Kenneth L. Brown

A molecular mechanics force field for the cobalt corrinoids

Abstract A force field for the cobalt (III) corrinoids (derivatives of vitamin B 12 ) for use with a modified version of the molecular mechanics program mm 2(87) has been developed empirically around 19 cobalt corrinoid crystal structures. Bond lengths, bond angles and torsional angles are reproduced with r.m.s. differences of 0.01 A, 2.4 °, and 4.2 °, respectively, within the standard deviation of the mean of these parameters found in the solid state. The axial ligand occupying the lower coordination site in the cobalamins, 5,6-dimethylbenzimidazole, is shown to have very limited rotational freedom and is constrained by the downward-pointing b and d propionamide side chains of the corrin ring. Strain-energy profiles for rotation of the side chains of the corrin ring show the existence of several local energy minima and this explains the observed variability in the orientations of these side chains in the solid state. The known change in conformation which occurs in the C ring when the e side chain is epimerized from the lower to the upper face of the corrin ring in cyano-13- epico balamin is correctly predicted, provided the starting conformation of the C ring is unbiased. A study of cyano-8- epico balamin indicates that an analogous conformational change does not occur in the B ring and the epimerized d side chain assumes an equatorial orientation relative to the corrin ring. Parameters for the Coue5f8C bond in alkylcobalamins were developed and the structure of methyl- and adenosylcobalamin are accurately reproduced. An examination of the strain energy consequences of rotation of the adenosyl ligand about the Coue5f8C bond identifies a number of low-energy conformations at least two of which, in which adenosyl lies over the “southern” and “eastern” portions of the corrin ring, respectively, have been previously deduced from NMR observations. Coordinated neopentyl in neopentylcobalamin is much more hindered to rotation about the Coue5f8C bond and the lowest conformation finds two γ(C) atoms straddling the upwardly projecting C46 methyl group of the corrin.

CONFORMATIONAL FLEXIBILITY IN COENZYME B12 : A NUCLEAR MAGNETIC RESONANCE AND MOLECULAR MECHANICS STUDY

Abstract The conformation of 5′-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) with respect to rotation of the Ado ligand about the Co C bond has been used studied by rotating-frame Overhauser enhancement NMR spectroscopy. Previous molecular-mechanics calculations suggest that in addition to the conformation seen in the solid state, in which the Ado ligand lies over the “southern” quadrant of the corrin ring, relatively low-energy conformations exist in which the Ado lies over the “eastern”, “northern” and “western” quadrants as well. At 27°C, 12 NOE cross peaks are observed between Ado protons and protons elsewhere on the molecule, all of which are consistent with the calculated “southern” or “eastern” conformations, or both. However, at 60°C, several of the Ado protons undergo significant chemical shift changes and of the 27 observed NOE cross peaks, eight are compatible only with the “northern” or “western” conformations or both. The effect of the apparent population of these additional conformations at elevated temperatures on the activation parameters for thermal carbon-cobalt bond homolysis is discussed in the light of molecular-mechanics calculations on the influence of Co C bond geometry and acetamide side-chain rotational motions on the energetivs of this reaction.

The inhibition of corrinoid-catalyzed oxidation of mercaptoethanol by methyl iodide: Mechanistic implications

The cobalamin coenzymes (5-deoxyadenosyl- and methylcobalamin) and their cobinamide counterparts (5-deoxyadenosyl- and methylcobinamide) catalyze the oxidation of 2-mercaptoethanol to its disulfide with hydrogen peroxide formation under aerobic conditions. The reactions are blocked by methyl iodide. Inhibition by methyl iodide is apparently due to the formation of the trans dialkyl corrinoids: methyl(adenosyl)cobalamin, dimethylcobalamin, methyl(adenosyl)cobinamide, and dimethylcobinamide, respectively. When the reaction system is illuminated with visible light, inhibition is released and a dramatic enhancement in the rate of oxygen consumption occurs. For reactions catalyzed by adenosyl- and methylcobalamin and then inhibited by methyl iodide, the rates observed during photolysis approach those obtained with aquacobalamin. For reactions catalyzed by adenosyl- and methylcobinamide and then inhibited by methyl iodide, the rates observed during photlysis approach those obtained with diaquacobinamide. Thus, both trans axial carbon-cobalt bonds in the putative dialkyl corrinoid are homolyzed during photolysis. In contrast to these results, the catalysis of the aerobic oxidation of 2-mercaptoethanol by aquacobalamin is only weakly inhibited by methyl iodide. This observation suggests that aquacob(II)alamin is produced during the catalysis of this reaction. Superoxide, the anticipated product of the reaction between aquacob(II)alamin and dioxygen, is formed during aquacobalamin-catalyzed 2-mercaptoethanol oxidation since superoxide dismutase decreases the rate of oxygen consumption by 50%. However, the enzyme has no effect on oxygen uptake during reactions catalyzed by cobalamin coenzymes and their cobinamide counterparts. These corrinoid catalysts apparently transfer two electrons to dioxygen from cobalt(I) intermediates formed during the reactions. Nitrogenous bases inhibit corrinoid-catalyzed thiol oxidation by competing with 2-mercaptoethanol for axial-ligand coordination sites on the catalyst. In contrast to the inhibition observed with methyl iodide, visible light has no effect on the inhibition obtained with nitrogenous bases.

Epimerization of cyanocobalamin. Improved synthesis of cyano-8-epicobalamin and its characterization by x-ray crystallography, and 1H, 13C, and 15N NMR

Abstract The structure of cyano-8-epicobalamin (CN-8-epiCbl), the C8 epimer of vitamin B12 (cyanocobalamin, CNCbl) has been determined by X-ray crystallography, complete assignment of its 1H and 13C NMR spectra, as well as observation and assignment of its amide 1H and 15N NMR spectra in DMSO-d6. This compound was previously reported as the poorly characterized product of the amidation of its c-monocarboxylate, obtained as a minor product of the borohydride reduction of cobalamin-c-lactone. The conditions for the key epimerization step have now been changed to provide a tripling of the yield of the 8-epi-c-monocarboxylate. Along with substantial improvement in the procedures for synthesizing cobalamin-c-lactone and for amidating the monocarboxylate, this modified method for epimerization of cyanocobalamin now provides CN-8-epiCbl in sufficient quantities for thorough characterization and studies of its properties as a vitamin B12 analog. The crystal structure of CN-8-epiCbl has been determined. The main difference between the crystal structure of CNCbl and CN-8-epiCbl is that the d side chain (at C8) is “down-wardly” axial in CNCbl, but pseudo-equatorial in CN-8-epiCbl. In addition, the B pyrrole ring in CN-8-epiCbl is twisted by about 5°, the axial benzimidazole moiety is tilled about 7° toward the A and D rings, and the corrin macrocycle fold angle along the Co … C10 axis (23.8°) is more severe than that in CNCbl (17.7°). Fourteen water molecules are well located in the crystal structure and four others occur at half-occupancy. The epimeric relationship between CN-8-epiCbl and CNCbl and the configuration at C8 are also evident in observed 1H NMR nuclear Overhauser enhancements and differences in amide 1H and 15N chemical shifts at the d amide between CNCbl and CN-8-epiCbl. Measurements of the amide proton chemical shift thermal gradients strongly suggest the presence of a hydrogen bond in CN-8-epiCbl involving the c amide syn proton and the d amide carbonyl in solution. This intramolecular hydrogen bonding is not present in the crystal structure in which the d amide carbonyl is hydrogen bonded to two water molecules and the c amide nitrogen is hydrogen bonded to the b amide carbonyl oxygen of a symmetry related molecule. Comparison of the 13C spectra of CNCbl and CN-8-epiCbl shows that significant chemical shift changes are not restricted to the B ring (the site of epimerization) but are more global in nature, reflecting the changes in corrin ring conformation.

Placing hydroxide in the thermodynamic trans influence order of the cobalt corrinoids: equilibrium constants for the reaction of some ligands with aquahydroxocobinamide

Abstract Diaquacobinamide (DAC) was prepared by reducing aquacyanocobinamide (Factor B) with zinc and oxidising in aqueous HCl. The macroscopic acid dissociation constants p K Co1 and p K Co2 for coordinated H 2 O in DAC were found spectrophotometrically to be 5.91 ± 0.04 and 10.30 ±0.24 at 25 °C; from their temperature dependence it was found that Δ H =45±4 and 27±2 kJ mol −1 , and Δ S =40±14 and −109±8 J K −1 mol −1 , for p K Co1 and p K Co2 , respectively. Equilibrium constants for the reaction of the cobinamide with cyanide were determined in the pH range 8–12 at 25 °C, μ=1.0 M (NaClO 4 ). Two CN − ligands bind to aquahydroxocobinamide and pH dependence of the observed equilibrium constants can be accounted for by assuming that both dihydroxocobinamide and hydroxocyanocobinamide are inert, while equilibrium constants for reaction of the labile species aquahydroxocobinamide and aquacyanocobinamide with HCN are insignificantly small compared to those for reaction with CN − anion. A fit of the data shows that the macroscopic equilibrium constant, log β 2 , for reaction of 2CN − with the two diastereomers of aquahydroxocobinamide to produce dicyanocobinamide is 19.0±0.1. Hence the macroscopic log K for binding of CN − to the two diastereomers of aquahydroxocobinamide is 11 since log K for binding of CN − to aquacyanocobinamide is 8. Equilibrium constants for binding of azide, pyridine, N - methylimidazole and 3-amino-l-propanol to aquahydroxocobinamide were determined at 25 °C, μ=1.0 M (NaClO 4 ), pH 12. In these cases only a single ligand is bound; the macroscopic log K values are N -methylimidazole, 6.06±0.04; 3-amino-l-propanol, 4.66±0.04; pyridine, 4.19±0.01; and azide, 3.45±0.04. A comparison of the present results with others available shows that hydroxide is above water and 5, 6-dimethylbenzimidazole, but below cyanide, sulfite and alkyl ligands in the trans influence order of the cobalt corrinoids.

Substituent effect analysis in organocobalt corrinoids: binding constants for cyanide, azide and the pendent 5,6-dimethylbenzimidazole nucleotide

Binding constants for cyanide ion to eight alkylcobinamides (including four β-alkylcobinamides in which the organic ligand is in the ‘upper’ axial ligand position and four α-alkylcobinamides in which the organic ligand is in the ‘lower’ axial ligand position) have been determined. Unlike all other alkylcobinamides investigated, which undergo rapid cyanolysis to form dicyanocobinamide in the dark, these eight complexes were sufficiently stable towards cleavage by cyanide to permit accurate measurement of the binding constants. The values obtained vary by 6.4×104-fold from 4.33 M−1 for β-ethylcobinamide to 2.79×105 M−1 for β-cyanomethylcobinamide. In contrast, these alkylcobinamides are stable in the presence of excess azide ion, but bind this ligand much more weakly, the binding constants varying from <1 M−1 for β-ethylcobinamide to 15.8 M−1 for β-cyanomethylcobinamide. A multiparameter substituent effect equation, including inductive, resonance and steric terms, has been successfully used to correlate the intramolecular equilibrium constants for the coordination of the pendent 5,6-dimethylbenzimidazole nucleotide to the metal atom in a series of ten alkylcobalamins. The significance of this correlation with respect to similar correlations recently obtained for alkylcobaloximes is discussed.

Acid—base chemistry of α-alkylcobalamins. Determination of additional formation constants for the ‘tuck-in’ species of base-off cobalamins

Abstract Potentiometric titration of five α-alkylcobalamins, in which the axial organic ligand is in the ‘lower’ (α) axial position preventing coordination of the axial nucleotide, has provided the first values of the p K a of the conjugate acid of the pendent, but uncoordinated, benzimidazole nucleotide in cobalamins. In every case, these values are lower than the p K a of the conjugate acid-of the free nucleoside, α-ribazole, at the same temperature. This suggests that, as is the case with base-off dicyanocobalamin and base-off but benzimidazole deprotonated β- alkylcobalamins, the free base nucleotide in the α-alkylcobalamins is associated with a corrin ring side chain to form what is known as the ‘tuck-in’ species. The data permit calculation of apparent equilibrium constants for formation of the ‘tuck-in’ species of the α-alkylcobalamins which vary from about 0.3 to 1.3, but are essentially temperature independent for a given complex. The isoenthalpic nature of this equilibrium is consistent with the principle interaction in the ‘tuck-in’ species being a hydrogen bond between the nucleotide B3 nitrogen and a side chain amide N-H, as is the case for dicyanocobalamin and the base-off β-alkylcobalamins. Further evidence for formation of ‘tuck-in’ species in α-alkylcobalamins has been obtained from comparison of the 13 C NMR resonances of the pendent nucleotide of these complexes with those of the free nucleotide, α-ribazole-3′-phosphate, and with those of dicyanocobalamin, in which the ‘tuck-in’ species is well characterized. These spectral comparisons suggest that formation of the ‘tuck-in’ species may be accompanied by a change in conformation about the nucleotide N -glycosidic bond, and that the conformation of the nucleotide ribose moiety in the ‘tuck-in’ species of the α-alkylcobalamins may be different from that in dicyanocobalamin.

Heteronuclear NMR studies of cobalt corrinoids—17. characterization of neopentylcobinamide and neopentyl-13-epicobinamide by 1H and 13C NMR spectroscopy: Inferred corrin ring conformations from chemical shift differentials☆☆☆

Abstract While the 1H and 13C NMR spectra of neopentylcobalamin (NpCbl) and its epimer at corrin ring C(13), neopentyl-13-epiCbl (Np-13-epiCbl), are extremely broad, apparently due to chemical exchange between the base-on and base-off species, the cobinamide derivatives, neopentyl cobinamide (NpCbi+) and neopentyl-13-epicobinamide (Np-13-epiCbi+), have sharp, well-resolved NMR spectra. The 1H and 13C NMR spectra of NpCbi+ and Np-13-epiCbi+ have now been completely assigned by modern two-dimensional NMR methodologies. Comparison of the 13C spectra of these two complexes shows that significant chemical shift differences occur at a variety of corrin ring and peripheral carbon atoms and are not localized near the site of epimerization. Similarly, comparison of the 13C NMR spectra of NpCbi+ and 5′-deoxyadenosylcobinamide (AdoCbi+) shows differences at many corrin ring and peripheral carbons. A first attempt at discerning differences in corrin ring conformation from such differences in 13C chemical shift has been made by comparing the X-ray crystal structures and 13C NMR spectra of 5′-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) and cyanocobalamin (vitamin B12, CNCbl). After elimination of carbon atoms whose chemical shifts are likely to be significantly affected by differences in the inductive effect of the Ado and CN ligands, and after consideration of differential anisotropic shielding effects in the two complexes due to the presence or absence of the Ado ligand, the difference in magnetic anisotropy of the central cobalt atom, the change in position of the axial nucleotide and differences in the magnetic anisotropy of the corrin ligand, 15 peripheral carbon atoms [C(2), C(18), C(20), C(25), C(26), C(30), C(36), C(37), C(41), C(46), C(47), C(48), C(54), C(55), C(60)] emerge as candidate “reporter” carbons whose 13C chemical shifts may be useful in deducing conformational differences in cobalt corrinoids. Application of this method to adeninylpropylcobalamin (AdePrCbl), for which the X-ray crystal structure and absolute NMR assignments are known, correctly predicts the gross conformational differences between the corrin ring of AdePrCbl and that of CNCbl. Use of these reporter carbon chemical shifts suggests that in NpCbi+, the fold angle, defined as the angle between the “northern” and “southern” planes of the corrin ring, is reduced relative to AdoCbi+. Comparison of the chemical shifts of the reporter carbon atoms in NpCbi+ and Np-13-epiCbi+ suggests that the fold angle in the former is larger than that for the latter.