Chemoenzymatic synthesis of enantiomerically enriched α-hydroxyamides

A study on a chemoenzymatic synthesis of model α-hydroxyamide was performed. Special attention was paid to the optimization of the enzymatic process, both on the selection of enzyme and cosolvent. An intriguing influence of cosolvent on the enantioselectivity of Wheat Germ Lipase and Amano PS Lipase catalyzed hydrolysis was observed, as the results obtained proved that enzyme's enantioselectivity is directly correlated with cosolvent's hydrophobicity. In the best example (Wheat Germ lipase, Et₂O used as a cosolvent), the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7%ee.     

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.     

Synthesis of CuPF6‐(S)‐BINAP loaded resin and its enantioselectivity toward phenylalanine enantiomers

A type of resin‐anchored CuPF₆‐(S )‐BINAP was synthesized and identified. The PS‐CuPF₆‐(S )‐BINAP resin was used to adsorb the phenylalanine enantiomers. The results showed that the adsorption capacity of PS‐CuPF₆‐(S )‐BINAP resin toward L‐phenylalanine was higher than that of resin toward D‐phenylalanine. PS‐CuPF₆‐(S )‐BINAP resin exhibited good enantioselectivity toward L‐phenylalanine and D‐phenylalanine. The influence of phenylalanine concentration, pH, adsorption time, and temperature on the enantioselectivity of the resin were investigated. The results showed that the enantioselectivity of the resin increased with increasing the phenylalanine concentration, pH, and adsorption time, while it decreased with an increase in temperature. The causes for these influences are discussed. The highest enantioselectivity (α = 2.81) was obtained when the condition of phenylalanine concentration was 0.05 mmol/mL, pH was 8, adsorption time was 12 h, and temperature 5°C. The desorption test for removing D/L‐phenylalanine on PS‐CuPF₆‐(S )‐BINAP resin was also investigated. The desorption ratios of D‐phenylalanine and L‐phenylalanine at pH of 1 were 95.7% and 94.3%, respectively. This result indicated that the PS‐CuPF₆‐(S )‐BINAP resin could be regenerated by shaking with an acidic solution. The reusability of the PS‐CuPF₆‐(S )‐BINAP resin was also assessed and the resin exhibited considerable reusability.     

Lack of Enantioselectivity in the SULT1A3‐catalyzed Sulfoconjugation of Normetanephrine Enantiomers: An In Vitro and Computational Study

(1R)‐Normetanephrine is the natural stereoisomeric substrate for sulfotransferase 1A3 (SULT1A3)‐catalyzed sulfonation. Nothing appears known on the enantioselectivity of the reaction despite its potential significance in the metabolism of adrenergic amines and in clinical biochemistry. We confronted the kinetic parameters of the sulfoconjugation of synthetic (1R)‐normetanephrine and (1S)‐normetanephrine by recombinant human SULT1A3 to a docking model of each normetanephrine enantiomer with SULT1A3 and the 3′‐phosphoadenosine‐5′‐phosphosulfate cofactor on the basis of molecular modeling and molecular dynamics simulations of the stability of the complexes. The K_M, V_max, and k_cat values for the sulfonation of (1R)‐normetanephrine, (1S)‐normetanephrine, and racemic normetanephrine were similar. In silico models were consistent with these findings as they showed that the binding modes of the two enantiomers were almost identical. In conclusion, SULT1A3 is not substrate‐enantioselective toward normetanephrine, an unexpected finding explainable by a mutual adaptability between the ligands and SULT1A3 through an “induced‐fit model” in the catalytic pocket. Chirality, 25:28‐34, 2012.© 2012 Wiley Periodicals, Inc.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

Substituent effects on chiral resolutions of derivatized 1‐phenylalkylamines by heptakis(2,3‐di‐O‐methyl‐6‐O‐tert‐butyldimethylsilyl)‐β‐cyclodextrin GC stationary phase

Chiral resolutions of trifluoroacetyl‐derivatized 1‐phenylalkylamines with different type and position of substituent were investigated by capillary gas chromatography by using heptakis(2,3‐di‐O‐methyl‐6‐O‐tert‐butyldimethylsilyl)‐β‐cyclodextrin diluted in OV‐1701 as a chiral stationary phase. The influence of column temperature on retention and enantioselectivity was examined. All enantiomers of meta‐substituted analytes as well as fluoro‐substituted analytes could be resolved. Temperature had a favorable influence on enantioselectivity for small amines with substituents at the ortho‐position. The type of substituent at the stereogenic center of amines also had a crucial effect as the ethyl group led to poor enantioseparation. Among all analytes studied, trifluoroacetyl‐derivatized 1‐(2′‐fluorophenyl)ethylamine exhibited baseline resolution with the shortest analysis time.     

Crystal structures of halohydrin hydrogen‐halide‐lyases from Corynebacterium sp. N‐1074

Halohydrin hydrogen‐halide‐lyase (H‐Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2‐diol. Until now, six different H‐Lyases have been studied. These H‐Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N‐1074 has two different isozymes of H‐Lyase, HheA (A‐type) and HheB (B‐type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H‐Lyases. Among the B‐type H‐Lyases, HheB shows relatively high enantioselectivity compared to that of HheB_GP1. This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3‐dicyano‐2‐propanol at the catalytic site in the crystal structure of the HheB‐DiCN complex suggests that the product should be (R)‐epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity. Proteins 2015; 83:2230–2239. © 2015 Wiley Periodicals, Inc.     

Novel Magnetic Cross‐Linked Lipase Aggregates for Improving the Resolution of (R,S)‐2‐octanol

Novel magnetic cross‐linked lipase aggregates were fabricated by immobilizing the cross‐linked lipase aggregates onto magnetic particles with a high number of ‐NH₂ terminal groups using p‐benzoquinone as the cross‐linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross‐linked lipase aggregates were achieved. The magnetic cross‐linked lipase aggregates were able to efficiently resolve (R, S)‐2‐octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross‐linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross‐linking. These results provide a great potential for industrial applications of the magnetic cross‐linked lipase aggregates. Chirality 27:199–204, 2015. © 2014 Wiley Periodicals, Inc.     

Improvement of natural isolates of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for synthesis of a chiral building block using classic genetics

The asymmetric bio-reduction of 4-chloro-acetoacetic-acid-ethyl-ester to the pharmaceutical building block (S)-4-chloro-3-hydroxybutanoate-ethyl-ester requires the utilization of an enantioselective robust biocatalyst. Some of the natural Saccharomyces cerevisiae strains, isolated from Mount Carmel National Park in Israel, were characterized as resistant to environmental stress. Nevertheless, these strains showed relatively low enantiomeric-excess (ee), while a laboratory strain, Y103, exhibited a selectivity of 98% ee. The enantioselective lab strain was crossed with the multi-stress resistant environmental isolate (93% ee) followed by backcross with Y103, to subsequently obtain a haploid offspring of backcross-1, exhibiting both high multi-stress resistance and high enantioselectivity (98% ee). Introducing osmotic (1 M NaCl), oxidative (0.6 mM H₂O₂) and thermal stress (44°C) to growing cultures of the enantioselective parent, resulted in a decrease of 24–32% in specific activity, while the enantioselectivity of the stress-resistant parent decreased by 4–12% ee. Unlike its original parental strains, the new strain maintained constant specific activity and enantioselectivity when introduced to the various stress factors. This work shows that the classic introgression method, can serve as a viable approach for creating a robust enantioselective biocatalyst, designed for industrial production of chiral compounds.     

Cloning,screening and characterization of enantioselective ester hydrolases from <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

Thirty-one ester hydrolases were cloned from Escherichia coli K-12 and an efficient screening strategy was applied to screen and characterize them, emphasizing on their enantioselectivity. We are the first to investigate the enantioselectivity of these enzymes, although their activity had been reported by other researchers. The enzyme XL3 from gene b0349, XL10 from gene b0494, XL15 from gene b3412, XL27 from gene b2154 and XL31 from gene b3825 exhibited high activity towards p-nitrophenyl esters with short chain. The enzyme XL15 from gene b3412 was demonstrated for the first time to show high enantioselectivity to (R)-1-phenylethyl acetate both in hydrolysis and esterification with enantioselectivity value (E) > 100 at the conversion of 31.2 and 36.8%, respectively.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).     

Alcohols as enantioselective inhibitors in a lipase catalysed esterification of a chiral acyl donor

Summary Increased reaction rates and increased enantioselectivities were observed with decreased concentrations of n-alkanols when resolving 2-methyldecanoic acid by esterification catalysed by immobilised lipase from Candida rugosa at controlled water activities in cyclohexane. The enantioselectivity was found to be independent of the water activity in the reaction medium at the n-heptanol concentrations investigated. However, when n-decanol was used as the acyl acceptor, not only the alcohol concentration but also the water activity in the reaction medium, influenced the enantioselectivity. The results obtained showed that the low enantioselectivity seen at a high alcohol concentration could be explained by the alcohol influencing the apparent V _max ^S and V _max ^R differently.     

Application of a new amidophosphite ligand to Rh‐catalyzed asymmetric hydrogenation of β‐dehydroamino acid derivatives in supercritical carbon dioxide: Activation effect of protic co‐solvents

New chiral amidophosphite ligand was synthesized and tested in the Rh‐catalyzed asymmetric hydrogenation of (Z)‐β‐(acylamino)acrylates in protic solvents and supercritical carbon dioxide (scCO₂) The catalytic performance is affected greatly by the acidity of the solvents. Better enantioselectivity (up to 88% ee) was achieved in scCO₂ containing 1,1,1,3,3,3‐hexafluoro‐2‐propanol, compared to neat protic solvents. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Enantioselective carbonylation reactions of para-substituted 2-phenylpropenes

The enantioselective hydroformylation catalyzed by [(R,R)-Diop]Pt(SnCl₃)Cl7 and the enantioselective hydroisopropoxycarbonylation catalyzed by [(R,R)-Diop]PdCl₂8 or by [(R,R)-Diop-dbp]PdCl₂9 of some para-substituted 2-phenylpropenes (para substituents = NO₂, H, CH₃O, Cl, CF₃) was investigated in order to recognize possible electronic influences on the regioselectivity and on the enantioface selection which take place in such carbonylation reactions. The catalytic systems used gave no carbonylation products when the nitro compound was the substrate. 7 and 8 show similar regioselectivities, the less branched isomer being exclusively formed for all substrates except p-methoxy-2-phenylpropene which gave small amounts of the alternative regioisomer. The enantioselectivity depends on the σ_p effect of the substituent in both cases; the differences are, however, rather small and the trend is opposite in the two cases. The regioselectivity displayed by 9 is still in favour of the less branched isomer but it is high only in the case of p-trifluoromethyl-2-phenylpropene. Larger differences with respect to the other catalytic systems were also observed for enantioselectivity but the trend for both regioselectivity and enantioselectivity is not linear.     

Noncovalent Immobilization of Ionic‐tagged Box‐Cu(OAc)2 Complex and Its Application in Asymmetric Henry Reaction

Immobilized Cu(OAc)₂‐bis(oxazolines) via hydrogen bonding by SBA‐15 was applied to asymmetric Henry reaction, and good enantioselectivities were obtained (up to 83% ee) between 2‐methoxybenzaldehyde and CH₃NO₂ in isopropyl alcohol (iPrOH). The catalyst could be reused seven times without any obvious loss in enantioselectivity. For the first time, this facile and clean immobilization method is applied to the use of bis(oxazolines) complexes. Chirality 24:1092‐1095, 2012. © 2012 Wiley Periodicals, Inc.     

Liquid and subcritical fluid chromatographic enantioseparation of Nα‐Fmoc proteinogenic amino acids on Quinidine‐based zwitterionic and anion‐exchanger type chiral stationary phases. A comparative study

Stereoselective high‐performance liquid chromatographic and subcritical fluid chromatographic separations of 19 N^α‐Fmoc proteinogenic amino acid enantiomers were carried out by using Quinidine ‐based zwitterionic and anion‐exchanger‐type chiral stationary phases Chiralpak ZWIX(−) and QD‐AX. For optimization of retention and enantioselectivity, the ratio of bulk solvent components (MeOH/MeCN, H₂O/MeOH, or CO₂/MeOH) and the nature and concentration of the acid and base additives (counter‐ and co‐ions) were systematically varied. The effect of column temperature on the enantioseparation was investigated and thermodynamic parameters were calculated from the van't Hoff plots ln α vs. 1/T. The thermodynamic parameters revealed that the enantioseparations were enthalpy‐driven. The elution sequence was determined in all cases and with the exception of Fmoc‐Cys(Trt)‐OH, it was identical on both chiral stationary phases whereby the L‐enantiomers eluted before the D‐enantiomers.     

Modeling the αIIbβ3 integrin solution conformation

The α_IIbβ₃ platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of α and β subunits. Electron microscopic (EM) images of the α_IIbβ₃ complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross-linking studies rather suggest a somewhat more compact conformation for the α_IIbβ₃ complex. We have studied the shape of detergent-solubilized α_IIbβ₃ by employing a low-resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three-dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and -18% from the experimental values, determined, respectively, in octyl glucoside and Triton X-100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by -7% (octyl glucoside) to -6% (Triton X-100). Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent-solubilized α_IIbβ₃ complex, where the relative spatial location of specific domains the size of 5–10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.     

Enantioselectivity of Epoxide Formation from Halohydrins by Means of Flavobacterium Rigense

The formation of epoxides from several halohydrins was achieved using resting cells from Flavobacterium rigense. The reaction showed a high substrate specificity for halohydrins with a terminal halogen atom but only low enantioselectivity (12–58% e.e.). The epoxides always had the (S)-configuration. Substrates which in the halogen atom was replaced by another leaving group (-O-SO₂CH₃, -O-Tos, -N₃) were not accepted. An attempt to improve the enantioselectivity by using a two phase system consisting of an aqueous and an organic solvent phase was not successful.     

Resolution of (R,S)‐ibuprofen catalyzed by immobilized Novozym40086 in organic phase

The enantioselective esterification of ibuprofen catalyzed by Novozym40086 was successfully conducted in organic solvent. Removing‐water reagent was added into the reaction mixture to remove water produced in the esterification. The effects of temperature, n‐hexanol concentration, ibuprofen concentration, and loading of enzymes were investigated. Under the condition of equilibrium, the thermodynamic equilibrium constant (K) of 7.697 and enantioselectivity (E) of 8.512 were obtained. The esterification reaction achieved its equilibrium in approximately 30 hours with conversion of 56% and ee_S of 93.78%. The predicted values of X and ee_S were 67.90% and 95.60%, respectively. The experimental value is approximately equal to the theoretical value, which indicates the feasibility of ideal models.     

Enantioselective esterification of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-2-methylalkanoic acid with <Emphasis Type="Italic">Carica papaya</Emphasis> lipase in organic solvents

Isooctane was the best reaction medium for the enantioselective esterification of (R,S)-2-methylalkanoic acid with n-butanol using Carica papaya lipase as catalyst. Increasing linear alkyl-chain length of racemic 2-methylalkanoic acids from ethyl to hexyl increased the enantioselectivity (E) from 2.1 to 98.2 for the esterification of racemic 2-methylalkanoic acids with n-butanol at 35°C. Decreasing reaction temperature from 40 to 20°C increased the enantioselectivity (E) from 14 to 33 for the esterification of racemic 2-methylhexanoic acids with n-butanol. We obtained a maximum enantioselectivity, of E = 24.3, for the enantioselective esterification of racemic 2-methylhexanoic acids with n-butanol in isooctane at water activity 0.33, and at 35°C.