Asymmetric reductions of ethyl 2-(benzamidomethyl)-3-oxobutanoate by yeasts

The stereoselective reduction of ethyl 2-(benzamidomethyl)-3-oxobutanoate 1 using yeasts was investigated among a restricted number (12) of yeasts. Kluyveromyces marxianus var. lactis CL69 diastereoselectively produced (2R,3S)-ethyl 2-(benzamidomethyl)-3-hydroxybutanoate 2, whereas Pichia glucozyma CBS 5766 gave (2S,3S)-2 as the major stereoisomer. The biotransformations were independently optimized for minimizing by-product formation and maximizing the diastereoselectivity. Under optimized conditions, K. marxianus var. lactis CL 69 gave the (2R,3S)-ethyl 2-(benzamidomethyl)-3-hydroxybutanoate 2 with ee > 99% and de = 98%, while P. glucozyma CBS 5766 allowed for the production of (2S,3S)-2 with ee > 99% and de = 86%.     

Resolution of racemic 4-hydroxy-2-cyclopentenone with immobilized penicillin G acylase

Enantiomerically pure 4-oxo-2-cyclopentenyl derivatives were prepared by kinetic resolution with penicillin G acylase (EC 3.5.1.11) immobilized on an epoxy activated polymer. The enzyme selectively hydrolyzes the phenylacetyl ester of the (S)-enantiomer to give the (S)-alcohol. The enantioselectivity E increases from 12.4 in 0.05 M phosphate buffer, to E = 100–110 in acetonitrile-buffer (20% v/v) and E >200 in diisopropyl ether. The immobilized enzyme is stable and retains >90% of its activity after 10 recycles over one week at room temperature.     

Lipase catalyzed enantioselective desymmetrization of a prochiral pentane-1,3,5-triol derivative

The enantioselective desymmetrization of the prochiral 3-O-silyl protected pentanetriol derivative 3 was carefully investigated. At −10 °C, the bacterial lipase from Burkholderia cepacia immobilized on ceramic particles led to monoacetate (S)-4 in 52% yield and >99.9% ee. At a reaction temperature of −40 °C the yield and enantioselectivity were even higher, but the reaction time was very long. Theoretical simulations of the reaction progress indicated an enantioselectivity of 25:1 at −10 °C and 35:1 at −40 °C. (S)-4 was converted into the enantiomerically pure building block 5-azidopentane-1,3-diol (S)-7 in two steps. The absolute configuration of (S)-7 was determined by exciton-coupled circular dichroism (ECCD) of diester (S)-8.     

New chemical and chemo-enzymatic routes for the synthesis of (RS)- and (S)-enciprazine

The chemo-enzymatic synthesis of racemic and enantiopure (RS)- and (S)-enciprazine 1, a non-benzodiazepine anxiolytic drug, is described herein. The synthesis started from 1-(2-methoxyphenyl) piperazine 3, which was treated with 2-(chloromethyl) oxirane (RS)-4 using lithium bromide to afford a racemic alcohol, 1-chloro-3-(4-(2-methoxyphenyl) piperazin-1-yl) propan-2-ol (RS)-6 in 85% yield. Intermediate (S)-6 was synthesized from racemic alcohol (RS)-6 using Candida rugosa lipase (CRL) with vinyl acetate as the acyl donor. Various reaction parameters such as temperature, time, substrate, enzyme concentration, and the effect of the reaction medium on the conversion and enantiomeric excess for the transesterification of (RS)-6 by CRL were optimized. It was observed that 10 mM of (RS)-6, 50 mg/mL of CRL in 4.0 mL of toluene with vinyl acetate (5.4 mmol) as acyl donor at 30 °C gave good conversion (C = 49.4%) and enantiomeric excess (ee_P = 98.4% and ee_S = 96%) after 9 h of reaction. Compound (S)-6 is a key intermediate for the synthesis of enantiopure (S)-1. The (RS)- and (S)-enciprazine drug 1 was synthesized by treating (RS)- and (S)-6 with 3,4,5-trimethoxyphenol 5 using MeCN as a solvent and K₂CO₃ as a base.     

Synthesis of the (1S,2S)- and (1R,2S)-stereoisomers of the respective E- and Z-isomers of ethyl 4-[(2-hydroxycyclohexyl)methyl]phenoxy-3-methyl-2-butenoate using yeast whole cell bioreduction of the parent ketones

Saccharomyces cerevisiae, strain DBM 2115, was successfully employed in the reduction of the separated Z- and E-isomers of ethyl 4-[(2-oxocyclohexyl)methyl]phenoxy-3-methyl-2-butenoates 1 and 2, in order to prepare the (1S,2S)- and (1R,2S)-enantiomers of the corresponding ethyl 4-[(2-hydroxycyclohexyl)methyl]phenoxy-3-methyl-2-butenoates 3–6. The products were obtained with the required absolute configuration: (1S,2S)-3 (ee = 98%; yield 48%), (1R,2S)-4 (ee = >99%; yield 45%), (1S,2S)-5 (ee = 98.5%; yield 47%), and (1R,2S)-6 (ee = >99%; chemical yield 44%).     

Effective synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol by asymmetric enzymatic reduction

The synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol, a pharmaceutically important alcohol intermediate for the synthesis of NK-1 receptor antagonists, was demonstrated from a ketone via asymmetric enzymatic reduction. The isolated enzyme alcohol dehydrogenase from Rhodococcus erythropolis reduced the poorly water soluble substrate with excellent ee (>99.9%) and good conversion (>98%). The optimized process was demonstrated up to pilot scale using high substrate concentration (390 mM) using a straightforward isolation process achieving excellent isolation yields (>90%) and effective space time yield (100–110 g/L d). Process improvements, demonstrated at preparative scale, increased the substrate concentration to 580 mM achieving a space time yield of 260 g/L d.     

A green resolution–separation process for aliphatic secondary alcohols

In order to obtain both enantiomers of aliphatic secondary alcohols via a greener method, the four-step resolution–separation process involving lipase-catalyzed enantioselective esterification and hydrolysis as well as two separation procedures both via heterogeneous azeotropic distillation was developed. (S)-2-Pentanol (ee = 98.6%), (R)-2-pentanol (ee >99%), (S)-2-octanol (ee = 98.2%), and (R)-2-octanol (ee = 98.5%) were all produced in high purity (>98%) and high yield (>90%). In addition to the two model substrates, this method could also be applied to the resolution of other aliphatic secondary alcohols.     

Enantioselective hydrolysis of styrene oxide with the epoxide hydrolase of Sphingomonas sp. HXN-200

The soluble bacterial epoxide hydrolase (EH) from Sphingomonas sp. HXN-200 catalyzed the enantioselective hydrolysis of racemic styrene oxide to give (S)-styrene oxide with an enantiomeric ratio (E) of 21–23 in aqueous buffer, better than any reported native EHs. The ring opening of the styrene oxide with this EH was only at the terminal position for the (S)-enantiomer and at the terminal and benzylic position in an 87:13 ratio for the (R)-enantiomer. Enzymatic hydrolysis of the styrene oxide in a two-liquid phase system significantly reduced autohydrolysis, thus improving the E to 26–29. Hydrolysis of 160 mM styrene oxide with cell-free extract (CFE) of Sphingomonas sp. HXN-200 (10 mg protein/mL) in aqueous buffer and n-hexane (1:1) for 30.7 h afforded 39.2% (62.7 mM) of (S)-styrene oxide in >99.9% ee. The lyophilized CFE was proven to be stable, while the rehydrated lyophilized CFE powder was successfully used for the hydrolysis of 320 mM styrene oxide in the two-liquid phase system, yielding 40.2% (128.6 mM) of (S)-styrene oxide in >99.9% ee after 13.8 h. No inhibitory effect of the diol product on the hydrolysis was observed when the diol concentration was lower than 476 mM, suggesting a straightforward process for the hydrolysis of up to 1 M styrene oxide.     

Enantioselective transesterification of (RS)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol using Pseudomonas aeruginosa lipases

Lipases from the bacterial strain, Pseudomonas aeruginosa, isolated from the soil by enrichment techniques, are assessed for the enantioselective transesterification of (RS)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) to (R)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol, a key intermediate in the synthesis of the chiral drug (S)-Lubeluzole. The lipases produced by the organism yielded the (S)-ester and the (R)-alcohol as the remaining substrate with an excellent yield (>49.9%) and almost complete enantioselectivity (ee >99.9%) in the presence of vinyl butyrate as an acyl donor in an organic medium. In contrast, purified and expensive commercially available lipases of Candida rugosa and porcine pancreas achieved much lower conversion with enantioselectivities of 15% and 5%, respectively. A well-mixed (∼1000 rev min⁻¹) batch reactor having the aqueous phase finally dispersed in hexane was used in these studies. The parameters of the transesterification reaction were optimized and the optimal concentrations of rac-CDPP and vinyl butyrate were found to be 5 and 150 mM at 30 °C. A preparative-scale reaction yielded the (S)-ester at 42% conversion and ee >99%.     

Approaches to (R)- and (S)-1′-(1-aminoethyl)ferrocene-1-carboxylic acid derivatives

Lipase-catalyzed esterification of (±)-methyl 1′-(1-hydroxyethyl)ferrocene-1-carboxylate 4 afforded its (R)-acetate (−)-5 (ee = 99%) and (S)-(+)-4 (ee = 90%). Stereoretentive azidation/amination/acetylation of (R)-(−)-5 gave (R)-(+)-methyl 1′-(1-acetamidoethyl)ferrocene-1-carboxylate (R)-3 (ee = 98%). In a similar manner (S)-(+)-4 was converted into (S)-(−)-3 (ee = 84%). Both enantiomers of 3 were obtained in high chemical yields without a loss of enantiomeric purity. The title compounds can be coupled with natural amino acids and peptides on both C- and N-termini.     

Asymmetric hydrogenation of α,β-unsaturated ketones catalyzed by Ru–TPPTS–(S,S)-DPENDS complex in ionic liquids

The asymmetric hydrogenation of α,β-unsaturated ketones catalyzed by the achiral ruthenium monophosphine complex RuCl₂(TPPTS)₂ [TPPTS: P(m-C₆H₄SO₃Na)₃] modified by (S,S)-DPENDS [disodium salt of sulfonated (S,S)-1,2-diphenyl-1,2-ethylene-diamine] was investigated in ionic liquid [RMIM]Ts (1-alkyl-3-methylimidazolium p-methylphenylsulfonates, R = ethyl, butyl, octyl, dodecyl). Chemoselectivity of 100% and 75.9% ee were obtained for benzalacetone under the optimized conditions. The resulting products can be easily separated from the catalyst immobilized in ionic liquid [EMIM]Ts by extraction with n-hexane, while the catalyst can be reused seven times without the loss of catalytic activity and enantioselectivity. Especially, the addition of water can improve the performance of the catalyst.     

Synthesis of β-adrenergic blockers (R)-(−)-nifenalol and (S)-(+)-sotalol via a highly efficient resolution of a bromohydrin precursor

(R)- and (S)-2-bromo-1-(4-nitrophenyl)ethanol are precursors of important β-adrenergic receptor blocking drugs (R)-nifenalol and (S)-sotalol, respectively. Both were obtained in enantiomeric pure forms via a single highly efficient enzymatic transesterification reaction of (±)-2-bromo-1-(4-nitrophenyl)ethanol using immobilized lipase PS-C-II (E >1000; concn 200 g/L), while PS lipase completely failed to react. On the other hand, the hydrolytic method also produced enantiorich precursors though relatively less efficient (PS-C-II, E = 5.1). Out of all the approaches employed the transesterification method proved to be the most efficient.     

Response surface methodology as an optimization strategy for the light-controlled asymmetric hydrogenation of 4-(trimethylsilyl)-3-butyn-2-one by photosynthetic bacteria

Enantiomerically pure (S)-4-(trimethylsilyl)-3-butyn-2-ol {(S)-TMSBL} is a key intermediate for the synthesis of many biologically and structurally interesting compounds and pharmaceuticals. Herein we propose a new light-controlled asymmetric hydrogenation of 4-(trimethylsilyl)-3-butyn-2-one (TMSBO) to enantiopure (S)-TMSBL by photosynthetic bacteria Rhodobacter sphaeroides, which is a newly isolated photosynthetic bacteria strain that has the capacity to capture light energy and to generate NADPH through photosynthetic electron-transfer reactions; no oxygen or other metabolic intermediates were used. Response Surface Methodology (RSM) was used to investigate the effects of substrate concentration, pH, and temperature on the reaction yield. A 3³ factorial design was performed to optimize the production of (S)-TMSBL. The optimum conditions were: cell concentration (200 g/L), shaking speed (140 rpm), pH (6.9), substrate concentration (14.4 mmol/L), and temperature (33.6 °C). This optimization strategy led to an increase of the yield from 88.9% to 94.5%.     

The combi-CLEA approach: enzymatic cascade synthesis of enantiomerically pure (S)-mandelic acid

Enantiomerically pure (S)-mandelic acid was synthesised from benzaldehyde by sequential hydrocyanation and hydrolysis in a bienzymatic cascade at starting concentrations up to 0.25 M. A cross-linked enzyme aggregate (CLEA) composed of the (S)-selective oxynitrilase from Manihot esculenta and the non-selective nitrilase from Pseudomonas fluorescens EBC 191 was employed as the biocatalyst. The nitrilase produces approx. equal amounts of (S)-mandelic acid and (S)-mandelic amide from (S)-mandelonitrile under standard conditions, but we surprisingly found that high (up to 0.5 M) concentrations of HCN induced a marked drift towards amide production. By including the amidase from Rhodococcus erythopolis in the CLEA we obtained (S)-mandelic acid as the sole product in 90% yield and >99% enantiomeric purity.     

Novel chiral copper complexes of N,P-ferrocenyl ligands with central and planar chirality as efficient catalyst for asymmetric addition of diethylzinc to imines

A new type of chiral copper complexes of N,P-ferrocenyl ligands with central and planar chirality as efficient catalyst was applied to the enantioselective addition of diethylzinc to N-diphenylphosphinoylimines. The (R)- and (S)-enantiomers of the addition reaction were obtained for this transformation. In the presence of 6 mol % of bidentate ligand 1 and 12 mol % of Cu(OTf)₂, the asymmetric addition process affords N-diphenylphosphinoylamides in up to 97% ee and 95% yields.     

Synthesis of rigid and C2-symmetric 18-crown-6 type macrocycles bearing diamide–diester groups: enantiomeric recognition for α-(1-naphthyl)ethylammonium perchlorate salts

A series of rigid and chiral C₂-symmetric 18-crown-6 type macrocycles (S,S)-4, (S,S)-5, (S,S)-6 and (R,R)-2 bearing diamide–ester groups were synthesized. The binding properties of these macrocycles were examined for α-(1-naphthyl)ethylammonium perchlorates salts by an ¹H NMR titration method. Taking into account the host employed, important differences were observed in the K_a values of (R)- and (S)-enantiomers of guests for macrocycles (S,S)-4 and (S,S)-6, K_S/K_R = 3.6, and K_S/K_R = 0.1 (K_R/K_S = 10.3) ΔΔG = 3.19 and ΔΔG = ?5.77 kJ mol^?1, respectively. The results indicated excellent enantioselectivity of macrocyclic (S,S)-6 towards the enantiomers of α-(1-naphthyl)ethylammonium perchlorate salts.     

(R)- or (S)-Bi-2-naphthol assisted,l-proline catalyzed direct aldol reaction

Chiral Brønsted acids (R)- and (S)-BINOL were employed as additives in the classic l-proline catalyzed direct aldol reaction. Eighteen substrates were tested with 0.5 mol % (R)-BINOL loading and 1 mol % of (S)-BINOL loading, and the enantioselectivity was improved from 72% ee without additive to 98% ee. In the proposed transition state, the chiral Brønsted acid promoted the reaction through hydrogen bonding, which not only activated the carbonyl group but also stabilized the transition state.     

Efficient synthesis of an ε-hydroxy ester in a space–time yield of 1580 g L−1 d−1 by a newly identified reductase RhCR

A new NADH-dependent carbonyl reductase RhCR capable of efficiently reducing the ε-ketoester ethyl 8-chloro-6-oxooctanoate (ECOO) to give ethyl (S)-8-chloro-6-hydroxyoctanoate [(S)-ECHO], an important chiral precursor for the synthesis of (R)-α-lipoic acid, was identified from Rhodococcus sp. ECU1014. Using recombinant Escherichia coli cells expressing RhCR and glucose dehydrogenase used for the regeneration of cofactor, 440 g L⁻¹ (2 M) of ECOO were stoichiometrically converted to (S)-ECHO in a space–time yield of 1580 g L⁻¹ d⁻¹ without the external addition of any expensive cofactor.     

Physicochemical properties of {1-methyl piperazine (1) + water (2)} system at T = (298.15 to 343.15) K and atmospheric pressure

Densities (ρ) and viscosities (η) of aqueous 1-methylpiperazine (1-MPZ) solutions are reported at T = (298.15 to 343.15) K. Refractive indices (n_D) are reported at T = (293.15 to 333.15) K, and surface tensions (γ) are reported at T = (298.15 to 333.15) K. Derived excess properties, except excess viscosities (Δη), are found to be negative over the entire composition range. The addition of 1-MPZ reduces drastically the surface tension of water. The temperature dependence of surface tensions is explained in terms of surface entropy (S^S) and enthalpy (H^S). The measured and derived properties are used to probe the microscopic liquid structure of the bulk and surface of the aqueous amine solutions.     

Synthesis of enantiomerically pure glycidol via a fully enantioselective lipase-catalyzed resolution

The efficient enzymatic synthesis of enantiopure 2,3-epoxypropanol (glycidol) has been achieved. The racemic glycidyl butyrate was successfully resolved by enzymatic hydrolysis using a strategy that combines different immobilization protocols and different experimental reaction conditions. A new enzyme (25 kDa lipase)—which is a lipase-like enzyme purified from the pancreatic porcine lipase (PPL) extract—immobilized on DEAE–Sepharose was selected as the optimal biocatalyst. The optimal results were obtained at pH 7, 25 °C and 10% dioxane using this biocatalyst and a very high enantioselectivity for the enzyme was displayed, obtaining both (R)-(−)-glycidyl butyrate and (R)-(+)-glycidol with enantiomeric excesses >99% (E >100). The hydrolysis of (R)-(−)-glycidyl butyrate produced pure (S)-(−)-glycidol.