Synthesis and Immunological Studies of Linear Oligosaccharides of β-Glucan As Antigens for Antifungal Vaccine Development

Article abstract of DOI:10.1021/bc500575a

Antifungal vaccines have recently engendered considerable excitement for counteracting the resurgence of fungal infections. In this context, β-glucan, which is abundantly expressed on all fungal cell surfaces, functionally necessary for fungi, and immunologically active, is an attractive target antigen. Aiming at the development of effective antifungal vaccines based on β-glucan, a series of its oligosaccharide derivatives was designed, synthesized, and coupled with a carrier protein, keyhole limpet hemocyanin (KLH), to form new semisynthetic glycoconjugate vaccines. In this article, a convergent and effective synthetic strategy using preactivation-based iterative glycosylation was developed for the designed oligosaccharides. The strategy can be widely useful for rapid construction of large oligo-β-glucans with shorter oligosaccharides as building blocks. The KLH conjugates of the synthesized β-glucan hexa-, octa-, deca-, and dodecasaccharides were demonstrated to elicit high titers of antigen-specific total and IgG antibodies in mice, suggesting the induction of functional T cell-mediated immunity. Moreover, it was revealed that octa-, deca-, and dodeca-β-glucans were much more immunogenic than the hexamer and that the octamer was the best among these. The results suggested that the optimal oligosaccharide sequence of β-glucan required for exceptional immunogenicity was a hepta- or octamer and that longer glucans are not necessarily better antigens, a finding that may be of general importance. Most importantly, the octa-β-glucan-KLH conjugate provoked protective immunity against Candida albicans infection in a systemic challenge model in mice, suggesting the great potential of this glycoconjugate as a clinically useful immunoprophylactic antifungal vaccine. (Chemical Equation Presented).

Full text of DOI:10.1021/bc500575a

Article
pubs.acs.org/bc
Synthesis and Immunological Studies of Linear Oligosaccharides of
β‑Glucan As Antigens for Antifungal Vaccine Development
Guochao Liao,†,∥ Zhifang Zhou,†,∥ Srinivas Burgula,† Jun Liao,†,‡ Cheng Yuan,§ Qiuye Wu,‡
and Zhongwu Guo*,†
†Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States
‡School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
§Department of Pharmacy, Shanghai East Hospital, Tongji University, 150 Jimo Road, Shanghai 200120, China
S
* Supporting Information
ABSTRACT: Antifungal vaccines have recently engendered
considerable excitement for counteracting the resurgence of
fungal infections. In this context, β-glucan, which is abundantly
expressed on all fungal cell surfaces, functionally necessary for
fungi, and immunologically active, is an attractive target
antigen. Aiming at the development of effective antifungal
vaccines based on β-glucan, a series of its oligosaccharide
derivatives was designed, synthesized, and coupled with a
carrier protein, keyhole limpet hemocyanin (KLH), to form
new semisynthetic glycoconjugate vaccines. In this article, a convergent and effective synthetic strategy using preactivation-based
iterative glycosylation was developed for the designed oligosaccharides. The strategy can be widely useful for rapid construction
of large oligo-β-glucans with shorter oligosaccharides as building blocks. The KLH conjugates of the synthesized β-glucan hexa-,
octa-, deca-, and dodecasaccharides were demonstrated to elicit high titers of antigen-specific total and IgG antibodies in mice,
suggesting the induction of functional T cell-mediated immunity. Moreover, it was revealed that octa-, deca-, and dodeca-β-
glucans were much more immunogenic than the hexamer and that the octamer was the best among these. The results suggested
that the optimal oligosaccharide sequence of β-glucan required for exceptional immunogenicity was a hepta- or octamer and that
longer glucans are not necessarily better antigens, a finding that may be of general importance. Most importantly, the octa-β-
glucan−KLH conjugate provoked protective immunity against Candida albicans infection in a systemic challenge model in mice,
suggesting the great potential of this glycoconjugate as a clinically useful immunoprophylactic antifungal vaccine.
explored and shown to elicit protection against Candida in a
mouse model.9−11 Recent studies suggested that linear β-glucan
and its short oligosaccharides could elicit immune responses
and protection against C. albicans.12−14 Furthermore, it has
been demonstrated that the CMR197 conjugate of a synthetic β-
glucan hexasaccharide elicited stronger glucan-specific antibody
responses than the conjugate of natural β-glucan. As an
antifungal vaccine, the hexasaccharide conjugate was proposed
to be more promising than other synthetic or natural β-glucan
conjugates.12−14
Developing conjugate vaccines using synthetic oligosacchar-
ide antigens is a relatively new concept. One of the successful
stories of this approach was the development of a semisynthetic
anti-Haemophilus influenzae type b (Hib) vaccine.15 This type
of vaccine has some advantages. For example, its synthetic
antigens have defined chemical structures, which would
facilitate detailed immunological and structure−activity rela-
tionship studies to help gain more insight into the function of
INTRODUCTION
■
Fungal infection poses a great threat to human health, and its
cases grow rapidly year by year1 owing to the limitations of
current antifungal drugs and, especially, the emergence of drug-
resistant strains. As a result, deep-seated infections in
nosocomial settings have a high mortality even after treatment
with antifungal drugs.2,3 Moreover, many commensal and
opportunistic fungi, previously thought to be nonpathogenic,
have emerged as pathogens in immunocompromised pa-
tients.4,5 To meet the urgent medical need for antifungal
therapies, development of prophylactic and/or therapeutic
antifungal vaccines is increasingly considered to be one of the
most attractive and appropriate strategies.6
Beta-(1,3)-glucan (β-glucan) with sporadic branches linked
to the 6-O-position is an essential cell wall component of
various fungi.7 This biopolymer is exposed on the surface of
fungal cells and is functionally necessary; thus, it is an excellent
target antigen for the development of broadly useful antifungal
vaccines. It has been demonstrated that β-glucan can provoke
immunogenic protection against Candida albicans in mice.8
Therefore, a series of vaccines based on natural β-glucans, such
as their conjugates with diphtheria toxin CMR197, has been
Received: December 7, 2014
Revised: January 20, 2015
© XXXX American Chemical Society
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the vaccine and to optimize vaccine design. The reaction sites
and/or linkage positions of the carbohydrate antigens are well-
defined and predictable, which would improve vaccine quality
control. Therefore, as promising candidate antigens for vaccine
design, oligomeric β-glucans have received considerable
attention. For example, several groups have recently synthe-
sized linear tetra,16 penta,17 hexa,14,18,19 dodeca,20 hexadeca,21
and branched heptadeca oligosaccharides21 of β-glucans by
various methods. However, most conjugate vaccines studied so
far are made of heterogeneous natural β-glucans or
oligosaccharides derived from natural β-glucans, and there are
very few reports about conjugate vaccines derived from
synthetic β-glucan derivatives.12−14
In an effort to systematically explore the structure−
immunogenicity relationships of linear β-glucans and to
optimize the design of the antigen for creating more efficacious
antifungal vaccines, we have (1) developed a highly convergent
and effective method for the synthesis of oligosaccharides of β-
glucan with varied chain lengths, (2) coupled them with
keyhole limpet hemocyanin (KLH), and (3) evaluated the
immunological properties of resulting glycoconjugates and their
capability to elicit protective immune responses against C.
albicans in mice.
dicyano-1,4-benzoquinone (DDQ).22,23 Removal of the 3-O-
NAP group in 11 with DDQ was straightforward, giving 12 in
excellent yield (92%).26,27 Thereafter, 11 was coupled with 12
via preactivation glycosylation28,29 to obtain 13. Specifically,
glycosyl donor 11 was first activated with p-toluenesulfenyl
triflate (p-TolSOTf) that was generated in situ from the
reaction between p-toluenesulfenyl chloride (p-TolSCl) and
silver triflate (AgOTf) at −78 °C. Then, glycosyl acceptor 12
was added to furnish glycosylation, resulting in desired β-
disaccharide 13 (J1,2 = 7.5 Hz, 90% yield) in a stereospecific
manner, due to neighboring group participation. Compound 13
was used as one of the common glycosyl donors for subsequent
carbohydrate chain elongation. Removal of the NAP group in
13 with DDQ provided 14. A convergent [2 + 2] glycosylation
reaction between 13 and 14 by the same preactivation protocol
yielded tetrasaccharide 15 (86%) as a glycosyl donor for more
complex oligosaccharide assembly. Preactivated glycosylation of
2-azidoethanol with 13, followed by removal of the NAP group
with DDQ, afforded 16 (91%), which carried an azido group at
the nonreducing end. The azido group would be reduced to
form a primary amine later on to enable a selective reaction
with the linker and then coupling with carrier proteins.
Moreover, since the preactivation-based glycosylation reaction
was clean and high yielding and the donor and acceptor were
almost completely consumed, this allowed us to move on to the
next step after glycosylation, i.e., removal of the NAP group,
without purification of the reaction intermediate. Similarly,
preactivation-based glycosylation of 16 with 13 and then
removal of the NAP group produced tetrasaccharide 17. On the
basis of 17, the sugar chain was further elongated successfully
via preactivation-based glycosylation to achieve all of the
designed β-glucan oligosaccharides. Coupling of 17 with
disaccharide 13 and tetrasaccharide 15, followed by selective
removal of the NAP group, afforded hexasaccharide 18 and
octasaccharide 19, respectively. Subsequently, 19 was coupled
with 13 and 15, which was followed by NAP group removal to
produce decasaccharide 20 and dodecasaccharide 21. Notably,
the glycosylation yields were not significantly affected by the
increased size of the involved building blocks. All of the
synthetic intermediates and final products were fully charac-
terized, proving that the glycosylation reactions were β-specific.
Attempts to globally deprotect 18−21 via saponification
followed by hydrogenation or via hydrogenation followed by
saponification in one pot were unsuccessful, mainly due to the
poor mutual solubility of 18−21 and the partially deprotected
products in various solvent combinations. Eventually, they were
fully deprotected in a stepwise manner using the proper solvent
or solvent combination for each transformation, following the
order of Zn-mediated reduction of the azido group in
dichloromethane (DCM), acidic cleavage of all benzylidene
groups in acetic acid and water (5:1), and finally sodium
hydroxide-promoted removal of all benzoate groups in t-butyl
alcohol and water (4:1). The final products were purified with a
Sephadex-G25 size-exclusion column with distilled water as the
eluent to afford 22−25 as white fluffy solids upon
lyophilization. The final products were fully characterized by
both NMR and MS.
RESULTS AND DISCUSSION
■
On the basis of reports that a hexasaccharide of β-glucan was
highly immunogenic12−14 and that at least an octa- or
nonasaccharide may be required to generate special 3D
structures,22 we planned to prepare and compare hexa-, octa-,
deca- and dodecasaccharides of β-glucan (Figure 1). These
Figure 1. Structure of designed β-glucan oligosaccharides and their
protein conjugates 1−8.
oligosaccharides were coupled with KLH to form vaccines 1−4.
KLH is one of most commonly used and effective protein
carriers for glycoconjugate vaccine development.23 In the
meantime, the oligosaccharides were also coupled with human
serum albumin (HSA) to provide conjugates 5−8 that were
used as capture reagents for detecting β-glucan-specific
antibodies by enzyme-linked immunosorbent assay (ELISA).
Oligosaccharide Synthesis. As depicted in Scheme 1, the
designed β-glucan oligosaccharides were achieved through
preactivation-based iterative glycosylation with p-toluenethio-
glycosides as glycosyl donors and disaccharide 13 as a key
building block. The synthesis was commenced with the
preparation of 9 from D-glucose according to a literature
procedure in four steps and in 40% overall yield.24,25 Treatment
of 9 with dibutyltin oxide to furnish the stannylene acetal-
directed regioselective protection of the 3-O-position with a 2-
naphthylmethyl (NAP) group26,27 was followed by 2-O-
benzoylation of the resultant 10 to afford thioglycoside donor
11. Here, the NAP group was employed as a temporary
protecting group instead of the common para-methoxybenzyl
(PMB) group because the former is more stable under the
acidic conditions involved in glycosylation reactions, although
both groups can be readily removed with 2,3-dichloro-5,6-
Conjugation of Oligosaccharides 22−25 with Carrier
Proteins. Free oligosaccharides 22−25 were conjugated with
carrier proteins KLH and HSA through the bifunctional glutaryl
group, as this conjugation can be easily and effectively realized
via activated glutaryl ester and, not like other complex linkers,
this simple linker would not affect the immunological activity of
B
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a
Scheme 1. Synthesis of β-Glucan Oligosaccharides 22−25
a
Reagents and conditions: (a) Bu2SnO, toluene, reflux, 6 h; then 2-naphthylmethyl bromide, CsF, DMF, 70 °C, 12 h, 72%; (b) BzCl, Et3N, CH2Cl2,
rt, 12 h, 96%; (c) DDQ, CH2Cl2/H2O (18:1), rt, 8 h, 92% for 12, 95% for 14; (d) AgOTf, TTBP, p-TolSCl, CH2Cl2, −78 °C to rt, 4 h, 90% for 13,
86% for 15; (e) AgOTf, TTBP, p-TolSCl, CH2Cl2, −78 °C, rt, 4 h; then DDQ, CH2Cl2/H2O (18:1), rt, 8 h, 91% for 16, 90% for 17, 87% for 18,
81% for 19, 80% for 20, 85% for 21; (f) Zn, AcOH, CH2Cl2, 24 h, rt; then AcOH/H2O (5:1), 60 °C, 24 h; finally NaOH, t-BuOH/H2O, 40 °C, 24
h, 80% for 22, 88% for 23, 85% for 24, 88% for 25.
a
Scheme 2. Preparation of Oligo-β-glucan−Protein Conjugates 1−8
a
Reagents and conditions: (a) DSG, DMF, and PBS buffer (4:1), rt, 4 h; (b) KLH or HSA, PBS buffer, rt, 2.5 days.
conjugates.30,31 A two-step procedure was used to furnish the
conjugation (Scheme 2). First, reaction between the free amino
group in 22−25 and a large excess of active ester disuccinimidal
glutarate (DSG) gave the corresponding monoactivated esters
26−29 in quantitative yields. Then, 26−29 were coupled with
KLH or HSA in 0.1 M phosphate-buffered saline (PBS) to
afford desired glycoprotein conjugates 1−8, which were
purified with a Biogel A0.5 column to remove remaining free
sugars. The conjugate-containing fractions were dialyzed
against distilled water and lyophilized to give 1−8. Finally,
the glucose content of each conjugate was analyzed by the
phenol−sulfuric acid method following a reported protocol.32
The glucose content of the KLH and HSA conjugates was 7.5−
9.1% and 10.5−25.8%, respectively (Table 1), showing that the
coupling reactions were efficient and the antigen loading levels
were in the desired range for glycoconjugate vaccines.33 The
sugar loading of HSA conjugates was also confirmed by
MALDI-TOF mass spectrometry.
C
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The above immunological results revealed that, overall,
conjugates 2−4 induced significantly higher titers of both total
(anti-kappa) and IgG1 antibodies than 1 (P ≪ 0.01, Figure 3),
indicating that 2−4 were much more immunogenic and
provoked much stronger immune responses in mice than 1.
Further analysis of the immune responses showed that the
IgG1 antibody titer induced by 2 was significantly higher than
that induced by 3 and 4 (P < 0.01, Figure 3B) as well. Although
the total antibody titer for 2 was also slightly higher than that
for 3 and 4 (Figure 3A), this difference was less significant (P >
0.05 and <0.01, respectively). There are several factors that may
affect the immune response to a glycoconjugate, such as
carbohydrate loading,38,39 conjugation method, immunization
protocol, and carbohydrate antigen structure. The carbohydrate
loadings of 1−4 were very similar, and their conjugation
method and immunization protocol were identical. Therefore,
the different immunological properties for these glycoconju-
gates were because of their different carbohydrate structures,
and, among the oligosaccharides investigated here, octa-β-
glucan seemed to be the most immunogenic and the most
promising antigen for vaccine development.
Protection against Fungal Infection in Mice. To
ultimately prove the efficacy of the new glycoconjugate vaccine
to protect against fungal infections, conjugate 2, which elicited
the strongest immune responses in the above studies, was
evaluated in a fungal challenge experiment in mice. The fungus
used was C. albicans (strain SC5314), one of the most common
and important pathogenic fungi in clinic.40 In this experiment,
each group of 11 mice was immunized with 2 or PBS (the
control group) 4 times on days 1, 14, 21, and 28 according to
the above-mentioned protocols. On day 38, a predetermined
lethal dose of C. albicans (7.5 × 105 cells/mouse in 200 μL
Table 1. Carbohydrate Loading of Glycoconjugates 1−8
KLH conjugates
HSA conjugates
sample
1
2
3
4
5
6
7
8
loading (%)
8.3
7.8
7.5
9.1
10.5
11.0
14.3
25.8
Immunological Studies of Glycoconjugate Vaccines
1−4. The immunological properties of KLH conjugates 1−4
were investigated in female C57BL/6J mice. For this purpose,
each conjugate was thoroughly mixed with Titermax Gold
adjuvant, and the resulting emulsion was then injected
intramuscularly (i.m.) into mice. Following the initial
immunization, mice were boosted 4 times on days 14, 21, 28,
and 38 by subcutaneous (s.c.) injection of the same vaccine
emulsion. Blood samples of each mouse were collected through
the leg veins prior to the initial immunization on day 0 and after
immunizations on days 27, 38, and 48. Antisera were obtained
from clotted blood samples and were stored at −80 °C before
use. ELISA using the corresponding HSA conjugates as capture
reagents for plate coating was employed to determine antibody
titers, which reflected the elicited immune responses. Antibody
titers were defined as the dilution number yielding an OD value
of 0.2, and the results are shown in Figure 2.
Evidently, all of the KLH conjugates 1−4 elicited high titers
of antigen-specific total (kappa) antibodies, indicating strong
immune responses. More importantly, high titers of IgG1
antibodies were observed for all glycoconjugates, suggesting
memorable T cell-dependent immunities.34,35 IgG1 antibody is
usually considered to be the protective antibody isotype;36,37
thus, these conjugates were believed to elicit protective immune
responses and have great potential for being developed into
clinically functional vaccines against fungal infections.
Figure 2. ELISA results of the day 48 antisera obtained with conjugates 1 (A), 2 (B), 3 (C), and 4 (D) combined with Titermax Gold adjuvant,
respectively. The titers of corresponding antigen-specific antibodies are displayed. Each dot represents the antibody titer of an individual mouse, and
the black bar shows the average titer.
D
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Figure 3. Comparison of the average antibody titers of corresponding antigen-specific (A) total (anti-kappa) antibody and (B) IgG1 antibody in the
day 48 pooled antisera of mice immunized with conjugates 1−4. Each error bar is the standard deviation of three parallel experiments. *, P ≪ 0.01
#
compared to 1; , P < 0.05 compared to 2.
PBS) was given by i.v. injection to each mouse. The responses
of these mice were observed under normal feeding and care
conditions. As shown in Figure 4, mice in the control group
influence on the glycosylation efficiency. Furthermore, with the
help of neighboring group participation, all of the glycosylations
were highly stereoselective to create the desired β-anomer.
Therefore, this synthetic strategy can be widely applicable to
larger and more complex β-glucan derivatives via [n + n] or [n
+ (n + 1)] glycosylations.
Second, the synthesized oligosaccharides had a reactive
amino group at their reducing ends, enabling their effective
coupling with carrier proteins, such as KLH, through a
bifunctional linker. The resultant conjugates contained
structurally defined carbohydrate antigens to facilitate detailed
and in-depth investigation of glycoconjugate vaccines, such as
structure−activity relationship analysis. Although a number of
β-glucan oligosaccharides have been synthesized previously,
only a few have been conjugated with a carrier protein and
investigated as vaccines. On the other hand, conjugate vaccines
currently employed for biological studies are typically made of
heterogeneous natural β-glucans or oligosaccharides derived
from natural β-glucans.
Third, immunological studies of glycoconjugate vaccines 1−4
revealed that while all of them could elicit robust T cell-
dependent immune responses, octa-, deca-, and dodeca-β-
glucans were much more immunogenic than hexa-β-glucan,
which was different from the literature results.14 Close
structure−activity relationship analysis further revealed that
the immunogenicity decreased in the order of octa-, deca-, and
dodeca-β-glucans. These results suggest that at least an octamer
is necessary for oligo-β-glucans to be optimal antigens for
eliciting functional immune responses. However, this does not
necessarily mean that the longer the better for an
oligosaccharide antigen. As a result, an octa- or nona-β-glucan
was identified as the most promising antigen for designing and
developing β-glucan-based antifungal vaccines, which is of
general significance.
Figure 4. Survival time of mice immunized with 2 compared with mice
immunized with PBS after i.v. injection of C. albicans (7.5 × 105 cells
per mouse and 11 mice per group).
started to die of infection on day 6 after the fungal injection,
and all died within 4 days (on day 10). In comparison, mice in
the 2-immunized group did not have a fatal incident until day 8,
and on day 14, the animal survival rate was about 55%. At the
end of this experiment (on day 32), there were still 4 mice
(about 34%) in the immunized group unaffected, suggesting
complete protection of these mice from C. albicans infection.
These results proved that glycoconjugate 2 could elicit
protective immunity in mice against lethal systemic challenge
with C. albicans.
CONCLUSIONS
■
In summary, a series of β-glucan oligosaccharides was
synthesized and coupled with KLH to generate glycoconjugates
that contained structurally well-defined carbohydrate antigens.
These glycoconjugates were shown to elicit robust T cell-
dependent and protective immune responses in mice, which
helped to identify some promising antifungal vaccines. This
work is distinguished from previous studies in the area in
several aspects. First, a highly convergent, effective, and
potentially broadly applicable strategy was developed for the
synthesis of structurally well-defined β-glucans. Large oligo-
saccharides could be rapidly assembled from short oligosac-
charide segments by the preactivation-based glycosylation
protocol that had significantly reduced the number of steps
for anomeric manipulation. It was further observed that the size
of the oligosaccharide segments used for the synthesis had little
Finally and most importantly, we have demonstrated in a
mouse model that the conjugate of KLH and octa-β-glucan,
namely, 2, could elicit protective immune responses against the
deadly pathogen, C. albicans. This result is highly relevant to
clinic application. Therefore, this work has paved the
foundation for developing an effective and clinically useful
antifungal vaccine.
EXPERIMENTAL SECTION
■
General Experimental Methods. Chemicals and materials
were obtained from commercial sources and were used as
received without further purification unless otherwise noted.
MS 4 Å were flame-dried under high vacuum and used
immediately after cooling under a N2 atmosphere. Analytical
E
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TLC was carried out on silica gel 60 Å F254 plates with
detection by a UV detector and/or by charring with 15% (v/v)
H2SO4 in EtOH. NMR spectra were recorded on a 400, 500, or
600 MHz machine, with chemical shifts reported in ppm (δ)
downfield from tetramethylsilane (TMS), which was used as an
internal reference.
CH2Cl2. The combined organic layers were washed three times
with saturated aqueous NaHCO3 solution and dried over
Na2SO4. After removal of the solvent in vacuum, the product
was purified by silica gel chromatography.
p-Tolyl 2-O-Benzoyl-4,6-O-benzylidene-1-thio-β-D-gluco-
pyranoside (12). Compound 12 (5.6 g, 92%) was prepared
from 11 (7.88 g, 12.73 mmol) and DDQ (5.78 g, 25.47 mmol)
according to the general procedure for deprotection of
naphthylmethyl ethers and was purified by flash column
chromatography (toluene/EtOAc 15:1 to 10:1). 1H NMR
(500 MHz, CDCl3): δ 8.12 (d, J = 8.24 Hz, 2H), 7.64−7.61 (m,
1H), 7.51−7.48 (m, 4H), 7.40−7.36 (m, 5H), 7.13 (d, J = 7.93
Hz, 2H), 5.56 (s, 1H Ph-CH), 5.14 (dd, J = 9.77, 8.85 Hz, 1H),
4.84 (d, J = 10.07 Hz, 1H, H-1), 4.43 (dd, J = 10.68, 4.88 Hz,
1H), 4.06−4.02 (m, 1H), 3.82 (t, J = 10.38 Hz, 1H), 3.62−3.53
(m, 2H), 2.83 (br s, 1H), 2.36 (s, 3H). 13C NMR (125 MHz,
CDCl3): δ 165.9, 138.7, 136.8, 133.8, 133.5, 130.1, 129.8, 129.6,
129.4, 128.5, 128.4, 127.9, 126.3, 101.9 (Ph-CH), 86.8 (C-1),
p-Tolyl 4,6-O-Benzylidene-3-O-(naphthalene-2-ylmethyl)-
1-thio-β-D-glucopyranoside (10). A mixture of diol 9 (9.0 g,
24.03 mmol) and Bu2SnO (7.18 g, 28.84 mmol) in anhydrous
toluene (400 mL) was refluxed in a flask equipped with a
Dean−Stark device for 6 h. After the mixture was cooled to
room temperature, the residual solvent was removed under
vacuum. CsF (7.99 g, 52.87 mmol), 2-bromomethylnaphtha-
lene (10.10 g, 45.66 mmol), and DMF (60 mL) were added,
and the resulting solution was stirred at 70 °C for 12 h, when
TLC showed completion of the reaction. After DMF was
removed under vacuum, the residue was dissolved in CH2Cl2
and washed with 1 M NaF aqueous solution. The organic phase
was dried over Na2SO4 and purified by flash column
chromatography (toluene/EtOAc 10:1) to give 10 (8.9 g,
25
80.6, 73.7, 73.3, 70.4, 68.5, 21.2. [α]D = −45.2° (c 0.5,
CHCl3). HRMS (ESI-TOF) m/z: calcd for C27H26NaO6S [M +
1
72%) as a white solid. H NMR (400 MHz, CDCl3): δ 7.85−
Na]+, 501.1348; found, 501.1345.
7.70 (m, 4H), 7.51−7.40 (m, 10H), 7.15 (d, J = 7.83 Hz, 2H),
5.60 (s, 1H Ph-CH), 5.12 (d, J = 11.74 Hz, 1H), 4.99 (d, J =
11.74 Hz, 1H), 4.57 (d, J = 9.29 Hz, 1H, H-1), 4.40 (dd, J =
10.27, 4.89 Hz, 1H), 3.81 (t, J = 10.27 Hz, 1H), 3.75 (t, J = 9.29
Hz, 1H), 3.68 (t, J = 9.29 Hz, 1H), 3.56−3.48 (m, 2H), 2.61
(br s, 1H), 2.36 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
138.8, 137.2, 135.6, 133.8, 135.3, 133.0, 129.9, 129.1, 128.3,
128.3, 127.9, 127.7, 127.1, 126.9, 126.1, 126.0, 125.9, 101.3
(Ph-CH), 88.64 (C-1), 81.4, 81.1, 74.8, 72.2, 70.7, 68.6, 21.2.
[α]D25 = −27.2° (c 0.5, CHCl3). HRMS (ESI-TOF) m/z: calcd
for C31H30NaO5S [M + Na]+, 537.1712; found, 537.1709.
p-Tolyl 2-O-Benzoyl-4,6-O-benzylidene-3-O-(naphtha-
lene-2-ylmethyl)-1-thio-β-D-glucopyranoside (11). A solution
of 10 (8.6 g, 16.71 mmol), triethyl amine (5.8 mL, 41.77
mmol), benzoyl chloride (2.33 mL, 20.05 mmol), and a
catalytic amount of N,N-dimethylaminopyridine in anhydrous
CH2Cl2 (150 mL) was stirred at room temperature overnight.
The reaction mixture was washed with saturated aqueous
NaHCO3 solution (3 × 150 mL), and the organic layer was
dried over Na2SO4. The desired product 11 was obtained (9.9
g, 96%) after purification by flash column chromatography
General Procedure for Preactivation-Based Glycosy-
lation Reactions. After the mixture of a glycosyl donor (1
mmol) and 4 Å MS (1.5 g) in CH2Cl2 (20 mL) was stirred at
room temperature for 1 h, it was cooled to −78 °C, and AgOTf
(3 mol in 6 mL dry acetonitrile) was added, followed by
addition of p-toluene sulfenyl chloride (p-TolSCl) (1 mmol)
via a microsyringe 10 min later. The mixture was stirred at −78
°C for an additional 15 min, when TLC showed that the donor
was completely consumed. A solution of the acceptor (1 mmol)
and 2,4,6-tri-tert-butyl pyrimidine (TTBP) (1 mmol) in
CH2Cl2 (5 mL) was added. The resulting mixture was stirred
for 20 min and warmed to room temperature, followed by
filtration to remove MS. The filtrate was washed with saturated
aqueous NaHCO3 solution and brine, dried over Na2SO4, and
concentrated under vacuum. The resultant crude product was
purified by silica gel flash column chromatography to get the
desired compound.
p-Tolyl [2-O-Benzoyl-4,6-O-benzylidene-3-O-(naphtha-
lene-2-ylmethyl)-β-D-glucopyranosyl]-(1→3)-2-O-benzoyl-
4,6-O-benzylidene-1-thio-β-D-glucopyranoside (13). Com-
pound 13 (6.15 g, 90%) was prepared from glycosyl donor
11 (4.37 g, 7.05 mmol) and acceptor 12 (3.375 g, 7.05 mmol)
according to the general procedure for preactivation-based
glycosylation and was purified by flash column chromatography
1
(hexanes/EtOAc/CH2Cl2 6:1:1) as a white solid. H NMR
(400 MHz, CDCl3): δ 7.99 (d, J = 7.34 Hz, 2H), 7.71−7.68 (d,
J = 8.80 Hz, 1H), 7.62−7.54 (m, 5H), 7.47−7.34 (m, 10H),
7.23 (d, J = 8.31 Hz, 1H), 7.10 (d, J = 7.83 Hz, 2H), 5.65 (s,
1H Ph-CH), 5.32 (dd, J = 9.78, 8.80 Hz, 1H), 4.99 (d, J = 12.23
Hz, 1H), 4.86 (d, J = 12.23 Hz, 1H), 4.78 (d, J = 9.78 Hz, 1H,
H-1), 4.45 (dd, J = 10.76, 5.38 Hz, 1H), 3.95 (t, J = 8.80 Hz,
1H), 3.87 (t, J = 10.27 Hz, 1H), 3.86 (t, J = 9.29 Hz, 1H),
3.60−3.54 (m, 1H), 2.34 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 165.0, 138.5, 137.2, 135.2, 133.6, 133.2, 133.0, 132.9,
129.9, 129.8, 129.7, 129.1, 128.3, 128.3, 128.2, 128.0, 127.8,
127.6, 127.0, 126.2, 126.1, 125.9, 125.7, 101.3 (Ph-CH), 87.2
(C-1), 81.5, 79.1, 74.3, 72.0, 70.6, 68.6, 21.2. [α]D25 = +19.5° (c
0.5, CHCl3). HRMS (ESI-TOF) m/z: calcd for C38H34NaO6S
[M + Na]+, 641.1974; found, 641.1979.
General Procedure for Deprotection of 2-Naphthyl-
methyl Ethers. To the stirred solution of a 2-naphthylmethyl
ether compound (1 mmol) in CH2Cl2 (18 mL) and water (1
mL) was added DDQ (2 mmol) at room temperature. After the
reaction was stirred for 8 h, saturated aqueous NaHCO3
solution was added, and the product was extracted with
1
(toluene/EtOAc 15:1). H NMR (500 MHz, CDCl3): δ 7.85
(d, J = 7.32 Hz, 2H), 7.70 (d, J = 7.93 Hz, 1H), 7.58−7.48 (m,
8H), 7.46−7.33 (m, 12H), 7.30−7.16 (m, 6H), 7.08 (d, J =
7.93 Hz, 2H), 5.58 (s, 1H Ph-CH), 5.33 (s, 1H Ph-CH), 5.29
(t, J = 7.02 Hz, 1H), 5.28 (dd, J = 9.77, 9.16 Hz, 1H), 4.89 (d, J
= 7.52 Hz, 1H H-1′), 4.87 (d, J = 12.51 Hz, 1H), 4.79 (d, J =
11.60 Hz, 1H), 4.78 (d, J = 10.07 Hz, 1H, H-1), 4.41 (dd, J =
10.38, 4.88 Hz, 1H), 4.21 (t, J = 9.16 Hz, 1H), 4.17 (dd, J =
10.68, 4.88 Hz, 1H), 3.95 (dd, J = 9.46, 9.16 Hz, 1H), 3.84 (t, J
= 10.38 Hz, 1H), 3.81 (dd, J = 9.46, 9.16 Hz, 1H), 3.75 (dd, J =
8.55, 7.63 Hz, 1H), 3.70 (dd, J = 10.38, 10.07 Hz, 1H), 3.61−
3.56 (m, 1H), 3.42−3.37 (m, 1H), 2.34 (s, 3H). 13C NMR (125
MHz, CDCl3): δ 164.7, 164.6, 138.5, 137.3, 137.1, 135.3, 133.5,
133.0, 132.8, 132.7, 129.9, 129.7, 129.7, 129.5, 129.5, 129.4,
129.1, 129.0, 128.4, 128.4, 128.3, 128.2, 128.1, 127.9, 127.6,
126.7, 126.2, 126.1, 126.0, 125.8, 125.7, 101.6 (Ph-CH), 101.0
(Ph-CH), 100.5 (C-1′), 87.4 (C-1), 80.7, 79.3, 79.0, 78.2, 73.7,
25
73.4, 72.3, 70.7, 68.7, 68.6, 65.9, 21.2. [α]D = +40.0° (c 0.5,
F
DOI: 10.1021/bc500575a
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
CHCl3). HRMS (ESI-TOF) m/z: calcd for C58H52NaO12S [M
+ Na]+, 995.3077; found, 995.3040.
according to the general procedure for preactivation-based
glycosylation, and the crude product was directly subjected to
deprotection by the general procedure to remove the
naphthylmethyl group to afford 16 (2.5 g, 91%), which was
purified by flash column chromatography (toluene/EtOAc 5:1).
1H NMR (400 MHz, CDCl3): δ 7.84 (d, J = 7.83 Hz, 2H), 7.74
(d, J = 7.83 Hz, 2H), 7.55−7.49 (m, 4H), 7.43−7.24 (m, 12H),
5.56 (s, 1H Ph-CH), 5.36 (s, 1H Ph-CH), 5.27 (dd, J = 8.31,
7.34 Hz, 1H), 5.12 (dd, J = 8.31, 7.34 Hz, 1H), 4.95 (d, J = 6.85
Hz, 1H, H-1′), 4.67 (d, J = 7.34 Hz, 1H, H-1), 4.37 (dd, J =
10.27, 4.89 Hz, 1H), 4.24−4.19 (m, 2H), 3.88−3.79 (m, 4H),
3.70 (dd, J = 10.76, 10.27 Hz, 1H), 3.66 (t, J = 9.29 Hz, 1H),
3.61−3.49 (m, 2H), 3.43−3.37 (m, 1H), 3.28−3.18 (m, 2H),
2.60 (br d, J = 3.91 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ
165.6, 164.6, 137.1, 136.9, 133.1, 133.0, 129.8, 129.7, 129.3,
129.3, 129.2, 129.0, 128.3, 128.3, 128.2, 126.3, 126.1, 101.7
(Ph-CH), 101.4 (Ph-CH), 101.1 (C-1), 100.29 (C-1′), 80.5,
79.1, 77.8, 75.2, 73.2, 72.6, 68.7, 68.6, 67.7, 66.5, 66.0, 50.6.
p-Tolyl [2-O-Benzoyl-4,6-O-benzylidene-β-D-glucopyrano-
syl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-gluco-
pyranoside (14). Compound 14 (1.63 g, 95%) was prepared
from 13 (2.0 g, 2.05 mmol) and DDQ (0.93 g, 4.11 mmol)
according to the general procedure for naphthylmethyl ether
deprotection and purified by flash column chromatography
(toluene/EtOAc 7:1). 1H NMR (500 MHz, CDCl3): δ 7.85 (d,
J = 8.24 Hz, 2H), 7.70 (d, J = 8.24 Hz, 2H), 7.56−7.50 (m,
4H), 7.43−7.27 (m, 14H), 7.08 (d, J = 7.63 Hz, 2H), 5.57 (s,
1H Ph-CH), 5.33 (s, 1H Ph-CH), 5.26 (dd, J = 9.77, 8.85 Hz,
1H), 5.09 (dd, J = 7.63, 7.32 Hz, 1H), 4.93 (d, J = 7.02 Hz, 1H,
H-1′), 4.79 (d, J = 10.07 Hz, 1H, H-1), 4.41 (dd, J = 10.38, 4.88
Hz, 1H), 4.25 (t, J = 8.85 Hz, 1H), 4.17 (dd, J = 10.38, 4.88 Hz,
1H), 3.86−3.77 (m, 3H), 3.70−3.66 (m, 2H), 3.61−3.56 (m,
1H), 3.39−3.34 (m, 1H), 2.63 (br s, 1H), 2.33 (s, 3H). 13C
NMR (125 MHz, CDCl3): δ 165.5, 164.7, 138.5, 137.1, 136.9,
133.5, 133.1, 133.0, 129.8, 129.8, 129.7, 129.5, 129.4, 129.3,
129.2, 128.3, 128.3, 128.2, 126.3, 126.2, 101.6 (Ph-CH), 101.56
(Ph-CH), 100.30 (C-1′), 87.36 (C-1), 80.4, 79.3, 79.1, 75.4,
72.6, 72.3, 70.7, 68.6, 68.6, 66.0, 21.2. HRMS (ESI-TOF) m/z:
calcd for C47H44NaO12S [M + Na]+, 855.2451; found,
855.2425.
25
[α]D = −22.0° (c 1, CHCl3). HRMS (ESI-TOF) m/z: calcd
for C42H42N3O13 [M + H]+, 796.2718; found, 796.2697.
2-Azidoethyl 3-O-[2-O-Benzoyl-4,6-O-benzylidene-β-D-glu-
copyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (17). Compound 17 (1.53 g, 90%) was
prepared from glycosyl donor 13 (1.10 g, 1.13 mmol) and
acceptor 16 (0.905 g, 1.13 mmol) according to the general
procedure for preactivation-based glycosylation and was
purified by flash column chromatography (toluene/EtOAc
p-Tolyl [2-O-Benzoyl-4,6-O-benzylidene-3-O-(naphtha-
lene-2-ylmethyl)-β-D-glucopyranosyl]-(1→3)-[2-O-benzoyl-
4,6-O-benzylidene-β-D-glucopyranosyl]-(1→3)-[2-O-benzoyl-
4,6-O-benzylidene-β-D-glucopyranosyl]-(1→3)-2-O-benzoyl-
4,6-O-benzylidene-3-O-1-thio-β-D-glucopyranoside (15).
Compound 15 (3.3 g, 86%) was prepared from glycosyl
donor 13 (2.23 g, 2.29 mmol) and acceptor 14 (1.91 g, 2.29
mmol) according to the general procedure for preactivation-
based glycosylation and was purified by flash column
1
4:1). H NMR (400 MHz, CDCl3): δ 7.92 (d, J = 7.83 Hz,
2H), 7.87 (d, J = 7.83 Hz, 2H), 7.79 (d, J = 7.83 Hz, 2H), 7.67
(d, J = 7.83 Hz, 2H), 7.57−7.44 (m, 7H), 7.42−7.30 (m, 17H),
7.28−7.18 (m, 8H), 5.54 (s, 1H), 5.42 (s, 1H), 5.18 (t, J = 7.34
Hz, 1H), 5.17 (t, J = 8.80 Hz, 1H), 5.05 (d, J = 7.83 Hz, 1H
anomeric), 5.00 (d, J = 5.38 Hz, 1H anomeric), 4.94 (dd, J =
8.31, 7.83 Hz, 1H), 4.87 (s, 1H), 4.83 (t, J = 8.80 Hz, 1H), 4.82
(d, J = 5.38 Hz, 1H anomeric), 4.76 (s, 1H), 4.56 (d, J = 7.83
Hz, 1H anomeric), 4.34 (dd, J = 10.27, 4.40 Hz, 1H), 4.21 (dd,
J = 10.76, 4.89 Hz, 1H), 4.15−4.06 (m, 4H), 4.00−3.92 (m,
3H), 3.89−3.84 (m, 1H), 3.76−3.63 (m, 3H), 3.60−3.42 (m,
8H), 3.37 (t, J = 9.29 Hz, 1H), 3.30−3.20 (m, 2H), 2.66 (br d, J
= 3.42 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 165.8, 164.7,
164.6, 164.6, 137.3, 137.2, 137.1, 137.0, 133.6, 133.4, 133.2,
133.1, 129.9, 129.8, 129.7, 129.7, 129.4, 129.3, 129.3, 129.1,
128.9, 128.6, 128.6, 128.4, 128.4, 128.3, 128.3, 128.2, 128.1,
126.4, 126.3, 126.3, 126.1, 125.3, 101.8, 101.8, 101.3, 101.1,
100.8 (anomeric), 98.8 (anomeric), 98.4 (anomeric), 97.0
1
chromatography (toluene/EtOAc 12:1). H NMR (500 MHz,
CDCl3): δ 7.89 (d, J = 7.63 Hz, 2H), 7.82 (d, J = 7.93 Hz, 2H),
7.78 (d, J = 7.93 Hz, 2H), 7.71 (d, J = 7.93 Hz, 1H), 7.65 (d, J
= 7.63 Hz, 2H), 7.59−7.44 (m, 11H), 7.43−7.25 (m, 25H),
7.23−7.19 (m, 4H), 7.09 (d, J = 7.93 Hz, 2H), 5.55 (s, 1H Ph-
CH), 5.46 (s, 1H Ph-CH), 5.35 (dd, J = 7.93, 7.63 Hz, 1H),
5.19 (dd, J = 5.80, 5.49 Hz, 1H), 5.01 (d, J = 6.41 Hz, 1H
anomeric), 4.99 (d, J = 7.93 Hz, 1H anomeric), 4.97 (s, 1H),
4.92 (d, J = 12.21 Hz, 1H), 4.87−4.79 (m, 4H, 1H anomeric),
4.69 (d, J = 10.07 Hz, 1H, H-1), 4.63 (s, 1H Ph-CH), 4.37 (dd,
J = 10.38, 4.58 Hz, 1H), 4.23 (dd, J = 10.38, 4.58 Hz, 1H),
4.16−4.07 (m, 4H), 3.97 (dd, J = 8.24, 7.93 Hz, 1H), 3.93−
3.90 (m, 2H), 3.85 (dd, J = 8.85, 8.24 Hz, 1H), 3.77−3.71 (m,
2H), 3.62−3.57 (m, 3H), 3.49−3.41 (m, 4H), 3.27 (dd, J =
9.46, 9.16 Hz, 1H), 2.34 (s, 3H). 13C NMR (125 MHz,
CDCl3): δ 165.0, 164.8, 164.6, 164.6, 138.3, 137.4, 137.3, 137.3,
137.9, 135.4, 133.5, 133.3, 133.2, 133.1, 133.1, 132.9, 132.8,
129.8, 129.8, 129.8, 129.7, 129.7, 129.6, 129.5, 129.4, 129.4,
129.1, 129.0, 129.0, 128.9, 128.6, 128.5, 128.4, 128.4, 128.3,
128.1, 128.0, 127.9, 127.9, 127.6, 126.7, 126.4, 126.3, 126.1,
126.0, 125.8, 125.6, 102.0, 101.1, 100.9, 100.9, 99.3 (anomeric),
98.0 (anomeric), 97.0 (anomeric), 87.9 (C-1), 81.3, 78.6, 78.5,
78.2, 77.1, 75.7, 74.3, 73.7, 73.5, 73.2, 73.0, 72.7, 70.7, 68.7,
(anomeric), 80.8, 78.7, 78.3, 77.5, 76.9, 75.0, 74.8, 74.4, 73.7,
25
73.4, 72.6, 72.5, 68.7, 68.6, 67.9, 66.5, 66.0, 65.5, 50.6. [α]D
=
+6.0° (c 0.5, CHCl3). HRMS (ESI-TOF) m/z: calcd for
C82H81N4O25 [M + Na]+, 1521.5190; found, 1521.5153.
2-Azidoethyl [2-O-Benzoyl-4,6-O-benzylidene-β-D-gluco-
pyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-glu-
copyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (18). Compound 18 (0.606 g, 87%) was
prepared from glycosyl donor 13 (0.306 g, 0.315 mmol) and
acceptor 17 (0.475 g, 0.315 mmol) by the same synthetic
procedure as that for 17 and was purified by flash column
25
68.7, 68.5, 66.0, 65.7, 65.4, 21.2. [α]D = +43.0° (c 0.5,
CHCl3). HRMS (ESI-TOF) m/z: calcd for C98H89O24S [M +
H]+, 1681.5465; found, 1681.5461.
2-Azidoethyl [2-O-Benzoyl-4,6-O-benzylidene-β-D-gluco-
pyranosyl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-gluco-
pyranoside (16). The reaction between 13 (3.5 g, 3.60 mmol)
and 2-azidoethanol (0.5 g, 5.65 mmol) was carried out
1
chromatography (toluene/EtOAc 10:1 to 6:1). H NMR (600
MHz, CDCl3): δ 7.95 (d, J = 7.2 Hz, 2H), 7.86 (d, J = 7.3 Hz,
G
DOI: 10.1021/bc500575a
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
2H), 7.81−7.77 (m, 5H), 7.65 (d, J = 7.3 Hz, 2H), 7.59 (d, J =
7.4 Hz, 2H), 7.51 (m, 2H), 7.47−7.17 (m, 45H), 5.52 (s, 1H),
5.43 (s, 1H), 5.22−5.17 (m, 2H), 5.10 (d, J = 7.5 Hz, 1H),
5.03−4.96 (m, 2H), 4.94−4.85 (m, 6H), 4.79 (m, 4H), 4.55 (d,
J = 7.6 Hz, 1H), 4.33 (dd, J = 10.4, 4.8 Hz, 1H), 4.22 (dd, J =
10.3, 4.9 Hz, 1H), 4.18−4.07 (m, 6H), 4.00 (m, 4H), 3.92 (dd,
J = 6.8, 4.4 Hz, 1H), 3.88−3.84 (m, 1H), 3.69 (m, 3H), 3.60
(m, 1H), 3.56−3.38 (m, 12H), 3.33−3.27 (m, 2H), 3.23 (m,
2H). 13C NMR (150 MHz, CDCl3): δ 165.86, 164.84, 164.6,
164.5, 137.4, 137.27, 137.25, 137.1, 137.0, 133.8, 133.6, 133.56,
133.5, 133.2, 133.1, 129.9, 129.74, 129.7, 129.6, 129.5, 129.4,
129.34, 129.31, 129.23, 129.17, 129.2, 129.1, 129.0, 128.97,
128.8, 128.7, 128.6, 128.58, 128.55, 128.4, 128.3, 128.27, 128.2,
128.1, 127.99, 126.4, 126.36, 126.33, 126.3, 126.1, 125.3, 101.9,
101.8, 101.3, 101.24, 101.21, 101.19, 100.7, 98.43, 98.39, 97.2,
97.1, 96.9, 80.8, 79.1, 78.7, 78.1, 77.9, 77.88, 77.5, 76.6, 75.0,
74.8, 74.6, 74.3, 73.7, 73.4, 73.2, 72.9, 72.5, 72.4, 68.7, 68.6,
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (20). Compound 20 (0.156 g, 80%) was
prepared from glycosyl donor 13 (108 mg, 0.11 mmol) and
acceptor 19 (158 mg, 0.05 mmol) by the same synthetic
procedure as that for 17 and was purified by flash column
1
chromatography (toluene/EtOAc 10:1 to 4:1). H NMR (500
MHz, CDCl3): δ 7.99 (d, J = 7.63 Hz, 2H), 7.89−7.79 (m,
15H), 7.68 (d, J = 7.93 Hz, 2H), 7.61 (d, J = 7.63 Hz, 2H),
7.57−7.17 (m, 79H), 5.53 (s, 1H), 5.46 (s, 1H), 5.24 (t, J =
7.93 Hz, 1H), 5.23 (t, J = 7.93 Hz, 1H), 5.15 (d, J = 7.63 Hz,
1H), 5.05 (d, J = 4.58 Hz, 1H), 5.00−4.77 (m, 23H), 4.56 (d, J
= 7.63 Hz, 1H), 4.35 (dd, J = 9.77, 4.27 Hz, 1H), 4.25 (dd, J =
10.38, 4.58 Hz, 1H), 4.18−4.09 (m, 10H), 4.07−3.96 (m, 8H),
3.94−3.86 (m, 2H), 3.76−3.68 (m, 3H), 3.66−3.41 (m, 22H),
3.40−3.23 (m, 7H), 2.64 (br s, 1H). 13C NMR (125 MHz,
CDCl3): δ 165.9, 164.9, 164.8, 164.77, 164.75, 164.72, 164.70,
164.68, 164.5, 137.8, 137.4, 137.36, 137.34, 137.31, 137.2,
137.1, 133.8, 133.7, 133.6, 133.58, 133.52, 133.19, 133.16,
129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.37, 129.33, 129.23,
129.19, 129.17, 129.1, 128.8, 128.7, 128.65, 128.58, 128.4,
128.3, 128.26, 128.18, 128.14, 128.1, 128.0, 126.5, 126.39,
126.36, 126.33, 126.1, 125.3, 105.0, 102.0, 101.8, 101.4, 101.33,
101.29, 101.26, 101.2, 101.1, 100.6, 98.4, 98.2, 97.2, 97.1, 97.02,
96.98(2C), 96.91, 96.86, 96.8, 80.9, 78.7, 78.1, 78.0, 77.94,
77.91, 77.88, 77.8, 77.5, 76.6, 74.8, 74.5, 74.4, 74.4, 74.2, 74.1,
73.8, 73.5, 73.1, 73.0, 72.95, 72.9, 72.6, 72.4, 68.7, 68.6, 68.0,
66.48, 66.06, 65.60, 65.55, 65.50, 65.47, 50.67. [α]D25 = +31.2°
(c 0.5, CHCl3). MS (MALDI TOF) m/z: calcd for
25
67.9, 66.5, 66.0, 65.6, 65.5, 65.48, 50.6. [α]D = +22.0° (c 0.5,
CHCl3). MS (MALDI TOF) m/z: calcd. For C122H114N3O37
[M + H]+, 2212.7131; found, 2212.7119.
2-Azidoethyl [2-O-Benzoyl-4,6-O-benzylidene-β-D-gluco-
pyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-glu-
copyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (19). Compound 19 (0.74 g, 81%) was
prepared from glycosyl donor 15 (0.53 g, 0.315 mmol) and
acceptor 17 (0.47 g, 0.315 mmol) by the same synthetic
procedure as that for 17 and purified by flash column
C
202H185N3NaO61 [M + Na]+, 3651.13; found, 3651.50.
1
chromatography (toluene/EtOAc 8:1 to 4:1). H NMR (400
2-Azidoethyl [2-O-Benzoyl-4,6-O-benzylidene-β-D-gluco-
MHz, CDCl3): δ 7.97 (d, J = 6.85 Hz, 2H), 7.87−7.77 (m,
12H), 7.66 (d, J = 7.83 Hz, 2H), 7.58 (d, J = 7.34 Hz, 2H),
7.55−7.16 (m, 62H), 5.51 (s, 1H), 5.45 (s, 1H), 5.22 (t, J =
7.83 Hz, 1H), 5.21 (t, J = 8.31 Hz, 1H), 5.12 (d, J = 7.83 Hz,
1H), 5.02 (d, J = 4.89 Hz, 1H), 4.94−4.79 (m, 16H), 4.77 (d, J
= 8.31 Hz, 1H), 4.55 (d, J = 7.34 Hz, 1H), 4.33 (dd, J = 10.76,
4.89 Hz, 1H), 4.23 (dd, J = 10.27, 4.89 Hz, 1H), 4.16−4.08 (m,
8H), 4.04−3.96 (m, 6H), 3.91−3.84 (m, 2H), 3.75−3.67 (m,
3H), 3.62−3.34 (m, 19H), 3.32−3.23 (m, 4H), 2.61 (br s, 1H).
13C NMR (100 MHz, CDCl3): δ 165.9, 164.9, 164.8, 164.7,
164.7, 164.7, 164.6, 137.4, 137.4, 137.4, 137.3, 137.2, 137.1,
133.8, 133.8, 133.7, 133.7, 133.9, 133.7, 133.64, 133.63, 133.56,
133.55, 133.22, 133.20, 133.19, 123.0, 129.8, 129.7, 129.66,
129.56, 129.44, 129.41, 129.34, 129.27, 129.25, 129.2, 129.13,
129.09, 129.06, 129.0, 128.9, 128.7, 128.6, 128.58, 128.44,
128.4, 128.33, 128.30, 128.2, 128.1, 128.05, 126.5, 126.48,
126.42, 126.38, 126.2, 125.4, 102.0, 101.8, 101.4, 101.39,
101.32, 101.27, 101.2, 100.7, 98.5, 98.32, 97.2, 97.2, 97.14,
97.05, 97.0, 96.9, 80.9, 79.0, 78.7, 78.1, 78.0, 77.9, 77.9, 76.6,
74.9, 74.8, 74.6, 74.5, 74.4, 74.3, 74.2, 73.8, 73.5, 73.2, 73.12,
73.11, 73.05, 72.95, 72.94, 72.5, 68.8, 68.7, 68.66, 68.6, 68.0,
pyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-glu-
copyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranoside (21). Compound 21 (0.380 g, 85%) was
prepared from glycosyl donor 15 (218 mg, 0.13 mmol) and
acceptor 19 (300 mg, 0.10 mmol) by the same synthetic
procedure as that for 17 and was purified by flash column
1
chromatography (toluene/EtOAc 12:1 to 3:1). H NMR (500
MHz, CDCl3): δ 7.97 (d, J = 7.02 Hz, 2H), 7.87−7.77 (m,
17H), 7.66 (d, J = 7.32 Hz, 2H), 7.59 (d, J = 7.32 Hz, 2H),
7.55−7.15 (m, 97H), 5.51 (s, 1H), 5.45 (s, 1H), 5.22 (t, J =
8.55 Hz, 1H), 5.21 (t, J = 7.32 Hz, 1H), 5.13 (d, J = 7.63 Hz,
1H), 5.02 (d, J = 5.19 Hz, 1H), 4.92−4.73 (m, 29H), 4.55 (d, J
= 7.63 Hz, 1H), 4.33 (dd, J = 10.38, 4.88 Hz, 1H), 4.24 (dd, J =
10.38, 4.88 Hz, 1H), 4.16−4.07 (m, 11H), 4.04−3.95 (m,
10H), 3.91−3.84 (m, 2H), 3.75−3.67 (m, 3H), 3.64−3.34 (m,
30H), 3.33−3.20 (m, 7H). 13C NMR (125 MHz, CDCl3): δ
165.9, 164.9, 164.8, 164.79, 164.78, 164.77, 164.76, 164.74,
164.72, 164.7, 164.6, 164.5, 137.9, 137.4, 137.33, 137.30, 137.2,
137.0, 133.8, 133.7, 133.6, 133.2, 129.9, 129.8, 129.7, 129.6,
129.5, 129.38, 129.35, 129.3, 129.23, 129.2, 129.1, 129.0, 128.8,
128.7, 128.6, 128.4, 128.3, 128.2, 128.18, 128.13, 128.08, 127.9,
25
66.5, 66.1, 65.6, 65.58, 65.51, 50.7. [α]D = +27.0° (c 0.5,
CHCl3). MS (MALDI TOF) m/z: calcd for C162H149N3NaO49
[M + Na]+, 2944.89; found, 2944.71.
2-Azidoethyl [2-O-Benzoyl-4,6-O-benzylidene-β-D-gluco-
pyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-glu-
copyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl]-(1→3)-[2-O-benzoyl-4,6-O-benzylidene-β-D-
H
DOI: 10.1021/bc500575a
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
126.5, 126.4, 126.35, 126.32, 126.1, 125.3, 102.0, 101.8, 101.4,
101.33, 101.30, 101.26, 101.24, 101.22, 101.20, 101.18, 101.1,
100.6, 98.4, 98.2, 97.1, 97.12, 97.09, 96.98 (2C), 96.91, 96.88,
96.85, 96.84, 96.8, 80.9, 78.7, 78.1, 78.0, 77.96, 77.9, 77.88,
77.8, 77.5, 77.3, 76.59, 76.58, 74.8, 74.5, 74.4, 74.3, 74.2, 73.8,
73.5, 73.2, 73.1, 73.0, 72.9, 72.8, 72.6, 72.4, 68.8, 68.7, 68.67,
68.62, 68.0, 66.5, 66.1, 65.6, 65.6, 65.5, 50.7. [α]D25 = +36.0° (c
0.5, CHCl3). MS (MALDI TOF) m/z: calcd for
C242H221N3NaO73 [M + H]+, 4359.35; found, 4360.53.
General Procedure for Global Deprotection of 18−21.
To a solution of 18, 19, 20, or 21 (23 μmol) in CH2Cl2 (12
mL) were added acetic acid (8 drops) and zinc powder (80
mg). The mixture was vigorously stirred at room temperature
for 24 h and then filtered through a pad of Celite. The filtrate
was condensed in vacuum, and the resulting residue was
dissolved in AcOH and H2O (5:1, 60 mL) and heated at 60 °C
for 24 h. The solvents were removed in vacuum, and the
residue was coevaporated with toluene 5 times. After the
product was dissolved in t-BuOH and H2O (4:1, 60 mL),
NaOH (120 mg) in H2O (6.0 mL) was added in portions. The
mixture was stirred at 40 °C for 24 h, neutralized with 0.25 N
HCl at 0 °C, and lyophilized. The crude product was purified
on a sephadex G-25 gel filtration column using water as the
eluent, and the product fractions were lyophilized to afford the
desired free oligosaccharides.
60.6, 60.3, 60.0, 39.4. HRMS (ESI-TOF) m/z: calcd for
C62H108NO51 [M + H]+, 1682.5888; found, 1682.5787.
2-Aminoethyl β-D-Glucopyranosyl-(1→3)-β-D-glucopyra-
nosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-
(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→
3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl]-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glu-
copyranosyl-(1→3)-β-D-glucopyranoside (25). Compound 25
(10.4 mg) was prepared from 21 (25.6 mg, 5.9 μmol) by the
1
above general procedure in 88% yield. H NMR (600 MHz,
D2O): δ 4.65−4.59 (m, 11H), 4.39 (d, J = 7.83 Hz, 1H), 3.98−
3.93 (m, 1H), 3.80−3.74 (m, 13H), 3.65−3.54 (m, 24H),
3.42−3.31 (m, 32H), 3.27−3.18 (m, 4H), 3.08−3.06 (m, 2H).
13C NMR (151 MHz, D2O): δ 102.7, 102.4, 101.8, 84.1, 84.1,
83.9, 75.9, 75.5, 73.4, 73.2, 72.7, 71.6, 69.6, 69.5, 69.3, 68.0,
65.8, 64.8, 62.4, 60.6, 60.4, 60.3, 60.0, 39.3. HRMS (ESI-TOF)
m/z: calcd for C74H128NNaO61 [M + H + Na]2+, 1014.8421;
found, 1014.8417.
General Procedure for Activation of Amino Oligosac-
charides 22−25. Each oligosaccharide was dissolved in DMF
and 0.1 M PBS buffer (4:1, 0.5 mL), and then disuccinimidal
glutarate (15 equiv) was added to the solution. The reaction
was kept under gentle stirring at room temperature for 4 h,
followed by removal of the solvents under vacuum. The
excessive reagent was removed from the reaction by
precipitation with 9 volumes of EtOAc, and the precipitates
were washed 10 times with EtOAc followed by drying under
vacuum to give activated oligosaccharides 26−29.
General Procedure for Conjugating Activated Oligo-
saccharides 26−29 with KLH and HSA. After solutions of
26−29 and KLH or HSA in a molar ratio of 30:1 in 0.1 M PBS
buffer (0.35 mL) were gently stirred at room temperature for 3
days, they were applied to a Biogel A0.5 column to separate the
resultant glycoconjgates 1−8 from unreacted oligosaccharides
using 0.1 M PBS buffer (I = 0.1, pH 7.8) as the eluent.
Fractions containing glycoconjugates, which were confirmed by
the bicinchoninic acid (BCA) assay for protein and the
phenol−sulfuric acid assay for carbohydrate, were combined
and dialyzed against distilled water for 2 days. The solutions
were lyophilized to obtain 1−8 as white solids.
Analysis of the Carbohydrate Loadings of Glyco-
conjugates 1−8.32 Aliquots of a standard D-glucose solution
(1 mg/mL) in water were added in ten dry 10 mL test tubes in
5 μL increments to form standard samples that contained 0−50
μg of glucose. In another test tube, an accurately weighed
sample of to-be-analyzed glycoconjugate 1−8 was placed. The
content of glucose in each tested sample was estimated to be
also in the range of 0−50 μg. To all of the tubes were then
added 500 μL of 4% phenol and 2.5 mL of 96% sulfuric acid at
room temperature. About 20 min later, the resultant colored
solutions were transferred into cuvettes, and their absorption at
490 nm wavelength (A490) was measured. A sugar calibration
curve was created by plotting the A490 values of standard
samples against their glucose content (in micrograms), which
was employed to calculate the glucose content of each tested
glycoconjugate sample based on its A490 value. The
carbohydrate loading of each glycoconjugate was calculated
according to the following equation, and the results are shown
in Table 1.
2-Aminoethyl β-D-Glucopyranosyl-(1→3)-β-D-glucopyra-
nosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-
(1→3)-β-D-glucopyranosyl-β-D-glucopyranoside (22). Com-
pound 22 (18.7 mg) was prepared from 18 (50.0 mg, 23 μmol)
1
by the above general procedure in 80% yield. H NMR (600
MHz, D2O): δ 4.59 (m, 5H), 4.39 (d, J = 8.1 Hz, 1H), 3.97
(dd, J = 10.9, 5.1 Hz, 1H, 1/2OCH2CH2), 3.85−3.74 (m, 6H),
3.68−3.55 (m, 10H), 3.47−3.30 (m, 13H), 3.29−3.16 (m, 4H),
3.15−3.09 (m, 2H, CH2N3). 13C NMR (150 MHz, D2O): δ
102.7, 102.5, 101.8, 84.2, 84.0, 76.0, 75.6, 75.5, 73.4, 73.2, 72.7,
69.5, 68.0, 65.8, 60.6, 60.5, 60.03, 60.02, 39.4. HRMS (ESI-
TOF,) m/z: calcd for C50H88NO41 [M + H]+, 1304.3775;
found, 1034.3727.
2-Aminoethyl β-D-Glucopyranosyl-(1→3)-β-D-glucopyra-
nosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-
(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→
3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranoside (23).
Compound 23 (10.2 mg) was prepared from 19 (25 mg, 8.5
1
μmol) by the above general procedure in 88% yield. H NMR
(600 MHz, D2O): δ 4.57 (m, 7H), 4.35 (d, J = 8.0 Hz, 1H),
3.93 (m, 1H), 3.78−3.71 (m, 8H), 3.62−3.50 (m, 16H), 3.40−
3.26 (m, 22H), 3.19 (m, 3H), 3.08 (t, J = 4.9 Hz, 2H). 13C
NMR (150 MHz, D2O): δ 102.7, 102.4, 101.8, 84.1, 84.1, 84.0,
75.9, 75.5, 75.5, 73.4, 73.2, 73.1, 72.7, 69.5, 68.0, 67.9, 65.8,
60.6, 60.4, 39.3. HRMS (ESI-TOF) m/z: calcd for C50H88NO41
[M + H]+, 1358.4832; found, 1358.4775.
2-Aminoethyl β-D-Glucopyranosyl-(1→3)-β-D-glucopyra-
nosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-
(1→3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→
3)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→3)-β-D-
glucopyranosyl]-(1→3)-β-D-glucopyranoside (24). Com-
pound 24 (11.0 mg) was prepared from 20 (27.8 mg, 7.6
1
μmol) by the above general procedure in 85% yield. H NMR
(600 MHz, D2O): δ 4.66−4.59 (m, 9H), 4.39 (d, J = 8.24 Hz,
1H), 4.00−3.96 (m, 1H), 3.82−3.74 (m, 12H), 3.65−3.55 (m,
19H), 3.42−3.31 (m, 26H), 3.27−3.19 (m, 4H), 3.13−3.11 (m,
2H). 13C NMR (150 MHz, D2O): δ 102.7, 102.5, 101.8, 84.1,
84.0, 75.9, 75.6, 73.4, 73.2, 72.7, 71.6, 69.5, 69.4, 68.0, 65.78,
sugar weight in a tested sample
total weight of the sample
carbohydrate loading % =
× 100%
I
DOI: 10.1021/bc500575a
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Immunization of Mouse. After each glycoconjugate (2.17
mg of 1, 2.32 mg of 3, 2.40 mg of 5, or 1.98 mg of 7) was
dissolved in 0.3 mL of 10× PBS buffer, it was diluted with water
to form a 2× PBS solution (1.5 mL). The solution was well-
mixed with 1.5 mL of Titermax Gold adjuvant (1:1, v/v) to
form an emulsion according to the protocol given by the
manufacturer. Each group of six female C57BL/6J mice was
initially immunized (day 1) by i.m. injection of 0.1 mL of the
emulsion described above. Following the initial immunization,
mice were boosted 4 times on days 14, 21, 28, and 38 by s.c.
injection of the same conjugate emulsion. Therefore, each
injected dose of glycoconjugate contained about 6 μg of the
carbohydrate antigen. Mouse blood samples were collected
through the leg veins of each mouse on day 0 prior to the initial
immunization and on days 27, 38, and 48 after the boost
immunizations. Finally, antisera were obtained from the clotted
blood samples and stored at −80 °C before use.
The ELISA Protocol. Each well in the ELISA plate was
treated with 100 μL of a solution of an individual HSA
conjugate 5, 6, 7, or 8 (2 μg/mL) dissolved in coating buffer
(0.1 M bicarbonate, pH 9.6) at 4 °C overnight and then at 37
°C for 1 h, which was followed by washing (3 times) with PBS
buffer containing 0.05% Tween-20 (PBST) and treatment with
blocking buffer (10% BSA in PBS buffer containing NaN3) at
room temperature for 1 h. After 3 times of washing with PBST,
half-log serially diluted solutions (from 1:300 to 1:656 100) of a
pooled or an individual mouse antiserum in PBS were added to
the coated ELISA plates (100 μL/well), followed by incubation
at 37 °C for 2 h. The plates were then washed with PBST and
incubated at room temperature for another 1 h with a 1:1000
diluted solution of alkaline phosphatase (AP)-linked goat anti-
mouse kappa, IgG1, IgG2a, or IgM antibody (100 μL/well),
respectively. Finally, the plates were washed with PBST and
developed with 100 μL of p-nitrophenylphosphate (PNPP)
solution (1.67 mg/mL in buffer) for 30 min at room
temperature, which was followed by colorimetric readout at
405 nm using a microplate reader. The optical density (OD)
values were plotted against the logarithmic scale of antiserum
dilution values, and a best-fit line was obtained. The equation of
the line was employed to calculate the dilution value at which
an OD of 0.2 was achieved, and the antibody titer was obtained
as the inverse of the dilution value.
ASSOCIATED CONTENT
■
S
* Supporting Information
Calculated antibody titers from various ELISA experiments and
NMR and MS spectra of synthesized compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: zwguo@chem.wayne.edu.
Author Contributions
∥G.L. and Z.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by an NIH/NCI grant (R01
CA95142) and by the National Major Scientific and
Technological Special Project for “Significant New Drugs
Development” of China (grant no. 2012ZX09502001-005) and
the National High Technology Research and Development
Program of China (grant no. 2012AA021500). The authors
thank Dr. B. Ksebati for performing some of the 2D NMR
measurements. The 600 MHz NMR instrument used in this
study was financed by an NSF grant (CHE-0840413).
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DOI: 10.1021/bc500575a
Bioconjugate Chem. XXXX, XXX, XXX−XXX