Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

The role of different anions in ionic liquids on Pseudomonas cepacia lipase catalyzed transesterification and hydrolysis

Lipase Pseudomonas cepacia (PS) catalyzed transesterification of ethyl 3-phenylpropanoate with eleven alcohols was investigated in three ionic liquids [ILs], [Bmim]BF₄, [Bmim]PF₆, and [Bmim]Tf₂N, consisting of an identical cation and different anions. The yields were higher in hydrophobic ILs [Bmim]Tf₂N (55–96%) and [Bmim]PF₆ (22–95%), than in hydrophilic [Bmim]BF₄ (0–19%). The incubation of lipase PS in hydrophobic ILs for a period of 20–300 days at room temperature resulted in an increased yield of 62–98% in [Bmim]Tf₂N and 45–98% in [Bmim]PF₆, respectively. The lipase PS-hydrophobic IL mixture was recycled five times without any decrease in the yield of the products. In another set of experiments, the hydrolytic activity of the enzyme was determined after incubation in each of the three ILs and in hexane for 20 days at room temperature. It was found to be 1.8- and 1.6-fold higher in [Bmim]Tf₂N and [Bmim]PF₆, respectively, remained unchanged in [Bmim]BF₄ and was 1.6 times lower in hexane as compared to the non-incubated enzyme.     

On the nature of ionic liquids and their effects on lipases that catalyze ester synthesis

Free and immobilized lipases from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) were used as catalysts in the synthesis of butyl propionate by transesterification in reaction media consisting in nine different ionic liquids. Enzyme activities were clearly dependent on the nature of the ions, the results being improving as the alkyl chain length of the imidazolium cation increased, and as a function of the type of anion ([PF₆], [BF₄] or [ethylsulphate]). The best synthetic activity (655.5 U/mg protein at 40 °C) was obtained when free CALB were assayed in the water-miscible IL cocosalkyl pentaethoxy methyl ammonium methosulfate ([CPMA][MS]), and was clearly related with the water content of the medium. The synthetic activity of free CALB in [CPMA][MS] was enhanced with the increase in temperature, while practically no effect was obtained for TLL. The ability of free CALB to synthesize aliphatic esters of different alkyl chain lengths, using different alkyl vinyl esters and 1-alkanols as substrates, was also studied in [CPMA][MS], the best results (4500 U/mg protein) being obtained for the synthesis of hexyl butyrate.     

Pseudomonas cepacia lipase catalyzed esterification and transesterification of 3-(furan-2-yl) propanoic acid/ethyl ester: A comparison in ionic liquids vs hexane

A comparison of the Pseudomonas cepacia lipase (lipase PS) catalyzed esterification of 3-(furan-2-yl) propanoic acid and transesterification of ethyl 3-(furan-2-yl) propanoate with six straight chain alcohols (propanol to octanol) in ionic liquids and hexane was carried out. The ionic liquids selected, [Bmim]BF₄, [Bmim]PF₆, and [Bmim]Tf₂N, consisted of an identical cation and different anions. This is the first report on the biocatalyzed synthesis of these esters. In all the media, lipase PS catalyzed esterification of 3-(furan-2-yl) propanoic acid resulted in high yields of the esters compared to the transesterification of ethyl 3-(furan-2-yl) propanoate. [Bmim]Tf₂N proved to be the best; yielding 98–67% of the product by lipase PS catalyzed esterification. The lipase PS–[Bmim]Tf₂N and lipase PS–[Bmim]PF₆ mixture was recycled five times without any decrease in the yields of the products and was found to be operationally stable up to 10 months at room temperature.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.     

Biocatalytic synthesis of poly(ε‐caprolactone) using modified lipase in ionic liquid media

Ionic liquids have been used as exceptional nonaqueous reaction media for enzymatic transformation. The ring‐opening polymerization of ε‐caprolactone catalyzed by Novozyme‐435 lipase was successfully conducted in 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([Bmim]PF₆) ionic liquid. ¹H‐NMR and MALDI‐TOF analyses of poly(ε‐caprolactone) (PCL) formed by Novozyme‐435 lipase‐catalyzed reaction revealed an asymmetric telechelic α‐hydroxy‐ω‐carboxylic acid end group. The effects of enzyme concentration, temperature, reaction time, and water activities on monomer conversion and M_n were systematically evaluated. Through the optimization of reaction conditions, PCL was produced in 85% monomer conversion, with an M_n of 5942, in [Bmim]PF₆ at 60°C for 48 h. DSC results demonstrated that high‐molecular‐weight PCL exhibited an excellent thermal property. SEM results showed that PCL had a clear spherulites structure, which could provide a large surface area for cell adhesion. These results showed that [Bmim]PF₆ ionic liquid was suitable for the biocatalytic synthesis of PCL using Novozyme‐435 lipase, and could be used as alternative environmentally friendly media to replace the traditional organic solvents.     

Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the Presence of the Ionic Liquids [BMIM][BF4] and [BMIM][PF6] and Organic Solvents

The influence of the two most commonly used ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate, [BMIM][BF₄], 1-butyl-3-methyl imidazolium hexafluorophosphate, [BMIM][PF₆]) and three selected organic solvents (dimethylsulfoxide, ethanol, methanol) on the growth of Escherichia coli, Pichia pastoris and Bacillus cereus was investigated. [BMIM][BF₄] was toxic at 1% (v/v) on all three microorganisms. The minimal inhibitory concentration (MIC) of [BMIM][BF₄] on E. coli growth was between 0.7 and 1% (v/v). In contrast, [BMIM][PF₆] was less toxic for P. pastoris and B. cereus, whereas E. coli was not able to tolerate [BMIM][PF₆] (MIC value: 0.3–0.7% v/v). Growth of P. pastoris was unaffected by [BMIM][PF₆] at 10% (v/v). Similar results were found for dimethylsulfoxide. Thus, ionic liquids (ILs) can have substantial inhibitory effects on the growth of microorganisms, which should be taken into account for environmental reasons as well as for the use of ILs as co-solvents in biotransformations. Revisions requested 2 November 2005; Revisions received 20 December 2005     

Effect of ionic liquid [BMIM][PF<Subscript>6</Subscript>] on asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]) on the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) to synthesize optical active ethyl 2-hydroxy-4-phenylbutyrate (EHPB) catalyzed by Saccharomyces cerevisiae was investigated. (R)-EHPB [70.4%, e.e.(R)] is obtained using ethyl ether or benzene as the solvent. The main product is (S)-EHPB [27.7%, e.e.(S)] in [BMIM][PF₆]. However, in ionic liquid-water (10:1, v/v) biphasic system, the enantioselectivity of the reduction is shifted towards (R)-side, and e.e.(R) is increased from 6.6 to 82.5% with the addition of ethanol (1%, v/v). The effect of the use of [BMIM][PF₆] as an additive in relatively small amounts on the reduction was also studied. We find that there is a decline in the enantioselectivity of the reduction in benzene. In addition, a decrease in the conversion of EOPB and the yield of EHPB with increasing [BMIM][PF₆] concentrations occurs in either organic solvent–water biphasic systems or benzene.     

Effect of replacing [NTf2] by [PF6] anion on the [BMIm][NTf2] ionic liquid confined by gold

The effect of replacing bis(trifluoromethylsulphonyl)imide ([NTf₂]) by hexafluorophosphate ([PF₆]) in room temperature ionic liquid (IL) 1-butyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide ([BMIm][NTf₂]) confined between two gold interfaces is herein reported through molecular dynamics simulations using all-atom non-polarisable force-fields. Five systems were studied ranging from pure [BMIm][NTf₂] to pure [BMIm][PF₆], with [PF₆] molar fractions of 0, 0.125, 0.25, 0.375 and 0.5. Special attention was drawn to investigate the impact of the [PF₆] anion on the IL, in particular on the first layers of the liquid in close contact with the solid gold surface.     

Hydrogenation of C–C double bonds in an ionic liquid reaction system using the obligate anaerobe, <Emphasis Type="Italic">Sporomusa termitida</Emphasis>

Sporomusa termitida reduced caffeate (1mM) in anaerobic, two-liquid phase, reaction systems containing either tetradecane or 1-butyl-3-methylimidazolium hexafluorophosphate {[bmim][PF₆]} (20% v/v). The initial rate and final product yield were 20 and 7% lower, respectively, in [bmim][PF₆]. Since caffeate partitioned only into the aqueous phase, the lower rate cannot be attributed to mass transfer barriers. Therefore, [bmim][PF₆] inhibited the biocatalyst, perhaps unsurprisingly since it is very polar and hydrolyses to produce HF.Revisions requested 30 September 2004; Revisions received 2 December 2004     

Improvement of trans-sialylation versus hydrolysis activity of an engineered sialidase from Trypanosoma rangeli by use of co-solvents

Biocatalytic trans-sialylation is relevant for the design of biomimetic oligosaccharides such as human milk oligosaccharides. t-Butanol and ionic liquids, EAN (ethylammonium nitrate), [MMIm][MeSO₄] (1,3-dimethylimidazolium methyl sulfate), and [C₂OHMIm][PF₆] (1-(2-hydroxyethyl)-3-methylimidazolium hexafluorophosphate), were examined as co-solvents for the improvement of the synthesis versus hydrolysis ratio in the trans-sialylation of lactose, catalysed by an engineered sialidase from Trypanosoma rangeli. The use of 25 % (v/v) t-butanol as co-solvent significantly increased 3′-sialyllactose production by 40 % from 1.04 ± 0.09 to 1.47 ± 0.01 mM. The synthesis versus hydrolysis ratio increased correspondingly by 1.2-times. 1–2.5 % (v/v) EAN or [C₂OHMIm][PF₆] improved the synthesis versus hydrolysis ratio up to 2.5-times but simultaneously decreased the 3′-sialyllactose yield, probably due to enzyme inactivation caused by the ionic liquid. [MMIm][MeSO₄] had a detrimental effect on the trans-sialylation yield and on the ratio between synthesis and hydrolysis.     

Enantioselective acylation of (RS)-phenylethylamine catalysed by lipases

The enzymatic acylation of (RS)-phenylethylamine with different acyl donors catalysed by lipases, was studied in organic solvents with different hydrophobicities and in mixtures with ionic liquids ((ILs); [BMIm][BF₄], [BMIm][SCN], [BMIm][Cl] and [BMIm][PF₆]). Using lipases from Candida antarctica B (CAL-B) and from Aspergillus niger higher conversion degrees and E-values were obtained with ethyl acetate as the acyl donor. When CAL-B was used as the biocatalyst, in a two-phase system formed by [BMIm][X]/dichloromethane or [BMIm][X]/chloroform, the selectivity was better than that obtained in pure organic solvents. The selectivity was found to be related to individual anions in ILs. In this reaction, the ion effectiveness in enhancing the enzyme selectivity followed the series: Cl⁻ > SCN⁻ > BF₄⁻ > PF₆⁻ in mixtures with dichloromethane, and PF₆⁻ > BF₄⁻ > SCN⁻ > Cl⁻ in mixtures with chloroform.     

Chemoenzymatic synthesis of feruloylated monoacyl- and diacyl-glycerols in ionic liquids

Feruloylated monoacyl- and diacyl-glycerols (FMAGs and FDAGs) are lipophilic antioxidants and potential UV absorbers. FMAGs and FDAGs were synthesized by a novel chemoenzymatic method: firstly, ferulic acid was esterified with glycerol to synthesize glyceryl ferulate, using p-toluenesulfonic acid as chemical catalyst in 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF₄); secondly, glyceryl ferulate was esterified with oleic acid to synthesize FMAGs and FDAGs, using Novozym 435 as biocatalyst in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF₆). The conversion of ferulic acid and yield of glyceryl ferulate in the first reaction were both 98%. The yields of FMAGs and FDAGs in the second reaction reached 34 ± 2% and 66 ± 3%, respectively.     

Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquids mixtures

Novozym 435-catalyzed synthesis of 6-O-lauroyl-d-glucose in ionic liquids (ILs) was investigated. The highest lipase activity was obtained in water-miscible [Bmim][TfO] which can dissolve high concentration of glucose, while the highest stability of lipase was shown in hydrophobic [Bmim][Tf(2)N]. The optimal activity and stability of lipase could be obtained in [Bmim][TfO] and [Bmim][Tf(2)N] mixture (1:1, v/v). Specifically, the activity of lipase was increased from 1.1 to 2.9 micromolmin(-1)g(-1) by using supersaturated glucose solution in this mixture, compared with reaction using saturated solution. After 5 times reuse of lipase, 86% of initial activity was remained in this mixture, while the residual activity in pure [Bmim][TfO] was 36%. Therefore, the productivity obtained by using ILs mixtures was higher than those in pure ILs.     

A Novel Imidazole Nucleoside Containing a Diaminodihydro-S-triazine as a Substituent: Inhibitory Activity Against the West Nile Virus NTPase/Helicase

The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2′-deoxy-β-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected product, 1-(2′-deoxy-β-D-erythropentofuranosyl)-5-(2,4-diamino-3,6-dihydro-1,3,5-triazin-6-yl)imidazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed promising in vitro anti-helicase activity against the West Nile virus NTPase/ helicase with an IC ₅₀ of 3-10 μg/mL.     

Novel Synthesis of 4′C-Aryl-Branched Acyclic Nucleoside Using [3,3]-Sigmatropic Rearrangement

Abstract

A very efficient synthetic route for preparing a novel 4′-C-aryl branched-1′,2′-seco-2′,3′-dideoxy-2′,3′-didehydro-nucleoside is described. Mesylate 7 was successfully synthesized via a Horner-Wadsworth-Emmons reaction and a [3,3]-sigmatropic rearrangement, with which an adenine base was coupled by nucleophilic substitution conditions (K₂CO₃, 18-Crown-6, DMF) to give the target nucleoside 9.     

Synthesis of New 5″-Sulfonylamido Derivatives of 3″-Azido-3″-Deoxythymidine (AZT)

Abstract

A series of 5′-N-methanesulfonyl derivatives of 3′-azido-5′-(alkylamino)-3′,5′-dideoxythymidine was synthesised. The first step of the synthesis involved the reaction of 1-(2,5-dideoxy-5-O-tosyl-β-D-threo-pentofuranosyl)thymine 1 with an appropriate amine to give 1-[5-(alkylamino)-2,5-dideoxy-β-D-threo-pentofuranosyl]thymines 2a-e and 1-(2,5-dideoxy-β-threo-pent-4-enofuranosyl)thymine 3 as a by-product. Compounds 2a-e were treated with an excess of methanesulfonyl chloride to yield intermediates 1-[5-(dimethylamino)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-pentofuranosyl]-thymine 4a and 1-[5-(N-alkyl-N-methanesulfonyl)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-penfuranosyl]thymines 4b-e. The reaction of 4a-e with lithium azide in dimethyl-formamide afforded the final compounds 1-[3-azido-5-(N-methyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymine 5a and 1-[3-azido-5-(N-alkyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymines 5b-e. The independent synthesis of 4′,5′-unsaturated product 3 was also described.     

Acyclo C-Nucleoside Analogs. Regioselective Annellation of a Triazole Ring to 5-Methyl-1,2,4-Triazino[5,6-b]Indole and Formation of Certain 3-Poly Hydroxyalkyl Derivatives

ABSTRACT

Cyclodehydrogenation of the ethylidene derivative of (5-methyl-1,2,4-triazino[5,6-b]indol-3-yl)hydrazine (1) gave the angular isomer, 1,10-dimethyl-1,2,4-triazolo[3′,4′:3,4][1,2,4]triazino[5,6-b]indole (4). The linear isomer, 3,10-dimethyl-1,2,4-triazolo[4′,3′:2,3][1,2,4]triazino[5,6-b]indole (7) could be prepared regioselectively by the cyclodehydration of the acetyl derivative of 1. The cyclodehydrogenation was extended to the monosaccharide derivatives of 1. The role of the N-methyl group on the site of annellation has been discussed.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.