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There is much debate whether the fatty acid substrate of lipoxygenase binds "carboxylate-end first" or "methyl-end first" in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steady-state and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly pi-pi interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind "carboxylate-end first" with sLO-1 and not "methyl-end first" as has been proposed for human 15-lipoxygenase.

Herein, we report on the role of the allosteric site in the activation mechanism of soybean lipoxygenase-1 utilizing stopped-flow inhibition kinetic studies. The K(D) for the activation was determined to be 25.9 +/- 2.3 microM and the rate constant for the oxidation of the iron cofactor, k(2), to be 182 +/- 4 s(-1). Two inhibitors were employed in this study, (Z)-9-octadecenyl sulfate (OS) and (Z)-9-palmitoleyl sulfate (PS), of which OS is an allosteric inhibitor of the turnover process, while PS is a linear mixed inhibitor with a K(i) of 13.7 +/- 1.3 microM for the catalytic site and a K(i)' of 140 +/- 9 microM for the allosteric site. It was found that OS does not inhibit the activation of soybean lipoxygenase-1, while PS acts as a competitive inhibitor versus the product, 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid, with a K(i) of 17.5 +/- 3.8 microM. These results suggest that OS binds to an allosteric site that is separate from the catalytic iron site. We further observed that the allosteric site binding selectivity is sensitive to inhibitor length as seen by its preference for OS over that of PS, which is two carbons longer than PS.

The spectroscopic and kinetic evidence is presented for the reaction of methyl [beta]-methylthio-[alpha]-nitrocinnamate with piperidine, morpholine and hydroxide ion. It is found that a nucleophilic vinylic substitution by strongly basic amines leads to intermediate accumulation and that there is no accumulation in the reaction with hydroxide

A kinetic study of the reversible deprotonation of benzofuran-3(2H)-one (3H-O) and benzothiophene-3(2H)-one (3H-S) by amines and hydroxide ion in water is presented. The results have shown that aromatic stabilization of the transition state is ahead of proton transfer, which is in contrast with simple resonance effects.

A study was conducted to determine whether the nucleophilic substitution of the p-nitrophenoxy group in [(CO).sub.5]Cr=C(O[C.sub.6][H.sub.4]-N[O.sub.2])Ph by a series of substituted phenoxide ions is concerted or stepwise. The tetrahedral intermediates formed in the reactions of [(CO).sub.5]Cr=C(O[C.sub.6][H.sub.4]-N[O.sub.2])Ph are much more stable than those in acyl transfers.

An investigation of how steric effects affect the rates of hydrolysis of [alpha]-alkyl-[alpha]-(methylthio)methylene Meldrum's acids is reported. The kinetic results confirm that crowding at the transition state is an important effect but also show that for very large R groups destabilization of the substrate reverses the decreasing trend in the rate constants.

The replacement of the methylthio group of substituted methylthiobenzylidene Meldrum's acids (2-SMe-Z) by secondary alicyclic amines occurs by a three-step mechanism. A detailed structure-reactivity analysis indicates that for the reaction with piperazine, 1-(2-hydroxythyl)- piperazine, and morpholine it is deprotonation of T(sub A)(super plus or minus) to products is rate limiting.

The results relating to the deprotonation of 1-benzyl-1-methoxy-2-nitroethylenes, by amines, which include structural features in the nitroalkanes are reported. The question addressed is whether the special structural features affect the actual size of the transition state imbalance and/or its manifestation in terms of alpha and beta values.

The deprotonation of substituted (methylthiophenylcarbene)pentacarbonyl tungsten(0) ((CO)5W=C(SC6H4Z)CH3) and (benzoxymethylcarbene)pentacarbonyl tungsten(0) ((CO)5W=C(OCH2C6H4Z)CH3) in aqueous acetonitrile is studied. This indicates a transition state imbalance in which the loss of the carbene complex lags behind the proton transfer.

Proton-transfer reactions of 3H-Z get characterized by high intrinsic barriers or low intrinsic rate constants. Manifestation of imbalance is marked by the pi-donor effect of methoxyl group.

Research into carbon-to-carbon identity proton transfer from ketene, allene, thioketene and ketenimine to their respective conjugate anions in the gas phase is presented. It was demonstrated that the acidities are all higher while the barriers are lower than for the corresponding CH3CH=X series.

Results reveal that the transition states of the proton transfer reactions are not in balance despite the absence of correlation between the degree of imbalance and the pi-acceptor strength of the Y group.

The reversible formation of cyclic adducts by the intramolecular nucleophilic reactions is reported.

A study of the acid-catalyzed loss of alkoxide and thiolate ions from alkoxide and thiolate ion adducts is presented. The acid-catalyzed breakdown of the intermediate toward products and reactants has been analyzed.

The nucleophilic vinylic substitution S(sub N)V intermediate was not detected during the reaction of beta-methoxy-alpha-nitrostilbene with cyanamide (CNA) in 50% DMSO-50% water. This despite the high reactivity of 1-OMe and CNA- which was supposed to favor the presence of the S(sub N) V intermediate. What was observed is that the CNA anion is the reactive species for pH values from 8.5 to 12.4. However, competing hydrolysis prevented the reaction of neutral CNA at low pH values.

A study of the kinetic and thermodynamic acidities of Fischer carbene complexes has confirmed that thioalkoxy carbene complexes are acidic than the corresponding alkoxy complexes. The change in the pi-donor group from MeO to MeS has been shown to result in a strong increase in the thermodynamic acidity, an increase in the dependence of the pK(sub a) on the nature of the metal, and declines in the intrinsic rate constants for proton transfer as well as the dependence of the intrinsic rate constant on the metal. Results have also demonstrated that the weakened pi-donor ability of the MeS group gives rise to other important differences in the reactivity of methoxy and thiomethoxy carbene complexes.

Intrinsic rate constants were determined for proton transfer from monoketones to a family of amine bases. Specifically, the reaction of secondary alicyclic amines with 2-acetyl-1-methylpyridinium ion was examined. The intrinsic rate constants obtained were found to be lower than for any sterically unhindered carbon acids except for nitroalkanes. The results suggest the presence of a strong pi-acceptor character of the CO group and strong salvation in the enolate ion.

The pKa values of five Fischer carbene complexes in acetonitrile have been measured. Fischer carbene complexes with one or several hydrogen atoms in the alpha-carbon are moderately acidic. Pure acetonitrile was used because the carbene complexes are stable in this solvent. Its polarity is large enough such that ion pairing does not pose a problem and pKa values up to 36 can be measured. Results show that the pKa values in pure acetonitrile are 10.2 units higher than in 50% MeCN-50% water. This is due to the weaker solvation of the hydrogen ion in pure acetonitrile.

The synthesis of oligoguanylate on a polycytidylate template has been undertaken. The oligomerization starts with the dimerization of the first primer (k2) that is followed by the elongation of a preformed primer (ki) and ends with the hydrolysis of the monomer to form the deactivated material (kh). It has been observed that ki' does not depend on i when i is greater than or equal to 4 or less than or equal to 16. The rate constants k3' and ki' for oligoguanylate elongation agree with the template-directed model and enhances next-neighbor assistance.

Research was conducted to examine kinetics of the reactions of methoxybenzylidene Meldrum's acid with thiolate ions, alkoxide ions, OH-, and water in aqueous DMSO. The objective was to detect the S(sub N)V intermediate in the reaction of Meldrum's acid with nucleophiles and to determine the rate constants. Results indicate that the corresponding intermediates accumulate to detectable levels in the reactions with RS- and RO- but not in those with OH- or H2O.

An approach that combines kinetic and thermodynamic data is used to study the proton-transfer kinetics of the reaction of (phenylthio)nitromethane, PhSCH2NO2, and 1-nitro-2-phenylethane, PhCH2CH2NO2 with amines in water and 90% DMSO-10% water. It is shown that the effect of the phenylthio group on the intrinsic rate constants of proton transfer can be the basis of a distinction between various interpretations of the acidifying effect of sulfur groups. The effects of various factors on the intrinsic rate constants for proton transfer are analyzed.

Proton transfers such as the replacement of PhCO group in phenylnitromethane and hydrogen in nitromethane are observed to have a strong acidifying effect upon carbon acids. This may be attributed to factors such as solvation effects, inductive/field and resonance. In a test involving the deprotonation of PhCoCh(Ph)NO2, the behavior of the said compound is similar to phenylnitromethane as seen from intrinsic rate constants.

The pKa values for transition metal carbene complexes (CO2)5Cr=C(OMe)CH2CH3 (3-Et), (CO)5(Cr=C(OMe)CH2CH2CH3) (3-Bu) and (CO)5CR=C(OMe)CH(CH3)2 (3-Pr) in aqueous acetonitrile are reported as 12.62, 12.84 and 12.27 respectively. They are all more acidic than (CO5)Cr=C(OMe)CH3 (3-Me). In contrast to their thermodynamic acidities, 3-Et, E-Bu and 3-PR all exhibit greater rate constants than 3-Me. Other research observations are made. Kinetic reaction parameters are given together with data from spectrophotometer analysis.

A study was conducted on the thermodynamic and kinetic acidity of (2-oxacyclopentylidene)pentacarbonylchromium(0) (compound 3) in aqueous acetonitrile. The findings indicate that the expected intrinsic rate lowering effect due to the increased resonance stabilization of the compound 3 is offset by an intrinsic rate enhancing preorganization effect. Moreover, compound 3 was found to be less acidic than its acyclic analog, methoxymethylcarbenepentacarbonylchromium(0).

An ab initio study of the identity of carbon-to-carbon proton transfers between nitromethane and nitromethide anion and between oxygen protonated nitromethane and aci-nitromethane was reported. Transition state imbalances were shown to cause slight decrease in intrinsic gas phase barriers. Dependence of the barriers on the pi-acceptor was observed to be a result of complex reactions among resonance, inductive and hydrogen bonding effects.

Kinetic studies were done to characterize the first stage of the hydrolysis of (CO)5Cr=C(OMe)Ph and (CO)5Cr=(OMe)CH=CHPh in aqueous acetonitrile. It was demonstrated that the rate limiting step is the nucleophilic attack by OH- at high pH and by water at low pH that lead to the formation of a tetrahedral intermediate. In the presence of buffers, there was an observed general base catalysis of water addition.

Rate constants for the deprotonation of 2-nitro-4-X-phenylacetonitrile, 2-X (X = NO2, SO2CH3, CN, CF3, Br and Cl) by piperidine and morpholine and for the reverse reaction were determined in 90% Me2SO-10% water, 50% Me2SO-50% water and water (X = NO2, SO2CH3, CN only). There was an imbalance which is consistent with a transition state in which delocalization of the negative charge into the 2-nitrophenyl moiety lags behind proton transfer.

The identity carbon-to-carbon proton transfer between oxygen-protonated acetaldehyde (syn and anti) and acetaldehyde enol (syn and anti) was examined by ab initio methods at the 6-311+G**//6-311+G**, MP2/6-311+G**/6-311+G**, and MP2/6-311+G** levels. The transition states of all reactions under study indicate a strong imbalance because the charge shift in the product enol lags behind proton transfer and the charge shift in the reactant enol is ahead of proton transfer.

The kinetic data on the deprotonation of 2-nitroethanol and 2-nitro-1,3-propanediol which concerned the rate of reversible carbon deprotonation by hydroxide ion, water, aminews, and carboxylate ions and pKa values for the ionization at carbon, pK(super CH)(sub a), and oxygen, pK(super OH)(sub a) and ionization of the aci-forms pK(super NOH)(sub a) at 25 degree celsius is reported. The rate constants in the order nitromethane > metroethane > 2-nitropropane is also reported. The rate of proton transfer induced by the substitution of one hydrogen in CH3NO2 by a CH2OH group is also discussed.

The reaction of CF3CH2O-adduct of alpha-nitro-beta-(2,2,2-trifluoroethoxy) stilbene with aqueous acid leads to carbon protonation, generating the corresponding acetal. The reaction of acid with the MeO-adduct of beta-methoxy-alpha-nitrostilbene leads to acid catalyzed CH3O-expulsion. A mixture of acetals are produced from a reaction between acid and CF3CH2O-adducts of beta-methoxy-alpha-nitrostilbene. Kinetic studies help determine the rate constants for these competition reactions.

Kinetic studies of the nucleophilic vinylic substitution reactions of alpha-nitro-beta-stilbenes with various nucleophiles enable the determination of the rate constants for the elementary steps associated with the addition-elimination mechanism. Spectroscopic studies of the reaction of beta-methoxy-alpha-nitrostilbene with CF3CH2O- and of alpha-nitro-beta-(2,2,2-trifluoroethoxy)stilbene with HOCH2CH2S- enable the identification of intermediates. Resonance, steric, anomeric and electronic effects account for the influence of structure on the rate constants.

1H NMR and reactivity data help structural characterization of aniline adducts of alpha-nitrostilbenes. Analysis of dissociation kinetics of aniline adducts into benzalanilines and arylnitromethane anions reveals an unimolecular mechanism for dissociation. Buffer concentration and pH do not influence the rate constants. Estimates of the influence of aniline substituents on the equilibrium constant help calculate structure-reactivity parameters that indicate significant transition state imbalance for the dissociation.

Kinetic studies of reactions of various phenyl-substituted benzylidenemalonodialdehydes with water and OH(-) at various pH help determine rate and equilibrium constant for OH(-) and water addition, pKa of the enol form and the equilibrium constant of addition reaction between OH(-) and a carbonyl group. Correlation of kinetic parameters reveals the loss of resonance stabilization during the conversion of olefin into the adduct. Bronsted-type plots for p-NMe2 and p-OMe substituents reveal reduced intrinsic rate constants due to a transition state in which resonance loss precedes bond formation.

Stopped-flow method helps determine the kinetics of reactions of phenyl nitromethane with various carbanions. Reaction between phenyl nitromethane and anions of 9-cyanofluorene and 4-nitrophenylacetonitrile involves oxygen to carbon and carbon to carbon proton transfers. Fast reaction between phenylnitromethane and anions of 1,3-indandione involves oxygen to oxygen proton transfer while slow reactions with various anions involve C to O and C to C proton transfer.

Two kinetic processes, observed in the hydrolysis of benzylideneacetylacetone in a strong basic medium, include fast step in the reversible OH- addition to the substrate to form T(sub OH)(super -) and the rate-determining step in the carbon protonation of T(sub OH)(super -) to form the products. pH jump experiments determine rate constants for all forward and reversible steps and are compared with those of the benzylidenemalondialdehyde and benzylidene Meldrum's acid hydrolysis.

Ab initio calculations are employed to analyze the imbalance in the transition state of proton transfer step in the acetaldehyde to its enolate ion reaction. A charge imbalance toward charge delocalization is observed in the transition state of the gas phase reaction due to tightness and large positive charge on the protons. Imbalance in solution phase is higher than the gas phase due to solvation.

Major transition state imbalances such as those evident in the deposition of ArCH2NO2 are also evident in the water and OH(super -) addition to NS-Z and the breakdown of T(O-) to benzaldehyde and ArCH double bond NO2-. This is evident from a kinetic analysis of the hydrolysis of substituted alpha-nitrostilbenes. Intrinsic rate constants for the K, OH and K4 processes in the hydrolysis of other olefins of the type PhCH double bond CXY are high.

An analysis of the physical organic chemistry of transition metal carbene complexes involved the measurement of rate constants for proton transfer from (methoxymethylcarbene)pentacarbonylchromium(0) to OH- and amine bases, and from (benzylmethoxycarbene)pentacarbonylchromium(0), to OH- amines, carboxylate ions and H2O. Aqueous acetonitrile ensured 2- and 4.8-fold enhancements in the deprotonation rate of (CO)5Cr double bond C(OCH3)CH3 by OH- and piperidine.

The oligomerization kinetics of guanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpG, in the presence of polycytidylate, poly (C), functioning as a template at 23 degrees Celsius, were analyzed. The presence of additional next-neighbor monomer units aids bond formation between two monomer units or between a primer and a monomer.

Researchers conducted kinetic analysis of the reversible deprotonation of 9-cyanofluorene (2), 1,3-indandione (3), 4-nitrophenyl-acetonitrile (4), (3-nitrophenyl) nitromethane (5), and (4-nitrophenyl) nitromethane (6) by the aminos of replaced benzylmalononitrile (1-X) in 90% Me2SO-10% water (v/v) at 20 degrees centigrade. Extrapolation or interpolation of Bronsted plots, whose slopes (beta) are all close to 0.5, identified the intrinsic rate constants and hindrances in these reactions.

The kinetics of aminolysis of a Fischer metal carbene complex, (methoxy(phenyl)carbene)pentacarbonylchromium(O) in a polar solvent is examined. The transition metal carbene complex composed of five alipathic amines is measured in 20% acetonitrile and 80% water at 25 degrees centigrade. Results indicate a general base catalysis since the second order constants increased as amine and hydroxide ion concentrations increased. The mechanisms involved in the process of aminolysis are discussed.

The deprotonation of arylnitromethanes by alkyl thiolate and aryloxide ions serves as basis for a study of factors affecting proton abstraction from a carbon acid. The kinetic study points to the hydrogen-bonding ability of the central atom and, where the former is negligible, the demand for partial transition state desolvation of the base as the two strongest such factors. The similarityin the transition state imbalances of this reaction with that of amines suggestthat they are not characteristic of the thiolate ions but of the acid-base interactions that affect nucleophilic additions but not proton transfers.

The kinetic data on the reactions of mercaptoethanol's and methyl mercaptoacetate's anions with Z-substituted beta-nitrostyrenes indicate that the reaction does not follow a radicaloid transition state. Moreover, the results show that non-pi-donor groups have log K1 values that are consistent with the Hammett plots. However, the pi-donor groups are found to create negative deviations, which result from olefin resonance stabilization, in the Hammett plots' equilibrium constants. In addition, the analysis of the beta-nitrostyrene's and 3-cyano-beta-nitrostyrene's RS- basicity leads to the identification of the Bronsted parameters and intrinsic rate constants.

The reactions of phenyl-substituted beta-nitrostyrenes with hydroxides and nitromethides in different Me2SO-water mixtures have been examined in order to determine the kinetics that characterize the nucleophilic addition. An increase in the resonance stabilization of substituted beta-nitrostyrenes results in a decrease in the equilibrium constants of hydroxide and nitromethide ions. In addition, the through resonance-induced preorganization of the electronic structure promotes a negative charge delocalization of the transitional state's nitro group and an increase in the reaction's intrinsic rate constant.

The benzoate ion promoted deprotonation reactions of arylnitromethanes in acetonitrile solution are investigated. Solvent effects on the second-order rate constants results from the combined equilibrium solvation of the transition state and various Principle of Nonperfect Synchronization (PNS) effects generated by the nonequilibrium solvation of different portions of the transition state. A PNS analysis of the conversion of Me2SOn to MeCN suggests that the observed rate increase is an indication of the large contribution of equilibrium solvation effect.