Enzymatic Kinetic Resolution Research Articles

Evaluation of gem-Diacetates as Alternative Reagents for Enzymatic Regio- and Stereoselective Acylation of Alcohols

Geminal diacetates have been used as sustainable acyl donors for enzymatic acylation of chiral and nonchiral alcohols. Especially, it was revealed that geminal diacetates showed higher reactivity than vinyl acetate for hydrolases that are sensitive to acetaldehyde. Under optimized conditions for enzymatic acylation, several synthetically relevant saturated and unsaturated acetates of various primary alcohols were obtained in very high yields up to 98% without E/Z isomerization of the double bond. Subsequently, the acyl donor was recreated from the resulting aldehyde and reused constantly in acylation. Therefore, the developed process is characterized by high atomic efficiency. Moreover, it was shown that acylation using geminal diacetates resulted in remarkable regioselectivity by discriminating among the primary and secondary hydroxyl groups in 1-phenyl-1,3-propanediol providing exclusively 3-acetoxy-1-phenyl-propan-1-ol in good yield. Further, enzymatic kinetic resolution (EKR) and chemoenzymatic dynamic kinetic resolution (DKR) protocols were developed using geminal diacetate as an acylating agent, resulting in chiral acetates in high yields up to 94% with enantiomeric excesses exceeding 99%.

Engineering of a borneol dehydrogenase from P. putida for the enzymatic resolution of camphor

Several thousand different terpenoid structures are known so far, and many of them are interesting for applications as pharmaceuticals, flavors, fragrances, biofuels, insecticides, or fine chemical intermediates. One prominent example is camphor, which has been utilized since ancient times in medical applications. Especially (−)-camphor is gaining more and more interest for pharmaceutical applications. Hence, a commercial reliable source is needed. The natural sources for (−)-camphor are limited, and the oxidation of precious (−)-borneol would be too costly. Hence, synthesis of (−)-camphor from renewable alpha-pinene would be an inexpensive alternative. As the currently used route for the conversion of alpha-pinene to camphor produces a mixture of both enantiomers, preferably catalytic methods for the separation of this racemate are demanded to yield enantiopure camphor. Enzymatic kinetic resolution is a sustainable way to solve this challenge but requires suitable enzymes. In this study, the first borneol dehydrogenase from Pseudomonas sp. ATCC 17453, capable of catalyzing the stereoselective reduction of camphor, was examined. By using a targeted enzyme engineering approach, enantioselective enzyme variants were created with E-values > 100. The best variant was used for the enzymatic kinetic resolution of camphor racemate, yielding 79% of (−)-camphor with an ee of > 99%.Key points• Characterization of a novel borneol dehydrogenase (BDH) from P. putida.• Development of enantioselective BDH variants for the reduction of camphor.• Enzymatic kinetic resolution of camphor with borneol dehydrogenase.Graphical abstract

Synthesis, Absolute Configurations, and Biological Activities of Floral Scent Compounds from Night-Blooming Araceae.

The uncommon jasmone derivatives dehydrojasmone, isojasmol, and isojasmyl acetate, floral scent compounds from night-blooming Araceae, were synthesized in a scalable synthesis employing conjugate addition with a selenoacetal as the key step. The stereoselective strategy with subsequent enzymatic kinetic resolution allowed determining the absolute configuration of the natural compounds by GC on a chiral phase. The homoterpene (E)-4,8-dimethyl-1,3,7-nonatrien-5-yl acetate, another uncommon scent compound, was obtained by α-regioselective aldehyde prenylation. The biological activities of dehydrojasmone and isojasmol were investigated in field assays, showing that these unique volatiles are able to selectively attract specific cyclocephaline scarab beetle pollinators.

Chemoenzymatic enantioselective and stereo-convergent syntheses of lisofylline enantiomers via lipase-catalyzed kinetic resolution and optical inversion approach

• The first successful lipase-catalyzed chemoenzymatic synthesis of enantiomerically pure lisofylline has been elaborated. • Optimal biocatalytic system consisted of Candida antarctica lipase B suspended in a vinyl acetate-ethyl acetate mixture. • Stereoinversion of ( S )-lisofylline was obtained via acetolysis of ( S )-mesylate using AcOCs and hydrolysis of the acetate. • XRD analyses were performed for both lisofylline enantiomers. Highly enantioselective enzymatic kinetic resolution (EKR) of racemic lisofylline is presented for the first time. A comprehensive optimization of the key parameters of lipase-catalyzed transesterification of racemic lisofylline revealed that optimal biocatalytic system consisted of immobilized lipase type B from Candida antarctica (Chirazyme L-2, C-3) suspended in a mixture of 3 equiv of vinyl acetate as an acetyl donor and ethyl acetate as a solvent. Under optimal reaction conditions, the 1 g-scale (Chirazyme L-2, C-3)-catalyzed kinetic resolution of racemic lisofylline furnished both the EKR products in a homochiral form (>99 % ee) with the 50 % conv., and the highest possible enantioselectivity. The best results in terms of the reaction yields (47–50 %) and enantiomeric purity of the kinetically-resolved optically active products were achieved when the preparative-scale EKR was carried out for 2 h at 60 °C. In addition, stereoinversion of the less biologically-relevant ( S )-lisofylline into its ( R )-enantiomer was successfully achieved via acetolysis of the respective optically pure ( S )-mesylate by using 2 equiv of ceasium acetate and catalytic amount of 18-Crown-6 in dry toluene, followed by K 2 CO 3 -mediated methanolysis of ( R )-acetate. The elaborated EKR methodology together with enantioconvergent strategy provided a useful chemoenzymatic protocol for the synthesis of complementary enantiomers of titled API. Moreover, we report on the first single-crystal X-ray diffraction (XRD) analyses performed for the synthesized lisofylline enantiomers. Insight into the source of CAL-B stereoselectivity toward racemic lisofylline was gained by molecular docking experiments. In silico theoretical predictions matched very well with experimental results.

Enantioselective extraction of unprotected amino acids coupled with racemization

Scalable and economical methods for the production of optically pure amino acids, both natural and unnatural, are essential for their use as synthetic building blocks. Currently, enzymatic dynamic kinetic resolution (DKR) underpins some of the most effective processes. Here we report the development of enantioselective extraction coupled with racemization (EECR) for the chirality conversion of underivatized amino acids. In this process, the catalytic racemization of amino acids in a basic aqueous solution is coupled with the selective extraction of one enantiomer into an organic layer. Back-extraction from the organic layer to an acidic aqueous solution then completes the deracemization of the amino acid. The automation of the EECR process in a recycling flow reactor is also demonstrated. Continuous EECR is made possible by the sterically hindered chiral ketone extractant 5, which prevents the coextraction of the copper racemization catalyst because of its nonplanar geometry. Furthermore, the extractant 5 unexpectedly forms imines with amino acids faster and with greater enantioselectivity than less bulky derivatives, even though 5 cannot participate in intramolecular resonance-assisted hydrogen bonding. These features may allow EECR to challenge the preponderance of enzymatic DKR in the production of enantiomerically enriched amino acids.

The sustainable synthesis of levetiracetam by an enzymatic dynamic kinetic resolution and an ex-cell anodic oxidation

Levetiracetam is an active pharmaceutical ingredient widely used to treat epilepsy.

Overview: Enzyme-catalyzed Enantioselective Biotransformation of Chiral Active Compounds Used in Hypertension Treatment

Enzymatic kinetic resolution is one of the methods which allows for the synthesis of enantiomerically pure various active pharmaceutical ingredients. In contrast to chemical routes, enzymatic reactions have characteristics, including mild reaction conditions, a few byproducts, and relatively high activity of the used enzymes. β-adrenolytic drugs are widely used in the treatment of hypertension and cardiovascular disorders. Due to the fact that β- blockers possess an asymmetric carbon atom in their structure, they are presented in two enantiomeric forms. It was reported by many studies that only the (S)-enantiomers of these drugs possess the desired therapeutic effect, whereas the administration of the racemate may cause dangerous side effects, such as bronchoconstriction or diabetes. Nevertheless, β- blockers are still commercially available drugs mainly used in medicine as racemates, whereas there are several methods that are widely used in order to obtain enantiomerically pure compounds.

Enzymatic Kinetic Resolution by Addition of Oxygen.

Kinetic resolution using biocatalysis has proven to be an excellent complementary technique to traditional asymmetric catalysis for the production of enantioenriched compounds. Resolution using oxidative enzymes produces valuable oxygenated structures for use in synthetic route development. This Minireview focuses on enzymes which catalyse the insertion of an oxygen atom into the substrate and, in so doing, can achieve oxidative kinetic resolution. The Baeyer–Villiger rearrangement, epoxidation, and hydroxylation are included, and biological advancements in enzyme development, and applications of these key enantioenriched intermediates in natural product synthesis are discussed.

Enzymatic Kinetic Resolution by Addition of Oxygen

AbstractKinetic resolution using biocatalysis has proven to be an excellent complementary technique to traditional asymmetric catalysis for the production of enantioenriched compounds. Resolution using oxidative enzymes produces valuable oxygenated structures for use in synthetic route development. This Minireview focuses on enzymes which catalyse the insertion of an oxygen atom into the substrate and, in so doing, can achieve oxidative kinetic resolution. The Baeyer–Villiger rearrangement, epoxidation, and hydroxylation are included, and biological advancements in enzyme development, and applications of these key enantioenriched intermediates in natural product synthesis are discussed.

The production of enantiomerically pure 1-phenylethanol by enzymatic kinetic resolution method using response surface methodology.

As the enantiomers of 1-phenylethanol are valuable intermediates in several industries, the lipase catalyzed kinetic resolution of (R,S) -1-phenylethanol is a relevant research topic. In this study, the goal was to determine the optimum reaction parameters to produce enantiomerically pure 1-phenylethanol by lipase (Novozyme 435) catalyzed kinetic resolution using response surface methodology (RSM). Reactions were performed with 40–400 mM (R,S)-1-phenylethanol, 120–1200 mM vinyl acetate and 2–22 mg/mL biocatalyst concentrations (BC L ), at 20–60 °C and with a stirring rate of 50–400 rpm for 5–120 min. The samples were analyzed using high performance liquid chromatography (HPLC) with a Chiralcel OB column. Optimum reaction parameters to reach 100% enantiomeric excess for the substrate ( ee s ) were determined as follows: substrate concentration (C s ): 240 mM, BC L : 11 mg/mL, at 42 °C with a reaction time of 75 min. Model validation was performed using these conditions and ee s was calculated as 100%, which indicates the predicted model was efficient and accurate. When compared to the literature, it was observed that the reaction time decreased significantly. This is an important result considering the industrial scale perspective.

Immobilization of Candida antarctica Lipase on Nanomaterials and Investigation of the Enzyme Activity and Enantioselectivity.

This study defines the lipase immobilization protocol and enzymatic kinetic resolution of 1-phenyl ethanol with the use of immobilized lipases (LI) as a biocatalyst. Commercially available lipase Candida antarctica B (Cal-B) was immobilized onto graphene oxide (GO), iron oxide (Fe3O4) nanoparticles, and graphene oxide/iron oxide (GO/Fe3O4) nanocomposites. Characterization of pure and enzyme-loaded supports was carried out by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The influences of pH, temperature, immobilization time, crosslinker concentration, glutaraldehyde (GLA), epichlorohydrin (EPH), and surfactant concentrations (Tween 80 and Triton X-100) on the catalytic activity were evaluated for these three immobilized biocatalysts. The highest immobilized enzyme activities were 15.03U/mg, 14.72U/mg, and 13.56U/mg for GO-GLA-CalB, Fe3O4-GLA-CalB, and GO/Fe3O4-GLA-CalB, respectively. Moreover, enantioselectivity and reusability of these immobilized lipases were compared for the kinetic resolution of 1-phenyl ethanol, using toluene as organic solvent and vinyl acetate as acyl donor. The highest values of enantiomeric excess (ees = 99%), enantioselectivity (E = 507.74), and conversion (c = 50.73%) were obtained by using lipase immobilized onto graphene oxide (GO-GLA-CalB). It was obtained that this enzymatic process may be repeated five times without important loss of enantioselectivity.

Isolation of ibuprofen enantiomers and racemic esters through electrodialysis

The electrodialysis of pure rac-ibuprofen and the products of the enzymatic kinetic resolution of the profen were investigated in order to establish the best conditions for the separation of the ibuprofen from the ethyl esters. The enzymatic esterification of rac-ibuprofen with ethanol (used as solvent and acyl-aceptor) was catalyzed with Novozym®435 at the optimum conditions investigated previously. The reaction product mixtures possess about 60% of ibuprofen (enriched in the S-enantiomer) and 40% of the racemic esters.An electrodialysis cell composed by four compartments separated by cation-, anion- and cation-exchange membranes was developed allowing the selective migration of ibuprofen avoiding its decomposition on the electrodes. In fact, the stainless steel electrodes submerged in ammonium formate solutions were separated from the compartments containing the profen (~200 ppm in ethanol, pH 6.81–6.34) with cation-exchange membranes. The best separation performance was reached after 6 h of operation at 60 V with an anion-exchange membrane that led up to 57% extraction of ibuprofen from the products of the enzymatic esterification in ethanol media. The membrane allowed the selective migration of both stereoisomers and a negligible quantity of the ethyl esters.

Enzymatic kinetic resolution of desmethylphosphinothricin indicates that phosphinic group is a bioisostere of carboxyl group

Escherichia coli glutamate decarboxylase (EcGadB), a pyridoxal 5’-phosphate (PLP)-dependent enzyme, is highly specific for L-glutamate and was demonstrated to be effectively immobilised for the production of γ-aminobutyric acid (GABA), its decarboxylation product. Herein we show that EcGadB quantitatively decarboxylates the L-isomer of D,L-2-amino-4-(hydroxyphosphinyl)butyric acid (D,L-Glu-γ-PH), a phosphinic analogue of glutamate containing C-P-H bonds. This yields 3-aminopropylphosphinic acid (GABA-PH), a known GABAB receptor agonist and provides previously unknown D-Glu-γ-PH, allowing us to demonstrate that L-Glu-γ-PH, but not D-Glu-γ-PH, is responsible for D,L-Glu-γ-PH antibacterial activity. Furthermore, using GABase, a preparation of GABA-transaminase and succinic semialdehyde dehydrogenase, we show that GABA-PH is converted to 3-(hydroxyphosphinyl)propionic acid (Succinate-PH). Hence, PLP-dependent and NADP+-dependent enzymes are herein shown to recognise and metabolise phosphinic compounds, leaving unaffected the P-H bond. We therefore suggest that the phosphinic group is a bioisostere of the carboxyl group and the metabolic transformations of phosphinic compounds may offer a ground for prodrug design.

Modulation of lipase B from Candida antarctica properties via covalent immobilization on eco-friendly support for enzymatic kinetic resolution of rac-indanyl acetate.

In this study, the modulation of enzymatic biocatalysts were developed by the use of lipase B from Candida antarctica covalently immobilized on an eco-friendly support, cashew apple bagasse, activated with 10% glycidol-ethylenediamine-glutaraldehyde (GEG) under different immobilization strategies (5mM or 100mM ionic strength and in absence or presence of 0.5% (v/v) Triton X-100). The biocatalysts were characterized for thermal and organic solvents stabilities and compared with the soluble enzyme. The biocatalysts were then applied to the hydrolysis of the rac-indanyl acetate (2:1 ratio enzyme/substrate) at pH 7.0 and 30°C for 24h. For all the strategies evaluated, GEG promoted kinetic resolution of rac-indanyl acetate with maximum conversion (50%) and led to (R)-indanol with excellent enantiomeric excess (97%), maintaining the maximum conversion for five consecutive cycles of hydrolysis. Therefore, the use of cashew apple bagasse has proved to be a promising eco-friendly support for enzyme immobilization, since it resulted in stable biocatalysts for enzymatic kinetic resolution.

Synthesis of Chiral 1,3‐keto‐acetates through Enzymatic Kinetic Resolution with Amano Lipase from Pseudomonas fluorescens

AbstractThis study provides an enzymatic kinetic resolution (EKR) method for the biocatalytic enantioselective synthesis of chiral β‐keto acetates. For the first time, the attitude of several lipase enzymes has been examined under different conditions for the EKR of the racemic 1,3‐keto alcohols. We found that the Amano lipase from Pseudomonas fluorescens (AL‐PF) lipase showed the best results for the resolution of the racemic 1,3‐keto alcohols. We developed a highly effective EKR method by changing the enzyme, temperature, solvent, and enzyme amount in order to synthesize the chiral 1,3‐keto esters, which are useful intermediates in organic synthesis. This study presents an efficient method for the synthesis of 1,3‐keto acetates with a good yield and high enantioselectivity with the R configuration.

A Sequential Umpolung/Enzymatic Dynamic Kinetic Resolution Strategy for the Synthesis of γ-Lactones.

Combining biological and small-molecule catalysts under a chemoenzymatic manifold presents a series of significant advantages to the synthetic community. We report herein the successful development of a two-step/single flask synthesis of γ-lactones through the merger of Umpolung catalysis with a ketoreductase-catalyzed dynamic kinetic resolution, reduction, and cyclization. This combined approach delivers highly enantio- and diastereoenriched heterocycles and demonstrates the feasibility of integrating NHC catalysis with enzymatic processes.

Lipase mediated enzymatic kinetic resolution of phenylethyl halohydrins acetates: A case of study and rationalization

Racemic phenylethyl halohydrins acetates containing several groups attached to the aromatic ring were resolved via hydrolysis reaction in the presence of lipase B from Candida antarctica (Novozym® 435). In all cases, the kinetic resolution was highly selective (E > 200) leading to the corresponding (S)-β-halohydrin with ee > 99 %. However, the time required for an ideal 50 % conversion ranged from 15 min for 2,4-dichlorophenyl chlorohydrin acetate to 216 h for 2-chlorophenyl bromohydrin acetate. Six chlorohydrins and five bromohydrins were evaluated, the latter being less reactive. For the β-brominated substrates, steric hindrance on the aromatic ring played a crucial role, which was not observed for the β-chlorinated derivatives. To shed light on the different reaction rates, docking studies were carried out with all the substrates using MD simulations. The computational data obtained for the β-brominated substrates, based on the parameters analysed such as NAC (near attack conformation), distance between Ser-O and carbonyl-C and oxyanion site stabilization were in agreement with the experimental results. On the other hand, the data obtained for β-chlorinated substrates suggested that physical aspects such as high hydrophobicity or induced change in the conformation of the enzymatic active site are more relevant aspects when compared to steric hindrance effects.

Efficient and Stable Magnetic Chitosan-Lipase B from Candida Antarctica Bioconjugates in the Enzymatic Kinetic Resolution of Racemic Heteroarylethanols.

Lipase B from Candida antarctica immobilized by covalent binding on sebacoyl-activated chitosan-coated magnetic nanoparticles proved to be an efficient biocatalyst (49.2–50% conversion in 3–16 h and >96% enantiomeric excess) for the enzymatic kinetic resolution of some racemic heteroarylethanols through transesterification with vinyl acetate. Under optimal conditions (vinyl acetate, n-hexane, 45 °C), the biocatalyst remains active after 10 cycles.

An entry to non-racemic β-tertiary-β-amino alcohols, building blocks for the synthesis of aziridine, piperazine, and morpholine scaffolds.

A method for the preparation of enantiopure β-tert-amino alcohols bearing a tetrasubstituted C-stereocenter, as well as their conversion into selected medicinally privileged heterocyclic systems (morpholines, aziridines, piperazines) is reported. These compounds were obtained through enantiospecific sigmatropic rearrangement of allyl carbamates as a key step. The latter were prepared from the corresponding β,β'-dialkyl-substituted non-racemic allyl alcohols. In addition, an asymmetric synthesis of such highly substituted allylic alcohols via either enantioselective 1,2-reduction of enones, enzymatic kinetic resolution, or a functionalization of chiral propargyl alcohols, with discussion of scope and limitations of each method is reported.

Stereoselectivity-tailored chemo-enzymatic synthesis of enantiocomplementary poly (ω-substituted-δ-valerolactone) enabled by engineered lipase

The synthesis of chiral polyesters with customized (R)- or (S)-configurations is of great significance but challenging. Despite many advances in enzymatic stereoselective polymerization, the limitations of inherent stereoselectivity and the narrow substrate scope of the biocatalysts used should be overcome. Herein, we combined chemical polymerization and highly selective enzymatic dynamic kinetic resolution (DKR) with tailor-made (R)- or (S)-selectivity enabled by engineered lipase to offer an efficient chemo-enzymatic route for the synthesis of enantiocomplementary (R)- or (S)-poly(ω-substituted-δ-valerolactone) (ω-PVL). In the DKR of homoallylic alcohols, (R)-selective wild-type Candida antarctic lipase B (CALB) and an (S)-selective CALB mutant (W104V/A281L/A282K) were used, providing enantiocomplementary homoallylic alcohol esters with 99% conversion and up to 99% enantiomeric excess (ee), which were further transformed into enantiopure lactone monomers. Using these monomers, ring-opening polymerizations catalyzed by a diphenyl phosphate system successfully provided (R)- or (S)-ω-PVL with aromatic and aliphatic side chains. Additionally, their structure and thermal properties were characterized by nuclear magnetic resonance (NMR), circular dichroism (CD) spectra, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). (R)- or (S)-ω-PVL exhibited vertically mirrored negative and positive CD signals, and were thermally stable while having different decomposition temperatures (Td), which are highly dependent on their molecular weight and side chain structure. It was concluded that stereoregularity has significant influence on the crystallization of the polyesters.