Interaction between super-reduced cobalamin and selenite

The kinetics of the reaction between the two-electron reduced form of cobalamin (super-reduced cobalamin, cob(I)alamin, or Cbl(I)) and sodium selenite in an alkaline medium is studied spectrophotometrically. It is shown that the selenite rapidly oxidizes Cbl(I) to cob(II)alamin (Cbl(II)). It is established that the active form of the oxidant is the protonated selenite anion (HSeO₃^-), which receives six electrons during the reaction and transforms into HSe^–. The reactions of cob(I)alamin oxidation by selenite and sulfite are compared.     

the Mechanism of the Oxidation of Cob(I)alamins

Abstract

Spectroelectrochemical investigations of the reoxidation sequence of the reduced cob(I)alamin to the oxidized cob(III)alamin show that two different cob(II)alamin intermediates are formed during the processes which appear to correlate to base-on and base-off cob(II)-alamin species.     

Characterization of alkyl-cobalamins formed on trapping of epoxide metabolites of 1,3-butadiene

Analytical methods facilitating studies of electrophilically reactive and genotoxic compounds in vitro and in vivo are needed. The strong nucleophile, cob(I)alamin, formed by reduction of Vitamin B12 [cob(III)alamin], may be used for trapping and analysis of 1,2-epoxides and other electrophiles. In the present study, cob(I)alamin is evaluated as an analytical tool for 1,2-epoxide metabolites (oxiranes) of 1,3-butadiene. Products of reaction of cob(I)alamin with 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EBdiol) have been analyzed by reversed phase high performance liquid chromatography (HPLC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) and ultraviolet diode array detection (UV-DAD). It was shown that a specific alkyl-CbI complex is formed for each metabolite and that it was possible to discriminate between the products by HPLC-UV and by LC-MS. Quantification of DEB with the method by use of another 1,2-epoxide as an internal standard was successfully performed. The possibility of using cob(I)alamin for trapping and analysis of the three oxirane metabolites of 1,3-butadiene will facilitate quantitative comparisons of species in vitro with regard to metabolism of 1,3-butadiene.     

Cobalamin as an analytical tool for analysis of oxirane metabolites of 1,3-butadiene: development and validation of the method

The reduced form of vitamin B12 [cob(I)alamin] is known to be a supernucleophile, with the ability to react 10(5) times faster than standard nucleophiles. Procedures have been developed where cob(I)alamin is used as an analytical tool for the trapping of electrophilically reactive compounds. In the present work, a sensitive and accurate method for determination of reactive metabolites produced in vitro has been developed and validated. Diepoxybutane (DEB), a metabolite of 1,3-butadiene, was used as a model compound. The intermediate precursor 1,2-epoxybutene (EB) was incubated in a mouse liver S9 metabolic system and the formation of DEB was studied. Samples were taken at different times from the incubation mixture and added to the cob(I)alamin. The alkyl-cobalamins (alkyl-Cbl) formed were directly analysed by a miniaturized LC-MS/MS method and column switching. The assay was linear over the concentration range of 1.5-500 microM with acceptable precision and accuracy.     

Cob(I)alamin als Katalysator. 6. Mitteilung [1]. Bildung und Fragmentierung von Alkylcobalaminen,ein Gleichgewichtsprozess zwischen nukleophiler Addition und reduktiver Fragmentierung

Cob(I)alamin as Catalyst. 6. Communication [1]. Formation and Fragmentation of Alkylcobalamins: the Nucleophilic Addition – Reductive Fragmentation Equilibrium Isolated olefines can be saturated using catalytic amounts of cob(I)alamin in aqueous acetic acid; as electron source an excess of zinc dust is added to the solution containing the homogeneous catalyst. During this overall hydrogenation of isolated double bonds intermediate alkylcobalamins are formed (compare e.g. Schemes 2, 4, 5, 7 and 12). Clear evidence is presented that the nucleophilic attack on the isolated double bond is carried out by cob(I)alamin and not by cob(II)alamin also present in the system (see Scheme 3b and 3c). As this catalytic saturation of olefins depends on the pH of the solution, characterized by a slow reaction at pH = 7.0 compared to the same reduction in aqueous acetic acid (see Scheme 2, 2 → 4 , and Scheme 3a), it is reasonable to accept the participation of an electrophilic attack by a proton during the generation of alkylcobalamins. – We use the term nucleophilic addition to describe the formation of alkylcobalamins from a proton, an olefin and cob(I)alamin (compare Schemes 4–7 and 12). A special sequence of experiments showed the nucleophilic addition to be regioselective. Preferentially the higher substituted alkylcobalamin revealed to be produced. Therefore, the nucleophilic addition of cob(I)alamin follows the Markownikoff rule (compare chap. 4: formation and fragmentation of β-hydroxyalkylcobalamins). Under the reaction conditions applied the intermediate alkylcobalamins can be present in base-on and base-off forms. They are known to exist as octahedral complexes and might also be stable to some extent as tetragonal-pyramidal species. In addition the base-off forms can partially be protonated at the dimethylbenzimidazole moiety in aqueous acetic acid (compare Scheme 12). From this equilibrium of intermediate alkylcobalamins three modes of decay disclosed to be possible: (i) The reductive fragmentation leading to an olefin, a proton, and cob(I)alamin is the formal retro-reaction of the nucleophilic addition (see Schemes 2, 4 and 6–12). This equilibrium of an associated alkylcobalamin and the corresponding dissociation products revealed to be a fast process compared to the reductive cleavage of the Co, C-bond cited below (s. (iii)). (ii) As the second reaction pattern an oxidative fragmentation producing an olefin, a hydroxy anion (or water, respectively) and cob (III)alamin has been observed (see Schemes 7, 8, 10 and 12). (iii) The slow reductive cleavage of the Co, C-bond, initiated by addition of electrons (see [1a] [24]), was the third reaction path observed (see Schemes 2, 4–8 and 10–12). – The stereochemistry of the three transformations originating from the intermediate alkylcobalamins is unknown up to now. The antiperiplanar pattern of the fragmentation reactions presented in the Schemes has been chosen arbitrarily (see e.g. Scheme 12).     

Validated assay for the simultaneous determination of the anti-cancer agent gemcitabine and its metabolite 2',2'-difluorodeoxyuridine in human plasma by high-performance liquid chromatography with tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the quantitative determination of gemcitabine (dFdC) and its metabolite 2',2'-difluorodeoxyuridine (dFdU) is presented. A 200-microL aliquot of human plasma was spiked with a mixture of internal standards, didanosine, lamivudine and fludarabine, and extracted using solid-phase extraction. Dried extracts were reconstituted in 1 mM ammonium acetate/acetonitrile (97:3, v/v) and 10-microL volumes were injected onto the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 bonded phase endcapped with polar groups (Synergi Hydro-RP column) using an eluent composed of 1 mM ammonium acetate (pH 6.8)/acetonitrile (94:6, v/v). Detection was performed by positive ion electrospray ionization followed by MS/MS. The assay quantifies a range from 0.5 to 1000 ng/mL for gemcitabine and from 5 to 10,000 ng/mL for dFdU using 200 microL of human plasma sample. Validation results demonstrate that gemcitabine and dFdU concentrations can be accurately and precisely quantified in human plasma. This assay is used to support clinical pharmacologic studies with gemcitabine.     

Thermodynamic trans-Effects of the Nucleotide Base in the B12 Coenzymes

The thermodynamic effects of the nucleotide coordination on the Co-C bond strengths in the B ₁₂ coenzymes were analyzed. Methyl group transfer reactions from methylcob( III )inamides to cob( II )inamides and cob( I )inamides in neutral aqueous solution were used in equilibration experiments to determine the effect fo the intramolecular coordination of the nucleotide function on the Co-C bond dissociation energies of methylcob( III )alamin ( 4 ). In the equilibrium between 4 , cob( I )inamide ( 11 ), cob( I )alamin ( 10 ) and methylcob( III )inamide 6 (Scheme 2), 4 and 11 were found to predominate ( 4 + 11 ? 10 + 6 , equilibrium constant K_I/III≈0.004), while the equilibrium between 4 , cob( II )inamide 9 , cob( II )alamin ( 5 ), and 6 (Scheme 1) proved to be well balanced ( 4 + 9 ? 5 + 6 , equilibrium constant K_II/III=0.60). These equilibrium values indicate the nucleotide coordination to stabilize the Co–C bond in 4 both against homolysis (slight effect) and against nucleophilic heterolysis (considerable effect). They reflect a stabilization of the complete corrins 4 and 5 by the nucleotide coordination, which is also indicated for 4 and 5 by their (nucleotide) basicity. The latter information, where available for other organocobalamins, allows the analysis of the thermodynamicnucleotide trans effect there as well: e.g. in coenzyme B ₁₂ ( 1 ), the nucleotide coordination is found this way to weaken the Co–C bond towards homolysis by ca. 0.7 kcal/mol.     

Ion chromatography tandem mass spectrometry for simultaneous confirmation and determination of indandione rodenticides in serum

This paper describes a simple method for the simultaneous determination and confirmation of the indandione rodenticides in serum. After samples were extracted with 10% (v/v) methanol in acetonitrile and cleaned by solid‐phase extraction, chromatographic separation was performed on an IonPac® AS11 analytical column (250 × 4.0 mm) using gradient KOH eluent with 10% (v/v) methanol as organic modifier. Confirmation was depended on the extensive fragmentation of the indandione molecule under MS/MS conditions which provides sufficient structural information. Quantification was performed by negative electrospray ionization in multiple reaction monitoring mode. All the method parameters were validated. It was confirmed that this method could be used in clinical diagnosis and forensic toxicology. Copyright © 2009 John Wiley & Sons, Ltd.     

Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy.     

Reduction‐Labile Organo‐cob(III)alamins via Cob(II)alamin: Efficient Synthesis and Solution and Crystal Structures of [(Methoxycarbonyl)methyl]cob(III)alamin

An efficient synthesis of Coβ‐[(methoxycarbonyl)methyl]cob(III)alamin ( 6 ) is reported as an example of a new method for the preparation of some easily reducible organo‐cob(III)alamins via the alkylation of cob(II)alamin. The procedure represents a considerable improvement compared to earlier methods that were based on an alkylation of cob(I)alamin. Thus, aquacob(III)alamin chloride ( 5 ⁺?Cl) was reduced to cob(II)alamin ( 4 ), either by controlled potential electrolytic reduction or with an excess of sodium formate as reducing agent. The solution of 4 was then treated with an excess of methyl bromoacetate while being reductively poised potentiostatically or kept reduced by the formate, to give crystalline 6 in a yield of up to 91%. The structure of 6 in aqueous solution was mainly established by the completely assigned ¹H‐ and ¹³CNMR spectra (Table 1). The NOE data (Table 2) were best rationalized by the presence of a single main conformation of the (methoxycarbonyl)methyl ligand. Single crystals of 6 were obtained by crystallization from an aqueous solution, and the crystal structure was determined by X‐ray analysis at cryotemperatures. The NMR and crystallographic data of 6 indicated similar structures in aqueous solution and in the crystal with the (methoxycarbonyl)methyl ligand preferring a ‘southern' orientation in each case.     

Cob(I)alamin als Katalysator 2. Mitteilung [1]. Reduktion von gesättigten Nitrilen in wasserfreier Lösung

Cob (I)alamin as Catalyst 2. Communication [1]. Reduction of Saturated Nitriles in Anhydrous Solution Using cob (I)alamin as homogenous catalyst in glacial acetic acid saturated nitriles are reduced following the path of a reductive amination. The results prove the presence of an intermediate imine during the reduction of saturated nitriles with cob (I)alamine.     

Mechanistic studies on the reaction between cob(II)alamin and peroxynitrite: evidence for a dual role for cob(II)alamin as a scavenger of peroxynitrous acid and nitrogen dioxide

Peroxynitrite/peroxynitrous acid (ONOO(-)/ONOOH; pK(a(ONOOH)) =6.8) is implicated in multiple chronic inflammatory and neurodegenerative diseases. Both mammalian B(12)-dependent enzymes are inactivated under oxidative stress conditions. We report studies on the kinetics of the reaction between peroxynitrite/peroxynitrous acid and a major intracellular vitamin B(12) form, cob(II)alamin (Cbl(II)), using stopped-flow spectroscopy. The pH dependence of the reaction is consistent with peroxynitrous acid reacting directly with Cbl(II) to give cob(III)alamin (Cbl(III)) and (.)NO(2) , followed by a subsequent rapid reaction between (.)NO(2) and a second molecule of Cbl(II) to primarily form nitrocobalamin. In support of this mechanism, a Cbl(II)/ONOO(H) stoichiometry of 2:1 is observed at pH 7.35 and 12.0. The final major Cbl(III) product observed (nitrocobalamin or hydroxycobalamin) depends on the solution pH. Analysis of the reaction products in the presence of tyrosine-a well-established (.)NO(2) scavenger-reveals that Cbl(II) reacts with (.)NO(2) at least an order of magnitude faster than tyrosine itself. Given that protein-bound Cbl is accessible to small molecules, it is likely that enzyme-bound and free intracellular Cbl(II) molecules are rapidly oxidized to inactive Cbl(III) upon exposure to peroxynitrite or (.)NO(2).     

Cob(I)alamin als Katalysator, 5. Mitteilung [1]. Enantioselektive Reduktion α,β-ungesättigter Carbonylderivate

Cob(I)alamin as Catalyst. 5. Communication [1]. Enantioselective Reduction of α,β-Unsaturated Carbonyl Derivatives The cob(I)alamin-catalyzed reduction of an α,β-unsaturated ethyl ester in aqueous acetic acid produced the (S)-configurated saturated derivative 2 with an enantiomeric excess of 21%. The starting material 1 is not reduced at pH = 7.0 in the presence of catalytic amounts of cob(I)alamin (see Scheme 2). It is shown that the attack of cob(I)alamin and not of cob(II)alamin, also present in Zn/CH₃COOH/H₂O, accounts for the enantioselective reduction observed. All the (Z)-configurated starting materials 1 , 3 , 5 , 7 , 9 and 11 have been transformed to the corresponding (S)-configurated saturated derivatives 2 , 4 , 6 , 8 , 10 and 12 , respectively. The highest enantiomeric excess revealed to be present in the saturated product 12 (32,7%, S) derived from the (Z)-configurated methyl ketone 11 (see Scheme 3 and Table 1). The reduction of the (E)-configurated starting materials led mainly to racemic products. A saturated product having the (R)-configuration with a rather weak enantiomeric excess (5.9%) has been obtained starting from the (E)-configurated methyl ketone 23 (see Scheme 5 and Table 2). The allylic alcohols 16 and 24 have been reduced to the saturated racemic derivative 17 .     

Thermal Methyl-Group Transfer between Methylcobalt(III) Corrinates and Cobalt(II) Corrinates. Equilibration Experiments with Heptamethyl Cobyrinates and Cobalamins

Separate neutral aqueous solutions of either (a) methylcob(III)alamin ( 2 ) and (heptamethyl cob(II)yrinate) perchlorate ( 3 ) or of b) cob(II)alamin ( = vitamin B_12r; ( 4 ) and [Coβ-methyl(heptamethyl cob(III)yrinate)] perchlorate ( 5 ) equilibrated thermally at r.t. according to 2 + 3 ? 4 + 5 . The corresponding equilibrium constant K_e was determined (K_e = 0.63 ± 0.15). This equilibration experiment indicates that the coordination of the nucleotide function in methylcob(II)alamin ( 2 ) hardly affects the thermodynamics of the Co? C bond homolysis in aqeous solution when compared to nucleotide-free methylcorrinoids such as 5 .     

Identification of In Vitro Metabolites of Amoxicillin in Human Liver Microsomes by LC–ESI/MS

Amoxicillin (AMOX) metabolism in human liver microsomes was studied in vitro using liquid chromatography–mass spectrometry (LC/MS). Amoxicillin was incubated with human liver microsomes along with NADPH, and the reaction mixture was analyzed by LC/MS to obtain the specific metabolic profile of the studied antibiotic drug. Positive electrospray ionization was employed as the ionization source. An ACE C18-column (4.6 mm × 150 mm, 3 μm) was implemented with acetonitrile and water (+0.1 % formic acid) in isocratic mode as the mobile phase at the flow 0.4 mL min^?1. The chemical structures of metabolites were proposed on the basis of the accurate mass measurement of the protonated molecule as well as their main product. Six phase I and one phase II metabolites were detected and structurally described. The metabolism of AMOX occurred via oxidation, hydroxylation and oxidative deamination, as well as through combination of these reactions. Compound M7, with glucuronic acid was also observed as phase II metabolite. Neither sulfate nor glutathione conjugates were detected. This study presents novel information about the chemical structure of the potential AMOX metabolites and provides vital data for further pharmacokinetic and in vivo metabolism studies.     

Mass spectrometric and tandem mass spectrometric behavior of nitrocatechol glucuronides: A comparison of atmospheric pressure chemical ionization and electrospray ionization

The mass spectrometric (MS) and tandem mass spectrometric (MS/MS) behavior of six nitrocatechol-type glucuronides using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was systematically studied, and the effect of operation parameters on the fragmentations are presented. The positive ion APCI- and ESI-MS spectra showed an intense protonated molecule and the respective negative ion spectra a deprotonated molecule with minimal fragmentation. The main fragment ions in the MS/MS spectra of the protonated and deprotonated molecules were [M + H - Glu]+ and [M - H - Glu]-, respectively, formed by the loss of the glucuronide moiety. The measured limits of detection indicated that ESI is a significantly more efficient ionization method than APCI in the negative and positive ion modes for the compounds studied. MS/MS was found to be less sensitive, but more reliable and simple than MS due to the absence of chemical noise.     

Characterization of chlorovinylcobalamin,a putative intermediate in reductive degradation of chlorinated ethylenes

The first X-ray structure of a vinylcobalamin is reported. Chlorovinylcobalamin is formed in the reaction of cob(I)alamin with chloroacetylene. Subsequently, cob(I)alamin catalyzes the reduction of chlorovinylcobalamin to vinylcobalamin in the presence of excess titanium(III)citrate. Introduction of a chlorine onto the vinyl group of vinylcobalamin greatly changes its reduction potential. These results are discussed with respect to vitamin B12-catalyzed dechlorination of perchloroethylene, a pollutant on the priority list of the EPA.     

APPI-MS: Effects of mobile phases and VUV lamps on the detection of PAH compounds

The technique of atmospheric pressure photoionization (APPI) has several advantages over electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), including efficient ionization of nonpolar or low charge affinity compounds, reduced susceptibility to ion suppression, high sensitivity, and large linear dynamic range. These benefits are greatest at low flow rates (i.e., 相似文献   

Mechanistic investigation of a novel vitamin B(12)-catalyzed carbon [bond] carbon bond forming reaction, the reductive dimerization of arylalkenes

In the presence of catalytic vitamin B(12) and a reducing agent such as Ti(III)citrate or Zn, arylalkenes are dimerized with unusual regioselectivity forming a carbon [bond] carbon bond between the benzylic carbons of each coupling partner. Dimerization products were obtained in good to excellent yields for mono- and 1,1-disubstituted alkenes. Dienes containing one aryl alkene underwent intramolecular cyclization in good yields. However, 1,2-disubstituted and trisubstituted alkenes were unreactive. Mechanistic investigations using radical traps suggest the involvement of benzylic radicals, and the lack of diastereoselectivity in the product distribution is consistent with dimerization of two such reactive intermediates. A strong reducing agent is required for the reaction and fulfills two roles. It returns the Co(II) form of the catalyst generated after the reaction to the active Co(I) state, and by removing Co(II) it also prevents the nonproductive recombination of alkyl radicals with cob(II)alamin. The mechanism of the formation of benzylic radicals from arylalkenes and cob(I)alamin poses an interesting problem. The results with a one-electron transfer probe indicate that radical generation is not likely to involve an electron transfer. Several alternative mechanisms are discussed.     

Separation of acidic compounds by strong anion-exchange capillary electrochromatography

Separation of the acidic compounds in the ion-exchange capillary electrochromatography (IE-CEC) with strong anion-exchange packing as the stationary phase was studied. It was observed that the electroosmotic flow (EOF) in strong anion-exchange CEC moderately changed with increase of the eluent ionic strength and decrease of the eluent pH, but the acetonitrile concentration in the eluent had almost no effect on the EOF. The EOF in strong anion-exchange CEC with eluent of low pH value was much larger than that in RP-CEC with Spherisorb-ODS as the stationary phase. The retention of acidic compounds on the strong anion-exchange packing was relatively weak due to only partial ionization of them, and both chromatographic and electrophoretic processes contributed to separation. It was observed that the retention values of acidic compounds decreased with the increase of phosphate buffer and acetonitrile concentration in the eluent as well as the decrease of the applied voltage, and even the acidic compounds could elute before the void time. These factors also made an important contribution to the separation selectivity for tested acidic compounds, which could be separated rapidly with high column efficiency of more than 220000 plates/m under the optimized separation conditions.