Towards a novel explanation of Pseudomonas cepacia lipase enantioselectivity via molecular modelling of the enantiomer trajectory into the active site

In the transesterification reaction between (RS)-2-bromophenyl acetic acid ethyl ester and 1-octanol in n-octane, Pseudomonas cepacia lipase enantioselectivity towards the (R)-isomer is 57. Two strategies are described to investigate the structural basis involved in this enzyme enantioselectivity. Molecular modelling of the tetrahedral intermediate mimicking the transition state enables the identification of two potentially productive substrate-binding modes for each enantiomer. However, the conformations obtained with the faster and slower-reacting enantiomers have equivalent potential energies and most of them possess the hydrogen bonds essential for catalysis. On this basis, it is not possible to distinguish the diastereomeric complexes. The second approach is original and consists in a simple but robust protocol of pseudomolecular dynamics simulations under constraints to map the probable trajectory of the enantiomers in the active site. Enzyme/substrate interaction energy is always found to be lower for the faster-reacting enantiomer, which satisfactorily corroborates the experimental results. Energy differences are attributed to specific interactions of these substrates with a network of hydrophobic residues lining the access path. Furthermore, mechanistic details suggest that the pivoting side chains of the hydrophobic residues act in a concerted step–tooth gear motion whose basic role is to select and guide the substrates towards the active site. With this type of lipase, such dynamic features could be the key explanation of this as yet unexplored enantiorecognition. For the slower-reacting enantiomer, it appears that the concerted motion of the side chains is perturbed when the substrate passes through a bottleneck formed by Val266 and Leu17. The enantioselectivity of mutant Val266Leu with a more bulky side chain at this position supports our assumption: by narrowing the bottleneck, the enantioselectivity was considerably enhanced as much as up to 200.     

Theoretical study on the origin of enantioselectivity in the primary amine–Brønsted acid catalyzed epoxidation of cyclic enones

Computational studies to determine the origin of enantioselectivity in the (1R,2R)-1,2-diphenylethane-1,2-diamine (DEPN)–Brønsted acid catalyzed epoxidation of 2-cyclohexen-1-one have been performed using density functional theory. Transition states for conjugate addition and ring closure steps of the epoxidations catalyzed by three different catalyst systems were characterized. Our calculations show that the C_sp2H?O H-bond interaction between the benzene ring of the catalyst and H₂O is mainly responsible for the chiral discrimination observed. The Brønsted acid counterion plays a very important role in ensuring high enantioselectivity by improving the rigidity of the transition state structures to allow the efficient formation of the C_sp2H?O H-bond. Moreover, we explain why these two diamine catalysts (1S,2S)-DACH and (1R,2R)-DPEN display consistent enantioselectivities in the catalytic epoxidation of 2-cyclohexen-1-one when combining with three different cocatalysts; achiral TFA, and chiral (R)- and (S)-TRIP.     

Efficient resolution of profen ethyl ester racemates by engineered Yarrowia lipolytica Lip2p lipase

Enzyme-catalyzed enantiomer discrimination is still a great challenge for the development of industrial pharmaceutical processes. For the resolution of ibuprofen, naproxen and ketoprofen racemates, three major anti-inflammatory drugs, only lipases from Candida rugosa present a high selectivity if solvent and surfactant use is discarded. However, their catalytic activities are too low. In the present work, we demonstrate that the lipase Lip2p from the yeast Yarrowia lipolytica has a higher catalytic activity than C. rugosa lipases to hydrolyze the ethyl esters of ibuprofen, naproxen and ketoprofen, but its selectivity is not sufficient [E = 52 (S); 11 (S) and 1.5 (R) respectively]. The enantioselectivity was further improved by site-directed mutagenesis, targeted at the substrate binding site and guided by molecular modelling studies. By investigating the binding modes of the (R)- and (S)-enantiomers in the active site, two amino acid residues located in the hydrophobic substrate binding site of the lipase, namely residues 232 and 235, were identified as crucial for enantiomer discrimination and enzyme activity. The (S) enantioselectivity of Lip2p towards ethyl ibuprofen esters was rendered infinite (E ? 300) by replacing V232 by an A or C residue. Substitution of V235 by C, M, S, or T amino acids led to a great increase in the (S)-enantioselectivity (E ? 300) towards naproxen ethyl ester. Finally, the variant V232F enabled the efficient kinetic resolution of ethyl ketoprofen ester enantiomers [(R)-enantiopreference; E ? 300]. In addition to the increase in selectivity, a remarkable increase in velocity by 2.6, 2.7 and 2.5 times, respectively, was found for ibuprofen, naproxen and ketoprofen ethyl esters.     

A structural examination of the impact of oxygenated side chains in Ephedra compounds in the catalytic asymmetric addition of diethylzinc to aldehydes

An investigation of the impact of oxygenated side chains in Ephedra compounds on the catalytic asymmetric addition of diethylzinc to aldehydes has been conducted. (1R,2S)-Ephedrine and (1S,2S)-pseudoephedrine were alkylated with either alkyl halides or β-alkoxyalkyl halides to afford a series of ligands 9a–h and 10a–h. These compounds were employed in the enantioselective addition of diethylzinc to a variety of aldehydes. It was determined that the presence of oxygen could have a negative effect in terms of obtaining high levels of enantiomeric discrimination, but the effect is diminished with higher levels of substitution near the oxygen.     

Synthesis of new chiral fluorescent sensors and their applications in enantioselective discrimination

Five novel sensors (R,R)-3–6 and (S, S)-6 were synthesized and developed for enantioselective recognition of chiral compounds. Sensor 6 with two thiourea groups and steric π-conjugation frameworks could discriminate different chiral substrates, including acidic compounds, basic compounds, and neutral compounds. These results disclosed that the outstanding performance of enantioselective discrimination could be attributed to the thiourea group which acted as a hydrogen-bonding donor and the bulky steric moiety of the hosts which provided appropriate chiral environment. This result will be of great practical value in the designation of chiral sensors and high-throughput assay of chiral products.     

Chiral ligand derived from (1S,2R)-norephedrine as a catalyst for enantioselective prochiral ketone reduction

Chiral oxazaborolidines derived from (1S,2R)-(+)-norephedrine and substituted salicylaldehydes were employed in the asymmetric reduction of prochiral ketones using borane dimethyl sulfide as a reducing agent. The secondary alcohols were formed in excellent yields (45–99.8%) with enantioselectivities up to 99.8%. The effect of the substitution in the aromatic ring of the ligand was discussed with the enantioselectivity of the product.     

Enzyme-catalyzed kinetic resolution of N-Boc-trans-3-hydroxy-4-phenylpyrrolidine

The first enzyme-catalyzed kinetic resolution of tert-butyl-3-hydroxy-4-phenylpyrrolidine-1-carboxylate is presented. Enzyme, solvent and temperature optimization resulted in a new resolution method with E = 40 enantioselectivity. The acetate derivative of the (+)-(3S,4R) enantiomer formed while the (?)-(3R,4S) isomer remained intact. Very good enantioselectivities (E > 200) were achieved in the enzyme-catalyzed alcoholysis of the racemic acetate in i-propanol and t-butanol where the (+)-(3S,4R) enantiomer was prepared in pure form (ee > 99.7%). Absolute configuration of the (?)-(3R,4S)-enantiomer was determined by single crystal X-ray diffraction method.      

Resolution of non-protein amino acids via the microbial protease-catalyzed enantioselective hydrolysis of their N-unprotected esters

In the Aspergillus oryzae protease-catalyzed ester hydrolysis, substitution of N-unprotected amino acid esters for the corresponding N-protected amino acid esters resulted in a large enhancement of the hydrolysis rate, while the enantioselectivity was deteriorated strikingly when the substrates employed were the conventional methyl esters. This difficulty was overcome by employing esters bearing a longer alkyl chain such as the isobutyl ester. Utilizing this ester, amino acids carrying an aromatic side chain were resolved with excellent enantioselectivities (E=50 to >200). With amino acids bearing an aliphatic side chain also, good results in terms of the hydrolysis rate and enantioselectivity were obtained by employing such an ester as the isobutyl ester. Moreover, the enantioselectivity proved to be enhanced further by conducting the reaction at low temperature. This procedure was applicable to the case where the enantioselectivity was not high enough even by the use of the isobutyl ester.     

Chirality control in the enantioselective arylation of aromatic aldehydes catalyzed by cis-(1R,2S)-2-benzamidocyclohexanecarboxylic acid derived 1,3-aminoalcohols

A series of chiral 1,3-aminoalcohols derived from cis-(1R,2S)-2-benzamidocyclohexanecarboxylic acid were synthesized and applied to the enantioselective arylation of aromatic aldehydes. The reactions exhibited good yields (up to 90%) and moderate to high enantioselectivities (up to 99%). Not only the enantioselectivity but also the stereochemistry of the product were controlled by the substituent effect of the chiral ligands.     

Novel tetradentate bisoxazoline ligands from l-serine and β-DDB for asymmetric pinacol coupling reactions of aromatic aldehydes

A variety of novel hydroxyl-containing tetradentate bisoxazolines were successfully synthesized from natural l-serine and β-DDB. The applications of these ligands in the asymmetric pinacol coupling of aromatic aldehydes revealed that the absolute configurations of the resulting pinacols were entirely dominated by the axial chirality of the biphenyl component and that the bulky substituent adjacent to the hydroxyl group was favorable for achieving both high diastereoselectivities and enantioselectivities. Among the ligands screened, (S,aR,S)-1c exhibited much better asymmetric induction capacity to furnish the (R,R)-pinacols with diastereoselectivities up to 99/1 and with 63–89% enantiomeric excess. A plausible mechanism of the asymmetric pinacol coupling was also suggested.     

Enantiomer discrimination by mass spectrometry: noncovalent interactions of an N-derivatized dipeptide with various cinchona alkaloid derivatives and comparison with enantioselective liquid-phase separations

The enantiomer discrimination properties of cinchona alkaloid derived chiral selectors (CSs) towards a dipeptide analyte are examined by electrospray ionization mass spectrometry. The complexes formed between the CSs and the analyte enantiomers owing to various noncovalent interactions are analyzed and the magnitudes of enantiomer discrimination are determined from the complexes mass spectrometric intensities. The influence of different structural features of the CSs on enantioselectivity is discussed. The enantiomer discrimination results obtained by mass spectrometry are compared with those from related liquid chromatography enantiomer separations. A certain coherence between the chromatographic and mass spectrometric enantioselectivities could be established and the enantiomer discrimination patterns, i.e., the relative binding strengths, were identical for the two techniques. Thus, the use of mass spectrometry as a screening tool in the development of new CSs for chromatographic applications seems feasible.     

Inclusion of aliphatic alcohols in pockets of (S)-threonyl-(S)-phenylglycine using grinding method

Inclusion compounds of a dipeptide, (S)-threonyl-(S)-phenylglycine (Thr-Phg), with several aliphatic alcohols were easily prepared by grinding them in a mortar. Thr-Phg molecules arranged in antiparallel to construct a sheet, and guest alcohols were accommodated in a chiral pocket between the sheets. 3-Butyn-2-ol and 2-butanol were included with moderate enantioselectivity, 57% ee (R) and 49% ee (R), respectively. The role of the hydroxy group of Thr-Phg is not only to construct the unique pocket but also to capture guest alcohols by hydrogen bonding.     

Origins of Enantiopreference of Mycobacterium smegmatis Acyl Transferase: A Computational Analysis

Acyl transferase from Mycobacterium smegmatis (MsAcT) is a promising biocatalyst because it catalyzes an acyl transfer reaction in aqueous solution, thereby accepting many primary and secondary alcohols as substrates. MsAcT also exhibits high enantioselectivity for a selected number of secondary alcohols. To increase the applicability of this enzyme for the production of optically active compounds, a detailed understanding of the reaction mechanism and the factors that affect enantioselectivity is essential. Herein, quantum chemical calculations are employed to study the reactions of two secondary alcohols, 1-isopropyl propargyl alcohol and 2-hydroxy propanenitrile, for which the enzyme displays opposite enantiopreference, favoring the S enantiomer in the former case and R enantiomer in the latter. A model of the active site has been designed and for both substrates various binding modes are evaluated and the intermediates and transition states along the reaction path are then located. The calculated energy profiles agree with the experimental observations, and reproduce the selectivity outcome. Through a detailed analysis of the geometries of key transition states, insights into the origins of the enantiopreference are obtained.     

Synthesis of chiral ligands containing the N-(S)-α-phenylethyl group and their evaluation as activators in the enantioselective addition of Et2Zn to benzaldehyde

The synthesis of chiral ligands 4–18 derived from N-[(S)-α-phenylethyl]-trans-β-aminocyclohexanols (S,S,S)-1a and (R,R,S)-2 is described. Addition of diethylzinc to benzaldehyde catalyzed by ligands 4–18 (6 mol %) proceeds in fair to good yield (45–86%), and low to good enantioselectivities (1–76% ee). Highest enantioselectivities were induced by ligands (S,S,S)-4 and (S,S,S,S,R,R)-18 (76% and 68% ee, respectively). The configuration of the major enantiomer of carbinol 3 is (R) in both cases.     

Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Ophiostoma piliferum,esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes

Recent advances in biochemistry and molecular biology have simplified the discovery and preparation of new hydrolases. Although these hydrolases might solve problems in organic synthesis, measuring their selectivity, especially enantioselectivity, remains tedious and time consuming. Recently, we developed a colorimetric screening method to measure the enantioselectivity of hydrolases. Here we apply this rapid screening method to map the substrate selectivity of four new hydrolases: lipases from the thermophilic Bacillus thermocatenulatus (DSM 730, BTL2) and a filamentous fungus Ophiostoma piliferum (NRRL 18917, OPL) and esterases from two bacteria, Pseudomonas fluorescens (SIK-W1, esterase I, PFE) and Streptomyces diastatochromogenes (Tü 20, SDE). We screened a general library of 29 substrates and a chiral library of 23 pairs of enantiomers. All four hydrolases catalysed the hydrolysis of unnatural substrates, but the two lipases accepted a broader range of substrates than the two esterases. As expected, the two lipases favoured more hydrophobic substrates, while the two esterases showed a preference for smaller substrates. Several moderately enantioselective reactions were identified for the solketal esters: BTL2, butyrate, E=7.9 (R); octanoate, E=4.9 (R) and 3-bromo-2-methyl propionate methyl esters, PFE, E=12 (S); SDE, E=5.6 (S). OPL showed low enantioselectivity toward all substrates tested. The current colorimetric screen could not measure the selectivity for several slow-reacting substrates. Traditional screening identified high enantioselectivity of BTL2 and PFE toward one of these slow substrates, 1-phenylethyl acetate (E>50).     

Synthesis of Chiral Sulfoximines via Iridium-Catalyzed Regio- and Enantioselective C−H Borylation: A Remarkable Sidearm Effect of Ligand

Transition metal-catalyzed enantioselective C−H activation of prochiral sulfoximines for non-annulated products remains a formidable challenge. We herein report iridium-catalyzed enantioselective C−H borylation of N-silyl diaryl sulfoximines using a well-designed chiral bidentate boryl ligand with a bulky side arm. This method is capable of accommodating a broad range of substrates under mild reaction conditions, affording a vast array of chiral sulfoximines with high enantioselectivities. We also demonstrated the synthetic utility on a preparative-scale C−H borylation for diverse downstream transformations, including the synthesis of chiral version of bioactive molecules. Computational studies showed that the bulky side arm of the ligand confers high regio- and enantioselectivity through steric effect.     

A DFT exploration of the enantioselective rearrangement of cyclohexene oxide to cyclohexenol

In this paper, we present computational results for the (1S,3R,4R)-3-(pyrrolidinyl)-methyl-2-azabicyclo[2.2.1]heptane mediated rearrangement of cyclohexene oxide. The results nicely explain the differences in enantioselectivities between catalytic and stoichiometric mode between different ligands, and provides a rational for the identification of non-stereospecific background reactions as the major cause of decreased enantioselectivity in catalytic reactions for sterically hindered diamines.     

Rhodium/Chiral Diene‐Catalyzed Asymmetric 1,4‐Addition of Arylboronic Acids to Chromones: A Highly Enantioselective Pathway for Accessing Chiral Flavanones

Chromone has been noted to be one of the most challenging substrates in the asymmetric 1,4‐addition of α,β‐unsaturated carbonyl compounds. By employing the rhodium complex associated with a chiral diene ligand, (R,R)‐Ph‐bod*, the 1,4‐addition of a variety of arylboronic acids was realized to give high yields of the corresponding flavanones with excellent enantioselectivities (≥97 % ee, 99 % ee for most substrates). Ring‐opening side products, which would lead to erosion of product enantioselectivity, were not observed under the stated reaction conditions.     

Switched enantiopreference of Humicola lipase for 2-phenoxyalkanoic acid ester homologs can be rationalized by different substrate binding modes

Humicola lanuginosa lipase was used for enantioselective hydrolyses of a series of homologous 2-phenoxyalkanoic acid ethyl esters. The enantioselectivity (E-value) of the enzyme changed from an (R)-enantiomer preference for the smallest substrate, 2-phenoxypropanoic acid ester, to an (S)-enantiomer preference for the homologous esters with longer acyl moieties. The E-values span the range from E=13 (R) to E=56 (S). A molecular modeling study identified two different substrate-binding modes for each enantiomer. We found that the enantiomers favored different modes. This discovery provided a model that offered a rational explanation for the observed switch in enantioselectivity.     

Lipase-catalyzed resolution of 4-aryl-substituted β-lactams: effect of substitution on the 4-aryl ring

Pseudomonas cepacia lipase (PS-30) was used in hydrolytic resolution of 3-acetoxy-4-aryl-substituted azetidin-2-ones (>97% ee). Twenty-three β-lactam substrates with varied substituents at the C-4 center of the ring were synthesized and subjected to lipase-PS catalyzed hydrolysis in phosphate buffer (pH 7.2, 0.2 M) at 25°C. The reactions occurred with high enantioselectivity and substrate conversion. The effect of substitution on the C-4 aryl ring on lipase hydrolytic activity was dependent upon the steric and electronic nature of the substituent and its position on the aryl ring. The stereopreference of the lipase PS-30 for the (3S,4R) enantiomer was rationalized using a known active site model. Absolute stereochemistry of the enantiomers was established using single crystal X-ray crystallographic techniques.