Exploring Rigid and Flexible Core Trivalent Sialosides for Influenza Virus Inhibition

Article abstract of DOI:10.1002/chem.201804826

Herein, the chemical synthesis and binding analysis of functionalizable rigid and flexible core trivalent sialosides bearing oligoethylene glycol (OEG) spacers interacting with spike proteins of influenza A virus (IAV) X31 is described. Although the flexible Tris-based trivalent sialosides achieved micromolar binding constants, a trivalent binder based on a rigid adamantane core dominated flexible tripodal compounds with micromolar binding and hemagglutination inhibition constants. Simulation studies indicated increased conformational penalties for long OEG spacers. Using a systematic approach with molecular modeling and simulations as well as biophysical analysis, these findings emphasize on the importance of the scaffold rigidity and the challenges associated with the spacer length optimization.

Full text of DOI:10.1002/chem.201804826

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Title: Exploring rigid and flexible core trivalent sialosides for influenza
virus inhibition
Authors: Rainer Haag, Pallavi Kiran, Sumati Bhatia, Daniel Lauster,
Stevan Aleksić, Carsten Fleck, Natalija Peric, Wolfgang
Maison, Susanne Liese, Bettina G. Keller, and Andreas
Herrmann
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Chemistry - A European Journal
Exploring rigid and flexible core trivalent sialosides for
influenza virus inhibition
Pallavi Kiran,a,1 Sumati Bhatia,a,1 Daniel Lauster,b Stevan Aleksić,c Carsten Fleckd, Natalija
Pericd, Wolfgang Maison,d Susanne Liese,e Bettina G. Keller,c Andreas Herrmann,b Rainer
Haaga,*
aInstitut für Chemie und Biochemie Organische Chemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany
bInstitut für Biologie, Molekulare Biophysik, IRI Life Sciences, Humboldt-Universität zu Berlin, Invalidenstr. 42, 10115 Berlin, Germany
cInstitut für Chemie und Biochemie, Physikalische und Theoretische Chemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany
dFachbereich Chemie, Institut für Pharmazie, Universität Hamburg, Bundesstr. 45, 20146 Hamburg, Germany
eDepartment of Mathematics, University of Oslo, P.O box 1053 Blinder, 0316 Oslo, Norway
1Authors contributed equally
*Corresponding author
Rainer Haag: haag@zedat.fu-berlin.de
Abstract
We describe the chemical synthesis and binding analysis of functionalizable rigid and flexible core trivalent sialosides bearing
oligoethylene glycol (OEG) spacers interacting with spike proteins of influenza A virus (IAV) X31. Although the flexible Tris-based
trivalent sialosides achieved micromolar binding constants, a trivalent binder based on a rigid adamantane core dominated flexible
tripodal compounds with micromolar binding and hemagglutination inhibition constants. Simulation studies indicated increased
conformational penalties for long OEG spacers. Using a systematic approach with molecular modeling and simulations as well
as biophysical analysis, our findings emphasize on the importance of the scaffold rigidity and the challenges associated with the
spacer length optimization.
Key words: trivalent sialoside, adamantane core, influenza inhibitor, oligoethylene glycol
Introduction
Annually, seasonal influenza infections pose a great burden to global health and economy affecting up to 15% of the
world population resulting in more than 500,000 deaths per year worldwide.[1] High mutation rates of this virus limit the
use of available antiviral agents for a long-lasting application. Thus, there is urgent need for the development of novel
antiviral drugs and therapies. A well-known strategy is the application of biomimetic binding inhibitors, acting as decoy
molecules to prevent viral attachment to host cells. The latter is mediated by multivalent interaction of the homotrimeric
hemagglutinin (HA) spike protein that is highly abundant on the viral surface with terminal sialic acids of glycostructures
on the host cell plasma membrane.[2] Each monomer of the HA trimer has a receptor binding site (RBS) for sialic acid
(SA). Inhibitors displaying SA moieties and thus competing with virus receptors on host cells, have been successfully
implemented in various studies.[2b] Recently, Bandlow et al. reported on micromolar binding constants with bivalent
sialyllactose derivatives based on DNA-PNA complexes.[3] Further, Bhatia et al. reported on nanomolar inhibitors of
influenza virus infection with linear and dendritic polyglycerol based multivalent sialosides.[4] However, only few examples
demonstrated the design of a trivalent binder, which is based on a rational design to match the geometric and topological
situation on HA.[3, 5]
As revealed from the crystal structure of HA from a human pathogenic H3N2 strain (pdb:1hgg), the planar distance
between two SA-binding sites on a single HA trimer is around 4-5 nm (Figure 1).[6] HA trimers are densely packed in the
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10.1002/chem.201804826
Chemistry - A European Journal
viral envelope and two adjacent HA3 are 10-12 nm centrally apart as determined from electron microscopy imaging.[7]
Considering this spatial HA organization, a high affinity trivalent SA binder can be hypothetically designed by choosing
an appropriate spacer to bring three covalently connected SA units in the right geometry matching that of the RBSs of
HA3. Waldmann et al. explored trivalent SA presenting glycopeptide conjugates for targeting HA trimers of influenza A
virus type H5 and obtained a binding constant of 446 nM from surface plasmon resonance (SPR) measurements, which
is a 4000-fold increase compared to the monomeric binder methyl 2-methyl-α-d-N-acetylneuraminide (Neu5Acα2Me) or
methyl sialic acid respectively. However, the inhibition values were still weak and in the micromolar range in saturation-
transfer difference (STD) NMR based competition experiments. Nishimura and colleagues devised a glycopeptide
possessing a cyclic peptides as scaffold [Neu5Acα(2,3)Galβ(1,4)Glc] to display three sialotrisaccharide units at the
termini.[8] In further studies, Nishimura et al. prepared trivalent sialyllactosides using trisphenol and trisaniline as cores
and extended the length of the spacer with ethylenglycol trimers (EG3), glutamine, and C6 to partially compensate for the
smaller scaffold.[9] These studies indicated that the design of trivalent binders is still challenging and that there are still
several unknown parameters to finetune a trivalent binder for an optimal interaction with HA.
In this study, we address the relevance of the flexibility of the anchor point carrying the sialoside residues for optimal
interaction with HA. Furthermore, we varied the spacer length on both a rigid and a flexible core system to adjust the
receptor spacing to match the given RBS distribution on HA trimers. The OEG spacers have been used due to their
hydrophilicity and biocompatibility. However, the drawback of long OEG spacers is their high tendency to form globular
structures, hampering an orchestral receptor orientation. OEG spacer containing HA-binders were implemented first by
Knowles et al. for the synthesis of bivalent sialosides that showed 100-fold increased hemagglutination inhibition against
influenza A virus X31 (H3N2) as compared to monovalent sialic acid.[10]
In the present study, we systematically explored trivalent sialosides with different OEG lengths using two differently
functionalizable core structure with either (i) an adamantane core, and (ii) a commercially available Tris i.e. 4-
(((benzyloxy)carbonyl)amino)-4-(2-carboxyethly)heptanedioic acid core. Due to a restricted degree of rotational freedom
of the anchorage points on the different scaffolds, adamantane can be considered to be more rigid compared to Tris.
Here we present the synthesis and comparative analysis of rigid and flexible core based trivalent sialosides with different
spacer lengths for binding to IAV-X31 (subtype Aichi) using microscale thermophoresis (MST) and hemagglutination
inhibition (HAI) assays. Despite the challenges associated with the spacer length optimization, we could identify an
inhibitor against X31 with a HAI value in the micromolar range, thus being approximately 3000-fold more potent compared
to monomeric SA. The present study reveals insights into the scaffold selection and spacer length optimization and
emphasizes their relevance for the design of trivalent HA binder.
Results and Discussion
Based on the crystal structure of a single HA trimer (pdb: 1hgg), we determined the projected distance between the
receptor binding sites, and the trimer midpoint of 2.6 nm. The binding site is found about 0.9 nm below the top of the HA
surface, which leads to an effective distance of 3.5 nm between the receptor binding sites and the HA midpoint. The part
of the tripod has a length of approximately 1.2 nm. Hence the OEG- spacer has to bridge a distance of approximately 2
nm. The length distribution of flexible OEG chains that we obtained from our previous MD simulations,[11] suggested that
the OEG-linker must consist of at least 6-14 ethylene glycol units to span the distance between the ligand core and the
binding sites (c.f. Supporting Information)
2
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Figure 1. a) Top and b) side view of the HA -region (pdb: 1hgg). Bound sialic acid is highlighted in green.
Synthesis of adamantane based trivalent and bivalent sialosides
Heterobifunctional oligoethylene
glycols,[12]
tris(2-carboxymethyl)aminoadamantane
amine,[13]
bis(2-
carboxymethyl)aminoadamantaneamine and cbz-protected adamantane triacid,[14] and diacid[15] were prepared as
reported in literature. SA-conjugated trivalent derivative with only an ethylene spacer 4 was prepared by using amide
coupling with an ethanolamine sialic acid derivative 3 (Scheme 1). While reactions of adamantane triacid were less
efficient when done by direct amide coupling of bigger length EG14 spacer. Therefore, coupling of cbz-protected
adamantane triacid 1 with propargylamine provided an easy way to couple heterobifunctional oligoethylene glycols by a
click chemistry approach. The addition of extra triazole units by amide bonds provided the limited flexibility and decreased
the length requirements of a flexible OEG spacer. Heterobifunctional azido-tosylated EG6 and EG14 were synthesized as
short and long OEG spacers to achieve the predicted OEG requirements. The prop-2-ynyl α-thiosialoside was synthesized
using procedure reported by Roy et al[16] and a slightly modified procedure of Ogura et al.[17] The prop-2-ynyl α-
thiosialoside was conjugated by click chemistry to trivalent adamantane derivatives 6 and 7 (Scheme 2). For the
preparation of a divalent adamantane sialoside, a heterobifunctional EG6 spacer bearing azide and sialic acid residues
13 was synthesized and coupled to divalent propargylated adamantane 12. The adamantane divalent sialoside was
prepared to study the dependency of affinity on the presence of 2 vs 3 sialic acid valencies on the rigid core (Scheme 3).
Scheme 1. Synthesis of adamantane core trivalent sialoside with an EG1 spacer.
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Scheme 2. Synthesis of adamantane core trivalent sialosides with EG6 and EG14 spacers.
Scheme 3. Synthesis of adamantane core divalent sialoside with EG6 spacer.
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Synthesis of Tris-based trivalent sialosides
4-(((benzyloxy)carbonyl)amino)-4-(2-carboxyethly)heptanedioic acid 16 was prepared according to literature procedures
from di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 15[17] followed by coupling with propargylamine to
produce tripropagylated derivative 19. The triazido derivatives cbzTris(EG4N3)3 22 and cbzTris(EGN3)3 20 were prepared
in two consecutive steps by conjugating heterobifunctional azido-tosyl EG4 and EG1 units followed by in situ azidation
(Scheme 5). Prop-2-ynyl α-thiosialoside was then clicked in a similar fashion as done before to afford the final trivalent
sialosides with a flexible core 21 and 23. Heterobifunctional amino-azido EG7 was easily obtained from Polypure which
was then coupled with 4-(((benzyloxy)carbonyl)amino)-4-(2-carboxyethly)heptanedioic acid 16 using amide coupling
(Scheme 4). Prop-2-ynyl α-thiosialoside was clicked with cbzTris(EG7N3)3 17 as described before to afford
cbzTris(EG7SA)3 18.
Scheme 4. Synthesis of Tris core trivalent sialoside with EG7 spacer.
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Scheme 5. Synthesis of Tris core trivalent sialosides with an EG1 and EG4 spacer.
Influenza virus binding studies
Binding of all synthesized sialosides and control molecules to influenza A virus X31 was studied first by an
hemagglutination inhibition assay. Unfunctionalized core molecules (1 and 16), and core molecules carrying only OEG
spacers (24 and 17) did not show any significant hemagglutination binding inhibition through-out the tested concentration
range (Figure 2). Among the adamantane based compounds, only the trivalent sialoside with EG6 spacer length, i.e.,
cbzAd(EG6SA)3 8 showed inhibition in the micromolar range. This suggests a divalent interaction, which was subsequently
validated by synthesizing a divalent sialoside cbzAd(EG6SA)2 14. Binding inhibition values for both cbzAd(EG6SA)3 8 and
divalent cbzAd(EG6SA)2 14 were in the micromolar range, i.e., 30 and 100 µM, respectively. The values based on SA are
100 and 200 µM, respectively. The slight difference between both values can be rationalized by the cooperativity factor
which is discussed in the next section.
Next, microscale thermophoresis (MST) was applied to determine the affinity constants of all constructs using rhodamine
(R18) labeled intact X31 virus. The apparent Kd,app values for both trivalent and divalent rigid core-based adamantane
sialosides 8 and 14 were almost in the same micromolar range, i.e., 58 and 71 µM respectively. Other adamantane based
constructs did not show any affinity for the virus which is in agreement with the results from the HAI assay.
The flexible Tris-based core sialosides showed HAI values only in the millimolar range, 6.67 mM was lowest for the one
with EG4 spacer, i.e., cbzTris(EG4SA)3 23. In contrast, the affinity constant values KiHAI for the Tris core trivalent sialosides
measured by MST were in the micromolar range. cbzTris(EG4SA)3 23 showed enhanced affinity towards virus particles
with apparent Kd,app around 16 µM. A shorter or longer spacer caused around 10 times lower affinity. The disagreement
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between the results of HAI and MST measurements might be due to additional non-specific interactions of the compounds
with the red blood cells or viral surface at sites other than the sialic acid binding pockets of HA.
Both cores (Ad and Tris) are similar in size. However, the dissociation constant of inhibitor 8 and 23, which have similar
spacer lengths but different cores, differ significantly. We attribute this difference to the flexibility of the inhibitor cores. A
binding event is always associated with a restriction of conformational degrees of freedom, which weakens the binding
affinity. In case of the more rigid Ad-core, this conformational restriction is less pronounced compared to the more flexible
Tris-core, which leads to a stronger binding affinity for Ad-based inhibitors.
Table 1: Summary of hemagglutination inhibition values (KiHAI) and apparent dissociation constants (Kd,app) as determined by microscale
thermophoresis (MST). Data from the HAI are given in respect to SA or tripod concentration. For Kd,app values the mean (N≥4) and SEM are given.
Kd,app (µM)
Compound
KiHAI (mM) SA
KiHAI (mM) tripod
tripod
2,6-sialyl lactose
cbzAd(EG1SA)3 4
cbzAd(EG6SA)3 8
cbzAd(EG14SA)3 9
cbzAd(EG6SA)2 14
100
>20
0.10
>15
0.20
-
ND
>6.7
0.03
>5
no binding
58±15
no binding
71±11
0.10
cbzAd(COOH)3(Control) 1
cbzAd(EG6OH)3(Control) 24
cbzTris(EG1SA)3 21
no inhibition
no inhibition
>20
>10
no binding
no binding
169±21
-
>6.7
6.7
cbzTris(EG4SA)3 23
20
16±4
cbzTris(EG7SA)3 18
>50
>16.7
124±21
cbzTris(COOH)3(Control) 16
cbzTris(EG7N3)3(Control) 17
no inhibition
no inhibition
no binding
no binding
-
-
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Figure 2. Microscale thermophoresis measurements with intact R18 labeled X31 virus against a) different adamantane-based or b) Tris-based
constructs. The change in fluorescence intensity during thermophoresis is shown as a function of the construct’s concentration. Data points are
one-sited fit. Error bars indicate the SEM (N≥4).
Model for trivalent vs. divalent binding
The HA inhibition measurements reveal a binding affinity of the trivalent inhibitor 8 that is by about a factor of three
stronger than the binding affinity of the divalent inhibitor 14.To further investigate the size range in which trivalent inhibitors
have the potential to outperform their divalent counterparts, we theoretically determined the ratio of the trivalent and
divalent dissociation constant in dependence of the average spacer length. The dissociation constant, Kn, of a n-valent
ligands that is bound with all n binding moieties to an n-valent receptor reads[18]
퐾m푛ono휔푛
퐾n =
, (1)
퐶푛
where each binding moiety, i.e. each bound sialic acid, contributes with Kmono, the monovalent dissociation constant, to
the multivalent dissociation constant. We set Kmono = 2.5 mM[19] for the monovalent sialic acid. The angular restriction
factor ⍵ accounts for the reduced rotational degrees of freedom due to steric repulsion between the sialic acid and the
OEG spacer. This parameter ⍵ describes the angular space available to each sialic acid compared to an unbound
monovalent sialic acid unit. We assumed a value of ⍵=0.03, which was found for pentavalent inhibitors against cholera
toxin and heptavalent inhibitors against the anthrax receptor.[18] The impact of the inhibitor scaffold on the multivalent
dissociation constant is quantified by the cooperativity factor Cn, which describes the probability that all n sialic acid units
are simultaneously bound to the HA receptor. The cooperativity factor accounts for the ligand conformation as well as the
number of binding permutations. For both the trivalent as well as the divalent ligand there are six equivalent binding
permutations by which the ligands can bind to a trivalent receptor. For the divalent inhibitor, Cn is equivalent to the effective
concentration. A detailed discussion of the influence of inhibitor core size and spacer length on the cooperativity factor is
presented in the Supporting Information. According to Eq. 1 the ratio between the dissociation constant of a fully bound
trivalent and a fully bound divalent inhibitor is obtained as:
퐾3
퐾2
퐶2
퐶3
= 퐾mono
휔
, (2)
which is shown in Figure 3 depending on the average spacer length rete. Over a wide range of spacer lengths, the
dissociation constant of di- and trivalent inhibitors differ by less than one order of magnitude. While for very short spacers,
divalent inhibitors bind stronger than trivalent inhibitors, in the size range of spacer length that is optimal for HA binding,
i.e., for spacers with a length of around 1.4 nm, the binding affinity of trivalent inhibitors surpasses the divalent inhibitors,
due to a favorable binding cooperativity.
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Figure 3. Trivalent vs. divalent binding: The ratio of the trivalent dissociation constant K3 and the divalent dissociation constant K2, according to Eq.
2. The distance between 1.4 nm and 2.3 nm that has to be bridged by the inhibitor spacer to reach HA binding pocket, is indicated by a dashed
vertical line.
Computer simulation studies
To answer the question why only compound 8 exhibited a significant affinity, we set up molecular dynamics simulations
of the monovalent counterparts of compounds 8 and 9. This simulation protocol was chosen in order to minimize
computational costs. Still, we were able to determine the behavior of the entire inhibitor, since the extensions of the
individual OEG spacers are independent of each other. To trace the conformational behavior of the simulated ligands, we
measured the distance distributions for several atom pairs as indicated in Figure 4A. First, we were interested if addition
of the OEG spacer influenced the conformational behavior of the adamantane core. As presented in Figure 4B, the C8-
N5 atom pair distance distributions of both investigated molecules overlapped and were mostly located in the range of
0.7 nm to 0.9 nm. This finding was not surprising, as the planarity of the amide bond and adjacent triazole ring provided
the limited flexibility of this building block. In Figures 4C and 4D respectively, the distribution of the distance between
outermost OEG oxygens, of and the distance between the adamantane core and the sialic acid is shown. Both
distributions overlap, but the C8-S1 distribution was much narrower, which suggests that the sialic acid and the inhibitor
core were in close contact without a receptor, i.e. the binding pocket. Therefore, we investigated the formation of the
intramolecular hydrogen bonds between all the pairs of hydrogen bond donors and acceptors. Our analysis revealed that
sialic acid remained free to potentially interact with the respective binding site of HA. In both simulated systems, the
bonded atom pair N2-H20 was the most dominant hydrogen bond donor. It holds the OEG spacer and imidazole ring
adjacent to the sialic acid in proximity to the adamantane core through the formation of several transient hydrogen bonds.
In the case of compound 8, N2-H20 donor participated in the intramolecular hydrogen bonding for 34.5% of the simulation
time, whereas it acted for compound 9 for the half of the simulation time. These findings would suggest that these two
compounds should have bound to HA with almost similar affinity. Intuitively one notices from Figures 4E and 4G the
increase in the length of OEG spacer is correlated with the increase in the size of the respective OEG coil. To quantify
the size of OEG coils, we used the radius of gyration as a measure. As shown in Figure 4G, the size of OEG coils of
compound 9 is indeed larger compared to compound 8. If we now consider that all three OEG arms adopted the OEG-
coil conformation, then the whole molecule would have been in a rather globular /compact conformation resulting in
sterical repulsion from the receptor. Having the larger overall size of the OEG-coils, long spacer inhibitor 9 would repel
more often from the surface of the receptor, thus the probability for the binding event is reduced compared to short spacer
inhibitor 8.
Consequently, this suggests that compound 8 unfolds from the coil-like structure easier than compound 9, which might
explain the inhibitory constant in the micromolar range.
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Figure 4. (A) Pymol representations of the monovalent counterparts of compound 10 (left panel) and compound 11 (right panel). The atoms used
to characterize the conformational dynamics of the ligands are marked according to the Acpype parametrization. (B) The distance distribution of
the C8-N5 atom pair capturing the dynamics of the rigid part of the ligands. (C) The distance distribution of the C8-S1 atom pair capturing the distance
between the rigid core and sialic acid. (D) The distance distribution of the outermost oxygen atoms of OEG spacers. (E) Pymol representation of
the most stable intramolecular hydrogen bond of compound 8. (F) Pymol representation of the most stable intramolecular hydrogen bond of
compound 9. (G) Distribution of the radius of gyration compounds 8 and 9.
Conclusion
We have presented a quantitative comparison of the rigid and flexible core trivalent sialosides for influenza virus inhibition.
The rigid adamantane core trivalent sialoside with hexamethylene glycol spacer is the most potent candidate against
IAV/X31, showing an inhibitory constant in the micromolar range. On the contrary, no binding was observed with a longer
OEG spacer, which is consistent with the simulation data that suggests increased conformational penalties when the OEG
length is increased from EG6 to EG14. We also observed that trivalent cbzAd(EG6SA)3 4 (Ki = 30 µM) was slightly more
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potent than the divalent cbzAd(EG6SA)2 14 (Ki = 100 µM) in the HAI assay, which could be assigned to a somewhat
higher rebinding probability for the trivalent sialoside. However, both binding constants indicate a bivalent binding between
ligand and receptor. Flexible Tris core-based systems did not show any potent inhibitions in the HAI assay. The observed
micromolar range affinity constant values in MST for the Tris core sialosides might be assigned to an interaction other
than HA with the virus particles. Following, non-specific interaction of Tris-based compounds with cell membranes or
binding to virus structures other than HA needs to be further investigated. Overall, our findings show that optimizing a
significantly active trivalent sialoside with a non-toxic and structurally simple spacer like OEG is a challenging task
because of high conformational entropic penalties and the chance of intramolecular hydrogen bonding with the spacer
groups on the core itself. Nevertheless, we could design an adamantane-based trivalent sialoside which was significantly
more active in the HAI assay compared to monovalent SA or other adamantane derivatives used in this study. Trivalent
cluster units, such as cbzAd(EG6SA)3, could be further used for a multimeric presentation on larger scaffolds to achieve
even better inhibition constants against the influenza A virus.
Experimental Section
Materials and Methods
Synthesis
All reagents and solvents were purchased from commercial suppliers and used without further purification. Analytical
grade PEG600 (~EG14OH), hexa-, tetraethylene glycol, and azido-amino EG7 were purchased from Fischer Chemical,
Sigma Aldrich, Acros Organic, and Polypure respectively. Reactions requiring dry or oxygen-free conditions were carried
out under argon in Schlenk glassware. 1H and 13C NMR spectra were recorded on Bruker AMX 500 (500 and 125 MHz
for 1H and 13C NMR, respectively) and Delta Joel Eclipse 700 (700 and 175 MHz for 1H and 13C NMR, respectively)
spectrometer at 25 oC and calibrated by using the deuterated solvent peak. Infrared (IR) spectra were recorded with a
Nicolet AVATAR 320 FT-IR 5 SXC (Thermo Fisher Scientific, Waltham, MA, USA) with a DTGS detector from 4000 to
650 cm−1. A TSQ 7000 (Finnigan Mat) instrument was used for ESI measurements and a JEOL JMS-SX-102A
spectrometer was used for the high-resolution mass spectra. Naturally occurring sialic acids constitute a family of more
than 50 structurally distinct nine-carbon 3-deoxy-ulosonic acids, the most widespread derivative being 5-N-acetyl-
neuraminic acid (Neu5Ac) was used as starting material for the preparation of prop-2-ynyl α-thiosialoside. We used the
abbreviation of sialic acid (SA) for Neu5Ac. Similarly ‘Tris’ was used as an abbreviation for the commercially available 4-
(((benzyloxy)carbonyl)amino)-4-(2-carboxyethyl)heptanedioic acid based flexible core.
Biological assay
Virus material
X31 virus (influenza strain A/Aichi/2/68 H3N2) was harvested from allantoic fluid of embryonated chicken eggs. Virus
isolates were clarified upon low speed centrifugation (300 x g, 10 min). For binding (inhibition) experiments clarified
allantoic fluid was further concentrated by ultracentrifugation (100,000 x g, 1 h).
Synthesis and characterization
cbzAd(CH2CH2-NHS)3 2
cbzAd(CH2CH2COOH)3 1[17] (510 mg,1 mmol), EDC·HCl (868 mg, 4.5 mmol) and NHS-OH (520 mg, 4.5 mmol) were
dissolved in 20 mL abs. DMF and stirred for 12 h at rt. The solvent was removed in vacuo and the residue was dissolved
in 50 mL EtOAc. The solution was washed with water (3 x 50 mL), dried over Na2SO4, and filtered. The solvent was
removed in vacuo and the residue recrystallized from PrOH to give the title compound 2 (729 mg, 92 %) as a colorless
solid. 1H NMR (600 MHz, CDCl3): δ (ppm) = 7.39 – 7.29 (m, 5H, aryl H), 5.04 (s, 2H, benzyl H), 4.87 (s, 1H, -NH-), 2.82
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(br s, 12H, 9-H), 2.60 (t, 6H, 3J = 7.6 Hz, 6-H), 1.70 (t, 6H, 3J = 7.6 Hz, 5-H), 1.64 (s, 6H, 2-H), 1.22 (d, 3H, 2J = 12.0 Hz,
4-Ha), 1.14 (d, 3H, 2J = 12.0 Hz, 4-He); 13C NMR (150 MHz, CDCl3): δ (ppm) = 169.3, 169.2, 154.8, 136.9, 128.7, 128.2,
128.2, 66.3, 52.7, 44.8, 44.8, 36.8, 35.2, 25.7, 25.3; IR (film): ν = 2974, 2860, 1724, 1460, 1070, 910 cm-1; HRMS (ESI)
= m/z calcd. for C39H44N4O14: 793.2927 [M+H+], found 793.2925.
cbzAd(CH2CH2SA)3 4
O- benzyloxycarbonylaminoethoxy sialic acid 3 (166 mg, 0.33 mmol) was dissolved in 5 ml H2O/MeOH (v/v = 1/4) and
treated with LiOH (26 mg, 1.10 mmol). The solution was stirred at rt for 2 h and neutralized with Amberlite IR-120. After
filtration, the solution was freeze dried and the residue was dissolved in 20 mL MeOH/H2O (v:v = 1:1). The solution was
treated with 10 mg Pd/C under H2-atmosphere (balloon) and stirred for 24 h at rt. After filtration, the solvent was removed
in vacuo and the residue was dissolved in abs. DMSO and treated with NEt3 (50 µL, 0.36 mmol). To this solution, NHS-
ester 2 (75 mg, 0.09 mmol) was added and the mixture was stirred for 48 h at rt. The solvent was removed in vacuo and
the crude product was purified by column chromatography on RP-18 silica (H2O/MeCN 1:0 → 0:1). The title compound 4
(37 mg, 35 %) was obtained as a colorless solid. 1H NMR (600 MHz, D2O): δ (ppm) = 7.47 – 7.37 (m, 5H, aryl H), 5.07 (s,
2H, benzyl H), 3.89 – 3.78 (m, 12H, 18-H, 16-H, 14-H), 3.72 – 3.66 (m, 6H, 13-H, 17-H), 3.64 (td, 3H, 2J = 10.1 Hz, 3J =
5.1 Hz, 9-Ha), 3.58 (br d, 3H, 3J = 9.2 Hz, 15-H), 3.51 (td, 3H, 2J = 10.1 Hz, 3J = 5.1 Hz, 9-Ha), 3.36 – 3.31 (m, 6H, 8-H),
2.73 (dd, 3H, 2J = 12.3 Hz, 3J = 4.8 Hz, 12-He), 2.22 (br s, 6H, 6-H), 2.03 (s, 9H, -NHCOCH3), 1.68 (dd, 3H, 2J = 12.3 Hz,
3J = 12.3 Hz, 12-Ha), 1.54 (s, 6H, 2-H), 1.49 (br s, 6H, 5-H), 1.17 (d, 3H, 2J = 12.0 Hz, 4-Ha), 1.11 (d, 3H, 2J = 12.0 Hz,
4-Hb); 13C NMR (150 MHz, D2O): δ (ppm) = 177.8, 175.1, 173.6, 136.7, 128.8, 128.2, 127.6, 100.5, 72.6, 71.6, 68.2, 68.2,
66.3, 62.64, 62.61, 52.9, 51.9, 44.3, 44.1, 40.2, 39.4, 38.4, 34.6, 30.0, 22.0; IR (film): ν = 2920, 1751, 1670, 1527, 1369,
1226, 1076, 1039, 848 cm-1; HRMS (ESI): m/z calcd. for C66H101N7O32: 1504.6564 [M+H+], found 1504.6535.
Synthesis of the compound cbzAd(propargyl)3 5
cbz-Adamantane triacid 1 (0.7 g, 1.4 mmol) was dissolved in dry DMF (100 mL) and cooled to 0 oC (ice bath). DIPEA (0.9
mL, 5.3 mmol) was added to the reaction mixture and stirred for 5 min followed by addition of EDC.HCl (1.0 g, 5.3 mmol)
and HOBt (0.7 g, 5.3 mmol). The reaction mixture was stirred for 30 min. Then propargyl amine (0.3 mL, 5.3 mmol) was
added and stirred for 72 h at room temperature. DMF was removed and the residue was dissolved in ethyl acetate (250
mL) and washed with 1 M HCl (3 × 100 mL), water (100 mL), and brine (2 × 50 mL). The organic phase was dried over
Na2SO4, and the solvent was removed under reduced pressure. The resulting crude product was purified by flash
chromatography on silica gel (CHCl3/methanol) to afford the propargylated product 5 (453.68 mg, 53 %).1H NMR (400
4
MHz, CD3OD): δ (ppm) = 7.34-7.26 (m, 5H, aryl H), 5.01 (s, 2H, benzyl H), 3.93 (d, 6H, J = 4.0 Hz, -NHCOCH2CCH),
2.57 (t, 3H, 4J = 4.0 Hz, -NHCOCH2CCH), 2.17 (t, 6H, 3J = 4.0 Hz, AdCH2CH2CONH-), 1.58 (s, 6H, AdH), 1.49 (t, 6H, 3J
= 8.0 Hz, AdCH2CH2CONH-), 1.19-1.09 (m, 6H, AdH); 13C NMR (100 MHz, CD3OD): δ (ppm) = 176.2, 153.4, 138.6, 129.4,
128.9, 128.7, 80.7, 72.1, 53.7, 46.3, 45.9, 39.7, 36.1, 30.8, 29.4; IR (film): ν = 3282, 2913, 1645, 1529, 1454, 1420, 1385,
1353, 1307, 1235, 1024 cm−1; HRMS (ESI) = m/z calcd. for C36H44N4O5 : 635.3209 [M+Na+]; found: 635.3200.
Synthesis of the compound cbzAd(EG6N3)3 6 and cbzAd(EG14N3)3 7
The cbz protected adamantane (propargyl)3 5 (50.0 mg, 0.081 mmol) and N3EG6OTs (Scheme S3) (188.0 mg, 0.041
.
mmol) was dissolved in DMF (10mL). CuSO4 5H2O (12.2 mg, 0.048 mmol) was dissolved in 0.2 mL of H2O and added to
the solution of sodium ascorbate (97.2 mg, 0.48 mmol) in 0.4 mL H2O. The resultant solution of salts in water was added
drop wise to the solution containing cbzAd(propargyl)3 5 and N3EG6OTs. The reaction mixture was degassed with argon
for 10 minutes and then allowed to stir at room temperature for 48 h. The completion of reaction was monitored by
checking TLC and ESI. HRMS(ESI) = m/z calcd. for C93H137N13O26S3: 2019.8739 [M+Na+]; found: 2019.8884 to ensure
o
the reaction completion. It was followed by in situ azidation with NaN3 (31.8 mg, 0.489 mmol), at 70 C for 24 h. The
stirring was stopped, and solvent was removed under reduced pressure. Reaction mixture was first filtered through celite
12
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and washed with CHCl3 to remove excess of DMF. The organic solvent was evaporated. The crude product was purified
by flash column chromatography on silica gel (CHCl3/MeOH) to afford the pure product as colorless oil (65.0 mg, 50 %).
cbzAd(EG6N3)3 6
1H NMR (500 MHz, CDCl3): δ (ppm)= 7.69 (s, 3H, -C=CH-), 7.32-7.28 (br m, 4H, aryl H), 6.92 (s, 1H, aryl H ), 4.96 (s, 2H,
benzyl H), 4.47 (t, 6H, J = 10 Hz, -TrzCH2CH2O-), 4.44 (d, 6H, J = 10 Hz, -NHCH2Trz-), 3.81 (t, 6H, J = 10 Hz, -
TrzCH2CH2O-), 3.64 - 3.57 (m, 66 H, -OCH2CH2N3, EG spacer), 3.35 (t, 6H, J = 10 Hz, -OCH2CH2N3), 2.08 (t, 6H, J = 10
Hz, AdCH2CH2CONH-), 1.47-1.44 (br m, 12H, AdH and AdCH2CH2CONH-), 1.06-0.94 (br m, 6H, AdH); 13C NMR (125
MHz, CDCl3): δ (ppm) = 173.4, 154.4, 144.6, 136.7, 128.5, 128.0, 123.4, 70.5, 70.0, 69.4, 65.9, 52.7, 50.6, 50.2, 45.1,
38.0, 34.9, 30.1, 29.6, 22.8; IR (film): ν = 3301, 2906, 2360, 2105, 1715, 1654, 1540, 1455, 1349, 1302, 1242, 1117, 940,
736 cm−1; HRMS (ESI) = m/z calcd. for C72H116N22O20: 1631.8634 [M+Na+]; found: 1631.8717.
cbzAd(EG14N3)3 7
Synthesis was carried out by the above-reported procedure. The crude product was purified by dialysis with MWCO 2kDa
in CHCl3 to afford the pure product as colorless oil (125.77 mg, 58.5 %), 1H NMR (500 MHz, CD3OD-CD2Cl2): δ (ppm) =
7.75 (s, 3H, -C=CH-), 7.26 (br s, 4H, aryl H ), 7.13 (s, 1H, aryl H ), 4.94 (s, 2H, benzyl H), 4.90-4.47 (br m, 12H, -
TrzCH2CH2O- and -NHCH2Trz-), 4.35 (s, 6H, -TrzCH2CH2O-), 3.81 (s, 6H, -OCH2CH2N3), 3.58-3.33 (m, 156 H, -
OCH2CH2N3, EG spacer), 1.49-1.43 (m, 12H, AdH and AdCH2CH2CONH-), 1.10-1.01 (br m, 6H, AdH); 13C NMR (125
MHz, CDCl3): δ (ppm) = 173.5, 154.5, 144.7, 136.6, 128.6, 128.1, 123.5, 70.6, 70.1, 69.4, 66.0, 52.8, 50.7, 50.3, 45.3,
38.1, 35.0, 34.1, 29.7, 22.9; IR (film): ν = 3318, 2910, 2868, 2103, 1769, 1717, 1651, 1534, 1455, 1349, 1283, 1240,
1113, 1035, 992, 932, 841, 777 cm−1; HRMS (ESI) = m/z calcd. for C120H212N22O44: 2705.4698 [M+K+]; found: 2705.4340.
Synthesis of the compound cbzAd(EG6SA)3 8 and cbzAd(EG14SA)3 9
cbzAd(EG6SA)3 8
cbzAd(EG6N3)3 6 (0.05 g, 0.03 mmol) and prop-2-ynyl α-thiosialoside (0.06 g, 0.11 mmol) was dissolved in DMF (5 mL).
.
The CuSO4 5H2O (0.77 mg, 0.003 mmol) was dissolved in 0.2 mL of H2O and added to the solution of sodium ascorbate
(6.2 mg, 0.03 mmol) in 0.4 mL H2O. The resultant solution of salts in water was added drop wise to the solution containing
cbzAd(EG6N3)3 6 and prop-2-ynyl α-thiosialoside. The reaction mixture was degassed with argon for 10 minutes and then
allowed to stir at room temperature for 48 h. The completion of reaction was monitored by IR for the disappearance of the
N3 frequency at 2100 cm-1. After the reaction completion, DMF was evaporated over a rota-vapor. Then 2M NaOH (5 mL)
solution was added to the left-over residue and stirred for 2 h at room temperature. The solution was dialyzed against
water for 3 days using dialysis membrane with MWCO 2 kDa to afford the pure product as white solid (70.56 mg, 84 %).
1H NMR (700 MHz, D2O): δ (ppm) = 7.87 (s, 3H, -C=CH-), 7.26 (br s, 5H, aryl H ), 4.91 (s, 2H, benzyl H), 4.49 (br s, 12H,
-TrzCH2CH2O- and -OCH2CH2Trz-), 4.34 (br s, 6H, -NHCH2Trz-), 3.95 - 3.48 [m, 87H, -TrzCH2CH2O-, -OCH2CH2Trz-,
SA (8-H, NH, 7-H, 4-H, 9-H, 5-H, 6-H), SCH2C=C, EG spacer], 2.72 (d, 3H, J = 7 Hz, SA H-3e), 2.10 (br s, 6H,
AdCH2CH2CONH-), 1.94 (s, 3H, SA NHAc), 1.69 (s, 3H, SA H-3a), 1.35 (br s, 12H, AdH and AdCH2CH2CONH-), 0.94 (br
s, 6H, AdH); 13C NMR (175 MHz, D2O): δ (ppm) = 177.0 175.1, 156.0, 145.0, 136.6, 128.7, 128.2, 127.6, 124.8, 124.5,
74.8, 71.7, 69.5, 68.7, 68.7, 68.5, 68.1, 65.9, 62.5, 52.6, 51.7, 50.0, 44.3, 44.2, 40.6, 38.0, 34.6, 34.4, 29.9, 23.5, 22.0;
IR (film): ν = 3268, 2915, 1644, 1608, 1550, 1372, 1133, 1028, 951, 834 cm−1; HRMS (ESI) = m/z calcd. for
C114H179N25O44S3: 2721.1553 [M+Na+-H+], found: 2721.1324; calcd 2737.1292 [M+K+-H+], found: 2731.1120.
cbzAd(EG14SA)3 9
Synthesis was carried out by the above-reported procedure. The crude product was purified by dialysis with MWCO 2kDa
in CHCl3 to afford the pure product as colorless oil (69.83 mg, 62 %). 1H NMR (700 MHz, D2O): δ (ppm) = 7.97 (s, 3H, -
C=CH-), 7.36 (br s, 5H, aryl H ), 5.02 (s, 2H, benzyl H), 4.57 (br s, 12H, -TrzCH2CH2O- and -OCH2CH2Trz-), 4.41 (br s,
6H, -NHCH2Trz-), 4.04 - 3.52 [m, 180H, -TrzCH2CH2O-, -OCH2CH2Trz-, SA (8-H, 7-H, 4-H, 9-H, 5-H, 6-H), SCH2C=C, EG
spacer], 2.79 (d, 3H, J = 7 Hz, SA H-3e), 2.18 - 2.16 (br m, 6H, AdCH2CH2CONH-), 2.01 (s, 3H, SA NHAc), 1.77 (s, 3H,
13
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Chemistry - A European Journal
SA H-3a), 1.44-142 (m, 12H, AdH and AdCH2CH2CONH-), 1.05-0.99 (br m, 6H, AdH); 13C NMR (175 MHz, D2O): δ (ppm)
= 177.0, 175.1, 156.0, 145.0, 136.6, 128.7, 128.2, 127.6, 124.8, 124.5, 74.8, 71.7, 69.5, 68.7, 68.7, 68.5, 68.1, 65.9, 62.5,
52.6, 51.7, 50.0, 44.3, 44.2, 40.6, 38.0, 34.6, 34.4, 29.9, 23.5, 22.0; m/z calcd. for C162H272N25NaO68S3: 3777.79 [M+Na+];
GPC (H2O): Mn = 4071 Da, Mw = 4756 kDa, PDI = 1.16; Elemental analysis: calcd. 51.79% C, 7.38% H, 9.32% N, 2.56%
S, found. 51.62% C, 7.40% H, 9.29% N, 2.60% S. IR (film): ν = 3274, 2872, 1607, 1549, 1454, 1350, 1245, 1080, 950,
892, 834 cm−1.
Synthesis of the compound cbzAd(COOH)2 11
Bis(2-carboxyethyl) aminoadamantane 10 (2 g, 6.78 mmol) was dissolved in dioxane (30 mL) followed by addition of
aqueous NaHCO3 (850 mg, 10.12 mmol, 30 mL) solution. NaOH (2M) was added to the reaction mixture to achieve the
pH of 9 and the reaction mixture was cooled to 0 oC (ice bath). Benzyl chloroformate (1.73 mL, 12.10 mmol) was added
dropwise to the stirred solution. Stirring was continued at room temperature overnight. The reaction mixture was washed
three times with CH2Cl2 (30 mL) and three times with EtOAc (30 mL), acidified to pH 1, and extracted six times with EtOAc.
The combined organic layers were dried over Na2SO4, filtered, and concentrated to give the intermediate cbz-protected
diacid (1.78 g, 62 %). 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.37 - 7.31 (m, 5H, aryl H), 5.04 (s, 2H, benzyl H), 2.30 (t,
4H, 3J = 10 Hz, 9’-H), 2.20 (br t, 1H, 6’-H), 1.80 (br. s, 2H, 7’-H), 1.65 - 1.58 (m, 4H, 2’-H), 1.53 (t, 4H, 3J = 10 Hz, 8’-H),
1.39 - 1.32 (m, 4H, 5’-H), 1.15 (br s, 2H, 4’-H); 13C NMR (125 MHz, CDCl3): δ (ppm) = 179.7, 128.6, 128.2, 45.6, 40.4,
37.5, 34.7, 29.6, 28.1; IR (film): ν = 3032, 2915, 2850, 1704, 1587, 1497, 1454, 1392, 1341, 1302, 1261, 1025, 941, 909,
823, 788 cm−1; HRMS (ESI) = m/z calcd. for C24H31NO6 : 452.2049 [M+Na+]; found: 452.2083.
Synthesis of the compound cbzAd(propargyl)2 12
cbz-protected diacid 11 (350 mg, 0.82 mmol) was dissolved in 15 mL dry DMF and cooled to 0 oC (ice bath). Then DCC
(336 mg, 1.63 mmol) and N-hydroxysuccinimide (196 mg, 1.70 mmol) were added, and the reaction mixture was stirred
at room temperature for 12 h. The completion of reaction was monitored by mass. Then propargyl amine (0.14 mL, 2.18
mmol) was added and stirred for 72 h at rt. The solution was diluted with ethyl acetate (70 mL) and washed with 1M HCl
(3 × 20 mL), water (40 mL), and brine (2 × 20 mL). The organic phase was dried over Na2SO4, and the solvent was
removed under reduced pressure. The crude product was purified by column chromatography with a DCM/methanol
mixture. (233.18 mg, 57 %). 1H NMR (700 MHz, CD3OD): δ (ppm) = 7.33 - 7.26 (m, 5H, aryl H), 4.99 (s, 2H, benzyl H),
3.91 (d, 4H, 4J = 7 Hz, 11’-H), 2.34 (t, 2H, 4J = 7 Hz, 12’-H), 2.16 - 2.12 (m, 5H, 9’-H, 6’-H ), 1.79 (br s, 2H, 7’-H), 1.62 -
1.55 (m, 4H, 2’-H), 1.48 - 1.44 (m, 4H, 8’-H), 1.39 - 1.29 (m, 4H, 5’-H); 1.14 (br s, 2H, 4’-H); 13C NMR (175 MHz, CD3OD):
δ (ppm) = 175.5, 128.9, 128.5, 128.2, 80.0, 71.5, 52.6, 46.1, 45.8, 40.9, 39.2, 35.2, 30.3, 29.2; IR (film): ν = 3288, 3060,
2910, 2847, 2358, 1701, 1646, 1536, 1454, 1420, 1343, 1301, 1235, 1138, 1113, 1045, 1031, 928, 736, 697 cm−1; HRMS
(ESI) = m/z calcd. for C30H37N3O4 : 527.2682 [M+1+Na+]; found: 527.2755.
Synthesis of the compound SAEG6N3 13
TsEG6N3 was synthesized from EG6 diol. The TsEG6N3 (500 mg, 1.08 mmol) and prop-2-ynyl α-thiosialoside (914 mg,
.
1.68 mmol) were dissolved in DMF. CuSO4 5H2O (0.06 mg, 0.24 mmol) was dissolved in 0.2 mL of H2O and added to the
solution of sodium ascorbate (511 mg, 2.57 mmol) in 0.4 mL H2O. The resultant solution of salts in water was added drop
wise to the solution containing TsEG6N3 and prop-2-ynyl α-thiosialoside. The reaction mixture was degassed with argon
for 10 minutes and then allowed to stir at 40 oC for 48 h. The completion of reaction was monitored by IR spectroscopy
for the disappearance of azide peak. It was followed by in situ azidation with NaN3 (716 mg, 11.02 mmol), at 70 oC for 24
h. The stirring was stopped, and solvent was removed under reduced pressure. The crude compound was purified by
column chromatography to obtain the pure product SAEG6N3 13. (691.40 mg, 73 %). 1H NMR (500 MHz, CD2Cl2): δ (ppm)
= 7.66 (s, 1H, -C=CH-), 5.59 (m, 1H, 8-H), 5.43 - 5.39 (m, 1H, 7-H), 4.91 - 4.86 (m, 1H, 4-H), 4.49 (t, J = 10 Hz, 2H, -
TrzCH2CH2O-), 4.27 (dd, 1H, 2J = 10 Hz, 3J = 5 Hz, 9-Ha), 4.09 - 3.98 (m, 4H, 5-H, 9-He, -SCH2C=CH-), 3.92 - 3.88 (m,
2H, 6-H, 5-H), 3.85 (t, 2H, -TrzCH2CH2O-), 3.71 ( s, 3H, -OCH3), 3.65 – 3.57 (m, 18H, EG spacer), 3.36 (t, 2H, 3J = 5 Hz,
14
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Chemistry - A European Journal
-CH2CH2N3-), 2.74 (dd, 1H, 2J = 10 Hz, 3J = 5 Hz, 3-He), 2.14 (s, 3H, -OCOCH3), 2.11 (s, 3H, -OCOCH3), 1.99 (s, 3H, -
OCOCH3), 1.98 (s, 3H, -OCOCH3), 1.83 (s, 3H, -NHOCH3); 13C NMR (500 MHz, CD2Cl2): δ (ppm)= 171.0, 170.9, 170.5,
170.3, 168.7, 143.5, 123.9, 83.5, 74.5, 71.0, 71.0, 71.0, 70.9, 70.8, 70.3, 69.9, 68.6, 67.9, 62.7, 53.5, 51.2, 50.7, 49.5,
38.1, 23.8, 23.4, 21.5, 21.1, 21.1, 21.0; IR (film): ν = 3274, 2869, 2103, 1736, 1682, 1662, 1543, 1437, 1368, 1215, 1121,
1032, 946 cm−1; HRMS (ESI) = m/z calcd. for C35H55N7O17S : 900.3273 [M+Na+]; found: 900.3270.
Synthesis of the compound cbzAd(EG6SA)2 14
.
The SAEG6N3 (174 mg, 0.20 mmol) and cbzAd(propargyl)2 12 (50 mg, 0.099 mmol) were dissolved in DMF, CuSO4 5H2O
(9.9 mg, 0.03 mmol) was dissolved in 0.2 mL of H2O and added to the solution of sodium ascorbate (78 mg, 0.39 mmol)
in 0.4 mL H2O. The resultant solution of salts in water was added drop wise to the solution containing SAEG6N3 and
cbzAd(propargyl)2. The reaction mixture was degassed with argon for 10 minutes and then allowed to stir at 40 oC for 48
h. The completion of reaction was monitored by IR spectroscopy for the disappearance of azide peak. The stirring was
stopped, and solvent was removed under reduced pressure. 2M NaOH (20 mL) was added to the residue and stirred at
room temperature for 12 h. The reaction mixture was dialyzed first against water and aqueous EDTA solution for 2 days
and again using only water for 4 days. The aqueous solution obtained after dialysis was lyophilized to get the pure product
cbzAd(EG6SA)2 14 (146.24 mg, 78 %). 1H NMR (700 MHz, D2O): δ (ppm) = 8.30 (br s, 4H, -C=CH-), 7.41 - 7.38 (m, 5H,
aryl H), 5.04 (s, 2H, benzyl H), 4.65 - 4.62 (m, 4H, -TrzCH2CH2O- and -OCH2CH2Trz-), 4.47 (br s, 4H, -NHCH2Trz-), 3.98
- 3.49 [m, 56H, -TrzCH2CH2O-, -OCH2CH2Trz-, SA (8-H, 7-H, 4-H, 9-H, 5-H, 6-H), SCH2C=C, EG spacer], 2.89 (br s, 2H,
SA H-3e), 2.28 (br s, 4H, AdCH2CH2CONH-), 2.13 (br s, 1H, 6’-H), 2.04 (s, 6H, SA NHAc), 1.73 (br s, 2H, Ad 7’-H), 1.54
- 1.48 (m, 8H, Ad 2’-H, AdCH2CH2CONH-), 1.28 (br d, 4H, Ad 5’-H), 1.11 (br s, 2H, Ad 4’-H); 13C NMR (175 MHz, D2O):
δ (ppm) = 177.0, 174.9, 156.0, 136.9, 128.7, 128.2, 127.6, 74.8, 71.8, 69.5, 68.5, 68.1, 62.4, 51.6, 50.4, 44.7, 40.6, 39.5,
38.3, 34.3, 29.2, 22.0; IR (film): ν = 3279, 2905, 2360, 1698, 1614, 1551, 1428, 1349, 1235, 1109, 1023, 948, 890, 834
cm−1; HRMS (ESI) = m/z calcd. for C82H127N17O30S2: 1915.8274 [M+Na+-1]; found: 1915.8152.
Synthesis of the compound cbzTris(EG7N3)3 17
o
cbz-protected triacid 16 (231 mg, 0.61 mmol) was dissolved in 15 mL of dry DMF and cooled to 0 C (ice bath). Then
triethylamine (0.35 mL, 2.51 mmol), EDC.HCl (493 mg, 2.57 mmol) and DMAP (169 mg, 1.38 mmol) were added, and the
reaction mixture was stirred at 0 oC for 20 minutes. Then N3EG7NH2 (902 mg, 2.58 mmol) was added and stirred at room
temperature for 72 h. The solution was diluted with ethyl acetate (70mL) and washed with 1 M HCl (3 × 20 mL), water (40
mL), and brine (2 × 20 mL). The organic phase was dried over Na2SO4, and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography with a DCM/methanol mixture to afford the pure
cbzTris(EG7N3)3 17 (49.55 mg, 59 %). 1H NMR (500 MHz, DMSO-d6): δ (ppm) = 7.87 (t, 3J = 5 Hz, 3H,- NHCH2CH2-),
7.34 - 7.29 (m, 5H, aryl H ), 4.97 (s, 2H, benzyl H), 3.60 (t, 3J = 5 Hz, 6H, -NHCH2CH2-), 3.54 - 3.50 (m, 60H, EG spacer),
3.39 (t, 3J = 5 Hz, 12H, -NHCH2CH2, -CH2CH2N3-), 3.18 - 3.16 (m, 6H, -CH2CH2N3-), 2.01 (t, 3J = 10 Hz, 6H, -
CH2CH2CONH-); 1.76 (br t, 6H, -CH2CH2CONH-); 13C NMR (125 MHz, DMSO-d6) : δ (ppm) = 172.1, 154.1, 137.3, 128.3,
127.4, 69.7-69.1, 64.5, 56.1, 50.0, 30.3, 29.5; IR (film): ν = 3301, 2921, 2868, 2106, 1717, 1650, 1533, 1455, 1348, 935,
746 cm−1; HRMS (ESI): m/z: calcd. for C60H107N13O23: 1400.7500 [M+Na+]; found 1400.7518.
Synthesis of the compound cbzTris(EG7SA)3 18
The prop-2-ynyl α-thiosialoside (154 mg, 0.42 mmol) and cbzTris(EG7N3)3 (100 mg, 0.07 mmol) were dissolved in DMF.
.
CuSO4 5H2O (21 mg, 0.08 mmol) was dissolved in 0.2 mL of H2O and added to the solution of sodium ascorbate (217
mg, 1.10 mmol) in 0.4 mL H2O. The resultant solution of salts in water was added drop wise to the solution containing
prop-2-ynyl α-thiosialoside and cbzTris(EG7N3). The reaction mixture was degassed with argon for 10 minutes and then
allowed to stir at 40 oC for 48 h. The completion of reaction was monitored by IR spectroscopy for the disappearance of
azide peak. The stirring was stopped, and solvent was removed under reduced pressure 2M NaOH (20 mL) was added
to the residue and stirred at room temperature for 12 h. The reaction mixture was dialyzed against water and aqueous
15
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Chemistry - A European Journal
EDTA solution. The aqueous solution obtained after dialysis was lyophilized to get the pure product cbzTris(EG7SA)3 18.
(125.85 mg, 73 %). 1H NMR (500 MHz, D2O) : δ (ppm) = 7.96 (s, 3H, -C=CH-), 7.42 - 7.36 (m, 5H, aryl H ), 5.03 (s, 2H,
benzyl H), 4.57 (br t, 6 H, -NHCH2CH2-), 4.02 - 3.49 (m, 99H, SA (8-H, NH, 7-H, 4-H, 9-H, 5-H, 6-H,) -SCH2C=C-, EG
spacer), 3.33 (br t, 6H, -CH2CH2N3-), 2.78 (d, 3J = 10 Hz, 3H, SA 3-He), 2.19 (br t, 6H, - CH2CH2CONH-), 2.00 (s, 9H, SA
3
-NHCOCH3), 1.88 (m, 6H, -CH2CH2CONH-), 1.75 (t, 3H, J = 10 Hz, SA 3-Ha); 13C NMR (125 MHz, D2O) : δ (ppm) =
175.9, 128.9, 124.9, 74.9, 71.9, 69.7-69.6, 68.9, 68.8,68.7, 68.2, 62.67, 51.8, 50.2, 40.8, 39.2, 38.0 30.0, 23.9, 23.8, 22.1;
IR (film): ν = 3350, 2923, 1597, 1446, 1371, 1349, 1113, 1078, 986, 935, 903, 857 cm−1; HRMS (ESI): m/z: calcd. for
C120H170N16O47S3: 2550.9982 [M+2Na++K+-1]; found 2550.9737.
Synthesis of the compound cbzTris(propargyl)3 19
cbz-protected triacid (950 mg, 2.49 mmol) was dissolved in 15 mL of dry DMF and cooled to 0 oC (ice bath). Then
triethylamine (1.56 mL, 11.18 mmol), EDC.HCl (2.14 g, 11.16 mmol) and DMAP (638 mg, 5.22 mmol) were added, and
o
the reaction mixture was stirred at 0 C for 20 minutes. Then propargyl amine (0.717 mL,11.19 mmol) was added and
stirred for 72 h at room temperature. The solution was diluted with ethyl acetate (70 mL) and washed with 1 M HCl (3 ×
20 mL), water (40 mL), and brine (2 × 20 mL). The organic phase was dried over Na2SO4, and the solvent was removed
under reduced pressure. The crude product was purified by column chromatography with a DCM/methanol mixture. (732
mg, 60 %). 1H NMR (500 MHz, DMSO-d6) : δ (ppm) = 8.24 (t, 3J = 10 Hz, 3H, -NHCH2-), 7.36 - 7.29 (m, 5H, aryl H), 4.98
5
(s, 2H, benzyl H), 3.83 - 3.82 (m, 6H, -CH2CCH-), 3.08 (t, J = 5 Hz, 3H, -CH2CCH-), 2.03 (br t, 6H, -CH2CH2CONH-),
1.77 (br t, 6H, -CH2CH2CONH-); 13C NMR (125 MHz, CDCl3) : δ (ppm) = 173.4, 155.2, 137.3, 128.9, 128.4, 80.0, 71.9,
66.6, 57.5, 31.2, 30.7, 29.5; IR (film): ν = 3289, 3057, 2939, 1704, 1644, 1525, 1242, 1057, 1027, 926, 732 cm−1; HRMS
(ESI): m/z: calcd. for C27H32N4O5: 515.5658 [M+Na+]; 531.5397 [M+K+]; found: 512.2291, 531.2023 respectively.
Synthesis of the compound cbzTris(EG1N3)3 20
The coupling of cbzTris(propargyl)3 (240 mg, 0.56 mmol) and azido ethyl tosylate (459 mg, 1.90 mmol) using copper
assisted click reaction followed by azidation was performed like 22. (342.28 mg, 75 %). 1H NMR (500 MHz, DMSO-d6) :
δ (ppm) = 7.83 (s, 3H, -C=CH-), 7.26 - 7.18 (m, 5H, aryl H), 4.92 (s, 2H, benzyl H), 4.46 (t, 3J = 5 Hz, 6H, -CH2CH2N3);
4.34 (s, 6H, -CONHCH2-), 3.71 (t, 3J = 5 Hz, 6H, -CH2CH2N3); 2.14 (t, 6H, 3J = 5 Hz, -CH2CH2CONH-), 1.87 (t, 3J = 5 Hz,
6H, -CH2CH2CONH-); 13C NMR (125 MHz, CD3OD) : δ (ppm) = 175.6, 156.6, 146.5, 129.4, 128.7, 124.8 66.9, 57.9 51.7,
50.5, 35.6, 31.5, 31.0; IR (film): ν = 3287, 2925, 2107, 1712, 1678, 1529, 1455, 1240, 1061 cm−1; HRMS (ESI): m/z: calcd.
for C33H44N22O5: 851.3763 [M+Na+]; found: 851.3788.
Synthesis of the compound cbzTris(EG1SA)3 21
The coupling of cbzTris(EG1N3)3 20 (32 mg, 0.04 mmol) and prop-2-ynyl α-thiosialoside (81.9 mg, 0.015 mmol) using
1
copper-assisted click reaction and further deprotection was performed similarly to 18. (49.28 mg, 69 %). H NMR (500
MHz, D2O) : δ (ppm) = 7.81 (br s, 3H, -C=CH-), 7.58 (br s, 3H, -C=CH-), 7.26-7.22 (m, 5H, aryl H), 4.89 (s, 2H, benzyl H
), 4.22 - 3.43 [m, 42H SA (8-H, 7-H, 4-H, 9-H, 5-H, 6-H,) SCH2C=C, -CONHCH2-, ethylene spacer], 2.68 (br s, 3H, SA 3-
He), 2.13 (br s, 6H, -CH2CH2CONH-), 1.91 (s, 9H, SA -NHCOCH3), 1.80 (br s, 7H, -CH2CH2CONH-, SA 3-Ha); 13C NMR
(125 MHz, D2O) : δ (ppm) = 175.7, 174.9, 136.7, 130.2, 129.4, 128.6, 128.1, 127.6, 74.8, 71.3, 68.0, 62.7, 51.6, 49.8,
40.2, 34.5, 29.7, 22.0; IR (film): ν = 3317, 2971, 1647, 1557, 1374, 1364, 1032, 1013 cm−1; HRMS (ESI): m/z: calcd. for
C75H107N25O29S3: 1960.6613 [M+2Na+-3]; found: 1960.6398.
Synthesis of the compound cbzTris(EG4N3)3 22
The TsOEG4N3 was synthesized by reported procedure of Xuyi Yue et al.[12a] The cbzTris(propargyl)3 19 (200 mg, 0.47
.
mmol) and TsOEG4N3 (590 mg, 1.58 mmol) were dissolved in DMF. CuSO4 5H2O (21 mg, 0.08 mmol) was dissolved in
0.2 mL of H2O and added to the solution of sodium ascorbate (217 mg, 1.10 mmol) in 0.4 mL H2O. The resultant solution
of salts in water was added drop wise to the solution containing TsOEG4N3 and cbzTris(popargyl)3. The reaction mixture
was degassed with argon for 10 minutes and then allowed to stir at 40 oC for 48 h. The completion of reaction was
monitored by TLC. It was followed by in situ azidation with NaN3 (308 mg, 4.74 mmol), at 70 oC for 24 h. The stirring was
stopped, and solvent was removed under reduced pressure. The crude product was purified by column chromatography
16
This article is protected by copyright. All rights reserved.

10.1002/chem.201804826
Chemistry - A European Journal
with a DCM/methanol mixture. (414.2 mg, 72 %). 1H NMR (700 MHz, MeOH-d4) δ: (ppm) = 7.90 (s, 3H, -C=CH- ), 7.34 -
7.26 (m, 5 H, aryl H), 5.00 (s, 2H, benzyl H), 4.54 (t, 3J = 7 Hz, 6H, -TrzCH2CH2O-); 4.41 (s, 6H, -CONHCH2-), 3.87 (t, 3J
= 7 Hz, 6H, -TrzCH2CH2O-), 3.64 - 3.59 (m, 24H, EG spacer), 3.35 (t, 6H, 3J = 7 Hz, -CH2CH2N3), 2.22 (br t, 6H, -
CH2CH2CONH-), 1.95 (br t, 6H, -CH2CH2CONH-); 13C NMR (125 MHz, CD3OD-d6) : δ (ppm) = 175.4, 156.5, 145.9, 138.4,
129.4 128.9, 128.7, 124.9, 171.5-71.0, 70.3, 66.8, 57.8, 51.7, 51.3, 35.7, 31.6, 31.0; IR (film): ν = 3301, 2921, 2868, 2106,
1717, 1650, 1533, 1455, 1244, 1098, 1058, 935 cm−1; HRMS (ESI): m/z: calcd. for C51H80N22O14: 1247.6122 [M+Na+];
found: 1247.6217.
Synthesis of the compound cbzTris(EG4SA)3 23
The coupling of cbzTris(EG4N3)3 22 (108 mg, 0.088 mmol) and prop-2-ynyl α-thiosialoside (187 mg, 0.34 mmol) using
copper assisted click reaction and further deprotection was performed similarly to 18. (154 mg, 77 %). 1H NMR (500 MHz,
D2O) : δ (ppm) = 7.90 (s, 3H, -C=CH-), 7.86 (s, 3H, -C=CH-), 7.32 - 7.29 (m, 5H, aryl H), 4.96 (s, 2H, benzyl H), 4.54 (t,
12H, -TrzCH2CH2O-, -OCH2CH2Trz-), 4.36 (br s, 6H, -NHCH2Trz-), 4.01 - 3.48 [m, 63H, SA (8-H, NH, 7-H, 4-H, 9-H, 5-H,
6-H,) SCH2C=C, EG spacer], 2.75 (dd, 3J = 14 Hz , 2J = 7 Hz , 3H, SA 3-He), 2.19 (br t, 6H, -CH2CH2CONH-), 2.01 (s,
9H, SA, -NHCOCH3), 1.89 (br s, 6H, -CH2CH2CONH-), 1.79 (t, 3J = 14 Hz, 3H, SA 3-Ha); 13C NMR (125 MHz, D2O) : δ
(ppm) = 175.67, 175.17, 172.72, 144.59, 128.86, 128.38, 127.62, 124.9, 124.5, 84.3, 75.0, 71.4, 69.8, 69.6, 68.8, 68.7,
68.2, 62.9, 56.9, 51.8, 40.3, 34.6, 29.9, 23.3, 22.2; IR (film): ν = 3280, 2927, 1705, 1645, 1548, 1114, 1059, 1027, 954
cm−1; HRMS (ESI): m/z: calculated for C93H143N25O38S3: 2335.9080 [M+Na+-1]; found: 2335.8764.
Hemagglutinnation inhibition assay
Inhibitors were twofold serially diluted in PBS. Then, 4 HAU X31 virus containing approximately 4∙107 virus particles were
added to all the wells. The amount of virus particles per volume was estimated as published by Desselberger et al.[20]
After 30 min incubation under slight agitation at rt, 50 µl of a 1 % human erythrocyte solution (German Red Cross, ~2∙106
cells/µl) was added, gently mixed, and incubated for 60 min at rt. The inhibitor constant KiHAI, reflects the lowest inhibitor
concentration, which is necessary to achieve full inhibition of virus induced hemagglutination. To check for full
hemagglutination inhibition, the microtiter plate was tilted by 60° to cause droplet formation from the red blood cell pellet.[21]
Microscale thermophoresis (MST)
Measurements have been performed using a Monolith NT.115 instrument (Nanotemper) and standard glass capillaries.
For safety reasons, X31 was inactivated with UV light for 5 min on ice. Then the envelope of viruses (1 mg/ml protein)
was labeled at 20 µM R18 for 30 min at rt under gentle shaking. Unbound R18 was removed under centrifugation at
20,000 x g for 5 min. The resuspended virus was filtered through a 0.45 µm filter (Millipore) to remove virus aggregates.
To quantify the amount of virus and to check whether the prepared virus was still able to bind, a hemagglutination assay
was performed. Each inhibitor was twofold serially diluted and mixed with equal amounts of X31 (4 HAU). After a short
incubation, then the inhibitor-virus mix was loaded on glass capillaries and measured at rt for initial fluorescence (5 s) and
change in fluorescence over time (30 s) with the thermophoresis measurement (MST power: 80%, green LED power
100%). The T-jump with thermophoresis was used for analysis. Data were analyzed by with Graph pad Prism 5 using a
one-sided fit with a cooperativity factor of 1. In order to determine the noise level, triplicates of the same virus dilution
were measured leading to ΔF = 5 FU. For safety reasons X31 was inactivated with UV light for 5 min on ice. The change
in fluorescence was expressed as ΔFnorm (‰) and represented values after subtraction of background and correlation
to the initial fluorescence before IR laser activation.
Molecular Dynamics Simulation Set-up
17
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10.1002/chem.201804826
Chemistry - A European Journal
All-atom molecular dynamics (MD) simulations were performed for the monovalent counterparts of compounds 8 and 9
(later referred as ligands 8 and 9, respectively) in explicit water (TIP3P water model),[22] using the GROMACS 5.0.2
simulation package.[23] The initial structures of the ligands were drawn in Marwin Sketch (MarvinSketch (version 6.2.2,
calculation
module
developed
by
ChemAxon),
http://www.chemaxon.com/products/marvin/marvinsketch/, 2014). Both ligands were parametrized in Acpype.[24] The
topologies of the ligands 8 and 9 were generated according to the General Amber Force Field (GAFF).[25] The semi-
emipirical quantum chemistry program SQM[26] was used to assign the partial charges with the AM1-BC level of theory.
The systems were minimized in vacuum with the steepest decent algorithm[27] (emtol = 1000.0 (kJ/mol)/nm, nsteps =
50,000). 4069 and 14441 water molecules were added to solvate the ligands 8 and 9, respectively, in a dodecahedron
box (volume of the ligand 8 box 130.51 nm3, volume of the ligand 9 box 443.76 nm3). Then solvated ligands were brought
to another round of minimization with the same parameters, followed by two equilibration runs, first in the NVT ensemble
at 300 K (V-rescale thermostat,[28] time constant = 0.1 ps), and then in NPT ensemble (Parrinello-Rahman barostat,[29]
reference pressure = 1 bar, time constant = 2 ps) for 100 ps respectively. Five starting structures per ligand were randomly
taken from the NPT equilibration run and later used in the subsequent MD runs. The production runs were simulated in
the NPT ensemble (temperature 300 K, pressure 1 bar). The covalent bonds to all hydrogen atoms were constrained with
the LINCS algorithm[30] (lincs iter = 1, lincs order = 4), allowing for an integration time step of 2 fs. Newton’s equations of
motion were integrated with a leap-frog scheme. The cut-off for Lennard-Jones interactions was set to 1 nm. The
electrostatic interactions were treated with the particle-mesh ewald (PME) algorithm[31] with a real space cut off 1 nm, a
Fourier grid spacing of 0.16 nm, and an interpolation order of 4. Periodic boundary conditions were applied in all three
dimensions. The solute coordinates were written to the trajectory file every 1 ps. In total, 1 µs of the simulations were
obtained for each ligand.
Molecular Dynamics Data Analysis
The respective distances used to characterize ligands 8 and 9 were obtained with the GROMACS command g_mindist.
Then, they were visualized with an in-house MATLAB (Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 10089
(https://doi.org/10.1063/1.464397) script. The intramolecular hydrogen bond networks were computed with GROMACS
command g_hond, while the radius of gyration was extracted from the respective trajectories with GROMACS command
g_gyrate.
Supporting Information
Details on the synthesis of heterobifunctional oligoethylene spacers, sialic acid derivative, and control molecule 24. Model
for trivalent and divalent sialoside binding.
Acknowledgement
This contribution was generously supported by the Deutsche Forschungsgemeinschaft DFG via grants from SFB 765 and
Marie Curie Funding. We thank Professor Dr. Joachim Heberle for giving us the opportunity to perform MST
measurements in his lab. We acknowledge Dr. Pamela Winchester for careful language polishing of the manuscript. The
computer facilities of the Freie Universität Berlin (ZEDAT) are acknowledged for the computer time.
[1]
[2]
a) E. Vanderlinden, L. Naesens, Medicinal Research Reviews 2014, 34, 301-339; b) C. A. Russell, T. C. Jones,
I. G. Barr, N. J. Cox, R. J. Garten, V. Gregory, I. D. Gust, A. W. Hampson, A. J. Hay, A. C. Hurt, J. C. de Jong,
A. Kelso, A. I. Klimov, T. Kageyama, N. Komadina, A. S. Lapedes, Y. P. Lin, A. Mosterin, M. Obuchi, T. Odagiri,
A. D. M. E. Osterhaus, G. F. Rimmelzwaan, M. W. Shaw, E. Skepner, K. Stohr, M. Tashiro, R. A. M. Fouchier,
D. J. Smith, Science 2008, 320, 340-346.
a) J. J. Skehel, D. C. Wiley, Annual Review of Biochemistry 2000, 69, 531-569; b) M. Mammen, S.-K. Choi, G.
M. Whitesides, Angewandte Chemie International Edition 1998, 37, 2754-2794; c) A. Bernardi, J. Jiménez-
18
This article is protected by copyright. All rights reserved.

10.1002/chem.201804826
Chemistry - A European Journal
Barbero, A. Casnati, C. De Castro, T. Darbre, F. Fieschi, J. Finne, H. Funken, K.-E. Jaeger, M. Lahmann, T. K.
Lindhorst, M. Marradi, P. Messner, A. Molinaro, P. V. Murphy, C. Nativi, S. Oscarson, S. Penadés, F. Peri, R.
J. Pieters, O. Renaudet, J.-L. Reymond, B. Richichi, J. Rojo, F. Sansone, C. Schäffer, W. B. Turnbull, T.
Velasco-Torrijos, S. Vidal, S. Vincent, T. Wennekes, H. Zuilhof, A. Imberty, Chemical Society reviews 2013, 42,
4709-4727; d) W. Weis, J. H. Brown, S. Cusack, J. C. Paulson, J. J. Skehel, D. C. Wiley, Nature 1988, 333,
426-431.
[3]
[4]
[5]
V. Bandlow, S. Liese, D. Lauster, K. Ludwig, R. R. Netz, A. Herrmann, O. Seitz, Journal of the American
Chemical Society 2017, 139, 16389-16397.
S. Bhatia, D. Lauster, M. Bardua, K. Ludwig, S. Angioletti-Uberti, N. Popp, U. Hoffmann, F. Paulus, M. Budt, M.
Stadtmüller, T. Wolff, A. Hamann, C. Böttcher, A. Herrmann, R. Haag, Biomaterials 2017, 138, 22-34.
a) M. Waldmann, R. Jirmann, K. Hoelscher, M. Wienke, F. C. Niemeyer, D. Rehders, B. Meyer, Journal of the
American Chemical Society 2014, 136, 783-788; b) M. Yamabe, K. Kaihatsu, Y. Ebara, Bioconjugate Chemistry
2018, 29, 1490-1494.
[6]
[7]
I. A. Wilson, J. J. Skehel, D. C. Wiley, Nature 1981, 289, 366.
A. Harris, M. J. Borgnia, D. Shi, A. Bartesaghi, H. He, R. Pejchal, Y. Kang, R. Depetris, A. J. Marozsan, R. W.
Sanders, P. J. Klasse, J. L. S. Milne, I. A. Wilson, W. C. Olson, J. P. Moore, S. Subramaniam, Proceedings of
the National Academy of Sciences 2011, 108, 11440-11445.
[8]
T. Ohta, N. Miura, N. Fujitani, F. Nakajima, K. Niikura, R. Sadamoto, C. T. Guo, T. Suzuki, Y. Suzuki, K. Monde,
S. I. Nishimura, Angewandte Chemie International Edition 2003, 42, 5186-5189.
F. Feng, N. Miura, N. Isoda, Y. Sakoda, M. Okamatsu, H. Kida, S.-I. Nishimura, Bioorganic & Medicinal
Chemistry Letters 2010, 20, 3772-3776.
[9]
[10]
[11]
G. D. Glick, P. L. Toogood, D. C. Wiley, J. J. Skehel, J. R. Knowles, The Journal of biological chemistry 1991,
266, 23660-23669.
S. Liese, M. Gensler, S. Krysiak, R. Schwarzl, A. Achazi, B. Paulus, T. Hugel, J. P. Rabe, R. R. Netz, ACS Nano
2017, 11, 702-712.
[12]
[13]
[14]
X. Yue, Y. Feng, Y. B. Yu, Journal of Fluorine Chemistry 2013, 152, 173-181.
W. Maison, J. V. Frangioni, N. Pannier, Organic Letters 2004, 6, 4567-4569.
E. Franzmann, F. Khalil, C. Weidmann, M. Schröder, M. Rohnke, J. Janek, B. M. Smarsly, W. Maison, Chemistry
– A European Journal 2011, 17, 8596-8603.
[15]
[16]
[17]
[18]
[19]
C. Dorith, H. Malte, C. Nadine, M. Wolfgang, European Journal of Organic Chemistry 2013, 2013, 6361-6371.
Z. Gan, R. Roy, Canadian Journal of Chemistry 2002, 80, 908-916.
H. Ogura, K. Furuhata, M. Itoh, Y. Shitori, Carbohydrate Research 1986, 158, 37-51.
S. Liese, R. R. Netz, ACS Nano 2018, 12, 4140-4147.
W. J. Lees, A. Spaltenstein, J. E. Kingery-Wood, G. M. Whitesides, Journal of Medicinal Chemistry 1994, 37,
3419-3433.
[20]
[21]
[22]
U. Desselberger, Archives of Virology 1975, 49, 365-372.
G. Cross, Seminars in Avian and Exotic Pet Medicine 2002, 11, 15-18.
W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein, The Journal of Chemical Physics
1983, 79, 926-935.
[23]
D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark, H. J. Berendsen, Journal of Computational
Chemistry 2005, 26, 1701-1718.
[24]
[25]
A. W. Sousa da Silva, W. F. Vranken, BMC Research Notes 2012, 5, 367.
J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman, D. A. Case, Journal of Computational Chemistry 2004, 25,
1157-1174.
[26]
[27]
[28]
[29]
[30]
R. C. Walker, M. F. Crowley, D. A. Case, Journal of Computational Chemistry 2008, 29, 1019-1031.
R. S. Dumont, Journal of Computational Chemistry 1991, 12, 391-401.
G. Bussi, D. Donadio, M. Parrinello, The Journal of Chemical Physics 2007, 126, 014101.
M. Parrinello, A. Rahman, Journal of Applied Physics 1981, 52, 7182-7190.
B. Hess, H. Bekker, H. J. C. Berendsen, J. G. E. M. Fraaije, Journal of Computational Chemistry 1997, 18, 1463-
1472.
[31]
T. Darden, D. York, L. Pedersen, The Journal of Chemical Physics 1993, 98, 10089-10092.
19
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