University of Massachusetts Boston ScholarWorks at UMass Boston Graduate Doctoral Dissertations Doctoral Dissertations and Masters Theses 6-1-2013 Biochemistry of 1, 2-Dehydro-N-Acetyldopamine Derivatives Adal T. Abebe University of Massachusetts Boston Follow this and additional works at: http://scholarworks.umb.edu/doctoral_dissertations Part of the Molecular Biology Commons Recommended Citation Abebe, Adal T., "Biochemistry of 1, 2-Dehydro-N-Acetyldopamine Derivatives" (2013). Graduate Doctoral Dissertations. Paper 106. This Open Access Dissertation is brought to you for free and open access by the Doctoral Dissertations and Masters Theses at ScholarWorks at UMass Boston. It has been accepted for inclusion in Graduate Doctoral Dissertations by an authorized administrator of ScholarWorks at UMass Boston. For more information, please contact [email protected]. BIOCHEMISTRY OF 1, 2-DEHYDRO-N-ACETYLDOPAMINE DERIVATIVES A Dissertation Presented by ADAL T. ABEBE Submitted to the Office of Graduate Studies, University of Massachusetts Boston, in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY June 2013 Molecular, Cellular and Organismal Biology Program © 2013 by Adal T. Abebe All rights reserved BIOCHEMISTRY OF 1, 2-DEHYDRO-N-ACETYLDOPAMINE DERIVATIVES A Dissertation Presented by ADAL T. ABEBE Approved as to style and content by: ________________________________________________ Manickam Sugumaran, Professor Chairperson of Committee ________________________________________________ Steven Ackerman, Associate Professor Member ________________________________________________ Jason Evans, Associate Professor Member ________________________________________________ William Robinson, Professor Member ________________________________________________ Alexey Veraksa, Associate Professor Member _________________________________________ Linda Huang, Program Director Biology Graduate Program _________________________________________ Richard Kesseli, Chairperson Biology Department ABSTRACT BIOCHEMISTRY OF 1,2-DEHYDRO-N-ACETYLDOPAMINE DERIVATIVES June 2013 Adal T Abebe, B.S., Addis Ababa University, Addis Ababa, Ethiopia M.S., University of Massachusetts Boston Ph.D., University of Massachusetts Boston Directed by Professor Manickam Sugumaran Dehydrodopa/dopamine derivatives form an important group of biomolecules participating in sclerotization of all arthropod cuticles, gluing and cementing mussels and related organisms to solid surfaces, and defense reactions of countless marine and invertebrate organisms. Yet very little information is available on the biochemistry of these highly reactive and unstable molecules. To understand their physiological role, I conducted a thorough biochemical study on three representative compounds that cover the entire plethora of dehydrodopa/dopamine derivatives. Employing diode array UV- visible spectroscopy, HPLC, liquid chromatography-mass spectrometry, and electrospray ionization tandem mass spectrometry, I investigated the oxidation chemistry of 1,2- dehydro-N-acetyldopamine (dehydro NADA), 1,2-dehydro-N-acetyldopa and 1,2- dehydro-N-acetyldopa methyl ester. Tyrosinase converted dehydro NADA to a reactive iv quinone methide that formed oligomeric products with the parent compound. The sister enzyme laccase, produced semiquinone radicals that exhibited a novel coupling reaction producing just dimers. Nonenzymatic oxidation of dehydro NADA also produced semiquinone radicals that formed oligomeric products. Moreover, nonenzymatic oxidation resulted in the production of superoxide anions that could function in defense reactions. The nonenzymatic oxidation studies on dehydro NADA at mild alkaline conditions revealed the mechanisms of defense reactions and tunic formation in a vast array of tunicates. Oxidative transformations of 1,2-dehydro-N-acetyldopa indicated a new route for the biosynthesis of a vast array of bioactive marine molecules possessing dihydroxycoumarin skeleton. In addition, it revealed new transformations of coumarins to oligomeric products via highly reactive quinone methide intermediates. Biochemical studies on 1,2-dehydro-N-acetyldopa methyl ester revealed a new Diels Alder type condensation of its quinone with the parent compound. This reaction shed light on the mode of gluing of mussels and other bivalves to solid surfaces as well as the hardening reactions occurring in their periostracum. I also examined the oxidation chemistry of dehydro NADA with a model nucleophile, N-acetylcysteine and discovered yet another new addition reaction of dehydro NADA that has tremendous biological significance. Finally, I investigated the mechanism of dehydro NADA binding to insect cuticle using labeled compounds and established that they could uniquely produce ketocatecholic compound, arterenone upon hydrolysis. The biochemical significances of all these new reactions are discussed in the dissertation. v ACKNOWLEDGEMENTS Completing my Ph.D. degree is probably the most challenging activity of my life. The best and worst moments of my journey have been shared with many people. It has been a great privilege to spend several years in the department of Biology at the University of Massachusetts Boston, and its members will always remain dear to me. Foremost, I will like to express my sincere gratitude to my advisor professor Manickam Sugumaran for the continuous support of my Ph.D. study and research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have asked for better role model and mentor for my study. Special thanks to my thesis committee, Associate Professor Jason Evans, without his help in mass spectrometer study I could have not completed my research. Thanks also to Associate Professor Alexey Veraksa for helpful discussion and suggestion. I will also like to thank Associate Professor Steven Ackerman and Professor William Robinson for their support and guidance. I will also like to thank Zheng Dong and Qun Kung for their help in mass spectrometer analysis and friendship. I wish to thank my wife Tiruwork Abebe, whose love and encouragement allowed me finish this journey. My mom Kebebush and my siblings their love provided me inspiration and was my driving force. Finally to my friends in Boston, Washington D.C, Ethiopian and other parts of the world, they were source of laughter and joy. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... vi LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii LIST OF ABBRREVIATIONS ........................................................................................ xx CHAPTER Page 1. INTRODUCTION .............................................................................................. 1 1.1 Dehydrodopas in insect cuticle sclerotization, in small molecules and peptides…….…………………………………………………...1 1.2 Research objectives...……...….………….…….…………………….25 2. TYROSINASE CATALYZED OXIDATIONOF 1,2-DEHYDRO-N- ACETYL DOPAMINE…………..………………………………………..27 2.1. Chapter summary .............................................................................. 27 2.2. Introduction ....................................................................................... 28 2.3. Materials and methods… .................................................................. 33 2.4. Results ............................................................................................... 37 2.5. Discussion ......................................................................................... 53 2.6. Conclusion ......................................................................................... 55 3. LACCASE CATALYZED OXIDATION OF 1,2-DEHYDRO-N- ACETYL DOPAMINE ................................................................................ 56 3.1. Chapter summary ................................................................................ 56 3.2. Introduction ......................................................................................... 57 3.3. Materials and methods ........................................................................ 59 3.4. Results ................................................................................................. 61 3.5. Discussion ........................................................................................... 69 3.6. Conclusion ........................................................................................... 72 vii CHAPTER Page 4. NON ENZYMATIC OXIDATION OF 1,2-DEHYDRO-N- ACETYL DOPAMINE .................................................................................. 75 4.1. Chapter summary ................................................................................... 75 4.2. Introduction ............................................................................................ 76 4.3. Materials and methods ........................................................................... 80 4.4. Results .................................................................................................... 84 4.5. Discussion ............................................................................................ 106 4.6. Conclusion ..........................................................................................