Synthesis and comparative lectin-binding affinity of mannosyl-coated β-cyclodextrin-dendrimer constructs

Article abstract of DOI:10.1039/b003765f

Targeted drug delivery systems have been built from β-cyclodextrin by monoconjugation with mannosyl-coated dendritic branches following an iterative thiourea-forming convergent strategy; the multivalent adducts showed high Concanavalin A lectin binding ability and intact inclusion capabilities.

Full text of DOI:10.1039/b003765f

Synthesis and comparative lectin-binding affinity of mannosyl-coated
b-cyclodextrin-dendrimer constructs†
Isabelle Baussanne,a Juan M. Benito,b Carmen Ortiz Mellet,b José M. García Fernández,*c Ho Lawa and
Jacques Defaye*a
a CNRS (UMR 5063) and Université Joseph Fourier-Grenoble 1, Departement de Pharmacochimie
Moléculaire-Glucides, BP 138, F-38243 Meylan, France. E-mail: Jacques.Defaye@ujf-grenoble.fr
b Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Aptdo. 553, E-41071 Sevilla,
Spain
c
Instituto de Investigaciones Químicas, CSIC, Américo Vespucio s/n, Isla de la Cartuja, E-41092 Sevilla, Spain.
E-mail: jogarcia@cica.es
Received (in Liverpool, UK) 8th May 2000, Accepted 15th June 2000
Published on the Web 19th July 2000
Targeted drug delivery systems have been built from b-
cyclodextrin by monoconjugation with mannosyl-coated
dendritic branches following an iterative thiourea-forming
convergent strategy; the multivalent adducts showed high
Concanavalin A lectin binding ability and intact inclusion
capabilities.
carbohydrate coating and covalent attachment to the b-CD core
through a convergent iterative methodology that exploits the
reactivity of isothiocyanate and amine functionalised mono-
Grafting biorecognisable saccharide epitopes onto suitable
molecular carriers may provide new vectors for site-specific
delivery of therapeutics. Several examples of oligosaccharide-
branched cyclodextrins (CDs) have been reported in recent
years for this purpose, taking advantage of the known ability of
CDs to include a variety of hydrophobic compounds via host-
guest complexation.1 The majority of such conjugates are
monosubstituted derivatives at a single primary hydroxy group
position of the CDs.2 A few per-(C-6)-substituted branched-
CDs have also been synthesised to comply with the need for a
multivalent presentation pattern of the saccharide markers.2a,d,3
However, although lectin-binding capacity is substantially
increased when compared with the monovalent counterparts,
polysubstitution at the primary face of CDs may seriously
impair inclusion and complex stabilisation of potential guests.
Our own results indicated approximately a 10-fold decrease in
the water solubilisation power of branched b-CDs, measured for
the anticancer drug Taxotère®, upon heptafunctionalisa-
tion.2c,d
As an attempt to combine both high biological receptor
binding ability and efficient inclusion capabilities, a series of
monosubstituted multivalent b-CD carriers have now been
synthesised. For this purpose, we have developed an efficient
preparation of dendritic wedges suitable for sequential external
Scheme 1 Reagents and conditions: i, 6-azidohexanoyl chloride, 1:1 DMF–
collidine, rt, 96%; ii, 1 1 TFA–water, rt, 2 h, 98%; iii, pyridine, 24 h, 87%;
iv, PPh3, CS2, toluene, rt, 16 h, 90%; v, 7 + 5, 1 1 water–acetone, pH 8
(NaHCO3), 55%; vi, 4, 1 1 water–acetone, pH 8 (NaHCO3), 70%.
† Dedicated to Dr R. U. Lemieux, Emeritus Professor, University of Alberta
(Edmonton, Canada), on the occasion of his 80th birthday.
DOI: 10.1039/b003765f
Chem. Commun., 2000, 1489–1490
This journal is © The Royal Society of Chemistry 2000
1489

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mers.4 The key templates are 6I-amino-6I-deoxy-b-CD (1), for
which an improved synthetic route has been recently elabo-
rated,‡5 the selectively protected 1,2,3-triaminopropane
branching element 2,6 and the isothiocyanate functionalised a-
g, 30 mmol) in water (750 mL) and NaOH (10 g, 250 mmol) in water (250
mL) were successively added. After 10 min, tosyl chloride (15 g, 79 mmol)
in acetonitrile (100 mL) was added dropwise within 1 h. The mixture was
stirred for 4.5 h, then neutralised (1 M HCl, 50 mL), the salts were filtered
2
off and the solution was concentrated by freeze-drying to of its original
3
D
-mannopyranosyl derivatives 34 and 4. The latter was prepared
volume. The crystallised solid was washed with acetone (2 3 40 mL), ether
(2 3 30 mL) and dried. After two recrystallisations from water, pure
monotosyl b-CD (6.33 g, 48%) was obtained.
§ Coupling yields 45–75%. Removal of the acetyl groups was effected by a
mixed transterification–saponification process (see ref. 2d). In the cases of
compounds 12, 13 and 15 this step was performed at 0 °C to avoid
anomerization of the external mannosylthioureido subunits (see ref. 10).
¶ All new compounds gave microanalytical, mass spectral (FAB or
MALDI-TOF) and 13C NMR data (125.7 MHz, D2O) in agreement with the
proposed structures.
Selected data for 12: [a]D +107.1 (c 0.7, H2O); 13C NMR d 182.8 (CS),
102.6–101.9 (C-1I–VII), 82.8 (C-1A), 46.3 (C-6I). For 13: [a]D +105.2 (c 1,
H2O); 13C NMR d 183.3 (CS), 177.3 (CO), 102.7–101.8 (C-1I–VII), 83.0 (C-
1A). For 14: [a]D +74.0 (c 1.1, H2O); 13C NMR d 182.2 (CS), 102.7–102.0
(C-1I–VII), 100.8 (C-1A) 46.8 (C-6I). For 15: [a]D +54.6 (c 1.4, H2O); 13C
NMR d 182.5 (CS), 177.0 (CO), 102.1–101.7 (C-1I–VII), 82.5 (C-1A). For 16:
[a]D +32.0 (c 1.1, H2O); 13C NMR d 182.9 (CS), 177.3 (CO), 102.1–101.9
(C-1I–VII), 100.9 (C-1A).
by reaction of the known tris-a-mannopyranoside amine7 with
thiophosgene. Intercalation of a six-carbon spacer has also been
considered to ensure the accessibility of the grafted bioactive
components to molecular recognition events.
Mono- and trivalent mannosylated thioureido b-CDs 12 and
14 were obtained in high yield by direct coupling of 1 with 3 and
4, respectively, followed by removal of the O-acetyl groups.§
Reaction of 2 with 6-azidohexanoyl chloride and further TFA-
catalysed hydrolysis of the Boc N-protecting groups afforded
dendron 5, which was subsequently reacted with 3 and 4 to give
the di- and hexavalent ligands 6 and 10 (Scheme 1). Aza-Wittig
type isothiocyanation reaction of the terminal azido group using
the triphenylphosphine–CS2 system led to the corresponding
bridging armed glycosyl clusters 7 and 11, which were
conjugated with monoamine 1 and deacetylated to yield the
mannosyl labelled b-CD carriers 13 and 16. The potential of the
approach was further examined by constructing the second-
generation tetravalent homologue 15 via nucleophilic addition
of diamine 5 to the divalent isothiocyanate 7 (?8), iso-
thiocyanation of the resulting adduct (?9), conjugation with
the b-CD reagent 1 and final deacetylation.¶
1 S. A. Nepogodiev and J. F. Stoddart, in Carbohydrate Chemistry, ed.
G.-J. Boons, Blackie Academic & Professional, London, 1998, p. 322;
K. A. Connors, Chem. Rev., 1997, 97, 1325.
2 (a) K. Maysuda, T. Inazu, K. Haneda, M. Mizuno, T. Yamanoi, K.
Hattori, K. Yamamoto and H. Kumagai, Bioorg. Med. Chem. Lett.,
1997, 7, 2353; (b) R. Kassab, C. Felix, H. Parrot-Lopez and R. Bonaly,
Tetrahedron Lett., 1997, 38, 7555; (c) J. M. García Fernández, C. Ortiz
Mellet, S Maciejewski and J. Defaye, Chem. Commun., 1996, 2741; (d)
C. Ortiz Mellet, J. M. Benito, J. M. García Fernández, H. Law, K.
Chmurski, J. Defaye, M. L. O’Sullivan and H. N. Caro, Chem. Eur. J.,
1998, 4, 2523.
3 J. J. García-López, F. Santoyo-González, A. Vargas-Berenguel and J. J.
Giménez-Martínez, Chem Eur. J., 1999, 5, 1775.
4 J. M. García Fernández and C. Ortiz Mellet, Adv. Carbohydr. Chem.
Biochem., 2000, 55, 35.
Comparative protein-affinity evaluation of the mannosyl-
coated thioureido b-CDs 12–16 towards horseradish perox-
idase-labelled concanavalin A (Con A) was effected by
performing the enzyme-linked lectin assay (ELLA) test.8 The
corresponding IC50 values for inhibition of Con A-yeast
mannan binding9 reflected the expected amplification of lectin-
binding strength for the higher-valent representatives. Nonethe-
less, preliminary Taxotère® solubilisation experiments in water
showed solubility values (e.g. 4.5 g L21 in a 50 mM solution of
14) that were similar to those obtained for monobranched
CDs.
5 J. Defaye, S. Crouzy, N. Evrard and H. Law, PCT Int. Appl. WO 99
61,483; Chem. Abstr., 2000, 132, 24077a.
6 E. Benoist, A. Loussouarn, P. Remaud, J.-C. Chatal and J.-F. Gestin,
Synthesis, 1998, 1113.
This research was supported by the European Comission DG
XII under the FAIR programme (contract no. FAIR CT95-
0300) and the DGICYT (grant no. PB 97/0747).
7 P. R. Ashton, E. F. Hounsell, N. Jayaraman, T. M. Nilsen, N. Spencer,
J. F. Stoddart and M. Young, J. Org. Chem., 1998, 63, 3429.
8 M.-C. Shao and C. C. Q. Chin, Methods Enzymol., 1994, 247, 253.
9 Binding inhibition of horseradish peroxidase-labeled Con A to yeast
mannan by mannosylated monosubstituted b-CDs (IC50 values): 12, 800
mM; 13, 780 mM; 14, 91 mM; 15, 110 mM; 16, 8 mM.
Notes and references
‡ A much better yield for the key precursor of 1, namely the corresponding
C-6 monotosyl derivative, was achieved by using the following procedure:
To a solution of b-CD (11.35 g, 10 mmol) in water (500 mL), CuSO4 (7.5
10 J. M. Benito, C. Ortiz Mellet, K. Sadalapure, T. K. Lindhorst, J. Defaye
and J. M. García Fernández, Carbohydr. Res., 1999, 320, 37.
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Chem. Commun., 2000, 1489–1490