Synthesis and Biological Evaluation of α-D-Mannopyranoside-Containing Dendrimers

Article abstract of DOI:10.1021/jo9804184

The extension of dendritic synthetic principles to the preparation of new kinds of dendrimer-based glycoconjugate systems has been investigated. A convergent growth approach to the synthesis of α-D-mannopyranoside-containing dendrimers of various generations has been used successfully to obtain high molecular weight and monodisperse dendrimers in which 3-36 identical copies of the mannopyranoside residues are located at their peripheries. A repetitive synthetic sequence for forming amide bonds is employed in the preparation of various dendritic wedges and their attachment to a 1,3,5-trisubstituted benzenoid core in the last step. The protecting groups on the saccharide residues are then finally removed to obtain free saccharide-containing dendrimers. The efficacy of these dendrimers in inhibiting the binding of concanavalin A lectin to a purified yeast mannan fraction (as measured by an enzyme-linked lectin assay) is shown to be about four times more pronounced on a molar basis for the dendrimers possessing nine, 18, and 36 mannose residues, when compared to a dendritic wedge possessing three mannose residues and methyl α-D-mannopyranoside as control inhibitors.

Full text of DOI:10.1021/jo9804184

J . Org. Chem. 1998, 63, 3429-3437
3429
Syn th esis a n d Biologica l Eva lu a tion of
r-D-Ma n n op yr a n osid e-Con ta in in g Den d r im er s†
Peter R. Ashton,‡ Elizabeth F. Hounsell,*,§ Narayanaswamy J ayaraman,‡ Torill M. Nilsen,§
Neil Spencer,‡ J . Fraser Stoddart,*,‡ and Mia Young§
School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K., and
Department of Biochemistry and Molecular Biology, University College, Gower Street,
London WC1E 6BT, U.K.
Received March 5, 1998
The extension of dendritic synthetic principles to the preparation of new kinds of dendrimer-based
glycoconjugate systems has been investigated. A convergent growth approach to the synthesis of
R-D-mannopyranoside-containing dendrimers of various generations has been used successfully to
obtain high molecular weight and monodisperse dendrimers in which 3-36 identical copies of the
mannopyranoside residues are located at their peripheries. A repetitive synthetic sequence for
forming amide bonds is employed in the preparation of various dendritic wedges and their
attachment to a 1,3,5-trisubstituted benzenoid core in the last step. The protecting groups on the
saccharide residues are then finally removed to obtain free saccharide-containing dendrimers. The
efficacy of these dendrimers in inhibiting the binding of concanavalin A lectin to a purified yeast
mannan fraction (as measured by an enzyme-linked lectin assay) is shown to be about four times
more pronounced on a molar basis for the dendrimers possessing nine, 18, and 36 mannose residues,
when compared to a dendritic wedge possessing three mannose residues and methyl R-D-
mannopyranoside as control inhibitors.
In tr od u ction
phenomenon, referred to as the “glycoside cluster effect”
has been well documented and verified for many neogly-
coconjugates, i.e., synthetic analogues of naturally oc-
curring oligosaccharide chains containing the active
mono- or oligosaccharide components only.6 The impor-
tance of intersaccharide distances and the branching
mode in cluster glycosides has been demonstrated ele-
gently by Lee et al.,6c and these requirements for syn-
thetic cluster glycosides have been confirmed further by
van Boom et al.7 in the studies involving the attachment
of cluster galactosides with the hepatic asialoglycoprotein
receptor. In the quest to obtain multivalent cluster
glycosides, strategies employed commonly include at-
tachment of determinant sugar ligands to carriers de-
rived from proteins8 and polymers.9 On the basis of the
idea that the rapidly emerging field of dendrimer chem-
istry10 could be utilized to create new and novel forms of
glycomimetics, we11 and others12 have been developing
synthetic routes to present multiple copies of sugar
ligands on the surfaces of dendrimers and dendritic-like
structures. Following the development of numerous
A large number of biological events are related to the
functional aspects of protein-bound saccharides.1 The
carbohydrate portions of glycoproteins and glycolipids on
cell surfaces participate at a macromolecular level in
many biological recognition processes,2 such as those
involving immune defense response, viral replication,
parasite infection, cell-cell adhesion, and inflammation.3
The specific biological functions of the oligosaccharide
chains in glycoproteins are mediated by a range of
carbohydrate-binding proteinssthe so-called lectins. Gen-
erally, multiple copies of saccharide ligands are necessary
in order to generate strong binding affinities to their
constituent lectin receptor sites,4 e.g., several endoge-
neous selectin ligands having multiple, densely spaced
O-linked sugars with very high surface densities.5 The
* Address correspondence to Dr. J . Fraser Stoddart, Department of
Chemistry and Biochemistry, University of California at Los Angeles,
405 Hilgard Avenue, Los Angeles, CA 90095-1569.
† Synthesis of Carbohydrate-Containing Dendrimers. 6. For part
5, see: J ayaraman, N.; Stoddart, J . F. Tetrahedron Lett. 1997, 38,
6767-6770.
‡ University of Birmingham.
(4) (a) Lee, Y. C., Lee, R. T., Eds. Neoglycoconjugates, Preparation
and Applications; Academic Press: San Diego, 1994; pp 23-50. (b)
Spillmann, D. Glycoconjugate J . 1994, 11, 169-171.
(5) (a) Van der Merwe, P. A.; Barclay, A. N. Trends Biochem. Sci.
1994, 19, 354-358. (b) Tedder, T. F.; Steeber, D. A.; Chen, A.; Engel,
P. FASEB J . 1995, 9, 866-873.
(6) (a) Lee, Y. C.; Lee, R. T.; Rice, K.; Ichikawa, Y.; Wong, T. C.
Pure Appl. Chem. 1991, 63, 499-506. (b) Lee, Y. C.; Lee, R. T. Acc.
Chem. Res. 1995, 28, 321-327. (c) Lee, Y. C.; Townsend, R. R.; Hardy,
M. R.; Lo¨nngren, J .; Arnarp, J .; Haraldsson, M.; Lo¨nn, H. J . Biol. Chem.
1983, 258, 199-202.
(7) (a) Valentijn, A. R. P. M.; van der Marel, G. A.; Sliedregt, L. A.
J . M.; van Berkel, Th. J . C,; Biessen, E. A. L.; van Boom, J . H.
Tetrahedron 1997, 53, 759-770. (b) Biessen, E. A. L.; Benting, D. M.;
Roelen, H. C. P. F.; van der Marel, G. A.; van Boom, J . H.; van Berkel,
Th. J . C. J . Med. Chem. 1995, 38, 1538-1546.
§ University College London.
(1) (a) Varki, A. Glycobiology 1993, 3, 97-130. (b) Drickamer, K.
In Molecular Glycobiology; Fukuda, M., Hindsgaul, O., Eds.; IRL:
Oxford, 1994; pp 53-87. (c) Montreuil, J ., Vliengenthart, J . F. G.,
Schachter, H., Eds. Glycoproteins. New Comprehensive Biochemistry,
29a; Neuberger, A., Van Deenan, L. L. M., General Eds.; Elsevier:
Amsterdam, 1995. (d) Dwek, R. Chem. Rev. 1996, 96, 683-720.
(2) (a) Feizi, T.; Childs, R. A. Biochem. J . 1987, 245, 1-11. (b)
Rademacher, T. W.; Parekh, R. B.; Dwek, R. A. Annu. Rev. Biochem.
1988, 57, 785-838. (c) Karlsson, K.-A. Annu. Rev. Biochem. 1989, 58,
309-350. (d) Sharon, N.; Lis, H. Eur. J . Biochem. 1993, 218, 1-27.
(e) Sharon, N.; Lis, H. Sci. Am. 1993, 268, 82-89. (f) Feizi, T. Trends
Biochem. Sci. 1994, 233-234. (g) Fukuda, M. Bioorg. Med. Chem.
1995, 3, 207-215.
(3) (a) Lasky, L. A. Science 1992, 258, 964-969. (b) Harlan, J . M.,
Liu, D. Y., Eds. Adhesion: Its Role in Inflammatory Disease; W. H.
Freeman: New York, 1992. (c) Springer, T. A. Cell 1994, 76, 301-
314. (d) Parekh, R. B.; Edge, J . Ch. Trends Biotechnol. 1994, 12, 339-
345.
(8) (a) Stowell, C. P.; Lee, Y. C. Adv. Carbohydr. Chem. Biochem.
1980, 37, 225-281. (b) Lee, R. T.; Lee, Y. C. In Lectins and
Glycobiology; Gabius, H.-J ., Gabius, S., Eds.; Springer Laboratory,
Springer-Verlag: New York, 1993; pp 9-22.
S0022-3263(98)00418-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/30/1998

3430 J . Org. Chem., Vol. 63, No. 10, 1998
Ashton et al.
elegant synthetic routes for the synthesis of different
types of dendrimers,10 using various kinds of building
blocks, dendrimer chemistry now appears to offer the
most highly controllable methodology for synthesizing
large three-dimensional macromolecules with very pre-
cise chemical constitutions and increasingly well-defined
molecular structures.
assay (ELLA), involving the inhibition of concanavalin
A (Con A) binding to a purified yeast mannan fraction
Sc500.14 This fraction is of interest because of its strong
binding of antibodies found in the serum of patients with
Crohn’s disease14,15 and also because of its implication
in the allergy to yeast mannan of bakers and brewers
who are overexposed to yeast in their work.14 Dendri-
mers of the type discussed herein may therefore find a
use in the clinic when the optimum branching densities
and lengths of branches have been determined. This
investigation addresses the first question by reference
to Con A binding that is known to be inhibited, if weakly,
at the monosaccharide level.
We report herein the synthesis of a series of R-D-
mannopyranose-containing dendrimers in which these
monosaccharides are located at the peripheries of the
dendrimers with peptidic-like interiors. Central to the
synthesis of these functionalized dendrimers is the choice
of the appropriate synthetic methodology that will allow
their facile preparation. Of the various methods that
have now been identified, the so-called “convergent
synthetic route” and the “divergent synthetic route” have
been applied widely in the synthesis of many different
dendrimers.10 In view of the structural characteristics
of carbohydrate-containing dendrimers, the convergent
synthetic route has been applied by us in their synthesis
in order to obtain the component dendritic wedges and
the final dendrimers. These have extremely high purities
and structural homogeneities associated with their mac-
romolecules and also boast unique chemical constitutions.
Recently, Roy et al.13 have reported the synthesis and
studies of mannosylated dendrimers, prepared by the
modification of the peripheries of PAMAM dendrimers
(generation 0-3) and also other dendritic structures.
Following the convergent synthesis of these new R-D-
mannopyranoside-containing dendrimers, we have evalu-
ated their biological potencies by an enzyme-linked lectin
Resu lts a n d Discu ssion
The convergent synthetic methodology involves es-
sentially the synthesis of dendritic wedges possessing
carbohydratesspreferably lining a surfacesas one of the
structural components, followed by the attachment of
these wedges to further branching components and then
finally to core components in order to obtain the desired
dendrimers. The structural components that constitute
the branching regions of the dendritic wedges have been
derived from (i) tris(hydroxymethyl)aminomethane (Tris)
and (ii) 3,3′-iminodipropionic acid, while the core has
been derived from 1,3,5-benzenetricarbonyl chloride. The
desired synthetic sequences using these components rely
on (i) glycosylation of the three hydroxyl groups in Tris
so as to obtain a tris-branched saccharide-containing
wedge, (ii) attachment of these wedges to the branched
3,3′-iminodipropionic acid derivatives by the formation
of amide bonds, affording highly branched saccharide-
containing dendritic wedges, and finally, (iii) attachment
of these branched wedges to the core component derived
from 1,3,5-benzenetricarbonyl chloride, once again by the
formation of amide bonds. The syntheses of these den-
drimers have been carried out by first constructing the
respective dendritic wedges, followed by the coupling of
these wedges to the central core component.
Syn th esis of Tr is-Ma n n op yr a n osid e-Con ta in in g
Den d r it ic Wed ge (7). The tris-branched mannopy-
ranoside derivative 7 was readily obtained (Scheme 1)
by the reaction of 2,3,4,6-tetra-O-benzoyl-R-D-mannopy-
ranosyl bromide (2) with the Z-Tris derivative 1 in the
presence of AgOTf and 2,4,6-collidine in CH2Cl2/MeNO2.
Under these conditions, the tris-glycosylation was ste-
reoselective and afforded the Tris-R-D-mannopyranoside
derivative 3 in 84% yield after column chromatography.
The first step in the elaboration of the dendritic wedge 3
involved the modification of the protecting groups on the
mannopyranoside residues. Removal of the O-benzoyl
protecting groups in 3 using the Zemple´n procedure gave
4, which was protected with O-acetyl functions using
Ac2O/C5H5N to give 5 in 93% yield overall. This modi-
fication was required in order to reduce the steric
bulkiness associated with the O-benzoyl protecting groups
that, otherwise, would slow the reactions of the wedge
components in the later stages of the dendrimer synthe-
sis. The amine protecting group in 5 was then removed
by hydrogenolysis (10% Pd on C) to give 7 in excellent
yield. The completely deprotected “3-mer” wedge 6 was
(9) (a) Byramova, N. E.; Mochalova, L. V.; Belyanchi, I. M.; Ma-
trosovich, M. W.; Bovin, N. V. J . Carbohydr. Chem. 1991, 10, 691-
700. (b) Glick, G. D.; Knowles, J . R. J . Am. Chem. Soc. 1991, 113,
4701-4703. (c) Sabesan, S.; Du¨us, J .; Neira, S.; Domaille, P.; Kelm,
S.; Paulson, J . C.; Bock, K. J . Am. Chem. Soc., 1992, 114, 8363-8375.
(d) Roy, R.; Tropper, F. D.; Romanowska, A. J . Chem. Soc., Chem.
Commun. 1992, 1611-1613. (e) Nishimura, S. I.; Furuike, T.; Mat-
suoka, K.; Maruyama, K.; Nagata, K.; Kurita, K.; Nishi, N.; Tokura,
S. Macromolecules 1994, 27, 4876-4880. (f) Sigal, G. B.; Mammen,
M.; Dahmann, G.; Whitesides, G. M. J . Am. Chem. Soc. 1996, 118,
3789-3880. (g) Mortell, K. H.; Weathermann, R. S.; Kiessling, L. L.
J . Am. Chem. Soc. 1996, 118, 2297-2298.
(10) (a) Tomalia, D. A.; Durst, H. D. Top. Curr. Chem. 1993, 165,
193-313. (b) Ardoin, N.; Astruc, D. Bull. Soc. Chim. Fr. 1995, 132,
875-909. (c) Fre´chet, J . M. J .; Hawker, C. J . In Comprehensive
Polymer Science; Aggarwal, S. L., Russo, S., Eds.; Pergamon: Oxford,
1996; pp 140-201. (d) Newkome, G. R.; Moorefield, C. N.; Vo¨gtle, F.
Dendritic Molecules, Concepts, Syntheses, Perspectives; VCH: Wein-
heim, 1996.
(11) (a) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; J ayaraman, N.;
Nepogodiev, S. A.; Stoddart, J . F. Chem. Eur. J . 1996, 2, 1115-1128.
(b) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; J ayaraman, N.; Stoddart,
J . F. Angew. Chem., Int. Ed. Engl. 1997, 36, 732-735. (c) Ashton, P.
R.; Boyd, S. E.; Brown, C. L.; Nepogodiev, S. A.; Stoddart, J . F. Chem.
Eur. J . 1997, 3, 974-984. (d) J ayaraman, N.; Nepogodiev, S. A.;
Stoddart, J . F. Chem. Eur. J . 1997, 3, 1193-1199. (e) J ayaraman,
N.; Stoddart, J . F.; Tetrahedron Lett. 1997, 38, 6767-6770.
(12) (a) Roy, R.; Zanini, D.; Meunier, S. J .; Romanowska, A. J . Chem.
Soc., Chem. Commun. 1993, 1869-1872. (b) Roy, R.; Zanini, D.;
Meunier, S. J .; Romanowska, A. Am. Chem. Soc. Symp. Ser. 1994, 560,
104-119. (c) Zanini, D.; Roy, R. J . Am. Chem. Soc. 1997, 119, 2088-
2095. (d) Roy, R. Polymer News 1996, 21, 226-232. (e) Lindhorst, T.
K.; Kieburg, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 1953-1956. (f)
Kieburg, C.; Lindhorst, T. K. Tetrahedron Lett. 1997, 38, 3885-3888.
(g) Aoi, K.; Itoh, K.; Okada, M. Macromolecules 1995, 28, 5391-5393.
(h) Kretzschmar, G.; Sprengard, U.; Kunz, H.; Bartnik, E.; Schmidt,
W.; Toepfer, A.; Ho¨rsch, B.; Krause, M.; Seiffge, D. Tetrahedron 1995,
51, 13015-13030. (i) Lehmann, J .; Weitzel, U. P. Carbohydr. Res.
1996, 294, 65-94. (j) Patch, R. J .; Chen, H.; Pandit, C. R.; Kichler, J .;
Schuber, F. Glycoconj. J . 1995, 12, 275-281. (k) Hansen, H. C.;
Haataja, S.; Finne, J .; Magnusson, G. J . Am. Chem. Soc. 1997, 119,
6974-6979.
(14) Young, M.; Haavik, S.; Smestad-Paulsen, B.; Bro¨ker, M.;
Barnes, R. M. R. Carbohydr. Polym. 1996, 30, 243-252.
(15) Young, M.; Davies, M. J .; Bailey, D.; Gradwell, M. J .; Smestad-
Paulsen, B.; Wold, J .; Barness, R. M. R.; Hounsell, E. F. Glycoconj. J .,
in press.
(13) (a) Page´, D.; Zanini, D.; Roy, R. Bioorg. Med. Chem. 1996, 4,
1949-1961. (b) Page´, D.; Aravind, S.; Roy, R. Chem. Commun. 1996,
1913-1914. (c). Page´, D.; Roy, R. Bioconjugate Chem. 1997, 8, 714-
723.

Synthesis of R-D-Mannopyranoside-Containing Dendrimers
J . Org. Chem., Vol. 63, No. 10, 1998 3431
Sch em e 1
Sch em e 3
Sch em e 2
70
obtained by the hydrogenolytic removal of the protecting
group from 4.
wedges 11 and 16, respectively, in good yields. All these
dendritic wedges were purified by silica gel column
chromatography and are stable with the exception of the
free amine wedges, which lead to the formation of some
intractable products when kept in solution for long
periods of time.
Syn th esis of th e “9-Mer ” 19. The tricarboxylic acid
17, obtained by the reaction of 1,3,5-benzenetricarbonyl
chloride with Gly-OMe, followed by hydrolysis of the ester
functions, was used as the core for the attachment of the
dendritic wedges. Amide bond formations were carried
out in the presence of DCC/HOBT as the coupling
reagents. The 9-mer 18 was obtained (Scheme 4) after
reaction of 7 (3.3 mol equiv) with 17 (1.0 mol equiv).
Removal of the O-acetyl protecting groups in 18 using
NaOMe/MeOH afforded the final free R-D-mannopy-
ranoside-containing 9-mer 19.
Syn t h esis of t h e H igh ly Br a n ch ed Ma n n op y-
r an oside-Con tain in g Den dr itic Wedges. These higher
generation dendritic wedges were synthesized subse-
quently by the reaction of 7 with branched carboxylic acid
derivatives acting as sources of further branching com-
ponents. The amide bond-forming reactions were carried
out in the presence of the DCC/HOBT coupling reagents.
Thus, the reaction of 7 with 9 afforded (Scheme 2) the
amino-protected “6-mer” wedge 10 in 70% yield.
The tetracarboxylic acid derivative 14 was obtained
(Scheme 3) as follows: reaction of 2.0 mol equiv of 12
with 1.0 mol equiv of 9 afforded the tetramethyl ester
13, which was then subjected to ester hydrolysis to give
14. Amide bond formation between 7 and the tetracar-
boxylic acid 14 proceeded smoothly to give the amino-
group protected “12-mer” wedge 15 in 73% yield. The
amine protecting groups in 10 and 15 could both be easily
removed by hydrogenolysis to produce the free amine
Syn th esis of th e “18-Mer ” 21. Following the con-
vergent synthetic protocol, involving the minimum num-

3432 J . Org. Chem., Vol. 63, No. 10, 1998
Ashton et al.
Sch em e 4
to each other using a particular set of elution conditions
and have not been calibrated to any commercially known
standards. A similar trend of the elution profile was
observed in the case of free saccharide-containing den-
drimers also.
The structural characterizations of all the new com-
pounds were achieved using common physical methods.
The chirooptical properties, as measured by the specific
and molar optical rotations of the dendritic wedges and
dendrimers, do not change very significantly. They were
found to be approximately proportional to the number
of monosaccharide residues each compound possesses.
The elemental analyses of all the compounds were in good
agreement with the expected values.
Liquid secondary ion (LSI) and matrix-assisted laser-
desorption time-of-flight (MALDI-TOF) mass spectrom-
etries have been used to confirm the molecular weights
of the dendritic wedges and dendrimers. While LSI-MS
has been useful in the detection of the lower molecular
weight compounds, the high molecular weight ones have
been characterized by MALDI-TOF-MS. The MALDI-
TOF mass spectrum of the 36-mer 22 is shown in Figure
2, wherein the protonated molecular ion peak, as well
as its sodium adduct, can be observed.
1H and 13C NMR spectroscopies have also been indis-
pensable in the characterization of all the dendritic
wedges and the dendrimers. The protons associated with
mannopyranosyl units, as well as with the nonsaccharide
1
units, can be observed clearly in their H NMR spectra.
In keeping with our previous observations on glucopy-
ranoside-containing dendrimers,11a,b the room-tempera-
1
ture H NMR spectra of the 18-mer 20 and the 36-mer
22 were very broad, and elevated temperatures within
the probe were necessary in order to obtain sharp and
well-resolved resonances. 1H and 13C NMR spectra of the
completely deprotected dendrimers 19, 21, and 23 were
ber of reactions at the final stage of dendrimer synthesis,
the preparation of the 18-mer 21 was carried out (Scheme
5) using 11 and 17 as the precursors. Reaction of the
6-mer 11 with 17, in the presence of DCC/HOBT, afforded
the 18-mer 20 in 88% yield after GPC. The facile nature
of this coupling reaction was illustrated by the formation
of the desired 18-mer as the exclusive product of the
reaction. The free R-D-mannopyranoside-containing 18-
mer 21 was obtained by deprotection (NaOMe/MeOH/
H2O).
1
recorded in D2O. A comparison of the H and 13C NMR
chemical shift values of the dendritic wedge 6 and the
dendrimers 19, 21, and 23 are given in Tables1 and 2,
respectively. Broad-band decoupled 13C NMR spectra of
all dendritic wedges and the dendrimers shared the
1
common features of the H NMR spectra in that all of
the resonances were sharp and “simple”. In the case of
1
Syn th esis of th e “36-Mer ” 23. The synthesis of the
36-mer 23 was achieved (Scheme 6) by the so-called 12
× 3 reaction sequence, which involves the reaction of 16
(3.5 mol equiv) with 17 (1.0 mol equiv), once again in the
presence of DCC/HOBT: it led to the isolation of the
desired 36-mer dendrimer 22 in 39% yield, after GPC.
The disubstituted product was also isolated in 30% yield.
Deprotection of 22 afforded the free R-D-mannopyrano-
side-containing 36-mer 23, which was subjected to GPC
in order to remove salts and other trace impurities.
Str u ctu r al Ch ar acter ization s of Den dr itic Wedges
a n d Den d r im er s. In their protected forms, all the
dendritic wedges and dendrimers are soluble in a variety
of organic solvents (CH2Cl2, EtOAc, MeOH, and THF),
rendering their purifications by either column chromato-
graphic (silica gel) or GPC (phenogel) techniques routine.
In the case of the high molecular weight dendrimers,
purification by GPC appears to be more suitable, since
the molecular weight differences between the desired
dendrimer products, other byproducts, and reagents
allows their facile separation. The behavior of the elution
profiles of the 36-mer 22, 18-mer 20, and the 9-mer 18
is shown in Figure 1. These retention times are relative
the 18-mer 20 and the 36-mer 22, the H and 13C NMR
chemical shift assignments were confirmed further by
heteronuclear multiple quantum coherence (HMQC)
spectroscopy. Although all the resonances for the carbon
atoms of lower molecular weight dendritic wedges and
dendrimers could be observed clearly, those associated
with the inner skeleton methylenes arising from glycine
and propionamide residues in the higher molecular
weight derivatives were difficult to detect because of their
relatively small signal intensities. The observed chemical
shift values and coupling constants values supported the
assigned R-anomeric configuration to the D-mannopy-
ranosyl residues present in all the dendritic wedges and
dendrimers.
Biologica l Eva lu a tion of th e Den d r im er s 6, 19, 21,
a n d 23. Figure 3 shows the results of the ELLA with
the various dendrimers as inhibitors of binding of Con A
to the yeast mannan fraction Sc500. This biological
evaluation of the dendrimers compares the inhibition
activity to that of methyl R-D-mannopyranoside, which,
in several assays, has given 50% inhibition (IC50) with
0.5 mg mL-1 (2.5 mM). This inhibition activity of methyl
R-D-mannopyranoside approximates to the activity shown

Synthesis of R-D-Mannopyranoside-Containing Dendrimers
Sch em e 5
J . Org. Chem., Vol. 63, No. 10, 1998 3433
time (min)
F igu r e 1. GPC traces of the O-acetylated dendrimers 9-mer
18, the 18-mer 20, and the 36-mer 22.
F igu r e 2. MALDI-TOF mass spectrum of the 36-mer 22.
acid residues at the periphery of polylysine dendritic
in Figure 3 for the dendritic wedge 6 on a molar basis.
The 9-mer 19 was the most active inhibitor at the mg
mL-1 level with an IC50 of 0.65 mM. Thus, the inhibition
potential of the 9-mer 19 is four times that of the 3-mer
6 and methyl R-D-mannopyranoside. This IC50 value of
the 9-mer 19 compares very favorably with the increase
in potency that other researchers have found for the
inhibition of lectins: e.g., for sialic acid inhibition of
hemagglutination of human erythrocytes by influenza
virus, where the so-called “16-mer” possessing 16 sialic
backbone, was twice as active as the “8-mer”, which was
found to be twice as active as the “4-mer”, which, in turn,
was twice as active as the dimer.12a Also, for Con A
binding to unfractionated yeast mannan, inhibition using
mannosylated poly(amidoamine) starburst dendrimers,
which are most similar to the dendrimers reported in this
paper, showed an increase in activity by a factor of 3.6
in going from a tri- to an octasaccharide derivative.13c In
the present study, the 18-mer 21 and the 36-mer 23 both
have similar activities when compared on a molar basis

3434 J . Org. Chem., Vol. 63, No. 10, 1998
Ashton et al.
Sch em e 6
Ta ble 1. 1H NMR Sp ectr oscop ic Da ta (δ Va lu es) of r-D-Ma n n op yr a n osid e-Con ta in in g Den d r im er s 6, 19, 21, a n d 23 a t
300 MHz (6 a n d 19) a n d 500 MHz (21 a n d 23) in D2O
6
19
21
23
4.76
3.40-4.20
H-1
4.74
3.40-3.90
4.76
4.72-4.76
3.42-3.92, 4.16
H-2-H-6, CH2C(quat),
GlyCH2, and/or CH2CH2
Ph
3.53-3.94, 4.03
8.35
8.40
8.40
CH2CH2
2.40-2.64
2.32-2.90
with each other. Their activities are not much greater
than the 9-mer 19, suggesting that with the homoge-
neous, monodisperse, “complete” dendrimers reported
here the clustering effect is most pronounced between
the 9-mer 19 and the 18-mer 21. Previous molecular
modeling studies had shown that the peripheral monosac-
charide residues cover the internal aglycon scaffold of
dendrimers.11a,b Thus, the 9-mer 19 and the 18-mer 21
represent suitable model compounds for future studies
on dendrimers having oligosaccharide sequences greater
than one monosaccharide residue on each antenna for
inhibition of antibodies that have a more specific binding
site for functional groups, usually on tri- to heptasac-
charides,16 although some disaccharides may be active.17
In the present investigation, we evaluated the perfor-
mance of the newly synthesized dendrimers as inhibitors
of the binding of antibodies found in the serum of Crohn’s
patients, which recognize15 the yeast mannan fraction
Sc500. With a maximum dilution of 0.2 mg mL-1 of the
dendrimers as inhibitors, we did not observe any inhibi-
tion. This observation was not surprising, given the fact
(16) Feizi, T, Hounsell, E. F. Alais, J . Veyrie´res, A.; David, S.
Carbohydr. Res. 1992, 228, 289-297.
(17) (a) Bundle, D. R.; Baumann, H.; Brisson, J .-R.; Gagne´, A.;
Zdanov, A.; Cygler, M. Biochemistry 1994, 33, 5183-5192. (b) Evans,
S. V.; Sigurskjold, B. W.; J ennings, H. J .; Brisson, J .-R.; To, R.; Tse,
W. C.; Altman, E.; Frosch, M.; Weisgerber, C.; Kratzin, H. D.; Klebert,
S.; Vaesen, M.; Bitter-Suermann, D.; Rose, D. R.; Young, N. M.; Bundle,
D. R. Biochemistry 1995, 34, 6737-6744.

Synthesis of R-D-Mannopyranoside-Containing Dendrimers
J . Org. Chem., Vol. 63, No. 10, 1998 3435
Ta ble 2. 13C NMR Sp ectr oscop ic Da ta (δ Va lu es) of r-D-Ma n n op yr a n osid e-Con ta in in g Den d r im er s 6, 19, 21, a n d 23 a t
75.5 MHz (6 a n d 19) a n d 125.5 MHz (21 a n d 23) in D2O
6
19
103.0
21
23
C-1
C-2
C-3
C-4
C-5
C-6
CH2C(quat)
C(quat)
102.9
72.5
73.2
69.5
75.5
63.5
71.5
56.5
102.9
72.5
73.2
69.3
75.6
63.4
68.1
62.1
102.9
72.5
73.2
68.9
75.6
63.4
68.2
62.1
72.6
73.3
69.5
75.7
63.6
68.6
56.5
46.2
GlyCH2 and/or CH2CH2
36.7, 37.4, 43.5,
45.2,45.7, 46.2
33.2, 33.8, 36.8, 37.5,
37.7, 44.8,45.1, 45.4,
46.0, 46.9, 47.2
Ph
CONH
132.2, 137.0
171.5, 173.4
132.3, 136.8
171.6, 172.5, 174.1,
175.3,176.1
130.3, 131.4
175.1, 175.3, 175.8,
176.0, 176.1
dendrimers been obtained in good to excellent yields, but
also a number of potentially useful multivalent dendritic
wedge structures have been prepared. It should be
possible, using this methodology, to synthesize very large
dendritic structures by introducing additional building
blocks, derived from saccharides or otherwise. The
biological potencies of the 3-mer 6, the 9-mer 19, the 18-
mer 21, and the 36-mer 23 have been evaluated using
an enzyme-linked lectin assay procedure and have been
shown to exhibit most pronounced inhibitory effects
between the 9-mer 19 and the 18-mer 21 in this particu-
lar assay, when compared with methyl R-D-mannopy-
ranoside, the monomer analogue of the R-D-mannopy-
ranoside-containing dendrimers.
Exp er im en ta l Section
Gen er a l Meth od s. Chemicals were purchased from Ald-
rich and used as received. Solvents were either used as
purchased or dried according to procedures described in the
literature.18 TLC was carried out using aluminum sheets
precoated with silica gel 60F. The plates were inspected by
UV light or by treatment with 5% H2SO4 in EtOH, followed
by heating the plates. Column chromatography was carried
out using silica gel 60F (230-400 mesh). Gel permeation
chromatography (GPC) of the fully protected dendrimers was
carried out on a Phenogel (500 and 1000 Å) semipreparative
columns (300 × 7.80 mm) attached to a high-performance
liquid chromatography system fitted with a UV-detector.
Detection was carried out at 254, nm and GPC-grade THF was
used for the elutions. The fully deprotected dendrimers were
purified on a Fractogel (25-40 µm) column (100 × 0.25 cm)
fitted with a differential refractometer with deionized and
degassed water as eluant. Microanalysis were performed by
the University of North London Microanalytical Service.
Liquid secondary ion mass spectra (LSI-MS) were recorded
using m-nitrobenzyl alcohol matrix. Matrix-assisted laser
desorption ionization-time-of-flight mass spectra (MALDI-
TOF-MS) was performed using a trans-indoleacrylic acid
matrix and an average of 50 laser shots per sample. Optical
rotations were performed at 23 °C. 1H NMR and 13C NMR
spectra were recorded on a 300 and 75.5 MHz spectrometer, a
400 and 100.6 MHz spectrometer, or a 500 and 125.5 MHz
spectrometer with either the residual solvent or TMS as
internal standards. For studies in D2O, TSP was used as the
external reference. The chemical shifts are expressed in ppm,
F igu r e 3. Inhibition of binding of biotin-labeled ConA to yeast
mannan Sc500 by the free-saccharide 3-mer wedge (~) and
the free-saccharide dendrimers 9-mer (19) ([), 18-mer (21),
(9) and 36-mer (23) (]).
that more than a monosaccharide is recognized by these
antibodies; all the antigenic activity is lost on acetolysis,
very mild acid hydrolysis, or mild alkali elimination,
which leaves significant amounts of mannopyranose
residues linked to the remaining polymer, but we pre-
sume no intact Man 1 f 3 or 1 f 2 side chains. In
addition to their involvement in Crohn’s disease, yeast
mannans are also a source of allergies for bakers and
brewers who are overexposed to the yeast Saccharomyces
cerevisiae. Although the exact nature of this epitope has
not yet been established, it is most strongly expressed
on the Sc500 yeast mannan fraction used in the present
investigation and is therefore likely to be similar to the
antigen present in Crohn’s patients. The route of expo-
sure would dictate which type of immunogenicity was
invoked. In the case of the bakers’ and brewers’ allergies,
the IgE hypersensitivity reaction can be tested by den-
drimers having the appropriate di- or trisaccharide
sequences in multivalent forms, and if shown to be active,
these compounds could also be tried in clinical desensi-
tization.
1
and the coupling constants from the H NMR spectra in hertz
(Hz) and are within a ca. 0.2 Hz error range. The following
abbreviations were used to explain the multiplicities: s,
singlet; d, doublet; t, triplet; m, multiplet; dd, double doublet;
app d, apparent doublet; app t, apparent triplet; band, several
overlapping signals; br, broad. The 1H and 13C NMR chemical
Con clu sion s
R-D-Mannopyranoside-containing dendrimers, carrying
3-36 copies of these monosaccharide units, have been
synthesized using a convergent growth approach. By
employing this methodology, not only have the desired
(18) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals, 3rd ed.; Pergamon Press: Oxford, 1988.

3436 J . Org. Chem., Vol. 63, No. 10, 1998
Ashton et al.
shift values of deprotected dendritic wedge 6 and deprotected
dendrimers 19, 21, and 23 are given in Tables 1 and 2,
respectively.
120 (H+ form) ion-exchange resin and filtered. The solvents
were then removed in vacuo, the resulting residue was
dissolved in a minimum amount of H2O and extracted with
Et2O, and the aqueous portion was evaporated in vacuo. The
resulting residue was subjected to GPC purification to afford
the completely deacylated dendritic wedges and dendrimers.
Tr is[(r-D-m a n n op yr a n osyloxy)m et h yl]m et h yla m in e
3-Mer (6). A suspension of 4 (0.250 g) in MeOH/H2O (8:2)
(10 mL) containing 10% Pd/C (0.10 g) was subjected to
hydrogenolysis using a balloon filled with H2 gas for 16 h. After
filtration over Celite, and the solvents were removed in vacuo
to afford 6 (0.160 g, 76%): [R]D +58.8° (c 0.9, H2O); MALDI-
TOF-MS m/z 630 [M + Na]+.
Gen er a l P r oced u r e for th e Am id e Bon d F or m a tion .
A solution of the amine component (1.0-1.2 mol equiv) in CH2-
Cl2 was added to a stirred solution of the carboxylic acid
component, DCC (1.0-1.2 mol equiv), and HOBT (1.0-1.2 mol
equiv) in CH2Cl2/DMF (2:1) at 0 °C under a N2 blanket. The
reaction mixture was stirred at room temperature and filtered,
and the solvents were evaporated off. The resulting residue
was dissolved in EtOAc and washed successively with 5%
aqueous HCl solution (30 mL), saturated NaHCO3 solution (30
mL), and H2O (15 mL), before being dried. The solvents were
evaporated off in vacuo, and the crude product was purified
either by column chromatography or by gel permeation chro-
matography.
N-(Ben zyloxyca r bon yl)tr is[[(2,3,4,6-tetr a -O-ben zoyl-r-
D-m a n n op yr a n osyl)oxy]m eth yl]m eth yla m in e (3) A solu-
tion of 2,3,4,6-tetra-O-benzoyl-R-D-mannopyranosyl bromide
(2)19 (6.0 g, 9.10 mmol) in CH2Cl2 (10 mL) was added dropwise
over a period of 0.5 h to a stirring mixture of 111a (0.67 g, 2.63
mmol), AgOTf (2.34 g, 9.10 mmol), and 2,4,6-collidine (0.943
g, 7.78 mmol) in CH2Cl2 (20 mL) and MeNO2 (20 mL) at -25
to -30 °C. Stirring was continued for 0.5 h at the same
temperature, and then the reaction mixture was allowed to
reach room temperature before being stirred overnight. C5H5N
(2.0 mL) was then added to the reaction mixture, which was
diluted with CH2Cl2 (50 mL) before being filtered over Celite.
The filtrate was washed successively with aqueous Na2S2O3
(1 M) solution (3 × 30 mL), aqueous NaHCO3 (1 M) solution
(3 × 30 mL), and H2O (2 × 20 mL) and dried, before the
solvents were evaporated off in vacuo. Purification by column
chromatography (PhMe/EtOAc 95:5) afforded 3 (4.36 g, 84%)
as a white foamy solid: TLC Rf (PhMe/EtOAc 9:1) 0.58 (UV,
H2SO4); [R]D -19.2° (c 1, CHCl3); 1H NMR (300 MHz, CD3-
COCD3) δ 4.38 (3H, d, J ) 10.3 Hz), 4.67 (3H, d, J ) 10.3 Hz),
4.73 (3H, dd, J ) 2.5, 12.4 Hz), 4.88 (6H, band), 5.17 (1H, d,
J ) 12.1 Hz), 5.25 (1H, d, J ) 12.1 Hz), 5.48 (3H, app s), 5.77
(1H, br), 5.97 (3H, app.t), 6.09 (3H, dd, J ) 2.94, 10.1 Hz),
6.31 (3H, app t, J ) 10.1 Hz), 7.03-8.30 (65H, m); 13C NMR
(75.5 MHz, CD3COCD3) δ 59.9, 63.4, 67.1, 67.3, 68.0, 70.4, 71.2,
71.6, 99.6, 126.1-138.4, 156.6, 165.8-166.4; MALDI-TOF-MS
m/z 2012 [M + Na]+. Anal. Calcd for C114H95NO32: C, 68.78;
H, 4.78; N, 0.70. Found: C, 68.84; H, 4.75; N, 0.74.
6-Mer Am in e Wed ge (11). A solution of 7 (0.60 g, 0.54
mmol) in CH2Cl2 (15 mL) was added dropwise to a stirred
solution of 911a (0.091 g, 0.26 mmol), DCC (0.112 g, 0.54 mmol),
and HOBT (0.073 g, 0.54 mmol) in CH2Cl2/DMF (2:1) (40 mL)
at 0 °C under a N2 blanket. The reaction mixture was allowed
to stir at room temperature for 55 h before being worked up
as described in the general procedure to obtain the N-
(benzyloxycarbonyl) group-protected derivative 10 (0.470 g,
70%). A suspension of 10 (0.450 g) in EtOAc/MeOH (2:1) (15
mL) containing 10% Pd/C (0.250 g) was subjected to hydro-
genolysis as described for 7 to afford 11 (0.40 g, 93%): [R]D
N-(Ben zyloxyca r bon yl)tr is[(r-D-m a n n op yr a n osyloxy)-
m eth yl]m eth yla m in e (4). A solution of 3 (4.14 g, 2.08 mmol)
in 0.1 M methanolic NaOMe (250 mL) was stirred at room
temperature for 15 h before being neutralized with Amberlite
IR-120 (H+ form) ion-exchange resin and filtered. After the
solvents had been removed in vacuo, the resulting mixture was
dissolved in H2O (15 mL) and extracted with Et2O (2 × 20
mL), and the aqueous portion was evaporated before being
dried thoroughly to obtain 4 (1.46 g, 95%): [R]D +61.4° (c 1,
H2O); 1H NMR (300 MHz, D2O) δ 3.42-3.92 (39H, band), 5.05
(2H, s), 7.24-7.42 (5H, m); 13C NMR (75.5 MHz, CD3COCD3)
δ 61.1, 63.4, 68.5, 69.3, 72.5, 73.2, 75.6, 102.9, 130.3-139.1,
158.2; MALDI-TOF-MS m/z 764 [M + Na]+. Anal. Calcd for
1
+49.4° (c 1, CHCl3); H NMR (400 MHz, CD3COCD3) δ 1.93,
2.01, 2.03, 2.09 (72H, 4 × s), 2.57 (4H, br), 3.59 (4H, br), 3.90
(6H, d, J ) 9.9 Hz), 3.98-4.23 (20H, band), 4.29 (6H, dd, J )
3.7, 11.6 Hz), 4.89 (6H, d, J ) 2.9 Hz), 5.19-5.31 (18H, band),
7.47 (2H, b); 13C NMR (75.5 MHz, CD3COCD3) δ 20.3, 20.37,
20.41, 20.47, 25.5, 26.2, 34.3, 54.7, 59.8, 62.6, 66.3, 69.6, 69.7,
98.8, 169.9, 170.1, 170.2, 170.3, 170.6; MALDI-TOF-MS m/z
2428 [M + Na]+. Anal. Calcd for C100H140N4O63: C, 49.92; H,
5.82; N, 2.33. Found: C, 49.88; H, 5.91; N, 2.30.
C
30H47NO20: C, 48.58; H, 6.34; N, 1.89. Found: C, 48.61; H,
6.44; N, 1.87.
Tetr a ca r boxylic Acid Wed ge (14). Et3N (1.67 mL, 12.0
mmol) was added to a stirred suspension of 12·HCl (2.0 g, 8.89
mmol) in CH2Cl2 (15 mL) at 0 °C. After 0.75 h, Et2O was
added, the reaction mixture was filtered, and the solvents were
removed to obtain 3,3’-iminobis(methyl propionate) 12 as an
oil (1.42 g, 85%). A solution of 12 (1.14 g, 6.03 mmol) in CH2-
Cl2 was added dropwise to a stirred solution of 9 (0.91 g, 2.50
mmol), DCC (1.13 g, 5.50 mmol), and HOBT (0.74 g, 5.50
mmol) in CH2Cl2/DMF (2:1) (15 mL) at 0 °C. The reaction
mixture was stirred at room temperature for 30 h before being
worked up as described in the general procedure to obtain 13
(1.68 g, 93%). A solution of 13 (1.60 g, 2.30 mmol) in MeOH
(35 mL) was added to a 2 M aqueous NaOH solution (9.7 mL)
at 0 °C, and then it was stirred for 5 h. The precipitated
material was dissolved in a minimum amount of H2O. The
aqueous solution was neutralized with Amberlite (H+ form)
ion-exchange resin, filtered, evaporated, and dried thoroughly
to afford 14 (1.42 g, 95%) as an oil: 1H NMR (300 MHz, CD3-
SOCD3) δ 2.45 (4H, br t), 2.58 (8H, br), 3.49 (8H, br), 3.59 (4H,
br), 3.93 (2H, d), 5.05 (2H, s), 7.20 (1H, br t), 7.38 (5H, br s);
13C NMR (75.5 MHz, CD3SOCD3) δ 30.6, 31.4, 32.1, 32.4, 33.0,
33.5, 41.5, 42.0, 42.4, 43.4, 51.5, 65.5, 127.8, 128.5, 137.2, 156.5,
168.6, 170.3, 171.7, 172.0, 172.8, 173.2; LSI-MS m/z 639 [M +
H]+.
Tr is[[(2,3,4,6-t e t r a -O-a ce t yl-r-D -m a n n op yr a n osyl)-
oxy]m eth yl]m eth yla m in e (7). Acetic anhydride (4.2 mL)
was added to a solution of 4 (1.1) in C5H5N (7.24 mL), which
was stirred at room temperature for 8 h before being evapo-
rated in vacuo. The resulting residue was dissolved in EtOAc
(70 mL), washed with saturated aqueous NaHCO3 solution (3
× 25 mL) and H2O (2 × 25 mL), and dried before the solvents
were evaporated off to give 5 (1.72 g, 93%) as a foamy powder.
A suspension of 5 (1.50 g) in EtOAc/MeOH (2:1) (50 mL) and
10% Pd/C (0.75 g) was subjected to hydrogenolysis by using a
balloon filled with H2 gas for 12 h. After filtration over Celite,
the solvents were removed in vacuo to yield 7 (1.25 g, 94%) as
a white foamy powder: TLC Rf (EtOAc) 0.19 (H2SO4); [R]D
1
+54.8° (c 1, CHCl3); H NMR (500 MHz, CD3COCD3) δ 1.93,
2.01, 2.03, 2.09 (36H, 4 × s), 3.52 (3H, d, J ) 10.0 Hz), 3.80
(3H, d, 10.0 Hz), 4.07-4.13 (6H, band), 4.28 (3H, dd, J ) 5.5,
12.0 Hz), 4.91 (3H, d, J ) 1.8 Hz), 5.22-5.30 (9H, band); 13C
NMR (125.5 MHz, CD3COCD3) δ 20.6-20.7, 56.4, 63.1, 66.8,
69.8, 70.0, 70.1, 70.8, 99.1, 170.2-170.8; MALDI-TOF-MS m/z
1112 [M + H]+. Anal. Calcd for C46H65NO30: C, 49.73; H, 5.86;
N, 1.26. Found: C, 49.75; H, 6.06; N, 1.27.
Gen er a l P r oced u r e for th e Dea cyla tion s. A solution of
the acylated dendritic wedges or dendrimers in MeOH or in
MeOH/H2O was treated with 1 M NaOMe and left stirring at
room temperature before being neutralized with Amberlite IR-
12-Mer Am in e Wed ge (16). A solution of 7 (0.90 g, 0.81
mmol) in CH2Cl2 (20 mL) was added with stirring to a solution
of 14 (0.123 g, 0.193 mmol), DCC (0.167 g, 0.81 mmol), and
HOBT (0.110 g, 0.81 mmol) in CH2Cl2/DMF (2:1) (60 mL)
under a N2 blanket at 0 °C. The reaction mixture was stirred
(19) Ness, R. K.; Fletcher, H. G., J r.; Hudson, C. S. J . Am. Chem.
Soc. 1950, 72, 2200-2204.

Synthesis of R-D-Mannopyranoside-Containing Dendrimers
J . Org. Chem., Vol. 63, No. 10, 1998 3437
at room temperature for 72 h before being worked up as
described in the general procedure to obtain 15 (0.70 g, 73%).
A suspension of 15 (0.50 g) and 10% Pd/C (0.40 g) in EtOAc/
MeOH (2:1) (25 mL) was subjected to hydrogenolysis for 30 h
as described for 7 to afford 16 (0.402 g, 82%): [R]D +52.8° (c
1.24, CHCl3); 1H NMR (400 MHz, CD3COCD3) δ 1.95, 2.02,
2.05, 2.10 (144H, 4 × s), 2.43, 2.58 (12H, 2 × br), 3.60 (12H,
br), 3.86-3.97 (12H, band), 4.0-4.21 (38H, band), 4.29 (12H,
dd, J ) 5.20, 12.30 Hz), 4.95 (12H, br), 5.20-5.30 (36H, band),
7.42 (4H, br); 13C NMR (75.5 MHz, CD3COCD3) δ 20.5, 20.6,
20.7, 25.6, 26.4, 34.4, 56.1, 60.0, 62.8, 62.9, 66.5, 66.6, 69.6,
69.7, 69.9, 70.0, 99.0, 170.1-170.8; LSI-MS m/z 4881 [M + H]+.
Anal. Calcd for C204H284N8O127: C, 50.21; H, 5.87; N, 2.30.
Found: C, 50.15; H, 5.75; N, 2.21.
(125.5 MHz, CD3SOCD3) δ 20.2, 20.4, 21.0, 33.6, 43.4, 58.4,
61.5, 65.2, 65.6, 68.2, 68.5, 68.6, 97.4, 128.0, 139.1, 169.3, 169.4,
169.5, 169.9, 171.3; MALDI-TOF-MS m/z 14969 [M + H]+.
Anal. Calcd for C627H861N27O387: C, 50.31; H, 5.80; N, 2.53.
Found: C, 50.38; H, 5.74; N, 2.61.
36-Mer Den d r im er (23). The deacetylation of 22 (0.10 g,
0.007 mmol) in MeOH/H2O (1:1) (10 mL) was performed using
a 1 M methanolic NaOMe solution (1.0 mL) for 30 h, followed
by workup and purification as described in the general
procedure for deacylations, to afford 23 (0.025 g, 42%) as a
glassy solid: [R]D +50.2° (c 1.35 H2O).
En zym e-Lin ked Im m u n o Sor ben t Assay (ELISA). Wells
of Nunc Maxisorp U96 microtiter plates were coated with a
100 µL solution of antigen (Sc500, 100 ng mL-1 in PBS, pH
7.5). The plate was incubated for 16-20 h at 4 °C (in the
refrigerator). The coating solution was removed, and the wells
were blocked by the addition of 200 µL 0.5% BSA in PBS, pH
7.5, with incubation for 4 h at 4 °C. The blocking solution
was removed, and serial dilutions of the dendrimers were
added in 0.5% BSA in PBS, 50 µL to each well, followed by 50
µL solution of (monoclonal antibody or) serum-containing
antibodies (diluted 1:200 in 0.5% BSA in PBS). The plate was
incubated for 16-24 h at 4 °C, after 1 h of shaking at 20 °C.
The excess of the solution was removed, and the wells were
washed with 2 × 200 µL PBS, pH 7.5. The peroxidase-labeled
second antibody (100 µL of anti-IgG solution diluted 1:1000
in 0.5% BSA in PBS, pH 7.5) was added to each well and the
plate incubated for 4 h at 20 °C. The solutions were removed
and the wells washed with 3 × 200 µL PBS, pH 7.5. A freshly
prepared solution (100 µL) of p-nitrophenyl phosphate (1 mg
mL-1) (Sigma alkaline phosphatase substrate) in 0.1 M Tris,
pH 10 was added to each well. The plate was incubated at 20
°C until an appropriate color was generated (30-60 min,
highest value ca. 1) and the absorbance read at 405 nm using
a microplate reader.
En zym e-Lin k ed Lectin Assa y (ELLA) w ith Con A.
Wells of Nunc Maxisorp U96 microtiter plates were coated
with a 100 µL solution of S. cerevisiae mannan (100 ng mL-1
in PBS, pH 7.5). The plate was incubated for 16-20 h at 4 °C
(in the refrigerator). The coating solution was removed, and
the wells were blocked by the addition of 200 µL 0.5% BSA in
PBS, pH 7.5, with incubation for 4 h at 4 °C. Serial dilutions
of the dendrimers or methyl R-D-mannopyranoside were made
up in 0.5% BSA in PBS, pH 7.5, and 50 µL added to each well,
followed by 50 µL of biotin-labeled ConA (10 µg mL-1 in 0.5%
BSA in PBS, pH 7.5, containing 1 mM CaCl2 and 1 mM MgCl2).
The plate was again incubated for 16-24 h at 40 °C, after 1 h
of shaking at 20 °C. The excess of the solution was removed,
and the wells were washed with 2 × 200 µL PBS, pH 7.5. To
each well was added 100 µL of antibiotin solution diluted
1:15 000 in 0.5% BSA, pH 7.5, and containing 1 mM CaCl2
and 1 mM MgCl2, and the plate was incubated for 4 h at room
temperature (20 °C). The solutions were removed and the
wells washed with 3 × 200 µL of PBS, pH 7.5. To each well
was added 100 µL of a freashly prepared solution of p-
nitrophenyl phosphate (1 mg mL-1 in 0.1 M Tris, pH 10). The
plate was incubated until an appropriate color was generated
(30-90 min) at 20 °C and the absorbance read at 405 nm on
a microplate reader.
P er a cetyla ted 9-Mer Den d r im er (18). A solution of 7
(0.30 g, 0.270 mmol) in CH2Cl2 (10 mL) was added dropwise
to a stirred solution of 1711a (0.031 g, 0.082 mmol), DCC (0.052
g, 0.253 mmol), and HOBT (0.034 g, 0.253 mmol) in CH2Cl2/
DMF (2:1) (15 mL) at 0 °C under an N2 blanket. The reaction
mixture was left stirring at room temperature for 30 h before
being worked up as described in the general procedure to
obtain the acetylated dendrimer 18 (0.290 g, 96%): [R]D +30.7°
1
(c 0.7, CHCl3); H NMR (400 MHz, CD3COCD3) δ 1.95, 1.97,
2.02, 2.06 (108H, 4 × s), 3.92 (9H, d, J ) 9.8 Hz), 4.01-4.14
(24H, band), 4.18 (9H, d, J ) 9.8 Hz), 4.26 (9H, dd, J ) 5.6,
12.0 Hz), 4.90 (9H, app s), 5.19-5.29 (27H, band), 7.48 (3H,
s), 8.02 (3H, br t), 8.53 (3H, s); 13C NMR (75.5 MHz, CD3-
COCD3) δ 20.4-20.5, 43.8, 60.1, 62.7, 66.2, 66.3, 69.6, 69.7,
98.7, 128.8, 135.5, 166.4, 170.0-170.4; MALDI-TOF-MS m/z
3684 [M + Na]+. Anal. Calcd for C153H204N6O96: C, 50.12; H,
5.57; N, 2.29. Found: C, 49.95; H, 5.57; N, 2.34.
9-Mer Den d r im er (19). Deacetylation of 18 (0.150 g, 0.041
mmol) was carried out in 0.1 M methanolic NaOMe solution
(20 mL) for 6 h, followed by workup and purification as
described in the general procedure for deacylations to afford
19 (0.060 g, 68%) as a glassy solid: [R]D +58.3° (c 1.25, H2O);
MALDI-TOF-MS m/z 2171 [M + Na]+.
P er a cetyla ted 18-Mer Den d r im er (20). A solution of 11
(0.50 g, 0.208 mmol) in CH2Cl2 (10 mL) was added dropwise
to a stirred solution of 17 (0.024 g, 0.063 mmol), DCC (0.043
g, 0.208 mmol), and HOBT (0.028 g, 0.208 mmol) in CH2Cl2/
DMF (2:1) (30 mL) at 0 °C under an N2 blanket. The reaction
mixture was left stirring at room temperature for 60 h and
then worked up as described in the general procedure to obtain
the acetylated dendrimer 20 (0.420 g, 88%) as a foamy powder
that was purified by GPC: [R]D +34.4° (c 0.7, CHCl3); 1H NMR
(500 MHz, CD3SOCD3 353 K) δ 1.92, 1.98, 2.01, 2.09 (∼216H,
4 × s), 2.36 (12H, br), 3.48 (12H, br), 3.76 (18H, d, J ) 10.0
Hz), 3.91-4.22 (84H, band), 4.82 (18H, app s), 5.08-5.19 (54H,
band), 7.62 (6H, br), 8.50 (3H, s), 8.55-8.72 (6H, br); 13C NMR
(125.5 MHz, CD3SOCD3) δ 20.2, 20.5, 20.7, 20.9, 33.6, 42.4,
58.5, 61.6, 65.2, 65.3, 68.5, 68.6, 97.3, 128.0, 139.1, 169.3, 169.4,
169.5, 169.9, 170.4; MALDI-TOF-MS m/z 7580 [M + Na]+.
Anal. Calcd for C315H429N15O195: C, 50.16; H, 5.73; N, 2.79.
Found: C, 50.45; H, 5.80; N, 2.81.
18-Mer Den d r im er (21). The deacetylation of 20 (0.150
g, 0.020 mmol) in MeOH/H2O (1:1) (20 mL) was performed
using 1 M NaOMe in MeOH (0.8 mL) 24 h, followed by workup
and purification as described in the general procedure for
deacylations to afford 21 (0.042 g, 45%) as a glassy solid: [R]D
+54.7° (c 1.12, H2O).
Ack n ow led gm en t . This research at Birmingham
University was supported by the Engineering and
Physical Sciences Research Council in the UK. E.F.H.
is supported by the UK Medical Research Council.
T.M.N. was a Visiting Student from the Department of
Pharmacognosy at the University of Oslo. Studies of
Sc500 mannan are collaborative with Professor B.
Smestad-Paulsen (Oslo), and Ms. Mia Young has re-
ceived funding from the EU Human Capital and Mobil-
ity Grant and COST D7 Chemistry. Serum IgG was
kindly supplied by Dr. R. Barnes (University of
Liverpool).
P er a cetyla ted 36-Mer Den d r im er (22). A solution of 16
(0.450 g, 0.092 mmol) in CH2Cl2 (15 mL) was added dropwise
to a stirred solution of 17 (0.01 g, 0.026 mmol), DCC (0.031 g,
0.082 mmol), and HOBT (0.011 g, 0.082 mmol) in CH2Cl2/DMF
(2:1) (50 mL) at 0 °C under an N2 blanket. The reaction
mixture was left stirring at room temperature for 5 days and
then worked up as described in the general procedure to obtain
the O-acetylated dendrimer 20, which was purified by GPC
1
to provide 22 (0.154 g, 39%): [R]D +36.4° (c 0.9, CHCl3); H
NMR (500 MHz, CD3SOCD3, 353 K) δ 1.92, 1.97, 2.01, 2.08
(432H, 4 × s), 2.41 (24H, br), 2.68 (12H, br), 3.46 (36H, br),
3.77 (36H, d, J ) 9.5 Hz), 3.95 (84H, br), 4.07 (36H, app d, J
) 12.0 Hz), 4.20 (36H, dd, J ) 5, 12.0 Hz), 4.82 (36H, app s),
5.08-5.18 (108H, band), 7.58 (12H, br), 8.50 (9H, br); 13C NMR
J O9804184