Multivalent neoglycoconjugates: Solid-phase synthesis of N-linked α-sialodendrimers

Article abstract of DOI:10.1039/a703679e

A series of N-linked α-sialodendrimers with valencies of 2, 4 and 8 has been scaffolded on an N,N-bis(3-aminopropyl)succinamic acid core using Wang resin and 9-fluorenylmethoxycarbonyl and 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluronium tetrafluorobora

Full text of DOI:10.1039/a703679e

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Multivalent neoglycoconjugates: solid-phase synthesis of N-linked
a-sialodendrimers
Muriel Llinares and Rene´ Roy*
Department of Chemistry, University of Ottawa, Ottawa, ON, K1N 6N5 Canada
A series of N-linked a-sialodendrimers with valencies of 2, 4
and has been scaffolded on an N,N-bis(3-
aminopropyl)succinamic acid core using Wang resin and
9-fluorenylmethoxycarbonyl and 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate solid-phase
chemistry.
phase synthesis of a new family of symmetrical N-linked
sialodendrimers based on N,N-bis(3-aminopropyl)succinamic
acid 4 core is described.
8
In order to provide a strategy which could be used with other
carbohydrate ligands, a divergent approach was used to build
these novel poly(amidoamine) dendrimers on a solid-phase.
Suitably acid-functionalized carbohydrate derivatives could
then be added onto polyamino dendrimers to afford glycoden-
drimes with even valencies. The dendritic core was prepared as
follows (Scheme 1). The primary amine groups of
N-(3-aminopropyl)propane-1,3-diamine 1 were regioselec-
tively protected with benzyloxycarbonyl (Z) groups using
benzyl cyanoformate in CH2Cl214 to give secondary amine 2 in
72% yield. Acylation of the residual amine with succinic
anhydride (triethylamine, CH2Cl2, 96%) provided key building
block 3 having a divalent structure with a bis-Z-protected
monoacid core. In order to use the Fmoc strategy, the
Z-protecting groups were removed by catalytic hydrogenation
(10% Pd–C, MeOH) to give 4, which was then treated with
9-fluorenylmethyl chloroformate (FmocCl, 10% Na2CO3–
dioxane, 0 °C) to afford 5† in 72% yield. The dendritic
polyamine scaffolds were built on a b-alanine spacer attached to
Wang resin [4-(hydroxymethyl)phenoxymethyl–co-poly(sty-
rene–1% divinylbenzene), 0.58 mmol g21], using the Fmoc-
strategy and performing the deprotection–coupling cycles as
follow: (i) Fmoc deprotection by treatment with 20% piperidine
in DMF (1 3 5 min, 1 3 15 min), (ii) coupling of Fmoc–
protected acid 5 (2 equiv.) using 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (2
equiv.) as activating agent and Pri2EtN (4 equiv.) in DMF
during 30 min. Coupling completion was determined by a
ninhydrin test for residual amino groups.15 To characterize the
dendrimers, 30 mg of resin from each sequential generation was
Fmoc-deprotected and the dendrimers were released from the
solid support following conventional acid treatment (95% aq.
TFA, 2 h, 30–79% yield).
Dendrimers are attractive monodispersed macromolecules with
broad spectrum applications from physics to medicine.1 The
design of novel dendrimers represents an active research area
and their syntheses are considered challenging owing to their
size and highly branched structures. Traditionally, divergent2
and convergent3 strategies have been used to synthesize
dendrimers. However, more recently a double exponential
growth strategy4 and a self-assembling method5 have been
developed. To date, the synthesis of most dendrimers has been
carried out in solution, although Tam6 has built hyperbranched
l-lysine dendrimers on a solid-phase for multiple antigen
peptide (MAP) presentation. Because of the critical role of
carbohydrate-containing macromolecules in many cellular
proceses, glycodendrimers have also become a growing re-
search area.7 There is increasing evidence that the multivalent
effect displayed by these novel biopolymers may overcome the
characteristic low-affinity binding interactions between carbo-
hydrate ligands and proteins.8,9
Potent l-lysine-based dendritic a-thiosialoside inhibitors of
Influenza virus’s hemagglutination to human erythrocytes have
been previously described.10 Similarly, different series of
symmetrical dendrimers containing N-acetylneuraminic acid
(NeuAc or sialic acid), representing one of the most widespread
mammalian cell surface carbohydrate ligands,11 have been
successfully synthesized in solution.12,13 Therefore, it was
attractive to build a new series of sialodendrimers on a solid-
phase. The present approach differs from previous S-linked
sialosides10,11 by the use of N-linked sialylated derivatives
which are also expected to be sialidase resistant. Thus, the solid-
Glycodendrimer synthesis required sialic acid residues
suitably functionalized at the anomeric position for coupling
with amine-terminated dendrimers. The readily available
a-sialic acid azide derivative 816 was reduced by catalytic
hydrogenation (10% Pd–C, MeOH) to provide a-sialic acid
amine in quantitative yield (Scheme 2). To minimize anomeri-
sation, the next acylation step was performed within 1 h. To
RHN
N
R′
RHN
1 R = R′ = H
i
ii
2 R = Z, R′ = H
3 R = Z, R′ = COCH2CH2CO2H
4 R = H, R′ = COCH2CH2CO2H
5 R = Fmoc, R′ = COCH2CH2CO2H
iii
iv
OAc
CO2Me
OAc
AcO
O
N3
O
RHN
RHN
AcHN
NH
N
AcO
8
O
O
i, ii
6 R = Fmoc
7 R = H
vi
OAc
CO2Me
OAc
AcHN
AcO
= Wang resin
NHCO(CH2)8COX
O
AcO
Scheme 1 Reagents and conditions: i, BnO2CCN, CH2Cl2, 2 h, 72%; ii,
succinic anhydride, Et3N, CH2Cl2, 1 h, 96%; iii, H2, 10% Pd–C, MeOH, 2
h, quant.; iv, FmocCl, 10% Na2CO3–dioxane, 12 h, 72%; v, H-bAla-Wang
resin, TBTU, Pri2EtN, DMF, 30 min; vi, 20% piperidine–DMF (1 3 5 min,
1 3 15 min)
9 X = Cl
10 X = OH
iii
Scheme 2 Reagents and conditions: i, H2, 10% Pd–C, MeOH, 1 h, quant.;
ii, ClCO(CH2)8COCl, Pri2EtN, CH2Cl2, 3 h; iii, H2O, 53% overall yield
Chem. Commun., 1997
2119

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R1O
O
OR1
OR1
Fmoc-HN
CO2R2
AcNH
R1O
O
O
HN
R1O
O
OR1
OR1
(
)
8
i–iv
HN
CO2R2
NH
AcNH
R1O
O
HN
N
O
(
)
8
OR1
OR1
R1O
O
O
O
CO2R2
O
O
NH
AcNH
O
R1O
O
NH
(
)
N
8
HN
HN
NH
OR1
O
N
CO2R2
NH
OR1
R1O
O
O
11, 14
(
)
8
HN
O
AcNH
OR1
O
O
O
OR1
O
N
NH
O
HN
O
CO2R2
OR1
O
O
O
N
N
R1O
O
12, 15
O
(
)
NH
HN
AcNH
N
8
NH
OR1
O
HN
HN
CO2R2
NH
(
)
R1O
8
OR1
AcNH
O
R1O
O
13, 16
O
O
O
11, 12, 13 R1 = Ac, R2 = Me
OR1
O
(
N
O
= Wang resin
NH
CO2R2
R1O
)
OR1
v–vii
8
NH
R1O
O
HN
AcNH
OR1
R1O
14 (Dimer), 15 (Tetramer), 16 (Octamer)
R1 = R2 = H,
= OH
(
)
O
CO2R2
NH
O
8
OR1
O
R1O
AcNH
OR1
Scheme 3 Reagents and conditions: i, 20% piperidine–DMF (1 3 5 min, 1 3 15 min); ii, 5, TBTU, Pri2EtN, DMF, 30 min; iii, repeat cycle or 20% piperidine–
DMF; iv, 10, TBTU, Pri2EtN, DMF, 0.5–8 h; v, 95% aq. TFA, 2 h; vi, 1 m NaOMe, MeOH, 2–8 h; vii, 0.05 m NaOH, 2–8 h
g-CH2s), 1.87 (s, 3 H, NAc), 2.07 (m, 1 H, H-3ax), 2.74 (m, 1 H, H-3eq), 3.74
offer suitable carbohydrate accessibility, introduction of a
(s, 3 H, OCH3), 5.35 (dd, 1 H, J6,7 2.2, J7,8 6.1, H-7); dC 23.14 (NAc), 24.58
(g-C), 52.9 (OCH3), 83.3 (C-2); FAB-MS (positive): calc. for
spacer arm between the sialic acid residues and the dendrimer
was carried out using excess ClCO(CH2)8COCl (CH2Cl2,
C30H46N2O15: 674.7. Found: 675.4 (M + 1). For 14: dH(D2O) 1.62 (m, b-,
Pri2EtN, 0 °C) to give unstable monoacid chloride 9 which was
g-CH2s), 1.84 (m, 6 H, b-CH2, H-3ax), 2.08 (s, 6 H, NAc), 2.44 (m, 2 H,
immediately hydrolysed to afford 10 in 53% overall yield.
Attempts to isolate 9 for subsequent attachment to the resin
b-alanyl a-CH2), 2.73 [m, 4 H, succinyl CH2C(O)N, H-3eq], 4.16 (d, 2 H,
J 9.9, H-7); dC 21.5 (NAc), 24.5 (g-C), 26 and 27 (b-Cs), 36.2 (g-Cs, b
were unsuccessful. a-Sialic acid derivative 10 was then ready to
be coupled to the dendritic polyamine core on the solid-phase
using the same TBTU coupling strategy described to scaffold
the dendrimer generations. After coupling, peracetylated sialo-
dendrimers with the valencies of 2, 4 and 8 (11, 12 and 13) were
released from the resin by treatment with 95% TFA. The
sialodendrimers were then deprotected by sequential ester
hydrolysis [(i) de-O-acetylation with 1 m NaOMe in MeOH, (ii)
0.005 m NaOH]. Purification of each independent generation by
size exclusion chromatography over Biogel-P2 (H2O as eluent)
provided pure deprotected glycodendrimers 14, 15 and 16 in
moderate yields (25–56%) (Scheme 3). The purity of each
compound was readily established from the relative integration
alanyl a-C and b-C), 72.4 (C-7), 84.4 (C-2); FAB-MS (positive): calc. for
C55H94N8O24: 1250.64. Found: 1251.5 (M+ + 1, 1%); MALDI-TOF
(negative): Found: 1250.03 (M 2 1)2. For 15: MALDI-TOF (negative):
calc. for C117H200N18O48: 2625.37. Found: 2626 (M 2 1)2. For 16: dH
(D2O) 1.86 (m, 28 H, b-CH2s), 2.11 (s, 24 H, NAc), 4.18 (m, 8 H, H-7); dC
21.57 (NAc), 26.2 (b-Cs), 72.5 (C-7), 85.2 (C-2); MALDI-TOF (negative)
calc. for C241H412N38O96: 5,374.85. Found: 5374 (M 2 1)2.
1 D. Astruc, C. R. Acad. Sci. Paris, 1996, 322, 757.
2 D. A. Tomalia and H. D. Durst, Top. Curr. Chem., 1993, 165, 193.
3 C. J. Hawker and J. M. J. Fre´chet, J. Am. Chem. Soc., 1990, 112,
7638.
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6 J. P. Tam, Proc. Natl. Acad. Sci. USA, 1988, 85, 5409.
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187, 241; K. Aoi, K. Itoh and M. Okada, Macromolecules, 1995, 28,
5391, and references therein.
8 E. J. Toone, Curr. Opin. Struct. Biol., 1994, 4, 719.
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therein.
10 R. Roy, D. Zanini, S. J. Meunier and A. Romanoska, J. Chem. Soc.,
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1
of key signals in the H NMR spectrum. MALDI-TOF mass
spectral data (negative mode) further confirmed the integrity of
the dendrimers. Biological properties of these novel N-linked
sialodendrimers will be reported in due course.
We thank Qing Quan Wu and Denis Carrie`re for providing
a-sialic acid azide derivative 8. We are also grateful to Drs P.
Thibault and D. Krajcarski from NRC, Ottawa, for running the
MALDI-TOF experiments on a Perspective Elite-STR instru-
ments.
11 J. C. Paulson, in The Receptors, ed. M. Conn, Academic Press, Orlando,
1985, vol. 2, p. 131.
Footnotes and References
12 D. Zanini and R. Roy, J. Org. Chem., 1996, 61, 7348.
13 D. Zanini and R. Roy, J. Am. Chem. Soc., 1997, 119, 2088.
14 S.-I. Murahashi, T. Naota and N. Nakajima, Chem. Lett., 1987, 879.
15 E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook, Anal.
Biochem., 1971, 34, 595.
* E-mail: rroy@science.uottawa.ca
† All compounds showed consistent NMR and mass spectral data. Because
of dendrimers’ repetitive structures, only selected data are reported.
Selected data for 5: dH([2H6]acetone) 1.69 and 1.91 (2 m, 4 H, b-CH2), 2.6
[m, 2 H, succinyl CH2C(O)OH], 4.3 (dd, 4 H, Fmoc-CH2s); dC 28.7 and
30.5 (b-C), 29.8 [succinyl CH2C(O)OH]; FAB-MS (positive): calc. for
16 F. Tropper, F. O. Andersson, S. Braun and R. Roy, Synthesis, 1992,
618.
C40H41N3O7: 675.7. Found: 676.3 (M + 1). For 10 (spacer identification
from anomeric position to acid is a to h): dH(CDCl3) 1.59 (m, 4 H, b-,
Received in Corvallis, OR, USA, 27th May 1997; 7/03679E
2120
Chem. Commun., 1997