Synthesis of Methylenebis(Phosphonate) Analogues of Nucleotide Coenzymes. A Novel Coupling Mechanism

Synthesis of P¹, P²-disubstituted methylenebis(phosphonate)s as inhibitors of inosine monophosphate dehydrogenase is presented.     

超高效液相色谱-质谱联用法同时快速测定细胞中的4种吡啶核苷酸辅酶

建立了超高效液相色谱-质谱联用法同时测定细胞内4种吡啶核苷酸辅酶(烟酰胺腺嘌呤二核苷酸(NAD+)和烟酰胺腺嘌呤二核苷酸磷酸(NADP+)以及它们的还原态(NADH和NADPH))的方法.样品经甲醇低温快速提取后,在新型混合模式Atlantis PREMIER BEH C18 AX色谱柱上分离,10 mmol/L甲酸铵...     

Replication Experiments with Nucleotide Base Analogues

The principles governing the replication fidelity of genomes are not fully understood yet. Watson and Crick's base-pairing principle for matched deoxyribonucleotide (DNA) bases can explain why the guanine–cytosine and adenine-thymine base pairs are approximately one hundred times more stable thermodynamically than mismatched combinations. In vitro, DNA polymerases reduce the number of mismatched base pairs to about 10^?6 per Watson–Crick base pair. Replication fidelity can further be enhanced to a mutation probability of 10^?10or less in vivo if optimal conditions for DNA synthesis are provided by polymerase–assisting proteins and DNA-repairing enzymes. The precise reasons for the formation of mismatched base pairs (mispairs), which are responsible for a substantial part of DNA mutations, are still in debate. Although it is agreed that a template-directed “reading” of the hydrogen-substitution pattern in the heterocyclic bases is crucial for proper base pairing during DNA synthesis, it is not clear which type of “misreading” leads to mispairs. Misreading may be due to a non-Watson–Crick base pairing as well as to a change in the hydrogen-substitution pattern, leading to Watson-Crick-like mispairs. The surprising discovery of the selective and quantitative DNA-polymerase-catalyzed formation of a pyridine-pyrimidine base pair (involving a nucleotide base analogue) indicated that rare tautomeric forms in template DNA strands can lead to Watson-Crick-like mispairings that are hardly recognized by the polymerase's proofreading activity. This reveals new pathways for substitution mutations (replication-dependent DNA point mutations) and suggests a new type of mutagen in vivo.     

Pseudocoenzyme B12 and Adenosyl‐Factor A: Electrochemical Synthesis and Spectroscopic Analysis of Two Natural B12 Coenzymes with Predominantly ‘Base‐off' Constitution

Pseudocoenzyme B₁₂ (=Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(adenin‐7‐yl)cobamide; 1 ) and adenosyl‐factor A (=Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(2‐methyladenin‐7‐yl)cobamide; 3 ) are two natural analogues of coenzyme B₁₂ (=adenosylcobalamin‐Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(5,6‐dimethyl‐1H‐benzimidazolyl)cobamide; 2 ), where the Co‐coordinating 5,6‐dimethyl‐1H‐benzimidazole nucleotide base of 2 is replaced by the purine bases adenine and 2‐methyladenine. In contrast to 2 , which exists solely in the ‘base‐on' form, UV/VIS spectroscopy qualitatively indicates ‘base‐off' constitution for 1 and 3 in aqueous solution. (cf. the established ‘base‐off' form as unexpected binding mode of B₁₂ cofactors in several B₁₂‐dependent enzymes, such as in methionine synthase from Escherichia coli and in glutamate mutase from Clostridium cochlearium). In the present work, pseudocoenzyme B₁₂ ( 1 ) was synthesized in 85% yield by alkylation with 5′‐O‐tosyladenosine of (adenin‐7‐yl)cob(I)amide, which was produced electrochemically from pseudovitamin B₁₂ (Coβ‐cyano‐(adenin‐7‐yl)cobamide). Likewise, adenosyl‐factor A ( 3 ) was prepared in ca. 70% yield from factor A (=Coβ‐cyano‐(2‐methyladenin‐7‐yl)cobamide; 5 ). All the spectroscopic properties of 1 and 3 in aqueous solution indicated that these two Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(adenin‐7‐yl)cobamides exist predominantly in a ‘base‐off' constitution, with minor but significant contributions of the ‘base‐on' form. From the UV/VIS spectra, the temperature‐dependent equilibrium constants of the ‘base‐off'/‘base‐on' reconstitution reaction were determined as K_on ( 1 )=0.30 and K_on ( 3 )=0.48 at 25°, corresponding to a contribution of the ‘base‐on' forms of 23% for 1 and of 32% for 3 .     

Thermodynamic Stability of Ribonuclease B

The thermodynamic stability of pancreatic ribonuclease B (RNase B), which possesses identical protein structure of pancreatic ribonuclease A (RNase A), but differs by the presence of a carbohydrate chain attached to Asn 34, was studied by means of differential scanning calorimetry (DSC) at different pH conditions. The comparison between the two proteins has shown a little but significant stabilization of RNase B with respect to the unglycosylated one at pH values higher than 7.0. The thermodynamic analysis reveals the carbohydrate moiety to have a small stabilization effect of 3 kJ mol^–1 at pH 8.0 and 63°C on the protein. This revised version was published online in July 2006 with corrections to the Cover Date.     

四氢叶酸辅酶仿生化学的进展

本文简要地介绍了四氢叶酸辅酶仿生化学研究的进展,包括仿生有机合成、酶促反应模型和反应机理等方面。     

B12-retro-riboswitches: guanosyl-induced constitutional switching of B12 coenzymes

Complete B12 derivatives are natural "molecular switches" as a result of the coordinative switch ("base on" or "base off") of the natural nucleotide base. Certain predesigned B12-nucleotide conjugates were shown recently to behave as "retro riboswitches", in which the nucleotide environment modified the equilibrium between these two isomeric B12 structures. In contrast, the "reverse" situation has been discovered in natural B12 riboswitches, in which the binding of coenzyme B12 induces a conformational switch in the RNA species. The first (predesigned) B12-retro-riboswitches were DNA conjugates of methylcobalamin. We describe herein two representative B12-retro-riboswitches, in which an appended (RNA) nucleotide is used to destabilize the base-on form and induce the base-on to base-off switch. Through use of heterogeneous solid-phase synthetic methods, Co(beta)-cyanocobalamin-(3'-->2')-2'-methoxyguaninyl-3'-ate was prepared first as the crucial covalent RNA conjugate of vitamin B12. This cyanocorrinoid opened the door to two organometallic B12-nucleotide conjugates, which were made by electrosynthetic means: the cyanocorrinoid was cleanly methylated or adenosylated at the cobalt center to furnish covalent RNA conjugates of the organometallic B12 cofactors methylcobalamin and coenzyme B12, respectively. At room temperature, aqueous solutions of both of these organometallic RNA-B12 conjugates exhibited properties indicative of significant weakening of the axial (Co--N) bond (of their base-on forms) and of an enhanced formation of the base-off species. The base-on to base-off switch was studied by UV/Vis and NMR spectroscopic studies, which showed that the switch was very temperature-dependent and was accentuated with increasing temperatures. Thermodynamic data of the two organometallic RNA-B12 conjugates revealed an important contribution of entropic effects to the observed base-on to base-off switch. The two organometallic RNA-B12 conjugates thus acted as B12-retro-riboswitches and allowed the observation of a temperature-dependent reverse switch in the B12 cofactor moiety, induced by the appended nucleotide moiety. This behavior may be of interest in the "RNA-world" hypothesis, in which (simple) B12 derivatives are thought to act as possible catalytic enhancers ("cofactors") in RNA-based "B12 ribozymes".     

酚醛型吸附树脂吸附VB12的热力学研究

研究了酚醛型附树脂JDW-2对VB12的吸附。在304-322K和研究的浓度范围内，JDW-2对VB12吸附平衡数据符合Freundilch吸附等温方程。Freundilich吸附等温线和等量吸附焓表明：JDW-2对VB12吸附是吸热过程。测试了VB12在JDW-2上的吸附焓、自由能和吸附熵，并对吸附行为作了合理解释。     

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[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 B(12) (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 B(12) 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 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) 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 A 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 B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.     

Reorientational dynamics of the dodecahydro-closo-dodecaborate anion in Cs2B12H12

Rapid reorientational motions of the B(12)H(12)(2-) icosahedral anion, a key intermediate in borohydride dehydrogenation, are revealed by quasielastic neutron scattering (QENS) measurements of Cs(2)B(12)H(12) between 430 and 530 K. At 430 K, over the range of momentum transfers collected, the elastic incoherent structure factor (EISF) is consistent with a model for reorientational jumps about a single molecular axis. At temperatures of 480 K and higher, however, the EISF suggests the emergence of multiaxis reorientation by dynamically similar, independent jumps about two axes, on average, preserving crystallographic order. Alternatively, if one assumes that the anions are undergoing temperature-dependent rotational trapping, then the EISF is also consistent with a jump model involving a temperature-dependent mobile fraction of anions statistically tumbling between discrete crystallographic sites. Although neutron vibrational spectra demonstrate that the anion torsional modes soften dramatically with increasing temperature, the QENS-derived activation energy of 333 meV for reorientation clearly shows that the anions are not undergoing isotropic rotational diffusion.     

Electrochemistry of vitamin B12: Part VII. Role of the base-off/base-on reaction in the oxido-reduction in the B12r-B12s couple in non-aqueous solvents

The cyclic voltammetric study of vitamin B_12r in DMSO shows the importance of the base-on/base-off reaction in the electrochemical reduction mechanism. Depending upon the flux of electrons flowing through the system. part of the base-on complex is reduced through prior opening of the nucleotide side-chain which gives rise to the more easily reduced DMSO-Co(II) complex. The quantitative analysis of the variations of the peak heights with the sweep rate allows the thermodynamic and kinetic characterization of the base-on/base-off reactions to be determined. DMSO thus appears as a stronger ligand toward Co(II) than water, leading to an increased participation of the base-off complex in the reduction process. The greater stability of the DMSO complex is also related to the observation that electron transfer is significantly slower than in the case of the water complex. The importance of the ligand exchange reactions in the reduction of B_12r is confirmed by the effect of pyridine additions. Three complexes then participate in the reduction process, their reduction potentials lying in the order DMSO >Py >Bzm. The reduction mechanism involving the interconversion of the three complexes is described as a function of the electrode potential, the flux of electrons and the pyridine concentration. An estimation of the equilibrium and rate constants of the three ligand exchange reactions is made, based on the variations of the cyclic voltammograms with the sweep rate and the pyridine concentration.     

The electronic structure ofα-B12, B12P2 and B12AS2

The electronic band structures of the rhombohedral-based boron compounds -B₁₂, B₁₂P₂ and B₁₂ As₂ have been investigated along all symmetry directions. The calculations show that the band gap, in all cases, is of the order of 2 eV, which correlates with the known color of -rhombohedral boron. The materials should be intrinsic semi-conductors, as has recently been shown experimentally. The states around the band gap in -B₁₂ are dominated by the boron 2p atomic states. The bonding in the icosahedra, as illuminated by cluster calculations, is shown to be rather similar to that in the isolated B₁₂ icosahedron. Of the intericosahedron interactions, those between B(2) and B(2) atoms are the strongest and have a bond index just above unity. In B₁₂P₂ the orbitals of the P₂ moiety make a significant contribution to the valence band edge states and the conduction band edge states also incorporate considerable (55%) phosphorus 3d orbital character. In B₁₂As₂ the arsenic 4d orbitals do not have as much effect in that crystal as do the 3d orbitals in B₁₂P₂.     

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.