Exploring the potential of the vitamin B12 derivative azidocobalamin to undergo Huisgen 1,3-dipolar azide-alkyne cycloaddition reactions.

There is considerable interest in using the metalloprotein cofactor vitamin B12 as a vehicle to deliver drugs and diagnostic agents into mammalian or bacterial cells by exploiting the B12-specific active uptake pathways. Conjugation of the cargo via the ß-axial site or the 5'-OH of the ribose of the nucleotide are the most desirable sites, to maximise intracellular uptake. Herein we show the potential of conjugation at the beta-azido ligand of the vitamin B12 derivative azidocobalamin via a click-type azide-alkyne 1,3-dipolar cycloaddition (Huisgen cycloaddition) reaction. Reacting azidocobalamin with dimethyl acetylenedicarboxylate at 40 °C results in essentially stoichiometric conversion of azidocobalamin to the corresponding triazolato complex. The stability of the complex as a function of pH and in the presence of cyanide were investigated. The complex is stable in pD 7.0 phosphate buffer for 24 h. The rate of beta-axial ligand substitution was found to be one order of magnitude slower for the triazolatocobalamin complex compared with azidocobalamin.

Exploring the Potential of 2-(2-Nitrophenyl)ethyl-Caged N-Hydroxysulfonamides for the Photoactivated Release of Nitroxyl (HNO).

The emergence of nitroxyl (HNO) as a biological signaling molecule is attracting increasing attention. HNO-based prodrugs show considerable potential in treating congestive heart failure, with HNO reacting rapidly with metal centers and protein-bound and free thiols. A new class of 2-(2-nitrophenyl)ethyl (2-NPE)-photocaged N-hydroxysulfonamides has been developed, and the mechanisms of photodecomposition have been investigated. Three photodecomposition pathways are observed: the desired concomitant C-O/N-S bond cleavage to generate HNO, sulfinate, and 2-nitrostyrene, C-O bond cleavage to give the parent sulfohydroxamic acid and 2-nitrostyrene, and O-N bond cleavage to release a sulfonamide and 2-nitrophenylacetaldehyde. Laser flash photolysis experiments provide support for a Norrish type II mechanism involving 1,5-hydrogen atom abstraction to generate an aci-nitro species. A mechanism is proposed in which the (Z)-aci-nitro intermediate undergoes either C-O bond cleavage to release RSO2NHO(H), concerted C-O/N-S bond cleavage to generate sulfinate and HNO, or isomerization to the (E)-isomer prior to O-N bond cleavage. The pKa of the N(H) of the N-hydroxysulfonamide plays a key role in determining whether C-O or concerted C-O/N-S bond cleavage occurs. Deprotonating this site favors the desired C-O/N-S bond cleavage at the expense of an increased level of undesired O-N bond cleavage. Triplet state quenchers have no effect on the observed photoproducts.

Mechanistic Insights into Rapid Generation of Nitroxyl from a Photocaged N-Hydroxysulfonamide Incorporating the (6-Hydroxynaphthalen-2-yl)methyl Chromophore.

HNO is a highly reactive molecule that shows promise in treating heart failure. Molecules that rapidly release HNO with precise spatial and temporal control are needed to investigate the biology of this signaling molecule. (Hydroxynaphthalen-2-yl)methyl-photocaged N-hydroxysulfonamides are a new class of photoactive HNO generators. Recently, it was shown that a (6-hydroxynaphthalen-2-yl)methyl (6,2-HNM)-photocaged derivative of N-hydroxysulfonamide incorporating the trifluoromethanesulfonamidoxy group (1) quantitatively generates HNO. Mechanistic studies have now been carried out on this system and reveal that the ground state protonation state plays a key role in whether concerted heterolytic C-O/N-S bond cleavage to release HNO occurs versus undesired O-N bond cleavage. N-Deprotonation of 1 can be achieved by adding an aqueous buffer or a carboxylate salt to an aprotic solvent. Evidence is presented for C-O/N-S bond heterolysis occurring directly from the singlet excited state of the N-deprotonated parent molecule on the picosecond time scale, using femtosecond time-resolved transient absorption spectroscopy, to give a carbocation and 1NO-. This is consistent with the observation of significant fluorescence quenching when HNO is generated. The carbocation intermediate reacts rapidly with nucleophiles including water, MeOH, or even (H)NO in the absence of a molecule that reacts rapidly with (H)NO to give an oxime.

Mechanistic Studies on the Reaction between Aquacobalamin and the HNO Donor Piloty's Acid over a Wide pH Range in Aqueous Solution.

Detailed kinetic and mechanistic studies have been carried out on the reaction between aquacobalamin/hydroxocobalamin (CblOH2+/CblOH) and nitroxyl (HNO) generated by Piloty's acid (PA, N-hydroxybenzenesulfonamide) over a wide pH range (3.5-13). The resulting data showed that in a basic solution HNO can react with hydroxocobalamin to form nitrosylcobalamin despite the inert nature of CblOH. It was shown that at low PA concentrations the rate-determining step is the decomposition of PhSO2NHO- to release HNO, whereas the reaction between CblOH and HNO becomes the rate-determining step at high PA concentrations. Data from kinetic studies on the reaction of CblOH with an excess of HNO enabled us to experimentally determine the pKa(HNO) value from initial rate data as a function of pH, giving pKa(HNO) = 11.47 ± 0.04. An especially interesting observation was made in the neutral pH range, where PA is stable and does not produce HNO. Under such conditions, rapid formation of CblNO was observed in the studied system. The obtained data suggest that CblOH2+ reacts directly with PA to form a Piloty's acid-bound cobalamin intermediate, which deprotonates rapidly at neutral pH followed by rate-determining S-N bond cleavage to give CblNO and release PhSO2-.

Stoichiometric Nitroxyl Photorelease Using the (6-Hydroxy-2-naphthalenyl)methyl Phototrigger.

The design and synthesis of a photoactivatable HNO donor incorporating the (6-hydroxynaphthalen-2-yl)methyl (6,2-HNM) photocage coupled to the trifluoromethanesulfonamidoxy analogue of the well-established HNO generator Piloty's acid is described. The photoactive HNO donor stoichiometrically generates HNO (∼98%) at neutral pH conditions, and evidence for concerted C-O and N-S bond cleavage was obtained. The methanesulfonamidoxy analogue primarily undergoes undesired N-O bond cleavage.

Synthesis and HNO Donating Properties of the Piloty's Acid Analogue Trifluoromethanesulphonylhydroxamic Acid: Evidence for Quantitative Release of HNO at Neutral pH Conditions.

Trifluoromethanesulphonylhydroxamic acid, CF3 SO2 NHOH, is shown to release HNO under physiological pH conditions. A two-step synthesis is presented with the first complete characterization of CF3 SO2 NHOH. This molecule rapidly decomposes in neutral aqueous solution to cleanly release HNO and CF3 SO2- , which was demonstrated using the HNO traps TXPTS and HOCbl, and by 19 F NMR spectroscopy.

Rapid Photoactivated Generation of Nitroxyl (HNO) under Neutral pH Conditions.

Directly obtaining kinetic and mechanistic data for the reactions of nitroxyl (HNO) with biomolecules (k≈103 -107 m-1 s-1 ) is not feasible for many systems because of slow HNO release from HNO donor molecules (t1/2 is typically minutes to hours). To address this limitation, we have developed a photoactivatable HNO donor incorporating the (3-hydroxy-2-naphthalenyl)methyl phototrigger, which rapidly releases HNO on demand. A "proof of concept" study is reported, which demonstrates that, upon continuous xenon light excitation, rapid decomposition of the HNO donor occurs within seconds. The amount of HNO generated is strongly dependent on solvent and the rate of the reaction is dependent on the light intensity.

Kinetic studies on the reaction of cob(II)alamin with hypochlorous acid: Evidence for one electron oxidation of the metal center and corrin ring destruction.

Kinetic and mechanistic studies on the reaction of a major intracellular vitamin B12 form, cob(II)alamin (Cbl(II)), with hypochlorous acid/hypochlorite (HOCl/OCl-) have been carried out. Cbl(II) (Co(II)) is rapidly oxidized by HOCl to predominately aquacobalamin/hydroxycobalamin (Cbl(III), Co(III)) with a second-order rate constant of 2.4×107M-1s-1 (25.0°C). The stoichiometry of the reaction is 1:1. UHPLC/HRMS analysis of the product mixture of the reaction of Cbl(II) with 0.9mol equiv. HOCl provides support for HOCl being initially reduced to Cl and subsequent H atom abstraction from the corrin macrocycle occurring, resulting in small amounts of corrinoid species with two or four H atoms fewer than the parent cobalamin. Upon the addition of excess (H)OCl further slower reactions are observed. Finally, SDS-PAGE experiments show that HOCl-induced damage to bovine serum albumin does not occur in the presence of Cbl(II), providing support for Cbl(II) being an efficient HOCl trapping agent.

Mechanistic studies of the reactions of the reduced vitamin B12 derivatives with the HNO donor Piloty's acid: further evidence for oxidation of cob(I)alamin by (H)NO.

There is accumulating evidence for the existence of HNO in biological systems. Compared with NO (˙NO), much less is known about the chemical and biochemical reactivity of HNO. Kinetic and mechanistic studies have been carried out on the reaction between the vitamin B12-derived radical complex cob(II)alamin (Cbl(II)˙, Cbl(II)) with the widely used HNO donor Piloty's acid (PA). A stoichiometry of 1 : 2 Cbl(II) : PA was obtained and PA decomposition to HNO and benzenesulfinate (C6H5SO2(-)) is the rate-determining step. No evidence was found for nitrite (Griess assay), ammonia (Nessler's test) or NH2OH (indooxine test) in the product solution, and it is likely that HNO is instead reduced to N2. A mechanism is proposed in which reduction of Cbl(II) by (H)NO results in formation of cob(I)alamin (Cbl(I)(-)) and ˙NO. The Cbl(I)(-) intermediate is subsequently oxidized back to Cbl(II) by a second (H)NO molecule, and Cbl(II) reacts rapidly with ˙NO to form nitroxylcobalamin (NOCbl). Separate studies on the reaction between Cbl(I)(-) and PA shows that this system involves an additional step in which Cbl(I)(-) is first oxidized by (H)NO to Cbl(II), which reacts further with (H)NO to form NOCbl, with an overall stoichiometry of 1 : 3 Cbl(I)(-) : PA. Experiments in the presence of nitrite for both systems support the involvement of a Cbl(I)(-) intermediate in the Cbl(II)/PA reaction. These systems provide the second example of oxidation of cob(I)alamin by (H)NO.

Changes in Methionine Metabolism and Histone H3 Trimethylation Are Linked to Mitochondrial Defects in Multiple Sclerosis.

Mitochondrial changes, including decreased expression of electron transport chain subunit genes and impaired energetic, have been reported in multiple sclerosis (MS), but the mechanisms involved in these changes are not clear. To determine whether epigenetic mechanisms are involved, we measured the concentrations of methionine metabolites by liquid chromatography tandem mass spectrometry, histone H3 methylation patterns, and markers of mitochondrial respiration in gray matter from postmortem MS and control cortical samples. We found decreases in respiratory markers as well as decreased concentrations of the methionine metabolites S-adenosylmethionine, betaine, and cystathionine in MS gray matter. We also found expression of the enzyme betaine homocysteine methyltransferase in cortical neurons. This enzyme catalyzes the remethylation of homocysteine to methionine, with betaine as the methyl donor, and has previously been thought to be restricted to liver and kidney in the adult human. Decreases in the concentration of the methyl donor betaine were correlated with decreases in histone H3 trimethylation (H3K4me3) in NeuN+ neuronal nuclei in MS cortex compared with controls. Mechanistic studies demonstrated that H3K4me3 levels and mitochondrial respiration were reduced in SH-SY5Y cells after exposure to the nitric oxide donor sodium nitroprusside, and betaine was able to rescue H3K4me3 levels and respiratory capacity in these cells. Chromatin immunoprecipitation experiments showed that betaine regulates metabolic genes in human SH-SY5Y neuroblastoma cells. These data suggest that changes to methionine metabolism may be mechanistically linked to changes in neuronal energetics in MS cortex. SIGNIFICANCE STATEMENT: For decades, it has been observed that vitamin B12 deficiency and multiple sclerosis (MS) share certain pathological changes, including conduction disturbances. In the present study, we have found that vitamin B12-dependent methionine metabolism is dysregulated in the MS brain. We found that concentrations of the methyl donor betaine are decreased in MS cortex and are correlated with reduced levels of the histone H3 methyl mark H3K4me3 in neurons. Cell culture and chromatin immunoprecipitation-seq data suggest that these changes may lead to defects in mitochondria and impact neuronal energetics. These data have uncovered a novel pathway linking methionine metabolism with mitochondrial respiration and have important implications for understanding mechanisms involved in neurodegeneration in MS.

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 2.0×10(9) M(-1) s(-1) . The reaction of CO3 (.) (-) with hydroxycobalamin proceeds in two steps. The second-order rate constant for the first reaction is 4.3×10(8) M(-1) s(-1) . The rate of the second reaction is independent of the hydroxycobalamin concentration and is approximately 3.0×10(3) 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.

Pulse radiolysis studies of the reactions of nitrogen dioxide with the vitamin B12 complexes cob(II)alamin and nitrocobalamin.

Although now recognized to be an important reactive nitrogen species in biological systems that modifies the structures of proteins, DNA and lipids, there are few studies on the reactivity of NO2, including the reactions between NO2 and transition metal complexes. We report kinetic studies on the reactions of NO2 with two forms of vitamin B12 - cob(II)alamin and nitrocobalamin. UV-visible spectroscopy and HPLC analysis of the product solution show that NO2 cleanly oxidizes the metal center of cob(II)alamin to form nitrocobalamin, with a second-order rate constant of (3.5±0.3)×10(8)M(-1)s(-1) (pH7.0 and 9.0, room temperature, I=0.20M). The stoichiometry of the reaction is 1:1. No reaction is detected by UV-visible spectroscopy and HPLC analysis of the product solution when nitrocobalamin is exposed to up to 2.0molequiv. NO2.

Kinetic and mechanistic studies on the reaction of the vitamin B12 complex aquacobalamin with the HNO donor Angeli's salt: Angeli's salt and HNO react with aquacobalamin.

We report the first studies on the reaction between an HNO donor compound and vitamin B12 complexes. Kinetic and mechanistic studies have been carried out on the reaction between the vitamin B12 derivative aquacobalamin (H2OCbl(+)/HOCbl; pKa = 7.8) and the HNO donor Angeli's salt. Studies were carried out with aquacobalamin in excess, since nitrite also reacts with aquacobalamin to form nitrocobalamin (NO2Cbl). At pH <9.90 aquacobalamin reacts directly with the monoprotonated form of Angeli's salt, HN2O3(-), to form nitroxylcobalamin (NO(-)-Cbl(III); NOCbl) and nitrite. At pH >10.80 the reaction instead switches predominantly to a mechanism in which spontaneous decomposition of Angeli's salt to give HNO and nitrite becomes the rate-determining step, followed by the rapid reaction between aquacobalamin and HNO/NO(-) to again give NOCbl. Both reactions proceed with a 1:1 stoichiometry and formation of nitrite is confirmed using the Griess assay.

Mechanistic studies on the reaction of nitroxylcobalamin with dioxygen: evidence for formation of a peroxynitritocob(III)alamin intermediate.

Studies by others suggest that the reduced vitamin B12 complex, cob(II)alamin, scavenges nitric oxide to form air-sensitive nitroxylcobalamin (NO(-)-Cbl(III); NOCbl) in vivo. The fate of newly formed NOCbl is not known. A detailed mechanistic investigation of the oxidation of NOCbl by oxygen is presented. Only base-on NOCbl reacts with O2, and the reaction proceeds via an associative mechanism involving a peroxynitritocob(III)alamin intermediate, Co(III)-N(O)OO(-). The intermediate undergoes O-O bond homolysis and ligand isomerization to ultimately yield NO2Cbl and H2OCbl(+)/HOCbl, respectively. Ligand isomerization may potentially occur independent of O-O bond homolysis. Formation of (•)OH and (•)NO2 intermediates from O-O bond homolysis is demonstrated using phenol and tyrosine radical traps and the characterization of small amounts of a corrinoid product with minor modifications to the corrin ring.

Pulse radiolysis studies on the reaction of the reduced vitamin B12 complex Cob(II)alamin with superoxide.

O2.- scavenger: The rate constant for the rapid reaction of the ROS superoxide with the reduced vitamin B12 radical complex cob(II)alamin was directly determined to be 3.8×10(8) M⁻¹ s⁻¹. This rate was independent of pH over the range 5.5-8.7. These results have implications for studying the use of B12 supplements to combat diseases associated with oxidative stress.

Mechanistic Studies on the Reaction of Nitrocobalamin with Glutathione: Kinetic evidence for formation of an aquacobalamin intermediate.

The essential but also toxic gaseous signaling molecule nitric oxide is scavenged by the reduced vitamin B12 complex cob(II)alamin. The resulting complex, nitroxylcobalamin (NO--Cbl(III)), is rapidly oxidized to nitrocobalamin (NO2Cbl) in the presence of oxygen; however it is unlikely that nitrocobalamin is itself stable in biological systems. Kinetic studies on the reaction between NO2Cbl and the important intracellular antioxidant, glutathione (GSH), are reported. In this study, a reaction pathway is proposed in which the ß-axial ligand of NO2Cbl is first substituted by water to give aquacobalamin (H2OCbl+), which then reacts further with GSH to form glutathionylcobalamin (GSCbl). Independent measurements of the four associated rate constants k1, k-1, k2, and k-2 support the proposed mechanism. These findings provide insight into the fundamental mechanism of ligand substitution reactions of cob(III)alamins with inorganic ligands at the ß-axial site.

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).

Vitamin B12 protects against superoxide-induced cell injury in human aortic endothelial cells.

Superoxide (O(2)(•-)) is implicated in inflammatory states including arteriosclerosis and ischemia-reperfusion injury. Cobalamin (Cbl) supplementation is beneficial for treating many inflammatory diseases and also provides protection in oxidative-stress-associated pathologies. Reduced Cbl reacts with O(2)(•-) at rates approaching that of superoxide dismutase (SOD), suggesting a plausible mechanism for its anti-inflammatory properties. Elevated homocysteine (Hcy) is an independent risk factor for cardiovascular disease and endothelial dysfunction. Hcy increases O(2)(•-) levels in human aortic endothelial cells (HAEC). Here, we explore the protective effects of Cbl in HAEC exposed to various O(2)(•-) sources, including increased Hcy levels. Hcy increased O(2)(•-) levels (1.6-fold) in HAEC, concomitant with a 20% reduction in cell viability and a 1.5-fold increase in apoptotic death. Pretreatment of HAEC with physiologically relevant concentrations of cyanocobalamin (CNCbl) (10-50nM) prevented Hcy-induced increases in O(2)(•-) and cell death. CNCbl inhibited both Hcy and rotenone-induced mitochondrial O(2)(•-) production. Similarly, HAEC challenged with paraquat showed a 1.5-fold increase in O(2)(•-) levels and a 30% decrease in cell viability, both of which were prevented with CNCbl pretreatment. CNCbl also attenuated elevated O(2)(•-) levels after exposure of cells to a Cu/Zn-SOD inhibitor. Our data suggest that Cbl acts as an efficient intracellular O(2)(•-) scavenger.

Redetermination of the X-ray structure of nitroxylcobalamin: base-on nitroxylcobalamin exhibits a remarkably long Co-N(dimethylbenzimidazole) bond distance.

The X-ray structures of three new crystals of nitroxylcobalamin (NOCbl) have been determined. Unlike our earlier reported structure in which NOCbl was partially oxidized (L. Hannibal, C. A. Smith, D. W. Jacobsen and N. E. Brasch, Angew. Chem., Int. Ed. 2007, 46, 5140), the O atom of the nitroxyl ligand is located in a single position with a N=O bond distance of 1.12-1.14 Å, consistent with a double bond. The Co-N-O angle is in the 118.9-120.3 Å range. The α-axial Co-N(dimethylbenzimidazole) (Co-NB3) bond distance is a remarkable 2.32-2.35 Å in length, ~0.1 Å longer than that reported for all other cobalamin structures. The change in the Gibbs free energy for the base-on/base-off equilibrium now correlates extremely well with the Co-NB3 bond distance, as observed for other cobalamins.