1 an ABSTRACT of the THESIS of David L. Blanchard for the Degree of Doctor of Philosophy in Pharmacy Presented on March 18, 2008
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1 AN ABSTRACT OF THE THESIS OF David L. Blanchard for the degree of Doctor of Philosophy in Pharmacy presented on March 18, 2008. Title: Advanced Studies on Cyclic Amino Acids Involved in GABAergic Neurotransmission and Peptide Antibiotics Abstract approved:_______________________________________________ T. Mark Zabriskie L-pipecolic acid (L-PA) is the higher homolog of proline. It occurs naturally in many organisms, including primates, as an intermediate in lysine degradation. The pathway by which lysine is converted into L-pipecolic acid employs the enzyme L- pipecolate oxidase (L-PO), and appears to be tissue specific to the central nervous system (CNS). The oxidation facilitated by L-PO is the rate limiting step of lysine degradation in the CNS. For this reason, the mechanism of action for L-PO may be useful in the development of neuromodulation. This thesis describes efforts to probe the mechanism of action of L-PO through the synthesis of substrate analogs as alternate substrates and inhibitors of L-PO. Analogs that contain heteroatoms and other functionalities at key positions have been synthesized and analyzed as both alternate substrates and inhibitors of L-PO. The 4,5-methanopipecolic acid has been shown to be an excellent substrate for L-PO. The 5- keto analog was not a substrate or inhibitor of the enzyme. The 5-hydroxy and 5,5- 2 difluoro analogs have been shown to be weak inhibitors of L-PO. 6S-methyl-L-pipecolic acid (35) was shown to be a weak substrate and strong inhibitor while the enantiomeric 6R methyl-D-pipecolic acid (36) was neither a substrate nor inhibitor. These results suggest flexibility within the binding pocket of L-pipecolate oxidase toward analogs containing substituents at the 5-position. Additionally, these studies demonstrate the potential to develop mechanism-based inhibitors that could be used to control the rate of L-pipecolic acid consumption as well as the production of downstream products. A hallmark of peptide antibiotics are the varied and unique amino acids they employ. Capreomycidine and enduracididine are two such examples found in the peptide antibiotics muraymycin and enduracidin, respectively. Both are cyclic amino acids derived from arginine. Muraymycin is produced by Streptomyces sp. 30471 and has been shown to be active against a number of Gram-positive bacteria including Methicillin-resistant Staphylococcus aureus (MRSA). In an effort to further our understanding of this antibiotic, efforts to isolate the muraymycin gene cluster have begun. A genomic library has been constructed in the pCCFos1 Copy Control vector. Efforts to identify the genes encoding for the enzyme involved in the conversion of L-arginine to capreomycidine are described. 3 Enduracidin is another peptide antibiotic which contains a cyclized form of arginine, enduracididine. Enduracidin has potent activity against Gram-positive bacteria including Methicillin-resistant Staphylococcus aureus. The enduracidin gene cluster has been cloned and sequenced by Yin and Zabriskie . Efforts to express and characterized the genes involve in the biosynthesis of enduracididine are described. Labeled feeding studies were also employed to determine the precursor to enduracididine. 13C-γ- hydroxyarginine was synthesized and β- and γ-hydroxyarginine were used in feeding experiments to determine which, if either, would be incorporated into enduracidin. Labeled enduracidin was isolated and characterized by LC-MS from feeding studies using 13C-γ-hydroxyarginine, identifying γ-hydroxyarginine as a precursor to enduracididine. Additional studies were performed using 3-fluoro-L-tyrosine. The fluoro-tyrosine was converted to 3-fluoro-4-hydroxyphenylglycine through a pathway utilized for hydroxyphenylglycine biosynthesis. The 3-fluoro-4-hydroxyphenylglycine was found to be incorporated into multiple positions within enduracidin by LC-MS. 4 ©Copyright by David L. Blanchard March 18, 2008 All Rights Reserved Advanced Studies on Cyclic Amino Acids in Neurological Signaling and Peptide Antibiotics by David L. Blanchard A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented March 18, 2008 Commencement June 2009 2 Doctor of Philosophy thesis of David L. Blanchard presented on March 18, 2008 APPROVED: ______________________________________________________________ Major Professor, representing Pharmacy ______________________________________________________________ Dean of the College of Pharmacy ______________________________________________________________ Dean of the Graduate School I understand that my thesis will become a part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. ______________________________________________________________ David L. Blanchard, Author 3 ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Mark Zabriskie, for his patience, support, and mentorship. I would also like to thank my committee members: Dr. Philip Proteau, Dr. Taifo Mahmud, Dr. Xihou Yin, and Dr. Thomas Savage for their proofreading of the manuscript and for their services. Special thanks go to Dr. Michael Jackson, Dr. Patricia Flatt, and Dr. Kerry McPhail for their guidance and friendship. I would also like to thank Rodger Kohnert for sharing his knowledge in the area of NMR spectroscopy. I am very grateful to Brian Arbogast and Jeff Morré for their help and expertise in mass spectroscopy. The Center for Genome Research and Biocomputing core labs are acknowledged for their sequencing services and use of their equipment. I am indebted to the Oregon National Primate Research Center for providing the liver samples used to isolate L-pipecolate oxidase. I would also like to acknowledge the friendships of the members of the Zabriskie group: Xiaobo Fei, Dr. Ling Zhang, and Dr. Laura Grochowski; Proteau group: Chanokporn (Shiny) Phaosiri and Youn-Hi Woo; McPhail and Gerwick group: Dr. Lisa Nogle and Rebecca Medina; Mahmud group: Dr. Serge Fatso and XiuMei Wu. I am most grateful to my wife, Jamie, and children Chase and Madelynn. They have sacrificed much for me to be able to complete this work and I am truly thankful for the support and encouragement that they have shown me throughout this process. I dedicate this work to them. Finally, I thank the National Institutes of Health, Oregon Medical Research Foundation, and the College of Pharmacy for their financial support of these studies. 4 TABLE OF CONTENTS Page Forward 1 Medicinal Chemistry Research ......................................................................1 I. Mechanistic Probes of L-Pipecolate Oxidase Background and Introduction ........................................................................5 Discovery of L-Pipecolic Acid ......................................................................5 Lysine Biosynthesis .......................................................................................7 Lysine Degradation ........................................................................................12 L-Pipecolate Oxidase .....................................................................................15 Pharmacological and Physiological Role of L-Pipecolate Oxidase ...............20 Alternate Substrates and Inhibitors in Enzymological Studies ......................25 References ......................................................................................................25 II. Synthesis of Substrate Analogs of L-Pipecolate Oxidase Inhibitor and Alternate Substrate Design .......................................................33 Mechanism Models for L-Pipecolate Oxidase ...............................................35 Target Compound Design and Synthesis .......................................................38 Methods..........................................................................................................56 General ...............................................................................................56 Synthesis of Pipecolate Analogs ........................................................57 5 TABLE OF CONTENTS (Continued) Page D, L-Baikiain (6) ....................................................................57 1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridine -2-carboxylic acid (7) .............................................................58 1-tert-butyl 2-methyl 2,3-dihydropyridine-1,2(6H) -dicarboxylate (8) ..................................................................59 cis-2,3-Methano-1,4-butanediol (11) .....................................60 cis-1,4-Dibromo-2,3-methano-butane (12) ............................60 4,5-Methanopipecolic acid (15) .............................................61 1-tert-Butyl 2-methyl 5-hydroxypiperidine -1,2-dicarboxylate (19) .........................................................63 1-tert-Butyl 2-methyl 5-oxopiperidine- 1,2-dicarboxylate (20) ............................................................63 1-tert-Butyl 2-methyl 5,5-difluoropiperidine- 1,2-dicarboxylate (22) ............................................................64 General procedure for deprotection of N-Bocamines and methyl esters: ..................................................................64 5-Hydroxypiperidine-2-carboxylic acid (24) .........................65 5-Oxopiperidine-2-carboxylic acid (26) ................................66 5,5-Difluoropiperidine-2-carboxylic acid (29) ......................66 2-Cyano-6-phenoxazolopiperidine