Solution,Crystal and in Silico Structures of the Organometallic Vitamin B12-Derivative Acetylcobalamin and of its Novel Rhodium-Analogue Acetylrhodibalamin

The natural vitamin B₁₂-derivatives are intriguing complexes of cobalt that entrap the metal within the strikingly skewed and ring-contracted corrin ligand. Here, we describe the synthesis of the Rh(III)-corrin acetylrhodibalamin ( AcRhbl ) from biotechnologically produced metal-free hydrogenobyric acid and analyze the effect of the replacement of the cobalt-center of the organometallic vitamin B₁₂-derivative acetylcobalamin ( AcCbl ) with its group-IX homologue rhodium, to give AcRhbl . The structures of AcCbl and AcRhbl were thoroughly analyzed in aqueous solution, in crystals and by in silico methods, in order to gain detailed insights into the structural adaptations to the two homologous metals. Indeed, the common, nucleotide-appended corrin-ligand in these two metal corrins features extensive structural similarity. Thus, the rhodium-corrin AcRhbl joins the small group of B₁₂-mimics classified as ‘antivitamins B₁₂’, isostructural metal analogues of the natural cobalt-corrins that hold significant potential in biological and biomedical applications as selective inhibitors of key cellular processes.     

The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12 Cofactors for Catalysis

The B₁₂ cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt‐free B₁₂ corrin moiety, hydrogenobyric acid ( Hby ), a compound crafted through pathway redesign. Detailed insights from single‐crystal X‐ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized “entatic” states, thereby promoting the activation of B₁₂‐cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B₁₂.     

7‐Decarboxymethyl‐cobyrinates: Vitamin B12‐Derivatives that Lack the c‐Side Chain

The synthesis of cobyrinic acid derivatives by reduction of dehydrocobyrinates is largely unexplored. It is, however, a rational path to B₁₂ analogues that lack specific substituents of the corrin moiety of natural B₁₂ derivatives. The partial syntheses of four epimeric 7‐decarboxymethyl‐cobyrinates is described, which is achieved by reduction of Δ7‐dehydro‐7‐de[carboxymethyl]‐cobyrinate with zinc or with the ‘prebiotic’ reducing agent formic acid. A direct and remarkably efficient route was found to 7‐decarboxymethyl‐cobyrinates, which are cobyrinic acid derivatives in which the c‐side chain at ring B of vitamin B₁₂ is missing. The structures of the hexamethyl‐7‐decarboxymethyl‐cobyrinates were characterized and the stereochemical and conformational properties at their newly saturated ring B were analyzed. The stereochemical outcome of the reduction was found to depend strongly on the reaction conditions. In 7‐decarboxymethyl‐cobyrinates, both peripheral carbon centres of ring B carry a hydrogen atom, and the characteristic quaternary carbon centre at C7 of the cobyrinic acid moiety of vitamin B₁₂ is lacking. The still highly substituted 7‐decarboxymethyl‐cobyrinates are readily dehydrogenated in the presence of dioxygen, furnishing 7‐de[carboxymethyl]‐Δ⁷‐dehydro‐cobyrinate as the common, unsaturated oxidation product. The noted stability of vitamin B₁₂ and of other Co^III‐cobyrinates in the presence of air is a consequence of their highly substituted corrin macrocycle, a finding of interest in the context of chemical rationalizations of the B₁₂ structure.     

Fragmentation of vitamin B12 in aerosol matrix-assisted laser desorption ionization

Aerosol matrix-assisted laser desorption ionization (MALDI) with a reflection time-of-flight mass spectrometer was used to study fragmentation of vitamin B₁₂. Six MALDI matrices were used: 2,5-di-hydroxy benzoic acid (gentisic acid), 4-nitroaniline, 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapic acid), 3,4-di-hydroxy cinnamic acid (caffeic acid), trans-4-hydroxy-3-methoxy cinnamic acid (ferulic acid), and α-cyano-4-hydroxy cinnamic acid (4-HCCA). Mass spectra were obtained with a 355-nm pulsed Nd:YAG laser at irradiances between 0. 1 and 5 GW/cm² (between 3- and 150-mJ pulse energy). Loss of CN was a major product of prompt ion source fragmentation and the ratio of fragmented to intact analyte was found to be strongly dependent on matrix and weakly dependent on laser irradiance. Additionally, free cobalt ions and cobalt ions bound to small methanol clusters were observed in the mass spectra. The cobalt removal from the corrin ring of vitamin B₁₂ results from direct photon absorption by vitamin B₁₂, but is enhanced by the presence of matrix.     

Replacement of the Cobalt Center of Vitamin B12 by Nickel: Nibalamin and Nibyric Acid Prepared from Metal-Free B12 Ligands Hydrogenobalamin and Hydrogenobyric Acid

The (formal) replacement of Co in cobalamin ( Cbl ) by Ni^II generates nibalamin ( Nibl ), a new transition-metal analogue of vitamin B₁₂. Described here is Nibl , synthesized by incorporation of a Ni^II ion into the metal-free B₁₂ ligand hydrogenobalamin ( Hbl ), itself prepared from hydrogenobyric acid ( Hby ). The related Ni^II corrin nibyric acid ( Niby ) was similarly synthesized from Hby , the metal-free cobyric acid ligand. The solution structures of Hbl , and Niby and Nibl , were characterized by spectroscopic studies. Hbl features two inner protons bound at N2 and N4 of the corrin ligand, as discovered in Hby . X-ray analysis of Niby shows the structural adaptation of the corrin ligand to Ni^II ions and the coordination behavior of Ni^II. The diamagnetic Niby and Nibl , and corresponding isoelectronic Co^I corrins, were deduced to be isostructural. Nibl is a structural mimic of four-coordinate base-off Cbls , as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase.     

Homocoenzyme B12 and Bishomocoenzyme B12: Covalent Structural Mimics for Homolyzed,Enzyme‐Bound Coenzyme B12

Efficient electrochemical syntheses of “homocoenzyme B₁₂” ( 2 , Co_β‐(5′‐deoxy‐5′‐adenosyl‐methyl)‐cob(III )alamin) and “bishomocoenzyme B₁₂” ( 3 , Co_β‐[2‐(5′‐deoxy‐5′‐adenosyl)‐ethyl]‐cob(III )alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in‐depth investigations of the structures of 2 and 3 in solution were carried out and a high‐resolution crystal structure of 2 was obtained. The two homologues of coenzyme B₁₂ ( 2 and 3 ) are suggested to function as covalent structural mimics of the hypothetical enzyme‐bound “activated” (that is, “stretched” or even homolytically cleaved) states of the B₁₂ cofactor. From crude molecular models, the crucial distances from the corrin‐bound cobalt center to the C5′ atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 Å, respectively. These values are roughly the same as those found in the two “activated” forms of coenzyme B₁₂ in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2 , the cobalt center was observed to be at a distance of 2.99 Å from the C5′ atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B₁₂. The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.     

New developments in the field of vitamin B12: enzymatic reactions dependent upon corrins and coenzyme B12.

Simple corrins such as vitamin B₁₂ and vitamin B₁₂ coenzyme catalyze a variety of unusual enzymatic reactions of which some are still without analogy in organic or organometallic chemistry. The mechanisms of these reactions are currently the subject of lively discussion. The present review focuses attention on new ideas about the mode of action of vitamin B₁₂ coenzymes in enzymatic reactions.     

Vitamin B12: Experiments Concerning the Origin of Its Molecular Structure

Following the chemical synthesis of vitamin B₁₂, a search was begun for a potentially biomimetic “dark” variant of the photochemical A/D-secocorrin → corrin cycloisomerization, the central ring-closure step in one of the two cobyric acid syntheses. Significantly, not just one but a whole family of such variants was discovered. According to what is currently known, one of these variants can indeed be regarded as a chemical model for the reaction path followed by Nature in the biosynthetic construction of the corrin ring. These chemical studies of vitamin B₁₂ biosynthesis had revealed that the A/D-ring junction, regarded as the main obstacle to a chemical vitamin B₁₂ synthesis at the outset, is in fact a structural element that is formed readily and in a variety of ways from structurally appropriate precursors. More recent investigations have shown that the same holds for other specific structural elements of the vitamin B₁₂ molecule, including the characteristic arrangement of double bonds in the corrin chromophore, the special dimension of the macrocyclic ring of the corrin ligand, the specific attachment of the nucleotide loop to the propionic acid side chain of ring D, and the characteristic constitutional arrangement of the side chains around the ligand periphery (which vitamin B₁₂ shares with all uroporphinoid cofactors). All these outwardly complex structural elements are found to “self-assemble” with surprising ease under structurally appropriate preconditions; the amount of “external instruction” required for their formation turns out to be surprisingly small in view of the complexity and specificity of these structural elements. We view these findings as steps on the way toward a chemical rationalization of the vitamin B₁₂ structure. The goal is to arrive experimentally at a perception of the biomolecule's intrinsic potential for structural self-assembly. This potential, together with the specific type of reactivity related to the biological function, is considered to be responsible for the biomolecule having been chosen by natural selection. The chemical rationalization of the structure of biomolecules is an objective of organic natural product chemistry. The field of natural product synthesis provides appropriate conceptual and methodological tools to approach this objective experimentally.     

TiO2‐assisted Photocatalytic Degradation of Vitamin B12 in Aqueous Solution:Influencing Factors,Kinetics, and Reaction Investigations

The photodegradation (λ=365 nm) of the biomolecule vitamin B₁₂, catalyzed by the photocatalyst TiO₂ nanoparticles (NPs), has been investigated in aqueous suspension. The photodegradation process of vitamin B₁₂ has been monitored by means of electronic absorption (Abs), Fourier‐transform infrared (FT‐IR), and resonance Raman (RR) spectroscopies, respectively. The results show that only under UV illumination in the presence of TiO₂ is there effective degradation, and the photocatalytic degradation of vitamin B₁₂ is strongly influenced by the amount of TiO₂ NPs, the pH, and the initial concentration of vitamin B₁₂. The photocatalytic reaction kinetics of vitamin B₁₂ conforms to a Langmuir‐Hinshelwood isotherm model. Changes involving the three structural units of the carbon‐metal bond C–Co, the organic corrin macrocycle combined with the benzimidazole nucleotide, and the inorganic CN in the vitamin B₁₂ molecule during the photocatalytic degradation are also discussed.     

Zu den Kristallstrukturen der Übergangsmetall(II)‐Dodekahydro‐closo‐Dodekaborat‐Hydrate Cu(H2O)5,5[B12H12]·2,5 H2O und Zn(H2O)6[B12H12]·6 H2O

On the Crystal Structures of the Transition‐Metal(II) Dodecahydro‐closo‐Dodecaborate Hydrates Cu(H₂O)_5.5[B₁₂H₁₂]·2.5 H₂O and Zn(H₂O)₆[B₁₂H₁₂]·6 H₂O By neutralization of an aqueous solution of the free acid (H₃O)₂[B₁₂H₁₂] with basic copper(II) carbonate or zinc carbonate, blue lath‐shaped single crystals of the octahydrate Cu[B₁₂H₁₂]·8 H₂O (≡ Cu(H₂O)_5.5[B₁₂H₁₂]·2.5 H₂O) and colourless face‐rich single crystals of the dodecahydrate Zn[B₁₂H₁₂]·12 H₂O (≡ Zn(H₂O)₆[B₁₂H₁₂]·6 H₂O) could be isolated after isothermic evaporation. Copper(II) dodecahydro‐closo‐dodecaborate octahydrate crystallizes at room temperature in the monoclinic system with the non‐centrosymmetric space group Pm (Cu(H₂O)_5.5[B₁₂H₁₂]·2.5 H₂O: a = 768.23(5), b = 1434.48(9), c = 777.31(5) pm, β = 90.894(6)°; Z = 2), whereas zinc dodecahydro‐closo‐dodecaborate dodecahydrate crystallizes cubic in the likewise non‐centrosymmetric space group F23 (Zn(H₂O)₆[B₁₂H₁₂]·6 H₂O: a = 1637.43(9) pm; Z = 8). The crystal structure of Cu(H₂O)_5.5[B₁₂H₁₂]·2.5 H₂O can be described as a monoclinic distortion variant of the CsCl‐type arrangement. As characteristic feature the formation of isolated [Cu₂(H₂O)₁₁]⁴⁺ units as a condensate of two corner‐linked Jahn‐Teller distorted [Cu(H₂O)₆]²⁺ octahedra via an oxygen atom of crystal water can be considered. Since “zeolitic” water of hydratation is also present, obviously both classical H–O^δ?···^+δH–O and non‐classical B–H^δ?···^+δH–O hydrogen bonds play a significant role for the stabilization of the structure. A direct coordinative influence of the quasi‐icosahedral [B₁₂H₁₂]^2? anions on the Cu²⁺ cations has not been determined. The zinc compound Zn(H₂O)₆[B₁₂H₁₂]·6 H₂O crystallizes in a NaTl‐type related structure. Two crystallographically different [Zn(H₂O)₆]²⁺ octahedra are present, which only differ in their relative orientation within the packing of the [B₁₂H₁₂]^2? anions. The stabilization of the crystal structure takes place mainly via H–O^δ?···^+δH–O hydrogen bonds, since again the hydrogen atoms of the [B₁₂H₁₂]^2? anions have no direct coordinative influence on the Zn²⁺ cations.     

PHOTOOXYGENOLYSIS OF VITAMIN B12 and RELATED CORRINS: COBALT(III)CORRINS AS SUBSTRATES and QUENCHERS FOR SINGLET OXYGEN (1Δg)*

Under photooxygenolytic conditions, vitamin B₁₂ can be degraded to two isomeric dioxosec-ocorrins by a regioselective oxygenolytic cleavage of the corrin macrocycle (preliminary report in Krautler and Stepanek (1985), Angew. Chem. 97 ,71–73; Angew. Chem. Intl. Ed. Engl. 24 ,62–64): irradiation of an oxygen saturated solution of vitamin B₁₂ ( 1 ), of KCN and of methylene blue (molar ratio (1:1:0.005) in CD₃OD at ca 200 K with visible light led to a selective oxygenolysis of the vitamin. The two products, potassium Co_αCo_β-dicyano-5,6-dioxo-5,6-seco-5′6′-dimethylbenzimidazolyl-cobam-idate ( 3 , 10% yield) and potassium Co_αCo_β-dicyano-14,15-dioxo-14,15-seco-5′6′-dimetliylbenzirnida-zolyl-cobamidate (4, 25% yield), and 1 (31% yield) were chromatographically separated and isolated. The structures were established spectroanalytically, and by the help of an acid catalyzed methanolysis of 3 and 4 to the related Co_αCo_β -dicyano-heptamethyl- dioxosecocobyrinates (6 and 7). When irradiated with visible light in any oxygen saturated methanolic solution containing methylene blue, vitamin B₁₂ itself exhibited considerable inertness. Only upon addition of a stoichiometric amount of KCN to such a solution (to afford the adduct 2) did the photolysis lead to oxygenation of the cobaltcorrin within a useful time. The regioselectivity of the oxygenolysis of 2 and the ratio of products formed are comparable to the outcome of the photooxygenolysis of the structurally related heptamethylcobyrinate 5 (Kräutler (1982) Helv. Chim. Acta 65 ,1941–1948). The reaction at the methyl-substituted meso-positions of the corrin macrocycle is indicated to involve singlet oxygen (¹Δ_g). In support of this interpretation, the preparative photolysis of 2 at 200 K also turned out to be about 7 times slower when CH₃OH was used instead of CD₃OD.     

Ultrasensitive Determination of Vitamin B12 Using Disposable Graphite Screen‐Printed Electrodes and Anodic Adsorptive Voltammetry

A facile, rapid and ultra‐sensitive method for the determination of vitamin B₁₂ (cyanocobalamin) at the sub‐nanomolar concentration range by using low‐cost, disposable graphite screen‐printed electrodes is described. The method is based on the cathodic preconcentration of square planar vitamin B_12s, as occurred due to the electro reduction of Co(III) center in vitamin B_12a to Co(I), at ?1.3 V versus Ag/AgCl/3 M KCl for 40 s. Then, an anodic square wave scan was applied and the height of the peak appeared at ca. ?0.73 V versus Ag/AgCl/3 M KCl, due to the oxidation of Co(I) to Co(II) in the adsorbed molecule, was related to the concentration of the vitamin B₁₂ in the sample. EDTA was found to serve as a key‐component of the electrolyte by eliminating the background signal caused by metal cations impurities contained in the electrolyte (0.1 M phosphate buffer in 0.1 M KCl, pH 3). It also blocks trace metals contained in real samples, thus eliminating their interference effect. The method was optimized to various working parameters and under the selected conditions the calibration curve was linear over the range 1×10^?10–8×10^?9 mol L^?1 vitamin B₁₂ (R²=0.994), while the limit of detection for a signal‐to‐noise ratio of 3 (7×10^?11 mol L^?1 vitamin B₁₂) is the lowest value of any reported in the literature for the electrochemical determination of vitamin B₁₂. The sensors were successfully applied to the determination of vitamin B₁₂ in pharmaceutical products.     

Dodecahydro‐closo‐undecaborate [B11H12]–

DFT‐calculations of the geometries of the closo‐anion [B₁₁H₁₁]^2– in its ground state and in the transition state of its skeletal rearrangement and of the protonated species [B₁₁H₁₂]^– in its ground state were performed at the B3LYP/6‐31++G(d,p) level. The corresponding NMR shifts were computed on the basis of the optimized geometry by the GIAO method at the same level. Calculated and observed NMR data are in good agreement and thus prove the structure of [B₁₁H₁₂]^–, previously deduced from 2 D‐NMR spectra. The addition of water, ethanol, and pyridine to [B₁₁H₁₂]^– at low temperature gave the nido‐species [B₁₁H₁₃(OH)]^–, [B₁₁H₁₃(OEt)]^–, and [B₁₁H₁₂(py)]^–, respectively. The structures of these anions were investigated by NMR methods and the last two of them by crystal structure analyses of appropriate salts. The course of the addition reactions can be rationalized on the basis of the structurally characterized reaction components.     

Quantum-chemical simulation of the active center of vitamin B12

The electronic structure of porphin and corrin complexes of cobalt differing with respect to the oxidation state of the central ion has been investigated by the MO-LCAO-SCF-CNDO method with the Kai-Nishimoto parameters for transition metals. On the basis of an analysis of the distribution of the electron density and the structure of the energy spectrum, it has been shown that the oxidation-reduction processes of the complexes are accompanied by restructuring of the energy spectrum, and the differences between the electronic structures of porphin and corrin complexes have been discussed. It has been established that cobalt(I) porphin has stronger nucleophilic properties than does cobalt(I) corrin. The electronic structure of hexacoordinate complexes in which an imidazole molecule and a molecule of L (L = H₂O, CH₃ ⁺, CN^–) are axially coordinated has been calculated. The mechanisms of the dissociation of cobalt alkyl complexes and the differences between the processes of the heterolytic dissociation of porphin and corrin complexes have been discussed. It has been shown that the elimination of a CH₃ ⁺ cation, which plays an important role in biomethylation reactions, is more favorable in corrin complexes.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 22, No. 4, pp. 400–409. July–August, 1986.     

Synthesis and Irradiation of Vitamin‐B12‐Derived Complexes Incorporating Peripheral G⋅C Base Pairs

The vitamin‐B₁₂ derivative 11 , incorporating a peripheral N⁴‐acetylcytosine moiety, was alkylated under reductive conditions with 2‐(iodomethyl)‐2‐methylmonothiomalonate 8 bearing the complementary guanine moiety. The reaction yielded a mixture of vitamin‐B₁₂‐derived complexes with variations in the cytosine moiety: products 16 – 18 with a cytosine, a N⁴‐acetylated cytosine, and a N⁴‐acetylated reduced cytosine moiety were formed (see Scheme 5). The complexes were photolyzed in CHCl₃/MeCN to yield the dimethylmalonate derivative 22 (Scheme 6) but not the rearranged succinate, in contrast to the results obtained earlier with complexes incorporating the A⋅T base pair (see Scheme 1).     

Determination of vitamin B<Subscript>12</Subscript> using a chemiluminescence flow system

A method to determine vitamin B₁₂ by measuring the chemiluminescence (CL) intensities using a flow injection (FI) system has been proposed. It is based on the catalytic effect of cobalt(II) in vitamin B₁₂ on the CL reaction between luminol and hydrogen peroxide in a basic medium. The increment of the CL intensity is proportional to the concentration of vitamin B₁₂ in the range 8.68–86.9 ng/mL (r ² = 0.9984) with a detection limit (3σ) of 0.89 ng/mL. The CL response is obtained in 10 s at a flow rate of 3.0 mL/min with a relative standard deviation (RSD) of less than 2.5% (n = 6). The method has been successfully applied to the determination of vitamin B₁₂ in pharmaceutical injections. The text was submitted by the authors in English.     

Tetrahedron report : Concerning the biosynthesis of vitamin B12

The use of ¹³C-Fourier transform nuclear magnetic resonance (FT-NMR) has led to the observation that while 8 molecules of [2-¹³C]-ALA are incorporated into vitamin B₁₂ in P. shermanii, [5-¹³C]-ALA labels only seven of the carbon atoms of cyanocobalamin; i.e. one of the amino methyl groups of ALA is “lost” in the process. It has also been confirmed that seven of the methyl groups of vitamin B₁₂ are derived from [¹³CH₃]-enriched methionine and further that the chirality of the gem-dimethyl grouping at C₁₂ labeled with [¹³CH₃]-methionine is R. A soluble enzyme mixture from the 37,000 g or 100,000 g supernatant of disrupted cells of P. shermanii converts both ¹⁴C-labeled ALA and ¹⁴C-uro'gen III to cobyrinic acid, the simplest corrinoid material on the pathway to vitamin B₁₂ and the coenzyme, in presence of NADPH, Co²⁺, Mg²⁺, S-adenosylmethionine and glutathione. Multiply-labeled uro'gens (¹³C, ¹⁴C and ³H) have been used to show that incorporation takes place without randomization. A sequence for corrin synthesis from uro'gen III, involving as the first step decarboxylation of the ring-C acetic acid side chain, is proposed.     

Chemiluminescent Determination of Vitamin B12 Using Charge Coupled Device (CCD)

A method was developed for the determination of vitamin B₁₂ based on the enhancement of cobalt (II) on the chemiluminescence (CL) reaction between luminol and percarbonate (powerful source of hydrogen peroxide). The release of cobalt (II) from the vitamin B₁₂ was reached by a simple and fast microwave digestion (20 s microwave digestion time and a mix of nitric acid and hydrogen peroxide). A charge coupled device (CCD) photodetector, directly connected to the cell, coupled with a simple continuous flow system was used to obtain the full spectral characteristics of cobalt (II) catalyzed luminol-percarbonate reaction.

The optima experimental conditions were established: 8.0 m mol L^?1 luminol in a 0.075 mol L^?1 carbonate buffer (pH 10.0) and 0.15 mol L^?1 sodium percarbonate, in addition to others experimental parameters as 0.33 mL s^?1 flow rate and 2 s integration time, were the experimental conditions which proportionate the optimum CL emission intensity. The emission data were best fitted with a second-order calibration graph over the cobalt (II) concentration range from 4.00 to 300 µ g L^?1 (r² = 0.9990), with a detection limit of 0.42 µ g L^?1. The proposed method was successfully applied to the determination of vitamin B₁₂ in pharmaceuticals.     

Pulse Radiolysis and Ultra‐High‐Performance Liquid Chromatography/High‐Resolution Mass Spectrometry Studies on the Reactions of the Carbonate Radical with Vitamin B12 Derivatives

The reactions of the carbonate radical anion (CO₃ ^{. ?}) with vitamin B₁₂ derivatives were studied by pulse radiolysis. The carbonate radical anion directly oxidizes the metal center of cob(II)alamin quantitively to give hydroxycobalamin, with a bimolecular rate constant of 2.0×10⁹ M ^?1 s^?1. The reaction of CO₃ ^{. ?} with hydroxycobalamin proceeds in two steps. The second‐order rate constant for the first reaction is 4.3×10⁸ M ^?1 s^?1. The rate of the second reaction is independent of the hydroxycobalamin concentration and is approximately 3.0×10³ s^?1. Evidence for formation of corrinoid complexes differing from cobalamin by the abstraction of two or four hydrogen atoms from the corrin macrocycle and lactone ring formation has been obtained by ultra‐high‐performance liquid chromatography/high‐resolution mass spectrometry (UHPLC/HRMS). A mechanism is proposed in which abstraction of a hydrogen atom by CO₃ ^{. ?} from a carbon atom not involved in the π conjugation system of the corrin occurs in the first step, resulting in formation of a Co^III C‐centered radical that undergoes rapid intramolecular electron transfer to form the corresponding Co^II carbocation complex for about 50 % of these complexes. Subsequent competing pathways lead to formation of corrinoid complexes with two fewer hydrogen atoms and lactone derivatives of B₁₂. Our results demonstrate the potential of UHPLC combined with HRMS in the separation and identification of tetrapyrrole macrocycles with minor modifications from their parent molecule.     

Experimental Study of Hydrogen Bonding Potentially Stabilizing the 5′‐Deoxyadenosyl Radical from Coenzyme B12

Coenzyme B₁₂ can assist radical enzymes that accomplish the vicinal interchange of a hydrogen atom with a functional group. It has been proposed that the Co? C bond homolysis of coenzyme B₁₂ to cob(II)alamin and the 5′‐deoxyadenosyl radical is aided by hydrogen bonding of the corrin C19? H to the 3′‐O of the ribose moiety of the incipient 5′‐deoxyadenosyl radical, which is stabilized by 30 kJ mol^?1 (B. Durbeej et al., Chem. Eur. J. 2009 , 15, 8578–8585). The diastereoisomers (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin were used as models for coenzyme B₁₂. A downfield shift of the NMR signal for the C19? H proton was observed for the (R)‐isomer (δ=4.45 versus 4.01 ppm for the (S)‐isomer) and can be ascribed to an intramolecular hydrogen bond between the C19? H and the oxygen of CHOH. Crystal structures of (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin showed C19? H???O distances of 3.214(7) Å (R‐isomer) and 3.281(11) Å (S‐isomer), which suggest weak hydrogen‐bond interactions (?ΔG<6 kJ mol^?1) between the CHOH of the dihydroxypropyl ligand and the C19? H. Exchange of the C19? H, which is dependent on the cobalt redox state, was investigated with cob(I)alamin, cob(II)alamin, and cob(III)alamin by using NMR spectroscopy to monitor the uptake of deuterium from deuterated water in the pH range 3–11. No exchange was found for any of the cobalt oxidation states. 3′,5′‐Dideoxyadenosylcobalamin, but not the 2′,5′‐isomer, was found to act as a coenzyme for glutamate mutase, with a 15‐fold lower k_cat/K_M than 5′‐deoxyadenosylcobalamin. This indicates that stabilization of the 5′‐deoxyadenosyl radical by a hydrogen bond that involves the C19? H and the 3′‐OH group of the cofactor is, at most, 7 kJ mol^?1 (?ΔG). Examination of the crystal structure of glutamate mutase revealed additional stabilizing factors: hydrogen bonds between both the 2′‐OH and 3′‐OH groups and glutamate 330. The actual strength of a hydrogen bond between the C19? H and the 3′‐O of the ribose moiety of the 5′‐deoxyadenosyl group is concluded not to exceed 6 kJ mol^?1 (?ΔG).