Lipases in the Enantioselective Biocatalysis Process to Obtain Enantiomerically Pure Racemic Products

Schuenck-Rodrigues RA

Published on: 2022-05-28

Abstract

Obtaining enantiomerically pure substances is of great industrial interest, since enantiomers can present differences in odor, taste, toxicity and, mainly, biological action. From this, several methodologies have been developed for the stereoselective synthesis of organic compounds (asymmetric synthesis), among which biocatalysis using lipases as biocatalysts stands out, as they present a versatility of method and applications. The present study aimed to present the different applications and productions of racemic products and the use of enzymes as biocatalysts by enantioselectivity process to obtain enantiomerically pure compounds. From the study, it was possible to observe that lipases are important biocatalysts in industrial processes and are becoming an important tool for advances in biotechnology. It was possible to conclude that the lipases show promise in the process of purification of racemic products in order to obtain enantiomerically pure substances, as they present selectivity in the presence of specific substrates in order to solve challenging chiral substrate problems.

Keywords

Lipases; Enantioselectivity; Enantiomers; Racemate

Introduction

Most of the industrial enzyme market is occupied by hydrolytic enzymes such as proteases, amylases, amidases, esterases and lipases. In recent times, lipases (EC 3.1.1.3 triacylglycerol acyl hydrolase) have emerged as key enzymes in biotechnology, due to their diverse properties, being used in different industries such as food technology, detergents, chemistry and biomedical sciences [1-3].

Lipases are hydrolases that act under aqueous conditions on the ester bonds present in the carboxyl triacylglycerols to release fatty acids and glycerol. The natural substrates are long-chain triacylglycerol lipases, which have a very low solubility in water and the reaction is catalyzed at the lipid/water interface. Under micro-aqueous conditions, lipases have the unique ability to carry out the reverse reaction, leading to esterification, alcoholysis and acidolysis. In addition to being lipolytic, lipases also possess stereolytic activity and thus possess a very diverse range of substrates, although they are highly specific as chemo, regio and enantioselective [4-6].

In the pharmaceutical industry, stereoselective interactions are extremely important, since one or the other enantiomer (stereoisomer) in a mixture of two (racemic) forms of a chiral drug can have unexpected or unsuccessful beneficial effects. Nowadays, pharmaceutical companies shift their research to the development of drugs with a single pure enantiomer, which in turn has created an immense need for a robust, reliable, high-yield, environmentally benign, and technically economical and viable solution for separation. of chiral compounds allowing their availability on a commercial scale [7-9].

The growing importance of lipases within biotechnological perspectives can be easily predicted by its comprehensive aspects as an extremely versatile biocatalyst, its high selectivity for the production of new pure compounds, which makes it extremely promising, especially regarding the pharmaceutical industry in racemic products, demonstrating its biotechnological applications. In this sense, the present study is based on elucidating the applications and the aquatics of lipases through their enantioselective processes for the production of new enantiomerically pure substances.

Methodology

The qualitative approach study reviewed the literature in question for a better understanding of lipases of microbial origin in the application and production of enantiomerically pure racemic products by stereoselective enzymatic processes. It was decided to carry out a systematic review, defined as an instrument for obtaining, identifying, analyzing and synthesizing the literature directed to the specific topic. It also allows for a broad analysis of the literature, including discussions on methods and results of publications. Articles, monographs, dissertations and books published on the topic were consulted, such as the sites Scielo, Science Direct and Medline.

To identify the study designs, the following terms lipases, enantioselectivity, enantiomers and racemic products were used. The pre-selection of studies was based on reading their title and/or abstract, and, when necessary, the full text. The search was scheduled for the period from January 2022 to May 2022, with data dating from 1989 to 2022 being found. The use of articles was analyzed in consensus, with those that did not present specific data about the research being rejected. Articles based on studies of stereoselectives, racemic mixtures and lipases were obtained. Of the 134 articles analyzed, 87 were excluded from the research because they did not present specific content for carrying out the work and 47 presented essential data for carrying out the bibliographic survey.

The data will be reported following the recommendations of the JBI Manual for Evidence Synthesis and PRISMA for Scoping Reviews (PRISMA ScR) and initially presented through a flow diagram recommended by PRISMA ScR to present the evidence search flow. Then, the tables with the information extracted from the included articles will be presented, taking into account the population, concept and context. From the analysis of the tables, graphs will be plotted to present the correlations obtained in a didactic way. After presenting the data, they will be discussed in depth in order to list future research gaps and the limitations of the studies that will serve as a basis for conducting new research focused on the analysis of this review.

Results And Discussion

After the selection process, 47 studies met the inclusion criteria. The study selection process is shown in a flow diagram (Figure 1), according to the PRISMA standards.

Figure 1: Flowchart with study steps adapted from Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA).

Biocatalysis

Biocatalysis is a process in which a biocatalyst increases the rate of a chemical reaction by reducing the activation energy of the reaction. The biocatalyst itself is not consumed or altered in the process. Biocatalysis covers the areas of catalysis that use whole cell enzymes, enzymes, individual catalytic antibodies, and ribozymes as biocatalysts. According to the types of reactions they catalyze, enzymes are divided into six classes: Oxidoreductases 1., 2., 3. Transferases, hydrolases, lyases, 4. 5. 6. isomerases and ligases [9,10] Among the types of enzymes, lipases that belong to the class of hydrolases, which are enantiopure compounds, are of high value in the pharmaceutical industry. Consequently, enormous efforts are invested in the development of viable methods for their production. Biocatalysis provides powerful tools for the production of enantiomerically pure compounds that are versatile under ambient conditions. Biocatalysts alone or in combination with chemical reaction steps (chemoenzymatic synthesis) are widely explored in the field of research, increasingly also in industrial applications. Several strategies have been used to realize new efficient biocatalytic methods for the preparation of secondary alcohol and primary amine enantiomers. In addition to chemotherapy, regio- and enantioselectivities and the substrate specificity of a biocatalyst, factors include catalyst availability, cost, ease of handling and reusability, as well as substrate and product solubility and ease of product recovery [1,11].

Biocatalysis as a Promising Expansion Tool

Over the last few years, the use of enzymes as biocatalysts for the introduction of enantimerically pure active compounds has become a process of interest to the pharmaceutical industries. One of the main challenges facing organic chemistry is the rational synthesis of an increasing number of complex, optically active natural products and their analogues. The regulation of production of chiral drugs, agrochemicals, fine chemicals was left in enantiomerically pure form, because it often happens that only one of the two enantiomers shows the desired therapeutic effect [9].

The separation of enantiomers has always been considered one of the most difficult problems in chemistry. The use of enzymatic catalysts clearly becomes a very promising alternative. The separation is based on the kinetic resolution of the racemic mixture. The enzyme when enantioselective reacts faster with one enantiomer than with the other. Regarding the duplication of racemic esters, most of the proposed methods are based on the use of lipases, esterases or proteases that catalyze hydrolysis or synthesis reactions. These enzymes are generally used as additives for detergents or in the food industry and are available in large quantities at low cost [12-16].

Therefore, the use of biocatalysts is attractive due to the intelligent insertion of chirality and selectivity with regard to region and stereochemistry in simple molecules and the use in technical production units and the need for rigorous research for further advances.

Lipases

Triacylglycerol lipases (ester hydrolases) are ubiquitous enzymes, which belong to the family of hydrolytic enzymes. These enzymes are able to catalyze the hydrolysis of fats and oils from free fatty acids and glycerol. Since the reaction is reversible, it can also catalyze the formation of acylglycerols from free fatty acids and glycerol. Therefore, lipases are the most commonly used enzymes in synthetic organic chemistry for the hydrolysis of carboxylic acid esters in aqueous solvents or transesterification in organic solvents [6].

Lipases do not require cofactors, can be used free or immobilized, are inexpensive, readily available and accept a wide variety of substrates, maintaining their high chemo-, regio- and stereoselectivity. All these properties make them a useful tool in organic synthesis [16].

Furthermore, lipases function at the water interface of lipids and therefore do not require a water-soluble substrate and can function efficiently in the hydrophobic medium of organic solvents. They also have the ability to catalyze reactions under mild conditions (room temperature, around neutral pH). Lipases have been widely used to catalyze three main types of asymmetric transformation, racemic alcohols and carboxylic acids, enantiotopic group differentiation of meso-diols and mesodicarboxylic acids, and enantiotopic group differentiation of prochiraldicarboxylic acids and diols [16,17].

Lipases in biocatalysis occur according to the so-called “ping-pong” mechanism, whereby two substrates (RCON u1 and Nu2H) react to give two products (RCON u2 and Nu1H) in such a way that the first product (Nu1H ) is released prior to the binding of the second substrate (Nu2H). At the active site of all lipases, serine, histidine and aspartate or glutamate residues form the so-called catalytic triad that initiates catalysis (Figure 2) [17,18].

The orientation of these amino acid residues lowers the pKa value of the serine hydroxyl group, allowing it to act as a nucleophile which then attacks the acyl donor carbonyl carbon (RCONu2) in the first step of the reaction. The reaction proceeds through a tetrahedral transition state (T1) which reacts further releasing the first product (Nu1H) and constitutes the acyl-intermediate enzyme. In the second reaction step, the nucleophile (Nu2H) attacks the carbonyl carbon of the acyl-enzyme intermediate and the reaction proceeds again through a tetrahedral transition state (T2) in the formation of the second product (RCONu2) and the regeneration of the free enzyme [19].

Figure 2: Mechanism of the lipase-catalyzed reaction.

Source: Adapted from Kazlauskas, Patti [17,18].

Lipases have promising applications in organic chemical processing, detergent formulations, biosurfactant synthesis, the chemical industry, the dairy industry, the agrochemical industry, papermaking, nutrition, cosmetics, pharmaceuticals and processing. The development of lipase-based technologies for the synthesis of new compounds rapidly expands the uses of these enzymes [20].

A limiting factor is the lack of lipases with the specific processing characteristics required. An increasing number of lipases with suitable properties are becoming available and efforts are underway to commercialize biotransformation and lipase-based syntheses [6].

Lipase Applications

Lipases are of great importance in the industry due to their stability in organic solvents, their wide variety of substrates, their selectivity and their ability to catalyze reactions without the addition of expensive cofactors. Furthermore, they are also easily produced and active under ambient conditions. Some of the lipases of microbial origin commercialized in the world are shown in Table 1.

Table 1: Some of the commercially available lipases from microbial origin produced by different companies.

Source	Commercial name	Manufacturer	Industrial Applications
Bacteria
Alcaligenes sp.	Lipase PL	Meito Sangyo, Co	Food processing
Chromobacteriumviscosum	Lipase CV	Genzyme	Health care and diagnosis
Pseudomonas cepacia	Lipase SL	Amano	Food processing
Psudomonasmenodocina	Lumafast	Genencor International	Detergent
Fungi
Aspergillus niger	Lipase DS	Amano	Biopharmaceutical
Aspergillus niger	Lipase	Sigma	Food processing
Rhizopusoryzae	Lipopan^® Fa	Novozyme	Cooking and food processing
	Lipomod^TM 627P-L627P		Cooking and food processing
	Lipomod^TM 36P-L036P		Biopharmaceutical
Rhizomucormiehe	Palatase^®a	Biocatalysts	Dairy
Rhizomucormiehe	Palatase^®a	Novozyme	Dairy
Yeasts
Candida cylindracea	Lipase MY	Meito Sangyo, Co.	Biopharmaceutical
Candida cylindracea	Resinase^®a	Novozyme	Forest products
Candida antarctica	Novozym^® 435a		Food processing
Candida antarctica	Noopazyme^®a		Cereals
Candida cylindracea	Lipomod^TM 29P-L029P	Biocatalysts Ltd	Flavor / Dairy

Therefore, lipases are used in many different industrial areas, such as:

In the food industry: includes the production of dairy products such as cheese, modification of oils and fats, the production of baby foods and structured lipids with unique properties. They are used as emulsifiers in the improvement of baked goods and pasta, as additives in animal feed and also used to modify flavors and produce aroma compounds [21-23].

In the detergent and cleaning agent’s industries they are used as additives, as they are active and stable at high temperatures and an alkaline pH. They are also essential in the production of soap, dishwashing products, dry cleaning solvents and cleaning contact lenses [24,25].

In the pharmaceutical industry for the production of pure enantiomers by resolving racemic mixtures. Chiral molecules are also used as herbicides in the agrochemical industry. In the perfume and cosmetics industry, they are used to produce surfactants and aromas, and as emollients in personal care products [23,26].

In medical applications, they are diagnostic tools, as their presence and level can indicate an infection or disease, and as new drugs for digestive treatment and high cholesterol levels [25].

In the paper industry they are used to remove triglycerides and waxes. In addition, its presence increases whiteness and reduces pollution in wastewater.

In bioremediation and environmental processes, they are used for the treatment of oil-rich wastewater, the degradation of organic remains and sewage treatment from a wide range of industries. They are also used to degrade petroleum hydrocarbons in oil spills [25].

In the energy industry, they are used for the production of lubricants, biodiesel from renewable sources by transesterification of vegetable/animal oils and for the production of additives that decrease the viscosity of biodiesel [26].

Other possible applications include the production of biopolymers such as polyphenols, polysaccharides and polyesters, lipase-mediated lipophilization, the production of biosensors and modification of phenolic acids and antioxidants. Textile industry also uses lipases for enzymatic washing and denim treatment [23].

The Phenomenon of Chiral Molecules

Chirality plays a decisive role in the life processes of animals and plants. In fact, most naturally occurring organic molecules are chiral. They are involved in many biological processes, such as membrane transport, cell recognition phenomena (interaction with a receptor), metabolic reactions, among others. The biological activities of both enantiomers can be very different as shown in Figure 3. For example, they can interact separately with olfactory receptors. Thus, (R)-limonene, used in the perfumery industry, has an orange odor while (S)-limonene has a lemon aroma. It's the same interaction with the taste receptors. (S)-asparagine is naturally occurring in young asparagus that imparts a characteristic bitter taste when the (R)-asparagine enantiomer has a sweet taste. This rather pleasant property in the case of taste or smell, can become problematic even when it comes to drugs [27].

Thalidomide was marketed as a racemic mixture in several countries as prescribed hypnotics for its antiemetic properties. In a pharmacological study in animals it showed that the teratogenic activity was mainly due to the (S) enantiomer, however, in other studies indicate that thalidomide is readily racemized in the blood, which shows that even the therapeutic use of thalidomide (R) was doomed to failure [27,28].

However, this case of thalidomide was an impact on the pharmaceutical industry, with a focus on the risks associated with the use of a racemic drug. Indeed, often only one enantiomer has the desired therapeutic activity, the other is at best inactive and at worst toxic [28,29].

Figure 3: Examples of chiral molecules whose properties depend on their absolute configuration. The position of asymmetric centers is identified by an asterisk (*).

Source: Adapted from Vedovello [27].

Therefore, legislation has progressively imposed strict controls on the use of racemic mixtures. The FDA (Food and Drug Administration) does not require the systematic use of an enantiomer, but does require extensive biological study. This restriction induces most pharmaceutical companies to market the drug as a single enantiomer. The separation of chiral molecules has become a challenge for the pharmaceutical industry, as evidenced by the development of this area of research during the last decade [30-32].

Factors Affecting Lipase Activity and Selectivity

There are a number of factors that affect the activity and selectivity of lipase-catalyzed reactions, including the nature of the acylating agent, temperature, pH, and solvent selection. Since most organic compounds are hydrophobic in nature, it is necessary to carry out biocatalytic transformations in a non-aqueous medium. Furthermore, carrying out biocatalytic transformations in the presence of water leads to secondary reactions such as hydrolysis, decomposition, racemization and polymerization and makes it difficult to remove water after completion of the reaction [25].

This also provides the advantages of better product yield, greater substrate solubility, and ease of enzyme recovery after completion of the reaction. However, the use of enzymes in organic solvents sometimes leads to a decrease in catalytic activity (due to the heterogeneous system), reaction rate and enantioselectivity [33].

Production of Enantiomerically Pure Molecules

The role of stereochemistry in drug development has evolved and its importance in drug action has been fully considered [34]. In the 1970s, the field of pharmaceutical research was dominated by the synthesis of racemates. It was in the late 1980s when the technology for isolating, manufacturing, and detecting pure enantiomers became generally available. Advances in chiral technology and the ability to produce enantiomerically pure compounds have a major impact on drug design, research and development, and the strategies and policies of the pharmaceutical industry. Thus, from 1989 to 2000, the percentage of chiral drugs approved worldwide increased from 25% to over 50% [35,36].

As a consequence of the importance of chirality of chemical compounds and their biological activity, the new chiral-switch concept has to be considered. This term is used to describe chiral drugs that have already been claimed, approved and marketed in the form of racemates or as mixtures of diastereomers, but which have been reconstructed as individual enantiomers. Some important chiral-switch cases are drugs such as omeprazole and ibuprofen (Figure 4).

Figure 4: Chiral-switch concept for ibuprofen (Adapted from Agranat, Caner & Caldwell, [34]).

The enantiomerically pure molecules are obtained mainly chemically or biologically by three methods: (i) from chiral precursors, (ii) by asymmetric synthesis.

Enantiomerically pure natural substances, such as amino acids, alkaloids, terpenes, sugars and derivatives thereof, or carboxylic acids, can be used as precursors for the synthesis of enantiomerically pure derivatives. An example is Taxotere® (Taxol analogue) a drug used to fight cancer. The natural intermediate used is 10-deacetyl-baccatin III [36.37].

Asymmetric Synthesis

The synthesis of enantiomerically pure compounds from an achiral starting material requires the use of a chiral auxiliary (Figure 5). The chiral auxiliary is used in a catalytic amount, or associated with the substrate to create a stereoselective carbon associated with the compound to react with the prochiral carbon substrate. At the end of the synthesis, the chiral auxiliary can be separated from its support and recovered for reuse [38,39].

Figure 5: Synthesis of enantiomerically pure compounds from prochiral and chiral auxiliary reagents.

Source: Adapted from Crosby [38].

However, the stereoselectivity of the reaction is not always easy to predict. Furthermore, the use of stoichiometric amounts of chiral auxiliary can be expensive. This method requires two additional synthesis steps to attach and remove the chiral helper in the event that the latter is attached to the substrate. Examples of asymmetric synthesis developed on an industrial scale include the synthesis of (S)-naproxen (non-steroidal anti-inflammatory drug) [38,39].

In asymmetric syntheses, they can also be catalyzed by enzymes or microorganisms. They are often used in ketone reductions [28,39,40]. For example, the production of an intermediate for the synthesis of Montelukast (Sinclaire®: antiasthmatics) by enantioselective reduction of the corresponding ketone by means of the strain Microbacterium campoquemadoensis can be mentioned (Figure 6).

Figure 6: Synthesis of a Montelukast Synthesis Intermediate [41]..

Duplication of Racemic Mixtures

In addition to the increasingly frequent asymmetric syntheses, the method of doubling the resolution of racemic mixtures is by far the most used in the industry for the preparation of enantiomerically pure compounds. The duplication method provides access to both enantiomers, which is an advantage, for example, in the pharmaceutical industry, where pre-clinical studies are needed for both forms. The techniques used for the separation of racemates are mainly divided into four groups: (i) preferential or selective crystallization, (ii) crystallization of diastereomeric salts, (iii) separation by chromatography and (iv) kinetic resolution [28,39].

Crystallization

This technique consists of the selective crystallization of an enantiomer. In a saturated racemic solution is seeded with an optically active crystal and the nature of the seeds. The other enantiomer then remains in solution. This is an unbalanced process that occurs only in a few racemics called conglomerates. They have the particularity that in forming crystals that contain only one enantiomer socket of easy separation from optical isomers. Approximately 10% of all racemates have this property. The duplication procedure generally requires careful temperature control and the use of extremely pure seed crystals to induce crystallization and obtain good yields [42,43].

Separation by Chromatography

The separation is based on the difference in interaction of the two enantiomers with a homochiral adsorbent. The non-covalent binding between enantiomers and the adsorbent complex forms of diastereomers that have energies of various bonds. The balance between the limit and the free state is therefore distinct for each of the enantiomers. The result is a cleaner migration rate for each of the enantiomers, which allows for chromatographic separation. Although in principle it has been known and understood for some time, it has only recently been the progress of chromatography techniques and optically pure adsorbent preparation to allow the practical realization of such separations. However, the high price of stationary phases remains the main disadvantage of this technique [43,44].

Kinetic Duplication

This is another way of using separation of each enantiomer reaction rate difference with an optically pure reagent. The energies of the transition states of each of the enantiomers are different because both states are diastereoisomers (R and S), hence the difference in speed. Thus, chemical modification of one of the two enantiomers will secure the racemic by conventional separation methods used in organic chemistry. A yield not exceeding 50% is the intrinsic limitation of this technique. To circumvent this problem, dynamic kinetic resolution strategies were developed using simultaneous unconverted substrate racemization reactions. The substrate thus remains a racemic until conversion is complete [44,45].

As in the case of asymmetric synthesis processes, the resolution of racemic mixtures can be enzymatically catalyzed. One of the enantiomers will be converted faster than the other. The properties of the residual substrate and those of the product are then sufficiently different for a conventional separation, for example by chromatography. The three-dimensional structure, there are specific sites called active sites(s). The active site is composed of a small number of amino acids that are generally not contiguous in the sequence of the polypeptide chain. The unique combination of these amino acids is the reason for the stereospecificity of recognition between the active site and substrate(s) [20,46].

They allow for less polluting methods, catalyze reactions under mild application conditions in terms of pH and temperature, and have a high specificity towards chemical catalysts. Indeed, enzymes are chemo-, regio- and stereoselective. The hydrolases that represent 75% of the enzymes used in the industry are the most frequently used in the methods of solving racemic mixtures of enzymes [46,47].

Final Considerations

In conclusion, the present study demonstrates that:

Biocatalysis is an integral part of the industrial process and is becoming a powerful tool in biotechnology, having a major impact on food supply, environmental protection and sustainable fuel production. Enzymes are gaining a prominent place in future scenarios, with different advanced techniques being applied;

Lipases have several applications such as food (flavor modification), fine chemicals (ester synthesis), detergents (fat hydrolysis), fat modification, biodiesel production;

The enzymes of microbial origin, especially lipases, show to be promising biocatalysts in the process of purifying racemic products in order to obtain enantiomerically pure substances, as they present selectivity in the presence of specific substrates;

The availability of this enantioselective process becomes a tool that allows comparing the trajectories of enantiomeric pairs;

Thus, research is being carried out for a wider application of lipase biocatalysis in the production of enantiomerically pure substances by different methods in order to solve challenging chiral substrate problems.

This study will serve as a basis for the elucidation of new research gaps around this theme and will contribute to the knowledge for the production of racemates biocatalyzed by lipases through enantioselectivity processes to obtain enantiomerically pure composts, enabling the elucidation of the art of innovations for alternatives not developed and Remodulation of new medications.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

References

Muller GW. Thalidomide: From tragedy to new drug discovery. CHEMTECH. 1997; 27: 21-25.
Naaman R, Paltiel Y, Waldeck DH. Chiral Induced Spin Selectivity and Its Implications for Biological Functions. Annual Review of Biophysics. 2022; 51: 1.
Patel RN. Biocatalysis: synthesis of chiral intermediates for drugs. Curr Opin Drug Discv Devel. 2006; 9: 741-764.
Stinson SC. Chiral pharmaceuticals. Chem Eng News. 2001; 79: 79-97.
Thayer AM. Centering on Chirality: Chemists are finding asymmetric synthesis increasingly handy for making pharmaceutical compounds at large scale. Chem Eng News. 2007; 85: 11-19.
Solanki K. Gupta MN. A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents. Bioorg Med Chem Lett. 2011; 21: 2934-2936.
Agranat I, Caner H, Caldwell J. Putting chirality to work: the strategy of chiral switches. Nat Rev. 2002; 1: 753-768.
Camp WHD. Biotransformations in Organic Chemistry: A Textbook. Chirality. 1989; 1: 97-98.
Enespa, Chandra P, Singh DP. Sources, purification, immobilization and industrial applications of microbial lipases: An overview. Critical Reviews in Food Science and Nutrition.
Gou DM, Liu YC, Chen CS. A pratical chemoenzymatic synthesis of the Taxol C-13 side chain N-benzoyl-(2R,3S)-3-phenylisoserine. J Org Chem. 1993; 58: 1287-1289.
Crosby J. Chirality in industry. In Chirality in industry: the commercial manufacture and applications of optically active compounds, Collins AN, Sheldrake GN, Crosby J (Eds.) John Wiley & Sons. 1992 Ltd. 2-66.
Hall M. Enzymatic strategies for asymmetric synthesis. 2021; 2: 958-989.
Nakamura K, Yamanaka R, Matsumada T, Harada T. Recent developments in asymmetric reduction of ketones with biocatalysts. Tetrahedron: Asym. 2003; 14: 2659-2681.
Shafiee A, Motamedi H, King A. Purification, characterization and immobilization of an NADPH-dependent enzyme involved in the chiral specific reduction of the keto ester M, an intermediate in the synthesis of an anti-asthma drug, Montelukast, from Microbacterium campoquemadoensis (MB5614). Appl Microbiol Biotechnol. 1999; 49: 709-717.
Wood WML. Crystal science in manufactue of chiral compounds. In Chirality in Industry II: Developments in the Commercial Manufacture and Applications of Optically Active Compounds, Ed. Collins AN, Sheldrake GN, Crosby J. (Eds.) John Wiley & Sons Ltd. 1997; 118-156.
Koperwas K, Tu W, Affouard F, Adrjanowicz K, Kaskosz F, Paluch M. Pressure Dependence of the Crystallization Rate for the S-Enantiomer and a Racemic Mixture of Ibuprofen. Crystal Growth Design. 2021; 21: 7075-7086.
Martin-Matute B, Backvall JE. Dynamic kinetic resolution catalysed by enzymes and metals. Curr Opin Chem Biol. 2007; 11: 226-232.
Pellissier H. Dynamic kinetic resolution. Tetrahedron. 2003; 59: 8291-8327.
Trapp C, Barková K, Pecyna MJ, Herrmann C, Fuchs A, Greif D, et al. Deracemization of diastereomerically pure syn- and anti-α-substituted β-hydroxyesters by Novozyme 435 lipase and determination of their absolute configuration by NMR spectroscopy. Molecular Catalysis. 2021; 509: 111578.
Faber K. Biotransformation in organic chemistry, 4e éd., (Eds.) Springer-Verlag. 2000.