A new strategy for the solid-phase synthesis of glycoconjugate biomolecules

Article abstract of DOI:10.1055/s-2001-11391

A simple and efficient bidirectional solid-phase synthesis, based on the use of a Tentagel solid support, functionalized with a suitably protected 2-amino sugar residue, is proposed for the preparation of a variety of glycoconjugates, including glycopeptides and nucleo-glycopeptides.

Full text of DOI:10.1055/s-2001-11391

LETTER
341
A New Strategy for the Solid-Phase Synthesis of Glycoconjugate Biomolecules
Giovanni Di Fabio,a Antonia De Capua,b Lorenzo De Napoli,*a Daniela Montesarchio,a Gennaro Piccialli,c Filomena
Rossi,b Ettore Benedettib
aDipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli “Federico II”, Complesso Universitario di Monte S. An-
gelo, via Cinthia 4, 80126 Napoli, Italy
bCentro di Studio di Biocristallografia, Dipartimento di Chimica, Via Mezzocannone 4, 80134 Napoli, Italy
cUniversità del Molise, Facoltà di Scienze, Via Mazzini 8, 86170 Isernia, Italy
Fax +39(081)674393; E-mail: denapoli@cds.unina.it
Received 5 December 2000
tionalized support 4. This support contains an intact ami-
Abstract: A simple and efficient bidirectional solid-phase synthe-
nocarbohydrate epitope bearing orthogonally removable
sis, based on the use of a Tentagel solid support, functionalized with
protecting groups [4,4’-dimethoxytrityl (DMT) and
a suitably protected 2-amino sugar residue, is proposed for the prep-
aration of a variety of glycoconjugates, including glycopeptides and
Fmoc] on the sugar,13 thus allowing the bidirectional elon-
nucleo-glycopeptides.
gation of different kinds of chimeric biomolecules. For
example, it can form stable amide bonds with carboxyl-
containing molecules exploiting the previously deprotect-
ed 2-NH2; furthermore, after DMT removal, the 6-OH
function can be used to link oligosaccharide or oligonu-
cleotide chains by forming either glycosidic or phospho-
diester bonds, respectively.
Key words: conjugation, glycosylation, glycopeptides, oligonucle-
otides, solid-phase synthesis
Since the biomedical potential of oligosaccharides and re-
lated glycoconjugates has been well recognized1-5 the de-
velopment of effective methodologies for their rapid
assembly is attracting current attention. Glycoconjugates
are among the most functionally and structurally diverse
molecules in nature and it is now well established that pro-
tein- and lipid-bound saccharides play essential roles in
many molecular processes impacting eukaryotic biology
and disease. The presence of a sugar moiety is believed to
influence the transport of the drug through biological
membranes as well as to increase its resistance to hydro-
lytic enzymes.
In our synthetic strategy a suitable methyl- -D-glu-
cosamine derivative 214 was anchored to the solid support
through a succinic bridge, involving the 3-hydroxyl group
of the sugar residue and the native amino function of the
solid support (Tentagel-NH2 resin, 0.23 meq/g). Com-
pound 2 was synthesized starting from methyl-2-amino-2-
deoxy- -D-glucopyranoside 115 which was selectively
protected at the 2-amino and 6-hydroxy functions by treat-
ment with 9-fluorenylmethyl-N-succinimidyl carbonate
and DMTCl, respectively, and successively 3-O-succinyl-
ated by reaction with succinic anhydride (Scheme 1). In-
corporation of 2 in the solid matrix was achieved using
DCC/HOBt in CH2Cl2/DMF solution giving sugar deriva-
tized support 3; capping of the 4-OH function and of un-
reacted amino groups on the polymer, performed by
standard acetic anhydride treatment, gave support 4 (0.17
meq/g by DMT cation quantitation).
The synthesis and modification of oligosaccharide units
and their coupling with appropriate lipids, phospholipids,
oligonucleotides and proteins are essential to extend the
knowledge on the molecular mode of action of glycocon-
jugates and, furthermore, to get a further insight into their
physiological activity. In this context the polymer sup-
ported approach is particularly promising in principle. A
large number of studies on the solid-phase synthesis of
glycopeptides have been reported6,7 and since the polymer
supported synthesis of peptides is a well established pro-
cess,8 the majority of progress in the synthesis of glyco-
peptides has involved the linkage of the glycopeptide to
the polymer support via the peptide moiety rather than via
the carbohydrate moiety. Recently several papers have
been reported in the literature dealing with the synthesis
of glyco-oligonucleotide conjugates in which the sugar
residue is either attached to the nucleotide base before the
oligonucleotide assembly,9 or used as separate building
block in an “on line” solid phase strategy.10-12
To test the efficiency of support 4 in the synthesis of gly-
coconjugated peptides, we first prepared hybrid molecule
6 (Scheme 2). 2-Amino function in 4 was deprotected and
reacted with Fmoc-Leu-OH in the presence of DCC/
HOBt in CH2Cl2/DMF, following a standard Fmoc proto-
col,16 affording 5. After Fmoc removal and acetylation of
the Leu-NH2 group, detachment from the support was
achieved by treatment with conc. aq. ammonia solution (6
h, 50 °C) giving gluco-leucine derivative 6. TLC and
HPLC analyses of the detached crude material showed in
all cases the presence of 6 as a sole product, obtained in
1
97% after purification, which was characterized by H,
13C NMR and FAB-MS data. These results indicated a
high reactivity of the 2-amino function in the amide bond
formation and the resistance of the succinic bridge to the
treatment required for Fmoc removal.17
In an effort to develop a new and versatile approach to car-
bohydrate mimetics and glycoconjugate synthesis, we re-
port herein a general synthetic scheme to obtain a variety
of glycoconjugates by exploiting new amino sugar func-
Synlett 2001 No. 3, 341–344 ISSN 0936-5214 © Thieme Stuttgart · New York

342
G. Di Fabio et al.
LETTER
HO
DMTO
HO
DMTO
RO
O
O
O
i
ii
O
HO
HO
-
O
O
O
NH3
+
Cl
Fmoc NH
Fmoc NH
HO
OMe
OMe
OMe
O
1
2
S
O
N
H
3 R = H
iii
4 R = Ac
Scheme 1 Reagents and conditions: i. (a) FmocOSuccinimidyl carbonate (0.95 eq.), 9% K2CO3, H2O/DMF (1:1, v/v), 0 °C - r.t., 2 h, 85%,
(b) DMTCl (1.2 eq.), pyridine, 90% (c) succinic anhydride (3 eq.), pyridine, r.t., 4 h, 85%; ii.
(10 eq.) in CH2Cl2/DMF (1:1, v/v), r.t., 7h; iii. Ac2O/pyridine (1:1, v/v), r.t., 0.5 h.
(0.23 meq/g), DCC (10 eq.), HOBt
S
NH
2
DMTO
AcO
DMTO
O
O
O
i
HO
HO
ii
O
NH
NH
HN
S
OMe
OMe
O
O
O
FmocHN
AcHN
6 (97%)
5
HO
O
a. 20% piperidine in DMF
b. Fmoc Amino acid
+ HOBt + DCC
HO
HO
NH
OMe
4
O
iii
N
H
Phe -Gly -Gly -Tyr -H
7 (85%)
NH2
N
O
HO
O
O
N
HO
P
O
iv or v
O
O
8 (95%)
HO
HO
AcHN
OMe
NH2
N
N
HO
N
O
O
N
iv
HO
P
5
O
O
O
HO
HO
9 (93%)
HN
OMe
O
AcHN
Scheme 2 Reagents and conditions: i. (a) piperidine/DMF (1:4, v/v); (b) Fmoc-Leu-OH (10 eq.), DCC (10 eq.), HOBt (10 eq.) in CH2Cl2/
DMF (1:1, v/v), r.t., 7 h; ii. (a) piperidine/DMF (1:4, v/v), (b) Ac2O/pyridine (1:1, v/v), r.t., 0.5 h; (c) conc. NH4OH, 50 °C, 6 h; iii. (a) 2% DCA
in CH2Cl2 (w/w), (b) conc. NH4OH, 50 °C, 6 h; iv. (a) piperidine/DMF (1:4, v/v), (b) Ac2O/pyridine, (c) 2% DCA in CH2Cl2 (w/w), (d) com-
plete coupling cycle with dC (for 8) and dA-phosphoramidite (for 9) and final detritylation on an automated DNA synthesizer, (e) conc.
NH4OH, 50 °C, 6 h; v. (a) 2% DCA in CH2Cl2 (w/w), (b) complete coupling cycle with dC-phosphoramidite and final detritylation on an au-
tomated DNA synthesizer; (c) piperidine/DMF (1:4, v/v), (d) Ac2O/pyridine, (e) conc. NH4OH, 50 °C, 6 h.
Synlett 2001, No. 3, 341–344 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
A New Strategy for the Solid-Phase Synthesis of Glycoconjugate Biomolecules
343
We then used support 4 in the automated solid phase syn- co-oligonucleotides, as well as other yet unexplored
thesis of new glycosylated enkephalin18 derivative 7 classes of chimeric molecules. Further studies are current-
which was assembled following a standard Fmoc proto- ly in progress to extend this methodology to the solid
col. The peptide chain was prepared using DCC/HOBt as phase synthesis of new glycomimetics such as carbonu-
coupling agents, with each monomer addition being mon- cleotoids and carbopeptoids.21
itored by Kaiser test.19 Releasing of fully deprotected gly-
copeptide 7 from the resin was realised by aq. ammonia
treatment as for 6. The crude detached material was ana-
Acknowledgement
We thank MURST, Università degli Studi di Napoli “Federico II”
(Progetto Giovani Ricercatori), CNR and Regione Campania (Leg-
ge 41) for grants in support of this investigation and C.I.M.C.F.,
Università di Napoli “Federico II”, for the NMR facilities.
lysed and purified by reverse phase HPLC and the identity
and purity of 7, isolated in 85% yield, were ascertained by
1H NMR and MS data.
Support 4 was then employed in the synthesis of glyconu-
cleotide 8 and nucleo-glyco-amino acid 9. Compound 8
was prepared performing on 4 one coupling cycle with 5’-
O-(4,4’-dimethoxytrityl)-N-4-benzoyl-2’-deoxycytidine-
3’-O-(2-cyanoethyl)-phosphoramidite (dC phosphor-
amidite, 40mg/mL) in the presence of 0.1 M tetrazole in
anhydrous CH3CN, followed by a standard oxidative I2/
Py/THF treatment.20 The coupling yield was almost quan-
titative as checked by quantitation of the DMT cation re-
leased from the nucleotide 5’-OH function in the final
detritylation step with 2% dichloroacetic acid (DCA) in
CH2Cl2 solution. After piperidine treatment and acetyl-
ation, the cleavage and deprotection of 8 were carried out
as for 6.
References and Notes
(1) Kobata, A. Acc. Chem. Res. 1993, 26, 319.
(2) Varki, A. Glycobiology 1993, 3, 97.
(3) Dwek, R.A. Chem. Rev. 1996, 96, 683.
(4) Rudd, P.M.; Wormald, M.R.; Stanfield, R.L.; Huang, M.;
Mattsson, N.; Speir, J.A.; Di Gennaro, J.A.; Fetrow, J.S.;
Dwek, R.A.; Wilson, I.A. J. Mol. Biol. 1999, 293, 351.
(5) Ernst, B.; Hart, G.W.; Sinay, P. Carbohydrates in Chemistry,
Wiley-VCH: Weinheim, 2000.
(6) Osborn, H.M.I.; Khan, T.H. Tetrahedron 1999, 55, 1807.
(7) Hojo, H.; Nakahara, Y. Current Protein and Peptide Science
2000, 1, 23.
(8) Kunz, H. Pure and Appl. Chem. 1993, 65, 1223.
(9) de Kort, M.; Ebrahimi, E.; Wijsman, E.R.; van der Marel,
G.A.; van Boom, J. H. Eur. J. Org. Chem. 1999, 2337.
(10) Akhtar, S.; Routledge, A.; Patel, R.; Gardiner, J.M.
Tetrahedron Lett. 1995, 36, 7333.
In order to demonstrate the orthogonality of DMT and
Fmoc protecting groups in support 4, compound 8 was ob-
tained from 4 also following an alternative route, which
gave absolutely comparable yields. First the 6-OH func-
tion in 4 was deprotected, coupled with the dC phosphor-
amidite building block, as above reported, followed by
removal of the 5’-DMT group, acetylation, final deprotec-
tion and detachment from the support. Analysis of the
crude detached material in both strategies leading to 8 al-
ways showed the presence of a sole product, identified by
1H NMR and MS data. The absence of side products
proved also that in no case acetyl migration occurred after
DMT removal in 4.
(11) Adinolfi, M.; Barone, G.; De Napoli, L.; Iadonisi, A.;
Piccialli, G. Tetrahedron Lett. 1998, 39, 1953.
(12) Adinolfi, M.; Barone, G.; De Napoli, L.; Guariniello, L.;
Iadonisi, A.; Piccialli, G. Tetrahedron Lett. 1999, 40, 2607.
(13) Many recent efforts have been devoted to the design and
synthesis of aminosaccharide scaffolds having orthogonally
removable protecting groups; for example see: Sofia, M.J.;
Hunter, R.; Chan, T.Y.; Vaughan, A.; Dulina, R.; Wang, H.;
Gange, D. J. Org. Chem. 1998, 63, 2802.
(14) Selected data for 2, 6-9: 2 mp 137-141 °C (C6H6-CH3OH); Rf
0.60 (CHCl3 / CH3OH 95:5); [ ]D+30.0 (c 0.1, CH3OH);
H (400 MHz, CDCl3) 7.76 – 6.80 (21H, complex signals,
aromatic DMT and Fmoc protons) 5.36 (1H, d, NH) 5.15 (1H,
t, H-3) 4.76 (1H, d, J1,2 3.0, H-2) 4.41 (1H, t, CH-CH2 Fmoc)
4.39-4.04 (4H, complex signals, H-2, H-4, CH-CH2 Fmoc)
3.99 (1H, m, H-5) 3.68 (6H, s, 2 OCH3 DMT) 3.41 (3H, s,
1-OCH3) 3.38 (2H, m, H2-6) 2.57 (4H, m, succinic protons);
HRMS (FAB+): calc. for C47H48NO12+, 818.3177; found
818.3198;
The synthesis of hybrid trimer 9 was accomplished start-
ing from functionalized support 5 which, after deprotec-
tion and acetylation of the Leu-NH2 function, was
subjected, in the DNA synthesizer, to the coupling
with 5’-O-(4,4’-dimethoxytrityl)-N-6-benzoyl-2’-deoxy-
adenosine-3’-O-(2-cyanoethyl)-phosphoramidite
(dA
6: Rf 0.75 (CHCl3 / CH3OH 9:1); [ ]D+5.0 (c 0.08, CHCl3);
H (400 MHz, CD3OD) 7.50 – 6.80 (13H, complex signals,
aromatic DMT protons) 4.73 (1H, d, J1,2 3.3, H-1) 4.45 (1H,
dd, CH Leu) 3.92 (1H, dd, H-2) 3.80 (6H, s, 2 OCH3 DMT)
3.76, 3.63, 3.22 (1H each, m’s, H-3, H-4, H-5) 3.46 (2H,
further coupled AB system, partially submerged H2-6) 3.45
(3H, s, 1-OCH3) 2.01 (3H, s, CH3CO) 1.68 (1H, m, CH side
chain Leu) 1.60 (2H, m, CH2 side chain Leu) 0.94 and 0.97
(3H each, d’s, 2 CH3 side chain Leu); HRMS (FAB+): calc. for
phosphoramidite, 40 mg/mL) in the presence of 0.1 M tet-
razole in anhydrous CH3CN, followed by a standard oxi-
dative I2/Py/THF treatment.20 Final DMT removal was
achieved by standard DCA treatment. Addition of conc.
aq. ammonia gave deprotected 9. Products 8 and 9 were
purified by reverse phase HPLC (95 and 93% recovery
yields, respectively) and their structures confirmed by 1H
NMR and FAB MS data.
+
C36H47N2O9 , 651.3282; found 651.3306.
In conclusion, we have reported the synthesis of a new
sugar-functionalized solid support, characterized by the
presence of orthogonally protected amino and hydroxy
functions, which allow the facile and high yielding prepa-
ration of glycoconjugates such as glycopeptides and gly-
7: analysis by HPLC Rt. 24.5 min (Nucleosil NH, 250 5 mm,
5 m), gradient 5% B in A for 5 min. then from 5% to 30% B
in A in 30 min., solvents: A 0.1% TFA in H2O, B 0.1% TFA
in CH3CN, 210 and 260 nm, flow 1 mLmin-1; [ ]D+10.5 (c
0.27, CH3OH); H (400 MHz, D2O) significative signals at
7.38 – 7.28 (5 H, m’s, aromatic Ph protons) 7.11 and 6.28 (2H
Synlett 2001, No. 3, 341–344 ISSN 0936-5214 © Thieme Stuttgart · New York

344
G. Di Fabio et al.
LETTER
Leu); HRMS (FAB+): calc. for C25H41N7O12P+ 662.2551;
each, d’s, aromatic Tyr protons) 4.67 (1H, d, H-1) 3.37 (3H, s,
1-OCH3 glucose) 3.10 (2H, d, H2-6 glucose) 2.96 (1H, dd, H-
5 glucose) 0.80 and 0.85 (3H, each, d’s, 2 CH3 side chain Leu);
MS (MALDI-TOF): m/z 731.5 (M+H)+ 753.4 (M+Na)+ 769.4
(M+K)+.
8: Rf 0.5 (1-butanol / acetic acid / water 65:15:25); [ ]D+20.9
(c 0.1, CH3OH); H (400 MHz, CD3OD) 7.86 (1H, d, H-6 dC)
6.28 (1H, dd, H-1’ dC) 5.97 (1H, d, J 7.4, H-5 dC) 4.61 (1H,
d, J1,2 3.4, H-1 glucose) 4.20 (1H, m, H-4’ dC) 4.10 (2H, m,
H2-6 glucose) 3.99, 3.89 (1H each, m’s, H-2 and H-4 glucose)
3.61, 3.50 (1H each, m’s, H-3 and H-5 glucose) 3.37 (3H, s,
1-OCH3) 3.39 (2H, m, partially submerged, H2-5’ dC) 2.62,
2.30 (1H each, m’s, Ha-2’ and Hb-2’ dC) 1.97 (3H, s, CH3CO);
HRMS (FAB+): calc. for C18H30N4O12P+ 525.1598; found
525.1618.
9: analysis by HPLC Rt. 19.1 min (Nucleosil NH, 250 5 mm,
5 m) gradient from 0 to 100% B in A in 90 min., solvents: A
H2O, B CH3CN, 260 nm, flow 1 mLmin-1; [ ]D+6.25 (c 0.03,
CH3OH); H (400 MHz, CD3OD) 8.34 (1H, s, H-2 dA) 8.18
(1H, s, H-8 dA) 6.45 (1H, dd, H-1’ dA) 5.04 (1H, m, H-3’ dA)
4.62 (1H, d, J 3.5, H-1 glucose) 4.42 (1H, m, CH Leu) 4.36
(1H, m, H-4’ dA) 4.13 (2H, m, H2-6 glucose) 3.85 (1H, d, H-
2 glucose) 3.85 (1H, m, H-3 glucose) 3.65 (1H, m, H-4
glucose) 3.47 (2H, m, H2-5’ dA) 3.34 (3H, s, 1-OCH3 glucose)
3.31 (1H, m, submerged by the residual solvent, H-5 glucose)
2.87 and 1.97 (1H each, m’s, Ha-2’ and Hb-2’ dA) 1.97 (3H, s,
CH3CO) 1.67 (1H, m, CH side chain Leu) 1.58 (2H, m, CH2
side chain Leu) 0.95 and 0.90 (6H each, d’s, 2 CH3 side chain
found 662.2580.
(15) Takeda, R.; Ryu, S.Y.; Park, J.H.; Nakanishi, K. Tetrahedron
1990, 46, 5533.
(16) Atherton, E.; Sheppard, R.C. Eds., Solid Phase Peptide
Synthesis: A Practical Approach., IRL Press: Oxford, 1991.
(17) Other succinate linkers have been previously reported in the
literature to attach a glucose moiety to polymeric supports; see
for example: Douglas, S.P.; Whitfield D.M.; Krepinsky, J.J. J.
Am. Chem. Soc. 1991, 113, 5095; Adinolfi, M.; Barone, G.;
De Napoli, L.; Iadonisi, A.; Piccialli, G. Tetrahedron Lett.
1996, 37, 5007; Zhu, T.; Boons, G.J. Angew. Chem., Int. Ed.
Engl. 1998, 37, 1898.
(18) For example see: Varga-Defterdarovic, L.; Horvat, S.;
Schiller, P.W. Int. J. Pept. Protein Res. 1992, 39, 12; Horvat,
S.; Horvat, J.; Varga-Defterdarovic, L.; Pavelic, K.; Chung,
N.N.; Schiller, P.W. Int. J. Pept. Protein Res. 1993, 41, 399.
(19) Kaiser, E; Colescott, R.L.; Bossinger, C.D.; Cook, P.I. Anal.
Biochem., 1970, 34, 595.
(20) Eckstein, F. Ed. Oligonucleotides and Analogues: A Practical
Approach, IRL Press: Oxford, 1991.
(21) Nicolaou, K.C.; Flörke, H.; Egan, M.G.; Barth, T.; Estevez,
V.A. Tetrahedron Lett. 1995, 36, 1775.
Article Identifier:
1437-2096,E;2001,0,03,0341,0344,ftx,en;G24500ST.pdf
Synlett 2001, No. 3, 341–344 ISSN 0936-5214 © Thieme Stuttgart · New York