New Developments in the Field of Vitamin B12: Reactions of the Cobalt Atom in Corrins and in Vitamin B12 Model Compounds

Studies with vitamin B₁₂ model compounds such as the cobaloximes provide a basis for the understanding of the mode of action of corrinoid coenzymes in enzymatic reactions. They also widen our knowledge of the properties and reactions of organocobalt compounds. The present article outlines the most important nonenzymatic reactions of cobalt in vitamin B₁₂ and in model compounds of the cobaloxime type.     

Radiometric microbiological estimation of vitamin B12

Estimation of vitamin B₁₂ in blood is very important to determine in decifiency and diagnosis of anemic patients. Vitamin B₁₂ in blood can be estimated by spectrochemical, enzymatic, radioisotopic and microbiological methods. In the present study vitamin B₁₂ was determined in 48 normal subjects of Rawalpindi/Islamabad by radiometric microbiological assay (RMA) technique using a very rapid, sensitive and automated instrument Bactec 460. In this procedure¹⁴C-glucose media and microorganismsLactobacillus leichmannii were used. The sensitivity of the method for vitamin B₁₂ is 1 pg/ml and the vitamin B₁₂ found in normal subjects was in the range of 105–535 pg/ml with a median value of 246±6 pg/ml.     

Determination of Vitamin B12 in Multivitamin Tablets by High Performance Liquid Chromatography

Abstract

Two procedures for separation and determination of vitamin B₁₂ in multivitamin tablets by reversed phase high performance liquid chromatography are proposed. Sample preparation is very simple: tablets are dissolved in distilled water, centrifuged and filtered. The sample solution is directly applied in the sample loop injector and chromatograms are obtained with gradient elution using water-methanol and water-acetonitrile as solvents. The peak of vitamin B₁₂ from samples of B-complex tablets is well separated with the two procedures. For multivitamin tablets, however, only the procedure with water and methanol as solvents was good for separation and quantification of vitamin B₁₂. Both procedures were verified by the standard addition method and also compared to a previously developed method using electrothermal atomic absorption spectrometry for vitamin B₁₂ determination.     

Antivitamins B12—A Structure‐ and Reactivity‐Based Concept

B₁₂‐antimetabolites are compounds that counteract the physiological effects of vitamin B₁₂ and related natural cobalamins. Presented here is a structure‐ and reactivity‐based concept of the specific ′antivitamins B₁₂′: it refers to analogues of vitamin B₁₂ that display high structural similarity to the vitamin and are ′locked chemically′ to prevent their metabolic conversion into the crucial organometallic B₁₂‐cofactors. Application of antivitamins B₁₂ to healthy laboratory animals is, thus, expected to induce symptoms of B₁₂‐deficiency. Antivitamins B₁₂ may, hence, be helpful in elucidating still largely puzzling pathophysiological phenomena associated with B₁₂‐deficiency, and also in recognizing physiological roles of B₁₂ that probably still remain to be discovered.     

Mild olefin formation via bio-inspired vitamin B12 photocatalysis

Dehydrohalogenation, or elimination of hydrogen-halide equivalents, remains one of the simplest methods for the installation of the biologically-important olefin functionality. However, this transformation often requires harsh, strongly-basic conditions, rare noble metals, or both, limiting its applicability in the synthesis of complex molecules. Nature has pursued a complementary approach in the novel vitamin B₁₂-dependent photoreceptor CarH, where photolysis of a cobalt–carbon bond leads to selective olefin formation under mild, physiologically-relevant conditions. Herein we report a light-driven B₁₂-based catalytic system that leverages this reactivity to convert alkyl electrophiles to olefins under incredibly mild conditions using only earth abundant elements. Further, this process exhibits a high level of regioselectivity, producing terminal olefins in moderate to excellent yield and exceptional selectivity. Finally, we are able to access a hitherto-unknown transformation, remote elimination, using two cobalt catalysts in tandem to produce subterminal olefins with excellent regioselectivity. Together, we show vitamin B₁₂ to be a powerful platform for developing mild olefin-forming reactions.

Terminal or subterminal olefins can be selectively formed from alkyl electrophiles via bio-inspired vitamin B₁₂ photocatalysis.     

Antivitamins B12—Some Inaugural Milestones

The recently delineated structure- and reactivity-based concept of antivitamins B₁₂ has begun to bear fruit by the generation, and study, of a range of such B₁₂-dummies, either vitamin B₁₂-derived, or transition metal analogues that also represent potential antivitamins B₁₂ or specific B₁₂-antimetabolites. As reviewed here, this has opened up new research avenues in organometallic B₁₂-chemistry and bioinorganic coordination chemistry. Exploratory studies with antivitamins B₁₂ have, furthermore, revealed some of their potential, as pharmacologically interesting compounds, for inducing B₁₂-deficiency in a range of organisms, from hospital resistant bacteria to laboratory mice. The derived capacity of antivitamins B₁₂ to induce functional B₁₂-deficiency in mammalian cells and organs also suggest their valuable potential as growth inhibitors of cancerous human and animal cells.     

Study on methane fermentation and production of vitamin B12 from alcohol waste slurry

We studied biogas fermentation from alcohol waste fluid to evaluate the anaerobic digestion process and the production of vitamin B₁₂ as a byproduct. Anaerobic digestion using acclimated methanogens was performed using the continuously stirred tank reactor (CSTR) and fixed-bed reactor packed with rock wool as carrier material at 55°C. We also studied the effects of metal ions added to the culture broth on methane and vitamin B₁₂ formation. Vitamin B₁₂ production was 2.92 mg/L in the broth of the fixed-bed reactor, twice that of the CSTR. The optimum concentrations of trace metal ions added to the culture liquid for methane and vitamin B₁₂ production were 1.0 and 8 mL/L for the CSTR and fixed-bed reactor, respectively. Furthermore, an effective method for extracting and purifying vitamin B₁₂ from digested fluid was developed.     

Glycerol as a Substrate and Inactivator of Coenzyme B12-Dependent Diol Dehydratase

Diol dehydratase, dependent on coenzyme B₁₂ (B₁₂-dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B₁₂-independent glycerol dehydratase (B₁₂-iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B₁₂-dDDH and that of resistance of B₁₂-iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B₁₂-dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B₁₂-iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.     

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.     

Miniaturized HPTLC of Vitamin B12 Compounds in Foods

High-performance thin-layer chromatography (HPTLC) is a separation technique commonly used to identify and quantify compounds in chemical mixtures. Preliminary experiments indicated that Lichrospher silica gel 60 F254s HPTLC sheets were the most suitable for analyzing vitamin B₁₂ compounds. This study revealed the advantages of miniaturized Lichrospher HPTLC for analyzing authentic vitamin B₁₂ compounds as short migration distances (5 cm) and short development times (<45 min) in combination with high separation efficiency and sensitivity (>25 pmol at 254 nm). The practicability of using miniaturized HPTLC was demonstrated by the separation and identification of vitamin B₁₂ compounds purified from foods using an immunoaffinity column.     

Radioassay of serum vitamin B12 using a competitive protein binding technique

A method has been developed for the radiassay of serum vitamin B₁₂ using a competitive binding technique. By this method, vitamin B₁₂ levels as low as 50 pg per ml of serum can be estimated in test samples. Egg yolk has been used as a test binding protein, which has several advantages over serum or plasma protein binding agents, as described in this paper. This binder is quite suitable and reliable for the assay of vitamin B₁₂.     

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.     

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.     

Exploring the Biosynthetic Potential of TsrM,a B12-dependent Radical SAM Methyltransferase Catalyzing Non-radical Reactions

B₁₂-dependent radical SAM enzymes are an emerging enzyme family with approximately 200,000 proteins. These enzymes have been shown to catalyze chemically challenging reactions such as methyl transfer to sp2- and sp3-hybridized carbon atoms. However, to date we have little information regarding their complex mechanisms and their biosynthetic potential. Here we show, using X-ray absorption spectroscopy, mutagenesis and synthetic probes that the vitamin B₁₂-dependent radical SAM enzyme TsrM catalyzes not only C- but also N-methyl transfer reactions further expanding its synthetic versatility. We also demonstrate that TsrM has the unique ability to directly transfer a methyl group to the benzyl core of tryptophan, including the least reactive position C4. Collectively, our study supports that TsrM catalyzes non-radical reactions and establishes the usefulness of radical SAM enzymes for novel biosynthetic schemes including serial alkylation reactions at particularly inert C−H bonds.     

Vitamin B12 Phosphate Conjugation and Its Effect on Binding to the Human B12‐Binding Proteins Intrinsic Factor and Haptocorrin

The binding of vitamin B₁₂ derivatives to human B₁₂ transporter proteins is strongly influenced by the type and site of modification of the cobalamin original structure. We have prepared the first cobalamin derivative modified at the phosphate moiety. The reaction conditions were fully optimized and its limitations examined. The resulting derivatives, particularly those bearing terminal alkyne and azide groups, were isolated and used in copper‐catalyzed alkyne–azide cycloaddition reactions (CuAAC). Their sensitivity towards light revealed their potential as photocleavable molecules. The binding abilities of selected derivatives were examined and compared with cyanocobalamin. The interaction of the alkylated derivatives with haptocorrin was less affected than the interaction with intrinsic factor. Furthermore, the configuration of the phosphate moiety was irrelevant to the binding process.     

Antivitamin B12 Inhibition of the Human B12-Processing Enzyme CblC: Crystal Structure of an Inactive Ternary Complex with Glutathione as the Cosubstrate

B₁₂ antivitamins are important and robust tools for investigating the biological roles of vitamin B₁₂. Here, the potential antivitamin B₁₂ 2,4-difluorophenylethynylcobalamin (F2PhEtyCbl) was prepared, and its 3D structure was studied in solution and in the crystal. Chemically inert F2PhEtyCbl resisted thermolysis of its Co−C bond at 100 °C, was stable in bright daylight, and also remained intact upon prolonged storage in aqueous solution at room temperature. It binds to the human B₁₂-processing enzyme CblC with high affinity (K_D=130 nm ) in the presence of the cosubstrate glutathione (GSH). F2PhEtyCbl withstood tailoring by CblC, and it also stabilized the ternary complex with GSH. The crystal structure of this inactivated assembly provides first insight into the binding interactions between an antivitamin B₁₂ and CblC, as well as into the organization of GSH and a base-off cobalamin in the active site of this enzyme.     

A solid-phase enzyme-linked assay for vitamin B12

A new solid-phase enzyme-linked competitive binding assay for vitamin B₁₂ (cyanocobalamin) is described. The assay is based on the competition between analyte B₁₂ molecules and a glucose-6-phosphate dehydrogenase-vitamin B₁₂ conjugate for a limited number of R-protein binding sites immobilized on sepharose particles. After appropriate incubation and washing steps, the enzyme activity bound to the solid-phase is inversely related to the concentration of B₁₂ in the sample. Under optimized conditions, the method can detect B₁₂ in the range of 3×10^–10–1×10^–8 M (using 100l sample) with high selectivity over other biological molecules.     

Zinc Substitution of Cobalt in Vitamin B12: Zincobyric acid and Zincobalamin as Luminescent Structural B12‐Mimics

Replacing the central cobalt ion of vitamin B₁₂ by other metals has been a long‐held aspiration within the B₁₂‐field. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid ( Znby ) and zincobalamin ( Znbl ), the Zn‐analogues of the natural cobalt‐corrins cobyric acid and vitamin B₁₂, respectively. The solution structures of Znby and Znbl were studied by NMR‐spectroscopy. Single crystals of Znby were produced, providing the first X‐ray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding Co^II‐corrins, making such Zn‐corrins potentially useful for investigations of B₁₂‐dependent processes. The singlet excited state of Znby had a short life‐time, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (E^T=190 kJ mol^?1) and was found to be an excellent photo‐sensitizer for ¹O₂ (Φ_Δ=0.70).     

Infrarotspektroskopische Untersuchung des Borcarbids und seiner isotypen Derivate B12O2, B12P2 und B12As2

Infrared Spectra of Boron Carbide and its Isotypic Derivatives B₁₂O₂, B₁₂P₂, and B₁₂As₂ From very finely powdered samples of B₄C [ = B₁₁C(CBC)], B₁₂O₂, B₁₂P₂, and B₁₂As₂ infrared spectra with very well shaped bands could be obtained. The spectrum of B₁₂O₂ with the smallest number of bands was assigned with the aid of a normal coordinate analysis. This gave a good basis for the interpretation of the spectra of normal and ¹⁰B-enriched B₄C and of B₁₂P₂. Both are exhibiting some bands in addition to those expected for the ideal structures, which are explained by the assumption of disordering within the C? B? C resp. P? P groups. At boron carbide this disorder seems to depend on the ratio B:C within its range of homogeneity.