Biantennary oligoglycines and glyco-oligoglycines self-associating in aqueous medium

Article abstract of DOI:10.3762/bjoc.10.140

Oligoglycines designed in a star-like fashion, so-called tri-and tetraantennary molecules, were found to form highly ordered supramers in aqueous medium. The formation of these supramers occurred either spontaneously or due to the assistance of a mica surface. The driving force of the supramer formation is hydrogen bonding, the polypeptide chain conformation is related to the folding of helical polyglycine II (PG II). Tri-and tetraantennary molecules are capable of association if the antenna length reach 7 glycine (Gly) residues. Properties of similar biantennary molecules have not been investigated yet, and we compared their selfaggregating potency with similar tri-and tetraantennary analogs. Here, we synthesized oligoglycines of the general formula R-Gly _n-?-Gly_n-R (X =-HN-(₂)_m-NH-, m = 2, 4, 10; n = 1-7) without pendant ligands (R = H) and with two pendant sialoligands (R = sialic acid or sialooligosaccharide). Biantennary oligoglycines formed PG II aggregates, their properties, however, differ from those of the corresponding tri-and tetraantennary oligoglycines. In particular, the tendency to aggregate starts from Gly₄ motifs instead of Gly ₇. The antiviral activity of end-glycosylated peptides was studied, and all capable of assembling glycopeptides demonstrated an antiviral potency which was up to 50 times higher than the activity of peptide-free glycans. 2014 Tsygankova et al;.

Full text of DOI:10.3762/bjoc.10.140

Biantennary oligoglycines and glyco-oligoglycines
self-associating in aqueous medium
Svetlana V. Tsygankova1, Alexander A. Chinarev1, Alexander B. Tuzikov1,
Nikolai Severin2, Alexey A. Kalachev3, Juergen P. Rabe2,
Alexandra S. Gambaryan4 and Nicolai V. Bovin*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1372–1382.
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, ul.
Miklukho-Maklaya 16/10 Moscow V-437, 117997, Russia,
2Department of Physics, Humboldt University Berlin, Newtonstr. 15,
D-12489, Berlin, Germany, 3Plasmachem GmbH, Rudower Chaussee
29, D-12489 Berlin, Germany and 4M. P. Chumakov Institute of
Poliomyelitis and Viral Encephalitides, 142782 Moscow Region,
Russia
doi:10.3762/bjoc.10.140
Received: 28 February 2014
Accepted: 09 May 2014
Published: 17 June 2014
This article is part of the Thematic Series "Multivalent glycosystems for
nanoscience". A part of this work was presented at 17th European
Carbohydrate Symposium “EuroCarb17” [1].
Email:
Nicolai V. Bovin* - bovin@carb.ibch.ru
Guest Editor: B. Turnbull
* Corresponding author
© 2014 Tsygankova et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
glycopeptides; influenza virus; multivalent glycosystems; oligoglycine;
polyglycine II; self-assembling; tectomers
Abstract
Oligoglycines designed in a star-like fashion, so-called tri- and tetraantennary molecules, were found to form highly ordered
supramers in aqueous medium. The formation of these supramers occurred either spontaneously or due to the assistance of a mica
surface. The driving force of the supramer formation is hydrogen bonding, the polypeptide chain conformation is related to the
folding of helical polyglycine II (PG II). Tri- and tetraantennary molecules are capable of association if the antenna length reach
7 glycine (Gly) residues. Properties of similar biantennary molecules have not been investigated yet, and we compared their self-
aggregating potency with similar tri- and tetraantennary analogs. Here, we synthesized oligoglycines of the general formula
R-Glyn-Х-Glyn-R (X = -HN-(СН2)m-NH-, m = 2, 4, 10; n = 1–7) without pendant ligands (R = H) and with two pendant sialo-
ligands (R = sialic acid or sialooligosaccharide). Biantennary oligoglycines formed PG II aggregates, their properties, however,
differ from those of the corresponding tri- and tetraantennary oligoglycines. In particular, the tendency to aggregate starts from
Gly4 motifs instead of Gly7. The antiviral activity of end-glycosylated peptides was studied, and all capable of assembling glyco-
peptides demonstrated an antiviral potency which was up to 50 times higher than the activity of peptide-free glycans.
Introduction
Recently, we have synthesized and described tetraantennary [2] These layers are one or two molecules thick. The stability was
and triantennary [3] oligoglycines capable of spontaneous or attributed to the formation of a network of hydrogen bonds.
surface-promoted formation of flat layers in aqueous medium. This class of supramers has been called tectomers. Tectomers in
1372

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
a layer are packed by polyglycine II type (PG II) [4,5]. Their tural motifs (core), where the antennae are connected to each
helical polypeptide chain fundamentally differs from the canon- other. The knowledge of the rules found for the unsubstituted
ical α-helix. The association of symmetrical tetra- and trianten- assembly of oligoglycines may be suitable for us for the design
nary (star-like) oligoglycines spontaneously proceeds only of corresponding sialo derivartives, which are candidate thera-
when the number of glycine (Gly) residues in a chain (n) is peutics for the blocking of the influenza virus [6].
equal or greater to seven. Oligoglycines with an antennae size
Results and Discussion
less than seven do either not associate at all or require
Synthesis of biantennary oligoglycines and
their glyco derivatives
The synthesized biantennary oligoglycines and their glyco
derivatives are presented in Figure 1. Analogously to tri- and
tetraantennary molecules, oligoglycine antennas are connected
according to the ‘head-to-head’ principle, i.e., by their
C-termini, so that the two amino groups are terminal. The
extremely favorable conditions, in particular surface promotion.
Yet, the properties of similar biantennary molecules were not
investigated. Here, we synthesized biantennary oligoglycines
and studied them in order to determine the necessary and suffi-
cient conditions for self-association. More specifically, we
investigated the combination of structure elements, such as the
n value, the type of terminal substituents, and the type of struc-
Figure 1: The structure of biantennary oligoglycines and their glyco derivatives (sp = spacer group).
1373

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
obtained compounds differ threefold. Firstly, they differ by core neuraminic acid (Sug-ONp), where the carboxyl group of the
X nature: hydrophilic oligoethylene glycol (OEG), hydrophobic spacer was activated with 4-nitrophenol (Np) (Scheme 1). The
flexible decamethylene (C10), or short ethylene (C2). Secondly, synthesis of these compounds was described in [10,11]. Owing
the length of oligoglycine antennas, i.e., the number of to the poor solubility of the diamine form of oligoglycines with
glycine residues in a chain (n = 1–7) is different. Thirdly, the cores C2 and C10 in DMSO, the reaction was carried out in a
substances differ by the presence or absence of carbohydrate saturated aqueous solution of lithium bromide, which prevented
fragment (Sug), containing α-N-acetylneuraminic moiety the formation of hydrogen bonds and thus increased the solu-
(Neu5Acα).
bility of oligoglycines. Glycopeptides were isolated from the
reaction mixture by gel-permeation chromatography (yields
Diamines NH2–X–NH2 were the starting substances for the 70–75%). The peptide modification by the amino group with
synthesis, oligoethylene glycol diamine was obtained from di- mono- or oligosaccharides dramatically increased their solu-
tosylate as described in [7,8]. The synthesis of biantennary bility in water. This may support their antiviral action (see
oligoglycines was carried out by means of the activated esters below), because glycopeptides act topically, in the respiratory
method (Scheme 1) [9]. The glycine chains were elongated tract, and are administered as a spray.
stepwise by their N-terminus by using N-oxysuccinimidyl
esters (BocGlyONSu or BocGly2ONSu, Boc = tert-butyloxy- We then investigated the ability of synthesized biantennary
carbonyl). Boc-peptides were isolated from the reaction mix- oligoglycines to assemble in aqueous media as well as the
ture by the removal of the solvent and the re-crystallization of antiviral activity of glycoderivatives.
the reaction product from aqueous methanol (yields 75–95%).
Study of biantennary oligoglycines associ-
In the case of poorly soluble products the impurities and starting
materials were washed off with methanol (yields 60–90%). In ation in solution by dynamic light scattering
the case of an oil-like substance (Х = ОEG, n = 2) chromatog- The size of the particles formed by the biantennary oligo-
raphy on silica gel was performed. The quantitative removal of glycines in solution was measured with the dynamic light scat-
Boc groups was achieved by the treatment of the obtained tering method (DLS). We found that the ability of association
peptides with trifluoroacetic acid. Salt forms (trifluoroacetates depends on the number of the glycine units in the antennae, the
or hydrochlorides) of diamino derivatives were obtained by nature of the core, the pH, and the peptide concentration.
sedimentation from an aqueous solution by methanol (yield
≥95%). At later stages of elongation the salts were converted to It is known that the charge of terminal amino groups of the
the respective free bases by treatment with a slight excess of protonated form of oligoglycines hinders association. To over-
triethylamine. The preparation of oligoglycines with a chain come this obstacle an equimolar quantity of NaHCO3 or
length exceeding five glycine residues for the derivatives with Nа2CO3 was added to aqueous solutions of oligoglycine salts.
core C10 and six residues for core C2 failed due to their low In the absence of the base, pH values of oligoglycine salt solu-
solubility and, consequently, the impossibility of separating tions varied from 3.5 to 4.5 (hereinafter denoted as рН < 5). In
them from the intermediates of the synthesis.
the case of the addition of one base equivalent per one amino
group the solution becomes neutral (рН 6.5), in the case of two
Sialo conjugates of biantennary oligoglycines were obtained Nа2CO3 equivalents the pH value is more than 8.5 (basic solu-
from the corresponding diamines and derivatives of α-N-acetyl- tion, denoted as pH > 8.5).
Scheme 1: Synthesis of biantennary oligoglycines and their glycoderivatives.
1374

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
At n < 4 peptides with an oligoethylene glycol core and the tapping mode in air. The contact mode of scanning was used for
cores С2 and С10 did not form associates in aqueous medium in experiments in a liquid cell. Experiments in a liquid cell
all the studied ranges of pH and concentration (0.1–1.0 mg/mL). allowed us to study the kinetics of the process without a
possible distortion of the nanostructures resulting from the
Biantennary oligoglycines, cores С2 and С10, n ≥ 4, are drying of the sample.
capable of forming associates (700–900 nm) in acidic
solutions in the studied concentration range, except for The Raman spectra (Figure 3) of biantennary oligoglycines
Н-Gly4NH(СН2)10NHGly4-Н·2HCl, which associates in capable of association as well as the spectra of tri- and tetraan-
concentrations ≥0.5 mg/mL.
tennary peptides described earlier display bands at 884, 1261,
1382, 1424 and 1654 cm−1, which are characteristic and
Molecules with the core С2 (n = 4–6) and C10 (n = 5) associate specific for crystalline PG II. Based on the presence of these
so rapidly in neutral and basic media that a precipitate is formed bands we conclude that the structure organization of associates
(data for peptide with n = 5 are given in Figure 2a). Only the formed in solution corresponds to PG II. The sensitivity of
peptide Н-Gly4NH(СН2)10NHGly4-Н in concentration routine Raman scattering method is insufficient for the work
≤0.1 mg/mL is capable of forming associates (800–1200 nm) with oligoglycine monolayers, so indirect methods were used in
stable in aqueous media (Figure 2a,b).
order to attribute formed material to a PG II structure. More
specifically, geometrical parameters of the layers were deter-
mined by using SFM and compared with: 1) those for tectomers
(attributed to PG II, see above) formed in solution and 2) calcu-
Study of biantennary peptides association
using scanning force microscopy
Scanning force microscopy (SFM) elucidates information not lated values for different, not only PG II, models. As shown
only about the association process both in solution and on a below for particular examples, in most cases spontaneous and
surface, but also about fine details of the formed architectures. surface-mediated assembly led to associates of PG II structure,
Of particular interest are cases characterized by the active parti- i.e., tectomers.
cipation of the surface in accelerating the self-assembly. To
discriminate the processes taking place on the surface from The formation of the PG II structure for oligoglycines with a
similar processes in liquid volume, measurements were carried short rigid spacer С2 is only possible if the molecule is
out immediately after the deprotonation of oligoglycine salts at extended, i.e., antennas are pointing in opposite directions (con-
incubation times insufficient for a spontaneous association in formation “1 + 1”, Figure 4а). The presence of a flexible core
solution (found out as ≤1 min). The solution was placed on a (C10, OEG) allows the molecule to adopt the conformation
freshly cleaved surface of mica or graphite, exposed for fixed ”2 + 0”, which is characterized by unidirectional oligoglycine
time intervals (denoted as texp), followed by the removal of the antennas. The hydrophobic side of the tectomer should initiate
liquid phase from the surface and the scanning of the sample in the formation of the second layer with the opposite orientation
Figure 2: Dynamics of associate formation by biantennary oligoglycines Н-Glyn-NH(СН2)10NH-Glyn-Н. a) n = 4–5, in aqueous solution at рН 6.5 and
a concentration of 0.1 mg/mL at t = 0 and рН < 5 before the addition of base and at t > 0 and рН 6.5 after the addition of base. The region of sedimen-
tation is marked with a dotted line. b) n = 4, in aqueous solution at рН 6.5 and 8.5.
1375

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
of the monomer in aqueous solutions (Figure 4b) in order to
minimize the thermodynamically unfavorable contact with
water.
It was demonstrated for biantennary oligoglycines that a
concentration of 0.1 mg/mL is optimal for the study of the
dynamics of tectomer growth on a mica surface. At higher
concentrations the growth both in solution and on the surface
proceeded so rapidly that the dynamics study was considered
impossible. No tectomer structure was observed under
acidic conditions (рН < 5), whereas under neutral and
basic conditions the reaction proceeded similarly in terms
of both the velocity and the morphology of formed
tectomers. The oligoethylene glycol derivatives Н-Glyn-
NH(СН2СН2О)3СН2СН2NH-Glyn-Н (n = 2–7), non-associ-
ating in aqueous solutions as well as oligoglycines with cores
С2 and С10 (n < 4) did not form associates on a mica surface
under all studied ranges of pH (from 4.5 to 8.5).
According to dynamic light scattering data (see above) only
peptide Н-Gly4NH(СН2)10NHGly4-Н was capable of forming
tectomers in neutral and basic solutions which were unchanged
in an aqueous phase for a long time. Figure 5 demonstrates the
dynamics of layer growth on mica with the characteristic forma-
tion of islet structures (texp = 0.5 min, Figure 5а), growing later-
ally (texp = 1 min, Figure 5b), and covering the whole surface
with an even layer (texp = 2 min, Figure 5c). Presumably, longer
times (texp > 2 min) are characterized by the appearance of
multilayer tectomers resulting from the sorption of associates
formed in solution. The multilayer tectomers can be readily
removed by washing with buffer solution (рН 6.5 or 9.0). The
morphology of the first layer remains unchanged and the avail-
able defects (‘holes’) are preserved. The layer height is
3.7–4.0 nm, which may correspond to both mono- and bilayer
(conformations “1 + 1” and “2 + 0”, respectively, see Figure 4).
Figure 3: Raman spectra of a) [H-Gly7-NHCH2]4C; b) H-Gly4-
NH(CH2)2NH-Gly4-H; c) H-Gly4-NH(CH2)10NH-Gly4-H. The spectra
contain characteristic bands at 884, 1261, 1382, 1424 and 1654 cm−1,
corresponding to the structure PG II [12]. Spectra were recorded for
the samples in solid phase.
Figure 4: Model of the formation of tectomer layers by biantennary oligoglycines on a mica surface. The heights are given for
Н-Gly4NH(СН2)10NHGly4-Н.
1376

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
Figure 5: Growth of the tectomer formed by the peptide Н-Gly4-NH(СН2)10-NHGly4-Н (concentration 0.1 mg/mL) on a mica surface at рН 6.5, with
tapping mode, SFM on air, and a tеxp of а) 0.5 min, b) 1 min, c) 2 min. Here, the shown field is completely covered with tectomer layer; its roughness
found to be ± 0.1 nm. Т indicates the tectomer layer, M the uncovered mica regions.
The dynamics of Н-Gly4NH(СН2)10NHGly4-Н association was dimensions of 500–700 nm (Figure 7a). The compound with
studied in more detail in a liquid cell (Figure 6). After 3 min the core C10 associated more rapidly (Figure 7b), though the
surface was virtually completely covered with a uniform defect- surface was not completely covered (texp = 10 min). This is in
free layer. It should be noted that the stepwise surface profile contrast to the structure analog with four glycines in the
(typical for bilayer structures) was not observed in a liquid cell. antenna, where a time period of only two minutes was suffi-
The layer morphology was identical to the one observed in cient for complete covering. The measured heights fit the model
experiments in air (Figure 5).
“1 + 1”.
Dynamic light scattering data give evidence that in neutral The obtained data give evidence that mica promotes the forma-
aqueous solutions the association of compounds Н-Gly5-NH-X- tion of tectomers from biantennary oligoglycines in neutral and
NH-Gly5-Н (cores С2 and С10) leads to the formation of large basic solutions. Layer growth proceeds due to the surface
aggregates. By means of SFM it was demonstrated that the co-participation. In the case of the molecule Н-Gly4-
peptide with core C2 formed islet-like tectomers on mica NH(СН2)10NH-Gly4-Н growth continues until the surface is
surface (texp = 10 min) with a height of 3.3 nm and planar completely covered, whereas in the bulk of the liquid phase
Figure 6: Growth of the tectomer formed by peptide Н-Gly4-NH(СН2)10NH-Gly4-Н (concentration 0.1 mg/mL) on a mica surface in a liquid cell at рН
6.5. Phase SFM images were taken with a) texp = 1 min, b) texp = 2 min and c) texp = 3 min where texp is the time after the experiment was started. Т
indicates the tectomer layer, M the uncovered mica regions.
1377

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
Figure 7: SFM images of associates formed by peptides а) Н-Gly5-NH(СН2)2NH-Gly5-Н and b) Н-Gly5-NH(СН2)10NH-Gly5-Н (concentration
0.1 mg/mL) on a mica surface at рН 6.5 with tеxp = 10 min SFM tapping mode in air, and an incubation time in solution of 1 min. Surface profiles,
schematic layer models, and their calculated heights are given on the right.
Minimal size of Glyn fragment providing
association
dimensions remain unchanged over time according to dynamic
light scattering data.
In the case of biantennary molecules association formally starts
There are no direct data pointing at the particular conformation from the value n = 4, but, in fact, this value is supposedly equal
(“1 + 1” or ”2 + 0”) the peptide monomer has in the layer to 8 because biantennary peptides form a polyglycine structure
(Figure 4). The height value does not allow for the unam- in the extended conformation ”1 + 1”. It is noteworthy that
biguous assignment of one of the proposed models. Neverthe- related polymers, nylons with the formula -NH(CH2)xCO- are
less, all intrinsic data supports the model ”1 + 1”: 1) the steps known to form a PG II structure [14], i.e., additional methylene
typical for a bilayer profile are not present in the SFM images, groups (-(CH2)x- instead of -CH2-) have no significant influ-
2) intense washing does not lead to the formation of half-height ence on its ability to form PG II. The association of tri- and
structures, 3) the lack of any association on a graphite surface tetraantennary peptides leads to structures with the monomer
where the formation of ”2 + 0” is expected to be preferable (see conformations ”2 + 2” and ”3 + 0”, respectively. The associ-
below).
ation starts from n = 7. Presumably, the first and closest to
forking Gly residue takes a distorted conformation and does not
Mica promotes the assembling of amino-terminated chains due take part in the formation of hydrogen bonds with neighboring
to the negative charge of the surface. In contrast, graphite did residues. In the case of tetraantennary oligoglycines the plane of
not participate in the association of biantennary oligoglycines, one pair of antennas is rotated by 90° [8] with respect to another
we observed only irregular associates formed in solution. This pair, so that the glycines cannot form a continuous chain. On
experimental result is unexpected, because the formation of a the other hand, in the biantennary analog the chain Gly4-Х-Gly4
monolayer with the monomer conformation ”2 + 0” is favor- has the ability to form a PG II structure despite the core frag-
able according to molecular dynamics simulations [13].
ment -(CH2)n-.
1378

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
The nature of the core fragment Х in
H-Glyn-Х-Glyn-H
bling of monomer layers into surface tectomer layers or into
long-living associates in solution. 4) Oligoethylene glycol core
Biantennary molecules with core С10 form tectomers on a mica ‘inhibits’ the association both in the liquid phase and on a mica
surface more readily than the molecules with core C2 and an surface.
equal number of glycines. The more flexible core C10 should
lead to a entropy driven destabilization. The opposite effect Antiviral activity of glycoderivatives
observed in reality is most probably caused by van der Waals The idea of antiadhesion influenza virus therapy is based on the
interactions of hydrophobic fragments C10 closely situated in inhibition or the blocking of the binding of the influenza virus
the PG II structure. The oligoethylene glycol core abolishes the with target cells [15]. Monovalent oligosaccharides are inca-
formation of a PG II structure, presumably due to competitive pable of an efficient competition for analogous glycans on the
hydrogen bonding with spatially close oligoglycine fragments. cell surface due to the low binding constant with viral hem-
agglutinin. An attractive way of increasing the affinity of a
Spontaneous and surface-promoted
association
blocker (inhibitor) is the design of multivalent receptor analogs
such as the oligoglycine-based tectomers described above. A
The formation of tectomers on mica proceeds considerably first success for an application in this regard was achieved by
more rapidly than association in solution (the formation of asso- inhibiting the influenza virus by sialo derivatives of the associ-
ciates in solutions just starts when assembling on the surface is ating tetraantennary peptides, which demonstrated an antiviral
already finished), i.e., the mica surface obviously plays an activity three orders of magnitude higher than the activity of
active role in the process. Tectomer growth starts from the for- non-associating analogs [2]. Similar triantennary molecules
mation of islet structures that increase in lateral direction and with sialo-glycan located in the molecule “head”, however,
cover the whole surface in an even layer. This growth is limited appear to display a low activity [16]. Thus, it was interesting to
only by the dimensions of the support itself. It should be noted study the antiviral activity of sialo derivatives of biantennary
that graphite, in contrast to mica, does not promote association. oligoglycines in relation to their propensity to associate in
aqueous solutions.
Effect of рН value
Depending on the pH value, the free terminal group of the The fact that a sialylated biantennary peptide is capable of
oligoglycine chain can be heavily charged, weakly charged, or association in an aqueous solution similarly to glycan-free
neutral. In acidic solutions antennas are repulsed due to the peptides is confirmed by DLC data. The average size of the
positive charge, which hinders tectomer assembly or even abol- sialoglycopeptide aggregates in aqueous solution was about
ishes it. The pH value effects not only the ability to assemble 1 μm (data not shown).
but also the morphology of forming supramers. Thus, in neutral
solutions biantennary oligoglycines form multilayer tectomers. The antiviral activity of biantennary glycopeptides was studied
The process is unstoppable at the stage of the monolayer forma- by means of a fetuin binding inhibition test (FBI-test) [17] (the
tion. At the same time, monolayer tectomers are exclusively glycoprotein fetuin contains several sialylated carbohydrate
formed in basic solutions.
chains). In this test, the glycopeptides inhibited the binding of a
fetuin peroxidase conjugate to a virus immobilized on a plastic
(related to the corresponding monomer). Results are given in
Concentration range
Most parts of the experiments were carried out in the concentra- Table 1. The activity of associating sialooligoglycines with core
tion range of 0.1–1.0 mg/mL. A concentration of 0.1 mg/mL C2 was only 3–6 times higher than the activities of their non-
was used for the adequate comparison of association of all associated counterparts (n = 2–4) and the monomeric reference
investigated peptides. The association in the liquid phase sialoside, Neu5AcαBn. The compound with core С10 and n = 4
proceeds slower at low concentrations leading to an increased demonstrated the highest activity from the studied biantennary
size of the formed supramers.
glycopeptides, which was 50 times higher than the activity of
the monomer. The activity of bivalent derivatives with core
In summary, based on our investigations related to unglycosy- OEG and n = 2–5 did not exceed that of monovalent sialoside.
lated molecules we can conclude that the association of bianten- However, the activity increased dramatically when n = 6 (up to
nary oligoglycines is affected by several factors. 1) Mica but 50 times), although it was still orders of magnitude smaller
not graphite promotes the formation of tectomers. 2) The spatial compared to the high activity of polymeric inhibitors [18]. As
organization of oligoglycine molecules in supramers corre- sialooligoglycines of the OEG series did not associate in
sponds to PG II conformation. 3) Not less than four glycine aqueous solution, we suppose that the reason for the increased
residues in each of two antennas are required for the assem- activity is related to a critical distance, which facilitates the real-
1379

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
Table 1: Relative activity of biantennary glycopeptides in the influenza virus receptor binding inhibition assay [17].
Core, Х (n, number of glycine residues in
Compounda
Virus
Relative activity
antenna)
Neu5AcαOBn
1(150)b
С2 (2–4)
С2 (5)
1
Neu5Acα-sp1-Glyn-Х-Glyn-sp1-Neu5Acα
3
(A/Н3N2/29/90)
С2 (6)
6
C10 (1–3)
С10 (4)
С10 (5)
1
Neu5Acα-sp1-Glyn-Х-Glyn-sp1-Neu5Acα
6’SLN
50
25
1(150)b
OEG (2, 4)
ОEG (5)
ОEG (6)
1
(A/H1N1/NIB23)
(A/H5N2)
6’SLN-sp2-Glyn-Х-Glyn-sp2-6’SLN
3’SL
1
40
1(150)b
ОEG (2, 4)
ОEG (5)
ОEG (6)
1
1
3’SL-sp3-Glyn-Х-Glyn-sp3-3’SL
50
aAbbreviations: sp1 = -OCH2(p-C6H4)NHCOCH2NH-CO(CH2)4CO; Bn – benzyl; sp2 = -O(CH2)3NHCO(CH2)4CO; 6’SLN = Neu5Acα2-6Galβ1-
4GlcNAcβ; sp3 = -NHCOCH2NHCO(CH2)4CO; 3’SL = Neu5Acα2-3Galβ1-4Glcβ. bValues of IC50, μM, for monomeric Neu5AcαOBn, 6'SLN and 3’SL
are given in parentheses.
ization of a divalent interaction of this bivalent molecule with a signals in 1Н NMR spectra are given in ppm and spin–spin
viral hemagglutinin. Indeed, a simple calculation demonstrates coupling constants (J) in Hz. The scale of the chemical shifts
that this distance in a maximally extended molecule with n = 6 was calibrated against the signals of residual protons of solvents
is about 100 Å. This distance value corresponds to the distance (CDCl3: δ 7.26 ppm; DMSO-d6: δ 2.50 ppm; D2O: δ 4.75 ppm).
between the carbohydrate binding sites in one molecule of a Mass-spectra were recorded on the time-of-flight spectrometer
hemagglutinin homotrimer and slightly exceeds the distance Vision-2000 (Thermo Bioanalysis, UK) with MALDI with 2,6-
between a couple of hemagglutinin trimmers, which are closely dihydroxybenzoic acid as reference. Raman spectra were
situated on the virion surface.
recorded on a spectrometer Ramanor HG-2S (Jobin Yvon) with
the monochromator Anaspec 300S and argon (λ = 514.5 nm,
Spectra Physics, model 164-03).
Experimental
Reagents and solvents were bought from Merck and
Sigma–Aldrich. Activated esters BocGlyONSu and Synthesis of biantennary oligoglycines
BocGly2ONSu were prepared as described earlier [9] from Protocol 1: Elongation of the oligoglycine chain (Boc-GlynNH-
glycine or glycylglycine (Acros). Ethylenediamine and 1,10- X-NHGlyn-Boc; n = 1–7, X = С2, С10 and OEG). Et3N
diaminodecane were supplied from Sigma-Aldrich, and di- (8 mmol) followed by BocGlyONSu or BocGly2ONSu
amine NH2(CH2CH2O)3CH2CH2NH2 (1) was synthesized from (3 mmol) were added to a solution of diamine (1 mmol) in
ditosylate TosO(CH2CH2O)3CH2CH2OTos (Sigma–Aldrich) dimethyl sulfoxide (DMSO; 5 mL). The reaction mixture was
according to the described methods [7,8].
stirred until the disappearance of the starting diamine (1–24 h,
TLC control) and the solvent was removed under vacuum. The
Silica gel (Kieselgel 60, Merck, Germany) was used for low- dry residue was suspended in methanol, filtered, dissolved in
pressure column chromatography. Sephadex LH-20 (Pharmacia water, sedimentated with methanol, and dried in vacuo.
Biotech, Austria) was employed for gel chromatography. Thin-
layer chromatography (TLC) was performed on foil plates Protocol 2: Preparation of oligoglycines (HCl·GlynNH-X-
covered with silica gel (Kieselgel 60, Merck, Germany).
NHGlyn·HCl; n = 1–7, X = С2, С10 and OEG). The Boc-deriva-
tive (0.5 mmol) was dissolved in trifluoroacetic acid (5 mL), the
1Н NMR spectra were recorded on a Bruker spectrometer (600, reaction mixture was kept for 2 h at room temperature, co-evap-
700, 800 MHz) at 303 K. Chemical shifts (δ) for characteristic orated with toluene (2 × 10 mL) and 1 M HCl aqueous solution
1380

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
(1–2 mL), and finally with a mixture iPrOH/methanol 1:1 specified period of time within the range of 0 to 10 min. Liquid
(2 × 10 mL). The obtained product was sedimentated from the was removed from the surface by spin coating or in nitrogen
aqueous solution by the addition of methanol and dried in flow. Structures formed on the surface were visualized in
vacuo.
tapping mode SFM.
Synthesis of associating glycopeptides
Scanning in liquid cell. A plate of freshly cleaved mica
Protocol 3: Neu5Acα-sp1-ONp, 3'SL-sp3-ONp or 6'SLN-sp2- (1 × 1 cm2) was placed in a liquid cell. The cell was filled with
ONp (4 μmol) were added to a solution of diamine (1 μmol) in water (25 μL) and the instrument was set up. Then, water was
DMSO or saturated aqueous solution of LiBr (200 μL). NEt3 changed with a freshly prepared solution of deprotonated
(4 μmol) was added until a рН of 8 was reached, and the mix- peptide (see scanning in air above) and the surface was scanned
ture was stirred for 24 h at room temperature. Exclusion chro- in contact mode SFM in fixed time periods.
matography on Sephadex LH-20 (eluent: 0.1 М solution of NH3
in the mixture acetonitrile/water, 1:1). Fractions containing pure The influenza virus receptor-binding inhibition assay was
product were combined and evaporated. Dry residue was carried out as described in [17].
dissolved in water and freeze-dryed.
Supporting Information
Dynamic light scattering experiments
The light scattering of aqueous solutions of biantennary oligo-
Supporting Information File 1
glycines was studied with an analyzer of submicron particle size
“Malvern HPPS” (UK). After the preparation of aqueous
(Milli-Q) solutions of oigoglycine salts in a concentration of
0.01–0.1 mg/mL, the instrument reading was recorded (t = 0,
pH < 5). Then, 1–2 equiv of base (0.1 М aqueous solution of
NaHCO3 or Na2CO3) per amino group was added to the solu-
tion of the analyzed oligoglycine salt (t > 0, pH 6–8), and
instrument readings were recorded in fixed periods of time. In
the case of the formation of large associates (intense opales-
cence, sedimentation), whose dimensions exceeded the working
limit of the instrument, the experiment was stopped.
Descriptions of the synthesis of individual compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-140-S1.pdf]
Acknowledgments
This study was partly supported by the Russian Academy of
Sciences Presidium Program Molecular and Cell Biology. The
authors would like to thank Dr. Ekaterina A. Obraztsova for the
Raman analysis and Dr. Galina V. Pazynina for valuable discus-
sions about the experimental design and the results.
References
For experiments with biantennary sialooligoglycines their
aqueous (Milli-Q) solutions with a concentration of 0.1 mg/mL
were used.
1. Tsygankova, S. V.; Chinarev, A. A.; Tuzikov, A. B.; Gambaryan, A. S.;
Bovin, N. V. “Self-assembling glycopeptides” presented at the 17th
European Carbohydrate Symposium “EuroCarb17”, Tel Aviv, Israel,
July 7–11, 2013, P-55.
Scanning force microscopy (SFM)
2. Tuzikov, A. B.; Chinarev, A. A.; Gambaryan, A. S.; Oleinikov, V. A.;
Klinov, D. V.; Matsko, N. B.; Kadykov, V. A.; Ermishov, M. A.;
Demin, I. V.; Demin, V. V.; Rye, P. D.; Bovin, N. V. ChemBioChem
2003, 4, 147–154. doi:10.1002/cbic.200390025
The samples were imaged with a Nanoscope IIIa instrument
(Digital Instruments, USA). Commercial silicon nitride
cantilevers with force constants of 0.06, 0.12, and 0.32 Nm−1
were used for the measurements in contact mode in liquid cell.
Cantilevers with a resonance frequency of about 300 kHz and a
force constant of 42 Nm−1 were used for the SFM tapping mode
in air. Software WSxM (Nanotec Electronica, Spain) was
employed for the image treatment. Pure water (Fluka) was used
for the preparation of solutions.
3. Bovin, N. V.; Tuzikov, A. B.; Chinarev, A. A.
Nanotechnologies in Russia 2008, 3, 291–302.
doi:10.1134/S1995078008050042
4. Bamford, C. H.; Brown, L.; Cant, E. M.; Elliott, A.; Hanby, W. E.;
Malcolm, B. R. Nature 1955, 176, 396–397. doi:10.1038/176396a0
5. Crick, F. H. C.; Rich, A. Nature 1955, 176, 780–781.
doi:10.1038/176780a0
6. Bovin, N. V.; Tuzikov, A. B.; Chinarev, A. A.; Gambaryan, A. S.
Glycoconjugate J. 2004, 21, 471–478. doi:10.1007/s10719-004-5537-3
7. Katritzky, A. R.; Singh, S. K.; Meher, N. K.; Doskocz, J.; Suzuki, K.;
Jiang, R.; Sommen, G. L.; Ciaramitaro, D. A.; Steel, P. J. ARKIVOC
2006, (v), 43–62. doi:10.3998/ark.5550190.0007.505
Scanning in air. 1–2 equiv of 0.1 M of aqueous solution of
NaHCO3 or Na2CO3 per amino group (pH ~ 6–8) was added for
the deprotonation to a freshly prepared solution of oligoglycine
salt (0.1–1.0 mg/mL; pH < 5), and incubated for a specified
time period in the range of 0 to 90 min. Then the solution was
applied on the freshly cleaved mica or graphite, and kept for a
1381

Beilstein J. Org. Chem. 2014, 10, 1372–1382.
8. Katritzky, A. R.; Meher, N. K.; Hanci, S.; Gyanda, R.; Tala, S. R.;
Mathai, S.; Duran, R. S.; Bernard, S.; Sabri, F.; Singh, S. K.;
Doskocz, J.; Ciaramitaro, D. A. J. Polym. Sci., Part A: Polym. Chem.
2008, 46, 238–256. doi:10.1002/pola.22376
9. Gershkovich, A. A.; Kibirev, V. C. Chemical synthesis of peptides;
Naukova Dumka: Kiev, Ukraine, 1992.
10.Chinarev, A. A.; Tuzikov, A. B.; Gambaryan, A. S.; Matrosovich, M. N.;
Imberty, A.; Bovin, N. V. In Sialobiology and Other Novel Forms of
Glycosylation; Ynoue, Y.; Lee, Y. C.; Troy, F. A., II., Eds.; Gakushin
Publishing Co.: Osaka, 1999; pp 135–143.
11.Bovin, N. V.; Chinarev, A. A.; Tuzikov, A. B. Multiligand constructs. WO
Patent WO/2010/043230, Oct 13, 2008.
12.Krimm, S.; Bandekar, J. In Advanced Protein Chemistry;
Anfinsen, C. B.; Edsall, J. T.; Richards, F. M., Eds.; Academic Press:
New York; Vol. 38, pp 181–364.
13.Gus’kova, O. A.; Khalatur, P. G.; Khokhlov, A. R.; Chinarev, A. A.;
Tsygankova, S. V.; Bovin, N. V. Russ. J. Bioorg. Chem. 2010, 36,
574–580. doi:10.1134/S1068162010050043
14.Bella, J.; Puiggali, J.; Subirana, J. A. Polymer 1994, 35, 1291–1297.
doi:10.1016/0032-3861(94)90026-4
15.Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed.
1998, 37, 2754–2794.
doi:10.1002/(SICI)1521-3773(19981102)37:20<2754::AID-ANIE2754>3
.0.CO;2-3
16.Chugunov, P. A.; Chinarev, A. A.; Tuzikov, A. B.; Formanovsky, A. A.;
Prokhorov, V. V.; Gambaryan, A. S.; Bovin, N. V. Mendeleev Commun.
2009, 19, 62–63. doi:10.1016/j.mencom.2009.03.002
17.Gambaryan, A. S.; Matrosovich, M. N. J. Virol. Methods 1992, 39,
111–123. doi:10.1016/0166-0934(92)90130-6
18.Gambaryan, A. S.; Tuzikov, A. B.; Piskarev, V. E.; Yamnikova, S. S.;
Lvov, D. K.; Robertson, J. S.; Bovin, N. V.; Matrosovich, M. N. Virology
1997, 232, 345–350. doi:10.1006/viro.1997.8572
License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.10.140
1382