Organometals and Organometalloids - American Chemical Society

5

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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 of lead are less stable than the corresponding compounds of the lighter group IVb elements, 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 Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

66

ORGANOMETALS AND ORGANOMETALLOIDS

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~ Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 6, 2018 | https://pubs.acs.org Publication Date: January 12, 1979 | doi: 10.1021/bk-1978-0082.ch005

/ 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

2 M e

2

P b X

L/X , X/L I ^X/L^| solvent _ Me-Pb Pb-Me 2 molecules t ^ X / ^ X

e

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.

Brinckman and Bellama; Organometals and Organometalloids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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 measurements we used a mixed b a c t e r i a p o p u l a t i o n . 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 . At h i g h l e a d c o n c e n t r a t i o n s no e x p o n e n t i a l a c c e l e r a t i o n i s observed; c e l l d e n s i t y r i s e s o n l y 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

0.8 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 95

0.2 0.4

Et : P b C l

3

lag 6 6.5 10 6.5 13 12 25 30

2

log 1.3 2.4 0.5 2.4 0.36 1.1 0.25 0.42 0.03 0.06

2

lag

log

6 6.5 18 8 30 15 50 25

1.3 2.4 1.4 0.84 0.23 0.64 0.14 0.37 0.05 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 compounds i n the s o l u t i o n was not too h i g h (otherwise ELjS produced by the anaerobes p r e c i p i t a t e s PbS, l e a v i n g only 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 . Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 6, 2018 | https://pubs.acs.org Publication Date: January 12, 1979 | doi: 10.1021/bk-1978-0082.ch005

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 * were analyzed (13), which a c c o r d i n g t o [2] correspond t o 0.3 ymol Me^Pb produced c h e m i c a l l y ; we found 2.1 ymol P b , which a c c o r d i n g t o [3] correspond t o an a d d i t i o n a l 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 +

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 , i s biomethylated t o g i v e Me,Pb. Considering the d i f f e r e n t modes o f production (Et^Pb c h e m i c a l l y , 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

MePbXo

2 Me^Pb

MeX

•

!

PbX-

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 that T l i s biomethylated to M e T l under i n crease of the o x i d a t i o n number shows that " o x i d a t i v e m e t h y l a t i o n " i s not unique w i t h P b . I n t h i s context one has t o r e c a l l t h a t 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

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 . Monomethylthallium compounds a r e 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 + GT^OH 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 s of the products of d i s p r o p o r t i o n a t i o n according to equation [ 9 ] : 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"

ORGANOMETALS A N D ORGANOMETALLOIDS

+ 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 , L . J . , Wood, J.M., Science (1977) 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^ . We have p o s i t i v e r e s u l t s w i t h S n and negative r e s u l t s w i t h Pb . +

+

2 +

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.


Organometals and Organometalloids - American Chemical Society

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