Screening of Neu5Acα(2-6)gal isomer preferences of siglecs with a sialic acid microarray

Article abstract of DOI:10.1039/c6ob01688j

Sialic acids (Sias) are important terminal sugars on cell surfaces involved in a wide range of protein-carbohydrate interactions. Hence, agents modulating sias-mediated protein interactions are promising inhibitors or vaccine candidates. Here, we report t

Full text of DOI:10.1039/c6ob01688j

Organic &
Biomolecular Chemistry
View Article Online
View Journal
COMMUNICATION
Screening of Neu5Acα(2–6)gal isomer preferences
of siglecs with a sialic acid microarray†
Cite this: DOI: 10.1039/c6ob01688j
Rohan Yadav,a Shani Leviatan Ben-Arye,b Balamurugan Subramani,a
Received 5th August 2016,
Accepted 14th September 2016
Vered Padler-Karavani*b and Raghavendra Kikkeri*a
DOI: 10.1039/c6ob01688j
www.rsc.org/obc
Sialic acids (Sias) are important terminal sugars on cell surfaces To this end, the C-9 and C-4 positions of sialic acids were
involved in a wide range of protein–carbohydrate interactions. modified with aromatic and sulphate derivatives to increase
Hence, agents modulating sias-mediated protein interactions are the Siglec-2 binding affinity.7 The synthetic glycan microarray
promising inhibitors or vaccine candidates. Here, we report the analysis of these glycans revealed that biphenyl substitution at
synthesis of Neu5Acα(2–6)Gal structural analogs and their binding the C-9 position and sulphate at the C-4 position revealed
to a series of siglecs. The results showed distinct binding patterns 750 000-fold higher affinity to hCD22 compared to the native
with conserved siglecs (hCD22 and mCD22) compared to rapid sialic acid ligands.8 Similarly, potential modification on sialic
evolving siglecs (Siglecs -3 & -10).
acid has been studied to elucidate the role of siglecs in a bio-
logical system. However, the basic differences between the con-
servative and evolutionary siglec selectivity remains largely
unclear and it is unknown what factors are responsible for the
specificity. To understand how siglecs generate specificity for
sias glycans, it is essential to construct structural analogs of
Neu5Acα(2–6)Gal to modulate the distinct binding patterns.
Here, we have systematically investigated the effect of sialic
acid as terminal sugar and galactose/glucose/mannose as
penultimate sugar to study the major human siglecs’ binding
patterns. Based on the binding affinities, it could be possible
to deduce how nature differs with the evolutionary and con-
served siglec-ligand selectivity.
Sialic acids are the terminal sugars of glycoproteins and glycolipids
on cell surfaces that are characterized by a 9-carbon backbone.1 To
date, nearly fifty different forms of Sias have been identified.
Among them, N-acetylneuraminic acid and its N-glycolyl derivatives
are the most prominent.2 Given their structural diversity, Sias act
in several biological processes. Sias on the cell surfaces are
involved in regulation of the cell cycle and autoimmune disorders,
and are also involved in pathogen recognition.3 Sialic acid binding
lectins (Siglecs) are the major homologous subfamily of I-type
lectins that mediate Sia recognition via immunoglobulin (Ig)-like
domains.4 Expression of specific siglecs modulate immune
responses, inflammation, tumorigenesis and apopotosis.5
The sialic acid disaccharides (1, 3 and 5) were obtained
from fully protected disaccharides 8, 9 and 10 respectively. The
esters, ether and azide functionalities on 8–10 were succes-
sively removed by the use of a global deprotection protocol
(Scheme 1). The sialic acid donor (11) and galactose/mannose/
glucose acceptors (12–14) were synthesized according to a
modified literature procedure.9 The glycosylation of a donor
and acceptor were carried out by NIS/TfOH at −40 °C which
gave a protected form of disaccharides in moderate to good
yield. The 4,6-benzylidine protection of galactose and glucose
thiotoluene pentahydroxy derivatives and their successive C-2,
C-3 benzoylation afforded a protected form of galactose and
glucose thiotoluene. The stereodirecting benzoyl group at the
C-2 position was chosen to take advantage of the beta con-
figuration which is usually found on the natural linkage of
glycans. The glycosylation of galactose and glucose derivatives
with a linker in the presence of NIS/TfOH at −40 °C and suc-
cessive regioselective ring opening by PhBCl2/Et3SiH afforded
acceptors 12 and 13 with good yield. Simultaneously the regio-
Siglecs are classified into two subgroups, CD33-related sub-
groups, (Siglec-3, -5, -6, -7 & -10), which appear to be rapidly
evolving with complex expansion in different mammalian
lineages and the second subgroup, which includes the evolu-
tionarily conserved ones (Siglec-1, -2 and -4).6 In general,
siglecs bind to common epitopes containing Neu5Acα(2–6)gal
and/or Neu5Acα(2–3)gal linkages. Various siglecs exhibit over-
lapping actions as a result of similar glycan binding prefer-
ences that complicate the assessment of distinct siglec
functions. Hence, identifying the distinct Sias glycans’ reco-
gnition patterns is essential for revealing the siglec functions.
aIndian Institute of Science Education and Research, Pashan, Pune 411008, India.
E-mail: rkikkeri@iiserpune.ac.in; Fax: +91-20-25899790; Tel: +91-20-25908207
bTel-Aviv University, Department of Cell Research and Immunology, Tel-Aviv,
69978 Israel. E-mail: vkaravani@post.tau.ac.il; Tel: +972-3-640-6737
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6ob01688j
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
Scheme 2 Retrosynthesis of sialic acid trisaccharides.
Scheme 1 Retrosynthesis of sialic acid disaccharides.
Next, the synthetic di- and tri-saccharides were printed onto
selective 4,6 benzylidine protection and 2,3 benzylation gave a epoxide-functionalized microarray slides at 100 µM in replica-
thiophenol mannose derivative which was then converted into tion of 4 as described in the Experimental section. Human (H)
its sulfoxide in the presence of m-CPBA at lower temperature to and mouse (m) Siglec-Fc chimeras (H-Siglec-2, m-Siglec-2,
afford the highly active mannose-sulfoxide thiophenol donor. H-Siglec-3 and -10) were incubated on the slide at three con-
The mannose donor was glycosylated with a linker in the pres- centrations (5 ng µl−1, 10 ng µl−1 and 20 ng µl−1,) in PBS with
ence of Tf2O/TTBP, at −78 °C and the benzylidine ring cleavage 1% ovalbumin, followed by the secondary antibody (Cy3-anti-
by an acid catalyst afforded acceptor 14 in moderate yield.
human IgG). Slides were scanned and the binding was deter-
The trisaccharide analogs of Neu5Acα(2–6)Galβ(1–4)GlcNAc mined by the fluorescence intensity as described in the
were obtained by global deprotection of fully protected trisac- Experimental section. Initially, the sialic acid microarray was
charides 15–17 in moderate yield. A synthetic convergent analyzed by incubating with control lectins of a known
approach involves a sialic acid dibutylphosphate donor (18) binding specificity. As expected, sambucus nigra lectin (SNA),
and C6-OH thiotoluene, thiophenol acceptors of galactose (19) which is specific of Neu5Acα(2–6)gal bound to 1 and 2 and dis-
glucose (20), mannose (21) and glucosamine (22) at the non- played no binding with 3–6 (Fig. 1A). On confirming the
reducing end (Scheme 1). The donor 18 was synthesized from binding affinity with control lectin, H-Siglec-2 and m-Siglec-2
N-acetylneuraminic acid in 7 steps according to the literature binding in the microarray was established. H-Siglec-2 dis-
protocol.10 Synthesis of acceptors 19 and 20 was achieved in played weak binding with 1 and 2 compared to m-Siglec-2
four steps involving 4,6-benzylidine protection and then selec- (Fig. 1B and C). Interestingly, isomers of compounds 1 and 2
tive C-3 benzylation with nBu2SnO/BnBr followed by C-2 ben- did not show any binding while H-siglec-3 and H-siglec-10
zoylation and finally, regioselective selective benzylidine ring bound to 1 and 2. Remarkably, the isomers of 1 and 2 dis-
opening with PhBCl2/Et3SiH afforded the C6-OH acceptor in played better binding to Siglec-3 and -10 than the native form
good yield. Similarly mannose C6-OH acceptor (21) was synthesized (Fig. 1D and E). According to the microarray studies, com-
from its pentahydroxy thiophenol derivative by following temporary pounds 1–6 sialic acid glycans possess two distinct binding
C6-OH TBDMS protection, then benzylation of the remaining patterns. The evolutionarily conserved H-Siglec-2 or m-Siglec-2
hydroxyl-groups, and finally the removal of temporary silyl protec- displayed selective binding towards 1 and 2, indicating that
tion by p-TSA gave the desired compound in good yield. The syn- the interaction between the penultimate sugar (galactose) and
thesis of building block 22 was challenging and successfully the specific amino acid sequence in Siglec-2 is conserved, thus
achieved in 11 steps by following the published protocol.11 Sialic generating the specific binding motif. On the other hand, the
acid donor 18 was glycosylated with acceptors 19–21 in the pres- rapidly evolving siglec-3 and 10 displayed spatial flexibility,
ence of TMSOTf at −78 °C and gave the expected disaccharides, allowing them to bind galactose isomers (mannose and
which subsequently glycosylated with acceptor 22 in the presence glucose). Overall, the identification of these detailed binding
of NIS/TfOH at −40 to −20 °C yielding fully protected trisaccharide preferences provides the basis for understanding and targeting
analogs (15–17). Finally ester, Troc, and oxazolidinone protecting siglec selectively. The mannose isomers of sialic acid glycans
groups were removed by the global deprotection protocol and later are the common side products of β-mannosidosis12 disease
the removal of benzyl ether and simultaneous azide to amine and the binding between 5 and 6 with siglecs-3 and 10 reveals
reduction was achieved by hydrogenolysis to afford fully depro- the possible mechanism of immune responses in the human
tected trisaccharides (2, 4 and 6) in moderate yield (Scheme 2).
system.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Organic & Biomolecular Chemistry
Communication
Fig. 1 Microarray analysis of sialic acid-binding lectin derivatives (1–6) (at 20 ng µl−1).
2 (a) T. Aganta and A. Varki, Chem. Rev., 2002, 102, 439–
469; (b) X. Chen and A. Varki, ACS Chem. Biol., 2010, 5,
163.
Conclusion
We have synthesized a library of sialic acid glycans by modify-
ing the penultimate sugar and immobilized them on the
glycan microarray to determine siglec binding preferences.
Comparison of the siglec binding patterns allows further
understanding of the basic differences between the conserved
and evolving siglecs, and constitutes a valuable tool for design-
ing siglec specific molecules for therapeutic applications.
3 (a) K. Furukawa, K. Hamamura, W. Aixinjueluo and
K. Furukawa, Ann. N. Y. Acad. Sci., 2006, 1086, 185–198;
(b) I. Bucior and M. M. Burger, Curr. Opin. Struct. Biol.,
2004, 14, 631–637; (c) R. Schauer, Curr. Opin. Struct. Biol.,
2009, 19, 507–514; (d) S. I. Hakomori and Y. Zhang, Chem.
Biol., 1997, 4, 97–104; (e) R. D. Astronomo and
D. R. Burton, Nat. Rev. Drug Discovery, 2010, 9, 308–
324; (f) M. M. Fuster and J. D. Esko, Nat. Rev. Cancer,
2005, 5, 526–542; (g) R. A. C. Hughes and
D. R. Cornblath, Lancet, 2005, 366, 1653–1666;
(h) C. S. Berenson, K. B. Sayles, J. Huang, V. N. Reinhold,
M. A. Garlipp and H. C. Yohe, FEMS Immunol.
Med. Microbiol., 2005, 45, 171–182; (i) J. Fantini,
D. Hammache, G. Pieroni and N. Yahi, Glycoconjugate J.,
2000, 17, 199–204.
4 (a) H. Cao and P. R. Crocker, Immunology, 2011, 132, 18–26;
(b) P. H. Lopez and R. L. Schnaar, Curr Opin Struct Biol.,
2009, 19, 549–557.
5 (a) M. S. Macauley, P. R. Crocker and J. C. Paulson, Nat.
Rev. Immunol., 2014, 14, 653–666; (b) S. Pillai,
I. A. Netravali, A. Cariappa and H. Mattoo, Annu. Rev.
Immunol., 2012, 30, 357–392.
Acknowledgements
Financial support from the IISER, Pune, the Max-Planck
partner group and DST (Grant No. SB/S1/C-46/2014) is grate-
fully acknowledged. R.Y and B.S acknowledge CSIR-SRF for
supporting their fellowships. Supported by the European Union
Seventh Framework Program grants (FP7/2007-2013; 603049), by
the Israeli Cancer Research Foundation and by the Israeli
National Nanotechnology Initiative and Helmsley Charitable
Trust for a Focal Technology Area on Nanomedicines for
Personalized Theranostics (to V.P-K.).
References
6 (a) A. Varki and T. Angata, Glycobiology, 2006, 1R–27R;
(b) P. R. Crocker, J. C. Paulson and A. Varki, Nat. Rev.
Immunol., 2007, 7, 255–266.
7 (a) T. Angata, C. M. Nycholat and M. S. Macauley, Trends
Pharmacol. Sci., 2015, 36, 645–660; (b) H. Prescher,
A. Schweizer, E. Kuhfeldt, L. Nitschke and R. Brossmer,
1 (a) Essentials of Glycobiology, ed. A. Varki, R. Cummings,
J. Esko, H. Freeze, G. Hart and J. Martht, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 2009; (b) X. Chen and
A. Varki, ACS Chem. Biol., 2010, 5, 164–176; (c) D. B. Werz,
R. Ranzinger, S. Herget, A. Adibekian, C.-W. Von der Lieth
and P. H. Seeberger, ACS Chem. Biol., 2007, 2, 685–691.
This journal is © The Royal Society of Chemistry 2016
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
ACS Chem. Biol., 2014, 9, 1444–1450; (c) C. D. Rillahan,
M. S. Macauley, E. Schwartz, Y. He, R. McBride,
B. M. Arlian, J. Rangarajan, V. V. Fokin and J. C. Paulson,
Chem. Sci., 2014, 5, 2398–2406.
8 (a) O. Blixt, S. Han, L. Liao, Y. Zeng, J. Hoffmann,
S. Futakawa and J. C. Paulson, J. Am. Chem. Soc., 2008, 130,
9 (a) D. Crich and W. Li, J. Org. Chem., 2007, 72, 2387–2391;
(b) C. Wang, Q. Li, H. Wang, L. H. Zhang and X. S. Yi,
Tetrahedron, 2006, 62, 11657–11662; (c) M. Sakagami and
H. Hamana, Tetrahedron Lett., 2000, 41, 5547–5551;
(d) S. Serna, J. Etxebarria, N. Ruiz, M. Martin-Lomas and
C. Reichardt, Chem. – Eur. J., 2010, 16, 13163–13175.
6680–6681; (b) V. Padler-Karavani, X. Song, H. Yu, 10 (a) H. Y. Liao, C. H. Hsu, S. C. Wang, C. H. Liang,
N.
Hurtado-Ziola,
S.
Huang,
S.
Muthana,
H. Y. Yen, C. Y. Su, C. H. Chen, J. T. Jan, C. T. Ren,
C. H. Chen, T. J. Cheng, C. Y. Wu and C. H. Wong, J. Am.
Chem. Soc., 2010, 132, 14849–14856.
H. A. Chokhawala, J. Cheng, A. Verhagen, M. A. Langereis,
R. Kleene, M. Schachner, R. J. De Groot, Y. Lasanajak,
H. Matsuda, R. Schwab, X. Chen, D. F. Smith, 11 C. H. Wang, K. Tony, K. Mong and C. Y. Huang, J. Org.
R. D. Cummings and A. Varki, J. Biol. Chem., 2012, 287, Chem., 2003, 68, 2135–2142.
22593–22608; (c) J. Q. Gerlach, M. Kilcoyne and L. Joshi, 12 J. C. Michalski and A. Klein, Biochim. Biophys. Acta, 1999,
Anal. Methods, 2014, 6, 440–449.
1455, 69–84.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2016