John 5. Thayer University of Cincinnati Cincinnati, Ohio 45221
Some Biological Aspects of Organometdi~Chemistry
Organometallic compounds have been known for many years to be intimately connected with chemical processes in living organisms. Living organisms consist predominantly of water; to survive they must maintain a delicate, incredibly complex equilibrium among hundreds of simultaneous reactions. Introduction of a water-reactive organometal, such as methyllithium, into such a system would cause havoc and quite probably death. As a consequence, only those organometals which are stable (or at least unreactive) toward water have major biochemical importance. Research into the biological effects of organometals may be divided into two major categories with relatively little overlap. These categories are based on the origin of the compound being investigated. First are those species prepared by standard laboratory techniques and introduced into the living organism; these will be termed in vitro organometals. The second category includes those organometals actually generated within an organism by biochemical processes; these will be termed in wivo organometals. The two areas have evolved separately, and only very recently has their interrelation become apparent. In Vitw Organometals
With a very few exceptions, organometallic compounds are toxic toward living organisms. This toxicity varies enormously, and has numerous important applications. Early workers suffered, often severely, from their contact with dangerous organometals ( 1 3 ) . Bunsen during his classic researches on cacodyl "had to breathe through long glass tubes and a t one stage of his investigation an explosion occurred [November 9, 18361which caused a permanent injury to one eye (do)." Later he nearly died from careless handling of the deadly cacodyl cyanide, (CH&AsCN (db). Frankland (3) noted: "the vapor of this compound [dimetbylzinc] is highly poisonous, producing after its incautious inhalation all the symptoms of poisoning by zinc. It decomposes water with as much violence as potassium." Similar reports appeared throughout organometal literature of the nineteenth century. The idea that such biocidal effectsmight have practical usage arose from the researches of Paul Ehrlich (1853-1915) on organoarsenical chemotherapy (4). His work was based on the following statement of principle C o r p o ~ amn agunt nigi jEzata (bodies do not act unless fixed).
Thii means that parasites are killed only by those substances far
...
I call such substances which they have a certain aEnity parmitropic (4a) All substances which are wed for the destruotion of parasites we also poisons-i.e., they have an affinity for vitally important organs and are thus, at the same time, also
...
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organotropie only those substances in which the orgsnotropic and parasitropic affinitiesstand in the right relation to one another can be used as remedies (4b).
Ehrlich and his associates started their researches with "atoxyl" (p-NH2C6HAsO3HNa) and studied a long list of organor~rsenicals. Three of these are shown in the figure; the numbers refer to their relative positions on Ehrlich's list. Salvarsan met the criteria much better than any of its predecessors. For many years it was used in treatment of diseases caused by spirochetes (e.g., sypb~hs),trypanosomes (e.g., sleeping sickness), and similar organisms. Neosalvarsan proved slightly better because of enhanced water solubility. Salvarsan was originally thought to be an arsenic analog of azobenzene; the earlier literature show this compound as a dimer with an As-As double bond. Recently arsenobenzene itself was found to be a cyclic bexamer (@, so it seems likely that salvarsan and neosalvarsan will be cyclic species also. Ehrlich, remarkably farsighted in his observations, noted that certain strains of organisms showed lower sensitivity than other strains, and warned about the possible development of completely resistant strains-a warning that has proved all too tragically pertinent in recent years. Salvarsan and its derivatives continued in use until they were displaced by the safer and more effectiveorganic antibiotics. Shortly after Ehrlich's work, organoarsenicals found another, much grimmer biological application: toxic gases in World War I. Most notorious among these was Lewisite @-chlorovinyldicbloroarsine). Lewisite was used primarily as an irritant (6) which attacked skin and respiratory system, making its victims more susceptible to other deadlier gases. Organomercurials comprise another group of compounds used for their biocidal effects. The antiseptics mercuricbrome and merthiolate, shown in the figure, are still widely usedin the treatment of external wounds. This past year there was a report on the effects of organomercury compounds on photosynthesis of plankton (7). Organomercurials have occasionally been used to control micro-organisms of various sorts, along with weeds, fungi, and other forms of plant life that men might consider undesirable. Organo compounds of Sn, Pb, and P have also been employed for this purpose. For example, tributyltin oxide is often coated onto hospital bed linen and mattresses to destroy infectious bacteria. Much of these biocidal aspects and applications have been reviewed recently (8). The toxicity of organo compounds of As, Sb, Hg, TI, Pb, etc., arise from the metal itself; i.e., organomercurials are poisonous becanse mercury is poisonous. I n the case of Si and Ge, the elements and most of their inorganic derivatives are nontoxic, but certain of their
organo derivatives show marked biocidal effects. The toxicity of Group IV organo species may be summarized in the following manner 1) Pb >> Sn > Ge > Si 2 ) Alkyls are more toxic than aryls
3) Trialkylmetal compound show greatest effects 4 ) Toxicity varies with chain length of dkyl, reaching a maximum at about n-butyl (though this varies from orgitnism to organism), then decressing; branched alkyls are less effective than straight-chain alkyh of the sitme number of carbons 5) Inorganic ligsnds present h w e no effect, unless they themselves are toxic (i.e., oyanide)
At present, the exact reason for these effects is unknown, but it is believed that trialklgermanium and trialkltin compounds interfere with oxidative phosphorylation. Organosilicon compounds, unless they have a toxic ligand that can by hydrolyzed off, are generally harmless. In fact, the silicones have had a number of therapeutic applications that make use of their complete inertness to bodily processes. One class of organosilanes, however, shows considerable toxicity: the silatranes, (8, 9) organosilicon esters of triethanolamine RSi[(OCH2CH&N]. Even here, the magnitude of the effect depends on R. The phenyl derivative is 5000 times more deadly than the methoxy derivative and is twice as toxic as the alkaloid strychnine. Fessenden, et al. (lo), have performed some very interesting work on the effect of silicon substitution into biologically active molecules. While ordinarily this substitution has virtually no effect, occasionally there does appear a great variation. One such is shown in the table. Such variations are potentially extremely Relative Effect of Silicon Substitution
Compound
Nature of effect
mso
(mice).
useful for providing insight into the specific mechanism of reaction that makes the drug important. Unfortunately such potential is not easy to realize because the compounds are quite difficultto synthesize. In Vivo Organomelals
During the nineteenth century there appeared numerous reports of malodorous gases containing arsenic being produced from moldy wallpaper. The arsenic came from pigmentation (11). As early as 1839, workers recognized that the molds played the vital role in producing these gases; Selmi in 1874 suggested that the gas was ASH$(12). Gosio reported in 1891 that molds grown in a potato-arsenic oxide culture medium produced volatile Akcoutaining compounds (13). One of these molds was Scopulariopsis breuicaulis (formerly Penicillium brevicaule), related to the molds from which penicillin is obtained. Bigiuelli found that the gas formed a complex with mercuric chloride; he assigned its formula as (CzHs)zAsH.2HgClr (11). Around the same period appeared reports that ingestion of K2Te03or bismuth carbonate (containing traces of
tellurium) by animals or humans caused exhalation of a similarly malodorous gas. This was tentatively identified in 1894 as (CH&Te (11). The definitive work came in a long series of papers by Challenger and his associates, beginning in 1931 (11, 13). They found that various molds and other organisms would convert inorganic or organo compounds of arsenic, selenium, tellurium and (in a few specific cases) sulfur to the methyl derivatives. The gases were analyzed by passing them into a dilute HC1/HgC12 solution and analyzing quantitatively the solid precipitate. Arsenic oxide and other inorganic arsenic compounds gave trimethylarsine exclusively, as did sodium methylarsonate, CHaAs03Na, and sodium cacodylate, (CH&AsO2Na. Other alkylarsonates gave the mixed methylalkylarsines.
Similar reactions were observed for selenium and tellurium. Since methyl groups were invariably and exclusively introduced regardless of what had been originally present, Challenger termed this process "biological methylation." Research then began to focus on its chemical source and mechanism, Various precursors, such as formaldehyde or acetic acid, were suggested. Challenger solved this problem by using 1%-labeling. He defined a "methylation percentage" % Methylstion
=
100 ['Product] nf ['Source]
where n = number of methyl groups per molecule of product, f = fraction of labeled methyl groups actually labile, [*I = molar radioactivity of methylated product or methyl source. Challenger tested sodium formate, choline chloride [(CH3)3NCH2CH20H]+CI-,betaine (CH~)&CHGO~-, and methionine CHaSCHzCHr (NH2)C02H. In the first three compounds, the methylation percentage was 1-5% and much of the 14C appeared as "C02. For methionine, however, methylation percentages ranged 25 to 95, and much less "CO2 was produced. From this Challenger concluded that the methyl groups in biological methylation originated from methionine, and suggested that methionine itself came from homocysteine, HSCH2CH,CH(NH,)COIH. Determination of the actual mechanism came from another area of research. Investigations into the biochemistry of Vitamin B12("cohalamin") showed that one coenzyme form of this species actually had a Co-C sigma bond (14-16) !!! Because of the complicated structure of cobalamin itself, a number of "model" compounds, related in their structure immediately around cobalt but simpler to prepare and characterize, have been studied. Most important among these are the derivatives of bis(dimethylglyoximato)cobalt(III), the cobaloximes. The figure shows the basic structure. The two trans-axial positions, A and B, are occupied by various groups. There is a marked resemblance in the chemistry of cobaloximes to cobalamin. Most significantly, it has been shown that an alkyl group bonded to the cobalt atom shows considerably greater chemical stability than sigma-bonded organocobalt compounds formed in the laboratory (16). Here a living organism Volume 48, Number 7 2, December 1971
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(-ks-), (--As-), Arsenophenylglycine Salvarsan
(418)
(--As-), Neosalvarsan
(606)
(914)
NaO Br@O
s\HscH2cH, Merthiolate
Therefore, labile bonds, so often a bane of the laboratory chemist, become a major asset in biological processes! Very recently biological methylation has become important in a different and unexpected context: mercury pollution! Mercury metal, though poisonous, is very unreactive. In consequence, excess metal has often been disposed of by simple dumping. Likewise, compounds of mercni-y are also occasionally included among waste effluents. Since mercury is toxic, aquatic organisms (bacteria, sheash, fishes, etc.) protect themselves by converting mercury or its inorganic compounds to gaseous (CH.&Hg. In higher organisms, especially fishes, the water-soluble CHaHg+ also forms (18). This collects in the tissues. Mercury concentrations thus build up far above the level of the surrounding water; eventually the organisms die. If land animals (including humans) eat such organisms, they themselves will ingest the mercury and be poisoned (7, 18). Mercury can also be methylated by a nonenzymatic pathway (19). Recently it has been reported that 2-aminoethylphosphonic acid, NH2CH2CH,PO(OH),, and related compounds with P--C bonds were formed within living organisms, presumably by biological processes (90). Conclusions
Some important medicinal orgmometols.
utilizes a chemical principle only recently learned by chemists: the reactivity of a given metal-ligand bond in a coordination compound can vary considerably depending on the nature of other ligands present. Much of the chemistry of cobaloximes (and by extension, of cobalamin in living organisms) arises from ready interconversion among diierent oxidation states in cobalt. Cobalamin has been shown to react with homocysteine (17) (Go)
+ +
CHsSCHnCH,CH(NH2)C031 H f (Co)- (2) I n this form, the reaction goes irreversibly. However, methionine can be demethylated by reaction of the reduced form of cobalamin, (Co)-, and S-adenosylmethionine. This would indicate that methylcobalamin serves as the actual methylating agent in biological methylation, a t least for some organisms (there is evidence that this may occasionally proceed by alternative pathways) (17). An important but subtle point: the importance of methylcobalamin comes from the fact that the Co-C bond is stable enough for existence in the body but labile enough for easy reaction. Another organometal that forms readily in living organisms is carboxyhemoglobin, in which a molecule of carbon monoxide bonds to a coordination site on the iron in hemoglobin. This Re-C linkage is so strong, so stable, that it precludes the formation of oxyhemoglobin, the necessary intermediate for oxygen transport throughout the body. Consequently, oxygen starvation and death result.
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Thus it can be seen that organometals, whether formed within or without a living organism, play an important role in biochemical processes. I n most cases this arises from the toxicity of the species or the lability of the metal-carbon bond. The unexpected role of cobalamin leads to speculation about the possible existence of other organometals that serve as active intermediates. There has been no evidence that other organic groups are transferred in the same way as methyls, but then research in this area is still quite recent. It seems quite possible, even probable, that further work will reveal new applications of organometallic compounds to the vast area of biological chemistry. Literature Cited (1) T m r m . J. S.. J. CHEU. EDUC.. 43, 594 (1966). (2) (a) Vna K~oowsn.H . 6.. J. CHEW. ED"... 28.359 (1951); (b) LoOKEY A W N . G . , AND OBBPBR,R. E,,J. CHEW.EDOC., 32, 456 (1955). J. S., J. C ~ E XEDUO.. . 46, 764 (1968). (3) TXATER, (4) (a) H r m e b w m ~ F. , (Editm). "The Collected Papers of PaulEhrlich, Psrgarnon Preas, London. 1960, 111. p. 505; (b) ibid., p. 282. (5) Hennsaa, K.. H u o m s . E. W.. A N D WABER,J.. A d a CIY~~.. 14. 369 ,1961>~ ~.---,. (6) J*caaolr. K.E., awn JACRSON, M. A,, C h m . Rev.. 16,439 (1935). R. C..WHITE, D. B..AND MACFAELANE, R. 11.. Science, 170, (7) H*BRISB. 737 (1970). , M.,A m M ~ o o sI., , Orgonomat. Chern. Rw., 16, 438 (1936), (8) I h a N ~ s J. (9) VORONBOY. M. G..J . PUI. AppI Chcm., 19, 399 (1969). (10) FEBBENDBN,R.J.. i i * ~COON,M. D., J . Madicind Chsn.,8,604 (1965). (n)CEUb~ewols.F.. Chem. RBY.,36, 315 (1945). (12) Smrr. F.,Chem. Be..7, 1642 (1874). (13) CH*fimwaa, F.. Quorf.Rm.. 9, 255 (1955). F., Ann. RBY.Biochem.. 35. 405 (1966). (14) WAONGR. D. (15) C n o w r o o ~ H o o o m ~ ,.Proc. Roy. Soc. London. A288, 294 (1965). n , N.. Accfs. Chem. ~escorch.1, 97 (1967). (16) S c n n ~ u ~ eG. (17) GDEST.J. R., et ol.. Nofur., 195, 340 (1962). A,, Nafwe, 223,753 (1969). (18) JENSBN. 8.. AND JBRNE~~OY. (19) Wooo. J. M.,KBNNEDY.F. S., A N D Roam, C. S., NCUUIB, 220. 173 ,I(IRP,
