Selectivity is an Important Characteristic of Lipases (Acylglycerol Hydrolases)

Lipases are enzymes that usually hydrolyze acylglycerols, but will hydrolyze the carboxylic esters in many other compounds. They also catalyze esteriftcations and transesterifications. In addition to specificity for carboxylic esters, the lipases are selective for lipid classes and show selectivity for primary vs. secondary alcohols (positional or regio-), fatty acids, enantiomers (chirality of either the acid or alcohol residue) and combinations of these. Uses of the enzymes have depended to some extent on regio- and fatty acid selectivities. Newer applications, such as ester synthesis and asymmetric hydrolysis, may not be based on selectivities. Factors affecting selectivities are discussed and some areas for research are mentioned.     

Lipases catalyzed enantioselective hydrolysis of (R,S)-methyl 1,4-benzodioxan-2-carboxylate intermediate for (S)-doxazosin mesylate

(S)-1,4-benzodioxan-2-carboxylic acid-1 is used as starting compound for the production of the more effective (S) enantiomer of the drug doxazosin mesylate. The catalytic ability of some commercial lipases for preparations of (S) enantiomer of 1 from (±) methyl 1,4-benzodioxin-2-carboxylate-2 is reported. Lipases from bacterial sources were more successful in resolving the ester than those from the yeast lipases. About 85% enantiomerically pure ester was achieved by lipase from alcaligenes sp.     

二氢杨梅素硬脂酸酯的合成及其抗油脂氧化特性研究

二氢杨梅素是药食两用植物———藤茶中的主体活性成分,具有很好的水溶性抗氧化作用,但其难溶于油的特性限制了其在油脂上的应用。本研究通过对其化学修饰,制备了既能溶于油脂又具有较好抗氧化作用的二氢杨梅素硬脂酸酯。     

Lipase Catalyzed Resolution of (±)-2-Substituted 1-Propanol Derivatives Possessing an Aromatic Ring

For the purpose of the chiral synthesis of natural products, lipase-catalyzed kinetic resolutions of three types of 2-substituted 1-propanol derivatives (each having an aromatic ring) was investigated. In every case, chiral recognition of the primary alcohol unit took place to provide the corresponding alcohols and the acetates in high optical purity. The (2S,3S)-5-aryl-2-methyl-3-hydroxy-4E-pentenol 5 was formally converted into the antibiotic, (-)-oudemansin X.     

(S)-3-Pentyn-2-ol production through microbial enzyme-catalyzed, highly enantioselective hydrolysis of racemic 3-pentyn-2-ol esters

Air-dried cells of Hansenula nonfermentans AKU 4332 catalyzed the production of (S)-3-pentyn-2-ol from (RS)-3- pentyn-2-ol acetate ester at 10% (v/v). The product was formed at 96.6% e.e. with a molar yield of 45% in 24 h. © Rapid Science Ltd. 1998     

Studies of the Heterogeneity of a Candida cylindracea (rugosa) Lipase: Monitoring of Esterolytic Activity and Enantioselectivity by Chiral Liquid Chromatography

A method for the determination of lipase activity in terms of rate and enantioselectivity of hydrolysis of a chiral ester substrate has been developed. When this method was applied to fractions, isolated from preparative, column chromatographic separations (anion-exchange, molecular sieve) of the lipase, significant differences in enantioselectivity (E) was found between the fractions. The highest enantioselectivity was found in the first main peak obtained on DEAE-Sepharose chromatography, meaning that the enzyme with the highest isoelectric point shows the highest esterolytic enantioselectivity.

The experimental results are discussed in the light of some earlier reported results and with respect to the possible existence of subunit aggregates and isoenzymes.     

番茄醇酰基转移酶基因SlAAT1克隆、序列分析和原核表达

酯类物质是许多果实香气的主要成分。醇酰基转移酶(AATs)是酯类化合物合成的关键酶。本研究通过反转录PCR,从番茄的成熟果实中克隆了SlAAT1基因(GenBank登录号为JQ070977),其编码一个含有442个氨基酸残基的蛋白,含有醇酰转移酶BAHD家族的H-x-x-x-D和DFGWG保守基序。系统进化分析表明,SlAAT1与苹果MpAAT1,山字草的BEBT及烟草Hsr201等聚在同一分支,进化关系较近。SDS-PAGE电泳分析表明,转化SlAAT1基因的大肠杆菌BL21(DE3)在22℃、0.8 mmol·L^-1 IPTG条件下可获得大量的可溶性目标蛋白。同时,纯化的SlAAT1大肠杆菌重组蛋白的体外酶活性分析表明了SlAAT1重组蛋白具有醇酰基转移酶活性,可能参与了酯类挥发性成分的合成。     

Synthesis of glycosides in which the aglycon is an N-(Hydroxymethyl)amino-1,3,5-triazine derivative

The synthesis of analogues of the anti-tumour drug 2-[N-(hydroxymethyl)methylamino]-4,6-bis (dimethylamino)-1,3,5-triazine (HMPMM) in which the OH or a dimethylamino group is replaced by a carbohydrate has been explored. Triazinyl β-glycosides were readily prepared by reaction of sugars with trimethyl-triazinylammonium salts. These were made with one or two methylamino groups on the triazine for reaction with formaldehyde to give the cytotoxic NMeCH2OH group. However, reaction of the triazinyl glycosides with formaldehyde gave complex intractable mixtures. When the carbohydrate portion was changed to the fully protected 2,3,4,6-tetra-O-acetyl glucose a good yield of the 2-[N-(hydroxymethyl)methylamino]-4-(dimethylamino)-1,3,5-triazin-2-yl tetra-O-acetyl β-glucoside was obtained. However, de-acetylation using sodium methoxide also removed the N–CH2OH group. We are investigating protection of the base-sensitive N–CH2OH group as trialkylsilyl and benzyl ethers and are looking at de-acetylation methods that are more selective. We have prepared glycosides in which the sugar is joined through the oxygen of the NMeCH2OH group. Coupling of acetobromoglucose with HMPMM catalysed by silver salts was not successful. Although methyl and cyclohexyl derivatives of HMPMM may be produced in high yields by reaction of HMPMM with methyl and cyclohexyl alcohols under acidic catalysis, production of glycosides in this way gave poor yields. MNDO calculations on reactions of HMPMM helped us devise improved reaction conditions for the condensation of 2,3,4,6-tetra-O-acetyl glucose with HMPMM and its derivatives. The best procedure to generate one of the target glycosides is to react 2,3,4,6-tetra-O-acetyl glucose and formaldehyde with 2-methylamino- 4,6-bis(dimethylamino)-1,3,5-triazine. The β-glycoside product was de-acetylated using potassium carbonate in dry methanol. Abbreviations: HMM, hexamethylmelamine (2) or 2,4,6-tris(dimethylamino)-1,3,5-triazine; HMPMM, hydroxymethylpentamethylmelamine or 2-[N-(hydroxymethyl)-methylamino]-4,6-bis(dimethylamino)-1,3,5-triazine; PMM, Pentamethylmelamine or 2-methylamino-4,6-bis(dimethylamino)-1,3,5-triazine; TBMS, t-Butyldimethylsilyl; p-TSA, p-Toluenesulphonic acid This revised version was published online in November 2006 with corrections to the Cover Date.     

Synthesis of the (9R,13R)-isomer of LDS1, a flower-inducing oxylipin isolated from Lemna paucicostata

The first synthesis of the (9R,13R)-stereoisomer of LDS1, a flower-inducing oxylipin isolated from Lemna paucicostata, has been achieved from a known allylic alcohol by a seven-step sequence that involves the Horner–Wadsworth–Emmons olefination to construct its full carbon framework and an enzymatic hydrolysis of a penultimate methyl ester intermediate to provide the target molecule.     

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides ofDl-alanine and N-(tert-butoxycarbonyl)-Dl-alanine

Summary The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-Dl-alanine, a 40% enantiomeric excess of thel isomer was incorporated at the internal 2 site and the positions of equilibrium for the 23 migration reaction differed for theD andl enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2(3)>internal 2>5, and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotectedDl-alanine led to an excess of theD isomer at the internal 2 site, while reaction with the N-carboxy anhydride ofDl-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.     

The Beneficial Effect of Organic Solvents on the Enzymatic Synthesis of Nucleoside Analogues Using N-Deoxyribosyltransferases from Lactobacillus leichmannii (E.C. 2.4.2.6)

Addition of ethylene glycol (10% v/v) has a beneficial effect on the synthesis of 2'-deoxynucleosides catalysed by crude preparations of N-deoxyribosyltransferases from Lactobacillus leichmannii. In the absence of added organic solvent decomposition of products and starting materials by deamination or hydrolysis occurs giving rise to poor yields of products if the transfer reaction is slow. The glycosyl transfer reaction is unaffected by addition of organic solvent but decomposition of products and starting materials is largely suppressed. The organic solvent appears to inhibit selectively contaminating enzymes in the crude N-deoxyribosyltransferase preparation as the purified transferase does not possess hydrolytic or deaminating activity. Different concentrations of ethylene glycol and other organic solvents have been examined as inhibitors of the side reactions but 10% (v/v) appears to be the most effective. Using the N-deoxyribosyltransferase in the presence of ethylene glycol, a number of 2'-deoxynucleosides of 6-substituted nucleosides have been obtained in high yield on a preparative scale.     

Separation of (R)- and (S)-tert-butyl 2-tert-butyl-4-methoxy-2,5-dihydro-1,3-imidazole-1-carboxylate (building block for amino acid synthesis) by preparative high performance liquid chromatography on a polysaccharide stationary phase

The preparative separation of the enantiomers of the title compound, a versatile chiral building block for the synthesis of unnatural amino acid esters, by high performance liquid chromatography on a chiral stationary phase (CSP), is reported for the first time. The CSP consists of amylose-(3,5-dimethylphenyl-carbamate), which has been coated onto the surface of macroporous aminopropyl-functionalized silica gel. The effect of mobile phase composition and the amount of amylose derivative on the silica gel has been thoroughly investigated. Using 2-propanol as organic modifier in hexane as mobile phase, on a semi-preparative column (200 mm × 40 mm ID, containing 192 g of stationary phase) about 200 mg of the racemate was separated per injection. Running the equipment under automatic conditions with repetitive injection mode allowed for the separation of 30 g per day. Both enantiomers were obtained with enantiopurities >99.75:0.25. Chirality 10:217–222, 1998. © 1998 Wiley-Liss, Inc.     

Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli. The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.     

First microwave-assisted synthesis of an electron-rich phosphane and its coordination chemistry with platinum(II) and palladium(II)

The P-O ligand 3-(di(2-methoxyphenyl)phosphanyl)propionic acid (HL) was synthesized by a microwave-assisted reaction of a secondary phosphane. The coordination of HL to Pt^II yielded the neutral mononuclear complex trans-[PtCl(κ²-P,O-L)(κ-P-HL)] (1), while the reaction of PdClMe(η⁴-COD) (COD = 1,4-cyclooctadiene) with HL in the presence of NEt₃ gave the anionic Pd^II compound of the formula (HNEt₃)[PdClMe(κ²-P,O-L)] (2). Upon crystallization of the latter compound the neutral chloride-bridged dimetallic compound cis-[Pd(μ-Cl)Me(HL)]₂ (3) was obtained. HL, 1 and 3·CH₂Cl₂ have been characterized by single crystal X-ray structure analyses.     

Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth

A protein fraction purified from bovine brain myelin, previously called arretin because of its ability to inhibit neurite outgrowth, has been identified as consisting predominantly of oligodendrocyte-myelin glycoprotein (OMgp). We show that it is a potent inhibitor of neurite outgrowth from rat cerebellar granule and hippocampal cells; from dorsal root ganglion explants in which growth cone collapse was observed; from rat retinal ganglion neurons; and from NG108 and PC12 cells. OMgp purified by a different procedure from both mouse and human myelin behaves identically in all bioassays tested.     

Synthesis,characterization, and evaluation of (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate as a biological agent and an anion sensor

An amino acid based and bidentate Schiff base, (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate (ligand), was synthesized from the reaction of glycine-methyl ester hydrochloride with 2-hydroxy-1-naphthaldehyde. Characterization of the ligand was carried out using theoretical quantum–mechanical calculations and experimental spectroscopic methods. The molecular structure of the compound was confirmed using X-ray single-crystal data, NMR, FTIR and UV–Visible spectroscopy, which were in good agreement with the structure predicted by the theoretical calculations using density functional theory (DFT). Antimicrobial activity of the ligand was investigated for its minimum inhibitory concentration (MIC) to several bacteria and yeast cultures. UV–Visible spectroscopy studies also shown that the ligand can bind calf thymus DNA (CT-DNA) electrostatic binding. In addition, DNA cleavage study showed that the ligand cleaved DNA without the need for external agents. Energetically most favorable docked structures were obtained from the rigid molecular docking of the compound with DNA. The compound binds at the active site of the DNA proteins by weak non-covalent interactions. The colorimetric response of the ligand in DMSO to the addition of equivalent amount of anions (F⁻, Br⁻, I⁻, CN⁻, SCN⁻, ClO₄⁻, HSO₄⁻, AcO⁻, H₂PO₄⁻, N₃⁻ and OH⁻) was investigated and the ligand was shown to be sensitive to CN⁻ anion.     

Interaction of copper(II) complexes with bis(p-nitrophenyl)phosphate: Structural and spectral studies

When the complexes [Cu(L1)(H₂O)](ClO₄)₂1, where L1 = 4-methyl-1-(pyrid-2-ylmethyl)-1,4-diazacycloheptane, and [Cu(L2)Cl₂] 2, where L2 = 4-methyl-1-(quinol-2-ylmethyl)-1,4-diazacycloheptane are interacted with one/two equivalents of bis(p-nitrophenylphosphate, (p-NO₂Ph)₂PO₂, BNP), no hydrolysis of BNP is observed. From the solution the adducts of copper(II) complexes [Cu₂(L1)₂((p-NO₂Ph)₂PO₂)₂]-(ClO₄)₂3 and [Cu(L2)((p-NO₂Ph)₂PO₂)₂]·H₂O 4 have been isolated and structurally characterised. The X-ray crystal structure of 3 contains two Cu(L1) units bridged by two BNP molecules. The Cu···Cu distance (5.1 Å) reveals no Cu-Cu interaction. On the other hand, the complex 4 is mononuclear with Cu(II) coordinated to the 3N ligand as well as BNP molecules through phosphate oxygen. The trigonality index (τ, 0.37) observed for 4 is high suggesting the presence of significant trigonal distortion in the coordination geometry around copper(II). The complexes are further characterized by spectral and electrochemical studies.     

Substrate specificity of an esterase from the archaeon Sulfolobus tokodaii bearing a GGG(A)X motif

A GGG(A)X-type esterase (Est0071) from an archaeon catalyzes asymmetric hydrolysis of prochiral bulky malonic diesters in good enantioselectivity. The selectivity of Est0071 was for the opposite enantiomer to that previously shown for pig liver esterase, and the resulting enantiomeric excess of the products was higher. Est0071 could also catalyze the hydrolysis of various acetates of secondary alcohols, and showed moderate enantioselectivity in these reactions.     

Purification and immunological properties of an NAD(H) dependent glutamate dehydrogenase from soybean cells (Glycine max L.)

Studies were carried out on glutamate dehydrogenase (GDH, EC 1.4.1.2) isolated from the SB1 and SB3 soybean (Glyciene max L. cv. Mandarin) cell cultures. The NAD(H) dependent enzyme from SB1 and SB3 cells was purified to homogeneity, and that from the SB3 cells studied in detail. It was shown to be activated by calcium. The molecular weight of the native enzyme was found to be 263 000 ± 12 000. The molecular weight of the subunits was shown to be 41 000 ± 2000, which indicates that the enzyme has a hexameric structure. Anti-GDH antibodies were produced in rabbits, to GDH purified to homogeneity from both cell cultures. Each antibody preparation reacted with the purified enzyme produced from either cell culture. Antibodies to GDH from SB3 cells were utilized to study the apparent induction of GDH, which occurs when these cells are grown in a medium with ammonium ions as the sole nitrogen source. The increase in GDH activity was shown to be due to de-novo protein synthesis. The anti-SB3-GDH antibody preparation was also tested for cross reactivity with crude GDH preparations from a number of plant sources, and purified GDH from a number of other organisms. The antibody was shown to cross react with a number of the GDH preparations.     

Vitreal dihydroxyphenylacetic acid (DOPAC) as an index of retinal dopamine release

Dopamine is generally accepted as a major neurotransmitter associated with light-adaptive processes in the retina. However, little is known about its precise release pattern in vivo, largely due to the lack of an unambiguous method for the determination of dopamine release. We have found that vitreal levels of dihydroxyphenylacetic acid (DOPAC) reflect the rate of dopamine release in chickens. Blocking re-uptake with nomifensine significantly lowered vitreal DOPAC and retinal dopamine, confirming the retinal origin and reliance of vitreal DOPAC on intact re-uptake mechanisms. Further, inhibition of monoamine oxidase with pargyline reduced vitreal as well as retinal DOPAC levels, confirming that the DOPAC detected is generated by monoamine oxidase. Finally, we found that DOPAC diffused freely into and out of isolated vitreous bodies and we found the vitreous to be metabolically inert with respect to DOPAC, supporting the idea that vitreal levels of DOPAC are consequential to the retinal metabolism of dopamine. Exposure to light, which is known to increase retinal dopamine release, readily increased vitreal DOPAC levels. The accumulation of DOPAC in the vitreous over 6 h light fitted a mathematical model of DOPAC accumulation based on zero-order influx (proportional to dopamine release rates) and diffusion driven, first-order efflux.