LACCASE IN ORGANIC SYNTHESIS AND ITS APPLICATIONS A Dissertation Presented to The Academic Faculty by Suteera Witayakran In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Chemistry and Biochemistry Georgia Institute of Technology December 2008 LACCASE IN ORGANIC SYNTHESIS AND ITS APPLICATIONS Approved by: Dr. Arthur J. Ragauskas, Advisor Dr. John Cairney School of Chemistry and Biochemistry School of Biology Georgia Institute of Technology Georgia Institute of Technology Dr. David M. Collard Dr. Preet M. Singh School of Chemistry and Biochemistry School of Mechanical Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Uwe H. F. Bunz School of Chemistry and Biochemistry Georgia Institute of Technology Date Approved: October 22, 2008 ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. Arthur J. Ragauskas, for his instruction, encouragement, advice, and support throughout my graduate education. I also would like to thank my thesis committee, Dr. David M. Collard, Dr. Uwe H. F. Bunz, Dr. John Cairney, and Dr. Preet M. Singh for their insightful comments and support throughout this project. I would like to especially thank Dr. Leslie Gelbaum. His guidance and support in teaching me the techniques of NMR spectroscopy has helped me immensely in this research. I also would like to thank Dr. Robert Braga for conducting the IR experiments in the synthesis of p-naphthoquinone project. I also appreciate the assistance of all of my co-workers in Dr. Ragauskas’ lab that allowed me to complete my research tasks, and especially, I would like to thank the following colleagues: Dr. Yunqiao Pu for his kind help in NMR spectroscopy; Lenong Allison for her strong support through my study; Dr. Kristina Knutson for her training in how to measure laccase activity; Dong Ho Kim for his assistance in bulk acid group testing and handsheet making; Shaobo Pan and Shoujian Hu for their kind help in taking SEM images; Dr. Nan Jiang for his helpful advice for my synthetic work; Lee Goetz and Poulomi Sannigrahi for their friendship and support during my study; Rajalaxmi Dash for her help in FT-IR experiments; and Abdullah Zettili, summer undergrad student, for helping me in the synthesis of ortho-naphthoquinone project. iii I am pleased to acknowledge the IPST endowment fund and the Royal Thai Government Scholarship for providing me with financial support throughout my years of study. Ms. Tuwanda Strowbridge is appreciated for her guidance and help in the paper testing lab. Lastly, I would like to thank my parents, family, and friends for always supporting and encouraging me in my education. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS……………………………………………………………..iii LIST OF TABLES…………………………………………………………………….. xii LIST OF FIGURES………………………………………………………………….…xiv LIST OF EQUATIONS………………………………………………………………..xxii NOMENCLATURE…………………………………………………………………. xxiii SUMMARY…………………………………………………………………………...xxvi CHAPTER 1 INTRODUCTION………………………………………………………………. 1 1.1 Introduction……………………………………………………………… 1 1.2 Objectives………………………………………………………………...3 2 LITERATURE REVIEW………………………………………………………...5 2.1 Green Chemistry………………………………………………………… 5 2.1.1 Definition of Green Chemistry……………………………………. 5 2.1.2 Twelve Principles of Green Chemistry……………………………. 5 2.2 Water as Solvent in Organic Synthesis………………………………….. 8 2.2.1 Diels-Alder Reaction ……………………………………………..10 2.2.1.1 Quinone Diels-Alder reaction……………………………… 15 2.2.1.2 Uncatalyzed Diels-Alder Reaction in Aqueous Medium….. 22 2.2.1.3 Lewis-Acid-Catalyzed Diels-Alder Reaction in Aqueous Medium…………………………………………. 30 2.3 Biocatalysis…………………………………………………………….. 35 2.3.1 Enzymes………………………………………………………….. 35 v 2.3.1.1 Nomenclature and Classification…………………………... 35 2.3.1.2 Enzyme Mechanism………………………………………... 36 2.3.1.3 Enzyme Kinetics…………………………………………… 38 2.3.1.4 Advantages and Disadvantages of Biocatalyst…………….. 40 2.3.2 Enzymes in Domino Reactions…………………………………... 41 2.3.2.1 Enzyme-Triggered Diels-Alder Reaction………………….. 42 2.3.2.2 Enzyme-Triggered Rearrangement………………………… 44 2.3.2.3 Enzyme-Triggered Fragmentation…………………………. 47 2.3.2.4 Enzyme-Triggered Intramolecular Substitution Affecting Cyclization…………………………………………………. 48 2.3.2.5 Enzyme-Triggered Other Type of Ractions………………... 52 2.3.2.6 Multienzymatic One Pot Reaction…………………………. 53 2.4 Laccase…………………………………………………………………. 56 2.4.1 Distribution in Nature……………………………………………. 56 2.4.2 Laccase Structure………………………………………………… 57 2.4.3 Catalytic Mechanism and Properties……………………………...59 2.4.4 Laccase in Organic Synthesis……………………………………. 64 2.4.4.1 Laccase-Catalyzed Oxidation Reaction……………………. 65 2.4.4.2 Laccase-Mediated Formation of Intermediate Quinones In Organic Synthesis……………………………………….. 80 2.4.4.3 Laccase-Catalyzed Polymerization Reaction………………. 84 2.4.5 Laccase in Fiber Modification…………………………………… 88 2.4.5.1 Lignocellulosic Fibers……………………………………… 89 2.4.5.2 Laccase Application in Fiber Modification………………. 105 2.4.6 Conclusions……………………………………………………... 114 vi 2.5 Lipase…………………………………………………………………. 115 2.5.1 A General Account………………………………………………115 2.5.2 Lipase-Catalyzed Michael Reaction……………………………. 119 3 EXPERIMENTAL MATERIALS AND PROCEDURES…………………….124 3.1 Materials……………………………………………………………… 124 3.1.1 Chemicals……………………………………………………….. 124 3.1.2 Enzymes………………………………………………………… 124 3.1.3 Pulp……………………………………………………………... 126 3.2 Experimantal Procedures for the Use of Laccase in Organic Synthesis……………………………………………………………… 127 3.2.1 General Information…………………………………………….. 127 3.2.2 Analytical Analysis Procedures………………………………… 128 3.2.3 General Procedure of the Synthesis of 1,4-Naphthoquinones and Related Structures…………………………………………...129 3.2.4 General Procedure of the Synthesis of o-Naphthoquinones……..130 3.2.5 General Procedure of the Synthesis of Benzofuran Derivatives via Laccase Oxidation-Michael Addition………………………. 131 3.2.6 General Procedure of the Synthesis of Benzofuran Derivatives Using Laccase-Lipase Co-catalytic System…………………….. 132 3.2.7 General Procedure of the Reaction of Catechols and Anilines Catalyzed by Laccase-Lipase Co-catalytic System……………...132 3.3 Experimental Procedures for the Use of Laccase in Fiber Modification…………………………………………………………...133 3.3.1 Pulp Treatment………………………………………………….. 133 3.3.2 Bulk Acid Group Measurement………………………………… 133 vii 3.3.3 Pulp Refining and Handsheet Formation……………………….. 135 3.3.4 Paper Physical Tests……………………………………………..137 3.3.5 Nitrogen Analysis………………………………………………..138 3.3.6 Scanning Electron Microscope (SEM)…………………………..139 4 ONE POT SYNTHESIS OF 1,4-NAPHTHOQUINONES AND RELATED STRUCTURES WITH LACCASE…………………………………………... 140 4.1 Introduction…………………………………………………………… 140 4.2 Experimental Section…………………………………………………. 143 4.2.1 Meterials…………………………………………………………143 4.2.2 Enzyme Assay…………………………………………………... 144 4.2.3 General Procedure for the Study of the Effect of Laccase Dose and Temperature………………………………………………... 144 4.2.4 General Procedure of the Synthesis of 1,4-Naphthoquinones and Related Structures…………………………………………...145 4.2.5 Product Characterization………………………………………... 146 4.3 Results and Discussion………………………………………………...152 4.3.1 Preliminary Study of the Reaction System……………………... 152 4.3.2 The Effect of Laccase Dose…………………………………….. 154 4.3.3 The Effect of Temperature……………………………………… 157 4.3.4 The Reaction of p-Hydroquinone and Dienes…………………...159 4.4 Conclusions…………………………………………………………… 162 5 LACCASE-GENERATED QUINONES IN 1,2-NAPHTHOQUINONE SYNTHESIS VIA DIELS-ALDER REACTION............................................... 163 5.1 Introduction…………………………………………………………… 163 viii 5.2 Experimental Section…………………………………………………. 166 5.2.1 Enzyme Assay…………………………………………………... 166 5.2.2 General Procedure of the Synthesis of o-Naphthoquinones……..166 5.2.3 Typical Experimental Procedure for p-Naphthoquinone Synthesis………………………………………………………... 166 5.2.4 Product Characterization………………………………………... 167 5.3 Results and Discussion………………………………………………...171 5.4 Conclusions…………………………………………………………… 180 6 CASCADE SYNTHESIS OF BENZOFURAN DERIVATIVES VIA LACCASE OXIDATION-MICHAEL ADDITION………………………….. 181 6.1 Introduction…………………………………………………………… 181 6.2 Experimental Section…………………………………………………. 183 6.2.1 General Information…………………………………………….. 183 6.2.2 Enzyme Assay…………………………………………………... 183 6.2.3 General Procedure of the Synthesis of Benzofuran Derivatives via Laccase Oxidation-Michael Addition………………………. 183 6.2.4 Product Characterization………………………………………... 184 6.3 Results and Discussion………………………………………………...186 6.3.1 Preliminary Study and the Effect of pH on the Reaction System.186 6.3.2 The Effect of the Lewis Bases on the Reaction System…………187 6.3.3 The Effect of the Lewis Acids on the Reaction System…………189 6.3.4 The Synthesis of Benzofuran Derivatives………………………. 190 6.3.5 The Recyclability of the Laccase/Sc(OTf)3-Catalytic System….. 194 6.4 Conclusions…………………………………………………………… 196 ix 7 CO-CATALYTIC ENZYME SYSTEM FOR THE MICHAEL ADDITION REACTION OF IN SITU-GENERATED ORTHO QUINONES……………. 197 7.1 Introduction…………………………………………………………… 197 7.2 Experimental Section…………………………………………………. 199 7.2.1 General Information…………………………………………….. 199 7.2.2 Enzyme Assay…………………………………………………... 200 7.2.3 General Procedure of the Synthesis of Benzofuran Derivatives Using Laccase-Lipase Co-catalytic System…………………….. 200 7.2.4 Procedure for the Study of the Reaction of 5a and 8a (with and without Lipase)………………………………………. 200 7.2.5 General Procedure of the Reaction of Catechols and Anilines Catalyzed by Laccase-Lipase Co-catalytic System……………...201 7.2.6 Product Characterization………………………………………..