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University Microfilms International A Beil & Howell Information Company 300 North Z eeb Road Ann Arbor Ml 48106-1346 USA 313/614/00 800 5310600 Order Number 8907259 Synthesis and physical studies of thiophospholipids using nuclear magnetic resonance Loffredo, William Michael, Ph.D. The Ohio State University, 1988 UMI 300 N, Zecb Rd. Ann Aibor, MI 48106 SYNTHESIS AND PHYSICAL STUDIES OF THIOPHOSPHOLIPIDS USING NUCLEAR MAGNETIC RESONANCE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By William Michael Loffredo, B.S. ****** The Ohio State University 1988 Dissertation Committee: Approved by v M.-D. Tsai M.D. Caffrey Adviser R.M. Mayer Department of Chemistry To my Dad "Therefore, since we are surrounded by such a great cloud of witnesses..." (Hebrews 12:1) ii ACKNOWLEDGEMENTS I want to thank my Father in Heaven, who has comforted, supported and encouraged me through this stage in my life. I thank Him for imparting His Wisdom and Knowledge to me, that I might complete such a great task. I want to thank my wife, Barbara and my son, Daniel for their love, support and understanding. I wish to thank my mother and brothers for their prayers and encouragement. I acknowledge the prayers of my other family members and those of the saints of God in my life that have upheld me through the course of this event. I express appreciation to Dr. Ming-Daw Tsai for his guidance throughout this research. Thanks also goes to the other members of my committee, Drs. Robert M. Mayer and Martin D. Caffrey for their suggestions and comments. I especially want to thank Dr. Ru-Tai Jiang, Ted, for taking me under his wing and teaching me the techniques valuable to completing this research, and also for being my very good friend. Thanks goes to Mr. Gialih Lin for his helpful discussions in synthesis. I also want to thank Dr. Charles C. Cottrell and Mr. Carl Engelman for their expertise and generosity in instrument time. VITA May 8 , 1960 Born to William R. and Carmella Loffredo, Pittsburgh,Pennsylvania 1980 Research Associate Lebanon Valley College, Annville, Pennsylvania 1982 B.S. in Chemistry with Certification in Teaching by The State of Pennsylvania, Lebanon Valley College, Annville, Pennsylvania 1982-1988 Graduate Teaching or Research Associate, Department of Chemistry, The Ohio State University, Columbus, Ohio 1984-1988 Departmental Operator and Overseer of Bruker WP-200 Nuclear Magnetic Resonance Spectrometer, The Ohio State University, Columbus, Ohio September 6 , 1986 Married to Barbara R. Jonas, Columbus, Ohio April 26, 1988 Delivered Daniel Michael Loffredo at Saint Ann's Hospital, Columbus, Ohio iv PUBLICATIONS "Phospholipids Chiral at Phosphorus. Characterization of the Subgel Phase of Thiophosphatidylcholines by Use of X-ray Diffraction, Phosphorus-31 Nuclear Magnetic Resonance and Fourier Transform Infrared Spectroscopy." H.E. Sarvis, W.M. Loffredo, R.A. Dluhy, L. Hernqvist, D.A. Wisner, and M.D. Tsai, Biochemistry 27, 4625-4631 (1988) "Use of Short-chain Cyclopentano-phosphatidylcholines to Probe the Mode of Activation of Phospholipase A2 from Bovine Pancreas and Bee Venom." G. Lin, J.P. Noel, W. Loffredo, H.Z. Sable and M.D. Tsai, J. Biol. Chemistry 263, 13208-13214 (1988) "Phospholipids Chiral at Phosphorus. 15. Steric Course of Phosphatidylserine Synthases from E. coli and Yeast." C.R.H. Raetz, G.M. Carman, W. Dowhan, R.T. Jiang, W. Waszkuc, W. Loffredo and M.D. Tsai, Biochemistry 26, 4022-4027 (1987) "Synthesis of (Rp) and (Sp)-l,2-Dipalmitoyl-jn-glycero-3-thiophospho-L- serine." W.M. Loffredo and M.D. Tsai, Presented at The American Chemical Society 19th Central Regional Meeting, Columbus, Ohio, March, 1987 "Thermotropic Properties of Specifically Deuterated Chiral Thiophos­ phatidylcholines." W.M. Loffredo and M.D. Tsai, Presented at The American Chemical Society 19th Central Regional Meeting, Columbus, Ohio, March, 1987 "Amide Interactions in Aqueous and Organic Medium." J.N. Spencer, S.K. Berger, C.R. Powell, B.D. Henning, G.S. Furman, W.M. Loffredo, E.M. Rydberg, R.A. Neubert, C.E. Shoop and D.N. Btauch, J. Phys. Chem. 85, 1236-1241, (1981). FIELD OF STUDY Major Field: Chemistry Emphasis in Biochemistry, Professor Ming-Daw Tsai, Advisor v TABLE OF CONTENTS DEDICATION li ACKNOWLEDGEMENTS iii VITA iv LIST OF TABLES viii LIST OF FIGURES x LIST OF SCHEMES xiv CHAPTER PAGE I. INTRODUCTION 1 II. SYNTHESIS OF THIOPHOSPHATIDYLCHOLINES 7 Introduction 7 Experimental Procedures and Results 8 Discussion 45 References 47 III. SYNTHESIS AND ENZYMATIC REACTIONS OF THIOPHOSPH ATI DYL-L-SERINE 48 Introduction 48 Experimental Procedures and Results 51 Discussion 89 References 91 IV. 31P NMR OF PHOSPHATIDYLCHOLINE AND THIOPHOSPHATIDYLCHOLINES 92 Introduction 92 Materials and Methods 105 Results 107 Discussion 112 References 117 V. 2H NMR OF SPECIFICALLY LABELED THIOPHOSPHATIDYLCHOLINES 119 Introduction 119 Materials and Methods 142 Results 144 Discussion 184 References 192 BIBLIOGRAPHY 194 vii LIST OF TABLES 1. Chemical Shielding Anisotropy Values 2. Approximate Magnitudes of Various Spin Interactions 3. Summary of the Thermotropic Phase Properties of DPPC and the Diastereomers of DPPsC 4. 2H NMR Spectral Widths at Half-height and Quadrupolar Splittings versus Temperature for ad 2 -DPPsC 5. 2H NMR Order Parameters versus Temperature for ad 2 -DPPsC Diastereomers 6 . 2H NMR Spin-Lattice Relaxation Times versus Temperature for a d 2 -DPPsC Diastereomers 7. Activation Energies of Reorientation for ad 2 -DPPsC Diastereomers 8 . 2H NMR Spectral Widths at Half-height and Quadrupolar Splittings versus Temperature for pd 2 -DPPsC Diastereomers 9. 2H NMR Order Parameters versus Temperature for pd 2 -DPPsC Diastereomers 10. 2H NMR Spin-Lattice Relaxation Times versus Temperature for pd 2 -DPPsC Diastereomers 11. Activation Energies of Reorientation for pd 2-DPPsC Diastereomers 12. 2H NMR Spectral Widths at Half-height and Quadrupolar Splittings versus Temperature for yNdg-DPPsC Diastereomers 13. 2H NMR Order Parameters versus Temperature for yNdg-DPPsC Diastereomers viii 2H NMR Spin-Lattice Relaxation Times versus Temperature for yNdg-DPPsC Diastereomers Activation Energies of Reorientation for yNd^DPPsC Diastereomers LIST OF FIGURES 1. The Diastereomers of DPPsC 2. *H NMR (250 MHz) Spectrum of 1,2-Dipalmitin in CDCI 3 3. 13C NMR (62.9 MHz) Spectrum of 1,2-Dipalmitin in CDCI 3 4. Comparison of the *H NMR (200 MHz) Spectra of 2 , 2 -[2 H 2 )- Choline Tosylate and Choline Tosylate in CDCI 3 5. Comparison of the 'H NMR (200 MHz) Spectra of 1,1-[ 2 H2]- Choline Tosylate and Choline Tosylate in CDCI 3 6 . Comparison of the ]H NMR (200 MHz) Spectra of N,N,N- Trimethyl-[2 H 9 ]-Choline Tosylate and Choline Tosylate in CDCI 3 7. 3tP NMR (121.5 MHz) Spectrum of (Rp+Sp)-DPPsC in CDCI 3 8 . 1H NMR (500 MHz) Spectrum of (Rp+Sp)-DPPsC in CDCI 3 9. 13C NMR (75.4 MHz) Spectrum of (Rp+Sp)-DPPsC in CDCI 3 10. Homonuclear *H NMR (500 MHz) Spectrum of (Rp+Sp)-DPPsC in CDCI 3 11. Condensation Reaction Between Thiophosphoryl Chloride and Dipalmitin in CDCI 3 12. Condensation Reaction Between Excess Thiophosphoryl Chloride and Dipalmitin in CDCI 3 13. The Hydrolysis of Thiophosphoryl Chloride in CDCI 3 14. The Hydrolysis of Thiophosphatidic Acid Dichloride in CDCI 3 15. The 31P NMR Spectrum (121.5 MHz) 'Immediately' After the Addkion of L-Serine Benzyl Ester 16. The 31P NMR Spectrum (121.5 MHz) 30 Minutes After the Addition of L-Serine Benzyl Ester x 17. The 31P NMR Spectrum (121.5 MHz) 1.5 Hours After the Addition of L-Serine Benzyl Ester 18. The 3lP NMR Spectrum (121.5 MHz) 2.5 Hours After the Addition of L-Serine Benzyl Ester 19. The 31P NMR Spectrum (121.5 MHz) 6.5 Hours After the Addition of L-Serine Benzyl Ester 20. The 31P NMR Spectrum (121.5 MHz) ’ Immediately' After the Addition of Water to the L-Serine Benzyl Ester Reaction 21. The 31P NMR Spectrum (121.5 MHz) 10 Minutes After the Addition of Water to the L-Serine Benzyl Ester Reaction 22. The 31P NMR Spectrum (121.5 MHz) 30 Minutes After the Addition of Water to the L-Serine Benzyl Ester Reaction 23.