Synthesis of a New Series of Sialylated Homo- and Heterovalent Glycoclusters by using Orthogonal Ligations

Article abstract of DOI:10.1002/open.201600062

The synthesis of heteroglycoclusters (hGCs) is being subjected to rising interest, owing to their potential applications in glycobiology. In this paper, we report an efficient and straightforward convergent protocol based on orthogonal chemoselective ligations to prepare structurally well-defined cyclopeptide-based homo- and heterovalent glycoconjugates displaying 5-N-acetyl-neuraminic acid (Neu5Ac), galactose (Gal), and/or N-acetyl glucosamine (GlcNAc). We first used copper-catalyzed azide–alkyne cycloaddition and/or thiol-ene coupling to conjugate propargylated α-sialic acid 3, β-GlcNAc thiol 5, and β-Gal thiol 6 onto cyclopeptide scaffolds 7–9 to prepare tetravalent homoglycoclusters (10–12) and hGCs (13–14) with 2:2 combinations of sugars. In addition, we have demonstrated that 1,2-diethoxycyclobutene-3,4-dione can be used as a bivalent linker to prepare various octavalent hGCs (16, 19, and 20) in a controlled manner from these tetravalent structures.

Full text of DOI:10.1002/open.201600062

DOI: 10.1002/open.201600062
Synthesis of a New Series of Sialylated Homo- and
Heterovalent Glycoclusters by using Orthogonal
Ligations**
Gour Chand Daskhan+,[a] Carlo Pifferi+,[a] and Olivier Renaudet*[a, b]
The synthesis of heteroglycoclusters (hGCs) is being subjected
to rising interest, owing to their potential applications in glyco-
biology. In this paper, we report an efficient and straightfor-
ward convergent protocol based on orthogonal chemoselec-
tive ligations to prepare structurally well-defined cyclopeptide-
based homo- and heterovalent glycoconjugates displaying 5-
N-acetyl-neuraminic acid (Neu5Ac), galactose (Gal), and/or N-
acetyl glucosamine (GlcNAc). We first used copper-catalyzed
azide–alkyne cycloaddition and/or thiol-ene coupling to conju-
gate propargylated a-sialic acid 3, b-GlcNAc thiol 5, and b-Gal
thiol 6 onto cyclopeptide scaffolds 7–9 to prepare tetravalent
homoglycoclusters (10–12) and hGCs (13–14) with 2:2 combi-
nations of sugars. In addition, we have demonstrated that 1,2-
diethoxycyclobutene-3,4-dione can be used as a bivalent linker
to prepare various octavalent hGCs (16, 19, and 20) in a con-
trolled manner from these tetravalent structures.
1. Introduction
Multivalent interactions between carbohydrates and lectins
play key roles in the initiation and control of numerous biolog-
ical and pathological processes.[1] For these reasons, a large
panel of synthetic scaffolds[2] decorated with clusters of identi-
cal sugar residues, namely homoglycoclusters, have been de-
veloped over the last decade and successfully used as antiad-
hesive agents, immunomodulators, or diagnostic tools.[3] How-
ever, these compounds barely reflect the heterogeneity of the
glycocalix, which limits their utility to decipher the most com-
plex recognition processes and to develop more active and se-
lective probes.[4] Recently, a few reports highlighted the inter-
est of heteroglycoclusters (hGCs), which display different sugar
units, as they represent attractive tools to deepen the under-
standing of the influence of heterogeneous sugar display on
biological systems.[5] Unlike homoclusters, for which a number
of potent synthetic methods are available, the assembly of
structurally well-defined hGCs remains challenging, even
though a few procedures have been described so far. The
most efficient methods rely on the use of orthogonal chemo-
selective ligations, which allow the controlled conjugation of
sugar epitopes onto properly functionalized scaffolds. For ex-
ample, our group has previously described the synthesis of tet-
ravalent hGCs based on clickable cyclopeptide scaffolds deco-
rated with two non-identical sugar residues, either in 2:2 or
3:1 relative proportions, which were obtained in a stepwise
protocol combining oxime ligation (OL) and CuI-catalyzed
azide–alkyne cycloaddition (CuAAC).[6] Subsequently, we have
demonstrated the orthogonality of both thiol-ene and thiol-
chloroactetyl couplings (TEC and TCC, respectively) for the syn-
thesis of hexavalent hGCs that display two different sugars, in
a 4:2 relative proportion and in the two possible orientations
on the scaffold.[7] More recently, we have reported, for the first
time, a multi-click protocol involving four different conjugation
methods: OL, TEC, CuAAC, and TCC.[8] By using a proper reac-
tion order, this approach gave access to unprecedented tetra-
valent hGCs, displaying four different sugar motifs in a sequen-
tial one-pot assembly in excellent yields and with a single pu-
rification step.
With the aim to design a new series of cyclopeptide-based
glycoclusters, we synthesized diverse tetravalent homogly-
coclusters and hGCs, and then octavalent structures from
these building blocks were prepared by using a convergent
protocol. Owing to their involvement in numerous biological
and pathological recognition events, we focused our attention
on 5-N-acetyl-neuraminic acid (Neu5Ac), N-acetyl glucosamine
(GlcNAc), and galactose (Gal).[9] In fact, we have introduced
propargyl Neu5Ac on a cyclopeptide scaffold with four azide
groups through CuAAC, whereas GlcNAc thiol units have been
coupled on a scaffold displaying four allyloxycarbonyl (Alloc)
groups, using TEC. Alternatively, a combination of both reac-
tions have been used to generate tetravalent hGCs displaying
Neu5Ac, Gal, and/or GlcNAc (2:2 combination). Furthermore, to
obtain two series of octavalent hGCs (4–4 and 2:2–2:2 combi-
nations), 1,2-diethoxycyclobutene-3,4-dione, namely diethyl
squarate (DS), was used as a linker to undergo selective cou-
[a] Dr. G. C. Daskhan,+ C. Pifferi,+ Prof. Dr. O. Renaudet
Universitꢀ Grenoble Alpes, Dꢀpartement de Chimie Molꢀculaire (DCM)
CNRS, DCM, 38000 Grenoble (France)
Fax: (+33)4-56-52-08-33
E-mail: olivier.renaudet@univ-grenoble-alpes.fr
[b] Prof. Dr. O. Renaudet
Institut Universitaire de France
103 boulevard Saint-Michel, 75005 Paris (France)
[+] These authors contributed equally to this work
[**] This article is part of the Virtual Issue “Carbohydrates in the 21st Century:
Synthesis and Applications”
Supporting information and ORCIDs from the authors for this article are
available on the WWW under http://dx.doi.org/10.1002/open.201600062.
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plings with the pending amine of those tetravalent scaffolds.
The utilization of CuAAC, TEC, and DS coupling (DSC) in an or-
thogonal sequential protocol has not yet been reported.
acetate buffer pH 7.4, as recently used in our group.[13] Al-
though the reaction was successful with other propargyl glyco-
sides, no trace of the expected compound 10 was obtained by
using propargyl sialoside 4 under these experimental condi-
tions. We, thus, decided to perform the same reaction at room
temperature in phosphate-buffered saline (PBS) pH 7.4. The re-
action was followed by analytical HPLC and UPLC–MS, and was
found to be complete after 2 h, providing the tetravalent gly-
cocluster 10 in a low yield (ca. 10%) after preparative HPLC.
Surprisingly, the utilization of a mixture of DMF and PBS
(pH 7.4, 1:1, v/v) was found to be less efficient and led to prod-
uct decomposition after 2 h. It should be mentioned that,
unlike other carbohydrates, the preparation of sialylated conju-
gates is more frequently described with fully protected prop-
argyl sialoside, except in a few cases.[14] For example, Dondoni
and co-workers have reported the synthesis of calix[4]arene
appended sialoclusters from acetylated propargyl thiosialoside
and calixarene azides with good yields.[15] For this reason, we
hypothesized that the presence of the free carboxylic acid
group in 4 might be the reason of these difficulties. To confirm
this, we decided to perform the CuAAC reaction with the prop-
argyl sialoside (3) protected as a methyl ester instead of 4. As
expected, we were able to obtain the desired tetravalent gly-
cocluster 11 in the presence of Cu micropowder in PBS
(pH 7.4) in 69% yield after RP–HPLC purification. Finally, tetra-
valent homoglycocluster 12 was prepared in 83% yield by
treating cyclopeptide 8 with GlcNAc thiol 5 in the presence of
2,2-dimethoxy-2-phenylacetophenone (DMPA) under UV irradi-
ation (lmax 365 nm), as reported previously.[15,16]
2. Results and Discussion
2.1. Synthesis of Tetravalent Homo- and Heteroclusters 10–
14
Three clickable carbohydrate building blocks, namely propargyl
sialoside 4, GlcNAc thiol 5,[10] and galactose thiol 6,[10] have
been prepared. The synthesis of propargyl sialoside 4 was
adapted from the procedure described by Gan and Roy, who
have reported the synthesis of the acetyated derivative of a-
sialoside propargyl 3 by using a Koenigs–Knorr glycosylation
(Scheme 1).[11]
Commercially available N-acetyl neuraminic acid 1 was treat-
ed with dry MeOH in the presence of Dowex resin (H+), and
then successively with acetyl chloride (saturated with HCl gas)
at room temperature to give derivative 2 in good yield. Next,
a glycosylation of derivative 2 with distilled propargyl alcohol
in the presence of AgOTf and molecular sieves provided an
anomeric mixture of propargyl sialoside, from which the alpha
anomer 3 was obtained in 68% yield after successive recrystal-
lization in cold diethyl ether. The appearance of a characteristic
1
triplet in the H NMR spectrum at 2.45 ppm (J=4.0 Hz), corre-
sponding to the alkyne proton, and of a signal at 98.1 ppm in
the 13C NMR spectrum for C-2 confirmed the presence of the
a-linked propargyl functionality in compound 3. De-O-acetyla-
tion with NaOMe and methyl ester hydrolysis with LiOH afford-
ed compound 4 in 85% yield over two steps.
With these compounds in hand, we next synthesized several
cyclopeptide scaffolds by using solid-phase peptide synthesis
followed by cyclization of the linear peptide in solution. Two
scaffolds displaying four azide (7)[12] or allyloxycarbonyls (8)
groups and one scaffold with a combination of two allyloxycar-
Next, TEC and CuAAC were sequentially used to prepare the
hetero-tetravalent compounds 13 and 14, as the orthogonality
of these two reactions has been demonstrated previously by
Dondoni and co-workers.[15] In addition, we have determined
the best reaction sequence is to use both reactions in a one-
pot fashion.[8] Accordingly, we first performed the photoin-
duced (l=365 nm) addition of GlcNAc thiol 5 (1.5 equiv per
Alloc) on cyclopeptide 9 under UV irradiation in the presence
of 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a radical
initiator in water/DMF (2:1, v/v) for 45 min. The CuAAC ligation
was subsequently carried out by adding to the crude reaction
mixture propargyl sialoside 3 (1.6 equiv per azide) in the pres-
bonyl and azide groups
(Scheme 2).
9 have thus been prepared
We first decided to perform the CuAAC reaction with the
propargyl glycosides 4 and the scaffold 7 in the presence of
copper micropowder in a mixture of isopropanol and sodium
Scheme 1. Synthesis of compound 4 and structures of 5 and 6.
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Scheme 2. Synthesis of tetravalent homo- and heteroglycoclusters 10–14. Conditions for CuAAC: Cu micropowder, PBS pH 7.4, RT, 45 min; Conditions for
TEC: 2,2-dimethoxy-2-phenylacetophenone, DMF/H2O (2:1, v/v), hn (365 nm), RT, 45 min.
ence of Cu micropowder (4–5 equiv per azide) in PBS. This pro-
cess gave the desired tetravalent glycoclusters 13 and 14 in 71
and 69% overall yield after purification, respectively.
(1.1 equiv) in the presence of N,N-diisopropylethylamine
(DIPEA) at pH 8.5 in DMF at room temperature led to the for-
mation of mono-amido ester 15 as the unique product after
4 h of reaction with 88% yield (Scheme 3).
We next performed the second amidation reaction to afford
the octavalent compound 16 by using a slight excess of glyco-
cluster 11 (1.5 equiv) in sodium phosphate buffer (pH 9.5) at
378C. This reaction was found to be slow and low yielding;
2.2. Synthesis of Octavalent hGC 16
A large number of artificial scaffolds that include carbohy-
drates,[17] peptides,[18] proteins,[19] polymers,[20] liposomes,[21]
dendrimers,[22] nanostructures,[23] or calyx[4]arenes[24] have been
exployed to conjugate multiple copies of sialoglycans, and the
resulting glycoclusters have shown interesting binding efficien-
cies, particularly in the hemagglutination assay against influen-
za A viruses. In general, the strength of carbohydrate–protein
interactions is closely dependent on the ligand structure. Many
studies in this field have demonstrated that valency, linker
structure, and scaffold geometry are also important parameters
to consider in order to achieve high affinity. Our group has re-
cently investigated the influence of these parameters in the
binding efficiency between 4-, 16- and 64-valent series of gly-
cocyclopeptides and diverse vegetal and bacterial lectins.[25]
Here, we extended the scope to generate the structural diver-
sity of these compounds by preparing a new series of octava-
lent heteroclusters. For this, we focused our attention on DS,
which is used in a variety of sequential amidation reactions by
tuning the pH of the reaction mixture.[26] In fact, this linker ap-
pears ideal to selectively conjugate the previous scaffolds 11–
14 in two steps, which all contain a pending amine group. We
first studied the reaction between compound 12 and DS in
a mixture of Na2CO3 buffer (pH 8) and EtOH or DMF, as report-
ed by Kunz and co-workers.[27] However, we found this proce-
dure to be unsuccessful, presumably owing to solubility a prob-
lem. Instead, we determined that the treatment of 12 with DS
Scheme 3. Synthesis of sialylated octavalent hGC 16.
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moreover, the partial hydrolysis of methyl esters of Neu5Ac
moieties was also observed by UPLC analysis. To avoid this
problem, we decided to use sodium carbonate buffer pH 9.5
instead of sodium phosphate buffer to perform the second
amidation and concomitant full deprotection of the methyl
esters. Under these conditions, compound 16 was obtained in
a 63% yield after semi-preparative HPLC purification, along
with the formation of compound 10 in a 31% yield as a side
product.
3. Conclusions
An efficient and straightforward protocol for the controlled
synthesis of multivalent homo- and hGCs has been developed.
This convergent protocol is based on the use of three different
chemoselective ligations that are CuAAC, TEC, and squaric acid
diester as the central core. We first demonstrated that tetrava-
lent homo- and heteroglycocluster precursors can be synthe-
sized in high yield by using Cu-micropowder-catalyzed azide–
alkyne cycloadditon, thiol-ene coupling reaction, or both,
using propargyl sialoside 3 and GlcNAc thiol 5, or galactoside
6 and cyclopeptides 7, 8, and 9, respectively. During the pro-
cess, an alternative method to synthesize propargyl sialoside 4
through silver-triflate-mediated Koenigs–Knorr glycosylation
was also developed in a good yield. Furthermore, we also de-
scribed that squaric acid diester represents a simple and versa-
tile linker to connect two amine-terminated glycoclusters
through two consecutive coupling reactions at basic pH. The
overall protocol gave access to octavalent hGCs in good yields
and purity. This synthetic approach together with our previous
reports in this field represent versatile synthetic tools to devel-
op original series of structurally well-defined homo- and heter-
oglycodendrimers with promising biological properties. In par-
ticular, we are currently exploring the best chemoselective liga-
tion sequences to prepare multi-antigenic vaccines composed
of tumor-associated carbohydrate and peptide antigens.
With the aim to prepare Neu5Ac-containing heteroclusters
with different geometrical distributions to 16, we synthesized
compounds 19 and 20, displaying Neu5Ac with GlcNAc and
Gal residues, respectively, at alternating positions on the cyclo-
peptide scaffold, following the protocol described above
(Scheme 4).
Experimental Section
General Procedures
All chemical reagents were purchased from Aldrich (Saint Quentin
Fallavier, France) or Acros (Noisy-Le-Grand, France). All protected
amino acids and Fmoc-Gly-Sasrinꢁ resin were obtained from Ad-
vanced ChemTech Europe (Brussels, Belgium). For peptides and
glycopeptides, analytical RP-HPLC was performed on a Waters
system equipped with a Waters 2695 separations module and
a Waters 2487 Dual Absorbance UV/Vis Detector. Analysis was car-
ried out at 1.0 mLminÀ1 (EC 125/3 nucleosil 300–5 C18) with UV
monitoring at 214 and 250 nm by using a linear A–B gradient (buf-
fer A: 0.09% CF3CO2H in water; buffer B: 0.09% CF3CO2H in 90%
acetonitrile). Purifications were carried out at 22.0 mLminÀ1 (VP
250/21 nucleosil 100–7 C18) with UV monitoring at 214 and 250 nm
by using a linear A–B gradient. For carbohydrate derivatives, mois-
ture-sensitive reactions were performed under an argon atmos-
phere by using oven-dried glassware and reactions was monitored
by using TLC with silica gel 60 F254 precoated plates (Merck).
Spots were inspected by UV light and visualized by charring with
10% H2SO4 in EtOH for carbohydrates. Silica gel 60 (0.063–0.2 mm
Scheme 4. Synthesis of octavalent heteroclusters 19 and 20.
Compound 17 was first prepared in an 87% yield from hGC
13 (1 equiv) and 1,2-diethoxycyclobutene-3,4-dione (1.1 equiv)
in DMF at room temperature. After purification with semi-prep-
arative HPLC, the addition of a slight excess of compound 13
(1.5 equiv) in the presence of sodium carbonate buffer at 378C
afforded compound 19 in 67% yield. Similar treatment of com-
pound 14 under identical conditions afforded the desired het-
erocluster 20 in a 68% yield.
1
or 70–230 mesh, Merck) was used for column chromatography. H
and 13C NMR spectra were recorded on Bruker Avance 400 MHz or
Brucker Avance III 500 MHz spectrometers and chemical shifts (d)
were reported in parts per million (ppm). Spectra were referenced
to the residual proton solvent peaks relative to the signal of CDCl3
1
1
(d=7.26 and 77.0 ppm for H and 13C) and D2O (4.79 ppm for H),
assignments were performed by using GCOSY and GHMQC experi-
ments. Standard abbreviations s, d, t, dd, br s,m refer to singlet,
doublet, triplet, doublet of doublet, broad singlet, multiplet, re-
spectively. The anomeric configuration was established from J1,2
coupling constants. HRMS and ESI-MS spectra of peptides and gly-
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copeptides were measured on an Esquire 3000 spectrometer from
Bruker or on an Acquity UPLC/MS system from Waters equipped
with an SQ Detector 2. MALDI-TOF experiments were performed
on a AutoFlex I Bruker after sample pretreatment in an OligoR3 mi-
crocolumn (Applied Biosystems, USA) using 2,5-dihydroxybenzoic
acid matrix.
washed with Et2O (2ꢂ20 mL) and dried to give 3 (0.41 g, 92%) as
a white powder. HRMS (ESI+-TOF) m/z: calcd for C15H23NO9Na
[M+Na]+: 384.1271; found 384.1267.
Prop-2-ynyl 5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-2-
onulopyranose (4)
LiOH (62.0 mg, 2.59 mmol) was added to a solution of 3 (0.30 g,
0.86 mmol) in water (15 mL), and the reaction stirred at room tem-
perature for 1 h. The reaction mixture was neutralized with amber-
lite IR-120 resin (H+) and filtered; the aqueous solution was
washed with Et2O (2ꢂ20 mL) and freeze-dried to give 4 (275 mg,
92%) as a flocculent powder. (400 MHz, D2O): dH =4.25 (2H, ddd,
J=1.6, 8.0 20.8 Hz, H-8 NHAc), 3.84–3.72 (3H, m, H-2, H-7, H-4),
3.64–3.51 (7H, m, H-5, H-6a,b, H-9a,b, ÀCH2 ꢀ), 2.68 (1H, dd, J=4.0,
12.0 Hz, H-3a), 1.97 (3H, s, Me), 1.61 ppm (1H, t, J=12 Hz, H-3b);
(100 MHz, D2O): dC =174.6 (C=O), 173.9 (C-1), 95.3 (C-2), 70.6 (C-3),
70.5 (C-7), 68.3 (C-4), 66.4 (C-6), 63.1 (C-5), 52.0 (Me), 48.9
(ÀOCH2 ꢀ), 38.6 (C-3), 21.9 ppm (Me); HRMS (ESI+-TOF) m/z: calcd
for C14H21NO9 [M]+: 347.1216; found 347.1208.
Synthesis of Propargyl Sialoside (4)
Methyl (5-Acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-tri-
deoxy-d-glycereo-b-d-galacto-2-nonulopyranosid)onate (2)
Dowex-50WX2 resin (1 g, H+ form) was added to a solution of N-
acetyl neuraminic acid 1 (2 g, 6.5 mmol) in dry MeOH (100 mL) at
room temperature under an argon atmosphere. The reaction mix-
ture was stirred for 18 h and the resin was filtered through a Celite
pad, washed with dry methanol (10 mL), and the filtrate was
evaporated under vacuum to give a white solid. The crude reaction
mixture was used for next reaction without further purification.
Acetyl chloride (50 mL) was added to the crude reaction mixture at
room temperature under an HCl (gas) environment. The white sus-
pension gradually became pale pink and the reaction mixture was
stirred continuously for 12 h. The solvents were removed in vacuo,
co-evaporated with toluene (2ꢂ50 mL), and the residue was puri-
fied by flash chromatography (SiO2) to afford 2 (2.81 g, 85%) as
a white solid. Rf =0.33 (MeOH/CH2Cl2 =24:1). The NMR data is con-
sistent with literature.[28]
General Procedure for Solid-Phase Peptide Synthesis
Assembly of the protected linear peptide was performed manually
by employing a solid-phase peptide synthesis (SPPS) protocol
using the Fmoc/tBu strategy and the Fmoc-Gly-SasrinTM resin. Cou-
pling reactions were performed by using, relative to the resin load-
ing, 2 equivalents of N-Fmoc-protected amino acid activated in situ
with PyBOP (2 equiv) and DIPEA (5 equiv) in DMF (10 mLgÀ1 resin)
for 30 min. The coupling reaction was checked by using the TNBS
test with a solution of 1% trinitrobenzenesulfonic acid in DMF. N-
Fmoc protecting groups were removed upon treatment with piper-
idine–DMF (1:4, 10 mLgÀ1 resin) for 10 min. The process was re-
peated three times and the resin was further washed five times
with DMF (10 mLgÀ1 resin) for 2 min. The peptide was released
from the resin by treating ten times with a cleavage solution of
TFA/CH2Cl2 (1:99). The combined cleavage fractions were concen-
trated under reduced pressure; ice-cold Et2O was added to induce
precipitation and the linear peptide was obtained as a white
powder after filtration and used without any further purification.
Methyl (Prop-2-ynyl 5-acetamido-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosid)onate (3)
A mixture of 2 (2.81 g, 5.5 mmol), AgOTf (2.1 g, 8.2 mmol) and
powdered 4 ꢃ molecular sieves (4 g) in freshly distilled propargyl
alcohol (60 mL) was stirred at room temperature under an Ar at-
mosphere. The reaction mixture was protected from light and
stirred for 2 h in the dark. The crude mixture was filtered through
a Celite pad, washed with CH2Cl2 (2ꢂ50 mL), and the filtrate was
concentrated under vacuum to dryness. The residue was dissolved
in CH2Cl2 (500 mL), washed with water (2ꢂ200 mL), brine (100 mL),
and aqueous Na2S2O3 (10% m/v). The combined organics were
dried over MgSO4, filtered, and the solvent removed in vacuo. The
crude was purified by using flash chromatography (SiO2) and crys-
tallized from diethyl ether at À48C to give the peracetylated pre-
cursor of 3 (1.98 g, 68%) as a colorless solid. Rf =0.30 (MeOH/
CH2Cl2 =1:24); (400 MHz, CDCl3): dH =5.45–5.41 (1H, m, H-8), 5.33
(1H, dd, J=1.4, 8.0 Hz, H-7), 5.18 (1H, d, J=8.0 Hz, NHAc), 4.93–
4.86 (1H, m, H-4), 4.43 (1H, dd, J=4.0, 12.0 Hz, CH2aCꢀCH), 4.31
(1H, dd, J=4.0, 12.0 Hz, H-9’), 4.19 (1H, dd, J=4.0, 16.0 Hz,
CH2bCꢀCH), 4.13–4.09 (3H, m, H-9, H-5, H-6), 3.84 (3H, s, OCH3),
2.66 (1H, dd, J=4.0, 12.0 Hz, H-3a), 2.45 (1H, t, J=4.0 Hz, CH2 ꢀ
CH), 2.17–2.05 (4ꢂ3H, 4 s, 4ꢂOCOCH3), 1.90 (3H, s, NHCOCH3),
1.85 ppm (1H, m, H-3b); (100 MHz, CDCl3): dC =170.9–170.0 (4ꢂ
OCOCH3 and CONH), 167.7 (C-1), 98.1 (C-2), 78.9 (CꢀCH), 74.4 (Cꢀ
CH), 72.6 (C-6), 68.8 (C-4), 68.2 (C-8), 67.1 (C-7), 62.3 (C-9), 52.8
(OCH3), 52.7 (OCH2 ꢀ), 49.3 (C-5), 37.9 (C-3), 23.1 (NHCOCH3), 21.0,
20.8, 20.7, 20.5 ppm (4 x OCOCH3); HRMS (ESI+-TOF) m/z: calcd for
C23H31NO13Na [M+Na]+: 552.1693; found 552.1698.
General Procedure for Peptide Cyclization
All linear peptides were dissolved in CH2Cl2 (0.5 mm) and the pH of
the solution was adjusted to 8.5–9 by addition of DIPEA. PyBOP
(1.2 equiv) was added and the reaction mixture was stirred at
room temperature for 1 h. The solvent was removed under re-
duced pressure and precipitation from diethyl ether afforded de-
sired Boc-protected cyclic peptides as white solids. Boc-protected
cyclic peptides were treated with a solution of TFA/CH2Cl2 (10 mL,
3:2, v/v) for 1 h; the crude reaction mixture was concentrated to
afford -NH2 terminated cyclic peptides 7–9.
Compound 8
The linear peptide sequence A (0.49 mmol) (see Scheme 1 in the
Supporting Information) was synthesized following the general
procedure for SPPS, then cyclized, and finally the Boc group was
cleaved. Yield: 90% (over three steps, 569 mg) after purification by
preparative RP-HPLC and lyophilization; RP-HPLC: Rt =7.6 min (C18,
l=214 nm 5–100% B in 15 min); HRMS (ESI+-TOF) m/z: calcd for
C63H102N15O18 [M+H]+:1356.7527, found: 1356.7614
NaOMe/MeOH (200 mL, pH 9) was added to a solution of the pro-
tected precursor of 3 (0.65 g, 1.23 mmol) in MeOH (20 mL), and the
reaction mixture was stirred at room temperature for 3 h. Amber-
lite IR-120 resin (H+) was added to neutralize the reaction and the
solvent was removed under vacuum. The obtained residue was
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Compound 9
Compound 12
The linear peptide sequence B (0.40 mmol) (see Scheme 2 in the
Supporting Information) was synthesized following the general
procedure for SPPS, then cyclized, and finally the Boc group was
cleaved. Yield: 88% (over three steps, 569 mg) after purification by
preparative RP-HPLC and lyophilization; RP-HPLC: Rt =7.6 min (C18,
l=214 nm 5–100% B in 15 min); HRMS (ESI+-TOF) m/z: calcd for
C55H90N19O14 [M+H]+: 1240.6915, found 1240.6980.
Compound 12 was obtained by coupling 8 (11.0 mg, 8.5 mmol)
with 510 (12.1 mg, 51.0 mmol), following the general procedure for
TEC ligation, in 83% yield (16.3 mg) as a white foam after lyophili-
zation. RP-HPLC: Rt =9.4 min (C18, l=214 nm 0–40% B in 15 min);
ESI+-MS m/z: calcd for C95H162N19O38S4 [M+H]+: 2306.7, found:
2306.5; calcd for C95H163N19O38S4 [M+2H]2+: 1153.8, found: 1153.8.
Compound 13
General Procedure for CuAAC Ligation
Following the procedure for TEC ligation, compound 9 (10.0 mg,
8.3 mmol) was reacted with 510 (5.9 mg, 25.0 mmol) in the presence
A solution of cyclopeptide and propargyl sialoside (1.5–1.8 equiv/
N3) in PBS buffer (10 mm, pH 7.4) was degassed at room tempera-
ture under an Ar atmosphere for 15 min. Copper micropowder (4–
5 equiv/N3) was added to the reaction mixture under a nitrogen at-
mosphere and the solution was vigorously stirred at room temper-
ature. After 45 min, analytical HPLC indicated complete coupling.
ChelexTM resin was added to remove excess copper and the solu-
tion was filtered, concentrated, and purified by using RP-HPLC to
afford pure compound as a white powder after lyophilization.
of
2,2-dimethoxy-2-phenylacetophenone
(DMPA,
2.6 mg,
10.1 mmol). After 45 min, the crude reaction mixture was concen-
trated under reduced pressure, the residue was dissolved in PBS
buffer (1.0 mL, pH 7.4, 10 mm), and then 3 (10.8 mg, 29.9 mmol)
was added to this solution. The coupling was performed by follow-
ing the general procedure for CuAAC ligation. After crude mixture
purification through semi-preparative RP-HPLC, compound 13 was
obtained in 71% yield (14.4 mg) over two steps as a white foam
after lyophilization. RP-HPLC: Rt =8.9 min (C18, l=214 nm 0–40% B
in 15 min); ESI+-MS m/z: calcd for C101H166N23O42S2 [M+H]+: 2348.7,
found: 2348.4; calcd for C101H167N23O42S2 [M+2H]2+: 1219.9, found:
1219.7; calcd for C101H168N23O42S2 [M+3H]3+: 813.6, found: 813.4.
General Procedure for TEC Ligation
To a solution of cyclopeptide (1–5 mm) and thiol glycoside (1.5–
1.8 equiv/allyl) in a mixture DMF/H2O (2:1), 2,2-dimethoxy-2-phe-
nylacetophenobe (DMPA, 2–3 mg) was added and the solution was
irradiated at 365 nm for 45 min while stirring. The reaction was di-
rectly purified by using semi-preparative RP-HPLC to afford pure
compound as white foam after lyophilization.
Compound 14
Compound 14 was obtained in 70% yield (13.7 mg) from 9
(10.0 mg, 8.3 mmol), 6[10] (4.9 mg, 24.9 mmol) and
3 (9.6 mg,
26.6 mmol), following the same strategy used for compound 13.
RP-HPLC: Rt =8.4 min (C18, l=214 nm 0–40% B in 15 min); ESI+
-MS m/z: calcd for C97H160N21O42S2 [M+H]+: 2356.6, found: 2356.2;
calcd for C97H161N21O42S2 [M+2H]2+: 1178.8, found: 1178.5; calcd
for C97H162N21O42S2 [M+3H]3+: 786.2, found: 786.1.
General Procedure for DSC Ligation
Procedure A: mono-Amido Ester Formation
To a solution of amino-containing cyclopeptide (1–5 mm) and 1,2-
diethoxycyclobutene-3,4-dione (1.1 equiv) in DMF, DIPEA was
added at room temperature to reach pH 8.5. After 4 h of stirring,
analytical RP-HPLC showed mono-amide product formation, the
crude mixture was then purified by using semi-preparative
RP-HPLC to afford the desired compound as a white foam after
lyophilization.
Compound 15
Compound 15 was obtained by coupling 12 (12.0 mg, 5.2 mmol)
with 3,4-diethoxy-3-cyclobutene-1,2-dione (850 mL, 5.7 mmol), fol-
lowing procedure A for the DSC coupling, in 88% yield (11.1 mg)
as a white foam after lyophilization. RP-HPLC: Rt =8.0 min (C18, l=
214 nm 0–60%
B
in 15 min); ESI+-MS m/z: calcd for
C101H166N19O41S4 [M+H]+: 2430.8, found: 2429.9, calcd for
C101H167N19O41S4 [M+2H]2+
: 1215.9, found: 1215.8; calcd for
Procedure B: bis-Amido Ester Formation
C101H168N19O41S4 [M+3H]3+: 810.9, found: 810.7.
To a solution of squaric acid mono-amide compound (1–5 mm) in
sodium carbonate buffer (pH 9.5), amino-containing cyclopeptide
(1.1–1.5 equiv) was added at room temperature. The reaction mix-
ture was incubated at 378C and the evolution was monitored by
using analytical RP-HPLC. After 1 day, the crude reaction mixture
was purified by using semi-preparative RP-HPLC to give the de-
sired compound as a white foam after lyophilization.
Compound 16
Compound 16 was obtained by coupling compound 15 (3.9 mg,
1.6 mmol) with compound 11 (6.2 mg, 2.4 mmol), following proce-
dure A for the DSC coupling, in 63% yield (5.0 mg) as a white
foam after lyophilization. Compound 10 (1.5 mg, 0.5 mmol, 31%)
was also obtained as by-product. RP-HPLC: Rt =7.2 min (C18, l=
214 nm 0–60%
B
in 15 min); ESIÀ-MS m/z: calcd for
Compound 11
C202H318N46O86S4 [MÀ2H]2À
: 2447.6, found: 2447.6; calcd for
Compound 11 was obtained by coupling 7 (10.0 mg, 8.9 mmol)
with 3 (19.3 mg, 53.4 mmol), following the general procedure for
CuAAC ligation, in 69% yield (15.8 mg) as a white foam after lyo-
philization. RP-HPLC: Rt =8.6 min (C18, l=214 nm 0–40% B in
15 min); ESI+-MS m/z: calcd for C107H170N27O46 [M+H]+: 2570.6,
found: 2569.9; calcd for C107H171N27O46 [M+2H]2+: 1285.8, found:
1285.7.
C
202H317N46O86S4 [MÀ3H]3À: 1631.4, found: 1631.5.
Compound 17
Compound 17 was obtained by coupling 13 (4.5 mg, 1.8 mmol)
with 3,4-diethoxy-3-cyclobutene-1,2-dione (300 mL, 2.0 mmol), fol-
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Chiodo, I. Garcia, S. Penadꢆs, Chem. Soc. Rev. 2013, 42, 4728; j) N. Spine-
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D. M. Haddleton, Eur. Polym. J. 2004, 40, 431.
lowing procedure A for the DSC coupling, in 87% yield (4.0 mg) as
a white foam after lyophilization. RP-HPLC: Rt =7.9 min (C18, l=
214 nm 0–60%
B
in 15 min); ESI+-MS m/z: calcd for
C107H170N23O45S2 [M+H]+: 2562.8, found: 2562.3, calcd for
C107H171N23O45S2 [M+2H]2+: 1281.9, found: 1281.7.
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Marradi, P. Messner, A. Molinaro, P. Murphy, C. Nativi, S. Oscarson, S. Pe-
nadꢆs, F. Peri, R. J. Pieters, O. Renaudet, J.-L. Reymond, B. Richichi, J.
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Compound 18
Compound 18 was obtained by coupling 14 (6.0 mg, 2.6 mmol)
with 3,4-diethoxy-3-cyclobutene-1,2-dione (410 mL, 2.8 mmol), fol-
lowing procedure A for the DSC coupling, in 85% yield (5.5 mg) as
a white foam after lyophilization. RP-HPLC: Rt =7.6 min (C18, l=
214 nm 0–60%
B
in 15 min); ESI+-MS m/z: calcd for
C103H164N21O45S2 [M+H]+: 2480.6, found: 2480.5, calcd for
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b) R. J. Pieters, Org. Biomol. Chem. 2009, 7, 2013.
C103H165N21O45S2 [M+2H]2+
C103H166N21O45S2 [M+3H]3+: 827.6, found: 827.4
: 1240.8, found: 1240.6, calcd for
[5] a) C. Mꢄller, G. Despras, T. K. Lindhorst, Chem. Soc. Rev. 2016, 45, 3275;
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Compound 19
Compound 19 was obtained by coupling compound 17 (4.1 mg,
1.6 mmol) with compound 13 (5.6 mg, 2.3 mmol), following proce-
dure A for the DSC coupling, in 67% yield (5.2 mg) as a white
foam after lyophilization. RP-HPLC: Rt =7.2 min (C18, l=214 nm 0–
60%
B
in 15 min); ESIÀ-MS m/z: calcd for C202H318N46O86S4
[MÀ2H]2À
: 2447.6, found: 2446.6; calcd for C202H317N46O86S4
[MÀ3H]3À: 1631.4, found: 1631.1.
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Compound 20
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Compound 20 was obtained by coupling compound 18 (4.0 mg,
1.6 mmol) with compound 14 (5.9 mg, 2.5 mmol), following proce-
dure A for the DSC coupling, in 68% yield (5.1 mg) as a white
foam after lyophilization. RP-HPLC: Rt =7.3 min (C18, l=214 nm 0–
60%
B
in 15 min); ESIÀ-MS m/z: calcd for C194H306N42O86S4
[MÀ2H]2À
: 2365.5, found: 2364.7; calcd for C194H305N42O86S4
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The authors acknowledge support from the “Communautꢀ d’ag-
glomꢀration Grenoble-Alpes Mꢀtropole” (Nanobio program),
ICMG FR 2607, LabEx ARCANE (ANR-11-LABX-0003-01), the french
Agence Nationale de la Recherche (ANR-12-JS07-0001-01
“VacSyn”) and the European Research Council Consolidator Grant
“LEGO” (647938) to O.R.
Keywords: cyclopeptides
heteroglycoclusters · multivalency · orthogonal ligations
·
homoglycoclusters
·
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Received: June 15, 2016
Published online on && &&, 2016
&
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FULL PAPERS
Extending molecular diversity: A
straightforward convergent protocol
based on orthogonal chemoselective li-
gations is described to generate hetero-
multivalency in a controlled manner.
Copper-catalyzed azide–alkyne cycload-
dition (CuAAC) and thiol-ene coupling
(TEC) are first used to functionalize
cyclopeptide scaffolds with propargyl
and thiol glycosides to generate tetrava-
lent heteroglycoclusters with 2:2 combi-
nations of sugars. In addition, these
compounds can be easily (hetero)dimer-
ized by pH-dependent sequential cou-
pling with diethyl squarate to afford
various octavalent structures.
G. C. Daskhan, C. Pifferi, O. Renaudet*
&& – &&
Synthesis of a New Series of Sialylated
Homo- and Heterovalent Glycoclusters
by using Orthogonal Ligations
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&
These are not the final page numbers! ÞÞ