Chemoenzymatic dynamic kinetic resolution

During the past decade a new concept has appeared in asymmetric catalysis involving the combination of a biocatalyst and a chemocatalyst in one 'pot' leading to efficient deracemization via dynamic kinetic resolution (DKR). Here, we outline the different strategies that have been developed for efficient chemoenzymatic DKR, in particular the powerful combination of an enzyme and a metal catalyst.     

Synthesis of 4-phenylpyrrolidin-2-one via dynamic kinetic resolution catalyzed by ω-transaminases

Enantiomerically enriched 4-phenylpyrrolidin-2-one was prepared within only three steps starting from a commercial compound employing dynamic kinetic resolution (DKR) as the key asymmetric step. To the best of our knowledge, for the first time a DKR was performed involving an enzymatic enantioselective amination reaction catalyzed by ω-transaminases. Careful optimization of co-solvent and pH conditions allowed enhancing the enantioselectivity. The general method allows access to 4-arylpyrrolidin-2-ones derivatives, the cyclic analogues of γ-aminobutyric acid (GABA) derivatives.     

Enzyme‐assisted physicochemical enantioseparation processes—Part III: Overcoming yield limitations by dynamic kinetic resolution of asparagine via preferential crystallization and enzymatic racemization

The application of enantioseparation methods alone can only yield up to 50% of the desired chiral product. Thus enantioseparation becomes more attractive when accompanied by the racemization of the counter‐enantiomer. Here we present first results of dynamic kinetic resolution of L ‐asparagine (L ‐Asn) via preferential crystallization and enzymatic racemization from a racemic, supersaturated solution on a 20 mL scale. An enzyme lyophilisate (WT amino acid racemase from P. putida KT2440 (E.C. 5.1.1.10), overexpressed in E. coli BL21(DE3)) was used for in situ racemization (enzyme concentrations varying from 0 to 1 mg/mL). When preferential crystallization was applied without any enzyme, a total of 31 mg of L ‐Asn monohydrate could be crystallized, before crystal formation of d ‐Asn started. Crystallization experiments accompanied by enzymatic racemization led to a significant increase of crystallized L ‐Asn (198 mg L ‐Asn monohydrate; >92%ee) giving the first experimental proof for this new process concept of dynamic kinetic resolution via preferential crystallization and enzymatic racemization. Measurements of the racemase activity before and after the crystallization process showed no significant differences, which would allow for enzyme recovery and recycling. Biotechnol. Bioeng. 2009; 104: 1235–1239. © 2009 Wiley Periodicals, Inc.     

Dynamic kinetic resolution catalyzed by enzymes and metals

The development of new strategies to efficiently synthesize chiral compounds is of extreme importance. Dynamic kinetic resolution is a powerful tool to transform a racemic mixture into one enantiomer. This strategy overcomes the limitation of the maximum 50% yield in a kinetic resolution by combining it with an in situ racemization of the substrate. Recently, the coupling of enzymes and transition metals for dynamic kinetic resolution of a variety of molecules has attracted considerable attention and a deeper understanding of the compatibility of these two catalysts has been achieved.     

Integration of reactive membrane extraction with lipase-hydrolysis dynamic kinetic resolution of naproxen 2,2,2-trifluoroethyl thioester in isooctane

Lipases immobilized on polypropylene powders have been used as the biocatalyst in the enantioselective hydrolysis of (S)-naproxen from racemic naproxen thioesters in isooctane, in which trioctylamine was added to perform in situ racemization of the remaining (R)-thioester substrate. A detailed study of the kinetics for hydrolysis and racemization indicates that increasing the trioctylamine concentration can activate and stabilize the lipase as well as enhance the racemization and non-stereoselective hydrolysis of the thioester. Effects of the aqueous pH value and trioctylamine concentration on (S)-naproxen dissociation and partitioning in the aqueous phase as well as the transportation in a hollow fiber membrane were further investigated. Good agreements between the experimental data and theoretical results were obtained when the dynamic kinetic resolution process was integrated with a hollow fiber membrane to reactively extract the desired (S)-naproxen out of the reaction medium.     

Chemoenzymatic and microbial dynamic kinetic resolutions

This review tracks a decade of dynamic kinetic resolution developments with a biocatalytic inclination using enzymatic/microbial means for the resolution part followed by the racemization reactions either by means of enzymatic or chemocatalyst. These fast developments are due to the ability of the biocatalysts to significantly reduce the number of synthetic steps which are common for conventional synthesis. Future developments in novel reactions and products of dynamic kinetic resolutions should consider factors that are needed to be extracted at the early synthetic stage to avoid inhibition at scale-up stage have been highlighted.     

Dynamic kinetic resolution: alternative approach in optimizing S-ibuprofen production

A lipase catalysed enantioselective hydrolysis process under in situ racemization of the remaining (R)-ibuprofen ester substrate with sodium hydroxide as the catalyst was developed for the production of S-ibuprofen from (R,S)-ibuprofen ester in isooctane. Detailed investigations on parameters study indicated that 0.5 M NaOH, addition of 20% (v/v) co-solvent (dimethyl sulphoxide), operating temperature of 45 °C, and 40 mmol/L substrate gave 86% conversion and 99.4% optical purity of S-ibuprofen in dynamic kinetic resolution. Meanwhile, in common enzymatic kinetic resolution process, only 42% conversion of the racemate and 93% enantiomeric excess of the product was obtained which are of lower values as compared to dynamic kinetic resolution. The S-ibuprofen produced during each process was evaluated and approximately 50% increment in concentration of S-acid product was produced when dynamic kinetic resolution was applied into the process.     

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.     

Preparation of chiral 4-benzyloxymethyldihydrofuran-2-one using lipase-catalyzed kinetic resolution: synthesis of (-)-virginiae butanolide C (VB C)

Lipase-catalyzed kinetic resolution of the N,N-dialkyl-3-benzyloxymethyl-4-hydroxybutanamide 10a,b afforded the acetate 11a,b with (R) configuration, whereas the N-monoalkyl-3-benzyloxymethyl-4-hydroxybutanamide 10c-e gave the acetate 11c-e with (S) configuration. The butanamide 10 smoothly cyclized to give chiral 4-benzyloxymethyldihydrofuran-2-one 9 without racemization, which was effectively transformed into highly stereocontrolled virginiae butanolide C (VB C).     

How do reaction conditions affect the enantiopure synthesis of 2‐substituted‐1,4‐benzodioxane derivatives?

Several biologically active compounds structurally include the enantiopure 2‐substituted‐1,4‐benzodioxane scaffold. The straightforward racemization that affects reactions involving most of the common chemical reactives is thus a crucial issue. The developing of a completely stereo‐controlled synthetic route that does not affect the enantiomeric excess is consequently mandatory. It is also important to set up a reliable chiral HPLC method, able to follow the reaction, and to improve the synthetic performances. Here, we report the chiral investigation of two different synthons, we specifically evaluated the synthetic pathways that could be run in order to afford them, avoiding the racemization processes, which could normally occur in basic conditions. In addition, we developed peculiar chiral HPLC methods in order to resolve the enantiomers, define the enantiomeric excess, and fully characterize these compounds.     

Axial chirality in biaryl N,N-dialkylaminopyridine derivatives bearing an internal carboxy group

Axial chirality in N,N-dimethylaminopyridines as well as N,N-dipropylaminopyridines bearing an internal carboxy group were evaluated based on their racemization barriers and circular dichroism spectra. The half-life of racemization of N,N-dipropylaminopyridine derivative 2 was estimated to be 19.7 days at 20°C. Its enantiomers isolated as optically active forms showed positive-negative and negative-positive Cotton effects for (+)- 2 and (−)- 2 , respectively, from 310 to 210 nm. Furthermore, (−)- 2 was applied as a chiral nucleophilic catalyst and exhibited asymmetric induction in acylative kinetic resolution of 1-(1-naphthyl)ethane-1-ol.     

Process modeling of the lipase-catalyzed dynamic kinetic resolution of (<Emphasis Type="Italic">R</Emphasis>, <Emphasis Type="Italic">S</Emphasis>)-suprofen 2,2,2-trifluoroethyl thioester in a hollow-fiber membrane

A Candida rugosa lipase immobilized on polypropylene powder was employed as the biocatalyst for the enantioselective hydrolysis of (R, S)-suprofen 2,2,2-trifluorothioester in cyclohexane, in which trioctylamine was added as the catalyst to perform in situ racemization of the remaining (R)-thioester. A hollow-fiber membrane was also integrated with the dynamic kinetic resolution process in order to continuously extract the desired (S)-suprofen into an aqueous solution containing NaOH. A kinetic model for the whole process (operating in batch and feed-batch modes) was developed, in which enzymatic hydrolysis and deactivation, lipase activation, racemization and non-enantioselective hydrolysis of the substrate by trioctylamine, and reactive extraction of (R)- and (S)-suprofen into the aqueous phase in the membrane were considered. Theoretical predictions from the model for the time-course variations of substrate and product concentrations in each phase were compared with experimental data.     

Dynamic kinetic resolutions and asymmetric transformations by enzymes coupled with metal catalysis

The combination of enzyme and metal catalysis is described as a useful method for the synthesis of optically active compounds. A key feature of this new methodology is the use of metal catalysts for the in situ racemization of enzymatically unreactive enantiomers in the enzymatic resolution of racemic substrates. So far, two combinations - lipase-ruthenium and lipase-palladium - have been developed for the efficient dynamic kinetic resolution of alcohols and amines. The use of these combinations has also been extended to catalysis of the asymmetric transformation of ketones, their enol acetates, and ketoximes. In most cases, enzyme-metal combination catalysis has provided good yields and high optical purities.     

Asymmetric synthesis of β,γ-unsaturated α-amino acids via efficient kinetic resolution with cinchona alkaloids

The β,γ-unsaturated amino acids are versatile chiral building blocks and biologically interesting compounds. The asymmetric synthesis of β,γ-unsaturated amino acids presents a challenging task as these compounds are labile toward racemization as well as the undesirable double bond isomerization. An efficient, general and mild kinetic resolution with readily accessible and fully recyclable cinchona alkaloid catalysts has been developed to provide a reliably useful approach toward optically active β,γ-unsaturated amino acids.     

Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives

1,4-Dihydropyridines possess a broad range of biological activities, such as the ability to control the influx of calcium into cells, as well as neuroprotective, antineurodegenerative, cognition and memory enhancing, anti-inflammatory, antiviral and many other properties. Chirality plays an important role in the biological activity of 1,4-dihydropyridines. The chemoenzymatic synthesis of 1,4-dihydropyridine derivatives in enantiopure form as the key intermediates for the synthesis of enantiopure drugs and chiral analogues of symmetrical drugs has become an advantageous alternative to the other synthetic methods. Hydrolytic enzymes, as efficient chemo-, regio- and stereoselective biocatalysts have been successfully applied for the asymmetrisation or kinetic resolution of various 1,4-dihydropyridine derivatives. Several synthetic strategies to overcome the inactivity of hydrolytic enzymes towards 1,4-dihydropyridine carboxylic acids have been developed during the last decade, often based on the introduction of a spacer between an enzymatically labile group and the 1,4-DHP nucleus. Good to excellent enantioselectivities can be obtained by careful optimisation of the reaction temperature and the organic (co)solvent used in the enzymatic transformations.     

Racemization of undesired enantiomers: Immobilization of mandelate racemase and application in a fixed bed reactor

Production of optically pure products can be based on simple unselective synthesis of racemic mixtures combined with a subsequent separation of the enantiomers; however, this approach suffers from a 50% yield limitation which can be overcome by racemization of the undesired enantiomer and recycling. Application of biocatalyst for the racemization steps offers an attractive option for high‐yield manufacturing of commercially valuable compounds. Our work focuses on exploiting the potential of racemization with immobilized mandelate racemase. Immobilization of crude mandelate racemase via covalent attachment was optimized for two supports: Eupergit^® CM and CNBr‐activated Sepharose 4 Fast Flow. To allow coupling of enzymatic reaction with enantioselective chromatography, a mobile phase composition compatible with both processes was used in enzymatic reactor. Kinetic parameters obtained analyzing experiments carried out in a batch reactor could be successfully used to predict fixed‐bed reactor performance. The applicability of the immobilized enzyme and the determined kinetic parameters were validated in transient experiments recording responses to pulse injections of R‐mandelic acid. The approach investigated can be used for futher design and optimization of high yield combined resolution processes. The characterized fixed‐bed enzymatic reactor can be integrated e.g. with chromatographic single‐ or multicolumn steps in various configurations.     

Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives

1,4-Dihydropyridines possess a broad range of biological activities, such as the ability to control the influx of calcium into cells, as well as neuroprotective, antineurodegenerative, cognition and memory enhancing, anti-inflammatory, antiviral and many other properties. Chirality plays an important role in the biological activity of 1,4-dihydropyridines. The chemoenzymatic synthesis of 1,4-dihydropyridine derivatives in enantiopure form as the key intermediates for the synthesis of enantiopure drugs and chiral analogues of symmetrical drugs has become an advantageous alternative to the other synthetic methods. Hydrolytic enzymes, as efficient chemo-, regio- and stereoselective biocatalysts have been successfully applied for the asymmetrisation or kinetic resolution of various 1,4-dihydropyridine derivatives. Several synthetic strategies to overcome the inactivity of hydrolytic enzymes towards 1,4-dihydropyridine carboxylic acids have been developed during the last decade, often based on the introduction of a spacer between an enzymatically labile group and the 1,4-DHP nucleus. Good to excellent enantioselectivities can be obtained by careful optimisation of the reaction temperature and the organic (co)solvent used in the enzymatic transformations.     

Highly efficient one pot dynamic kinetic resolution of benzoins with entrapped Pseudomonas stutzeri lipase

The immobilisation of lipase from Pseudomonas stutzeri (Lipase TL^®) by different entrapment protocols (sol–gel, static emulsion-silicone and direct entrapment in silicone spheres) is described for the first time. As this not very common commercial lipase has been recently reported as able to catalyse the dynamic kinetic resolution of benzoins (1,2-diaryl-2-hydroxyethanone structures) combined with a transition metal catalyst, although suffering a deactivation at high temperatures, the different immobilisation methodologies were tested with the aim of enhance lipase activity and stability in the above mentioned process. The enzyme immobilisation by silicone spheres entrapment was the most appropriate method, resulting in a considerable activation of this lipase. Furthermore, the high stability of this immobilised lipase at 60 °C, allowed the development of a “one pot” benzoin DKR process, reaching high conversions in short time, with a 30-fold increase of the productivity of the process due to the possibility of recycling and reuse of the catalyst.     

Geochemical evolution of amino acids in dentine of Pleistocene bears

A linear correlation was established between aspartic acid racemization ratio from cave bear dentine collagen and absolute dating. The high correlation coefficient obtained allowed age calculation through amino acid racemization. Aspartic acid and glutamic acid racemization kinetics have also been explored in dentine from a North American black bear (Ursus americanus Pallas). Three sample sets were prepared for kinetic heating experiments in nitrogen atmosphere: one water soaked, one with a water-saturated nitrogen atmosphere, and one without any moisture. It was possible to show that the presence of water is a factor controlling amino acid racemization rate. The aspartic acid in a heating experiment at 105 degrees C shows an "apparent kinetics reversal" which can be explained by a progressive hydrolysis of amino acid chains (proteins and polypeptides). Because of the low potential of collagen preservation over long periods of time, the apparent kinetics reversal phenomenon will not affect the dating of old material where no traces of collagen remain. An apparent kinetics reversal was not observed in glutamic acid, which racemizates more slowly.     

Racemization of amino acid esters by aromatic aldehydes in basic non-aqueous solvents

Summary Aromatic aldehydes catalyze the racemization of amino acid esters dissolved in organic solvents. The racemization rate depends on the kind of aldehyde and is sensitive to general base catalysis. Active aldehydes bound to an insoluble support racemize amino acid esters in a heterogeneous phase system. Several aspects of the mechanism, together with possible applications of this reaction to the resolution of racemic mixtures of amino acids, are discussed.