The palladium(II) promoted hydrolysis of the methyl esters of glycyl-L-leucine,glycyl-L-alanine and L-alanylglycine

The palladium(II)-promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine have been studied at 25 °C and I=0.1 M in the pH range 4–5. At a 1:1 metal to ligand ratio the peptide esters act as tridentate ligands, donation occurring via the terminal amino group, the deprotonated amide nitrogen, and the carbonyl group of the ester. Due to the high Lewis acidity of Pd(II) rapid hydrolysis of the ester function by water and hydroxide ion occurs. Rate constants k_OH and k_H2O have been obtained for base hydrolysis and water hydrolysis of the coordinated peptide esters at 25 °C. The rate constants for base hydrolysis are 3.4 X 10⁶ M⁻¹ s⁻¹ (L-alaglyOMe), 6.4 X 10⁶ M⁻¹ s⁻¹ (gly-L-alaOMe) and 2.3 X 10⁷ M⁻¹ s⁻¹ (gly-L-leuOMe). Base hydrolysis of the coordinated peptide esters is at least 10⁶ times that of the free unprotonated ligand. Activation parameters have been obtained for both water and base hydrolysis of the Pd(II) complex of methyl L-alanylglycinate and possible mechanisms for the hydrolyses are considered.     

Striking susceptibility of a kinin-yielding pentadeca-peptide to tryptic hydrolysis

Hydrolysis of Lys-Arg-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser by trypsin (EC 3.4.21.4) yields lysyl-bradykinin by rupture of the Arg-Ser bond. The k_cat/K_m value found for this hydrolysis was 1.4 × 10¹⁰ M^?1 × sec^?1, which is 10^?5-fold higher than that obtained for the hydrolysis of bradykinyl-Ser-Val-Gln-Val-Ser. This effect was abolished by acetylation of the lysine amino groups of the pentadecapeptide. Contrarywise, the esterolytic activity of trypsin on bradykinin methyl ester was the same as in lysyl-bradykinin methyl ester. The high susceptibility of Lys-bradykinyl-Ser-Val-Gln-Val-Ser to trypsin catalysis is striking because: a) it constitutes the first example that an amino acid residue distant from the bond split may enhance trypsin catalysis; b) this pentadecapeptide is the best synthetic substrate so far described for trypsin and c) the value of k_cat/K_m for its hydrolysis is unusually high for proteases.     

Substrate activation of rat trypsins 1 and 2

Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of K_cat for N¹-tosyl-l-arginine methyl ester vs K_cat for N¹-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N¹-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N¹-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N¹-tosyl-l-arginine methyl ester 4.5 times faster than that of N¹-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.     

Enzymatic removal of a cephalosporin methyl ester blocking group

Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C₄ methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH₃ and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10^?10 mol min^?1 mg^?1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K_m value was 2.3 mg ml^?1. The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K₁ values of 6.63 and 0.188 mg ml^?1, respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.     

Endogenous gibberellins and inhibitors in the Douglas-fir

In young needles of the Douglas-fir (Pseudotsuga menziesii) GA₉ has been shown by GC and HPLC to be the main gibberellin. As minor compounds GA₇, GA₃ and GA₈ have been tentatively identified by HPLC. In addition to the free gibberellins small amounts of GA₉ glucosyl ester and a not yet identified ester of GA₂₀ have been isolated. From the group of endogenous inhibitors ABA has been identified by GC-MS and ABA glucosyl ester by HPLC. After enzymatic hydrolysis of the ester, ABA and glucose have been quantified by GC and GOD-POD reaction giving the ratio 1:1. Another plant growth inhibitor has been identified as the methyl ester of jasmonic acid.     

Metal-ion-catalyzed hydrolysis of monomethyl and dimethyl esters of 3-(2-imidazoleazo)phthalic acid: Bifunctional catalysis by carboxyl group and the metal ion in the hydrolysis of an alkyl ester

The Cu(II) or Ni(II) ion-catalyzed hydrolysis of methyl 2-carboxy-6-(2-imidazoleazo)benzoate (1) and the corresponding dimethyl ester (2) was studied kinetically at various pH values. For 2, the ester group located at the o position to the azo substiuent was hydrolyzed. From the rate data obtained at various metal concentrations, the values of k_cat and K_f were estimated at each pH value. For the Ni(II)-catalyzed hydrolysis of 1 at pH < 4, k_cat increases as pH is lowered, indicating bifunctional catalysis by the carboxyl group and the metal ion. For most of the reactions investigated under other conditions, the ester hydrolysis was subjected to sole catalysis by the metal ions. Detailed analysis of kinetic data obtained for these reactions indicated that the metal-ion catalysis involves the rate-determining breakdown of the tetrahedral intermediates formed by the addition of a water molecule or hydroxide ion. The bifunctional catalysis by the carboxyl group and Ni(II) ion can be considered as a model for carboxypeptidase A. The kinetic data indicate that the bifunctional catalysis proceeds through the nucleophilic attack of the carboxylate ion at the Ni(II)-coordinated carbonyl group.     

Effects of (15S)-15-methyl prostaglandin F2 methyl ester and estrogens upon the corpus luteum and conceptus of the rhesus monkey

The corpus luteum inhibiting properties of eighteen 15-methyl prostaglandin analogs were determined in the rhesus monkey during concomitant stimulation of the corpus luteum with chorionic gonadotropin. The methyl ester of (15S)-15-methyl PGF_2^α (15M-PGF_2^α, 12.5 mg/monkey) lowered serum progesterone to 12% of pretreatment values within 24 hours, however progesterone returned to normal limits within 48 hours. Elongation of the top side-chain by two carbons (2a,2b-dihomo-15M-PGF_2^α methyl ester, 13 mg/monkey), substitution of a hydroxymethyl group at carbon 1 (2-decarboxy-2-hydroxymethyl-15M-PGF_2^α, 12 mg/monkey), or the formation of the carbon 1 amide (15M-PGF_2^α amide, 12.5 mg/monkey) improved the inhibitory activity of 15M-PGF_2^α; serum progesterone for these 3 analogs was depressed to 15–30% of pretreatment levels within 24 hours, and did not return to control values. Luteal function was not inhibited (12 or more mg/monkey) when the 15-methyl group was placed in the R configuration, the top side chain was shortened by two carbons, an amino group was substituted for carbon 1, the 5-oxa modification was added, or the 1,9-lactone was formed. Some other modifications of 15M-PGF_2^α were also inactive, although not all were tested at equivalent doses: 2,2-difluoro; 4,5-cis-didehydro; 9,11-dideoxy-9α,11α-dichloro; 11-deoxy; 17-phenyl; 1,15-lactone; and the p-benzamidophenyl ester of 2a,2b-dihomo-15M-PGF_2^α. (15S)-15-Methyl PGE₂ methyl ester (1 mg/monkey) depressed serum progesterone concentrations to 42% of pretreatment values within 24 hours; 2a,2b-dihomo-11-deoxy-(15S)-15-methyl PGE₂ methyl ester was inactive (5 mg/monkey). A corpus luteum inhibiting action of certain 15-methyl prostaglandins can be demonstrated in the rhesus monkey.     

Inhibition of the monkey corpus luteum with 15-methyl prostaglandins

The corpus luteum inhibiting properties of eighteen 15-methyl prostaglandin analogs were determined in the rhesus monkey during concomitant stimulation of the corpus luteum with chorionic gonadotropin. The methyl ester of (15S)-15-methyl PGF_2^α (15M-PGF_2^α, 12.5 mg/monkey) lowered serum progesterone to 12% of pretreatment values within 24 hours, however progesterone returned to normal limits within 48 hours. Elongation of the top side-chain by two carbons (2a,2b-dihomo-15M-PGF_2^α methyl ester, 13 mg/monkey), substitution of a hydroxymethyl group at carbon 1 (2-decarboxy-2-hydroxymethyl-15M-PGF_2^α, 12 mg/monkey), or the formation of the carbon 1 amide (15M-PGF_2^α amide, 12.5 mg/monkey) improved the inhibitory activity of 15M-PGF_2^α; serum progesterone for these 3 analogs was depressed to 15–30% of pretreatment levels within 24 hours, and did not return to control values. Luteal function was not inhibited (12 or more mg/monkey) when the 15-methyl group was placed in the R configuration, the top side chain was shortened by two carbons, an amino group was substituted for carbon 1, the 5-oxa modification was added, or the 1,9-lactone was formed. Some other modifications of 15M-PGF_2^α were also inactive, although not all were tested at equivalent doses: 2,2-difluoro; 4,5-cis-didehydro; 9,11-dideoxy-9α,11α-dichloro; 11-deoxy; 17-phenyl; 1,15-lactone; and the p-benzamidophenyl ester of 2a,2b-dihomo-15M-PGF_2^α. (15S)-15-Methyl PGE₂ methyl ester (1 mg/monkey) depressed serum progesterone concentrations to 42% of pretreatment values within 24 hours; 2a,2b-dihomo-11-deoxy-(15S)-15-methyl PGE₂ methyl ester was inactive (5 mg/monkey). A corpus luteum inhibiting action of certain 15-methyl prostaglandins can be demonstrated in the rhesus monkey.     

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway

[5-³H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB³H₄ and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem.15, 428–435). The [5-³H]shikimate has been converted enzymatically to [5-³H]chorismate and [5-³H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatograhic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.     

Enhanced hydrolysis of a phosphonate ester by mono-aquo metal cation complexes

The hydrolysis of the ester 2,4-dinitrophenyl- ethyl methylphosphonate has been examined by both stop-flow spectrophotometric and pH-stat techniques. These reactions have been carried out in the presence of several nucleophiles including simple non-labile (w.r.t. substitution) mono-aquo metal ion complexes. Comparison of reaction rates of the metal complexes with sterically hindered organic nucleophiles has led to the conclusion that the metal ions function predominantly as general base catalysts in dilute aqueous solution. Reaction rates for the various nucleophiles studied are tabulated together with solvolysis constants for hydroxide ion and water at 35 °C and I=0.1 mol dm⁻³ (KNO₃). These later two values are respectively 32.7 mol⁻¹ dm³ s⁻¹ and 1.37 x 10⁻⁴ s⁻¹. A Brönsted β value of 0.52 for the phosphonate ester studied has also been derived.     

Gibberellin metabolism in excised lettuce hypocotyls: Evidence for the formation of gibberellin A1 glucosyl conjugates

The properties of the water-soluble metabolites of [³H]gibberellin A₁ ([³H]GA₁) from lettuce (Lactuca sativa L.) hypocotyls were compared with those of authentic samples of gibberellin (GA) glucosyl esters and ethers. Partitioning against l-butanol at high and low pH was not an efficient method of differentiating between ester and ether conjugates of GA₁ or GA₃. Extraction into l-butanol at pH 2.5 was, however, useful as a group purification step. Gel-filtration on acrylamide indicated a mean molecular weight of ca. 600 for the polar material and high-voltage electrophoresis separated two compounds (LH 1 and LH 2) with differing charge properties. Both metabolites incorporated ¹⁴C from glucose and ³H from GA₁. Subsequent enzymatic hydrolysis of LH 1 released material with identical properties to [¹⁴C]glucose together with a second uncharacterised component. Feeding with [³H]GA₁ methyl ester greatly reduced the formation of LH 1 but not LH 2. The metabolites were provisionally identified as GA₁-glucosyl ester (LH 1) and GA₁-glucosyl ether (LH 2).Abbreviations GA gibberellin - LH1 GA₃-glucosyl ester - LH2 GA₁-glucosyl ether - HVE high voltage paper electrophoresis - TLC thin-layer chromatography     

油包水微乳液中抗体酶催化布洛芬酯选择性水解的酶学特性

根据过渡态理论设计和合成了能诱导产生催化选择性水解布洛芬甲酯的催化抗体的四面体硫酸盐半抗原,并与牛血清白蛋白(BSA)偶联制备成免疫源,通过免疫手段成功筛选出具有加速选择性水解生成S-布洛芬的特异性催化抗体.其Kcat,app/Kuncat,app达1.6x104.进一步地将催化抗体运用到W/O微乳体系(反胶束)中进行布洛芬酯的选择性水解研究,其动力学研究证明其催化过程同样遵循Michaelis.Menten方程.考察了pH值和温度对催化初速度影响,Wo(体系中水和琥珀酸二辛酯磺酸钠(AOT)的摩尔比)对催化初速度影响呈现为钟罩型,最适的Wo.为21.     

Simultaneous quantitation of indole 3-acetic Acid and abscisic Acid in small samples of plant tissue by gas chromatography/mass spectrometry/selected ion monitoring

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [²H₄]IAA (m/z 194, 134) or [²H₅]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [²H₃]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue.     

The chemical structure of the polysaccharide from Dasyclonium incisum

The polysaccharide from Dasyclonium incisum was found to be an agar-type polymer, in which the 2-hydroxyl group on the 3,6-anhydrogalactosyl unit is partially substituted by a methyl ether, and the 4-hydroxyl group on the galactosyl unit is almost completely replaced by a sulphate ester group. Minor levels of other substitution patterns were also detected, including single branching xylose residues. Techniques used in determining the structure include selective hydrolysis, reductive hydrolysis followed by saccharide composition and methylation analysis, infrared and ¹³C-NMR spectroscopy.     

On the structures of some adducts of biotin with electrophiles: Does sulfur transannular interaction with the carbonyl group play a role in the chemistry or biochemistry of biotin?

Biotin methyl ester (1), 1′-N-carbomethoxy biotin methyl ester (2) and 1′-N-trifluoroacetylbiotin methyl ester (3) were treated with boron trifluoride etherate and triethyloxonium tetrafluoroborate. Whereas no reaction could be observed in the case of 3, coordination of the electrophiles to the urea oxygen could be deduced from the IR, ¹H-NMR, and ¹³C-NMR spectra of the adducts obtained from 1 and 2. X-Ray analysis of the 1/BF₃-adduct (4) confirmed the O-coordination, but showed no transannular sulfur-carbonyl interaction. From comparison of the spectral data obtained for all addition products it is concluded that none of them shows a significant transannular sulfur-carbonyl interaction. Reaction of 3 with SbF₅ also formed an O-coordinated adduct (10) without sulfur transannular bonding. In magic acid (FSO₃H/SbF₅/SO₂) both 1 and 3 added a proton to the urea oxygen, to the sulfur atom, and to the ester group, as reported previously for biotin itself. The relationship of these findings to the kinetics of acid-catalyzed NH exchange in biotin, and to possible mechanisms of biochemical biotin-catalyzed reactions, are discussed.     

Enzymatic preparation of carboxyl oxygen-18 labeled prostaglandin F2α and utility for quantitative mass spectrometry

A nonspecific liver esterase was found to not only catalyze the hydrolysis of the methyl ester of prostaglandin F2α (PGF2α) but also to catalyze the exchange of the carboxyl oxygen atoms with water leading to the production of [¹⁸O₂]PGF2α when the enzymatic hydrolysis is carried out in H₂¹⁸O. The kinetics of this oxygen-18 exchange reaction are briefly discussed. The [¹⁸O₂]PGF2α was found to be relative stable toward back exchange in methanol, aqueous buffer, and urine, but rapidly back exchanged to the native PGF2α in plasma with a half-life of 1 h. The [¹⁸O₂]PGF2α was relatively stable in plasma to which alcohol had been added. The utility of the oxygen-18 labeled prostaglandin as an internal standard in a gas chromatography-mass spectrometry assay was demonstrated at the picomole range.     

Kinetic resolution of 2-chloro-3,3,3-trifluoropropanoic acid esters catalyzed by lipase from Candida rugosa

Abstract

By screening around 30 commercially available lipases and esterases, two enzymes, C. rugosa lipase and P. fluorescens esterase, were found to posess catalytic activity and enantioselectivity (E?10) for the hydrolysis of 2-chloro-3,3,3-trifluoropropanoic acid (CTFPA) methyl and ethyl ester. Both enzymes were tentatively assigned to be (S)-selective based on the assumption that they have the same stereopreference as in the hydrolysis of methyl 2-chloropropanoate, which is a non-fluorinated analogue of CTFPA. The enzymes were applied in the kinetic resolution of CTFPA ethyl ester and 95% ee of the remaining ester could be achieved at 60% conversion. The crosslinked enzyme aggregate (CLEA) of C. rugosa lipase was found to catalyze enantioselective transesterification (E?40) of CTFPA methyl ester with ethanol. By conducting the transesterification in a 10-mL packed-bed reactor containing CLEA, it was possible to convert racemic CTFPA methyl ester into the mixture of (S)-methyl and (R)-ethyl esters with 82% and 90% ee, respectively, at 4.0 g/L^-1/h^-1 space-time yield, which decreased to 1.0 g/L^-1/h^-1 after four repetitive batches.     

Stereospecific hydrolysis of 3-(4-methoxyphenyl)glycidic ester in supercritical carbon dioxide by immobilized lipase

In the hydrolysis of racemic 3-(4-methoxyphenyl)glycidic acid methyl ester by immobilized Mucor miehel lipase in supercritical CO₂ the initial hydrolysis rate of the (2S,3R)-form was faster than the rate of the (2R,3S) -form. The stereoisomeric excess of the (2R,3S)-form reached 87 % at 53 % total conversion level. The water content of the reaction mixture and the initial concentration of the 3-(4-methoxyphenyl) glycidic acid methyl ester had no effect on isomeric purity. The reaction rate in supercritical CO₂ was considerably faster than in toluene/water -mixture.     

Synthesis of esters of saturated and unsaturated sixteen-carbon acids deuterated at the terminal or penultimate carbons

General syntheses of saturated and unsaturated fatty acids, specifically trideuterated at the terminal carbon or dideuterated at the penultimate carbon, from ω-hydroxy esters, have been developed. Methyl [16-²H₃]hexadecanoate was synthesized from methyl 16-hydroxyhexadecanoate. The hydroxyl group was protected as the tetrahydropyranyl ether and the ester group reduced with lithium aluminum deuteride, first to an alcohol and then, by way of the derived mesylate, to a trideuteromethyl group. The new ester group was formed by oxidation of the hydroxyl group. Methyl 16-hydroxy[2-²H₂]hexadecanoate was prepared, from 16-hydroxy-hexadecanoate, by exchange of the α protons and, by the reductive route above, with lithium aluminum hydride, gave methyl [15-²H₂]hexadecanoate. Methyl 16-hydroxy-7-hexadecynoate was synthesized from 6-chlorohexanol and was converted, by means of the above reactions, to methyl [16-²H₃]- and [15-²H₂]-9-hexadecynoates. Lindlar reduction gave methyl [16-²H₃]- and [15-²H₂]cis-9-hexadecenoates. Overall yields ranged from 30% to 38%.