Efficient convergent synthesis of Bi-, Tri-, and tetra-antennary complex type N-glycans and their HIV-1 antigenicity

Article abstract of DOI:10.1021/ja409097c

The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We r

Full text of DOI:10.1021/ja409097c

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Article
Efficient Convergent Synthesis of Bi-, Tri-, and Tetraantennary
Complex Type N-Glycans and Their HIV-1 Antigenecity
SACHIN SHANKAR SHIVATARE, Shih-Huang Chang, Tsung-I Tsai, Chien-Tai Ren, Hong-
Yang Chuang, Li Hsu, Chih-Wei Lin, Shiou-Ting Li, Chung-Yi Wu, and Chi-Huey Wong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja409097c • Publication Date (Web): 13 Sep 2013
Downloaded from http://pubs.acs.org on September 14, 2013
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Efficient Convergent Synthesis of Biꢀ, Triꢀ, and Tetraantennary Complex Type NꢀGlycans
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and Their HIVꢀ1 Antigenicity
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Sachin S Shivatare,†, , ShihꢀHuang Chang,†, TsungꢀI Tsai,† ChienꢀTai Ren,† HongꢀYang
‡ §
§
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Chuang,†,∞ Li Hsu,†,∞ ChihꢀWei Lin,†,∞ ShiouꢀTing Li,† ChungꢀYi Wu,*,†, and ChiꢀHuey
‡
Wong*,†
†. Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang,
Taipei 115, Taiwan
‡. Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program,
Academia Sinica, Nankang, Taipei 115, Taiwan
§
. Institute of Biochemical Sciences, National Taiwan University, 1 Roosevelt Road, Section
4, Taipei, 106, Taiwan
∞. Department of Chemistry and Institute of Biotechnology, National Taiwan University, 1
Roosevelt Road, Section 4, Taipei, 106, Taiwan
*To whom correspondence should be addressed: C.ꢀY. Wu, Tel: +886ꢀ2ꢀ27871263, Fax: +886ꢀ
2ꢀ27898771, Eꢀmail: cyiwu@gate.sinica.edu.tw or C.ꢀH. Wong, Tel: +886ꢀ2ꢀ27899400, Fax:
+886ꢀ2ꢀ27853852, Eꢀmail: chwong@gate.sinica.edu.tw (or wong@scripps.edu).
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Abstract
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The structural diversity of glycoproteins often comes from posttranslational glycosylation
with heterogeneous Nꢀglycans. Understanding the complexity of glycans related to various
biochemical processes demands a wellꢀdefined synthetic sugar library. We report herein a
unified convergent strategy for the rapid production of biꢀ, triꢀ, and tetraꢀantennary complex type
Nꢀglycans with and without terminal Nꢀacetylneuraminic acid residues connected via the αꢀ2,6 or
αꢀ2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic
steps through the use of shared intermediates to simplify the complicated procedures associated
with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides
were compiled to create a glycan array for the profiling of HIVꢀ1 broadly neutralizing antibodies
PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16,
we identified potential natural and unnatural glycan ligands, which may facilitate the design of
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carbohydrateꢀbased
immunogens
and
fasten
the
HIV
vaccine
development.
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Introduction
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The process of Nꢀlinked glycosylation of HIVꢀ1 envelope gp120 and gp41 glycoproteins
results in diverse carbohydrate structures, masking the underlying glycoprotein domains and thus
forming a "glycan shield" that can evade immune recognition.1 The outer envelope of
glycoprotein gp120 contains 24 glycosylation sites. Among those, 13 are linked to complex type
Nꢀglycans, and 11 are occupied by Nꢀglycans of the highꢀmannose or hybrid type, which in fact
account for half of the mass of gp120.2 The biosynthesis of Nꢀglycans begins in endoplasmic
reticulum (ER), where the Nꢀlinked glycan precursor, Glc3Man9GlcNAc2, is transferred coꢀ
translationally to the nascent gp160 polypeptide.3 The terminal glucose moieties are then
trimmed by ER resident glucosidase I and II, freeing gp160 from calnexin/calreticulin to exit ER.
As the gp160 transported from ER to Golgi, the glycan moieties get trimmed further by
mannosidase to form Man5GlcNAc2, a substrate for various Golgi resident cellular enzymes.4 Nꢀ
acetyl glucosamine transferase (GnT) catalyses the transfers of GlcNAc to the D1 arm of
Man5GlcNAc2 substrate, converting it to a hybrid type glycan. This hybrid glycoform is then a
substrate for modification into complex glycans, in which the D2 and D3 arm mannose is
cleaved, allowing addition of GlcNAc moieties by a series of GnT family enzymes to form
multiantennary mature oligosaccharides (Figure 1a). The occurrences of biꢀ, triꢀ, and tetraꢀ
antennary structures with various degrees of sialylations on gp120 surface were characterized by
mass spectrometric analysis.2, 5
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A series of broadly neutralizing antibodies (bNAbs) that recognize a cluster of
oligomannose type glycans in the V3 loop of gp120 is recently described.6 This epitope has
fueled considerable interests in exploiting oligomannose clusters as possible carbohydrateꢀbased
vaccines. However, most of the oligomannoseꢀbased glycoconjugate vaccines so far are unable
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to achieve exact mimicry of the carbohydrate epitope on cell surface and, therefore, failed to
elicit effective neutralizing antibodies.7 In contrast to previously identified oligomannose
targeting bNAbs, Mouquet et al. reported the first example of complex type Nꢀglycans binding
bNAb PGT121,8 which emphasized the role of complex type Nꢀglycans in eliciting neutralizing
antibodies.6 Thereafter, two studies demonstrated the dependence of sialylated complex and
hybrid type Nꢀglycans at a secondary binding site in addition to Man5GlcNAc2 at the primary
binding site recognized by two somatically related bNAbs, PG9 and PG16.9 However, the
binding preference of PG9 and PG16 to isolated carbohydrates in the absence of peptide
backbone has not been attempted, so there is no binding study for glycans only. As a part of our
interests in the development of carbohydrateꢀbased HIVꢀ1 vaccines, we developed a glycan
microarray of synthetic Nꢀlinked oligosaccharides found on gp120 for use to investigate the
glycan binding specificities of newly discovered HIVꢀ1 bNAbs. Our preliminary results showed
that without multiantennary glycans, we could not achieve a complete profiling since we know
the existence and abundance of the multiantennary glycans on gp120 surface. We established
that access to biꢀ, triꢀ, and tetraꢀantennary glycans would allow validation of the glycan
processing in the biosynthesis pathway to analyze the impact of various glycoforms on antibody
recognition.
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Much of the complexity associated with multiantennary oligosaccharide synthesis was
addressed by Danishefsky,10 Unverzagt,11 Huang12 and Reichardt12 in developing synthesis
strategies and methods. Not surprisingly, due to the difficulty of sialic acid chemistry, only a few
examples of fully sialylated triꢀ and tetraꢀantennary Nꢀglycans have been reported.14
Consequently, building a comprehensive complex type Nꢀglycanꢀbased sugar library for the
study of HIV glycosylation and related immune response would be a major challenge. Thus, an
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efficient strategy that generates diverse complex oligosaccharides would be advantageous.
Recently, Boons and coworkers developed a general strategy to chemoenzymatically synthesize
a library of unsymmetrical multiꢀantennary Nꢀglycans.13 Despite the advancement in synthetic
methodologies, the complete access to all possible HIVꢀgp120 related Nꢀlinked oligosaccharide
structures is still not avaliable to date. In this study, we described the development of a
convergent strategy that utilized oligosaccharyl fluoride as a versatile donor to cleanly conjugate
highly branched carbohydrate building blocks in a highly stereoꢀ and regioselective manner to
prepare unnatural and naturally occurring versions of multiantennary glycans. Then,
sialyltransferases were used to perform terminal sialylation that permitted access to both αꢀ2,6
and αꢀ2,3ꢀlinked glycans of desired purity to avoid complications in chemical synthesis. With a
larger and more complete sugar library, we conducted a glycan microarrayꢀbased profiling of
PG9 and PG16 to understand the binding specificity of these antibodies and the information was
used for the design of carbohydrateꢀbased vaccines.
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Results and Discussion
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Biꢀ, triꢀ and tetraꢀantennary complex glycans are composed of a central trisaccharide core
and equipped with two, three, and four antennae, respectively, with varying degree of sialylation
(Figure 1b). The analysis of strategic bond disconnection provides a guiding principle of total
chemical synthesis that tetraꢀantennary complex glycans (structure VII) could be assembled by
conjugation of three building units in two glycosylation events, followed by one enzymatic
sialylation (Figure 1c). Our general synthetic strategy envisioned the double regioꢀ and
stereoselective glycosylations at the 3ꢀO and 6ꢀO positions of the core trisaccharide with
strategically protected triꢀ/pentasaccharide donors. The resulting galactose terminated systems
would be further elaborated by sialyl transferases mediated enzymatic extension to recruit
diverse complex structures to our library. At the reducing end, the amino pentyl linker, which
was aimed at preserving the native glycan structure, could serve as a handle for covalent
immobilization on NHSꢀcoated glass slides for microarray analysis and also for conjugation to a
carrier protein.
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Based on previous reports, the most challenging phase of complex oligosaccharide
synthesis involves the installation of highly branched triꢀ or pentasaccharide antennae at the
sterically hindered sites in the central core. Danishefsky and coworkers recently accomplished
the total synthesis of fully sialylated triꢀantennary Nꢀglycan and elaborated the intricacy of its
assembly.14ꢀ15 Dealing with the branched architecture, our strategy is to first gain access to
antennae 7, 8, and 9 (Scheme 1). The synthesis of key intermediate 3, which was commenced
with NIS/TMSOTf16 catalyzed coupling of glucosamine 1 and properly functionalized galactosyl
thioglycoside donor17 2 (See Supporting Information), required the complete βꢀselectivity.
Condensation of disaccharide donor 3 with mannosyl acceptors 4, 5, and 6 under the promotion
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of NIS and TMSOTf provided pentasaccharides 7, 8, and trisaccharide 9, respectively, in
excellent yield. It is noteworthy that the disaccharide 3, without further manipulation, was used
directly as a donor for a single or double glycosylation to facilitate antennae assembly in just two
glycosylation events from appropriate monosaccharide building blocks. Anticipating upcoming
difficulties associated with incorporation of antennae to the core, the D1/D2 arm building blocks
7, 8, and 9 were transformed into anomeric trichloroacetimidates and fluorides. The
trichloroacetimidate donors 7a, 8a, and 9a were prepared by selective cleavage of anomeric allyl
or pꢀmethoxyphenyl (PMP) ethers and subsequent reaction with trichloroacetonitrile in the
presence of DBU. Similarly, the synthesis of glycosyl fluorides 7b, 8b, and 9b was approached
through the exposure of anomeric ꢀOH to DAST18. With two kinds of glycosyl donors in hand,
we next sought to evaluate the compatibility of these branched oligosaccharide donors for regioꢀ
and stereoselective glycosylation.
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The key intermediate 10, a chitobiose trisaccharide precursor, was synthesized on
multigram scales through a sequence of coupling of the reducing end diꢀglucosamine acceptor
with a glucoꢀtype donor. Next, the βꢀMannoside structure was accessed via 2ꢀOTf mediated SN2
inversion of βꢀglucoside (See Supporting Information). With all required elements in place, the
assembly of skeleton was initiated by 3ꢀO glycosylation of the core trisaccharide. We initially
used Schmidt's19 trichloroacetimidate donor, which was extensively utilized by Unverzagt12 in
the synthesis of multiantennary glycans. Installation of the D1 arm trisaccharide 9a via
glycosidation at the 3ꢀO position of core 10 in the presence of BF3ꢁEt2O afforded desired
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hexasaccharide 11 in 60% yield. However, using fluoride donor 9b in Cp2HfCl2/AgOTf
promoted glycosylation, we observed a slight improvement in yield (Scheme 2). Then, pꢀtoluene
sulfonic acid mediated reductive benzyledene ring opening of 11 afforded diol 12, which served
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as a common precursor in the construction of biꢀ and triꢀantennary glycans. Then, diol 12 was
glycosidated with donor 9a in the presence of BF3ꢁEt2O or TMSOTf to afford nonasaccharide 13
in low yield (Table 1). Likewise, glycosidation with 7a under similar conditions led to donor
decomposition. We suspected that trichloroacetimidates may experience the steric hindrance at
the attachment point and the low stability of donor in acidic conditions, resulting in the formation
of side products from the donor. On the other hand, engagement of fluorides 7b and 9b in the
reaction pathway to supply the corresponding biꢀ and triꢀantennary glycans 13 and 15,
respectively, proceeded very efficiently without forming any major side products. The
stereochemistry of all the glycosidic linkages was confirmed by NMR analysis (see Supporting
Information).
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After establishing the effective use of fluoride donor, we proceeded to obtain highly
branched tetraꢀantennary glycans. The preparative routes for undecaꢀ and triꢀdecasaccharide
derivatives are via regioselective 3ꢀO glycosylation of 10 with 8b catalyzed by
Cp2HfCl2/AgOTf22 to furnish the desired octasaccharide 17 in 68% yield (Scheme 3). Using
another trichloroacetimidate donor 8a and catalyzed by BF3ꢁEt2O, the desired octasaccharide 17
was obtained in 25% with minor acceptor recovery (Table 1). Compound 17 was undergone
reductive ring opening in the presence of pꢀtoluene sulfonic acid to afford diol 19. To exploit the
higher reactivity of primary ꢀOH, compound 19 was condensed with the D2 arm 9b and
pentasaccharide 7b in the presence of Cp2HfCl2/AgOTf. The triꢀantennary 20 and tetraꢀantennary
22 glycans were obtained in satisfactory yields (Scheme 3). Our results showed that
glycosylation with fluorides (7b, 8b, and 9b) under Cp2HfCl2/AgOTf mediated condition
proceeded very efficiently with short reaction time and less side products (Table 1). Throughout
the aboveꢀmentioned protocols, we utilized a versatile glycosyl fluoride donor and exclusive
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glycosylation condition (Cp2HfCl2/AgOTf) to prepare multiple target products, which effectively
reduce the synthetic steps.
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With compounds 13, 15, 17, 20, and 22 in hand, we next proceeded to the stage of global
deprotection. The process commenced with removal of phthalimide protection by using nꢀbutyl
alcohol and ethylene diamine at 90 °C. This mixture was then exposed to Nꢀacetylation of the
free amine followed by deacetylation using sodium methoxide. Finally, the compounds were
subjected to hydrogenolysis to supply 14, 16, 18, 21, and 23, respectively. Throughout the global
deprotection process, and three rounds of column purifications, the products were, however,
obtained in relatively low yields. Structure studies are done by extensive NMR analysis (See
Supporting Information II for spectra). Motivated by Man4GlcNAc2 (D1 arm of Man9GlcNAc2)
recognition by HIVꢀ1 bNAb 2G12,23 we also prepared the unnatural form 18 for comparative
study.
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The chemical installation of sialic acidꢀcontaining antennae to assemble complex
oligosaccharides is particularly challenging due to the lack of participating group to achieve the
desired stereochemistry and the complexity of tedious multistep protection and deꢀprotection
processes24. However, the enzymatic glycosylation with glycosyl transferases is a single step
process with high regioꢀ and stereoꢀspecificity in excellent yield. As a result, the enzymatic
method of sialylation,20,21 which was well demonstrated previously25 especially through the
synthesis of sLex, was implemented in our strategy with regeneration of sugar nucleotide.26 In
general, bacterial enzymes are more stable and easier to express in Escherichia coli, and bacterial
glycosyltransferases have broader acceptorꢀsubstrate specificity than mammalianꢀderived
enzymes27. Yamamoto and coworkers have reported that αꢀ2,3ꢀsialyltransferase from Vibrio sp.
bacterium JTꢀFAJꢀ1628 and αꢀ2,6ꢀsialyltransferase from photobacterium sp. JTꢀISHꢀ22429 have
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very broad acceptor specificity, excellent productivity, high specific activity and no intrinsic
sialidase activity. The unique properties of these sialyltranferases provide a powerful tool to
construct the library of αꢀ2,3/2,6 sialosides. Therefore, the galactose terminated acceptor
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substrates 14, 16, 18, 21, and 23 were subjected to sialylation by using CMPꢀNeuAc as donor in
the presence of αꢀ2,6 and αꢀ2,3 sialyltransferases (see Supporting Information I) to furnish the
desired sialosides 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33, respectively, in respectable yields
(Scheme 4). Overall, our chemoenzymatic synthesis offers an efficient and convenient way to
access both αꢀ2,6 and αꢀ2,3 linked glycans of highly branched structures.
Profiling glycan specificities of HIVꢀ1 bNAbs PG9 and PG16. A series of broadly
neutralizing antibodies (bNAbs) that recognize a cluster of oligomannose type glycans in the V3
loop of gp120 have been described.6 This glycan recognition phenomenon was well documented
by Burton and Wilson through their study of bNAbs 2G12 and PGTs 127/128, which recognize
oligomannose clusters in the V3 loop of gp120.6,9,30 Previous structural and biochemical studies
showed the necessity of both the carbohydrate and peptide domains in forming an epitope of
PG9,9a,31 PG16,9b and PGT128,30b in contrast to 2G12, which apparently does not interact with
the gp120 peptide backbone. However, past work has largely relied on recombinant gp120,
supplied as mixtures of glycoforms that might not represent the actual glycosylation pattern on
native envelope. The glycan heterogeneity complicates the precise correlation between glycan
structure and immunoactivity. In the present study, we attempted to reveal the glycan specificity
of PG9 and PG16 by using the glycan array platform.
In order to examine the glycan binding specificity of antibodies PG9 and PG16 using
carbohydrate microarray, the synthetic multiantennary Nꢀglycan ligands were printed on Nꢀ
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hydroxy succinimideꢀactivated glass slides through covalent immobilization. The slides were
spotted by taking an aliquot from stock solution of individual glycans (100 ꢂm) (Fig. 2). Each
sample was printed with 10 replicates horizontally to form an array of 15 x 10 spots in each sub
array, and the slide image was obtained from a fluorescence scan after incubation with
DyLight649ꢀconjugated donkey antiꢀHuman IgG antibody. Our microarray analysis resulted in
almost no detectable binding for PG9, probably due to (1) very weak binding affinity towards
protein/peptide free glycans; (2) the requirement of closely spaced Man5GlcNAc2 (N160) and
complex type glycan (N156/163) as PG9 epitopes; and (3) the heterogeneous distribution of
NHS groups on glass slides resulting in uneven and low density glycan array. However, using
the glycan array on aluminumꢀoxide coated glass slides, we were able to identify the ligands
recognized by PG9 and the results will be published separately. On the other hand, PG16 showed
detectable binding to only αꢀ2,6ꢀlinked sialic acid terminated complex oligosaccharides,
implying significant structural specificity. Moreover, we observed that PG16 exhibited high
affinity binding to the triꢀantennary glycans 26, 30, and the tetraꢀantennary glycan 32 in addition
to the biꢀantennary glycan 28, a major complex type glycan observed on gp120, probably the
increase in number of antennae would offer additional binding sites for PG16.
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Determination of dissociation constants (KD.surf). To determine the dissociation
constants of αꢀ2,6ꢀsialylated complex glycans 24, 26, 30, and 32, that are bound on array
surface to interact with PG16, we used various concentrations (1.25ꢀ0.039 μM) of PG16 and
printed sugars (100ꢀ12.5 μM) to obtain binding curves, which were analyzed by the Langmuir
isotherm to generate surface dissociation constants (K D, surf.).32 To calculate the KD for glycan 32,
we plotted PG16 concentrations against the fluorescence intensities at various printing
concentrations (Figure 4). The dissociation constants (KD) and relative binding affinity of PG16
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for additional carbohydrate structures were calculated using the same method (Table 2, Figure S5,
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and Table S2 SI). Our data revealed that the tetraꢀantennary 32 (KD = 0.1 μM) and triꢀantennary
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30 (KD = 0.093 μM) exhibit highest binding affinity. Interestingly, the unnatural glycan 28 (KD
= 0.117 μM), a truncated version of tetraꢀantennary 32, showed at least threeꢀfold higher
affinity compared to its structural surrogate, biꢀantennary glycans 24 (KD = 0.327 μ M).
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However, it should be pointed out that the relative binding affinity of PG16 towards biꢀantennary
complex type glycan 24 (KD = 0.327μM) was found to be significantly higher than the glycan
affinity (KD = 1.19 mM) reported by Pancera et. al.9b by using STD NMR, probably due to
multivalent interactions between PG16 and glycan 24 printed on array. Careful structural
observation of 28 revealed that deletion of one mannose residue of naturally occurring biꢀ
antennary complex type structure might impose restriction on the flexibility of antennae. Such
restricted orientation may project two antennae at appropriate distance necessary for strong PG16
binding. Our detailed survey of structures and binding constants (KD) suggest that glycan 28
could be considered as an important unnatural glycan ligand of HIVꢀ1 bNAb PG16 in relation to
Man4GlcNAc2 (an important unnatural ligand of HIVꢀ1 bNAb 2G12). Further studies are needed
to understand the exact mode of binding of 28 to PG16 in comparison with the natural form 24.
Theoretically, immunization with vaccine constructs bearing different copies of unnatural glycan
28 would induce antibodies that may not target the host glycoproteins on their surfaces. In
principle, glycan 28 is structurally simpler and its synthesis could be achieved in fewer steps than
other glycans in the multiantennary series. For these reasons, we select glycan 28 as a potential
candidate for the development of carbohydrateꢀbased HIVꢀ1 vaccine and immune response
studies.
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Conjugation of carbohydrate antigen to carrier protein. Carbohydrate immunogenicity
can be enhanced by their covalent attachment to antigenic carrier proteins such as tetanus toxoid
(TT), diphtheria toxoid (DT), keyhole lympet hamocyanin (KLH), etc. Our group has recently
demonstrated that for Globo H (GH) based cancer vaccine, GHꢀDT adjuvanted with glycolipid
C34 showed the highest enhancement of antiꢀGH IgG.33 Thus, we explored DT as a potential
carrier protein for our carbohydrateꢀbased HIVꢀ1 vaccine candidate. We adopted the thiolꢀ
maleimide coupling chemistry to increase the carbohydrate epitope ratio on carrier protein to
reduce the amount of precious glycosyl thiol. Thus, glycan 28 with amino pentyl spacer at the
reducing end was modified with 3,3’ꢀdithiobis (sulfosuccinimidylꢀ propionate) (DTSSP) in
phosphate buffer (pH 7.2). Then, the disulfide bond was reduced by dithiothreitol (DTT) to
afford the desired Michael donor 34 after P2ꢀGel column purification. Surface modification of
DT with thioꢀactive maleimide functional group was performed by reaction with an excess of Nꢀ
(ꢀεꢀmaleimidocaproyloxy) sulfosuccinimide ester (SulfoꢀEMCS) in phosphate buffer (pH 7.2)
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for 2 h (Scheme 5). The loading of maleimide linkers on DT was confirmed by MALDIꢀTOF
mass analysis. Finally, maleimide derivatized DT was incubated with 5 and 20 equivalents of
thiolated decasaccharide 34 to afford glycoconjugates 36 with epitope ratio of four and 37 with
epitope ratio of eleven respectively. The unreacted maleimide functional groups on DT were
deactivated by excess amount of thioethanol (supporting information).
To assess the affinity of PG16ꢀglycoconjugate antigens, we used synthetic glycoconjugates
as a coating antigen to detect PG16 IgG antibody. The synthetic constructs were immobilized on
microtiter plate and titrated against serial PG16 dilutions. We found that DT modified with
carbohydrates could detect PG16 in a clear dose response (Figure 5). We also observed some
dose dependent nonꢀspecific binding of PG16 to carrier protein DT. A glycoconjugate construct
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37 with high carbohydrate content showed enhancement in sensitivity of the detection in
comparison with 36, perhaps due to multivalent interactions between PG16 and the glycans on
carrier protein. It should be noted that the relative binding affinity of our synthetic
glycoconjugates is still much lower than the affinity of PG16 to gp120. Taken together, the
present study provides the synthetic glycoconjugate constructs in an attempt to mimic the
putative epitope of PG16 for HIVꢀ1 vaccine design.
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Conclusion
We have successfully developed a highly efficient strategy based on the use of oligosaccharyl
fluorides for the convergent installation of branched galactose terminated antennae to achieve
high regioꢀ and stereoselectvity with excellent yields. Combining with sialyltransferases
mediated sialylation, this method allows easy access to αꢀ2,6 and αꢀ2,3ꢀsialylated glycans of biꢀ,
triꢀ and tetraꢀantennary complex type. With the glycans found on gp120 available from this
synthetic approach, a glycan array was thus prepared on glass slides, and the binding specificity
of PG16 towards a panel of complex Nꢀglycans in the carbohydrate microarray was used to
define the glycan composition required for strong antibody binding. Furthermore, the potential
candidate 28 was conjugated to the carrier protein DT for antibody binding studies. The results
confirmed that the glycoconjugates indeed bind to PG16.This study also revealed the problem of
glycan array on glass slides, which led to the development of glycan array on aluminum oxideꢀ
coated glass slides for use to identify mixed ligands in PG9ꢀglycan interaction, and the result will
be reported separately.
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Supporting Information.
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Materials, supplementary schemes, synthetic methods, spectroscopic and analytical data for new
compounds, and experimental protocols.
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Acknowledgments.
This work was supported by National Science Council (grant no. NSC 101ꢀ2321ꢀBꢀ001ꢀ024),
Academia Sinica and International Aids Vaccine Initiative (IVAI). The authors thank Prof. Peter
Kwong for providing broadly neutralizing antibodies PG9 and PG16, Mrs. Huang YiꢀPing for
her help with NMR analysis of synthetic compounds, and Mrs. Jennifer Chu for critically reading
the manuscript.
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Figure 1. (a) Golgi dependent glycan biosynthesis pathway ; (b) Representation of biꢀ, triꢀ and
tetraꢀantennary Nꢀglycan structures selected for synthesis; (c) Overall synthetic strategy. D1/D2
building blocks could be installed in two glycosylation steps, followed by enzymatic sialylation.
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Figure 2. Pictorial representation of multiantennary complex type Nꢀglycan structures printed on
NHSꢀactivated glass slide through amide bond formation.
Figure 3. (a) Slide obtained from fluorescent scan after secondary antibody incubation; (b)
Binding of PG16 to a panel of glycans in glycan microarray represented in bar chart.
Figure 4. Binding curves for glycan 32 printed on microarray surface at various concentrations
(100, 50, 25, and 12.5 ꢂM). The curves were obtained by using DyLight649ꢀconjugated donkey
antiꢀHuman IgG secondary antibody. Error bars indicate the SEM of KD values obtained from
two independent experiments while each experiment was performed in triplicates and average of
fluorescent intensity was taken.
Figure 5. ELISA binding of PG16 to glycoconjugate constructs in serial antibody dilutions.
Scheme 1. Reagents and Conditions : (a) NIS, TMSOTf, 4 Å MS, CH2Cl2, ꢀ50°C, 3: 82%, 7:
85%, 8: 76%, 9: 72%; (b) (1) CAN, ACN:toluene:H2O (4 : 2 : 1), 1 h, (2) DBU, CCl3CN, CH2Cl2,
0 °C, 3h, 7a: 75%, 8a: 70%, over 2 steps; (c) (1) CAN, ACN : toluene : H2O (4 : 2 : 1), 1 h, (2)
DAST, CH2Cl2, ꢀ10°C, 1 h, 7b: 72%, 8b: 69%, over 2 steps; (d) (1) PdCl2, CH2Cl2 : MeOH (1/1),
3 h, (2) DBU, CCl3CN, CH2Cl2, 0 °C, 85% over 2 steps; (e) (1) PdCl2, CH2Cl2 : MeOH (1/1), 3
h, (2) DAST, CH2Cl2, ꢀ10 °C, 1 h , 76% over 2 steps. NIS : Nꢀiodosuccinimide; TMSOTf :
trimethylsilyl trifluoromethanesulfonate; CAN : cerium ammonium nitrate; DBU : 1,8ꢀ
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Diazabicyclo[5.4.0]undecꢀ7ꢀene; CCl3CN : trichloroacetonitrile; DAST : diethylaminosulfur
trifluoride.
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Scheme 2. Reagents and Conditions : (a) 9b, AgOTf, Cp2HfCl2, 4 Å MS, toluene, ꢀ40 °C, 2 h,
71%; (b) pꢀTSA, acetonotrile, rt, 3 h, 70%; (c) 9b, AgOTf, Cp2HfCl2, 4 Å MS, toluene, ꢀ40 °C, 3
h, 69%; (d) 7b, AgOTf, Cp2HfCl2, 4 Å MS, toluene, ꢀ40 °C, 2 h, 51%; (e) (1) NH2CH2CH2NH2,
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o
nBuOH, 90 C, overnight; (2) Ac2O, pyridine, overnight; (3) NaOMe, MeOH, overnight; (4)
Pd(OH)2, MeOH : H2O : HCOOH (5 : 3 : 2), H2, 14: 60%; 16: 39%. Cp2HfCl2 : Bis
(cyclopentadienyl) hafnium Dichloride, AgOTf : silver trifluromethanesulfonate.
Scheme 3. Reagents and Conditions : (a) 8b, AgOTf, Cp2HfCl2, 4 Å MS, toluene, ꢀ50 °C to ꢀ
30 °C, 2 h, 68%; (b) pꢀTSA, acetonotrile, rt, 8 h, 72%; (c) 9b, AgOTf, Cp2HfCl2, 4 Å MS,
toluene, ꢀ40 °C, 3 h, 64%; (d) 7b, AgOTf, Cp2HfCl2, 4 Å MS, toluene, ꢀ50 °C to ꢀ30 °C, 2 h,
o
73%; (e) (1) NH2CH2CH2NH2, nꢀBuOH, 90 C, overnight; (2) Ac2O, pyridine, overnight; (3)
NaOMe, MeOH, overnight; (4) Pd(OH)2, MeOH : H2O : HCOOH (5 : 3 : 2), H2, 18: 51%; 21:
37%; 23: 27%.
Scheme 4. Reagents and Conditions : (a) CMPꢀNeu5Ac, αꢀ2,6/2,3ꢀ sialyltransferase, alkaline
phosphatase, 2 d for αꢀ2,6 and 8 d for αꢀ2,3, 24: 72%; 25: 75%; 26: 66%; 27: 67%; 28: 55%;
29: 70%; 30: 64%; 31: 66%; 32: 60%; 33: 70%.
Scheme 5. Reagents and Conditions : (a) (1) DTSSP, PBS, pH 7.4, rt, overnight; (2) DTT, PBS,
pH 7.4, 40°C, 2 h ; (b) SulfoꢀEMCS, PBS, pH 8.0, rt, 2 h ; (c) (1) PBS, pH 7.2, rt, 2 h; (2)
thioethanol, PBS, pH 7.2, rt, overnight.
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Scheme 1. Synthesis of D1/D2 arm triꢀ and pentasaccharides.
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OBn
O
OBn
O
BnO
BnO
BnO
BnO
BnO
BnO
HO
a)
O
O
STol
STol
STol
O
BnO
+
BnO
NPhth
OAc
NPhth
OAc
2
3
1
OH
O
HO
BnO
BnO
OH
O
OH
O
BnO
HO
BnO
BnO
BnO
BnO
a)
a)
a)
OPMP
OPMP
OAllyl
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5
OBn
OBn
BnO
OBn
O
BnO
BnO
BnO
BnO
O
BnO
BnO
BnO
O
BnO
O
O
O
O
O
O
O
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O
BnO
BnO
BnO
OAc
NPhth
OAc
OAc
NPhth
NPhth
OBn
O
BnO
BnO
BnO
OBn
O
BnO
BnO
O
BnO
BnO
BnO
O
BnO
O
BnO
BnO
BnO
O
O
BnO
O
O
O
O
OAc
PhthN
BnO
BnO
OAc
NPhth
R
R
R
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: R = -O-allyl
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: R = -OPMP
7
: R = -OPMP
d)
e)
9a : R = -O(C=NH)CCl3
9b : R = -F
b)
c)
b)
c)
8a : R = -O(C=NH)CCl3
8b : R = -F
7a : R = -O(C=NH)CCl3
7b : R = -F
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Scheme 2. Synthesis of biꢀ and triꢀantennary glycans 14 and 16.
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Scheme 3. Synthesis Triꢀ and tetraantennary glycans 21 and 23.
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