Chemical and chemoenzymatic synthesis of S-linked ganglioside analogues and their protein conjugates for use as immunogens

Article abstract of DOI:10.1002/chem.200500518

Analogues of the tumor-associated gangliosides GM₃ and GM ₂ containing terminal S-linked neuraminic acid residues and an amino terminated, truncated ceramide homologue have been synthesized and conjugated to a protein. The synthesis

Full text of DOI:10.1002/chem.200500518

FULL PAPER
DOI: 10.1002/chem.200500518
Chemical and Chemoenzymatic Synthesis of S-Linked Ganglioside
Analogues and Their Protein Conjugates for Use as Immunogens
Jamie R. Rich,[a] Warren W. Wakarchuk,[b] and David R. Bundle*[a]
Abstract: Analogues of the tumor-asso-
ciated gangliosides GM3 and GM2 con-
taining terminal S-linked neuraminic
acid residues and an amino terminated,
truncated ceramide homologue have
been synthesized and conjugated to a
protein. The synthesis involved cou-
pling of a S-linked sialyl a(2!3) galac-
tose disaccharide with a glucosyl sphin-
gosine analogue, followed by elabora-
tion and deprotection to give amino-
terminated glycosyl ceramide 1. Glyco-
syltransferase-catalyzed extension of
the trisaccharide 1 provided access to
the modified GM2 tetrasaccharide 2 or
sulphur-containing GD3 analogue 30.
Owing to their potentially enhanced re-
sistance to endogenous exo-glycoside
hydrolases and their inherent non-self
character, carbohydrate antigens con-
taining non-reducing terminal thiogly-
cosidic linkages may be more immuno-
genic than O-linked antigens and may
stimulate the production of antibodies
capable of recognizing naturally occur-
ring oligosaccharides. Our initial results
suggest that in fact these antigens are
viable immunogens and furthermore,
that immune sera cross reacts with O-
gangliosides in the context of a heterol-
ogous glycoprotein conjugate.
Keywords:
gangliosides
carbohydrates
glycoconjugates
·
·
·
oligosaccharides · vaccines
Introduction
glycoconjugate vaccine against Haemophilus influenzae type
b.[9,10] However, in most cases additional, complementary
strategies for the generation of carbohydrate specific
immune responses remain in demand.
Complex carbohydrates decorate the surfaces of bacteria,
parasites, viruses, tumors and numerous other targets for
which vaccine development is an important and active area
of research.[1–3] Owing to the prominent location of these
molecules at the interface of the cell and its surroundings,
the generation of a carbohydrate specific immune response
is an attractive means of developing therapies to combat the
effects of pathogens such as HIV,[4] and malaria,[5] as well as
diseases such as cancer.[6,7] The conjugation of defined oligo-
saccharide epitopes to immunogenic carrier proteins has
been advanced as a means to overcome the generally poor
immune response to otherwise T-cell independent carbohy-
drate antigens.[8] The feasibility of this approach has recently
been demonstrated in the development of a fully synthetic
Carbohydrate immunogens and other therapeutics are
susceptible to in vivo degradation by glycoside hydrolases.
The incorporation of unnatural, hydrolysis resistant carbohy-
drate analogues into vaccine constructs has been proposed
as a means to enhance the bioavailability of the antigen to
the immune system, but would be of use only if the resultant
immune response were to target the naturally occurring
structure.[11,12] To this end, hydrolysis resistant carbohydrate
analogues must maintain conformations similar to those of
the target oligosaccharides.
Substitution of the glycosidic oxygen atom by sulfur or
carbon is a well known method to enhance the stability of
the glycosidic linkage towards hydrolysis by either chemical
or enzymatic means, and in fact, these molecules have been
demonstrated to inhibit the activity of glycosidases and
other carbohydrate-binding proteins in some cases.[13–18] It
has been demonstrated that while S-glycosides are signifi-
cantly more flexible about the glycosidic linkage and are
consequentially more prone to adopt the high energy anti
conformations, in many cases they sample similar conforma-
tional space to O-glycosides.[19–26] Although S-glycosides, in
the form of a thiooligosaccharide–protein conjugate and thi-
oglycopeptide, have been evaluated for their in vitro immu-
[a] J. R. Rich, Prof. Dr. D. R. Bundle
Alberta Ingenuity Centre for Carbohydrate Science
Department of Chemistry, University of Alberta
Edmonton, AB T6G 2G2 (Canada)
Fax : (+1)780-492-7705
E-mail: dave.bundle@ualberta.ca
[b] Dr. W. W. Wakarchuk
Institute for Biological Sciences
National Research Council of Canada
100 Sussex Drive
Ottawa, ON K1A 0R6 (Canada)
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nostimulatory activity,[12,27] we are unaware of any instance
in which these molecules have been evaluated as immuno-
gens in vivo.
GM3 and GM2 and synthesized the corresponding carbohy-
drate-protein conjugates for evaluation as immunogens in
mice (Figure 2). Only the terminal sialosyl linkage was tar-
geted for stabilization as the majority of glycoside hydrolas-
es function via cleavage of residues at the non-reducing
glycan terminus (exo-glycosidases). Glycosides of N-acetyl-
neuraminic acid are known to be susceptible to enzymatic
and acidic hydrolysis,[33] and sialidase levels have been sug-
gested to be up-regulated in the sera of cancer patients.[34–36]
The plasma membrane sialidase Neu3 is a ganglioside-spe-
cific hydrolase that has been demonstrated to exert activity
toward the surface of neighbouring cells, synthetic glycopro-
tein conjugates, and GM3.[37,38] It should be noted that glyco-
sylation adjacent to the point of sialic acid attachment sig-
nificantly reduces the susceptibility of this residue to neura-
minidase.[39,40] In keeping with other extracellular sialidases,
Neu3 does not desialylate gan-
Many ganglioside glycolipids have been identified as
tumor-associated antigens, which as a consequence of their
vast over expression on the surface of tumor cells may serve
as targets for cancer immunotherapy. It is proposed that
tumor-associated ganglioside containing conjugate vaccines
will induce anti-ganglioside antibodies that have the poten-
tial to effect clearance of circulating tumor cells and thus in-
hibit further metastasis.[28,29] Several clinical trials have
shown promising results in this regard.[30] In particular the
tri- to pentasaccharides GM3, GM2, GD3, and GD2
(Figure 1), have received attention for use as immunogens
in active immunotherapy against melanoma and other can-
cers.[30–32]
glioside GM2, which is degrad-
ed lysosomally. However, we
have constructed S-GM2 in
order to investigate the conse-
quence of an adjacent branch-
ing residue on the thiosialoside
epitope. Our initial results indi-
cate that S-gangliosides conju-
gated to tetanus toxoid (TT)
induce an IgG response in
Figure 1. Structures of the tumor associated gangliosides GM3, GM2, GD3, and GD2.
mice and that the immune sera
cross react with O-ganglio-
sides.[41]
While the structural similarity of O- and S-linked ganglio-
sides is critical to this concept, paradoxically their necessary
differences may also serve to identify S-glycosides as non-
self antigens and thus further enhance their immunogenicity
Results and Discussion
relative to O-glycosides. Towards these ends we have devel-
oped syntheses of sulfur containing analogues (1 and 2) of
Retrosynthesis: The target neoglycolipids were synthesized
from the derivatives outlined in the retrosynthetic Scheme
1a. Modifications to the naturally occurring glycolipids were
limited to substitution of the terminal sialosyl glycosidic
oxygen by sulfur and use of a truncated ceramide aglycon
(9) to facilitate coupling to protein and enhance the water
solubility of the antigens. We have previously communicated
the synthesis of S-GM3 derivatives by a new route involving
assembly of the thiosialyllactose trisaccharide followed by
coupling with a truncated azidosphingosine and subsequent
elaboration to the glycosyl ceramide (Scheme 1b).[42] How-
ever, in order to secure fragments of the glycolipids suitable
for evaluation of antisera by ELISA, we chose to employ an
entirely new synthetic route centred around S-GM4 disac-
charide donor 4 and the truncated glucosyl sphingosine ac-
ceptor 5.
Briefly, S-GM2 (2) was potentially available from S-GM3
(1) by glycosyltransferase catalyzed addition of N-acetylga-
lactosamine. A protecting group strategy was selected that
would allow for a chemical synthesis of the S-GM2 tetrasac-
charide from S-GM3 in the event that this approach was un-
successful. Although we have recently demonstrated the use
of carbohydrate thiols as acceptors in glycosyltransferase
Figure 2. Target S-gangliosides and glycoprotein conjugates.
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Glycoproteins
FULL PAPER
Scheme 2. Synthesis of protected thiosialosyl galactoside 19. a) NaBH4,
H2O, H + resin, 08C; b) Ac2O, pyridine; c) 45% HBr/HOAc, CH2Cl2,
08C!RT; d) AllOH, Drierite, Hg(CN)2, HgBr2; e) cat. NaOMe, MeOH,
73% over 3 steps; f) PhCH(OMe)2, DMF/MeCN, cat. pTSA·H2O, 73%;
g) pyridine, BzCl, CH2Cl2, 08C, 94%; h) Tf2O, pyridine, CH2Cl2, ꢀ20!
ꢀ58C; i) KSAc, DMF, 508C, 94% over 2 steps; j) hydrazine acetate,
DMF, 98%; k) NaH, DMF, then 6, Kryptofix 21, 90%.
acetal in a mixture of DMF and acetonitrile containing cata-
lytic acid yielded the diol 14. When this reaction was con-
ducted by using either solvent alone, small quantities of the
diastereomeric benzylidene acetal contaminated the reaction
mixture.[51] Regioselective benzoylation at the more reactive
equatorial hydroxyl yielded the axial alcohol 15 and a trace
of dibenzoate 16. The alcohol was smoothly converted to
the thioester 17 by reaction of the corresponding triflate
with potassium thioacetate in DMF. De-S-acetylation with
hydrazine acetate gave the thiol and small amounts of disul-
fide. Condensation of the 18 with 2-chlorosialic acid deriva-
tive 6 under basic conditions afforded the thiodisaccharide
19 in high yield.[52]
Scheme 1. Retrosynthetic analysis of a) S-linked gangliosides 1 and 2,
and b) protected S-GM3 trisaccharide 3b.
catalyzed glycosylations, 3’-thiolactosides have proven un-
suitable substrates for sialyltransferases that we have stud-
ied, necessitating a chemical synthesis of S-GM3.[43] The pro-
tected trisaccharide 3a was constructed via the coupling of
the S-GM4 disaccharide 4 with glucoside 5.
Synthesis of protected glucosyl sphingosine analogues 5 and
23 (Scheme 3): Traditional approaches to the preparation of
glycosyl ceramide–protein conjugates have involved isola-
tion or total synthesis of the glycolipid followed by degrada-
tive ozonolysis of the olefin to yield the aldehyde, which is
then coupled to protein via reductive amination. Our group
has described the use of modified ceramide aglycons derived
from an azidosphingosine homologue 9.[42,53,54] This strategy
allows for an abridged synthesis, facilitates glycoconjugate
formation, enhances water solubility of the neoglycolipids,
and simplifies handling of the chemical intermediates while
maintaining the stereochemical integrity and many of the
important structural features found in naturally occurring
ceramides. Conjugation to protein may be achieved by
either elaboration of the terminal alkene, for example via
the photoaddition of cysteamine and subsequent reaction of
the resulting amine, or by installation of a suitably function-
alized N-acyl group after reduction of the azide function.
We and others have demonstrated that such truncated mole-
cules may be efficiently elaborated to the full-length sphin-
gosines by olefin cross metathesis.[55–58]
Synthesis of the S-GM4 disaccharide 19 (Scheme 2): The a-
(2!3) linked sialyl galactose disaccharide is a fragment cen-
tral to all gangliosides, is present as a repeating unit in
higher gangliosides, and is common to numerous other gly-
coconjugates. 3-Thiogalactosides have previously been syn-
thesized from d-gulopyranosides,[14,42,44–46] and d-gulofurano-
sides.[47] High yielding inversions at C-3 of gulopyranosides
require protection of the 4,6-diol as a cyclic acetal and are
favoured by use of ester, rather than ether protecting groups
at O-2.[42,48] Two synthetic routes to substrates meeting these
requirements were identified and are outlined in Scheme 2.
d-Gulose, available on large scale and in excellent yield
by reduction of d-gulono-g-lactone with sodium borohy-
dride,[49] is readily converted to the 4,6-O-isopropylidene
protected triol 11.[50] Unfortunately, attempts to alkylate the
anomeric position of the triol or the related 2,3-diacetate by
reaction with allyl bromide and base resulted in poor stereo-
selectivities and moderate yields. Instead, the allyl guloside
13 was prepared, with the expectation of future conversion
to a glycosyl donor or elaboration of the terminal olefin for
the preparation of glycoconjugates. Subsequent installation
of a 4,6-benzylidene acetal with benzaldehyde dimethyl
Reaction of the glucosyl trichloroacetimidate 8[59] with the
azido alcohol 9 (Scheme 3) afforded the glycoside 20 in high
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847

D. R. Bundle et al.
Scheme 4. Synthesis of protected S-GM3 trisaccharide 26. a) PdCl2,
NaOAc, HOAc/H2O 19:1, ))), <308C, 81%; b) K2CO3 or DBU, Cl3CCN,
CH2Cl2, ~70%; c) 5, 4 Š MS, BF3·Et2O, 0 8C!RT, 64%; d) PPh3, pyri-
dine/H2O 9:1, 508C; e) 6-(benzyloxy carbonylamino)hexanoic acid succi-
nimidyl ester, 508C, 90%; f) 23, 4 Š MS, BF3·Et2O, 0 8C!RT, 52 %.
Scheme 3. Construction of protected glucosyl ceramide analogues 5 and
23. a) 4 Š MS, CH2Cl2, TMSOTf, ꢀ508C!RT, 90%; b) cat. NaOMe,
MeOH; c) NaH, BnBr, DMF, 0 8C!RT, 92%; d) 3 Š MS, NaCNBH3,
HCl, Et2O, THF, 94%; e) PPh3, pyridine/H2O 9:1; f) 6-(benzyloxycarbo-
nylamino)hexanoic acid succinimidyl ester, 458C, 79% for 23, 85% for
24.
selective protection of the resulting primary alcohol 27 or
regioselective cleavage of the acetal to give 28 (see
Figure 3). Prior to investigation of this route, protected S-
GM3 was fully deblocked (26 ! 29 ! 1, Scheme 5) to test
as a substrate for glycosylation by a b(1,4) N-acetylgalacto-
saminyl transferase. It is noteworthy that substantial losses
of the deprotected trisaccharide 1 were incurred when con-
ventional SepPak or C18 silica gel HPLC columns were em-
ployed. This problem was overcome by use of a polymeric
reverse phase column from Hamilton (see Experimental
Section), or by inclusion of ammonium hydroxide in the
mobile phase.
yield along with a small amount of the acetylated acceptor
21. Exchange of ester protecting groups for benzyl ethers
was followed by regioselective benzylidene acetal opening
to furnished the acceptor 5 and a small amount of benzyl
2,3,6-tri-O-benzyl-a-d-glucopyranoside (<2%).[60] Reduc-
tion of the azide and N-acylation provided the alternative
acceptor 23.
Coupling of S-GM4 disaccharide and glucosyl sphingosines
(Scheme 4): Removal of the allyl protecting group at the
anomeric centre proved problematic. Reaction of 19 with
tris(triphenylphosphine) rhodium chloride gave only small
amounts of the isomerized product (<10%).[61] A one-pot
deprotection was attempted by using palladium chloride in
methanol. Under these conditions product decomposition
occurred before significant quantities of starting material
were consumed. Exchange of methanol for buffered acetic
acid[62] gave slightly better yields (~40%), but decomposi-
tion predominated. Fortunately, a significant rate enhance-
ment was realized when the reaction mixture was immersed
in an ultrasonic cleaning bath (<308C), and the disaccharide
hemiacetal 25 could be isolated as a mixture of isomers in
reasonable yield (81%) after only 5 h. Conversion of the
hemiacetal to the trichloroacetimidate 4 was effected by
using either potassium carbonate or DBU as base.[63] Glyco-
sylation of acceptor 5 with 4 by using boron trifluoride di-
ethyletherate or trimethylsilyl trifluoromethanesulfonate as
promoter in dichloromethane gave moderate yields of the
trisaccharide 3a. One-pot reduction of the azide and acyla-
tion of the resulting amine with 6-amino-N-carboxybenzyl
caproic acid NHS ester[64] yielded protected GM3 analogue
26. Similarly, glycosylation of the N-acylated acceptor 23
gave 26 directly, albeit in slightly reduced overall yield.
Scheme 5. Deprotection of S-GM3 and enzymatic elaboration to S-GM2
(2) and O,S-GD3 (30). a) cat. NaOMe, MeOH, 558C; b) NaOH, MeOH,
408C, 94%; c) Na, NH3, TH F,ꢀ788C, 83%; d) b(1,4) GalNAc transfer-
ase, UDP-GlcNAc 4-epimerase, UDP-GlcNAc, 74%; e) a(2,3/8) Neu5Ac
transferase, CMP-Neu5Ac, 38%.
Deprotection of S-GM3 and extension to thiotetrasaccharide
S-GM2 (Scheme 5/Figure 3): It was envisaged that a chemi-
cal synthesis of S-GM2 from 26 could be accomplished fol-
lowing either hydrolysis of the acetal protecting group and
Figure 3. Potential intermediates in the chemical synthesis of S-GM2.
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Glycoproteins
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Despite its thioglycosidic linkage we found that 1 served
as an acceptor for glycosyltransferase catalyzed elaboration
to 2 (Scheme 5). Several examples of a Leloir glycosyltrans-
ferase-mediated glycosylation of a thiooligosaccharide ac-
ceptor have been reported.[43,65,66] Bacterial transglucosylas-
es,[67–69] as well as sulfotransferases and acyltransferases from
Sinorhizobium meliloti,[70] have also been shown to utilize
thiooligosaccharides as acceptors. The reaction was carried
out in the presence of two previously cloned enzymes, a bac-
terial b(1,4) N-acetylgalactosaminyl transferase from Cam-
pylobacter jejeuni O:36,[71] and a UDP-GlcNAc 4-epimer-
ase[72] from C. jejuni NCTC 11168 that catalyzes the conver-
sion of UDP-GlcNAc to the more expensive UDP-GalNAc.
Treatment of 1 with a cloned bifunctional a(2,3/8) sialyl-
transferase[73] and the appropriate sugar nucleotide donor
resulted in conversion to the sulfur containing GD3-ana-
logue 30. Successful glycosylation of the thiooligosaccharide
by two different enzymes encourages the conclusion that 1
readily adopts conformations similar to that of O-sialyllac-
tose. With the amino-terminated thioglycosides 1 and 2 in
hand, coupling to protein could be undertaken.
Scheme 6. Conjugation of 1 and 2 to tetanus toxoid and bovine serum al-
bumin via adipate linker 33. a) 33, DMF, Et3N, 81% for 1, 56% for 2; b)
tetanus toxoid or bovine serum albumin, phosphate buffered saline, pH
7.2. Average hapten incorporation levels were determined by MALDI-
TOF.
Amino terminated GM4 disaccharide 45 was obtained via
enzymatic sialylation[78] of 6-azidohexyl b-d-galactopyrano-
side[79] followed by reduction of the azide 44 with triphenyl-
phosphine. Coupling was achieved by reaction of the amines
1,2,40–43 and 45 with 0.95 equivalents of diethyl squarate in
methanol followed by removal of solvent and reaction with
BSA in borate buffer at pH9.0. Average hapten incorpora-
tion as determined by MALDI TOF mass spectrometry is
outlined in Table 1.
Synthesis of carbohydrate–protein conjugates for immuniza-
tion and screening of immune sera: Recently, our lab has
obtained high IgG titres against TT–carbohydrate conju-
gates in rabbits,[74] and monomeric TT was selected as the
carrier protein for this study. Diethyl squarate has gained
favour as an efficient cross linking agent for the preparation
of glycoconjugates.[75] However, work in our group has iden-
tified linker specific antibody responses and as a conse-
quence we have discontinued its use for synthesis of immu-
nogens.[74] Instead, we prefer a linear adipic acid derived ho-
mobifunctional linker that can be employed for the genera-
tion of oligosaccharide–protein conjugates.[76] Conjugation
via either squarate or adipate linkers requires reaction of
the activated ester with an amine terminated deprotected
oligosaccharide such as 1 or 2, followed by reaction of the
product with protein in aqueous solution. Screening antigens
were coupled to BSA via diethyl squarate, to enable quanti-
tation of the immune response by ELISA while avoiding
measurement of protein or linker specific antibodies.
Table 1. Hapten incorporation for BSA conjugates using diethyl squarate
as coupling reagent.
Amine
Incorporation (n)
1
2
5
4
40
41
42
43
45
10
6
7
20
6
The conjugation of thiooligosaccharides 1 and 2 to TT and
BSA is outlined in Scheme 6. The amines 1 and 2 were
treated with an excess of linker 33 in anhydrous DMF con-
taining triethylamine. Following precipitation of the excess
coupling agent with 1% acetic acid solution, the activated
esters 34 and 35 were purified by HPLC. Coupling with teta-
nus toxoid and BSA was carried out as previously report-
ed,[76] by using an activated saccharide to protein ration of
30:1 to yield conjugates 36–39. Incorporation levels are indi-
cated in Scheme 6.
Structures of the amine terminated mono-, di- and oligo-
saccharides conjugated to BSA with diethyl squarate are
outlined in Figure 4. The synthesis of O-GM3 (40), O-GM2
(41) and lactosyl ceramide (42) analogues will be reported
elsewhere.[77] The cerebroside analogue 43 was available
after dissolving metal reduction of protected glucoside 24.
Figure 4. Amine functionalized O- and S-glycosides for conjugation to
BSA.
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D. R. Bundle et al.
GM4-type disaccharides 46 and 48 (Scheme 7) were de-
signed to serve as soluble ligands for inhibition of immune
sera binding to immobilized glycoconjugates and potentially
for elaboration to glycoconjugates themselves by functional-
ization of the terminal olefin. The O-linked GM4 disacchar-
ide 46 was prepared enzymatically from allyl b-d-galactopyr-
anose, while the S-linked analogue 48 was prepared from 19
by hydrolysis of the benzylidene acetal followed by Zem-
plØn transesterification and saponification.
conjugated via a squarate linker to BSA. In addition, three
of five mice gave good Ig titers in the range of 103–104
against the O-GM3-BSA conjugate as shown in Figure 5.
The preponderance of IgG subtype observed is indicative of
a class switch and recruitment of T cell help.
Conclusion
The chemical and chemoenzymatic synthesis of a number of
ganglioside analogues containing S-linked sialic acid resi-
dues and a truncated ceramide aglycon have been reported.
We have successfully demonstrated that the thiosialoside 1
may serve as an acceptor for bacterial enzymes which cata-
lyze the addition of N-acetylgalactosamine (to give GM2 an-
alogue 2) and N-acetylneuraminic acid (to give GD3 ana-
logue 30), and we have described the conjugation of the O-
and S-gangliosides and related molecules to tetanus toxoid
and bovine serum albumin. We are currently evaluating the
potential of S-linked oligosaccharide–TT conjugates to elicit
a carbohydrate specific immune response, and we are ana-
lyzing the ability of immune sera to recognize the corre-
sponding O-glycosides. Our initial results indicate that S-gly-
coside containing tetanus toxoid conjugates are immunogen-
ic and that these antigens stimulate the production of anti-
bodies that recognize O-glycosides.
Scheme 7. Synthesis and deprotection of allyl-functionalized disacchar-
ides. a) a(2,3) sialyltransferase, CMP-Neu5Ac, 68%; b) 80% HOAc,
558C, 85%; c) NaOMe (cat.), MeOH, RT; d) NaOH, MeOH, 408C, 81%
from 19.
Immunization: The synthetic sulfur containing GM3 ana-
logue has been tested for its ability to generate antibodies in
mice and we have examined the ability of these antibodies
to bind to O-glycosides. Five BALB/c mice were immunized
with S-GM3 tetanus toxoid conjugate 36 at days 1, 21, and
28 before collecting sera on day 38. The sera were titrated
against an immobilized O-GM3 BSA conjugate (amine 40,
Table 1) to identify cross reactivity with O-glycosides. A
strong antigen specific IgG response was observed when
immune sera were screened against thiooligosaccharide 1
Experimental Section
Chemical synthesis: All chemical reagents were of analytical grade, used
as supplied from Sigma-Aldrich unless indicated. Solvents used in non-
hydrolytic reactions were distilled under an inert atmosphere, except
N,N-dimethylformamide (stored over 3 Š molecular sieves and subjected
to reduced pressure for about fifteen minutes (<0.5 mm Hg) prior to
use) and pyridine (dried over solid potassium hydroxide and used with-
out further purification). Unless otherwise noted, all reactions were car-
ried out at room temperature and non-hydrolytic reactions were per-
formed under a positive pressure of argon. Solvents were removed be-
tween 20 and 408C (bath temperature) unless indicated otherwise. Mo-
lecular sieves were stored in an oven (>1708C) and cooled in vacuo
prior to use. Ammonia was distilled once from sodium metal prior to use.
Rexyn-101 (H+ form) was used where acidic ion-exchange resin is indi-
cated. Analytical thin layer chromatography (TLC) was conducted on
silica gel 60-F254 (Merck). Plates were visualized under UV light, and/or
by treatment with either acidic cerium ammonium molybdate or 5%
ethanolic sulfuric acid, followed by heating.
Enzymatic synthesis: Clones of the following recombinant enzymes
CMP-Neu5Ac synthetase from N. meningitidis,[80] UDP N-acetylglucosa-
mine 4-epimerase from C. jejuni NCTC 11168,[72] b(1,4) N-acetylgalacto-
saminyl transferase from C. jejuni O:36,[73] a(2,3) sialyltransferase[78] and
a(2,3/8) sialyltransferase[73] from Campylobacter jejuni OH4384 were ex-
pressed in E. coli, isolated by separation of the pellet and supernatant
following cell lysis, diluted or resuspended in the appropriate buffer and
used without further purification. The activity of the a(2,3) sialyltransfer-
ase and b(1,4) N-acetylgalactosaminyl transferases were determined by
radiochemical assay by using 8-methoxylcarbonyloctyl b-d-galactopyra-
nosyl-(1!4)-b-d-glucopyranoside and 8-methoxylcarbonyloctyl (5-acet-
amido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulopyranosylonic acid)-
(2!3)-b-d-galactopyranosyl)-(1!4)-b-d-glucopyranoside, respectively, as
acceptor substrates.[81] CMP-Neu5Ac was prepared as described.[80] Fol-
lowing removal of the protein by centrifugation of the CMP-Neu5Ac
Figure 5. Immunization with S-GM3 tetanus toxoid conjugate 36. Antisera
from five BALB/c mice immunized with 36 were titrated against immobi-
lized O-GM3-BSA. Data are shown for three mice that showed apprecia-
&
*
ble cross reactivity with the O-glycoside (mouse 1: , mouse 2: , mouse
~
3: ).
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Glycoproteins
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containing solution, the supernatant was lyophilized and the crude solid
obtained was used directly in reactions assuming 90% conversion of
starting material. A unit of enzyme activity is the amount that catalyzes
the formation of 1 mmol of product per minute at 378C using the speci-
fied acceptor. Alkaline phosphatase was obtained from Roche Diagnos-
tics.
(w/v); 60 mL). After allowing the mixture to warm to RT over 2 h, the
solution was evaporated to dryness and then concentrated from toluene
(3”60 mL). The yellow residue was then dissolved in dichloromethane
(250 mL) and washed with equal volumes of saturated sodium bicarbon-
ate solution followed by water. The organic layers were dried over
sodium sulfate, filtered, and concentrated. The syrup obtained was dis-
solved in allyl alcohol (150 mL) and Drierite (20 g) was added. After stir-
ring the mixture for 30 min, mercury(ii) cyanide (7.0 g, 27.7 mmol,
0.54 equiv) and mercury(ii) bromide (0.74 g, 2.1 mmol, 0.04 equiv) were
added. The reaction mixture was stirred at room temperature under
Argon overnight and then concentrated. The residue was suspended in
dichloromethane (250 mL), filtered, and washed with 10% potassium
bromide solution and water, then dried over sodium sulfate, filtered and
concentrated. Column chromatography using a gradient of hexanes and
Chromatography: Medium pressure chromatography was conducted on
silica gel (230–400 mesh, SiliCycle, Montreal) or Iatrobeads 6RS-8060
(Iatron Laboratories Inc., Japan), using a ratio between 50:1 and 125:1
(w/w) of silica gel/crude product at flow rates of 5–10 mLminꢀ1. Linear
gradients were generated using a Q-Grad gradient mixer from Lab Alli-
ance (State College, PA, USA). Reverse phase C18 silica gel cartridges
(tC18 SepPak) were obtained from Waters Corp. (Milford, USA). HPLC
purification was conducted on a Waters Delta 600 system by using a UV
absorbance detector. Separations were performed on Beckman C18-silica
semi-preparative or Hamilton PRP-1 reverse phase semi-preparative col-
umns as noted, with combinations of water and methanol (flow rate 1.5–
2.5 mLminꢀ1) as eluents.
ethyl acetate 3:1 ! 5:2 afforded 12 (14.76 g, 38.0 mmol, 74%). [a]D
=
ꢀ29 (c = 1.21, CHCl3); 1HNMR (600 MHz, CDCl 3): d = 5.90 (dddd,
1H, J=5.0, 6.0, 10.5, 17.2 Hz, H-b), 5.41 (dd, 1H, J3,2 =J3,4 =3.7 Hz, H-3),
5.30 (dddd, 1H, J=1.6, 1.6, 1.6, 17.2 Hz, H-c), 5.21 (dddd, 1H, J=1.4,
1.4, 1.4, 10.5 Hz, H-d), 5.05 (dd, 1H, J2,1 =8.0 Hz, H-2), 4.98 (dd, 1H,
Analytical methods: 1Hand 13C NMR spectra were recorded on Varian
INOVA 400, 500 and 600 MHz spectrometers. 1HNMR chemical shifts
are reported in d (ppm) units by using signals from residual solvents as a
reference. 13C NMR chemical shifts are referenced to internal solvent or
to external acetone in the case of D2O. 1Hand 13C NMR spectra were
tentatively assigned with the assistance of COSY, HMQC, HMBC,
TOCSY and TROESY spectra as necessary. NMR data for allyl glyco-
sides or compounds containing sphingosine (sph) or ceramide and deriva-
tives as the aglycon were labelled as indicated below.
J
4,5 =1.7 Hz, H-4), 4.81 (d, 1H, H-1), 4.36 (dddd, 1H, J=1.5, 1.5, 5.0,
13.1 Hz, H-a), 4.19–4.16 (m, 3H, H-5, H-6a, H-6b), 4.13 (ddd, 1H, J=1.4,
1.4, 6.0, 13.1 Hz, H-a’); 13C NMR (100 MHz, CDCl3): d = 171.4, 170.5,
170.3, 169.8, 134.3, 118.1, 98.0, 70.8, 70.2, 68.8, 68.1, 68.0, 62.2, 20.84,
20.81, 20.79, 20.7; ESI HRMS: m/z: calcd for C17H24O10Na: 411.1261;
found: 411.1262 [M+Na+]; elemental analysis calcd (%) for C17H24O10: C
52.57, H6.23; found: C 52.96, H6.14.
Allyl b-d-gulopyranoside (13): Tetraacetate 12 (1.488 g, 3.9 mmol) was
suspended in anhydrous methanol (10 mL) under a stream of argon. A
small piece of sodium metal was added, and the reaction mixture was
stirred overnight. The solution was then made neutral by addition of
acidic ion exchange resin, and the solids were removed by filtration. A
colourless solid was obtained after evaporation of the solvents (0.825 g,
3.7 mmol, 98%). A small portion of this material was further purified for
characterization as follows. Silica gel (2 g) was added to a solution of the
tetrol (~100 mg) in methanol and the suspension was evaporated to dry-
ness. The solid was applied to the top of a silica gel column, which was
then eluted with ethyl acetate/methanol 12:1 to afford pure 13 (87 mg).
Electrospray ionization mass spectra were recorded on a Micromass Zab-
spec TOF mass spectrometer. For high resolution mass determination,
spectra were obtained by voltage scan over a narrow range at a resolu-
tion of approximately 104. MALDI TOF mass spectra were recorded on
a Voyager Elite spectrometer from Applied Biosystems. Optical rotations
were determined with a Perkin–Elmer model 241 polarimeter at 22ꢁ
1
[a]D = ꢀ58 (c = 0.27, MeOH); HNMR (600 MHz, CD 3OD): d = 5.96
(dddd, 1H, J=5.3, 6.1, 10.6, 17.2 Hz, H-b), 5.31 (dddd, 1H, J=1.8, 1.8,
1.8, 17.2 Hz, H-c), 5.14 (dddd, J=1.4, 1.4, 1.8, 10.5 Hz, H-d), 4.63 (d, 1H,
28C, and are reported in units of degrees mLgꢀ1 dmꢀ1
.
J
1,2 =8.2 Hz, H-1), 4.36 (dddd, 1H, J=1.6, 1.6, 5.2, 12.9 Hz, H-a), (dddd,
1H, J=1.4, 1.4, 6.0, 12.9 Hz, H-a’), 3.94 (dd, 1H, J3,2 =J3,4 =3.6 Hz, H-3),
3.88–3.86 (m, 1H, H-5), 3.74 (dd, 1H, J6a,5 =6.8, J6a,6b =11.5 Hz, H-6a),
3.71–3.67 (m, 2H, H-4, H-6b), 3.63 (dd, 1H, H-2); 13C NMR (100 MHz,
CDCl3): d = 136.0, 117.2, 101.5, 75.0, 73.2, 71.3, 70.8, 69.7, 62.7; ESI
HRMS: m/z: calcd for C9H16O6Na: 243.0839; found: 243.0838 [M+Na+].
Immunization: Eight week old BALB/c mice were immunized by inter-
peritoneal injection of tetanus toxoid conjugate 36 (50 mg) in a 1:1 mix-
ture of PBS and complete Freundꢁs adjuvant. Subsequent injections
(50 mg) on days 21 and 28 were made subcutaneously in PBS and incom-
plete Freundꢁs adjuvant. Mice were bled on day 38 and sera were isolated
by centrifugation and then frozen at ꢀ208C.
Allyl 4,6-O-benzylidene-b-d-gulopyranoside (14): Guloside 13 (0.170 g,
0.77 mmol) was dissolved in N,N-dimethylformamide (3.0 mL) and aceto-
nitrile (3.0 mL). Benzaldehyde dimethyl acetal (0.127 mL, 0.85 mmol,
1.1 equiv) was added, followed by p-toluenesulfonic acid monohydrate
(6 mg), and the mixture was heated at 508C overnight. The reaction mix-
ture was made basic by addition of triethylamine, then concentrated. The
residue was dissolved in dichloromethane and acetone, mixed with silica
gel (1 g), and concentrated. The residue was applied to a silica gel
column and eluted with a linear gradient of hexanes and ethyl acetate
(2:1!1:1) to afford 14 (0.175 g, 0.56 mmol, 73%). [a]D = ꢀ81 (c = 1.06,
CHCl3); 1HNMR (500 MHz, CDCl 3): d = 7.53–7.48 (m, 2H, Ar), 7.39–
7.31 (m, 3H, Ar), 5.96 (ddd, 1H, J=5.3, 6.5, 10.4, 17.0 Hz, H-b), 5.32
(dddd, 1H, J=1.6, 1.6, 1.6, 17.1 Hz, H-c), 5.25–5.21 (m, 1H, H-d), 4.75
(d, 1H, J1,2 =8.2 Hz, H-1), 4.46 (m, 1H, H-a), 4.35 (dd, 1H, J6a,5 =1.1,
Enzyme
linked
immunosorbent
assay:
O-GM3-squarate-BSA
(10 mgmLꢀ1; 100 mL; 48C, overnight) was used to coat 96-well ELISA
plates (MaxiSorp, Nunc). The plate was washed (Molecular Devices Skan
Washer 400) five times with PBST (PBS containing Tween 20, 0.05%
v/v). Mouse sera were diluted with PBST containing 0.15 BSA and the
solutions were added to the plate and incubated at room temperature for
2 h. The plate was washed with PBST (5”) and goat anti-mouse IgG an-
tibody conjugated to horseradish peroxidase (Kirkegaard & Perry Labo-
ratories; 1:2000 dilution in PBST) was added (100 mL) and incubated for
a period of 1 h. The plate was washed five times with PBST before addi-
tion of a 1:1 mixture of 3,3’,5,5’-tetramethylbenzidine (0.4 gLꢀ1) and
0.02% H2O2 solution (Kirkegaard and Perry Laboratories; 100 mL).
After 2 min the reaction was stopped by addition of 1m phosphoric acid
(100 mL). Absorbance was read at 450 nm (Molecular Devices Spectra
Max 190 plate reader).
J
6a,6b =12.4 Hz, H-6a), 4.21 (dd, 1H, J3,2 =J3,4 =3.2 Hz, H-3), 4.15–4.06 (m,
3H, H-a’, H-4, H-6b), 3.87 (dd, 1H, H-2), 3.79 (brs, 1H, H-5); 13C NMR
(125 MHz, CDCl3): d = 137.7, 134.0, 129.1, 128.2, 126.3, 117.9, 101.3,
99.1, 75.9, 70.0, 69.9, 69.3, 68.9, 66.0; ESI HRMS: m/z: calcd for
C16H20O6Na: 331.1152; found: 331.1155 [M+Na+]; elemental analysis
calcd (%) for C16H20O6: C 62.33, H6.54; found: C 62.14, H6.65.
Compounds:
Allyl 2,3,4,6-tetra-O-acetyl-b-d-gulopyranoside (12): 1,2,3,4,6-Penta-O-
acetyl-b-d-gulopyranose (20.0 g, 51.2 mmol) was evaporated from toluene
(150 mL) and concentrated. The syrup was dissolved in dry dichlorome-
thane (60 mL) and cooled to 08C before adding HBr in acetic acid (45%
A small amount of the product was acetylated to identify the position of
the free hydroxyl groups, yielding allyl 2,3-di-O-acetyl-4,6-O-benzyl-
Chem. Eur. J. 2006, 12, 845 – 858
ꢀ 2006 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
www.chemeurj.org
851

D. R. Bundle et al.
idene-b-d-gulopyranoside: 1HNMR (500 MHz, CDCl 3): d = 7.54–7.50
(m, 2H, Ar), 7.40–7.34 (m, 3H, Ar), 5.91 (dddd, 1H, J=4.9, 5.8, 10.5,
17.3 Hz, H-b), 5.54 (s, 1H, CHPh), 5.49 (dd, 1H, J3,2 =J3,4 =3.4 Hz, H-3),
5.30 (dddd, 1H, J=1.7, 1.7, 1.7, 17.3 Hz, H-c), 5.21 (dd, 1H, J2,1 =8.6 Hz,
H-2), 5.21–5.16 (m, 1H, H-d), 4.88 (d, 1H, H-1), 4.40 (dddd, 1H, J=1.6,
1.6, 5.0, 13.2 Hz, H-a), 4.35 (dd, 1H, J6a,5 =1.5, J6a,6b =12.6 Hz, H6a), 4.13
(dddd, 1H, J=1.4, 1.4, 5.9, 13.2 Hz, H-a’), 4.08 (dd, 1H, J6b,5 =1.9 Hz, H-
6b), 4.01 (dd, 1H, J4,5 =1.3 Hz, H-4), 3.75 (m, 1H, H-5), 2.15 (s, 3H,
C(O)CH3), 2.03 (s, 3H, C(O)CH3).
68.6, 66.3; ESI HRMS: m/z: calcd for C30H28O8Na: 539.1676; found:
539.1679; elemental analysis calcd (%) for C30H28O8: C 69.76, H5.46;
found: C 69.66, H5.65.
Allyl 3-thioacetyl-2-O-benzoyl-4,6-O-benzylidene-3S-b-d-galactopyrano-
side (17): Alcohol 15 (2.20 g, 5.3 mmol) was dissolved in dichloromethane
(40 mL) containing pyridine (2.10 mL, 27 mmol, 5 equiv). The solution
was cooled to ꢀ208C and trifluoromethanesulfonic anhydride (1.20 mL,
7.1 mmol, 1.34 equiv) was added over a period of one minute. The mix-
ture was allowed to warm to ꢀ58C over 1.5 h, and was then stirred at
room temperature for a further 1.5 h. After diluting with dichlorome-
thane (40 mL), the reaction mixture was washed with an equal volume of
water, dried over sodium sulfate, filtered, and concentrated. The residue
was dissolved in N,N-dimethylformamide (50 mL), potassium thioacetate
(1.30 g, 11.4 mmol, 2 equiv) was added, and mixture was immersed in an
oil bath at 508C. After stirring overnight, the black solution was concen-
trated. The syrup was dissolved in dichloromethane (50 mL) and washed
with an equal volume of water, dried over sodium sulfate, filtered and
concentrated. The yellow residue was chromatographed on a silica gel
column (3.5 cm”30 cm) capped by a 2 cm deep plug of decolourising
carbon by using hexanes/ethyl acetate 2:1 as eluent to yield 17 (2.36 g,
5.0 mmol, 94%). [a]D = 156 (c = 0.48, CHCl3); 1HNMR (600 MHz,
CDCl3): d = 8.01–7.99 (m, 2H, Ar), 7.58–7.52 (m, 3H, Ar), 7.46–7.42 (m,
2H, Ar), 7.41–7.36 (m, 3H, Ar), 5.77 (dddd, 1H, J=4.8, 6.2, 10.8,
17.2 Hz, H-b), 5.56 (s, 1H, CHPh), 5.49 (dd, 1H, J2,1 =7.7, J2,3 =11.5 Hz,
H-2), 5.20 (dddd, 1H, J=1.7, 1.7, 1.7, 17.2 Hz, H-c), 5.09 (dddd, 1H, J=
1.4, 1.4, 1.4, 10.8 Hz, H-d), 4.78 (d, 1H, H-1), 4.39–4.35 (m, 2H, H-6a, H-
a), 4.25 (dd, 1H, J3,4=3.3 Hz, H-3), 4.17–4.07 (m, 3H, H-4, H-6b, H-a’),
3.70 (m, 1H, H-5), 2.21 (s, 3H, C(O)CH3); 13C NMR (125 MHz, CDCl3):
d = 194.9, 165.2, 137.4, 133.8, 133.0, 129.8, 129.7, 129.0, 128.3, 128.2,
126.3, 117.2, 101.3, 101.1, 75.9, 69.3, 69.04, 69.01, 68.5, 47.0, 30.5; ESI
HRMS: m/z: calcd for C25H26O7SNa: 493.129695; found: 493.129366
[M+Na+]; elemental analysis calcd (%) for C25H26O7S: C 63.81, H5.57;
found: C 63.95, H5.51.
When the initial reaction was carried out under identical conditions using
N,N-dimethylformamide or acetonitrile as the lone solvent, slightly lower
yields were realized, and a second isomer (~5%), identified as allyl (R)-
4,6-O-benzylidene-b-d-gulopyranoside could be recrystallized (ethyl ace-
tate/hexanes) after chromatography as described above. [a]D = ꢀ81 (c =
0.89, CHCl3); 1HNMR (500 MHz, CDCl 3): d = 7.50–7.46 (m, 2H, Ar),
7.44–7.39 (m, 2H, Ar), 7.37–7.34 (m, 1H, Ar), 6.14 (s, 1H, CHPh), 5.97
(dddd, 1H, J=5.6, 6.4, 10.4, 16.9 Hz, H-b), 5.33 (dddd, 1H, J=1.6, 1.6,
1.6, 17.1 Hz, H-c), 5.24 (dddd, 1H, J=10.3, 1.3, 1.3, 1.3 Hz, H-d), 4.71 (d,
1H, J1,2 =7.7 Hz, H-1), 4.46 (m, 1H, H-a), 4.26 (dd, 1H, J3,2 =J3,4 =2.5 Hz,
H-3), 4.12 (m, 1H, H-a’), 4.06–4.01 (m, 3H, H-4, H-6a, H -6), 3.95 (dd,
b
1H, H-2), 3.78 (m, 1H, H-5), 2.55 (brs, 1H, OH), 2.49 (brs, 1H, OH);
13C NMR (125 MHz, CDCl3): d = 137.7, 134.0, 129.1, 128.2, 126.3, 117.9,
101.3, 99.1, 75.9, 70.0, 69.9, 69.3, 68.9, 66.0; ESI HRMS: m/z: calcd for
C16H20O6Na: 331.1152; found: 331.1154 [M+Na+]; elemental analysis
calcd (%) for C16H20O6: C 62.33, H6.54; found: C 62.09, H6.51.
A small amount of the product was acetylated to identify the position of
the free hydroxyl groups, yielding allyl 2,3-di-O-acetyl-(R)-4,6-O-benzyl-
idene-b-d-gulopyranoside: 1HNMR (600 MHz, CDCl 3): d = 7.47–7.44
(m, 2H, Ar), 7.39–7.32 (m, 3H, Ar), 5.99 (s, 1H, CHPh), 5.95–5.87 (m,
2H, H-3, H-b), 5.33 (dddd, 1H, J=1.6, 1.6, 1.6, 17.4 Hz, H-c), 5.30 (dd,
1H, J2,3 =3.4, J2,1 =4.4 Hz, H-2), 5.24 (dddd, 1H, J=1.4, 1.4, 1.4, 10.5 Hz,
H-d), 4.82 (d, 1H,
J1,2 =4.5 Hz, H-1), 4.34–4.24 (m, 3H, H-4,
OCH2CHCH2, H-6a), 4.19 (m, 1H, H-5), 4.12–4.04 (m, 2H, H-6b,
OCH2CHCH2), 2.12, 2.08 (2s, 6H, 2”C(O)CH3).
Allyl 2-O-benzoyl-4,6-O-benzylidene-3-thio-b-d-galactopyranoside (18):
Thioester 17 (0.320 g, 0.68 mmol) was dissolved in N,N-dimethylforma-
mide (5.0 mL), and argon was bubbled through the solution for 20 min
before hydrazine acetate (0.113 g, 1.23 mmol, 1.7 equiv) was added. The
mixture was stirred for 2 h, diluted with ethyl acetate (50 mL), washed
with water (2”40 mL), and the organic layers dried over sodium sulfate,
filtered and concentrated to afford 18 (0.286 g, 0.67 mmol, 98%).
1HNMR (400 MHz, CDCl 3): d = 8.09–8.05 (m, 2H, Ar), 7.59–7.55 (m,
3H, Ar), 7.49–7.37 (m, 5H, Ar), 5.79 (dddd, 1H, J=4.8, 6.2, 11.0,
17.3 Hz, H-b), 5.61 (s, 1H, CHPh), 5.37 (dd, 1H, J2,1 =7.8, J2,3 =11.2 Hz,
H-2), 5.21 (dddd, 1H, J=1.7, 1.7, 1.7, 17.3 Hz, H-c), 5.09 (dddd, 1H, J=
1.4, 1.4, 1.4, 10.5 Hz, H-d), 4.66 (d, 1H, H-1), 4.38 (dd, 1H, J6a,6b =12.4,
Allyl 2-O-benzoyl-4,6-O-benzylidene-b-d-gulopyranoside (15): Diol 14
(4.00 g, 13.0 mmol) was dissolved in a mixture of dry dichloromethane
and pyridine (4:1; 60 mL), and the flask was immersed in an ice bath.
Benzoyl chloride (1.66 mL, 14.3 mmol, 1.1 equiv) was added, and after
40 min the reaction mixture was quenched by addition of methanol
(5 mL). After stirring for 20 min at room temperature the solution was
concentrated. The residue was dissolved in dichloromethane (100 mL),
washed successively with 1m HCl, saturated sodium bicarbonate, and
water, then dried over sodium sulfate, filtered and concentrated. Column
chromatography in hexanes/ethyl acetate 2:1 yielded 15 (5.01 g,
12.1 mmol, 94%). [a]D = ꢀ40 (c = 0.76, CHCl3); 1HNMR (600 MHz,
CDCl3): d = 8.07–8.04 (m, 2H, Ar), 7.62–7.58 (m, 1H, Ar), 7.57–7.54 (m,
2H, Ar), 7.49–7.45 (m, 2H, Ar), 7.41–7.35 (m, 3H, Ar), 5.85 (dddd, 1H,
J=5.0, 6.1, 10.6, 17 Hz, H-b), 5.59 (s, 1H, CHPh), 5.41 (dd, 1H, J2,1 =8.5,
J
6a,5 =1.5 Hz, H-6a), 4.39–4.33 (m, 1H, H-a), 4.17–4.09 (m, 3H, H-4, H-a’,
H-6b), 3.61 (m, 1H, H-5), 3.14 (ddd, 1H, J3,4 =3.2, J3,2 =11.0, J3,SH
=
11.0 Hz, H-3), 2.24 (d, 1H, SH); 13C NMR (125 MHz, CDCl3): d = 165.4,
137.5, 133.9, 133.0, 130.1, 129.8, 129.1, 128.3, 128.2, 128.3, 117.2, 101.5,
101.0, 76.6, 72.5, 69.2, 69.03, 68.96, 43.8; ESI HRMS: m/z: calcd for
C23H24O6SNa: 451.1191; found: 451.1191 [M+Na+].
J
2,3 =3.3 Hz, H-2), 5.26 (dddd, 1H, J=1.6, 1.6, 1.6, 17.2 Hz, H-c), 5.13
(dddd, 1H, J=1.4, 1.4, 1.4, 10.6 Hz, H-d), 5.11 (d, 1H, H-1), 4.44–4.36
(m, 2H, H-a, H-6a), 4.19–4.10 (m, 2H, H-a’, H-6b), 3.89 (m, 1H, H-5);
13C NMR (125 MHz, CDCl3): d
=
165.1, 137.6, 134.1, 133.3, 129.82,
The title compound was slowly converted to allyl 2-O-benzoyl-4,6-O-ben-
zylidene-3-thio-b-d-galactopyranoside disulfide in solution: 1HNMR
(500 MHz, CDCl3): d = 8.09–8.06 (m, 2H, Ar), 7.60–7.53 (m, 3H, Ar),
7.47–7.43 (m, 2H, Ar), 7.40–7.34 (m, 3H, Ar), 5.78 (dddd, 1H, J=4.8,
129.77, 129.1, 128.5, 128.2, 126.4, 117.1, 101.3, 96.9, 76.3, 71.3, 69.5, 69.28,
69.27, 65.7; ESI HRMS: m/z: calcd for C23H24O7Na: 435.1414; found:
435.1414 [M+Na+]; elemental analysis calcd (%) for C23H24O7: C 66.98,
H5.87; found: C 67.06, H5.79.
6.1, 10.6, 17.2 Hz, H-b), 5.60 (s, 1H, CHPh), 5.59 (dd, 1H, J2,1 =7.8, J2,3
=
11.2 Hz, H-2), 5.20 (dddd, 1H, J=1.6, 1.6, 1.6, 17.2 Hz, H-c), 5.08 (dddd,
1H, J=1.6, 1.6, 1.6, 10.6 Hz, H-d), 4.71 (d, 1H, H-1), 4.39–4.34 (m, 2H,
H-6a, H-a), 4.33 (dd, 1H, J=0.9, 3.2 Hz, H-4), 4.16–4.09 (m, 2H, H-6b,
H-a’), 3.56 (brs, 1H, H-5), 2.99 (dd, 1H, H-3); 13C NMR (125 MHz,
CDCl3): d = 165.2, 137.6, 133.9, 133.0, 130.1, 129.8, 128.9, 128.4, 128.1,
126.3, 117.1, 101.4, 101.2, 78.4, 69.9, 69.2, 69.1, 68.9, 49.3; ESI HRMS:
m/z: calcd for C46H46O12S2Na: 877.2328; found: 877.2327 [M+Na+]; ele-
mental analysis calcd (%) for C46H46O12S2: C 62.93, H5.28; found: C
63.18, H5.40.
A second, less polar compound allyl 2,3-di-O-benzoyl-4,6-O-benzylidene-
b-d-gulopyranoside (16) was also isolated (0.064 g, 0.12 mmol, 1%). [a]D
= ꢀ88 (c = 0.35, CHCl3); 1HNMR (500 MHz, CDCl 3): d = 8.08–8.05
(m, 2H, Ar), 7.92–7.87 (m, 2H, Ar), 7.67–7.62 (m, 1H, Ar), 7.60–7.56 (m,
2H, Ar), 7.53–7.47 (m, 3H, Ar), 7.43–7.36 (m, 3H, Ar), 7.35–7.29 (m,
2H, Ar), 5.93–5.83 (m, 2H, H-3, H-b), 5.63 (dd, 1H, J2,1 =8.4, J2,3
=
3.5 Hz, H-2), 5.62 (s, 1H, CHPh), 5.29 (dddd, 1H, J=1.7, 1.7, 1.7,
17.2 Hz, H-c), 5.23 (d, 1H, H-1), 5.14 (dddd, 1H, J=1.4, 1.4, 1.4, 10.6 Hz,
H-d), 4.49–4.39 (m, 2H, H-a, H-6a), 4.26–4.19 (m, 2H, H-4, H-a’), 4.14
(m, 1H, H-6b), 3.93 (m, 1H, H-5); 13C NMR (125 MHz, CDCl3): d =
164.93, 164.91, 137.3, 134.0, 133.6, 133.0, 129.8, 129.71, 129.69, 129.4,
129.2, 128.7, 128.25, 128.22, 126.4, 117.0, 101.3, 97.7, 74.3, 70.5, 69.4, 69.1,
Allyl S-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-
a-d-galacto-non-2-ulopyranosylonate)-(2!3)-2-O-benzoyl-(S)-4,6-O-
benzylidene-3-thio-b-d-galactopyranoside (19): Thiol 18 (0.613 g,
852
www.chemeurj.org
ꢀ 2006 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 845 – 858

Glycoproteins
FULL PAPER
1.43 mmol) was dissolved in N,N-dimethylformamide (15 mL) and argon
was bubbled through for one hour prior to cooling the solution to 08C.
Sodium hydride (dispersion in mineral oil; 60% by mass; 0.074 g,
1.85 mmol, 1.3 equiv) was added in portions and stirring was continued
until the evolution of gas had ceased. A solution of methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-2-chloro-3,5-dideoxy-d-glycero-b-d-galacto-2-nonu-
lopyranosonate[82] (1.30 g, 2.55 mmol, 1.8 equiv) and 1,4,10-trioxa-7,13-di-
azacyclopentadecane (0.062 g, 0.29 mmol, 0.2 equiv) in N,N-dimethylfor-
mamide (5 mL) was added via canula and the mixture was stirred for
40 min, after which time the mixture was neutralized with acetic acid.
The solution was diluted with ethyl acetate (150 mL) and washed with an
equal volume of water, then dried over sodium sulfate, filtered and con-
centrated. Column chromatography on silica gel with a linear gradient of
hexanes/ethyl acetate/acetone 4:3:1!4:3:2) afforded 19 (1.220 g,
1.35 mmol, 95%). [a]D = 50 (c = 0.27, CHCl3); 1HNMR (500 MHz,
CDCl3): d = 8.16–8.12 (m, 2H, Ar), 7.58–7.52 (m, 3H, Ar), 7.49–7.45 (m,
2H, Ar), 7.39–7.31 (m, 3H, Ar), 5.79 (dddd, 1H, J=5.1, 5.9, 10.5,
17.2 Hz, H-b), 5.63 (ddd, 1H, J=2.5, 5.6, J8’,7’ =10.0 Hz, H-8’), 5.48 (s,
1H, CHPh), 5.30 (dd, 1H, J2,1 =7.7, J2,3 =11.8 Hz, H-2), 5.23 (dd, 1H,
2,3-Di-O-benzyl-4,6-O-benzylidene-b-d-glucopyranosyl-(1!1)-(2S,3R)-2-
azido-3-O-benzyl-pent-4-ene-1,3-diol (22): Diacetate 20 (1.053 g,
1.81 mmol) was dissolved in dry methanol (30 mL) and a small piece of
sodium was added. After 3 h, the mixture was neutralized with acidic
cation exchange resin, filtered, and concentrated. The residue was dis-
solved in anhydrous N,N-dimethylformamide (25 mL) and cooled to 08C.
Sodium hydride (0.434 g of a 60% dispersion in oil; 10.9 mmol, 6 equiv)
was added in portions, followed by benzyl bromide (1.3 mL, 10.9 mmol,
6 equiv). After 4 h methanol was added followed by acidic cation ex-
change resin. The neutral solution was filtered and concentrated, then re-
dissolved in dichloromethane (100 mL) and washed with water (2”
60 mL), dried and concentrated. The residue was chromatographed on
silica gel with hexanes/ethyl acetate 10:1 as eluent to give 22 (1.136 g,
1.71 mmol, 94%). [a]D = ꢀ43 (c = 1.21, CHCl3); 1HNMR (500 MHz,
CDCl3): d = 7.51–7.48 (m, 2H, Ar), 7.41–7.25 (m, 18H, Ar), 5.84 (ddd,
1H, J=7.9, 10.4, 18.3 Hz, H-4sph), 5.57 (s, 1H, CHPh), 5.44–5.41 (m, 1H,
H-5asph), 5.38–5.33 (m, 1H, H-5bsph), 4.92 (d, 1H, J=11.4 Hz, CH2Ph),
4.84 (d, 1H, J=11.0 Hz, CH2Ph), 4.80 (d, 1H, J=11.4 Hz, CH2Ph), 4.78
(d, 1H, J=11.0 Hz, CH2Ph), 4.62 (d, 1H, J=11.9 Hz, CH2Ph), 4.51 (d,
1H, J1,2 =7.6 Hz, H-1), 4.38 (d, 1H, J=11.4 Hz, CH2Ph), 4.35 (dd, 1H,
J=10.4, 5.0 Hz, H-6a), 4.00 (dd, 1H, J=6.7, 10.0 Hz, H-1asph), 3.95 (dd,
1H, J=5.3, 7.9 Hz, H-3sph), 3.81–3.67 (m, H-6b, H-1bsph, H-3, H-4, H-
2sph), 3.48 (dd, 1H, J2,3 =8.3 Hz, H-2), 3.44–3.37 (m, 1H, H-5); 13C NMR
(125 MHz, CDCl3): d = 138.5, 138.3, 137.8, 137.3, 134.3, 128.9, 128.4,
128.34, 128.28, 128.23, 127.99, 127.96, 127.72, 127.68, 127.63, 127.61, 126.0,
120.8, 103.9, 101.2, 82.1, 81.5, 80.9, 79.8, 75.4, 75.1, 70.5, 68.8, 68.7, 66.1,
64.2; ESI HRMS: m/z: calcd for C39H41N3O7Na: 686.2837; found:
686.2838 [M+Na+]; elemental analysis calcd (%) for C39H41N3O7: C
70.57, H6.23, N 6.33; found: C 70.94, H6.40, N 6.07.
J
7’,6’ =2.2 Hz, H-7’), 5.18 (dddd, 1H, J=1.7, 1.7, 1.7, 17.2 Hz, H-c), 5.06–
5.01 (m, 2H, H-1, H-d), 4.83 (ddd, 1H, J4’,3’eq =4.6, J4’,5’ =10.4, J4’,3’ax
=
11.6 Hz, H-4’), 4.38–4.31 (m, 3H, H-9a’, H-6a, H-a), 4.15 (dddd, 1H, J=
1.4, 1.4, 6.0, 13.1 Hz, H-a’), 4.12–4.05 (m, 2H, H-9b’, H-6b), 3.93–3.85 (m,
2H, H-5’, H-3), 3.80–3.73 (m, 3H, H-5, H-4, H-6’), 3.77 (s, 3H, CO2CH3),
2.58 (dd, 1H, J3eq,3ax =12.9 Hz, H-3’eq), 1.91 (dd, 1H, H-3’ax), 2.21, 2.07,
1.97, 1.84, 1.70 (5s, 5”3H, 5”C(O)CH3); 13C NMR (125 MHz, CDCl3):
d = 170.9, 170.7, 170.18, 170.14, 170.12, 169.8, 165.9, 137.6, 134.1, 132.8,
130.6, 130.1, 128.9, 128.2, 128.1, 126.3, 116.9, 101.1, 100.9, 81.0, 76.3, 73.6,
69.6, 69.4, 69.2, 69.1, 67.9, 67.5, 66.9, 62.4, 53.0, 49.3, 45.6, 37.4, 23.2, 21.5,
20.8, 20.6; ESI HRMS: m/z: calcd for C43H51NO18SNa: 924.2719; found:
924.2720 [M+Na+]; elemental analysis calcd (%) for C43H51NO18S: C
57.26, H5.70, N 1.55; found: C 56.87, H5.56, N 1.40.
2,3-Di-O-benzyl-4,6-O-benzylidene-b-d-glucopyranosyl-(1!1)-(2S,3R)-2-
(N-carboxybenzyl-6-aminohexanamido)-3-O-benzyl-pent-4-ene-1,3-diol
(24): Azide 22 (0.098 g, 0.15 mmol) was dissolved in a mixture of pyridine
and water (20:1; 5 mL) and triphenylphosphine (0.078 g, 0.29 mmol,
2.0 equiv) was added. The solution was heated at 508C for 4.5 h and then
cooled to room temperature. 6-(Benzyloxycarbonylamino)hexanoic acid
succinimidyl ester (0.107 g, 0.29 mmol, 2.0 equiv) was added to the flask,
and after heating overnight at 508C the mixture was concentrated. Chro-
matography on silica gel using a linear gradient of methanol in dichloro-
methane (5.0!6.2%) as eluent afforded 24 (0.112 g, 0.13 mmol, 85%).
[a]D = ꢀ25.09 (c = 0.31, CHCl3); 1HNMR (600 MHz, CDCl 3): d =
7.51–7.48 (m, 2H, Ar), 7.41–7.25 (m, 23H, Ar), 5.75 (ddd, 1H, J=7.9,
10.3, 17.3 Hz, H-4sph), 5.71 (d, 1H, J=9.4 Hz, NHC(O)CH2), 5.58 (s, 1H,
CHPh), 5.31–5.27 (m, 1H, H-5asph), 5.23–5.18 (m, 1H, H-5bsph), 5.09 (s,
2H, CH2Ph), 4.94 (d, 1H, J=11.5 Hz, CH2Ph), 4.83 (d, 1H, J=11.1 Hz,
CH2Ph), 4.80 (d, 1H, J=11.5 Hz, CH2Ph), 4.76 (brt, 1H, NHCO2Bn),
4.69 (d, 1H, J=11.1 Hz, CH2Ph), 4.62 (d, 1H, J=11.9 Hz, CH2Ph), 4.45
(d, 1H, J1,2 =7.8 Hz, H-1), 4.35 (d, 1H, J=11.9 Hz, CH2Ph), 4.34 (dd,
2,3-Di-O-acetyl-4,6-O-benzylidene-b-d-glucopyranosyl-(1!1)-(2S,3R)-2-
azido-3-O-benzoyl-pent-4-ene-1,3-diol (20): A suspension of the imidate
8 (0.301 g, 0.61 mmol, 1.28 equiv), (2S,3R)-2-azido-3-O-benzoyl-pent-4-
ene-1,3-diol (9) (0.117 g, 0.47 mmol, 1.00 equiv) and powdered 4 Š mo-
lecular sieves (100 mg) were combined in freshly distilled dichlorome-
thane (10.0 mL) and stirred for 30 min under an argon atmosphere at
room temperature. The mixture was then cooled to ꢀ508C and trimethyl-
silyl trifluoromethanesulfonate (20 mL in 0.5 mL CH2Cl2) was added. The
reaction mixture was allowed to warm to room temperature and stirring
was continued for 6 h before triethylamine was added to neutralize the
mixture. After filtration and concentration, the residue was chromato-
graphed on silica gel by using hexanes/ethyl acetate 4:1 as eluent to
afford 20 (0.248 g, 4.3 mmol, 90%). [a]D
=
ꢀ59 (c
= 1.52, CHCl3);
1HNMR (500 MHz, CDCl 3): d = 8.09–8.06 (m, 2H, Ar), 7.62–7.57 (m,
1H, Ar), 7.50–7.41 (m, Ar, 4H), 7.37–7.33 (m, 3H, Ar), 5.96 (ddd, 1H,
J=7.1, 10.6, 17.2 Hz, H-4sph), 5.67–5.63 (m, 1H, H-3sph), 5.52–5.46 (m,
1H, J6a,5 =5.0, J6a,6b =10.4 Hz, H-6a), 4.29 (dd, 1Hx, J1a,2 =3.9, J1a,1b
10.5 Hz, H-6a), 4.23 (m, 1H, H-2sph), 3.85 (dd, 1H, J3,2 =J3,4 =7.6 Hz, H-
sph), 3.80–3.75 (m, 2H, H-3, H-6b), 3.70 (dd, 1H, J4,3 =J4,5 =9.4 Hz, H-4),
=
2H, CHPh, H-5sph), 5.45–5.40 (m, 1H, H-5sph), 5.31 (dd, 1H, J3,2 =J3,4
9.4 Hz, H-3), 5.04 (dd, 1H, J2,1 =7.8 Hz, H-2), 4.29 (dd, 1H, J6a,6b =10.5,
6a,5 =5.0 Hz, H-6a), 4.01–3.90 (m, 2H, C-2sph, C-1sph), 3.75–3.64 (m, 3H,
=
3
3.61 (dd, 1H, J1,2 =3.7 Hz, H-1bsph), 3.44 (dd, 1H, J2,3 =8.7 Hz, H-2), 3.39
(ddd, 1H, J5,6b =9.6 Hz, H-5), 3.16–3.11 (m, 2H, CH2NHCO2Bn), 1.89–
1.75 (m, 2H, C(O)CH2), 1.49–1.38 (m, 4H, 2”CH2), 1.22–1.15 (m, 2H,
CH2); 13C NMR (125 MHz, CDCl3): d = 172.3, 156.4, 138.3, 138.1 (2C),
137.3, 136.6, 135.7, 129.0, 128.51, 128.46, 128.35, 128.33, 128.25, 128.11,
128.06, 128.04, 127.87, 127.84, 127.80, 127.70, 127.66, 126.0, 119.6, 104.4,
101.2, 82.0, 81.7, 81.1, 79.5, 75.4, 75.0, 70.6, 68.8, 68.7, 66.6, 66.1, 51.7,
40.9, 36.2, 29.6, 26.2, 25.0; ESI HRMS: m/z: calcd for C53H60N2O10Na:
907.4146; found: 907.4145 [M+Na+]; elemental analysis calcd (%) for
C53H60N2O10: C 71.92, H6.83, N 3.17; found: C 71.73, H6.71, N 2.96.
J
H-4, H-6b, C-1sph), 3.53 (ddd, 1H, J=5.0, 9.9, 9.9 Hz, H-5), 2.10 (s, 3H,
C(O)CH3) 2.05 (s, 3H, C(O)CH3); 13C NMR (125 MHz, CDCl3): d =
170.1, 169.5, 165.0, 136.7, 133.4, 131.4, 129.8, 129.7, 129.2, 128.5, 128.2,
126.1, 120.8, 101.5, 101.0, 78.2, 74.4, 72.1, 71.8, 68.4, 68.2, 66.5, 63.2, 20.8,
20.7; ESI HRMS: m/z: calcd for C29H31N3O10Na: 604.1902; found:
604.1907 [M+Na+]; elemental analysis calcd (%) for C29H31N3O10: C
59.89, H5.37, N 7.23; found: C 59.61, H5.20, N 7.09.
A side product, (2S,3R)-1-O-acetyl-2-azido-3-O-benzoyl-pent-4-ene-1,3-
diol (21) was isolated (0.007 g, 0.001 mmol, 4%). 1HNMR (400 MHz,
CDCl3): d = 8.10–8.06 (m, 2H, Ar), 7.63–7.57 (m, 1H, Ar), 7.51–7.45 (m,
2H, Ar), 6.01–5.91 (m, 1H, H-4), 5.69–5.64 (m, 1H, H-3), 5.54–5.47 (m,
2,3,6-Tri-O-benzyl-b-d-glucopyranosyl-(1!1)-(2S,3R)-2-azido-3-O-
benzyl-pent-4-ene-1,3-diol (5): Azide 22 (0.720 g, 1.08 mmol) was concen-
trated twice from toluene and the residue was dissolved in tetrahydrofur-
an (20 mL) containing powdered 3 Š molecular sieves. After stirring for
20 min, sodium cyanoborohydride (0.681 g, 10.84 mmol, 10 equiv) was
added, followed by a crystal of methyl orange. A saturated solution of
hydrochloric acid in diethyl ether was added to the mixture, dropwise,
until a pink colour persisted. After 90 min, an additional solution of the
acid (3 mL) was added. After a total of 2.5 h, the reaction mixture was
1H, H-5a), 5.45–5.41 (m, 1H, H-5b), 4.28 (dd, 1H, J1a,2 =4.6, J1a,1b
=
11.6 Hz, H-1a), 4.19 (dd, 1H, J1b,2 =7.8 Hz, H-1b), 4.02 (ddd, 1H, J=4.5,
4.5, 7.8 Hz, H-2), 2.12 (s, 3H, C(O)CH3); 13C NMR (100 MHz, CDCl3): d
= 170.4, 165.1, 133.5, 131.2, 129.8, 129.4, 128.5, 120.8, 74.3, 62.9, 62.6,
20.7; ESI HRMS: m/z: calcd for C14H15N3O4Na: 312.09548; found:
312.09526 [M+Na+].
Chem. Eur. J. 2006, 12, 845 – 858
ꢀ 2006 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
www.chemeurj.org
853

D. R. Bundle et al.
filtered into a separatory funnel containing a 1:1 mixture of dichlorome-
thane and water (150 mL). The organic layer was removed, dried over
sodium sulfate and concentrated. Chromatography of the residue, by
using hexanes/ethyl acetate 5:1 as eluent afforded 5 (0.685 g, 1.03 mmol,
95%). [a]D = ꢀ34.30 (c = 0.52, CHCl3); 1HNMR (500 MHz, CDCl 3): d
vacuo. The residue was then taken up in dichloromethane (10.0 mL) and
then trichloroacetonitrile (1.0 mL, 10 mmol, 15.4 equiv) and 1,8-diazabi-
cyclo-[5.4.0]-undec-7-ene (0.050 mL, 0.33 mmol, 0.5 equiv) were added.
After 2 h, the reaction mixture was concentrated and the residue chroma-
tographed on silica gel with ethyl acetate/hexanes 7:1 containing triethyl-
amine (2%) as eluent to afford 4 (0.458 g, 0.46 mmol, ~70%) and some
minor contaminants (<10%). ESI HRMS: m/z: calcd for
C42H47N2O18SCl3Na: 1027.1505; found: 1027.1502 [M+Na+].
=
7.36–7.24 (m, 24H, Ar), 5.83 (m, 1H, J=8.0, 10.4, 17.4 Hz,
OCH2CHCH2), 5.42–5.39 (m, 1H, OCH2CHCH2), 5.35–5.31 (m, 1H,
OCH2CHCH2), 4.92 (d, 1H, J=11.6 Hz, OCH2Ph), 4.87 (d, 1H, J=
11.3 Hz, OCH2Ph), 4.74 (d, 1H, J=11.6 Hz, OCH2Ph), 4.71 (d, 1H, J=
11.2 Hz, OCH2Ph), 4.62 (d, 1H, J=11.8 Hz, OCH2Ph), 4.59 (d, 1H, J=
S-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-non-2-ulopyranosylonate)-(2!3)-(2-O-benzoyl-4,6-O-benzyli-
dene-3-thio-b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-benzyl-b-d-glucopyr-
anosyl-(1!1)-(2S,3R)-2-azido-3-O-benzyl-pent-4-ene-1,3-diol (3a): Ac-
12.0 Hz, OCH2Ph), 4.55 (d, 1H, J=12.0 Hz, OCH2Ph), 4.41 (d, 1H, J1,2
7.3 Hz, H-1), 4.38 (d, 1H, J=11.8 Hz, OCH2Ph), 4.02 (dd, 1H, J6a,5 =6.7,
6a,6b =10.2 Hz, H-6a), 3.96 (dd, 1H, J=5.3, 7.9 Hz, H-3sph), 3.78–3.60 (m,
=
J
ceptor
5 (0.110 g, 0.17 mmol) and imidate 4 (0.250 g, 0.25 mmol,
5H, H-4, H-5, H-6b, H-2sph, H-1asph), 3.49–3.40 (m, 3H, H-1bsph, H-2, H-
3); 13C NMR (125 MHz, CDCl3): d = 138.6, 138.4, 137.87 (2C), 134.3,
128.5, 128.42, 128.38, 128.37, 128.0, 127.9, 127.8, 127.7, 127.68, 127.67,
127.65, 127.62, 120.7, 103.5, 84.0, 81.7, 79.8, 75.3, 74.8, 74.1, 73.7, 71.6,
70.4, 70.2, 68.4, 64.2; ESI HRMS: m/z: calcd for C34H36O6Na: 688.2993;
found: 688.3001 [M+Na+]; elemental analysis calcd (%) for C34H36O6: C
70.36, H6.51, N 6.31; found: C 70.46, H6.68, N 6.06.
1.5 equiv) were combined and concentrated from toluene at room tem-
perature. The residue was dissolved in freshly distilled dichloromethane
(12 mL) containing powdered 4 Š molecular sieves, and the mixture was
stirred under argon for 45 min at room temperature before being cooled
to 08C. BF3·Et2O (0.030 mL, 0.24 mmol, 1.5 equiv) was added and the re-
action mixture was allowed to warm to room temperature. After 1.5 h, an
additional 0.5 equivalents of BF3·Et2O was added and stirring was contin-
ued for 2 h before the mixture was made basic with triethylamine, fil-
tered, and concentrated. Chromatography of the residue on silica gel
with a gradient of hexanes/ethyl acetate/acetone (8:6:1!8:6:2) afforded
3a (0.158 g, 0.10 mmol, 64%). [a]D = 57 (c = 0.40, CHCl3); 1HNMR
(600 MHz, CDCl3): d = 8.22–8.19 (m, 2H, Ar), 7.57–7.53 (m, 1H, Ar),
7.51–7.45 (m, 4H, Ar), 7.35–7.30 (m, 5H, Ar), 7.28–7.15 (m, 18H, Ar),
5.77 (ddd, 1H, J=8.0, 10.4, 17.4 Hz, H-4sph), 5.69 (ddd, 1H, J8’’,9a’’ =2.5,
2,3,6-Tri-O-benzyl-b-d-glucopyranosyl-(1!1)-(2S,3R)-2-(N-carboxyben-
zyl-6-aminohexanamido)-3-O-benzyl-pent-4-ene-1,3-diol (23): Azido al-
cohol 5 (0.201 g, 0.30 mmol) was taken up in pyridine/water 9:1 (10 mL)
and triphenylphosphine (160 mg, 0.61 mmol, 2 equiv) was added. The
mixture was stirred at 458C for 3 h and then cooled to room temperature.
6-(Benzyloxycarbonylamino)-hexanoic acid succinimidyl ester (0.383 g,
3.5 equiv) was added and the reaction mixture was stirred at 458C for a
further 2.5 h, then concentrated. The residue was chromatographed on
silica gel by using a linear gradient of dichloromethane (15:1 ! 12:1) as
eluent to yield 23 (0.211 g, 0.24 mmol, 79%). [a]D = ꢀ17.30 (c = 0.23,
CHCl3); 1HNMR (600 MHz, CDCl 3): d = 7.39–7.31 (m, 25H, Ar), 5.79
(d, 1H, NHC(O)CH2), 5.74 (ddd, 1H, J=7.9, 10.5, 17.5 Hz, H-4sph), 5.28
(brd, 1H, J=10.6 Hz, H-5asph), 5.20 (brd, 1H, J=17.1 Hz, H-5bsph), 5.09
(s, 2H, CH2Ph), 4.82 (d, 1H, J=11.9 Hz, CH2Ph), 4.80 (d, 1H, J=
11.9 Hz, CH2Ph), 4.79 (brs, 1H, CH2NH), 4.76 (s, 2H, CH2Ph), 4.61 (d,
1H, J=12.0 Hz, CH2Ph), 4.58 (d, 1H, J=11.9 Hz, CH2Ph), 4.54 (d, 1H,
J=11.9 Hz, CH2Ph), 4.36 (d, 1H, J1,2 =7.4 Hz, H-1), 4.35 (d, 1H, J=
11.9 Hz, CH2Ph), 4.25 (dd, 1H, J=4.5, 10.4 Hz, H-1asph), 4.27–4.18 (m,
1H, H-2sph), 3.88 (dd, 1H, J3,4 =7.3 Hz, H-3sph), 3.75 (dd, 1H, J6a,5 =4.2,
J
8’’,9b’’ =6.1, J8’’,7’’ =10.0 Hz, H-8’’), 5.39 (s, 1H, CHPh), 5.35–5.32 (m, 1H,
H-5asph), 5.29–5.23 (m, 3H, H-1’, H -’2, H-5bsph), 5.22 (dd, 1H, J7’’,6’’
2.3 Hz, H-7’’), 5.12 (d, 1H, J=11.7 Hz, CH2Ph), 5.00 (d, 1H, JNH,5’’
10.1 Hz, NH), 4.89 (d, 1H, J=11.7 Hz, CH2Ph), 4.79 (ddd, 1H, J4’’,5’’
=
=
=
11.4,
J4’’,3’’ax =12.8, J4’’,3’’eq =4.6 Hz, H-4’’), 4.76 (d, 1H, J=11.3 Hz,
CH2Ph), 4.63 (d, 1H, J=11.3 Hz, CH2Ph), 4.55 (d, 1H, J=11.9 Hz,
CH2Ph), 4.35 (dd, 1H, J9a’’,9b’’ =12.5 Hz, H-9a’’), 4.31 (d, 1H, J=11.9 Hz,
CH2Ph), 4.28 (d, 1H, J1,2 =7.8 Hz, H-1), 4.18 (d, 1H, CH2Ph), 4.15 (d,
1H, CH2Ph), 4.05 (dd, 1H, H-9b’’), 4.01 (dd, 1H, J6a’,6b’ =12.5, J6a’,5’
~0.5 Hz, H-6a’), 3.94–3.87 (m, 4H, H-4, H-1asph, H -5’’, H -3 ), 3.84 (dd,
sph
1H, J3’,2’ =10.7, J3’,4’ =3.2 Hz, H-3’), 3.78–3.69 (m, 3H, H-6b’, H -2 , H -
sph
6’’), 3.74 (s, 3H, CO2CH3), 3.66 (dd, 1H, J6a,6b =10.7 Hz, H-6a), 3.64 (dd,
1H, J3,2 =J3,4 =8.8 Hz, H-3), 3.61 (d, 1H, H-4’), 3.57–3.53 (m, 2H, H-5’,
H-1bsph), 3.43 (dd, 1H, J6b,5 =6.8 Hz, H-6b), 3.40–3.34 (m, 2H, H-2, H-5),
2.56 (dd, 1H, J3eq,3ax =12.8 Hz, H-3’’eq), 2.18 (s, 3H, C(O)CH3), 2.04 (s,
3H, C(O)CH3), 1.96 (s, 3H, C(O)CH3), 1.87 (dd, 1H, H-3’’ax), 1.84 (s,
3H, C(O)CH3), 1.73 (s, 3H, C(O)CH3); 13C NMR (125 MHz, CDCl3): d
= 170.8, 170.7, 170.3, 170.1, 170.0, 169.7, 165.6, 139.3, 138.7, 138.4, 137.9,
137.8, 134.1, 133.1, 130.27, 130.25, 128.7, 128.5, 128.3, 128.2, 128.1, 128.00,
127.94, 127.87, 127.6 (2C), 127.4, 127.18, 127.17, 127.16, 126.7, 126.2,
120.7, 103.1, 101.9, 101.2, 83.2, 82.0, 81.2, 79.9, 77.7, 75.7, 75.0, 74.68,
74.66, 73.6, 72.8, 70.4, 69.8, 69.6, 69.4, 68.7, 68.3, 68.1, 67.1, 67.0, 64.2,
62.6, 53.0, 49.2, 46.0, 37.5, 23.2, 21.5, 20.84, 20.77, 20.68; ESI HRMS:
m/z: calcd for C79H88N4O24S Na: 1531.5406; found: 1531.5407 [M+Na+];
elemental analysis calcd (%) for C79H88N4O24S: C 62.85, H5.88, N 3.71;
found: C 62.61, H6.02, N 3.55.
J
6a,6b =10.3 Hz, H-6a), 3.71 (dd, 1H, J6b,5 =5.1 Hz, H-6b), 3.67–3.62 (m,
2H, H-1bsph, H-4), 3.48 (dd, 1H, J3,2 =J3,4 =9.0 Hz, H-3), 3.42 (ddd, 1H,
5,4 =9.4 Hz, H-5), 3.38 (dd, 1H, H-2), 3.16–3.09 (m, 2H, CH2NH2), 1.91–
J
1.79 (m, 2H), 1.51–1.37 (m, 4H), 1.22–1.17 (m, 2H); 13C NMR
(125 MHz, CDCl3): d = 172.4, 156.4, 138.5, 138.23, 138.17, 137.7, 136.6,
135.7, 128.53, 128.50, 128.46, 128.45 (2C), 128.3, 128.10, 128.07, 127.96,
127.85, 127.81, 127.78, 127.76, 127.70, 127.6, 119.4, 104.0, 84.2, 81.7, 79.5,
75.3, 74.8, 74.0, 73.7, 71.9, 70.6, 70.2, 68.4, 66.6, 52.0, 40.9, 36.2, 29.6, 26.2,
25.0; ESI HRMS: m/z: calcd for C53H62N2O10Na: 909.4297; found:
909.4298 [M+Na+]; elemental analysis calcd (%) for C53H62N2O10: C
71.77, H7.04, N 3.16; found: C 71.73, H6.71, N 2.96.
S-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-non-2-ulopyranosylonate)-(2!3)-2-O-benzoyl-4,6-O-benzyli-
dene-3-thio-d-galactopyranose
(25):
Disaccharide
19
(0.950 g,
S-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-non-2-ulopyranosylonate)-(2!3)-(2-O-benzoyl-4,6-O-benzyli-
dene-3-thio-b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-benzyl-b-d-glucopyr-
anosyl-(1!1)-(2S,3R)-2-(N-carboxybenzyl-6-aminohexanamido)-3-O-
benzyl-pent-4-ene-1,3-diol (26): Azide 3a (0.150 g, 0.10 mmol) was dis-
solved in pyridine and water 9:1 (10 mL) then triphenylphosphine
(0.052 g, 0.20 mmol, 2 equiv) was added and the mixture was heated at
508C for 4 h before being allowed to cool to room temperature. 6-(Ben-
zyloxycarbonylamino)hexanoic acid succinimidyl ester (0.180 g,
0.50 mmol, 5 equiv) was added and the solution was heated at 508C over-
night and then concentrated. Chromatography of the residue on silica gel
using hexanes-ethyl acetate-acetone (4:3:2!3:3:2) as eluent afforded 26
1.05 mmol), palladium chloride (0.265 g, 1.49 mmol, 1.4 equiv), and
sodium acetate (0.360 g, 4.39 mmol, 4.2 equiv) were combined and added
to acetic acid/water 19:1 (30 mL). The reaction was immersed in an ultra-
sonic cleaning bath (Branson model B-32) and water temperature was
maintained below 308C by portionwise addition of ice. After 5 h, the
mixture was filtered through Celite, diluted with ethyl acetate (80 mL),
washed with an equal volume of water, dried over sodium sulfate, filtered
and concentrated. The residue was chromatographed by using hexanes/
ethyl acetate/acetone 3:3:2 as eluent to yield 25 (0.735 g, 0.85 mmol,
81%) as an inseparable mixture of a and b isomers. ESI HRMS: m/z:
calcd for C40H47NO18SNa: 884.2406; found: 884.2405 [M+Na+].
(0.155 g, 0.09 mmol, 90%). [a]D
(600 MHz, CDCl3): d = 8.21–8.18 (m, 2H, Ar), 7.58–7.54 (m, 1H, Ar),
7.51–7.45 (m, 4H, Ar), 7.36–7.12 (m, 28H, Ar), 5.80 (d, 1H, JNH,H2sph
8.9 Hz, NHsph), 5.70–5.62 (m, 2H, H-8’’, H -s4ph), 5.41 (s, 1H, CHPh),
= 53 (c =
0.41, CHCl3); 1HNMR
S-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-non-2-ulopyranosylonate)-(2!3)-2-O-benzoyl-4,6-O-benzyli-
dene-3-thio-d-galactopyranosyl trichloroacetimidate (4): Hemiacetal 25
(0.560 g, 0.65 mmol) was concentrated from toluene (2x) and dried in
=
854
www.chemeurj.org
ꢀ 2006 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 845 – 858

Glycoproteins
FULL PAPER
5.29–5.24 (m, 2H, H-1’, H -2’), 5.23 (dd, 1H, J7’’,8’’ =2.3, J7’’,6’’ =10.1 Hz, H-
7’’), 5.19 (dd, 1H, J=10.4, 1.5 Hz, H-5asph), 5.16 (d, 1H, J=11.4 Hz,
syl-(1!1)-(2S,3R)-2-(6-aminohexanamido)-pent-4-ene-1,3-diol (1): Tri-
saccharide 29 (0.115 g, 0.08 mmol) was dissolved in anhydrous tetrahy-
CH2Ph), 5.12–5.06 (m, 2H, CH2Ph, H-5bsph), 5.01 (d, 1H, JNH,H5’’
=
drofuran (8.0 mL) and added via cannula to
a solution of sodium
10.0 Hz, NHAc), 4.85 (d, 1H, J=11.4 Hz, CH2Ph), 4.82–4.75 (m, 2H, H-
4’’, NHZ), 4.68 (d, 1H, CH2Ph), 4.63 (d, 1H, CH2Ph), 4.54 (d, 1H, J=
11.9 Hz, CH2Ph), 4.34 (dd, 1H, J9a’’,8’’ =2.6, J9a’’,9b’’ =12.4 Hz, H-9a’’), 4.30
(0.090 g) in liquid ammonia at ꢀ788C. Sodium metal (0.060 g) was
added, and the dark blue solution was stirred for 1 h, then quenched with
methanol (15 mL) and acetic acid (0.50 mL). The mixture was allowed to
warm to room temperature and was stirred overnight to allow ammonia
to evaporate. The methanolic solution was combined with Iatrobeads
(~1 g) and concentrated. The solid material was applied to the top of a
column of Iatrobeads, and eluted by application of a linear gradient of
methanol/isopropanol/water 1:1:0!41:41:18. HPLC purification (Hamil-
ton PRP column; water/methanol gradient) of the material obtained
(d, 1H, J=11.9 Hz, CH2Ph), 4.24 (brs, 2H, CH2Ph), 4.21 (d, 1H, J1,2
=
7.9 Hz, H-1), 4.17 (dd, 1H, J1a,2 =4.6, J1a,1b =10.9 Hz, H-1asph), 4.11–4.06
(m, 2H, H-2sph, H-6a’), 4.05 (dd, 1H, J9’’b,8’’ =6.6 Hz, H-9b’’), 4.00 (dd, 1H,
J
4,3 =8.8, J4,5 =9.6 Hz, H-4), 3.92–3.84 (m, 2H, H-5’’, H -3’), 3.83–3.78 (m,
2H, H-6b’, H -3 ), 3.75–3.72 (m, 1H, H-6’’), 3.74 (s, 3H, CO2CH3), 3.67
sph
(dd, 1H, J6a,5 =1.6, J6a,6b =10.9 Hz, H-6a), 3.64 (brd, 1H, H-4’), 3.63 (dd,
1H, J3,2 =9.0 Hz, H-3), 3.59 (brs, 1H, H-5’), 3.56 (dd, 1H, J1b,2 =6.3 Hz,
H-1bsph), 3.52 (dd, 1H, J6b,5 =6.3 Hz, H-6b), 3.34 (dd, 1H, H-2), 3.30 (m,
yielded 1 (0.0571 g, 0.07 mmol, 83%). [a]D
= 33 (c = 1.0, H2O);
1HNMR (600 MHz, D 2O): d = 5.85 (ddd, 1H, J4,3 =7.0, J4,5a =17.1, J4,5b
=
1H, H-5), 3.10 (m, 2H, CH2NHZ), 2.57 (dd, 1H, J3’’eq,4’’ =4.6, J3’’eq,3’’ax
12.8 Hz, H-3’’eq), 2.16, 2.05, 1.97, (3”s, 3”3H, C(O)CH3), 1.88 (dd, 1H,
’’ax,4’’ =12.0 Hz, H-3’’ax), 1.85–1.68 (m, 2H, CH2), 1.84, 1.73, (2”s, 3”3H,
=
10.4 Hz, H-4sph), 5.32 (brd, 1H, H-5asph), 5.26 (brd, 1H, H-5bsph), 4.53 (d,
1H, J1’,2’ =7.2 Hz, H-1’), 4.47 (d, 1H, J1,2 =8.0 Hz, H-1), 4.24–4.20 (m, 1H,
H-3sph), 4.11 (ddd, 1H, J2,1a =6.7, J2,1b =3.6, J2,3 =3.6 Hz, H-2sph), 4.04 (dd,
1H, J1a,1b =10.5 Hz, H-1asph), 4.00 (dd, 1H, J6a,6b =12.3, J6a,5 =2.2 Hz, H-
6a), 3.92 (dd, 1H, J=2.5, 6.2, 9.2 Hz, H-8’’), 3.88–3.82 (m, 4H, H-4’, H -
9a’’, H -5’’, H-6b), 3.79 (dd, 1H, H-1b), 3.78–3.75 (m, 1H, H-5’), 3.74–3.70
(m, 2H, H-6a’, H-6b’), 3.70–3.63 (m, 3H, H-4, H-4’’, H -7’’), 3.64 (dd, 1H,
J ~J ~8.7 Hz, H-3), 3.60–3.56 (m, 3H, H-6’’, H-5, H-9b’’), 3.39 (dd, 1H,
J
C(O)CH3), 1.42–1.34 (m, 4H, 2”CH2), 1.16–1.08 (m, 2H, CH2);
13C NMR (125 MHz, CDCl3): d = 172.3, 170.8, 170.7, 170.2, 170.1, 170.0,
169.7, 165.6, 156.3, 139.1, 138.5, 138.3, 138.2, 137.7, 136.7, 135.7, 133.1,
130.23, 130.21, 128.7, 128.489, 128.486, 128.478, 128.31, 128.29, 128.14,
128.13, 128.02, 128.00, 127.8, 127.7, 127.57, 127.53, 127.50, 127.3, 127.1,
126.9, 126.2, 119.3, 103.8, 101.5, 101.2, 83.4, 81.9, 81.2, 79.4, 75.6, 75.2,
74.8, 74.5, 73.6, 72.7, 70.5, 69.8, 69.5, 69.4, 68.8, 68.5, 68.1, 67.1, 66.9, 66.5,
62.5, 53.0, 52.2, 49.2, 45.9, 40.8, 37.5, 36.0, 29.7, 29.6, 26.2, 24.9, 23.2, 21.5,
20.9, 20.8, 20.7; ESI HRMS: m/z: calcd for C93H107N3O29SNa: m/z:
1752.6714; found: 1752.6710 [M+Na+]; elemental analysis calcd (%) for
C93H107N3O29S: C 64.53, H6.23, N 2.43; found: C 64.54, H6.39, N 2.25.
J
2’,3’ =11.4 Hz, H-2’), 3.37 (dd, 1H, J3’,4’ =2.5 Hz, H-3’), 3.32 (dd, 1H, H-
2), 3.00–2.96 (m, 2H, CH2NH2), 2.80 (dd, 1H, J3eq,3ax =12.7, J3’’eq,4’’
=
4.8 Hz, H-3eq’’), 2.28 (dd, 2H, NC(O)CH2), 2.02 (s, 3H, NHC(O)CH3),
1.81 (dd, 1H, J3ax,4’’ =11.3 Hz, H-3’’ax), 1.70–1.58 (m, 4H, 2”CH2), 1.41–
1.34 (m, 2H, CH2); 13C NMR (125 MHz, D2O): d = 177.6, 175.9, 175.4,
137.2, 118.9, 105.0, 103.2, 85.0, 79.1, 78.7, 75.9, 75.7, 75.1, 73.8, 72.82,
72.77, 69.7, 69.5, 69.35 (2C), 69.0, 63.5, 62.1, 60.9, 53.9, 52.5, 51.5, 41.5,
40.2, 36.4, 27.4, 26.0, 25.6, 22.9; ESI HRMS: m/z: calcd for
C34H59N3O20SNa: 884.3316; found: 884.3310 [M+Na+].
S-(5-Acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulopyranosy-
lonic acid)-(2!3)-(4,6-O-benzylidene-3-thio-b-d-galactopyranosyl)-(1!
4)-2,3,6-tri-O-benzyl-b-d-glucopyranosyl-(1!1)-(2S,3R)-2-(N-carboxy-
benzyl-6-aminohexanamido)-3-O-benzylpent-4-ene-1,3-diol (29): Trisac-
charide 26 (0.140 g, 0.08 mmol) was combined with methanol (7.0 mL)
and a small piece of sodium was added. After evolution of gas had
ceased, the mixture was heated at 558C for 7 h and then cooled to room
temperature. Water (0.350 mL) was added and the solution was heated at
408C overnight. After acidification with acetic acid, the reaction mixture
was concentrated and the residue chromatographed on silica gel using a
gradient of methanol and dichloromethane (0:100!25:75). The residue
was dissolved in 50% methanol and loaded on a SepPak cartridge (5 g),
then eluted with 80–90% methanol to afford 29 (0.108 g, 0.08 mmol,
94%). [a]D = 10 (c = 0.34, CHCl3); 1HNMR (600 MHz, CDCl 3): d =
7.43–7.38 (m, 4H, Ar), 7.36–7.34 (m, 2H, Ar), 7.33–7.28 (m, 10H, Ar),
7.28–7.20 (m, 11H, Ar), 7.10–7.04 (m, 3H, Ar), 5.74 (ddd, 1H, H-4sph),
5.50 (s, 1H, CHPh), 5.30–5.24 (m, 2H, H-5asph, H-5bsph), 5.16 (d, 1H, J=
10.7 Hz, CH2Ph), 5.03 (brs, 2H, CH2Ph), 4.78 (d, 1H, J=11.4 Hz,
CH2Ph), 4.63 (brd, 3H, 3”CH2Ph), 4.58–4.54 (m, 3H, CH2Ph, CH2Ph,
H-1’), 4.43 (d, 1H, J1,2 =7.8 Hz, H-1), 4.38 (d, 1H, J1,2 =11.8 Hz, CH2Ph),
S-(5-Acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulopyranosy-
lonic acid)-(2!3)-[2-acetamido-2-deoxy-b-d-galactopyranosyl-(1!4)]-(3-
thio-b-d-galactopyranosyl)-(1!4)-b-d-glucopyranosyl-(1!1)-(2S,3R)-2-
(6-aminohexanamido)-pent-4-ene-1,3-diol (2): UDP-N-acetylglucosamine
disodium salt (0.022 g, 0.02 mmol, 1.5 equiv) was added to a falcon tube
containing 1 (15.2 mg, 18 mmol, 1.0 equiv). Freshly prepared b(1,4) N-ace-
tylgalactosaminyl transferase (2.0 mL) in 50 mm HEPES, pH 7.5 contain-
ing 10% glycerol (v/v) and UDP N-acetylglucosamine 4-epimerase
(2.0 mL) in 0.2m NaCl, 1.0 mm EDTA, 20 mm HEPES, 5 mm b-mercap-
toethanol, pH7.0 were added to the mixture, followed by 4.0 mL of 0.5m
manganese(ii) chloride and 12 mL (12 units) of alkaline phosphatase. The
mixture was tumbled a total of 24 h and then centrifuged for 30 min at
14000 rpm. The supernatant was applied to a SepPak cartridge (5 g)
which had been conditioned with methanol (10 mL) followed by 1% am-
monia solution (20 mL). After application of the sample, the cartridge
was washed with 1% ammonia (25 mL) and eluted with 10% methanol
containing 1% ammonia. HPLC of the residue thus obtained (Hamilton
PRP-1 semi-preparative column; water/methanol gradient elution) af-
4.17 (ddd, 1H, J1a,2 =6.4, J=6.6, 3.8 Hz, H-2sph), 4.12 (dd, 1H, J1a,1b
10.1 Hz, H-1asph), 4.09 (dd, 1H, J4,3 =3.5, J4,5 =0.6 Hz, H-4’), 4.06 (dd, 1H,
6a’,5’ =1.4, J6a’,6b’ =12.4 Hz, H-6a’), 4.04–3.99 (m, 3H, H-8’’, H-4, H-6a),
3.95 (dd, 1H, J6a,5 =1.6, J6a,6b =11.0 Hz, H-6b), 3.93–3.87 (m, 2H, H-3sph
=
forded the desired GM2 analogue 2 (18.3 mg, 0.017 mmol, 94%). [a]D
22 (c = 0.20, H2O); HNMR (600 MHz, D 2O): d = 5.85 (ddd, 1H, J4,3
=
J
1
=
,
7.0, J4,5a =17.3, J4,5b =10.5 Hz, H-4sph), 5.35–5.30 (m, 1H, H-5asph), 5.28–
5.25 (m, 1H, H-5bsph), 4.75 (obscured, 1H, H-1’’’), 4.53 (d, 1H, J1’,2’
7.6 Hz, H-1’), 4.47 (d, 1H, J1,2 =7.9 Hz, H-1), 4.24–4.20 (m, 1H, H-3sph),
4.11 (ddd, 1H, J2,1a =6.6, J2,1b =3.6, J2,3 =3.6 Hz, H-2sph), 4.06 (dd, 1H,
H-6b’), 3.82 (dd, 1H, J9a’’,8’’ =2.8, J9a’’,9b’’ =11.4 Hz, H-9a’’), 3.77–3.73 (m,
3H, H-1bsph, H -5’’, H -4’’), 3.69 (dd, 1H, J9b’’,8’’ =4.4 Hz, H-9b’’), 3.65 (dd,
1H, J3’,4’ =3.4, J3’,2’ =11.0 Hz, H-3’), 3.62 (dd, 1H, J3,2 =J3,4 =9.1 Hz, H-3),
3.59 (dd, 1H, J=1.8, 9.2 Hz, H-7’’), 3.56–3.52 (m, 2H, H-6’’, H-5), 3.47
(dd, 1H, J2’,1’ =7.6, J2’,3’ =11.0 Hz, H-2’), 3.29 (m, 1H, H-2), 3.17 (brs, 1H,
H-5’), 3.01 (m, 2H, CH2NHZ), 2.94 (m, 1H, H-3’’eq), 2.00 (s, 3H,
NC(O)CH3), 2.00 (m, 2H, CH2), 1.70 (m, 1H, H-3’’ax), 1.47 (m, 2H,
CH2), 1.39 (m, 2H, CH2), 1.21 (m, 2H, CH2); 13C NMR (125 MHz,
CDCl3): d = 178.6, 175.7, 175.4, 175.0, 140.2, 140.1, 140.0, 139.9, 139.8,
139.6, 136.9, 129.8, 129.6, 129.45, 129.39, 129.34, 129.28, 129.09, 129.03,
128.99, 128.98, 128.93, 128.85, 128.81, 128.7, 128.6, 128.5, 128.3, 127.4,
120.1, 105.3, 104.7, 102.2, 85.8, 84.6, 83.0, 80.8, 78.1, 77.6, 76.9, 76.0, 75.9,
74.2, 72.5, 71.8, 70.8, 70.4, 70.0, 69.81, 69.78, 69.6, 69.1, 67.3, 64.4, 54.0,
53.7, 50.9, 49.3, 42.9, 41.7, 37.1, 30.6, 27.3, 26.6, 22.6; ESI HRMS: m/z:
calcd for C77H93N3O22SNa: 1466.5868; found: 1466.5869 [M+Na+].
=
J
1a,1b =10.6 Hz, H-1asph), 4.01–3.98 (m, 2H, H-4’, H-6a), 3.92–3.55 (m,
21H, H-3, H-4, H-5, H-6b, H-5’, H-6a’, H-6b’, H -4’’, H -5’’, H -6’’, H -7’’, H -
8’’, H-9a’’, H-9b’’, H -2’’’, H -3’’’, H -4’’’, H -5’’’, H-6a’’’, H-6b’’’, H-1bsph), 3.42
(dd, 1H, J3’,2’ =11.5, J3’,4’ =2.4 Hz, H-3’), 3.32 (dd, 1H, J2,3 =9.1 Hz, H-2),
3.21 (dd, 1H, H-2’), 3.00–2.97 (m, 2H, CH2NH2), 2.80 (dd, 1H, J3eq,3ax
=
12.6, J3’’eq,4’’ =4.8 Hz, H-3’’eq), 2.28 (dd, 2H, NC(O)CH2), 2.09 (s, 3H,
NHC(O)CH3), 2.03 (s, 3H, NHC(O)CH3), 1.84 (dd, 1H, J3ax,4’’ =11.6 Hz,
H-3’’ax), 1.70–1.59 (m, 4H, 2”CH2), 1.40–1.35 (m, 2H, CH2); 13C NMR
(125 MHz, CDCl3): d = 177.6, 175.8, 175.5, 175.3, 137.2, 118.9, 105.0,
104.1, 103.2, 84.6, 79.2, 77.6, 77.0, 75.72, 75.69, 75.5, 75.0, 73.7, 73.0, 72.7,
71.5, 69.44, 69.37, 69.2, 69.1, 68.6, 63.5, 61.9, 61.6, 60.9, 53.8, 53.4, 52.5,
50.5, 41.5, 40.2, 36.4, 27.4, 25.9, 25.6, 23.5, 22.9; ESI HRMS: m/z: calcd
for C42H72N4O25SNa: 1065.428417; found: 1065.428462 [M+Na+].
S-(5-Acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulopyranosy-
lonic acid)-(2!3)-(3-thio-b-d-galactopyranosyl)-(1!4)-b-d-glucopyrano-
Chem. Eur. J. 2006, 12, 845 – 858
ꢀ 2006 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
www.chemeurj.org
855

D. R. Bundle et al.
5-Acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulopyranosylonic
acid-(2!8)-[S-(5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-
ulopyranosylonic acid)]-(2!3)-(3-thio-b-d-galactopyranosyl)-(1!4)-b-d-
glucopyranosyl-(1!1)-(2S,3R)-2-(6-aminohexanamido) -pent-4-ene-1,3-
diol (30): GM3 analogue 1 (2.09 mg, 2.4 mmol) and crude CMP-Neu5Ac
(21 mg, 7.3 mmol, 3.0 equiv) were combined as solids. To this mixture was
added successively freshly prepared a(2–3/8) sialyltransferase in 20 mm
Tris buffer, pH8.3 (2.0 mL, 1.33 UmL ꢀ1), MnCl2 (20 mL of a 0.5m solu-
tion), and alkaline phosphatase (2 mL; 2U). The mixture was tumbled
overnight at room temperature before centrifugation (30 min at
14000 rpm). The supernatant was loaded directly onto a Biogel P4
column (2”50 cm) and eluted with 5% methanol at a flow rate of 0.4
mLminꢀ1. Fractions containing the tetrasaccharide were purified further
by HPLC (Hamilton PRP-1 semipreparative column, water-methanol
gradient) to afford 30 (1.1 mg, 1.0 mmol, 39%): 1HNMR (500 MHz,
D2O): d = [note: a(2!3) and a(2!8) linked Neu5Ac residues could
not be unambiguously identified, but individual spin systems are denoted
by H# and H*) 5.86 (ddd, 1H, J=6.9, 10.5, 17.2 Hz, H-4sph), 5.33 (ddd,
1H, J=1.2, 1.2, 17.2 Hz, H-5asph), 5.28 (ddd, 1H, H-5bsph), 4.54 (d, 1H,
corresponding to the Neu5Ac residue were broadened significantly. Se-
lected 1HNMR resonances are reported here (600 MHz, CD 3OD): d =
8.29 (m, 2H, Ar), 8.06 (brd, 1H, NH), 7.94 (brt, 1H, NH), 7.89 (d, 1H,
NH), 7.37 (m, 2H, Ar), 5.86 (ddd, 1H, J=6.6, 10.6, 17.3 Hz, H-4sph), 4.63
(brs, 1H, H-1’’), 4.43 (d, 1H, J1’,2’ =7.2 Hz, H-1’), 4.29 (d, 1H, J1,2
=
7.7 Hz, H-1), 1.98 (brs, 3H, NHC(O)CH3); ESI HRMS: m/z: calcd for
C46H70N4O25SNa: 1336.4741; found: 1336.4742 [M+Na+].
b-d-Glucopyranosyl-(1!1)-(2S,3R)-2-(6-aminohexanamido)-pent-4-ene-
1,3-diol (43): Glucoside 24 (0.068 g, 0.08 mmol) in tetrahydrofuran
(6 mL) was added to a solution of sodium (0.090 g) in ammonia (50 mL)
at ꢀ788C. After 1 h, methanol (5 mL) and acetic acid (0.5 mL) were
added, and ammonia was removed by evaporation with a stream of
argon. The residue was concentrated and dissolved in water, then applied
to a SepPak cartridge (5 g) that had been preconditioned with methanol
followed by 1% ammonia. After washing with 1% ammonia, the product
was eluted with 20% methanol containing the same and concentrated.
The residue was purified by HPLC (Hamilton PRP-1 column, water-
methanol gradient) to afford 43 (0.026 g, 0.07 mmol, 85%). [a]D = ꢀ1 (c
= 0.44, CHCl3); 1HNMR (600 MHz, CD 3OD): d = 5.87 (ddd, 1H, J=
6.6, 10.4, 17.1 Hz, H-4sph), 5.27 (ddd, 1H, J=1.5, 1.6, 17.1 Hz, H-5asph),
5.14 (ddd, 1H, J=1.2, 1.6, 10.4 Hz, H-5bsph), 4.26 (d, 1H, J1,2 =7.8 Hz, H-
1), 4.16–4.13 (m, 1H, H-3sph), 4.10 (dd, 1H, J1a,1b =10.2, J1a,2 =5.7 Hz, H-
J
1’,2’ =7.0 Hz, H-1’), 4.48 (d, 1H, J1,2 =8.0 Hz, H-1), 4.25 (dd, 1H, J9a*8* =
4.1, J9a*,9b* =12.4 Hz, H-9a*), 4.24–4.21 (m, 1H, H-3sph), 4.17–4.10 (m, 2H,
H-8*, H-2sph), 4.07–4.01 (m, 2H, H-1asph, H-6a), 3.95–3.89 (m, 1H, H-8#),
3.89–3.78 (m, 8H, H-9a#, H -4’, H-6b, H-5*, H-7*, H-1bsph, H -5, H -5’),
#
1asph), 4.04 (ddd, 1H, J2,1b =3.4, J2,3 =7.4 Hz, H-2sph), 3.87 (dd, 1H, J6a,5
=
3.74–3.53 (m, 12H, H-6a’, H-6b’, H-4, H-4*, H-9b*, H-3, H-9b#, H-5, H-
1.7, J6a,6b =11.8 Hz, H-6a), 3.68–3.64 (m, 1H, H-6b), 3.65 (dd, 1H, H-
1bsph), 3.37–3.30 (m, 1H, H-3), 3.28–3.25 (m, 2H, H-4, H-5), 3.18 (dd,
1H, H-2), 2.89 (m, 2H, CH2), 2.23 (m, 2H, CH2), 1.70–1.60 (m, 4H, CH2,
CH2), 1.40 (m, 2H, CH2); 13C NMR (125 MHz, CD3OD): d = 175.8,
139.5, 117.0, 104.6, 78.01, 77.95, 75.2, 73.3, 71.6, 69.6, 62.6, 54.7, 40.5, 36.7,
28.3, 26.9, 26.2; ESI HRMS: m/z: calcd for C17H33N2O8: 393.22314;
found: 393.22307 [M+Na+].
6#, H-6*, H-7#, H -4), 3.42 (dd, 1H, J3’,4’ =2.3, J3’,2’ =11.5 Hz, H-3’), 3.38
#
(dd, 1H, H-2’), 3.33 (dd, 1H, J2,3 =8.9 Hz, H-2), 3.02–2.97 (m, 2H,
CH2NH2), 2.78 (dd, 1H, J3eq*,4* =4.7, J3eq*,3ax* =12.3 Hz, H-3eq*), 2.75 (dd,
1H, J3eq#,4# =4.3, J3eq#,3ax# =12.3 Hz, H-3eq#), 2.32–2.27 (m, 2H, C(O)CH2),
2.07 (s, 3H, NHC(O)CH3), 2.04 (s, 3H, NHC(O)CH3), 1.77 (dd, 1H,
J
3ax#,4# =12.0 Hz, H-3ax#), 1.73 77 (dd, 1H, J3ax*,4* =12.1 Hz, H-3ax*), 1.70–
1.59 (m, 4H), 1.43–1.36 (m, 2H); ESI HRMS: m/z: calcd for
C45H75N4O28SNa3: 1220.3971; found: 610.1989 [M+2Na]2+
.
6-Azidohexyl (5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulo-
pyranosylonic acid)-(2!3)-b-d-galactopyranoside (44): 6-Azidohexyl b-
d-galactopyranoside[79] (0.017 g, 0.06 mmol) was combined with crude
CMP-Neu5Ac (crude mass 180 mg, estimate 0.09 mmol, 1.5 equiv). a(2,3)
Sialyl transferase (CST-04 in 50 mm HEPES, pH 7.5, 10% glycerol (v/v);
0.14 UmLꢀ1; 4.75 mL), MnCl2 and MgCl2 (100 mL of a 0.5m solution;
final conc. 10 mm), dithiothreitol (38 mL, 0.4m; final conc. 3 mm) and al-
kaline phosphatase (12 mL) were added and the mixture was tumbled
overnight. After centrifugation (30 min at 14000 rpm) the supernatant
was loaded onto a SepPak cartridge (5 g) and eluted with a gradient of
water and methanol to afford 44 (0.0311 g, 0.05 mmol, 94%). [a]D = ꢀ3
p-Nitrophenyl ester of 1 (34): Amine 1 (10.5 mg, 0.01 mmol) was dis-
solved in N,N-dimethylformamide (1.5 mL) and triethylamine (3.7 mL;
0.02 mmol, 2.2 equiv). The p-nitrophenyl diester 33 (0.047 g, 0.12 mmol,
10 equiv) was added, and reaction progress was monitored by TLC (di-
chloromethane/methanol/water/acetic acid 4:5:1:0.5). After 90 min, sever-
al drops of acetic acid were added, and the mixture was concentrated at
room temperature from toluene (3”) on a rotary evaporator. The residue
was suspended in 2% acetic acid solution (3 mL) and filtered through
cotton. The filtrate was applied to a SepPak cartridge (0.4 g), washed
with 2% acetic acid (10.0 mL), and eluted with 2% acetic acid in metha-
nol. Concentration of the eluent yielded a clear solid which was purified
by HPLC (Hamilton PRP-1 semi-preparative column; water-methanol
gradient elution) to yield the activated oligosaccharide 34 (0.011 g,
10 mmol, 81%). 1HNMR spectra were acquired in CD 3OD. Although
visible, resonances corresponding to the Neu5Ac residue were broadened
significantly. Selected 1HNMR resonances are reported here (600 MHz,
CD3OD): d = 8.29 (m, 2H, Ar), 8.14 (brs, 1H, NH), 7.94 (brs, 1H, NH),
7.89 (brs, 1H, NH), 7.37 (m, 2H, Ar), 5.86 (ddd, 1H, J=6.7, 10.5,
(c = 0.52, H2O); 1HNMR (600 MHz, D 2O): d = 4.46 (d, 1H, J1,2
8.0 Hz, H-1), 4.08 (dd, 1H, J3,2 =9.8, J3,4 =3.3 Hz, H-3), 3.95 (d, 1H, J3,4
3.3 Hz, H-4), 3.94–3.85 (m, 3H, OCH2, H -8’, H-9a’), 3.84 (dd, 1H, J5’,6’
=
=
=
J
5’,6’ =10.1 Hz, H-5’), 3.77–3.72 (m, 2H, H-6a, H-6b), 3.72–3.61 (m, 5H,
H-4’, H-5, OCH2, H -6’, H-9b’), 3.59 (dd, 1H, J=1.8, 9.1 Hz, H-7’), 3.53
(dd, 1H, H-2), 3.32 (dd, 2H, CH2N3), 2.76 (dd, 1H, J3eq’,4’ =4.7, J3eq’,3ax’
=
12.4 Hz, H-3’eq), 2.03 (s, 3H, C(O)CH3), 1.80 (dd, 1H, J3ax’,4’ =12.2 Hz, H-
3’ax), 1.67–1.58 (m, 4H, CH2CH2N3, OCH2CH2), 1.43–1.37 (m, 4H,
CH2CH2); 13C NMR (125 MHz, D2O): d = 175.9 (2C), 103.4, 100.7, 76.8,
75.7, 73.7, 72.6, 71.2, 70.0, 69.2, 69.0, 68.4, 63.5, 61.8, 52.6, 52.0, 40.6, 29.5,
28.8, 26.6, 25.5, 22.9; ESI HRMS: m/z: calcd for C23H39N4O14: 641.22527;
found: 641.22510 [M+Na+].
17.1 Hz, H-4sph), 4.45 (d, 1H, J1’,2’ =7.1 Hz, H-1’), 4.29 (dd, 1H, J1,2
=
7.8 Hz, H-1), 1.99 (brs, 3H, NHC(O)CH3); ESI HRMS: m/z: calcd for
C46H70N4O25SNa: 1133.3948; found: 1133.3948 [M+Na+]. [Note: addition
of an amine (for example diallylamine) to the NMR tube resulted in res-
1
olution of the HNMR signals corresponding to the Neu5Ac residue].
6-Aminohexyl
(5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-
ulopyranosylonic acid)-(2!3)-b-d-galactopyranoside (45): Azide 44
(0.024 g, 0.04 mmol) was combined with triphenylphosphine (0.053 g,
0.2 mmol, 5.0 equiv) and a mixture of pyridine and water 9:1 (5 mL) was
added. The suspension was stirred at 658C for 36 h, then diluted with
water and filtered through a plug of sand and cotton. The filtrate was
concentrated and then passed through a tC18 SepPak cartridge (0.4 g)
preconditioned with ~1% NH4OHbefore final purification by HPLC
(Hamilton PRP-1 column, methanol/water gradient) to give 45 (0.0186 g,
0.03 mmol, 78%). [a]D = ꢀ2.6 (c = 0.40, H2O); 1HNMR (600 MHz,
p-Nitrophenyl ester of 2 (35): Amine 2 (4.5 mg, 4.2 mmol) was dissolved
in N,N-dimethylformamide (1.5 mL) and triethylamine (2.0 mL, 9.2 mmol,
2.2 equiv). The p-nitrophenyl diester 33 (0.017 g, 42 mmol, 10 equiv) was
added, and reaction progress was monitored by TLC (dichloromethane/
methanol/water/acetic acid 4:5:1:0.5). After 2 h, several drops of acetic
acid were added, and the mixture was concentrated at room temperature
from toluene (3”) on a rotary evaporator. The residue was suspended in
2% acetic acid solution (4 mL) and filtered through cotton. The filtrate
was applied to a SepPak cartridge (0.4 g), washed with 2% acetic acid
(5 mL), and eluted with 2% acetic acid in methanol. Concentration of
the eluent yielded a clear solid that was purified by HPLC (Hamilton
PRP-1 semi-preparative column; water-methanol gradient elution) to
yield the activated thiotetrasaccharide 35 (0.0031 g, 2.3 mmol, 56%):
1HNMR spectra were acquired in CD 3OD. Although visible, resonances
D2O): d = 4.46 (d, 1H, J1,2 =8.0 Hz, H-1), 4.08 (dd, 1H, J3,2 =9.7, J3,4
3.2 Hz, H-3), 3.95 (d, 1H, J3,4 =3.2 Hz, H-4), 3.93 (ddd, 1H, J=J=3.1,
9.8 Hz, OCH2), 3.90–3.86 (m, 2H, H-8’, H-9a’), 3.84 (dd, 1H, J5’,6’ =J5’,6’
=
=
10.2 Hz, H-5’), 3.77–3.62 (m, 7H, H-6a, H-6b, H-4’, H-5, OCH2, H -6’, H -
9b’), 3.60 (dd, 1H, J=1.7, 9.1 Hz, H-7’), 3.54 (dd, 1H, H-2), 3.00 (dd, 2H,
856
www.chemeurj.org
ꢀ 2006 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 845 – 858

Glycoproteins
FULL PAPER
CH2NH2), 2.76 (dd, 1H, J3eq’,4’ =4.8, J3eq’,3ax’ =12.5 Hz, H-3’eq), 2.03 (s, 3H,
solid which was loaded onto a SepPak cartridge (5 g) in a minimum
volume of water. Elution with water afforded 49 (0.049 g, 0.09 mmol,
81%). [a]D = 50 (c = 0.68, MeOH); 1HNMR (500 MHz, D 2O): d =
6.04–5.96 (m, 1H, H-b), 5.42–5.37 (m, 1H, H-c), 5.30–5.27 (m, 1H, H-d),
4.53 (d, 1H, J1,2 =7.3 Hz, H-1), 4.43–4.39 (m, 1H, H-a), 4.26–4.22 (m, 1H,
H-a’), 3.93–3.82 (m, 4H, H-8’, H-9a’, H-4, H-5’), 3.75–3.71 (m, 3H, H-5,
H-6a, H-6b), 3.70–3.62 (m, 2H, H-4’, H-9b’), 3.60–3.56 (m, 2H, H-6’, H -
7’), 3.39 (dd, 1H, J2,3 =11.4 Hz, H-2), 3.35 (dd, 1H, J3,4 =2.7 Hz, H-3),
C(O)CH3), 1.80 (dd, 1H,
J3ax,4’ =12.0 Hz, H-3’ax), 1.70–1.61 (m, 4H,
CH2CH2NH2, OCH2CH2), 1.45–1.39 (m, 4H, CH2CH2); 13C NMR
(125 MHz, D2O): d = 175.9, 174.7, 103.4, 100.7, 76.8, 75.8, 73.8, 72.7,
71.1, 70.0, 69.2, 69.0, 68.4, 63.5, 61.9, 52.6, 52.0, 40.6, 40.3, 29.3, 27.6, 26.1,
25.4, 22.9; ESI HRMS: m/z: calcd for C23H42N2O14: 593.2528; found:
593.2537 [M+Na+].
Allyl (5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulopyrano-
sylonic acid)-(2!3)-b-d-galactopyranoside (46): Allyl b-d-galactopyrano-
side (0.020 g, 0.09 mmol) was combined with crude CMP-Neu5Ac (crude
mass 290 mg, estimate 0.10 mmol, 1.1 equiv). a(2,3) Sialyltransferase
2.81 (dd, 1H,
J
3eq,4 =4.8,
J3eq,3ax =12.7 Hz, H-3’eq), 2.03 (s, 3H,
NHC(O)CH3), 1.82 (dd, 1H, J3ax,4 =11.7 Hz, H-3’ax); 13C NMR (125 MHz,
D2O): d = 175.9, 175.4, 134.3, 119.6, 104.0, 85.1, 78.3, 75.8, 72.8, 71.4,
69.7, 69.4, 69.1, 69.0, 63.4, 62.1, 52.5, 51.8, 41.6, 22.9; ESI HRMS: m/z:
calcd for C20H32NO13S: 526.1589; found: 526.1585 [M+Na+].
(50 mm HEPES, pH 7.5, 10% glycerol (v/v); 0.4 UmLꢀ1
;
1.353 mL),
buffer (50 mm HEPES, pH 7.5, 10% glycerol (v/v); 1.5 mL) MnCl2 and
MgCl2 (60 mL of 0.5m solution; final conc. 10 mm), dithiothreitol
Preparation of TT and BSA conjugates
a
(38 mL, 0.4m; final conc. 3 mm) and alkaline phosphatase (3 mL; 3U)
were added and the mixture was tumbled overnight. After centrifugation
(30 min at 14000 rpm) the supernatant was loaded onto a Biogel P2
column (2.5”30 cm) and eluted with water. Final purification by reverse
phase HPLC (Beckman C18 column, water-methanol gradient) afforded
Procedure for preparation of TT conjugates 36 and 37 with adipate
linker: Purified tetanus toxoid (~6.6 mgmLꢀ1) in PBS, pH7.2, was added
to the activated oligosaccharide (34 or 35) (~30 mol equiv). The mixture
was tumbled gently overnight before being dialyzed against PBS (5”2L)
at 48C.
46 (0.0311 g, 0.06 mmol, 67%). [a]D
= 0 (c =
0.40, H2O); 1HNMR
Procedure for preparation of BSA conjugates 38 and 39 with adipate
linker: Bovine serum albumin (Sigma) (~3.5 mgmLꢀ1) in PBS, pH7.2,
was added to the adipate activated oligosaccharide (34 or 35) (~30 mol
equiv). The mixture was tumbled gently overnight, dialyzed against
milliQ water (5”2 L) at 48C, and then lyophilized. The glycoconjugate
was stored at ꢀ208C prior to use.
Procedure for preparation of BSA conjugates with diethyl squarate:
Sugar amines (1, 2, 40–43 and 45) were dissolved in methanol and 3,4-di-
ethoxy-3-cyclobutene-1,2-dione (0.9 equiv) was added in methanol. Reac-
tion progress was monitored by TLC, and when necessary, sodium meth-
oxide was added. When all starting material had been consumed as
judged by TLC, the mixture was transferred to an Epindorph tube and
solvent was removed with a stream of argon. The residue was dried in
vacuo for 1 h, after which time a solution of BSA (5 mgmLꢀ1) in borate
buffer, pH9.0 was added. The solutions were tumbled overnight and the
conjugates processed and stored as described for 38 and 39.
(600 MHz, D2O): d = 5.98 (dddd, 1H, J=J=5.9, 10.6, ~17 Hz, H-b),
5.37 (brd, 1H, J=17 Hz, H-c), 5.26 (brd, 1H, J=10.6 Hz, H-d), 4.50 (d,
1H, J1,2 =8.0 Hz, H-1), 4.38 (dd, 1H, J=5.7, 12.6 Hz, H-a), 4.21 (dd, 1H,
J=6.5, 12.6 Hz, H-a’), 4.07 (dd, 1H, J3,2 =9.8, J3,4 =3.1 Hz, H-3), 3.93 (d,
1H, J3,4 =3.1 Hz, H-4), 3.88–3.83 (m, 2H, H-8’, H-9a’), 3.82 (dd, 1H,
J
5’,6’ =J5’,6’ =10.2 Hz, H-5’), 3.77–3.71 (m, 2H, H-6a, H-6b), 3.70–3.57 (m,
5H, H-4’, H-5, H-6’, H-9b’, H -’7), 3.55 (dd, 1H, H-2), 2.74 (dd, 1H,
J
3eq’,4’ =4.5, J3eq’,3ax’ =12.4 Hz, H-3’eq), 2.02 (s, 3H, C(O)CH3), 1.78 (dd,
1H, J3ax’,4’ =12.0 Hz, H-3’eq); 13C NMR (125 MHz, D2O): d = 175.9, 174.8,
134.3. 119.7, 102.5, 100.7, 76.8, 75.8, 73.7, 72.7, 71.4, 70.0, 69.2, 69.0, 68.4,
63.5, 61.8, 52.6, 40.5, 22.9; ESI HRMS: m/z: calcd for C20H32NO14
510.1817; found: 510.1812 [M+Na+].
:
Allyl S-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-
a-d-galacto-non-2-ulopyranosylonate)-(2!3)-2-O-benzoyl-3-thio-b-d-
galactopyranoside (47): Disaccharide 19 (0.285 g, 0.32 mmol) was dis-
solved in 80% acetic acid (10 mL) and the mixture was heated at 558C
overnight with stirring. After evaporation of the volatiles, the residue was
concentrated from toluene several times. Chromatography of the residue
on silica gel by using dichloromethane/methanol (96:4) as eluent yielded
47 (0.217 g, 0.27 mmol, 85%). [a]D = 22 (c = 1.15, CHCl3); 1HNMR
(400 MHz, CDCl3): d = 8.15–8.11 (m, 2H, Ar), 7.59–7.54 (m, 1H, Ar),
7.49–7.43 (m, 2H, Ar), 5.84–5.73 (m, 1H, H-b), 5.58 (ddd, 1H, J=2.8,
5.7, J8’,7’ =9.8 Hz, H-8’), 5.24–5.16 (m, 3H, H-2, H-7’, H-c), 5.10 (brd, 1H,
NH), 5.05 (ddd, 1H, J=1.4, 1.4, 1.4, 10.5 Hz, H-d), 4.97 (d, 1H, J=
7.7 Hz, H-1), 4.80 (ddd, 1H, J4’,3’eq =4.6, J4’,3’ax =11.7, J4’,5’ =10.3 Hz, H-4’),
4.37–4.29 (m, 2H, H-9’a, H-a), 4.15 (dddd, 1H, J=1.4, 1.4, 5.9, 13.2 Hz,
H-a’), 4.06 (dd, 1H, J9’b,8’ =5.8, J9’b,9’a =12.5 Hz, H-9’b), 3.95–3.83 (m, 4H,
H-6a, H-6b, H-5, H-5’), 3.79–3.74 (m, 1H, H-6’), 3.77 (s, 3H, CO2CH3),
3.71 (dd, 1H, J3,4 =2.7, J3,2 =11.7 Hz, H-3), 3.62 (brd, 1H, H-4), 2.60 (dd,
1H, J3eq,3ax =12.8 Hz, H-3’eq), 2.4–2.2 (b, 2H, 2”OH), 2.19, 2.07, 1.98, (3 s,
3”3H, 3”C(O)CH3), 1.94 (dd, 1H, H-3ax), 1.83, 1.70 (2 s, 2”3H, 2”
Acknowledgements
The authors would like to thank Sandra Jacques for assistance in prepa-
ration of O-glycosides 40–42 and for expression of enzymes, Joanna
Sadowska for ELISA work, Chang-Chun Ling for helpful discussions,
Michel Gilbert at the National Research Council of Canada (Institute for
Biological Science) for providing clones of enzymes used in this work,
Angie Morales-Izquierdo for acquiring mass spectra and Blake Zheng
for enzyme expression and assay of glycosyltransferase activity. J.R.
thanks the Natural Sciences and Engineering Research Council of
Canada (NSERC) and the Alberta Heritage Foundation for Medical Re-
search (AHFMR) for postgraduate studentships. This work was funded
by grants to D.R.B. from NSERC and the Alberta Ingenuity Centre for
Carbohydrate Science.
C(O)CH3); 13C NMR (100 MHz, CDCl3):
d = 170.9, 170.8, 170.25,
170.20, 170.12, 169.4, 165.8, 133.9, 132.9, 130.3, 130.1, 128.3, 117.1, 101.4,
81.4, 75.2, 73.6, 70.3, 70.2, 69.3, 69.1, 67.4, 66.8, 63.2, 62.4, 53.2, 49.1, 48.2,
37.9, 23.1, 21.4, 20.8 (2C), 20.5; ESI HRMS: m/z: calcd for
C43H51NO18SNa: m/z: 836.2406; found: 836.2402 [M+Na+]; elemental
analysis calcd (%) for C36H47NO18S: C 53.13, H5.82, N 1.72; found: C
52.95, H5.99, N 1.66.
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Received: May 10, 2005
Published online: September 30, 2005
858
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