Candida enantioselectivity

Lipases from C. antarctica and P. cepacia showed higher enantioselectivity in the two ionic liquids l-ethyl-3-methylimidazolium tetrafluoroborate and l-butyl-3-methylimidazolium hexafluoroborate than in THE and toluene, in the kinetic resolution of several secondary alcohols [49]. Similarly, with lipases from Pseudomonas species and Alcaligenes species, increased enantioselectivity was observed in the resolution of 1 -phenylethanol in several ionic liquids as compared to methyl tert-butyl ether [50]. Another study has demonstrated that lipase from Candida rugosa is at least 100% more selective in l-butyl-3-methylimidazolium hexafluoroborate and l-octyl-3-nonylimidazolium hexafluorophosphate than in n-hexane, in the resolution of racemic 2-chloro-propanoic acid [51]. 

In recent years, a great variety of primary chiral amines have been obtained in enantiomerically pure form through this methodology. A representative example is the KR of some 2-phenylcycloalkanamines that has been performed by means of aminolysis reactions catalyzed by lipases (Scheme 7.17) [34]. Kazlauskas rule has been followed in all cases. The size of the cycle and the stereochemistry of the chiral centers of the amines had a strong influence on both the enantiomeric ratio and the reaction rate of these aminolysis processes. CALB showed excellent enantioselec-tivities toward frans-2-phenylcyclohexanamine in a variety of reaction conditions ( >150), but the reaction was markedly slower and occurred with very poor enantioselectivity with the cis-isomer, whereas Candida antarctica lipase A (GALA) was the best catalyst for the acylation of cis-2-phenylcyclohexanamine ( = 34) and frans-2-phenylcyclopropanamine ( =7). Resolution of both cis- and frans-2-phenyl-cyclopentanamine was efficiently catalyzed by CALB obtaining all stereoisomers with high enantiomeric excess. 

The chiral intermediate (S)-l-(2 -bromo-4 -fluorophenyl) ethanol was prepared by the enantioselective microbial reduction of 2-bromo-4-fiuoroacetophenone [lObj. Organisms from genus Candida, Hansmula, Pichia, Rhodotcnda, Saccharomyces, Sphingomonas, and baker s yeast reduced the ketone to the corresponding alcohol in... 

For reduction of acetylenic ketones, two oxidoreductases were used [25]. Lactobacillus brevis alcohol dehydrogenase (LBADH) gave the (R)-alcohols and Candida parapsilosis carbonyl reductase (CPCR) afforded the (S)-isomer, both in good yield and excellent enantioselectivity. By changing the steric demand of the substituents, the enantiomeric excess values can be adjusted and even the configurations of the products can be altered (Figure 8.34). 

Other biocatalysts were also used to perform the dynamic kinetic resolution through reduction. For example, Thermoanaerobium brockii reduced the aldehyde with a moderate enantioselectivity [30b,c], and Candida humicola was found, as a result of screening from 107 microorganisms, to give the (Jl)-alcohol with 98.2% ee when ester group was methyl [30dj. 

A combination of an enzymatic kinetic resolution and an intramolecular Diels-Alder has recently been described by Kita and coworkers [23]. In the first step of this domino process, the racemic alcohols ( )-8-55 are esterified in the presence of a Candida antarctica lipase (CALB) by using the functionalized alkenyl ester 8-56 to give (R)-8-57, which in the subsequent Diels-Alder reaction led to 8-58 in high enantioselectivity of 95 and 91 % ee, respectively and 81 % yield (Scheme 8.15). In-... 

Two interesting yeast carbonyl reductases, one from Candida magnoliae (CMCR) [33,54] and the other from Sporobolomyces salmonicolor (SSCR) [55], were found to catalyze the reduction of ethyl 4-chloro-3-oxobutanoate to give ethyl (5)-4-chloro-3-hydroxybutanoate, a useful chiral building block. In an effort to search for carbonyl reductases with anti-Prelog enantioselectivity, the activity and enantioselectivity of CMCR and SSCR have been evaluated toward the reduction of various ketones, including a- and /3-ketoesters, and their application potential in the synthesis of pharmaceutically important chiral alcohol intermediates have been explored [56-58]. 

The carbonyl reductase from Candida magnoliae catalyzed the enantioselective reduction of a diversity of ketones, including aliphatic and aromatic ketones and a- and /3-ketoesters (Figure 7.17), to anti-Prelog configurated alcohols in excellent optical purity (99% ee or higher) [56]. 

Zhu, D., Yang, Y. and Hua, L. (2006) Stereoselective enzymatic synthesis of chiral alcohols with the use of a carbonyl reductase from Candida magnoliae with anti-Prelog enantioselectivity. The Journal of Organic Chemistry, 71 (11), 4202-4205. 

Biocatalysts also operate in ionic liquids [28]. The ones that have been most widely investigated are the lipase family of enzymes. For example, Candida Antarctica lipase B immobilized in [bmim][BF4] or [bmim][PFe] under anhydrous conditions is able to catalyse transesterifications at rates comparable to those observed in other solvents. Certain lipase mediated enantioselective acylations have even resulted in considerable improvements in enantiomeric excesses... 

The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Among them, two commercially available Upases, Candida antarctica lipase B (CALB trade name, Novozym-435) and Pseudomonas cepacia lipase (PCL trade name. Lipase PS-C), are particularly useful because they have broad substrate specificity and high enantioselectivity. They display satisfactory activity and good stability in organic media. In particular, CALB is highly thermostable so that it can be used at elevated temperature up to 100 °C. 

This process has many benefits in the context of green chemistry it involves two enzymatic steps, in a one-pot procedure, in water as solvent at ambient temperature. It has one shortcoming, however-penicillin acylase generally works well only with amines containing an aromatic moiety and poor enantioselectivities are often observed with simple aliphatic amines. Hence, for the easy-on/easy-off resolution of aliphatic amines a hybrid form was developed in which a hpase [Candida antarctica hpase B (CALB)] was used for the acylation step and peniciUin acylase for the deacylahon step [22]. The structure of the acyl donor was also optimized to combine a high enanhoselectivity in the first step with facile deacylation in the second step. It was found that pyridyl-3-acetic acid esters gave optimum results (see Scheme 6.8). 

Although [BMIM]BF4 has been evaluated as an isolation medium for lipase-catalyzed biotransformations, the general experience with it has not been favorable, relative to that with other ionic liquids, such as [BMIM]PF6. However, excellent performance was recently reported for [BDMIM]BF4 when it was used to host Candida antarctica (Novozym 435) for the enantioselective transesterification of 5-phenyl-l-penten-3-ol (+ and -) with vinyl acetate. The working hypothesis was that the oligomerization of acetaldehyde may be caused by the C2 proton of the [BMIM] ion because of the unfavorable acidity of this group (226). In contrast, the cation in [BDMIM]BF4 lacks this acidity. 

Chemoenzymatic polymerizations have the potential to further increase macro-molecular complexity by overcoming these limitations. Their combination with other polymerization techniques can give access to such structures. Depending on the mutual compatibility, multistep reactions as well as cascade reactions have been reported for the synthesis of polymer architectures and will be reviewed in the first part of this article. A unique feature of enzymes is their selectivity, such as regio-, chemo-, and in particular enantioselectivity. This offers oppormnities to synthesize novel chiral polymers and polymer architectures when combined with chemical catalysis. This will be discussed in the second part of this article. Generally, we will focus on the developments of the last 5-8 years. Unless otherwise noted, the term enzyme or lipase in this chapter refers to Candida antarctica Lipase B (CALB) or Novozym 435 (CALB immobilized on macroporous resin). 

Hansen, T.V., Waagen, V., Partali, V., Anthonsen, H.W. and Anthonsen, T. (1995) Cosolvent enhancement of enantioselectivity in lipase-catalysed hydrolysis of racemic esters. A process for production of homochiral C-3 building blocks using lipase B from Candida antarctica. Tetrahedron Asymmetry, 6, 499-504. 

Persichetti, R-A., Lalonde, J.J., Govardhan, C.P., Khalaf, N.K. and Margolin, A.L. (1996) Candida-ragosa lipase—enantioselectivity enhancements in organic solvents. 

Candida antarctica lipase B has been employed to resolve kinetically a series of bicyclic 1 -heteroaryl primary amines by enantioselective acetylation <2003JOC3546>. 

In a photometric assay NADP(H)-dependent LBADH (see above) [9] and NAD(H) -dependent Candida parapsilosis carbonyl reductase (CPCR) [40] were identified as suitable catalysts accepting a broad range of ynones as substrates. Both enzymes catalyze the reduction of various aryl alkynones 21 with high enantioselectivity and efficiency (Scheme 2.2.7.13) [41]. 

The enantioselective hydrolysis of racemic menthyl benzoate (industrially key compound) by recombinant Candida rugosa lipase LIPl leads to optically pure l-(-)-menthol ee>99% [21]. This pathway is part of a menthol synthesis developed by the flavour industry. 

Scheme 2.2 Enantioselective kinetic resolution of cis/ttoris (IR,5R)-bicyclo[3.2.0]hept-6-ylidene-acetate ethyl ester, 1, catalyzed by immobilized lipase from Candida antarctica (Novozyme 435). The reaction is run in a pH-stat at pH 8.0 and...

Genetic engineering. The X-ray structures are known for many hydrolases, allowing for modeling of the substrate in the active site as well as structurally based, random or rational protein mutation to magnify or invert enantioselectivity. An example of the latter is provided by the rational design of a mutant of Candida antarctica lipase (CALB), which, instead of the wild-type R-selectivity, displayed... 

It is known that enantioselectivity of enzymes depends on many different parameters such as temperature, substrate structure, reaction medium, and presence of water. Enantiopreference of enzymes can be greatly affected, even reversed, by changing the reaction solvent. Such an example was reported by Ueji et al. in 1992 for Candida cylindracea lipase-catalyzed esterification of ( )-2-phenoxy propionic acid with 1-butanol [29]. 

Candida antarctica (enantioselective) Candida cylindracea lipase (enantioselective)... 

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