Lipase-catalyzed enantioselective esterification of mono-aza-benzo-15-crown-5-ether derivatives in organic media

For the purpose of developing a new chiral crown ether unit as a chiral synthon, three racemic mono azabenzo-15-crown-5-ethers, i.e. (R,S)-1-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-yl)-propan-2-ol, (R,S)-2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-yl)-1-phenyl-ethanol and (R , S)-1-[2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-ylmethyl)-phenyl]-ethanol were esterified with vinyl acetate using a lipase from Candida antarctica. The enzymatic acylation of alcohols produced monoacylated products. Two optically active azacrown ethers, (R)-propionic acid 1-methyl-2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-yl)-ethyl ester and (R)-acetic acid 1-[2-(6,7,9,10,12,13,15,16-octahydro-5,8,14,17-tetraoxa-11-aza-benzocyclopentadecen-11-ylmethyl)-phenyl]-ethyl ester were obtained within 48% and 36% yields, respectively and, at an enantiometric excess of over 99% in each case.     

Rat small-intestinal β-galactosidases: Influence of pH on the hydrolysis of different substrates

1. The influence of pH and the kind of buffer on the hydrolysis of lactose and four hetero-β-galactosides (phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside) by homogenates of rat small-intestinal mucosa has been studied. 2. There are at least two β-galactosidases present in the homogenates, one with optimum pH3–4 and another with optimum pH5–6. 3. The enzyme with the lower pH optimum is mainly a heterogalactosidase. It hydrolyses lactose slowly. The other enzyme is mainly a disaccharidase, since it hydrolyses lactose much more rapidly than the heterogalactosides. 4. Under the conditions used, citrate had an inhibitory effect on the 6-bromo-2-naphthyl β-galactosidase activity at pH3–4, but did not influence the 6-bromo-2-naphthyl β-galactosidase activity at pH5–6 or the hydrolysis of the other substrates at any pH.     

Preparation of Single-Enantiomer Biofunctional Molecules with (S)-2-Methoxy-2-(1-naphthyl)propanoic Acid

(RS)-β-Ionol and (RS)-2-methyl-4-octanol were resolved by using (S)-2-methoxy-2-(1-naphthyl)propanoic acid [(S)-MαNP acid]. The specific stereochemistry of each MαNP ester was elucidated by 2D NMR analyses, and shielding by the 1-naphthyl group was observed in both the ¹H- and ¹³C-NMR spectra. Solvolysis of the individual (S)-MαNP esters gave four single-enantiomer alcohols. The normal-phase HPLC elution order of each MαNP ester is also discussed.     

Acaricidal activity of the aggregation pheromone of Japanese pine sawyer against two-spotted spider mite

Japanese pine sawyer (Monochamus alternatus) (Coleoptera: Cerambycidae), a vector of pine wilt disease, produces male-produced aggregation pheromone called monochamol, 2-(1-undecyloxy)-1-ethanol (C₁₁OEtOH). After the identification of unique nematicidal activity of the synthetic monochamol and its homologues (ROEtOH: R = C₇-C₁₁) against Bursaphelenchus xylophilus nematode, their acaricidal activities were evaluated against two spotted spider mite (TSSM, Tetranychus urticae Koch) (Acari: Tetranychidae). In a leaf disc bioassay method, 100 mgL⁻¹ of monochamol and 2-(1-decyloxy)-1-ethanol (C₁₀OEtOH) exhibited 100% adult mortality and zero fecundity. Along with C₁₁OEtOH and C₁₀OEtOH, 2-(1-octyloxy)-1-ethanol (C₈OEtOH) and 2-(1-nonyloxy)-1-ethanol (C₉OEtOH) were also 100% acaricidal with complete oviposition inhibition at increased concentration of 1000 mgL⁻¹. When the TSSM eggs on leaf discs were immersed in 1000 mgL⁻¹ solutions, none of the components were ovicidal. Egg hatching was completely inhibited by three components (C₉OEtOH, C₁₀OEtOH and C₁₁OEtOH) at 6000 mgL⁻¹ and by four components (C₈OEtOH, C₉OEtOH, C₁₀OEtOH and C₁₁OEtOH) at 8000 mgL⁻¹. However, none of the compounds showed acaricidal effects in 0.5 h and 24 h residual toxicity tests at 1000 mgL⁻¹. This study highlights the potential acaricidal activity of monochamol and its homologues. Notably, the monochamol and its C₁₀-homologue could be listed as efficient acaricidal agents.     

Studies on cellulolytic enzymes I: The use of 3,4-dinitrophenyl glycosides as substrates

The syntheses of 3,4-dinitrophenyl β-d-glucoside, β-cellobioside, β-cellotrioside, and β-cellotetraoside and their use to monitor the purification of two enzymes from a crude commercial cellulase preparation from Trichoderma viride are described. The enzymes isolated are an endo-β-1,4-d-glucan glucanohydrolase (EI) of molecular weight ca. 12 000 which catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenol-β-cellotetraoside, and an enzyme of molecular weight about 76 000 which catalysed the hydrolysis of 3,4-dinitrophenyl β-d-glucoside (EII) and is probably a cellobiase or exo-β-1,4-d-glucan glucohydrolase. Kinetic parameters are reported for the hydrolyses of 3,4-dinitrophenyl β-cellobioside, β-cellotrioside, and β-cellotetraoside catalysed by enzyme EI. In the presence of cellotriose, cellotetraose, or cellopentaose 3,4-dinitrophenyl β-d-glucoside underwent induced hydrolyses by EI. Similar but faster induced hydrolyses were shown by 3,4-dinitrophenyl β-d-xyloside and 3,4-dinitrophenyl β-d-6-deoxyglucoside; 3,4-dinitrophenyl 6-chloro-6-deoxy-β-d-glucoside and 3,4-dinitrophenyl 6-O-methyl-β-d-glucoside underwent slower induced hydrolyses than the glucoside. p-Nitrophenyl β-d-glucoside also underwent an induced hydrolysis in the presence of cellopentaose and the enzyme EI, but p-nitrophenyl 2-deoxy-β-d-glucoside did not. These results are discussed and compared with the results obtained previously on induced hydrolyses found with lysozyme. Kinetic parameters are reported for the hydrolysis of 3,4-dinitrophenyl and p-nitrophenyl β-d-glucosides catalysed by the enzyme EII. 3,4-Dinitrophenyl 6-deoxy-β-d-glucoside, β-d-xyloside, 6-chloro-6-deoxy-β-d-glucoside, 6-O-methyl-β-d-glucoside and p-nitrophenyl-β-d-galactopyranoside and 2-deoxy-β-d-glucopyranoside were hydrolysed 10² to 10³ times slower by EII than the corresponding glucosides, but 3,4-dinitrophenyl 2-acetamido-2-deoxy-β-d-glucoside was only hydrolysed about 25 times slower than 3,4-dinitrophenyl β-d-glucoside. The significance of these results is discussed. EII catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenyl β-cellobioside, β-cellobioside, and β-cellotetraoside, but these reactions showed induction periods which are consistent with stepwise removal of glucose residues from the oligosaccharide chains before release of the phenol.     

Some observations on the chemical and stereochemical specificity of the de-alkylation of organophosphorus esters by a hepatic glutathione transferase

Hepatic glutathione (GSH) S-methyl transferase from rabbit, pig and dog demethylates dimethyl phosphate triesters. No stereospecificity towards racemic ethyl methyl 2-chloro-1-(2,4-dichlorophenyl)vinyl phosphate could be demonstrated but the enzyme exhibited some selectivity towards the (+) and (?) forms of O-methyl S-methyl 1-naphthyl phosphorothiolate. Pig liver enzyme, purified 30-fold, demethylated the prochiral substrate dimethyl 1-naphthyl phosphorothionate with 90% stereo-selectivity.     

Bitter phenylpropanoid glycosides from Conandron ramoidioides

A new bitter glycoside, conandroside and a known glycoside, acteoside were isolated from Conandron ramoidioides. On the basis of the chemical and spectral evidence, conandroside was shown to be 2-(3′,4′-dihydroxy-phenyl)-ethanol 1-O-β-D-xylosyl-(1 → 3)-β-D-(4-caffeyl)-glucoside.     

1,3,5-Triazine multiselector systems: New tools for chiral discrimination

2-Hexylamino-4-[(S)-1-(1-naphthyl)ethylamino]-6-L-valyl-L-valyl-L-valine isopropylester-1,3,5-triazine (1), a molecule characterized by two different chiral selectors, and 2-hexylamino-4,6-bis-L-valyl-L-valyl-L-valine isopropylester-1,3,5-triazine (2) and 2-ethoxy-4-hexylamino-6-[(S)-1-(1-naphthyl) ethylamino]-1,3,5-triazine (3), systems in which a single kind of chiral selector is present, have been prepared. The enantiodiscriminating ability in solution of the three compounds toward the N-3,5-dinitrobenzoyl derivatives of 1-phenylethylamine (4) or valine methylester (5) has been investigated by ¹H nuclear magnetic resonance (NMR) spectroscopy: 1 shows an improved versatility, relative to 2 and 3, as a chiral solvating agent for NMR spectroscopy. On the basis of the indications obtained, the usefulness of 2-chloro-4-[(S)-1-(1-naphthyl)ethylamino]-6-L-val-L-val-L-valine isopropylester-1,3,5-triazine (1a), a direct precursor of 1, as chiral solvating agent for the determination by NMR of the enantiomeric compositions of derivatives of amines, amino alcohols, amino acids, and carboxyl acids bearing a 3,5-dinitrophenyl moiety, has been demonstrated. Chirality 9:113–121, 1997. © 1997 Wiley-Liss, Inc.     

6-Bicycloaryl substituted (S)- and (R)-5,6-dihydro-2H-pyran-2-ones: Asymmetric synthesis,and anti-proliferative properties

(R)-Goniothalamin, is a member of styryl lactones, possesses selective cytotoxicity against cancer cell lines. In this work, replacement of styryl substituent with 2-naphthyl and 3-quinoyl gave new analogues which may have less conformational changes compared to the lead compound. Anti-proliferative tests indicated that 2-naphthyl substituted (R)-5,6-dihydro-2H-pyran-2-one has slightly better cytotoxicity than (R)-goniothalamin. To clarify the effect of 2-naphthyl substituent additional aryl substituted (R)-5,6-dihydro-2H-pyran-2-ones have been synthesized enantioselectively and tested against PC-3 and MCF-7 cell lines.     

Enzymatic synthesis of structural analogs of PAF-acether by phospholipase D-catalysed transphosphatidylation.

PAF-acether can be transformed into analogs by the phospholipase D enzyme activity of Streptomyces sp. In this reaction choline is replaced by primary cyclic alcohols (acceptors). The reaction products, cyclic phospholipid and phosphatidic acid, were separated by silicic acid chromatography. This procedure enabled us to synthetize five analogs of PAF-acether, with a cyclic ring structure. The primary cyclic alcohols used in this work were: 3-(2-hydroxyethyl)-indol, OH-Et-I; 2-(hydroxymethyl)-1,4-benzodioxan, OH-Met-BZD; N-(2-hydroxyethyl)-phthalimide, OH-Et-PHT; 2-(2-thienyl)-ethanol, Th-EtOH; (1-R)-(-)-Nopol, R-NOP.     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从 11株微生物中筛选出 4株具有不对称还原 2′ 氯 苯乙酮能力的酵母 ,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对 2′ 氯 苯乙酮还原的最佳反应时间为 2 4h ;最佳pH 8 0 ;最佳反应温度为2 5℃ ;最佳共底物为 5 % (体积比 )乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为 10 75mg mL(细胞干重 反应体积 )的酿酒酵母B5可将 6 47mmol L的 2′ 氯 苯乙酮10 0 %地转化为R 2′ 氯 1 苯乙醇 ,其对映体选择性为 10 0 %。酿酒酵母B5可重复利用的特点可提高产物的产量。     

Convenient method for determining the absolute configuration of chiral alcohols with racemic 1H NMR anisotropy reagent,M(alpha)NP acid: use of HPLC-CD detector

A convenient method for determining the absolute configuration of chiral secondary alcohols using the racemic NMR anisotropy reagent, (+/-)-2-methoxy-2-(1-naphthyl)propionic acid [(+/-)-M(alpha)NP acid], and an HPLC-CD detector was developed. The method was successfully applied to some chiral alcohols derived from (-)-alpha-santonin.     

The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, α- and β-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20–80). It showed pronounced activity towards p-nitrophenyl and α- and β-naphthyl esters of C₁₂–C₁₆. Higher activity was observed with α-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 °C) and thermal stability. CD analysis revealed predominance of α-helical structure (54% α-helix, 21% β-sheet) and a T_m value at 66 °C.     

Preparation of single-enantiomer biofunctional molecules with (S)-2-methoxy-2-(1-naphthyl)propanoic acid

(RS)-beta-Ionol and (RS)-2-methyl-4-octanol were resolved by using (S)-2-methoxy-2-(1-naphthyl)propanoic acid [(S)-MalphaNP acid]. The specific stereochemistry of each MalphaNP ester was elucidated by 2D NMR analyses, and shielding by the 1-naphthyl group was observed in both the 1H- and 13C-NMR spectra. Solvolysis of the individual (S)-MalphaNP esters gave four single-enantiomer alcohols. The normal-phase HPLC elution order of each MalphaNP ester is also discussed.     

Lipase-catalyzed optical resolution of trifluoro(aryl)ethanols

Optical resolutions of racemic 2,2,2-trifluoro-1-(aryl)ethanols — (1-naphthyl), (2-naphthyl), (4-methylnaphthyl), (phenyl), (1-pyrenyl) — were achieved by lipase-catalyzed enantioselective acetylations with vinyl acetate as an acetyl donor in octane, and (S)-acetates and (R)-alcohols were obtained. Among the lipases tested, lipase from Pseudomonas aeruginosa (lipase LIP, Toyobo) showed good enantioselectivity for above ethanols. However, no acetylation occurred with sterically hindered alcohols — (9-phenanthryl), (9-anthryl), (2-methylnaphthyl), (2, 4, 6-trimethylphenyl) — by various lipases. The resolutions of the three alcohols were carried out by the enantioselective alcoholysis or hydrolysis of their chloroacetates by lipase LIP.     

Enzymic degradation of chemically modified extracellular polysaccharides from Rhizobia

Extracellular polysaccharides from Rhizobium trifolii, U226, Coryn and Bart A; Rhizobium phaseoli, U453; Rhizobium leguminosarum, U331; and Rhizobium meliloti, U27, after chemical modification, become substrates for certain β-d-glucan hydrolases. The Streptomyces (1 → 4)-β-d-glucan endohydrolase (EC 3.2.1.4) hydrolyses reduced and deacetylated rhizobial polysaccharides, both before and after removal of carboxyethylidene substituents, to produce a series of oligosaccharides. The Rhizopus arrhizus (1 → 3)-β-d-glucan endohydrolase (EC 3.2.1.6) hydrolyses only fully modified polysaccharides to yield, in the case of R. meliloti U27, laminarabiose, and, in all other instances, a disaccharide identified β-d-Gal-(1 → 3)-D-Glc. The same disaccharides are released by the Rhizopus enzyme from oligosaccharides produced by the action of the Streptomyces enzyme on fully modified polysaccharides. The results are discussed in relation to the available data for the structure of the polysaccharides and the specificity of the enzymes.     

Enantioselective reduction of ortho-substituted acetophenones by bacterial strains isolated from medium enriched with biphenyl or diesel fuel

Application of 21 new bacterial strains from natural environments (coastal plain of Santos and Atlantic Rain Forest, São Paulo, Brazil) in the asymmetric reduction of acetophenone derivatives is described. The bioreduction was carried out with whole bacterial cells leading to (S)-chiral alcohols in up to ≥99% e.e. The (S)-(−)-1-(2-bromo-phenyl)-ethanol was employed in the preparation of chiral tellurium derivatives.     

Reactions of nitrosamines in the Udenfriend system: Principal products and biological activity

Reaction of diethylnitrosamine in the non-enzymatic, ascorbic acid-dependent hydroxylating system of Udenfriend yielded N-nitroso-2-(ethylamino)-ethanol as the major product extracted into methylene chloride. The major product derived from nitrosopiperidine in the same system was N-nitroso-4-piperidone. These products, however, were mutagenic to Salmonella typhimurium TA 1535 only when activated by a rat liver microsomal preparation.     

Glycerogalactolipids from the fruit of Lycium barbarum

Four glycerogalactolipids (1-4), together with 11 other previously known homologues were isolated from the fruit of Lycium barbarum. Their structures were elucidated by chemical analyses including regio-selective enzymatic, alkaline and acidic hydrolyses and spectroscopic methods involving GCMS, HRESIMS and 1D and 2D NMR, respectively.     

[Dmt1]DALDA analogues with enhanced μ opioid agonist potency and with a mixed μ/κ opioid activity profile

Analogues of [Dmt¹]DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂; Dmt = 2′,6′-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity, were prepared by replacing Phe³ with various 2′,6′-dialkylated Phe analogues, including 2′,6′-dimethylphenylalanine (Dmp), 2′,4′,6′-trimethylphenylalanine (Tmp), 2′-isopropyl-6′-methylphenylalanine (Imp) and 2′-ethyl-6′-methylphenylalanine (Emp), or with the bulky amino acids 3′-(1-naphthyl)alanine (1-Nal), 3′-(2-naphthyl)alanine (2-Nal) or Trp. Several compounds showed significantly increased μ agonist potency, retained μ receptor selectivity and are of interest as drug candidates for neuropathic pain treatment. Surprisingly, the Dmp³-, Imp³-, Emp³- and 1-Nal³-containing analogues showed much increased κ receptor binding affinity and had mixed μ/κ properties. In these cases, molecular dynamics studies indicated conformational preorganization of the unbound peptide ligands due to rotational restriction around the C^βC^γ bond of the Xxx³ residue, in correlation with the observed κ receptor binding enhancement. Compounds with a mixed μ/κ opioid activity profile are known to have therapeutic potential for treatment of cocaine abuse.