Advances in Supramolecular Chemistry. Volume 7

",4

---t -
10 6 ions s-l (channel open) is too fast to measure. Ideally, the data should exhibit a series of rectangular pulses of constant amplitude and should be of random duration. Each integral transition is representative of a single channel opening. When channels are open, ions go through the membrane according to the difference in the voltage and the ion concentration on both sides of the membrane. The ions passing through the channel are observed as a channel current. It is therefore possible to calculate the ion flux (conductance, normally in picoSiemens) for the channel molecule by dividing the observed peak height (current) by the applied voltage, i.e. conductance = current/voltage. Conductance is dependent on many factors including the pore size and the pore length.

10

GREGORY J. KIRKOVITS and C. DENNIS HALL

Determining Ion Selectivity Many channels discriminate between ions and transfer biological signals across cell membranes by only allowing the passage of specific ions. This selectivity is dependent on various factors including pore size, electrostatic interaction between the ions and the pore-forming amino acids, and the dehydration energy of the ions involved. 14Such selectivity can vary from broad discrimination between anions and cations or more specifically cations of the same charge, e.g. Na + and K +. As noted above, one of the advantages of the planar bilayer technique is the ability to introduce ionic concentration gradients across the two chambers. Consequently, this permits the determination of channel permeability for one particular ion over another by measurement of the reversal potential (Vrev). This is simply the potential at which there is no net ionic current in the presence of the transmembrane concentration gradient. For example, if the only electrolyte present is KC1 at a concentration of Ccis on one side of the bilayer and of concentration Ct~ans on the other then the reversal potential, Vrev of a channel in a bilayer can be expressed by the Goldman-Hodgkin-Katz equation 1 as:

RT [PKCtrans+Pc]Ccisl

Vrev= 7

In PKCcis +

PcICuans

(1)

where PK and Pcl are the relative permeabilities of K + and C1- ions, respectively. Thus the permeability ratio for the two ions can be calculated by: PK _ I0~Ctrans - Ccisl PcI

Ctrans- Ccis

where c~ = exp(FVrev/RT ). Alternatively, Equation 1 can be used to determine the permeability ratio of ions of like charge when their concentrations are equal on either side of the bilayer. In this manner, ionic selectivity sequences can be established for a channel former.

3.2. Patch-Clamping The method of patch-clamping permits monitoring of the activity of single channels in cell membranes. 3 Experimentally, a glass micropipette (ca. 1 gtm in diameter) is applied to the surface of a cell, and by the use of slight suction, a piece of the cell membrane can be drawn into the pipette. Alternatively, this patch can be detached from the membrane. In so doing, the isolated piece of membrane may contain only one or at most a few channels. Consequently, the patch, seals off the end of the micropipette. The pipette can then be immersed in bathing solutions of different composition and different solutions may be introduced into the body of the pipette to permit control of the aqueous environments. The setup is completed by applying an electrical potential across the membrane patch. In order for the

Natural and Artificial Ion Channels

(i)

On-cell

11

(ii) Whole-cell

suck

~pull (iii) i n s i d e - o u ~

~ pull (iv)

Outside-ou~

Figure 5. The four-patch clamp recording methods. In each example, the cell is shown to the left, the micropipette to the right. For a discussion of the methods see text. Reproduced with permission from Hille, B., "Ionic channels of excitable membranes" 1st ed., Sinauer, Sunderland, MA (1984) p. 217.

method to be successful, the patch must form an extremely tight (high resistance) barrier across the aperture of the pipette. This is achieved by fire-polishing the pipette. Records of channel activity can then be monitored like the planar bilayer method, as current versus time traces. There are four protocols for performing the patch-clamp experiment (Figure 5). These are listed as follows: (i) On-cell in which the pipette is pressed against the membrane and left in place, thereby allowing the conductance of a tiny patch to be measured; (ii) Whole-cell where suction breaks the membrane thus enabling measurements of the entire cell; (iii) Inside-out and (iv) Outside-out: in both these cases, the pipette is pulled out of the membrane thus isolating that patch.

4. SYNTHETIC PEPTIDE ION CHANNEL MODELS As discussed above, the majority of known natural channel-forming structures involve large protein aggregates that contain a number of helical transmembrane segments acting in concert to control transmembrane ion and potential gradients. Naturally, the size and complexity of these molecules has meant that elucidation of the mechanisms governing their function has been difficult. One approach to this problem has been to study much smaller synthetic peptides, and by using those amino acids implicated in channel function, effectively "recreate" the channel pore structure with membrane spanning amphiphilic and/or hydrophobic o~-helical

12

GREGORY J. KIRKOVITS and C. DENNIS HALL

motifs. Of particular relevance has been the development of methods to control the assembly of peptides to form a more defined structure. Mutter and Montal synthesized template-assembled synthetic proteins (TASP) to control transmembrane assembly of helical peptides. 14 The TASP were designed to adopt a globular, four-helix bundle channel pore structure which produced conductance events characteristic of channel-forming peptides, while the shortest analogue elicited only erratic current fluctuations. The helical segments of natural proteins usually consist of multiple nonidentical helices, displaying sophisticated channel functions. To this end, Futaki has used the template approach to organize nonidentical helical peptide segments to form channel structures. 15 DeGrado et al. 16a'17prepared 21-residue amphiphilic peptides composed of only leucine (L) and serine (S) residues. Leucine was chosen to provide the apolar face of the helix because of its hydrophobicity and helix-forming propensity, while serine was chosen to provide the polar, yet uncharged face of the helix. The length of the helix corresponded to that of the hydrophobic portion of a membrane bilayer and the two sequences, (LSSLLSL)3 and (LSLLLSL) 3 gave different ion channel activity. The former peptide was found to produce cation selectivity similar to that of the acetylcholine receptor, while the latter was found to be proton-selective. Energy-minimization modeling suggested that this was due to the formation of parallel hexamer- and tetramer-helix bundles, respectively, in the membrane and attempts were made to stabilize the four-helix bundle structure by using a porphyrin template. 16b The subsequent results of planar bilayer studies showed that the template exerted a major influence on open lifetime and voltage dependence and that the channels were unimolecular. The transmembrane m-helices formed by Ac-(LSSLLSL) 3 (Ac = acetyl; Cterminus = CONH2), although formally neutral, have partial charges of +0.5 and -0.5 near their N- and C-termini, respectively, due to alignment of their amide groups. 18 This electrostatic asymmetry can account not only for the preferred transmembrane orientation of the peptide but also the nonlinear single-channel current-voltage curves (rectification) observed for Ac-(LSSLLSL) 3. DeGrado et al. extended this model by the addition of formal charged residues to just one end of the helix. In so doing, they were able to demonstrate how the electrostatic environment at the mouth of the channel can have a large effect on conductance and cation selectivity. By introducing a crown ether unit at the C-terminal region of hydrophobic helical peptides, Otoda et al. 19 were able to demonstrate increased stabilization of the peptide aggregate in the membrane by the formation of sandwich-type complexes with large cations. Ion channel activity was also increased due to the ability of the crown peptide to bind ions to the terminal portion of the hydrophobic helix bundle at the water-lipid interface. Ueda et al. 2~considered the problem of insolubility of hydrophobic peptides which restricts the distribution of peptides in water to a phospholipid bilayer membrane. In consequence they constructed a hydrophobic helix bundle shielded by hydrophilic peptides that acts rather like an umbrella.

Natural and Artificial Ion Channels

13

5. ENGINEERED NATURAL ION CHANNELS Another approach to the study of the mechanisms of transport has been to alter the performance of some of the more simplistic natural ion channels by structural changes. By adding a positively charged group to the C-terminal end of a gramicidin monomer near the mouth of the channel as exemplified by 1, Woolley et al. 2l reasoned that cation conductance could be modulated depending on the proximity of the charge to the channel entrance. They observed, not only single-channel currents of 1 that were of reduced amplitude relative to gramicidin, but more importantly, different current levels during the lifetime of the gramicidin-ethylenediamine dimer of 1. These different levels were due to the charged-NH~ group whose effect varied with cis-trans isomerization of the carbamate functionalities. In fact carbamate conformation was found to govern the proximity of the positive charge to the channel mouth, thereby inducing partial blocking of the entrance to the channel. By incorporating the photoisomerizable azobenzene unit in 2 they achieved a further degree of control over the current levels via photochemical trans cis isomerization of the azo linkage. 22 Schreiber et al. 23a connected two gramicidin units using tartaric acid derivatives (dioxolane) to form a covalently linked hybrid, thereby eliminating channel disruption as seen in the native channel. However, the ion channel conducting properties of these molecules were observed to be dependent on the stereochemistry of the tartaric acid linker. In particular, the (R,R)-dioxolane linker (a stereochemical mismatch with the helix geometry of gramicidin A) exhibited rapid interruptions in current (flickers). However, by introducing sterically demanding substituents onto the central linker, they were able to eliminate this behavior thus demonstrating that the observed gating phenomenon was due to flipping of the dioxolane ring into and out of the channel (Figure 6). 23b Sansom and co-workers 24 attached redox-active ferrocene units to the C-terminal end of alamethicin to explore the effect of oxidation on channel formation. Briefly, alamethicin is an antibiotic peptide, composed of 20 amino acids, which forms ion channels by self-association in lipid bilayers, in a voltage-dependent manner. Evidence supports the "barrel-stave" model of channel formation, in which czhelical monomers of the peptide associate to form an aggregate transmembrane pore. Different sized aggregates are observed to give multiple conductance states of alamethicin. The single-channel properties and voltage sensitivity of 3 and 4 were similar to alamethicin, although higher cis-positive potentials were required

o

o

9

1

NH~

2 (trans-meta-azobenzene)

14

GREGORY J. KIRKOVITS and C. DENNIS HALL

Figure 6. Schematic representation of the intramolecular linked gramicidin dimer showing flipping of the (R,R)-dioxolane ring into and out of the conduction pore. Reproduced with permission from Stankovic, C. J.; Heinemann, S. H.; Schreiber, S. L., J. Am. Chem. Soc. 1990, 112, 3703.

to elicit activity. This was thought to be due to steric and/or conformational demands imposed on the peptides by the bulky substituents. In addition, 4 exhibited a transition with unusually protracted open lifetimes. The effect of oxidation on channel formation was investigated by treatment with the oxidizing agent ceric ammonium nitrate (CAN). The in situ addition of CAN to 3 caused a time-dependent decrease in channel formation at constant potential, consistent with the presence of positively charged head groups decreasing the propensity of alamethicin peptides to form channels. The long lifetimes of 4 were selectivity eliminated, whereas alamethicin itself was unaffected by CAN. Premixing of the peptides with CAN produced similar results. Indeed, approximately twice the concentration of peptide was required to induce channel activity as compared to peptide in the absence of CAN. Conductance histograms showed that the oxidized peptides spent a greater percentage of their time in the closed state when compared to the reduced forms.

3

4

Natural and Artificial Ion Channels

15

6. CLASSIFICATION OF ARTIFICIAL (NON-PEPTIDE) ION CHANNEL MODELS Within the last 20 years the curiosity of the organic chemist has spawned an interest in the study of non-peptide channel models. These have been primarily designed to incorporate the structural elements of natural ion channels required for channel activity. The aim has been to contribute to the mechanistic understanding of the significantly more complex natural channel proteins. Perhaps a more realistic target however, is their application to the development of new pharmaceuticals and drug-delivery systems. 25Synthetic peptide models of ion channels function mainly as well-defined aggregates which create an assembled pore for ion transport. 17 Non-peptide channel models, however, can be loosely categorized into two functional modes 26'27 as follows: (i) unimolecular membrane-spanning tunnels, which provide "relay" elements for cation transport (commonly macrocyclic crown ethers), and (ii) transport through self-assembled aggregates. Usually amphiphilic in nature, the latter form the transmembrane tunnel by recognition and organization within the membrane bilayer. Both systems require the application of principles evolved in studies of supramolecular chemistry.

6.1. Early Examples The earliest example of a non-peptidic channel model was prepared by Tabushi et al. 28 It consisted of a ~-cyclodextrin, attached to four hydrophobic tails designed to afford a "half-channel?' The transport of copper and cobalt was assessed in artificial liposomes (kco(u) = 4.5 x 10-4s-Z), the rate being much faster than in the absence of the half-channel. In the same year, Lehn 29 reported a solid-state model of a molecular tunnel consisting of stacked macrocyclic polyethers, with K § ions located alternatively inside and on top of successive macrocycles. Following the theme of the macrocyclic tunnel model, Nolte 3~ prepared an oligomer, 5, of isocyanide with benzo-18-crown-6 side chains which possessed a rigid helical backbone with four repeating units per helical turn, thereby generating four tunnel-like tubes composed of the macrocyclic tings aligned on top of one another and spaced ca. 4 ,/k apart. The system displayed features akin to natural

N=C

'~

-

""c~f'c- 'R

.N II

R'

v

-0

L

0

o.J

16

GREGORY J. KIRKOVITS and C. DENNIS HALL

ionophores comprising (i) pores with a polar interior and apolar exterior; (ii) a hydrophilic top and bottom to face the aqueous medium inside and outside the membrane, and (iii) a chain length of ca. 40/~ to bridge a typical membrane bilayer. Vesicles of dihexadecyl phosphate were prepared with a UV-active dye trapped in the inner aqueous volume. The rate of Co 2§ transport into the vesicles was determined by monitoring the increase in absorbance of the C02§ complex using UV-vis spectrophotometry (kco(u)= 10-4s-I). No Co 2§ transport was detected in the absence of 5. 6.2.

Proton Channels

In an attempt to prepare flux-promoting compounds Menger 31 synthesized phosphatidylcholine derivatives incorporating a polyether side chain. These proved incapable of promoting ion movement across bilayers, but intermediates in their synthesis bearing the general structure RO(CH2CH20),,R' proved to be active. Proton transport across phospholipid bilayers was assessed using a pH-fluorescence spectroscopy technique by incorporating acid-responsive pyranine dye trapped within the vesicles. By varying the three sections of the general structure, Menger was able to maximize proton transport, which was achieved with CHa(CH2)10 COO(CH2CH20)sCH2Ph, 6. The three sections comprised a hydrophobic polymethylene chain, a relatively hydrophilic ester plus ethyleneoxy groups and a benzyl head group, presumably to serve as a membrane anchor. It was recognized that a single molecule of 6 was of insufficient length to span the membrane and that a minimum of two molecules would need to align in order to traverse the entire lipid bilayer and promote proton passage. The transport ability of 6 exceeded that of gramicidin and the simple cartier molecule 18-crown-6 proved inactive under identical conditions. Dubowchik 32 synthesized a linear bola-amphiphilic octamine 7, in which each nitrogen was attached to an adamantylmethyl group terminated at either end by a propylsulfonate headgroup. Fully extended, 7 was designed to be 48 A in length, thus capable of spanning a typical membrane bilayer. Nonaggregated and essentially non-protonated 7 was intended to dissipate a proton gradient with proton transfer occurring along the length of the chain and with the backbone effectively acting as a proton wire. Phosphatidylcholine vesicles were prepared, the pH of the

7

Natural and Artificial Ion Channels

17

external aqueous solution was raised to 7.5-8.0 and the change in pH over a period of time was monitored inthe presence of 7. The pH dropped steadily over two hours almost immediately upon addition of 7. Control experiments showed there was only a very slow leakage of protons most probably due to lysis of a small fraction of defective liposomes. The results were consistent with the transporter acting as designed, but membrane defects caused by other kinds of interaction with 7 were also considered possible. Using the principle of ion pair formation between ammonium cations and the phosphate anions of lipids, Matile et al. 33 prepared 8, an amphiphilic polyamine dendrimer. Rather than acting as a membrane channel, 8 was expected to form reversible membrane defects in the lipid bilayer. The steroid moiety was expected to act as the hydrophobic anchor for bilayer orientation and steric bulk was expected to prevent the polyamine penetrating the bilayer. Proton transport was assessed in unilamellar vesicles using the pH-fluorescence technique in which the external pH was increased to 7.8 relative to the internal pH at 7.4. The results demonstrated that 8 was almost as active as gramicidin, and maximal flux was achieved in ca. 20 s.

HaN~NH.

fi

/

§

J 9 "J H3N

"'q 8"WA"

NH

8

Menger had previously shown that the ion flux across synthetic phospholipids could be promoted by attaching alkyl groups of various sizes to the hydrophobic portion of the lipid, presumably by creating reversible defects in the bilayer. 34 The rigid molecule, 9 was designed to resemble the polyene and polyol segments of amphotericin B (AmB) (Figure 7), but with the charged terminal anchoring group absent. 35 Briefly, AmB is a low molecular weight polyene antibiotic that forms channels by the self-assembly of 8-10 monomers. The model is that of the "barrel-stave", in which the hydroxylated portion of each molecule faces into the channel pore and extends across the bilayer. Oligo(p-phenylene) units were chosen for the backbone because of their hydrophobicity, conductivity, conformational flexibility, and luminescence properties. Small unilamellar vesicles containing. entrapped HPTS (8-hydroxypyrene-l,3,6-trisulfonic acid) were used to monitor proton transport again using pH-fluorescence spectroscopy. In the presence of a pH

18

GREGORY J. KIRKOVITS and C. DENNIS HALL

H r, 3"

~

~

~

~

~

~

~

.

H3C

H.J

/.. OH

OH

-

OH

NH2

OH

amphoteriein O

0

OH HO

OHHO

O0

OH HO

OHHO

O0

OH HO

OHHO

O0

OH HO

OH

0

Figure 7. Structure of amphotericin B (top) and oligo(phenylene) 9 (bottom). gradient, changes in internal pH facilitated by 9 showed that the length of the rigid rod must match that of the bilayer for optimal activity. 35a Compound 9 also demonstrated significant H § > K § selectivity. Without 9 and in the presence of the selective potassium cartier, valinomycin ([K§ = [K§ only minor increases in HPTS emission were seen. However, in the presence of 9 the flux rate was 16-fold higher implying that under these conditions 9 mediates H§ § exchange with H § > K § selectivity. Transport was explained by the hydrogen-bonded chain (HBC) mechanism. This is a two-step "hop-and-turn" process in which proton passage occurs via successive hypothetical "hops" followed by sequential "turns" in order for the new O - H bond to return to the initial configuration and acquire another proton. Fluorescence quenching experiments using spin-labeled lipids confirmed the transmembrane orientation of octamer 9. 35b Overall, this presents a picture of a proton-selective membrane-spanning and presumably unimolecular channel model. Matile reported poor bilayer solubility of rigid-rod 9 so tackled this problem by incorporating lateral propylene side chains. 35r This was expected to increase lipophilicity and also to improve the flexibility of the hydroxyl groups thus facilitating proton transport by the HBC mechanism. The structural modifications did not appear to have a detrimental effect on the active structure and increased ion flux rates were seen for the propylene-substituted oligo(phenylenes).

6.3. Macrocycle Based Mimics The key to Fyles original design 36 for an artificial ion channel (Figure 8) was based on a central rigidifying macro-ring using polycarboxylate 18-crown-6 de-

Natural and Artificial Ion Channels

19

rived from tartaric acid [COOH-(CHOH)2-COOH ]. Fyles exploited the availability of tartaric acid in its optically active and meso forms to direct "wall" units from the core. These units incorporated either or both polar and nonpolar functionalities to provide structural control (i.e. alignment with the bilayer phospholipids) and a tunnel-like path for transport within the bilayer. The crown ether was composed of one, two, or three tartaric acid units such that the structure consisted of either two, four, or six wall units. To each wall unit was attached a hydrophilic head (H) group (glucose, 2-mercaptoacetic acid or 3-mercaptopropanol) to serve as an anchor and to assist in establishing the transmembrane configuration. Cation flux was studied using the pH-stat method for a variety of compounds with systematic structural variations. Subunits that were well represented at the lower end of activity included mercaptopropanol (head group), the long wall unit (dodecanediol chains) and the lipophilic wall group {-(OCHECH2)30-chains }. In a group of otherwise identical structures, changes in the head group from glucose through mercaptopropanol to ethanol resulted in an increase in proton flux. Four wall units appeared to be most conducive to activity, but somewhat surprisingly the sixth wall unit was found predominantly at the lower end of the scale. The best wall unit was a hydrophilic and lipophilic balance, composed of both octyl and -(OCHECH2)30- chains (Figure 8). This provided the extended molecule with a length appropriate to span the bilayer. Interestingly, a combination of kinetic and inhibition studies showed that several of the molecules studied actually functioned as carders. Based on similar ideas, Lehn et al. 37 constructed an artificial ion channel utilizing a central tetracarboxy-18-crown-6 unit for labile alkali-metal complexation. Substitution of two pairs of axially oriented lipophilic dendrimer-like arrays with terminal carboxyl groups for anchoring at water-membrane interfaces, was expected to provide a so-called "bouquet" molecule with an extended transmembrane orientation. The central macrocyclic nucleus was also replaced with ~cyclodextrin to provide a larger rigid hole of internal diameter, ca. 6/~ (Figure 9). Alkali metal ion NMR spectroscopy was employed to assess cation transport across the phospholipid bilayer of liposomes. Briefly, the presence of alkali metal ions in the outer aqueous solution can be distinguished from internal ions by the presence of a shift reagent. Two resonance signals were therefore seen which corresponded to internal (unshifted signal) and external (shifted signal) metal ions. Vesicles with external shift reagent, were created with opposing gradients in Na § (outside) and Li § (inside) concentration. Transport of the cations down their concentration gradients due to the presence of the bouquet in the membrane was therefore followed directly using 23Na and 7Li NMR spectroscopy as a function of time. Ion transport activities were seen to be approximately the same for the two systems, and it was concluded that ion transfer proceeded by a one-for-one Na § ion for Li § ion exchange (cation-cation antiport). Rates of transport for the ~cyclodextrin derivative through gel-state membranes were similar to those observed in thefluid state, a result consistent with channel function.

20

GREGORY J. KIRKOVITS and C. DENNIS HALL 140

head

H

OI4

"-

+

=

o4-%o

H

oo

~

~o

c-o

-'-~o.]=~ core ~~176

o~~--o, o - ~ -,-

_~o

;o,c~o

%o.J

o

~

o~~

o-~o~,

-4H

H

Figure & Generalized structure and example of the channel mimics prepared by Fyles and co-workers. In 1990 Gokel and co-workers published evidence of a membrane-insertable, sodium cation-conducting channel active in large unilamellar vesicles (LUVs) using a synthetic compound based upon diaza-18-crown-6. 38a Several design principles were applied to the preparation of tris(macrocycle) 10, which contained a central macrocycle similar to the model systems developed by Lehn and Fyles but designed to be flexible (Figure 10). First, the channel former had to correspond to the length of a typical phospholipid bilayer (ca. 30/~). Dodecyl alkyl chains were chosen for this purpose and also to serve as connective units. Second, head groups were required to provide a point of cation entry and exit and to anchor the structure in an extended conformation in the bilayer. Work undertaken in the same laboratory demonstrated that amphiphilic crown ethers could aggregate into stable vesicles 39 and that bola-amphiphilic crowns (crown-hydrocarbon chain crown) could form monolayer lipid membranes. 4~ Hence crown ethers were selected to employ an elementary principle of cation selectivity; in addition, the choice of diaza-18crown-6 permitted derivatization at nitrogen while retaining conformational flexi-

Natural and Artificial Ion Channels

21

RO~O '~OR

OR

O

o~

o

~

OR

o

~

o

o__,y-o---~oLT -~o o~ o "~,,"-" o

F o ~F ~

J

0"

f

oF/

~

~--~,,~ "'~ ~

beta'cycl~

. j o--.-J/)"- ,H

o)

o/

O

?

OR

o

L.. OCH ~" - - 7

,._.,

O~o RO

o

5

o

o~

OR

o

o

Figure 9. Structures of the bouquet molecules incorporating an 18-crown-6 center (left) and a 13-cyclodextrin center (right).

bility. Third, a central unit to capture and relay the cation through the bilayer was required. For synthetic simplicity a diazacrown was also chosen such that the overall structure would afford a hypothetical "tunnel." By this approach the ether oxygen atoms of the three macro-rings would play a central role as relay elements. Cation flux rates were assessed for 10 directly in phospholipid vesicles using a dynamic 23Na NMR method. In this procedure, addition of a Dy 3+ shift reagent to an aqueous vesicle solution prepared in the presence of Na § results in an external Na § signal shifted with respect to internal Na § With the incorporation of a channel former, the line width broadens, reflecting dynamic exchange between internal and external Na + through the membrane channel. Consequently, the linewidth varies directly with cation flux and a plot of [channel] versus linewidth permits determi-

22

GREGORY J. KIRKOVITS and C. DENNIS HALL

/"'X

C~ Y

/--"x

0- 3

C~

N-(CH2)~3-N

0- 3

C~

N--(CHz)~N

CoM.../0-2 Cok...../09 10, Y=N(CH2)llCH 3 14, Y - N" y / ~ A

A

11, Y=NH

NO 2

Y

CoX_.._/0-2

12, Y=O

15, Y - N" y /

0- 3

~

13, Y=NCH2C6H 5

OCHaI6'

Y--N

H

Figure 10. Tris(macrocycles) prepared by Gokel and co-workers.

nation of the rate constant from the slope of the graph. The rate for 10 was found to be 13.5 s-l, ca. 40-fold greater than the simple carrier molecule, N,N'-di-dodecyl4,13-diaza-18-crown-6, but ca. 100-fold poorer than gramicidin at the same concentration. The initial findings were followed by a comprehensive study in which a series of bis- and tris-(macrocyclic) compounds with structural variations were prepared. 38b Initially, proton transport was monitored across phospholipid bilayers by using a fluorescence technique. In this method a pH-sensitive pyranine dye is trapped within vesicles. If the external pH is rapidly lowered, in the presence of a proton conductor, protons will flow into the vesicles. The pH of the internal solution is thus lowered and this is reflected by a decay in the corresponding fluorescence signal of the dye. When the covalent spacer units were simply changed from alkyl -(CH2)12- in 10, to oxyethylene -(CH2CH20)3CH2CH 2- proton flux showed a marked fall. This result was most unexpected as it had been anticipated that a larger number of donor atoms would increase the cation flux by lowering the transport energy needed in passage. It was deduced that more donor atoms lead to stronger binding whereas the dynamics of channel activity require weak complexation. Cation flux rates of the tris(macrocycles) were determined by 23Na NMR spectroscopy as described above and were standardized relative to gramicidin which was given an arbitrary value of 100. The original design hypothesis envisaged the three macro-rings lying parallel to one another, thus providing a tunnel-like path. However, when the central diaza-18-crown-6 ring was replaced by diaza-15crown-5 and subsequently by open-chained O(CH2CH20)3 , transport rates were respectively 28, 25, and 14. The results suggested that although the central ring is important, the cation does not necessarily pass through it. It was thus postulated that the central ring of 10 lies parallel to the phospholipids thereby lowering the polarity in the bilayer midplane and reducing the distance a cation is required to travel unaided by donor atoms. Removing the dodecyl side chains (as illustrated by

Natural and Artificial Ion Channels

23

11) did not affect the rate but when the head group diazacrowns were replaced with aza-18-crown-6 (e.g. 12), activity was lost (50 units), short-chain oligomers, and cyclic oligomers of (R)-3-hydroxybutyrate to solvate salts. Seebach et al. 65'66 prepared crown ester complexes from cyclic trimers (triolides) of (R)-3-hydroxybutyrate and sodium thiocyanate; Biirger and Seebach 67 demonstrated that cyclic oligolides and oligomers of (R)-3hydroxybutyrate (OHBs) transport alkali and alkaline earth picrates across methylene chloride layers in U-tubes; Reusch and Reusch 64 prepared conducting complexes from PHB and its homologue, poly-(R)-3-hydroxyvalerate, with lithium perchlorate; and Fritz et al. 68 showed that OHBs can transport Ca 2§ into liposomes in the presence of a Ca 2§gradient. Finally, as discussed below, PHBs make bilayers permeant to ions.

3.3. Channels in Planar Lipid Bilayers The ability of PHBs and OHBs to conduct salts across phospholipids bilayers was examined in a planar bilayer voltage-clamp setup. 69'7~In this system, a bilayer is formed between two aqueous solutions by "painting" a decane solution of phospholipids across a small aperture (ca. 0.2 mm in diameter) in a partition separating two chambers containing aqueous salt solutions. The hydrocarbon drains away and the phospholipids spontaneously arrange themselves into a bilayer (black lipid membrane or BLM). The aqueous solution on one side (cis side) of the bilayer

PHB and PolyP Ion Transport

59

Figure 5. Planar bilayer setup. The system consists of two aqueous solutions, labeled cis and trans, separated by a planar bilayer. External voltage commands are applied to the cis side, with the trans side maintained at ~round (defined as zero voltage).

OSC--oscilloscope; VCR--recording tape system.

represents the cell cytoplasm and the solution on the other side (trans) represents the aqueous environment outside the cell. The trans side is maintained as ground and external voltage steps are applied to the cis side (Figure 5). Seebach et al. 71'72 prepared monodisperse oligomers (MwlM n = 1.0) of (R)-3-hydroxybutyrate using an exponential fragment-coupling strategy, and incorporated them individually into planar lipid bilayers of synthetic 1,palmitoyl-2,oleoyl-phosphatidyl choline (16:0,18:1 PC) between symmetric solutions of 60 mM RbC1, 5 mM MgC12, 10 mM Hepes CsOH, pH 7.2. 73 Channel-forming activity was observed for OHBs of 16, 32, 64, and 96 monomer units when concentrations of these oligomers were 0.1-1.0% of bilayer lipids (w/w) (Figure 6) and a voltage _>60 mV was applied to the cis side. The stepwise fluctuations were also observed under these conditions when natural short-chain PHBs (-60 units), obtained by acid-catalyzed degradation of PHB granules, were incorporated into bilayers of the same composition at concentrations of 5% (w/w). Stereoregularity was not essential; channel activity was observed with low molecular weight polymers containing 39% isotactic diads (Mw ~ 4 x 103, M n -- 6 x 102, Mw/M n = 6.7) at concentrations of 5% (w/w). In all cases, the channel conductances were highly variable. It was possible to obtain linear current-voltage relationships for a given single channel in the bilayer, but measurements of single channels formed with the same sample in a fresh bilayer yielded quite different conductances. Many of the single channels exhibited high open probabilities, indicating they were essentially open pores. As expected, they did not discriminate well among cations. The current records were similar to those observed with ionic and nonionic detergents, such as Triton X- 100, octyl glucoside, or sodium dodecyl sulfate, in planar bilayers. 74'75

60

ROSETTA N. REUSCH

I

t

~

H

o

80 mV OH pO =0.51 96

200~------] 3pAl /

N.620

o

|.l

17

26

3s [psi

Figure 6. Single-channel currents of synthetic PHB96. Left:. Representative current fluctuations obtained when the given voltage was applied at a planar bilayer made from 1-palmitoyl, 2-oleoyl, phosphatidylcholine (16:0,18:1, PC) containing 0.1 to 1% of 96met of PHB between symmetric solutions of 60 mM RbCI, 5 mM MgCI2, 10 mM Hepes CsOH, pH 7.2. The solid horizontal bar in each record indicates the current level with the channel closed, pO is the probability that the channel is in the open state. Right:. Corresponding conductivi~ histograms. N indicates the total number of observations that have been analyzed. '~ In addition to high concentration, oligomer size and end group structure proved to be important factors in channel formation. High molecular weight PHBs from natural sources (>3000 units) and very small synthetic oligomers of 16 units. 72'77 It is presumed that each PHB molecule crosses the hydrophobic region and is stabilized at each end by the formation of hydrogen bonds from the terminal hydroxyl and carboxyl groups to the ester groups of the phospholipids (Figure 7). These data imply that chains >16 units fold back on themselves. Considering the high concentrations of the polyesters required to form channels, it seems probable that the pores are formed by aggregates of several molecules of appropriate size. Seebach et al. 71 propose that the oligomers form islands of lamellar crystallites in the bilayer, and that ion permeability results from areas of mismatch at the interfacial regions between phospholipids and the oligomers. When the oligomers are of mixed lengths, higher concentrations are required, suggesting that there is selection for oligomers of approprirte size.

PHB and Polyp Ion Transport

61

Figure 7. Schematic representation of (R)-3-hydroxY7butyrateoligomers (OHBs) of 32 units incorporated into planar phospholipid bilayer. 3

Fritz et al.68 examined the channel-forming activity of synthetic oligomers of 8, 16, 32, and 64 units in bilayers of liposomes, which encapsulated the fluorescent dye, Quin-2. Ca 2§ transport into the vesicles was followed by observing the Ca2§ Quin-2 absorption at 264 nm. In the presence of a concentration gradient, the 32mers and 64mers, but not the 8mers or 16mers, transported Ca 2§ into vesicles at a rate comparable to the ionophore, calcimycin. 78 The OHB-mediated Ca 2§ transport was inhibited by the presence of La 3§ A carrier-like mechanism was suggested, with Ca 2§ ions hopping between complexing carbonyl groups. Transport of other cations was not examined; hence specificity for Ca 2§ was not established. More insights into the relationship between polyester size and bilayer width were provided by Das et al., 79 who examined the channel-forming ability of synthetic oligomers of (R)-3-hydroxybutyrate of M n 1670, Mw/Mn 1.2, and isotacticity 94%. Since these oligomers have an average of 19 residues by M n measurements and 23 residues by M w measurements, they are referred to as OHBI9t23. The oligomers were prepared synthetically by Jedlinsky et al. 8~ via regioselective ring-opening polymerization of (S)-13-butyrolactone, catalyzed by a supramolecular complex of a crown ether and sodium (R)-3-hydroxybutyrate. This process is less laborious than the exponential fragment-coupling process above. Although polymers or oligomers formed by this method do not have the advantage of identical length, the size range for a given preparation is relatively narrow and the end groups (OH and COOH groups) are the same as those present in natural PHB, as shown by NMR and ESI-MS spectroscopy, sl OHBI9~ did not form channels in bilayers of 16:0 18:1 PC and cholesterol (5:1 w/w) at concentrations up to 2.5%. 79 This failure was attributed to a mismatch between oligomer length and bilayer width. Solid-state measurements based on the above 2 l helix conformation indicate that the length of oligomers with 19 units and 23 units are ca. 57/~ and 69/~, respectively. Geometric considerations indicate that at least six oligomers must assemble to form a pore large enough to conduct ions. 6a For OHB 19t23, the probability is high that most of these oligomers will be too short to fold properly and too long to remain fully extended in a bilayer of 48/~. To test this hypothesis, the complexes were incorporated into bilayers of 1,2 dieicosenoyl phosphatidyl choline (di20:l PC), cholesterol (5:1 w/w), and separately into bilayers of 1,2 dierucoyl

62

ROSE-IrA N. REUSCH AO

16000

15 O t-.

w :3 r

O

LI.

OJ

6

"26o ~o"s6o Time, ms

0

86o

......

| w, I I

8

12

1e

32000

15 O

Q.

r-

g

tO

4

Amplitude, pA

BO

1% ofphospholipids (w/w), OHB 19t23displayed activity in both bilayers; however, channel formation was observed much more frequently in di22:1 PC. Representative current records of the dominant channels observed in each bilayer are shown in Figure 8A. The channels in both cases were essentially open pores of fluctuating conductance, similar to those formed by the synthetic oligomers above (Figure 6). As shown by the all points amplitude histograms 83 (Figure 8B), channels in di22:1 PC had a lower average conductance as well as a much narrower amplitude distribution than in di20:1 PC (61 + 4 vs. 109 + 13 pS), suggesting that more organized channels were formed in the wider bilayer. Over long periods of recording (>10 min), full closures were brief (order of ms) and extremely rare (0.99). The channels formed by OHBI9/23 displayed no cation selectivity. This was tested by determining the reversal potential (zero-current potential) when the channels were incorporated into bilayers formed between aqueous solutions of unequal ion composition; Ca2§ dominant on one side and Na§ dominant on the opposite side. The Nernst equilibrium potentials (calculated from concentrations) 84 are Eca= -82 mV, Ecl= +9 mV, and ENa = +76 mV so that preference for Ca2§ would be demonstrated by a negative reversal potential and a preference for Na§ as a positive reversal potential. As shown in Figure 8C, the reversal potential, estimated graphically from the single-channel current-voltage relationships, was zero indicating no preference for divalent or monovalent cations. In summary, OHBs form nonselective ion channels in planar lipid bilayers, provided that the ratio of OHBs to lipid is high and there is a rough correspondence between oligomer length and bilayer width. The data suggest that OHBs longer than bilayer width can bend back in hairpin turns to fit within the confines of the hydrophobic region, but the folded segments should each be of approximately bilayer length. It is postulated that short-chain OHBs aggregate in clusters, with molecules arranged perpendicular to the bilayer and parallel to fatty acyl chains. The results demonstrate the ability of oligomers and polymers of (R)-3-hydroxybutyrate to make bilayers permeable to ions; however, the OHB "channels" cannot select among cations by charge, size, or coordination geometry. Like polyP, PHB has some of the essential properties of physiological ion channels but not others. Auspiciously, the two polymers complement each other, and together they possess all the requisite characteristics of well-regulated ion-selective channels.

4. COMPLEXES OF PHB A N D POLYP 4.1. PHB/polyPComplexes in Bacterial Membranes PHB/polyP complexes were fn-st discovered in the plasma membranes of bacteria by Reusch and Sadoff22-25'85during spectrofluorometric studies of membrane structure, using the hydrophobic probe, N-phenyl- 1-naphthylamine (NPN). When NPN is added to cell suspensions, it partitions into the hydrocarbon region of the cell membranes and

64

ROSETTA N. REUSCH

responds to changes in the viscosity or polarity of the bilayer by a change in fluorescence intensity. This procedure, developed by Overath and Traiible, s6 results in minimal disturbance of cell processes, and reports lipid transitions specifically and with reasonable agreement with transitions determined by light-scattering and X-ray diffraction. The major transition in membranes of log-phase cells is the broad gel to liquid-crystalline transition of the phospholipids that begins below 0 ~ and ends at 20-24 ~ While observing the thermotropic transitions in bacterial membranes at stages of cell

>Ior) z Lu I-z ,..,,,

uJ (/) z uJ

(b

O0 ILl nO :::) .J IJ. UJ ~>

b -

a

i

I..J IJJ n-

0

I

10

1

20

I

30

1

40

|

50

i

60

T E M P E R A T U R E (~

Figure 9. A. Thermotropic fluorescence spectra of E. coil DH1 cells using the hydrophobic probe, N-phenyl-l-naphthylamine (NPN). (a) Mid-log phase cells; (b) stationary lphase cells; (c) cells made genetically transformable by the method of Hanahan. 46 NPN was added to 4 mL of cell culture to a final concentration of 1 ~M and the thermotropic fluorescence spectra were recorded. 24 Measurements were made at increasing temperature (ca. 2 ~ per min). Excitation: 360 nm; emission: 410 nm. Measurements were made at increasing temperature (ca. 2 ~ per rain). B. Effects of physical treatments on the thermotropic transitions in genetically competent E. coil DH1. (a) Thermotropic transitions at descending temperature; (b) cells pelleted at low speed and suspended in supernatant; (c) as in b but suspended in equal volume of distilled water; (d) as in (b) but suspended in 10 mM phosphate buffer, pH 7.4. Excitation: 360 nm; emission: 410 nm. Fluorescent probe was NPN. Measurement (a) was made at decreasing temperature and (b), (c), (d) at increasing temperatures (ca. 2 ~ per min).

PHB and PolyP Ion Transport

65

l,m (1t z w tzI lal v) z u tn lal 0

w

.I w

L

I0

|

20

I

30

TEMPERATURE

l

l

40

50

_

1

60

('C)

Figure 9. Continued. differentiation, a new relatively sharp transition at --56 ~ was noted. The fluorescence peak at --56 ~ was weak or imperceptible in cells during mid-log phase growth, became more evident in stationary-phase cells, and was most intense in genetically transformable cells, i.e. cells that showed high competence to take up exogenous DNA 23-25 (Figure 9A). The transition was irreversible (Figure 9Ba). When the cells were collected and resuspended in the original growth medium, in water, or in buffer, the sharp transition diminished in intensity or disappeared and was replaced with broad fluorescence at lower temperatures (Figure 9Bb,c,d). This suggested that the structure responsible for the transition is labile and composed of lipid-soluble and aqueous-soluble components. The lipidic component was identified as PHB by chemical analysis of the isolated plasma membranes. 22-25 The MW was estimated as --13,000 Da (-150 units) by viscosity measurements 22 and --12,000 Da (-140 units) by nonaqueous size-exclusion chromatography. 19 The water-soluble component was identified as polyP by chemical analysis, 22-2s and its size was estimated as 55-65 residues (MW ca. 5000 Da) by acrylamide gel electrophoresis. 87 The neutralizing cations for polyP were determined to be Ca 2§ by graphite furnace atomic absorption spectrometry. 25 It was suggested that the sharp rise in fluorescence is caused by the dissociation of PHB/Ca(polyP) complexes. As the polymers separate, the polar polyP molecules move to the aqueous interface and the PHB chains are released into the bilayer, effecting an increase in

66

ROSETTA N. REUSCH

bilayer viscosity and a consequent increase in NPN fluorescence. Since the polymers cannot reassociate when temperatures are lowered, the viscosity of the bilayer (and fluorescence intensity) increases at decreasing temperatures until the liquidcrystalline to gel transitions of the phospholipids begin. Freeze-fracture electron microscopy studies of the membranes of E. coli and A. vinelandii by Reusch et al. 24 provide evidence of structural changes that support the fluorescence data (Figure 10). Freeze-fracture micrographs of log-phase cells show a typical mosaic of particles and pits on both concave and convex surfaces of the plasma membranes. However, as complexed PHB was increasingly incorporated into the membranes, as determined by analysis of the purified membranes and evidenced by the intensity of the thermotropic transition at - 56 ~ the micrographs revealed the formation of small semi-regular plaques in the plasma membranes (arrows) that possess shallow particles. The plaques grew in size and frequency as the concentration of membrane PHB and intensity of the PHB/polyP transition increased.

4.2. E. coli PHB/polyP Complexes as Ion Channels The ability of E. coli PHB/polyP complexes to form calcium-selective channels in planar bilayers was investigated in the planar bilayer system described above (Figure 5). E. coli DH5~ cells were made genetically competent to increase the concentration of PHB/polyP in the membranes. Then vesicles were prepared from the cell envelopes, and added to the cis side of a planar bilayer formed by synthetic 16:0, 18:1 PC between symmetric bathing solutions of 250 mM CaCI 2, 5 mM MgC12, 10 mM Tris Hepes, pH 7.3. The complexes were allowed to insert spontaneously into the bilayer. 27 No activity was observed in the absence of an applied

Figure 10. Representative freeze-fracture electron micrograph of competent E. coli DH1. The micrograph shows the typical appearance of small semi-regular plaques (arrows) in the plasma membranes of E. coli DH1 cells after treatment to make them genetically transformable by the method of Hanahan. 146 These cells have sharp thermotropic transitions at -56 ~ when examined as in Figure 9A. 24

PHB and Polyp Ion Transport CHCI3

80 mV

PM

120 m V

PM

_0_.

~

$0 mV ..II_.

~ . j ' ~ - ~ ]~:

Tr-I I I* 'T I'q _+ ~

-_--,:_ -

--

. . . . . . . . . . .

67

~

p

~

.

l

~

r

-,

,~. . . . . . . . .

r

..-

|~

Figure 11. Profiles of calcium channels from E. coiL Single-channel currents observed in membrane vesicles and extracts of E. coli competent cells when incorporated into planar bilayers of 16:0,18:1, PC between symmetric aqueous bathing solutions of 250 mM CaCI2, 5 mM MgCI2 in 10 mM Iris Hepes, pH 7.3. 27 Line 1. Membrane vesicles of whole cells (WC) were added to the cis bathing solution and the solution was gently stirred to allow spontaneous incorporation of the vesicles into the bilayer. Single-channel currents were activated by a voltage step >60 mV. A representative current record at 100 mV is shown. Lines 2-3. Plasma membrane vesicles (PM) were incorporated into the bilayer and single-channel currents were activated as above. Representative records at 80 mV and 120 mV are shown. Line 4. A chloroform extract of genetically competent cells was added to a decane solution of 16:0, 18:1, PC. The chloroform was removed by evaporation, and the remaining lipid mixture was used to form the bilayer. Single-channel currents were activated as above. A representative current record at 80 mV is shown.

voltage, but when a potential of>60 mV was held for several minutes, single-channel currents were observed signifying the presence of Ca2+-permeant channels (Figure 11, line 1). The envelopes were then separated on sucrose gradients into plasma and outer membranes. Each was tested in a similar manner, but channel activity was observed only with the plasma membrane vesicles (Figure 11, lines 2,3). This is consistent with earlier findings, by chemical analysis and freeze fracture electron microscopy, that PHB/polyP complexes are confined to the plasma membranes. 23,24 The calcium channel activity observed in plasma membrane vesicles of competent E. coli was essentially the same as that for PHB/polyP complexes extracted from plasma membranes into chloroform solution (Figure 1 l, line 4). The molecu-

68

ROSETTA N. REUSCH

lar weight of the extracted PHB/polyP complexes was estimated as 17,000 + 4000 Da by size-exclusion chromatography. 27 This measurement, together with the molecular weights of the component polymers, indicates that the complexes are formed from one strand of PHB (~ 12,000 Da) and one strand of Ca(polyP) (~5000 Da). To establish the composition of the channels still further, the PHB/Ca(polyP) complexes were reconstituted from PHB recovered from E. coli and Ca(polyP) prepared from commercial sodium polyphosphate and calcium chloride. This solution was then premixed with phospholipid and used to form a bilayer. Finally, the complexes were formed in situ. PHB was mixed with phospholipids before painting the bilayer; Ca(polyP) was then added to the aqueous bathing solutions, and a potential was applied to induce the formation of the complexes within the bilayer. For all preparations, the concentration of PHB in the bilayer was restricted to one-hundredth or less of the amount required to form PHB channels. Singlechannel currents similar to those described above were obtained with each of these procedures. 27

4.3. Synthetic Ion Channels from PHB128 and PolyPs To resolve remaining doubts as to whether the channel activity was indeed effected by PHB/polyP complexes and not by trace protein contaminants, Das et al. 2s performed a total synthesis of the channel complex from (R)-3-hydroxybutanoic acid, sodium polyphosphate, and calcium chloride. Lengweiler et al. 26 prepared a 128mer of (R)-3-hydroxybutyrate (PHB 12s)by an exponential fragmentcoupling strategy. This synthetic polymer has a MW of 11.4 kDa, which is close to that of E. coli PHB (~ 12 kDa). Calcium polyphosphate was prepared from sodium polyphosphate glass (av residue number 65) and calcium chloride. PHB128/polyP complexes were formed by adding a chloroform solution of PHBI28 to an excess of dry Ca(polyP) (av 65 units). After evaporation of the chloroform, the dry mixture of polymers was heated briefly in a microwave oven, and then suspended in chloroform and gently mixed in a bath ultrasonicator. PolyPs are highly insoluble in chloroform; hence, only polyP complexed with PHB12s is found in solution. Uncomplexed PHBI28 will also be present, but the concentration of the complexes in the bilayer was maintained at concentrations too low (< 0.01% of phospholipid w/w) for the formation of PHBI2 s channels. The size of complexed polyP was determined by acrylamide gel electrophoresis to be in the same range (50-70 residues) as that in the E. coli complexes. 27 The current records of channels formed in planar bilayers by the synthetic complexes were indistinguishable from those of PHB/polyP complexes extracted from E. coli (Figure 12), and the conductances of the synthetic and E. coli channels were equivalent; 101 + 6 pS and 104 + 12 pS (Figure 13), respectively. These results show clearly that protein is not essential to the observed channel activity.

PHB and PolyP Ion Transport

69

Figure 12. Representative single-channel current fluctuations of synthetic and E. coli PHB/POlyP complexes at various clamping potentials. Left: Synthetic PHB128/polyP; Right: Channels extracted from competent cells of E. coil DH5a. Complexes were incorporated into planar lipid bilayers composed of 16:0, 18:1, PC and cholesterol (5:1; w/w) between aqueous bathing solutions of 200 mM CaCI2, 5 mM MgCl2, 10 mM Tris Hepes, pH 7.4 at 22 ~ The bars at the side of each profile indicate the fully closed state of the channel. Clamping potentials with respect to ground are indicated at the left side.28

70

ROSETTA N. REUSCH

g

i

....

, .......

-120 -80

A~ 6

AO 0

0 . _0 . . . . . .

40

g 0_5

40

80

120

-10 -15

Holding Potential, mV Figure 13. Current-voltage relations for synthetic PHB128/polyP (E3) and E. coli PHB/polyP channel complexes (A). The conductance of the channel for Ca 2+ in symmetric solutions, under the experimental conditions described in Figure 12, is 101 + 6 pS for the synthetic channels and 104 + 12 pS for the E. coli channels. The data points represent mean values of 10 observations. 28

4.4. Synthetic Ion Channels from OHB19/23 and PolyPs Ion channels were also prepared from synthetic oligomers and polyPs. Das et al. 79 formed complexes from synthetic OHB19/23 with Ca(polyP) (av 65 units) following the procedure used to prepare the synthetic complexes of PHBl28 and Ca(polyP) above. The channel activity of the complexes was examined in bilayers of di22:1 PC and cholesterol (5:1 w/w) between symmetric bathing solutions of 200 mM CaC12, 5 mM MgC12, 10 mM Tris-Hepes, and pH 7.4 at 22 ~ The current records of OHB19/2a/polyE shown in Figure 14A, display high conductance and complex channel activity. All points amplitude histograms for single channel records at different clamping potentials indicate a major fully open state with a conductance of 260 _+8 pS and a minor open state with conductance of 153 _ 3 pS (Figure 14B). These conductances were substantially higher than the -61 pS conductance of OHB 19/23in the same bilayer, or the ~ 100 pS conductance reported for the major open state of the biological or synthetic PHB/polyP complexes. The channel structure is unknown but it is proposed that polyP stretches across the bilayer, encircled and solvated by an indeterminate number of OHBs. The polyP polyanion, with its high negative charge and conformational polymorphism, would reasonably be responsive to voltage change. The space between the two polymers, lined with ester carbonyl oxygens on one side and phosphoryl oxygens on the other, can accommodate multiple conductive pathways for cations. The presence of multiple lanes for ion transport may explain

PHB and PolyP Ion Transport

71

Figure 14. Characteristics of PHBIgI23/POlyP complexes in di22:1 PC/cholesterol bilayers. A. Profiles of single channel currents. PHB19123/polyP complexes were incorporated into planar lipid bilayers, composed of synthetic di22:1 PC/cholesterol (5:1 w/w), between symmetric bathing solutions of 200 mM CaCI2, 5 mM MgCI2, I 0 mM Tris-Hepes, pH 7.4 at 22 ~ Data was filtered at I kHz. B. Current-voltage relations for PHB19123/polyPchannel complexes. All points amplitude histograms were constructed for single channel records at each indicated potential. 81 Data was filtered at 2 kHz. The points show the mean peak position of Gaussian distributions, fit by a simplex least-square procedure at respective clamping potentials. The data shown here are mean amplitudes of the major open state from several experiments. Best fit obtained by linear regression yields a single-channel conductance of 260 + 8 pS. Each symbol represents an independent experiment.

72

ROSETTAN. REUSCH 260pS

BO

30.

o,,~

20.


3. While the crowned spirobenzopyrans 48-51, in which the spirobenzopyran moiety was much further removed from the crown ether units by longer alkyl spacers than in 41-44, showed a small selective coloration for large alkali metal cations such as K § and Cs § the molar absorptivities in the presence of alkali metal iodides were considerably smaller than those of 41-44. The low coloring efficiency might result from unfavorable entropy effects: the probability of the existence of complexed cations in the neighborhood of the phenolate oxygen of the merocyanines is reduced, and the electrostatic interaction between the complexed large univalent cations and thep-nitrophenolate dipole of the merocyanines is weak. Taking into account the above points, Inouye and co-workers developed cryptand and bibrachial lariat type crowned spirobenzopyrans 62-64 and 65 and 66, respectively (Schemes 3 and 4). 117'118 Cryptand-containing spirobenzopyrans 62-64 (Scheme 3) exhibited no absorption bands above 400 nm in nonhydroxylic solvents indicating the closed spiropyran form. Indeed, this interpretation was verified by an NMR study. The absorption spectra were scarcely affected upon addition of any alkali metal iodides in CH3CN. In the 1H NMR spectra of 63 in CD3CN, however, downfield shifts (for aromatic and crown ring protons), splitting (crown ring and alkyl protons), and sharpening (aromatic protons) of the signals in the spiropyran form were observed after the addition of KI. This result clearly indicated that the alkali metal cations were bound to the macrocycle moiety of 63, and that the colorless form was a result of weak electrostatic interactions between the complexed univalent cations and the p-nitrophenolate dipole of the merocyanines. On the other hand, addition of alkaline earth metal iodides to these CH3CN solutions gave rise to changes in their spectra with 62 giving the most intense coloration for Ca 2§ and 63 for S1"2§ Titration

Functionalized Macrocyclic Ligands

115

Me_ Me NO2

0

NO=

Me Me,~ MIz

0

0

62' 63' 64'

62 n = l 63 n=2 64 n=3

Scheme 3.

experiments demonstrated that about 1 equiv of SrI 2 is enough to obtain the maximum coloration of 63. Unfortunately, 64 showed little change and poor selectivity in its absorption spectrum under the same conditions. This may be because the crown ether ring is too flexible to interact strongly with the cations. As expected, little change in the spectra of 41-44 and 48-51 occurred under the same conditions. Isomerization of cryptand-containing spirobenzopyran 63 (Scheme 3) to the open-colored merocyanine 63' in the presence of SrI 2 has been studied by 1H NMR spectroscopy at 500 MHz. In several deuterated solvents (e.g. CDC13, CD3CN, DMSO-d 6) the 1H NMR signals of 63 were considerably broadened at room temperature. High-temperature NMR (85 ~ in DMSO-d 6 succeeded in resolv-

Me

NO2 NO2

O

n

Me MI2

O

Me #

-=

O

62' 63' 64'

62 n = 1 63 n=2 64 n=3 Scheme 4.

116

XUE, SAVAGE, BRADSHAW, ZHANG, and IZATT

ing the identifiable species. (In contrast, the spectra of 62 and 64 at room temperature in CDCI 3 were well-resolved.) To a solution of 63 (20 lxmol) in CD3CN (0.7 mL) was added SrI 2 (40 lxmol), and the 1H NMR spectrum was measured as a function of time in the dark. After 30 rain, new resonances were detected which were assigned to merocyanine 63', and the remaining signals of spiropyran form 63 sharpened and shifted. Equilibrium was reached after 3 h (>80% conversion). These observations suggest fast and strong binding of Sr 2+ by 63, and a slow isomerization of the resulting complex (63-Sr 2§ to the merocyanine form 63'. The fast and strong binding of Sr 2§ by 63 was corroborated on the basis of FAB MS experiments. Before addition of SrI 2, ion peaks for (M + H) § and (M + Na) § were detected; after the addition, these signals decreased and finally disappeared, while peaks for (M - H + Sr § and (M + Sr + I) § appeared and increased. Coloration of bibrachial lariat type crowned spirobenzopyrans 65 and 66 (Scheme 4) was examined. The new crowned spirobenzopyrans were designed to recognize divalent cations in which the complexed cations would interact with the two phenolate oxygens of the open merocyocnines 65' and 66' and the crown ether tings (Scheme 4). Although the molar absorptivities of 65 and 66 in the presence of 1 equiv of alkaline earth metal iodides in CH3CN were small compared to those of 62 and 63, 65, and 66 revealed the highest coloration for Mg 2§ (~, max = 513 nm, e = 700) and Ca 2§ (~, max = 524 nm, e = 3500), respectively. These metal ions were smaller than those used for 62 and 63 which possess the same type of crown ethers. This observation was satisfactorily explained by CPK molecular models, which indicated that the radius of the cavity of (N,N'-diacetyl)diaza-crown ethers (in 65 and 66) was smaller than that of the corresponding (N-monoacetyl)diaza-crown ethers (in 62 and 63). More recently, a spirobenzopyran dimer bridged by a diaza-18-crown-6 moiety through the 8-position (67) was developed by Kimura and co-workers, llg-121 Crowned bis(spirobenzopyran) 67 shows a similar coloration selectivity to that of 63. Complexation of multivalent metals, especially Ca 2§ and La 3§ by 67 enhanced the isomerization of the spirobenzopyran moiety to the corresponding merocyanine form due to an effective intramolecular interaction between a crown-complexed cation and the two phenolate anions in the cation complexes of the merocyanine form. In this section, the sensing properties of the cryptand and bibrachial lariat-type crowned spirobenzopyrans were presented. In the spirobenzopyrans, coloration was efficiently induced in the presence of the alkaline earth metal cations. The cryptand and bibrachial lariat-type crowned spirobenzopyrans represent highly sensitive and selective chemosensors for alkaline earth metal cations.

FunctionalizedMacrocyclic Ligands

117

Me. Me

~

N

0 2 Me C["N

/---N O

O--~ O

67 3.3. AzacrownEthersFunctionalizedwith 8-Hydroxyquinolineand Its Derivativesas Metal-Ion Sensors Our interest in chemosensors has been focused on a series of azacrown ethers functionalized with 8-hydroxyquinoline and its derivatives. 122-125 Evaluation of stability constants for interactions of these ligands with metal ions indicates that the ligands form stable complexes with alkali, alkaline earth, and transition metal ions, which is a prerequisite for their use as chemosensors. 8-Hydroxyquinoline is an analytical reagent containing a phenol-like function wherein ligand fluorescence is moderated upon complexation with certain metal ions. 126 5-Chloro-8-hydroxyquinoline (CHQ)-substituted diaza- 18-crown-6 ligands prepared in our laboratory, 68 and 69, exhibit greatly improved ion-complexing ability and selectivity for certain metal ions compared to unsubstituted diaza-18-crown-6. ]22'123Ligands 68 and 69 have the CHQ sidearms attached through different CHQ positions to determine if positions of attachment would alter metal-ion complexation by 68 and 69. Indeed, the site of attachment has profound consequences for cation complexation properties of these ligands. Thermodynamic data in MeOH show that ligand 68 selectively binds Mg 2+ (log K = 6.82) over other alkali and alkaline earth cations (log K = 2.89-5.31).123 UV-vis

c, Co o_ 68

Cl

co

Cl

c~

X---ox_jo--/

OH

~

69

118

XUE, SAVAGE, BRADSHAW, ZHANG, and IZATT

spectra ofligand 68 and its complexes with Mg 2§ Ca 2§ and K § in MeOH are shown in Figures 3 and 4. The UV-vis spectral data for other metal ions have also been reported. 123 Among the alkali and alkaline earth metal ions, the UV-vis spectrum of the Mg 2+ complex is unique. A new peak at 265 nm is observed for the 68-Mg 2+ complex. Neither Mg 2+ nor the ligand itself has absorption in that region and the other alkali and alkaline earth metal cations do not cause such an absorption. ~23As observed in Figure 3, the presence of Mg 2+ causes both CHQ and ligand 69 to exhibit new peaks in the vicinity of 265 nm but the peak intensities are much weaker than that of the 68-Mg 2+ complex. Therefore, the unique UV peak at 265 nm for the 68-Mg 2+ system may provide a promising method for Mg 2§ analysis in samples where an excess amount of alkali and alkaline earth metal cations are present. Figures 5 and 6 show the UV-vis spectra of two analogues, 70 and 71124 of 68. In these cases, Mg 2+ also causes the new peaks in the vicinity of 265 nm (see curve c in both figures) but the peak intensities are lower than that of the 68-Mg 2+complex. The luminescence properties of ligand 68 and its complexes have been examined. 125 As shown in Figure 7, uncomplexed 68 exhibits a very weak luminescence band (@ < 5 x 10-5, t < 0.5 ns) centered at 540 nm in MeOH/H20 (1:1 v:v), which is consistent with the luminescence behavior of 8-hydroxyquinoline in protic solvents. Also, no appreciable luminescence intensity increase was observed from pH 2 to 13 with uncomplexed 68. However, addition of Mg 2+ to 68 (5 x 10-5 M) in a neutral (1:1 v:v) MeOH/H20 solution (pH 7.2) results in a strong enhancement of the luminescence band (~ = 0.042, t = 7.4 ns). Upon complexation with Mg 2+ and excitation at 393 nm, the fluorescence intensity of 68 is increased by a factor of 1000. The excitation spectrum of the complex strictly matches the absorption spectrum of 68-Mg 2+, suggesting that the observed fluorescence is due to neutral complex formation.

~

265

~1 200

397

3OO 400 Wavelength (X, rim)

Figure 3. UVovis spectra of Mg 2+ complexes with CHQ (dotted line), 68 (solid line) and 69 (point-dash line) in MeOH; [CHQ] = 3.1 x 10-5 M; [68] = 1.1 x 10-5 M; [69] = 2.0 x 10-s M; [Mg 2§ is 40 times the ligand concentration in each case.

119

F u n c t i o n a l i z e d /vlacrocyclic Ligands 1.25

1.00

0.75

0.50

0.25

200

250

300

350

400

450

Wavelength (;L,nrn) Figure 4. UV-vis spectra of free and complexed 68 in MeOH; [68] (solid line) = 1.0 x 10 -5 M; [K +] (point-dash l i n e ) = [Ca 2+] (dotted l i n e ) = 5.0 x 10-4 M.

Contrary to what was observed with Mg 2+, no luminescence increase was detected upon addition of K § Na +, Ca 2+, Sr 2+, or Ba 2+ to 68 (5 x 10 -5 M) at pH 7.2 in the 1:1 MeOH/H20 solvent. 125 The lack of luminescence increase can be attributed to the absence of formation of neutral complexes. Although Cu 2+ and Ni 2+ are able to form neutral complexes with 68, addition of Cu 2§ or Ni 2§ into a solution of 68 does not result in a luminescence increase. In these complexes, energy and electron transfer processes are accessible providing a fast deactivation route of the excited state to the ground state. The complexes of Cu 2§ and Ni 2+ with other 8hydroxyquinoline derivatives have also exhibited a lack of fluorescence. 127'128 Addition of Zn 2§ to a solution of 68 resulted in a luminescence complex. 125 However, the fluorescence quantum yield was 8 times lower than that of the

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