Synthesis and immunochemical characterization of 5-linked glycoconjugate vaccines against Candida albicans

Article abstract of DOI:10.1002/chem.200800352

Replacement of the glycoside oxygen atom by a sulphur atom is a promising technique for creating glycoconjugates with increased resistance to hydrolysis by endogenous glycosidases. The synthesis and antigenic properties of two distinct (1 →2)-β-mannan trisaccharides with inter residue-S-linked mannopyranose residues are described. Syntheses were based on an oxidationreduction strategy to construct the O-_linked β-mannopyranoside bonds and a S_N2 inversion to provide 1-thio-β-mannopyranoside residues. Subsequently the allyl trisaccharide glycosides were subjected to photo addition with cysteine amine and coupled to tetanus toxoid and bovine serum albumin with good efficiency via an adipic acid tether. Rabbit immunization studies revealed that the antibodies elicited by the two glycoconjugates were able to recognize the corresponding O-linked trisaccharide epitope conjugated to BSA and the native cell wall antigen of Candida albicans.

Full text of DOI:10.1002/chem.200800352

DOI: 10.1002/chem.200800352
Synthesis and Immunochemical characterization of S-linked Glycoconjugate
Vaccines against Candida albicans
Xiangyang Wu, Tomasz Lipinski, Eugenia Paszkiewicz, and David R. Bundle*[a]
Abstract: Replacement of the glycosi-
dic oxygen atom by a sulphur atom is a
promising technique for creating glyco-
conjugates with increased resistance to
hydrolysis by endogenous glycosidases.
The synthesis and antigenic properties
of two distinct (1!2)-b-mannan trisac-
charides with inter residue-S-linked
mannopyranose residues are described.
Syntheses were based on an oxidation–
reduction strategy to construct the O-
linked b-mannopyranoside bonds and a
SN2 inversion to provide 1-thio-b-man-
nopyranoside residues. Subsequently
the allyl trisaccharide glycosides were
subjected to photo addition with cys-
teine amine and coupled to tetanus
toxoid and bovine serum albumin with
good efficiency via an adipic acid
tether. Rabbit immunization studies re-
vealed that the antibodies elicited by
the two glycoconjugates were able to
recognize the corresponding O-linked
trisaccharide epitope conjugated to
BSA and the native cell wall antigen of
Candida albicans.
Keywords:
antibodies
·
Candida albicans · glycoconjugates ·
oligosaccharides · vaccines
Introduction
patients,as well as those undergoing long term antibiotic
treatment.[6] Cutler and co-workers have shown the protec-
tive potential of monoclonal antibodies specific for the (1!
2)-b-mannan trisaccharide antigen present in the C. albicans
cell wall phosphomannan[7] in a disseminated candidiasis
mouse model. A glycoconjugate vaccine prepared from this
trisaccharide attached to tetanus toxoid was shown to be a
highly effective immunogen in rabbits,although immuniza-
tion of mice has so far failed to demonstrate a robust
immune response.[8]
We have demonstrated that thiooligosaccharide conjugate
vaccines could evoke antibodies specific for native antigens
in mice.[9,10] For example synthetic ganglioside antigens con-
taining a terminal S-linked sialic acid were able to generate
antibodies that recognized the corresponding O-linked anti-
gen. As part of a detailed study of the immunochemistry of
the protective (1!2)-b-mannan antigen of Candida albicans
we have investigated the synthesis of C. albicans trisacchar-
ide vaccine candidates composed of oligosaccharides con-
taining terminal and an internal interglycosidic S-linkages.
Epitopes I and II appeared to be a good model to study the
immunological properties of isosteric conjugates with in-
creased resistance to catabolic processing by glycosidases.
The 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.[11,12]
Conjugate vaccines composed of microbial polysaccharides
conjugated to immunogenic proteins have been shown to be
an attractive and cost effective strategy to prevent deadly in-
fectious diseases.[1–3] These successes have created renewed
interest in the wider potential of glycoconjugate vaccines for
both prophylactic and therapeutic applications. Completely
synthetic carbohydrate based vaccines to combat Haemophi-
lus influenzae type b have recently been reported and
shown to be as effective as their semi-synthetic counterparts
that are derived from bacterial fermentation,extraction of
the polysaccharide and subsequent conjugation to carrier
protein.[4] Strategies to create completely synthetic vaccines
are therefore attracting attention.
A particularly attractive epitope for consideration as a
synthetic vaccine is the C. albicans,(1 !2)-b-mannan trisac-
charide. C. albicans is the most common etiologic agent of
candidiasis,[5] and commonly affects immunocompromised
[a] Dr. X. Wu,Dr. T. Lipinski,Dr. E. Paszkiewicz,
Prof. Dr. D. R. Bundle
Alberta Ingenuity Centre for Carbohydrate Science
Department of Chemistry,University of Alberta
Edmonton,AB T6G2G2 (Canada)
Fax : (+1)780-492-7705
E-mail: dave.bundle@ualberta.ca
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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A significant number of publications report the synthesis
of S-linked oligosaccharides.[11–13] The most convenient
access to thioglycosides is based on SN2 displacement of hal-
ogenoses because of the high nucleophilicity of thiolate
groups.[14] Crich and co-workers have reported a direct ste-
reoselective synthesis of b-thiomannosides based on the cou-
pling of anomeric S-phenyl sulfoxide with sugar thiol.[15] Our
group has successfully synthesized a S-linked tetrasaccharide
with a novel enzyme-catalyzed technique employing a lac-
tose thiol acceptor and UDP-gal donor.[16] Here,we intro-
duce a related but simplified approach based on the glycosy-
lation of a 2-deoxy-2-thiomannopyranoside acceptor by a
glucopyranosyl trichloroacetimidate donor and an oxida-
tion–reduction strategy to achieve the epimerization at C-2
to obtain S-linked b-mannose oligomers suitably functional-
ized for covalent attachment to immunogenic proteins.
Scheme 1. a) Tf2O,CH 2Cl2,pyridine; b) KSAc,DMF,70
8C; c)
NH2NH2·HOAc,THF; d) TMSOTf,CH 2Cl2, ꢀ208C; e) NaOMe,MeOH;
f) DMSO,Ac 2O; then L-selectride,THF; g) TMSOTf,CH 2Cl2, ꢀ108C;
h) NaOMe,MeOH; and DMSO,Ac 2O; then L-selectride,THF; i) 10%
Results and Discussion
Pd/C,H .
2
Synthesis of (1!2)-b-linked inter-thio trisaccharide (12):
Building block 1 was synthesized according to a published
procedure.[17,18] The reaction between alcohol 1 and trifluor-
omethanesulfonic anhydrate (Tf2O) was performed in the
presence of pyridine as base in CH2Cl2 at 08C to give triflate
2 in 94% yield. Inversion of configuration with potassium
thioacetate (KSAc)[17] furnished the desired protected thiol
3 in excellent yield (Scheme 1).
1
by H NMR. Replacement of the glycosidic oxygen atom by
sulfur did not lead to either decreased yield or poor selectiv-
ity. Hydrogenolysis of trisaccharide 11 using 0.15 equivalent
of palladium charcoal (10% Pd) furnished the deprotected
trisaccharide 12 in 89% yield.
Synthesis of (1!2)-b-linked terminal-thio trisaccharide (22):
Building blocks 13 and 14 were synthesized according to
published procedures (Scheme 2).[17] Reaction of glycosyl
donor 14 with the monosaccharide acceptor 13 in CH2Cl2 at
ꢀ108C in the presence of TMSOTf as catalyst (0.02 equiv)
afforded disaccharide 15 in 83% yield. Transesterification of
Interestingly,the sec-triflate 2 is sufficiently stable to be
purified by chromatography without detectable decomposi-
tion. The treatment of 3 with hydrazine acetate in DMF af-
forded thiol 4 in 87% yield. It should be mentioned that
compound 4 was not oxidized to the corresponding disulfide
even when exposed to air for a long time,possibly due to
the steric hindrance caused by the 4,6-O-benzylidene pro-
tecting group. The glycosylation of acceptor 4 by imidate
donor 5 was performed in the presence of trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf) (0.03 equiv) in CH2Cl2
at ꢀ208C to give disaccharide 6 in good yield. Transesterifi-
cation gave the desired alcohol 7 in quantitative yield,and
this was oxidized to the corresponding keto derivative by
DMSO and Ac2O (2:1). Subsequent stereoselective reduc-
tion with L-selectride at ꢀ788C in THF afforded disacchar-
ide 8 in 80% yield over two steps. Repetition of the glycosy-
lation with donor 5 followed by transesterification,oxida-
tion and reduction gave trisaccharide 11 in 69% yield over
four steps. Most importantly,excellent diastereoselectivity
was observed in this strategy since in this reduction reaction
only trace amounts of the b-gluco epimer could be detected
15 in MeOH,followed by triflation with Tf O and pyridine
2
in CH2Cl2 at 08C furnished 16 in high yield. The reaction of
16 with potassium thioacetate in DMF at 708C gave acety-
lated thiol 17. Reaction of compound 17 with hydrazine ace-
tate in DMF afforded the acceptor 18,and subsequent gly-
cosylation by glycosyl donor 5 in the presence of TMSOTf
(0.02 equiv) furnished trisaccharide 19 in 62% yield. Finally,
deacetylation,oxidation and reduction afforded the desired
trisaccharide 21 in 50% yield over three steps. Hydrogenoly-
sis of trisaccharide 21 using 0.15 equivalents of palladium/
charcoal activated (10% Pd) furnished the unprotected tri-
saccharide 22 in 85% yield.
Synthesis of tether halfesters 26 and 27: For the conjugation
of deprotected oligosaccharides to proteins,a terminal
amine was chosen as a versatile handle from which glyco-
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6475

D. R. Bundle et al.
Scheme 3. a) 2-Aminoethanethiol hydrochloride,MeOH,CH 2Cl2, hn
365 nm; b) Na/NH3, tBuOH,THF.
Formation of neoglycoproteins: With the required half
esters at our disposal,coupling of 26 and 27 to BSA was per-
formed by an 18 h incubation in buffer (pH 7.5) at ambient
temperature. The BSA conjugates 28 and 29 were obtained
as white powders after dialysis against deionized water fol-
lowed by lyophilization (Scheme 4). In the same way, 26 and
27 were conjugated to tetanus toxoid (TT) in phosphate
buffer (pH 7.2) overnight at ambient temperature. After di-
alysis against phosphate buffer saline (PBS) pH 7.2,the con-
jugates 30 and 31 were ready for vaccine formulation. Tar-
geted and observed incorporations are tabulated below
(Table 1). The degree of incorporation of the oligosacchar-
Scheme 2. a) TMSOTf,CH 2Cl2, ꢀ108C; b) NaOMe,MeOH; then Tf 2O,
CH2Cl2,pyridine; c) KSAc,DMF,70 8C; d) NH2NH2·HOAc,THF; e)
TMSOTf,CH 2Cl2, ꢀ108C; f) NaOMe,MeOH; and DMSO,Ac 2O; then
L-selectride,THF; g) 10% Pd/C,H
.
2
conjugates could be readily generated.[17] The protected oli-
gosaccharides 11 and 21 were elaborated via photoaddition
of 2-aminoethanethiol to the allyl glycosides to give the
amine-functionalized glycosides,[17] then subsequently depro-
tection under Birch conditions to give the desired amino-
functionalized glycosides 23 and 24 in good yields. For effi-
cient attachment of oligosaccharides to proteins,a highly ef-
ficient conjugation strategy is required. Previously,coupling
of such compounds to bovine serum albumin (BSA) protein
was achieved through a squarate linker.[17] While conjugates
prepared in this manner are perfectly acceptable for use in
ELISA screening,its use in conjugate vaccine applications
has been correlated with antibody response to the squarate
residue itself.[17,19] Here,half esters of adipic acid phenyl
ester were prepared according to our recently published
procedure.[8,19,20] The oligosaccharide amines 23 and 24 were
treated with five equivalents of the homobifunctional p-ni-
trophenyl ester 25 in dry DMF at room temperature for 5 h,
affording the corresponding half esters 26 and 27 in good
yields after purification on a reverse phase column,as
shown in Scheme 3. The reaction is readily monitored by
TLC or UV spectroscopy,and the half esters are stable
during chromatography purifications on silica gel and re-
verse-phase under acidic conditions. Excess linker could be
easily removed by washing with dichloromethane and the
yields of this reaction were in the range of 60–65%.
Table 1. BSA and tetanus toxoid mannopyranan conjugates.
Product Saccharide Molar ratio of pro- Number of
Incorporation
efficiency
[%]
[mg]
tein/ester
haptens
28
29
30
31
2.0
0.5
1.2
0.6
1:30
1:20
1:30
1:30
11
36.7
39
24
7.8
7.2
8.7
29
ides on BSA and TT was established by MALDI-TOF MS
using sinapinic acid as the matrix. Conjugation efficiencies
of between 24 and 39% were achieved,corresponding to
the incorporation of 7–12 ligands on TT or BSA with a 30-
fold molar excess of activated oligosaccharides similar to
those published for the coupling of oligosaccharides to
BSA.[8,19,20]
Immunization studies in rabbits: To show the ability of syn-
thetic S-analogues to mimic the corresponding O-linked
native antigen we analyzed the binding of compounds 12
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Glycoconjugate Vaccines
FULL PAPER
tively small binding site of the
monoclonal antibody C3.1 that
we believed to be complemen-
tary to a trisaccharide and the
rather rigid antigen conforma-
tion that occurs in b1,2-manno
oligomers.[22] Larger oligosac-
charide such as the tetra
through hexsaccharide bind
with significantly reduced affin-
ity.[22] We attributed this to the
helical conformation of these
antigens which places the
Scheme 4. Synthesis of neoglycoproteins.
fourth residue close to the first
mannose.[22] Since the native
and 22 to monoclonal antibody C3.1 which is specific for the
b-mannan oligosaccharide epitope of C. albicans phospho-
mannan. Figure 1 shows the results of the inhibition EIA.
structure is relatively rigid there is likely a steric clash at the
periphery of the binding site,which account for the lower
activity of larger structures. The thio-linked tetrasaccharide
is able to release this steric interaction because thio glycosi-
dic bonds are far less constrained than the corresponding O-
linked antigen.[17,22]
Recent binding site mapping studies on a disaccharide
epitope (Nycholat and Bundle,unpublished results) have
suggested that the preferred binding epitope is possibly the
terminal reducing disaccharide rather than a terminal non-
reducing disaccharide. In this context 22 can provide this
binding element whereas trisaccharide 12 cannot. In addi-
tion because the terminal S-linked mannose residue has
greater flexibility there is the potential for mutual fitting of
this third residue to create higher affinity interactions than
is the case for the native trisaccharide. On going modeling
studies of the C3.1 binding site are intended to clarify this
point.
Further studies established immunogenic properties of
compounds 23 and 24 conjugated to TT. Groups of three
rabbits were immunized with both glycoconjugates. The im-
munization results were evaluated by ELISA using plates
coated with either O-linked trisaccharide–BSA conjugate or
C. albicans cell wall extract. The results for the titrations of
sera obtained after two immunizations with both conjugates
are shown in Figure 2. The average titre of immune sera
(IgG subclass) reached 1:300000 for 30 and 1:100000 for 31
when evaluated against native trisaccharide–BSA conjugate.
The titre against cell wall antigen was similar for both com-
pounds and reached about 1:12000. Similar results were ob-
served for immunization with O-linked trisaccharide–TT
conjugate (unpublished data). The lower titre against the
cell wall antigen suggests that a portion of serum antibodies
recognize an epitope consisting of oligosaccharide plus at
least part of the tether. The data show the tether effect ap-
plies similarly to S- and native O-linked oligosaccharides.
&
&
Figure 1. Inhibition assay (ELISA) for compounds 12 ( ,left) and 22 ( ,
right) with b-mannan specific monoclonal Ab (C3.1). Plates were coated
with trisaccharide–BSA conjugate and co-incubated with a constant con-
centration of monoclonal Ab and serially diluted 12 and 22.
Plates were coated with b-mannan trisaccharide–BSA conju-
gate and co-incubated with monoclonal antibody C3.1 and
serially diluted 12 and 22 S-analogues. Both compounds
were able to inhibit binding of antibodies to cell wall anti-
gen. Trisaccharide 22 (IC50 =3.7 mm) proved to be the tighter
binding inhibitor compared with 12 (IC50 =56 mm). While
this result confirms the close conformational similarity of
the sulphur containing oligosaccharides to native O-linked
oligosaccharide it raises the question why 22 is so much
more active than 12 or the native trisaccharide.
We previously reported a tetrasaccharide also bearing a
terminal S-linked mannose residue (compound 33 of ref.
[14]). It exhibited only slightly better inhibition than the cor-
responding O-linked tetrasaccharide (ꢁ1.7-fold more
active).[17,21,22] Initially we attributed this activity to the rela-
Conclusion
Two analogues of thio-linked (1!2)-b-mannanpyranan tri-
saccharide were synthesized employing a oxidation-reduc-
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6477

D. R. Bundle et al.
2.27 mmol) was added dropwise,then the reaction mixture was warmed
up to room temperature and stirred for 2.0 h. The solution was diluted
with dichloromethane,washed with aqueous sodium bicarbonate,dried
over sodium sulfate,and concentrated to a brown oil. The residue was
purified by chromatography with ethyl acetate/hexane 1:4 to give product
2 (610 mg,94%). [ a]D = ꢀ45.58 (c = 1.0,CHCl 3); 1H NMR (300 MHz,
CDCl3): d=7.46–7.22 (m,10H,Ar),5.97–5.84 (m,1H,OCH
5.57 (s,1H,PhC H),5.37–5.24 (m,2H,OCH 2CH=CH2),4.92–4.71 (dd,
2H,C H2Ph),4.68–4.62 (m,2H,1- H,2- H),4.41–4.34 (m,2H,6- H,
2CH=CH2),
OCH2CH=CH2),4.19–4.12 (m,1H,OC H2CH=CH2),3.91–3.73 (m,3H,
3-H,4- H, 6’-H),3.51–3.43 ppm (m,1H,5- H); ESI HRMS: m/z: calcd for
C24H25O8F3SNa: 553.11145; found: 553.11155.
Allyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-thioacetyl-b-d-mannopyra-
noside (3): Compound 2 (106 mg,0.2 mmol) and potassium thioacetate
(456 mg,4 mmol) were put together and DMF (5 mL) was added under
argon. The resulting mixture was stirred at 708C for 30 min. TLC check
indicated that the starting material disappeared. The reaction mixture
was diluted with toluene (50 mL),washed several times with water and
brine,dried with sodium sulfate and concentrated to a brown oil. Purifi-
cation by chromatography with ethyl acetate/hexane 1:4 gave 3 (83 mg,
Figure 2. Titration of rabbit sera obtained by immunization with glyco-
njugates 30, 31 and O-linked trisaccharide–tetanus toxoid conjugate. Sera
were titrated against Candida cell wall antigen and O-linked trisacchar-
ide-BSA conjugate. Filled bars represents serum titre of individual rab-
bits,patterned bars show the geometric mean of titres for groups of ani-
mals,error bars represent the standard deviation. A) rabbits (1–3) immu-
nized with 30,B) rabbits (4–6) immunized with 31,C) rabbits immunized
with O-linked trisaccharide–tetanus toxoid conjugate (this group of eight
animals was a part of another study,not yet published,for clarity individ-
ual titres are not shown).
1
91%). [a]D = ꢀ53.38 (c = 1.0,CHCl ); H NMR (600 MHz,CDCl ): d=
3
3
7.48–7.24 (m,10H,Ar),5.86–5.83 (m,1H,OCH
2CH=CH2),5.59 (s,1H,
PhCH),5.32–5.20 (m,2H,OCH 2CH=CH2),4.77 (m,1H,1- H),4.75–4.62
(dd,2H,CH 2Ph),4.61 (m,1H,2- H),4.36–4.33 (m,1H,OC H2CH=CH2),
4.31 (m,1H,6- H),4.15–4.11 (m,1H,OC H2CH=CH2),3.97 (dd, 3J=4.67,
9.71 Hz,1H,3- H),3.83 (t, 3J=10.2 Hz,1H,6 ’-H),3.69 (t, 3J=9.6 Hz,
1H,4- H),3.40 ppm (m,1H,5H); ESI HRMS:
C25H28O6SNa: 479.14988; found: 479.14986.
m/z: calcd for
tion methodology for the O-linked b-mannopranoside and
SN2 inversion for 1-thio-b-mannopyranoside. Conjugation of
these oligosaccharides to BSA or tetanus toxoid using a
linear homobifunctional linker was accomplished with high
efficiency under mild conditions. Inhibition data show that
the two S-linked trisaccharides are effective inhibitors of a
monoclonal antibody generated to the native cell wall b-
mannan,implying that these oligosaccharides can adopt con-
formations similar to the O-linked trisaccharide. Remarka-
bly,antibody induced by the tetanus toxiod glycoconjugates
30 and 31 was cross-reactive with the native trisaccharide
antigen and also exhibited affinity and titres for the cell wall
of C. albicans similar to those induced by the corresponding
O-linked conjugate.
Allyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-thio-b-d-mannopyranoside
(4): Hydrazine acetate (35 mg,0.34 mmol) was added to a solution of
compound 3 (104 mg,0.23 mmol) in THF (5 mL),and the mixture was
stirred for 3 h. TLC check indicated that the reaction was complete. The
mixture was diluted with ethyl acetate (30 mL),washed with water then
brine and dried with sodium sulfate followed by concentration. The resi-
due was purified by chromatography on silica gel using ethyl acetate/
hexane 1:4 to give 4 (83 mg,87%). [ a]D = ꢀ63.18 (c = 1.0,CHCl 3);
1H NMR (600 MHz,CDCl 3): d=7.54–7.24,5.96–5.77 (m,1H,OCH 2CH=
CH2),5.62 (s,1H,PhC H),5.36–5.22 (m,2H,OCH 2CH=CH2),4.80–4.74
(m,2H,C H2Ph),4.72 (s,1H,1- H),4.38–4.41 (m,1H,OC H2CH=CH2),
4.35–4.32 (m,1H,6- H),4.26 (t, 3J=9.0 Hz,1H,4- H),4.15–4.11 (m,1H,
OCH2CH=CH2),3.92 (t,1H,6
’-H),3.87 (m,2H,3-
H,2- H),3.41–
3.37 ppm (m,1H,5- H); ESI HRMS: m/z: calcd for C23H26O5SNa:
437.13932; found: 437.13909.
Allyl
(2-O-acetyl-3,4,6-tri-O-benzyl-b-d-glucopyranosyl)-(1!2)-3-O-
benzyl-4,6-O-benzylidene-2-deoxy-2-thio-b-d-mannopyranoside (6): Gly-
cosyl donor 5 (140 mg,0.21 mmol),monosaccharide acceptor 4 (67 mg,
0.162 mmol) and activated 4 Š molecular sieves (20 mg) were dried to-
gether in a pear-shaped flask (10 mL) under vacuum for one hour then
dichloromethane (3 mL)was added. The suspension was stirred for
10 min. at room temperature under argon,and then the temperature was
reduced to ꢀ408C and 0.1m trimethylsilyl trifluoromethanesulfonate so-
lution in dichloromethane (80 mL) was added dropwise. After 30 min.,
the reaction mixture was neutralized with triethylamine and concentrated
in vacuum. The residue was purified by flash chromatography (n-hexane/
Experimental Section
General methods: 1H NMR spectra were recorded at either 400,500,or
600 MHz,and are referenced to the residual protonated solvent peaks;
dH 7.24 ppm for solutions in CDCl3,and 0.1% external acetone ( dH
2.225) for solutions in D2O. Mass analysis was performed by positive-
mode electrospray ionization on a hybrid sector-TOF mass spectrometer
and for protein glycoconjugates by MALDI mass analysis,employing si-
napinic acid as matrix. Analytical thin-layer chromatography (TLC) was
performed on silica gel 60-F254 (Merck). TLC detection was achieved by
charring with 5% sulfuric acid in ethanol. All commercial reagents were
used as supplied. Column chromatography used silica gel (SiliCycle,
Quebec City,Quebec,230–400 mesh,60 Š),and redistilled solvents.
HPLC separations were performed on a Beckmann C18 semipreparative
reversed-phase column with a combination of methanol and water con-
taining 0.1% HOAC as eluents. Photoadditions were carried out using a
spectroline model ENF-260C UV lamp and cylindrical quartz vessels.
ethyl acetate 6:1) to afford 6 (115 mg,80%) as a white foam. [ a]D
=
ꢀ49.58 (c = 1.0,CHCl 3); 1H NMR (300 MHz,CDCl 3): d=7.5–7.2 (m,
25H,Ar),5.95–5.86 (m,1H,OCH 2CH=CH2),5.56 (s,1H,PhC H),5.36–
5.20 (m,2H,OCH 2CH=CH2),5.09–5.05 (t, 3J=10 Hz,1H,2b- H),4.92–
4.78 (m,4H,1b- H,3/2 CH Ph),4.72–4.70 (d, 2J=12.0 Hz,1H, 1= CH2Ph),
2
2
4.65 (s,1H,1a- H),4.58–4.47 (m,4H,2CH
2Ph),4.42–4.34 (m,1H,
OCH2CH=CH2),4.28 (m,1H,6a- H),4.10–4.02 (m,1H,OC H2CH=CH2),
3.90 (dd,1H,2a- H),3.80–3.87 (m,2H,3a- H,6b- H),3.78–3.62 (m,5H,
6’a-H, 6’b-H,3b- H,4a- H,4b- H),3.54–3.48 (m,1-H,5b- H),3.38–3.30 ppm
(m,1H,5a- H); 13C NMR (125 MHz,CDCl 3): d = 138.1–126.2,116.9,
101.5,100.8,84.5,84.1,79.1,78.1,75.2,75.22,75.1,73.4,73.1,69.7,69.4,
68.6,67.7 ppm; ESI HRMS: m/z: calcd for C52H56O11SNa: 911.34356;
found: 911.34321.
Allyl
3-O-benzyl-4,6-O-benzylidene-2-O-trifluoromethanesulfonyl-b-d-
glucopyranoside (2): Alcohol 1 (485 mg,1.28 mmol) was dissolved in di-
chloromethane (10 mL) and pyridine (570 mL) was added followed by
N,N-dimethyl-4-aminopyridine (50 mg,0.4 mmol). The resulting solution
was cooled to 08C and trifluoromethanesulfonic anhydride (384 mL,
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Glycoconjugate Vaccines
FULL PAPER
Allyl 3-O-benzyl-2-O-(3,4,6-tri-O-benzyl-b-d-glucopyranosyl)-4,6-O-ben-
zylidene-2-deoxy-2-thio-b-d-mannopyranoside (7): Sodium methoxide
(0.7 mg,0.013 mmol) was added to a solution of 6 (115 mg,0.13 mmol) in
methanol (5 mL) and the mixture was stirred at room temperature over-
night. Then it was neutralized with IR 120 (H+ form),and concentrated
in vacuum. The residue was purified by flash chromatography (n-hexane/
d = 138.6–117.7,101.6,101.2,101.1,84.4,83.1,81.3,79.9,78.7,75.5,75.3,
75.0,74.8,74.7,74.5,73.8,73.5,73.4,73.3,70.9,70.3,69.8,69.7,69.4,68.5,
68.1 ppm; ESI HRMS: m/z: calcd for C79H84O16SNa: 1343.53723; found:
1343.53749.
Allyl (3,4,6-tri-O-benzyl-b-d-glucopyranosyl)-(1!2)-(3,4,6-tri-O-benzyl-
b-d-mannopyranosyl)-(1!2)-3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-
thio-b-d-mannopyranoside (10): Compound 10 was prepared according
to the procedure for preparation of 7 using trisaccharide 9 (90 mg,
0.068 mmol),sodium methoxide (0.4 mg),dichloromethane (2 mL),meth-
anol (1 mL). Column chromatography in n-hexane/ethyl acetate 3:1 gave
ethyl acetate 4:1) to afford 7 (110 mg,100%) as a white foam. [ a]D
=
ꢀ38.58 (c = 1.0,CHCl 3); 1H NMR (600 MHz,CDCl 3): d=7.5–7.2 (m,
25H,Ar),5.90–5.82 (m,1H,OCH 2CH=CH2),5.60 (s,1H,PhC H),5.36–
5.30 (m,1H,OCH 2CH=CH2),5.20–5.16 (m,1H,OCH 2CH=CH2),5.00
(d, J=11.4 Hz,1H, 1= CH2Ph),4.90–4.76 (m,4H,2CH 2Ph),4.64 (s,1H,
2
2
trisaccharide 10 (87 mg,100%).
[
a]D
=
ꢀ1.28 (c
= 1.0,CHCl 3);
2J=11.4 Hz,1H,
1H NMR (600 MHz,CDCl 3): d=7.50–7.38 (m,40H,Ar),5.86–5.80 (m,
1a-H),4.58–4.54 (m,2H,CH
2Ph),4.49–4.46 (d,
1= CH2Ph),4.40–4.34 (m,2H,1b- H,OC H2CH=CH2),4.32–4.28 (m,1H,
2
1H,OCH 2CH=CH2),5.62 (s,1H,PhCH),5.23–5.19 (m,1H,OCH
2CH=
6a-H),4.14–4.08 (m,1H,OC H2CH=CH2),3.90–3.82 (m,3H,3a- H,4a- H,
6’a-H),3.75 (m,2H,6b- H, 6’b-H),3.68–3.58 (m,4H,2a- H,2b- H,3b- H,
4b-H),3.50 (m,1H,5b- H),3.44 (s,1H,OH),3.37–3.32 ppm (m,1H,5a-
H); 13C NMR (125 MHz,CDCl 3): d = 139.1–126.0,117.8,101.6,99.9,
86.7,85.7,80.3,79.7,76.8,75.7,75.1,75.0,74.0,73.7,72.6,69.9,69.3,68.6,
67.7 ppm; ESI HRMS: m/z: calcd for C50H54O10SNa: 869.33299; found:
869.33291.
1
CH2),5.14–5.10 (m,2H,OCH 2CH=CH2), = CH2Ph),5.07 (s,1H,1a- H),
2
4.98–4.81 (m,6H,3 CH 2Ph),4.75 (d, 3J=7.2 Hz,1H,1c- H),4.69 (d, 3J=
1.2 Hz,1H,1b- H),4.56–4.44 (m,9H,4 CH 2Ph,2a- H),4.40–4.39 (m,2H,
1= CH2Ph,OC H2CH=CH2),4.31 (m,1H,6b-
H),4.07–4.04 (m,1H,
2
OCH2CH=CH2),3.95 (dd, 3J=1.2,4.8 Hz,1 H,2b- H),3.91–3.86 (m,3H,
3b-H,4a- H, 6’b-H),3.82 (t, 3J=7.8,9.0 Hz,1H,2c- H),3.78–3.68 (m,5H,
3c-H,4b- H,6a- H, 6’a-H, 6’c-H),3.66–3.58 (m,3H,4c- H,5c- H,6c- H),
3.52 (dd, 3J=3.0,9.0 Hz,1H,3a- H),3.48 (m,1H,5a- H),3.38 (m,1H,
Allyl
3-O-benzyl-2-O-(3,4,6-tri-O-benzyl-b-d-mannopyranosyl)-4,6-O-
5b-H),3.22 ppm (br,1H,OH); 13C NMR (125 MHz,CDCl ): d = 139.3–
benzylidene-2-deoxy-2-thio-b-d-mannopyranoside (8): Disaccharide
7
3
(46 mg,0.054 mmol) was dissolved in freshly distilled DMSO (4 mL) and
acetic anhydride (2 mL) was added. The resulting solution was stirred for
18 h at room temperature then diluted with ethyl acetate,washed with
water,aqueous sodium bicarbonate and a brine solution. Finally,the so-
lution was concentrated at low pressure to give a yellow syrup. It was dis-
solved in THF (5 mL),cooled to ꢀ788C under argon,then L-selectride
(1m THF,0.5 mL) was added dropwise and the reaction mixture was
stirred for 15 min. The cooling bath was removed and the solution was al-
lowed to warm up to room temperature. The reaction was quenched
after 15 min with methanol (0.5 mL),and the mixture was diluted with
dichloromethane. Washings with a solution of hydrogen peroxide (5%)
and sodium hydroxide (1m) followed by sodium thiosulfate (5%) and
sodium chloride solutions gave a clear organic solution. It was dried over
magnesium sulfate and concentrated to a colourless oil which was puri-
fied by flash chromatography (n-hexane/ethyl acetate 3:1) to afford 8
117.8,103.7,101.6–101.1,85.2,83.7,81.4,79.9,78.8,76.9,75.7,75.5,74.9,
74.88,74.81,74.2,74.1,73.5,73.3,70.2,69.9,69.7,69.6,68.5,68.1 ppm;
ESI HRMS: m/z: calcd for C77H82O15SNa: 1301.52667; found:
1301.52639.
Allyl (3,4,6-tri-O-benzyl-b-d-mannopyranosyl)-(1!2)-(3,4,6-tri-O-benzyl-
b-d-mannopyranosyl)-(1!2)-3-O-benzyl-4,6-O-benylidene-2-deoxy-2-
thio-b-d-mannopyranoside (11): The procedure used was analogous to
the preparation of 8 using trisaccharide 10 (60 mg,0.047 mmol),DMSO
(4 mL),acetic anhydride (2 mL),THF (3 mL),L-selectride (1 m,0.5 mL).
Column chromatography in n-hexane/ethyl acetate 5:2 gave trisaccharide
11 (50 mg,83%). [ a]D = ꢀ68.48 (c = 1.0,CHCl 3); 1H NMR (600 MHz,
CDCl3): d=7.50–7.38 (m,40H,Ar),5.86–5.80 (m,1H,OCH
2CH=CH2),
2CH=CH2),5.16–5.14 (m,
H),5.00–4.91 (m,5H,1c- H,
5.62 (s,1H,PhCH),5.24–5.21 (m,1H,OCH
1H,OCH 2CH=CH2),5.08 (s,1H,1b-
2CH2Ph),4.83–4.81 (d, 2J=12.0 Hz,1H, 1= CH2Ph),4.71 (d, 3J=1.4 Hz,
2
(37 mg,80%) as a clear syrup. [ a]D
=
ꢀ45.68 (c
= 1.0,CHCl 3);
1H,1a- H),4.67 (d,
3J=3.4 Hz,1H,2b-
H),4.62–4.41 (m,10H,
1H NMR (600 MHz,CDCl 3): d=7.46–7.20 (m,25H,Ar),5.90–5.83 (m,
OCH2CH=CH2,4CH 2Ph, 1= CH2Ph),4.38–4.36 (d, 2J=12.0 Hz,1H,
2
1H,OCH 2CH=CH2),5.56 (s,1H,PhCH),5.27–5.22 (m,1H,OCH
2CH=
1= CH2Ph),4.32 (m,1H,6b- H),4.10 (m,1H,OC H2CH=CH2),4.05 (dd,
2
CH2),5.20–5.16 (m,1H,OCH 2CH=CH2),5.04 (s,1H,1b- H),4.92 (m,
3J=4.9,1.4 Hz,1H,2a- H),3.97 (t, 3J=9.5 Hz,1H,4c- H),3.91 (dd, 3J=
4.9,9.9 Hz,1H,3a- H),3.86 (t, 3J=10.4 Hz,1H,4b- H),3.82–3.78 (m,2H,
6a-H,6c- H),3.75–3.68 (m,2H,6 ’a-H, 6’c-H),3.66–3.62 (m,2H,4a- H,
6’b-H),3.58 (dd, 3J=2.9,9.1 Hz,1H,3c- H),3.56–3.50 (m,3H,3b- H,5a-
2H,CH 2Ph),4.76 (d, 2J=12 Hz,1H, 1= CH2Ph),4.67 (s,1H,1a- H),4.5–
2
4.67 (m,5H, 5= CH2Ph),4.44–4.40 (m,1H,OC
H2CH=CH2),4.31 (dd,
2
3J=10.2,4.8 Hz,1H,4a- H),4.25 (t, 3J=3.6 Hz,1H,2b- H),4.09–4.06 (m,
1H,OC H2CH=CH2),3.94 (dd, 3J=4.8,1.8 Hz,1H,2a- H),3.90–3.84 (m,
3H,3a- H,4b- H,6a- H),3.80–3.72 (m,3H,6b- H, 6’a-H, 6’b-H),3.59 (dd,
3J=9.6,3.6 Hz,1H,3b- H),3.51 (m,1H,5b- H),3.37 ppm (m,1H,5a- H);
13C NMR (125 MHz,CDCl 3): d = 138.2–126.1,117.6,101.6,100.9,83.6,
82.7,79.7,79.0,75.4,75.0,74.4,73.5,71.3,70.1,69.9,69.8,68.9,68.6,
67.9 ppm; ESI HRMS: m/z: calcd for C50H54O10SNa: 869.33299; found:
869.33308.
H,5c- H),3.39ppm (m,1H,5b-H);
13C NMR (125 MHz,CDCl 3): d =
138.4–137.3,133.3,128.9–126.1,117.7,101.6,101.1,99.6,83.5,81.5,81.4,
79.7,78.7,77.3,77.0,76.8,75.3,75.2,75.1,74.9,74.3,74.2,73.3,73.2,71.8,
70.7,703,,70.2,69.5,69.4,68.5,67.9,67.8 ppm; ESI HRMS:
for C77H82O15SNa: 1301.52667; found: 1301.52639.
m/z: calcd
Propyl (b-d-mannopyranosyl)-(1!2)-(1-thio-b-d-mannopyranosyl)-(1!
2)-2-deoxy-2-thio-b-d-mannopyranoside (12): Compound 11 (27 mg,
0.02 mmol) and Pd/C-activated (10% Pd) (50 mg) were put in the flask.
The whole system was vacuumed for 10 min. followed by the addition of
hydrogen. Methanol (5 mL) was added and the reaction mixture was
stirred overnight. TLC check indicated that the starting material disap-
peared. The reaction mixture was filtered through Celite to give the clear
solution which was concentrated. The resulting residue was purified by
HPLC on C18 silica to give product 12 (10 mg,89%). 1H NMR
Allyl (2-O-acetyl-3,4,6-tri-O-benzyl-b-d-glucopyranosyl)-(1!2)-(3,4,6-tri-
O-benzyl-b-d-mannopyranosyl)-(1!2)-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-2-thio-b-d-mannopyranoside (9): The procedure used was analo-
gous to the preparation of 6,using glycosyl donor 5 (63 mg,0.1 mmol),
disaccharide 8 (70 mg,0.083 mmol),dichloromethane (3 mL),trimethyl-
silyl trifluoromethanesulfonate (33 mL) (0.05m CH2Cl2),activated 4 Š
molecular sieves (20 mg) and the temperature was ꢀ108C. Column chro-
matography in n-hexane/ethyl acetate 4:1 gave trisaccharide 9 (90 mg,
1
(400 MHz,CDCl ): d=5.02 (s,1H,1b- H),4.88 (s,1H,1c- H),4.87 (s,1H,
3
83%). [a]D = ꢀ40.68 (c = 1.0,CHCl ); H NMR (600 MHz,CDCl ): d=
3
3
1a-H),4.33 (d, 3J=3.0 Hz,1H,2b- H),4.21 (d, 3J=4.8 Hz,1H,2a- H),
3.95–3.57 (m,14H,OC H2,3a- H,3b- H,3c- H,4a- H,4b- H,6a- H, 6’a-H,
6b-H, 6’b-H,6c- H, 6’c-H,2c- H),3.44–3.34 (m,3H,5a- H,5b- H,5c- H),
7.54–7.20 (m,40H,Ar),5.80 (m,1H,OCH
2CH=CH2),5.62 (s,1H,
PhCH),5.21–5.17 (m,2H,2c-
OCH2CH=CH2),5.10–5.08 (m,1H,OCH 2CH=CH2),4.98–4.77 (m,8H,
1a-H,1c- H,3CH 2Ph),4.68 (s,1H,1b- H),4.60–4.41 (m,10H,2a- H,
OCH2CH=CH2,4CH 2Ph),4.33 (m,1H,6b- H),4.06–4.02 (m,1H,
OCH2CH=CH2),3.93 (t, 3J=10.2 Hz,1H,4b- H),3.89–3.80 (m,4H,3b-
H,OCH 2CH=CH2),5.20–5.16 (m,1H,
3.30 (t,1H,4c- H),1.61 (m,2H,OCH
2CH2CH3),0.91 ppm (t,3H,
OCH2CH2CH3); ESI HRMS: m/z: calcd for C21H38O15SNa: 585.18236;
found: 585.18243.
Allyl
2-O-(2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-b-d-glucopyrano-
H,2b- H,3c- H,6c- H),3.72–3.62 (m,7H,4a- H,4c- H,5c- H,6a- H, 6’a-H,
6’c-H, 6’b-H),3.50 (m,1H,5a- H),3.47 (dd, 3J=3.0,9.0 Hz,1H,3a- H),
3.38 (m,1H,5b- H),1.98 ppm (s,3H,Ac); 13C NMR (125 MHz,CDCl 3):
syl)-3,4,6-tri-O-benzyl-b-d-mannopyranoside (15): The procedure used
here was analogous to the preparation of 6. Glycosyl donor 14 (470 mg,
Chem. Eur. J. 2008, 14,6474 – 6482
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
6479

D. R. Bundle et al.
0.86 mmol),acceptor 13 (350 mg,0.72 mmol) were used and dichlorome-
thane (8 mL),trimethylsilyl trifluoromethanesulfonate (4.6 mL),activated
4 Š molecular sieves (100 mg) and the temperature was ꢀ108C. Column
chromatography in n-hexane/ethyl acetate 4:1 gave trisaccharide 15
(520 mg,83%). [ a]D = ꢀ24.48 (c = 1.0,CHCl 3); 1H NMR (600 MHz,
75.2,74.2,73.4,72.0,71.2,70.5,70.1,69.5,68.7,67.8 ppm; ESI HRMS:
z: calcd for C50H54O10SNa 869.33299; found: 869.33323.
Allyl
(2-O-acetyl-3,4,6-tri-O-benzyl-b-d-glucopyranosyl)-(1!2)-(3-O-
m/
benzyl-4,6-O-benzylidene-2-deoxy-2-thio-b-d-mannopyranosyl)-(1!2)-
3,4,6-tri-O-benzyl-b-d-mannopyranoside (19): The procedure used was
analogous to the preparation of 6. Glycosyl donor 5 (120 mg,0.19 mmol)
and disaccharide 18 (81 mg,0.096 mmol) were used,dichloromethane
(3 mL),trimethylsilyl trifluoromethanesulfonate (38 mL; 0.05m CH2Cl2),
activated 4 Š molecular sieves (20 mg) and the temperature was ꢀ108C.
Column chromatography in n-hexane/ethyl acetate 5:1 gave trisaccharide
19 (78 mg,62%). [ a]D = ꢀ57.88 (c = 1.0,CHCl 3); 1H NMR (600 MHz,
CDCl3): d=7.56–7.24 (m,25H,Ar),5.98–5.92 (m,1H,OCH
5.62 (s,1H,PhCH),5.38–5.34 (m,1H,OCH
2H,2b-H,OCH 2CH=CH2),5.05 (d, 3J=8.4 Hz,1H,1b- H),4.98–4.54 (m,
2CH=CH2),
2CH=CH2),5.25–5.20 (m,
8H,4CH Ph),4.45–4.40 (m,3H,1a-H,6b-H,OC
H2CH=CH2),4.25 (d,
2
3J=3.0 Hz,1H,2a- H),4.48–4.45 (m,1H,OC H2CH=CH2),3.94–3.82 (m,
4H,4b- H,3b- H, 6’b-H,6a- H),3.72–3.64 (m,2H,4a- H,6a- H),3.57–3.48
(m,3H,3a- H,5a- H,5b- H),2.12 ppm (s,3H,Ac); ESI HRMS:
m/z:
CDCl3): d=7.56–7.10 (m,40H,Ar),5.92–5.86 (m,1H,OCH
5.56 (s,1H,PhCH),5.29 (m,1H,1c- H),5.27–5.23 (m,1H,OCH 2CH=
CH2),5.18–5.14 (m,3H,1b- H,2c- H,OCH 2CH=CH2),5.00–4.38 (m,
2CH=CH2),
calcd for C52H56O12Na: 895.36640; found: 895.36597.
Allyl 3,4,6-tri-O-benzyl-2-O-(3-O-benzyl-4,6-O-benzylidene-2-O-trifluoro-
methanesulfonyl-b-d-glucopyranosyl)-b-d-mannopyranoside (16): Sodium
methoxide (3.24 mg,0.06 mmol) was added to a solution of 15 (520 mg,
0.60 mmol) in methanol (10 mL) and the resulting mixture was stirred
overnight at room temperature. Then it was neutralized with IR 120 (H+
form),and concentrated in vacuum. The crude product was dissolved in
dichloromethane (10 mL) and pyridine (270 mL) was added followed by
N,N-dimethyl-4-aminopyridine (24 mg,0.19 mmol). The resulting solution
was cooled to 08C and trifluoromethanesulfonic anhydride (180 mL,
1.08 mmol) was added dropwise,then the reaction mixture was warmed
up to room temperature and stirred for 2.0 h. The solution was diluted
with dichloromethane,washed with aqueous sodium bicarbonate,dried
over sodium sulfate,and concentrated to a brown oil. The residue was
purified by chromatography with ethyl acetate/hexane 1:4 to give 16
(520 mg,90%). [ a]D = ꢀ28.48 (c = 1.0,CHCl 3); 1H NMR (600 MHz,
17H,8CH Ph,OC H2CH=CH2),4.20 (m,1H,6b- H),4.16 (m,1H,2b- H),
2
4.07–4.03 (m,1H,OC H2CH=CH2),3.91–3.85 (m,3H,3c- H,3b- H,4a- H),
3.83–3.74 (m,6H,4c- H, 6’b-H,6a- H, 6’a-H,6c- H, 6’c-H),3.72–3.68 (m,
2H,4b- H,5c- H),3.60 (dd, 3J=3.0,9.0 Hz,1H,3a- H),3.45 (m,1H,5a-
H),3.38 (m,1H,5b-H),1.72 ppm (s,3H,Ac);
13C NMR (125 MHz,
CDCl3):d = 138.5–126.2,117.1,102.3,101.5,100.2,85.0,84.4,80.8,78.9,
78.6,78.5,75.5,75.4,75.1,75.1,75.0,73.4,73.1,73.0,70.3,69.8,69.6,69.5,
69.3,68.6,67.9 ppm; ESI HRMS:
1343.53723; found: 1343.53728.
m/z: calcd for C79H84O16SNa:
Allyl (3,4,6-tri-O-benzyl-b-d-glucopyranosyl)-(1!2)-(3-O-benzyl-4,6-O-
benzylidene-2-deoxy-2-thio-b-d-mannopyranosyl)-(1!2)-3,4,6-tri-O-
benzyl-b-d-mannopyranoside (20): The procedure used was analogous to
the preparation of 7 using trisaccharide 19 (75 mg,0.057 mmol),sodium
methoxide (0.3 mg),dichloromethane (2 mL) and methanol (1 mL).
Column chromatography in n-hexane/ethyl acetate 4:1 gave trisaccharide
CDCl3): d=7.42–7.21 (m,25H,Ar),5.96–5.90 (m,1H,OCH
5.54 (s,1H,PhCH),5.29–5.20 (m,3H,OCH 2CH=CH2,1b- H),4.95–4.90
(m,2H,CH 2Ph),4.82–4.75 (m,3H,2b- H,CH 2Ph),4.66–4.50 (m,4H,
2CH2Ph),4.47 (s,1H,1a-H),4.44–4.40 (m,1H,OC H2CH=CH2),4.38
(m,1H,6b-H),4.33 (d,1H,2a- H),4.06–4.02 (m,1H,OC H2CH=CH2),
3.92 (t, 3J=9.0 Hz,1H,3b- H),3.86–3.80 (m,2H,4b- H,6a- H),3.77 (t,
2CH=CH2),
1
20 (72 mg,100%). [ a]D = ꢀ57.88 (c = 1.0,CHCl ); H NMR (600 MHz,
3
CDCl3): d=7.52–7.20 (m,40H,Ar),5.92–5.86 (m,1H,OCH
2CH=CH2),
5.59 (s,1H,PhCH),5.31 (s,1H,1b-
H),5.26–5.18 (m,2H,OCH 2CH=
CH2),5.05 (d, 2J=10.8 Hz,1H, 1= CH2Ph),5.0–4.84 (m,4H,2CH 2Ph),
2
4.75 (d, 3J=10.2 Hz,1H,1c- H),4.68–4.52 (m,8H,2a- H, = CH2Ph),4.5–
7
2
4.38 (m,5H,1a- H,CH 2Ph,OC H2CH=CH2),4.35 (m,1H,6b- H),4.07–
4.0 (m,3H,2b-H,4a-H,OC H2CH=CH2),3.92–3.76 (m,5H,3b- H,4b- H,
6’b-H,6a- H, 6’a-H),3.73–3.66 (m,3H,3c- H,4c- H,6c- H),3.60–3.54 (m,
1H,6 ’b-H),3.67–3.62 (m,2H,4a-H,6
H,5a- H,5b- H).
’a-H0,3.58–3.46 ppm (m,3H,3a-
Allyl
2-O-3,4,6-tri-O-benzyl-(3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-
3H,3a- H,2c- H,5c- H),3.45–3.38 ppm (m,3H,5a-
H,5b- H, 6’c-H);
13C NMR (125 MHz,CDCl 3): d = 138.9–126.2,117.5,101.6,100.4,100.3,
thioacetyl-b-d-mannopyranosyl)-b-d-mannopyranoside (17): The proce-
dure used here was analogous to the preparation of 3,using compound
16 (1.2 g,1.25 mmol),potassium thioacetate (2.84 g,24.9 mmol),and
DMF (30 mL). Column chromatography in n-hexane/ethyl acetate 6:1
gave trisaccharide 17 (700 mg,63%). [ a]D = ꢀ77.08 (c = 1.0,CHCl 3);
1H NMR (500 MHz,CDCl 3): d=7.52–7.20 (m,25H,Ar),5.96–5.90 (m,
89.6,87.2,80.5,79.4,78.9,76.9,75.7,75.5,75.4,75.2,75.1,75.0,74.5,73.6,
73.4,70.9,70.6,70.3,69.9,69.8,69.5,68.7,67.9 ppm; ESI HRMS:
calcd for C77H82O15SNa: 1301.52667; found: 1301.52659.
m/z:
Allyl (3,4,6-tri-O-benzyl-b-d-mannopyranosyl)-(1!2)-(3-O-benzyl-4,6-O-
benzylidene-2-deoxy-2-thio-b-d-mannopyranosyl)-(1!2)-3,4,6-tri-O-
benzyl-b-d-mannopyranoside (21): The synthesis of 21 was accomplished
using the procedure for preparation of 8. Trisaccharide 20 (60 mg,
0.047 mmol),dimethyl sulfoxide (4 mL),acetic anhydride (2 mL),THF
(3 mL) and L-selectride (1m,0.5 mL) were used. Column chromatogra-
phy in n-hexane/ethyl acetate 5:2 gave trisaccharide 21 (30 mg,50%).
[a]D = ꢀ33.88 (c = 1.0,CHCl 3); 1H NMR (600 MHz,CDCl 3): d=7.50–
1H,OCH 2CH=CH2),5.55 (s,1H,PhCH),5.29 (s,1H,1b-H),5.28–5.18
3
(m,2H,OCH 2CH=CH2),4.90–4.74 (m,4H,2b- H, = CH2Ph),4.64–4.50
2
(m,5H, 5= CH2Ph),4.46–4.42 (m,1H,OC H2CH=CH2),4.41 (s,1H,1a-
2
H),4.35 (d, 3J=3.0 Hz,1H,2a- H),4.29 (m,1H,6a- H),4.04–4.00 (m,
1H,OC H2CH=CH2),3.96 (dd, 3J=4.5,9.5 Hz,1H,3b- H),3.82–3.68 (m,
5H,4a- H,4b- H, 6’a-H,6b- H, 6’b-H),3.54 (dd, 3J=3.0,9.0 Hz,1H,3a-
H),3.46–3.40 (m,2H,5a-
H,5b- H),2.22 ppm (s,3H,SAc);
13C NMR
7.05 (m,40H,Ar),5.89–5.82 (m,1H,OCH
CHPh),5.36 (s,1H,1c- H),5.32 (s,1H,1b-
2CH=CH2),5.57 (s,1H,
H),5.24–5.14 (m,2H,
(125 MHz,CDCl 3): d = 138.6–126.1,117.1,101.5,99.9,99.6,80.6,80.2,
75.9,75.5,75.1,74.6,73.4,72.1,71.5,70.4,69.9,69.6,68.7,67.6 ppm; ESI
HRMS: m/z: calcd for C52H56O11SNa: 911.34356; found: 911.34366.
OCH2CH=CH2),4.93–4.8 (m,4H,2 CH 2Ph),4.56–4.46 (m,8H,2a- H,7/2
3
CH2Ph),4.43 (s,1H,1a- H),4.41–4.3 (m,4H,OC H2CH=CH2, = CH2Ph),
2
4.23 (d,1H, 1= CH2Ph),4.14–4.13 (m,2H,2b-
H,2c- H),4.00 (m,1H,
H,4a- H,4c- H),3.80 (t, 3J=
Allyl
3,4,6-tri-O-benzyl-2-O-(3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-
2
thio-b-d-mannopyranosyl)-b-d-mannopyranoside (18): The procedure
used was analogous to the preparation of 4 using compound 17 (0.7 g,
0.79 mmol),hydrazine acetate (165 mg,1.58 mmol),and THF (20 mL).
OCH2CH=CH2),3.92–3.86 (m,3H,3b-
11.4 Hz,1H,4b- H),3.74–3.59 (m,8H,6a- H, 6’a-H,6b- H, 6’b-H,6c- H,
6’c-H,5a- H,3a- H),3.58 (dd, 3J=3.0,10.8 Hz,1H,3c- H),3.43–3.37 ppm
(m,2H,5b- H,5c- H); 13C NMR (125 MHz,CDCl 3): d = 138.5–126.1,
117.4,101.6,100.6,83.7,83.5,80.6,79.5,79.1,77.3,77.1,76.8,75.6,75.4,
75.3,75.2,75.1,74.5,73.5,73.4,71.6,70.9,70.5,70.2,69.7,69.2,68.7,68.6,
68.1 ppm; ESI HRMS: m/z: calcd for C77H82O15SNa: 1301.52667; found:
1301.52664.
Column chromatography in n-hexane/ethyl acetate 5:1 gave trisaccharide
1
18 (560 mg,84%). [ a]D = ꢀ77.08 (c = 1.0,CHCl ); H NMR (600 MHz,
3
CDCl3): d=7.48–7.20 (m,25H,Ar),5.94–5.86 (m,1H,OCH
2CH=CH2),
5.62 (s,1H,PhCH),5.26–5.19 (m,2H,OCH 2CH=CH2),5.18 (s,1H,1b-
H),4.92–4.52 (m,8H,4CH 2Ph),4.45–4.40 (m,3H,1a-H,2a-H,
OCH2CH=CH2),4.34 (t,1H,4b-H),4.29 (m,1H,6b-H),4.17 (m,1H,
2b-H),4.05 (m,1H,OC H2CH=CH2),3.90–3.80 (m,4H,4a-H,3b-H,6
Propyl (1-thio-b-d-mannopyranosyl)-(1!2)-(2-deoxy-2-thio-b-d-manno-
pyranosyl)-(1!2)-b-d-mannopyranoside (22): Compound 22 was ob-
tained according to the procedure described for preparation of 12 using
trisaccharide 21 (30 mg,0.023 mmol),activated 10% Pd/C (50 mg) and
MeOH (5 mL). Purification by HPLC on C18 silica provided product 22
’b-
H,6a-H),3.74 (m,1H,6a- H),3.58 (dd, 3J=3.0,9.0 Hz,1H,3a- H),3.48–
3.40 (m,2H,5a- H,5b- H),2.40 ppm (s,1H,SH);
CDCl3): d = 138.4–126.1,117.3,101.6,99.9,99.6,80.7,78.1,76.0,75.7,
13C NMR (125 MHz,
6480
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Chem. Eur. J. 2008, 14,6474 – 6482

Glycoconjugate Vaccines
FULL PAPER
(11 mg,85%). 1H NMR (600 MHz,D 2O): d=5.13 (s,1H,1c- H),5.05 (s,
1H,1b- H),4.76 (s,1H,1a- H),4.27 (d, 3J=3.6 Hz,1H,2a- H),4.10 (d,
3J=3.0 Hz,1H,2c- H),3.96–3.58 (m,13H,OCH 2,2b- H,3b- H,4b- H,3c-
H,3a- H,6a- H, 6’a-H,6b- H, 6’b-H,6c- H, 6’c-H),3.45 (t,1H,4a-H),3.42–
(m,2H,C 6H2),7.38–7.36 (m,2H,C 6H2),5.04 (s,1H,1c- H),4.93 (1H,
1b-H),4.58 (s,1H,1a- H),4.13 (d, 3J=3.6 Hz,1H,2a- H),4.03–4.01 (m,
2H,2c- H,OCH ),3.90–3.16 (m,19H,2b-H,3a-H,3b-H,3c-H,4a-H,4b-
2
H,4c-H,5a-H,5b-H,5c-H,6a-H,6
’a-H,6b-H,6 ’b-H,6c-H,6 ’c-H,OC H2,
3.34 (m,3H,5a- H,5b- H,5c- H),3.26 (t,1H,4c-
H),1.62 (m,2H,
CH2CO),2.67–2.62 (m,6H,NHCOC H2,CH 2CH2NH,COC H2CH2),2.26
OCH2CH2CH3),0.92 ppm (t,3H,OCH 2CH2CH3); ESI HRMS: m/z:
(t,2H,SC
H2CH2NH),1.97–1.87 (m,2H,OCH
2CH2CH2S),1.78–
calcd for C21H38O15SNa: 585.18236; found: 585.18243.
1.72 ppm (m,4H,CH
2CH2CH2CH2); ESI HRMS: m/z: calcd for
C35H54N2O20S2Na: 909.94; found: 909.28.
3-(2-Aminoethylthio)-propyl (b-d-mannopyranosyl)-(1!2)-(1-thio-b-d-
mannopyranosyl)-(1!2)-2-deoxy-2-thio-b-d-mannopyranoside (23): Pho-
toaddition of 2-aminoethanethiol (164 mg,1.45 mmol) to compound 11
(37 mg,0.029 mmol) in dichloromethane (1 mL) and methanol (5 mL)
with subsequent debenzylation under Birch condition as reported by lit-
erature[16] afforded free amine 23 (12 mg,65%). 1H NMR (600 MHz,
D2O): d=5.0 (s,1H,1b- H),4.90 (s,1H,1c- H),4.86 (s,1H,1a-H),4.33
(d, 3J=3.0 Hz,1H,2b- H),4.21 (d, 3J=3.6 Hz,1H,2a- H),4.00 (m,1H,
Glycoconjugates: The general procedure for generating protein-carbohy-
drate conjugates employing the hapten/protein molar ratios of Table 1
was as follows: BSA (10 mg) was dissolved in phosphate buffer pH 7.5
(2 mL),and the half ester dissolved in DMF (100 mL) was slowly injected
into the reaction medium,and the reaction mixture was left for one day
at room temperature. The reaction mixture was then diluted with deion-
ized water and dialysed against deionized water (2 L,changed 5”). The
tetanus toxoid (TT) (0.5 mL at 10 mgs/mL in PBS pH 7.2) was conjugat-
ed as described for BSA and after 24 h were dialyzed against PBS. The
solution of BSA conjugate was lyophilized to provide the product as a
white solid,while the TT conjugates were not freeze dried. MALDI-MS
(positive mode,matrix sinapinic acid,H 2O): inter-thio-trisaccharide–BSA
conjugate 28 (74677); terminal-thio-trisaccharide–BSA conjugate 29
(72247); inter-thio-trisaccharide–TT conjugate 30 (158430); terminal-
thio-trisaccharide–TT conjugate 31 (159542).
OCHa),3.95–3.6 (m,13H,OCHb,2c-
H,3a- H,3b- H,3c- H,4a- H,4b- H,
6a-H,6b- H,6c- H, 6’a-H, 6’b-H, 6’c-H),3.46–3.32 (m,3H,5a- H,5b- H,5c-
H),3.30 (t,1H,4c- H),3.28–3.22 (m,1H,SCH 2CH2NH2),2.58 (t,1H,
SCH2CH2NH2),2.55–2.52 (m,4H,CH 2CH2SCH2CH2NH2),1.92 ppm (m,
2H,OCH 2CH2CH2S); ESI HRMS: m/z: calcd for C23H44NO15S2Na:
638.21469; found: 638.21474.
3-(2-Aminoethylthio)-propyl
deoxy-2-thio-b-d-mannopyranosyl)-(1!2)-b-d-mannopyranoside
(1-thio-b-d-mannopyranosyl)-(1!2)-(2-
(24):
Compound 21 (80 mg,0.063 mmol) and 2-aminoethanethiol (500 mg) in
methanol/dichloromethane 5:1 (6 mL) were irradiated with UV light
(385 nm) and the product was subjected to subsequent debenzylation re-
action under Birch condition as reported by literature[16] to give free
amine 24 (58 mg,73%). 1H NMR (600 MHz,D 2O): d=5.13 (s,1H,1c-
H),5.07 (s,1H,1b- H),4.76 (s,1H,1a- H),4.29 (d, 3J=3.0 Hz,1H,2b- H),
4.11 (d, 3J=3.0 Hz,1H,2c- H),4.02 (m,1H,OC Ha),3.93 (dd,1H,1H,
3a-H),3.92–3.86 (m,3H,6a- H,6b- H,6c- H),3.83 (d, 3J=3.0 Hz,1H,2a-
H),3.76–3.56 (m,7H,OC Hb,3b- H,3c- H, 6’b-H, 6’c-H, 6’a-H,4b- H),
3.45 (t,1H,4a- H),3.42–3.34 (m,3H,5a- H,5b- H,5c- H),3.28–3.22 (m,
Preparation of antigens: Glycoconjugates were absorbed on alum by
mixing a PBS solution of TT conjugate (0.723 mL conjugate 30,0.663 mL
conjugate 31) with a PBS suspension of alum (3.337 and 3.277 mL,re-
spectively) and the mixture was rotated for at least 2 h or overnight at
48C. Each vaccine formulation contained 300 mg of conjugate per 1 mL
dose.
Immunization of rabbits: Groups of three New Zeeland white rabbits
(weighing approximately 3 kg) were given injections of 1 mL of alum de-
posited glycoconjugate formulation (0.2 mL into quadriceps/posterior
thigh,lumbar muscles (both sides) and 3 subcutaneous sites,0.2 mL
each). A booster injection was given on day 21 and blood samples were
drawn on days 0 and 31.
2H,4c- H,SCH CH2NH2),2.58 (t,1H,SCH 2CH2NH2),2.55–2.52 (m,4H,
2
CH2CH2SCH2CH2NH2),1.92 ppm (m,2H,OCH
2CH2CH2S); ESI
HRMS: m/z: calcd for C23H44NO15S2Na: 638.21469; found: 638.21463.
ELISA experiments: BSA–carbohydrate–protein conjugates (10 mgmLꢀ1
in PBS) were used to coat 96-well microtiter plates (MaxiSorp,Nunc)
overnight at 48C. The plate was washed 5” with PBST (PBS containing
0.05% (v/v) Tween 20). Sera were diluted with PBST containing 0.1%
BSA. The solutions were distributed on the coated microtiter plate and
incubated at room temperature for 2 h. The plate was washed with PBST
(5”) and goat anti-mouse IgG antibody conjugated to horseradish perox-
idase (Kirkegaard & Perry Laboratories; 1:2000 dilution in 0.1% BSA/
PBST; 100 mL per well) was added. The mixture was then incubated for 1
hour. The plate was washed 5” with PBST before addition of a 1:1 mix-
ture 3,3’,5,5’-tetramethylbenzidine (0.4 gLꢀ) and 0.02% H2O2 solution
(Kirkegaard & Perry Laboratories; 100 mL per well). After 2 min,the re-
action was stopped by addition of 1m phosphoric acid (100 mL per well).
Absorbance was read at 450 nm. Titres are recorded as the dilution
giving an absorbance 0.2 above background.
Inhibition EIA: O-linked trisaccharide–BSA conjugate (5 mgmLꢀ1 in
PBS) was used to coat 96-well microtiter plates (MaxiSorp,Nunc) over-
night at 48C. The plate was washed 5 times with PBST (PBS containing
0.05% (v/v) Tween 20). The IgG monoclonal antibody (C3.1) was pre-
mixed with various concentrations of inhibitors and added to wells
(100 mL per well) in triplicates,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 0.1% BSA/PBST; 100 mL per well) was
added. The mixture was then incubated for 1 h. The plate was washed 5
times with PBST before addition of a 1:1 mixture 3,3’,5,5’-tetramethyl-
benzidine (0.4 gLꢀ1) and 0.02% H2O2 solution (Kirkegaard & Perry Lab-
oratories; 100 mL per well). After 2 min,the reaction was stopped by ad-
dition of 1m phosphoric acid (100 mL per well). Absorbance was read at
450 nm and percent inhibition was calculated relative to wells containing
sera without inhibitor.
7-Aza-8,13-dioxo-13-(4-nitrophenoxy)-4-thiatridecanyl (b-d-mannopyra-
nosyl)-(1!2)-(1-thio-b-d-mannopyranosyl)-(1!2)-2-deoxy-2-thio-b-d-
mannopyranoside (26): To a solution of free amine 23 (5 mg,7.85 mmol)
in dry DMF (1 mL),diester 25 (30 mg,78.5 mmol) was added under
argon and the mixture was stirred for 5.0 h then TLC indicated the reac-
tion was almost complete. Finally,the reaction mixture was co-evaporat-
ed with toluene to remove DMF and the residue was dissolved in CH2Cl2
(10 mL),washed with H 2O (10 mL) containing 0.3% acetic acid. The
water solution was then passed through a C18-Sep-Pac cartridge and
eluted with methanol containing 0.3% acetic acid,to remove any com-
pound that would be irreversibly absorbed to the reverse phase silica
column. The solution was concentrated at low pressure to afford crude
product as a solid. Final purification on reverse phase silica (C18) was ac-
complished with a water methanol mixture containing 0.3% acetic acid
gradient to yield pure half ester 26 (4.1 mg,60%). 1H NMR (600 MHz,
CD3OD): d=8.30–8.28 (m,2H,C 6H2),7.38–7.36 (m,2H,C 6H2),4.91 (s,
1H,1b- H),4.75 (1H,1c- H),4.73 (s,1H,1a- H),4.20 (d, 3J=3.6 Hz,1H,
2b-H),4.06 (d, 3J=3.6 Hz,1H,2c- H),4.00 (m,1H,OC Ha),3.89–3.83
(m,3H,6a-H,6b-H,6c-H),3.76 (dd,
3J=4.2,9.0 Hz,1H,3a- H),3.70–
3.62 (m,4H,OC Hb,4b- H,4c- H, 6’a-H),3.60 (d, 3J=4.2 Hz,1H,2a- H),
3.55–3.51 (m,2H,6 ’b-H, 6’c-H),3.47–3.43 (m,2H,3b- H,3c- H),3.37 (t,
2H,C H2COO),3.30–3.19 (m,4H,4a- H,5a- H,5b- H,5c- H),2.67–2.62
(m,6H,NHCOC
SCH2CH2NH),1.97–1.87 (m,2H,OCH
H2,CH 2CH2NH,COC H2CH2),2.26 (t,2H,
2CH2CH2S),1.78–1.72 ppm (m,
4H,CH 2CH2CH2CH2); ESI HRMS: m/z: calcd for C35H54N2O20S2Na:
909.94; found: 909.28.
7-Aza-8,13-dioxo-13-(4-nitrophenoxy)-4-thiatridecanyl (1-thio-b-d-man-
nopyranosyl)-(1!2)-(2-deoxy-2-thio-b-d-mannopyranosyl)-(1!2)-b-d-
mannopyranoside (27): The procedure used was analogous to the prepa-
ration of 26 using free amine 24 (2.7 mg,4.2 mmol),diester 25 (16 mg,
42 mmol) and DMF (0.5 mL). Purification by HPLC on C18 silica gave
product 27 (2.4 mg,65%). 1H NMR (600 MHz,CD 3OD): d=8.30–8.28
Chem. Eur. J. 2008, 14,6474 – 6482
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
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Acknowledgement
This work was funded by the Alberta Ingenuity Centre for Carbohydrate
Science and the Canadian Institutes of Health Research (CIHR).
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6482
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Chem. Eur. J. 2008, 14,6474 – 6482