Solvent-induced anomeric diastereoselectivity switching using a single glycosyl donor

Highly diastereoselective glycosylation reactions have been developed; however, not all glycosylation reactions are diastereoselective and these reactions have probably not been reported. For some fucosylation reactions, unusually low or abnormally opposite selectivities have been demonstrated. In the present study, the fucosylation reaction of long-chain hydrocarbon alcohols, ethyl 9-hydroxynonanoate and decanol using a series of the 2-O-benzyl-protected fucopyranosyl donors were investigated. The resulting products demonstrated the solvent-induced diastereoselectivity switching using diethyl ether (Et₂O) or dichloromethane (CH₂Cl₂). Practical α-selectivities were observed using ether solvents. In contrast, practical β-selectivities were observed using CH₂Cl₂. The anomeric diastereoselectivity switching was similarly observed in the alcohol galactosylation reaction. The larger spin-lattice relaxation time constant (T₁) actually indicated that molecular motion of ethyl 9-hydroxynonanoate was more vigorous in Et₂O than in CH₂Cl₂, suggesting its dissociation in Et₂O and association in CH₂Cl₂. The bulkiness of the associated alcohols is most likely responsible for the observed diastereoselectivity.     

Kinetics of batch-wise enzymatic cycling system for mass production of chiral compound

Kinetics of batch-wise enzymatic cycling system (oxidoreductase-catalyzed reaction system involving enzyme-coupled cofactor regeneration) has been studied covering a broad range of the conserved total cofactor concentration, [C]₀ (=NAD(P)⁺?+?NAD(P)H), based on reasonable several assumptions. It is composed of two elementary reactions, i.e. product synthesis reaction and cofactor regeneration reaction, both of which have been expressed by Michaelis–Menten type rate equations. A novel dimensionless variable, r, has been introduced, which is defined as the concentration of one of the two cofactor components, [X] (NADH⁺ or NADPH⁺), divided by [C]₀, i.e. r .e[X]/[C]₀. The following results have been obtained. (1) The fundamental equation of the batch-wise enzymatic cycling system has been transformed to a differential equation whose formula is: dr/dT?=?N(r)/D(r) (N(r) and D(r) are quadratic equations of r having different coefficients). (2) It has been elucidated that the batch-wise enzymatic cycling system has two phases, an early short transient phase followed by a long phase in quasi-steady state (QSS). (3) In the enzymatic cycling system, r converges to a definite level regardless of any initial value of r. (4) In QSS, the definite level of r nearly equals the singular solution, r_singular, of the differential equation. (5) The actual rate of the targeted product (chiral compound) formation can be calculated by Michaelis–Menten equation in which the cofactor concentration is [C]₀×r_singular instead of [C]₀. r_singular has been proposed to name “redistribution factor”. (6) It is recommended that the “unit” of the cofactor regeneration enzyme be 2–3 times more used than the “unit” of the synthesis enzyme and that [C]₀ be 15–25 times more than the K_m value. Four special cases relating to the batch-wise enzymatic cycling system have been discussed.     

Rate Equations and Product Yields of Serine Proteinase Catalysed Transfer Reactions

The steady state rate equations of transfer reactions catalysed by enzymes that follow the serine proteinase reaction mechanism in their hydrolysis reactions, have been solved and integrated. The integrated equations allow for calculations of maximal yields of product and of the time, t_max, at which that yield is present in a given reaction mixture. These important quantities have not been dealt with in previous theoretical studies of such systems.     

A mathematical model for the synthesis of Gly-Phe by papain in an aqueous-organic system

The synthesis of the protected dipeptide BocGlyPheOMe, has been modellised when working in an aqueousorganic biphasic system, with papain as a catalyst. The mathematical model takes into account that one of the substrates, PheOMe, has parallel hydrolysis reactions and that the reaction only takes place in the aqueous phase while the whole reaction system is biphasic. The reaction system has been modellised when working in batch as well as when working in fed-batch mode, achieving a good prediction of the product evolution for both working strategies. When working in fed-batch mode, the extension of the undesired parallel reactions has been diminished, the model has been used for a computer aided optimisation of the addition sequence of PheOMe. The results obtained led to a process operation strategy with a compromise between yield and productivity.List of Symbols [i] concentration of any component i - [i] _aq concentration of i in the aqueous phase - [i] _bi concentration of i in the biphasic system - [E] ₀ initial concentration of enzyme - k _e, k_q first order kinetic constants - K _A, K_B equilibrium constants - r _m maximum rate of reaction This worked was financed by the Interministerial Commission for Science and Technology (CICYT)from the Spanish Government under projects number BIO/88-370 and SAF92-0261-CO2-02.     

Reactivity of Nitric Oxide with Cytochrome c Oxidase: Interactions with the Binuclear Centre and Mechanism of Inhibition

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the K _i being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized Cu_B leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from Cu_B to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.     

Kinetics and Mechanism of the Peptide Synthesis in Solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: = kC _N ^a C _AE ^b , where k is the reaction rate constant, C _N is the concentration of leucinamide, and C _AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.     

Stepped water activity control for efficient enzymatic interesterification

The benefits of controlling water activity, a _w, during enzymatically catalysed synthesis reactions, such as reverse-hydrolytic reactions promoted by lipases, are now well recognized. Numerous techniques for controlling a _w in the laboratory and their implementation in continuous reactors have been discussed in the published literature. However, in enzymatic interesterification reactions, such as acidolysis and transesterification, it is not appropriate merely to maintain the a _w of the reaction system at one value since the two stages of the reaction, namely the cleavage of the original acyl bond and the formation of a new one, are best carried out at different levels of water activity – the former at a high a _w and the latter at a lower one. The use of a continuous packed-bed hollow-fibre reactor has been described in this article for carrying out solvent-free acidolysis of ethyl laurate with octanoic acid with in situ a _w control, using air that has been pre-equilibrated with saturated salt solutions to the desired a _w. At a single optimum (a _w = 0.54), the highest steady-state conversion to ethyl octanoate was 32%. However, it is possible to obtain a steady-state conversion of 46% by operating the reactor with a step change in the water activity, from an initial value of unity to 0.23. Received: 10 February 1998 / Received revision: 2 June 1998 / Accepted: 7 June 1998     

Gas-Phase Reactions in Extraterrestrial Environments: Laboratory Investigations by Crossed Molecular Beams

We have investigated gas-phase reactions of N(²D) with the most abundant hydrocarbons in the atmosphere of Titan by the crossed molecular beam technique. In all cases, molecular products containing a novel CN bond are formed, thus suggesting possible routes of formation of gas-phase nitriles in the atmosphere of Titan and primordial Earth. The same approach has been recently extended to the study of radical–radical reactions, such as the reaction of atomic oxygen with the CH₃ and C₃H₅ radicals. Products other than those already considered in the modeling of planetary atmospheres and interstellar medium have been identified. Presented at: National Workshop On Astrobiology: Search For Life In The Solar System, Capri, Italy, 26 to 28 October, 2005.     

The kinetics of linear systems with special reference to periodic reactions

It is shown on the basis of (1) conservation of mass, (2) positive concentrations, and (3) the principle of detail balancing that periodic reactions cannot occur in a closed system described bylinear differential equations. The matrix,A, of the rate equations must be such that |A|=0,a _ij>0 fori≠j,a _ii<0, andVAV ⁻¹=B, whereV is diagonal andB is symmetric. These properties ofA imply that the latent roots are real and non-positive and that neither catalysis nor inhibition can be described bylinear equations. It is further shown that periodic reactions cannot occur in anopen system for which the matrix associated with the chemical reactions has the above properties and in which thesimple law of diffusion is obeyed. The relation of these results to Onsager's reciprocal relations and to previous work on periodic and cyclic chemical reactions is discussed. The utility of certain of these results for the treatment of isotope kinetics is indicated. A portion of this work was performed while the author was in the Department of Physiology, University of Chicago, and was supported by a grant from the Dr. Wallace C. and Clara A. Abbott Memorial Fund.     

Interrelationship among specific rates of cell growth,substrate consumption,and metabolite formation in some simple microbial reactions producing primary metabolites

Applying the carbon balance principle, the interrelationship between ν = μ/Y + m (μ is the specific growth rate of microorganism, v is the specific substrate consumption rate) and π = Aμ B (Luedeking–Piret eqyuation, π is the specific metabolite formation rate) has been established for three types of simple microbial reactions. Equations for the kinetic parameters A and B have been proposed for each of the three types of microbial reactions, Expresses in terms of γ_x, γ_s and γ_p (carbon contents of dry cell, mass, major carbon energy source, and metabolite) as well as the parameters Y and m. Values of both A and B calculated from the proposed equations were compared with their experimental data for lactic acid fragmentation, aerobic SCP production, and alcohol fermentation. The estimated values agreed with the observed ones with reasonably small deviations.     

Mechanism for unwinding of double-helical polynucleotides by formaldehyde

We have formulated a mathematical model for the unwinding process of polynucleotides induced by formaldehyde by making use of the master equation approach. Our model assumes that the formaldehyde kinetics follows the helix–coil theory of Zimm and Bragg. The model incorporates experimental features such as the interplay between pH-dependent and -independent reactions and the dependence of the initial and final helix stability on the unwinding. That is, our model incorporates the existence of the critical point such that if the pH of the system is high enough (i.e., pH ? 7), the unwinding of polynucleotides occurs by means of two separate chemical reactions, either by the HCHO–imino reaction alone or by the HCHO–amino reaction alone. The critical point depends on the ionic strength, temperature, and the formaldehyde concentration satisfying the relation s = 1 + K_Uλ, where s is the helix stability parameter, K_U is the binding constant for the HCHO–imino reaction, and λ is the formaldehyde concentration. Thus above the critical point, i.e., s < (1 + K_Uλ), the unwinding is due to the reaction of imino groups with formaldehyde, and below the critical point, i.e., s > (1 + K_Uλ), the unwinding is due to the reaction of amino groups with formaldehyde. Although, below the critical point, the imino group does not participate directly in the rate-determining step, it participates indirectly in such a manner that it reduces the initial helix stability parameter s to s/(1 + K_Uλ) by forming a complex. The kinetic constants in the master equation have been determined by making use of the principle of detailed balance and the breathing mechanism proposed in the past. With this model we have quantitatively explained the anomalous temperature dependence observed in the kinetics of formaldehyde-induced poly[d(A-T)] and the salt dependence observed in poly(A·U).     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates

Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox‐dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non‐invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H₂ and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase‐mediated reactions simultaneously. Using the proposed system, we confirmed that H₂ (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D₂/H₂O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (k_out) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.     

Possibility of the Nonenzymatic Browning (Maillard) Reaction in the ISM

The possibility of the occurrence of the nonenzymatic browning reaction in the gaseous phase in the interstellar medium has been investigated by using Density Functional Theory computations. Mechanisms for the reactions between formaldehyde (Fald) + glycine (Gly), Fald + NH ₃ and Fald + methylamine (MeAm) have been proposed, and the possibility of the formation of different compounds in the proposed mechanisms has been evaluated through calculating the Gibb's free energy changes for different steps of the reaction, by following the total mass balance. The Fald + Gly reaction under basic conditions is found as the most favorable for producing 1-methyl-amino methene or 1-methyl-amino methelene (MAM). The reaction under acidic conditions is found to be the least favorable for producing MAM. The Fald + NH ₃ reaction is found to be plausible for the production of MeAm, which can participate by reaction with Fald, resulting in the formation of MAM.     

Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants

Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T₁ progeny contained T-DNA. Molecular characterization of one primary transformant and its T₁ and T₂ progeny plants established that T-DNA was integrated into the host genome.     

Synthesis of thiazol-2-imines from the reduction of single enantiomer 2-imino-thiazolidin-4-ones followed by a spontaneous water elimination

Chiral hemiaminals ( 5-8RR and 5-8SS ) have been synthesized from the corresponding 2-iminothiazolidine-4-ones ( 1-4RR and 1-4SS ) by LiAlH₄ reductions stereoselectively and were then converted to single enantiomer thiazol-2-imines ( 9-12RR and 9-12SS ) by a water elimination reaction. The kinetics of the dehydration reactions which occurred spontaneously both in the solid state and in the solution have been followed by time dependent ¹H nuclear magnetic resonance spectroscopy. The corresponding first order rate constants and free energies of activation values for the conversions have been reported.     

Catalytic reactions in the solar nebula: Implications for interstellar molecules and organic compounds in meteorites

Organic compounds in meteorites seem to have formed by Fischer-Tropsch-type, catalytic reactions of CO, H₂, and NH₃ in the solar nebula, at 360–400K and (4–10)×10^–6 atm. The onset of these reactions was triggered by the formation of catalytically active grains of magnetite and serpentine at these temperatures.Laboratory experiments show that the Fischer-Tropsch reaction gives a large kineticisotope fractionation of C¹²/C¹³, duplicating the hitherto unexplained fractionation in meteorites. All of the principal compound classes in meteorites are produced by this reaction, or a variant involving a brief excursion to higher temperatures. (1) normal, mono-, and dimethylalkanes (2)arenes andalkylarenes; (3) dimericisoprenoids from C₉ to C₁₄; (4)purines andpyrimidines, such as adenine, guanine, uracil, thymine, xanthine, etc.; (5)amino acids, including tyrosine and histidine; (6)porphyrin-like pigments; (7) aromaticpolymer with –OH and –COOH groups.These reactions may also have played a major role in the evolution of life: first, by converting carbon to a sufficiently non-volatile form to permit its accretion by the inner planets; second, by synthesizing organic compounds on the primitive planets whenever CO, H₂, NH₃, and clay or magnetite particles came together at the right temperature. Similar reactions in other solar nebulae may be the source of interstellar molecules, as first suggested by G. H. Herbig. Ten of the twelve polyatomic interstellar molecules have in fact been seen in these syntheses or in meteorites.This paper is a revised and abridged version of the author's article inScience 182 (1973), 781.     

Theoretical studies on identity SN2 reactions of lithium halide and methyl halide: A microhydration model

Reactions of lithium halide (LiX, X = F, Cl, Br and I) and methyl halide (CH₃X, X = F, Cl, Br and I) have been investigated at the B3LYP/6-31G(d) level of theory using the microhydration model. Beginning with hydrated lithium ion, four or two water molecules have been conveniently introduced to these aqueous-phase halogen-exchange S_N2 reactions. These water molecules coordinated with the center metal lithium ion, and also interacted with entering and leaving halogen anion via hydrogen bond in complexes and transition state, which to some extent compensated hydration of halogen anion. At 298 K the reaction profiles all involve central barriers ΔE _cent which are found to decrease in the order F > Cl > Br > I. The same trend is also found for the overall barriers (ΔE _ovr ) of the title reaction. In the S_N2 reaction of sodium iodide and methyl iodide, the activation energy agrees well with the aqueous conductometric investigation.     

A Non-radioisotopic Anion-exchange Chromatographic Method to Measure the CO2/O2 Specificity Factor for Ribulose Bisphosphate Carboxylase/Oxygenase

A non-radioisotopic anion-exchange ion chromatographic method for measuring the carboxylation/ oxygenation specificity (τ) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is presented. The assay measures the amounts of fixation products at varying [CO₂]/[O₂] ratios to measure the relative rates of CO₂ and O₂ fixation reactions. The amount of 3-phosphoglycerate (3-PGA) and phosphoglycolate (PG) in the reaction mixture were measured with a conductivity detector and the specific factor was calculated using the following equations: ν_c = ([3-PGA] – [PG])/2 and ν_o = [PG]. By this method, specificity factors for RubisCOs were measured without using radioactive reagents.