Recent advances in biotechnological applications of alcohol dehydrogenases

Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.

     

Trends and innovations in industrial biocatalysis for the production of fine chemicals

Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure alpha- and beta-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective.     

Chemoenzymatic preparation of optically active hydroxy azido butyric acids

Summary The syntheses of some azido hydroxy butanoates and azido hydroxy butandioates in enantiomerically pure form are presented. The racemic, diastereomerically pure educts are prepared in few steps. These racemates are resolved with the aid of lipases fromPseudomonas fluorescens (P) andCandida cylindracea (CC).     

Recent progress in biocatalyst discovery and optimization

The use of enzymes in industrial catalysis continues to grow because of the considerable advantages of natural catalytic systems. The need for enantiomerically pure fine chemicals and the movement away from chemically burdened technologies will drive the acceptance of enzyme-assisted processes. New technologies for enzyme discovery and optimization have enabled the application of enzymes in harsh industrial conditions and in processes demanding stringent selectivity. These discovery and laboratory evolution methods entail genomic approaches that by their nature engender screening of extremely large numbers of gene types and variants. By extension, the fitness of an individual high-throughput screen requires an intelligent, process-targeted assay amenable to a chosen screening platform.     

HPLC-CD selectivity assay for alcohol dehydrogenases

Enantioselective reductions are a key to successful target-oriented syntheses. Finding the most suitable conditions is often a tedious work that is especially hampered by the time-consuming analytical investigation. A possible solution is the combined use of high-performance liquid chromatography and circular dichroism to find a suitable system for providing enantiomerically pure alcohols. This investigation led to an efficient protocol for the alcohol dehydrogenase-catalyzed reduction of 1-phenyl-2-propyn-3-trimethylsilyl-1-on (1).     

A novel method for preparing Eligulstat through chiral resolution

Eliglustat is a ceramide glucosyltransferase inhibitor work as first line oral therapy for adults with Gaucher disease type 1 (a rare disease) at present. Although the eliglustat in enantiomerically pure forms is obtained by asymmetric syntheses, the reported methods suffer from many limits when it comes to industrial applications. Therefore, the preparation of a racemic mixture followed by resolution can still be a viable and straightforward alternative, especially when it could be adapted to large scale. Herein, we developed an effective and practical synthetic route to prepare stereoisomers mixture of eliglustat, and a novel chiral resolution method to prepare eliglustat. Using 1,1′-Binaphthyl-2,2′-diyl -hydrogenphosphate (BNDHP) as resolution reagent, optical pure eliglustat (e.e. >99%, 13.97% total yield) could be obtained after recrystallization.     

Lipase mediated resolution of γ-azidoalcohols in aqueous and organic media: Synthesis of (R)- and (S)-fluoxetine and duloxetine

A simple and efficient method for the synthesis of optically active γ-azidoalcohols is described. The lipase catalyzed kinetic resolutions of acetates of γ-azidoalcohols in aqueous as well as organic media have been studied. The enantiomerically pure γ-azidoalcohols obtained by the kinetic resolution in high enantiopurity have been utilized towards the synthesis of enantiomeric pairs of anti-depressant drugs, fluoxetine and duloxetine.     

Syntheses of phosphatidyl-beta-D-glucoside analogues to probe antigen selectivity of monoclonal antibody 'DIM21'

Herein, we report the chemical syntheses of a series of phosphatidyl-beta-D-glucoside (PtdGlc) analogues, including 6-O-Ac, sn-2-O-Me, phosphorothioate as well as phosphatidylgalactoside and -mannoside derivatives. In the key step, beta-glycosyl H-phosphonate was condensed with enantiomerically pure diacylglycerol. Comparison of spectroscopic data with mono-acetylated PtdGlc from natural source confirmed the presence of an acetyl moiety at position 6. Furthermore, the reactivity of PtdGlc and its analogues toward monoclonal antibody 'DIM21' (MAb DIM21) was evaluated, revealing the crucial structural antigen features for successful MAb DIM21 binding.     

Mass balancing in kinetic resolution: calculating yield and enantiomeric excess using chiral balance

When kinetic resolution is applied for the production of enantiomerically pure compounds, process options may be used which involve more than one chiral substrate and one chiral product, such as sequential or parallel enzymatic kinetic resolutions or hydrolysis of diastereomers. Although the relation between the yields (y) of the chiral compounds is straightforward in these cases, the relation between their enantiomeric excess (ee) values is not. Combining mass balances into a so-called chiral balance (Sigma y . ee(R) = 0) provides the relation between enantiomeric excess values in a useful manner. This chiral balance easily shows which nonmeasured enantiomeric excess values and yields can be calculated from measured values. The chiral balance is only valid when configurations at chiral centers are conserved. (c) 1995 John Wiley & Sons, Inc.     

Effect of mass transfer limitations on the enzymatic kinetic resolution of epoxides in a two-liquid-phase system

Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases. To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system. A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter. A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model. It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase. Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted. Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide. Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30%.     

Single enantiomeric β-blockers—The existing technologies

A number of β-blocking drugs are available in the world market, only few compounds are found as single enantiomers. The need to use the single enantiomeric β-blockers affects development of drugs and technology. Many processes have been exploited to replace the existing racemates. Two main routes are established: (1) asymmetric syntheses and (2) racemic resolutions. The syntheses give medium-high yields and excellent enantiomeric excess, but the resolutions are limited by 50% yield. Both technologies involve new techniques such as dynamic kinetic resolution (DKR) and membrane-based extraction. The synthetic ways utilise various substrates and catalysts. A simultaneous formation is also afforded by these processes. They offer oriented alternatives to the single enantiomeric β-blockers. Resolutions of the racemates appear with many attractive separation methods. Direct or indirect resolutions show excellent characteristics and produce high enantiomeric excess. The existing processes operate continuously at mild operating temperatures compared to the asymmetric synthesis. In situ separation is also exploited. Development of the single enantiomeric β-blockers using the DKR based on enzymatic membrane(s) is encouraged. Integration of acetylation, racemisation and hydrolysis followed by separation of the enantiomers in the enzymatic membrane reactors could be a better option in resolution and separation of the β-blocker racemates.     

Direct micellar systems as a tool to improve the efficiency of aromatic substrate conversion for fine chemicals production

Whole-cell bioconversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Escherichia coli JM109(pPS1778) recombinant strain, carrying naphthalene dioxygenase and regulatory genes cloned from Pseudomonas fluorescens N3, in direct micellar systems is optimized as an example of fine chemicals bioproduction from scarcely water-soluble substrates. The oxygen insertion into the aromatic substrate, which stops at the enantiomerically pure cis dihydroxylated product, is performed in direct microemulsion systems, where a non-ionic surfactant stabilizes naphthalene containing oil droplets in an aqueous medium. These media provide an increased substrate solubility so that a homogeneous reaction can be carried out, while not affecting bacteria viability and performances. The influence of the chemical nature of the oil is investigated. The phase behavior of the direct microemulsion system was monitored for three different oils as a function their volume fraction and characterized through light scattering. The addition of isopropyl palmitate, oleic acid, or glyceryl trioleate, 0.6-1.2% v/v to the micellar systems, led to an increase of the substrate concentration in the solution and particularly its bioavailability, allowing faster catalytic conversions. All these systems resulted in being suitable for catalytic conversions of aromatic compounds. Although the nature of the oil does have a deep effect on the phase behavior of the micellar systems, in the present investigation no differences in the yields and in the rates of product formation of the enzymatic system were observed on changing the oil, thus showing that in this case the substrate concentration or bioavailability is not the rate-limiting step.     

N-succinimidyl methoxyphenylacetic acid ester, an amine-directed chiral derivatizing reagent suitable for enzymatic scale resolutions

A chiral derivatizing reagent, N-succinimidyl-2-(S)-methoxy-2-phenylacetic acid ester (SMPA), directed toward reaction with primary amine-containing compounds has been synthesized and characterized. This reagent is suitable for HPLC resolution from enzymatic-scale reactions where only microgram quantities of chiral products may be obtainable. SMPA derivatization was shown to be effective in the resolution of the enantiomers of a number of different racemic compounds. SMPA was used to resolve the diastereoisomeric derivatives of a previously unknown enzymatically oxygenated product, allowing determination of the stereochemical course of the enzymatic reaction. SMPA is easily prepared from an inexpensive, commercially available, and enantiomerically pure precursor with the formation of a shelf-stable crystalline product which is utilizable in water-containing solutions. In addition to its usefulness for micro-determinations, SMPA is useful for preparative-scale resolutions of enantiomers since the reagent is cleaved from the diastereoisomeric derivative by acid hydrolysis.     

Enantioselective chemoenzymatic synthesis of (R)-γ-valerolactone from levulinic acid

A number of chemicals with high industrial value can be synthesized from levulinic acid, a feasible building block readily available from cellulosic biomass. Among them, γ-valerolactone is a versatile chemical precursor for the synthesis of value-added products including bio-active molecules, bio-fuels, and carbon-based chemicals. In this study, a novel two-step chemoenzymatic conversion of levulinic acid to (R)-γ-valerolactone via 4-hydroxyvaleric acid was investigated. For that purpose, an engineered 3-hydroxybutyrate dehydrogenase (e3HBDH) with improved catalytic activity toward levulinic acid was employed in the first-step reaction, and dehydration with 1 % (v/v) sulfuric acid was applied for the lactonization of 4-hydroxyvaleric acid to γ-valerolactone in the second step. As a result, enantiomerically pure (R)-γ-valerolactone (>99 % ee) was successfully produced from the free acid form of levulinic acid with the maximum yield of approximately 100 %.     

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

Stereoselectivity in the Salt–Cocrystal Products formed by Phenylglycinol or Phenylglycine with their Respective Sodium or Hydrochloride Salts

The salt and stereoselective cocrystal phenomena associated with 2‐phenylglycinol and 2‐phenylglycine have been studied using X‐ray powder diffraction and differential scanning calorimetry. The chiral identities of the free acids and their sodium salts, or the free bases and their chloride salts, were found to play a determining role as to whether a salt–cocrystal product could or could not be formed. In particular, when cocrystallization of an enantiomerically pure basic or zwitterionic substance with its enantiomerically pure acid addition salt was attempted, a salt–cocrystal was only obtained when the absolute configuration of the two reactants is opposite. On the other hand, it has been found that no stereoselectivity in salt–cocrystal formation existed in the cocrystallization of an enantiomerically pure acidic or zwitterionic substance with its enantiomerically pure base addition salt. Chirality 25:8–15, 2013. © 2012 Wiley Periodicals, Inc.     

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.     

Stereospecific cleavage of carbon-phosphorus bonds: stereochemical course of the phosphinoyl curtius (Harger) reaction

The homochiral phosphinic azides (R,R)-1 and (S,S)-1 were prepared in enantiomerically pure form by resolution of diastereomeric phosphinamides derived from (S)-l-phenylethylamine and (R)-phenylglycine. Irradiation of the azides in methanol induced a photo-Curtius rearrangement to phosphonamidates in which the stereogenic carbon unit migrated to a nitrogen atom. Hydrolysis of the phosphonamidates produced 1-phenylethylamine, which was 99.0% e.e. and of the same configuration as the carbon unit in the starting azide (99.0% retention).     

An Enantiospecific Polyene Cyclization Initiated by an Enantiomerically Pure Bromonium Ion

Dimethylaluminum triflate‐mediated activation of tetrafluorobenzoates of enantiomerically pure bromohydrins results in enantiospecific polyene cyclizations. The initiation of cyclization by enantiomerically pure bromonium ions and subsequent propagation is not subject to catastrophic erosion of enantiomeric purity by any intramolecular or intermolecular bromonium ion‐to‐alkene transfer. Chirality 25:692–700, 2013. © 2013 Wiley Periodicals, Inc.     

Solubility phase diagram of praziquantel enantiomeric system

The solubility behaviour of praziquantel enantiomeric system at ?15°C, 4°C, and 15°C is presented. This system exhibits both a pseudoracemate behaviour and one which leads to the formation of enantiomerically pure crystals. For solutions with lower enantiomeric purity, the pseudoracemate behaviour is more prominent. Solid solutions are formed when the mixtures of the enantiomers are crystallised. The phase diagram shows a complete absence of a eutetic. For mixtures with higher enantiomeric purity, pure crystals of the excess enantiomer are obtained when the solution is crystallised. Temperature dependency of this polymorphism is not significant. With these phase diagrams, a method is suggested to obtain enantiomerically pure isomers of praziquantel from a partially resolved mixture. © 1995 Wiley-Liss, Inc.