Modulating Biocatalytic Activity toward Sterically Bulky Substrates in

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Modulating biocatalytic activity towards sterically bulky substrates in CO-expanded bio-based liquids by tuning physicochemical properties 2

Hai Nam Hoang, Emanuel Granero-Fernandez, Shinjiro Yamada, Shuichi Mori, Hiroyuki Kagechika, Yaocihuatl Medina-Gonzalez, and Tomoko Matsuda ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03018 • Publication Date (Web): 14 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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ACS Sustainable Chemistry & Engineering

Modulating biocatalytic activity towards sterically bulky substrates in CO2-expanded bio-based liquids by tuning physicochemical properties Hai N. Hoang,1 Emanuel Granero-Fernandez,2 Shinjiro Yamada,1 Shuichi Mori,3 Hiroyuki Kagechika,3 Yaocihuatl Medina-Gonzalez,2* Tomoko Matsuda1* 1

Department of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-

cho, Midori-ku, Yokohama, Kanagawa, 226-8501, JAPAN 2

Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INPT, UPS, 4 Allée Emile

Monso, CS84234, F-31432 Toulouse Cedex 4, FRANCE 3

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10

Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, JAPAN #

*

Equally contributed. Corresponding

authors:

[email protected]

(Yaocihuatl

Medina-

Gonzalez); [email protected] (Tomoko Matsuda).

ACS Paragon Plus Environment

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KEYWORDS. Pressurized carbon dioxide, Candida antarctica lipase B, 2methyltetrahydrofuran, transesterification, kinetic resolution, substrate specificity, solvatochromism, green solvent engineering.

ABSTRACT. The study of CO2-expanded liquids using a green component such as a bio-based solvent has been recently raised as a new concept for an alternative solvent, and yet been largely unexplored in literature for neither fundamental nor application studies. On the other hand, structural bulkiness of substrates remains one of the main limitations to promote enzymes as an efficient versatile catalytic tool for organic synthesis, especially biocatalysis in non-conventional solvents. Herein, we report a detailed investigation of CO2-expanded bio-based liquids as reaction media for improved biocatalysis of sterically hindered compounds. We have found that CO2 acts as a crucial trigger for various lipases to catalyse transesterification of challenging bulky alcohols in CO2-expanded 2-methyltetrahydrofuran (MeTHF). Furthermore, this study determines physicochemical and transport properties of CO2-expanded MeTHF for the first time, which were then utilized for modulating biocatalytic activity. It was found that lipase activity increased with the accordingly decrease of the dipolarity of CO2-expanded MeTHF, which is tunable by altering the concentration of CO2 in the solvent system.

INTRODUCTION Solvents, which play an important role in chemical and pharmaceutical industries, have often been claimed as a major source of waste generation and associated environmental and economic


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burdens.1-3 With eco-environmental urge of developing sustainable processes, academic and industrial intentions have been focused both on minimizing the overall solvent usage and on replacing traditional organic solvents by more environmental-friendly alternatives.4-6 A novel class of alternative solvents is gas-expanded liquids,7-9 and more particularly CO2expanded liquids (CXLs).10 They are considered as tunable solvents and very promising as they inherit the advantages of pressurized CO2 and of traditional solvents in an optimal fashion: (1) the dissolved CO2 can tune the physicochemical and transport properties of the solvent such as viscosity, density, diffusivity across a large polarity range, and (2) the component organic solvent improves the solubility of polar compounds (Figure 1). The use of CXLs, on the other hand, can overcome the sustainable drawbacks of the required high working pressure of supercritical CO2, and subsequently reduces the installation cost of equipment and energy consumption of operation.11 Other important environmental advantages include the reduced waste of organic solvents because CXLs are generated by substantial replacing the organic solvents with the environmentally benign dense CO2.

Viscosity Diffusivity Solubility of polar compounds

Organic solvent

CO2expanded liquid

Liquid or scCO2

Amount of CO2 Waste of solvent generation Working pressure

Figure 1. The green and sustainable use of carbon dioxide to tune physical properties of solvents.


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On the other hand, biocatalytic processes, which has been well established as a topic of chemical researches,12-14 shares many attractive features in the context of green chemistry and sustainability.15 Traditionally, biocatalysts have been used in aqueous solvents, which sometimes results in inevitable side reactions and low solubility towards hydrophobic compounds. The scope of biocatalysis, especially in organic synthesis,14 has been extended through the use of enzymes in organic solvents16 and in non-conventional solvents17,18 such as supercritical19 and liquid20-22 CO2, ionic liquids23 and deep eutectic solvents.24,25 To date, however, the study of CXLs as reaction media for a biocatalysis process has remained less explored. Although the use of CXLs adhere to some principles of green chemistry and sustainability, such as reduced waste of organic solvents and energy cost; usually a CXL system employs a petroleum-sourced volatile organic compound, which is depletable, and often harmful to the environment. Therefore, classical CXLs cannot be adequately satisfactory as green solvents. An alternative way-out is to replace the conventional harmful organic solvents with environmentally-friendly bio-based liquids.26-28 Previously we have reported CXLs using biobased liquids such as 2-methyltetrahydrofuran (MeTHF) as reaction media for Candida anctarctica lipase B (Novozym 435, CALB) to effectively catalyse kinetic resolution of various sec-alcohols.29 MeTHF, which can be derived from lignocellulosic biomass, has recently gained much interests as a promising and an emerging bio-based solvent for various synthesis applications30-32 including biocatalysis.33,34 However, as there has been no study on solvent properties of CO2-expanded MeTHF reported, the biocatalytic behaviour in this novel medium has not yet addressed.29 In this present study, by using 1-adamantylethanol 1a as a challenging bulky model substrate it was found out that CO2 acts as a crucial trigger for various lipases to catalyse transesterification


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in a wide range of CO2-expanded bio-based liquids (Scheme 1). In order to explain our observation, physicochemical and transport properties of CO2-expanded MeTHF were studied both experimentally and by molecular modelling. Thus, vapour-liquid equilibrium and polarity of the expanded phase were determined experimentally, in particular, polarity of the expanded phase was determined by solvatochromic measurements. Density and viscosity of the CO2expanded phase were obtained by molecular dynamics. The improved lipase activity can be attributed to important changes in solvent properties, of which transport properties are enhanced in CO2-expanded MeTHF by the decrease of viscosity and density, while dipolarity/polarizability parameter naturally decreases to a certain degree that is ideal for the lipase-catalysed reaction. Significantly, this study shows for the first time that the activity of a biocatalysis process in CO 2expanded MeTHF at different temperatures was closely correlated to physicochemical properties, which are modulated by tuning the CO2 molar fraction (XCO₂).

Scheme 1. Kinetic resolution of rac-1-adamantylethanol 1a by lipases in CO2-expanded biobased liquids.


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RESULTS AND DISCUSSION Lipase-catalyzed transesterification of 1a First, transesterification of rac-1-adamatylethanol 1a using vinyl acetate as an acyl donor was investigated in MeTHF, and CO2-expanded MeTHF using various lipases. Because MeTHF has a low hydrophobicity (log P =1.0, Supplementary Table S1), which could be unfavorable for lipase-catalyzed reaction in organic solvents,35 we also performed the same reactions in hexane (log P = 3.5, Supplementary Table S1) and CO2-expanded hexane for comparison (Table 1). Vinyl acetate was used in a small volume (50 L) compared to a bulk amount of MeTHF or hexane to minimize the solvent effect of vinyl acetate, which can also contribute as a co-solvent if used in a large amount. As can be seen from Table 1, most of the used enzymes exhibited no or very low catalytic activity for the transesterification of 1a, although they have potent catalytic activity towards general sec-alcohols such as 1-phenylethanol. The low reactivity of those lipases Table 1. Lipase-catalyzed transesterification of rac-1-adamatylethanol 1a. CO₂-expanded MeTHF

Neat MeTHF Lipase (Organism, carrier)

Conversion (%)

E-value

Conversion (%)

E-value

CO₂-expanded hexane

Neat hexane Conversion (%)

E-value

Conversion (%)

E-value

CALB (Candida antarctica, acrylic 200 3.1a N.d. 29 >200 resin) TL (Pseudomonas 47 >200 45 >200 45 >200 36 >200 stutzeri, beige powder) PS-D (Burkholderia 200 6.9 >200 200 5.1 >200 12 >200 cepacia, ceramic) LIP 301 (Pseudomonas 6.3 >200 4.5 >200 22 >200 200 2.1 >200 14 >200

Modulating Biocatalytic Activity toward Sterically Bulky Substrates in

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