SYNTHESES AND BIOLOGICAL BEHAVIOR OF ADDITIONAL CORRINOID COENZYMES

Otto Muller and Konrad Bernhauer Lehrstzihl f i i r Biochemk der Terhnischen Hochschule,

Sttittgart, Gcrniany

The biological behavior of synthetic corrinoid coenzyme analogues gives an insight into the mode of action and the biochemical significance of certain structural elements of the coenzyme forms. While cobalamin co- enzyme and cobinamide coenzyme were shown to be active in the methyl- malonate isomerase system, Co-5’-deoxyinosylcobalamin was inactive.14 This points to a special function of the nucleoside ligand. The nucleotide portion on the other hand is not necessary. In order to examine the struc- tural specificity, the synthesis of further cobalamin coenzyme analogues was undertaken. Co-5’-dexoyguanosylcobalamin already could be pre- pared.

Following the principle of coenzyme ~ y n t h e s i s ~ J ~ ~ * ~ ~ ~ ~ we tried to isolate 5’-tosyl-2’,3’-isopropylidene-guanosine in the pure form. But this was not possible probably because of intramolecular reaction of the C ( 5 ’ ) and N( 3) position of the guanine, forming the cyclic nucleoside. As is known, the cyclic nucleoside is formed, as a by-product, by the synthesis of 2’,3‘- isopropylideneS’-to~yladenosine.~ The greater tendency of the guanosine derivative to form the cyclic nucleoside appears to be clear, for its N(3) is more nucleophilic than the N(3) of the corresponding adenosine de- rivative on account of the neighboring amino-group on C(2) . But the synthesis of Co-5’-deoxyguanosyl-cobalamin is possible when the cobala- min-hydride is allowed to react with the crude mixture of the tosylation of 2’,3’-isopropylidene-guanosine in pyridine/dimethylformamide and sub- sequent removal of the isopropylidene-group by dilute sulphuric acid. On treatment with cyanide ions in the dark the splitting takes place in the same way as in the case of cobalamin coenzymei and Cod’-deoxyinosyl- cobalamin. The cyano-cobalamin and guanine obtained, were identified by means of chromatography, electrophoresis, and spectrophotometry.

The discovery of the method for synthesizing corrinoid coenzymes and analogues opens another possibility for the study of the biochemical func- tions of corrinoids. The enzymatic transfer of a methyl-group from Co- methylcobalamin to homocysteine8 demonstrates that not only the co- enzyme forms of the corrinoids have a biochemical function. It is possible that the corrinoids also participate in other transfer-reactions, the inter- mediates of which can not be isolated because of their rapid transforma- tion in biological systems. Nowadays it is possible to synthesize and to test supposed intermediates of biochemical transfer-reactions.

Coenzyme analogues of cobalamin, for example, Co-5‘-deoxyinosyl- cobalamin, are transformed relatively rapidly, coenzyme analogues of cobinamide relatively slowly into the coenzyme forms by P. shermanii (FIGURE 1 ) . We presume the transformation to take place by an exchange

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576 Annals New York Academy of Sciences

R P. shermanii 61 % - Co bal amin - coenzyme

ucleotide

Cobalamin-deriv.

R P. shermanii 5% I - Co binamide- coenzyme

Cobinamide -deriv.

R = 5'- deoxyinosyl- group FIGURE 1. Conversion of coenzyme analogues into coenzymes by Propionibacterium

shermanii ( 10 days).

of the ligands. In the presence of 5,6-dimethylbenzimidazole the greater part of coenzyme analogues of cobinamide and 2-methyladeninecobamide is first transformed into the equivalent analogues of cobalamin and after- wards into cobalamin coenzyme ( FIGURE 2 ) . This demonstrates that the

HO OH I t

+ 5.6- DBI P. shermanii L

4 2 % c o balamin- coenzyme

58% Co-5'-deoxyinosyl- cobalamin

FIGURE 2. Conversion of Co-5'-deoxyinosyl-cobinamide into cobalamin by PTO- pionibacteriurn shermanii ( 10 days).

biosynthesis of the nucleotide and the exchange of the bases are also possible with other forms than the native products, the corrinoid co- enzymes.

The synthetic cobinamide coenzyme is identical with the natural product and the product derived from cobalamin coenzyme by Cer( 111)-

MGller & Bernhauer: Synthesis of Corrinoid 577 hydroxide degradation. Accordingly, the hydride formation follows exclu- sively on the “upper” coordination position, that is on the side of the acetamide groups ( FIGURE 3) .

The different kinds of reaction of the two coordination positions of the Co-atom, oriented vertically, in respect of the corrin-skeleton, are shown in the following examples: the “upper” cyano group of the dicyano cobyric acid is bound loosely and the “lower” one tightly, as the investigation of

HO OH I I

Cobinamide- hydride Cobinamide-coenzyme FIGURE 3. Formation of cobinamide coenzyme.

the monocyano cobyric acid5 has showno (FIGURE 4 , ) . On forming the cobinamide hydride only the “upper” coordination position reacts. In the case of cobalamin derivatives the “upper” ligand has considerable influ- ence on the strength of the coordinative Co-nucleotide bond. The open- ing of this bond by protonation of the baselo in acid soIution requires a decrease of pH in the following order: cobalamin coenzyme, Co-methyl- cobalamin, cobalamin-Co-carbonic acid ethyl-ester. The cyano form of cobinamide and the cobinamide coenzyme can be more easily reduced by zinc in NH4C1-solution than the corresponding derivatives of cobalamin. This agrees with the polarographic behavior of these compounds. Cobinamide coenzyme is not as light sensitive as cobalamin coenzyme. But on being treated with cyanide cobinamide, coenzyme is split more rapidly than cobalamin coenzyme. The exchange of the same ligands of coenzyme analogues by P. shcmuznii during the formation of the respective coenzymes is slower with the derivatives of cobinamide than with de- rivatives of c0ba1amin.l~ The already mentioned differences in the re- actions of the two coordination positions, oriented vertically to the corrin skeleton of the Co-atom, point to the fact that the centre of the negative charge is displaced a little towards the “upper” coordination position if cobalt has the same ligands. The structural reasons for this are not known.

578 Annals New York Academy of Sciences

Cobyric acid

O H 2 I H I I

pI@ Reduct. L ,Fd0 t

Ho O H 2 Ho O H 2

Cobinamide

R R

ucleotide

Co bakmin

R = 5'-deoxyadenosyl-, CH3-, - C O O C 2 H s

FIGURE 4. Different kinds of reaction of the two coordination positions of the Co-atom, oriented vertically, in respect of the corrin skeleton.

But we can perhaps argue, that they are due to the corrin skeleton being not completely planar.llsga

BlZr and cobalamin hydride are stable to cyanide in the absence of oxygen. Cobalamin coenzyme on the contrary becomes cyano-cobalamin when treated with cyanide in the presence as well as in the absence of oxygen. Under the same conditions Co-acetyl-cobalamin can also be transformed into cyano-cobalamin. On treatment with 0.5 N acetic acid in the presence as well as in the absence of oxygen hydroxo-cobalamin has been obtained. Bla, is stable under these conditions in the absence of oxygen. Therefore cobalamin hydride and BIZ, can not be intermediates of the cleavage of cobalamin coenzyme and of Co-acetyl-cobalamin. Ob- viously cyano-cobalamin and hydroxo-cobalamin respectively are formed by exchange of ligands only.

Muller & Bernhauer : Synthesis of Corrinoid 579 The cobalt to carbon bond of corrinoid coenzymes and analogues is

also split by i ~ d i n e . ~ By cleavage of cobalamin coenzyme in alcoholic solution iodo-cobalamin and 5’-iodine-5’-deoxyadenosine were obtained. Iodo-cobalamin is hydrolysed at once into hydroxo-cobalamin by water. The cleavage of the cobalt-carbon bond by iodine takes place at a decreas- ing rate in the following order: Co-methyl-cobalamin, Co-ethyl-cobala- min, cobalamin coenzyme, isopropylidene cobalamin coenzyme.

As synthesis13 and b io~ynthes is~ ,~~ of cobinamide coenzyme lead to the same compound it is possible that they have the same principle of reaction. Here arises the question of what kind of natural compounds transfers the 5’-deoxyadenosyl-group to cobinamide and cobalamin. Enzymatic ex- periments have shown that the 5‘-deoxyadenosyl-group of cobalamin coenzyme is derived from ATP.lG But it is improbable that ATP reacts directly with cobalamin. There is no non-enzymatic reaction between ATP and cobalamin hydride. As sulfonium-compounds are electrophilic it seems to be possible that compounds react during the biosynthesis of corrinoid coenzymes the active centres of which have a sulfonium struc- ture. The “activation” of adenosine for the reaction with cobalamin hydride could also consist in the formation of a double bond between C(4’) and C ( S ’ ) , perhaps by cleavage of pyrophosphate and phosphate from ATP.G By reacting cobalamin hydride with the extracts of P . sher- mad, however, no cobalamin coenzyme was obtained.

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