UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Synthesis of cholesterol based model glycolipids Sather, Paula Joan 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1990_A6_7 S34_5.pdf [ 3.53MB ]
Metadata
JSON: 831-1.0059693.json
JSON-LD: 831-1.0059693-ld.json
RDF/XML (Pretty): 831-1.0059693-rdf.xml
RDF/JSON: 831-1.0059693-rdf.json
Turtle: 831-1.0059693-turtle.txt
N-Triples: 831-1.0059693-rdf-ntriples.txt
Original Record: 831-1.0059693-source.json
Full Text
831-1.0059693-fulltext.txt
Citation
831-1.0059693.ris

Full Text

SYNTHESIS OF CHOLESTEROL BASED MODEL GLYCOLIPIDS By PAULA JOAN SATHER B.Sc,  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  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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  DE-6 (2/88)  Qrf  (Q  fad  ABSTRACT The synthesis of g l y c o l i p i d s containing a variable length polyethylene glycol spacer group between a glucuronic acid (glu) headgroup and a cholesterol (chol) t a i l glu-0CH (CH 0CH 2  2  2  )nCH 0-chol 2  i s described. The homologs (n=2,3,5) were prepared by reaction of an excess of commercially available t r i , t e t r a and hexaethylene g l y c o l s 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 y i e l d s were dependent on the amount of excess used. The headgroup was prepared by e s t e r i f i c a t i o n and a c e t y l a t i o n of glucuronolactone to produce methyl (1, 2, 3, 4-tetra-0-acetyl-/3-Dglucopyran)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 g l y c o l s by a Koenig Knorr type reaction using f r e s h l y prepared s i l v e r carbonate as the catalyst. Methyl[3-0-(3,6-dioxaoctyl)cholest-5-en-3/3-y1-2,3,4-tri-0acetyl-/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 - t r i - 0 -acetyl-/3-D-glucopyranosid] uronate (7) were produced with y i e l d s 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 a c i d i f i c a t i o n 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 g l y c o l and dodecaethylene g l y c o l were prepared using a s o l i d supported synthesis. The s o l i d polymer used was a t r i t y l chloride f u n c t i o n a l i z e d polystyrene 1% d i v i n y l benzene. Mono protected tetraethylene g l y c o l 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 g l y c o l was prepared using sodium hydride i n DMF. The tetraethylene g l y c o l bound resin was added, and reaction continued at 120 °C f o r 24 hours. Cleavage of the resultant product from the polymer support yielded octaethylene g l y c o l . Repetition of the mesylation and elongation steps followed by cleavage yielded dodecaethylene g l y c o l . The oligoethylene g l y c o l s were p u r i f i e d by passage through a Fractogel 40S gel permeation column. Two d i f f e r e n t protecting groups f o r the tetraethylene g l y c o l were t r i e d . T r i a l k y l s i l y l groups were f i r s t attempted, but were abandoned due to reduced r e a c t i v i t y 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.  ii  TABLE OF CONTENTS Abstract  ii  List of Tables  iv  List of Figures  v  Acknowledgements  vi  Chapter One 1.1  1.2  1 Introduction  1  1.1.1  1  Glycocalyx  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 2.3.2  Advantages and Disadvantages of the Polymer Supported Synthesis Protecting Groups in the Polymer Supported Synthesis  2.4  25  Conclusions  27 33  Experimental  34  General Details  34  Solution Chemistry  35  Solid Phase Chemistry  45  References Appendix  53 55  iii  LIST OF TABLES Table I: Table II:  Table III:  Influence of molar excess of glycol on yield  triethylene  S i l y l removal conditions on the polymer backbone using 0.5 M Bu^NF in THF Acetate removal conditions on the polymer backbone  iv  14 31  33  LIST OF FIGURES Figure 1:  Structure of targeted glycolipid  Figure 2:  Targeted lipids and glycolipids: n=2,3,5  10  Figure 3:  Synthesis of methyl (2,3,4-tri-O-acetyl-a-Dglucopyranosyl bromide)uronate (1_)  12  Figure 4:  Synthesis of  3  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  Figure 8:  Reaction of monoprotected tetraethylene glycol  19  to polymer bound t r i t y l 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)  Figure 12:  Acid cleavage of polymer bound glycols  Figure 13:  Synthesis of mono t-butyl dimethyl s i l y l  . 24 25  tetraethylene glycol (1J5)  28  Figure 14:  Indirect analysis of s i l y l group removal  29  Figure 15:  Synthesis of monoacetyl tetraethylene glycol (16)  v  . . . 32  ACKNOWLEDGEMENTS  I would l i k e to express my a p p r e c i a t i o n to my s u p e r v i s o r , Don Brooks, f o r h i s p a t i e n c e the o t h e r  members o f h i s group f o r t h e i r  s t a f f and f a c u l t y Victoria  and a s s i s t a n c e throughout t h i s p r o j e c t , and to support.  Thanks a l s o to the  o f the Chemistry Department a t the U n i v e r s i t y o f  and p a r t i c u l a r l y  their kind h o s p i t a l i t y  to Tom F y l e s and the members o f h i s group f o r  and p r a c t i c a l  a s s i s t a n c e d u r i n g my s t a y i n  Victoria.  vi  CHAPTER ONE  1.1  INTRODUCTION The g l y c o c a l y x  cell  membrane.  glycolipids glycocalyx  It  extending is  system  therefore  complex  glycocalyx  develop  and  of  of  a cell  embedded  i n many c e l l u l a r and d i f f i c u l t  its  one  to  such  and  mixture  in  the  functions  to it  assist was  the  outwards  of  from  and phenomena. in situ. in  the  glycoproteins  p l a s m a membrane.  understand  in order  functions,  and s y n t h e s i z e  extending  a complicated  lipids  desirable  The g l y c o c a l y x complicated.  It  is  cell-substrate  antigen-antibody physical  is  the  and  The These  A simple  model  understanding  aim of  this  project  of to  system.  reactions  phenomena  of  the  glycocalyx  information  to  in  these  influences  the  cell  and c e l l u l a r  occur  resulting  of  that  i n many c e l l u l a r  interaction  u n d e r s t a n d i n g many o f  this  a region  involved  in  use  region  Glycocalyx  1.1.1  or  from  involved  are  the  the  consists  phenomena is  is  the  phenomena  mobility.  adhesion is  physical  chemically  functions  glycocalyx  cellular  the  develop  in  is  to  as  such as  cell-cell  Interactions well  as  such  as  reactions  or  and r e p u l s i o n .  determine  measurements  and r e f i n e  very  theoretical  how  A step  the  made o n models  structure  it, of  and  to  the  glycocalyx. Two s p e c i f i c determining are In  the the  some o f  phenomena the  electrophoretic first  of  these  which  illustrate  structure/function m o b i l i t y and the  examples  the  difficulties  relationships  of  the  p a r t i t i o n i n g behavior  classical  1  the  theoretical  models  of glycocalyx of  cells.  which  describe  the electrophoretic  mobility of erythrocytes often predict much  higher m o b i l i t i e s than are observed (1).  This i s thought to be because  the hydrodynamic drag caused by the charged groups of the glycocalyx usually either ignored or the charges are assumed to be at the b i l a y e r surface  (2).  is  lipid  More complicated theoretical models are the r e s u l t  of experimental r e s u l t s from erythrocytes and model c e l l s composed of gangliosides  and phospholipids (1).  further refined and corrected The  These new  second example of these d i f f i c u l t i e s i s i l l u s t r a t e d by  in a phase system of two  the  Large p a r t i c l e s such as c e l l s , when mixed  immiscible polymer solutions, w i l l  d i s t r i b u t e themselves between one phase and  often  the interface between the  Their p o s i t i o n r e f l e c t s their r e l a t i v e a t t r a c t i o n for one  over the other based on t h e i r r e l a t i v e surface contact with each l i q u i d .  be  with a more extensive model c e l l system.  p a r t i t i o n i n g behavior of c e l l s .  phases.  t h e o r e t i c a l models could  two  phase  free energies when i n  The positioning can be influenced  by changing  the e l e c t r o s t a t i c potential between the two phases with the a d d i t i o n of s a l t s which p a r t i t i o n s l i g h t l y in favour of one phase.  The p o s i t i o n i n g  of  the p a r t i c l e s and hence the difference in surface free energy i n each phase can be calculated by measuring the contact angle of the p a r t i c l e at the interface (3).  The  structure of the glycocalyx,  including  the  positioning and numbers of charges within i t , plays a large r o l e i n the behavior of these p a r t i c l e s in the two phase systems (4).  As i n  electrophoretic mobility work, theoretic models are being used to attempt to interpret experimental r e s u l t s ( 5 ) , and a more extensive model system would aid t h i s work. The examples described  above are two of the many areas i n which a  more thorough understanding i s needed of how  2  the structure of  the  glycocalyx of a cell influences its behavior.  In order to further refine  the current theories of these behaviors a simpler model c e l l 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 c e l 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 the headgroup attachment,  (6,8).  Potentially, based on  the liposome carrier could be targeted to a  3  specific tissue in the body.  No s y n t h e s i s or  application o f 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 i t 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. headgroup was chosen for a variety of reasons.  This  Because i t contains a  carboxylic acid i t 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 i a l i c 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  oligoethylene glycol.  used in the model glycocalyx synthesis is  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. these specific oligomers it is possible to buy heterodisperse glycol fractions of average molecular weight 400, 600 etc.  Besides polyethylene  "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 f i r s t 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.  5  However,  such a system was The  not  available to  next i d e a was  T h i s i d e a had  to s y n t h e s i z e v a r i o u s o l i g o m e r s  some precedence.  g l y c o l have been s y n t h e s i z e d n o n a e t h y l e n e g l y c o l and  us.  Hexaethylene g l y c o l and  (10,11), and  decaethylene  difficulties  g l y c o l has  obstacles,  molecular  to a c q u i r e  oligoethylene g l y c o l s centred  (12).  weight Poor y i e l d s and  p u r i f i c a t i o n presented  so a s o l i d s t a t e s y n t h e s i s was  Most of the e f f o r t s  been r e p o r t e d  none were v e r y s u c c e s s f u l .  w i t h chromatography and  octaethylene  r e c e n t l y the s y n t h e s i s of  S e v e r a l attempts were made to produce h i g h e r o l i g o e t h y l e n e g l y c o l s , but  specifically.  the  major  introduced.  l o n g e r c h a i n monodisperse  around u s i n g a s o l i d polymer  supported  s y n t h e s i s to produce s p e c i f i c o l i g o e t h y l e n e g l y c o l c h a i n s from m u l t i p l e s of tetraethylene g l y c o l . c o u l d be prepared  on  Using  t h i s method the o l i g o e t h y l e n e g l y c o l s  the polymer.  The  final  p u r i f i c a t i o n c o u l d be done  the g e l permeation column because t h i s column does b a s e l i n e ethylene  1.2.3  g l y c o l oligomers  separate  d i f f e r i n g by f o u r u n i t s .  Polymer Supported  Synthesis  Polymer supported  s y n t h e s i s i s a s y n t h e t i c method i n which  substrate  i s attached  on  to an  i n s o l u b l e polymer.  Reactions  the  are c a r r i e d  out  on the polymer bound s u b s t r a t e ; at the c o n c l u s i o n of the s y n t h e s i s  the  f i n a l product  Polymer  i s c l e a v e d from the polymer f o r f i n a l p u r i f i c a t i o n .  bound r e a g e n t s (13).  and  In a l l cases  polymer bound c a t a l y s t s are a l s o used i n s y n t h e t i c work the polymer o f f e r s  These advantages c e n t r e around the f a c t be  i t s u b s t r a t e , reagent  s p e c i e s present  or c a t a l y s t  the same types of advantages. t h a t the polymer bound compound -  - i s e a s i l y separable  i n the r e a c t i o n mixture  6  (14,15).  The  from the  advantages a r e  other well  illustrated in the case of the polymer bound substrate. is done simply by f i l t e r i n g the polymer from the solvent, biproducts.  Product work up reagents and  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 f i r s t 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 i f the f i r s t peptide could be  attached to an insoluble support the isolation and purification of intermediate products could be accomplished with f i l t r a t i o n and rinsing, therefore that product losses would be reduced and time would be saved (17).  Merrifield's f i r s t 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  i t 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. synthetic schemes.  However, they have also been used in many other  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. dimerizes and loses its activity.  The titanocene by i t s e l f rapidly  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 i t 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 f i l t e r i n g away the excess reagents solved the problem of separating the unreacted tetraethylene glycol from the product.  The f l e x i b i l i t y of  the method gives i t potential for use in the synthesis of model glycocalyxes with various different spacer groups.  It is also possible  that i t could be developed for other syntheses such as crown ether synthesis.  8  In  summary,  attempted used  using  to link  the synthesis  two d i f f e r e n t  together  supported  synthesis  potential  spacer  methods  could  structure  o f the d e s i r e d model g l y c o c a l y x e s  methods.  the separate  f o r these  be f u r t h e r d e v e l o p e d  f o r many f u t u r e  solution synthesis  s e c t i o n s o f t h e compounds.  of oligoethylene  groups  Standard  g l y c o l s was  compounds.  p h y s i c a l chemical  9  developed  The  that  more g l y c o l i p i d s  studies.  was polymer  t o make  It i s possible  to produce  was  of  both various  CHAPTER TWO  2.1  INTRODUCTION  The goal of this project was to synthesize a series of lipids and glycolipids.  Figure 2:  Their structures are:  Targeted lipids and glycolipids:  n=2,3,5  The synthetic work of this project may be divided roughly into two parts. The f i r s t part was the solution synthesis of the above compounds using available ethylene glycols. steps.  This synthesis was carried out in four major  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 i r s t 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. was the coupling of the spacer group to the l i p i d base.  The second  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 i r s t major step in this synthesis was the production of the protected and brominated glucuronic acid (see Figure 3). was the starting material. occurred in a single vessel.  Glucuronolactone  The reaction had three parts, two of which 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 a l . (23). The f i r s t 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. 11  Based on  H NMR data the product  was t h e 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 lAc  Ac 0, py  OAc  methyl t e t r a - 0 - a c e t y l - £ - D glucopyranuronate  glucuronolactone  Br  Figure 3: Synthesis of methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl bromide)-uronate  (compound 1_)  The second step of the synthesis was the coupling of the oligoethylene glycol "spacer" group to cholesterol.  This was f i r s t 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 i c a gel column in 73% yield.  The product  was a thick syrup which could be neither d i s t i l l e d nor crystallized. 12  Thin  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 triethylene glycol.  H NMR spectrum of pure  The mass spectrum showed the expected molecular ion  at m/e 517 and logical fragment ions, for example: peak) is the mass of the cholesta-3,5-diene  m/e 368 (the 100%  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, i t was found that smaller excesses led to lower yields Table I).  (see  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  13  tosylate.  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 number 1 2 3,4  Molar ratios triethylene glycol 25 10 5  cholesteryl-ptoluene sulfonate 1 1 1  14  Yields (purified) 73% 63%  50%  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, fragment ions.  there is nothing to indicate that these are not simply 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), to (JJ (Figure 6).  (3) and (4)  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 d i s t i l l e d nor  crystallized and the purification was done using a s i l i c a 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).  A l l three  reactions were carried out in a similar manner and the products a l l behaved similarly during the purification.  16  .C0 Me, 2  A c O ^ ^ v  (1)  OAc] Br  n=2,3,5 Ag C0 2  3  benzene  .C0 Me, 2  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-0acetyl-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. 17  The f i r s t attempt  was removal of the acetate groups using sodium methoxide in anhydrous 1 methanol followed by ion exchange with Amberlite. step was successful: visible.  H NMR showed that this  the three acetate hydrogen peaks were no longer  The methyl ester was removed using sodium hydroxide in water  followed by ion exchange to remove the sodium.  1  H 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 d i s t i l l e d 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 a l . 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 t e r the precipitate from the supernatant salt solution.  Instead, the supernatant was decanted and the  (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  precipitate was rinsed.  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  n=2,  (5),(6)  [3-0-(3,6-dioxaoctyl)cholest-5-en-3/3-yl-£-D-glucopyranosid] 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 problems.  straightforward to purify. There are, however,  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 i t would have required a preparative scale gel permeation column on an HPLC apparatus. at the time of the experiments.  This equipment was not available  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 a l .  (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.  20  Based on gas chromatography  data, yields were low in these and other experiments and purification was difficult,  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 i f t h 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 t r i t y l chloride active sites. Polysciences  The polymer was obtained from  (lot 953-7) and was treated with butyl lithium followed by  reaction with benzophenone to produce t r i t y l alcohol active sites. sites were then chlorinated with acetyl chloride.  These  The polymer was  titrated against 5% HNO^ and found to have 0.55 mmol chloride per gram of polymer.  21  The f i r s t step of the synthesis was to attach the f i r s t 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 t r i t y 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 t r i t y 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 d i f f i c u l t , and it is described in some detail in section 2.4.2 of this discussion.  22  ©  Tr—0  Figure 9:  r{\  k 0 J  3  ®P ) T r — 0  OPG  K 0 J  OH  (10)  Production of polymer bound tetraethylene glycol  (10)  After having attached the f i r s t 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  Figure 10:  r \ /n ko  OH  MsCl  bz, py  © T r — 0  I 0 )  3  OMs ( i i )  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. means.  These products were analysed by various  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 i n i t i a l l y .  Subsequently, i t 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\\  I0 J  HO  (P)Tr—0  l[\ 3 3  OH  r|\ / n .  NaH DMF  HO  n  J  3  0  r(\ /|-\  I 0 J OMs 3  DMF  HO  I0  ^ o)  3  Figure 11:  (P)Tr—0  I0 >  7  OH(12)  o  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.  A l l the hydrogens are similar and have similar shifts so they  appear together as a broad singlet or multiplet at 3.7 ppm. 24  Infrared  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.  © Tr—0  r\\ in I 0 Jn  n=7,11  OH  r(\ in  HCI dioxane  HO  I 0 Jn OH  n=7, octaetylene glycol  (13)  n=8, dodecaethylene glycol Figure 12:  (14)  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 f i l t r a t i o n of the polymer and its attached compounds. starting materials and many impurities are washed away.  Solvents,  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 f l e x i b i l i t y 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 d i f f i c u l t i e s . Major among these is monitoring the reactions occuring on the polymer and analysing the products of those reactions. be utilized. mixture.  Some ordinary methods cannot  The polymer backbone is very much the dominant part of the  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 f a c i l i t i e s for this, but i t 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.  26  This problem often results  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 t r i a l and error  (13).  In this project both the d i f f i c u l t y 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 A.  Protecting groups in the polymer supported synthesis Trialkyl s i l y l groups The f i r s t protecting group that was used for protection of one of  the hydroxyl groups of the tetraethylene glycol was t-butyl dimethyl s i l y l chloride.  Monoprotected tetraethylene glycol was synthesized (see Figure  13), and the resulting mono t-butyl dimethyl s i l y l tetraethylene glycol I  (15) was purified by d i s t i l l a t i o n .  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 l y 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). protecting group for alcohols, gentle, quite neutral and fast.  Also, the group is a flexible  in part because the removal conditions are 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).  CH, (CH ) C— Si—CI 3  3  CH t-butyl dimethyl s i l y l chloride 3  DMF  (CH ) C—Si—0 3  r(\  UJ  3  in OH  CH  HO k o > tetraethylene glycol Figure 13:  r\ ir\  i  imidazole  3  OH (15)  3  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 l y l protecting group.  The s i l y 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 t e l l the extent of the previous removal.  Finally, an indirect  method was developed (see Figure 14). After the attempted deprotection the 28  freed hydroxyls were acetylated with acetic anhydride in pyridine.  The  acetate group is easily seen in IR spectra, and i t 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 t e l l the extent of the acetylation and thus the extent of the removal of the s i l y l group.  a) (CH ) 3  3  i r\ C :— Si—0 I 0 J,  HO  k 0  OTr(P) +  Bu NF 4  OTr(?)  THF  CH-  Si—0  rf\ / h k 0 >  3  OTr ( ? )  b) -0  r{\ iY\ k  0 )  O T r ®  3  Si—0  k 0 )  :0  k 0 )  OTr ( ? )  3  py HO  k  0 )  3  OTr (P)  c) i—0  U ;  r{\ ir\  O T r ®  3  HO  k  AcO  fr\  3  OH  dioxane ^c0  I 0 J , OTr  a)  removal of s i l y l protecting group  b)  acetylation of free hydroxyl groups  c)  cleavage of glycols from polymer support  Figure 14:  0 ) +  HCI  r{\  O T r ®  3  Indirect analysis of s i l y l group removal  29  k  0  OH  The removal of the trialkyl s i l y 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 i r s t , because of the monitoring difficulties, i t 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 l y 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 i t was more convenient and less expensive i t 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. II.  30  Results are summarized in Table  Table II:  S i l y l removal conditions on the polymer backbone using 0.5 M Bu NF in THF 4  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 t r i a l k y l s i l y 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 d i s t i l l a t i o n and analysed by spectroscopy and elemental analysis.  H NMR, IR, mass  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 found.  was a l s o  in  agreement with that which was  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  HO  ^  I 0 J , OH •  Figure 15:  Ac 0 2  — py  2  Synthesis of  mono  r\\ fr\  HO 1 0 ) , OAc 3  (16)  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. cm^  The acetate carbonyl appears at  and disappears as the deprotection  1730  progresses.  The f i r s t 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 l y 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. rinsed.  After 16 hours at 50 °C the polymer was filtered and  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. 16 hours at 85 °C.  The reaction went again for  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 f i l t e r i n g 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 f i l t e r i n g 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 concentration (M) 0.43 4.60 neat  Solvent  Temperature (°C)  pyridine pyridine  115 115 160  —  Time (days)  1730 cm * peak (IR spectra)  1 1 4  present present absent  2.4 CONCLUSIONS The synthesis of (5), were produced.  (6) and (7) was developed and these compounds  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. synthesized by this method.  Octa and dodecaethylene glycol were  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. 3 4  Carbon spectra were recorded at 62.9 MHz ^  (WM 250) in MeOD-d^ or CDCl^ with the central solvent line as standard (49.0  (MeOD-d ) and 77.0 (CDC1 ) relative to Me Si). 4  3  4  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. Elmer 283, Pye Unicam SP1100, spectrometers.  Infrared spectra were recorded on Perkin Bomem MB 100 and Perkin Elmer 783 infrared  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 d i s t i l l e d prior to use.  Dimethyl formamide was  d i s t i l l e d from C a ^ and stored over freshly activated 4A molecular sieves. Tetrahydrofuran was d i s t i l l e d from potassium benzophenone ketyl under inert atmosphere immediately prior to use.  Triethylene glycol and  tetraethylene glycol were d i s t i l l e d 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 t r i t y l alcohol  34  (32).  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 a l . (23).  The reagents and amounts used were:  glucuronolactone  20 g, 0.11 mol  sodium hydroxide  0.06 g, 1.5 mmol  anhydrous methanol acetic anhydride pyridine  150 mL 65 mL and 10 mL  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) CDC1  3  (d,  2.03 (2s, 9H), 2.12 (s, 3H), 3.75 (s, 3H), 4.18  IH), 5.14 (t, IH), 5.27 (m, 2H), 5.78 (d, IH)  Infrared: 1770,  Nujol mull, NaCl plates  1750 cm" C=0 1  Elem. Anal.  stretch; 1215 cm  Calculated for  c  H 1 5  2o ii 0  :  C-0 stretch  _1  C  4 8  -  0 9 %  >  H  5.35%  Found: C 47.87%, H 5.36%' Mass Spec.  DCI: m/e 394 (M + NH ) , m/e 334 (M-42) , +  4  +  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) chloroform  36 mL  25 mL  The product was recrystallized from 15 mL 100% ethanol.  The yield was  4.48g, 11.0 mmol. 1  ti NMR (400 MHz) CDC1  3  (d,  2.06 (2s, 6H), 2.10 (s, 3H), 3.77 (s, 3H), 4.59  IH), 4.86 (d of d, IH), 5.25 (t,  Infrared:  IH), 5.62 (t, IH), 6.52 (d, IH),  Nujol mull, NaCl plates  -1 1770 cm C=0 Elem. Anal.  -1  stretch; 1225 cm  C-0 stretch  Calculated for C H 0 B r : 13  Found: Mass Spec. EI  m/e  17  g  C 39.31%, H 4.31%  C 39.33%, H 4.51% 396,398  (M) ,  m/e  +  356,354  (M-42) , +  337,339 (M-60) , m/e 317 (M-Br) , m/e 274 (317-43) , +  m/e  +  215 (257-42) , m/e 197 +  +  m/e  (257-60) , m/e 155 (197-43) +  m/e  257  (317-60) , +  +  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, was evaporated under reduced pressure.  filtered and the f i l t r a t e  The residue was loaded onto a  s i l i c a 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 c a 60, ethyl acetate/chloroform 50:50). 1  H NMR (250 MHz) CDC1 0.65 (s, 3H), 0.84 (d of d, 6H), 0.89  (d,  3  0.98 (s, 3H), 1.00-2.40 (~29H), 3.16 (m, IH), 3.65 (~16H), 5.31  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,  3  11.9,  18.8,  (d, IH),  19.4, 21.1, 22.6, 22.8, 23.9, 24.2,  1 3  C NMR CDC1  3H),  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" OH stretch; 1120 cm" C-0 stretch (ether) 1  1  Elem. Anal.  Calculated for C__H 0 •1.25 H 0: co  Found:  C 73.01%,  C 73.22%, H 11.27%  o  33 bo 4  Z  H 11.30%  Mass Spec EI  m/e 517 (M-l) , m/e 368 (M-150)  Mass Spec DCI  m/e 536 (M + NH ) , m/e 369 (M-150)  +  +  +  +  4  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. tetraethylene glycol  5g, 9.2  37  mmol  The amounts used were:  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) CDC1  0.68 (s, 3H), 0.87 (d of d, 6H), 0.92  3  (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 3500 cm  -1 1130 cm C-0 stretch (ether)  OH stretch;  Elem. Anal.  Calculated for C^H^JD,, • 0.25 H„0: 35 62 5 2 Found:  Mass Spec EI  C 74.09%,  H 11.10%  C 74.14%, H 11.20%  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 t r 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) CDC1  3  1.01  0.69 (s, 3H), 0.87 (d of d, 6H),  0.93  (s, 3H), 1.00-2.60 (~29H), 3.20 (m, IH), 3.60-3.69 (~22H),  (d,  3H),  3.75  (t,  2H), 5.35 (d, IH) Mass Spec DCI m/e 667, 685, 697 (M + nNH ) n = 1,2,3 +  4  282)  ;  m/e  367  (M -  +  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 f r 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 s i l i c a 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 i c a 60, ethyl acetate/hexane/chloroform 1  H NMR (300 MHz) CDC1  3  60:20:20).  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,  39.  IH) 5.22  (t,  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)  cholest-5-en-3/3-yl-2,3,4-tri-0-acetyl-£-D-glucopyranosid]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 f o i l .  After 48 hours the mixture was filtered and  the f i l t r a t e was evaporated under reduced pressure to remove the solvent. The residue was separated on a s i l i c a gel column (100 g) packed with ethyl acetate/pentane/chloroform  60:20:20.  acetate/pentane/chloroform  60:20:20 and 10 mL fractions were collected and  monitored with tic.  The column was eluted with ethyl  Those containing the product were combined.  solvents were removed by evaporation under reduced pressure. purified (6) was about 650 mg, 25% (Rf = 0.27, acetate/pentane/chloroform 1  H NMR (250 MHz) CDC1  3  The  The yield of  s i l i c a 60, ethyl  60:20:20). 0.65 (s, 3H), 0.83,  (s, 3H), 1.00-2.40 (~29H), 2.00,  2.02 (2s,  0.89 (d of d, d,  s, 9H),  3.15  (m,  9H),  0.98  IH),  3.62  (~16H), 3.74 (s, 3H), 4.02 (d, IH), 4.64 (d, IH), 4.98 (t, IH), 2H), 5.31 1 3  5.21  (m,  (d, IH)  C NMR CDC1  3  11.9,  18.8,  19.4, 20.5, 20.7, 21.2, 22.6, 22.8,  28.0, 28.3, 28.5, 32.0, 35.8, 36.2, 36.9, 37.2, 39.2, 39.5, 39.8,  40  24.3, 42.2,  50.2,  52.8,  53.5, 56.2, 56.8,  70.9,71.3, 72.1, 72.7, 79.4, Infrared:  67.3, 6 9 . 3 , 69.5,  70.2, 70.5,  70.8,  103, 1 2 1 , 142, 167.2, 169.2, 169.3,  170.1  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 Found:  Mass Spec DCI  C. H_ 0 •2H 0: 4o fo 14 c. o  o  o  C 63.00%, H 9.03%  C 63.23%, H 8.53%  m/e 962,945,930,914,898 (M + nNH ) n=5,4,3,2,l;  m/e  +  4  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 (1_) (2.0 g, 5.0 mmol), hexaethoxycholesterol  (7)  ( 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 i c a gel column (100 mL s i l i c a , 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 i c a 60, ethyl  acetate/pentane/chloroform  50:25:25). X  H NMR (250 MHz) CDC1  3  0.58  12H), 0.91-2.35 (~28H), 1.93,  (s, 3 H ) , 0.78, 1.96 (2s,  0.83,  s, 9H), 3.09  0.90 (d of (m,  ~24H), 3.66 (s, 3H), 3.97 (d, I H ) , 4.59 (d, IH), 4.92 (t, 2H), 5.25  (d, IH)  41  d,  d,  s,  IH),  3.55  (m,  IH),  5.14  (m,  1 3  C NMR CDC1  11.9,  3  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__H .0 Found:  Mass Spec (DCI): 367 (M - 600)  Preparation  a)  •1H_0:  o  z>iL  oo  16  C 63.39%, H 9.00%  Z  C 63.06%, H 8.87%  m/e 997, 983 (M + nNH ) n=2,l; m/e 616 (M - 351) ; m/e +  +  4  +  of glycolipids  Preparation of  [3-0-(3,6-dioxaoctyl)cholest-5-en-3£-yl-  8-D-glucopyranosid]uronic acid (8) (5) (350 mg, 0.42 mmol) was dissolved in dichoromethane (6 mL).  anhydrous ether (6 mL) and  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. total of 4 hours at room temperature. to 3 with the addition of 10% HCl.  Stirring was continued for a  The pH of the solution was adjusted  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 d i s t i l l e d water and centrifuged again.  42  The precipitate was  dissolved in MeOH d^. *H NMR (250 MHz) CD 0D 3  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), (t, 1 3  3.52  IH), 3.64 (m, -12), 3.80 (d, IH), 4.37 (d, IH), 5.35 (d, IH)  C NMR CD 0D 3  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) CDC1  3  0.65(s, 3 H ) , 0.84(d of d, 6H),  0.97(s, 3H), 1.00-2.40 (~30H), 3 . 2 (broad m, I H ) , 3.60(d,  0.89  (d, 3H),  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 CDC1  3  11.9, 18.9, 19.4, 2 1 . 2 , 2 2 . 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 -1 (acid); 1220, 1085 cm C-0 stretch  Elem. Anal.  Calculated for  Mass Spec (DCI):  c i l  ^  7 0  °  (ester); 1660 cm  stretch  - 5 H 1 1  2  C=0  stretch  °  m/e 774,756,738 (M + nNH ) n=2,l,0; m/e 581 (M - 157) ; +  +  4  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 f r i t f i l t e r funnel and the resin is then rinsed with the following series of solvents: 100% ethanol  10-20 mL/g resin  3 times  d i s t i l l e d 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 b y removal of water by azeotropic d i s t i l l a t i o n 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 l e d with 4A molecular sieves. thimble apparatus was topped with a condenser.  The  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 t r a t e 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 t y 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  300 mL of 107. solution in dry benzene).  (giving  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). then air dried and evacuated to remove volatile Infrared:  KBr pellet  The polymer was  components..  (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.  46  The sample  was then poured into a 125 mL Erlenmeyer flask.  5% n i t r i c 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. to ice water (1 L). solution.  Concentrated sulfuric acid (250 mL) was added  The reaction mixture was poured into the acid  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. crude product was purified by vacuum d i s t i l l a t i o n . distillate 1  (47% of  The  The yield was 71 g of  theoretical).  H NMR (400 MHz) CDC1  3  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 3500 cm  -1 0-H stretch; 1760 cm  -1 C-0 stretch (carbonyl); 1260 cm  C-0  stretch (ether) Elem Anal.  Calculated for C,_H„_0,: 1U  Found: Mass Spec (DCI):  C 50.84%,  H 8.53%,  ZU 6  C 50.67%, H 8.53% m/e 254 (M + NH ) ; m/e 237 (M + H) ; m/e 279 (diacetyl +  +  4  tetraethylene glycol impurity + H) ; m/e 296 (279 + NH ) +  47  +  Preparation of mono t r i a l k y l s i l y l tetraethylene glycol a) t-butyl dimethyl s i l y 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 s t i r 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 f i l t r a t i o n was repeated to remove a l l of the precipitate and then the dichloromethane was removed by rotoevaporation.  The products were  purified by d i s t i l l a t i o n . a)  t-butyl dimethyl s i l y l tetraethylene glycol 1  H NMR (90 MHz) CDC1 : 3  0.50 (s, 6 H ) , 1.32 (s, 9H), 4.08  (s,  16H) Infrared: neat, NaCl plates: 3470 cm * OH stretch Mass Spec (CI): m/e 423 ( d i s i l y l ion: r e l . int. 38); m/e 309 (M-3) b)  +  (rel. int.  100)  trimethyl s i l y l tetraethylene glycol 1  H NMR (90 MHz):  0.74  (s, 9H),  48  4.25 (s, 16H)  Addition of mono acetyl tetraethylene  glycol to polymer bound t r i t y l  chloride Polymer bound t r i t y l chloride resin (10 g, 5.5 mmol CI) was dried in the usual fashion. round bottom flask.  After f i l t r a t i o n the resin was placed in a 250 mL Dry pyridine (50 mL) and monoacetyl tetraethylene  glycol (2.14g, 9.1 mmol) were added. at room temperature for 96 hours. the usual manner.  The flask was stoppered and stirred  The mixture was filtered and rinsed in  One gram of resin was cleaved and yielded approximately  90 mg of monoacetyl tetraethylene glycol. -1 -1 1730 cm C=0 stretch; 1220 cm C-0  Infrared: KBr pellet (uncorrected) stretch  Addition of mono t r i a l k y l s i l y l tetraethylene glycol to polymer bound t r i t y l chloride: Polymer bound t r i t y l chloride resin (20 g , l l mmol CI) was dried in the  usual fashion.  round bottom flask.  After f i l t r a t i o n the resin was placed in a 250 mL 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. hours.  The flask was stoppered and stirred at room temperature for 96  The mixture was then filtered and rinsed in the usual fashion. trialkyl:  a) t-butyl dimethyl s i l y 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 t e r i n g , the mixture  was cooled to room temperature and dry pyridine (50 mL) was added to i t (giving a 3:1 mixture of dry benzene and dry pyridine). chloride (6.5 mL, 84 mmol) was also added.  Methanesulfonyl  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. Infrared: KBr pellet  (uncorrected)  1350  -1 cm  -1 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. Infrared: KBr pellet  -1 1350 cm  -1 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. attached to the other.  One neck was stoppered and a gas line was  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. decanted.  This rinse was repeated two more times.  The pentane was  Distilled  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 nitrogen.  system was s t i l l being flushed with  After 2-3 minutes stirring at room temperature the bubbling  stopped and the solution was clear. mesylate (2 g,  1 mmol) was added.  Polymer  bound tetraethylene glycol  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 f i l t r a t e  was evaporated under reduced pressure.  51  The f i l t r a t e was then diluted  approximately 1:1 with d i s t i l l e d 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 d i s t i l l e d 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) CDC1  3  2.65 (s, water impurity) 3.61  (t,  4H)  3.65  (d,  3.65  (d,  ~24H) 3.74 (t, 4H) -1 Infrared:  neat, NaCl plates (uncorrected)  3390 cm  OH stretch;  1105 cm" C-0 stretch 1  Elem. Anal  Calculated for  C^U^„0i  Found: Mass Spec (DCI): b)  1.75H 0: 2  C 47.81%, H 9.40%  o  16 34 9  C 47.77%, H 9.37% m/e 388 (M + NH ) ; m/e 371 (M + H) +  +  4  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) CDC1  3  2.65 (s, water impurity) 3.61  (t,  4H)  ~40H) 3.74 (t, 4H) Infrared:  neat, NaCl plates (uncorrected)  3390 cm" OH stretch; 2915, 2865 cm" CH stretch; 1462, 1355 cm" CH bend; 1  1105 cm  -1  1  1  C-0 stretch  Elem. Anal.  Calculated for Found:  Mass Spec (DCI):  c  4 5 H  2  0 0  i3'  2 H  2°  :  C  4 9  -  4 7 %  >  H  C 49.45%, H 9.22%  m/e 564 (M + NH ) ; m/e 547 (M + H) +  4  52  +  9  -  3  4  %  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. McLaughlin, S., Biophysical Journal. 49, 741 (1986) .  3.  Brooks, D . E . , Sharp, K . A . , Fisher, D., Theoretical aspects of partitioning. In: H. Walter, D.E. Brooks, D. Fisher (Eds), Partitioning in Aqueous Two Phase Systems: Theory, Methods, Uses and Applications to Biotechnology. Academic Press, p. 11-85. (1985).  4.  Walter, H . , Surface Properties of Cells Reflected by Partitioning: Red Blood Cells as a Model. In: H. Walter, D.E. Brooks, D. Fisher (Eds), Partitioning in Aqueous Two Phase Systems: Theory. Methods, Uses and Applications to Biotechnology. Academic Press, p. 328-372. (1985).  5.  Gast, A . P . , Leibler, L . , Macromolecules 19, 686, (1986).  6.  Patel, K.R., L i , M.P., Schuh, J . R . , Baldeschwieler, et Biopysica Acta. 797, 20 (1984).  7.  Brockerhoff, H . , Ramsammy, L . S . , Biochimica et Biophysica Acta, 691. 227 (1982).  8.  Patel, K.R., L i , M.P., Schuh, J . R . , Baldeschwieler, et Biophysica Acta 814, 256 (1985).  9.  Goodrich, R.P., Handel, T . M . , Baldeschwieler, Biophysica Acta, 938. 143 (1988).  J . D . , Biochimica  J . D . , Biochimica  J . D . , Biochimica et  10. Nakutsuji, Y . , Kameda, N . , Okahara, M., Syntheses. (1987) .  March, 280  11. Bartsch, R.A., Cason, C . V . , Czech, B.P., Journal of Organic Chemistry, 54, 857, (1989). 12. V i t a l i , C.A., Masci, B . , Tetrahedron, 45(7), 2201, (1989). 13. Hodge, P., Organic Reactions using Polymer-Supported Catalysts, Reagents or Substrates, in Syntheses and Separations Using Functional Polymers, ed. Sherrington, D . C , Hodge, P., John Wiley and Sons, Chinchester. 1988. 14. Akelah, A. Sherrington, D . C , Chemical Reviews, 81, 557, (1981). 15. Frechet, J . M . J . , Tetrahedron, 37, 663, (1981).  53  16. Merrifield, R.B., Journal of the American Chemical Society. 85, 2149 (1963) in Solid Phase Synthesis, ed. Blossey E . C . , Neckers, D . C , John Wiley and Sons, Inc. (1975). 17. Henahan, J . F . , Chemical and Engineering News. Aug. 2, 22 (1971) in Solid Phase Synthesis, ed. Blossey E . C . , Neckers, D . C , John Wiley and Sons, Inc. (1975). 18. Gutte, B . , Merrifield, R . B . , Journal of Biological Chemistry. 246(6), 1922 (1971) in Solid Phase Synthesis, ed. Blossey E . C , Neckers, D . C , John Wiley and Sons, Inc. (1975). 19. Letsinger, R . L . , Mahadeuan, V . , Journal of the American Chemical Society, 87(15), 3526, (1965) in Solid Phase Synthesis, ed. Blossey E . C , Neckers, D . C , John Wiley and Sons, Inc. (1975). 20. Excoffier, g., Gagnaire, D., U t i l l e , J . P . , Vignon, M., Tetrahedron Letters, No. 50, 5065, (1972) in Solid Phase Synthesis, ed. Blossey E . C . , . Neckers, D . C , John Wiley and Sons, Inc. (1975). 21. Bonds, W.D., Brubaker, C . H . , Chandrasekaran, E . S . , Gibbons, C . , Grubbs, R . H . , Kroll, L . C , Journal of the American Chemical Society, 97, 2128 (1978). 22. Worster, P.M., McArthur, C R . , Leznoff, C . C , Angew Chem. International Edition, 18, 221 (1979). 23. Bollenback, G.N., Long, J.W., Benjamin, D.G., Lindquist, J . A . , Journal of the American Chemical Society, 77, 3310 (1955). 24. Simons, W.W., (ed), The Stadtler Handbook of Proton NMR Spectra. Stadtler Research Laboratories Inc. Philadelphia. 1978. 25. Fieser, L . , Fieser, M., Reagents in Organic Synthesis, vol. 2, 363, Wiley-Interscience, New York. 1969. 26. Schneider, (1969).  J . J . , Bhacca, N.S., Journal of Organic Chemistry, 34, 1990  27. Johnson, L . F . , Jankowski, W . C , Carbon-13 NMR Spectra, Wiley, New York, 1972. 28. Pfeffer, P . E . , Valentine, L , M . , Parrish, F.W., Journal of the American Chemical Society. 101, 1265, (1979). 29. Olgivie, K . K . , Iwacha, D . J . , Tettrahedron Letters. No. 4, 317 (1973). 30. Corey, E . J . , Venkateswarlu, A . , Journal of the American Chemical Society, 94(17), 6190 (1972). 31. Lalonde, M . , Chan, T . H . , Synthesis, September, 817, (1985). 32. Fyles, T.M. "The Solid Phase Synthesis of Insect Sex Attractants." PhD Thesis, York University (Canada) (1977).  54  APPENDIX I  Selected Spectra  1  H NMR compound (3)  56  H NMR compound (7)  57  1 3  1  C NMR compound (7)  58  H NMR compound (8)  1 3  59  C NMR compound (8)  60  IR  polymer bound t r i t y 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  55  65  compound  loo  7  fcle-*^  C0,Me OAc protected  glycollpid  1 ~i I;  1  1  1  r  C 3 O  a.  e o o  p4"V>o.»l  compound  8  • W/ftnr\ &  T V C G . fi I cA>*o  compound  8 iiCy  a, o  1  i  1  1  1  1  1  i>  1  1  o  rf  *l  c-*n  ©TrCI (p)=polystyrene 0.5  W. d l v l n y l  mmol C l / g r a m  polymer  benzene  CONDITIONS: FRACTOGEL 40S COLUMN ELUENT: RATE:  DISTILLED WATER 0.2  ML/MIN  CHART SPEED: READOUT:  2 ML/MIN  2 ML/CM  COLUMN HEIGHT: COLUMN DIAMETER:  60 CM 1. 5 CM  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0059693/manifest

Comment

Related Items