Complementary and synergistic roles in enzyme-catalyzed regioselective and complete hydrolytic deprotection of O-acetylated ß-D-glucopyranosides of N-arylacetohydroxamic acids.

An efficient chemoenzymatic synthesis of ß-D-glucopyranosides of N-arylacetohydroxamic acids 3a-c was achieved by the chemoselective O-deacetylation of 1a-c under mild, neutral conditions, with no accompanying N-deacetylation. Lipase AS Amano from Aspergillus niger (LAS) and carboxylesterase from Streptomyces rochei (CSR) played complementary, synergistic roles in the O-deacetylation of 1a and its partially O-deacetylated intermediates. An intramolecular O-acetyl migration, which proceeded simultaneously, also accelerated the overall reaction rate. Under weakly acidic conditions at pH 5.0, where the intramolecular O-acetyl migration is markedly slower, LAS, CSR, and porcine liver esterase (PLE) exhibited different regioselective O-deacetylation activities. LAS and PLE showed regioselective 3-O-deacetylation and 2-O-deacetylation activity, respectively, for 1a and its tri-O-acetyl derivatives (4-7). CSR showed marked preferences for 3-O-deacetylation of 2,3,6-tri-O-acetyl intermediate 5 and 4-O-deacetylation of 2,4,6-tri-O-acetyl intermediate 6. In contrast, CSR showed almost no O-deacetylation activity toward the other tri-O-acetyl intermediates 4 and 7, which were efficiently O-deacetylated by LAS in a complementary manner. Using these enzyme-catalyzed regioselective O-deacetylation as well as chemical methods, we could synthesize all 14 partially O-acetylated intermediates (4-17) derived from 1a.

Structure-activity relationships for the degradation reaction of 1-beta-O-acyl glucuronides. Part 2: Electronic and steric descriptors predicting the reactivity of 1-beta-O-acyl glucuronides derived from benzoic acids.

Glucuronidation of carboxylic acid drugs has been found to be a metabolic activation pathway, possibly leading to covalent binding of the resultant 1-beta-O-acyl glucuronides (beta GAs) to proteins. Previous studies on the structure-activity relationships (SARs) of the degradation rate constants (k values) of beta GAs have revealed that the electrophilicity of and steric hindrance around the 1-beta-O-acyl linkages cause the diversity and complexity of the observed k values. To evaluate these effects and ultimately predict the k values of structurally diverse beta GAs, we derived further SARs for k values of 18 1-beta-O-benzoyl glucuronides with o-, m-, and p-substituents (BAGAs). In single regression analyses of 10 m- and p-substituted BAGAs, the log k values were well-predicted using an electronic parameter of Hammett's sigma constant, pK(a), (1)H NMR chemical shift (delta(COOH)), computed delta(COOH), or computed partial atomic charge (H(PAC) or O(PAC)) of the parent benzoic acids. The log k values of eight o-substituted BAGAs, although showing a correlation with the (13)C NMR chemical shift of the parent benzoic acids [delta(C=O)OH)], were well-predicted using multiple regression analyses; some combinations of electronic (delta(COOH), H(PAC), or calculated pK(a)) and steric [delta(C=O)OH) or Es] descriptors predicted the 18 observed k values with a high degree of certainty. The standard partial regression coefficients indicate that steric effects affected the k values as strongly as electronic effects, indicating that the k values increase as the acidity of the parent acids increases and as the steric bulkiness around the 1-beta-O-acyl linkages decreases. These single and multiple regression equations, using different electronic and/or steric descriptors of the parent benzoic acids, are expected to be useful for predicting the k values of BAGAs. The applicability domain and mechanistic interpretation of the derived SAR models are also discussed together with the relevant toxicology of beta GAs.

Structure-activity relationships for the degradation reaction of 1-beta-O-acyl glucuronides. Part 3: Electronic and steric descriptors predicting the reactivity of aralkyl carboxylic acid 1-beta-O-acyl glucuronides.

Since 1-beta-O-acyl glucuronides (betaGAs) are thought to be chemically reactive metabolites capable of binding to tissue proteins, possibly leading to adverse drug reactions of the parent carboxylic acid drugs, we have initiated research efforts to derive structure-activity relationships (SARs) of betaGAs, with a focus on finding appropriate descriptors that predict their intrinsic electrophilic reactivity or degradation rate constants (k values). Our previous SAR studies on the k values of betaGAs derived from o-, m-, and p-substituted benzoic acids demonstrated that the diversity and complexity of the k values were controlled by the electronic and/or steric effects of the parent carboxylic acids. In the present study, we performed further SAR studies on the k values of 13 betaGAs derived from aralkyl carboxylic acids, focusing on the substituents and stereochemistry at the alpha-position of the parent carboxylic acids. In single regression analyses, the pKa and (1)H NMR chemical shifts (delta(COOH)) of the parent carboxylic acids correlated well with the log k values of seven betaGAs derived from five arylacetic and two (R)-2-arylpropionic acids, whereas the (13)C NMR chemical shifts [delta(C horizontal lineO) and delta((C horizontal lineO)OH)] correlated with the log k values of another seven betaGAs derived from the five arylacetic and two (S)-2-arylpropionic acids. Excellent correlations were also obtained between the log k values of four betaGAs with a common 4-phenylbenzyl moiety and the partial atomic charges (natural type) of the corresponding carboxylic hydrogen atoms (Hpac), the molar volume (MV), and the molar refractivity (MR). In multiple regression analyses, appropriate combinations of electronic (delta(COOH) or pKa) and steric [Taft's steric constant (Es) or delta((C horizontal lineO)OH)] descriptors could predict the log k values of betaGAs; electron-withdrawing 1-beta-O-acyl groups increased the k values, while increasing steric hindrance around the linkages decreased them. The standard partial regression coefficients indicated that the steric effects of the 1-beta-O-acyl groups of betaGAs affected the k values as strongly as the electronic effects. External validation of the derived SAR models is also discussed.

Structure-activity relationships for degradation reaction of 1-beta-o-acyl glucuronides: kinetic description and prediction of intrinsic electrophilic reactivity under physiological conditions.

1-beta-O-Acyl glucuronides (betaGAs) are potentially reactive metabolites capable of binding to proteins, and they have been implicated in adverse drug reactions of the carboxylic acid drugs. To explore their electrophilic reactivity, we studied structure-activity relationships (SARs) to characterize the factors affecting the degradation rate constants (k values) of betaGAs and ultimately to predict k values of structurally diverse betaGAs. Twenty-seven betaGAs and four related compounds were synthesized, and their k values were determined under physiological conditions (pH 7.4 and 37 degrees C). 1-beta-O-Benzoyl glucuronide (BAGA) and glucopyranoside (BAG) showed almost the same k values, whereas their 1-alpha-O-benzoyl isomers degraded approximately 40-fold faster than BAGA and BAG. BAGA methyl ester showed almost the same rate constant as BAGA in the cleavage of their 1-beta-O-benzoyl linkages. A pH-log k profile obtained indicated kinetics catalyzed by both specific and general bases. The log k of betaGAs derived from m- and p-substituted benzoic acids correlated with Hammett's sigma constants. A similar correlation was observed with delta(COOH), (1)H NMR chemical shifts of the parent benzoic acids including ones with less sterically bulky o-substituents. Alternative descriptors of delta(CO) and delta((CO)OH), (13)C chemical shifts for ester carbonyl carbons of betaGAs and for carbonyl carbons of the parent benzoic acids, respectively, correlated well with the log k of all 16 betaGAs derived from benzoic acids including ones with bulkier o-substituents. Of the betaGA isomers derived from (2R)- and (2S)-alpha-methyl-4-biphenylylacetic acid, the (2R)-isomer degraded approximately 2-fold faster than the (2S)-isomer. The alpha-methyl group in the (2S)-isomer would encumber the intramolecular acyl migration. The log k of betaGAs derived from n-aralkyl carboxylic acids and of the (2R)-isomer correlated with their delta(COOH). However, the log k of betaGAs derived from alpha,alpha-dimethyl- and alpha,alpha-diethyl-4-biphenylylacetic acids deviated downward from the regression line, probably due to a steric effect. The diversity and complexity of k values were discussed with respect to the electrophilicity of the ester carbonyl carbons of betaGAs and the steric hindrance around them.

Chemo-Enzymatic Synthesis, Structural and Stereochemical Characterization, and Intrinsic Degradation Kinetics of Diastereomers of 1-ß-O-Acyl Glucuronides Derived from Racemic 2-{4-[(2-Methylprop-2-en-1-yl)amino]phenyl}propanoic Acid.

Alminoprofen, (RS)-2-{4-[(2-methylprop-2-en-1-yl)amino]phenyl}propanoic acid (ALP) 1, is a racemic drug categorized as a 2-arylpropanoic acid-class nonsteroidal anti-inflammatory drug. Pharmacokinetic studies of 1 in patients have revealed that the corresponding acyl glucuronide 5 is a major urinary metabolite, but little is known about the structure and stereochemistry of 5. The present work describes the synthesis of a diastereomeric mixture of 1-ß-O-acyl glucuronides (2RS)-5 from 1 and methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-α-d-glucopyranuronate 2 using our chemo-enzymatic method that has complete specificity for the ß-configuration. The structure of (2RS)-5 was characterized by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry as well as by complete hydrolysis by ß-glucuronidase. The absolute stereochemistry of (2RS)-5 was determined by comparison with (2R)-5 synthesized alternatively from (2R)-1 and 2. Compound (2R)-1 was prepared in two steps starting from chiral (R)-2-(4-nitrophenyl)propanoic acid (2R)-6. Chiral resolution of (2RS)-1 was achieved using a chiral high-performance liquid chromatography column, and its stereochemistry was determined by comparison with (2R)-1. The intrinsic degradation rate constant of (2R)-5 was 0.405 ± 0.002 h-1, which is approximately twice that of (2S)-5 (the k value was 0.226 ± 0.002 h-1) under physiological conditions (pH 7.40, 37 °C).

Enzymatic and mechanistic studies on the formation of N-phenylglycolohydroxamic acid from nitrosobenzene and pyruvate in spinach leaf homogenate.

The biotransformation mechanism of an unknown metabolite formed enzymatically from nitrosobenzene (NOB) and pyruvate in spinach (Spinacea oleracea L.) was investigated using spinach leaf homogenate. The unknown metabolite was identified as N-phenylglycolohydroxamic acid (PGA). The activity of PGA formation was decreased by l-alanine, increased by l-serine, and completely inhibited by aminooxyacetic acid, an inhibitor of transaminases. These results indicate that the transaminase participates in PGA formation. Indeed, hydroxypyruvate and alanine were produced in the transamination between pyruvate and serine. Hydroxypyruvate served as a direct-acting glycoloyl donor for PGA formation. A good correlation between the activities of the 200 g supernatant of spinach homogenate and commercial yeast transketolase for PGA formation from several glycoloyl donors was obtained. These results suggest the following mechanism for PGA formation from NOB and pyruvate: transamination of l-serine into hydroxypyruvate, which serves as a glycoloyl donor to NOB.

An improved chemo-enzymatic synthesis of 1-beta-O-acyl glucuronides: highly chemoselective enzymatic removal of protecting groups from corresponding methyl acetyl derivatives.

An improved and widely applicable chemo-enzymatic method for the synthesis of a series of 1-beta-O-acyl glucuronides 5a-f has been developed from the corresponding methyl acetyl derivatives 3a-f, which were stereospecifically synthesized from cesium salts of carboxylic acids 1a-f and methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate (2). Chemoselectivity of lipase AS Amano (LAS) in the hydrolytic removal of O-acetyl groups of 3a-f to provide methyl esters 4a-f was influenced by the nature of their 1-beta-O-acyl groups; high selectivity was evident only for 3b and 3f. Carboxylesterase from Streptomyces rochei (CSR), newly screened as an alternative to LAS, showed much greater chemoselectivity toward the O-acetyl groups than LAS; 3a, 3d, and 3e were chemoselectively hydrolyzed only by CSR. The combination of CSR with LAS yielded better results in the hydrolysis of 3c and 3f than did single usage of CSR. Final deprotection of the methyl ester groups of 4a-f to provide 5a-f was chemoselectively achieved by using lipase from Candida antarctica type B (CAL-B) as well as esterase from porcine liver (PLE), although CAL-B possessed higher chemoselectivity and catalytic efficiency than did PLE. CSR also exhibited high chemoselectivity in the synthesis of (S)-naproxen 1-beta-O-acyl glucopyranoside (7) from its 2,3,4,6-tetra-O-acetyl derivative 6.

Synthesis of 1-beta-O-acyl glucuronides of diclofenac, mefenamic acid and (S)-naproxen by the chemo-selective enzymatic removal of protecting groups from the corresponding methyl acetyl derivatives.

Using a straightforward chemo-enzymatic procedure, 1-beta-O-acyl glucuronides of three non-steroidal anti-inflammatory drugs, diclofenac (DF) 5, mefenamic acid (MF) 6 and (S)-naproxen (NP) 7, were prepared. Caesium salts of these carboxylic acid drugs reacted with commercially available methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate 4 to give exclusively the corresponding 1-beta-O-acyl glucuronides 8-10 in moderate yields. The protecting acetyl (for -OH group) and methyl ester (for -CO2H group) groups of each sugar moiety were easily removed to provide the corresponding free 1-beta-O-acyl glucuronides 1-3 in high yields. Deprotection was achieved through effective enzyme-catalysed chemo-selective hydrolyses of the acetyl groups using lipase AS Amano (LAS), and of the methyl ester group using esterase from porcine liver (PLE).

Microsomal oxidation of tribromoethylene and reactions of tribromoethylene oxide.

Halogenated olefins are of interest because of their widespread use in industry and their potential toxicity to humans. Epoxides are among the enzymatic oxidation products and have been studied in regard to their toxicity. Most of the attention has been given to chlorinated epoxides, and we have previously studied the reactions of the mono-, di-, tri-, and tetrachloroethylene oxides. To further test some hypotheses concerning the reactivity of these compounds, we prepared tribromoethylene (TBE) oxide and compared it to trichloroethylene (TCE) oxide and other chlorinated epoxides. TBE oxide reacted with H(2)O about 3 times faster than did TCE oxide. Several hydrolysis products of TBE oxide were the same as formed from TCE oxide, i.e., glyoxylic acid, CO, and HCO(2)H. Br(2)CHCO(2)H was formed from TBE oxide; the yield was higher than for Cl(2)CHCO(2)H formed in the hydrolysis of TCE oxide. The yield of tribromoacetaldehyde was < 0.4% in aqueous buffer (pH 7.4). In rat liver microsomal incubations containing TBE and NADPH, Br(2)CHCO(2)H was a major product, and tribromoacetaldehyde was a minor product. These results are consistent with schemes previously developed for halogenated epoxides, with migration of bromine being more favorable than for chlorine. Reaction of TBE oxide with lysine yielded relatively more N-dihaloacetyllysine and less N-formyllysine than in the case of TCE oxide. This same pattern was observed in the products of the reaction of TBE oxide with the lysine residues in bovine serum albumin. We conclude that the proposed scheme of hydrolysis of halogenated epoxides follows the expected halide order and that this can be used to rationalize patterns of hydrolysis and reactivity of other halogenated epoxides.

Tetrachloroethylene oxide: hydrolytic products and reactions with phosphate and lysine.

Tetrachloroethylene, or perchloroethylene (PCE), has considerable industrial use and is of toxicological interest because of a variety of effects. Most of the existing literature presents PCE oxide as a critical intermediate in the oxidative metabolism of PCE to Cl(3)CCO(2)H, oxalic acid, and products covalently bound to proteins, including trichloroacetyl derivatives of lysine. PCE oxide was synthesized by photochemical oxidation of PCE and characterized. Decomposition at neutral pH (t(1/2) = 7.9 min at 0 degrees C, 5.8 min at 23 degrees C, 2.6 min at 37 degrees C) yielded only trace ( approximately 1%) Cl(3)CCO(2)H; the major products identified were CO (73% yield) and CO(2) (63% yield). In phosphate buffer (0.10 M) a major product was identified as oxalyl phosphate. Oxalyl chloride also reacted to form CO and CO(2) in aqueous solution and to form oxalyl phosphate in neutral phosphate buffer. Oxalyl phosphate decomposed to oxalic acid (t(1/2) = 53 min at 37 degrees C) but did not react with lysine. Reaction of PCE oxide with free lysine yielded the oxalic acid amide derivatives of lysine plus lysine dimers in which cross-linking of the amino groups involved oxalo linkage. The reaction of PCE oxide with albumin yielded mainly N(6)-oxalolysine and some (<5%) N(6)-trichloroacetyllysine. We propose a reaction pathway for PCE oxide based on our previous studies with trichloroethylene oxide, in which C-C bond scission is a major product of reaction in aqueous buffer and yields CO and CO(2). Oxalyl species are proposed as intermediates and prominent acylating species formed in the reactions of the epoxide. The formation of Cl(3)CCO(2)H in cytochrome P450 reactions is postulated to result from intramolecular migration within an enzyme intermediate.