Methyl (R)‐o‐chloromandelate [(R)‐ 1 ], which is an intermediate for a platelet aggregation inhibitor named clopidogrel, was obtained in >99% ee by the asymmetric reduction of methyl o‐chlorobenzoylformate ( 2 ) with recombinant Escherichia coli overproducing a versatile carbonyl reductase. A remarkable temperature effect on productivity was observed in the whole‐cell reduction of 2 , and the optimum productivity as high as 178 g/L was attained at 20 °C on a 2‐g scale (1.0 M). The optimized reaction could be scaled up easily to transform 20 g of 2 in 100 mL of buffer. Three synthetic methods for 2 are compared. 相似文献
It was found that Δ4,5‐steroid 5β‐reductases are capable of reducing also small molecules bearing an activated CC double bond such as monocyclic enones and acyclic enoate esters. As preferred Δ4,5‐steroid 5β‐reductase (5β‐StR) for this purpose, 5β‐StR from Arabidopsis thaliana was used. In part, enzyme activities are even higher than that for progesterone. Successful preliminary biotransformations with enzymatic in situ cofactor recycling were also carried out. When using the prochiral compound isophorone as a substrate, a high enantioselective reaction course (>99% ee) was observed. 相似文献
(S)‐Mandelic acid is a useful and high value chemical with many synthetic applications, but its synthesis often requires the use of toxic cyanide. Here, we report the development of several cyanide‐free methods to prepare (S)‐mandelic acid via cascade biotransformation. Enhanced production of (S)‐mandelic acid from styrene via 4‐step enzyme cascades was achieved with Escherichia coli (A‐M1_R‐M2) cells, giving 118–144 mM (18–21.9 g/L, 72–79% yield) product. The process was scaled up to 1.5 L to produce 140 mM (21.3 g/L) of (S)‐mandelic acid in 70% yield. A strategy with the recycling and reuse of Escherichia coli cells, unreacted substrate, and organic solvent was developed to enhance the productivity of (S)‐mandelic acid through repeated batches, affording 190 mM (95% yield) and 328 mM (82% yield) product in two and four batches, respectively. (S)‐mandelic acid was also produced from bio‐derived L‐phenylalanine via 6‐step enzyme cascades. Biotransformation of L‐phenylalanine with Escherichia coli (LZ37) cells expressing all enzymes for the reactions gave 160 mM (S)‐mandelic acid in 80% yield. Moreover, (S)‐mandelic acid were synthesized from glycerol or glucose via L‐phenylalanine biosynthesis pathway and the 6‐step enzyme cascade. Coupling of Escherichia coli (NST74‐Phe) with Escherichia coli (LZ37) enabled the sustainable production of 63 mM (10 g/L) or 52 mM (8 g/L) (S)‐mandelic acid from renewable feedstocks glycerol and glucose, respectively.
Various ω‐transaminases were tested for the synthesis of enantiomerically pure amines from the corresponding ketones employing D ‐ or L ‐alanine as amino donor and lactate dehydrogenase to remove the side‐product pyruvate to shift the unfavourable reaction equilibrium to the product side. Both enantiomers, (R)‐ and (S)‐amines, could be prepared with up to 99% ee and >99% conversions within 24 h at 50 mM substrate concentration. The activity and stereoselectivity of the amination reaction depended on the ω‐transaminase and substrate employed; furthermore the co‐solvent significantly influenced both the stereoselectivity and activity of the transaminases. Best results were obtained by employing ATA‐117 to obtain the (R)‐enantiomer and ATA‐113 or ATA‐103 to access the (S)‐enantiomer with 15% v v−1 DMSO. 相似文献
The concise synthesis of a pharmaceutical candidate is described. The chiral core of the molecule is assembled using an aza‐benzoin condensation and a dynamic kinetic resolution (DKR) as the key reactions. This enables superb control of the regio‐, diastereo‐ and enantioselectivity of the synthesis. Both biocatalysts and transition metal catalysts are remarkably effective in the key asymmetric reduction step. Similar approaches could be considered in the synthesis of other 1,2‐amino alcohols where traditional approaches based on functionalization of alkenes, epoxides or aziridines may suffer from selectivity issues.
Over the last decades biocatalysis has emerged as an indispensable and versatile tool for the asymmetric synthesis of active pharmaceutical ingredients (APIs). In this context, especially transaminases (TAs) have been successfully used for the preparation of numerous α‐chiral, optically pure amines, serving as important building blocks for APIs. Here we elaborate on the development of transaminases recognizing the α‐chiral centre adjacent to an aldehyde moiety with aliphatic residues, opening up concepts for novel synthetic routes to the antiepileptic drugs Brivaracetam and Pregabalin. The transformation proceeded via dynamic kinetic resolution (DKR) based on the bio‐induced racemisation of the aldehyde enantiomers, enabling the amination of the racemic substrates with quantitative conversions. Medium, substrate as well as enzyme engineering gave access to both (R)‐ and (S)‐enantiomers of the amine precursors of the stereocomplementary drugs in high optical purity, representing a short route to mentioned APIs.
Highly enantioselective benzylic hydroxylations of benzene derivatives ( 1–4 ) containing reactive functional groups were achieved for the first time with Pseudomonas monteilii TA‐5 as biocatalyst, giving the corresponding (R)‐benzylic alcohols 5 – 8 in 93–99% ee as the only products. Preparative biotransformations were demonstrated by the biohydroxylation of 1 and 2 with resting cells of P. monteilii TA‐5 to afford (R)‐ 5 in 94% ee and 66% yield and (R)‐ 6 in 94% ee and 56% yield, respectively. The highly enantioselective biohydroxylations represent a simple access to (R)‐benzylic alcohols containing reactive functional groups that are useful pharmaceutical intermediates and versatile chiral building blocks. 相似文献
A simplified procedure for cell‐free biocatalytic reductions of conjugated CC double bonds using old yellow enzymes (OYEs) is reported. Instead of indirectly regenerating YqjM (an OYE homologue from B. subtilis) or NemA (N‐ethylmaleimide reductase from E. coli) via regeneration of reduced nicotinamide cofactors, we demonstrate that direct regeneration of catalytically active reduced flavins is an efficient and convenient approach. Reducing equivalents are provided from simple sacrificial electron donors such as ethylenediaminetetraacetate (EDTA), formate, or phosphite via photocatalytic oxidation. This novel photoenzymatic reaction scheme was characterized. Up to 65% rates of the NADH‐driven reaction were obtained while preserving enantioselectivity. The chemoselectivity of the novel approach was exclusive. Even when using crude cell extracts as biocatalyst preparations, only CC bond reduction was observed while ketone and aldehyde groups remained unaltered. Overall, a simple and practical approach for photobiocatalytic reductions is presented. 相似文献
A new type of planar chiral Shvo catalysts, where the chirality is based solely on different substitution flanking the C O function, was prepared and used for transfer hydrogenation of imines and ketones. The reduction of ketimines represented by N‐(1‐phenylethylidene)aniline and prochiral ketones such as phenyl trifluoromethyl ketone with 2‐propanol was efficiently catalyzed by 0.5 mol% of the chiral Shvo catalyst to give high yields of the corresponding reduction products with the enantioselectivities in the range 45% to 64% ee.
The amination of racemic α‐chiral aldehydes, 2‐phenylpropanal derivatives, was investigated employing ω‐transaminases. By medium and substrate engineering the optical purity of the resulting β‐chiral chiral amine could be enhanced to reach optical purities up to 99% ee. Using enantiocomplementary ω‐transaminases allowed us to access the (R)‐ as well as the (S)‐enantiomer in most cases. It is important to note that the stereopreference of the ω‐transaminases found for α‐chiral aldehydes did not correlate with the stereopreference previously observed for the amination of methyl ketones. In one case the stereopreference switched even upon exchanging a methyl substituent to a methoxy group.
Candida antarctica lipase B (CAL-B) catalyzes the regioselective acylation of natural thymidine with oxime esters and also the regioselective acylation of an analogue, 3',5'-diamino-3',5'-dideoxythymidine with nonactivated esters. In both cases, acylation favors the less hindered 5'-position over the 3'-position by upto 80-fold. Computer modeling of phosphonate transition-state analogues for the acylation of thymidine suggests that CAL-B favors acylation of the 5'-position because this orientation allows the thymine ring to bind in a hydrophobic pocket and forms stronger key hydrogen bonds than acylation of the 3'-position. On the other hand, computer modeling of phosphonamidate analogues of the transition states for acylation of either the 3'- or 5'-amino groups in 3',5'-diamino-3',5'-dideoxythymidine shows similar orientations and hydrogen bonds and, thus, does not explain the high regioselectivity. However, computer modeling of inverse structures, in which the acyl chain binds in the nucleophile pocket and vice versa, does rationalize the observed regioselectivity. The inverse structures fit the 5'-, but not the 3'-intermediate thymine ring, into the hydrophobic pocket, and form a weak new hydrogen bond between the O-2 carbonyl atom of the thymine and the nucleophile amine only for the 5'-intermediate. A water molecule might transfer a proton from the ammonium group to the active-site histidine. As a test of this inverse orientation, we compared the acylation of thymidine and 3',5'-diamino-3',5'-dideoxythymidine with butyryl acyl donors and with isosteric methoxyacetyl acyl donors. Both acyl donors reacted at equal rates with thymidine, but the methoxyacetyl acyl donor reacted four times faster than the butyryl acyl donor with 3',5'-diamino-3',5'-dideoxythymidine. This faster rate is consistent with an inverse orientation for 3',5'-diamino-3',5'-dideoxythymidine, in which the ether oxygen atom of the methoxyacetyl group can form a similar hydrogen bond to the nucleophilic amine. This combination of modeling and experiments suggests that such lipase-catalyzed reactions of apparently close substrate analogues like alcohols and amines might follow different pathways. 相似文献
The challenging bioamination of hydrophobic substrates has been attained through the employment of a disperse system consisting of a combination of a low polarity solvent (e. g. isooctane or methyl‐tert‐butylether), a non‐ionic surfactant and a minimal amount of water. In these conditions, amine transaminases (ATA) were shown to efficiently carry out the reductive amination of variously substituted cyclohexanones, providing good conversions often coupled with a superior stereoselectivity if compared with the corresponding chemical reductive amination. An array of synthetically useful 4‐substituted aminocyclohexanes was consequentially synthesized through biocatalysis, analyzed and stereochemically characterized.
Two microbial epoxide hydrolases – i.e., Aspergillus niger (AnEH) and Rhodococcus erythropolis (the so‐called “Limonene EH”: LEH) were used to achieve, for the first time, the biocatalysed hydrolytic kinetic resolution (BHKR) of spiroepoxide rac‐ 1 . This compound is a strategic key building block allowing the synthesis of 11‐heterosteroids. Interestingly enough, the two enzymes exhibited opposite and therefore complementary enantioselectivity allowing us to isolate the residual (R,R)‐ 1 (from AnEH) and the residual (S,S)‐ 1 (from LEH) in nearly enantiopure forms (>98 %). Their absolute configurations were determined by X‐ray crystallography. An opposite regioselectivity of the oxirane ring opening for both enantiomers of substrate 1 , determined using H218O labelling and chiral GC‐MS analysis, was also observed, corresponding to an attack at the less substituted carbon atom using AnEH, and at the most substituted carbon atom using LEH. A chemical process‐improving methodology was also developed. This allowed us to obtain both enantiomers of the substrate in high enantiomeric purity (99 %) and optimised quantity. In the case of the AnEH, the use of a biphasic (water/isooctane) reaction medium allowed us to increase the global substrate concentration up to 200 g/ L. The preparation of both enantiomers of 1 clearly paves the way to the preparative scale synthesis and biochemical evaluation of the corresponding 11‐heterosteroid enantiomers. 相似文献
Lipase from Pseudomonas cepacia (PCL) surprisingly favors acylation of the secondary hydroxyl at the 3'-position over the primary hydroxyl at the 5'-position in 2'-deoxynucleosides by up to >98:1. Catalytically productive tetrahedral intermediate analogues for both orientations were found by molecular modeling. However, acylation of the 3'-hydroxyl places the thymine base in the alternate hydrophobic pocket of PCL's substrate-binding site where it can hydrogen bond to the side-chain hydroxyls of Tyr23 and Tyr29 and the main chain carbonyl of Leu17. Conversely, acylation of the 5'-hydroxyl leaves the thymine base in the solvent where there is no favorable binding to the enzyme. We propose that these remote stabilizing interactions between the thymine base and PCL's substrate-binding site stabilize the 3'-acylation transition state and thus account for the unusual regioselectivity. 相似文献
Enantiopure d ‐phenylglycine and its derivatives are an important group of chiral amino acids with broad applications in thepharmaceutical industry. However, the existing synthetic methods for d ‐phenylglycine mainly rely on toxic cyanide chemistry and multistep processes. To provide green and safe alternatives, we envisaged cascade biocatalysis for the one‐pot synthesis of d ‐phenylglycine from racemic mandelic acid, styrene, and biobased l ‐phenylalanine, respectively. Recombinant Escherichia coli (LZ110) was engineered to coexpress four enzymes to catalyze a 3‐step reaction in one pot, transforming mandelic acid (210 mM) to give enantiopure d ‐phenylglycine in 29.5 g L−1 (195 mM) with 93% conversion. Using the same whole‐cell catalyst, twelve other d ‐phenylglycine derivatives were also produced from the corresponding mandelic acid derivatives in high conversion (58–94%) and very high ee (93–99%). E. coli (LZ116) expressing seven enzymes was constructed for the transformation of styrene to enantiopure d ‐phenylglycine in 80% conversion via a one‐pot 6‐step cascade biotransformation. Twelve substituted d ‐phenylglycines were also produced from the corresponding styrene derivatives in high conversion (45–90%) and very high ee (92–99%) via the same cascade reactions. A nine‐enzymeexpressing E. coli (LZ143) was engineered to transform biobased l ‐phenylalanine to enantiopure d ‐phenylglycine in 83% conversion via a one‐pot 8‐step transformation. Preparative biotransformations were also demonstrated. The high‐yielding synthetic methods use cheap and green reagents (ammonia, glucose, and/or oxygen), and E. coli whole‐cell catalysts, thus providing green and useful alternative methods for manufacturing d ‐phenylglycine.
L ‐Ornithine could serve as an intermediate in the biobased production of 1,4‐diaminobutane from L ‐arginine. Using the concept of biorefinery, L ‐arginine could become widely available from biomass waste streams via the nitrogen storage polypeptide cyanophycin. Selective hydrolysis of L ‐arginine to L ‐ornithine is difficult to perform chemically, therefore the stabilization and immobilization of Bacillus subtilis arginase (EC 3.5.3.1) was studied in a continuously stirred membrane reactor system. Initial pH of the substrate solution, addition of L ‐aspartic acid and reducing agents all appeared to have an effect on the operational stability of B. subtilis arginase. A remarkably good operational stability (total turnover number, TTN=1.13⋅108) at the pH of arginine free base (pH 11.0) was observed, which was further improved with the addition of sodium dithionite to the substrate solution (TTN>1⋅109). B. subtilis arginase was successfully immobilized on three commercially available epoxy‐activated supports. Immobilization on Sepabeads EC‐EP was most promising, resulting in a recovered activity of 75% and enhanced thermostability. In conclusion, the stabilization and immobilization of B. subtilis arginase has opened up possibilities for its application in the biobased production of nitrogen‐containing chemicals as an alternative to the petrochemical production. 相似文献
Oxidative biocatalytic reactions were performed on solid‐supported substrates, thus expanding the repertoire of biotransformations that can be carried out on the solid phase. Various phenylacetic and benzoic acid analogues were attached to controlled pore glass beads via an enzyme‐cleavable linker. Reactions catalyzed by peroxidases (soybean and chloro), tyrosinase, and alcohol oxidase/dehydrogenase gave a range of products, including oligophenols, halogenated aromatics, catechols, and aryl aldehydes. The resulting products were recovered following cleavage from the beads using α‐chymotrypsin to selectively hydrolyze a chemically non‐labile amide linkage. Controlled pore glass (CPG) modified with a polyethylene glycol (PEG) linker afforded substantially higher product yields than non‐PEGylated CPG or non‐swellable polymeric resins. This work represents the first attempt to combine solid‐phase oxidative biotransformations with subsequent protease‐catalyzed cleavage, and serves to further expand the use of biocatalysis in synthetic and medicinal chemistry. 相似文献
