-glucopyranoside. J. Liquid Chrom. 10(1987) 1589-1596. S. Einarsson, B. Josefsson, P. Moller and D. Sanchez: Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography, ylwfl/. Chem. 59(1987) 1191-1198. J. Csapo, Z. Csapo-Kiss, L. Wagner, T. Talos, T.G., Martin, S. Nemetiiy, S.Folestad and A. Tivesten, Hydrolysis of proteins performed at high temperatures and for short times with reduced racemization, in order to determine the enantiomers of D- and L-amino acids. Analytica ChimicaActa 339 (1997) 99-107. S. Moore and W.H. Stein: Chromatographic determination of amino acids by the use of automatic recording equippment. In: Methods in Enzymology (Eds. S.P. Colowick and N.O. Kaplan), 6 (1963) 819831. J. Csapo, I. Toth-Posfai and Z. Csapo-Kiss, Optimization of hydrolysis at determination of amino acid content in food and feed products. Acta Alimentaria 15 (1986) 3-21.


Chapter 7 Enantiomeric Enrichment in Nonracemic Conglomerates. A Possible Component of the Solution to the Problem of the Origin of Biochirality Stanley I. Goldberg Department of Chemistry, University of New Orleans, New Orleans, Louisiana, 70148, USA sgoldber@uno. edu

1.

Introduction There is a growing conviction, based on theoretical and experimental studies, reviewed by Bonner [1], that polyribonucleic acids had to have been initially homochiral in order to have been able to carry out the essential tasks of replication and translation. With even the slightest deviation from homochirality the two helical strands of polyribonucleic acids cannot achieve complete pairing of complimentary bases required for these processes. Homochirality, therefore, is not only an indispensable condition of contemporary life, but it also appears to have been a requirement for the initial emergence of life. Any solution to the biochirality problem, therefore, must provide either enantiopure prebiotic chiral biomolecules or, as suggested by Wald [2], racemic prebiotic homochiral biopolymers. Schemes involving partially resolved material which proceed on to living systems before they achieve the homochiral condition of contemporary life are unacceptable. The present report provides a possible component to a solution in accord with the enantiopure prebiotic stereochemical constraint. It combines the observational evidence for the presence of nonracemic samples of several amino acids in 4.5 billion year old Murchison meteorite, which fell in Australia in 1969 [3, 4, 5], and in the 1949 Murray Kentucky carbonaceous meteorite [6] with experimentally determined properties of partially resolved conglomerates [7] to show how global accumulations of enantiopure biologically relevant material could have formed on prebiotic Earth.

2.

Extraterrestrial Nonracemic Chiral Influence The discovery of nonracemic material in the Murchison and Murray meteorites is tantamount to the discovery of the earlier action of a nonracemic chiral influence that was extraterrestrial and gave rise to selective enantiomer depletions in prebiotic racemic material prior to its dehvery to Earth's surface. Whether that chiral influence was the circularly polarized portion of the synchrotron radiation (ultraviolet) associated with neutron stars, as postulated by [8], or the infrared (and presumed ultraviolet) circular polarization recently

80


observed in the Orion OMC-1 star formation by [9] (or some other source) will have to await ftiture clarification. The important fact is that large amounts of nonracemic chiral material was available on prebiotic Earth. Although the meteorites' more abundant enantiomer in every case was found to an L-amino acid, the levels of enantiomeric purity were low, below the 100% level required to meet the homochirality prerequisite discussed above. What was needed therefore was a readily available natural prebiotic process to transform that partially resolved material into global accumulations of enantiopure biomaterial.

3.

High Amplifications of Enantiomeric Purity by Crystallizations from Solutions of Nonracemic Conglomerates Although it is true that in the absence of a nonracemic chiral influence enantiomers are identical, this is not the case when an enantiomer on the molecular level is compared to an aggregated state of enantiomers within the same system. In such circumstances, the enantiomer in excess may be discriminated from those in the aggregated or associated state. This was experimentally demonstrated in the ^H-nuclear magnetic resonance spectra of highly structured chiral monolayers [10] and in partially resolved samples of strongly hydrogenbonded compounds by [11]. Molecular aggregation reaches a maximum, of course, in the crystalline state, and most enantiomers exhibit properties different from those of the crystalline racemic modifications with which they exist in equilibrium. Of these, the most important is solubility. In partially resolved conglomerates, it is the enantiomer in excess that is always first to crystallize [7] and the crystals may be obtained in near enantiomeric purity. Thus in 1956 Amiard [12] allowed aqueous solutions of nonracemic threonine to cool from 80"^ to 20''C and obtained crystals enriched in the more abundant enantiomer in amounts which, on average, were about two-thirds greater than the original excess. A single recrystallization gave enantiopure threonine. Watanabe and Noyori [13] found when they gradually cooled an aqueous solution of glutamic acid hydrochloride (13% enriched with the L enantiomer) they rapidly obtained almost enantiopure crystals, (98.7% L enantiomer). Nohira et al [14] used 20 mg samples of enantiopure crystals to seed solutions containing 15.5 g of racemic chiral amine salts to obtain, after only one recrystallization of initially deposited precipitate, between 1.6 and 3.4 g of enantiomerically pure material. Also to be noted is the rather wide spread occurrence of conglomerates. By examining 1308 compounds in Beilstein, [15] estimated that between 5 and 10% of all chiral neutral organic compounds crystallize as conglomerates. These facts when combined with current models of comets and the comments added below constitute an element of the solution to the biochirality problem. 4.

Mechanisms for Formation and Isolation of Deposits with High Enantiomeric Purity from Nonracemic Aqueous Solutions of Conglomerates Nondestructive delivery [16, 17, 18] of organic compounds, including chiral material along with huge amounts of water, principally by comets, to the Earth's surface during its first billion years is considered [16, 17, 19] to have been the major source of the planet's biomass. Some of these aqueous solutions, containing nonracemic chiral substances among the dissolved material, would flow into surface features where they would exist as extensive shallow lakes (Figure l.A), eventually depositing their solutes or part of their solutes (crystallization or fractional crystallization. Figurel.B), owing to evaporative concentration

Enantiomeric Enrichment in Nonracemic Conglomerates 81

6

D

Figure 1. Possible mechanisms for formation and isolation of highly enantiomerically enriched precipitatesfromnon-racemic aqueous solutions of conglomerates on early Earth. A - Initial aqueous solution fills an extensive hallow depression; B - concentration of solution owing to evaporation brings about deposition of material highly enriched in the originally more abundant enantiomer; C tectonic slippage or collision of a small impactor removes part of the rim of depression, allowing the supernatant to flow away; or, D - an earthquake like disturbance shifts solid material into the depression, absorbing the supernatant and forming an isolated stratum of enantiomerically enriched material

and/or temperature lowering. Earthquakes, avalanches, or an impacting meteors could breech the banks of the lakes, allowing the supematents to drain away leaving isolated deposited material (Figure l.C), or shift solid material into the depression, absorbing the supernatant and forming a stratum (Figure 1. D). In some cases, these isolated deposits will include, in a highly enriched state, the more abundant enantiomer of partially resolved conglomerates. Thus, during the long era when there were continuous, nondestructive deliveries of nonracemic chiral material to the surface of early Earth, one could expect such processes to be repeated many times in many different locations, resulting in substantial global accumulations of enantiopure biomaterial and setting the stage for the emergence of homochiral life.

82


References [1] W. A. Bonner, in: D-Amino Acids in Sequences of Secreted Peptides of Multicellular Organisms, (Ed. P. JoUes) Birkhauser Verlag, Basel, Switzerland 1998, pp. 159-188. [2] G. W2\d,Ann. N. Y. Acad Sci. 69 (1957) 352-368. [3] M.H. Engel and B. Nagy, Nature 296 (1982) 837-840. [4] M.H. Engel and S.A. Macko, Nature 389 (1997) 265-268. [5] J.R. Cronin and S. Pizzarello, Science 275 (1997) 951-954. [6] J.R. Cronin and S. Pizzarello, in: Abstracts of the 9th ISSOL Meeting, San Diego, California (USA) 11-16 July, 1999, p. 41. [7] J. Jacques, A. Collet and S.H. Wilen, Enantiomers, Racemates, and Resolutions, John Wiley & Sons, New York, 1981, pp. 181-182. [8] E. Rubenstein, W.A. Bonner, H.P. Noyes and G.S. Brown, Nature 306 (1983) 118. [9] J. Bailey, A. Chrysostomou, J.H. Hough, T.M. Gledhill, A. McCall, S. Claik, F. Menard and M. Tamma, Science 281 (1998) 672-674. [10] M.V. Stewart and EM. Amett in: Topics in Stereochemistry (Eds. N.L. Allinger, E.L. Eliel and S.H. Wilen) John Wiley & Sons. New York, 13 (1982) 195-262. [11] B.S. Jursic and S.I. Goldberg, J. Org Chem. 57 (1992) 7172-7174. [12] G. Amiard, Bull. Soc. Chim. France (1956) 447. [13] T. Watanabe and G. Noyori, Kogyo Zasshi 70 (1967) 2174-7; Chem. Ahstr. 69 (1968) 874431. [14] H. Nohira, M. Kai, M. Nohira. J. Nishikawa. T. Hoshiko, and K. Saigo, Chemistry Letters (1981) 951-952. [15] J. Jacques, M. Leclercq and M.-J. Brienne, Tetrahedron 37 (1981) 1727-1733. [16] C.F. Chyba, Nature 343 (1990) 129-133. [17] C. F. Chyba, P.J. Thomas, L. Brookshaw and C Sagan, Science 249 (1990) 366-373. [18] J.M. Greenberg, A. Kouchi, W. Niessen. H. Irth, J. van Paradijs, M. de Groot and W. Hermsen, J. Biol. Fhys. 20 {1994) 61-10. [19] J.M. Greenberg, in: Physical Origin of Homochirality in Life, (Ed. D.B. Cline) American Institute of Physics. Woodbury, New York. 1996, pp. 185-210.


Chapter 8 Genetic Code: a Self-Referential and Functional Model Romeu Cardoso Guimaraes'* and Carlos Henrique Costa Moreira^ '^Dept Biologia Geral, Inst. Ciencias Biologicas, Universidade Federal de Minas Gerais, 3J270-901 BeloHorizonteMG, Brazil romeucg@icb. ufmg. br ^Dept. Matemdtica, Inst Ciencias Exatas, Universidade Federal de Minas Gerais, 31123-970 Belo Horizonte MG, Brazil

1.

Introduction Fundamental constituents of living beings are the polymers proteins and nucleic acids. Their sequences are mutually correspondent through the genetic code, a defining pillar of life's homogeneity [1]. The code is the set of correspondence rules between the amino acids and nucleotide base triplets. In cells, sequences of segments of DNA are transcribed into messenger RNA, which are translated into proteins. In this context, the code is a mechanism for translation. It can be said that the symbol or signal (significant) resides in the nucleic acid, the encoded (signifier) 'meaning' being the correspondent amino acids and proteins. In the context of an isolated system of protein synthesis, it is not necessary to consider DNA. Translation involves only RNA, and mRNA, can be considered genes [2]. This text presents a fijll model for construction of the code but remains synthetic in various more technical sections whose analytical treatment is forwarded to other presentations. The model indicates that the system was originally only coding for protein synthesis, not decoding pre-existing mRNAs. The presentation is organized in two topics. It starts describing the matrix of correspondences and the three basic constituents of the system. It is indicated that the clustering of similar attributions in the matrix can be well described by its division into sectors and quadrants. Some rules prevailing in the development of the system are individually reviewed and others introduced: amino acid types and biosynthesis routes, and their relationships with aRS classes; the locations of aRS classes and of the specific punctuation system in the sectors and quadrants. The topic is closed with the presentation of a scheme for the temporal order in the occupation of boxes containing more than one attribution, derived fi'om a rule of precedence of purines over pyrimidines in the development of attributions and in the evolution of variant sets, and of a mechanism involving the participation of tRNA pairs in protein synthesis. It is then described the systemic rationale followed in devising the temporal succession of attributions entering the code. Our analysis of the hydropathy correlation could point out the first attributions, which were also shown to belong in the main protein-stabilizing and RNA-binding set of amino acids, thereby able to

84


build a stabilized nucleoprotein working system. The development of a nonspecific punctuation system could be proposed, based on the distribution, along strings, of proteinstabilizing then destabilizing amino acids, followed by the introduction of the specific punctuation system, derived from the tRNA pairing mechanism. Amino acid sets characteristic of the protein secondary structures and of the DNA-binding motifs were adequately placed in the temporal scheme. The model was able to propose that genes were originated during the process of development of the coding system and defined in simultaneity with the definition of the products of the code, at the moments of their mutual coupling.

2.

Ingredients

2.1 Correspondence between triplets and amino acids Correspondences in the standard code are established by a system of 20 catalysts, the aminoacyl-tRNA synthetases (synthetases, aRS), one for each of the amino acid substrates. They are proteins, so that the notion of self-reference should already be introduced [3]. At some point in the evolution of the system, products became catalysts, participating in the process of their own formation; the system became cyclic and auto-catalytic. Other substrates are, according to the kind of organisms or organelles, 22-45 tRNAs (transfer RNA, transporters or adaptors). Each reaction synthesizes an aminoacyl-tRNA (ac-tRNA). The problem of deciphering the formation of the system is then a '3-body problem', at the heart of the process of building the cellular nucleoprotein system [4]. Nevertheless, much of present research is still devoted to the hypothesis that the code was formed by direct interactions between RNA and amino acids, as a '2-body problem' [5-7]. Such approaches may be relevant to a part of the process but certainly not to its totality. They usually privilege thermodynamic aspects and consider important the concentration of amino acids abundant in pre-biotic syntheses, partially overlapping the precursor or of independent biosynthetic origin, in the 3' C row (GNC triplets; Val, Ala, Gly, Asp, Glu) [6, 8]. The first four in this list were highlighted [9] on the basis of their wide variety of properties, which would be adequate for formation of all protein conformations together. Ninio [10], among others, valued the homogeneity of simple boxes in the core (Pro, Arg, Ala, Gly), but the two first ones are biosynthetically derived. A 'hidden' pattern of mRNA triplets, which would help ribosomes to maintain the regularity of reading in the triplet frame [11], was updated to GCU [12], indicating the origin of the coding system by Ala codons, composing the first 'words' of mRNA. Our systemic '3-body' approach accepts important participation of thermodynamic stability of triplets, but does not privilege this entirely. Thermodynamic data [13] referring to tRNA dimers but supposedly applicable to codon:anticodon minihelices, indicated that stability of the paired anticodon loops is much greater than what would be expected from helices of common RNA triplets. They are equivalent to helices of heptamers and relatively independent from the base composition of the triplets included. Most frequently, this text will refer to anticodons, their set being the genetic anticode. The standard anticode is considered the largest, with 45 anticodons, and containing only standard bases. The triplets generated by combinations of the 4 bases are 64 (4^) codons. The order of codons along the mRNA sequences dictates that of amino acids in proteins through pairing of anticodons with codons. Three codons do not have their correspondent tRNAs. They may be called non-sense, due to

Genetic Code: a Self-Referential and Functional Model

85

not encoding an amino acid, but determine the end of synthesis of a protein and are called terminators or stop (X). Codes containing more than one signal or symbol (codons, anticodons) for a meaning (amino acid, punctuation) are called degenerate. These can be read without ambiguity in one direction only; in the present case, from codons or anticodons to amino acids. The reading is made by synthetases, recognizing in the class of tRNA corresponding to one amino acid the attributes adequate to the class and accepting the ambiguity or degeneracy. A greater portion of the development of complexity, with reduced degeneracy, resides in the 5' position. Three of the 8 tetracodonic boxes (Ser, Leu, Arg) belong in the group of hexacodonic attributions. Tricodonics are He, with 5' (R, U) ambiguity, and X, with complex constitution: 3' A plus central and 5' Y. The other 4 split boxes are central U, 5' R or Y. Met and Trp are monocodonic, utilizing 5' C. Both tRNAs of Met, for elongation, and of fMet, for initiation (this with a slipped pDiN CAU; Section 3.14), are read by the same synthetase. In Table 1, each box shows the 4 bases in the 5' position of the triplets. These are necessary for identification of attributions in the 8 complex boxes, containing more than one attribution. Simple boxes contain only one attribution. In the standard anticode, only the bases in bold show up. Base A in this position is rare [14], here indicating only the occurrence of its complement (U) in codons. In X, 5' in parenthesis, only the codons occur, anticodons having been deleted. The pDiN approach is not a revival of the '2 bases out of 3' proposal [8, 15] for formation of the early code. It just describes main components of the triplets. 2.2 Matrix ofpDiN The order of bases follows their hydropathy (Figure 1), from hydrophobic to hydrophilic, A-G-C-U [6, 16]. This is different from the one sedimented in the traditional bibliography, AG-U-C. Nonetheless, the order through the aqueous interactions is the physiologic one. This is also indicated by various other symmetries depicted by the hydropathic matrix. An order in the distribution of simple and complex boxes can be described by the pDiN constitution (Figure IB). The 8 simple boxes are in the core (G and C in the pDiN) and in the non-axial boxes with central R, the 8 complex in the tips (A and U in the pDiN) and in the non-axial Table 1. Genetic anticode matrix. Read through columns the central base, through rows the 3' base, of the principal dinucleotides (pDiN; central -^3') of anticodons, which define the boxes of the matrix A G C U A G C U A G C U A G C U

AA

AG

AC

AU

Phe Phe Leu Leu Leu Leu Leu Leu Val Val Val Val He He Met, fMet He

A G C U A G C U A G C U A G C U

GA

GG

GC

GU

Ser Ser Ser Ser Pro Pro Pro Pro Ala Ala Ala Ala Thr Thr Thr Thr

A G C (U) A G C U A G C U A G C U

CA

CG

CC

CU

Cys Cys Trp X Arg Arg Arg Arg Gly Gly Gly Gly Ser Ser Arg Arg

A G (C) (U) A G C U A G C U A G C U

UA

UG

UC

uu

Tyr Tyr X X His His Ghi Gin Asp Asp Glu Glu Asn Asn Lys Lys

86


Ho RR

AA

Mx YR

AA AG

GA GG

CA CG

UA UG

AC AU

GC GU

cc cu

UC

RY

uu YY Ho

AU

"-.^ ^

GA

~" CA

AG 1 AC

GG

CG

GC

X

UA "^«« **

UG 1

^C

CC

,^^ "^ GU _ . . CU

(A)

" UU

(B)

Figure 1. Symmetries in the pDiN matrix and the precedence of central purines. (A) The 16 pDiN boxes are separated in homogeneous (Ho) and mixed (Mx) sectors, each of these divided into quadrants, respectively, RR : YY, and RY : YR. Axial and non-axial boxes are distinguished: Ho axis (bases repeated) bold, Mx axis (bases self-complementary) underlined. (B) The core of the axes or of the matrix is constituted by G and C bases, the tips by A and U. Non-axial boxes, italics, build a circle around the core and internal to the tips. Simple (tetracodonic) boxes in bold (core and non-axial with central R); complex boxes are the tips and non-axial with central Y

boxes with central Y. The core versus tips contrast may be related t o thermodynamic properties [6, 10] but the central purine properties involved should be related to their reactivity or specificity, e. g. bulkiness and hydrophobicity, in the interactions with the synthetase active centers.

2.3 Amino acid types and biosynthesis routes An important advance in understanding the process o f structuring the code w a s introduced by W o n g [17, 18], indicating that amino acids belonging to a biosynthesis route were regionalized, localized close to each other and regularly along the matrix. The indication originated from the proposal o f determination by biosynthesis routes is that the code was

Class 2 Group

6

5

4

3

pDiN 2

1 Ser Thr Pro

His Gly Ala

GA GU GG UG CC GC

Asp UC Asn UU

b Lys Phe

Class 1 1

AG AC AU AU CA CG

3

4

5

Group

Leu Val lie Met Cys Arg

Glu UG

UU

AA

2

Ghi

b (Lys)

CA UA

Trp Tyr

c

Figure 2. Synthetase classes and groups. The schematic, adapted from [26], is an attempted consensus maximizing the balanced sizes of the groups. The rare LysRSl is in parenthesis. Attributions in the CU box and Leu^^, involved with the hexacodonic attributions, are not shown

Genetic Code: a Self-Referential and Functional Model (A) ^ Asp(Ho2) ^ ^

(B)

Asn (Ho2) Lys(Ho2^'>) ThrrM.9. Thr (Mx2) ^
Arg(Mxl) [orAspÂrg]

Ser (H02)

87

Cys (Mxl) Trp (Mxl)

Phe (Ho2)

— • Tyr (Mxl)

Ser (Ho2)

— • Gly (Ho2)

Glu(Hol) or Gin (Mxl)

— ^ His (Mx2)

(C)

s

c,w

P

R — E D

I,M

T

N,K

Figure 3. Amino acid biosynthesis routes. Sectors, Ho and Mx; classes numbered; in parenthesis, the rare LysRS class 1. (A) Precursors and their derivations, in famiUes. (B) Amino acids of independent origin and nonconsensual derivationsfromothers belonging in the code. All propositions, except the derivation of His, are consistent with our model and summarized in (C) formed gradually, in co-evolution with the successive abundance of available amino acids, thereafter becoming incorporated into the synthetase system. Attributions of precursor amino acids were fixed and, when their derivations became abundant, these were attributed to tRNAs similar to the previous one. So, the code was established inside a metabolic system already in a complexification process and availability of amino acids was a limiting factor. It became open to discussion if such clustering, driven by biosynthesis relatedness, was more significant than those due to synthetase properties. The latter should be more akin to amino acid constitution, reflecting either similarity in chemical reactivity or steric properties. Some authors [19, 20] admit that the latter seems to be more coherent with groups inside the synthetase classes (Figure 2). Other aspects of the regionalization will be discussed in Section 2.5. Amino acid properties are multitude and may lead to different classifications [7^ 21], usually attempting quantification of attributes, ranking and averaging of different qualities, with questionable consensuses. Our choice was for some qualitative attributes thought most relevant for formation of the code, biosynthesis relatedness being one of them. Biosynthesis routes have received many revisions [22-24]. Only the consensual routes are adopted here (Figure 3). Amino acids of independent origin or with non-consensual derivations are 7, so

88

Progress in Biological Chiralit\' Table 2. Distribution of synthetase classes and punctuation signs. *, deviants from de aRS class / pDiN type rule; •**, the hexacodonic aRSl expansions and the rare LysRSl; the Ho sector bold Tyr 1 AA 2^' 1 UA CA Phe GA Cys Ser 2 X 1 Leu Trp X 2* His 1* Arg UG AG 1 Leu GG 2 CG Pro Gin 1 2 AC 1 Val Asp GC 2* 2 Ala Gly 1* Glu 1 Asn 2 AU He Ser 2 GU Thr 2* 2(1*) Met 1 Lys l^-> Arg fMet

cc cu

uc uu

that models obeying the biosynthesis derivation rule are constrained only by the relationships in the families of 3 precursors and 10 derived. Class transfers from precursors to derived are: class 2 -> 1 (n=5) and class 1 -> 2 (Glu -> Pro). 2.4 Aminoacyl-tRNA synthetases The 20 synthetases of the standard code are divided in 2 classes or families (Figure 2), with internal homogeneity, formed by duplications of an original enzyme. Each class contains 10 components, distributed in the matrix as in Table 2. LysRS is atypical, class 1 in some organisms. These duplications, followed by variations enabling adequation of the new enzyme to new substrates, should have been limiting factors in configurating the system. Class 1 aminoacylates the 2' hydroxyl of the terminal adenosine of tRNA, while aRS2 does so on the 3' hydroxyl, PheRS being also atypical, aminoacylating in the aRSl fashion [25]. The presentation in the form of dendrograms reflects degrees of similarity of sequences of the proteins. The most similar to each other are in level 1, the most different from all others in levels 5 or 6. The most common interpretation is phylogenetic, saying that the proximity to the root would indicate longer time of divergence from an ancestor to the class or group, and distance from the base more recent branches. Such interpretation should not be taken at face value. Criticisms may be valid, due to the algorithms generating the trees being dependent on not firmly based assumptions. The phylogenetic trees would depend on divergence rates being regular, while these are generally not well known and depend on choices of adequate informative sites', leaving aside some special types of mutations. The maximized numerical symmetry of groups (Figure 2) is only approximate, being better qualified as balance or equilibration. In the standard code, the classes are composed of 10 enzymes each. Variations are of 11 class 1 to 9 class 2, in organisms where LysRSl replaces LysRS2 [27], but may be wider when AsnRS or GlnRS are dispensed with, such amino acids being obtained by amidation of the respective Asp-tRNA and Glu-tRNA. A synthetase is known with double attribution, for Pro and Cys [28]. Inside the groups of each class, variations may be more complex due to formation of functional subgroups, more valued by other authors. In [29], Arg-, Glu- and GlnRS are grouped together due to their tRNA-binding before that of amino acids; GlyRS is the simplest and PheRS the most complex, this being atypical in the acylation mode. In a consensual phylogeny [30], it is proposed to group GlyRS with PheRS, and AlaRS with HisRS, the most basal in the class. SerRS2 and AlaRS2, together with LeuRSl are also special due to not binding the anticodon stem-loops of tRNA [31-33]. It is clear from the distribution in the sectors, that aRS2 shows preference for the Ho and aRSl for the Mx sector.


89

Synthetases not obeying this mle sum 25-30% in each class, and are called deviants. Hexacodonic expansions of aRSl are not counted as deviants and LysRS is class 1 in a few organisms, not being counted as standard. Other regularities are apparent, most evident the preference of aRS2 for central G and of aRSl for central A. It will be shown below that the code organization followed the aRS preferences for sectors, other regularities being consequent and secondary. A compelling evidence for antiquity of aRS2, relative to aRSl [34-35], stems from the observation that the former corresponds to simpler and smaller amino acids, the latter to bulkier and complex ones (Table 3). Class 2 corresponds also to a greater number of amino acids precursor to biosynthesis routes, protein N-end stabilizers, characteristic of simpler protein conformations and nucleic acid binders, and to more degenerate attributions (Table 4). The ensemble of these data indicated the temporal order, starting the construction of the code by the Ho sector and aRS2. Class 2 predominates over class 1 (7/1 amino acids) below a threshold of around 70 A, the reverse (1/9) above the threshold. The 2 others, the largest of aRS2, are activated by the atypical aRS systems. It is indicated that LysRS2 and PheRS2 active center pockets were not fully adequate to receive such bulky amino acids and with extreme hydropathy so that, in some organisms, LysRS 1 replaced the usual class 2 and PheRS2 adopted exceptional conformation, resulting in aminoacylation on the hydroxyl typical of aRSl. 2.5 Regionalization and the diagonal route The traditional explanation for the processes leading to the regionalized distribution of attributions in the matrix (clustering of similar amino acids and codons), has been that of Table 3. Amino acid side chain volume and amino acid protein residue hydropathy. Volumesfrom[36], hydropathy from [37]. The rare LysRS 1 in parenthesis, acylation site not known. The exceptional PheRS2, acylating the T hydroxyl of the terminal adenosine of the tRNA, in the aRSl mode, is marked aRS2

Hydropathy

A

Gly Ala Ser Pro Asp

0.423 0.258 0.508 0.677 0.962

3 31 32 32.5 54

Asn Thr

0.809 0.438

56 61

His

0.573

aRSl

Hydropathy

A

Cys

0.111

55

Glu Val Ghi

0.935 0.069 0.841

83 84 85

Met He Leu (Lys) Arg

0.059 0.029 0.066

105 111 111

0.982

124

Tyr Tip

0.361 0,325

136 170

96

Lys

1.000

119

Phe^

0.000

132

90


selection against errors derived from the non-clustered distributions (supposed to have occurred), which is the 'minimization of errors' hypothesis, reviewed in [23, 38]. Determinist contributions from the co-evolution of the routes of amino acid biosynthesis and of tRNA derivation [17], and from aRS specificities, tend to reduce the weight of the minimization hypothesis. The iterative selection process might have combined some properties distributed mainly along columns with others concentrated along rows, thereby reaching some demarcation of homogeneous islands, which could resemble the sectors and quadrants. Properties distinctively distributed along columns are, e. g., hydrophobic amino acids concentrated along the central A and hydrophilic along the central U, and small amino acids along the central G while, through rows, main regularities are the concentration of small amino acids and precursors to biosynthesis families along the 3' C, and of the tendency of biosynthesis families to spread along rows. Our approach through the diagonals [39-40], is more easily conductive to the regionalization, now in quadrants, offering a simpler mechanism, less costly in the number of iterations. The space available for distribution of variations was reduced from the beginning, thereby making easier the work of selection. The diagonal path is like following 'the midle way', of the hypothenusa of triangles instead of the cathets. Table 4 depicts a synthesis of the properties of amino acids belonging in sectors and quadrants of the matrix. Each of the properties will be detailed in the correspondent sections. The distinction of sectors stems from the formation of palindromic tRNA pairs, which remain restricted to the realm of each sector (Section 2.11). The complementarity of pDiN, in pairs, is correlated to some divergent or 'opposite' properties of the corresponding amino acids, such as the hydropathy (Section 3.2). Since synthetase classes were organized coherently with the sectors and these are divided into complementary quadrants, each class developed divergent properties, following the complementarity. It is expected, due to the 25-30% overlap between sectors in each class, that the averages of amino acid properties in the classes should be less divergent than the averages of sectors (Table 4). It can be said that the code organization resulted from close interaction between tRNA and Table 4. Regionalization in quadrants. Data on quadrants, sectors and classes are shown as averages (with decimals) or as numbers of occiurences. Highestratiosshown Degeneracy (Table 1) RR (4.0) / any of the central Y quadrants, YY or YR (2.3) = 1.7 Amino acids in aRS classes (Table 2) YY (5 aRS2 /1 aRSl) / YR the central Y quadrants (1 aRS2 / 5 aRSl) Amino acid hydrophilicity (Table 3) YY (0.773) / RY (0.171) = 4.52, the 3' Y quadrants Amino acid side chain volume (Table 3) YR (111.0) / YY (57.8) = 1.92. the central Y quadrants Destabilization of protein N-ends (Table 9) YR (6.8) / RY (2.4) = 2.83. the quadrants of the Mx sector Amino acids characteristic of protein conformations (Table 12) Aperiodic (coils and turns): Ho or aRS2 (5) / Mx or aRSl (0) Helices: YR (3) / RR (1), the 3' R quadrants; also Mx or aRSl (5) / Ho or aRS2 (3) Strands: Mx (6) / Ho (1); also aRSl (5) > aRS2 (2) Nucleic acid-binding amino acids (Table 11) RNA-binding: RR (1.55) / YR (0), the 3' R quadrants; also Ho or aRS2 > Mx or aRSl DNA-binding: RY (0.6) / RR (0), the central R quadrants; also Mx > Ho, but aRS2 > aRSl Binding both equally: Mx or aRSl (5) / Ho or aRS2 (0)


91

synthetases, both strong determinants of the regionalized distribution of amino acids through the quadrants. It is a simplification to state that the code was constructed only by the synthetases since their distribution in the matrix followed tRNA complementarity. The distribution of degeneracy reflects the participation of these two principal components. Quadrants were the divisions most frequently producing higher contrasts (8 of the 11 characters). The rule of preference of aRS classes 1 and 2 for, respectively, the sectors of Mx and Ho pDiN, was generally obeyed, with the exception at the amino acids characteristic of DNA-binding motifs, indicating that this character was associated to the attributions deviant from the rule. 2.6 Variant codes The standard code was once thought universal but the finding of evolutionary variants showed that it is only canonic. Variants did not change significantly the standard (Table 5). They contributed, otherwise, to a general rule saying that the sites where they incided upon were evolutionarily labile and also considered of late introduction. The attribution they lead to is considered the original; the variant would be returning to the primary one. Events 'forcing' the changes were, mainly, the development of genomes with disequilibrated base with high G+C contents as in Micrococcus luteus, or high A+U as in organelles or some organisms such as mycoplasmas [14]. Variations found in eukaryotes occurred mainly in unicellulars (ciliates, yeasts, algae) and incided upon the same sites found, more frequently, in bacteria. Our evaluation showed that the most labile sites are the boxes involved with the punctuation system, indicating its late introduction and the involvement of various protein factors, forming a complex system, with more sites of variation. Another labile site is the CU box, especially the Arg dicodonic attribution. Among all sites, most labile are the 5' Y attributions. 2.7 First settlers of complex boxes The rule of purine precedence, firmed for the distribution of simple and complex boxes, can be extended to the definition of the first occupiers of complex boxes, the ones presently attributed to 5' R triplets. It is indicated the first occupier developed tetracodonic degeneracy, retained the 5' R and conceded 5' Y to new occupiers. This is not intended to mean that the structure with simple boxes-only ever existed. The fifth box entering the code (UC), right after the Gly, Pro and Ser group (Section 3.10), already developed complexity (Asp, Glu). Evidences supporting this conclusion stem from various considerations. (1) In the 3 boxes Table 5. Variant codes. Compiled from [41]. Some changes were not completely clarified as to the destination of the change, only knowing that the cited one was lost, and are not detailed 5'Y(13U,4C,5Y) Specific punctuation (11) CA(4) X->W, C UA(4) X ^ Y , Q , L , A AU(3) I(5'U)T

discarded the hypothesis of inversion but provided an explanation for the relationship among all three boxes containing the specific punctuation signs [79]. 3.14.1 Initiation The fMet pDiN is different from that of Met. The latter, utilized in elongation, maintains the standard configuration (central base - ^ 3 ' , CAU) but the fMet pDiN is slipped, becoming 5' "> central (CAU): the same occurs with the respective codons. Initiation codons are various while the anticodon is unique, configurating a new kind of 'degeneracy'. Alignment of the initiation codons demonstrates the slippage (Figure 7). There are various consequences of the slippage. Translation of mRNA is placed in phase, according to the precise modules of

Initiation

Elongation

Codons

5'

N[UG1

Anticodons

3'

"U [A C\

Codons

5'

NfUG]

G U

N

Anticodons

3'

Û [A C1

C A

N^ N_N

Amino acids

A C

N

^ w

fMet

-

Thr

N N

N

N_J^

N"'

~ N N

N N^

fMet ~ Val ~ Amino acids Figure 7. Initiation pDiN slippage. The second amino acids in the chains shown are either Thr, the preferred in this position (Section 3.7), or Val, the other strong stabihzer of the RY quadrant (Section 3.5), and preferred in the N-ends of proteins (Section 3.6). Standard pDiN underlined, initiation pDiN in brackets. W, wobble position

Genetic Code: a Self-Referential and Functional Model 109

triplets, in a thermodynamically strong way. The inverted' position of the fMet pDiN, recalling the inverted phosphodiester bond in the eukaryotic mRNA cap structure, eliminates the wobble position between the first two triplets. The two pDiN become contiguous, forming a tetranucleotide with all standard base pairs, two of them G:C. In the case of fMet-Thr, the two pDiN form a direct repeat structure, with inverted positions in the two strands; in the case of fMet-Val, the structure is of inverted repeats. Before the introduction of this process, determination of the reading frame would have to rely only upon the correct positioning of tRNA in the ribosomal decoding sites. Initiation is described, instead of the mere entry of fMet, as the bimolecular reaction between the two first ac-tRNAs, with second order kinetics, forming the first peptide bond.

5' 3'

A. Conflicts between the main] initiation codon and anticodons of the YR quadrant Initiation codon A [U G] G A [U G] A [U G] A [U G] A Anticodons of the YR quadrant A C A A c A Cys A C A A C A P P p P p A C G A c G A C G A c G w P P P p A c C Trp A c C A C c A c C P p P P p P A c X A c U A c u A c U u P p w P p P w Tyr

A A

X

A A

Arg

u u

A

u p

G

A

u p

C P U w

A

u

A

u u p

A

G

c

G

c

G

G

c

G

c

C P U w

G

c

A

G

G

G G

c u u

G

c u p

G

G

u p

u

C

u

P U w

A P G w C

p

c

G Gin

u

A

G

G His

u

G

u

A P G w C U A P G w C

p

G

u

U

^Figure 8. Part I

A

u

P w A u P w A U P w A U P w G w G w G w G w G w G w G w G w

A G C U P

C P

A

c p c

G C

p

c p

U P

u

A

w U G w U C w U U w P

A

u

P A P A P A P

w

G w G w G w G w G w G w G w G w

u

w U w U w

A G C P U w

C P

A

c p c

G

p

C P U w

u

A

p

c w

u

G

w

C P u U w w

u w

110

Progress in Biological Chiiality B. Pairings between the initiation anticodon and the stop codons Initiation anticodon [C A] U [C A] U [C A] U Stop codons A G U A G U A G U w P P A A U A A U A A U P P G A u G A u G A U

5'

C. Pairings between the main initiation codon and the stop codons Initiation codon A [U G] A [U G] A [U Stop codons A A G U A G U w w P A A U A A U A P P w G G A U G A U

G] G

U

A

U

A

u

D. Pairings between the initiation anticodon and anticodons of the YR quadrant Initiation anticodon _5;_ [C A] U [C A] U [C A] U Anticodons of the YR quadrant A C N Cys, Trp, X A C N A C N Tyr,X

A

U

N

A

U N

A

U N

N

G

C

N

G

C

N

R p, w Y

G

U

R

G

U

R

P Arg His

G P G P G

c u

P

p

p

G U Y Y /,P Figure 8. Part II. Fishing of anticodons correspondent to stop codons by the initation system. Standard (p) and G:U (w) base pairs; elongation pDiN underlined, initiation (slipped) pDiN in square brackets. The conflictive triplets considered decisive and meaningful (bold) involve a standard base pair ^ ) with the central base of the initiation codon. The second and third columns consider the different dinucleotides of the triplets. In (A), the 5' bases of the Thr (A) and Val (G) codons are added, respectively, to the third and fourth columns Gin

u

G

U

3.14.2 Termination Specific termination is necessary for precise configuration of protein tails. We could demonstrate that the stop signs were generated and localized through fishing by the initiation anticodon, with the slipped pDiN. The mechanism was coherent and superimposed upon the properties of the amino acids inhabiting the termination boxes, of protein N-end destabilization (Section 3.5). The search for relationships or possible interactions between the pDiN of initiation and termination boxes required tests with pairings not only between triplets but also between


111

dinucleotides, and considering the two possibilities of dinucleotide slippage (Figure 8). Pairs meaningful for the physiology of punctuation should necessarily be coherent for the 3 boxes of the system. A single conflictive situation was observed, coherent for both termination boxes and the initiation system: the slipped pDiN of the initiation codon was found to be able to pair with the pDiN of tRNA correspondent to the boxes containing the stop signs, these in the standard configuration, thereby competing with the initiation anticodon (Figure 8.A). The conflict was defined by pairings of doublets of the initiation and termination partners, allowing for G:U base pairs, and requiring formation of a standard base pair in the central position, but could be extended to triplets, incorporating the 5' base of the second codon. The conflict was eliminated and solved with deletion of the tRNA correspondent to stop codons. Other mechanisms intervened in determination of the precise constitution of the punctuation triplets, such as in the choice of 5' C, monospecific in the wobble rules, for Met, fMet and Trp. The specific punctuation system may have extended the polymers. The initiation fMet may have been added to the N-end, instead of being only a modification of Met pre-existing in this position. Stop codons may have been added to the mRNA, instead of their tRNA having been only substituted by the Release Factor 1. In fact, repetitions of stop codons are frequent. 5.15 Genetic and systemic information A 'systemic concept of the gene' and the concept that 'the system defines the gene' were expressed earlier [99]. Nonetheless, even a decade after the original proposal [100], another study [101] was not able to provide complete clearness and precision to the proposal. Now it becomes possible to advance a systemic definition of primordial genes. The scenario constructed in the present work (Section 3.4) is that both RNAs and peptides participating in the primitive protein synthesis system formed wide 'spaces' of variation, with extensive heterogeneity of types. Among the RNAs, some participated more directly in the process; also among peptides, some were m.ore stable, others better RNA-binders, others even participating in the process of their own synthesis. The components becoming relevant for formation of a self-feeding system were mutually identified as genes and products through a cognitive process [102]. A sub-class of peptides combining, simultaneously, the properties of stability, RNA binding and participation in the protein synthesis process was able to bind, cognitively, to the same RNA that participated in their synthesis. At the cognitive meeting, the proteins, 'surprised', 'said' to 'their' R N A - you are my genes! -, and these, also 'surprised', 'said' to them-youaremy life! (Figure 9). In this way, the system became stabilized and selffeeding, auto-catalytic. In the simultaneity of the event, RNA became genes and the peptides recognizing them became products. Definition of genes and products occurs in the process of their mutual recognition, partners defining each other at the same time. The 'chicken or egg?' problem is dissolved. During the construction of the code, genes arose in consequence of the binding promoted by proteins. The event may be located at the time of Stage 2 of the code development, where there is already the possibility of linear ordering in the peptide sequences, dictated by the stability properties, and also of the arisal of genetic 'string memories'. Three stages are delineated in mRNA structuration. The triplet reading-frame is defined by the SrRNA decoding site, together with the anticodon modules. The 'hidden' auxiliary RNY triplet pattern will be examined in a separate study. Finally, the stage of ordering the types oftripletsinthe sequences, initially defined by the properties of protein

112 Progress in Biological Chirality

Figure 9. Systemic definition of gene. When the cognitive meeting occurrecL and RNA, 'surprised', recognized the 'others' to 'themselves' N-end stabilization. Information is defined as the set of interactive properties of elements [103]. In the interactions, information is exchanged or shared. The system, the organized collectivity, gives sense and reality to the information in the elements. While these are dispersed, not organized, their interactive properties may fill the whole lists of possibilities, where all potentials are variably and continuosly shaped and reshaped, unconstrained. When the linear order of protein sequences, which participated in the system, became fixed in genes, thereby guaranteeing a repetitive memory (source), it can be said that a part of the information located in various parts of the system became sedimented in the strings [1]. Other genes, which were added on top of the basic translation process, building a wider system, should be all coherent and participant in refeeding and stabilizing the basic modules. 3.16 Chemistry, history, self-organization and selection Three terms in this title are borrowed from [23], to which we add self-organization. They are discussed together, in order to organize various themes in the text and to introduce the two latter ones. The path described in development of the code obeys some strict rules of chemistry. Firstly, that of tRNA fishing in palindromic pairs, following from the Ho pDiN sector to the Mx sector. Then the rules of precedence of aRS2 over aRS 1, of triplets with higher over those with lower thermodynamic stability, and of central and 5' purines over pyrimidines. Any concessions to other rationalities would have to incorporate many ad hoc arguments. The order resulted adequate in relation to functional criteria. With guaranteed stability of peptides and their binding to RNA, in Stage 1, such properties do not constrain the next stages. Amino acids entering in that order produced satisfactory coherence with the development of protein conformations, fi"om aperiodic structures to helices then strands. Historic events became evident already in Stage 2. Data on Proline biosynthesis were incorporated, introducing a sugestion for improvement of Stage 1. Synthetase specificities were also successfully accommodated in the two pairs of boxes of Stage 2. Selection is an obligatory component of all evolutionary processes. In fact, it was introduced since the beginnings of studies on the organization of the matrix [69]. A main argument in the Darwinian proposition is the observation of adaptafions, in the sense of adequacy between structures and function. It becomes easy but almost trivialized to propose that the mechanism underlying adequacies was the selection of variants, among which the more adaptive, the successful ones in producing function, remained. The observation that the code is organized is usually described by the regionalization, which would result in minimization of mutational damage, or optimization [21, 104-106]. Mutafions, occurring at replication, transcription or


113

translation would, most frequently, in the regionalized matrix, lead to substitution of an amino acid by another similar one. Many statistical tests have been elaborated showing that the standard code is situated among 'the best' in sets composed of various alternative codes artificially generated. Otherwise, such reasoning remains empty if the components of the process were not explicated. As strange as it may seem to selectionists, clarification of the molecular processes involved introduces chemical determinisms, which resuh in reduction of the space available for selection. For instance, the very mechanisms of tRNA fishing and recruitment of synthetases of coherent classes can be better classified as self-organized, as well as those of thermodynamic stability of the triplet pairs. 3,17 Selection focus So much obvious as the operation of selection, it is important to point out the steps where it should be claimed with emphasis. It acts upon the functionality of the system, its 'phenotype'. Most fimdamental should be the types of amino acids composing the code and the proteins derived from it. It can be said that the standard code became partially 'frozen' at some stage, but without loss of evolvability. This is evidenced by the addition of some important monocodonic attributions, best known being those of seleno-cysteine and pyrrolysine [107], and by the evolution of variant codes. Some amino acid substitutions can be proposed in the course of evolution of the standard code, as we did in the case of Gly^^ by Pro and, following earlier propositions, indicating a simpler alternative (Ala) for the predecessor to Arg. The main modus operandi of selection should have been through differential codon and amino acid usage, but its study is of high complexity. At a most basic level, is the process of organization of the linear sequence of codon and amino acid types, which define the specificity of biologic functions. This is called genetic information, a fraction of the information which was distributed in the system and became stored in genes [103]. Our model of sequential addition of amino acids to the system indicates that a polar organization of sequences, with concentration of protein-stabilizing and RNA-binding amino acids in the amino-terminal portions was among the first focus of selection. Further development should have been upon protein conformations. After the aperiodic structures, coinciding with the attributions referred to in the first stage, helices then strands were added. The last are the most complex, with larger and more hydrophobic amino acids, to inhabit the inner microenvironments of proteins. Differential codon usage should also be advocated to explain the allocation of (a) monocodonic attributions with 5' C, the least wobbler of standard bases, exactly to amino acids which became directly involved with initiation (Met) or conceding the neighboring triplet (Trp) to one of the stop signs; (b) concessions of 5' Y to the hexacodonic expansions, Phe to Leu and Ser^^ to Arg. The procedures of sequential addition of attributions and of disposing off of an mRNA to be translated make the process eminently constructive, of assemblage and addition of parts. The constructive process is like the bottomup building of pyramids [4]; the construction starting with the presence of an mRNA from the beginning, to be translated, would be like a top-down building of pyramids. A complex space of codons to be filled [108] was not real from the beginning but only virtual, a universe of possibilities. Our model becomes, then, one of high probability. As corollary, it can be indicated that calculations of probabilities, applied to the organization of the matrix, are not relevant as 'tests' of the hypothesis.

114


4.

Acknowledgments Support from FAPEMIG and CNPq; discussions and help from Andre Ribeiro de Oliveira Cavalcanti, Luiz Jose Delaye Arredondo, Marcello Barbieri and Savio Torres de Farias.

5.

References

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Chapter 9 Specific Symmetry of Living Systems Victor A. Gusev Sobolev Institute ofMathematics, Siberia Division Russian Academy of Science, 4 Koptyuga str., Novosibirsk 630090, Russia vgus@math. nsc. ru

1.

Introduction At the microscopic scales the morphological grace of living objects is transformed into formless heap. On the other hand, nonliving crystals on micro and macro scales have graceful geometric shapes. There are many myths created by people about effects of minerals on the fate and health of the owners. Are living systems deprived of this magic feature? Our aim is to find where and what kind of symmetry presents into living systems. And for this purpose, i.e. to discover specific living symmetry, we will examine life from the point of view non-biology but mathematics and physics. 2.

About Fibonacci Numbers, Golden Section and Five-Symmetry It is common knowledge [1] that most tree's twigs are arranged in a spiral and the same applies to cone's scales of conifers and others plants. The ratios object's and spiral's turns are: 2/1, 3/1, 5/2, 8/3, 13/5, 21/8, 34/13, 55/21... (Ser. 1). All these are numbers of Fibonacci series: 0, 1, 1, 2, 3, 5, 8, 13, 21, 34, 55, ..., mn, (mn+mn-i), ... (Ser. 2). As it is well known, m , \ + \l5 . lim—^=^:= 1.6180339..., where q = On the other hand, q is the coefficient of self-similarity in the golden section defined by the equation of the solution (f = q -^ 1. The members of Ser. 1 can be described by — ^ = —ii__«d_ « (^r^ = ^ +1. We call attention to Fig. 1. If we divide the big pentagon as related as L// = ^ + 1. We call attention to the fact that the angle between two nearest corners of the big and small pentagons (Fig. 1) is equal to 27i/10. After 10 moves the initial pentagon makes a full rotation. The same number of nucleotides and molecules of deoxyriboses baring to one turn of DNA helix. Recall that deoxyribose contains pentoses. Nonliving natural crystals have axial symmetry by second, third, fourth and sixth, but not fifth order. Living systems have in addition fifth ordinal symmetry. Why and what purpose?

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Figure 1. Schematic illustration of pentagonal symmetry, Fft)onacci numbers and golden section

3.

The Symmetries of the Genetic Code To look for specific symmetry in the genetic code we must use methods of discrete mathematics - arithmetic, algebra and matrices*. The basic idea, which gave impetus to research of genetic code by group-theoretic method was formulated by Rumer [2]". He discovered that purine-pyrimidine inversion CA, GÛ(T) (Rumer transformation) transforms two octet groups of standard genetic code list to each other. The essence of grouptheoretical method is to search for symmetry and regularities in the structure of the genetic code after its matrix mapping [3-12]. Notice that search of symmetries and integer characteristics are not defined by aesthetic prejudices of investigators. As well known, presence of any symmetry of structure or process indicates presence of selection rules or the rules of exclusion [13]. In other words, the presence of symmetry indicates presence of conservation law, but its physical sense is not always clear. Papers [4, 5] introduced vector notation for nucleotides as column vector.

and congruent row vector:

v^

=(CGUA)*

The exterior multiplication of these vectors gives the doublet-matrix:

^CC CG CU CA ^ GC GG GU GA UC UG UU UA I ÂC AG AU AA j

(1)

In the next step the authors [4, 5] define the nucleotide's "power" d as dc=4, da=3, dA=2, du=l, and doublet-nucleotide power as dij=di+dj, where /,y—»• A, U, G, C. The matrix (1) can be represented in digital form after the definition:

7654 6543 V5432y

(2)

The main properties of the matrix: 'the symmetry of this matrix is so high that it is singular, ' "Now, concerning the interpretation(s) of this new kind of small numbers coincidences, we have proposed some personal thought coming from a theoretical physicist which could be, perhaps, one of the theoretical physicists undergoing the great migration towards Biology, Erwin Schrodinger announced a long time ago " [9]. " Danckwerts and Neubert [3] rediscovered this purine-pyrimidine symmetry of genetic code in 1975. "" This definition of vectors clearly views the analogy with matrix notation of quantum mechanics of Dirac.

Specific Symmetry of Living Systems

121

i.e. its determinant is equal to zero detDÔ, the rank and defect are equal to two. This follows from exterior multiplication (1). It is remarkable that spur trD=20. This value is equal to the sum of numbers in the side-diagonal. Notice that in the side-diagonal and in parallel lines are ^'power-equaT' doublets. The inversion of matrix around the side-diagonal, amounts to a purine-pyrimidine inversion, no exchanges matrix ofproperty'' [4]. We do not give here all symmetries and digital regularities of genetic code are obtained in papers [4, 5], only cite the author's conclusion: ''Thus, algebraic method provides insight into root of genetic code. Symmetry into genetic code is manifested in selection of triplets for relevant amino acids. The model is constructed on the base of differ power of nucleotides C, G, U, A unique determines of relevant their amino acids'\ The author's conclusion is great argument that genetic code is no frozen accident. Really, it difficult to image a "nature plan" providing genetic code of symmetry [12]. ''The concept of nucleotides' power-determination introduced here allows imaging the genetic texts as sequence number from 1 to 4 {really in quaternary number system)"". [4], In refs. [8-11] the author analyzes the structure of genetic code by group-theoretical method. He analyzes genetic code as chemical structure of nucleotides by this method. One of the goals is finding connection between chemical contents of nucleotides and structure of genetic code. We do not analyze all the interesting resuhs about genetic code symmetry represented by Negadi, citing only one. The total sum of the matrix pattern elements transformed with each other after purine-pyrimidine transformation is characterized by numbers 44, 128, 84 and others. The author notices that the same "magic" numbers (44, 128, 84) are presented in modem physical theory of super-gravitation. May it be coincidence or chance? But in general terms the physical magic numbers of quantum mechanics, for example, magic numbers of nucleons in the nucleus and numbers of electrons in molecules, correspond to stable states. We can assume that the coincidence of biological and physical magic numbers is nature's prompt as this genetic code must be originated in our Universe but not in an other. The obvious conclusion springs to mind: The genetic code is not a product of chemical and prebiotic evolutions'"" but of the chemical structure of 4 nucleotides, 20 canonical amino acids and mapping the triplets to amino acids are determined by primary particles and chemical elements of their own origin in the observable Universe. This point of view of the genetic code might be formulated as a fundamental law of nature. If so, the structure of genetic code obtains the same status of noninterpretation as the physical laws of conservation of energy, linear momentum and angular momentum. The three last laws are implication of time's symmetry and space's homogeneity and isotropy, according to Noether's theorem [15]. The time, space, energy, linear momentum and angular momentum are degree values. On the other hand, all of symmetries and arithmetic and algebraic curiosities discovered in the genetic code are pure abstract numbers. We can propose that to understand the sense of Life and its attribute the genetic code and how they originated, it is necessary to find the symmetries connecting the genetic code with dimensionless properties of the Universe.

4.

About Chirality As it is well known, natural non-biological molecules are forming crystal structures as a

' From an other concept, but to the same conclusion reached by M. Eigen [13].

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racemic mixture. All stmctural elements are involved in the process of transforming the genetic information presented by chiral molecules D sugars and L amino acids. In such a manner, in living systems the principal equivalence of right and left is broken. In other words, in living systems one of the critically important laws of nature is broken. All the biochemical processes are time-unidirectional - "from the cradle to grave, but no inverse" by figurative expression. In other words, time flies only one way in living systems. If so, the enzymes' geometric structure must be chiral, right or left, based on the fundamental physical CPTtheorem [16]. Enzymes direct all of biochemical processes. Hence it follows that DNA and RNA must be chiral too. The choice of right or left chirality in this case is predetermined by the enzymes' chirality. In order that enzymes make their key biochemical fianctions for muhiplication of living systems - replication, transcription and translation - they must be combined with DNA and RNA and move along the polymers. A necessary condition can be realized if enzymes and DNA/RNA polymers have the same chirality. By analogy: the nut (peptide) of right thread might be screwed on the right but not on the left screw (DNA/RNA). Thus, chirality of peptides and DNA/RNA and its monomer amino acids and nucleotides is determined by time-unidirectional biochemical processes. The requirement of time-unidirectionality i.e. of cause-and-effect self-coordination in biochemical processes in the living systems produces the need for structural elements, informational molecules (DNA, RNA and peptides) must consist of atoms, which allow the easy formation of chiral polymers [16]. It follows that the choice of carbon as a base of life on the Earth is determined too.

5.

Conclusion Apparently, the life phenomenon and its origin can not be adequately realized merely by biological categories. In other words, fundamental physical laws in observed Universe define not only the structure of micro and macro world and evolution of a nonliving matter, but also control the living matter by the determination of genetic code uniformity and monochirality of informational polymers. Only with the presence of living systems {Homo sapiens as a final step of biological evolution) can the Universe be observed - it is a single and principle criteria for its physical reality. Physical laws determine the origin of Life in our Universe. The genetic code's curiosities described above do not appear as a chance from this point of view. On the contrary, they force scientists to look for deep connection between laws of living and nonliving nature. '7« fact, argument is that: would be understood what is the Universe without before understanding what is the Life? " [17]. To summarize: 1. The biological objects have two global specific types symmetry - structural and functional. 2. The structural symmetry subdivided into micro and macro symmetries. 3. The macro symmetry represented by Fibonacci numbers, golden section and fivesymmetry. 4. The micro symmetry represented by monochirality of informational molecules and genetic code's symmetry. 5. The functional symmetry determined by cause-and-effect connections and timeunidirectional biochemical process.

Specific Symmetry of Living Systems

123

6. The time-unidirectional and monochirality symmetries of DNA/RNA and peptides are linked together and determined by global space-time symmetry of our Universe.

6.

Epilogue Our analyses of specific biological symmetries would be incomplete if the author does not express his own point of view on these problems. On the basis of discussion of the life phenomena and its attributes, the genetic code may be considered as a fundamental law in the same way as laws of conservation of energy, linear momentum and angular momentum. Only under this condition we can have a little hope to repeat the natural experiment, i.e. to construct in vitro something similar to a cell of microorganism. Otherwise the chance of success is near zero.

7,

Acknowledgement

The Siberia Division of RAS fimd of interdisciplinary research, project N142, 2003, Russia is acknowledged for financial support.

8. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

References V.A. Kizel, Physical Causes Dissymetry of Living Systems, Science, Moscow, 1985 (in Russian). Yu.B. Rumer, Dokl. Akad Nauk SSSR 21 (1966) 1393-1394 (in Russian). H.J. Danckwerts and D. Neubert, J. Mot. Evol. 5 (1975) 327-332. D. Duplij and S. DupUj, Biophys. Bull. Kharkov Univ. 488 (2000) 60 (in Russian). D. Duplij and S. Duplij, Biophys. Bull. Kharkov Univ. 497 (2000) 1 (in Russian). M.A. Jimenez-Montano, C.R. de la Mora-Basanez and T. Poschel, BioSystems 39 (1996) 117-125. M.A. Jimenez-Montano, BioSystems 54 (1999) 47-64. T. Negadi, PREPRINTLPTO/negadi/NoDS101/J4/No01/2002. T. Negadi 2 / * Meeting of the International Colloquium on Group Theoretical Methods in Physics, Paris, July 15-20, 2002. T. Negadi, PREPRINTLPTO/negadi/NoD3}01/14/No02/2003. T. Negadi, Int J. Quant Chem. 94 (2003) 65-74. V.I. Shcherbak, BioSystems 70 (2003) 187-209. J.P. Elliott and P.G. Dawber, Eds., Symmetry in Physics, Macmillan Press Ltd, London, 1979. A. Babloyantz, Ed., Molecules, Dynamics, and Life, John Wiley & Sons, New York, Chichester, Bribaa Toronto, Singapore, 1986. L. Landau and E. Lifshitz, Eds., Course of Theoretical Physics, vol. 3, Science, Moscow, 1990 (in Russian). V. Gusev, Fundamentals of Life (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002, pp. 545-552. A.D. Linde, Ed. Elementary Particle Physics and Inflationary Cosmology, Science, Moscow, 1990 (in Russian).


Chapter 10 Origin of Biomolecules - Origin of Homochirality C. Hajdu* and L. Kesztheiyi** "^Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen 40 JO, Hungary hajducs@tigris. kite, hu ** Institute of Biophysics, Biological Research Centre, Szeged H 6701, Hungary kl@nucleus. szbk u-szeged. hu

1.

Introduction The problem, how life originated on Earth and probably elsewhere in the Universe, has been and is still a fundamental question [1]. Answers are numerous starting from religious scriptures to attempts based on scientific information. Two trends dominated the thinking about origin of life until the 19^^ Century. The Platonic trend, dead matter was made living by some spiritual principle and the Democritian trend, spontaneous generation of life from dead matter. The Democritian trend in its simple terms, however, had to be discarded after the famous experiments of Spallanzani and Pasteur that demonstrated that life from non-living entities did not emerge. As we will see later presently a more sophisticated combination of the two trends might be the answer. In 1848 Pasteur discovered a phenomenon intimately connected with the living organisms what he named "dissimilarity" and we call it after 175 years homochirality of biomolecules. Homochirality of biomolecules means, in simple terms, that only L-amino acids and D-sugars occur in living systems with negligible exceptions though L- and D-forms (enantiomers) of these molecules are synthesized in equal number if one starts from achiral precursors. Pasteur's works opened up the way to investigate how did the complex, information-rich molecules that govern the complicated processes of life, arise from the inorganic matter. Major steps were made in 1920-ies by Oparin and Haldane developing the concept of "primordial soup" that contains large quantity of preformed building blocks of the molecules playing role in life. They assumed that the building blocks have been synthesized from inorganic matter directed by the laws of physics and chemistry. Oparin stated that the origin of life is an integral part of the evolution of Universe [2]. As science developed in the 20* Century his note became more and more authentic. It is now well accepted that to understand the origin of life the facts about the Universe, the laws that direct its evolution, the events occurring in it, all have to be included in the search for the origin of life. Conservative estimates date the appearance of the first living organisms on Earth to 3.8-3.5 billion years ago [3], This event was surely preceded by the accumulation of their building

126


blocks, the biomolecules (amino acids, carbohydrates, lipids and heterocyclic compounds [4]) in substantial quantity, as conceived by Oparin and Haldane. It is generally accepted that homochirality of chiral biomolecules (i. e. L-amino acids and D-sugars) is and has been a uniqueness of life. Theoretical arguments suggest that the homochirality preceded life,, i. e. it emerged during the accumulation of the biomolecules, though observational proofs do not exist. In this paper we try to follow how the Universe developed from Big Bang, when immense energies were acting, to reach such stage of evolution when low energy processes could synthesize molecules. The environments for syntheses are the interstellar space or our habitat, the planet Earth or, possibly, other planets. Thus, first we present a timeline of the evolution of Universe, Solar System and Earth then we survey the recent ideas about the origin of biomolecules and homochirality. A large number of experiments demonstrate that the biomolecules could have been synthesized on Earth. As the space technology advanced more and more research and imitation experiments were devoted to show that they might have arrived from outside sources. Connecting the origin of homochirality to the origin of biomolecules the following possibilities arise: 1) biomolecules and homochirality were established on Earth, 2) biomolecules came from space, homochirality evolved on Earth, or 3) both came from space.

2.

Timeline of the Evolution of Universe, Solar System and Earth Physicists, cosmologists formulated a rather real theory on the time course of the Universe from Big Bang (about 12-15 billion years ago) to present sketched in Fig. 1 [5]. The time region and the assigned events are well founded with theories and observations regarding the elemental composition of the known matter. Uncertainties exist for the inflation period (earlier than lO'^s from Big Bang) and also from the calculations that the hitherto identified matter is not enough to understand the gravitation effects in the Universe. The missing matter (dark matter) and energy (dark energy) amount to about 95 % of what is necessary to keep the Universe in its present expanding state [6]. Great effort is devoted to search of this dark matter. We do not know whether the findings will help to understand life. In Fig. 1 an arrow shows the time of the origin of our Solar System 5 billion years ago. It 10-15x10 year \

modern galaxies

/~

--SxiO'yr 10%ear

\

early galaxies \

/

star formation \

/

first atoms \ \

synth. of nuclei

/ /

3'yr 3 min

/

\ protons, / \ neutrons/

10"'s lO^'s

\ Big Bang J L

/ inflation Planck age

10-^^s

Figure 1. Timeline of the evolution of the Universe from Big Bang to present Arrow signs the formation of the Solar System at 5 billion years ago

Origin of Biomolecules - Origin of Homochirality

127

1 -5

-4.6

t

1

1

1 Origin of i Solar System Earth

-4

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-3|5

1

1

•!

1

1

I'

0

xlO'yr

1

1

Solid Earth • First organic I First cells molecules 1 -^chemical biological evolution 1 Meteorites Space molecules

Figure 2. Timeline of the evolution of Solar System, Earth and life happened in the period when the Universe already contained modem galaxies like ours. Fig. 2 follows the origin of Earth, 4.6 billion years ago, its evolution and the timeline of the origin of life and its evolution to present. The organic molecules appeared within about 200 million years after the solidification of the Earth crest (4 billion years ago) and accumulated for additional 300 million years when living cells already existed [3]. This period, the chemical evolution, was followed with the biological evolution that is still going on. Zero year, the present time is characterized with new developments, discovering biomolecules in meteorites and also in space. We restrict our review to chemical evolution: try to summarize the information on the origin of biomolecules and the homochirality.

3. Origin of Biomolecules 3. J Origin on Earth Surveying the research of the origin of biomolecules the following strategy may be recognized: 1) selecting possible environment; 2) finding energy source, similar to that existing in Nature; 3) choosing a procedure how to imitate the synthesis of compounds; 4) performing reliable experiments. In the first successful experiments it was assumed that H2, H2O, CO2, N2 and CH4 were present in the Earth atmosphere as starting materials for synthesis of the first organic molecules [7]. A very important feature of this environment was that in contrast to the present it did not contain oxygen. For energy source and procedure electric discharge through the mixture of the above gases was chosen. The electric discharge imitated the lightning. Surprisingly, the product contained different amino acids. Following this pioneering study solar light [8], X-ray [9], P-[10] and y-radiation [11] and heat [12] were applied. All these energy sources were surely present on Earth 4 billion years ago. As a result of these experiments formation of organic molecules, even with complicated structure, amino acids, carbohydrates, lipids and heterocyclic compounds [4], was detected.

128


In the late 1970-ies more and more information accumulated that the Earth atmosphere was not reducing but oxidizing containing mainly CO, CO2, N2, H2O gases [13]. In such environment the above processes could not occur. Kobayashi's group irradiated such gas mixture by protons and other high-energy particles imitating cosmic radiation that surely bombarded the Earth. They found amino acids and other biomolecules in the products [14]. The late 1970-ies revealed other important possible environment on Earth: the deep see hydrothermal vents [15]. Wachtershauser elaborated a detailed theory how pyrite, NiS, FeS compounds, abundant at such places could catalyze the production of biologically important molecules in presence of heat energy. Experiments confirmed the ideas: even dimers of amino acids were synthesized under circumstances imitating the deep see hydrothermal vents [16]. These and other experiments [17] emphasize the importance of this terrestrial environment for syntheses in presence of high heat source and catalysts. 3.2 Biomolecules from space Oro in a seminal paper from 1961 pointed out that biomolecules could have arrived from space [18]. Since that time more and more research is devoted to collect information not only about biomolecules but even living organisms on planet Mars and moons Europa and Titan not mentioning the program of Search for Extraterrestrial Intelligence (SETI). The Solar System could have passed through interstellar clouds, the early Earth was bombarded by comets and meteorites. As we now know (see Fig. 2) all these objects contain biologically important molecules [19-21]. Infrared spectroscopy found about hundred molecules in these clouds. Similar molecules have been produced in laboratories imitating the possible cosmic circumstances [22]. Molecules in comets were in situ studied at the comet Halley [23]. Amino acids were found in meteorites [21]. Recent investigations try to avoid criticism that terrestrial impurities plaid role in measurement by observing the C - C ratio (expressed as 5^^C = [(^^C/^^C)organic / (^^C/^^C)i„organ,c - 1] X 10^) 5^^C is in the average -30 from the earliest studied kerogens (in the Issua finding) through the present living systems on Earth while that for amino acids found in Murchison meteorite is « + 30 [23, 24]. Many authors accept that enough evidence has been accumulated that the first organic molecules on the Earth were of cosmic origin from the interstellar grains, comets and meteorites [25, 26]. According to the published estimations the quantity of these molecules were sufficient to start higher order processes like polymerization, compartmentalization and building up information [27]. At the end of this paragraph we present estimation about the evolution of the complexity of molecules. In Fig. 3 the complexity is characterised by the mass of the largest molecules (for example DNA).

4. Origin of Homochirality 4.1 Origin on Earth In general, ideas about the origin of homochirality may be divided into two groups: it happened via chance process or it was caused by some asymmetric physical force. In establishing homochirality the first step is to have a small asymmetry between the quantity of enantiomers the so-called enantiomeric excess (e.e.) which gets amplified by other processes. The discussions whether homochirality originated by chance or it was the result of a certain

Origin of Biomoiecules - Origin of Homochirality

129

10 T

6

8

10

12

14

16

Time (billion years)

Figure 3. Estimated timeline of the evolution of molecules. M means the mass of molecules in Daltons asymmetric physical agent will continue for long time probably until other, extraterrestrial living systems will be contacted. If living organisms based on D-amino acids and L-sugars will be detected then chance origin becomes almost certain. Without this development, efforts to find an effective physical force will continuously be pursued. The theoretical background of the chance process was thoroughly discussed in recent works [28, 29]. These studies clearly demonstrated that homochirality could evolve in a developing system where chiral molecules were formed from achiral sources, grown autocatalitically and competed against each other for the material resources. Though for this bifurcation theory, convincing experimental demonstration with biomoiecules, has not yet been reported. The recent work of the Soai group may be considered the closest approach to bifurcation [30]. The outcome of this process, however, is decided by the actual state of fluctuation at the bifurcation point, i. e. by chance. The requirements of the bifurcation processes probably exclude their effectiveness in space. Considering the asymmetric physical forces we have to distinguish two groups, global forces acting mainly on Earth and universal forces, that produce asymmetry everywhere in the Universe. These forces might cause a small e. e. that could be amplified. The first group involve asymmetric adsorption; circularly polarized light; electric, magnetic and gravitational fields: light and magnetic field, electric and magnetic fields, gravitation and rotation; Gilat's idea (magnetic moments around amino acids) [31]. Presently only one asymmetric universal force is known: the parity violating weak interaction that may act in two ways. The beta radiations from radioactive decays are polarized. Their spin points backward for P" particles and forward for P^ particles. Their interaction was expected different for L- and D-molecules. After the first investigators of this possibility it is named Vester-Ulbricht process. Until now no reliable experiment was reported. The other way of parity violating weak interaction is via the parity violating weak nuclear current that contributes, though in extremely small measure, to all interactions in molecules (for details see ref [33] and references therein). One of the authors of this paper (L. K.) recently reviewed the different works on the above topics [31-33] therefore we will deal only with the latest results. A quite intriguing result was reported by Shinitzky et al. [34] that D-tyrosine crystallizes

130

Progress in Biological Chirality Table 1, Distribution of Left and Right Quartz Crystals Number of crystals

Left

Right

Refs.

16807 27053

8607(50.5%) 13481 (49.8%)

8320(49.5%) 13572 (50.2 %)

[41] [42]

from saturated solutions much faster than L-tyrosine. Independent confirmations of these results would be highly desirable. Another very interesting and also surprising study was reported by Ribo et al. [35]. In water solution of achiral diprotonated porphyrins chiral homoassociates are formed. Repeating the experiments many times showed that the signs are statistical. When the solutions are stirred the chiral signs of the resulting homoassociates depend about 85% on the rotation direction. These experiments are somehow similar to those performed by Kondepudi's group crystallizing NaC104 and other molecules under stirring [36, 37], In these studies, however, the distribution of chiral signs was statistical. The preference in chiral selection by the direction of stirring the solution should be reinforced by independent investigators. If correct then one could imagine that vortices appearing in water could act as stirrers. Such vortices could also form around hydrothermal vents. One of the recurrent ideas for inducing homochirality is the adsorption on asymmetric surfaces like quartz crystals found everywhere on Earth. L-amino acids are adsorbed more on L-quartz crystals and vice-versa [38]. The problem, however, is the distribution of L- and D-quartz crystals on Earth. This problem was surveyed by Klabunovski and Thiemann [39]. Table 1 shows that the deviation from 50 - 50 % is small and even opposite in the two studies. Thus, this new compilation indicates that asymmetric adsorption on quartz crystals is not a real source of homochirality though Soai's group demonstrated that small excess of one crystal form can induce large asymmetry in autocatalytic systems [40]. In the area of Vester-Ulbricht process no new report appeared in the reviewed period which probably means a diminishing interest. There were, however, important new developments in the field of parity-violating neutral current. In theory, new calculation of the parity-violating energy difference between enantiomers (PVED) [43, 44] ended up with values of an order of magnitude larger than the values obtained earlier [45]. New relativistic calculation confirmed the dependence of PVED on the 6th power of theatomic number Z in the asymmetry centre [46]. The theory also pointed out that the effect of parity-violation could be even larger in chemical reaction [47]. Experimental confirmation of these calculations is important not only from the point of view of biological homochirality but also from basic knowledge about parity-violating effects. Because of the strong Z dependence it is advantageous to use those molecules that have high Z atoms in the asymmetry centre. We may divide the experimental approaches into two groups: microscopic and macroscopic measurements. In the first group the energy difference is measured with some very high-resolution spectroscopic method. Lasers [43], nuclear magnetic resonance (NMR [48] and IVlossbauer-effect [49]) were discussed as possible experimental approaches. A Mossbauer experiment on L- and D-tris(l,2-ethanediamine) iridium (III) complex provided only an upper limit of 4x10'^ eV being 3.6x10^ times larger than PVED [49]. Table 2 collects data on possible Mossbauer nuclides. PVED values are given according to the new calculations and are related to the line centre determined as 10% of the line width. It is seen that the best candidate for a successful study is the nuclide 73Ta^^\


131

Table 2. Mossbauer-nuclides for Possible Measurement of Parity-Violating Energy Difference (PVEP). (Central value means 10% of line width) Nuclide

Linewidth

y-energy inkeV

73Ta'^' 32Ge^^ .3Np^' iilr'"' soZn"^' 26Fe"

6.23 13.3 59.6 73 93.3 14.4

PVED/ central value

Putative PVED ineV

rineV

6.2x10'^ 2.2x10-^ 2.5x10' 4.2x10-^ 4.2x10"^ 1.5x10'^

8.1x10-^^ 6x10-^' 3.5x10-" l.lxlO" 3.9x10"^' 1.5x10-^'

1.3x10-^^ 2.7x10-^^ 1.4x10-^ 2.6x10"^ 9.3x10" 9.6x10-^

It should be noted, however, that calculations how PVED appears in the isomer shift of a Mossbauer line are not known. In a recently reported Mossbauer measurements on chiral iron complexes a difference of (1.9±1.0)xl0-10 eV was found [50]. The difference does not seem statistically significant as seen in Fig. 4 where we delineated the published data with error. Nevertheless, that difference may be considered an upper limit for PVED. Using Table 2 this upper limit is « 104xPVED. In this measurement, however, the difference was 2%, 5 times less than the conservative estimate of 10 % of the line width in Table 2. Sophisticated infrared spectroscopy on vibrational modes of CHFClBr enantiomers has shown an upper limit for the difference of 13 Hz while theory predicted 2 mHz [51]. Another high-resolution spectroscopic method (NMR) was also considered as a possible way to measure PVED directly [48]. The authors concluded that in cases of high Z value spin one half nuclei such as Pt, Tl, Xe in the asymmetry centre of the enantiomers the difference could be measured with ultra-high-resolution technique. Suitable microscopic or, with other word, direct method to measure PVED are only in fijture plans therefore macroscopic or indirect methods may be used at least to demonstrate the theoretically forecasted PVED. Macroscopic methods, like polymerization or

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132


crystallization of enantiomers amplify the small e. e. [52, 53] and simulate the mode how could PVED generate asymmetry during chemical evolution. In our approach we deah with crystallization of racemic mixture of L- and D forms of sodium ammonium tartrate, tris(l,2ethanediamine) cobak(III) and tris(l,2-ethanediamine) iridium(III) complexes [54]. Performing a large number of crystallizations the distribution measured by circular dichroism was shifted from zero value outside error for Co and Ir complexes while the distribution for tartrate crystallization did not show shift larger than error (Fig. 5). This study appears to have provided an experimental demonstration of the parity-violating weak interaction in molecules. According the main critics of such macroscopic experiments it is questionable whether the preferential crystallization is due to the parity-violating weak interaction or to some unknown chiral impurity [43]. For the actual experiment, in which many controls closed out the influence of chiral impurities, this reasoning is not acceptable even more because the influence of impurities surely does not increase with atomic number as was found in experiment (Fig. 5) and expectedfromtheory. In spite of this result we consider PVED an open question as a determining factor for the origin of homochirality. 4. 2 Homochiralityfromspace As the information on possible space source of biomolecules evolved suggestion for homochirality from space also appeared. Bonner and coworkers assumed that the circularly polarized light of neutron stars could induce e.e. on molecules synthesized at the interstellar grains and then transport it to Earth [55-58]. The space origin of homochirality gained fiirther support from two observations. The meteorites contained more L- than D- amino acids [23, 24] and circularly polarized infrared radiation was discovered in the Orion Nebula [59]. Infrared radiation has low energy to decompose molecules therefore it was inferred that the Orion Nebula contains also circularly polarized u. v. light. The e. e. in meteorites was assigned to the effect of these circularly polarized light sources. This hypothesis was criticized in ref [33] emphasizing its negligible probability. Therefore it seems to be usefiil to

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Figure 5. Relative shifts of the distributions of ciystallisation depending on the atomic number of atom in the asymmetry centre (data takenfromref [54])


13 3

call attention to alternative explanations. Salam pointed out that quantum mechanical cooperative and condensation phenomena might give rise to second order phase transition below a critical temperature Tc [60, 61] (transformation of D-amino acids to L-amino acids that have lower energy due to parityviolating energy differences). Tc is probably very low. The experimental verification of this phenomenon has not proved successful under terrestrial conditions [62, 63]. The negative results of the experiments performed in laboratories at very low temperatures for short time do not mean, however, that such transitions may not occur in the Cosmos during very long time and at low temperatures. This possibility, offering an understanding of e. e. of amino acids in meteorites was already considered by Figureau et al. [62]. Thus, presently the best candidate for inducing e. e. in meteorites is the Salam-process based on parity-violating weak interaction.

5.

Conclusion In the introduction questions were raised about the location (Earth or space) of origin of biomolecules and homochirality and also about philosophical {Platonic or Democritiah) trends. There are no indisputable arguments to make decision in these questions. We favor the 13

terrestrial origin. In the case of biomolecules the value of 8 C in meteorites could advice us. Accepting the value as a characteristics of the extraterrestrial organic matter it is difficult to 13

understand the immediate change in the value of 5 C from +30 to -30 (note that the present living organisms accomplish this change from 0 to -30 [64, 65]). It is easier to return to the terrestrial origin of biomolecules. In the case of homochirality we already considered the low probability of space origin. We think that the whole paper demonstrates the more complicated way of the Democritian trend, living from not living matter. Here only the first events were considered, even these are complicated and not yet clearly known. The most important events, like the origin of molecules with information, their repHcation and the formation of first cell, were not touched. The ambition of Science is to understand the origin of life based on the known Universe (matter and laws). But: not all matter is known (only about 5%), not all laws are known (? %). The final problem is the origin of matter and its laws. We have to assume, as a modern version of Plutonian trendy a Creator or God who established the harmony of matter and its laws. 6.

References

[1] G. Palyi, C. Zucchi and L. Caglioti, Eds., Fundamentals of Life, Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002. [2] A.J. Oparin, The Origin ofPrebiological Systems (Ed. S.W. Fox) Academic Press, New York, 1965, pp. 91-96. [3] (a) J.W. Schopf, A.B. Kudryavtsev, D.G. Agresti, T.J. Wdowiak and A.D. Czaja, Nature 416 (2002) 7376. (b) M.D. Brasier, O.R. Green, A.P. Jephcoat, A.K. Kleppe, M.J. Van Kranendonk, J.F. Lindsay, A. Steele and N.V. Grassineau, Nature 416 (2002) 76-81. [4] J. Brooks and G. Shaw, Origin and Development of Living Systems, Acad. Press, London-New York, 1973. [5] M. Rees, Sci. Am., Separate Issue on the Frontiers of Space (2003) 82-85. [6] D.B. Cline, Sci. Am. 288/3 (2003) 28-35. [7] S. L. Miller, Science 117 (1953) 528-529.

13 4 [8 [9 [10 [11 [12 [13 [14 [15 [16 [17 [18 [19 [20 [21 [22 [23 [24 [25 [26 [27 [28 [29 [30 [31 [32 [33 [34 [35 [36 137 [38 [39 [40 [41

[42 [43 [44 [45 [46 [47 [48 [49 [50 [51 [52

Progress in Biological Chirality K. Bahadur. Nature 173 (1954) 1141-1143. K. Dose and B. Rajewsky, Biochim. Biophys. Acta 25 (1957) 225-226. T. Hasselstrom, M.C. Heniy and B. Murr, Science 125 (1957) 350-351. R. Paschke, R.W.H. Chang and D. Young, Science 125 (1957) 881-883. S.J. Fox and K. Harada J. Am. Chem. Soc. 82 (1960) 3745-3751. J.M. Kasting, Orig. Life. Evol. Biosphere 20 (1990) 199-231. K. Kobayashi, M. Tsuchija, T. Oshima and H. Yanagawa, Orig. Life. Evol. Biosphere 20 (1990) 99-109. J.B. Corliss, J. Diamond, L.I. Gordon. J.M. Edmond, R.P. van Hertzen, R.D. Ballard, K.K. Green, D. Williams, A. Bainbridge, K. Crane and T.H. van Andel, Science 203 (1979) 1073-1083. C. Huber and G. Wachtershauser, Science 216 (1997) 245-247. H. Oasawara, A. Yoshida, E. Imai, H. Honda, K. Halori and K. Malsuno, Orig. Life. Evol. Biosphere 30 (2000)519-526. J. Oro, Nature 190 (1961) 389-390. A.G.G.M. Tielens and S.B. Charley, Orig. Life. Evol. Biosphere 27 (1997) 23-51. P. Ehrenfreund and S. B. Charmley, Annu. Rev. Astronom. Astr. 38 (2000) 427-460. M.H. Engel and B. Nagy, Nature 296 (1982) 837-840. J.M. Greenberg, Sci. Am. 283/6 (2000) 70-75. JR. Cronin and S. Pizzarello, Science 275 (1997) 951-955. M.H. Engel and S. A. Macko, Nature 389 (1997) 265-268. J.M. Greenberg, Sci. Am. 250/6 (1984) 124-130. C.F. Chyba, P.J. Thomas, L. Brookshaw and C. Sagan, Science 249 (1990) 366-373. C.F. Chyba and C. Sagan, Nature 355 (1992) 125-132. V.A. Avetisov and V.I. Goldanskii, Physics- Uspekhi 39/8 (1996) 819-831. V.A. Avetisov and V.I. Goldanskii, Proc. Natl. Acad Sci. USA 94 (1996) 11435-11442. K. Soai, I. Sato, T. Shibata, S. Koniya, M. Hayashi, Y. Matsueda, H. Inamura, T. Hayase, H. Morioka, H. Tabira, J. Yamamoto and Y. Kowata, Tetrahedron Asymmetry 14 (2003) 185-188. L. Keszthelyi, Quart. Rev. Biophys. 28 (1995) 437-504. L. Keszthelyi, in: Fundamentals of Life (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002, pp. 379-387. L. Keszthelyi. Orig. Life Evol. Biosphere 31 (2001) 249-256. M. Shinitzky, F. Nudelman, Y. Barda, R. Haimovitz, Effie Chen and D. W. Deamer, Orig. Life Evol. Biosphere 32 (2002) 285-297. J.M. Ribo, J. Crusat, F. Sagues, J. Claret and R. Rubires, Science 292 (2001) 2063-2066. D.K. Kondepudi, R. J Kaufman and N. Singh. Science 250 (1990) 975-976. K. Asakura, A. Ikumo, K. Kurihara, S. Osanai and D.K. Kondepudi, J. Phys. Chem. 104 (2000) 26892694. W.A. Bonner, Orig Life Evol. Biosphere 21 (1991) 59-111. E. Klabunovski aod W. Thiemann, Orig. Life Evol. Biosphere 30 (2000) 431-434. K. Soai, S. Osanai, K. Kadowaki, S. Yonekubo, T. Shibata, I. Sato, J. Am. Chem. Soc 121 (1999) 11235. C. Palache, G. B. Berman and C. Frondel, Relative frequences of left andrightquartz, in C. Frondel ed. The System of Mineralogy of J. D. Dana and E. S. Dana, Yale Univ. 1837, 1892. 7th ed. V. 3 Silica minerals. J. Wiley and Sons Inc. New York, London, 1962, p. 17. C. Frundel, American Mineralogist, 63 (1978) 17-27. (a) M. QaacKAngew. Chem. Int. Ed 41 (2002) 4618-4630. (b) D.A. Singleton, L.K. Vo, J. Am. Chem. Soc. 124 (2002) 10010-10011. F. Faglioni and P. Lazzeretti, Phys. Rev. E65 (2002) 11904. A. MacDermott, Orig. Life Evol. Biosphere 25 (1995) 191-199. J.K. Laerdahl and P. Schwerdtfeger, Phys. Rev A 60 (1999) 4439-4453. A. Soncini, A. Ligabue and P. Laz^xretti, Phys. Rev. E 62 (2000) 8395-8399. J.B. Robert and AI. Barra, Chirality 13 (2001) 699-702. L. Keszthelyi, J. Biol. Phys. 20 (1994) 241-245. A S . Lahamer, S.M. Mahurin, R.N. Compton. D. House, J.K. Laerdahl. M. Lein and P. Schwerdtfeger, Phys. Rev. Lett. 85 (2000) 4470-4473. A. Daussy, T. Marrel, A. Amy-Klain, C.T. Nguyen, C.J. Borde and C. Chardonnet, Phys. Rev. Lett. 83 (1999) 1554. Y. Yamagata, J. Theor. Biol. 11 (1966)495-498.

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[53] Y. Yamagata, H. Sakihama and K. Nakano, Orig. Life 10 (1980) 349-355. [54] (a) A. Szabo-Nagy and L. Keszthelyi, Proc. Natl. Acad. Sci. USA 96 (1999) 4252^255. (b) A. SzaboNagy and L. Keszthelyi, in: Advances in BioChirality (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier, Amsterdam, 1999, 367-376. [55] E. Rubinsteia W A. Bonner, H.P. Noyes and G.S. Brown, Nature 306 (1983) 118-119. [56] W.A. Bonner and E. Rubinstein, BioSystems, 20 (1987) 99-111. [57] J.M. Greenberg, A. Kouchi, W. Niessen, H. Irth, J. van Paradijs, M. de Groot and W. Hermsea J- Biol. Phys. 20(1994)61. [58] W.A Bonner, J.M. Greenberg and E. Rubinstein, Orig. Life Evol Biosphere 29 (1999) 215-219. [59] J. Bailey, A. Chrysosyomou, J.H. Hough, T.M Gledhill, A. McCall, S. Clark, F. Menard and M. Tamura, S'c/e/ice 281 (1998) 672. [60] A. Salam,y. Mol. Evol. 33 (1991) 105-113. [61] A. Salam, in: Chemical Evolution: Origin of Life (Eds. C. Ponnamperuma and J. A. Chela-Flores) Deepak Publishing, Hampton, Virginia, 1993, pp. 101-117. [62] A. Figureau, E. Duval and A. Boukenter, in: Chemical Evolution: Origin of Life (Eds. C. Ponnamperuma, and J. A. Chela-Flores) Deepak Publishing, Hampton, Virginia, 1993, pp. 157-164. [63] R. Navarro-Gonzales, R.K. Khaima, and C. Ponnamperuma, in: Chemical Evolution: Origin of Life (Eds. C. Ponnamperuma, and J.A. Chela-Flores) Deepak Publishing, Hampton, Virginia, 1993, pp. 135-155. [64] M. Schidlowski, in: Fundamentals of Life (Eds. G. Palyi, C. Zucchi and L. Caghoti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris 2002, pp. 307-329. [65] S.J. Mojzsis, G. Arrhenius, K.D. McKeegan, T.M. Harrison, A.P. Nutman and C.R.L. Friend, Nature 384 (1996)55-59.


Chapter 11 Chiral Crystal Faces of Common Rock-Forming Minerals Robert M. Hazen Geophysical Laboratory and NASA Astrobiology Institute, Carnegie Institution of Washington, 5251 Broad Branch Road NW, Washington, DC 20015-1305, USA r. hazen@gl. ciw. edu

1.

Introduction Chiral crystalline surfaces provide effective environments for chiral molecular discrimination in both natural and industrial contexts [1]. Such surfaces have been cited for almost 70 years in reference to their possible role in the origins of biochemical homochirality [2-7]. In the past decade, fiirthermore, chiral crystal surfaces have received attention for their potential applications in the chiral selection and purification of pharmaceuticals and other molecular products [8-12]. Many recent studies have focused on the behavior of chiral surfaces of cubic close-packed (CCP) metals, including copper, silver, gold and platinum [13-23]. Single crystals of these metals, which can be modified by cutting, polishing and annealing faces with high Miller indices, display surfaces with chiral "kink" sites, even though the three-dimensional CCP structure is intrinsically achiral. Theoretical studies of these metal surfaces have demonstrated the potential for significant differences in adsorption energies of D- versus Lmolecules [14, 21-23], while experiments provide indirect evidence for chiral selectivity [13, 15-19]. Considerably less attention has been focused on the wide variety of chiral oxide and silicate mineral surfaces, which are ubiquitous in Earth's crust. Such surfaces provide the most abundant and accessible local chiral geochemical environments, and thus represent logical sites for the prebiotic chiral selection and organization of essential biomolecules. This chapter summarizes the geological occurrence, physical properties, crystal morphology and surface structures of some of the most common of these natural surfaces, including crystal faces of quartz (Si02), alkali feldspar [(Na,K)AlSi308], clinopyroxene [(Ca,Mg,Fe)Si03], and calcite (CaC03). One or more of these minerals is present in most common rocks in Earth's crust, as well as on the Moon, Mars and other terrestrial bodies, so chiral crystal environments are correspondingly ubiquitous [24, 25].

2.

Chiral Environments on Mineral Surfaces: General Considerations Many natural crystals are "euhedral" - bounded by a set of planar faces. These natural

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Figure 1. Crystals commonly display three types of chiral surface featm-es, illustrated here in idealized drawings, (a) A periodic two-dimensional chiral arrangement of atoms in a plane; these atoms may be coplanar or they may occur at slightly different heights, (b) A terrace step that is chiral along a step edge (red line) (c) A kink site that provides a chiral center (X)

crystal growth surfaces, or "terminations," may be represented as the intersection of a plane with a three-dimensional periodic atomic structure. Such surfaces are usually defined in terms of a set of three integers, known as Miller indices, which relate the orientation of the terminal plane to integral intercepts of the three crystallographic axes [26, 27]. For a given unit cell, every possible planar termination has a unique corresponding set of Miller indices. A chiral crystal surface is defined as any terminal arrangement of atoms that cannot be superimposed on its reflection in a mirror perpendicular to the surface. Such crystal surfaces display three common types of chiral environments. Some atomic surfaces are chiral because the periodic two-dimensional structure of the exposed surface lacks mirror symmetry (Figure la). These surface atoms may be coplanar or they may display significant topography. In either case such a surface, if chiral, is not superimposable on its reflection in a perpendicular mirror. Many crystal surfaces possess perpendicular mirror symmetry and thus are inherently achiral. Nevertheless, such faces often feature steps in the atomic structure that intersect the mirror symmetry operator at other than right angles (Figure lb). Under these circumstances, local environments immediately along the step edge are chiral, even though most of the crystal face is achiral. A third type of chiral environment, a local "chiral center," may occur on any crystal face. Chiral centers commonly arise on surfaces in which both planar regions and steps possess mirror symmetry, as in the case of face-centered cubic metals. In these cases the steps may be "kinked" to provide a chiral center at the kink site (Figure Ic) [13]. Two distinct types of symmetry conditions lead to chiral crystal surfaces. A few minerals are inherently chiral because their crystallographic space group lacks any of the so-called "improper" symmetry operators, including mirrors, glide planes, an inversion center or a rotoinversion operator [26, 27]. Thus, in minerals such as quartz (space group P3i21 or P3221) every surface is chiral and there exist so-called left- and right-handed structural variants, which are not superimposable and thus related to each other by mirror symmetry [28, 29]. Most minerals possess space group symmetries that incorporate at least one mirror symmetry operator, and thus the mineral is intrinsically achiral. Nevertheless, as noted above, a crystal termination will be chiral if no perpendicular mirror symmetry operator intersects

Chiral Crystal Faces of Common Rock-Forming Minerals

13 9

that termination. This condition is met by one or more common crystal growth surfaces of many common rock-forming minerals. These faces, which have received little attention in terms of their chiral properties, provide the primary focus of this chapter. In addition to chiral planes, most crystal surfaces possess etch pits, growth steps, twin boundaries, dislocations or other nonperiodic features that provide numerous local chiral centers on an otherwise achiral surface environment. These ubiquitous local chiral features may have been important in fostering chiral molecular processes, but they are not in the scope of this review. Before examining the characteristics of specific chiral mineral surfaces, it is important to emphasize that all of these natural chiral surface environments occur in both left- and righthanded variants in approximately equal proportions. No evidence exists for an enantiomeric excess of any chiral mineral feature [30, 31]. Nevertheless, the widespread occurrence of local chiral environments provided the prebiotic Earth with innumerable sites for experiments in chiral selection and organization - experiments that may have led, through a process of chiral amplification [32-35], to a fortuitous, self-replicating homochiral entity. These minerals.

Figure 2. Common crystal forms of quartz include the hexagonal prism m (100), the dominant rhombohedron r (101) and the secondary rhombohedron z (Oil). Left- and right-handed quartz (a and b, respectively) may be distinguished by two additional forms, denoted ^ (111) and x (511). Most crystals, such as the 3.2-cm diameter specimen from Montgomery County, Arkansas (c), display only the /w, r, and z faces. Less common specimens, such as the 3.5-cm diameter right-handed crystal from Betroka, Madagascar (d), develop the additional forms

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furthermore, represent an untapped library of chiral surfaces for possible industrial applications. The following section examines four common groups of rock-forming minerals that routinely display chiral crystal growth faces.

3.

Common Chiral Crystal Faces of Minerals

3. J Quartz Quartz (SiOi, trigonal space group P3i21 or P3221, a = 4.91 A, c = 5.41 A) is the predominant colorless mineral in most beach sand and is a principal component of many igneous, sedimentary and metamorphic rocks. Quartz is the only common rock-forming mineral that occurs in both right and left-handed variants. This structural distinction arises fi-om the silicatefi*ameworkthat incorporates either right- or left-handed helices of comerlinked Si04 tetrahedra [28, 36]. Three common crystal faces, illustrated in Figure 2, provide important chiral surfaces for study [37]: the ubiquitous (100) prism faces (denoted m in Figure 2), the dominant (101) rhombohedral termination (r), and the (Oil) rhombohedral termination (z), which is typically less well developed than (101). Note, however, that these three crystal forms are generally insufficient to distinguish right- fi-om left-handed specimens. This distinction can be made,

Figure 3. The (100), (101) and (Oil) surface structures of quartz (Si02), viewed from above (a, c, and e, respectively) and tilted 3° from horizontal (b, d, and f, respectively). Oxygen and silicon atoms are shown in red and blue, respectively. Positions of terminal oxygen atoms are indicatai by yellow Xs. In each drawing the caxis projection is vertical and each drawing presents an area 15 x 15 A

Chiral Crystal Faces of Common Rock-Forming Minerals 141 however, if the (111) and (511) faces (s and x, respectively) are present (Figures 2a and 2b, respectively). The surface structures of the three common quartz forms (/w, r, and z), while all chiral, are markedly different from each other, as illustrated in Figure 3. Above the point of zero charge of quartz (pH « 2.5), the quartz surface charge is negative [38-40]. In addition, silicon atoms typically remain tetrahedrally coordinated, so oxygen atoms (perhaps bonded to H, depending on pH) are expected to define the crystal terminations [41, 42]. Given this assumption, the surface structures of the (100), (101), and (011) faces are well constrained. The (100) prism face has zigzag bands of oxygen atoms separated by channels approximately 1.5 A wide and 2.0 A deep (Figure 3a). Note, therefore (Figure 3b), that the "surface" oxygen atoms are not coplanar. This feature is of critical importance in modeling surface interaction of quartz and other minerals. By contrast, the (101) face can be modeled with a more planar surface with a distribution of oxygen atoms that is much closer to an achiral array (Figure 3c and d). The (011) face presents yet a different character, with a denser chiral array of surface oxygen atoms (Figure 3e and f). These three faces also differ in the coordination of terminal oxygen atoms. On the (101) face all oxygen atoms are coordinated to a single silicon atom, whereas all oxygen atoms on the (Oil) face are coordinated to two silicon atoms. The (100) face, by contrast, features both one- and two-coordinated oxygen atoms. These marked differences in surface distribution of oxygen atoms explain, for example, the dramatically different adsorption characteristics of hematite (Fe203) on (101) versus (Oil) rhombohedral faces of some natural quartz crystals (Figure 4). These differences also point to the necessity of studying any surface interactions, such as selective adsorption of organic molecules, on individual faces rather than on powdered material. Given the striking differences in surface structures, the adsorption behavior of a molecule on one surface can bear little relationship to adsorption on any other face.

...^^:; •

^

^

'

;

fi§^

' " ;{P-- ^

Figure 4. Hematite (Fe203) preferentially deposits on (101) faces of quartz, while (011) faces remain largely micoated (-^1-mm diameter crystals from Paterson, New Jersey). This phenomenon results from significant differences in the surface structures of these two rhombohedral faces (see Figure 3c and d versus 3e and f)

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J-'X

m

Figure 5. Common caystal faces of feldspar include the chiral form m (110), which is often well developed in orthoclase (a) and albite (b). The 7- x 7-cm specimen of alkah feldspar (c) from Ethiopia displays these faces

3.2 Alkali Feldspar Feldspars, including the alkali feldspar series (Na,K)AlSi308 and the plagioclase feldspar series (NaSi,CaAl)AlSi208, are among the most common rock-forming minerals in Earth's crust [24, 25]. These framework aluminosilicates are major constituents of most igneous rocks and they provide the principal repositories of alkali and alkaline earth cations. Feldspars form significantfractionsof many sedimentary and metamorphic rocks, as well. A variety of alkali feldspars, including both orthoclase (Figure 5a: KAlSiaOg, monoclinic space group C2/m, a = 8.56 A, b = 13.0 A, c = 7.19 A, p = 89.1°) and albite (Figure 5b: NaAlSiaOg, triclinic space group CI, a = 8.14 A, ^ = 12.8 A, c = 7.16 A, a = 94.3^ P = 116.5", y = 87.7"), commonly have well-developed (110) faces. This form occurs in enantiomeric pairs in many natural crystals (Figure 5c). The surface structures of feldspar are less well constrained than those of quartz because of uncertainties in the terminal oxygen configurations near alkali cations. It is likely, for example, that oxygen coordination of alkali cations near the crystal surface in an aqueous environment will vary as a fimction of pH. Uncertainty also arises from the occurrence of different ordered distributions of silicon and aluminum atoms in tetrahedral coordination, as well as the facile exchange of alkali and alkaline earth cations between the crystal surface and aqueous solution [43]. Given these uncertainties, one possible configuration of oxygen atoms at the (110) chiral surface is illustrated in Figure 6. In this example of an orthoclase surface structure with, potassium atoms retaining their full 7-coordination, oxygen atoms are arrayed in rows approximately parallel to [001], as illustrated in Figure 6a. This surface displays significantly nonplanar topography as a consequence of the oxygen atoms selected (Figure 6b). A different choice of terminal oxygen atoms (for example removing the highest rows of atoms in Figure 6b) would significantly increase the surface exposure of positively-charged alkali cations. 3.3 Clinopyroxene Clinopyroxenes, the most common of all ferromagnesian rock-forming minerals, incorporate a diverse group of species with the general formula (Ca,Mg,Fe)Si03 [36]. Pyroxenes are major components in many igneous and metamorphic rocks in both the Earth's crust and upper mantle. They occur commonly in both orthorhombic and monoclinic varieties, but it is the latter that most commonly offer chiral crystal growth faces. The most


143

Figure 6. One possible (110) chiral surface structure of orthoclase, which is a member of the alkaU feldspar group. Silicon, oxygen and potassium atoms are shown in blue, red and turquoise, respectively. Terminal oxygen atoms are marked with yellow Xs. The [001] axis is vertical and the area is 15 x 15 A. Note that terminal oxygen atoms are chosen in this model so that potassimn is fully coordinated, which effectively shields potassium atomsfromthe surface

common clinopyroxene structure, as typified by the mineral diopside (CaMgSi206, monoclinic space group C2/c, a = 9.75 A, b = 8.90 X c = 5.25 A, P = 105.6"), features chains of comer-linked silicate tetrahedral that are crossed-linked by divalent Mg and Ca cations in 6- and 8-coordination, respectively. The most common chiral clinopyroxene face is the ubiquitous (110) perfect cleavage plane, which is designated m (Figure 7a). This face also occurs on crystals, occasionally in

Figure 7. Clinopyroxene [(Ca,Mg,Fe)Si03] displays several chiral faces (a and b), including the common (110) cleavage plane (designated w), and occasionally the (111), (221) and (2 21) forms (designated w, o, and v, respectively), (c) The 1.3-cm diameter crystal of diopside (CaMgSi206)fromXinjiang, Uygur Province, China, displays both the (110) and the (111) chiral forms

144


combination with the ( H I ) , (221) and ( 2 21) chiral faces [37]. In addition, four (llO)-type faces often combine with pairs of (100) and (010) faces to form an 8-sided crystal prism (Figure 7c). Such elongated crystals, which parallel the silicate chain, represent a distinctive morphology of clinopyroxenes. The fact that the (110) surface is also a perfect cleavage surface in clinopyroxene raises the possibility of obtaining large, freshly exposed chiral surfaces from cleaved samples for studies of chiral molecular interactions. Ambiguity arises when attempting to model the (110) surface structure of clinopyroxene. As in quartz and feldspar, the silicon atoms are assumed to remain tetrahedrally coordinated. The coordination of divalent cations, however, is less certain and will likely vary depending on the environment of the crystal. Figure 8 illustrates three different possible terminal atomic arrangements for the (110) surface of diopside. In the first configuration (Figure 8a and b) calcium atoms near the surface are coordinated to seven rather than eight oxygen atoms, thus exposing both positively-charged calcium and negatively-charged oxygen atoms at the surface. Alternatively, magnesium may be partially coordinated near the surface in at least two possible configurations (Figure 8c through f). If Mg is four-coordinated near the surface, then a quasi-linear pattern of approximately planar surface atoms results (Figure 8c and d). If magnesium is five-coordinated near the surface, then a more complex surface structure results, with both positively-charged magnesium atoms and oxygen atoms at three different

Figure 8. Three possible terminations for the (110) surface of diopside (CaMgSi206). Ca, Mg, Si and O are turquoise, green, blue, and red, respectively. Each 15 x 15 A drawing has the [001]-axis projection vertical. X and + indicate O atoms and cations near the surface. Small Xs in (e) are O atoms that are significantly below other surface atoms, but may participate in surface binding


145

heights relative to the surface (Figure 8e and f). The adsorption characteristics of (110), consequently, will depend critically on the as yet unknown cation coordination at the surface. Calcite (CaC03; rhombohedral space group R3c), the principal mineral of limestone and marble, is of special interest in studies of chiral selection by mineral surfaces. Calcite was one of the most abundant marine minerals on the early Earth and calcite crystal surfaces would 3.4 Calcite have been widely present in prebiotic environments [44, 45]. Calcite is also one of the most common biominerals; it is strongly bonded to proteins in the shells of many invertebrates [46,47]. The potential for calcite to interact with chiral molecular species has been underscored by studies of surface growth topology, which may be strongly affected by the presence of L versus D amino acids [48]. The literature on calcite is confused by the common use of four different axial systems, each of which results in a different set of Miller indices for any given plane [36]. Two of these sets of axes are based on inconvenient rhombohedral unit cells (in which one axial length and one interaxial angle are specified). Most authors prefer the simpler hexagonal setting (in which two orthogonal axial lengths, a and c, are specified) and that convention is used in this chapter. However, additional confusion arises from the existence of two different axial conventions for the hexagonal unit cell. One set of axes, based on the classic morphology of the calcite cleavage rhomb, results in the so-called "cleavage rhomb unit cell" or "morphological unit cell" (a = 10 A; c = 8.5 A in the hexagonal setting). This cell is invariably used to describe twinning, cleavage, and crystal forms [36,37]. In this setting, the Miller indices for the common cleavage rhomb are (101). Alternatively, the so-called "structural unit-cell" (a = 5 A; c = 17 A in the hexagonal setting) is the minimal unit cell determined by x-ray methods. In this case the axial orientations are identical to the morphological cell, but the a axis is halved and the c axis is doubled. Thus, for example, Miller indices for the cleavage face (101) in the hexagonal morphological setting become (104) in the hexagonal structural setting. When working with calcite surfaces, therefore, it is critical to specify both the unit cell and the Miller indices in order to avoid ambiguity. The most common calcite crystal form is the scalenohedron, in which adjacent faces have mirror-related surface structures (Figure 9). This form, with Miller indices (211) in the hexagonal morphological cell or (214) in the structural cell, is of special interest because of its ability to adsorb D and L amino acids selectively [7]. Modeling the (211) scalenohedral surface is complicated by the nature of the calcite structure, which has a halite or NaCl-type face-centered cubic arrangement of alternating Ca cations and rigid CO3 anions. A few surfaces, such as the perfect rhombohedral cleavage [(101) or (104) in the morphological or structural settings, respectively], present a uniform surface structure of coplanar Ca and CO3 (Figure 10). This surface incorporates a gHde plane operator and is thus achiral. Most calcite crystal surfaces, however, intersect coplanar arrays of Ca and CO3 groups so that the surface must incorporate steps and kinks, in much the same way as high-index planes of face-centered cubic metals are stepped and kinked [13]. Thus, the common calcite scalenohedral faces [(211) or (214) in the hexagonal morphological or structural settings, respectively] display a complex chiral surface topology that is not easily, or unambiguously, modeled. Figure 11 displays a possible surface configuration, based on the assumption that all surface oxygen atoms are associated with CO3 groups. This requirement leads to prominent

146


Figure 9. Calcite, CaCOs,frequentiyoccurs with (a) the chiial scalenohedral form [designated v; (211) or (214) in the hexagonal morphological or structural setting, respectively], as well as (b) the rhombohedral form [designated r; (101) or (104), respectively], which is also the common cleavage plane, (c) A doubly-terminated crystal from Elmwood Mine, Tennessee, displays a well-formed scalenohedron

2-A high surface steps (Figure lib, arrow). These steps are parallel to [018] in the hexagonal structural setting (or [012 ] in the hexagonal morphological setting), and are spaced approximately 12 A apart. This topology, with its linear array of chiral binding sites, may provide a natural template for the synthesis of homochiral polypeptides [7].

Figure 10. (a) The calcite rhombohedral cleavage [(101) or (104) in the hexagonal morphological or structural settings, respectively] presents a surface in which Ca cations andrigidCO3 anions alternate. The surface has glide plane symmetry (vertical yellow lines) and so is achiral. (b) The cleavage surface topology is revealed in a view that is tilted 6°fromthe horizontal. Ca, C and O atoms are turquoise, blue and red, respectively. Each drawing is approximately 15 x 15 A, and the c-axis projections are vertical


147

Figure 11. (a) The structm-e of the scalenohedral face of calcite [(211) or (214) in the hexagonal morphological or structmBl settings, respectively] features a chiral arrangement of positive (+) and negative (X) charge centers near the crystal termination. Ca, C and O atoms are turquoise, blue and red, respectively. In this 20 x 20 A view the ( 0 1 8 ) axis in the hexagonal structural setting [equivalent to the (012) axis in the hexagonal morphological setting] is vertical ~ an orientation that provides a useful image of the surface structure, (b) A view of this surface tilted 3° from horizontal (projected almost down the [01 8 ] axis) reveals the irregular surface topology, including 2-A-deep steps (yellow arrow) that result from the oblique intersection of layers of Ca and rigid CO3 groups with the surface (yellow line)

3.5 Other common chiralfaces of rock-forming minerals In addition to quartz, feldspar, pyroxene and calcite, numerous other minerals display chiral surfaces. Most of these species are rare or their chiral forms are seldom expressed. However, two other particularly common minerals, amphibole and gypsum, deserve mention with regard to their common chiral crystal faces. The amphibole minerals include a varied suite of hydrous chain silicates that are often found in igneous and metamorphic rocks [36]. This compositionally diverse group commonly conforms to the formula (Na,K,Ca)2(Mg,Fe,Al)5Si8022(OH)2. Amphiboles are structurally related to pyroxenes and, like pyroxenes, occur in both orthorhombic and monoclinic forms. The latter clinoamphibole group frequently displays chiral crystal faces and cleavage surfaces. These amphiboles, such as tremolite, Ca2Mg5Si8022(OH)2, and actinolite, Ca2(Mg,Fe)5Si8022(OH)2 (both monoclinic space group C2/m; a = 9 . 8 A , b = 1 8 . l A , c = 5.3 A, P= 104.7*'), routinely develop the chiral (110) and (Oil) forms, designated m and r, respectively, in Figure 12, as well as less common (120) form [37]. In addition, the (110) plane is a perfect cleavage in all clinoamphibole species and so offers the potential for exposing fresh chiral surfaces for study. However, as with clinopyroxenes, the detailed surface structure of clinoamphiboles with be strongly dependent on coordination of monoand divalent cations at the surface. Further characterization of these faces thus represents a promising research opportunity. Gypsum (CaS04 2H20: monoclinic space group C2/c; a = 5.7 A, b = 15.2 A, c = 6.3 A, p=

148


Figure 12. (a) The (110) and (011) forms of clinoamphibole, designated m and r, respectively, are relatively common chiral crystal surfaces, (b) The chiral (120) form (designated e) also occurs, though infrequentiy. (c) The 2-cm diameter crystal of actinolite [nominally Ca2(Mg,Fe)5Si8022(OH)2]fromMpwa-Mpwa, Tanzania, displays both the m and r forms

113.8*'), the most abundant natural sulfate, is a common marine evaporite mineral that readily forms euhedral crystals with chiral (110) and (111) faces, as illustrated in Figure 13 [36,37]. Thick gypsum deposits are found around the globe in many regressive sedimentary sequences. Crystal growth is extremely rapid under appropriate evaporative conditions; natural euhedral crystals may achieve lengths in excess of 10 cm in several days (R.Lavinski, personal communications). Gypsum has proven to be of special interest in studies of the interactions of chiral surfaces with chiral molecules. Growth of (110) and (111) faces, in particular, are dramatically influenced by the presence of chiral solute molecules [49]. Thus, for example, D and L alanine have been shown to thwart the growth of enantiomeric (110) faces, producing highly

ar^> b

Figure 13. (a) Gypsum, CaS04 2H2O, conunonly developstiiechiral (110) and (111) forms, which are designated m and / respectively, (b) The (1 11) form (designated n) is also seen occasionally, (c) A euhedral gypsum crystalfromGui Lin, Guanxi Province, China, 7.5 x 2.5 cm


149

distorted crystals. This phenomenon has been invoked to explain the occurrence of uniformly asymmetric gypsum crystals from a Miocene evaporite deposit in Poland - an environment presumably dominated by L amino acids [50]. However, in spite of these intriguing morphological curiosities, the chiral surfaces of gypsum remain difficult to study because of the mineral's high degree of solubility in water.

4.

Conclusions This brief overview points to several important conclusions regarding chiral crystalline surfaces. 1. Chiral crystalline faces are ubiquitous in nature. Quartz, feldspar, pyroxene, calcite, amphibole and gypsum provide a weahh of enantiomeric atomic surfaces in virtually every common crustal rock on Earth and other terrestrial bodies. Furthermore, any irregular mineral fracture surface will provide an additional variety of local chiral environments. In addition, hundreds of other candidate crystal growth faces also occur in nature. Most of those surfaces occur either on relatively rare minerals [i.e., the (111) and (221) faces of topaz, Al2Si04(OH)2, which occasionally forms crystals > 1 m in length] or are crystal forms that are rare in well-developed specimens [i.e., the unusual (111) form of the common mineral olivine, (Mg,Fe)2Si04]. A few of these less common surfaces will be present in most geochemical settings. Each of these surfaces has a specific atomic structure that represents a possible location for the selection, concentration and assembly of chiral organic species from the indiscriminately racemic prebiotic molecular soup into the homochiral macromolecules of life. 2. Most mineral surfaces are not chiral: With the exceptions of quartz (for which all crystal faces are inherently chiral) and calcite (for which the predominant scalenohedral face is chiral), most crystal grov^h surfaces on most minerals are achiral. Care must be taken, therefore, when studying minerals for their ability to induce chiral molecular separations. In this regard, special note should be made of layer hydroxides and layer silicates, including micas, chlorites and clays, which have been invoked in prebiotic processes of molecular selection and organization [51-54]. All layer silicates develop primarily the achiral (001) basal surfaces and therefore cannot impose a chiral environment. 3. Different forms of a crystal typically display very different surface structures: The (100), (101) and (Oil) forms of quartz are dramatically different. Each form has a different chiral surface distribution of atoms and a different atomic topography. Chiral interactions of molecules, therefore, are expected to differ for these different surfaces. In this regard it is significant that several previous experiments have employed powdered minerals (notably left- or right-handed quartz) in the hopes of inducing a chiral selective effect [2-5,55]. While such experiments may yield fortuitous enantiomeric excesses in the product suite, this use of powdered material greatly reduces the hope of discerning a structural mechanism for the observed chiral effect. The use of well-documented chiral crystalline surfaces is therefore much to be preferred. 4. Some surfaces are ''more chiral" than others: The distribution of surface charges on some chiral faces, such as the (101) form of quartz, closely approximates an achiral

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configuration. Other faces, such as those of the calcite scalenohedron, deviate significantly from their enantiomer. These differences point to the possible utility of a "chirality index" that measures the misfit of a chiral surface with its enantiomer [56]. 5. In some instances the surface structure is ambiguous: The surface structures of feldspar and clinopyroxene, for example, depend on the coordination numbers of monovalent and divalent cations at or near the surface - structural details that will depend strongly on the surface environment. Small changes in cation coordination can result in significant changes of the chiral surface charge distribution. 6. So-called "flat" crystal faces may be stepped: The surface of the calcite scalenohedron typifies an atomic configuration in which coplanar structural elements intersect a surface obliquely. This situation results in a stepped surface that may provide a linear array of chiral centers. Such an array may facilitate the condensation of homochiral polymers. These distinctive attributes of chiral mineral surfaces point to significant opportunities for future studies at the dynamic interface between crystals and their environments.

5.

Acknowledgements I am grateful to Aravind Asthagiri, Robert Downs, Gozen Ertem, Mary Ewell, Andrew Gellman, James Kubicki, David Sholl and Henry Teng for useful discussions and constructive reviews of the manuscript. All crystal structure drawings were made with the program XtalDraw [57], courtesy of Robert Downs. Photographs of mineral specimens were generously supplied by Dr. Robert Lavinsky, President of Arkenstone in Garland, Texas.

6. [1] [2] [3] [4] |5] [6] [7] [8] [9] [10] Ill] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

References R.M. Hazen and D.S. Sholl, Nature Materials 2 (2003) 367-374. R Tsuchida. M. Kobayashi and A. Nakamura, J. Chem. Soc. Japan 56 (1935) 1339-1341. G. Karagounis and G. Coumoulos, Nature 142 (1938) 162-163. A. Amariglio, H. Amarigho and X. Duval Helv. Chim. Acta 51 (1968) 2110-2111. W.A. Bonner, P.R. Kavasmaneck, F.S. Martin and J.J. Flores, Ohg. Life 6 (1975) 367-376. N. Lahav, Biogenesis: Tfieories of Life's Origin, Oxford University Press, NY, 1999. R.M. Hazen, T.R. Filley and G.A. Goodfreind, Proc. Natl. Acad. Sci. USA 98 (2001) 5487-5490. B. Kahr, S. Lovell and J.A. Subramony, Chirality 10 (1998) 66-71. B. Kahr and R.W. Gumey, Chem. Rev. 101 (2001) 893-951. S.C. Stinson, Chem. Eng. News 79 (May 14, 2001) 45-46. M. Jacoby, Chem. Eng News 80 (March 25, 2002) 43-46. AM. Rouhi, Chem. Eng. News 80 (June 10. 2002) 43-44. C.F. McFadden, P.S. Cremer and A.J. Gelhnan, Langmuir 12 (1996) 2483-2487. D.S. ShoU. Langmuir 14 (1998) 862-867. A. Ahmadi, G. Attard, J. Feliu and A. Rodes, Langmuir 15 (1999) 2420-2424. G.A. Attard, J. Phys. Chem. B 105 (2001) 3158-3167. D.S. Sholl, A. Asthagiri and T.D. Power,./. Phys. Chem. B 105 (2001) 4771-4782. J.D. Horvath and A.J. Gellman, J. Am. Chem Soc. 123 (2001) 7953-7954. J.D. Horvath and A.J. Gellman, J. Am. Chem. Soc. 124 (2002) 2384-2392. A. Kuhnle, T.R. Linderoth. B. Hammer and F. Besenbacher. Nature 415 (2002) 891-893. Z. Sljivaneanin, K.V. Gothelf and B. Hammer. J. Am. Chem. Soc. 124 (2002) 14789-14794. T.D. Power and D.S. SholL Top. Catal. 18 (2002) 201-208. T.D. Power, A. Asthagiri and D.S. ShoU, Langmuir 18 (2002) 3737-3748. F.J. Turner and J. Verhoogen, Igneous andMetamorphic Petrology, Mc-Graw-Hill, NY, 1960.


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[25] F.J. Pettijohn, Sedimentary Rocks, Harper & Row, NY, 1957. [26] F.D. Bloss, Crystallography and Crystal Chemistry, Holt, Reinhart and Winston, NY, 1971. [27] M.B. Boisen, Jr. and G.V. Gibbs, Mathematical Crystallography, Mineralogical Society of America, Washington, 1985. [28] L. Bragg, G.F. Claringbull and W.H. Taylor, Crystal Structures of Minerals, Cornell University Press, Ithaca, NY, 1965. [29] J.V. Smith, Geometrical and Structural Crystallography, John Wiley, NY, 1982. [30] C. Frondel,v4m. Mineral 63 (1978) 17-27. [31] E. Evgenii and T. Wolfram, Orig Life Evol. Biosphere 30 (2000) 431-434. [32] R.D. Murphy and T.M. El-Agez, Indian J. Chem. 35 A (1996) 546-549. [33] B.L. Feringa and R.A. van Delden, Angew. Chem., Int. Ed 38 (1999) 3418-3438. [34] D.Z. Lippmann and J. Dix, in: Advances in BioChirality (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier, Amsterdam, 1999, 85-98. [35] H. Zepik, E. Shavit, M. Tang, T.R. Jensen, K. Kjaer, G. Bolbach, L. Leiserowitz, I. Weissbuch and M. Lahav, Science 295 (2002) 1266-1269. [36] W.A. Deer, R.A. Howie and J. Zussman, An Introduction to the Rock-Forming Minerals, John Wiley, NY, 1971. [37] E.S. Dana, A Textbook of Mineralogy, John Wiley, NY, 1949. [38] R. Parsons, Surface Sci. 24 (1964) 418-826. [39] J.A. Davis and D.B. Kent, Rev. Mineral. 23 (1990) 177-259. [40] H. Chm-chill, H. Teng and R.M. Hazen, Am. Mineral, in press. [41] M.C. Goldberg, E.R. Weiner and P.M. Boymel, J. Chem. Soc. Faraday Trans. 80 (1984) 1491-1498. [42] Y. Xiao and A.C. Lasaga, Geochim. Cosmoch. Acta 60 (1996) 2283-2295. [43] P.H. Ribbe, Feldspar Mineralogy, T'^ Edition, Mineralogical Society of America, Washington, DC, 1983. [44] A.W. Bailey and A.R. Pahner, Eds., The Geology of North America: An Overview, Geological Society of America, Boulder, Colorado, 1989. [45] D.W. S\xmmx,Am. J. Sci. 297 (1997) 455-487. [46] H. Lowenstam and S. Weiner, On Biomineralization, Oxford University Press, Oxford, UK, 1989. [47] S. Weiner and L. Addadi, J. Mater. Chem. 1 (1997) 689-702. [48] H. Teng, P.M. Dove, C.A. Orme and J.J. DeYoreo, Science 282 (1998) 724-727. [49] A.M. Cody and R.D. Cody, /. Crystal Growth 113 (1991) 508-519. [50] M. Babel,^rc/z. Mineral. 44 (1990) 103-135, plates 1-9. [51] A.G. Cairns-Smith, Clay Minerals and the Origin of Life, Cambridge University Press, 1986. [52] H. Hartman, G. Sposito, A. Yang, S. Manne, S.A.C. Gould and P.K. Hansma, Clays & ClayMin. 38 (1990)337-342. [53] J.P. Ferris, C.-H. Huang and W.J. Hagan, Jr., Orig Life Evol. Biosphere 18 (1988) 121-128. [54] L.E. Orgel, Orig Life Evol. Biosphere 28 (1998) 227-234. [55] K. Soai, S. Osanai, K. Kadowaki, S. Yonekubo and I. Sato, /. Am. Chem. Soc. 121 (1999) 11235-11236. [56] R.T. Downs and R.M. Hazen, J. Mol. Catal. in press. [57] R.T. Downs and M. Hall-Wallace, Am. Mineral 88 (2003) 247-250.


Chapter 12 Implication of Polya's Urn Experiment in Biochirality and Cerebral Lateralization Noboru Hokkyo Senjikan Institute, Igarsi 2-847S-16, Niigata 950 -2102, Japan noboru@icn. ne.jp

1. Introduction Everything is dependently co-arisen Nagarjuna(BC250-150)[l] A large majority of chiral molecules in living organisms is present in only one enantiometric form, while the abiotic synthesis of organic compounds yield exclusively racemic mixtures. Presently we do not know exceptions to the homochirality rule in DNA or RNA. Yet, there is a rapidly increasing number of biochemical studies [2] detecting the sign of "a record of the past from a racemic world" in living organisms [3]. Examples of macroscopic manifestations of chiral feature in the spatial structures and behaviors of vertebrate are also known. Here we propose to understand the evolutionary origin of chiral purity and impurity of the biological world on the same footing from molecular level to macroscopic level in terms of Polya's urn experiment (1923), a toy experiment of stochastic population growth with feed back. The recent neurophysioiogical observations indicate the exclusive use of a hand in humans requires an active ("enantioselective") neuro-functional organization developed during postnatal brain maturation to prevent mirror movements in ("racemic") infants.

2. Polya's Experiment and Chiral Evolution of a Racemic Pair of Molecular Strands To be an Error and to be Cast out is a part of God's design W.Blake (1757-1827) [4] In 1923 Eggenberger and Polya [5, 6] proposed the following "urn scheme" as a model for the stochastic population growth and the development of contageous phenomenon. Consider a cell, or an urn, initially containing one black ball and one white ball from which one ball is drawn at random. Whatever its color, it is returned to the urn together with a fresh ball of the same color. Repeating the same procedure many times and observing the percentage of, for example, black balls in the urn, we find that the percentage can approach any value between 0 and 100 depending on the outcome of the first few draws.

154


50

1 1

500

1000

Figure 1. Percentage of black balls vs. number of draws in three separate Polya experiments

In Fig.l three separate experiments are plotted, each up to 1000 draws. In all of them an initial stage of fluctuations is followed by a stable behavior, which differs in each case. It can be shown that there is an equal probability for the behavior converging to any percentage of black balls. In Oparin's theory [4] early life began by the successive accumulation of more and more complicated molecular populations within a naturally occurring cell, such as a droplet of coacervate evolving into self-sustaining metabolic cycles. Polya's thought experiment indicates the corresponding model for the stochastic chiral evolution defined by the following assumptions. 1. Early life originated within an urn, such as a fine pore in rock [7], with small openings flooded by an aqueous solution of energy-rich monomers that diffuse in and out of the urn. 2. Early life originated from a racemic doublet of monomer strands, L and D, each serving as a template for self-replication by assembling monomers within the urn. 3. Each strand has a life time of a replication cycle. 4. Environmental factors (temperature, humidity, pH, etc.) and insufficient energy and monomer supply to the urn, as well as the geometrical constraint of the openings, make the simultaneous replication of L and D strands improbable. Fig. 2 shows the first four generations of a progeny of the racemic doublet LD. It is seen that the racemic doublet LD produces, in the first generation, a pair of chiral triplets, LLD and LDD, with equal probability 1/2, each of which, in turn, generates a pair of quartets, LLLD, LLDD and LLDD,LDDD with branching ratio 2/3,1/3, and 1/3, 2/3, and so on. The chiral asymmetry is beginning to be seen in the progeny in the 4th generation. 3. Polya Model of Cerabral Lateralization Any man who, upon looking down at his bare feet, doesn 7 laugh, has either no sense of symmetry or no sense of humour. Descartes (1596-1650) [8]

Implication of Polya' s Urn Experiment in Biochirality and Cerebral Lateralization

LLLLLD

LLLLDD

IXLDDD

LLLDDD

LLDDDD

155

LDDDDD

Figure 2. Development of chiral asymmetry in the progeny of the racemic doublet LD

The evolutionary and ontogenetic basis of the asymmetric specialization of the cerebral hemispheres is notfiiUyexplored. But if the neural hardware in each sphere is prespecified, it would be difficult to account for the ample evidence of the ability of each hemisphere to assume a specific mental operation of the other where the latter is damaged or totally excised. Mirror movements are involuntary movements on one side of the body that occur as reversals of an intended movements contralaterally. They are present physiologically in infants and commonly persists as normal phenomenon in young children. They usually disappear after the first decade of life, coinciding with the completion of the corpus callosum connecting the two hemisphere. It is therefore conceivable that the exclusive ("enantioselective") use of a hand in infants ("racemites") requires lateralized functional organization- developed during postnatal brain maturation to prevent mirror movements [9]. Fig. 3a compares the blood-oxigenization-level-dependent contrast functional magnetic resonance images (BOLD-fMRI of activated premotor cortex of a normal right-handed subject (left) and a left-handed subject (right), both instructed to perform self-paced grasp motions with right hands; Fig. 3b compares a native English speaker (left) and native Japanese speaker (right) instructed to read the same English text. We may define a Polya model for cerebral lateralization by the following assumptions to be examined by experiments. 1. Brain of vertebrate bifurcated into left and right hemispheres, L and R, to resolve conflicts between the evolutionary pressure of expanding cerebral cortex and the limited cranical capacity of the skull. 2. Human brain uses L and D as a binary decisions system detecting the difference in similar objects and the sameness in different objects, respectively. 3. Once activated, the decision system triggers successively higher level systems until a behavioral command to motor system is elicited in the premotor cortex. 4. The neuro-flinctional organization preventing the bilateral movements, developed during postnatal brain maturation by an exclusive use of a hand, suppresses the synchronous activation of L and D unless instructed. 5. The synchronous activation of L and D occurs in the process of perceptual binding ("creative synthesis") of what is known and what is to be known.

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R

L

R

L

Figure 3. a: Brain activities of left-handed subjects, constrained to be right-handed (right) and unconstrained (left), using left hands; b: Native Enghsh speaker (left) and native Japanese speaker (right) reading the same English text [9]

4.

Conclusion It is often remarked that biochirality originates from a chiral molecule abiotically produced by chance in a droplet or a fine pore in terrestrial rock or comet of extra terrestrial origin. But the well-founded CPT symmetry of the physical universe between positive and negative charges (C), between left and right (P) and between past and future (T) makes it more likely that the chiral world of modern life evolved from an abiotically bom racemic pair of organic compounds. In the light of recent development of functional MRI evidences are being accumulated, attributing the origin of cerebral lateralization of humans to exclusive ("enanioselective") use of a hand suppressing ("racemic") mirror movements in infants during the postnatal development of the binary decision system, reminiscient of the Polya experiment in which only one or other out of a racemic mixture of L and D balls in the urn can be dawn at a time.

5.

References

[1] J. Garfield, The Fundamental Wisdom of the Middle Way, Oxford University Press, Oxford, 1995. [2] See papers in the Special Issue on the Origin of Chirahty and D-amino Acids in Biological World. Viva Origino 30 (2002). [3] G. Palyi, C. Zucchi and L. Caghoti, Eds., in: Advances in Biochirality, Elsevier, Amsterdam, 1999. [4] F. Dyson, / Mol EvoL 18 (1982)340-350. [5] F. Eggenberger and G Polya, Zeit. Angew. Math. Mech. (1932) 279-289. [6] F.J. Varela, Principles ofBiological Autonomy, North Holland, New York, 1979, p. 172. [7] H. Kuhn and J. Waser, in Biophysics, (Eds. W. Hoppe et al.) Springer, Berlin 1983. [8] A. Salam, J. Mol Evol. 33 (1991) 105. [9] T. Nakata, et al., Neuroscience Research 32 (1998) 355-362.

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Appendix to Chapter 12 Evolutionary Origin of Binary Decision System and Pharmaceutics

When the world ceases to be the stage for personal hopes and desires, where we, as free beings, behold it in wonder to question and to contemplate there we enter the realm of science and art. A. Einstein [1879-1955] In "What the Frog's Eye Tells the Frog's Brain" [Al] Lettvin, Maturana, McCuUoch and Pitts discovered two major populations of neurons in frog's visual system, sometimes called 'sameness neurons' and 'newness neurons' distinguishing between retinal images of known and unknown objects. The newness neurons are supposed to be excited to bring the animal into an attentive state in search of unknown stimuli. The known objects are further classified by higher level neurons into two groups, biologically favorable and unfavorable [A2]. Quickly moving small objects (preys) and slowly approaching dark dim objects (predators) are examples of favorable and unfavorable objects. It is conceivable that a 'creative synthesis' of the external image of upper Hmbs of amphibian falling in the field of vision with an internal image of an animal on land propelled its own motor behavior. Next, there must occur the partial release of motor activity from genetically determined reflexive movement to voluntary movement. Human brain can be regarded as a device to transform a stream of sensory information from dual world, external and internal, into improved behavioral response and world image. Depending on the urgency to respond, information is immediately converted to instinctive/intuitive (preconscious) response, while some information is momentarily stored as a short-term memory and elicits slowly developing change in the internal attitude conductive of emotional (subconscious) response. The information can acquire an adequate conceptual(conscious) meaning only as far as it requires a critical evaluation and updating of

Figure lA. Frog's binary decision system distinguishing between visual images of what is known and what is to be known

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Figure 2A. Proto-semantic responses to sensory information biologically favourable (upper hemisphere) and unfavourable (lower hemisphere), divided into specific emotive responses according to three biological values, self-preserving, species-preserving and exploring. Emotionally neutral information felling on the equator does not ehcit conscious evaluation of the information conductive of behavioural response [A2]

our inner picture of the external world stored in the long-term memory reservoir reachable by introspection (meditation or transcendental consciousness). Since, in human, hypocampal formation within the emotive brain is innervated by both extro- and intero-ceptive nervous systems, it is in a position to store, compare, and synthesize externally and internally derived information. In mammals the reflexive and emotive brains still regulate autonomic (unconscious) activity of internal organs and reflexive behavioral responses, but somato-muscular organs in higher mammal (hand and speech) fall under voluntary control mechanisms involved in protosemantic communication within species. By means of such voluntary movement, we can use language in speech. The human use of symbols, which is not under genetic control, affords unlimited boundaries to create 'conceptual space' transforming physical reality into transphysical one (virtual reality).'Eureka pleasure' associated with a sudden leap of an innovative scientific idea is likely to be generated when the threat of unknown (newness), is converted to emerging deja vu feeling of reality (sameness). It is tempting to hypothesize that the dominant (left) cerebral hemisphere is devoted to detect difference in similar objects (articulation of the world), and the non-dominant (right) hemisphere to discover similarity in different objects (grasping of the world in its coherent whole). It is hoped that the chemical neuro-transmitter substances which are involved in each of the cerebral hemisphere will eventually be identified. The accuracy and stability of long-term memories which depend upon the specificity of those neuro-transmitters would therefore be affected in precise ways by changes in the chemical balance of the transmitters in a given individual. [Al] L. Lettvin et al. Proc. IRE, 47 (1959) 1940. [A2] N. Hokkyo, in: Biocomputers (Eds. T. Kaminuma and G. Matsimioto; tr. N. Cook) Chapman & Hall, London, 1991.


Chapter 13 Theory of Hierarchical Homochirality Dilip K. Kondepudi Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA [email protected]

I dedicate this article to Ilya Prigogine whose vision of the fundamental nature of irreversible processes inspired so many.

1.

Introduction The ubiquity of chiral asymmetry in nature, from matter's most fundamental constituents to the morphology of mammals, makes it obvious to us that there are physico-chemical processes that generate and propagate chiral asymmetry. But vv^hat these processes are and what mechanisms propagate the asymmetry is far from obvious. Nevertheless, based on symmetry considerations, and using elementary group theory, one can construct a general theory of spontaneous generation of chiral asymmetry and elucidate general features of how asymmetry can propagate from one structure to another. In this article I shall present such a general theory that is based on the work I did with George Nelson [1,2]. In developing a general theory of chiral asymmetry, one must keep in mind the fact that structures on different scales are somewhat independent. In simple terms, the shape of a spiral staircase does not depend on the shape of bricks with which it is built. Similarly, spiral spatial patterns that arise as a consequence of chemical reactions and diffusion are unrelated to the structure of molecules. Conversely, the vorticity of fluid motion could have no observable effect on the outcome of chemical reactions involving chiral molecules. After all, we cannot feel the effect of the Coriolis force due to Earth's rotation while riding a bicycle: the scales of motion are widely separated and one has an insignificant effect on the other. This is because the physical laws that govern processes at various spatial scales are somewhat independent: nuclear states have very little effect on chemistry and the basic laws of hydrodynamics are quite independent of molecular structure. So it is only natural to think that the asymmetry we observe at the level of elementary particles is unrelated to that at the level of biomolecules and that the biomolecular asymmetry is unrelated to the morphological asymmetry we see in mammals. Must the asymmetries at different levels be always independent? Is it possible that there are indeed processes that connect asymmetries at various levels in certain situations? In the following sections I will outline a basic theory that enables us to see how asymmetry at various levels could be connected. This theory provides us with a framework for

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understanding the connection between molecular asymmetry and morphological asymmetry and the connection between fluid vorticity and molecular aggregation that was recently reported [3].

2.

The Roots of Morphological Homochirality In the case of morphologies of mammals, the organ placement is asymmetric: in humans, the heart is a little to the left, the liver is on the right, the stomach on the left and so on. But this asymmetry, apart from rare exceptions [4], is the same in all humans; it is inherited. Why this "homochirality"? If the process that establishes asymmetry is independent of the asymmetry at the molecular level, and is established during morphogenesis of each human, we must expect statistically equal number of people with mirror-image morphologies - as we do in conglomerate crystallization. Since the homochirality of organ placement is inherited [5], might it not be coded in the genetic sequence of DNA just like all other inherited traits? Could it not be that a particular sequence of A, T, G and C codes for the morphological asymmetry? The answer is no: It is impossible to code handedness in a linear sequence of letters, ATTGGCATGA Richard Feynman [6] and Martin Gadner [7], have noted the fact that it is impossible to communicate what one means by "left" or "righf (without using basic parity violating laws of nature that distinguishes between the two) by sending and receiving messages, in other words with strings of letters. There is a simple proof for this assertion using reductio ad absurdum. Let us suppose there exists a physical process without any inherent bias towards the left or the rights that takes as its input a sequence ATTGGCATGA... and produces a chiral structure, say a right-handed helix. (If we do not require that the process is "without any inherent bias towards the left or right" then the problem does not exist: the bias is itself the code for what is meant by "left" or "right".) The requirement "without any inherent bias towards the left or right" means that the process is either achiral or chirally symmetric, i.e., it should be indistinguishable from its mirror image. Processes that are governed by the laws of mechanics and electromagnetism satisfy this requirements. The sequence ATTGGCATGA... is assumed to provide the right-handed bias that drives the system to generate a right-handed helix. Using the assumed process, one could communicate what is meant by a right-handed helix, as opposed to left-handed helix. Why is such a process not possible? Let us look at the process in a mirror, as show in Fig. 1, which is also realizable because the laws of mechanics and electromagnetism are mirror-symmetric. Since the mirror-image process is also realizable, we have to conclude that the same process must also produce a left-helical structure using the same input ATTGGCATGA... This contradicts our original assumption that the sequence ATTGGCATGA...carries a right-handed bias and produces right-helical structure. The mirror-image process tells us that the same sequence must also carry a lefthanded bias and produce a left-helical structure. Hence an achiral or symmetric process that produces only a right-handed helix with the input ATTGGCATGA... cannot exist. It is often tacitly assumed that computer codes can carry any type of information. Since computer codes are ultimately strings of zeros and ones, the above lemma shows that it is impossible to design a code that can specify what one means by "left": the chiral objects that we are able to display on a computer screen are left- or right-handed only because the display

Theory of Hierarchical Homochirality

161

A ATTGGCATGA

MIRROR

SYMMETRIC PROCESS

ATTGGCATGA

\J 0 Figure 1. Is it possible for a symmetric process to generate only a "right-handed" object using a linear sequence of letters such as ATTGGCATGA .... as input? If such a process existed, by looking at the processes in a mirror, we must conclude that the same process must also generate a "left-handed" object. Hence, a process that takes as input a linear sequence such as ATTGGCATGA .... and generates only a "right-handed" object cannot exist window is scanned, or pixels identified, with a particular convention, from left to right or vice versa. Whether an object on the screen is right- or left-handed depends on this convention, not the computer code. So the morphological asymmetry cannot be inherited through a genetic sequence. It must be coded in some three-dimensional structure in which a particular handedness is coded. What might this structure be? Can its chirality be traced all the way down to the homochirality of Lamino acids of proteins or the D-sugars? Recent investigations on the origin of morphological asymmetry in chick embryos suggest that the asymmetry originates in the asymmetric motion of cilia during development [8]. This asymmetric motion must clearly be due to the some chiral aspect of its macromolecular structure which is likely to have its origin in L-amino acids. If we have an understanding of how chiral asymmetry propagates from one level to another, at this stage we could at least see in principle how the morphological homochirality could have its roots in the homochirality of the amino acids.

3.

Theory of Spontaneous Chiral Symmetry Breaking Using group theory, a general theory of spontaneous chiral symmetry breaking can be formulated [1]. In this theory, the state of the system, be it chemical or other, is specified by a n-vector X=[xi, X2, ... x j . In the case of a chemical system, Xk would be the concentrations; for a macromolecules, XR would be the variables that specify its structure. In general, the time-

162


evolution of this system is described by a set of nonlinear equations: ^ = F[X,A] at

(3.1)

in which F is a nonlinear operator; >. is a parameter that represents a constraint on the system; in an open chemical system, for example, it could be a flow rate, or a concentration that is fixed. In general, for any given system there will be several such parameters but, to present the main features of the formulation, we may consider explicitly only one parameter that is relevant to chiral symmetry breaking; the other parameters can be thought of as implicit in the definition of F. The state of the system, X, also defines its chirality: X could be a chirally symmetric state or a chirally asymmetric state. We shall use P for the parity operator that transforms X to its "mirror-image state" (strictly speaking, mirror-image state is the result of P followed by a rotation). Thus, X and PX represent mirror-image states. If the state of the system is symmetric, then X = PX. In the case of a chemical system in which the state is represented by the concentrations of chiral compounds, X would be symmetric if the concentrations of the enantiomers are equal; under the operation P, the enantiomers are interchanged (L-enantiomer becomes D-enantiomer and vice versa). If X is not symmetric, then X ^ PX. The processes, such as chemical kinetics, difftision, forces between the constituents of a macromolecule etc., that govern the time-evolution of X are assumed to be chirally symmetric. In mathematical term this means: PF[X,;i]=F[PX,?i]

(3.2)

The parameter X is generally achiral (as for instance, a flow rate or a concentration of an achiral reactant) so P does not alter X. That the processes that determine the state of the system, X, are chirally symmetric is expressed by equation (3.2). Spontaneous "breaking" of chiral symmetry is in the sense that, tough the processes F(X,>.) that determine the state X are chirally symmetric, the state itself may not be symmetric (X ^ PX), i.e., chirally symmetric processes generate a chirally asymmetric state. As a consequence of (3.2), if X is a solution to (3.1) then PX is also a solution. Hence, if X ^ PX, there are two independent solutions. The general features of spontaneous symmetry breaking are as follows: for every value of the parameter X, and appropriate initial and boundary conditions, there corresponds a steady state Xs, for which dXs/dt = 0. (In some cases, the trivial Xs=0 may be the only steady state). This steady state is symmetric for a range of values of X, but at a "critical point" Xc, this steady state becomes unstable to small perturbations in X; as a consequence, an appropriate fluctuation can drive the system to an asymmetric state. In mathematical terms, the transition from symmetric to asymmetric states is because of "bifiarcation" of new states, which is described as follows. In the vicinity of the critical point, the asymmetric state can be represented by: X = Xs + aXA

(3.3)

in which XA is an asymmetric state and a its "amplitude". Using very general arguments, it is possible to obtain an equation for the time-evolution of the amplitude a of the form:


163

Figure 2. Diagram showing the bifiircation of asymmetric steady states (09^) from the symmetric steady state (a=0) at Xc. The symmetric steady state is unstable when X>Xc

da dt

G[a,A]

(3.4)

When X< Xc, the only steady state that is stable corresponds to a=0; when X>Xc, states with a^Ô are also possible. Equation (3.4) is sometimes called the "bifurcation equation" because it tells us how a new solution (3.3) "bifurcates" from the solution Xs when X>Xc. The asymmetric state, XA, has the property PXA = -XA. As a consequence, since PXs=Xs and PX is also a solution of (3.1), PX = Xs - aXA is also a solution. Consequently, if a is a solution of G[a,A,]=0, then - a is also a solution. This symmetry property restricts the possible forms of G[a,^]. Using the general theory of bifurcation and group theory one can show that, in the vicinity of Ac, (3.4) is of the form: —-=-AQr^+B(A-X,)Qr dt

(3.5)

in which A and B are constants that depend on the particular features of the system, such as kinetic rate constants or force constants (as will be made clear in the example below). For a given F[X, X], the constants A and B can be calculated using general procedures[l]. dct The steady states of (3.5) (for which — = 0) as a fiinction of X shows the "bifurcation" dt of asymmetric states (Fig. 2). When X<Xc, it can easily be seen that a=0 is the only real steady-state solution; when X>Xc, two solutions are possible:

a = ±^B(X-Xc)/A

(3.6)

These general features can be illustrated in a simple chemical system which is an extension of a model proposed by Frank in 1953. The extension puts the model in a thermodynamic

164

Progress in Biological Chiialiw

framework so that spontaneous generation of asymmetric state can be understood as the appearance of a far-from-equilibrium dissipative structure [2, 9]. The model chemical system that shows spontaneous chiral symmetry breaking is: S+T

(3.7a) (3.7b) (3.7c) (3.7d) (3.7e)

X I , + XD

Here S and T are achiral reactants that produce chiral product X in the L- and Dconfirmations. The rate constants for the forward and reverse reactions are denoted by kif and kir and so on. The equality of the rate constants of reactions (3.7a) and (3.7c), and (3.7b) and (3.7d), reflects the chiral symmetry of the processes. As indicated by reactions (3.7b) and (3.7.d), the chiral species X is autocatalytic. In addition, the two enantiomers of X react to form an inactive species P. Reaction scheme: S + T -> S+T ^

S + T + Xj ;f^ 2Xj Xj,

S+T+Xj,^

XL+XD ^

?L=[S][T]

S T

2XD

P

a = ([XLl-[XD])/2 S T

XT

-

XD

A,Xc

Figure 3. A chemical model to illustrate the general theory of spontaneous chiral symmetry breaking. With the reaction scheme shown on top. in an open system with inflow of S and T and an out flow of P, the system can spontaneously make a transition to a state in which XL^XD even though the kinetics of the two enantiomers are identical. The general behavior of all such systems is shown in the bifurcation diagram


165

As shown in Fig. 3, we consider an open system into which there is inflow of S and T and an outflow of P such that the concentrations of S and T are maintained at a fixed level in the reactor. With these flow constraints, the system could be described by a set of two rate equations:

™

dt

- kif[S][T]- k J X J + k,,[S][T][XJ-k,,[X,f -k3[XJ[XJ

(3.8a)

= k JS][T]-k J X J + kJS][T][XJ- k,,[XJ^ - k3[XJ[XJ

(3.8b)

in which [S] denotes the concentration of species S. These equations correspond to the general time-evolution equation (3.1). In this model reaction scheme, by setting the parameter X =[S][T], it is possible to obtain the generic equation (3.5) in which the "amplitude" of the asymmetric state a=([XL]-[XD])/2:

dt in which, Xc, the critical value of X. at which asymmetric solutions bifurcate is given by:

Xc

(2 s + p-4kifk?,f

(3.9)

2kL S=2k3,k, + 4(k^^k„)/(k3-k,,)

Explicit expressions for the coefficients A and B are:

k3(k2'^C-kir)

B = k2f _iS2L

V

2kifk2r kjAc klr.

(3.10)

The details of obtaining these expressions using the general formalism can be found in ref 1. The same formalism can also be used to describe structural transition from an achiral structure to a chiral structure. As a simple example, let us consider the following structural transition shown in Fig. 4. In this model, the potential energy between the two arms of the molecule depend on the distance X and angle 9 between them: V(A,}ic, the stable steady state is 0=0; for \Xc the equilibrium stmcture is synunetric; when X<Xc the equilibrium structure is asymmetric


167

chiral structures, the chiral influence may be from the lower level of the building blocks of the macromolecule which posses an asymmetry. For example, if we consider an achiral polymer that can fold in a helical form, the transition from a linear structure to helix is a case of spontaneous chiral symmetry breaking. Making one of the monomers chiral will introduce a chiral influence that will bias the system to favor one of the two helical secondary structures. This in turn may influence the chirality of the tertiary structure an so on. In the following section, we shall discuss hierarchical nature of structural chirality in the framework of the theory presented in this section. The origin of the chiral interaction and its magnitude depends on the particular conditions of the system under consideration but, as in the previous section, it is possible to formulate a general theory of how a chiral interaction might influence a system when it makes a symmetry-breaking transition. To be sure, we expect it to be sensitive to even a small chiral influence. A quantitative theory should specify what exactly is meant by "small" and how to quantify the system's sensitivity. In analyzing the influence of a chiral interaction on chiral-symmetry-breaking transition, we found it necessary to distinguish between two types of time evolution as shown in Figure 5A. In the first type (I), the system is initially below the critical value Xc and evolves to a value larger than the critical value. We shall assume that X starts with some initial value A,o, which is below the critical point, and evolves to a value above the critical point at a rate y: ?i-?io + rt

(4.1)

During this process, the system makes a transition to the asymmetric state, a>0 or aA,c and, because the symmetric state is unstable, makes a transition to one of the asymmetric states.

Figure 5. Evolution to an asymmetric state. A. Two types of evolutions, I and IL are shown. In type I, the system is initially below Xc and evolves to a higher value. During this process, it makes a transitionfroma=0 to a>0 or aXc and a=^0 and evolves to a>0 or a0 state: the state a>0 is reached with a probability' P+


Systematic chiral interactions are usually very small. In this context, the notion of "small" is with respect to the size (root-mean-square value) of random fluctuations in a that are present in every physical and chemical system. When the random fluctuations and the systematic chiral interaction are included, the general equation changes to [1, 2]: — - - A a ' + B(>. - ^c )a + Cg + Vef(t) dt

(4.2)

in which C is a constant that depends on the system parameters, g is a parameter that gives us measure of the strength of the chiral interaction; for example, if the chiral interaction causes a difference in the reaction rates of the enantiomers, this difference may be expressed as:

ki,=ai+g)=^H-||]

(4.3)

in which AG is the difference in the Gibbs energy of the transition state of the enantiomers. 8 is mean-square value of the random fluctuations denoted by f(t) (one may assume f(t) to be Gaussian white noise for simplicity). The bifurcation diagram and a typical evolution of a when Cg>0 is shown in Figure 5B. The sensitivity of the system to the chiral interaction can be quantified by the probability, P+, that the system will make a transition to the asymmetric state favored by the chiral interaction or bias. At first sight, it might seem that the influence of the chiral bias will be small and unobservable if CgnR rate = kgLRn]

(8)

and dissociation of oligomers: Sn^nS rate =^ kgLSn]

Possible Mechanisms for Production of Large Enantiomeric Excess

175

If [S] is greater than [R], Sn will be produced more rapidly than Rn, and the difference ([S] "^ i^[Sn]) - ([R] - n[Rn]) will increase with time. If the oligomers are more stable than the monomers, formation of oligomers will reduce the effects of reactions (4) and (5). If the oligomers are more stable than the monomers and are also catalysts for the production of their respective monomers, H + S„->S + Sn rate = k9[H][Sn]

and and

H + Rn->R + Rn rate = k9[H][R,,],

(9)

then catalysis by the oligomers will be more effective at increasing enantiomeric excess than autocatalysis by the monomers. Avetisov has pointed out that production of even a single large homochiral molecule is extremely improbable unless there is already a large difference between the concentrations of the enantiomeric monomers [3]. For example, if the mixture of monomers is racemic and if S and R add randomly to growing chains, the probability that a 50-mer will be homochiral, S50 or R50, is {Mlf^ = 2 X 10"^^ and the probability that a 100-mer will be homochiral is only 2 x 10'^^. If homochiral addition is faster than random addition, then the probability of forming homochiral oligomers is increased. Nevertheless, it is apparent that a large enantiomeric excess would have been necessary for production of homochiral oligomers as large as modern enzymes. The oligomers in reactions (7), (8), and (9) must have been small. There is a possible exception to the conclusion that the earliest catalytic oligomers must have been small. Suppose that the probability of forming a moderately large homochiral molecule, Sn or Rn, is so small that the rate of formation is not represented accurately by a continuous rate equation. Eventually a single Sn or Rn molecule is produced. Assume that it is Sn. If it is a catalyst for production of S from H, the rates of production of S from H and Sn from S increase initially, but as the concentration of H decreases the rates of reactions (7) and (9) decrease until they reach the limit imposed by the rate of production of H from L. The rate of uncatalyzed production of R from H decreases as the concentration of H decreases, and the probability that even one Rn molecule will form becomes even smaller than it was initially. The increased concentration of S and the decreased concentration of R increase the probability that additional homochiral Sn molecules will be produced. Kondepudi and his coworkers have carried out experimental studies of a system analogous to the one described in the previous paragraph [4, 5, 6, 7]. They started with an aqueous sodium chlorate solution. The individual sodium ions and chlorate ions in the solution are not chiral, but solid sodium chlorate crystals are chiral. Water was evaporated until the solution became highly supersaturated with respect to macroscopic crystals but only slightly supersaturated with respect to microscopic crystals. Eventually a single microscopic crystal formed spontaneously. It grew rapidly by incorporating ions from the solution. Rapid stirring caused microscopic daughter crystals to break away from the growing crystal, and the daughter crystals also grew and produced new generations of microscopic crystals. The stirring caused the decrease in concentrations of free sodium ions and chlorate ions to be uniform throughout the solution. As water continued to evaporate, the solution became and remained only slightly supersaturated with respect to macroscopic crystals but unsaturated with respect to microscopic crystals, so that only crystals with the same chirality as the first one could be formed. Repeated experiments produced either S or R crystals randomly, but only one enantiomer was produced in each experiment.

176


Mutual destruction of enantiomers: The most plausible mutual destmction process is formation, decomposition, and decay of mixed dimers. S + R -> SR rate = kio[S][R] (10) SR ~> S + R rate = kii[SR] (11) SR->2L rate = k,2[SR] (12) Frank demonstrated that a combination of autocatalysis and mutual destruction would cause enantiomeric excess to approach unity if the decay reaction (4), the inversion reaction (5), and the decomposition reaction (11) were negligible [2]. In our previous paper we showed that mutual destruction alone can cause the enantiomeric excess to approach unity if the supply of H is finite, even if the only difference between the rates of production of S and R is random fluctuation [1]. If the supply of H is infinite, random fluctuations will cause the enantiomeric excess to swing between positive values (excess S) and negative values (excess R). As [S] and [R] increase, larger absolute values of the enantiomeric excess will become more common and last longer. Mutual inhibition of chiral catalysts: Let Cs and Cr represent a pair of enantiomeric catalysts. Cs catalyzes production of S, and Cr catalyzes production of R. [Cs] and [Cr] represent concentrations if Cs and Cr are homogeneous catalysts or number of active sites if Cs and Cr are heterogeneous catalysts. [Cs] and [Cr] are equal initially. Then the following reactions occur: H + Cs ^ HCs rate = k,3[H][Cs]

and and

H + Cr -> HCr rate =k,3[H][Cr]

(13)

HCs -> H + Cs rate = k,4[HCs]

and and

HCr -> H + Cr rate = kM[HCr]

(14)

HCs —^ SCs rate = ki5[HCs]

and and

H C r - > RCr rate= k,5[HCr]

(15)

SCs —^ HCs rate= ki^SCs]

and and

RCr -> HCr rate-ki6[RCr]

(16)

SCs -> S + Cs rate = k n [SCs]

and and

RCr -> R + Cr rate = k]7[RCr]

(17)

S + Cs -> SCs rate = k,8[S][Cs]

and and

R + Cr -> RCr rate = ki8[R][Cr]

(18)

S + Cr ^ SCr rate = k,9[S][Cr]

and and

R + Cs -> RCs rate = ki9[R][Cs]

(19)


SCr -> S + Cr rate = k2o[SCr]

and and

RCs --> R + Cs rate = k2o[RCs]

177

(20)

If ki9 is larger than kig, or kio is smaller than kn, or both, each enantiomer will inhibit catalytic production of the other more than it inhibits its own production, and if there is an excess of either enantiomer, that enantiomer will be produced faster than the other.

3.

Limits to Enantiomeric Excess Any of the processes discussed in the previous section, or any combination of them, will amplify an initial difference between [S] and [R], but the effect will be limited. The reactions that increase the differences between [S] and [R] and between [Sn] and [Rn] become slower as H is consumed. The reactions that tend to equalize the concentrations of S and R and the concentrations of Sn and Rn by converting S, R, Sn and Rn to L accelerate as the concentrations of S, R, Sn and Rn increase. The inversion reactions (5) decrease the absolute value of ([S] - [R]). Catalysis may not be perfectly enantioselective, even if the catalyst is chiral [8]. We have not considered imperfectly enantiospecific catalysis explicitly because it is equivalent to the inversion reactions. Mutual inhibition of racemic catalysts is self limiting, because as more and more Cs and Cr are inactivated by conversion to RCs and SCr the rates of catalyzed production of S and R both decrease. The extreme case of this would occur if SCr and RCs never decomposed (k2o - 0). Even if there were a large difference between [S] and [R], eventually both Cs and Cr would be completely converted to RCs and SCr and the difference between [S] and [R] would decay. Even with the concentration of H maintained above its equilibrium value by an external source of free energy, enantiomeric excess can only increase to a maximum value less than unity and then either remain constant or decrease as a steady state is approached. However, an enantiomeric excess close to unity can be attained by successive mutations of a catalytic species. Suppose that an alteration in the structure of a single S, R, Sn, or R„ molecule increases the catalytic efficiency of that molecule or makes it more stable. Copies of the altered molecule will gradually replace the unaltered molecules and will become more numerous than the unaltered molecules were. Such favorable mutations will occur randomly, but the rate of mutation will be proportional to the concentration of the species that is modified by the mutation. Successive favorable mutations will cause one species to replace the original S, R, Sn and Rn almost completely. Darwinian evolution can occur, even at the prebiotic stages.

4.

Numerical Examples The sets of simultaneous differential equations that describe the time evolution of an initially racemic system were solved numerically for a number of different combinations of rate constants. Reactions that are the reverse of reactions (3), (4), and (6) were not included. These reverse reactions would have large positive standard free energy changes, so the rates of these reverse reactions would be negligible relative to the rates of their respective forward reactions. The algorithm used was NDSolve of Mathematica 4. These computations confirmed the qualitative arguments that were presented in the preceding sections. The resuhs of four of these computations are shown in Tables 1, 2, 3, and 4. The units of

178

Progress in Biological ChiraliU

concentration, time, and the rate constants are arbitrary, but they were consistent over all the calculations. Table 1. Number of amplification Rate Constants

Initial Concentrations

k,= 1000 k.= 1 1.01 k3S = 1 k3R = 1 k4 = 0.1 ks-

[L] = 1000000 [H]= 0 [S]= 1 [R]= 1

Steady State Concentrations [L] = 999.003 [H] =331895.0 [S] =334938 [R] =332170

Table 2. Catalysis by dimers Rate Constants


k,= 1000 1 k2 = 1.01 k3S ~ 1 ksR = 1 k4 = 0.1 ks = k.= 1 1 k8 = ^10000

[L] =1000000 [H] = 0 1 [S] = IR] = 1 0 ISzl^ 0 lS2l = 0 [R2I-

Steady State Concentrations [L] = 50587.5 [H] = 0.0107 [S] 4340.56 [R] = 2935.81 [S2] = 325885 [S2] = 325885 [R2l = 145184

Table 3. Mutual destruction Rate Constants


k,= 1000 k.= 1 1.01 k3S = 1 ksR = 1 k,= 0.1 ks = kio = 1000 1 kn = 1 ki2-

[L] =1000000 [H]= 0 tS]= 1 [R]= 1 [SR] = 0

Steady State Concentrations [L] = 999.003 [H]= 857821.0 [S]= 698.131 [R] = 1.3294 [SR] = 70241.3

Table 4. Catalysis by dimers, self protection by formation of dimers and mutual destruction Rate Constants


1000 1 1.01 k3S = 1 k3R = 1 k4 = 0.1 ks = 100 k7 = 0.001 k8 = 100 k9 = 0.001 kn = 0.001 ki2 =

|LJ = 1000000 0 [H] = 1 [Sl = [R] = 1 0 tS2] = 0 1R2] = 0 [SR1 =

ki =

k.=

Steady State Concentrations [L] = 46.4344 [H]= 0.001164 [S] = 15.2067 [R] = 2.5538 [82] = 364793 [R2]= 34096.7 [SR] =101079


179

5.

Conclusions There are several mechanisms that can cause enantiomeric excess to increase in a system that is initially racemic or achiral. None of these can increase the enantiomeric excess to unity, and some of them produce only a transient increase that decays to a steady state having zero or very small enantiomeric excess. Mutual destruction is capable of producing enatiomeric excess that is close to unity, but mutual destruction causes the steady state concentrations of both S and R to be small. One of the most effective mechanisms for production of enantiomeric excess close to unity is random formation of a single homochiral oUgomer molecule that catalyzes production of its own monomers. Two or more mechanisms acting together are more effective than any one of them alone. Even a small or transient enantiomeric excess can lead to a large, permanent one because the chiral species can undergo favorable mutations, and the more abundant enantiomers will have greater probability of mutating. The relative importance of the several amplification mechanisms depends on the values of the rate constants. These values will have to be determined by experiment for specific reactions.

6.

Acknowledgements This work was partially supported by the Ilya Prigogine Center for Statistical Mechanics and Complex Systems, University of Texas at Austin and the Department of Mathematics, Southwest Texas State University.

7.

References

[1] D.Z. Lippmann and J. Dix, Possible mechanisms for spontaneous production of enantiomeric excess. In: Advances in BioChirality (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier, Amsterdam, 1999, pp. 85-97. [2] F.C. Frank, Biochim. Biophys. Acta 11 (1953) 459-463. [3] V.A. Avetisov, Origin of biological homochirality: directed selection or random motion? In: Advances in BioChirality (Eds. G. Palyi, C. Zucchi and L. Caghoti) Elsevier, Amsterdam, 1999, pp. 69-83. [4] D.K. Kondepudi, R.J. Kaufman and N. Singh, Science 250 (1990) 975-977. [5] D.K. Kondepudi, K.L. Bullock, J. A. Digits, J.K. Hall and J.M. Miller, J. Am. Chem. Soc. 115 (1993) 10211-10216. [6] D.K. Kondepudi and C. Sabanayagam, Chem. Phys. Lett. Ill (1994) 364-368. [7] D.K. Kondepudi, K.L. Bullock, J. A. Digits and P.D. Yarborough, J. Am. Chem. Soc. 117 (1995) 401-404. [8] V.A. Avetisov, in: Physical Origin of Homochirality in Life (Ed. D. Cline) AIP Press, Woodbuiy, New York, 1996, p. 141.

Progress in Biological Chirality G. Palyi, C. Zucchi and L. Caglioti (Editors) © 2004 Elsevier Ltd. Allrightsreserved.

Chapter 15 Sugar C-suifonic Acids Andras Liptak Research Group for Carbohydrates of the Hungarian Academy of Sciences, H-4010 Debrecen, P.O. Box 55, Hungary liptaka@tigris. kite, hu

1.

Introduction "Glycolipids are glycosyl derivatives of lipids such as acylglycerols, ceramides and prenols. The term glycolipid designates any compound containing one or more monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety such as an acylglycerol, a sphingoid, a ceramide (N-acylsphingoid) or a prenyl phosphate" [1]. This chapter focuses mainly on the occurrence of a special group of glycodiacylglycerolipids, namely the sulfoquinovosyldiacylglycerols (SQDG). The SQDGs differ from each other only in their fatty acid composition. The first representative of the SQDGs was isolated in 1959 from Chlorellapyranoidosa [2]. Additional sources were found in photosynthetic organisms, such as algae, cyanobacteria [20], sponge [11], starfish [12] and surprisingly in the bacteroid forms of Bradyrhizobium Japonicum [13], because this plant glycolipid is rarely found in bacteria. Structural studies [14-17] showed that these compounds were l,2-di-0-acyl-3-0-(6-deoxy6-sulfo-a-D-glucopyranosyl)-L-glycerols. These structures were confirmed by X-ray crystallographic studies [18] of the rubidium salts of the deacylated glycolipids which were also verified by the ^^C-NMR spectra [19]. The fatty acid distribution in the natural lipid from various sources has been examined [20-22] and as in most phospholipids and glycolipids [23], a wide spectrum of fatty acids was found. Hexadecanoic acid was found to be present predominantly at the C-2 OH group of the glycerol moiety and mono-, di- and trienic derivatives of octadecanoic acid occurred preferentially at the OH-1 of glycerol. 2.

Synthesis Miyano and Benson [17, 19] reported the first synthesis of 6-deoxy-6-sulfo-Dglucopyranose, as well as that of 3-0-(6-deoxy-6-sulfo-a-D-glucopyranosyl)-L-glycerol. The first preparation of 3-0-(6-deoxy-6-sulfo-a-D-glucopyranosyl)-1,2-di-O-hexadecanoyl-Lglycerol, the "sulfoquinovosyl diglyceride" was described by R. Gigg and his co-workers [24]. During the last decade, it turned out that the sulfoquinovosyl mono- (SQMG) and


Figure 1 diacylglycerols (SQDG) indicated strong inhibitory activities against eukaryotic DNA polymerase a and b [25, 26], They possessed extensive biological activities such as antitumour effects [27], P-selectin receptor inhibition [28], inhibition of HN-RT [25, 29] and AIDS-antiviral [10] properties. These biological indications initiated the syntheses of more than ten SQDG and SQMG compounds [24,30-32] and their biological investigations were achieved (Figure 1). The structure elucidation and the synthesis of 3-0-(6-deoxy-6-sulfo-a-D-glucopyranosyl)1,2-di-O-acylglycerol required a strong synthetic capacity and different processes were worked out for the preparation of 6-deoxy-6-sulfo-D-glucose. Partially or fully protected 6-0sulfonyl(tosyl [15, 16], mesyl, triflyl)-D-glucose or -glycoside were treated with nucleophilic reagents, such as NaHSOs [15, 16, 19, 34, 35, 37] or CH3COSK [24, 30, 31, 32, 38, 39]. In the first case 6-C-sulfonyl compounds were directly obtained, and in the second reaction the 6-Sacetyl intermediates were oxidized to 6-C-sulfonic acids by peracids, H2O2, Oxone or (NH4)6Mo7024.4H20. In another approach alkyl 6-deoxy-D-x);/o-hex-5-enopyranosides were treated either with Na2S03 or with CH3COSH under free-radical addition conditions to obtain directly the glucose-6-suIfonic acid or glucose-6-thioacetate which was oxidized to glucose-6C-sulfonic acid (Figure 2). Sulfoquinovose is the defining constituent of photosynthesizing organisms and an important component of the biological sulfur cycle [40]. The presence of other sugar sulfonic

Sugar C-sulfonic Acids 183 •

PO'

^SAc

SO3 Na O @

^^"Ai-*"*T,

J

'c )

S 20 C LU

i.

^

^

#

0

o

•^

t

0 i

1

20

1

40

60

__-l 80

J 1

100

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120

Time [ min ] Figure 1. Time developments of enantiomeric excess for three cases of starting with 50 mM solution of pure L-alanine (solid circle), pure D-alanine (open circle), and complete racemic mixture of L- and D-alanine (solid square). The temperature of the high tenq)erature chamber reached the designated 230 °C about 20 minutes after the start of theflow-reactoroperation. Error bar denotes a half of the standard deviation of measured enantiomeric excess estimated out of three tofiveindependent experiments

Racemization of Amino Acids in Hydrotheraial Environments: a Contribution of...

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(pH 5.2) was added. The specimen of 250JLI1 of the solution was then taken for the HPLC analysis. The fluorescence detector equipped with a lOjiil flow cell was used with an excitation wave at 320nm and an emission wave at 440nm. The column used was the reversed phase YMC ODS-M80 (4.6mm x 250mm). The mobile phase was 27.5% methanol and 50mM sodium acetate (pH 2.5).

4.

Results Typical time developments of enantiomeric excess for alanine are displayed in Figure 1, in which the concentration of alanine was maintained at 50mM. Enantiomeric excess is denoted as the numerical figure |[L]-[D]|xlOO/([L]+[D]) [%l where [L] and [D] represent the concentrations of L- and D-alanine. We showed three cases of starting with pure L-alanine, pure D-alanine, and complete racemic mixture of L- and D-alanine. The initial pH measured at room temperature was 5.7 for both pure L- and D-alanine, while the final pH after 120 minutes operation of the flow-reactor measured again at room temperature was 7.6 for the Lalanine case and 7.8 for D-alanine. There was observed a sizable difference of enantiomeric excess whether the reaction solution started with pure L-alanine or D-alanine. In order to see the contribution of the pH value on enantiomeric excess, we measured the similar time developments for the pH valued fixed at 4.0 by adjusting the amount HCl to be added (Figure 2a), and at 8.0 by adjusting the amount of NaOH to be added (Figure 2b). The pH value was constantly monitored at the low temperature chamber maintained at 0 °C. We

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206

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Figure 3. Contribution of ionic strengths to enantiomeric excess. The excesses were measured after 120 minutes operation of theflowreactor starting with either pure L-alanine (solid circle) or pure D-alanine (open circle) with the controlled amount of added sodium chloride. The concentration of alanine was 50mM for either case. Error bar denotes a half of the standard deviation of each measurement estimated out of three tofiveindependent experiments

could not however observe a significant difference in the contribution of the pH value to the racemization of alanine whether it started with pure L-alanine or D-alanine. One more candidate for influencing the racemization may be ionic strengths of the reaction solution. The effect of added sodium chloride on enantiomeric excess is then displayed in Figure 3, demonstrating that the reaction solution starting with L-alanine had slightly greater enantiomeric excess compared to the case starting with D-alanine. One remarkable difference between the two cases of starting with L-alanine and with D-alanine is that L-alanine is quite sensitive to the concentration of sodium chloride for its racemization, while D-alanine remains rather indifferent. In addition, there is one subtle point to be noted. When there was no added sodium chloride (see Fig. 1), the reaction solution starting with pure L-alanine had slightly smaller enantiomeric excess compared with the case starting with D-alanine. The conversion from enantiomeric excess in favor of D-alanine to L-alanine occurred in the narrow region of ionic strength between 0.0 and 1 /i M of sodium chloride.

5.

Discussion Although optical purity of amino acids [11, 12] could almost fully be lost through racemization in thermal equilibrium at high temperatures available near hydrothermal vents, the actual hydrothermal environments in the ocean are not in thermal equilibrium because of the presence of constant temperature gradient between the hot water inside the vents and the cold water in the surroundings. Such temperature gradient may provide a likelihood of enantioselectivity that could not be expected in thermal equilibrium. One indication of

Racemization of Amino Acids in Hydrothermal Environments: a Contribution of...

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enantioselectivity of nonequilibrium origin thermally can be seen in the fate of the racemization of each individual amino acid molecule experiencing the temperature gradient. Our observation of enantioselectivity of L- over D-alanine in racemization in the flow reactor facilitating a sharp temperature gradient, as demonstrated in the case of low concentration of sodium chloride, manifests that the rate of an individual L-alanine molecule to be transformed into a D-alanine molecule is smaller than the similar rate of the reversed transformation. So long as the reaction environment is maintained in the presence of such a temperature gradient as in the actual hydrothermal environments, the survival probability of L-alanine relative to that of D-alanine could be enhanced with time because of its multiplicative, instead of merely additive, nature of the very survival in time. Of course, even racemization for both reaction solutions starting with L- and with Dalanine could be expected when these reactants visit the region at high temperatures as with our high temperature chamber maintained at 230 °C. The unevenness of enantiomeric excess between L- and D-alanine acquired as traversing the accompanied temperature gradient would certainly be mitigated when the reactants visit and stay in the high temperature region. What is significant to such hydrothermal environments is that there is expected a situation enhancing enantiomeric excess in favor of L-alanine in the presence of sodium chloride or in favor of D-alanine in its absence, even locally both spatially and temporarily. Prebiotic synthesis of amino acid molecules might have almost completely been racemic because of the random nature of energy sources available effectively at high temperatures. Nonetheless, enantioselectivity of L-alanine in racemization while experiencing temperature gradient in the presence of sodium chloride could have served as an evolutionary screen for enhancing the relative population of L-alanine in prebiotic or protobiotic organizations insofar as the environments in the vicinity of robust hydrothermal vents were persistently maintained on the sea floor in the ocean.

6.

Concluding Remarks The difference of the rates of transition from an L- to D-amino acid and its reverse in thermal equilibrium is almost insignificant because of the absence of energetic difference between the enantiomeric isomers. Only an extremely weak asymmetric influence of physical origin such as parity-violating jS -decay, electroweak force mediated by a neutral boson Z and magnetic field from a rotating neutron star [13] could account for the difference of the rates of racemization internal to the enantiomeric isomers. If so, it would be required to justify how such an extremely small difference of the rates of racemization imputed to parityviolating physical processes, if any, could be amplified up to the detectable level as in our observation with use of the flow reactor simulating a hydrothermal circulation of seawater through hot vents. A rationale for the enormous enhancement of the difference of the apparent rates of racemization between different enantiomers rests upon the rapid quenching of the reactants across temperature gradient [14]. When a tiny solution droplet including the reactants suddenly happens to suffer its temperature drop as traversing the interface separating between the hot and the cold region, the droplet would actualize only the quickest temperature drop out of possible alternatives. In this temperature drop, the reactants in the droplet would come to transform themselves so as to accommodate them to the sudden temperature drop outside. In fact, the transformation facilitating the quickest temperature drop comes to take over there

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since there is no room for the temperature to further drop once the quickest one has been in place. When amino acid molecules quickly traverse the narrow interface zone between hot and cold regions, there could be four possibilities of transformation and no-transformation such as L to L, L to D, D to L and D to D. Among those four possibilities, the alternative that may survive will be the ones dropping their temperature fastest while crossing the interface. Enantiomeric selectivity conceivable in the interface between hot and cold regions as expected in hydrothermal environments in the ocean is for those enantiomeric isomers substantiating the quickest temperature drop as traversing the interface from the hot to the cold, in spite of the fact that racemization carrying no such selectivity persistently would have proceeded outside the interface zone. In particular, L-alanine has been demonstrated to have less heat capacity compared with D-alanine [13, 15]). Less heat capacity means more rapid in quenching if other conditions are equal. This observation would come to suggest that Lalanine would be more favored in the hydrothermal environments in the presence of sharp temperature gradients. In summary, in a simulated hydrothermal environment allowing fluid circulation between hot and cold regions repeatedly, L- and D-alanine molecules were racemized differently depending upon the quantities of salt ions available there. When there were no added salt ions, the rate of racemization starting with pure L-alanine was slightly greater than that starting with pure D-alanine. When there were added salt ions, on the other hand, the rate of racemization starting with L-alanine was slightly smaller than that starting with D-alanine. Hydrothermal environments in the primitive ocean could have maintained the capacity of selectively retaining enantiomeric excess in favor of either L-amino acids or D-amino acids depending upon the quantities of salt ions available there.

7.

Acknowledgments Experimental work reported in this article has been done in collaboration with Minako Horie, Eiichi Imai, Hajime Honda and Kuniyuki Hatori.

8. [1] [21 |3] [4] [5] [6] [7] [8] [91 [10] [11] [12] [13] [14] [15]

References K. Kawamura and M. Yukioka, Thermochim. Acta 375 (2001) 9-16. J.L. Bada S.L. Miller and M. Zhao, Origins Life Evol. Biosphere 25 (1995) 111-118. M.J. Russell and A.J. Hall. J. Geol. Soc. London 154 (1997) 377-402. G.D. Cody, N.Z. Boctor, R.M. Hazen, J.A. Brandes, H.J. Morowitz and H.S. Yonder, Jr., Geochim. Cosmochim. Acta 65 (2001) 3557-3576. V.A. Basink, Ad\^ Space Res. 27 (2001) 335-340. K. Matsuno, Viva Origino 25 (1997) 191-204. E. Imai. H. Honda. K. Halori. A. Brack and K. Matsuno. Science 283 (1999) 831-833. A. Nemoto. E. Imai, H. Honda, K. Halori and K. Matsuno, Viva Origino 29 (2001) 168-173. A. Nemoto, M. Horie, E. Imai, H. Honda, K. Hatori and K. Matsuno, Origins Life Evol. Biosphere, in press. D.'W. Aswad, Anal. Biochem. 137(1984)405-409. JR. Cronin and S. Pizzarello, Science 275 (1997) 951-955. G.L.J.A. Rikken and E. Raupach. Nature 405 (2000) 932-935. W. Wang, F. Yi, Y. Ni, Z. Zhao, X. Jin and Y. J. Biol. Phys. 26 (2000) 51-65. K. Matsuno and R Swenson. BioSystenis 51 (1999) 53-61. R. Sullivan, M. Pyda, J. Pak. B. Wunderlich, J. R. Thompson, R. Pagi, H. Pan, P. Schwerdtfeger, and R. Compton,./. Phys. Chem. A 107 (2003) 6674-6680.

Progress in Biological Chiralit>' G. Palyi, C. Zucchi and L. Caglioti (Editors) © 2004 Elsevier Ltd. All rights reserved.

Chapter 17 The Theory of Chirality Induction and Chirality Reduction in Biomolecules Paul G. Mezey'^'' ""Albert Szent-Gyorgyi Professor, Department of Organic Chemistry, Eotvos Lordnd University of Budapest, H-1117 Budapest, Pdzmdny Peter Setdny 1/A, Hungary ^Canada Research Chair in Scientific Modelling and Simulation, Department of Chemistry and Department of Physics and Physical Oceanography, Memorial University of Newfoundland, Saint John % NF, CANADA A IB 3X7 [email protected], mezeypg@hotmailcom, [email protected]

1.

Introduction In this study two aspects of molecular chirality are discussed: chirality induction and chirality reduction. Both processes occur on both the local and global levels of molecules. Some of the fundamental interactions within molecules have the power to extend the chiral influence of any small region throughout the entire molecule, and the ability of these interactions to induce chirality in remote parts of the molecule have important practical consequences, especially in large molecules. Whereas much of the importance of the general relations are already evident in small molecules, as we shall demonstrate this in the next section, nevertheless, our main emphasis will be on the role of these relations in those chiral molecules which are perhaps of the greatest significance: chiral biomolecules. In this context, evolution will be one of the aspects often referred to, nevertheless, several of the conclusions we shall reach appear equally valid for organic as well as inorganic compounds. Whereas some of the discussions will rely on results obtained using somewhat special mathematical methods not routinely involved in biomolecular studies, here we shall not elaborate on the mathematical detail and the reader will be referred to the published literature for precise derivations. The fundamental principles of chirality induction and chirality reduction are very general and also universal: they provide constraints for practically all processes involving chiral molecules. Some of these constraints may be regarded as very natural, and are often taken for granted, yet they may also appear as being in conflict with some other, also natural expectations concerning biomolecules. In certain sense both chirality induction and chirality reduction may be regarded as natural trends, following either from overwhelming experimental evidence or from well-established natural laws, but the two opposing processes represent a conflict that appears to have essential connections to life processes. As a consequence of the wide scope of phenomena involving chirality induction and chirality

210


reduction, the examples include applications of the principles to parts of small molecules, to building blocks of larger biomolecules, and also to complex molecular systems involved in living organisms. Specifically, we shall discuss the following topics: (i) chirality induction in potentially achiral parts of electron density clouds within chiral molecules, and the theoretical basis of chirality reduction accompanying molecular decompositions; (ii) chirality induction by selection mechanisms and the combinatorial under-utilization of choices in the building processes of large biomolecules, influenced by type (i) of chirality induction; (iii) chirality induction and chirality reduction as a simple analogy for the entropy balance of living organisms, and the chirality induction and reduction processes of complex systems of biomolecules.

2. Some Fundamental Relations Governing Chirality Induction and Chirality Reduction in Molecules In the molecular context, both chirality induction and chirality reduction are processes involving interactions between various parts of molecules. It is of some interest to investigate, how large parts are involved in such interactions, and how the chirality of some molecular part is detected and processed by another molecular part, leading either to chirality induction or to chirality reduction (or, possibly, to no net change of chirality) there. Ahhough in the traditional viewpoint molecular chirality is often considered to require some minimal molecular size, nevertheless, in reality, even single atomic neighbourhoods, or even smaller, but positive volume parts of the electron density cloud may be chiral. Using the old concept of chiral carbon centers of organic chemistry, at least four different substituents about some carbon nucleus have been regarded as necessary for that type of chirality, that, of course, implied at least four nuclei for that type of chiral entity. As molecular modelling has become more refined and detailed electron density modelling and reliable quantum chemistry calculations have become more widely used, the focus has been narrowed and it has become increasingly possible and useful to consider the local features of chiral electron density clouds on a much smaller scale. Evidently, these clouds reflect all the chirality aspects of molecules, and even small parts of this cloud show evidently chiral features in any chiral molecule. Based on these developments, one natural question has been the following, how accurately can such local electron density ranges represent the chirality features of complete molecules, or at least, of those dominant parts of molecules which are primarily responsible for chiral, interactions with other molecules, or with polarized light in chemical or physical experiments. A simple answer can be given to the above question, relying on a general result of molecular physics: any small, positive volume part of the electron density cloud fiilly represents all chirality features of the entire molecule. (Strictly speaking, this statement has been rigorously proven only for molecules in their non-degenerate ground electronic states). The fundamental theorem that implies the above result for chirality underscores the importance of molecular electron density as the primary information carrier in molecules, and this includes chirality information. The origin of this fundamental theorem is the well-known foundation of density functional theory, a resuh on electron densities as established by the Hohenberg-Kohn Theorem [1], stating that for the non-degenerate electronic ground state of any quantum system of

The Theory of Chirality Induction and Chirality Reduction in Biomolecules

211

electrons, such as the electron density cloud of a molecule, the energy is a unique functional of the electron density. Since through the Hamiltonian operator of energy all other properties are also determined, it follows that for any molecule of a non-degenerate electronic ground state all properties are determined by the electron density. In fact, this result of the Hohenberg-Kohn Theorem is not surprising, since a molecule contains no other "material" than the atomic nuclei and the electron density cloud, and knowing that the nuclear distribution is fully reflected in the distribution of the electron density cloud, the latter must in fact determine all properties of the molecule. If the molecular electron density clouds were confined to a finite volume, that is, if molecular electron density clouds would be of finite size and they would have boundaries (what they actually cannot have), then an early result of J. Riess and W. Miinch [2] implies that local electron densities can be extended in a unique way to this finite and bounded density cloud. In a more recent development, both of these results have been extended to apply to local ranges of electron density clouds of actual, boundaryless electron densities of real molecules. This more recent result, as established by the proof of the Holographic Electron Density Theorem[3], states that the local electron densities of molecules are already sufficient to have a fully deterministic role in determining all molecular properties: any nonzero volume part of a molecular electron density in a non-degenerate ground state contains the complete information about all properties of the entire, boundaryless molecule. The scope of the Holographic Electron Density Theorem extends to both the exhibited and the latent properties [4] of molecules. In particular, various consequences [4] of this theorem provide new tools for Molecular Informatics and for the prediction of a whole range of latent molecular properties, such as those exhibited in different circumstances, in alternative conformations or in excited electronic states. The Holographic Electron Density Theorem also provide the justification and the tools for local molecular shape analysis as employed in shape activity correlations, where the activity often involves the complete molecules, or molecular parts not well identified. According to the "Holographic Electron Density Theorem" it is meaningful to use such local shape evaluations in correlations with observed molecular properties even if the actual mechanism and details of the molecular effect is not well understood, and if one cannot safely assign the observed property to any local range of the molecule. Recent reviews of various aspects of molecular shape analysis can be found in references [5-11], whereas the original developments and early applications, some in fact starting with knot-algebraic description of molecular chirality [12], can be found in references [12-42]. In fact, the methods originally developed for the electron density shape analysis of complete molecules [13-17] are now applicable to local molecular moieties [43-51]. Another development has coincided with the introduction of the above local shape analysis approaches: it has become possible to compute ab initio quality electron densities [53, 54] and other properties [55], such as electrostatic potentials [56], molecular energies [57], and forces acting upon individual nuclei [54] for truly large molecules, such as proteins of well over thousand atoms. This has been achieved using a linear-scaling algorithm (one that has computer time requirements directly proportional with molecular size, as opposed to conventional ab initio methods where the computer time requirement grows with the fourth power of the number of electrons). This method, the Adjustable Density Matrix Assembler (ADMA) method [53, 54], based on the Additive Fuzzy Density Fragmentafion (AFDF) approach, also provides efficient tools for the study of local ranges of macromolecules.

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Whereas an early, numerical density representation approach was suitable only for macromolecular density computations [58-63], the more advanced ADMA approach [53, 54] provides the tools for the computation of many more macromolecular properties [53-57]. Ab initio quality quantum chemistry computations of macromolecular conformation changes, computational studies of forces acting on individual nuclei within a protein, the analysis of latent properties, detailed local shape analysis of complex macromolecules, including macromolecular shape - activity correlations in large biochemical systems, specifically, the study of chiral shape features of biomolecules, both on the global and on the local levels are now possible using the linear-scaling ADMA method and the additive fuzzy density fragmentation (AFDF) approach applied to large systems [53, 54].

3.

Chirality Measures as Symmetry Deficiency Measures In order to be able to quantify chirality induction and chirality reduction in the context of individual molecules, it is advantageous to be able to say whether chirality has increased or decreased in a given process. A natural approach is to use some type of measure for the degree of chirality. Whereas several such chirality measures have been proposed, here we shall focus on one type of measure, that is based on the concept of symmetry deficiency. It is clear that in the three-dimensional context, chirality is a manifestation of a special type of symmetry deficiency: the lack of mirror planes and of improper rotation axes of the S(2n) type. It is of special significance that the definition of chirality, in terms of nonsuperimposability of mirror images, is in fact equivalent to the definition relying on the lack of these two types of symmetry elements in three dimensions. Nevertheless, we may formulate the particular chirality measure in the more general context of symmetry deficiency, that is, in fact, applicable to any symmetry element R, not just those of mirror planes and of improper rotation axes of the S(2n) type, whose lack implies chirality. For simplicity in describing the concepts, first we shall discuss classical, macroscopic objects instead of quantum mechanical molecules. We say that a set D is an R-set if this set D has the symmetry element R, that is, if by carrying out the symmetry operation corresponding to the symmetry element R, the object D is transformed into an object that is indistinguishable from the original object D. Consider a three-dimensional rigid object A with a well-defined boundary and a particular symmetry element R that either is or is not present for the given object A. Consider all the R-sets which are contained within the object A, and also all R-sets which contain the object A. Since the classical, geometrical object A has a well-defined volume V(A), as well, all the above R-sets also have well-defined volumes, there must exist at least one set B that is a maximum volume R-subset of set A, as well as at least one set C that is a minimum volume R-superset of set A. Note that neither the maximum R-subset B nor the minimum R-superset C needs to be unique for a given set A, nevertheless, their volumes V(B) and V(C) are both unique, well-defined values for any given set A. These three volumes, V(A), V(B), and V(C) serve as the basis of two symmetry deficiency measures. The internal symmetry deficiency measure I(A,R) of set A with respect to symmetry element R is defined as: I(A,R) = (V(A)-V(B))A^(A), (1)


213

whereas the external symmetry deficiency measure E(A,R) of set A with respect to symmetry element R is defined as: E(A,R) = (V(C)-V(A))/V(A).

(2)

Note that these quantities I(A,R) and E(A,R) are scaled by the volume V( A) of set A, that provides a natural normalization appropriate for symmetry deficiency measures. Clearly, if the object A actually has the symmetry element R, then both sets B and C are equal to set A, A = B = C,

(3)

hence both the internal and the external symmetry deficiency measures of set A with respect to symmetry element R give zero, I(A,R) = E(A,R) = 0,

(4)

that is, no R-symmetry deficiency occurs for object A. On the other hand, if sets A, B, and C are different, then (with the exception of degenerate, zero volume differences, such as that occurs when having a line protruding from set A that without this line would have the symmetry element R, where this line, of course, is not present in maximum R-subset B) the symmetry deficiencies of set A with respect to symmetry element R will have some positive values. Note that the internal and external symmetry deficiency measures are typically (but not necessarily) different for an R-symmetry deficient object A. The internal symmetry deficiency measure I(A,R) expresses how much of the volume of A must be taken away in order to achieve symmetry R, whereas the external symmetry deficiency measure E(A,R) expresses how much additional volume must be added to the object A in order to obtain an object that already has the symmetry element R. Here we assume that the volumes are scaled so that the volume V(A) of object A is considered as the reference. Consider now a molecule M and a particular symmetry element R that either is or is not present for the given molecule M. Since molecules are not classical objects with precisely defined boundary and volume, the simplest application of the above method can be achieved if one considers isodensity contour surfaces of the molecule, in order to provide a representation analogous to a macroscopic body with boundary surface and volume. For example, an isodensity contour surface A = A(M,0.01)

(5)

of molecule M, taken at the density value of 0.01 a.u. (atomic units) provides a suitable representation. Such isodensity contours do have volumes V(A), hence the above described approach is, indeed, applicable. Note that a more general approach, involving pointwise density differences and an integral measure extending over the whole space can also be used, effectively replacing the classical volume concept v^th a quantum mechanically more appropriate integral measure. For some alternative approaches, the reader may find information in references [64, 65], and for there broader context of chirality manifestations.

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ultimately related to stmctural concepts, in references [66, 67]. If one is to consider several symmetry deficiencies simultaneously, one may generate new measures. Take, for example, a family F of symmetry elements, F = R(l),R(2),...R(k),

(6)

and evaluate the internal and external symmetry deficiency measures for the given object A according to each of these symmetry elements: I(A,R(1)), I(A,R(2)),

I(A,R(k)),

(7)

and E(A,R(1)), E(A,R(2)),

E(A,R(k)).

(8)

One can then compute, for example, the minimal, maximal, and average symmetry deficiency measures of object A with respect to the entire family F of symmetry elements: Imin(A,F) = min { I(A,R(1)), I(A,R(2)),

I(A,R(k)) },

Imax(A,F) = max { I(A,R(1)), I(A,R(2)), Iav(A,F) = [ I(A,R(1)) + I(A,R(2)) +

(9)

I(A,R(k))},

(10)

+ I(A,R(k)) ] / k,

(11)

Emin(A,F) = min { E(A,R(1)), E(A,R(2)), Emax(A,F) = max { E(A,R(1)), E(A,R(2)),

E(A,R(k)) },

(12)

E(A,R(k))},

(13)

+ E(A,R(k)) ] / k .

(14)

and Eav(A,F) = [ E(A,R(1)) + E(A,R(2)) +

Depending on what aspect of symmetry deficiency is of relevance, one may choose the appropriate symmetry deficiency measure from the alternatives (9) - (14). In the case of chirality, considered as symmetry deficiency of all mirror planes and all improper rotations of the S2n type, the family F of symmetry elements considered is precisely the set of all mirror planes and all improper rotations of the S2n type. According to our choice, the molecular object considered is the molecular isodensity contour A. This object A is achiral if any one of the symmetry elements from the family F is present. On the other hand, fi-om a given chiral isodensity surface A one may obtain an achiral subset or achiral superset the simplest way, that is, by the smallest volume change, if the symmetry element R(i) of the least actual symmetry deficiency measure is considered, either by removing or adding the smallest necessary volume. Consequently, for a chirality measure, taken as an "achirality deficiency measure", it is natural to consider the two minimal measures from the choices (9) - (14), that is, the measures Imin(A,F) and Emin(A,F).


215

In terms of these symmetry family deficiency measures, the internal chirality measure Ic(M,0.01) and external chirality measure Ec(M,0.01) of molecule M, as represented by the isodensity contour surface at the density value of 0.01 a.u. are defined as Ic(M,0.01)-Imin(A,FX

(15)

Ec(M,0.01) = Emin(A,F),

(16)

and where F is the family of all mirror planes and of all improper rotations of the S2n type. These chirality measures are numerical expressions of "how much" relative volume change is required as a minimum, in order to obtain a subset or a superset that is already achiral. It is of some interest to pinpoint which actual "achirality inducing" symmetry element R is the one that is requiring the smallest volume change for the object in order to turn it into an achiral object.

4.

Chirality Induction and Chirality Reduction within Individual Molecules Whereas chirality induction and chirality reduction are perhaps of the greatest significance in the context of biochemistry and biology, a similar phenomenon of chirality induction and chirality reduction also exists on a smaller, non-biological scale within a given molecule. As a consequence of the interactions of local ranges of electron density clouds within a molecule, in a chiral molecule all, potentially achiral fiinctional groups or other groups of atoms are also chiral. A rigorous proof has been given earlier, asserting that local-only achirality cannot exist in any molecule: for example, a local mirror plane of a methyl group may exist only, if the entire molecule has a mirror plane: in fact, the entire molecule must have the same mirror plane. One may view this as chirality induction: if the molecule is chiral, that induces chirality also in such potentially achiral functional groups as a methyl group. On the other hand, if the molecule is decomposed, and the methyl group becomes an independent, non-interacting CH3 radical, then it becomes achiral. Hence, reduction of complexity, the decomposition of the original molecule may be accompanied by chirality reduction.

5.

Chirality Induction and Chirality Reduction in Biomolecules The structure, stability, function, and versatility of biomolecules are the resuh of molecular evolution. All of these features are influenced by various contributions to the chirality of biomolecules and are often fine tuned (but sometimes only roughly tuned) by evolution. Evolution is an opportunistic process where Nature usually takes advantage of variety, and make selections from a pool of all of the options available. However, the high dominance of homochirality in biomolecules implies that, somewhat uncharacteristically, evolution did not find advantageous to exploit the very high degree of combinatorial variability offered by mixing, for example, L- and D- amino acids in the largest family of biomolecules where such amino acids occur: peptides and proteins. There exists a very high degree of combinatorial under-utilization of choices. Apparently, structural regularities, that is, limitations to variety offered by homochiral amino acids when combined into larger molecular structures, have been more important in the course of evolution than the opportunities offered by the astronomical variety of potential peptide-like molecules generated fi-om a pool of non-

216


restricted combinations of L- and D- forms. This fact lends support to the idea that already in early evolution, the generation of complex systems necessary for life benefited from a rather general molecule-building mechanism that has relied on homochirality. Such a mechanism, in effect, provided chirality induction, by enhancing the overall chirality of a biomolecule built from chiral units of a given homochirality class. As the complexity of life forms have increased during evolution, this chirality induction process has become more dominant. Evolution of life has been accompanied by chirality induction. A similar chirality induction process accompanies the development of an individual multicell organism from a single cell to a complex, full-grown individual. On the other hand, death of an organism involves chirality reduction, since many of the ultimate decomposition products of biomolecules are small molecules, such as CO2 and N2, that are achiral. Of course, many chiral biomolecules become re-used by various life forms without decomposition to such small molecules, nevertheless, a significant amount of biomolecules lead to achiral decomposition products after the death of an individual organism. Death is usually accompanied by chirality reduction. As Professor Albert Szent-Gyorgyi has often stated, "life needs energy to fight entropy". The entropy of a live organism is lower than the system the same set of atoms will have after the death of the organism; in this sense an unusually low entropy is one characteristic of life. With respect to entropy, any (possibly inorganic) process that selectively favors homochiral arrangements also shows a reduction relative to the potential randomness when other, nonhomochiral building blocks are available. In this context, homochiral selectivity, even if demonstrated by some inorganic mechanism, may be regarded as a primitive model for life. In addition, many of the ultimate product molecules of metabolism generated by the normal life processes of a healthy individual are also achiral. Much of the intake of most terrestrial life forms is water, an achiral molecule, that predominantly plays the role of solvent, and much of this water is secreted without changing achirality. However, for most other molecules the situation is different. For many animals, metabolism involves the intake of chiral molecules and the uhimate generation of achiral ones, notably CO2. Whereas for many plants some important molecules in the intake are achiral, (again, one may think of CO2), that are used to build chiral biomolecules, nevertheless, the ultimate decomposition product of dead plant, if left to itself without biological utilization by other organisms, are also achiral. In analogy with Szent-Gyorgyi's statement, life needs mechanisms for chirality enhancement to fight achiral death. Within the context of chirality and life on Earth, we may think of life as chirality induction and death as chirality reduction.

6.

Acknowledgement The financial support of NSERC of Canada and the Albert Szent-Gyorgyi Award of Hungary are gratefully acknowledged.

7.

References

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The Theory of Chirality Induction and Chiralily Reduction in Biomolecules

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[31] G. A. Arteca and P.G. Mezey, Similarities between the efFects of configurational changes and applied electric fields on the shape of electron densities. J. Moi Struct. Theochem 256 (1992) 125-134 [special issue: Electrostatics in Molecules (Ed. G. Naray-Szabo and W.J. Orwille Thomas)]. [32] P.G. Mezey, Shape-similarity measures for molecular bodies: a 3D topological approach to QShAR. J. Chem. Inf. Comp. Sci. 32 (1992) 650-656. [33] P.G. Mezey, Dynamic shape analysis of biomolccules using topological shape codes. In: The Role of Computational Models and Theories in Biotechnology (Ed. J. Bertran) BUuwer Academic Publishers, Dordrecht, 1992, pp. 83-104. [34] P.G. Mezey, Dynamic Shape analysis of molecules in restricted domains of a configuration space. J. Math. Chem. 13 (1993) 59-70. [35] P.G. Mezey, Topological shape analysis of chain molecules: an application of the GSTE principle. J. Math. Chem. 12 (1993) 365-373. [36] X. Luo, G. A. Arteca and P.G. Mezey. Shape similarity and shape stability along reaction paths. The case of the ppo to opp isomerization,//?/. J. Quantum Chem. 42 {1992) 459-414. [37] P.D. Walker, G.A. Arteca and P.G. Mezey, Shape groups of the electronic isodensity surfaces of small molecules: the shapes of 10-electron hydrides. J. Comput. Chem. 14 (1993) 1172-1183. [38] P.G. Mezey, Methods of Molecular shape-similarity analysis and topological shape desiga In: Molecular Similarity in Drug Design (Ed. P.M. Dean) Chapman & Hall - Blackie Publishers, Glasgow, U.K., 1995, pp. 241-268. [39] P.G. Mezey, Molecular similarity measures for assessing reactivity. In: Molecular Similarity and Reactivity: From Quantum Chemical to Phenomenological Approaches (Ed. R. Carbo) Kluwer Academic Publ., Dordrecht The Netherlands, 1995, pp.57-76. [40] P.G. Mezey, Dynamic Shape group theory' of molecular nuclear potentials. In: Chemical Group Theory (Eds. D. Bonchev and D.H. Rouvray) Gordon and Breach Publ. Group, Reading, U.K., 1995, pp. 163-189. [41] P.G. Mezey, Semi-similarity of molecular bodies: scaling-nesting similarity measures. Int J. Quantum CAe'w. 51(1994)255-264. [42] P.G. Mezey, Quantum chemical shape: new density domain relations for the topology of molecular bodies, functional groups, and chemical bonding. Can. J. Chem. 72 (1994) 928-935 (Special issue dedicated to Prof J.C. Polanyi). [43] P.G. Mezey, Local shape analysis of macromolecular electron densities. In: Computational Chemistry: Reviews and Current Trends. Vol.1 (Ed. J. Leszczynski) Worid Scientific Publ., Singapore, 1996, pp. 109137. [44] P.G. Mezey, Functional groups in quantum chemistry. Adv. Quantum Chemistry 27 (19%) 163-222. [45] P.G. Mezey, Local electron densities andftmctionalgroups in quantimi chemistry. In: Topics in Current Chemistry, Vol. 203 "Correlationand Localization" (Ed. PR. Surjan) Springer-Veriag, Berlin, Heidelberg, New York, 1999, pp. 167-186. [46] P.G. Mezey, Holographic electron density shape theorem and its role in drug design and toxicological risk assessment. J. Chem. Inf Comp Sci. 39 (1999) 224-230. [47] P.G. Mezey, Combinatorial aspects of biomolecular shape analysis. Bolyai Soc. Math. Stud. 1 (1999) 323332. [48] P.G. Mezey, K. Fukui and S. Arimoto, A treatment of small deformations of polyhedral shapes of ftmctional group distributions in biomolccules. Int J. Quant Chem. 76 (2000) 756-761. [49] P.G. Mezey, Topological methods of molecular shape analysis: continuum models and discretization, DIMACS Series in Discrete Mathematics and Theoretical Computer Science 51 (2000) 267-278. [50] P.G. Mezey, Shape-similarity relations based on topological resolution. J. Math. Chem. 27 (2000) 61-69. [51] P.G. Mezey, Topological similarity of molecules and the consequences of the holographic electron density theorem, an extension of the Hohenberg-Kohn theorem. In: Fundamentals ofMolecular Similarity, (Eds. R. Carbo-Dorca, X. Girones and P.G. Mezey) Kluwer Academic/Plenum, New York, 2001, pp. 113-124. [52] P.G. Mezey, Theor>' and detailed computer modelling of biomolecules. In: Fundamentals o/(Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionalc di Scienze, Lettere ed Arti (Modcna), Paris, 2002, pp. 401-416. [53] P.G. Mezey, Macromolecular density matrices and electron densities with adjustable nuclear geometries. J. Math. Chem. 18 (1995) 141-168. [54] P.G. Mezey, Quantum similarity measures and Lowdin's transform for approximate density matrices and macromolecular forces. Inl J. Quantum Chem. 63 (1997) 39-48.


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Chapter 18 Transfer of the Chiral Information of Natural Amino Acids in Biomimetic Organic Syntheses Karoly Micskei,^* Csongor Hajdu,'*'' Tamas Patonay,'' Claudia Zucchi/ Luciano Caglioti^ and Gyula Palyi"* ^Department of Inorganic and Analytical Chemistry, ^Department of Organic Chemistry University of Debrecen, Egyetem ter I, H-4010 Debrecen, Hungary kmicskei@delfin. kite, hu ^Department of Chemistry, University ofModena and Reggio Emilia, Via Campi 183,1-41100 Modena, Italy [email protected] '^Department of Chemistry and Technology of Biologically Active Compounds, University "La Sapienza*', P.leA. Moro 5,1-00I85, Roma, Italy

1.

Introduction Biological processes are very important sources of organic compounds. These procedures called biosyntheses [1] are typically characterized by a very high level of chemo-, regio-, diastereo- and enantioselectivity. Biosynthetic processes are operating in aqueous medium and in narrow temperature and pH ranges. Their key elements are enzymes (biocatalysts) which operate using a vast variety of "chemical tools" for achieving an exceptional level of efficiency. This capability is a result of a very fme, concerted tuning determined by several factors such as hydrophilic/hydrophobic groups, macromolecular properties, complexed metal ions or chiral structural elements, H-bonds, TC-TT stackings, electrostatic interactions. The formation of a highly organized "chiral space" is considered essential for the exceptional level of enantioselectivity. Today quite much is known about these processes and about their structural requirements but we are only at the beginning of understanding biosyntheses. However, on the basis of the available information and experience biotechnological industry produces many organic molecules, some of which are practically unavailable through other methods. These "other methods" are the organic chemical syntheses [2] which were developed starting from observation and imitation of biological processes. But then the stereoselective syntheses took an independent way of evolution utilizing conditions which are very far from the biological ones. Such conditions are the use of non-aqueous, often strictly anhydrous solvents, strong acidic (protonating) or basic reagents, inert atmosphere, much wider temperature ranges than the biological conditions — to list only a few of them. These conditions allow several reactions impossible by biosyntheses but the transformations are characterized by lower yields and selectivities than those typical for biological processes. One

222 Progress in Biological Chiralib of the major successes of synthetic organic chemistry in the last decades is the use of transition metal containing catalysts [3]. These derivatives provided nice tools to avoid drastic reaction conditions by lowering the activation energy barriers dramatically and, consequently, making possible to obtain substantially higher selectivities. Prominent achievements in this field were obtained in the enantioselective catalytic synthesis of chiral, non-racemic organic compounds by the use of transition metal complexes of chiral organic ligands, recognized also by the Nobel Prize in Chemistry in 2001 [4]. Another outstanding result in this field is the discovery of chiral autocatalysis [5], which is also the topic of Chapter 29 of this book. Comparison of the biochemical discoveries with the most recent resuhs of synthetic organic chemistry prompted several groups to initiate a new direction in synthetic chemistry by amalgamating these two fields. This trend led to the development of so-called biomimetic chemistry [6] characterized by searching and finding synthetic methods which are using experience from biochemistry as much as possible and similar experimental conditions (aqueous media, near-neutral pH, more or less ambient temperature, atmospheric pressure, etc.), as well. Our groups in the last decade joined this trend by investigating the chemistry of transition metal complex reagents containing typical "bio-organic" molecules (mostly amino acids) as ligands and which could be used under the near-biological conditions listed above. We summarize these resuhs in the present paper, discussing also the possibilities of future developments.

2.

Precedents Previous studies have shown that various Cr(Il) complexes with aminopolycarboxylate ligands (e.g. IDA, NTA, EDTA, etc.) can be used as highly selective reagents for the (reductive) transformation of natural organic substances (monosaccharides, morphine alkaloids) [7, 8]. Later, these reagents were found to be useful in a modified variant [9] of the Nozaki-Hiyama reaction [10]. This variant allowed the formation of carbon-carbon bond in neutral aqueous medium. A similar reagent was also used for the generation of ferrocenylketyl radicals [11] with unusual structure and stability. These results prompted us to focus on the use of natural amino acids as ligands in the Cr(II) complex reagents [12]. Natural amino acids are one of the most abundant and, therefore, usually relatively cheap natural sources of chirality. However, in spite of these attractive features L-amino acids themselves are only rarely used as chiral ligands in transition metal complex-catalyzed or -assisted enantioselective transformations; in this field the best resuhs were obtained by using non-natural amino acids [13] which are much more expensive [14] or commercially not available. On the other hand, naturally occurring amino acids play an important role in the stereoselective synthesis in the form of their derivatives such as 1,2-amino alcohols, bisisoxazolines, oxazaborolidines, etc. [13, 15]. We believe, the major obstacle for the use of amino acids in their original form is that they and their complexes are only sparingly soluble in most of organic solvents (used generally as reaction medium). In aqueous media, however, most natural amino acids are nicely soluble and form many — well characterized — complexes with transition metal [16]. One of the most studied metal ion is chromium in its various oxidation states. Although chromium(II) is a broadly used reducing reagent in organic chemistry [17] nothing has been published on the utilization of the amino acid complexes in the synthetic organic chemistry.

Transfer of the Chiral Information of Natural Amino Acids in Biomimetic Organic Syntheses

3.

223

Amino Acid Complex Reagents in Aqueous Media In their complexes, natural amino acids are typically coordinated through the a-amino and the carboxyl(ate) groups as bidentate ligands forming stable five-membered rings. The stability, structure, reactivity and solubility of these complexes is influenced in a fairly broad range by the structure of the a-substituent ("side chain"). Several classifications based on the structure of these side chains has been done which are widely used in biochemistry to interpret (or predict) the behaviour of amino acids, as well as their oligo- and polycondensates. One of the most important features is the polarity of the side chain [18]. The following natural amino acids can be regarded typically apolar: Ala, Val, Leu, He, Pro, Phe, Tyr, Trp, Cys, Met. These representatives are characterized by low solubility and micelle-formation in water. The members of other group (Lys, Arg, His, Asp, Glu, Asn and Gin) bear polar, mostly dissociable groups in their side chain which assures a good solvation in water and (consequently) higher solubility. In spite of the fact that the majority (14) of the natural amino acids possesses groups with typical ligand properties in the side chain, in aqueous solution these are only weakly or not coordinated. Only three natural amino acids can be regarded as tridentate (His, Asp, and Cys) [16]. All natural amino acids with the exception of Gly contain at least one sterogenic center (the a-carbon atom) and belong to the L-series in an overwhelming majority of cases [19]. The structure of the side chains widely influences the applicability of their complexes as synthetic reagents. Until now, mostly reagents from amino acids with apolar side chains were studied [13, 15c] predominantly because of their solubilities in organic solvents. In aqueous media the use of non-derivatized amino acids (as inductors of chirality) is much less common [20, 21]. Recently, some uses of natural amino acids as sources of chirality in the enantioselective synthesis of carbon-carbon bonds were reported [22, 23]. The formation of amino acid complexes 1, 2 is accompanied by a fairly complicated network of equilibria (Figure 1), In aqueous solutions amino acids 4 undergo

R-CH

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Figure 1. Solution equilibria of the transition metal ion - amino acid complexes in aqueous medium

224

Progress in Biological Chirality [Cr2+]^Q^ = 1.0

10.00 m M

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=

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Figure 2. Formation of chromium(II)-L-alamne-complexes in aqueous medium

deprotonation/reprotonation processes (Eq. 1), resulting charged and zwitterionic species 3, 6 and 5, respectively. The deprotonated form 3 can react with the metal ion as a bidentate ligand, resulting a 1:1 complex 1 (Eq. 2). Then, the next ligand 3 can easily be attached to the metal forming a 1:2 complex 2 (Eq. 3). Under aqueous and basic conditions hydroxide ions as ligands can be coordinated (parallel or concurrently) to the metal (Eq. 4). Noteworthy, that deprotonation is crucial for the coordination since the carboxyl group itself gets much weaker ligated. All these aspects should be quantitatively considered in the planning of the reagent. Detailed knowledge of the equilibrium constants [24] and production of suitable computer programs [25] are crucial. The distribution of various complexes in the aqueous Cr(II)/Ala system are shown as an example in Figure 2, diagrams of other complexes are of similar shape. The most important practical conclusion from these studies is that the Cr(II)/amino acid reagents should be very carefully planned and prepared otherwise the composition of these systems cannot be controlled. The narrow operative ranges (as well as the fact that these ranges appear just between pH-5 and -9) and the sensitivity towards changes in the experimental conditions are very similar to the behaviour of enzymatic systems.

Chirality Transfer of Natural Amino Acids 4,1 Reduction ofC=0 double bond At the beginning of our joint research efforts we investigated whether the chirality of natural amino acids coordinated to Cr(n) could be transferred to simple organic compounds obtained by reduction with Cr(II) from prochiral precursors. Acetophenone (8) was chosen as a model substrate [26] (Scheme 1). Indeed, it has been found that treatment of ketone 8 with solutions of Cr(II)/amino acid 1:2


O

HO H CH3

225

H OH

Cr(ll)L* (2 equiv.) H2O/DMF. pH = 6-9

R-9

S-9 L* = natural L-amino acid Scheme 1

complexes (2, M = Cr) afforded 1-phenylethanol (9) with >95% conversion. The analysis of the enantiomeric ratio of the product 9 showed that the chiral information [27] of the ligand amino acids appeared in the product. The transfer of the chiral information resulted in different enantioselectivities and different absolute configuration of the major enantiomer depending on the structure of the amino acid used (Figure 3). The highest enantiomeric excess (75% e.e.) was obtained with Cr(L-Val)2 complex at pH-'9. While bidentate L-amino acids such as Ala, Pro, Asn yielded excesses of the R-9, typical tridentate L-amino acids (Asp, His) induced S preference. In accordance with the pH optimum in the formation of Cr(L-Ala)2 the increase of pH from 6-7 to 9 resulted in a slight increase of e.e. values. A few control experiments showed that: (i) without amino acid ligand there is no detectable reduction of 8, (ii) with achiral ligands (EDTA) there is no detectable enantiomeric excess of 9, and (iii) corresponding D-isomers of the amino acid ligands induced the formation of the enantioselective reduction of a broad scale of various ketones, with excellent chemical yields (Ala, His, Asp) and low-to-moderate e.e. (5-55%). Again, both enantiomers could be achieved by using natural L-amino acids. The structure

t

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226

Progress in Biological Chirality o

o

a^ 10

14

15

of the amino acid ligand has a decisive influence on the absolute configuration of the product. Bidentate ligands usually furnished S preference ahhough in the case of Leu both S and R enantiomers could be obtained depending on the structure of the substrate. Similarly, the tridentate ligand His gave S enantiomers as the predominant isomer for all substrates but in the case of Asp R enantiomers were the major products in the reduction of cyclic substrates 16,18,19. The remaining starting ketones afforded S preference, again. Our data revealed that the enantioselectivity of the reduction was also influenced by the substrate which could be divided into two classes on the basis of the conformational properties. In the series of alkyl aryl ketones 8-14 and the conformationally more flexible benzosuberone (17) His provided the best e.e.'s but it gave poor enantioselectivity in the reduction of the more rigid benzocyclanones 16, 18, 19. The reverse effect was observed for Ala. These observations clearly show that the structure of the starting ketone also influences the

ketones

Figure 4. Enantioselectivity of the Cr(II)/Z-amino acid/H20/DMF system in ketone reduction


227

Stereochemical outcome of the asymmetric reduction Optimization experiments with chromanone (18) supported the important role of pH, e.e.'s and conversions slowly decreased in the range of pH 10 and 8 followed by a sharp breakdown between 7 and 8. Solvent dependence has also been found. The best yields and enantioselectivities were found using DMF and formamide/water mixtures, other donor solvents gave poorer results. These differences could be interpreted in terms of the structure and reducing potential of the Cr(II)/amino acid complexes. Consequently, the efficiency of our system can be highly tuned by the modification of the composition of the reducing species and of the conditions used. Analysis of the preparative results, the study of the model system [11], spectroscopic (UVVIS and CD) studies of the reaction mixtures [29] as well as comparison of these results with some relevant literature reports [30, 31] allowed us to deduce a hypothesis (Scheme 3) about the possible steps of the reduction. Steps of this mechanism include the one-electron formation of a ketyl radical-anion (22) (Eq. 5), its (fast) protonation (Eq. 6), the reaction of the radical 23 with the chromium(II) complex 21 under the formation of an organochromium intermediate 24 (Eq. 7) and the subsequent proton-metal exchange (Eq. 8). The crucial step, where the evolvement of the chirality happens, is the formation of the intermediate 24. The discrimination leading to

)=0

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(5)

22 R^ > R (CIP priority order)

.0

H«

R (6)

-OH 23

22

)ÔH

+

Cr"(L*)n

SET

21

23

R.

Cr"'(L*)n

R'

OH

R"X Ri

H--Y-Cr'"(L*)n TS1

TS2

©

®

OH

OH

(7) OH

S-24

R{ R

H I

CrJ"(L*)n

R

R-24 inversion

retention

Rl.

I R..

OH

R'

H

R-25 Scheme 3

inversion

R\ R H---y--CH"{L*)n OH (8)

Tsr Rl.

OH

A R

H

S-25

228

Progress in Biological Chirality NOH ^CH3

NH

"

H2N H Cr(ll)L*(2equiv.)^ ^

Cr(ll)L*(2equiv.)

H2O/DMF, pH - 6 *

H2O/DMF, pH - 6 26

Y

"CH3

L S-28

27 hydrolysis

HO

O CH3

L* = natural L-amino acid '

Cr(ll)L*(2equiv.)^ | ^ H2O/DMF, pH - 6 *

8

Y ^ ^ ^ 3

U ^

RS-9

Scheme 4

different amounts of R- and S-24 products is probably governed by steric factors (different steric demand of R, R^ groups and the amino acid side chain) and internal hydrogen bonding between hydroxyl functionality of the intermediate 24 and the amino acid ligands. The enantiopurity of the R- and S-25 depends on the discriminating power of these interactions since it is retained in the proton-metal exchange step taking place with inversion. On the other hand, we can not exclude the attack of a proton coming from the inner ligand sphere. This step may take place with retention leading to the opposite enantiomer and, therefore, decrease the enantiomeric excess. 4.2 Reduction of C=N double bond The next step in our joint research was to attempt the reduction of C=N double bonds by the Cr(II)/amino acid reagents. For practical reasons oximes were chosen as substrates. No literature precedents of this reaction are known by us [32]. As our first model reaction the reduction of acetophenone oxime (26) was investigated. In these experiments [33] we found that Cr(II)/L-amino acid complexes could be used for the enantioselective reduction of the C=N double bond. The reaction could be performed with medium-to-excellent yields (50 - >90%) but only with low-to-moderate (--5 - 50%) enantiomeric excesses (Scheme 4, Figure 5).

ee% 20

amino acid Figure 5. Enantiomeric excesses (e.e.) of the reaction of acetophenone oxime (26) and Cr(n)/L-amino acid complexes

Transfer of the Chiral Information of Natural Amino Acids in Biomimetic Organic Syntheses 229 The reduction of oximes represents a more complicated problem as compared to that of the ketones (Scheme 4). Our studies have indicated that there is a four-electron reduction and a hydrolytic side reaction of the intermediate imine 27 yielding the corresponding ketone 1. The reduction of the by-product 8 leads to alcohol 9 as described earlier (Scheme 1). This complication can be (partly) avoided by using much higher (-7) Cr(n)/substrate ratio than applied in the case of the ketone. This modification allows to obtain 1-phenylethyl amine (28) in almost quantitative (>90%) yield. The pathway outhned above is in accordance with the observation of Corey and Richman [34] who reported the formation of the corresponding ketones from oxime 0-acetates upon treatment with Cr(II) acetate in aqueous THF, clearly by the hydrolysis of the imine intermediate formed in the first reduction step. Model experiments have shown that: (i) aqueous Cr(II) solution (containing presumably [CR(II)(H20)6]) does not give any reduction product, and (ii) Cr(II) ions in the presence of achiral aminopolycarboxylate ligands such as IDA, NT A, EDTA) do not give any enantioselectivity. This latter system, however, could be advantageously applied for the preparation of amines (as racemic mixtures) from the corresponding oximes 29-34 shown by Scheme 5 in moderate (9-55%) yields and poor (-3:2) diastereomeric (33, 34) ratios [35]. Experiments on the enantioselective reduction of these latter substrates are in progress. NOH

29

NOH

NOH

NOH

NOH

NOH

30

However, stereochemical outcome of the reactions with bi- and tridentate L-amino acids is not so clearly separated as in the ketone reduction. The highest e.e. (-50%) was observed with tridentate L-Asp (7^-28). At the same time, now L-Ala gave the same preferred absolute configuration in the product 28 but in the case of the similarly bidentate, somewhat more apolar L-Val a small but reproducible excess of S-28 was found. The rational analysis of the preparative results, the spectroscopic (UV-VIS and CD) studies on the analogous reduction of a-oximino carboxylic acids (vide infra) (Figure 6), as well as some relevant literature reports [31, 36, 37] seem to support the most important steps of our suggested mechanism shown in Scheme 6. The reaction course of oximes (Scheme 6) is clearly much more complicated than the previous one of ketones shown by Scheme 2, this fact is also reflected in the problems to achieve better chemical and enantiomeric yields, as well as in the different influence of bidentate v^. tridentate ligands on the absolute configuration, in comparison with ketones. The first steps of the mechanism (Eq. 9-11) results in the imine intermediate 38 by a reductive dehydroxylation. Again, the key element of the enantioselective reduction is the formation of the a-alkylchromium intermediate 40 (Eq. 13) which gives the final product 41 in a hydrolytic step by inversion (Eq. 14). As before, we can not exclude the possibility of a retention. The UV-VIS/CD /spectra shown in Figure 6 indicate:

230


500

600

800

Wavelength (nm)

500

600 Wavatongth (nm)

Figure 6. (i) UV-VIS and (ii) CD spectra of (a) L-Ala; (b) Cr(n); (c) Crai)+L-Ala; (d) Cr(II)+L-Ala+a-oximino-phenylacetic acid in water solution

(i) an inner sphere coordination of the ligand amino acid prior to the addition of oxime which phenomenon is verified by the presence of low energy d-d bands, (ii) formation of the chiral organochromium intermediate 40 upon addition of oxime, and (iii) chiral perturbation of the central chromium ion in both cases as verified by the appearance of the low energy CD bands. The experience obtained in the asymmetric reduction of acetophenone oxime (26) prompted us to set ourselves a more ambitious aim, the enantioselective preparation of a-amino acids from achiral precursors using Cr(n)/L-amino acid complexes as reducing agent and source of chirality. This goal could be achieved [38] by reducing oximes of a-ketocarboxylic acids with Cr(II)/amino acid 1:2 complexes (42) in aqueous solution (or water/DMF mixture) in pH range 8.9-9.4. These reaction took place with:

Transfer of the Chiral Information of Natural Amino Acids in Biomimetic Organic Syntheses )=NOH +

Cr"(L*)n

35

SET

^ N *

+

CrJ"(L*)n + :0H^

231 (9)

36

21

R^ > R (CIP priority order) )=N*

+

CH'(L*)n

SET

(10)

21

36

)=N

37

+ H2O

>=NH

+

(11)

Cr"'(L*)n + :OH®

38

37 1.SET 38

2. H2O

)^NH2 R^^ 39

SET

R.

21

)^NH2

+

Cr"(L*)n

^

Cr'"(L*)n

R^

21

39

(12)

+

R1

Cr"'(L*)n

A

"^

NH2

R

R-40

NH2 S-40 inversion

inversion

retention

TS3

K >r"'(L*)n R---K p,i

NH2

TS4

R1 R H---y--CrJ"(L*)n

Ri R H--y:-CrV)n

NH2

H I

(13)

R

NH2

R^^H

R-41 Scheme 6

J

L

NH2 (14)

TS3'

R1 R

NH2 H

S-41

(i) nearly quantitative (>90%) chemical yields, and (ii) low-to-moderate (-5-50%) e.e. for such systems where (iii) the ligand amino acids were different from the products expected in the reduction of the substrate (Scheme 7). Transformation of oxime 43 by Cr(II) complexes of L-Asp, L-His, L-Val, L-Ala, L-Phe, afforded a-phenylglycine (46) in excellent chemical and various enantiomeric yields. The major enantiomer was always S, independently of the nature of the ligand (Figure 7). Transformation of oxime 43byCr(II) complexes of L-Asp, L-His, L-Val, L-Ala, L-Phe,

232


O COOH R-CH

(n-2)®

cr^® R-CH

NH2

h,-^^' NH2

4

42

CH-R '0^0 / w O

O 42 (4 equiv.; OH

OH

NOH

NH2

43-45

46-48

43,46

44,47

45,48

C3H5

C6H5CH2

CH3

Scheme 7

afforded a-phenylglycine (46) in excellent chemical and various enantiomeric yields. The major enantiomer was always S, independently of the nature of the ligand (Figure 7). Further experiments with other oximes such as a-oximino-ji-phenylpropanoic acid (44) and a-oximinopropanoic acid (45) led to similar resuhs (Figure 9). The only qualitative difference was that in the case of these substrates the absolute configuration of the major enantiomer depended on the structure of the L-amino acid ligands, again, in accordance with the highly complicated character of the reduction process. We are carrying on our efforts in the directions as follows: (a) to develop analytical techniques which render the reliable analysis of reaction mixtures possible in those cases when the ligand and product amino acids are the same, (b) to improve the enantiomeric yields, and (c) to find catalytic variants.

his

val amino acid

Figure 7. Enantiomeric excess (e.e.) in the reaction of a-oximinophenylacetic acid (43) and Cr(IiyL-amino acid complexes


asp

val

ala

his

233

phe

amino acid Figure 8. Enantiomeric excess (e.e.) obtained in the reaction of a-oximinophenylacetic acid (43), a-oximino-p-phenylpropanoic acid (44) and a-oximinopropanoic acid (45) with Cr(II)/L-amino acid complexes

At the present stage we believe that the most important result of this research is that the chirality of natural amino acid ligands could be transferred to a-amino acids prepared from prochiralprecursors by reducing these with complexes of the former amino acids. These results are highly relevant to problems of the so-called biological homochirality [39]. This is the phenomenon that all living organisms use chiral molecules of very high enantiomeric excesses which in the whole living nature (on Earth) are belonging to the same series of configuration (L-amino acids, D-sugars, etc.). The origin of this phenomenon is not yet clear. Several theories speculate that it arises from an autocatalytic amplification of any casually or accidentally [19d, 39] formed and originally small excess of these enantiomers. Such autocatalytic amplification, however, has been reported in a single reaction (alkylation of aldehydes by iPr2Zn in organic solvents [5]) so far and its conditions are fairly far from the usual operational mode of living organisms. We hope that the system based on the aqueousphase preparation of chiral, non-racemic and naturally occurring amino acids by metal complexes with natural amino acids as ligands might represent the first step in the development of a system which is autocatalytic in the sense of chirality of the product amino acids.

6.

Acknowledgements This joint research project was supported by the [Hungarian] Scientific Research Foundation (Grant OTKA, No. T33130, T32429), the [Hungarian] Ministry of Education (Grant: FKFP 0614/2000), the [Italian] Ministry of University and Research, the [Italian] National Research Council (CNR) and the Italian-Hungarian Erasmus/Socrates exchange

234


program. Help in the initial steps of the analysis of amino acids is gratefully acknowledged to Prof Miklos HoUosi, Dr. Gyula Szokan and Dr. Sandor Szabo (Budapest).

7. [I]

References

(a) R.H. Garrett and CM. Grisham, Biochemistry, Saunders College Publ., Fort Worth, 1995. (b) K. Drauz, H. Waldman, Eds., Enzyme Catalysis in Organic Synthesis, VCH, Weinheim, 1995. [2] B.M. Trost and I. Fleming, Eds., Comprehensive Organic Synthesis, Pergamon Press, London, 1991. [3] B. Comils and W.A. Hermann, Eds., Applied Homogeneous Catalysis with Organometallic Compounds, Vol. 1-2., VCH, Weinheim, 1996. [4] (a) W.S. Knowles, Angew. Chem., Int. Ed. 41 (2002) 1998-2007. (b) R. Noyori, ibid 41 (2002) 2008-2022. (c) K.B. Sharpless, ibid. 41 (2002) 2024-2032. [5] K. Soai, T. Shibata, H. Morioka and K. Choji, Nature 378 (1995) 767-768. (b) K. Soai and T. Shibata, in: Advances in Biochirality (Eds. G. P^yi, C. Zucchi and L. Caglioti) Elsevier, Amsterdam, 1999, pp. 125136. (c) K. Soai, T. Shibata, I. Ssio,Acc. Chem. Res. 33 (2000) 382-390. (d) K. Soai, in: Fundamentals of Life (Eds. G. Pdlyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002, pp. 427-435. [6] (a) B. Honig, K. Sharp and A.-S. Yang, J. Phys. Chem. 97 (1993) 1101-1109. (b) A. Lubineau, J. Auge and Y. (Jueneau, Synthesis (1994) 741-760. (c) P. A. Grieco, Organic Synthesis in Water, Thomson Science, London, 1997. [7] G. Kov^cs, J. Gyarmati, L. Soms^ and K. Micskei, Tetrahedron Lett. 37 (1996) 1293-1296. [8] K. Micskei, J. Gyarmati, G. Kov^cs, S. Makleit, C. Simon, Z. Szabo, J. Marton, S. Hosztafi, H. Reinke and H-J. Drexler, £:wr. J. Org Chem. (1999) 149-153. [9] K. Micskei, A. Kiss-Szikszai, J. Cârmati and C. Hajdu, Tetrahedron Lett. 42 (2001) 7711-7713. [10] Leading references: (a) Y. Okude, S. Hirano, T. Hiyama and H. Nozaki, J. Am. Chem. Soc. 99 (1977) 31793181. (b) K. Takai, K. Kimura, T. Kuroda, T. Hiyama and H. Nozaki, Tetrahedron Lett. 24 (1983) 52815284. [II] Z. Ratkovic, L. Somsak, K. Micskei, C. Zucchi and G. Palyi, J. Organomet Chem. 637 (2001) 813-819. [12] K. Micskei, O. Holczknecht, C. Hajdu, M. Meo, T. Patonay, C. Zucchi and G. P^yi, 9^^ Internal Conf on Circular Dicroism in Chemistry and Life Sciences (Aug. 31-Sept. 4, 2003, Budapest, Hungary), Abstracts L20p.43. [13] A. Studer, 5yw//2g5;5 (1996) 793-815. [14] (a) Fluka, Chiral Complounds Chemistry, Fluka Chemie AG, Buchs (CH) 1994. (b) Sigma-Aldrich, Chiral Nonracemic Compounds, Sigma-Aldrich, Milwaukee (WI, USA), 1998. [15] (a) A. Togni, A, L. M. \Qr\2axn, Angew. Chem., Int Ed 33 (1994) 497-526 (b) L. Deloux, M. Srebnik, Chem. Rev. 93 (1993) 763-784. (c) K. Drauz, A. Kleemann and J. Usntns, Angew. Chem. 94 (1982) 590613. [16] T. Kiss, in: Biocoordination Chemistry: Coordination Equilibria in Biologically Active Systems (Ed. K. Burger) Elhs Horwood (Publ), New York, 1990, pp. 57-134. [17] Leading references (a) L.A. Wessjohann, G. Scheid, Synthesis (1999) 1-36. (b) A. Fiirstner, Chem. Rev. 99 (1999) 991-1045. [18] L. Stryer, Biochemistry, 4* ed., W. H. Freeman and Co., New York, 1995, p. 17-23. [19] See for example: (a) P. JoU^s, Ed., D-Amino Acids in Sequences of Secreted Peptides of Multicellular Organisms, Birkhauser Verl., Basel, 1998. (b) Y. Nagata, in: Advances in BioChirality (Eds. G. P^yi, C. Zucchi and L. CagUoti) Elsevier, Amsterdam, 1999, pp. 271-283. (c) G. KreU, ibid. pp. 297-304; Annu. Rev. Biochem. 66 (1997) 337-345. (d) G. P^yi, K. Micskei, L. Bencze and C. Zucchi, Magyar Kern. Lapja 58(2003)218-223. [20] U.M. LindstrOm, Chem. Rev. 102 (2002) 2751-2771. [21] A. Lubineau, J. Aug6 and Y. (Jueneau, Water-Promoted Organic Reactions, in: Houben-WeylMethods of Organic Chemistry (Eds. G. Helmchen, R. W. Hoflftnann, J. Mulzer and E. Schaumann) G. Thieme (Publ.), Stuttgart-New York, 1995,4* ed. Vol. E21d., pp. 741-760. [22] S. Otto, G. Boccaletti, J.B.F.N. Engberts, J. Am. Chem. Soc. 120 (1998) 4238-4239. [23] S. Otto and J.B.F.N. Engberts, J. Am. Chem. Soc. 121 (1999) 6798-6806. [24] (a) K. Micskei, F. Debreczeni and I. Nagypdl, J. Chem. Soc., Dalton Trans. (1983) 1335-1338. (b) K. Micskei and 1. NagypM, ibid (1986) 2721-2723.


235

[25] L. Z^kany and I. Nagypal, in: Computational Methods for the Determination of Formation Constants (Ed. DJ. Leggett) Plenum Press, New York, 1985, pp. 291-353. [26] J. Gyarmati, C. Hajdu, Z. Dinya, K. Micskei, C. Zucchi and G. P%i, J. Organomet. Chem. 586 (1999) 106-109. [27] G. Palyi, C. Zucchi, L. Bencze and L. Caglioti, in: Bioinformatics (Eds. J. Seckbach and E. Rubin) Kluwer Academic Publ., Dordrecht (NL) 2004, in press. [28] T. Patonay, C. Hajdu, J. Jekd, A. Levai, K. Micskei, C. Zucchi, Tetrahedron Lett. 40 (1999) 1373-1374. [29] J. Telek-Gyarmati, PhD Thesis, University of Debrecen, 2000. [30] (a) R.S. Nohr and J.H. Espenson, J. Am. Chem. Soc. 97 (1975) 3392-3396. (b) G.W. Kirker, A. Bakac and J.H. Espenson, ibid 104 (1982) 1249-1255. [31] J.H. Espenson, in: Advances in Inorganic andBioinorganicMechanisms, Ed. A.G. Sykes, Academic Press, London-New York, 1982, Vol. 1. pp. 1-63. [32] T. K^gl and L. Koll^ in: Encyclopedia of Catalysis (Ed. I.T. Horvath) Wiley-Interscience, Hoboken (NJ, USA), 2003, Vol. 2. pp. 509-534. (b) ibid. Vol. 6. pp. 697-770 [index], (c) J. McCleverty and T.J. Meyer, Eds., Comprehensive Coordination Chemistry II. Vol. 10, Applications of Coordination Chemistry, Elsevier, Oxford,UK, 2004, in press. [33] K. Micskei, C. Hajdu, V. Marchis, O. Holczknecht, T. Patonay, A. Uvai, M. Meo, C. Zucchi and G. Palyi, submitted for publication, 2004. [34] E. J. Corey, J. E. Richman, J. Amer. Chem. Soc. 92 (1970) 5276-5277. [35] T. Patonay, A. Levai, V. Marchis, K. Micskei, unpublished results. [36] (a) J.K. Kochi andD.D. Davis, J. Am. Chem. Soc. 86 (1964) 5264-5271. (b) A. Bakac, V. Butkovic, J.H. Espenson, R. Marcec and M. Ohranovic, Inorg. Chem. 25 (1986) 2562-2566. (c) P. Huston, J.H. Espenson and A. Bakac, Inorg Chem. 30 (1991) 4826-4830. (d) J.H. Espenson, ^cc. Chem. Res. 25 (1992) 222-227. [37] (a) G.H. Timms and E. Wildsmith, Tetrahedron Lett. 2 (1971) 195-198. (b) K. Takai, N. Katsura and Y. Kunisada, Chem. Commun. (2001) 1724-1725. [38] K. Micskei, O. Holczknecht, C. Hajdu, T. Patonay, V. Marchis, M. Meo, C. Zucchi and G. Palyi, J. Organomet Chem. 682 (2003) 143-148. [39] Reviews: (a) L. Keszthelyi, Quart Rev. Biophys. 28 (1995) 473-507. (b) Marko, L. Diss. Savariensis 24 (1998) 1-64. (c) G. P^yi, C. Zucchi and L. Caglioti, in: Advances in BioChirality (Eds. G. Palyi, C. Zucchi, L. Caglioti) Elsevier, Amsterdam, 1999, pp. 3-12. (d) L. Keszthelyi, ibid, pp. 99-103. (e) idem. Fundamentals of Life (Eds. G. P^yi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002, pp. 379-387.

Progress in Biological Chirality G. P^yi, C. Zucchi and L. Caglioti (Editors) © 2004 Elsevier Ltd. All rights reserved.

Chapter 19 CD and Visual Science Koji Nakanishi,* Nathan Fishkin and Nina Berova* Department of Chemistry, Columbia University, New York, NY 10027, USA kn5@columbia. edu, ndhl@columbia. edu

1.

Introduction The cross section of a human eye is shown in Fig. 1. The estimated 7 million cone cells, responsible for color ("photopic") vision are clustered around the fovea where the incoming image is focused most sharply. The cone cells contain three kinds of rhodopsins (Rh) or visual pigments absorbing around 450 nm (blue light), 530 nm (green light) and 560 nm (red light). A yellow solution absorbs the blue light at 450 nm so that the light that enters our eyes is green and red, which when mixed gives a yellow color. On the other hand, the 100 million rod cells responsible for black and white ("scopotic") vision are mostly distributed in the peripheral area and absorb at 500 nm. Since solar energy is strongest at 400-700 nm, the human eye is taking full advantage of the solar energy distribution. The rod/cone cell ratio depends on the animal: in nocturnal rats it is 4000, humans 20, goldfish 15, frogs 1, while owls only have rod cells. Although not nocturnal, cows and sheep (4 million rods), horse, and dog only have rods and hence do not have color vision. retina

•

rods (bl/wh)

ros

hv

Na*

\

cones (color)

\ disks

\^miBmm\\uim^

rhodopsln Figure 1. Cross section of the eye

sensation yellow

purple

bl.-green

238


The outer segments of rod cells (rod outer segment or ROS) consist of about 1500 thin membranes called disks that are formed at the basis of the rod visual cells by pinching off sections of the plasma membrane. The disks, which embed the 40 kDa Rh molecules consistin of 348 amino acids, move towards the tip of the cell in about 15 days where they are phagocytosed. Phagocytosis of debris by the retinal pigment epithelial cells (RPE) leads to accumulation of orange fluorescent pigments, A2E and others, resulting in age-related macular degeneration (AMD). Visual transduction is the process by which visual cells convert light into a neural signal, which in turn is transmitted to the brain via the optic nerve. In the dark, Na^ ions flow from the rod inner segment (RIS) into the rod outer segment (ROS) (see Fig. 1). Upon absorption of one photon of light, the flow of >million Na^ ions is blocked. Namely, absorption of a single photon activates -1000 molecules of G protein (guanyl-nucleotide binding protein, in the case of vision the term transducin is used), which via activation of phosphodiesterase, hydrolyzes 100,000 molecules of cyclic GMP. This drop in cGMP closes the cation-specific ion channels and leads to a build-up of electric potential that is picked up by the optic nerve.

2.

The 11-cis Retinoid Chromophore The unique attributes of the 11-c/^-retinal that serves as the skeleton for all visual pigments are summarized in Figure 2. The C20 1 l-c/^-retinal, biosynthesized from the C40 P-carotene, is the basic structure of all visual pigments. The P-carotene produces C20 vitamin A (?i\\-trans retinol), the precursor of retinal. However, since humans cannot biosynthesize carotenoids, we have to depend on exogenous sources for vision. The visual chromophore of salmons is 11-c/^-retinal in fresh water but during their migratory period in the ocean, the

Why 11 'Cis-retinal ?

I

The polyene chain is linked to a protonated Schiff base linkage. This allows the positive charge of SBH"" to delocalize depending on: Distance of counter ion and local electrostatic field within binding site. The chromophore is nonplanar around bonds 6/7 and 12/13, the degree of nonplanarity depending on the receptor opsin. The extent of nonplanahty in turn enables the pigment to adjust its Xâx to suit environment. 9-Me is needed for hydrophobic binding and for changing the Rh shape after bleaching; the transducin activation of 9-desmethyl Rh is weak. Double bond or OH can be added to adjust >.rnax to suit environment. Retinal is readily available from carotenoids. A single chromophore, aided by its receptor, can cover the range from 350 to 680 nm! It is a most unique chromophore that is central to all vision. Figure 2. Attributes of 11-cis-retinal as the visual chromophore

CD and Visual Science

239

becomes 3,4-dehydro-ll-cz5'-retinal to induce a red shift in the vision more suited for the ocean where there is less light. The chromophore of the tadpole is also 3,4-dehydro-ll-c/6'retinal, which again becomes 11-c/i'-retinal in the frog. Squids have an additional 4-ahydroxyl group [1] while insects have 3p-hydroxy-11-c/^-retinal [2], the chromophore possibly responsible for their UV sensitivity.

3.

The Visual Transduction Cycle The rhodopsin visual cycle, the result of immense amount of studies over the years, is outlined in Figure 3 together with the involvement of vitamin A. Irradiation of Rh at 500 nm causes 11 -cis -^trans isomerization to yield photo-Rh by femtosecond order photochemistry during which the 11-cz^ becomes a highly distorted trans geometry [3],[4]. This is the only light-triggered change that occurs in Rh, the subsequent changes to batho-Rh and others being caused by relaxation of the protein to relieve the high strain caused by the ''trans'' 11 -ene of the chromophore/protein complex. The strain energy, estimated to be 30-36 kcal/mol, [5] is spectra measured in liquid He, a temperature at which it can be sequestered [7]. Microscopic rate constants coupled with time-resolved spectroscopy led to the finding that a blue-shifted intermediate (BSI, not shown) is in equilibrium with batho-Rh. After batho-Rh the pigment reflected in the red-shifted absorption maxima of photo-Rh (570 nm) and batho-Rh- (543 nm).

OCOR all-trans retinyl esters carotenoids caroten

\ 11-cis-retinal

1

bovine rhodopsin hv

CHO

498 nm

lumi transition. Furthermore, the transducin bioassay showed that the rhodopsin pigment containing 4 exhibited 80% activity of the native chromophore implying that the conformation around the 6-s-cis bond remains

1 — ret-7: binds smoothly

^ ^^^

ret-67-a-locked:

^HO

2

^—^

CHO

ret-8: binds with difficulty

O (llZ)-diazol<eto ret

CHO ret-7-p-CPR: binds defines 11,12,13 conformation

CHO

P-locked does not bind cdamantyl allene ionone binding site is lenient (i)

this side flips oveP; (ii) chromophore conformation in Rh

CHO

opsin "binding cleft" Figure 13. Retinal analogs used to determine chromophore conformation in dark Rh and chromophore movement in the various bleaching intermediates

CHO

250


unchanged in the native Rh bleaching process. This may not be surprising in view of the observation that even adamantyl allenic retinal 6 could be bound to opsin [20]. The fact that ret-8 2 was difficult to incorporate, and only retCPr 3, but not its enantiomeric form yielded a pigment, show that as the retinoid enters the intradiscal (N-terminal) side of the receptor from the side indicated by arrows in Figure 13-i, the conformation of the bottom side of the 7-membered ring in 3 can fit into the potential cleft in opsin; however, this is not the case for its enantiomer. Namely, we infer that the methylene portion of the sevenmembered ring in 3 is most likely predisposed with the proper conformation that can be accomodated by the receptor binding pocket. This allows us to conclude that the retinoids enter the Rh binding site from the side of the 5-methyl and aldehyde groups (see arrows in Fig. 13-i). Finally, as described in Figure 9, the bound conformation of the chromophore in Rh can be represented as in Figure 13-ii.

14.

Acknowledgements These studies were supported by NIH grants GM-36564 and GM-34509 (to K.N. and N.B.) and an NIH Vision training grant EY 13933-03 (to N.F.)

15. References [I] Y. Katsuta, M. Ito, K. Yoshihara, K. Nakanishi, T. Kikkawa and T. Fujiwara, Synthesis of (+)-(4S)- and (-)-(4R)-(l lZ)-4-hydroxyretinals and determination of the absolute stereochemistry of a visual pigment chromophore in the firefly squid, Watasenia scintillans. J. Org. Chem. 59 (1994) 6917-21. [2] M. Ito, T. Hiroshima, K. Tsukida, Y. Shichida and T. Yoshizawa, A novel rhodopsin analogue possessing the conformationally 6-s-67.s-rixed retinylidene chromophore. J. Chem. Soc, Chem. Commim. (1985) 14431444. [3] Y. Shichida, S. Matuoka and T. Yoshizawa, Formation of photorhodopsin, a precursor of bathorodopsin, detected by picosecond laser photolysis at room temperature. Photochem. Photobiophysics 7 (1984) 221228. [4] R.W. Schoenlein, L.A. Peteanu, R.A. Mathies and C.V. Shank, The first step in vision: Femtosecond isomerization of rhodopsin. Science 254 (1991) 412-415. [5] A. Cooper, Energy uptake in the first step of visual excitation. Nature 254 (1979) 531-533. [6] T. Yoshizawa an^ Y. Kito, Chemistry of the rhodopsin sycle. Nature 182 (1958) 1604-1605. [7] T. Yoshizawa and G. Wald, Pre-lumirhodopsin and the bleaching of visual pigments. Nature 197 (1963) 1279-1286. [8] D. Kliger and J. Lewis, Spectral and kinetic characterization of visual pigment photointermediates. Isr. J. Chem. 35(1995)289-307. [9] R. Rando, The Biochemistry of the Visual Cycle. Chem. Rev. 101 (2001) 1881-1896. [10] Y. Shichida and H. Imai, Visual pigment: G-protein-coupled receptor for light signals. Cellular & Mol. Life Sciences 54 (1998) 1299-1315. [II] T. Nakayama and H.G. Khorana, Orientation of Retinal in Bovine Rhodopsin Determined by Cross-linking Using a Photoactivable Analog of 1 l-c/5-Retinal. J. Biol. Chem. 265 (1990) 15762-15769. [12] T.A. Nakayama and H.G. Khorana, Mapping of the amino-acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin. J. Biol. Chem. 266 (1991) 4269-4275. [13] H. Zhang, K.A. Lerro, T. Yamamoto, T.H. Lien, L. Sastry, M.A. Gawinowicz and K. Nakanishi, The Location of the Chromophore in Rhodopsin: A Photoaffinity Study. J. Am. Chem. Soc. 116 (1994) 1016510173. [14] K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, 1. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto and M. Miyano, Crystal structure of rhodopsin: A G protein-coupled receptor. Science, 289 (2000) 739-45.

CD and Visual Science

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[15] T. Okada, Y. Fujiyoshi, M. Silow, J. Navarro, E.M. Landau, and Y. Shichida, Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Proc. Natl. Acad. Sci. USA 99 (2002) 5982-5987. [16] B. Borhan, R. Kunz, A.Y. Wang, K. Nakanishi, N. Bojkova and K. Yoshihara, Chemoenzymatic synthesis of 11-cis-retinal photoaffinity analog by use of squid retinochrome. J. Am. Chem. Soc. 119 (1997) 57585759. [17] T. Hara and R. Hara, Rhodopsin and retinochrome in the squid retina. Nature 2\A (1967) 573-575. [18] T. Hara and R. Hara, Retinochrome and rhodopsin in the extraocular photoreceptor of the squid, Todarodes. J. Gen. Physiol. 75 (1980) 1-19. [19] B. Borhan, M.L. Souto, J.M. Um, B. Zhou and K. Nakanishi, Efficient Synthesis of 1 l-c/^-Retinoids. Chem. Eur. J. 5 (1999) 1172-1175. [20] R.A. Blatchly, J.D. Carriker, N.V. Balogh and K. Nakanishi, Adamantyl allenic rhodopsin. Leniency of the ring binding site in bovine opsin. J. Am. Chem. Soc. 102 (1980) 2495-2497. [21] B. Borhan, M.L. Souto, H. Imai, Y. Shichida and K. Nakanishi, Movement of Retinal Along the Visual Transduction Path. Science 288 (2000) 2209-2212. [22] Y. Fujimoto, R. Xie, S.T. Tully, N. Berova and K. Nakanishi, Synthesis of 1 l-c/^-locked bicyclo [5.1.0]octanyl retinal and an enantioselective binding to bovine opsin. Chirality 14 (2002) 340-346. [23] Y. Fujimoto, J. Ishihara, S. Maki, N. Fujioka, T. Wang, T. Furuta, N. Fishkin, B. Borhan, N. Berova and K. Nakanishi, On the bioactive conformation of the rhodopsin chromophore: absolute sense of twist around the 6-s-cis bond. Chem. Eur. J. 1 (2001) 4198-4204. [24] Y. Fujimoto, N. Fishkin, G. Pescitelli, J. Decatur, N. Berova and K. Nakanishi, Solution and biologically relevant conformations of enantiomeric 1 l-cw-locked cyclopropyl retinals. J. Am. Chem. Soc. 124 (2002) 7294-7302. [25] T. Ebrey, M. Tsuda, G. Sassenrath, J.L. West and W.H. Waddel, Light activation of bovine rod phosphodiesterase by non-physiological visual pigments. FEES Lett. 116 (1980) 217-219. [26] J. Buczylko, J.C. Saari, R.K. Crouch and K. Palczewski, Mechanisms of opsin activation. J. Biol. Chem. 271 (1996)20621-20630. [27] Q. Tan, K. Nakanishi and R. Crouch, Mechanism of transient dark activity of 13-desmethylretinal/rod opsin complex. J. Am. Chem. Soc. 120 (1998) 12357-12358. [28] T. Okada, T. Matsuda, H. Kandori, Y. Fukada, T. Yoshizawa and Y. Shichida, Circular dichorism of metaiodopsin-II and its binding to transducin - A comparative study between meta-II intermediates of iodopsin and rhodopsin. Biochemistry 33 (1994) 4940-4946. [29] H. Akita, S. Tanis, M. Adams, V. Balogh-Nair and K. Nakanishi, Nonbleachable rhodopsins retaining the full natural chromophore. J. Am. Chem. Soc. 102 (1980) 6370-6372. [30] S. Hu, P.J. Franklin, J. Wang, B.E.R. Silva, F. Derguini and K. Nakanishi, Unbleachable rhodopsin with an 11-cis-locked eight-membered ring retinal: The visual transduction process. Biochemistry 33 (1994) 408416.

Progress in Biological Chirality G. Palyi, C. Zucchi aiid L. Caglioti (Editors) © 2004 Elsevier Ltd. All rights reserved.

Chapter 20 Volatile Compounds in Food Aroma: Biosynthesis and Biotransformations Franco Bellesia,^ Adriano Pinetti,^'* Ugo M. Pagnoni,^ Riccardo Rinaldi,^ Claudia Zucchi^ Luciano Caglioti'' and Gyula Palyi^ "^Department of Chemistry, University ofModena andReggio Emilia, Via Campi 183,141100Modena, Italy pinetti. adriano@unimo, it AITEL, Associazione Tecnici del Latte, Via Galli 18,1-41100 Modena, Italy "" Department of Chemistry and Technology of Natural Substances, University 'Xa Sapienza' Piazzale Aldo Moro 5,1-00185 Roma, Italy

Flavouring compound analysis has widely grown with the introduction of easy sampling methods such as solid phase microextraction (SPME) and purge and trap (P&T) [1]. The high quantity of achievable data makes possible the research of some manufacture and ageing markers, or the finding of information about the biosynthesis and biotransformations occurring during the shelf-life. In this case some suggestions about their use in the production of asymmetric compounds may arise. Enz-B H

Enz

^N^CH3

H^

H\

H ; ^ N ^ C H 3

Tyrosine-piridoxal adduct

^0PO^

H'' ^l/^CHj

Tyrosine

Phenol Scheme 1

254

Progress in Biological Chiralitv

Serine + PMP +NH3 H+

/

While studying the composition of the volatile organic compounds (VOCs), a mixture responsible for the flavour [2] and for the fat alteration products [3] in Parmesan cheese, we found a release of phenol, which could arise by the action of tyrosine-phenol liase (TPL) [4, 5, 6] through an intermediate complex between tyrosine itself and pyridoxal phosphate (Scheme

1). Such an intermediate is also formed in the nucleophilic displacement of the serine hydroxy group in many transformations (Scheme 2), occurring both in animal and plant kingdom, leading to very important compounds, for example to thyptophan and L-DOPA when an indolyl anion or catechol respectively act as nucleophiles; Lactobacilli and Streptococci, featuring components of the bacterial flora of cheeses, can thus be used to obtain the above mentioned compounds, or many others having pharmacological or industrial appeal, by fermentation methods, instead of using expensive gene-modified microrganisms as biocatalysts.

Rib H

Rib H

Rib

H 3 C ^ ^ N % " H ^ H 3 C " ^ N 1 Y ^

H 3 C ^ ^ N ^ ^ H

FADH2

FAD

Ambrettolide

'^^

'^CH^Q''^

Rib ^ ÔO H H3C^^^v^Ny^^0 H3(X^^N^j^.H HoO^ H3C^^N+Y""H

H I O OH

H3C^^r:J^Nô

Rib Scheme 3

Volatile Compounds in Food Aroma: Biosynthesis and Biotransformations O ^

255

—HOO)

r>XrÔNH2 V5J

Methyl ketones

L-^1 ^ j

(H3.

A-o^V R ) _|

O

Methyl esters

NAD"" Scheme 4

O Acetic esters

Other biotransformations encountered in this research involve some Bayer-Villiger microbial oxidations [7, 8, 9] occurring on methyl ketones, arising from an altered fatty acid degradation pathway, and some compounds, as muscone, coming from the feeding of cows. While in the oxidation of the former compounds to methyl esters no chiral centre is formed, on the latter ones a new stereocentre is created (Scheme 3). So even in this case it would be possible to use the normal milk bacterial flora in some fermentations to produce, for example, flavouring lactones [10, 11] enantiomerically pure from cyclic asymmetric ketones. Only methyl esters of even carbon atom number short chain fatty acids are found within the VOC mixture, indicating that the oxidation of methyl ketones occurs with high regioselectivity, supporting a reaction pathway similar to the one depicted in Scheme 3, with the peroxy-intermediate linked to the enzyme co-factor. Other pathways, like the one involving the release of a free hydroperoxyde anion by molecular oxygen through a NADH intervention (Scheme 4), seem not to work with sufficient selectivity. During the detection of VOC-s in various samples of Parmesan cheese, many a discontinuity have been found. A first source of error arises from the sample size. For SPME or P&T methods, few milligrams of cheese are required, a quantity too low to be really representative. This problem can be avoided by collecting the samples from a larger amount (about 5 grams) of homogeneously finely grated cheese. A further discontinuity is due to the complex migration process of small molecules inside the form during ageing. This accounts for differences in composition found among the various parts of the bulk. The processes are most likely linked to the hydrophilicity-lipophilicity of the migrating compounds (the rim is more "greasy" than the inside parts). A previous study [12] has brought to the suggestion that an ideal surface of the same concentration of each migrating product (flavours, free amino acids, free fatty acids etc..) might have the shape of a "red blood cell" (Figure 1). As all the releases of these molecules are a frmction of ageing, probably the distance between the rim and the surface, relative to a particular compound concentration or to a certain ratio of compounds, may be taken as a ripening marker. This concept can also be applied to the levels of epimerization of L- to D-amino acids, whose D/L ratio could be represented by an analogous set of surfaces.

256


Figure 1

Acknowledgement Financial support is acknowledged to the town council of Serramazzoni (Modena, Italy), to the Comunita Montana del Frignano (Modena, Italy), to the [Italian] Ministry of Instruction, University and Research (MUIR) and to the [Italian] National Research Council (CNR).

References [1] (a) F. Bellesia, A. Bianchi, A. Pinetti and B. Tirillini, Fl. Fragr. J. 11 (1996) 239. (b) Idem, Riv. Ital E.P.P.O.S. 23 (1997) 41. (c) Idem, Fl. Fragr. J. 13 (1998) 56. (d) Idem, J. Ess. Oil Res. 10 (1999) 483. [2] F. Bellesia. A. Pinetti, U. M. Pagnoni. R. Rinaldi. C. Zucchi, L. Caglioti and G. Palyi, FoodChem. 83 (2003) 55. 13] C. Zucchi, F. Bellesia, A. Pinetti, L. Simon-Sarkadi, B. Weimer, L. Caglioti and G. Palyi, Featuring compounds of the greasy base of the Parmesan cheese aroma 3'''^ Italian-French Meeting on Organic Chemistry. Organic Chemistry towards Interfaces. Pisa, 20-23/11/2002 Abstract p. 60. 14] S.V. Plevnev, M.N. Isupov, Z. Dauter, K.S. Wilson, N.G. Faleev, E.G. Harutyunyan and T.V. Demidkina, Biochem. Mol. Biol. Int. 38 (1996) 37. [5] H.Q. Smith and R.L. Somerville, J. Bacteriol. 179 (1997) 5914. 16] H. Chen and R.S. Phillips, Biochemistry 32 (1993) 11591. 17] (a) V. Massey, J. Biol. Chem. 269 (1994) 22459. (b) C.T. Walsh and Y.-C. Jack Chen, Angew. Chem., Int. Ed Engl. 28 (1988) 333. 18] S. Rissom. U. Schwarzhnek, M. Vogel, V.I. Tishkov and U. Kragl. Tetrahedron: Asymmetry 8 (1997) 2523. 19] M.T. Bes, R. ViUa, S.M. Roberts, RW.H. Wan and A. Willetts. J. Mol. Cat. B 1 (1996) 127. 110] C Fuganti and G. Zucchi, Chim. Ind 79 (1997) 745. HI] G. Feron, P. Bonnarme and A. Durand, Trends FoodSci. Tech. 1 (1996) 285. [12] C Zucchi, F. Bellesia, A. Pinetti, L. Simon-Sarkadi, B. Weimer and G. Palyi, unpublished results.


Chapter 21 Origin of the Biological Chirality N. M. Chemavskaya, D. S. Chernavsky, R. F. Polishchuk Lebedev Physical Institute of the Russian Academy of Sciences rpol@asc. rssi. ru

Life is a negentropy current providing self-correction by the genetic code and supporting itself by free energy affluent [1]. Origin of the biological chirality in frame of the biosphere is an example of the spontaneous breaking of symmetry due to the energetic advantage criteria. Several organic molecules have a right (D-) and left (L-) chirality (helicity) and turn a light polarization vector to the right or left direction respectively. Liquids and crystals have a form of the D-junctions or L-junctions. More complex organic junctions may include fragments with both chiralities. The inorganic part of nature has a racemic "mixture" of its elements with the both chirality forms. In organic nature this symmetry gets to be broken. In nature all the amino acids (except glycine) are present in the chiral L-form. But L-form of its main fragments may be combined with the helicity of other fragments. All molecules of the C-5 sugar units (ribose) in the main chain of the ribonucleonic acids have a right helicity. Most of the nucleotides of the lateral ribopolymer groups have a left helicity. Why is the racemic mixture absent in living nature? Because a racemic mixture needs a double composition of proteins. All the chemical reactions in organisms are catalysed by enzyme-proteins. Each of these proteins have an active centre that is complementary for the substrate. Biological organisms with the double compositions of proteins are too cumbersome for successfial competition for the maintenance of life. We must take in account also one fact that for the proteins, catalysing reactions with substrates of some chiral protein-antipodes are acids, inhibitors. These inhibitor effects for DNA (desoxiribonucleonic acids) are considered in [2]. It was demonstrated that in a racemic mixture of nucleotides the enantiopure DNA-chains only with 50-100 links can be formed. Fore the longer chains the probability of the additional antipodes is increasing by factorial law. In modern biosphere the DNA-chain which codes for one fijnctional protein (approximately 200 amino acids) must contain more than 600 nucleotides. That is why in work [2] are introduced biological (number of links is more than 600) and chemical (less than 50) levels of complexity. In a racemic world non-enantiopure DNA with only chemical complexity level can duplicate. The choice of chirality type may be connected with (weak) external asymmetric influence. Polarized neutrino flows promote D-sugars to be synthesed and L-amino acids [2]. But this

258


influence is too weak to provide observable asymmetry. It is a more realistic hypothesis to suppose the spontaneous breaking of symmetry [3]. In the work [3] the following informational dynamic system is proposed: dii^ Idt- 2/, / r, + Z,^ .b,.uM, - a,w ^ + Z), Aw,

Here w, is a number of the /-th element (a concentration) in a family of w possible types, i; is a characteristic of the autoreproduction time. The term btj Ut tij describes the antagonistic interaction of the different elements. The term a^w,^ corresponds to the "narrowness" effect; D, is a diffusion coefficient. This dynamical system is multistable. Its development is dividing into separate phases: the origin of clean regions (clusters) in phase space, its extension and then a "victory" of one cluster. In the case of the biological chirality the number of possible clusters is two. This model describes also a chemical reaction of the synthesis of molecules with one centre of chirality from a racemic mixture of molecules with the different chiralities with two conditions: (a) new chiral molecules stimulate the synthesis of new molecules with the same chirality (autocatalysis), (b) the meeting of two antipodes removes both of these antipodes from the reaction sphere (antagonistic interaction). If the racemic state is nonstable then one of the chiral molecules will form. Its chirality type is accidental. Near a bifurcation point the evolution of a dynamical system may be described by the following equation: dTj/dt=ST]-rj\

7] = (C. - C.)/(C, + C.)

where rj is a chirality parameter, C+ and C. are concentrations of the D- and L-enantiomers, respectively, ^ is a (small) bifurcation parameter. If s 0 there are stable non-racemic states TJ = ±4s . For the biological systems we have s ~ 1 and 77 = ±1 giving non-racemic products. Non-catalytic reactions are excluded. A very important problem of the origin of biological chirality is the time of its appearance: is it in the biological period or already in pre-biological one? We are convinced of the appearance of biological chirality in the period of the primitive hypercycles. In fact, the length of DNA-molecules in this period was 30-50 units. These molecules did not perform at this period any biological coding function but they were performing the chemical function of heterogeneous catalysts. Probably these molecules were in the homochiral form. The enantiopurity of the proteins was important in this phase. The primitive hypercycle processes could take place in racemic nucleotide mixtures: external homochirality was not needed for this. Also non-autocatalytic synthesis of these DNA-molecules can practically be excluded. These reactions were of the middle-level complexity - between the biological complexity level and the chemical one. The two "antipode" forms of the enantiopure hypercycles were arising in this period with equal probability. Then one of these antipodes was squeezed out by an other one. In this case the antagonistic interaction was more strong than it was between the different biological codes: one of the antipodes was "eaten" by the other one and it was eliminating itself due to this poisoning.

Origin of the Biological Chirality 259

The products of biological organisms are nutritious substances for other organisms. Of course these substances do not necessarily have chiral symmetry. The production of the opposite chiral isomers often has a protection function. As a consequence of the above argumentation the non-stability of the symmetrical state is a principal cause of the biological asymmetry. An external influence is not needed here. There are however some intermediate variants, considering the influence of the neutrino flux, which are discussed in [4]. These variants are possible if the parameter s changes gradually from negative to positive value and if the concentrations of antipodes are sufficient. Near the bifurcation point {s = 0) even a little external influence can change the choice of the finite state. In this case the Lyapunov parameters (in exponential evolution law for the dynamical system) are small and even fast fluctuations may be averaged yielding small s >0. Then our equation has the following form

drj /dt=S + 6rj-7]\

ri«\

In this case there is a stable evolution branch where a bifurcation for s = 0 is absent and for s> 0 stable and non-stable (a separative) branches are appearing. For s « 1 these branches approach (as 5^'^ ) to the main stable evolution branch. Formally here an external factor is the cause of the evolution picture but this variant is not realistic. More real is the situation when the concentration of hypercycle is small but its relative fluctuations are large and the antagonistic interaction is strong. Here a symmetric state is strongly non-stable and the Lyapunov's parameters are large. Then fluctuations of concentrations can not be averaged to the fast evolution. Here external factors as a neutrino influence are not important and the choice of an evolving variant is determined by the initial fluctuations accidentally. References [1] R.F. Polishchuk, in: Fundamentals of Life (Eds. G. Palyi, C. Zucchi and L. Caglioti) Elsevier and Accademia Nazionale di Scienze, Lettere ed Arti (Modena), Paris, 2002, p. 141-151. [2] V.A. Avetisov and V.I. Gol'dansky, Uspekhi Fiz. Nauk 160 (1996) 873-890 (in Russian). [3] D.S. Chemavsky, Synergetics and Information, "Znanie", Moscow, 1990 (in Russian). [4] D.K. Kondepudi and G.W. Nelson, Physics A: Statistical Mech. Appl. 125 A (1984) 465-496.


Chapter 22 Chiral Spaces in Encapsulation Complexes Alessandro Scarso, Alexander Shivanyuk, Osamu Hayashida and Julius Rebek, Jr. * The Skaggs Institute for Chemical Biology and The Department ofChemistiy, The Scripps Research Institute, MB'26, 10550 North Torrey Pines Road, LaJolla, CA 92037 (USA) jrebek@scripps. edu

Encapsulation complexes are assemblies in which small molecule guests are completely surrounded by large molecule hosts [1, 2, 3, 4]. The hosts are made up of subunits held together by intermolecular forces: hydrogen bonds, van der Waal's forces and metal-Hgand contacts. The assemblies are formed reversibly and are dynamic; they come together and dissipate on time scales ranging from milliseconds to days, long enough for their study by NMR methods. When multiple hosts can assemble from a given set of subunits, template effects can be expected and they have recently been reported [5, 6, 7, 8, 9], The synthesis of capsules with asymmetric spaces has also been accomplished, but enantioselection of guests within these hosts is inadequate, whether the capsules are held together with covalent bonds [10], hydrogen bonds [11] or metal/ligand interactions [12, 13]. Moreover, the syntheses are lengthy and problematic and in this chapter we report alternatives. The first involves creation of capsules from achiral precursors and imprinting them chiral guests. The second involves the space left in a large achiral host when a small chiral guest is encapsulated. While the enantioselectivity is still low, the methods may have wider applicability. We consider the capsule la-la (Figure la [5]), formed when two self-complementary subunits la dimerize in organic solvents. A seam of eight hydrogen bonds holds the subunits together. The dimer has only C2 axes and exists as a pair of enantiomers even though each subunit features a plane of symmetry. The cavity of the capsule is chiral and asymmetric guests generally prefer one enantiomer of the capsule to its mirror image. The enantiomeric capsules racemize by complete dissociation and recombination of their subunits - a slow process - but guest exchange does not require this dissociation. Rather, guests get in and out of these capsules through flaps opened by the breaking of hydrogen bonds - a faster process [14, 15, 16]. A related structure lb with additional hydrogen bonding possibilities was prepared [17]. It has a slower rate of racemization when it dimerizes into capsules lb-lb. Parts of the NMR spectrum of lb-lb alone and with added chiral guest, (+)-pinanediol (+)2 are shown in Figure 2 (Figure lb). The broad signals of Ib^lb in /?-xylene-J;o are characteristic of unspecific aggregation. On the addition of 3 equivalents (+)2 a sharp spectrum is obtained in which the

262

Progress in Biological Chirality O

Y 0 R = n-heptylphenyl

H. Y

Y

OH

?" OH

^-*Ny

H-2

(+)-2

Figure 1. (Top) Line drawing of the monomers 1. (Middle) Energy-minimized [18] model of the dimeric assembly lb»lb. Some hydrogens and the /i-heptylphenyl groups of the dimer are omitted for clarity. (Bottom) Guests used in the study

A B

tJJi^

'>A«**^*ww^^«x/^^v'>nA'

X

B BfB* >H*vwyw-.v-*f

8.65

8.50

8.45

WH#«

2.90

i

T

B+B-

A+AI 2.75

2.60

Figure 2. Selected NH resonances of ^H-NMR spectra showing of the dimer (8.65-8.45 ppm) and Ha peaks for the guest in the enantiomeric capsules (2.90-2.60 ppm). The labels A and A* are the thermodynamically more stable complexes of (+)2 and (-)2, respectively, while B and B* are the less stable complexes, a) Ib^lb alone in /7-xylene-c/io (0.825 mM); b) 4 min and c) 120 h after addition of 3 eq of (+)2; d) 17 min and e) 191 h after addition of 30 eq of (-)2 other than those of the acids

Chiral Spaces in Encapsulation Complexes

263

two diastereomeric complexes are observed in approximately equal amounts (Figure 2b). After a few days, the system reaches its thermodynamic equilibrium at a 50% diastereomeric excess of the favored isomer (Figure 2c). The half-life for equilibration --20 hours) is the lifetime of the assembly. When 30 equivalents of the enantiomer (-)2 was added to the equilibrated mixture, the ratio of the two diastereomers partially inverts. This indicates a situation - unique to reversibly formed assemblies - in which a temporary excess of the less stable diastereomeric complex exists (Figure 2d). This occurs because the guest exchange process is much faster than dissociation of the two halves of the capsule. The rate of guest exchange (ti/2 ~ 1 min) is in line with that observed in similar systems [16]. The new guest (-)2 is now present in the capsule that formed preferentially around (+)2. Eventually the mixture of diastereomers returns to equilibrium. The half-life is -- 10 hours and the original 50% d.e. is achieved (Figure 2e). The rate is twice as fast as that measured for the initial equilibration, and is likely due to the high concentration of the polar, hydrogen-bonding 2 in the exchange mixture [19]. Additional competition experiments established the situation scheme shown in Figure 3. The memory, or "ghost" persists through multiple guest exchanges. For example, a solution of lb»lb templated with (+)-2. The solvent and the excess guest was removed, leaving only lb»(+)2»lb. The complex was redissolved in benzene and excess 1-adamantanol, 3, was added. This displaced the guest (+)2 into the solution. The solvent was removed and extraction gave a solution free of (+)-2. However, we were unable to detect any optical activity by polarimetry of this solution. When this solution was treated with excess (-)2, the NMR spectrum again showed a 2:1 excess of the less stable diastereomeric complex. Accordingly, that the chiral memory was maintained even in the absence of chiral guests. The memory of the capsule reflects the relative rates of guest and monomer exchange. The

V^«10-20h

Figure 3. Cartoon representations of the exchange equilibria. Guest exchanges are the horizontal equilibria and monomer exchanges are the vertical equilibria. The cartoons not intended to imply the assign a stereochemistry to the assemblies

264


Figure 4. (Left) Cartoon for monomer exchange by dissociation; (center) guest exchange by disrupting hydrogen bonds; (right) energy-minimized structure of the intermediate showing the openings for guest exchange exchange of subunits l b requires the complete dissociation of the capsule (Figure 4a), while guest exchange proceeds through openings that only disrupt a part of the seam of hydrogen bonds that hold the capsule together (Figure 4b). The inversion of the pyridazinyl ring of l b creates openings in the dimer that are large enough to allow passage of incoming and outgoing guests (Figure 4c [15]). Many thousands of molecules enter and depart the capsule during its lifetime, but each guest will experience the imprinted asymmetric microenvironment of the capsule. Examples of nonracemic chiral encapsulation complexes are rare [20, 21, 22] and the imprinting [23, 24] described here provides another route to such systems. It is likely that similar behaviors will also emerge from capsule held together by metal-ligand interactions [8]. The second system showing chiral spaces requires the encapsulation of two different guests. Consider placing a chiral object in an achiral space, a glove in a box for example (Figure 5). Since the glove is chiral the space remaining in the box becomes chiral. Now if a second chiral object enters the box, some selection is expected, especially if the two objects interact. That space should distinguish between enantiomers of the second guest. Relevant here for the molecular scale is the notion of "peristatic" chirality [25]. Reversibly formed capsules capable of surrounding two guest molecules - coencapsulation - are recent inventions [26] that can show of new forms of stereoisomerism [27], suggest possibilities for data storage [28] and permit encapsulated bimolecular reactions [29]. The guests of a coencapsulation complex are confined in space and time; they have much longer contact times and more defined orientations than the diffusion complexes in bulk solution. Diastereomeric complexes can be formed with two chiral guests, and either guest alone in the capsule would leave a chiral remaining space, but capsules are not formed if they are not properly filled. Instead, capsules are known to select guests that provide good fits. A good fit for the liquid phase means filling -55% of the cavity [30]. We explored the concept with the

^j-

•

Figure 5. A chiral object in an achiral container leaves a chiral space

Chiral Spaces in Encapsulation Complexes H.

265

0

Figure 6. (Top) Line drawing of the snbnnit and the ball and stick representation of capsule 4«4. Long, peripheral pendant chains have been removed. Cartoon representation used elsewhere in this work is shown on the left. Bottom: Coencapsulation complexes. Size matching (left) Coencapsulation of the small cyclopropane occurs when the large p-xylene is available. The enantiomers of cyclohexane diol (center) are slightly favored over two identical molecules (right)

cylindrical capsule of Figure 6. Earlier we found that an enantiomeric pair of cyclohexane diols fills the space in a capsule slightly better than two molecules of the same handedness (Figure 6) [31]. This observation may be related to the preference in nature for centrosymmetric crystals or, alternatively stated, the higher melting points of racemates vs enantiopure compounds. This generality is far from absolute, as we shall relate below. The capsule is too short to accommodate two p-xylenes and cyclopropane alone is not encapsulated. But a mixture of cyclopropane and p-xylene, 4*4 results in the coencapsulation complex [32]. The NMR spectrum of this arrangement shows different signals for the methyl groups of the encapsulated xylene. The separate methyl signals result from two restricted motions of the p-xylene guest: tumbling is slow on the NMR timescale and the two guests are too large to slip past each other while within the capsule. Shorter aromatics tumble rapidly on the NMR timescale inside the capsule [33]. The dimensions of the capsule select appropriately sized combinations and its shape can be used to fix rigid molecules. Polar functions are attracted to the seam of hydrogen bonds that holds the capsule together. These inherent characteristics of the capsule were useful for the enantioselection. A combination of (R)-styrene oxide 5 and isopropyl chloride in a solution of 4*4 in mesitylene-di2 gives an NMR spectrum showing the coencapsulation complex that features

266


*+x

II

Krl, I —

-2.0

-2.4

-2.8

d X

,|(

1 '^ i

>^' -2.4

-2.6

-2.8

-2.0

IMJJ^ -3.0

-4.0

8 Figure 7. Upfield region of the ^H-NMR spectra (600 MHz, 300 K) of coencapsulation complexes of 4*4 in mesitylene-di2 and various liquid guests, a) (R) - styrene oxide with i-PrCl; b) phenylcyclopropane with i-PrCl; c) (R) - styrene oxide with (±) 2-BuCl: •, * the 4- and 1- methyl groups, respectively, of 2-BuCl encapsulated with (R) - styrene oxide; the signals marked x and • are the 4- and 1-methyl groups, respectively, of the capsule containing two 2-BuCl guests [34]; d) (S)-mandelic acid with (±)- 2-BuOH: • is the 4-methyl group of (R) 2BuOH and • is the 4-methyl group of (S) 2-BuOH coencapsulated with the acid; x represents the 1-methyl groups of both diastereomeric complexes; the signals maiiced * and • are the 1 and 4 methyl groups, respectively, of the capsule containing two 2-BuOH guests

diastereotopic methyl groups of the smaller guest (Fig. 7). The large upfield shifts place its methyls near the end of the capsule while the observed magnetic anisotropy place the epoxide function near the capsule's center. In contrast, the coencapsulation of phenylcyclopropane and isopropyl chloride showed a complex with only one doublet at -2.61 ppm (Fig. 7b). The chiral epoxide with coguests racemic 2-butyl chloride, 2- butanol or 2-pentanol gave capsules including one molecule of each guest. Two sets of signals in 1:1 ratio correspond to the methyl protons of the encapsulated halide (Fig. 7c), and the chemical shifts indicate that the ethyl group of the halide is near the end of the capsule. No diastereoselection was observed, even though the asymmetric features of both guests are near the capsule's center, because no attractive forces are in play between the two guests. Their interface lacks steric contacts and they stay as far apart as possible (Figure 8) [35]. We had expected that racemic 2-butanol or 2-pentanol with (R)-styrene oxide would provide hydrogen bonds between the coguests but no diastereoselectivity was observed. With these alcohols, other diastereomeric complexes are also present. These arise from the encapsulation of two molecules of 2-butanol or 2-pentanol (i.e. R+S, R+R and S+S couples). Even so, no stereoselection is detected, yet the asymmetric centers are near the middle of the capsule and intermolecular hydrogen bonding between the two alcohols should exist. We obtained some better results with the superior donor (S)-mandelic acid 6. The ^H-NMR spectrum showed the formation of two diastereomeric capsules (Figure 7d), and their ratios


267

Figure 8. (Left) High packing coefficients (large spheres) force the interdigitation of large, medium and small groups. (Right) Smaller coguests result in a more remote arrangement of asymmetric centers and lower enantioselection

varied with temperature: 1.1 at 303 K and 1.3 at 283 K. Coencapsulation of 6 with (R)-2butanol established the identity of the diastereomeric complexes. This enantiomer was the better guest for the coecapsulation with (S)-mandelic acid. The contacts between mandelic acid and butanol guests were found in the energy minimized structures shown in Fig. 9. This arrangement brings the asymmetric centers closer but they are still at too far away from one another.

Figure 9. Structures (obtained from the MM^ forcefieldcalculations [36]) of the coencapsulation complexes. (R)-styrene oxide with i-PrCl (left); (R)-styrene and (R)-2-Cl-butane (middle); (S)-mandelic acid and (R)-2butanol (right)

Table 1. Guest (±)-2-bromo-3-methylbutyric acid (±)-2-bromovaleric acid (±)-2-bromobutyric acid

Enant Ratio (298 K) 1.5 1.3 1.6

268 Progress in Biological Chirality *

-3.0

•

-4.0

-3.5

6

Figure 10. Upfield region of the 'H-NMR spectra (600 MHz) of encapsulation complexes of U and guests in mesitylene-di2 at 298 K. a) (±)-2-bromo-3-methylbutyric acid (* and • are 4 and 3-methyl groups of 2-bromo-3methylbutyric acid); b) (±)-2-bromovaleric acid (• are 4-methyl groups of 2-bromovaleric acid); c) (±)-2bromobutyric acid (• are 4-methyl groups of 2-bromo-butyric acid)

Carboxylic acids gave clearly interpretable spectra, but the two molecules of 3hydroxybutyric acid 7 were encapsulated without diastereoselectivity. This guest gave modest selectivities in complexes with aromatics at 273K: (S)-mandelic acid showed 18% de, while (R)-mandelonitrile 8 and (R)-l-phenylethanol 9 each showed 21% de. The isomeric 2-OH butyric acid, with the asymmetric center nearer the coguest, poorly resolved spectra. Instead, we found that small a-bromo acids were good guests. Two molecules are encapsulated and with modest selectivity (Table 1 and Figure 10). Little differences in the length or in the shape of the acids cause differences in stereoselection. We assume that interguest hydrogen bonding occurs and that leaves the stereocenters at some distance. Figure 11 shows the results of Molecular Modeling MM+ and emphasizes that the acids' centers are, on average, further apart than the alcohols'. The acid appears important for good interactions between guests and two acids are also effective. This is unexpected as the hydrogen bonded dimers place their asymmetric centers no closer than 6.7 A (Fig 11). The diastereoselectivities are poor: with partners that have weak

^ Br

0-I+-0.

V A

\/wjl 6.7

Figure 11. Asymmetric centers of hydrogen bonded alcohols can be closer to each other than those of the acids


269

attractions or with alcohols there is no selectivity; only with carboxylic acids can some (up to 25% de) be observed. The volume of the capsule translates into ~4 M concentration of each guest inside and the lifetime of the complex is on the order of 1 second. The guests are isolated in space and in time and a chiral guest has ample opportunity to provide an effective asymmetric magnetic environment for its partner. Perhaps the two-point connections and the stronger hydrogen bonds of the dimeric acids increase the lifetimes of complexes. As a final observation, 2-bromo-3-methyl butyric acid (commercially available in optically active and racemic forms) gave the best stereoselection with itself, rather than its mirror image. Are these attractions between dipoles? Steric and magnetic environments appear insufficient for diastereoselection; multiple attractive contacts between guests should be more effective and provide the challenge.

Acknowledgements We are grateful to the Skaggs Foundation and the National Institutes of Health (GM 50174) for financial support. References [I] [2] [3] [4] [5] [6]

M.M. Conn and J. Rebek Jr., Chem. Rev. 97 (1997) 1647-1668. T. Kusukawa and M. Fujita, J. Am, Chem. Soa 121 (1999) 1397-1398. T.N. Parac, D.L. Caulder and K.N. Raymond, J. Am. Chem. Soc. 120 (1998) 8003-8004. R.G. Chapman and J.C. Sherman, J. Am. Chem. Soc. 120 (1998) 9818-9826. J.M. Rivera, T. Martin, J. Rebek Jr., Science 279 (1998) 1021-1023. (a) M. Scherer, D.L. Caulder, D.W. Johnson and K.N. Raymond,^wgew. Chem. I l l (1999) 1690-1694. (b) M. Scherer, D.L. Caulder, D. W. Johnson and K.N. Raymond, Angew. Chem., Int. Ed. 38 (1999) 15881592. [7] F. Ibukuro, T. Kusukawa and M. Fujita, J. Am. Chem. Soc. 120 (1998) 8561-8562. [8] S. Hiraoka and M. Fujita, J. Am. Chem. Soc. 121 (1999) 10239-10240. [9] H. Hof, C. Nuckolls and J. Rebek Jr., J. Am. Chem. Soc. Ill (2000) 4251-4252. [10] (a) J. Canceill, L. Lacombe and A. Collet, J. Am. Chem. Soc. 107 (1985) 6993-6996. (b) J. Yoon and D.J. Cram, J. Am. Chem. Soc. 119 (1997) 11796-11806. (c) J.K. Judice and D.J. Cram, J. Am. Chem. Soc. 113 (1991)2790-2791. [II] (a) J.M. Rivera, T. Martin and J. Rebek Jr., J. Am Chem. Soc. 123 (2001) 5213-5220. (b) J. Tokunaga and J. Rebek Jr., J. Am Chem. Soc. 120 (1998) 66-69. (c) F. Hof, S.L. Craig, C. Nuckolls and J. Rebek Jr., Angew. Chem., Int. Ed. 41 (2002) 1488-1508. (d) R.K. CasteUano, B.H. Kim and J. Rebek Jr., /. Am. Chem. Soc. 119(1997)12671-12672. [12] (a) M. Fujita, K. Umemoto, M. Yoshizawa, N. Fujita, T. Kusukawa and K. Biradha, Chem. Comm. (2001) 509-518. (b) S. Hirakoa and M. Fujita, / . Am. Chem. Soc. Ill (1999) 10239-10240. (c) T. Kusukawa and M. Fujita, J. Am. Chem. Soc. 124 (2002) 13576-13582. [13] D.L. Caulder and K.N. Raymond, ^cc. Chem. Res. 32 (1999) 975-892. [14] T. Szabo, G. Hilmersson and J. Rebek Jr., J. Am. Chem. Soc. 120 (1998) 6193-6194. [15] X. Wang and K.N. Houk, Org Lett. 1 (1999) 591-594. [16] J. Santamaria, T. Martin, G. Hilmersson, S.L. Craig and J. Rebek Jr., Proc. Natl. Acad Sci. USA 96 (1999) 8344-8347. [17] J.M. Rivera, S.L. Craig, T. Martin and J. Rebek Ir., Angew. Chem., Int. Ed 39 (2000) 2130-2132. [18] F. Mohamadi, N.G.J. Richards, W.C. Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T. Hendrickson and W.C. Still, J. Comp. Chem. 11 (1990)440-467. [19] Even low concentrations of hydrogen-bonding molecules can have dramatic effects on the rates of assembly processes. R.K. CasteUano, S.L. Craig, C. Nuckolls and J. Rebek Jr., J. Am. Chem. Soc. Ill (2000) 78767822.

270 [20] [21] [22] [23]

Progress in Biological Chiralily

R.K. Castellano, B.H. Kim and J. Rebek Jr.. 1 Am. Chem. Soc. 119 (1997) 12671-12672. R.K. Castellano, C. Nuckolls and J. Rebek, Jr., J. Am. Chem. Soc. Ill (1999) 11156-11163. C. Nuckolls, F. Hof, T. Martin and J. Rebek Jr., J. Am. Chem. Soc. 121 (1999) 10281-10285. For examples of imprinting in covalent materials, see: (a) K.J. Shea, Trends Polym. Sci. 2 (1994) 166-173. (b) G. Wulff, Angew. Chem. 107 (1995) 1958-1979; G. Wulff, Angew. Chem., Int. Ed Engl. 34 (1995) 1812-1832. (c) K. Mosbach and O. Ramstroem, Biotechnology 14 (1996) 163-170. (d) H. Shi, W.-B. Tsai, M.D. Garrisoa S. Ferrari and B.D. Ratner, Nature 398 (1999) 593-597. (e) K. Polbom and K. Severin, Chem. Commun. 24 (1999) 2481-2482. (f) K. Dabulis and A.M. Klibanov, Biotechnol. Bioeng 39 (1992) 176-185. [241 For examples of memory effects in noncovalent materials, see: (a) Y. Furusho, T. Kimura Y. Mizuno and T. Aida J. Am. Chem. Soc. 119 (1997) 5267-5268. (b) E. Yashima, K. Maeda and Y. Okamoto, Nature 399 (1999)449-451. [25] E. Graf, R. Graff, M.W. Hosseini, C. Huguenard and F. Taulelle, Chem. Commun. (1997) 1458-1460. [26] (a) T. Heinz, D. Rudkevich and J. Rebek Jr., Nature, 394 (1998) 764-766. (b) M.K. Ebbing, M.-J. Villa JM. Malpuesta, P. Prados and J. de Mendoza, Proa Natl. Acad Sci. USA 99 (2002) 4962-4966. (c) A. Shivanyiik and J. Rebek Jr., Chem. Commun. (2001) 2424-2425. [27] A. Shivanyuk and J. Rebek Jr., J. Am. Chem. Soc. 124 (2002) 12074-12075. [28] A. Shivan>iik and J. Rebek h., Angew. Chem., Int. Ed 42 (2003) 684-686. [29] J. Chen and J. Rebek Jr., Org. Lett. 4 (2002) 327-329. [30] S. Mecozzi and J. Rebek Jr., Chem.-A Eur. J. 4 (1998) 1016-1022. [31] T. Heinz, DM. Rudkevich and J. Rebek h.. Angew. Chem., Int. Ed 38 (1999) 1136-1139. [32] A. Shivanyuk, A. Scarso and J. Rebek Jr., Chem. Comm. (2003) 1230-1231. [33] S.K. Komer, F.C. Tucci, DM. Rudkevich, T. Heinz and J. Rebek Jr., Chem. Eur. J. 6 (2000) 187-195. [34] Under these conditions, 2-Cl-butane, 2-butanol and 2-pentanol all form encapsulation complexes with 2 guests inside. The complexes are diastereomeric: one meso-form including two guests of opposite handedness and those with two molecules of R or S 2-butanol. The diastereomeric capsules are formed in equal amounts. [35] J. Rebek Jr., B. Askew, M. Doa and P. Ballester, J. Am. Chem. Soc. 109 (1987) 4119-4120. [36] Hyperchem TM. Release 7. Hypercube Inc. 2002.

Progress in Biological Chirality G. Palyi, C. Zucchi and L. Caglioti (Editors) © 2004 Published by Elsevier Ltd.

Chapter 23 Serum Albumin and Natural Products Lorenzo Di Bari, Silvia Ripoli and Piero Salvadori* AmbiSEN- Centro di Alta Tecnologiaper lo Studio degli Effetti di Agenti Nocivi, Dipartimento di Chimica e Chimica Industriale - Universitd di Pisa, Via Risorgimento 35, 1-56126 PISA, Italy psalva@dcci. unipi. it

1.

Introduction The fundamental role of serum albumin (from now on SA, HSA for the human protein, and BSA for the bovine [1, 2]) as a vector for both endogenous and exogenous compounds has been recognized for a long time. By non-covalent binding to poorly soluble organic molecules or sometimes to metal complexes, it may cause meaningful increase in the blood concentration of these systems, permitting absorption after ingestion or inhalation. Conversely, it buffers the concentration of soluble drugs, by reducing the amount of free molecules. This issue is particularly relevant for pharmaceutical research: lead molecules that show high activity in vitro may be less effective in vivo when binding to serum albumin is relevant. Knowing and predicting interactions between drugs upon co-administration is a key step for setting up therapeutic protocols; its understanding on a molecular basis may save time and experimentation on living organisms. For example, it was demonstrated that one of the reasons for the adverse effects resulting from co-administration of phenylbutazone and warfarin, derives from their competition for SA binding. The plasma concentration of free warfarin is normally kept very low because it is largely (up to 99%) engaged in albumin binding on site I (vide infra), thus, it displays only a fractional anticoagulant activity. When also phenylbutazone is present in the blood, it competes for SA site I, displacing warfarin, that becomes more available for its receptors, ultimately enhancing the risk of haemorrhage [3]. All of this matter has been enucleated and reviewed [4], but we shall address a specific issue, which does not appear to be completely focussed in the literature: what is the impact of substances commonly assumed in the diet on the binding sites of SA and consequently what may be the relevance of quality and quantity of food introduced during our meals towards the absorption of drugs. While progress has been made [4, 5] and can be envisaged in the field of pharmaceuticals (thus on drug-drug interactions), the role of molecules present in food needs to be stressed, which is the stimulus for this contribution. The characterization of the thermodynamics of ligand association to serum albumin has been faced with many experimental techniques, ranging from classical biochemical methods like ultracentrifugation to chromatography and to spectroscopy. Summarizing this enormous

272


wealth of work in a brief contribution is impossible and drastic choices are necessary: we shall limit our discussion to a few techniques, which respond selectively only to the mole fraction of SA-bound molecules, that is, which are intrinsically blind to a solution of the ligand alone. Since one of the most successful spectroscopies applied to investigate SA interactions is circular dichroism (CD), the issue of molecular chirality and even more specifically of induced chirality after the binding is of primary interest. Only a couple of other methods very recently introduced will be discussed at the end of the chapter in order to open up the perspectives for widening the knowledge in the field, through innovation. The text will be divided into two parts: sections, 2, 3 and 4 contain introductory information: the general framework of ligand-protein binding (and the chirality related issues), and some of the best characterized cases where albumin is involved in the transport of molecules contained in the food. In the second part, sections 5 to 7, the three experimental techniques, we chose to focus on, will be discussed by means of selected examples, which demonstrate some meaningful features of the methods themselves.

2.

Serum Albumin: Multiple Binding Sites Serum albumin (SA) is a globular heart-shaped protein made of three main domains, subject to some conformational freedom. It contains various grooves and pockets where smaller molecules can be hosted, ensuring its role of carrier protein [4]. These binding sites can exhibit more or less pronounced affinity to specific ligands: a given compound may fit particularly well in one pocket, leading to very stable non-covalent binding, but at the same time display secondary binding elsewhere. Moreover, different compounds may be allocated simultaneously in different sites: as a general reminder. Figure 1 depicts the matter.

cys34 metal ions

domain III

subdomain IIIA (Sudlow site II) ibuprofen digitoxin diazepam

domain I

subdomain IIA (Sudlow site I) warfarin phenylbutazone

domain 11

Figure 1. Structure of human serum albumin HSA with the two most important sites for drug binding [2]

Serum Albumin and Natural Products

273

Multiple binding deserves a brief discussion of the network of complex solution equilibria, which can take place when the ligands X and Y are in the presence of S A. If X or Y are mixed with SA separately, one can write Kx SA + X ^ ^ SA'X

..

Ky SA^Y ::;^=±^SA*Y While, when both are simultaneously present, on top of the one-to-one equilibria, one may expect also multiple binding, leading to the formation of the trimolecular adduct: SA+X^Y

:z^=^ SA^X^Y

^2)

Provided KXY >0, we can distinguish the three cases KxY = KXKY KXY > KXKY KXY < KXKY

Independent binding Cooperative binding Anticooperative binding

(3)

The latter two cases are examples of allosteric binding, which must be associated to a variation of the properties of one site upon the occupancy of the other one. This is usually made possible by a conformational rearrangement, following one of the interactions, whereby the macromolecule becomes better (cooperative binding) or poorer (anticooperative) fit to the other ligand. The extreme case KXY = 0, provided both KX,KY > 0, means that the ternary adduct SA^X^Y cannot exist, which indicates that X and Y insist on the same site, thus leading to competitive binding and to a displacement of one for the other ligand. Interestingly, multiple binding can be observed also for one ligand alone. A slight modification of the above equations holds ^; SA+X :;;=±r SA^X^ (4) ^2

SA+X—^SA^X^

(5)

Kl2 SA+X~^SA*XJ'X2 (6) SA*X^ and SA^X^ represent the adducts where ligand occupies site 7 or site 2 on SA, respectively. Once more the two binding processes can be independent or (anti)cooperative. In the case of multiple binding sites for one ligand, it is worth recalling that the two processes described by equations (4) and (5) are equilibria with common species (SA and X): therefore the ratio [SA^X'] K h -4 = —- = constant (7) [SA-X'] K, is independent of the concentration of X or of the molar ratio. This implies that any instrumental response where the signals of SA^X^ and of SA*X^ are superimposed will not change its appearance during the course of a titration and that even sophisticated methods like factor analysis will fail detecting the co-presence of different species: deviations from this

274


situation will be manifest only at the onset of the ternary adduct SA*X^*X\ whose concentration has a completely different dependence on fXJ. The approximation of independent binding should be used with great caution for SA, because its various sites may have very different affinities for a given ligand. Nevertheless, it is often employed through more or less questionable approaches. The most convincing method [6] is to plot the ratio of the moles of total protein (SAtot) to moles of bound ligand [By versus the reciprocal concentration of free ligand, l/[F\. If a linear relationship is found, statistical factors {viz. perfect independence and equivalence of the sites) are predominant in distributing the ligand among the n sites:

^

=^ ^ . i

(8)

[B] n [F] n All of these equations have been used for interpreting the spectroscopic titration data, in order to obtain the apparent affinity constant, or the number of sites, as well as for evaluating the kind of interaction between different compounds (independent/competitive/allosteric). 2.1 Binding kinetics A rough picture of the ligand/SA interaction process from a dynamic point of view is necessary to better understand some of the techniques used to investigate the freeôund equilibrium. Indeed, most spectroscopic techniques (notably optical absorption and emission spectroscopy) are surely/ay/ in the sense that the spectrum of a mixture containing the ligand and the protein are the weighted sum of contributions, arising from the free molecule and from the complex(es). By means of suitable methods, like stopped flow or T-jump, one can monitor the evolution of the spectra toward thermodynamic equilibrium and thus obtain the kinetic association/dissociation rates. On the contrary, NMR may be on the edge: depending on several factors discussed below, the dynamic process may appear fast or slow. The non-covalent ligand binding of SA is made ftilly reversible by a comparatively fast association/dissociation kinetics. Although dynamic data are much less abundant than those concerning equilibrium constants, in many cases dissociation rates in the range 1-20 s" (at room or physiological temperature) have been reported. This implies that the ligand can be released promptly from SA in proximity of a receptor with higher affinity, which ensures a role of neutral carrier for HSA. Among the methods for studying the dynamic association/dissociation, there are stopped flow [7], chromatography, NMR [8] and EPR [9]. Much more recently, the equilibrium process has been investigated through various types of sensors: they constitute a breakthrough in the field of drug/protein binding which is likely to bear major consequences in thefixture.A brief review of these applications will be found at the end of this chapter. The dissociation and association rate constants, A:"'"^ and F"", are linked together with the equilibrium constant through the relation: lass

K =^

(9)

' In the case of multiple binding the concentration of bound Ugand equals the sum of the occupancy of all sites [B] = ^[SA^X']-^^ [SA •X' •X^]-\-.. Here the first term refers to single occupation, the second term to the ternary adduct and - at least in principle - this should go on with all possible higher orders. For the concentration of free ligand, we have [F] = [X].


275

Assuming that the association process is controlled by diffusion, we can assess jâss ^ io^M"^s\ which allows us to have a first estimate of A:^' ^' ^ from the knowledge of the equilibrium constant only [10]. However, it should be borne in mind that this argument might be valid only if binding does not involve a more complex process, like, for example a structural rearrangement of either the ligand or SA, which would sizably reduce F^^ NMR is surely one of the most popular spectroscopic techniques in these days, in spite of the fact that high field instrumentation may be very expensive. A given dynamic process is defined slow or fast on the NMR timescale if its rate constant is respectively small or large compared to half the difference in angular frequency (measured in Hz), nAv^ between the resonances corresponding to the same nucleus in the two exchanging forms. In the case of fast exchange, one observes an NMR spectrum with only one set of resonances, where each line is located at the weighted average between those of the two forms; on the contrary, for slow exchange, one has two distinct sets of lines, with integral rafios equal to the mole ratio of free and bound. A typical complexation shifl; (difference between the chemical shift of the bound and free forms) for a proton can be of the order of O.lppm, which at 500MHz means TtAv =J60Hz, implying that the free/bound exchange rate constants of about 100 - 200 s"^ are likely to fall in the so-called intermediate exchange, where neither of the two situations depicted above is quite correct and one observes extensive line broadening. Naturally, the boundary for intermediate exchange depends on the complexation shifts. In fact, the same process may in principle appear slow for a given nucleus and fast for another. Moreover, it should not be forgotten that the frequency difference quoted above is a function of the external magnetic field and that on increasing its strength, fast processes may appear intermediate and finally slow.

3.

Albumin and Food Although the role of albumin in the circulation of drugs in the human body was largely investigated [4], less attention has been addressed to the constituents of food. Here we shall briefly summarize what is known about the interaction between SA and organic compounds often taken up during a meal; in particular flavors, toxins and antioxidants. The ability of SA to bind reversibly small molecules and its abundance in the blood gives this protein the crucial role of bio-carrier of nutrients. This is particularly relevant for two classes of molecules: first, apolar ligands, that may be practically insoluble in water, and whose enhanced plasma concentration arises from protein binding (we remind that the SA pockets have a hydrophobic character); second, degradation-prone molecules, which may be protected - notably from oxidation - by the polypeptidic environment of SA. Another important active role of SA is to mask some properties of the guest, by buffering its concentration [11]. On a more academic ground, it must be considered that, since the binding properties and the structure of albumin are well described in the literature, this protein constitutes a very good model to reproduce food proteic matrix. For this reason, albumin was chosen in investigations of flavors-protein interactions [12, 13]. The rate of release of volatile components is one of the aspects (beyond their composition) determining the perception of a fragrance and a taste, and consequently also the acceptability of food. Proteins are generally tasteless but they do influence the flavor of food by modulating the concentration of free (volatile) odorants through binding [13]. A work of Burova et ah [14] very recently brought our attention onto two interesting aspects of the flavor-protein interactions, which will deserve

276

Progress in Biological Chirality CHO

CHO

4,5,6

vanillin vanilla essence

„ ketones ^'3' '!^'?'^ components of cheese or oil

benzaldehyde ^^^^ ^,^ J„^

Figure 2. Chemical structures of some flavours, whose HS A interactions were studied

future investigations: a) prediction of the protein conformational changes in solution can provide information about the accessibility to its hydrophobic pockets, influencing the reversibility of odorant binding; b) little is known about the competition between proteins in mixture for a particular odorant and vice versa about that of odorants for a single protein. These authors demonstrated the effects of the acidic and thermal denaturation and aggregation of proteins (processes typically occurring during the yogurt production), on the binding of volatile compounds. As specific examples, the studies about alkylketons can be considered. Their interaction with SA was studied by means of different techniques from optical spectroscopies to differential scanning calorimetry (DSC) or diffusion NMR (see also below) [12-14]. From these studies, it can be inferred that the hydrophobic nature of the ligand-S A binding is the driving factor in the case of alkylketons, so that 2-nonanone has major affinity for SA with respect to shorter chain or internal ketones [12]. A few compounds whose interactions with SA have been studied are displayed in Figure 2. Although it may appear surprising, toxins are very common in our food. They may have very different origins: they may be products of more or less desirable fermentations or of cooking processes, or be present as residues of food preservatives or of pesticides, just to make a few examples. A particular interest was devoted to the ubiquitous contaminants ochratoxins (Figure 3) [15]. These compounds, derivatives of isocoumarin and Lphenylalanine, are released by Aspergillus and Pennicillum fungi found in badly stored food or feed. Ochratoxins are responsible for toxic effects like carcinogenity, mutagencity or nephrotoxicity; a wide literature on this topic is cited in reference 15. The high affinity of ochratoxins for plasma proteins (Kaff >10''^ M"^) causes two very relevant effects [15b,c]. First, is the prolongation of their half-life time in plasma, which is an example of the protection operated by SA binding toward metabolic evolution (and degradation) of ligated species. Second, their passive absorption from the digestive system is active also when the plasma concentration of the toxins is higher than in the intestine, as a consequence of the fact that the largest fraction of these compounds in plasma is captured by SA and subtracted by this way to the free osmotic equilibrium through the intestinal epithelium. These findings very recently stimulated Simon et al. to study the interaction between HSA and ochratoxin A (OTA), the most toxic of this class, and its derivatives by means of optical (absorption and fluorescence) spectroscopy [15b-d]. The main results of these studies can be summarized: • OTA binds to HSA as a dianion; • only two sites of HSA are available for OTA-dianion and each site can host only one

Serum Albumin and Natural Products [ f ^

COOH O

OH

277

O

Ochratoxin A (OTA)

f r %

COOH O

OH

O

Ochratoxin B (OTB) f f ^

COOEt O

OH

O

Ochratoxin C (OTC)

Figure 3. Structures of ochratoxins

molecule. The affinity constants for the two sites differ by one order of magnitude (K^=5' 10^M-*;K^=1-10^M-^); • through Trp214 fluorescence quenching and through warfarin competition, subdomain IIA was identified as the primary binding site, sub-domain IIIA as the secondary one. Another example is offered by uremic toxins (for example indole-3-acetic acid, indoxyl sulfate or hippuric acid), which accumulate in plasma of patients with chronic renal diseases [16]. This accumulation was related to SA binding (some uremic toxins bind to site II but also site I can be occupied, that is the case of 3-carboxy-4-methyl-5-propyl-2-fljran propanoic acid) and explains why minor plasma concentration of drugs as warfarin and furosemide is found in these patients. The study of Sakai et al [16b] shows that, in addition to the competitive displacement, other mechanisms (allosteric interaction or site-site interaction) can take place. Vegetable food and fruit may be very rich in antioxidants like e.g. flavonoids (and more in general polyphenols, see Figure 4) or carotenoids, and through this way they enter our daily diet. The attention of scientific research was directed to antioxidants largely because of their potential impact on health [17]. The protective action of vegetable food against cancer and coronary diseases has been associated with the intake of these compounds, notably acting as radical scavengers [18]. In order to better understand the activity of these compounds, some main aspects must be taken into account: their bioavailability, their metabolism, and the masking of their antioxidant activity. Serum albumin plays a crucial role in the first and third points. It is known that, in spite of the presence of polar groups, flavonoids are very scarcely soluble in water and their transport in plasma is conveniently mediated by albumin [19]; the same happens for carotenoids, essentially water insoluble [20, 21]. This would Hmit their availability in aqueous medium and thus in plasma, without a good carrier as it is albumin.

278


flavonol-glycoside anthocyanidtn

Figure 4. Structures of main flavonoids

Moreover, it was shown that albumin or other proteins can decrease the antioxidant activity of flavonoids [19]. These findings throw the basis for more detailed investigations on the flavonoids-SA interaction in order to identify which factors promote flavonoids absorption without reducing their activity. Site I (see Figure 1) is responsible for the binding of flavonoid, as well as of natural coumarins, which indicates possible interference between these compounds, and warfarin, phenylbutazone and other drugs which have affinity that site. The equilibria in a ternary solution HSA/warfarin/quercetin (a flavonol) have been investigated by means of fluorescence spectroscopy and demonstrate that the interaction is not a pure competition, and indicate that in the same site warfarin and quercetin may be both simultaneously accommodated [17c]. In the case of carotenoids it was found that the presence of carboxylic groups promotes the binding to HSA. Carotenoids form structured aggregates where HSA can act as asymmetric template on which a chiral self-assembly of carotenoids grows with increasing the carotenoid/HSA ratio [22]. This result is promising for achieving the transport of higher amounts of carotenoid derivates in aqueous phases. More on carotenoids will be discussed in section 5.3 below. Another important class of diet-related compounds that bind S A are fatty acids. There are many sites on this protein where this interaction can take place, and the number of long alkyl chain molecules that can be allocated on SA is variable, depending primarily on the length of the chain. X-ray crystallography determined the location of these sites and the conformation of the fatty acid molecule, which appears often bent or folded [23]. This binding is of utmost relevance, because of possible competition with drugs and of allosteric interaction with the other sites. Wainer and Noctor demonstrated these effects by means of affmity HPLC,


279

showing that site II is the primary binding site for octanoic acid, thus interference with profens (by direct competition) has to be expected, while allosteric effects with site I (e.g. hosting warfarin) can also occur [24]. Moreover, the effect of fatty acid binding induces a conformational change in SA, which exposes Cys-34 SH-groups to oxidation. As shown in Figure 1, this is one of the sites where metal cations, and in particular Cu(II), are hosted (often called site V). It has been demonstrated that the oxidized SH-Cys34/Cu(II) is an effective catalytical oxidant towards, e.g., ascorbate. The role of radical scavenger recognized for SA is thus reverted into a prooxidant [25].

4.

Albumin and Chiraiity In a book on biochirality, the issue of the role of molecular dissymmetry must occupy a central role. In fact, as more or less explicit, many of the molecules transported by albumin are chiral and the other ones assume a temporary chiraiity when they are bound to the polypeptide. Surely, a majority of drugs, nutrients, flavors are dissymmetric and usually the two enantiomers exhibit different biological activity, because of selective interaction with the receptors as well as with the transporters. We may recall a couple of facts; 1. Usually albumin displays high selectivity towards enantiomeric ligands, the affinity being very different for the two enantiomers. Indeed, the two mirror image structures of a drug may find place on different sites, as is the case of (R) and (S)-warfarin [26]. Owing to the broad spectrum of organic and inorganic systems that can be accommodated in the various grooves, pockets or simply on the surface of this protein, the scope of chiral discrimination is unusually wide. This fact, coupled with the long term stability of S A justifies the use of this protein in liquid chromatography as a modifier to obtain chiral stationary phases. Indeed, HPLC [4] and related separation techniques are most common for studying SA binding, and this whole subject will not be treated in the present contribution. It is worth observing that two enantiomers are nothing else than two different molecules and that the proper context to describe their SA binding is that discussed in section 2, with the equations (1), (2) and (3). 2. Albumin can induce chiraiity on intrinsically symmetrical ligands, by providing a symmetry-breaking environment. In both cases, induced chiraiity follows, which is a subject that will be discussed below. Although in normal physiology albumin plays the role of a molecular chaperon, deputated not to alter the transported compounds, in some cases it may exhibit interesting properties in reactivity. It has been reported that BSA binds differently the two enantiomers of binaphthol and ketoprofen, altering their photochemistry. As the absorption spectra of the diastereomeric adducts BSA/enantiomer are different, it is possible to irradiate selectively only one species, causing its photodegradation. The other isomer remains virtually intact and by dissociating the HSA/drug complex, the unreacted enantiomer can be collected [27]. This finding does not seem to have met further application to date.

5.

Induced Circular Dichroism Circular dichroism (CD, measured through Ae) is a property of chiral nonracemic molecules, because it is equal to the difference in molar extinction toward left and right

280 Progress in Biological Chiralit> circularly polarized light, / and €^, respectively: only a species with well-defined handedness can discriminate between the two chiral components of the electromagnetic radiation. Such a difference can be found non-vanishing only when s^,6^>0, that is, in proximity of an absorption band. Moreover, it can assume positive or negative sign depending on the relative magnitudes o f / and 8^ [28]. These features will account for the large popularity of electronic CD (i.e. relative to the spectrum of electronic transitions) in the field of ligand/SA interactions [29]. SA displays itself a strong CD spectrum below 300 nm, being essentially transparent at longer wavelengths. On the contrary, many conjugated organic chromophores are endowed with absorption bands well above that limit, thus do not suffer from interference. Achiral ligands do not show any CD when they are in the free state; on the contrary, once they are bound to SA, they are surrounded by a chiral environment. In the case of achiral ligands CD responds selectively only to the mole fraction of bound drug. For chiral non-racemic molecules, we can notice that CD is generally more sensitive than isotropic absorption to variations of the molecular environment. Thus, by taking the difference between the spectra of the ligand in the presence and in the absence of S A one can extract the contributions of the bound form. Finally, the sensitivity of CD to conformational variations of proteins [30] opens the way to the simultaneous investigation of the binding process and of the structural rearrangements occurring on SA. We shall concentrate on the case of an achiral chromophoric ligand, discussing the various processes that may be responsible for optical activity of the SA-bound form. The three mechanisms detailed below are not mutually exclusive and in certain cases may concur in determining the observed spectrum. 5.7 Coupling between albumin and the host Whenever an intrinsically symmetrical chromophore is embedded in a chiral environment, its electronic transitions may exhibit a CD (induced CD or ICD). Various mechanisms can be invoked to account for this phenomenon, but the one which often appears dominating is the coupling between electric or magnetic dipole transitions of the chromophore and those (mainly electric) localized on its macromolecular surroundings. In this case, the main contribution must be expected stemming from the aromatic groups on the amino acids side chains of SA. If this mechanism holds on an isolated transition of the ligand, a monosignated Cotton effect must be envisaged." Ligands characterized by a complex absorption spectrum, featuring several transitions, are expected to give rise to separate - and possibly of different sign Cotton effects, where the maxima in absorption and CD practically coincide, as shown in Figure 5. This may be no longer true when an absorption band is the envelope of several contributions. A special case is offered by magnetic-dipole-allowed electric-dipole-forbidden transitions: they are usually endowed with very small extinction, to the point that they may not show up in the isotropic UV-VIS spectrum, but, once the ligand is surrounded by the chiral environment of SA, they may give rise to intense Cotton effects. Predicting the sign of " A fundamental conservation law of chiroptical spectroscopy predicts that the total integral through all the spectrum vanishes. In the case of the coupling between albumin aromatic side chains and chromophoric ligands considered here, two contribution of opposite signs canceling each other are expected: one is centered around the wavelengths where the Ugand absorbs, while the counterpart will be located together with the CD of the protein and it is often not observed, possibly being offset by the intrinsic contribution of SA.


281

CD [mdeg] absorption

wavelength [nm]

Figure 5. ICD (solid line) and absorption (dashed line) spectra of the 1:1 complex between a synthetic coumarin and HSA. A single Cotton efifect is apparent alUed to the 400 nm transition of the conjugated chromophore [31]

the SA-ligand coupling is in general hard: it may depend on the structure of the macromolecular pocket and the detailed orientations of the aromatic sidechains around it. Usually, a heuristic approach is taken, based on the observation of the onset of the spectrum: from the mere existence of an induced CD, one has the qualitative evidence of a ligand/protein interaction; thereafter, one can quantitatively analyze the magnitude of the Cotton effects as a fiinction of the Hgand concentration (or of the ligand/albumin mole ratio) [32]. The analysis of ICD data is a very powerful tool for analyzing the ligand/SA interactions. Especially by means of the probes indicated in Figure 1, it becomes possible to identify the site where the ligand X is allocated and its possible allosteric interactions. To this end, the ICD band due to some electronic transition located on X in a mixture X/SA is followed while a specific probe, Y, is added stepwise. If the two ligands have affmity for the same site, a competitive equilibrium will take place and one must ultimately expect the complete disappearance of ICD of the ^S^^-Xadduct, with the simultaneous growth of that of SA^Y. This case represents the displacement of X operated by Y. The position of this equilibrium in the various steps of a titration depends on the relative affinities of S A for the two ligands, i.e. on the ratio Kx/Ky. If X and Y bind on different sites, one of the three cases represented in Equations (3) will be found. To have a full picture, cross titrations can be performed: the 1:1 mixture SA/X is titrated with Y, and/or the mixture SA/Y is titrated with X. The resuhs are compared with the direct titrations with X and Y separately. The CD data must be followed at wavelengths characteristic for SA»X or SA*Y or appropriate difference spectra must be taken. The plots of ICD versus the concentration of X or Y in the cross and direct titration are compared: if they match, and independent binding must be envisaged, if deviations are observed, allosteric interaction (cooperative or anticooperative) must be invoked. Quantitative analysis of the magnitude of ICD as a function of the concentration of the added ligand may yield the association constant, in analogy with spectrophotometry. Owing to the fact that, at wavelengths where SA is transparent, ICD is selectively sensitive to the bound ligand, one can write ICD =k[SA*X] (10) with k a. proportionality constant dependent on the pathlengts and on the molar dichroism,

282


here treated as a phenomenological parameter. By observing that the total concentration of SA, CA, and of ligand, cx, must be conserved, we obtain fSA*XJ=-ICD/k fSAJ = CA'ICD/k [X] = cx-ICD/k

(11)

we can transform equation (1) into K-

(^^^ (c^-ICDIk)(c,.-ICDIk)

(12)

which yields ICD = | [ c , + c , + / : - ' - 4{c, + c, +K'y-

4c,c^ j

(13)

A titration curve of ICD versus cx (at constant CA) can thus be fed into a least square fitting software, which can derive the affinity constant K. It must be observed that when association is very strong, K^ may become vanishingly small compared to (CA+CX): in such a case the equation above reduces to ICD = kcx which describes a linear growth of the ICD amplitude and does not allow one to determine K. One can also rearrange equation (12) and neglecting the term (ICD/kf obtain Â^X _ 1 ^ ( ^ t f x l ICD kK k

(14)

Thus, plotting the first term versus (CA^CX), a straight line with slope 1/k and intercept 1 (kK) is expected. This linearization is possible only if (ICD/k) = [SA*X] is small, i.e. when the association is not very strong. Therefore the two methods, namely the fitting through the ftill equation (13) and through its linearization (14) have the same limitation with respect to the relative size of AT, CA and cx. 5.2 Exciton coupling I: conformational chirality The most popular approach to understanding CD is exciton coupling, which is a typical feature of two (or more) interacting chromophores, as shown in Figure 6. In the extreme, paradigmatic case of two identical chromophores, this leads to a split dissignated CD band, with positive and negative components. It is called an exciton couplet and defined positive or negative according to the sign of the low energy (long wavelength) component. The origin of this phenomenon is a through-space dipolar interaction between electric dipole allowed transitions, which may thus be located on the same as well as on two different molecules. In this first part, we shall discuss the intramolecular case. In the context of SA binding, the most notable example is offered by bilirubin, one of the products of the degradation of the heme porphyrin.

Serum Albumin and Natural Products ^ 3 9 5 (+38)

-

283

CD

/ \ negative couplet

\ I \

i

\ 1

\l

L-

A

43t (-72)

UVAîs

Wavelength (nm)

Figure 6. A typical bisignate CD spectrum due to exciton coupling in a bischromophoric system, obtained by derivatizing ponasterone A with a coumarin derivative [33]. We can appreciate that the negative chirality defined by the chromophores is reflected in a negative couplet

Bilirubin

Bilirubin is an achiral molecule made of two symmetrical moieties with extended conjugation, held together by a conformationally flexible bridge. Owing to intramolecular hydrogen bond, it assumes a bent chiral conformation, referred to as ridge-tile, whose two enantiomers are in fast exchange. In the presence of HSA, a 1:1 adduct is formed with one enantiomeric conformation of bilirubin: this displaces the equilibrium in an asymmetric transformation and a neat ICD is observed, with the shape of a positive exciton couplet. This sign is unambiguously related to the sense of twist of the transition dipoles located on the two chromophores, as depicted in Figure 7, and this indicates that the P conformer of bilirubin has higher affmity for HSA [34]. A closely similar situation is also found for curcumin, the major yellow dye of turmeric [35]. The powdered rhizome of this plant is a common ingredient in Asian food and used in traditional oriental medicine.

Figure 7. Bilirubin: enantiomeric conformations in equilibrium (reproduced with permission from Ref [34b])

284


Curcumin features a P-diketone connecting two symmetrical and conjugated moieties. Since it is most stable in its tautomeric form, electron derealization covers the whole molecule, which determines a long wavelength absorption, centered at 460 nm at neutral pH.

Curcumin

It forms a 1:1 adduct with HSA, which at high pH (pH=9) displays an exciton-splitted CD, shown in Figure 8, whose crossover point is about the absorption maximum of neutral curcumin. This spectral feature clearly calls for a coupled dipoles mechanism and a superficial interpretation may indicate that two molecules of curcumin are hosted on the same HSA, leading to a supramolecular chiral aggregate, as will be discussed in the next section. Such a view clashes against the simple evidence of a 1:1 complex, whereby no intermolecular

31

30

29

28

350

27

26

Wavenumber/10''(cm") 25 24 23 22 21

400

20

19

18 17

600

Wavelength (nm)

Figure 8. pH-dependent CD and absorption spectra of curcumin bound to HAS (reproduced with permission from Reference [35b])


285

coupling can be invoked. The ultimate explanation of the CD spectra is that: [35] • upon binding to HSA in alkaline solution, curcumin must adopt a folded conformation where the P-diketone is preserved in order to ensure the CH2 hinge between the two molecular halves; • as in the case of bilirubin, this conformation is chiral and one enantiomer is preferred; • accordingly, in the HSA adduct, delocalization is restricted with respect to the free, linear and fully conjugated curcumin and a blue-shifted absorption maximum (and CD crossover) should be expected; • owing to the high pH a deprotonation of the phenol hydroxyls occurs, therefore the two interacting molecular halves must be represented by the corresponding anions; • the absorption spectrum of the anionic moiety is accidentally similar to that of whole, neutral curcumin. This points justify the position of crossover, as the result of the concurrent blueshift (operated by conjugation restriction) and redshift (caused by deprotonation). As depicted in Figure 6, a left-handed arrangement of the transition dipoles must be envisaged, in agreement with the positive couplet at 460 nm. The optimal conformation of curcumin bound to HSA at high pH, proposed on the basis of the CD spectra and from a docking investigation on the HSA site I is represented in Figure 9. Interestingly, at neutral pH, a completely different situation is found, where the ligand exhibits a very weak ICD. The interpretation of this spectrum is still unclear: Reddy et al [36] reported that two molecules of curcumin may find place on HSA on two sites with very different affinity: the one characterized by a K=2.0'10^M'^ that is detected only through fluorescence, the secondary site is revealed through ICD (which appears insensitive to the occupancy of the other one). This situation looks particularly intriguing and may deserve fiirther investigation, although it should be observed that in the determination of the affinity constant by ICD, a modified Hill equation was used, thereby deriving log K from a logarithmic plot. This requires great confidence in the data, because a small error in log units implies a very large variation in the assessed K. Moreover, Hill equation is a questionable choice for albumin, owing to observed the strong correlation between the occupancy of the various sites (allostery) and the large differences in affinity constants. 5.3 Exciton coupling 11: supramolecular chirality As we have already seen, albumin is a multisite carrier, able to bind several guests

. ^ i ^ ^ ^

J^ «*^

Figure 9. Most likely structure of curcumin bound to HSA site I (reproduced with permissionfromReference [35a] and [35b])

286


simultaneously. In such an occurrence two identical or diflferent ligands can be accommodated in a close neighborhood, with a well-defmed geometrical arrangement. It is well known, for example, that SA is a fatty acids carrier and indeed, X-ray diffraction of albumin saturated with a large excess of different fatty acids revealed that there are at least seven sites distributed onto all the three domains of SA [23]. These locations when occupied, induce a supramolecular chirality of the ligands. The saturated alkyl chains of fatty acids have vanishing absorption above 300 nm and cannot give rise to Cotton effects. Polyene chains, as found in carotenoids, may be considered as having more or less the same structural and binding characteristics of fatty acids, were it not for the pronounced flexibility of the latter ones as opposed to the stiffness of the former ones. From the UV-VIS spectroscopy point of view, carotenoids are excellent chromophores, with very intense and red-shifted absorption bands, due to electric-dipole allowed K- 7t* transitions. If two (or more) carotenoid molecules are hosted in nearby sites on SA, intermolecular exciton coupling can occur, giving rise to the bisignated couplet feature. This is what found for crocetin, one of the components of a common spice, saffron, obtained from Crocus sativiis.

Crocetin is a dicarboxylic acid, with a 16-carbon atoms conjugated chain connecting the two terminal groups; a good starting point for predicting its binding sites on SA is to look for the seven locations where XRD determines palmitic acid (also a hexadecanoic system) [37]. Observing that in the seventh site (in the classification of reference [23]), palmitic acid is in a strongly folded conformation, which is unrealizable for the stiff carotenoid crocetin, its role can be excluded. In the other six, Simonyi and coworkers docked crocetin molecules and, assuming that what dominates the ICD spectrum (Figure 10) is a pairwise interaction (i.e. neglecting higher orders), they evaluated the chirality of the geometric arrangements between all possible pairs. The result of such investigation is that two pairs of sites, namely (3,4) and (4,5) can be held responsible for most of the exciton-coupled ICD seen with crocetin/SA. Interestingly, on displacing most of the carotenoid with palmitic acid, the bisignated doublet vanishes, leaving place to a monosignated Cotton effect, centered on the absorption maximum of crocetin, i.e. where the doublet had its crossover: this is what one expects from one chromophoric molecule bound to SA, as described above.

6.

NMR Methods: Slow Tumbling and Diffusion One of the most dramatic changes brought about on a ligand by interacting with SA is about the motional dynamics. Both translational and tumbling motions of free small organic molecules can be slowed down by 1 to 3 orders of magnitude in SA complex. The consequences become apparent only in spectroscopies sensitive to the translational or orientational diffusion, like are some contemporary high resolution NMR techniques: a long rotational correlation time is associated with high relaxation rates (manifested through broad lines and short Ti); moreover, it is nowadays very common to equip NMR probes of high field instruments with gradient coils, which allows the easy access to diffusion coefficients.


287

Figure 10. Absorption and CD spectra of mixures of HSA and crocetin as a function of the molar ratio of the two species (reproduced with permission from Reference [37])

When the ligand is in fast (on the NMR timescale) exchange between a free (F) and a bound (B) state one can write for a given property ^

r^'-^(l-x)'f^x'i^.

(15)

where x is the mole fraction of the bound form. Taking advantage of this equation (when it can be correctly applied), and setting up appropriate experimental protocols for extracting the diffusion coefficients or relaxation rates (which are dynamic properties), one can assess the thermodynamic affinity constant. Translational diffusion coefficients, D, can be determined through pulsed gradient spin echo (PGSE) experiments, also referred to as DOSY (Diffusion Ordered SpesctroscopY) [38]. In this case, the limiting quantity D^ can be approximated by the one of the protein alone, i.e. assuming that the bound ligand assumes the same translational diffusion of SA. The mole fraction x of equation (15) is dictated from the equilibrium constant, e.g. through an equation closely related to (13). A global fitting of titration data as a function of total albumin and ligand concentration affords the affinity constant. Such an approach has been used for salycilate,"' which is known to have several binding sites on SA. In a first approximation, these /i-sites were considered fully equivalent and independent: this amounts to saying that the effective concentration of the host is w-times the stoichiometric albumin concentration CA, thus in equation (13) one should replace n*CA for CA and determine n, as well (or equivalently consider CA as a fitting parameter). The results reported for this analysis are not completely satisfactory: a very large number («>30) of sites with very low affinity is "* In the case of salycilate, D^ is found concentration-dependent, and must be described through an empirical polynomial of the form

^FC^X)-^

^p^x^

where Gp are coefficients to be determined by a fitting procedure and cx is the total ligand concentration.

288


found, while the experimental trend of the data is poorly reproduced by the fitting. Apparently, there are two orders of problems in this approach: first, the approximation on equivalent and independent sites is very crude for a molecule known to have sites of very high or rather low specificity and to undergo profound conformational rearrangements with ligand binding, which justifies its allosteric behavior; second, the marked concentration dependence of the diffusion coefficient of free salycilate demonstrates that this molecule is subjected to other solution equilibria (e.g. dimerization), which are completely overlooked. These observations do not diminish the importance and scope of diffusion measurements for protein binding, which will likely strongly increase in the fiiture, owing to the bursting number of NMR probes equipped with gradients and to the growing interest toward DOSY. Measuring proton longitudinal relaxation rates by inversion recovery is one of the easiest NMR experiments, still, on observing that Ti is a function of the molecular tumbling rate, we realize that it must assume completely different values for the free and bound ligand. In order to perform a rapid screening of pharmaceutical lead molecules, it has been proposed to use a competition essay for HS A with tryptophan as a probe. A set of solutions containing 1 part of HSA, a 12.5-fold excess of L-tryptophan, and the same amount (4-fold) of each lead was subjected to a selective inversion recovery (i.e. an experiment when only the Trp C2-H is inverted by means of a shaped pulse and after a fixed mixing time the whole spectrum is acquired using a hard, non selective 90° pulse). The inverted tryptophan resonance has a faster recovery (shorter Ti) in the presence of a larger fraction of bound Trp: the lead molecules which displace it more efficiently from HSA will induce a lengthening of Ti of the probe resonances. Consequently, they will appear increasingly negative, as shown in Figure 11 [39]. The slowed tumbling of bound ligand has been exploited also to derive information of the structural rearrangement a ligand may undergo on protein binding, with the so-called transferred-NOE measurements (e.g. tr-NOESY) [38]. It is known that magnetization transfer processes are much more effective in slow reorienting systems than in small free organic molecules, in fact for the latter ones small and positive (i.e. of opposite sign to the diagonal in a NOESY) NOE's are detected, while they are large and negative for the former ones. If two protons I and S are far enough (4A or more) in the free ligand, they are expected to have a

R«for«nc«

27$583 276708

K o a 11 ^ M ( 9 8 . 2 % ) KO=10MM(98.4%)

Ko « 9 M M (98.5%) Ko a $ MM (99.0%) KD s 5 M M (99.2%) Ko ~ 2 M M (99.716) KD « 1 M M (99.8%)

Figure 11. Recovery of the C2-H resonance of tryptophan after a selective inversion pulse followed by a delay T=0.96 S, in the presence of the same amounts of the lead molecules indicated by the company codes and of HSA. The dissociation constants of each lead from HSA are reported below. The leftmost spectrum, labelled with an asterisk was obtained in the absence of any lead and used as a reference. Reproduced with permission from Reference [39]


289

vanishing cross-relaxation rate ois^^^, manifested by a null steady state NOE or no cross peak in a NOESY-like spectrum. If upon binding to SA a ligand rearrangement occurs, bringing I and S nearer, ais^''""'*

7.4 7.2 7.0

O 6.8

• D-tyrosine in H2O A L-tyrosine in H2O

j J1

a U 6.6 6.4

r

•

4

^

i

t

•

i ^

1

6.2

T i

I

P99%, >99.5% ee 10th round: >99%, >99.5% ee Obtained alcohol was used as an asymmetric autocatalyst for the next round. Scheme 2

Asymmetric Autocatalysis, Absolute Asymmetric Synthesis and Origin of Homochirality of Biomolecules 357 asymmetric autocatalysis. When (*S)-2-(2-/-butylethynyl)-5-pyrimidyl alkanol 2c with >99.5% ee was used as an asymmetric autocatalyst, (S)-2c with >99.5% ee was formed in a yield of >99% [12]. One of the avantages of asymmetric autocatalysis is that the structures of the asymmetric autocatalyst and the product are the same. Thus, the product and the initial autocatalyst of the first round was used as an asymmetric autocatalyst for the next round. Again, the product (S)-2c and the initial autocatalyst had ee of >99.5% and the yield of the newly formed (S)-2c was >99%. Even after tenth round, the yield of 2c was >99% and the ee was >99.5%. The amount of (5)2c has automultipHed by a factor of ca. 60 million times during these 10 rounds. Thus, 2alkynylpyrimidyl alkanol 2c was found to be an asymmetric autocatalyst with almost complete enantioselectivity and very high catalytic activity. 3.

Amplification of Enantiomeric Excess by Asymmetric Autocatalysis Surprisingly, the ee of pyrimidyl alkanol was found to increase in asymmetric autocatalysis [9a]. When pyrimidyl alkanol with low ee was used as an asymmetric autocatalyst, i.e., the ee of the product (including the original autocatalyst) was higher than that of the original catalyst. To take advantage of asymmetric autocatalysis with amplification of ee over nonautocatalytic amplification of ee [13], the product of one run was used as an asymmetric autocatalyst for the next run. Thus, extremely low ee of pyrimidyl alkanol was amplified to very high ee by consecutive asymmetric autocatalysis. Asymmetric autocatalysis of (*S)-pyrimidyl alkanol 2a with 2% ee gave (iS)-2a with 10% ee (Scheme 3). When, (*S)-2a with 10% ee was used as an asymmetric autocatalyst for the next asymmetric autocatalysis, pyrimidyl alkanol 2a with an increased ee of 57% was obtained. The subsequent consecutive asymmetric autocatalysis using the product as an asymmetric autocatalyst for the next round gave (^-pyrimidyl alkanol 2a with 81 and 88% ee, respectively. Thus, the overall process is the asymmetric autocatalysis of (*S)-2a starting from 2% ee with significant amplification of ee to 88% and with the increase in the amount without the need for any other chiral auxiliary [9a]. This stands as the first example of an asymmetric

Asymmetric autocatalysis with amplification of ee

2a-c 2c:R=f-Bu-^

lowee ca. 0.00005% ee (2c)

[Asymmetric autocatalyst]

2a-c high ee >99.5%ee(2c)

1 +

/-Pr2Zn Scheme 3

358


-yA

1

|i(S)-2c

==-

^(R)-2c

(Hi^ 600000 H

o

1 400000 H

1 200000 A

J yT

n i'mmmty. initial conditions

fmmsss'. r run 1 57% ee

/v..

_ 1

iffly.

WL)

run 2 run 3 99% ee >99.5% ee

ca. 0.00005% ee Fig. 1. Asymmetric autocatalysis with amplification of ee autocatalysis with amplification of ee. One-pot asymmetric autocatalysis of pyrimidyl alkanol 2b also significantly increased the ee from 0.28% ee to 87% ee [14a]. We also found the efficient amplification of chirality by using 2-alkynylpyrimidyl alkanol 2c from as low as ca. 0.00005%o ee to almost enantiomerically pure (>99.5% ee) product in only three consecutive asymmetric autocatalysis (Scheme 3, Fig. 1) [14b]. The first asymmetric autocatalysis with (S)-lz of ca. 0.00005% ee gave (^-2c in 96% yield with an enhanced ee of 57%. The second asymmetric autocatalyses with the autocatalyst of 57% ee afforded (*S)-2c with 99% ee, and the ee reached >99.5% ee by the third asymmetric autocatalysis. During the three consecutive asymmetric autocatalyses, the initially major (5)enantiomer of 2c has automuhiplied by a factor of 630,000 times, whereas the initially minor (i?)-enantiomer of 2c by a factor of only less than 1,000 times. As described, pyrimidyl alkanols 2 act as highly efficient asymmetric autocatalysts with amplification of ee. 3-Quinolyl alkanol [15a] and 5-carbamoyl-3-pyridyl alkanol [15b] were also found to act as asymmetric autocatalysts with amplification of ee.

Asymmetric Autocatalysis, Absolute Asymmetric Synthesis and Origin of Homochirality of Biomolecules

359

4. The Role of Asymmetric Autocatalysis as a Linkage Between the Origin of Chirality and Homochirality of Biomolecules 4.1 Asymmetric autocatalysis triggered by organic compounds with low enantiomeric excess It is known that circularly polarized light (CPL) is one of the origins of chirality of organic compounds [16]. Asymmetric photolysis of racemic leucine by right circularly polarized light (CPL, 213 nm) affords L-leucine with only 2% ee [16a]. Hexahelicene with low (90% yield (Scheme 5) [21]. On the other hand, in the presence of /-NaClOa, {Ryic with 98% ee was formed. It should be noted that crystallization of a stirred solution of NaClOa affords either d- or /-enriched form [22]. As described, a chiral organic compound with high ee is formed using chiral inorganic crystals in conjunction with asymmetric autocatalysis. 4.3 Absolute (spontaneous) asymmetric synthesis Based on the theory of statistics, it is considered that small fluctuations in the ratio of the two enantiomers are present if chiral molecules are produced from achiral starting materials under conditions under which the probability of formation of the enantiomers is equal. So-

(Chiral inorganic crystals ) d-Quartz d-NaClOa

CHO Zn/-Quartz /-NaClOa

ee Scheme 5


361

called racemate doesn't usually contain the exact numbers of (S) and (/?)-enantiomers, although the ee is below the detection level by contemporary analytical methods. However, if the small fluctuation of chiralty is amplified by asymmetric autocatalyses, one would obtain enantiomerically enriched compound with well above the detection level [23]. The significant ability of amplification of enantiomeric excess by the asymmetric autocatalysis of pyrimidyl alkanol prompted us to examine the absolute asymmetric synthesis, i.e., the reaction of achiral pyrimidine-5-carbaldehyde with diisopropylzinc without adding any chiral substance in combination with the subsequent asymmetric autocatalysis with amplification of ee. Pyrimidine-5-carbaldehyde was reacted with diisopropylzinc, and the resulting pyrimidyl alkanol was used as an asymmetric autocatalyst for the next asymmetric autocatalysis. The subsequent consecutive asymmetric autocatalysis afforded pyrimidyl alkanol of either S or R configuration with enantiomeric enrichment well above the detection level.

[Absolute (Spontaneous) Asymmetric Synthesis]

-^jJ^

+ /-Pr2Zn

1c

Scheme 6

100 Enantiomeric excess (% ee)

Absolute configuration and ee of pyrimidyl alkanol Fig. 2. Absolute (spontaneous) asymmetric synthesis of pyrimidyl alkanol 2c in combination with asymmetric autocatalysis

362


The effect of the substituent on the 2-position of 2-alkynylpyrimidine-5-carbaldehyde and the effect of solvent are significant. Reaction of 2-alkynylpyrimidine-5-carbaldehyde with /Pr2Zn in a mixed solvent of ether and toluene or in a mixed solvent of ether and dibutylether and the following one-pot asymmetric autocatalysis with amplification of ee gave enantiomerically enriched pyrimidyl alkanol well above the detection level [24]. The absolute configurations of the pyrimidyl alkanol formed show an approximate stochastic distribution of ^ and R enantiomers (19 times formation ofS and 18 times R). The approximate stochastic behavior in the formation of pyrimidyl alkanols form one of the conditions necessary for absolute (spontaneous) asymmetric synthesis [25].

5.

Conclusions We found that chiral 5-pyrimidyl alkanol 2, 3-quinolyl alkanol and 5-carbamoyl-3-pyridyl alkanol are highly enantioselective asymmetric autocatalysts for the addition of /-Pr2Zn to pyrimidine-5-carbaldehyde 1, 3-quinolinecarbaldehyde and 5-carbamoyl-3pyridinecarbaldehyde, respectively. Among these, 2-alkyny 1-5-pyrimidyl alkanol 2c is a highly efficient asymmetric autocatalyst with >99.5% enantioselectivity. Moreover, asymmetric autocatalysis with amplification of ee from extremely low ee to >99.5% ee was realized for the first time by consecutive asymmetric autocatalysis without the need for any other chiral auxiliary. Kinetic analysis of pyrimidyl alkanol suggested that the reaction proceeds via second order of the zinc monoalkoxide of pyrimidyl alkanol [26]. For the mechanism of the amplification from very low ee, the presence of additional mechanism as well as the second order mechanism of the zinc monoalkoxide of pyrimidyl alkanol is postulated [26b]. The elucidation of the actual reactive species remains as a future subject. Chiral organic compounds with low ee which are induced by CPL serve as chiral triggers of the asymmetric autocatalysis. The overall process correlates, for the first time, the chirality of CPL with an organic compound with very high ee. Very recently, chirality of CPL was directly correlated with the chirality of pyrimidyl alkanol with high ee by asymmetric photolysis of racemic pyrimidyl alkanol in combination with asymmetric autocatalysis [27]. Chiral inorganic crystals such as quartz and sodium chlorate act as chiral triggers and regulate the sense of the asymmetric autocatalysis. The process correlates, for the first time, the chirality of inorganic crystals with an organic compound with very high ee. Absolute (spontaneous) asymmetric synthesis is described in the formation of enantiomerically enriched pyrimidyl alkanol from the reaction of pyrimidine-5-carbaldehyde and /-Pr2Zn without adding chiral substance in combination with asymmetric autocatalysis. The approximate stochastic distribution of the absolute configurations of pyrimidyl alkanols strongly suggests that the reaction is an absolute (spontaneous) asymmetric synthesis. As described, asymmetric autocatalysis is closely related with the origin of homochirality of organic compounds.

6.

Acknowledgements Special gratitude is expressed to the coworkers whose names appear in the papers. Financial support from the Ministry of Education, Culture, Sports, Science and Technology, New Energy and Industrial Technology Development Organization (NEDO) and the Japan Space Forum is gratefully acknowledged.


7.

363

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Chapter 30 Charophyte Gyrogonites, the Result of Enantioselective Influence 250 Million Years Ago Ingeborg Soulie-Marsche Laboratoire de Paleobotanique, Institnt des Sciences de VEvolution, Universite Montpellier II, CP. 062, Place E. Bataillon, 34095 MontpelUer-Cedex 5, France marsche@i sent, univ-montp2.fr

I dedicate this paper to Jacques Vautier, who unfortunately departed in December 2003, as a sign of gratitude for his inestimable help with the writing of this article and as an expression of my sincere thanks for his continuous encouragement of my work on charophytes.

1.

Introduction Charophytes (stoneworts) are macrophytes growing entirely submerged in freshwater and brackish water. They become fossilised through their calcified oospores termed gyrogonites. These reproductive organs are an equivalent to the seeds of land plants. The gyrogonites of all modern species fit to a single basic structure consisting in five enveloping cells, which are twisted clockwise around an inner ovoid egg cell. This simple but characteristic pattern was recognised to be unique in the plant kingdom and also proved to be absolutely constant. One has to go back into the Palaeozoic Era to find different morphological types of gyrogonites. The structure of these ancestral gyrogonites was much more complex than the modern type. Based on the fossil record, the charophytes constitute a polyphyletic group that appeared in the Late Silurian (c. 400 Mya) with already two very distinct morphologies of calcified fructifications: a group with vertical and even transversally segmented enveloping cells and a group with numerous counter clockwise twisted enveloping cells. Both types were originally described fi-om the "Uppermost Silurian" in Podolia, Ukraine [1, 2]. It can be assumed that charophyte plants existed already much earlier but did not provide fossil remains as these ancestors probably did not yet form calcified fructifications. The hitherto earliest known gyrogonites with clockwise twisted enveloping cells came from the Middle Devonian of Canada [3]. The Devonian to Permian periods then saw the coexistence of both counter clockwise and clockwise twisted cells. At the end of the Palaeozoic, however, the counter clockwise twisted forms got extinct and since 250 Million years the morphological structure of the gyrogonites has been reduced to the single modern type with strictly five clockwise twisted cells. The aim of the present paper is to highlight the important change in chirality that occurred

366

Progress in Biological Chiralit>

Mirror line

Figure 1. Outline of a modem Charophyte gyrogonite and its theoretical mirror image. The gyrogonites of all living species fit to this unique pattern composed of five sinistrally (clockwise) twisted spiral cells surrounding an inner ovoid egg cell

for the charophyte gyrogonites at the Permian-Triassic boundary (PTB) and to discuss the possible causes of the loss of one type of handedness.

2. Structure of the Gyrogonites Charophytes provide striking examples of biological chirality not only in the female reproductive organ, the gyrogonite, but also in the plant's architecture. At macroscopic scale, the bud displacement follows a helical along the main axis. At microscopic scale, conspicuous asymmetry characterises the gyrogonites. Both structures can be explained by the ontogenetic development of the cells that follows the rules of 2/5^^ spiral phyllotaxis, well known for Embryophytes [4]. 2.1 Coiling direction: a definition The gyrogonites of all living Charophyte species are twisted clockwise with the cells ascending from right to left. Usually less than 1.5 mm in size, they represent homo-chiral bodies at microscopic scale (Fig. 1). The coiling direction of the spiral cells is determined by the growth dynamics ascending from the base to the apex. The polarity of the spiral can be accurately determined because the basal pole always shows a clearly determined basal structure in the form of a pentagonal opening or a pentagonal basal plate. Detailed analysis of the structure allowing the orientation of the gyrogonites was described earlier [4]. This coiling pattern of the gyrogonites is conventionally called sinistral in accordance with the definition traditionally employed by botanists. The botanical term "sinistrorse" points to plants "climbing upwards in a spiral form from right to left or clockwise"[5]. The Palaeozoic gyrogonites could be either sinistral or dextral. On the latter, the spiral cells are twisted counter clockwise, ascending from lower left side to upper right. The dextral or sinistral coiling thus is best seen in lateral views whereas in the basal and apical views of gyrogonites, the handedness of the spiral appears as it were exactly the opposite (Fig. 2). Martin-Closas pointed to the confusion that might result from the botanical definition being exactly the opposite of dextrogyrous and levogyrous as commonly used in other fields

Charophyte Gyrogonites, the Result of Enantioselective Influence 250 Million Years Ago

To the left = sinistral

^Si^^,^ ^ ^

^^^^ ^ ^

367

^^ ^^^ "Sh* = dextral

Figure 2. Comparison of a sinistral and dextral gyrogonite in basal view. a. Eochara wickenendi Choquette; b. Moellerina greenii Ulrich. Bothfromthe Devonian. Arrows indicate coiling (hrection startingfromthe basal opening. Scale bar 100 fim

[6, 7]. However, for more than a century, the gyrogonites have been described with the botanical meaning of "left" and "right". We will use these terms in this conventional sense throughout the text. 2,2 Morphological types of Palaeozoic Charophytes Palaeozoic Charophytes have been little studied and relevant literature mostly dealt with systematics and their stratigraphical value for the continental realm. In the light of phylogeny, the Charophytes represent a polyphyletic group with two well differentiated morphological types being present among the oldest known finds. A branch that displayed fructifications with numerous, up to 18, vertically arranged enveloping cells and a second branch with up to 12 slightly dextrally twisted gyrogonites [8, 9]. The existence of dextral gyrogonites was long known although not particularly underlined for its interest for the study of asymmetry. Abundant and well preserved gyrogonites from the Ural region of Russia described in 1906 [10] and from Ukraine definitely solved the question of whether or not these microfossils should belong to the Charophytes [11]. Amazingly, the very first description of dextral gyrogonites from Indiana, USA, published as Moellerina greenii Ulrich 1886 [12], was long disregarded because the material had been attributed to the "Foraminifera" and had been figured incorrectly as a sinistrally coiled body. The types of this material were preserved in the United States National Museum. Reexamination by R. Brown finally revealed the spirals to be dextral [13]. The original author, "Dr. Ulrich explained that the sinistral spirals were an inadvertence, he having drawn the illustrations by camera lucida directly on lithographic limestone. Consequently, in printing, a reversed image of the original resulted" [13, 14]. According to the rules of botanical nomenclature. Peck and Morales rehabilitated Ulrich's Moellerina greenii as the type species. Most of the dextral forms are currently attributed to genus Moellerina [15]. A first synthetic chart by L. Grambast showed the great variety of morphological types [8, 9]- The general evolufionary trends in the Palaeozoic Charophytes were suggested to proceed from a primitive type with vertical enveloping cells, the Sycidiaceae Peck 1934, to the

368


dextrally (counter clockwise) coiled gyrogonites whereas the first known sinistrally coiled forms come from the Middle Devonian of Alberta, Canada [3]. This scheme has to be modified according to later finds from eastern Europe. Indeed, fructifications with vertical and dextrally coiled cells were already present concomitantly in the "Uppermost Silurian", in the same geological formation, the Skaly unit in Podolia, Ukraine, and thus appeared simultaneously [1, 2]. One type is genus Primochara^ represented by Phmochara calvata Ishchenko and Saydakovskiy 1974, whose gyrogonites are illustrated with counter clockwise helicity. The other type, represented by Praesycidium sjluricum Ishchenko and Ishchenko 1982, belongs to the order of Sycidiales, characterised by the presence of vertical and even segmented enveloping cells, a structure out of purpose for the present paper. Both types also occurred together in the Devonian of northern Russia (St. Petersburg and Ural region) [10]. The forms with vertical cells became extinct in the Early Carboniferous and were considered a non adaptive lineage [9]. The dextrally coiled Moellerinales persisted up to the Late Permian and coexisted with the sinistrally coiled forms. The global trend for dextral as well as sinistral forms was a reduction of the variability and a reduction of the number of enveloping cells forming roughly a chronological suite [8, 9, 16]. Dextral and sinistral Devonian gyrogonites were approximately the same size (Fig. 3). These non ornamented species display large variation in shape to the point that individual specimens can be found that appear the mirror image one to the other suggesting they could represent "enantiomorphic", dextral and sinistral variants of a same species [4]. The current classification of the Palaeozoic Charophytes makes use of categories of the number of spiral cells [15]. The dextral forms are divided into three main types: (/) a goup with 8 to 12 cells (genus Moellerina Ulrich 1886), (//) a group with 8-9 cells (genus Gemmichara Wang Zhen 1984), (///) a group with 5-7 cells (genus Pseudomoellerina Wang Zhen 1984). The Palaeozoic sinistrally coiled gyrogonites comprise three categories as follows: (/) a group with 8-13, mostly 10-12 enveloping cells (genus Eochara Choquette 1956), (//) a group with 6 or 7 cells (genus Palaeochara Bell 1922), (///) a group with strictly five spiral cells, (genus Leonardosia Sommer 1954 and the large family of the Porocharaceae Grambast 1962).

Figure 3. Comparison of Devonian gyrogonites in lateral view. a. sinistral Eochara wickenendi Choquette. clockwise twisted, ascending from lower right to upper left {bot.: sinistrorse); b. dextisA Moellerina greenii Ulrich, counter clockwise twisted, ascending from lower left to upper right (bot.: dextrorse); Scale bars 100 ^m

Charophyte Gyrogonites, the Result of Enantioselective Influence 250 Million Years Ago

369

The Porocharaceae, defined by the presence of an apical opening, provide the basis of all post-Palaeozoic Charophytes. Depending on the geographical area and on the classification used by individual authors, these early Charales were split into a number of different genera [17]. Given the tight resemblance of these forms, the distinction of so many taxa, however, seems not always justified and the systematics of these forms claims revision. At the end of the Palaeozoic, all dextral forms got extinct. Only the Charophytes with five sinistrally coiled cells came to survive into the Mesozoic Era. 2,3 Evolution of asymmetry in the Charophytes Asymmetry of the gyrogonites appeared with anti-clockwise arrangement of a variable number of enveloping cells. Given the high number of cells (8-12), only a slight torsion was necessary to cover the internal egg. The shape of each single cell corresponds roughly to a reversed S as shown by Eochara wickenendi and Moellerina greenii (Fig. 3). Progressive reduction of the number of cells increased the degree of twisting. The sinistrally coiled forms underwent the same process in the opposite direction and rapidly remained with a fixed number of five cells. A number of specimens with opposite handedness can be found that represent the mirror image one to the other suggesting they could represent enantiomorphous pairs [4]. It was also suggested that a single mutation could have been responsible for the reverse coiling direction [9]. The general evolutionary trend was toward reduction and fixation of the number of spiral elements leading to increased spiralisation. Whereas the dextral five-celled gyrogonites, classified as Pseudomoellerina, made only a short appearance in the Devonian, the five-celled sinistral gyrogonites appeared in the Carboniferous and became dominant in the Permian. From the PTB onwards no dextrally coiled gyrogonite has ever been found. The review of the distribution of the Palaeozoic Charophytes makes evidence that the sinistral forms expanded rapidly whereas the dextral ones regressed. Up to the Late Permian, dextral species with 8-9 cells and sinistral forms with five cells still coexisted in North China and grew even at the same place in Liaoning Province [18, 19]. Although the number of their spiral cells is different, their outline, size and shape are very close (Fig. 4). From the botanical standpoint the sinistral forms seem to have had better fitness or an adaptive advantage over the dextral forms. Possibly, plants of the dextral Gemmichara with a

Figure 4. Comparison of the latest coexisting dextral and sinistral Charophyte species, a. Gemmichara sinensis Z. Wang; b. Leonardosiajinxiensis Z. Wang. Both from the Late Permian in Liaoning Province, North China. Redrawn from photographs [18,19]

370 Progress in Biological Chirality variable number of cells were less inter-fertile than the strictly five celled species. Indeed, it cannot be established if the gyrogonites with 8 cells and those with nine cells could be produced by the same plant or if they correspond to different plants of the same species. In contrast to examples of chiral structures in animal species Hke Foraminifera or snails, the coiling direction of the gyrogonites suffers no exception. The five-celled sinistrally coiled structure of the gyrogonites obviously represents an inheritable coiling pattern. However, the molecular background of the phenomenon has not yet been investigated.

3. Palaeogeographic Distribution of Palaeozoic and Triassic Charophytes During the Devonian period, the dextral forms were largely dominant. Their known distribution concentrates on the palaeo-equatorial latitudes (Fig. 5) The type species of the earliest dextral Charophytes, Moellerina greenii wasfi-equentin the northern states of the USA [14, 22-24] where it was already present in the Late Silurian [25]. Abundant material was also described from northern and eastern Europe including Russia and Ukraine [10, 11]. The northern most occurrence was found in Spitzberg [26, 27]. Finds in Asia were hitherto located only in South China [19, 28]. Devonian sinistrally coiled gyrogonites are known only from today's Alberta, Canada [3] located at that time at the western rim of the Euramerican landmass. During the following Carboniferous period, sinistral forms occupied continental waters in Canada and the Unites States [14] and expanded also over North China [19]. One notes the discrete appearance of the five-celled sinistral Porocharaceae. Charophyte finds are rare in the Carboniferous. This was worldwide a period with extensive wetlands. However, the water must have been acid due to the rotting of high amounts of organic material (the basic material for the coal measures) creating unfavourable condition for the growth and preservation of calcareous organisms.

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Figure 5. Palaeogeographical distribution of known Devonian Charophyte localities. Base map for palaeogeography and plate positions 390 My ago redrawn and simplified from the PALEOMAP-project [20, 21]

Charophyte Gyrogonites, the Result of Enantioselective Influence 250 Million Years Ago 371 Siberia )

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