THE VITAMIN B12 COENZYME

D. DoLPHIN, A. W. JoHNSON, R. RoDRIGO and N. SHAW Department of Chemistry, University of Nottingham, U.K.

INTRODUCTION In 19•58 Barker and his associatesl-3 recognized a new coenzyme which controlled the conversion of glutamate into ß-methylaspartate by Clostridium tetanomorpkim. The coenzyme was shown4 to be related to !f;-vitamin B12, i.e. contair ing an adenine nucleotide grouping in place of the 5,6-dimethyl­ benziminawle nucleotide of vitamin B12, although similar coenzymes con­ taining btnziminazole or 5,6-dimethylbenziminazole were produced by growing C. tetanomorphum in the presence of the a ppropriate base5. Other variations of the nucleotide base have been achieved using Propionibacterium arabinosum in the presence of other purines and benziminazoles6• The pres­ ence of;:he coenzymes in a wide variety of micro-organisms such as several species of Actinomycetes including Streptomyces olivaceus and S. griseus has been dem( mstrated by the glutamate isomerase assay7 or by isolation. I t appears thü Vitamin B12 and its analogues are always biosynthesized in the form of their coenzymes. Preliminary physical and chemical studies sug­ gested that in the 5,6-dimethylbenzirninazolyl cobamide coenzyme the cyanide gr )up of vitamin B12, cyanocobalamin, was replaced by an adenine nucleoside':, 5, 8 and the determination9 of the complete structure (I; R = 5'-de·)xyadenosyl) of the coenzyme by X-ray analysis revealed the existence c f an essentially covalent bond between the cobalt atom and the S'.. carbon üom of the additional 5'-deoxyadenosine group. The molecule

Me CH 2• CO· NH2

In the vitamin 8 12 coenzyme R =5' - deoxyadenosyl = Me Me

539 D. DOLPHIN, A. W. JOHNSO~, R. RODRIGO and N. SHAW is thus an organametallic compound and is the first naturally occurring compound containing a metal-carbon bond. I t is presumed that the adeninyl and benziminazolyl cobamide coenzymes have similar structures apart from the nature of the nucleotide base. In all of these compounds the cobalt-carbon bond is readily broken and several methods, including irradiation of aqueous solutions, exist for replacing the 5'-deoxyadenosyl group by hydroxo (i.e. forma_tion of vitamin B12b and the action of cyanide on the coenzymes in the absence of light gives cyano­ cobalamin (initially in its dicyano form). These reactions will be discussed below in greater detail. PARTIAL SYNTHESIS A partial synthesis of the coenzyme was achievedl0-13 by treating a re­ duced form ofvitamin B12b or B12 itselfwith 5'-tosyl-2', 3'-0-isopropylidene­ adenosine14 followed by hydrolytic removal of the isopropylidene grouping. Sodium borohydride, zinc and acetic acid, or chromaus acetate were suitable reducing agents and appeared to convert vitamin B12b to a grey-green reduction product, the so-called Vitamin B12s, which under acid or alkaline conditions was found to react with a variety of alkylating agents to give good yields of coenzyme analogues. The product from the reaction with 5'­ tosyl-2',3'-isopropylidene-adenosine, after hydrolytic removal of the iso­ propylidene group and purification was identical with the 5,6-dimethyl­ benziminazolyl cobamide coenzyme (henceforward referred to as the vitamin B12 coenzyme) bothin physical, chemical and biological properties (for example, the enzyme assay of Abeles and Lee15). Analogausexperiments using 2',3'-0-isopropylidene-5'-tosylinosine or 2',3'-0-isopropylidene-5'­ tosyluridine yielded crystalline products having physical and chemical pro­ perties similar to those of the coenzyme (I), but containing hypoxanthine and uracil residues respectively in place of adenine. In later experiments it was found that protection of the 2',3'-hydroxyl groups ofthe sugarwas not essential. A wide range of alkyl coenzyme analogues has been prepared from re­ duced hydroxocobalamin using alkyl halides, dimethyl sulphate and triethyl phosphate as alkylating agents. Reactions with ethylene bromohydrin, chloroacetic acid, and acetyl chloride or acetic anhydride also gave related products (II; R = CH2·CH20H, CH2·C02H or Ac respectively). It there­ fore seems that a wide variety of electrophilic reagents will react with the reduction product of hydroxocobalamin, steric conditions permitting, to give compounds of the B12 coenzyme type. Bromobenzene and t-butyl bromide did not react to an appreciable extent, probably for steric reasons. The crystalline alkyl-cobalt derivatives were stable at room temperaturein the absence of strong light. They are almost the first representatives of this class of compound to be made, although alkyl-cobalt intermediates probably have a transient existence in the Fischer-Tropsch synthesis16. An understanding of the reaction mechanism involved in the above alkylation reactions clearly requires a knowledge of the constitution of the reduction products ofvitamin B12b, hydroxocobalamin. It is agreed generally that the cobalamins are complexes of trivalent cobalt17-20, Intermediate between the cobalamins and Vitamin B128 is the vitamin B12r21-23 which has 540 THE VITAMIN B12 COENZYME been varioHsly stated to contain divalent22-24 and rnonovalent25 cobalt. We regard vitamin B12r as a derivative of divalent cobalt on the basis of the quantitative~ hydrogenation ofhydroxocobalamin in the presence ofplatinum when O·S mol ofhydrogen is absorbed during the formation ofvitamin B12r:

Coiii + iH2 ~ Coii + H+. Vitamin B12s is believed to be a cobalt hydride 11, 13, 25, 26 and as such it can be ad

Treatment ,)fvitamin B12s with cyanogen bromide yields27 cyanocobalamin, vitamin Bt2· The acidity of the hydride is emphasized by its reaction with diazomethane to give the methyl analoguell, 23 and these reactions of vitamin B12~ suggest that the product can best be formulated as an equili­ bri.Im: H- H+

Coiii L_ __ --"7 Coi.

Moreover, Ne have found28 that quantitative oxidation of vitamin B12s to B12b requin s 0·5 mol of oxygen: OH2

H+ H 20 t Co1 + !02 --~ Co1II + OH-

BIOSYNTHESIS The cobalt-hydride intermediate, vitamin B12s, does not appear to be involved in the biological synthesis of the vitamin B12 coenzymes. Cell-free extracts of Propionibacterium shermanii or Clostridium tetanomorphum, for example, required to be supplemented with adenosine triphosphate, a sulphydryl com.pound, such as glutathione or ß-mercaptoethanol, reduced flavin and manganese ions. By the use of isotope-labelled adenosirre triphosphate (ATP) it w 1s shown29, ao that both the adenine and sugar fragments of the 5'-deoxyadt:nosyl group ofthe coenzyme were derived from ATP. Although the precise mechanism of this conversion was not established it is clear that a reduct]on step is involved andin this senseit parallels the purely chemical synthesis al though the cobalt-hydride intermediate is probably not con­ cerned in tl Le biosynthesis. ln an eJ<.amination of the reaction of hydroxocobalamin with various thiols we h we recently shown31 that violet coloured products are initially fon:ned and that these may be transformed to brown compounds. The 541 2Q D. DOLPHIN, A. W. JOHNSON, R. RODRIGG and N. SHAW spectra of solutions of the brown products closely resembles that of vitamin B12r22, 23 and it is believed that they contain cobalt-sulphur bonds and are formed from the violet compounds by reduction. The reaction of hydroxo­ cobalamin with sodium hydrogen sulphide was examined in detail and the brown product, unlike the violet intermediate, was found to react very rapidly with methyl iodide to yield methylcobalamin in the absence oflight. When the solution was photolysed the methylcobalamin was converted back to hydroxocobalamin and in the presence of excess sulphide and methyl iodide the cycle could be repeated many times.

2 ~H RSH -~R Mel [le ] ~e Coiii ----+ Coll ----+ Coll ----+ Coiii

~. __h_v ______,/ The displacement of the sulphur Iigand by an alkyl group is more sus­ ceptible to steric hindrance than the corresponding hydride displacement although many examples have been provided. With ethyl iodide for example the reaction is markedly slower than with methyl iodide and the formation of the brown intermediate (spectrum) can be clearly observed. These reactions appear to be analogous to the biological syntheses of the co­ enzymes although the exact parallel of the biosynthesis, i.e. the use of ATP as alkylating agent, does not appear to operate under in vitro conditions.

PROPERTIES AND REACTIONS OF THE COENZYMES AND THEIR ALKYL ANALOGDES Although the results ofmagnetic measurements on the coenzyme reported from different schools are at variance32, the observed chemical reactions of the coenzyme are best interpreted by regarding them as complexes of trivalent cobalt. Quantitative hydrogenation of the methyl analogue in the presence ofplatinum resulted in the formation ofvitamin B12r and an increase in volume of 0·5 mol caused by the Iiberation of methane28, The methaue was identified by gas-liquid chromatography. CHa OH2 I H20 t Co + !H2 ---+ Co + CH4 The coenzyme itself cannot be hydrogenated in a similar manner probably because the increased size of the nucleoside substituent renders the cobalt atom more inaccessible to the reducing agent.

PHOTOLYSIS OF THE COENZYMESAND THEIR ALKYL ANALOGDES In the absence of oxygen (I0-6 mm) the alkyl coenzyme analogues are stable in light of moderate intensity; in the presence of small quantities öf air however (cu. I0-4 mm), photolysis occurs giving vitamin B12r and a mixture of olefins and paraffins. Finally, in presence of excess oxygen, 542 THE VITAMIN B12 COENZYME vitamin B12r· is oxidized to vitamin B12b and the radical undergoes some oxidation to the corresponding aldehyde28. The photolytic fission is regarded as a. hon10lytic process although evidence other than the nature of the products is ~ tilllacking. Et

I hv;02 Coiii ----- Coii + [Ee] ----31- C2H 4, (but no appreciable amounts of ""' C4H10 or C2H6) "- hv; . "----> Coiii + [Ee] -----3>- CHa·CHO Excess 0~, In rhe case of the coenzyme itself the reactions are analogous except that photolysis occurs even at I0-6 mm when the intermediate free radical under­ goes cycliza tion to the so-called nucleoside33 A (II), while in presence of oxygen the free radical is oxidized to the corresponding acid32 (111) or aldehydea4 ( Figure 1).

+

( Il )

Figure 1 When the :1Jhotolytical decompositions are carried out in presence of a radical acc( ptor such as a thiol the radical is captured before the trans­ forrnations described above can occur35 and similar enzymic controlled reactions have also been reported36. Thus photolysis of the methyl co­ enzyme a.nalogue in presence of either cysteine or homocysteine yields S-tnethykysteine or methionine respectively. A similar reaction of the vitamin B12 coenzyme in presence of homocysteine gave S-adenosylhomo­ cysteine (IV), a reaction of significance in the biogenesis of active methionine. It was not f.mnd possible to transfer the adenosyl group to methionine itself (Figure 2:. 543 D. DOLPHIN, A. W. JOHNSON, R. RODRIGO and N. SHAW

HO OH I I c--c jH \ CH 2·CH /C~ I ~/ N + Colll 0 <\ü (I) NH2

OH ~ + con

Figure 2

REACTION WITH ACIDS A striking difference between cyano- and hydroxo-cobalamins and the coenzymes or their alkyl analogues is the relative ease of protonation of the latter compounds. This reaction is characterized by a red to yellow colour shift and a consequent major change in the absorption spectrum25, 37. Although dilute mineralacidswill suffice to convert the coenzyme series to the corresponding conjugate acids, the cobalamins normally require con­ centrated sulphuric or perchloric acidstobring about this change. Analysis of the spectra of the yellow protonated forms shows that the bond between the nucleotide and the metal has been broken and that a proton is located on the nucleotide base. However, this displacement of the nucleotide base is in itself not sufficient to account for the marked colour change, and the observed red to yellow colour change at pK 3·3 probably involves an addition of a proton to the chromophoric system. According to Williams

H ·H

/ ( l~Me ',, N~Me ...... ___ ,

Figure 3 544 THE VITAMIN B12 COENZYME and his co-v\orkers25, the meso position, C1o in the chromophore is a likely site for this '.ddition to occur (Figure 3). The highc~r electron density at C1o in the coenzymes and their alkyl analogues a~ compared with the cobalamins correlates with the observed course of re1ction with chlorine (see below). An estimation of the pKa values of a sc~ries of alkyl coenzyme analogues has shown a relation between the obser:ec values and the inductive effect of the alkyl group ( Table 1). Thi~; suggestf that thereactivityat C1ois tosome extent controlled by thenature ofthe substi1uent R (V; R = alkyl, CN, H 20 etc.) which recalls the pheno­ menon of perpendicular conjugation38,

Table 1

Nature rif cobalt-alkyl pKa group (R in V)

CH3- 2·7 n-C.1H9- 3·93 n-C7Hl5- 4·01 Coenzyme 3·52 2',3'-Isopropylidene-coenzyme 2·94 H02 GCH2- 1·05

Free ad.enme is released from the 5'-deoxyadenosyl group ofthe coenzymes by the action of 0·1 N hydrochloric acid at 100 o for 90 minutes5, 8, 32. In the same reaction vitamin B12b is formed and the sugar is liberated as n-erythro- 2,3-dihydrmypent-4-enal (VI), formed by an elimination reaction32 (Figure 4):

OH 2 ~ + CH2 :CH·CHOH·CHOH·CHO Co (VI)

Figure 4 The struc ture of (VI) was determined by Rogenkamp and Barker39 and confirmed32 by a synthesis of erythro-pent-4-en-1,2,3-triol, the racemic form of the prodt ct of sodium borohydride reduction of the sugar: 0

/-~ C 2H 2 H·C03 H CH2·CH·Cl[2Cl----+ HC: C·CH:CH·CH20H ------+ H2 HC: C·(CHOH)2·CH20H ----+ CH2 :CH·(CHOH)2·CH20H OH- (as monoformate) 545 D. DOLPHIN, A. W. JOHNSON, R. RODRIGO and N. SHAW REACTION WITH CYANIDE Vitamin B12b, hydroxocobalamin, as weil as its coenzyme, react readily with cyanide when the nucleotide base is detached from the cobalt in an elimination reaction similar tothat brought about by hot dilute acid (above). The products formed from the coenzyme with cyanideS, 32, 39 are dicyano­ cobalamin40 (VII), the cyanhydrin of (VI) and free adenine (Figure 5).

CN I ..,Co /I / CN \,~~e 'N~Me',,, (VII)

Figure 5

On the other hand, the simple alkyl coenzyme analogues do not react with aqueous potassium cyanide when light is excluded. A slow displacement reaction occurs with hydrogen cyanide and it therefore seems that an initial protonation of the nucleotide base facilitates the substitution reaction.

CYCLIZATIONS OF THE RING B ACETAMIDE SUBSTITUENT Of the other known reactions of the cobalamins the cyclizations of the ring B acetamide substituent41 are of special interest. The action of one equivalent of chlorine (as chloramine T) on cyanocobalamin causes the formation of a stable fused Iactone (VIII; R = H) and only the further action of chlorirre brings about substitution, possibly at C1o, with the forma­ tion of a chlorolactone (VIII; R = Cl) (Figure 6).

H Me c~co l

(VIII) Figure 6 546 THE VITAMIN B12 COENZYME The reaction of the methyl coenzyme analogue took a different course and the first equiva.lent of chloramine T caused substitution without cycliza­ tion and tl.e formation of a 10( ?)-monochloro compound. However the reaction of excess chloramine T caused the formation of a monochloro­ lactone of the cobalt-methyl derivative, revealed by its characteristic infi·ared absorption at 1778 cm-1 and by its electrophoretic behaviour. Photolys:.s ofthe product in presence ofair caused the formation ofthe corre­ sponding hydroxocobalamin derivative. This with cyanide gave the same chlorolactone (VI1 I; R = Cl) as had been obtained previously41 by treat­ ment of cy<.nocobz.lamin with excess chlorine (Figure 7).

(1 mole)

hv

Figure 7

The cycl ization of the ring B acetamide side chain of cyanocobalamin to a lactant41 is not parallelled in the coenzyme series. Certain of the results of the chlorination experiments were originally interpreted42 as supporting a theory that the chromophore of the coenzyme contained one double bond less (I witl C9-10 bond saturated) than that of the vitamin. However this theory has now :::>een rejected43, 44 and the coenzymes and their alkyl analogues are regarded as containing the normal cobalamin chromophore. There thus exists a large series of cobalamins varying from vitamin B12 (I.: R = CN) to th~ alkylcobalamins (I; R = alkyl) including the coenzyme, and the pr<,perties ofa particular cobalamin depends markedly on the nature of the subs:ituent R. Other partially synthetic cobalamins are known which show p1·operties intermediate between these extremes. For example, sul­ phitocohahmin25, 45, which is converted to hydroxocobalamin by aerobic photolysis, is readily protonated by dilute acid and behaves like methylcoba­ lainin on

BIOCHEMICAL FUNCTION As mentioned above, the vitamin B12 coenzymes were discovered through their ahili :y to bring about the conversion of glutamate into ß-methyl• aspartate1• 2• 3, Apart from this reaction they also appear tobe involved in 547 D. DOLPHIN, A. W. JOHNSON, R. RODRIGO and N. SHAW the biological interconversion of C-methylmalonate and succinate46-48, and of propane-1,2-diol and propionaldehyde15, and a number of related reactions, e.g. the interconversion of glycerol and ß-hydroxypropional• dehyde49. The precise mechanism of these reactions has not yet been established, although deuteriwn studies have shown that the rearrangements are intramolecular and stereospecific. An early free radical mechanistic theory for the rearrangement of C-methylmalonate to succinate46 has now been abandoned50. The recent demonstration of the participation of com­ pounds of the vitamin B12 coenzyme type in methanc production51 is also of interest.

It is a pleasure to acknowledge the friendly collaboration we have enjoyed throughout this work with Dr E. Lester Smith, F.R.S. and Dr L. Mervyn of Glaxo Laboratories Ltd.

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549