Synthesis of multitopic neoglycopeptides displaying recognition and detection motifs

Article abstract of DOI:10.1016/j.bmcl.2005.05.072

We describe herein the synthesis of cyclic decapeptide template displaying clustered carbohydrate recognition motifs and detection agent on spatially separated domains. Such multitopic labeled neoglycopeptides represent attractive tools for binding assays

Full text of DOI:10.1016/j.bmcl.2005.05.072

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3619–3622
Synthesis of multitopic neoglycopeptides displaying recognition
and detection motifs
Olivier Renaudet and Pascal Dumy*
´ ´
LEDSS, UMR-CNRS 5616 & ICMG FR 2607, Universite Joseph Fourier, BP 53 38041 Grenoble Cedex 9, France
Received 22 February 2005; revised 3 May 2005; accepted 11 May 2005
Abstract—We describe herein the synthesis of cyclic decapeptide template displaying clustered carbohydrate recognition motifs and
detection agent on spatially separated domains. Such multitopic labeled neoglycopeptides represent attractive tools for binding
assays with carbohydrate binding proteins in glycomic research.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Many biological processes including fertilization, tissue
formation, cell adhesion, antigen/antibody interactions,
cancer metastasis, and infection of viruses or bacteria
involve polyvalent interactions between oligomeric pro-
teins and recognition motifs.1 The design of molecules
displaying multiple binding proteins elements,2 particu-
larly carbohydrate-based ligands, represents an
emerging challenge to investigate these fundamental rec-
ognition processes3 and to discover new active and selec-
tive therapeutics.4 In the past few years, considerable
synthetic efforts have been expended in this direction,
leading to the development of a diverse supply of glyco-
conjugates,5 such as calixarenes, cyclodextrines, dendri-
mers, or carbohydrate-based combinatorial libraries,6
which have shown enhancement of affinity with target
proteins through multivalent interactions. However, ac-
cess to well-defined multitopic structures displaying
independent probes (e.g., fluorescent agent, dye, or bio-
tin) and clustered recognition motifs remains of great
interest, enabling the high-throughput screening with
unlabeled target proteins by multiple binding assay
formats.7
assembly of carbohydrate-based conjugates remains
fastidious due to a number of manipulations of orthog-
onal protecting groups.8 As a convenient alternative,
we have recently reported that regioselectively address-
able functionalized templates (RAFTs) provide a useful
platform for the incorporation of multiple copies of
peptide9 or sugar10 recognition motifs through an
oxime-based strategy.
RAFT molecules are commonly composed of a back-
bone-cyclized decapeptide containing two prolines-gly-
cines as b-turn inducers that stabilize the conformation
in solution. While four lysine side chains were previously
used as addressable sites for multivalent presentation of
carbohydrates10 or protein de novo design,11 two addi-
tional lysines9,12 can be incorporated to the sequence,
providing molecules that allow functional components
to be assembled and directed in well-defined and con-
trolled spatial orientations.13 Taking advantage of this
topological feature, we report herein the preparation
of well-defined RAFT molecules displaying two separat-
ed addressable domains to get multitopic labeled neogly-
coconjugates. One domain of the molecule is devoted to
the presentation of multiple copies of carbohydrate,
which ensure the recognition properties of our system
(recognition domain, Fig. 1). On the second domain, a
molecular probe, such as fluorescein or biotin, is incor-
porated to characterize the interaction with the target
protein (detection domain, Fig. 1). Both detection and
carbohydrate moieties are incorporated into the mole-
cules sequentially, by using orthogonal protecting
groups Boc (lysines 3, 5, 8, and 10) and Aloc (lysines 4
and 9).9,12
The crucial point of this approach focuses on control-
ling the molecular assembly processes, which is essen-
tial to warrant the multitopic structure of the target
molecule. While many chemical or chemoenzymatic
methodologies including solution and solid-phase
synthesis protocols have been reported so far, the
Keywords: Oxime ligation; Template; Multivalency; Glycopeptide.
*
Corresponding author. Tel.: +33 476635545; fax: +33 476514382;
e-mail: pascal.dumy@ujf-grenoble.fr
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.072

3620
O. Renaudet, P. Dumy / Bioorg. Med. Chem. Lett. 15 (2005) 3619–3622
ylamine (DIEA) in dimethylformamide under high
dilution (0.5 mM) to prevent oligomerization. After
30 min at room temperature, the corresponding pro-
tected cyclodecapeptide 3 is obtained in 86% yield
after precipitation from diethyl ether. The sequential
topological functionalization of the template is readily
performed after regioselective removal of orthogonal
protections (Scheme 2). Lysines at positions 4 and 9
bearing Aloc are first deprotected following a typical
procedure, by treatment of a catalytic amount of
Pd(Ph3P)4 and a large excess of PhSiH3 in CH2Cl2 un-
der nitrogen gas.15 The cyclopeptide 4 displaying two
free amino side chains on the detection face is ob-
tained with 79% yield after semi-preparative reverse-
phase HPLC purification.
At this stage, we introduce two different detection agents
(Scheme 2). Biotin is first chosen, as it can easily be used
for binding studies with a target protein as an immobi-
lization agent on avidin/streptavidin-coated surfaces,
as well as to detect the formation of a ligand/receptor
complex with streptavidin coupled with peroxidase or
alkaline phosphatase.7a,b The coupling reaction between
4 and biotin is thus performed with PyBOP and DIEA
in DMF and the progress of the reaction is monitored
by an RP-HPLC analysis. The reaction cleanly proceeds
in 1 h and gives a white powder corresponding to the
bis-biotinylated RAFT molecule 5 after precipitation
from a mixture of dichloromethane and diethyl ether
(87% yield).
Figure 1. Solution strategy for the preparation of topological
template.
The linear orthogonally protected decapeptide 2 is first
prepared, following the standard Fmoc/t-Bu strategy
from the highly acid-labile Fmoc-Gly-SASRINꢁ resin
1 using a parallel peptide synthesizer (Scheme 1).
After the removal of N-terminal Fmoc-protecting
group, the linear peptide 2 is cleaved from the support
by treatment of a mixture 1% trifluoroacetic acid
(TFA) in dichloromethane. Further head-to-tail cycli-
zation of the template takes place with PyBOP14 as
the coupling reagent in the presence of diisopropyleth-
As a complementary alternative, we incorporate a
fluorescent agent to dispose of detectable molecules
suitable for carbohydrate microarrays and for recogni-
tion assays in solution using fluorescence technolo-
gies.16 Indeed, previous studies have shown that
spatial proximity of two fluorescent probes pointing
to the lower face of a template has no effect on the
fluorescence of the molecule since no self-quenching
was clearly observed.9 For this purpose, we introduce
fluorescein at both free amino positions on the detec-
tion domain of the template 4 by treatment of fluores-
cein isothiocyanate (FITC) in DMF under basic
conditions. After 1 h, the conversion is complete and
the fluorescent RAFT compound 6 was obtained in
75% yield.
The subsequent incorporation of carbohydrate recogni-
tion motifs is realized, following an oxime-based strate-
gy from aminooxylated modified sugar derivatives that
were prepared according to the protocol that we have
described previously.17 Moreover, to prevent any risk
of side reactions during the oxidative cleavage common-
ly required to generate aldehyde functions from serine,18
we decide to employ levulinic acid 7 as a more direct
route to introduce the ketone functions needed for the
oxime ligation with aminooxy sugars, providing at the
same time a suitable spacer between the anchoring site
and the template.
Scheme 1. Reagents and conditions: (a) successive cycles of (i) 20%
piperidine/DMF, 3 · 10 min; (ii) Fmoc-aa-OH (4 equiv), PyBOP
(4 equiv), DIEA (8 equiv), DMF, 30 min, 87%; (b) PyBOP, DIEA,
DMF, 86%.
The Boc-protecting groups of biotinylated and fluores-
cent products 5 and 6 are first removed with a solution
of 50% TFA in dichloromethane to quantitatively

O. Renaudet, P. Dumy / Bioorg. Med. Chem. Lett. 15 (2005) 3619–3622
3621
Scheme 2. Reagents and conditions: (a) (i) PhSiH3, Pd(Ph3P)4, DMF, 79%; (ii) biotin, PyBOP, DIEA, DMF, 87% or FITC, DIEA, DMF, 75%; (b)
pentafluorophenol, DCC, CH2Cl2, 99%; (c) (i) TFA/CH2Cl2 (1/1), 95%; (ii) 8, DIEA, DMF, 70%; (d) Aminooxy sugar (Man-a-ONH2, GalNAc-a-
ONH2, Gal-a-ONH2 or Gal-b-ONH2), AcONa buffer 0.1 M pH 4.0, 40–60% after semi-preparative RP-HPLC purification.
Table 1. Analytical data of glycopeptides 11–16
obtain RAFT molecules presenting four free side chains
at positions 3, 5, 8, and 10 of the recognition domains,
which are used without further purification. The levulin-
ic acid 7 is first activated as the pentafluorophenol ester
8 using a standard procedure by treatment with dicyclo-
carbodiimide (DCC) in dichloromethane. The incorpo-
ration of four copies of 8 into the deprotected RAFTs
5 and 6 takes place under basic conditions in DMF to
get 9 and 10 with 70% yield after semi-preparative
RP-HPLC purification.
Compound
Rta
Mcalcd
m/zb
11
12
13
14
15
18.9
18.9
20.9
20.4
19.1
2631.0
2795.2
2957.2
3121.4
2957.2
877.9 [M+3H]3+
932.3 [M+3H]3+
986.1 [M+3H]3+
1041.0 [M+3H]3+
986.5 [M+3H]3+
a Reverse-phase HPLC retention time is given in minutes: linear gra-
˚
dient 95:5 A:B to 40:60 A:B in 30 min (Column: nucleosil 100 A 5 lm
C18 particles, 250 · 4.6 mm; solvent B: 0.09% TFA in 90% acetoni-
trile and solvent A: 0.09% TFA; flow rate: 1 mL/min; detection:
k = 214 nm for biotinylated conjugates, k = 214 and 499 nm for
fluorescent conjugates).
The chemoselective functionalization of the recogni-
tion domain takes place with aminooxylated a-man-
nose, a-N-acetylgalactose (mucin-related TN-antigen),
and a/b-galactose17, which are relevant for diagnostic
or therapeutic applications. The oxime ligation oc-
curred in sodium acetate buffer, pH 4.0, at room tem-
perature and the progress of the reaction is monitored
by RP-HPLC analysis. Whereas the conversion is
found to be incomplete after 2 days compared to
our previous studies,10 six new tetravalent RAFT-
ligated glycoconjugates 11–16 are yet to be obtained
after HPLC purification in satisfactory isolated yield
(ꢀ50%). These glycosylated compounds are character-
ized by electrospray ionization mass spectrometry (en-
tries 1–6, Table 1). Additionally, two-dimensional
TOCSY and ROESY NMR experiments performed
on product 1219 permit the assignment of each proton
and also confirm the sequence of the peptidic
backbone.
b Mass spectrometry analysis was performed by electrospray ionization
In summary, we report, in this paper, the preparation
of multitopic neoglycopeptides presenting clustered
carbohydrates as recognition motifs and molecular
probes for biological characterizations with various
carbohydrate binding proteins. These molecules dis-
playing both recognition and detection properties on
two separated addressable domains represent attrac-
tive tools to investigate the functional and structural
features of the multivalent interactions between pro-
teins and oligosaccharides. In addition, the easy chem-
ical access of such neoglycoconjugates using oxime
ligation strategy combined with the development of
a fully solid-phase protocol for synthesis and screening

3622
O. Renaudet, P. Dumy / Bioorg. Med. Chem. Lett. 15 (2005) 3619–3622
10. (a) Renaudet, O.; Dumy, P. Org. Lett. 2003, 5, 243; (b)
Renaudet, O.; Dumy, P. Tetrahedron Lett. 2004, 45,
65.
11. Peluso, S.; Dumy, P.; Nkubana, C.; Yokokawa, Y.;
Mutter, M. J. Org. Chem. 1999, 64, 7114, and references
cited therein.
with target proteins might enable the rapid discovery
of new active and selective carbohydrate-based thera-
peutics. This is currently explored in the laboratory
and will be reported in due course.
12. (a) Scheibler, L.; Dumy, P.; Boncheva, M.; Leufgen, K.;
Mathieu, H.-J.; Mutter, M.; Vogel, H. Angew. Chem., Int.
Ed. 1999, 38, 696; (b) Scheibler, L.; Dumy, P.; Stamou, D.;
Duschl, C.; Vogel, H.; Mutter, M. Tetrahedron 1998, 54,
3725.
13. (a) Dumy, P.; Eggleston, I. M.; Esposito, G.; Nicula, S.;
Mutter, M. Biopolymers 1996, 39, 297; (b) Peluso, S.;
Ruckle, T.; Lehmann, C.; Mutter, M.; Peggion, C.;
¨
Acknowledgments
This work was supported by the Association pour la
Recherche contre le Cancer (ARC), the Centre National
pour la Recherche Scientifique (CNRS) for specific ac-
tion (AIP), and the Institut Universitaire de France
(IUF). We thank Prof. G. Esposito from the University
of Udinese, Italy, and Dr. C. Fontaine for NMR studies
of compound 12.
Crisma, M. ChemBioChem 2001, 2, 432.
14. Coste, J.; Le Nguyen, D.; Castro, B. Tetrahedron Lett.
1990, 31, 205.
´
15. (a) Thieriet, N.; Alsina, J.; Girald, E.; Guibe, F.; Alberi-
cio, F. Tetrahedron Lett. 1997, 38, 7275; (b) Dangles, O.;
´
Guibe, F.; Balavoine, G.; Lavielle, S.; Marquet, A. J. Am.
Chem. Soc. 1987, 52, 4984.
References and notes
16. For a recent review about fluorescence technologies for
analysis of protein interactions, see: Yan, Y.; Marriott, G.
Curr. Opin. Chem. Biol. 2003, 7, 1.
1. Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew.
Chem., Int. Ed. 1998, 37, 2754.
2. For selected review about peptide and protein recognition
by designed molecules, see: Peczuh, M. W.; Hamilton, A.
D. Chem. Rev. 2000, 100, 2479.
3. (a) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321;
(b) Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102,
555; (c) Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.;
Oetjen, K. A.; Kiessling, L. L. J. Am. Chem. Soc. 2002,
124, 14922.
4. (a) Sears, P.; Wong, C.-H. Angew. Chem., Int. Ed. 1999,
38, 2300; (b) Yarema, K. J.; Bertozzi, C. R. Curr. Opin.
Chem. Biol. 1998, 2, 49; (c) Danishefsky, S. J.; Allen, J. R.
Angew. Chem., Int. Ed. 2000, 39, 836; (d) Toyokuni, T.;
Singhal, A. K. Chem. Soc. Rev. 1995, 231; (e) Bertozzi, C.
R.; Kiessling, L. L. Science 2001, 291, 2357.
5. (a) For selected publications, see: Fulton, D. A.; Stoddart,
J. F. Bioconjugate Chem. 2001, 12, 655; (b) Roy, R. Curr.
Opin. Struct. Biol. 1996, 6, 692; (c) Sprengard, U.;
Schudok, M.; Kretzschmar, G.; Kunz, H. Angew. Chem.,
Int. Ed. 1996, 35, 321; (d) Wittmann, V.; Seeberger, S.
Angew. Chem., Int. Ed. 2000, 39, 4348; (e) Veprek, P.;
Jezek, J. J. Peptide Sci. 1999, 5, 5.
17. Renaudet, O.; Dumy, P. Tetrahedron Lett. 2001, 42,
7575.
18. Geoghegan, K. F.; Stroh, J. G. Bioconjugate Chem. 1992,
3, 138.
19. 1H NMR data of glycopeptide 12 (500 MHz, H2O/D2O,
303 K): d ppm 8.60 (2H, NH-G1,6), 8.43 (2H, NH-K4,9),
8.16 (2H, 2· NHAc GalNAc), 8.10 (2H, 2· NHAc
GalNAc), 8.07 (2H, NHe-K5,10), 7.98 (2H, NHe-K3,8),
7.94 (2H, NHe-K4,9), 7.93 (2H, NH-K5,10), 7.82 (2H, NH-
K3,8), 6.47 (2H, 2· NH biotin), 6.41 (2H, 2· NH biotin),
5.46 (2H, 2· H-10 GalNAc), 5.44 (2H, 2· H-10 GalNAc),
4.73 (2H, Ha-K3,8), 4.65 (2H, 2· H biotin), 4.47 (2H, 2· H
biotin), 4.44 (2H, Ha-K10,5), 4.41 (2H, Ha-P2,7), 4.36 (2H,
Ha-K4,9), 4.35 (2H, 2· H-20 GalNAc), 4.32 (2H, 2· H-20
GalNAc), 4.16 (2H, Ha-G1,6), 4.08 (4H, 4· H-40 GalNAc),
4.06 (2H, 2· H-30 GalNAc), 4.05 (2H, 2· H-30 GalNAc),
3.95 (4H, 4· H-50 GalNAc), 3.85 (2H, Ha0-G1,6), 3.84 (2H,
Hd-P2,7), 3.76 (8H, 8· H-60 GalNAc), 3.69 (2H, Hd0-P2,7),
3.37 (2H, 2· H biotin), 3.24 (2H, He-K5,10), 3.23 (4H, He-
K4,9), 3.20 (4H, He-K3,8), 3.18 (2H, He0-K5,10), 3.04 (2H,
2·H biotin), 2.87 (4H, 2·CH2 levul.-K3,8), 2.83 (2H, 2· H
biotin), 2.82 (2H, CH2 levul.-K5,10), 2.71 (2H, CH2 levul.-
K5,10), 2.53 (2H, CH2 levul.-K3,8), 2.51 (4H, CH2 levul.-
K5,10), 2.48 (CH2 levul.-K3,8), 2.35 (2H, Hb-P2,7), 2.29
(4H, 4· H biotin), 2.14 (2H, Hc-P2,7), 2.10 (6H, 2· NHAc
GalNAc), 2.07 (6H, 2· NHAc GalNAc), 2.05 (2H, Hc0-
P2,7), 2.00 (6H, N@C–Me), 1.98 (2H, Hb0-P2,7), 1.94 (6H,
N@C–Me), 1.88 (4H, Hb-K4,9 and Hc-K4,9), 1.82 (4H,
Hb-K3,8, Hc-K3,8), 1.81 (4H, Hb-K5,10 and Hc-K5,10), 1.80
(4H, Hb0-K4,9 and Hc0-K4,9), 1.76 (2H, 2· H biotin), 1.67
(2H, 2· H biotin), 1.66 (4H, Hb0-K3,8 and Hc0-K3,8), 1.65
(2H, 2· H biotin), 1.63 (2H, 2· H biotin), 1.57 (4H, Hb0-
K5,10 and Hc0-K5,10), 1.55 (2H, Hd-K4,9), 1.53 (2H, Hd-
K3,8), 1.52 (2H, Hd-K5,10), 1.44 (2H, 2· H biotin), 1.42
(2H, 2· H biotin), 1.38 (2H, Hd0-K3,8), 1.37 (4H, Hd0-K4,9
and Hd0-K5,10).
6. (a) St. Hilaire, P. M.; Meldal, M. Angew. Chem., Int. Ed.
2000, 39, 1162; (b) Osborn, H. M. I.; Khan, T. H.
Tetrahedron 1999, 55, 1807.
7. (a) Love, K. R.; Seeberger, P. H. Angew. Chem., Int. Ed.
´
2002, 41, 3583; (b) Ortiz Mellet, C.; Garcıa Fernandez, J.
M. ChemBioChem 2002, 3, 819; (c) Hirabayashi, J. Trends
Biotechnol. 2003, 21, 141; (d) Jelinek, R.; Kolusheva, S.
Chem. Rev. 2004, 104, 5987; (e) Shin, I.; Park, S.; Lee, M.
Chem. Eur. J. 2005, 11, 2894.
8. (a) For selected reviews, see: Herzner, H.; Reipen, T.;
Schultz, M.; Kunz, H. Chem. Rev. 2000, 100, 4495; (b)
Seitz, O. ChemBioChem 2000, 1, 214; (c) Davis, B. G.
Chem. Rev. 2002, 102, 579.
9. Boturyn, D.; Coll, J.-L.; Garanger, E.; Favrot, M.-C.;
Dumy, P. J. Am. Chem. Soc. 2004, 126, 5730.