Microwave-assisted cobinamide synthesis.

We present a new method for the preparation of cobinamide (CN)2Cbi, a vitamin B12 precursor, that should allow its broader utility. Treatment of vitamin B12 with only NaCN and heating in a microwave reactor affords (CN)2Cbi as the sole product. The purification procedure was greatly simplified, allowing for easy isolation of the product in 94% yield. The use of microwave heating proved beneficial also for (CN)2Cbi(c-lactone) synthesis. Treatment of (CN)2Cbi with triethanolamine led to (CN)2Cbi(c-lactam).

Interactions between coenzyme B12 analogs and adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum.

Adenosylcobalamin (AdoCbl)-dependent glutamate mutase from Clostridium tetanomorphum comprises two weakly-associating subunits, MutS and MutE, which combine with AdoCbl to form the active holo-enzyme. Three coenzyme analogs, methylcobinamide (MeCbi), adenosylcobinamide (AdoCbi) and adeosylcobinamide-GDP (AdoCbi-GDP), were synthesized at milligram scale. Equilibrium dialysis was used to measure the binding of coenzyme B(12) analogs to glutamate mutase. Our results show that, unlike AdoCbl-dependent methylmalonyl CoA mutase, the ratio k(cat)/K(m) decreased approximately 10(4)-fold in both cases when AdoCbi or AdoCbi-GDP was used as the cofactor. The coenzyme analog-binding studies show that, in the absence of the ribonucleotide tail of AdoCbl, the enzyme's active site cannot correctly accommodate the coenzyme analog AdoCbi. The results presented here shed some light on the cobalt-carbon cleavage mechanism of B(12).

Cobinamides as structural probes in B12-biosynthesis: synthesis and spectroscopic analysis of isomers of Co alpha,Co beta-dicyanocobinamide.

Vitamin B(12) and its coenzyme forms are cobalamins (i.e., cobamides, 'complete' with a 5,6-dimethylbenzimidazole nucleotide base), in which the particular corrinoid moiety of the cobinamides is conjugated to alpha-ribazole-3'-phosphate via a phosphate-diester group. Aside of being provided with their particular reactivity, required for their functions as organometallic cofactors in B(12)-dependent enzymes, the cobalamins also depend upon their specific three-dimensional buildup, to be able to adapt the unique constitution of 'base-on' corrinoids by intramolecular Co-coordination of the nucleotide base. We report rational partial syntheses and detailed spectral analyses of three close cobinamide isomers in their Co(alpha),Co(beta)-dicyano forms: of 13-epicobinamide (also called neocobinamide), of 176(S)-epicobinamide, and of 176-isocobinamide. Neocobinamide was obtained under acidic conditions as a degradation product of vitamin B(12). 176(S)-Epicobinamide and 176-isocobinamide were prepared by condensation of cobyric acid with (2S)-1-aminopropan-2-ol and with 3-aminopropan-1-ol, respectively. Natural cobinamide represents the corrinoid nucleus produced by proper microbial biosynthesis (as intermediate for the further assembly of the 'complete' corrinoid cofactors) or is required in some microorganisms, such as Escherichia coli, as an exogenously supplied unit for further biosynthetic buildup. The three compounds may thus be of use as structural probes for the biosynthetic capacity and tolerance in microorganisms, and (some of them) may serve as substrates as well, for further biosynthetic 'completion' of corrinoid cofactors or their analogues.

Nitrosyl-cobinamide, a new and direct nitric oxide releasing drug effective in vivo.

A limited number of nitric oxide (NO)-generating drugs are available for clinical use for acute and chronic conditions. Most of these agents are organic nitrates, which do not directly release NO; tolerance to the drugs develops, in part, as a consequence of their conversion to NO. We synthesized nitrosyl-cobinamide (NO-Cbi) from cobinamide, a structural analog of cobalamin (vitamin B12). NO-Cbi is a direct NO-releasing agent that we found was stable in water, but under physiologic conditions, it released NO with a half-life of 30 mins to 1 h. We show in five different biological systems that NO-Cbi is an effective NO-releasing drug. First, in cultured rat vascular smooth muscle cells, NO-Cbi induced phosphorylation of vasodilator-stimulated phosphoprotein, a downstream target of cGMP and cGMP-dependent protein kinase. Second, in isolated Drosophila melanogaster Malpighian tubules, NO-Cbi-stimulated fluid secretion was similar to that stimulated by Deta-NONOate and a cGMP analog. Third, in isolated mouse hearts, NO-Cbi increased coronary flow much more potently than nitroglycerin. Fourth, in contracted mouse aortic rings, NO-Cbi induced relaxation, albeit to a lesser extent than sodium nitroprusside. Fifth, in intact mice, a single NO-Cbi injection rapidly reduced blood pressure, and blood pressure returned to normal after 45 mins; repeated NO-Cbi injections induced the expected fall in blood pressure. These studies indicate that NO-Cbi is a useful NO donor that can be used experimentally in the laboratory; moreover, it could be developed into a vasodilating drug for treating hypertension and potentially other diseases such as angina and congestive heart failure.

Synthesis, spectroscopic characterization, axial base coordination equilibrium and photolytic kinetics studies of a new coenzyme B12 analogue-3'-deoxy-2',3'-anhydrothymidylcobalamin.

A new coenzyme B12 (AdoCbl) analogue, 3'-deoxy-2',3'-didehydrothymidylcobalamin (2',3'-anThyCbl) was prepared by the reaction of 5'-iodo-3'-deoxy-2',3'-dihydrothmidine with reduced B12a, and characterized by UV-Vis, CD, ESI-MS and NMR spectroscopies. Its axial base (dbzm) coordination equilibria with pH's and temperatures were investigated and showed similar features to those of coenzyme B12. Photolytic dynamics studies under homolytic and heterolytic conditions demonstrated that the Co-C bond of the analogue is slightly more photolabile relative to coenzyme B12.

Searching for intermediates in the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate catalyzed by coenzyme B12-dependent 2-methyleneglutarate mutase from Eubacterium barkeri.

Coenzyme B(12)-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations in the highly conserved coenzyme binding-motif DXH(X)(2)G(X)(41)GG (D483N and H485Q) exhibited decreased substrate turnover by 2000-fold and >4000-fold, respectively. These findings are consistent with the notion of H485 hydrogen-bonded to D483 being the lower axial ligand of adenosylcobalamin in 2-methyleneglutarate mutase. (E)- and (Z)-2-methylpent-2-enedioate and all four stereoisomers of 1-methylcyclopropane-1,2-dicarboxylate were synthesized and tested, along with acrylate, with respect to their inhibitory potential. Acrylate and the 2-methylpent-2-enedioates were noninhibitory. Among the 1-methylcyclopropane-1,2-dicarboxylates only the (1R,2R)-isomer displayed weak inhibition (noncompetitive, K(i) = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with 2-methyleneglutarate under anaerobic conditions generated an electron paramagnetic resonance (EPR) signal (g(xy) approximately 2.1; g(z) approximately 2.0), which by analogy with the findings on glutamate mutase from Clostridium cochlearium [Biochemistry, 1998, 37, 4105-4113] was assigned to cob(II)alamin coupled to a carbon-centered radical. At longer incubation times (>1 h), inactivation of the mutase occurred concomitant with the formation of oxygen-insensitive cob(II)alamin (g(xy) approximately 2.25; g(z) approximately 2.0). In order to identify the carbon-centered radical, various (13)C- and one (2)H-labeled substrate/product molecules were synthesized. Broadening (0.5 mT) of the EPR signal around g = 2.1 was observed only when C2 and/or C4 of 2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-(13)C]adenosylcobalamin was used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-elimination and fragmentation-recombination mechanisms proposed for 2-methyleneglutarate mutase.

Synthesis of adenosylcobinamide 2-chlorophenyl phosphate, a zwitterionic cobinamide phosphate analog of adenosylcobalamin en route to crystallizable cobinamides.

The design and implementation of a new, higher yield synthetic method for synthesizing zwitterionic cobinamide phosphates is described. Adenosylcobinamide 2-chlorophenyl phosphate, beta-AdoCbi-PAr -- a 5,6-dimethylbenzimidazole-free adenosylcobalamin analog, where a 2-chlorophenyl group replaces the ribofuranose and 5,6-dimethylbenzimidazole moieties -- is prepared in tens of milligram quantities, quantities sufficient for crystallization and enzyme trials, amounts 100-fold greater than previously available. The use of (31)P NMR spectroscopy to follow reactions directly, the use of control reactions to learn how to reduce reactant water content, and the use of reaction solvents that completely dissolved the corrinoid reactants were crucial for developing this new synthetic route. beta-AdoCbi-PAr was synthesized in 10% overall isolated yield from cyanocobinamide. Cyanocobinamide was converted to cyanocobinamide 2-chlorophenyl phosphate by direct phosphorylation with 2-chlorophenyl phosphodi-(1,2,4-triazolide) in 25% isolated yield and > or = 98% purity. Sodium borohydride reduction of cyanocobinamide 2-chlorophenyl phosphate and reaction with 5'-chloro-5'-deoxy-adenosine produced beta-AdoCbi-PAr in 42% yield and > or = 98% purity. These compounds were characterized by HPLC, (1)H and (31)P NMR, UV-visible spectroscopy, and liquid secondary ionization mass spectroscopy.

The synthesis and characterization of 8-methoxy-5'-deoxyadenosylcobalamin: a coenzyme B(12) analog which, following Co-C bond homolysis, avoids cyclization of the 8-methoxy-5'-deoxyadenosyl radical.

The compound 8-methoxy-5'-deoxyadenosylcobalamin (8-MeOAdoCbl), has been synthesized in 37% yield and > or = 95% purity by HPLC, monitored at both 254 and 525 nm, or 90+/-2% purity as judged by the (1)H NMR spectrum of the aromatic cobalamin region. This is the first synthesis of this complex in which sufficient details are reported, where a yield and purity are reported, and where key problems in the synthesis and purification are overcome, so that 8-MeOAdoCbl can actually be obtained for use in other studies. Also demonstrated is the clean Co-C bond homolysis of 8-MeOAdoCbl to give initially 8-MeOAdoCbl and Co(II)Cbl in a UV-visible thermolysis experiment at 110 degrees C, results which show that the 8-MeO moiety suppresses the cyclization to the 8,5'-anhydro-adenosine otherwise seen for the adenosyl radical (Ado)*. Suppression of this cyclization pathway makes 8-MeOAdoCbl invaluable for studying the kinetic isotope effect (KIE) of the Ado* plus substrate H* abstraction reaction, a component of the first definitive test of Klinman's hypothesis that the optimization of enzyme catalysis may entail strategies that increase the probability of tunneling and thereby accelerate H* atom abstraction reaction rates.

Thermal and photochemical epimerization/equilibration of carbohydrate cobaloximes.

Epimeric carbohydrate alkyl cobaloximes 4:5, 9:10, and 12:13 can be equilibrated thermally or photochemically. In each case, one isomer is strongly favored: exo-3-deoxy-3-pyridyldimethylglyoximatocobalt-1,2:5,6-di-O-isopropylidene-alpha-D-glucofuranose 4 for the 4:5 epimer pair, exo-3-deoxy-3-pyridyldimethylglyoximatocobalt-5-O-carboxymethyl-1,2-O-isopropylidene-alpha-D-xylofuranose 9 for the 9:10 epimer pair, and equatorial 1-deoxy-1-pyridyldimethylglyoximatocobalt-2,3,4,6-tetra-O-benzyl-beta-D-glucopyranose 12 for the 12:13 epimer pair. These data indicate that there is a strong facial preference for the coupling of py(dmgH)(2)Co(*) radicals with alkyl R(*) free radicals, with the preferred kinetic path leading to the more stable product.

Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation.

Ribonucleotide reductases (RNRs) catalyze the rate-determining step in DNA biosynthesis: conversion of nucleotides to deoxynucleotides. The RNR from Lactobacillus leichmannii utilizes adenosylcobalamin (AdoCbl) as a cofactor and, in addition to nucleotide reduction, catalyzes the exchange of tritium from [5'-3H]-AdoCbl with solvent. Examination of this exchange reaction offers a unique opportunity to investigate the early stages in the nucleotide reduction process [Licht S. S., Gerfen, G. J., and Stubbe, J. (1996) Science 271, 477-481]. The kinetics of and requirements for this exchange reaction have been examined in detail. The turnover number for 3H washout is 0.3 s-1, and it requires an allosteric effector dGTP (Km = 17 +/- 3 microM), AdoCbl (Km = 60 +/- 9 microM) and no external reductant. The effects of active-site mutants of RTPR (C119S, C419S, C731S, C736S, and C408S) on the rate of the exchange reaction have been determined, and only C408 is essential for this process. The exchange reaction has previously been monitored by stopped-flow UV-vis spectroscopy, and cob(II)alamin was shown to be formed with a rate constant of 40 s-1 [Tamao, Y., and Blakley, R. L. (1973) Biochemistry 12, 24-34]. This rate constant has now been measured in D2O, with [5'-2H2]-AdoCbl in H2O, and with [5'-2H2]-AdoCbl in D2O. A comparison of these results with those for AdoCbl in H2O revealed kH/kD of 1.6, 1.7, and 2.7, respectively. The absolute amounts of cob(II)alamin generated with [5'-2H2]-AdoCbl in D2O in comparison with AdoCbl in H2O reveal twice as much cob(II)alamin in the former case. Similar transient kinetic studies with C408S RTPR reveal no cob(II)alamin formation. These experiments allow proposal of a minimal mechanism for this exchange reaction in which RNR catalyzes homolysis of the carbon-cobalt bond in a concerted fashion, to generate a thiyl radical on C408, cob(II)alamin, and 5'-deoxyadenosine.

Structural and enzymatic studies of a new analogue of coenzyme B12 with an alpha-adenosyl upper axial ligand.

A new analogue of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl), in which the configuration of the N-glycosidic bond in the Ado ligand is inverted [(alpha-ribo)AdoCbl], has been synthesized and its crystal structure determined by X-ray diffraction [MoKalpha, lambda = 0.71073 A, monoclinic P212121, a = 16.132(12) A, b = 21. 684(15) A, c = 27.30(3) A, 9611 independent reflections, R1 = 0. 0708]. As suggested by molecular mechanics modeling before the structure was known, the Ado ligand lies over the southern quadrant of the molecule, as is the case for AdoCbl. The most striking feature of the structure is disorder in the orientation of the adenine (Ade) moiety relative to the ribose of the Ado ligand. This was resolved with a two-state model in which in the major (0.57 occupancy) conformer the A16(O)-A11-A9(N)-A8 dihedral angle is 1.9 degrees and the Ade is virtually perpendicular to the corrin ring; in the minor conformer, the Ade is tilted down, and this dihedral is -48.7 degrees. The Co-C and axial Co-N bond lengths and the Co-C-C bond angle are quite similar to those in AdoCbl. The corrin ring is considerably flatter than that of AdoCbl, with a fold angle of 11.7 degrees. The molecule was successfully modeled by molecular mechanics (MM), and rotation of the Ado ligand relative to the corrin gave rise to four locally minimum structures with the Ado in the southern, eastern, northern, or western quadrant, with the southern conformation as the global minimum, as is the case with AdoCbl itself. Nuclear Overhauser effects (nOe's) observed by two-dimensional (2D) NMR were incorporated as restraints in molecular dynamics (MD) and simulated annealing (SA) calculations. A MD simulation at 300 K showed that only the southern conformation is populated with the Ado ligand confined to an arc from over C15 to over C12, while the Ade ring oscillates from perpendicular to parallel to the corrin ring. Twenty-seven structures were collected by MD-SA. Most of these annealed into the southern conformation, but examples of the other conformations were also found. The new analogue is a partially active coenzyme for the ribonucleotide reductase from Lactobacillus leichmanii with maximal activity that is 9.7% of that of AdoCbl itself, and a very high Km value (245 microM compared to 0.54 microM for AdoCbl). In addition, the rate constant for enzyme-induced carbon-cobalt bond cleavage of (alpha-ribo)AdoCbl is 160-fold smaller than that for AdoCbl, and only 1/3 as much cob(II)alamin is produced at the active site.

Synthesis of 4-aza-5,6-dimethylbenzimidazole and biosynthetic preparation of 4- and 7-aza-5,6-dimethylbenzimidazolylcobamide.

We report on the preparation of 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide. These vitamin B12-analogs were required as reference compounds for comparison with a corrinoid previously isolated in small amounts for Eubacterium limosum grown in the presence of 4(5)-aminoimidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was synthesized from N-1-benzyl-4-nitroimidazole which was reduced to N-1-benzyl-4-aminoimidazole and condensed with 1-dimethylamino-2-methylbutan-3-one to yield N-1-benzyl-4-aza-5,6-dimethylbenzimidazole. The benzyl group of this compound was split off by catalytic hydrogenation to form 4(7)-aza-5,6-dimethylbenzimidazole. 4(7)-Aza-5,6-dimethylbenzimidazole was transformed by a growing culture of Propionibacterium shermanii into 4-aza-5,6-dimethylbenzimidazolylcobamide and 5,6-dimethyl-7-azabenzimidazolylcobamide. Both vitamin B12-analogs were almost as active as Vitamin B12 in a growth test with the vitamin B12-dependent Escherichia coli-mutant DSM 4261.

Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: implications for the mechanism.

The transfer of tritium between adenosylcobalamin and substrate in the reaction catalyzed by glutamate mutase was examined to investigate the possibility of a protein-based radical intermediate. There was no evidence that tritium was transferred to the protein during the reaction, as tritium neither became stably bound to the protein nor exchanged with water. The kinetics of tritium transfer from adenosylcobalamin to 3-methylaspartate was investigated. Both the transfer of tritium to product and the exchange of enzyme-bound and free coenzyme contribute to the kinetics of tritium loss from adenosylcobalamin. By varying the experimental conditions, the rates of both coenzyme exchange and tritium transfer could be measured. Exchange of adenosylcobalamin with enzyme is very slow, k off = 0.01 s-1, which may reflect a conformational change in the coenzyme and/or protein involved in forming active holo enzyme. The rate constants for the loss of tritium from adenosylcobalamin and the appearance of tritium in 3-methylaspartate are much faster and very similar, k = 0.67 +/- 0.05 s-1 and k = 0.50 +/- 0.05 s-1, respectively, consistent with the transfer of tritium occurring directly between coenzyme and substrate. The isotope effect, calculated from the rate constants for tritium transfer, and kcat, determined for the overall reaction under the same conditions, are between 13.5 and 18. These values are typical of primary isotope effects seen for enzymes in which hydrogen transfer is substantially rate limiting. A protein radical, therefore, appears unlikely to feature in the mechanism of this enzyme.

The synthesis of a pyridyl analog of adenosylcobalamin and its coenzymic function in the diol dehydratase reaction.

A novel analog of adenosylcobalamin in which 5,6-dimethylbenzimidazole and D-ribose moieties of the nucleotide loop are replaced by pyridine and the trimethylene group, respectively, was synthesized and examined for coenzymic function. The coordination of pyridine to the cobalt atom in this analog was stronger than that of 5,6-dimethylbenzimidazole in the corresponding homolog. The adenosyl form of pyridyl analog served as partially active coenzyme for diol dehydratase. The kcat/Km values calculated from the initial velocity indicate that this analog is a better coenzyme than the 5,6-dimethylbenzimidazolyl or imidazolyl counterpart. However, the reaction with the pyridyl analog as coenzyme was accompanied with a concomitant inactivation during catalysis, with a kcat/Kinact value 50-100 times lower than that for adenosylcobalamin or the 5,6-dimethylbenzimidazolyl analog. Therefore, it can be concluded that the 5,6-dimethylbenzimidazole moiety of adenosylcobalamin is important for continuous progress of a catalytic cycle by protecting the reactive intermediates from side reactions.

The preparation and characterization of the diaquo- forms of several incomplete corrinoids: cobyric acid, cobinamide, and three isomeric cobinic acid pentaamides.

The preparation of the diaquo- forms of the incomplete corrinoids, cobyric acid, cobinamide, and three isomeric cobinic acid pentaamides, from the corresponding aquocyanocorrinoids has been accomplished. The compounds were characterized by electronic absorption spectra, pka values, and HPLC/TLC behavior. Upon titration, the acids exhibited three pKa values, but only the pKa corresponding to the carboxylic acid involved the transfer of a full equivalent of protons. In addition, the progress of diaquo- to dihydroxo-titration, and the spectrum of the final products indicated a complex process. These diaquocorrinoids appear to be unstable, and should be stored in solution, frozen, under an inert atmosphere.

The synthesis of adenine-modified analogs of adenosylcobalamin and their coenzymic function in the reaction catalyzed by diol dehydrase.

Five analogs of adenosylcobalamin modified in the adenine moiety of the Co beta ligand were synthesized and tested for coenzymic function with diol dehydrase of Klebsiella pneumoniae ATCC 8724. 1-Deaza and 3-deaza analogs of adenosylcobalamin were active as coenzyme, whereas 7-deaza and N6,N6-dimethyl derivatives and guanosylcobalamin did not show detectable coenzymic activity. 7-Deaza and N6,N6-dimethyl analogs acted as strong competitive inhibitors with respect to adenosylcobalamin. The formation of cob(II)alamin as intermediate in the catalytic reaction was spectroscopically observed with catalytically active complexes of the enzyme with 1-deaza and 3-deaza analogs in the presence of 1,2-propanediol, but not with complexes with the inactive analogs. Oxygen sensitivity of the enzyme-analog complexes suggests that the carbon-cobalt bond of 1-deaza and 3-deaza analogs becomes activated by the enzyme even in the absence of substrate. These results indicate that the importance of the nitrogen atoms in the adenine moiety of the coenzyme for manifestation of catalytic function and for activation of the carbon-cobalt bond decreases in the following order: N-7 greater than 6-NH2 greater than N-3 greater than N-1. The dissociation constant for 5'-deoxyadenosine determined by equilibrium dialysis at 37 degrees C was about 23 microM.

[Deamination and reductive deamination of (2-amino-5, 6-dimethylbenzimidazolyl)cobamide. Preparation of (2-hydroxy-5, 6-dimethylbenzimidazolyl)cobamide and (5, 6-dimethyl-[2(-14)C]-benzimidazolyl)cobamide]. / Desaminierung und reduktive Desaminierung von (2-Amino-5, 6-dimethylbenzimidazolyl)cobamid. Darstellung von (2-Hydroxy-5 6-dimethylbenzimidazolyl)cobamid und (5, 6-Dimethyl-[2(-14)C] benzimidazolyl)cobamid.

(2-Amino-5, 6-dimethylbenzimidazolyl)-cobamide (III) is transformed to (2-hydroxy-5, 6-dimethylbenzimidazolyl) cobamide (IV) by nitrous acid. Exchange of the NH2-group by hydrogen with nitrous acid/hypophosphorous acid yields vitamin B12 (I). This reaction completes a cycle vitamin B12 (I)----[carboxy(2-cyanoamino-4,5-dimethylphenyl)amino]cobamide+ ++ (II)----(2-amino-5,6-dimethylbenzimidazolyl)cobamide (III)----vitamin B12 (I), which allows chemical 14C-labelling of vitamin B12. In this procedure cyanogen bromide, which is necessary for the first step, was labelled with [14C] cyanide. By the following reactions a vitamin B12 was formed in which C-2 of the 5, 6-dimethylbenzimidazole moiety is labelled.