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Chapter 35 : The Use of Enzymes for Nonaqueous Organic Transformations

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Abstract:

This chapter focuses on synthetic applications of enzymes in monophasic organic solvents and is intended to illustrate many types of transformations that can be catalyzed by enzymes in organic solvents. Nonaqueous enzyme systems can be divided into two classes: homogeneous systems in which enzymes are modified to be soluble, and heterogeneous systems in which the catalyst is in an insoluble form. Colyophilisates for use with enzymes in organic solvents can be categorized according to three primary acting mechanisms: activating salts, molecular imprinting agents/molds, and lyoprotectants. This chapter summarizes many applications of enzymes in organic solvents, with focus placed on three classes of enzymes that have found a wide measure of use in organic solvents: hydrolases (EC 3), lyases (EC 4), and oxidoreductases (EC 1). Hydrolase enzymes (EC 3) comprise the predominant bio-catalysts for transformations reported in organic solvents, and two groups, lipases and proteases, enjoy widespread use. Chiral cyanohydrins produced by hydroxynitrile lyases (HNLs) in organic solvents have been used as building blocks in the synthesis of various bioactive compounds, such as epinephrine derivatives. Enzymes are not only active in organic solvents, they often display high regio-, chemo-, and enantioselectivities, making them particularly suited for the selective modification of complex molecules. Novel enzyme preparation methods and system conditions, along with multienzyme processes and the ability to engineer enzymes with improved properties for synthesis, will lead to new synthetic routes and ensure an expanding role of organic-phase biocatalysis in the synthetic chemist’s repertoire.

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35

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Candida rugosa
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Alcohol Dehydrogenase
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Escherichia coli
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Rhodococcus ruber
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Escherichia coli
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Image of FIGURE 1
FIGURE 1

Enzyme preparations used in organic solvents. (A) Lyophilized powder. (B) Immobilized enzymes (left to right): single-point covalent attachment, multipoint covalent attachment, and physisorption. (C) Directly solubilized as a cluster via a surfactant. (D) Colyophilized powder containing an excipient. (E) Covalently modified with PEG. (F) Reverse micelle encapsulation with retained water. (G) Surfactant-paired, extracted single-enzyme molecules.

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 2
FIGURE 2

Doxorubicin derivatives generated with solubi-lized and salt-activated lipase and subtilisin preparations. All preparations could acylate the 14-O hydroxyl of doxorubicin (black arrow) in toluene, but only salt-activated subtilisin Carlsberg could modify the amino and hydroxyl groups indicated by the white arrows ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 3
FIGURE 3

Recommended enzymes for catalyzing various reactions in organic solvents. The recommended systems are those shown to give the best performance for a certain reaction (high enzyme stability, reactivity, and selectivity) based on the literature cited in this chapter.

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 4
FIGURE 4

A simple scheme for the hydrolase-catalyzed resolution of an alcohol. Vinyl acetate is a common acyl donor in alcohol resolutions. Upon enzyme acylation by vinyl acetate, vinyl alcohol is released, which rapidly tautomerizes to the nonnucleophilic acetaldehyde. The acyl-enzyme intermediate is then attacked preferentially by the () or ()-alcohol enantiomer.

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 5
FIGURE 5

These alcohols were resolved enantioselectively [()-preference] by acylation with vinyl acetate in diiso-propyl ether by three lipases. values are for CALB. Adapted from reference .

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 6
FIGURE 6

Increasing the chain length of acyl donor increased from 33 to 65 in the subtilisin Carlsberg-catalyzed acylation of 1-phenyl ethanol in THF. Adapted from reference .

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 7
FIGURE 7

To facilitate resolution of cyclo-hexenol, the bulky benzylthiol was added. After resolution by CALB in diisopropyl ether (di-IPE), the benzylthiol was removed. Adapted from reference .

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 8
FIGURE 8

Due to different active-site geometries, lipases and proteases show opposite enantiopreference in the resolution of secondary alcohols if the substituents differ in size ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 9
FIGURE 9

Multistep lipase-catalyzed resolution of a primary alcohol with distant stereocenter. di-IPE, diisopropyl ether. Adapted from reference .

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 10
FIGURE 10

Enantioselectivities of suspended CALB and suspended subtilisin Carlsberg against various acyl acceptors in organic solvents. CALB reaction was in neat ethyl acetate, which was also the acyl donor ( ). Subtilisin reactions were in 3-methyl-3-pentanol, with trifluoroethyl butyrate as the acyl donor ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 11
FIGURE 11

A dynamic kinetic resolution scheme. The lipase CALB and ruthenium and palladium catalysts are compatible with resolutions in organic solvents, affording fast racemization and 98% product yield ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 12
FIGURE 12

Regioselective acylations or alcoholysis of steroids, flavonoids, and nucleosides by hydrolases in organic solvents. (A) A model steroid ( ). (B) The flavonoid morin ( ). (C) Adenosine ( ). (Note: No amino acylations are observed.) (D) The antioxidant bergenin ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 13
FIGURE 13

Loss of chemoselectivity by PCL when the solvent is changed. The enzyme was 20-fold more selective at hydroxyl acylation in dichloroethane than in -butanol ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 14
FIGURE 14

Medium engineering to favor CALB-catalyzed Michael addition () over aminolysis () in various anhydrous organic solvents. Adapted from reference .

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 15
FIGURE 15

The Markovnikov addition of imidazoles to vinyl esters in dimethyl sulfoxide by several acylases ( ) (top), and the asymmetric aldol addition of ketones and aldehydes catalyzed by PPL in acetone ( ) (bottom). di-IPE, diisopropyl ether.

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 16
FIGURE 16

Asymmetric hydrocyanic addition of prochiral benzaldehyde is a popular model reaction for HNLs.

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 17
FIGURE 17

A one-pot synthesis involving Celite-HbHNL and CALB in water-saturated toluene. The water present was used by CALB to generate acetic acid, which deactivated the HbHNL. Adapted from reference .

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 18
FIGURE 18

Alternative reaction paths to optically active α-cyano-3-phenoxybenzyl alcohol, a building block in pyrethrin insecticides. In the top path, an ()-selective HNL is used for direct cyanoaddition ( ). In the bottom path, nonselective cyanoaddition is coupled with enantioselective lipase acylation in a dynamic kinetic resolution ( ). di-IPE, diisopropyl ether.

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 19
FIGURE 19

Polyphenol oxidase conversion of benzalcohols to diquinones in chloroform. This reaction was difficult in aqueous buffer because both the enzyme and quinone were unstable ( ). In a subsequent study, the enzyme-generated quinone was subjected to various olefins to prepare bicyclooctenones ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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Image of FIGURE 20
FIGURE 20

Horse liver alcohol dehydrogenase catalyzes the selective reduction of a racemic aldehyde with the consumption of NADH in isopropyl ether. The enzyme and NADH cofactor were coimmobilized on a glass support. The NADH was regenerated by the same enzyme in the oxidation of etha-nol to acetaldehyde ( ).

Citation: Hudson E, Liszka M, Clark D. 2010. The Use of Enzymes for Nonaqueous Organic Transformations, p 509-523. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch35
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