Increased Selectivity of Novozym 435 in the Asymmetric Hydrolysis of a Substrate with High Hydrophobicity Through the Use of Deep Eutectic Solvents and High Substrate Concentrations.

Yerko Fredes,Zaida Cabrera,Lesly Chamorro

doi:10.3390/molecules24040792

Abstract

The effects of the reaction medium and substrate concentration were studied on the selectivity of Novozym 435 using the asymmetric hydrolysis of dimethyl-3-phenylglutarate as a model reaction. Results show that the use of choline chloride ChCl:urea/phosphate buffer 50% (v/v) as a reaction medium increased the selectivity of Novozym 435 by 16% (e.e = 88%) with respect to the one in 100% phosphate buffer (e.e = 76%). Best results were obtained when high substrate concentrations (well above the solubility limit, 27-fold) and ChCl:urea/phosphate buffer 50% (v/v) as reaction medium at pH 7 and 30 °C were used. Under such conditions, the R-monoester was produced with an enantiomeric purity of 99%. Novozym 435 was more stable in ChCl:urea/phosphate buffer 50% (v/v) than in phosphate buffer, retaining a 50% of its initial activity after 27 h of incubation at pH 7 and 40 °C. Results suggest that the use of deep eutectic solvents (ChCl:urea/phosphate buffer) in an heterogeneous reaction system (high substrate concentration) is a viable and promising strategy for the synthesis of chiral drugs from highly hydrophobic substrates.

Highlights

The enantiomeric purity of a chiral drug is an essential requirement for its commercialization, because even though both optical isomers are chemically similar, the organism is able to distinguish between both enantiomeric forms and recognize only one of them [1,2,3]
choline chloride (ChCl):urea and ChCl:glycerol were previously selected to perform this investigation, because they are within simples deep eutectic solvents (DESs) most utilized with CALB lipase [11,15]
CHCl-glycerol was subsequently discarded because it showed low substrate solubility (0.87 mM of DMFG) in DES-buffer mixture compared with ChCl:urea (2.02 mM)

Summary

Introduction

The enantiomeric purity of a chiral drug is an essential requirement for its commercialization, because even though both optical isomers are chemically similar, the organism is able to distinguish between both enantiomeric forms and recognize only one of them [1,2,3]. The physiological responses in the organism will be quite different depending on the isomer consumed, and in some cases, they could have opposite effects. In this context, the pharmaceutical industry urgently requires the development of novel production strategies for the synthesis of pure enantiomers, taking into consideration the viability of the process at the industrial level and its environmental benignity. The asymmetric synthesis of prochiral compounds by enzyme biocatalysis is very promising, by producing essentially pure enantiomers in a single reaction step [4,5]. The use of organic solvents allows increasing the solubility of hydrophobic substrates, but notoriously affects the stability of the enzyme [6,7,8]

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Conclusion