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SYNTHETIC ROUTES TO HIGHER-ORDER OLIGOSACCHARIDES
CORRESPONDING TO THE CELL-WALL POLYSACCHARIDE
OF THE j9-HAEMOLYTIC STREPTOCOCCI GROUP A
John Schofield Andrews
B.Sc (Hons.) University College of Swansea, Wales, U.K., 1986
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIRMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in the Department
0 f
Chemistry
@ John Schof ield Andrews 1989 SIMON FRASER UNIVERSITY
January 1989
All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author.
i i
APPROVAL
Name : John S. Andrews
Degree : M.Sc. Chemistry
Title of thesis: Synthetic Routes To Higher-Order Oligosaccharides Corresponding To The
Cell-Wall Polysaccharide Of The f3-Haemolytic Streptococci Group A
Examining Committee: Chairperson: P. Percival, Professor
B. M. Pinto, Senior Supervisor, Assistant Professor
.< - f
-- R.J. Cushley, Professor
r L .
/ M.J. Gresser, Ass iate Professor
,
External Examiner: A.C.
Date Approved: March 29th, 1989
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T i t l e o f Thes i s/Project/Extended Essay
SYNTHETIC ROUTES TO HIGHER-ORDER OLIGOSACCHARIDES
, OF THE 8-HAEMOLYTIC STREPToCoCCI GROUP A.
Author: - -
s ignature)
JOHN S. ANDREWS
( name 1
3rd MARCH 1989
(date)
iii
ABSTRACT
The synthesis of a fully functional linear trisaccharide and its
use in the synthesis of a branched tetrasaccharide corresponding to a
portion of the cell-wall polysaccharide of the /3-haemolytic
Streptococci Group A is described. The acetate groups on the
disaccharide, allyl 3-0-(3',4',6'-tri-O-acetyl-2'-deoxy-2'-phthal mido < /3-D-glucopyranosyl)-2-O-benzoyl-4-O-benzyl-a-L-rhamnopyranoside, w 2 re transesterified and the resulting free alcohol groups were protected
using 2-(trimethylsilyl)ethoxymethyl chloride. Removal of the allyl
group was accomplished by isomerisation to the prop-1-enyl group,
followed by hydrolysis of the vinyl ether to give the hemiacetals. The
hemiacetals were then treated with the Vilsmeier-Haack reagent, N,N-
dimethyl(chloromethy1ene)ammonium chloride, affording the
functionalised disaccharide chloride as a glycosyl donor. Reaction of
the glycosyl donor and the monosaccharide allyl 2,4-di-0-benzyl-a-L-
rhamnopyranoside under Konigs-Knorr conditions afforded the afore-
mentioned linear trisaccharide, allyl 3-0-(2'-0-benzoyl-4'-O-benzyl-3'-
0-(2"-deoxy-2''-phtha1imido-3'',4'',6''-tri-0-2-[trimethy1si1y1]-
ethoxpethyl-/3-D-glucopyranosyl)-a-l-rhamnopyranosyl)-2,4-di-O-benzyl-
a-L-rhamnopyranoside. Selective removal of the 2'-0-benzoate yielded a
fully functional trisaccharide for use in the synthesis of the branched
tetrasaccharide. Reaction with the glycosyl donor, 2,3,4-tri-0-acetyl-
a-L-rhamnopyranosyl bromide under modified Konigs-Knorr conditions
afforded the fully blocked tetrasaccharide as its allyl glycoside.
Deprotection was accomplished by transesterification, followed by
hydrogenolysis, hydrazinolysis, and selective N-acetylation to give the
iv
pure tetrasaccharide as its propyl glycoside. The tetrasaccharide may
be used as a hapten in binding studies with complementary monoclonal
antibodies raised against the natural antigen.
v
DEDICATION
To D and M , the other two.
vi
ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr. Mario Pinto for giving me
the opportunity to carry out interesting research, and for his
invaluable guidance and constructive criticism of my work and in the
preparation of this manuscript. I would also like to thank Marcy
Tracey and Kerry Reimer for recording numerous routine and two-
dimensional NMR spectra, and M.K. Yang for microanalyses.
vi i
TABLE OF CONTENTS --
Page Number
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Approval Page ii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abstract iii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dedication v
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements vi
Table Of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List Of Tables :. viii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List Of Figures ix
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . List Of Abbreviations xi
Introduction
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
. . . . . . * * . . . . . . . . . . . . . . . . . . c; Methods Of Glycosylation
Synthetic Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Results and Discussion
Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NMR Analysis 27
. . . . . . Comments and suggestions for future research 47
Experimental
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Procedures 49
Specific Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
v i i i
LIST OF TABLES --
Page Number
Table 1. Important react ion parameters a f fec t ing
s e l e c t i v i t y and y i e ld ( i n glycosylations) . . . . . . 13
Table 2 . Attempted reactions t o synthesise
branched tetrasaccharide 44 . . . . . . . . . . . . . . . . . . . . 25
Table 3 . ' H NMR data fo r the r ing protons i n
the t r isacchar ide 39 . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Table 4 .
Table 5 .
Table 6.
I3c NMR data fo r the r ing carbons i n
the t r isacchar ide 39 . . . . . . . . . . . . . . . . . . . . . . . . . . .
H NMR data f o r the r ing protons i n
the tetrasaccharide 44 . . . . . . . . . . . . . . . . . . . . . . . . .
l 3 C NMR data fo r the r ing carbons i n
. . . . . . . . . . . . . . . . . . . . . . . . . the tetrasaccharide 44 45
Table 7 . 'H NMR data fo r the r ing protons i n
. . . . . . . . . . . . . the deprotected tetrasaccharide 45 46
ix
LIST OF FIGURES --
Page Number
1. Schematic representation of the structure of
the streptococcal cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Group specific carbohydrates of the
P-haemolytic Streptococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3 . Synthesis of the artificial antigen by the
acyl azide coupling reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. Representation of a general glycosylation route
(eg. for sugars in the rhamno-series) . . . . . . . . . . . . . . . . . . 6
5. Neighbouring group assisted procedure showing
1,2-trans linkages for sugars in the D-manno-
and D-gluco-series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6. Application of the phthalimido group in preparation
. . . . . . . . . . . . . . . . . . . . . . of sugars in the P-D-gluco-series 9
7. In situ anomerisation procedure (in absence of
neighbouring group) showing preferential formation
of the 1,2-cis-glycoside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
8. Structure of the cell-wall polysaccharide
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . of Streptococci Group A 15
9. Previously synthesised oligosaccharides corresponding
to portions of the cell-wall polysaccharide
of Streptococci Group A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
10. 400.13 MHz ~D-'H NMR COSY spectrum of
trisaccharide 39 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
X
11. Expanded region of the 2D-IH NMR COSY spectrum of
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . trisaccharide 39 32
12. 2D-I3c-l H chemical-shift correlated spectrum of
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . trisaccharide 39 33
1 3 . Expanded region of the 2I1-l C-I H chemical-shift
. . . . . . . . . . . . . . . . correlated spectrum of trisaccharide 39 34
14. 400.13 MHz 2 ~ - I H NMR COSY spectrum of
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . tetrasaccharide 44 35
15. Expanded region of the ~D-'H NMR COSY spectrum of
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . tetrasaccharide 44 36
16. 2 ~ - I C-I H chemical-shift correlated spectrum of
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . tetrasaccharide 44 37
17. Expanded region of the 2 ~ - I C-I H chemical-shift
. . . . . . . . . . . . . . correlated spectrum of tetrasaccharide 44 38
18. 400.13 MHz 2 ~ - I H NMR COSY spectrum of
. . . . . . . . . . . . . . . . . . . . . . . . . deprotected tetrasaccharide 45 39
19. Expanded region of the 2D-IH NMR COSY spectrum of
. . . . . . . . . . . . . . . . . . . . . . . . . deprotected tetrasaccharide 45 40
1
1. INTRODUCTION -
Background
The B-haemolytic Streptococci Group A is widely recognised as the
organism responsible for causing streptococcal pharyngitis or strep
throat in humans .' Whilst not being a serious condition on its own, it
may lead to the additional complications of rheumatic fever and acute
glomerulonephritis. Streptococci Group A have also been implicated in
the induction of rheumatoid arthritis and chronic rheumatic heart
disease; the latter may proceed to the more serious condition of
chronic rheumatic valvular disease. The immunological cross -reaction
between the Streptococci Group A cell-wall polysaccharide and the
structural glycoproteins of heart valves has also been e~tablished;~
polysaccharide from Group A Streptococci induce the production of
antibodies which then may react against the host's own valvular
glycoproteins.
Ca~sule
Cell wall
3, - Protein
I ' Hucopep t ide
h Protoolast membrane
Figure 1. Schematic representation of the structure of the streptococcal cell
The streptococcal cell is surrounded by an outermost layer of
capsular polysaccharide, beneath which is the cell-wall comprising a 21
layer of protein, group specific carbohydrate, and mucopeptide. Below \ /
the cell-wall is the protoplast, or cell-membrane (Figure 1). The
serological classification of Streptococci is categorised according to
the structure of the cell-wall carbohydrates. These are classified
into three main groups, Groups A, A-variant, and C (Figure 2).
Group A
Group C
Figure 2 Group-specific carbohydrates of p-haemolytic Streptococci
Structural studies4 have shown that group-specific carbohydrates
of p-haemolytic Streptococci are composed of a common linear poly-
rhamnopyranosyl backbone of alternating a-L-(1-2) and a-L-(1-3)
linkages. The Group A-variant carbohydrates consist solely of this
alternating backbone whereas the Group A contain ,f3-D-N-acetyl-
glucosamine residues attached to the 3-position and the Group C contain
3-O-a-D-N-acetylgalactosaminosyl-a-D-N-acetylgalactosamine disaccharide
bacterial serogroups only Group A is clinically significant. The
immunodominant group of Group A carbohydrates is the GlcpNAc monomer as
indicated by the fact that GlcpNAc readily inhibits the immunological
reaction between Group A carbohydrate and Group A antibodies.'~~
The diagnosis of streptococcal pharyngitis has been traditionally
accomplished by standard culture techniques or by fluorescent antibody
procedures,7s8 both of which may take a day or longer, and require some
degree of technical sophistication. Hence, antibiotics are either
prescribed before confirmation of a diagnosis by a positive culture
test, or after a positive test and the bacterial infection is already
well established. Since prompt antibiotic treatment reduces the
morbidity from group A streptococcal pharyngitis, a quick and simple
test for the diagnosis of the dtsease is desirable. Recently, several
rapid detection methods hzve been devehped9 which cax detect groclp A
streptococcal antigen directly from a swab of the patient's pharynx.
The test assays involve the extraction of the group antigen (with
nitrous acid or enzyme) from the cell-walls of group A Streptococci and
reaction of the extract with the specific antibody linked to latex
particles (latex agglutination). Test kits'' employing both of these
methods have been developed, and their clinical evaluation1 - has
indicated good specificity as compared with the culture method.
However, in two of these studies1 ' I2 the sensitivity of the test was
found to be significantly lower than that obtained using the standard
culture techniques, requiring the confirmation of negative tests
obtained with the test kits.
If portions of the Group A Streptococci cell-wall carbohydrates
could be chemically synthesised, the corresponding glycoconjugates
could be prepared by coupling them to a carrier protein, enzyme, or
immunoadsorbent via the 8-methoxycarbonyloctyl linking arm,14 using the
acyl azide methodology. 15
H2 NNH2 . EtOH a-L-Rhap
\a - L- ~ha~--a - L- Rhap-0 (CH~ )8 CONHNH2 /3-D-GlcpNAc /
I N204 in DMF
a-L-Rhap \ ~ - L - R ~ ~ ~ - - ~ - L - R ~ ~ ~ - O ( C H ~ )8 CON^
/3-D-GlcpNAc / I
1 Protein solution 1 -L-Rhap\ / c~-L-Rhap-a-L-Rhap]-O(CH~)~C0 "NH-Protein
/3-D-GlcpNAc i 1 Figure 3. Synthesis of the artificial antigen by the acyl
azide coupling reaction
The artificial antigens thus obtained could be used in the latex
agglutination method to test for anti-Streptococci Group A antibodies
in patients' sera. Alternatively, specific monoclonal antibodies of
highly defined specificity could be raised against the artificial
antigens using the hybrid-myeloma technique. l6 The monoclonal
antibodies produced could then be used to improve or replace existing
diagnostic reagents and detect Streptococci Group A antigens by enzyme-
linked immunoadsorbent assay (ELISA). The alternative kits utilising
specific monoclonal antibodies would be both more sensitive and more
5
specific than methods currently available.
This thesis describes the synthesis of oligosaccharides that can
be used for the aforementioned purposes. Specifically, we propose to
synthesise a fully functional linear trisaccharide for use in the
synthesis of tetrasaccharide and higher-order oligosaccharide portions
of the cell-wall polysaccharide of the /I-haemolytic Streptococci Group
A. The oligosaccharides could then be used as haptens in binding
studies, or as antigens after covalent coupling to protein.1 Specific
monoclonal antibodies raised against the antigens will be used,
together with natural antigen in competitive binding studies. The
oligosaccharide which gives the maximum inhibition and hence maximum
specificity will be used to identify the surface unique to the
Streptococci Group A organism, and may be used immunodiagnostically, as
previously described.
Methods of Glycosylation
In recent years there has been a resurgence in the interest in the
chemistry and biochemistry of carbohydrates. It has been found that
the oligosaccharide residues of glycoconjugates are of importance in
many recogniton processes such as those involved in antibody-antigen
recognition.
The chemical syntheses of complex oligosaccharides have been
reviewed in the literature17 and only the important methods of
glycosylation are presented in this section. Glycosylation reactions
generally involve the selective coupling of two polyfunctional sugar
units. The reaction strategy involves the selective blocking or
I protec t a l l
1 but one OH
I protec t a l l OH X=good leaving
I group on the anomeric carbon
GLYCOSYL ACCEPTOR GLYCOSYL DONOR
cis o r trans l i n k a g e depending upon na tu re o f R3and c a t a l y s t
F igu re 4 . Representa t ion o f a genera l g l y c o s y l a t i o n r o u t e (eg. f o r sugars i n the rhanno- se r i es )
7
deblocking of hydroxyl groups depending on their involvement in
coupling. The blocking groups used must be stable to a multitude of
conditions (acidic and basic), yet amenable to selective removal under
conditions sufficiently mild so as not to cleave inter-glycosidic
linkages. In glycosylation reactions the anomeric centre of one of the
sugar units must be functionalised with a good leaving group, often a
halide (glycosyl donor), and reacted with its partner on which usually
all but one hydroxyl group have been protected (glycosyl acceptor)*.
The reactivities of the donor and acceptor are very dependent upon the
choice of leaving and protecting groups which are very often the
decisive factors in glycosylation reactions (Figure 4).
The stereochemical outcome of any glycosylation reaction must be
predictable, since only the oligosaccharide of specified stereo-
chemistry is desired. The stereochemistry at newly formed glycosidic
centres is governed by the reaction conditions, the reactivities of the
two partners, and the presence or absence of neighbouring groups (to
the anomeric centre). There are two general types of glycosidic
linkage; 1,2-cis and 1,2-trans, in which the aglyconic oxygen and the
2-hydroxyl are cis and trans to each other, respectively. 1,2-Cis
linkages are involved in the formation of a-glycosidic bonds in the
gluco- and galacto- series, and p-glycosidic bonds in the manno-
series. Conversely, 1,2-trans linkages are involved in the formation
of 8-glycosidic bonds in the gluco- and galacto- series, and a-
glycosidic bonds in the manno- series. The chemical synthesis of
oligosaccharides involving the formation of these two types of linkages
*Glycosyl donors and acceptors are not termed as such due to their donation/acceptance of electrons as in modern organic chemistry. Historically, they are donors or acceptors in the context of a glycosylation reaction.
9
is very different, requiring distinct reaction conditions, and
therefore, will be discussed separately.
1,2-Trans Glycosylations
Linkages of this type are generally formed from the stable a-D-
glycosyl halides with neighbouring groups such as acetates or benzoates
at C-2. A catalyst with receptor qualities causes the expulsion of
halide yielding stable cyclic dioxocarbenium ions via the participation
of the neighbouring group with the oxocarbocation.'8 This newly formed
cyclic dioxocarbenium ion regulates the stereochemical outcome of the
following glycosylation reaction by effectively blocking the cis face
to attack by the incoming alcohol. It is possible therefore, to obtain
exclusively the trans product from a glycosylation, for e'xample, sugars
in the 8-D-gluco- or a-D-manno- series (Figure 5). Common promotors
used to facilitate neighbouring group participation include Hg(CN\ ' 2 '
HgClZ, HgBrZ, AgC104, and Ag triflate. Another useful method for the
synthesis of 1,2-trans-linked 2-amino sugars employs the use of the
Figure 6. Application of the phthalimido group in the preparation of sugars in the fl-0-gluco- series.
10
phthalimido group as a non participating substituent at the 2 -
position'9 (Figure 6). Whilst the cyclic dioxocarbenium ion is not
formed in this case, the steric bulk of the phthalimido group directs
the incoming nucleophile to the /?-face.
1,2-Cis Glycosylations
The synthesis of this type of linkage, for example, those in a-D-
glucopyranosides and /3-D-mannopyranosides is far more difficult. The
presence of a participating group at the 2-position would cause the
formation of a 1,2-trans linkage. Therefore it is necessary to have a
non-participating group such as N3, NO2, Bzl, or 0COCC13 at the 2 -
position. Synthesis of a-D-glucopyranosides via /I-halides would, in
theory, induce reaction with inversion of configuration. In practice
though, the unstable /I-halides anomerise to the more stable a-anomers
resulting in anomeric mixtures of glycosldes . Lemieux et 21. devised
a method, referred to as the in situ anomerisation procedure, to bypass
this problem (Figure 7). The method utilises the more stable a-D-
pyranosyl halide with no neighbouring group, which in the presence of a
Lewis-catalyst such as a tetraalkylammonium halide or metal salt,
causes an equilibrium between the a-D and b-D-halides proceeding via
different ion pairs. The equilibrium strongly favours the a-D-halide
which is stabilised by the anomeric effect,21 albeit the reaction of
the /?-D-halide to the a-D-glucopyranoside is favoured kinetically to
such an extent that this reaction can be accomplished with great
stereoselectivity.
This method, however, cannot be used to synthesise /?-D-
mannopyranosides, since the kinetically favoured reaction of the /I-D-
% OR'
Figure 7. In s i t u anomerisation procedure (in absence of oeighbouring group) showing preferential formation of the 1,2-cis glycoside.
12
halide would lead to the formation of the a-D-mannopyranoside.
Reaction of the stable a-D-halide, containing no neighbouring group,
with inversion of configuration should, in theory, give rise to the
desired product. The heterogenous phase reaction employing silver
carbonate, silver silicate or silver salicilate as catalysts has been
used sucessfully to prepare p-D-mannopyranosides. 2 2
In oligosaccharide synthesis the reactivity of the glycosyl
acceptor and donor depend upon several reaction parameters. In
general, there are few standard conditions for a given reaction. Each
individual reaction type requires specific reaction conditions. The
reaction parameters must therefore be adjusted for individual reactions
in order to maximise selectivity and yield. For example, the choice of
type and number of hydroxyl protecting groups and the nature of the
halide leaving group have profound effects on the reactivity of the
glycosyl halide. 1 7 c * 2 3 Selection of the c ~ r r e c t catalyst i s also
crucial in obtaining the desired reaction and must be chosen according
to catalyst reactivity and reaction type. In addition, the solvating
ability of the solvent also appears to play a crucial role. Varying
the reactivity of the hydroxyl group generally presents the most
problems since the structure of the glycosyl acceptor is usually
predetermined, although primary hydroxyl groups are more reactive than
secondary. Table 1 shows the most important reaction parameters and
lists them according to reactivity.
The methods of glycosylation reviewed so far all employ a halide
as leaving group on the glycosyl donor. Alternative leaving groups
include 0-acetyl functions which, whilst being less reactive than
halides, can be removed with Lewis acid catalysts and are suitable for
13
use in the neighbouring group assisted procedures. 24 The use of
Table 1. Important reaction parameters affecting selectivity and yield
Donor Acceptor Promoter
Et4NBr/4A molecular sieves
Hg(CW2
Hg ( CN) 2 /HgBr2
R HgBr2/4A molecular sieves
R-Bz l>Bz>Ac lo-OH>2"-OH Ag triflate, Ag2C03
X-I>Br>Cl 6-OH>>3-OH>2-OH>4-OH Ag triflate/4A mol. sieves
trichloroacetoimidate as a leaving group has been developed by Schmidt
et alZ5 employing BF3-ether as catalyst. Whilst being less reactive
than halide, the trichloroacetoimidate is far more reactive than O-
acetyl and is suitable for use in compounds with or without neighbour-
ing groups, to give both 1,2-trans and 1,2-cis linkages.
Other methods employ stable thoiglycosides as glycosyl donors
(with or without neighbouring group activated substituent at C-2); the
reactions are activated by either methyl triflate,26 NBS,27 or dimethyl
(methylthio) sulphonium trif late. The nucleophilicity of the bivalent
sulphur atom towards these reagents generates an unstable sulphonium
species from S-glycosides that react with the nucleophilic glycosyl
acceptor. The disadvantage of these methods are low yields, long
14
reaction times, and the high toxicity and difficulty in handling of the
reagents.
To circumvent these problems Pozsgay and ~ e n n i n ~ s ~ ~ have used
nitrosyl tetrafluoroborate to activate methyl and phenyl thio-
glycosides, both of which are readily available. In reactions where
either a participating neighbouring group or a phthalimido group at C-2
are present, the exclusive formation of a 1,2-trans-0-glycosidic
linkage occurs. Conversely, a 1,2-cis-0-glycosidic linkage is formed
when a non-participating group at C-2 is present. The mechanism for
the glycosylation is thought to proceed via the intermediate S-nitroso
species which leads, in turn, to the cyclic dioxocarbenium ion via the
participation of a neighbouring group with the previously formed
reactive oxocarbocation. The cyclic intermediate then effectively
blocks the cis face to attack by the glycosyl acceptor at C-1 leading
to che exclusive f~rmation of the trans linkage.
Synthetic objectives
The objective of this research project was to synthesise a fully
functional trisaccharide for use in the synthesis of tetrasaccharide
and higher-order oligosaccharide portions of the cell wall poly-
saccharide of Streptococcus Group A. The oligosaccharides together
with previously synthesised ~li~osaccharides~~ (of different chain
length and degree of branching) could then be used immunodiagnostic-
ally, as previously described.
Previous workers in our laboratory have synthesised various
oligosaccharides corresponding to segments of the cell wall
polysaccharides of Streptococci Group A (Figure 8). These are the
Figure 8. Structure of the cell-wall polysacchar ide o f Streptococci Group A .
disaccharides3' BC (1: and 2), and linear3' ABC (3 and 4) and branched3'
B(C)A1 (2 and 6 ) trisaccharides, synthesised as their n-propyl and 8 -
methoxycarbonyloctyl glycosides (Figure 9). However, none of these
synthetic efforts afforded a fully functionalised oligosaccharide block
that could be used for the elaboration of higher-order structures. For
example, initial attempts to synthesise a linear trisaccharide from
the disaccharide 2 (Scheme 1) were unsuccessful, the glycosylation
reactions proceeded with low stereoselectivity and/or yielded
elimination product. Compound 12 (obtained from 10 by deacetylation)
would have served as a suitable glycosyl acceptor in future
glycosylations. This route was abandoned as a general synthetic route
16
although the disaccharide L was used in the terminal synthesis
of the branched trisaccharides 5 and 5.31
OH NHAc
HO
Figure 9. Previously synthesised 01 igosacchar ides corresponding to port ions of the cell-wall polysaccharide of Streptococci Group A.
It was desirable therefore, to develop a general synthetic route
to higher-order structures. This thesis describes such a route, in
particular, the synthesis of a suitably functionalised trisaccharide
(ABC) and its use for the synthesis of a branched tetrasaccharide
(AB(C)A').
Investigation into the use of the 2-(trimethylsilyl)ethoxymethyl
(SEM) group32 as a viable alcohol protecting group in carbohydrate
chemistry 'has been carried out in our laboratory. 3 3 It was discovered
that SEM ethers were stable during the removal of benzoate esters and
during the isomerisation and subsequent hydrolysis of the 1-0-ally1
NPhth
Bz 10
\ OBzl OBz 1
BzlO
\
BzlO 4iZl
Scheme 1. P r e v i o u s l y a t tempted syn thes i s o f t r i s a c c h a r i d e acceptors .
BzlO
group. The effect of SEM ethers (on glucosamine) on the reactivity of
a glycosyl donor, and its stability during glycosylation reactions
however, was unknown.
sEM$EM OSEM
B z l O * - BzlO R=CH zCH=CH 2
NPhth R= (CH 2) a CO 2CH 3
18 19 RI R i =CH = (CH 2CH=CH 2) aC0 2CH 2 3 R 2432 R S E ~ 2=Bz
20 R i =CH 2CH=CH 2 R 2=H OSEM 21 RI = (CH 2) aC0 2CH 3 R 2=H
SEMO
Scheme 2. Proposed synthesis of branched te t rasacchar ides
19
We envisaged that it would be possible to synthesise the
disaccharide (BC) used in previous syntheses3 with SEM protecting
groups at the 3,4 and 6 positions of N-phthalimidoglucosamine (l4)
(instead of previously used acetate or benzyl groups). Removal of the
1-0-ally1 group on rhamnose could be effected by isomerisation to the
prop - 1 - enyl group3 4 8 * and subsequent hydrolysis to afford the hemi -
a~etals.~' Formation of the glycosyl halide 15 could then be possible
using Vilsmeier-Haack reagents ,36 rendering the disaccharide a
useful glycosyl donor.
Synthesis of a fully functional trisaccharide as the allyl and 8-
methoxycarbonyloctyl glycosides 18 and l9, from and monosaccharides
16 and 17 respectively, followed by selective removal of the 2 ' - -
benzoate in the presence of benzyl and SEM ethers should then be
feasible giving the selectively functionalised trisaccharides 20 and 21
as glycosyl acceptors. Synthesis of the tetrasacchzride as the ally1
glycoside 22, followed by stepwise deprotection36a 38 should afford the
appropriate hapten 24, whereas the corresponding 8-methoxycarbonyl-
octyl glycoside 23 would afford the tetrasaccharide 25 in a form
suitable for the preparation of an artificial antigen (Scheme 2).
The synthesis of a branched hexasaccharide AB(C)A'B1(C') should
also be possible. Glycosylation of 20 or 21 with the trisaccharide
2630 would afford the hexasaccharide as the allyl (27) and 8-methoxy- -
carbonyloctyl (28) glycosides, respectively. Deprotection, as before,
would then afford 29 and 3 (Scheme 3).
a 7 0 cum
2 1
2. RESULTS AND DISCUSSION -
Synthesis
The first stage in the synthesis was the removal of the acetate
groups in the disaccharide x3O with 3% HC1 in methanol to give 32 in 81% yield. Protection of the alcohol groups with SEN was accomplished
with 2-(trimethylsilyl)ethoxymethyl chloride and hindered base
diisopropylethylamine32 giving 33 in 70% yield (Scheme 4).
Bzlo+-? OBZ
NPhth
AcO 8 ____C
NPhth
SCHEME 4.
Isomerisation of the ally1 group was achieved using tris(tri-
phenylphosphine) rhodium I chloride as catalyst3 a 1 giving the prop - 1 -
enyl glycosides 2. These were then hydrolysed to the hemiacetals 35
using mercuric chloride-mercuric oxide35 in an overall yield of 7 6 % .
The disaccharide was now needed as a glycosyl donor in the
following glycosylation to prepare the trisaccharide. This Was
achieved by making the glycosyl halide of correct reactivity, namely
22
the a-glycosyl chloride 36, prepared from the mixture of hemiacetals
using the Vilsmeier-Haack reagent, N,N-dimethylamine(chloromethy1ene)-
ammonium chloride. a
OSEM OSEM
SCHEME 5.
The trisaccharide 3 was synthesised in 81% yield by a Kdnigs-
Knorr reaction between the glycosyl chloride donor 36 and acceptor 3733
using silver trifluoromethanesulphonate as promoter and 1,1,3,3-
tetramethylurea39 as proton accceptor. The stereoselective formation
of the a-glycoside was achieved by the neighbouring group participation
of the benzoate group during the glycosylation. The reactive cyclic
dioxocarbenium intermediate 38 formed effectively blocks the /I-face of
the disaccharide to attack from the incoming alcohol 37 (Scheme 5 ) .
Selective removal of the 2'-benzoate was accomplished with 0.1 M
sodium methoxide solution to yield a fully functionalised trisaccharide
acceptor 40 (81% yield) for use in the preparation of the
tetrasaccharide.
Numerous reactions to synthesise the tetrasaccharide were
attempted (see Table 2). The most succesful reaction was that of 40
with 2 , 3 , 4 - tri-G-zcetyl-0-5=rhamn~pyranosj:1 bromide u4 zs donor and a
mixture of mercuric cyanide and mercuric bromide as Lewis acid
promotors, to give 44 in 48% yield (Scheme 6, R X ~ IV Table 2).
The glycosylation reaction (I) was initially envisaged to go in
high yield since a very similar reaction during the synthesis of the
branched trisaccharide 5 by a previous worker was achieved in 81%
yield.31 This was a K6nigs-Knorr reaction between ally1 3-0- (3' ,4' ,6' -
tri-0-benzyl-2'-deoxy-2'-phthalimido-~~-~l~~0~~r~n0~~l)-4-O-benz~l-~-
L-rhamnopyranoside (synthesised from the corresponding 2-0-acetate) and
2-O-acetyl-3,4-di-O-benzyl-a-l-rhamnopyr0~1 chloride42 using silver
trif luoromethanesulphonate as promoter and 1,1,3,3- tetra~neth~lurea~~ as
proton acceptor.
The poor yields of tetrasaccharide obtained in all the reactions
x OBz 1
0 Ac
SEMO
OSEM AcO
SCHEME 6.
Table 2:
Attempted reactionsa to synthesise branched tetrasaccharide 44.
Monosaccharide
RxN
. Lewis acid
Base
hN
. temp.
% donorb
No.
promoterC
' range
"C
yieldd
I
Silver
TMU
-78
-25
8
.5
414
trif late
43e
VI I
Silver triflate
TMU
-78
-25
0
'In
anhydrous dichloromethane.
in 3 molar equivalents to tri-
saccharide alcohol(40).
'Ratios
of Hg(CN)2:HgO/HgBr2
are 1:l where
appropriate. d~ields of tetrasaccharide are based on the trisaccharide
alcohol (40). ePrepared by bubbling HC1 gas through a solution of 1
,2,3
,4-
tetra-0-acetyl-a-L-rhamnopyranoside in glacial acetic acid.
could be due to the presence of a very hindered trisaccharide alcohol
40. In support of this hypothesis, removal of the 2'-benzoate from the -
trisaccharide 39 takes typically 50-60 hours in 0.1 M NaOMe solution
compared to 2-3 hours for the removal from the mono~accharide~~ ally1
2-O-benzoyl-4-O-benzyl-3-0-2-(trimethylsilyl)ethoxpethyl-a-L-rhamno-
pyranoside (in 0.15-2.0 M NaOMe). Further evidence is provided by the
fact that, contrary to normal, the alcohol 40 in this case is less
polar than the corresponding blocked tetrasaccharide 44. This suggests
that the alcohol function is in a sterically hindered environment
causing a decrease in the overall polarity of the molecule. The steric
hindrance is presumably due to the presence of the large SEM groups.
In addition, the larger than normal C{ H) coupling constants for
C-1" and C-1"' support this theory (see NMR discussion).
The glycosyl halides are very sensitive to the reaction conditions
and are broken down (to the hemiacetaisj before an appreciable amount
of the glycosylation reaction takes place. In reaction VI the less
reactive tri-0-acetyl-a-L-rhamnopyranosyl chloride 4343 was used in the
hope of overcoming this complication. However, no reaction took place.
Future reactions that might be worth attempting are those between
the trisaccharide alcohol 40 and ethyl 2,3,4-tri-0-acetyl-1-thio-a-L-
rhamn~~~ranoside~~ or ethyl 2 -0-acetyl-3,4-di-0-benzyl- 1- t h i o - d -
rhamn~pyranoside,~~ activated by either nitrosonium tetrafl~oroborate~~
or methyl trif luorome thanesulphonate . The postulate of the presence of a sterically hindered alcohol
could be tested by obtaining a two dimensional nuclear Overhauser
enhancement (NOESY) NMR spectrum of the By observation of
nOe effects between protons on different saccharide units across
2 7
glycosidic linkages, an idea of the solution conformati~n~~ and hence,
the degree of steric hindrance about the hydroxyl group may then be
inferred.
~ e ~ r o t e c t i o n ~ ~ ~ ~ ~ of the tetrasaccharide 44 by 1) removal of SEM
and acetate groups using 3% HC1 in methanol, 2) palladium-catalysed
hydrogenation of the benzyl and ally1 ethers, 3) removal of the
phthalimido group with 100% hydrazine monohydrate, and 4) selective N-
acetylation of the resulting free amino group with acetic anhydride in
methanol afforded the deprotected tetrasaccharide as its propyl
glycoside 45 in 67% yield (Scheme 6). This may be used as a hapten in
binding studies as described previously.
NMR analysis
The assigned structures were in accord with their 'H and I3c NMR
spectral data. Compounds were characterised by use of routine 'H,
and c (I H) spectra. H-Homonuclear chemical-shif t correlated (COSY)
experiments49 were performed on compounds 33, 39, 44, and 45, and C-
H chemical-shif t correlated experiments5 were performed on compounds
39 and 44.
The vicinal coupling constants of the ring-protons in the mono-
saccharide units within oligosaccharides were found to be consistent
with a 4 ~ 1 (D) conformation for the N-acetylglucosamine ring and with a
C4 (L) con•’ iguration for the rhamnopyranosyl units.
The stereochemical integrity of the tetrasaccharide 44 was
confirmed by examination of the one-bond C-I H coupling constants,
1 J13C-1H, for the anomeric carbons. The value for the rhamnosyl
anomeric carbons of rings A and B (168 Hz) were consistent with the
2 8
presence of an a-L-conf iguration about the rhamnosyl residues. Both
values obtained for the remaining rhamnosyl and glucosamine anomeric
carbons were found to be unusually high, 177 Hz and 165 Hz,
respectively. The expected value for glucosamine is 160- 162 Hz. The
unexpectedly high values obtained may be explained by the fact that the
glycosidic linkages between rings B-C and B-A' are strained due to
steric hindrance. It has been found that 1 3 ~ { 1 ~ ) couplings are
directly proportional to the degree of s-character in the carbon-
hydrogen bond.52 I propose that the increased bond strain causes the
anomeric carbon to be more sp2 like in nature (as opposed to sp3) thus
giving rise to a greater 3 ~ { H I coupling value. The values of 177 Hz
and 165 Hz are still consistent therefore, with an a-L-configuration
about the rhamnosyl residue, and a /I-D-configuration about the
glucosamine residue, respectively.
Owing tc c o q l e x overlap of signals, assignment of the i H NMR
spectra of 39, 44, and 45 was facilitated by the examination of the
COSY spectra (Figures 10 and 11, 14 and 15, and 18 and 19,
respectively) . Assignment of the H NMR spectra of 33, 39, and 44 was
further complicated owing to the overlap of the SEM diastereotopic
protons with the ring-protons. The chemical-shift values for
individual ring-proton signals within a multiplet were obtained from
the COSY cross-peak pattern. Individual vicinal coupling constants were
determined from separated signals in the one -dimensional H NMR
spectra. A detailed account of the attribution of signals to the
individual rings for compounds 39, 44, and 45 follows.
By following the coupled and cross-peak patterns on a COSY
spectrum it is possible to identify groups of signals of a given ring.
29
Therefore, if one can unambiguously assign one of these signals within
a group to a particular ring, the complete assignment of signals to
individual rings is possible. For compound 39 the assignment of the
glucosamine (C-ring) protons is straightforward due to the
characteristic splitting patterns and coupling constants. The signal
at 64.76 in the spectrum was assigned to H-1 of the A-ring, based on
the expected shielding of H-1 when C-1 is attached to an acyclic
aglycone. Similarly, the signal at 65.60 was assigned to H-2 of the B-
ring, based on the deshielding effect of the 2'-0-benzoyl group. This
assignment was verified by the examinat ion of the one - dimens ional H
NMR spectrum of the trisaccharide alcohol 40 from which the 2'-0-
benzoyl group had been selectively removed. The signal at 65.60 was no
longer present, and the signal at 64.19 was assigned to H-2 of the B-
ring. Having assigned these marker signals to a particular ring, all
of the ring-proton signals c m l d now be iinequivocaily ascribed to its'
appropriate ring. Following assignment of the I H NMR spectrum of 39,
the I3C('~) -NMR signals were assigned in a straightforward manner by
examination of the C - I H chemical- shif t correlated spectrum (Figures
12 and 13).
A similar analysis was performed for the spectrum of the blocked
tetrasaccharide 44. Thus, a COSY spectrum permitted the identification
of the individual spin systems (Figures 14 and 15). The ring proton
signals for the glucosamine ring were again clearly identified by their
characteristic coupling constants and splitting patterns. The tri-0-
acetyl-a-L-rhamnopyranosyl (A'-ring) resonances were clearly identified
by the fact that the protons H-2, H-3, and H-4 at 65'41, 65.32, and
65.02, respectively, were deshielded. Again, the A-ring was assigned
3 0
based on the fact that the H-1 signal (64.77) was shielded by the
acyclic aglycone; furthermore, the signal at 63.65 was assigned to the
H-2 of the A-ring, based on the relative shielding effect of the 1-0-
benzyl group. The remaining group of ring-proton signals was assigned
to the B-ring. As before, the 1 3 ~ ( 1 ~ ) - ~ ~ ~ signals were assigned by
examination of the I3 C-I H chemical-shif t correlated spectrum (Figures
16 and 17).
A COSY spectrum of the deprotected tetrasaccharide 45 was also
used to identify the individual spin systems (Figures 18 and 19). As
before, the ring proton signals for the N-acetylglucosamine ring were
clearly identified by their characteristic coupling constants and
splitting patterns. The signal at 64.72 was assigned to H-1 of the A-
ring, based on the expected shielding of H-1 when C-1 is attached to an
acyclic aglycone. Similarly, the signal at 65.14 was assigned to H-1
of the A' ring based on chemical-shift cnrrelations with F A - b ---+-..-I-. A"Ua y
synthesised related structures, and the natural polymer. 5 3 The
assignment of the protons to the B-ring followed readily.
Expanded region -
shown in Figure 11.
Figure 10. 400.13 MHz 21)-I H NMR COSY spectrum of trisaccharide 2
-7-7-J- 8 4.50 4 . 2 0 3.90 3.68
PPH
Figure 11. Expanded region of the 2 ~ - ' l l NMR COSY spectrum of trisaccharide 39
Expanded region ' shown i n Figure 1 3 .
U- Figure 12. ~D-'~c-'H chemical-shift B correlated spectrum of
trisaccharide 2 I I I I
Figure 13. Expanded region of the ~D-'~c-' H chemical- shift correlated spectrum of trisaccharide 39
0 - 10 0
06'5 0Q'S 06'b
- - -
I Figure 14.
HJd 96'5 0v'S 06'7 0v.Z BG'I 9v'I I
L~LLLLLLL~LLL!..I.I.I.I.I.L
.. i I I I I I
e , Expanded region I shown in Figure 1 5 .
i00.13 MHz ~ D - ' H NMR COSY spectrum of tetrasaccharide 44
Figure 15. Expanded region of the tetrasaccharide 44
~ D - ' H NMR COSY spectrum of
Figure 16. Partial region of the 2 ~ - ' 3 ~ - 1 H chemical-shift correlated spectrum of tetrasaccharide 44
Wdd
Figure 17 . Anomeric region of the 2 ~ - ' ~ C-I H chemical-shift correlated spectrum of tetrasaccharide 44
-. Expanded region shown in Figure 19.
Figure 18. 400.13 MHz ~ D - ' H NMR COSY spectrum of tetrasaccharide 45
Figure 19. Expanded region of the ~ D - ' H NMR COSY spectrum of tetrasaccharide 45
Table 3: 'H
NMR dataas for the ring protons in the trisaccharide 39
Ring
H-1
H-2
H-3
H-4
H- 5
H- 6
a~
n
CDC13. The numbers in brackets denote coupling constants, in Hz.
signals:
- 6,, (400.13 MHz):
-0.27 (9H, s, 0-CH20CH2CH2Si(CH3)3),
-0.07 (lH, m, 0-CH20CH2CH,Hb-
Si(CH3)3),
-0.01 (18H, s, ~XO-CH~OCH~CH~S~(CH~)~),
0.27 (lH, m, 0-CH20CH2CH,CHbSi(CH3)3),
0.82, 0.93 (2x2H, m, 0-CH20CH2CH2Si(CH3)3),
2.99 (2H, m, 0-CH20CH2CH2Si(CH3)3),
3.38 (lH,
m, 0-CH20CHaHbCH,Si(CH3)3), 3.50 (O-CH20CHaHbCH2Si(CH3)3), 3.60 (0-CH20CH2CH2Si(CH3),),
3.86 (lH, m, 0-CH,HbCH-CH2), 4.08 (lH, m, 0-CHaHbCH=CH2), 4.32, 4.50 (
AB
q's, J=12.0 Hz,
0-CH2Ph), 4.34 (2H, t, 0-CH2Ph or 0-CH20CH2CH2Si(CH3)3),
4.62 (2H, s, 0-CH2Ph or O-
CH20CH2CH2Si(CH3)3), 4.64, 5.10 (AB q's, 5-12.0 Hz, 0-CH2Ph), 4.70, 4.72 (
AB
q's, J=6.3
Hz, 0-CH20CH2CH2Si(CH3)3),
4.72 (2H, s, 0-CH2Ph or 0-CH20CH2CH2Si(CH3)3), 5.10 (lH, m,
JcIs=10.5 Hz, 0-CH2CH=CHZHE), 5.18 (lH, m, JTRANS=17.5
Hz, 0-CH2CH=CHzHE), 5.80 (lH, m,
0-CH2CH=CH2). 'These
values are the sum of the individual coupling constants, LAX+&,.
Table
5 cont. :
H NMR dataa
for ring protons in the tetrasaccharide 44
Ring
H- 1
H- 2
H- 3
H-4
H- 5
H-6
a~
n
CDC13. The numbers in brackets denote coupling constants, in Hz.
signals: d
,, (400.13 MHz): -0.27 (9H, s, 0-CH20CH2CH2Si(CH3)3),
- 0.13 (lH, m, 0-CH20CH2CH,HbSi(CH3)3), -0.01 (18H, s, 2x0-CH20CH2CH2-
Si(CH3)3),
0.28 (lH, m, 0-CH20CH2CHaCHbSi(CH3)3), 0.80, 0.90 (2x2H, m,
0-CH20CH2CH2Si(CH3)3), 3.00, 3.39 (2x2H, m, 0-CH20CH2CH2Si(CH3)3),
3.57
(O-CH20CHaHbCH2Si(CH3)3), 3.67 (O-CH20CHaHbCH2Si(CH3)3), 3.87 (lH, m, O-
CHaHb-CH=CH2), 4.08 (lH, m, 0-CHaHbCH=CH2), 4.16, 4.29 (AB q's, 5-12.0
Hz, 0-CH2Ph), 4.42, 4.45 (AB q's, J =7.0 Hz, 0-CH20CH2CH2Si(CH3)3),
4.53, 4.77
(AB
q's, 5-11.5 Hz, 0-CH2Ph), 4.61 (2H, s, 0-CH2Ph or O-
CH20CH2CH2Si(CH3)3), 4.68, 4.71 (AB q's, J=6.0 Hz, 0-CH20CH2CH2-
Si(CH3)3),
4.77 (2H, s, 0-CH2Ph or 0-CH20CH2CH2Si(CH3)3),
5.10 (lH, m,
J,,,=10.5
Hz, 0-CH2CH=CHZHE), 5.18 (lH, m, JTRANS=17.5
HZ, O-
CH2CH=CHzH,),
5.79 (lH, m, 0-CH2CH=CHZ). '~hese values are the sum of
the individual coupling constants,
Table 6 : 13c NMR dataat for the ring carbons in the tetrasaccharide 44.
Ring
C-1
C-2
C-3
C - 4
C-5
C-6
'1n
CDC~~
signals: 6, (100.6 MHz): -1.8 (O-CH20CH2CH2Si(CH3)3), -1.4
(~xO-CH,OCH,CH,S~(CH~)~), 17.1 (O-CH20CH2CH2Si(CH3)3), 18.1 (2x0-CH20CH2CH2-
Si(CH3),),
20.8 (2x0-COCH3), 21.1 (0-Corn3), 64.9, 65.7, 66.3 (3x0-CH20CH2-
CH2Si(CH3)3),
67.8 (0-CH2CH=CH2), 72.5, 74.4, 75.3 (3x0-CH2Ph), 95.2, 96.5,
97.1 (~XO-CH~CH,CH,S~(CH,>~), 116.9 (0-CH2CH=CH2), 133.5 (0-CH2CH=CH2),
165.7 (0-CH2C6H5 ) , 169.7, 169.9, 170.14 (0-COCH~)
.'values
in parentheses are
the ' J
- ,, c
oupling constants in Hz. d
# e~ssignments may be interchanged.
'obscured
by CDC13 peaks.
Comments and suggestions for future research
The tetrasaccharide 44 may be used as a hapten in binding studies
with monoclonal antibodies raised against the natural antigen. A
synthetic route to a tetrasaccharide 25 based on a similar strategy
should be possible. The tetrasaccharide 25 may be coupled, via the 8 -
me thoxycarbonyloc tyl linking arm1 to protein, using the simplified
acyl azide methodology15 to give an artificial antigen for use in
immunochemical studies.
Although the desired compounds were obtainable using the described
synthetic routes, complications in synthesising the tetrasaccharide 44
and presumably 25 and higher-order oligosaccharides are evident. The
fully functional trisaccharide 40, whilst synthesised in good yield is
not ideally suitable for the synthesis of higher-order oligo-
saccharides, owing to the sterically hindered nature of the alcohol
fwiction. The relatively low glyzosylation p'lelds obtained with
monosaccharide 42 would suggest greater problems for the glycosylation
with trisaccharide to give hexasaccharide 29, assuming the steric
hindrance theory to be correct.
Alternate synthetic routes would probably exclude the use of SEM
groups in favour of smaller alcohol protecting groups. For example, a
trisaccharide similar to that of 39, employing benzyl ether instead of
SEM protecting groups on glucosamine could be synthesised and
selectively deprotected to give a fully functional trisaccharide.
Glycosylation with monosaccharides 41 or 42 in reasonable yield might
then be feasible . Other alternatives could include the use of different glycosyl
donors such as various thioglycosides ,44 mentioned previously; the
reactions may be activated by either nitrosonium tetraflu~roborate~~ or
methyl triflate . 4 6
4 9
3. EXPERIMENTAL -
General procedures
'H NMR (400.13 MHz) and I3c NKR (100.6 MHz) spectra were recorded
with a Bruker WM-400 NMR spectrometer. Spectra were measured in
deuteriochloroform for solutions of protected compounds and in
deuterium oxide for solutions of deprotected compounds; the chemical
shifts are given in p.p.m. downfield from Me4Si and 2,2-dimethyl-2-
silapentane-5-sulphonate (DSS), respectively. Chemical shifts and
coupling constants were obtained from a first-order analysis of the
spectra. Optical rotations were measured on a Perkin-Elmer P22
spectropolarimeter.
Analytical t.1.c. was performed on pre-coated aluminium foil
plates with Merck silica gel 6O-FZs4 as the adsorbent. The
developed plates were air-dried, exposed to U.V. light and/or sprayed
with 10% sulphuric acid i:: ethanol and hzated fit 150•‹C. All compourlds
were purified by medium pressure column chromatography on Kieselgel 60
(230-400 mesh) according to a published procedure .54 High-performance
liquid chromatography (h.p.1.c.) was performed at 4 MPa on a Waters
Associates Prep LC/system 500 instrument with two Prep PAK-500 silica
gel normal-phase columns and a refractive-index detector.
Solvents were distilled before use and were dried, as necessary,
by literature procedures. Work-up of solutions involved evaporation
under reduced pressure below 40•‹C.
Reactions performed under nitrogen were carried out in
deoxygenated solvents. Transfers under nitrogen were effected by means
of standard Schlenk-tube techniques.
Specific procedures
Ally1 2-O-benzovl-4-O-benzvl-3-0-(2'-deoxv-2'-vhthalimido-6-D-
jzlucovvranosvl)-a-L-rhamnopvranoside =.---A solution of the
disaccharide ( 3 1 ) ~ ' (2.2 g, 2.7 mmol) in methanolic HCl (3%, 80 ml)
[prepared by treating anhydrous methanol (80 ml) with acetyl chloride
(4.6 ml)] was stirred under nitrogen for 10 h. T.1.c. (hexane-ethyl
acetate-methanol 4:4:0.5) indicated that the starting material had been
consumed. The reaction mixture was neutralised with triethylamine and
solvent removed to give a syrup and triethylamine salt. The mixture
was dissolved in ethyl acetate and washed with distilled water (X2) to
remove the salt, and dried (Na2S04). Evaporation of the solvent gave a
syrup that was chromatographed with hexane-ethyl acetate-methanol
(4:4:0.5 Rf0.26) as eluent. The title compound (32) was obtained as a
white foam (1.53 g, 81%) ; [a]D22 -89.5' (c 1.05 in CH2C12) ;
S~(100.6 &HZ): 17.9 ( C - 6 ) , 50.8 (C-2'1, 61.8 ( C - 5 ' ) . 57.5 (C-51,
68.3 (0-CH2CH=CH2), 71.3 (C-4'), 71.8 ( C-3'), 72.6 (C-2), 74.5 (0-
CH2Ph), 76.3 (C-5'), 79.1 (C-3), 80.2 (C-4), 96.5 (C-1), 99.3 (C-l'),
117.9 (0-CH2CH=CH2), 133.6 (0-CH2CH=CH2), 167.18 (0-COPh), 168.3
(phthaloyl C=O).
SH(400.13 MHz): 1.12 (3H, d, J5#6-6.3 HZ, 6-H3), 3.36 (lH, t,
J3,4 + J4,5, -18.0 Hz, 4-H), 3.45 (lH, t, J31,41 + J4',51 -19.0 HZ, 4 ' -
H), 3.50 (lH, ddd, J4,15,=9.75 HZ, J5,,61a=2.0 HZ, J5' bIbG5.0 HZ, 5'- ,
H), 3.59 (lH, dd, J5, 61b=5.0 HZ, J61b,61a "12.5 HZ, 6'b-H), 3.73 (lH, ,
dq, J4 5=9.0 HZ, J5,656.3 Hz, 5-H), 3.88 (IH, dd, J5, 6,a-2.0 HZ, ,
J61a,61b-12.5 HZ, 6'a-H), 3.97 (IH, m, 0-CHaHbCH-CH2), 4.12 (IH,
obscured m, 0-CHaHbCH-CH2), 4.15 (lH, dd, J2,3-=3.3 Hz, J314-9.5 Hz, 3-
H), 4.18 (lH, dd, J2, 3,=11.0 HZ, J3,,4,-9.0 HZ, 3'-H), 4.32, 4.34 (AB ,
5 1
q's, 5-12.0 HZ, 0-CH2Ph), 4.76 (lH, S, Jl12-1.8 HZ, 1-H), 5.22 (lH, m,
J,,s-10.5 Hz, 0-CH2CH-CH,HE), 5.30 (lH, m, JTRANs-17.5 Hz, O-
CH2CH=CH,H,), 5.53 (lH, d, J1,,21-8.0 HZ, 1'-H), 5.68 (lH, dd, JIr2-1.8
Hz, J2,3==3.3 Hz, 2-H), 5.92 (lH, m, 0-CH2CH-CH2).
(Found: C, 64.27; H, 5.70; N, 1.98. C3,H3,NOI2 Requires C, 64.27;
H, 5.70; N, 2.03%).
Ally1 2-O-benzovl-4-O-benzyl-3-0-(2'-deoxv-2'-~hthalimido
3',4'.6'.-tri-0-2-~trimeth~1sil~11ethoxp-methv1-6-D-~1uc0v~ran0sv1~-a-
L-rhamno~yranoside (33).---A mixture of the disaccharide (32) (1.77 g,
2.56 mmol), diisopropylethylamine (3.1 ml, 17.8 mmol), 2-(trimethyl-
sily1)ethoxymethyl chloride (2.1 ml, 11.9 mmol) in anhydrous
dichloromethane (4 ml) was stirred under nitrogen at room temperature
for 55 h. The reaction mixture was diluted with dichloromethane and
washed successively with 0.5M HC1 (x2), water, saturated sodium
bicarbonate solution and aqueous sodium chloride solution. The organic
layer was dried (Na2S04) and concentrated to give a syrup that was
chromatrographed with hexane-ethyl acetate (4:1, Rf0.35) to yield the
title compound (33) as a white foam (1.93 g, 70%). [a]p22 +8.8'
(c-1.29 in CH2C12) ;
6c(100.6 MHz): -1.7, -1.4, -1.3 ( ~ X O - C H ~ O C H ~ C H ~ S ~ ( C H ~ ) ~ ) , 17.2
(O-CH20CH2CH2Si(CH3)3), 17.9 (C-6), 18.1 ( ~ X O - C H ~ O C H ~ C H ~ S ~ ( C H ~ ) ~ ) , 56.2
(C-2'), 65.2, 65.6, 66.4 (3x0-CH20CH2CH2Si(CH3)), 67.3 (C-6'), 67.5
5 2
SH(400.13 MHz): -0.26, -0.01, 0.02 (3~9H, S , 0-CH20CH2CH2-
Si(CH3)3), -0.06 (lH, m, 0-CH20CH2CHaHbSi(CH3),), 0.28 (lH, m, O-
CH20CH2CHaHbSi(CH3),>, 0.91 (4H, m, ~xO-CH~OCH~CH~S~(CH,)~), 1.09 (3H,
d, J516=6.0 Hz, 6-H), 2.99 (2H, m, 0-CH20CH2CH2Si(CH3)3), 3.45 (1-H, t,
J,,, + J,15=18.0 Hz, 4-H), 3.49-3.76 (9H, complex multiplet, 3.53 [O-
CH20CH2CH2- Si(CH3)3], 3.54 [J3,,4, + J4,151-19.0 HZ, 4'-H], 3.58 [5'-
HI, 3.60 [O-CH20CH2CH2Si(CH3)3], 3.68 [5-HI, 3.73 [6',-HI, 3.76 [6lb-
HI, 3.97 (lH, m, 0-CHaHbCH=CH2), 4.13 (lH, m, 0-CHaHbCH=CH2), 4.18 (lH,
dd, J2,,==3.5 HZ, J3,,+=9.5 Hz, 3-H), 4.20 (IH, dd, J1,,21=7.5 HZ,
J2,,31=11.0 Hz, 2'-H), 4.28, 4.42 (AB q ' s , 5-12.0 Hz, 0-CH2Ph), 4.32
(lH, dd, J2,,3,-11.0 Hz, J3, =8.0 Hz, 3'-H), 4.46, 4.53 (AB q's, J 14'
-7.0 Hz, 0-CH20CH2CH2Si(CH3)3), 4.61 (2H, s, 0-CH20CH2CH2Si(CH3)3),
4.71, 4.79 (AB q's, J-6.0 Hz, 0-CH20CH2CH2Si(CH3)3), 4.90 (ZH, d,
JIl2=1.6 HZ, 1-H), 5.20 (lH, m, Jc,,-10.5 Hz, 0-CH2CH-CHZH,), 5.30 (lH,
m, JiKAN3-17 3 HZ 0-CH2CH-CHIHE ) , 5.44 (I!!, dd, 5 , , 2-1.6 HZ, J2, 3=3. 5
Hz, 2-H), 5.45 (lH, d, J1, 2,=7.5 Hz, 1'-H), 5.91 (lH, m, 0-CH2CH=CH2). ,
(Found: C, 61.13; H, 7.53; N, 1.36. C55H81N015Si3 requires C,
61.14; H, 7.56; N, 1.30%)
rhamnovpranoside 0 . - - - T r i s ( t r i p h e n y 1 p h o s p h i n e ) r h o d i w n (I) chloride
(0.166 g, 0.179 mmol) and 1,4-diazabicyclo[2.2.2]octane (0.086 g, 0.768
mmol) were added to a solution of the ally1 glycoside (33) (1.93 g,
1.786 mmol) in ethanol-water (9:1, 70 ml) and the mixture was refluxed
lightly for 12 h under an atmosphere of nitrogen. The solvent was
removed by evaporation to give a dark brown residue which was taken up
in ethyl acetate and filtered through a short column of silica gel.
Removal of the solvent gave a light brown foam ( 3 4 ) which was dissolved
in 90% aqueous acetone (98 ml) and the solution was stirred. Yellow
mercury (11) oxide (0.387 g, 1.78 mmol) was added followed by the
dropwise addition, over 5 minutes, of a solution of mercury (11)
chloride (0.392 g, 1.78 mmol) in 90% aqueous acetone (5 ml). The
mixture was stirred for 12 h, the solvent was evaporated and the
resulting residue was dissolved in ethyl acetate and filtered through
celite. The filtrate was washed successively with aqueous potassium
iodide (x2), aqueous sodium thiosulphate (x2) and water (x2). The
organic layer was dried (Na2S04), the solvent was evaporated and the
resulting yellow foam was chromatographed using hexane-ethyl acetate
(2:l) Rf0.25. The title compound (35) was obtained as a white foam
(1.42 g, 76% yield - based on ally1 glycoside).
6,,(400.13 MHz): -0.27, -0.02, 0.02 (3~9H, S, 0-CH20CH2CH2-
Si(CH3)3), -0.07 (lH, m, 0-CH20CH2CH,HbSi(CH3)3), 0.27 (lH, m, O-
CH20CH2CHaHbSi(CH3)3), 0.92 (4H, m, ~XO-CH,OCH,CH,S~.(CH~)~), 1.08 (3H,
d, J5,6=6.2 Hz, 6-H), 3.00 (2H, m, 0-CH2OCH2CH2Si(CH3),), 3.45 (lH, t,
J31 4 + J4,5=18.4 Hz, 4-H), 3.49-3.70 (7H, complex multiplet, 4'-H, 5 ' -
H, 6'b-H, 2~ ROCH20CH,CH2Si(CH3),), 3.70 (IH, dd, JS1 6#bq4.5 HZ, I
J61a161~a11.5 HZ, 6'b-H), 3.79 (IH, dd, J5, ,6,,-1.8 HZ, JgIa,6,b=11.5
Hz, 6'a-H), 3.91 (lh, m, J415=9.3 HZ, J5,6=6.2 HZ, 5-H), 4.19 (lH, dd,
J,,12r=8.5 HZ, J2r13r=ll.0 HZ, 2'-H), 4.25 (lH, dd, J213=3.5 HZ,
J314-9.2 HZ, 3-H), 4.27, 4.40 (AB q's, 5-12.0 HZ, 0-CH2Ph), 4.33 (ZH,
d, J2' 3,-11.0 HZ, J3,,4,=8.5 HZ, 3'-H), 4.54, 4.60 (AB q's, 5-6.5 HZ, I
0-CH20CH2CH2Si(CH3)3), 4.61 (2H, s, 0-CH20CH2CH2Si(CH3)3), 4.27, 4.77
5 4
(AB qls, 5-6.5 HZ, 0-CH20CH2CH2SI(CH3)3), 5.26 (lH, br 9 , 1-HI, 5.45
(lH, d, J,, 21-8.5 HZ, 1'-H), 5.48 (IH, dd, J1,2a1.9 HZ, J2,3"3.5 HZ, #
2-H) .
Allvl 3-0-(2'-0-benzoyl-4'-0-benzpl-3'-0-(2"-deox~-2"-
phthalimido-3",4".6"-tri-O-2-ltrimeth~lsilvllethoxmethvl-6-D-
gluco~vranosvl)-a-L-rhamnopvranosvlb2.4-di-O-benzvl-a-L-
rhamnovvranoside 0 . - - - O x a l y l chloride (0.06 ml, 0.685 mmol) was
added to a stirred solution of DMF (0.055 ml, 0.685 mmol) in anhydrous
dichloromethane (0.5 ml) and was stirred under nitrogen for 5 min. The
solvent was evaporated under reduced pressure and the white salt was
dried in vacuo for 1 h. The N,N-dimethyl(chloromethylene)ammonium
chloride was then dissolved in anhydrous dichloromethane (2 ml) and
pyridine (0.06 ml) and a solution of the hemiacetais (35j (0.143 g,
0.137 mmol) in anhydrous dichloromethane (1 ml) was transferred to the
flask under nitrogen by means of a cannula. The flask was rinsed with
additional portions of solvent and transferred as before. The mixture
was stirred under nitrogen for 2 h, and then the reaction was quenched
by the addition of cold aqueous sodium bicarbonate (10 ml). The
organic layer was diluted with dichloromethane and washed successively
with 0.5 M hydrochloric acid, aqueous sodium bicarbonate and aqueous
sodium chloride. The organic layer was dried over anhydrous potassium
carbonate and the solvent evaporated to give the disaccharide chloride
(36) as a dark brown syrup which was then dried in vacuo for 5 h.
A mixture of ally1 2,4-di-0-benzyl-a-L-rhamnopyranoside (37)33
(0.036 g, 0.094 mmol), silver trifluoromethanesulphonate (0.042 g,
0.164 mmoi) , and 4A molecular sieves in anhydrous dichloromethane
(1 ml) was stirred in the dark under an atmosphere of nitrogen for
0.5 h in a Schlenk tube fitted with a dropping funnel which was
equipped with a cooling jacket. A solution of the glycosyl chloride
(36), in anhyrous dichloromethane (1 ml), previously stirred with 4A
molecular sieves for 0.5 h under nitrogen, was transferred to the
dropping funnel by means of a cannula. The flask was rinsed with
additional portions of anhydrous dichloromethane (3 x 0.5 ml) and
transferred as before. The solution of the glycosyl chloride was
cooled to -78'C (acetone/dry ice) and added dropwise, during 10 min, to
the cooled ( - 7 8 • ‹ C ) solution of (37). The dropping funnel was rinsed
with additional portions of anhydrous dichloromethane (2 x 0.5 ml).
The mixture was stirred under an atmosphere of nitrogen in the dark and
allowed to slowly attain room temperture. After 40 h, t.1.c (hexane-
ethyl zcetate 3:1) indictate& the reaction to be complete. The solids
were removed by filtration and the filtrate diluted with
dichloromethane and washed successively with 0.2M hydrochloric acid,
aqueous sodium bicarbonate and aqueous sodium chloride solutions. The
organic layer was dried (Na2S04) and the solvent was evaporated to
yield a syrup which was chromatographed using hexane:ethyl acetate
(4:l) as eluent (Rf0.40). The title compound (39) was obtained as a
white foam (0.106 g, 81%).
6c(100.6 MHz):- see Table 4.
6,,(400.13 MHz):- see Table 3.
(Found: C, 63.95; H, 7.30; N, 1.33. C7,H1,,N01,Si, requires C,
64.03; H, 7.38; N, 0.99%).
56
Ally1 2.4-di-0-benz~l-3-0-(31-0-(2'P-de~~p-2"a1imid0-
3".4".6"-tri-0-2-ltrimethvlsilvllethox~ethv1-6-D-~luc0~vran0sv1)-
4 ' - O - b e n z v l - a - L - r h a m n o ~ ~ n o s ~ l ) - a - l - r h a 0 ~ 0 ~ d (40).---A sample
of (39) (0.439 g, 0.312 mmol) was dissolved in freshly prepared 0.1 M
NaOMe (5 ml) and stirred at room temperature under nitrogen for 47 h.
T.1.c (hexane-ethyl acetate 3:l) showed most of the starting material
to be consumed and the presence of a relatively highly polar side
product (removal of SEM groups, RfO.O) and desired product (Rf0.38).
The reaction was worked up by quenching in ice cold 0.2 M HC1,
extracting with dichloromethane (4 x 15 ml) and washing successively
with aqueous sodium hydrogen carbonate, aqueous sodium chloride
followed by drying (Na2S04). The solvent was removed to yield a light
yellow syrup which was purified by column chromatography using hexane-
ethyl acetate (3:l Rf0.38) as eluent to yield the title compound (40)
as a white foam (0.330 g, 8 1 % ) . - 3 . 7 ' ( c 1.29 in C H 2 C 1 , )
Jc(100.6 MHz): -1.8, -1.5, -1.4 (~XO-CH,OCH,CH,S~(CH~)~), 17.2
(O-CH20CH2CH2Si(CH3)3), 17.7 (C-6'), 17.84 (C-6), 17.96, 18.05 (2x0-
CH20CH2CH2Si(CH3)3), 55.7 (C-2"), 65.2, 65.7, 66.4 (3x0-CH20CH2CH2-
Si(CH3)3), 66.9 (C-6"), 67.6, 67.7 (0-CH2CH-CH, + C-5'), 68.2 (C-5),
69.8 (C-2'), 72.5 (0-CH2Ph), 74.4 (C-5"+ 0-CH2Ph), 75.1 (0-CH2Ph),
77.5 (C-4"), 77.8 (C-2 + C-3), 79.0 (C-4')' 79.8 (C-3"), 80.8 (C-4),
82.9 (C-3'), 95.3, 96.6 (~xO-CH,OCH,CH,S~(CH,)~, 96.7 (C-1), 97.1 (0-
CH20CH2CH2Si(CH3)3), 98.1 (C-I"), 101.1 (C-l'), 116.9 (0-CH2CH=CH2),
133.6 (0-CH2CH-CH,).
6" (400.13 MHz): -0.26 (9H, s, 0-CH20CH2CH2Si(CH3)3), -0.07 (IH,
m, 0-CH20CH2CH,HbSi(CH3)3), 0.02 (18H, s, ~XO-CH~OCH,CH,-S~(CH~)~),
0.03 (lH, m, 0-CH20CH2CH,H,Si(CH3)3), 0.90, 0.95 (2x2H, m, O-
complex multiplet, 2-H, 4-H, 4'-H, 4"-H, 5-H, 5'-H, 5"-H, 6",-H,
6",-H, 0-CH20CHaHbCH2Si(CH,)3, 0-CH20CH2CH2Si(CH3)3), 3.83 (lH, m, 0-
CHaHbCH-CH,), 3.87 (lH, m, 0-CHaHbCH-CH,), 3.95 (lH, dd, J213==3.1 Hz,
J3,4-9.0 Hz, 3-H), 4.09 (2H, complex multiplet, Jz1 3,=3.4 Hz, I
J31,4r=9.1 HZ, 3'-H, 0-CH20CH-CH2), 4.19 (lH, br s, 2'-H), 4.25-4.35
(2H, complex multiplet, [J111,2,,=8.1 HZ, J21,,3,,=9.5 Hz, 2"-HI, 3"-
H), 4.27, 4.50 (AB q's, 5-12.0 Hz, 0-CH2Ph), 4.62, 4.87 (AB q's, J=6.7
Hz, 0-CH20CH2CH2Si(CH3)3), 4.64, 4.68 (2x2H, s, 0-CH2Ph or 0-
CH20CH2CH2Si(CH3)3), 4.74, 4.82 (AB q's, 5-6.5 Hz, 0-CH20CH2CH2-
Si(CH3)3), 4.77 (lH, d, J1,,=1.8 Hz, 1-H), 5.12 (lH, m, JcIs==10.5 Hz,
0-CH2CH-CHZHE), 5.17 (lH, m [partially obscured by 1-HI, 0-
CH2CH-CH2HE), 5.21 (1H; s: 1'-H), 5,10 (IH, d , J i , , , 2 , ,=8.0 Hz, 1''-u\ - I ,
5.81 (lH, m, 0-CH2CH=CH2).
(Found: C, 63.95; H, 7.30; N, 1.33. C75H103N019Si3 requires C,
64.03; H, 7.38; N, 0.99%).
2.4-di-0-benzvl-a-L-rhamnopvranoside 0 . - - - A mixture of the
trisaccharide alcohol (40) (0.048 g, 0.037 mmol), Hg(CN2) (0.029 g,
0.112 mmol), HgBr, (0.403 g, 0.112 mmol), and 4A molecular sieves in
anhydrous dichloromethane (1 ml) was stirred in the dark under an
58
atmosphere of nitrogen for 0.5 h in a Schlenk tube fitted with a
dropping funnel which was equipped with a cooling jacket. A solution
of 2,3,4-tri-0-acetyl-a-L-rhamnopyranosyl bromide in anhydrous
dichloromethane (1 ml), previously stirred with 4A molecular sieves for
0.5 h under nitrogen, was transferred under nitrogen by means of a
cannula. The flask was rinsed with additional portions of anhydrous
dichloromethane (3 x 0.5 ml) and transferred as before. The solution
of the glycosyl bromide was cooled to -78OC (acetone/dry ice) and added
dropwise, during 10 min, to the cooled (-78•‹C) solution of (40). The
dropping funnel was rinsed with additional portions of anhydrous
dichloromethane (2 x 0.5 ml). The mixture was stirred under an
atmosphere of nitrogen in the dark and allowed to slowly attain room
temperature. After 24 h, t.1.c (hexane-ethyl acetate 3:l) indictated
the reaction to be complete. The solids were removed by filtration and
tho filtrate diiuted with dichloromethane and washed successively with
aqueous potassium iodide solution and aqueous sodium chloride. The
organic layer was dried (Na2S04) and the solvent was evaporated to
yield a syrup which was chromatographed using hexane-ethyl acetate
(3:l) as eluent, Rf0.35. The title compound (44) was obtained as a
clear syrup (0.028 g, 48%) [alDZ2 -10.6' (c 0.9 in CH2C12)
bc(100.6 MHz):- see Table 6.
6,,(400.13 MHz) : - see Table 5.
(Found: C, 60.86; H, 7.14; N, 0.77. C80H115N025Si3 requires C,
5 9
Pro~vl 3-0-(3'-0-(2"-acetamido-2'b-deoxv-8-D-~luco~vranosvl)-2~-
O-(a-L-rhamno~vranosv~~-a-L-rhamno~vranosv~~-a-L-rhamno~vranoside
145).---The protected tetrasaccharide (44) (0.037 g, 0.0235 mmol) was
dissolved in 3% HC1 in methanol (3 cm3) and stirred under nitrogen at
room temperature. T.1.c. (hexane:ethyl acetate:methanol 2:3:1) after
14 h showed the reaction to be complete. The reaction was worked up by
diluting with methanol and neutralising with Rexyn 201 (OH) beads.
After filtering, the solution was concentrated to give an amorphous - solid that was chromatographed on silica gel using hexane:ethyl
acetate:methanol (0.2:4:0.5; Rf0.27) to yield the partially deprotected
tetrasaccharide (acetate and SEM groups removed; 0.019 g, 76% yield).
This was then dissolved in 80% aqueous acetic acid (2.5 cm3 ) and 90%
ethanol (lcm3 ) and hydrogenolysed over 10% palladium-carbon (0.027 g)
at a hydrogen pressure of 51 p.s.i. for 65 h. The mixture was filtered
through celite and concentrated to an amorphous solid, the acetic ~ c l d
was removed by repeated codistillation with 100% ethanol. The solid
was dissolved in 100% ethanol (2.5 cm3) containing 98% hydrazine
monohydrate (0.001 cm3) and refluxed under nitrogen. After 14 h t. 1 .c
(ethyl acetate:methanol:water 7:2:0.5) indicated the reaction to be
complete (RfO.l). The solids were removed by filtration through celite
and solvent removed to give a yellow glass which was dissolved in
methanol (1.5 cm3 ) containing acetic anhydride (0.15 cm3 ) and stirred
for 1.5 h. Excess acetic anhydride was removed at 30•‹C by repeated
codistillation with methanol and the acetic acid formed was removed as
usual to give a white amorphous solid which was purified by column
chromatography on silica gel using ethyl acetate:methanol:water (7:2:1)
as eluent (Rf0.37). Solvent removal afforded the title compound (45)
61
REFERENCES
I. Goldstein, L. Scebat, J , Renais, P. Hadjinsky and J. D~tartre,
Isr. J. Med. Sci., 1983, 1 9 , 483; I. olds stein, B. Halpern and L.
Robert, Nature (London), 1967, 44; I. Goldstein, P. Rebeyrotte, J . Parlebasand B.Halpern, Nature(~ondon),l968,866.
J.E. Coligan, T.J. Kindt and R.M. Krause, Immunochemistry, 1978,
15, 755.
R.M. Krause and M. McCarty, J. Exp. Med. 1962, 115, 49.
M. McCarty, The Harvey Lectures, Series 65. Academic Press, New
York, 73.
R.R. Facklam, 1979, Isolation and identification of Streptococci.
Part 2. Department of Health, Education, and Welfare, Centre for
Disease Control, Atlanta.
G. Peter and A.L. Smith, N. Engl. J. Med., 1977, 297, 365.
The Medical Letter. 1985, 27, 49.
See, for example, R. Rosenstein, K.P Aspden and P. Stopa, Eur.
Pat. Appl. EP 153 477 1985 (Chem. Abstr. 1986, 104, 3136a); J.W.
Holland and R.H. Walter, Eur. Pat. Appl., EP 0150 7A2, 1985.
R.A. Venesia, A. Ryan, S. Alward and W.A. Kostun, J. Clin.
Microbiol., 1985, 21, 395.
B.G. Miceika, A.S. Vitous and K.D. Th~pson, J. Clin.
1985, 21, 467.
R.M. Centor, F.A. Meier and H.P. Dalton, Ann. Int. Med -..
Microbiol.,
14. R.U. Lemieux, D.R. Bundle and D.A. Baker, J. Am. Chem. Soc., 1975,
97, 4076; R.u. Lemieux, D.A. Baker and D.R. Bundle, Can- J.
Biochem., 1977, 55, 507.
15. B.M. Pinto and D.R. Bundle, ~arbohydr. Res., 1983, 124, 313.
16. C. Milstein, Angew. Chem. Eng. Ed., 1985, 24, 816; G . Kohler and
C. Milstein, Eur. J. Immunol., 1976, 6, 511; G. Kohler and C.
Milstein, Nature, 1975, 256, 495.
17. H. Paulsen, 1985, in Organic Synthesis fith IUPAC Symposium,
eds. J. Streith, H. Prinzback and G. Schill, Blackwell Scientific
Publications, Oxford, p.317; N.K. Kochetkov, Pure Appl. Chem.,
1984, 56, 923; H. Paulsen, Angew. Chem. Int. Ed. Engl., 1982, 21,
155.
18. A.S. Perlin, Can. J . Chem., (1963), 41, 399.
19. R.U. Lemiecx, T. Takeda and B.Y. Chung, PIE. Chem. Sm. Sy~pcsium
Series., 1976, 39, 90.
20. R.U. Lemieux, K.B. Hendriks, R.V. Stick and K. James, J. Am. Chem.
SOC., 1975, 97, 4056.
21. J.T. Edward, Chem. Ind. (London), 1955, 1022; R.U. Lemieux, 1964,
in Molecular Rearrangements, ed. P. de Mayo, Interscience, New
York p.709; R.U. Lemieux, Pure Appl. Chem., 1971, 25, 527;
Anomeric Effect Origin and Consequences, Am. Chem. Soc. Symposium
Series No. 87, 1979, eds. W.A. Szarek and D. Horton, Washington,
D.C.; S. Wolfe, M.H. Whangbo and D.J. Mitchell, Carbohydr. Res.,
1979, 69, 1; A.J. Kirby, 1983, The Anomeric Effect and Related
Stereoelectronic Effects at Oxygen, Springer Verlag, Berlin.
22. H. Paulsen and 0. Lockhoff, Tetrahedron Lett., 1987, 42, 4027.
63
23. H. Paulsen, Angew Chem., 1982, 94, 184.
24. H . Paulsen, T . Hasenkamp and M . Paa l , Carbohydr. Res . , 1985, 144,
45.
25. R.R. Schmidt and J . Michel, Angew Chem., 1983, 92, 763; Angew
Chem., I n t . Ed. Engl . , 1980, 19 , 731.
26. H . Lonn, Carbohydr. Res. , 39 1985, 139, 105 and 115.
27. K . C . Nicolaou, S.P. S e i t z and D.P. Papaha t j i s , J. Am. Chem. Soc . ,
1983, 105, 2430.
28. P. Fiiugedi and P. Garegg, Carbohydr. Res . , 1986, 149, C9.
29. V . Pozsgay and H . J . Jennings, J. O r g . Chem., 1987, 52, 4635.
30. K . B . Reimer and B.M. P in to , J. Chem. Soc . , Perkin Trans. I , 1988,
2103.
31. A . T i x i d r e , K.B. Reimer, and B.M. P in to , manuscript i n p repa ra t ion .
32. B.H. Lipshutz and J . J . Pegram, Tetrahedron L e t t . , 1980, 21, 3343.
3 3 ; M. Buiting, and B.M. Pinte, mer?uscript ix preparation.
34a. E . J . Corey and J . W . Suggs, J. O r g . Chem., 1973, 38, 3224.
34b. A. Emery, A . C . Oehlschlager and A.M. Unrau, Tetrahedron L e t t . , 1970,
50, 4401.
35. R . Gigg and C . D . Warren, J. Chem. Soc. C., 1968, 1903.
36. B.M. P in to , D . G . Mor isse t te and D . R . Bundle, J. Chem. Soc . , Perkin
Trans. I , 1987, 9.
37. H . H . Bosshard, R . Mory, M . Schmid and H . Zo l l inge r , Helv. Chirn.
Acta . , 1959, 42, 1653; T . Iversen and D.R. Bundle, Carbohydr. Res . ,
1982, 103, 29; R.U. Lemieux, S.Z. Abbas and B.Y. Chung, Can. J.
Chem., 1982, 60 , 58.
38. T . Iversen , S. Josephson and D.R. Bundle, J. Chem. Soc . , Perkin
Trans. I, 1981, 2379.
H. Paulsen, organic Synthesis, 5th IUPAC Symposium, Edited by G.
Schill, ~ l ~ ~ k ~ ~ l l scientific Publications, Oxford, 1985, 317.
E. Fischer, M. Bergmann and A. Rabe, Ber., 920, 53, 2362.
S. Josephson and D.R. Bundle, J. Chem. Soc., Perkin Trans. I.,
1980, 297.
D.R. Bundle and S. Josephson, Carbohydr. Res., 1986, 80, 75.
V. Pozsgay and H.J. Jennings, J. Org. Chem., 1988, 53, 4042.
V. Pozsgay and H.J. Jennings, J. Org. Chem., 1987, 52, 4635.
H. Lonn, Dissertation University of Stockholm 1984; Abstr. XIIth
Int. Carbohydr. Symp., 1984, 88, 116; Carbohydr. Res., 1985,
139, 105; Carbohydr. Res., 1985, 139, 115.
J.H. Noggle and R.E. Shirmer, The Nuclear Overhauser Effect,
Academic Press, New York, 1971.
48, K. Bock, Pure Appl. Chem., 1983, 55, 605; D.A. Cmiiing and J.P.
Carver, Biochemistry, 1987, 26, 6664; S.W. Homans, R.A. Dwek and
T.W. Rademacher, Biochemistry. 1987, 26, 6571.
49. A. Bax and R. Freeman, J. Magn. Res., 1981, 44, 542.
50. F.A. Wilde and P.H. Bolton, J. Magn. Res., 1984, 59, 343.
51. K. Bock and C. Pedersen, J. Chem. Soc., Perkin Trans. I., 1974,
293; K. Bock and C. Pedersen, Acta Chem. Scand., 1978, B29, 258.
52. J. Auge and S. David, Nouv. J. Chim., 1977, 1, 57.
53. D.H. Huang, N.R. Krishna and D.G. Pritchard, Carbohydr. Res.,
54. D.R. Bundle, T. Iversen and S. Josephson, Am. Laboratory, 1980,
12, 93.
