Sialic acid and sialyl-lactose glyco-conjugates: Design, synthesis and binding assays to lectins and swine influenza H1N1 virus

Article abstract of DOI:10.1002/psc.1415

The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC₄), to formulate human and avian receptor mimics, respectively. SOC₄, formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N^εH₂ groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC₄-glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC₄-glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC₄-glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC₄[(Ac)₂,(3'SL-Aoa)₂]-NH₂ 5 and Ac-SOC₄[(Ac)₂,(6'SL-Aoa)₂]-NH₂ 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions.

Full text of DOI:10.1002/psc.1415

Research Article
Received: 6 April 2011
Revised: 28 July 2011
Accepted: 1 August 2011
Published online in Wiley Online Library: 3 November 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1415
Sialic acid and sialyl-lactose glyco-conjugates:
design, synthesis and binding assays to lectins
and swine influenza H1N1 virus
Stella Zevgiti,a* Juliana Gonzalez Zabala,b Ayub Darji,b,c Ursula Dietrich,d
Eugenia Panou-Pomonisa and Maria Sakarellos-Daitsiotisa
The terminal parts of the influenza hemagglutinin (HA) receptors a2,6- and a2,3-sialyllactoses were conjugated to an artificial
carrier, named sequential oligopeptide carrier (SOC4), to formulate human and avian receptor mimics, respectively. SOC4,
formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of
biomolecules to the Lys-N«H2 groups. By doing so, it preserves their initial conformations and functionalities of the epitopes.
We report that SOC4-glyco-conjugate bearing two copies of the a2,6-sialyllactose is specifically recognized by the biotinylated
Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an a2,6-linkage. SOC4-glyco-conjugate
bearing two copies of the a2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its
well-known a2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both
the SOC4-glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2 5
and Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6 mimic the HA receptors. These findings could be useful for easy screening of binding
and inhibition assays of virus–receptor interactions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: influenza virus; sequential oligopeptide carrier (SOC4); chemoselective oxime ligation; SOC4-sialo-conjugates; HA-binding
receptor mimics; lectin binding assays; immune blot binding of H1N1
Carbohydrate recognition is crucial in numerous biological
properties such as stability, activity, binding, affinity and specific-
Introduction
The possibility of an outbreak of H5N1 avian influenza virus is not
considered a remote possibility if uncontrolled. Therefore, there
is a great deal of interest in examining the various factors
important for entry of the virus into host cells, the interspecies
transmission and the host restrictions of influenza virus [1].
Human and avian influenza A viruses differ in their recognition
of host cell receptors: the former preferentially recognize recep-
tors with saccharides terminating in sialic acid-a2,6-galactose
(SAa2,6Gal), whereas the latter prefer those ending in SAa2,3Gal.
It is the receptor-binding site of hemagglutinin (HA) protein that
recognizes SAa2,3Gal or SAa2,6Gal on the host cell surface and
initiates the virus attachment. A conversion from SAa2,3Gal to
SAa2,6Gal recognition is thought to be one of the changes that
must occur before avian influenza viruses can replicate efficiently
in humans and acquire the potential to cause a pandemic [2–4].
Computational studies based on the crystal structure of H5N1
HA showed that SAa2,3Gal has multiple strong hydrophobic
and hydrogen bond interactions in its trans conformation with
HA, whereas the SAa2,6Gal only shows weak interactions in a
cis-type conformation [5]. In an another study, it was shown that
a characteristic structural topology – and not the a2,6-linkage
itself – enables specific binding of HA to a2,6-sialylated glycans
and that recognition of this topology may be critical for adapta-
tion of HA to bind glycans in the upper respiratory tract of
humans [6]. In agreement with this study, it was suggested that
the distribution and detection of SAa2,3Gal and SAa2,6Gal-linked
receptors within the respiratory tract might not be as clear-cut as
has been reported [7,8].
ity for other biomolecules [9]. The importance of HA binding to
the host receptor at the sialyl-saccharide termination has led to
an ongoing interest in the formation of N-linked glycopeptides.
Several approaches to the chemical synthesis of glycopeptides
have been reported. For example, peptides have been glycosy-
lated with synthetic or naturally isolated oligosaccharides, and
glycosylated amino acids have been isolated from naturally
occurring glycoproteins and used in chemical conjugation of
polypeptides. The hydroxyl groups of the carbohydrate moiety
have been protected or left unprotected. After removal of the
C-terminal protection, the glycosyl amino acids have been used
as active esters or with the use of in situ coupling reagents.
Recent developments in the ligation of unprotected peptide frag-
ments by chemoselective methods have opened new routes to
*
Correspondence to: Stella Zevgiti, Department of Chemistry, Section of Organic
Chemistry and Biochemistry, University of Ioannina, 45110 Ioannina, Greece.
E-mail: stella.zevgiti@gmail.com
a Department of Chemistry, Section of Organic Chemistry and Biochemistry,
University of Ioannina, 45110 Ioannina, Greece
b Centre de Recerca en Sanitat Animal (CReSA), UAB Campus de la Universitat
Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
c
Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain
d Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Strasse
42-44, 60596 Frankfurt, Germany
J. Pept. Sci. 2012; 18: 52–58
Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

SIALYL-SACCHARIDE SOC4 CONJUGATES
the synthesis of glycopeptides. However, the regiospecific chem-
ical synthesis of glycopeptides remains a difficult task because of
the chemical lability of the glycosidic linkage [9–11].
and Rink Amide AM resin were purchased from GL Biochem
(Shanghai, China). DIEA was from Merck-Schuchardt (Munich
Germany). Triisopropylsilane (TIS) was obtained from Sigma
(St. Louis, MO, USA), whereas HPLC-grade acetonitrile, DCM
and DMF were from LABSCAN (Dublin, Ireland). Piperidine,
DIC, Boc-amino-oxyacetic acid (Boc-Aoa), TFA, TFE and sialic
acid (N-acetyl-neuraminic acid, Neu5Ac) were from Fluka
(Buchs, Switzerland). 6′-sialyllactose (6′-N-acetyl-neuraminyl-lactose,
Neu5Aca2,6Galb1,4Glc) and 3′-sialyllactose (3′-N-acetyl-neuraminyl-
lactose, Neu5Aca2,3Galb1,4Glc) ammonium salts were obtained
from IsoSep (Tullinge, Sweden). Biotinylated lectins Maackia
amurensis Lectin II (MAL II) and SNA were purchased from Vector
Laboratories (Burlingame, CA, USA). Microtiter plates (NUNC
Immuno Plates) were from NUNC A/S (Roskilde, Denmark)
(Denmark), whereas ExtrAvidin/alkaline phosphatase solution
and p-nitrophenyl phosphate were from Sigma.
Our objective here is to design, synthesize and use models of
the terminal segment of the receptor to validate their binding to
different lectins as well as to appropriate influenza viruses. For this
purpose, sialic acid (SA), 3′-sialyllactose (3′SL) and 6′-sialyllactose
(6′SL) were linked independently, in two and four copies, to
SOC4, a synthetic carrier developed in our laboratory, which is
formed by having three repeats of the tripeptide unit Lys-Aib-Gly
in tandem. The saccharides were conjugated to the Lys-NeΗ2
groups of the carrier via a chemoselective ligation approach, which
generates an oxime bond between the H2N-O-groups of the
modified lysine residues and the reducing end of each saccharide.
1H NMR, computational and CD studies have shown that the carrier
adopts a helical conformation, which is retained even after the
attachment of various biomolecules to the Lys-NeH2 groups of
SOC4. Moreover, it was found that the attached biomolecules do
not interact with the carrier or with each other and preserve their
conformation and initial bioactivity [12–14].
Peptide Synthesis
The SOC4 carriers, H-SOC4(Aoa)4-NH2 1 and Ac-SOC4[(Ac)2,
(Aoa)2]-NH2 2 [15], bearing four or two amino-oxyacetyl (Aoa)
groups, respectively, were synthesized. The following SOC4-
glyco-conjugates: H-SOC4(SA-Aoa)4-NH2 3, Ac-SOC4[(Ac)2,
(SA-Aoa)2]-NH2 4, Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2 5 and Ac-SOC4
[(Ac)2,(6′SL-Aoa)2]-NH2 6 were also prepared. Their conforma-
tional properties were studied by CD, and their ability to recognize
lectins was tested in a microtiter plate binding assay. Because of their
specificity to different carbohydrates, lectins are often used as tools
for the structural and functional study of glycans [16].
Our study pointed out that Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6,
bearing two copies of the 6′-sialyllactose, is specifically recog-
nized by the biotinylated Sambucus nigra (SNA) lectin, which
binds preferentially to sialic acid attached to a2,6Gal – the termi-
nal part of the human HA receptor. However, SOC4[(Ac)2,(3′SL-
Aoa)2]-NH2 5 is not recognized by Maackia amurensis (MAL II)
biotinylated lectin, although its known specificity to the a2,
3-sialyl bond. However, both compounds bind to H1N1 virus in
an immune blot experiment.
Solid phase peptide synthesis [17,18] was carried out manually
following the Fmoc/tBu methodology on a Rink Amide AM resin
(0.67 mmol/g). Fmoc groups were removed using 20% piperidine/
DMF. The protected fragment of the SOC4 carrier, where SOC4 is
(K-Aib-G)4, was synthesized by coupling each Fmoc-amino acid
(3 mol equiv.) in the presence of HBTU/HOBt/DIEA (2.9 : 3 : 6 molar
ratio) in a DMF/DCM mixture. Completion of couplings and depro-
tection reactions were monitored using the Kaiser ninhydrin test.
The synthesized compounds are shown in Table 1.
For the synthesis of compound 1, lysines were introduced as
Fmoc-Lys(Mtt)-OH. The Mtt groups were removed by 1.8% TFA/
DCM, whereas Boc-amino-oxyacetic acid was coupled to the
Lys-NeH2 groups using DIC and HOBt (three molar excess).
Deprotection of the N-terminal Lys of the carrier (Fmoc removal)
was also performed. Compound 2 was synthesized as previously
reported [15]. Briefly, lysines at the fourth and tenth positions
were introduced as Fmoc-Lys(Ac)-OH, whereas lysines at the
first and seventh positions were introduced as Fmoc-Lys(Mtt)-
OH. The N-terminal group of the carrier was acetylated using
acetic anhydride in pyridine.
Materials and Methods
The side chain deprotection and the resin cleavage were
achieved by treatment of each peptidyl resin with TFA/TIS/H2O
(95/2.5/2.5, v/v/v, 4 h). The resin was removed by filtration, the
filtrate was evaporated under reduced pressure and the product
was precipitated with cold diethyl ether. The precipitate was
Chemicals
Na-Fmoc protected amino acids, 2-(1H-benzotriazole-1-yl)-
1,1,3,3,-tetramethyluronium hexafluorophospate (HBTU), HOBt
Table 1. Parameters of the synthesis, purification and characterization of the synthesized compounds 1–6
No.
Compound
Yield (%)a
HPLC gradient elution (% B)b
tR (min)
ESI-MS
Molecular ion
Calculated
Found
1
2
3
4
5
6
H-SOC4(Aoa)4-NH2
45
38
25
56
18
29
5–70
10–50
5–70
13
[M + H]+
1391.6
1371.6
1276.8
977.6
1392.3
1372.4
1276.9
978.1
[M + H]+
c
Ac-SOC4[(Ac)2,(Aoa)2]-NH2
H-SOC4(SA-Aoa)4-NH2
13
11.5
11
[M-2H]-2
Ac-SOC4[(Ac)2,(SA-Aoa)2]-NH2
Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2
Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2
10–50
10–50
10–50
[M + 2H]+2
[M + 2H]+2
[M + 2H]+2
15
1301.8
1301.8
1302.2
1302.3
15
aThe purity of the final products was analyzed according to HPLC peak integrals at 214 nm on analytical HPLC and was estimated >95%.
bEluent B: CH3CN/0.1% TFA; eluent A: H2O/0.1% TFA.
cReference [15].
J. Pept. Sci. 2012; 18: 52–58 Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

ZEVGITI ET AL.
filtered, dissolved in 2 N aqueous acetic acid and lyophilized to
obtain the crude peptide.
Waters (Milford, USA) instrument equipped with a Waters 616
pump and a Waters 2487 dual l absorbance detector. Eluent A
was 0.1% TFA in water, and eluent B was 0.1% TFA in acetonitrile.
A linear gradient for compounds 1 and 3 was 5%–70% B at a flow
rate of 1 ml/min for 30 min and for compounds 2, 4, 5 and 6 was
10%–50% B at a flow rate of 1 ml/min for 30 min.
Crude peptides were purified by semipreparative HPLC. Aceto-
nitrile was evaporated, and the fractions were lyophilized to
obtain the pure peptide. The purity of the peptides was checked
using analytical HPLC and characterized using ESI-MS. The
calculated molecular mass-to-charge ratios were in agreement
with those found using ESI-MS (Table 1).
Semipreparative HPLC
Semipreparative HPLC purification was carried out on a SHIMADZU
(Shimadzu Corporation, Tokyo, Japan) semipreparative instrument
equipped with a SHIMADZU LC-10 AD VP pump and a SHIMADZU
SPD-10A VP UV-VIS detector, using a Supelco Discovery C18 (25 cm
10 mm, 5 mm) reverse phase column, with the same linear gradient
as for analytical HPLC at a flow rate of 4.7ml/min. Detection was
carried out at l = 214 nm.
Chemoselective Ligation with Sialic Acid, 3′-sialyllactose and
6′-sialyllactose
SOC4-glyco-conjugates 3–6 (Table 1) were synthesized in the
liquid phase using the chemoselective ligation approach
[10,19,20], which leads to the formation of an oxime bond
between each aminooxy group of the SOC4 carrier (1 and 2)
and the corresponding saccharide moiety (Figure 1).
For compound 3, sialic acid (10 mmol) was added to the stirred
compound 1 solution (20 mM in 0.1 M acetate buffer pH 4.0), and
the reaction was allowed to proceed at 35–40 ꢀC for 27h. For
compounds 4–6, a solution of each carbohydrate (50 mM in 0.1 M ac-
etate buffer pH 4.0) was added dropwise to the stirred compound 2
solution (10 mM in the previous buffer), and the reaction was allowed
to proceed at 35–40 ꢀC for 24, 72 and 48 h, respectively (Figure 1).
The glyco-conjugates 3–6 were purified by semipreparative
HPLC, acetonitrile was evaporated and the fractions were lyophi-
lized to obtain the pure product. Purity was checked by analytical
HPLC and characterization was performed by ESI-MS (Figure 2).
The calculated molecular mass-to-charge ratios were in agree-
ment with those found using ESI-MS (Table 1).
Mass Spectrometry
Positive or negative ion ESI-MS analyses of synthesized compounds
1–6 were performed on a Micromass Platform (Waters Corporation,
Milford, USA) Quandrupole LC-MS. Capillary and cone voltages
were set to 3 kV and 35–75 V, respectively. Samples were dissolved
in water/acetonitrile/trifluoroacetic acid (1/1/0.05 v/v/v).
Circular Dichroism
The CD spectra were recorded at 25 ꢀC on a Jasco (Jasco Corpo-
ration, Japan) J-815 spectropolarimeter equipped with a thermo-
electric temperature controller. Spectra were obtained using a
quartz cell of 1 mm path length, and the concentration of the
tested compounds was 10À4 M. Experiments were performed in
TFE/H2O mixtures (0%, 50% and 100%). Spectra were obtained
with a 1 nm bandwidth, a scan speed of 50 nm/min and a response
of 1 s. The signal-to-noise ratio was improved by accumulating
three scans. All CD spectra are reported in terms of mean residue
Analytical HPLC
Analytical HPLC of the synthesized compounds 1–6 (Table 1) was
performed using a Supelco (Sigma Aldrich, Bellefonte, USA) Dis-
covery C18 (25 cm  4.6 mm, 5 mm) reverse phase column with a
Figure 1. Coupling reaction of 6′-sialyllactose to Ac-SOC4[(Ac)2,(Aoa)2]-NH2 2. R = SAa2,6Gal.
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SIALYL-SACCHARIDE SOC4 CONJUGATES
Figure 2. ESI-MS mass spectrum and analytical HPLC of the purified Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6.
molar ellipticity [Y]R in degrees cm2/dmol-1residue-1. The percent-
age helical content was estimated on the basis of the [Y]222 nm
Results
Sequential oligopeptide carriers (SOCn, n = 2–7), a novel foldamer
values, in different environments, as described by Chen et al. [21].
class of artificial carriers, was introduced by our laboratory as a
tool for the construction of reconstituted protein mimics with
various functionalities [25–28]. Here, the terminal parts, sialic acid
and sialyllactose, of the HA receptor, critical for the recognition of
the influenza virus, were linked independently to SOC4 to recon-
stitute mimics of the receptor. The SOC4-glyco-conjugates were
synthesized in two steps: (i) solid phase synthesis of the SOC4 car-
rier bearing two or four amino-oxy-acetyl (H2N-O-CH2CO-) groups
and (ii) chemoselective ligation of the saccharide through the for-
mation of an oxime bond. Derivatization reactions were carried
out in acetate buffer (0.1 M) at pH 4.0 and in excess of the carbo-
hydrate. All final products were obtained in sufficient yields and
high purity as confirmed by HPLC and ESI-MS (Table 1).
The CD spectra of the amino-oxy-acetyl SOC4 carrier, Ac-SOC4
[(Ac)2,(Aoa)2]-NH2 2, in TFE/H2O (50/50 v/v) and 100% TFE, exhib-
ited a positive band at 192 nm and two negative bands at 205
and 222 nm typical of helical structure. The helical content of
the carrier, estimated on the basis of the [Y]222 value, was found
12% and 10% at 50% and 100% TFE, respectively [20]. These
helical features, in agreement with our previous studies, were
conserved even after the attachment of the saccharides to
the carrier. For example, Ac-SOC4[(Ac)2,(SA-Aoa)2]-NH2 4 and
Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6 exhibited 15% and 17% helical
conformation at 50% TFE and 21% and 16% at 100% TFE,
respectively, confirming the persistence of the helical structure
of the carrier in its conjugated form (Figure 3).
Microtiter Plate Binding Assay of Lectins to SOC4-
glyco-conjugates
The 96-well polystyrene plates were coated overnight at 4 ꢀC
with 5 mg/ml of each compound in carbonate buffer, pH 9.6 (50
ml/well). After washing of the plates with Tris Buffered Saline
(TBS) (0.05 M Tris-HCl buffer/0.15 M NaCl, pH 7.4) containing
0.05% Tween 20 (TBS-T), biotinylated lectins (MAL II and SNA)
were added and incubated at room temperature for 30 min (50
ml/well). The plates were washed, and the ExtrAvidin/alkaline
phosphatase solution (diluted 1 : 10 000 in TBS-T) was added (50
ml/well). After 1 h at room temperature, the plates were exten-
sively washed and incubated with p-nitrophenyl phosphate in
carbonate buffer, pH 9.6, containing 1 mM MgCl2 (1 mg/ml, 50
ml/well). The absorbance was read at 405 nm in a microtiter plate
reader after 30 min incubation with the substrate at 37 ꢀC [22,23].
Binding Assay of H1N1 Virus to Immobilized SOC4-glyco-
conjugates
The attachment of virus to SOC4-glyco-conjugates 5 and 6 was
performed using immune blot assay as previously described with
some modifications [24]. Briefly, nitrocellulose membrane was
washed, and SOC4-glyco-conjugates 5 and 6 as well as acetylated
SOC4 carrier (control) were immobilized by adding 2 ml of the solution
containing 100 mg/ml of each compound for 15 min and blocked
with 1% blocking buffer (BSA in Phosphate Buffered Saline (PBS))
for 1h at 37ꢀC. After several washes with PBS-Tween20, 0.1% mem-
brane was further incubated with 1 : 100 dilutions of the H1N1 virus
for 1h at 37ꢀC, washed and incubated with swine influenza H1N1-
positive serum (dilution 1 : 50) for 2 h at 37 ꢀC. Peroxidase conjugated
secondary anti-pig antibody (1 : 20 000) was used in chemilumine-
sence ECL system (GE Healthcare Munich, Germany) to detect the
binding of virus to SOC4-glyco-conjugates.
The binding of lectins to SOC4-glyco-conjugates was tested in
a biotin/avidin-mediated binding assay. SNA lectin binds prefer-
entially to sialic acid attached to terminal a2,6Gal, whereas MAL
II lectin appears to bind sialic acid in an a2,3Gal linkage. Lectins
were used to differentiate the functional properties of SOC4-
glyco-conjugates because of their carbohydrate specificity.
Figure 4 represents the binding of lectins to microtiter plates
coated with sialic acid conjugates and sialyllactose conjugates.
J. Pept. Sci. 2012; 18: 52–58 Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

ZEVGITI ET AL.
that is specific for a2,6Gal. More important, the binding of SNA
lectin to compound 6 is dose-dependent (10, 100, and 200 mg/
well of lectin) indicating the specificity of the synthesized glyco-
conjugate. A similar pattern was obtained when microtiter plates
were coated with different concentrations of glyco-conjugate 6
ranging from 1 to 10 mg/ml.
Preliminary immune blot assays showed that the SOC4-glyco-
conjugates 5 and 6 are both well recognized by H1N1 virus onto
nitrocellulose membrane, whereas the acetylated SOC4 carrier is
not recognized (Figure 5). These findings indicate that the virus
binds both a2,6 and a2,3Gal bonds contrary to lectins.
Discussion
Sequential oligopeptide carrier, SOC4, is an artificial carrier intro-
duced by our laboratory with the aim of optimizing the
Figure 3. CD spectra of Ac-SOC4[(Ac)2,(Aoa)2]-NH2 2, Ac-SOC4[(Ac)2,
(SA-Aoa)2]-NH2 4 and Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6 in 50% TFE/H2O.
SOC4-conjugates bearing four and two copies of sialic acid (3 and
4) were not recognized by the lectins. The same is true for SOC4-
conjugate 5 with two copies of 3′-sialyllactose. On the contrary,
compound Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6, which incorporates
two copies of 6′-sialyllactose, was well recognized by SNA lectin
Figure 4. (A) Binding of H-SOC4(SA-Aoa)4-NH2 3, Ac-SOC4[(Ac)2,(SA-Aoa)
2]-NH2 4, Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2 5 and Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-
NH2 6 to biotinylated SNA and MAL II lectins (10 mg/ml). (B) Binding of
serially diluted biotinylated SNA lectin to Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2
6. Concentration of substrates: 5 mg/ml. Control: acetylated SOC4 carrier.
Figure 5. Immune blot with immobilized SOC4-glyco-conjugates 5 and 6
showing binding with H1N1 virus (A) and without H1N1 virus (B) onto
nitrocellulose membrane. Concentration of substrates: 100 mg/ml. Acetylated
SOC4 carrier with and without the H1N1 virus are indicated as control.
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SIALYL-SACCHARIDE SOC4 CONJUGATES
presentation of the anchored biomolecules and help in the re-
constitution of protein mimics. SOC4 is a sterically constrained
scaffold comprising a-aminoisobutyric acid (Aib), a strongly heli-
cogenic Ca-tetrasubstituted amino acid. It was found that the
beneficial structural elements of SOC4 (helical conformation), at-
tributed mainly to the inclusion of Aib, induce a favorable ar-
(National Institute of Research and Food Technology), RTA2010-
00084-C02-02 (to A.D). It was also supported in part by the INIA
(National Institute of Research and Food Technology), RTA2010-
00084-C02-02 (to A.D).
rangement of the conjugated biomolecules, which retain their References
initial ‘active’ conformation and their potential for site-specific
1 Longini IM, Nizam A, Xu K, Ungchusak K, Hanshaowarakul W,
Cummings DA, Halloran ME. Containing pandemic influenza at the
source. Science 2005; 309: 1083–1087.
2 Rogers GN, Paulson LC. Receptor determinants of human and animal
influenza virus isolates: differences in receptor specificity of the H3
haemagglutinin based on species of origin. Virology 1983; 127:
361–373.
interactions [25–28]. Sialic acid, 3′-sialyllactose and 6′-sialyllac-
tose, which are the terminal and most critical parts of the HA re-
ceptor for its recognition of influenza viruses, were coupled to
SOC4 to formulate simplified reconstituted mimics of the
receptors.
Conformational studies by CD pointed out that the helical
features of the carrier are retained even after the attachment of
the saccharides to the carrier. It is expected that the coupled
terminal parts of HA receptor might preserve, in agreement with
our previous results, their initial topology, which is critical for the
virus recognition.
3 Yamada S, Suzuki Y, Suzuki T, Le MQ, Nidom CA, Sakai-Tagawa Y,
Muramoto Y, Ito M, Kiso M, Horimoto T, Shinya K, Sawada T, Kiso M,
Usui T, Murata T, Lin Y, Hay A, Haire LF, Stevens DJ, Russell RJ, Gamblin
SJ, Skehel JJ, Kawaoka Y. Haemagglutinin mutations responsible for
the binding of H5N1 influenza A viruses to human-type receptors.
Nature 2006; 444: 378–382.
4 Rogers GN, Paulson JC, Daniels RS, Skehel JJ, Wilson IA, Wiley DC.
Single amino acid substitutions in influenza haemagglutinin change
receptor binding specificity. Nature 1983; 304: 76–78.
5 Li M, Wang B. Computational studies of H5N1 haemagglutinin bind-
ing with SAa2,3Gal and SAa2,6Gal. Biochem. Biophys. Res. Commun.
2006; 347: 662–668.
6 Chandrasekaran A, Srinivasan A, Raman R, Viswanathan K, Raguram S,
Tumpey TM, Sasisekharan V, Sasisekharan R. Glycan topology deter-
mines human adaptation of avian H5N1 virus haemagglutinin. Nat.
Biotechnol. 2008; 26: 107–113.
7 Nicholls JM, Chan RWY, Russell RJ, Air GM, Peiris JSM. Evolving com-
plexities of influenza virus and its receptors. Trends Microbiol. 2008;
16: 149–157.
8 Qi L, Kash C, Dugan VG, Wang R, Jin G, Cunningham RE, Taubenberger
JK. Role of sialic acid binding specificity of the 1918 influenza virus
heamagglutinin protein in virulence and pathogenesis for mice. J.
Virol. 2009; 83: 3754–3761.
9 Jimenez-Castells C, de la Torre BG, Andreu D, Gutierrez-Gallego R.
Neo-glycopeptides: the importance of sugar core conformation in
oxime-liked glycoprobes. Glycoconj. J. 2008; 25: 879–887.
10 Cervigni SE, Dumy P, Mutter MS. Synthesis of glycopeptides and lipo-
peptides by chemoselective ligation. Angew. Chem. Int. Ed Engl. 1996;
35: 1230–1232.
11 Christiansen-Brams I, Meldal M, Bock K. Protected-mode synthesis of
N-linked glycopeptides: single-step preparation of building blocks as
peracetyl glycosylated Na-Fmoc asparagine Opfp esters. J. Chem.
Soc. Perkin Trans. 1 1993; 13: 1461–1472.
12 Tsikaris V, Sakarellos C, Sakarellos-Daitsiotis M, Orlewski P, Marraud M,
Cung MT, Vatzaki E, Tzartos S. Construction and application of a new
class of sequential oligopeptide carriers (SOCn) for multiple anchoring
of antigenic peptides-application to the acetylcholine receptor (AChR)
main immunogenic region. Int. J. Biol. Macromol. 1996; 19: 195–205.
13 Krikorian D, Panou-Pomonis E, Voitharou Ch, Sakarellos C, Sakarellos-
Daitsiotis M. A peptide carrier with a built-in vaccine adjuvant: construc-
tion of immunogenic conjugates. Bioconjug. Chem. 2005; 16: 812–819.
14 Kargakis M, Zevgiti S, Krikorian D, Sakarellos-Daitsiotis M, Sakarellos C,
Panou-Pomonis E. A palmitoyl-tailed sequential oligopeptide carrier for
engineering immunogenic conjugates. Vaccine 2007; 25: 6708–6712.
15 Skarlas Th, Zevgiti S, Droebner K, Panou-Pomonis E, Planz O, Sakarellos-
Daitsiotis M. Influenza virus H5N1 hemagglutinin (HA) T-cell epitope
conjugates: design, synthesis and immunogenicity. J. Pept. Sci. 2011;
17: 226–232
With the aim to test the potential of SOC4-glyco-conjugates as
simplified models of the human and avian HA receptor, their
binding to lectins was studied. Lectins are very often used as
tools to investigate the carbohydrate specificity. Sialic acid conju-
gates were not recognized by lectins indicating that sialic acid
alone is not sufficient for interacting with lectins. Although
Maackia amurensis (MAL) II lectin is thought to be highly specific
for the a2,3Gal linkage, Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2 5 did not
bind to this lectin. It seems likely that the a2,3Gal linkage is not
sufficient for binding to MAL II lectin and that some other
structural features might be required, as for example hydropho-
bic and hydrogen bond interactions in a trans-type conformation
with lectin. However, in contrast to what is observed for com-
pound 5, SOC4-conjugate that bears two copies of 6′-sialyllactose,
Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6, is well recognized by SNA lectin
confirming the carbohydrate specificity of this lectin for the
a2,6Gal bond. Apparently, the predetermined conformation of
compound 6 fulfils the cis-type structural requirements for a
specific interaction with SNA lectin [5,6]. Moreover, these find-
ings indicate that Ac-SOC4[(Ac)2,(6′SL-Aoa)2]-NH2 6 adopts the
appropriate orientation that allows the coupled 6′-sialyllactoses
to obtain a characteristic structural topology that enables the
specific binding to SNA lectin.
The binding of H1N1 virus onto immobilized Ac-SOC4[(Ac)2,(3′
SL-Aoa)2]-NH2 5 suggests a relatively low sensitivity of Maackia
amurensis (MAL II) lectin toward the a2,3Gal bond. However,
H1N1 virus showed a specific binding both to Ac-SOC4[(Ac)2,(6′
SL-Aoa)2]-NH2 6 as well as to Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2 5.
We assume that this discrepancy of virus binding to both the
SOC4-glyco-conjugates could be because of the form of virus
propagation because it was obtained initially from allantoic
cavity of Specific Pathogen Free (SPF) eggs. Virus propagation
in the eggs may have shifted the binding of H1N1 virus also to-
ward Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2 5. Similar observation has
been reported previously [29,30]. Our results indicate a potential
use of Ac-SOC4[(Ac)2,(3′SL-Aoa)2]-NH2 5 and Ac-SOC4[(Ac)2,(6′SL-
Aoa)2]-NH2 6 in easy screening of binding and inhibition assays
of virus–receptor interactions.
16 Hirabayashi J. Lectin based structural glycomics, glycoproteomics,
and glycan profiling. Glycoconj. J. 2004; 21: 35–40.
17 Bodansky M, Bodansky A. Activation and coupling. In The Practice of
Peptide Synthesis, 2nd edn, Bodansky M, Bodansky A (eds). Springer-
Verlag: Berlin, Heidelberg, 1994; 75–125.
18 Giralt E, Albericio F. Synthesis of peptides on solid supports. In Synthe-
sis of Peptide and Peptidomimetics, Volume E22a: Synthesis of
Peptides, Goodman M, Felix A, Moroder L, Toniolo G (eds). Georg
Thieme Verlag: Stuttgard, NY, 2003; 665–824.
19 Zhao Y, Kent SBH, Chait BT. Rapid, sensitive structure analysis of oligo-
saccharides. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1629–1633.
Acknowledgments
This study was supported by the EU (FP6 «EUROFLU» project,
SP5B-CT-2007-044098). It was also supported in part by the INIA
J. Pept. Sci. 2012; 18: 52–58 Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

ZEVGITI ET AL.
20 Carrasco MR, Nguyen MJ, Burnell DR, MacLaren MD, Hengel SM.
Synthesis of neoglycopeptides by chemoselective reaction of carbo-
hydrates with peptides containing a novel N′-methyl-aminooxy amino
acid. Tetrahedron Lett. 2002; 43: 5727–5729.
26 Sakarellos-Daitsiotis M, Alexopoulos Ch, Sakarellos C. Sequential
oligopeptide carriers, SOCn, as scaffolds for the reconstitution of anti-
genic proteins: applications in solid phase immunoassays. J. Pharm.
Biomed. Anal. 2004; 34: 761–769.
21 Chen YH,Yang JT, Martinez H. Determination of the secondary struc-
tures of proteins by circular dichroism and optical rotatory dispertion.
Biochemistry 1972; 11: 4120–4131.
22 Wu AM, Wu JH, Liu J-H, Singh T. Recognition profile of Bauhinia
purpurea agglutinin (BPA). Life Sci. 2004; 74: 1763–1779.
23 Duk M, Lisowska E, Wu JH, Wu AM. The biotin/avidin-mediated micro-
titer plate lectin assay with the use of chemically modified glycopro-
tein ligand. Anal. Biochem. 1994; 221: 266–272.
24 Ferraz N, Carnevia D, Nande G, Rossotti M, Miguez M, Last J, Gonzalez-
Sapienza G. Specific immunoassays for endocrine disruptor monitor-
ing using recombinant antigens cloned by degenerated primer PCR.
Anal. Bioanal. Chem. 2007; 389: 2195–2202.
27 Voitharou C, Krikorian D, Sakarellos C, Sakarellos-Daitsiotis M, Panou-
Pomonis E. A complementary La/SSB epitope anchored to sequential
oligopeptide carrier regulates the anti-La/SSB response in immunized
animals. J. Pept. Sci. 2008; 14: 1069–1076.
28 Sakarellos-Daitsiotis M, Krikorian D, Panou-Pomonis E, Sakarellos C.
Artificial carriers: a strategy for constructing antigenic/immunogenic
conjugates. Curr. Top. Med. Chem. 2006; 6: 1715–1735.
29 Ito T, Suzuki Y, Takada A, Kawamoto A, Otsuki K, Masuda H, Yamada
M, Suzuki T, Kida H, Kawaoka Y. Differences in sialic acid-galactose
linkages in the chicken egg amnion and allantois influence human
influenza virus receptor specificity and variant selection. J. Virol.
1997; 71: 3357–3362.
25 Alexopoulos Ch, Sakarellos-Daitsiotis M, Sakarellos C. Synthetic
carriers: sequential oligopeptide carriers SOCn-I and SOCn-II as an
innovative and multifunctional approach. Curr. Med. Chem. 2005; 12:
1469–1479.
30 Gambaryan AS, Karasin AI, Tuzikov AB, Chinarev AA, Pazynina GV,
Bovin NV, Matrosovich MN, Olsen CW, Klimov AI. Receptor-binding
properties of swine influenza viruses isolated and propagated in
MDCK cells. Virus Res. 2005; 114: 15–22.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 52–58