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Synthesis of cholesterol based model glycolipids Sather, Paula Joan 1990

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SYNTHESIS OF CHOLESTEROL BASED MODEL GLYCOLIPIDS By PAULA JOAN SATHER B . S c , University of British Columbia, Canada, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1990 © Paula Joan Sather, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of CAo/w\/<fjy/ The University of British Columbia Vancouver, Canada Date Qr f (Q fad DE-6 (2/88) ABSTRACT The synthesis of glycolipids containing a variable length polyethylene glycol spacer group between a glucuronic acid (glu) headgroup and a cholesterol (chol) t a i l glu-0CH2(CH20CH2 )nCH 20-chol is described. The homologs (n=2,3,5) were prepared by reaction of an excess of commercially available t r i , tetra and hexaethylene glycols with cholesteryl-p-toluene sulfonate. 3-0-(8-hydroxy-3,6-dioxaoctyl) cholest-5-ene (2), 3-0-(ll-hydroxy-3,6,9-trioxaundecyl)cholest-5-ene (3) and 3-0-(17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl)cholest-5-ene (4) were produced, and yields were dependent on the amount of excess used. The headgroup was prepared by esterification and acetylation of glucuronolactone to produce methyl (1, 2, 3, 4-tetra-0-acetyl-/3-D-glucopyran)uronate which was then brominated at the anomeric carbon to produce methyl (2, 3, 4-tri-O-acetyl-oc-D-glucopyranosyl bromide)uronate (_1). The headgroup was coupled to the cholesteroxy oligoethylene glycols by a Koenig Knorr type reaction using freshly prepared silver carbonate as the catalyst. Methyl[3-0-(3,6-dioxaoctyl)cholest-5-en-3/3-y1-2,3,4-tri-0-acetyl-/3-D-glucopyranosid] uronate (5), Methyl[3-0-(3,6,9-trioxaundecyl) cholest-5-en-3£-yl-2,3,4-tri-0-acetyl-£-D-glucopyranosid] uronate (6), and Methyl[3-0-(3,6,9,12,15-pentaoxaheptadecyl)cholest-5-en-3/3-yl-2, 3, 4-tri-0 -acetyl-/3-D-glucopyranosid] uronate (7) were produced with yields of up to 30%. The removal of the methyl ester and acetate protecting groups on the headgroup was accomplished using NaOH in a mixture of solvents followed by acidification with HCl to produce 3-0-(3,6-dioxaoctyl)cholest-5-en-3£-yl-£-D-glucopyranosiduronic acid (8) and 3-0-(3,6,9-trioxaundecyl)cholest-5-en-3£-yl-/3-D-glucopyranosiduronic acid (9). Octaethylene glycol and dodecaethylene glycol were prepared using a solid supported synthesis. The solid polymer used was a t r i t y l chloride functionalized polystyrene 1% divinyl benzene. Mono protected tetraethylene glycol was prepared and attached to the polymer. The protecting group was removed, and the hydroxy terminal was converted to a mesylate leaving group by reaction with methane sulfonyl chloride. To elongate the chain, the anion of tetraethylene glycol was prepared using sodium hydride in DMF. The tetraethylene glycol bound resin was added, and reaction continued at 120 °C for 24 hours. Cleavage of the resultant product from the polymer support yielded octaethylene glycol. Repetition of the mesylation and elongation steps followed by cleavage yielded dodecaethylene glycol. The oligoethylene glycols were purified by passage through a Fractogel 40S gel permeation column. Two different protecting groups for the tetraethylene glycol were tried. Trialkyl s i l y l groups were f i r s t attempted, but were abandoned due to reduced reactivity and monitoring d i f f i c u l t i e s during the deprotection. An acetate protecting group was f i n a l l y used and deprotection was monitored with infrared spectroscopy. i i TABLE OF CONTENTS Abstract i i List of Tables iv List of Figures v Acknowledgements vi Chapter One 1 1.1 Introduction 1 1.1.1 Glycocalyx 1 1.2 Synthesis of Model Glycolipid 3 1.2.1 Headgroups 4 1.2.2 Oligoethylene glycols 5 1.2.3 Polymer Supported Synthesis 6 Chapter Two 10 2.1 Introduction 10 2.2 Solution Synthesis 11 2.3 Polymer Supported Synthesis 21 2.3.1 Advantages and Disadvantages of the Polymer Supported Synthesis 25 2.3.2 Protecting Groups in the Polymer Supported Synthesis 27 2.4 Conclusions 33 Experimental 34 General Details 34 Solution Chemistry 35 Solid Phase Chemistry 45 References 53 Appendix 55 i i i LIST OF TABLES Table I: Influence of molar excess of triethylene glycol on yield 14 Table II: S i ly l removal conditions on the polymer backbone using 0.5 M Bu^ NF in THF 31 Table III: Acetate removal conditions on the polymer backbone 33 iv LIST OF FIGURES Figure 1: Structure of targeted glycolipid 3 Figure 2: Targeted lipids and glycolipids: n=2,3,5 10 Figure 3: Synthesis of methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl bromide)uronate (1_) 12 Figure 4: Synthesis of 3-0-(8-hydroxy-3,6-dioxaoctyl) cholest-5-ene (2) 14 Figure 5: Compounds (3) (n=3) and (4) (n=5) 15 Figure 6: Coupling reaction to produce protected glycolipids . . 17 Figure 7: Production of the target glycolipids 19 Figure 8: Reaction of monoprotected tetraethylene glycol to polymer bound trityl chloride 22 Figure 9: Production of polymer bound tetraethylene glycol [10) 23 Figure 10: Synthesis of polymer bound tetraethylene glycol mesylate 23 Figure 11: Synthesis of polymer bound octaethylene glycol (12) . 24 Figure 12: Acid cleavage of polymer bound glycols 25 Figure 13: Synthesis of mono t-butyl dimethyl s i l y l tetraethylene glycol (1J5) 28 Figure 14: Indirect analysis of s i ly l group removal 29 Figure 15: Synthesis of monoacetyl tetraethylene glycol (16) . . . 32 v ACKNOWLEDGEMENTS I would l i k e to express my appreciation to my supervisor, Don Brooks, f o r h i s patience and assistance throughout t h i s project, and to the other members of h i s group f o r t h e i r support. Thanks also to the s t a f f and f a c u l t y of the Chemistry Department at the U n i v e r s i t y of V i c t o r i a and p a r t i c u l a r l y to Tom Fyles and the members of h i s group f o r t h e i r kind h o s p i t a l i t y and p r a c t i c a l assistance during my stay in V i c t o r i a . v i CHAPTER ONE 1.1 INTRODUCTION The g l y c o c a l y x i s the r e g i o n o f a c e l l e x t e n d i n g o u t w a r d s f r o m the c e l l membrane. I t c o n s i s t s o f a c o m p l i c a t e d m i x t u r e o f g l y c o p r o t e i n s and g l y c o l i p i d s e x t e n d i n g f r o m l i p i d s embedded i n t h e p l a s m a membrane. The g l y c o c a l y x i s i n v o l v e d i n many c e l l u l a r f u n c t i o n s and phenomena. T h e s e phenomena a r e complex and d i f f i c u l t t o u n d e r s t a n d i n s i t u . A s i m p l e model s y s t e m i s t h e r e f o r e d e s i r a b l e i n o r d e r to a s s i s t i n the u n d e r s t a n d i n g o f the g l y c o c a l y x and i t s f u n c t i o n s , and i t was the a im o f t h i s p r o j e c t to d e v e l o p and s y n t h e s i z e one s u c h s y s t e m . 1.1.1 G l y c o c a l y x The g l y c o c a l y x i s a r e g i o n o f the c e l l t h a t i s c h e m i c a l l y v e r y c o m p l i c a t e d . I t i s i n v o l v e d i n many c e l l u l a r f u n c t i o n s s u c h as c e l l - c e l l o r c e l l - s u b s t r a t e i n t e r a c t i o n and c e l l u l a r m o b i l i t y . I n t e r a c t i o n s s u c h as a n t i g e n - a n t i b o d y r e a c t i o n s o c c u r i n the g l y c o c a l y x as w e l l as r e a c t i o n s o r p h y s i c a l phenomena r e s u l t i n g i n c e l l u l a r a d h e s i o n and r e p u l s i o n . A s t e p i n u n d e r s t a n d i n g many o f t h e s e phenomena i s t o d e t e r m i n e how the s t r u c t u r e o f the g l y c o c a l y x i n f l u e n c e s the p h y s i c a l measurements made on i t , and to use t h i s i n f o r m a t i o n to d e v e l o p and r e f i n e t h e o r e t i c a l mode ls o f the g l y c o c a l y x . Two s p e c i f i c phenomena w h i c h i l l u s t r a t e the d i f f i c u l t i e s o f d e t e r m i n i n g some o f t h e s t r u c t u r e / f u n c t i o n r e l a t i o n s h i p s o f t h e g l y c o c a l y x a r e t h e e l e c t r o p h o r e t i c m o b i l i t y and t h e p a r t i t i o n i n g b e h a v i o r o f c e l l s . I n t h e f i r s t o f t h e s e examples the c l a s s i c a l t h e o r e t i c a l mode l s w h i c h 1 describe the electrophoretic mobility of erythrocytes often predict much higher mobilities than are observed (1). This is thought to be because the hydrodynamic drag caused by the charged groups of the glycocalyx is usually either ignored or the charges are assumed to be at the l i p i d bilayer surface (2). More complicated theoretical models are the result of experimental results from erythrocytes and model c e l l s composed of gangliosides and phospholipids (1). These new theoretical models could be further refined and corrected with a more extensive model c e l l system. The second example of these d i f f i c u l t i e s is i l l u s t r a t e d by the partitioning behavior of ce l l s . Large particles such as c e l l s , when mixed in a phase system of two immiscible polymer solutions, w i l l often distribute themselves between one phase and the interface between the two phases. Their position reflects their relative attraction for one phase over the other based on their relative surface free energies when in contact with each liquid. The positioning can be influenced by changing the electrostatic potential between the two phases with the addition of salts which partition s l i g h t l y in favour of one phase. The positioning of the particles and hence the difference in surface free energy in each phase can be calculated by measuring the contact angle of the particle at the interface (3). The structure of the glycocalyx, including the positioning and numbers of charges within i t , plays a large role in the behavior of these particles in the two phase systems (4). As in electrophoretic mobility work, theoretic models are being used to attempt to interpret experimental results ( 5 ) , and a more extensive model system would aid this work. The examples described above are two of the many areas in which a more thorough understanding is needed of how the structure of the 2 glycocalyx of a cell influences its behavior. In order to further refine the current theories of these behaviors a simpler model cell system is required. 1.2 SYNTHESIS OF MODEL GLYCOLIPID It was the aim of this project to develop and synthesize a series of glycolipids that could be used to create a model glycocalyx for a model cel l . The targeted compounds have a cholesterol base, an oligoethylene glycol spacer group and a glucuronic acid headgroup (see Figure 1). These compounds could be formed into model cells in mixtures with phospholipids to be used in both electrophoretic experiments and partitioning experiments. headgroup spacer group base Figure 1: Structure of targeted glycolipid It has been reported ( 6 , 7 ) that cholesterol coupled to oligoethylene glycol units can be formed into liposomes: closed bilayer vesicles. These liposomes and derivatives of them have been used to investigate their possible use as drug delivery systems ( 6 , 8 ) . Potentially, based on the headgroup attachment, the liposome carrier could be targeted to a 3 specific tissue in the body. No synthesis or application of such molecules with a charged headgroup has been reported, however. The synthesis involves coupling the various parts of the desired product together. First, the oligoethylene glycol is coupled to the cholesterol by displacing a tosylate group from cholesteryl tosylate. The headgroup can be added by protecting and brominating i t , and then coupling it to the hydroxyl end group of the cholesteroxy oligoethyene glycol ( 9 ) . The final step is the removal of the headgroup protecting groups. The advantage of synthesizing this type of glycolipid for a model glycocalyx over that of simply mixing gangliosides with phospholipid bilayers is that the structure of the model glycocalyx may be systematically varied. The variation may be produced in several ways. One method would be to vary the concentration of the headgroups in the liposomes by varying the compositions of the mixtures of the synthesized product and the phosphlipid. Another would be to change the distance of the headgroup from the surface of the liposome by varying the length of the oligoethylene glycol spacer group (n in Figure 1). Finally the charge on the headgroup may be manipulated by varying pH. 1.2.1 Headgroups The headgroup used in this project is glucuronic acid. This headgroup was chosen for a variety of reasons. Because it contains a carboxylic acid it has the potential to be negatively charged. The negative charge will make a model glycocalyx more useful in electrophoretic and partitioning experiments than that which could be formed from glycolipids made using neutral sugars such as glucose and galactose. Yet glucuronic acid is s t i l l a simple sugar, and thus is 4 relatively easy to work with. Possibilities for other model cell variation include using different headgroups. Most of the headgroups would be saccharides of varying complexity and charge. An example might be s ial ic acid which would give a headgroup that is known to be involved in biological recognition phenomena. Other choices could include using oligosaccharides as headgroups. Finally, the model glycocalyxes could also be varied by embedding a charged group within the spacer group. 1.2.3 Oligoethylene glycols The spacer group used in the model glycocalyx synthesis is oligoethylene glycol. Oligoethylene glycols are available commercially from a variety of companies. It is possible to buy specific oligomers up to heptaethylene glycol in amounts useful for synthetic work. Besides these specific oligomers it is possible to buy heterodisperse polyethylene glycol fractions of average molecular weight 400, 600 etc. "Monodisperse" oligoethylene glycols higher than heptaethylene glycol must be synthesized or separated. For this project, it was hoped to use only monodisperse spacer groups, or as close to that ideal as possible, for oligoethylene glycols under molecular weight 1000, so some effort was put into developing a method to acquire these units. Purification of some of the heterodisperse fractions was the first idea pursued. Separation could be achieved to some extent using a Fractogel 40S aqueous gel permeation column with a low pressure chromatographic system and a refractive index detector, but the separation was not near the baseline. Preparative scale HPLC with a size exclusion column could be used and would probably be successful. However, 5 such a system was not a v a i l a b l e t o u s . The next idea was to synthesize various oligomers s p e c i f i c a l l y . This idea had some precedence. Hexaethylene g l y c o l and octaethylene g l y c o l have been synthesized (10,11), and recently the synthesis of nonaethylene g l y c o l and decaethylene g l y c o l has been reported (12). Several attempts were made to produce higher molecular weight oligoethylene g l y c o l s , but none were very successful. Poor y i e l d s and the d i f f i c u l t i e s with chromatography and p u r i f i c a t i o n presented major obstacles, so a s o l i d state synthesis was introduced. Most of the e f f o r t s to acquire longer chain monodisperse oligoethylene g l y c o l s centred around using a s o l i d polymer supported synthesis to produce s p e c i f i c oligoethylene g l y c o l chains from multiples of tetraethylene g l y c o l . Using t h i s method the oligoethylene g l y c o l s could be prepared on the polymer. The f i n a l p u r i f i c a t i o n could be done on the gel permeation column because t h i s column does baseline separate ethylene g l y c o l oligomers d i f f e r i n g by four units. 1.2.3 Polymer Supported Synthesis Polymer supported synthesis i s a synthetic method i n which the substrate i s attached to an insoluble polymer. Reactions are c a r r i e d out on the polymer bound substrate; at the conclusion of the synthesis the f i n a l product i s cleaved from the polymer f o r f i n a l p u r i f i c a t i o n . Polymer bound reagents and polymer bound c a t a l y s t s are also used i n synthetic work (13). In a l l cases the polymer o f f e r s the same types of advantages. These advantages centre around the f a c t that the polymer bound compound -be i t substrate, reagent or c a t a l y s t - i s e a s i l y separable from the other species present i n the reaction mixture (14,15). The advantages are well 6 illustrated in the case of the polymer bound substrate. Product work up is done simply by filtering the polymer from the solvent, reagents and biproducts. A series of reactions can be carried out without lengthy isolation and purification procedures between each step. Yields are often high because there are no losses due to work up and purification, so a lengthy synthesis often has a much higher overall yield than is possible using solution synthesis. Also, large excesses of reagents may be used without causing separation problems. The polymer is reusable so the reactions can often be quite economical. Finally, the reaction sequence has potential to be automated (14,15). Polymer supported synthesis was first developed in 1963 by R. B. Merrifield. He was involved in the field of polypeptide synthesis and was concerned with the problem of synthesizing long chain polypeptides. Technical problems with solubility and purification were the major obstacles which had limited the length of polypeptide chains until this time (16). Merrifield realized that if the first peptide could be attached to an insoluble support the isolation and purification of intermediate products could be accomplished with fi ltration and rinsing, therefore that product losses would be reduced and time would be saved (17). Merrifield's first published project in this area was the production of the tetrapeptide L-leucyl-L-alanylglycyl-L-valine (16). Refinements in the chemical methods, followed by the development of a machine to automate the process, culminated in the synthesis of the 124 residue bovine pancreatic ribonuclease A in 1971 (18). Since that time polymer supports have been used in a great variety of different applications. Peptide synthesis has been revolutionized, and it is now a routine procedure to produce very long polypeptide chains 7 (14,15). Other repetitive synthetic projects have included polynucleotides and oligosaccharides (19,20). Polymer supported catalysts also have been used and studied extensively, and polymer supported reagents such as ion exchange resins and oxidizing agents are well known (13). Polymer supports have found their most dramatic uses in automated repetitive synthesis. However, they have also been used in many other synthetic schemes. The polymer offers the advantage of high dilution because the active sites on the polymer isolate one molecule from another. An example of this feature's advantages is provided by polymer bound titanocene as a hydrogenation catalyst. The titanocene by itself rapidly dimerizes and loses its activity. The dimerization is prevented on the polymer support (21). Asymmetric syntheses can sometimes be achieved because of the microenvironment of the substrate molecule on the polymer. An example of the use of this feature is the synthesis of (S)-2-methylcyclohexanone which was prepared in 95% optical yield on a polymer support (22). Because of the many advantages of polymer supports it was thought that the method would be useful for the synthesis of some longer oligoethylene glycols. Tetraethylene glycol was available as an inexpensive building block and could be used in excess. The workup of simply fi ltering away the excess reagents solved the problem of separating the unreacted tetraethylene glycol from the product. The f lexibi l i ty of the method gives it potential for use in the synthesis of model glycocalyxes with various different spacer groups. It is also possible that it could be developed for other syntheses such as crown ether synthesis. 8 In summary, the s y n t h e s i s o f the d e s i r e d model g l y c o c a l y x e s was attempted u s i n g two d i f f e r e n t methods. S t a n d a r d s o l u t i o n s y n t h e s i s was used t o l i n k t o g e t h e r the s e p a r a t e s e c t i o n s o f the compounds. The polymer s u p p o r t e d s y n t h e s i s o f o l i g o e t h y l e n e g l y c o l s was d e v e l o p e d to make p o t e n t i a l s p a c e r groups f o r these compounds. I t i s p o s s i b l e t h a t b o t h methods c o u l d be f u r t h e r d e v e l o p e d to produce more g l y c o l i p i d s o f v a r i o u s s t r u c t u r e f o r many f u t u r e p h y s i c a l c h e m i c a l s t u d i e s . 9 CHAPTER TWO 2.1 INTRODUCTION The goal of this project was to synthesize a series of lipids and glycolipids. Their structures are: Figure 2: Targeted lipids and glycolipids: n=2,3,5 The synthetic work of this project may be divided roughly into two parts. The f irst part was the solution synthesis of the above compounds using available ethylene glycols. This synthesis was carried out in four major steps. The second part was the development of a polymer supported synthesis of ethylene glycol oligomers that are not readily available. This five step synthesis was used to produce bctaethylene glycol and dodecaethylene glycol. 10 2.2 SOLUTION SYNTHESIS The f irst part of this project was the synthesis of the complete lipids and glycolipids. The first of the four major steps in this synthesis was the protection and bromination of the headgroup. The second was the coupling of the spacer group to the l ipid base. The third step was the coupling of this new lipid to the headgroup, and the final step was the removal of the protecting groups from the headgroup of the protected glycolipid. This synthesis is staightforward and most of the reactions are easy to do. However, yields of some of the reactions are quite low or require large excesses of one substrate. Also, the protecting groups are difficult to remove. The f irst major step in this synthesis was the production of the protected and brominated glucuronic acid (see Figure 3). Glucuronolactone was the starting material. The reaction had three parts, two of which occurred in a single vessel. These parts were: the base catalysed esterification of the glucuronolactone to produce methyl glucuronate, the acetylation of the hydroxyl groups of the methyl glucuronate and the bromination at the anomeric carbon of the resulting methyl tetra-O-acetyl-p-D-glucopyranuronate. These reactions were a l l done following published procedures. The procedure to synthesize the methyl tetra-O-acetyl-p-D-glucopyranuronate is described in Bollenback et al . (23). The f irst product to crystallize was the fi anomer in 50% yield. It 1 was the only anomer to be isolated. The H NMR spectrum shows a doublet at 5.8 ppm with a coupling constant of 9 Hz which indicates that the CI and the C2 hydrogens are both axial (ie. the product is the fi anomer). The bromination step was done according to a procedure also published by Bollenback et a l . (23), and yields of up to 80% were attained. Based on 11 H NMR data the product was the a anomer. The CI hydrogen doublet is at 6.65 ppm and has a coupling constant of 4 Hz indicating an equatorial/axial relationship of the CI and C2 hydrogens. C=0 OH. MeOH ^  Ac 0, py Ac OAc lAc glucuronolactone methyl t e t r a - 0 - a c e t y l - £ - D -glucopyranuronate Br Figure 3: Synthesis of methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl The second step of the synthesis was the coupling of the oligoethylene glycol "spacer" group to cholesterol. This was first done using triethylene glycol as described by Patel et al. (6) (see Figure 4). Triethylene glycol and cholesteryl p-toluene sulfonate in a 25:1 molar ratio were dissolved in dioxane and stirred together at reflux under a nitrogen atmosphere. The resulting 38-0-(8-hydroxy-3,6-dioxaoctyl)cholest-5-ene (compound 2) was isolated and purified by passage through a s i l ica gel column in 73% yield. The product was a thick syrup which could be neither disti l led nor crystallized. Thin bromide)-uronate (compound 1_) 12 layer chromatography confirmed the presence of a single compound. Analysis by *H NMR showed the presence of oligoethylene glycol hydrogens and cholesteryl hydrogens in approximately a 1:4 ratio which is expected as there are 12 hydrogens associated with the non terminal oligoethylene glycol positions and there are 46 cholesteryl hydrogens. The peaks were assigned on the basis of comparison with standard spectra (24), and 1 triethylene glycol peaks obtained from the H NMR spectrum of pure triethylene glycol. The mass spectrum showed the expected molecular ion at m/e 517 and logical fragment ions, for example: m/e 368 (the 100% peak) is the mass of the cholesta-3,5-diene fragment and the m/e 149 peak indicates the triethylene glycol fragment. This coupling reaction was tried at several different molar excesses in an attempt to reduce the amount of triethylene glycol required. As expected, it was found that smaller excesses led to lower yields (see Table I). It was also found that yields dropped as the oligomer length was increased. For instance, in an analogous reaction a five fold molar excess of hexaethylene glycol gave only a 26% yield. Cost was a consideration in accepting the lower yield as the higher ethylene glycols rapidly become more expensive than the cholesterol tosylate. 13 Figure 4: Synthesis of 3-0-(8-hydroxy-3,6-dioxaoctyl)cholest-5-ene (compound 2) Table I: Influence of molar excess of triethylene glycol on yield Reaction Molar ratios Yields number triethylene glycol cholesteryl-p-toluene sulfonate (purified) 1 25 1 73% 2 10 1 63% 3 , 4 5 1 50% 14 3-0-(1l-hydroxy-3,6,9-trioxaundecyl)cholest-5-ene (compound 3) and 3-0-(17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl)cholest-5-ene (compound 4) were synthesized using a five fold molar excess of the ethylene glycol oligomer (see Figure 5). The mass spectra show molecular ion peaks and a 1 similar fragment pattern to that seen for (2), and the H NMR spectra are a l l similar to the spectrum of (2) showing the presence of both cholesteryl and the oligoethylene glycol portions with approximately correct ratios of oligoethylene glycol hydrogens and cholesteryl hydrogens. Figure 5: Compounds (3) (n=3) and (4) (n=5) There is no indication of large impurities of other ethylene glycol oligomers in the product or of the dicholesteryl product. The impurities in the length of oligomer are difficult either to prove or rule out. For example, though the mass spectra of (2) and (3) show very small peaks that could correspond to the molecular ion peaks of the di and penta analogues respectively, there is nothing to indicate that these are not simply fragment ions. It was assumed that the impurities in the products would not be significantly higher than in the original oligoethylene glycol 15 used. These purities were indicated by the manufacturer (Aldrich) to be at least 98%. The presence of a small amount of the dicholesteryl product in a sample of (2) is indicated by a small peak at m/e 906 in the DCI mass spectrum of that product. That the amount is small (<5%) is also indicated because a large amount would begin to show itself in altered NMR integrations and a higher carbon percentage in elemental analysis. Similar peaks were not observed in the DCI mass spectrum of (4) or the EI mass spectrum of (3). The third step in the synthesis was the coupling of (2), (3) and (4) to (JJ (Figure 6). The reactions were Koenig Knorr type reactions. Silver carbonate catalyst was freshly synthesized for the reactions according to the instructions found in Feiser and Feiser (24) in about 10% scale. The products of the coupling reactions were isolated and purified in up to 30% yield. Again, these products could be neither disti l led nor crystallized and the purification was done using a s i l ica gel column with the products emerging between the two starting compounds. The identity of 1 the products was confirmed using H NMR as well as mass spectrometry, infrared spectroscopy and elemental analysis. The NMR spectra show peaks of the cholesteryl, the oligoethylene glycol and the methyl tri-0-acetyl-/3-D-glucopyranuronate portions of the compound, and were assigned by comparison with standard spectra (24,27,28). Al l three reactions were carried out in a similar manner and the products a l l behaved similarly during the purification. 16 .C02Me, A c O ^ ^ v (1) OAc] Br n=2,3,5 .C0 2 Me, Ag2C03 benzene A C C ^ > \ r\\ i[\ OAc n=2, methyl [3-0-(3,6-dioxaoctyl)cholest-5-en-3f3-yl-2,3,4-tri-0-acetyl-/3-D glucopyranosid]uronate (5) n=3, methyl [3-0-(3,6,9-trioxaundecyl)cholest-5-en-3/3-yl-2,3,4-tri-0-acety p-D-glucopyranosid]uronate ( 6 ) n=4, methyl [3-0-(3,6,9,12,15-heptadecyl)cholest-5-en-3|3-yl-2,3,4-tri-0-acetyl-p-D-glucopyranosid]uronate (7) Figure 6: Coupling reaction to produce protected glycolipids The fourth and final step in the production of the final target compounds was the removal of the acetate and methyl ester protecting groups of the glucuronate portion of the compounds (see Figure 7). Because the final product is a powerful surfactant, and difficult to handle, a clean protecting group removal is required. The first attempt 17 was removal of the acetate groups using sodium methoxide in anhydrous 1 methanol followed by ion exchange with Amberlite. H NMR showed that this step was successful: the three acetate hydrogen peaks were no longer visible. The methyl ester was removed using sodium hydroxide in water followed by ion exchange to remove the sodium. 1H NMR showed that this step did remove the methyl ester, but the resulting product was not pure. The base concentration was lowered, and the Amberlite resin was more carefully cleaned by repeated rinsing with disti l led water in an effort to improve the purity of the final product. Also, the acetate removal was combined with the methyl ester removal in one step. None of these measures was successful in providing clean spectra of the products. The removal of the acetate groups and the methyl ester was finally accomplished by adapting the method described by Harris et al . in 1969 (26). The acetate removal occurred in an ether, dichloromethane solvent mixture using 0.1 molar sodium hydroxide in anhydrous methanol. After three hours tetrahydrofuran was added and the methyl ester was removed by the addition of aqueous 1.0 molar sodium hydroxide, methanol and more water. The mixture was acidified and concentrated. The product precipitated, but it was not possible to f i l ter the precipitate from the supernatant salt solution. Instead, the supernatant was decanted and the precipitate was rinsed. (4) and ( 5 ) were both successfully treated with 1 13 this procedure to produce (8) and (9). Both H NMR and C NMR were used to characterize the products. There are no extra peaks in either spectrum 13 and every peak in the C NMR can be identified. Peak identification for both the cholesteryl and glucuronic acid portions of the compounds were made by comparisons with literature spectra (24,27,28). The other peaks in the spectra were found to correspond both in number, location and 18 integration to those expected for the oligoethylene glycol portion of the compounds. Full characterization of the tetra analogue was carried out. Because the final surfactants are potentially more biologically active than their protected analogues it was decided to leave any other compounds protected until shortly before use. n=2,3 (5),(6) n=2, [3-0- (3 ,6-d ioxaocty l )choles t -5-en-3 /3-y l -£-D-g lucopyranos id] uronic acid (8) n=3, [3-0-(3, 6, 9-trioxaundecyl)cholest-5-en-3/3-yl-/3-D-glucopyranosid] uronic acid (9) Figure 7: Production of the target glycolipids 19 The above reaction procedure is simple and has several attractive points. The methyl tetraacetyl glucuronate is stable on storage, so it may be prepared in a large batch and used as stock. The two coupling reactions - the coupling of the ethylene glycol oligomer to the cholesterol tosylate and the Koenig-Knorr reaction - are both easy to do and the products are straightforward to purify. There are, however, problems. The yields of the coupling reactions were not high, especially the Koenig Knorr reaction where the yield was about 30%. The other major limitation was the lack of readily available monodisperse oligoethylene glycols, which limited the length of the spacer chain. The problem of producing longer chain oligoethylene glycols could be solved in a variety of ways, including separation from mixtures and synthesis. Separation of individual oligomers from average molecular weight mixtures containing sequential oligomers was a possibility, but was not pursued because it would have required a preparative scale gel permeation column on an HPLC apparatus. This equipment was not available at the time of the experiments. Synthesis of longer specific oligomers from shorter, commercially available monodisperse oligomers was attempted. Bi and t r i molecular reactions were tried. For example, pentaethylene glycol was condensed with itself in an attempt to produce decaethylene glycol according to the synthesis described by Nakutsuji et al . (10). Also, condensation of tetraethylene glycol to each end of tetraethoxyditosylate to produce dodecaethylene glycol was attempted. Decaethylene glycol was probably produced in low to moderate yield, but the dodecaethylene glycol experiment failed. Based on gas chromatography 20 data, yields were low in these and other experiments and purification was diff icult , so this work was not pursued. 2.3 POLYMER SUPPORTED SYNTHESIS It was decided to try to develop a polymer supported synthesis that would produce certain longer chain ethylene glycol oligomers. The synthesis of octaethylene glycol occured in five major steps. First, the substrate was added to the polymer and then deprotected. The third step was the conversion of the hydroxyl group to a mesylate group. The fourth step was the extension of the tetraethylene glycol, and the f ifth step was the removal of the octaethylene glycol from the polymer support. Dodecaethylene glycol could also be produced by repeating the third and forth steps before cleaving the product from the polymer. This synthesis of specific oligomers had many advantages over the solution synthetic attemps described briefly above. However, it also has some unique problems concerning the monitoring of the reactions as well as problems of changed reactivities between substrates bound to the polymers and those same substrates not bound to polymers. This last problem was most obviously illustrated in the problems of protecting group removal with trialkyl s i l y l and acetate protecting groups. The polymer used in this synthesis was polystyrene 1% divinyl benzene with tr i ty l chloride active sites. The polymer was obtained from Polysciences (lot 953-7) and was treated with butyl lithium followed by reaction with benzophenone to produce tr i ty l alcohol active sites. These sites were then chlorinated with acetyl chloride. The polymer was titrated against 5% HNO^  and found to have 0.55 mmol chloride per gram of polymer. 2 1 The f irst step of the synthesis was to attach the f irst tetraethylene glycol unit to the polymer (see Figure 8). The chloride on the polymer is the leaving group in this reaction and the hydroxyl group of the tetraethylene glycol is the nucleophile. Because tetraethylene glycol has two hydroxyl groups it is possible for a tetraethylene glycol molecule to react with two different tr i ty l chloride groups. Further reactions on this particular tetraethylene glycol would be prevented, and the overall synthesis would have a lowered yield. In order to avoid this problem the tetraethylene glycol was monoprotected. The synthesis of the monoprotected tetraethylene glycol is described more thoroughly in section 2.4.2 of this discussion. The attachment of the monoprotected tetraethylene glycol to the polymer was successful. Acid cleavage of one gram of the resultant polymer resulted in the recovery of 85 mg (0.5 mmol) of tetraethylene glycol. (^=polystyrene 1% divinyl benzene PG=protecting group Figure 8: Reaction of monoprotected tetraethylene glycol to polymer bound tr i ty l chloride Before continuing with the reaction the protecting group on the polymer bound tetraethylene glycol had to be removed (see Figure 9) This step proved to be quite difficult, and it is described in some detail in section 2.4.2 of this discussion. 22 © r{\ T r —0 k 0 J 3 OPG P ) T r — 0 K 0 J ® OH (10) Figure 9: Production of polymer bound tetraethylene glycol (10) After having attached the first tetraethylene glycol unit and removed the protecting group the next step was to convert the hydroxyl terminal of the tetraethylene glycol to a good leaving group. This was done by reacting the polymer bound tetraethylene glycol with methanesulfonyl chloride to produce the polymer bound tetraethylene glycol mesylate (see Figure 10). The product of this reaction could be observed by infrared spectroscopy. The sulfur-oxygen stretch shows at 1350 cm © T r — 0 r \ / n k o OH MsCl bz, py © T r — 0 I 0 ) 3 OMs ( i i ) Figure 10: Synthesis ofpolymer boundtetraethylene glycol mesylate(11) The next step was to extend the polymer bound tetraethylene glycol chain to polymer bound octaethylene glycol. This was done by making the anion of tetraethylene glycol in solution and reacting it with the polymer bound tetraethylene glycol mesylate (see Figure 11). The reaction was evaluated by analysing the products of acid cleavage of some of the polymer after the reaction. These products were analysed by various means. Some form of chromatography was necessary to separate different 23 5 oligomers of the same structure. Gas chromatography was attempted; it proved to be useful and was used ini t ia l ly . Subsequently, it was found that gel permeation chromatography would work to separate different oligomers and could be made to do so on a preparative scale. Fractogel 40S was able to baseline separate oligoethylene glycols four units apart. For example, tetraethylene glycol and octaethylene glycol can be separated. r\\ l[\ NaH r|\ / n . HO I 0 J 3 OH HO I 0 J 3 0 3 DMF ( P ) T r —0 r(\ /|-\ I 0 J 3 OMs DMF n HO ^ o ) 3 o ( P ) T r —0 I 0 > 7 OH(12) Figure 11: Synthesis of polymer bound octaethylene glycol (12) The mesylation of the hydroxy terminal and the extention of the oligoethylene glycol chain were repeated prior to product cleavage from the polymer to produce dodecaethylene glycol. Dodecaethylene glycol is 1 also separable on the fractogel column. H NMR and infrared spectroscopy were used to confirm the structure of the octa and dodeca ethylene glycols. Al l the hydrogens are similar and have similar shifts so they appear together as a broad singlet or multiplet at 3.7 ppm. Infrared 24 spectra show OH stretching bands and C-0 stretching bands. The molecular weights of the products were confirmed with DCI mass spectrometry, both of which show strong (M + NH^)+ peaks. The final step is acid cleavage of the chain from the polymer support (see Figure 12). This was usually accomplished using about 5 mL/g polymer of dry hydrochloric acid in dioxane (0.5 M), but could also be done, though not as efficiently, with several drops of concentrated hydrochloric acid in dioxane. © r\\ in Tr—0 I 0 Jn OH HCI dioxane n=7,11 r(\ in HO I 0 Jn OH n=7, octaetylene glycol (13) n=8, dodecaethylene glycol (14) Figure 12: Acid cleavage of polymer bound glycols 2 . 3 . 1 Advantages and Disadvantages of Polymer Supported Synthesis The use of polymer supported synthesis in the production of oligoethylene glycols offers several advantages over the attempts to make these compounds in solution. The work up in polymer supported synthesis is simply fi ltration of the polymer and its attached compounds. Solvents, starting materials and many impurities are washed away. This is an attractive feature when making oligomers of ethylene glycol. Excess tetraethylene glycol used to make octaethylene glycol is easily rinsed away without having to either d i s t i l l the excess tetraethylene glycol and thus create more impurities in the stillpot residue or attempt a lengthy large scale fractogel column separation. Another advantage is that purification need only be done at the last step after cleavage of the 25 product from the polymer. This eliminates the need for time consuming and yield depleting purification procedures on the intermediate products. The final advantage lies in the flexibility of the method. Once developed, the method may be used for other syntheses. Along with a l l the advantages of the polymer supported synthesis of the oligoethylene glycols, the method also presents some difficulties. Major among these is monitoring the reactions occuring on the polymer and analysing the products of those reactions. Some ordinary methods cannot be utilized. The polymer backbone is very much the dominant part of the mixture. Mass spectrometry cannot be used as the polymer fragment ions would overwhelm any substrate peaks. Elemental analysis of carbon, hydrogen and oxygen would be dominated in the same way. Routine NMR is also impossible because of the insolubility of the polymer in any solvents. Analysis techniques that are possible are solid state NMR spectroscopy and infrared spectroscopy. Solid state NMR was not pursued as most of the work for this project was done where there are no faci l i t ies for this, but it is a potentially valuable aid to this synthesis. Infrared spectroscopy using KBr pellets was used most often for this project. This method was very valuable for compounds that contained infrared active functional groups. Finally, the polymer bound substrates may be cleaved from the polymer and analysed fully. This was done with some compounds, however, many intermediates do not survive the cleavage step. Another major problem to be overcome in a polymer supported synthesis is that substrates bound to polymers often have different reactivities than the substrates in solution. This problem often results 26 in reduced reactivity which can sometimes be improved by changing solvents or temperatures as well as increasing the length of reaction. The proper reaction conditions for the polymer bound reactions are often best found by tr ia l and error (13). In this project both the difficulty of monitoring the reaction and the difficulty of lowered reactivities became problems when removing the protecting group from the polymer bound tetraethylene glycol. 2.3.4 Protecting groups in the polymer supported synthesis A . Trialkyl s i l y l groups The f irst protecting group that was used for protection of one of the hydroxyl groups of the tetraethylene glycol was t-butyl dimethyl s i ly l chloride. Monoprotected tetraethylene glycol was synthesized (see Figure 13), and the resulting mono t-butyl dimethyl s i ly l tetraethylene glycol I (15) was purified by disti l lation. H NMR, infrared and mass spectroscopy showed the product to be primarily mono protected (80% mono). The small amount of the diprotected product was not a problem because this product could not react with the polymer and would be filtered from the polymer at the end of the reaction. The t-butyl dimethyl s i ly l group was chosen as the protecting group for two major reasons. The group was easy to attach to the tetraethylene glycol, and the removal conditions are neutral and mild. The monoprotected tetraethylene glycol was easily produced, isolated and purified in a large amount (lOOmL). Also, the group is a flexible protecting group for alcohols, in part because the removal conditions are gentle, quite neutral and fast. For example, the s i l y l group can be completely removed at 22 °C in 1M tetrabutyl ammonium fluoride (Bu.NF) in 27 THF in 30 minutes (29), and other mild conditions are sometimes possible (30,31). i r\ ir\ (CH3)3C—Si—0 U J 3 OH (15) CH3 Figure 13: Synthesis of mono t-butyl dimethyl s i l y l tetraethylene glycol (15) Compound (1_5) was successfully added to the polymer support as described in section 2.3. The major limitation with this protecting group was the problem of monitoring the removal of the s i ly l protecting group. The s i ly l groups are not visible in the IR range so IR could not be used. Solid state NMR was not available where most of this work was done so direct analysis was available only by elemental analysis of silicon. This required that samples be sent away to be analysed, which was both expensive and slow. Acid cleavage of (.15) from the polymer also cleaved the protecting group from the tetraethylene glycol; so it was not possible to analyse cleavage products to te l l the extent of the previous removal. Finally, an indirect method was developed (see Figure 14). After the attempted deprotection the 28 CH, (CH 3) 3C— Si—CI CH3 t-butyl dimethyl s i l y l chloride imidazole DMF HO r(\ in k o > OH tetraethylene glycol freed hydroxyls were acetylated with acetic anhydride in pyridine. The acetate group is easily seen in IR spectra, and it was found that the acetate group was stable to the acid cleavage conditions. The acetylated tetraethylene glycol was acid cleaved from the polymer and *H NMR spectra were taken. Integrations of oligoethylene hydrogen and acetate hydrogen peak heights could be compared to tel l the extent of the acetylation and thus the extent of the removal of the s i l y l group. a) ( C H 3 ) 3 C i r\ :— Si—0 I 0 J , O T r ( ? ) C H -Bu4NF THF HO k 0 O T r ( P ) + rf\ /h S i — 0 k 0 >3 O T r ( ? ) b) r{\ iY\ -0 k 0 ) 3 O T r ® HO k 0 )3 O T r ( P ) py S i — 0 k 0 )3 O T r ( ? ) :0 k 0 )3 O T r ® c) i—0 U ; 3 O T r ® r{\ fr\ I 0 J , OT r A c O HCI dioxane r{\ ir\ HO k 0 )3 OH + c^0 k 0 OH a) removal of s i ly l protecting group b) acetylation of free hydroxyl groups c) cleavage of glycols from polymer support Figure 14: Indirect analysis of s i ly l group removal 29 The removal of the trialkyl s i ly l group was attempted on (.15) in solution, and based on the results obtained it was decided to try the following conditions for the removal of the trialkyl s i l y l group from the material bound to the polymer: 25 °C, 0.5M Bu^ NF in THF (10 mL/g resin) for two hours. Unfortunately, the conditions needed were not this gentle. At f irst , because of the monitoring difficulties, it was simply assumed that the deprotection had occured completely, and subsequent reactions were tried. None of these worked, so the deprotection step was more completely investigated. The conditions were made harsher by increasing the time of the reactions and by increasing the temperature from room temperature to 65 °C (reflux) in THF. DMF was also tried as solvent. The indirect analysis method (described above) was developed, and samples of the polymer that had been subjected to a certain series of deprotection conditions were sent for elemental analysis. Other attempts were made using the trimethylsilyl analogue of this protecting group. This analogue is generally more easily cleaved than the t-butyl dimethyl s i l y l group (31). The trimethyl s i ly l group was also subjected to varying removal conditions, and was analysed by the indirect method. In no case was the protecting group found to be completely removed by the treatment. Even after 48 hours at room temperature and 24 hours at reflux (65 °C) repeated twice, elemental analysis showed that about 60% of the original silicon remained. The indirect method was in agreement with these findings, and as it was more convenient and less expensive it was used exclusively to analyse for the trimethylsilyl group. Even this more labile group could not be removed from the polymer bound tetraethylene glycol under the conditions attempted. Results are summarized in Table II. 30 Table II: S i ly l 0.5 M removal conditions on the Bu4NF in THF polymer backbone using cleavage conditions elemental analysis (% silicon) indirect: acetylation of unprotected hydroxyls no cleavage 1.51 24 hr 65 °C (2X) 1.01 small acetate peaks only 48 hr 25 °C, 24 hr 65 °C (2X) 0. 88 small acetate peaks only B.Acetate protecting group Because of the failure to remove the silicon protecting groups, acetate was tried instead. This protecting group is not as attractive as the tr ialkyls i ly l groups because it is less selective than they are. Another important limitation is that the group is removed in base. For the production of oligoethylene glycol this is not a difficulty, but for other syntheses where other functional groups or protecting groups may be involved the acetate removal conditions may not be as satisfactory as those for trialkyl s i l y l protecting groups. Mono acetyl tetraethylene glycol (1_6) was made by reaction of tetraethylene glycol with acetic anhydride in pyridine (see Figure 15). 1 The product was purified by disti l lation and analysed by H NMR, IR, mass spectroscopy and elemental analysis. Based on the molecular ion peak heights in the mass spectrum, the product contained approximately 10% of the diacetyl tetraethylene glycol. Elemental analysis calculated to 31 include 10°/ of this impurity was a l s o i n agreement with that which was found. As before, the presence of this impurity is not considered serious as the diacetyl tetraethylene glycol cannot react with the polymer and is filtered away at the end of the reaction. r\\ / n ^ r\\ fr\ HO I 0 J , OH Ac20 — HO 1 0 ) , OAc (16) • 2 py 3 Figure 15: Synthesis of mono acetyl tetraethylene glycol (16) Monitoring the extent of deprotection was much more satisfactory with the acetate protecting group than with the trialkyl s i l y l protecting groups. The monitoring of the removal of the acetate protecting group was done using infrared spectroscopy. The acetate carbonyl appears at 1730 cm^  and disappears as the deprotection progresses. The f irst conditions tried for the removal of the acetate protecting group on the polymer were 0.5 M benzyl amine in pyridine, 6 mL/g polymer. As with the trialkyl s i ly l groups, the acetate group was much harder to remove than was expected. The final conditions required were found after trying the series listed in Table III. Nine grams of monoacetyl tetraethylene glycol bound to polymer was treated with 53 mL 0.5 M benzyl amine in pyridine. After 16 hours at 50 °C the polymer was filtered and rinsed. KBr IR showed a large acetate peak. The same polymer was then treated as before with the addition of 0.55 g dimethyl amino pyridine in the 53 mL of 0.5 M benzyl amine in pyridine. The reaction went again for 16 hours at 85 °C. The IR spectrum of the filtered and rinsed polymer 32 s t i l l showed the acetate peak at 1730 cm This second set of conditions was repeated on the same polymer, again with the same results. The next attempt used the same polymer with 3 mL per gram of a 1:1 v/v mixture of pyridine and benzyl amine mixture (4.7 M) left for 16 hours at reflux (120 °C). After fi ltering and rinsing the acetate peak, though slightly smaller, was s t i l l present. The final conditions tried were using the same polymer (9 grams) in 20 mL of neat benzyl amine at 160 °C for 65 hours. After fi ltering and rinsing, the acetate peak at 1730 cm '''was absent and the synthesis could be continued. Table III: Acetate removal conditions on the polymer backbone Benzyl amine Solvent Temperature Time 1730 cm * peak concentration (M) (°C) (days) (IR spectra) 0.43 pyridine 115 1 present 4.60 pyridine 115 1 present neat — 160 4 absent 2.4 CONCLUSIONS The synthesis of (5), (6) and (7) was developed and these compounds were produced. A method for the deprotection of (5) and (6) was developed which possibly could be used with other protected analogues. A polymer supported synthesis was developed to synthesize oligoethylene glycols higher than heptaethylene glycol. Octa and dodecaethylene glycol were synthesized by this method. The polymer supported synthesis has potential for further use in synthesizing products for this project. 33 EXPERIMENTAL General Details Proton NMR spectra were recorded on Bruker WH-400 MHz, Varian XL-300 MHz, Brucker WM 250.13 MHz, and Perkin Elmer R32 90 MHz spectrometers in CDCT„ or MeOD-d. as the solvent. Carbon spectra were recorded at 62.9 MHz 3 4 ^ (WM 250) in MeOD-d^  or CDCl^ with the central solvent line as standard (49.0 (MeOD-d4) and 77.0 (CDC13) relative to Me4Si). Mass spectra were recorded on Kratos MS80 RFA with 70eV (EI), Delsi/Nermag R10-10C with the heating current at 7mA/s with ammonia (DCI), and Finnegan 2200 GC-MS with methane (CI) mass spectrometers. Infrared spectra were recorded on Perkin Elmer 283, Pye Unicam SP1100, Bomem MB 100 and Perkin Elmer 783 infrared spectrometers. Elemental analyses were carried out by Guelph Chemical Laboratories Ltd. (Silicon analyses) and by P. Borda of UBC Microanalytical Service (carbon, hydrogen analyses). Solvents and reagents were used as purchased with the exception of those noted below. Dichloromethane was disti l led prior to use. Dimethyl formamide was dist i l led from C a ^ and stored over freshly activated 4A molecular sieves. Tetrahydrofuran was disti l led from potassium benzophenone ketyl under inert atmosphere immediately prior to use. Triethylene glycol and tetraethylene glycol were disti l led under vacuum. The polymer used was divinyl benzene styrene copolymer (1%) from Polysciences lot #935-7. The polymer was functionalized by reaction with n-butyl lithium and N,N,N',N'-tetramethylethylenediamine followed by quenching with benzophenone to produce the polymer bound tr i ty l alcohol (32). 34 SOLUTION CHEMISTRY Preparation of methyl (l,2,3,4-tetra-0-acetyl-|3-D-glucopyran)uronate This reaction was carried out using the pyridine catalysed procedure described by Bollenback et al . (23). The reagents and amounts used were: glucuronolactone 20 g, 0.11 mol sodium hydroxide 0.06 g, 1.5 mmol anhydrous methanol 150 mL acetic anhydride 65 mL and 10 mL pyridine 50 mL The product was recrystallized from 100% ethanol. The yield was 11.8 g, 0.032mol of the pure |3 anomer. 1 H NMR (400 MHz) CDC13 2.03 (2s, 9H), 2.12 (s, 3H), 3.75 (s, 3H), 4.18 (d, IH), 5.14 (t, IH), 5.27 (m, 2H), 5.78 (d, IH) Infrared: Nujol mull, NaCl plates 1770, 1750 cm"1 C=0 stretch; 1215 cm_ 1 C-0 stretch Elem. Anal. Calculated for c 1 5 H 2 o 0 i i : C 4 8 - 0 9 % > H 5.35% Found: C 47.87%, H 5.36%' Mass Spec. DCI: m/e 394 (M + NH 4) +, m/e 334 (M-42)+, m/e 317 (M-59)+, m/e 274 (317-43)+, m/e 257 (317-59)+ Preparation of methyl (2,3,4-tri-0-acetyl-a-D-Glucopyranosyl bromide) uronate (1) This reaction was carried out using the procedure described by Bollenback et a l . (23). The reagents and amounts used were: 35 methyl (1,2,3,4-tetra-0-acetyl-/3-D-glucopyran)uronate 6.0 g, 16 mmol hydrobromic acid (30% in acetic acid) 36 mL chloroform 25 mL The product was recrystallized from 15 mL 100% ethanol. The yield was 4.48g, 11.0 mmol. 1ti NMR (400 MHz) CDC13 2.06 (2s, 6H), 2.10 (s, 3H), 3.77 (s, 3H), 4.59 (d, IH), 4.86 (d of d, IH), 5.25 (t, IH), 5.62 (t, IH), 6.52 (d, IH), Infrared: Nujol mull, NaCl plates -1 -1 1770 cm C=0 stretch; 1225 cm C-0 stretch Elem. Anal. Calculated for C 1 3 H 1 7 0 g Br: C 39.31%, H 4.31% Found: C 39.33%, H 4.51% Mass Spec. EI m/e 396,398 (M)+, m/e 356,354 (M-42)+, m/e 337,339 (M-60)+, m/e 317 (M-Br)+, m/e 274 (317-43)+, m/e 257 (317-60)+, m/e 215 (257-42)+, m/e 197 (257-60)+, m/e 155 (197-43)+ Preparation of cholesteroxy oligoethylene glycols a) Preparation of 3-0-(8-hydroxy-3,6-dioxaoctyl)cholest-5-ene (2) Triethylene glycol (53g, 353 mmol), cholesteryl-p-toluene sulphonate(7.5g, 13.9 mmol) and dioxane (135 mL) were added to a 500 mL round bottom flask. A condenser was added and the mixture was stirred at reflux under a nitrogen atmosphere for 24 hours. The dioxane was removed by rotoevaporation and the residue was dissolved in 150 mL water. The mixture was added to a 1 L separatory funnel and extracted with diethyl ether (5 X 150 mL). The organic extracts were combined, washed with 10% sodium carbonate solution (1 X 75 mL) and then with water (5 X 100 mL). 36 The organic phase was dried over sodium sulfate, filtered and the fi ltrate was evaporated under reduced pressure. The residue was loaded onto a s i l ica gel column (100 g) packed with 50:50 ethyl acetate/chloroform. The column was eluted with ethyl acetate/chloroform 50:50, and the product was collected in 10 mL fractions. The combined fractions were evaporated under reduced pressure to give 8g, 14 mmol of product. (R .^=0.12; s i l i ca 60, ethyl acetate/chloroform 50:50). 1 H NMR (250 MHz) CDC13 0.65 (s, 3H), 0.84 (d of d, 6H), 0.89 (d, 3H), 0.98 (s, 3H), 1.00-2.40 (~29H), 3.16 (m, IH), 3.65 (~16H), 5.31 (d, IH), 1 3 C NMR CDC13 11.9, 18.8, 19.4, 21.1, 22.6, 22.8, 23.9, 24.2, 28.0, 28.2, 28.3, 32.0, 32.1, 35.8, 36.1, 36.9, 37.2, 38.9, 39.5, 39.8, 42.2, 50.2, 56.2, 56.9, 61.9, 67.2, 70.4, 70.8, 70.9, 72.5, 79.6, 121, 142 Infrared: neat, NaCl plates 3470 cm"1 OH stretch; 1120 cm"1 C-0 stretch (ether) Elem. Anal. Calculated for C__Hco0 •1.25 Ho0: C 73.22%, H 11.27% 33 bo 4 Z Found: C 73.01%, H 11.30% Mass Spec EI m/e 517 (M-l) + , m/e 368 (M-150)+ Mass Spec DCI m/e 536 (M + NH 4) +, m/e 369 (M-150)+ also present: m/e 906 (small) which indicates the presence of the dicholesteryl product b) Preparation of 3-0-(1l-hydroxy-3,6,9-trioxaundecyl) cholest-5-ene (3) The preparation of (3) was carried out in the same manner as described above except a five times molar excess of tetraethylene glycol was used instead of a 25 times molar excess. The amounts used were: tetraethylene glycol 5g, 9.2 mmol 37 cholesteryl-p-toluene sulfonate Sg, 41.2 mmol dioxane 50 mL The purification was identical to that for (2) and the yield was 3.0 g, 40 mmol. 1 H NMR (400 MHz) CDC13 0.68 (s, 3H), 0.87 (d of d, 6H), 0.92 (d, 3H), 1.00 (s, 3H), 1.02-2.41 (~ 29H), 3.20 (m, IH), 3.60-3.75 (~17H), 5.34 (d, IH) Infrared: neat, NaCl plates, (uncorrected values, 1601 peak at 1615 cm *) -1 -1 3500 cm OH stretch; 1130 cm C-0 stretch (ether) Elem. Anal. Calculated for C^H^JD,, • 0.25 H„0: C 74.09%, H 11.10% 35 62 5 2 Found: C 74.14%, H 11.20% Mass Spec EI m/e 560 (M-2)+, m/e 472 (M-90)+, m/e 428 (472-44)+, m/e 368 (M-194)+ c) Preparation of 3-0-(17-hydroxy-3, 6, 9, 12,15-pentaoxaheptadecyl) cholest-5-ene (4) The preparation of (4) was carried out in the same manner as for tetraethoxycholesterol using the following quantities: hexaethylene glycol 25 g, 88.7 mmol cholesteryl-p-toluene sulfonate 9.6 g, 17.7 mmol dioxane 100 mL The reaction was carried out as for the tr i and tetraethoxycholesterol reactions except that the reaction was left at 101 °C for 60 hours. The purification was identical to that for tetraethoxycholesterol, and the yield was 3.0 g, 4.6 mmol. 38 H NMR (400 MHz) CDC13 0.69 (s, 3H), 0.87 (d of d, 6H), 0.93 (d, 3H), 1.01 (s, 3H), 1.00-2.60 (~29H), 3.20 (m, IH), 3.60-3.69 (~22H), 3.75 (t, 2H), 5.35 (d, IH) Mass Spec DCI m/e 667, 685, 697 (M + nNH4)+ n = 1,2,3 ; m/e 367 (M -282) + Preparation of protected glycolipids a) Preparation of methyl [3-0-(3, 6-dioxaoctyl )cholest-5-en-3/3-yl-2,3,4-tri-0-acetyl-8-D-glucopyranosid]uronate(5) (I) (3.2g, 7.9 mmol), (2) (5.2g, 9.0 mmol), silver carbonate (1.5g), 4A molecular sieves (3.3g), a few crystals of iodine, and chloroform (50 mL) were added to a 50 mL round bottom flask. The flask was stoppered, and stirred for 96 hours at room temperature in the dark. The reaction mixture was then filtered through a glass fr i t to remove the silver salts and the molecular sieves. Then the solvent was removed by evaporation under reduced pressure. The residue was a mixture containing both starting materials and the product. The product was isolated and purified by chromatography on a si l ica gel column (100 g) packed with ethyl acetate/hexane/chloroform 33:33:33. -The column was eluted with ethyl acetate/hexane/chloroform 33:33:33, and the eluent collected in 10 mL fractions. Those fractions containing the product were combined and evaporated under reduced pressure. The yield was 1.36g, 21 % (R .^=0.27; s i l ica 60, ethyl acetate/hexane/chloroform 60:20:20). 1 H NMR (300 MHz) CDC13 0.66 (s, 3H), 0.85 (d, 6H), 0.91 (d, 3H), 0.98 (s, 3H), 1.00-2.40 (~29H), 2.01 (2s, 6H), 2.03 (s, 3H), 3.16 (m, IH), 3.60 (s, ~12H), 3.73 (s, 3H), 4.02 (d, IH), 4.65 (d, IH) 4.99 (t, IH) 5.22 (t, 39. 2H), 5.32 (d, IH) Infrared solution cell (CHCl^ ) uncorrected -1 - 1 1745 cm C=0 stretch; 1210 cm C-0 stretch (ether) Mass Spec EI m/e 835 (M) + , m/e 775 (M-60)+, m/e 715 (775-60)+, m/e 683 (715-32)+, m/e 655 (714-59)+, m/e 353 (368-14)+, m/e 339 (353-14)+, m/e 517 (M-317)+, b) Preparation of methyl [3-0-(3,6,9-trioxaundecyl) cho les t -5 -en-3 /3 -y l -2 ,3 ,4 - t r i -0 -ace ty l -£ -D-g lucopyranos id ]uronate (6 ) (3) (2 g, 3.6 mmol), (1_) (1.2 g, 3.0 mmol), freshly prepared silver carbonate/Celite (3 g), a few crystals of iodine and dry benzene (50 mL) were stirred together at room temperature in a 50 mL round bottom flask coated with aluminum fo i l . After 48 hours the mixture was filtered and the f i l trate was evaporated under reduced pressure to remove the solvent. The residue was separated on a s i l ica gel column (100 g) packed with ethyl acetate/pentane/chloroform 60:20:20. The column was eluted with ethyl acetate/pentane/chloroform 60:20:20 and 10 mL fractions were collected and monitored with tic. Those containing the product were combined. The solvents were removed by evaporation under reduced pressure. The yield of purified (6) was about 650 mg, 25% (Rf = 0.27, s i l ica 60, ethyl acetate/pentane/chloroform 60:20:20). 1 H NMR (250 MHz) CDC13 0.65 (s, 3H), 0.83, 0.89 (d of d, d, 9H), 0.98 (s, 3H), 1.00-2.40 (~29H), 2.00, 2.02 (2s, s, 9H), 3.15 (m, IH), 3.62 (~16H), 3.74 (s, 3H), 4.02 (d, IH), 4.64 (d, IH), 4.98 (t, IH), 5.21 (m, 2H), 5.31 (d, IH) 1 3 C NMR CDC13 11.9, 18.8, 19.4, 20.5, 20.7, 21.2, 22.6, 22.8, 24.3, 28.0, 28.3, 28.5, 32.0, 35.8, 36.2, 36.9, 37.2, 39.2, 39.5, 39.8, 42.2, 40 50.2, 52.8, 53.5, 56.2, 56.8, 67.3, 6 9 . 3 , 69.5, 70.2, 70.5, 70.8, 70.9,71.3, 72.1, 72.7, 79.4, 103, 1 2 1 , 142, 167.2, 169.2, 169.3, 170.1 Infrared: neat, NaCl plates, uncorrected values (1601 cm * peak at 1615 -1 -1 -1 cm ) 1770 cm C=0 stretches; 1240 cm C-0 stretch Elem. Anal. Calculated for C. oH_ o0 •2 H o 0 : C 63.00%, H 9.03% 4o fo 14 c. Found: C 63.23%, H 8.53% Mass Spec DCI m/e 962,945,930,914,898 (M + nNH4)+ n=5,4,3,2,l; m/e 528 (M - 351)+; m/e 367 (M-512)+ c) Preparation of methyl [3-0-(3,6,9,12,15-pentaoxaheptadecyl) cholest-5-en-30-yl-2,3,4-tri-0-acetyl-/3-D-glucopyranosid]uronate (7) (1_) (2.0 g, 5.0 mmol), hexaethoxycholesterol (2 g, 3.1 mmol), dry benzene (40 mL), freshly prepared silver carbonate (3 g) and a few grains of iodine were stirred together at room temperature in a 100 mL round bottom flask in the dark The mixture was filtered, the solvent was evaporated under reduced pressure and the product was loaded onto a s i l ica gel column (100 mL si l ica, 12 inch column) packed with ethyl acetate/pentane/chloroform 50:25:25. The column was eluted with the same solvent mixture and the product was collected in 10 mL fractions. The solvents were removed by evaporation under reduced pressure to give 0.40 g, 0.4 mmol,(Rf=0.27, s i l ica 60, ethyl acetate/pentane/chloroform 50:25:25). XH NMR (250 MHz) CDC13 0.58 (s, 3 H ) , 0.78, 0.83, 0.90 (d of d, d, s, 12H), 0.91-2.35 (~28H), 1.93, 1.96 (2s, s, 9H), 3.09 (m, IH), 3.55 (m, ~24H), 3.66 (s, 3H), 3.97 (d, I H ) , 4.59 (d, IH), 4.92 (t, IH), 5.14 (m, 2H), 5.25 (d, IH) 41 1 3 C NMR CDC13 11.9, 18.5, 19.2, 20.3, 20.5, 20.9, 22.4, 22.6, 23.7, 24.1, 27.8, 28.0, 28.2, 31.7, 35.5, 35.9, 36.7, 37.1, 38.9, 39.2, 39.7, 42.1, 50.0, 52.6, 56.1, 56.6, 67.1, 69.1, 69.3, 70.0, 70.4, 70.7, 71.0, 71.9, 72.4, 76.5, 77.0, 77.5, 79.2, 103, 121, 141, 167.0, 169.1, 169.2, 169.8 -1 Infrared: neat, NaCl plates, uncorrected values (1601 cm peak at 1615 -1 -1 -1 cm ) 1770 cm C=0 stretch; 1240 cm C-0 stretch Elem. Anal. Calculated for C__Ho.0 •1H_0: C 63.39%, H 9.00% z>iL o o 1 6 Z Found: C 63.06%, H 8.87% Mass Spec (DCI): m/e 997, 983 (M + nNH4)+ n=2,l; m/e 616 (M - 351)+; m/e 367 (M - 600)+ P r e p a r a t i o n o f g l y c o l i p i d s a) Preparation of [ 3 - 0 - ( 3 , 6 - d i o x a o c t y l ) c h o l e s t - 5 - e n - 3 £ - y l -8-D-glucopyranosid]uronic acid (8) (5) (350 mg, 0.42 mmol) was dissolved in anhydrous ether (6 mL) and dichoromethane (6 mL). 0.1N sodium hydroxide in anhydrous methanol (1.2 mL) was added and the mixture was left for 3 hours at room temperature. THF (40 mL) was added and stirring commenced. Aqueous 1 M sodium hydroxide (12 mL) was then added in one portion followed by methanol (35 mL). Then water (75 mL) was added slowly. Stirring was continued for a total of 4 hours at room temperature. The pH of the solution was adjusted to 3 with the addition of 10% HCl. The solution was then concentrated by evaporation under reduced pressure to about 10-20 mL. The precipitate was centrifuged and the supernatant was poured off. The precipitate was rinsed with dist i l led water and centrifuged again. The precipitate was 42 dissolved in MeOH d^. *H NMR (250 MHz) CD30D 0.71 (s, 3H), 0.88 (d of d, 6H), 0.94 (d, 3H), 1.02 (s, 3H), 1.03-2.45 (~30H), 3.21, 3.25 (m, t, 2H), 3.39 ( t , IH), 3.52 ( t , IH), 3.64 (m, -12), 3.80 (d, IH), 4.37 (d, IH), 5.35 (d, IH) 1 3 C NMR CD30D 12.2, 19.2, 19.9, 22.1, 22.9, 23.2, 24.9, 25.3, 29.0, 29.3, 29.4, 33.0, 33.2, 37.2, 37.4, 38.0, 38.4, 40.1, 40.7, 41.1, 43.5, 51.8, 57.6, 58.2, 68.3, 70.0, 71.2, 71.3, 71.8, 73.1, 74.7, 76.7, 77.4, 80.9, 104, 122, 142, 174 b) Preparation of [3-0-(3,6,9-trioxaundecyl)cholest-5-en-3B-yl-8-D-glucopyranosid]uronic acid (9) (6) (200 mg, 0.23 mmol) was treated as for (5) above using half the solvents and reagents.- After adjusting the pH of the solution to 3 the mixture was concentrated by evaporation under reduced pressure and methanol (20 mL) was added and the mixture was stored at -1 °C for 4 days. The solvent was decanted and the precipitate was dissolved in chloroform-d. 1 H NMR (250 MHz) CDC13 0.65(s, 3H) , 0.84(d of d, 6H), 0.89 (d, 3H), 0.97(s, 3H), 1.00-2.40 (~30H), 3 . 2 (broad m, I H ) , 3.60(d, H), 3.50(buried broad peaks) 3.85 (broad s, I H ) , 3.95 (broad s, I H ) , 4.45 (broad s, IH), 5.35 (broad d, IH) 1 3 C NMR CDC13 11.9, 18.9, 19.4, 2 1 . 2 , 22.5, 22.9, 24.0, 24.3, 28.1, 28.4, 32.0, 35.7, 36.2, 37.0, 37.3, 39.0, 39.5, 39.8, 42.3, 50.1, 56.2, 56.8, 67.2, 68.9, 70.2, 70.4, 70.7, 71.4, 72.9, 74.3, 75.6, 79.5, 104, 122, 141, 171 Infrared: neat, NaCl plates (uncorrected) 43 3380 cm OH stretch; 1730 cm C=0 s t r e t c h (ester); 1660 cm C=0 stretch -1 (acid); 1220, 1085 cm C-0 stretch Elem. Anal. Calculated for c i l ^ 7 0 ° 1 1 - 5 H 2 ° Mass Spec (DCI): m/e 774,756,738 (M + nNH4)+ n=2,l,0; m/e 581 (M - 157)+; m/e 383 (cholesteryl)+; m/e 369 (cholestadiene)+; m/e 195 (tetraethylene glycol) 44 SOLID PHASE CHEMISTRY General rinsing procedure (32): The reaction mixture was filtered through a 40-60 mesh glass fr i t f i l ter funnel and the resin is then rinsed with the following series of solvents: 100% ethanol 10-20 mL/g resin 3 times dist i l led water 5-10 mL/g resin 5 times 100% ethanol 5-10 mL/g resin 3 times tetrahydrofuran 10-20 mL/g resin 2 times diethyl ether 5-10 mL/g resin 3 times The resin was then air dried. General drying procedure (32): The polymer resin was dried by removal of water by azeotropic dist i l lat ion with benzene. The resin was placed in a 500 mL round bottom flask, and benzene (usually about 250 mL) was added. The flask was topped with a soxhlet with the thimble f i l led with 4A molecular sieves. The thimble apparatus was topped with a condenser. Reflux with stirring under a nitrogen atmosphere was commenced and continued for at least 12 hours. Unless otherwise noted the polymer was then filtered from the benzene and used without further drying. 45 General cleavage procedure (32): To cleave bound glycols from the polymer the resin was placed in a roundbottom flask. 0.5 M HCI (5-10 mL) in dioxane per gram of polymer resin was added to the flask. The mixture was stirred at room temperature for 24 hours. The mixture was filtered through a glass f r i t and the f i l trate was evaporated under reduced pressure to remove the dioxane. Then the crude residue could be analysed and purified, and the polymer reused. Chlorination of polymer bound t r i ty l alcohol (32) 20 g of resin was dried in the usual fashion using approximately 270 mL benzene. Acetyl chloride (30 mL) was added to the suspension (giving 300 mL of 107. solution in dry benzene). The mixture was stirred at reflux (oilbath temperature 100 °C) under a nitrogen atmosphere. After 16 hours the mixture was filtered and the polymer was rinsed with dry benzene (150 mL) followed by rinsing with anhydrous ether (150 mL). The polymer was then air dried and evacuated to remove volatile components.. Infrared: KBr pellet (uncorrected): s -1 -1 3050, 2900 cm CH stretch; 1940,1860,1800 cm substituted benzene -1 -1 stretches; 1590 cm C=C stretch; 1485, 1440 cm CH bend Titration of polymer to determine amount of chlorine active sites A polymer sample was prepared by heating resin (200 mg) in pyridine (3 mL) in a test tube with a ground glass top. The test tube was topped with a condenser and heated to about 80 °C for three hours. The sample 46 was then poured into a 125 mL Erlenmeyer flask. 5% nitric acid (50 mL) IV was added along with 5 mL of 0.1 M AsNO^ and Fe indicator (10 drops). The mixture was back titrated with standard 0.1 M NaSCN (sodium thiocyanate) to a persistent red endpoint. It was shown that the polymer had 0.55 mmol chloride per gram of resin. Preparation of monoacetyl tetraethylene glycol Tetraethylene glycol (125.3 g, 646 mmol), acetic anhydride (65.9 g, 646 mmol) and pyridine (200 mL) were placed in a 1 L round bottom flask. The mixture was heated to reflux (110 °C) under a nitrogen atmosphere, and was refluxed for 16 hours. Concentrated sulfuric acid (250 mL) was added to ice water (1 L). The reaction mixture was poured into the acid solution. This new mixture was poured into a separatory funnel and extracted with dichloromethane (5 X 200 mL). The dichloromethane extracts were combined and the dichloromethane was removed by rotoevaporation. The crude product was purified by vacuum disti l lation. The yield was 71 g of dist i l late (47% of theoretical). 1 H NMR (400 MHz) CDC13 2.19 (s, 3H), 3.06 (broad s, IH), 3.62 (m, 2H), 3.65-3.75 (m, 12H), 4.23 (sextet, 2H) Infrared Neat NaCl plates (uncorrected - 1601 peak at 1640 cm -1 -1 -1 3500 cm 0-H stretch; 1760 cm C-0 stretch (carbonyl); 1260 cm C-0 stretch (ether) Elem Anal. Calculated for C,_H„_0,: C 50.84%, H 8.53%, 1U ZU 6 Found: C 50.67%, H 8.53% Mass Spec (DCI): m/e 254 (M + NH 4) +; m/e 237 (M + H) +; m/e 279 (diacetyl tetraethylene glycol impurity + H) +; m/e 296 (279 + NH ) + 47 Preparation of mono trialkyl s i l y l tetraethylene glycol a) t-butyl dimethyl s i ly l protecting group b) trimethyl s i l y l protecting group A 500 mL round bottom flask was charged with tetraethylene glycol (19.4 g, 100 mmol), imidazole (8.2 g, 120 mmol), DMF (200 mL) and either a) t-butyl dimethyl s i l y l chloride (15.1 g, 100 mmol) OR b) trimethyl s i l y l chloride (10.86 g, 100 mmol). The reaction mixture was stirred under nitrogen atmosphere at 35-40 °C for 10 hours and then left to st ir for 12 hours at room temperature. The DMF was evaporated under reduced pressure and the residue dissoved in dichloromethane (250 mL). A precipitate formed and the mixture was then filtered through a glass f r i t . The fi ltration was repeated to remove a l l of the precipitate and then the dichloromethane was removed by rotoevaporation. The products were purified by disti l lation. a) t-butyl dimethyl s i ly l tetraethylene glycol 1 H NMR (90 MHz) CDC13: 0.50 (s, 6H) , 1.32 (s, 9H), 4.08 (s, 16H) Infrared: neat, NaCl plates: 3470 cm * OH stretch Mass Spec (CI): m/e 423 (disilyl ion: rel . int. 38); m/e 309 (M-3)+ (rel. int. 100) b) trimethyl s i l y l tetraethylene glycol 1 H NMR (90 MHz): 0.74 (s, 9H) , 4.25 (s, 16H) 48 Addition of mono acetyl tetraethylene glycol to polymer bound t r i t y l chloride Polymer bound tr i ty l chloride resin (10 g, 5.5 mmol CI) was dried in the usual fashion. After f i l tration the resin was placed in a 250 mL round bottom flask. Dry pyridine (50 mL) and monoacetyl tetraethylene glycol (2.14g, 9.1 mmol) were added. The flask was stoppered and stirred at room temperature for 96 hours. The mixture was filtered and rinsed in the usual manner. One gram of resin was cleaved and yielded approximately 90 mg of monoacetyl tetraethylene glycol. -1 -1 Infrared: KBr pellet (uncorrected) 1730 cm C=0 stretch; 1220 cm C-0 stretch Addition of mono trialkylsilyl tetraethylene glycol to polymer bound tr i t y l chloride: Polymer bound tr i ty l chloride resin (20 g , l l mmol CI) was dried in the usual fashion. After f i ltration the resin was placed in a 250 mL round bottom flask. Dry pyridine (100 mL) and mono trialkyl s i l y l tetraethylene glycol a)(5 g, 17 mmol) or b)(4.6 g, 17 mmol) were added to the flask. The flask was stoppered and stirred at room temperature for 96 hours. The mixture was then filtered and rinsed in the usual fashion. trialkyl: a) t-butyl dimethyl s i ly l b) trimethyl s i l y l 1 Analyzed by examining cleavage products by H NMR. In each case the cleavage of one gram of resin yielded approximately 85 mg of tetraethylene glycol. 49 Removal of the acetyl protecting group: Polymer bound monoacetyl tetraethylene glycol resin (9 g, 4.5 mmol) was placed in a 50 mL round bottom flask. Benzyl amine (20 mL, 20.4 g, 190 mmol) was added. The flask was topped with a condenser and stirred at 120 °C for 65 hours. The mixture was then filtered and rinsed in the usual manner. Infrared spectroscopy indicated the complete removal of the acetate. Preparation of polymer bound tetraethylene glycol mesylate: a) Polymer bound tetraethylene glycol resin (5 g) was dried in the usual fashion in benzene (about 50 mL). Instead of f i l tering, the mixture was cooled to room temperature and dry pyridine (50 mL) was added to it (giving a 3:1 mixture of dry benzene and dry pyridine). Methanesulfonyl chloride (6.5 mL, 84 mmol) was also added. The mixture was topped with a calcium chloride drying tube and stirred for 48 hours. The mixture was then filtered and rinsed in the usual manner. -1 -1 Infrared: KBr pellet (uncorrected) 1350 cm S=0 stretch; 1220 cm C-0 stretch b) The above reaction was also carried out in exactly the same proportions of reagents using polymer bound octaethylene glycol in place of the polymer bound tetraethylene glycol. -1 -1 Infrared: KBr pellet 1350 cm S=0 stretch; 1220 cm C-0 stretch 50 Preparation of polymer bound octaethylene glycol 60% sodium hydride in o i l suspension (0.16g, 4 mmol) was placed in a 2 necked round bottom flask. One neck was stoppered and a gas line was attached to the other. The system was flushed with nitrogen atmosphere. Pentane (10 mL) was added to the sodium hydride. The mixture was stirred and then the sodium hydride solid was left to settle. The pentane was decanted. This rinse was repeated two more times. Disti l led tetraethylene glycol (1.94 g, 10 mmol) was dissolved in dry DMF (20 mL). The tetraethylene glycol and DMF solution was added to the round bottom flask through one neck while the system was s t i l l being flushed with nitrogen. After 2-3 minutes stirring at room temperature the bubbling stopped and the solution was clear. Polymer bound tetraethylene glycol mesylate (2 g, 1 mmol) was added. The flask was then topped with a condenser and the reaction mixture was stirred under nitrogen at 120 °C for 24 hours. The mixture was then filtered and rinsed in the usual manner. The reaction was repeated in exactly the same manner using polymer bound octaethylene glycol mesylate in place of polymer bound tetraethylene glycol mesylate to produce polymer bound dodecaethylene glycol. Preparation of Oligoethylene glycols a) Preparation of octaethylene glycol 5 g of octaethylene glycol bound resin were subjected to the usual cleavage conditions. The reaction mixture was filtered and the fi ltrate was evaporated under reduced pressure. The fi ltrate was then diluted 51 approximately 1:1 with disti l led water and 0.5 mL portions were purified by passage through a Fractogel 40S gel permeation column (1.5 cm diameter x 60 cm length) with a Laboratory Data Control Refractomonitor III detector using disti l led water as the buffer; 48 two mL fractions were collected. Fractions 33-35 were combined and evaporated under reduced pressure. 1 H NMR (400 MHz) CDC13 2.65 (s, water impurity) 3.61 (t, 4H) 3.65 (d, ~24H) 3.74 (t, 4H) -1 Infrared: neat, NaCl plates (uncorrected) 3390 cm OH stretch; 1105 cm"1 C-0 stretch Elem. Anal Calculated for C^U^„0i 1.75Ho0: C 47.81%, H 9.40% 16 34 9 2 Found: C 47.77%, H 9.37% Mass Spec (DCI): m/e 388 (M + NH 4) +; m/e 371 (M + H) + b) Preparation of dodecaethylene glycol Dodecaethylene glycol was recovered in the same manner as octaethylene glycol except that fractions 29-32 were combined and evaporated under reduced pressure instead of fractions 33-35. *H NMR (400 MHz) CDC13 2.65 (s, water impurity) 3.61 (t, 4H) 3.65 (d, ~40H) 3.74 (t, 4H) Infrared: neat, NaCl plates (uncorrected) 3390 cm"1 OH stretch; 2915, 2865 cm"1 CH stretch; 1462, 1355 cm"1 CH bend; 1105 cm - 1 C-0 stretch Elem. Anal. Calculated for c 2 4 H 5 0 0 i 3 ' 2 H 2 ° : C 4 9 - 4 7 % > H 9 - 3 4 % Found: C 49.45%, H 9.22% Mass Spec (DCI): m/e 564 (M + NH 4) +; m/e 547 (M + H) + 52 REFERENCES 1. Sharp, K.A., Brooks, D.E. , Biopysical Journal. 47, 563 (1985). 2. McDaniel, R.V., Sharp, K. , Brooks, D.E. , McLaughlin, A . C , Winiski, A.P. , Cafiso, D. 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PhD Thesis, York University (Canada) (1977). 54 APPENDIX I Selected Spectra H NMR compound (3) 56 1 H NMR compound (7) 57 1 3 C NMR compound (7) 58 1 H NMR compound (8) 59 1 3 C NMR compound (8) 60 IR polymer bound tr i ty l alcohol 61 IR polymer bound acetyl tetraethylene glycol 62 IR polymer bound tetraethylene glycol 63 IR polymer bound tetraethylene glycol mesylate 64 GPC chromatogram crude octaethylene glycol (top) and dodecaethylene glycol (bottom) preparations 65 55 compound 7 C0,Me OAc p r o t e c t e d g l y c o l l p i d 1 loo fcle-*^ ~ i 1 1 1 r I; C 3 O a. e o o c o m p o u n d 8 p 4 " V > o . » l • W / f t n r \ & c o m p o u n d 8 a, o 1 i 1 1 1 1 1 1 1 T V C G . fi I cA>*o i> iiCy o rf * l c-*n ©TrCI ( p ) = p o l y s t y r e n e W. d l v l n y l b e n z e n e 0 . 5 mmol C l / g r a m p o l y m e r CONDITIONS: FRACTOGEL 40S COLUMN ELUENT: DISTILLED WATER RATE: 0.2 ML/MIN CHART SPEED: 2 ML/MIN READOUT: 2 ML/CM COLUMN HEIGHT: 60 CM COLUMN DIAMETER: 1. 5 CM 

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