One-pot synthesis of N-acetyl- and N-glycolylneuraminic acid capped trisaccharides and evaluation of their influenza A(H1 N1) inhibition

Article abstract of DOI:10.1002/anie.201309646

Human lung epithelial cells natively offer terminal N-acetylneuraminic acid (Neu5Ac) α(2→6)-linked to galactose (Gal) as binding sites for influenza virus hemagglutinin. N-Glycolylneuraminic acid (Neu5Gc) in place of Neu5Ac is known to affect hemagglutinin binding in other species. Not normally generated by humans, Neu5Gc may find its way to human cells from dietary sources. To compare their influence in influenza virus infection, six trisaccharides with Neu5Ac or Neu5Gc α(2→6) linked to Gal and with different reducing end sugar units were prepared using one-pot assembly and divergent transformation. The sugar assembly made use of an N-phthaloyl- protected sialyl imidate for chemoselective activation and α- stereoselective coupling with a thiogalactoside. Assessment of cytopathic effect showed that the Neu5Gc-capped trisaccharides inhibited the viral infection better than their Neu5Ac counterparts. Flu the coop: A stereoselective one-pot assembly and divergent transformation enabled the synthesis of six N-acetyl- and N-glycolylneuraminic-acid-capped trisaccharides. Two of the N-glycolylneuraminic-acid-capped trisaccharides showed inhibitory activities against a common human influenza virus. Bz=benzoyl, NIS=N-iodosuccinimide, Tf=trifluoromethanesulfonyl, TMS=trimethylsilyl. Copyright

Full text of DOI:10.1002/anie.201309646

Angewandte
Chemie
DOI: 10.1002/anie.201309646
Carbohydrates
One-Pot Synthesis of N-Acetyl- and N-Glycolylneuraminic Acid
Capped Trisaccharides and Evaluation of Their Influenza A(H1N1)
Inhibition**
Yun Hsu, Hsiu-Hwa Ma, Larry S. Lico, Jia-Tsrong Jan, Koichi Fukase, Yosuke Uchinashi,
Medel Manuel L. Zulueta, and Shang-Cheng Hung*
Abstract: Human lung epithelial cells natively offer terminal
N-acetylneuraminic acid (Neu5Ac) a(2!6)-linked to galac-
tose (Gal) as binding sites for influenza virus hemagglutinin.
N-Glycolylneuraminic acid (Neu5Gc) in place of Neu5Ac is
known to affect hemagglutinin binding in other species. Not
normally generated by humans, Neu5Gc may find its way to
human cells from dietary sources. To compare their influence
in influenza virus infection, six trisaccharides with Neu5Ac or
Neu5Gc a(2!6) linked to Gal and with different reducing end
sugar units were prepared using one-pot assembly and
divergent transformation. The sugar assembly made use of
an N-phthaloyl-protected sialyl imidate for chemoselective
activation and a-stereoselective coupling with a thiogalactoside.
Assessment of cytopathic effect showed that the Neu5Gc-
capped trisaccharides inhibited the viral infection better than
their Neu5Ac counterparts.
interspecies transmission.[6] For instance, humans are suscep-
tible to flu viruses that prefer the a(2!6)-linkage, which are
dominant in respiratory epithelial cells, and are shielded from
avian flu viruses by lung mucins, which are rich in a(2!3)-
linkages.[7] Thus, human transmission of avian viruses is
relatively rare. Viruses recognizing both linkage types can
however, infect swine.[8]
Other glycan features also contribute to hemagglutinin
affinity. The sugar unit at the reducing side of Gal can be
either glucose (Glc), N-acetylglucosamine (GlcNAc), or N-
acetylgalactosamine. Studies showed the positive influence of
GlcNAc in the binding of many human flu viruses.[9] The sialic
acid forms common in vertebrates are N-acetylneuraminic
acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc).
Many avian flu viruses recognize Neu5Gc-capped glycans[10]
and those strains can be transmitted to horses[11] and pigs.[12]
Humans, in their evolutionary past, lost the capacity to form
Neu5Gc and exclusively generate Neu5Ac for glycan biosyn-
thesis under normal conditions.[13] Hence, pathogens that
prefer Neu5Gc cannot readily infect humans,[14] though some
human influenza viruses also attach to Neu5Gc-capped
glycans.[15] Recent reports suggested that human cells can
acquire Neu5Gc from dietary sources, and may increase
susceptibility to a wider range of pathogens and diseases.[16]
With much focus on Neu5Ac-containing glycans,[17] chem-
ical strategies aimed at their Neu5Gc counterparts[18] are
uncommon and even more uncommon are their biological
evaluations.[15] Accordingly, we prepared a set of Neu5Ac-
and Neu5Gc-containing trisaccharides having a(2!6)-link-
ages for evaluation with a common human influenza virus
(Scheme 1). The reducing end sugar was also varied. For
efficient synthesis, we developed a one-pot method for the
assembly of trisaccharide precursors amenable to divergent
transformations. The trisaccharide skeletons were generated
stereoselectively using the building blocks 1–5. Such a strategy
should reduce the effort and waste for the overall synthetic
process.
I
nfluenza is a persistent global health concern.[1] Apart from
the seasonal flu, humans are at times hit by potent viral strains
of animal origin. The latest of these include the A(H1N1) flu
virus from swine[2] and the avian A(H1N1)[3] and A(H7N9)[4]
strains. The attachment of hemagglutinin, a viral envelope
glycoprotein, to complementary sialosides on the host cell
surface triggers conformational changes, which ultimately
result in host cell entry and viral replication.[5] Sialic acids
mainly occur at terminal glycan positions linked to galactose
(Gal) in either an a(2!3) or a(2!6) manner. Prevalence of
one linkage type over the other in critical infection sites limits
[*] Dr. Y. Hsu, H.-H. Ma, L. S. Lico, Dr. J.-T. Jan, Dr. M. M. L. Zulueta,
Prof. Dr. S.-C. Hung
Genomics Research Center, Academia Sinica
No. 128 Academia Road, Section 2, Taipei 115 (Taiwan)
E-mail: schung@gate.sinica.edu.tw
Prof. Dr. K. Fukase, Dr. Y. Uchinashi
Department of Chemistry, Graduate School of Science
Osaka University, Osaka 560-0043 (Japan)
Dr. Y. Hsu
The difficult a sialylation and the poor reactivity of sialyl
donors are challenges in the one-pot assembly of glycans with
terminal sialic acid units.[19] Various approaches to a sialyla-
tion exploit the modulating effect of functionalities at C5 of
the sialyl donor.[20] In one such effort, the phthalimido group
was found to favor the a isomer as a result of its fixed dipole
effect on the oxocarbenium ion.[21] Poor reactivity prevents p-
tolyl thiosialoside from being used in reactivity-based, one-
pot assembly.[19b] To bypass this problem, more-reactive
leaving groups, or those that can be selectively activated
Department of Chemistry, National Tsing Hua University
No. 101, Section 2, Kuang-Fu Road, Hsinchu 300 (Taiwan)
L. S. Lico
Institute of Chemistry, University of the Philippines
Diliman, Quezon City 1101 (Philippines)
[**] This work was supported by the National Science Council (NSC 100-
2113-M-001-019-MY3 and NSC 101-2628-M-001-006-MY3) and
National Health Research Institutes (NHRI-EX101-10146NI).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201309646.
Angew. Chem. Int. Ed. 2014, 53, 2413 –2416
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2413

.
Angewandte
Communications
yield was markedly reduced, even with BF3·Et2O as an
activator (entries 2 and 3). Successive donor addition
improved the yield, the best of which was attained when
1.0 equivalents of 1 was further supplemented with 0.8 equiv-
alents after 30 minutes of reaction (entry 5). These reaction
conditions resulted in complete a selectivity with 75% yield
for the adduct 6.
With suitable sialylation conditions in hand, we moved
forward on the one-pot assembly (Scheme 2). Thus,
30 minutes after the second addition of 1 at ꢀ788C, either
the acceptor 3,[24] 4,[25] or 5[26] was added together with N-
iodosuccinimide (NIS) and TMSOTf to activate the thio-
Scheme 1. Retrosynthetic approach to the sialic-acid-capped trisacchar-
ides. Bz=benzoyl, Tol=4-tolyl.
were employed.[18b,22] Here, we chose the sialyl donor 1,[21]
which carries 5-N-phthaloyl protection, to direct a stereose-
lectivity and the N-phenyltrifluoroacetimidate[23] leaving
group to enable chemoselective activation using plain trime-
thylsilyl triflate (TMSOTf). The orthogonal phthaloyl pro-
tecting group also permits the installation of either acetyl or
glycolyl units during the final transformation.
We first examined sialylation conditions best suited for
the formation of the disaccharide 6 using the known
thiogalactoside 2[22b] as acceptor (Table 1). Good yield but
poor stereoselectivity was observed when 1 was activated with
TMSOTf in CH2Cl2 at ꢀ788C (entry 1). Here, the orientation
of the newly formed glycosidic bond was confirmed by using
Scheme 2. One-pot assembly of the fully protected sialic-acid-capped
trisaccharides. Reagents and conditions: a) 1. TMSOTf, EtCN, ꢀ788C,
30 min; 2. 1, ꢀ788C, 30 min; b) NIS, TMSOTf, ꢀ788C to ꢀ408C,
2.5 h.
3
the JC1,H3ax values for the sialic acid unit.[19a] Changing the
solvent from CH2Cl2 to EtCN gave full a selectivity, but the
Table 1: Glycosylation with the sialyl donor 1.
toluene group of the intermediate disaccharide 6a. Neigh-
boring-group participation of the 2-O-benzoyl group in the
galactosyl unit and the EtCN solvent effect ensured b glyco-
sylation. This one-pot process delivered the target sialyl-
capped trisaccharide backbones 7, 8, and 9 in 46, 64, and 45%
yields, respectively.
Functional-group transformations toward the final prod-
ucts started with cleavage of the acetate, benzoate, and
methyl esters by saponification (Scheme 3). Ethylenediamine
treatment then removed the N-phthaloyl groups to form the
intermediates 10, 11, and 12. With the free amine available on
the sialic acid unit, either the acetyl or the glycolyl function-
ality was introduced. Hydrogenation exposed the hydroxy
groups after benzyl removal. It also reduced the azido group
in the glucosamine-derived moiety to the amine, which was
further selectively acetylated with Ac2O in MeOH. Accord-
ingly, the trisaccharides 13–18 with Neu5Ac and Neu5Gc caps
and with Glc, GlcNAc, or Gal reducing end sugar units were
acquired in four or five steps in good overall yields.
Entry Donor
(equiv)
t [h]
Promoter Solvent Yield a/b[b]
[%][a]
1
2
3
4
5
1.2
1.2
1.2
3.0
0.5
0.5
TMSOTf
TMSOTf
CH2Cl2
EtCN
70
21
18
52
75
1:1.8
a only
a only
a only
a only
BF3·Et2O EtCN
1.0 + 0.5[c] 0.5 + 0.5[c] TMSOTf
1.0 + 0.8[c] 0.5 + 0.5[c] TMSOTf
EtCN
EtCN
[a] Isolated yields.[b] Determined by 1H NMR analysis. [c] Initial use of
1.0 equiv of donor, then additional amount added after 30 min, with
quenching 30 min later.
2414
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 2413 –2416

Angewandte
Chemie
Scheme 3. Synthesis of Neu5Ac- and Neu5Gc-containing trisaccharides. Reagents and conditions: a) NaOMe, MeOH, RT, 10 min; H2O, reflux,
18 h; b) NH2CH2CH2NH2, tBuOH, reflux, 24 h; c) Ac2O, MeOH, RT, 5 h; d) H2, Pd/C, MeOH, RT, 2 d; e) BnOCH2COCl, Et3N, MeOH, RT, 2 h;
f) Ac2O, MeOH, RT, 0.5 h.
The synthetic sialotrisaccharides were analyzed for their
anti-influenza activity through the cytopathic effect (CPE).
Mardin–Darby canine kidney (MDCK) cells were used
against the common human influenza A/WSN/33(H1N1)
virus as a model in this evaluation. MDCK cell surfaces
contain a(2!6)-type sialoside linkages.[27] In the absence of
multivalent interactions, binding of sialosides to hemaggluti-
nin typically occur in the millimolar range.[28] Thus, MDCK
cells were treated with the viral suspension preincubated with
1 mm of the sialotrisaccharides. Among the six compounds
tested, only the Neu5Gc-capped trisaccharides 14 and 18
displayed inhibitory activities against the virus (Figure 1b and
c). The cells corresponding to these compounds appear
normal and did not display agglutination, which is indicative
of successful viral infection and were evident in the controls
(Figure 1a) as well as those cells corresponding to compounds
13, 15, 16, and 17. These results signify the enhanced affinity
of hemagglutinin in the tested human influenza strain with
Neu5Gc units compared to Neu5Ac. The Glc or Gal units
occupying the reducing end also boosted the interaction. In
Figure 1. CPE inhibition assay of the synthesized sialotrisaccharides
against influenza A/WSN/33 (H1N1) virus. a) Control (cells infected
with the virus). b) Cells treated with the virus preincubated with 1 mm
of 14. c) Cells treated with the virus preincubated with 1 mm of 18.
the case of Neu5Ac-capped trisaccharides as well as com-
pound 16, the concentration applied is probably just too low
to exhibit any inhibitory effect.
In summary, we have developed a one-pot and stereose-
lective glycosylation strategy for the construction of Neu5Ac-
and Neu5Gc-capped trisaccharides. The monosaccharide
building blocks and the protecting groups used in the
reactions were strategically chosen not only to aid in the
introduction of the target substituents but also to help control
the stereoselectivity of the glycosylation. Neu5Gc-containing
trisaccharides with Glc or Gal units at the reducing end
position displayed inhibitory activities against a common
human A(H1N1) virus. This evaluation shows preliminary
evidence of the Neu5Gc sway on human influenza virus
Angew. Chem. Int. Ed. 2014, 53, 2413 –2416
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2415

.
Angewandte
Communications
Fukunaga, M. Ohuchi, T. Toyoda, A. Ishihama, Y. Kawaoka,
Y. Suzuki, FEBS Lett. 1999, 464, 71 – 74.
binding. Future studies will focus on the observed enhanced
binding ability in greater detail.
[16] a) P. Tangvoranuntakul, P. Gagneux, S. Diaz, M. Bardor, N.
Varki, A. Varki, E. Muchmore, Proc. Natl. Acad. Sci. USA 2003,
100, 12045 – 12050; b) E. Byres, A. W. Paton, J. C. Paton, J. C.
Lofling, D. F. Smith, M. C. J. Wilce, U. M. Talbot, D. C. Chong,
H. Yu, S. Huang, X. Chen, N. M. Varki, A. Varki, J. Rossjohn, T.
Beddoe, Nature 2008, 456, 648 – 652; c) A. Varki, Proc. Natl.
Acad. Sci. USA 2010, 107, 8939 – 8946.
[17] a) C.-C. Wang, S. S. Kulkarni, M. M. L. Zulueta, S.-C. Hung in
The Molecular Immunology of Complex Carbohydrates-3,
Vol. 705 (Ed.: A. M. Wu), Springer, Heidelberg, 2011, pp. 691 –
726; b) A. K. Adak, C.-C. Yu, C.-F. Liang, C.-C. Lin, Curr. Opin.
Chem. Biol. 2013, 17, 1030 – 1038.
[18] a) K. Fukunaga, T. Toyoda, H. Ishida, M. Kiso, J. Carbohydr.
Chem. 2003, 22, 919 – 937; b) D. Crich, B. Wu, Org. Lett. 2008, 10,
4033 – 4035; c) H. Tamai, H. Ando, H. Ishida, M. Kiso, Org. Lett.
2012, 14, 6342 – 6345; d) G. Pazynina, T. Tyrtysh, V. Nasonov, I.
Belyanchikov, A. Paramonov, N. Malysheva, A. Zinin, L.
Kononov, N. Bovin, Synlett 2013, 226 – 230.
[19] a) G.-J. Boons, A. V. Demchenko, Chem. Rev. 2000, 100, 4539 –
4565; b) C.-H. Hsu, S.-C. Hung, C.-Y. Wu, C.-H. Wong, Angew.
Chem. 2011, 123, 12076 – 12129; Angew. Chem. Int. Ed. 2011, 50,
11872 – 11923.
[20] C. De Meo, U. Priyadarshani, Carbohydr. Res. 2008, 343, 1540 –
1552.
[21] a) K. Tanaka, T. Goi, K. Fukase, Synlett 2005, 2958 – 2962; b) S.-i.
Tanaka, T. Goi, K. Tanaka, K. Fukase, J. Carbohydr. Chem. 2007,
26, 369 – 394.
[22] a) H. Tanaka, M. Adachi, T. Takahashi, Chem. Eur. J. 2005, 11,
849 – 862; b) C.-C. Wang, J.-C. Lee, S.-Y. Luo, S. S. Kulkarni, Y.-
W. Huang, C.-C. Lee, K.-L. Chang, S.-C. Hung, Nature 2007, 446,
896 – 899; c) C.-H. Hsu, K.-C. Chu, Y.-S. Lin, J.-L. Han, Y.-S.
Peng, C.-T. Ren, C.-Y. Wu, C.-H. Wong, Chem. Eur. J. 2010, 16,
1754 – 1760.
[23] a) B. Yu, H. Tao, Tetrahedron Lett. 2001, 42, 2405 – 2407;
b) X. M. Zhu, R. R. Schmidt, Angew. Chem. 2009, 121, 1932 –
1967; Angew. Chem. Int. Ed. 2009, 48, 1900 – 1934.
Received: November 6, 2013
Revised: December 25, 2013
Published online: January 31, 2014
Keywords: carbohydrates · glycosylation · stereoselectivity ·
.
synthetic methods · viruses
[1] J. S. Robertson, S. C. Inglis, Virus Res. 2011, 162, 39 – 46.
[2] M. Khanna, N. Gupta, A. Gupta, V. K. Vijayan, J. Biosci. 2009,
34, 481 – 489.
[3] Y. Watanabe, M. S. Ibrahim, Y. Suzuki, K. Ikuta, Trends
Microbiol. 2012, 20, 11 – 20.
[4] H. Zhu, D. Wang, D. J. Kelvin, L. Li, Z. Zheng, S. W. Yoon, S. S.
Wong, A. Farooqui, J. Wang, D. Banner, R. Chen, R. Zheng, J.
Zhou, Y. Zhang, W. Hong, W. Dong, Q. Cai, M. H. Roehrl, S. S.
Huang, A. A. Kelvin, T. Yao, B. Zhou, X. Chen, G. M. Leung,
L. L. Poon, R. G. Webster, R. J. Webby, J. S. Peiris, Y. Guan, Y.
Shu, Science 2013, 341, 183 – 186.
[5] a) J. J. Skehel, D. C. Wiley, Annu. Rev. Biochem. 2000, 69, 531 –
569; b) H.-Y. Liao, C.-H. Hsu, S.-C. Wang, C.-H. Liang, H.-Y.
Yen, C.-Y. Su, C.-H. Chen, J.-T. Jan, C.-T. Ren, C.-H. Chen, T.-
J. R. Cheng, C.-Y. Wu, C.-H. Wong, J. Am. Chem. Soc. 2010, 132,
14849 – 14856.
[6] a) M. N. Matrosovich, A. S. Gambaryan, S. Teneberg, V. E.
Piskarev, S. S. Yamnikova, D. K. Lvov, J. S. Robertson, K. A.
Karlsson, Virology 1997, 233, 224 – 234; b) N. M. Varki, A. Varki,
Lab. Invest. 2007, 87, 851 – 857; c) M. Imai, Y. Kawaoka, Curr.
Opin. Virol. 2012, 2, 160 – 167.
[7] a) J. N. S. S. Couceiro, J. C. Paulson, L. G. Baum, Virus Res. 1993,
29, 155 – 165; b) K. Shinya, M. Ebina, S. Yamada, M. Ono, N.
Kasai, Y. Kawaoka, Nature 2006, 440, 435 – 436.
[8] T. Ito, J. N. S. S. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R.
Castrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster, Y.
Kawaoka, J. Virol. 1998, 72, 7367 – 7373.
[9] A. S. Gambaryan, A. B. Tuzikov, V. E. Piskarev, S. S. Yamni-
kova, D. K. Lvov, J. S. Robertson, N. V. Bovin, M. N. Matroso-
vich, Virology 1997, 232, 345 – 350.
[10] T. Ito, Y. Suzuki, T. Suzuki, A. Takada, T. Horimoto, K. Wells, H.
Kida, K. Otsuki, M. Kiso, H. Ishida, Y. Kawaoka, J. Virol. 2000,
74, 9300 – 9305.
[24] C.-R. Shie, Z.-H. Tzeng, S. S. Kulkarni, B.-J. Uang, C.-Y. Hsu, S.-
C. Hung, Angew. Chem. 2005, 117, 1693 – 1696; Angew. Chem.
Int. Ed. 2005, 44, 1665 – 1668.
[25] A. Pastore, S. Valerio, M. Adinolfi, A. Iadonisi, Chem. Eur. J.
2011, 17, 5881 – 5889.
[26] C.-R. Shie, Z.-H. Tzeng, C.-C. Wang, S.-C. Hung, J. Chin. Chem.
Soc. 2009, 56, 510 – 523.
[11] Y. Suzuki, T. Ito, T. Suzuki, R. E. Holland, T. M. Chambers, M.
Kiso, H. Ishida, Y. Kawaoka, J. Virol. 2000, 74, 11825 – 11831.
[12] T. Suzuki, G. Horiike, Y. Yamazaki, K. Kawabe, H. Masuda, D.
Miyamoto, M. Matsuda, S.-I. Nishimura, T. Yamagata, T. Ito, H.
Kida, Y. Kawaoka, Y. Suzuki, FEBS Lett. 1997, 404, 192 – 196.
[13] A. Varki, Biochimie 2001, 83, 615 – 622.
[14] N. M. Varki, E. Strobert, E. J. Dick, K. Benirschke, A. Varki,
Annu. Rev. Pathol. Mech. Dis. 2011, 6, 365 – 393.
[15] H. Masuda, T. Suzuki, Y. Sugiyama, G. Horiike, K. Murakami, D.
Miyamoto, K. I.-P. J. Hidari, T. Ito, H. Kida, M. Kiso, K.
[27] S. Hatakeyama, Y. Sakai-Tagawa, M. Kiso, H. Goto, C.
Kawakami, K. Mitamura, N. Sugaya, Y. Suzuki, Y. Kawaoka, J.
Clin. Microbiol. 2005, 43, 4139 – 4146.
[28] a) N. K. Sauter, M. D. Bednarski, B. A. Wurzburg, J. E. Hanson,
G. M. Whitesides, J. J. Skehel, D. C. Wiley, Biochemistry 1989,
28, 8388 – 8396; b) C.-C. Wang, J.-R. Chen, Y.-C. Tseng, C.-H.
Hsu, Y.-F. Hung, S.-W. Chen, C.-M. Chen, K.-H. Khoo, T.-J.
Cheng, Y.-S. E. Cheng, J.-T. Jan, C.-Y. Wu, C. Ma, C.-H. Wong,
Proc. Natl. Acad. Sci. USA 2009, 106, 18137 – 18142.
2416
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 2413 –2416