A thermodynamic study of the ketoreductase-catalyzed reduction of 2-alkanones in non-aqueous solvents

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Abstract

Equilibrium constants K have been measured for the reactions (2-alkanone + 2-propanol = 2-alkanol + acetone), where 2-alkanone = 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, and 2-octanone and 2-alkanol = 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, and 2-octanol. The solvents used were n-hexane, toluene, methyl tert-butyl ether (MTBE), and supercritical carbon dioxide SCCO2 (pressure P = 10.0 MPa). The temperature range was T = (288.15 to 308.27) K. Chiral analysis of the reaction products showed that the enzyme used in this study was stereoselective for the 2-octanone reaction system, i.e. only (S)-(+)-2-octanol was formed. For the reactions involving butanone, pentanone, and hexanone, the products were racemic mixtures of the respective (S)-(+)-2-alkanol and the (R)-(−)-2-alkanol. Chiral analysis showed that for the 2-heptanone reaction system, the 2-alkanol product was a mixture of (S)-(+)-2-heptanol and (R)-(−)-2-heptanol, at the respective mole fractions of 0.95 and 0.05. The equilibrium constant for the reaction system involving 2-butanone carried out in n-hexane was measured at several temperatures. For this reaction, the values for the thermodynamic reaction quantities at T = 298.15 K are: K = 0.838±0.013; the standard molar Gibbs free energy change ΔrGm=(0.44±0.040)kJ·mol-1; the standard molar enthalpy change ΔrHm=-(1.2±1.7)kJ·mol-1, and the standard molar entropy change ΔrSm=-(5.5±5.7)J·K-1·mol1. Interestingly, inspection of the values of the equilibrium constants for these reactions carried out in n-hexane, toluene, MTBE, and SCCO2 shows that these values are comparable and have little dependence on the solvent used to carry out the reaction. The values of the equilibrium constants decrease monotonically with increasing value of the number of carbons NC and trend towards a limiting value of ≈0.30 for NC > 8.

Introduction

Enzyme-catalyzed reactions in organic solvents have proven to be useful for the stereoselective synthesis of chiral intermediates for pharmaceuticals and agrochemicals [1], [2], [3]. Lipase-catalyzed esterification [4], [5], [6] and transesterification [1], [7] reactions in organic solvents have been commonly used for the enantioselective resolution of racemic mixtures. Recently, ketoreductase-catalyzed reactions [8], [9], [10], [11], [12] have been used for the production of chiral secondary alcohols from the corresponding ketones. These chiral alcohols are useful intermediates for the pharmaceutical, agrochemical, and perfume industries. For several years, our laboratory has been interested in the thermodynamics [13], [14], [15], [16], [17], [18] of enzyme-catalyzed reactions in organic solvents. Recently, our studies have been extended to include enzyme-catalyzed reactions carried out in supercritical carbon dioxide (SCCO2) [19]. Supercritical carbon dioxide is an attractive solvent for biocatalysis [20], [21], [22] because of its easy removal from the reaction mixture, non-toxicity, non-flammability, abundant availability, environmental friendliness, and recyclability. The low viscosity and high diffusivity of SCCO2 also help to provide favorable mass transfer properties, which can enhance the catalysis. The thermodynamic results obtained in these studies are essential for the basic understanding of the energetics of these reactions and also for the practical utilization of these enzyme-catalyzed reactions in these solvents.

In the present investigation, we report results of equilibrium measurements for the following ketoreductase-catalyzed transhydroxylation reactions (see figure 1)2-butanone(soln)+2-propanol(soln)=2-butanol(soln)+acetone(soln),2-pentanone(soln)+2-propanol(soln)=2-pentanol(soln)+acetone(soln),2-hexanone(soln)+2-propanol(soln)=2-hexanol(soln)+acetone(soln),2-heptanone(soln)+2-propanol(soln)=2-heptanol(soln)+acetone(soln),2-octanone(soln)+2-propanol(soln)=2-octanol(soln)+acetone(soln).Here “soln” denotes any of the four organic solvents used in this study.

The activity of the ketoreductase depends on the presence of a small catalytic amount of β-nicotinamide-adenine dinucleotide (reduced) {NADP(red)}. The ketoreductase-catalyzed reaction proceeds in two steps. In the first step, the 2-alkanone is reduced to the corresponding 2-alkanol and NADP(ox). In the second step, the NADP(ox) is reduced to NADP(red):2NADP(red)+2-alkanone=2-alkanol+2NADP(ox),2NADP(ox)+2-propanol=2NADP(red)+acetone.

Combination of equations (6), (7) gives the overall reaction for the reduction of the 2-alkanone2-alkanone+2-propanol=2-alkanol+acetone.

The principal aims of this study were to determine how the values of the equilibrium constants for the above reactions vary as a function of the number of carbons in the respective alkanones and also to examine how the values of these equilibrium constants depend on the solvent. The equilibrium constants for reaction (1) were measured by using n-hexane, toluene, methyl tert-butyl ether (MTBE), and supercritical carbon dioxide (SCCO2) as the solvents. The temperature dependency of the equilibrium constant for reaction (1) in n-hexane was also measured. Reaction (2) was studied by using hexane and SCCO2 as solvents. Reactions (3), (4) were studied by using n-hexane as the solvent. Finally, reaction (5) was studied by using n-hexane, toluene, and MTBE as the solvents. These solvents were selected based upon their current and potential use in non-aqueous enzymology [3].

In all cases, the chirality of the above reactions was investigated. Chiral analysis of the 2-alkanols in reactions (1), (2), (3) showed that the alkanols produced in these reactions were racemic mixtures (equal mole fractions) of both the (S)-(+)-2-alkanol and the (R)-(−)-2-alkanol. However, reaction (4) had a much greater degree of stereoselectivity and the product was primarily the S-(+)-2-heptanol. For reaction (5), the only product was (S)-(+)-2-octanol.

Section snippets

Materials

The substances used in this study, their Chemical Abstract Service (CAS) registry numbers, empirical formulas, molar masses, sources, and purities as determined by gas chromatography (g.c.) are given in table 1.

Thermodynamic formalism

The respective equilibrium constants for reactions (1) through (5) are:K=c(2-butanol)·c(acetone)/{c(2-butanone)·c(2-propanol)},K=c(2-pentanol)·c(acetone)/{c(2-pentanone)·c(2-propanol)},K=c(2-hexanol)·c(acetone)/{c(2-hexanone)·c(2-propanol)},K=c(2-heptanol)·c(acetone)/{c(2-heptanone)·c(2-propanol)},K=c(2-octanol)·c(acetone)/{c(2-octanone)·c(2-propanol)}.Here, c is the concentration of the indicated substance. In organic solvents, the substrates involved in these reactions are in a non-ionized

Acknowledgment

We thank Dr. Robert N. Goldberg for helpful discussions and suggestions.

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