Organometals and Organometalloids - ACS Publications - American

1 Biosynthesis of Organometallic and Organometalloidal Compounds FREDERICK C H A L L E N G E R

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19 Elm Avenue, Beeston, Nottingham, NG9 1BU, U. K.

I f e e l g r e a t l y honoured to have been asked to send to the Symposium an i n t r o d u c t o r y communication which s h a l l form a keynote 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 by the fundamental work of Vincent du Vigneaud (5). There we have remained ever s i n c e . I r e a l i z e that my metaphors are very mixed here, but the personal excitement of 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

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

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

CH :CHCH As(CH ) 2

2

C H

R

*>sO(OH)

>

3

2

2

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

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) , t o c u l t u r e s of two s t r a i n s of j>. b r e v i c a u l i s gave no dimethylsulphide. These negative r e s u l t s are i n t e r e s t i n g , 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


ORGANOMETALLOIDS

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 s methyl d e r i v a t i v e s such as c h o l i n e and c r e a t i n e are produced." No p a r t i c u l a r compound was suggested as 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 , and Wood (25) and 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 by whole c e l l s of a Methanobacterium ( s t r a i n MoH) i n present of methylcobalamin (methyl-B-^) as a methyldonor. 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 n which Sb i s an a n i o n , m e t h y l a t i o n s might be expected but none was observed. A s p i r a t i o n of v o l a t i l e 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 almost every aspect of selenium metabolism has been admirably discussed by D i p l o c k (40) i n a review of 57 c l o s e l y p r i n t e d pages w i t h 430 r e f e r e n c e s . 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 HOOCCH(NH )CH SeCH 2

2

3

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 forms of Co-enzyme M ( I I ) were present together i n the r e a c t i o n medium. The onium compound (IV) would not methylate 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 product o f the h y d r o l y s i s o f t h e c e l l w a l l s o f a 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 n view of i t s impending use, an i n v e s t i g a 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 l A d v i s o r y S e r v i c e revealed a h i g h c o n c e n t r a t i o n of heavy metals, c h i e f l y z i n c , copper, and n i c k e l , 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 s and i n human t i s s u e s , where i t may i n c r e a s e w i t h age, causing h i g h blood pressure and c a r d i a c d i s ease. The cadmium content of normal s o i l s has been v a r i o u s l y 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 confined 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, increase w i t h depth, probably owing to a decrease i n the organic matter of the s o i l . The lead content of the s o i l has l i t t l e e f f e c t on i t s uptake 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 Foreback (70) have reported the occurrence of a r senate, a r s e n i t e , methylarsonate, and dimethylarsonate ( c a c o d y l 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 Wood and Professor Thayer f o r the encourage ment and help they have accorded me d u r i n g i t s p r e p a r a t i o n .

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., Unpublished o b s e r v a t i o n s . A x e l r o d , J., and D a l y , J., Science (1965) 150, 892-3. 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. (1975) 62, 357 (Note e s p e c i a l l y 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. 36, 315 (1945)] which i s a masterpiece, not o n l y i n terms of the chemistry, but a l s o i n terms of the h i s 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 volume f o r a d d i t i o n a l d i s c u s s i o n of charge on Sb and p o t e n t i a l f o r m e t h y l a t i o n - Eds.] 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.


Organometals and Organometalloids - ACS Publications - American

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