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Stereochemistry and mechanism of phospholipase A2 and phosphatidylinositide-specific phospholipase C
Lin, Gialih Hoffmann, Ph.D.
The Ohio State University, 1989
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 STEREOCHEMISTRY AND MECHANISM OF PHOSPHOLIPASE A2 AND PHOSPHATIDYLINOSITIDE- SPECIFIC PHOSPHOLIPASE C
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Giaiih Hoffmann Lin, B.S.,
^ ^ ^
The Ohio State University
1989
Dissertation Committee: Approved
M.-D. Tsai
G. A. Fraenkel Advisor Department of Chemistry A. W. Czarnik To My Grandparents and Parents ACKNOWLEDGMENTS
I express sincere appreciation to Dr. Ming-Daw Tsai for his guidance and insight throughout the research. Thanks go to the other member of my committee, Drs. Gideon A. Fraenkel and Anthony W. Czarnik, for their suggestions and comments. To Mr. Joseph Noel, I offer sincere thanks for the kinetic results in Chapter III. To Dr. Henry Z. Sable, I thank you for providing the starting material for the synthesis of cyclopentanoid analogues of phosphatidylcholines. To Dr. William Loffredo, I thank you for the great assistance in NMR works. To my wife, Chun-Zu, I offer many thanks for your understanding. VITA February 8, 1959 Bom, Taiwan
June, 1981 . . . B.S., The National Taiwan
University, Taipei, Taiwan 1983-Present . . Teaching assistant and
Research assistant in
Chemistry Department of
the Ohio State University
PUBLICATIONS
1. Use of Short-chain Cyclopentano-phosphatidylcholines to Probe the Mode of Activation of Phospholipase A2 from Bovine Pancreas and Bee Venom: Gialih Lin, Joseph Noel,
William Loffredo, Henry Z. Sable, and Ming-Daw Tsai, J. Biol. Chem. 263,13208-
13214 (1988) 2. Phospholipids Chiral at Phosphorus. 18. Stereochemistry of Phosphatidylinositide-
Specific Phospholipase C: Gialih Lin and Ming-Daw Tsai, J. Am. Chem. Soc. 111.
3099-3101 (1989)
FIELDS OF STUDY
Major Field: Chemistry Studies in (Organic Synthesis, Phosphorus Chemistry, and Enzyme
Stereochemistry) TABLE OF CONTENTS
ACKNOWLEDGMENTS ii VITA iii
LIST OF ABBREVIATIONS iv
LIST OF FIGURES xiii
LIST OF SCHEMES xvii
LIST OF TABLES xviii CHAPTER PAGE
I. INTRODUCTION 1
Introduction 1
Backgrounds of Lipid Chemistry 2
Introduction to Part I: the Cyclopentanoid Analogues
of Short-chain Phosphatidylcholines 5
Introduction to Part II: the Stereochemistry of
Phosphatidylinositide-specific Phospholipase C 16
Reference 32
II. SYNTHESIS OF SHORT-CHAIN CYCLOPENTANOID
ANALOGUES OF PHOSPHOLIPIDS 39
Introduction 39
Results 40
Experimental Section 43 Discussion 64
Reference 65
HI. USE OF SHORT-CHAIN CYCLOPENTANOID
v ANALOGUES OF PHOSPHATIDYLCHOLINES TO
PROBE THE MODE OF PHOSPHOLIPASE A2 67 Introduction 67
Experimental Section 68
Results and Discussion 68
Reference 86
IV. SYNTHESIS AND CONFIGURATIONAL ANALYSIS
OF THIOPHOSPHATIDYLINOSITOL 88 Introduction 88
Results 89
Experimental Section 99
Discussion 117
Reference 120
V. THE STERIC COURSE OF THE REACTION CATALYZED BY PHOSPHATE)YLINOSITIDE-SPECIFIC
PHOSPHOLIPASE C 122
Introduction 122
Results 123
Experimental Section 138
Discussion 148
Reference 159
VI. SYNTHESIS OF SOME DERIVATIVES OF MYO-INOSITOL 161 Introduction 161
Results 161
Experimental Section 167
vi Reference
BIBLIOGRAPHY LIST OF ABBREVIATIONS
AA arachidonic acid bs broad singlet BSA baine serum albumin
CDP-DG cytidine diphosphate-1,2-diacyl-sn-
glycerol CDP-DGsyn cytidine diphosphate-1,2-diacyl-sn-
glycerol synthase clPhyd myo-inositol 1,2-cyclic phosphate
hydrolase cmc critical micelle concentration
COSY correlated spectroscopy
Cp- cyclopentanoid- Cp-DC4PC cyclopentanoid analogue of 1,2-
dibutyryl-sn-glycero-3-
phosphocholine Cp-DC6PC cyclopentanoid analogue of 1,2-
dihexanoyl-sn-glyceio-3- phosphocholine Cp-DC8PC cyclopentanoid analogue of 1,2-
dioctanoyl-sn-glycero-3 -
phosphocholine Cp-DPPC cyclopentanoid analogue of 1,2-
dipalmitoyl-sn-glycero-3-
phosphocholine
Cs lipid concentration 5 chemical shift d doublet DCnPC l,2-diacyl-sn-glycero-3-
phosphocholine dd doublet of doublets ddd doublet of doublet of doublets de diastereomeric excess
DEPT distortionless enhancement by
polarization transfer
DMAP 4-dimethylaminopyridine DG 1,2-diacylglycerol
DG kinase diacylglycerol kinase
DPPC l,2-dipalmitoyl-sn-glycero-3-
phosphocholine
DPPE l,2-dipalmitoyl-sn-glycero-3-
phosphoethanolamine
DPPS l,2-dipalmitoyl-sn-glycero-3-
phosphoserine
DPPsC 1,2-dipalmitoyl-sn-glycero-3-
thiophosphocholine DPPsE 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine
DPPsS 1,2-dipalmitoyl-sn-glycero-3-
thiophosphoserine
dt doublet of triplets
FAB fast atom bombardment
GC gas chromatography
h hour
IP myo-inositol phosphate
l:2c-IP myo-inositol l:2cyclic phosphate 1»4-IP2 myo-inositol 1,4-bisphosphate
1:2c,4-IP2 myo-inositol l:2cyclic,4-
bisphosphate l,4,5-n>3 myo-inositol 1,4,5-trisphosphate
1:2c,4,5-IP3 myo-inositol l:2cyclic,4,5-
trisphosphate
IPase myo-inositol-1 -phosphatase
IPs phosphorothioate analogues of myo
inositol phosphate
l:2c-IPs phosphorothioate analogues of myo
inositol 1:2 cyclic phosphate
IPsyn L-myo-inositol-l-phosphate synthase LUV large unilamellar vesicles m multiplet
MG monoacylglycerol MLV multilamellar vesicles
MM- molecular mechanics-
MOMQ chloromethyl methyl ether
mp melting point
MPLC medium pressure liquid
chromatography
MS mass spectroscopy
NMR nuclear magnetic resonance
NOE nuclear Oveihauser effect
NOESY nuclear Overhauser enhancement and
exchange spectroscopy
PA phosphatidic acid PAF patelet activating factor
PC phosphatidylcholine (lecithin)
PC-PLC phosphatidylcholine-hydrolyzing phospholipase C
PE phosphatidylethanolamine
PG phosphatidylglycerol
PI phosphaddylinositol
PI kinase phosphaddylinositol kinase
PI-PLC phosphatidylinositide-specific
phospholipase C
PIP phosphaddylinositol
4-monophosphate PIP2 phosphaddylinositol 4,5-bisphosphate
PEPase phosphatidylinositol 4-phosphate 4-
phosphatase PIP2ase phosphatidylinositol 4,5-
bisphosphate 5-phosphatase
PIP kinase phosphatidylinositol 4-phosphate
kinase
PIsyn myo -inositol by phosphatidylinositol
synthetase
PLA1 phospholipase A1
PLA2 phospholipase A2
PLC phospholipase C
PLD phospholipase D
PPi pyrophosphate rt room temperature
s singlet
sn stereospecific numbering
Sph sphingomyelin
Sph-PLC sphingomyelin-hydrolyzing
phospholipase C
SUV small unilamellar vesicles
t triplet
TLC thin layer chromatography
TsOH p-toluenesulfonic acid LIST OF FIGURES
1. Phospholipases and phospholipids 4
2. Hydrolysis of dioctanoylphosphatidylcholine by bovine pancreatic PLA2 7
3. The structure of aggregated phospholipids 8
4. The diastereoisomeric cyclopentane-l,2,3-triols 10
5. The Tentative Rules, Sundaralingam numbering system
and sn numbering system 13
6. Phospholipids and their phosphorothioate analogues
(thiophospholipids) 15
7. The metabolism of inositol phospholipids 17
8. The D-and L-Numbering system of myo-inositol 19
9. Stereospecificities of phospholipase toward thiophospholipids 25
10. Three methods to prepare the known configuration
substrates of PI-PLC 27
11. Three methods to determine the absolute configuration at
the phosphorus of the products from PI-PLC hydrolysis 29
12. Determination of the absolute configuration at phosphorus
of non-cyclic inositol phosphate from PI-PLC 31 13. *H NMR spectrum of Cp-DCgPC 51
14.13C and 31P NMR spectra of Cp-DC8PC 52
15. W H COSY NMR spectrum of Cp-DC8PC 53
16.2D J-resolved NMR spectrum of Cp-DC8PC 54
17. NOESY spectrum of Cp-DC8PC 55
18. Mass (FAB) spectrum of Cp-DC8PC 57
xiii 19. The fragments of short-chain cyclopentanoid analogues
of phosphatidylcholines 58 20. *11 NMR spectrum of Cp-DC4PC 60
2 1 .13C NMR spectrum of Cp-DC4PC 61
22. Mass (FAB) spectrum of Cp-DC4PC 62
23. The a-methylene multiplet of DQPC 70
24. Determination of the cmc of DC6PC 71
25. The a-methylene multiplet of DCgPC 72
26. Determination of the cmc of DCgPC 73
27. The a-methylene multiplet of Cp-DCgPC 74 28. Determination of the cmc of Cp-DCgPC 75
29. a-Methylene and t-methyl regions of the *H NMR spectra 76
30. Determination of the cmc of Cp-DCgPC in the presence
ofBSA 77 31. Determination of % de of compound 12 by 13C NMR 93 32. Determination of % de of compound 12 by GC 94
33. The 31P NMR spectra of compound lfi 95
34. The 31P NMR spectrum of compound DPPsI 97
35. The hydrolysis of DPPsI catalyzed by bee venom
PLA2 monitored by 31P NMR 98
36. The *14 NMR spectrum of DPPsI 111 37. The COSY spectrum of DPPsI 112
38. The expansion spectrum of Figure 46 113
39. The partial 13C DEPT spectra of DPPsI 114
40. The mass (FAB) fragments of DPPsI 116
xiv 41. The 1,2-phosphoryl migration of DPPsI 118
42. The absolute configuration of //zyo-inositol
l:2cyclic phosphorothioate 124
43. Hydrolysis of DPPsI catalyzed by PI-PLC
monitored by 31P NMR 125
44. The stability of 1,3,2-dioxaphospholanes 129
45. The structure of phosphites 28a and 28h
from MM-2 calculation 130 46. The conformations of 22a (exo) and 29b (endo) (obtained from MM-2 energy minimization) 132
47. Another view of Figure 46: The H(l)-C(l)-0-P dihedral angles of 22a and 29b 134 48. NOESY spectrum of 29a (exo) 135
49. NOESY spectrum of 22b (endo) 136
50. Determination of the absolute configuration of the product
of the hydrolysis of DPPsI catalyzed by PI-PLC 137
51. The steric course of the reaction catalyzed by
Bacillus cereus PI-PLC 138
52. The NMR spectrum of compound 29a 147 53. The 31P decoupling *H NMR spectrum of compound 22a 148 54. The 2D J-resolved *H NMR spectrum of compound 22a 149 55. The NMR spectrum of compound 22b 150 56. The 31P decoupling NMR spectrum of compound 29b 151
57. The 2D J-resolved !H NMR spectrum of compound 29b 152
58. Possible Mechanisms of PI-PLC (A to C) 156
xv 59. Possible Mechanisms of PI-PLC (D to F) 157
xvi LIST OF SCHEMES
1. Synthesis of (l,3/2)-l-0-benzyl-cyclopentane-l,2,3-triol 41
2. Synthesis of short-chain cyclopentanoid analogues of phosphatidylcholines and phosphatidic acids 42
3. Synthesis and resolution of D- and L- myo-inositol precursors 90
4. Synthesis of l,2-dipalmitoyl-sn-glycerol-3-thiophosphoinositol 91
5. Synthesis of DL- myo-inositol l:2cyclic phosphorothioate 127 6. Synthesis of D-2,3:4,5-di-0-cyclohexylidene-6-0-P-
trimethylsilylethoxymethyl-myo-inositol 162 7. Synthesis of D-2,3:5,6-di-0-cyclohexylidene-4-0-p-
trimethylsilylethoxymethyl-myo-inositol and D-4-O-benzyl-1,2-0-
cyclohexylidene-myo-inositol 164
8. Synthesis of D-4,5-di-0-acetyl-3-0-benzyl-6-(-)-camphanoyl-
myo-inositol and D-3-O-benzyl-l ,2-0-cyclohexylidene-myo-inositol 165
9. Synthesis of DL-3,4,5,6-tetra-0-butyryl-l,2-0-cyclohexylidene-myo-inositol 166 LIST OF TABLES
Summary of kinetic data of PLA2 hydrolysis Comparison of A8 values between monomers of Cp-compounds and corresponding open-chain compounds
xviii CHAPTER I
INTRODUCTION
Introduction
In this dissertation, two mechanistic questions of phospholipases are investigated. In
the first part (Chapter II and HI), we try to answer the question: Is the substrate
conformation model an effect for the interfacial activation between monomers and micelles
of the hydrolysis catalyzed by phospholipase A2 (PLA2)? To probe this question, we use
a conformationally restricted cyclopentanoid analogue of phosphatidylcholine. Chapter II deals with the synthesis of the short-chain cyclopentanoid analogues of phosphatidylcholine and phosphatidic acid. Chapter ID discusses the use of cyclopentanoid analogues of phosphatidylcholine to probe the mode of activation of PLA2.
This chapter reports that the hydrolysis of the cyclopentanoid analogue of phosphatidylcholine catalyzed by the pancreatic PLA2 lacks the interfacial activation from a monomer to a micelle, arid this chapter supports the substrate conformation model (Wells,
1974): the interfacial activation of the PLA2 hydrolysis is because of a conformational change of the substrate from monomers to micelles.
The second part (Chapter IV to VI) of this dissertation deals with the stereochemistry of phosphatidylinositide-specific phospholipase C (PI-PLC). We use the substrate and the
1 2
product phosphorothioate analogues of the PI-PLC to probe this question. Chapter IV
deals with the synthesis of the phosphorothioate analogue of the substrate of PI-PLC, 1,2-
dipalmitoyl-sn-glycero-3-thiophosphoinositol (DPPsI) and the determination of its absolute
configuration at phosphorus by the action of bee venom PLA2. Chapter V reports the
synthesis and configurational analysis of the phosphorothioate analogues of the product of
PI-PLC, myo-inositol l:2cyclic phosphorothioate (l:2c-IPs). Chapter VI deals with the synthesis of some precursors for the total synthesis of inositol containing compounds. The
second part concludes that the steric course of the reaction catalyzed by PI-PLC proceeds
with complete inversion of configuration at phosphoms. In other words, the reaction
mechanism involves a single-step displacement.
Background of Lipid Chemistry
Lipid
Including fatty acids and their derivatives, steroids, terpenes, carotenoids and bile
acids, lipid is a class of natural products which are soluble in organic solvents such as
diethyl ether, hexane, benzene, chloroform or methanol. Today, lipid is always restricted to fatty acids and their ester or amide derivatives and to compounds biosynthetically related to fatty acids. Lipids can be divided into four main classes: fatty acid and alcohol components, which contain simple fatty acid, long-chain alcohol, sterol, mono-, di-, and triglycerides, cholesterol and cholesterol esters, and wax esters; glycerolphospholipids
(or phosphoglycerides) which yield glycerol, fatty acids and inorganic phosphate on hydrolysis; glycoglycerolipids (or glycosyldiglycerides), which yield glycerol, fatty acids and carbohydrates on hydrolysis; and sphingolipids, which contain a long-chain
base, fatty acids inorganic phosphate, carbohydrates or other complex organic compounds.
Phospholipids
Phospholipids contain glycerophospholipids such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI),
polyphosphatidylinositide, phosphatidylserine (PS), phosphatidylglycerol (PG), ether
phospholipids, platelet activating factor (PAF), and sphingomyelin (Sph) (Figure 1). Unless otherwise specified, the abbreviation DCnPC represents the L-isomer of 1,2-
diacyl(n-carbon fatty acid)-sn-glycerol-3-phosphocholine. When n=16 (palmitoyl), the abbreviation DP- is used instead of DC16-. For the phosphorothioate analogues, Ps is used
to replace P (phospho) in the abbreviation. For the cyclopentanoid analogues of glycerol,
the prefix, Cp-, is used to represent 1,3/2 (all trans)-l,2,3-cyclopentane-triol in the abbreviation.
Phospholipase
According to the particular ester bond being hydrolyzed, phospholipases are divided
into phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC) and
phospholipase D (PLD) (Figure 1). PLA1 (EC 3.1.1.32) and PLA2 (EC 3.1.1.4)
respectively catalyze the hydrolysis of diacylglycerolphospholipids at sn-1 and sn-2
positions. The products of both PLA's are lysophospholipids (sn-l-lyso for PLA1 and sn-
2-lyso for PLA2; the term lyso, if not specified, usually means sn-2-lysophospholipid that
yield from the PLA2 hydrolysis of phospholipids such as lysoPC, lysoPI, etc.). Because the liberation of unsaturated fatty acids such as dihomogammalinolenic acid, arachidonic PLA1 (EC 3.1.1.32)
O s n 1 PLD (EC 3.1.4.4) C —I O j Ri R,— c —o » 4 sn-2 o *2 i! i n ii t I------o — P— OY sn-3 A | cr PLA2 f (EC 3.1.1.4) pT r Pi-specific : (EC 3.1.4.10) PC-hydrolyzing: (EC 3.1.4.3) Y= CH2CH2N(CH3)3+: PC; Y= CH2CH2NH3+: PE; Y= CH2CH(OH)CH2OH: PG; Y=H: PA; Y= CH2CH(COO' )NH3+: PS;
OH
OH Y= HO
OW ZO
W = Z = H : PI; W= H, Z= P032*: PIP; W =Z=P032': PIP2
Figure 1: Phospholipids and phospholipases acid (AA) or eicosapentanenoic acid initiates the formation of many physiologically active
metabolites, a great deal of attention has been focused on the liberation of unsaturated fatty
acids from sn-2 position of various phospholipids catalyzed by PLA2. These fatty acids
are substrates for cyclooxygenase and lipoxygenases (Hamberg et al., 1975; Taylor and
Morris, 1983). The hydrolysis of glycerolphosphate ester bond in phospholipids is
catalyzed by PLC. There are three types of PLC (Ikezawa and Taguchi, 1986):
phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC, EC 3.1.4.3) catalyzes the
hydrolysis of PC, PE, PS, lysoPC, PA and Sph; sphingomyelin-hydrolyzing
phospholipase C (Sph-PLC, EC 3.1.4.12) catalyzes the hydrolysis of Sph and lysoPC;
and phosphatidylinositide (or phosphoinositide)-specific phospholipase C (PI-PLC, EC
3.1.4.10) catalyzes the hydrolysis of PI,.lyso-PI, phosphatidylinositiol 4-monophosphate
(PIP), and phosphatidylinositol 4,5-bisphosphosphate (PIP 2). The products of PI-PLC
hydrolysis are 1,2-diacylglycerol (DG) and the corresponding inositol phosphates.
Introduction to Part I : The Cyclopentanoid Analogues of Short-chain Phosphatidylcholines
Phospholipase A2
PLA2 catalyzes the hydrolysis of the fatty acid ester at the sn-2 position of phospholipids (Figure 1). The enzyme was found in a large variety of snake venoms, pancreatic juice and mammalian exocrine gland secretions, where it serves a digestive role. The venom and pancreatic enzymes are small, water-soluble and about 14,000 molecular weight. The enzymes are stable probably due to their seven disulfide
bonds. The enzyme requires Ca2+ for its optimal activity. The mechanism of PLA2 has
been investigated extensively (For review: see Dennis, 1983). It is most active in the
hydrolysis of PC, PA, and PE, less active in that of PS, PI, and PG, much less active in
that of the plasmalogens, and inactive in that of sphingolipids. The specificity of the brain
particulate PLA2 is different from those enzymes mentioned above in the fact that its
maximal rate in the catalysis is to PI followed in order by its rates to PC, PA, PE, and PS.
(Shum et al., 1979; Gray and Strickland, 1982). The physical properties of the substrate
markedly influence the activity of PLA2 (Wells, 1974,1978). Earlier studies (deHaas et al., 1971; Pieterson, 1973) have shown that the aggregated short chain lecithins are considerably better substrates than the monomeric forms. This is so called the interfacial
activation (Figure 2).
Physical State of Phospholipid
Figure 3 illustrates the different physical states of phospholipids. In aqueous solution
without addition of detergents, synthetic short chain phospholipids form monomers at the
concentration below the critical micelle concentration (cmc) and form micelles above cmc.
There are two types of micelles: the first micelle (micelle I) and the second micelle (micelle n). Micelle I is proposed to be an oblate ellipsoid as is described by Tanford (1973 and 1977). Micelle II is proposed to arise from micelle I through a surface transition (Tanford,
1973,1977). As shown in Figure 3, this transition produces a micelle with spherical ends connected by a long ribbonlike structure. In some mixed solvent systems, such as ether- water, natural phospholipids form reverse micelles with a small amount of water in the central core. In the presence of a detergent, the phospholipids are solubilized by forming 4001
v 300- \ i mol/ min mg •
200 -
cm c
100 -
0.000 0.100 0.200 0.300 0.400 0.500 0.600 C s (mM)
Figure2: Hydrolysis of dioctanoylphosphatidylcholine by bovine pancreatic PLA2 (Lin et al., 1988)
~4 ^aaaP^ = phospholipid Q = nonionic detergent H20
A Reverse Micelle
MLV LUV SUV
Mixed Micelle
«/\AA/\Bk
H,0 Micelle II Micelle I
Figure 3: The structures of aggregated phospholipids 9 mixed micelles (Dennis, 1983). In the presence of water natural phosphatidylcholine can form vesicles. For vesicles, multilamellar vesicles (MLV) and large unilamellar vesicles
(LUV) have large diameters and small unilamellar vesicles (SUV) have small diameters and the outer surface is highly curved. Vesicles exist in different phases at different temperatures.
Interfacial Activation
In 1974, deHaas and co-workers (Pieterson et al., 1974) suggested that the pancreatic phospholipase A2 acts poorly on monomeric phospholipids and optionally on micelles. As shown in Figure 2, the rate enhancement above cmc is so called “interfacial activation.”. Over the years, 5 models have been proposed to explain this phenomenon
(Veger et al., 1976; Verheij et al., 1981; Dennis, 1983 ):
(1) Substrate conformation model From a monomer to an aggregate, packing a phospholipid substrate induces a conformational change and produces productive encounters with active site.(Wells, 1974,1978).
(2) Interfacial recognition site model An interface-induced conformational change occurs in the enzyme and results in a more active enzyme (Pieterson et al,1974;
Volwerk et al., 1974).
(3) Dual phospholipid model It was suggested by Dennis and co-workers. The model is based principally on kinetic studies on phospholipids in mixed micelles. The enzyme first binds to Ca and undergoes a conformational change that allows it to bind to substrate. The presence of interfacial phospholipid causes the enzyme to form an asymmetric dimer. One subunit of this dimer is responsible for binding to the interface while the other hydrolyzes an accessible phospholipid (Roberts et al.,1977). 10 (4) Product effect model The release of products is rate-determining and is aided by the presence of interfaces (Tinker et al., 1978,1979,1980).
(5) Acylated enzyme model The enzyme was acylated by the substrate at two Lys residues (Lys-7 and Lys-10) during the early stage of catalysis. The irreversible acylation enhances the specific activity of the enzyme toward substrate (Cho et al., 1988).
Which of these models or what combinations of these is the most important for the mechanism of phospholipase action at lipid-water interfaces becomes a central question in lipid enzymology. One way to test the substrate conformation model is to use a designed
“rigid” phospholipid analogue whose conformation does not change from monomers to micelles, fit other words, a “rigid” phospholipid analogue resists steric deformation during aggregation and may show little or no enhancement in the rate of PLA2 hydrolysis according to substrate conformation model. It occurs to us that the restricted conformation at the glycerol backbone of cyclopentanoid phosphatidylcholine could be a good candidate for this study.
Cyclopentanoid Analogues of Phospholipid
A. Rationale of Design of Cyclopentanoid Analogues of Glycerides
Because of the free rotation about C-C single bonds, the glycerol backbone of natural lipids may adopt a large number of confoimers. Therefore, it is difficult to study the relationship between those conformers and physiological interaction. However, the three isomeric cyclopentane-l,2,3-triols (Figure 4) are plausible analogues of glycerides.
Because of the restriction of a free rotation by the ring, alicyclic triols may be used as analogues of rotameric state of glycerol. Cyclopropanoid and cyclobutanoid systems are rigid and present special synthetic problems due to their inherent instabilities. With 4 extra 11
HO OH OH OH OH OH OH OH OH
(1,2,3/0) (1,2/3) (1,3/2) all-cis cis-trans all-trans
Figure 4: The diastereoisomeric cyclopentane-l,2,3-trio!s 12
carbons, cyclohexanoid systems are too big and perhaps also too rigid due to fixed
chair/boat conformations. In the cyclopentanoid system, the conformational equilibrium is
rarely restricted to one, or even two forms, and the conformers are much less rigid,
allowing a certain amount of transformation of the ring from the preferred form (Tolbert et
al., 1967; Altona et al., 1967). This flexibility could allow an induced fit between
cyclopentanoid analogue and a neighboring protein or lipid molecule, or within the active
site of a lipolytic enzyme.
B. Nomenclature of Cyclopentanoid Glycerides and Phospholipids
Cyclic compounds described in this dissertation are named according to the Tentative
Rules for Nomenclature of Cyclitols (IUBAC-IUB, 1968a). The names are derived from
those of the parent cyclanes of which they are formal derivatives; the location and disposition of the hydroxyl groups are indicated by a configurational fraction in which all
the substituents on one side of the plane of the cyclane ring are assembled in the numerator,
and those on the other side are assembled in the denominator. Thus the configurations
depicted in Figure 4 are denoted by 1,2,3/0,1,2/3 and 1,3/2. The lowest possible numbers
are used in each case, and no absolute configuration is implied by this fraction. When
absolute configuration must be specified, a separate convention relates the lowest numbered
asymmetric center to D- or L-glyceraldehyde. Unless otherwise specified, the abbreviation
DCjjPC represents the L-isomer of glycerol-phosphatidylcholine, and the abbreviation Cp-
DCnPC represents the DL-(l,3/2-lP) isomer of cyclopentanoid phosphatidylcholine
(Figure 5). In contrast to sn system for glycerol phospholipids (IUPAC-IUB, 1977), the
Tentative Rules for Nomenclature of cyclitols gives number 1 to the phosphate group; 13
0 — P — 0 + s / ' N(CH3)3
sn -1 s n - 3
0
R
Figure 5: (A) The Tententative rules' or Sundaralingam's nomenclature and IUPAC
nomenclature (sn-system) (B) Sundaralingam's nomenclature 14 however, it agrees with Sundaralingam (1972) system. Therefore, we use
Sundaralingam's nomenclature for phospholipids (Figure 5).
DPPC represents l,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPE represents l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DPPS represents 1,2-dipalmitoyl-sn- glycero-3-phosphoserine; DPPIrepresents l,2-dipalmitoyl-sn-glycero-3-phosphoinositol. Phosphorothioate analogues of phospholipids are shown in Figure 6. Thus, DPPsC represents l,2-dipalmitoyl-sn-glycero-3-thiophosphocholine; DPPsE represents 1,2- dipalmitoyl-sn-glycero-3-thiophosphoethanolamine; DPPsS represents 1,2-dipalmitoyl-sn- glycero-3-thiophosphoserine; DPPsI represents l,2-dipalmitoyl-sn-glycero-3- thiophosphoinositol.
C. Previous Studies of Cyclopentanoid Glycerides
The cyclopentanoid glycerophospholipid have been proposed to be analogues of phospholipid with restricted conformation at glycero-backbone (Hancock, 1981; Hancock et al., 1982; Pajouhsh & Hancock, 1983; Pajouhsh & Hancock, 1984; Lister, 1985). In the phosphohydrolase activity of canine lung surfactant material, Hancock and Benson
(unpublished result) have shown that only all-trans-(l,3/2-1 P)-cyclopentano-phosphatidic acid is an effective substrate for the enzyme (Lister,1985). It has been shown that PLA2 from Crotalus Adamanteus has a high degree of selectivity toward the all-trans-(l,3/2-lP)- cyclopentanoid DPPC among all of the isomers (Lister, 1985). Therefore, it has been suggested that PLA2 shows a high degree of configurational and conformational selectivity toward the substrates.
D. Use of Cyclopentanoid Analogues of Phosphatidylcholines to Probe the Mode of Activation of Phospholipase A2 DPPsC: := CH2CH2N(CH3)3+; DPPsE: := CH2CH2NH3+; DPPsG: := CH2CH(OH)CH2OH; DPPsA: C=H; DPPsS: :=CH2CH(CX)0*)NH3+;
OH
OH DPPsI: HO
OH H 0‘
Figure 6: Phosphorothioate analogues of Phospholipids 16
Although cyclopentanoid chemistry have been well developed, there are still many
questions still unknown. In cyclopentanoid analogues of phosphatidylcholines, the
cyclopentane ring fixes the conformation of glycero-backbone and conformational change
may not occur between monomers and micelles. As mentioned before, substrate
conformation model suggested that the interfacial activation is due to the conformational
change from monomers to micelles. If the substrate conformation model is an effect for the
interfacial activation of PLA2, then PLA2 may not show interfacial activation between
monomers and micelles toward cyclopentanoid phosphatidylcholine.
Introduction to Part II: The Stereochemistry of Phosphatidyinositide-specific Phospholipase C
The Metabolism of Inositol Phospholipids
In 1930, Anderson and Roberts (1930) first reported that inositol is a phospholipid
constituent from avian tubercle bacillus. Klent and Sakai (1939) discovered the inositol
lipid from soybean oil. Folch and Woolley (1942) found a brain phospholipid containing
inositol. Of the possible stereoisomers, only myo-inositol has been found so far in the
naturally occurring phospholipids.
The metabolism of inositol phospholipids is summarized in Figure 7. Among those products from PI-PLC hydrolysis, DG, myo-inositol 1,4,5-trisphosphate (1,4,5-IP3) and
myo-inositol l:2cyclic,4,5-trisphosphate (l:2c,4,5-IP3) are potent second messengers
(Berridge, 1986). The myo-inositol phosphates are degraded to myo-inositol phosphate by phosphatases, then to myo-inositol by myo-inositol-l-phosphatase (IPase, EC 3.1.3.25;
Takimoto et al., 1985) and myo-inositol cyclic phosphates are degraded to myo-inositol 17 AA ► prostaglandins
PLA2- l:2c-IP *------1:2c,4-IP, ■ 1:2c,4,5-IP-
cIPhyd ATP ATP PI kinase PIP kinase PIsyn ML PIP PIP- Plase PIPase
stoi D;IP 1,4,5-IP; L-IP DG Lipase jy j DG
CDP-DG DG kinase IPsyn
PA
calmodulin inositol D-glucose PLA2 and C-kinase kinase 6-phosphate PI-PLC ^ IPase
protein protein P protein extracellular agoms^^l
ceUula^esponse|
I plasma membrane PI-PLC receptor
acetyl choline esterase alkaline phospho alkaline diesterase I phosphatase 5-nucleotidase
Figure 7: The metabolism of inositol phospholipids l:2cyclic phosphate by phosphatase and then to myo-inositol by myo-inositol 1,2-cyclic
phosphate hydrolase (cIPhyd; Dawson and Clarke, 1972). IPase can hydrolyze both D and
L enantiomers of myo-inositol-l-phosphate (Parthasarathy and Eisenberg, 1986). L-myo-
inositol-l-phosphate is irreversibly biosynthesized from D-glucose 6-phosphate by L-myo-
inositol-l-phosphate synthase (IPsyn, EC 5.5.1.4). DG either is hydrolyzed by lipase (EC
3.1.1.3) to monoacylglycerol (MG) and then to free arachidonic acid (AA) and glycerol or
is phosphorylated by diacylglycerol kinase (DG kinase, EC 2.7.1.-) (Kanoch and Ohno,
1976; Hosaka et al.,1977; Polokoff and Bell, 1980) to form phosphatidic acid (PA), which then formed CDP-1,2-diacyl-sn-glycerol (CDP-DG) with CTP by CDP-DG synthetase
(CDP-DGsyn, EC 2.7.7.41) (Sumida and Mudd, 1970; Sparrow and Raetz, 1985).
Phosphatidylinositol (PI) is biosynthesized from CDP-DG and myo -inositol by
phosphatidylinositol synthetase (PIsyn, EC 2.7.8.11) (Bishop and Strickland, 1970;
Parries and Hokin-Neaverson, 1984). Phosphatidylinositol 4-phosphate (PIP) and
phosphatidylinositol 4,5-bisphosphate (PIP 2) are biosynthesized by phosphatidylinositol
kinase (PI kinase, EC 2.7.1.67) and phosphatidylinositol 4-phosphate kinase (PIP kinase,
EC 2.7.1.68) (Stubbs et al., 1988; Hou et al.,1988; Sommarin and Sandelius 1988). PIP 2
and PIP can be degraded to PIP and PI by phosphatidylinositol 4,5-bisphosphate 5- phosphatase (PH^ase) and phosphatidylinositol 4-phosphate 4-phosphatase (PIPase)
(Abdel-Latif, 1983).
Nomenclature
myo -inositol
Numbering proceeds anticlockwise on the axial OH side of myo-inositol ring of the
D-configuration and clockwise of the L-configuration as shown in Figure 8 (IUPAC, 19
OH OH
OH
HO OH HO
The D-numbering System
OH OH
OH
HO OH HO
The L-numbering System
Figure 8 : The D- and L-numbering systems for myo -inositol 20
1968b). According to this nomenclature, D -myo -inositol 1-phosphate (IP) is the same as
L-myo -inositol 3-phosphate but the convention dictates the use of the lower available number, i.e. D -myo -inositol 1-phosphate.
Phosphoinositides
Floch and Woolley (1942) introduced the term phosphoinositides for those inositol lipids. Three common phosphoinositides are phosphatidylinositol (PI), phosphatidylinositol 4-phosphate [(4) PIP or PIP] and phosphatidylinositol 4,5- bisphosphate [(4,5)PIP2 or p n y .
myo -Inositol Polyphosphates PI-PLC hydrolyzes phosphoinositides at glycerol-phosphate ester bond and yields myo-inositol 1-phosphate (IP) and myo-inositol l:2cyclic phosphate (l:2c-IP) from PI, myo-inositol 1,4-bisphosphate (M - n y and myo-inositol l:2cyclic,4-bisphosphate (1:2c,4-IP2) from PIP, and myo-inositol 1,4,5-trisphosphate (1,4,5-Ip3) and myo-insitol l:2cyclic,4,5-trisphosphate (l:2c,4,5-IP3) from PIP 2. The abbreviations for inositol phosphates and phosphoinositides followed the Chilton Convention (Agranoff et al.,
1985). Phosphorothioate Analogues
In this dissertation, the phosphorothioate analogues will be abbreviated as Ps. For example, the phosphorothioate analogue of DPPI is DPPsI and that of l:2c-IP is l:2c-IPs.
Phosphatidvlinositide-Specific Phospholipases C 21
Phosphoinositides constitute 2 to 8% of the lipid in cell membranes in eukaryotic cells and are essential for cell survival (Heniy et al., 1977, Esko and Raetz 1980).
Phosphoinositides are hydrolyzed by a PI-PLC to form DG and the various inositol phosphates. Because of the possible role of this enzyme in the stimulated phosphoinositide turnover in cell membranes, many investigators have investigated its properties in a variety of tissues, including brain, liver, intestinal mucosa, kidney, smooth muscle, platelets, lymphocytes, and erythrocytes (Abdel-Latif, 1983). This enzyme is also found in microorganisms with molecular weight from 23,000 to 30,000 (Ikezawa and Taguchi,
1981; Low, 1981). The bacterial enzyme is an acidic protein with an isoelectric point of
5.4 to 6.0 (Ikezawa and Taguchi, 1981). PI-PLC from culture supernatants of Bacillus cereus, Staphylococcus aureus, Clostridium novyi, Bacillus thuringiensis does not require
Ca2+ and is, therefore, unaffected by EDTA. In serval instances Ca2+ (above 10"3 M),
Mg2+, Zn2+, NaCl and KC1 can be inhibitory (Ikezawa and Taguchi, 1981). PI-PLC of higher plants require Ca2+ (Irvine et al., 1980). PI-PLC from bacterial culture supernatants hydrolyzes PI and lyso-PI specifically without any detectable attack on any other phospholipids (Ikezawa and Taguchi, 1981; Low, 1981). PIP and Pn*2 are not hydrolyzed by Staphylococcus aureus PI-PLC (Shukla, 1982). An approximate molecular weight of 68,000-70,000 was found for PI-PLC from guinea pig intestinal mucosa and from rat liver supernatant and that of 36,000 was found for the enzyme from rat brain supernatant (Quin, 1973; Takenawa andNagai, 1977; Atherton and Hawthorne, 1968).
PI-PLC's from guinea pig intestinal mucosa, iris smooth muscle, human and horse platelets, rat brain and liver lysosome, and human and ram seminal vesicles hydrolyze PI,
PIP and PIP 2 (Majerus et al., 1986; Ribbes et al., 1987). With the exception of PG, PI-
PLC's do not hydrolyze other phospholipids (Majerus et al, 1986). Two distinct soluble 22 enzymes have been found in ram seminal vesicles. The first enzyme has a molecular weight of 65,000, and the second enzyme has a molecular weight of 85,000. (Hofmann and Majerus, 1982). Low et al.(1986) reported that platelets have three forms of PLC made up of two different types of peptides of molecular weight 140,000 and 95,000.
Banno and co-workers (1986) also reported three different platelet enzymes of molecular weight 120,000,70,000, and 65,000. Wang et al. (1986) reported a molecular of 70,000 for two PI-PLC enzymes from murine thymocytes. The products of the reaction of PI- PLC are 1,2-diacylglycerol (DG), inositol phosphates (IP, 1,4-0*2 or l»4,5-n*3) and inositol l:2cyclic phosphates (l:2c-IP, 1:2c,4-0*2 or l:2c,4,5-D*3). Dawson et al. (1971) studied the effects of pH on the proportions of 1:2c-D* and D* produced by the enzyme in vitro. At pH 4.4,88% of the product was the cyclic product; at pH 6.8, the percentage decreased to 45%. These observations are consistent with the idea that either the 2-position hydroxyl on the myo-inositol ring or free OH“ in solution may attack the phosphorus. As the pH increases, hydrolysis is preferred over formation of a cyclic phosphate by transfer of phosphate to the 2-hydroxyl on the myo-inositol ring. These results suggest that PI-
PLC produces simultaneously two products rather than sequentially forming myo-inositol l:2cyclic phosphate as an intermediate with myo-inositol phosphate as the product.
There are two major effects for the hydrolysis of phosphoinositides by PI-PLC.
First, it will increase the intracellular concentration of Ca2+, which acts as a mediator for subsequent cellular response by the action of protein kinase C (C-kinase) or calmodulin
(see next section). The second effect is the release of membrane-bound enzymes such as acetylcholine esterase, alkaline phosphatase, 5-nucleotidase and alkaline phosphodiesterase I from the membrane (Ikezawa, 1986; Low and Saltiel, 1988).
The Second Messengers Among those products from PI-PLC hydrolysis, DG, 1,4,5-IP3, l:2c-IP and
1:2c,4,5-IP3 are putative second messengers. It is believed that 1,4,5-IP3 and DG are two
interacting second messengers (for review see Berridge 1987). A characteristic feature of
calcium-mobilizing receptors is that they initiate a signal cascade leading to the formation of
a number of putative second messengers (for review see Berridge 1986 and Abdel-Latif 1986). The initial event in this cascade is the hydrolysis of PIP2 to yield 1,4,5-IP3 and
DG. The action of 1.43-IP3 is to initiate an intermediate release of calcium ions from
intracellular stores into the cytosol. The elevated calcium ion concentration can activate
calcium-calmodulin dependent phosphorylation for one set of cellular proteins.
Meanwhile, DG activates a calcium-phospholipid depend C-kinase (Filbum and Udhida,
1985; Niedel and Blackshear, 1986) which occurs in most mammalian tissues, and
phosphorylates a different set of cellular protein. Majerus et al. (1986) suggested that
1:2c,4,5-IP3 may also be the second messenger on the basis of the fact that the releasing
45Ca2+ is as potent as 1,4,5-IP3. Recently Saltiel et al.(1986) isolated a derivative of 1:2c-
IP from cultured myocytes and proposed it to be one of the second messengers of insulin
action on cyclic AMP metabolism.
Phosphorothioate Analogues of Phosphates
Phosphorothioate analogues of phospholipids have been shown to be very useful in the studies of the mechanisms of phospholipases (Bruzik et al., 1983; Tsai et al., 1985; Jiang et al., 1984; Orr et al., 1982) as well as nonperturbing probes of aggregated phospholipid organization (Tsai et al., 1983; Tsai et al., 1984; Chang et al., 1986; Sarvis et al., 1988). The phosphorothioate analogues of PA was the first analogues of thiophospholipids and was synthesized by Predvoditelev et al. (1979). DPPsE has been synthesized by Orr et al. (1982) and Brazik et al. (1983). DPPsC has been synthesized by
Bruzik et al.(l982,1983, and 1986). Lately, phosphorothioate analogues of PAF have
been synthesized by Rosario Jansen et al.(1988) and Lamant et al. (1987), DPPsS have
also been synthesized by Lofffedo (1988), and phosphorothioate analogues of Sph have
been synthesized by Bruzik (1988). The most interesting effect of these phosphorothioate
analogues is that they are steieospecifically hydrolyzed by various phospholipases (Figure 9). For example, both DPPsC and DPPsE were steieospecifically hydrolyzed by PLA2
(prefers the Rp isomer), PC-PLC (prefers the Sp isomer) (Orr et al., 1982; Bruzik et al.
1983) and PLD (prefers the Sp isomer) (Jiang et al., 1984). Because PLA2 specificity for the head groups is broad, it can be used to resolve the Rp and Sp isomers of many
phosphorothioate analogues of phospholipids. For example, phosphorothioate analogues
of sn-1 ether PC (Rosario et al., 1988), PS (Loffredo, 1988), and PI (see Chapter V) can be resolved into Rp and Sp isomers by PLA2 from bee venom. Thus, PLA2 specifically
hydrolyzed Rp isomer of DPPsC, DPPsE, DPPsS, and DPPsG. However, it should be
noted that due to a change in priority the relative configuration of (Rp)- and (Sp)-DPPsI correspond to that of Sp and Rp isomers, respectively, of thiophosphatidylcholine. Thus
PLA2 should be specific to the Sp isomer of DPPsL Meanwhile, PLC also specifically hydrolyzed one isomer of phosphorothioate analogues of PC, PE (Orr et al., 1982; Bruzik et al., 1983), Sph (Brazik, 1988), and PI (see Chapter IV); PLD specifically transphosphorylated Sp isomers of DPPsC (Jiang et al., 1984) (Figure 9).
Phosphorothioate analogues of /Myo-inositol 1,4,5-trisphosphorothioate and myo inositol 1,4-bisphosphorothioate have been synthesized and shown to display the phosphatase-resistant character by Potter’s group recently (Cooke et al., 1987 and Hamblin et al., 1987). Lately, phosphorothioate analogues of l:2c-IP and IP, l:2c-IPs and IPs have 25
PLC PLD
R—C-O- R -c -o - ii O ox s t OX PLA2
Sp isomer for PC, sn-1 ether PC, Rp isomer for PC, sn-1 ether PC, PE, PS, and PAF analogues; PE, PS, and PAF analogues; Rp isomer for PI analogues. Sp isomer for PI analogues.
PLC PLA2
t.
R-C-OH HO- II O— P. O \ S > 0 -OH OX •O— OX DG Lyso
PLC
H2n+ic ri N—C—CH2-C nH2n+ 1 I II H O
Sp-phosphorothioate analogue of SPH
Figure 9: Stereospecificities of phospholipases 26 also been synthesized by Jastorff s group (Schultz et al., 1988 and Metschies et al., 1988), but the configuration of l:2c-IP has not been determined.
Stereochemistry of Phospholipases
Stereospecificity at the chiral center of phosphorothioate toward PLA2, PC-PLC, and
PLD on phosphorothioate analogues of phospholipids has been discussed before. So far, the steric courses of membrane-bound enzymes, PLD (Bruzik and Tsai, 1984b), phosphatidylserine synthase (EC 2.7.8.S) (Raetz et al., 1987), and phosphatidylserine decarboxylase (No et al.,1988) have been elucidated.
Approaches to Study the Stereochemistry of Phosphatidvlinositide-specific Phospholipase C
To elucidate the steric course of the reaction catalyzed by PI-PLC, we need to know the absolute configurations at phosphorus of both the substrate and the products of PI-
PLC.
The absolute configuration at phosphorus of the substrate of PI-PLC can be elucidated by the following three methods: (a) As shown in Figure 10a, known configuration [170 , 180]-DPPE (Bruzik and Tsai, 1984a) can be transphosphorylated to
[170 . 180]-DPPI by spinach PLD (Mandal et al., 1980; Kaplun et al., 1983). The steric course of spinach PLD can be determined by transphosphorylated known configuration
[170 . 180]-DPPC (Bruzik and Tsai, 1984b) to [170 , 180]-DPPE. Possibly, the steric course of spinach PLD is the same as other PLD's with a retention of configuration at phosphorus (Bruzik and Tsai, 1984b). Therefore, known configuration [170 , 180]-DPPI 27 • = 180 a n d 0 = 170 RCOO—i
RCOO-, RCOO— ___ spinach PLD «—O -P a RCOO- 0 OH \ •O— P, myo-inositol OH HO- HO' OH
RCOO—i RCOO—i
RCOO—I O s HO- ? O L 0 - P A -O-P b (Rp+Sp)-DPPsl OH \ OH \ pLA2 L ^ P -— r-OH OH HQ-i— HO 7 H o ^ - O H HO' ^ — OH
Rp-DPPsI Sp-Lyso Psl
RCOO—i RCOO—| RCOO— RCOO- 2 > * > s H20 - O - P —O-P OH \ OH \ -► Br2 r 0H HO- HO— -— 7 HO OH HO OH
Figure 10: Three methods to prepare the known configuration substrates of PI-PLC can be obtained by this method. The synthesis of DPPI from DPPE by
transphosphorylation is a low yield step (Mendal et al., 1980); therefore, it is not practical
to use a large amount of [170 , 180]-DPPE to achieve this purpose, (b) As shown in Figure
10b, the phosphorothioate analogue of PI, DPPsI can be synthesized and its absolute configuration can be determined by the action of bee venom PLA2 (prefers the Sp isomer).
This approach is used in this dissertation, (c) As shown in Figure 10c, [180]-DPPsI can
be synthesized and its absolute configuration can be determined by the action of bee venom
PLA2. To substitute the S atom in [180]-DPPsI with a 170 label is a stereochemically
known (inversion) step (Jiang et al., 1984 and references cited in). Therefore, known configuration DPPI can be obtained by this method. To Incorporate an 180 isotope on
DPPsI, a phosphorylating agent with an 180 label need to be prepared. It is not practical to
use a large amount of expensive 180-methanol to achieve this purpose. So far, the most
suitable way for us to approach is to use method b.
There are also three methods to determine the absolute configuration at phosphorus of
the products of the PI-PLC hydrolysis (IP and l:2c-IP): (a) As shown in Figure 1 la,
[17Q, 180]-IP can be transphosphorylated to (S)-propnane 1,2-diol by alkaline phosphatase
(Buchwald et al., 1982). After cyclization (inversion configuration at phosphorus), the
absolute configuration at phosphorus of [170 , 180]-IP can be elucidated by Knowles'
method (Abbott et al., 1979). The absolute configuration of the other product, 1:2c-IP can
be determined by cyclizing the configurationally known compound, [170 , 180]-IP. This
method is limited by the low yield of transphosphorylation by alkaline phosphatase, (b) By
using Lowe's procedures (Cullis and Lowe, 1981), known configuration [170 , 180]-IP can be asymmetrically synthesized (Figure 1 lb). The absolute configuration of the other product, 1:2c-IP can be determined by cyclizing known configuration [170 , 180]-IP. This 29
• = 180 a n d 0 = 170
.OH 0 0 f HO P O ^ ^ O H r-O H HO—* r« alkaline OH ^ ^ O H HO phosphatase
Knowles' method
oy f ° JCx OH HO H r OH /V V ,— ^0 Lowe's method v RO
OR ra» C ' \ , RO-H^t - n A r RO ^ — OR
NOE cf NOE
X-ray or OH OH HO HO NMR method HO OH HO OH
Figure 11: Three methods to determine the absolute configuration of the products from PI-PLC hydrolysis 30 method is limited by the low yield of reaction between the hydroxyl group of inositol and
170-P0C13. (c ) The third method is to determine the absolute configuration of l:2c-IPs by
X-ray and/or NMR (Figure 1 lc). This method is used in this dissertation; however, the absolute configuration of noncyclic product (IP) can not be determined by this method. In order to determine the absolute configuration of the other product (IP), the hydrolysis of DPPsI need to be carried out in H2180 (Figure 12); the product can be cyclized to configurationally known l:2c-IPs and [180]-l:2c-IPs (Figure 11c). RCOO—| • = 180
RCOO— 0 S L—O -P (0 )« -P HO \ H2 » HO \ + 1 :2c-IPs ~ T T ^ —r OH V ^ t oh PI-PLC HO- HO OH HO OH
Rp-DPPsI
H2i
PI-PLC O
1:2c-lPs HO 7 + HO- HO' OH HO OH
% > * <0) (O)* O -P \ / S HO \ V
— r-OH ~ 0 * ~ OH HO v - 7 S \ HO- HO- OH HO ^ — OH HOf x :OH HO
Figure 12: Determination of the absolute configuration of the non-cgclic product from the PI-PLC hydrolysis References
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Synthesis of Short-chain Cyclopentanoid Analogues of phospholipids
Introduction
The cyclopentanoid glycerophospholipid have been proposed to be analogues of
phospholipid with restricted conformation at glycero-backbone (Hancock, 1981; Hancock et al., 1982; Pajouhsh & Hancock, 1983; Pajouhsh & Hancock, 1984; Lister, 1985). It has
been shown that PLA2 from Crotalus Adamanteus has a high degree of selectivity toward
the all-trans-(l,3/2-lP)-cyclopentanoid DPPC among all of the isomers (Lister, 1985).
Therefore, it has been suggested that PLA2 shows a high degree of configurational and
conformational selectivity toward the substrates. As mentioned before, substrate
conformation model suggested that the interfacial activation is due to the conformational
change from monomers to micelles. In cyclopentanoid analogues of phosphatidylcholines, the cyclopentane ring fixes the conformation of glycero-backbone and conformational
change may not occur between monomers and micelles. Cp-DPPC has a sub-micromolar cmc which is beyond the sensitivities of PLA2 assays (Lister, 1985); therefore,we plan to synthesize the short-chain cyclopentanoid phosphatidylcholines. Cp-DPPC has been synthesized by Lister (1985). Cp-DC6PC has also been synthesized by Barlow et al. (1988a). The total yields of these two syntheses were quite
39 40 low because of the low yield on the phosphorylation steps (~40%). Recently, Bruzik and co-workers (1986) have successfully adapted the phosphorylation method from DNA synthesis by using chloro(N,N-diisopropylamino)methoxyphosphine to the phospholipid synthesis. By using this method, the yield of phosphorylation (from 60 to 80%) is higher than other methods; moreover, the phosphite intermediate can easily be oxidized to phosphate and phosphorothioate. Therefore, we synthesized Cp-DCgPC and Cp-DC4PC by applying Bruzik's method.
Starting from DL-(l,3/2)-l-0-benzyl-cyclopentane-l,2,3-triol (Scheme 1), we synthesized DL-(l,3/2)-2,3-di-0-octanoyl-l-0-phosphocholine-cyclopentane-l,2,3-triol (Cp-DCgPC) andDL-(l,3/2)-2,3-di-0-butyryl-l-0-phosphocholine-cyclopentane-l,2,3- triol (Cp-DC 4PC) by using this method (Scheme 2). DL-(l,3/2)-2,3-Di-0-butyryl-l-0- phosphoacid-cyclopentane-1,2,3-triol (Cp-DC 4PA) has also been synthesized by P(V) phosphorylating agent (Scheme 2).
Results
The synthesis of Cp-DCgPC, Cp-DC4PC and Cp-DC4PA were outlined in Scheme 2.
DL-(l,3/2)-l-0-benzyl-2,3-di-0-octanoyl-cyclopentane-l,2,3-triol (2 b) was obtained from the reaction of DL-(l,3/2)-l-0-benzyl-cyclopentane-l,2,3-triol (1) with octanoyl chloride in 79% yield. DL-(l,3/2)-l-0-benzyl-2,3-di-0-butyryl-cyclopentane-l,2,3-triol (2a) was obtained from the reaction of DL-(l,3/2)-l-0-benzyl-cyclopentane-l,2,3-triol (1) with butyryl chloride in 97% yield. DL-(l,3/2)-l-0-benzyl-2,3-di-0-palmitoyl-cyclopentane- 1,2,3-triol (2sD was also obtained from the reaction of DL-(l,3/2)-l-0-benzyl- cyclopentane-1,2,3-triol (1) with palmitoyl chloride in 95% yield. DL-(l,3/2)-2,3-di-0- octanoyl-cyclopentane-1,2,3-triol (3b) was obtained by the hydrogenolysis of 2b in 100% Scheme.l: Synthesis of (1,3/2)-1-O-benzylcyclopentane- 1,2,3-triol (Hancock, 1981)
NaHCO, O Q Cl PhCH2a
Q — r = \ O Q lO O H OH OBn c i Bn = CH2Ph
Q * Q OBn OBn
OH l,p-N 02C6H4C0Cl 2, fract. ciyst. 3, saponify (O H ') HO*
OH OBn OBn Schem e.2: Synthesis of short-chain cyclopentanoid analogues of phosphatidylcholines and phosphatidic adds
° j OH
1 Y — V ' — V 1 7 — 0 1 / 2. a: R=C3H7 > = 0 r s t s f e
? ° H3 II ° “ n 4 5n ^ pn R”” \ 4 5 ^F> / \ > n (ch 3)3+ 9/ \? N(i-Pr)2 Qj— vP H O ^ c 3^ d 3U.0 2 21
p ir o p h 0 / vO II OH
Reagents: a) RC0C1, 4-pyrtolidinopyridene/ pyridine b) H2, Pd-C7 EtOAc c) ClP(OCH3)N(i-Pr)2, Et3N/ CHC13 d) i)choline tosylate, tetrazole/ THF- CH3CN ii) t-BuOOH/ 2,2,4-trimethyIpentane-toluene iii) Me 3N/ toluene e) (Ph0) 2P(0)Cl/ pyridine-Et20 0 H2, Pt 20 / CH3COOH 43 yield. DL-( 1,3/2)-2,3-di-O-butyryl-cyclopentane-1,2,3-triol (2a) and DL-(l,3/2)-2,3-di-
O-palmitoyl-cyclopentane-1,2,3-triol (3c) were obtained by hydrogenolysis from 2a and 2£ in quantitative yield. The synthesis of Cp-DCgPC and Cp-DC4PC was adapted the
method of Bruzik et al. (1986). The phosphite intermediates, DL-( 1,3/2)-1-0-N,N-
diisopropylaminomethoxyphosphine-2.3-di-0-octanovl-cvclopentane-1.2.3-triol(4a)and DL-(l,3/2)-2,3-di-0-butyryl-l-0-N,N-diisopropylaminomethoxyphosphine-cyclopentane-
1,2,3-triol (4b) were obtained from the reaction of 2a and 2b with chloro(N,N- diisopropylamino)methoxyphosphine. Cp-DCgPC and Cp-DC4PC were obtained from the
phosphite intermediates by further two step reaction in 80% and 58% yield, respectively,
based on 2a and 2b- The first step was the oxidation of the phosphite intermediates with tert-butyl hydroperoxide to give the phosphotriester adducts. The second step was the
demethylation of the phosphotriesters with trimethylamine to give the final products. DL-
(l,3/2)-2,3-di-0-butyryl-l-0-diphenylphospho-cyclopentane-l,2,3-triol (£) was obtained from the reaction of 2a with diphenylphosphorylchloride in 89% yield. DL-(l,3/2)-2,3-di-
O-butyryl-l-O-phosphoacid-cyclopentane- 1,2 ,3-triol (2) was obtained from the hydrogenolysis of £ in 73% yield.
Experimental Section
Materials and Instrumental Methods
The starting material, DL-(l,3/2)-l-0-benzyl-cyclopentane-l,2,3-triol was provided by professor Henry Z. Sable at Case Western Reserve University. As shown in Scheme 1, this compound has been synthesized by elegant efforts of Sable, Hancock and co-workers 44 during the last two decades (Hancock, 1981). All other chemicals were purchased from either chemical stores of the Ohio State University or Aldrich. Other biochemicals were purchased from Sigma. Silica gel used in liquid chromatography (200-400 mesh) and thin layer chromatography (60 F 254) was obtained from EM reagent (Merck). Most liquid chromatography was preformed under moderate pressure (about 20 psi). *H NMR spectra were recorded on Bruker WP-200, AM-250 or AM-500 spectrometers. 13C and 31P NMR spectra were obtained at 62.89 and 101.25 MHz, respectively on a Bruker AM-250 spectrometer, with % decoupling. 31P chemical shifts are referenced to external 85%
H3PO4. The *H and 13C chemical shifts are referenced to external sodium 3-
(trimethylsilyl)-l-propanesulfonate for D20 samples and to Me4Si for samples in organic solvents. Fast atom bombardment (FAB) mass spectra were recorded on a VG-70-250S mass spectrometer. Melting point was recorded on a Thomas Hoover capillary melting point apparatus without calibration.
DL-(1,3/2)-1 -0-benzyl-2,3-di-0-acyl-cyc!opentane-1,2,3-triols (2)
DL-(l,3/2)-l-0-benzyl-2,3-di-0-octanoyl-cyclopentane-l,2,3-triol (2h): To 1.0654 g of DL-(l,3/2)-l-0-benzyl-cyclopentane-l,2,3-triol (1) (obtained from Dr. H. Z. Sable), toluene 20 ml was added. The mixture was then evaporated to dryness. This drying procedure was repeated twice. To this dry precursor (1.0407 g, 5 mmole), 4-pyrrolidino- pyridine 1.4023 g (9.5 mmole) was added. The mixture was evaporated with 5 ml of dry pyridine and was then dried in vacuum (0.25 mmHg) for 10 hours. To this mixture, 15 ml of dry pyridine and 1.27 mmole of octanoyl choride were added through a dry syringe at
0°C. Let this reaction mixture warm to room temperature and stir for 48 hours. The reaction was stop by adding 1 g of ice. To this reaction mixture, 25 ml of chloroform and 25 ml of water were added. Wash the organic layer with (1) 2 x 12.5 ml of water, (2) 2 x
12.5 ml of 0.2N H 2S04, and (3) 2.5 ml of saturated sodium bicarbonate. Dry the organic
layer over Na^C^ for 10 hours. Removal of solvent gave an oil. This crude product was
purified by flash chromatography on silica gel (chloroform/diethyl ether, 20/1, v/v). Final product (2b: 1.816 g, 0.395 m mole, 79%) is a colorless liquid. TLC: Rf = 0.7
(chloroform/ diethyl ether, 20/1, v/v). This compound was further characterized by !H
NMR. The resonances at 7.3,4.7 and 4.6 ppm were the characters of the 1-O-benzyl protons. The triplet at 0.9,1.6,2.3 and 1.3 ppm was assigned to be terminal-methyl, p-
methylene, a-methylene and all methylene protons of 1,2-di-O-octanoyl groups,
respectively, by references to octanoyl chloride and octanoic acid. These assignments were
further confirmed by decoupling experiments. The other resonances were the
cyclopentane ring protons. Without substituents, H 2C(4) and H 2C(5) should resonate at
the highest field of all cyclopentane ring resonances. Thus, the multiplet between 2.2 and
1.6 ppm was assigned to be H 2C(4) and H 2C(5). The resonances at 3.8,5.0 and 5.2 ppm
were assigned to be HC(1), HC(2) and HC(3). According to Shoolery's rules (Williams
and Fleming, 1980), the HC(2) resonance should be the most down field shift and the
HC(3) resonance should be the second down field shift of the three protons. Thus, the
resonances at 5.2,5.0, and 3.8 ppm were assigned to be HC(2), HC(3), and HC(1), respectively, and these assignments were further confirmed by decoupling
experiments. *H NMR (CDC13, 200 MHz): 8 7.3 (m, 5H, phenyl), 5.2 (t, J = 3.4 Hz,
1H, HC(2)) (Carbon numbering according to Figure 5), 5.0 (m, 1H, HC(3)), 4.7 and 4.6
(AB, J = 12 Hz, 2H, CH2 of benzyl), 3.8 (m, 1H, HC(1)), 2.3 (t, J=7.4 Hz, 4H,
H2C(22) and H2C(32)), 2.2-1.6 (m, 4H, H 2C(4) and H 2C(5)), 1.6 (m, 4H, H2C(23) and 46
H2C(33)), 1.3 (broad singlet, 16H, H2C(24) to H2C(27) and H 2C(34) to H 2C(37)), 0.9
(t, J=7.4 Hz, 6H, H3C(28) and H3C(38)).
DL-(l,3/2)-l-0-benzyl-2,3-di-O-butyryl-cyclopentane-l,2,3-triol (2a): 2 a was obtained from 1 and butyryl chloride in 97% yield by a similar procedure. TLC: Rf = 0.6
(hexane/ethyl acetate, 5/1, v/v). This compound was further characterized by *H NMR.
The assignments of the *H NMR spectrum were made by references to Zh, butyryl chloride, and butyric acid. The assignments of the 13C NMR spectrum were made by comparing both JH coupling and *H decoupling spectra and by reference to butyric acid.
!H NMR (CDC13, 200 M Hz): 5 7.3 (m, 5H, phenyl), 5.2 (t, J = 3.6 Hz, 1H, HC(2)), 5.0
(m, 1H, HC(3)), 4.7 and 4.6 (AB, J = 12 Hz, 2H, CH 2 of benzyl), 3.8 (m, 1H, HC(1)),
2.2 (t, J=7.3 Hz, 4H, H2C(22) and H2C(32)), 2.2-1.6 (m, 4H, H 2C(4) and H 2C(5)), 1.6
(m, 4H, H2C(23) and H 2C(33)), 0.9 (two triplets, A 8 = 2.0 Hz, J = 7.3 Hz, 6H, H3C(28) and H3C(38)).13C NMR (CDC1 3, 75.44 MHz): 8 173.8, 173.2 (C=0) 138.9, 129.0,
128.3 (phenyl of benzyl) 82.9, 82.6 and 66.5 (C(l), C(2) and C(3)), 71.9 (CH 2 of benzyl), 36.9 (H 2C(22) and H2C(32)), 29.6, 29.4 (H 2C(4) and H 2C(5)), 19.1(H2C(23) and H 2C(33)), 14.3 (H3C(28) and H3C(38)).
DL-(l,3/2)-l-0-benzyl-2,3-di-0-palmitoyl-cyclopentane-l,2,3-triol (2c): By a similar procedure, Z& was obtained from the reaction of 1 with palmitoyl chloride in 95% yield. TLC: R p 0.52 (hexane/ethyl acetate, 5/1, v/v). mp = 42-44°C. This compound was further characterized by *H NMR. The assignments of the *H NMR spectrum were made by references to Zh, palmitoyl chloride and palmitic acid. *H NMR (CDC13, 200
M H z): 8 7.2 (m, 5H, phenyl), 5.2 (t, J = 3.8 Hz, 1H, HC(2)), 4.6 (m, 1H, HC(3)), 4.7 and 4.6 (AB, J = 12 Hz, 2H, CH 2 of benzyl), 3.8 (m, 1H, HC(1)), 2.2 (t, J = 7.3 Hz,
4H, H2C(22) and H2C(32)), 2.2-1.6 (m, 4H, H 2C(4) and H 2C(5)), 1.6 (m, 4H, H2C(23) 47
and H 2C(33)), 1.2 (m, 48H, H2C(24) to H2C(2,16) and H 2C(34) to H2C(3,16)), 0.9 (t, J
= 6.7 Hz, 6H, H3C(2,16) and H3C(3,16)).
DL-(1,3/2)-2,3-di-0-acyl-cyclopentane-1,2,3-triols (3.)
DL-(l,3/2)-2,3-di-0-octanoyl-cyclopentane-l,2,3-triol (3b): To a two necked flask equipped with a gas inlet and a balloon, 338.3 mg of 10% Pd-C was added. Fill with
hydrogen gas and connect to the vacuum. Repeat this procedure for three times to assure
there is no moisture and oxygen in the flask. To this dry mixture, 660 mg (1.43 mol) of
2b in 20 ml of ethyl acetate was added. Let the reaction mixture fill with hydrogen gas and
stir at room temperature for 48 hours. Removal of solvent from the filtrate gave an oil
liquid (538 mg, 100%). TLC: Rf=0.1 (hexane: ethyl acetate, 5:1, v/v). This compound
was further characterized by *H NMR. The assignments of the XH NMR spectrum were made by references to 2b> by decoupling experiments, and by the extra splittings of HC(1) from 1-OH. *H NMR (CDC13, 200 MHz): 5 5.1 (m, 1H, HC(2)), 4.8 (t, J = 4.5
Hz, 1H, HC(3)), 4.0 (m, 1H, HC(1)), 2.2 (m, 4H, H2C(22) and H2C(32)), 2.2-1 .6 (m,
4H, H2C(4) and H 2C(5)), 1.6 (t, J=6.5 Hz, 4H, H2C(23) and H 2C(33)), 1.4 (broad
singlet, 16H, H2C(24) to H2C(27) and H 2C(34) to H 2C(37)), 0.9 (t, J=7 Hz, 6H,
H3C(28) and H3C(38)).
DL-(l,3/2)-2,3-di-0-butyryl-cyclopentane-l,2,3-triol (2a): By the procedure described in 2b» 2a can be obtained from 2a in 100 % yield. This compound was further characterized by *H NMR. The assignments of the *H NMR spectrum were made by references to 2a and 2b and by the extra splittings of HC(1) from 1-OH. TLC: Rf = 0.1
(hexane/ethyl acetate, 5/1, v/v). !H NMR (CDC13, 200 MHz): 5 5.1 (m, 1H, HC(2)), 4.8
(t, J = 4.5 Hz, 1H, HC(3)), 4.0 (m, 1H, HC(1)), 3.4 (d, J = 10 Hz, 1H, OH), 2.3 (two 48 triplets, AS=7.4 Hz, J = 7.4 Hz, H, H 2C(22) and H2C(32)), 2.2-1 .6 (m, 4H, H2C(4) and
H2C(5)), 1.6 (two sextets, J = 7.4, A8 = 1.6 Hz, 4H, H2C(23) and H 2C(33)), 0.9 (two triplets, J = 7.3 Hz, A 8 = 1.6 Hz, 6H, H3C(24) and H 3C(34)).
DL-(l,3/2)-2,3-di-0-palmitoyl-cyclopentane-l,2,3-triol (3c): 2fi were also obtained from 2& by the method described in 2b in 100 % yield. TLC: Rf=0.1 (hexane: ethyl acetate, 5:1, v/v). This compound was further characterized by *H NMR. The assignments of the *H NMR spectrum were made by references to 2£ and 2b and by the fact that 1-OH causes the extra splittings of HC(1). *H NMR (CDC13, 200 MHz): 8 5.1
(m, 1H, HC(2)), 4.7 (t, J = 4.8 Hz, 1H, HC(3)), 4.0 (m, 1H, HC(1)), 2.3 (m, H,
H2C(22) and H2C(32)), 2.2-1.6 (m, 4H, H 2C(4) and H 2C(5)), 1.6 (m, 4H, H2C(23) and
H2C(33)), 1.2 (m, 48H, H2C(24) to H2C(2,16) and H 2C(34) to H2C(3,16)), 0.9 (t, J =
7.0 Hz, 6H, H3C(2,16) and H3C(3,16)).
Short-chain Cyclopentanoid phosphatidylcholines (£)
DL-( 1,3/2)-2,3-di-0-octanoyl- 1-O-phosphocholine-cyclopentane-1,2,3 -triol (Cp- DCgPC, 5b): Introduction of the phosphocholine group to 2b was carried out by a procedure adapted from Brazik et al. (1986). The reactions were carried out in a two necked flask. Compound 2b (0.46 mmol) was dried by rotary evaporation with 3 x 5 ml of dry toluene and then added with 5 ml of chloroform (dried over P 20 5) and 1 mmol of triethylamine (dried over NaH) through vacuum transfer. The reaction mixture was filled with argon. While the mixture being stirred at room temperature, chloro(N,N- diisopropylamino)methoxyphosphine (0.55 mmol) was added to the flask through a dry syringe. After the reaction was complete (about 15 minutes) on the basis of the disappearance of 2b on TLC, the solvent and excess triethylamine were removed under 49 vacuum, and the reaction mixture was further dried in vacuo (0.2 mm Hg) for 4 hours.
The product 4b was not isolated and was subjected to the following steps directly.
Thoroughly dried tetrazole (1.84 mmol) and choline tosylate (1.38 mmol, prepared by refluxing the mixture of choline chloride and p-toulene sulfonic acid monohydrate in toulene) were dissolved in tetrahydrofuran-acetonitrile ( 1:1, v/v) through vacuum transfer.
The mixture was filled with argon. After warming to 40°C to assure all compounds were dissolved, the solution was added to 4b through a syringe under argon. After being stirred at room temperature for 17 hours, the reaction was complete on the basis of TLC. The solvents were removed by rotary evaporation and replaced with 10 ml of dry toluene (over
NaH). This heterogeneous mixture was cooled to -80°C and added with 0.92 mmol of t- butyl hydroperoxide (3 M solution in 2,2,4-trimethylpentane). The suspension was stirred at room temperature for 22 hours. The reaction mixture was then washed with 5 ml of 1.5
M trimethylammonium bicarbonate (pH 7); the organic phase was repetitively evaporated to dryness with anhydrous toulene. To this anhydrous semisolid, anhydrous toluene (3 ml) and anhydrous trimethylamine (0.5 ml) were added by vacuum transfer. The resulting solution was stirred at room temperature for 30 hours. After the deprotection reaction was complete, excess trimethylamine and solvents were evaporated. The crude product was extracted successively with chloroform-methanol-water (10 ml/20 ml/8 ml) and with chloroform-water (10 ml/10 ml). The crude product from the organic layer was concentrated, dissolved in 1 ml of chloroform-methanol-water (66/33/4, v/v/v), and purified by flash chromatography on silica gel with chloroform-methanol-water (66/33/4, v/v/v) as the eluting solvent. The white solid product was then precipitated from acetone and dried in vacuo to give 212 mg of 5b (80% yield from 2b)- TLC: Rf = 0.2
(chloroform: methanol: water, 66:33:4, v/v/v). This compound was further characterized 50
by NMR. Except the resonances of HC(1), HC(2), HC(3), H 2C(4) and H 2C(5), the *H
and 13C NMR spectra of Cp-DCgPC and DCgPC were identical. Except those five
resonances, the assignments to the other *H and 13C resonances of Cp-DC 8PC.(Figures 13
and 14B) were made by references to DCgPC. The assignments to those five proton
resonances were made by reference to 2b and confirmed by the 2D correlation
experiment (Figure 15) and by 2D J-resolved 1H NMR experiment (Figure 16). Thus, the
multiplet between 2.26 and 1.80 ppm was assigned to be H 2C(4) and H 2C(5). The most
downfield shift resonance was assigned to be HC(2), the second one was HC(3), and the
third one was HC(1). Because most resonances were second order, the 2D J-resolved
experiment could not resolve most J-J coupling constants. The assignments of the 13C NMR spectrum were made by references to DCgPC (Bums and Roberts, 1980) and DC16PC (Birdsall et al., 1972). The assignments of the acyl chain 13C NMR peaks were
made by applying Bengsch et al.'s rales (1986). As shown in Figure 17, the NOESY spectrum of Cp-DCgPC confirmed the results of those assignments we made before. The resonance x in Figure 17 represented the NOE of two a-methylene groups. The resonance
y indicated the NOE between choline N(CH 3)3 and acyl chain methylene groups of the two
acyl chains. The resonance z indicated that HC(1) and H(3) of the cyclopentane may prefer
the di-pseudo-axial to di-pseudo-equatorial position. *H NMR (CD 3OD, 250 MHz)
(Figure 13): 8 5.15 (t, J = 3.7 Hz, 1H, HC(2)), 5.05 (m, 1H, HC(1)), 4.50 (m, 1H,
HC(3)), 4.26 (m, 2H, H2C(11) (choline-POCH2)), 3.61 (m, 2H, H 2C(12) (choline-
CH2N)), 3.21 (s, 9H, choline-N(CH3)3), 2.34 (t, J = 7.2 Hz, 2H, H 2C(22)), 2.29 (t, J =
7.4 Hz, 2H, H2C(32)), 2.26-1.80 (m, 4H, H 2C(4) and H 2C(5)), 1.59 (m, 4H, H2C(23)
and H 2C(33)), 1.3 (b, 16H, H2C(24) to H2C(27) and H 2C(34) to H 2C(37)), 0.89 (t, J =
6.8 Hz, 6H, H3C(28) and H3C(38)). 13C NMR (CD 3OD, 62.896 MHz) (Figure 14B): 8 ^ j T 0-. o / l 0 “ a
0 - TrCH2CH2(CH2)4CH3
4*H 5-H 2-H 3*H 1-H //
PPM
Figure 13: The *H NMR spectrum (250 MHz, CD 3OD) of Cp-DC 8PC. 7
5 O - P - O
H3C4(H2C)H2CH2C, o -jr ch 2c h 2(ch 2)4c h 3 o a a y t
T •.» ».• 77
Figure 14: The 13C and 31P NMR spectra of Cp-DC 8 PC. (A) The 31P NMR spectrum (101.256 MHz, CDjOD) (B) Thel3CNMR spectrum (62.869 MHz, CD 3OD) 5 O - P - 0 y — N(CH3)3+
y l r ' T ‘
4 * H 0 ir^LlX_JIJULI l
- 0 .
- 1.
- 2.
i _ 5. j
.. 4.
PPM 1 ■—■—-r—•-*-—•*—t ...... r - — -i— 8 0 £.0 4.0 3.0 2.0 1.0 0.0 Figure 15: The COSY spectrum (500 MHz, CDC13) of Cp-DCgPC 54
4*H 5-H 1-H a b JL i
■'-I f ' ■ r I ' I I ' 3.5 3.0 2.5 2.0 1• S
Figure 16: The 2D J-resolved lH NMR spectrum (500 MHz, CDC13) of Cp-DC8PC 2 Q-H 2^3-H >H * V.
.u.
r j h - I - j. 7.5
3.?
3.3
S. *-W H-,C,(H,C)HJCH,C^x° $ ^ ^ 1 3 ► X 'a >* ; 7 p a o 3 0-n-C H 2CH2(CH2)4CH3 r 4 *• , ° a P Y t
rr=J- ; s.s
3.5 5.0 4.5 4.0 3 5 3.0 J.S 7.3 i.* 1.0 .5 0 9
Figure 17: The NOESY spectrum (500 MHz, CDC13) of Cp-DC8PC 56
174.79 and 174.52 (C(21) and C(31)), 84.26 (d, 3Jcp = 8.1 Hz, C( 2)), 80.03 (d, 2Jcp =
5.7 Hz, C(l)), 78.88 (C(3)), 67.54, (m, C(12)), 60.37 (d, 2JCP = 5.0 Hz, C (ll)), 54.74
(t, ^CN = 3.8 Hz, C(13), C(14) and C(15)), 35.03 (C(22) and C(32)), 32.84 (C(26) and
C(36)), 31.02, 30.98, 30.09, 30.06, and 29.15 (C(24), C(25), C(34), and C(35)), 25.97
(C(23) and C(33)), 23.64 (C(27) and C(37», and 14.38 (C(28) and C(38)). 31P NMR
(CD3OD, 101.256 MHz) (Figure 14A): 8 1.04. MS (FAB) (Figure 18): The peaks at m/z
1072,1071,537 and 536 were assigned to be. [2M]+, [2M-1]+, [M+l]+ and M+, respectively. The assignments to some other peaks were shown in Figure 19. The
assignments to the peaks at m/z 184,166 and 104 were made by references to DPPC
(Gasgupta et al., 1987 and Ayanoglu et al., 1984). Thus, m/z at 184 was assigned to be
choline phosphate ion. Loss of water from choline phosphate ion gave an ion at m/z 166.
The peak at m/z 104 was assigned to be choline ion. The peaks at m/z 127 and 99 were
assigned to be C 7 H15CO+ and C 7H15+. Loss of water from choline ion gave an ion at m/z
86 .
DL-(1,3/2)-2,3-di-0-butyryl-1 -O-phosphocholine-cyclopentane- 1,2,3-triol (Cp-DC4 PC, £a):
If the procedures of Cp-DCgPC were applied, the yield of £a from 2a was only 8 %.
A better yield (58.4%) was obtained by the following modifications: In work-up procedures, the Et 3N-H2C 03 wash was omitted. In purification, liquid chromatography was performed on the silica gel which was treated with 5 % weight of triethylamine and eluted with 100 ml of chloroform, 100 ml of chloroform-methanol ( 2/ 1, v/v), 100 ml of chloroform-methanol (1/1, v/v), and 200 ml of chloroform-methanol (1/2, v/v). Final elution with chloroform-methanol ( 1/2, v/v) gave the product which was characterized as 8 0 ..
6 0 .
,4 (2 M -I] 1071
• . i f ! y ■ 11 i ■ • t I , T— r. m ss 400 600 800 1000 1800 Figure 18: The Mass spectrum (FAB) of Cp-DC8PC Ul •J 58 o o - i • o ^ o ^ o V 1^ o - H+ I Y° _ HO— / A ^ / n (CH3)3+ O ° > = 0 m/z 184 5. a: R=C3H 7 m/z 424 1 b: R=C 7 H 15 m/z 536 t
O IT II H3CO = P ^ o / \ y N(CH3)2 HO-P^oy \s/ N(CH3)2 -H ,0 OCH, m/z 160 m/z 184
-PO,
H* N(CH3)2 / A ^/ N(CH3)3+ HaCO/A y ^ HO
m/z 104 I 1
H2Cn / N(CH3)3+
m/z 86
Figure 19 : The fragments of short-chain cyclopentanoid analogues of phosphatidylcholines follows: TLC: Rf = 0.22 (chloroform/methanol/water, 66/25/4, v/v/v), Rf = 0.55
(chloroform/methanol/water, 5/4/1, v/v/v). The assignments of *H NMR spectram were made by references to 5b, DC 4PC, DC8PC, Cp-DCgPC and DC16PC and confirmed by the decoupling experiments. The assignments of 13C NMR spectrum were made by references to DC 4PC, DCgPC, Cp-DC8PC and DC 16PC. *H NMR (CD 3OD, 500 MHz)
(Figure 20): 8 5.13 (t, J = 3.2 Hz, 1H, HC(2)), 4.99 (m, 1H, HC(1)), 4.49 (m, 1H,
HC(3)), 4.27 (m, 2H, H2C(11)), 3.64 (m, 2H, H2C(12)), 3.22 (s, 9H, choline-NMe3),
2.33 (t, J = 7.3 Hz, 2H, H2C(22)), 2.28 ((t, J = 7.3 Hz, 2H, H2C(32)), 2.2-1 .8 (m, 4H,
H2C(4) and H 2C(5)), 1.65 (t, J = 7.1 Hz, 2H) and 1.64 (t, J = 7.1 Hz, 2H) (H2C(23) and
H2C(33)), 0.95 (t, J = 6.0 Hz, 2H) and 0.94 (t, J = 7.3 Hz, 2H) (H2C(24) and H 2C(34)).
13C NMR (CDC13, 62.896 MHz) (Figure 21): 5 173.04 and 172.83 (C(21) and C(31)),
82.88 (C(2)), 78.30 and 77.00 (C(l) and C(3)), 66.40 (C(12)), 59.17 (C(ll)); 54.368
(C(13), C(14), and C(15)), 36.147 and 36.093 (C(22) and C(32)), 30.035 and 28.185
(C(4) and C(5)), 18.348 (C(23) and C(33)); 13.574 (C(24) and C(34)). 31P NMR
(CD3OD, 101.256 MHz): 8 -2.4. MS (FAB) (Figure 22): The peaks at m/z 848,847,
425 and 424 were assigned to be [2M]+, [2M-1]+, [M+l]+ and M+, respectively. The peaks at m/z 446 was assigned to be M+Na ion. The assignments to some other peaks were shown in Figure 19. Thus, m/z at 184 was assigned to be choline phosphate ion.
Loss of water from choline phosphate ion gave an ion at m/z 166. The peak at m/z 104 was assigned to be choline ion. The peaks at m/z 71 and 43 were assigned to be C 3H7CO+ and C 3H7+. Loss of water from choline ion gave an ion at m/z 86 .
Short-chain Cyclopentanoid phosphatidic Acid r • h 3c h 2c h 2c—c— [ S / V N(CH3)3+ t P a
v2« ° a p t ‘ o — c —ch 2ch 2ch 3
2-H I 3-H 1-H 1 a J
-T-l-r— - T ...... , , , , ■ , , ■ .. 5. -I *.S 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM
Figure 20: The !H NMR spectrum (500 MHz, CD 3OD) of CP-DC 4PC
8 0 b 1 5 O -P —O ^ / — N(CH3)3+ v c A- HaCHzCHzC^A.^/ R a O ^ 0 -n”CH 2CH2CH3 1 p o a P t c=o 4 5 n
170
»■ -If HIM*1 •)tulf
.. ■ ■ I ■ • ■ I ■ ■ '' i • ~r~ go so 70 60 SO 40 30 20 10 PPM
Figure 21: The 13C NMR spectrum (62.869 MHz, CDC13) of Cp-DC4PC 100
MfiSS .300 400 500 £00 700 1000 0 \ Figure 22: Hie Mass spectrum (FAB) of Cp-DC4PC to 63
DL-( 1,3/2)-2,3-di-0-butyryl-l -O-diphenylphosphoryl-cyclopentane-1,2,3 -triol (£):
To a pyridine-diethyl ether (1:1, v/v) solution of 2a (570 mg, 2.2 mmol), 0.91 ml (4.4 mmol) of diphenylphosphoryl chloride was added and stirred at room temperature for 30 hours. The reaction mixture was evaporated to dryness then was partitioned between chloroform (30 ml) and water (20 ml). Chloroform layer was washed with 2x10 ml of water, 3x10 ml of 2N H 2S04, and 10 ml of saturated NaHC0 3 then evaporated to dryness to give 956.5 mg (88.5%) of £. TLC: Rf = 0.6 (CC14: EtOAc, 4:1, v/v). This compound was further characterized by NMR. The assignments of the *H NMR spectrum were made by reference to 2a and by decoupling experiments. The assignments of 13C NMR spectrum were made by reference to 2a* *H NMR (CD 3OD, 250 MHz): 8 7.4-7.2 (m,
10H, phenyl), 5.3 (t, J = 4.1 Hz, 1H, HC(2)), 5.1 (m, 1H, HC(1)), 5.0 (m, 1H, HC(3)),
2.3 (m, 4H, H2C(22) and H2C(32)), 2.2-1.6 (m, 4H, H 2C(4) and H 2C(5)), 1.6 (m, 4H,
H2C(23) and H 2C(33)), 0.9 (two triplets, A 8 = 6.5 Hz, J = 7.3 Hz, 6H, H3C(24) and
H3C(34)). 13C NMR (CD3OD, 62.896 M Hz): 8 174.29 and 173.73 (C(21) and C(31)),
151.65, 131.05, 126.88,121.13 and 121.05 (phenyl), 83.67 (d, 3Jcp = 6.3 Hz, C(2)),
82.80 (d, 2JCP = 6.9 Hz, C(l)), 77.64 (C(3)), 36.83 and 36.63 (C(22) and C(32)), 30.13
(d, 3JCP = 3.8 Hz, C(5)), 28.73 (C(4)), 19.30 and 19.17 (C(23) and C(33)), 13.86
(C(24) and C(34)). 31P NMR (CD 3OD, 101.256 MHz): 8 -13.989.
DL-(l,3/2)-2,3-di-0-butyryl-l-phosphoacid-cyclopentane-l,2,3-triol (2): To a solution of £ (798 mg, 1.6 mmol in 20 ml of acetic acid), 100 mg of Pt0 2 was added and hydrogen gas (1 atm) was filled. After 41 hours, the reaction mixture was evaporated to dryness and partitioned in 5 ml of chloroform, 5 ml of methanol and 4.5 ml of 0.2 N
H2S04 Chloroform layer was washed with methanol-water (5 ml/4 ml) then evaporated to 64
dryness to yield 402.1 mg (73%) of 2- This compound was further characterized by
NMR. The assignments of the *H and 13C NMR spectra were made by reference to £. *H
NMR (CDC13, 250 M Hz): 8 5.2 (m, 1H, HC(2)), 5.0 (m, 1H, HC(1)), 4.6 (m, 1H,
HC(3)), 2.3 (m, 4H, HZC(22) and H2C(32)), 2.2-1.7 (m, 4H, H 2C(4) and H 2C(5)), 1.6
(m, 4H, H2C(23) and H 2C(33)), 0.9 (t, J = 7.5 Hz, 6H, H3C(24) and H 3C(34)). 13C
NMR (CDC13, 62.896 M Hz): 8 173.79 and 173.18 (C(21) and C(31)), 82.49 (C(2)),
80.44 (C(l)), 76.67 (C(3)), 36.07, 36.04 and 35.71 (C(22) and C(32)), 29.35 and 28.01 (C(4) and C(5)), 18.30 and 18.20 (C(23) and C(33)), 13.53 and 13.48 (C(24) and C(34)).
31PNMR (CDC13, 101.256 MHz): 8 -1.0932.
Discussion
Cp-DC 8PC, Cp-DC4PC and Cp-DC4PA have successfully been synthesized
(Scheme 2). Synthesis of phosphatidylcholine by using Bruzik et al.'s (1986) method has
been proven to give better yield than by using other methods; for example, the yield of phosphorylation by this method for Cp-DC8PC and Cp-DC4PC is 80% and 50 %,
respectively, which is higher than that of Cp-DC6PC (40%, Barlow, 1988a). However,
some problems occurred during this synthesis. First, we tried in vain to modify the procedures for the synthesis of the starting material in Scheme 1. Second, the Cp-DC4PC
was unstable during the purification on an ordinary silica gel column. This was avoided by using the silica gel pretreated with triethylamine. Third, Cp-DC8PC and Cp-DC4PC were decomposed during regular ionization in order to obtain a mass spectrum. This can be prevented by using FAB mass spectroscopy. Reference
Ayanoglu, E., Wegmann, A., Pilet, O., Marbury, D. G., Hass, J. R., & Djerassi, C.
(1984) J. Am. Chem. Soc. 106, 5246-5251.
Barlow, P. N., Lister, M. D., Hancock, A. J., & Sigler, P. B.(1988a) Chem. Phys.
Lipids 46, 157-164.
Barlow, P. N., Lister, M. D., Sigler, P. B., & Dennis, E. A. (1988b) J. Biol. Chem. 236,
12954-12958.
Bengsch, E., Perly, B., Deleuze, C., & Valero, A. (1986) J. Mag. Res. 6 8 , 1-13.
Birdsall, N. J., Feeney, J., Lee, A. G., Levine, Y. K., & Metcalfe, J. C. (1972) J. Chem.
Soc. Perkin Trans I I 1441-1445.
Bruzik, K. S., Salamonczyk, G., & Stec, W. J. (1986) J. Org. Chem. 51, 2368-2370.
Bums, R. A. and Roberts, M. F. (1980) Biochemistry 19, 3100-3106.
Gasgupta, A., Ayanoglu, E., Tomer, K. B., & Djerassi, C. (1987) Chem. Phys. Lipids
43, 101-111.
Hancock, A. J. (1981) Methods Enzymol. 72, 640-672.
Hancock, A. J., Lister, M. D., & Sable, H. Z. (1982) J. Lipid Res. 23, 183-189.
65 66 Lin, G., Noel, J., Loffiredo, W., Sable, H. Z., & Tsai, M.-D. (1988) J. Biol. Chem. 263, 13208-13214.
Lister, M. D. (1985) Ph. D. Dissertation, University of Missouri.
Pajouhesh, H. and Hancock, A. J. (1983) J. Lipid Res. 24, 645-651.
Pajouhesh, H. and Hancock, A. J. (1984) J. Lipid Res. 25,294-303.
Williams, D.H. and Fleming, L (1980) in “Spectroscopic Methods in Organic Chemistry”,
second edition, pp 83-146, William Clowes & Sons, Limited, London, The Great Britain. CHAPTER III Use of Short-chain Cyclopentanoid Analogues of Phosphatidylcholines to Probe the Mode of Phospholipase A2
Introduction
The mode of activation of PL A2 is an important and controversial problem. Except
for the bee venom PLA2 (Shipolini et al., 1971) and the proenzyme of pancreatic PLA2, it
has been well established that the activity of a substrate is greatly enhanced in the reaction
catalyzed by PLA2 when the substrate concentration passes the cmc. Many different (and
opposing) models have been proposed to explain this phenomenon (see Chapter I).
According to the substrate conformation model (the one supported by the results of this
chapter), PLA2 interacts with aggregated or monomeric substrate in essentially the same manner. An interface-induced conformational change is not significant, and the so called
“interfacial activation” is due to a conformational change of the substrate in an interface which allows for a higher fraction of productive interactions with the enzyme.
In contrast to the properties of pancreatic and snake venom PLA2's, Shipolini et al. (1971) reported that the activity of DC6PC as a substrate of bee venom PLA2 increases linearly below cmc but levels off at cmc (14 mM). They suggested that the activity of micelles for this enzyme is negligible compared to that of monomers. Since the bee venom
67 68
PLA2 has been an unpopular enzyme, this unusual behavior has been ignored in most discussions concerning the interfacial activation of PLA2, except the report that the bee venom PLA2 shows a tendency to denature at the air-water interface (Cohen et al., 1976) and that it is activated by fatty acid or by acylation (Lawrence and Moores, 1975; Drainas et al., 1978; and Drainas and Lawrence, 1978).
This chapter deals with the use of the cyclopentanoid analogues of phosphatidylcholines to probe the mode of activation of PLA2's from bovine pancreas and bee venom. Therefore, kinetic studies, conformational analysis, and determination of cmc's of cyclopentanoid phosphatidylcholines are reported in this chapter.
Experimental Section
The cmc determination and conformational analysis were performed on an AM-500
NMR spectrometer. The samples were exchanged with 99.996% D20 (from Aldrich) 2-3 times. The chemical shifts were referenced to internal HCOONa. DC 4PC, DCgPC, and
DCgPC were purchased from Avanti Polar Lipids, Inc. Bovine serum albumin (BSA) and
3-(trimethylsilyl)-l-propanesulfonic acid, sodium salt were purchased from Sigma. All other chemicals were obtained from either chemical stores of the Ohio State University or
Aldrich. The kinetic works of this chapter were preformed by Joseph Noel according to
Lin et al. (1988).
Result and Discussion
Determination of critical micelle concentration by 1H NMR 69
The cmc of DCgPC and Cp-DC8PC were determined by NMR in D 20 , on the
basis of the changes in the resonances of the H 2C(22) and H2C(32) groups, as described in
Hershberg et al. (1976). As shown in Figure 23, the a-methylene multiplet of DCgPC was dependent on
concentration. Therefore, the cmc value of DCgPC was estimated to be 14 mM from
Figure 24A and 24B (taken from Hershberg et al., 1976). The cmc value of DCgPC was
also estimated to be 14 mM from the plot of AS(a-methylene) versus lipid concentration
(Cs) as shown in Figure 24C. As shown in Figure 25, the a-methylene multiplet of DCgPC was also dependent on
concentration. The cmc of DCgPC was estimated to be 0.28 mM from the plot of 8 (sn-l a-
methylene) versus Cs (Figure 26A), 0.25 mM from the plot of 8(sn-2 a-methylene) versus
Cs (Figure 26B) and 0.30 mM from the plot of A 8 (a-CH2) versus Cs (Figure 26C). In
good agreement with results from Tausk 0.2 mM (Tausk et al., 1974), Roberts 0.3 mM
(Roberts et al., 1978), Pieterson 0.19 mM (Pieterson, 1973), and Wells 0.17 ± 0.02 mM (Wells, 1974), the cmc from this analysis was 0.28 ± 0.02 mM.
As shown in Figure 27, the a-methylene multiplet of Cp-DCgPC was also dependent on concentration. The cmc of Cp-DCgPC was estimated to be 0.65 mM from the plot of
8 (sn-l a-methylene) versus Cs (Figure 28A), 0.70 mM from the plot of 8(sn-2 a- methylene) versus Cs (Figure 28B), or 0.75 mM from the plot of A 8 (a-methylene) versus
Cs (Figure 28C). The cmc from this analysis was 0.70 ± 0.05 mM. The criterion of the magnetic nonequivalence of terminal methyl groups in micelle occurred in both DCgPC and
Cp-DCgPC, and this criterion confirmed the cmc values that we obtained (see Figure 29).
To use more convincingly the cmc data obtained, we must assume that the presence of enzyme does not affect the cmc. As shown in Figure 30, we also determined the cmc of 70
snl sn - 2
OD B2D OSO 040 080 MO
Figure 23: Concentration dependence of the a-methylene multiplet (220 MHz, D 20 ) of
DC6PC taken from Hershberg et al. (1976). The scale of these spectra is in Hz from
internal formate. 71
020
US
* OSS - r '
043 041 037
(OHEXANOTUCCITHtt) Motor
HZ 26 ■;
.1 1 10 100 1000 Cs (mM)
Figure 24: Determination of the cmc of DCgPC by the plot of (A) a -methyiene
versus lipid concentration (Cs), (B) a.methylenc versus Cs ((A) and (B) are
taken from Hershberg et al. (1976)], and (C) A5aMne(hykne versus Cs (C) sn-2
Figure 25: Concentration dependence of the a-methylene proton resonances (500 MHz,
D20 ) of DC8PC at concentration (A) 0.50 mM, (B) 0.45 mM, (C) 0.25 mM, (D)
0.20 mM, and (E) 0.15 mM. All spectra have a same scale. 1280-1 73
1270 - ‘ A HZ
1260 tI \ X a s n -1 cm c-0.28 mM A 1250 " r i i .1 C s(m M )
1285-t
1283 HZ 1282
1281 cm c-0.25 mM ♦ s n -2 1280-
1278 .1 1
C s(m M )
2 4 -
□ AS
cmc - 0.3 mM
C s(m M )
Figure 26: Determination of the cmc of DCgPC by the plot of (A)
a-m cthyicnc versus lipid concentration (Cs), (B) 6sn.2 a.methylene
versus Cs, and (C) A5 0.mclhyicnc versus Cs 74
Figure 27: Concentration dependence of the a-methylene proton resonances (500 MHz,
D20 ) of Cp-DC8PC at concentration (A) 0.10 mM, (B) 0.29 mM, (C) 0.48 mM, (D)
0.91 mM, (E) 1.38 mM, (F) 1.67 mM, and (G) 2.00 mM. All spectra have a same
scale. 1218
1212 -
1210 1 10 Cs(m M )
1244 1243
1242
HZ 1241 1240 1239
1238- cmc «= 0.7 mM 1237- 1 1 10
Cs(m M )
3 0 -
Hz 2 6 - 2 4 -
2 2 -
cmc « 0.75 mM
1 10 Cs (mM)
Figure 28: Determination of the cmc of Cp-DC8PC by the plot of (A)
5 sn-i a-m c[hyicne versus lipid concentration (Cs), (B) 5sn.2a.
methylene versus Cs, and (C) A 8 a.mC|}jyjene versus Cs a -C H t-CH3 76 sn-21 I s n-i
^ A .
L
. , T " ' T' 2.50 2.40 .90 PPM PPM
Figure 29: The a-methylene and terminal-methyl proton resonances
(500 MHz, D20 ) of (A) monomeric DC 8PC, (B) micellar DCgPC,
(C) monomeric Cp-DCgPC, and (D) micellar Cp-DC8PC 19 n
AS
Hz
1 1 10
C s(m M )
Figure 30: Determination of the cmc of Cp-DCgPC in the presence of 6
jig of BS A by the plot of A5a.methylene versus lipid concentration
(Cs) 78
Cp-DC8PC in the presence of 6 |ig of “non-reactive” protein bovine serum albumin (BSA).
The cmc of Cp-DC8PC with BSA was 0.50 ± 0.05 mM and was about the same as that without BSA within the experimental error. Barlow et al. (1988b) reported that the cmc values of DCgPC and Cp-DC6PC are 10 and 11.2 mM, respectively. In these two cases, the cmc values of phosphatidylcholines are smaller than those of cyclopentanoid analogues.
This is somewhat unexpected since the extra two methylene groups (H 2C(4) and H 2C(5)) of the cyclopentanoid analogues are expected to make the molecules more hydrophobic and to lower the cmc.
Hydrolysis of Short-chain Phosphatidylcholines and Their
Cyclopentanoid Analogues by Bovine Pancreatic and Snake Venom PLA2's
Table 1 shows the Vmax's of PLA2's toward substrates DCnPC and Cp-DCnPC at concentrations (Cs) below and around cmc. As expected, DCgPC and DCgPC induced the interfacial activation of bovine pancreatic PLA2 and N. n. naja PLA2.
In contrast to DCgPC and DCgPC, Cp-DCgPC and Cp-DCgPC behaved like a single species across the entire concentration range without showing an interfacial activation. In any case, it is clear that micellar cyclopentanoid analogues of phosphatidylcholines do not induce an activation of bovine pancreatic or snake venom PLA2.
Possible Explanation for the Kinetic Property of Cp«DC8PC
A straightforward explanation of the lack of activation in cyclopentanoid analogues would be that no conformational change could occur between monomers and micelles of Cp- DCgPC due to the restricted conformation of the glycerol backbone. Such an explanation strongly supports the substrate conformation model. This suggests that the interfacial activation is mediated by the conformation of the substrate, as has been advocated by Wells
(1974) and Allgyer and Wells (1979). 79
Table l.Summary of Kinetic Data
Vmax PLA2 Source Substrate State fyimol minting"1} Source
Bovine pancreas DCgPC monomer 38 Lin et al., 1988
Bovine pancreas DCgPC micelle 2510 Lin etal., 1988
Bovine pancreas Cp-DCgPC monomer 0.81 Lin et al., 1988
Bovine pancreas Cp-DCgPC micelle 0.48 Lin et al., 1988
Bee venom DCgPC - 4000 Lin etal., 1988
Bee venom Cp-DCgPC - 110 Lin et al., 1988
C. Adamanteus DC16PC - 500 Lister, 1985
C. Adamanteus Cp-DC16PC - 4.3 Lister, 1985
N.n. naja DC6PC monomer 200 Barlow et al., 1988
N.n. naja d c 6pc micelle 4000 Barlow et al., 1988
N.n. naja Cp-DC6PC monomer 200 Barlow et al., 1988
N.n. naja Cp-DCgPC micelle 200 Barlow et al., 1988 80
Two questions, however, could be raised concerning the above interpretation. The first question is that since the activity of Cp-DCgPC is lower than that of either monomeric
or micellar DCgPC, we don't know whether the conformation of Cp-DCgPC resembles that
of monomeric DCgPC (thus behaves like monomers even above cmc) or resembles that of
micellar DCgPC (thus behaves like micelles even below cmc). The second question is that
!H NMR analysis and other spectroscopic studies suggested only minor changes in the
glycerol backbone conformations of DCgPC and DC 7PC between monomers and micelles
(Roberts et al. 1978; Hauser et al., 1980; Bums et al., 1982). Then, why should a restriction in glycerol backbone conformation result in the observed change in the kinetic
pattern? These two questions have been addressed by *H NMR analysis (at 500 MHz) of
DCgPC, DC4PC, Cp-DCgPC, and Cp-DC4PC as described in the next section.
Conformational Analysis by *H NMR
According to several previous reports on the *H NMR studies of phosphatidylcholines (Hershberg et al., 1976; Roberts et al., 1978; Hauser et al., 1980;
Bums et al. 1982, Lin et al., 1987), the most notable changes between monomers and micelles are the following: (i) In monomers the H 2C(22) (sn-2 a-methylene) (see Figure 5 for numbering system) and H2C(32) (sn-1 a-methylene) groups are only slightly different
(two overlapping triplets in *H NMR). Upon micellization, the chemical shift difference
(A8 ) increases from ^ 0.02 ppm to 0.05-0.1 ppm. The H2C(32) group resides in a more hydrophobic environment and resonates at the higher field. The 8 value or A8 value is a function of lipid concentration; therefore, the cmc value can be estimated by the plot of lipid concentration (Cs) versus chemical shift ( 8 ) of sn-2 a-methylene protons or by the plot of
Cs versus chemical shift difference (AS) between sn-1 and sn-2 a-methylene protons, (ii)
The carbonyl region of the sn-2 acyl chain is so rigid that the two protons of the H 2C(22) 81
group become magnetically nonequivalent and give rise to two slightly separated triplets (the AB part of an ABX2). Such a magnetic nonequivalence occurs in most micelle
systems, but not in any monomer, (iii) The chemical environment of the two acyl chains
becomes so different in micelles that the two terminal methyl groups become distinguishable (two triplets) in *11 NMR spectra of the short-chain phospholipids DCgPC
and DC 7PC.
Figure 29 shows the partial *H NMR spectra of monomeric DCgPC (A), micellar
DC8PC (B), monomeric Cp-DCgPC (C), and micellar Cp-DCgPC (D). In Figure 29C, the
HZC(22) (sn-2 a-methylene) and H2C(32) (sn-1 a-methylene) resonances are well
separated, and two sn-2 a-methylene protons are magnetically nonequivalent (the AB part
of an ABX2). These satisfy criteria (i) and (ii) mentioned above for micelles of
phosphatidylcholines. Thus, the monomeric Cp-DCgPC is conformationally
restricted in the carbonyl region of the acyl chains; moreover, this
restriction resembles that of glycerol phosphatidylcholine micelles. Two
structural factors could be responsible for such a conformation: (a) The two additional
CH2 groups, H 2C(4) and H 2C(5) may pose steric hindrance to the random motion of the two acyl chains, as revealed from model building, (b) The trans configuration of the two ester groups may help the two acyl chains to orient in a micelle-like conformation. It is still unclear to us which of these two factors may predominate. We have further generalized this phenomenon by showing that the A 8 values of monomeric Cp-compounds are always larger than those of the corresponding glycerol phospholipids under a variety of conditions, as summarized in Table 2.
Properties of Cp-DC8PC Micelles 82
Table.2. Comparison of A 8 values between monomers of Cp-compounds and the corresponding open-chain compounds.
Pattern of Pattern of Compounds Solvent H2C(32) H2C(22) AS(ppm) terminal CH 3
Cp-DCgPC D 2O ti dt2 0.040 t
DCgPC D2O t t 0.029
Cp-DCgPC CD3OD t t 0.048 t
DCgPC CD3OD t t 0.024 t
Cp-DCgPC 3 d 2o t t 0.038 t
DCgPC d 2o t t 0.028 t
Cp-DC4PC D 20 t t 0.035 dt
DC4PC D20 t t 0.028 dt
Cp-DC4PC CD 30D t t 0.047 dt
DC4PC CD30D t t 0.022 dt
*The A2 part of an A 2 X.
2The AB part of an ABX 2 .
3Taken from Barlow et al., 1988b. 83 An alternative interpretation for the lack of changes in activity at the cmc of Cp- DCgPC is that Cp-DCgPC forms poor micelles which cannot interact properly with the
interfacial recognition site of PLA2. However, we have added Triton X-100 to Cp-DC8PC
attempting to improve the quality of the micelle surface, and found no significant increase in activity. Even though Cp-DCgPC posses micelle-like conformation in monomeric form, it
does undergo a transition from monomers t micelles as evidenced by proton NMR
experiments. The nonequivalence (A 8 ) between H 2C(22) and H2C(32) increased from
0.045 to 0.058 ppm at cmc for Cp-DCgPC and from 0.029 to 0.038 ppm for DCgPC.
Both continued to increase slowly above cmc. The chain-terminal methyl groups become nonequivalent immediately after cmc for Cp-DCgPC and at higher concentration for
DCgPC. In addition, both compounds showed an additional set of broader peaks (possibly
due to formation of larger micelles or small vesicles) when the concentration was well
above cmc. Thus, the differences in the proton NMR properties of monomers and micelles are quantitatively similar between Cp-DCgPC and DCgPC. This suggests that the different kinetic behavior of the two compounds is not due to differences in their surface properties.
Hydrolysis of DCgPC and Cp-DCgPC by PLA2 from Bee Venom
Since monomeric Cp-DCgPC mimics the conformation of micellar DCgPC, it can be used to probe Shipolini's suggestion: bee venom PLA2 hydrolyzes monomers but not micelles. Plots of the activity of bee venom toward DCgPC and Cp-DCgPC (Lin et al.,
1988) resembles that of DC 6PC (Shipolini et al., 1971) in showing a sharp, linear increase before cmc and leveling off after cmc. The results suggest that DCgPC and Cp-DCgPC behave similarly as substrates of PLA2 in terms of the kinetic pattern of monomers and 84 micelles. The difference in activity (by ~37x) is most likely a structural rather than a
conformational effect on the basis of the above discussions. This would suggest that the
catalytic mechanism of bee venom PLA2 is not sensitive to the conformation of monomers
and micelles.
Thus, our interpretation of the unusual kinetic behavior of bee venom PLA2 is that
this enzyme does not differentiate the conformation of substrates between monomers and
micelles in activation. In other words, either they can both activate the enzyme or the
enzyme already exists in the activated form before substrate binding. According to this
interpretation, micelles should also be good substrates. This is supported by the previous
observation that the mixed micelles of long-chain lecithins are good substrates for bee
venom PLA2 (Upreti and Jain, 1978; Bruzik et al., 1983; Tsai et al., 1985), and that long-
chain lecithin bilayers (sonicated and unsonicated) can also be hydrolyzed by bee venom
PLA2 (Upreti and Jain, 1978; Wilschut et al., 1978), although with low activity.
Alternative Interpretations
Although the above results strongly favor the substrate conformation model for the explanation of the interfacial activation of PLA2 from bovine pancreas, they do not necessarily argue against other models. An alternative interpretation of our results on pancreatic PLA2 is that the monomeric Cp-DCgPC, possessing micelle-like conformation, can bind to the interfacial recognition site and activate the enzyme. A precedent of this behavior is that the PLA2 from Naji melanoleuca has been shown to be activated by forming lipid-protein aggregates at sub-micellar concentrations (van Eijk et al., 1983,
1984). Such a model may also be applied to the bee venom PLA2. In other words, we can conclude that the bee venom PLA2 accepts micelles and monomers as opposed to the 85 suggestion of Shipolini et al. (1971), but we cannot differentiate whether the enzyme preexists in the activated form (thus insensitive to the conformation of substrates) or it can bind to and be activated by monomers and micelles. It is also possible that more than a single model may function simultaneously and that different PLA2's may function by different models. References
Allgyer, T. T. and Wells, M. A. (1979) Biochemistry 18,4354-4361.
Barlow, P. N., Lister, M. D., Sigler, P. B., & Dennis, E. A. (1988b) J. Biol. Chem.
263, 12954-12958.
Bruzik, K., Jiang, R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486.
Bums, R. A., Roberts, M. F., Dluhy, R., & Mendelsohn, R. (1982) J. Am. Chem.Soc.
104, 430-438.
Cohen, H., Shen, B. W., Snyder, W. R., Law, J. H., & Kdzdy, F. J. (1976) J. Colloid
Interface Sci. 56, 240.
Drainas, D. and Lawerence, A. J. (1978) Eur. J. Biochem. 91, 131-138.
Drainas, D., Moores, G. R., & Lawrence, A. J. (1978) FEBS Lett. 86 , 49-52.
Hauser, H., Pascher, I., Pearson, R.H., & Sundell, S. (1980) Biochim. Biophys. Acta
650, 21-51.
Hershberg, R. D., Reed, G. H., Slotboom, A. J., & de Haas, G. H. (1976) Biochim.
Biophys. Acta 424, 73-81.
Lawrence, A. J. and Moores G. R. (1975) FEBS Lett. 49,287-291.
Lin, T.-L., Chen, S.-H., & Roberts, M. F. (1987) J. Am. Chem. Soc. 109, 2321-2328.
Lin, G., Noel, J., Lofffedo, W., Sable, H. Z., & Tsai, M.-D. (1988) J. Biol. Chem. 263,
13208-13214.
86 87
Pieterson, W. A. (1973) Ph. D. Thesis, Rijksuniversiteit, Utrecht, The Netherlands.
Roberts, M. F., Bothner-By, A. A., & Dennis, E. A. (1978) Biochemistry 17, 935-942.
Shipolini, R. A., Callewaert, G. L., Cottrell, R. C., Doonan, S., Vernon, C. A., &
Banks, B. E. C. (1971) Eur. J. Biochem. 20, 459-468.
Tausk, R. J. M., Karmiggelt, J., Oudshoom, C., & Overbeek, J. T. G. (1974a)
Biophys. Chem. 1, 175-183.
Tausk, R. J. M., Oudshoom, C., & Overbeek, J. T. G. (1974b) Biophys. Chem. 2,53-
63. Tsai, T.-C., Hart, J., Jiang, R.-T., Bruzik, K., & Tsai, M.-D. (1985) Biochemistry 24,
3180-3188.
Upreti, G. C. and Jain, M. K. (1978) Arch. Biochem. Biophys. 188,364-375. van Eijk, J. H., Verheij, H. M., Dijkman, R., & de Haas, G. H. (1983) Eur. J. Biochem.
132, 183-188. van Eijk, J. H., Verheij, H. M. R., & de Haas, G. H. (1984) Eur. J. Biochem. 139,51-
57. Wells, M. A. (1974) Biochemistry 13,2248-2257.
Wells, M. A. (1978) in “Advances in Prostaglandin and Thromboxane Research”, C.Galli,
et al., eds., Vol. 3, pp. 39-45, Raven Press, New York, USA.
Wilschut, J. C., Regti, J., Westenberg, H., & Scherphof, G. (1978) Biochim.Biophys.
Acta 508,185-196. CHAPTER IV
Synthesis and Configurational Analysis of Thiophosphatidylinositol
Introduction
There are three challenges in the synthesis of the phosphorothioate analogue of
phosphatidylinositol, l,2-dipalmitoyl-sn-glycero-3-thiophosphoinositol (DPPsI). The first
challenge is to make a protective precursor containing a free 1-hydroxy group and
protective groups at the other 5 positions of the inositol ring. This involves not only the
selectivity of the 6 hydroxyl groups of the inositol ring but also the resolution of D- and L-
isomers of inositol precursors. The second challenge is that the reactivity of a secondary
hydroxyl group of the inositol ring is very low toward phosphorylation. Finally, there are
some limitations in choosing the protective groups for this synthesis. For example, the benzyl protective group can not be used in the presence of both the phosphorothioate and the carboxyl ester functions. One reason is that the hydrogenolytic catalyst for debenzylation is poisoned by the sulfur atom of a phosphorothioate group. The other reason is that debenzylation by the dissolving metal method (Na or Li in liquid NH3) will also hydrolyze two acyl chains and produce new problems in reacylation.
88 There are many ways to resolved a D-form inositol precursor from myo-inositol.
Among those methods, the resolution by deriving with (-)-camphanic acid chloride was
most successful (Billington et al., 1987; Vacca et al., 1987). By using this method, we can
obtain a free 1-OH inositol precursor with the D-configuration. Billington's method is
discussed in this chapter and Vacca's method is discussed in Chapter VI. The second
problem can be solved by using a highly reactive phosphorylation agent. Under this
circumstance, a commercially available reagent, chloro(N, N-
diisopropylamino)methoxyphosphine, is the best candidate on the basis of its high activity
and easily introducing sulfur (Bruzik et al., 1986). The third problem can be solved by
avoiding using the benzyl-type protective groups after introducing sulfur.
Results
Synthesis of 1,2-dipalmitoyl-sn-glycero-3-thiophosphoinositol (Schemes 3 & 4) Compound 12d and 121 are synthesized according to Scheme 3. Treatment of myo inositol with 1-ethoxycyclohexene and a catalytic amount of p-toluenesulfonic acid (TsOH) in N,N-dimethylformide yields 2,3:5,6( 1,2:4,5)-Di-0-cyclohexylidene-myo-inositol (£), 2,3:4,5(l,2:5,6)-di-0-cyclohexylidene-myo-inositol (2), and 3,4:5,6(l,6:4,5)-di-0- cyclohexylidene-myo-insitol (JJD (the D-form numbering system was used, the numbering in parentheses indicated the L system) (Garegg et al., 1984). Partial benzylation of fi yields monobenzyl ethers, H (Billington et al., 1987). Esterification of the free hydroxyl group with (S)-(-)-camphanic acid chloride yielded a mixture of two diastereomers that were separated by a combined method of fractional recrystallization and chromatography to give 12d (mp 226-228 °C) and 12L(mp 225-226 °C) (Billington et al., 1987). If careful separation of these two diastereomers (for examples, two fractional recrystallizations 90 Scheme.3: Synthesis of D- and L-1-0-benzyl>4-0-(-)-camphanoyl- 2,3:5,6-di-0-cyclohexylidene-myo-inositol
OH 1-elhoxycyclohexene \ L*0 . H o - f c ~ r ° H ------H & a & s T ) T s O H I ip DMF, 90-100°C, 5 myo-inositol 2h 24% & (46%)
Ok? , OH HO A—/
2 (42%) i a ( 1 2 %)
® OBn (-) camphanic PhCH2Br, NaH Q acid chloride ^
toluene, reflux, HO ® DMAP, Et3N, overnight, 65% t . . CH 2Cl2 ,r t, 11 tULJ 17h> g2%
Bn = CH2Ph
BnO OE + EO3 > - 0
m (50%) 121 (50%) Scheme.4: Synthesis of 1,2-diplamitoyl-sn-glycero-3 thiophosphoinositol
LiOH 1 2 d ------T H F - H 2 0 7 MO iPr«NF.r ru nru n ^—O 2:1, rt, 2Sh, H0 CH30CH20 99% l t d CH2Ch. ^ rt, 4.5h, 11 90% / s CHa° \ Li Q
NHj-THF, pnc h ,no 2 r.ThV ^ ^ ^ - oo V 'W ^ Et E.3NCH2C12,3 N , C H 2 CI 51 -78 C, l/2h. CH3°°Ha0 n. l/2h CH30CH2O- 1 0 0 % 1 4 1 5 r— O R 1,1,2-diplamitoyl-sn- RO^ / no D n / — O H glycerol, tetrazole, rw n S ROj. / THF-CH3CN, rt, 1 ^ 1 = H= ° X s V * H ° Z -V HO ^ P - 2411 ~ » - v l " Q i)6NHC' H 0
2, S8, toluene, rt, CH3 OCH2 o ' ^ “ ° 47h ») N M e 3 OH R =C i5H31CO HO 73% 83% from to 1£ 12 (Rp+Sp)-DPPsI 1 b.
■ O R Bacillus cereus s O H° V i H PI-PLC t HO V o - ' 0 s n * \ V m ^«enom _ h o —OH 12. (Sp)-lyso-PsI o rLu\ZPLA2 k l _ 0 H + . , OR H 0-l-7^i” (Sp)-DPPsI V k H H O ^ “ OH X < v ? s " ° V HO ^ P ~ 0 - l:2 c -IP s OH H O - ^ j OH UK (Rp)-DPPsI HO 92
followed by two purifications of medium pressure liquid chromatography (MPLC)) the %
of diastereomeric excess (% de) is 90 based on the 13C NMR analysis and 100 based on the GC analysis. Compound 12d from two fractional ieciystallizations and two MPLC purifications gave >90% de based on the carbony 13C resonance at 178 ppm (Figure 31 A).
The % de of this compound was calculated to be 100 based on the intensity of GC (Figure 32A). Compound 12d from one fractional recrystallization and one MPLC purification gave 90% de based on GC (Figure 32B). Compound 121 from one fractional
reciystallization and one MPLC column gave 74% de based on 13C NMR (Figure 31B). For practical reasons, we used a large amount of compound 12d with 75-90% de (obtained from one fractional recrystallization and one MPLC). DPPsI was synthesized from 12d
according to Scheme 4. Basic hydrolysis of 12d gave lid in quantitative yield.
Reprotection of H i with chloromethyl methyl ether (MOMC1) (Corey et al., 1982a,b; Stork and Takahashi, 1977) gave 12 in 90 % yield. Deportection of 12 by hydrogenolysis on 10 % Pd-C or by reductive debenzylation (Li metal in tetrahydrofuran and liquid NH3)
which was modified from Potter and Lowe (1981) gave 14 in 97 % yield.
Phosphorylating 14 with chloro(N,N-diisopropylamino)methoxyphosphine gave 15.
Compound 12 reacted with 1.2 equivalents of 1,2-dipalmitoyl-sn-glycerol and 4 equivalents of tetrazole in tetrahydrofuran-acetonitrile at 25 °C for 24 hours. The resulting amidotriester was treated with an excess of S 8 in toluene at 25 °C for 47 hours to give 12.
The overall yield from 14 to 12 was 83%. The presence of two diastereomers of 12 was characterized by two equal intensity resonances in 31P NMR at 67.63 and 67.93 ppm (Figure 33A). A separate sample of 12 obtained from L-H gave two signals at 67.69 and 67.86 ppm (Figure 33B). As shown in Figure 33, the %de of the inositol moiety in both A and B is about 83. (Rp+Sp)-DPPsI (12) was obtained by deprotection of two ketal groups by acid hydrolysis and demethylation with trimethylamine (Scheme 4), and gave 93
A
/v ty V
I&5 -----\T%~Jl—____ U- PPM
Figure 31: Use of 13C NMR (75.436 MHz, CDC13) to determine the %de of
compound 12d. and JL21. The C=0 resonances at 178 ppm of (A) >90% de 12d
and (B) 74% de 12L- fibuntlar.ee Abundance C.9C! C; . 4 nr i r i r n - i o .QE5 + 6 1 i or or UL l U l L U . Ui • r • i. U i 0 L+B- l j 30.0 ~> Figure 32: The GC traces of (A) 100% de 12d and (B) 90% de 12d. de 90% (B) and 12d de 100% (A) of traces GC The 32: Figure
\ / 1
2 I oo -‘w.t-1 .0 a TTC TTf. / 12 n + n-f / d e i roime o 34.0 e i me) a I .c_> nfiTA:AA 10604.fi
HATR: n
m i n .) in. in. H 0 I J 6 0 6 0 6 3 36.0 . 3 . Ti 38.0 38
~r i B .0 95
• i t i i 89.0 68.9 60.0 67.9 67.0 PPM
Figure 33: The 31P NMR spectra (101.25 MHz, CDC13) of 1£ (A) D-form and (B) In
form. two resonances in 31P NMR (101.256 MHz, D 20) at 55.32 and 55.71 ppm (Figure 34).
One small peak (55.56 ppm) between those two major peaks was one of the DPPsI from L-
isomers. Besides those peaks, two small peaks at 57.13 and 57.02 ppm in 31P NMR were
also observed and could be the products of 1,2 -phosphoryl migration (18) (see
Discussion). These two isomeric impurities were not separated from DPPsI by liquid
chromatography. However, they presented no problem to the spectral assignments and the
biochemical studies of DPPsI.
Determination of the Absolute Configuration at Phosphorus of 1,2- dipalmitoyl-sn-glycero-3-thiophosphoinositol by Bee Venom PLA2
It has been established previously that PLA2 from various sources specifically hydrolyze the Rp isomer of phosphorothioate analogues of PC, PE, sn-1 ether PC and PS
(see Chapter I). Since PLA2 has a broad specificity toward different types of
phospholipids, it can be used to assign the absolute configuration at phosphorus of DPPsI. It should be noted that due to a change in priority, the relative configuration of (Rp)- and
(Sp)-DPPsI correspond to that of Sp and Rp isomers, respectively, of DPPsC. As shown
in Figure 35A, (Rp+Sp)-DPPsI show two distinct 31P NMR (101.256 MHz, D 20) resonances at 55.32 and 55.71 ppm. Two minor peaks a and b (55.56 and 55.33 ppm) can
be attributed to DPPsI derived from L-H. Upon addition of PLA2 from bee venom, the down field resonance disappeared and was thus assigned to the Sp isomer. The resonance of the product Sp-lyso-PsI (19) happened to coincide with that of (Rp)-DPPsI (17R). as shown in Figure 35B. A trace of (Rp)-lyso-PsI was also detectable in Figure 35B (55.20 ppm), which grows upon prolonged incubation as shown in Figure 35C. It is interesting to note that the DPPsI from L-isomers was also specifically hydrolyzed by PLA2: peak b in
Figure 35 A was converted to peak c in Figure 35B. One of the 1,2-phosphoryl migration «! IS I c s
c
sa.o ss.s 3S.0 94.3 3 4 .0
Figure 34: The 31P NMR spectra (101.25 MHz, D 20 ) of DPPsI. Peaks (a) and (c) are D- (Rp+Sp)-DPPsI; peak (b) is L-DPPsI; and peak (d) is 2-DPPsI. 98
DPPkl
Rn-DPPsI
5 6 .0 S S .5 5 5 .0 5 4 .5 PPM
Figure 35: The hydrolysis of DPPsI catalyzed by bee venom PLA2 monitored by 31P
NMR (101.25 MHz, D20): (a) 10 mg of (Rp+ Sp)-DPPsI, (b) spectrum taken within
the first 30 minutes after addition of 1.3 mg of bee venom PLA2, and (c) spectrum
taken after further incubation at 25°C for 12 hours. 99 products was also specifically hydrolyzed by PLA2. The peak at 57.13 ppm decreased
while a new peak at 56.83 ppm increased (not shown). Therefore, the peak at 57.13 ppm in Figure 34 was assigned to be the Sp isomer of l,2-dipalmitoyl-sn-glycero-3-
thiophospho-2-myo-inositol (18S1 (see Discussion).
Expe rimental Section
Materials and Instrumental Methods
Phosphatidylinositol (sodium salt) from bovine liver was purchased from Avanti
polar lipids. Bee Venom PLA2 was purchased from Boehringer Mannheim. All other
chemicals were purchased either from chemical stores of the Ohio State University or from
Aldrich. Other biochemicals were purchased from Sigma. Silica gel used in liquid
chromatography (200-400 mesh) and thin layer chromatography (60 F 254) was obtained
from EM reagent (Merck). Most liquid chromatography was preformed under moderate pressure (about 20 psi). Medium pressure liquid chromatography was preformed on a
Licorpre Silica 60 (Merck) column. *H NMR spectra were recorded on Bruker WM-300,
AM-250 or AM-500 spectrometers. With Waltz-16 *H decoupling, 13C spectra were obtained at 75.44,62.89 and 125.76 MHz on Bruker WM-300, AM-250 or AM-500 spectrometers and 31P NMR spectra were obtained at 101.25 MHz on a Bruker AM-250 spectrometer. 31P chemical shifts are referenced to external 85% H 3P04. The *H and 13C chemical shifts for D20 samples are reference to external sodium 3-(trimethylsilyl)-l- propanesulfonate and to Me4Si for organic solvent sample. FAB mass spectra were recorded on a VG-70-250S mass spectrometer. Melting point was recorded on a Thomas 100 Hoover capillary melting point apparatus without calibration. Gas chromatography was
preformed on a HP 5890 machine.
Synthesis of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoinositol
2,3:5,6(1,2:4,5)-Di-0 -cyclohexylidene- 7nyo-inositol (&), 2,3:4,5(l,2:5,6)-di-0-
cyclohexylidene-myo-inositol (2), and 3,4:5,6(l,6:4,5)-di-0-cyclohexylidene-/nyo-insitol
(10) (the D-form numbering system was used, the numbering system in parentheses indicated the L system, Garegg et al., 1984): A solution of wyo-inositol (9.0 g, 50 mmol),
1-ethoxy-l-cyclohexene (16.5 g, 130 mmol), and p-toluenesulfonic acid monohydrate
(0.25 g, 1.2 mmol) in N, N-dimethylformamide (DMF) (125 ml) was heated at 95-100°C
for 2 hours. The mixture was cooled to room temperature, diluted with dichloromethane
(250 ml), and washed with a saturated solution of aqueous NaHC0 3 (100 ml) and water (2
x 100 ml). The dichloromethane layer was collected and the solvents (dichloromethane and
DMF) were evaporated to a syrup which, after dissolution in acetone-light petroleum (1:1,
v/v), gradually deposited crystalline (2) (1.58 g, 10.47%) m.p. 170-172°C
(literature(Garegg et al., 1984) m.p. 172-174°C). The remaining mother liquid was evaporated to a syrup which was chromatographed on silica gel (eluted by solvent gradient from chloroform to chloroform-acetone 2 :1, v/v) to yield 2 (200 mg); 2 (1-4 g, 10.3%), mp 134-137°C (literature 130-131°C); and M (0.45 g, 3 %), m.p. 152-154°C (literature 157-159°C). These compounds were further characterized by TLC and NMR. 2 : R f=
0.65 (benzene-acetone, 4/7, v/v). The assignments of the *H NMR spectrum were according to the following criteria. The 6 inositol ring protons resonated between 3.0 and
5.0 ppm. The peaks at 4.47 ppm was assigned to be equatorial 2-H based on the facts that this is the most downfield shift of the sue protons and that the 3Jh-h coupling constants are 101
in the ranges of axial-equatorial couplings of a chair-form cyclohexane. From the 1H-1H
decoupling experiments, the resonances between 4.05 and 3.96 ppm were assigned to be
3-H and 1-H and the resonances between 3.91 and 3.78 ppm were assigned to be 4-H and
6-H of the inositol ring protons. The most upfield resonance of the inositol ring protons
was then assigned to be 5-H. This assignment was confirmed by 1H-1H decoupling
experiments. ^H NMR (CDCI 3, 250 MHz) S 4.47 (t, Jh -C (2)-C(1)-h = Jh-C(2)-C(3) - h = 4.7
Hz, 1H, 2-H), 4.05-3.96 (m, 2H, 1-H and 3-H), 3.91-3.78 (m, 2H, 4-H and 6 -H), 3.30
(dd, J= 10.5 Hz, J= 9.5 Hz, 1H, 5-H), 2.91 (d, J = 2.6 Hz, 1H, OH), 2.58 (d, J = 9.0
Hz, 1H, OH), 1.62-1.40 (m, 20H, CH2 of 2,3: 5,6-di-O-cyclohexylidene). 13C NMR
(CDCI3, 75.46 MHz) 8113.27 (tertiary C-l of 5,6-O-cyclohexylidene), 110.86 ( tertiary
C-l of 2,3-O-cyclohexylidene) (This was assigned by reference to the compound only
containing 2,3-O-cyclohexylidene group.), 81.56 ,77.91,77.77 , 77.27,75.42 and
70.183 (6 inositol carbons), 37.81,36.49 and 35.06 (C-2 and C -6 of 2,3: 5,6-di-O-
cyclohexylidene), 24.98, 24.90, 24.03, 23.70 and 23.65 (C-3, C-4 and C-5 of 2,3: 5,6- di-O-cyclohexylidene). 2 : Rf = 0.55 (benzene-acetone, 4/7, v/v). *H NMR (CDC13, 250
MHz): 8 4.48 (dd, J= 6.4 Hz, J= 4.2 Hz, 1H), 4.32 (dd, J = 8.0 Hz, J = 6.4 Hz, 1H),
4.05 (dd, J = 8.7 Hz, J=4.6 Hz, 1H), 3.95 (dd, J= 10.3 Hz, J =8.1 Hz, 1H), 3.85
(unresolved dd, J =3.5 Hz, 1H), 3.40 (dd, J= 10.4 Hz, J = 8.8 Hz, 1H), 2.80 (broad singlet, 1H, 4-OH), 2.62 (d, J = 3.4 Hz, 1H, 3-OH), 1.64-1.41 (m, 20H). 13C NMR
(CDCI3, 75.46 MHz): 8 113.15,111.23 (tertiary C-l of 2,3:4,5-di-O-cyclohexylidene)
(This was assigned by reference to the compound containing only 2,3-O-cyclohexylidene group.), 78.24,77.83, 76.45, 75.64 , 74.62 and 73.53 (6 inositol carbons), 37.16 and
36.58 (C-2 and C -6 of 4,5-O-cyclohexylidene), 36.51 and 34.20 (C-2 and C -6 of 2,3-O- cyclohexylidene), 25.03, 25.00, 23.91, 23.64 and 23.47 (C-3, C-4 and C-5 of 2,3: 4,5- di-O-cyclohexylidene). 12: Rf = 0.44 (benzene-acetone, 4/7, v/v). 102
DL-1 -O-benzyl-2,3: 5,6-di-O-cyclohexylidene-ttry 0-inositol (H ) (Billington et al.,
1987): To a solution of S (10 mmol in 600 ml of toluene), 11 mmol of benzyl bromide
and 15 mmol of NaH were added and heated overnight under reflux. The filtrate was
washed with H20 (2 x 200 ml), saturated NaHC0 3 (200 ml), and brine (200 ml), dried
over CaCl2, and evaporated to dryness. Purification was performed by liquid
chromatography on silica gel (chloroform-acetone, 10/1, v/v) to give 2.74 g (65.20 % yield) of U (Rf= 0.53, chloroform-acetone, 10/1, v/v). This compound was further
characterized by NMR. The assignments to specific resonances and coupling constants
were made by reference to S. In *H NMR spectrum, the most upfield resonance was assigned to be 2-H, the doublet of doublet of doublets (ddd) at 3.77 ppm was assigned to
be 4-H based on the additional splittings from 4-OH, and the other 3 resonances of the
inositol ring protons were assigned by comparing the coupling constants of 4-H, 2-H and
5-H. *H NMR (CDC13, 250 MHz): 8 7.38-7.19 (m,5H, phenyl of 1-O-benzyl), 4.82 and
4.74 (AB, J = 12.5 Hz, 2H, CH2 of 1-O-benzyl), 4.29 (t, J h - c (2)-C(3)-h = Jh-c(2)-C(1)-h =
4.4 Hz, 1H, 2-H), 3.96 (dd, J h -C (6)-C(1)-h = Jh-c(6)-C(5)-h = 10.0 Hz, 1H, 6-H ), 3.85 (dd,
JH-C(i)-C(6)-H = 9.5 Hz, J h - c (1)-C(2)- h = 4.9 Hz, 1H, 1-H), 3.77 (ddd, J h - c (4)-C(5)-h - 9.6
Hz, J h -C (4)-C(3)-H = 10.2 Hz, % c(4)-0 -H = 2-5 Hz, 1H, 4-H), 3.70 (dd, J h -C (3)-C(2)-H = 4.2
Hz, J h - c (3)-C(4) - h = 10.2 Hz, 1H, 3-H), 3.18 (dd, J h - c (5) - c ( 4)-h = 9.6 Hz, J h - c (5) - c ( 6)-h =
10.1 Hz, 1H, 5-H), 2.55 (d, JH-C(4)-0 - h = 2.6 Hz, 1H, OH), 1.58-1.35 (m, 20H, CH 2 of
2,3:5,6-di-O-cyclohexylidene). 13C NMR (CDC13, 62.896 MHz): 8 138.08 (phenyl C-l of 1-O-benzyl), 128.35 (phenyl C-2 and C -6 of 1-O-benzyl), 128.15 (phenyl C-3 and C-5 of 1-O-benzyl), 127.74 (phenyl C-4 of 1-O-benzyl), 113.02 (tertiary C-l of 5,6-0- cyclohexylidene), 110.76 (tertiary C-l of 2,3-O-cyclohexylidene), 81.35 (CH 2 of 1-O- benzyl), 78.21, 76.78, 76.18, 74.99, 74.76 and 71.64 (6 inositol carbons), 37.78, 36.55
, 36.33 and 35.18 (C-2 and C -6 of 2,3:5,6-di-O-cyclohexylidene), 25.01,24.97, 23.94, 103 23.77 and 23.59 (C-3,C-4 and C-5 of 2,3:5,6-di-O-cyclohexylidene).
D-l-0-benzyl-4-0-(-)-camphanoyl-2,3:5,6-di-0-cyclohexylidene-myo-inositol(12d) andL(D)-3(l)-0-benzyl-6(4)-0-(-)-camphanoyl-l,2:4,5(2,3:5,6)-di-0-cyclohexylidene- myo-inositol (1211 (the numbering system in parentheses indicated the L system)
(Billington et al., 1987): To a solution of H (3 mmol in 100 ml of dichloromethane), 3.3
mmol of (-)-camphanic acid chloride, 3.3 mmol of 4-dimethylaminopyridine (DMAP) and 2 ml of Et3N were added and stirred at room temperature for 17 hrs. The reaction was
stopped by adding 5 g of wet Rexyn®-I300 (IR-300) ion exchange bead and partitioned in
200 ml of dichloromethane and 200 ml of water. The dichloromethane layer was washed
with 200 ml of H 20 , 200 ml of saturated NaHC03, and 200 ml of brine, dried over
Na2S0 4, and evaporated to dryness. The more polar diastereomer (Rf = 0.55, light
petroleum ether-ethyl acetate, 3:1, v/v) was obtained by recrystallization (light petroleum
ether-ethyl acetate, 2:1, v/v) 4 times from the mixture to give 628 mg of 121 (35 %). The less polar diastereomer (Rf = 0.62, light petroleum ether-ethyl acetate, 3:1, v/v) was recovered from combined mother liquors by liquid chromatography on silica gel (light petroleum ether-ethyl acetate, 3:1, v/v) to give 797 mg of 12d (44 %). These compounds were further characterized by NMR. The assignments of specific resonances and coupling constants were made by reference to 11 and camphanic acid, and by decoupling experiments. 12d: The assignments of the *H NMR spectrum were according to the following criteria. By reference to 11, the most upfield shift resonance was assigned to be
5-H. This assignment to this resonance was confirmed by its two axial-axial coupling constants (9.4 and 11.1 Hz) and by ^ ^ H decoupling experiments. Saturation of 5-H collapsed two resonances at 5.36 and 4.15 ppm. The resonance at 5.36 ppm was assigned to be 6-H on the basis of the downfield shift character of the ester derivative at C -6 according to Shoolery's rules (Williams and Fleming, 1980). Thus, the resonance at 4.15 104 ppm was assigned to- be 4-H. The resonance at 4.36 ppm was assigned to be 2-H based on
two identical cis-equatorial-axial couplings (4.4 Hz). The other resonances of the inositol
ring protons were assigned by comparing the known coupling constants and confirmed by
^ ^ H decoupling experiments. Thus, the resonances at 4.15,4.11 and 3.78 ppm were
assigned to be 4-H, 1-H and 3-H, respectively. *H NMR (CDC13, 250 MHz): 8 7.43-7.26
(m, 5H, phenyl of 1-O-benzyl), 5.36 (dd, J h - c (6)-C(1)-h = 7.0 Hz, Jh-c(6)-C(5)-h = H-2 Hz,
1H, 6-H), 4.94 and 4.86 (AB, J = 12.4 Hz, 2H, CH 2 of 1-O-benzyl), 4.36 (t, JH.C( 2)-C(i)-
h = j h - c ( 2) - c ( 3) - h = 4.4 Hz, 1H, 2-H), 4.15 (dd, Jh-c(4)-c(5)-h -9.6 Hz, %C(4)-C(3)-H =
10.2 Hz, 1H, 4-H), 4.11 (dd, J H- c (1)-C(2)- h = 4.1 Hz, JH-c(1)-C(6)-h =7.2 Hz, 1H, 1-H),
3.78 (dd, J h -C (3) - c ( 2) - h = 4.1 Hz, J h - c (3)-C(4)- h =10.3 Hz, 1H, 3-H), 3.38 (dd,JH_C( 5)_c(4)-
H =9.4 Hz, J h -C (5)-c (6)-h =11.1 Hz, 1H, 5-H), 2.48-1.70 (m, 4H, CH 2 of camphanoyl),
1.58-1.42 (m, 20H, CH 2 of 2,3:5,6-di-O-cyclohexylidene), 1.11 (s, 3H, camphanoyl 9-
CH3), 1.04 (s, 3H, camphanoyl 8 -CH3), 0.99 (s, 3H, camphanoyl 10-CH3). 13C NMR
(CDC13, 75.44 MHz) (Figure 37): 8 178.27 (camphanoyl C -ll), 166.44 (camphanoyl C-
2), 137.83 (phenyl C-l of 1-O-benzyl), 128.33 and 128.15 (phenyl C-3 and C-5 of 1-O-
benzyl), 127.95 (phenyl C-2 and C -6 of 1-O-benzyl), 127.46 (phenyl C-4 of 1-O-benzyl),
113.22 (tertiary C-l of 5,6-O-cyclohexylidene) and 111.10 (tertiary C-l of 2,3-O-
cyclohexylidene) (The assignments of this resonance was made by reference to the
compound containing only a 2,3-cyclohexylidene group.), 91.10 (camphanoyl C-4),
76.90, 76.32, 78.25,75.98, 75.74, 74.38 and 71.71 (CH 2 of 1-O-benzyl and 6 inositol carbons), 54.78 and 54.68 (camphanoyl C-l and C-7), 37.50, 36.33, 36.18 and 35.20 (C-
2 and C -6 of 2,3:5,6-di-O-cyclohexylidene), 24.91, 24.86,23.78,23.60 and 23.58 (C-3,
C-4 and C-5 of 2,3:5,6-di-O-cyclohexylidene), 30.28 (camphanoyl C- 6), 28.90
(camphanoyl C-5), 16.54 (camphanoyl C-9), 16.33 (camphanoyl C- 8 ), 9.67 (camphanoyl
C-10). 121: The assignments of the *H and 13C NMR spectra were similar to 12d. !H NMR (CDCI3, 250 MHz): 8 7.36-7.20 (m, 5H, phenyl of 1-O-benzyl), 5.32 (dd, JH_C(6).
c ( i ) - H = 7.0 Hz, JH-C(6)-C(5)-H =11-2 Hz, 1H, 6 -H), 4.82 and 4.73 (AB, J = 12.4 Hz, 2H,
CH2 of 1-O-benzyl), 4.27 (t, Jh-c(2)-C(1)-h = 7h-C(2)-C(3)-h = 4.1 Hz, 1H, 2-H), 4.08 (t, Jjj_
C(4)-C(3)-H = jh-C(4)-C(5)-h =9.8 Hz, 1H, 4-H), 3.97 (dd,JH.C(1).C(2).H = 4.9 Hz, Jh-c(1)-C(6)-
h =6.7 Hz, 1H, 1-H), 3.71 (dd, Jh-c(3)-c(2)-h “ 4.0 Hz, Jh-c(3)-C(4)-h ~ 10.2 Hz, 1H, 3-
H), 3.31 (t, Jh-C(5)-C(4)-h = Jh-C(5)-C(6)-h= 10.6 Hz, 1H, 5-H), 2.39 and 2.12-1.70 (m,
4H, CH2 of camphanoyl), 1.53-1.40 (m, 20H, CH 2 of 2,3: 5,6-di-O-cyclohexylidene),
1.04 (s, 3H, camphanoyl 9-CH3), 0.98 (s, 3H, camphanoyl 8 -CH3), 0.93 (s,3H,
camphanoyl 10-CH3). 13C NMR (CDC13, 62.896 MHz): 8 178.05 (camphanoyl C-l 1),
166.21 (camphanoyl C-2), 137.80 (phenyl C-l of 1-O-benzyl), 128.31 (phenyl C-3 and
C-5 of 1-O-benzyl),128.14 (phenyl C-2 and C -6 of 1-O-benzyl), 127.77 (phenyl C-4 of
1-O-benzyl), 113.30 (tertiary C-l of 5,6-O-cyclohexylidene), 111.16 (tertiary C-l of 2,3-
O-cyclohexylidene), 91.03 (camphanoyl C-4), 78.38, 77.03 ,76.31,75.96, 75.64,74.18
and 71.70 (CH 2 of 1-O-benzyl and 6 inositol carbons), 54.69 (camphanoyl C-l and C-7),
37.21,36.28, 36.20 and 35.28 (C-2 and C -6 of 2,3: 5,6-di-O-cyclohexylidene), 24.89,
24.84,23.78 and 23.58 (C-3, C-4 and C-5 of 2,3: 5,6-di-O-cyclohexylidene), 30.16
(camphanoyl C- 6), 28.98 (camphanoyl C-5), 16.47 (camphanoyl C-9), 16.22
(camphanoyl C- 8 ), 9.63 (camphanoyl C-10). The % de of these two diastereomers was calculated by the relative intensities of the camphanoyl C-l 1 resonance from 13C
NMR spectra (Figure 32) under the following nonsaturating conditions: 75.44 MHz,
CDC13, 45° pulse and 5 seconds delay. The % de was also calculated by the relative intensities from the GC traces (Figure 32). The % de of these products after one fractional recrystallizations and one MPLC purification is about 75-90. The % de of these products after two fractional recrystallizations and two MPLC purifications is about 90-100. In GC, 106 the retention time for 12d is 33.5 minutes and that of 121 is 31.0 minutes. MS for both
isomers: m/z 610 (M*), 397,199 and 91.
D-l-0-benzyl-2,3:5,6-di-0-cyclohexylidene-myo-inositol (lid'): A mixture of 12d
(145 mg, 0.241 mmol), 25 ml tetrahydrofuran, 100 mg (10 equivalent) of LiOH-H 20 , and
12.5 ml of water was stirred at room temperature for 25 hours. To the reaction mixture,
100 ml of dichloromethane and 100 ml of water were added. The organic layer was washed with 100 ml of saturated NaCl, 100 ml of saturated NaHCC^ and 100 ml of water.
The crude product was further purified by flash chromatography on silica gel (chloroform:
Acetone, 10 :1, v/v) to give a semi-solid (100 mg, 98.8% yield). Both TLC and
NMR of this product were the same as that of DL form.
D-l-0-benzyl-2,3:5,6-di-0-cyclohexylidene-4-methoxymethyl-myo-inositol(12): To U d (100 mg, 0.238 mmol) in dichloromethane (25 ml), 0.96 mmol of iPr2NEt and
0.96 mmol of chloromethyl methyl ether (MOMC1) were added and stirred at room temperature for 4.5 hours. Excess MOMC1, iPr2NEt and solvent were removed by rotary evaporation. The crude product was purified by flash chromatography on silica gel in hexane-ethyl acetate (5/1, v/v) to give a white solid (100 mg, 90% yield), mp 110-113°C. TLC Rf = 0.5 (hexane/ethyl acetate, 5/1, v/v). This compound was further characterized by NMR. The assignments of specific resonances and coupling constants were made by reference to 12. Thus, two AB systems were assigned to be the methylene protons of 1-0- benzyl and 4-O-MOM. The more down field shift methylene was assigned to be the methylene protons of 4-O-MOM based on Shoolery's rules. The more upfield AB system, therefore, was assigned to be the methylene protons of 1-O-benzyl group. This assignment was confirmed by references to the coupling constants of benzyl methylene of 11 and 12-
The resonance at 4.33 ppm was assigned to be 2-H on the basis of its cis-equatorial-axial coupling constants and its down field shift character. NMR (CDC13, 250 MHz): 8 7.45-7.26 (m, 5H, phenyl of 1-O-benzyl), 4.90 and 4.78 (AB, J = 6.4 Hz, 2H, CH 2 of 4-
O-MOM), 4.89 and 4.82 (AB, J = 12.5 Hz, 2H, CH 2 of 1-O-benzyl), 4.33 (t, JH-c( 2)-C(i)-
ji = Jh-C(2)-C(3)-h = 4*4 Hz, 1H, 2-H), 4.10-3.88 (m, 3H) and 3.73 (dd,J = 4.2 Hz, 10.1
Hz, 1H) (1-H, 3-H, 4-H and 6-H), 3.43 (s, 3H, CH3 of 4-O-MOM), 3.26 (dd, J = 10.3,
9.4 Hz, 1H, 5-H), 1.82-0.77 (m, 20H, CH 2 of 2,3:5,6-di-0-cyclohexylidene). 13C NMR
(CDC13, 62.896 MHz): 5 138.08 (phenyl C-l of 1-O-benzyl), 128.32 (phenyl C-2 and C-
6 of 1-O-benzyl), 128.16 (phenyl C-3 and C-5 of 1-O-benzyl), 127.71 (phenyl C-4 of 1-
O-benzyl), 112.74 (C-l of5,6-0-cyclohexylidene), 110.50 (C-l of 2,3-0-
cyclohexylidene), 95.20 (CH 2 of 4-O-MOM), 80.26, 78.01,76.94 , 76.66 , 76.18 ,
74.56 and 71.60 (CH 2 of 1-O-benzyl and 6 inositol carbons), 55.23 (CH 3 of 4-O-MOM),
37.57,36.42 and 35.33 (C-2 and C -6 of 2,3: 5,6-di-O-cyclohexylidene), 25.04,24.98,
23.90,23.78 and 23.58 (C-3, C-4 and C-5 of 2,3: 5,6-di-O-cyclohexylidene).
D-2,3:5,6-di-0-cyclohexylidene-4-0-methoxymethyl-myo-inositol (141: Hydrogenolysis method: To 12 (100 mg, 0.215 mmol) in 40 ml of ethyl acetate, 159 mg of 10% Pd in activated carbon was added at room temperature. The solution was connected to hydrogen gas (1 atm) and stirred at room temperature for 24 hours. The filtrate (from celite) was evaporated to dtyness and gave 80 mg (97% yield) of a white solid after purification by flash chromatography on silica gel in chloroform-acetone ( 10/ 1, v/v).
Li or Na-NH 3 method: To a dry tetrahydrofuran (15 ml, over Na) solution of 12 (232 mg, 0.5 mmol), Li or Na (about 160 mg) was added at -78°C (dry ice-tetrahydrofuran bath). Dry NH 3 (over Na) was distilled into the reaction mixture and stirred at -78°C for 1 hour. To this reaction mixture, 300 mg of NH 4C1 was added at -78°C and stirred from -
78°C to room temperature. The reaction mixture was diluted with 200 ml of light petroleum ether. The organic layer was washed with 3 x 50 ml of water, 100 ml of saturated NaCl, and 100 ml of water and dried over CaCl2. The product was purified by 108
flash chromatography on silica gel in chloroform-acetone ( 10/ 1, v/v) to give a white semi solid (190mg, 100% yield). TLC R f= 0.14 (hexane/ethyl acetate, 5/1, v/v). This
compound was further characterized by NMR. The assignments were made by reference to 12 and MOMC1. *H NMR (CDC13, 250 MHz) 5 4.90 and 4.78 (AB, J = 6.4 Hz, 2H,
CH2 of 4-O-MOM), 4.44 (t, Jh-C(2)-C(1)-h = Jh-C(2)-C(3)-h = 4.7 Hz, 1H, 2-H), 4.11 (dd,J
= 5.1 Hz, J = 6.6 Hz, 1H, 3-H) and 3.94-3.79 (m, 3H) (1-H, 3-H, 4-H and 6-H), 3.43
(s, 3H, CH3 of 4-O-MOM), 3.31 (dd, J = 10.3, 9.4 Hz, 1H, 5-H), 1.74-0.76 (m, 20H,
CH2 o f2,3:5,6-di-O-cyclohexylidene). 13C NMR (CDC13, 62.896 MHz): 8 112.99 (C-
1 of 5,6-O-cyclohexylidene), 110.58 (C-l of 2,3-O-cyclohexylidene), 95.18 (CH 2 of 4-O-
MOM), 80.41 ,77.92,77.24 ,77.09 ,77.00 and 69.98 (6 inositol carbons), 55.24 (CH 3
of 4-O-MOM), 37.59,36.53,36.29 and 35.18 (C-2 and C -6 of 2,3:5,6-di-O-
cyclohexylidene), 24.97,24.87, 23.84, 23.76 and 23.68 (C-3, C-4 and C-5 of 2,3: 5,6-
di-O-cyclohexylidene).
l,2-Dipalmitoyl-sn-glycero-3-(0-methylthiophospho)-2,3:5,6-di-0-cyclohexylidene-
4-0-methoxymethyl-/wyo-inositol (161: Compound H (0.47 mmol) was dried three times
by rotary evaporation with 5 ml of dry toluene. To this dry compound, 5 ml of
dichloromethane (dried over Na 2S0 4) and an excess of triethylamine (dried over NaH)
were added through vacuum transfer. To this reaction mixture, chloro(N,N-
diisopropylamino)methoxyphosphine (0.70 mmol) was added through a dry syringe under
the argon atmosphere. After the reaction was complete (about 25 minutes) on the basis of the disappearance of U on TLC, the solvent and excess triethylamine were removed under vacuum, and the reaction mixture was further dried in vacuo (0.2 mm Hg) for 4 hours.
Thoroughly dried tetrazole (1.88 mmol) and 1,2-dipalmitoyl-sn-glycerol (0.56 mmol) were dissolved in tetrahydrofuran-acetonitrile (1:1, v/v) through vacuum transfer. After warming to 40°C to make sure all compounds were dissolved, the solution was added to the above reaction mixture by a syringe under the argon atmosphere. After stirring for 18
hours at room temperature, the reaction was complete on the basis of TLC. The solvents
were evaporated to dryness and replaced with 10 ml of dry toluene (distilled over NaH).
To this heterogeneous mixture, an excess of S 8 (powder, sublimed) was added at room
temperature. The suspension was stirred at room temperature for 2 days. The reaction
mixture was then washed with 25 ml of saturated NaHCC >3 and the organic phase was
evaporated to dryness. The crude product was purified by liquid chromatography on silica gel in dichloromethane-diethyl ether (95/ 5, v/v) to give an oil (405 mg, mw=1035, 83% yield based on 14). TLC (dichloromethane/ diethyl ether, 95/ 5, v/v): Rf = 0.7. The
product was characterized by NMR. The assignments to specific resonances and coupling
constants were made by reference to 14, phosphatidylinositol (Shibata et al., 1984) and
l,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (Hauser et al., 1980b), and31P
decoupling *H NMR. !H NMR (CD 3OD, 250 MHz): 8 5.24 (m, 1H, glycero-sn-2 CH),
4.88 and 4.76 (AB, J = 6.4 Hz, 2H, CH 2 of 4-O-MOM), 4.82 and 4.35 (m, 2H, glycero-
sn-1 CH2), 4.52 (dd, J= 4.5, 9.0 Hz, 1H, 1-H), 4.26-3.99 (m, 4H, 4-H, 6-H and
glycero-sn-3 CH2), 3.81 and 3.75 (two doublets, 3JP.H = 5.2 Hz, 3H, Rp- and Sp-
POCH3), 3.36 (t, J = 10.2 Hz, 1H, 5-H), 2.31 (t, J = 7.5 Hz, 2H, a-CH2 of sn-2 acyl),
2.28 (t, J = 7.4 Hz, 2H, a-CH 2 of sn-1 acyl), 1.60 (m, 4H, P-CH 2 of two acyl chains),
1.8-1.1 (m, 72H, all the other CH 2 protons), 0.86 (t, J = 7.4 Hz, 6H, terminal-CH 3 of
two acyl chains). 13C NMR (CD 3OD, 62.896 MHz): 8 173.14 and 172.71 (C=0 of two
acyl chains), 113.20 and 110.77 (tertiary C-l of 2,3:5, 6-di-O-cyclohexylidene), 95.15
(CH2 of 4-O-MOM), 80.31, 75.94,75.65,75.58, 75.09 and 74.99 (6 inositol carbons),
69.15 (glycero-sn-2), 65.51 (glycero-sn-3), 61.81 (glycero-sn-1), 55.24 (CH 3 of 4-O-
MOM), 54.76 and 54.44 (dd, 3Jpc= 6.9 Hz), 37.55, 36.33, 36.12 and 35.15 (C-2 and C-
6 of 2,3:5,6-di-O-cyclohexylidene), 34.15 (a-CH 2 of sn-2 acyl), 34.00 (a-CH 2 of sn-1 acyl), 31.68 (14-CH2 of two acyl chains), 29.63,29.43,29.30, 29.24 and 29.08 (4- to
13-CH2 of two acyl chains), 24.88,23.87,23.78 and 23.56 (C-3, C-4 and C-5 of 2,3:
5,6-di-O-cyclohexylidene), 24.81 (P-CH 2 of two acyl chains), 22.63 (15-CH2 of two acyl
chains), 14.04 (terminal-CH 3 of two acyl chains). 31P NMR (CD 3OD, 101.256 MHz)
16d (Figure 34A): 8 67.93, 67.63 (D form, intensity = 100%), 67.86,67.69 (L form,
intensity = 18 %). % de = 82. ISL (Figure 34B): % de = 83. With the exception of 31P NMR, both *H and 13C NMR cannot distinguish these diastereomers. Rp- and Sp-l,2-Dipalmitoyl-sn-glycero-3-thiophosphoinositol (Rp- and Sp-DPPsI,
12): To 110 mg of 1£, 4 ml of tetrahydrofuran and 2 ml of 6N HC1 were added. The
resulting solution was stirred at room temperature for 3 hours. Remove the solvents and HC1 by rotary evaporation. To this dry solid, toluene (over NaH, 10 ml) and anhydrous
trimethylamine (over NaH, 5 ml) were added through vacuum transfer. The resulting
solution was stirred at room temperature for 3 days. After the deprotection reaction was
complete, trimethylamine and solvents were evaporated to dryness and dissolved in 100 ml
of chloroform. The organic layer was washed with 3x 50 ml of saturated NaCl and was
concentrated to about 1 ml. This compound was purified by liquid chromatography on
silica gel with chloroform-methanol (2:1, v/v) as the eluting solvent to give 62.4 mg of a
white solid 12 (73 % yield). TLC: Rf=0.2 (chloroform/ methanol/ H 20 , 66/ 33/ 4,
v/v/v). The product was further characterized by NMR. The assignments to specific resonances and coupling constants of the *H NMR spectrum (Figure 36) were made by reference to and phosphatidylinositol (Shibata et al., 1984) and confirmed by ^ ^ H decoupling experiments. The assignments of 13C NMR spectrum were made by the *H and 13C COSY spectrum (Figure 37 and 38).and by 13C DEPT spectra (separation C, CH,
CH2, and CH 3 13C resonances) (Figure 39). JH NMR (500 MHz,CD 3OD-
CDC13,1:2,v/v) (Figure 36): 8 4.89 (m, 1H, sn-2 CH), 4.05,3.83 (m, 2H, glycero-sn-1 I l l
sn-i sn-3 2*H l-H n
sn-2 J
A _lL^lA . U KAkAk
i ■! |‘ i i t | i I ] i 'i'ii | l 11 *i1 |»'i* i-r1! r-i-i r l Ti |-|* i is » p 'i*t i'|*i i n | n 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 .3 0.0 PPM
Figure 36: The !H NMR spectrum (500 MHz, CDCI 3-CD3OD) of DPPsI 1 ■ -« - A 1 1 A . 1 1 1
-.8 O S ~ 0 — C— CH2CH2(CH2) 12c h 3 0.0 H3C(CH2)12CH2CH2 — C —-0 sn -2 O a p Y t f * .8 Sw 0 'sn -3 "*11 g) *• bt 1.0 HO p - 0 T y ^ 6 p 1.8 OH HO*-**—7 y \ » 2.0 6 H O ^ — OH a 2.8
>.0
1.0
4 .0
. 4 .8
. 8 .0
. 8.8
PPM 1 I ■ ■ ri-fT. 1 t p m pTiTfi r..V. ■ 1 » | ■ 1 i| 1 1 n | ■ ■■I n rrriTT rrm vi» 11 | 1 ■ H|H IS 10 79 70 OS 80 88 90 49 40 19 M 28 20 IB 10 ppm K> Figure 37: The 'H -^C correction NMR spectrum (500 MHz, CD 3OD-CDCI3) of DPPsI sn -1
R—C sn-3 HO OH HO. -OH
1 FPU T* ii •’—r i T l TT T" *T* ~r ~r T* i 00 70 70 74 72 70 00 (I •4 02 oo M 90 94 Figure 38: The expansion of Figure 46 S p 'sn -3 HO V -0 2 L v ° 6
J — OH
vw4>« I
CH3 OK.V
-CH 2 CH2 (CH2) 1 0 CH2 CH2 CH3 II 0 a P y 8 e t 5 i »n-t I #n-;I VA»
CH ONLY
C-2
C-6 C-3 C-4
C, -5 1 ' /
•n -2 c '1 1 L r VY>VI^ I w V l r f | 1| fy I ‘ y f tiii i V r 1* I f ‘ il u i^ lijl* ^ ^ - ir|>iln |‘\ y
C13 aa AM-299-2
Wvd" W.VV* I . . . . I . . • ■ I . r .-. r.T I 1 1 1 1 I ' 'I'” '1 1 I ■ ■»',-■ <0 79 70 OS 00 BO BO 49 40 99 30 29 20 19
Figure 39: The partial ,3C DEPT spectra (62.896 MHz, CD 3OD-CDCI3) of DPPsI CH2), 3.87-3.73 (m, 4H, 2-H, glycero-sn-3 CH 2 and 1-H), 3.42 (m, 1H, inositol 6-H),
3.23 (m, 1H, inositol 4-H), 3.06 (m, 1H, inositol 3-H), 2.89 (m, 1H, inositol 5-H), 1.95
(t, J = 7.4 Hz, 2H, oc-CH2 of sn-2 acyl), 1.93 (t, J = 7.4 Hz, 2H, 1.22 (m, 4H, p-CH 2 of two acyl chains), 0.90 (b, 48H, all the other CH 2 protons ), 0.50 (t, J = 7.4 Hz, 6H, terminal-CH 3 of two acyl chains). 13C NMR (CD 3OD, 125.759 MHz, ): 5 173.43 and 173.13 (C=0 of two acyl chains), 76.53 (inositol C-l), 73.91 (inositol C- 5), 71.92 (inositol C-4), 71.16 (inositol C- 6), 70.91, 70.78 (inositol C-2), 70.70 (inositol C-3), 69.94, 69.85 (glycero-sn-2), 63.61, 63.34 (glycero-sn-3), 62.13 (glycero-sn-1), 33.51 (sn-2 a-CH2), 33.39 (sn-1 a-CH2), 31.19 (14-CH2 of two acyl chains), 28.96, 28.79,28.62,28.45, (4- to 13-CH2 of two acyl chains), 24.19 ((3-CH 2 of two acyl chains), 21.91 (15-CH2 of two acyl chains), 12.96 (terminal-CH 3 of two acyl chains). 31P NMR (CD30D-CDC13, 1:2, v/v, 101.256 MHz) (Figure 34): 8 55.934, 57.01 (Rp-D form and Sp-D form, intensity = 1), 56.35 (Rp-L form and Sp-L form, intensity = 0.14), 57.88 (IS, intensity= 0.20). The impurity, IS was assumed to be the products of the 1,2- phosphoryl migration (see Discussion). The % de of XL obtained from 31P NMR were the same as that of 1£if IS was not counted. With the exception of 31P NMR, *H and 13C NMR failed to distinguish the diastereomers. MS (FAB): The peaks at m/z 849 and 850 were the molecular ion [M+ Na]+ and [M+ Na+ H]+. The assignments to other peaks were made by references to PI (Jugalwala, 1985; Jensen, 1987). The assignments to special fragments were outlined in Figure 40. m/z 850,849 ,552,551,331,315,275 and 179. The Hydrolysis of DPPsI by PLA2 The use of 31P NMR to monitor the hydrolysis of DPPsI by bee venom PLA2 was preformed at 37 °C on 15 mg of (Rp+Sp)-DPPsI and 1.3 mg of bee venom PLA2 in D20 116 Na+ -o P-°G HO OH HO HO OH O-P-O DPPsI m/z 848 t OCOC15H31 O-P-O inositol o c o c 15h 31 phosphorothioate F + C3H5O c h 2 m/z 331 m/z 551 m/z 275 T HO OH OH HO OH m/z 179 Figure 40: The mass (FAB) fragments of DPPsI 117 containing 5 % Triton X-100,50 mM HEPES buffer (pH 7.2), 2.5 mM Ca2+, and 0.25 mMEDTA. 1,2-Phosphoryl migration of phosphatidylinositol The reaction was carried out in a NMR tube containing 12 mg of phosphatidylinositol and 600 |il CDC13. Phosphatidylinositol showed a broad peak at 5.31 ppm. After adding 300 pi of 6N HC1, the peak at 5.31 ppm was decreased while a new sharp peak at 7.71 ppm was increased. This migration was monitored by 31P NMR with !H decoupling. PiS.SU53j.Qn, DPPsI was synthesized according to Scheme 3 and Scheme 4. However, two types of isomeric impurities were still present after final purification. The first type of impurity came from the L-inositol part. This impurity could be reduced by starting from purer D- inositol precursor. So far, our best result contained about 14 % of this impurity. Because there are 8 steps to synthesize DPPsI from the D-inositol precursor, we need a large amount of pure D-inositol precursor to get pure DPPsI. The second type of isomeric impurities were proposed to be the 2-position (of inositol) isomers (2-DPPsI, IS) which arose from the acid deprotection (Before acid deprotection, compound IS contained no such impurity). This interpretation was further confirmed by the fact that PI underwent the 1,2-phosphoryl migration in the acid condition. Although we did not isolate the migration product, we could rule out the possibilities of acyl chain migration and acyl chain hydrolysis because both lyso-PI and glycero-PI resonated at higher field than PI in 31P NMR. The proposed mechanism of 1,2- phosphoryl migration was based on the sn-3,sn-2 migration of lyso-PC as suggested by Pliickthun and Dennis (1982). As shown in Figure 4 1 ,1,2-phosphoryl migration could o f DG H+ O-DG HO H02i ^ r 0H -OH OH HO HO 7 S ^ - o h 1 1 (Sp) DPPsI 21 ° \ ) JD-DG \ 'P 0 ^==»S h T OH -*► ho ^ - oh 2 1 p .DG > «4S H o f e - H O ^ - O H US. (Sp) 2-DPPsI Figure 41: The 1,2-Phosphoryl Migration of DPPsI 119 occur through apentacoordinated intermediate ( 21) followed by a pseudorotation to give (22)- If stereochemistry is applied, the migration through a pseudorotation should be retention in its configuration at phosphorus. It was interesting to know that the Sp isomer of this migration product (2-DPPsI) was also specifically hydrolyzed by bee venom PLA2. References Billington, D. C., Baker, R., Kulagowski, J., & Mawer, I. M. (1987) J. Chem. Soc. Chem. Commun. 314-316. Bruzik, K. S., Salamonczyk, G., & Stec, W. J. (1986) J. Org. Chem. 51, 2368-2370. Cooke, A. C., Gigg, R., & Potter, B. V. L. (1987) J. Chem. Soc. Chem. Commun. 1525-1526. Corey, E. J., Pan, B.-C., Hua, D. H., & Deardorff, D. R. (1982a) J. Am. Chem. Soc. 104, 6816-6818. Corey, E. J., Hua, D. H., Pan, B.-C., & Steitz, S. P. (1982b) J. Am. Chem. Soc. 104, 6818-6820. Garegg, P. J., Iversen, T., Johansson, R., & Lindberg, B. (1984) Carbohyd. Res. 130, 322-326. Hauser, H., Guyer, W., Pascher, I., Skrabal, P., & Sundell, S. (1980b) Biochemistry 19, 366-373. Jensen, N. J., Tomer, K. B., & Gross, M. L. (1987) Lipids 22, 480-489. Jugalwala, F. B. (1985) in “Phospholipids in Nervous Tissues”, Eichberg, J. ed., pp 1- 44, John Wiley & Sons, Inc. press, N.Y., USA. Pliickthun, A. and Dennis, E. A. (1982) Biochemistry 21,1743-1750. 120 121 Potter, B. V. L. and Lowe, G. (1981) Biochem. J. 199, 693-698. Shibata, T., Uzawa, J., Sugiura, Y., Hayashi, K., & Takizawa, T. (1984) Chem. Phys. Lipids 34,107-113. Stork, G.and Takahashi, T. (1977) J. Am. Chem. Soc. 99,1275-1276. Vacca, J. P., deSolms, S. J., & Huff, J. R. (1987) J. Am. Chem. Soc. 109, 3478-3479. Williams, D.H. and Fleming, L (1980) in “Spectroscopic Methods in Organic Chemistry”, second edition, pp 83-146, William Clowes & Sons, Limited, London, The Great Britain. CHAPTER V The Steric Course of the Reaction Catalyzed by Phosphatidylinositide-Specific Phospholipase C Introduction To elucidate the steric course of PI-PLC, we need to know the absolute configuration at phosphorus of the substrate and that of the product, hi chapter IV, the absolute configuration at phosphorus of the substrate for PI-PLC, DPPsI, has been determined by the action of PLA2. In this chapter, we will elucidate the steric course of the reaction catalyzed by PI-PLC by determination of the absolute configuration at phosphorus of the products. There are several possible methods to determine the absolute configuration at phosphorus of the products of the PI-PLC hydrolysis (see Chapter I). Among these methods, the determination of the absolute configuration at phosphorus of the product, 1:2c-IPs by X-ray and/or NMR would be most easy and straightforward. The X-ray method is the most convincing evidence for this study. However, so far no crystal structure of inositol phosphates (either open or cyclic) has been published. This problem may partially be due to the difficulty of crystallizing the water soluble inositol phosphates. We, therefore, chose the NMR method. To use this method, we need to use a protecting precursor instead of l:2c-IPs because the latter two isomers will not show much differentiation in NMR. One of the D-form diastereomers [D-Sp(endo)- and D-Rp(exo)- 122 123 l:2c-IPs] has the same stereochemistry as one of the products of PI-PLC hydrolysis. Therefore, if we separated and determined configurationally these two D-form diastereomers, we can known the absolute configuration of the cyclic phosphorothioate product from PI-PLC hydrolysis. In practice, DL-mixture can be used since we can separate endo enatiomers (D-Sp and L-Rp) and exo enantiomers (D-Rp and L-Sp) by chromatography from 4 DL-isomers. Endo- and exo-isomers are diastereomers to each other as shown in Figure 42. Two endo-isomers have the same NMR character as pure D- endo isomer (D-Sp-l:2c-IPs) which can be the cyclic phosphorothioate product of PI-PLC hydrolysis, and two exo-isomers have the same NMR character as pure D-exo isomer (D- Rp-l:2c-IPs) which can also be the cyclic phosphorothioate product of PI-PLC hydrolysis. Two DL-diastereomers of wyo-inositol l:2cyclic phosphorothioate (l:2c-IPs) have been synthesized by Schultz et al. (1988) but their absolute configuration at phosphoms has not yet been determined. They used PSC1 3 to prepare the cyclic phosphorothioate ring. However, the yield of this method was low (20%). Therefore, we were prompted to develop a new method to synthesize l:2c-IPs. Results. The Stereospecificity of DPPsI toward PI-PLC In contrast to PLA2 which accepts most phospholipids as substrates, Bacillus cereus PI-PLC (EC 3.1.4.10) catalyzes the hydrolysis of phosphatidylinositides only (PI,.lyso- PI, PIP, and PIP 2). PI-PLC is distinctly different from the phosphatidylcholine- hydrolyzing phospholipase C (PC-PLC) in its substrate specificity, structure, and function. As shown in Figure 43, the PI-PLC from Bacillus cereus stereospecifically hydrolyzed the Rp isomer of DPPsI and the product, wyo-inositol l:2cyclic phosphorothioate (l:2c-IPs) was found at 69.89 ppm from the 31P NMR spectrum. The PI-PLC from guinea pig 124 % - ^ S (1) (2 )0 * ' \ D-Sp(endo)-l:2c-IPs L-Rp(endo)-l:2c-IPs diastereomers - o O i-“ •'/ # > 0 (2) (3)° OH HO HO OH D-Rp(exo)-l:2c-IPs L-Sp(exo)-l:2c-IPs enantiomers Figure 42: The absolute configuration of 1:2c-IPs (The numbers shown in parentheses are the priorities) Sp Rp 125 J u B /V* 1 I "" 1 '■ m.o n.o • Xo J and o 7 0 .• «9.o Figure 43: The Hydrolysis of 10 mg of DPPsI by Bacillus cereus PI-PLC monitored by 31P NMR (101.25 MHz) in DjOcontaining 5% Triton X-100,50 mM HEPES buffer, pH 7.2,2.5 mM CaCl 2 and 0.25 mM EDTA. Spectrum taken at (A) Oh and (B) 39h. The 31P NMR spectra (101.25 MHz) containing 0.12M NH 4C1 in 50% D20 ) of (C) exo-l:2c-IPs and (D) endo-l:2c-EPs. 126 uterus also stereospecifically hydrolyzed the Rp isomer of DPPsI and two products, 1:2c- IPs and IPs were found at 69.93 and 43.33 ppm, respectively, from the 31P NMR spectrum. It has been shown that PC-PLC specifically hydrolyzes the Sp isomers of DPPsC and DPPsE (see Chapter I). Bruzik (1988) also observed that PLC (PC-PLC or Sph-PLC) also specifically hydrolyzed the Sp-isomer of the phosphorothioate analogues of Sph. It should be noted that due to a change in priority the relative configuration of (Rp)- and (Sp)-DPPsI correspond to that of Sp and Rp isomers, respectively, of DPPsC. So far, all PLC's have the same steric requirement on the phosphorothioate analogues. Synthesis of of myo-lnositol 1:2cyc!ic Phosphorothioate It is easy to monophosphorylate the first hydroxyl group of a diol system with chloro(N,N-diisopropylamino)methoxyphosphine. Then, intramolecular cyclization to form cyclic phosphite can easily occur if the N,N-diisopropylamino group is protonated to become a leaving group. The resulting cyclic phosphite can easily be oxidized by oxygen, sulfur or selenium. To the best of our knowledge, no application of this reagent in forming a 1,3,2-dioxaphospholane ring (5 membered ring phosphite, phosphate, and phosphorothioate) has been reported. We first used this methodology to form 1,3,2- dioxaphospholanes in a high yield. The synthesis of l:2c-IPs was shown in Scheme 5. Dibenzyl ether 23 was prepared by an ordinary procedure from l,2:4,5-di-0-cyclohexylidene-myo-inositol, & (Garegg et al., 1984). Selective removal of the 4,5-cyclohexylidene ketal of 22 by 1 equivalent of ethylene glycol and 3 % weight of anhydrous p-toluenesulfonic acid (TsOH) in dichloromethane gave 24 in 74 % yield (Ozaki et al., 1986). Tetrabenzyl ether 25 was prepared from the reaction of 24 with 6 equivalents of benzylbromide. Hydrolytic removal of the residual cyclohexylidene group of 25 gave 24 in 74 % yield. 24 was then monophosphorylated with 1.2 equivalents of chloro(N,N- Scheme 5: Synthesis of DL-myo-inositol 1 :2cyclic 127 phosphorothioate 2.5 eq. BnBr, 1 eq. ethylene 3 eq. NaH, 2 glycol. BnO OBn & toluene, 3% weight v * reflux, ofT sO H , h S ^ oh overnight, CHnCli, rt 5 71% l/2h, 73% D L 2 4 DL 4 (^ v S) D L 21 eq. BnBr, 6NHC1- OH ClP(OCH3)N(iPr)2 , eq. NaH, < /\ ol 1 OH Et3N. 0 THF, 2; OBn Bn o i ^ r OBn toluene, BnO rt, overnight, BnO'^'^OBn i f e CH 2 CI2 , rt, reflux, BnO' 74% l/2h overnight, DL 2 k 71% D L 2 5 Q /O C H 3 c h 3o n CH30 , P — N iPr, \ tetrazole, P — N iPr, I CH3CN-THF, HO I ■■ ■' ■■ OBn ° P H u a , ® - OBn rt, 17h BO n O 4 n OBn n . ^ N - n R r BnO'4"J“ OBn BnO' ' “ OBn 25a. (endo) 2 7 a 22k OCH, c h 3o . S CH30 , V V \ 0 Sg, toluene, 0* 6 n OBn OBn *♦ BnO B „ 0 ^ C 6 5 % &om2kto BnO4 5 OB BnO OBn B nO ' — OBn 21 22a (exo) (8%) 29b (endo) (92%) 2 8 b (exo) / Li, THF- Li, THF. NH 3, -78°C, N H 3, -78 C, Sv - P 0 % - #S 5 min, 60% 5 min, 60% Y V f b OH HO H O ' OH H O ^ O H 3 0 a (exo) 30 b (endo) 128 diisopropylamino)methoxyphosphine in the presence of 1.5 equivalents of triethylamine to give 2Z • After removal of the excess triethylamine, 4 equivalents of tetrazole in tetrahydrofuran-acetonitrile were added to the reaction mixture. The phosphite intermediates of 22 were oxidized by elemental sulfur (S8) and gave 22a and 29b. The products, 22a and 22b were separated by medium pressure liquid chromatography and resulted in 5 and 60 % yield, respectively, based on 26- Assignments of the !H NMR spectra of 22a and 29b were made by reference to myo-inositol 1:2cyclic phosphate (Cerdan et al., 1986), by its splitting patterns, by decoupling experiments, by 31P decoupling *H NMR, by 2D J-resolved, and by NOESY (see Experiment Section). Compound 30a and 30b were obtained from the deprotection of 22a and 29h. respectively, with Li metal in tetrahydrofuran-liquid NH 3 at -78°C for 5 minutes (the procedure was modified from Potter and Lowe, 1981). Determination of the Absolute Configuration at Phosphorus of 29a and 29h The absolute configuration at phosphorus of 22a and 22b was assigned based on the following four criteria: (1) The predominant conformation of phosphite 21 (Figure 44) should be the sterically less hindered and more stable trans-form, 21b (McEven, 1965; Denney et al., 1969; Bentrude and Tan, 1976); therefore, the more stable conformer of 28a and 28b was assigned to be exo-28b. This assignment was further confirmed by the MM-2 calculation (MODEL and MMX programs were used, and the minimization energies of 28a and 28b were 88.5 kcal/mol and 85.3 kcal/mol, respectively.) (Figure 45). The oxidation or sulfurization of phosphite proceeded with retention in the configuration of phosphorus; moreover, the cis/trans ratio of phosphite (the reactant) was the same as that of phosphate or phosphorothioate (the product).(McEven, 1965; Denney et al., 1969; Bentrude and Tan, 129 R5 1 R5 yD C H 3 A 5 -Q OCH, R4 R4 \/r n / *0 ‘O 3 3 31a (cis) 32a (trans) less stable isomers R5 A5 \ > R4 Py'r 2 n OCH / ‘OCH, 3 31b (trans) 32b (cis) more stable isomers Figure 44: The Stability of 1,3,2-dioxaphospholanes 130 Figure 45: The most stable conformation of phosphite 28a (endo) (A) and 28b (exo) (B) obtained from MM-2 energy minimization. 131 1976; Mikolajczyk et al., 1976; Mikolajczyk and Witczak, 1977). Thus, the cis/trans ratio of 28a and 28b is the same as that of the resulting products 22a and 29b. Since 22b is the major product (60 % yield from 28) and 22a is the minor product (5 % yield from 28), 22b should be from the more stable phosphite exo-28b: 29a should be from the less stable phosphite endo-28a. Thus, 29b and 22a are assigned to endo and exo, respectively (note that the endo and exo notations of 22 are opposite to those of 28 due to the presence of sulfur in 291 (2) The second evidence supporting these assignments was derived from the chemical shift difference in the 31P NMR spectra. In 31P NMR, the trans isomer of 22 resonated at lower field than the cis isomer by 0.5-2.5 ppm (Mikolajczyk et al., 1976; Mikolajczyk and Witczak, 1977). 22a and 22b was observed 31P resonances at 84.41 and 82.65 ppm, respectively; therefore, 22a was assigned to be the exo configuration and 29b was the endo configuration. (3) The assignments were further confirmed by the three-bond coupling constants of P and 1-H. Like H-C-C-H, P-X-C-H (X= C, O, N or S) also has the Karplus relationship (Bentrude and Setzer, 1987); therefore, the relationship between 3Jp.o-c-H aud P-O-C-H dihedral angle (<)>) can be written as the equation 1. 3Jpoch = 15.3 cos 2 According to the criteria of Haake et al.(1968) (a twist envelope conformation of 1,3,2- dioxaphospholanes), the criteria of Marynoff et al., (1979) ( In the 1,3,2- dioxaphospholanes, the OCH 3 or OPh substitutent at 2-position (P) is in the pseudoaxial position and the other substituent, lone pair, sp 2 O, or sp 2 S is at the pseudoequatorial position.), the data of Mikolajczyk et al.(1976) (some x-ray crystal structures of 1,3,2- dioxaphospholanes), the data of Cerdan et al.(1986) NMR data of inositol l:2cyclicphosphate), and the MM-2 energy minimization (Chem 3D program), the H-C(l)- O-P dihedral angles of 22a and 22b were 150° and 124°, respectively (Figures 46 and 132 Figure 46: The conformations of (A) 22a (exo) and (B) 22fc (endo) (obtained from MM-2 energy minimization) 133 47). Therefore, the Jh-C(1)-0 -p values of 22a and 29b should be 18.3 and 9.8 Hz, respectively, according to equation 1. From the *H NMR spectra (see experimental section), the observed 3Jp.o-c(i)-H values of 22a and 29b were 18.4 and 9.7 Hz, respectively; therefore, 22a was the exo configuration and 29b was the endo configuration. (4) The fourth evidence which supports these assignments is based on the analyses of the NOES Y spectra of 22a and 29b (Figures 48 and 49). In 29b but not in 29a. a detectable NOE is observed between 2-H and the O-methyl proton resonance. This suggests that the O-methyl group is in the opposite sides with 2-H of the inositol ring in 29a and that the O-methyl group is at the same sides with 2-H of the inositol ring in 29b . Therefore, 29a has the exo configuration and 29b has the endo configuration. Elucidation the Steric Course of Phosphoinositide-specific Phospholipase C The PI-PLC from Bacillus cereus hydrolyzed the Rp isomer of DPPsI and produced one isomer of l:2c-IPs (Figure 43). In consistence with the natural substrate for PI-PLC from Bacillus cereus, the formation of inositol 1-phosphorothioate (IPs) was undetectable (Michell, 1975). After adding 30b (8 31P = 69.00 ppm) to the reaction mixture of PI-PLC hydrolysis, we found that 30b is not the product (Figure 50). Adding 30a to the above solution, we found that 2fia ( 8 31P = 69.85 ppm) coincided with the product. Therefore, the absolute configuration at phosphorus of the product l:2c-IPs of PI-PLC from Bacillus cereus was the exo form ('30a'). The PI-PLC from guinea pig uterus also hydrolyzed the Rp isomer of DPPsI and also produced exo-l:2c-IPs ( 8 31P = 69.93 ppm). Therefore, the stereochemical course of the consequence of PI to l:2c-IP catalyzed by PI-PLC was inversion in the configuration of phosphorus as shown in Figure 51. 134 Figure 47: Another view of Figure 46: The H(l)-C(l)-0-P dihedral angles of (A) 22a and (B) 29b 4-H 5-H (b, 5-H) ( -H , -H) i 3 5 3.4 o - (2-H , 3-H ) (bl 3-H) (a. 4-H ) • ra --J> . (4-H . 6-H) (b, 4-H) 4.0 (b, 6H) ^ / 0CH: 4.6 JO CXO 4.6 OChUPh 5.4 PPM i •------1------«------1------r — «------1------1------«------1------1------•------1------»------1------«------1------•------1----- «— 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 Figure 48: The NOES Y spectrum (500 MHz, CDC13) of 22a (exo) ^ 2-H 3-H 1-H 6-H 4-H 'A a . _ 3 —e — 6> (3-H, 5-H) i (1-H, 5-H) (4-H, 5-H) / - 3 (b, 3-H); (2-H, 3-H) J(1-H, 3-H) (3-H, 4-H) '• ------_T(a, 2-H) i . 3 (b, 4-H) (4-H, 6-H) » (b, 6-H) (1-H' 6^ (1-H, 2-H) ty n Lk. endo _ 4 y PPM —i i i i i ! i i i i I i r* i i ? i 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.] 4.0 3.9 3.8 3.7 3.6 3.5 3.4 PPM Figure 49: The NOESY spectrum (500 MHz, CDC13) of 22k (endo) 137 70.0 6 9 .0 PPH 70.0 69.0 t—i—|—r 70.0 69.S 69.0 PPM Figure 50: Determination of the absolute configuration at phosphorus of the product from PI-PLC hydrolysis of DPPsI by 31P NMR (101.25 MHz, D 2O). (A) the product from PI-PLC hydrolysis; (B) left: the spectrum of 30b (endo). right: the spectrum taken after addition of 30b (endo) to the mixture (A); (C) left: the spectrum of 30a (exo), and right: spectrum taken after addition of 20a (exo) to the mixture (B)-right. O-DG PI-PLC DG S C ° ‘ HO > * 0 O H02i ^ r ° H Q% HO l-OH OH Rp-DPPsI L- transition state - 1 exo-l:2c-IPs Figure 51: The steric course of the reaction catalyzed by PI-PLC Experimental Section All chemicals were purchased either from chemical stores of the Ohio State University or from Aldrich. Other biochemicals and PI-PLC from Bacillus cereus were purchased from Sigma. Bacillus cereus PLC from Sigma contained three enzymes, the first one was PC-PLC, the second one was SPH-PLC and the third one is PI-PLC. PI-PLC from guinea pig uterus was obtained from Dr. C. Frank Bennett at Isis Pharamceutical Inc. Silica gel used in liquid chromatography (200-400 mesh) and thin layer chromatography (60 F254) was obtained from EM reagent (Merck). Most liquid chromatography was preformed under moderate pressure (about 20 psi). Medium pressure liquid chromatography was preformed on a Licorpre Silica 60 (Merck) column. !H NMR spectra were recorded on Bruker AM-250 or AM-500 spectrometers. 13C spectra were obtained at 62.89 MHz on a Bruker AM-250 spectrometer, and 31P NMR spectra were obtained at 101.25 MHz on a Bruker AM-250 spectrometer, with Waltz !H decoupling. 31P chemical shifts are referenced to external 85% H 3P04. The *H chemical shifts for D20 samples are referenced to external sodium 3-(trimethylsilyl)-l-propanesulfonate. Melting point was recorded on a Thomas Hoover capillary melting point apparatus without calibration. The MM-2 energy minimization was calculated by using a combination of MODEL and CHEMX, a combination of MacroModel and CHEMX, and Chem 3D Version 2.0. 140 The Hydrolysis of DPPsI Catalyzed by PI-PLC Use of 31P NMR to monitor the hydrolysis of DPPsI by PI-PLC from Bacillus cereus was preformed at 37°C on 7.5 mg of (Rp+Sp)-DPPsI and 6.8 mg of enzyme (from Sigma without purification or purified by fast protein liquid chromatography) in 50 % D20 contain 5 % Triton X-100,50 mM HEPES buffer, pH 7.2,2.5 mM Ca2+, and 0.25 mM EDTA. Use of 31P NMR to monitor the hydrolysis of DPPsI by guinea pig uterus PI-PLC was preformed at 37°C on 3 mg of (Rp+Sp)-DPPsI and 2.5 mg of enzyme (60 % pure, 210 n mol/mg/min) in 50 % D20 contain 5 % Triton X-100,50 mM HEPES buffer, pH 7.2,2.5 mM Ca2+, 0.25 mM EDTA, and 3 mM deoxycholate. Synthesis of myo-inositol 1:2cyclic phosphorothioate DL-3,6-di-0-benzyl-l,2:4,5-di-0-cyclohexylidene-myo-inositol (231 (Ozaki et al., 1986): To a solution of & (5 mmol in 300 ml of toluene), 2.5 equivalents of benzyl bromide and 3 equivalents of NaH were added and heated overnight under reflux. The filtrate was washed with H20 (2 x 200 ml), saturated NaHC0 3 (200 ml), and brine (200 ml), dried over CaCl2, and evaporated to dryness. Purification was performed by liquid chromatography on silica gel (hexane-ethyl acetate, 5 :1, v/v) to give 1.80 g (70.7% yield) of 22 (Rf= 0.50, hexane-ethyl acetate, 5 :1 , v/v). This compound was further characterized by NMR. The assignments of the !H NMR spectrum were according to the following criteria. The resonances at 4.82 and 4.70 ppm were assigned to be the AB system of one of the methylene protons of 3,6-di-O-benzyl groups. The other benzyl methylene protons were found at 4.78 ppm as an A 2 system. The resonance at 4.24 ppm was assigned to be 2-H on the basis of its most down field shift character of the 6 inositol ring protons and its cis-equatorial-axial couplings (4.5 Hz). The resonance at 3.25 ppm 141 was assigned to be 5-H on the basis of its most upfield shift character of the 6 inositol ring protons. Two doublet of doublets with a cis-equatorial-axial coupling at 3.96 and 3.66 ppm were assigned to be 1-H and 3-H. In myo-inositol, the chemical shift of 1-H and 3-H are the same, so are the 4-H and 6-H. In 22, C-l has an 0-C(CH 2-)(CH2-)(0-) substituent and C-3 has an O-benzyl substituent. According to Shoolery's mles (Williams and Fleming, 1980), the resonance of 1-H should be more down field shift than that of 3- H. Thus, the resonance at 3.96 and 3.66 ppm were assigned to be 1-H and 3-H, respectively. The other two doublets at 4.24 and 3.93 ppm were assigned to be 2-H and 4- H, respectively, based on Shoolery's rule and coupling constants. !H NMR (CDC13, 250 MHz) 8 7.36-7.06 (m, 10H, phenyl of 3,6-di-O-benzyl), 4.82,4.70 (AB, J = 14 Hz, 2H) and 4.78 (A 2,2H, CH2 of 3,6-di-O-benzyl), 4.24 (t, Jh-c(2)-c(3)-h = ^h-C(2)-c(1)-h = 4*5 Hz, 1H, 2-H), 3.96 (dd, Jh-C(6)-C(1)-h = 6.6 Hz, Jh-C(1)-C(2)-h = 4.7 Hz, 1H, 1-H ), 3.93 (l» j h-C(3)-C(4)-h = j h-C(4)-C(5)-h = 9.6 Hz, 1H, 4-H), 3.66 (dd, % c(2)-C(3)-h = 4-1 Hz, JH_ C(4)-C(3)-H = 10.1 Hz, 1H, 3-H), 3.57 (dd, Jh-c(1)-c(6)-h = 6.6 Hz, Jh-c(5)-C(6)-h = 10-6 Hz, 1H, 6-H), 3.25 (dd, Jh-C(5)-C(4)-h = 9.4 Hz, Jh-c(5)-C(6)-h = 10.5 Hz, 1H, 5-H), 1.58- I.35 (m, 20H, CH2 of 1,2:4,5-di-O-cyclohexyhdene); 13C NMR (CDC13, 62.896 MHz) 8 138.37,138.15, 128.31,128.14, 127.98, 127.69 and 127.40 (phenyl of 3,6-di-O- benzyl), 112.66 (tertiary C-l of 4,5-O-cyclohexylidene) (This was assigned by reference to 24, containing only a 1,2-O-cyclohexylidene group.), 110.39 (C-l of 1,2-0- cyclohexylidene), 80.63 and 79.97 (CH 2 of 3,6-di-O-benzyl), 78.72 , 76.79 , 76.22, 74.64,71.93 and 71.59 (6 inositol carbons), 37.48,36.48 and 35.28 (C-2 and C -6 of 1,2: 4,5-di-O-cyclohexylidene), 25.110,25.02,24.01, 23.93, 23.86 and 23.55 (C-3, C-4 and C-5 of 1,2:4,5-di-O-cyclohexylidene). 142 DL-3,6-di-0-benzyl 1,2-0-cyclohexylidene-wyo-inositol (24) (Ozaki et al., 1986): To a solution of 22 (2 mmol in 50 ml of dichloromethane), 1 equivalent of ethylene glycol and 3 % weight of anhydrous TsOH were added. Then, the reaction mixture was stirred at room temperature for 1/2 hour. The reaction was terminated by adding 2 ml of pyridine. The reaction mixture was evaporated to dryness. Purification was performed by liquid chromatography on silica gel (chlorofoim-acetone, 2 :1 , v/v) to give 650 mg (73 % yield) of 24 (Rf= 0.60, chloroform-acetone, 2 :1 , v/v, mp 145-147°C). This compound was further characterized by NMR. The assignments of the *H NMR spectrum were according to the following criteria. The most downfield shift resonance (4.33 ppm) of 6 inositol ring protons was assigned to be 2-H based on the cis-equatorial-axial couplings and based on the references to myo-inositol and 22- The most upfield shift resonance (3.37 ppm) of those 6 protons was assigned to be 5-H by reference to 22- The doublet of triplets at 3.94 ppm was assigned to be 4-H based on the extra splittings from 4-OH. According to Shoolery's rules, 1-H should have a lower field shift than 3-H and 6-H. unlike 6-H, 3-H should have a small cis-equatorial-axial coupling constant (4.0 Hz) and a trans-axial-axial coupling constant (9.5 Hz). Thus, the resonances at 4.09, 3.55 and 3.53 ppm were assigned to be 1-H, 6-H and 3-H, respectively. *H NMR (CDC13, 250 MHz): 8 7.43- 7.25 (m, 10H, phenyl of 3,6-di-O-benzyl), 4.97,4.68 (AB, J = 11.6 Hz, 2H), and 4.78, 4.76 (AB, J = 12.2 Hz, 2H) (CH2 of 3,6-di-O-benzyl), 4.33 (dd, JH-c(2)-C(3)-h = 4-l Hz, j h -C (2)-C(1) - h = 5.1 Hz, 1H, 2-H), 4.09 (dd, Jh-C(6)-c(1)-h = 6-9 Hz, Jh-c(1)-C(2)-h = 5-l Hz, 1H, 1-H ), 3.94 (dt, J h - c (3)-C(4)-h = Jh-C(4)-C(5)-h = 9.4 Hz, J h - c (4) - o - h = 1-6 Hz, 1H, 4-H), 3.55 (dd, J h -C (1)-C(6) - h = 2.9 Hz, Jh-c(5)-C(6)-h = 9.6 Hz, 1H, 6-H), 3.53 (dd, JH_ C(2)-C(3)-H = 4-0 Hz, Jh-c(4)-C(3)-h = 9.5 Hz, 1H, 3-H), 3.37 (dt, Jh-c(5)-C(4)-h = J H -c (5 )- 143 C(6)-H = 9.4 Hz, JH-c (5 )-0 -h = 1-8 Hz, 1H, 5-H), 1.77-1.35 (m, 10H, CH 2 of 1,2-0- cyclohexylidene); 13C NMR (CDC13, 62.896 MHz) 6 138.26,137.92,128.49,128.39, 128.03,127.99 and 127.75 (phenyl of 3,6-di-O-benzyl), 110.60 (tertiary C-l of 1,2-0- cyclohexylidene), 82.48 and 78.90 (CH 2 of 3,6-di-O-benzyl), 77.24 ,73.43, 72.97 , 72.31 and 71.46 (6 inositol carbons), 37.77 and 35.15 (C-2 and C -6 of 1,2-0- cyclohexylidene), 24.98,23.94 and 23.67 (C-3, C-4 and C-5 of 1,2-O-cyclohexylidene). DL-3,4,5,6-tetra-0-benzyl 1,2-O-cyclohexylidene-wyo-inositol (25): To a solution of 24 (0.75 mmol in 100 ml of toluene), 6 equivalents of benzyl bromide and 6 equivalents of NaH were added and heated overnight under reflux. The filtrate was washed with H20 (3x100 ml), saturated NaHC0 3 (100 ml), and brine (100 ml), dried over CaCl2, and evaporated to diyness. Purification was performed by liquid chromatography on silica gel (hexane-ethyl acetate, 5 : 1, v/v) to give 175 mg of 2 2 (Rf = 0.36, hexane- ethyl acetate, 5 :1, v/v; 71 % yield). This compound was further characterized by NMR. The assignments to specific resonances and coupling constants were made by reference to 24- *HNMR (CDC13, 250 MHz) 8 7.46-7.15 (m,20H, phenyl of 3,4,5, 6-tetra-O- benzyl), 4.98-4.77 (m, 8 H, CH2 of 3,4,5,6-tetra-O-benzyl), 4.34 (dd, J h-C(2)-C(3)-h = 3.8 Hz, J h - c (2)-C(1) - h = 5.5 Hz, 1H, 2-H), 4.16 (dd, Jh-C(6)-C(1)-h = 6.9 Hz, Jh-c(1)-C(2)-h = 5.7 Hz, 1H, 1-H ), 3.98 (t, Jh-c(3)-c(4)-h = Jh-C(4)-c(5)-h= 8.4 Hz, 1H, 4-H), 3.89 (dd, J H -C (i)-C (6)-H = 7.0 Hz, J h - c (5)-c ( 6)- h = 9-6 Hz, 1H, 6-H), 3.75 (dd, % C(2)-C(3)-H = 3.8 Hz, J h - c (4)-C(3) -h = 8.4 Hz, 1H, 3-H), 3.47 (dd, J h - c (5)-C(4)-h = 8-3 Hz, J h - c (5) - c ( 6)-h = 9.5 Hz, 1H, 5-H), 1.85-1.27 (m, 10H, CH 2 of 1,2-O-cyclohexylidene). and 13C NMR (CDC13, 62.896 MHz) 8 138.64, 138.58, 138.27, 130.71, 130.10, 129.66, 128.30, 128.23, 128.19, 127.92, 127.70, 127.55, 127.47, 127.42 and 126.29 (phenyl of 3,4,5, 6- 144 tetra-O-benzyl), 110.39 (tertiary C-l of 1,2-O-cyclohexylidene), 82.85,82.10,80.89 and 78.74 (CH 2 of 3,4,5, 6-tetra-O-benzyl), 77.24 , 75.17,75.01, 73.98 , 73.90 and 73.06 (6 inositol carbons), 37.35 and 35.00 (C-2 and C -6 of 1,2-O-cyclohexylidene), 25.03,23.89 and 23.63 (C-3, C-4 and C-5 of 1,2-O-cyclohexylidene). DL-3,4,5,6 -tetra-O-benzyl myo-inositol (26): To a solution of 25 (0.49 mmol in 20 ml of tetrahydrofuran, 10 ml of 6 N HC1 was added. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was evaporated to dryness. Purification was performed by liquid chromatography on silica gel (chloroform-acetone, 10 : 1, v/v) to give 194 mg (74% yield) of 24 (Rf= 0.45, chloroform-acetone, 10 :1, v/v, mp 111-113°C). This compound was further characterized by NMR. The assignments to specific resonances and coupling constants were made by reference to 25- *H NMR (CDC13, 250 MHz) 8 7.33-7.26 (m, 20H, phenyl of 3,4,5, 6-tetra-O-benzyl), 4.98-4.72 (m, 8 H, CH2 of 3,4,5, 6-tetra-O-benzyl), 4.21 (t, Jh -c (2)-C(3)-h = ^H-C(2)-C(i)-H = 2.6 Hz, 1H, 2-H), 3.98 (t, J = 9.5 Hz, 1H), 3.84 (t, J = 9.5 Hz, 1H) and 3.52-3.45 (m, 3H)[1- H, 3-H, 4-H, 5-H and 6-H], 2.53 (s, 1H) and 2.44 (d, J = 4.3 Hz, 1H) [1-OH and 2- OH]. 13C NMR (CDCI3, 62.896 MHz): 8 138.62, 138.49,137.78,128.57, 128.56, 128.38,127.94,127.88,127.81 and 127.61 (phenyl of 3,4,5, 6-tetra-O-benzyl), 83.21, 81.65,81.30 and 80.01 (CH 2 of 3,4,5, 6-tetra-O-benzyl), 75.93, 75.69, 75.59 , 72.75, 71.75 and 69.18 (6 inositol carbons). DL-3,4,5,6-tetra-0-benzyl-l:2cyclic-0-endo-methylphosphorothioate-myo-inositol (22a) and DL-3,4,5,6-tetra-0-benzyl-l :2cyclic-0-exo-methylphosphorothioate-myo- inositol (29b): Compound 25 (0.156 mmol) was dried three times by rotary evaporation 145 with 2 ml of dry toluene. To this dry material, 5 ml of dichloromethane (over Na 2S0 4) and an excess of triethylamine (over NaH) were added through vacuum transfer. While the mixture was still cold, chloro(N,N-diisopropylamino)methoxyphosphine (1.25 equivalents, 0.195 mmol) was added to the flask through a dry syringe. The reaction was completed about 30 minutes later on the basis of TLC, and the solvent and excess triethylamine were removed under vacuum. The reaction mixture was further dried in vacuo (0.001 mm Hg) for 1/2 hour. Thoroughly dried tetrazole (4 equivalents, 0.624 mmol, Aldrich gold label) was dissolved in tetrahydrofuran-acetonitrile (1:1, v/v, 20 ml, over NaH) through vacuum transfer. After warming to 40°C to make sure all compounds were dissolved, this solution was added to the above reaction mixture through a syringe under the argon atmosphere. The reaction mixture was stirred at room temperature for 4 hours and then evaporated to dryness. To this mixture, 50 ml of dry toluene (dried over NaH) and an excess of S 8 (powder, sublimed) were added at room temperature. The suspension was stirred at room temperature for 6 days. The reaction mixture was then washed with 25 ml of saturated NaHC03.and evaporated to dryness. The crude products were purified by MPLC on silica gel in hexane-ethyl acetate (5:1, v/v) to give an oil 22a (5 mg, 5 % yield based on 26) Rf = 0.31 (TLC, hexane-ethyl acetate, 5:1, v/v) and an oil 29b (42 mg, 60 % yield based on 26) Rf = 0.26 (TLC, hexane-ethyl acetate, 5:1, v/v). These two isomers were further characterized by *H NMR, ^ ^ H decoupling experiments, and 31P decoupling !H NMR, 2D J-resolved *H NMR, 2D-NOE *H NMR, and 31P NMR. 22a (exo): The assignments to 6 inositol ring protons were made by reference to l:2c-IP (Cerdan et al., 1986), by their splitting patterns of the *H NMR spectrum (Figure 52), by the ^ ^ H decoupling experiments, by the 31P decoupling !H NMR spectrum (Figure 53), by the 2D J-resolved *H NMR spectrum (Figure 54), and by the NOESY spectrum (Figure 48). The vicinal coupling constants, Jh -C(3)-C(4)-h > % -C (5> c (6> h Jh -c (6)-c (1)-h were assigned by the *H NMR spectrum and were confirmed by the 2D J-resolved *H NMR spectra (Figure 54). The P-O-methyl protons, 1-H, 2-H and 3-H resonances, Jh -c (1)-C(2)-h > j h -c (2)-c (3)-h > Jh -C (1)-0-p a n d Jp-o-c-H(methyi)were assigned by the !H NMR spectra with (Figure 53) and without (Figure 52) 31P decoupling and were confirmed by the 2D J-resolved *H NMR spectrum (Figure 54). Jh -c (2)-o -p was assigned by 2D J-resolved *H NMR spectmm (Figure 54). The methylene protons of 3,4,5, 6-tetra- O-benzyl groups were assigned from the NOESY spectrum (Figure 48) and from the 2D J- resolved XH NMR spectmm (Figure 54). 31P NMR (CDC13, 101.256 MHz): 8 84.41. XH NMR (CDC13, 500 MHz): 8 7.35-7.24 (m, 20H, phenyl of 3,4,5, 6-tetra-O-benzyl), 4.87 (ddd, Jh-C(2)-C(1)-h = 6.5 Hz, Jh-c(2)-C(3)-h = 4.0 Hz, 3Jp.o-c(2)-H = 3.7 Hz, 1H, 2- H), 4.85 and 4.75 (AB, J = 11.2 Hz, 2H, CH 2 of 6-O-benzyl), 4.76 and 4.70 (AB, J = 11.0 Hz, 2H, CH2 of 5-O-benzyl), 4.77 and 4.69 (AB, J =11.0 Hz, 2H, CH 2 of 3-O- benzyl), 4.68 and 4.66 (AB, J = 11.0 Hz, 2H, CH 2 of 4-O-benzyl), 4.59 (ddd, JH-c(i)- C(2)-H = 6-3 Hz, Jh-c(1)-C(6)-h = 8.1 Hz, 3Jp.o-c(i)-H = 18.4 Hz, 1H, 1-H), 4.20 (dd, JH_ C(6)-C(5)-H = 9.7 Hz, Jh-c(6)-c(1)-h = 7.9 Hz, 1H, 6-H), 3.86 (dd, Jh-c(4)-c(3)-h = 7.3 Hz, Jh-C(4)-C(5)-h = 7.1 Hz, 1H, 4-H), 3.76 (d, 3JP.aC-H = 15.0 Hz, 3H, CH3OP), 3.73 (dd, j h-C(3)-c(4)-h = 7.5 Hz, Jh-c(3)-C(2)-h = 3.9 Hz, 1H, 3-H), 3.45 (dd, Jh-c(5)-C(6)-h - 9.7 Hz, J h - c (5)-C(4) - h = 7-0 Hz, 1H, 5-H). 29b (endo): The inositol ring protons of 29b were assigned by comparing with the *H NMR spectra of 29a. by its splitting patterns of *H NMR spectmm (Figure 55), by the ^ ^ H decoupling experiments, by 31P decoupling !H NMR spectmm (Figure 56), by the 2D J-resolved !H NMR spectmm (Figure 57), and by the NOESY spectmm (Figure 49). Jh-c(3)-c(4)-h and Jh-C(2)-C(3)-h were assigned by the _ i - U ^ T “ OCH2Ph PhCH20 <^ ^ — OCH2Ph 4-H 1-H 6H 5-H 2-H 3-H PPM 3.9 3.9 3.7 3.3 3.4 3.3 Figure 52: The !H NMR spectrum (250 MHz, CDC 13) of 29a fexrri -j a \ v O C H 3 . y , PhCH20 2 PhCH20 4 ^ — OCH2Ph 5 1 H 2 H , - —.—, r —I------I— —I— «.e 4.7 4.5 4.5 4.4 4.3 4,3 4.1 PPH 4.0 3.9 3.6 3.7 3.C 3.5 3.4 3.3 Figure 53: The 31P decoupling *H NMR spectrum (250 MHz, CDCI 3) of 22*L(exo) ^ / 0CH: }J — ■ 2-H-P 00- FH 3-H * VA 4*° 3<* 1.7 1,1 l.S 3.4 Figure 54: The 2D J-resolved *H NMR spectrum (500 MHz, CDC13) of 22a (exo). 2-H [4-H 3-H 6-H 1-H «•« «•« »•» >:• 3 .1 3 * i j Figure 55: The *H NMR spectrum (250 MHz, CDC13) of 22k_(endo) h3co. a Y ° f6 i b OCH2Ph PhCH,0'4^— OCH2Ph 5 ~ 1 ~ * » I I I I 1_ _ _ J 'I *" ™ |" '■■■ | I | 1 | , 1 1 | <•» 4.7 4.« 4.9 4 ,4 4 .) 4.1 4.1 4 .0 3.0 3.1 3.7 3.0 3.9 3 .4 PPM Figure 56: The 31P decoupling *H NMR spectrum (250 MHz, CDC13) of29hfendol h3co, 2 L l / ^ 6 b % ^ - r— OCH2Ph PI'C% C H p^S^ — OCH2Ph h 5 M . v A j j l A -10 >H 4-H 2-H HERTZ ■nr~ T ~T“ V T T i T “ T“ I T I 5.0 4.8 4 .0 ' 4 .2 4.0 3.8 3 .8 PPM Ul Figure 57: The 2D J-resolved *H NMR spectrum (500 MHz, CDC13) of 22k (endo). 153 !H NMR spectmm and were confirmed by the 2D J-resolved *H NMR spectra (Figure 57). The P-O-methyl protons, 1-H, 2-H, 3-H and 4-H resonances, Jh .c(5)-C(6)-h> j h-C(1)-C(6)-h» Jh -C (2)-C(3)-h» %-C(3)-C(4)-H Jh -C (4)-C(5)-h couplings were assigned from the *H NMR spectra with (Figure 56) and without (Figure 55) 31P decoupling and were confirmed by the 2D J-resolved ^H NMR spectmm (Figure 57). The Jh -c (1)-c (6)-h » ^h -C(1)-C(2)-h > Jh -c (1)- 0.p and Jh -c (2)-o -p couplings were assigned from the 2D J-resolved *H NMR spectmm (Figure 57). The methylene protons of 3,4,5,6-tetra-O-benzyl groups were assigned from the NOESY spectrum (Figure 49) and the 2D J-resolved *H NMR spectmm (Figure 57). 31P NMR (CDC13, 101.256 MHz): 8 82.65. XH NMR (CDC13, 500 MHz): 8 7.44-7.22 (m, 20H, phenyl of 3,4,5,6-tetra-O-benzyl), 4.92 and 4.82 (AB, J = 11.1 Hz, 2H, CH2 of 6-O-benzyl), 4.83 and 4.73 (AB, J = 12.2 Hz, 2H, CH2 of 3-O-benzyl), 4.79 and 4.71 (AB, J = 11.1 Hz, 2H, CH2 of 5-O-benzyl), 4.73 (ddd, Jh -c (2)-c (1)-h = Hz, Jh_c(2)- C(3)-H = 3*4 Hz, 3Jp-oc(2)-H = Hz> 2-H), 4.60 (ddd, Jh -c (1)-C (2)-h = 6.6 Hz, JH_ C(i)-C(6)-H = 8.2 Hz, 3Jp.o-c(i)-H = 9-7 Hz, 1H, 1-H), 4.56 (A2., 2H, CH2 of 4-O-benzyl), 4.35 (dd, J H-C(6)-C(5)-H = 10*0 Hz, Jh -c (6)-C (1)-h = 7.8 Hz, 1H, 6-H), 3.87 (dd, Jh -c (4)- C(5)-H = 6.3 Hz, Jh -c (4)-C (3)-h = 5.3 Hz, 1H, 4-H), 3.85 (d, 3Jp-o-c-H= 14.6 Hz, 3H, CH3OP), 3.76 (ddJTH-C(2)-C(3)-H = 3.2 Hz, Jh .C(4)-C(3)-h = 5.7 Hz, 1H, 3-H), 3.46 (dd, JH-C(5)-C(6)-H = 10-2 JH-C(5)-C(4)-H = 6 .6 Hz, 1H, 5-H). DL-exo-myo-inositol l:2cyclic phosphorothioate (DL-exo-l:2c-IPs, 30al and DL- endo-wyo-inositol l:2cyclic phosphorothioate (DL-endo-l:2c-IPs, 30al: 30a: To a dry tetrahydrofuran (2 ml, over Na) solution of 22a (14 mg, 0.022 mmol), Li (12 mg, 60 equivalents) was added at -78°C (dry ice-tetrahydrofuran bath). Dry NH3 (over Na) was distilled into the above reaction mixture. The reaction mixture was stirred at -78°C for 5 minutes. To this reaction mixture, 3 mg of NH4C1 was added at-78°C. The reaction mixture was stirred from -78°C to room temperature and dissolved in 2 ml of D 20. This product (60 % yield based on 31P NMR) was not purified and directly characterized by 31P 154 NMR. 31P NMR (D20 , 101.256 MHz): 8 69.85 ppm. 30b was prepared by the same procedure in 60 % yield. 31P NMR (D20 , 101.256 MHz): 8 69.00 ppm. Discussion So far, there is no reliable method to prepare 1,3,2-dioxaphospholanes. Although conventional phosphorus reagents, such as PC13, POCl3, and PSC1 3 have a high reactivity toward the first hydroxy group of a diol system, they suffer a low selectivity and reactivity toward the second hydroxy group (intermolecular phosphorylation is the major side reaction). For the first time, a phosphorylation agent, chloro(N,N- diisopropylamino)methoxyphosphine was utilized for the synthesis of 1,3,2- dioxaphospholanes. Thus, phosphorylation of the first hydroxyl group of 26 produced in 27a and 27b. Because the nucleophilic reactivity of 2-OH was higher than that of 1-OH (Watanabe et al., 1988), 2212 may be the major product in the first step. The second step, intramolecular cyclization at phosphorus occurred when the N,N-diisopropylamino group was protonated to become a leaving group and the concentration of the substrate was very low (avoiding the intermolecular condensation). Two resulting phosphite products, 22a and 2212 were subjected to oxidation by sulfur to give the expected cyclic phosphorothioate, 22a and 29b with retention in the configuration of phosphorus (McEven, 1965 and Bentrude et al., 1976). Subsequent deblocking using lithium in liquid ammonia removes all protective groups and gives 211a and 30b. It is believed that the benzyl groups are removed by a radical reductive cleavage and the methyl group is removed by a SN 2 nucleophilic attack of ammonia. Both reactions would not effect the stereochemistry at phosphorus (see for example, Cullis and Lowe, 1981). This procedure must be done within 5 minutes. If the 155 reaction time is longer than 5 minutes, the ring opening and desulfurization products are dominated. As shown in Figures 46 and 47, the most stable conformation of exo form in solution is 22a and that of endo form is 22b. It has been shown that the Jh-c( 1)-o-p °f 1: 2 c-EP was 19.5 Hz (Cerdan et al., 1985). According to equation 1, the dihedral angle of H-C(l)-0-P in l:2c-IP was 154°. Therefore, the most stable conformation of l:2c-IP is similar to that of 22a (^H-CM-O-P = 150°). If we assume that the conformation of 1,3,2- dioxaphospholane rings in 22a is retained after deblocking, it is reasonable to believe that the exo conformation of 211a was similar to that of l:2c-IP. We still do not know the conformation of 2012. Therefore, the conformation of 22b can resemble either that of 22b or that of 29a. PI-PLC specifically catalyzed the hydrolysis of Rp-DPPsI. Two possibilities can be made according to this stereospecificity. One possible reason is that the enzyme requires metal in its active site as from mammalian source. In this case, replacement of an O by S at the phosphate center will change the binding character. Therefore, the stereospecificity of this enzyme is because of different binding characters between Rp and Sp isomers. The other possibility is that the enzyme does not require any metal for the catalysis (as from bacterial source) but the catalysis requires the binding between the Rp-phosphorothioate and a positive charge site of the enzyme (including the enzyme-water-Rp-phosphorothioate binding) or the enzyme holes can only be fitted by the Rp-isomer. In order to explain the stereospecificity of PI-PLC, further kinetic studies on different metal toward Rp- and Sp- isomer of DPPsI.need to be done. Figures 58 and 59 summarizes the six possible mechanisms for the formation of IP and l:2c-IP from DPPI, along with the predicted stereochemical outcomes (assuming every single substitution at phosphorus is inversion, as has been the case in all known enzymatic reactions). Mechanisms A and B are parallel reactions (at the same active site), and are (A) Parallel reactions, Direct displacement IP (Inversion) n2o y y o *attack ^ 2-OH attackMkVUVil OH HO ^ l:2c-IP (Inversion) HO OH (B) Parallel reactions, Covalent E-S intermediate h ,h 0 O .DG v Enzyme f c - ) OH / attack O .DG P l O IP (Retention) h 2o y >q attack .2-OH attack ^ l:2c-IP (Retention) Figure 58: Possible mechanisms of PI-PLC, mechanisms A and B (C) Sequential reactions, l:2c-IP to IP, Direct displacement 2-OH H20 EDPPI E l:2 c -IP — q -*- EIP E + IP (Retention) attack 4 attack E+ l:2c-IP (Inversion) (D) Sequential reactions, l:2c-IP to IP, Covalent E-S intermediate Enzyme 2-OH E • DPPI — 0 -*- E-X-P-I —q E- l:2c-IP E + IP (Inversion) attack attack ^ E + l:2c-IP (Retention) (E) Sequential reactions, IP to l:2c-IP, Direct displacement h 20 E ‘ DPPI — q E‘ IP E + IP (Inversion) attack jp E + l:2c-IP ------►- E + l:2c-IP (Retention + solvent incorportion) (F) Sequential reactions, IP to l:2c-IP, Covalent E-S intermediate Enzyme H20 E ' DPPI — 0 -**- E-X-P-I - q »» E-IP ► E+ IP (Retention) attack attack y ^-OH attack E'l:2c-IP ►- e + l:2c-IP (Inversion + solvent incorportion) Figure 59: Possible mechanisms of PI-PLC, mechanisms D, E, and F favored by some in the field (Dawson et al., 1971; Michell, 1975; Quinn, 1978; Majerus et al., 1986). Mechanism C mimics the mechanism of ribonuclease A, but ribonuclease A does not release the cyclic intermediate. Mechanism D differs from C in that a covalent enzyme-phosphoinositol intermediate has been formed. In mechanisms C and D the conversion of l:2c-IP to IP should be enzyme-mediated, since when l:2c-IP is subjected to chemical hydrolysis, both 1-IP and 2-IP should be (and have been) obtained. It has been shown that both BP and l:2c-IP are released from the substrate simultaneously and there is no subsequent interconversion of l:2c-IP into IP or vice versa (Dawson et al., 1971, Allan and Michell, 1974; Lapetina and Michell, 1973; Michell, 1975; Quinn, 1978; Majerus et al., 1986). Therefore, these two mechanisms (C and D) seem less likely. Mechanisms E and F differ from C and D, respectively, in the order of formation of IP and 1:2c-EP. These two mechanisms (E and F) are unlikely, and can be ruled out because no lsO was incorporated into l:2c-IP when the reaction was carried out in 180 water (Wilson et al., 1984 and 1985). The stereochemical course of the consequence of PI to l:2c-IP is inversion in the configuration of phosphorus, and that is the same as the predicted stereochemical outcome of mechanisms A, C, and F. Therefore, mechanism A of Figure 58 may be the mechanism for PI-PLC catalysis. PI-PLC from Bacillus cereus gave only cyclic products from both PI (Michell, 1975) and DPPsI (this dissertation). However, PI-PLC from guinea pig uterus gave both cyclic and noncyclic products from PI (BP: l:2c-BP, 1:1) and DPPsI (IPs: l:2c-IPs, 1:2). The steric course of the consequence of PI to l:2c-BP is inversion in the configuration of phosphorus; however, the steric course of the consequence of PI to IP is still unknown. To determine the steric course of the consequence of PI to IP, a new method shown in Figure 12 of Chapter I need to be investigated. R eferences Allan, D. and Michell, R. H. (1974) Biochem J. 142,591-597. Bentrude, W. G. and Tan, H.-W. (1976) J. Am. Chem. Soc. 98, 1850-1859. Bentrude, W. G., Hargis, J. H., Nelson, A. J., Tae, B. M., Rusek, P. E., Tan, H.-W., & Wielesek, R. A. (1976) J. Am. Chem. Soc. 98, 5348-5357. Bentrude, W. G. and Setzer, W. N. in “Phosphorus-31 NMR Spectroscopy in Stereochemical Analysis”, Verkade, J. G. and Quin, L. D. eds., pp 365-389, VCH publisher, Inc. Press, USA. Bruzik, K. S. (1988) J. Chem. Soc. Perkin Trans. 1423-431. Cerdan, S., Hansen, C. A., Johanson, R., Inubushi, T., & Williamson, J. (1986) J. Biol. Chem. 261, 14676-14680. Cullis, P. M. and Lowe, G. (1981) J. Chem. Soc. Perkin Trans. I 2317-2321. Dawson, R. M., Freinkel, N., Jugalwala, F. B., & Clarke, N. (1971) Biochem. J. 122, 605-607. Denney, D. Z., Chen, G. Y., & Denney, D. B. (1969) J. Am. Chem. Soc. 91, 6838- 6841. Haake, P., MacNeal, J. P., & Goldsmith, E. J.(1968) J. Am. Chem. Soc. 90, 715-720. Lapetina, E. G. and Michell, R. H. (1973) Biochem. J. 131,433-442. Majerus, P. W., Connolly, T. M., Deckmyn, H., Ross, T. S., Brass, T. E., Ishii, H., Bansal, V. S., & Wilson, D. B. (1986) Science 234,1519-1526. Marynoff, B. E., Hutchins, R. O., & Marynoff, C. A. (1979) Topics Stereochem. 11, 187-326. 159 160 McEven, W. C. (1965) Topics Phosphorus Chem. 2,1-41. Michell, R. H.(1975) Biochim. Biophys. Acta 415, 81-147. Mikolajczyk, M., Witczak, M., Wieczorek, M., Bokij, N. G., & Struchkov, Y. T. (1976) J. Chem. Soc. Perkin Trans. 1 371-377. Mikolajczyk, M. and Witczak, M. (1977) J. Chem. Soc. Perkin Trans. 12213-2222. Ozaki, S., Watanabe, Y., Ogasawara, T., Konodo, Y., Shiotani, N., Nishii, H., & Matsuki, T. (1986) Tetrahedron Lett. 27,3157-3160. Potter, B. V. L. and Lowe, G. (1981) Biochem. J. 199, 693-698. Quinn, P. J. (1978) in “Cyclitols and phosphoinositides”, Wells, W. W. and Eisengerg, F., eds. Academic Press, USA. Schultz, C.,Matschies, T., & Jastorff, B. (1988) Tetrahedron Lett. 29,3919-3920 Watanabe, Y., Ogasawara, T., Nakahira, H., Matsuki, T., & Ozaki, S. (1988) Tetrahedron Lett. 29,5259-5262. Williams, D.H. and Fleming, I. (1980) in “Spectroscopic Methods in Organic Chemistry”, second edition, pp 83-146, William Clowes & Sons, Limited, London, The Great Britain. Wilson, D. B., Bross, T. E., Hofmann, S. L., & Majerus, P. W. (1984) J. Biol. Chem. 259, 11718-11724. Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A., & Majerus, P. W. (1985) Proc. Natl. Acad. Sci. USA 82,4013-4017. CHAPTER VI Synthesis of Some Derivatives of myo-lnositol introduction Selectively protecting some hydroxyl groups in myoinositol is an important subject in carbohydrate chemistry. The natural occurring inositol lipids, such as PI, PIP and PIP 2 and inositol phosphates such as IP, 1,4-EP 2, 1 ,4 ,5 -IP3, l:2c-IP, l:2c,4-IP2 and 1:2c,4,5- IP3 are the derivatives of myoinositol at the specific positions. In this chapter, many precursors for the total synthesis of the natural occurring inositol compounds and their analogues are synthesized. Results and Discussion As mentioned in Chapter IV, two compounds have been resolved by their champhony derivatives with their X-ray crystal structures. In chapter IV, the resolution of compound 1 1 was preformed by following the procedures of Billington's (Billington et al., 1987). In this chapter, compound 2 2 was also resolved by champanic acid chloride (Vacca et al., 1987). The synthesis of 42 was summarized in Scheme 6 . Partial benzylation of 2 under the condition of two-phase transfer catalysis yields of monobenzyl ethers, 22 (Garegg et al., 1984) in 43% yield. Esterification of the free hydroxyl group of 22 with (S)-(-)- camphanic acid chloride yielded a mixture of two diastereomers that were separated by chromatography to give 39d (41% yield, mp 152-153 °C) and 221.(40% yield, mp 120- 161 162 Schem e 6: Synthesis of D-2,3:4,5-di-0-cyclohexylidene-6-0- p-trimethylsilylethoxymethyl-myo-Inositol OH ,OCH2Ph PhCH 2 Br, O k -OCH2Ph Q l OC1 OH OH H & r " ' o s 0 + 2 1 11BU4NHSO4, (43%) CH 2 CI2 , reflux, 'a 20h, 92% (-)-camphanic n r u Dh acid chloride, 0CH2Ph DMAP, + 3 M Et3 N , CH 2 C12, PhCH ( j rt, 24h, 81% 21 391 H2 (1 atm), SEMC1, 10%Pd-C, O iPr2 NEt, ► EtOAc, rt, CH 2 C12, 19h, 89% 40°C, 18h, 78% 3 9 d ^ v / ^ S i M e a a k v 0H A v / ^ S i M e , UAIH 4 , Et2 0 , 0 C to rt, 2 2 h, 5 : 92% 41 123 °C) (Scheme 6, Vacca et al., 1987). Hydrogenolysis of 39d give 4ft in 89% yield. Protection 4ft or 2ft with 2,3-dihydrofuran was not successful. 42 was obtained from 41 by the reductive cleavage with LiAlH 4 in 92 % yield. The synthesis of DPPI and DPPsI from 42 by using chloro(N, N-diisopropylamino)methoxyphosphine or chlorodimorpholinophosphine as a phosphorylating agent was not successful. We tried in vain to debenzylate the phosphite or phosphorothioate product. Thus, 2Z was phosphorylated with chloro(N, N-diisopropylamino)methoxyphosphine and then condensed with 1,2-diplamitoyl-sn-glycerol to give a phosphite adduct. However, the hydrogenolysis of this phosphite was failed. Oxidation of this phosphite by S 8 gave the phosphorothioate product. The debenzylation of this phosphorothioate compound was also failed (also see for examples: Loffredo, 1988 and Schultz et al., 1988). This may be because the lone pare electrons of phosphite or the sulfur atom of phosphorothioate poisoned the catalytic hydrogenolysis. Compounds 44 and 4ft were synthesized according to Scheme 7. Protection lid with 2-trimethylsilylethoxymethyl chloride (SEMC1) gave 42 in 90% yield (Scheme 7). Hydrogenolysis of 42 gave 44 in low yield (18%). This may be because the SEM ether is unstable under the condition of hydrogenolysis. Compound 4ft was synthesized according to Vacca et al.'s method (1987). In order to synthesize the analogues of wyo-inositol cyclicphosphates such as /Myo inositol l:2cyclic phosphorothioate (l:2c-IPs) and /Myo-inositol l:2cyclic,4,5- trisphosphorothioate (1 :2 c,4,5-IP s3), the compounds with two free hydroxy groups at 1- and 2-position need to be prepared. In Chapter V, l:2c-IPs has been synthesized from 3,4,5,6-tetra-0-benzyl-/nyo-inositol via a novel intramolecular cyclization process. In this chapter, more compounds which can serve as the starting materials for the synthesis of inositol cyclicphosphates have also been synthesized (Schemes 7 ,8 and 9). Treatment of 121 with one equivalent of ethylene glycol gave 4Z in 43% yield. Esterification of two free 164 Scheme 7: Synthesis of D-2,3:5,6-di-0-cyclohexyHdene-4-0-p- trimethylsilylethoxymethyl-myo-inositol and D-4-O-benzyl- 1,2-0 -cyclohexylidene-/nyo-inositol SEMC1, iPr2 NEt, CH 2 C12, HO 4 40°C, 20h, JLM 98% SiMe* .OH H 2 (latm ), 10% Pd-C, EtOAc, rt, 72h, 18% SiMeq c a t HC1 gas, MeOH-EtOAc (l:5,v/v), rt, 2h o ? » H PhCH20 4 ^ OH PhCH20 LiOH, THF- H20(2:1,v/v), rt, l / 2 h PhCH 165 Schem e 8 : Synthesis of D-4,5-dI-0-acetyl-3-0-benzyl-6-(-)- camphanoyl-myoinositol and D-3-0-benzyl-1,2-0- cyclohexylidene-myo-inositol leq. ethylene P glycol, cat PhCH20 TsOH, PhCH20, CH2C12, rt, lh , 43% A c20 , py, THF-6NHC1 DMAP, (2:1, v/v), r t r t 25h l/2h * ------ 74% 89% IS HO 1 OH PhCH20 OAc LiOH, THF- H20(2:1,v/v), rt, l/2h PhCH20 1 2 Scheme 9: Synthesis of DL-3,4,5,6-tetra-0-butyryl-1,2-0- cyclohexylidene-myo-inositol butyric anhydride, HO OH DMAP, CH2C12, rt, 68 h, n a " 86 % 1 eq. ethylene ( y glycol, biityric 5.7 % weight of I annydride, TS°H' » ^ 0s^f0Y^ >» S HiC5h2; 0 » 73% H 98% 167 hydroxyl groups of 4Z with acetic anhydride yielded 48 (74%). Acid hydrolysis of 48 gave 42 in 89% yield. Basic hydrolysis of 4Z gave 58100% yield. Tribenzylation of 48 and 58 was not successful. Esterification of two free hydroxyl groups of 8 with butyric anhydride yielded 51 (86 %) (Scheme 9). Treatment of 51 with one equivalent of ethylene glycol gave 52 in 73% yield. Esterification of two free hydroxyl groups of 52 with butyric anhydride yielded 52 (98%). Intramolecular cyclization of 42 with chloro(N,N- diisopropylamino)methoxyphosphine was not successful. Under an acid condition, it has been reported that the palmitoyl group can undergo 1,2-migration (Pliickthun and Dennis, 1982). Therefore, it is possible that the acyl groups may migrate under the condensation condition catalyzed by a weak acid tetrazole. Experimental Section DL-l,6-Di-O-benzyl- 2,3:4,5-di-O-cyclohexylidene-my 0-inositol (36), DL-6-0- benzyl-2,3:4,5-di-0-cyclohexylidene-wyo-inositol (2Z) and DL-l-0-benzyl-2,3:4,5-di-0- cyclohexylidene-wyo-inositol (28) (Garegg et al., 1984): 125 ml of 5% sodium hydroxide aqueous solution was added to a solution of 3,4:5,6-di-O-cyclohexylidene-wyo- inositol (2) (2.5 g, 7 mmol), tetrabutylammonium hydrogensulfate (2.5 g, 7 mmol), and benzyl bromide (2.0 g, 12 mmol) in dichloromethane (125 ml). The mixture was boiled 20 hours under reflux, cooled to room temperature, and the two layers were separated. The dichloromethane layer was washed with water and evaporated. Chromatography on silica gel (hexanes-ethyl acetate, 5:1, v/v) yielded syrupy 28 (1-44 g, 39%), Rf = 0.65 (hexanes-ethyl acetate 5:1, v/v), 2Z (1-27 g, 43%), Rf = 0.5 (hexanes-ethyl acetate 5:1, v/v), and 28 (310 mg, 10%), Rf = 0.05 (hexanes-ethyl acetate 5:1, v/v). These three compounds were characterized by *H NMR and 13C NMR as the followings. 28: *H NMR (CDC13, 250 MHz): 8 7.26 (m, 10H, phenyl of 1,6-di-O-benzyl), 4.62,4.56 (AB, J = 12.3 Hz, 2H), 4.58,4.45 (AB, J = 11.8 Hz, 2H) [CH 2 of l,6-di-0-benzyl], 4.28 (dd, J= 6.9, 3.7 Hz, 1H), 4.23 (t, J = 6.9 Hz, 1H), 4.05 (dd, J= 10.6, 7.3 Hz, 1H), 3.78 (dd, J= 7.7, 2.8 Hz, 1H), 3.68 (t, J= 3.2 Hz, 1H) and 3.42 (dd, J = 10.6, 7.8 Hz, 1H) [6 inositol ring protons], 1.57-1.32 (m, 2H, CH 2 of 2,3:4,5-di-0-cyclohexylidene). 13C NMR (CDC13, 62.896 MHz): 8 138.17,128.31,127.84,127.69 and 127.63 (phenyl of 1,6-di-O-benzyl), 112.82 and 111.57 (tertiary C-l of 2,3:4,5-di-O-cyclohexylidene), 79.70 and 79.34 (CH 2 of 1,6-di-O-benzyl), 78.712, 77.57, 77.00, 75.08, 73.234 and 71.70 (6 inositol ring carbons), 36.64, 36.39 , 34.64, 25.18, 25.08, 23.96, 23.80, 23.73 and 23.63 (CH 2 of 2,3:4,5-di-O-cyclohexylidene). 2Z: *H NMR (CDC13, 300 MHz): 8 7.26 (m, 5H, phenyl of 6-O-benzyl), 4.68 and 4.56 (AB, J = 11.8 Hz, 2H, CH 2 of 6-O- benzyl), 4.34 (m,lH), 4.25 (t, J = 7.5 Hz, 1H), 4.08 (dd, J= 10.7, 7.4 Hz, 1H), 3.93 (t, J= 1.7 Hz, 1H), 3.80 (dd, J = 7.9, 2.0 Hz, 1H) and 3.45 (dd, J= 10.6, 7.9 Hz, 1H) [6 inositol ring protons], 2.48 (d, J = 1.0 Hz, 1H, 1-OH), 1.55-1.31 (m, 20H, CH 2 of 2,3:4,5-di-O-cyclohexylidene). 13C NMR (CDC13, 75.46 MHz): 8 138.00 (phenyl C-l of 6-O-benzyl), 128.38 (phenyl C-3 and C-5 of 6-O-benzyl), 127.83 (phenyl C-4 of 6-O- benzyl), 127.69 (phenyl C-2 and C -6 of 6-O-benzyl), 112.96 and 111.28 (tertiary C-l of 2,3:4,5-di-O-cyclohexylidene), 79.92 (CH 2 of 6-O-benzyl), 78.94,77.51, 76.69,75.31, 72.37 and 71.69 (6 inositol ring carbons), 36.67, 36.62 , 36.34, 33.49, 25.10, 25.07, 23.84.23.70 and 23.39 (CH 2 of 2,3:4,5-di-O-cyclohexylidene). 2fi: *HNMR (CDC13, 250 MHz): 8 7.30 (m, 5H, phenyl of 1-O-benzyl), 4.61 and 4.51 (AB, J = 11.9 Hz, 2H, CH2 of 1-O-benzyl), 4.33 (t, J = 5.3 Hz, 1H), 4.14 (dd, J = 8.4, 5.3 Hz, 1H), 4.01 (ddd, J= 10.5, 6.9 Hz, J = 2.4 Hz, 1H), 3.72 (dd, J= 10.2, 8.5 Hz, 1H), 3.50 (dd, J= 6.8 , 5.4 Hz, 1H,) and 3.24 (t, J = 10.6 Hz, 1H) [6 inositol ring protons], 2.48 (d, J=2.2 Hz, 1H, 6-OH), 1.57-1.32 (m, 20H, CH2 of 2,3:4,5-di-O-cyclohexylidene). 13C NMR (CDCI3, 62.896 MHz) 8 137.77,128.51,128.03,127.64 and 126.97 (phenyl of 1-O- benzyl), 112.90 and 111.10 (tertiary C-l of 2,3:4,5-di-O-cyclohexylidene), 80.89 (CH 2 of 169 1-O-benzyl), 78.68, 77.32, 76.10, 74.16,72.76,72.27 (6 inositol ring carbons), 37.70, 36.50 , 36.37, 34.98, 29.67, 29.27, 25.05,24.97, 23.92, 23.67, 23.61 and 23.56 (CH 2 of 2,3:4,5-di-O-cyclohexylidene).. D-6-0-benzyl-l-0-(-)camphanoyl-2,3:4,5-di-0-cyclohexylidene-myo-inositolQ2d) andL(D)-6(4)-0-benzyl-l(3)-0-(-)camphanoyl-2,3:5,6(l,2:5,6)-di-0-cyclohexylidene- myo-inositol (391) (The numbering system in parentheses indicates the D numbering system) (Vacca et al., 1987): To a dry mixture of 2Z (420 mg, 1 mmol), 4- dimethylaminopyridine (DMAP) (163 mg, 1.1 mmol) and (S)-(-)camphanic acid chloride (239 mg, 1.1 mmol), 15 ml of dichloromethane and 1 ml of triethylamine were added through vacuum transfer. After filled with argon gas, the mixture was warmed to room temperature and stirred for 24 hours. After dichloromethane and excess triethylamine were evaporated, a mixture of two diastereomers were chromatographically separated on silica gel [eluted by solvent gradient from dichloromethane to dichloromethane-diethyl ether, 98/5, v/v) to give 39d (41% yield), mp 152-153°C, Rf = 0.60 ( dichloromethane-diethyl ether, 95/5, v/v) and 221 (40% yield), mp 120-123°, Rf = 0.72 (dichloromethane-diethyl ether, 95/5, v/v). These two compounds were characterized by *H and 13C NMR. The assignments to specific resonances and coupling constants were made by reference to 22. 36.12d and 121. The assignments of the 6 inositol ring protons were according to the following criteria. The resonance at 5.26 ppm was assigned to be 2-H based on its down field shift and small coupling constants. Two doublet doublets at 4.60 and 3.85 ppm with a small coupling constant (4.9 Hz) were assigned to be 1-H and 3-H. According to Shoolery’s rules, 1-H should resonate at lower field than 3-H. Therefore, the resonance at 4.60 ppm was assigned to be 1-H. The most upfield resonance at 3.51 ppm was assigned to be 5-H on the basis of its up field shift as most compounds in Chapter V and VI. 39d: !H NMR (CDC13, 250 MHz): 8 7.35 (m, 5H, phenyl of 6-O-benzyl), 4.84 and 4.73 (AB, J = 1 1 .9 Hz, 2H, CH2 o f 6-O-benzyl), 5.26 (t, Jh -C (2)-C(1)-h ~ Jh-c(2)-C(3)-h = 4 .9 Hz, 1H, 170 2-H), 4.60 (dd, Jh -c (1)-c (2)-h = 4.7 Hz, Jh -C(1)-C(6)-h = 6.1 Hz, 1H, 1-H), 4.32 (dd, JH_ C(i)-C(6)-H = 6.3 Hz, Jh -c (6)-C(5)-h = 8-3 Hz, 1H, 6-H), 3.85 (dd, Jh -C(3)-c (4)-h - 8-7 Hz, j h -C(3)-C(2)-h = 6-0 H z , 1H, 3-H), 3.85 (dd, Jh -c (4)-c (3)-h = 8-5 Hz, Jh -c (4)-C(5)-h = 10.4 Hz , 1H, 4-H), 3.51 (dd, Jh -C(5)-c (6)-h = 8,8 Hz, Jh -c (5)-C(4)-h = 10.5 Hz, 1H, 5-H), 2.36 (m,lH), 1.95 (m, 3H) and 1.18 (m, 1H) [camphanoyl CH2],1.65 (m, 16H) and 1.39 (m, 4H) [CH2 of 2,3:4,5-di-O-cyclohexylidene], 1.10 (s, 3H, camphanoyl 9-CH3), 1.05 (s, 3H, camphanoyl 8-CH3), 0.91 (s, 3H, camphanoyl 10-CH3). 13C NMR (CDC13, 62.896 MHz) 8 177.82 (camphanoyl C-l 1), 166.61 (camphanoyl C-2), 137.758 (phenyl C-l of 6- O-benzyl),128.33 (phenyl C-3 and C-5 of 6-O-benzyl), 127.79 (phenyl C-4 of 6-o- benzyl), 127.72 (phenyl C-2 and C-6 of 6-O-benzyl), 113.14,111.54 (tertiary C-l of 2,3:4,5-di-O-cyclohexylidene), 90.72 (camphanoyl tertiary C-4), 77.94,78.16,77.89, 75.96,74.21,73.23 and 72.13 (CH2 of 6-O-benzyl and 6 inositol ring carbons), 54.76 (camphanoyl C-l), 54.40 (camphanoyl C-7), 37.03, 36.54 , 36.46, 34.46, 24.99, 23.81, 23.75,23.71 and 23.48 (CH2 of 2,3:4,5-di-O-cyclohexylidene),30.66 (camphanoyl C-6), 28.99 (camphanoyl C-5), 16.60 (camphanoyl C-9), 16.55 (camphanoyl C-8), 9.66 (camphanoyl C-10). 221: !H NMR (CDC13, 250 MHz): 8 7.36 (m, 5H, phenyl of 6-O- benzyl), 5.27 (dd, Jh-c(2)-C(3)-h = % -C(2)-C(i)-H = 5.2 Hz, 1H, 2-H), 4.82 and 4.74 (AB, J — 12 Hz, 2H, CH2of 6-O-benzyl), 4.60 (dd, Jh-C(1)-C(2)-h = 4.6 Hz, Jh-c(1)-C(6)-h = 6.2 Hz,lH, 1-H), 4.32 (dd, Jh-c(6)-C(1)-h = 6.4 Hz, Jh-c(6)-C(5)-h = 8-3 Hz, 1H, 6-H), 3.86 (dd, 1H, Jh-c(3)-C(2)-h = 5.1 Hz, Jh-c(3)-C(4)-h = 8*6 Hz, 1H, 3-H ), 3.85 (dd, 1H, J h -c (4 )- C (3)-H = 8-3 Hz, Jh-c(4)-c(5)-h = 10.4 Hz, 1H, 4-H ) , 3.53 (dd, Jh-C(5)-C(6)-h = 8-6 Hz, JH_ C(5)-C(4)-H = 10-5 Hz, 1H, 5-H), 2.44 (m,lH), 1.96 (m, 3H) and 1.20 (m, 1H) [camphanoyl CH2], 1.65 (m, 16H) and 1.39 (m, 4H) [CH2 of 2,3:4,5-di-o- cyclohexylidene], 1.10 (s, 3H, camphanoyl 9-CH3), 1.00 (s, 3H, camphanoyl 8-CH3), 0.92 (s, 3H, camphanoyl 10-CH3). 13C NMR (CDC13, 62.896 MHz): 8 177.80 (camphanoyl C -ll), 166.48 (camphanoyl tertiary C-2), 137.67 (phenyl C-l of 6-0- 171 benzyl), 128.31 (phenyl C-3 and C-5 of benzyl), 127.74 (phenyl C-4 of 6-O-benzyl), 127.66 (phenyl C-2 and C-6 of 6-O-benzyl), 113.11 and 111.43 (tertiary C-l of 2,3:4,5- di-O-cyclohexylidene), 90.86 (camphanoyl tertiary C-4), 77.96,78.18,77.79,75.89, 74.22,73.13 and 72.05 (CH2 of 6-O-benzyl and 6 inositol ring carbons), 54.80 (camphanoyl C-l), 54.25 (camphanoyl C-7), 36.90, 36.58 , 36.43, 34.19, 24.96, 23.80, 23.71,23.68 and 23.50 (CH2 of 2,3:4,5-di-O-cyclohexylidene), 30.64 (camphanoyl C-6), 28.91 (camphanoyl C-5),16.87 (camphanoyl C-9), 16.68 (camphanoyl C-8), 9.66 (camphanoyl C-10). L-6-0-benzyl-2,3:4^-di-0-cyclohexylidene-wyo-inositol (2Z1) and D-6-O-benzyl- 2,3:4,5-di-0-cyclohexylidene-wyo-inositol (2Zd) (Vacca et al., 1987): To a solution of 1,2-dimethoxyethane (DME)-H20 (2:1, v/v), 380 mg of 221 (0.63 mmol) and 6.3 mmol of LiOH’H20 were added at room temperature. The reaction was monitored by TLC. After 2 hours the reaction was completed. 100 ml of dichloromethane and 100 ml of water were added to the reaction mixture. The dichloromethane layer was dried over sodium sulfate and evaporated to give 240 mg of 2Z1 (92%). TLC: R f=0.5 (hexanes-ethyl acetate, 5/1, v/v). 2Zd can be obtained by the same procedure from 39d in 93 % yield. These products were characterized by ^ NMR, and the NMR spectrum of 221 and 37d were the same as DL form (371. D-l-0-(-)-camphanoyl-2,3:4,5-di-0-cyclohexylidene-/wyo-inositol (42): 820 mg of 10% Pd-C was dried with heat gun in vacuo (2.7 mmHg) and the system was filled with hydrogen gas (1 atm). To this mixture, 278 mg of 39d (0.463 mmol) in 50 ml of dry ethyl acetate was added. The reaction mixture was stirred at room temperature for 19 hours. Purification by flash chromatography on silica gel in dichloromethane-acetone (4:1, v/v) gave a white semi-solid (210 mg, 89%). This compound was characterized by TLC and NMR. TLC: R f=0.69 in dichloromethane-acetone (4:1, v/v), Rf = 0.05 in hexanes- ethyl acetate (5:1, v/v). The assignments of the 6 inositol ring protons were according to 172 the following criteria. The triplet at 4.5 ppm with a small coupling constant (5 Hz) was assigned to be 2-H. The resonances at 5.0 and 3.9 ppm were assigned to be 1-H and 3-H, respectively, based on the coupling constants and Shoolery’s rules. The other resonances were assigned by comparing the known coupling constants. *H NMR (CD3OD, 200 MHz): 8 5.0 (dd, J = 5,10 Hz, 1H, 1-H), 4.5 (t, J = 5 Hz, 1H, 2-H), 3.9 (dd, J = 5,7 Hz, 1H, 3-H), 3.6 (t, J= 10 Hz, 1H, 6-H), 3.4 (dd, J = 11,10 Hz, 1H, 5-H) and 3.2 (m,lH, 4-H), 2.5-1.3 (m, 24H, CH2 of 2,3:4,5-di-O-cyclohexylidene and CH2 of camphanoyl), 1.1 (s, 3H, camphanoyl C-9), 1.0 (s, 3H, camphanoyl C-8), 0.9 (s, 3H, camphanoyl C-10). D-l-0-(-)-camphanoyl-2,3:4,5-di-0-cyclohexylidene-6-0-P- trimethysilylethoxymethyl-wyo-inositol (4 1 ) : To a dry semi-solid of 411 (140 mg, 0.27 mmol), 10 ml of dichloromethane, 5 equivalents (200 ml) of iPr2NEt and 3 equivalents of 2-trimethylsilylethoxymethyl chloride (SEMC1) were added by a syringe under the argon atmosphere. The solution was stirred at 40°C for 18 hours. Purification by column chromatography on silica gel (hexanes-ethyl acetate, 5/1, v/v) gave a white powder (136 mg; 78%; mp = 192-194°C; Rf = 0.33 in hexanes: ethyl acetate (5/1, v/v). This compound was characterized by *H and 13C NMR. The assignments to *H NMR spectrum was made by reference to 411- *H NMR (CDC13, 250 MHz) 8 4.84 (s, 2H ,- OCH20- of 6-O-SEM), 5.18 (dd, J = 5.3,4.7 Hz,lH, 2-H), 4.59 (dd, J = 4.8, 6.1 Hz, 1H, 1-H), 4.31 (dd, J= 6.1, 8.3 Hz, 1H, 6-H), 4.10 (dd, J = 5.6, 8.7 Hz,lH, 3-H), 3.86 (dd, J= 8.4, 10.4 Hz,lH, 5-H) and 3.44 (dd, J= 8.7, 10.5 Hz, 1H, 4-H), 3.74 and 3.60 (the AB part of an ABX2, = 10.7 Hz, = JBX = 10.7 Hz, 2H, 0-CH2-CH2- SiMe3), 2.43 (m,lH), 1.98 (m, 2H) and 1.18(m,lH) [camphanoyl CH2],1.58-1.31 (m, 20H, CH2 of 2,3:4,5-di-O-cyclohexylidene), 1.12 (s, 3H, camphanoyl C-9), 1.11 (s, 3H, camphanoyl C-8), 0.99 (s, 3H camphanoyl C-10), 0.99 (m, 2H, 0-CH2CH2-SiMe3), 0.02 (s,9H, SiMe3). 13C NMR (CDC13, 62.896 MHz) 8 177.84 (camphanoyl C -ll), 173 166.53 (camphanoyl C-2), 113.04,111.56 (tertiary C-l of 2,3:4,5-di-O- dicyclohexylidene), 94.00 (0-CH2-0 of 6-O-SEM), 90.76 (camphanoyl C-4), 78.05, 76.00.75.279.74.69 and 73.23 (inositol ring carbons), 65.65 (0-CH2-CH2-SiMe3), 54.80 (camphanoyl C-l), 54.49 (camphanoyl C-7), 37.15, 36.47 , 36.57,24.98, 23.81, 23.67 and 23.47 (CH2 of 2,3:4,5-di-0-dicyclohexylidene), 30.66 (camphanoyl C-6), 29.03 (camphanoyl C-5), 18.00 (0-CH2-CH2-SiMe3), 16.67 (camphanoyl C-9), 16.58 (camphanoyl C-8), 9.70 (camphanoyl C-10), -1.40 (SiMe3). D-2,3:4,5-Di-0-cyclohexylidene-6-0-P-trimethysilylethoxymethyl-/«yo-inositol (42): To a dry ether solution of 41 (77 mg, 0.12 mmol), 22 equivalents (157 mg) of UA1H4 was added. The reaction mixture was stirred from 0°C to room temperature for 22 hours. From TLC, the starting material (Rf = 0.30, hexanes/ethyl acetate, 5/1, v/v) disappeared and a new spot appeared at Rf = 0.34. Excess LiAlH4 was destroyed by adding 200 ml of wet ether (saturated with water) in an ice bath. The filtrate was washed with 200 ml of water, dried over Na2S04, and then evaporated to give 50 mg of 42 (92 % yield). This compound was characterized by XH NMR and 13C NMR. JH NMR (CDC13, 250 MHz): 8 4.87 and 4.84 (AB, J = 6.9 Hz, 2H, 0-CHr 0 - of 6-O-SEM), 4.45 (dd, J = 4.0, 6.6 Hz,lH), 4.30 (dd, J = 6.6, 7.9 Hz, 1H), 4.02 (m, 2H), 3.93 (m,lH) and 3.44 (dd, J = 10.5, 8.6 Hz, 1H) [6 inositol ring protons ], 3.74 and 3.60 (AB part of ABX2, Jab = 10.4 Hz, Jax = Jbx = 9.7 Hz, 2H, 0-CH2-CH2-SiMe3),3.18 (1-OH), 2.28-1.30 (m, 20H, CH2 of 2,3:4,5-di-O-cyclohexylidene), 0.98 (m, 2H, 0-CH2CH2-SiMe3),0.02 (s,9H, SiMe3). 13C NMR (CDC13, 62.896 MHz): 8 112.85 and 111.10 (tertiary C-l of 2,3;4,5-di-O-dicyclohexylidene), 94.26 (0-CH2-0 of 6-O-SEM), 78.48,77.41, 77.00, 76.36,75.73 and 72.74 (6 inositol ring carbons), 65.70 (0-CH2-CH2-SiMe3), 36.92, 36.57 , 36.48, 34.03, 25.03, 24.99, 23.84, 23.67, 23.63 and 23.39 (CH2 of 2,3:4,5-di- O-cyclohexylidene), 18.05 (0-CH2-CH2-SiMe3), -1.46 (SiMe3). 174 D-l-0-benzyl-2,3:5,6-di-0-cyclohexylidene-4-0-p-trimethysilylethoxymethyl-/M}'o- inositol ( 1 2 ) : To a solution of 12d (0.67 mmol in 10 ml of dichloromethane), 10 equivalents of KOH in 100 ml of EtOH was added. The reaction mixture was stirred at room temperature for 30 minutes. The solvents were removed and the residue was dissolved into 200 ml of dichloroform, washed with 3x100 ml of water, 100 ml of saturated NaHC03, and 100 ml of brine, and dried over NajSO*. The crude product (100% yield) was was used in the next step reaction without further purification. To a solution of this compound, 3 equivalents of SEMC1 and 5 equivalents of iPr2NEt were added and stirred at 40-42° C for 20 hours. The reaction mixture was purified by liquid chromatography on silica gel (hexanes-ethyl acetate, 5:1, v/v) gave 343.6 mg (90 %) of 42. This compound was characterized by TLC and NMR. The assignments to specific resonances and coupling constants were made by reference to 12d and 41. TLC: Rf = 0.53 (hexanes-ethyl acetate, 5:1, v/v). The assignments of the *H NMR spectrum were according to the following criteria. The assignments to two AB systems were made according t o Shoolery's rules. Thus, the 0-CH2-0 of SEM resonates at lower field than 0-CH2Ph. This assignments were confirmed by reference to 44- The triplet at 4.33 ppm was assigned to be 2-H on the basis of its small coupling constants (4.3 Hz) and its down field shift. Two doublet doublets at 3.95 and 3.74 ppm were assigned to be 3-H and 1-H, respectively, based on Shoolery's rules. Other inositol ring protons were assigned by comparing the known coupling constants. *H NMR (CDC13, 250 MHz): 8 7.45-7.26 (m, 5H, phenyl of 1-O-benzyl), 4.93 and 4.90 (AB, J = 6.6 Hz, 2H, 0-CH2-0 of 4-O-SEM), 4.89 and 4.82 (AB, J — 12.5 Hz, 2H, CH2 of 1-O-benzyl), 4.33 (t, Jh -c (2)-C(1)-h ~ Jh -C(2)- C(3)-H = 4.3 Hz, 1H, 2-H), 4.06 (dd, Jh -c (4)-c (3)-h = 10.0 Hz, Jh -c (4)-c (5)-h = 9-5 Hz, 1H, 4-H), 3.97 (t, Jh -c (6)-c (1)-h = j h -C(6)-C(5)-h =10.0 Hz, 1H, 6-H), 3.95 (ddJH.C(3).C( 2)-H = 4.2 Hz, Jh - c (3)-C (4)-h = 10.0 Hz, 1H, 1-H), 3.74 (dd, Jh -c (1)-C (2)-h = 4.2 Hz, Jh -c (1)-C (6)-h =10.2 Hz, 1H, 1-H), 3.78-3.66 (AB part of ABX2,2H, 0-CH2-CH2-Si Me3), 3.26 (dd, 175 Jh -c (5)-C (6)-h = 10-3 Hz, Jh -C(5)-C(4)-h = 9.6 Hz, 1H, 5-H), 2.39 and 1.76-0.86 (m, 22H, CH2 of 2,3:4,5-di-O-cyclohexylidene and CH2 of 4-O-SEM), 0.02 (s, 9H, Si Me3). 13C NMR (CDC13, 62.896 MHz): 8138.12 (phenyl C-l of 1-O-benzyl), 128.29 (phenyl C-3 and C-5 of l-o-benzyl),128.11 (phenyl C-2 and C-6 of 1-O-benzyl), 127.66 (phenyl C-4 of 1-O-benzyl), 113.64 and 111.41 (tertiary C-l of 2,3;4,5-di-O-cyclohexylidene), 91.64 (0-CH2-0 of 4-O-SEM), 80.51 (CH2 of 1-O-benzyl), 76.89,76.76 , 76.50, 76.18, 74.64 and 65.01 (6 inositol ring carbons), 71.66 (0-CH2-CH2-SiMe3), 37.62, 36.43, 36.20, 35.30, 29.63, 25.04, 24.98, 23.96, 23.87, 23.78 and 23.56 (CH2 of 2,3:4,5-di-O- cyclohexylidene), 17.92 (0-CH2-CH2-SiMe3), -1.43 (SiMe3). D-2.3:5,6-di-0-cvclohexvlidene-4-0-P-trimethvsilvlethoxvmethvl-/nvQ-inositol(44): To a solution of 42 (0.424 mmol in 30 ml of ethyl acetate), 253 mg of dry 10% Pd-C was added. The mixture was stirred at room temperature under H2 gas flux (1 atm) for 72 hours. The reaction mixture was filtrated and purified by liquid chromatography on silica gel (hexane-ethyl acetate 2:1 v/v) to give 36 mg of 44 (18% yield). This compound was characterized by TLC and NMR. TLC: Rf = 0.53 (hexane-ethyl acetate, 2:1, v/v). The assignments to 1-H, 2-H and 3-H were made by reference to 42- The assignment to 1-H was confirmed by the extra splittings from 1-OH. All the other protons of the inositol ring were assigned by comparing to the known coupling constants. !H NMR (CDC13, 250 MHz) 8 4.91 and 4.87 (AB, J = 6.6 Hz, 2H, 0-CH2-0 of 4-O-SEM), 4.44 (t, JH-C( 2)- C (i)-H = Jh -C(2)-C(3)-h = 4-7 Hz, 1H, 2-H), 4.11 (dd, Jh -c (3)-C(2)-h = 5.2 Hz, Jh -c (3)-C (4)-h = 6.4 Hz, 1H, 3-H), 3.96 (dt, Jh -C(1)-c (2)-h = 4-5 Hz, Jh -c (1)-c (6)-h = % c(i)-o-H = 100 Hz, 1H, 1-H), 3.92 (dd, Jh -c (4)-C (3)-h = 6-4 Hz, Jh -c (4)-C (5)-h =10.6 Hz, 1H, 4-H), 3.82 (dd, Jh -c (6)-C (1)-h = 10.0 Hz, Jh -C(6)-C(5)-h =9.3 Hz, 1H, 6-H), 3.68 (AB part of ABX2, 2H, 0-CH2-CH2-Si (Me)3), 3.30 (dd, Jh -C(5)-C(4)-h = 10.7 Hz, Jh -c (5)-C (6)-h = 9.3 Hz, 1H, 5-H), 2.44 (d, Jh.c( 1).o-h = l°-° Hz), 1.8-0.7 (m, 22H, CH2 of 2,3:5,6-di-0- cyclohexylidene and CH2Si(Me)3), 0.02 (s, 9H, Si Me3). 13C NMR (CDC13, 62.896 m MHz) 8 112.92 and 111.53 (tertiary C-l of 2,3:5,6-di-0-cyclohexylidene), 91.64 (O- CH2-0 of 4-O-SEM), 80.69,77.89,77.503,77.23, 76.49 and 65.06 (6 inositol ring carbons), 70.07 (0-CH2-CH2-SiMe3), 37.67, 36.54, 36.35, 35.16, 29.66, 29.40, 24.99,24.01,23.74 and 23.60 (CH2 carbons of 2,3:5,6-di-0-cyclohexylidene), 17.97 (O- CH2-CH2-SiMe3), -1.42 (SiMe3). D-4-0-benzyl-3-0-(-)-camphanoyl-l,2-0-cyclohexylidene-/wyo-inositol (45) (Vacca et al., 1987): To a solution of 600 mg of 221 (0.98 mmol) and 30 ml of methanol-ethyl acetate (1:5, v/v), 300 pi of solution A was added and stirred at room temperature for 2 hours. Solution A was prepared by bubbling HC1 gas into 5 ml of methanol-ethyl acetate (1/5, v/v) for 1 mimute. The reaction mixture was washed with 100 ml of saturated NaHC03 solution. After purification by flash chromatography on silica gel (chloroform- acetone, 2/1, v/v) gave a while solid (500 mg, 99%). This compound was characterized by TLC and mp. TLC: Rf = 0.34 (chloroform-acetone, 2:1, v/v), mp = 174-176°C (literature 176-178°C, Vacca et al., 1987). D-4-O-benzyl-1,2-O-cyclohexylidene-myo-inositol (4 0 : To a solution of 500 mg of 45 (0.98 mmol) and 20 ml of tetrahydrofuran, 10 mmol of LiOH in 10 ml of water was added and stirred at room temperature for 1/2 hour. After purification by flash chromatography on silica gel (chloroform-acetone, 2:1, v/v) gave a while solid (250 mg, 71%). This compound was characterized by TLC and mp. TLC: Rf = 0.5 in chloroform- acetone (2:1, v/v), mp = 135-137°C (literature 137-139°C, Vacca et al., 1987). D-3-0-benzyl-6-0-(-)-camphanoyl-l ,2-0-cyclohexylidene-/nyo-inositol (4Z) (The procedures were adapted from Ozaki et al., 1986.): To a solution of 121 (0.98 mmol in 15 ml of dichloromethane), 1 equivalent of ethylene glycol and 6 mg of anhydrous p- toluenesulfonic acid (TsOH) were added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was terminated by washing with 50 ml of saturated NaHC03 solution and 50 ml of saturated NaCl solution, and the organic layer Ill was evaporated to dryness. Purification was performed by flash chromatography on silica gel (eluted by solvent gradient from chloroform to chloroform-acetone, 2:1, v/v) to give 225 mg of 4Z (43 % yield). This compound was characterized by TLC and NMR. TLC: Rf = 0.60 (chloroform-acetone, 2/1, v/v). The assignments to specific resonances and coupling constants were made by reference to 121 and were confirmed by the extra splittings in 4-H and 5-H from OH. *H NMR (CDC13, 250 MHz): 8 7.42-7.26 (m, 10H, phenyl of 3-O-benzyl), 5.24 (dd, Jh-c(6)-C(1)-h = 8*0 Hz, Jh.c(6)-C(5)-h =10.6 Hz, 1H, 6- H), 4.77 and 4.74 (AB, J = 12.0 Hz, 2H, CH2 of 3-O-benzyl), 4.31 (t, JH-c( 2)-C(1)-h =%- C(2)-C(3)-H = 4.3 Hz, 1H, 2-H), 4.00 (dd,JH.c(i)-c( 2)-H = 4.7 Hz, Jh-c( 1)-C(6)-h =7-8 Hz, 1H, 1-H), 3.94 (dt, Jh-C(4)-C(3)-h = j h-C(4)-C(5)-h = 9 .6 Hz, J h-c(4)-o-h = I - 6 Hz, 1H, 4- H), 3.52 (dd, Jh-c(3)-C(2)-h = 4-0 Hz, Jh-c(3)-C(4)-h = 9 .6 Hz, 1H, 3-H), 3.42 (dt, J h -c (5 )- C(4)-H = J H-C(5)-C(6)-H = 10-3 Hz, Jh-C(5)-0-H = 3-2 Hz, 1H, 5-H), 3.02 (d, Jh-C(4)-0-H = 2 0 Hz, 1H, 4-OH), 2.93 (d, JH-c( 4)-o-H = 3-2 Hz, 1H, 3-OH), 2.52-1.41 (m, 14H, CH2 of camphanoyl and of 1,2-O-cyclohexylidene), 1.10 (s, 3H, camphanoyl 9-CH3), 1.04 (s, 3H, camphanoyl 8-CH3), 1.00 (s,3H, camphanoyl 10-CH3). 13C NMR (CDC13, 62.896 MHz) 8 178.34 (camphanoyl C -ll), 166.90 (camphanoyl C-2), 137.67 ,128.56 and 128.12 (phenyl of 3-O-benzyl), 111.24 (tertiary C-l of 1,2-O-cyclohexylidene), 91.21 (camphanoyl C-4), 77.68 ,75.94, 73.07,72.04,72.09 and 71.10 (CH2 of 1-O-benzyl and 6 inositol carbons), 54.83 and 54.70 (camphanoyl C-l and C-7), 37.38 and 35.29 (C- 2 and C-6 of 1,2-O-cyclohexylidene), 30.39 (camphanoyl C-6), 28.94 (camphanoyl C-5), 24.91,23.86 and 23.65 (C-3, C-4 and C-5 of 1,2-O-cyclohexylidene), 16.52 (camphanoyl C-9), 16.37 (camphanoyl C-8), 9.69 (camphanoyl C-10). D-4,5-di-0-acetyl-3-0-benzyl-6-0-(-)-camphanoyl-l,2-0-cyclohexylidene-myo- inositol (48): Compound 4Z*was dried by evaporating with 10 ml of dry pyridine (over NaH) three times. To a solution of 42 (0.325 mmol in 10 ml of pyridine), 159 mg of DMAP and 123 |il of acetic anhydride were added and stirred at room temperature for 25 hours. The product was purified by flash chromatography on silica gel (eluted by solvent gradient from chloroform to chloroform-acetone, 10/1, v/v) to give a white solid of (145 mg, 74% yield). This product was characterized by TLC and NMR. TLC: Rf = 0.7 (chloroform-acetone, 10/1, v/v). The assignments to specific resonances and coupling constants were made by reference to 4Z. The assignments of the *H NMR spectrum were according to the following criteria. The triplet at 4.35 ppm with a small coupling constant was assigned to be 2-H. The resonances at 3.77 and 4.12 ppm with small coupling coupling constants were assigned to be 3-H and 1-H, respectively, based on Shoolery's rules. The other inositol ring protons were assigned by comparing to the known coupling constants. *H NMR (CDC13, 250 MHz): 8 7.35-7.26 (m, 10H, phenyl of 3-O-benzyl), 5.49 (dd, Jh-c(6)-C(1)-h = 7 -5 Hz, Jh-c(6)-c(5)-h = 10-1 Hz, 1H, 6-H), 5.44 (t, Jh-c(4)-c(3)-h = j h-C(4)-C(5)-h = 8.6 Hz, 1H, 4-H), 5.00 (dd, Jh-c(5)-C(4)-h = 8.3 Hz, Jh-c(5)-C(6)-h = 1 0 - ° Hz, 1H, 5-H), 4.71 (A2,2H, CH2 of 3-O-benzyl) (A2 represents two hydrogens HA with same chemical shift.), 4.35 (t, Jh-C(2)-C(1)-h = 6-4 Hz, Jh-c(2)-C(3)-h = Hz, 1H, 2-H), 4.12 (dd,JH.C(1).C(2).H = 5.6 Hz, Jh -c (1)-c (6)-h = 7-4 Hz, 1H, 1-H), 3.77 (dd, Jh -c (3)-c (2>- H = 3.8 Hz, JH-c (3)-C(4)-H = 8.8 Hz, 1H, 3-H), 2.01 and 1.98 (CH3CO), 2.43-1.39 (m, 14H, CH2 of camphanoyl and of 1,2-O-cyclohexylidene), 1.10 (s, 3H, camphanoyl 9- CH3), 1.01 (s, 3H, camphanoyl 8-CH3), 0.95 (s,3H, camphanoyl 10-CH3). 13C NMR (CDC13, 62.896 MHz): 8 177.89 (camphanoyl C -ll), 169.59 and 169.43 (acetyl C=0), 166.30 (camphanoyl C-2), 137.60,128.43,127.94 and 127.74 (phenyl of 3-O-benzyl), 111.67 (tertiary C-l of 1,2-O-cyclohexylidene), 90.75 (camphanoyl C-4), 75.39, 74.57,74.02,73.22,72.66,71.45 and 70.90 (CH2 of 1-O-benzyl and 6 inositol carbons), 54.75 and 54.25 (camphanoyl C-l and C-7), 36.89 and 34.89 (C-2 and C-6 of 1,2-O- cyclohexylidene), 30.62 (camphanoyl C-6), 28.84 (camphanoyl C-5), 24.92,23.84 and 23.60 (C-3, C-4 and C-5 of 1,2-O-cyclohexylidene), 20.67 and 20.54 (CH3CO), 16.59 (camphanoyl C-9), 16.39 (camphanoyl C-8), 9.62 (camphanoyl C-10). D-4,5-di-0-acetyl-3-0-benzyl-6-0-(-)-camphanoyl-wyo-inositol (4 2 ): To 10 ml tetrahydrofuran solution of 4 8 (0.18 mmol), 3 ml of 6N HC1 was added. The reaction was stirred at room temperature for 1.5 hours. The product was purified by flash chromatography on silica gel (chloroform-acetone, 2/1, v/v) to give a white solid of 42 (84 mg, 89% yield). This product was characterized by TLC and NMR. TLC: Rf = 0.6 (chloroform-acetone, 2/1, v/v). The assignments of the !H NMR spectrum were according to the following criteria. The triplet at 5.50 ppm was assigned to be 6-H by reference to 4 8 - The triplet at 4.22 with a small coupling constant (2.7 Hz) was assigned to be 2-H. The resonances at 5.46 and 5.09 ppm were assigned to be 4-H and 5-H, respectively, by reference to 4 8 - The resonances at 3.52 and 3.62 ppm were assigned to be 3-H and 1-H, respectively, by comparing to the known coupling constants. *H NMR (CDC13, 250 MHz): 8 7.38-7.25 (m, 10H, phenyl of 3-O-benzyl), 5.50 (t, Jh-C(6)-C(1)-h = ^H-C(6)-C(5)-H = 9.9 Hz, 1H, 6-H), 5.46 (t, Jh-c(4)-C(3)-h = Jh-c(4)-C(5)-h= 9-8 Hz, 1H, 4-H), 5.09 (t, JH. C(5)-C(4)-H = Jh-C(5)-C(6)-h = 9.9 Hz, 1H, 5-H), 4.65 and 4.55 (AB, J = 12.0 Hz, 2H, CH2 of 3-O-benzyl), 4.22 (t, Jh-C(2)-C(1)-h = Jh-C(2)-C(3)-h= 2.7 Hz, 1H, 2-H), 3.62 (m, 1H, 1- H), 3.52 (dd, Jh-c(3)-C(2)-h = 2.6 Hz, J h - c (3)-C(4)-h = 9*9 Hz, 1H, 3-H), 3.12 and 2.86 (OH), 1.974 and 1.969 (CH3CO), 2.43-1.60 (m, 4H, CH2 of camphanoyl), 1.08 (s, 3H, camphanoyl 9-CH3), 1.00 (s, 3H, camphanoyl 8-CH3), 0.94 (s,3H, camphanoyl 10- CH3). 13C NMR (CDC13, 62.896 MHz): 5 178.03 (camphanoyl C -ll), 169.93 and 169.82 (acetyl C=0), 167.19 (camphanoyl C-2), 137.07,128.62,128.25 and 127.80 (phenyl of 3-O-benzyl), 91.04 (camphanoyl C-4), 76.85, 73.47, 72.54, 71.32, 70.44, 70.24 and 69.35 (CH2 of 1-O-benzyl and 6 inositol carbons), 54.83 and 54.12 (camphanoyl C-l and C-l), 30.78 (camphanoyl C-6), 28.84 (camphanoyl C-5), 20.72 and 20.58 (CH3CO), 16.62 (camphanoyl C-9), 16.40 (camphanoyl C-8), 9.61 (camphanoyl DL-3,6-di-O-butyryl-l,2:4,5-di-O-cyclohexylidene-wy0-inositol (£1): & was dried by evaporating with 10 ml of dry pyridine (over NaH) three times. To a solution of 8 (4 mmol in 15 ml of dichloromethane), 1.95 g of DMAP and 2.6 ml of butyric anhydride were added and stirred at room temperature for 68 hours. The reaction was terminated by adding 5 g of IR-300 (ion exchange resin). The product was purified by flash chromatography on silica gel (hexane-ethyl acetate, 5/1, v/v) to give a white solid of £1 (mp 119-121°C, 1.65 g, 86% yield). This product was characterized by TLC and NMR. TLC: Rf = 0.5 (hexane-ethyl acetate, 5:1, v/v). The assignments to specific proton resonances and coupling constants were made by reference to 8 and by decoupling experiments. The triplet at 4.58 ppm was assigned to be 2-H on the basis of its small coupling constant (4.5 Hz). The resonances at 5.07 and 4.09 ppm with a small coupling constant (4.5 Hz) were assigned to be 3-H and 1-H respective, based on Shoolery's rales. Other inositol ring protons were assigned by comparing to the known coupling constants. The assignments to 6 inositol ring protons were further confirmed by ^ ^ H decoupling experiments. *H NMR (CDC13, 250 MHz): 8 5.26 (dd, Jh -c (6)-c (1)-h = 7-° Hz, JH-c(6)- C(5)-H = 11*1 Hz, 1H, 6-H), 5.07 (dd, Jh -c (3)-c (2)-h = 4.3 Hz, Jh -c (3)-C(4)-h = 10.6 Hz, 1H, 3-H), 4.58 (t, Jh -c (2)-C(1)-h = %C(2)-C(3)-h = 4-5 Hz, 1H, 2-H), 4.12 (dd, Jh -c (4)-C(3)- h = H .l Hz, Jh - c (4)-c (5)-h = 9.2 Hz, 1H, 4-H), 4.09 (dd, Jh -c (1)-c (2)-h = 4.5 Hz, Jh -c (1)- C(6)-H = 6.6 Hz, 1H, 1-H), 3.44 (dd, Jh -c (5)-C(4)-h = 9.5 Hz, Jh -c (5)-C(6)-h =11.1 Hz, 1H, 5-H), 2.39 (t, J = 7.5 Hz, 2H) and 2.35 (t, J = 7.3 Hz, 2H) [a-CH2 of 3,6-di-O-butyryl], 1.74-1.35 (m, 24H, CH2of 1,2:4,5-di-O-cyclohexylidene and p-CH2 of 3,6-di-O- butyryl), 0.96 (t, J = 7.4 Hz, 3H) and 0.95 (t, J = 7.4 Hz, 3H) [terminal-CH3 of 3,6-di-O- butyryl]. 13C NMR (CDC13, 75.46 MHz): 8 173.20 and 172.37 (C=0 of 3,6-di-O- butyryl), 113.92 (C-l of 4,5-O-cyclohexylidene), 111.02 (C-l of 1,2-O-cyclohexylidene) (assigned by reference to the compound which only has 1,2-di-O-cyclohexylidene), 78.91 , 76.01, 74.66 , 74.54, 74.06, 70.55 (6 inositol carbons), 37.34, 36.29, 36.19, 36.16, 181 36.04,35.00 (C-2 and C-6 of l,2:4,5-di-0-cyclohexylidene and a-CH2 of 3,6-di-O- butyryl), 24.88, 24.84, 23.81, 23.65, 23.60, 23.55 (C-3, C-4 and C-5 of l,2:4,5-di-0- cyclohexylidene), 18.50 and 18.45 (p-CH2 of 3,6-di-O-butyryl), 13.44 and 13.30 (terminal-CH3 of 3,6-di-O-butyryl). DL-3,6-di-O-butyiyl-1,2-O-cyclohexylidene-wyo-inositol (52) (The procedures were adapted from Ozaki et al., 1986): To a solution of 51 (1.338 g, 2.79 mmol in 50 ml of dichloromethane), 1 equivalent of ethylene glycol and 77 mg of anhydrous TsOH were added. The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was terminated by adding 2 ml of pyridine. The mixture was evaporated to dryness. Purification was performed by flash chromatography on silica gel [eluted by solvent gradient from chloroform-acetone (10/1, v/v)to chloroform-acetone(2/l, v/v)] to give a white solid of 52 (810 mg, mp 121-124°C, 73 % yield). This compound was characterized by TLC and NMR. TLC: R p 0.70 (chloroform-acetone, 2:1, v/v). The assignments to specific resonances and coupling constants were made by reference to 51- *H NMR (CDC13, 250 MHz) 8 5.00 (m, 2H), 4.40 (unresolved triplets, 1H), 4.07 (dd, J = 5.1 Hz, J = 7.0 Hz, 1H), 3.92 (t, J= 9.0 Hz, 1H) and 3.36 (t, J= 9.0 Hz, 1H) [6 inositol ring protons], 3.74 (bs, 1H) and 3.57 (broad singlet, 1H) [2 hydroxy protons], 2.37 (t, J = 7.5 Hz, 2H) and 2.34 (t, J= 7.5 Hz, 2H) [cc-CH2 of 3,6-di-O-butyryl], 1.73- 1.33 (m, 14H, CH2of 1,2-O-cyclohexylidene and |3-CH2 of 3,6-di-O-butyryl), 0.93 (m, 6H, terminal-CH3 of 3,6-di-O-butyryl). 13C NMR (CDC13, 75.46 MHz): 8 173.71 and 173.55 (C=0 of 3,6-di-O-butyryl), 111.03 (C-l of 1,2-O-cyclohexylidene), 75.96 , 75.31, 73.22 , 72.16, 71.15, 70.75 (6 inositol carbons), 37.37 and 35.07 (C-2 and C-6 of 1,2-O-cyclohexylidene), 36.08 and 35.98 (a-CH2 of 3,6-di-O-butyryl), 24.80,23.74 and 23.47 (C-3, C-4 and C-5 of 1,2-O-cyclohexylidene), 18.39 and 18.32 (|3-CH2 of 3,6-di- O-butyryl), 13.39 (terminal-CH3 of 3,6-di-O-butyryl). DL-3,4 ,5 ,6 -tetra-O-butyryl-l,2-O-cyclohexylidene-wy 0-inositol (53): Compound 52 was dried under vacuum (0.2 mmHg) for 18 hours. To 52 (354 mg, 0.885 mmol), dichloromethane (15 ml), 4 equivalents of DMAP, and 4 equivalents of butyric anhydride were added. The reaction mixture was stirred at room temperature for 21 hours. The reaction was terminated by adding 10 g of IR-300. The product was purified by flash chromatography on silica gel (hexane-ethyl acetate, 5/1, v/v) to give a white semi-solid of 52 (470 mg, 98% yield). This product was characterized by TLC and NMR. TLC: Rf = 0.55 (hexane-ethyl acetate, 5/1, v/v); The assignments to specific resonances and coupling constants were made by reference to 51 and 52- The doublet of doublets (J = 5.1 and 4.1 Hz) was assigned to be 2-H on the basis of two small cis-equatorial-axial coupling constants. Two doublet of doublets with a small coupling constant at 5.15 and 4.14 ppm were assigned to be 3-H and 1-H, respectively, based on Shoolery's rules. Other inositol ring protons were assigned by comparing to the known coupling constant. JH NMR (CDCI3, 250 MHz): 8 5.50 (dd, Jh-C(4)-C(3)-h = 10.0 Hz, Jh-c(4)-C(5)-h = 9.0 Hz, 1H, 4- H), 5.25 (dd, Jh-c(6)-c(1)-h = 7-1 Hz, Jh-c(6)-c(5)-h = 9-4 Hz, 1H, 6-H), 5.15 (dd, J h -c (3 )- C (2)-H = 4.0 Hz, Jh-C(3)-C(4)-H = 10-0 Hz, 1H, 3-H), 5.01 (t, Jh-C(5)-C(4)-H = % C(5)-C(6)-H = 9.1 Hz, 1H, 5-H), 4.45 (dd, Jh-C(2)-C(1)-h = 5.1 Hz, Jh-C(2)-C(3)-h = 4.1 Hz, 1H, 2-H), 4.14 (dd, Jh-C(1)-C(2)-h = 5.3 Hz, Jh-C(1)-c(6)-h = 7.0 Hz, 1H, 1-H), 2.30-2.11 (a-CH2 of 3,4,5,6-tetra-O-butyryl), 1.65-1.31 (m, 18H, CHz of 1,2-O-cyclohexylidene and p-CH 2 of 3,4,5,6-tetra-O-butyryl), 0.96 (t, J = 7.4 Hz, 3H) and 0.93-0.80 (m, 12H, terminal- CH3 of 3,4,5,6-tetra-O-butyryl). 13C NMR (CDCI 3, 75.46 MHz): 6 172.64,172.14, 171.90 and 171.86 (C=0 of 3,4,5,6-tetra-O-butyryl), 111.51 (C-l of 1,2-O- cyclohexylidene) , 75.52 ,72.75, 72.34 , 70.49, 69.05, 68.88 (6 inositol carbons), 36.91 and 34.70 (C-2 and C -6 of 1,2-O-cyclohexylidene), 35.81 and 35.77 (a-CH 2 of 3,4,5,6- tetra-O-butyryl), 24.75,23.65,23.44 (C-3, C-4 and C-5 of 1,2-O-cyclohexylidene), 18.26,18.18,18.09 and 18.02 (p-CH2 of 3,4,5,6-tetra-O-butyryl), 13.40 and 13.34 (terminal-CH3 of 3,4,5,6-tetra-O-butyryl). 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