Liquid carbon dioxide as a superior solvent for candida antarctica lipase b catalyzed kinetic resolution of sec alcohols

Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology Department of Bioengineering Matsuda Laboratory Liquid carbon dioxide as a superior solvent for Candida antarctica lipase B catalyzed kinetic resolution of sec-alcohols Thesis submitted for the degree of Master of Engineering by Hoang Nam Hai July 2015 Index Index Index i Abbreviations iii Chapter Introduction Chapter Candia antarctica lipase B catalyzed kinetic resolution of rac-1-phenylethanol in liquid carbon dioxide 2.1 CAL-B catalyzed KR of 1-phenylethanol in organic solvents and in liquid CO2 2.2 The use of liquid CO2 in continuous flow reactors for large-scale biosynthesis of optically pure compounds 2.2.1 KR of 1-phenylethanol in a continuous-flow stirred-tank reactor (CSTR) using liquid CO2 2.2.2 KR of 1-phenylethanol in a continuous packed – bed reactor (CPBR) using liquid CO2 12 2.3 Comparison of the productivity of continuous-flow and batch reactors using liquid CO2 as an medium 14 Chapter Expanding substrate scope of Candida antarctica lipase B using liquid carbon dioxide 18 3.1 Substrate specificity of CAL-B catalyzed transesterification of alcohols 18 3.2 Expanding substrate scope of CAL-B using liquid CO2 19 Chapter Conclusion and perspective 25 Chapter Experimental section 26 4.1 Chemicals and instrument 26 4.2 General procedure for preparation of rac-alcohols 27 4.3 General procedure for preparation of acetates 29 4.4 General procedure for enzymatic reaction in organic solvents 32 4.5 General procedure for enzymatic reaction in dense carbon dioxide 33 i Index 4.6 General procedure for determination of absolute configuration of products 33 4.7 Data analysis 35 Publications 39 References 40 Acknowledgments 43 Appendix 44 ii Abbreviations Abbreviations BSTR batch stirred – tank reactor CAL-B Candida antarctica lipase B CPBR continuous packed-bed reactor CSTR continuous stirred – tank reactor ee enantiomeric excess eep enantiomeric excess of product ees enantiomeric excess of substrate GC gas chromatography KR kinetic resolution NMR nuclear magnetic resonance scCO2 supercritical carbon dioxide iii Chapter Chapter Introduction Chapter Chapter Introduction In nature, there are numerous biologically active and structurally complex compounds which have attracted and challenged the organic chemists for centuries One of the greatest difficulties is that caused by matters of stereochemistry.1 Basically there are three main approaches2 to obtain enantiomerically pure compounds based on the type of starting material used (Scheme 1) With remarkable chemo-, regio-, and enantioselectivity, enzymes (biocatalysts) are effectively used as a powerful tool for the synthesis of enantiopure compounds: through asymmetric synthesis from prochiral compounds (maximum 100% yield) or kinetic resolution (KR) of racemates (maximum 50% yield) Chiral pool Racemates Prochiral compounds resolution asymmetric synthesis synthesis kinetic crystallization chromatography (bio) catalysis (bio) catalysis Enantiomerically pure compounds Scheme Methods to obtain enantiomerically pure compounds Enzymes are used in two pathways (i) asymmetric synthesis from prochiral substrates or (ii) KR of racemates Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3), have been well established as a valuable catalyst for the preparation of enantiomerically enriched compounds because of their stability, broad substrate range, non-requirement of co-factors, compatibility under mild reaction conditions.3 Among them, the lipase B from Candida antarctica (CAL-B) has been found to be a particularly useful biocatalyst for the kinetic resolution of sec-alcohols and related compounds.4 CAL-B is supplied as a recombinant protein patented by Novo-Nordisk and is Chapter commercially available in immobilized form under Novozym 435® trade name This lipase accepts a broad range of non-natural substrates and in many cases it exhibit good to excellent stereoselectivity.5 Furthermore, CAL-B retains most of its activity and robustness in nonaquenous media, allowing the efficient use of the enzyme in esterification and transesterification reactions, in addition to the traditional hydrolysis reactions.6 On the other hand, the scope of biotransformation in organic synthesis has been extended through the use of enzymes in organic solvents7 as well as in “green” nonaqueous media, such as ionic liquids8 and supercritical fluids.9 Carbon dioxide, which has a number of positive impacts on green chemistry as a nonflammable, nontoxic, abundant, and generally chemically inert source, has been intensively studied in its supercritical phase as an alternative solvent for enzymatic reactions.10 Some of the advantages of using supercritical carbon dioxide (scCO2) as a solvent include low viscosity, low surface tension, and ease of product recovery At below critical temperature (31oC), carbon dioxide can exist in a liquid phase, which can also be employed as a solvent for chemical reactions.10-11 However, to the best of our knowledge, no reports exist on liquid CO2 as a practical solvent for biocatalysts Carbon dioxide in a liquid phase is intrinsically different from its supercritical phase in many physical properties as a solvent, the enzymatic behaviors in liquid and supercritical CO2 could be very different The apparent benefit of using liquid CO2 is that it can be maintained under relatively modest pressure (e.g 4.5 MPa at 10oC) that reduces the cost of specialized equipment for high-pressure (over 7.4 MPa) reaction in supercritical CO2 Furthermore, it can be employed at low temperature, which has the potential to enhance the enatioselectivity by the low-temperature strategy.12 This study is the first investigation to exploit the potential of liquid CO2 as an alternative solvent for biotransformation By using Candida antarctica lipase B catalyzed the kinetic resolution of Chapter sec-alcohols as the model reaction, the feasibility and compatibility of liquid CO2 as a medium for biosynthesis was comprehensively studied in a batch reactor and two extreme continuousflow reactors (Chapter 2) Surprisingly, the substrate scope the lipase was found to be expanded toward bulky phenyl alkyl sec-alcohols by just using it in liquid CO2 (Chapter 3) The outline of this thesis is as the follow (Scheme 2) Liquid CO2 as a superior solvent for CAL-B catalyzed kinetic resolution of sec-alcohols Chapter Chapter Kinetic resolution of Investigation of rac-1-phenylethanol substrate specificity Comparison between liquid Liquid CO2 in flow reactors Expanding CO2 and organic solvents for large-scale biosynthesis substrate scope using a batch reactor Comparison of productivity between reactors Scheme Outline of this study Chapter Chapter Candia antarctica lipase B catalyzed kinetic resolution of rac-1-phenylethanol in liquid carbon dioxide Chapter Chapter Candia antarctica lipase B catalyzed kinetic resolution of rac-1phenylethanol in liquid carbon dioxide In this chapter, liquid CO2 was studied first time as a solvent for a biocatalyzed reaction The KR of typical substrate 1-phenylethanol by the immobilized lipase B from C antarctica (Novozym 435®) using vinyl acetate as an acyl donor was investigated as the model reaction in liquid CO2 and in conventional organic solvents The practical potential of liquid CO2 fluid was also examined with continuous flow bioreactors Parts of the results have been published.13 2.1 CAL-B catalyzed KR of 1-phenylethanol in organic solvents and in liquid CO2 First, the KR of rac-1-phenylethanol catalyzed by CAL-B was performed in liquid CO2 and in various conventional organic solvents using a batch reactor (Scheme 3) Vinyl acetate was used as the acylating agent for the purpose of an irreversible transesterification, because the formed vinyl alcohol irreversibly tautomerizes to acetaldehyde For gaining impression on the accuracy, all reactions were performed in the same shape reactor, and vigorously stirred with a magnetic bar to eliminate the mass transfer effects Scheme KR of rac-1-phenylethanol by CAL-B As shown in Figure 1, as expected, the activity of the lipase is closely related to the solvent hydrophobicity (represented in log P value) where the more hydrophobic solvents generally showed higher yields Interestingly, the CAL-B exhibited the highest transesterification activity with excellent enatioselectivity (eep>99%) in liquid CO2, followed by hexane and toluene Appendix 1b 52 Appendix 2b 53 Appendix rac-3b 54 Appendix rac-4b 55 Appendix rac-5b 56 Appendix rac-6b 57 Appendix rac-7b 58 Appendix rac-8b 59 Appendix rac-9b 60 Appendix rac-10b 61 Appendix rac-11b 62 Appendix rac-12b 63 Appendix rac-13b 64 Appendix rac-14b 65 Appendix rac-15b 66 ... lipase B using liquid carbon dioxide Candida antarctica lipase B is a robust and versatile biocatalyst that has been mostly used for highly enantioselective kinetic resolution of wide range of. .. optimization in the operation 17 Chapter Chapter Expanding substrate scope of Candida antarctica lipase B using liquid carbon dioxide Chapter Chapter Expanding substrate scope of Candida antarctica. .. carbon dioxide Chapter Chapter Candia antarctica lipase B catalyzed kinetic resolution of rac-1phenylethanol in liquid carbon dioxide In this chapter, liquid CO2 was studied first time as a solvent