Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3′-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3′,6′-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3′-O-acetylasperulosidic acid (2a), 3′,6′-O-diacetylasperulosidic acid (2b), 4′-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6″-O-acetylpuerarin (3a), 6″-O-decanoylpuerarin (3b), 6″-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Complete and regioselective deacetylation of peracetylated uridines using a lipase

Lipase-catalysed alcoholysis and hydrolysis of 2',3',5'-tri-O-acetyluridine (1a) and 2',3',5'-tri-O-acetyl-2'-C-methyluridine (1b) were studied. Conditions for full and regioselective deacetylation of 1aand 1b are shown in the present work. New compound 2',3'-di-O-acetyl-2'-C-methyluridine (3b) was prepared by regioselective lipase-catalysed deacetylation.     

Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.     

Regio- and enantio-selective acetylation of 3,3,3-trifluoro-2-arylpropane-1,2-diols using Candida antarctica lipase

Of nine commercially available lipases, lipase SP 435 from Candida antarctica, showed moderate enantioselectivity (E=17) for acetylation of racemic 3,3,3-trifluoro-2-phenylpropane-1,2-diol, 2, with vinyl acetate in diisopropyl ether (S selectivity). The other eight had low selectivities, with E values below 10. The selectivity and reactivity of SP 435 for 2 was markedly improved in dichloroethane (E=41). Moreover, SP 435 had moderate to high selectivity for the related compounds 3,3,3-trifluoro-2-(1-naphthyl)-propane-1,2-diol, 4, (E=20), 3,3,3-trifluoro-2-(indol-3-yl)propane-1,2-diol, 6, (E=80), and 3,3,3-trifluoro-2-(pyrrol-2-yl)-propane-1,2-diol, 8, (E=17).     

Enzymatic resolution of rac-1,1-dimethyl-1-sila-cyclohexan-2-ol by ester hydrolysis or transesterification using a crude lipase preparation of Candida cylindracea

Summary rac-2-Acetoxy-1,1-dimethyl-1-sila-cyclohexane (rac-2) was synthesized by esterification of rac-1,1-dimethyl-1-sila-cyclohexan-2-ol (rac-1) with acetic anhydride. Enantioselective hydrolysis of rac-2 in aqueous solution, catalysed by a crude lipase preparation of Candida cylindracea (EC 3.1.1.3), led to the formation of (S)-1 (95% ee). Enantioselective transesterification of rac-1 with triacetin in isooctane, catalysed by the same enzyme preparation, yielded (S)-2 (95% ee), which was separated by chromatography from non-reacted (R)-1 (96% ee). Recrystallization led to an improvement of the enantiomeric purity of (R)-1 and (S)-1 up to >98% ee. Thus the enantiomers of rac-1 were prepared (100 mg scale) with high enantiomeric purities by the use of two different types of enzyme-catalysed reaction.     

Hormonal control of lipase activity in oilseed rape germinants

In oilseeds, storage lipids provide the respiratory fuel for seedling growth. The enzyme responsible for their initial hydrolysis is lipase (triacylglycerol acylhydrolase; EC 3.1.1.3). We investigated the possibility that lipase is regulated by gibberellins (GAs). In four oilseed rape cultivars of Brassica napus and B. rapa, seed imbibition in 10^?6 to 10^?3M GA₃ increased lipase activity 1.5- to 7-fold over control levels. Conversely, imbibition in 10^?7 to 10^?5M abscisic acid or 10^?6 to 10^?4M paclobutrazol, an inhibitor of GA biosynthesis, markedly decreased lipase activity. While lipase activity in B. napus cv. Parkland increased during the first 5 days following imbibition, concentrations of endogenous GA₁, GA₈ and GA₁₉ (as measured by GC-selected ion monitoring using [²H₂]GA internal standards) were relatively constant and GA₂₀, a precursor of GA₁, decreased. Levels of endogenous GA₃ were apparently variable. Thus, lipase activity was not correlated with GA₁ concentration, but the inverse correlation with GA₂₀ concentration suggests that GA turnover could be positively correlated with lipase activity. Lipase activity was also examined in three genotypes of rapid cycling B. rapa that vary in endogenous GA content: rosette, a GA-deficient dwarf, a normal line and elongated internode, a tall mutant with high GA content. The three genotypes showed similar patterns of lipase activity during the first 4 days following imbibition and the subcellular distribution of lipase activity was also similar in the three genotypes. Although GA may be involved in the regulation of lipase in oilseed rape germinants, it is not the sole regulatory factor.     

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli

The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K _m and V _max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 μM and 217 ± 22.5 μmol min⁻¹ mg⁻¹ protein, respectively.     

Design, synthesis and in vitro evaluation of novel chroman-4-one, chroman, and 2H-chromene derivatives as human rhinovirus capsid-binding inhibitors

As part of an effort to generate broad-spectrum inhibitors of rhinovirus replication, novel series of (E)-3-[(E)-3-phenylallylidene]chroman-4-ones 1a–e, (E)-3-(3-phenylprop-2-yn-1-ylidene)chroman-4-ones 2a and 2b, (Z)-3-[(E)-3-phenylallylidene]chromans 3a–e, and (E)-3-(3-phenylprop-1-en-1-yl)-2H-chromenes 4a–d were designed and synthesized. All the compounds were tested in vitro for their efficacy against infection by human rhinovirus (HRV) 1B and 14, two representative serotypes for rhinovirus group B and A, respectively. Most of the analogues were found to be potent and selective inhibitors of both HRVs, although HRV 1B was generally more susceptible than HRV 14. Mechanism of action studies of (E)-6-chloro-3-(3-phenylprop-1-en-1-yl)-2H-chromene 4b, the most potent compound on HRV 1B infection, suggested that 4b behaves as a capsid-binder probably acting at the uncoating level.     

高山被孢霉中二酰甘油酰基转移酶2同源基因的克隆、表达和活性分析

[背景] 高山被孢霉（Mortierella alpina）是一种可积累大量花生四烯酸（Arachidonic Acid,AA）的产油丝状真菌,其所产脂肪酸主要被组装到甘油骨架上以三酰甘油（Triacylglycerol,TAG）形式存在。二酰甘油酰基转移酶（Diacylglycerol Acyltransferase,DGAT）是TAG生物合成途径的关键酶,对于高山被孢霉TAG的生产具有重要意义。[目的] 通过探究高山被孢霉DGAT2在TAG生物合成方面的功能特点,以期为提高产油真菌的TAG产量及改善TAG的脂肪酸组成提供参考。[方法] 利用序列比对在高山被孢霉ATCC32222基因组中筛选出2个编码DGAT2的候选基因MaDGAT2A/2B,在酿酒酵母（Saccharomyces cerevisiae）中异源表达后进行功能分析,并在外源添加AA条件下通过检测TAG产量进一步分析MaDGAT2A/2B的活性,最后在高山被孢霉中同源过表达MaDGAT2A/2B,通过检测重组菌总脂肪酸产量及组分以分析MaDGAT2A/2B的体内活性。[结果] MaDGAT2A在S. cerevisiae中异源表达时,重组酵母菌TAG的产量达到细胞干重的3.06%,为对照组的4.91倍;而MaDGAT2B未明显提高重组酵母菌TAG的产量。在外源添加AA时,MaDGAT2A/2B均可显著促进重组酵母菌中TAG合成,表达MaDGAT2A的重组酵母菌TAG含量为对照组的3.67倍,表达MaDGAT2B的重组酵母菌TAG含量为对照组的2.61倍。MaDGAT2A/2B在高山被孢霉中过表达对其总脂肪酸产量无显著影响,但可显著提高总脂肪酸中AA的含量,AA占总脂肪酸比例最高达到39.15%,相比对照组提高16.14%。[结论] MaDGAT2A/2B可以参与TAG的生物合成,表明2个候选基因编码的蛋白具有DGAT活性,并且可提高高山被孢霉脂肪酸中AA的含量,对于改善产油真菌的脂肪酸组成从而提高其应用价值具有重要意义。     

Resolution of 2-octanol via immobilized Pseudomonas sp. lipase in organic medium

Abstract

Pseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol. The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 ± 5% and 2.5:1, respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess > 99%) could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in its activity and enantioselectivity.     

Two additives to improve stability of immobilized lipase

Abstract

The presence of two different additives during non-covalent immobilization of lipase was studied. Lipase was immobilized via hydrophobic interactions on an amorphous silica with large pore size bearing octyl groups on the surface. Polyethyleneglycol (PEG) with different molecular weights (MW: 1500, 3000 and 10,000) were added to the suspension during enzyme immobilization, in an enzyme to PEG molar ratio of 1:10, and also 1:20 in the case of PEG1500. The activity after 15 d increased from 10% (absence of PEG) to values close to 40% in samples with PEG except the catalyst immobilized in the presence of 1:10 PEG1500, which kept fully active after 15 d incubation in toluene at 70?°C. The presence of water during storage of immobilized enzymes leads to significant activity loss. Saturated solutions of salts controlling the water activity of the systems were used to reduce in a controlled fashion the moisture of the systems: CaCl₂ (a_w=?0.037), MgCl₂ (a_w=?0.328), Mg(NO₃)₂ (a_w=?0.529), Na₂PO₄.12H₂O (a_w=?0.74) and KCl (a_w=?0.84). The immobilized lipase was suspended in saturated solutions of these salts, and then filtered and incubated in desiccators in the presence of the corresponding saturated salt solutions. Catalysts suspended and incubated in KCl or only suspended in phosphate kept some 20% activity after 33 d incubation whereas the maximal stability was achieved when the catalyst was suspended in phosphate and kept in a desiccator without salt solution. This catalyst kept around 50% activity after 33 d incubation. An inversely proportional relationship can be established between the stability achieved by the enzyme and the water content of the system.     

Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta

Protein tyrosine phosphatase 1B (PTP1B) appears to be an attractive target for the development of new drugs for type 2 diabetes and obesity. In our preliminary test, a MeOH extract of the stem barks of Sorbus commixta Hedl. (Rosaceae) showed strong PTP1B inhibitory activity. Bioassay?guided fractionation of the MeOH extract resulted in the isolation of two lupane?type triterpenes, lupenone (1) and lupeol (2). Compounds 1 and 2 inhibited PTP1B with IC₅₀ values of 13.7 ± 2.1 and 5.6 ± 0.9 μM, respectively. Kinetic studies revealed that both the compounds 1 and 2 are non?competitive inhibitors of PTP1B that decrease V_max values with no effect on K_m values.     

PTP1B inhibitory activity of kaurane diterpenes isolated from Siegesbeckia glabrescens

Protein tyrosine phosphatase 1B (PTP1B) is considered as a therapeutic target for the treatment of diabetes and obesity. In our preliminary screening study, a MeOH extract of the aerial part of Siegesbeckia glabrescens was found to inhibit PTP1B activity at 30 μg/mL. Bioassay‐guided fractionation led to the isolation of two active diterpenes, ent-16βH,17-isobutyryloxy-kauran-19-oic acid (1) and ent-16βH,17-acetoxy-18-isobutyryloxy-kauran-19-oic acid (2), along with ent-16βH,17-hydroxy-kauran-19-oic acid (3). Compounds 1 and 2 inhibited the PTP1B activity with IC₅₀ values of 8.7 ± 0.9 and 30.6 ± 2.1 μM, respectively. Kinetic studies suggest that both 1 and 2 are non-competitive inhibitors of PTP1B. However, compound 3 substituted with a hydroxyl group at C-17 in kaurane-type showed no inhibitory effects towards PTP1B.     

Overexpression of lipase A and B of Geotrichum candidum in Pichia pastoris: High-level production and some properties of functional expressed lipase B

Lipase A and B from Geotrichum candidum CMICC 335426 were functionally expressed and secreted in Pichia pastoris. Lipase B exhibits a unique substrate specificity for long-chain cis-9 unsaturated triacylglycerols. Thus, using different cultivation conditions, the production of recombinant lipase B was optimised, yielding 200 000 Units per liter culture. After overexpression lipase B was purified by diafiltration (372 U/mg). The investigated enzyme properties were comparable to those reported for the native enzyme.     

Effects of carbon sources on extracellular lipase production and lipA transcription in Acinetobacter calcoaceticus

  The effects of lactic acid, oleic acid, gum arabic and their mutual interactions, on the production of extracellular lipase and the regulation of the expression of the lipase encoding gene (lipA) in Acinetobacter calcoaceticus were investigated. Formation of extracellular lipase was measured in culture supernatants of wild-type strain BD413 and expression of the lipA gene was monitored in vivo with a chromosomal fusion of lipA to lacZ. At the level of lipA expression only oleic acid had a significant effect; it lowered expression. Neither gum arabic nor lactic acid had any effect on lipA expression. On the other hand, the yield of extracellular lipase increased 2–5 times by the addition of gum arabic, possibly due to the release of cell surface-bound lipase. An interaction between oleic acid and lactic acid was also detected. Journal of Industrial Microbiology & Biotechnology (2000) 24, 25–30. Received 03 May 1999/ Accepted in revised form 04 September 1999     

中国猪种布鲁氏菌分子流行病学特征分析

【目的】调查猪种布鲁氏菌的基因多态性和分子流行病学特征。【方法】用经典分型方法对菌株的生物型进行鉴定,分析菌株的地理分布特点;用MLVA-16分型方法对60株猪种布鲁氏菌进行基因分型,采用在线软件评估分型方法的分辨率和位点的多态性,用BioNumerics 5.0软件进行聚类分析。【结果】我国流行的猪种布鲁氏菌主要是猪种生物1型(33株)、2型(3株)和3型菌(24株);分布范围较广,包括广东、广西、内蒙古、北京、吉林、宁夏和西藏等地。MLVA-16分型方法对猪种布鲁氏菌具有极高的分辨力,多态性指数为0.992;Panel1、MLVA-11和Panel 2B均具有较高的分辨率,多态性指数分别为0.884、0.916和0.979。60株猪种布鲁氏菌聚为6大类52个基因型,5个共享基因型(GT24,GT25,GT26,GT28,GT29)包括13株布鲁氏菌,各基因型菌株间有潜在的流行病学关联,可能是分别来自相同传染源的暴发流行;另47株布鲁氏菌呈现独特的基因型,表明菌株来自无流行病学关联的零星散发病例。猪种布鲁氏菌的最小生成树表明我国菌株分别与美国、法国和波兰的菌株有完全相同的MLVA-15基因型。【结论】中国猪种布鲁氏菌有较高的遗传多态性,并与美国、法国和阿根廷的菌株有较高的遗传相似性。我国猪种布病以零星散发为主。     

黄皮种子中酰胺类生物碱及其杀线虫活性研究

为了解黄皮种子中的酰胺类生物碱及其杀线虫活性,运用多种色谱学及波谱学方法分离并鉴定了10个酰胺类生物碱,分别为:N-甲基桂皮酰胺(1),clausenalansamide A(2),3-dehydroxy-3-methoxyl-clausenalansamide A(3),clausenalansamide B(4),黄皮新肉桂酰胺B(5),N-(2-苯乙基)肉桂酰胺(6),2′-dehydroxy-2′-oxo-clausenalansamide B(7),lansamide-7(8),homoclausenamide(9),1,5-dihydro-5-hydroxy-1-methyl-3,5-diphenyl-2H-pyrrol-2-one(10)。其中,化合物3,7,10为新天然产物。首次对黄皮种子中的酰胺类生物碱2~8进行全齿复活线虫致死活性的测试,发现所测化合物均有致死活性,其中,化合物2,3,5和8有较强的致死活性,且均优于阳性对照除线磷,可为相关农药的研发提供科学依据。     

A Lichenological Excursion to the West of Scotland

ABSTRACT

Background: Hybridisation can be a threat for the survival of a rare species because, in the case of insufficient numbers of appropriate mates, a rare form is much more likely to cross with a widespread taxon.

Aims: In the present study, we tested hypotheses concerning the level of hybridisation between endangered Betula humilis and its widespread congeners: B. pendula and B. pubescens as a function of habitat conditions.

Methods: We genotyped 312 individuals of three species using AFLP markers. B. humilis specimens were sampled in populations with low and high groundwater levels. Morphological identification of B. pubescens and B. pendula was verified using the Atkinson discriminant function.

Results: Altogether, 15 individuals (4.8%) were indicated as putative hybrids. The B. humilis hybrids were found in dry habitats and they could be classified as F1 or F2 generation. Tree hybrids could represent backcrosses to either B. pendula or B. pubescens.

Conclusions: Genetic analyses contradicted the idea that hybridisation between B. humilis and its close relatives was extensive. On the other hand, the presence of introgressed individuals in the populations in areas with low groundwater levels implied that pollen swamping might be a threat for declining B. humilis stands.     

研究报告: EIF2B5表达下调的人星形胶质细胞与人神经元在内质网应激后细胞存活及miRNA表达的差异

目的 探讨白质消融性白质脑病中胶质细胞选择性受累而神经元受累轻微的原因。方法 将EIF2B5-RNAi表达载体转染至人星形胶质细胞和人神经元,检测基础状态下及内质网应激（endoplasmic reticulum stress,ERS）后细胞凋亡和活力,检测参与ERS调控的已知和未知miRNA,筛选EIF2B5-RNAi人星形胶质细胞在ERS后miRNA变化。结果 与EIF2B5-RNAi人神经元相比,星形胶质细胞自发凋亡及细胞活力下降。较之神经元,更多miRNA参与星形胶质细胞ERS刺激后的调控,EIF2B5-RNAi组参与调控的miRNA数目显著减少。聚类分析发现,5条已知miRNA是通路连接的关键组分。结论 人星形胶质细胞在ERS后可能更加依赖众多促细胞增殖分化的miRNA修复,而EIF2B5-RNAi人星形胶质细胞存在自发凋亡,ERS后严重减少的miRNA可能导致细胞无法存活。     

Analysis of CYP450 gene allelic variants can predict ifosfamide toxicity in Mexican paediatric patients

Abstract

Context: Ifosfamide (IFA) is an effective antineoplastic for solid tumours in children, although it is associated with high levels of systemic toxicity and causes death in some cases.

Objective: The aim of this study was to determine whether the presence of certain allelic variants of genes CYP2B6, CYP2C9, CYP3A4 and CYP3A5 increases the risk of toxicity in children with solid tumours treated with ifosfamide.

Materials and methods: A total of 131 DNA samples were genotyped by real-time polymerase chain reaction (RT-PCR) using TaqMan probes. Toxicity was assessed using WHO criteria, and survival analysis was performed using Kaplan–Meier curves.

Results: The rs3745274 allelic variant in CYP2B6 was associated with haematological toxicity, affecting neutrophils; CYP3A4 variant rs2740574 was also associated with toxicity, affecting both leukocytes and neutrophils. Additionally, the CYP3A5 gene variant rs776746 was found to affect haemoglobin.

Conclusions: Our results show that allelic variants rs3745274 (CYP2B6), rs2740574 (CYP34) and rs776746 (CYP3A5) increase the risk for high haematological toxicity.

Clinical trial registration: 068/2013