Organometals and organometalloids: Occurrence and fate in the environment

Organometals and Organometalloids In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; Am...

Author: F. E. Brinckman | J. M. Bellama

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Organometals and Organometalloids

In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


Organometals and Organometalloids Occurrence and Fate in the Environment F. E . Brinckman,

EDITOR

National Bureau of Standards J . M . Bellama,

EDITOR

University of Maryland

Based on a symposium sponsored by the Division of Inorganic Chemistry at the 175th Meeting of the American Chemical Society, Anaheim, California, March 13-17,

1978.

ACS SYMPOSIUM SERIES 82

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1978


Library of Congress

Data

Organometals and organometalloids. (ACS symposium series; 82 ISSN 0097-6156) Includes bibliographies and index. 1. Organometallic compounds—Environmental as pects—Congresses. I.Brinckman, F. Ε. II. Bellama, Jon M., 1938III. American Chemical Society. Division of Inorganic Chemistry. IV. Title: Organometalloids: occurrence and fate in the environment. V. Series: American Chemical Society. ACS symposium series; 82. QH545.074073 ISBN 0-8412-0461-6

574.5'2 79-24316 ACSMC8-82 1-447 1979

Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, repro duce, use, or sell any patented invention or copyrighted work that may in any way be related thereto.


ACS Symposium Series Robert F . G o u l d , Editor

Advisory Board Kenneth B. Bischoff

James P. Lodge

Donald G . Crosby

John L. Margrave

Robert E. Feeney

Leon Petrakis

Jeremiah P. Freeman

F. Sherwood Rowland

E. Desmond Goddard

Alan C. Sartorelli

Jack Halpern

Raymond B. Seymour

Robert A. Hofstader

Aaron Wold

James D. Idol, Jr.

Gunter Zweig


FOREWORD T h e A C S S Y M P O S I U M SERIES was founded in 1974

to provide

a medium for publishin format of the Series parallels that of the continuing A D V A N C E S IN C H E M I S T R Y SERIES except that in order t ô save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. and

Both reviews

reports of research are acceptable since symposia may

embrace both types of presentation.


To Frederick Challenger


EMERITUS

PROFESSOR

O F ORGANIC

CHEMISTRY

UNIVERSITY O F LEEDS


DEDICATION he significance of the metal-carbon or metalloid-carbon bond to that Α

rather vague area of descriptive science variously termed "environ

mental chemistry," "bioinorgani now perceived as basic to the assessment of man's activities and future on our planet, and there is one individual who stands out as the pioneer in this field that encompasses so many disciplines. More than 40 years ago, long before chemists recognized the implications of sterically con strained donor sites or enjoyed spectrometric means for virtually non destructive characterization

of trace materials, this researcher single-

handedly applied all available chemical forces to a unified study of the biogenesis of organometalloids. His work still stands as a beacon i n the field, and even today a report rarely appears without citing one or more of the many papers of Professor Frederick Challenger—papers that span 60 years of personal research. Today technological, social, and political pressures give urgency to expanded studies on the occurrence and fate of organometals and their implications for mankind: this volume is an attempt to highlight such work. It is obvious that no attempt to give a topical perspective on our present state of knowledge concerning problems in the field could succeed without the most direct recognition of Professor Challenger s contribu tions and views. The

editors deem themselves and all readers most fortunate that

Professor Challenger, now enjoying more than 90 summers of health, could and would give this symposium volume a keynote paper.

Most

happily, all contributors to this volume take this opportunity as fitting and proper to recognize his past and present primal role in the field of environmental organometallic chemistry. W e dedicate this book to h i m and to his work. F. E .

BRINCKMAN

J. M . B E L L A M A

National Bureau of Standards


Washington, D C

College Park, M D xi



PREFACE he inorganic chemist today is confronted with an intriguing and challenging prospect which will significantly advance our understanding of chemical reactions and transport of toxic elements in the environment.

T h e opportunity lies partly in extending fundamental

studies of bioactive sigma carbon-metal bonds or other similar toxic heteroatomic

combinations that are formed in polar transport

media

such as water. T h e challenge also exists for inorganic chemists to inject their ideas and findings described as "environmental chemistry" and thereby to profit from such an interchange by finding exciting new problems to solve. Our current perception of mobilization and transport of certain combinations of organometals and organometalloids is that certain toxic moieties can be and are relocated on a global scale. Recent unambiguous findings show that even simple, labile methylmetal species occur in the environment as a result of biogenic processes.

Moreover, anthropogenic

inputs of organometals parallel growing technological and agricultural demands. Matching these increased demands are amplified requirements to understand the potential, or lack of it, for wastewater treatment of refractory organometals and organometalloids by chlorination or ozonation, a neglected study area. Similarly, study and discussion are necessary for us to understand the means b y which man introduces organometals and organometalloids into the environment, and the pathways by which the environment can pass on ( return ) such bioactive metal species to the human organism. Consequently, not only from pragmatic environmental quality or public health perspectives, but also from basic needs to understand biogeochemical cycles, participation of inorganic chemists in the dialogue is timely and is needed urgently. T h e symposium on inorganic chemical problems in the environment could not deal with the entire range of such identifiable problems amenable to inorganic research in a brief two-day meeting.

Therefore, the

symposium focused on two main objectives: a) definition, by example, of the latest research concerned with organometal and organometalloid chemistry relevant to environmental concerns, with particular emphasis on aqueous reactions and transport mechanisms; and b) dialogue, with interested and competent colleagues outside the inorganic community, who can best transmit the current needs and consequences of alternative courses for future research relevant to biological implications of organometallic chemistry. xiii In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Therefore, this symposium comprises a series of invited research and review papers by researchers active internationally in both biological and organometallic chemistry. A t the welcome suggestion of reviewers, additional papers were solicited from highly competent colleagues to achieve better balance and topical presentation for this volume. T h e editors assume full responsibility for the fact that additional areas in the title field are not discussed. Such omissions were dictated by our personal biases, constraints of publication costs, and the obvious need to produce similar symposia in the near future. W e have not attempted to arrange papers in chronological order, e.g. in the symposium format.

Rather, we have exercised editorial preroga-

tive and (with apologies to authors) have assembled the volume into three broad categories of needed and ongoing research: biogenesis of organometals and organometalloids try and mechanisms; and the nature of entry, transport, or uptake of organometals and organometalloids into environmental compartments. Clearly, many papers overlap these classifications, just as expected for such an interdisciplinary dialogue. Just as clearly, some readers will find useful a structuring which aids them in placing their own interests into perspective with others. T h e editors also chose to adopt a symposium format which devoted approximately 30%

of available meeting time to discussion.

(Authors

were asked to complete their manuscripts after the symposium to exploit this feedback feature.)

W e have recorded and edited these additional

verbal interactions between symposium participants, and have attached these discussions to the end of each paper. Again, rigorous, and hopefully equitable, editing was imposed to provide the best presentation of each commentator's remarks while adhering to minimum length. T h e editors hope that readers will find the discussions to be useful supplements to the formal papers, particularly from the standpoint of highlighting untouched research topics in this fascinating field. In addition to the authors who have steadfastly supported the attempts of the editors to produce a volume of excellent papers, and the referees (those from the American Chemical Society ( A C S ) as well as those who critically read the individual papers), we owe debts of gratitude to many others.

W e are deeply grateful to S. Sisk (University of

Maryland) and to C . L a m b (National Bureau of Standards ( N B S ) ) who unstintingly provided the secretarial critical to a book in this format.

and professional typing support

W e especially thank our colleagues,

particularly Professors J. M . W o o d , M . L . Good, J. S. Thayer, J. K . Kochi, and W . R. Cullen, who sparked and sustained our effort for so many months. In the final analysis, these colleagues gave their talents to conducting and pacing symposium sessions, without which realization of this volume would have been impossible. xiv In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Finally, we are grateful to our sponsors who both financially a n d spiritually supported our effort. W e thank the A C S for many valuable suggestions and aids i n producing the symposium. W e are indebted to the National Measurements Laboratory, N B S , and the Division of Inorganic Chemistry, A C S , for assistance in organizing and defraying costs for invited authors. F. E . BRINCKMAN

J. M . B E L L A M A

Center for Materials Science

Department of Chemistry

National Bureau of Standards


Washington, D C 20234

College Park, M D 20742

August, 1978

xv


1 Biosynthesis of Organometallic and Organometalloidal Compounds FREDERICK C H A L L E N G E R 19 Elm Avenue, Beeston, Nottingham, NG9 1BU, U. K.

I f e e l g r e a t l y honoure Symposium an i n t r o d u c t o r to the proceedings. The programme covers almost every aspect of the b i o l o g i c a l methylation of a l a r g e number of elements and the p r o p e r t i e s of s e v e r a l other o r g a n o - m e t a l l i c compounds e . g . those of tin. C l e a r l y , only a b r i e f survey of c e r t a i n areas of t h i s wide field i s p o s s i b l e , and t h i s paper will be confined to those aspects of the Symposium which approach most c l o s e l y to the work of my students and myself, e s p e c i a l l y on the b i o l o g i c a l m e t h y l a t i o n of compounds of a r s e n i c , selenium, and t e l l u r i u m (1931-1953) at the U n i v e r s i t y of Leeds. A strictly historical treatment takes us back to the o l d l a b o r a t o r i e s at U n i v e r s i t y C o l l e g e , Nottingham (now merged in the Trent P o l y t e c h n i c ) where between 1900 and 1944 Kipping laid the foundations of the organic chemistry of silicon. I t was my privilege to work with him f o r 2½ years and to succeed i n the o p t i c a l r e s o l u t i o n of D , L - d i b e n z y l e t h y l p r o p y l s i l a n e monosulphonic a c i d by means of brucine i n December 1909. The study of the S i - C bond (though strictly speaking silicon i s n e i t h e r metal nor a m e t a l l o i d ) gave me a budding i n t e r e s t i n the metal-carbon link i n general, and p a r t i c u l a r l y i n o r g a n o - d e r i v a t i v e s of bismuth which l a t e r was extended to a r s e n i c . I t i s impossible here f o r me to emphasize s u f f i c i e n t l y my deep indebtedness to Professor F r e d e r i c k Stanley K i p p i n g , which I have endeavoured partially to discharge i n an a p p r e c i a t i o n of the man and h i s work, published i n 1950 and 1951 (1-2). His exacting l a b o r a t o r y methods were based on those of von Baeyer ( i n whose l a b o r a t o r y he had worked) and r e q u i r e d little but beakers, f l a s k s , t e s t tubes and g l a s s r o d s . They were at once the d e s p a i r and the i n s p i r a t i o n of h i s research students. I t was not s o l e l y an i n t e r e s t i n o r g a n o - m e t a l l i c compounds that l e d to our work on the a r s e n i c a l Gosio-gas but an e q u a l l y keen a t t r a c t i o n , m i c r o b i o l o g i c a l chemistry, of which I gained some rudimentary knowledge i n the l a b o r a t o r y of Professor A l f r e d Koch in Gottingen (1910-1912) and maintained i n Manchester. In 1931 i t

0-8412-0461-6/78/47-082-001$05.00/0 © 1978 American Chemical Society In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

ORGANOMETALS AND

ORGANOMETALLOIDS

became p o s s i b l e , w i t h the a s s i s t a n c e of Constance Higginbottom and Louis E l l i s (3),to combine these two i n t e r e s t s . I was a t t r a c t e d by the e a r l y work on the unknown composition of Gosio-gas which was evolved from a r s e n i c a l wallpaper c o n t a i n i n g the m i n e r a l p i g ments Scheele's green and P a r i s green. L a t e r on, t h i s vapour was found by Gosio (4) to be evolved from pure c u l t u r e s of the mould Pénicillium b r e v i c a u l i s (now d e s i g nated S c o p u l a r i o p s i s b r e v i c a u l i s ) c o n t a i n i n g arsenious oxide. At the time i t appeared that the p o s s i b l e i d e n t i f i c a t i o n of Gosio-gas would, w h i l e i n t e r e s t i n g , r e l a t e to a s m a l l area of m y c o l o g i c a l chemistry w i t h , p o s s i b l y , a q u i t e r e s t r i c t e d s i g n i f i c a n c e . When we found that Gosio-gas was pure t r i m e t h y l a r s i n e , we were suddenly e j e c t e d from our s m a l l corner and p i t c h f o r k e d d i r e c t l y i n t o the growing f i e l d of Transmethylation, then i n the e a r l y stages of i t s development i n animals b th fundamental k f Vincent d Vigneaud (5). There we my metaphors are very mixe persona the i n v e s t i g a t i o n s which f o l l o w e d may perhaps be accepted as an excuse Î B i o l o g i c a l M e t h y l a t i o n of A r s e n i t e and Arsenate The e a r l i e s t attempts t o i d e n t i f y Gosio-gas were made by B i g i n e l l i who passed the gases from aerated c u l t u r e s of S^. b r e v i c a u l i s c o n t a i n i n g AsÔ^ through H g C l i n d i l u t e h y d r o c h l o r i c a c i d . He regarded the r e s u l t i n g p r e c i p i t a t e as (CH^CH^ AsH* 2 H g C l , but a study i n my l a b o r a t o r y at the U n i v e r s i t y of Leeds showed f t to be a mixture of the mono- and d i m e r c u r i c h l o r i d e s of t r i m e t h y l a r s i n e . The f i r s t c l u e to the i d e n t i t y of Gosio-gas was obtained, however, by passage through a l c o h o l i c b e n z y l c h l o r i d e , formation of trimethylbenzylarsonium p i c r a t e , and comparison w i t h an a u t h e n t i c specimen prepared some years p r e v i o u s l y by I n g o l d , Shaw, and Wilson at Leeds i n research (6) on the o r i e n t i n g i n f l u e n c e of posi t i v e poles i n aromatic s u b s t i t u t i o n . The i d e n t i t y was confirmed by the formation of s e v e r a l other d e r i v a t i v e s , see below. Sodium methylarsenate CHÂsO(ONa) and sodium cacodylate (CH^) AsO(ONa) when added to the mould c u l t u r e s on bread crumbs a l s o gave t r i m e t h y l a r s i n e . This r e a c t i o n was, however, ambiguous owing to the p o s s i b i l i t y of the f i s s i o n of the As-C l i n k i n these a c i d s and formation of A s 0 ^ . However, when s e v e r a l mono- or d i a l k y l a r s o n i c a c i d s RAsO(OH) and RR'AsO-OH or t h e i r sodium s a l t s were added t o bread c u l t u r e s of S. b r e v i c a u l i s , m e t h y l a t i o n occurred g i v i n g e t h y l d i m e t h y l a r s i n e , n - p r o p y l d i m e t h y l a r s i n e , a l l y l d i m e t h y l a r s i n e and m e t h y l e t h y l n-propylarsine. These were c h a r a c t e r i z e d by formation of v a r i o u s d e r i v a t i v e s such as the m e r c u r i c h l o r i d e , the b e n z y l t r i a l k y l a r sonium p i c r a t e and the h y d r o x y t r i a l k y l a r s o n i u m p i c r a t e , and comp a r i s o n s w i t h a u t h e n t i c specimens. I t was t h e r e f o r e c l e a r t h a t the methyl group was s u p p l i e d by the mould (7) as summarized i n the scheme below. 2

2

2

2

2

2

2


1.

CHALLENGER

Biosynthesis

B i o l o g i c a l M e t h y l a t i o n of A l k y l and D i a l k y l a r s o n i c A c i d s CH AsO(OH) 3

2

(CH ) As 3

3

(CH ) AsO(OH) 3

2

CH CH As(CH ) 3

RAsO(OH)

2

3

2

> CH CH CH As(CH )

2

3

2

2

3

2

CH :CHCH As(CH )

C H

R

*>sO(OH)

>

3

CH.CH Âs 0

R

y

3

CH CH CH 3

2

2

M e t h y l a t i o n o f Selenate, S e l e n i t e , and T e l l u r i t e S e v e r a l workers had already drawn a t t e n t i o n t o the odourous products evolved from c u l t u r e s of S_. b r e v i c a u l i s c o n t a i n i n g v a r i o u s oxy-acids of selenium and t e l l u r i u m , but without i d e n t i f y i n g them. Using s i m i l a r methods t o those employed f o r a r s e n i c com pounds, the odours were i d e n t i f i e d by Harry North (8) and M a r j o r i e B i r d (9) as due t o d i m e t h y l s e l e n i d e and d i m e t h y l t e l l u r i d e . These were c h a r a c t e r i s e d by v a r i o u s d e r i v a t i v e s such as the m e r c u r i c h l o r i d e , mercuribromide, p l a t i n o c h l o r i d e ( ( C H ) S e - P t C J l ) , hydroxyselenonium n i t r a t e (CH > Se(OH)·Ν0 and the p i c r a t e s prepared u s i n g b e n z y l c h l o r i d e as before. Dimethylselenide and - t e l l u r i d e were a l s o evolved from v a r i o u s c u l t u r e s of Pénicillium chrysogenum, JP. notaturn, and a mould c l o s e l y a l l i e d t o P. notatum. A s p e r g i l l u s n i g e r gave (CH^KSe w i t h s e l e n a t e . D i m e t h y l t e l l u r i d e could only be detected (except by i t s powerf u l odour) when the c u l t u r e s were grown i n t e s t tubes i n s e r i e s t o minimise atmospheric o x i d a t i o n and the minimum of absorbent was used. 3

3

2

2

2

3>

The Metabolism of Some Sulphur Compounds i n Mould C u l t u r e s S t r i c t l y speaking, t h i s subject i s not w i t h i n the purview of t h i s Symposium, but as the r e s u l t s now t o be summarised a r e of r a t h e r general a p p l i c a t i o n , some account of them may be j u s t i f i e d .


4

ORGANOMETALS AND

ORGANOMETALLOIDS

S_. b r e v i c a u l i s was a l s o shown by Alan Rawlings (10), P h i l i p C h a r l ton (11), and Stanley Blackburn (12) to cause f i s s i o n of d i m e t h y l , d i e t h y l , d i - n - p r o p y l and d i - n - b u t y l d i s u l p h i d e s , RS-SR, g i v i n g RSH and RSCïL. No f i s s i o n was observed w i t h d i b e n z y l - and d i p h e n y l d i s u l p h i a e s , i n accord w i t h the r e s u l t s obtained w i t h phenyl-and b e n z y l a r s o n i c a c i d s which g i v e no methylated a r s i n e s i n the mould c u l t u r e s . Another i n t e r e s t i n g f i s s i o n r e a c t i o n was observed by C h a r l t o n (11). b r e v i c a u l i s , i n bread c u l t u r e s , produces a l k y l t h i o l and alkylmethy 1 sulphide from S-methyl-, S - e t h y l - and S-n-prop y l c y s t e i n e s , RSCH CHNH C00H. The sulphur compounds were charact e r i s e d by formation of well-known a u t h e n t i c compounds. These f i s s i o n r e a c t i o n s have w e l l - e s t a b l i s h e d analogies i n animal b i o chemistry. A d d i t i o n of sulphur, Na S0«, Na S„0«, t h i o u r e a , sodium e t h anesulphinate and sulphonate and sodium t h i o d i g l y e o l l a t e S(CH C00Na) , to culture gave no dimethylsulphide and at present the author knows of only one mould which w i l l methy l a t e simple i n o r g a n i c compounds of sulphur, namely Schizophyllum commune, a higher fungus which destroys wood and which was shown by Birkinshaw, F i n d l a y , and Webb (13) to g i v e methane t h i o l and t r a c e s of hydrogen sulphide when grown on a glucose medium cont a i n i n g sulphate. We found a t Leeds t h a t d i m e t h y l s u l p h i d e and - d i s u l p h i d e are a l s o evolved, and t h a t c u l t u r e s on wort or bread without a d d i t i o n of sulphate evolve methanethiol due to sulphur compounds i n the medium. When the c u l t u r e s are almost odourless, a d d i t i o n of sodium selenate gave d i m e t h y l s e l e n i d e i n s m a l l amount. Only f a i n t odours were observed on adding a r s e n i t e , t e l l u r i t e , or methyl- or n-propylarsonic a c i d s to s i m i l a r c u l t u r e s . F i s s i o n to the corresponding t h i o l , RSH, was observed w i t h d i m e t h y l , d i e t h y l , and d i - n - b u t y l d i s u l p h i d e s . With d i m e t h y l d i s u l p h i d e some dimethylsulphide was detected. A c a r e f u l study of the metabolism of t h i s fungus might y i e l d i n t e r e s t i n g r e s u l t s . 2

2

2

2

2

2

Selective Methylation B i r d et a l . (14) s t u d i e d the c a p a c i t y of a number of other moulds to methylate i n o r g a n i c and organic compounds of a r s e n i c , selenium, and t e l l u r i u m . A. n i g e r , V_. notatum and P. chrysogenum i n bread c u l t u r e s gave no t r i m e t h y l a r s i n e w i t h a r s e n i t e , but w i t h methyl- and dimethylarsonic a c i d s as sodium s a l t s , t r i m e t h y l a r s i n e was evolved. A. n i g e r gave e t h y l d i m e t h y l a r s i n e w i t h sodium e t h y l arsonate. The same mould w i t h selenate and the two P e n i c i l l i a w i t h s e l e n i t e gave d i m e t h y l s e l e n i d e . A. v e r s i c o l o r and A. glaucus gave t r i m e t h y l a r s i n e w i t h a r s e n i t e and sodium methylarsonate, but no dimethylselenide or - t e l l u r i d e w i t h s e l e n i t e or t e l l u r i t e . Attempts at an e x p l a n a t i o n of s e l e c t i v e m e t h y l a t i o n were made by B i r d et a l . (14), but no d e f i n i t e c o n c l u s i o n s were reached.


1.

CHALLENGER

Biosynthesis

5

The Development of Ideas on The Mechanism of B i o l o g i c a l M e t h y l ation I n 1887 i t was shown by H i s (15) t h a t p y r i d i n e acetate admin i s t e r e d t o dogs and t u r t l e s i s e x c r e t e d as methylpyridinium ace t a t e . An analogous r e a c t i o n occurs w i t h q u i n o l i n e i n dogs. A s t r o n g g a r l i c odour was observed by Gmelin (15) and by Hansen (16) i n animals and man, r e s p e c t i v e l y , a f t e r doses of potassium t e l l u r i t e . T h i s odour was regarded by Hofmeister (15) as being due t o d i m e t h y l t e l l u r i d e , though without p r o o f . I n h i s a r t i c l e we can recognize the f i r s t r a t h e r vague conception of a p o s s i b l e t r a n s f e r of a methyl group. He suggested that "the methyl group i s a l r e a d y present i n the t i s s u e s which possess the c a p a c i t y f o r m e t h y l a t i o n . I n presence of p y r i d i n e and t e l l u r i u m these are methylated, where as under normal c o n d i t i o n c r e a t i n e are produced. the source of the methyl group. T h i s conception was c a r r i e d much f u r t h e r by R i e s s e r i n 1913 (17) who considered that methyl groups of the (assumed) d i m e t h y l t e l l u r i d e , formed i n the animal body a f t e r t e l l u r i t e a d m i n i s t r a t i o n , probably arose from c h o l i n e or bet a i n e . He based t h i s suggestion p a r t l y on h i s o b s e r v a t i o n t h a t , when t e l l u r i t e was heated w i t h sodium formate and e i t h e r c h o l i n e c h l o r i d e or b e t a i n e h y d r o c h l o r i d e , an odour resembling d i m e t h y l t e l l u r i d e was evolved. Challenger et a l . (18) extended t h i s r e a c t i o n t o i n c l u d e sodium s e l e n i t e and s u l p h i t e , which on h e a t i n g w i t h b e t a i n e f r e e from h y d r o c h l o r i d e and without formate, gave d i m e t h y l s e l e n i d e and-sulphide which were i d e n t i f i e d by formation of d e r i v a t i v e s . Under s i m i l a r c o n d i t i o n s pure b e t a i n e , when heat ed w i t h primary aromatic amines, gave RNHCH^. Phenol and β-napht h o l gave the corresponding methyl e t h e r s , an i m i t a t i o n at h i g h temperatures of many b i o l o g i c a l m e t h y l a t i o n s . In 1935 C h a l l e n g e r and Higginbottom (19) s t a t e d " I t i s not impossible that some i n g r e d i e n t of the c e l l substance c o n t a i n i n g a methylated n i t r o g e n atom may, under the s p e c i a l c o n d i t i o n s ob t a i n i n g i n the c e l l , l o s e a methyl group which i f i t be e l i m i n a t e d w i t h a p o s i t i v e charge could be e a s i l y co-ordinated by the un shared e l e c t r o n s of t e r v a l e n t a r s e n i c o r q u a d r i v a l e n t selenium and t e l l u r i u m . " The u n d e r l i n i n g i n d i c a t e s the degree t o which t h i s suggestion extends those of Hofmeister and R i e s s e r . C h a l l e n g e r , i n s e v e r a l p u b l i c a t i o n s , d i s c u s s e d t h i s conception i n d e t a i l and proposed a s e r i e s of r e a c t i o n s f o r the m e t h y l a t i o n of arsenate or a r s e n i t e , s e l e n a t e o r s e l e n i t e which f o r b r e v i t y may be summarised on the next page. The scheme i n v o l v e s i o n i s a t i o n , r e d u c t i o n , and the c o o r d i n a t i o n of a p o s i t i v e methyl group. The suggested i n t e r m e d i a t e a r s e n i c compounds were not found i n the medium but were a l l conver ted t o t r i m e t h y l a r s i n e i n bread c u l t u r e s of J3. b r e v i c a u l i s . Met h y l a r s o n a t e , however, was found by McBride and Wolfe (20) as an i n t e r m e d i a t e compound i n the b i o l o g i c a l formation of d i m e t h y l a r s i n e by a methanobacterium from c a n a l mud. The potassium s a l t s of methane-, ethane-, and n-propanesel-


ORGANOMETALS AND ORGANOMETALLOIDS

6

e n i n i c a c i d s , RSeC^H, r e a d i l y gave the corresponding m e t h y l a l k y l s e l e n i d e s i n bread c u l t u r e s o f SL b r e v i c a u l i s . The potassium s a l t s o f methane-, ethane- and n-propaneselenonic a c i d s , RSeCÔH, unexpectedly gave o n l y d i m e t h y l s e l e n i d e due t o h y d r o l y s i s t o s e l e n i t e . Dimethylselenone, (CH«) SeO«, has not been prepared, but methylselenoxide n i t r a t e , (ciL^SefoH)(ONC^), a l s o gave d i m e t h y l selenide. ?

B i o l o g i c a l M e t h y l a t i o n of A r s e n i t e and Selenate

1.

As(OH)

> CH AsO(OH)

3

3

(CH ) AsO 3

2.

Se0 (0H) 2

> (CH > AsO(OH)

2

3

> (CH ) As

3

3

3

> CH SeO(OH)

2

>(CH > Se0

3

(CH ) SeO 3

>

2

3

2

>

2

> (CH ) Se

2

3

2

The r e a c t i o n s s e t out above may be represented as the a d d i t i o n s o f a methylcarbonium i o n , CH +, t o a n e u t r a l molecule, f o l lowed by e x p u l s i o n of a proton: CH

+ 3

+ :As(OH)

> CH As(OH>

3

3

>CH AsO(OH> + H

3

3

+

2

In recent years much a t t e n t i o n has been d i r e c t e d towards methion i n e . I n l i v e r o r kidney enzyme systems which can e f f e c t methyla t i o n , Cantoni (21) found i n 1952 t h a t added methionine forms a sulphonium compound, S-adenosylmethionine, or " a c t i v e methionine", as i t was f i r s t c a l l e d . I t s formula and involvement i n b i o l o g i c a l m e t h y l a t i o n a r e so,well-known as t o need no comment. I f i t i s represented as RR sCH , and i f we assume that methionine i s s i m i l a r l y " a c t i v a t e d " i n moulds the b i o m e t h y l a t i o n of a r s e n i t e could be represented: + + RR SCH + :As(OH) > [ R R S < — CH :As(OH> ] > f

3

f

l

3

3

3

1

RSR + [CH As(OH) ] 3

3

3

> CH AsO(OH> + H 3

+

2

The a t t r a c t i o n of the p o s i t i v e sulphur center f o r the e l e c t r o n s of the -S-CH l i n k might a l l o w n u c l e o p h i l i c a t t a c k on the methyl group by the a r s e n i c atom w i t h i t s unshared e l e c t r o n s . The r e s u l t ing t r a n s i t i o n s t a t e would l e a d t o a n e u t r a l s u l p h i d e (R*S*R ), a p r o t o n , and methylarsonic a c i d without formation of a f r e e p o s i t i v e methyl i o n a t any s t a t e . This e x p l a n a t i o n i s p r e f e r r e d by some of my c o l l e a g u e s . I t was put forward by Challenger i n 1955 3

f


1.

CHALLENGER

7

Biosynthesis

and 1959 i n two reviews (22, 23) and i s s t i l l regarded as a s a t i s f a c t o r y explanation. The f a i l u r e of the author and co-workers (24) i n t h e i r adm i t t e d l y p r e l i m i n a r y experiments of 1935 t o observe any methyla t i o n of mercuric oxide by S^. b r e v i c a u l i s i s e x p l i c a b l e because the Hg^ i o n would not behave as a n u c l e o p h i l e . For the l a t e r r e s u l t s of other workers see p. 16 of t h i s review. I t may, perhaps, be emphasised here that the fundamental demo n s t r a t i o n of the a c t i v a t i o n of methionine p r i o r t o the t r a n s f e r of i t s methyl group (transmethylation) was f i r s t made i n 1952 by Cantoni (21). My suggestion (22, 23) t h a t methionine might be s i m i l a r l y a c t i v a t e d i n moulds and t h a t t h i s might precede the m e t h y l a t i o n of a r s e n i c was an adaption of Cantoni*s work a t a much l a t e r date. This h i s t o r i c a l p o i n t i s n o t , perhaps, made q u i t e c l e a r by R i d l e y , D i z i k e s Cheh, and Wood (26). +

14 M e t h y l a t i o n of Selenium u s i n g Methionine L a b e l l e d w i t h L i q u i d C u l t u r e s of A s p e r g i l l u s n i g e r

C- i n

In an extended i n v e s t i g a t i o n w i t h P h i l i p D r a n s f i e l d and Denis L i s l e (27, 28), the author showed t h a t i n l i q u i d c u l t u r e s of A. n i g e r c o n t a i n i n g sucrose, g l y c i n e , i n o r g a n i c s a l t s , s e l e n a t e , and e i t h e r DL-, L- or D-[Me C]methionine, over 90% of the methyl groups of the evolved d i m e t h y l s e l e n i d e were d e r i v e d from the l a b e l l e d methionine. The d i m e t h y l s e l e n i d e was c o l l e c t e d i n aqueous mercuric c h l o r i d e and counted as the m e r c u r i c h l o r i d e adduct, ( C H ) S e - H g C l , m.p. 153-154°. A. n i g e r does not methylate A s ^ , but i n one experiment w i t h S^. b r e v i c a u l i s i n bread c u l t u r e s cont a i n i n g arsenious oxide and DL-[Me-^C]methionine, the m e t h y l a t i o n percentage was 28.3%, a v e r y much lower f i g u r e than was obtained w i t h A. n i g e r and s e l e n a t e . 3

2

2

Search f o r Methanol i n A r s e n i c a l L i q u i d C u l t u r e s of S. b r e v i c a u l i s I f a f r e e p o s i t i v e CIL group i s the m e t h y l a t i n g agent i n j[. b r e v i c a u l i s c u l t u r e s , i t might be expected t o r e a c t w i t h the water of the medium t o g i v e methanol. Attempts were made t o detect the methanol i n 2-3 l i t r e s of a r s e n i c a l medium upon which the mould had grown f o r 46 days. A f t e r c a r e f u l f r a c t i o n a l d i s t i l l a t i o n , the f i r s t runnings were t e s t e d f o r methanol by Wright's method (29). T h i s depends on o x i d a t i o n t o formaldehyde w i t h potassium permanganate, and i t s d e t e c t i o n by S c h i f f s reagent. The r e s u l t s obtained by Douglas Barnard (30) suggested the presence of not more than 0.001 mL per l i t r e of medium. This f i g u r e was q u i t e i n s u f f i c i e n t to a l l o w any c o n c l u s i o n t o be drawn as to the mechanism of format i o n or even of the a c t u a l presence of methanol. L a t e r experiments by Fernand K i e f f e r (31) were e q u a l l y i n c o n c l u s i v e . More d e l i c a t e methods employed by A x e l r o d and Daly i n 1965 (32, 33) have shown t h a t an enzyme o c c u r r i n g i n the p i t u i t a r y f


ORGANOMETALS AND

8

ORGANOMETALLOIDS

XH

gland of s e v e r a l mammals can form methanol from [Me C] -S-adenosylmethionine. I n p a r a l l e l experiments u s i n g the same enzyme p r e p a r a t i o n and [ 2 - ^ C ] S-adenosylmethionine, the demethylated p r o duct [2--^C] S-adenosylhomocysteine was formed. The methanol and the homocysteine d e r i v a t i v e were formed e n z y m i c a l l y and i n s t o i chiometrical proportions. The methanol was detected by conversion to i t s 3 , 5 - d i n i t r o benzoate which was r a d i o a c t i v e and was i d e n t i f i e d by t h i n l a y e r chromatography a f t e r d i l u t i o n w i t h an i n a c t i v e specimen and c r y s t a l l i s a t i o n t o constant s p e c i f i c a c t i v i t y . The authors s t a t e " t h i s r e a c t i o n proceeds by m e t h y l a t i o n of water or by h y d r o l y s i s of S—adenosylmethionine". McBride and Wolfe (20) showed t h a t arsenate i s reduced and methylated under anaerobic c o n d i t i o n s ( i n a hydrogen atmosphere) by c e l l e x t r a c t s and b MoH) i n present of methylcobalami The organism was i s o l a t e d from the mud of a c a n a l near D e l f t . The gaseous product was regarded as d i m e t h y l a r s i n e , and m e t h y l a r s o n i c a c i d CH AsO(OH) was i d e n t i f i e d by e l e c t r o p h o r e s i s . Dimethyla r s o n i c (cacodylic) a c i d was not found i n the medium, but both i t and m e t h y l a r s o n i c a c i d gave d i m e t h y l a r s i n e i n c e l l e x t r a c t s of the bacterium. The r e a c t i o n was shown t o be enzymic. The authors p o i n t e d out t h a t a mixture of CILAslL and (CH-KAs would g i v e the same r a t i o of CH^ t o As as was found by a n a l y s i s of the v o l a t i l e product. However, v a r i a t i o n i n the c o n c e n t r a t i o n of the methyl donor d i d not a f f e c t t h i s r a t i o . In another paper of t h i s symposium Dr. McBride and h i s c o l leagues d i s c u s s t h e i r c o n s i d e r a b l e l a t e r work i n which dimethy1a r s i n e i s produced by anaerobic organisms, and t r i m e t h y l a r s i n e i s produced by a e r o b i c organisms. A l l the b i o l o g i c a l l y methylated a r s i n e s described by Challenger were produced i n w e l l - a e r a t e d mould c u l t u r e s which n e v e r t h e l e s s e x h i b i t e d a s t r o n g reducing a c t i o n . The authors s t a t e t h a t they are not aware of any other references t o the b a c t e r i a l s y n t h e s i s of a r s i n e or i t s a l k y l a t e d d e r i v a t i v e s . T h i s r e c a l l s the n e g a t i v e r e s u l t s of Challenger and Higginbottom (19) w i t h AsÔ^ and s e v e r a l b a c t e r i a , a p o i n t emphas i s e d by Challenger (34) at the B r u s s e l s Biochemical Congress i n 1955. 3

2

Attempts to Methylate D e r i v a t i v e s of Elements other than A r s e n i c Antimony I n Vienna a case of c h r o n i c antimony p o i s o n i n g occurred i n a house c o n t a i n i n g s i l k c u r t a i n s mordanted w i t h a compound of antimony. A s e r i e s of experiments were performed i n 1913 by K n a f f l - L e n z (35) to detect the p o s s i b l e formation of v o l a t i l e compounds of antimony. Thus, S^. b r e v i c a u l i s was grown on media c o n t a i n i n g one percent of t a r t a r emetic (potassium antimonyl t a r t r a t e , 0=Sb-0-C0·CHOH·CHOH·CO·OK), w i t h the v o l a t i l e products being a s p i r a t e d through concentrated n i t r i c a c i d which was l a t e r t e s t e d f o r antimony w i t h n e g a t i v e r e s u l t s . L a t e r , s i m i l a r work by


1.

CHALLENGER

Biosynthesis

9

Tiegs (36) and a l s o by Smith and Cameron (36) was a l s o unsuccessf u l . The l a s t named authors s t a t e t h a t antimony compounds do not i n t e r f e r e w i t h the b i o l o g i c a l t e s t f o r a r s e n i c u s i n g j>. b r e v i c a u l i s . Challenger and E l l i s (36) found that bread o r l i q u i d c u l tures of S^. b r e v i c a u l i s c o n t a i n i n g t a r t a r emetic gave no odour or any p r e c i p i t a t e i n a c i d i f i e d mercuric c h l o r i d e s o l u t i o n . When the c u l t u r e s were l e f t ' f o r 9 months, a l a r g e amount of antimony t r i oxide Sb 0^ was deposited i n the mycelium. An attempt t o i s o l a t e t r i m e t h y l s t i b i n e oxide as the p i c r a t e from these mould c u l t u r e s f a i l e d . T a r t a r emetic and Sb 0~ c o n t a i n antimony as a c a t i o n . M e t h y l a t i o n by n u c l e o p h i l i c mechanism analogous to that e x e r t e d by j>. b r e v i c a u l i s i n a r s e n i c a l media would t h e r e f o r e not be expected. In experiments by Barnard (37) w i t h Î5. b r e v i c a u l i s and Έ_. no t a turn c u l t u r e s c o n t a i n i n g p h e n y l s t i b o n i c a c i d C^H^SbOiOH)^ or potassium meta-antimoniate KSbO^ i whic i anion methylation might be expected but non products from the c u l t u r e s i n t o concentrated n i t r i c a c i d , evapor a t i o n of the a c i d and a p p l i c a t i o n of the G u t z e i t or Marsh t e s t to the r e s i d u e gave v a r y i n g r e s u l t s . The amount of v o l a t i l e antimony compound was, however, f a r too s m a l l to a l l o w any c o n c l u s i o n s as t o i t s nature or o r i g i n . The p o s i t i o n does not seem t o have a l t e r e d s i n c e these exper iments were performed i n 1933 and 1947. P a r r i s and Brinckman (38) s t a t e "At t h i s time i t has not been demonstrated that methyls t i b i n e s are m e t a b o l i t e s of microorganisms a c t i n g on i n o r g a n i c antimony compounds, but the e x t e n s i v e s i m i l a r i t y of the chemistry of a r s e n i c and antimony g i v e s reasons t o b e l i e v e t h a t antimony can be b i o l o g i c a l l y methylated." In a l a t e r paper (39) these authors say "There i s no obvious thermodynamic or k i n e t i c b a r r i e r to b i o m e t h y l a t i o n and the chem i c a l s i m i l a r i t i e s between Sb and Sn, Pb, As, Se, and Te, which l i t e r a l l y surround Sb i n the P e r i o d i c Table, and a l l of which have been shown to be s u b j e c t t o b i o m e t h y l a t i o n , would suggest b i o m e t h y l a t i o n pathways f o r antimony." The authors then r e f e r t o the use of i n o r g a n i c and o r g a n i c compounds of antimony along w i t h halogenated hydrocarbons i n f i r e r e t a r d a n t systems. Should b i o m e t h y l a t i o n o c c u r , the antimony i n v a r i o u s commercial products would become more s o l u b l e i n water and become a p o t e n t i a l hazard. The occurrence of arsenious oxide i n antimony p r e p a r a t i o n s should a l s o be borne i n mind. An unusual outbread of antimony p o i s o n i n g (not due, however, t o b i o m e t h y l a t i o n ) occurred s e v e r a l years ago i n a l a r g e shop i n Newcastle-upon-Tyne. On a v e r y hot day the employees were g i v e n lemonade, presumably " s y n t h e t i c " lemonade, i n l a r g e enamelled jugs. The enamel, as i n many other products, contained Sb 0- which d i s solved i n the t a r t a r i c (or c i t r i c ) a c i d of the lemonade to g i v e t a r t a r emetic or an analogous compound. The r e s u l t can be im agined . P a r r i s and Brinckman (38) a l s o r e f e r t o the atmospheric o x i d a t i o n of a s o l u t i o n of t r i m e t h y l a r s i n e i n methanol and the f o r 2

2

2


ORGANOMETALS AND

10

ORGANOMETALLOIDS

mation of c a c o d y l i c a c i d and t r i m e t h y l a r s i n e o x i d e , (CH~)~AsO, detected by NMR. This was a l s o observed by E l l i s (36) i n e t h e r . The c a c o d y l i c a c i d was separated and the oxide i s o l a t e d as the p i c r a t e . D i m e t h y l a l l y l a r s i n e behaved i n a s i m i l a r manner. Metabolism

of Selenium Compounds

Any study of the b i o c h e m i s t r y of selenium compounds w i l l i n v o l v e not o n l y t h e i r m e t h y l a t i o n but a l s o t h e i r uptake by p l a n t s and t r a n s f e r to animals. This has been d i s c u s s e d i n very many p u b l i c a t i o n s and need not be d e a l t w i t h i n d e t a i l here. Many selenium analogues of b i o l o g i c a l l y important amino a c i d s c o n t a i n ing sulphur have been found i n p l a n t s growing on s e l e n i f e r o u s soils. The l i t e r a t u r e on seleniu metabolis has been admirably discusse c l o s e l y p r i n t e d pages w i t Mention may here be made of a statement by Cerwenka and Cooper (41) that the odours produced i n animals by a d m i n i s t r a t i o n of s e l e n i t e and of t e l l u r i t e are s i m i l a r . The odour of d i m e t h y l t e l l u r i d e resembles that of t r i m e t h y l a r s i n e which i s , however, q u i t e d i f f e r e n t from that of d i m e t h y l s e l e n i d e . Here i t may be s a i d that the well-known m e t h y l a t i o n of i n o r g a n i c t e l l u r i u m compounds i n man and animals, so much more pronounced at s i m i l a r conc e n t r a t i o n s than that of selenium, w i l l not be d e a l t w i t h f u r t h e r i n t h i s review. I t s h i s t o r y and chemistry have been discussed p r e v i o u s l y by the author (22, 23). The e x h a l a t i o n of d i m e t h y l s e l e n i d e a f t e r i n j e c t i o n of r a d i o a c t i v e ( S e ) sodium s e l e n a t e i n t o r a t s has been reported by McConnell and Portman (42). Much a t t e n t i o n i s now being p a i d t o the e f f e c t of compounds of a second element on the biogenesis of o r g a n o m e t a l l i c or -métalloidal d e r i v a t i v e s . D i p l o c k (40) d i s c u s s e s i n d e t a i l the e f f e c t of a r s e n i c , cadmium, mercury, s i l v e r , and t h a l l i u m on the t o x i c i t y of selenium compounds i n animals. R e s u l t s up to the present are not always easy t o i n t e r p r e t and f u r t h e r advances w i l l be awaited w i t h interest. An important step forward was made by Byard (43) i n the course of a study of the metabolism of sodium s e l e n i t e c o n t a i n i n g t r a c e s of H ^Se0« a f t e r o r a l a d m i n i s t r a t i o n to r a t s . He found that the u r i n e contained the trimethylselenonium i o n Me Se . This ion was i s o l a t e d a f t e r i o n exchange chromatography as tne r e i n e c kate and converted to the c h l o r i d e (CH-^SeCJl. The i d e n t i t y of t h i s s a l t was e s t a b l i s h e d by paper chromatography, NMR, and mass spectrometry. I t was a l s o detected i n the bladder 30 minutes a f t e r i n j e c t i o n of s e l e n i t e and was t h e r e f o r e not a product of b a c t e r i a l a c t i o n . A second compound of selenium was i s o l a t e d but not i d e n t i f i e d . Almost simultaneously, Palmer et a l . (44) i s o l a t e d t r i m e t h y l selenonium c h l o r i d e from the u r i n e of r a t s a f t e r i n j e c t i o n w i t h [ S e ] s e l e n i t e by methods very s i m i l a r t o those employed by 2

75


CHALLENGER

1.

Biosynthesis

11

Byard. They used the r e i n e c k a t e , and by c o - c r y s t a l l i s a t i o n w i t h the a u t h e n t i c trimethylselenonium s a l t , i d e n t i f i e d t h e i r specimen as the corresponding c h l o r i d e , a c o n c l u s i o n confirmed by the NMR spectrum. In l a t e r s t u d i e s , Palmer elt a l . (45) i n j e c t e d r a t s i n t r a p e r i t o n e a l l y w i t h sodium selenate c o n t a i n i n g H '^SeO^. Several seleno-amino a c i d s were a l s o used. 2

Amino-Acids Containing Selenium

HOOCCH(NH )CH Se-SeCH CH(NH )COOH 2

2

2

2

Selenocystine

Selenomethylselenocysteine HOOCCH(NH )CH CH SeCH 2

2

2

3

Selenomethionine 75 Some of the selenomethionine was l a b e l l e d w i t h Se. Coc r y s t a l l i s a t i o n s of the r e i n e c k a t e s were again used and a l l the selenoamino a c i d s gave the same t r i m e t h y l s e l e n i u m c h l o r i d e , (CH ) SeC£, which was a l s o formed on feeding the r a t s w i t h s e l e n i f e r o u s wheat grown on s o i l c o n t a i n i n g s e l e n i t e o r s e l e n a t e . 3

3

Co-Enzyme M (2-Mercaptoethanesulphonic

Acid)

Recent work by McBride, Wolfe, and t h e i r colleagues (46, 47, 48, 49, 50) has provided much i n f o r m a t i o n about a h e a t - s t a b l e cof a c t o r f o r methyl t r a n s f e r p r i o r t o methane formation i n c e l l ext r a c t s of Methanobacterium s t r a i n M.O.H. grown i n hydrogen. The c o - f a c t o r i s a c i d i c and d i a l y s a b l e . I t a l s o occurs i n rumen f l u i d and has been designated Co-enzyme M. I t contains phosphate when f i r s t i s o l a t e d , and t h i s i s removable by prolonged a c i d h y d r o l y s i s . I t s r e l a t i o n t o the r e s t of the molecule has not been establ i s h e d . A f t e r prolonged p u r i f i c a t i o n of c e l l e x t r a c t s by i o n exchange, o r on a s m a l l e r s c a l e by d i a l y s i s , chemical a n a l y s i s and numerous s p e c t r o s c o p i c s t u d i e s showed t h a t the product was 2 : 2 - d i t h i o d i e t h a n e s u l p h o n i c a c i d ( I ) , a s t r u c t u r e v e r i f i e d by comparison w i t h a s y n t h e t i c specimen prepared from sodium 2-bromoethanesulphonate and H S i n ammoniacal s o l u t i o n . This procedure gave the corresponding -SH compound ( I I ) , which was converted t o the d i s u l p h i d e by gaseous oxygen. By u s i n g methanethiol i n s t e a d of H S, the S-methylated product ( I I I ) was obtained which was i d e n t i c a l w i t h the product formed b i o l o g i c a l l y from Co-enzyme M ,

2

2



12

Co-Enzyme M and D e r i v a t i v e s HOS0 CH CH SSCH CH S0 OH 2

2

2

2

2

(I)

2

CH SCH CH S0 OH 3

2

2

(III)

2

HSCH CH S0 OH 2

2

(II)

2

(CH ) t-CH CH S0 5 3

2

2

2

(IV)

2

( t h i o l form) and methyl-B^ i n c e l l e x t r a c t s of the Methanobacterium. The enzyme r e s p o n s i b l e was named methylcobalamin Coenzyme M methyl t r a n s f e r a s e , and was p u r i f i e d 100 times. By r e a c t i o n w i t h methyl i o d i d e i n methanol, the -SCH d e r i v a t i v e gave the corresponding dimethylsulphonium ethanesulphonate (IV). T h i s was found t o be i n e r t as a methyl donor i n the methyl r e d u c t a s e - c a t a l y z e d r e a c t i o n i n i n c u b a t i o n periods up t o 70 minu t e s . No methane production was observed when the onium compound (IV) and the t h i o l form i n the r e a c t i o n medium ate the t h i o l t o y i e l d b i o l o g i c a l l y a c t i v e methylCo-enzyme M ( I I I ) . T h i s s p e c i e s c o u l d not be f u r t h e r methylated by the enzyme methylt r a n s f erase. I t i s probable t h a t the d i s u l p h i d e form of Co-enzyme M (I) i s an a r t i f a c t a r i s i n g d u r i n g the p u r i f i c a t i o n process, and that the e f f e c t i v e Co-enzyme M i s the t h i o l form ( I I ) . I n b i o l o g i c a l systems which o b t a i n i n the mud of swamps, r i v e r s , and l a k e s , t h i s i s methylated t o t h e -SCH compound ( I I I ) which by enzymatic r e d u c t i o n g i v e s methane. 2

3

3

Speculations on the B i o s y n t h e s i s of Co-enzyme M A compound o f c l o s e l y r e l a t e d s t r u c t u r e i s i s e t h i o n i c a c i d H0CH CH «S0 0H. Some s p e c u l a t i o n s on the p o s s i b l e biochemical s i g n i f i c a n c e o f t h i s sulphonic a c i d were made by the author (51, 52) i n 1970 i n another connection. By r e a c t i o n w i t h H S, i s e t h i o n i c a c i d might g i v e the t h i o l form of Co-enzyme M, a r e a c t i o n analogous t o the enzymatic conv e r s i o n of s e r i n e t o c y s t e i n e i n yeast by H S, and the enzyme formerly known as s e r i n e s u l f h y d r a s e . Dagley and Nicholson (53) name the enzyme " L - s e r i n e hydrolyase, adding ILS". The modern name f o r t h i s enzyme (because of one of i t s c h i e f r e a c t i o n s ) i s c y s t a t h i o n i n e 3-synthase[L-serine-hydro-lyase (adding homoc y s t e i n e ) E.G.4.2.1.21.] (54). S i m i l a r l y , O-phosphohomoserine w i t h H S and a s u l f h y d r a s e gives homocysteine (55). I s e t h i o n i c a c i d i s connected w i t h c y s t e i n e by a s e r i e s of r e a c t i o n s s e t out below. Cysteine i s , no doubt, present i n the c e l l p r o t e i n of the methanobacterium. I t can be b i o l o g i c a l l y converted t o t a u r i n e , H ^ C I L ' C H ^ S O ^ H , which y i e l d s i s e t h i o n i c a c i d by enzymatic o x i d a t i v e deamination f o l l o w e d by r e d u c t i o n . Some of these r e a c t i o n s were c i t e d by Challenger i n 1970 (51, 5 2 ) . I have not access t o a l l my f i l e s a t present, but I t h i n k the c o n v e r s i o n of c y s t e i n e t o t a u r i n e i s w e l l e s t a b l i s h e d ; I f o r g e t the circumstances. I t h i n k i t i s described i n M e i s t e r s "Amino-acids"(2nd e d i t i o n ) . 2

2

2

2

2

2

1


1.

CHALLENGER

Biosynthesis

HSCH CH(NH )COOH 2

> H0 SC^CH (NH ) COOH

2

2

Cysteine

2

2

~ ° s H0 S · CH CH NH C

2

2

Cysteinesulphinic Acid

HOS0 CH CH NH 2

13

2

> [HOS0 CH CHO]

2

2

2

2

2

Hypotaurine

>HOS0 CH CH OH — 2 —

2

2

Taurine

2

Isethionic

2

acid

-> HOS0 'CH CH SH 2

2

2

Co-enzyme M

Taurine i s a produc J>. s u b t i l i s mutant (56). T h i s i s i n t e r e s t i n g i n view o f the f o r mation o f Co-enzyme M i n e x t r a c t s of s o n i c a t e d c e l l s o f Methanobacterium. When t a u r i n e i s present as s o l e source o f sulphur i n A. n i g e r c u l t u r e s , i s e t h i o n i c a c i d i s formed (57). Another p o s s i b l e (though not e s t a b l i s h e d ) source o f i s e t h i o n i c a c i d and so of Co-enzyme M i s 3 - L - s u l f o l a c t i c a c i d , HO^SCHp·CHOH*COOH, which has been p r o v i s i o n a l l y i d e n t i f i e d i n 1969 as a major sulphur compound i n jB. s u b t i l i s spores by Bonsen e t a l . (58). By a w e l l recognized b i o c h e m i c a l r e a c t i o n t h i s might g i v e formic a c i d and HO^S'CH'CHO which by r e d u c t i o n would y i e l d i s e t h i o n i c a c i d . I have not f o l l o w e d the f u r t h e r work ( i f any) on s u l f o l a c t i c a c i d , but the c l e a r a s s o c i a t i o n of s u l p h o n i c a c i d s w i t h c e l l w a l l s , d i s r u p t e d c e l l s , and spores suggests f u r t h e r study o f these r e l a t i o n s h i p s . I have always thought that i s e t h i o n i c a c i d i s worthy o f more a t t e n t i o n by b i o c h e m i s t s , and the f a c t s set out above may be s a i d to strengthen t h i s view. T h i s i s , I t h i n k , t r u e f o r s u l f o n i c a c i d s i n general. A b i l i t y of Metals f o r I n c o r p o r a t i o n i n t o L i v i n g Systems In a most u s e f u l p u b l i c a t i o n (59) summarising t h e r e s u l t s o f s i x o r seven y e a r s work, Wood d i s c u s s e s t h e a v a i l a b i l i t y o f numerous metals f o r i n c o r p o r a t i o n i n t o l i v i n g systems. He p o i n t s out that t i t a n i u m and aluminum are not so a v a i l a b l e because of the i n s o l u b i l i t y of t h e i r hydroxides ; and that n i c k e l and chromium a r e almost absent from b i o l o g i c a l systems, owing t o t h e s t a b i l i t y o f these c a t i o n s i n o c t a h e d r a l s i t e s i n s i l i c a t e s . These l a s t two elements do not form complexes w i t h p r o t e i n s f o r g e o m e t r i c a l reasons. The l i m i t s imposed by space and t h e s u b j e c t o f h i s r e view, " B i o l o g i c a l Cycles f o r Elements i n the Environment", unf o r t u n a t e l y do not permit him t o d i s c u s s these i n t e r e s t i n g observ a t i o n s i n d e t a i l . He r e f e r s , however, t o ten metals which, a f t e r 1


14

ORGANOMETALS AND

ORGANOMETALLOIDS

t r a n s p o r t i n t o the c e l l by s u i t a b l e c h e l a t i n g agents, bind to v a r i o u s l i g a n d s , and mentions the molybdenum-phosphorus l i n k . This r e c a l l s the ready formation of the y e l l o w phosphomolybdate p r e c i p i t a t e i n q u a l i t a t i v e a n a l y s i s . The requirements of many enzyme systems f o r magnesium, z i n c , and manganese are w e l l - r e c o g n i z e d . Having d i g r e s s e d s l i g h t l y i n t o the realm of i n o r g a n i c b i o chemistry, d e t a i l e d reference may now be made to a somewhat s i m i l a r i n v e s t i g a t i o n i n t o some aspects of the behaviour of t o x i c metals i n s o i l s . The r e s u l t s were p u b l i s h e d i n 1975 and only very r e c e n t l y came to the author's n o t i c e . Much v a l u a b l e work was published i n 1975 (60, 61) as the r e s u l t of a survey of the metal content of the s o i l of the s i t e f o r a s a t e l l i t e town at Beaumont-Leys, two m i l e s from L e i c e s t e r . P r i o r to 1964 the s i t e was used as a sewage farm, a f t e r which i t was l e t to farmers. In 1970, i vie f i t impendin investiga t i o n by the N a t i o n a l A g r i c u l t u r a c o n c e n t r a t i o n of heavy metals y copper on the s i t e . A v a l u e , based on the content of these three metals, known as the z i n c e q u i v a l e n t was used f o r a s s e s s i n g t h e i r content i n the s o i l , and 250 p a r t s per m i l l i o n (ppm) was regarded as perm i s s i b l e . Maximum values of 1000-6000 ppm were obtained f o r s o i l s from Beaumont-Leys. Moreover, the z i n c content of g r a i n grown on the e s t a t e was found to be 115 ppm, the maximum p e r m i s s i b l e v a l u e being 50 ppm. The study was then extended to i n c l u d e other more poisonous metals such as cadmium, a r s e n i c , and l e a d . The r e s u l t s were of p a r t i c u l a r i n t e r e s t because of the imminent development of the s i t e which was known to be contaminated (a) w i t h sewage sludge and (b) by sewage e f f l u e n t which flowed o f f a f t e r d e p o s i t i o n of the sludge. The r e s u l t s of t h i s i n v e s t i g a t i o n , r e p r e s e n t i n g much ext e n s i v e and d e t a i l e d work by E. R. P i k e , the L e i c e s t e r s h i r e County A n a l y s t , Miss L. C. Graham ( h i s deputy), and M. W. Fogden, are s e t out (60, 61) i n two p u b l i c a t i o n s . P a r t I deals w i t h z i n c , copper, and n i c k e l , P a r t I I w i t h l e a d , cadmium, a r s e n i c , and chromium. Some of the c o n c l u s i o n s w i l l be discussed l a t e r . In consequence, a r e - d i s t r i b u t i o n of the proposed s i t e s f o r houses and gardens, r e l a t i v e to other non-domestic b u i l d i n g , was found necessary. The q u e s t i o n of the uptake of t o x i c elements by vegetables a l s o had to be considered. I am g r e a t l y indebted to Mr. P i k e and h i s colleagues f o r sending me r e p r i n t s of t h e i r two papers and a l i s t of r e f e r e n c e s . The m i c r o b i o l o g i c a l and biomethy l a t i o n aspects of the s u b j e c t were not i n v e s t i g a t e d . The main l i n e s of study pursued by P i k e et a l . i n c l u d e d (1) determination of t o t a l metal p o l l u t i o n i n the s o i l by (a) sewage sludge d e p o s i t s and (b) sewage e f f l u e n t ; (2) determination of " a v a i l a b l e " metal i n the s o i l by e x t r a c t i o n w i t h 0.5 M a c e t i c a c i d . The " n o n - a v a i l a b l e " metal i s probably h e l d by r e a c t i o n w i t h the organic matter of the s o i l . Most of the analyses were c a r r i e d out by atomic a b s o r p t i o n spectrophotometry. A t h i r d l i n e of i n v e s t i g a t i o n i n v o l v e d the determination of t o x i c metal content i n


1.

CHALLENGER

Biosynthesis

15

vegetables grown on both (a) and (b) types of s o i l , both at Beaumont-Leys and on s i t e s remote therefrom. For d e t a i l s r e g a r d i n g most metals, r e f e r e n c e must be made t o the o r i g i n a l papers, but because of work on the uptake of cadmium by an American o y s t e r (62) and by c e r t a i n b a c t e r i a (63), r e s u l t s obtained f o r t h i s metal may be given i n some d e t a i l . At Beaumont-Leys, however, z i n c appears t o be present i n the g r e a t e s t c o n c e n t r a t i o n . The symptoms of the I t a i - I t a i d i s e a s e (Japan, 1939-1945) are d e s c r i b e d and a t t r i b u t e d to the cadmium content of r i v e r water t h a t r e c e i v e d waste\from a z i n c , l e a d , and cadmium mine and which was used f o r d r i n k i n g purposes and f o r i r r i g a t i o n of r i c e f i e l d s . The symptoms are probably due t o an i n h i b i t i o n of enzymes r e q u i r i n g z i n c . Cadmium hazards i n Great B r i t a i n have a r i s e n from fumes produced d u r i n g the welding of cadmium i n badly v e n t i l a t e d p l a c e s . I t has been found i n f o o d s t u f f d i huma t i s s u e s wher i t i n c r e a s e w i t h age, causin ease. The cadmium conten given as 0.01-5.00 ppm. In s p i t e of the h i g h z i n c content of s o i l at Beaumont-Leys, the t o t a l cadmium ranged from l e s s than 5 ppm ( e f f l u e n t area) t o 50 ppm i n the sludge areas. The corresponding f i g u r e s f o r a v a i l a b l e cadmium were l e s s than 5 ppm, r i s i n g to 25 ppm, on sludge ground. Those f i g u r e s were based on a v e r y l a r g e number of samples (sludge areas over 1200 samples, e f f l u e n t areas over 1700 samples). The r a t i o of a v a i l a b l e t o t o t a l cadmium (30%), and the t o t a l cadmium do not a l t e r g r e a t l y w i t h the depth of the s o i l . Lead presents a d i f f e r e n t p i c t u r e w i t h a p o s s i b l e a v a i l a b l e percentage of only 1.0. The mode of f i x a t i o n of compounds of metals to p r o t e i n and humus of the s o i l and t o v a r i o u s a c i d s , e.g. phosphoric, or t o s i l i c a t e s would present an i n t e r e s t i n g s u b j e c t f o r a l e i s u r e l y study beginning w i t h the s i m p l e s t analogues. The cadmium content of many garden and a l l o t m e n t s o i l s remote from Beaumont-Leys i s l i t t l e d i f f e r e n t from t h a t of the s o i l s which have r e c e i v e d e f f l u e n t , but much lower than t h a t i n those r e c e i v i n g sludge. As regards the uptake of cadmium by v e g e t a b l e s , a c a r e f u l study of seven garden v a r i e t i e s grown i n s o i l s c o n t a i n i n g cadmium r e v e a l e d a content of 0.05 to 0.5 ppm, but u s u a l l y nearer the lower l i m i t . The f i g u r e s obtained a t Beaumont-Leys and elsewhere may represent a normal cadmium content. A r s e n i c does not appear t o be a problem a t Beaumont-Leys. The h i g h e s t observed f i g u r e i s 59 ppm and then o n l y i n the areas h e a v i l y p o l l u t e d by sludge. I t s c o n c e n t r a t i o n i n the s o i l of most of the area i s s i m i l a r t o t h a t i n the gardens and a l l o t m e n t s a l r e a d y mentioned as remote from the s i t e . On c a r e f u l a n a l y s i s of 10 types of vegetab l e s grown on p l o t s at Beaumont-Leys only t r a c e s of a r s e n i c were found i n l e t t u c e s and r a d i s h e s and no d e t e c t a b l e amounts i n the others. The r e s u l t s at Beaumont-Leys c o n f i r m those c i t e d by MonierW i l l i a m s (64) t h a t only n e g l i g i b l e amounts of a r s e n i c are taken up by p l a n t s even from h i g h l y a r s e n i c a l s o i l s .


ORGANOMETALS AND

16

ORGANOMETALLOIDS

Lead The a n a l y t i c a l r e s u l t s of a study of the d i s t r i b u t i o n of l e a d on the Beaumont-Leys s i t e are i l l u s t r a t e d by two maps f o r (1) t o t a l and (2) a v a i l a b l e l e a d . Land r e c e i v i n g only e f f l u e n t t r e a t ment had a lead content not g r e a t l y d i f f e r e n t from the 200 ppm g i v e n by Swaine (65) f o r n a t u r a l s o i l . H i s s t u d i e s on the metal content of s o i l s are f r e q u e n t l y quoted by P i k e et a l . I t seems t h a t i t i s the content of " a v a i l a b l e " l e a d which i s s i g n i f i c a n t i n the uptake of t h i s metal by p l a n t s , much l e a d becoming i n s o l u b l e due t o r e a c t i o n w i t h p r o t e i n and other compounds present i n the s o i l a f t e r sludge d e p o s i t i o n . The presence of l e a d appears t o be confine d t o the f i r s t two f e e t of s o i l , below which the f i g u r e approaches that of the normal l e a d content of the area. The " a v a i l a b l e " l e a d can, however increas w i t h depth probabl owin to a decrease i n the organi The lead content o uptak by the u s u a l vegetables employed i n t h i s study, and i n a l l cases was much l e s s than the 2 ppm allowed f o r lead i n Food R e g u l a t i o n s (48, 53, 66). The paper presented t o the Symposium by Dr. G. K. Pagenkopf on the t r a n s p o r t of ions of t r a n s i t i o n and heavy m e t a l s , mediated by f u l v i c and humic a c i d s , i s v e r y r e l e v a n t t o the quest i o n of " a v a i l a b l e " and " n o n - a v a i l a b l e " metals ions t o which P i k e et a l . make such frequent r e f e r e n c e i n t h e i r d i s c u s s i o n of the Beaumont-Leys s o i l . Although Pagenkopf appears t o be concerned mainly w i t h r e a c t i o n s i n aqueous media, h i s f u l l paper w i l l be read w i t h much i n t e r e s t by a l l a g r i c u l t u r i s t s . The B i o l o g i c a l Degradation of Organo-derivatives of Mercury Wood (59), when r e f e r r i n g t o h i s well-known work on the m e t h y l a t i o n of compounds of mercury by methyl-B-^* p o i n t s out t h a t m e t h y l a t i o n proceeds more r a p i d l y than degradation by other organisms. Nelson, Brinckman et a l . (67), working i n a c l o s e d system, f i n d that benzene and mercury vapours are produced from phenylmercury (C^H HgOCOCH ) by c u l t u r e s of m e r c u r y - r e s i s t a n t b a c t e r i a . Under n a t u r a l c o n d i t i o n s the mercury so produced may p o s s i b l y , owing t o i t s v o l a t i l i t y , escape i n t o the atmosphere. D e t a i l s are given of the c u l t i v a t i o n of m e r c u r y - r e s i s t a n t b a c t e r i a on media c o n t a i n i n g mercuric c h l o r i d e and phenylmercury a c e t a t e , and a l s o r e f e r e n c e s t o t h e i r i d e n t i f i c a t i o n as t o genus. The phenylmercury a c e t a t e was l a b e l l e d w i t h H g and f u l l d e t a i l s of the analyses f o r Hg and benzene are provided and the apparatus d e s c r i b e d . References are a l s o given t o the conversion of C.H^HgOCOCH^ to mercury and diphenylmercury by a e r o b i c b a c t e r i a , which a l s o conv e r t CH«Hg and C H H g t o methane, ethane, and mercury. The work of Spangler et a l . (68) has shown t h a t , i n l a k e sediments c o n t a i n i n g Hg , formation of CH Hg occurs followed by a f a l l i n c o n c e n t r a t i o n and formation of m e t a l l i c mercury. Four p u r i f i e d c u l t u r e s of what appeared to be a Pseudomonas were 2 0 3

+

2

5

+

3


1.

CHALLENGER

Biosynthesis

17

+

i s o l a t e d from the sediment. These a l s o converted CH~Hg t o methane and mercury. These products were i d e n t i f i e d i n the head-space gases by flame i o n i s a t i o n gas chromatography and mass spectromet r y , r e s p e c t i v e l y , thus e x p l a i n i n g why CILHg " has been so d i f f i c u l t t o i d e n t i f y i n n a t u r a l sediments o r r i v e r s . Methane formation was only observed w i t h c u l t u r e s that degraded CHgHg under i d e n t i c a l c o n d i t i o n s . Somewhat s i m i l a r r e s u l t s were obtained by Edwards and McBride (69) who s t u d i e d the b i o s y n t h e s i s and degradation o f CH^Hg " i n human f e c e s . Formation o f methane i s mentioned s e v e r a l times but an extended d i s c u s s i o n of i t s o r i g i n would have been i n t e r e s t i n g . 4

+

4

The N a t u r a l Occurrence of Methylated

Compounds of A r s e n i c :

Braman and Forebac senate, a r s e n i t e , methylarsonate ate) i n many environmental samples i n c l u d i n g s e a - s h e l l s , egg s h e l l s , n a t u r a l waters and human u r i n e . The methods o f a n a l y s i s devised f o r the purpose a r e discussed and a l s o e a r l i e r methods s u i t a b l e only f o r t o t a l a r s e n i c . A r s e n i t e i s reduced t o AsH^ by sodium borohydride a t pH 4-9. Arsenate i s s t a b l e t o t h i s reagent and must f i r s t be reduced t o a r s e n i t e by sodium cyanoborohydride a t pH 1-2, followed by r e d u c t i o n w i t h NaBH^ a t pH 1-2. Methylarsonate and cacodylate gave the corresponding a r s i n e s w i t h NaBH, a t pH 1-2. The AsH^, CILAsIL, and ( C H ^ A s H were c o l l e c t e d i n g l a s s beads cooled i n l i q u i d n i t r o g e n and then passed a f t e r f r a c t i o n a l v o l a t i l i s a t i o n through an e l e c t r i c a l discharge g i v i n g a r s e n i c emission l i n e s which were examined p h o t o m e t r i c a l l y . Methylarsonic a c i d and/or c a c o d y l i c a c i d were s i m i l i a r l y found i n human u r i n e , and the authors suggest t h a t i n o r g a n i c a r s e n i c i s methylated by methylcobalamin or methionine i n the body. A l l these a r s e n i c compounds were found i n nanogram q u a n t i t i e s . The m e t h y l a t i o n of i n o r g a n i c a r s e n i c t o v o l a t i l e d i - o r t r i m e t h y l a r s i n e i n the human body has, t o the author's knowledge, never been r i g i d l y e s t a b l i s h e d . M e t h y l a t i o n may, o f course, cease at the c a c o d y l i c a c i d s t a t e , o r the methylarsines may be o x i d i s e d i n the body. The evidence was discussed i n 1945 by Challenger (71), and l a t e r work may have escaped h i s n o t i c e . The author would a p p r e c i a t e any references which h i s colleagues might send him. At the most the amount v o l a t i l i s e d must be minute as m e d i c i n a l doses of a r s e n i t e produce h a r d l y any odour, whereas that a r i s i n g from absorption o f t r a c e s o f t e l l u r i t e i s i n t e n s e . 4

Acknowledgement s I need h a r d l y say how much I am indebted t o my research c o l l a b o r a t o r s , the r e s u l t s o f whose labours f o r over 20 y e a r s , o f t e n w i t h unpleasant compounds, have added so g r e a t l y t o my enjoyment of U n i v e r s i t y l i f e and work.


18


Dr. D. Barnard has k i n d l y allowed me to mention some of h i s unpublished work on antimony compounds, and h i s research f o r meth a n o l i n mould c u l t u r e s [Ph.D. T h e s i s , U n i v e r s i t y of L e e d s ] . My thanks are a l s o due to D r . P . A . B r i s c o e and the l a t e D r . J . W. Baker f o r v a l u a b l e d i s c u s s i o n s on t h e o r e t i c a l p o i n t s . D i s c u s s i o n i n t h i s review on the development of ideas on b i o l o g i c a l m e t h y l a t i o n i s based on p a r t s of pages 164 and 170-173 of the a u t h o r ' s book "Aspects of the Organic Chemistry of S u l f u r " . I am indebted to my p u b l i s h e r s , Messrs. Butterworths, London, f o r permission to use t h i s m a t e r i a l . In c o n c l u s i o n , may I once more thank the o r g a n i s e r s of t h i s Symposium f o r a s k i n g me t o w r i t e t h i s review, and p a r t i c u l a r l y D r . Brinckman, Professor Woo ment and help they hav

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

C h a l l e n g e r , F., Obituary N o t i c e s of Fellows of the Royal Soc iety, (1950) 7, 183. C h a l l e n g e r , F., J. Chem. Soc. (1951) 849. C h a l l e n g e r , F., Higginbottom, Miss C., and Ellis, L., J. Chem. Soc. (1933) 95. G o s i o , Β., A r c h . Ital. Biol., (1901) 35, 201. duVigneaud, V., "A Trail of Research". C o r n e l l U n i v . P r e s s , I t h a c a , New York. I n g o l d , C.K., Shaw, F.R., and W i l s o n , C., J. Chem. Soc. (1928), 1280. C h a l l e n g e r , F., "Aspects of the Organic Chemistry of Sulphur". 165. Butterworths, London, 1959. C h a l l e n g e r , F., and N o r t h , H.E., J. Chem. Soc. (1934) 68. B i r d , M.L., and C h a l l e n g e r , F., J. Chem. Soc. (1939) 163. C h a l l e n g e r , F., and Rawlings, A.A., J. Chem. Soc. (1937) 868. C h a l l e n g e r , F., and C h a r l t o n , P.T., J. Chem. Soc. (1947) 424. B l a c k b u r n , S . , and C h a l l e n g e r , F., J. Chem. Soc. (1938) 1872. B i r k i n s h a w , J.H., F i n d l a y , W . P . K . , and Webb, R.A., Biochem. J. (1942) 36, 526. B i r d , M.L., C h a l l e n g e r , F., C h a r l t o n , P.T., and Smith, J.O., Biochem. J. (1948) 43, 78. C h a l l e n g e r , F., Quart. Rev. Chem. Soc. (1955) 9, 255. Hansen, Α . , Ann. der Chemie (1853) 86, 213. R i e s s e r , O., Z . p h y s i o l . Chem. (1913) 86, 440. C h a l l e n g e r , F., T a y l o r , P., and T a y l o r , B., J. Chem. Soc. (1942) 48. C h a l l e n g e r , F., and Higginbottom, C., Biochem. J. (1935) 29, 1757. McBride, B.C., and Wolfe, R.S., Biochemistry (1971) 10, 4312. C a n t o n i , G.L., J. Amer. Chem. Soc. (1952) 74, 2942.


1.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

CHALLENGER

Biosynthesis

19

See reference 7. See reference 15. C h a l l e n g e r , F., J. Soc. Chem. I n d . , London (1935) 54, 657. R i d l e y , W . P . , D i z i k e s , L.J., and Wood, J.M., Science (1977) 197, 329. R i d l e y , W . P . , Dizikes, L.J., Cheh, Α . , and Wood, J.M., E n v i r o n . Health P e r s p e c t i v e s (1977) 1 9 , 4 3 . D r a n s f i e l d , P.B., and C h a l l e n g e r , F., J. Chem. Soc. (1955) 1153. C h a l l e n g e r , F., Lisle, D.B., and D r a n s f i e l d , P.B., Chem. Soc. (1954) 1760. W r i g h t , J., I n d . & Eng. Chem., (1927) 19, 750. Barnard, D . , T h e s i s U n i v of Leeds (1947) 92-93 K i e f f e r , F., Unpublishe A x e l r o d , J., and D a l y , J., (1965) , A x e l r o d , J., in "Transmethylation and Methionine B i o s y n t h e sis", (Shapiro, S.R. and Schlenk F., e d s . ) U n i v . o f Chicago P r e s s , Chicago, 1965, p p . 71-83. C h a l l e n g e r , F., C o n f é r e n c e s e t Rapports, 3éme Congrès I n t e r n . de B i o c h i m i e , B r u x e l l e s (1955) 238. K n a f f l - L e n z , Arch. exp. P a t h . Pharm., (1913) 72, 224. C h a l l e n g e r , F., and Ellis, L., J. Chem. Soc. (1935) 396. Barnard, D . , T h e s i s , U n i v . o f Leeds, (1947) 16-29. P a r r i s , G . E . and Brinckman, F.E., J. Org. Chem. (1975) 40, 3801. P a r r i s , G.E., and Brinckman, F.E., E n v i r o n . S c i Technol. (1976) 10, 1128. D i p l o c k , A., Critical Review in T o x i c o l o g y , Chemical Rubber C o . , C l e v e l a n d , Ohio (1976) 271. Cerwenka, E.A., and Cooper, W . C . , A r c h i v . E n v i r o n . H e a l t h (1961) 3 , 189. McConnell, K.P., and Portman, O.W., J. Biol. Chem. (1952) 195, 277. Byard, J.L., A r c h . Biochem. Biophys. (1969) 130, 556. Palmer, I.S., F i s c h e r , D . D . , Halverson, A . W . , and O l s o n , O.E. Biochem. Biophys. A c t a (1969) 177, 336. Palmer, I.S., Gunsalus, R.P., H a l v e r s o n , A . W . , and O l s o n , O.E., Biochem. Biophys. A c t a (1970) 208, 260. McBride, B.C., and Wolfe, R.S., Biochemistry (1971) 10, 2317. T a y l o r , C . D . , and Wolfe, R.S., J. Biol. Chem. (1974) 249, 4879. T a y l o r , C . D . , and Wolfe, R.S., J. Biol. Chem (1974) 249, 4886. T a y l o r , C . D . , McBride, B.C., Wolfe, R.S., and Bryant, M.P., J. Bacteriol. (1974) 120, 974. F r i c k , T., F r a n c i a , M . D . , and Wood, J.M., Biochem. Biophys. A c t a (1976) 428, 808.


20

51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72.

73. 74. 75.


C h a l l e n g e r , F., Biochem. J. (1970) 117, 65P. C h a l l e n g e r , F., Komrower, G . M . , and Robins, A.J., Quart. Rep. S u l f u r Chem. (1970) 5, 91. Dagley, S.J., and N i c h o l s o n , D.E., "An I n t r o d u c t i o n to Met a b o l i c Pathways", B l a c k w e l l , Oxford (1970), p.212. T a l l a n , H.H., Sturman, J.Α., P a s c a l , T.A., and G a u l l , G.E., Biochem. Med. (1974) 9, 90. Datko, A.H., Mudd, S . H . , Giovanelli, J., J . Biol. Chem. (1977) 252, 3436. K e l l y , A.P., and Weed, L.L., J. Biol. Chem. (1965) 240, 2519. Braun, R., and Fromageot, P., Biochem. Biophys. Acta (1962) 62, 548. Bonsen, P.P.M., Spudich, J.Α., N e l s o n , D.L., and Kornberg,A., J. Bacteriol. (1969) 98, 62. Wood, J.M., Naturwissensch page 358.) P i k e , E.R., Graham, L.C., and Fogden, N . W . , J. Assoc. P u b l . A n a l y s t s (1975) 13, 19. P i k e , E.R., Graham, L.C., and Fogden, N . W . , J. Assoc. P u b l A n a l y s t s (1975) 13, 48. Zaroogian, G.E., and Cheer, S . , Nature (1976) 261, 408. Doyle, J.J., M a r s h a l l , R.T., and Pfander, W . H . , A p p l . M i c r o biol. (1975) 29, 562. M o n i e r - W i l l i a m s , G.W., "Trace Elements in Foods", Chapman and Hall, London (1949), p . 168. Swaine, D.N., Comm. Bur. Soil Sci., Tech. Bull. No. 48, (1955). The Lead i n Food R e g u l a t i o n s , S . I . 1961, No. 1931, H . M . S . O . (London) 1961. N e l s o n , J.D., Blair, W., Brinckman, F.E., C o l w e l l , R . R . , and I v e r s o n , W.P., A p p l . Microbiol. (1973) 26, 321. Spangler, W.J., Spigarelli, J.L., Rose, J.M., and Miller, H . M . , Science (1973) 180, 192. Edwards, T., and McBride, B.C., Nature (1975) 253, 462. Braman, R.S., and Foreback, C.C., Science (1973) 182, 1247. C h a l l e n g e r , F., Chem. Rev. (1945) 36, 326. Thayer, J.S., "Organometallic Chemistry: A Historical P e r s p e c t i v e . " Advances in O r g a n o - M e t a l l i c Chemistry, (Stone, F.G.A., and West, R . , e d s . ) , vol. 13, Academic P r e s s , New York (1975), p . 1. Thayer, J.S., "Organometallic Compounds and L i v i n g Organisms" J. Organometal. Chem. (1974) 76, 265. Thayer, J.S., "Teaching Bio-Organometal Chemistry. I . The Metalloids." J . Chem. E d . (1977) 54, 604. Thayer, J.S., "Teaching Bio-Organometal Chemistry. I I . The M e t a l s . J. Chem. Ed. (1977) 54, 662.


1.

CHALLENGER

Biosynthesis

21

Discussion J . S. THAYER ( U n i v e r s i t y of C i n c i n n a t i ) : I would l i k e t o add j u s t a b i t of h i s t o r i c a l p e r s p e c t i v e i n p o i n t i n g out that C h a l l e n g e r s work had o r i g i n i n the r e a l problem that "Gosio-gas" was emitted i n p o o r l y v e n t i l a t e d housing, and q u i t e a number of people d i e d or s u f f e r e d from a r s e n i c p o i s o n i n g . In England, i n 1930, there was a r a t h e r n o t o r i o u s case t h a t l e d d i r e c t l y to C h a l l e n g e r s i n t e r e s t i n t h i s area. He t r i e d t o methylate mercury u s i n g t h i s same route and u n f o r t u n a t e l y f a i l e d by a very narrow margin; otherwise, the s i t u a t i o n that developed i n the 50 s and l a t e r might have a t l e a s t been a n t i c i p a t e d . 1

1

1

J . M. WOOD ( U n i v e r s i t y of Minnesota): I n 1945, Challenger wrote a review [Chem. Rev not o n l y i n terms of th t o r y of the Gosio-gas p o i s o n i n g cases. [ I t d e s c r i b e d the] h i s t o r y of c h i l d r e n i n the Forest of Dean who were poisoned by t r i m e t h y l a r s i n e when a farmer put some a r s e n i c compounds i n a p a r t i c u l a r area i n the f o r e s t . He g i v e s d e t a i l s of the c l i n i c a l h i s t o r y as w e l l as the chemistry. That review i s almost as long as h i s present paper, but i t ' s worth the e f f o r t to s i t down and read i t . W. R. CULLEN ( U n i v e r s i t y of B r i t i s h Columbia): Yes, I can add t h a t the r e f e r e n c e to a r s e n i c i n concrete was t h a t the house the c h i l d r e n were l i v i n g i n had a r s e n i c i n the f l y ash used f o r the concrete. T h i s i s what the molds worked on t o produce t r i methylarsine. F. E. BRINCKMAN ( N a t i o n a l Bureau of Standards): From C h a l lenger s h i s t o r y and commentary on h i s n u c l e o p h i l i c r e a c t i o n shown i n F i g u r e 2, I'd l i k e to address a simple question t h a t ' s of great i n t e r e s t to us. In the f i n a l step he p o i n t s out formation of t r i m e t h y l a r s i n e , which i s the observed v o l a t i l e product. There i s a t r i m e t h y l a r s i n e oxide intermediate step and t h i s of course i n v o l v e s the two e l e c t r o n r e d u c t i o n t o form u l t i m a t e l y the observed v o l a t i l e s p e c i e s . Dr. P a r r i s and I , s e v e r a l years ago, looked at the r a t e s of o x i d a t i o n of t r i m e t h y l a r s i n e i n aqueous media and i n a i r [Environ. S c i . Technol. (1976), 10, 1128]. I t was f a i r l y c l e a r t h a t w h i l e comparatively slow as a chemical r e a c t i o n , i t proceeded r a p i d l y compared to b i o l o g i c a l r a t e s of f o r m a t i o n , a t l e a s t our estimates of b i o l o g i c a l r a t e s of formation. I'm r a t h e r c u r i o u s i f anybody has a comment on t h a t f i n a l r e d u c t i o n s t e p , whether t h a t i s an a b i o t i c or whether i t i s a b i o g e n i c step. 1

CULLEN: I t h i n k I can answer t h a t q u e s t i o n . We have a c t u a l l y used t r i m e t h y l a r s i n e oxide as a s u b s t r a t e f o r Candida humicola and have obtained t r i m e t h y l a r s i n e from i t . So there i s some f u n g a l r e d u c t i o n anyway. I t h i n k we can comment f u r t h e r on t h a t when P r o f e s s o r McBride speaks l a t e r today.


22


F. CHALLENGER (in absentia): Regarding the matter of p o s s i b l e b i o m e t h y l a t i o n of antimony, the question of i t s charge i n t a r t a r emetic bears f u r t h e r note. R e i h l e n and Hezel [Ann. (1931) 487, 213], a f t e r much experimental work, have proposed a formula f o r t a r t a r emetic

i n which antimony appears i n the anion. This work escaped my n o t i c e u n t i l r e c e n t l y . The few experiments o f Barnard were i n s u f f i c i e n t t o a l l o w f o r a d i s c u s s i o n o f the i m p l i c a t i o n s o f t h i s f o r mula f o r the p o s s i b l e m e t h y l a t i o n o f antimony, [ c f . paper by G.E. P a r r i s i n t h i s volum Sb and p o t e n t i a l f o r m e t h y l a t i o F. CHALLENGER (in absentia): Regarding methylation of i n organic a r s e n i c i n t h e body, a f u r t h e r comment i s a p p r o p r i a t e . I n a s e r i e s of important s t u d i e s , C r e c e l i u s [Environmental Health Pers p e c t i v e s (1977) 19, 147] demonstrated, f o r example, that human i n g e s t i o n of A s ^ - r i c h wine r e s u l t s w i t h i n 5-10 hours i n about a 5 - f o l d increase i n u r i n a r y l e v e l s of A s and As , methylarsonic a c i d (MAA), and dimethy l a r s o n i c a c i d (DMAA). Most (~ 80%) of the ingested a r s e n i c i s excreted i n u r i n e over s e v e r a l days. Other experiments r e v e a l t h a t both As and A s ^ a r e excreted from t h e body, c h i e f l y i n methylated forms. I n g e s t i o n o f "marine a r s e n i c " i n crab meat r e s u l t s i n u r i n a r y e l i m i n a t i o n , but the o r g a n i c a l l y bound As e n t e r i n g t h e body apparently undergoes no m e t h y l a t i o n . I n a l l of these s t u d i e s , the r e d u c t i v e v o l a t i l i z a t i o n method of Braman and Forelock [ c f . reference 70] was used. Consequently, my e a r l i e r statement that methylation of i n o r g a n i c a r s e n i c t o v o l a t i l e d i - or t r i m e t h y l a r s i n e i n the human body i s not e s t a b l i s h e d cannot y e t be r e v i s e d . +

J

+

RECEIVED September 15, 1978.


2 Biotransformations οf Sulfur as Evolutionary Prototypes for Metabolism of Metals and Metalloids GEORGE E. PARRIS Division of Chemical Technology HFF-424, Food and Drug Administration, Washington, DC 20204

Sulfur, like phosphorus earth's crust and sulfu early in the process of evolution. Some non-metals and metalloids ( e . g . , Se, Te, As) have chemical properties similar to metabolicly active forms of sulfur and/or phosphorus. There is always a possibility that an organism w i l l encounter an element, compound or ion in its environment which will act as a pseudo-sulfur or pseudo-phosphorus nutrient. Pseudo-nutrients may or may not be actively toxic, but they can inhibit c e l l functions as they compete with true nutrients in respiration and assimilation. It is observed that pseudo-nutrients are often transformed in vivo into products which no longer encumber the c e l l ' s function. Ideally, such a transformation w i l l produce a material which has few labile valences capable of combining with enzymes, nucleic acids or constituents of the cytoplasm, is not i t s e l f a mimic of a normal metabolite, and has low polarity so that i t can passively diffuse directly out of the cytoplasm through the c e l l membrane. Whether these transformations are merely serendipitous or are evolved specifically for the purpose of dealing with pseudonutrients may be debatable. Because the analogy between the chemistry of S, Se and Te is rather obvious and has been reviewed at length (1), attention in this manuscript is focused on arsenic and other elements where current understanding is relatively incomplete. Evolution of Respiration Chains It is convenient to f i r s t consider biotransformation of sulfur in the respiration of cells which obtain energy from the oxidation of sulfur compounds. In 1 9 4 5 » Horowitz ( 2 J stated a hypothesis which is summarized as follows: The primordial organism is assumed to be capable of a simple form of respiration

0-8412-0461-6/78/47-082-023$05.00/0 © 1978 American Chemical Society



24

i n v o l v i n g one enzyme.

Within the environment o f the organism

substrate a

Enzyme A

»

products + energy

there are other substances such as s u b s t r a t e "b" which could be converted to substrate " a " , but the primordial organism cannot u t i l i z e substrate " b " . There may be, however, spontaneous mutants of the primordial s t r a i n which possess the genetic b l u e p r i n t f o r "Enzyme B" which could convert "b" to " a " i n t r a c e l l u l a r ^ , perhaps even with a small r e l e a s e o f useful energy. Under the s t r e s s o f Malthusian growth, the primordial organisms become so numerous that s u b s t r a t e " a " i s consumed as f a s t as i t enters the organisms' environment. When the a v a i l a b i l i t y of substrate " a " becomes c r i t i c a l , the b a s i c s t r a i n o f the primordial organism becomes e x t i n c t and i s replaced cal transformations. Thus, a chain o f r e s p i r a t o r y enzymes i s R

b —

A

a

» .products

+

energy

g r a d u a l l y b u i l t up. G e n e r a l l y , the primeval enzyme systems w i l l be r e t a i n e d i n more h i g h l y evolved and s p e c i a l i z e d organisms. However, a new group o f enzymes, F , G, H, e t c . , may evolve that i s capable o f c a r r y i n g out these o r other e n e r g y - r e l e a s i n g r e a c t i o n s without r e s o r t i n g to use o f the primeval enzyme system A, B, C, e t c . In such a c a s e , an e n t i r e l y new r e s p i r a t o r y system may evolve as the n o w - s u p e r f i c i a l primeval system i s l o s t p i e c e meal i n subsequent mutations ( 2 , 4 , 5 , ) . Unlike the development o f a products

+

energy

r e s p i r a t i o n sequence i n an e v o l v i n g f a m i l y o f organisms, the i n t r o d u c t i o n o f a defensive d e t o x i f i c a t i o n system can l o g i c a l l y s p r i n g from any o f the e x i s t i n g enzymes. A l s o , the organisms w i l l seldom be so lucky as to be able to d e r i v e any net useful energy from d e t o x i f i c a t i o n and i t i s l i k e l y t h a t d e t o x i f i c a t i o n w i l l a c t u a l l y amount to a considerable tax on the normal energy pools o f the organism. There are two types o f biochemical o x i d a t i o n r e a c t i o n s : dehydrogenations and oxygen i n s e r t i o n s . Dehydrogenations are b e l i e v e d to have evolved i n the time before molecular oxygen was a v a i l a b l e i n q u a n t i t y i n the atmosphere. Oxygen i n s e r t i o n r e a c t i o n s could not have been o f much importance u n t i l a f t e r the e v o l u t i o n o f photosynthetic blue-green a l g a e . The dehydrogenation process o f most organisms are s i m i l a r . T y p i c a l l y , an organism has a r e s p i r a t o r y chain which f a c i l i t a t e s dehydrogenation o f some s u b s t r a t e (DH ) ( £ ) . Each time the hydride (H:~ or H- + e") i s 2


2.

PARRIS

25

Biotransformations of Sulfur carrier 2 red.

c a r r i e r 1 red. enzyme 1

c a r r i e r η red.

enzyme 2

carrier 1 oxid.

enzyme η

c a r r i e r 2 oxid.

carrier η oxid.

1

t r a n s f e r r e d , chemical p o t e n t i a l i s made a v a i l a b l e t o the c e l l . This chemical p o t e n t i a l i s u t i l i z e d by coupling the o x i d a t i o n steps t o phosphorylation steps which u l t i m a t e l y form ATP from ADP + Ρ · (inorganic phosphate). The f i r s t e l e c t r o n c a r r i e r s i n the r e s p i r a t i o n chain are adapted to the type o f substrate being o x i d i z e d ( e . g . , dehydrogenated). Nicotinamide adenine d i n u c l e o t i d e (NAD , Figure 1 ) i s a common r e c e p t o r which functions as a coenzyme i n the o x i d a t i o n o f many organic compounds. NAD"" can be reduced t o NADH by t r a n s f e r o f a hydrid nicotinamide. A very s i m i l a r coenzyme NADP has a t h i r d phosphate group a t the C - 2 hydroxy! group o f the ribose moiety o f the adenine p o r t i o n o f the i o n . There are d i f f e r e n t enzymes and coenzyme receptors f o r o x i d a t i o n o f d i f f e r e n t s u l f u r species (e.g., sulfur, sulfide, thiosulfite, sulfite). Once the e l e c t r o n s have been removed from the s u b s t r a t e , they can be passed i n t o a c a r r i e r chain which i s made up o f a s e r i e s o f cytochromes. Cytochromes are complexes o f metals (such as i r o n o r copper) which have convenient o x i d a t i o n p o t e n t i a l s and favorable e l e c t r o n t r a n s f e r k i n e t i c s f o r enzymatic coupling to phosphorylation r e a c t i o n s producing ATP. As discussed above, the major elements of t h i s chain may be common to many s p e c i e s . Complexes o f Ί

+

Substrate A — » C« Substrate Β Substrate

C,

C— • C — *

C-| · CoQ

C —• C 2

3

C — » 0, n

Substrate Ν — » C , Ν ubiquinones (coenzyme Q) with cytochromes are known t o be the focal point f o r e l e c t r o n s a r r i v i n g from d i f f e r e n t s u b s t r a t e branches o f r e s p i r a t o r y chains (]J. Respiration in T h i o b a c i l l i There are several genera o f b a c t e r i a which o x i d i z e i n o r g a n i c s u l f u r compounds. The genus T h i o b a c i l l u s has been the most thoroughly s t u d i e d . T h i o b a c i l l i o b t a i n T l l t h e i r carbon from C 0 and a l l t h e i r energy from r e a c t i o n o f various reduced forms o f

2


26


s u l f u r (Sg, S " , S O 3 " , S 2 O 3 " ) with oxygen or an oxygen donor ( N O 3 - ) to form o x i d i z e d forms o f s u l f u r ( S Û 4 " ) ( 8 ) . T h i o b a c i l l u s thiooxidans derives i t s energy a u t o t r o p h ! c l y by o x i d i z i n g elemental s u l f u r to s u l f a t e . The e f f i c i e n c y with which 2

2

2

2

1/8 S

+

g

3/2 0 =

AG

2

+

H0 J = ±

H S0

2

2

4

-596 kJ

t h i s energy f i x e s carbon to form c e l l u l a r material has been c a l c u l a t e d to be about 6%. Nearly 32 gm o f s u l f u r are o x i d i z e d f o r every gram o f carbon a s s i m i l a t e d i n t o c e l l components. Cytochromes and coenzyme Q have been i d e n t i f i e d i n T . thiooxidans and i t has been observed t n a t low concentrations o f c y a n i d e , azide or carbon monoxide ( a l l o x i d a t i o n i n t h i s organism to be comparable t o t h a t i n b a c t e r i a which are not s p e c i a l i z e d sulfur oxidizers. Thus, the cytochrome-!inked phosphorylation was concluded to be an i n t e g r a l part o f the r e s p i r a t o r y machinery o f the organism (9,10). T h i o b a c i l l u s t h i o p a r u s , u n l i k e T . t h i o o x i d a n s , does not appear capable o f using n a t i v e s u l f u r as a substrate s i n c e when i t i s grown on t h i o s u l f a t e , i t accumulates elemental s u l f u r and produces s u l f a t e . A cytochrome (cyt s , t e n t a t i v e l y c l a s s i f i e d as a c - t y p e cytochrome) has been i s o l a t e d from T . t h i o p a r u s . Nonetheless, evidence seems to i n d i c a t e t h a t the c l a s s i c a l cytochrome-!linked o x i d a t i v e phosphorylation chain may have been supplemented, i n the course o f e v o l u t i o n a r y s p e c i a l i z a t i o n , by a d i r e c t o x i d a t i o n sequence which involves formation o f high energy AP^S (APS, adenosine 5*-phosphosulfate). Notice that i n 8

(1)

S 0=

+

?

c

2 H

+

+

2 e"

0

(2)

2 HS

(3)

SO

+

2

=

+

0 AMP

2

thiosulfate^ reductase

sulfide o x i d a s

e >

S

2

2 e~

+

s

=

o

+

J

H

S c

2 H 0 2

cyt

(ox)

II

0

APS

APS-reductase 2 e" ρ c y t (red)

r e a c t i o n 3 a high energy s u l f a t e bond has been formed i n APS" analogous to ADP . The h y d r o l y s i s of APS to AMP + S. i s s a i d to be about 42 kJ more exothermic than the h y d r o l y s i s of ADP to AMP + P j . While f o r some energy r e q u i r i n g functions of the c e l l , APS may be used d i r e c t l y , a more conventional view i n d i c a t e s t h a t APS i s p r i m a r i l y used as a substrate to form ADP . Ultimately, =

=


2.

(4)

PARRIS

APS

27

Biotransformations of Sulfur

=

+

P.

ADP-sulfurylase

A

Ί

D

p

+

s

-

=

1

the conventional c e l l u l a r energy source ATP i s formed. (5)

2 ADP

adenylic kinase

Peck and

AMP + ATP

F i s h e r and Stulberg noted t h a t reactions 3 and 4 may be capable o f s u p p l y i n g the necessary ATP f o r a l l c e l l u l a r energy requirements (11,12). However, i t i s u n l i k e l y t h a t cvtochrome-1inked phosphorylation ( i n i t i a t e d i n r e a c t i o n 3) i s completely supplanted

(11,14).

Involvement of Arsenic i A r s e n i t e i s not only a p s e u d o - s u l f i t e n u t r i e n t , i t i s very t o x i c to c e l l s because i t combines i n d i s c r i m i n a t e l y with t h i o l groups causing mass d i s r u p t i o n i n metabolism. Arsenate i s a pseudo-phosphate n u t r i e n t , but s i n c e i t does not r e a d i l y r e a c t with t h i o l s , i t only " c l o g s - u p " c e r t a i n phosphate-processing enzymes r a t h e r than rendering enzymes n o n f u n c t i o n a l . T h u s , the o x i d a t i o n o f a r s e n i t e to arsenate can be viewed as a defensive mechanism. The f i r s t report o f b a c t e r i a l o x i d a t i o n o f a r s e n i t e t o arsenate dates to 1918 when an organism known as B a c i l l u s arsenoxydans was observed i n c a t t l e d i p p i n g preparations based on arsenite. However, the organism was never s t u d i e d s y s t e m a t i c l y and has s i n c e been l o s t . In 1949 Turner (15,16) r e i n v e s t i g a t e d organisms responsible f o r the o x i d a t i o n o f a r s e n i t e t o arsenate i n sheep d i p and i s o l a t e d f i f t e e n s t r a i n s o f h e t e r o t r o p h i c b a c t e r i a which both t o l e r a t e d up t o 0.1 M a r s e n i t e and accomplished the o x i d a t i o n to arsenate. The b a c t e r i a were b e l i e v e d t o be s t r a i n s of Pseudomonas. The biochemical functions o f one s t r a i n were studied. An optimal pH f o r c e l l growth was found to be between 6.0 and 6.5 where a r s e n i t e i s protonated (pK 9 . 1 2 ) . The optimum o x i d a t i o n rate was 90 μΜ a r s e n i t e per hour per mg dry weight o f cells. Oxidation was completely i n h i b i t e d o u t s i d e pH 3 - 1 1 . The a r s e n i t e oxidation was i n h i b i t e d by c y a n i d e , azide and carbon monoxide. The i n h i b i t i o n by CO was only i n the dark. These observations are t y p i c a l o f cytochrome e l e c t r o n t r a n s f e r systems. In p a r t i c u l a r , they are suggestive o f the s o - c a l l e d P-450 cytochrome system. The r e v e r s i b l e i n h i b i t i o n by CO i s due to the e q u i l i b r i u m below. The a c t i v e s i t e o f P-450 i s b e l i e v e d to a

P-450(Fe ) 2+

C

Q

»

P-450(Fe )-C0 2+


28

ORGANOMETALS AND

ORGANOMETALLOIDS

i n v o l v e a pentacoordinate i r o n moiety which has been r e c e n t l y discussed (17,18^19.)· While e l e c t r o n s given up by As ( 111 ) i n t h i s process are apparently funneled i n t o the normal cytochrome r e s p i r a t i o n c h a i n , there was no evidence that any useful energy i s derived from a r s e n i t e o x i d a t i o n as measured by growth rate o f cultures. Turner regarded the transformation as purely defensive. In a study r e l a t e d to b i o l o g i c a l o x i d a t i o n o f a r s e n i t e to arsenate, Johnson and Pi 1 son (20) have examined o x i d a t i o n o f a r s e n i t e i n s t e r i l i z e d sea water. The s t e r i l i z a t i o n was accomplished by f i l t r a t i o n (0.1 micron) to remove l i v e c e l l s . Thus, the sea water can be regarded as a d i l u t e c e l l - f r e e e x t r a c t s i n c e rupture o f nonviable c e l l s i n a population under natural c o n d i t i o n s undoubtedly frees a whole range o f enzymes. The observed a b i o t i c ( i . e . , e x t r a - c e l l u l a r ) o x i d a t i o n rate o f As(111) was s u f f i c i e n t to account f o r most of the a r s e n i c i n sea water each year (0.023 micromole At t h i s time no organism has been found which o x i d i z e s a r s e n i t e f o r r e l e a s e o f useful energy. Arsenate i s an uncoupler o f o x i d a t i v e phosphorylations. I t i s b e l i e v e d t h a t i n (unadapted) c e l l s arsenate i s mistaken f o r phosphate and unstable ADPÂs products are formed which are hydrolyzed before they can be used as ATP (ADP^P) analogues. In the t h i o s u l f a t e o x i d i z i n g b a c t e r i a T. t h i o p a r u s , arsenate was b e l i e v e d to function i n place o f pïïosphate i n the l i b e r a t i o n o f S 0 from APS (see Reaction 4 ) . 4

[Ado-0P0 0S0 ] 2

3

+

=

As0

4

=

ADP-sulfurylasç

^

A M P

^

+

A s

S

0

4

=

There has apparently been no i n v e s t i g a t i o n o f the p o s s i b l e o x i d a t i o n of a r s e n i t e by J \ thioparus i n analogy to s u l f i t e oxidation. Thermodynamicly the o x i d a t i o n o f s u l f i t e i s s l i g h t l y more favorable than the o x i d a t i o n o f a r s e n i t e . H S0 2

+

3

H As0 3

Reduction of

1/2 0 +

3

> H S0

2

2

1/2 0

2

»

H

3

A

s

0

A G = -205 kJ

4

δ

4

6

"

=

1 3 0

k J

Sulfate

Adenosine-5'-phosphosulfate (APS) a l s o plays a c e n t r a l r o l e in the a s s i m i l a t i o n of s u l f a t e ( $ · ) Q 4 ) . Reaction 6 l i e s very Ί

(6) '

ATP

+

x

S. ι

*

" = ATP-sulfurylase Λ Τ Π

Ί Χ

APS

+

PP. ι

much i n favor of ATP + S. ( A G * + 4 6 k J ) . Some o f t h i s energy can be recouped ini vivo by h y d r o l y s i s o f ΡΡ ·. Regardless, APS i s Ί

( 7 )

P P

i

=

+

¥

pyrophosphatase'

2

V

+

2

H +


2.

PARRIS

29


trapped i n a usable form f o r a s s i m i l a t o r y reactions by phosphorylation o f the 3 h y d r o x y l . Reaction 8 i s favored by 1

(8)

APS +

ATP • χ , . *PAPS * APS-kinase

+ ADP

Λ η

about 21 kJ* Reactions 6 and 8 are found i n a l l c e l l s which utilize 3*-Phosphoadenosine-5'-phosphosulfate (PAPS, Figure 2) can be reduced by NADPH to s u l f i t e . (9)

PAPS

+

NADPH

*PAP PAPS-reductase

+

S0

o

+

=

NADP

PAPS i s also the s u l f u r source i n formation o f s u l f a t e R-OH

+

PAPS

+

+

H

+

ό

esters.

»

An inorganic reduction o f s u l f i t e to s u l f i d e has been p o s t u l a t e d by Wool fork (21):

(10)

0 0 it II * "0-S—S-0

2 HSO "

+

H«0

0 (metabisulfite) -0 (11)

SJ) *

+

H

0

y

9

S-S

0

4

V

+

H 0 9

(dithionite) (12)

S 0 2

4

2

"

+

H

2

>

S 0 2

3

2

*

+

H 0 2

(thiosulfate) (13)

S 0 2

3

2

"

+

H

2

>

HS0 " 3

+ HS"

2S i m i l a r pathways i n v o l v i n g t r i t h i o n a t e S 0 c have a l s o been proposed (22). An enzymatic scheme f o r s u l f a t e reduction t o enzyme bound -SH v i a PAPS has been o u t l i n e d (23). 3

Reduction o f Arsenate L i t t l e i f any biochemical experimentation has been d i r e c t e d at b i o l o g i c a l reduction o f arsenate, or formation o f a r s e n i c



30

hydrogen bonds. Many s u l f a t e reducing b a c t e r i a ( D e s u l f o v i b r i o ) are known. The production of hydrogen s u l f i d e i s usually observed under anaerobic c o n d i t i o n s . Reduction o f arsenate to a r s e n i t e commonly occurs i n aerobic media, though formation of (CH ) AsH has only been reported under anaerobic c o n d i t i o n s (24,25,26;. Johnson and Pi 1 son (20,27) have pointed out that the presence o f a r s e n i t e i n sea water i s undoubtedly due to a c t i v i t y of arsenate reducers. The absence o f any organism which produces A s H i s probably due to the f a c t t h a t t h i s compound i s one of the most potent biochemical toxins known. 3

3

3

A l k y l a t i o n o f Metals and M e t a l l o i d s With regard to the c r i t e r i a l i s t e d i n the i n t r o d u c t i o n f o r an ideal d e t o x i f i c a t i o n process d e s i r a b l e f o r b a c t e r i a an (metal = Sn,Pb,Sb,Hg) and c a r b o n - m e t a l l o i d ( m e t a l l o i d = S e , T e , A s , S i ) bonds u s u a l l y unreactive towards -OH, -SH and -NH groups i n the cytoplasm of c e l l s , organometal and organometallofds are f r e q u e n t l y l i p o p h i l i c enough to d i f f u s e through the c e l l ' s membrane and escape more o r l e s s permanently from the c e l l ' s environment. In complex organisms, l i p o p h i l i c i t y leads to undesirable t o x i c e f f e c t s s i n c e f a t - s o l u b l e compounds breach the body's compartmentalization o f s e n s i t i v e systems. The non-methyl c a r b o n - s u l f u r bonds i n p r o t e i n s are formed by r e a c t i o n s i n which carbon-oxygen bonds are d i s p l a c e d . The s u l f u r n u c l e o p h i l e i s g e n e r a l l y s u l f i d e ( S ) or hydrosulfide (HS~)(V4). These powerful n u c l e o p h i l e s are produced i n t r a c e l l u l a r ^ by enzymatic reduction o f s u l f i t e . NADPH i s the most common reducing coenzyme. L i t t l e i s known about the d e t a i l s o f the r e a c t i o n because the s u l f u r intermediates are u s u a l l y p r o t e i n bound. T h i o s u l f a t e may be i n v o l v e d . Reactions 11 and 12 account f o r the introduction of s u l f i d e into proteins. 2

=

(14)

HO-protein

+

(15)

AcO-pro te i n

acetylCoA +

HS 2

>

AcO-protein

%» HS-protein enzyme^

0

r

e n z y m e

+

AcOH

r

The formation of m e t h y l - s u l f u r bonds i s a common biochemical transformation and i t appears t h a t biomethylation of metals can be t r a c e d t o the methylation o f s u l f u r . The b i o s y n t h e s i s o f methionine from aspartate by way o f c y s t e i n e has been o u t l i n e d by Mahler and Cordes (28). For the c u r r e n t d i s c u s s i o n , only the l a s t major transformation i n which homocysteine i s methylated to form methionine i s important. HSCH CH CHNH C0 " 2

2

3

homocysteine

+

2

CH SCH CH CHNH C0 " 3

2

2

3

+

2

methionine


2.

PARRis


31

There are a t l e a s t two enzymatic pathways by which t h i s methylat i o n occurs. In species o f organisms which lack vitamin Βτο» there are r e l a t i v e l y i n e f f i c i e n t transmethylases which bind methyl donor p o l y - L - g l u t a n a t e - N - m e t h y l t e t r a h y d r o f o l a t e (N -methyl-H4f o l a t e ( G l U ] , G l U o , e t c . ) Figure 3) and homocysteine and promote methyl t r a n s f e r t o form methionine and t e t r a h y d r o f o l a t e (hLfolate). Most enzyme systems o f t h i s type do not f u n c t i o n with the m o n o - L - g l u t a m a t e - N - m e t h y l - H - f o l a t e . The main function o f the glutanate moieties appears to be to provide a s u i t a b l e binding s i t e f o r the methyl donor to the enzyme. There i s l i t t l e i f any information a v a i l a b l e concerning the l a b i l i z a t i o n o f the methyl group i n t h i s t r a n s f e r . The turnover rate f o r one o f these d i r e c t transmethylase r e a c t i o n s i s only 14 moles o f N -methyl groups per minute per mole o f enzyme (0.25 mole/sec-mole enzyme)(29). The second and more e f f i c i e n t transmethylase enzyme system depends upon vitamin Β ^ of vitamin B ^ chemistry (30). In vitamin B i ? , c o b a l t i s complexed by a porphyrin-iTîce c o r r i n r i n g . The f i f t h c o o r d i n a t i o n s i t e i s occupied by a 5,6-benzimidazole (Bz) r i n g and the s i x t h s i t e can be occupied by a methyl group o r a v a r i e t y o f other ligands. Unlike the d i r e c t methyl transferase described above, the CHj-B-jp mediated methyl t r a n s f e r process requires a reducing (anaerobic) mediurn. The CHo-B^-enzyme system t r a n s f e r s methyl groups from N - m e t h y l - H a - f o l a t e (Glu-i, e t c . ) to homocysteine a t a steady rate o f about 800 moles per mi η per mole o f enzyme (13 mole CH /sec-mole enzyme)(31). Weissbach and T a y l o r (32) summarized the work o f a number o f others t o describe the r o l e o f vitamin B-J2 the synthesis o f methionine. The scheme which evolved from a v a r i e t y o f e x p e r i mental observations i s o u t l i n e d i n Figure 4. Here 'ΈΝΖ" stands f o r the " N - m e t h y l t e t r a h y d r o f o l a t e homocysteine cobalamin methyl t r a n s f e r a s e " which has a molecular weight o f about 140,000 and has u s u a l l y been i s o l a t e d from s t r a i n s of E. c o l i . The enzyme i t s e l f may o r may not be involved i n any other biomethylation r e a c t i o n s ; but the o v e r a l l scheme, where "ENZ" stands f o r some other enzyme and di f f e r e n t methyl donors and r e c e i v e r s are i n v o l v e d , appears t o be adaptable to many s i t u a t i o n s where the methyl group i s donated as an i n c i p i e n t carbon c a t i o n producing a reduced form o f the Βίο-enzyme complex ( B ^ s ) - The mode o f B - E N Z binding i n the methionine synthetase system has been discussed by Law and Wood (33) who point out t h a t the p h o t o s t a b i l i t y o f the CHo-Co bond i n t h i s C F ^ - B ^ - E N Z complex weighs against a s u l f u r Tigand r e p l a c i n g the benzimidazole base i n s p i t e o f the f a c t that CH3-B12 w i l l not bind t o the enzyme i f a l l the enzyme's s u l f h y d r y l groups have been p r e v i o u s l y blocked. The r o l e o f S-adenosylmethionine (SAM), i n t h i s scheme, i s to provide a r e l a t i v e l y high-energy methyl donor which, i n the presence o f a s u i t a b l e reducing agent, w i l l methyl ate the o x i d i z e d , i n a c t i v e , enzyme-bound Β ^ · SAM w i l l also methyl ate the r e d u c e d - r e a c t i v e , enzyme-bound B moiety but i t i s more than 1 0 times slower than 5

5

5

4

5

5

3

i

n

5

12

1 2

2


32

ORGANOMETALS A N D ORGANOMETALLOIDS

Figure 1. Nicotinamide adenine dinu cleotide (NAD")

0

0

"0- S-O-P-0—CH,

II Figure 2. 3'-Phosphoadenosine5'phosphosulfate (PAPS )

ADENINE

A. 0 I

OH P 0

3~

Figure 3. Tetrahydrofolic acid


2.

ρARRIS


33

N f - m e t h y l - H - f o l a t e i n t h i s r e a c t i o n . Thus, i n the presence o f N - m e t h y l - H 4 - f o l a t e , l i t t l e SAM i s consumed except to regenerate the a c t i v e methyl-B-jo-ENZ from the o x i d i z e d form. It i s note worthy t h a t SAM can be replaced by other a l k y l a t i n g aaents such as CH3I i n the presence o f a s u i t a b l e reducing agent (34). 4

5

Formation o f Carbon-Metal and Carbon-Metalloid Bonds Alkylcobalamins are a t t r a c t i v e models f o r b i o l o g i c a l a l k y l donors where the a l k y l r e c e i v e r i s a metal o r m e t a l l o i d . Depending upon the a b i l i t y o f the ligands ( e . g . , enzyme) bound to cobalt t o s t a b i l i z e the product o x i d a t i o n s t a t e ( C o , C o o r Co ) , the a l k y l - c o b a l t bond can be broken i n three ways (35*36, 37): 1

1 1

1 1 1

R-B

1 2

Ν

»

R.

+

B

1 2 r

>

R"

+

B

1 2

(Co ) n

(Co

l n

)

The a b i l i t y o f C H - B to y i e l d an i n c i p i e n t methyl anion may mean that cobalamin p l a y s a unique r o l e as a coenzyme i n the b i o m e t h y l a t i o n - o f metals i n high o x i d a t i o n s t a t e s ( e . g . , Hg , Sn4 , S b , P t , P b * ) . In v i t r o transmethylations i n v o l v i n g C H o - B have been studied e x t e n s i v e l y . In these cases the methyl i s t r a n s f e r r e d as a carbon anion i n an S 2 type mechanism. The o v e r a l l r e a c t i o n 3

1 2

1

+

5 +

2

4

+

1 2

E

M -CH I

3

+

M

+

\

H

Γ CH Π L M ' " ^ M J

+

M

r

, +

+

CH -M 3

has an i n t e r e s t i n g complication i n that the metals which have been studied as methyl receptors compete with the c o b a l t f o r the benzimidazole l i g a n d . This r e a c t i o n has an e q u i l i b r i u m constant CH 3

B L 1 £

BZ

+

M

-J±CH -B 3

1 2

k^Bz-M base-on

base-off

on the order o f 10 and the " b a s e - o f f " complex i s formed more r a p i d l y than methyl t r a n s f e r from the "base-on" complex to the metal M. The " b a s e - o f f " complex i s probably a c u l - d e - s a c s i n c e , with the e l e c t r o n donating benzimidazole group gone, the c o b a l t would develop a very high e f f e c t i v e p o s i t i v e charge d e n s i t y i f the methyl group l e f t as an anion. Thus, the ultimate productive reaction i s hampered by the d i v e r s i o n o f the r e a c t i v e substrate ( C H B j base-on)(38). Hughes has discussed the observation t h a t the alEylcobalamins behave as though they are Co(II) r a t h e r than 3

2



34

Co(III) s i n c e l i g a n d exchange a t the f i f t h c o o r d i n a t i o n s i t e i s r a p i d and there appears t o be an e q u i l i b r i u m between 5 and 6 coordinate species i n the alkylcobalamins. Normally, Co(III) has very slow l i g a n d exchange and i t i s s t r i c t l y 6 coordinate (39). There i s a host o f p o t e n t i a l coenzymes f o r t r a n s f e r o f i n c i p i e n t a l k y l cations to metals and m e t a l l o i d s i n low o x i d a t i o n s t a t e s ( e . g . , A s 3 , S b , S n , P t , H g ° , P b , d i v a l e n t S , Se and T e , and I"). The mechanism most l i k e l y to account f o r a l k y l t r a n s f e r i n these cases can be c a l l e d " o x i d a t i v e a d d i t i o n " and presumably i s r e l a t e d to the well-known non-enzymatic S 2 mechanisms (40). In t h i s r e a c t i o n a l e a v i n g group (X) on carbon i s d i s p l a c e d with i t s e l e c t r o n s as a n u c l e o p h i l e (N) forms a new +

3 +

2 +

2 +

2 +

N

R

bond t o carbon. The t r a n s i t i o n s t a t e i s s t a b i l i z e d by p o l a r i z a b l e Ν and X groups which can form r e l a t i v e l y strong bonds even when the N-C and X-C bond distances are rather l o n g . Bulky s u b s t i t u ents (R) on carbon i n h i b i t the r e a c t i o n by s t e r i c crowding. S-Adenosylmethionine (Figure 5) comes t o mind i n t h i s c l a s s of C H donating coenzymes. There has been some work which suggests t h a t the enzymatic t r a n s f e r o f methyl from S - a d e n o s y l methionine to 3,4-dihydroxyacetophenone by r a t l i v e r e x t r a c t i n v o l v e s a r a t e l i m i t i n g S^2 s t e p . I t i s not known whether o r not the methyl i s t r a n s f e r r e d to the enzyme at any p o i n t during the r e a c t i o n (41)· ATP a l k y l a t e s (adenosylates) methionine with ΡΡΡ · a c t i n g as a l e a v i n g group (42). Other phosphate e s t e r s may have s i m i l a r a l k y l a t i n g p o t e n t i a l . Acetate has been mentioned as a po t e n t i a l l e a v i n g group ( r e a c t i o n 15 above). S u l f a t e e s t e r s , R0S0 ; would appear t o be p o t e n t i a l l y very good R donor coenzymes. N - m e t h y l - f y - f o l a t e (Figure 3) has been mentioned as a methyl donor i n n o n - B formation o f methionine (29). E t h y l a t i o n has not been s e r i o u s l y considered s i n c e Challenger refuted G o s i o ' s c l a i m f o r d i e t h y l a r s i n e formation by fungi (43,44). G o s i o ' s proposal might not have been so r e a d i l y o v e r t u r n e d T f the production o f e t h i o n i n e by E . c o l i (45) and the formation o f S-adenosylethionine (46) had been recognized i n 1930. The nature o f the metal o r m e t a l l o i d n u c l e o p h i l e i s as important as the l e a v i n g group i n a l k y l t r a n s f e r s . March (40) and Pocker and Parker (47) should be consulted f o r general d i s c u s s i o n s about n u c l e o p h i l i c i t y . S u l f u r seems to be b i o a l k y l a t e d only a f t e r reduction to a powerful RS" o r HS" n u c l e o p h i l e (Equation 12 and Figure 5 ) . Selenium and t e l l u r i u m may a l s o be reduced t o the d i v a l e n t s t a t e p r i o r t o d i s p l a c i n g a l e a v i n g group from carbon. The reduction o f a r s e n i t e to arsenide does not seem to be l i k e l y . I t i s noteworthy that s u l f i t e i s a n u c l e o p h i l e which i s capable 3

+

Ί

3

5

1 2


2.

PARRIS

35


PPP,

ATP

8AM sγ

v

reoucod r levin

EN Ζ Homocysteine

N-methyl-tj^folete

Bthlonine -

oxidatio

Figure 4.

B

lg

System for methyhtion of homocys teine

ADENINE

Figure 5. S-Adenosylmethionine

Figure 6. Structure of anti mony (III) tartrate showing pseudotrigonal bipyrimid (ψtbp) coordination


36


of displacing halides from alkyl halides (48,49). Sel enite, t e l l u r i t e and arsenite should have similar nucleophilic capabilities, and they are reasonable choices as alkyl acceptors in biological systems. Antimonite probably is less nucleophilic than arsenite because its lone pair of electrons tends to be in a more s-type molecular o r b i t a l . Nonetheless, trimethyl stibine displaces halogens from alkyl halides (50), even though the lone pair of electrons is in an orbital with somewhat more than 25% s character and the bonding orbitals C-Sb have somewhat more than 75% ρ character ( e . g . , C-Sb-C a n g l e s « 1 0 9 ° ) ( 5 1 ) . The polarizability of the low oxidation states of heavy metals ( e . g . , Pb2 , Sb3 ) may make them reasonably nucleophilic when the lone pair occupies a pseudo-pendent site on the coordinated metal ion, e.g., as in antimony tartrat to observe biomethylatio taken too negatively since trimethyl stibine wouTJ not survive long enough in the aerobic systems investigated to allow detection by the usual technique of headspace gases analysis (55). +

+

Literature Cited 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15.

Klayman, D. L. and Gunther, W. Η. Η . , "Organic Selenium Compounds: Their Chemistry and Biology", John Wiley and Sons, New York, N . Y . , 1973. Horowitz, Ν. H . , Proc. Natl. Acad. S c i . U.S. (1945) 31, 153157. Foster, J . W., "Chemical Activities of Fungi", 207-208, Academic Press, New York, N . Y . , 1949. Calvin, M . , Amer. Scientist (1975) 63, 169-177. Lock, D. Μ., "Enzymes-The Agents of Life", 177-207, Crown Publishers, New York, N . Y . , 1969. Mahler, H. R. and Cordes, Ε. Η . , "Biological Chemistry, 2nd Ed.", 632, Harper and Row, New York, N . Y . , 1971. Mahler, H. R. and Cordes, Ε. H . , loc. cit., 778-781. Zajic, J . Ε., "Microbial Biogeochemistry", 46-78, Academic Press, New York, N . Y . , 1969. Cook; T. M. and Umbreit, W. W., Biochem. (1963) 2, 194-196. London, J., Science (1963) 140, 409-410. Peck, H. D . , J r . and Fisher, E., Jr., J . Biological Chem. (1962) 237, 190-197. Peck, H. D . , J r . and Stulberg, M. P . , J. Biological Chem. (1962) 237, 1648-1652. Bowen, T. J., Happold, F. C. and Taylor, B. F., Biochim. Biophys. Acta (1966) 118, 566-576. Peck, H. D . , Jr., Sulfur Requirements and Metabolism of Microorganisms,"Symposium: Sulfur in Nutrition", Muth, O. H. and Oldfield, J . Ε., editors, 61-79, Avi Publishing C o . , Westport, Conn., 1970. Turner, A. W., Nature (1949) 164, 76-77.


2.

PARRIS

16. 17.

Turner, A. W., A u s t r a l . J. B i o l o g i c a l Sci. (1954) 7 , 452. Collman, J. P . , Sorrell, T . N. and Hoffman, Β. Μ., J. Amer. Chem. Soc. (1975) 9 7 , 913-914. Koch, S . , Tang, S. C., Holm, R. H. and F r a n k e l , R. Β . , J. Amer. Chem. Soc. (1975) 9 7 , 914-916. Koch, S . , Tang, S. C., Holm, R. Η . , F r a n k e l , R. B. and I b e r s , J. Α . , J. Amer. Chem. Soc. (1975) 97, 916-918. Johnson, D. L. and P i l s o n , M. E . Q . , E n v i r o n . L e t s . (1975) 8, 157-171. Woolfork, C. Α . , J. B a c t e r i o l . (1962) 84, 659. F i n d l e y , J. E . and A k a g i , J. Μ., Biochem. Biophys. Res. Commun. (1969) 36, 266. Roy, A. B. and T r u d i n g e r , P. Α . , "The Biochemistry o f Inorganic Compounds o f S u l f u r " , 256, Cambridge a t the U n i v e r s i t y Press, London, 1970. Z a j i c , J. E., l o c Johnson, D. L., Nature (1972) 240, 44-45. McBride, B. C. and Wolf, R. S., Biochemistry (1971) 10, 4312-4317. Pilson, M. E . Q . , Limnol. Oceanog. (1974) 19, 339-341. Mahler, H. R. and Cordes, Ε. H . , l o c . cit., 778-781. W h i t f i e l d , C. D. and Weissbach, H . , J. Biol. Chem. (1970) 245, 402. P r a t t , J. Μ., "Inorganic Chemistry o f Vitamin B ", Academic P r e s s , New York, N . Y . , 1972. T a y l o r , R. T . and Hanna, M. L., A r c h . Biochem. Biophys. (1970) 137, 453. Weisbach, H. and T a y l o r , R. T., Roles o f Vitamin B and F o l i c A c i d i n Methionine S y n t h e s i s , "Vitamins and Hormones", H a r r i s , R. S., Munson, P. L. and P i c z f a l u s y , E., e d i t o r s , 415-440, Academic P r e s s , New York, N . Y . , 1970. Law, P. Y. and Wood, J. Μ., J. Amer. Chem. Soc. (1973) 95, 914-919. Penley, M. W., Brown, D. G. and Wood, J. Μ., Biochem. (1970) 9, 4302-4310. B r o d i e , J. D . , Proc. N a t l . Acad. Sci. U.S. (1969) 6 2 , 461.

18. 19. 20. 21. 22. 23.

24. 25. 26. 27. 28. 29. 30. 31. 32.

33. 34. 35. 36. 37. 38. 39.

40. 41. 42. 43.


37

12

12

Schrauzer, G. Ν . , Accounts Chem. Res, (1968) 1, 9 7 103. Silverman, R. B. and Dolphin, D . , J. Amer. Chem. Soc. (1974) 96, 7094-7096. S c o v e l l , W. Μ., J. Amer. Chem. Soc. (1974) 9 6 , 3451-3456. Hughes, M. N . , "The Inorganic Chemistry o f B i o l o g i c a l P r o c e s s e s " , 186-187, John Wiley and Sons, New York, N . Y . , 1972. March, J., "Advanced Organic Chemistry", 251-375, McGraw-Hill Book C o . , New York, N . Y . , 1968. Hegazi, M. F., Borchardt, R. T . and Schowen, R. L., J. Amer. Chem. Soc. (1976) 9 8 , 3048-3049. Mahler, H. R. and Cordes, Ε. H . , loc. cit., 382. Gosio, Β . , Arch. Ital. B i o l . (1893) 18, 253.



38

44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

54. 55.

Challenger, F . , Higginbottom, C. and Ellis, L., J. Chem. Soc. (1933), 95-101. Fisher, J . F. and Mallette, M. F., J. Gen. Physiol. (1961) 45, 1. Farber, E., Adv. Cancer Res. (1963) 7, 383 Pocker, Y. and Parker, A. J., J. Org. Chem. (1966) 31, 1526. March, J., loc. cit., 330. Gilbert, Ε. Ε., "Sulfonation and Related Reactions", 136-163, Interscience Publishers, Inc., New York, N . Y . , 1965. Parris, G. E. and Brinckman, F. E., J. Org. Chem. (1975) 40, 3801-3803. B r i l l , Τ. Β., Parris, G. E., Long, G. G. and Bowen, L . Η . , Inorg. Chem. (1973) 12, 1888-1891. Poore, M. C. and Russell, D. R . , J. Chem. Soc. A (1971), 18. Cotton, F. A. and Wilkinson G . "Advanced Inorgani Chemistry; 3rd E d . " N.Y., 1972. Challenger, F. and Ellis, L., J. Chem. Soc. (1935), 396. Parris, G. E. and Brinckman, F. Ε., Environ. S c i . Tech. (1976) 10, 1128-1134.

RECEIVED August 22, 1978.


3 Occurrence of Biological Methylation of Elements in the Environment Y. K. CHAU and P. T. S. WONG

Canada Centre for Inland Waters, Burlington, Ontario, Canada

Biological methylatio ing biochemical processes involved wit l i v i n g systems s phenomenon was observed in the early nineteenth century (1815) when several cases of arsenical poisoning occurred in Germany due to the use of domestic wallpapers containing arsenic pigments. In 1839 Gmelin (1) noted that a garlic odor was present in rooms associated with the incident. The mystery was not thoroughly unveiled until 1893 when Gosio (2) observed the evolution of a garlic odor gas from a mould-infected sample of mashed potato on exposure to a i r . The gas was called Gosio gas which was later identified by Challenger as trimethylarsine. The term "biological methylation" was f i r s t used by Challenger (3) to describe the replacement of the οxy-groups of arsenic, selenium and tellurium compounds by methyl groups through the action of moulds, resulting in the formation of organometalloids or organometallic compounds. It has since been shown that bio logical methylation is a general process for l i v i n g organisms (4). Available information indicates that microorganisms, especially bacteria and fungi play an important role in the transformation. While the biological function of methylation is not clearly known, it has been proposed to be a detoxification process. Alternately, it may be energetically preferable for some organisms to trans methyl ate metal rather than to synthesize methane (5). Studies on the environmental impacts of biological methyla tion have gained much momentum since the discovery that micro organisms in a natural lake sediment were able to methylate mercury to a highly-neurotoxic methylmercury species (6). Because methylmercury is produced at a rate faster than organisms can accomplish its degradation, i t may accumulate in fish and so poses a threat to public health (7). Indeed, several incidents of environmental catastrophies caused by mercury have been documented f8.9). Methylation in the environment results in the formation of organometalloids or organometals which are generally more toxic and easily bioaccumulated; it plays an important role in the mobilization of elements from sediment to water; i t may cause 0-8412-0461-6/78/47-082-039$05.00/0 © 1978 American Chemical Society


40

organometals and organometalloids

transmethylation of other elements. Attempts have been made to predict the p o s s i b i l i t y of methylation of other elements by the relative ease of formation of metal-carbon bonds (7) and by the reduction potential of the elements (10). The metals Hg, Sn, Pd, Pt, Au and T l and metalloids As, Se, Te and S have been postulated to accept methyl groups from methyl-cob alamin in biological systems. However, Pb, Cd and Zn have been predicted not to be methylated because of the extreme i n s t a b i l i t y of their monoalkyl derivatives in aqueous systems. Methylation studies pertaining to environmental impact began when Jensen and Jernelov (6) investigated the transformation of HgCl in bottom sediments from freshwater aquaria. Then Cox and Alexander (11) studied the transformation of methylated arsenic and selenium compounds from their inorganic salts by fungi isolated from raw sewage and grow (12,13) investigated methylatio species isolated from Chesapeake Bay. A l l these investigations were carried out with environmentally originated microorganisms grown in laboratory media. More r e a l i s t i c approaches were adopted by Bramen (14), Langley (15), and Wong et a l . (16) who used systems containing natural waters and sediments to investigate respectively the methylation of As, Hg, and Pb. A l l these investigations, however, bear certain relevance and significance to the environment and can be extrapolated to living ecosystems. Methylation of mercury in the environment has been well established and documented by other workers. 2

Experimental A sediment-lake water system is used for the investigation of methylation of metals and metalloids in the aquatic environment. In each study, 50 g of sediment and 150 ml of lake water were placed in a 250 ml f i l t e r flask. Nutrient broth (0.5%), glucose (0.1%), and yeast extract (0.1%) were added to stimulate microbial growth. The compounds to be tested for methylation were added to the sediment (5 mg/Jt) and the flasks were capped and incubated at 20°C for 7-10 days. The headspace gas was analyzed for volatile methylated compounds and the lake water was analyzed for the presence of methylated species of the element. A specially-developed Gas Chromâtograph-Atomic Absorption technique was used for the analyses of volatile alkyllead compounds (17), methyl selenides (18), methylarsines and methylarsenic acids (19). Sediments from several lakes in Ontario were used for the studies. In a l l these experiments, appropriate controls were prepared either by omitting the test compounds or by s t e r i l i z i n g the medium by autoclaving. The toxicity of the v o l a t i l e alkyl metals (Pb) and metalloids (As, Se) on algal growth was investigated by using the biologically generated alkyls since most of the methylated products are highly


3.

CHAU AND WONG

Biological Methylation

41

v o l a t i l e and i n s o l u b l e i n water, making the dosing o f the compounds t o b i o t a d i f f i c u l t . The b i o l o g i c a l generator (Figure 1) c o n s i s t s o f a 4 £ c u l t u r e f l a s k c o n t a i n i n g a b a c t e r i a l inoculum (Aeromonas sp. 150 ml) and 1350 ml o f the f r e s h n u t r i e n t medium w i t h and without the d e s i r e d compound (at 5 ppm l e v e l as the element) f o r generation o f the v o l a t i l e methylated products. When sediment was used, 500 g sediment and one l i t r e of l a k e water w i t h and without the compound were incubated w i t h glucose (0.1%). A f t e r about 10 days i n c u b a t i o n , the headspace gases were analyzed f o r the presence of the methyl d e r i v a t i v e s before the c u l t u r e f l a s k was connected t o a t e s t f l a s k (4 l i t r e ) c o n t a i n i n g 1.4 £ o f f r e s h CHU-10 medium and 100 ml of a l g a l inoculum. The b i o l o g i c a l l y - g e n e r a t e d methylated product was sucked through the a l g a l c u l t u r e f l a s k by a p e r i s t a l t i c pump. The setup without the a d d i t i o n o f the compound was used as a c o n t r o l . product on a l g a l growt Results and

Discussion

Methylation of lead. Wong et a l . (16) presented the f i r s t evidence that under l a b o r a t o r y c o n d i t i o n s microorganisms i n s e d i ments from s e v e r a l Canadian lakes would transform c e r t a i n i n o r g a n i c and organic lead compounds i n t o a v o l a t i l e and h i g h l y t o x i c tetramethyllead. I t was a l s o observed t h a t i n c u b a t i o n o f c e r t a i n lake sediments produced Me^Pb even without the a d d i t i o n o f extraneous lead compound. There was no d i r e c t r e l a t i o n s h i p between lead concentrations i n the sediment and the amount o f Me^Pb produced (Table I ) . The conversion o f MeaPbOAc to Me^Pb was observed i n a l l experiments but that o f lead n i t r a t e was only sporadic. Subsequently, J a r v i e et a l . (20) confirmed the methylation o f MeaPbOAc t o Mei*Pb and proposed a mechanism i n v o l v i n g hydrogen s u l f i d e complexed w i t h MeaPbOAc f o l l o w e d by decomposition t o Mei*Pb. These workers suggested t h a t lead methylation was a chemical process, without c o n s i d e r i n g the m i c r o b i a l production o f s u l f i d e i n sediment as being a b i o l o g i c a l process. Later Schmidt and Huber (21) demonstrated t h a t not only t r i m e t h y l l e a d , but a l s o lead a c e t a t e , could be methylated t o t e t r a m e t h y l l e a d i n aquarium water. The mechanism o f methylation i s yet to be established. Chemical d i s p r o p o r t i o n a t i o n r e a c t i o n s o f Me3Pb s a l t s are known to produce Me^Pb. The experiments s e t up t o determine the p o r t i o n o f Mei+Pb due t o chemical d i s p r o p o r t i o n a t i o n r e a c t i o n s c o n s i s t e d of a s e r i e s of c u l t u r e of Aeromonas species i n n u t r i e n t b r o t h w i t h a d d i t i o n o f 0-100 mg Pb/£ o f Me PbOAc. An i d e n t i c a l set o f samples was s t e r i l i z e d f o r determining the MeiiPb produced by chemical d i s p r o p o r t i o n a t i o n r e a c t i o n s . A f t e r three weeks i n c u b a t i o n , the b a c t e r i a growth was measured and the amounts o f Me^Pb produced i n the headspace of the chemical and b i o l o g i c a l systems were q u a n t i f i e d . The amounts o f Me^Pb generated +

3



4.7 4.0 21.0 7.6 2.4 1.4 6.4

0.09 1.10 0.14 2.10 0.55 0 0 0.13

0 1.80 0.16 0 0.71 0 0 0.01

116 285 47 48 47 48 43

Long Lake

K e l l y Lake

Lunch Lake

Robinson Lake

D i l l Lake

Norway Lake

Babine Lake

Hamilton Harbor

4.

5.

6.

7.

8.

9.

10.

11.

50 gm sediment ( w e t w t . ) , 150 ml l a k e water, 0.5% n u t r i e n t broth and 0.1% glucose with and without the a d d i t i o n o f 1 mg Pb as Pb (NO3)2 o r Me PbOAc. F i n a l Pb c o n c e n t r a t i o n 5 ppm.

3

8.6

0

0

69

P o r t Stanley

3.

273

2.9 5.2

0

0

60

E r i e a u Harbor

2.

4.7

2.20

1.20

110

3

Me PbOAc

M i t c h e l Bay

2

Pb(N0 )

No a d d i t i o n 3

yg Me^Pb generated from sediment supplemented w i t h

(mg/kg dry wt.)

T o t a l Pb cone. i n sediment

M e t h y l a t i o n o f l e a d i n l a k e sediments.

1.

Lake

Table I .

3.

CHAU AND WONG

43


b i o l o g i c a l l y and c h e m i c a l l y a t d i f f e r e n t concentrations o f MeaPbOAc and t h e i r r e l a t i o n s h i p s t o b a c t e r i a l growth a r e i l l u s t r a t e d i n Figure 2. At any c o n c e n t r a t i o n o f MeaPbOAc where t h e microorganisms were a c t i v e l y growing, the Me^Pb generated chemic a l l y only represented about 15-20% o f t h e t o t a l MeifPb produced i n the b i o l o g i c a l system. When growth was i n h i b i t e d a t 100 ppm o f MeaPbOAc, the Me^Pb generated i n the system was s o l e l y due t o chemical d i s p r o p o r t i o n a t i o n . U l t r a v i o l e t i r r a d i a t i o n d i d n o t cause f u r t h e r chemical conversion o f Me PbOAc t o Me^Pb i n t h e absence o f microorganisms. D i r e c t chemical s y n t h e s i s o f methyllead compounds through a l k y l a t i o n o f i n o r g a n i c lead i s very d i f f i c u l t because o f t h e extreme i n s t a b i l i t y o f t h e p o s t u l a t e d f i r s t i n t e r m e d i a t e monoa l k y l l e a d s a l t ( M e P b ) . The d i f f i c u l t i e s have a l s o been e x p l a i n e d i n terms o f o x i d a t i o n - r e d u c t i o b i o m e t h y l a t i o n (10). However i t i s not e n t i r e l y unreasonable t o envisage l i g a n d systems which could form s t a b l e monomethyllead complexes before t h e s u c c e s s i v e methylation steps occur. Such may have been the case i n t h e observations o f methylation o f lead n i t r a t e and lead c h l o r i d e t o Me^Pb i n some sediments. The t o x i c i t y o f Mei*Pb on an a l g a (Scenedesmus quadricauda) was s t u d i e d by b u b b l i n g t h e b i o l o g i c a l l y - g e n e r a t e d Mei»Pb i n t o t h e c u l t u r e medium (22). I t was estimated t h a t l e s s than 0.5 mg (as Me^Pb) had passed through t h e c u l t u r e medium. The primary product i v i t y ( C technique) and c e l l growth (dry weight) decreased by 85% and 32%, r e s p e c t i v e l y , as compared w i t h t h e c o n t r o l s without exposure t o Mei*Pb. Furthermore, c e l l s exposed t o Me»»Pb tended t o clump together. S i m i l a r r e s u l t s were obtained w i t h Ankistrodesmus f a l c a t u s . To o b t a i n s i m i l a r e f f e c t s , t w i c e as much lead i n t h e form o f MesPbOAc, and twenty times as much lead n i t r a t e would be required. Lead methylation i s analogous t o mercury i n s e v e r a l aspects. I t i s dependent on temperature, pH and m i c r o b i a l a c t i v i t i e s o f t h e medium but independent o f t h e c o n c e n t r a t i o n o f l e a d i n t h e sediment. So f a r t h e r e are o n l y three r e p o r t s o f l a b o r a t o r y experiments on lead methylation. Evidence o f i t s occurrence i n t h e environment i s s t i l l l a c k i n g . However, e x i s t e n c e o f s i g n i f i c a n t l y h i g h r a t i o s o f t e t r a a l k y l l e a d t o t o t a l lead i n c e r t a i n f i s h e r y products i n d i c a t e s the p o s s i b i l i t y o f methylation i n sediment o r i n f i s h t i s s u e s (23). The f a c t o r s and t h e occurrence o f i n s i t u l e a d methylation i n t h e environment and t h e mechanisms o f methylation a r e being f u r t h e r investigated. 3

+3

llf

M e t h y l a t i o n o f Selenium. The production o f v o l a t i l e selenium compounds by microorganisms has been acknowledged f o r decades (24). I t i s known t h a t v o l a t i l e selenium compounds (Me Se and Me Se ) a r e produced through methylation by f u n g i , b a c t e r i a , r a t s , and h i g h e r p l a n t s (25). Not much i s known about t h e methylation o f selenium i n the a q u a t i c environment. Under l a b o r a t o r y c o n d i t i o n s , Chau e t 2


2

2


VOLATILE ALKYL-METAL GENERATOR Figure 1.

O

BIOASSAY FLASK

PUMP

Biological generation of volatile methyl alkyls for algal toxicity testing

20

40

Meg Pb OAc (mg

60

80

100

Pb-Γ)

Figure 2. Production of Me Ph by chemical disproportionation and by biological methylation and their rehtionships to growth of Aeromonas species. Growth was measured by optical density. h


3. CHAU AND WONG

45


a l . (26) observed the production of Me Se and Me Se from s e v e r a l sediment and soil samples w i t h and without enrichment w i t h the f o l l o w i n g selenium compounds: sodium s e l e n i t e , sodium s e l e n a t e , s e l e n o c y s t i n e , selenourea, and selenomethionine. In many cases, an u n i d e n t i f i e d v o l a t i l e selenium compound was produced w i t h a r e t e n t i o n time between t h a t o f Me Se and Me Se i n the gas chromatogram. The production o f v o l a t i l e selenium compounds was observed to be a s s o c i a t e d w i t h m i c r o b i o l o g i c a l growth and was temperature dependent. A summary o f the i n v e s t i g a t i o n s w i t h l a k e sediments w i t h and without a d d i t i o n of sodium s e l e n i t e and s e l e n ate i s given i n Table I I . I t has been shown t h a t s i g n i f i c a n t concentrations of selenium e x i s t i n both f r e s h and s a l t water f i s h (27). In studying the r e l a t i v e t o x i c i t y o f organic and i n o r g a n i c selenium compounds t o f i s h , N i i m i and LaHam (28 selenium l e v e l s i n the t e s t o the m i c r o b i a l methylation o f the i n o r g a n i c selenium t o a v o l a t i l e organoselenium compound which was more t o x i c t o f i s h . Selenium i s of p a r t i c u l a r i n t e r e s t as a p o t e n t i a l environmental t o x i c a n t because o f the small s a f e t y margin between the l e v e l s necessary i n the d i e t and the concentrations hazardous t o man (29). Organic selenium compounds are more t o x i c and have longer r e t e n t i o n times than the i n o r g a n i c selenium s a l t s (30). Thus i t i s of considerable i n t e r e s t t o study the methylation o f selenium i n the aquatic environment. 2

2

2

2

2

2

Methylation of arsenic. M e t h y l a t i o n o f a r s e n i c by f u n g i and b a c t e r i a has been known f o r s e v e r a l decades. Challenger and co-workers (3) e x t e n s i v e l y i n v e s t i g a t e d the a b i l i t y o f S c o p u l a r i opsis b r e v i c a u l i s t o methylate organic and i n o r g a n i c a r s e n i c compounds. Cox and Alexander (11) found 3 sewage f u n g i t h a t would methylate v a r i o u s a r e s e n i c compounds used as p e s t i c i d e s t o form t r i m e t h y l a r s i n e . McBride et_ a l . (31) a l s o showed that aerobic microorganisms produced t r i m e t h y l a r s i n e whereas the anaerobic methanogenic b a c t e r i a produced d i m e t h y l a r s i n e when incubated i n the presence of pentavalent and t r i v a l e n t a r s e n i c d e r i v a t i v e s . In our experiments w i t h lake water and sediment, we have demonstrated t h a t i n c e r t a i n sediments c o n t a i n i n g high a r s e n i c l e v e l s , such as those from the Moira R i v e r area i n O n t a r i o , nonv o l a t i l e methylated a r s e n i c compounds are detected i n the overl a y i n g lake water without the a d d i t i o n of extraneous a r s e n i c compounds (Table I I I ) . In other sediments w i t h low a r s e n i c l e v e l s , a d d i t i o n o f a r s e n i c compounds was r e q u i r e d f o r methylation. However i n most o f these experiments, no v o l a t i l e methylated a r s i n e s were detected i n the headspace of the i n c u b a t i o n f l a s k s . I n s e v e r a l experiments w i t h the sediments from the Moira R i v e r , v o l a t i l e methylated a r s i n e s (Me AsH,, Me3As) were detected. Factors which c o n t r o l a r s e n i c methylation are not completely understood. Adenosine t r i p h o s p h a t e and hydrogen were found t o be e s s e n t i a l f o r the formation of d i m e t h y l a r s i n e by c e l l e x t r a c t s o f 2



i

0.90

3.Kelley

4.Long

2

0

0

0

0

6.3

0

0.65

0.67

0.44

0.53

0.55

0.67

7. Windy

8. Moose

9. Kukagami

lO.Nepewassi

11.Johnnie

12.George

0

0.52

0

1.7

2.7

0

34

6.Vermillion

16.28

1.64

20.48

2.Ramsey

0.48

5.Simons

3

No A d d i t i o n 3

0

0

0

0

0

0

0

0

0

3.3

0

0

2

0

3.7

0

0

0

0

0

0

0

2.3

0

3.3

yg/g dry ( C H ) S e (CH )ζSe Unknown wt.

1.Elbow

Lake

Se i n Sediment 2

56.8

43.3

25.3

33.3

5

12.3

20.3

18.7

27.3

14

0

33.3

3

(CH ) Se 3

0

16.5

0

0

0

0

0

0

0

0

0

0

2

(CH ) Se 2

Na s e l e n i t e 3

2

8.7

0

0

0

0

0

18

0

0

0

0

94.7

94.6

28.7

23.4

10.5

0

9.7

20

24.7

20

33.6

34

11

19.4

0

0

0

0

8

4.7

0

3.3

0

0

3

2

Unknown ( C H ) S e ( C H ) S e

Na selenate

Table I I . M e t h y l a t i o n o f selenium compounds i n 12 Sudbury area l a k e sediment samples.

2

7.3

54.8

0

0

2

0

5.3

19.7

0

5.3

0

3.3

Unknown

£2-

5 g?

2.

Sample

3* CD

ET —*

20.40 3.73

3.96

arsenite

17.92 7.19

arsenate

8.89

6.03

none

7.59

trimethy1 a r s i n e oxide

2.93 0.63

0.52

3.27

0.50

0.45

arsenate

arsenite

2.29

7.84

9.57

3.25

dime t h y l a r s i n i c acid

3.75

0.80

1.03

2.14

methylarsonic acid

1.16

0.80

1.92

As(III) As(V)

none

arsenite

arsenate

* - below 0.1 ng d e t e c t i o n l i m i t

Pond sediment

ça q *2 ~ R i v e r sediment

§

CD

3

R~

Ο

none

Arsenic a d d i t i o n (5 mg/1)

A r s e n i c i n medium (ug/l)

Table I I I . Methylations o f a r s e n i c compounds i n sediment samples from Moira R i v e r area, O n t a r i o .

2?La -, Lake sediment 3 I 3

"•"*· cr%



limit

dimethylarsinic acid

-

9.10

arsenite

methylarsonic a c i d

4.89

arsenate


-

0.14 3.76

12.60

-

1.18

-

1.0

0.40

-

0.5

1.1

-

0.4

-

0.6

0.2

0.5

0.8

Arsine

-

-

-

trimethyl arsine oxide

10.9

-

—

113.0

-

Trimethyl-

V o l a t i l e As i n headspace (ng)

6.15

0.47

-

11.73

1.79

1.67

0.09

8.09

arsenite

methylarsonic a c i d

0\51

0.08

7.61

arsenate

below 0.1 ng d e t e c t i o n

Flavobacterium sp

E. C o l i

2.72

-

2.88

-

9.63


*

0.78

-


_

8.92

arsenite

-

methylarsonic acid

(mg/£)

me t h y l a r s o n i c a c i d

8.59

arsenate

Aeromonas sp

As(III) As (V)

As i n medium

o f a r s e n i c compounds by pure b a c t e r i a .

Arsenic addition (10 mg/£)

Methylation

Bacterial species

Table IV.

ο

> ο

Ο Ο » Ο

>

r

g >

> ο

w ο

Ο

3.

CHAU AND WONG

49


Methane-bacterium (32). Phosphate, s e l e n a t e and t e l l u r a t e , however, i n h i b i t e d the conversion o f arsenate t o t r i m e t h y l a r s i n e by a fungus (11). Since sediment i s a complex b i o l o g i c a l and chemical mosaic, i t i s d i f f i c u l t t o understand what organisms(s) and r e a c t i o n s are r e s p o n s i b l e f o r the a r s e n i c m e t h y l a t i o n . In an attempt t o s i m p l i f y the s i t u a t i o n , we have found two pure b a c t e r i a l c u l t u r e s Aeromonas and Flavobacterium sp., i s o l a t e d from lake water and another bacterium E s c h e r i c h i a c o l i , commonly found i n the i n t e s t i n e o f an organism and i n p o l l u t e d water, had the a b i l i t y t o methylate a r s e n i c compounds when grown i n a medium o f 0.5% n u t r i e n t b r o t h , 0.1% glucose and 10 mg/£ a r s e n i c compound (as As) at 20°C under aerobic c o n d i t i o n s (Table I V ) . Results show t h a t the a d d i t i o n s o f a r s e n i c compounds t o the medium would g e n e r a l l y r e s u l t i n the formation o f d i m e t h y l a r s e n i a c i i medium Trimethylarsin oxide was seldom detected a r s i n e and t r i m e t h y l a r s i n e were a l s o found i n the headspace o f the culture flasks. The t o x i c i t y o f a mixture o f b i o l o g i c a l l y - g e n e r a t e d v o l a t i l e a r s i n e s on an a l g a ( C h l o r e l l a pyrenoidosa) was i n v e s t i g a t e d by the p r e v i o u s l y mentioned technique. The primary p r o d u c t i v i t y ( C technique) and c e l l growth ( c e l l count) decreased by 45% and 44%, r e s p e c t i v e l y , as compared w i t h the c o n t r o l algae without exposure to a r s i n e s . Chemical analyses o f the a l g a l c e l l s r e v e a l e d t h a t the t o t a l As l e v e l s i n the exposed c e l l s were 10 times more than t h a t i n the unexposed c e l l s . McBride et a l . (31), u s i n g c e l l - f r e e e x t r a c t s o f the a e r o b i c s o i l organism, Candida humicola, demonstrated t h a t i n the presence o f S-adenosylmethionine and NADPH, the e x t r a c t s could s y n t h e s i z e a r s e n i t e , methylarsonate and d i m e t h y l a r s i n a t e from arsenate. Whether such r e a c t i o n s occur i n sediments r e q u i r e s f u r t h e r investigation. llf


50


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2.

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7.

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8.

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9.

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Scienc

10.

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L.J.

and Wood, J . M .

11.

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12.

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13.

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14.

Braman, R . S .

Bull.

E n v i r o n . Contam. T o x i c o l .

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pp. 108-123.

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15.

Langley, D.G. J. Water

16.

Wong, P.T.S., Chau, Y . K . and Luxon, P . L . Nature (1975) 253, 263. Chau, Y.K., Wong, P . T . S . and Goulden, P . D . A n a l . Chim. Acta (1976) 85, 421.

17.

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CHAU AND WONG


51

18.

Chau, Y.K., Wong, P . T . S . and Goulden, P . D . (1975) 47, 2279.

A n a l . Chem.

19.

Wong, P.T.S., Chau, Y.K., Luxon, P . L . and Bengert, G.A. Proc. 11th Ann. Conf. "Trace Substances i n E n v i r o n . Health X I " M i s s o u r i . Ed. D.D. H e m p h i l l , pp. 100-106 (1977).

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Nature

(1975) 225, 217. 21.

Schmidt, U . and Huber, F .

Nature (1976)

22. 23.

S i l v e r b e r g , B.A., Wong P . T . S d Chau Y . K A r c h E n v i r o n Contam. T o x i c o l S i r o t a , G.R. and Uthe, J.F. A n a l . Chem. (1977) 49, 823.

24.

C h a l l e n g e r , F.

25.

C h a l l e n g e r , F. Adv. Enzymology and r e l a t e d areas o f molecular biol. (1951) 12, 429.

26.

Chau, Y.K., Wong, P.T.S., S i l v e r b e r g , B.A., Luxon, P . L . and Bengert, G.A. Science (1976) 192, 1130.

27.

Oelschlager, V.W. and Menke, K . H . Ernahrungswiss (1969)

Chem. and Ind. (1935)

259, 159.

54, 657.

9, 216. 28.

N i i m i , A.J. and LaHam, Q.N.

Can. J. Z o o l .

(1976) 54, 501.

29.

Copeland, R.

30. 31.

Schroeder, H.A., F r o s t , D.A. and B a l a s s a , J.J. J . Chronic Disease (1970) 23, 227. McBride, B.C., Reimer, M.M. and C u l l e n , W.R. 175th ACS N a t i o n a l Meeting, Anaheim, Calif. (1978) A b s t r a c t o f Papers, I n o r g a n i c , no. 116.

32.

McBride, B . C . and Wolfe, R . S .

Limnos (1970) 3, 7.

Biochem. (1971) 10, 4312.

Discussion J . M . WOOD ( U n i v e r s i t y of M i n n e s o t a ) : One of the t h i n g s I f i n d p u z z l i n g about the m e t h y l a t i o n of l e a d i s what s o r t of comp l e x has to be formed i n b i o l o g i c a l systems to s t a b l i z e a s i n g l e lead-carbon bond i n water? T h i s i s a tremendous problem from a thermodynamic p o i n t of v i e w . Y e t , i t appears that somehow t h i s happens because t e t r a m e t h y l l e a d i s produced. I wonder whether


52

ORGANOMETALS AND

ORGANOMETALLOIDS

anybody here has any idea on what s o r t of l i g a n d i s l i k e y t o coo r d i n a t e lead to s t a b l i z e a s i n g l e lead-carbon bond i n water. CHAU: We are working on a number of b i o l o g i c a l l i g a n d s , name l y , g l u t a t h i o n e p o l y c a r b o x y l i c a c i d s , and s u l f u r l i g a n d s , t r y i n g t o s t a b l i z e the monomethyllead complex. F. HUBER ( U n i v e r s i t y of Dortmund): Did you add v i t a m i n B t o your s o l u t i o n s when studying the production of t e t r a m e t h y l l e a d from l e a d ( I I ) compounds? 1 2

CHAU: Yes, we d i d add B^» use no B^2«

but i n our general experiments we

HUBER: So there i s no i n f l u e n c e on the t e t r a m e t h y l l e a d d u c t i o n when you add v i t a m i CHAU: No,

pro

there was no enhancement.

HUBER: This i s about the same r e s u l t we got, e s p e c i a l l y when we t r i e d i t w i t h t h a l l i u m . You t a l k e d about the problem of the p r o p o r t i o n where you found about 15%-20%; we f i n d about 80% chemi c a l l y produced t e t r a m e t h y l l e a d . I t might be p o s s i b l e that the compositions of the s o l u t i o n s are d i f f e r e n t . We show t h a t , depending on the composition of the s o l u t i o n , there i s a tremendous i n f l u e n c e on the r a t e of r e d i s t r i b u t i o n r e a c t i o n s of organolead compounds· J . J . ZUCKERMAN ( U n i v e r s i t y of Oklahoma): In some cases you s p e c i f i e d the a c e t a t e ; I take i t that these were s o l u b l e l e a d compounds , and t h a t these engaged i n the r e d i s t r i b u t i o n r e a c t i o n . Did you t r y c o l l o i d a l or i n s o l u b l e lead compounds to see i f they could be m o b i l i z e d by the b i o l o g i c a l species i n these r e a c t i o n s ? CHAU: We t r i e d s e v e r a l i n s o l u b l e compounds, such as l e a d hydroxide , lead c h l o r i d e , and s p a r i n g l y s o l u b l e l e a d carbonate. There was no m e t h y l a t i o n . But even w i t h l e a d n i t r a t e we had d i f f i c u l t y g e t t i n g c o n s i s t e n t r e s u l t s , so I'm not s u r p r i s e d that those i n s o l u b l e compounds d i d n ' t r e a c t . W. P. RIDLEY ( U n i v e r s i t y of Minnesota): I'm i n t e r e s t e d i n your experiments on the methylation of l e a d i n f i s h i n t e s t i n e s . Have you examined the t r a n s p o r t of the methylated a r s e n i c species across the i n t e s t i n a l w a l l of the f i s h ? CHAU: No, we haven't. We used f i s h i n t e s t i n e cut i n t o pieces and mixed i t w i t h an i n o r g a n i c a r s e n i c s o l u t i o n . The b a c t e r i a from the f i s h i n t e s t i n e could transform the arsenate and a r s e n i t e i n t o methyl and dimethyl a r s e n i c compounds.


3.

CHAU AND WONG

Biological Methyfotion

53

K. IRGOLIC (Texas A & M U n i v e r s i t y ) : You showed data on a r s e n i c compounds which you i d e n t i f i e d as d i m e t h y l a r s i n i c and met h y l a r s o n i c a c i d s . As you pointed out, i t i s not n e c e s s a r i l y t r u e t h a t the d i m e t h y l a r s i n e or monomethylarsine you detected need come from these compounds. They could come from an arsenobetaine. I would l i k e t o warn that use of d i m e t h y l a r s i n i c a c i d or methylars o n i c a c i d as a standard i n a n a l y t i c a l procedures does not mean t h a t you have these compounds i n s o l u t i o n . I would l i k e to ask what k i n d of s t r u c t u r e was suggested f o r the v o l a t i l e unknown selenium intermediate i n the methylation of the selenium. WOOD: I n a model system, we were l o o k i n g at the methylation of selenium by dimethylmercury. Under the r i g h t c o n d i t i o n s , you can get methyl t r a n s f e r from dimethylmercury. We i s o l a t e d and c h a r a c t e r i z e d a seleniu f a c t , the predominant produc r a p i d l y converted to dimethylselenide by b e t a - e l i m i n a t i o n . Dr. Chau had i s o l a t e d something s i m i l a r and I sent him the mass spect r a l data and the mar data. I j u s t learned today when you t a l k e d t h a t these t h i n g s are i d e n t i c a l . CHAU:

Yes, they are i d e n t i c a l .

E. BEVAGE ( U n i v e r s a l O i l P r o d u c t s ) : I s i t your o p i n i o n t h a t the m e t h y l a t i o n of these elements i s given by a wide number o f species of b a c t e r i a or do you f e e l i t i s l i m i t e d t o f a i r l y few? I n o t i c e d you had data on E. c o l i . CHAU: Dr. Wong has i d e n t i f i e d s e v e r a l b a c t e r i a l s p e c i e s , namely Aeromonas and Pseudomonas, very common i n l a k e sediments. They do c a r r y out methylation. I t h i n k we have i d e n t i f i e d f o u r s p e c i e s of b a c t e r i a which could c a r r y out t h i s m e t h y l a t i o n . It is more f a v o r a b l e i n anaerobic s i t u a t i o n s . Sometimes m e t h y l a t i o n occurs w i t h e i t h e r aerobic or anaerobic c o n d i t i o n s as w i t h a r s e n i c . RECEIVED August 22,

1978.


4 Kinetic and Mechanistic Studies on B -Dependent Methyl Transfer to Certain Toxic Metal Ions 12

Y.-T. FANCHIANG, W. P. RIDLEY, and J. M . WOOD Freshwater Biological Institute/Department of Biochemistry, College of Biological Sciences, University of Minnesota, P.O. Box 100, Navarre, M N 55392 Introduction In the 1930's Challenger discovered the biomethylation of arsenic, and provided us with the f i r s t example of how biological systems possess the capability for synthesizing very toxic organo-arsenic compounds from less toxic inorganic substrates [1,2]. Even in those early days Challenger recognized that biomethylation could only present a local environmental hazard, if the methylated product is produced in significant concentration so as to exert its toxic effect and if the methylated product is stable to hydrolysis. Oxidation-reduction reactions were not well understood in the 1930's; in fact, they are not too well understood to this day, especially in biological systems. For example, biochemists and chemists do not understand the fundamental processes of nitrogen fixation, hydrogen evolution and fixation, photosynthesis, sulfite and n i t r i t e reduction, e t c . , etc. These processes all use electron transfer systems which contain transition metal ions such as molybdenum, iron, manganese, e t c . , for catalysis, and most of these systems are sensitive to molecular oxygen. A major, as yet unanswered question i s : "How did such a great variety of single electron transfer reactions evolve and remain active in a global system which is bathed in molecular oxygen?" Clearly, the "redox" conditions in a specific environment must select for the preferred reaction mechanism, but also control the kinetics for environmentally significant reactions such as biomethylation. Dynamic aspects of these reactions are of c r i t i c a l importance, because even though most methylated metals are thermodynamically unstable in water, many of them are kinetically stable. In fact, i t can be shown that metals which are lower in their periodic groups form metal-alkyls which are kinetically more stable. For example, mercury, platinum and possibly lead offer potentially stable systems whereas palladium, chromium and cadmium do not. In this brief report we examine the different mechanisms for 0-8412-0461-6/78/47-082-054$05.00/0 © 1978 American Chemical Society In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

4.

F A N C H i A N G E T AL.

55

B n-Dependent Methyl Transfer

Bj^-dependent m e t h y l - t r a n s f e r t o a s e l e c t e d group o f t o x i c e l e ments p l a c i n g s p e c i a l emphasis on "redox" c o n d i t i o n s . Four i n v i t r o mechanisms have been e l u c i d a t e d f o r B^2""dependent m e t h y l - t r a n s f e r to date: (1) h e t e r o l y t i c cleavage o f the Co-C bond w i t h t h e t r a n s f e r o f a carbanion t o the a t t a c k i n g metal i o n ; ( 2 ) h e t e r o l y t i c cleavage o f the Co-C bond w i t h the t r a n s f e r o f a carbonium ion t o an a t t a c k i n g n u c l e o p h i l e ; ( 3 ) homolytic cleavage o f t h e Co-C bond w i t h t r a n s f e r o f a m e t h y l - r a d i c a l t o an a t t a c k i n g f r e e r a d i c a l ; and ( 4 ) "Redox-Switch", a mechanism where metal ions complex w i t h the c o r r i n macrocycle t o l a b i l i z e the Co-C bond t o a t t a c k by weak e l e c t r o p h i l e s [ 3 , 4 , 5 , 6 ] . The t r a n s f e r o f CH3" o r CH3* have been found t o be the most predominant r e a c t i o n mecha nisms f o r a number of metals and m e t a l l o i d s . (1) E l e c t r o p h i l i M e t h y l - t r a n s f e r from methyI bond. The methyl t o c o b a l t bond can break under d i f f e r e n t con d i t i o n s t o g i v e a carbanion, a r a d i c a l o r a carbonium i o n (Figure 1). We have shown that the carbanion and r a d i c a l forms are the p r i n c i p a l species i n v o l v e d i n m e t h y l - t r a n s f e r t o metals and m e t a l l o i d s [7J . The r e a c t i o n between mercuric i o n and methyl c o r r i n o i d s i s an example o f carbanion m e t h y l - t r a n s f e r ( F i g u r e 2 ) . Because mercuric i o n i s a good e l e c t r o p h i l e , i t a l s o coordinates t o t h e n i t r o g e n of the 5,6-dimethylbenzimidazole base t o g i v e a mixture of "base o f f " and "base on" methy1-Βχ2. The "base on" species r e a c t s 1000 times f a s t e r than the "base o f f " s p e c i e s t o g i v e methylmercury as the product [ 3 J ( F i g u r e 2 ) . Other metals which are known t o r e a c t w i t h methyI-B12 by a s i m i l a r mechanism t o mercuric i o n are l e a d ( P b ) , t h a l l i u m ( T l ) and p a l l a d i u m (Μ") Γ8.9.10Ί. The r e a c t i o n s d e s c r i b e d above a l l i n v o l v e the displacement of a carbanion from the c o b a l t atom o f methy l - B - ^ . These r e a c t i o n s occur under aerobic c o n d i t i o n s w i t h r a t e constants i n the order o f m i l l i s e c o n d s . I t i s apparent t h a t metals which r e a c t by e l e c t r o p h i l i c a t t a c k on the Co-C bond (SE2 mechanism) occur w i t h the more o x i d i z e d s t a t e of the metal ( i . e . P b , T l , Hg , Pd , etc.). I V

1 1 1

I V

1 1 1

1 1

1 1

(2) Free R a d i c a l A t t a c k on the Co-C bond o f Methyl-Βτ 9 . A second general mechanism f o r b i o m e t h y l a t i o n i n v o l v e s the d i s placement o f a methyl r a d i c a l from methyI-B12 by the a t t a c k i n g m e t a l l i c s p e c i e s . Homolytic cleavage o f the Co-C bond occurs w i t h the t r a n s f e r o f the methyl r a d i c a l . This r e a c t i o n can be viewed as one e l e c t r o n o x i d a t i v e a d d i t i o n [ 5 , 7 ] . Reactions o f t h i s k i n d r e q u i r e the generation o f a r a d i c a l i n t e r m e d i a t e . This r a d i c a l intermediate can be produced under anaerobic c o n d i t i o n s i n the l a b o r a t o r y e i t h e r by adding a s i n g l e e l e c t r o n oxidant t o a metal i o n i n the reduced s t a t e o f a redox couple (e.g. Sn-*-* — > S I I I + e) o r by adding a s i n g l e e l e c t r o n reductant t o a m e t a l n


ORGANOMETALS

Codll)

α u\ ϋ

A N D ORGANOMETALLOIDS

•

CHi

+

CH *

•

CH

CHl

Codll)

1

θζ

C o d I)

Χ

1

3

Βζ

Co(l)

ί—Βζ Βζ = 5,6-dimethylbenzimidazole Figure 1. Methyl transfer from methyl-B requires cleav age of the Co-C bond. The methyl-to-cobalt bold can break under different conditions giving a carbanion, a radical, or a carbonium ion. 12

CH

CH

3

3

H 0 + Hg > >Co+: Κ 1

2

N

H'° H L

BzHg

2+

Fast reaction

ν

+

-Hg'

H

s

Slow reaction

Η +

>Co< + CH Hg + Hg

CH Hg +

2+

3

3

L

Bz

Figure 2. The reaction between mercuric ion and methyl corrinoids is an example of carbanion methyl transfer.


4.

B12-Dependent Methyl Transfer

FANCHiANG E T AL.

57 1

1

i o n i n the o x i d i z e d s t a t e o f a redox couple (e.g. A u * + e — > A u ) [11]. I n the case o f t i n we have shown that r e d u c t i v e Co-C bond cleavage of methy l - B ^ occurs by a t r a n s i e n t Sn*** r a d i c a l which i s generated by one e q u i v a l e n t o x i d a t i o n o f Sn** (Figure 3) [5*7]. I n the case o f gold we have shown that cleavage o f the Co-C bond r e q u i r e s the generation of Au** by s i n g l e e l e c t r o n r e d u c t i o n of Au*** (Figure 4) [11]. For t h i s r e a c t i o n preincubat i o n o f the Au*** s a l t w i t h the Fe** s a l t i s r e q u i r e d before a r e a c t i o n w i t h methyl-B^2 proceeds. S i m i l a r r e a c t i o n mechanisms have been demonstrated f o r methy1-transfer t o s u i f h y d r y 1 groups [12] and t o chromium (Cr**) [13]. The standard r e d u c t i o n p o t e n t i a l s (E° v o l t s ) f o r elements known t o be methylated by methyl-B^2 shown i n Table I . I t i s c l e a r t h a t those metals w i t h a h i g h r e d u c t i o n p o t e n t i a l ( o x i d i z i n g agents) r e a c t by a those w i t h a low r e d u c t i o a r e d u c t i v e mechanism (Type I I ) [5,7]. This connection between standard r e d u c t i o n p o t e n t i a l and mechanism f o r b i o m e t h y l a t i o n seems h i g h l y r a t i o n a l , because E° d e s c r i b e s the r e l a t i v e thermodynamic tendency f o r the metals i n v o l v e d t o accept or donate electrons. 11

v

a

r

e

Table I . R e l a t i o n s h i p Between Standard Reduction P o t e n t i a l (E°) and the Mechanism of M e t h y l a t i o n f o r Selected Elements. Redox Couple Pb(IV)/Pb(II) T1(III)/T1(I) Se(VI)/Se(IV) a c i d Pd(II)/Pd(0) Hg(II)/Hg(0) Au(III)/Au(I) Pt(IV)/Pt(II) As(V)/As(III) acid Au(III)/Au(II) Sn(IV)/Sn(II) Se(VI)/Se(IV) base Cys-S-S Cys/2Cys-SH Cr(III)/Cr(II) A s ( V ) / A s ( I I I ) base

E° ( v o l t s ) +1.46 +1.26 +1.15 +0.987 +0.854 +0.805 +0.760 +0.559 +0.50* +0.154 +0.05 -0.22 -0.41 -0.67

Mechanism of M e t h y l a t i o n Type I [7,8] Type I [9]

* The A u ( I I I ) / A u ( I I ) couple i s estimated

Type I [10] Type I [3,7] Redox Switch [14] Redox Switch [14] Type I I [ 6 J Type I I [5,7] Type I I [12] Type I I [13]

[19].

Those metals which r e a c t by e l e c t r o p h i l i c a t t a c k (Type I mechanism) have standard r e d u c t i o n p o t e n t i a l s g r e a t e r than +0.80 v o l t s , whereas those metals which r e a c t by r e d u c t i v e homolytic cleavage (Type I I mechanism) tend to have standard r e d u c t i o n p o t e n t i a l s l e s s than +0.56. Reactions which are not c l e a r l y defined occur c l o s e t o the standard r e d u c t i o n p o t e n t i a l f o r molecular oxygen (+0.68 v o l t s ) , and t h i s group o f metals have been c a l l e d "Redox


58

ORGANOMETALS AND

ORGANOMETALLOIDS

Switch" metals [14] because both o x i d a t i o n s t a t e s of the metal are r e q u i r e d f o r b i o m e t h y l a t i o n to occur. I n the f o l l o w i n g sec t i o n we w i l l present recent data on t h i s "Redox S w i t c h " mechanism. (3) The "Redox Switch" Mechanism f o r A t t a c k on the Co-C Bond of Methyl-B-p. In 1972, Brown and Wood [15] showed t h a t 2 3 - i s o p r o p y l i d e n e - 5 - d e o x y - 3 - ( D ) r i b o x y l cobinamide c o u l d e x i s t as two s t a b l e c o r r i n - r i n g isomers. This i s o m e r i z a t i o n was shown to be pH dependent and s o l v e n t dependent. The y e l l o w c o l o r e d isomer (Y) had a X at 420 nm and the Co-C bond was shown t o be s t a b l e to v i s i b l e l i g h t , but the red isomer (R) had a X at 475 m\i and was shown t o be p h o t o l a b i l e . A 220 MHz NMR study of these two isomers gave markedly d i f f e r e n t s p e c t r a . For (R) the v i n y l proton at C^Q was assigned at σ = 6.7 s h i f t i n g t o α = 7.48 i n the (Y) form. The (Y r e v e r s i b l e . T h i s researc i n f l u e n c e of c o r r i n - r i n g conformation on the s t a b i l i t y of the Co-C bond. At that time we suggested t h a t the i n t e r a c t i o n of Βχ2 w i t h p r o t e i n s could lead t o c o r r i n - r i n g i s o m e r i z a t i o n s which may represent an important f e a t u r e i n understanding s u b s t r a t e directed l a b i l i z a t i o n of the Co-C bond i n the Bi2-enzymes [15]« Certainly, i t seems reasonable t h a t the propionamide s i d e chains on Βχ2" coenzymes could hydrogen bond to p r o t e i n s to cause some d i s t o r t i o n of the c o r r i n macrocycle. In 1975, f i r s t Hogenkamp et a l . , [16] and then Cockle et a l . , [17] used 270 MHz NMR to demonstrate that a s i m i l a r r e d - y e l l o w s h i f t t o t h a t observed by Brown and Wood [15] could be e x p l a i n e d by c o r r i n - r i n g i s o m e r i z a t i o n of cobalamins. R e c e n t l y , Abeles et a l . , [18] showed t h a t a red to y e l l o w isomer i z a t i o n occurs i n the Bj^-enzyme d i o l dehydrase when a mono-ester d e r i v a t i v e of Bi2~coenzyme i s s u b s t i t u t e d f o r 5'deoxyadenosylcobalamin. This mono-ester coenzyme analog shows a s u b s t r a t e dependent red t o y e l l o w t r a n s i t i o n , and the y e l l o w form of the coenzyme analog i s about 5% as a c t i v e as 5 deoxyadenosylcobalamin itself. Each of the above examples demonstrate t h a t the red (R) to y e l l o w (Y) i s o m e r i z a t i o n r e a c t i o n f o r f r e e Βχ2 analogs as w e l l as f o r a Bi2 analog bound to the enzyme d i o l dehydrase, r e s u l t s i n s t a b i l i z a t i o n of the Co-C bond to h o m o l y t i c cleavage. Recently we have shown t h a t P t H C l ^ - forms a one to one complex w i t h the c o r r i n macrocycle and l a b i l i z e s the Co-C bond to a t t a c k by weak e l e c t r o p h i l e s such as P t , A s and S e ( F i g u r e 5 ) . No r e a c t i o n occurs w i t h P t , A s or S e alone. U s i n g 270 MHz NMR together w i t h a d e t a i l e d k i n e t i c and e q u i l i b r i u m study of t h i s r e a c t i o n the f o l l o w i n g f e a t u r e s emerge: 1. P t ^ C l ^ " forms a one t o one "outer sphere" complex w i t h methyI-B12· 2. There i s no displacement or i n t e r a c t i o n w i t h the benz i m i d a z o l e moiety nor any other r e g i o n of the molecule which pro j e c t s below the plane of the c o r r i n macrocycle. 3. The pKa f o r the displacement of benzimidazole i n c r e a s e s 1

f

1

m a x

m a x

1

I V

I V

v

v

V I

V I

2


Β ι'-Dependent

FANCHiANG ET AL.

FE11

+

SN

ς n

+

/ ^

d

FE*

Figure 3. Reductive Co-C bond cleavage of methyl-B, occurs by a transient Sn radicflZ which is generated by one equivalent oxidation of Sn . 2

h \ · 0

ν ^

*

L- Bz

FE

•

Η

v

•m S

• SN*

1

Methyl Transfer

ch

3

Sn

L-5

+ Au

m

111

x

11

Z

* Au*

Figure 4. Cleavage of the Co-C bond requires the gen eration of Au by single-elec tron reduction of Au . 11

m

IL Q_

CH*

Co 1 1

+

PT C L É

= ^

Bz

H

Co

L

B

z

H H

CHz

_ ο

\ Ι**/Ρτ C L É

Y

«κ 9-

Co^

+

Ρτ θ |

+

Η

2

0 - ^

L^BZ

^Βζ

-π 5 -

Co^ +

Ρ Α ή

+

Ε CH3PT

CL§

+

CL"

V

Figure 5. Vt Cl?~ forms a one-to-one complex with the corrin macrocycle and labilizes the Co—C bond to attack by weak electrolytes such as ?t , As , and Se . u

IV

v

VI


60


I

2

by 0.6 u n i t s i n the P t * C l 4 ~ - m e t h y l - B i 2 complex. 4. Complex formation i s s e n s i t i v e to both i o n i c s t r e n g t h and the nature of the anion i n s o l u t i o n . 5. A downfield s h i f t of 0.064 ppm i s observed f o r the methyl group σ bonded t o the c o b a l t atom of M e B ^ - P t ^ C l ^ - com p l e x i n d i c a t i n g a change i n e l e c t r o n d e n s i t y on the methyl-group making i t more s u s c e p t i b l e t o e l e c t r o p h i l i c a t t a c k . 6. The Co-C bond i n the Ρt^Cl^'-methyl-Bj^ complex i s more p h o t o l a b i l e than f o r methy1-Βχ2 alone ( i . e . the bond i s a p p r o x i mately h a l f as s t a b l e t o l i g h t i n the complex). A d e t a i l e d r e p o r t on the k i n e t i c s and mechanism f o r methylt r a n s f e r to platinum i s i n press [ 6 J . The major f e a t u r e s of the r e a c t i o n mechanism are presented i n F i g u r e 5. Although we do not know the d e t a i l e d s t r u c t u r e of the P t C l 4 " - m e t h y l - B i 2 complex, i t i s c l e a r that a c t i v a t i o formation of t h i s complex MHz NMR study of t h i s i n t e r a c t i o n i s underway w i t h a view to determination of the p r e c i s e s t r u c t u r e of t h i s " a c t i v a t e d " methy1-B^2 s p e c i e s . 2

II

2

Conclusions The mechanisms f o r Β-^-dependent m e t h y l - t r a n s f e r to a number of metals and m e t a l l o i d s are shown t o be determined by the s t a n dard r e d u c t i o n p o t e n t i a l (E°) f o r the a t t a c k i n g i n o r g a n i c s a l t . The standard r e d u c t i o n p o t e n t i a l f o r molecular oxygen separates those complexes which cleave the Co-C bond by h e t e r o l y s i s from those which cleave t h i s bond by homolysis. I n the case of p l a t i num, which has an E° f o r the P t ^ / P t couple c l o s e t o that f o r molecular oxygen, we have discovered the formation of an "outer sphere" complex between the P t s a l t and the c o r r i n macrocycle which l a b i l i z e s the Co-C bond t o e l e c t r o p h i l i c a t t a c k by P t . This mechanism has been c a l l e d a "Redox Switch" and has many s i m i l a r i t i e s t o current mechanisms being proposed f o r l a b i l i z a t i o n of the Co-C bond i n the B^-enzymes. We b e l i e v e that a fundamen t a l study of k i n e t i c s and mechanism f o r B^-dependent methylation of metals and m e t a l l o i d s has not only helped t o d e f i n e e n v i r o n mental c o n d i t i o n s f o r b i o m e t h y l a t i o n r e a c t i o n s , but has a l s o shed some l i g h t on the p r e r e q u i s i t e s r e q u i r e d f o r " a c t i v a t i o n " of the Co-C bond i n Bi2-enzyme c a t a l y z e d r e a c t i o n . 1 1

1 1

i v

Acknowledgements Some of the research supported i n t h i s report was s u b s i d i z e d by the U.S. P u b l i c Health S e r v i c e AM 18101, the I n t e r n a t i o n a l Lead Z i n c Research O r g a n i z a t i o n and the Northwest Area Foundation.


4. FANCHIANG ET AL.

B -Dependent 12

Methyl Transfer

61

References 1. C h a l l e n g e r , F . Chem. Rev. (1945) 36, 315. 2. R i d l e y , W . P . , Dizikes, L.J., Cheh, Α . , and Wood, J.M. Env. Health P e r s p e c t i v e s (1977) 1 9 , 4 3 . 3. DeSimone, R.E., P e n l e y , M.W., Charbonneau, L., Smith, S.G., Wood, J.M., Hill, H . A . O . , Pratt, J.M., R i d s d a l e , S . , and W i l l i a m s , R.J.P. Biochim. Biophys. A c t a (1973) 304, 851. 4. Schrauzer, G.N., Sibert, J . W . , and Windgassen, R.J. J. Amer. Chem. Soc. (1968) 90, 6681. 5. D i z i k e s , L.J., R i d l e y , W . P . , and Wood, J.M. J. Amer. Chem. Soc. (1978) 1 0 0 : 3 , 1010. 6. Fanchiang, Y.-T., R i d l e y , W . P . , and Wood, J . M . J. Amer. Chem. Soc. (1978) 100, 1010. 7. R i d l e y , W . P . , Dizikes, L.J., d Wood J.M. Scienc (1977) 197, 329. 8. Wood, J.M. (1978) ceedings of an I n t e r n a t i o n a l Conference on Lead in the Marine Environment. R o v i n j , Y u g o s l a v i a . M . B r a n i c a , e d i t o r . 9. Agnes, G., Hill, H . A . O . , Pratt, J.M., R i d s d a l e , S.C., Kennedy, F.S., and W i l l i a m s , R.J.P. Biochim. Biophys. A c t a (1971) 252, 207: 10. S c o v e l l , W.H. J. Amer. Chem. Soc. (1974) 96, 3451. 11. Fanchiang, Y.-T., R i d l e y , W . P . , and Wood, J.M. Chem. Communs (1978) (submitted) March. 12. F r i c k , T., F r a n c i a , M . D . , and Wood, J.M. Biochim. Biophys. A c t a (1976) 428, 808. 13. Espenson, J.H., and S e e l e r s , T . D . J . Amer. Chem. Soc. (1974) 96, 94. 14. Agnes, G., Bendle, Β., Hill, H . A . O . , W i l l i a m s , F.R., and Williams, R.J.P. Chem. Communs. (1971) 850. 15. Brown, D.G., and Wood, J.M. S t r u c t u r e and Bonding (1972) 11, 47. 16. Hogenkamp, H.P.C., V e r g a m i n i , P.J., and Matwiyoff, N . A . J. Chem. Soc. D a l t o n Trans. (1975) 2628. 17. C o c k l e , S.A., Hensens, O . D . , Hill, H . A . O . , and W i l l i a m s , R.J.P. J . Chem. Soc. D a l t o n Trans. (1975) 2633. 18. A b e l e s , R . H . Current Status of the Mechanism of B -coenzyme. B i o l o g i c a l Aspects o f I n o r g a n i c Chemistry. Wiley I n t e r s c i e n c e , E d . D . H . Dolphin 1977) 245. 12

19.

R i c h , R.L., and Taube, H . J. Phys. Chem. (1954) 58, 1,6.

Discussion J . J . ZUCKERMAN ( U n i v e r s i t y of Oklahoma): I s there evidence for b i o a l k y l a t i o n where the a l k y l group i s other than methyl? WOOD: On the question of B^-dependent r e a c t i o n s , e t h y l c o balamin has been i s o l a t e d and c h a r a c t e r i z e d as a n a t u r a l product by a group of German workers, but i t ' s there i n v e r y s m a l l concen-


62


t r a t i o n s . I t may be p o s s i b l e t o get e t h y l t r a n s f e r r e a c t i o n s . I f t h a t ' s the case, I would t h i n k that ethylmercury should have been t u r n i n g up i n some b i o l o g i c a l systems; however, i t ' s only been t u r n i n g up where people have s p i l l e d e t h y l l e a d . I f you have a c h l o r i n e p l a n t next t o an a l k y H e a d - p r o d u c i n g p l a n t , you get d i s p r o p o r t i o n a t i o n i n the s y n t h e s i s of ethylmercury compound. ZUCKERMAN: I r e c a l l that marsh gas has an odor and i s proba b l y a mixture of a l a r g e number of hydrocarbons. Are these d i r e c t l y incorporated onto a r s e n i c by b i o l o g i c a l organisms? WOOD: The s y n t h e s i s o f the a r s i n e s and the s y n t h e s i s o f v o l a t i l e selenium compounds i s w e l l known; the s y n t h e s i s of v o l a t i l e dime thy lmercury i s w e l l known. So, v o l a t i l i z a t i o n o f metal and m e t a l l o i d a l k y l s i n thes occurs. A l s o , Brinckman' methyItins. [Proc. I n t e r n a t . Con. on Transport o f P e r s i s t e n t Chemicals i n Aquatic Ecosystems. N a t i o n a l Research C o u n c i l , Ottawa, Canada, 1974, p.11-73.] ZUCKERMAN: With respect t o the a l k y l a t i o n o f t i n , has i t been demonstrated t o occur i n environmental c o n d i t i o n s of pH, tempe r a t u r e , etc? F. E. BRINCKMAN ( N a t i o n a l Bureau of Standards): There i s a c o m p l i c a t i o n here. S a l i n i t y i s a very l a r g e f a c t o r , but the r e s u l t s of a change i n the m i c r o b i a l p o p u l a t i o n may be more important as C o l w e l l showed [ M i c r o b i a l Ecology (1975), 1, 191]. She made i t q u i t e c l e a r that the Pseudomonas p o p u l a t i o n , which i s the p r i n c i p l e a c t o r i n t h i s p a r t i c u l a r case i n v o l v i n g the mercury o r the t i n t r a n s f o r m a t i o n s , was very s u s c e p t i b l e t o such changes. So I t h i n k there i s an a d d i t i o n a l f a c t o r o f the a v a i l a b i l i t y o f growth. The growth k i n e t i c s of the microorganisms themselves w i l l then of course a f f e c t the apparent r a t e even f o r the e x o c e l l u l a r production of methylcobalamin. I t i s not y e t c l e a r whether the methylcobalamin might be i n v o l v e d i n the e n d o c e l l u l a r o r an exoc e l l u l a r v i s - a - v i s these metal ions such as t i n o r mercury. ZUCKERMAN: I f I r e c a l l your paper i n J . Amer. Chem. Soc. [(1978) 100, 1010] the c o n d i t i o n s were r a t h e r more severe than b i o l o g i c a l environmental c o n d i t i o n s . WOOD: The k i n e t i c s experiments were run a t low pH f o r the obvious reason o f keeping the t i n i n s o l u t i o n . For the t i n t a r t r a t e complex, s i m i l a r k i n e t i c s are obtained f o r r e a c t i o n s a t amb i e n t pH and temperature. We chose t o do the study under these c o n d i t i o n s so t h a t we could do k i n e t i c s more r e a d i l y w i t h t i n i o n . I t i s not a simple problem, but i f you make the t a r t r a t e complex you can do i t . There are many complexes i n b i o l o g y t h a t could mimic t a r t a r i c a c i d .


4. FANCHIANG E T AL.

Bi>-Dependent Methyl Transfer

63

G. E. PARRIS (Food & Drug A d m i n i s t r a t i o n ) : With regard t o the formation of other a l k y l m e t a l compounds, I read an ACS p u b l i c a t i o n e n t i t l e d "Chemical Carcinogens" [ACS "Advances", No. 173]. In a s e c t i o n r e g a r d i n g n a t u r a l l y o c c u r r i n g carcinogens, i t was r e ported that E. c o l i produced e t h i o n i n e and even the adenosyl der i v a t i v e of e t h i o n i n e analogous to methionine, which a l s o p r o v i d e s another p o s s i b l e a l k y l a t i n g agent. WOOD: I n that case, you have to l o o k a t some n u c l e o p h i l e . So the m e t a l l o i d s would be good candidates f o r r e a c t i o n s of that k i n d . PARRIS: I f i t i s a b i m o l e c u l a r n u c l e o p h i l i c r e a c t i o n , as the methyl t r a n s f e r might be i n e i t h e r enzymatic or non-enzymatic cases, the e t h y l t r a n s f e r would s u f f e r c o n s i d e r a b l y , r e l a t i v e t o methyl t r a n s f e r , i n term f WOOD: When you do these k i n e t i c s s t u d i e s , you f i n d the e t h y l always s u f f e r s a p p r e c i a b l y . PARRIS: I t h i n k t h e r e i s an i m p l i c a t i o n t h a t cobalamin i s preeminent i n the methyl t r a n s f e r r e a c t i o n i n v i v o , as w e l l as i n i n v i t r o s t u d i e s you have done. Would you comment? WOOD: Yes, I can put t h i s i n p e r s p e c t i v e . Many microorgan isms do i n f a c t produce Β ^ · Almost a l l of the blue-green algae s y n t h e s i z e l a r g e amounts of B ^ . M e t h y l - B ^ has been shown t o be a coenzyme i n a number of enzyme-catalyzed r e a c t i o n s . Therefore, t h i s coenzyne does t u r n over, and so you have t h i s c o n s t a n t l y r e generated m e t h y l a t i n g agent j u s t l i k e you have w i t h S-adenosylmethionine. B a s i c a l l y , i f you want t o look a t m e t h y l a t i o n problems i n the environment, you are always chasing the k i n e t i c parameters. The i n t r i g u i n g t h i n g about methylcobalamin i s that you are l o o k i n g at r e a c t i o n s w i t h r e s p e c t a b l e r e a c t i o n r a t e s . In f a c t , the r e a c t i o n r a t e f o r the m e t h y l a t i o n of mercury i s a l i t t l e b e t t e r than the turnover number f o r B-^-dependent methionine s y n t h e s i s , which i s a c r u c i a l B^-dependent enzyme. T h i s means every time a mer cury i o n gets i n the v i c i n i t y of methyl-B-^ bound i n the methionine enzyme, you are more l i k e l y t o make methylmercury than you are methionine. PARRIS: With regard t o metals other than the e l e c t r o p h i l i c mercury, or m e t a l l o i d s , do you regard B ^ preeminent methyl donor, or would you then regard S-adenosylmethionine as more l i k e l y source of methyl? a

s a

WOOD: I t h i n k i t ' s v e r y important t o do these experiments w i t h i s o t o p e s . We do model s t u d i e s and we ask that people working i n the environmental s c i e n c e s do t h i s , f o r example, w i t h l e a d . I f you want to f i n d out whether lead i s methylated, see where the C ^ -methyl group comes from i n these extremely complex experiments


64

ORGANOMETALS AND

ORGANOMETALLOIDS

where you have complex media i n v o l v i n g mixed b a c t e r i a l c u l t u r e s , sludge, e t c . I t ' s c r i t i c a l l y important to do i s o t o p e experiments to f i n d out where the methyl group i s coming from. I f t h a t ' s done, then you can w r i t e a t e n t a t i v e mechanism. I f you can w r i t e a tent a t i v e mechanism, you can t e s t i t . What we t r y t o do i s t o g i v e people a c l u e about the s o r t of mechanism that may occur i n the environment so t h a t they can go and see whether i t does. That has always been our p o s i t i o n . 1

PARRIS: One l a s t comment w i t h regard to the other a l k y l groups. I n support of the suggestion that t r a n s f e r from something l i k e S-adenosylmethione t o a m e t a l l o i d would be a n u c l e o p h i l i c r e a c t i o n . There i s one p u b l i c a t i o n i n _J. Amer. Chem. Soc. [(1976), 98, 3048] concerning the o - a l k y l a t i o n of dihydroxyacetophenone, and i n t h a t case the autho i u s i n deuteriu i s o t o p e suggested that the r e a c t i o the c r i t i c a l step i n the r e a c t i o n was b i m o l e c u l a r . WOOD: That's the only example i n the l i t e r a t u r e . RECEIVED

August 22, 1978.


5 Aqueous Chemistry of Organolead and Organothallium Compounds in the Presence of Microorganisms F. HUBER, U. SCHMIDT, and H. KIRCHMANN Chemistry Department, University of Dortmund, D 4600 Dortmund 50, Federal Republic of Germany

Organocompounds o corresponding compound following the decreasing strength of the central atom-carbon bond. Their stability is strongly dependent on the nature and also on the number of the organic groups, R, bound to lead. Alkyllead compounds are, i n general, d i s t i n c t l y less stable than aryllead compounds, and their s t a b i l i t y decreases with decreasing number of R. In aqueous solution, tri- and dialkyllead compounds, R PbX and R PbX (X = anion), show a more or less marked tendency to decompose, and monoalkyllead compounds, RPbX are actually unknown [there is only one report that Pb(OAc ) (OAc = acetate) reacts with alkylpentafluorosilicates to give (impure) RPbF (R = CH , C H , CH =CH) (1)]. Tetraalkyllead compounds are only very s l i g h t l y soluble i n water. For a general review see (2). Alkylthallium compounds, R T1X and RT1X , are i n general more stable than the corresponding lead compounds, but only a rather limited number of monoalkylthallium compounds, RT1X , are known. Trialkylthallium compounds, R T1, hydrolyze to give R T1 + OH and RH (3). 3

2

2

3

4

3

3

2

5

2

2

2

2

3

+

-

2

Decomposition of Organolead Compounds i n Water Me^PbXg. We have investigated quantitatively the decompos i t i o n of Me PbX (Me = CH ) i n aqueous solution and i n aqueous salt solutions between 20 and 60°C (4). The reaction proceeds i r r e v e r s i b l y according to equation [1] 2 Me PbX 2

2

Me PbX + PbX 3

2

+ MeX

[1]

and the stoichiometry i s not influenced by the type or the concentration of s a l t added to the solution. (The redistribution of Me ~PbX [see below] i s much slower, so i t does not appreciably interfere with [1].) The reaction rate, which i n a l l cases corresponds to f i r s t order, increases strongly with increasing salt concentration, the anions displaying much stronger influence 0-8412-0461-6/78/47-082-065$05.00/0 © 1978 American Chemical Society In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

66


than the c a t i o n s . The anions arranged according t o t h e i r a b i l i t y to i n c r e a s e the r e a c t i o n r a t e k g i v e a s e r i e s r e f l e c t i n g t h e i r polarizability: X : OAc V" Cl~ / k

:

C10

0 , 0 2

130) < (~10 )

(Concentrations^-0.036 mol Me PbX«.L "S NaX and KX r e s p e c t i v e l y 2.7 - 0.7 mol.lT ). The r e s u l t s i n d i c a t e that MeJPbX i n a f i r s t step g i v e s an intermediate which i s bridged by anions X and/or s o l v e n t molecules L: ?

2

L/X , X/L I ^X/L^| 2 M e

2

P b X

solvent _ 2 molecules t

e

X / ^

e

In a second rate-determining s t e p , t r a n s a l k y l a t i o n i s e f f e c t e d when t h i s intermediate r e d i s t r i b u t e s t o give Me^PbX and MePbX~; the l a t t e r decomposes immediately and i r r e v e r s i b l y t o PbX and MeX. B r i d g i n g i n organolead compounds i s not unusual; e.g., P h P b C l (5) or Ph PbX (Ph = C ^ ; X = CI, Br) (6) are h a l i d e bridged polymers i n the s o l i d s t a t e ; a l s o , emf measurements w i t h the system P h P b / i " i n d i c a t e d the formation of b i n u c l e a r complexes, e.g. P h P b I , P l ^ P b ^ , o r P h P b I i n a d d i t i o n t o the u s u a l mononuclear complexes ( 7 ) . 2

2

2

3

2 +

2

2 +

4

MeJPbXto [2]

2

2

4

2

4

Me^PbX r e d i s t r i b u t e s i n aqueous s o l u t i o n according

J

3

2Me.PbX

a t b

Me,Pb + Me PbX 0

2

0

[2]

2

Me PbX i s an educt of [ 1 ] , but s i n c e [2] i s r e v e r s i b l e , [2b] competes w i t h [ 1 ] . The i r r e v e r s i b i l i t y of [ 1 ] , however, f i n a l l y causes the decomposition of Me^PbX according t o [3] (=[2]+[l]) 2

2

3Me PbX + 2Me,Pb + PbX + MeX 3 4 2 0

0

[3]

The r e a c t i o n r a t e s o f [2a] and of [3] a r e a p p r e c i a b l y s m a l l e r than t h a t of [1] so the c o n c e n t r a t i o n of Me PbX« i s always s m a l l i n such s o l u t i o n s . [2b] i s much f a s t e r tnan f l ] . The r a t e law i s r a t h e r complicated, as d i s s o c i a t i o n and complex e q u i l i b r i a p l a y an important r o l e . The r a t e o f [3] i s increased i n the same way as mentioned f o r [ 1 ] , the dependency on the type of anion, however, being s m a l l e r . This can be explained by the f a c t t h a t the comproportionation [2b] i s i n f l u e n c e d by the same a c c e l e r a t i n g f a c t o r s . R e s u l t s of experiments on r e d i s t r i b u t i o n r e a c t i o n s of m e t h y l t h a l l i u m compounds s h a l l be discussed i n a subsequent chapter.


5. HUBER E T AL.

Organolead and Organothallium

67

T o x i c i t y of Organolead and Organothallium Compounds During our r e d i s t r i b u t i o n experiments, we became i n t e r e s t e d i n the t o x i c i t y o f organolead and o r g a n o t h a l l i u m compounds toward b a c t e r i a . The two t o p i c s , apparently having nothing t o do w i t h each o t h e r , f i n a l l y brought us t o study b i o m e t h y l a t i o n . Organolead Compounds. Organolead compounds act as b i o c i d e s (8, 9 ) ; i n the main, i n t e r e s t i n i n v e s t i g a t i o n o f t h i s property had been d i r e c t e d towards determining minimum concentrations necessary t o prevent m i c r o b i a l o r f u n g a l growth, and very few data are a v a i l a b l e on e f f e c t s o f s u b l e t h a l doses and on the chemical f a t e o f the organolead s p e c i e s . Even l e s s was known regarding the t o x i c i t y o f o r g a n o t h a l l i u m compounds. For our measurement C u l t u r e media were prepared i n BOD b o t t l e s . Lead compounds and n u t r i e n t s (peptone, yeast e x t r a c t ) d i s s o l v e d i n d i l u t i o n water (10, 11) were p i p e t t e d i n t o the f l a s k s , simultaneously i n o c u l a t e d w i t h 10 m l o f aquarium water ( c e l l d e n s i t y 10^ - 10-* c e l l s / m L ; from an aerated aquarium, which was repeatedly i n o c u l a t e d w i t h s u r f a c e water from a freshwater l a k e ) , f i l l e d w i t h d i l u t i o n water to the top, and s e a l e d . The b o t t l e s stood i n the dark a t 20 C. R e s u l t s o f the BOD- and BOD measurements are l i s t e d i n t a b l e I . I n other experiments, d i s s o l v e d 0 was analyzed c o n t i n u o u s l y w i t h an oxygen e l e c t r o d e . As growth parameters we determined the

Table I . Dependency o f I n h i b i t i o n o f B a c t e r i a l Growth a f t e r 5 Days (or 24 h*) on Type o f Lead Compound Added t o C u l t u r e ( N u t r i e n t : peptone, 5 mg/L; i n 24 h experiments 15 mg/L) Compound

N.I. T.I. [mg Pb/L]

Compound

Me^PbCl Me PbCl Et^PbOAc

1 0.5 % 1 0.05

Et PbCl Et PbCl

°· * 0.01

Bu Pb0Ac J Bu Pb(0Ac) PluPbCl PtuPbCl, Pb6l

3

2

2

(a)

o

1

?

0 1

2

10

2

Me - CH , E t = C H , Bu = n - C ^ , 3

2

2

5

N.I. [mg * 0.05 0.1 0.01 0.01 0.1 1

T.I. Pb/L] * 5 10 5-8 10 (a) 50

Ph = C ^ , OAc = CH C00 3

N.I. = no i n h i b i t i o n , T.I. = t o t a l i n h i b i t i o n (a) No t o t a l i n h i b i t i o n i n s a t u r a t e d s o l u t i o n o f organolead s a l t ( S o l u b i l i t i e s a t 20°C: Me PbCl ça. 130 g Pb/L, Ph PbCl ça. 0.5 g Pb/L) 3


68

ORGANOMETALS AND

ORGANOMETALLOIDS

d u r a t i o n of the i n i t i a l l a g phase and the slope of the 0 consumption r a t e during the beginning of the l o g phase, l i n e a r l y approximated. A t y p i c a l set of growth curves i s shown i n F i g . 1; data of growth parameters at v a r i o u s l e a d and n u t r i e n t concentrations are given i n t a b l e I I . 9

At comparatively low l e a d concentrations the l a g phase i s prolonged, w h i l e the l o g phase seems to be u n a f f e c t e d ; the c e l l count of 10 - l C r cells/mL a f t e r 24 h i s equal t o Pb-free samples. At medium l e a d c o n c e n t r a t i o n s , the l a g phase i s more prolonged; the l o g phase slope decreases and up t o 20% n e c r o t i c c e l l s are observed a f t e r 24 h. I t i s important t o note t h a t the l a g phase sometimes i s so prolonged, t h a t short term BOD values would I n d i c a t e t o t a l i n h i b i t i o n exponential acceleratio to about 10"* cells/mL (ca. 50% n e c r o t i c ) and decreases a f t e r about 2 days. 2+ The measurements show t h a t Pb i s l e s s t o x i c than organol e a d compounds and t h a t , i n g e n e r a l , R^PbX^ compounds are more t o x i c than R^PbX compounds, though there are e x c e p t i o n s . The e f f e c t of Me PbCl i s a s t o n i s h i n g l y s m a l l , compared w i t h t h a t of other compounds. Tetraorganoleads are more t o x i c than R-PbX or R^PbX^; the corresponding e f f e c t s on b a c t e r i a l growth were observed at concentrations one or two orders of magnitude lower ( i n the case of P b , at concentrations one order of magnitude h i g h e r ) . The r u l e that the t o x i c i t y of organolead compounds i n c r e a s e s w i t h i n c r e a s i n g c h a i n lengths of R (8) proved not to be t r u e under a l l c o n d i t i o n s ; apparently n u t r i e n t and a l s o organol e a d concentrations are of a p p r e c i a b l e i n f l u e n c e (see t a b l e I I ) . 2 +

Organothallium Compounds. The r e s u l t s of the measurements of the t o x i c i t y of T l compounds showed a s t o n i s h i n g d i f f e r e n c e s compared to the r e s u l t s w i t h Pb compounds. T l i s more t o x i c than the R^TIX compounds i n v e s t i g a t e d ; the s m a l l e s t c o n c e n t r a t i o n at which i n h i b i t i o n was observed i s 0.01 mg T l /L, t o t a l i n h i b i t i o n was found at 1000 mg T l /L. As F i g . 2 shows, the i n h i b i t i o n of b a c t e r i a l growth i n v a r i o u s cases was greater at lower R T1X c o n c e n t r a t i o n s , and there a l s o i s no s t r a i g h t f o r w a r d r e l a t i o n s h i p of c h a i n length of R and t o x i c i t y . 2

2+ Biomethylation of Pb

and Organolead Compounds.

Considering the f a c t s t h a t organolead compounds r e d i s t r i b u t e i n s o l u t i o n and t h a t d i f f e r e n t species show d i f f e r e n t t o x i c i t i e s , i t was convient to study the i n f l u e n c e of r e d i s t r i b u t i o n on the t o x i c i t y and v i c e v e r s a t o c o n t r o l the s t o i c h i o m e t r y of the r e d i s t r i b u t i o n reactions i n a nonsterile solution.


HUBER E T AL.


mg CWt 1 g peptone/l

Figure 1, Typical growth curves at various Et PbCl concentra tions showing Ο consumption as a function of time a

g

lagphase [h] 20

(Nutrient

cone

: 1g/l

)

/ /

15 /

/

I 10 n-Prop

/

Me/ i-Prop

/ /

01

1

10

100

C o n c e n t r a t i o n of R T l 2

Figure 2.

+

1000

Img/l]

Inhibition of bacterial growth by diorganothallium compounds


70


TABLE I I . I n h i b i t i o n of B a c t e r i a l Growth by Organolead Compounds at Various Lead and N u t r i e n t Concentrations Concentr. a

3 PbCl lag log M e

b

Pb >

Nu >

0 0 0.1 0.1 1 1 10 10 100 100

0.2 1 0.2 1 0.2 1 0.2 1 0.2 1

7 5 7

2.9 3.7 3.4

12

2.9

80 30

0. 0.8

Me PbCl 2

lag

Et PbCl

2

log

7 5 20 10 45 23

2.9 3.7 2.4 2.2 2.7 2.6

95

0.4

Et : P b C l

3

lag 6 6.5 10 6.5 13 12 25

2

log 1.3 2.4 0.5 2.4 0.36 1.1 0.25

2

lag

log

6 6.5 18 8 30 15 50

1.3 2.4 1.4 0.84 0.23 0.64 0.14

0.06

0.05

l a g = p e r i o d of l a g phase ( h ) ; l o g = slope of l o g phase (mg 0 /L-h) 9

a) b)

Concentration of l e a d compound (mg Pb/L) Concentration o f n u t r i e n t (g/L)

In no case was there a n o t i c e a b l e e f f e c t o f r e d i s t r i b u t i o n on the growth curves, and w i t h r a t h e r low c e l l d e n s i t i e s no remark a b l e i n f l u e n c e on r e d i s t r i b u t i o n was observed. But i n c o n t i n u a l l y aerated c u l t u r e s w i t h h i g h e r c e l l d e n s i t i e s , e.g., correspond ing t o those i n the a c t i v a t e d sludge o f a sewage p l a n t , a very a p p r e c i a b l e i n c r e a s e i n the r e d i s t r i b u t i o n r a t e was observed. T h i s was accompanied by o x i d a t i v e degradation o f 50 - 60% o f the added methyllead s p e c i e s t o Pb . T h i s could be i n t e r p r e t e d as a detox i f i c a t i o n by the b a c t e r i a , as Pb i s l e s s t o x i c than the added Me^PbX o r Me PbX . Under discontinuous c o n d i t i o n s ( i n BOD f l a s k s ) the o x i d a t i v e degradation amounted t o about 15 - 20%. 2

2

2+ B i o m e t h y l a t i o n of Pb . When we f o l l o w e d t h e r e d i s t r i b u t i o n of Me^PbX i n anaerobic c u l t u r e s ( b a c t e r i a from the s u r f a c e of a n a t u r a l l a k e , grown under N2, o r from the anaerobic sedimen^ of a s m a l l pond), we a l s o observed a r a t e i n c r e a s e , but l e s s Pb and more Me,Pb than were expected from the s t o i c h i o m e t r y of equation [31.

2

+

I n f e r r i n e from the d e f i c i t of Pb and the e x t r a amount o f Me^b t h a t Pb might have been methylated by b a c t e r i a , we added Pb [as P b ( 0 A c ) J t o anaerobic c u l t u r e s i n gas wash b o t t l e s and c u l t i v a t e d these c u l t u r e s under N i n t h e dark a t 30 C. A f t e r 7 to 14 days v o l a t i l e products were f l u s h e d w i t h N i n t o a 0.2 Ν methanolic I scrubber s o l u t i o n . Ten mL ΚΙ·Ι s o l u t i o n were added t o ensure q u a n t i t a t i v e t r a n s f o r m a t i o n o f organolead species 2 +

2

2

2

2


5.

HUBER E T A L .


71

toPb .The l a t t e r was determined p h o t o m e t r i c a l l y a f t e r r e d u c t i o n of I w i t h Na«SO« i n ammoniacal b u f f e r s o l u t i o n and a f t e r complexa t i o n w i t h PAR [ 4 - ( 2 - p y r i d y l a z o ) - r e s o r c i n o l ] (12, 13). A blank and a l s o a s t e r i l e s o l u t i o n c o n t a i n i n g Pb o r methyllead compounds showed no Pb content i n the methanolic s o l u t i o n a f t e r the same treatment. We t h e r e f o r e concluded t h a t Me/Pb was the v o l a t i l e species produced i n the b i o m e t h y l a t i o n of P b by b a c t e r i a (14). We could f u r t h e r prove the i d e n t i t y of Me^Pb i n the head-space gas above the c u l t u r e s by GC a n a l y s i s . The production r a t e of Me^Pb was about 2.5 yg Pb/d. 2

9 + -

2 +

2+ The b i o m e t h y l a t i o n of Pb proceeded r e p r o d u c i b l y , provided a) the P b c o n c e n t r a t i o n was c o n t r o l l e d ( t a k i n g i n t o c o n s i d e r a t i o n the r e s u l t s of the t o x i c i t y measurements) b) the concentrat i o n of s u l f u r compound wise ELjS produced by th a c o n c e n t r a t i o n o f P b which i s too s m a l l f o r the generation of d e t e c t a b l e amounts of Me^Pb), c ) the inoculum was not more than 6 - 7 weeks o l d . Optimum r e s u l t s were obtained w i t h glucose and urea o r amino a c i d s as n u t r i e n t s (supply of s u l f u r i s maintained by SO," i n the d i l u t i o n water) and a t concentrations of 1 - 10 yg Pb2+ 7mL. Only s l i g h t l y s m a l l e r production r a t e s were u s u a l l y observed u s i n g concentrations o f ca. 100 yg P b /mL [ca. 15 mg Pb(OAc) /100 mL]. I t was f a v o r a b l e t o add CaCO^ t o avoid greater decrease of pH. 2 +

2 +

2 +

2

Biomethylation o f Me PbX. On a d d i t i o n of Me PbX t o the ana e r o b i c c u l t u r e s , the Me.Pb p r o d u c t i o n was much higher than from c u l t u r e s c o n t a i n i n g P b , and a l s o higher than from the r e d i s t r i b u t i o n of Me^PbX i n s t e r i l e s o l u t i o n s . T h i s i n d i c a t e d a h i g h prop o r t i o n o f Me^Pb production by chemical r e d i s t r i b u t i o n . A f t e r we had obtained these r e s u l t s , Wong, Chau and Luxon reported (15) that they had detected Me.Pb above the sediment o f a l a k e , and that a d d i t i o n o f MeaPbOAc and, i n some cases, of P b ( N 0 ) o r PbC^, increased Me^Pb p r o d u c t i o n ; pure species of b a c t e r i a l i s o l a t e s , however, were not able t o produce Me.Pb from PbX«. Another paper presented a c o n t r o v e r s i a l e x p l a n a t i o n denying a l k y l a t i o n o f P b by microorganisms (16): Me.Pb and Et.Pb (Et = CJH^) should be products of chemical a l k y l a t i o n of Me PbOAc and Et PbOAc, r e s p e c t i v e l y , i n an anaerobic sediment system. As a p o s s i b l e mechanism, i n i t i a l formation of ( R P b ) S f o l l o w e d by decomposition t o g i v e R^Pb as one product was proposed. 3

3

2 +

3

9

2

3

3

2

Our r e s u l t s e x p l a i n the observations u n e q u i v o c a l l y (see f i g u r e 3); i n an anaerobic b a c t e r i a l c u l t u r e , P b i s methylated t o g i v e Me.Pb ( r e a c t i o n [4] i n F i g . 3 ) . Since a l k y H e a d compounds r e d i s t r i b u t e i n aqueous s o l u t i o n according t o [1] and [2] t o g i v e Me^Pb and P b , i n a b a c t e r i a l c u l t u r e both chemical and b i o l o g i c a l production o f Me,Pb occurs. I n media c o n t a i n i n g h i g h concen2 +

2 +


72


t r a t i o n s o f s u l f u r compounds (which was the case i n the work of r e f . [16]), r e d i s t r i b u t i o n o f ILPbX proceeds r a t h e r f a s t , as s u l f i d e present has a h i g h p o l a r i z a b i l i t y ; P b , though produced s i multaneously i n a p p r e c i a b l e amounts, i s p r e c i p i t a t e d as PbS. Therefore, t h e amount o f Me^Pb produced by b i o m e t h y l a t i o n o n l y can be r a t h e r low i n such systems, and the Me^Pb p r o d u c t i o n e s s e n t i a l l y i s caused by chemical r e d i s t r i b u t i o n . 2 +

A rough estimate o f the r a t i o o f c h e m i c a l l y and b i o l o g i c a l l y produced Me^Pb from our c u l t u r e s (100 mL s o l u t i o n , c o n t a i n i n g 200 mg g l u c o s e , 10 mg urea; 56 mg Me^PbCl were added a f t e r 1 week o f i n c u b a t i o n ) i s p o s s i b l e by comparing concentrations o f Me^Pb, Me^PbX and PbX^, which were a) analyzed and b) c a l c u l a t e d from equations [2] and [ 3 ] : a f t e r 7 days we found 5.3 ymol Me^Pb; i n the s o l u t i o n 0.3 ymol Me Pb * analyzed (13) which a c c o r d i n t o [2] correspond t o 0. 2.1 ymol P b , which a c c o r d i n g [3] correspon amount o f 4.2 ymol Me^Pb produced c h e m i c a l l y . (The sediment cont a i n e d no d e t e c t a b l e amount of Pb.) The d i f f e r e n c e between the analyzed and the c a l c u l a t e d amount of Me^Pb i s 0.8 ymol. T h i s amount, however, must s t i l l be c o r r e c t e d i n two r e s p e c t s . Any Pb transformed b i o l o g i c a l l y t o Me,Pb was produced c h e m i c a l l y t o gether w i t h Me^Pb; on the other hand, one has t o make allowance f o r a c e r t a i n amount of Me^Pb produced by immediate b i o m e t h y l a t i o n of Me^PbX (see below). We assume that these two q u a n t i t i e s a r e about s i m i l a r . One t h e r e f o r e can estimate that not more than 0.8 ymol Me^Pb have been produced b i o l o g i c a l l y a f t e r 7 days, i . e . , not more than about 16% of the t o t a l amount of Me^Pb found. From f o u r a d d i t i o n a l experiments we obtained s i m i l a r estimates of 15, 16, 17, and 19% f o r the p o r t i o n of Me,Pb which was produced b i o l o g i c a l l y . The p r o d u c t i o n r a t e o f Me,Pb from s t e r i l e n u t r i e n t s o l u t i o n s (100 mL s o l u t i o n , c o n t a i n i n g 200 mg g l u c o s e , 10 mg u r e a , amino a c i d s ; 0.1 and 10 mg Pb r e s p e c t i v e l y , added as MeJPbCl o r MeJPbCl ) a f t e r 7 days was 2.9 and 3.4 yg Pb/d (Me PbCl) and 3.1 and 3.3 Ug Pb/d ( M e P b C l ) . I n analogous experiments s i m i l a r r a t e s were observed; i n autoclaved c u l t u r e s maximum r a t e s o f 6 - 8 yg Pb/d have been measured. The p r o d u c t i o n of MeJPb from c u l t u r e s c o n t a i n i n g MeJPbCl (56 mg Me PbCl/100 mL = 40 mg Pb/100 mL) and MeJPbCl (60 mg Me PbCl /100 m l ) , r e s p e c t i v e l y , v a r i e d between 85 and 157 yg/d and between 45 and 124 yg/d. 24

2 +

2

3

2

2

3

2

2

2

Biomethylation of Et^PbX. To check whether R~PbX i s a l s o b i o methylated by anaerobes, we added E t ^ P b C l (up t o 100 mg Pb/L) t o a 4 L c u l t u r e and observed a maxmium p r o d u c t i o n r a t e of 500 yg Pb/d. As n u t r i e n t , a s o l u t i o n of 0.1 g NH,C1, 0.052 g K HP0,'3H 0, 0.1 g MgCl '6H 0, 1 g EtOH i n d i l u t i o n water (10, 11) w i t h added amino a c i d s was used. The gas above the c u l t u r e s o l u t i o n was s l o w l y passed (using a slow N stream) through petroleum e t h e r t o absorb the t e t r a a l k y l l e a d compounds. The s o l u t i o n , which was separated by GC on a Chromosorb column w i t h Apiezon (15%), contained b e s i d e s 2

2

2

2


2

73


HUBER E T A L .

5.

s o l v e n t Et^Pb (83%), Et MePb (13%) and Me,Pb ( 3 - 4 % ; t o t a l amount of R _ R P b « 100%). P r a c t i c a l l y , n e i t h e r Et Me Pb nor EtMe Pb were found. (The composition o f the products from other e x p e r i ments was d i f f e r e n t . ) To exclude the p o s s i b i l i t y t h a t any com pounds i n the c u l t u r e s c a t a l y z e the r e d i s t r i b u t i o n of mixtures of Et^Pb and Me^Pb, 0 5 ml o f these compounds were placed i n s t e r i l e n u t r i e n t s o l u t i o n s and autoclaved c u l t u r e s ; a f t e r 2 weeks no change i n the composition o f the R^Pb mixture had occurred. Ac c o r d i n g t o these r e s u l t s which are summarized i n f i g u r e 4, Et^PbX i s biomethylated d i r e c t l y t o Et^MePb. Et,Pb i s produced chemical l y by r e d i s t r i b u t i o n of Et^PbX; E t P b X , the other r e d i s t r i b u t i o n product which could not be detected (13) i n the f i l t e r e d s o l u t i o n , apparently r e d i s t r i b u t e s (according t o [1])so f a s t , that no appre c i a b l e amount i s a v a i l a b l e f o r b i o m e t h y l a t i o n . P b , one of the f i n a l products of r e d i s t r i b u t i o n Considering the d i f f e r e n Et^MePb and Me^Pb b i o l o g i c a l l y ) the percentage of the d i f f e r e n t t e t r a a l k y l l e a d species i n the mixture roughly corresponds t o the estimate of c h e m i c a l l y and b i o l o g i c a l l y produced Me^Pb from c u l t u r e s c o n t a i n i n g Me^PbX. 3

4

n

n

2

2

3

?

2

2

2 +

Biomethylation o f T l * . An i n t r i g u i n g problem r a i s e d by the d i s c o v e r y of b i o m e t h y l a t i o n of P b i s the i n c r e a s e of the formal o x i d a t i o n number of Pb during i t s b i o l o g i c a l t r a n s f o r m a t i o n t o Me^Pb. For t h i s and other obvious reasons we sought t o f i n d out whether T l , i s o e l e c t r o n i c w i t h P b , i s a l s o subject t o biomethyl a t i o n i n anaerobic mixed b a c t e r i a l c u l t u r e s , during which process i t too increases i t s formal o x i d a t i o n number. I n the l i t e r a t u r e there were no r e p o r t s on b i o m e t h y l a t i o n o f T l . Model experiments showed that methylcobalamin i s not demethylated by T1(I) (17), y e t i s demethylated by T l ( I I I ) (17, 18); from s p e c t r a l t i t r a t i o n s i t was concluded t h a t M e T l i s produced (19). 2 +

+

2 +

2 +

+

The experimental c o n d i t i o n s we used t o biomethylate T l essen t i a l l y corresponded t o those we had a p p l i e d d u r i n g b i o m e t h y l a t i o n of P b . I n 250 mL gas wash b o t t l e s we incubated three types of s o l u t i o n s which had been i n o c u l a t e d w i t h 5 g anaerobic sediment. The s o l u t i o n s were prepared from 100 ml d i l u t i o n water (10, 11) and contained 1 g peptone ( s o l u t i o n A) o r 0.1 g NH.Cl, 0.052 g Κ ΗΡ0 ·3Η 0, 0.01 g MgCl -6H 0 and e i t h e r 1.0 g C a ( 0 A c ) ( s o l u t i o n B) o r 1.0 g C j OH ( s o l u t i o n C). 1.9 g s o l i d CaC0 were added t o n e u t r a l i z e a c i d from metabolic processes. Starting at the t h i r d day of i n c u b a t i o n , 100 mg TlOAc were added during 7 days. A f t e r 2-3 weeks, M e 2 T l could be detected i n samples as w e l l as T l ; that M e 2 T l was the only methylated t h a l l i u m species found i s understandable i n view o f the normal behavior of a l k y l t h a l l i u m compounds: MeTlX compounds i n general are unstable or tend t o decompose i n aqueous s o l u t i o n (20, 21): ΜββΤΙ decomposes i n s t a n t a neously i n water t o form s t a b l e M e T l and methane ( 3 ) . To determine the amount of M e 2 T l produced by b i o m e t h y l a t i o n 2 +

2

4

2

2

2

2

3

+

+

+

2

+

2

+



j 4 Me PbX 3

JiL

2 Me2PbX

+

2

2 Me^Pb

!

Bacteria alkylate η 3 M^PbX

Figure S.

Et PbCl

*

(2*n) Me^Pb • (1-n)PbX + MeX 2

Sources of Me Fb in an anaerobic bacterial culture k

R E D I S T R I B U T I 0 N

3

•

PbCl

BIOMETHYLATION

Figure 4.

2

•

EtCl

Et^Pb

•

ELMePb

Biomethylation of Et PbCl 3


5.


HUBER E T A L .

75

+

of T l , 10 mL samples a f t e r f i l t e r i n g through a diaphragm were mixed w i t h EDTA t o mask T l . Then l - ( 2 - p y r i d y l a z o ) - 2 - n a p h t h o l (PAN) was added a t pH 10-12 t o complex M e T l , and a f t e r e x t r a c t i o n w i t h CHClg the Me Tl-PAN c o n c e n t r a t i o n was measured photomet r i c a l l y a t 570 nm. [Masking of T l i s necessary as T l forms a complex w i t h PAN (22) which a l s o absorbs a t 570 nm.] +

+

2

2

+

+

The r e s u l t s showed t h a t i n the c u l t u r e s o f s o l u t i o n s Β and C a f t e r 21 days, 10 yg and 36 yg Me Tl /mL, r e s p e c t i v e l y , had been formed. I n s o l u t i o n A only about 1 - 2 yg Me Tl /mL were detected a f t e r 21 days; a low y i e l d of methylated species from c u l t u r e s c o n t a i n i n g peptone as n u t r i e n t was a l s o observed i n b i o m e t h y l a t i o n experiments w i t h Pb^+. +

2

+

2

+

+

The observation tha T l i biomethylated T l unde i n crease of the o x i d a t i o i s not unique w i t h P b an i n c r e a s e i n o x i d a t i o n number occurs a l s o during b i o m e t h y l a t i o n of As compounds (23). 2 +

Mechanistic

Considerations

The o v e r a l l process of the i n v i v o methylation of metal ions i s c e r t a i n l y complex. Hence, use o f r e s u l t s of mechanistic i n v i t r o experiments t o p o s t u l a t e general i n v i v o mechanisms should be done only w i t h great care. Moreover, well-founded knowledge i s s t i l l too scarce t o a l l o w one of the v a r i o u s pathways which a r e conceivable f o r " o x i d a t i v e m e t h y l a t i o n " t o be favored. One might assume t h a t m e t h y l a t i o n i n v o l v e s methylcobalamin which conveys Me" t o an e l e c t r o p h i l i c t h a l l i u m o r l e a d s p e c i e s ; a p r i n c i p a l question i s then whether o x i d a t i o n occurs f i r s t f o l l o w e d by m e t h y l a t i o n , o r v i c e v e r s a ; the two steps could a l s o be s i m u l taneous. I t i s hard t o see which o x i d i z i n g agent could overcome the h i g h o x i d a t i o n p o t e n t i a l of T l and P b (24), and t h e r e f o r e i t seems reasonable t o t h i n k o f m e t h y l a t i o n as the f i r s t s t e p , p a r t i c u l a r l y s i n c e MePb was r e c e n t l y reported as a product of the r e a c t i o n of a dimethylcobalt complex and Pb (25). I n t h i s case the primary methylation product of P b and T l could d i s p r o p o r t i o n a t e t o g i v e M e P b o r M e T l and the element. Since we could not detect elementary Pb o r T l i n the samples, we conclude t h a t dimethyl species are not formed by d i s p r o p o r t i o n a t i o n of interme d i a t e MePb* o r MeTl. We t h e r e f o r e would p r e f e r t o assume that T l and P b , present i n a p p r o p r i a t e complexed form a t s p e c i f i c s i t e s of the c e l l , are simultaneously o x i d i z e d and methylated t o M e T l ( I I I ) and MePb(IV) m o i e t i e s which are s t a b i l i z e d by complexa t i o n . The importance and e f f e c t i v e n e s s of complexation f o r s t a b i l i z i n g unstable monoorganolead compounds i s known: Organolead t r i h a l i d e s , RPbX (Χ φ F ) , are unknown; however, i t i s p o s s i b l e t o prepare r a t h e r s t a b l e complex d e r i v a t i v e s M [PhPbX4] and +

2 +

+

+

2 +

2 +

2

+

+

2

+

2 +

3

f


76


M^[PhPbX ] (Μ' « P h P , Ph As; M" = Me^N; X = CI, Br) (26). 5

4

4

Another pathway might s t a r t w i t h an e l e c t r o p h i l i c a t t a c k by CK^ , which c o u l d e a s i l y s o l v e the problem of o x i d a t i o n . I n t h i s case the r e a c t i o n of the " o x i d i z i n g agent" CHL w i t h the metal i o n ( i n a s p e c i f i c a l l y complexed form, mainly w i t h negative l i g a n d s ) would l e a d f o r m a l l y t o M e T l o r MePb . These could d i s p r o p o r t i o n a t e , o r , being s t r o n g electrophilés, could be f u r t h e r methylated a t a s p e c i f i c CH "-conveying methylating s i t e of the c e l l system. +

2 +

Behavior o f MeTlX^ i n S o l u t i o n . I n order not t o end t h i s paper w i t h s p e c u l a t i o n , some r e a c t i o n s i n aqueous s o l u t i o n r e l e vant t o c o n s i d e r a t i o n s on methyl t r a n s f e r i n b i o l o g i c a l systems by r e d i s t r i b u t i o n w i l l be described Decomposition of MeTlXp Monomethylthalliu compound unstable i n aqueous s o l u t i o n . The decomposition of MeTl(OAc) f o l l o w s a f i r s t order r a t e law (X = OAc) (21) and produces, accordi n g t o the r e a c t i o n sequence [ 5 ] , 2

MeTl(OAc) + TlOAc + CH.COOCH. 9

1

CH COOCH + H 0 t CH COOH + GTÔH 3

3

2

J

3

TlOAc, CH COOCH , and, as products of h y d r o l y s i s of the l a t t e r , CH COOH and CH OH (21, 27). Based on k i n e t i c and conductometric data and by comparison w i t h data c a l c u l a t e d from d e r i v e d r a t e equ a t i o n s , a b i m o l e c u l a r S 2 mechanism w i t h a r a t e determining step of the a t t a c k o f OAc" a t the Tl-bonded Me-group of the very e l e c t r o p h i l i c MeTlOAc*" could be e s t a b l i s h e d (21). Other n u c l e o p h i l i c agents a l s o a t t a c k MeTl (OAc) and can thereby be methylated: pyri d i n e and other N-bases r e a c t w i t h MeTl (OAc) t o N-methylated compounds a c c o r d i n g t o [ 6 ] : 3

3

3

3

N

2

2

MeTl (OAc )

0 2

+ N^ t \

Met"" + OAc" + TlOAc X

[6]

Since the n u c l e o p h i l i c c h a r a c t e r o f these N-bases i s higher than that o f OAc"", CH COOCH i s formed only i n minor amounts (28, 2 9 ) . 3

3

In methanolic s o l u t i o n MeTl (OAc) decomposes over a p e r i o d of s e v e r a l weeks t o TlOAc and CH COOCH (28). I f t h i o a n i s o l e i s added, the decomposition i s complete i n 2-3 days. This " c a t a l y t i c e f f e c t " i s caused by a two-step r e a c t i o n (29). A t f i r s t , MeTl (OAc) methylates t h i o a n i s o l e according t o [ 7 ] : 2

3

3

2

MeSPh + MeT10Ac

+

ί

+

[ M e S P h ] + TlOAc

and i n the second s t e p , OAc according t o [ 8 ] :

2

i s methylated

[7] by S-methy 1 t h i o a n i s o l e


5.

HUBER E T AL.

Organolead and

+

[Me SPh] + OAc"

î

2

Organothallium

MeSPh + CH COOCH 3

77 [8]

3

T h i s behavior of MeTl(OAc) , which i s shown f o r o t h e r monomethylt h a l l i u m compounds (28, 30;, a l s o helps to e x p l a i n why experiments t o prepare MePbX were doomed t o f a i l u r e : MePb^ , being s t i l l more e l e c t r o p h i l i c than M e T l , methylates most e f f e c t i v e l y s o l vent molecules or/and anions and i s decomposed d u r i n g t h i s r e a c t i o n to P b ; t h i s r e a c t i v i t y i s h i g h l y promoted by the " i n e r t p a i r effect". 2

+

3

2 +

2 +

D i s p r o p o r t i o n a t i o n of MeTlX^. D i s p r o p o r t i o n a t i o n r e a c t i o n s of monoalkylthallium compounds have not yet been r e p o r t e d i n the l i t e r a t u r e . In s o l u t i o n s of MeTl (OAc) i n CD C0CD /CH 0H (5:1; composition chosen to optimize c o n d i t i o n s f o r NMR measurements), the e q u i l i b r i u m c o n c e n t r a t i o n disproportion a t i o n according to equatio 2

2 MeTl (OAc)

2

Ζ

3

Me Tl(0Ac) + T l ( 0 A c ) 2

3

3

[9]

3

are too s m a l l to be detected. However, i f one adds a reducing agent to remove Tl(0Ac)«, one gets q u a n t i t a t i v e t r a n s f o r m a t i o n of MeTl (OAc) to Me Tl(0AcJ (30) according t o [ 9 ] . The r e a c t i o n of T l ( 0 A c ) ~ and (Meu)^P proceeds according t o [10]: 2

2

T l ( 0 A c ) + (MeO) P + CH 0H 3

3

TlOAc + (Me0) P0 +

3

3

HOAc + CH COOCH 3

[10]

3

The r e a c t i o n products have been i d e n t i f i e d by NMR spectroscopy and by GC. Equations [9] and [10] add to g i v e the o v e r a l l r e a c t i o n [11]: 2 MeTl (OAc)

+ (Me0) P + CH 0H +

2

3

3

[ η ]

Me Tl(0Ac) + TlOAc + (MeO) PO + HOAc + CH C00CH 2

3

3

3

Reaction [10] i s instantaneous, w h i l e [11] i s complete o n l y a f t e r some minutes. We t h e r e f o r e conclude t h a t the exchange of the methyl groups i n the r e d i s t r i b u t i o n r e a c t i o n [9] i s r a t e determin ing. Regarding the behavior of MeTl (OAc ) i n s o l u t i o n s , and p r e supposing that M e T l i s an intermediate of b i o m e t h y l a t i o n of T l , one a r r i v e s a t an important c o n c l u s i o n concerning the open ques t i o n as t o which pathways might be of s i g n i f i c a n c e . There are i n p r i n c i p l e two p o s s i b i l i t i e s f o r the t r a n s f o r m a t i o n of M e T l to M e T l , one chemical and one b i o l o g i c a l (the v e r t i c a l and the h o r i zontal branch r e s p e c t i v e l y of the f o l l o w i n g scheme) i n which [12] and [13] f o r m a l l y symbolize the complexation of the monomethylt h a l l i u m species i n s o l u t i o n : 2

2 +

+

2 +

+


>

78

+ +Me+

π

Tl

-2

e"


+ Me"

MeTl2* •X"

+>

Me2Tl+

[12]

MeTlX* MeTlX

^113]

[5]

MeX • T l X

2+ If one assumes t h a t methylations o f MeTl and o f the i s o electronic H g (31) proceed at. a s i m i l a r r a t e , the chemical path way f o r the formation of Me«Tl ( r e d i s t r i b u t i o n according t o [ 9 ] ) _ has no a p p r e c i a b l e chance o f competing w i t h the b i o t r a n s f e r of Me (via methylcobalamin), s i n c e the r a t e of [9] i s comparatively s m a l l , even i f T l X ^ i s removed extremely r a p i d l y from the e q u i l i b rium. An eventual c o m p e t i t i o n of the decomposition [5] (being f a s t e r than the non-catalyzed r e a c t i o n [9]) and b i o m e t h y l a t i o n (presumably much f a s t e r than [5]) can be neglected j u s t as w e l l . So, according t o these c o n s i d e r a t i o n s f o r the t r a n s f o r m a t i o n of M e T l t o M e T l i n b a c t e r i a l c u l t u r e s , b i o l o g i c a l pathways should be favored over chemical ones. 2 +

2 +

+

2

Analogous c o n s i d e r a t i o n s apply f o r the d i s c u s s i o n of d i f f e r ent pathways of the b i o m e t h y l a t i o n of P b . Since M e P b i s s t i l l more e l e c t r o p h i l i c than MeTl +, the r a t e of i t s m e t h y l a t i o n of Me might be s t i l l h i g h e r , as might the r a t e o f i t s decomposition. One can t h e r e f o r e expect that the b i o m e t h y l a t i o n of P b i s a l s o , on the whole, a stepwise b i o l o g i c a l m e t h y l a t i o n i n v o l v i n g no chemical steps v i a d i s p r o p o r t i o n a t i o n r e a c t i o n s . 2 +

3+

2

2 +

+ 2+ S i g n i f i c a n c e of B i o m e t h y l a t i o n o f T l and Pb +

+

Me«Tl compounds are l e s s t o x i c t o b a c t e r i a than T l , so b i o m e t h y l a t i o n of T l appears as a d e t o x i f i c a t i o n of the b a c t e r i a l environment (but only i n a r e l a t i v e sense, s i n c e T l remains i n the s o l u t i o n ) . This aspect, however, must not of n e c e s s i t y be the determining reason f o r the occurrence of m e t h y l a t i o n , as biometh y l a t i o n of P b leads t o Me^Pb, which i s much more t o x i c t o bac t e r i a l c u l t u r e s than P b . However, the s o l u b i l i t y o f Me^Pb i n water i s extremely low, and Pb i s t h e r e f o r e f i n a l l y removed from +

2 +

2 +


5.

HUBER ET AL.


79

the s o l u t i o n . Concerning c o n c l u s i o n s on the e c o l o g i c a l s i g n i f i c a n c e of the b i o m e t h y l a t i o n of T l and P b . one should remember that biomethy l a t i o n only has been observed i n an anaerobic medium, and that r e s u l t s on t h i s r e a c t i o n and those on t o x i c i t y of T l and Pb compounds were obtained from l a b o r a t o r y experiments and should be t r a n s f e r r e d to n a t u r a l c o n d i t i o n s o n l y w i t h great care and not without s p e c i f i c experimental examination. N e v e r t h e l e s s , one should be a l e r t t o (and i n v e s t i g a t e ) the p o s s i b i l i t y that P b ^ and T l , i n anaerobic regions of metal-contaminated n a t u r a l w a t e r s , are methylated and are t r a n s p o r t e d as o r g a n o m e t a l l i c compounds to other regions of the n a t u r a l system, there showing the d i f f e r e n t behavior of o r g a n o m e t a l l i c species or being reconverted t o P b ^ and Tl . +

2 +

+

+

+

+

Acknowledgement The support of t h i s work by Deutsche Forschungsgemeinschaft and Herbert-Quandt-Stiftung i s g r e a t f u l l y acknowledged. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

S h a p i r o , H., F r e y , F . W . , "The Organic Compounds of Lead", W i l e y - I n t e r s c i e n c e , New Y o r k , 1968. M ü l l e r , R . , R e i c h e l , S . , Dathe, C., I n o r g . N u c l . Chem. L e t t e r s (1967) 3, 125. H a r t , C . R . , I n g o l d , C.K., J. Chem. Soc. (1964) 4372 Haupt, H.-J., Huber, F., Gmehling, J., Z. anorg. allg. Chem. (1972) 390, 3 1 . Mammi, M., Busetti, V., D e l P r a , Α . , I n o r g . Chim. A c t a (1967) 1, 419. P r e u t , H., Huber, F., Z. anorg. allg. Chem. (1977) 435, 234. S t a f f o r d , S . , Haupt, H.-J., Huber, F., I n o r g . Chim. A c t a (1974) 1 1 , 207. Van der Kerk, G.J.M., B i o d e t e r i o r . M a t e r . , P r o c . I n t e r n a t . B i o d e t e r i o r . Symp. (1971, p u b l . 1972) 1. Lorenz, J., B i o d e t e r i o r . M a t e r . , P r o c . I n t e r n a t . B i o d e t e r i o r . Symp. (1971, p u b l . 1972) 443. Standard Methods f o r the Examination of Water and Wastewater, 13th ed., 489 ( e d i t , by APHA, AWWA, WPCF, Washington, 1971) Deutsche E i n h e i t s v e r f a h r e n zur Wasseruntersuchung, H 5, 6; L 12 ( e d i t , by GDCh, Fachgruppe Wasserchemie, V e r l a g Chemie, Weinheim, 1972) D a g n a l l , R . M . , West, T.S., Young, P., T a l a n t a (1965) 12, 583. Schmidt, U., Huber, F., A n a l . Chim. Acta (1978) 98, 147. Schmidt, U., Huber, F., Nature (1976) 259, 157. Wong, P.T.S., Chau, Y.K., Luxon, P.L., Nature (1975) 253, 263.


80 16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.


J a r v i e , A.W.P., M a r k a l l , R.M., P o t t e r , H.R., Nature (1975) 255, 217. Agnes, G., Bendle, S., Hill, H.A.O., W i l l i a m s , F.R., W i l l i a m s , R.J.P., Chem. Comm. (1971) 850. Agnes, G., Hill, H.A.O., Pratt, J.M., R i d s d a l e , S.C., Kennedy, F.S., W i l l i a m s , R.J.P., Biochim. Biophys. A c t a (1971) 252, 207. Abley, P., Dockal, E.R., Halpern, J., J. Amer. Chem. Soc. (1973) 95, 3166. Lee, A.G. "The Chemistry o f T h a l l i u m " , Elsevier, Amsterdam, London, New York, 1971. P o h l , U., Huber, F., J. Organometal. Chem. (1976) 116, 141. Cheng, K.L., Bray, R.H., A n a l . Chem. (1955) 27, 782. C h a l l e n g e r , F., Chem. Rev. (1945) 36, 315. R i d l e y , W.P., D i z i k e s 197, 329. Witman, M.W., Weber, J.H., Inorg. Chem. (1976) 15, 2375. Lindemann, Η., Huber, F., Z. anorg. allg. Chem. (1972) 394, 101. Kurosawa, H., Okawara, R., J. Organometal. Chem. (1967) 10, 211. P o h l , U., Huber, F., J. Organometal. Chem. (1977) 135, 301. K n i p s , U., Huber, F., unpublished r e s u l t s . P o h l , U., Huber, F., unpublished r e s u l t s . DeSimone, R.E., Penley, M.W., Charbonneau, L., Smith, S.G., Wood, J.M., Hill, H.A.O., Pratt, J.M., R i d s d a l e , S., W i l l i a m s , R.J.P., Biochim. Biophys. A c t a (1973) 304, 851.

Discussion J . H. WEBER ( U n i v e r s i t y o f New Hampshire): Concerning some of t h e products you got i n your r e a c t i o n s , p a r t i c u l a r l y w i t h the m i c r o b i o l o g i c a l a l k y l a t i o n o f l e a d , i f you a l k y l a t e d l e a d ( I I ) you could have an u n s t a b l e species o f d i m e t h y l l e a d ( I I ) . This i s not a well-known species and i s considered a t r a n s i e n t s p e c i e s which de composes t o t e t r a m e t h y l l e a d and l e a d metal. Have you thought o f t h i s p o s s i b i l i t y and looked f o r l e a d metal i n your r e a c t i o n s ? HUBER: Yes, we d i d and we d i d not f i n d l e a d . d i d not f i n d m e t a l l i c t h a l l i u m .

S i m i l a r l y , we

J . M. WOOD ( U n i v e r s i t y o f Minnesota): Do you assume 10 days i s a good time f o r steady s t a t e when you determine how much i s b i o l o g i c a l and how much i s chemical d i s p r o p o r t i o n a t i o n ? HUBER: I t i s a time which g i v e s r e p r o d u c i b l e r e s u l t s . Ex periments over a longer p e r i o d were s i m i l a r , so we chose t h e s h o r t e r time.


5.

HUBER E T A L .


81

WOOD: The microbes were i n the s t a t i o n a r y phase, and they were j u s t s i t t i n g there when you had these d i f f e r e n t chemical species i n s o l u t i o n a t the time you d i d the analyses? [HUBER: Y e s ] . Now, I t h i n k i t i s c r i t i c a l l y important t o s t a r t i s o t o p e experiments w i t h C-14 l a b e l s , probably C - 1 4 - l a b e l l e d methionine which should get i n t o the c e l l s . This w i l l e s t a b l i s h what s o r t o f methyl t r a n s f e r i s o c c u r r i n g i n these systems. I have a d i f f i c u l t y i n t r y i n g t o r a t i o n a l i z e a mechanism without knowing what the b i o l o g i c a l methyl donor i s i n the system. Do you p l a n t o do some i s o t o p e experiments and f i n d out? HUBER: Yes, we p l a n t o do so. We are s t u d y i n g the t h a l l i u m compound. C u r r e n t l y we are u s i n g methylcobaloxime and methylcobalamin t o see i f m e t h y l a t i o n occurs w i t h a d d i t i o n of an oxidant f o r the P b ^ and Sn^ . W negative r e s u l t s w i t h P +

+

WOOD: When you add t h a l l i u m ( I ) t o a complex system, have you any i d e a what the o x i d a t i o n s t a t e i s o f the a c t i v e t h a l l i u m species? We can r a t i o n a l i z e methyl t r a n s f e r of t h a l l i u m ( I I I ) . I n f a c t the r e a c t i o n goes q u i t e w e l l . T h a l l i u m ( I ) i s unusual; i f you look a t the standard r e d u c t i o n p o t e n t i a l idea f o r t h a l l i u m ( I I I ) t o t h a l l i u m ( 1 } , i t i s f a i r l y h i g h . I f , f o r example, methyl Β were i n v o l v e d i n your r e a c t i o n c o n d i t i o n s , I'm sure t h a t c o n d i t i o n s a r e so extreme t h a t the o x i d i z i n g agent would c e r t a i n l y break the cobalt-carbon bond anyway. Therefore, there i s a r e a l d i f f i c u l t y w i t h a CHg suggestion. 2

HUBER: I f we s t a r t w i t h t h a l l i u m ( I ) we cannot p o s t u l a t e t h a t we have an o x i d a t i o n i n the f i r s t p l a c e f o l l o w e d by m e t h y l a t i o n , because the o x i d a t i o n would g i v e T l ( I I I ) , and T l ( I I I ) would o x i d i z e the methylcobalt compound. Therefore, we have some k i n d o f simultaneous methylation and o x i d a t i o n , where two e l e c t r o n s a r e taken away and some k i n d of CH^~ r e s u l t s t o g i v e MeTl^"*". We have attempted t o r a t i o n a l i z e t h i s p o s s i b i l i t y i n a way which i s con s i s t e n t w i t h our experimental r e s u l t s , as shown i n the Scheme on page 12. RECEIVED

August 22,

1978.


6 Bioorganotin Chemistry: Stereo- and Situselectivity in the Monooxygenase Enzyme Reactions of Cyclohexyltin Compounds RICHARD H. FISH, JOHN E. CASIDA, and ELLA C. KIMMEL Pesticide Chemistry and Toxicology Laboratory, College of Natural Resources, Wellman Hall, University of California, Berkeley, CA 94720 The in vitro reaction oxygenase enzymes utilizin studied with tributyltin derivatives (1a,b). The results from that study confirmed carbon-hydroxylation as the primary bio chemical reaction occurring with these compounds. Furthermore, the tin-carbon sigma electrons were implicated in the possible stabilization of carbon free radicals generated on the α and β carbon atoms to the tin atom. Additionally, by using several criteria, we established that the metabolism of these tributyltin derivatives involved the interesting and biologically important cytochrome P-450 dependent monooxygenase enzyme system (1a). Recent studies on this system have concluded that a l l the available evidence points to a heme-iron-monooxygen complex which converts carbon-hydrogen bonds to carbon-hydroxyl bonds (2), Figure 1. The reaction has been shown to be highly stereospecific 3+ 3+ Fe

+

RH

- R H

>**' .RH

3+

2+ 2+

Fe " 1

[+o

- R O H

+ Fe:= Q 1 i

RH

2

2+ Fe0

+2H+ -H 0

_RH

2

2

RH

Figure 1. Mechanism of Cytochrome P-450 en zyme hydroxyhtion reaction

(3); however, surprisingly few investigations have been concerned with the cyclohexyl ring system (4). Our work i n this area was logically extended to cyclohexyltin compounds for several reasons. F i r s t l y , these compounds are used as agricultural miticides on 0-8412-0461-6/78/47-082-082$05.00/0 © 1978 American Chemical Society In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

6.

FISH E T A L .

Bioorganotin Chemistry

83

food crops and a study o f t h e i r monooxygenase enzyme r e a c t i o n s would be important f o r b i o l o g i c a l and t o x i c o l o g i c a l reasons. Secondly, we wanted t o a s c e r t a i n the s t e r e o as w e l l as s i t u s e l e c t i v i t i e s o f these r e a c t i o n s w i t h a p p r o p r i a t e c y c l o h e x y l t i n model compounds, s i n c e t h i s aspect, as f a r as we c o u l d e s t a b l i s h w i t h t h e cytochrome P-U50 monooxygenase enzyme system, has n o t been e l u c i d a t e d i n a d e f i n i t i v e manner. We decided i n i t i a l l y (j?) t o study c y c l o h e x y l t r i p h e n y l t i n , ^ because we found i n an e a r l i e r i n v e s t i g a t i o n ( l b ) t h a t the t r i p h e n y l t i n d e r i v a t i v e s were not h y d r o x y l a t e d under i n v i t r o r e a c t i o n c o n d i t i o n s . More i m p o r t a n t l y , the s y n t h e s i s o f potent i a l m e t a b o l i t e s , which w i t h the c y c l o h e x y l system i n v o l v e s t h e c i s - and t r a n s - h y d r o x y c y c l o h e x y l t i n isomers i n the 2,3> and *Jp o s i t i o n s ( t r i p h e n y l t i n being p o s i t i o n l ) , would be more conveni e n t u s i n g the t r i p h e n y l t i A d d i t i o n a l l y , the e x t e n s i o J^, would o n l y i n v o l v e e l e c t r o p h i l i c cleavage o f a phenyl group i n order t o prepare p o t e n t i a l m e t a b o l i t e s f o r t h i s model substrate ( 7 ) . A d i s c u s s i o n o f the s t e r e o and s i t u s e l e c t i v i t y i n v o l v e d i n the P-lj-50 monooxygenase r e a c t i o n s o f 1 and and the consequence o f t h e i r conformation a t the a c t i v e sTte w i l l be presented. Results The p r e p a r a t i o n o f [ l - ^ C ] c y c l o h e x y l t r i p h e n y l t i n , j ^ , was r e a d i l y accomplished by the r e a c t i o n o f [ l - ^ l e y c l o h e x y l magnesium bromide w i t h t r i p h e n y l t i n c h l o r i d e i n 3 3 $ y i e l d w i t h a s p e c i f i c a c t i v i t y o f 1 . 2 6 mCi/mmole (Eq l ) . The use o f [ C ] 1 4

h /

-

=

y f ^ B r

h»

1) Mg/THF 2>(©)snCI

/

^

- ^ 7 ^ S n ( ^

(1) 1

l a b e l l e d o r g a n o t i n s u b s t r a t e s i s mandatory i n these metabolism s t u d i e s , s i n c e the amount of metabolism i s g e n e r a l l y t o t h e extent o f 10% o f t h e s t a r t i n g r a d i o l a b e l l e d s u b s t r a t e . Thus, compound 1 ( 0 . 0 5 pmole) was incubated w i t h our source o f c y t o chrome P - Ç 5 0 , r a t l i v e r microsomes ( l a ) ( 1 0 . 6 mg p r o t e i n ) , f o r 1 h r a t 3 7 ° i n 2 . 0 ml phosphate b u f f e r (pH 7·*0 c o n t a i n i n g ΝΑΌΡΗ (2 Mmole) the e s s e n t i a l c o f a c t o r . A f t e r chloroform e x t r a c t i o n , we u t i l i z e d t h i n l a y e r chromatography (TLC) t o separate the m e t a b o l i t e s and l i q u i d s c i n t i l l a t i o n counting t o q u a n t i f y them. They were i d e n t i f i e d by a combination o f TLC cochromatography, p r e p a r a t i v e TLC i n c o n j u n c t i o n w i t h 360 MHz % FT nmr s p e c t r o scopy and by s p e c i f i c degradation r e a c t i o n s (Eq 2 ) . The


84

ORGANOMETALS

•Sn(®)

pH 7.4,

Ih


3

H

I

g (85.6%)

(2) 3 (6.5%)

4 (3.0%)

•Sn(®)

6 (1.9%)

3

+

5 (1.6%)

/ ^ ^ S n ( © )

3

7 (1.4%)

percentages i n Eq 2 represen m e t a b o l i t e s and account f o r 8 $ o f s t a r t i n g s u b s t r a t e , ^ The remainder was 1 ( 7 0 $ ) and u n i d e n t i f i e d m a t e r i a l s ( 2 2 $ ) . One important aspect of t h i s type o f work i s the a b i l i t y t o synthesize p o t e n t i a l m e t a b o l i t e s and t o understand t h e i r subsequent chemistry. This f a c i l i t a t e s t h e i r u l t i m a t e i d e n t i f i c a t i o n and allows t h e use o f cochromatography as one c r i t e r i o n f o r t h i s purpose. A l l the m e t a b o l i t e s , 2,-£, were separated from one another u s i n g n e u t r a l TLC s o l v e n t systems (5) and then cochromatographed w i t h s y n t h e t i c standards ( 6 j j ) . Furthermore, compound 2 was p u r i f i e d by p r e p a r a t i v e TLC and a 360 MHz % FT nmr spectrum was obtained ( 1 3 , 6 0 0 acquisitions,CDC1-> TMS) conf i r m i n g t h a t the major m e t a b o l i t e ( 8 5 . 6 $ ) was trans-4-hydroxyc y c l o h e x y l t r i p h e n y l t i n , 2 . The nmr spectrum showed t h e a x i a l methine proton on the carbon (ck) bearing the h y d r o x y l group as a 9 u n e m u l t i p l e t ( 3 . 5 8 ppm, J - J = 11.1 Hz; J - J = h.O Hz) c o n s i s t e n t w i t h a spectrum or the a u t h e n t i c compound 2 ( 6 , 7 ) · We were a b l e t o detect the corresponding c i s isomer o f ^15y t h i s nmr technique; however, none was observed i n the nmr spectrum of metabolite 2 . The c i l f - and trans-3-hydroxy m e t a b o l i t e s , ^ and j ^ , were i d e n t i f i e d o n l y by TLC cochromatography w i t h a u t h e n t i c compounds, because o f the low amounts produced i n the b i o l o g i c a l o x i d a t i o n r e a c t i o n . I n t h i s regard, we are c o n f i d e n t o f t h e i r assigned s t r u c t u r e s , s i n c e both isomers are r e a d i l y separable by t h i s technique. M e t a b o l i t e 5 was i d e n t i f i e d by TLC cochromatography and by a s p e c i f i c degradation r e a c t i o n . The m e t a b o l i t e mixture was a c i d i f i e d w i t h g l a c i a l a c e t i c a c i d and TLC a n a l y s i s showed the disappearance o f m e t a b o l i t e 5 . Experiments w i t h a u t h e n t i c ^ r e v e a l e d t h a t t h i s trans-2-Kydroxy compound undergoes a f a c i l e 1 , 2 - d e o x y s t a n n y l a t i o n r e a c t i o n g i v i n g cyclohexene, t r i p h e n y l t i n a c e t a t e and water. In agreement w i t h t h i s , we assume t h a t m e t a b o l i t e 5 r e a c t e d s i m i l a r l y under a c i d i c c o n d i t i o n s (Eq 3 ) · a

x

a x

e q


6.

FISH E T A L .


85

The t r a n s m e t a b o l i t e , but not t h e corresponding c i s isomer o f 5, can form eycloiiexene, s i n c e a t r a n s d i a x i a l con formation ( I ) i s needed f o r r e a c t i o n t o take p l a c e . The

corresponding c i s-methyl e t h e r , a model f o r t h e c i s - a l c o h o l we were not able t o s y n t h e s i z e , d i d not r e a c t even a f t e r t h r e e days w i t h g l a c i a l a c e t i c a c i d (Eq k).

^ H

CH

3

S n

(@),

HOAc Ίί^

hs.

3days

Η

_ ( @ ) SnOAc + CH OH /

+

Λ

3

(4)

Η

These r e s u l t s a r e a l s o c o n s i s t e n t w i t h those found w i t h t h e corresponding c i s - and t r a i i s - 2 - h y d r o x y c y c l o h e x y l t r i m e t h y l s i l i c o n d e r i v a t i v e s under weakly a c i d c o n d i t i o n s ( 8 ) . A c c o r d i n g l y , any cis-2-hydroxy m e t a b o l i t e t h a t might form would have been detected and was not. We a l s o found t h a t t h e ketones and J£ from t h e alcohols and ^ were produced, and v e r i f i e d t h i s r e s u l t by TIC cochromatography, but again, we a r e c o n f i d e n t o f t h e i r assigned s t r u c t u r e s u s i n g t h i s technique. Compound j ^ , 1 - h y d r o x y c y c l o h e x y l t r i p h e n y l t i n , a m e t a b o l i t e t h a t might a l s o be formed, c o u l d not be detected because o f i t s probable low c o n c e n t r a t i o n and experimental d i f f i c u l t i e s a s s o c i ated w i t h q u a n t i f y i n g i t . We found t h a t 1 - h y d r o x y a l k y l t i n d e r i v a t i v e s undergo e l e c t r o p h i l i c cleavage r e a c t i o n s o f t h e t i n carbon bond b e a r i n g t h e h y d r o x y l group w i t h h y d r o c h l o r i c a c i d t o g i v e t h e a l c o h o l and t h e corresponding d e a l k y l a t e d t i n d e r i v a t i v e ( l a ) . Consequently, r e a c t i o n o f j3 i f present w i t h hydro c h l o r i c a c i d would provide [ l - M2 ]cycïohexanol and t r i p h e n y l t i n c h l o r i d e (Eq 5)· I n c o n t r o l experiments w i t h [ l - ^ C l c y c l o h e x a nol,determined as i t s phenylcarbamate d e r i v a t i v e , we c o u l d detect l e v e l s o f t h i s d e r i v a t i v e down t o 2$ but not lower. S i m i l a r experiments w i t h t h e r e a c t i o n mixture gave no d e t e c t a b l e phenylcarbamate o f [ l - ^ C Jcyclohexanol and thus we r a t i o n a l i z e d 1


ORGANOMETALS

86

£ = J - H m

3

JSL

+

M

AND

ORGANOMETALLOIDS

S N C (

3

8 |©-N=C=0

(5)

t h a t < 2 $ o r none o f 8 was formed. The study o f [ l - J ^ C ] c y c l o h e x y l d i p h e n y l t i n a c e t a t e , in those monooxygenase enzyme r e a c t i o n s was accomplished by conv e r t i n g compounds J^-J^, p h e n y l t i n bromides. Fo propanol/chloroform t o g i v e an e x c e l l e n t y i e l d o f [ l - l ^ C ] c y c l o h e x y l d l p h e n y l t i n bromide (Eq 6 ) . P r e p a r a t i v e TLC u s i n g d i i s o -

p r o p y l e t h e r / a c e t i c a c i d (^9:1) converted t h e bromide t o t h e acetate, as analyzed by 90 MHz hi nmr spectroscopy. Reaction o f £ w i t t i * r a t l i v e r microsomes, as w i t h 1 , gave t h e f o l l o w i n g r e s u l t s a f t e r a c i d i f i c a t i o n o f the r e a c t i o n mixture and e x t r a c t i o n w i t h chloroform (Eq 7 ) . The percentages i n Eq 7 represent

(7)

normalized v a l u e s o f i d e n t i f i e d m e t a b o l i t e s which account f o r 1 0 $ o f s t a r t i n g compound, 9 . The remaining m a t e r i a l s were (82$) as w e l l as 8 $ u n i d e n t i f i e d compounds.


6.

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87

The corresponding ketones from and although a v a i l a b l e , were not p o s i t i v e l y i d e n t i f i e d as m e t a b o l i t e s because o f i n t e r f e r i n g compounds and i n c o n s i s t e n t cochromatography r e s u l t s , r e s p e c t i v e l y , upon TLC a n a l y s i s . We a l s o analyzed f o r [ l - ^ ! ] c y c l o h e x a n o l , as p r e v i o u s l y d e s c r i b e d f o r 1-hydroxycyclohexylt r i p h e n y l t i n , j ^ , but were not a b l e t o detect t h i s compound as i t s phenylcarbamate due t o experimental d i f f i c u l t i e s . Thus, l - h y d r o x y c y c l o h e x y l d i i ) h e n y l t i n acetate was not determined u s i n g t h i s method. Compounds and J £ were i d e n t i f i e d by TLC cochromatography, but u n f o r t u n a t e l y , s e p a r a t i o n o f both the c i s - and t r a n s - 3 or -U-hydroxyl isomers was not s u c c e s s f u l by TLC and t h e r e f o r e no assignments c o u l d be made. The t r a n s stereochemistry o f was a s c e r t a i n e d , as w i t h J^, by r e a c t i o n w i t h g l a c i a o f compounds ^ j ) - ^ was 10 c o u l d not be d i r e c t l y analyzed as was done w i t h m e t a b o l i t e 5· tïônsequently, t r a p p i n g o f the [ l - ^ C l c y c l o h e x e n e was e s s e n t i a l * * f o r q u a n t i f i c a t i o n o f JJjh T h i s was accomplished by r e a c t i o n o f the [ l - ^ C J c y c l o h e x e n e w i t h mercuric a c e t a t e i n methanol t o g i v e the oxymercuration product, 3^, which c o u l d be r eery s t a l i i zed t o constant s p e c i f i c a c t i v i t y and q u a n t i f i e d (Eq 8 ) . 1

13 Discussion Cytochrome P-^50 monooxygenase enzyme r e a c t i o n s have been w i d e l y s t u d i e d ; however, the use o f monosubstituted c y c l o h e x y l d e r i v a t i v e s as s u b s t r a t e s has r e c e i v e d o n l y l i m i t e d a t t e n t i o n . One such i n v i t r o study (k) concerned methylcyclohexane, ijjb and i t was shown t h a t P-^50 enzyme h y d r o x y l a t i o n gave the

14 f o l l o w i n g s i t u s e l e c t i v i t y on a per hydrogen b a s i s , i . e . , C^ : C : C : C o f 5 : 5 : 1 : 1 1 . No stereochemical l


ORGANOMETALS

88


assignments were made, u n f o r t u n a t e l y , t h u s , t h i s important aspect cannot be compared t o our r e s u l t s . In c o n t r a s t t o the observed s i t u s e l e c t i v i t y f o r j ^ , compound ] ^ presents a d r a m a t i c a l l y d i f f e r e n t r e s u l t . Thus, t h e s i t u s e l e c t i v i t y f o r 1 on a per hydrogen b a s i s f o r : C-d : C£ : C± i s 1 0 9 : 7 : 1 Τ 0 . T h i s s t r i k i n g s i t u s e l e c t i v i t y f o r 1 i s a l s o complemented by a h i g h degree o f s t e r e o s e l e c t i v i t y f o r predominantly e q u a t o r i a l h y d r o x y l a t e d m e t a b o l i t e s , w i t h compounds 2> ^ and representing 95$ o f the m e t a b o l i t e s formed and g i v i n g an e q u a t o r i a l / a x i a l r a t i o o f 59· While s t e r i c e f f e c t s c o u l d be invoked t o e x p l a i n the s i t u s e l e c t i v i t y d i f f e r e n c e s between ] ^ an<J i t should be noted t h a t the c a r b o n - t i n bond l e n g t h i s 2 . 1 8 A as compared t o a c a r bon-carbon bond l e n g t h o f 1.5*4- Κ and t h i s should a l l e v i a t e the s t e r i c b u l k o f the t r i p h e n y l t i account f o r the s i t u s e l e c t i v i t by which must b i n d t o t h e membrane i n p r o x i m i t y t o the P-^-50Fe=0 complex. By v i e w i n g D r i e d i n g models o f i t was r e a l i z e d t h a t the t r i p h e n y l t i n group c o u l d p r o v i d e a template f o r the p o s s i b l e p o s i t i o n i n g o f the c y c l o h e x y l group towards the Fe=0 complex as shown i n F i g u r e 2 .

Figure 2. One possible confor mation of 1 at the active un

i n t e r e s t i n g l y , removal o f one phenyl group, i . e . , compound £s p r o v i d e s a v a s t l y d i f f e r e n t s i t u s e l e c t i v i t y , w i t h the r a t i o per hydrogen f o r C V : CQ : C being 1 . 2 : 1 : 2 . 9 . E v i d e n t l y , the conformation o f Jg, w i t h respect t o b i n d i n g i n p r o x i m i t y t o the Fe=0 complex i s d i f f e r e n t than 1. I t i s h i g h l y conceivable t h a t w i t h the t r i o r g a n o t i n d e r i v a t i v e a t e t r a h e d r a l configura t i o n r a t h e r than a t r i g o n a l - b i p y r a m i d a l c o n f i g u r a t i o n i s more important. Since has a r e p l a c e a b l e a c e t a t e group, b i n d i n g may be d e p i c t e d as i n F i g u r e 3 . T h i s would make hydrogens on the 2 , 2

Figure 3. One pos sible conformation of 9 at the active enzyme site


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and 3 carbon atoms o f Jg, more a c c e s s i b l e than f o r comparable hydrogens i n ^ ( F i g u r e " 2 ) . We wished t o l e a r n more about the b i n d i n g d i f f e r e n c e s between compounds ] ^ and j ^ . V i s i b l e spectrophotometry has been u t i l i z e d t o determine the "finding c h a r a c t e r i s t i c s o f a wide v a r i e t y o f compounds i n p r o x i m i t y t o o x i d i z e d ( F e 3 ) F-kÔ ( 9 ) . We thought s i m i l a r s p e c t r a might show the u s u a l o p t i c a l d i f f e r e n c e s a s s o c i a t e d w i t h type I and I I b i n d i n g . F i g u r e k shows t h e o p t i c a l d i f f e r e n c e s p e c t r a f o r compounds and as w e l l as f o r t r i p h e n y l t i n and t r i b u t y l t i n c h l o r i d e s a t ~ 1 . 5 "x 1 0 " % w i t h r a t l i v e r microsomes (0Λ3 nmole F-kÔ/ml phosphate b u f f e r , pH 7·*0· Compounds and Jg. showed troughs a t 3 8 6 - 3 8 9 nm and a peak a t * 4 l 8 nm. T h i s was somewhat s i m i l a r t o t r i p h e n y l t i n c h l o r i d e , 385 and klk nm, but o p p o s i t e t o t r i b u t y l t i n c h l o r i d e which had a peak a t klO nm and a trough a ^25 type I I s p e c t r a (troug w h i l e the l a t t e r gave a s h i f t e d type I curve (peak a t 3 8 5 - 3 9 0 and trough a t ^-16-^20 nm). These dramatic d i f f e r e n c e s between the a r y l - and a l k y I t i n compounds might p r o v i d e one e x p l a n a t i o n as t o why the t r i p h e n y l t i n group does not get h y d r o x y l a t e d , i . e . , the b i n d i n g p l a c e s t h i s group away from the Fe=0 complex (Figure 2). The s p e c t r a l f e a t u r e s o f jL and ^ w h i l e s l i g h t l y d i f f e r e n t , cannot be d e f i n i t i v e l y i n t e r p r e t e d as evidence f o r the p o s s i b l e b i n d i n g changes we e n v i s i o n f o r both s u b s t r a t e s (Figures 2 and 3 ) · The s t e r e o s e l e c t i v i t y provides a t each carbon atom the thermodynamic a l l y most s t a b l e e q u a t o r i a l h y d r o x y l a t e d isomer. I n our previous s t u d i e s w i t h the t r i b u t y l t i n d e r i v a t i v e s , we p o s t u l a t e d from the observed s i t u s e l e c t i v e h y d r o x y l a t i o n p a t t e r n and other c r i t e r i a t h a t a f r e e r a d i c a l mechanism was o p e r a t i v e i n these b i o l o g i c a l o x i d a t i o n r e a c t i o n s ( l a ) . The present r e s u l t s w i t h compounds 1 α c l e a r l y demonstrate t h a t t h i s f r e e r a d i c a l process can be h i g h l y s s t e r e o s p e c i f i c (Eq 9 ) · +

S

In regards t o t h i s s p e c i f i c i t y , a r e c e n t l y r e p o r t e d r e s u l t (12.) u s i n g a p u r i f i e d P-l+50 enzyme p r e p a r a t i o n from r a b b i t l i v e r microsomes w i t h exo-D^-norbornane r e v e a l s a pronounced s t e r e o s e l e c t i v i t y f o r exo hydrogen a b s t r a c t i o n (k exo/k endo = 7 )


ORGANOMETALS

AND

ORGANOMETALLOIDS

Cyclohexyltriphenyltin +0.01-1

418 OH 386 -0.01 -L 350 nm

500 nm

Cyclohexyldiphenyltin Acetate +0.01· 418

1

-0.0I- 350 nm

389 500 nm Triphenyltin Chloride 414

+0.01 -ι

-0.01-L 350 nm

500 nm

Tributyltin Chloride +0.01 410

-0.0 l-L 350 nm Figure 4.

500 nm

Optical difference spectra with P-450 (Fe *) oxidized enzyme 3


6.

FISH E T

AL.


91

and a l a r g e k i n e t i c i s o t o p e e f f e c t (k^/k^ = 1 1 ) . Groves et a l . (10) i n t e r p r e t e d these r e s u l t s i n terms o f a homolytic C-H bond s c i s s i o n g i v i n g a l o n g l i v e d f r e e r a d i c a l capable o f e p i m e r i z i n g , i . e . , endo hydrogen a b s t r a c t i o n t o g i v e exo a l c o h o l (25$ o f exonorborneol had r e t a i n e d a l l deuterium). The important q u e s t i o n i n our case i s whether the Fe=0 complex h o m o l y t i c a l l y removes e q u a t o r i a l hydrogens i n a t o t a l l y s t e r e o s p e c i f i c r e a c t i o n (as i m p l i e d i n F i g u r e 2) or whether some o f the e q u a t o r i a l products come from a x i a l hydrogen a b s t r a c t i o n and can o n l y be a s c e r t a i n e d w i t h s p e c i f i c deuterium l a b e l l i n g . Our r e s u l t s and t h a t o f Groves et a l . (10) make the f r e e r a d i c a l h y d r o x y l a t i o n w i t h the V-kÔ Fe=0 complex a v i a b l e pathway and our r e s u l t s show t h a t t h i s can occur w i t h h i g h s t e r e o s e l e c t i v i t y . I n c o n c l u s i o n , the r e a c t i o n s o f c y c l o h e x y l t i n compounds, and 2j w i t h a P-^50 monooxygenas cyclohexyl metabolites i n d i c a t e t h a t the conformational aspects o f the s u b s t r a t e , upon b i n d i n g near the Fe=0 complex, can c o n t r o l these b i o t r a n s f o r m a tions . Abstract The r e a c t i o n s o f a cytochrome P-^50 monooxygenase enzyme system from r a t l i v e r microsomes w i t h c y c l o h e x y l t r i p h e n y l t i n and c y c l o h e x y l d i p h e n y l t i n a c e t a t e were s t u d i e d . The s t e r e o and s i t u s e l e c t i v i t y w i t h c y c l o h e x y l t r i p h e n y l t i n showed a strong preference f o r e q u a t o r i a l h y d r o x y l a t i o n o c c u r r i n g predominately a t the ^ - p o s i t i o n ( t r a n s - ^ - h y d r o x y c y c l o h e x y l t r i p h e n y l t i n ) . Thus, the conformation o f the c y c l o h e x y l group i n p r o x i m i t y t o the P-^50 f e r r y l oxygen complex seems t o be an important f a c t o r i n determining the s i t u s e l e c t i v i t y . Furthermore, by removing one p h e n y l group, i . e . , c y c l o h e x y l d i p h e n y l t i n a c e t a t e , the p a t t e r n o f carbon-hydroxylation i s more e q u a l l y d i s t r i b u t e d on the 2 , 3 and k carbon p o s i t i o n s . The stereochemistry i n the case o f t h i s l a t t e r system was o n l y v e r i f i e d f o r the trans-2-hydroxyc y c l o h e x y l d i p h e n y l t i n a c e t a t e m e t a b o l i t e . The r e s u l t s w i t h c y c l o h e x y l d i p h e n y l t i n a c e t a t e f u r t h e r s u b s t a n t i a t e the conformat i o n a l f a c t o r as c r i t i c a l f o r the observed s i t u s e l e c t i v i t y . Acknowledgment s We wish t o thank Dr. W. W. Conover o f the S t a n f o r d Magnetic Resonance Laboratory f o r o b t a i n i n g the 360 MHz H nmr spectrum o f m e t a b o l i t e 2 . SMRL i s supported by grants NSF GR 23633 and NIH RR 0 0 7 1 1 . * * The work r e p o r t e d h e r e i n was supported by the N a t i o n a l I n s t i t u t e o f Environmental H e a l t h Sciences (Grant P01 ES0CKA-9).


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References 1. a. R. H. Fish, E. C. Kimmel, J. E. Casida, J. Organometal. Chem. (1976) 118, 41. b. E . C. Kimmel, R. H . Fish, J. E. C a s i d a , J. A g r . Food Chem. (1977) 25, 1. 2 . J . H . Dawson, J. R. Trudell, G. B a r t h , R. E . Linden, E . Bunnenberg, C. Djerassi, R. C h i a n g , L . P . Hager, J. Amer. Chem. Soc. (1976) 98, 3707 and references t h e r e i n . 3· K. R. Hanson, I. A. Rose, Accounts Chem. Res. (1975) 8, 1. 4. U . Frommer, V . Ullrich, H . Staudinger, Hoppe-Seyler's Ζ. Physiol. Chem. (1970) 351, 903. 5. R. H . Fish, J. Lett. (1977 6. R. H . Fish, B . M . Broline, J. Organometal. Chem. (1977) 136, C41. 7. R. H. Fish, Β. M. Broline, I b i d . (1978) in p r e s s . 8. 9. 10.

M. De J e s u s , O. R o s a r i o , G. L . Larson, I b i d . (1977) 132, 301. A . P . K u l k a r n i , R. B . Mailman, E . Hodgson, J. A g r . Food Chem. (1975) 23, 177 and references t h e r e i n . J . T. Groves, G. A . McClusky, R. E . White, M . J. Coon, Biochem. Biophys. Res. Comm. (1978) 81, 154.

Discussion J . J . ZUCKERMAN ( U n i v e r s i t y of Oklahoma): Could there be another pathway w i t h the cleavage of the cyclohexane group from t i n , and would you know anything about the steps along that route? FISH:

You mean without h y d r o x y l a t i o n ?

ZUCKERMAN:

With or w i t h o u t , but w i t h s e p a r a t i o n from the

tin? FISH: I t h i n k t h a t you need h y d r o x y l a t i o n i n order to sepa r a t e the c y c l o h e x y l group; e i t h e r 1 - h y d r o x y l a t i o n (which we d i d n ' t see) t h a t would r e s u l t i n the l o s s of t i n or the c y c l o h e x y l group, and/or formation of the 2-hydroxy compound which i s a l s o l a b i l e under p r o t i c c o n d i t i o n s . F . HUBER ( U n i v e r s i t y of Dortmund): I would l i k e to comment on the mechanism you proposed. I f e e l a b i t uneasy that you p r o pose a c o o r d i n a t i o n number of f i v e (a t r i g o n a l bipyramid) when you have the h y d r o x y l a t i o n of the t r i p h e n y l t i n compound, and you p r o pose only a t e t r a h e d r a l c o o r d i n a t i o n when you have a d i p h e n y l t i n


6.


FISH E T A L .

1

a c e t a t e . I t s normal i n the gan ο t i n compounds that there dination with tetraorganotin d i n a t i o n increases when you at the t i n center.

93

c o o r d i n a t i o n chemistry o f these o r i s no great tendency f o r pentacoorcompounds, and the tendency f o r coor reduce the number o f organic l i g a n d s

FISH: You mean the mechanism o f the b i n d i n g ( c f . Figures 2 and 3 ) . I t h i n k t e t r a o r g a n o t i n compounds can, i n f a c t , form t r i gonal bipyramidal-type intermediates i n s o l u t i o n o r on a p r o t e i n s u r f a c e . You do b e l i e v e that the t r i o r g a n o t i n compounds can b i n d the way I depicted? HUBER: Yes, they c e r t a i n l y can but they show much g r e a t e r tendency t o have a t r i g o n a l b i p y r a m i d a l c o n f i g u r a t i o n than t h e other ones. ZUCKERMAN: We have i s o l a t e d a complex o f a t e t r a a l k y l t i n compound w i t h a bromide i o n . I t h i n k Dr. F i s h has t h i s interme d i a t e as a step i n a k i n e t i c pathway, and maybe i t wouldn't have to l a s t very long t o do i t s j o b . HUBER: I t ' s the same problem we have when we study r e a c t i o n s of t e t r a o r g a n o - t i n o r ^-lead compounds. We have t o assume some k i n d o f c o o r d i n a t i o n f o r a short time. A. J . CANTY ( U n i v e r s i t y o f Tasmania): You mentioned t h a t the phenyl groups do not get hydroxylated and t h i s has a l s o been shown f o r phenylmercury compounds i n r a t l i v e r . I s t h i s a general phe nomenon f o r a r y l organometallics? FISH: I'm not sure. I don't t h i n k a wide spectrum o f phen y l m e t a l compounds has been s t u d i e d . One of the problems i s t h e b i n d i n g ; i . e . , i n d i s c r i m i n a t e b i n d i n g a t other s i t e s . I t a l s o could be the mechanism o f h y d r o x y l a t i o n . J . M. WOOD ( U n i v e r s i t y o f Minnesota): The h y d r o x y l a t i o n of aromatic compounds by P450 i n the l i v e r i s w e l l known, i . e . , hy d r o x y l a t i o n of the s t e r o i d s . I t could be t h a t l i v e r i s a bad choice w i t h these metal complexes. I'm sure you could f i n d some m i c r o b i a l communities t h a t w i l l produce P450 and hydroxylate aromatic r i n g s attached t o metal i o n s . FISH: I t may happen i n v i v o , where we see t r i p h e n y l t i n being metabolized t o d i p h e n y l o r monophenyl, but i t ' s not apparent what the mechanism i s i f we can't do i t i n v i t r o . RECEIVED

August 22,

1978.


7 Anaerobic and Aerobic Alkylation of Arsenic BARRY C. McBRIDE, HEATHER MERILEES, WILLIAM R. CULLEN, and WENDY PICKETT Departments of Microbiology and Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1W5 Canada

Arsenic and i t s derivatives are important as herbicides and are used in, or are by-products of, many industrial processes. Large quantities of this potentially toxic compound are mobilized and placed i n new environments where they accumulate in concentrations which may exceed the normal arsenic burden of these ecosystems. The microflora i n these environments possess the metabolic machinery to transform these compounds into gaseous arsines. Challenger (1) described the formation of trimethylarsine by fungi. Cox and Alexander (2) have shown that s o i l organisms w i l l produce trimethylarsine and McBride and Wolfe (3) reported that the anaerobic methane bacteria synthesized dimethylarsine. A consequence of this microbial activity i s the modification of arsenic to new compounds possessing different chemical, physical, and biological properties. Methanogenic B a c t e r i a The methanogenic b a c t e r i a a r e a unique group of m i c r o organisms which produce methane as their principal m e t a b o l i c end product. They a r e found in l a r g e numbers in anaerobic ecosystems when o r g a n i c matter is decomposing. As a group they are morphol o g i c a l l y d i v e r s e , embracing c o c c a l , b a c i l l a r y and s p i r a l forms. They a r e extremely s e n s i t i v e t o 0 » a f a c t o r which has c o n t r i b uted t o our l i m i t e d understanding o f t h e i r biochemical a c t i v i t i e s . A r e s t r i c t e d number o f s u b s t r a t e s can be reduced t o methane these i n c l u d e : C 0 , formate, a c e t a t e , and methanol. Hydrogen i s the p r e f e r r e d source o f e l e c t r o n s . The p e r t i n e n t c h a r a c t e r i s t i c s of the methane b a c t e r i a a r e summarized i n Table I . There a r e s e v e r a l (4-8) reviews o f these organisms which d e a l w i t h t h e i r i s o l a t i o n , c h a r a c t e r i z a t i o n and b i o c h e m i s t r y . The methane b a c t e r i a f u n c t i o n as the t e r m i n a l members o f the 2

2


7.

MCBRiDE E T A L .

Aïkylation of Arsenic

95

Table I . C h a r a c t e r i s t i c s of the Methanogenic B a c t e r i a Organisms

Habitat

Morphology

Substrates

Methanobacterium ruminantium

rume sludge

short rods

formate

Methanobacterium s t r a i n M.oH

mud sludge

irregularly curved rods

H2+CO2

Methanobacterium formicicum

mud, sludge

irregularly curved rods

H2+CO2

Methanobacterium mobilie

rumen

short r o d , motile

H2"KX)2

Methanosarcina barkerii

mud, sludge

Methanococcus vannielii

mud

Methanospirilium hungatii

mud,sludge

sarcina

formate H2+CO2

methanol, acetate m o t i l e coccus

H2+CO2

formate

mud, sludge Methanobacterium thermoautotrophicum hot s p r i n g s

spirillum

H2+CO2

formate irregularly curved r o d

H2+CO2

formate


96

ORGANOMETALS

AND

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anaerobic food c h a i n , scrubbing the environment of p o t e n t i a l l y t o x i c components and r e l e a s i n g a non-toxic end product which e v e n t u a l l y escapes i n t o oxygenated environments. A g e n e r a l i z e d scheme (6) i l l u s t r a t i n g the e s s e n t i a l steps r e q u i r e d to synthesize a molecule of methane i s summarized i n F i g . 1. The scheme accounts f o r a l l the known methane precursors and i n d i c a t e s the r e a c t i o n s r e q u i r e d to form a completely reduced carbon molecule. Compound X i s a c a r r i e r which may represent one or more molecular s p e c i e s . Our understanding of the steps i n methane b i o s y n t h e s i s i s sketchy and i s almost e n t i r e l y l i m i t e d to the t e r m i n a l methyl t r a n s f e r r e a c t i o n s . No intermediates between CO2 and X - C H 3 , have been i d e n t i f i e d . The c a r r i e r molecule X has been p o s t u l a t e d to be r e q u i r e d because no f r e e reduced C - l intermediates have been i d e n t i f i e d . Progress has been mad i understandin th mechanis f th methyl t r a n s f e r r e a c t i o n s demonstrated that methy methane b i o s y n t h e s i s i n c e l l e x t r a c t s of Methanosarcina b a r k e r i i . Subsequently Wo l i n et_ a l (10) found that methylcobalamin was a s u b s t r a t e f o r methane b i o s y n t h e s i s i n Methanobacterium o m e l i a n s k i i . B l a y l o c k was able to r e s o l v e the Methanosarcina (11) system i n t o a number of components,one of which was a c o r r i n o i d c o n t a i n i n g p r o t e i n . Wood (12) was s u c c e s s f u l i n i s o l a t i n g a cobalamin c o n t a i n i n g p r o t e i n from M. o m e l i a n s k i i . The p r o t e i n s t i m u l a t e d CHi+ production when added to c e l l e x t r a c t s (13) of the organism. These s t u d i e s suggested that B12 might be the X f a c t o r i n v o l v e d i n methyl t r a n s f e r . This seemed p a r t i c u l a r l y f e a s i b l e i n l i g h t of the r o l e of C H 3 - B 1 2 i n the CH3 t r a n s f e r r e a c t i o n s l e a d i n g to methionine b i o s y n t h e s i s . U n f o r t u n a t e l y , Wood s c u l t u r e was found to c o n s i s t of two organisms; a. methanogen (Methanobacterium s t r a i n M.oH) which reduced CO2 to CHi* and a second organism which s u p p l i e d e l e c t r o n s f o r methanogenesis by o x i d i z i n g ethanol to acetate and hydrogen (14). R e s u l t s from i n v e s t i g a t i o n s w i t h the mixed c u l t u r e must be viewed w i t h c a u t i o n because i t i s impossible to i d e n t i f y w i t h c e r t a i n t y the g e n e t i c o r i g i n of the B12 p r o t e i n which s t i m u l a t e d methanogenesis. Biochemical s t u d i e s have been complicated by the l a b i l i t y of the e x t r a c t s and u n t i l a few years ago i t was not p o s s i b l e to f r a c t i o n a t e the c e l l s or r e s o l v e them f o r s p e c i f i c components of the methane s y n t h e s i z i n g system. As a consequence i t was necessary to use s t i m u l a t i o n of CHif b i o s y n t h e s i s as the assay f o r c o n s t i t u e n t s which had been f r a c t i o n a t e d from other e x t r a c t s . In t h i s type of assay the r e a c t i o n mixture was complete and would support a l i m i t e d amount of CHi* b i o s y n t h e s i s . Fractionated c e l l e x t r a c t was then added to the r e a c t i o n mixture and i f i t s t i m u l a t e d CHi* production i t was i m p l i c a t e d i n the t e r m i n a l methyl t r a n s f e r r e a c t i o n s . A d i f f i c u l t y w i t h t h i s type of assay i n a complex m u l t i s t e p r e a c t i o n i s that one can only measure s t i m u l a t i o n of a r a t e l i m i t i n g r e a c t i o n . In methane b i o s y n t h e s i s , 1


7.

97

Alkyhtion of Arsenic

MCBRIDE E T A L .

s t i m u l a t i o n could r e s u l t from i n c r e a s e d e l e c t r o n t r a n s p o r t , a c t i v a t i o n o f ATP,or t r a n s f e r of methyl groups. Thus the i m p l i c a t i o n o f a c o r r i n o i d p r o t e i n i n CHi» b i o s y n t h e s i s by Μ· o m e l i a n s k i i was based on r a t h e r tenuous evidence, but c e r t a i n l y the best evidence that could be developed a t that time. The isolâtion,of the pure c u l t u r e o f M.oH f a c i l i t a t e d the study of methanogenesis; c e l l e x t r a c t s were not as l a b i l e and c a t a l y z e d CHi* formation a t an i n c r e a s e d r a t e . I n a d d i t i o n these e x t r a c t s reduced C 0

t o CHi*

2

(7) .

Attempts t o i s o l a t e the c o r r i n o i d p r o t e i n from M.oH have been unsuccessful (15). I n f a c t no B 1 2 c o n t a i n i n g enzymes appear to be present i n t h i s organism. The red p r o t e i n i s o l a t e d by Wood was shown t o be synthesized by the non-methanogenic contaminant (15) and thus could not be a p a r t o f the i n v i v o methane s y n t h e s i z i n g system. Th cast some doubt on the importanc transfer reactions. The question was answered w i t h the discovery o f the methyl donor, C H 3 - C 0 M . CoM i s a low m o l e c u l a r weight, heat s t a b l e c o f a c t o r found i n a l l methane b a c t e r i a that have been examined (16). Chemically CoM i s 2,2 - d i t h i o d i e t h a n e s u l f o n i c a c i d (17). I t can be reduced and methylated c h e m i c a l l y o r b i o l o g i c a l l y t o form 2-(methylthio) e t h a n e s u l f o n i c a c i d . I n the presence of c e l l e x t r a c t , ATP, and H ,CH -CoM i s r e d u c t i v e l y cleaved t o y i e l d CH^ and CoM ( F i g . 2 ) . E x t r a c t s can be r e s o l v e d f o r CoM by anaerobic d i a l y s i s and w i l l synthesize CHi* only i f they a r e s u p p l i e d w i t h CoM. I t seems reasonable t o assume that Βχ2 i s n o t i n v o l v e d i n CHi» b i o s y n t h e s i s i n MoH, and that t h i s r e a c t i o n i s c a t a l y z e d by CoM. I t i s not c l e a r a t t h i s time whether Methanosarcina depends on B 1 2 , as t h i s organism has been shown t o possess CoM i n a d d i t i o n t o a c o r r i n o i d p r o t e i n . P o s s i b l y t h i s organism possesses 2 methane s y n t h e s i z i n g pathways,one dependent and one independent of Β 1 2 · f

2

3

A l k y l a t i o n o f Metals M.oH has been i m p l i c a t e d as an organism which w i l l a l k y l a t e mercury (18) and a r s e n i c ( 3 ) . A l k y l a t i o n o f metals by M.oH could t h e o r e t i c a l l y occur a t any b i o s y n t h e t i c step which generated a C - l group. Because the methanogenic b a c t e r i a process l a r g e numbers of C - l u n i t s i t i s not unreasonable to assume that the CH^ b i o s y n t h e t i c pathway could be important i n these r e a c t i o n s , and that CoM would be a molecule of i n t e r e s t . I t would be unwise t o f o r g e t that methyl groups must be generated f o r the s y n t h e s i s of methionine and that intermediates i n t h i s r e a c t i o n could be i n v o l v e d i n metal a l k y l a t i o n . Two methionine b i o s y n t h e t i c pathways operate i n b a c t e r i a . One system i n v o l v e s t r a n s f e r o f a methyl group from N -methyl-tetrahydrofolic acid (N , CH3THFA) to B and then t o homocysteine. The methylation of homocysteine r e q u i r e s c a t a l y t i c 5

5

1 2


ORGANOMETALS

98

AND

ORGANOMETALLOIDS

amounts of S-adenosyl methionine (SAM). The second system r e q u i r e s N , CH -THFA t r i - g l u t a m a t e and SAM. The i n a b i l i t y to f i n d a c o r r i n o i d p r o t e i n i n M.oH suggests that the l a t t e r mechanism may operate i n these organisms. 5

3

Arsine Biosynthesis C e l l e x t r a c t s of M.oH produce a s t r o n g g a r l i c odor when they are incubated w i t h arsenate. The f o l l o w i n g s e c t i o n of t h i s paper w i l l d e a l w i t h the b i o c h e m i c a l s t u d i e s which l e d to the i d e n t i f i c a t i o n of the g a r l i c s m e l l i n g compound and to a scheme f o r i t s b i o s y n t h e s i s . This w i l l be f o l l o w e d by a study of a r s e n i c t r a n s f o r m a t i o n i n n a t u r a l anaerobic systems . A l k y l a r s i n e Assays. The r e a c t i o n v e s s e l and c o n d i t i o n s used f o r the b i o s y n t h e s i s of a l k y l a r s i n e s i m i l a t thos described f o r methane b i o s y n t h e s i v e s s e l was connected by polyethylen g glas contained 2 mL of 2M HNO3. The t r a p p i n g tube was placed i n an e t h a n o l - i c e bath. A slow stream of H2 was passed i n t o the r e a c t i o n f l a s k and the v o l a t i l e a l k y l a r s i n e s were swept i n t o the n i t r i c a c i d , where they were condensed and trapped by o x i d a t i o n to n o n - v o l a t i l e a c i d s (20). The contents of the t r a p were assayed f o r a r s e n i c by atomic a b s o r p t i o n spectrometry and f o r C by counting i n Bray's s c i n t i l l a t i o n f l u i d . The i s o t o p e , *As was used to f o l l o w the formation of v o l a t i l e a l k y l a r s i n e . E x t r a c t s were incubated w i t h [ A s ] N a 2 H A s 0 i * and the v o l a t i l e methylated As was measured. Advantage was taken of the property of a l k y l a r s i n e s to r e a c t w i t h the red-rubber serum stoppers which were used to s e a l the r e a c t i o n f l a s k s . At the a p p r o p r i a t e times the r e a c t i o n mixture was i n a c t i v a t e d by h e a t i n g on a steam cone. The f l a s k s were then incubated f o r an a d d i t i o n a l 20 min to ensure t h a t a l l the v o l a t i l e d i m e t h y l a r s i n e was adsorbed onto the rubber stopper. The serum stopper was removed from the f l a s k , r i n s e d i n water, cut i n h a l f , and placed i n a s c i n t i l l a t i o n v i a l together w i t h 15 mL of Bray's s c i n t i l l a t i o n f l u i d . T h i s mixture was e i t h e r counted d i r e c t l y , as the rubber stopper d i d not quench the high-energy 3 p a r t i c l e s emitted by ks, or the stopper was removed a f t e r the **As had been leached from the rubber (1 h r . ) . The rubber stopper technique was found to be an e f f i c i e n t and s p e c i f i c means of s e p a r a t i n g [ C] dime thy l a r s i n e from [ C]CHi . lh

7l

7lf

7lf

7k

llf

1If

t

Requirements f o r B i o s y n t h e s i s . Conditions f o r the b i o s y n t h e s i s of a l k y l a r s i n e d e r i v a t i v e s are d e s c r i b e d i n Table I I . In these experiments c e l l e x t r a c t s were incubated under anaerobic c o n d i t i o n s w i t h [ * A s ] N a 2 H A s 0 i , and the formation of v o l a t i l e *ks compounds was measured. A methyl donor, H 2 , ATP, and arsenate were r e q u i r e d f o r the r e a c t i o n ; w i t h the exception of arsenate these same components are r e q u i r e d i n the CHi*s y n t h e s i z i n g system. A t o t a l of 34 yg of a r s e n i c was found i n 7l

f

7t


7.

MCBRiDE E T A L .

Alkyfotion of Arsenic

99

an a l k y l a r s i n e t r a p l i n k e d to a r e a c t i o n f l a s k which contained arsenate and c e l l e x t r a c t , p r o v i d i n g evidence that a v o l a t i l e a l k y l a r s i n e was b e i n g s y n t h e s i z e d . V o l a t i l e a l k y l a r s i n e compounds were not d e t e c t e d i n a s i m i l a r c o n t r o l r e a c t i o n mixture which contained arsenate and b o i l e d e x t r a c t . S u b s t r a t e s . C H 3 - B 1 2 and CO2 were shown to be methyl donors f o r a l k y l a r s i n e s y n t h e s i s . Arsenate, a r s e n i t e and m e t h y l a r s o n i c a c i d were reduced to d i m e t h y l a r s i n e i n the presence of a C - l donor. C a c o d y l i c a c i d was reduced i n the absence of a C - l donor. M e t h y l a r s o n i c a c i d was found i n e x t r a c t s incubated w i t h AsOi* "and a C - l donor. Whole C e l l s . The a b i l i t y of whole c e l l s to s y n t h e s i z e a l k y l a r s i n e i s shown i an a c t i v e l y growing c u l t u r 12-L fermentor and was incubated a n a e r o b i c a l l y under a H : C 0 atmosphere. 2

2

A n a l y s i s of the A l k y l a t e d A r s i n e . The s t r u c t u r e of the a l k y l a r s i n e formed i n c e l l e x t r a c t s was i n d i c a t e d by two experimental procedures. I n one procedure N a 2 H A s 0 i f was incubated w i t h [ C]methylcobalamin, and the trapped a l k y l a r s i n e was analyzed f o r a r s e n i c and C. The r e s u l t s of two such experiments are presented i n Table I I I . D i s s o l v e d [^CjCHt, was removed by b u b b l i n g CHi* through the t r a p p i n g s o l u t i o n a f t e r i t had been disconnected from the r e a c t i o n f l a s k . The number of methyl groups i n the trap was c a l c u l a t e d from the s p e c i f i c a c t i v i t y of [ C ] C H 3 - B i 2 . T h i s v a l u e was d i v i d e d by the amount of a r s e n i c to o b t a i n a methyl group to a r s e n i c r a t i o . The r a t i o s obtained (1.8:1 and 1.9:1) i n d i c a t e that the compound i s dimethylarsine. These r e s u l t s were s u b s t a n t i a t e d by a d o u b l e - l a b e l i n g experiment i n which [ *As]Na2HAs0if and [ C]methylcobalamin were s u b s t r a t e s . The r e s u l t s of two such experiments are shown i n Table IV. A l k y l a r s i n e was separated from contaminating [ C]CHi by t r a p p i n g i n a rubber serum stopper as d e s c r i b e d p r e v i o u s l y . The s p e c i f i c a c t i v i t y of the s u b s t r a t e s was used to c a l c u l a t e the micromoles of a r s e n i c as w e l l as methyl groups. The r a t i o s of methyl groups to a r s e n i c (2.1:1 and 1.9:1) suggest that the g a r l i c - s m e l l i n g compound i s d i m e t h y l a r s i n e . A pathway f o r d i m e t h y l a r s i n e s y n t h e s i s has been proposed (3) F i g . 4. Arsenate i s f i r s t reduced to a r s e n i t e which i s then methylated to form m e t h y l a r s o n i c a c i d . T h i s compound was found i n r e a c t i o n mixtures when e x t r a c t s were incubated w i t h e i t h e r arsenate o r a r s e n i t e and C H 3 - B 1 2 . M e t h y l a r s o n i c a c i d i s reduced and methylated to form d i m e t h y l a r s i n i c a c i d . The a c i d i s then reduced to form dime t h y l a r s i n e . A l l i n t e r m e d i a t e s have been shown to be converted to d i m e t h y l a r s i n e by c e l l e x t r a c t s . T h i s pathway i s based on s t u d i e s i n which C H 3 - B 1 2 was used as methyl llf

llf

7l

llf

llf

f


ORGANOMETALS


CO + XH a

ι X-COOH

ι ». X-CHO

1 I>

CH,OH

XCH OH 2

h

ChLCOOH

X-CH

Figure 1. Possible pathway for methane biosynthesis. C-2 of ace tate is preferentially reduced to methane.

3

H X + CH

(-S-CH CH S03 ) 2

1

2

h

CH As 0 3

3

-o "2

9p f_>

3

·· τ τ τ CH As O

2

i l J

3

V Ϊ (CH ) As 0 3

3

>

2

2e ——f

. ·· I I I (CH )As 3

-o 2

T h i s scheme was proposed on the b a s i s of the a b i l i t y of S. b r e v i c a u l i s t o produce t r i m e t h y l a r s i n e from a l l the arsenic(V) p r e cursors and t o produce l a b e l e d t r i m e t h y l a r s i n e from a r s e n i t e when C l a b e l e d D,L-methionine, *CH SCH CH CH(NH )C00H, was added to the c u l t u r e medium. The mechanism i s e s s e n t i a l l y the same as that of F i g . 4 except that the methyl donor i s i d e n t i f i e d as the chemically reasonably (29) methyl carbonium i o n and that the r e a c t i o n pro ceeds f u r t h e r , before the f i n a l r e d u c t i o n , i n the aerobic system. Challenger r e p o r t s that crude c e l l e x t r a c t s of S. b r e v i c a u l i s do not produce t r i m e t h y l a r s i n e when exposed t o a r s e n i c a l s . T h i s and other experiments (1) i n d i c a t e d that b i o l o g i c a l methylation by t h i s organism i s confined t o the mould c e l l , and does not take p l a c e i n the medium. I n t h i s connection some recent work on c e l l e x t r a c t s o f C. humicola a r e o f considerable i n t e r e s t (30). I t i s found that when the e x t r a c t i s incubated w i t h As l a b e l e d arsenate, SAM as methyl source, and NADPH as reducing agent, and the r e s u l t i n g supernatant l i q u i d a p p l i e d t o a Dowex 1 i o n exchange column, s e v e r a l d i s t i n c t a r s e n i c c o n t a i n i n g f r a c t i o n s can be e l u t e d as shown i n F i g . 7. These can be i d e n t i f i e d f o l l o w i n g e l e c t r o p h o r e s i s and autoradiography as being l a r g e l y a r s e n i t e , c a c o d y l i c a c i d , methylarsonic a c i d , and arsenate i n f r a c t i o n s 1 t o 4 r e s p e c t i v e l y . Experiments of t h i s s o r t can be r e f i n e d f u r t h e r and r e v e a l the presence o f other a r s e n i c c o n t a i n i n g f r a c t i o n s i n c l u d i n g t r i m e t h y l a r s i n e oxide. Thus, p r o v i d i n g SAM i s added, a l l the arsenic(V) intermediate i n Challenger's scheme can be i d e n t i f i e d i n c e l l e x t r a c t s of an organism which i s known to produce t r i m e t h y l a r s i n e . 11+

1I

3

2

2

2

7I+


7.

MCBRIDE E T

Alkylation of Arsenic

AL.

109

The r e s u l t of experiment 15, Table V I I i s p a r t i c u l a r l y i n t e r e s t i n g s i n c e , when e t h i o n i n e i s added i n place of methionine, no a r s i n e s are produced. The absence of e t h y l a r s i n e s argues a g a i n s t a p u r e l y chemical t r a n s f e r of an a l k y l group from sulphur to a r s e n i c , and the absence o f t r i m e t h y l a r s i n e argues s t r o n g l y f o r a methionine based enzyme i n v o l v e d s y n t h e t i c path s i n c e e t h i o n i n e i s a w e l l known antagonist to methionine (31). Along the same l i n e s i t has been found (.2,25) that phosphates i n h i b i t s the f o r mation of t r i m e t h y l a r s i n e from arsenate, a r s e n i t e , and methylarsonate, but not from cacodylate, by growing c u l t u r e s of C. humicola. The same organism can be p r e c o n d i t i o n e d by cacodyl a t e to produce t r i m e t h y l a r s i n e at a g r e a t e r than u s u a l r a t e from both arsenate and cacodylate (32). This i s seen i n F i g . 8. On the other hand,using the same organism, p r e c o n d i t i o n i n g w i t h arsenate r e s u l t s i n a dramati production from cacodylat production from arsenate. Model Studies Each step i n Challenger's mechanism i s c h e m i c a l l y reasona b l e . Indeed the a l k y l a t i o n steps are r e l a t e d to the w e l l known Meyer r e a c t i o n (20,32), which uses methyl i o d i d e or d i m e t h y l sulphate as the source of C H 3 + . Since SAM i s a sulphonium compound (34) other simpler analogues have been s t u d i e d w i t h respect to t h e i r a b i l i t y to methylate a r s e n i c (35). At pH 12 and 80°C (CH ) P"hPF ~ can be used f o r a l l the steps i n Challenger's scheme which i n v o l v e C H (these r e a c t i o n s can be monitored by NMR t e c h niques s i n c e the r e a c t a n t and products have d i s t i n c t chemical s h i f t s ) and each a r s e n i c ( V ) compound i n the scheme can be reduced to the a p p r o p r i a t e a r s e n i c ( I I I ) d e r i v a t i v e using S 0 . Thus the whole sequence can be e a s i l y d u p l i c a t e d . Methyl methionine i s o f t e n invoked as a model f o r SAM (34) and t h i s compound s l o w l y but i n c o m p l e t e l y , methylates CH AsO^~ at 25°C and a t the more r e a l i s t i c pH of 5.8. However a methyl sulphonium d e r i v a t i v e of CH -CoM under the same c o n d i t i o n s f a i l e d to t r a n s f e r i t s methyl group. 3

3

6

+

3

2

3

3

A r s e n i c Cycle i n the Environment Studies w i t h aerobic and anaerobic organisms have shown t h a t the former produce t r i m e t h y l a r s i n e whereas the l a t t e r produce the more c h e m i c a l l y r e a c t i v e d i m e t h y l a r s i n e . Taking these observat i o n s i n t o account, i t i s p o s s i b l e to p o s t u l a t e t h a t there i s an a r s e n i c c y c l e i n nature, a c y c l e that r e l i e s upon both b i o l o g i c a l and a b i o t i c r e a c t i o n s . Such a scheme i s o u t l i n e d i n F i g . 9. I t i s important not to view the r e a c t i o n s as being confined to sediment, water, or a i r but r a t h e r to ecosystems which are e i t h e r a e r o b i c or anaerobic because i t i s the a v a i l a b i l i t y of oxygen which w i l l determine the nature of the m i c r o b i a l f l o r a and w i l l i n f l u e n c e the f a t e and subsequent movement of the a r s i n e . Arsenate, a r s e n i t e , and methylarsonate r e a c t i n a s i m i l a r manner


ORGANOMETALS

AND

ORGANOMETALLOIDS

1400 1300 1000 ftoo

ο 4c «oo 400

1

Λ

200

0

/V'

10

30

20

AJ 40

50

4

60

FRACTION

Figure 7. Ion-exchange separation of C. humicola extracts incu bated with As-Na HAsOi, SAM, and NADPH. Peaks 1-4 are arsenite, cacodylic acid, methylarsonic acid, and arsenate, respec tively. 74

2

(CH ) As(nmote) 3 3

400"

Figure 8. Amount of (CH ) As in the headspace above growing 300· cultures of C. humicola precondi tioned in cacodylate (C and D) and not preconditioned (A and B). In Curves A and C the arsenical substrate is arsenate, in Β and D 0 it is cacodylate. s

s


7.

MCBRIDE E TA L .


ARSENIC

111

CYCLE

(

C H

a) * 3

,

"•(CH ) AiO(OH) 3

^

A»0(OH^=Â.(OH)3—•CH,>UO(OH) =*(CHÂ.io(OH)—i a

2

I •(CH,)Â«H (CH,)i.-S^X

ί Figure 9. Biological arsenic cycle. (=) aerobic or anaerobic; (· - -) aerobic biotic or abiotic; ( ) anaerobic; ( ) aerobic; (++) these reactions are prob ably abiotic.


112

ORGANOMETALS

AND ORGANOMETALLOIDS

i n both a e r o b i c and anerobic ecosystem. Cacodylate i s an important branch p o i n t ; aerobes reduce and methylate t h i s compound t o form t r i m e t h y l a r s i n e , anerobes reduce i t t o d i m e t h y l a r s i n e . The t r i m e t h y l a r s i n e i s e v e n t u a l l y o x i d i z e d t o cacodylate which can be i n c o r p o r a t e d d i r e c t l y i n t o the c y c l e o r f u r t h e r m o d i f i e d t o methylarsonate o r arsenate by m i c r o b i a l a c t i v i t y . D i m e t h y l a r s i n e can be o x i d i z e d t o cacodylate but i t may r e a c t w i t h other chemical c o n s t i t u e n t s i n i t s environment and thus i n i t i a t e an e n t i r e l y new s e t o f r e a c t i o n s . The l a t t e r i s a reasonable hypothesis when one considers the r e a c t i v i t y o f the a r s i n e and the e n v i r o n ment i n which i t i s produced. Acknowledgements T h i s paper i s a c o n t r i b u t i o n from the B i o i n o r g a n i c Chemistry Group supported i n p a r grants from N.R.C. andM.R.C BIBLIOGRAPHY 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

C h a l l e n g e r , F. (1945). Chem. Rev., 36, 315. Cox, D.P. and Alexander, M. (1973). A p p l i e d M i c r o . 25, 408. McBride, B.C. and Wolfe, R.S. (1971). B i o c h e m i s t r y , 10, 4312. Wolfe, R.S. (1971). Adv. in Microb. P h y s i o l . 6, 107. Stadtman, T.C. (1967). Ann. Rev. Microbiol. 21, 121. Barker, H.A. (1956). " B a c t e r i a l Fermentations", P. 1 John Wiley and Sons, I n c . , New York. McBride, B.C. and Wolfe, R.S. (1971). Adv. in Chem. S e r . 105, 11. Zeikus, J.G. (1977). B a c t e r i o l . Rev. 41, 514. B l a y l o c k , B.A. and Stadtman, T.C. (1966). Biochem. Biophys. Res. Commun. 11, 34. W o l i n , M.J., Wolin, E.A. and Wolfe, R.S. (1963). Biochem. Biophys. Res. Commun. 12, 465. B l a y l o c k , B.A. (1968). Archs. Biochem. Biophys. 124, 314. Wood, J.M. and Wolfe, R.S. (1966). Biochemistry 5, 3598. Roberton, A.M. and Wolfe, R.S. (1969). Biochim. Biophys. A c t a . 192, 420. Bryant, M.P., Wohn, E.A., Wolin, M.J. and Wolfe, R.S. (1967). Arch. M i k r o b i o l . 59, 20. McBride, B.C. (1970). D i s s e r t a t i o n . U n i v e r s i t y o f Illinois, Urbana, Illinois. McBride, B.C. and Wolfe, R.S. (1971). Biochemistry 10, 2317. T a y l o r , C.P. and Wolfe, R.S. (1974). J. Biol. Chem. 249, 4879. Wood, J.M., Kennedy, F.S. and Rosen, C.G. (1968). Nature 220, 173. Wolin, E.A., Wolin, M.J. and Wolfe, R.S. (1963). J. Biol. Chem. 238, 2882.


7.

20.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

35.

MCBRIDE

ET

AL.


113

R a i z i s s , G.W. and Gavron, J.L. (1923). Organic a r s e n i c a l compounds, New York, N.Y. The Chemical Catalog Co., pp. 38-49. Smith, P.H. and Mah, R.A. (1966). A p p l . M i c r o . 14, 368. Bauchop, T. (1967). J . Bact. 94, 171. Peoples, S.A. (1975). A r s e n i c a l P e s t i c i d e s A.C.S. Symposium S e r i e s #7, p. 1. A v e l i n o , N., C u l l e n , W.R., McBride, B.C. Unpublished results. Woolson, E.A., Ed. (1975). A r s e n i c a l P e s t i c i d e s A.C.S. Symposium S e r i e s #7, Washington, D.C. Woolson, E.A. and Kearney, P.C. (1973). E n v i r o n . Sci. Technol. 7, 47. Isensee, A.R., Kearney, P.C., Woolson, E.A., Jones, G.E. and W i l l i a m s , V.P (1973) E n v i r o n Sci. Technol 7, 841 C u l l e n , W.R., Froese D.J. and Reimer, M. (1977). J. Organometal. Chem. 139, 61. Zingaro, R.A. and Irgolic, K . J . (1975). Science 187, 7651. C u l l e n , W.R., McBride, B.C. and Pickett, A.W. Unpublished results. Simmonds, S., Keller, E.B., Chandler, J.P. and duVigneaud, V. (1950). J. Biol. Chem. 183, 191. C u l l e n , W.R., McBride, B.C. and Reimer, M. Bull. E n v i r o n . Contam. T o x i c o l . , in p r e s s . Quick, A.J. and Adams, R. (1922). J . Amer. Chem. Soc. 44, 805. S a l v a t o r e , F. Borek, E., Zappia, V., Williams-Ashman, H.G., Schlenk, F., Eds. (1977). The Biochemistry o f Adenosyl methionine, Columbia U n i v e r s i t y P r e s s , New York, 1977. Chopra, A.K., C u l l e n , W.R., D o l p h i n , D. Unpublished r e s u l t s .

Discussion J . M. WOOD ( U n i v e r s i t y of Minnesota): I want t o ask t h i s question on b e h a l f of P r o f e s s o r C h a l l e n g e r , because i n h i s review a r t i c l e he a l l u d e s t o the p o s s i b l e r o l e o f coenzyme M i n methyl a t i o n of a r s e n i c by Methanobacteria. He has a s p e c u l a t i v e p a r a graph i n which he suggests t h a t the dimethylsulphonium d e r i v a t i v e would be a very good candidate f o r m e t h y l a t i o n of a r s e n i c . The methyl coenzyme M i t s e l f i s a bad candidate. Have you any i d e a whether h i s suggestion i s a good one? McBRIDE: enzyme M.

We haven't t r i e d any other compound on methyl c o

W. R. CULLEN ( U n i v e r s i t y of B r i t i s h Columbia): I n model s y s tems, t h a t methyl t r a n s f e r does take p l a c e from the methyl coen zyme M t o a r s e n i c ( I I I ) . Any sulphonium compound w i t h a methyl transfers to arsenic.


114

ORGANOMETALS

AND

ORGANOMETALLOIDS

G. Ε. ΡARRIS (Food and Drug A d m i n i s t r a t i o n ) : Do you have k i n e t i c data f o r t h i s t r a n s f e r that you are t a l k i n g about?

this.

CULLEN: Yes, we have attempted t o measure some k i n e t i c s on I t ' s a v e r y d i f f i c u l t system t o work on.

M. 0. ANDREAE ( S c r i p p s I n s t i t u t e of Oceanography): Do you have evidence f o r the formation of l a r g e r compounds ; would you see them i f they were formed? McBRIDE:

By l a r g e r compounds do you mean complexes?

ANDREAE:

Yes, arseno-betaine or something l i k e t h a t .

McBRIDE: We don't hav d evidence but r e c e n t l ran a Sephadex column, a c t i v e f r a c t i o n t h a t i n d i c a t e s a h i g h molecular weight. ANDREAE: D i d you do a mass balance t h a t w i l l t e l l you how much of the l a b e l can be accounted f o r by arsenate and other com pounds? McBRIDE:

Yes, we can account f o r 100%.

ANDREAE: In your experiments w i t h the marine mud, were you u s i n g a rubber t r a p t o assay f o r the a r s i n e or was t h a t determined by head space a n a l y s i s ? McBRIDE:

No, there was a t r a p .

F. E. BRINCKMAN ( N a t i o n a l Bureau of Standards): I'm i n t r i g u e d by t h i s apparent complexation on the Dowex column. I s i t engendered by the b i o a c t i v i t y ? That i s , might there be a t h i r d component t h a t leads t o complexation of these a r s e n i c species? [McBRIDE: Yes] T y p i c a l l y , under the c o n d i t i o n s you use f o r e l u t i o n , these are an i o n i c species from the pKa v a l u e s . In our l a b o r a t o r y , f o r HPLC, we use both weak and s t r o n g anion and c a t i o n columns and can make s a t i s f a c t o r y s e p a r a t i o n s of demethylated me t a b o l i t e s from s o i l b a c t e r i a i n v o l v i n g the m e t h y l a r s e n i c a l p e s t i c i d e s . We see no evidence of t h i s k i n d of complexation. We are concerned about higher molecular weight e l u a n t s because of your work and t h a t of P r o f e s s o r I r g o l i c concerning the b e t a i n e or other p o s s i b l e a n i o n i c species w i t h a l a r g e molecule pendant. McBRIDE: I t seems t o be r e a l , and i t seems to be a s s o c i a t e d w i t h a b i o l o g i c a l l y a c t i v e e x t r a c t . I f you i n a c t i v a t e the e x t r a c t you don't see these. You can use l a b e l s (C-14 c a c o d y l , C-14 meth y l a r s o n i c a c i d , As-74 arsenate) and you do not see these complexes form. E v e r y t h i n g e l u t e s at the a p p r o p r i a t e p o i n t , but when you have a b i o l o g i c a l l y a c t i v e system, then you see t h i s change t o


7.

MCBRIDE E T A L .

Alkyhtion of Arsenic

115

what we t h i n k i s a complex formation. I t ' s very dramatic when you look at C-14 cacodyl as a s u b s t r a t e r a t h e r than as arsenate. A l most 99% of the m a t e r i a l moves i n t o a f r a c t i o n which e l u t e s w i t h water. BRINCKMAN: This i s a very c l e a r demonstration that c a u t i o n should be e x e r c i s e d i n s p e c i a t i n g these t r a c e m a t e r i a l s , metabol i t e s p a r t i c u l a r l y , when u s i n g methodology l i k e Braman's technique of r e d u c t i v e v o l a t i l i z a t i o n to form h y d r i d e s . You may l o s e a r a t h e r c r i t i c a l molecule which i s important f o r c o u p l i n g s e v e r a l i o n i c species and which may be i n d i c a t i v e of the mechanistic pathway. K. J . IRGOLIC (Texas A & M u n i v e r s i t y ) : You a l l u d e to the p o s s i b l e transformation f t r i m e t h y l a r s i n dimethylarsini acid i n some of your systems c o n d i t i o n s , i t ' s a r e l a t i v e l y slo r e a c t i o n , but you brea dow the compound, l o s e one methyl group, and end up w i t h d i a l k y l a r s i n i c s from t r i a l k y l a r s i n e s . ANDREAE: Your marine mud produces h a r d l y any methane. Where d i d you get t h a t mud? Some marine muds produce methane, some do not. McBRIDE: We d i d n ' t f i n d any marine mud that was producing l o t of methane although o b v i o u s l y they e x i s t . ANDREAE: i n the core?

Was

your mud

a

sample from a r e l a t i v e l y shallow l e v e l

McBRIDE: No, we went f a i r l y deep. There was a l o t of s u l f u r i n these muds, whether t h a t was i n f l u e n c i n g what was going on, I don't know. RECEIVED

August

22,

1978.


8 Arsenic Uptake and Metabolism by the Alga Tetraselmis Chui N. R. BOTTINO—Department of Biochemistry and Biophysics, Texas A & M University, College Station, TX 77843 E. R. COX—Department of Biology, Texas A & M University, College Station, TX 77843 1

K. J. IRGOLIC, S. MAEDA , W. J. McSHANE, R. A. STOCKTON, and R. A. ZINGARO—Department of Chemistry, Texas A & M University, College Station, TX 77843 Not too many years ag s c i e n t i f i c concern about substances present in very low concentrations in the a i r , water or soil. Knowledge about their presence was not at hand. Now, because of the efforts of many analytical chemists a number of these trace elements and their compounds have been identified and quantified. We know now that a whole spectrum of compounds i s present at trace level concentrations in the environment. We also know that many of them are, or may be potentially harmful. Arsenic i s one of the elements which is widely distributed in the earth's environment. Arsenic has a complicated chemistry. It forms a great variety of organic and inorganic compounds of variable potency with regard to their effects on l i v i n g organisms. Arsenic compounds are amenable to chemical transformation through interactions with biological systems. It has been known for almost one hundred years, that molds convert the arsenic present in green paint pigments into a volatile arsenic compound which was identified as trimethylarsine (1). The biological methylation of arsenic compounds producing methylarsines is now a well-established fact (2, 3, 4, 5). Methylcobalamin has been implicated as the methyl donor (2), but recently, Cullen and coworkers (5a) have made the Tmportant discovery that when L-methionine-methyl-d is added to certain microbial cultures, the CD label is incorporated into the evolved arsenic. This is good evidence that S-adenosylmethionine i s involved in the biological methylation process. Lunde (6) found arsenic compounds in the lipids of marine and limnetic organisms. Acid hydrolysis of the arsenic-containing l i p i d fractions produced water soluble, organic arsenic compounds. The isolation of arsenobetaine from rock lobsters was reported by Edmonds et al. (7) in 1977. These findings prove that transformations more complex than simple methylation do occur to 3

3

Present Address: Department of Applied Chemistry, Faculty of Engineering, Kagoshima University, Kagoshima 890, Japan. 1


8.

BOTTiNO E T A L .

Arsenic Uptake by Alga

a r s e n i c compounds i n b i o l o g i c a l

117

systems.

A r s e n i c Incorporation and Algal Growth. Three years ago an i n v e s t i g a t i o n on the metabolism o f arsenate by marine algae was i n i t i a t e d a t Texas A&M U n i v e r s i t y . The goal o f the project was and s t i l l i s the i s o l a t i o n , i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f organic a r s e n i c compounds formed by the algae from arsenate under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . Preliminary experiments with a v a r i e t y o f marine algae l e d to the use o f Tetraselmis ehui, a green f l a g e l l a t e (Chlorophyta), as a convenient experimental organism. This alga grows w e l l , takes up a r s e n i c e f f i c i e n t l y and i s r a t h e r i n s e n s i t i v e toward mechanical shock which accompanies the harvesting o p e r a t i o n s . T. ehui was found to t o l e r a t e well exposure to 10 ppm As(arsenate) when grown i n a r t i f i c i a l sea water, and i n f a c t , at the beginning o f the s t a t i o n a r y phase the growth medium contained The a r s e n i c uptake by T. ehui depended on the l i g h t i n t e n s i t y . At a l i g h t i n t e n s i t y o f 12,500 lux the a r s e n i c content o f the c e l l s increased f i r s t to a pronounced maximum w i t h i n several days and then decreased r a p i d l y to a very low l e v e l . At 7000 lux the a r s e n i c l e v e l i n the c e l l s remained at h a l f the concentration reached at 12,500 l u x . The d e t a i l e d causes f o r t h i s behavior remain unknown, but i t can be p o s t u l a t e d that s i n c e arsenate absorption i s an endergonic process (9) i t might compete with c e l l growth f o r the a v a i l a b l e photosynthetic energy. Large-Scale Algal Growth and A r s e n i c - P r o t e i n I n t e r a c t i o n s . The c h a r a c t e r i z a t i o n o f the a r s e n i c - c o n t a i n i n g compounds present i n τ. ehui r e q u i r e d the i s o l a t i o n o f 300-500 mg amounts o f these compounds f o r f u r t h e r p u r i f i c a t i o n and a n a l y s i s . This could not be done with the t e s t - t u b e c u l t u r e s used i n the preceding ex periments. Consequently, an alga growing f a c i l i t y was con s t r u c t e d t o r a i s e T. ehui under c o n t r o l l e d c o n d i t i o n s i n c l u d i n g p r o t e c t i o n from other organisms such as b a c t e r i a . The f a c i l i t y c o n s i s t s o f a converted c o l d storage room, which houses four 1 5 0 0 - l i t e r tanks. This c u l t u r e room i s a i r - c o n d i t i o n e d , well i n s u l a t e d and i s kept under p o s i t i v e a i r pressure. Ultraviolet l i g h t s are s t r a t e g i c a l l y placed to minimize b a c t e r i a l contamin ation. The tanks are i l l u m i n a t e d by banks o f f l u o r e s c e n t l i g h t s . B a l l a s t s are mounted outside the c u l t u r e room t o reduce the heat load on the a i r c o n d i t i o n e r . The growth medium i s mixed i n a c o n t r o l room. The tanks can be f i l l e d , i n o c u l a t e d , aerated, sampled and harvested without e n t e r i n g the c u l t u r e room, through an appropriate system o f pipes and v a l v e s . Each large tank produces approximately one l i t e r o f t i g h t l y packed algae w i t h i n 20 days. Figure 1 i l l u s t r a t e s the t y p i c a l type o f experiments run with the l a r g e - s c a l e growing f a c i l i t y . When A s - l a b e l e d d i s sodium hydrogen arsenate was added to the growth medium at the same time as the inoculum, the algae reproduced r a p i d l y f o l l o w i n g 7 4


ORGANOMETALS


lure 1. Large-scale growth and arsenic uptake by T. ehui instant ocean medium containing 10 ppm As (arsenate) added at the time of inoculation


8. ΒΟΤτίΝΟ E T A L .


119

an i n i t i a l l a g p e r i o d , i n a r t i f i c i a l sea water c o n t a i n i n g 10 ppm arsenate. In separate experiments, the arsenate was added to the growing c u l t u r e s during t h e i r exponential phase o r s t a t i o n a r y phase. In the experiment depicted i n Figure 1 the arsenate was added at zero time, i . e . , at the time the medium was i n o c u l a t e d . The curves show p e c u l i a r , but recurrent f l u c t u a t i o n s r e s u l t i n g from changes i n the rate o f i n c o r p o r a t i o n and e f f l u x o f a r s e n i c i n t o and out o f the c e l l s . The two phenomena seem to reach an e q u i l i b r i u m a t the time the a l g a l c e l l s reach the s t a t i o n a r y phase o f growth (about day 14). Other experiments (not shown) i n d i c a t e d that intake and e f f l u x o f ^ A s e q u i l i b r a t e d at approx imately the same l e v e l shown i n Figure 1, whether the ^ a r s e n a t e was added a t the time o f i n o c u l a t i o n , during the exponential growth phase, or during the s t a t i o n a r y phase. The a l g a l c e l l s were a l s o grown i n the presence o f arsenate, then c o l l e c t e d at a tim c e l l s per ml and the c e l l population d i d not change a p p r e c i a b l y for two or three days. The c e l l s were then c e n t r i f u g e d and washed repeatedly with an A s - f r e e medium to remove any arsenate adhering to the c e l l s u r f a c e s . The a l g a l c e l l s were then homogenized i n chloroform/methanol (2:1 v/v) and e x t r a c t e d repeatedly with t h i s solvent mixture u n t i l a l l the green pigments had been removed and the residue assumed a grayish-white appearance. Approximately one h a l f o f the a r s e n i c was i n the organic e x t r a c t ; the other h a l f remained i n the r e s i d u e . The a r s e n i c d i s t r i b u t i o n between these two phases v a r i e d somewhat from batch to batch. When the residue was t r e a t e d with 1 M HC1 and the r e s u l t i n g s o l u t i o n was d i s t i l l e d , a l l the a r s e n i c remained i n the d i s t i l l a t e as a r s e n i t e as i n d i c a t e d by polarography and by reduction to a r s i n e . Hot.water e x t r a c t e d considerable amounts o f a r s e n i c from the r e s i d u e . When four ml o f methanol were added t o one ml o f aqueous e x t r a c t the p r e c i p i t a t e contained a l l the a r s e n i c . When i n c r e a s i n g amounts o f Na2HAsÛ4 were added t o f i x e d volumes o f the water e x t r a c t and the r e s u l t i n g s o l u t i o n s were mixed with methanol, a l l the arsenate p r e c i p i t a t e d q u a n t i t a t i v e l y when 500 yg o f As or l e s s were added. A r s e n i c added i n excess o f 1 mg remained i n the supernatant. The a r s e n i c content of the p r e c i p i t a t e reached a maximum value o f approximately 1 mg As (Figure 2 ) , i n the p r e c i p i t a t e obtained from one ml of the aqueous e x t r a c t . Most o f the a l g a l growth experiments were c a r r i e d out with arsenate tagged with the gamma-emitting A s isotope. When the p r e c i p i t a t i o n s with methanol a f t e r a d d i t i o n o f n o n - r a d i o a c t i v e Na£HAs04 were repeated, but only the ' ^ A s - c o n t e n t o f the p r e c i p i t a t e s and the supernatants determined, the r e s u l t s summarized i n Figure 3 were obtained. Exchange o f n o n - r a d i o a c t i v e arsenate f o r the r a d i o a c t i v e a r s e n i c compounds occurred only a f t e r approximately 500 yg As(arsenate) had been added t o one ml o f aqueous e x t r a c t . Both the p r e c i p i t a t e formed upon methanol a d d i t i o n , and the supernatant gave p o s i t i v e ninhydrin and b i u r e t t e s t s i n d i c a t i n g the presence o f 7

7 4


120

ORGANOMETALS

AND

ORGANOMETALLOIDS

Figure 2. The arsenic content of the precipitates and supernatants obtained upon addition of arsenate and then methanol to the aqueous arsenic-containing extracts of the residue from the CHCls/CH OH extraction of T. ehui s


8.

BOTTiNO E T A L .


121

Figure S. The distribution of As activity between the precipitates and supernatants obtained upon addition of arsenate and then methanol to the aqueous arsenic-containing extracts of the residue from the CHCl /CH OH extraction of T. ehui 74

s

s


122

ORGANOMETALS


amino groups and p r o t e i n . These observations are c o n s i s t e n t with the working hypothesis that arsenate forms a complex with a p r o t e i n located i n the algal c e l l membrane p r i o r to i t s chemical transformation by the biochemical apparatus o f the c e l l . The separation o f t h i s complex by chromatographic techniques i s i n progress. I s o l a t i o n and C h a r a c t e r i z a t i o n o f A s - C o n t a i n i n g L i p i d s . The combined organic s o l u t i o n s obtained by homogenizing the a l g a l c e l l s i n chloroform/methanol were dark green. The removal o f these pigments from the a r s e n i c compounds was d i f f i c u l t . The separation scheme which l e d t o the i s o l a t i o n o f an a r s e n i c - c o n t a i n i n g l i p i d f r a c t i o n free o f green compounds i s summarized i n Figure 4. A d d i t i o n o f water t o the e x t r a c t produced a chloroform l a y e r which contained a l with acetone removed th the chloroform phase (10). Gel f i l t r a t i o n chromatography on Sephadex LH-20 o f the green p r e c i p i t a t e produced green, a r s e n i c c o n t a i n i n g bands, which were then chromatographed on DEAE C e l l ulose employing a sequence o f solvents ranging from chloroform, chloroform/methanol, (9:1 v/v) chloroform/methanol/acetic a c i d (3:1:1 v/v/v) to a c e t i c a c i d . The brownish-green a r s e n i c f r a c t i o n s were f u r t h e r p u r i f i e d by preparative high pressure l i q u i d chromatography on S i l i c a gel with chloroform/methanol/ a c e t i c acid/water (17:3.5:2:1 v/v/v/v). A s l i g h t l y brown o i l was obtained. A n a l y t i c a l HPLC o f t h i s o i l on S i l i c a gel using a Hitachi Zeeman Graphite Furnace Atomic Absorption Spectrometer as an a r s e n i c - s p e c i f i c detector showed that a t l e a s t two a r s e n i c compounds were present (Figure 5). T h i n - l a y e r chromatography on S i l i c a gel H using a mixture o f chloroform methanol a c e t i c a c i d water ( 5 0 : 2 5 : 8 : 4 , v/v) (11_) as the developing solvent showed two major a r s e n i c - c o n t a i n i n g com pounds with Rf values o f 0.41 (compound A) and 0.61 (compound B) and one major component without a r s e n i c (compound C) with an Rf o f 0.85. F r a c t i o n A was the l a r g e s t , followed by f r a c t i o n s Β and C. Compound A co-chromatographed with phosphatidyl c h o l i n e , compound Β with phosphatidyl s e r i n e and compound C with phospha t i d y l ethanolamine and monogalactosyl d i g l y c e r i d e . A standard o f sulfoquinovosyl d i g l y c e r i d e was not a v a i l a b l e at the time these experiments were r u n , but i t s Rf i n the solvent system used would have been considerably lower than 0.41. Further experiments are i n process which involve the use o f other solvent systems, appropriate standards and c o l o r reactions f o r the r e c o g n i t i o n o f s p e c i f i c chemical groups. The f a t t y a c i d compositions o f compounds A, Β and C were determined by g a s - l i q u i d chromatography and are shown i n Table I. The most remarkable c h a r a c t e r i s t i c o f these data i s the extremely low l e v e l o f C20 to C22 h i g h l y unsaturated f a t t y a c i d s . Such acids have been found at much higher concentrations i n the phospholipids o f several algae (12). It i s p o s s i b l e that the


BOTTiNO E T A L .


TETRASELMIS CHUI centrifugation

l ! =

ι Growth Medium

CELLS

homogeniz at i o n C/M 2:1

E = = ^

J

Water/Methanol

Filtrate

g e l - f i l t r a t i o n LH-20 C/M 3:1

I Discard

e

» As-CONTAINING BANDS

DEAE C e l l u l o s e Ion exchange C; C/M; C/M/A; A

1»

C M A W

= = = •

Chloroform Methanol Acetic Acid Water

Figure 4.

As-CONTAINING FRACTIONS s i l i c a gel C/M/A/W prep. HPLC H Discard

discard

ARSENIC COMPOUNDS

Scheme for the chromatographic isolation of an arsenic-containing phospholipid fraction


ORGANOMETALS


Figure 5. Chromatographic separation and detection of two arsenic compounds in a phospholipid fraction isolation from T. ehui. (HPLC Microporosil (25 cm); CHCl /CH OH/CH COOH/H O, 17:3.5:2:1;flowrate, 0.5 mL min^/l-min fractions; Hitachi-Zeeman AA Model 170-70,193.6 nm.) s

s

s

t


8.

ΒοττίΝΟ E T A L .

125


Table I . Major F a t t y Acids o f P o l a r L i p i d s from Tetraselmis ehui

Weigh Percent TLC f r a c t i o n s y

Unfractionated

d/

A

Β

d/ C

3.6

5.5

4.3

5.2

18:0

3.8

18.3

10.5

24:0

2.6

1.4

0.9

28.5

10.4

11.3

29.6

Fatty Acid 14:0

d/

16:0

13:1

12.8

e/ 16:1 w7 e/ 18:1 w9

17.5

20:1 w9

10.2

18:2 20:4

1.7 11.9 2.8

3.9

5.7

0.4

3.1

17.4

5.4

8.3 21.2 2.0

17.0 1.8

7.4

1.2 3.3 0.1

2.3 1.8

f/ UNKNOWN

— .Only f a t t y a c i d s present a t a l e v e l o f 2% o r more are i n c l u d e d . — Chain l e n g t h : number o f double bonds, w = p o s i t i o n o f f i r s t i double bond counting from the methyl end. — TLC = t h i n - l a y e r chromatography w i t h a mixture o f chloroform: methanol:acetic acid:water (50:25:8:4, v/v) as developing solvent. ~^R o f Fractions*. A = 0.41; Β = 0.61; C = 0.85. T / l t may c o n t a i n other isomers. — R e t e n t i o n time r e l a t i v e t o 18:1 = 0.60, on s i l i c o n i z e d d i e t h y l e n e g l y c o l s u c c i n a t e p o l y e s t e r a t 170°C. f


126

ORGANOMETALS

AND

ORGANOMETALLOIDS

prolonged e x t r a c t i o n procedure might have caused t h e i r l o s s . Elemental analyses o f the a r s e n i c - c o n t a i n i n g l i p i d s a f t e r chromatography on S i l i c a gel (Figure 4) showed a r s e n i c present at a l e v e l o f 0.5 percent and confirmed the presence of C, Η, Ρ and Ν i n r a t i o s c h a r a c t e r i s t i c o f those in phosphatidyl c h o l i n e . Pure phosphatidyl arsenocholine would have an a r s e n i c content o f 8.8 percent. HPLC separation and enzymatic and chemical h y d r o l y s i s experiments are in progress to i s o l a t e the a r s e n i c - c o n t a i n i n g components from the a l g a l l i p i d f r a c t i o n . In 1976 at the I n t e r n a t i o n a l Conference on Environmental Arsenic at Fort Lauderdale, we suggested (13) that a r s e n i c could replace nitrogen i n c h o l i n e . The arsenocholine I could then become part of a l i p i d 2 bonded to the phosphate group of a phosphatidyl r e s i d u e .

CH.

CH.

CH -As-CH C00 3 i c

CH -As^CH CH -0H o

2

o

2

o

CH.

CH.

CH -0-C-R 2

i

I

CH-O-C-R CH -0-f-0-CH CH -As(CHj) 2

2

2

3

0

The r e s u l t s obtained thus f a r with the a l g a l l i p i d s are c o n s i s t e n t with t h i s hypothesis. The i s o l a t i o n by Edmonds e t a l _ . , o f arsenobetaine 3 from rock l o b s t e r s (7) shows that an a r s e n i c


8.

BOTTINO E T A L .


127

compound very s i m i l a r to arsenocholine e x i s t s i n nature. I t has been observed (14, 15) that a r s e n i c - c o n t a i n i n g compounds from various organisms would y i e l d v o l a t i l e a r s i n e s upon reduction with sodium borohydride, but only a f t e r the sample had been d i gested with 2H sodium hydroxide. We subjected s y n t h e t i c samples o f arsenobetaine and arsenocholine f i r s t to sodium hydroxide d i g e s t i o n and then to sodium borohydride reduction t o determine i f methylarsines were generated. Arsenocholine, under these c o n d i t i o n s , was found to produce, at b e s t , only t i n y traces o f dimethylarsine or t r i m e t h y l a r s i n e , but arsenobetaine d i d r e a d i l y . The a r s e n i c - c o n t a i n i n g l i p i d s i s o l a t e d from T. ehui do form dimethylarsine or t r i m e t h y l a r s i n e under these c o n d i t i o n s . Be cause betaine i s not a common c o n s t i t u e n t o f l i p i d s , i t i s un l i k e l y that a r s e n i c i s incorporated i n t o l i p i d s i n the form o f arsenobetaine. Arsenocholine might, however, be capable o f being converted t o arsenobetaine conversion i s s i g n i f i c a n t i n organisms, can be answered a f t e r the a r s e n i c - c o n t a i n i n g l i p i d s have been i d e n t i f i e d . The chemical s t r u c t u r e o f the a r s e n i c compounds formed by T. ehui from arsenate should soon be known. The s t a r t i n g mat e r i a l and the end products o f t h i s b i o l o g i c a l conversion w i l l then have been i d e n t i f i e d . Future work w i l l have to be c a r r i e d out to e l u c i d a t e the biochemical pathway o f t h i s transformation. I n v e s t i g a t i o n s o f t h i s nature w i l l f i n a l l y lead to the develop ment o f metabolic charts f o r a r s e n i c and other t r a c e elements s i m i l a r to those now a v a i l a b l e f o r l i p i d s , carbohydrates, n u c l e i c acids and p r o t e i n s . U n t i l such t r a c e element metabolic pathways have been worked o u t , the d e t a i l s o f the i n t e r a c t i o n s o f t r a c e elements with l i v i n g organisms cannot be understood. Basic r e search that would e v e n t u a l l y achieve t h i s important goal has been i n i t i a t e d i n several laboratories. Acknowledgement: These i n v e s t i g a t i o n s were supported by the National I n s t i t u t e o f Environmental Health Sciences (Grant No. 5 ROI ES01125), by the Robert A. Welch Foundation o f Houston, Texas, and by the Texas A g r i c u l t u r a l Experiment S t a t i o n . Literature Cited 1. 2. 3.

4. 5.

Challenger, F . , Chem. R e v . , (1945) 36, 315. McBride, B. C., and Wolfe, R. S., Biochemistry, (1971) 10, 4312. Schrauzer, G. N . , Seek, J. Α . , H o l l a n d , R. J., Beckham, T . Μ., Rubin, Ε. Μ., and S i b e r t , J. W., B i o i n o r q . Chem., (1972) 2, 93. R i d l e y , W. P . , D i z i k e s , L., Chek, Α . , and Wood, J. Μ., Environ. Health P e r s p e c t . , (1977) 19, 43. Wood, J. Μ., S c i e n c e , (T974) 183, 1049.


128

ORGANOMETALS


5a. C u l l e n , W. R., Froese, C. L . , L u i , Α., McBride, B. C., Patmore, D. J., and Reimer, Μ., J. Organometal. Chem., (1977) 139, 61. 6. Lunde, G . , E n v i r o n . Health P e r s p e c t . , (1977) 19, 47. 7. Edmonds, J. S., F r a n c e s c o n i , Κ. Α . , Cannon, J. R., Raston, C. L . , S k e l t o n , B. W., and White, Α. Η., Tetrahedron L e t t . , (1977) (18) 1543. 8. B o t t i n o , N. R., Newman, R. D . , Cox, E . R., S t o c k t o n , R., Hoban, Μ., Zingaro, R. A. and Irgolic, K. J., J. Exp. Mar. Biol. Ecol., in p r e s s . 9. B l a s c o , F., P h y s i o l . V e g . , 13 (1975) 185. 10. Kates, Μ., "Techniques o f L i p i d o l o g y : I s o l a t i o n , Analysis and I d e n t i f i c a t i o n o f L i p i d s " . American E l s e v i e r P u b l i s h i n g Co., New York, Ν. Υ . , 1972; p. 393. 11. S k i p s k i , V. P . , Peterson, R. F., and B a r c l a y , Μ., Biochem. J., (1964) 90, 374 12. N i c h o l s , B. W., "Comparative L i p i d Biochemistry o f Photo s y n t h e t i c Organisms", i n Harborne, J. B . , e d . , "Phyto chemical Phylogeny". Academic P r e s s , London, 1970, p. 105. 13. I r g o l i c , K. J., Woolson, Ε. Α . , S t o c k t o n , R. Α . , Newman, R. D . , B o t t i n o , N. R., Zingaro, R. Α . , Kearney, P. C., P y l e s , R. Α . , Maeda, S . , McShane, W. J. and Cox, E. R., Environ. Health P e r s p e c t . , (1977) 19, 61. 14. C r e c e l i u s , Ε. Α . , Environ. Health P e r s p e c t . , (1977) 19, 147. 15. Edmonds, J. S . , and F r a n c e s c o n i , Κ. Α . , Nature, (1977) 265, 436.

Discussion Y. K. CHAU (Canada Centre f o r Inland Water Research): How do you separate the methylation due t o b a c t e r i a which generate the n e t h y l a r s e n i c compounds from c o n c e n t r a t i o n o f a r s e n i c a l s by algae? IRGOLIC: F i r s t of a l l , we t r y t o keep the b a c t e r i a out of the algae. We checked whether any t r i m e t h y l a r s i n e i s formed i n our a l g a l c u l t u r e s . We couldn't f i n d any w i t h As-74 t r a c e r s . These c u l t u r e s a r e not completely b a c t e r i a f r e e , but we b e l i e v e most of the transformation i s done w i t h i n the a l g a l c e l l and not by b a c t e r i a . M. 0. ANDREAE (Scripps I n s t i t u t e o f Oceanography): A word o f c o n f i r m a t i o n . We t r i e d t o see i f there i s a c o n t r i b u t i o n by bac t e r i a by comparing c u l t u r e s and monocultures of the same organism, rhere was no measurable d i f f e r e n c e , so I t h i n k that b a c t e r i a do aot c o n t r i b u t e s i g n i f i c a n t l y i n the k i n d of systems that you use. Did you make any attempt t o i d e n t i f y t h e substance that was released by your a l g a l system a f t e r 5 days o r whenever that b i g peak occurred? IRGOLIC: No, but there i s one experiment which I d i d n ' t de s c r i b e . We took these algae, grew them i n 10 p a r t s per m i l l i o n arsenate, and then determined t h e i r a r s e n i c l e v e l . We then r e suspended the algae i n arsenate-free medium and found that between 50% and 75% of the a r s e n i c comes r i g h t out. We would l i k e t o know


8.

BOTTINO E T A L .


129

what form the a r s e n i c i s ; i t could simply be arsenate. The a r s e nate has t o go through the c e l l w a l l , perhaps t o form an arsenate complex there. The c e l l does the t r a n s f o r m a t i o n ; i t i n c o r p o r a t e s the a r s e n i c perhaps i n t o the l i p i d s . I f you d i s t u r b the c e l l , the arsenate can migrate r i g h t back out. We i n t e n d t o do t h i s ; we have now the s e n s i t i v i t y t o determinate t h i s arsenate by p o l a r ography. ANDREAE: Did you t r y t o deacylate the f r a c t i o n i n your l i p i d s o l u b l e e x t r a c t and see i f i t could be i d e n t i f i e d by e l e c t r o p h o r e s i s o r chromatography? IRGOLIC: We t r i e d some phospholipases w i t h the a r s e n i c act i v i t i e s p a r t i t i o n e d between the organic phase and the aqueous phase. We have not yet i n t e r p r e t e d th r e s u l t s ANDREAE: We t r i e d some d e a c y l a t i o o a l g a l i p i d s , and used the water e x t r a c t . C o e l e c t r o p h o r e s i s w i t h arsenocholine o r a r senobetaine was u n s u c c e s s f u l . G. E. PARRIS (Food and Drug A d m i n i s t r a t i o n ) : I s there any comment o r suggestion regarding formation o f a bond between a r s e n i c and the two-carbon u n i t i n b e t a i n e o r arsenobetaine o r a r senocholine? How does i t occur? IRGOLIC: Somehow we have t o go from arsenate t o whatever that organic compound i s . I f i t ' s arsenocholine o r arsenobetaine, t h a t i s s i m i l a r . But i f there are many steps i n between, we r e a l l y can't t a l k i n t e l l i g e n t l y about what i s happening unless we understand that metabolic pathway. There i s some i n d i c a t i o n t h a t organoarsenic compounds produced by transformations i n marine o r ganisms, e.g., shrimp o r c r a b s , do not seem t o be t o x i c when i n gested by man, as shown by Dr. C r e c e l i u s ["Methods and Standards f o r Environmental Measurement", W. H. K i r c h h o f f , ed., Proc. E i g h t h M a t e r i a l s Res. Symp., NBS Spec. P u b l . 464, Washington, D.C., 1977, p. 495]. W. R. CULLEN ( U n i v e r s i t y o f B r i t i s h Columbia): A compound i s o l a t e d from A t l a n t i c f i s h by Environment Canada was repeatedly p u r i f i e d , and we got back an a n a l y s i s r e c e n t l y ; i t had no a r s e n i c i n i t . B a s i c a l l y , i t was b e t a i n e o r something very l i k e b e t a i n e . We suggest that somehow there i s probably an arsenate that gets a s s o c i a t e d w i t h the b e t a i n e i n some way and can be l o s t through purification. IRGOLIC: I t was i n t e r e s t i n g t o hear Dr. McBride mention the a s s o c i a t i o n which then breaks up. We might see something l i k e t h i s i n some o f the c e l l w a l l s ( e x t r a c t r e s i d u e s ) . U n f o r t u n a t e l y , I t h i n k we are i n the dark about these a s s o c i a t i o n s . RECEIVED

August 2 2 ,

1978.


9 The Chemistry of Organometallic Cations in Aqueous Media R. STUART TOBIAS Department of Chemistry, Purdue University, West Lafayette, IN 47907

A number of organometalli moderately stable in aqueou these are the following species: Group IVB, R Ge , R Sn , R Sn , R Pb , R Pb ; Group IIIB, R Ga , R In , R Tl ; Group IIB, RHg ; Group IB, R2Au ; Group VIIIA, R Pt . Several of the methyl derivatives, R = CH , can be produced by the action of methanogenic bacteria or by reaction of methylcobalamin with inorganic compounds of the appropriate metal, but data on methylation under environmental conditions are mainly limited to mercury(II). Most of the information available on the aqueous chemistry pertains to the methyl species since these have the highest s o l u b i l i t y . Characteri s t i c a l l y these ions are sigma-bonded carbanion complexes of the metal in i t s maximum oxidation state. In 1966, the aqueous chemistry of organometallic cations was reviewed (1), but interest in environmental effects of several of these species in recent years has stimulated a good deal of new work. Although the reactions of metal ions in biological systems are exceedingly complex, a knowledge of the hydrolysis constants and s t a b i l i t y constants with a limited number of ligand types will permit a number of predictions about the ionic binding and transport. [In this discussion, unless otherwise indicated, hydrolysis will refer to proton transfer from a coordinated water molecule rather than M-C or M-X bond cleavage in the presence of water.] Thermodynamics will play a large part in governing the reactions of these organometallic species. Without exception, the methyl derivatives are highly labile to substitution at the metal center. In certain cases with bulky alkyl groups, reactions may proceed more slowly because of steric effects. Heterogeneous reactions, e.g. dissolution, are slower with large alkyl groups. Because of their hydrophobic nature, they r e s t r i c t attack at the metal center. 3

2+

+

2

3

+

2+

+

2

3

+

2

2

+

+

2

+

3

3


9.

Organometallic Cations

TOBIAS

131

This review w i l l concentrate on the i n t e r a c t i o n of these organometallic c a t i o n s with the solvent water, and a t t e n t i o n w i l l be focused on those ions o f environmental i n t e r e s t , namely the d e r i v a t i v e s o f mercury(II), t i n ( I V ) , and l e a d ( I V ) . The i n t e r a c t i o n with water i s a strong one, i t i s not normally encountered i n the organometallic chemistry o f these elements, and i t has a major influence on r e a c t i o n s a t the metal c e n t e r . Some o f the more a c i d i c species e x h i b i t amphoteric behavior, i . e . the organometallic oxides d i s s o l v e i n a c i d to give c a t i o n i c and i n base to give a n i o n i c s p e c i e s . Strong solvent i n t e r a c t i o n s are responsible f o r the absence from the l i s t above o f organometallic c a t i o n s o f c e r t a i n p e r f e c t l y s t a b l e organometallic m o i e t i e s , e . g . R2Ge(IV) and RSn(IV). These i n t e r a c t so strongly with water that they normally are encountered only as the hydrated o x i d e s . An even more extreme exampl which e x i s t s i n s o l u t i o n only i n the neutral or a n i o n i c form, (CH ) As0 ". U n t i l about a decade ago, i t g e n e r a l l y was f e l t that these metal-carbon bonds were r a t h e r weak and that t h e i r s t a b i l i t y was l a r g e l y k i n e t i c and not thermodynamic (2). Recent s y n t h e t i c work on metal a l k y l s i n d i c a t e s that the metal-carbon sigma bonds o f even h i g h l y r e a c t i v e metal a l k y l s can be quite s t r o n g . For example, the mean bond d i s s o c i a t i o n energy o f the Ta-C bond i n TaiCHjîs has been determined to be 62 ± 2 kcal m o l " ( 3 ) . The observation o f b i o l o g i c a l methylation o f Hg(II) (4,5) showed t h a t the synthesis o f a l e a s t some metal-carbon bonds was p o s s i b l e even in aqueous media. Nevertheless, in s p i t e o f the strength o f the metal-carbon bonds, cleavage r e a c t i o n s i n v o l v i n g water g e n e r a l l y w i l l be thermodynamically f a v o r a b l e , because both metal-oxygen and carbon-hydrogen bonds are formed. 3

2

2

1

Reactions Involving Cleavage o f the Metal-Carbon Bonds Very l i t t l e information i s a v a i l a b l e on the pathways by which the metal-carbon bonds are cleaved i n aqueous systems. For example, most o f these c a t i o n s are s t r o n g l y r e s i s t a n t to concentrated aqueous a c i d s . For a n a l y s i s , CH3Hg(II) compounds have been decomposed by heating with concentrated n i t r i c a c i d i n a bomb a t 150° for an hour (6). Preparative data and mechanistic s t u d i e s with nonaqueous media, some o f which are described elsewhere in t h i s volume ( 7 J , can give some general g u i d e l i n e s to f a c t o r s a f f e c t i n g s t a b i l i t y . From studies on p r o t o n o l y s i s of d i a l k y l m e r c u r y ( I I ) compounds, i t has been found that the rate o f the r e a c t i o n depends upon the e l e c t r o n d e n s i t y i n the Hg-C bond being cleaved and the p o l a r i z a b i l i t y o f the metal center ( 8 ) . With, for example, the very s t a b l e l i n e a r d i a l k y l t i n ( I V ) c a t i o n s , p r o t o n o l y s i s should be slow because the R'Sn(IV) residue i s o f low p o l a r i z a b i l i t y .


ORGANOMETALS

132

R — S n — R '

—**R-H

+

AND

ORGANOMETALLOIDS

— S n — R'

s/ t\

[1]

l\

Η Χ X In a d d i t i o n , there are no strong e l e c t r o n i c e f f e c t s r e l e a s i n g e l e c t r o n density i n the t r a n s i t i o n s t a t e as occurs with c e r t a i n t r a n s i t i o n metal compounds that e x h i b i t strong trans e f f e c t s . For example, strong a c i d s , HX, cleave one a l k y l group r a p i d l y from R3AUL (9), but the product R AuXL i s s t a b l e so long as X i s a reasonable good l i g a n d . The high r e a c t i v i t y o f the t r i a l k y l g o l d ( I I I ) compounds i s due t o high trans e f f e c t o f a l k y l groups which f a c i l i t a t e s e l e c t r o n release to the proton i n the t r a n s i t i o n state i n attack on R3AUL. R Strong trans I e f f e c t ligan 2

R

R ——Air—R

•

R' — Η

+

— A u — R

[2]

H

Χ Χ Most o f the decomposition r e a c t i o n s that have been studied in organic solvents are d i s s o c i a t i v e and involve as the f i r s t step the loss o f one o r more o f the ligands besides the carbanion from the f i r s t c o o r d i n a t i o n sphere. R.ML RML, + L n--m n (m-l) n (m-l)—^Decomposition products R

R

ML

1

X

Γ

M L

3 L

ΐ J

An example i s the p r o t o n o l y s i s o f the R3AUL compounds discussed above which involves d i s s o c i a t i o n o f L i n the f i r s t s t e p . An a l t e r n a t i v e mechanism that has been suggested f o r p r o t o n o l y s i s involves an o x i d a t i v e a d d i t i o n o f HX, i . e . a p r i o r protonation o f an e l e c t r o n - r i c h metal c e n t e r . This i s not a reasonable path f o r o r g a n o - m e r c u r y ( I I ) , - t i n ( I V ) , o r - l e a d ( I V ) compounds, because there i s no stable o x i d a t i o n state two u n i t s higher. Such a mechanism may operate i n the r e a c t i o n o f a l k y l p l a t i n u m ( I I ) compounds with HC1 ( 1 0 ) . CH Pt ciL n

3

+

2

HC1—*>CH (H)Pt Cl L I V

3

Ptnci L 2

2

2

[4]

2

+ CH

4

Another type o f r e a c t i o n t h a t i s observed i n the decomp o s i t i o n o f a l k y l metal complexes i s reductive e l i m i n a t i o n , [ 5 ] . R ML n

*

m

R

n

M L

(m-l)

+

L

L5J n

(m-1 )

^

2

(n-2)

(m-1)

This Intramolecular mechanism requires a r e l a t i v e l y

stable


9.


TOBIAS

133

o x i d a t i o n state two units lower than that c h a r a c t e r i s t i c o f the a l k y l complex. For example R2AUL2"*" c a t i o n s decompose r a p i d l y to R2 + AuL-2 when L i s a r e l a t i v e l y weak donor (11 ). In aqueous s o l u t i o n , [ ( C H ^ A u f O ^ ) ? ] * decomposes a t 25° i n hours with the production o f c o l l o i d a l gold and ethane. The low thermodynamic s t a b i l i t y o f t i n ( I I ) r e l a t i v e to t i n ( I V ) makes t h i s decomposition path e n e r g e t i c a l l y unfavorable f o r a l k y l t i n ( I V ) compounds and accounts i n part f o r t h e i r high stability. I t i s p o s s i b l e that coupling occurs with d i a l k y l lead(IV) s o l u t i o n s which are much l e s s stable than t h e i r tin(IV) counterparts. The l e a d ( I I ) i o n i s one o f the observed decomposition products. Most o f the organometallic ions are q u i t e r e s i s t a n t to nucleophilic attack. In s t r o n g l y a l k a l i n e s o l u t i o n , d i m e t h y l t i n ( I V ) has been observed to decompose according to [6] (12). 3(CH ) Sn 3

2+

120H

3(CH ) Sn(0H)

ΊψΓ"

2

3

2

24

[6]

J Slow 2(CH ) SnOH 3

3

+

Sn(0H)g

40H"

2

Reactions i n Which the Metal-Carbon Bonds Remain Intact · Proton Transfer from Coordinated Solvent: Hydrolysis. The aqueous chemistry o f many o f these species i s dominated by the h y d r o l y s i s r e a c t i o n s , i . e . by proton t r a n s f e r from coordinated water molecules, and by subsequent condensation r e a c t i o n s o f the hydroxo complexes to form c l u s t e r s with several organometallic c a t i o n s . + The h y d r o l y s i s r e a c t i o n s o f the RHg , R3Sn , and R2Sn ions were studied i n some d e t a i l over ten years ago, and t h i s and other e a r l y work was reviewed i n 1966. (1) The hydrolyses o f the a l k y l m e r c u r y ( I I ) , - t i n ( I V ) , and-leadÎV) species are the only r e a c t i o n s o f organometallic c a t i o n s included i n the recent monograph on the h y d r o l y s i s o f c a t i o n s by Baes and Mesmer (13). These r e a c t i o n s are somewhat simpler than i s c h a r a c t e r i s t i c o f s t r o n g l y hydrolyzing monatomic metal s p e c i e s , because the a l k y l groups e f f e c t i v e l y block c o o r d i n a t i o n s i t e s a t the metal c e n t e r . Consequently, the c h a r a c t e r i s t i c p o l y condensation r e a c t i o n s tend to give smaller c l u s t e r s with fewer metal atoms. The a l k y l m e r c u r y ( I I ) ions are unusually strong acids f o r u n i v a l e n t c a t i o n s , comparable to H g , r e a c t i o n [ 7 ] . Α

+

+

2

2 +

R

"

H 9 +

(aq)

+

¥ ( 1 )

^=i=

R H

9° (aq) H

+

H

3 (aq) 0 +

^

E q u i l i b r i u m constants f o r R = CH3, C2H5, n-C^Hj, and n-C/jHg are c o l l e c t e d i n Table I. These ions have pK = 5 and are a c i d s comparable i n strength to the c a r b o x y l i c a c i d s . As a a


134

ORGANOMETALS

AND

ORGANOMETALLOIDS

consequence, a t pH 7 the r a t i o [C^HgOH]: [Ch^Hg ] very much favors the hydroxide, and the c a t i o n i s a r e l a t i v e l y unimportant species. At pH values i n the v i c i n i t y o f the p K , condensed species a l s o are important, r e a c t i o n [8]. The f r a c t i o n o f the a

C H

3 9 (aq) H

+

+

CF^HgOH ^ H C H g ( 0 H ) H g C H 3

3 ( a q )

log Κ = 2.37 [ 8 ] ( l £ )

+

organometal i n the polycondensed species depends upon both pH and t o t a l organometal c o n c e n t r a t i o n . Very concentrated s o l u t i o n s appear to contain small amounts o f (CH3Hg) 0 , but t h i s i s not s i g n i f i c a n t below 0.2 M (15). In general p o l y nuclear complexes are unimportant below t o t a l metal c o n c e n t r a t i o n s o f ca_. 1 0 * M, so they probably play l i t t l e p a r t i n the r e a c t i o n s of mercurials i n the environment. The d i s t r i b u t i o n o f CH Hg(II) species as a function of pH i s i l l u s t r a t e d i n Figure 1. 3

+

4

3

Table I.

H y d r o l y s i s Constants f o r Univalent Cations at 2 5 °

Organometallic

a

Species CH HgOH (CH Hg) 0H (CH Hg) 0

log * K

-4.59 . CH Hg + CH Hg0H^(CH Hg) 0H log Κ = 2.37 CH HgOH + ( C H H g ) 0 H ^ (CH Hg) 0* + H 0 log Κ = 0 . 3 - 0 . 7 -4.98 -5.12 -5.17 -6.60 -6.81 -9.1

2

3

3

+

7

9

3

2

3

3

3

3

3

5

3

+

3

C H HgOH n-C H Hg0H n-C4H HgOH (CH3) SnOH (C H5) SnOH (CH ) PbOH 2

(14) Q4)

b

3

3

Reference

p q

3

3

+

2

+

2

D

3

3

2

(15) (44) (44) (44.) (16) (16) (17)

a-The symbols used throughout f o r the e q u i l i b r i u m constants are those of the Chemical S o c i e t y Tables o f S t a b i l i t y Constants (37.); b - 20°. Most o f the other u n i v a l e n t organometallic ions are r a t h e r weak aquo a c i d s . T y p i c a l are the t r i a l k y l t i n ( I V ) species which behave as simple monoprotic a c i d s , r e a c t i o n [9]. These have R

3Sn ( +

a q )

+

H 0 2

( 1

j ^

R SnOH 3

( a q )

+

H 0 3

+

( a q )

[9]

Q_6j

pK values between 6 and 7, i . e . a c i d strengths comparable to the weak oxyacids, e . g . HC10. The pK values increase s l i g h t l y with higher a l k y l groups as a l s o i s observed with the RHg ions and as would be expected from i n d u c t i v e e f f e c t s . At neutral pH, the r a t i o [R3SnOH]:[R3Sn ] s t i l l favors the hydroxo complex as i s i l l u s t r a t e d i n the d i s t r i b u t i o n diagram for (CH3)3Sn(IV) i n Figure 1. Since the R3SnOH i s uncharged i t should f a c i l i t a t e a

a

+

+


9.

TOBIAS


135

Figure 1. Species distributions as a junction of pH for 10~ M solutions of (CH ) Sn\ CH Hg\ and (CH ) Pb\ The fraction of organometal in the binuclear complexes will increase with increasing organometal concentration. Distribution diagrams for more concentrated solutions of CH$Hg* and (CH ) Pb* can be found in Refs. 40 and 117, respectively. 3

s

3

s

s

s

3

3


136

ORGANOMETALS


the d i s t r i b u t i o n o f the organotin species into hydrophobic phases. T h i s may be o f importance f o r membrane t r a n s p o r t o f these s p e c i e s . There i s l i t t l e tendency to form polynuclear species. Data on the h y d r o l y s i s constants are c o l l e c t e d i n Table I. As i s to be expected, the t r i a l k y l l e a d ( I V ) c a t i o n s are weaker a c i d s than the t i n analogs and w i l l e x i s t as the aquated c a t i o n s a t neutral pH. For r e a c t i o n [10], the e q u i l i b r i u m (

C H

3)3Pb

+

( a q )

+

constant l o g * K

1 1

H 0 2

( 1

p-(CH ) PbOH 3

i s 9.1 (17).

3

( a q )

+

H 0 3

[10]

+

Some condensation has been

observed with concentrated s o l u t i o n s , r e a c t i o n

[11].

Figure 1 a l s o i l l u s t r a t e s the species d i s t r i b u t i o n f o r a m i l l i molar s o l u t i o n o f (CH3) Pb . The d i p o s i t i v e d i a l k y l t i n ( I V ) ions are much stronger aqup a c i d s than the t r i a l k y l s , and a t concentrations above ç a . 1 0 " M they form s i g n i f i c a n t f r a c t i o n s o f polynuclear h y d r o l y s i s products (1J5,18,19). In g e n e r a l , t h e i r strength as a c i d s i s comparable to that o f S n , and many s o l u t i o n p r o p e r t i e s o f the R2Sn ions are s i m i l a r to those o f Sn^ . The l o g value o f ( C H ) ? S n , £ 3 . 5 , (16), comparable to that f o r n i t r o u s a c i d . This does not t e l l the whole s t o r y , however, because much o f the hydrolyzed organotin i s i n the form o f species such as [ ( ( C H ) S n ) 2 ( 0 H ) 2 ] » [ ( ( C H ) S n ) ( 0 H ) ] , e t c . i n more concentrated s o l u t i o n s . Figure 2 i l l u s t r a t e s the e f f e c t o f c o n c e n t r a t i o n on the species d i s t r i b u t i o n , and Table II l i s t s the h y d r o l y s i s c o n s t a n t s . While these polynuclear complexes should be o f l i t t l e importance i n most environmental or b i o l o g i c a l systems, they w i l l make up a large f r a c t i o n o f the d i a l k y l t i n ( I V ) species i n r e a c t i o n s designed to model these systems a t 3 ^ pH ^ 8 i f the organotin concentration i s as high as m i l l i m o l a r . I t should a l s o be noted t h a t the sets o f e q u i l i b r i u m constants such as those i n Tables I and II normally do not include s o l u b i l i t y product d a t a . While a species d i s t r i b u t i o n can be c a l c u l a t e d with them f o r the e n t i r e pH range, i t w i l l be meaningless a t higher concentrations i f p r e c i p i t a t i o n o c c u r s . (See Figure 15.7 (a), r e f . 13.) This e f f e c t i s i l l u s t r a t e d i n Figure 2 f o r the 10 mM s o l u t i o n . This f i g u r e a l s o c l e a r l y i l l u s t r a t e s the amphoteric behavior o f (CH ) Sn *. Again, the analogous dimethyllead(IV) ions are much weaker a c i d s than the t i n analogs (20), and the e q u i l i b r i u m constants are c o l l e c t e d in Table I I . The p r a c t i c a l consequence o f the magnitude o f these constants i s t h a t very l i t t l e ( C H ) 2 P b e x i s t s i n s o l u t i o n a t pH 7. Approximately one equivalent o f a c i d must be t i t r a t e d to reach pH 7. 3

5

2

2+

2 +

3

3

3

2

1

2 +

2

S

3

2

4

6

2 +

2

3


2+

9.

TOBIAS

137


Figure 2.

Species distribution as a function of pH for 10 M and 10' M solutions of (CH ) Sn * 5

2

s

t

2


138

ORGANOMETALS

Table I I .

Summary o f ( C H ) S n 3

Species

3

+

2

2

2

2

3

4

[(CH )|M] (0H) (CH ) M(0H) (CH ) M(0H) 23

3

2

3

2

4

XoÔ -8.29 -4.83

3

2+

0

4

H y d r o l y s i s a t 25°

5-7.4 -15.54 -10.83

-24.31

2

6

2 +

pq

[(CH ) M] (0H) 2 3

2

ORGANOMETALLOIDS

log *K ,M=Pb(20)

pq

2

3

and ( C H ) P b

2 +

log *K ,M=Sn(18.)

(CH ) M(0H) (CH3)2M(0H)o [(CH ) M] (0H) ^ 3

2

AND

-16.17 -20.0 -32.2

-28.52

B. Complexation o Solution. Information on s t a b i l i t y constants of organometallic cations i n aqueous s o l u t i o n makes i t p o s s i b l e to p r e d i c t a number o f t h i n g s . If p a r t i c u l a r l y s t a b l e complexes are formed with a c e r t a i n f u n c t i o n a l group that i s present i n environmental o r b i o l o g i c a l systems, these groups are l i k e l y to be involved i n the t r a n s p o r t and p h y s i o l o g i c a l a c t i v i t y o f the organometal. Often organometals are introduced, as i n the case o f t i n ( I V ) compounds, as the h a l i d e s ; and a knowledge o f the s t a b i l i t y constants f o r complexes with h a l i d e ions w i l l permit a p r e d i c t i o n o f whether the h a l i d e w i l l p e r s i s t o r aquate i n natural waters. With organometallic compounds, syntheses often are c a r r i e d out with non-aqueous s o l v e n t s , a p r a c t i c e t h a t i s l e s s common with simple inorganic s p e c i e s . S t a b i l i t y data can allow p r e d i c t i o n s o f whether model compounds have s u f f i c i e n t s t a b i l i t y to be important species i n d i l u t e aqueous s o l u t i o n . Trends i n S t a b i l i t y : Hard and Soft A c i d Character. The organom e t a l l i c c a t i o n f o r which the most systematic studies on complex s t a b i l i t y have been made i s CH3Hg . It i s a very s o f t a c i d and tends to bind s t r o n g l y to s o f t bases, i . e . to heavier donor atoms ( 2 1 , 2 2 ) . This i s demonstrated by the e q u i l i b r i u m constants for r e a c t i o n [ 1 2 ] with X = halide i o n . These increase markedly +

C H

3 9 H

+

+

X'(aq)

C H

3

H g X

(aq)

[

1

2

]

with heavier h a l i d e s : log K i = 1.50 ( F " ) , 5 . 2 5 ( C I " ) , 6 . 6 2 ( B r " ) , 8 . 6 0 (I") (14). Because o f the high s t a b i l i t y o f the c h l o r i d e , bromide, and iodide complexes, these compounds have quite low aqueous s o l u b i l i t y . The p r i n c i p a l c o o r d i n a t i o n number o f mercury(II) i s two, and the CH3HgX molecules do not i n t e r a c t s t r o n g l y with water. A d d i t i o n o f C I " or B r " can be used to reverse the binding of CH3Hg to n u c l e i c a c i d s , e . g . i n agarose e l e c t r o p h o r e s i s with CH3HgOH as a denaturing agent ( 2 3 ) . +


9.


TOBIAS

139

In c o n t r a s t to the aqueous s o l u b i l i t y , the CI^HgX molecules should have reasonable l i p i d s o l u b i l i t y , and chloro complexing may be important i n f a c i l i t a t i n g transport o f methylmercury(II) in b i o l o g i c a l systems. The d i s t r i b u t i o n c o e f f i c i e n t o f CH3HgCl between toluene and an aqueous phase was found to be 1 1 . 1 , r e a c t i o n [13] (24). C H

3 9 H

C 1

(aq)

C H

3 9 H

C 1

(toluene)

™

The a l k y l t i n ( i v ) s p e c i e s behave as hard a c i d s , and i n t h i s respect are very d i f f e r e n t from the alkylmercury(II) ions which are prototype soft acids (21). Experimentally t h i s i s demonstrated by the trend i n the s t a b i l i t y constants o f the h a l i d e ion complexes, r e a c t i o n s [14]: log K] = 2.3 ( F " ) , -0.17 (CI") (25). The Me

!

+ Me

Me

+

X'

(

a

q

)

^=5:

{[(CH ) Sn(0H ) ] -x} 3

!

3

2

2

+

j|

(aq)

llr

-H 0

[14]

2

X i ,Sn

/ 1^ Me

I

%

(aq) Me

Me so small t h a t values have not been determined. I t i s l i k e l y that the complexation i n s o l u t i o n i s mainly o f the outer sphere type, because the formation o f inner sphere (CH3)3SnX requires dehydration and a s t r u c t u r a l change from a planar to a pyramidal skeleton f o r ( O ^ ^ S n * . These data together with the knowledge that s u b s t i t u t i o n a t the t i n center occurs r a p i d l y t e l l us that d i s s o l u t i o n o f ( C ^ ^ S n X , X = C I , B r , I, i n water w i l l y i e l d the same aquated species w i t h i n the time o f mixing. Even i n sea water, mean [Cl~] - 0.5 M o r blood 0.103 M, (CH3)3Sn w i l l be present p r i n c i p a l l y as the aquo c a t i o n a t pH < 5 and (CHj^SnOH at pH 7. The low e q u i l i b r i u m concentration o f (CH3)oSnCi may be important i n processes t h a t involve e x t r a c t i o n into l i p i d phases, e . g . i n membrane t r a n s p o r t , although the hydroxide w i l l a l s o be quite l i p i d s o l u b l e . Studies on the permeability o f mitochondrial membranes show that f^Sn ions mediate the transport o f C I " and OH" across the membrane and provide a means f o r r a p i d e q u i l i b r a t i o n o f pH (26). The d i s c u s s i o n o f the chemistry i n t h i s reference i s somewhat obscured by neglect o f the h y d r o l y s i s o f these ions which was discussed i n 4

+


140


the previous s e c t i o n . The R3Pb ions form only very unstable complexes with h a l i d e i o n s : Ki (not log) (CFh^Pb" ^ 6.5 ( F " ) , 2.1 ( C I " ) ; ( C H ) P b = 3.5 ( F " J , 3.7 (CI") (27,28). Also they are not so hard, since K P / K Q I i s only 3.1 for (CH3) Pb , while i t i s 3 χ 1 0 with ( C H ) S n . The R Sn^ ions form somewhat more stable complexes with a n i o n i c 1igands because o f t h e i r d i p o s i t i v e charge, but the h a l i d e complex s t a b i l i t y constants i n d i c a t e that they, too, are very hard a c i d s . For r e a c t i o n [15], the values l o g Ki are 3.70 ( F " l (25), 0.38 (CI") (29), < - 0 . 5 (Br") (29). In the ( C H ) S n - C I " system, Raman spectra demonstrate t h a t the complexing i s mainly o f the outer sphere type, and inner sphere complexing only occurs with very concentrated s o l u t i o n s where the water a c t i v i t y i s reduced to a low value. +

1

2

5

+

3

3

2

3

+

3

2

3

z

2

Me I

2+

'Sn:

+ X"

Ί

^[(CH ) Sn(0H ) ] -X"} 3

(aq)

11

2

Me

2

4

2 +

" ¥

[15]

[(CH ) SnX(0H ) f 3

2

2

n

These data c l e a r l y show the much l e s s favorable displacement o f water by the heavier h a l i d e s in the case o f R S n and R S n compared to the RHg i o n s . A p r a c t i c a l consequence i s t h a t the o r g a n o t i n ( I V ) c a t i o n s w i l l tend to i n t e r a c t r e l a t i v e l y more s t r o n g l y w i t h nitrogen and oxygen donors, p a r t i c u l a r l y n e g a t i v e l y charged ones, compared to s u l f u r donors, while the converse w i l l be true f o r the a l k y l m e r c u r y ( I I ) s p e c i e s . The R Pb* ions form much l e s s s t a b l e complexes than the t i n analogs: (CH ) Pb , 54.0 ( F " ) , 5.8 ( C I " ) ; ( C H ) ? P b 35.0 ( F " ) , 9.2 (CI") (£7,28). Thev a l s o are much l e s s hard than the t i n analogs. For~TCH ) Sn' K P / K Q ] i s C J K 2.1 χ 1 0 , while the r a t i o f o r ( C H ) P b is 9.3. S t a b i l i t y with Biomolecules and Related Ligands. The hard o r s o f t a c i d c h a r a c t e r i s o f use i n p r e d i c t i n g the types o f donors t h a t w i l l give the most s t a b l e complexes. Of a t l e a s t comparable importance i s the i n t r i n s i c b a s i c i t y o f the donor; f o r example, a mereaptide w i l l form much more stable complexes with C H H g than a t h i o e t h e r even though both ligands are s u l f u r donors. With a given type o f donor, reasonably good l i n e a r f r e e energy r e l a t i o n s e x i s t f o r protonation and m e t h y l m e r c u r i a t i o n as observed o r i g i n a l l y by Simpson (30) f o r nitrogen donors and examined more g e n e r a l l y by Erni and Geier (J31_,_32). T h i s i s i l l u s t r a t e d in Figure 3 f o r some oxygen and s u l f u r donors. As expected from the s o f t a c i d c h a r a c t e r o f CH3Hg , the i n t e r c e p t in the l o g K C H v s . log K H L P l o t i s much l a r g e r f o r the s u l f u r than the oxygen donors. S t a b i l i t y con stants f o r a number o f CH3Hg complexes are c o l l e c t e d in Table I I I . +

3

2 +

2

+

2

3

2

2 +

2

3

3

3

2

2

5

2 +

2 +

3

2 +

+

+

H

g

L

+


9.

TOBIAS



141

142

ORGANOMETALS

Table

III.

CHoHg

A N D ORG A N O M E T A L L O IDS

Selected S t a b i l i t y Constants o f Organometallic Cations with Organic Ligands.

H0CH CH S2

2

1 P H 1 6 ^ 6 3 " ' glutathionate C3H6Ô2NS-, c y s t e i n e t e C6H5S-, thiophenolate CCHÎOANOS-, 2,4-dinitrothiof (&2N)2C=S

C

N

S

16.1 15.9 15.7 14.7 10.5 6.9

(14) (45)

5.4 5.5 3.8 3.2 2.7 1.1

(49) (32) (32) (50) (50) (50)

11.8 7.1 8.2 7.6 5.0 7.9 7.2 4.7

(32) (32)

(45) (46) (47) (32)

S(CH?CH C S(CH CH 0H 2

2

2

^ 7 ^ 5 ^ 2 ~ tropolonate C5H5Û"phenolate C6H4O3N" 4 - n i t r o p h e n o l a t e CH C0 " HC0 C1 HC0 " 3

2

2

2

2

3 3 2 ~ imidazolate C H 4 N imidazole H2NCH?CH2NH2 CH3NH2 (Cfl3)3N N H 2 C H C 0 " glycinate 1 2 8 2 1,10-phenanthroline C5H5N p y r i d i n e

C

H

N

3

2

2

C

(CH ) Pb 3

3

+

H

2

N

C-ioHoNo 1,10-phenanthroline C6fl40 N- picolinate C5H70 "acetylacetonate

3.9 5.3 6.0

(35) (35) (35)

CH C0 "

2.6

(54)

2

2+

2

2

2

2

(CH ) Pb 3

2

2+

(52) (53)

1.2 (U) 0.97,0.54(1_7,54) 0.86 (II) 0.52 (U)

2

2

3

(51) (51) (51)

C c H 9 0 2 " pivalate CH C0 " HC0 ~ C1CH C0 " 3

(CH ) Sn

(14)

3

2

(log K

2


1.0)

9.

TOBIAS

Organometallie Cations

143

Few data are a v a i l a b l e f o r s t a b i l i t y constants o f other organometallic ions with such l i g a n d s . Recently, some data f o r complexes o f (CH3)3Pb with carboxylates have been reported by Sayer e t a]_. (17). A rough l i n e a r f r e e energy r e l a t i o n i s observed between complexàtion by ( C H j ^ P b * and p r o t o n a t i o n , and t h i s i s i l l u s t r a t e d i n Figure 4. S i m i l a r behavior i s to be expected f o r the R3$n i o n s , although the s t a b i l i t y constants with hard bases should be s l i g h t l y l a r g e r . Consistent with t h i s i s the observation t h a t phosphate b u f f e r s , pH 7 . 4 , decrease the binding o f ^ H c ^ S n * to r a t hemoglobin (33). Phosphate buffers a l s o were observed to decrease the e x t r a c t i o n o f (C2H5h Sn(IV) i n t o a chloroform phase. In a q u a l i t a t i v e study o f complexing o f R3Sn species with a number o f b i o l o g i c a l molecules, no evidence could be found f o r complexes with ATP, g l u t a t h i o n e , a r g i n i n e , o r l y s i n e i n a borate-EDTA buffer a t pH 8.4 (34). The R£Sn and R 2 P b species probably form a much l a r g e r v a r i e t y o f complexes than the t r i a l k y l s p e c i e s , but the extensive h y d r o l y s i s o f the c a t i o n s has made the accurate d e t e r mination o f s t a b i l i t y constants by p o t e n s o m e t r i c t i t r a t i o n s very d i f f i c u l t . While t h i s i s the c l a s s i c a l technique f o r such determinations, i t r e q u i r e s an accurate knowledge o f a l l the h y d r o l y s i s constants as well as the l i g a n d protonation constants. To complicate matters, mixed hydroxide-1igand complexes, e . g . [R2$n(0H) L ], often w i l l be formed g i v i n g a l a r g e number o f p o s s i b l e species and rendering the a n a l y s i s o f t i t r a t i o n data even more d i f f i c u l t . While CF^Hg"*" i s e x t e n s i v e l y hydrolyzed i n s o l u t i o n s with pH > 1, i t s e s s e n t i a l l y monofunctional c h a r a c t e r precludes the formation o f mixed hydroxo-1igand complexes and permits the r e l a t i v e l y s t r a i g h t forward determination o f s t a b i l i t y c o n s t a n t s . A few data have been obtained f o r complexes o f (CH3)2$n with bidentate l i g a n d s , using a computer to analyze the data (35,36). As i s expected from the hard a c i d c h a r a c t e r o f ( C H 3 l ^ S n , the most s t a b l e complex i s formed with the negative bidentate oxygen donor, a c e t y l acetonate, l o g Ki 6 . 0 ; and the l e a s t s t a b l e complex was formed with the neutral bidentate nitrogen donor 1,10 phenanthroline, l o g K] 3 . 9 . The s t a b i l i t y o f the a c e t y l acetonate complex i s s i m i l a r to t h a t o f the N i , complex, l o g Κ ι ^ 6.0 ( 3 7 ) » while the phenanthroline complex i s much l e s s s t a b l e than f o r N i , l o g Κ] ^ 8.6 (37). +

+

4

+

2+

2+

n

m

2+

2+

2 +

2 +

Future Prospects For the organometallic c a t i o n s t h a t are e x t e n s i v e l y hydrolyzed i n s o l u t i o n , s p e c t r o s c o p i c techniques can be used to determine both e q u i l i b r i u m constants and i n many cases the binding s i t e s . While n e i t h e r the organometallic c a t i o n s nor most o f the l i g a n d s o f i n t e r e s t have s u i t a b l e e l e c t r o n i c t r a n s i t i o n s , both Raman and nmr spectroscopy have been used s u c c e s s f u l l y . Resonances o f both the c a t i o n and


ORGANOMETALS

AND

pivalate

ORGANOMETALLOIDS

Ο

1.0

/ HC0 "

ί

2

Ο

I y

CH3C02"

)

-

acetylgiyc i n a t e - ^

/ /

Ο

0.5 '2.5

°

CICH C0 " 2

3.0

2

3.5

4.0 logK

4.5

50

H L

Figure 4. Stability constants for (CH ) Pb* with carboxylic acids; log K ) PbL vs. log K . Data from Kef. 17. s

(CHs

S

s

HL


TOBIAS

9.

Organometallie Cations

145

CHÔH

1201

572'

CH HgOH 3

2

• Ç

'CH^g-N^.H

α 1616 1 ^ 1 5 7 9 ^

Figure 5. Raman perturbation differ ence spectra for the CH Hg(II)-pyridine system at pH 8.4, 6.5, and 4.4. In each set, A is the CH Hg(H) + pyridine vs. CH Hg(II) difference spectrum, while Β is the CH Hg(II) + pyridine vs. pyridine difference. Rehtive ordinate expansions are indicated at the right. s

s

3

1800

1600

1400

1007 1200 1000 800' 600 ' FREQUENCY (CM-1)

s

400


ORGANOMETALS AND

146

ORGANOMETALLOIDS

1igand protons have been used to study many r e a c t i o n s o f CH3Hg with amino acids (38) and with the nucleoside i n o s i n e (39). Raman p e r t u r b a t i o n d i f f e r e n c e spectroscopy has been used to study the r e a c t i o n s o f CH^Hg" " with pyrimidine (40,42,43) and purine (39,41,42) n u c l e o t i d e s as well as with c a l f thymus DNA (43). Figure 5 i l l u s t r a t e s the a p p l i c a t i o n o f the Raman technique to the simple C H o H g i l l J - p y r i d i n e system. A t pH 8 . 4 , the CH Hg(II) + p y r i d i n e v s . CH Hg(II) d i f f e r e n c e gives j u s t the spectrum o f p y r i d i n e showing that no r e a c t i o n o f the methylmercury c a t i o n has taken p l a c e . S i m i l a r l y the CH3Hg(II)+ p y r i d i n e ys_. p y r i d i n e d i f f e r e n c e gives j u s t the spectrum o f CH3HgOH. At pH 6.5 and 4.4 both spectra show extensive perturbations. The 1021 c n H band o f the complex i s s u f f i c i e n t l y well resolved from the cm-1 band o f the pyridiniu concentrations o f unreacted 1igand using the Raman i n t e n s i t i e s . While 100% i s unreacted a t pH 8 . 4 , the values are 20% and ç a . 0%, r e s p e c t i v e l y a t pH 6.5 and 4 . 4 . This i s i n good agreement with values c a l c u l a t e d from the known s t a b i l i t y constant. The Raman d i f f e r e n c e technique i s p a r t i c u l a r l y s e n s i t i v e in d e t e c t i n g small amounts o f r e a c t i o r ^ a n d both Raman and nmr can be used to determine the product d i s t r i b u t i o n . These s p e c t r o s c o p i c techniques should be e q u a l l y s u i t a b l e f o r the study o f R Sn and R 2 S n i n t e r a c t i o n s , and a s t a r t a l r e a d y has been made with R3Pb chemistry (17). 1

3

3

2+

3

+

Acknow!edgements. This work has been supported, i n p a r t , by the National Science Foundation Grant CHE 76-18591 and by the P u b l i c Health S e r v i c e , Grant AM-16101 from the National I n s t i t u t e f o r A r t h r i t i s , Metabolism, and D i g e s t i v e Diseases. The author a l s o would l i k e to express h i s a p p r e c i a t i o n to the O f f i c e o f Naval Research, as p a r t o f t h i s material was presented a t a workshop on organotin chemistry i n February, 1978. Thanks are due Mary R. M o l l e r f o r the C H H g ( I I ) - p y r i d i n e spectra. 3

Literature Cited 1. 2. 3.

4. 5.

T o b i a s , R. S., Organometal. Chem. Revs. (1966), 1, 93. Coates, G. E. "Organometallic Compounds," p 72, Methuen, London, 1960. A d e d e j i , F . Α . , Connor, J. Α . , Skinner, Η. Α . , G a l y e r , L . and W i l k i n s o n , G . , J. Chem. Soc. Chem. Commun. (1976), 159. Wood, J. M . , Kennedy, F . S . , and Rosén, C . G . , Nature (London) (1968), 220, 173. Jensen, S. and Jernelöv, Α . , Nature (London) (1969), 223, 753.


9.

TOBIAS


6.

Β.

147

Schellenberg, M., Ph.D. Thesis, Eidgenössische Technische Hochschule Zürich, 1963. 7. Kochi, J. K., Factors Involved in the Stability of Alkyl— Metal Bonds, this volume. 8. Nugent, W. A. and Kochi, J. K., J. Amer. Chem. Soc. (1976), 98, 273. 9. Komiya S. and Kochi, J. K., J. Amer. Chem. Soc. (1976), 98, 7599. 10. Belluco, U., Giustiniani, M., and Graziani, M., J. Amer. Chem. Soc. (1967), 89, 6494. 11. Kuch, P. L. and Tobias, R. S., J. Organometal. Chem. (1976), 122, 429. 12. Tobias, R. S. and Freidline, C. E., Inorg. Chem. (1965), 4, 215. 13. Baes, C. F., J r . an Cations," Wiley, Ne 14. Schwarzenbach, G. and Schellenberg, M., Helv. Chim. Acta. (1965), 48, 28. 15. Rabenstein, D. L., Evans, C. Α., Tourangeau, M. C., and Fairhurst, M. T., Anal. Chem. (1975), 47, 338. 16. Tobias, R. S., Farrer, H. N., Hughes, M. B., and Nevett, Α., Inorg. Chem. (1966), 5, 2052. 17. Sayer, T. L., Backs, S., Evans, C. Α., M i l l a r , Ε. K., and Rabenstein, D. L., Can. J. Chem. (1977), 55, 3255. 18. Tobias, R. S. and Yasuda, M., J. Phys. Chem. (1964), 68, 1820. 19. Tobias, R. S., Ogrins, I., and Nevett, Β. Α., Inorg. Chem. (1962), 1, 638. 20. Freidline, C. E. and Tobias, R. S., Inorg. Chem. (1966), 5, 354. 21. "Hard and Soft Acids and Bases," R. G. Pearson, E d . , Dowden, Hutchinson, and Ross, Strondsburg, Pa., 1973. 22. Pearson, R. G., J. Chem. Educ. (1968), 45, 581. 23. Bailey, J. M. and Davidson, N., Anal. Biochem. (1976), 70, 75. 24. Gruenwedel, D. W. and Davidson, N., J. Mol. B i o l . (1966), 21, 129. 25. Cassol, A., Magon, L., and Barbieri, R., Inorg. Nuclear. Chem. Lett. (1967), 3, 25. 26. Selwyn, M. J., Dawson, A. P., Stockdale, M., and Gains, Ν., Eur. J. Biochem. (1970), 14, 120. 27. P i l l o n i , G. and Magno, F., Inorg. Chim. Acta. (1970), 4, 105. 28. P i l l o n i , G. and Magno, F., Inorg. Chim. Acta. (1971), 5, 30. 29. Farrer, H. N., McGrady, M. M., and Tobias, R. S., J. Amer. Chem. Soc. (1965), 87, 5019. 30. Simpson, R. B., J. Amer. Chem. Soc. (1964), 86, 2059. 31. Geier, G., Erni, J., and Steiner, R., Helv. Chim. Acta. (1977), 60, 9. American Chemical S o c i e t y Library 16th St. N . Brinckman, W. In Organometals1155 and Organometalloids; F., et al.; ACS Symposium Series; American Chemical Washington, DC, 1979. Washington, D. C. Society: 20036

ORGANOMETALS AND

148

32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42. 43.

44. 45. 46. 47. 48. 49. 50. 51.

52. 53. 54.

ORGANOMETALLOIDS

E r n i , I. W., Ph.D. T h e s i s , Eidgenössische Technische Hochschule Z u r i c h , 1977. Rose, M. S., Biochem. J. (1969), 111, 129. A l d r i d g e , W. N. and S t r e e t , B. W., Biochem. J. (1964), 91, 287. Tobias, R. S. and Yasuda, M., Inorg. Chem. (1963), 2, 1307. Yasuda, M. and Tobias, R. S., Inorg. Chem. (1963), 2 , 207. " S t a b i l i t y Constants o f Metal-Ion Complexes," L. G. S i l l é n and A. E. M a r t e l l , e d s . , Special P u b l i c a t i o n No. 17, 1964, and No. 25, 1971, The Chemical S o c i e t y , London. Rabenstein, D. L., A c c t s . Chem. Res. (1978), 11, 100. Mansy, S. and Tobias, R. S., Biochemistry (1975), 14, 2952. Mansy, S., Wood, T. E., Sprowles, J. C., and Tobias, R. S., J. Amer. Chem. Soc. (1974), 96, 1762. Mansy, S. and Tobias, R. S., J. Amer. Chem. Soc. (1974), 96, 6874. Mansy, S., F r i c k , J. P., and T o b i a s , R. S., Biochim. Biophys. A c t a . (1975), 378, 319. Chrisman, R. W., Mansy, S., P e r e s i e , H. J., Ranade, Α., Berg, Τ. Α., and Tobias, R. S., B i o i n o r g . Chem. (1977) 7, 245. Z a n e l l a , P., Plazzogna, G., and Tagliavini, G., Inorg. Chim. A c t a . (1968), 2, 340. Simpson, R. B., J. Amer. Chem. Soc. (1961), 8 3 , 4711. Schwarzenbach, G. and K a r l e n , U., quoted i n ref. 32. Gross, H., Diplomarbeit, ΕΤΗ Zürich, quoted i n ref. 32. Fehr, R., Diplomarbeit, ΕΤΗ Zürich, quoted i n ref. 32. S t e i n e r , R., quoted i n ref. 32. L i b i c h , S. and Rabenstein, D. L., A n a l . Chem. (1973), 45, 118. Rabenstein, D. L., Ozubko, R., L i b i c h , S., Evans, C. Α., F a i r h u r s t , M. T., and Suvanprakorn, C., J. Coordn. Chem. (1974), 3, 263. Anderegg, G., Helv. Chim. A c t a . (1974), 57, 1340. Hohl, H., Diplomarbeit, ΕΤΗ Zürich, (1968), quoted i n ref. 32. Pilloni, G., M i l a n i , F., Inorg. Chim. A c t a . (1969), 3, 689.

R E C E I V E D August 28,

1978.


10 Organosilanes as A q u a t i c A l k y l a t o r s of M e t a l Ions RICHARD E. DESIMONE Department of Chemistry, Wayne State University, Detroit,MI48202

Much of the chemistr f t thi symposiu ha i recent years received ver has been studied in dept fro variety o perspectives. Ques tions which are now being asked in these areas have become quite detailed and sophisticated, although admittedly much still re mains to be learned. In contrast to this situation, that sur rounding the element silicon, its environmental significance and relevant chemistry, is still in its infancy. Indeed the answer to the simple question "What, if any, is the environmental sig nificance of organosilicon compounds?" is far from clear at this point in time. It is the purpose of what follows to point out some basic and potentially relevant chemistry of silicon and to try to focus to some small degree on i t s environmental import. Silicon:

Occurance and Distribution

It is commonly known that silicon is one of the most abun dant of elements (TABLE I) and also one of the most widely dis tributed (1). The vast majority is tied up in silicate rocks TABLE I.

SILICON - HOW MUCH, AND WHERE? Earth's surface - land, sea and air Oxygen 53.3% Silicon - 15.9% Hydrogen - 15.1% Aluminum - 4.8% Average silicon content 2.77 χ 10 ppm in earth's crust 40 ppm in man 3 ppm in seawater 5


150

ORGANOMETALS AND

ORGANOMETALLOIDS

and m i n e r a l s where i t i s r e l a t i v e l y immobile and of l i t t l e concern to us. I n l i v i n g organisms, s i l i c o n p l a y s (and has played) an important r o l e i n n e a r l y a l l stages of e v o l u t i o n a r y development. From s i l i c a t e b a c t e r i a t o protozoa, algae, and the h i g h e r p l a n t and animal organisms, n e a r l y a l l c o n t a i n and use s i l i c o n i n one form or another. I t i s not the purpose of t h i s a r t i c l e to survey the expanding area of b i o - o r g a n o s i l i c o n chemistry (2) nor t o consider the innumerable occurrences of s i l i c o n i n l i v i n g organisms ( 3 ) . I t i s worth mentioning however, t h a t among the myriad of known s i l i c o n compounds i n a wide v a r i e t y of b i o t a , there i s a conspicuous absence of t r u e o r g a n o s i l i c o n molecules. In the higher animal organisms f o r example, s i l i c o n t y p i c a l l y occurs as o r t h o - and o l i g o s i l i c i c a c i d s and s i l i c a t e s , ortho and o l i g o s i l i c i c e s t e r s of carbohydrates, p r o t e i n s , s t e r o i d s , l i p i d s and p h o s p h o l i p i d s , and much has been l e a r n e d i ganisms a great d e a l c e r t a i n l y remains to be d i s c o v e r e d . We know very l i t t l e about the occurrence of n a t u r a l o r g a n o s i l i c o n compounds and we can by no means make the assumption t h a t they do not e x i s t . Much work i s needed i n t h i s area. At t h i s p o i n t i n time and f o r the purposes of t h i s a r t i c l e i t seems t h a t what should be of concern i s not the occurrence of t o x i c o r g a n o s i l i c o n compounds, but the p o s s i b i l i t y t h a t r e a c t i o n s of s i l i c o n compounds w i t h other,more g e n e r a l l y troublesome metals or m e t a l l o i d s , w i l l produce s p e c i e s which are i n f a c t s i g n i f i c a n t l y damaging from an environmental v i e w p o i n t . I n p a r t i c u l a r we w i l l focus on organo group t r a n s f e r r e a c t i o n s i n aqueous media. S i l i c o n as an Organo Group Donor Organo group t r a n s f e r from s i l i c o n has been known f o r some time, the f i r s t r e p o r t e d i n s t a n c e being i n 1896 w i t h the format i o n of an organomercurial, p-dimethylaminophenylmercurie c h l o r i d e ( 4 ) . The r e a c t i v i t y of the c h l o r o s i l a n e i s not s u r p r i s i n g

+

s i n c e these are among the more r e a c t i v e of s i l i c o n s p e c i e s . However, s i m i l a r organo group t r a n s f e r r e a c t i o n s occur i n aqueous media w i t h a l k y l a r y l s i l a n e s , w i t h t r a n s f e r of the a r y l group, and these proceed r e l a t i v e l y q u i c k l y to s u b s t a n t i a l completion (5).


10.

DESIMONE

151

Organosihnes

f—\ H

3

EtOH

Si

H

+ SC1

CH

— \0)— < 3>3

C

H C—(Ο)— 8 H

C 1

+

h

(CH ) SiOH

3

/—\ (O/ HCΛ -

2

3

>

Q

+ HCl

3

(2)

HOAc S 1 ( C H

+

3)3

Hg(0Ac)

>

2

7

H

3

^0)"

Ç((^$1C1 + 2

S i G 1

5

3

A few r e p r e s e n t a t i v e

(^^

+

2

3

+

+ GaCl

+

2

3

HCl
(CH ) SiCl

3

3

(CH ) Si - 0 - Si(CH ) 3

k

I

^Q^Sb0(0H)

(CH )i Si

S h C 1

3

+

3

+ GaCl

3

3

(CH ) SiCl 3

3

'

H 0

(4)

2

+ CH GaCl

2

> CH GaCl

2

3

3

+

(5)

((CH ) Si0) 3

2

(6)

n

(6) i s noteworthy i n t h a t the s i l i c o n compound, h e x a m e t h y l d i s i l oxane, i s u s u a l l y considered t o be the s i m p l e s t s i l i c o n e . Thayer has reported t h a t t h i s molecule r e a c t s - w i t h mercuric s a l t s i n a very complex r e a c t i o n y i e l d i n g ^20 products ( 9 ) . The other major c l a s s o f a l k y l a t i n g agents among s i l i c o n compounds i s the o r g a n o f l u o r o s i l i c a t e s . These have been extens i v e l y s t u d i e d by Wuller and co-workers (6, 10). Reaction w i t h H g C l , i n the presence o f NHi+Cl t o i n c r e a s e s o l u b i l i t y , occurs r e a d i l y t o g i v e monomethyl and dimethylmercurie s p e c i e s . 2

20°C HgCl + ( N H ) ( C H S i F ) 2

1+

2

3

5

> Η 0/ΝΗ^01

CH HgCl + (NH ) ( S i F C l ) 3

l+

2

5

2

(

7

)

100°C CH HgCl + ( N H ) ( C H S i F ) - ^ - ^ (CH ) Hg + (NH^) ( S i F C l ) 3

1 +

2

3

5

3

2

2


5

ORGANOMETALS AND

152

ORGANOMETALLOIDS

S i m i l a r l y , elements such as Sb and B i can be a l k y l a t e d or a r y l a ted w i t h s u b s t a n t i a l y i e l d s (11).

SbF + 3 ( N H ) ( C H S i F ) 3

4

2

3

> H0

5

Sb(CH ) 3

+ 3(ΝΗ ) (SiF )

3

4

2

6

(8)

2

Bi(OH)

/(")V-SiF

+ 3

3

\

( 10" M" s e c " f o r both DSS TSP, but more r a p i d l y w i t h TSP. For Hg(0Ac) and DSS k=6.6xl0" M" s e c " ( 9 ) . 3

3

2

2

3

2

1

6

1

1

2

1

t

1

1

2

3

2

1

1


10.

DESIMONE

Organosilanes

153

S p e c u l a t i o n on the mechanism has centered on two p o i n t s ; an i n t r a m o l e c u l a r acid-base i n t e r a c t i o n between the s i l i c o n and a t e r m i n a l oxygen of the anion, and the a c i d i t y of the a t t a c k i n g (and q u i t e v a r i a b l e ) mercuric s p e c i e s . The former (12) would be c o n s i s t e n t w i t h the f a c t that TSP always r e a c t s f a s t e r than DSS, which has an e x t r a carbon and would probably not adapt as w e l l to the required- conformation. The second p o i n t , the nature of the a t t a c k i n g mercuric s p e c i e s , i s g e n e r a l l y of great s i g n i f i c a n c e (for any metal). Anion dependent and pH dependent s t u d i e s r e v e a l dramatic e f f e c t s on r a t e f o r these environmental parameters (14). The d e t a i l e d understanding o f any transmethylation r e a c t i o n w i l l r e q u i r e adequate s p e c i a t i o n o f a l l r e a c t a n t s and products under any g i v e n s e t of c o n d i t i o n s . This has g e n e r a l l y been one of the major weaknesses of most s t u d i e s to date. In some work c u r r e n t l the f a c i l e t r a n s f e r of number of 1-organosilatranes i n p r o t i c or a p r o t i c media (15). >p 1 R-Si(OCH CH ) N 2

2

+

3

HgX

+

2

> RHgX

>t 1 X-Si(OCH CH ) N 2

2

(11)

3

I n t e r e s t i n g l y , the parent o r g a n o t r i a l k o x y s i l a n e s a r e i n e r t under s i m i l a r r e a c t i o n c o n d i t i o n s . Presumably the e x t r a e l e c t r o n dens i t y a t the s i l i c o n a c t i v a t e s the Si-C bond t o a t t a c k by H g ( I I ) . Environmental

Considerations

We come now t o the environmental s i g n i f i c a n c e of the chemis t r y j u s t discussed. I n the absence o f s i g n i f i c a n t knowledge of n a t u r a l l y o c c u r r i n g o r g a n o s i l i c o n compounds, i t seems worthw h i l e t o consider the impact of man-made o r g a n o s i l i c o n compounds. By f a r the l a r g e s t group of such substances a r e the s i l i c o n e s , which have found l i t e r a l l y hundreds of uses (Table I I ) , almost a l l i n s p i r e d because o f the chemical i n e r t n e s s of the polymeric m a t e r i a l (16, 17). The estimated s i l i c o n e market i n the United States i n 1973 was 91 m i l l i o n pounds. Most a p p l i c a t i o n s i n v o l v e complete r e l e a s e of the s i l i c o n e i n t o the environment. Most uses, except f o r the proposed use i n e l e c t r i c a l transformers as a replacement f o r PCB s, i n v o l v e very s m a l l q u a n t i t i e s (18). A l e g i t i m a t e question a t t h i s p o i n t seems to be What u l t i m a t e l y happens t o a l l of t h i s m a t e r i a l ? " There appears to be no published work on m i c r o b i a l demethylation of s i l i c o n ( e s ) and i t i s not c l e a r how v a r i o u s environmental f a c t o r s a f f e c t the degradation o f the polymeric m a t e r i a l . Dow-Corning has reported that moist s o i l seems t o be the most d e s t r u c t i v e environment t o f

M


154

ORGANOMETALS AND

ORGANOMETALLOIDS

TABLE I I . APPLICATIONS OF SILICONES (16)

1) 2) 3) 4) 5) 6)

Waxes and p o l i s h e s Foaming and antifoaming agents Release agents ( i n molding processes) P r o t e c t i v e coatings Lubricants Cosmetics

n)

Cooling

* a "bulk volume" a p p l i c a t i o n (pending)

the s i l i c o n e molecule (18). i t i s p o s t u l a t e d that the degradation process begins w i t h a d e - p o l y m e r i z a t i o n of the long s i l o x a n e chain to form v o l a t i l e c y c l i c s i l o x a n e s of 4-5 S i - 0 u n i t s , a process w i t h a h a l f l i f e of VLO days. Under the i n f l u e n c e of moisture, oxygen and u.v. l i g h t , the v o l a t i l e c y c l i c s i l o x a n e s are then presumed to degrade i n the atmosphere to SiU2, H2O, and CO2. D e t a i l s on these s t u d i e s are l a c k i n g . One i s c u r i o u s to know f o r example whether the s o i l i n the experiments was s t e r i l e , whether the process could be i n t e r r u p t e d or a l t e r e d by the pre sence of other substances such as methyl acceptors, and whether *C l a b e l i n g s t u d i e s have been used to t r a c e the methyl group carbon to product CO2. H o p e f u l l y these d e t a i l s w i l l be f o r t h coming . I n other experiments, Dow-Corning r e p o r t s that a 15% emul s i o n of s i l i c o n f l u i d subjected to the a c t i o n of a c t i v a t e d sewage sludge f o r a p e r i o d of 70 days showed no evidence of bio-degrada t i o n (18). However Bellama r e p o r t s that r e l a t i v e l y low v i s c o s i t y s i l i c o n e f l u i d s w i l l methylate H g ( I I ) ( 1 9 ) . I n summary, we have b a r e l y scratched the s u r f a c e of some very i n t e r e s t i n g and p o s s i b l y s i g n i f i c a n t chemistry. Much work remains to be done i n areas mentioned and s u r e l y i n others not yet discovered. ll

Literature Cited

1. 2.

Ochiai, Ε., "Bioinorganic Chemistry, An Introduction", 5-12, A l l y n and Bacon, Inc., Boston, 1977. Voronkov, M.G., Chem. Brit.(1973) 9, 411.


pp.

10.

DESIMONE

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Organosilanes

155

Voronkov, M.G., Zelchan, G.I., Lukevitz, E.J., " S i l i c o n and L i f e " , Zinatne Publishing House, Riga, 1971. Combes, C., Compt. Rend., (1896), 122, 622. Eaborn, C., "Organosilicon Compounds", Butterworths, London, 1960. Müller, R., Organometal. Chem. Revs., (1966), 1, 359. Jakubowitch, A. J., and Mozarew, G.W., J. Gen. Chem. USSR, (1953), 23, 1414. Schmidbaur, H., Angew. Chem., (1964), 76, 753. Thayer, J.S., personal communication. Müller, R. and Dathe, C., Chem. Ber. (1965), 98, 235. Müller, R. and Dathe, C., Chem. Ber. (1966), 99, 1609. DeSimone, R.E., J.C.S. Chem. Comm. (1972), 780. Bellama, J.M. and Nies, J.D., personal communication. Jewett, K.L., Brinckman paper. Bellama, J.M. and Nies, J.D., J.C.S. Chem. Comm., submitted. Howard, P.H., Durkin, P.R., and Hanchett, A., "Assessment of Liquid Siloxanes", Report # PB 247778, National Technical Information Service, U.S. Dept. of Commerce, 1974. Calandra, J.C., Keplinger, M.L., Hobbs, E.J., and Tyler, L.J. Polymer Preprints, (1976), 17, 1. Pollution Engineering, Aug., 1977, p. 41. Bellama, J.M., personal communication.

Discussion G. E. PARRIS (Food and Drug A d m i n i s t r a t i o n ) : Have there been d i r e c t t o x i c i t y s t u d i e s on s i l i c o n e s from the standpoint of e n v i ronmental impact?. DeSIMONE: There have been many s t u d i e s on d i r e c t t o x i c i t y , i n c l u d i n g intravenous s t u d i e s . Except f o r minor eye i r r i t a t i o n i n c e r t a i n cases, these appear harmless t o j u s t about e v e r y t h i n g . PARRIS: The r e a c t i o n s you showed suggest that s i l i c o n e s may be new m e t h y l a t i n g agents i n the environment. DeSIMONE: I t remains t o be found out whether i n f a c t enough o r g a n o s i l i c o n compounds do e x i s t i n the environment, where they e x i s t , and whether what you j u s t suggested i s t r u e . PARRIS: One of the c r i t i c a l p l a c e s w i l l be i f s i l i c o n e s f i n d more use as d i e l e c t r i c f l u i d s . I doubt that s i l i c o n e s w i l l be the major replacement f o r PCB's. I t h i n k t h a t most people who a r e d e a l i n g w i t h the t o x i c i t y o f s i l i c o n e s and the importance o f e n v i ronmental chemistry of s i l i c o n e s have not considered them as pot e n t i a l m e t h y l a t i n g agents. C. FREY (Dow Corning C o r p o r a t i o n ) :

I would l i k e t o make a


156

ORGANOMETALS AND

ORGANOMETALLOIDS

couple of comments on the i m p l i c a t i o n s of the paper. The data there showing 91,000,000 l b s . of s i l i c o n e s g a i n i n g entry i n t o the environment could very w e l l be an item f o r some concern, but the great b u l k of t h a t m a t e r i a l , as many people know, i s p o l y d i m e t h y l s i l o x a n e . I t ' s i n t e r e s t i n g to note that the subsequent work of Dr. Thayer [vide i n f r a ] seems to show that mercuric n i t r a t e , which was j u s t about the most potent m e c u r i a l c l e a v i n g agent, i s without e f f e c t on c y c l o d i m e t h y l s i l o x a n e s a f t e r s e v e r a l months of contact. So the n o t i o n t h a t the o r g a n o s i l i c o n compounds, which are not natu r a l l y - o c c u r r i n g but f i n d t h e i r way i n t o the environment, w i l l be cleaved by mercury i s probably d i f f i c u l t to s u s t a i n . De SIMONE : The question i s not whether mercury w i l l cleave these compounds, but r a t h e r during the decomposition ( i n moist s o i l as you suggest i s the most e f f i c i e n t wa to do i t ) what hap pens i f some methyl accepto cleavage products f l o a t i n g FREY: I t h i n k i t ' s important to d i f f e r e n t i a t e between what might be c a l l e d environmental chemistry and the chemistry of s p i l l s ; that i s , what happens i f you have l a r g e concentrations of s i l o x a n e i n contact w i t h l a r g e concentrations of some metal. On an environmental s c a l e , I t h i n k one has to be concerned w i t h the r e a c t i o n of m e t h y l s i l i c o n compounds w i t h n a t u r a l l y - o c c u r r i n g e l e ments or compounds. As f a r as the s o i l i s concerned, w e ' l l present the d e t a i l s t h i s summer at the 5th I n t e r n a t i o n a l O r g a n o s i l i con Symposium i n Karlsruhe [August, 1978]. There i s no unusual chemistry there t h a t i n v o l v e s the c a r b o n - s i l i c o n bonds themselves. J . J . ZUCKERMAN ( U n i v e r s i t y of Oklahoma): The h a l f - l i f e of 10 days, d i d that r e f e r t o the d e p o l y m e r i z a t i o n - c y c l i z a t i o n to the 5- or 4-membered r i n g s ? DeSIMONE: That was the impression I got. ZUCKERMAN: There was another r e a c t i o n you mentioned which i n d i c a t e d the decomposition w i t h respect t o u l t r a - v i o l e t l i g h t g i v i n g C0 2

DeSIMONE: I got t h i s impression from an a r t i c l e i n " P o l l u t ion Engineering" [Reference 18] , an i n t e r v i e w w i t h John Ryan. FREY: I t h i n k the question has something to do w i t h the r a t e s of s o i l - c a t a l y z e d r e a c t i o n , whatever that happens t o be. The d e t a i l s of that r e a c t i o n , l i k e a l l o t h e r s , depend on the circumstances, and w e ' l l be going i n t o that d e t a i l i n a paper t h a t i s i n preparation. J . M. BELLAMA ( U n i v e r s i t y of Maryland): About the nature of water-soluble o r g a n o s i l i c o n chemistry, the TSP and DSS to which you r e f e r r e d are extended chains; s e v e r a l people have proposed a h e a d - t o - t a i l i n t e r a c t i o n where the presence of an e l e c t r o n - r i c h species (an a v a i l a b l e Lewis base moiety somewhere remote i n a molecule from the s i l i c o n ) can bend around and j o i n . We have looked at these k i n d s of i n t e r a c t i o n s [Inorg. Chem. (1965), 14,


10.

DESIMONE

Organosifones

157

1618], and we have two f e e l i n g s about them. One i s that these i n t e r a c t i o n s can be important when η (the number of i n t e r v e n i n g methylene groups) i s 1 or 2. Models of these compounds show t h a t the i n t e r a c t i n g atoms are e s s e n t i a l l y i n contact. With longer chains (n = 3-5), there seem to be some i n t e r a c t i o n . When you get t o η = 6, i t looks l i k e no i n t e r a c t i o n remains. Secondly, the nature of the group on the s i l i c o n seems t o be very important. Craig's o r i g i n a l paper J j . Chem. Soc. (1954), 332] p o s t u l a t e s the n e c e s s i t y of inducing a p o s i t i v e charge on s i l i c o n . I t would seem t h a t organic groups such as methyls or phenyls are not going to be p a r t i c u l a r l y good i n t h i s r e s p e c t . I f you have s u b s t i t u e n t s on the s i l i c o n l i k e a c h l o r i n e , or perhaps l i k e a hydrogen, which i s very e l e c t r o n e g a t i v e w i t h respect t o the s i l i c o n , the chances f o r these kinds of i n t e r a c t i o n s are going to be f a r g r e a t e r . So, using water-soluble t r i m e t h y l s i l y l compounds suggests t h a t these kinds of i n t e r a c t i o n s ar ing a s i t e for attack b the case i f other s u b s t i t u e n t s were present on the s i l i c o n . DeSIMONE: The d i f f e r e n c e between the r a t e s of DSS and TSP may be a r e f l e c t i o n of t h a t e x t r a carbon; i t i s a f a c t o r of a couple orders of magnitude. BELLAMA: M u l l e r and Frey [Z. anorg. a l l g . Chem. (1969) 368, 113] p o s t u l a t e d the n e c e s s i t y f o r ammonium f l u o r i d e a d d i t i o n to the m e t h y l t r i c h l o r o s i l a n e p l u s mercuric c h l o r i d e . They c l a i m t h a t i t ' s necessary to have a m e t h y l p e n t a f l u o r o s i l i c a t e species as the intermediate and as the a c t i v e methylating agent. We have done t h i s r e a c t i o n without adding the ammonium f l u o r i d e . We don't know what intermediate i s present, or what i s a c t u a l l y doing the methyl a t i n g , but i t ' s not necessary t o add the ammonium f l u o r i d e i n order t o get t h i s k i n d of r e a c t i o n to occur. J . S. THAYER ( U n i v e r s i t y of C i n c i n n a t i ) : We have found t h a t the DeSimone r e a c t i o n [equation 10] i s not confined t o mercury. One gets s i m i l a r r e a c t i o n s w i t h t h a l l i u m t r i a c e t a t e , w i t h l e a d t e t r a a c e t a t e and, we b e l i e v e , w i t h potassium h e x a c h l o r o p l a t i n a t e . We s t u d i e d the k i n e t i c s of t h i s r e a c t i o n and our f i g u r e s agree moderately w e l l the ones that were quoted here. Secondly, the other r e a c t i o n was the d i r e c t r e a c t i o n between h e x a m e t h y l d i s i l o x ane and mecuric n i t r a t e . The two dozen products a l l u d e d t o are a s e r i e s of l i n e a r and c y c l i c s i l o x a n e s , formed here by a d i s p r o p o r t i o n t i o n r e a c t i o n , p l u s a v a r i e t y of species we have not yet i d e n t i f i e d . Our b e l i e f i s that t h i s r e a c t i o n proceeds by i n i t i a l removal of a methyl group by mercury, from the s i l o x a n e moiety f o l l o w e d by rearrangement. We f i n d t h a t a s i m i l a r r e a c t i o n occurs w i t h the germanium analog, hexamethyldigermoxane. RECEIVED August 22,

1978.


11 Influence of E n v i r o n m e n t a l Parameters o n T r a n s m e t h y l a t i o n between A q u a t e d M e t a l Ions K. L. JEWETT and F. E. BRINCKMAN Center for Materials Science, National Bureau of Standards, Washington, DC 20234 J. M. BELLAMA Department of Chemistry, University of Maryland, College Park, MD 20742 While advances hav genic methyl-metals an have relied upon partition coefficients favoring degassing of permethylated species from aqueous media (1,2). Unfortunately, little work has been reported on the basic chemical features of relevant equilibria (z-n)+

(z-n)+

MenM (aq) = MenM (atm), or on determining partition coefficients. Where neutral molecules are involved, e.g., z = n, strong solvation by water is probably not important even though the central atom may be capable of greater than n-fold coordination. For example, in either fresh or salt water, Wasik et al. found that Me Hg partitions nearly equally between solution and atmos phere above (3). Preference for partition into fresh water was noted; presumably more favorable ligation by H O over C1 prevails, the so-called salting-out effect for hydrophobic molecules. Though not yet resolved, there appears to be general agree ment that biomethylation of metals and metalloids follows a step wise process (4,5). A number of very important corollaries o r assessing influences of environmental parameters hinge upon this basic viewpoint: (a) polar, charged (n [ C l ~ ] , of the r e a c t i o n medium. As a model system f o r a s s e s s i n g e f f e c t s of environmental parameters on aqueous t r a n s m e t h y l a t i o n , we extended our e a r l i e r work (17) on the r e a c t i o n , +

2 +

2 +

+

Me Sn + Hg « Me Sn + MeHg , 3 aq aq 2 aq aq Q

(1)

0

because of i t s relevance t o the observed b i o m e t h y l a t i o n of Sn(IV) (33) and involvement of a charged methyl donor o f f e r i n g g r e a t e r s i m i l a r i t y t o p o l a r i n t e r m e d i a t e s c u r r e n t l y viewed as important t o b i o g e n e s i s of organometals. As Huber (15) and we (34) have shown, an a t t r a c t i v e a l t e r n a t i v e e x i s t s w i t h the r a p i d t r a n s m e t h y l a t i o n chemistry of aquated methyllead s p e c i e s , but k i n e t i c l i m i t a t i o n s are imposed by the NMR measurement scheme employed. Moreover, though necessary e q u i l i b r i u b u t i o n s of m e t h y l t i n s p e c i e adequate, s u f f i c i e n t formatio constants f o analogous l e a d ions have not. In Tables I I and I I I are c o l l e c t e d , r e s p e c t i v e l y , k i n e t i c parameters obtained f o r r e a c t i o n 1 under c o n d i t i o n s which i s o l a t e e f f e c t s of temperature or i o n i c s t r e n g t h (μ) and s a l i n i t y ( [ C l " ] ) . Over the range of experimental c o n d i t i o n s examined f o r r e a c t i o n 1 i n the present work, a simple b i o m o l e c u l a r r a t e law was obeyed rate - k (obs) W ^ S n ^ 1 4

2

t

o

t

a

1^\^

l

ο

ί

β

1

·

(2)

E x c e l l e n t l i n e a r f i t s of k i n e t i c data were obtained f o r a l l r e a c t i o n s s t u d i e d , each t y p i c a l l y exceeding 75 percent completion. Reaction 1 appears t o be e s s e n t i a l l y i r r e v e r s i b l e : precipitation of Me2Sn2+ or MeHg does not a l t e r the r a t e , nor do these products undergo f u r t h e r t r a n s m e t h y l a t i o n r e a c t i o n s d e t e c t a b l e by NMR. +

E f f e c t s of Temperature. The l o n g - f a m i l i a r A r r h e n i u s equation which r e l a t e s the r e a c t i o n r a t e constant w i t h temperature defines a q u a n t i t y , E*, regarded as the a c t i v a t i o n energy of a component o r o v e r a l l process: E

rate - k - A e ~ *

/ R T

(3a)

and, I n k = -E*/RT + I n A.

(2b)

I n aqueous s o l u t i o n s , e s p e c i a l l y , i t i s apparent (35,36) t h a t the e n e r g e t i c s (or a c t i v a t i o n parameters) of the r e a c t i o n c o o r d i n a t e are profoundly a f f e c t e d by r e o r g a n i z a t i o n of s o l v e n t molecules surrounding the r e a c t a n t molecules, p a r t i c u l a r l y as t h i s m o d i f i e s t h e i r charge d i s t r i b u t i o n . For an i o n i c b i o m o l e c u l a r r e a c t i o n


11.

JEWETT E T AL.

Aquated Metal Ions

163

TABLE I I E f f e c t s o f Temperature upon Transmethylation +

2 +

3 +

between M e S n and H g or T l 3 aq aq aq 0

RUN*

(a) 1 2 3 4 5

T, ° C

b

k (obs)±S.E. 2

+ 2+ Me Sn + Hg 3

10.511.1 20.110.6 29.010.3 40.010.2 49.810.4

1.7210.06 4.3310.10 9.3310.21 15.5610.32 42.4411.15

r = 0.944 (b) 1 2 3 4 5

6.32 5.50 4.78 3.95 3.26

6.37 5.44 4.67 4.16 3.16 E* - 14.2 1 0.9

20 14 13 14 12

57.4 71.6 79.5 88.9 83.5

17 16 17 13 13

78.5 72.0 81.8 74.0 90.4

In A = 18.9

+ 3+ Me Sn + T 1 J

3

10.010.4 20.110.4 27.810.5 41.310.8 52.610.4

0.7510.03 2.3410.15 9.0610.30 17.2910.71 72.8014.19 r = 0.988

a

&

E* - 19.1 1 1.7

F o r s e r i e s (a) μ = 0.051; f o r (b) μ = 0.17.

deviation f o r Ν observations. of r e a c t i o n , %. Ε

7.12 5.96 5.11 3.75 2.69

7.20 6.06 4.70 4.06 2.62

C

i n k c a l mol

In A = 26.7

b

T 1 standard

k ( o b s ) χ 10 M~ 0

s~ .

Extent

.


In Organometals and Organometalloids; Brinckman, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 4

f

4

2

3

3

2

2 -1 c from NaCIO =1/2 Em ζ i n M~V 0 b t a i n e d at 26 ± 1°; \ . , counterions i c s tersetn ig mt ha t-e dμ upper l i m i t based on maximum NMR R e a c t i o n between Me SnC10 + H g ( C 1;0 ) I,o n 1:1;

3

f

s i g n a l detected a f t e r 23 h r . r u n ; R e a c t i o n between Me SnN0 + H g C l , 1:1.

d

a

0.320 0.353 0.389 0.501 0.768 0.758 0.1023 0.1249 0.1517 0.251 0.594 0.575 0.506 0.387 0.420 0.525 0.788 0.742 0.2565 0.1499 0.1767 0.2761 0.6211 0.550

-1.99 -2.02 -2.11 -2.11 -2.36 -2.34

0.41 0.67 0.58 0.38 0.25 0.19

10.24 9.56 7.80 7.77 4.40 4.61

4.00 5.00 6.07 10.03 22.13 23.03

4 5 6 7 8 9

0.277 0.275 0.275 0.274 0.0767 0.0767 0.0759 0.0753 0.636 0.786 0.420 0.526

0.4041 0.6186 0.1766 0.2764

-2.09 -2.03 -2.02 -2.04

0.53 0.27 0.28 0.24

8.15 9.23 9.45 9.16

3.03 3.06 3,01 3.02

10 11 12 13

1

C H C 1

3

~

3

-> C H P t C l

1 2

3

+

3

Pt +

2 3

"+

C l ~

(3a)

2C1~

(3b)

We t e s t e d t h i s b y r u n n i n g t h e r e a c t i o n i n t h e p r e s e n c e o f f i n e l y powdered p l a t i n u m m e t a l . The r e a c t i o n shows a marked i n c r e a s e i n r a t e , and f o l l o w e d s t a n d a r d second order k i n e t i c s ( 1 0 ) . D

e

Oxidative

Reaction

with

Methylcobalamin

The r e p o r t t h a t S n ( I I ) s p e c i e s c a n b e c o n v e r t e d t o m e t h y l t i n ( I V ) compounds (13) p r o m p t e d u s t o l o o k at a variety of substrate tive methylation. Tabl with their reaction rate constants. Table VII.

Rate Constants f o r Oxidative Reaction o f M e t h y l c o b a l a m i n w i t h M e t a l Compounds

* Compound

# Runs

—ι —ι Κ (s

M

)

Hg

3

1.0 Χ ΙΟ*"

C5H5TI

2

3.3

CsGeCl (CH ) 3

6

2

3

Sn

2

6

5

3

1.2 X ΙΟ""**

2

3

10~

5.6 Χ ΙΟ""**

2

(C H ) P

Χ

5

4.0 Χ Ι Ο "

4

A l l compounds o n l y p a r t l y s o l u b l e i n r e a c t i o n medium; h e n c e , numbers l i s t e d a r e minimum v a l u e s . I n t h e c a s e o f C s G e C l 3 , one p o s s i b i l i t y i s c h l o r i n e methyl exchange, f o l l o w e d by d i s p r o p o r t i o n a t i o n (Eqn. 4 ) . The o t h e r compounds 2CH GeCl " 3

2

->

(CH ) G e C l 3

2

2

+

Ge

+

2Cl"

(4)

cannot proceed b y t h i s pathway. Investigations are now u n d e r way t o d e t e r m i n e t h e m e c h a n i s m ( s ) b y w h i c h t h e s e compounds r e a c t . Discussion

two

E a r l y workers q u i c k l y recognized that a t l e a s t r e a c t i o n pathways e x i s t e d f o r methylcobalamin r e -



196

a c t i o n s (2) · T h e m e t h y l - c o b a l t b o n d may c l e a v e i n a n y of t h r e e ways: CH

3

-

CO

1 1 1

->

Θ :CH •CH

+

Oco

+

G

1 1 1

(5a)

3

CH CH

3

3

- Co -

CO

1

1

1

1 1 1

-> ->-

(5b)

•Co

3

CH ® 3

:Co

I

(5c)

In t h e l a t t e r two c a s e s , t h e c o b a l t i s b e i n g r e d u c e d , and t h e p r o d u c t s ( B and B ) have q u i t e d i f f e r e n t a b s o r p t i o n s p e c t r a , fiaking t h l m e a s y t o d i s t i n g u i s h , at l e a s t i n p r i n c i p l e . The f i r s t c l e a v a g e o c c u r s without o x i d a t i o n , and t h e product i s aquocobalamin, a l s o w i t h a unique a b s o r p t i o n spectrum. Similar mechanisms c o u l d b R CH3S , t h a t have bee 1 2

1 2

2

A.

Metathetical

Demethylation

T h i s i s t h e m o s t common m e c h a n i s m f o u n d , a n d i s e s s e n t i a l l y a t r a n s f e r o f a methyl carbanion t o a metal, substrate. The s u b s t r a t e i s a l m o s t a l w a y s c a t i o n i c i n n a t u r e ; hence, t h e mechanism o f t h e r e a c t i o n i s a p p a r e n t l y Sg2(14) , p r o c e e d i n g through an i n t e r mediate o f t h e type

By c o r o l l a r y , a n y t h i n g t h a t a f f e c t s t h e e l e c t r o p h i l l i c i t y o f the metal w i l l a f f e c t t h e rate o f reaction. Organic groups, b e i n g good e l e c t r o n donors, s h o u l d lower the e l e c t r o p h i l i c i t y o f a metal; t h i s i s consis tent w i t h t h e observed decrease i n r e a c t i o n rates as t h e number o f o r g a n i c g r o u p s i n c r e a s e s . The r a t e s u p p r e s s i o n by anions (Table I I I ) r e s u l t s from e l e c t r o n - r i c h s p e c i e s c o m p e t i n g f o r t h e same m e t a l c a t i o n . Hence, any s p e c i e s t h a t i s e l e c t r o p h i l i c has t h e p o t e n t i a l o f r e a c t i n g w i t h methylcobalamin by t h i s pathway, even i f t h e i n i t i a l l y formed p r o d u c t subse q u e n t l y decomposes.


12.

THAYER

197

Methylcobalamin

Workers s t u d y i n g the r e a c t i o n o f m e r c u r i c a c e t a t e w i t h methylcobalamin found t h a t the mercury i n t e r a c t s with the benzimidazole nitrogen o f methylcobalamin t o f o r m t h e " b a s e - o f f " c o m p l e x (.1/3/5) l e a d i n g t o m o r e complex k i n e t i c s . This competing r e a c t i o n , which c a n b e e l i m i n a t e d b y a d d i t i o n o f h a l i d e i o n s (5), w a s a l s o o b s e r v e d f o r P d C l ^ " ( 6 ) . We d i d n o t o b s e r v e i t i n t h e r e a c t i o n s o f organometals with methylcobalamin, perhaps r e f l e c t i n g t h e i r reduced e l e c t r o p h i l i c c h a r a c t e r r e l a t i v e t o Hg(OAc) · 2

2

B.

Redox

Demethylation

The m o s t t h o r o u g h l y s t u d i e d s y s t e m o f t h i s t y p e i s the S n ( I I ) - C H B an o x i d i z i n g a g e n t f o r r e a c t i o n t o o c c u r , a n d the proposed mechanism h a s h o m o l y t i c c l e a v a g e o f the m e t h y l - c o b a l t bond a s i t s c r u c i a l step (13,15). 3

Arsenic, selenium, and t e l l u r i u m are methylated by a d i f f e r e n t pathway, w h i c h i n v o l v e s r e d u c t i o n o f the s u b s t r a t e by removal o f oxygen. The mechanism p r o p o s e d b y C h a l l e n g e r (16) a n d e l a b o r a t e d o n b y C u l l e n e t a l . (17) i n v o l v e s t r a n s f e r o f a m e t h y l c a r bon ium i o n : (HO) A s :

+

3

+

[CH As(OH) ] 3

3

3

(HO) A s : 2

+

->

3

3

2

[ C H A s (OH) ] 3

+ +

2 e" CH

+ 3

+

(

6

a

)

3

-> C H A s O ( O H )

3

CH AsO(OH) CH

CH

+

2

+

H

(6b)

-* C H ( H O ) A s : 3

+

2

2

0 "(6c)

+

[ ( C H ) A s (OH) 3 e t c . (6d) 3

2

2

I n t h e s e s y s t e m s m e t h y l c o b a l a m i n d o e s n o t seem t o b e the m e t h y l a t i n g agent, a t l e a s t not d i r e c t l y ; instead, t h e m e t h y l g r o u p comes f r o m a s u l f o n i u m s a l t ( 1 6 , 1 7 ) . N e v e r t h e l e s s , a s i m i l a r mechanism c o u l d be w r i t t e n for methylcobalamin reactions. The r e a c t i o n b e t w e e n P t ( I V ) o r A u ( I I I ) a n d methylcobalamin h a sbeen a l l e g e d t o r e q u i r e the p r e s ence o f the lower o x i d a t i o n s t a t e o f the metal ( 7 ) . T h i s has been termed a "redox s w i t c h " mechanism, a n d has t h e f o l l o w i n g form (9):


198

ORGANOMETALS AND

2

PtCli* " complex

+

+

CH B 3

->

1 2

3

+

(7a)

complex

CH PtCl

PtClg

ORGANOMETALLOIDS

B

+ CI

5

(7b)

1 2

The c o m p l e x , r e p o r t e d b y Wood a n d c o w o r k e r s (115) t o involve i n t e r a c t i o n of P t C l ^ " with a side chain of the c o r r i n r i n g system, i s c h a r a c t e r i z e d by h a v i n g a more l a b i l e m e t h y l - c a r b o n bond t h a n m e t h y l c o b a l a m i n itself. C o n s e q u e n t l y , i t r e a c t s more r e a d i l y . Side c h a i n complexes between m e t a l s and B derivatives h a v e b e e n p r o p o s e d ( 1 8 ) . The m e t a l s f o l l o w i n g t h e "redox s w i t c h " mechanis t e n t i a l for molecula s h o u l d b e n o t e d t h a t t h e P t C l " / P t p o t e n t i a l i s +0.75 ν ( 1 9 ) . Our o b s e r v a t i o n t h a t P t c a t a l y s e s t h a t r e a c t i o n of P t C l i ^ " with methylcobalamin suggests that t h i s couple a l s o f a l l s i n t o the "redox s w i t c h " c a t e gory. Whether the "redox s w i t c h " mechanism i s l i m i t ed s o l e l y t o p l a t i n u m and g o l d o r a p p l i e s t o a w i d e r r a n g e o f m e t a l s r e m a i n s t o be d e t e r m i n e d . 2

1 2

i +

2

C.

In vivo

kinetics

The v a l i d i t y w i t h w h i c h m o s t r e p o r t e d k i n e t i c a n d m e c h a n i s t i c s t u d i e s c a n be e x t e n d e d t o b i o l o g i c a l p r o cesses i s quite uncertain. V e r y few r a t e s t u d i e s have been r e p o r t e d f o r m e t h y l a t i o n by n a t u r a l o r g a n i s m s . _ T r i m e t h y l a r s i n e f o r m e d a t t h e r a t e o f 3.9 X 1 0 " Ms"" when C a n d i d a h u m i c o l a m e t h y l a t e d 5 X 1 0 " M A s 0 s o l u t i o n ( 1 7 ) . When o n e g r a m o f r a t c e c a l c o n t e n t s w e r e t r e a t e d w i t h 2.0 m l 7.4 X 1 0 " M H g C l solution, 17.6 n g CH H g C l w e r e f o r m e d o v e r a p e r i o d o f t w e n t y hours (20)? S i m i l a r slow r e a c t i o n s were found i n t h e b i o l o g i c a l m e t h y l a t i o n o f P b ( I I ) and T l (I) ( 2 1 ) . O b v i o u s l y , r a t e s o f m e t h y l a t i o n under b i o l o g i c a l c o n d i t i o n s a r e g o i n g t o b e much s l o w e r t h a n u n d e r t e s t tube c o n d i t i o n s . One r e a s o n i s t h e much l o w e r c o n centrations involved. A n o t h e r r e a s o n , p r o p o s e d by R o b i n s o n e t a l . (5), i s t h a t m e t h y l c o b a l a m i n i n b i o l o g i c a l systems e x i s t s i n h y d r o p h o b i c e n v i r o n m e n t s , and that surfactants w i l l g r e a t l y i n h i b i t rates of react ions. Much r e m a i n s t o b e d o n e i n t h i s a r e a . χ

1 2

3

2

3

6

2

Acknowledgments We w i s h t o t h a n k D r . J o h n Wood a n d t h e F r e s h w a t e r B i o l o g i c a l Laboratory (Navarre, Minnesota) f o r p r o v i d -


12.

Thayer

Methylcobalamin

199

i n g the means t o i n a u g u r a t e t h i s p r o j e c t . D r s . M. O r c h i n , J . B . Smart, and R. S. T o b i a s k i n d l y p r o v i d e d some o f the more e x o t i c compounds used i n t h i s s t u d y . D r . E s t e l Sprague p r o v i d e d some v e r y h e l p f u l d i s c u s s i o n on the k i n e t i c s i n v o l v e d i n t h e s e r e a c t i o n s . Literature Cited 1.

DeSimone, R. Ε., P e n l e y , M. W. Charbonneau, L., S m i t h , S. G., Wood, J. Μ . , Hill, H.A.O., Pratt, J. M . , R i d s d a l e , S . , W i l l i a m s , R. J. P., B i o c h i m . B i o p h y s . A c t a (1973), 304, 851.

2.

Bertilsson, 10, 2805.

3.

C h u , V . C . W . , Gruenwedel, (1977), 7, 169.

D. W . , B i o i n o r g . Chem.

4.

C h u , V . C . W . , Gruenwedel, (1976), 31C, 753.

D. W . , Z . N a t u r f o r s c h .

5.

R o b i n s o n , G . C., Amer. Chem. S o c .

6.

Scovell, 3451.

7.

Agnes, G., B e n d l e , S. Hill, H . A . O., W i l l i a m s , F . R . , W i l l i a m s , R. J. P., Chem. Comm. (1971), 850.

8.

T a y l o r , R. T., Hanna, M. (1976), A 1 1 , 201.

L.,

Environ.

9.

T a y l o r , R. 6, 281.

L.,

B i o i n o r g . Chem. (1976),

10.

Thayer,

11.

P o h l , U., Huber, 116, 141.

12.

Capellos, C., Bielski, B . H . J., " K i n e t i c Systems," 59-62, W i l e y - I n t e r s c i e n c e , New Y o r k , 1972.

13.

D i z i k e s , L . J., R i d l e y , W. P., Wood, Amer. Chem. S o c . (1978), 100, 1010.

14.

M a t t e s o n , D . S . , " O r g a n o m e t a l l i c R e a c t i o n Mecha n i s m s , " Academic P r e s s , New Y o r k , 1974.

L.,

N e u j a h r , Η.

Υ.,

Biochem.

Nome, F., F e n d l e r , (1977), 99, 4969.

W. M., J. Amer. Chem. S o c .

J.,

T.,

Hanna, M.

unpublished F.,

J.

J.

(1971),

H.,

J.

(1974),

Sci.

96,

Eng.

results. Organometal. Chem.

J.

(1976),

M.,


J.

200

organometals and organometalloids

15.

F a n c h i a n g , Y . T., R i d l e y , W. Chapter , t h i s volume.

16.

Challenger,

F.,

C h a p t e r 1,

P.,

this

Wood, J. Μ . ,

volume.

17.

C u l l e n , W. T., F r o e s e , C . L., Lui, Α., M c B r i d e , Β . C . Patmore, D . J., and Reimer, M., J. Organo m e t a l . Chem. (1977), 139, 61.

18.

P r a t t , J. Μ . , I n o r g a n i c C h e m i s t r y o f V i t a m i n 189-190, Academic P r e s s , New Y o r k , 1972.

19.

H a r t l e y , F . R., "The C h e m i s t r y o f P l a t i n u m and P a l l a d i u m , " 13 A p p l i e d S c i e n c e s London 1973

20.

Rowland, I., D a v i e s H e a l t h (1977), 24.

21.

Huber, F., S c h m i d t , this volume.

U.,

B ," 12

Kirchmann, Η . , C h a p t e r

Discussion J . J . ZUCKERMAN (University of Oklahoma): Concerning the l i s t of insoluble species reacting with the methylating agent: Is reaction occurring with soluble species (although the Ksp value for Sn02 is a very small number), or is i t in fact occurring on the surface? If so, are a l l these species in a slurry of similar paricle size? THAYER: In some cases there is a p a r t i a l s o l u b i l i t y , and we expect that many of these are reacting with the species actually in solution, even though we could not get complete s o l u b i l i t y . In some other cases, e.g., lead tetraacetate, which is hydrolyzed to lead dioxide or some sort of oxy-acetato-lead species, there is not appreciable s o l u b i l i t y . There you are probably getting a sur face reaction. This may be true for some other species as well. ZUCKERMAN: Was there some standardization in particle size in the l i s t that you gave? THAYER: I tried to make these solids as finely divided and as homogeneous as I could, but other than that I'm afraid there wasn't any. J . M. WOOD (University of Minnesota): When you are studying these reactions you also must look at problems related to compet ing reactions for the displacement of benzimidazole. You can have


12.

THAYER

Methylcobalamin

201

e q u i l i b r i a w i t h the base-on, w i t h base-off or protonated base-off. J u s t r e c e n t l y , we have been able t o show that by making a number of c o r r i n - r i n g d e r i v a t i v e s and comparing complexation w i t h a c o r r i n macro-cycle, one can now form outer-sphere complexes w i t h propionamide s i d e chains. Consequently, the k i n e t i c i n t e r p r e t a t i o n of your r e a c t i o n s can be extremely d i f f i c u l t because you can be l o o k i n g at f o u r or f i v e a l t e r n a t e e q u i l i b r i a i n these systems. THAYER: I agree. Not much can be s a i d yet about mechanisms f o r these r e a c t i o n s . But f o r most organometals I don't t h i n k comp l e x a t i o n e i t h e r w i t h the benzimidazole or the r i n g i s important. With many i n o r g a n i c s p e c i e s , i t i s . ZUCKERMAN: I f the metal species f i n d s i t s e l f i n a complex w i t h the benzimidazole, then i t i s a l s o on the wrong s i d e of the r i n g to e f f e c t m e t h y l a t i o n that? WOOD: I t has a profound e f f e c t on the k i n e t i c s f o r the r e a c t i o n . I f you are l o o k i n g at e l e c t r o p h i l i c a t t a c k on the c o b a l t carbon bond and you d i s p l a c e the benzimidazole, t a k i n g mercury as a simple example, you get about two orders of magnitude d i f f e r e n c e i n the r e a c t i o n r a t e because i t ' s d i f f i c u l t to d i s p l a c e the methyl group as a carbanion. But i f you consider the r e a c t i o n w i t h a s i n g l e - e l e c t r o n oxidant i n t i n ( I I ) , you look at a f r e e r a d i c a l displacement of the methyl group. This seems t o be i n s e n s i t i v e t o c o o r d i n a t i o n by benzimidazole and i s very i n s e n s i t i v e to pH i n these systems. Depending on whether the mechanism i s e l e c t r o p h i l i c a t t a c k or s i n g l e - e l e c t r o n o x i d a t i v e a d d i t i o n , an enormous change i n the r e a c t i o n r a t e s i s seen f o r t h i s r e a c t i o n . However, the chemistry i s not t r i v i a l because the benzimidazole can come o f f , i t can be protonated, or i t can r e a c t w i t h a metal i o n . The formation of a complex w i t h a c o r r i n macrocycle which changes the e l e c t r o n d e n s i t y on the cobalt-carbon bond can g r e a t l y change the r e a c t i v i t y of the methyl group to e i t h e r e l e c t r o p h i l i c a t t a c k or possibly free r a d i c a l attack. ZUCKERMAN: That would change the m o l e c u l a r i t y of the r a t e determining s t e p , i f one metal were t o complex w i t h the benzimida z o l e and a second metal species were t o a t t a c k an a p i c a l s i t e . So that would be revealed i n the data, provided one could i n t e r p r e t those data i n terms of m o l e c u l a r i t y . W. R. CULLEN ( U n i v e r s i t y of B r i t i s h Columbia): We are b r a i n washed by the i d e a that methyl B-^ i s i n v o l v e d i n a l l of these r e a c t i o n s . We f i r m l y b e l i e v e t h a t there i s no B^« i n v o l v e d i n a r s e n i c methylation. P r o f e s s o r Wood pointed out that the nature of the methylating species had to be e s t a b l i s h e d i n nature. Much of our d i s c u s s i o n now i n v o l v e s base-on, base-off d e t a i l s , yet the compound may not i n f a c t be the methylating agent. I suggest t h a t


ORGANOMETALS

202

AND

ORGANOMETALLOIDS

the most important t h i n g we can do i s t o i n v e s t i g a t e these r e a c t i o n s i n b i o l o g i c a l systems, t o f i n d out what i s going on and what i s doing the methylating. There i s one easy way of g e t t i n g o x i d a t i o n : that i s t o use CH^ as an a t t a c k i n g agent. Use of CH^~ w i t h o x i d a t i o n r e q u i r e s a d d i t i o n a l m a n i p u l a t i o n of e l e c t r o n s . CH~ i s a good methylating agent from nature and i s r e a d i l y a v a i l a b l e . I suggest we should t h i n k more about CH^ ; i t can come from methyl well. +

+

+

a s

WOOD: I agree. The value of t h i s model chemistry i s t o point up p o s s i b l e mechanisms f o r transmethylation r e a c t i o n s i n b i o l o g i c a l systems. But the important t h i n g i s t o e s t a b l i s h p r e c i s e l y which coenzyme intermediate i s i n v o l v e d i n the transmethylation r e a c t i o n . T h a t s why experiments w i t h isotopes are necessary i n complicated systems l i k l a k sediment d mixed m i c r o b i a l n i t i e s . I t ' s important Then you can s t a r t t h i n k i n g terms o mechanism. Fo the m e t a l l o i d s , you are p r i m a r i l y l o o k i n g a t n u c l e o p h i l i c a t t a c k on the carbon-sulfur bond of something l i k e S-adenosylmethionine, or e l s e something l i k e methylated co-enzyme M i n anaerobic systems. You can get methyl t r a n s f e r t o a r s e n i c i n a h i g h o x i d a t i o n s t a t e i f you a c t i v a t e the c o r r i n r i n g by a r e a c t i o n w i t h a platinum complex. That may not have very much s i g n i f i c a n c e t o b i o l o g i c a l systems, but from nmr data you see that platinum complex a c t i v a t e s the s y s tem. I t i s the same s o r t of chemical s h i f t that you see f o r B ^ when i t binds t o the enzymes, where you have t o supply 70 k i l o c a l o r i e s t o break t h a t bond f o r s u b s t r a t e rearrangement r e a c t i o n s i n the enzymes. So, we don't r e a l l y know what i s happening i n the 12 P * 1

B

r o t e i n s

v e t

R. H. FISH ( U n i v e r s i t y of C a l i f o r n i a , B e r k e l e y ) : I don't t h i n k you ever i d e n t i f i e d any compounds i n these r a t e s t u d i e s . that c o r r e c t ? THAYER:

Is

We have not i d e n t i f i e d any products.

FISH: The r e s u l t s seems t o be tenuous unless you i d e n t i f y the compounds. How t r i m e t h y l t i n forms i n the environment i s s t i l l c o n t r o v e r s i a l . P r o f e s s o r Wood shows that t i n ( I I ) goes t o methylt i n ( I V ) , but what happens a f t e r that? We have looked a t methyland d i m e t h y l t i n species r e a c t i n g w i t h methyl B-« and couldn't f i n d any r e a c t i o n o c c u r r i n g . Again, you must i d e n t i f y the products bef o r e you can t a l k about r a t e s of r e a c t i o n p e r t a i n i n g t o methylation* THAYER: Not n e c e s s a r i l y . I n the case of the t i n compounds, i t seems there i s a s u b s t a n t i a l d i f f e r e n c e i n r a t e s of r e a c t i o n depending on what i n o r g a n i c groups are present. With m e t h y l t i n t r i c h l o r i d e , I couldn't see any r e a c t i o n . With m e t h y l t i n t r i a c e t a t e v i r t u a l l y no r e a c t i o n occurred. However, d i m e t h y l t i n d i c h l o -


12.

THAYER

Methylcobalamin

203

r i d e r e a c t s c o n s i d e r a b l y f a s t e r than d i m e t h y l t i n d i a c e t a t e . I t i s w e l l known that t i n - o x y species w i l l form i n t e r m o l e c u l a r b r i d g i n g bonds r e s u l t i n g i n polymeric species p a r t i c u l a r l y where you have 2 oxy groups per t i n , or even more where you have t h r e e . This may i n f l u e n c e the r e a c t i o n of o r g a n o t i n compounds w i t h whatever methyla t i o n agent there may happen to be. There was one case where we could i d e n t i f y the product, the t r i m e t h y l t e l l u r o n i u m i o n . I found t e l l u r i u m metal and d i m e t h y l t e l l u r i d e . For tetramethylarsonium ion and methylcobalamim a very unpleasant odor which q u i t e proba b l y was t r i m e t h y l a r s i n e was given o f f . F. HUBER ( U n i v e r s i t y of Dortmund): We have p r e l i m i n a r y r e s u l t s on the r e a c t i o n of t i n ( I V ) , not w i t h methylcobalamin, but w i t h me thy lcobaloxime. From nmr measurements we found t h a t we probably get the m e t h y l t i methylcobaloxime. We a l s m e t h y l t i n . We have a problem because the p o s i t i o n s of the nmr s i g n a l s are not very constant. We have to look more at the coordi n a t i o n s i t u a t i o n i n these s o l u t i o n s . We have been l o o k i n g at oxygen systems w i t h these r e a c t i o n s , but we have to consider the n a t u r a l systems which c e r t a i n l y i n v o l v e s u l f u r l i g a n d s . When we make compounds of organolead and o r g a n o t i n compounds w i t h the c a r b o x y l i c a c i d s c o n t a i n i n g SH groups, we f i n d a preference f o r l e a d bonding t o s u l f u r , and not to oxygen. We can make c a r b o x y l a t e s which are bonded through s u l f u r to l e a d , but i t i s d i f f e r e n t w i t h t i n compounds. T h i s i s a very i n t e r e s t i n g f i e l d and we should t r y to i n v e s t i g a t e t h i s f i e l d of s u l f u r c o o r d i n a t i o n to organometal compounds. F. E. BRINCKMAN ( N a t i o n a l Bureau of Standards): This concerns the p r e s s i n g q u e s t i o n of t r a c e metal f l u x ; I'm s t r u c k t h a t there are so many c o r r i n o i d s a v a i l a b l e i n environmental d e t r i t u s , e i t h e r of anthropogenic or n a t u r a l b i o g e n i c o r i g i n , which c o u l d a c t as s o l u b i l i z i n g agents. With Ksp values f o r many of the oxides t h a t you i n d i c a t e d , such s o l u b i l i z a t i o n might be important t o metal f l u x e s . I'm p a r t i c u l a r l y concerned w i t h the t i n case; I'm concerned about i t s b i o a v a i l a b i l i t y . A d m i t t e d l y , methylcobalamin might be the wrong model, but i t i s an attempt to look at i t as a s o l u b i l i z ing agent, not n e c e s s a r i l y as a m e t h y l a t i n g agent, f o r uptake i n t o food webs. Have you looked a t some other t i n m i n e r a l s o l u b i l i t i e s i n your s o l u t i o n s of methylcobalamin? THAYER: No, we haven't. I've confined my t i n ( I V ) s i n c e oxy-metal s p e c i e s seem to undergo r e a d i l y than s p e c i e s w i t h other groups present. of t i n t e t r a a c e t a t e , but under these c o n d i t i o n s mediately hydrolyzed. BRINCKMAN: metal s u l f i d e s ?

a t t e n t i o n to t h i s r e a c t i o n more There i s a r e p o r t I'm sure i t ' s im-

Have you looked at any t i n s u l f i d e s or any other


204

ORGANOMETALS AND

ORGANOMETALLOIDS

THAYER: There i s only species w i t h a m e t a l - s u l f u r bond that I have looked a t . This was chloromercury-thiomethyl and - t h i o - t b u t y l . We got a very r a p i d r e a c t i o n w i t h methylcobalamin, but i t was the other group, the c h l o r i d e , that was being removed. In a c i d i c media, w i t h something l i k e bis(methylthio)mercury i n the presence of methylcobalamin, a r e a c t i o n probably would be r a t h e r slow, but I t h i n k you could observe i t . A l i t t l e b i t of b i s - t h i o l d e r i v a t i v e of mercury d i s s o l v e s i n d i l u t e a c i d (~ pH=4) and w i l l react. CULLEN: I want to emphasize that i t ' s very important that we look at s u l f u r involvement i n these compounds, e s p e c i a l l y i n the case of the m e t a l l o i d s , and indeed, i n a l l metals w i t h a h i g h s u l f u r a f f i n i t y . A l s o , nature a c t u a l l y doesn't do a l l these r a t h e r complicated r e a c t i o n s i n one step I t h i n k we ought to look f o r mechanisms that go slower I n other words, we don' a r s e n i c there i s an o x i d a t i o n and then of course a r e d u c t i o n . These are two d i f f e r e n t t h i n g s . WOOD: I n 1953, a f t e r the Minimata (Japan) d i s a s t e r , when people s t a r t e d l o o k i n g f o r the cause, the molecule that was i s o l a t e d from s h e l l f i s h i n Minimata Bay was methylmercurythiomethyl. I t h i n k i t ' s i n t e r e s t i n g that government agencies are s t i l l not a n a l y z i n g f o r i t i n s h e l l f i s h , as f a r as I'm aware. I t took almost four years to persuade the EPA to stop a n a l y z i n g f o r t o t a l mercury and s t a r t a n a l y z i n g f o r methylmercury. RECEIVED August 22,

1978.


13 Mechanisms for Alkyl Transfers in Organometals JAY K. KOCHI

Department of Chemistry, Indiana University, Bloomington, IN 47401

The class of vitami one of three major coenzyme transfer in biological systems, being particularly effective with inorganic substrates. Thus methylcobalamin has been i m p l i cated in methyl transfers to a variety of metal ions including mercury, lead, tin, and thallium, as well as platinum, palladium, and gold (1). The mechanism of methyl transfer from cobalt to another metal center, that is, transmetallation, is the subject of extensive study. Two general mechanisms have been proposed for the methylation of metal ions by methylcobalamin (2). In type I reactions, the metal ion acts as an electrophile during the transfer of a methyl anion equivalent. In type II reactions, methyl radicals are transferred between metal centers. Thus type I reactions are considered to involve heterolytic cleavage of the cobalt-carbon bond of methylcobalamin, whereas the homolytic cleavage of the same bond in type II reactions is induced by a reduced member of a redox couple. In a more general sense, type I and type II reactions can be considered as two-equivalent and one-equivalent processes, respectively. The concept of electron transfer relates two-equivalent processes with their one-equivalent counterparts. For example, consider the carbonium ion as the key reactive intermediate in solvolysis reactions, which historically have served as prototypes for numerous ionic processes. Electron transfer by one— equivalent reduction produces alkyl radicals, CH

+ 3

^

w

CH * 3

which are crucial to homolytic processes. The same interchange between ionic and radical species applies to electron transfer processes between carbanions and alkyl radicals. CH " 3

^F=*r CH 3



206

F i n a l l y , an o v e r a l l t w o - e q u i v a l e n t change i n t e r r e l a t e s c a r b o n i u m i o n s and c a r b a n i o n s . ^ 2e CH · CH 3

3

V i e w e d i n t h i s way, t h i s h y p o t h e t i c a l t r a n s f o r m a t i o n i s b e t t e r considered as a two-step p r o c e s s i n v o l v i n g s u c c e s s i v e e l e c t r o n t r a n s f e r s . T h u s , i n e l e c t r o c h e m i c a l p r o c e s s e s , o n l y one e l e c t r o n i s t r a n s f e r r e d i n a s i n g l e a c t s i n c e the s i m u l t a n e o u s t r a n s f e r of two e l e c t r o n s , l i k e a b i p h o t o n i c p r o c e s s , i s a m u c h l e s s p r o b a b l e event. A l t h o u g h the o b s e r v a t i o n of e l e c t r o n t r a n s f e r p r o c e s s e s between a l k y l r a d i c a l s and c a r b o n - c e n t e r e d i o n s a r e as y e t r e l a t i v e l y r a r e , those between m e t a l c o m p l e x e s of c o u r s e r e p r e s e n t a w e l l - e s t a b l i s h e d p a r t of i n o r g a n i c c h e m i s t r y . Outer-sphere and i n n e r - s p h e r e m e c h a n i s m excellent basis for electro m e t a l c o m p l e x e s (3.). T h e p r e s e n c e of a b i m e t a l l i c i n t e r m e d i a t e either as a p r e c u r s o r or successor (postcursor) complex can play an important r o l e i n inner-sphere electron transfer pro c e s s e s . O u t e r - and i n n e r - s p h e r e m e c h a n i s m s h a v e a l s o been a p p l i e d to the o x i d a t i o n - r e d u c t i o n r e a c t i o n s of a l k y l r a d i c a l s w i t h m e t a l c o m p l e x e s (4). H o w e v e r , t h e d e t a i l e d e x a m i n a t i o n of the m e c h a n i s m of o x i d a t i o n - r e d u c t i o n p r o c e s s e s w i t h a l k y l r a d i c a l s i s m a d e d i f f i c u l t b y t h e i r t r a n s i e n t n a t u r e . A s a r e s u l t , the m e c h a n i s m s have been d e r i v e d h e r e t o f o r e f r o m d e d u c t i o n s b a s e d on k i n e t i c o b s e r v a t i o n s and p r o d u c t a n a l y s e s , and a few i n t e r m e d i a t e s have only r e c e n t l y been d e t e c t e d . F o r e x a m p l e , the i n n e r - s p h e r e c o m p l e x between m e t h y l r a d i c a l s and c o p p e r ( l l ) , CH * 3

+

Cu

1

>-

CH3-CU

1

has been o b s e r v e d by flash p h o t o l y t i c t e c h n i q u e s and found to d e c a y w i t h f i r s t - o r d e r k i n e t i c s (k = 7χ 1 0 sec" ) i n aqueous s o l u t i o n s at 25° (.§.). Indeed, the a s s o c i a t i o n of a l k y l r a d i c a l s w i t h m e t a l c o m p l e x e s t h r o u g h i n n e r - s p h e r e c o m p l e x e s m a y be t h e r o u t e b y w h i c h m o s t , i f not a l l , o x i d a t i o n - r e d u c t i o n r e a c t i o n s of a l k y l r a d i c a l s w i t h m e t a l c o m p l e x e s o c c u r , i r r e s p e c t i v e of w h e t h e r they have been p r e v i o u s l y c l a s s i f i e d a s i n n e r - o r o u t e r s p h e r e p r o c e s s e s . In t h i s r e g a r d , the r o o t of the d i f f e r e n c e between w h o l l y i n o r g a n i c s y s t e m s and the h y b r i d a l k y l m e t a l s y s t e m s p r o b a b l y l i e s i n the g r e a t p r o p e n s i t y of the c a r b o n c e n t e r e d i o n s , both c a r b o n i u m i o n s and c a r b a n i o n s , to be h i g h l y s o l v a t e d , e i t h e r a s i o n - p a i r s or i n n e r - s p h e r e c o m p l e x e s . A n i n n e r - s p h e r e a l k y l m e t a l i n t e r m e d i a t e s u c h a s that i n the a b o v e equation m a y be d e r i v e d by an a l t e r n a t i v e r o u t e i n v o l v i n g e i t h e r o x i d a t i o n o r r e d u c t i o n of a s t a b l e a l k y l m e t a l c o m p l e x , e.g., 2

CH -Cu 3

tt

g

" >

CH -Cu

1

m

3

In t h i s i n s t a n c e , the p r e c u r s o r i t s e l f i s u n s t a b l e .

However,


13.

Alkyl Transfers

KOCHi

207

t h e r e a r e a v a r i e t y of other s t a b l e a l k y l m e t a l c o m p l e x e s extant f r o m which electron transfer is possible. Reversible d i s s o c i a tion of s u c h i n t e r m e d i a t e s then r e l a t e s a l k y l r a d i c a l s to o r g a n o metals derived by conventional two-equivalent processes, i . e . , R

+

M

+

=c=^

^

R-M

^-=^=

R-MÎ"

R-

+

M

+

T h i s d i c h o t o m y i s i n h e r e n t i n a l l of t h e s e p r o c e s s e s , and t h e r e i s a s e v e r e p r o b l e m of r i g o r o u s l y d e m o n s t r a t i n g how e a c h m a y participate i n a particular organometal reaction. S e v e r a l m a j o r q u e s t i o n s a r i s e i n the t r e a t m e n t of a l k y l m e t a l s as i n t e r m e d i a t e s i n t r a n s m e t a l l a t i o n s : (a) the m e c h a n i s t i c d i s t i n c t i o n between e l e c t r o p h i l i c and e l e c t r o n t r a n s f e r m e c h a n i s m s i n the c l e a v a g e of a n a l k y l - m e t a l bond, (b) the s e p a r a t i o n of c o n c e r t e d sive, one-equivalent processes m e t a l s e s p e c i a l l y with r e g a r d to e l e c t r o n t r a n s f e r p r o c e s s e s . B i n a r y a l k y l m e t a l s of m e r c u r y , l e a d and tin a r e u s e f u l m o d e l s for the study of t h e s e q u e s t i o n s s i n c e they a r e s u b s t i t u t i o n - s t a b l e c o m p o u n d s and g e n e r a l l y w e l l b e h a v e d i n s o l u t i o n for k i n e t i c s t u d i e s . In t h i s r e p o r t , we s h a l l d e s c r i b e how v a r i o u s a l k y l d e r i v a t i v e s c a n be e x a m i n e d s y s t e m a t i c a l l y to a l l o w d i r e c t photoe l e c t r o n s p e c t r o s c o p i c study of the b o n d i n g o r b i t a l s i n t h e s e organometals. T w o s e r i e s of w e l l d e l i n e a t e d c l e a v a g e s of a l k y l m e t a l s w i l l then be d i s c u s s e d . T h e s e i n c l u d e : (a) the e l e c t r o p h i l i c p r o t o n o l y s i s of d i a l k y l m e r c u r y compounds, R'HgR and

+

HOAc

RH +

R'HgOAc

(b) the e l e c t r o n t r a n s f e r o x i d a t i o n of the s a m e o r g a n o m e r c u r i a l s with h e x a c h l o r o i r i d a t e ( l V ) . R'HgR

+

IrCl " 6

Rt

H g

2

RÎ

f

a

s

t

^

R'HgR*

>.

R.

+

+

R Hg l

IrCl " 6

+

,

3

etc.

U s i n g t h e s e s y s t e m s , we w i l l c o m p a r e and c o n t r a s t e l e c t r o p h i l i c and e l e c t r o n t r a n s f e r m e c h a n i s m s i n the c l e a v a g e s of a l k y l - m e t a l bonds i n both m a i n g r o u p and t r a n s i t i o n m e t a l c o m p l e x e s . I.

O r g a n o m e t a l s as E l e c t r o n D o n o r s — I o n i z a t i o n P o t e n t i a l s

A. D i a l k y l m e r c u r y C o m p o u n d s . T h e He(l) photoelectron s p e c t r a of d i a l k y l m e r c u r y c o m p o u n d s show two p r i n c i p a l bands of i n t e r e s t ( £ . ) . T h e f i r s t i o n i z a t i o n p o t e n t i a l , Ij> o c c u r r i n g i n a r a n g e between 7.5 7 eV ( d i - t - b u t y l m e r c u r y ) and 9.33 eV ( d i methylmercury) is included i n a fairly broad, u n s y m m e t r i c a l b a n d . A s e c o n d , w e a k e r band o c c u r r i n g between 14.4 and 15.0 eV i s due to i o n i z a t i o n f r o m the m e r c u r y 5 d shell. T h e ionization e n e r g i e s for t h e s e two bands a r e tabulated i n T a b l e I. Repre1 0


208

ORGANOMETALS


T a b l e I. T h e F i r s t and 5 d V e r t i c a l I o n i z a t i o n P o t e n t i a l s (eV) of D i a l k y l m e r c u r y C o m p o u n d s . 1 0

R—Hg—R CH CH C H CH CH CH C H n-C H i-C H C H i-C H n-C H i-C H t—C H i-C H neo-C H 3

2

5

3

5

7

3

7

2

5

3

7

4

9

4

9

4

9

4

9

5

5

3

3

3

5

2

3

2

CH C H C H i-C H i-C H t-C H i-C H n-C H i-C H t—C H t—C n-C i-C H t-C H neo-C H neo-C H 3

3

2

First I

1

n

7

4

9

4

9

3

7

3

7

3

7

4

9

4

9

5

n

5

n

D

5d

1 0

I

9.33 8.84 8.45 8.47 8.75 8.32 8.18 8.29 8.03 8.06

14.93 14.85 14.71 14.86 14.74

8.30 7.57 8.33 8.30

14.47

14.61 14.63 14.46

14.49 14.41

s e n t a t i v e s p e c t r a of only the f i r s t band a r e r e p r o d u c e d i n F i g u r e 1 f o r one s e r i e s of a l k y l m e t h y l m e r c u r y c o m p o u n d s , i . e . , R-HgCH . T h e effect of a l k y l s u b s t i t u t i o n on the f i r s t i o n i z a t i o n p o t e n t i a l of a s e r i e s of a l k y l d e r i v a t i v e s i s a t t r i b u t e d p r i m a r i l y to p o l a r i z a t i o n effects i n the m o l e c u l a r i o n f i n a l state. It has been r e c o g n i z e d that s u c h e l e c t r o n i c effects a r e a d d i t i v e a l o n g the series: M e , E t , i - P r , and t - B u . T h u s , the e n e r g y effect of r e p l a c i n g M e b y E t i s e x p e c t e d to e q u a l that of r e p l a c i n g E t by i-Pr, or of r e p l a c i n g i - P r by t - B u . In e a c h c a s e , α - h y d r o g e n s in C H a r e being sequentially replaced by methyl groups. Addi t i v e e n e r g y effects have been u s e d by T a f t (7) as a c r i t e r i o n for i d e n t i f y i n g p o l a r effects as denoted by the e m p i r i c a l substituent constant σ * : 3

3

(σ*)

M e : Et : i - P r : t - B u

=

0 : 0.10

: 0.19 :

0.30

It has been shown that the T a f t r e l a t i o n s h i p holds for the i o n i z a tion p o t e n t i a l s of a l c o h o l s and other a l k y l c o m p o u n d s . Figure 2 i l l u s t r a t e s the l i n e a r r e l a t i o n s h i p between σ * v a l u e s and the i o n i z a t i o n p o t e n t i a l s of a s e r i e s of a l c o h o l s , a l k y l b r o m i d e s , a l k y l h y d r a z i n e s , and a l d e h y d e s . T h e c o r r e l a t i o n of the i o n i z a t i o n potentials of a s e r i e s of a l k y l m e r c u r i a l s R - H g C H a l s o plotted i n F i g u r e 2 i s d i s t i n c t l y n o n l i n e a r . T h e i n c r e m e n t a l changes i n energies become p r o g r e s s i v e l y s m a l l e r or ' ' s a t u r a t e d " as one p r o c e e d s f r o m m e t h y l to t e r t - b u t y l . M o r e o v e r , the s a m e p a t t e r n of s a t u r a t i o n obtains f o r the a n a l o g o u s s e r i e s of G r i g n a r d r e a g e n t s R - M g X and t r i 3


13.

KOCHi

Alkyl Transfers


209

210

ORGANOMETALS AND

ORGANOMETALLOIDS

Or*

0

OJ

0.2

03

No. OF α-METHYL GROUPS Inorganic Chemistry

Figure 2. Correlation of the first ionization po tential of various alkyl-substituted compounds vs. Taft's σ* constant (top scale) and number of α-methyl groups (bottom scale) (6).


13.

KOCHi

Alkyl Transfers

211

m e t h y l t i n c o m p o u n d s R S n M e m e a s u r e d i n d e p e n d e n t l y (8,2,). T h e d i f f e r e n c e between e n e r g y effects w h i c h a r e s a t u r a t e d and those that a r e a d d i t i v e m a y be r e l a t e d to the n a t u r e of the highest o c c u p i e d m o l e c u l a r o r b i t a l ( H O M O ) . F o r those s y s t e m s c o n t a i n i n g nonbonding e l e c t r o n s , the i o n i z a t i o n p r o c e e d s f r o m a H O M O w h i c h i s l a r g e l y o r t h o g o n a l to the σ b o n d i n g o r b i t a l s , p a r t i c u l a r l y those a s s o c i a t e d with the bonding of the h e t e r o a t o m to carbon. T h u s , although i n t e r a c t i o n s between the a l k y l g r o u p and the h e t e r o a t o m c a n be o b s e r v e d , i o n i z a t i o n f r o m the H O M O i s only w e a k l y c o u p l e d to the b o n d i n g s y s t e m and a d d i t i v i t y i s o b s e r v e d . In c o n t r a s t , the i o n i z a t i o n p r o c e s s i n o r g a n o m e t a l s such as M e H g p r o c e e d s f r o m a m o l e c u l a r o r b i t a l that has s u b s t a n t i a l m e t a l - c a r b o n bonding c h a r a c t e r . T h i s conclusion is portrayed with a s i m p l e L C B O d i a g r a m as: 3

2

Η 6ρ·0~ C £ R , + R 2

Ο —\ y

Inorganic Chemistry

w h e r e the H O M O i s f o r m e d f r o m the b o n d i n g c o m b i n a t i o n of the a n t i s y m m e t r i c c o m b i n a t i o n of the σ type g r o u p o r b i t a l s of the a l k y l f r a g m e n t s with the m e r c u r y 6p a t o m i c o r b i t a l . In fact, i o n i z a t i o n of the H O M O of a l k y l r a d i c a l s ( i . e . , R* — R -f e) s e r v e s as a r e a s o n a b l e m o d e l for the i o n i z a t i o n of the H O M O of organomercurials. S i g n i f i c a n t l y , both the m e a s u r e d i o n i z a t i o n p o t e n t i a l s of a l k y l r a d i c a l s (JJ)) and those obtained f r o m S C F - M O c a l c u l a t i o n s l i s t e d i n T a b l e II (\l ) show the c h a r a c t e r i s t i c s a t u r +

T a b l e II. E x p e r i m e n t a l and C a l c u l a t e d I o n i z a t i o n P o t e n t i a l s of A l k y l R a d i c a l s . A l k y l Radical Methyl Ethyl iso-Propyl tert-Butyl

E x p e r i m e n t a l (eV) 9.84 8.38 7.55 6.93

C a l c u l a t e d (eV) 9-95 8.56 7.60 6.83

a t i o n effect d e s c r i b e d a b o v e , and they c o r r e l a t e w e l l with the f i r s t v e r t i c a l i o n i z a t i o n potentials of the o r g a n o m e r c u r i a l s l i s t e d i n T a b l e I. T h u s , i n t h i s c a s e , i o n i z a t i o n f r o m the H O M O i s s t r o n g l y c o u p l e d to the bonding s y s t e m . In s u c h a s i t u a t i o n , c h a n g e s i n e l e c t r o n r e p u l s i o n m a y w e l l be l a r g e and not e f f e c t i v e l y constant as the a l k y l g r o u p i s s y s t e m a t i c a l l y v a r i e d f r o m M e to t - B u . T h e v a r i a t i o n i n the 5d i o n i z a t i o n p o t e n t i a l t h r o u g h the s e r i e s of d i a l k y l m e r c u r y c o m p o u n d s a r i s e s p r i m a r i l y f r o m c h a n g e s i n the g r o u n d state c h a r g e on the m e r c u r y a t o m . The


212


Ij)(5d) data c a n b e u s e d to c a l c u l a t e the c h a r g e on the m e r c u r y a t o m as p r e s e n t e d i n T a b l e III, together with the c a l c u l a t e d T a b l e III. C a l c u l a t e d C h a r g e s (q) on the M e r c u r y A t o m and A p p r o x i m a t e E l e c t r o n e g a t i v i t i e s of the A l k y l G r o u p s . Dialkylmercury

q

(CH ) Hg (C H } Hg (n-C H ) Hg (i-C H ) Hg (i-C H ) Hg (neo-C H ) Hg

+ 0.0234 - 0.0338 -0.0547 - 0.0990 - 0.0703 - 0.112

3

2

2

5

2

3

7

3

7

4

9

2

2

2

5

n

2

Electronegativity 1.98 1.81 1.75 1.62 1.70 1.58

(CH ) (C H ) (n-C H ) (i-C H ) (i-C H ) (neo-C H ) 3

2

5

3

7

3

7

4

9

5

n

v a l u e s of the e l e c t r o n e g a t i v i t i e 5 d bands c o u l d not be m e a s u r e d with a h i g h d e g r e e of a c c u r a c y . T h e r e f o r e , c a l c u l a t e d c h a r g e s and e l e c t r o n e g a t i v i t i e s of the a l k y l g r o u p s m u s t be r e g a r d e d a s h a v i n g m o r e q u a l i t a t i v e r a t h e r than q u a n t i t a t i v e s i g n i f i c a n c e at this point. N e v e r t h e l e s s , the a p p r o x i m a t e l y e q u a l c h a n g e s i n e l e c t r o n e g a t i v i t y on p r o c e e d i n g f r o m m e t h y l to ethyl (1.98 to 1.81) a n d f r o m ethyl to i s o p r o p y l (1.81 to 1.62) a r e at l e a s t c o n s i s t e n t with the a d d i t i v i t y c i t e d a b o v e . Among binary mercury(ll) derivatives, dialkylmercury c o m p o u n d s p o s s e s s the l o w e s t i o n i z a t i o n p o t e n t i a l s a s shown i n Table IV. 1 0

T a b l e IV. F i r s t V e r t i c a l I o n i z a t i o n P o t e n t i a l s (eV) of B i n a r y M e r c u r y ( l l ) D e r i v a t i v e s . CIHgCl

(11.37)

MeHgCl

(10.88)

BrHgBr

(10.62)

MeHgBr

(10.16)

IHgl

( 9.50)

MeHgl

( 9.25)

MeHgMe

(9.33)

B. T e t r a a l k y l l e a d C o m p o u n d s . T h e vapor phase photoel e c t r ô n ~ ¥ p ë ^ t r â ~ 1 5 I T n ^ ^ i . e . , neopentane, tetramethylsilane, tetramethylgermane, tetramethylstannane and t e t r a m e t h y l p l u m b a n e , have been s c r u t i n i z e d . W i t h the e x c e p t i o n of neopentane, the s p e c t r a a r e a l l s i m i l a r , e a c h s h o w i n g two b r o a d b a n d s . T h e h i g h e r e n e r g y band o c c u r s at about 14 eV, and c o m p a r i s o n w i t h s i m p l e c o m p o u n d s s u g g e s t s that this band i s a s s o c i a t e d with i o n i z a t i o n f r o m the o ( C - H ) b o n d i n g m o l e c u l a r o r b i t a l m a i n l y l o c a l i z e d on the m e t h y l f r a g m e n t . T h e threshold or a d i a b a t i c i o n i z a t i o n e n e r g y of the l o w e r e n e r g y band d e c r e a s e s i n the o r d e r ( C H ^ C > (CH ) Si > (CH ) Ge > (CH ) Sn > ( C H ) P b f r o m 10.25, 9.42, 9.38, 8.85, to 8.38 eV, r e s p e c t i v e l y , i n d i c a t i n g that i o n i z a t i o n i s a s s o c i a t e d with e l e c t r o n s l o c a l i z e d r e l a t i v e l y c l o s e to the m e t a l a t o m . T h e l o w e r b a n d has b e e n a s s i g n e d to i o n i z a t i o n f r o m the 3 t o r b i t a l d e r i v e d p r i n c i p a l l y 3

3

4

3

4

3

4

4

2


13.

Alkyl Transfers

KOCHi

213

f r o m t h e a ( M - C ) bonding o r b i t a l s . In t e t r a m e t h y l p l u m b a n e i t i s s p l i t i n t o two w e l l - r e s o l v e d bands. E x a m i n a t i o n of the pes f o r the s e r i e s of m e t h y l / e t h y l l e a d c o m p o u n d s i n T a b l e V r e v e a l s that T a b l e V. Et _ PbMe 4

n

n

I o n i z a t i o n and O x i d a t i o n P o t e n t i a l s of O r g a n o l e a d C o m p o u n d s . O x i d a t i o n P o t e n t i a l (V) I o n i z a t i o n P o t e n t i a l (eV)

Et Pb Et PbMe Et PbMe EtPbMe Me Pb

1.67 1.75 1.84 2.01 2.13

4

3

2

2

3

4

8.13 8.26 8.45 8.65 8.90

the i o n i z a t i o n p o t e n t i a l s u b s t i t u t i o n of e t h y l for m e t h y l g r o u p s a r o u n d the l e a d n u c l e u s . T h e r e g u l a r t r e n d noted o v e r the e n t i r e s e r i e s of m e t h y l / e t h y l l e a d c o m p o u n d s s u g g e s t s that s u b s t i t u t i o n of a n e t h y l g r o u p f o r a m e t h y l g r o u p i s l a r g e l y a n e l e c t r o n i c effect, and that s t e r i c i n t e r a c t i o n s between a l k y l g r o u p s a r o u n d the l e a d a t o m a r e not l a r g e . M o r e o v e r , i t i s i n t e r e s t i n g t o note that the c u m u l a t i v e e f f e c t s of α-methyl g r o u p s a r e s i m i l a r i n a c o m p a r i s o n of the s e r i e s of d i a l k y l m e r c u r y c o m p o u n d s l i s t e d i n T a b l e I w i t h t h o s e t e t r a a l k y l l e a d c o m p o u n d s l i s t e d i n T a b l e V. T h e a n o d i c o x i d a t i o n of the s a m e s e r i e s of m e t h y l / e t h y l l ead c o m p o u n d s has a l s o been e x a m i n e d i n a c e t o n i t r i l e s o l u t i o n s w i t h l i t h i u m f l u o r o b o r a t e a s a s u p p o r t i n g e l e c t r o l y t e (12). The n u m b e r o f e l e c t r o n s i n v o l v e d i n the a n o d i c p r o c e s s was d e t e r m i n e d t o be 1.0 by t h i n l a y e r c h r o n o p o t e n t i o m e t r y u s i n g a p l a t i num electrode for a l l the t e t r a a l k y l l e a d compounds examined. T h e a n o d i c o x i d a t i o n of e a c h of t h e t e t r a a l k y l l e a d c o m p o u n d s w a s found t o be i r r e v e r s i b l e b y c u r r e n t - r e v e r s a l c h r o n o p o t e n t i o m e t r y , s u g g e s t i n g that t h e a l k y l l e a d c a t i o n - r a d i c a l i s u n s t a b l e . E q 1 r e p r e s e n t s i t s m o d e of d e c o m p o s i t i o n . R Pbt 4

•

R Pb 3

+

+

R.

[1]

T h e p o t e n t i a l i n a m a g n e t i c a l l y - s t i r r e d s o l u t i o n of t e t r a a l k y l l e a d depended on the i d e n t i t y of the l e a d compound. S i n c e e a c h of t h e s e o x i d a t i v e p r o c e s s e s i s i r r e v e r s i b l e , the o b s e r v e d poten t i a l s s h i f t e d to m o r e p o s i t i v e v a l u e s a s the c u r r e n t d e n s i t y i n c r e a s e d , and no t h e o r e t i c a l s i g n i f i c a n c e c a n be p l a c e d on the a b s o l u t e v a l u e s of the m e a s u r e d p o t e n t i a l s . A t a g i v e n c u r r e n t d e n s i t y , h o w e v e r , the p o t e n t i a l s r e f l e c t the r e l a t i v e e a s e of r e m o v a l of a s i n g l e e l e c t r o n f r o m the s e r i e s of m e t h y l / e t h y l l e a d c o m p o u n d s e x a m i n e d i n t h i s study. T h e l a t t e r r e s t s on the p r e s u m p t i o n that a n o d i c o x i d a t i o n of t h e s e c l o s e l y r e l a t e d c o m p o u n d s p r o c e e d s v i a a c o m m o n m e c h a n i s m . Indeed, e l e c t r o n detachment f r o m t e t r a a l k y l l e a d m e a s u r e d i n the gas phase b y p h o t o e l e c t r o n


214


s p e c t r o s c o p y a l s o shows a s t r i k i n g r e l a t i o n s h i p with the c h e m i c a l oxidation potentials i n a c e t o n i t r i l e solution.

electro

II. E l e c t r o p h i l i c C l e a v a g e of O r g a n o m e t a l s — Q u a n t i t a t i v e E f f e c t s of A l k y l G r o u p s A c e t o l y s i s of d i a l k y l m e r c u r y c o m p o u n d s l i b e r a t e s one e q u i v a l e n t of a l k a n e and of a l k y l m e r c u r y a c e t a t e a c c o r d i n g to eqs 2 and 3 (13). y > E H + R'HgOAc [2] R-Hg-R + HOAc ( Ν ' • R H + RHgOAc [3] k

1

k

!

T h e f u r t h e r c l e a v a g e of a l k y l m e r c u r y a c e t a t e i s too s l o w to i n t e r f e r e w i t h the a c e t o l y s i s study of d i a l k y l m e r c u r y . T h e p s e u d o f i r s t - o r d e r rate constant w e r e d e t e r m i n e d f r o m the r a t e s of l i b e r a t i o n of a l k a n e s R H and R H, respectively. T h e p r o t o n o l y s i s of d i a l k y l m e r c u r y i n a c e t i c a c i d s o l u t i o n s proceeds by a r a t e - l i m i t i n g proton t r a n s f e r . T h e experimental v a l u e s of the k i n e t i c i s o t o p e effect of 9-11 a r e c l o s e to the t h e o r e t i c a l m a x i m u m expected for the t r a n s f e r of d e u t e r i u m r e l a t i v e to p r o t o n i n t h i s s y s t e m . T h e l a r g e v a l u e s of k j j / k £ ) a l s o suggest a r a t h e r l i n e a r t r a n s i t i o n state for p r o t o n t r a n s f e r i n w h i c h the c o n t r i b u t i o n f r o m the s y m m e t r i c s t r e t c h i n g m o d e i s s m a l l . S u c h a t r a n s f e r of a p r o t o n h a l f w a y i n the t r a n s i t i o n state p l a c e s a c o n s i d e r a b l e p o s i t i v e c h a r g e on m e r c u r y . T h e s e r e s u l t s together with the r e t e n t i o n of c o n f i g u r a t i o n d u r i n g p r o t o - d e m e r c u r a t i o n a r e c o n s i s t e n t w i t h a 3 - c e n t e r t r a n s i t i o n state of the type d e picted below. f

: LR

H—OAc

1

T h e m o r e or l e s s t r i a n g u l a r a r r a y of c a r b o n , m e r c u r y and the p r o t o n i n the t r a n s i t i o n state f o r p r o t o n o l y s i s was o r i g i n a l l y p r o p o s e d b y K r e e v o y and H a n s e n (14). T h e extent to w h i c h t h e r e i s n u c l e o p h i l i c a s s i s t a n c e d u r i n g a c e t o l y s i s of d i a l k y l m e r c u r y i s not t r e a t e d e x p l i c i t l y . Instead, f o r the a c e t o l y s i s i n eq 2, the effects of a l k y l g r o u p s on the c l e a v a g e r e a c t i o n c a n be c l a s s i f i e d i n t o two c a t e g o r i e s — n a m e l y , l e a v i n g g r o u p (HgR ) effects and c l e a v e d g r o u p (R) e f f e c t s . S t e r i c e f f e c t s due to the l e a v i n g g r o u p s a r e u n i m p o r t a n t i n a c e t o l y s i s , and i t helps to l i m i t the m e c h a n i s t i c c o n s i d e r a t i o n s to the i m m e d i a t e l o c u s of the r e a c t i o n s i t e . L e a v i n g G r o u p E f f e c t s (HgR ). T h e effect of l e a v i n g g r o u p s H g R on the c l e a v a g e of a p a r t i c u l a r a l k y l - m e r c u r y bond a c c e l e r ates i n the o r d e r : R = M e < Et < i - P r < t - B u . This reactivity s e q u e n c e r e p r e s e n t s the i n c r e a s i n g a b i l i t y of t h e s e a l k y l g r o u p s to a c c o m m o d a t e a p o s i t i v e c h a r g e when they a r e attached to the 1

1

1

1


13.

Alkyl Transfers

KOCHi

215 1

d e p a r t i n g c a t i o n i c m e r c u r y (HgR ). E l e c t r o n r e l e a s e b y v a r i o u s a l k y l g r o u p s i n r e s p o n s e to a f i x e d e l e c t r o n d e m a n d i n the ground state of C H H g R i s a l s o r e f l e c t e d i n the m a g n i t u d e s of the m e t h y l proton coupling constants, j ( H g - H ) . M o r e appropriately, e l e c t r o n r e l e a s e b y a l k y l g r o u p s i n the t r a n s i t i o n state f o r a c e t o l y s i s m a y be m o d e l e d b y the c a t i o n - r a d i c a l of d i a l k y l m e r c u r y . T h e l a t t e r i s p r o b e d i n d e p e n d e n t l y b y m e a s u r i n g the e n e r g e t i c s of e l e c t r o n d e t a c h m e n t f r o m a h o m o l o g o u s s e r i e s of RHgR , e.g., f

3

1 9 9

1

CH Hg-R

!

!

>-

3

[CH Hg-R ]

+

+ e

3

as d e s c r i b e d i n the f o r e g o i n g s e c t i o n . Indeed, t h e r e i s a l i n e a r c o r r e l a t i o n of l o g k f o r a c e t o l y s i s a n d the v e r t i c a l i o n i z a t i o n p o t e n t i a l s of a s e r i e s of C H H g - R Sinc photoelectro i o n i z a tion i s a v e r t i c a l process l a r g e l y f r e e of s t e r i c f a c t o r s support c o n c l u s i o n that s t e r i c e f f e c t s of l e a v i n g g r o u p s a r e u n i m p o r t a n t i n a c e t o l y s i s of t h i s s e r i e s of o r g a n o m e r c u r i a l s . T h e c o r r e l a t i o n between r a t e s of c l e a v a g e and i o n i z a t i o n p o t e n t i a l i s not r e s t r i c t e d to o r g a n o m e r c u r i a l s . T h e s a m e r e l a t i o n s h i p i s a l s o obtained i n 4 - c o o r d i n a t e o r g a n o l e a d c o m p o u n d s (15.16). f

k

lv[e

> < )> C H + M e - E t . P b O A c [4] — / v ^ t y > CH3CH3 4- M e E t - P b O A c [5] 4

M e E t . P b + HOAc n

4

n

n

1

4

n

n

3

n

T h u s , the r a t e of a c e t o l y s i s of the M e - P b bond [i.e., l o g k ( M e ) ] d e c r e a s e s l i n e a r l y w i t h the i o n i z a t i o n p o t e n t i a l of M e E t - P b , w h e r e η = 0, 1, 2, 3,4, and a p a r a l l e l r e l a t i o n s h i p i s obtained d u r i n g the c o n c o m i t a n t c l e a v a g e of the E t - P b bond [ l o g k ( E t ) ] . I n c r e a s i n g s t e r i c f a c t o r s i n the l e a v i n g g r o u p ( i . e . , t r i a l k y l l e a d ) p r e v e n t e x t e n s i o n to h i g h e r h o m o l o g s . In the a c e t o l y s i s of d i a l k y l m e r c u r y , the l e a v i n g g r o u p (HgR ) e f f e c t s [under c o n d i t i o n s of a c o n s t a n t c l e a v e d (R) group] can be e x p r e s s e d q u a n t i t a t i v e l y by the l i n e a r f r e e e n e r g y r e l a t i o n s h i p i n eq 6, l o g Κ/Κ = I. [6] n

4

n

1

0

w h e r e K i s the r a t e c o n s t a n t f o r a c e t o l y s i s of R-HgR i n eq 2 when R = M e , and Κ i s that f o r R = E t , i - P r o r t-Bu. I- i s a l e a v i n g g r o u p constant w h i c h has a c h a r a c t e r i s t i c v a l u e f o r e a c h HgR , a n d i t does not depend on the n a t u r e of the c l e a v e d g r o u p (R). N o r m a l i z a t i o n s of I· to I-(Et) = 0.10 a r e l i s t e d i n T a b l e V I as I· to a l l o w d i r e c t c o m p a r i s o n w i t h the T a f t σ* c o n s t a n t s . T h e r e i s a s t r i k i n g d i f f e r e n c e b e t w e e n the v a l u e s of I· and σ*, a l t h o u g h both a r e due to e l e c t r o n i c o r p o l a r e f f e c t s . T h u s , t h e r e i s a " s a t u r a t i o n " i n i n c r e m e n t a l c h a n g e s i n e n e r g y f o r 1» as e a c h h y d r o g e n i n R H g C H i s s e q u e n t i a l l y r e p l a c e d b y m e t h y l g r o u p s i n the s e r i e s ; R H g C H , R H g C H C H , R H g C H ( C H ) , and R H g C ( C H ) . On the other hand, the c o r r e s p o n d i n g changes i n σ* 1

0

1

1

1

1

1

3

3

3

2

3

3

2

3


216

O R G A N O M E T A L S AND

ORGANOMETALLOIDS

Table VI. Leaving Group P a r a m e t e r s i n Acetolysis of D i a l k y l m e r c u r y . C o m p a r i s o n w i t h T a f t σ*. σ* I.* L e a v i n g G r o u p (HgR ) I. 1

HgCH HgCH CH HgCH(CH ) HgC(CH ) 2

3

3

3

2

3

0 0.10 0.19 0.30

0 0.10 0.17 0.19

0 0.76 1.28 1.44

3

11

a r e "additive, i n c r e a s i n g l i n e a r l y f r o m CH , C H C H , ( C H ) C H to ( C H ) C . Indeed, T a f t has e m p l o y e d the a d d i t i v i t y r e q u i r e m e n t for identifying polar effects. T h e d i f f e r e n c e between e n e r g y e f f e c t s w h i c h a r e s a t u r a t e d and t h o s e that a r e a d d i t i v e p r o v i d e s the key to the u n d e r s t a n d i n g of s u b s t i t u e n t e f f e c t s i strong linear correlatio p o t e n t i a l s of a s e r i e s of a l c o h o l s , a l k y l h y d r a z i n e s , a l d e h y d e s and a l k y l h a l i d e s r e p r e s e n t e d by the p r o c e s s : RX — R X ^ " + e. On the other hand, the i o n i z a t i o n p o t e n t i a l s of a s e r i e s of o r g a n o m e r c u r i a l s C H H g R a l s o plotted a g a i n s t σ* show a s a t u r a t i o n effect e q u i v a l e n t to that i n a c e t o l y s i s . T h e s a t u r a t i o n a l s o obtains f o r i o n i z a t i o n f r o m the s a m e s e r i e s of G r i g n a r d r e a g e n t s and a l k y l t r i m e t h y l t i n c o m p o u n d s , ( C H ) S n R . T h e d i f f e r e n c e between s a t u r a t i o n and a d d i t i v i t y e f f e c t s can be e x p l a i n e d by c o n s i d e r i n g the h i g h e s t o c c u p i e d m o l e c u l a r o r b i t a l ( H O M O ) i n e a c h s e r i e s . F o r t h o s e c o m p o u n d s c o n t a i n i n g nonbonding e l e c t r o n s , the i o n i z a t i o n p r o c e e d s f r o m a H O M O w h i c h i s l a r g e l y o r t h o g o n a l to the o r b i t a l i n v o l v e d i n the b o n d i n g of X to c a r b o n i n R-X, and i t s effect on the e l e c t r o n d e n s i t y i n the bond i s m i n i m a l . In c o n t r a s t , the i o n i z a t i o n p r o c e s s i n o r g a n o m e t a l s s u c h as M e H g p r o c e e d s f r o m a b o n d i n g m o l e c u l a r o r b i t a l w i t h a node at m e r c u r y . C o n sequently, the e l e c t r o n d e n s i t y i n the bond to c a r b o n i s d i m i n i s h e d s u b s t a n t i a l l y , and the c a t i o n i c c h a r a c t e r of the α-carbon i s a c c o m p a n i e d by a d e c r e a s e i n e l e c t r o n r e p u l s i o n , w h i c h i s not e f f e c t i v e l y c o n s t a n t as the a l k y l g r o u p i s s y s t e m a t i c a l l y v a r i e d f r o m M e to t-Bu. Indeed, the v a l i d i t y of t h i s d e s c r i p t i o n i s shown by v a l u e s of the i o n i z a t i o n p o t e n t i a l s of a l k y l r a d i c a l s [i.e., R» — • R 4- e], w h i c h a g r e e r e m a r k a b l y w e l l w i t h those obtained f r o m S C F - M O c a l c u l a t i o n s ( l 7.18). S i g n i f i c a n t l y , the i o n i z a t i o n p o t e n t i a l s of a l k y l r a d i c a l s show the c h a r a c t e r i s t i c s a t u r a t i o n effect d e s c r i b e d above, and they a l s o c o r r e l a t e w e l l w i t h l o g k for a c e t o l y s i s and the i o n i z a t i o n p o t e n t i a l s of the o r g a n o m e r c u r i a l s . It i s n o t e w o r t h y that l e a v i n g g r o u p (HgR ) e f f e c t s due to sub s t i t u t i o n of m e t h y l g r o u p s i n the (3-position of the a l k y l c h a i n R a r e h i g h l y attenuated r e l a t i v e to that a c c o m p a n y i n g a - s u b s t i t u t i o n . F o r i n s t a n c e , the p s e u d o f i r s t - o r d e r r a t e c o n s t a n t f o r m e t h a n e e v o l u t i o n f r o m M e H g E t i s 2.35 χ 10" sec** and that f o r M e H g — i - B u i s 2.39 x 10" s e c " . T h u s , l e a v i n g g r o u p e f f e c t s a r e not s i m p l y r e l a t e d to the s i z e of the a l k y l g r o u p (R ). C l e a v e d G r o u p E f f e c t s (R). T h e r a t e s of a c e t o l y s i s of a l k y l 3

3

2

3

3

2

3

f

3

f

3

3

2

+

1

1

6

6

1

1

1


13.

217

Alkyl Transfers

KOCHi

g r o u p s f r o m d i a l k y l m e r c u r y d e c r e a s e i n the o r d e r : R = Et > i - P r > M e > t - B u . T h e c l e a v e d a l k y l g r o u p e f f e c t s [under c o n d i tions i n w h i c h the l e a v i n g g r o u p (HgR ) i s constant] c a n be ex p r e s s e d q u a n t i t a t i v e l y by eq 7, 1

K'A'o

log

=

[7]

C

w h e r e K i s the r a t e constant for a c e t o l y s i s of R - H g R i n eq 2 when R = M e , and K i s that for R = E t or i - P r . C is a cleaved a l k y l g r o u p constant w h i c h has a c h a r a c t e r i s t i c v a l u e for e a c h R, and i t does not depend on the l e a v i n g g r o u p H g R . T h e n o n - s y s t e m a t i c t r e n d i n the v a l u e s of C i n T a b l e VII f

1

0

1

1

Table VII. Cleaved Group P a r a m e t e r s in Acetolysi C l e a v e d A l k y l G r o u p (R)

C

CH CH CH (CH ) CH (CH ) C

0 0.55 0.29 ^-0.9

3

3

2

3

2

3

3

s u g g e s t s that t h e r e a r e at l e a s t two o p p o s i n g effects p r e s e n t i n the a c e t o l y s i s of a n a l k y l - m e r c u r y b o n d . T h e d e c r e a s e o b s e r v e d i n p r o c e e d i n g f r o m E t , i - P r to t - B u f o l l o w s f r o m the i n c r e a s e i n s t e r i c b u l k at the s i t e of p r o t o n a t i o n . O n the other hand, the i n c r e a s e f r o m M e to E t (and to i - P r ) i s i n a c c o r d with e l e c t r o n r e l e a s e f r o m t h e s e R g r o u p s a c c o m p a n y i n g p r o t o n o l y s i s , as described earlier. ( T h e v a l u e of C f o r the t - b u t y l g r o u p i s a p p r o x i m a t e. ) T h e r a t e s of p r o t o n o l y s i s of a l k y l m e r c u r y i o d i d e s (14) i n aqueous p e r c h l o r i c and s u l f u r i c a c i d f o l l o w the o r d e r expected f r o m a d o m i n a n c e of s t e r i c f a c t o r s , v i z . , M e : E t : i - P r : t - B u i n the r e l a t i v e order* 123 : 49 : 16 : 1.0. T h e s m a l l kinetic i s o t o p e effect (kpj/krj)) m e a s u r e d f r o m the p r o t o n o l y s i s of m e t h y l m e r c u r y i o d i d e m a y r e f l e c t e i t h e r a t r a n s i t i o n state i n w h i c h the bond to c a r b o n i s p o o r l y f o r m e d or one i n w h i c h i t i s a l m o s t c o m p l e t e . T h e l a t t e r c o u l d a c c o u n t f o r the r e a c t i v i t y p a t t e r n , but other u n c e r t a i n t i e s i n t h i s s y s t e m d i s c o u r a g e f u r t h e r d i s c u s s i o n . A s i m i l a r effect, h o w e v e r , c a n be o b s e r v e d d u r i n g the a c e t o l y s i s of a s e r i e s of w e l l - b e h a v e d m e t h y l - e t h y l l e a d c o m pounds [ M e E t - P b , when η = 0, 1, 2, 3] ( l 5 , 1 6 ) . If l e a v i n g g r o u p effects a r e taken i n t o account, the c l e a v a g e of M e - P b i s c o n s i s t ently 8.6 t i m e s m o r e f a c i l e than E t - P b c l e a v a g e i n a l l t h r e e i n t r a m o l e c u l a r c o m p e t i t i o n s as w e l l as i n the i n t e r m o l e c u l a r c o m p e t i t i o n u s i n g M e E t « P b (n = 1, 2, 3) and M e P b / E t P b , respectively. It i s n o t e w o r t h y that the M e / E t r e a c t i v i t y i n t e t r a a l k y l l e a d i s r e v e r s e d f r o m that i n d i a l k y l m e r c u r y [ k ( M e ) / k ( E t ) = 0.30], although the k i n e t i c i s o t o p e effect of 9 i n the a c e t o l y s i s of n

4

n

n

4

n

4

4


218

ORGANOMETALS


t e t r a e t h y l l e a d (]J2) i s c o m p a r a b l e to that o b s e r v e d with d i e t h y l mercury. T h e d i f f e r e n c e i s due to i n c r e a s e d s t e r i c h i n d r a n c e i n the 4 - c o o r d i n a t e o r g a n o l e a d c o m p o u n d s c o m p a r e d to the m o r e accessible 2-coordinate organomercury analogs. The severe s t e r i c r e s t r i c t i o n s i m p o s e d on t e t r a a l k y l l e a d c o m p o u n d s i s a l s o b o r n e out by the f a i l u r e to extend the l i n e a r f r e e e n e r g y r e l a t i o n ships to h i g h e r a l k y l h o m o l o g s i n p r o t o n o l y s i s s t u d i e s . G e n e r a l i z e d E q u a t i o n f o r P r o t o n o l y s i s of D i a l k y l m e r c u r y . T h e l i n e a r f r e e e n e r g y r e l a t i o n s h i p s i n eqs 6 and 7 f o r l e a v i n g g r o u p effects and c l e a v e d g r o u p e f f e c t s , r e s p e c t i v e l y , d u r i n g a c e t o l y s i s of d i a l k y l m e r c u r y suggest that a g e n e r a l i z e d r e l a t i o n s h i p i s p o s s i b l e w h i c h c o r r e l a t e s a l l the r a t e s u s i n g the e m p i r i c a l p a r a m e t e r s i n T a b l e s V I and V I I , i . e . , log k / k

=

[8]

w h e r e ko r e p r e s e n t s the r a t e constant for a c e t o l y s i s of M e H g and k i s that f o r any other R H g R . T h e v a l i d i t y of eq 8 i s shown by c o m p a r i n g the e x p e r i m e n t a l r a t e c o n s t a n t s with t h o s e c a l c u l a t e d f r o m the e q u a t i o n . T h e e m p i r i c a l constant C i n the g e n e r a l i z e d eq 8 f o r a c e t o l y s i s of d i a l k y l m e r c u r y takes into a c c o u n t any s t e r i c i n t e r a c t i o n s due to the c l e a v e d g r o u p (R) at the r e a c t i o n s i t e . In the a b s e n c e of s u c h s t e r i c e f f e c t s , the c l e a v e d g r o u p effect i n e l e c t r o p h i l i c s u b s t i t u t i o n should be i n f l u e n c e d p r i m a r i l y by e l e c t r o n r e l e a s e and thus p a r a l l e l the l e a v i n g g r o u p (HgR ) effects as d e s c r i b e d i n eq 6 and T a b l e V I . S i g n i f i c a n t l y , the r e l a t i v e r e a c t i v i t i e s of the c l e a v e d a l k y l groups i n electron transfer cleavages follow a pattern i n which the i n c r e m e n t a l changes i n e n e r g y f o r R = M e , E t , i - P r and t - B u show a s a t u r a t i o n " effect, w h i c h i s the s a m e as that o b s e r v e d i n the l e a v i n g g r o u p effect (HgR ) denoted b y I* i n T a b l e V I . Indeed, t h e r e i s a l i n e a r c o r r e l a t i o n between the o x i d a t i o n or i o n i z a t i o n potentials and I·· T h u s , the s a t u r a t i o n p a t t e r n f o r a l k y l g r o u p s i s independent of whether they a r e i n v o l v e d as c l e a v e d (R) g r o u p s or as l e a v i n g (HgR ) g r o u p s . It c l e a r l y r e l a t e s to an i n t r i n s i c p r o p e r t y of the a l k y l - m e r c u r y bonds and r e f l e c t s the m a n n e r i n w h i c h an a l k y l g r o u p r e s p o n d s to the p r e s e n c e of a p o s i t i v e c h a r g e on m e r c u r y . T h e s a t u r a t i o n p a t t e r n for a l k y l g r o u p s c a n be u s e d as a d i a g n o s t i c p r o b e f o r the m e c h a n i s m of c l e a v a g e i n organometals. A c e t o l y s i s studies on d i a l k y l m e r c u r y have d e m o n s t r a t e d not only the i m p o r t a n c e of the c l e a v e d g r o u p (R) but a l s o the l e a v i n g g r o u p (HgR ) i n e l e c t r o p h i l i c s u b s t i t u t i o n . A l k y l g r o u p s a r e e x c e l l e n t p r o b e s f o r m e a s u r i n g t h e s e e l e c t r o n i c effects q u a n t i t a t i v e l y , and the c o r r e l a t i o n s of the r a t e s with the i o n i z a t i o n p o t e n t i a l s show that a p o s i t i v e c h a r g e i s d e v e l o p e d on both the l e a v i n g g r o u p (R) and the c l e a v e d g r o u p (HgR ). T h e s e f a c e t s of the r e a c t i v i t y of d i a l k y l m e r c u r i a l s t o w a r d p r o t o n i c e l e c t r o p h i l e s m a y be extended m o r e g e n e r a l l y , s i n c e i t has l o n g b e e n r e c o g 2

1

1

11

1

1

1

1


13.

Alkyl Transfers

KOCHi

219

n i z e d that n u c l e o p h i l i c r e a c t i v i t y i s i n f l u e n c e d b y the " p o l a r i z a b i l i t y " of the n u c l e o p h i l e . T h u s , the E d w a r d s o x y b a s e equation c o n t a i n s both a t e r m r e l a t e d to the o x i d a t i o n p o t e n t i a l of the n u c l e o p h i l e as w e l l a s a t e r m r e l a t e d to i t s b a s i c i t y (20,2,2,). T h e m o l e c u l a r o r b i t a l a n a l o g of the E d w a r d s equation has been d e v e l oped by K l o p m a n , i n w h i c h e l e c t r o s t a t i c and c o v a l e n t t e r m s a r e the c o u n t e r p a r t s to b a s i c i t y and p o l a r i z a b i l i t y , r e s p e c t i v e l y (22, 23). O r g a n o m e t a l l i c n u c l e o p h i l e s a r e σ - d o n o r s and have n e g l i g i b l e b a s i c i t y i n the E d w a r d s s e n s e . T h u s , the n u c l e o p h i l i c r e a c t i v i t y of o r g a n o m e t a l s u s i n g e i t h e r the E d w a r d s or K l o p m a n m o d e l s h o u l d r e d u c e to an equation s u c h as eq 8, i n w h i c h e l e c t r o n r e l e a s e by a l k y l g r o u p s i s the i m p o r t a n t c o n s i d e r a t i o n . The l a t t e r , i n e s s e n c e , r e p r e s e n t s a " v i r t u a l ' i o n i z a t i o n of the c a r b o n - m e t a l bond b y the e l e c t r o p h i l e s i n c e it c a n be d i r e c t l y r e l a t e d to the e n e r g e t i c s of e l e c t r o n d e t a c h m e n t . The 3-center t r a n s i t i o n state r e p r e s e n t e juncture. 1

H I . E l e c t r o n T r a n s f e r C l e a v a g e of O r g a n o m e t a l s with H e x a c h l o r o i r i d a t e ( I V ) A. T e t r a a l k y l l e a d Compounds. T e t r a a l k y l l e a d compounds r e a c t r a p i d l y with h e x a c h l o r o i r i d a t e ( I V ) at 2 5 ° C i n a c e t o n i t r i l e or a c e t i c a c i d s o l u t i o n (L2). F o r e x a m p l e , the a d d i t i o n of t e t r a m e t h y l l e a d to a s o l u t i o n of I r C l " " i n a c e t i c a c i d r e s u l t s i n the i m m e d i a t e d i s c h a r g e of the d a r k r e d - b r o w n c o l o r . Methyl c h l o r i d e , t r i m e t h y l l e a d a c e t a t e and the two r e d u c e d i r i d i u m ( l l l ) p r o d u c t s a r e f o r m e d f r o m t e t r a m e t h y l l e a d and h e x a c h l o r o i r i d a t e (IV) i n a c e t i c a c i d s o l u t i o n s a c c o r d i n g to the s t o i c h i o m e t r y : 6

Me Pb+ 2IrCl 4

6

2

"

HOAc

2

>

where S = solvent

M

e

3

P

b

o

A

c

+ CH C1 + 3

IrCl ~+ IrCl (S) " 6

3

[9]

2

5

O n l y one a l k y l g r o u p i s r e a d i l y c l e a v e d f r o m e a c h t e t r a a l k y l l e a d compound. T h e mixed methyl/ethyllead derivatives afford m i x t u r e s of m e t h y l and e t h y l c h l o r i d e s , the y i e l d s of w h i c h depend on the o r g a n o l e a d c o m p o u n d . Me

r—> PbR

Et

2

+ 2IrCl

6

2

Me-Cl

+ EtPbR

2

+

,

etc.

,

etc.

" —(

[10] ν—> Et-Cl

+ MePbR

2

A f t e r n o r m a l i z a t i o n for e a c h type of a l k y l g r o u p i n the r e a c t a n t , the r e l a t i v e y i e l d s of ethyl c h l o r i d e and m e t h y l c h l o r i d e a r e r a t h e r constant at about 25 i n a c e t o n i t r i l e , but v a r y s o m e w h a t i n acetic a c i d . T e t r a a l k y l l e a d c o m p o u n d s r e a c t with h e x a c h l o r o i r i d a t e ( l V ) at d i f f e r i n g r a t e s , w h i c h w e r e f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y by the d i s a p p e a r a n c e of the a b s o r p t i o n b a n d s at 490 and 585 n m . T h e k i n e t i c s showed a f i r s t - o r d e r d e p e n d e n c e on t e t r a a l k y l l e a d


220


and h e x a c h l o r o i r i d a t e ( l V ) i n both a c e t o n i t r i l e and a c e t i c a c i d solution. T h e s e c o n d - o r d e r rate constants determined i n aceto-d[IrCl "]/dt 6

=

2

2 k[R Pb] [IrCl ~] 4

6

[11]

2

n i t r i l e solutions i n c r e a s e p r o g r e s s i v e l y f r o m M e P b , M e P b E t , M e P b E t , M e P b E t , to E t ^ P b as l i s t e d i n T a b l e VIII. 4

2

2

3

3

T a b l e VIII. T h e C o r r e l a t i o n of S e l e c t i v i t i e s and Rates of O x i d a t i v e C l e a v a g e of T e t r a a l k y l l e a d s b y H e x a c h l o r o i r i d a t e ( l V ) with the E n e r g e t i c s of E l e c t r o n D e t a c h m e n t P r o c e s s e s . PbMe Et . n

4

k

ID

( M " sec )

MeCl

(eV)

(V)

(cm- )

26 11 3.3 0.57 0.02

24 25 24

8.26 8.45 8.65 8.90

1.75 1.80 2.01 2.13

20,400 22,600 23,200 24,300

1

PbEt PbEt Me PbEt Me PbEtMe PbMe 4

3

2

2

3

4

Ε

EtCl

n

l

-

1

T w o i m p o r t a n t c r i t e r i a c a n be u s e d to d i s t i n g u i s h the r e a c tion of t e t r a a l k y l l e a d with h e x a c h l o r o i r i d a t e ( l V ) f r o m the m o r e c o n v e n t i o n a l e l e c t r o p h i l i c p r o c e s s e s , e . g . , those i n v o l v i n g B r o n s t e d a c i d s , s i l v e r ( l ) , c o p p e r ( l ) or c o p p e r ( l l ) c o m p l e x e s , etc. (15,19*24,25). F i r s t , the r a t e of r e a c t i o n of E t » P b M e w i t h h e x a c h l o r o i r i d a t e ( l V ) i n c r e a s e s s u c c e s s i v e l y as m e t h y l i s r e p l a c e d b y ethyl g r o u p s [ s e e η = 4 to 0 i n T a b l e VIII, c o l u m n 2]. S e c o n d , a g i v e n e t h y l g r o u p i s c l e a v e d a p p r o x i m a t e l y 25 t i m e s f a s t e r than a m e t h y l g r o u p [ c o l u m n 3]. Both of t h e s e r e a c t i v i t y t r e n d s a r e d i a m e t r i c a l l y opposed to a n e l e c t r o p h i l i c c l e a v a g e w h i c h o c c u r s d i r e c t l y at the l e s s h i n d e r e d m e t h y l site f a s t e r than at a n ethyl site u n d e r equivalent c o n d i t i o n s . T h e s e r e s u l t s suggest that the r a t e - l i m i t i n g step with h e x a c h l o r o i r i d a t e ( l V ) o c c u r s p r i o r to a l k y l t r a n s f e r . T h e m e c h a n i s m g i v e n i n S c h e m e I i n v o l v e s e l e c t r o n t r a n s f e r i n eq 12 a s the r a t e limiting process. 4

n

n

S c h e m e I; R Pb 4

+ Ir^Cl " 6

2

k

R Pbt

f

a

s

R« + I r ^ C l / "

f

a

s

4

t

t

>

R Pbt

>

R- + R P b

>

RC1 + I i F c i / ' , etc.

4

+ Ir Cl M

3

6

3

"

+

[12] [13]

[14]

Indeed, t h e r e i s a good l i n e a r c o r r e l a t i o n of the r a t e s (log k) with the o n e - e l e c t r o n o x i d a t i o n or i o n i z a t i o n potentials of t e t r a a l k y l l e a d c o m p o u n d s p r e s e n t e d i n T a b l e V . S e l e c t i v i t y i n the c l e a v a g e of a l k y l g r o u p s f r o m o r g a n o l e a d a c c o r d i n g to S c h e m e I o c c u r s d u r i n g f r a g m e n t a t i o n of the c a t i o n - r a d i c a l i n a fast subsequent


13.

Alkyl Transfers

KOCHi

221

s t e p 13, w h i c h i s c o n s i s t e n t w i t h the m a s s s p e c t r a l study. Thus, a q u a n t i t a t i v e d e t e r m i n a t i o n of the c r a c k i n g p a t t e r n s of the s e r i e s of P b M e E t . showed that s c i s s i o n of the E t - P b bond i s f a v o r e d o v e r the M e - P b bond i n the p a r e n t m o l e c u l a r i o n s , l a r g e l y due to bond e n e r g y d i f f e r e n c e s . n

4

n

Me

Me* + E t P b R >bR

2

2

(

f

Et

[15]

+

. ν—•

Et-

+ MePbR

[16]

2

E x a m i n a t i o n of the e l e c t r o n s p i n r e s o n a n c e s p e c t r u m d u r i n g the r e a c t i o n w i t h h e x a c h l o r o i r i d a t e ( l V ) d i d not r e v e a l the p r e s e n c e of the c a t i o n - r a d i c a l P b E t t , w h i c h m u s t be h i g h l y u n s t a b l e e v e n at t e m p e r a t u r e s a s l o w as - 2 0 ° C . N o n e t h e l e s s , the f o r m a t i o n of ethyl r a d i c a l s i n high y i e l d s was evident f r o m s p i n - t r a p p i n g experiments with nitrosoisobutan w h i c h the w e l l - r e s o l v e obtained. 4

T h e u s e of h e x a c h l o r o i r i d a t e ( l V ) as an e f f i c i e n t s c a v e n g e r f o r a l k y l r a d i c a l s i s i m p l i e d i n S c h e m e I b y the i s o l a t i o n of a l k y l c h l o r i d e s i n h i g h y i e l d s . In s u p p o r t , s e p a r a t e e x p e r i m e n t s do i n d e e d show that e t h y l r a d i c a l s g e n e r a t e d u n a m b i g u o u s l y f r o m the t h e r m o l y s i s of p r o p i o n y l p e r o x i d e a r e q u a n t i t a t i v e l y c o n v e r t e d by h e x a c h l o r o i r i d a t e ( l V ) to e t h y l c h l o r i d e i n eq 14. T h e r e i s an a l t e r n a t i v e p o s s i b i l i t y that a l k y l h a l i d e i s f o r m e d d i r e c t l y f r o m the c a t i o n - r a d i c a l without the i n t e r m e d i a c y of a n a l k y l r a d i c a l . R Pbt + Ir^Cl/' 4

R Pb 3

+

+ R - C l + Ir^Clg " , 2

etc.

T h e d i f f e r e n c e between t h i s f o r m u l a t i o n and that p r e s e n t e d i n eq 13 of S c h e m e I r e s t s on the d e g r e e of m e t a s t a b i l i t y of the c a t i o n r a d i c a l toward fragmentation. T h e f a i l u r e to o b s e r v e the e s r s p e c t r a of R Pb+ and the i r r e v e r s i b i l i t y of the o x i d a t i o n w a v e i n c h r o n o p o t e n t i o m e t r y s u g g e s t s that i t s l i f e t i m e i s s h o r t . 4

B. D i a l k y l m e r c u r y Compounds. Hexachloroiridate(lV) a l s o r e a d i l y c l e a v e s d i a l k y l m e r c u r y c o m p o u n d s by s e c o n d - o r d e r k i n e t i c s s i m i l a r to eq 15 f o r t e t r a a l k y l l e a d (26). M o r e o v e r , the p r o d u c t s , both o r g a n i c and i r i d i u m ( l l l ) , as w e l l as the s t o i c h i o m e t r y of the r e a c t i o n a r e a l s o e q u i v a l e n t to that g i v e n i n eq 9> viz., Me Hg + 2 I r C l " 2

2

MeCl + MeHg

+

+ IrCl " + IrCl (S) " 6

3

5

2

N m r s t u d i e s i n d i c a t e that M e H g i s bound to I r C l ( S ) " i n s o l u t i o n . T h e s a m e s t o i c h i o m e t r y a p p l i e s to the h i g h e r h o m o l o g s ; the only d i f f e r e n c e lies i n the c o m p l e x i o n of the p r o d u c t s , i n c r e a s i n g a m o u n t s of a l k e n e s and a l k y l a c e t a t e s b e i n g f o r m e d at the e x p e n s e of a l k y l c h l o r i d e s on going f r o m e t h y l , i s o p r o p y l to t - b u t y l . T h e c l e a v a g e of d i a l k y l m e r c u r y by h e x a c h l o r o i r i d a t e ( l V ) i s h i g h l y dependent on the s t r u c t u r e of the a l k y l g r o u p s . T h u s , i n +

5

2


222


the h o m o l o g o u s s e r i e s of R H g C H , the r e l a t i v e r a t e s of c l e a v a g e i n c r e a s e f r o m R = m e t h y l : ethyl : i s o p r o p y l : t e r t - b u t y l , roughly i n the o r d e r of 1 0 ° : 10 · 10 · 1 0 . T h e s e r e s u l t s r u n c o u n t e r to the p a t t e r n o b s e r v e d i n the e l e c t r o p h i l i c c l e a v a g e of the s a m e m e r c u r i a l s d e s c r i b e d i n the f o r e g o i n g s e c t i o n , or to expectations b a s e d on i n c r e a s i n g s t e r i c h i n d r a n c e . Instead, it s u g g e s t s that the r a t e - l i m i t i n g step o c c u r s p r i o r to a l k y l t r a n s f e r . 3

3

5

6

S c h e m e II; R Hg 2

+ Ir^Cl*" R Hgt 2

R. + I r ^ C l " 2

k

f

a

s

f a s t

t

>

R Hgt + Ir Cl

>

RHg

>»

m

2

R

+

6

3

"

[17]

+ R.

[18]

+ Ir ci X " m

o x

5

[19]

n

T h e k i n e t i c s , p r o d u c t s and s e l e c t i v i t y as w e l l as s p i n t r a p p i n g w i t h t - B u N O and 0 a l l a c c o r d w i t h the m e c h a n i s m i n S c h e m e II. T h e o b s e r v a t i o n of p a r a m a g n e t i c i n t e r m e d i a t e s by s p i n t r a p p i n g i n d i c a t e s that a l k y l r a d i c a l s a r e f o r m e d d u r i n g the c l e a v a g e of R H g b y I r C l * " . In fact, the q u a n t i t a t i v e a c c o u n t i n g of the a l k y l f r a g m e n t s as a l k y l p e r o x y p r o d u c t s , when the r e a c t i o n i s c a r r i e d out i n the p r e s e n c e of oxygen, shows that a l l of the a l k y l g r o u p s m u s t d e p a r t f r o m m e r c u r y a s f r e e r a d i c a l s a c c o r d i n g to eq 18. T h e l a t t e r i s s t r o n g l y s u p p o r t e d by the o b s e r v a t i o n that I r C l " d i s a p p e a r s u n d e r t h e s e c o n d i t i o n s at just o n e - h a l f the r a t e o b s e r v e d i n an i n e r t a t m o s p h e r e , as p r e d i c t e d b y S c h e m e II. T h e f a i l u r e to o b s e r v e d i r e c t l y the e l e c t r o n s p i n r e s o n a n c e s p e c t r u m of R H g t s u g g e s t s that i t s l i f e t i m e i s v e r y s h o r t . It i s p r e s e n t as one of the p r i n c i p a l s p e c i e s d u r i n g e l e c t r o n i m p a c t of R H g i n the gas p h a s e , and a m e r c u r y ( l l l ) s p e c i e s has been o b s e r v e d as t r a n s i e n t i n the e l e c t r o c h e m i c a l o x i d a t i o n of H g ( c y c l a m ) +. S e l e c t i v i t y i n the c l e a v a g e of a l k y l g r o u p s f r o m u n s y m m e t r i c a l d i a l k y l m e r c u r y b y I r C l " a c c o r d i n g to S c h e m e II o c c u r s d u r i n g f r a g m e n t a t i o n of R H g t r a d i c a l - c a t i o n subsequent to the r a t e - l i m i t i n g step. T h e u n i m o l e c u l a r d e c o m p o s i t i o n of ( C H ) H g i n the gas phase has been e x a m i n e d b y p h o t o e l e c t r o n photoion c o i n c i d e n c e s p e c t r o s c o p y (27). 2

2

2

6

2

2

2

2

2

2

3

2

+

CH Hg 3

CH HgCH t 3

( \->

3

+

CH Hg3

+ CH 3

[20]

+ CH

3

[21]

T h e t h r e s h o l d e n e r g y f o r f r a g m e n t a t i o n i n eq 20 i s found to be n e a r l y 2.5 v o l t s l o w e r than that f o r eq 21. T h e e x c l u s i v e cleavage of R = t - B u and i - P r and p r e f e r e n t i a l c l e a v a g e of R = E t i n the h o m o l o g o u s s e r i e s of R H g C H i s i n a c c o r d w i t h a w e a k e r a l k y l m e r c u r y c o m p a r e d to a m e t h y l - m e r c u r y b o n d . T h e p r e d o m i n a n t f a c t o r w h i c h d e t e r m i n e s a l k y l v s . m e t h y l c l e a v a g e a r e the s t r e n g t h s of the r e l e v a n t C - H g b o n d s . T h e s e v a l u e s c a n be e v a l uated f r o m the a v e r a g e bond e n e r g i e s f o r M e H g , E t H g , and 3

2

2


13.

Alkyl Transfers

KOCHi

223 1

i - P r H g w h i c h a r e 58, 48, and 42 k c a l m o l " , r e s p e c t i v e l y (28.29)* A c c o r d i n g to S c h e m e II, the i s o l a t i o n of a l k y l c h l o r i d e s i n h i g h y i e l d s i m p l i e s that h e x a c h l o r o i r i d a t e ( l V ) i s an e f f i c i e n t s c a v enger of a l k y l r a d i c a l s i n eq 19 (Rox = RC1, X = S). H o w e v e r , i n a d d i t i o n to the r e d o x t r a n s f e r of c h l o r i n e f r o m h e x a c h l o r o i r i d a t e (IV) i n eq 14, a n a d d i t i o n a l r e d o x step i s r e q u i r e d , e s p e c i a l l y f o r R = i s o p r o p y l and t - b u t y l . T h e o b s e r v a t i o n of i s o b u t y l e n e and t e r t - b u t y l a c e t a t e f r o m t e r t - b u t y l r a d i c a l s and h e x a c h l o r o i r i d a t e (IV) i s a n a l o g o u s to e l e c t r o n t r a n s f e r o x i d a t i o n of a l k y l r a d i c a l s (30). ( C H ) 0 + Ir C l " >- I r C l " + ( C H ) C , etc. 2

2

3

3

3

6

+

6

3

3

The t e r t - b u t y l cation f o r m e d under such c i r c u m s t a n c e s w i l l u n d e r g o s o l v a t i o n , f o r e x a m p l e to t e r t - b u t y l acetate, or l o s s of a β-proton to i s o b u t y l e n e then m a i n t a i n i t s c o o r d i n a t i o the d i s t r i b u t i o n of I r C l ^ " and I r C l ( C H C N ) " a m o n g r e d u c e d i r i d i u m ( l l l ) products formed f r o m various a l k y l m e r c u r i a l s i s p r e c i s e l y i n a c c o r d w i t h t h i s f o r m u l a t i o n . T h u s , the r e s u l t s c l e a r l y i n d i c a t e that m e t h y l and e t h y l r a d i c a l s r e a c t w i t h I r C l ~ i n a c e t o n i t r i l e , e x c l u s i v e l y by c h l o r i n e t r a n s f e r . F o r i s o p r o p y l and t e r t - b u t y l r a d i c a l s , a p p r o x i m a t e l y 85 and 50$, r e s p e c t i v e l y , of the r e a c t i o n p r o c e e d s by c h l o r i n e t r a n s f e r and the r e m a i n d e r by e l e c t r o n t r a n s f e r . T h e l a t t e r b e c o m e s m o r e i m p o r t a n t i n a c e t i c a c i d s o l u t i o n s . T h e d e c r e a s i n g t r e n d of a l k y l r a d i c a l s to r e a c t w i t h I r C l " by e l e c t r o n t r a n s f e r i n the o r d e r : t - B u > i - P r » E t > M e f o l l o w s the e a s e of i o n i z a t i o n of the r a d i c a l i n the o r d e r : t - B u < i - P r < E t < M e as l i s t e d i n T a b l e II. Further m o r e , the opposed t r e n d i n the y i e l d s of a l k y l c h l o r i d e s i s c o n s i s t e n t w i t h the g e n e r a l l y d e c r e a s i n g a l k y l - c h l o r i n e bond energies f r o m M e C l t h r o u g h t - B u C l . W h e t h e r c h l o r i n e t r a n s f e r and c a r b o n i u m i o n f o r m a t i o n r e p r e s e n t i n n e r - and o u t e r - s p h e r e r e d o x p r o c e s s e s , r e s p e c t i v e l y , f o r m s an i n t e r e s t i n g s p e c u l a t i o n . I n n e r - and o u t e r - s p h e r e m e c h a n i s m s m e r i t c o n s i d e r a t i o n f o r the p r o c e s s by w h i c h e l e c t r o n t r a n s f e r o c c u r s f r o m R H g to I r C l " i n the r a t e - l i m i t i n g step i n eq 1 7. A l i n e a r f r e e e n e r g y r e l a t i o n s h i p b e t w e e n l o g k of r e a c t i o n and Ij) of R H g i s e x p e c t e d f o r t h i s s y s t e m i f e l e c t r o n t r a n s f e r o c c u r s by an o u t e r - s p h e r e p r o c e s s . H o w e v e r , the n e g a t i v e d e v i a t i o n of d i - t e r t - b u t y l - , d i i s o p r o p y l - , and d i e t h y l m e r c u r y f r o m the l i n e a r plot s u g g e s t s that s t e r i c f a c t o r s can be i m p o r t a n t i n the e l e c t r o n t r a n s f e r to IrCl ". 5

3

2

6

2

2

2

2

2

6

C. D i a l k y l ( b i s - p h o s p h i n e ) p l a t i n u m ( l I ) C o m p l e x e s . The c l e a v a g e of o r g a n o p l a t i n u m ( I I ) c o m p l e x e s w i t h o u t e r - s p h e r e o x i dants was c a r r i e d out as a c o m p a r i s o n f o r the a l k y l s of the m a i n g r o u p e l e m e n t s , l e a d and m e r c u r y , d e s c r i b e d above. Indeed, cis-dialkyl(bis-phosphine)platinum(ll) complexes are readily oxi d i z e d by h e x a c h l o r o i r i d a t e ( l V ) to a f f o r d two p r i n c i p a l types of p r o d u c t s d e p e n d i n g on the s t r u c t u r e of the a l k y l g r o u p and the


224

O R G A N O M E T A L S AND

ORGANOMETALLOIDS

c o o r d i n a t e d p h o s p h i n e (31)· T h u s , the d i e t h y l analog, c i s - E t P t ( L X ) ( P M e P h ) , a f f o r d s E t C l and e t h y l p l a t i n u m ( l l ) s p e c i e s by o x i d a t i v e c l e a v a g e of the E t - P t bond, 2

2

2

2

Et P#L + 2IrCl " 2

2

+

•

6

3

EtP^L S+

EtCl + IrCl "+IrCl

2

6

where L = P M e P h , P P h ; S = 2

2

5

S"

solvent

3

w h e r e a s M e P t ( P M e P h ) u n d e r g o e s o x i d a t i o n to d i m e t h y l platinum(lV) species. 2

2

2

2

Me PR. ( \ — R * 1

+

R Hg f

+

+ RHg

A s expected, the r a t e of t h i s c l e a v a g e (log k g ^ ) i s linearlyr e l a t e d to the i o n i z a t i o n p o t e n t i a l of the m e r c u r i a l as shown i n F i g u r e 3. A s i m i l a r c o r r e l a t i o n i s shown by o r g a n o m e t a l s u n d e r g o i n g s u b s t i t u t i o n [ G r i g n a r d r e a g e n t and p e r o x i d e (35)] or i n s e r t i o n [ t e t r a a l k y l l e a d and T C N E (36)] v i a e l e c t r o n t r a n s f e r , and they a r e a l s o i n c l u d e d i n F i g u r e 1 f o r c o m p a r i s o n . T h e r a t e constant k g f o r the e l e c t r o p h i l i c p r o t o n o l y s i s of an a l k y l - m e r c u r y bond, R-HgR

1

+

H

+

k

E

>

R-H +

R»Hg

+

can be d i s s e c t e d i n t o tw the c l e a v e d g r o u p R , and I·, w h i c h depends only on the l e a v i n g g r o u p , R H g , as d e s c r i b e d by eq 8. F i g u r e 4 shows that I· r e s p o n d s l i n e a r l y to the i o n i z a t i o n p o t e n t i a l of the m e r c u r i a l . F u r t h e r m o r e , I· a l s o s t r o n g l y c o r r e l a t e s w i t h the T a f t p o l a r s u b stituent constant, σ*, f o r the a l k y l g r o u p s l i s t e d i n T a b l e X . T h e !

T a b l e X . C o r r e l a t i o n of I· and C P a r a m e t e r s w i t h T a f t P o l a r ( σ * ) and S t e r i c ( E ) C o n s t a n t s . s

σ*

R

0 0.10 0.20 0.30

CH CH3CH2 (CH ) CH (CH ) C 3

3

2

3

3

Leaving Group Effects RHg

+

CH Hg+ CH CH Hg (CH ) CHHg+ (CH )3CHg 3

3

2

3

+

2

+

3

Cleaved Group Effects R CH C H C H2 (CH ) CH (CH ) C 3

3

3

2

3

3

JL( expt.) 0 0.76 1.28 1.44

C (expt.) 0 0.55 0.29 — 1

0 -0.07 -0.47 -1.54 8.1 σ * + 0.65 (cale.)

E

s

0 0.75 1.30 1.45 8.1 σ * + 2.8 (cale.)

E

s

0 0.61 0.30 -1.9

c u r v a t u r e i n F i g u r e 4 f o r the c l e a v e d g r o u p constant, C, on p r o c e e d i n g f r o m m e t h y l to t - b u t y l can be a t t r i b u t e d to an i n c r e a s i n g s t e r i c effect as a r e s u l t of a d d i t i o n a l e n c u m b r a n c e by s u c c è s -


ORGANOMETALS

228

AND

ORGANOMETALLOIDS

3 * Log k

8.5

9.0

Log

Figure 3. Εlectron-transfer processes in electrophilic substitutions. Saturation ef fects followed by alkyl substituents in the cleavage of organometals during the treat ment with various electrophiles: scale left and bottom for ((B) tetraalkyllead with tetracyanoethylene and (Q) dialkylmercury with hexachloroiridate(IV). Scale right (Tafel potential) and top for Grignard reagents and di-tert-butyl peroxide (·).

2

L

Log k

Figure 4. Correlation of cleaved-group constant C and leaving group constant L in acetolysis with the ionization potential of the dialkylmercury compound

C Bii*-HgCH

s

8

9

IONIZATION POTENTIAL,

tV


13.

Alkyl Transfers

KOCHi

229

s i v e l y m o r e α - m e t h y l groups. T h e latter i s a l s o supported by the s i z e a b l e c o n t r i b u t i o n of the T a f t s t e r i c p a r a m e t e r E to the c o r r e l a t i o n w i t h C, as shown i n T a b l e X . B a r r i n g s t e r i c effects, both I· and C a r e thus s t r o n g l y dependent on the i o n i z a t i o n p o t e n t i a l of the m e r c u r i a l ; that i s , e l e c t r o p h i l i c a t t a c k at a n a l k y l m e t a l bond i s r e s p o n s i v e to e l e c t r o n a v a i l a b i l i t y , n a m e l y the H O M O e n e r g y , i n m u c h the s a m e w a y that the i o n i z a t i o n p o t e n t i a l is. D e s c r i b e d i n an a l t e r n a t i v e way, the t r a n s i t i o n state f o r e l e c t r o p h i l i c c l e a v a g e i n eq 28 c a n be c o n s i d e r e d a s a p e r t u r b a t i o n of the o r g a n o m e t a l b y an e l e c t r o p h i l e , w h i c h i s a k i n to a virtual ionization. T h e s e c o m p a r i s o n s show that e l e c t r o n t r a n s f e r and e l e c t r o p h i l i c a t t a c k both depend h e a v i l y on the e l e c t r o n a v a i l a b i l i t y i n the organometal. It f o l l o w s that a n y c o r r e l a t i o n of the r e a c t i v i t y ( i . e . , r a t e c o n s t a n t s ) w i t h the i o n i z a t i o n or o x i d a t i o n p o t e n t i a l s i s not s u f f i c i e n t to d i f f e r e n t i a t ence between the two i s l a r g e l y due to the v a r i a t i o n s i n the s t e r i c i n t e r a c t i o n s , w h i c h can be l a r g e i n a n i n n e r - s p h e r e , e l e c t r o p h i l i c p r o c e s s ( i . e . , f o r C but not I.) and l e s s i m p o r t a n t i n an o u t e r sphere, electron transfer process. T h i s c o n c l u s i o n s u p p o r t s the g e n e r a l f o r m u l a t i o n that e l e c t r o n t r a n s f e r and e l e c t r o p h i l i c p r o c e s s e s c a n s h a r e a c o m m o n t h e m e of c h a r g e t r a n s f e r i n t e r a c t i o n s . S u c h a c o n c l u s i o n a l s o b e a r s on the m u l t i p l i c i t y of a v a i l a b l e m e c h a n i s m s f o r the c l e a v a g e of o r g a n o m e t a l s . T h e ready a c c e s s i b i l i t y of s u c h c o n c e r t e d and s t e p w i s e p r o c e s s e s f o l l o w s n a t u r a l l y f r o m t h e i r b a s i c s i m i l a r i t y , and i t c a n m a k e the t a s k of differentiation in individual cases difficult. T h e s e s t u d i e s a l s o shed l i g h t on the e f f e c t s of p o l y a l k y l a tion of m e t a l s on t h e i r r e a c t i v i t y to e l e c t r o p h i l i c a n d e l e c t r o n transfer cleavages. T h u s the r a t e s of c l e a v a g e of a s i n g l e a l k y l l i g a n d f r o m G r o u p I V B o r g a n o m e t a l s a l w a y s d e c r e a s e i n the orderR M > R M C 1 > R M C 1 , and f o r the m e r c u r i a l s , R Hg » R H g C l , i n d e p e n d e n t of whether an e l e c t r o p h i l i c o r e l e c tron transfer process pertains. T h i s reactivity sequence natur a l l y f o l l o w s f r o m the a v a i l a b i l i t y of σ - b o n d i n g e l e c t r o n s i n the H O M O as l i s t e d i n T a b l e IV f o r the m e r c u r i a l s . s

4

3

2

2

2

V.

Homolytic Displacements in A l k y l Transfers

O r g a n o c o b a l t ( l I I ) c o m p l e x e s a r e r e a d i l y c l e a v e d by c h r o m o n s i o n i n aqueous p e r c h l o r i c a c i d s o l u t i o n (37). RCo^(DMG)

2

+ Cr + aq 2

• > ZH'

R-Cr*+ + 4 a

Co^DMG^

where D M G = dimethylglyoximate T h e a l k y l g r o u p i s t r a n s f e r r e d to c h r o m i u m ( l l ) e s s e n t i a l l y q u a n t i t a t i v e l y . T h e c l e a v a g e f o r m a l l y r e p r e s e n t s a t r a n s f e r of a n a l k y l r a d i c a l R« to C r + , that i s an o v e r a l l r e d u c t i v e c l e a v a g e of an a l k y l - c o b a l t (III) b o n d . T h e r a t e of t r a n s m e t a l l a t i o n f o l l o w s 2


230


second-order kinetics, -d[Cr]/dt

+

=

+

k [ R C o ] [ C r ] + k«[RCoH ] [ C r ]

[30]

1

w h e r e k and k r e l a t e to the c l e a v a g e of the n e u t r a l and protonated alkylcobalt(lll) species, respectively. T h e v a r i a t i o n i n k and k w i t h the a l k y l g r o u p s f o l l o w s t h e s a m e o r d e r , a n d d e c r e a s e s i n the o r d e r ; 1

R 1

1

k'fM" s e c " )

=

Me

=

1

10 ·

>

Et >

4

n-Pr >

1

2

ΙΟ" ·*

ΙΟ" ·*

i - P r> i-Bu ΙΟ"

4

4

10" ·

2

A l k y l c o b a l a m i n s a r e a l s o c l e a v e d b y c h r o m o u s i o n (38), +

+

RCo(corrin) + C r

RCr

+ B

[31]

with simple second-orde and i n d e p e n d e n t of p H b e t w e e n 0-2.3. T h e c l e a v a g e s of the m e t h y l [k = 3 . 6 x l 0 M s e c " ] and e t h y l [k = 4.4 M** s e c " ] d e r i v atives proceed with different activation parameters: Δ Η * - 3.8 (Me), 11 (Et) k c a l m o l " ; Δ S* = -34(Me), - 1 8 ( E t ) eu. A l k y l t r a n s f e r s f r o m c o b a l t ( l l l ) t o c h r o m i u m ( l l ) as de s c r i b e d a b o v e a r e a n a l o g o u s t o the r e v e r s i b l e e x c h a n g e b e t w e e n a l k y l c o b a l t ( H I ) and c o b a l t ( l l ) (32,40,41), 2

- 1

1

1

1

1

m

E

R C o ( l ) + Co (2)

C o ( l ) + RCo (2)

=F=*=

[32]

M

w h e r e C o ( l ) and Co(2) r e f e r t o c o b a l t c o m p l e x e s w i t h s l i g h t l y d i f f e r e n t c h e l a t i n g l i g a n d s s u c h a s d i m e t h y l g l y o x i m a t o and c y c l o h e x a n e d i o n e d i o x i m a t o . T h e o v e r a l l p r o c e s s i n eq 32, w h i c h i s e q u i v a l e n t to e l e c t r o n t r a n s f e r , a c t u a l l y o c c u r s b y t r a n s f e r of a n a l k y l g r o u p a s a r a d i c a l a s shown b y l a b e l l i n g t h e c o b a l t a t o m s w i t h d i f f e r e n t c h e l a t i n g l i g a n d s . T h e r a t e of e x c h a n g e f o l l o w s s e c o n d - o r d e r k i n e t i c s , f i r s t - o r d e r i n cobalt(ll) and f i r s t - o r d e r i n a l k y l c o b a l t ( l l l ) . T h e second-order rate constants d e c r e a s e i n the r e l a t i v e o r d e r [ k ( E t ) = 1.1 χ 10" M" s e c " ] : 1

R k

r e l

= =

Me (2>10 ·6) 2

>

Et >

1

1

n-Pr ~ 1

(10°)

2

η-Bu 1

(ΙΟ" · )

4

(ΙΟ" · )

>

i - P r> i-Bu 2

5

(10- · )

(10-3.3)

T h e t r a n s f e r of t h e e r y t h r o - P h C H D C H D - g r o u p o c c u r s w i t h i n v e r s i o n (42). C o u p l e d w i t h the r e a c t i v i t y t r e n d of a l k y l g r o u p s , the c l e a v a g e i s b e s t c o n s i d e r e d a s a h o m o l y t i c d i s p l a c e m e n t on the c a r b o n c e n t e r (43). T h e t r a n s i t i o n state,

τη

E

V

'

η

m

i *

[Co—C—Co J i s s i m i l a r to that i n e l e c t r o p h i l i c c l e a v a g e s o c c u r r i n g w i t h i n v e r s i o n , e x c e p t t h e p r o c e s s i n v o l v e s a o n e - e q u i v a l e n t r a t h e r than a t w o - e q u i v a l e n t change. H o w e v e r , t h e l a t t e r does not a p p e a r to be


13.

Alkyl Transfers

KOCHi

231

a d e c i s i v e factor, since a l k y l t r a n s f e r f r o m alkylcobalt(lll) to cobalt(l), i.e., cJfl) + RCo (2)

m

m

R C o ( l ) + Cc?(2)

[33]

o c c u r s w i t h r a t e s and s t e r e o c h e m i s t r y m u c h l i k e that of i t s c o b a l t ( l l ) c o u n t e r p a r t i n eq 32. In p a r t i c u l a r , i n v e r s i o n of c o n f i g u r a t i o n at c a r b o n o c c u r s d u r i n g a l k y l exchange, a n d the s e c o n d - o r d e r r a t e c o n s t a n t s d e c r e a s e i n the r e l a t i v e o r d e r [ k ( E t ) = 1CT M" s e c " ] : 1

R

k

r e l

1

1

=

=

Me 2

> E t > n - P r - η-Bu > i - B u 2

fclO - )

(10°) (ίο- · )

(ίο- · )

1 4

3

(l0- -9)

1 7

A t t e m p t s to m e a s u r e the a l k y l exchange of a l k y l c o b a l t ( i l l ) with cobalt (ill) were unfortunatel and thus s u s c e p t i b l e t l o w r e a c t i v i t y of c o b a l t ( l l l ) i s p r o b a b l y due to i t s s u b s t i t u t i o n s t a b i l i t y , w h i c h l i m i t s the a v a i l a b i l i t y of the a c t i v e 5 - c o o r d i n a t e e l e c t r o p h i l i c s p e c i e s . N o n e t h e l e s s , the t r a n s i t i o n s t a t e s f o r a l k y l e x c h a n g e s i n a l l t h r e e s y s t e m s a r e l i k e l y to b e s i m i l a r , e f f e c t i v e l y i n v o l v i n g a l i n e a r 3-atom c o n f i g u r a t i o n (see above). T h e s i m i l a r i t y i n the r a t e s of the c o b a l t ( l l ) a n d c o b a l t ( l ) r e a c t i o n s s u g g e s t s that the e x t r a 1 a n d 2 e l e c t r o n s , r e s p e c t i v e l y , a r e i n a nonbonding o r b i t a l c e n t e r e d on both c o b a l t a t o m s . T h e c l e a v a g e s of a l k y l - m e t a l bonds b y e a c h of the t h r e e c o b a l t c o m p l e x e s w i t h o x i d a t i o n s t a t e s I, II a n d III a r e r e p r e s e n t a t i v e of what i s c o m m o n l y c o n s i d e r e d t o b e e l e c t r o p h i l i c , h o m o l y t i c a n d n u c l e o p h i l i c p r o c e s s e s , r e s p e c t i v e l y , e.g., C

, °>

R-Co* + M °

LÇsL+. R - C o + M 1

R-M*

1

WïL R-Cc? + M Y e t t h e r e a d y i n t e r c o n v e r s i o n of e a c h c o b a l t s p e c i e s b y onee q u i v a l e n t changes, 1

Co

^ *

Co

^

Co

r a i s e s the i s s u e of w h e t h e r e l e c t r o n t r a n s f e r p r o c e s s e s a r e i n v o l v e d i n a l k y l t r a n s f e r a s d i s c u s s e d i n the p r e v i o u s s e c t i o n s . S u c h p r o c e s s e s a r e e s p e c i a l l y r e l e v a n t i n v i e w of the e a s e w i t h w h i c h the a l k y l c o b a l t c o m p l e x e s t h e m s e l v e s u n d e r g o o x i d a t i o n r e d u c t i o n (44). RCo

111

=

^

RCo^

F o r e x a m p l e , t h e c l e a v a g e i n eq 32 m a y i n v o l v e a t w o - s t e p p r o cess, i n which cobalt(ll) acts as a nucleophile leading to the i n i t i a l r e d u c t i o n of a l k y l c o b a l t ( l l l ) , f o l l o w e d b y e l e c t r o n t r a n s f e r .


232

ORGANOMETALS

R C o ( l ) + Cc?(2) m

Ccf(l)

+ RdF(2)

^P*=


Co(l) + R C o ^ ) C

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