An ab initio computational study of thiamin synthesis from gaseous reactants of the interstellar medium

A set of chemical reactions is proposed to account for the formation of thiamin derivatives from gaseous reactants that have been identified in the interstellar medium, and may have been relevant to a prebiotic atmosphere. The gaseous mixture consisted of methanimine, acetonitrile, cyanoacetylene, ammonia, acetylene, allylene, hydrogen sulfide, thioformaldehyde, and hydrogen in the presence of water. Most of the reactions appear to be exothermic. The reactions have been shown to be feasible from the overall enthalpy changes in the ZKE approximation at the HF and MP2/6-31G(*) level.     

Asymmetric oxidative coupling of hydroxycarbazoles: Facile synthesis of (+)-bi-2-hydroxy-3-methylcarbazole

Asymmetric oxidative coupling reactions of hydroxycarbazoles have been established using a chiral dinuclear vanadium complex. To demonstrate the utility of vanadium-catalyzed reactions, we have used them to synthesize (+)-bi-2-hydroxy-3-carbazole in three steps from cyclohexanone and commercially available aniline derivatives.     

Biochemistry of terminal deoxynucleotidyltransferase: identification, characterization, requirements, and active-site involvement in the catalysis of associated pyrophosphate exchange and pyrophosphorolytic activity

Terminal deoxynucleotidyltransferase (TdT) has been found to catalyze both pyrophosphate exchange and pyrophosphorolysis reactions. Both reactions are strongly inhibited by antiserum to TdT. The reactions require the presence of a divalent cation, a single- or double-stranded oligomeric or polymeric DNA or RNA, and deoxyribonucleoside triphosphates (for PPi exchange only). Of the three divalent cations tested, Mg2+ and Co2+ are equally effective, while Mn2+ neither is used for catalysis nor inhibits the Mg2+-catalyzed reactions. Ribonucleoside triphosphates have been found to support the PPi exchange reaction to a minor extent and have no inhibitory effect on the catalysis mediated by dNTPs. Inhibition studies, using SH group inhibitors, Zn chelator, and a substrate binding site specific reagent, revealed that PPi exchange and pyrophosphorolysis reactions may be distinguished by differences in their sensitivity to inhibition by various reagents. While the PPi exchange reaction is strongly inhibited by sulfhydryl reagents, o-phenanthroline, and pyridoxal phosphate, the pyrophosphorolysis reaction is insensitive to these reagents. In addition, the pyrophosphorolysis reaction is also found not to require a free 3'-OH terminus of a primer. This difference in the susceptibility of the two reactions indicates that discrete active-site structures exist in TdT which catalyze PPi exchange and pyrophosphorolysis reactions.     

Advances in studying bioinorganic reaction mechanisms: isotopic probes of activated oxygen intermediates in metalloenzymes

Metalloenzymes catalyze reactions of molecular oxygen and its reduced forms through the controlled formation of metal-bound, activated oxygen intermediates. These intermediates have been a challenge to characterize and new experimental approaches capable of relating structure to reactivity under physiologically relevant conditions are needed. The application of a competitive isotope fractionation technique has enabled changes in O-O bonding to be probed during enzyme-catalyzed reactions. The derived isotope effects provide insights into the reaction mechanisms of O2 and O2*-, which probably could not have been obtained using more conventional methods.     

Kinetic studies of mobilization of copper(II) from human serum albumin with chelating agents

The kinetics of the mobilizing reactions of five chelating agents for human serum albumin (HSA)-bound copper(II) [Cu(II)] have been studied spectrophotometrically. The decreasing sequence of reaction rate has been determined to be EDTA greater than DTPA greater than EGTA greater than NTA greater than IDA. A group of mathematical models were established to define the mechanisms of the competitive reactions between low-molecular-weight ligand and macromolecular ligand. All reactions of the five chelating agents follow a process involving the intermediate ternary complexes: (formula; see text) The reactions of DTPA and EDTA were found to be different from those of EGTA, NTA, and IDA. In the former cases, the reactions are likely following an overlapping mechanism in which the rate constant k1 was closed to k2. The reactions involving the other three chelators are different in k1 much greater than k2.     

Thermodynamic analysis of the interaction of the acridine dye proflavin with deoxytetranucleotides with various base sequences from (1)H-NMR data]

Temperature dependences of proton chemical shifts of the molecules in aqueous solutions of proflavine with deoxytetraribonucleoside triphosphates of different base sequence--5'-d(CGCG), 5'-d(GCGC), 5'-d(ACGT) and 5'-d(AGCT) have been studied by 500 MHz NMR spectroscopy. Methods of calculation of thermodynamical parameters of complex formation, giving an opportunity to differentiate the contributions of the reactions leading to the formation of different types of complexes in conditions of the composite equilibrium in solution, have been suggested. Enthalpies and entropies of the reactions of 1:1, 2:1, 1:2, 2:2 complexes formation between proflavine and tetranucleotides have been determined. Comparative analysis of the calculated thermodynamical parameters has been made and assumptions about the nature of intermolecular interactions responsible for the formation of dye complexes with different tetranucleotides have been proposed.     

Synthesis and biological evaluation of new opioid agonist and neurokinin-1 antagonist bivalent ligands

Newly designed bivalent ligands-opioid agonist/NK1-antagonists have been synthesized. The synthesis of new starting materials-carboxy-derivatives of Fentanyl (1a-1c) was developed. These products have been transformed to 'isoimidium perchlorates' (2a-c). The new isoimidium perchlorates have been successfully implemented in nucleophilic addition reactions, with l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester to give the target compounds-amides (3a-c). Perchlorates (2a-c) successfully undergo reactions with other nucleophiles such as alcohols, amines or hydrazines. The obtained compound 3b exhibited μ-opioid agonist activity and NK1-antagonist activity and may serve as a useful lead compound for the further design of a new series of opioid agonist/NK1-antagonist compounds.     

The disequilibrium pH: a tool for the localization of carbonic anhydrase

The disequilibrium pH is defined as any discrepancy between the measured pH and the pH which would exist if CO2-HCO3-H+ reactions were at equilibrium. Measurement of the disequilibrium pH can be used to assess the status of CO2-HCO3--H+ reactions and, in combination with carbonic anhydrase (CA) or CA inhibitor treatments, may also be used to localize CA. Renal physiologists have used disequilibrium experiments to determine that HCO3- reabsorption in the kidney tubule occurs via proton secretion, and that CA activity is available to ultrafiltrate CO2-HCO3-H+ reactions in the proximal convoluted tubule, but not the distal tubule. Disequilibrium experiments were also used in investigating the availability of CA to CO2-HCO3--H+ reactions in water at the fish gill; the opposing results obtained in two studies have not yet been resolved. Respiratory physiologists have used the disequilibrium technique in vivo and with saline-perfused preparations to assess the availability of CA to plasma CO2-HCO3--H+ reactions following gas exchange. Saline-perfused preparations enable direct localization of CA activity, while in vivo measurements encompass the numerous factors affecting CO2-HCO3--H+ equilibration in a multi-phase solution. Given the many organs in which membrane-bound CA activity has now been identified, the usefulness of the disequilibrium pH technique has increased beyond its original applications in renal and pulmonary physiology.     

Kinetic parameters governing the formation of eIF-2.methionyl-tRNAi complexes in protein synthesis

Published data have been analysed to determine the rate constants governing the exchange of GDP in the complex of the eukaryotic protein synthesis initiation factor eIF-2 with GDP, catalysed by eIF-2B. The interaction of eIF-2B with eIF-2.GDP appears to include a very high 'on' rate constant of up to 4 x 10(8) M-1 sec-1 - a value very similar to that found by others for the interaction of the bacterial elongation factors Tu and Ts. Assuming a substituted enzyme mechanism that leads to displacement of GDP and ultimately to formation of a quaternary complex eIF-2B.eIF-2.GTP.methionyl-tRNA, minimum rate constants have been estimated for the additional reactions assuming in vivo rates of protein synthesis. Rate constants for the other reactions are unexceptional.     

Anaerobic oxidation of aromatic compounds and hydrocarbons

Aromatic compounds and hydrocarbons have in common a great stability due to resonance energy and inertness of CbondH and CbondC bonds. It has been taken for granted that the metabolism of these compounds obligatorily depends on molecular oxygen. Oxygen is required first to introduce hydroxyl groups into the substrate and then to cleave the aromatic ring. However, newly discovered bacterial enzymes and reactions involved in oxidation of aromatic and hydrocarbon compounds to CO(2) in the complete absence of molecular oxygen have been discovered. Of special interest are two reactions: the reduction of the aromatic ring of benzoyl-coenzyme A and the addition of fumarate to hydrocarbons. These reactions transform aromatic rings and hydrocarbons into products that can be oxidized via more conventional beta-oxidation pathways.     

Hydroformylation of alkenes and alkynes using a heterobinuclear Rh---W catalyst

Hydroformylation reactions of a series of alkenes and alkynes have been carried out using the heteronuclear Rh---W catalyst, (CO)₄ hH(CO)(PPh₃) (1). The results of these reactions have been compared with corresponding reactions using [Rh(OAc)₂]₂ as catalyst. Catalysis of a reaction of styrene using 1 gave a very high yield of the branched chain aldehyde containing only a trace of the straight chain isomer. Reactions of the phosphinoalkene, Ph₂P(CH₂)₃CH=CH₂ (7) and the corresponding alkyne, Ph₂P(CH₂)₃CCH (11) gave similar products using either catalyst system with the alkryne reaction being significantly slower. Reaction of the alkenyl dithiane, H---CH₂CH=CH₂ (2), using the Rh---W catalyst (1) gave a higher ratio of linear to branched aldehydes (47 linear:53 branched) than the corresponding reaction using [Rh(OAc)₂]₂ (25 linear:75 branched). Reactions of vinyl acetate using 1 as catalyst gave a significant amount of linear aldehyde in contrast to reactions using [Rh(OAc)₂]₂ but reactions of allyl acetate gave similar products for both catalyst systems.     

A discussion of the chemistry of oxidative and nitrosative stress in cytotoxicity

Nitric oxide (NO) has been shown to be a key bioregulatory agent in a wide variety of biological processes, yet cytotoxic properties have been reported as well. This dichotomy has raised the question of how this potentially toxic species can be involved in so many fundamental physiological processes. We have investigated the effects of NO on a variety of toxic agents and correlated how its chemistry might pertain to the observed biology. The results generate a scheme termed the chemical biology of NO in which the pertinent reactions can be categorized into direct and indirect effects. The former involves the direct reaction of NO with its biological targets generally at low fluxes of NO. Indirect effects are reactions mediated by reactive nitrogen oxide species, such as those generated from the NO/O2 and NO/O2- reactions, which can lead to cellular damage via nitrosation or oxidation of biological components. This report discusses several examples of cytotoxicity involved with these stresses.     

Synthesis and cytotoxicity of new aminoterpenylquinones

Several 6(7)-alkyl-1,4-naphthoquinones (NQ) have been prepared by cycloaddition reactions between the monoterpene alpha-myrcene and p-benzoquinones and halogen and nitrogen-containing functional groups have been introduced at the C-2 position of the naphthoquinone ring via nucleophilic addition or substitution reactions. These substituents at positions 2/3 of the NQ clearly influence the cytotoxic potency of this type of compound. Of particular interest is substitution by arylamino, specifically p-oxyarylamino, groups, which considerably enhance their bioactivity and selectivity.     

The Reactions of Methanimine and Cyanogen with Carbon Monoxide in Prebiotic Molecular Evolution on Earth

A primeval atmosphere is proposedcontaining simple molecules such as formaldehyde, ammonia, carbon monoxide, cyanogen andhydrogen cyanide, which have been detected in space. Chemical reactions aredescribed for the formation ofaziridine-2-one and di-azirine-3-one derivatives aspotential precursors for the original synthesesis of amino-acids, proteins, pyrimidines,purines, nicotinamide and flavin. The reactions have been shown to be kinetically feasiblefrom the overall enthalpy changes in the ZKE approximation at the MP2/6-31G^* level.     

Carbenic vs. ionic mechanistic pathway in reaction of cyclohexanone with bromoform

The extensive computation study was done to elucidate the mechanism of formation dibromoepoxide from cyclohexanone and bromoform. In this reaction, the formation of dihaloepoxide 2 is postulated as a key step that determines the distribution and stereochemistry of products. Two mechanistic paths of reaction were investigated: the addition of dibromocarbene to carbonyl group of ketone, and the addition of tribromomethyl carbanion to the same (C=O) group. The mechanisms for the addition reactions of dibromocarbenes and tribromomethyl carbanions with cyclohexanone have been investigated using ab initio HF/6-311++G** and MP2/6-311+G* level of theory. Solvent effects on these reactions have been explored by calculations which included a continuum polarizable conductor model (CPCM) for the solvent (H(2)O). The calculations showed that both mechanisms are possible and are exothermic, but have markedly different activation energies.     

The properties of precipitation reactions between secretion products in the oviduct of pleurodeles: identification of a lectin

The properties of two precipitation reactions occurring between secretory products from the oviduct of Pleurodeles waltl have been studied. It has been demonstrated that a lectin is involved in one of the reactions. This lectin precipitated glycogen and starch and required calcium; the most potent saccharide inhibitors were 2-amino-2-deoxy-D-glucose and D-glucose, respectively. The other reaction was related to glycoproteins (probably sulfated glycoproteins) that contained sulphur. The properties of this reaction were not the same as purely ionic interactions; basic protein-acidic polysaccharide interactions have been compared. A lectin was probably implicated but this could not be demonstrated because no saccharide inhibitor was found. There are several similitudes between this reaction and the lectin-galactoside reaction which occurs in the reaction between cortical granule content and egg jellies in anurans.     

On-line mass spectrometry: membrane inlet sampling

Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O₂ evolution versus O₂ uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H₂ generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.

     

The oxidative part of the glucose-oxidase reaction

1. Kinetic parameters of the oxidative part of glucose-oxidase reaction have been measured with 16 different electron-acceptors and glucose as a substrate. 2. In each case, the rate-limiting portion of the oxidative part of reaction was the formation of the E-FADH2.Acceptor-complex; this rate was pH-independent around the pH-optimum of the enzyme. 3. In each case, E-FADH2 acceptor-complex was undetectable in the steady-state kinetics, with the exception of cytochrome-c. 4. The rates of redox reactions between various forms of reduced 5-ethyl-lumiflavin and five different electron-acceptors have been examined with a conventional spectrophotometry. In each case, it was found that the reactions proceeded at high rates whenever thermodynamically feasible, and were totally prevented in the opposite case. 5. Molecular oxygen was able to oxidize only the neutral form of 5-ethyl-1,5-dihydrolumiflavin to its radical form, at a moderate rate; all other forms of reduced 5-ethyl-lumiflavin were not oxidized by O2. 6. By the comparison of enzymatic and model redox reactions, it was possible to establish the minimal mechanism of the oxidative part of the glucose-oxidase catalytic cycle.     

Free radical reactions with proteins and enzymes: the inactivation of pepsin.

The reactions of free radicals produced by ionizing radiation with pepsin have been studied by steady-state inactivation measurements and by pulse radiolysis. In de-aerated solutions thehydroxyl radical has been found to be the most efficient of the primary free radicals generated from water in causing inactivation. The reactions of the more selective oxidizing inorganic radical anions Br2-. and (SCN)2-., with pepsin have also beenexamined. In the case of the thiocyanate radical anion (SCN)2-., the inactivation efficiency is found to depend on SCN- concentration, an effect shown to arise from a reversible redox reaction involving the tryptophan and (SCN)2-. radicals. The results demonstrate that tryptophan residue plays an essential role in the enzyme activity of pepsin.     

Kinetics of reactivation during refolding of guanidine-denatured pancreatic ribonuclease A

The method aforementioned (Liu, W. and Tsou, C.L. (1987) Biochim. Biophys. Acta 916, 455-464) for the study of the kinetics of irreversible modification of enzyme activity has been applied to the reactivation of guanidine-denatured ribonuclease A, by following the hydrolysis of cyclic CMP during refolding upon diluting a guanidine-denatured enzyme with a substrate-containing buffer. Appropriate equations have been derived to deal with the kinetics of the substrate reaction during the course of activation, while the product formed, 3'CMP, is a competitive inhibitor. When the overall process consists of multiple first-order reactions, the individual rate constants could be obtained by suitable semilogarithmic plots. Moreover, in certain cases, it can be distinguished from the shapes of the plots, whether the overall process consists of parallel or consecutive first-order reactions. The kinetics for the reactivation reaction has been compared to that for the refolding of the substrate binding site, as indicated by complex formation with the competitive inhibitor, 2'CMP, and for the refolding of the molecule as a whole. At pH 6.0 and 25 degrees C, only monophasic first-order reactions could be detected by manual mixing for both the reactivation and the refolding processes. At lower temperatures (0-10 degrees C), both processes consist of two first-order reactions. In all cases, the same rate constants have been obtained for the refolding and reactivation reactions.