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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. The 31P NMR Spectrum (121.5 MHz) 1 Hour After the Addition of Water to the L-Serine Benzyl Ester Reaction

24. ]H NMR Spectrum (250 MHz) of N-Trityl-L-Serine Methoxy Methyl Ester

25. 13C NMR Spectrum (62.9 MHz) of N-Trityl-L-Serine Methoxy Methyl Ester

26. 31P NMR Spectrum (101.3 MHz) of Dipalmitoyl Methylthiophosphatidyl-N-Trityl-L-Serine Methoxy Methyl Ester

27. *H NMR Spectrum (250 MHz) of Dipalmitoyl Methylthiophosphatidyl-N-Trityl-L-Serine Methoxy Methyl Ester

28. 31PNMR Spectrum (101.3 MHz) of DPPsS

29. 1H NMR Spectrum (250 MHz) of DPPsS

30. >H NMR Spectrum (250 MHz) of the Serine OCW 2 Protons and the 31P Decoupled *H NMR Spectrum (250 MHz) of the Serine OCH2 Protons

31. 31P NMR Spectrum (121.5 MHz) of the Reaction of (Rp+Sp)- DPPsS with PL A2

32. 31P NMR Spectrum (81 MHz) of the Ring Diastereomers of Dipalmitoyl Thiophosphatidyl-N-Benzyl-L-Serine Benzyl Ester

33. *H NMR Spectrum (500 MHz) of the Ring Compound of Dipalmitoyl Thiophosphatidyl-N-Benzyl-L-Serine Benzyl Ester

xi 34. *H NMR Spectrum (500 MHz) of the Ring Compounds of Dipalmitoyl Thiophosphatidyl-N-Benzyl-L-Serine Benzyl Ester in the Region 5.0 to 3.5 ppm

35. Thermotropic Properties of Bilayers Following Incubation at 0°C

36. DSC Traces of (Rp)-DPPsC of Greater Isomeric Purity (>99%), Showing the Time Dependence of the Metastable Gel Phase

37. Thermotropic Phase Transitions with the Corresponding Proposed Molecular and Hydrocarbon Chain Packing Arrangements of the Lamellar Phases of DPPC

38. Molecular Orientation of the 31P Chemical Shift Tensor in Barium Diethyl Phosphate

39. The Theoretical Powder Lineshape for an Axially Asymmetric Tensor

40. 3IP NMR Spectrum (36.5 MHz) of Nonsonicated Bilayers of DPPC with Broadband Proton Decoupling at 44°C

41. 3ip NMR Spectra (121.5 MHz) of DPPC and the Thiophospholipid Diastereomers at Temperatures Characteristic of the Various Thermotropic Phases

42. The Effect of the Zeeman Interaction Between the Nuclear Magnetic Moment and the Applied Magnetic Field

43. The Quadrupolar Perturbation on the Zeeman Energy Levels

44. The 2H NMR Spectrum for a Single Crystal of a Molecule with an Isolated 1 = 1 Nucleus

45. The 2H NMR Spectrum for a Polycrystalline Powdered Molecule with an Isolated 1=1 Nucleus

46. A Diagramatic Representation of the Quadrupolar Echo Pulse Sequence

47. A Molecular Model of DLPE Showing the sn-2 Chain Bend

48. Molecular Models Illustrating the 50°C Tilt of the Phosphate Moiety in DPPC Molecules

49. Conformation of the Head Group in Glycerylphosphorylcholine

50. 2H NMR Spectra of a d 2 -DPPsC Diastereomers Below Their Main Transition Temperatures

xii 51. 2H NMR Spectra of ctd 2-DPPC Well Below Its Main Transition Temperature Showing the Subgel Phase

52. 2H NMR Spectra of a d 2-DPPsC Diastereomers Above Their Main Transition Temperatures

53. 2H NMR Spectra of pd 2 -DPPsC Diastereomers Below Their Main Transition Temperatures

54. 2H NMR Spectrum of (Rp+Sp)-pd 2 -DPPsC at 60°C Showing the Assignments of the Labels A,B,C,D,E

55. 2H NMR Spectra of pd 2 *DPPsC Diastereomers Above Their Main Transition Temperatures

56. 2H NMR Spectra of yNd^DPPsC Diastereomers Below Their Main Transition Temperatures

57. 2H NMR Spectra of yNdg-DPPsC Diastereomers Above Their Main Transition Temperatures

xiii LIST OF SCHEMES

1 . The Synthesis of 1,2-Dipalmitoyl-sn-glycerol from D-mannitol

2. The Overall Synthesis of Specifically Deuterated Choline Tosylates

3. Synthetic Routes for the Synthesis of (Rp+Sp)-DPPsC

4. Resolution of the Diastereomers of DPPsC

5. A General Scheme for the Synthesis of the Diastereomers of DPPsS via a 2-thiono-1, 3,2-oxazaphosphilidine Ring Intermediate

6 . The Synthesis of DPPsS Using the Phosphite Method and N-Trityl-L-Serine Methoxy Methyl Ester

xiv CHAPTER I

INTRODUCTION

The main thrust of this dissertation is to provide insight into the importance of the configuration at phosphorus on the thermotropic ph ase properties of phospholipids using thiophosphatidylcholines as probes.

Thiophosphatidylcholines are sulfur analogs of naturally occurring phosphatidylcholines in which one of the prochiral phosphate oxygens is replaced by a sulfur atom. This substitution provides a sensitive probe, having minimal perturbations, for the study of model membrane systems.

The concentration of this work deals with the polar head group of the phospholipid, namely the phosphorylcholine moiety. Characterizing the differences between the diastereomers of chiral dipalmitoyl thiophosphatidylcholines (DPPsC) at various positions along the polar head group will provide insight into the molecular structure of the bilayer.

A recent study using differential scanning calorimetry (Wisner, et al.,

(1986)) has shown that (Sp)-DPPsC shows a pretransition temperature

(Tpt) at 43.7°C and a main transition temperature (Tm) at 45°C, as well as a subtransition temperature (Ts) at 22°C when the sample was annealed at

4°C for several days. Such a thermotropic property is similar to that of

DPPC (Chen, et al., (1980)), except that Tpt and Tm are shifted higher by a few degrees. The mixture of isomers, (Rp+Sp)-DPPsC, behaves similarly

1 2

to the Sp isomer. The Rp isomer, however, shows only a broad transition

at 45.9°C, even after being annealed at 4°C for a period of two weeks. In

separate experiments, Wisner, et al., (1986) showed that when (Rp)-

DPPsC was heated at 50-70°C, cooled to 25°C quickly, and scanned

immediately, a pretransition and a main transition similar to those of the Sp

isomer were observed. These results were interpreted to indicate that (Rp)-

DPPsC is metastable in the gel phase, that it relaxes to the subgel phase

rapidly at 25°C (t t /2 of hours, depending on the isomeric purity), and that

it remains in the subgel phase in the heating scan until Tm is reached. Such

results suggest that the configuration at phosphorus is important to the

structure of phospholipid bilayers.

Differential scanning calorimetry provides only a limited depth of

understanding about the structure of model membrane systems. The overall

macromolecular structural changes may be deduced from differential

scanning calorimetry, but an understanding of the microstructure is more

desired. No single experimental technique can provide all the information

necessary to fully understand a system, so many techniques are used and a

theoretical model is derived that can best explain all the data. In addition

to the differential scanning calorimetry studies on the diastereomers of

DPPsC, X-ray diffraction studies were also performed (Sarvis, et al.,

(1988)). The information provided from X-ray diffraction studies confirmed the phase assignments deduced from the differential scanning calorimetry data.

To obtain an even better understanding of the structure of the polar head group of phosphatidylcholines, various nuclear magnetic resonance (NMR) 3 studies were performed, namely 31P NMR and 2H NMR. These two nuclei were chosen because of the sensitivity of their respective tensors to molecular changes. 31 P NMR makes use of the naturally abundant phosphorus-31 nucleus in the phospholipid. The chemical shielding anisotropy factor of the 31 P nucleus has already been used to study the motion and average orientation of the phosphate group in lipid systems

(Seelig, (1978) and Griffin, (1981)). 2H NMR, on the other hand, requires synthetic incorporation due to the natural abundance of 2H compared to ]H. Whereas the chemical synthesis is long and tedious, it provides a means to ensure specific incorporation of the probe and therefore allowing data to be acquired at specific sites along the choline head group. The spin 1 nucleus of 2H provides insight into the motion and orientation of the deuterated segment due to the sensitivity of the electric field gradient tensor, associated with the spin 1 nucleus, to the local magnetic field.

Chapter II describes the syntheses of the thiophosphatidylcholines. The overall synthesis of the thiophosphatidylcholines and the reactions used to resolve the diastereomer are given in detail. The reactions used in synthesizing the three specifically deuterated choline head groups are also given in detail. Included also in this chapter is a mini study on the condensation of thiophosphoryl chloride and dipalmitin, which gives optimization of the bottleneck step in the synthetic pathway.

Chapter III describes the synthesis of another thiophospholipid analog, dipalmitoyl thiophosphatidyl-L-serine, DPPsS. The goal of this chapter was to provide a unique synthetic scheme which would produce pure diastereomer of a thiophospholipid. Many attempts were made to reach this

goal, with the only result being a diastereomeric mixture of

thiophosphatidyl-L-serines. Three different methods produced the DPPsS

mixture, but only one in substantial yield. That pathway made use of a

highly reactive phosphorylating agent and a nitrogen, carboxyl blocked L-

serine that was relatively easy to deblock. The reaction of the

diastereomeric mixture of DPPsS with phospholipase A2 resolved the Sp

isomer, but efforts to resolve the Rp isomer by reaction with phospholipase

C failed.

Also given in Chapter III are the unsuccessful attempts at synthesizing

DPPsS. The most notable being the synthetic route using N benzyl-L-

serine benzyl ester to produce the 2-thiono-1,3,2-oxazaphosphilidine ring

intermediate. This intermediate was extremely stable and resolution into diastereomers was possible. However, a variety of debenzylating procedures were unsuccessful.

Chapter IV reports the 31P NMR studies of DPPC and the diastereomers of DPPsC at temperatures corresponding to their various thermotropic phases. Confirmation of the observed thermotropic phase changes was provided along with noticeable differences between the individual diastereomers.

Chapter V reports the 2H NMR studies of the DPPsC diastereomers specifically deuterated in three places along the choline head group. All three segments show confirmation of the thermotropic phase assignments.

The most interesting result being at the (3 methylene carbon in which two conformationally enantiomeric head group configurations were observed. 5 Clearly this study provides great insight into the microstructure of the choline head group and more detail into the differences between the diastereomers. 6

REFERENCES

Griffin, R.G. (1981) in Methods Enzymology 72, 108-174.

Sarvis, H.E., Loffredo, W.M., Dluhy, R.A,, Hernqvist, L., Wisner, D.A. & Tsai, M.D. (1988) Biochemistry 27, 4625-4631.

Seelig, J. (1978) Biochim. Biophys. Acta 515, 105-140.

Wisner, D.A., Rosario-Jansen, T. & Tsai, M.D. (1986) J. Am. Chem. Soc. 108, 8064-8068. CHAPTER II

SYNTHESIS OF THIOPHOSPHATIDYLCHOLINES

II. 1 Introduction

The synthetic procedures used to prepare and purify separate diastereomers of 1,2-dipalmitoyl-s/i-glycero-3-thiopho$phocholine

(DPPsC) from D-mannitol are described. The thiophosphate analogs of phosphatidylcholine are useful compounds in biophysical studies of phospholipid bilayers due to their chirality at phosphorus. Replacing one of the prochiral (nonbridging or nonesterified) oxygens of the phosphate with sulfur presents a very minor structural change, thus producing a nonperturbing structural probe for the investigation of head-group interactions involving the phosphate group. The two diastereomers of

DPPsC are shown in figure 1.

Q jHjiOOO— Cl3H31COO— C i5H3|CO O — C.j H j .COO- o —o— \ — O o N (C H ,) 3 O

tty - d p p .c (Sp)- DPP.C

Figure 1: The diasteromers of DPPsC.

7 The synthesis of the diastereomers of DPPsC was achieved by two different routes. Vasilenko, et al., (1982) make use of thiophosphoryl chloride as the phosphorylating agent, while Bruzik, et al., (1986) make

use of a highly reactive phosphorylating agent, N,N,- diisopropylmethylphosphonamidic chloride. The differences in yield

between the two methods (40-50% by way of Vasilenko, et al., versus 70-

80% by way of Bruzik, et al.) has made the phosphite route the pathway of choice.

The DPPsC diastereomers thus produced were used to study the thermotropic phase properties by 31 P NMR in Chapter IV.

The synthesis of specifically deuterated choline tosylates is also described. The specifically deuterated diastereomers of DPPsC were then used to study the thermotropic phase properties of DPPsC using 2H NMR as described in Chapter V.

II.2 Experimental Procedures and Results

Synthesis of 1,2-dipalmitoyl-sn-glycerol

The overall synthesis of 1,2-dipalmitoyl-j/t-glycerol, dipalmitin, from

D-mannitol is shown in scheme 1. The synthesis is rather time consuming so consequently, dipalmitin was often purchased from Sigma Chemical Co.

The dipalmitin from Sigma was virtually free of the 1,3-dipalmitoyl-s/i- glycerol isomer. The isomeric purity of the dipalmitin synthesized via 9

HO— o — HO— >o — ZnCU Pb(OAc)4 HO— HO— ■OH (CH3)2CO — OH EtOAc ■OH ■OH — O N

\ ° ~ i °~l I - LiAlH, 0 - \ 1. Na

H ° 2. H30 + 2. PhCH2Br

10 % HOAc I” 0** C^COCl *—OCH2Ph HO— O C H 2Ph

r -O O C C 15H31 j OOCC15H31 C 13H 310 0 0 - ^ — ------^ c 13h31c o o - ] 1—OCH^Ph I—OH

Scheme 1: The synthesis of 1,2-dipalmitoyl-sn-glycerol from D-mannitol. 10 scheme 1 was judged to be >97% pure by TLC with the remainder being the

1,3 isomer.

The preparation of 1 ,2 ,5 ,6 -di-isopropylidene-D-mannitol was achieved

following the procedure of Bird & Chadha (1966). In a clean dry 100 ml

round bottom flask 3.40g (0.025 mol) of anhydrous zinc chloride and 25

ml of anhydrous acetone were stirred until homogeneity. At which time,

1.82g (0.01 mol) of anhydrous D-mannitol (Fischer) was added. The

mixture was allowed to stir at room temperature for 2 hours, during which

time it became clear with a slight yellow tint. In a 250 ml erlenmeyer

flask, 6 . 8 g of anhydrous potassium carbonate was dissolved in 5 ml

distilled water and after cooling, 35 ml of diethyl ether was added with

vigorous stirring. The D-mannitol reaction mixture was added in one

portion to the potassium carbonate solution and allowed to stir vigorously

for 1 hour. The solid zinc carbonate was filtered and washed with two

portions of diethyl ether. The filtrate was dried over molecular sieves 3A,

then evaporated to dryness. The crude crystals were recrystallized from n- butyl ether. The solubility of 1,2,5,6-di-isopropylidene-D-mannitol , DIM, in hot n-butyl ether was lOg per 30 ml. The product, 1,2,5, 6 -di- isopropylidene-D-mannitol (>47%), was characterized by NMR at 200

MHz in CDCI 3 : 6 1.37 ppm (s, 6 H, O-C-C//3, equatorial); 1.43 ppm (s,

6 H, O-C-C//3 , axial); 2.51 and 2.55 ppm (s,s, 2H, H-C-O//); 3.76 ppm (t,

2H, //-COH); 4.01 ppm (m,2H, H-C-OC); 4.16 and 4.19 ppm (m, 4H,

C//2*OC), and had a melting point of 117-119°C (lit. 119°C, Jensen &

Pitas, (1976)). 11 DIM was oxidatively cleaved using lead tetraacetate (Jensen & Pitas

(1976)). In a clean dry 100 ml round bottom flask 2.61g (0.01 mol) of

DIM was dissolved in 100 ml dry ethyl acetate at 50-60°C. The mixture was then cooled to room temperature and 5.32g (0.012 mol) of lead tetraacetate was added in small increments over the course of 45 minutes.

Upon the addition of the last increment, the reaction mixture was tested for excess lead tetraacetate by using starch-potassium iodide paper (excess lead tetraacetate was removed by adding an equivalent amount of DIM). The reaction was monitored using thin layer chromatography, TLC, in solvent system A, hexanes: diethyl ether, (v;v), 1:3. The Rf values in this solvent system are DIM = 0.0, and 1,2-isopropylidene-i/i-glyceraldehyde = 0.4.

The reaction mixture was filtered through a short column of diatomaceous earth under a nitrogen atmosphere and washed with three 10 ml portions of ethyl acetate. The product, l,2-isopropylidene-.rn-glyceraldehyde, was distilled under reduced pressure, bp = 58°C, 30 Torr in 97% yield (lit.

38°C, 8 Torr, Baer and Fischer, (1939)).

The reduction of 1,2-isopropylidene-sn-glyceraldehyde to 1,2- isopropylidene-sn-glycerol was achieved using lithium aluminum hydride

(Schlenk,(1965)). In a 250 ml 2-necked round bottom flask equipped with a condenser and a pressure equalizing addition funnel, 0.456g (0.012 mol) of lithium aluminum hydride was suspended in 30 ml of anhydrous diethyl ether. The solution of 1. 30g (0.01 mol) of 1,2-isopropylidene-sn- glyceraldehyde in 10 ml anhydrous diethyl ether was dropped into the suspension maintaining a gentle reflux. Refluxing was continued for one hour more. The reaction was quenched by the successive addition of 10 ml 12 ethyl acetate and 8 ml water. The metal salts were filtered off using a short diatomaceous earth column. The filtrate was evaporated to remove solvents and the product, 1 , 2 -isopropylidene-^n-glycerol, was isolated by distillation, bp = 78-80°C, 12 Torr in 73% yield (lit. 72°C, 8 Torr, Baer and Fischer, (1939)).

The 1,2-isopropylidene-.sn-glycerol was then converted to the 3-benzyl derivative (Howe & Malkin,(1951)). In a 100 ml round bottom flask equipped with a U-shaped claisen adapter, a condenser and a 125 ml * pressure equalizing addition funnel, 0.253g (0.011 mol) of freshly cut sodium metal was refluxed in 25 ml of anhydrous toluene until the sodium melted. To this refluxing mixture, a solution of 1.32g (0.01 mol) 1,2- isopropylidine-sn-glycerol in anhydrous toluene was slowly added. This mixture was allowed to reflux for one hour. A solution of 1.49 ml (0.0125 mol) of benzyl bromide in 10 ml anhydrous toluene was dropped into the refluxing sodium metal solution over a two hour period. The reaction mixture was allowed to reflux one hour more. After cooling to room temperature, the solution was filtered through a diatomaceous earth column and washed with two 10 ml portions of anhydrous diethyl ether. The product, 3-benzyl-1,2-isopropylidene-jn-glycerol was isolated by distillation under reduced pressure, bp = 95-105° C, 0.3 Torr in 81% yield

(lit. 93-96°C, 0. 1 Torr, Sowden and Fischer, (1941)).

Removal of the isopropylidene group and successive acylation using palmitoyl chloride was achieved using the procedures of Baer & Fischer,

(1945). A 10% solution of acetic acid was used to deblock the isopropylidene group from 3-benzyl-1,2-isopropylidene-in-glycerol. 13

To 8 ml of 10% acetic acid, 2.22g (0.01 mol) of 3-benzyl-l ,2-

isopropylidene-sn-glycerol was added and refluxed for two hours. The

solvents were evaporated under reduced pressure and the product was kept

under vacuum (< 0. 1 Torr) overnight to assure removal of acetic acid. The

yield of this reaction was quantitative.

The 3-benzyl-sn-glyceTol was acylated using palmitoyl chloride. In a

1 0 0 ml round bottom flask equipped with a pressure equalizing addition

funnel, 1.82g (0.01 mol) of 3-benzyl-sn-glycerol, 1.61 ml (0.02 mol) of

anhydrous pyridine and 10 ml of anhydrous carbon tetrachloride was

allowed to stir at 0°C. To this solution, 6.09 ml (0.02 mol) of palmitoyl

chloride in 10 ml of anhydrous carbon tetrachloride was dropped in over 30

minutes and allowed to stir at 0°C for 30 minutes more. The reaction

mixture was then warmed to room temperature, heated to 40°C and stirred

overnight. Pyridinium hydrochloride was removed by filtration and a

solution containing 0.5g of calcium chloride, 0.3 ml of triethylamine and

water was added. The calcium salts were removed by filtration and the

filtrate was evaporated under reduced pressure to dryness. The crude

product was recrystallized from absolute ethanol (87% yield).

The benzyl group was removed via catalytic hydrogenolysis in hexanes.

In a 250 ml round bottom flask equipped with a stopcock side arm, 6.58g

(0.01 mol) of 3-benzyl-1,2-dipalmitoyl-sn-glycerol, 1.32g of palladium on carbon (;Esar) (reactant to catalyst ratio of 5:1) and 100 ml of anhydrous

hexanes was allowed to stir. The reaction vessel was purged three times

with hydrogen gas, then allowed to stir at room temperature under atmospheric pressure overnight. The reaction was monitored by TLC in 1 4 solvent system B, 95:5, (v:v), chloroform:acetone. The Rf values were

0.94 for 3-benzyl-1,2-dipalmitoyl-sn-glycerol, 0.74 for 1,3-dipalmitoyl- jn-glycerol and 0.64 for 1,2-dipalmitoyl-fn-glycerol. The reaction mixture was filtered through a diatomaceous earth column, and washed three times with 5 ml portions of anhydrous diethyl ether. The solvents were removed by evaporation under reduced pressure, the crude product recrystallized from n-hexane (80% yield) and characterized by lH NMR (250 MHz) in

CDCI3 5: 0.88 ppm (t, J=7 Hz, 6 H, acyl chain I6 -CW3 ); 1.26 ppm (s,

48H, acyl chain \5-4-CHj)', 1.62 ppm (m, 4H, acyl chain 3 -CW2 ); 2.03 ppm (t, J = 6 Hz, 1H, glycerol 3 -CH 2O//); 2.32 ppm (m, 4H, acyl chain 2-

C//2 ); 3.73 ppm (dd, J = 5 Hz, 2H, glycerol 3-Ctf 2OH); 4.27 ppm (m, 2H, glycerol l-C// 20) and 5.09 ppm (dt, 1 = 5 Hz, 1H, glycerol 2-C/ZO) and l3C

NMR (62.9 MHz) in CDCI3 5: 14.04 ppm (acyl chain I6 -CH 3 ); 22.65 ppm

(acyl chain 15-CH2); 24.91 ppm (acyl chain 14-CH2); 29.60 ppm (acyl chain 13-3-CH2); 34.27 ppm and 34.08 ppm (acyl chain 2-CH2); 61.50 ppm (glycerol 3-CH 2OH); 62.07 ppm (glycerol 1-CH2); 72.13 ppm

(glycerol 2-CHO) and 173.4 ppm and 173.7 ppm (acyl chain C=0).

Figures 2 and 3 show the 'H and 13C NMR spectra of dipalmitin in CDCI 3 .

The overall yield of 1,2-dipalmitoyl-^n-glycerol, dipalmitin, from D- mannitol was 2 0 %.

Synthesis of Choline Tosylate

In a 500 ml round bottom flask equipped with a Dean-Stark adapter and a condenser, 20.94g ( 0.15 mol) of anhydrous choline chloride (Aldrich), J u J u l i

7. S 7.06.56.0 5.5 3 .0 4.54 .0 3.53.03 .5 1.5 1.0 .5 PPM

Figure 2: NMR (250 MHz) spectrum of l,2~dipalmitin in CDCh. Spectrometer parameters were sw=2381 Hz, aq=3.44 s, 01=3917 Hz and lb=0. r” " i ■ ■ ■-T— • ■ , ...... 160 140 120 100 BO 60 40 20 PPM

Figure 3: 13C NMR (62.9 MHz) spectrum of 1,2-dipalmitin in CDCI 3 . Spectrometer parameters were sw = 17857 Hz, aq=0.918 s, 01=3700, 02=4350 and lb=3 Hz. 17

28.55g ( 0. 15 mol) of p-toluenesulfonic acid monohydrate in 200 ml of

toluene was refluxed for four hours during which time the molecular equivalence of water was azeotropically removed. The reaction mixture

was cooled and the solvents evaporated under reduced pressure. The crude product was recrystallized from hot acetone, washed with anhydrous diethyl ether, dried in vacuo (95% yield) and characterized by NMR at

200 MHz in CDCI 3 : 5.2.31 ppm (s,3H, C//3 *phenyl); 3.24 ppm (s,9H,

N (CW3 )3 ); 3.62 ppm (m, 2H, C //2 N); 4.01 ppm (m, 2H, OCH2), 7.12 and

7.16 ppm (d,J=8.0 Hz , 2H, meta-phenyl); 7.67 and 7.71 ppm (d, J = 8.0

Hz, 2H, orr/io-phenyl). Choline tosylate was stored in an evacuated desiccator over phosphorus pentoxide.

* Synthesis of Specifically Deuterated Choline Tosylates

The overall synthesis of the specifically deuterated choline tosylates is given in scheme 2 .

Synthesis of 2,2-f2H2 ]-choline iodide

2,2-[2 H 2 1-choline iodide was synthesized according to the procedure of

Dauben and Gee (1952). In a dry 100 ml round bottom flask equipped with a pressure equalizing addition funnel and a condenser, 0.84g (0.020 mol) of lithium aluminum deuteride (Aldrich Chemical Co., 98 atom % 2 H) was suspended in 12 ml of anhydrous tetrahydrofuran (distilled from NaH). To «d2 o

(CH3>2 1 LiAl rt, HO

2 H3O* 3 CHjI

Pd;

D D Choline H O ^ X AgOTs CN N (CHj)j I

2 HjCf Tosylates

3 CH3I

N dj HO. *n (CDj), a —I

( Purchased from MSO Isotopes)

Scheme 2: The Overall Synthesis of Specifically Deuterated Choline Tosylates

00 19 this solution was added dropwise a solution containing 4.20 ml (0.030 mol) of N, N-dimethylglycine ethyl ester (Fluka Biochemicals) in anhydrous tetrahydrofuran. The rate of addition was such to maintain a gentle reflux.

After the addition was complete, the reaction mixture was heated to allow further refluxing for 3 hours. The reaction mixture was cooled to room temperature and quenched by the slow addition of 3 ml of water. The resulting gelatinous mixture was filtered through a short column of diatomaceous earth. The residue was washed with 1 0 ml of water and 10 ml of acetone. The filtTate was evaporated under reduced pressure and yielded a yellow oil. The crude product, N,N-dimethyl- 2 , 2 -[2 H 2 ]- ethanolamine was isolated once by fractional distillation (bp= 135-137°C, yield 27%). It was found later that isolation of the product was not necessary prior to methylation using methyl iodide and thus was no longer done. To a solution containing 2.73g (0.030 mol) of N, N-dimethyl- 2,2-

[2 H 2 ]-ethanolamine in 10 ml of absolute ethanol was added 9.3 ml (0. 150 mol) of methyl iodide (J.T Baker). The reaction mixture was heated to refluxing conditions for 1 hour. Upon cooling in an ice bath, whitish crystals of 2 , 2 -[2 H 2 ]choline iodide precipitated out (81% yield). The product was filtered, washed with anhydrous cold diethyl ether (dried over

NaH), dried under high vacuum, and characterized by ]H NMR at 200 MHz in CDCI3: 8 3.24 ppm (s, 9H, N(CW3)3); 3.51 ppm (s, 2 H, CH2N). 20

Synthesis of 1,1 -[2H2 }-choline iodide

1,1 [2 H 2 )-choline iodide was synthesized by the deuteride reduction of cyanomethyl benzoate to produce 1, l-[ 2 H 2 ]-ethanolamine and subsequent methylation using methyl iodide as outlined by Douglas and Burditt (1955).

Cyanomethyl benzoate was prepared by adding 24 ml (0.27 mol) of a 37% formalin solution (J.T. Baker) to a solution of 15.4g (0.29 mol) of sodium cyanide (MCB Chemicals) in 50 ml of water at 0°C. After 3 hours, 30 ml

(0.26 mol) of benzoyl chloride (MCB Chemicals) was added dropwise with stirring at 0°C. The reaction mixture was allowed to stir overnight at 0°C.

To this reaction mixture, 30 ml of 5% aqueous sodium carbonate was added. The product, cyanomethyl benzoate, was extracted using four 200 ml portions of diethyl ether. The ether extract was dried over molecular sieves 3A. The removal of diethyl ether under reduced pressure afforded an oil. The oil was solidified at -30°C and characterized by NMR at

200 MHz in CD 3 OD: 6 5. 1 ppm (s,2H, OC//2 CN); 7.52 ppm (m, 2H, meta - phenyl); 7.68 ppm (m,lH, para-phenyl); 8.04 ppm (m, 2H, ort/io-phenyl).

To a suspension of 3.2g (0.075 mol) of lithium aluminum deuteride

(Aldrich, 98 atom % 2 H) in 50 ml of anhydrous tetrahydrofuran (distilled from NaH) was added dropwise a solution containing 8 . lg (0.05 mol) of cyanomethyl benzoate in 10 ml of anhydrous tetrahydrofuran. The addition rate was fast enough to maintain a gentle reflux. The reaction mixture was then stirred for 5 hours more under gentle reflux conditions. After cooling to room temperature, 10 ml of water was added and allowed to stir for 30 minutes. The resulting gel was filtered through a short column of diatomaceous earth and washed with two 1 0 ml portions of acetone and two

10 ml portions of wdter. All of the solvents were removed by evaporation under reduced pressure and the resulting orange oil was dried under vacuum {<0.3 Torr) for a few days. The crude product, 1,1 -[ 2 3- ethanolamine, was used without purification or characterization because distillation afforded low yields and it was found unnecessary to purify it at this stage. To a solution of 2.3g (0.025mol) of 1,1-[ 2 H 2 ]-ethanolamine in

5 ml of chloroform was added 7.0 ml (0. 10 mol) of methyl iodide and 2.0g

(0 . 0 2 mol) of anhydrous potassium bicarbonate. The heterogeneous mixture was allowed to stir for 48 hours. The potassium salts were filtered off and washed with two 10 ml portions of absolute ethanol. The filtrate was evaporated under reduced pressure and the orangish crystals of 1 , 1 -

[2 H 2 ]-choline iodide (76% yield) were dried under vacuum. The product was characterized by *H NMR at 200 MHz in CD 3 OD: 6 3.24 ppm (s,9H,

N{C«3)3); 4-01 ppm (s,2H, HOCH2).

The isotopic enrichments of the deuterated choline tosylate salts were assessed using mass spectroscopy and high resolution NMR. Mass spectroscopy was performed on both the choline iodides and the tosylate salts. The data obtained from the spectra were not reliable because in the case of the iodide salts the compounds were decomposing too fast over the different ionization temperatures, thus causing the intensities of the molecular fragment peaks to be low. The first fragmentation produces the molecule, N,N-dimethylethanolamine. The mass ratio of the deutero to proteo compounds corresponding to m/e values of 91.0967 and 89.0841, respectively, was used to calculate % enrichment. With decomposition 22 occurring too quickly, these peaks were not very intense and their mass

ratios unreliable. Using the tosylate salts introduced another problem.

R.H. Wiley, (1970) studied the mass spectral fragmentation of p-

toluenesulfonic acid and found that it was sensitive to temperature and

volatization time. He noted that the molecular ion peak (m/e 172) often

gave way to fragments of m/e 107 ([C 7 H 7 0 +]) and m/e 91 ([C 7 H 7 +]).

Obviously, the peak at m/e 91 would interfere greatly with the choline

fragment under investigation. This was indeed the case because the m/e 91

fragment from p-toluenesulfonic acid was always more intense than the m/e

91 fragment of dideuterated N,N-dimethylethanolamine. The isotopic

enrichments for 2 , 2 -[2 H 2 ]-choline iodide and 1,1-[ 2 H 2 ]-choline iodide

were 8 6 % and 85%, respectively, using this method. The isotopic enrichments were alternatively assessed by !H NMR. The sensitivity of the

spectrometer was >97% and the absence of peaks corresponding to the

protons of 2, 2-( 2 H 2 J-choline iodide and 1,1 *[ 2 H 2 ]-choline iodide showed that both were >97% isotopically labeled.

N,N,N-Trimethyl-l2Hg]-choline chloride

N ,N,N-trimethyl-[2 H 9 }-choline chloride was purchased from MSD isotopes and used without further purification. The deuterium enrichment was 99. 2%. 23 Conversion of the above deuterated choline halides to their respective tosylates

The above choline halides were converted to their respective tosylate salts using silver tosylate. Silver tosylate was synthesized by a modified procedure of Dauben and Gee, (1952). In a 100 ml round bottom flask,

1.9g {0.012 mol) of p-toluenesulfonic acid monohydrate was dissolved in 8 ml of water. A solution containing 1.7g (0.01 mol) of silver nitrate in 6 ml of water was added dropwise to the vigorously stirring p-toluenesulfonic acid solution. The reaction mixture was cooled to 0°C and the white crystals of silver tosylate (>98% yield) were filtered, washed with cold acetone, dried under high vacuum in the absence of light. The product was characterized by *H NMR at 200 MHz in CDCI 3 : 8 2.26 ppm (s, 3H, CH 3 - phenyl); 7.08 and 7.12 ppm (d, 2H, J=7.4 Hz, mera-phenyl); 7.59 and

7.63 ppm (d, 2H, J = 7.4 Hz, orrho-phenyl). The *H NMR chemical shifts of silver tosylate and choline tosylate are slightly different and thus provided a means to judge the purity of the choline tosylates.

The respective choline halides were then converted to their tosylate salts using silver tosylate. Aqueous solutions containing equimolar equivalents of the choline halide and silver tosylate were mixed. The silver salts were filtered off and the choline tosylate was recrystallized from hot acetone after drying. The choline tosylates were stored in vacuo.

Figures 4, 5, and 6 show the comparison of the j H NMR spectra (200

MHz) of the deuterated choline tosylates to the protonated choline tosylates in CDCI 3 . 24

T—

Figure 4: Comparison of the *H NMR (200 MHz) spectra of 2 , 2 -[2 H 2 ]- choline tosylate (B) and choline tosylate (A) in CDCI 3 . 25

B X

f, o 5 * * 5 * 9 J 5 I 0 ? 'j ? o

Figure 5: Comparison of the lH NMR (200 MHz) spectra of 1,1 - £2 H ^ J - choline tosylate (B) and choline tosylate (A) in CDCI 3 . 26

T T 6 0 ^ 3 0 <5 <0 3 ti 30 ? 0

Figure 6 : Comparison of the JH NMR (200 MHz) spectra of N.N.N- trimethyl-[2 H 9 ]-choline tosylate (B), and choline tosylate (A) in CDCI 3 . 27

Synthesis of 1,2-Dipalmitoyl-.sn-glycero-3-thiophosphocholine

Using thiophosphoryl chloride

The synthesis of l,2-dipalmitoyl-.yn-glycero-3-thiophosphocholine,

DPPsC, was achieved via two different routes. Scheme 3 (A and B) shows the two synthetic routes. The two schemes differ in the phosphorus valence number associated with the two alcoholic condensations. Scheme

3A makes use of a pentavalent phosphorus compound, namely, thiophosphoryl chloride (Vasilenko, et al., (1982)). Scheme 3B makes use of a highly reactive trivalent phosphorus compound, namely, N,N- diisopropylmethylphosphonamidic chloride (Bruzik, et al., (1986)). In scheme 3A, a solution containing l.Og (1,76 mmol) of dipalmitin and 0.37 ml (2.64 mmol) of triethylamine in 15 ml of anhydrous ethanol-free chloroform is dropped into a solution containing 0.268 ml (2.64 mmol) of thiophosphoryl chloride in 2.5 ml of anhydrous ethanol-free chloroform.

The reaction mixture was allowed to stir at room temperature for 45 minutes, then at 45°C for 30 minutes. To this solution was added a solution of 0.968g (3. 52 mmol) anhydrous choline tosylate in 1.42 ml

(17.6 mmol) of anhydrous pyridine. The reaction mixture was stirred overnight at room temperature. The progress of the reaction was monitored by TLC using solvent system C, 66:33:4, (v:v:v), chloroform :methanol: water, Rf = 0.6. Upon completion, one to two milliliters of water were added and the reaction mixture stirred for 30 minutes. The solvents were 28

C,jH31COO- C13H3ICOO— 1. P S C 13 C15H3iCOO- C i;H3lCOO — 2. MO"*' -----^ (Ch 3)3 tS° ' — OH >—O-P-O'^'—N (Chj)3 I o ( Rp + Sp ) - DPP4C

B

C ISH „ C O O - ,N (i-Pr)j C,jH3iCOO— C l-p : 'O C H i C13H31C O O - C,j H3iCOO—

— OH —O—P—N (i-Pr>2 QCH3

1 Tso C,jH31COO— 2. Sa/Toluene c ish 31c o o - 3. (CH3)3 N

— 0 -PI -O '^ 'N

Scheme 3: Synthetic routes for the synthesis of (Rp+Sp)-DPPsC. (A) using thiophosphoryl chloride and (B) using N,N- diisopropylmethylphosphonamidic chloride as the phosphorylating agents. 29 evaporated under reduced pressure and the crude product purified by silica gel chromatography using solvent system C, Rf = 0.6, yield 40-50%

{Vasilenko, et al.,(1982) and Bruzik, et al., (1983)) and characterized by

31P NMR (121.5 MHz) in CDC13: 5 56.318 ppm, (Sp)-DPPsC and 56.271 ppm (Rp)-DPPsC; *H NMR (500 MHz) in CDC13: 6 0.88 ppm (t, J=7 Hz,

6 H, acyl chain I6 -C//3 ); 1.26 ppm (s, 48H, acyl chain 15-4-C//2); 1.58 ppm (m, 4H, acyl chain 'i-CHj)', 2.28 ppm (m, 4H, acyl chain 2-CHj);

3.38 ppm (s, 9H, choline N(C//3)3); 3.85 ppm (m, 2H, choline p-C/^N);

4.07 ppm (dd, J = 6 Hz, 2H, glycerol 3 -CH 2 OP); 4.15 ppm (dd, J=7 Hz,

2H, glycerol I-C// 2 O); 4.36 ppm (m, 2H, choline (X-CH2 ) and 5.24 ppm

(m, 1H, glycerol 2-CHO) and 13C NMR (75.4 MHz) in CDCI 3 : 6 14.08 ppm (acyl chain 16-CH3); 22.66 ppm (acyl chain I5 -CH 2 ); 24.93 ppm (acyl chain M -C H2 ); 29.68 ppm (acyl chain 13-4-CH2); 31.90 ppm (acyl chain

3 -CH 2 ); 34.14 ppm and 34.33 ppm (acyl chain 2 -CH 2 ); 54.74 ppm (choline

N(CH3)3); 59.35 ppm (choline 01-CH 2 ); 62.95 ppm (glycerol I-CH 2 O);

63.77 ppm (glycerol 3 -CH 2 OP); 70.23 ppm (choline P-CH 2 N); 70.32 ppm

(glycerol 2-CHO) and 173.25 ppm and 173.65 ppm (acyl chain C=0) , figures 7, 8 , and 9, respectively. Figure 10 shows the homonuclear decoupling *H NMR spectrum of the choline and glycerol regions. The glycerol 2-CHO proton was irradiated and the collapsing of the I-C / / 2 and

3 -CH2 O glycerol peaks allowed their assignments to be made. T T 1 "T I 56 8 56 .6 56. 4 56.2 56,0 55.0 55 . 6 55 . 4 55.2 55.0 PPH

Figure 7- 31P NMR (121.5 MHz) spectrum of (Rp + Sp)-DPPsC in CDCI 3 . Spectrometer parameters were sw = 2000 Hz, aq = 2.048 s, 01=6100 Hz, 02=4800 Hz, DP=22H, lb=-1.0 Hz and gb=0.08 Hz. Referenced to external 85% H 3 PO4 . o /

A

) r— r-—T— r —y —r p r i-p —i--1----- 1---- 1---- 1---- 1---- 1---- 1---- \ j p 1---- \---- 1----- p —*-f 1----- ,---- 1---- p---- ,---- ,---- ,---j— i ------,---- ,------, j----r —r —(--r - | -r 1-----1---- r — |— 1 5.5 5.0 A .5 4.0 3.5 3.0 2.5 2.0 1.5 1 0 PPM

Figure 8 : JH (500 MHz) spectrum of (Rp + Sp)-DPPsC in CDCI 3 . Spectrometer parameters were sw=6024 Hz, aq=1.36 s, 01=7500 Hz and lb=0.1 HZ. J

| • ■ , • i 1 ■ 1 ...... 160 140 120 100 60 60 40 20 PPM

Figure 9: 13C NMR (75.4 MHz) of (Rp+Sp)-DPPsC in CDCI3. Spectrometer parameters were sw = 15,625 Hz, aq= 1.049 s, 01=40148 Hz, 02=4800 Hz, DP = 22H and lb = 3.0 Hz. UJ M " T " ' ”T’" "T”' 5. 20 5.00 4. 00 4.60 4 .40 4.20 4 . o o 3.60 PPK

Figure 10: Homonuclear decoupling of the lH NMR (500 MHz) spectrum of (Rp+Sp)-DPPsC in CDCI 3 . Spectrum before irradiation, (A) and irradiation of the glycerol 2-CHO proton (B) was used to confirm the assignments of the glycerol I-CH 2 O and 3 - 0 ^ 0 protons. oj Oo 34

Using N,N-diisopropylmethylphosphonamidic chloride

Following the procedure in scheme 3B, a clean dry 100 ml round

bottom flask equipped with an injection port and vacuum adapter, l.OOg

(1.76 mmol) of dipalmitin was placed and allowed to dry overnight under

high vacuum (<0.01 Torr). Using the vacuum line, 0.3764g (3.73 mmol)

of freshly distilled triethylamine and approximately 10-15 ml of anhydrous

ethanol-free chloroform were distilled into the flask containing dipalmitin.

While the contents were still frozen, 0.411 ml (2.11 mmol) of N,N-

diisopropylmethylphosphonamidic chloride was added via syringe through

the injection port. The reaction mixture was allowed to warm to room

temperature and stirred for approximately 2.5 hours. The condensation

was judged complete by the disappearance of dipalmitin using TLC, solvent

system D, hexanes.diethyl ether, (v:v), 2:1, (dipalmitin Rf=0.2,

condensation product, Rf=0. 8 ). The reaction mixture was dried under

reduced pressure and allowed to dry under high vacuum overnight to assure

the removal of excess phosphorylating agent. To this reaction flask was

added a solution of 1.4626g (5.28 mmol) of anhydrous choline tosylate and

0.4928g (7.04 mmol) of lH-tetrazole (Aldrich) in 10 ml of 50:50, (v:v),

anhydrous tetrahydrofuran:acetonitrile. The reaction mixture, which was

anhydrous and under vacuum, was stirred at room temperature for over 5

hours. The reaction was judged complete by the disappearance of the first condensation product using TLC, solvent system D (first condensation

product Rf=0.8, second condensation product, Rf=0. 1). The solvents were

removed and the reaction mixture dried under high vacuum for more than three hours. To the reaction flask, 10 ml of anhydrous toluene was distilled in. After warming the flask to room temperature, the reaction

mixture was opened to the environment and dry elemental sulfur was

added. The reaction mixture was stirred at room temperature overnight.

The crude product was subjected to extraction with 1.5 M triethylamine

carbonic acid buffer, pH=7.0. The organic layer was dried under reduced

pressure and further dried under high vacuum overnight. To this flask, 10*

15 ml of anhydrous toluene was distilled in followed by 10 ml of

anhydrous trimethylamine. The reaction was stirred overnight at room

temperature. The reaction was judged complete by the appearance of

product on TLC in solvent system C, Rf=0.6. All solvents were removed and the crude product purified by silica gel chromatography, solvent system C, Rf=0.6, 70-80% yield. The 3 1 P, *H, and 13C NMR spectra were the same as those of figures 7, 8 , and 9 obtained by using the synthetic route in scheme 3A.

The deuterated diastereomers of DPPsC were synthesized by the phosphite method using the respectively deuterated choline tosylate in place of choline tosylate in the procedure.

Investigation of the Condensation Reaction Between Dipalmitin and

Thiophosphoryl Chloride

The reaction of thiophosphoryl chloride with one mole of alcohol yields

ROPSCh, but reaction of this with another mole of alcohol to produce

(RO)2 PSCl is very poor and not very well understood. Addition of a third 36

mole of alcohol to produce (RO^PS does not readily occur (Trotman-

Dickenson, (1973)). Due to the ambiguity in the condensation reaction of

alcohols with thiophosphoryl chloride, the condensation reactions were

thus further investigated so as to improve the yield and conditions.

Vasilenko and co-workers (1982) have stated that the reactivity of

diacylglycerol with thiophosphoryl chloride is much lower than the

reactivity with choline tosylate and thus the dipalmitin condensation was

chosen for further investigation. Several aspects of the condensation

reaction were studied utilizing 3IP NMR, namely, i) the molar ratio of

reagents, ii) the relative rate of condensation and iii) the relative rate of

hydrolysis.

A Bruker WP-200 NMR spectrometer was used with a phosphorus frequency of 81 MHz. Proton decoupling was achieved using broadband proton decoupling with 1.5-2.0 Watts of power. The spectrometer parameters used in figures 11 through 14 were SW=10,000 Hz, 01=87000

Hz, 02=3700 Hz, AQ=0.410 s, and LB = 3.0 Hz. Deuterium lock was used and all signals were referenced from external 85% H 3 PO4 .

A typical experiment involved dropping a solution of 0 .15g (0.264 mmol) of dipalmitin and 0.49 ml (0.352 mmol) of triethylamine in 2.25 ml of deutero-chloroform into a 10 mm NMR tube containing 0.036 ml (0.352 mmol) of thiophosphoryl chloride in 0.34 ml of deuterochloroform under a nitrogen atmosphere. Stirring was achieved by use of a vortex stirrer.

Figure 11A shows the 31P NMR resonance of thiophosphoryl chloride at 32 ppm. The absence of other peaks reveals the purity of the thiophosphoryl chloride used. Figure 11B, which is the reaction thirty 37

65 6 0 40 35 30 SO PPM

Figure 11: Condensation reaction between thiophosphoryl chloride and dipalmitin in CDCI 3 . Pure thiophosphoryl chloride, (A) and 30 minutes after addition of dipalmitin, (B), inset shows the proton coupled spectrum revealing coupling between the phosphorus and the glycerol 3 -CH 2 O protons. Spectrometer parameters are described in the text. minutes after the addition of the dipalmitin-triethylamine solution, reveals the appearance of a new resonance at 59.5 ppm. This resonance was assigned as the condensation of dipalmitin with thiophosphoryl chloride to produce 1,2-dipalmitoyl-.s/j-glycero-3-thiophosphoric acid dichloride,

DPPsACl2 - The insert of figure 1 1 B shows the proton coupled spectrum of

DPPsACh. with Jp_H= 1 0 Hz, which is consistent with that observed for

SP(OCH 2 CH 3 )Cl2 (Topics in Phosphorus Chemistry, vol. 5 p 364 (1967).

An optimal reaction time of 2.5 hours was obtained by observing the integrated ratios of thiophosphoryl chloride and thiophosphatidic acid dichloride.

The spectra in figure 12 are the reaction with an excess of thiophosphoryl chloride (4 times). After 2.5 hours the integrated ratio of the resonances at 59.5 and 32 ppm is 1:3. This ratio did not change over time. Increasing the amount of thiophosphoryl chloride did not appear to cause the rate of the reaction to increase significantly.

The spectra in figure 13A-C illustrate the rate of hydrolysis of thiophosphoryl chloride and DPPSACI 2 . The hydrolysis of thiophosphoryl chloride was complete within 1 hour. These spectra also reveal that the hydrolysis product, thiophosphatidic acid, resonates at a higher field than does the chlorinated compound. The effect of chlorine on the 31P NMR chemical shift can be illustrated by examining the chemical shifts for POCl3 and (P 0 4 >3* 3 Na+,( 3.0 and 6.0 ppm, respectively), and for PSCI 3 and

(SPO 3 )3- 3 Na+, (31 and 61 ppm, respectively) (Gorenstein & Shah,

(1984)). Some interesting points are noted in the comparisons. First, the difference between the chlorinated oxyphosphorus and ionic phosphate is 39

rnn rT~' "1" 6 5 60 55 30 49 30 PPM

Figure 12: Condensation reaction of excess thiophosphoryl chloride and dipalmitin in CDCI 3 . Reaction after 30 min (A), reaction after 2.5 hrs (B), and reaction after 8 hrs , showing some hydrolysis products around 45 ppm, (C). Spectrometer parameters are described in the text. ' T ’ " ’ "1 •*' ■ I ■ TTT TTT • r * 1 ’TT 'T~r 50 45 40 35 30 50 45 40 35 30 50 45 JO 35 30 PPM PPM PPM

Figure 13: The hydrolysis of thiophosphoryl chloride in CDCla. Pure thiophosphoryl chloride (A), 15 min after the addition of water (B), and 1 hour after the addition of water (C).

o 4 1 quite small compared to thiophosphoryl chloride and ionic thiophosphate.

Second, the substitution of chlorine with oxygen results in deshielding, which shifts the phosphorus resonance to lower magnetic field. The hydrolysis reaction of thiophosphatidic acid dichloride is shown in figure

14A-C. It is then quite clear that the hydrolysis of DPPsACli (59.5 ppm) to DPPsA (59.8 ppm) showing an increase in the 31P chemical shift is not only feasible but quite probable. These figures also show that the hydrolysis of DPPsACl 2 was also complete in approximately 1 hour.

The investigation of the condensation of dipalmitin with thiophosphoryl chloride yielded several conclusions: i) the molar ratio of dipalmitin to thiophosphoryl chloride which yielded the best result was 1:3, ii) the heating step was important but needed to be extended to 2.5 hours instead of 30 minutes and iii) the relative rates of hydrolysis did not become significant until after the 2.5 hour heating time. These changes were made in the procedure for this and other thiophosphatidyl lipids synthesized in this manner.

Resolution of the Diastereomers of DPPsC using PL A2

It has already been shown that PL A2 from various sources shows stereoselectivity towards the hydrolysis of DPPsC diastereomers (Bruzik, et al., (1982)). Only the Rp isomer of DPPsC was hydrolyzed to give lyso-Rp ((Rp)-Monopalmitoyl Thiophosphatidylcholine). The Sp isomer was not hydrolyzed. Scheme 4 shows the separation scheme. Typically,

300 mg (0.400 mmol) of (Rp+Sp)-DPPsC was dissolved in 15 ml of 42

70 63 60 53 3 0 43 40 35 30

Figure 14: The hydrolysis of thiophosphatidic acid dichloride in CDCI 3 . Thiophosphatidic acid dichloride and thiophosphoryl chloride (1:3) (A), 30 min after the addition of water (B), and 1 hour after the addition of water (C). 43

r— OOCCuHj,

C |jH 3lCOO— s I ,N (CH3)3 —o-p-o- I o

(Rp + Sp)-DPPsC

phospholipase A2

Silica Gal Chromatography

DPPsC — OOCCitHis n 3i > 98 % Sp [< 2% Rp HO—1

S ,N(CH5)3 O-P-O'i o (Rp)-MPPsC

(Ci5H3iC0)20

DMAP

(Rp)-DPPsC

Scheme 4: Resolution of the diastereomers of DPPsC using PL A2. 44

chloroform and 90 ml of diethyl ether. To this homogeneous solution was

added 1-2 mg ( 2400 U/mg ) of bee venom PL A2 (Boehringer Mannheim)

in 0.5ml HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

buffer, pH = 7.2. The reaction mixture was stirred at room temperature and

monitored by TLC, solvent system C, DPPsC Rf=0.6, MPPsC Rf=0.3. The

reaction was judged complete by observing the disappearance of (Rp)-

DPPsC using high resolution 31P NMR. The sensitivity of the instrument

was as good as the signal to noise ratio obtained for the standard sample,

which was typically taken as the intensity of the spinning side bands (1.5-

2%). An aliquot of the reaction mixture was taken, dried and redissolved

in deuterochloroform. The 31P NMR parameters were; SF=12I.497 MHz,

SW= 2000 Hz, AQ= 4.1 sec, GB= 0.08 Hz, LB= -3.0 Hz. (Rp)-DPPsC

resonated at 56.515 ppm, lyso- Rp at 56.615 ppm and (Sp)-DPPsC at

56.545 ppm. The width at half maximal height for (Rp)- and (Sp)-DPPsC

was < 2 Hz. The chemical shift difference between diastereomers in CDCI 3

was 9 Hz (0.03 ppm). Obtaining a high signal to noise ratio allowed

detection of (Rp)-DPPsC to 2-3%, thus the digestion yielded (Sp)-DPPsC

that was >97% diastereomerically pure. No lyso-Sp was ever detected.

Lyso-Rp was reacylated using palmitic anhydride and 4-N,N-

dimethylaminopyridine, DMAP, as a catalyst. Typically, lOOmg (0.196

mmol) of lyso-Rp was dissolved in 10 ml of anhydrous ethanol-free chloroform. To this mixture 200mg (0.404 mmol) of palmitic anhydride and 50mg (0.410 mmol) of DMAP were added. The reaction was judged complete by the disappearance of lyso-Rp using TLC, solvent system C,

lyso-Rp, Rf=0.3, (Rp)-DPPsC, Rf=0.6. The DMAP was removed by 45 adding a dual ion exchange resin, Rexyn-300 (Fisher Scientific) to the reaction mixture. The crude product was purified by silica gel chromatography in solvent system C, yield >95%. No acyl or phosphoryl migration was observed using TLC (one spot corresponding to DPPsC) and

31P NMR (resonance corresponding to Rp isomer, 1,3-DPPsC resonates at upfield from 1,2-DPPsC)

II.3 Discussion

The goal of this chapter was to use and modify published procedures to synthesize DPPsC and the various deuterated choline derivatives of DPPsC.

The final objective being to have diastereomerically pure DPPsC samples to use in various biophysical studies. The synthesis of dipalmitin was performed so as to familiarize myself with the organic techniques of synthesizing lipids.

The synthesis of DPPsC accomplished through two fundamentally different pathways has provided a more higher yielding reaction scheme.

The use of thiophosphoryl chloride was the method used in our laboratory in the past. But because of the low overall yield, (40-50%) the phosphite method has now become the method of choice. The phosphoryl chloride method was optimized studying the condensation of dipalmitin and thiophosphoryl chloride. This step proved to be the product rate limiting step. This was done because the synthesis of the specifically deuterated

DPPsC diastereomers was desired in high yield due to the many steps involved in synthesizing the deuterated choline tosylates. Obtaining only a 46

40-50% yield meant that a lot of the choline tosylates were needed. Using the phosphite method in which the yield was 70-80%, meant that the amount of choline tosylate needed was almost stoichiometric.

As was mentioned in the introduction, the other purpose behind the synthesis of the DPPsC diastereomers and derivatives was use in biophysical studies. The resolved diastereomers of non-deuterated DPPsC were used in the 31 P NMR studies. The resolved diastereomers of deuterated DPPsC were used in the biophysical studies employing 2H

NMR. 47

II. 4 References

Baer.E., & Fischer, H.O.L. (1939)7. Am. Chem. Soc. 61, 761-765.

Baer,E., & Fischer, H.O.L. (1945) 7. Am. Chem. Soc. 67, 2031-2037.

Bird, P.F., & Chadha, J.S. (1966) Tetrahedron Lett. 38, 4541-4546.

Bruzik, K., Gupte.S.M., &Tsai,M.-D. (1982) 7, Am. Chem. Soc. 104, 4682-4684.

Bruzik, K., Jiang, R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478- 2486.

Bruzik, K., Salamonczk, G., & Stec, J. (1986) 7. Org. Chem. 51, 2368- 2370.

Dauben, W.G., Gee, M. (1952) 7. Amer. Chem. Soc. 74, 1078-1079.

Douglas, D.E., & Burditt, A.M. (1955) Can. J. Chem. 33, 1 183-1184.

Gorenstein, D., and Shah, D. (1984) in Phosphorus-31 NMR, Principles and Applications 549-591.

Howe, R.J., & Malkin, T. (1951) 7. Chem. Soc. 2663-2667.

Jensen, R.G., & Pitas, R.E. (1976) Adv. Lipid Res. 14, 213-247.

Mark, V., Dungan, C.H., Crutchfield, M.M, & Van Wazer, J.R. (1967) Topics in Phosphorus Chemistry 364.5,

Schlenk, W. (1965) 7. Am. Oil Chem. Soc. 42, 945.

Sowden, J.C. & Fischer, H.O.L. (1941) 7. Am. Chem. Soc.63, 3244- 3248.

Vasilenko, I., DeKruijff, B., & Verkleij, A.J (1982) Biochim. Biophys. Acta 685,. 144-152.

Wiley, R.H. (1970) Organic Mass Spectrometry 4, 55-58. CHAPTER III SYNTHESIS AND ENZYMATIC REACTIONS

OF THIOPHOSPH ATIDYL-L-S ERINE

III. 1 Introduction

This chapter deals with the synthesis of 1,2-dipalmitoyl-.m-3-glycero-

thiophosphoryl-L-serine, DPPsS, and its reaction with two phospholipases.

The goal of this synthesis was to devise a totally synthetic scheme, scheme

5, to arrive at pure resolved diastereomers. This was envisioned to occur

through a ring intermediate. The ring intermediate should be stable enough

for isolation but labile enough for deprotection. The balance between these

two factors has presented the major obstacle in this synthesis. A ring

intermediate that was isolable and stable was formed, but could not be deprotected. Because of this, blocking groups that were easily removed

were sought, so as to provide a synthetic scheme that would yield the

diastereomeric mixture. Three different serine derivatives yielded the diastereomeric mixture, but only the route in scheme 6 produced a desirable

yield.

The diastereomeric mixture was then subjected to two phospholipases that have shown opposite affinities for the diastereomers of various thiophospholipids (Bruzik, etal., (1983)). Phospholipase A2, which is

48 C15H31CO O - c 13h 31o o o - 1. PSCl3 c 1sh 31o o o - c 15h 31o c o - S I l-O H 1-0— p—ci I Cl

HO c 13h 31o o o -,

c , 5h 31c o o h HNR. . . i COOR

COOR I R

1. Separation into C, sH3 iCOO— i

diasteromers C1SH 3,C 0 0 — S _ I .C O O - 2. Deblocking of R groups *— O—— O— P— P~ 3 Ri"8 Opening \QH +NHj

(Rp) & (Sp) DPPsS

Scheme 5: A general scheme for the synthesis of the diasteromers of DPPsS via a 2-thiono-l,3,2- oxazaphosphilidine ring intermediate(I). 50

c i5 h „o o o ^ C15H310 0 0 ^ -N (i-POj C ,5H j,COO— c i - p ; 'O C H i c 13h51c o o —

— OH — O -P -N ( i-Pr); o c h 3

1- H O

HN COOCHjOCHj T r

2. Sg/Toluene

C lSH3tC O O -

c !5t:3|000— S COOCHiOCHi I. HCl/Aceione —O—P—o OCH3 HNTr 2. (CH3)3N

c o o - — o —p—o

+ N H

(Rp+Sp)-DPPsS

Scheme 6 : The synthesis of DPPsS using the phosphite method and N- trityl-L-serine methoxy methyl ester. 5 1 specific for the Rp isomer, hydrolyzed it to yield pure Sp from the mixture.

Phospholipase C, which is specific for the Sp isomer, did not hydrolyze

the Sp isomer due to the decrease in activity towards phosphatidyl-L-serine

as a substrate and also the decrease due to the sulfur substitution.

While the mixture was a disappointment in terms of achieving pure

diastereomers, it did in effect establish the synthetic route for a

ph ospholipid analog that prior to this had never been created. For that I am

grateful and hope that some physical studies may be performed on the

mixture, while efforts on resolving the diastereomers are made.

III.2 Experimental Procedures and Results^

Synthesis of 1,2-Dipalmitoyl-.sn-3-glycerothiophosphoryl-L-serine benzyl

ester (DPPsS benzyl ester)

L-serine benzyl ester is commercially available from Sigma Chemical

Co. The three commercially available L-serine esters are benzyl, methyl, and ethyl. Out of those three, benzyl should be the easiest to remove.

The synthesis of DPPsS benzyl ester was monitored by 31P NMR to

assess the relative stability of the 2 -thiono- 1 ,3 , 2 -oxazaphosphilidine ring

formed. The structure of such a ring is shown in scheme 5.

The spectrometer parameters for figures 15 through 23 were SF=121.5

MHz, SW= 15,000 Hz, AQ=0.27 sec, 01=5000 Hz, 02=4800 Hz, DP=22H,

TE=297 K, LB= -1.0 Hz and GB=0.07 Hz. All resonances were referenced from external 85% H 3 PO4 . In a 100 ml flask fitted with a septum port. 52

0. 1743g (1.03 mmol) of thiophosphoryl chloride, 0.2315g (2.29 mmol) of triethylamine and 1 0 ml of anhydrous ethanol-free chloroform were distilled in under vacuum to 0. 200g (0.352 mmol) dipalmitin. The reaction mixture was allowed to stir at 45°C for two hours. After two hours the solvents were evaporated under reduced pressure and the contents were allowed to dry overnight under high vacuum (<0.001 Torr). To this flask a solution of 0.0817g (0.352 mmol) L-serine benzyl ester and 0.3214g (3.18 mmol) triethylamine in ~5 ml of anhydrous ethanol-free chloroform was syringed in. Immediately, approximately 1-2 ml of this reaction mixture was placed in a 10 mm NMR tube fitted with an insert containing deuterochloroforin for the spectrometer lock. Figure 15 shows the spectrum 1 immediately' after the addition of the L-serine benzyl ester solution. The spectrum reveals three main resonances. The resonance at

60.09 ppm represents dipalmitoyl thiophosphatidic acid dichloride. The two resonances at 87.44 ppm and 86.19 ppm represent the 2-thiono-1,3,2- oxazaphosphilidine ring diastereomers (for ease in writing, the 2 -thiono-

1 ,3 , 2 -oxazaphosphilidine ring diastereomers will be referred to as simply the 'ring' compound). The othfr resonances are side products from the reaction mixture. It is interesting to note the fast rate in which the ring compounds were formed. Figure 16 shows the reaction after 30 minutes.

The amounts of the ring compounds -87 ppm has increased tremendously, but there is another resonance at 60.33 ppm. This resonance is assigned as the ring opened compound, that is dipalmitoyl thiophosphatidyl-L-serine benzyl ester. The side products have also increased in intensity. The formation of the ’open’ compound must have come about from water [ 1 < t I [ I rr " r T J I I T J 1---T "------1 T t j 1------'“I------1------"T "■ —^ ---- 1------1------1— 1— *j -----r- T T r 85 80 75 70 65 60 PPM

Figure 15: 31P NMR spectrum (121.5 MHz) ' immediately' after the addition of L-serine benzyl ester to DPPsACh- I

as ao 75 70 65 60 PPM

Figure 16: 31P NMR spectrum (121.5 MHz) 30 min after the addition of L-serine benzyl ester to DPPsAC12- 55 introduced into the reaction through transferring the reaction mixture to the

NMR tube.

After 1.5 hours the amount of dipalmitoyl thiophosphatidic acid dichloride (60.09 ppm ) has decreased tremendously as is shown in figure

17. Figure 17 also shows the increase in the ring compounds (-87 ppm) and the open compound (60.33 ppm) at this time.

Figure 18 shows the reaction after 2.5 hours. The reaction mixture is at an equilibrium between the ring compounds and the open compound. At this time all of the dipalmitoyl thiophosphatidic acid dichloride has reacted as seen by the disappearance of the resonance at 60.09 ppm.

At 6.5 hours the spectrum, figure 19, has not changed, which shows the consumption of all the water that was introduced into the reaction mixture.

To this reaction mixture a small amount of water was added and the hydrolysis was followed by 31P NMR. Figure 20 shows the reaction immediately after the addition of water. There is a dramatic increase in the resonance at 60.35 ppm with a concomitant decrease in the ring compounds.

Figure 21 shows the reaction mixture 10 minutes after the addition of water. The intensities of the resonances corresponding to the ring compounds are drastically reduced.

After 30 minutes there is very little ring compound left and figure 22 shows that, plus the insert in figure 2 2 shows that there are two diastereomers present in the open compound. The diastereomer resonances are 60.38 ppm and 60.33 ppm. A difference of 0.05 ppm, which is very I 1 1 '— '— I— '— '— '— I— I— I— I— I— >— I— I— I— I— I— I— I— I— I— I— ,— I— ,— I— ,— r 85 80 75 70 65 60 PPM

Figure 17: 31P NMR spectrum (121.5 MHz) 1.5 hrs after the addition of L-serine benzyl ester to DPPsACl2. O' ] » i t i -j t n i f -'j i t i —i----1—■—i l------T------i—----1——i------—r------r ~ — r~— 85 80 75 70 55 60 PPM

Figure 18: 31P NMR spectrum (121.5 MHz) 2.5 hrs after the addition of L-serine benzyl ester to DPPsAC12. •"j _ r _ I I l 1 1 65 80 75 70 55 50 PPM

Figure 19: 31P NMR spectrum (121.5 MHz) 6.5 hrs after the addition of L-serine benzyl ester to DPPsAC12. vi 00 T TT TT T TTTTTT T T T —r T 85 80 75 70 55 60 PPM

Figure 20: 5lP NMR spectrum (121.5 MHz) 'immediately' after the addition of water to the L-serine benzyl ester reaction. —r~ I I I 85 80 75 70 65 60 PPM

Figure 21: 31P NMR spectrum (121.5 MHz) 10 min after the addition of water to the reaction.

CT' O I I I 85 60 75 70 65 60 PPM

Figure 22: 31P NMR spectrum (121.5 MHz) 30 min after the addition of water to the reaction. The insert shows the resolution of two diastereomers. 62 similar to the differences between the diastereomers of DPPsC (0.03 ppm) at the same magnetic field strength.

The hydrolysis is complete after 1 hour and the two diastereomer resonances can be seen clearly (figure 23). The open product was isolated using silica gel chromatography with the solvent system, 5:1, (v:v), chloroform;methanol, Rf=0.47, in 40% yield. The ring intermediate was both formed and hydrolyzed easily. This is not a desirable property if the goal is to be able to separate ring compounds in pure diastereomeric form.

The reaction conditions also formed a significant amount of side products.

Deblocking of DPPsS benzyl ester with TrimethylsilyI Iodide (TMSI)

DPPsS benzyl ester was debenzylated using TMSI according to the procedure described by Jung & Lyster, (1977). The reaction was attempted to be followed by *H NMR by observing the disappearance of the benzyl methylene protons at 4.3 ppm and the concomitant appearance of the benzyl iodide methylene protons at 4.4 ppm. This proved to be a difficult task seeing that other resonances of DPPsS benzyl ester are also within the chemical shift range of 4.5 to 3.0 ppm.

In a clean dry 5 mm NMR tube containing a solution of 0.028g (33.2 pmol) of DPPsS benzyl ester in 0.8 ml deuterochloroform, 6 pi (39.8 pmol) of TMSI was added. The temperature was 30°C. The appearance of the benzyl iodide methylene was noticeable after 10 min. The intensity continued to increase up until 3 hours. After this it decreased slightly and leveled off at 5 hours. At this time 2 pd more of TMSI was added to see if n u V

~1~ ~1~ 85 0 0 75 70 65 60 PPM

Figure 23: 31P NMR spectrum (121.5 MHz) 1 hr after the addition of water to the reaction. The insert shows the resolution of two diastereomers. 64 the reaction would go further. Two hours later the intensity once again

leveled off and was only slightly higher than that at 5 hours.

After the second addition had gone for two hours, 13 ill (8 eq.) of

methanol was added. The reaction mixture was dried on a vacuum line and

an orangish oil was present, which is benzyl iodide. Attempts were made

to remove the benzyl iodide by successive precipitations using hot acetone.

The precipitate was isolated but revealed a myriad of products by TLC in

5:1 (v:v) chloroform:methanol. The product DPPsS was isolated by prep-

TLC but was in low yield.

Although the product was obtained from this method, it was not the best choice of deblocking reagent. However, the TMSI reaction was the only debenzylating attempt that provided any form of product. The other debenzylating reagents tried were potassium carbonate in water- tetrahydrofuran (Huffman, et al.(1978)), aluminum trichloride in chloroform (Tsuji, et al., (9179)), copper (II) sulfate at pH 8 (Prestidge, et al.{1975)) and catalytic hydrogen transfer (Felix, et al. (1978)). Each of these were unsuccessful for a variety of reasons. The potassium carbonate reaction worked to about 90% using L-serine benzyl ester. But when the lipid was used there were severe solubility problems seeing how the lipid was not soluble in water and the potassium carbonate not being soluble in organic solvents. The same was true for the copper (II) sulfate reaction.

The reaction worked exceptionally well on L-serine benzyl ester but the lipid had solubility problems. The aluminum trichloride reaction was more extensively studied on another compound that contained both a benzyl ester and a benzyl amine. The results of that reaction will be discussed later in this chapter. Catalytic hydrogen transfer is a method of debenzylating

esters in the presence of sulfur. Sulfur atoms have been known to complex

to the palladium or platinum catalysts used in hydrogenation causing

'poisoning* or deactivation of the catalyst. The amount of ’ poisoning' a

catalyst experiences by a sulfur compound depends on the oxidation state

of the sulfur. Thiols are the strongest catalysts poisoners, while sulfates

are considerably more mild. The amount of poisoning capacity is directly

related to the availability of the nonbonding electrons on the sulfur atom.

Not knowing the full extent of the bonding order of the phosphorus-sulfur

bond, the catalytic hydrogen transfer was attempted. The reaction

produced the desulfurized product without debenzylation. This is the

common scenario for sulfur poisoning of a transition metal catalyst.

Synthesis of N-Trityl-L-serine diethyl ammonium salt

The general procedure of Stelakatos, et al., (1959) was used to

synthesize N-trityl-L-serine diethyl ammonium salt. In a 50 ml round

bottom flask l.OOg (9.52 mmol) of L-serine was dissolved in 4 ml of water

and 8 ml of 2-propanol. To this inhomogeneous solution was added 2.95

ml (28.56 mmol) of diethylamine. The now homogeneous mixture was

stirred vigorously while 2.66g (9.52 mmol) of trityl chloride was added in

twelve equal portions over one hour. Four minutes after the final addition of trityl chloride, 30 ml of water was added. This mixture was extracted with two 50 ml portions of chloroform. The chloroform solution was washed with 2 0 ml of water and dried over magnesium sulfate for at least 6 6 four hours. The chloroform solution was then decanted and dried in vacuo to afford a white foamy product. This crude product was dissolved in anhydrous ethyl ether and approximately 1 ml of diethylamine was added.

The solution was cooled to ~0°C overnight and the crystalline product was filtered off and washed with cold dry ethyl ether. N-trityl-L-serine dieihylammonium salt was produced in 2 1 % yield and characterized by ]H

NMR (200 MHz) in deuteromethanol 8 : 1.25 ppm (t, J=7 HZ,

(C/y3 CH 2 )2 N H 2); 2.96 ppm (q, J=7 HZ, (CH 3 C//2 h N H 2); 3. 15 ppm (m,

3H, HOCH2 CH) and 7.23 to 7.49 ppm (m, 15H, phenyl).

Synthesis of N-Trityl-DPPsS from N-Trityl-L-serine diethylammonium salt

The synthetic route that is shown in scheme 3B, in which the phosphorylating agent is the phosphite, N,N-diisopropylmethyl- ph osphonamidic chloride, was used to synthesize N-trityl-DPPsS. In a clean dry 1 0 0 ml round bottom flask fitted with a septum port containing

0. lOOg (0.176 mmol) dipalmitin, was distilled in 0.0447g (0.443 mmol) triethylamine and approximately 1 0 ml of anhydrous ethanol-free chloroform. To this flask was added via syringe 41 pi (0.211 mmol) of

N, N-diisopropylmethylphosphonamidic chloride. The reaction mixture was allowed to stir at room temperature for 2.5 hours, at which time the solvents were removed in vacuo and the contents dried under high vacuum

(<0.001 Torr) overnight. To this dried crude product was added a solution containing 0.2281g (0.528 mmol) of N-trityl-L-serine diethylammonium salt and 0.0493g (0.704 mmol) 1-H tetrazole in a 50:50, (v:v). 67 acetonitriiertetrahydrofuran. The reaction mixture was stirred at room temperature for 5 hours, at which time the solvents were removed in vacuo and the contents dried under high vacuum (<0.001 Torr) for 3 hours. To this flask was added 10 ml of anhydrous toluene and ~0.5g of sulfur by opening the flask to the environment after the addition of the toluene. This reaction mixture was allowed to react overnight at room temperature. The reaction mixture was washed with a 1.5M triethylamine carbonic acid solution buffered at pH=7. The organic layer was dried and redissolved in anhydrous toluene. To this solution, trimethylamine was distilled in and the reaction was allowed to proceed at room temperature overnight. The solvents were removed in vacuo and the crude product, dipalmitoyl thiophosphatidyl N-trityl-L-serine was subjected to detritylation using glacial acetic acid at 40°C. The final product, DPPsS, was isolated by silica gel chromatography using 7:1, (v:v), chloroformrmethanol, Rf=0.3.

The product was isolated in relatively low yield, -25%, compared to the synthesis of DPPsC using this method (70-80%). The low yield was due to the myriad of side products produced by the glacial acetic acid reaction.

The 31P NMR (121.5 MHz) spectrum in deuterochloroform revealed two resonances at 51.47 ppm and 51.45 ppm. The *H NMR spectra were not very well resolved, so the listing of those resonances will be reported in the discussion of the following synthetic scheme, which uses N-trityl-L- serine methoxy methyl ester.

The detritylation step was attempted in another manner by hydrogenation with palladium on carbon at the phosphite intermediate step.

This is the step just prior to the addition of sulfur to oxidize the phosphite 6 8

to a thiophosphate. It was not certain at the time of the attempt as to what

might transpire. What did happen was that the phosphite was hydrogenated

to the phosphonate and thereby not allowing the subsequent oxidation with

sulfur to take place.

Synthesis of DPPsS using N-Trityl-L-serine methoxy methyl ester

Synthesis of N-Trityl-L-serine methoxy methyl ester

The procedure described by Corey, et al., (1982) was adapted to the synthesis of N-trityl-L-serine methoxy methyl ester. In a 25 ml round bottom flask, 0.5C0g (1.2 mmol) of N-trityl-L-serine diethylammonium salt, 365 pi (4.8 mmol) of chloromethyl methyl ether and 2 ml (12 mmol) of triethylamine were dissolved in 5 ml of chloroform. The reaction was allowed to stir overnight at room temperature. The reaction mixture was dried in vacuo and the crude product purified on silica gel using 95:5,

(v:v), chloroform:acetone, Rf=0.5, 71% yield. The NMR spectrum

(250 MHz) in deuterochloroform is shown in figure 24, 5: 2.32 ppm (bs,

LH, CH 2 0//); 3.04 ppm (bs, 1H, CH-NW-Tr); 3.32 ppm (s, 3H, OC//3);

3.56 ppm (s, 2 H, HOC//2); 3.73 ppm (d, J=7 Hz, 1 H, C//-NH); 4.77 ppm and 4.93 ppm (dd, J = 7 Hz, 2H, 0-C // 2 -0) and 7.2 and 7.5 ppm (m, 15H, phenyl). Figure 25 shows the l3C NMR spectrum (62.9 MHz) in I ------I ...... ' I ------...... I ...... I ' . J • ■ B. 0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 PPM

Figure 24: *H NMR spectrum (250 MHz) of N-trityl-L-serine methoxy methyl ester in CDCI 3 .

O' sD il j l U

-,— ,— ,— -— .— .— p - — i— .— i— .— |— .— ,— ■— j - ~1— 1—’— 1— I— ’— 1— T" 1 6 0 1 4 0 120 100 80 60 40 20 PPM

Figure 25: I3C NMR spectrum (62.9 MHz) of N-trityl-L-serine methoxy methyl ester in CDClj.

o 71 deuterochloroform 8 : 57.56 ppm (CH 2 -CH-NH); 57.94 ppm (O-CH 3 ); t

64.83 ppm (HOCH 2 -); 71.04 ppm (C00-CH 2 -0); 91.06 ppm (-C(Ph)3);

126.63 ppm, 127.92 ppm, 128.74 ppm ( phenyl) and 145.59 ppm

(CHCOOCH 2 ).

Synthesis of DPPsS using N-Trityl-L-serine methoxy methyl ester

In a clean dry 100 ml round bottom flask, fitted with a septum port containing 0. 500g (0.88 mmol) of dipalmitin, was distilled in 0.750g

(0.75mmol) of triethylamine and approximately 10 ml of anhydrous ethanol-free chloroform. To this flask was added via syringe 210 jtl (1.05 mmol) of N,N-diisopropylmethyIphosphonamidic chloride. The reaction was allowed to stir at room temperature for 2 hours, at which time the solvents were removed in vacuo. To this crude product was added a solution containing 0.4235g (1.05 mmol) of N-trityl-L-serine methoxy methyl ester and 0.3077g (3.52 mmol) of 1-H tetrazole in an anhydrous mixture of dichloromethane and acetonitrile. The reaction mixture was stirred at room temperature for 1 2 hours, at which time all the solvents were removed in vacuo and the contents dried under high vacuum (< 0 . 0 0 1

Torr) for 2-3 hours. To this flask was added 10 ml of anhydrous toluene and -0.7g sulfur by opening the flask to the environment after the addition of toluene. This reaction mixture was allowed to stir at room temperature for 46 hours. The solvents were removed in vacuo and the resulting residue dissolved in 2 :1 , (v:v), hexanetdiethyl ether and applied to a silica 72 gel column. The product, dipalmitoyl methylthiophosphatidyl-N-trityl-L- serine methoxy methyl ester, was isolated in 70% yield with an Rf=0.5.

Figure 26 shows the 31P NMR spectrum (101.3 MHz) of the product in deuterochloroform, 5: 69.42 ppm and 69.33 ppm. Figure 27 shows the 'H

NMR (250 MHz) spectrum of the product in deuterochloroform, 5: 0.89 ppm (t, 6 H, J = 7 Hz, acyl chain I 6 -C//3 ); 1.27 ppm (s, 52H, acyl chain 15-

4 C //2); 1 6 0 ppm (bs, 4H, acyl chain 3-C//2); 2.30 ppm (m, 4H, acyl chain 2-C/y2); 2.81 ppm (d, 1H, serine N//); 3.30 ppm (s, 3H, ester

OC//3 ); 3.62 ppm (m, 2H, serine OC//2); 3.71 ppm (dd, 3H, J=13 Hz, P-

O-C//3 ); 4.15 ppm to 4.45 ppm (m, 5H, glycerol backbone 1 -CH2, 3-CH2 and serine -C//-NH); 4.60 ppm and 4.85 ppm (dd, 2H, J=5 Hz, ester -

OCH2-0); 5.29 ppm (bm, 1H, glycerol 2-C H) and 7.21 ppm to 7.55 ppm

(m, 1 5H, phenyl).

The N-trityl and methoxy methyl ester groups were removed by treatment of the above product with concentrated hydrochloric acid in dry acetone (Appelgate, et al.,(1979)). Specifically, 0.203g (0.194 mmol) of dipalmitoyl methylthiophosphatidyl-N-trityl-L-serine methoxy methyl ester was dissolved in 10 ml of a solution containing 3 ml of concentrated hydrochloric acid in 180 ml of dry acetone. The reaction mixture was allowed to stir at room temperature for 2.5 hours. The reaction was assessed of its completion by the disappearance of the reactant using TLC in 2:1, (v:v), hexane:diethyl ether, Rf=0.5 and of the appearance of a product that was not U.V. active in the solvent system 5:1, (v:v), chloroform: methanol. This reaction mixture was dried under high vacuum

(<0.001 Torr) overnight. To this flask was added approximately 5 ml of ’T'" ~ r - ■’T ’1 '^Tr TT T 80 70 60 50 40 30 20 10 PPM

Figure 26: 3lP NMR spectrum (101.3 MHz) of dipalmitoyl methylthiophosphatidyl-N-trityl-L-serine methoxy methyl ester in CDCI3 .

u> T T T ■’T " "T" T 'T '' e.o 7 .0 6.0 5 .0 4 .0 3 .0 2.0 1 . 0 PPM

Figure 27: NMR spectrum (250 MHz) of dipalmitoyl methylthiophosphatidyl-N-L-serine methoxy methyl ester in CDCI3 . anhydrous toluene and 20 ml of trimethylamine. The reaction mixture was stirred at room temperature overnight and the product was visualized using

TLC in 5:1, (v:v), chloroform: methanol, Rf=0.4, 70% yield. Figure 28 shows the 31P NMR spectrum (101.3 MHz) of DPPsS in deuterochloroform, 8 : 57.85 ppm. The two diastereomers could not be seen in chloroform, nor if methanol was added Also, as will be discussed below, the two diastereomers were not observable even in deuterated water with 5% triton X-100. Figure 29 shows the NMR spectrum (250 MHz) of DPPsS in deuterochloroform 8 : 0.88 ppm (t, 6 H, J=7 Hz, acyl chain 16-

CH3 ); 1.26 ppm (s, 52H, acyl chain 15-4 CH2 )', 1.60 ppm (bs, 4H, acyl chain 3 -C//2 ); 2.33 ppm (m, 4H, acyl chain 2 -C//2 ); 3.48 ppm (s, 2H, serine NH 2 ); 3.60 ppm (dd, 2H, Jp=17 Hz, Jh=9 Hz, serine OCH2 ), 4.00 ppm (m, 2H, glycerol 3-C//j); 4.20 ppm (m, 2H, glycerol I-CW 2 ); 4.40 ppm (m, 1H, serine -CH) and 5.3 ppm (bm, 1H, glycerol 2-C H). Figure

30 shows the phosphorus decoupling of the *H NMR spectrum of the serine

OCH 2 protons at 250 MHz. The two doublets collapsed to one with a coupling constant of 5 Hz upon irradiation.

The DPPsS product was synthesized as a mixture of diastereomers. The next step was to investigate the reaction of the diastereomeric mixture with two enzymes that have shown stereoselectivity towards the thio analogs of

DPPC and DPPE, namely phospholipase A2 (PL A2) and phospholipase C

(PL C) (Bruzik, et al., (1983)). 1 ' 1 ' | r ~~l 1 I | 1— 1 I---- » I »---- 1---- 1-----1 p ~ i >---- 1---- 1---- [---- 1---- 1---- 1---- 1------1— r--1--- I— r— t-----r —i-----1-----r— |---1---- 1------1-i— j — t— t — i----- r —j 75 70 65 60 55 50 45 40 35 PPM

Figure 28: 31P NMR spectrum (101.3 MHz) of DPPsS in CDCI 3 . .A_

[■■■T » | I r T ■ t - |' - 1 "T " r T 1 J"T ‘ T" T ‘ T J 1-----1 ' T 1 1 ' 1 '- ^ MT * "| " I----- T 1 1 ' 1 1— r—p-T 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1 . 0 PPM

Figure 29: ]H NMR spectrum (250 MHz) of DPPsS in CDCI 3 . T ' ' I ’ ' ■ ■ i ■ ■ ' ' I ' ' ’ ”r_ 3.70 3 .6 0 3.50 3.70 3.60 3.50 PPM PPM

Figure 30: *H NMR spectrum (250 MHz) of the serine OC / / 2 protons (A), and the 3tP decoupled ]H NMR spectrum (250 MHz) of the serine OCH2 protons (B) in CDCI 3 .

00 79

Reaction of (Rp+Sp)-DPPsS with PL A2 (bee venom)

The reaction between (Rp+Sp)-DPPsS and PL A2 from bee venom was

followed by 31P NMR (121.5 MHz). The spectral parameters were

SW=5000 Hz, AQ=0.409 sec, 01=6562 Hz, 02=5200 Hz, DP = 20H,

TE = 310 K, NS = 6000, LB = -3.0 Hz and GB=0.05 Hz. The reaction conditions were 32 mg of DPPsS was dissolved in 2 ml of a buffer solution containing 50 mM HEPES, pH=7.20, 0.25 mM disodium EDTA, 2.5 mM calcium chloride, 5% (w:v) triton X-100 and 50% (v:v) deuterated water.

Small amounts of PL A2 were added over a period of time so as to see the start of hydrolysis. Too much enzyme added in the beginning would not have allowed the opportunity to observe the rate of hydrolysis. After 204 pg of enzyme was added the hydrolysis reaction began. Figure 31 shows the progress of the hydrolysis over a period of time. From these spectra the Rp isomer can be assigned as the resonance at 58.73 ppm, the Sp isomer as the resonance at 58.84 ppm and lyso- Rp as the resonance at

58.01 ppm. The hydrolysis was complete in 38 hours as seen by the spectra in figure 31. The Sp resonance was asymmetric after the reaction and the asymmetry was not due to residual Rp because further addition of enzyme caused no further changes in the shape. 14 Hr

19 Hr

38 Hr

^ I—r ^ ’ I ’ — :— r 59.5 59. 0 58.5 50.0 57.5 PPM

Figure 31: 31P NMR spectra (121.5 MHz) of the reaction of (Rp+Sp)- DPPsS with PL A2. 8 1

Reaction of (Rp+Sp)-DPPsS with PL C ( B. cereus )

The assignment of the diastereomers couid be confirmed from the reaction of (Rp+Sp)-DPPsS with PL C. PL C has been shown to hydrolyze only the Sp isomer of the thiophospholipids (Bruzik, et al.,(1983)). The reaction conditions and spectrometer conditions were exactly the same as described above for the PL A2 reaction except 0.1 mg of zinc nitrate was added. The PL C obtained from B. cereus is a metalloenzyme that requires zinc. The enzyme as purchased from Sigma Chemical Co. contains three different PL Cs. One is phosphatidylinositol specific, another is phosphatidylcholine specific and the third is sphingomyelin specific. The enzyme mixture was separated into two fractions using FPLC, PI specific and PC specific. The PI specific fraction was used by someone else in our laboratory, while the PC specific was used in this experiment. After the addition of 450 pi of the PC specific PL C solution, and after 10 hours, no reaction had taken place. The activity of the PL C solution was checked by observing the hydrolysis of DPPC. In this check experiment, 30 mg of

DPPC was dissolved in the buffer and 50 pi of the enzyme solution added.

The reaction was complete within 2.5 hours. So, 600 pi more of the enzyme solution was added to the DPPsS reaction and after an additional 12 hours no reaction could be detected. The lack of reactivity of PL C towards DPPsS is due to two factors. One factor is that DPPS is a rather poor substrate for PL C compared to DPPC. It has been reported that the 82 enzyme velocity value for DPPS is only 10% of that for DPPC (Otnaess, et al., (1977)). The second factor is that the thio analogs have shown to be poorer substrates for various phospholipases by -50 fold. These two factors together have quenched the reactivity of PL C towards DPPsS.

Unsuccessful Attempts at Obtaining DPPsS

Transphosphatidylation reaction of DPPsC with L-serine using phospholipase D

PL D is known to catalyze the transphosphatidylation reaction between a phospholipid and an alcohol. It has already been shown that the Sp thio analog is a substrate for PL D in the presence of ethanolamine to form

(Sp)-DPPsE (Jiang, et al., (1984)). The transphosphatidylation reaction using L-serine as the alcohol and DPPC as the lipid has been shown to result in poor yield (40%) (Comfurius & Zwaal, (1977)). The reaction of

(Rp + Sp)-DPPsC and L-serine using PL D (cabbage) produced no product.

The rate of transphosphatidylation to produce DPPsS was very slow and no product was detected even after several days, as judged by TLC.

Using L-serine methyl ester

L-serine methyl ester was considered as a possibility for a serine derivative because of the apparent ease of demethylating reactions that could be used. DPPsS methyl ester was synthesized through the 83 thiophosphoryl chloride pathway (scheme 3A) in moderate yield. The attempts at deblocking the methyl group, however were unsuccessful. The use of lithium hydroxide deblocked L-serine methyl ester, but not DPPsS methyl ester. The obvious problem being the lipid was not sufficiently soluble in the aqueous solvents needed for the lithium hydroxide.

Changing the solvent system to be less aqueous did not result in the formation of product.

Other demethylating reagents that worked on L-serine methyl ester but not on the lipid because of solubility problems were copper (II) sulfate at pH 8 and potassium carbonate in water and tetrahydrofuran, which were discussed above under debenzylating attempts.

Using N-benzyl-L-serine benzyl ester

Synthesis of N-benzyl-L-serine benzyl ester

The synthesis of N-benzyl-L-serine benzyi ester was accomplished through the reductive amination of L-serine benzyl ester and benzaldehyde. This method of approach was used to assure that only monobenzylation of the amine occurred. If a benzyl halide was used, then this would predominantly yield the dibenzylamino derivative.

The reaction mechanism is as follows. First a Schiff base (imine) is formed between the amine of serine and the carbonyl of benzaldehyde.

This imine is then reduced by sodium cyanoborohydride, which changes the

C-N double bond to a C-N single bond. 84

A typical synthesis was 2.0g (8.62 mmol) of L-serine benzyl ester,

0.96 ml (9.48 mmol) of benzaldehyde and 0.65g (10.3 mmol) of sodium cyanoborohydride were dissolved in 40-50 ml of anhydrous methanol and

allowed to stir overnight. Upon completion of the reaction, concentrated

hydrochloric acid was added to bring the pH cl. Addition of acid destroyed

the unreacted cyanoborohydride. Water was added and the product extracted four times with 125 ml of diethyl ether (a continuous extractor is called for, but our laboratory did not have one). The ether layers were dried over anhydrous magnesium sulfate, decanted and dried under reduced pressure. The residue was dissolved in anhydrous methanol and dry hydrochloric acid gas was bubbled in to assure protonation of the amine.

After evaporation of solvents the white crystals were isolated in 40% yield, mp=141 -142°C.

Synthesis of the 2-Thiono-1,3,2-oxazaphosphilidine Ring Intermediate of

Dipalmitoyl Thiophosphatidyl-N-benzyl-L-serine benzyl ester

The pathway that is described in scheme 5 was used to synthesize the ring intermediate of dipalmitoyl thiophosphatidyl-N-benzyl-L-serine benzyl ester. In scheme 5 the two R groups are both benzyl. A typical synthesis was as follows. To a solution of l.OOg (0.176 mmol) of dipalmitin and

0.848g (0.840 mmol) of triethylamine in 10 ml of anhydrous ethanol-free chloroform, 0.070g (0.414 mmol) of thiophosphoryl chloride was added.

The reaction mixture was allowed to stir overnight after which the solvents were removed under reduced pressure. The reaction mixture was dissolved 85 in 2:1, (v:v), hexane:diethyl ether and applied to a silica gel column. The

two diastereomers were separated and designated as 'fast', Rf=0.4 and

’slow’, Rf=0.3, with respect to their elution times. The total yield of both diastereomers was 50%. The ring compounds were characterized by 31 P

NMR (200 MHz) in deuterochloroform figure 32, 5: 85.4 ppm ('fast') and

83.6 ppm ('slow') and by NMR (500 MHz) in deuterochloroform figure

33. Figure 34 shows an expansion of the region 5.0 to 3.5 ppm of the 'H

NMR spectra of the 1 fast', ' mixture' and 1 slow' samples of the ring compounds.

The ring compounds were hydrolyzed with water and hydrochloric acid gas in acetone. The ring opening hydrolysis took approximately 5-6 hours.

The resonances of the ring opened compounds were around 60 ppm in acetone. The amount of time involved in opening the ring shows the extreme stability of this 2-thiono-1,3,2-oxazaphosphilidine ring. The ring stability is a direct result of the benzyl group on the nitrogen atom donating electrons to the phosphorus nitrogen bond giving it more double bond character.

The exceptional stability of the N-benzyl group required the search for rigorous methods to deblock it. Some of these methods were undesirable due to the sensitivity of the other functionalities present. The first attempt was the classical hydrogenation reaction. As already discussed, this attempt failed because of the extreme affinity sulfur had for the catalyst.

Trimethylsilyl iodide was attempted in hopes of debenzylating the nitrogen. But as was discovered, although silylation occurred at the carboxyl site it did not occur at the nitrogen site and thus debenzylation did 86

I...... "T...... I...... I'" ...... " 08.0 86.0 84.0 82.0 80.0 PPM

Figure 32: 3lP NMR spectra (81 MHz) of the ring diastereomers of dipalmitoyl thiophosphatidyl-N-benzyl-L-serine benzyl ester, (A) is the mixture, (B) is the 'fast' isomer, and (C) is the 'slow' isomer. I > I 1 [ I I ' T I » ' t T | I I ’ 1 ; [ I 1 T 'f I I I I ] I T I I | I I 1 I ] 1 I T I | I T 1 I j T T T~T~| 1 I I I | I I T I ] I I I I | I I T T | 1 I I T J 8.0 7 PPM

Figure 33: *H NMR spectrum (500 MHz) of the ring compound of dipalmitoyl thiophosphatidyl- N-benzyl-L-serine benzyl ester in deuterochloroform. oo ■vj Figure 34: *H NMR spectrum (500 MHz) of the ring compounds of dipalmitoyl thiophosphatidyl-N- benzyl-L-serine benzyl ester in the region 5.0 to 3.5 ppm, which shows the diastereomeric ring protons. (A) is the 'fast' isomer, (B) is the mixture and (C) is the 'slow' isomer. 89 not occur. Also, it was found with the DPPsS benzyl ester synthesis, that even the benzyl ester was not easily removed.

Aluminum trichloride was attempted to try and debenzylate the amine.

This reagent as a Lewis acid was tried so as to help stabilize the benzyl cation formed from the reaction. In effect, the benzyl cation was never formed and the starting material was always recovered after the work up.

When conditions were made harsher (e.g. heating) this resulted in desulfurization.

An attempt was made to use cerium ammonium nitrate (CAN) to try the debenzylation reaction. This reagent was found out to be too strong of an oxidizing agent because the product was always desulfurized.

The last attempt was to change the benzyl group into an acetyl group by using acetic anhydride and sodium acetate. The result of this attempt was no reaction. The ring compounds were not changed.

III.3 Discussion

The synthesis of diastereomers of DPPsS was hoped to have gone through a chiral isolable ring intermediate that could be deblocked and result in pure diastereomers without relying on the stereospecificity of PL

A2 and PL C to resolve the diastereomers. Actually, the stable isolable ring intermediate was isolated in moderate yield, 50%, and the ring was opened using hydrochloric acid and water. The unfortunate result being the failure in debenzylating the nitrogen and the carboxyl groups. All of the 90

many attempts to debenzylate the doubly blocked compounds failed.

Because of this, another route was devised in which the diastereomeric

mixture could be obtained in high yield. This route makes use of the

highly reactive phosphorylating agent, N.N-diisopropylmethylphos-

phonamidic chloride, and an easily deblocked yet stable serine derivative,

N-trityl-L-serine methoxy methyl ester. The yield from this synthesis was

70%. The diastereomeric mixture was consequently subjected to the

hydrolysis reactions of phospholipase A2 and phospholipase C (B . cereus).

Phospholipase A2 did indeed hydrolyze the Rp isomer of DPPsS as was expected, but phospholipase C did not hydrolyze the Sp isomer as was hoped. In a final note, the synthesis of DPPsS in pure diastereomeric forms has proven to be quite an extensive organic synthesis problem. I have benefited from such a challenge as it has caused me to explore vastly different avenues to solve a problem. Maybe some day in the future I may finally solve it, but at this point in my career it must come to a close. III.4 REFERENCES

Appelgate, H.E., Cimarusti, C.M, Dolfini, J.E., Funke, P.T., Koster, W.H., Puar, M.S., Slusarchyk, W.A. & Young, M.G. (1979)7. Org. Chem. 44, 811-818.

Bruzik, K., Jiang, R.T. & Tsai, M.D. (1983) Biochemistry 22, 2478-2486.

Comfurius, P & Zwaal, R.F.A. (1977) Biochim. Biophys. Acta488, 36-42

Corey, E.J., Pan, B.C., Hua, D.H. & Deardorff, D.R. (1982) 7. Am. Chem. Soc. 104, 6816-6818.

Felix, A.M., Heimer, E.P., Lambros, T.J., Tzougraki, C. & Meienhofer, J. (1978) 7. Org. Chem. 43, 4194.

Huffman, W.F., Hall, R.F., Grant, J.A. & Holden, K.G. (1978) 7. Med Chem. 21, 413.

Jiang, R.T., Shyy, Y.J. & Tsai, M.D. (1984) Biochemistry 23, 1661- 1667.

Jung, M.E. & Lyster, M.A. (1977) 7. Am. Chem. Soc. 99, 968-969.

Otnaess, A.B., Little, C., Sletten, K., Wallin, R., Johnsen, S., Flensrud, R. & Prydz, H. (1977) Eur. J. Biochem. 79, 459-468.

Prestidge, R.L., Harding, D.R.K., Battersby, J.E. & Hancock, W.S. (1975) 7. Org. Chem. 40, 3287-3288.

Stelakatos, G.C., Theodoropoulis, D. M. & Zervas, L. (1959) 7. Am. Chem. Soc. 81, 2884-2887.

Tsuji, T., Kataoka, T., Yoshioka, M., Sendo, Y., Nishitani, Y., Hirai, S., Maeda, T. & Nagata, W. (1979) Tetrahedron Lett., 2793. CHAPTER IV

3 1 P Nuclear Magnetic Resonance of Phosphatidylcholine

and Thiophosphatidylcholines

IV. 1 Introduction

Background information on the thiophosphatidylcholines

The diastereomers of DPPsC have been used as biological membrane models in studying the role of the phosphate group in the structure and function of membranes (Tsai, M.-D., et al., (1983); Tsai, T.-C., et al.,

(1984); (1985)). These studies at best only touched on the role of the phosphate group, because no detailed study of the conformation or motion was examined. The first step in understanding the structural role of the phosphate group in the bilayer structures was provided by studies employing differential scanning calorimetry (DSC). DSC alone can yield information concerning the various thermotropic phase changes and the amount of energy involved. DSC can also yield information regarding the relative stability of these phases and the interconversion between phases.

Recently, Wisner, et al., (1986), have provided DSC studies involving

DPPsC diastereomers. The thermotropic properties of the diastereomers

92 93 were compared to DPPC and among themselves, as shown below in figure

35.

DPPC

C 10 20 SOJO ’C

Figure 35: Thermotropic properties of bilayers following incubation at 0°C. The subtransition is shown for DPPC after 4 days of incubation (a), (Rp + Sp)-DPPsC after 14 days (b), and (Sp)-DPPsC after 16 days (c). The subtransition is not seen for (Rp)-DPPsC even after 14 days (d) (Wisner, et al., (1986)).

(Sp)-DPPsC showed a pretransition at 43.7°C and a main transition at

45°C, as well as a subtransition at 22UC when annealed at 4°C for several days. (Rp+Sp)-DPPsC had similar thermotropic phase characteristics.

These thermotropic phase characteristics resembled those observed in

DPPC (Chen, et al., (1980)), except that the thio analogs exhibited thermotropic phase changes that were a few degrees higher than DPPC.

The Rp isomer, however, showed only a broad transition at 45.9°C even after being annealed at 4°C for a period of two weeks. In separate experiments, Wisner, et al., (1986) showed that when (Rp)-DPPsC was heated to 50-70°C, cooled quickly and scanned immediately, a pretransition 94 and a main transition similar to those of (Sp)-DPPsC were observed (figure

36).

Equilibration time Cp 10 Kcal K^mof1

i

50

Figure 36: DSC traces of (Rp)-DPPsC of greater isomeric purity (>99%), showing the time dependence of the metastable gel phase. The sample was heated to 70°C for 5 min and then equilibrated at 25°C for 0 (a), 0.25 (b), 0.5 (c), and 65 h (d). Thermal equilibration of the calorimeter with the sample was conducted at 38°C for 10-15 min just prior to scanning (Wisner, et al., (1986)).

These results were interpreted as that (Rp)-DPPsC is metastable in the gel

phase, that it relaxes to the subgel phase rapidly at 25°C (half-time of

hours, depending on the isomeric purity), and that it remains in the subgel

phase until 45°C, the main transition. With the macroscopic structural differences seen by DSC, a more clearer picture of the microscopic changes 95 was sought. One such technique to help locate structural regions of influence is X-ray diffraction. Ruocco and Shipley (1982) have proposed a unified structural interpretation of the changes occurring in the two- dimensional acyl chain packing modes during the transitions of the bilayers of DPPC between the subgel (Le), gel (Lp1), rippled gel (Pp’) and liquid crystalline (La) phases.

Figure 37 shows the structural features of the thermotropic changes.

5 0 c C

DSC •ndoihyrmic

CRYSTAL

X R«y iiam • • Bilftytr i0 1t iiuii rm n 4lMr44r*d onhorhombk b - Subctll hybrid fhombic h«iagon*l |iqu«t h y b r id

short 4 40 1 4 14 I «ni m i spacing* 3 47 Jl (Oil

c - chain packing ol§fe>!i (W soc o ^ A .rJ 10 20 30 40 Ttin|Mf«tur* (° Cl

Figure 37: Thermotropic phase transitions with the corresponding proposed molecular and hydrocarbon chain packing arrangements of the lamellar phases of DPPC. Proposed by and adapted from Ruocco and Shipley (1982).

Recently, Sarvis, et al. (1988) have reported the X-ray diffraction results for the diastereomers of DPPsC. They found that the short spacings of the 96 acyl chains for (Sp) and (Rp+Sp)-DPPsC were similar to those of DPPC in

the subgel, gel, rippled gel and liquid crystalline phases. These data along

with the DSC results has allowed them to place the diastereomers of DPPsC

under the same thermotropic classifications as DPPC. The Rp isomer

showed an X-ray diffraction pattern characteristic of the more ordered

subgel phase up to 45°C, the main transition. This observation was in

agreement with the DSC results stated earlier. The gel and rippled gel

phases of (Rp)-DPPsC could be observed at 25°C and 44°C, respectively,

by collecting the data immediately after heating the samples to 50°C for 5

minutes, then cooling to 25°C. These results were consistent with the previous DSC results showing that these two phases of the Rp isomer were metastable, but could be detected by rapid cooling from above the main transition temperature. The data of these two phases were consistent with those of the gel and rippled gel phases of (Sp)- and (Rp+Sp)-DPPsC.

31 P NMR Background

Physical Basis of 31P NMR

The physical basis of 31P NMR as applied to these systems can be briefly described as follows: the external magnetic field experienced by the phosphorus nucleus is reduced by the field induced by the surrounding electron clouds of the bonding electrons. This is called chemical shielding or electron shielding. What is observed experimentally is the chemical shift, which corresponds to a chemical shielding relative to a reference 97 compound. Since the electron density is not isotropic but depends on the

bonding order (pattern), the electronic shielding is effective to a different

extent along the various molecular directions, thus becoming a tensorial

property. The shielding is less along the axis with the lowest electron

density and greatest along the molecular axis with the highest electron

density. In the case of single crystals in a lattice, if the lattice orientation

was varied in respect to the magnetic field, then it is possible to determine

both the orientation and the magnitude of the principal tensor elements

which describe the static chemical shift tensor.

BDEP, Model Crystal Studies

The elucidation of the 31P chemical shielding tensor principal values for

ph ospholipids would be directly obtainable from single crystal samples of

the phospholipids, but unfortunately most phospholipids are not favorable

to crystallization. Because of this, the principal values of a shift tensor of

a crystallizable model compound are found and then used to relate its shift

tensor orientation to that of the crystalline powder sample of the

phospholipid. The most widely used model for phospholipid diesters is

barium diethylphosphate, BDEP. BDEP is a very good choice because 1) it

is a phosphodiester, 2) the principal values of its shift tensor are within 5% of those for crystalline powder samples of phospholipids and 3) the crystal

structure geometry of the phosphate moiety is very similar to those found in the crystallizable phospholipid molecules, DPLE acetate and L-a

glycerophosphorylcholine. The molecular orientation of the 3IP chemical

shift tensor in barium diethyl phosphate is shown in figure 38.

crt

foil

Figure 38: Molecular orientation of the 31P chemical shift tensor in barium diethyl phosphate. The tensor is shown relative to an orthogonal X,Y,Z reference frame chosen so that Z is perpendicular to the nonesterified O-P- O plane, and X bisects the O-P-O angle. The angles between the tensor and X,Y,Z frame are exaggerated for clarity (Herzfeld, et al., (1978)).

The principal shift tensors, On > <1 2 2 . and 0 3 3 are shown relative to an orthogonal reference frame x,y,z, so that the z axis is perpendicular to the nonesterified O-P-O plane, and the x axis bisects the O-P-O angle

(Herzfeld, et al., (1978)). The magnitudes of the principal elements of

BDEP crystals at 20°C are On = -76 ppm, 0 2 2 = -18 ppm and 0 3 3 = 110 ppm.

To assure that these single crystal values were representative of the rigid lattice tensor values, the principal values were also extracted from powder samples of BDEP at 20°C (-79, -19, and 113 ppm for O n , o 2 2 , and O33, respectively) and at -110°C (-80, -22, and 108 ppm for O n , 0 2 2 , and O33, respectively). 99

Recently, Hauser et al. (1988) have extracted the principal values and thus the orientation of the 31P chemical shielding tensor for a phospholipid analog, 1-hexadecyl-2-deoxyglycerophosphoric acid. This analog could be grown into NMR measurable crystals. The results are in good agreement with other phosphomonoesters studied, e.g., phosphoethanolamine and phosphoserine. The orientation of the chemical shielding tensor differs only by 7-13° from that of barium diethyl phosphate, the best model compound describing ph osphodiesters.

Phospholipid Powder Samples

Because phospholipids are not readily crystallizable, the principal values of the chemical shielding tensor are derived from the rigid lattice condition of dry powder samples. These values are then compared to the model compound, BDEP, to help define the orientation of the phosphate moiety in phospholipids (for representative reviews see Griffin, (1981) and

Seelig, (1978)). The difference between single crystals and unoriented crystalline powders is that where the single crystals have only a few possible orientations, crystalline powders have a large number of microcrystalline regions which have a random distribution of orientations with respect to the magnetic field. The spectrum, instead of being sharp lines as in the case of single crystals, becomes a superposition of resonance frequencies of the microcrystalline regions, the intensities of which are a weighted distribution function of the regions. The principal values of the tensor can be extracted from the edges of the powder 100 spectrum and from the discontinuity in the center. Figure 39 illustrates the theoretical powder lineshape for this axially asymmetric tensor.

Figure 39: The theoretical powder lineshape for an axially asymmetric tensor.

The principal elements of the shielding tensor for most phospholipids are almost identical and possess principal values of approximately Cn = -80,

0 2 2 = "20, and 0 3 3 = +110 ppm. An exception to this is anhydrous DPPC in which the principal values are off by 10-20 ppm (Griffin (1976)). The magnitudes of the principal values for a racemic mixture of DPPsC (racemic at C2 of the glycerol moiety and also at the phosphate moiety) (Vasilenko, et al., (1982)) were elucidated from what the authors termed an

'anhydrous' sample, but what is most likely the monohydrate. The principal values reported are O n = -123, 0 2 2 = -96, and 0 3 3 = + 5 4 ppm. The difference in the electronic structure of sulfur compared to oxygen causes a large change (up to 50 ppm or so) in the shielding of the phos ph orus nucleus which is directly reflected in the chemical shift of thiophosphates.

Not only are the principal values shifted in terms of chemical shift values, but the half width of the spectrum is some 2 0 ppm smaller in monohydrate

DPPsC as compared to monohydrate DPPC. 101

Addition of Water to Powder Phospholipids

The addition of one molecule of water causes an increase in the phosphate symmetry which is reflected in the approximately 40 ppm change between the tensorial values of anhydrous and monohydrate DPPC. The actual effect of this one molecule of water is not clearly understood. The reason it is not, is because the addition of more water, up to 1 0 wt. %, causes further narrowing of the spectrum and a reversal in sign. Further addition of water, up to 50 wt. % causes no additional changes in the spectrum. There are two possible explanations offered for this observation. Firstly, the water induced narrowing and subsequent sign reversal is due to the onset of rotation of the phosphate group (in DPPC).

It has been shown that rapid anisotropic motion about an arbitrary axis will reduce an axially asymmetric powder spectrum to an axially symmetric spectrum (Mehring et al., (1971)). Secondly, the narrowing of the spectrum could be caused by the effect of hydrogen bonding of water to the ph osphate, which consequently perturbs the tensor elements. It is known that protonation does alter 31P NMR chemical shifts but the magnitude and direction of these changes towards the shift tensor is difficult to predict since no detailed study of the effect of protonation on shift anisotropy tensors has been done (Griffin, (1976)).

A possible molecular picture of the observed changes on going from anhydrous to fully hydrated lipid could be that anhydrous DPPC represents a rigid lattice in which any molecular motion is slower than the 31P NMR 102

time scale so that the representative spectrum exhibits the widest possible

breadth. The addition of one molecule of water causes a hydrogen bonding

between it and the phosphate, thus influencing the shift tensor to become

more axially symmetric. This hydration effect probably does not induce a

large amount of rotational motion in the phosphate. This can be seen by

the fact that the spectral lineshape still appears axially asymmetric, but the

principal values are shifted ( 1 0 - 2 0 ppm) causing a narrowing in the

spectrum. As more water is added the degree of hydration increases until the phosphate experiences a fully hydrated state. At this state, the spectrum which has changed shape and width due to the onset of molecular rotation about an arbitrary axis, does not narrow anymore in spite an increase in the amount of water added.

Fully Hydrated Phospholipid Bilayers

Oriented Bilayers

While powder samples provide a wealth of information, there is still a necessity to elucidate the phosphorus chemical shielding tensor orientation in membrane-like models. The most widely used model system is fully hydrated phospholipid bilayer structures. The closest model of phospholipid bilayers to single crystals are oriented bilayers. Much effort has been exerted to use the tensor orientation of BDBP to better explain the orientation of the phosphate moiety in oriented phospholipid samples 103 (Griffin, et al., (1978); Seelig, (1978); Herzfeld, et al., (1978); Kohler

and Klein, (1977) and Buldt and Wohlgemuth, (1981)).

Randomly Oriented Bilayers

While oriented lipid systems can provide a more accurate interpretation

of the molecular orientation of the phosphate moiety, a need still arises to

study the unoriented randomly distributed lipid system. This randomly oriented system more closely resembles biological membranes and is thus

important in their understanding. In the case of randomly oriented

samples, powder-type spectra are observed. The simplest powder lineshape is that due to an axially symmetric shift tensor, e.g., the liquid crystal state of a phospholipid such as DPPC. In this case, On is equal to

0 2 2 * but not equal to 0 3 3 . With this powder spectrum a new notation is used to describe the "unique" ( I 3 3 shift tensor, af/ (sigma parallel), and

° h - °2 2 * CT± (sigma perpendicular). The use of O// and Ci provides a means to evaluate the chemical shielding anisotropy factor, Ac, which is equal to G/f-a±. The two shift tensors, Cj. a°d O//, are easily measured at the half-height of the upfield and downfield shoulders, respectively, of the powder pattern spectrum as illustrated in figure 40 for fully hydrated DPPC at 44°C (i.e. the La phase) (36.5 MHz) Gaily, et al. (1975). It is this observable Ac, that qualitatively reflects the degree of motional freedom experienced by the phosphate moiety. Oh or* ■*h0

Figure 40: 31P NMR spectrum (36.5 MHz) of nonsonicated bilayers of fully hydrated DPPC with broadband proton decoupling at 44°C (Gaily, et al., (1975)).

There are a variety of factors affecting the degree of motional freedom of the phosphate group. Such influences as degree of hydration, temperature, pressure, rate of axial diffusion and ionic interactions, effect the observable chemical shift anisotropy factor. The goals of this chapter are to use solid state 31P NMR techniques to confirm the thermotropic phase assignments of DPPC and the DPPsC diastereomers and to compare structural properties of DPPC and DPPsC diastereomers in the subgel and other phases. Since no one technique can adequately explain the orientation and configuration of a lipid system, the results here will be interpreted in light of X-ray diffraction and FT-IR data accumulated in this laboratory (Sarvis, et al., (1988)). 105 IV.2 Materials and Methods

Materials

1,2-dipalmitoyl-sn-gIycero-3-phosphocholine, DPPC, was purchased

from Avanti and was used without further purification. The diastereomers of DPPsC were prepared as previously described in scheme 4. The DPPsC

samples used in this section did not contain deuterium substitutions.

Gaily, et al., (1975) observed distorted 31P NMR spectra when using

DPPC deuterated in the N-methyl group even under strong 1 H-decoupling conditions. The presence of dipolar deuteron-phosphorus coupling prompted the use of non-deuterated DPPsC diastereomers for 31P NMR studies. The diastereomers of DPPsC were further chemically purified by repetitive precipitation from acetone/ethanol (6-9 times). The chemical purity was judged by measuring the width of the main transition at half maximal height using DSC (Wisner, et al., (1986)). The width at half maximal height for the (Rp+Sp) and (Sp) samples were typically between

0.2 and 0.4 °K, while (Rp)-DPPsC was between 7 and 8 °K.

Methods

The lipid samples were prepared by mixing 150-200 mg of dry lipid with 175-225 (il of 2 H 2 0 in a 5 mm NMR tube. The samples were repetitively heated to 60°C, mixed on a vortex stirrer for 2-3 minutes and 106 cooled until all the lipid was hydrated. The samples were annealed at 0-

4°C for more than one week.

31P NMR spectra were recorded on a Bruker MSL-300 spectrometer at a frequency of 121.497 MHz. In a review by Seelig (1978), it was shown that the chemical shielding anisotropy factor is dependent on the magnetic field strength, while the dipolar interactions are not. Phosphorus-proton dipolar interactions are 1-5 kHz depending on the motional freedom of the phosphate group and the nearest methylene group. The separation of the edges of the powder pattern of a chemical shielding anisotropy of 50 ppm will increase from 1 .8 kHz at 36 MHz to 6 .1 kHz at 121 MHz. So at higher external magnetic fields the non-decoupled powder type spectral edges will be dominated by the chemical shielding anisotropy. For this reason it was not necessary to employ very high proton decoupling during acquisition of the spectra. Other spectral distortions were avoided by the use of a Hahn echo pulse sequence (90° -x- 180° ). Ranee & Byrd, (1983) have demonstrated the types of distortions that may arise when using conventional single pulse techniques over spin-echo sequences. The 90 degree pulse length was typically 9 psec with a recovery time of 2 seconds.

The spectral width was 125 kHz and 16 watts of broadband *H-decoupling was used. Quadrature detection and phase cycling were also used in all spectra. 107 IV.3 Results

The 31P NMR spectra of the thiophospholipids and DPPC at temperatures characteristic of the various thermotropic phases are shown in figure 41. The magnitudes of the chemical shielding anisotropy factors of these phospholipids at these temperatures are given in table 1 .

3'P NMR results at 5°C

Comparison of the spectra at 5°C reveals that the magnitudes of the chemical shielding anisotropy factor for (Rp+Sp)-, (Sp)-, and (Rp)-DPPsC are much larger than that of DPPC. Also the spectra show that there is a reversal in the sign of the chemical shielding anisotropy factor for the thio analogs compared to DPPC. Among the thio analogs there is virtually no significant difference in the chemical shielding anisotropy sign or magnitude.

31P NMR results at 25°C

As the temperature is raised to 25°C, all of the phospholipids except

(Rp)-DPPsC experience a thermotropic phase change from the subgel phase to the gel phase. This observation is clearly shown in the spectra at 25°C.

The spectra of (Rp + Sp)- and (Sp)-DPPsC show a reversal of sign compared to their 5°C spectra and a narrowing of the chemical shielding anisotropy factor. The chemical shielding anisotropy factor for DPPC also B

50

100

T T 50 Q 100 100 0 PPM PPM PPM

T T 100 0 0 200 0 200 P Py PPM PPM PPM

Figure 41: 3lP NMR spectra (121.5 MHz) of DPPC and the thiophospholipid diastereomers at temperatures characteristic of the various thermotropic phases. DPPC (A), (Rp+Sp)-DPPsC (B), (Sp)- 108 DPPsC (C), and (Rp)-DPPsC (D). 109 Table 1: Chemical Shielding Anisotropy Values*

Anhydrous Monohydrate 50 wt. % Water Powder Powder 5°C 25°C 38°C 43°C 50°C

DPPC 232ppmb 191 ppm 8 1 ppm 68ppm 56ppm 46ppm rac-DPPsCc 167ppm

(Rp + Sp)-DPPsC 1 70ppm 83ppm 53ppm 3 1 ppm

(Sp)-DPPsC 154ppm 86ppm 56ppm 39ppm

(Rp)-DPPsCd 1 70ppm 170ppm 170ppm 36ppm -

aChemical Shielding Anisotropy Values are Act=Icti i -(J3 3 1 and Aa=lCT//-Oj_l bFrom Scelig (1978) crac-DPPsC is (Rp+Sp)-1.2-dipalmiioyl-rac-glycero-3-thiophosphocholine Vasilenko et al., (1982) dThe principal values are cti j =-1 35 ppm, C22=-94 ppm and CT33=+50 ppm (adapting the convention used in the literature) 1 1 0

experiences a narrowing in magnitude as seen values listed in table 1. The

magnitudes of the chemical shielding anisotropy factors for the thio analogs

is still larger than that of DPPC. (Rp)-DPPsC continues to exhibit a

spectrum indicative of the subgel phase at 25°C, which is consistent with

DSC and X-ray diffraction results.

3ip NMR results at 38°C and 43°C

As the temperature increases to that of the pretransition temperatures of

38°C and 43°C for DPPC and (Rp+Sp)- and (Sp)-DPPsC, respectively, the phospholipids experience a transition from the gel phase to the rippled gel ph ase. This change is observable by the decrease in the magnitudes of the chemical shielding anisotropy factors for DPPC and (Rp+Sp)- and (Sp)-

DPPsC. (Rp)-DPPsC exhibits a broad thermotropic phase transition, the

main’ transition, from the subgel phase to the liquid crystalline phase at approximately 46°C and at 43°C the spectrum reveals the onset of this transition by the appearance of more than one lineshape.

31 P NMR results at 50°C

At 50°C, all of the phospholipids are in the liquid crystalline phase, which is characterized by fast-limit powder spectra. Comparison of the magnitudes of the chemical shielding anisotropy factors again reveals differences between the thio analogs and DPPC. The magnitude of the chemical shielding anisotropy factor for DPPC is larger than those of the 111 thio analogs. Among the thio analogs is seen differences in the magnitudes of the chemical shielding anisotropy factors. (Sp)-DPPsC shows the largest magnitude, 39 ppm. (Rp)-DPPsC is next with 36 ppm and the mixture, (Rp + Sp)-DPPsC has the smallest magnitude, 31 ppm. These results are in agreement with an earlier study by Tsai, et al., (1983). 1 1 2 IV. 4 Discussion

One of the overall goals of this research is to study the conformation and motion of the head group in the rtiastereomers of DPPsC and compare this data to that of DPPC. Much work has been done on DPPC in order to elucidate the orientation of the phosphate moiety. The fully hydrated sample has been assumed to undergo axial diffusion of the phosphate moiety about the long axis of the lipid molecule, commonly called the director axis (Griffin, (1976)). Compared to the anhydrous state, which possesses an axially asymmetric tensor, this fully hydrated state is axially symmetric. Extraction of information from fully hydrated lipids concerning head group conformation is not possible because of this difference. With fully hydrated samples two order parameters are required to describe the tensor principal values but only one can be extracted from the spectrum.

What others have done (Kohler & Klein, (1976); Seelig & Gaily, (1976);

Seelig, et al., (1977) and Griffin, et al., (1978)) is to combine information concerning orientation from other sources with the phosphorus data to elucidate a structural model. Campbell, et al., (1979) have assumed a continuous diffusion model and have fit the spectral lineshapes of DPPC below 35°C to simulated spectra in this slow motion regime. Above 35°C, the spectra collapse to the fast-limit powder patterns and the phosphate moiety can then be fit with a continuous set of orientations and diffusion rates.

The interpretations of Campbell et al. will be used to describe the spectral lineshapes observed in figure 41. The thio analogs of DPPC at I 1 3 5°C show a spectral lineshapc that resembles the rigid lattice. This lineshape was obtained for DPPC at -50°C by Campbell et al. Campbell, et al., have shown that the lineshape does not change upon further cooling of

DPPC to -110°C and assigned this as the rigid lattice spectrum with On =

-100, <*2 2 = -30, and 0 3 3 = +130 ppm (negative is downfield ). These values are in excellent agreement with those of anhydrous DPPC. This rigid lattice is described by very slow diffusion rates about a fixed orientation of the phosphate moiety. Upon close examination of the 25°C spectrum of (Rp)-DPPsC, the principal values of the phosphate tensor can be extracted. They are found to be C n = -135, 0 2 2 = -94, and 0 3 3 = +50 ppm (signs are reversed to stay within published conventions) and are in excellent agreement with those of Vasilenko, et al. (1982) for solid DPPsC monohydrate as stated earlier. Comparing these spectra to the spectrum of

DPPC at 5°C shows that DPPC resembles a spectrum experiencing relatively fast molecular diffusion (approximately 10 4 Hz according to

Campbell et al., (1979)) while the DPPsC diastereomers reflect the rigid lattice limit. This comparison qualitatively shows that the subgel phases of

DPPsC diastereomers have a much more rigid structure than DPPC, for it has been established that DPPC at 5°C (with prior annealing) is in the subgel phase. It has been suggested by FT-IR and X-ray diffraction data, that the subgel phase of (Rp)-DPPsC may possess interactions in the carbonyl region of the acyl chains so as to exclude more water from the head group region, thus making it more rigid (Sarvis, et al., (1988)). This explanation is supported by Griffin’s studies on the effect of hydration on

DPPC 31P NMR lineshapes (Griffin, (1976)). He showed that the 1 1 4 exclusion of water caused spectral changes similar to temperature changes of fully hydrated samples, in which dehydration led to the rigid lattice limit. The Rp diastereomer stays in this rigid state until the ’main' transition into the liquid crystalline phase. (Rp+Sp)- and (Sp)-DPPsC most closely resemble DPPC in the structures at various thermotropic phases. In the Lp' gel phase, at 25°C, the thio analogs have chemical shielding anisotropy factor values near that of DPPC at 5°C. DPPC in the gel phase, at 25°C, most closely resembles the spectrum associated with a rotational motion on the order of 10 5 Hz, which is an order of magnitude faster than at 5°C. So in the Lp' gel phase (Rp+Sp)- and (Sp)-DPPsC exhibit a slower rotation about the director axis than their oxygen counterpart, DPPC.

The next step in thermotropic phase changes is what is commonly called the pretransition. This is a structural change of the Lp’ gel phase from a planar arrangement to a rippled structure (Pp' gel phase). Earlier experiments exploring the pretransition of DPPC biUyers were unable to detect a significant change in the polar head group, even though the polar head group was thought to be effected by the pretransition. However, much evidence was seen for the effect of the pretransition on the hydrocarbon chains, and it was assumed that these then were the only sites influenced by the pretransition. Close examination of earlier work done by

Campbell, et al. (1979) utilizing diffusion models to fit 31P NMR spectra at various temperatures ( as described above) and Shepherd & Buldt, (1978) employing dielectric dispersion techniques, showed that the pretransition effected the rotational motion of the polar head group. Recently, Akutsu 1 1 5 (1986) has used !H-31P cross-polarization efficiencies to detect the

pretransition phase change of DPPC bilayers. There are significant changes in the cross-polarization efficiencies and also the chemical

shielding anisotropy factor on going through the pretransition. The observations reported in this dissertation of the chemical shielding anisotropy factor narrowing on going through the pretransition for DPPC and DPPsC diastereomers also supports the idea of the pretransition effecting the polar head group. Akutsu (1986) also measured cross- relaxation times. He observed an increase in the cross-relaxation time, which suggests that the amount of motion experienced by the rotational axis of the polar head group is significantly enhanced through the pretransition.

The decrease of the chemical shielding anisotropy value on going through the pretransition has been attributed to not only an increase in relative motion, as seen by Akutsu, but also a structural change in the polar head group, as seen by Campbell, et al., (1979). Campbell, et al. (1979) have provided evidence for a change in the orientation of the diffusion axis and a change in the parallel component of the rotational diffusion tensor in their diffusion model studies, which implies a change in the head group conformation. However, the nature of this conformational change is still unclear. Note that the chemical shielding anisotropy values obtained for

(Rp + Sp)-, (Sp)-DPPsC and DPPC in the rippled gel phase are identical within experimental error.

The next phase under consideration is the liquid crystalline (La) phase.

Here the 31P NMR spectra are in the fast-limit regime of the chemical shielding anisotropy and detailed models are not available because of the 1 16 complexity of the system. The phase is characterized by rapid motions of the phosphate moiety about the director axis, which leads to a time- averaged axially symmetric shielding tensor. The phase transition corresponds to a cooperative melting of the hydrocarbon chains and occurs within a narrow temperature range as demonstrated by DSC. It has been shown that the changes in the head group are not as abrupt because of the slower decrease in the chemical shielding anisotropy values over a broader temperature range associated with the phase change (Seelig, (1978)). 1 1 7

IV.5 REFERENCES

Akutsu, H. (1986) J. Magn. Resort. 66, 250-263.

Buldt, G., & Wohlgemuth, R. (1981) J. Membrane Biol. 58, 81-100.

Campbell,R. F., Meirovitch,E., & Freed, J.H. (1979) J. Phys. Chem. 83, 525-533.

Chen, S.C., Sturtevant, J.M., & Gaffney, B.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5060-5063.

Gaily, H.-U., Neiderberger, W., & Seelig, J. (1975) Biochemsitry 14, 3647-3652.

Griffin, R.G. (1976) J. Am. Chem. Soc. 98, 851-853.

Griffin, R.G. (1981) in Methods in Enzymology 72 (J.M. Lowenstein, ed.) pp 138-139.

Griffin, R.G., Powers, L. & Pershan, P.S. (1978) Biochemistry 17, 2718- 2722.

Hauser, H., Radloff, C., Ernst, R.R., Sundell, S., & Pascher, I. (1988) J. Am. Chem. Soc. 110, 1054-1058.

Herzfeld, J., Griffin, R.G., & Haberkorn, R.A. (1978) Biochemistry 17, 271 1-2718.

Kohler,S.J., & Klein, M.P. (1977) Biochemistry 16, 519-526.

Mehring, M., Griffin,R.G., & Waugh, J.S. (1971) J. Chem. Phys. 55, 746.

Ranee, M., & Byrd, A. (1983) J. Magn. Reson. 52, 221-240.

Ruocco, M.J., & Shipley, G.G. (1982) Biochim. Biophys. Acta 691, 309- 320.

Sarvis, H.E., Loffredo, W.M., Dluhy, R.A., Hernqvist, L., Wisner, D.A., & Tsai, M.-D. (1988) Biochemistry 27, 4625-4631. 11 Seelig, J. (1978) Biochim. Biophys. Acta 515, 105-140.

Shepherd, J.C.W ., & Buldt, G. (1978) Biochim. Biophys. Acta514, 83.

Tsai, M.-D., Jiang, R.-T., & Bruzik, K. (1983)7. Am. Chem. Soc. 105, 2478-2480.

Tsai, T.-C., Hart, J., Jiang, R.-T., Bruzik, K., & Tsai, M.-D. (1985) Biochemistry 24, 3180-3188.

Tsai, T.-C., Jiang, R.-T., & Tsai, M.-D. (1984) Biochemistry 23, 5564- 5570.

Vasilenko, I., DeKruijff, B., & Verkleij, A.J. (1982) Biochim. Biophys. Acta 685, 144-152.

Wisner, D.A., Rosario-Jansen, T., & Tsai, M.-D. (1986) J. Am. Chem. Soc. 108, 8064-8068. CHAPTER V

2H NMR of Specifically Labeled ThiophosphatidyIcholines

V. 1 Introduction

Basic Theoretical Aspects of 2H NMR

The purpose of this section is to provide a fundamental understanding of basic solid state NMR theory and various useful parameters pertinent to

2H NMR. This section is not intended to supply an exhaustive compilation of equations and derivations, which can be found in a variety of sources

(Seelig, (1977); Chan et al., (1981); Griffin, (1981) and Davis, (1983)), but only to report the equations necessary for the interpretation of the results obtained.

There are five main interactions involving a nucleus with a magnetic moment greater than or equal to one half that may occur in the solid state.

These are: I) the Zeeman interaction with the applied magnetic field, 2) direct dipole-dipote interactions with neighboring nuclei, 3) magnetic shielding by the surrounding electron clouds causing the chemical shift(s),

4) scalar spin-spin couplings to other nuclei, and 5) Quadrupolar interactions (Fyfe, (1983)). A general Hamiltonian for the interactions

119 120 experienced by a spin 1 nucleus ( 2 H, e.g.) in the solid state may be expressed as in equation (V. 1).

H = H z + H D + H CS + H j + H q (VI)

The approximate magnitudes of the various spin interactions are shown in

Table 2. A brief description of each interaction and a more detailed discussion of Quadrupolar interactions will be given.

Table 2: Approximate Magnitudes of Various Spin Interactions (in Hz).

Zeeman 1 0 6 - 1 0 9

Dipolar 0 - 1 0 4

Chemical Shift 0 - 1 0 4

Scalar Coupling 0 - 1 0 2

Quadrupolar 0 - 1 0 9 *

1). The Zeeman interaction occurs between the magnetic moment of a nucleus and the applied magnetic field, H 0, yielding 21+1 energy levels

(where I is the nuclear spin quantum number) separated by hco 0 , which is equal to YfiH 0 - So, the Hamiltonian may be written as (V.2)

H z = Y *H 0 Iz (V.2) 1 2 1

where 7 is the magnetogyric ratio, and 0)o the Larmor frequency. This

interaction can be illustrated as in figure 42, for 1=1.

m , -I

Zeeman

Figure 42. The effect of the Zeeman interaction between the nuclear magnetic moment and the applied magnetic field.

The interaction is linear with the applied magnetic field, such that larger

separations of the energy levels occur at higher fields with a corresponding

increase in the population difference between them and also an increase in

the signal to noise ratio of the spectrum.

2). The dipolar interaction arises from the direct dipole-dipole

interactions between nuclei. The interactions may be between the same

nuclei or between different nuclei. The important characteristics of this

interaction are that it is dependent on the magnitude of the magnetic

moments, reflected in the magnetogyric ratios, and therefore is most

important for spin 1/2 nuclei possessing large 7 (i.e. *H, ,9F and possibly

3 , P). It is independent of the applied magnetic field, H 0 , which as was

stated in the 31P NMR chapter is one characteristic made use of to diminish

its affect on the total Hamiltonian by performing the experiments at high magnetic field, thus taking into consideration the linear dependence of the 122 chemical shift term, as will be stated below. Dipolar interactions are also dependent on the internuclear distances ( 1/r3).

3). The nature of the chemical shift interaction was discussed in detail in the 31P NMR chapter, but some salient points will be brought out here.

The chemical shift interaction is linear with the applied magnetic field thus allowing the chemical shift anisotropy factor to dominate the dipolar interaction at high magnetic fields, as was the case in the solid state 31P

NMR experiments. As stated in the 31P NMR chapter, the chemical shift interaction is the most sensitive interaction to the geometry and identity of the surrounding atoms of a particular nucleus and as such is the most chemically diagnostic tool in NMR in both solution and condensed matter systems.

4). Scalar spin-spin coupling interactions between nuclei of similar or different spins is very small compared to the other interactions. The nature of the interaction is field independent as can demonstrated by observing 'H

NMR spectra at different magnetic field strengths.

5). The quadrupolar interaction occurs only when the nuclear spin number is greater than one-half. The quadrupolar interaction results from the interaction of the electronic charge distribution surrounding the atom with the non-spherically symmetric field gradient of the nucleus. The quadrupolar interaction is field independent and the Hamiltonian may be presented as in equation (V.3).

— A ^ H q = I Q I (V.3) 123 The magnitude of the interaction is large enough that for most nuclei it

completely dominates the spectrum. The only interaction discussed above

that may influence the quadrupolar effect would be the dipolar interaction.

This interaction usually contributes to the spectrum as linebroadening. The

Quadrupolar Hamiltonian is thus usually treated as a first-order

perturbation on the Zeeman Hamiltonian. In understanding the Quadrupolar

Hamiltonian one must keep in mind that since I, the spin number, is a

vector quantity, the quadrupolar tensor (operator), Q, must be a 3 x 3

matrix or second rank tensor in order to describe the three-dimensional

nature of the quadrupolar interaction. The tensor Q is defined as in equation V.4,

Q = feQ/2I(2I-l)h]-V (V.4)

where eQ is the quadrupole moment and V is the electric field gradient tensor at the nuclear site. The components of V describe the orientation and magnitude of the electric field gradient. In a suitable coordinate system (principal axis system) V is converted to diagonal form where the three principal values, Vn , V2 2 and V 33 describe the size and shape of the field gradient. What is commonly done is that V 33 is defined as eq, the principal value of the electric field gradient. It should be noted that the principal axis system is oriented such that the z axis, corresponding to V 3 3 , is parallel to the C-2H bond, thus making the electric field gradient symmetrical. Vn, V22 and V 33 are related by a term called the asymmetry parameter, T] (equation V.5). 1 24

n = (V 11-V22W 33 (v. 5 )

Due to the transformation of the principal axis to the laboratory frame

defining V 33 along the C-2H bond, V n = V 2 2 » which makes q=0. This

axially symmetric interaction is indeed a good approximation for

interpreting lipid bilayer results. (The concept of ti can be somewhat

confusing at first, but let's think about it just a little further. Because Tl is defined from the principal axis transformation into the laboratory frame, it

must be zero for 2H systems like ours. If T)*0, then that would imply that the electric field gradient tensor is asymmetrical. If the electric field gradient tensor is asymmetrical, then at very low temperatures (no molecular motion) the spectrum would not be a powder pattern. Therefore, in section III of this chapter, the loss of powder pattern lineshape for the deuterated DPPsC isomers is not due to T] not equalling zero, but to molecular motion of the C-D bond). The Quadrupolar Hamiltonian may be written as (V. 6 )

H q = [e2 qQ/4 I(2 I-l)][V 3 3 (3 l3 2 -I2 )+(V 1 1 -V2 2 )(I12 .i 2 2 )] (V.6 )

where Q is the quadrupole moment of the nucleus, 1 3 , Ij, and I 2 are the spin vectors in the molecular frame and V 3 3 , Vji and V 22 are the principal components of the electric field gradient tensor. Since the quadrupolar interaction is treated as a first order perturbation on the Zeeman interaction, it is convenient to transform the Quadrupolar Hamiltonian to the laboratory 125 frame describing the Zeeman Hamiltonian. The Quadrupolar Hamiltonian

then becomes (V.7)

H q = [eQ/4I(2I-l)][3Iz2-l2]V„ (V.7)

where Wlz is the zz component of the electric field gradient tensor in the

laboratory frame and is related to the principal values in the molecular

frame by V. 8 .

V2z = Vi ] sin 2 0 cos 2 (|»+V2 2 sin 2 0 sin 2 4)+V3 3 cos 2 0 (V. 8)

where 0 and <{> are the Eulerian angles defining the orientation of the

electric field gradient tensor with respect to the magnetic field, H 0 . Since

we defined V33 = eq and t) = (V 11 -V22)/V33, then the Quadrupolar

Hamiltonian may be written as (V.9)

H q = Ue2 qQ)/8I(2I-l)][3Iz 2 -I2 ][(3cos 2 e-l)+Tlsin20cos 2

and with ti=0, reduces to (V. 10)

H q = [(e2 qQ/8I(2I- 1 )][3IZ2 -I2 ][3cos 2 0 - 1 ] (V. 10)

The overall Hamiltonian may now be written as (V.ll)

H = -y hH oIz+[(e2 qQ)/8I(2I-l)][3Iz-I 2 ][3cos 2 0-l] (V.ll) where -y hH0 Iz is the Zeeman Hamiltonian contribution. This Hamiltonian predicts the following energy levels (V. 12)

E(mi) = -YhH0mj + [e2 qQ/8I(2I-1 )][3mI 2 -I(I+1)][(3cos20 - 1)] (V. 12)

where mi = +1, 0 or -1. The effect of the quadrupolar interaction on the

Zeeman energy levels is shown in figure 43.

The energy change between allowed energy transitions (nij from 0 to M and mj from 0 to 1) can be written as equation V. 13.

AEq = 3/8 (e 2 qQ/h)(3cos2 0 -l) (V. 13)

m, -1

'"-P

------fl

Z cem on + Ouodrupotor

Figure 43: The Quadrupolar perturbation on the Zeeman energy levels. 1 27 AEq is the energy shift due to the quadrupolar interaction and 9 the angle between the principal component of the electric field gradient tensor and the magnetic field vector. The spectrum that results from this interaction of a single spin 1 nucleus with an axially symmetric field gradient consists of a doublet symmetrically displaced about the Zeeman frequency, o0, with peak separations given by equation V. 14 and is shown in figure 44.

Au = 3/4(e 2 qQ/h)(3cos2 0 - 1) (V. 14)

i—o

Figure 44; The 2H NMR spectrum for a single crystal of a molecule with an isolated 1=1 nucleus (Chan, et ah, (1981)).

The collective term, (e2 qQ/h), is known as the 'quadrupole coupling constant' and can be directly measured from NMR studies of single crystals. For a C-D bond in an alkyl chain the static quadrupole coupling constant is typically - 170 kHz (Burnett & Muller, (1971)). Figure 44 represents the 2H NMR spectrum obtained from a single deuteron crystal.

As was stated in the 31P NMR chapter, single crystals of lipids are not easy to achieve and thus powder samples must be used. When polycrystalline powders are used equation V.9 must be averaged over all possible crystal 128 orientations, yielding a spectrum as shown in figure 45, again for a single deuteron.

B B

C C

A

Figure 45: The 2H NMR spectrum for a polycrystalline powdered molecule with an isolated 1 = 1 nucleus (Fyfe, (1983)).

The outer 'steps' at the wings of the spectrum (A, A') correspond to 9=0°, the central pair of peaks (' wings' ,B ', B) to 0=90° and the mid point (C ',

C) to 9=54°44', where the two peaks of the spectrum coalesce and the quadrupolar interaction effectively vanishes. The separation of the central doublet (B*, B) is defined in equation V. 15 and the outer ' steps’ (A, A ') in equation V. 16.

= 3/4 (e2 qQ/h) (V. 15)

A\)2 = 3/2 (e2 qQ/h) (V. 16)

The discussion so far has focused on static deuterons in the magnetic field. As can be seen in equations V. 13 and V. 14, the quadrupolar interaction is sensitive to the orientation between the principal axis of the 1 29 electric field gradient, which is assumed to be the C-D vector, and the applied magnetic field. The goal of our research is to elucidate membrane structure and ordering, therefore we must study the quadrupolar interaction in light of the molecular motions causing a change in the orientation of the electric field gradient tensor.

Molecular Motion

In static systems the quadrupolar coupling constant was determined for

C-D segments to be 170 kHz (Burnett & Muller, (1971)). Observations made with lipid systems in which specifically deuterated fatty acids were intercalated into phospholipid multilayers oriented at various angles with respect to the external magnetic field showed quadrupolar splittings significantly smaller than 170 kHz, the static quadrupolar coupling constant

(Seelig & Niederberger, (1974)). The cause of the reduced splitting is motional averaging of the electric field gradient tensor. Molecular motion is a relative term and is dependent on the timescale of observation. The timescale of observation for quadrupolar interactions is given by

(3e2 qQ/4h)-*, which when 170 kHz is used for the static quadrupolar coupling constant yields a timescale of ~ 8 p.sec. So any processes which modulate the angular function in the Hamiltonian at a rate much faster than this timescale will provide an effective averaging mechanism. The equation describing this motional averaging term is completely analogous to equation

V. 14 and is given in equation V. 17. 130

A d = 3/4(e2qQ/h)(3cos2y-1) (V. 17)

Here y is the angle between the principal axis of the electric field gradient tensor (the C-D vector) and the axis of rotation.

What is very useful in describing molecular orientations of lipids is to provide a quantitative measure of the motional anisotropy of a particular C-

D bond. This term is referred to as the order parameter, Scd - The order parameter is related to the quadrupolar splitting by equation V. 18.

A dq = 3/4(e2 qQ/h)SCD (V. 18)

If 9 denotes the instantaneous angle between the C-D vector and the bilayer normal, then Scd is defined by equation V. 19.

S c d = l/2(3cos 2 0~l) (V. 19)

The bar indicates the time average of the angular fluctuations.

So far the equations used were describing a singly deuterated carbon atom. Now let's consider a CD 2 and CD 3 segment, as would be found in a deuterated lipid. For simplicity a homogeneously oriented membrane with the orientation specified by the director axis, z is assumed. Both deuterons rotate around this axis and since the asymmetry parameter is essentially zero, the effective quadrupolar splitting is defined as equation

V. 20. 131

Aug = 3/2(e 2qQ/h)Sco (V. 20)

The movements and hence the order parameters of the two C-D bonds would not be expected to be identical, due to the fact that the director axis would not directly bisect the D-C-D bond angle. This point can be illustrated by looking at an extreme case in which one of the C-D bonds

(CD1) is aligned parallel to the director axis and thus becomes the rotation axis for the other C-D bond vector (CD2). The order parameters could then be calculated for CD1 and CD2 using equation V. 19 and the fact that the bond angle in a tetrahedral molecule is 109.5°, to be 1 and -1/3, respectively, and two different quadrupole splittings would observed.

Before 1980, it was generally assumed that only one quadrupole splitting was observed for lipids such as DPPC and DPPE deuterated at the alpha position on the head group, thus concluding that the two deuterons were equivalent and characterized by the same order parameter. In 1980,

Browning and Seelig upon reexamination of these two compounds at higher magnetic field (SF < 2 H) = 61.4 MHz) observed two quadrupole splittings, thus showing that indeed the two deuterons were not equivalent. In the case of a-d 2 *DPPC the difference between the two order parameters was only 0.002. Other head groups, such as serine and glycerol labeled at the same carbon, show very pronounced differences in the two deuterons even at lower magnetic field.

The analysis of the motion of a CD 3 group is rather straightforward. If we transform the electric field gradients of the deuterons into a segment- fixed coordinate system in which one axis, called the p axis, is parallel 132 with the long molecular axis (the carbon skeleton), then the order parameter of the CD3 group, which possesses a threefold axis of symmetry, can be represented by equation V.21

Sp = ((3cos 2|}*-1)/2)-1Scd (V.21)

where (3* is the angle between the C-D vector and the p axis. For example, if we wanted to calculate the order parameter for the N-C symmetry axis of a choline methyl group, assuming tetrahedral bond angles, the order parameter would be according to equation V. 16, ISncI = l(3cos2(109.4)-

1)/2I1 Scd - 3Scd- While the quadrupole splittings of the various C-D bond vectors yield a quantitative description of the C-D bond, they are not sufficient to describe the overall movement of a CD 2 or CD 3 group and caution must be exercised in interpreting these order parameters.

As has been discussed, deuterium quadrupole splittings, A\) q , provide information concerning the time-averaged orientations of the phospholipid segments. In contrast to this, 2H NMR relaxation times, dominated by the quadrupolar relaxation mechanism, provide information on the rate of segmental motion. The correlation between the orientation information provided by ADq and the rate or mobility information provided by relaxation times is not well understood yet, and it is thus important to keep these two topics separate in describing the molecules. In the case of fast isotropic motion of the C-D bond, the spin-lattice relaxation can be related to the correlation time of reorientation by equation V.22 (Seelig & Seelig,

(1980)). 133

1/Tj = 3/8(e2qQ/h)2(l-SCD)Tc (V.22)

Equation V.22 assumes that there is a single type of segment reorientation that is described by a single correlation time. Equation V.22 only applies for reorientations that are classified as being in the short correlation regime, to 0 2 i c2 <

Seelig, (1980) suggest that if T! increases with increasing temperature, this implies that t c is in the short correlation time regime. These conditions are met in the case of vesicles and also the methyl rotor of the choline head group (Gaily, et al., (1975)). The case of anisotropic motions is more complicated and will not be discussed here. While the requirements to describe the rotational correlation time are stringent, some information can still be extracted from the spin-lattice relaxation time. Using the Arrhenius correlation between temperature and spin-lattice relaxation times, the apparent activation energy for the motions involved in the spin-lattice relaxation can be obtained using equation V.23.

InTi = InA - AE./RT (V.23)

A plot of lnTj vs T 1 yields a slope equal to -AE,/R (R= 8.314 kJ(mol-K ) 1 or 1.987 cal(mol-K)1). 134 If deuterium NMR offers so much valuable information, why has it only been recently that its applications have been used. There are two main disadvantages to 2H NMR that hindered its effectiveness as a tool in elucidating lipid structures. The first is deuterium has a low natural abundance (0.16%). The low natural abundance is a blessing in disguise because while it guarantees isotopic incorporation by use of chemical means, it enables site specific labeling thus adding a powerful dimension to its use in elucidating specific structural sites of motion. The second problem is technical in nature. The main spectroscopic problem was an initial loss of signal due to fast decay after the pulse and a receiver dead time that overlapped most of the signal. This problem was overcome by the use of a multipulse sampling sequence called the quadrupole echo sequence

(Davis, et al., (1976)). The quadrupole echo pulse sequence is depicted in figure 46.

9 0 , 9 0 ,

Figure 46: A diagramatic representation of the Quadrupole echo pulse sequence.

The pulse sequence consists of a pair of 90 degree rf pulses whose phase differs by 90 degrees (an X and a Y pulse) separated by a time, x. The first pulse produces the free induction decay and the second pulse refocuses the magnetization so that an echo occurs after a time, 2 x. 135

Receiver overload is avoided by making t sufficiently long (20-60 jisec) so that the receiver has recovered from the rf pulses. The exact parameters used in our studies will be given in section II, materials and methods.

With this foundational knowledge of 2H NMR laid, let us turn our attention to the specific data describing the orientation and motion of DPPC in bilayers.

A Brief Description of DPPC Bilayers

As stated earlier, DPPC is the reference compound for the thio analogs,DPPsC diastereomers. One of the goals of my research is to compare DPPsC diastereomers with DPPC. This section will briefly mention the salient points describing the orientation and motion of the

DPPC molecule in bilayer dispersions. The DPPC molecule will be discussed according to three main divisions; 1 ) the acyl chains, 2 ) the glycerol backbone and 3) the choline head group.

The Acyl Chains

The acyl chains are more commonly referred to as the hydrocarbon region. The phospholipid molecules are packed into bilayers and membranes such that the hydrocarbon regions create a hydrophobic layer separated by the hydrophilic polar head group regions. Chemically the nature of the two acyl chains are identical. But when the phospholipid is dispersed into bilayers, an inequivalence is created at various positions of 136 the hydrocarbon chains. This inequivalence is most pronounced at C2 of

the acyl chains. 2H NMR studies utilizing deuterated chains at C2 showed

three quadrupolar splittings (Seelig & Seelig, (1975)). This result

suggested that the chains were physically inequivalent and adopted different average conformations in the liquid-crystalline bilayer. This

interpretation was supported by other techniques such as X-ray diffraction

studies of crystalline bilayers. The molecular picture that is most widely accepted is one in which the C2 methylene group of the sn-2 acyl chain is oriented parallel to the bilayer normal while the .yn -1 acyl chain is extended perpendicular to the bilayer normal. This twist at C2 of the .sn-2 chain is sensed in other parts of the hydrocarbon chains, which is deduced from the observation of two quadrupolar splittings for the C3 and C10-C15 segments. Even though the differences between these splittings is rather small (2-3 kHz), this data clearly shows that the two chains are not completely equivalent. A model showing this bend in the sn-2 chain is given figure 47. 137

032 3J >11 >03- ’32 013 011

01* 012

Figure 47: A molecular model of DLPE showing the sn-2 chain bend

(Hitchcock et al., (1974)).

The inequivalence of the two chains is also born out by the observation that segments of the two chains show differing degrees of motion or flexibility as revealed by relaxation data. The major cause of this differing in flexibility is the difference in the distance from the segment to the lipid- water interface in which the segments experience different fluid like environments. The relaxation times of the C2 segments are short, indicating restricted motion. They are slightly longer than the glycerol backbone relaxation times, which are the most rigid part of the molecule and have the shortest relaxation times. The relaxation times of the acyl segment from C3 to C9 are fairly constant and are indicative of a slightly more flexible segment. From C l2 to C14 there is a marked increase in flexibility as indicated by an increase in the relaxation times until the acyl chain terminal methyl group, which is in the center of the bilayer and possesses the greatest amount of flexibility, even more than the choline methyl groups. All of the relaxation times increase with increasing 138 temperature. As mentioned above, this implies that the correlation time of the segment reorientation falls within the short correlation time regime and can be related to the spin-lattice relaxation time and the order parameter by equation V. 17. The correlation times of the acyl chain components of the bilayer in the liquid crystalline state were found to range from 8 x 1 0 " 11 sec in the C3-C9 segment to about 3xl0 u sec for the C15 methylene group

(Brown, Seelig & Haberlen, (1979)).

The Glycerol Backbone

While there are some differences associated with the two acyl chains, the overall description of the hydrocarbon region indicates that the imbedding of the acyl chains into the bilayer increases the amount of molecular motion experienced by this segment. While this hydrocarbon segment shows varying degrees of mobility, the glycerol backbone region does not. The glycerol backbone is described as being the most rigid part of the phospholipid. While the motion of this segment is very slow, there is nonetheless some degree of flexibility. This was shown by labeling studies done at C3 of the glycerol backbone. Calculation of the order parameter for this segment showed that while the motion is restricted, it is not completely anisotropic, suggesting a significant degree of flexibility in the bilayer. The orientation of the glycerol backbone has been described by

X-ray crystal structures of anhydrous racemic l, 2 -dilauryl- 5 n-glycero- 3 - phosphoethanolamine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine dihydrate (Hitchcock, et al., (1974), and Pearson & Pascher, (1979)). The 139 glycerol backbone is nearly perpendicular to the bilayer planes and parallel to the sn-1 chain (see figure 47).

Choline Head Group

The final segment of the phospholipid that needs described is the choline head group. As was mentioned in Chapter IV. 1, 31P NMR studies of DPPC monohydrate crystals have shown that the plane containing the nonesterified oxygens and the phosphorus is oriented approximately at a

50° angle with respect to the bilayer normal, and the P-O vector in that plane is approximately parallel to the bilayer plane (figure 48).

Figure 48: Molecular models illustrating the 50° tilt of the phosphate moiety in DPPC molecules. The rod represents the bilayer normal. The model on the left shows the 50° tilt, and the model on the right shows the O-P-O plane parallel to the normal (Griffin, (1981)). 1 40

Neutron diffraction studies on specifically deuterated DPPC head groups have shown that the 2H nuclei at Ca, C|3 and Cy are all located at nearly the same distance from the center of the bilayer, implying that the P-

N electric dipole moment is roughly parallel to the plane of the bilayer

(Buldt, et al., (1978); Buldt, et al., (1979) and Zaccai, et al., (1979)).

From these studies and other 2H NMR measurements (Gaily, et al., (1975) and Seelig, (1977)) a conformational picture of the head group is described

(figure 49).

114 .€

C,

Figure 49. Conformation of the Head Group in Glycerylphosphoryl- choline (Seelig, et al., (1977)).

The conformation is described as a boomerang, which is also in agreement with the above cited neutron diffraction studies. In this figure, Cl 1 is Ca,

Cl 2 is Cp and Cl 3 is Cy. The motion of the choline head group has been described by two

models. The first model assumes two schemes of simple uncoupled

rotations which can simulate the observed reduction of the 31P shielding

tensor (Kohler & Klein, (1977)). The first scheme consists of a fast

rotation about the P-Oll bond (figure 49), followed by a slower overall

rotation about the glycerol C1-C2 bond, followed by an even slower overall

rotation about the long molecular axis. The second scheme includes only

two rotations, a fast one about the P-Oll bond, followed by a slower one

about the long molecular axis. This model can have these two explanations

of the observed 31P phenomenon because it only takes into account one

parameter. The second model, developed by Srffelig, et al., (1977),

accounts for five independent deuterium and phosphorus anisotropies.

But, just because the model accounts for more parameters this does not justify its correctness. Support from other types of studies will be needed

to justify it. In their model, Seelig, et al., assume a fixed conformation

about the C l-O il and P-O ll bonds and allow the torsional angles, a l and

a2 to oscillate between specific values. From this model, Seelig, et al. conclude that the choline head group can neither be completely rigid nor completely flexible but jumps between two enantiomeric conformations that

are comprised of gauche-gauche bonds between the choline atoms.

The observations obtained from the thio analogs will be interpreted in

light of these conclusions of DPPC. In the discussion section of this chapter specific points will be expanded upon to better interpret the results, but here only a brief description was necessary. 142 V.2 Materials and Methods

The synthesis of the respectively deuterated choline tosylates and

subsequent DPPsC diastereomers is given in Chapter II. 2. As stated in

Chapter II, the samples were of high purity both chemically and

stereochemical ly.

Samples for the 2H NMR experiments typically consisted of between

50-100mg of lipid dispersed in an equal weight of 2 H-depleted water

(Aldrich), which were sealed in 5 mm o.d. glass tubes. The lipids were assured of full hydration by first heating the sample to 60°C, mixing vigorously with a vortex stirrer, then cooling to room temperature. This process was repeated from 5-9 times until the lipid was fully hydrated as judged by the complete dispersion of the of dry lipid in water. The lipid samples were then annealed at 0-4°C for at least 10 days. The 2H NMR experiments were performed on a Bruker MSL-300 spectrometer with a 2H frequency of 46.07 MHz. 2H NMR spectra were obtained by using a two pulse quadrupole echo sequence, 90°x-ii-90°y-X2-AQ (Davis, et al.,

(1976)), with a 90 degree pulse length of 4.5 psec, a pulse separation, ii, of 25 psec and a mixing time, X 2 , of 28 |isec. The acquisition and recovery time between sequences was >500 ms. For spectra below the main transition temperature a typical sweep width of 625 kHz was used and between 50,000 to 100,000 transients collected (depending upon the sample size). Above the main transition temperature the spectral sweep width was typically 62.5 kHz and between 1,000 to 10,000 transients collected.

Spin-lattice relaxation experiments were performed using a modified 143 inversion recovery pulse sequence. The recovery of magnetization after a

single 180° pulse was detected using a quadrupole echo sequence, 180°-

VD-90o*-Ti-90oy-,t2-AQ. The quadrupole echo parameters were the same

as described above. The value of %2 was different than each value of the

variable delay, in order to assure a maximum echo. All measurements for

the spin-lattice relaxation time, T ), were carried out above the main

transition temperature of the lipid, where axially symmetric fast-limit powder pattern spectra with well-defined perpendicular edges are observed.

Because the spin-lattice relaxation rate of lipids in the liquid crystalline phase is independent of orientation (Brown, et al., (1981)), the intensity of the perpendicular edge was used to provide a measure of the recovery rate.

On a typical spectrum, the perpendicular edges are those frequencies that have a significantly higher population at a particular angular orientation.

This can be visualized by referring to figure 45. The Tj value was obtained by a least-squares fit of the amplitude data. Phase cycling and quadrature detection were used in all NMR experiments.

After the NMR experiments were conducted, the sample was checked for chemical purity by TLC and was always found pure. 1 44 V.3 Results

Diastereomers of ad 2 -DPPsC

Below the main transition temperature

It has already been established (Wisner, et al.,(1986) and Sarvis, et al.,

(1988)) that (Rp+Sp)- and (Sp)-DPPsC behave thermotropically similar to

DPPC, while (Rp)-DPPsC behaves uniquely. Table 3 summarizes the thermotropic phase properties of these lipids. Figure 50 shows the spectra for a d 2 -DPPsC diastereomers below their main transition temperatures.

Table 4 lists the values of the widths at half maximal height for the spectra in figure 50.

At 5°C

As mentioned in section V.2, materials and methods, the phospholipid samples were annealed prior to running the spectra. This annealing is necessary to assure that the lipid is in the subgel phase, as was born out in the DSC studies by Wisner, et al., (1986). The subgel phase of ph ospholipids (speaking specifically of phosphatidylcholines) is not usually studied by means of solid state NMR. The reason being that the spectra are extremely broad and often shapeless. So in order to compare the diastereomers of DPPsC to DPPC, we found spectra of ad 2 -DPPC from

Ruocco, et al., (1985), as shown in figure 51. While they did not anneal 145 Table 3: Summary of the Thermotropic Phase Properties of DPPC and the Diastereomers of DPPsC.*

Transitions (°C)

Sub- Pre- Main

DPPC 19 35 41

(Rp + Sp)-DPPsC 21.7 43.8 44.8

(Sp)-DPPsC 2 2 . 0 43.7 45.0

(Rp)-DPPsC - - 45.9

Taken from Wisner, et al., (1986). B C

J5* 35*

’ I ' ' ---- ' T ’"1 ’ I ' ' '-I-' 5 0 0 0 0 o -5 0 0 0 0 5 0 0 0 0 o -5 0 0 0 0 5 0 0 0 0 0 -5 0 0 0 0 HERTZ HERTZ HE fll 2

Figure 50: JH NMR spectra of a d 2-DPPsC diasteromers below their main transition temperatures. Where (A) is the (Rp+Sp) mixture, (B) is the (Sp) and (C) is the (Rp) diasteromer.

-Ft o\ 147 Table 4: 2H NMR Spectral Widths at Half-height and Quadrupolar

Splittings versus Temperature for a-d 2 -DPPsC diastereomers.

Temp (°C) At)* (kHz)

W i,2 A B C (Rp+Sp) 5 56 25 15 35 13 48 6.4 3.0 2.1 50 6.4 3.0 2 . 1 55 6.4 3.1 2 .2 60 6.3 3. 1 2.3 65 6 . 2 3.1 2.4

(Sp) 5 32 25 16 35 11 49 6.5 1.7 52 6.5 1.9 55 6.5 1.9 60 6 . 6 2 . 2 65 6.4 2 .2

(Rp) 5 82 25 82 35 82 48 6.9 50 7. 1 1 .0 55 6.9 1.5 60 6 . 8 1 .6 65 6 . 6 1.8

* The letters A,B,C refer to the sharp fast-limit powder pattern edges with A having the largest quadrupole splitting value and C having the smallest. 148

B

-2 3

KX) 0 HOO

FREQUENCY CkH?]

20 -20

FREQUENCY CkHz)

Figure 51: NMR spectra of ad 2 *DPPC well below its main transition temperature showing the subgel phase, (A) and at temperatures characteristic of the various thermotropic phases (B). 149 the DPPC sample prior to acquiring the spectra, they did acquire spectra

well below the subtransition temperature, which clearly display the subgel

phase of this lipid. When a d 2 -DPPC was cooled to -23°C, without prior

annealing, the spectrum appeared as an amorphous lineshape with a width

at half maximal height of 93 kHz. This spectrum exhibits a rigid-lattice 2H

NMR tensor. This spectrum shows that the amount of motion experienced

by the 2H tensor is very slight but not zero. For a 2H NMR tensor with no

motion the spectrum is a completely rigid lattice spectrum that has the

characteristic powder pattern type splitting but only with the difference in

quadrupolar splittings near 170 kHz (see figure 45). The spectra in figure

50, showing the diastereomers at 5°C, show that all of the diastereomers

are in the subgel phase. The width at half maximal height for the Rp+Sp

sample is 56 kHz, the Sp isomer is 32 kHz and the Rp isomer is 82 kHz.

The spectra all exhibit amorphous lineshapes with a sharp component in the

center. This sharp component could be a result of many phenomena, such

as HDO, small unilamellar vesicles of lipids oriented at the magic angle. It

is most likely due to residual HDO since this would have the highest

concentration of all the above possibilities.

Comparing the diastereomers of DPPsC there are some differences that

arise. The Rp isomer seems to be axially more rigid than the mixture,

Rp + Sp, and the Sp isomer indicated by the larger width at half maximal

height. Between Rp + Sp and Sp, the mixture exhibits a more axially rigid

tensor than Sp, again indicated by a larger width at half maximal height.

These results support the DSC data obtained by Wisner, et al., (1986), in

which they observed that the Rp isomer relaxes into the subgel phase faster 150 than the others and that Rp+Sp requires a shorter incubation time than Sp.

It is interesting to note that the arithmetic average of the widths at half maximal height for Rp and Sp is equal to that of Rp+Sp. The interpretation of a result such as that is beyond the scope of this work.

At 25°C

The spectra at 25°C for the diastereomers of DPPsC show the effect of going through the subtransition. The 2H NMR spectral lineshapes for the

(Rp + Sp)- and (Sp)-DPPsC samples narrow considerably indicating a higher degree of mobility experienced by the C-D bond. The width at half maximal height for (Rp+Sp)-DPPsC is 14.8 kHz and for (Sp)-DPPsC is

15.8 kHz. These two values agree very well with the values reported for a d 2 -DPPC by Ruocco, et al., (1985) in figure 51. The width at half maximal height at 28°C is 13.3 kHz. The spectrum reported by them at

16°C has the same width at half maximal height because their sample was not annealed prior to running the spectrum. The Rp isomer remains in the subgel phase indicated by the lack of change in the spectrum at 25°C.

At 35°C

The spectra at 35°C show more narrowing of the width at half maximal height for (Rp + Sp)- and (Sp)-DPPsC. This narrowing is caused by an increase in the motion of the C-D bond. The Rp isomer remains in the subgel phase as seen by the repeating of the lineshape. 15 1 The spectra of the ad 2-DPPsC diastereomers annealed prior to running confirm the previous DSC and 31P NMR results compiled for these lipids.

Specifically, (Rp+Sp)- and (Sp)-DPPsC behave thermotropically similarly to DPPC, while (Rp)-DPPsC is unique and remains in the subgel phase at temperatures below its main transition.

Above the Main Transition Temperature

At 45°C

Figure 52 shows the 2H NMR spectra of the ad 2 -DPPsC diastereomers above their main transition temperature. Table 3 lists the temperatures corresponding to the thermotropic phase changes of the lipids discussed so far. (Rp + Sp)- and (Sp)-DPPsC undergo a pretransition at 44°C. The limitations of the temperature control of the probe used in obtaining the 2H

NMR spectra prohibited the confidence of distinguishing between the prc- and main transitions of these thiophosphorylcholines, seeing the two transitions differed only by one degree. The spectra of (Rp + Sp)- and (Sp)-

DPPsC at 45°C reveal the fact that the lipid is in transition. This can be visualized by the severe broadening of the powder patterns (more noticeable in the Rp + Sp mixture than in the Sp isomer). These broadened spectra are similar to the spectrum of a d 2 -DPPC at a temperature just prior to the main transition. The Rp isomer has a ’main’ transition at 46°C. The

DSC trace of this thermotropic change is broad (see figure 36). The broadness of this DSC trace explains why the 2H NMR spectrum at 48°C ss*

FmTT™ I ' " '"I...... 10000 o -10000 IOOOO -10000 HERTZ 0 1O0QO 0 10000 HERTZ H f : H T

Figure 52: 2H NMR spectra of a d 2-DPPsC diastereomers above their main transition temperatures. Where (A) is the (Rp+Sp) mixture. (B) is the (Sp) and (C) is the (Rp) diasteromer. la 153 has not completely transferred into the fast-limit powder pattern spectrum indicative of the liquid crystalline phase, where the C-D bond exhibits a high degree of motional freedom.

From 50°C to 65°C

Beyond the main transition temperature the spectra for all the lipids assume the fast-limit powder pattern spectra indicative of 2H tensors with relatively fast fluctuations. ad 2 -DPPC exhibits a spectrum with a quadrupolar coupling splitting value of 6 kHz (Ruocco, et al., (1985)).

The quadrupolar coupling constant does not change with increasing temperature, within experimental error. The DPPsC diastereomers likewise exhibit fast-limit powder patterns above their main transition temperatures.

There is a difference between the DPPsC diastereomers and DPPC. The spectra of the Sp and Rp isomers of DPPsC show two sets of quadrupolar splittings. The two quadrupolar splittings arise from the inequivalence of the two deuterons on the choline chain and not from the the larger splitting being the 0° orientation and the smaller splitting the 90°. If the two only differed in the orientation (assuming the two deuterons were magnetically equivalent), then according to equations V. 15 and V. 16 the 0° orientation quadrupolar splitting would be exactly twice the value of the 90° orientation. This indeed is not the case, as can be seen clearly in table 4, which lists the quadrupolar splittings. The inner splittings are between 1 and 2 kHz, while the outer are 7 kHz. Close inspection of the spectra in figure 52 reveals a low shoulder approximately 14 kHz wide, which 154 corresponds to the 0° orientation for the outer splitting. The 0° orientation for the inner splittings is which is betwen 2 and 4 kHz wide, appears as a slight shoulder on the main trunk of the spectra in figure 52. The inequivalence of the two deuterons next to a chiral center is not an unusual phenomenon. Browning & Seelig, (1980) reported the inequivalence of two deuterons on 1,2-dimyristoyl-.rrt-glycero-3-phospho-L-serine

(deuterated at the a carbon of the serine). The difference between these two quadrupolar splittings was ~13 kHz. Also, when they used DL-serine

{deuterated in the a position) they observed four sets of splittings with the

D-serine splits occurring as shoulders on the L-serine splittings. A more interesting note from that reference is that for ad 2 -DPPC they observed an inequivalence of those two deuterons at a higher magnetic field (61.4

MHz). The difference in splittings was only 300 Hz. Since the deuterons at the alpha position are far away from the sn -2 chiral center, we think the splitting (nonequivalence) observed for ad 2 -DPPC is more likely to be due to a "transient chiral phosphorus center” arising from a stereospecific interaction involving one of the two nonbridging oxygen atoms of the phosphate group. This is precisely why we continue to study the properties of DPPsC diastereomers.

(Rp+Sp)-DPPsC displays three sets of quadrupolar splittings above the main transition temperature. The outer splitting being the same as the outer splittings corresponding to the Sp and Rp isomers and the two inner splittings corresponding to the Sp and Rp isomers, respectively. As was the case with the individual isomers, the 0 ° orientation shoulder is noticeable outside the widest splitting. 155

The splittings of all the a d 2 -DPPsC diastereomers remain fairly constant with an increase in temperature. This result is consistent with a d 2 -DPPC. One way to compare molecules is to calculate order parameters for them. Equation V. 1 8 can be used to calculate the order parameters for the deuterons using their respective quadrupolar splittings. The order parameter for ad 2*DPPC is 0.047, for one deuterium. The molecular order parameter would be twice this value {see equation V. 19). For comparison to the a d 2 -DPPsC diastereomers we will use the order parameter for one deuteron. The values of the order parameters for the diastereomers of

DPPsC over a range of temperatures in the liquid crystalline phase are given in table 5. The order parameters for the outer splittings of all a d 2-

DPPsC isomers is equal to that of a d 2 -DPPC. The inner order parameters are at least half of the outer. Order parameters in general range from 0 to

1. An order parameter equal to one has the most restrictive environment and an order parameter equal to zero has the most amount of motional freedom (these values are absolute values). So with order parameters ranging from 0.05 to 0.01, we can conclude that even the most relatively restricted C-D bond is somewhat free to rotate according to the absolute limits of the order parameter. It is also evident that there is one deuteron that experiences more freedom of rotation than another.

Another parameter that describes the amount of motion of a molecule is the spin-lattice relaxation time. Table 6 gives the spin-lattice relaxation times for the a d 2 -DPPsC molecules over a range of temperatures in the liquid crystalline phase. All of the spin-lattice relaxation times increase with increasing temperature, indicating less restriction of the C-D bond. 156 Table 5: 2H NMR Order Parameters versus Temperature for

a -d 2-DPPsC diastereomers.

Temp (°C) S CD

AB C

(Rp+Sp) 48 0.050 0.023 0.016 50 0.050 0.024 0.016 55 0.050 0.024 0.017 60 0.049 0.024 0.018 65 0.048 0.024 0.019

(Sp) 49 0.051 0.013 52 0.051 0.015 55 0.051 0.015 60 0.051 0.017 65 0.050 0.017

(Rp) 48 0.054 50 0.055 0.008 55 0.054 0 . 0 1 1 60 0.053 0.013 65 0.051 0.014

* S c d is the Order Parameter for the C-D bond. Its relationship to the Quadrupolar Coupling constant by equation in the text. The letters A,B,C refer to the sharp fast-limit powder pattern edges with A having the largest quadrupole splitting value and C having the smallest. 157 Table 6 : 2H NMR Spin-Lattice Relaxation Times versus

Temperature for a-d 2 -DPPsC diastereomers.

Temp (°C) Ti*(msec)

A BC

(Rp+Sp) 48 17.6 17.4 18.2 50 19.6 18.3 18.9 55 22.7 22.4 22.5 60 27.8 25.9 26.3 65 32.7 31.7 30.9

(Sp) 49 16.0 16.2 52 15.8 17.7 55 19.5 2 2 . 1 60 26.6 23.8 65 26.4 30.8

(Rp) 48 50 17.4 18.2 55 19.6 2 0 .1 60 2 2 .1 23.9 65 26.6 28.4

The letters A,B,C refer to the sharp fast-limit powder pattern edges with A having the largest quadrupole splitting value and C having the smallest. 158

The spin-lattice relaxation time for a d 2 -DPPC is 30 msec at 50°C. The

a d 2 -DPPsC diastereomers are significantly lower than this value at 50°C,

which suggests that the DPPsC diastereomers are more restricted in motion

than DPPC. The spin-lattice relaxation times alone do not yield very

informative molecular data. What is commonly done is to fit the data to an

Arrhenius equation so as calculate an energy of activation for the

reorientation process. The Arrhenius equation used is given by equation V

23. The activation energy values obtained by this method are listed in table

7. The values for (Rp+Sp)- and (Sp)-ocd 2 -DPPsC agree quite well with the

value of DPPC. The values for (Rp)-ad 2 -DPPsC are just slightly lower

than that of a d 2 -DPPC.

In summary the following points were arrived at:

1 ) the a d 2 *DPPsC diastereomers exhibit a much more restricted subgel

phase than a d2 -DPPC,

2) (R p)-ad 2 -DPPsC continues in the subgel phase until its 'main'

transition,

3) in the gel phase, the Rp+Sp and Sp isomers show gel structures comparable to DPPC, according to lineshape analysis,

4) in the liquid crystalline phase the DPPsC diastereomers show a

noticeable difference of inequivalence of the two deuterons in the a position compared to DPPC and

5) from the order parameter and activation energy data DPPC and the

DPPsC diastereomers (at least one of the deuterons) experience similar environments. 159 Table 7: Activation Energies of Reorientation for 0td 2 -DPPsC Diastereomers and Arrhenius Correlation Coefficients.*

E, (kj/mol)*

A B C

DPPCt 30. 1

(Rp + Sp)-DPPsC 31.37 30.89 27.65

(.9981) (.9957) (.9980)

(Sp)-DPPsC 33.08 34.62

(.9476) (.9782)

(Rp)-DPPsC 24.55 26.69

(.9856) (.9863)

5 The Arrhenius Correlation Coefficients are given in parentheses.

* A,B,C refer to the sharp fast-limit powder edges of the 2H NMR spectra with A having the largest quadrupolar splitting and C the smallest.

+ Taken from Browning (1981). 160

Diastereomers of pd 2 *DPPsC

Below the main transition temperature

At 5°C

Figure 53 shows the spectra of (Rp+Sp)-, (Sp)- and (Rp)-Pd 2-DPPsC at temperatures below the main transition temperature. The spectra at 5°C show the amorphous lineshapes characteristic of a restricted motional environment. These lineshapes are similar as those found for the ad 2 isomers at 5°C in figure 50. The widths at half maximal height are given in table 8 . The width for (Rp+Sp)-(3d 2 -DPPsC is 39 kHz and (Sp)-Pd 2 -

DPPsC is 24 kHz. These values are approximately 10-20 kHz smaller than their a d 2 counterparts. These spectra in figure 53 show that the pd 2 diastereomers reflect a less restricted motion at the p carbon of the head group than at the a position in the subgel phase (a note: because of the unavailability of low temperature spectra for pd 2 -DPPC, the pd2 -DPPsC diastereomers can only be compared to themselves and the other deuterated positions, namely ad 2 and yd^. (Rp)-Pd 2 -DPPsC exhibits a spectral lineshape that is considerably broader than (Rp+Sp)- and (Sp)-pd 2-DPPsC by about 25-40 kHz. The width at half maximal height is6 6 kHz. This value is approximately 20 kHz smaller than (R p)-ad 2 -DPPsC at 5°C. The reduction in half widths is characteristic of all three pd 2 samples and B

3S* 35*

T T T ’ ’ T’ '' "T r ’ ’"T” " 50000 0 -50000 5 0 0 0 0 0 -5 0 0 0 0 5 0 0 0 0 0 -5 0 0 0 0 HERTZ HERTZ HERTZ

Figure 53: 2H NMR spectra of |Jd 2-DPPsC diastereomers below their main transition temperatures. Where (A) is the (Rp+Sp) mixture, (B) is the (Sp) and (C) is the (Rp) diasteromer. 6 1 16 162 Table 8: 2H NMR Spectral Widths at Half-height and Quadrupolar Splittings versus Temperature for [5-d2-DPPsC diastereomers.

Temp (°C) Au (kHz)

W112 AB C D E

(Rp+Sp) 5 39 25 14 35 10 49 6.1 2.8 1.8 52 5.7 2.8 1.8 55 5.8 2.8 1.8 60 5.6 2.9 2.0 1.1 0.43 65 5.6 2.9 2.1 0.76 0.32

(Sp) 5 24 25 10 35 8 49 1.5 0.72 52 0.81 0.30 55 0.70 0.15 60 0.55 0.04 65 0.44

(Rp) 5 66 25 66 35 66 49 7.1 1.2 0.55 52 7.0 1.6 0.92 0.31 55 7.0 1.6 0.85 0.19 60 7.0 1.7 0.70 65 7.0 1.9 0.52 25b 66

“The letters A,B,C,D,E refer to the sharp fast-limit powder pattern edges defined in figure 54. bThe (Rp) sample was cooled back to 25°C to show the metastability of the subgel phase. 163 indicates a higher degree of mobility at the P carbon compared to the a

carbon.

At 25°C and 35°C

On going through the subtransition temperature, (Rp+Sp)- and (Sp)-

Pd 2 -DPPsC show a significant narrowing in their lineshapes in figure 53,

which is to be expected since the subgel phase is characteristically more

rigid than the gel phase. From 25°C to 35°C there is only slight narrowing

in the values of the widths at half maximal height for the two samples,

(Rp+Sp)- and (Sp)-Pd 2 -DPPsC. (Sp)-pd 2 -DPPsC is somewhat narrow,

indicating relatively more motional freedom. (Rp)-Pd 2 -DPPsC once again maintains the subgel phase up until the 'main' transition temperature as seen by the repeating spectra at 25°C and 35°C.

Above the main transition temperature

At 45°C

Figure 54 shows the spectra for the Pd 2 diastereomers above the main transition temperature. Figure 55 shows the complete letter labeling designations for the quadrupolar splittings of (Rp+Sp)-Pd 2 *DPPsC at

60°C. (Rp + Sp)-, (Sp)- and (Rp)-(ld 2 -DPPsC all show slightly broadened fast-limit powder pattern spectra at 45°C. These are a result of the broad B

65* 65*

60* 60*

55 * 55*

' I ’ 5000 0 -5 0 0 0 5000 0 -5 000 5000 0 -5000 HERTZ HERTZ HERTZ

Figure 54: 2H NMR spectra of pd 2 -DPPsC diastereomers above their main transition temperatures. Where (A) is the (Rp+Sp) mixture, (B) is the (Sp) and (C) is the (Rp) diasteromer. os -fi 20 -2 0 -* 0 -M

Figure 55: 2H NMR spectrum of (Rp+Sp)-(id 2 -DPPsC at 60°C showing the assignments of the labels A,B,C,D,E. 165 166 thermotropic phase transitions, which make the completion of the phase

change at a temperature higher than the reported transition temperature.

From 52°C to 65°C

(Sp)-JJd 2 -DPPsC at 52°C shows a narrow spectrum with two

quadrupolar coupling constants. The larger of the two being a slightly

resolved shoulder. The quadrupolar coupling constants at 52°C are 1.5 kHz and 0.72 kHz for the outer and inner splittings, respectively. The existence of two quadrupolar couplings is indicative of the inequivalence of the two deuterons in the p position. This result is quite unique since the (5 position is two bonds removed from the chiral phosphorus center. A plausible explanation of this result could be that the p position is not entirely flexible, perhaps causing an inequivalence of the two deuterons.

This phenomenon was observed in ad 2 *DPPC, in which the two deuterons are 5 bonds removed from the nearest chiral center (C2 of the glycerol backbone) and yet show two inequivalent deuterons which have quadrupolar coupling constants differing by 300 Hz (Browning & Seelig,

(1980)). The (Sp)Pd 2 -DPPsC diastereomer shows a difference of only

800 Hz, which is not entirely unreasonable. This idea of partial flexibility will be explored in more detail when the results of (Rp+Sp)- and (Rp)-pd 2 -

DPPsC are discussed. Table 8 lists the quadrupolar coupling constants over a range of temperatures in the liquid crystalline phase. The (Sp)-pd 2 *

DPPsC quadrupolar splittings both exhibit temperature dependence and decrease as temperature increases. The degree of temperature dependence 1 67 can be seen more clearly when the order parameters are compared over the range of temperatures. Table 9 lists the order parameters over the range in temperatures in the liquid crystalline phase. The order parameters of (Sp)-

Pd 2 -DPPsC are relatively small compared to the a d 2 isomer and become even more disordered as the temperature increases. Table 10 lists the spin- lattice relaxation times for the [3d2-DPPsC diastereomers and table 11 lists the activation energies. The activation energy for the reorientation process of (Sp)-pd 2 'DPPsC is 14.05 kJ/mol. This value is relatively smaller than that obtained for pd 2 *DPPC, 27.1 kJ/mol (Browning, (1981)). This implies that the reorientation process of the inner quadrupolar resonance corresponding to one of the deuterons can occur with a minimal amount of energy. This is usually interpreted as that deuteron being in a motionally less restrictive environment.

Let us now consider the (Rp+Sp)- and (R p )P d 2 -DPPsC samples at temperatures between 52° and 65°C. The Rp isomer will be explained in detail and the (Rp+Sp)-Pd 2 -DPPsC sample will be a composite of the Rp and Sp results.

At 52°C (Rp)-pd 2 -DPPsC shows a spectrum that is quite unique.

Keeping in mind that the Rp isomer only has two deuterons and if each were inequivalent, as has been the case so far, then each would produce one set of quadrupolar splittings thus making a total of two. What the Rp isomer is revealing is four sets of quadrupolar splittings. What this suggests is that not only are the two deuterons inequivalent, but there must also be an inequivaleqce between two molecules which is reflected at the (1 carbon position. Recalling that Browning and Seelig (1980) observed Table 9: 2H NMR Order Parameters versus Temperature for p-d 2 -DPPsC diastereomers.

a Temp (°C) Scd

AB CD E

(Rp+Sp) 49 .047 .021 .014 52 .044 .022 .014 55 .045 .022 .014 60 .044 .022 .015 .009 .003 65 .044 .022 .016 .006 .003

(Sp) 49 .012 .006 52 .006 .002 55 .005 .001 60 .004 .0003 65 .003

(Rp) 49 .055 .009 .004 52 .055 .012 .007 .002 55 .055 .012 .007 .001 60 .055 .014 .005 65 .055 .015 .004

“The letters A,B,C,D,E refer to the sharp fast-limit powder pattern edges designated in figure 55. 169 Table 10: 2H NMR Spin-Lattice Relaxation Times versus Temperature for (3-d2-DPPsC diastereomers.

Temp (°C) Ti*(msec)

A B C

(Rp+Sp) 49 14.0 18.2 19.1 52 24.4 20.5 21.7 55 19.0 21.2 24.8 60 15.0 17.8 29.8 65 15.9 26.4 33.1

(Sp) 49 52 21.2 55 32.5 60 42.1

(Rp) 49 17. 1 52 17.7 55 60 21.3

‘The letters A,B,C,D,E refer to the sharp fast-limit powder pattern edges designated in figure 55. 170 Table 11: Activation Energies of Reorientation for (3d2-DPPsC Diastereomers and the Arrhenius Correlation Coefficients®.

Ea (kJ/mol)*

A BC

DPPCt 27.1

(Rp+Sp)-DPPsC 27.7 19.6 30.9

(.1667) (.9902) (.9963)

(Sp)-DPPsC 14. 1

(.9598)

(Rp)-DPPsC 17.3

(.9901)

®The Arrhenius Correlation Coefficients are given in parentheses.

*A,B,C refer to the sharp fast-limit powder edges of the 2H NMR spectra with A having the largest quadrupolar splitting and C the smallest. tTaken from Browning (1981). 17 1

inequivalence between the deuterons of ad 2 -DPPC, it is very unlikely that

these were magnetically inequivalent since they were 5 bonds away from

the nearest chiral center. What then could be a possible explanation for

this? In his work on deuterated DPPC molecules, Seelig (1977) explained

why he saw two quadrupolar splittings for the C2 methylene deuterons on

the sn-2 chain and also the two deuterons on the Cl carbon of the glycerol

backbone (Cl is the carbon that is connected to the phosphate moiety in his numbering scheme). Summarizing the results obtained by Seelig (1977), observing only one set of quadrupolar splittings for each segment of the choline head group, he concluded that the two deuterons were in the same environments. This suggested a rather flexible choline head group.

Another explanation could be that since the quadrupolar splittings, in principle, could be a measure of the averages of many different configurations of the choline head group, the single quadrupolar splitting could arise from the fact that the transitions between configurations is faster than the NMR timescale, thus giving only an average. In another paper dealing with the effect of adding one degree of unsaturation to the s/j-acyl chain on the 2H NMR spectra, Seelig & Seelig (1977) explained the data of observing two sets of splittings along positions of the acyl chains by assuming two enantiomeric configurations of the molecule. They observed that the incorporation of a double bond in the in -2 acyl chain causes a loss of flexibility in the sn-l chain positions nearest the double bond. Seelig (1977) then applied this reasoning to explain why there appears two sets of quadrupolar splittings at in-2 C2 and the glycerol backbone Cl carbons. The torsional angles that they obtained from the head group deuteron data were similar to those established for crystals of

L-a-glycerophosphorylcholine by Abrahamsson and Pascher (1966), which contains two enantiomeric molecules per unit cell. Seelig concludes that if this model of the head group being rather flexible is correct, then the choline group as a whole is rotating around the glycerol C1-C2 bond, with the choline group parallel to the bilayer normal. In explaining the existence of two quadrupolar splittings for the sn-2 methylene and the glycerol C-l deuterons, Seelig offers two possible explanations. First, two quadrupolar splittings could be produced from assuming a very rigid glycerol backbone in which the two deuterons are oriented at different angles with respect to the rotation axis. But in light of the temperature dependence of the deuterons, one being temperature dependent and the other independent, this explanation becomes improbable. A second explanation makes use of the idea of two conformational states of the molecule. The existence of two long-lived conformations of the lipid molecule leading to two different orientations of the glycerol backbone will also produce two sets of quadrupolar splittings. This then implies that on the 2H NMR timescale the conformational transitions are slow in the glycerol backbone and fast in the phosphorylcholine group. This indeed is the most probable explanation seeing that the result of Browning and Seelig, (1980) in which they observed two quadrupolar splittings for ad 2 -DPPC, confirms the probability of the two conformations. Since they only could observe the two splittings by improving the resolution of the spectrum, this suggests that perhaps the timescale of the choline head group rotations is not faster than the NMR timescale. 173

How does this apply to the observations seen here for the (Rp)-pd 2 -

DPPsC isomer? Based on the differences in temperature dependence and

also the appearance of four quadrupolar splittings instead of two, I believe

that these results support the interpretation of two conformational states

present in the bilayer. Perhaps the Rp isomer's configuration at the

phosphate puts the choline head group in a position that slows down the

conformational transitions relative to the 2H NMR timescale and thus

allowing the observation of two such states. Why do only the pd 2 isomers

reveal these states and not the ocd 2 isomers? A possible explanation is that

the conformational states under consideration are not due to rotations about

the glycerol C1-C2 bond but about the Ca-CJJ bond of the choline head

group. This type of rotation would not induce changes in the a carbon

deuterons but would induce a change in the 3 carbon deuterons. Also the yN methyl deuterons would still experience rapid rotation about the N-Cy

bond thus making them equivalent. The y-Nd 9 case will be discussed later.

This phenomenon of extra quadrupolar splittings arising was observed

again by Akustu and Seelig (1981) when they were studying the effect of metal ions on phosphatidylcholine bilayers using 2H NMR. They observed the appearance of a second set of quadrupolar splittings for Pd 2 -DPPC with samples containing a Europium III ion concentration >5 mM. The second set of quadrupolar splittings were much larger than the original.

Unfortunately, no absolute conclusions were made as to the origin of these new resonances.

(Rp+Sp)-[Jd 2 -DPPsC behaved similarly to the Rp isomer. Two sets of the quadrupolar splittings behaving like the Sp isomer, showing 174 temperature dependence and two sets being temperature independent. The spin-lattice relaxation data was difficult to obtain for those resonances not well resolved in the Rp and Rp+Sp samples. For this reason, only the central peak was measured in the Rp sample and three out of five in the mixture. The activation energy of i? central Rp resonance is lower than that of (3d2-DPPC and is comparable to set B of the mixture and the Sp isomer. These activation energies correspond to the quadrupolar splittings designated A,B, and C. Those resonances with the lower activation energy reflect a temperature dependence in the quadrupolar splittings, which indicates an environment that is increasing in flexibility thus making the activation energy of the conformational transition lower.

The largest and smallest resonances listed in table 9 for (Rp+Sp)-(5d2-

DPPsC have activation energies comparable to pd 2 -DPPC. Where the quadrupolar splittings of the |3d2-DPPC sample were found to be temperature dependent (Gaily, et al., (1975)), the quadrupolar splittings of these resonances for (Rp+Sp)-pd 2 *DPPsC were not.

It appears that the activation energy data presented along with the quadrupolar splitting data and the spectral lineshapes indeed suggest the possibility of two conformationally different states for (Rp+Sp)- and (Rp)- pd 2 -DPPsC existing in the bilayer. 175 yNd 9 *DPPsC Diastereomers

Below the main transition temperature

At 5°C

Figure 56 shows the spectra of (Rp+Sp)-, (Sp)-, and (Rp)-yNd 9 -DPPsC at 5°C. The spectra of (Rp + Sp)- and (Sp)-yNd 9 -DPPsC are Lorentzian lineshapes. These are in great contrast to the 5°C spectra of the respective a d 2 and 3^2 isomers shown in figures 50 and 53, which are amorphous lineshapes characteristic of a more restricted motional environment. The widths at half maximal height (table 12) are considerably narrower for all the yNd9 samples compared to both the a and 3 samples. This reflects the great amount of motional freedom experienced by the choline methyl groups in the bilayer. The Rp isomer produces a rather unique lineshape in the subgel phase between 5°C and 35°C. There is a slightly resolved quadrupolar splitting of about 7 kHz.

25°C and 35°C

(Rp + Sp)- and (Sp)-yNd 9 -DPPsC show some narrowing in the widths at half maximal height on passing through the subtransition. The Sp isomer begins to show a poorly resolved quadrupolar splitting at 25°C. At 35°C, the Sp splitting is more resolved and the Sp isomer is approximately one B

35'

■ ' I " ■ ■ -1 ■ ■ • ■T” 10000 0 10000 10000 0 -10000 10000 0 -10000 HERTI HERTZ HERTZ

Figure 56: 2H NMR spectra of YNd(>-DPPsC diastereomers below their main transition temperatures. Where (A) is the (Rp+Sp) mixture, (B) is the (Sp) and (C) is the (Rp) diasteromer. 1 76 177 Table 12: 2H NMR Spectral Widths at Half-height and Quadrupolar

Splittings versus Temperature for y-Nd 9 -DPPsC diastereomers.

Temp (°C) Au(kHz)

w 1/2 AU q

(Rp+Sp) 5 4.2 25 2.2 35 1.9 49 0.37 52 0.33 55 0.30 60 0.18 65 0.11

(Sp) 5 3.1 25 2.2 35 0.80 49 0.37 52 0.33 55 0.26 60 0. 18 65 0.11

(Rp) 5 6.2 25 6.2 35 6.2 49 0.44 52 0.44 55 0.41 60 0.33 65 0.22 70 0. 18 178

half as broad as the Rp+Sp mixture. The Rp isomer continues in the subgel

phase.

Above the Main Transition Temperature

Figure 57 shows the 2H NMR spectra of the yNd 9 -DPPsC diastereomers

above their main transition temperatures. Above the main transition

temperatures, the yNd? samples assume fast-limit powder pattern

lineshapes with quadrupolar splittings that are narrow. All the samples

show quadrupolar splittings that decrease as temperature increases, which

can also be seen in table 13 when the order parameters are inspected as a

function of temperature. The Rp+Sp mixture and Sp isomer seem to behave

very similarly with quadrupolar splittings and order parameters almost

identical. The Rp isomer is just a little broader. The spin-lattice relaxation

times and calculated Arrhenius activation energies are given in tables 14

and 15, respectively. Compared to yNd^-DPPC, the thio analogs have

activation energies that are slightly higher, which would suggest that the

thio analogs are slightly more restricted in the motion of the choline head

group. In general though the yNdg-DPPsC samples are the motionally less restricted segment of the choline chain, which is in agreement with those

results obtained for DPPC. B

6 5*

60* 60*

55*

5 V

iI

’ T ^ [ ' ' 1 ' 5000 0 -5000 5000 0 -5000 5000 0 -5 000 HEA7Z meat ; HERTZ

Figure 57: 2H NMR speectra of ^Nd^-DPPsC diastereomers above their main transition temperatures. Where (A) is the (Rp + Sp) mixture, (B) is the (Sp) and (C) is the (Rp) diasteromer. vC 180 Table 13: 2H NMR Order Parameters versus Temperature for Y-Ndg-DPPsC diastereomers.

Temp (°C) ScD

(Rp+Sp) 49 0.003 52 0.003 55 0.002 60 0.001 65 0.001

(Sp) 49 0.003 52 0.003 55 0.002 60 0.00! 65 0.001

(Rp) 52 0.003 55 0.003 60 0.003 65 0.002 70 0.001 181 Table 14: 2H NMR Spin-Lattice Relaxation Times versus

Temperature for y-Nd 9 *DPPsC diastereomers.

Temp (°C) Ti(msec)

(Rp+Sp) 49 50.4 52 56.8 55 62.3 60 68.9 65 72.5

(Sp) 49 61.2 52 73.2 55 77.7 60 90.5 65 98.8

(Rp) 52 57.7 55 67.1 60 78.4 65 87.1 70 92. 1 182 Table 15: Activation Energies of Reorientation for ydg-DPPsC Diastereomers and the Arrhenius Correlation Coefficients*.

E, (kJ/mol)

DPPCt 16.7

(Rp + Sp)-DPPsC 19.5

(.9783)

(Sp)-DPPsC 25.5

(.9825)

(Rp)-DPPsC 23.8

(.9814)

f Taken from Browning (1981).

* The Arrhenius Correlation Coefficients are given in parentheses. 183 V.4 Discussion

The overall goal of this dissertation was to provide a clearer insight into

the nature and possibly the structure of DPPsC diastereomers. In light of

this goal, this discussion section must try to bring together the fragments

of information gathered by a variety of sources. The danger in producing a

structural model of a molecular segment is that if only one or two pieces of

data are used just about any type of model can be used to interpret the data.

What will be done here is that 31 P NMR and various 2H NMR data will be

used to try and create a visualization of the structure of the choline segment

of the DPPsC molecule. Before that is done, we must look at the currently

accepted model of the choline head group for the DPPC molecule. The

model proposed by Seelig, et al., (1977) is currently the accepted model.

A summary of the salient points of this model will be given here.

A description of the phosphorylcholine segment

The conformational analysis of the phosphorylcholine head group is based on the same model that describes the phosphorylethanolamine head group. In this model the C1-C2 axis of the glycerol backbone is the axis of rotation for the phosphorylcholine moiety. Crystal structure data obtained on dilaurolyl-DL-phosphatidylethanolamine shows that the C1-C2 vector is aligned perpendicular to the bilayer plane (Hitchcock, et al., (1974)). This was further supported by neutron diffraction data of l,2-dimyristoyl-3-.sn- phosphatidylcholine (Worcester, (1976)). It was shown by 2H NMR 1 84 studies of DPPC deuterated at Cl on the glycerol backbone that in the liquid crystalline state the C1-C2 axis is not rigidly perpendicular to the bilayer plane because of the observance of two quadrupolar splittings of 27 and 29 kHz (Gaily, et al., (1975)). Both of these values are considerably smaller than that expected for a completely rigid 2H tensor. Since X-ray crystal data do not account for motion but do support an unhindered C1-C2 axis, the NMR data was explained by assuming a fast wobbling motion in the C1-C2 axis about the bilayer normal. What the authors then did was to formulate a theoretical model to characterize the observed 2H NMR, 3lP

NMR and crystal data. With their model, they explored the possibility of three different cases to try and find the best set of conditions to explain the experimental results. The first case considered was free rotation around all bonds of the phosphorylcholine backbone. This was quickly disqualified when the chemical shift anisotropy data was compared to the theoretical values obtained from the free rotation of all the torsional angles in the phosphorylcholine moiety. An absolute chemical shielding anisotropy value of 5.1 ppm was obtained from this model. This is a far cry from the observed value of 47 ppm. Also when the 2H NMR quadrupolar coupling values were calculated, they too were much smaller than the observed.

Therefore, a completely flexible model was ruled out.

The second case was the opposite extreme, a completely rigid model.

In this model, difficulties were encountered when the a and p segments of the choline head group deuterium data were fit to this model. According to this model, the observable magnitudes of the quadrupolar splittings were explained by a bent configuration of the phosphorylcholine group. But 185 when configurations containing all cis or all trans (the two extremes) were

assumed the experimental results still did not fit.

The third case considered was a two-state model. The model assumes

rapid equilibrium between two enantiomeric choline conformations, one

with all the torsional angles positive and the other with all the torsional

angles negative. If the molecule fluctuates between these two configurations, then the corresponding deuterons will exchange their orientation relative to the rotation axis. If the jump is sufficiently rapid

(>104 Hz), then the two deuterons will experience the same average electric field gradient, which will lead to the same quadrupolar splittings. If the jump is slowed down (<104 Hz), then the two deuterons will not experience an average electric field gradient but will experience different gradients thus leading to a second set of quadrupolar splittings. The experimental data support for the existence of two enantiomeric states comes from the crystal studies of L-a-glycerophosphorylcholine

(Abrahamsson & Pascher, (1966)). These two configurations seem to be carried along from the crystal state to the liquid crystal state. The authors then used this model to calculate the 31P NMR chemical shielding anisotropy value and the various 2H NMR quadrupolar splittings in a configurational space of ±30° of the crystallographic torsion angles. The agreement between the calculated and experimental results was excellent.

Refinement of the torsional angles used would bring the values closer than reported, but the point was proven as to the validity of this model describing the glycerophosphoryl moiety. An interesting aspect of this model is that it shows the sensitivity of the Au and Ac values to a variation 186 of the torsional angles. For example, changing the Cp-Ca torsional angle from ±81° to ±80° reduces the quadrupolar splitting of the p deuteron by approximately 1 kHz. This sensitivity to the variation in the torsional angle can be used to explain the sensitivity of the quadrupolar splittings to temperature. Very small changes in the torsional angles are needed to cause such changes in the splittings.

A description of the choline segment

The authors conclude their structural model by citing evidence for the configuration of the choline moiety with respect to the bilayer normal.

They cite three pieces of evidence that show that the choline moiety is oriented parallel to the bilayer plane. The first evidence is found by X-ray diffraction studies of oriented multilayers of DPPC containing various amounts of water. The electron density profiles show that the phosphorus and nitrogen atoms of the choline group are lying in a plane parallel to the surface at temperatures above and below the main transition temperature.

The second evidence comes from neutron diffraction studies involving

2H20-H 20 exchange in which the same conclusion was drawn. The third piece of evidence comes from neutron diffraction studies of deuterated lipids at various temperatures and hydration states. The measurements clearly show that the average distances of the Ca . Cp and CT carbons from the bilayer surface are nearly identical, regardless of the temperature or hydration state. This piece of evidence is probably the most direct evidence pointing to a choline head group oriented parallel to the bilayer 187 surface. They also showed that these distances were not dependent upon the presence or absence of cholesterol or if the acyl chains were saturated or unsaturated.

In conclusion then, the model that is supported the most and that explains the observed data the best consists of a partially flexible glycerol backbone C1-C2 axis that wobbles about the long molecular axis (the director axis) and a choline head group that lies parallel to the bilayer surface and jumps between two enantiomeric conformations. With this background then, the results obtained in this work can be fit to a model similar to the one just described.

For clarity's sake, the phosphate head group will be divided into two segments as discussed above, the glycerophosphoryl moiety and the choline head group.

Since we did not acquire 2H NMR data on the glycerol backbone carbons, only a discussion of the 31P NMR data can be made. Realizing that the chemical shift anisotropy values only reflect the amount of flexibility, the following comparisons can be made. The thio analogs of

DPPC undergo relatively faster rotations in the phosphorus chemical shielding tensor, than DPPC, which is demonstrated by the decrease in the chemical shielding anisotropy values for (Sp)-, (Rp)- and (Rp+Sp)-DPPsC.

There then appears to be a significant difference in phosphate environments between chiral phosphates and achiral phosphates. And among the chiral phosphates there appears to be evident an effect in the difference in configuration at phosphorus on the flexibility of the phosphate group. The

Rp isomer being more freedom of motion than the Sp isomer. It appears 188 then that the incorporation of sulfur on the phosphate causes significant differences in the motional freedom experienced by the phosphorus tensor.

Perhaps the increase in atomic size of the sulfur atom, plus the lack of ionization of the sulfur to produce a strong ionic attraction either inter or intramolecularly could in effect increase the relative distance between phosphate groups, thus allowing for more motional freedom.

The 2H NMR data collected on the choline head group appears to have generated the most interesting results in light of the two-state model discussed above. The 2H NMR data obtained for the a d 2 - DPPsC diastereomers can be explained by the above model similarly to a d 2 -DPPC.

The a d 2 -DPPsC diastereomers showed two sets of quadrupolar splittings for both (Sp)- and (Rp)-DPPsC, and even in the mixture (which showed three sets due to the overlap of one set in the individual isomers), which were temperature independent. This would suggest that the torsional angles of the P-O-Ca bonds were not changed by increasing the temperature. This translates into a description of the fluctuations being in the fast regime (>104 Hz) and therefore the two conformational states being averaged into one. This may be the picture, but some doubt arises in my mind because first, there are two quadrupolar splittings observed. This is most likely a result of the presence of a chiral center which causes the inequivalence of the two deuterons. But the magnitude of the effect of a chiral center on the quadrupolar couplings has not been determined, so the contribution that a chiral center has on the appearance of a second quadrupolar splitting is not well defined. The question is raised because you may recall the discussion on the observance of two very close 189

quadrupolar splittings observed for ad 2 -DPPC by Browning and Seelig,

(1981). Clearly these cannot be explained by a chiral center, since the

deuterons are five bonds away from the nearest chiral center. So perhaps

the a d 2 carbon oxygen axis is not very much into the fast regime or maybe

it is on the borderline. A distinction between these two effects cannot be

made based on the available data.

The most interesting result came from the pd 2 studies. There was a

marked difference between the two diastereomers. The Sp isomer produced two sets of quadrupolar splittings both being temperature dependent. The difference between the two sets was small, but the two were resolved.

Based on the model described above, these two sets of quadrupolar splittings arise from two enantiomeric conformational states with respect to the Coc-Cp bond of the choline head group. This is analogous to the results obtained by Akustu and Seelig, (1981) for Pd 2 -DPPC over a variation in temperature, exct,. y only observed one set of splittings.

The two sets of splittings arise from the rate of interconversion between the two enantiomeric forms. It appears then, that the thio analogs experience a much slower interconversion rate than their DPPC counterparts, which allows the observation of the two iong-lived states. So the two enantiomeric conformational states for the Sp isomer arise from changes in the torsional angle of the C a-C p bond.

The Rp isomer shows an even more interesting phenomenon. The Rp isomer produces four sets of quadrupolar splittings, two of which are temperature dependent and two temperature independent. According to our established model the temperature dependent quadrupolar splittings are a 190 result of a change in the torsional angle of the Ca-Cp bond. This is analogous to the Sp isomer. The temperature independent enantiomeric conformational states arise from a change in the torsional angle of one of the axes in the glycerophosphoryl moiety. The precise axis cannot be identified without looking at deuterated glycerol samples. A change in this torsional angle will cause two enantiomeric conformational states which do not have a change in the torsional angle of the Ca-CP bond.

The Rp isomer then contains two sets of enantiomeric conformational states, one caused by a change in the Ca-Cp bond and the other by a change in one of the glycerophosphoryl bonds.

The mixture behaved very similarly to the Rp isomer and so the same model interpretation can be applied.

The yNdg isomers showed an increase in rotational motion of the methyl rotors causing very narrow lines. The freedom of rotation is in agreement with the DPPC model studies and the existence of two conformational states cannot be observed at this position.

In summary then, the DPPsC molecule shows differences between the two diastereomers by a variety of techniques, DSC, 31P NMR and 2H

NMR. The greatest difference seen at the P carbon position of the choline head group, where the Rp isomer reveals the existence of two long-lived enantiomeric pairs of conformational states. A clearer picture of these states and a conformation of their existence is needed and could be provided by other techniques such as X-ray diffraction or neutron diffraction allowing the observation of the two states to be confirmed. 1 9 1

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