Highly regioselective enzymatic synthesis of 5′-O-stearate of 1-β-D-arabinofuranosylcytosine in binary organic solvent mixtures

In this paper, highly regioselective enzymatic acylations of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5′-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS–ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50°C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol·L⁻¹h⁻¹, 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13(¹³C) NMR and confirmed to be 5′-O-stearate of ara-C.     

A novel biocatalytic approach to acetylation of 1-β-<Emphasis Type="SmallCaps">d</Emphasis>-arabinofuranosylcytosine by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> whole cell in organic solvents

Biocatalytic acylation of 1-β-d-arabinofuranosylcytosine (ara-C) was developed using whole cell of Aspergillus oryzae as a novel catalyst. ¹³C nuclear magnetic resonance (NMR) analysis indicated that the whole-cell biocatalyst had more specific activity toward the 3′-hydroxyl group than 5′-hydroxyl group among the available hydroxyl groups in sugar moiety of ara-C. Except for glucose and maltose, 11 carbon sources supplemented to basal media, including Spans, Tweens, olive oil and oleic acid, exhibited notable enhancement effects on both the cell growth and the acylation reactions. It was suggested that the carbon sources containing controlled-release oleic acid were the important substrates for the production of fungal cell-bound lipase with specific activity, partially due to a gradual induction effect of their released oleic acid on the cell-bound lipase production. Despite the low initial reaction rate and substrate conversion, the addition of 2.0 g/l Span 80 resulted in a higher 3′-regioselectivity of the cells than 81%. By using Tween 85 at its optimum concentration of 5.0 g/l, however, the highest initial rates (3.2 mmol/l h) and substrate conversion (76%) of the whole-cell catalyzed acylation of ara-C can be achieved. It was also found that the 3′-regioselectivity of the cells showed observable increase by extending the culture time. And the activity of cell-bound lipase drastically increased in the early stage of cell growth and then declined in the late culture stage, whatever the culture media used. Our results thus indicated that A. oryzae whole cell was a promising green tool for biosynthesis of nucleoside esters with potential bioactivities.     

Novel and highly effective chemoenzymatic synthesis of (2<Emphasis Type="Italic">R</Emphasis>)-2-[4-(4-cyano-2-fluorophenoxy)phenoxy]butylpropanoate based on lipase mediated transesterification

(2R)-2-[4-(4-Cyano-2-fluorophenoxy)phenoxy]butylpropanoate (cyhalofop-butyl, CyB) was synthesized by a chemoenzymatic route involving enantioselective transesterification with Candida antarctica lipase B (Novozym 435). The optimum organic solvent, acyl donor, a _w , reaction temperature and shaking rate for the transesterification were acetonitrile, n-butanol, 0.11, 45°C and 200 rpm, respectively. Under the optimum conditions, the maximum substrate conversion and the enantiomeric purity of the product were 96.9 and >99%, respectively. The total yield and enantiomeric purity of CyB by this chemoenzymatic synthesis were 60.4 and >99%, respectively; 15.3 and 21% higher than that of the traditional way (45 and 78%).     

Application of organic solvent system for lipase-catalyzed regioselective benzoylation of 1-<Emphasis Type="Italic">β</Emphasis>-D-arabinofuranosylcytosine

In this paper, enzymatic regioselective acylation of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl benzoate (VB) using immobilized Candida antarctica lipase B in binary organic solvents was explored. It was found that the lipase showed high regioselectivity (> 99%) towards the 5′-OH of ara-C in the representative organic solvent mixture (hexane-pyridine). To understand the enzymatic processes and provide a fair comparison of hexane-pyridine with C₄MIm·PF₆-pyridine (the representative ionic liquid-containing system), the effect of each process variable on the reactions in hexane-pyridine was investigated. The results indicate that the optimum hexane content, initial a _w , molar ratio of VB to ara-C, and temperature were 28% (v/v), 0.11, 15, and 40°C, respectively. Under optimized conditions, the initial reaction rate in hexanepyridine (44.4 mM/h) was much higher than that in C₄MIm·PF₆-pyridine (29.4 mM/h) for each case. The maximum conversion yield, however, was increased when the reaction system was shifted from hexane-pyridine to C₄MIm·PF₆-pyridine. Further study revealed that the presence of an acidic by-product (benzoate acid, released during the acylation process) may cause rapid inactivation of the enzyme in hexane-pyridine, leading to a lower conversion rate, whereas the ionic liquid may have coating and protecting effects on the lipase during the reaction.     

Unexpected reversal of the regioselectivity in <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> lipase-catalyzed acylation of floxuridine

Unexpected inversion of the 3′:5′-regioselectivity was observed in the enzymatic methacryloylation, crotonylation and cinnamoylation of floxuridine (1.5:1, 2.3:1 and 4.4:1, respectively), where Thermomyces lanuginosus lipase preferentially catalyzed the acylation of 3′-hydroxyl rather than that of 5′-hydroxyl group. The possible reason might be the presence of a remote interaction between the unsaturated bond in the acyl group and the aromatic ring of amino acid residue Trp89 in the lid of the lipase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of β-apo-10′-carotenal from β-carotene by human β-carotene-9′,10′-oxygenase expressed in <Emphasis Type="Italic">E. coli</Emphasis>

The gene encoding human β-carotene-9′,10′-oxygenase, which cleaves the 9′,10′ double bond in β-carotene into β-apo-10′-carotenal, was cloned and expressed in Escherichia coli. Under aqueous conditions, the optimum organic solvent for the formation of detergent micelles was toluene. The optimum pH, temperature, detergent type, and the optimum concentrations of detergent, substrate, and enzyme for β-apo-10′-carotenal production were 8.0, 37°C, Tween 40, 2.4%, 300 mg β-carotene/l, and 0.25 U/ml, respectively. Under the optimum conditions, 43 mg β-apo-10′-carotenal/l was produced after 21 h with a conversion of 14%. This is the first report to describe the enzymatic production of β-apo-10′carotenal.     

Simple and efficient enzymatic transglycosylation of stevioside by β-cyclodextrin glucanotransferase from <Emphasis Type="Italic">Bacillus firmus</Emphasis>

Stevioside was subjected to 1,4-intermolecular transglycosylation using β-cyclodextrin glucanotransferase (β-CGtase) produced from an alkalophilic strain of Bacillus firmus. The reaction was carried out by traditional, ultrasound-assisted and microwave-assisted techniques. Reaction under microwave conditions was faster and was completed in 1 min yielding two 1,4 transglycosylated products, 4′-O-alpha-d-glycosyl stevioside (I) and 4′′-O-alpha-d-maltosyl stevioside (II) in 66% and 24%, respectively. The optimum transglycosylation occurred by using stevioside (1.24 mmol), β-CD (1.76 mmol) and β-CGtase (2 U/g) under microwave assisted reaction (MAR) in 5 ml sodium phosphate buffer (pH 7) at 50°C and 80 W power. MAR is therefore potentially a useful and economical method for faster transglycosylation of stevioside. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

Regioselective enzymatic procedure for preparing 3′-O-stearoyl-6-azauridine by using Burkholderia cepacia lipase

Previous studies on the lipase-mediated acylation of 6-azauridine with vinyl stearate in organic solvents revealed that while preparing a potential prodrug, 3′-O-stearoyl-6-azauridine, a lipase from Burkholderia cepacia showed high regioselectivity toward the second hydroxyl group. The most suitable reaction solvent, molar ratio of vinyl stearate to 6-azauridine, and reaction temperature were anhydrous acetone, 15:1, and 45°C, respectively. Under these conditions, the initial reaction rate, 3′-regioselectivity, and maximum substrate conversion were as high as 10.4 mM/h, 86.0, and 99.0%, respectively.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Highly regioselective galactosylation of floxuridine catalyzed by <Emphasis Type="Italic">β</Emphasis>-galactosidase from bovine liver

5′-O-β-d-Galactosyl-floxuridine, a potential novel prodrug, was synthesized with a yield of 75% through β-galactosidase-catalyzed transgalactosylation. This enzyme displayed absolute regioselectivity toward the 5′-position of floxuridine. For the reaction, the optimal conditions were pH 6.5 at 45°C for 60 h with floxuridine to o-nitrophenyl-β-d-galactoside at 2:1 (mol/mol). Under these conditions, the initial reaction rate and the maximum yield were 0.28 mM h⁻¹ and 75%, respectively.     

Enzymatic synthesis of nucleoside analogues using immobilized 2′-deoxyribosyltransferase from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Covalent attachment of recombinant Lactobacillus reuteri 2′-deoxyribosyltransferase to Sepabeads EC-EP303 leads to the immobilized biocatalyst SLrNDT4, which displayed an enzymatic activity of 65.4 IU/g of wet biocatalyst in 2′-deoxyadenosine synthesis from 2′-deoxyuridine and adenine at 40°C and pH 6.5. Response surface methodology was employed for the optimization of SLrNDT4 activity. Optimal conditions for SLrNDT4 highest activity were observed at 40°C and pH 6.5. Immobilized biocatalyst retained 50% of its maximal activity after 17.9 h at 60°C, whereas 96% activity was observed after storage at 40°C for 110 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of different natural and therapeutic nucleosides effective against cancer and viral diseases. Among these last products, enzymatic synthesis of therapeutic nucleosides such as 5-ethyl-2′-deoxyuridine and 5-trifluorothymidine has been carried out for the first time. Importantly for its potential application, SLrNDT4 could be recycled for 26 consecutive batch reactions in the synthesis of 2,6-diaminopurine-2′-deoxyriboside with negligible loss of catalytic activity.     

Two new dialkoxycarbonylated nucleosides obtained through a regioselective enzymatic alcoholysis

Two new compounds, 2′,3′-di-O-ethoxycarbonyluridine and 2′,3′-di-O-ethoxycarbonylinosine, were obtained through a Candida antarctica lipase B catalysed regioselective ethanolysis of the corresponding trialcoxycarbonylated nucleosides with benzyl alcohol in 1,4-dioxane at 30°C.     

Transaminase-catalyzed asymmetric synthesis of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid from achiral reactants

Asymmetric synthesis of an unnatural amino acid was demonstrated by ω-transaminase from Vibrio fluvialis JS17. l-2-Aminobutyric acid was synthesized from 2-oxobutyric acid and benzylamine with an enantiomeric excess higher than 99%. The reaction showed severe product inhibition by benzaldehyde, which was overcome by employing a biphasic reaction system to remove the inhibitory product from the aqueous phase. In a typical biphasic reaction (50 mM 2-oxobutyric acid, 70 mM benzylamine and 2.64 U/ml purified enzyme) using hexane as an extractant, conversion of 2-oxobutyric acid reached 96% in 5 h whereas only 39% conversion was obtained without the product extraction.     

<Superscript>1</Superscript>H NMR-based metabolic profiling and target analysis: a combined approach for the quality control of <Emphasis Type="Italic">Thymus vulgaris</Emphasis>

The characterization of T. vulgaris plant material for quality control purposes was performed by NMR-based methods. Direct extraction of 141 T. vulgaris samples with DMSO-d ₆ enabled the obtainment of crude extracts with a representative composition in terms of both volatile and non-volatile constituents. The acquisition of 600 MHz ¹H NMR spectra resulted in a dataset which was analyzed by a combination of metabolic profiling and target analysis approaches. Preliminary analysis of the ¹H NMR spectra was performed by principal component analysis, which revealed sample discrimination on a chemotype basis (thymol, carvacrol and linalool chemotypes). Further minor discriminative constituents were identified as p-cymene, γ-terpinene, rosmarinic acid, and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl. Metabolite identification was accomplished by 1D and 2D NMR techniques and supported by spiking experiments. Fast dereplication of constituents not available as reference compounds was performed by HPLC–SPE–NMR experiments. A targeted approach based on qHNMR was validated for quantification of the identified secondary metabolites. Validation was performed in terms of precision (intra-day RSD ≤ 4.51%, inter-day RSD ≤ 4.18%), repeatability (RSD ≤ 2.30%), accuracy (recovery rates within 93.4 and 103.4%), linearity (correlation coefficients ≥ 0.9990), robustness, and stability. The amount of the dominant monoterpene in thymol, carvacrol, and linalool chemotypes was respectively found to be within 0.4–2.6, 0.7–2.3, and 1.1–3.6% (w/w). Variable amounts of the precursors p-cymene and γ-terpinene were found in thymol and carvacrol chemotypes. The highest amount of rosmarinic acid and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl in the analyzed samples was respectively 4.6 and 0.4% (w/w). Since quantification is performed on a weight basis, the essential oil content can be estimated based on the sum of the quantified monoterpenes. The NMR-based analysis of T. vulgaris represents a more comprehensive approach in comparison to traditional chromatographic methods such as GC and LC, respectively employed for the analysis of volatile and non-volatile constituents. Further advantages lie in the simple sample preparation, rapidity and reproducibility of the NMR analysis.     

Optimization of Process Variables for Lipase Biosynthesis from <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> NRRL 5905 Using Evolutionary Operation Factorial Design Technique

Extracellular lipase was produced from Rhizopus oligosporus NRRL 5905 through solid state fermentation (SSF). To provide an optimum fermentation conditions for maximum lipase yield, five process variables (temperature, liquid–solid ratio, pH, incubation period and spore concentration) were optimized using evolutionary operation (EVOP) factorial design technique taking into account the interaction between the process variables. Optimization through EVOP resulted in around 3 fold increase in lipase activity (77 U gds⁻¹) at a liquid–solid ratio of 1.5:1, fermentation temperature of 35°C, initial fermentation pH 6, incubation period 5 days and a spore concentration of 10⁸ spores ml⁻¹.     

Resolution of 2-nitroalcohols by Burkholderia cepacia lipase-catalyzed enantioselective acylation

Racemic 2-nitro-1-phenylethanol was resolved by via enantioselective transesterification catalyzed by Burkholderia cepacia lipase. The reaction afforded excellent E values (E > 200) and enantioselectivity (up to >99% enantiomeric excesses [ee]) of both remaining substrates and acetylated product. Moreover, the lipase displayed high enantioselectivity in the resolution of additional 2-nitroalcohols (E up to >200). This method provides an efficient alternative for obtaining enantiopure 2-nitroalcohols.     

Optimization of Lipase Production from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> by Response Surface Methodology and Its Potential for Synthesis of Partial Glycerides Under Solvent Free Conditions

Aspergillus terreus produces lipase 7.01 IU/ml in 96 h after optimization by one variable at a time method. Using the significant factors i.e. corn oil (A), sodium nitrate (B), casein (C), agitation rate (D) and incubation period (E) RSM was carried out resulting in 19.65 IU/ml from the combination +1(A), −1(B), −1(C), +1(D) and 0(E). The interactions between sodium nitrate, casein and agitation with corn oil were most significant. Scale up of production from 250 ml shake flask to 30 l bioreactor resulted in increased productivity of 0.52 IU/ml/h as against 0.2 IU/ml/h obtained in shake flasks. This lipase could carryout solvent free synthesis of partial glycerides of oleic acid with 96% efficiency in 12 h.     

Synthesis of chiral α-hydroxy amides by two sequential enzymatic catalyzed reactions

Enantiomerically pure α-hydroxy amides have been prepared from the corresponding α-oxo esters by the use of a double sequence reaction involving in a first step the highly enantioselective Saccharomyces cerevisiae bioreduction and then in a second step, the resulting α-hydroxy esters followed a non-enantiospecific lipase catalyzed aminolysis with n-butylamine reaction. In the first non-organic solvent process, the moistened baker’s yeast reduced seven α-oxo esters with high conversions degree (93% for one substrate and >99% for the others) and high enantioselectivities [>99% for all the substrates except for ketopantoyl lactone, which gave 88% of enantiomeric excess (ee)]. At the same way, the isolated resulting chiral α-hydroxy esters were subjected to the second Candida antarctica lipase fraction B (CAL-B) catalyzed aminolysis in dioxane conducting to the corresponding chiral α-hydroxy amides with high conversions degree, between 88 and 99%. Both processes were carried out at 28–30°C.