Cobyrinates

Corrin is the porphyrinoid chromophore of the vitamin parent compound cobyrinic acid. Corrin itself has not yet been synthesized, but routes to cobyrinic acid and several other synthetic corrins have been described by A. Eschenmoser (1970, 1974) and R.B. Woodward (1967). 

It is conceivable that related ligands, e.g. dehydrocorrins, could be obtained from pyrrolic units using pathways similar to those used for porphyrins and could be hydrogenated to corrins. This has indeed been achieved (I.D. Dicker, 1971), but it is, of course, impossible to introduce the nine chiral centres of cobyrinic acid by such procedures. 

Scheme 20. Synthesis of cobyrinic acid abcdeg hexamethylester f nitrile 96.

Scheme 21 presents the successful sequence of reactions that solved the remaining two problems and led to the completion of the synthesis of cobyric acid. Exposure of 96 to concentrated sulfuric acid for one hour brings about a clean conversion of the nitrile grouping to the corresponding primary amide grouping. The stability of die corrin nucleus under these rather severe conditions is noteworthy. This new substance, intermediate 97, is identified as cobyrinic acid abcdeg hexamethylester f amide and it is produced along with a very similar substance which is epimeric to 97 at C-13. The action of sulfuric acid on 96 produces a diastereomeric... 

A methyl group can be placed on cobalt in dicyano[cobyrinic acid heptamethyl ester] when it is treated with excess methyl magnesium iodide (the ester side chains being converted into tertiary alcohol groups). The alkylation could also be achieved using lithium alkyls (176). Presumably this reaction would be successful with any corrinoid which is soluble in solvents compatible with Grignard reagents. 

Naturally, the biosynthesis of cobalamins themselves require delivery of Co ions at a particular point in the reaction scheme. Cobaltochelatase catalyzes the ATP-dependent insertion of Co11 into the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrifleans. Cobaltochelatase is a heterodimeric enzyme (140 KDA and 450 KDA subunits each inactive in isolation), and the two components have been isolated and purified to homogeneity.1119 The reaction product is divalent cobyrinic acid, demonstrating that hydrogenobyrinic acid and its diamide (255) are precursors of AdoCbl. 

The similarity between the structures of the corrinoids and the porphyrins becomes evident from comparison of cobyrinic acid (75) (the simplest of the corronoids so far isolated) with uroporphyrinogen III (70). The possibility of a biosynthetic relationship between these structures was suggested by Shemin, who reported the incorporation of [14C]ALA into vitamin Bn and confirmed by the subsequent demonstration that PBG was also incorporated. The ubiquitous precursorial role of uroporphyrinogen III in heme, chlorophyll and corrinoid biosynthesis proposed by Porra (65BBA(107)176) was, however, not substantiated by experimental evidence until much later, when under carefully controlled conditions cells of Propionibacterium shermanii were shown to incorporate radioactivity from [14C]uroporphyrinogen III into vitamin Bn (72JA8269). 

Confirmation of this result has been obtained from a number of experiments in which a variety of synthetic 13C and 14C isotopomers of uroporphyrinogen III were shown to be regiospecifically incorporated into cobyrinic acid (isolated as coester, 75a) in cell-free systems and into vitamin Bi2 in whole cells (Scheme 22) (78ACR29, 77JCS(P1)166). 

Subsequently both a monomethyl and a trimethyl isobacteriochlorin, factors I (78) and III (79), have been isolated from P. shermanii and shown by radiolabelling experiments to act as precursors of cobyrinic acid in cell-free systems. While sirohydrochlorin (77) and factor III (79) are incorporated when administered in their oxidized forms, factor I (78) and uroporphyrin III (80) (the oxidized form of uroporphyrinogen III) are only effectively incorporated when added in the reduced forms (78a) and (70), suggesting that the four compounds are intermediates only at their reduced levels and that a cellular reductase system exists which is capable of reducing (77) and (79) but not (78) and (80). 

Scheme 26 Conversion of radiolabelled factor III (79) to cobyrinic acid (analyzed as cobester, 75a) and acetic acid by cell-free extracts of P. shermanii and Clostridium tetenomorphum. Labelling patterns = 3H, = 14C or...

Scheme 29 Proposed sequence for the formation of vitamin Bj2 from cobyrinic acid...

The structure of a /t-iodo dimer, [ Con(cobyrinic acid hexamethyl ester) 2lll has been determined.253 The cobalt atoms are displaced from the N4 plane by 0.11 A and 0.13 A respectively, and the Co—I—Co group is slightly bent. The dimer is diamagnetic due to strong antiferromagnetic coupling between the two Co atoms. It exists in equilibrium with the monomer in benzene solution. 

ISEs Based on Cobyrinates. Vitamin B12 is hydrophilic and, therefore, it is necessary to modify it chemically in order to use it as ionophore in polymer membrane-based ISEs. Different hydrophobic derivatives that lack the nucleotide part of the vitamin (cobyrinates) have been prepared for this purpose. These compounds, although structurally similar to vitamin B12, have a quite different coordination behavior from that of the vitamin (7). 

A hydrophobic cobyrinate (Figure 2, structure 2) was used to prepare solvent polymeric membranes (10). The typical membrane composition was 1% (w/w) ionophore, 66% (w/w) plasticizer and 33% (w/w) polymer. Electrodes prepared with this ionophore, dioctyl sebacate (DOS) and poly(vinyl chloride) (PVC) presented, at pH 6.6, the selectivity pattern shown in Figure 3. The response of the electrodes was near-Nernstian for salicylate, thiocyanate, and nitrite. Their selectivity behavior clearly deviates from that of the Hofmeister series, with nitrite being the anion that presents the larger deviation. 

In dicyanocobalt(III) a,b,c,d,e,g-hexamethyl-f-stearylamide cobyrinate (derivative 3) the six peripheral amide groups of vitamin B12 have been replaced with methyl ester groups, and the proximal base of the vitamin at the f-position with a stearylamide group (11). Electrodes prepared with this ionophore and DOS as the plasticizer were also selective for thiocyanate and nitrite over the rest of the anions tested. The main anionic interferent was salicylate. In all cases, the response of the electrodes to the preferred anions was sub-Nemstian. Overall, the selectivity pattern obtained with ionophore 3 is similar to that of 2 and to that of the hydrophobic cobyrinate-based electrodes reported previously (5, 12, 13). This observation suggests that in all cobyrinate ionophores the anions interact with the cobalt(III) center, and that the side chains of the corrin ring have a small effect on the selectivity of this interaction. 

Although the ISEs based on cobyrinates have good selectivity for nitrite over several anions, they also respond to salicylate and thiocyanate. To eliminate this interference, the nitrite-selective electrode based on ionophore 2 was placed behind a microporous gas-permeable membrane (GPM) in a nitrogen oxide gas-sensor mode (75). NOx was generated from nitrite in the sample at pH 1.7 and, after crossing the GPM, was trapped as nitrite by an internal solution that was buffered at pH 5.5 (0.100 M MES-NaOH, pH 5.5, containing 0.100 M NaCl). The internal solution was "sandwiched" between the nitrite-selective electrode and the GPM. 

---