Cobinamide

Cobinic acid and cobinamide, which aie hnked to libose 3-phosphate, are known as cobamic acid and cobamide (5), respectively. 

Chemical Name Cobinamide hydroxide phosphate 3 -ester with 5,6-dimethyl-l-o-D-ribofuranosylbenzimidazole inner salt... 

CN cobinamide Co-(5 -deoxyadenosine-5 ) de riv. hydroxide dihydrogen phosphate (ester) inner salt 3 -ester with 5,6-dimethyl-1 -a-D-ribofuranosyl-1 //-benzimidazole... 

A related phenomenon, apparently caused by interaction of the organo-ligand with the corrin ring and its substituents, is the absence of free rotation around the Co—C bond revealed by the nonequivalence of the a and/or jS-protons in the NMR spectra of -propyl- and isopropylcobin-amide and 5-deoxyadenosylcobalamin and cobinamide, though free rotation occurs in ethylcobinamide (88, 35, 20). 

Most of the examples listed are pentacyanide, corrinoid, or DMG complexes. The axial ligands are not identified in the tables, but are as follows corrinoids, 5,6-dimethylbenziminazole (cobalamins), H O or none (cobinamides), (DMG)2, usually pyridine or H2O, less frequently NHj, imidazole, benzimidazole, PBuj, etc. The nature of the axial and equatorial ligands may have a striking effect on reactivity, but few direct comparisons are available these are discussed in the next section. 

The decomposition of 4-pyridylmethyl- and a-(2-pyridyl)ethylpenta-cyanide, which probably involves homolytic fission (see Section B,l,a), occurs only after the loss of one cyanide to give the (presumably trans) organotetracyanoaquo complexes 100,102), i.e., in this case we observe the order H2O > CN . The decomposition of corrinoids possessing secondary alkyl ligands is accelerated by the addition or presence of bases and cyanide. Isopropylcobalamin is more unstable than the cobinamide 61) cyclohexyl-... 

The organo-corrinoids show similar behavior, but also additional complications. Rate constants have been determined 84) for the attack of mercury(II) acetate on various organocobalt cobinamides (X = H2O or absent) and cobalamins (X = 5,6-dimethylbenziminazole). The first complication, which has to be born in mind when comparing the cobinamides with the cobalamins or DMG complexes, is that the organocobin-amides are partly (R = vinyl and methyl) or wholly (R = Et, -Pr, t-Pr,... 

Fukui et /. have found that acetylcobinamide reacts faster than the cobalamin with hydroxide (t,/2 = 4 and 30 min, respectively, in 0.05 N KOH) (67). It is not known whether the cobinamide is five- or six-coordinate, but these relative rates establish the labilizing effect of the trans-axial ligand for heterolysis to give Co(I) as HjO or none > 5,6-dimethylbenziminazole (see also Section B,3). 

Vitamin Bu coenzymes, Ann. N.Y. Acad, Set. 112, 547-921 (1964). I. Chemistry, chemical synthesis and biosynthesis of corrin coenzymes II. Enzymic roles of cobinamide coenzymes III. B 12-coenzymes in micro-organisms and animals. 

One obvious way in which to attach a nitroxide group to B12 is to simple alkylate Cob(I)aiamin with a suitable nitroxide derivative. This would result in having the nitroxide covalently bound to the corrinoid at the upper axial coordination position of the cobalt. Such a procedure is outlined in Fig. 19. In this reaction 4-bromoacetamido 2,2,6,6-tetra-methylpiperidine-N-oxyl is used to alkylate Cob(I)alamin. This results in a Co(III)-nitroxalkylcobalamin. The corresponding cobinamide can then be produced by hydrolyzing the ribose-phosphate linkage (119). 

Fig. 19. Alkylation of Bias with 4 bromoacetamido 2,2,6,6-tetramethylpiperidine-N-oxyl to give nitroxalkylcobalamin, and (after hydrolysis) the corresponding cobinamide...

Alternatively the cobinamide can be synthesized by first carrying out the hydrolysis of either aquo- or cyano-cobalamin followed by alkylation of the reduced cobinamide. 

The nitroxalkylcorrinoids are easy to characterize since they are basically alkyl corrinoids. The u.v.-visible absorption spectra of the nitroxalkylcorrinoids are quite similar to other alkylcorrinoids. The nitroxalkyl cobalamin has a spectrum with Amax at 525,357, and 329nm with relative extinctions of 1.1, 1.2, and 0.65 respectively. Spectra for a number of typical alkylcobalamins have been reported by Firth et al. (121). The corresponding cobinamide has absorption maxima at 455, 428, 360, and 325 nm with relative extinctions of 1.4, 1.0, 0.61, and 0.62 respectively. Aerobic photolysis of these compounds leaves the corresponding aquocobalamin or aquocobinamide. Likewise, addition of cyanide to the nitroxalkylcobinamide in base leaves dicyano cobinamide. 

The electron spin resonance of the nitroxalkylcorrinoids can be readily observed in aqueous solution at room temperature. Both the cobalamin and cobinamide show nitrogen hyperfine coupling constants of 17.2 gauss. A typical spectrum is shown in Fig. 20. The line widths for the low, intermediate, and high field peaks are 1.87, 1.87, and 2.20... 

Another chemically more interesting spin labeled B12 derivative involves coordinate attachment of the nitroxyl function to the cobalt atom of a cobinamide. Fig. 22 shows a reaction in which an alkyl cobin-amide is mixed with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. The nitroxide displaces water from the 6th coordination position very slowly and therefore this reaction is usually allowed to proceed for a few days with a large excess of nitroxide. From the properties of the coordinated nitroxide derivative discussed below, it is certain that the cobalt is coordinated by the N—O functional group. An analogous compound to that shown in Fig. 22 can be made with a similar nitroxide in which the 4-hydroxyl-group is missing. In this case the N—O-function is the only basic site on the molecule and therefore must be the position of attachment to the cobalt 119). 

The u.v.-visible spectrum of the 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl-methyl-cobinamide is very similar to methyl-cobin-amide itself and as a result this technique cannot be used to rigorously identify the spin labeled derivative. The spin labeled compound does show a spectral change with pH between pH 7.0 and pH 2.0 which methyl-cobinamide does not exhibit. Despite the similarities between methyl-cobinamide and nitroxylmethylcobinamide, the circular dichroism spectrum of the two derivatives are quite different. Fig. 23 shows the marked difference in C. D. spectra of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, methylcobinamide, and a methylcobinamide solution containing an equimolar amount of uncoordinated nitroxide. 

Fig. 23. C. D. Spectra of 4-hydroxy-2,2,6,6-piperidine-N-oxyl-methyl-cobinamide (—) and methyl-cobinamide plus free nitroxide (---—) at 3 x 10 5 M in ethanol...

As with the nitroxalkylcobalamins (119) and cobinamides, the co-binamides in which nitroxide is coordinated show electron spin resonance spectra very similar to the spectrum of free nitroxide. The high field line is not broadened as much as in the spectrum of a nitroxalkyl-cobinamide. No hyperfine splitting from methyl protons in the 2 or 6 positions can be observed for the bound nitroxide. However, treatment of the coordinate spin labeled compounds with cyanide releases the nitroxide. When this happens, the proton hyperfine can be observed (Fig. 25). Thus treatment with cyanide simply displaces the nitroxide and a spectrum for free nitroxide is observed. 

One of the somewhat surprising aspects of the coordinate nitroxyl-cobinamides is that they show no indication of cobalt hyperfine splitting even though the nitroxyl function is coordinated directly to cobalt I(59Co) = 7j2. This is contrary to what has been found in nitroxide adducts with AICI3 (124). 

Fig. 29. Decrease in intensity of nitroxide ESR signal npon addition of deuterated ethanolamine to ethanolamine ammonia lyase containing spin labeled cobinamide coenzyme. The two curves are for different concentrations of coenzyme to enzyme. The arrows indicate the point at which alcohol dehydrogenase and NADH was added to remove acetaldehyde from the enzyme. Note that full intensity is regained...

Fig. 31 shows 220 MHz spectra for methyl cobalamin and methyl cobinamide. Most of the features of the PMR work to be discussed below can be illustrated with these two spectra as references. (See Fig. 1 for references to nomenclature.)... 

Fig. 31. 220 MHz NMR spectrum of methyl cobalamin in DaO and 220 MHz NMR spectrum of methyl cobinamide in D2O...

Assignment of the remaining peaks in the cobalamin or cobinamide spectra is less straightforward. The most detailed assignments made thus far have been made by Brodie and Poe (130). These are outlined in Table 2 for methyl cobalamin. 

In the spectra of alkyl cobinamides two peaks have been observed at 3.89 and 4.42 which were assigned to the protons of a water molecule coordinated at the lower axial site (130). To confirm this assignment, it was found that addition of cyanide to methyl cobinamide, which displaces coordinated water, caused the peaks to disappear. Likewise, addition of excess D2O caused disappearance of the peaks through either ligand exchange or proton-deuteron exchange. 

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