Novel dendritic α-sialosides: Synthesis of glycodendrimers based on a 3,3′-iminobis(propylamine) core

Article abstract of DOI:10.1021/jo961047z

The cluster or multivalent effect has been recognized as an effective means by which to increase binding interactions between carbohydrates and proteins. In fact, it has been demonstrated that sialylated multibranched L-lysine dendrimers were potent inhibitors of hemagglutination of human erythrocytes by Influenza viruses. In a continuation of these studies, the synthesis of novel glycodendrimers with even valencies from 2 to 16 and ending with equidistant thiosialoside residues is described. These symmetrical dendrimers were more readily characterized by standard NMR spectral techniques than previously reported nonsymmetrical dendrimers of this general type. The synthesis of the dendritic core was based on the regioselective protection of the primary amines of 3,3′-iminobis(propylamine) (4) using benzyl cyanoformate. The resulting secondary amine 5 was alkylated with tert-butyl bromoacetate to provide divalent core structure 6 with Cbz protected amines and acid protected tert-butyl ester. Sequential deprotection by trifluoroacetolysis or hydrogenation afforded acid 7 or diamine 8 as key precursors, respectively. The two fragments were coupled using HOBt/DIC strategy to provide Cbz-protected dendrimers with valencies of 2, 4, 8, and 16 in the first, second, third, and fourth generations, respectively, in reasonable to good yields (42-82%). Cbz-protected precursors were efficiently transformed into N-chloroacetylated dendrimers by hydrogenolysis and treatment of the resulting amines with chloroacetic anhydride (82-91%). N-Chloroacetylated dendrimers were then treated with an excess of 2-thiosialic acid derivative 3 to give fully protected sialodendrimers in 76-96% yields. Deprotection of sialodendrimers under Zemplen conditions followed by methyl ester saponification and purification by gel permeation chromatography afforded symmetrical dendritic α-thiosialosides 21, 23, 25, and 27 in fair yields (47-58%). These novel sialodendrimers, in keeping with their design, are currently being evaluated as inhibitors of human erythrocyte hemagglutination by Influenza viruses.

Full text of DOI:10.1021/jo961047z

7348
J . Org. Chem. 1996, 61, 7348-7354
Novel Den d r itic r-Sia losid es: Syn th esis of Glycod en d r im er s Ba sed
on a 3,3′-Im in obis(p r op yla m in e) Cor e
Diana Zanini and Rene´ Roy*
Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 Canada
Received J une 3, 1996X
The cluster or multivalent effect has been recognized as an effective means by which to increase
binding interactions between carbohydrates and proteins. In fact, it has been demonstrated that
sialylated multibranched L-lysine dendrimers were potent inhibitors of hemagglutination of human
erythrocytes by Influenza viruses. In a continuation of these studies, the synthesis of novel
glycodendrimers with even valencies from 2 to 16 and ending with equidistant thiosialoside residues
is described. These symmetrical dendrimers were more readily characterized by standard NMR
spectral techniques than previously reported nonsymmetrical dendrimers of this general type. The
synthesis of the dendritic core was based on the regioselective protection of the primary amines of
3,3′-iminobis(propylamine) (4) using benzyl cyanoformate. The resulting secondary amine 5 was
alkylated with tert-butyl bromoacetate to provide divalent core structure 6 with Cbz protected amines
and acid protected tert-butyl ester. Sequential deprotection by trifluoroacetolysis or hydrogenation
afforded acid 7 or diamine 8 as key precursors, respectively. The two fragments were coupled
using HOBt/DIC strategy to provide Cbz-protected dendrimers with valencies of 2, 4, 8, and 16 in
the first, second, third, and fourth generations, respectively, in reasonable to good yields (42-
82%). Cbz-protected precursors were efficiently transformed into N-chloroacetylated dendrimers
by hydrogenolysis and treatment of the resulting amines with chloroacetic anhydride (82-91%).
N-Chloroacetylated dendrimers were then treated with an excess of 2-thiosialic acid derivative 3
to give fully protected sialodendrimers in 76-96% yields. Deprotection of sialodendrimers under
Zemple´n conditions followed by methyl ester saponification and purification by gel permeation
chromatography afforded symmetrical dendritic R-thiosialosides 21, 23, 25, and 27 in fair yields
(47-58%). These novel sialodendrimers, in keeping with their design, are currently being evaluated
as inhibitors of human erythrocyte hemagglutination by Influenza viruses.
In tr od u ction
oligosaccharides in the design of potent adhesion inhibi-
tors, a strategy based on the cluster effect was success-
fully proposed. Clusters of R-sialosides have been pre-
pared.7 In addition, neoglycoproteins and glycopolymers
have also been designed to improve the inhibitory
potential of glycoconjugates.8 These sialylated neogly-
coproteins and glycopolymers, by virtue of their multi-
valent epitopes, possess the fundamental features nec-
essary to compensate for the low affinity of individual
R-sialosides. Sialylated polymers, because of their high
valencies, have contributed to increased inhibitory activ-
ity by a factor of a few thousand (mM to µM range).9 Still,
neoglycoproteins and glycopolymers have ill defined
chemical structures and are immunogenic. Conse-
quently, they may not constitute ideal candidates for
therapeutic uses.
Carbohydrate-protein interactions are responsible for
a wide range of biological phenomena including cancer
cell metastasis, inflammation, and infections by bacteria
and viruses.1 For example, binding of the viral mem-
brane glycoprotein hemagglutinin (HA) to host cell sia-
lyloligosaccharides present on glycolipids and glycopro-
teins mediates the infections of mammalian host tissues.2
N-Acetylneuraminic acid (sialic acid, NeuAc) represents
the most ubiquitous member of the sialic acid family of
derivatives present on cell surface glycolipids and glyco-
proteins and constitutes the key epitope recognized as
being essential for virus binding. The critical role played
by R-sialosides has been confirmed by inhibition experi-
ments with synthetic R-sialosides,3 X-ray data,4 and
proton NMR spectroscopic studies.5
Unfortunately, carbohydrate-protein interactions, as
opposed to protein-protein interactions, usually have low
dissociation constants (KD’s) and, therefore, their overall
attractive forces are generally weak.6 NeuAc and viral
HA interactions appear to be no exception (KD ≈ 3 mM).5
In order to compensate for the low KD’s of natural
(7) (a) Unverzagt, C.; Kelm, S.; Paulson, J . C. Carbohydr. Res. 1994,
251, 285. (b) Sabesan, S.; Duus, J . Ø.; Neira, S.; Domaille, P.; Kelm,
S.; Paulson, J . C.; Bock, K. J . Am. Chem. Soc. 1992, 114, 8363. (c)
Glick, G. D.; Knowles, J . R. J . Am. Chem. Soc. 1991, 113, 4701.
(8) (a) Lee, R. T.; Lee, Y. C. Enhanced Biochemical Affinities of
Multivalent Neoglycoconjugates in Neoglycoconjugates: Preparation
and Applications; Lee, Y. C., Lee, R. T., Eds.; Academic Press, San
Diego, CA, 1994; p 23. (b) Roy, R. Design and Syntheses of Glycocon-
jugates. In Modern Methods in Carbohydrate Synthesis; Khan, S.-H.,
O’Neil, R., Eds.; Harwood Academic: Amsterdam, 1995; p 378. (c)
Magnusson, G.; Chernyak, A. Ya.; Kihlberg, J .; Kononov, L. O.
Enhanced Biochemical Affinities of Multivalent Neoglycoconjugates.
In Neoglycoconjugates: Preparation and Applications; Lee, Y. C., Lee,
R. T., Eds.; Academic Press: San Diego, CA, 1994; p 53.
X Abstract published in Advance ACS Abstracts, October 1, 1996.
(1) (a) Varki, A. Glycobiology 1993, 3, 97. (b) Dwek, R. A. Chem Rev.
1996, 96, 683 and references cited therein.
(2) Corfield, A. P.; Schauer, R. In Sialic Acids, Chemistry, Metabo-
lism, and Functions; Schauer, R., Ed.; Cell Biology Monograph, Vol.
10; Springer-Verlag: New York, 1982, p 5.
(3) Pritchett, T. J .; Brossner, R.; Rose, U.; Paulson, J . C. Virology
1987, 160, 502.
(4) Weiss, W.; Brown, J . H.; Cusack, S.; Paulson, J . C.; Skechel, J .
J .; Wiley, D. C. Nature 1988, 333, 426.
(5) Sauter, N. K.; Bednarski, M. D.; Wurzburg, B. A.; Hanson, J .
E.; Whitesides, G. M.; Skechel, J . J .; Wiley, D. C. Biochemistry 1989,
28, 8388.
(9) (a) Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M.
J . Am. Chem. Soc. 1996, 118, 3787. (b) Roy, R.; Andersson, F. O.;
Harms, G.; Kelm, S.; Schauer, R. Angew. Chem., Int. Ed. Engl. 1992,
31, 1478. (c) Byramova, N. E.; Mochalova, L. V.; Belyanchikov, J . M.;
Matrosovich, M. N.; Bovin, N. V. J . Carbohydr. Chem. 1991, 10, 691.
(d) Roy, R.; Laferrie`re, C. A. Carbohydr. Res. 1988, 177, C1 and
references cited therein.
(6) Toone, E. J . Curr. Opin. Struct. Biol. 1994, 4, 719.
(10) Roy, R. Polym. News 1996, 21, 226.
S0022-3263(96)01047-X CCC: $12.00 © 1996 American Chemical Society

Novel Dendritic R-Sialosides
J . Org. Chem., Vol. 61, No. 21, 1996 7349
Sch em e 1
On the other hand, dendrimers with covalently at-
tached carbohydrates represent chemically well defined
polymers with known densities.10 In general, they may
be prepared by divergent or convergent approaches and
may also be synthesized as polydispersed structures.11
Bidirectional dendrimers can be used advantageously as
models for cell surface multiantennary glycoproteins as
they share unique structural parities. It therefore seems
a natural extension to consider bidirectional dendritic
R-sialosides as potent inhibitors of viral hemagglutina-
tion.
In fact, it has already been demonstrated that sialy-
lated dendrimers scaffolded onto multibranched L-lysine
afforded conjugates that were as potent as polymers in
the inhibition of hemagglutination of human erythrocytes
by Influenza viruses.12 However, the lack of symmetry
within the poly-L-lysine dendrimers previously reported
have rendered high field proton NMR spectral charac-
terization rather cumbersome and although the fractal
topology of these L-lysine based-glycodendrimers is likely
to better mimic cell surface multiantennary glycopro-
teins, novel symmetrical “sialodendrimers” having chemi-
cally equivalent thiosialoside residues represent com-
pounds of interest with possible therapeutic advantages.
In attempts to generate such compounds, and in efforts
to meet difficult synthetic challenges, we present herein
the design and synthesis of a new family of symmetrical
dendrimers with even valencies between 2 and 16 based
on a 3,3′-iminobis(propylamine) (4) core.
Sch em e 2
Although, the dendrimers presented here bear some
resemblance to commercially available Starburst PAM-
AM dendrimers, they have the distinct advantage of not
being susceptible to base-catalyzed retro-Michael degra-
dations. In addition, this methodology eliminates the
need for large excesses of reagents used to ensure
complete conversion, a problem commonly associated
with the Starburst PAMAM dendrimer synthesis.
modification14 of our previously established methodology
(Scheme 1).12
First, second, third, and fourth generation N-chloro-
acetylated dendrimers 9, 13, 16, and 19 with valencies
of 2, 4, 8, and 16 were efficiently and conveniently
synthesized in solution15 using Cbz protecting group
strategy and carbodiimide-hydroxybenzotriazole (HOBt)
coupling chemistry. Key monomeric precursors 7 and 8
were prepared in the following manner.
The primary amines of 3,3′-iminobis(propylamine) (4)
were regioselectively protected using the method of
Murahashi et al.16 to give diamine 5 (benzyl cyanofor-
mate, CH2Cl2, 72% yield). Diamine 5 was then alkylated
with tert-butyl bromoacetate in good yield (CH3CN, 87%)
to provide divalent core structure 6 with Cbz protected
amines and tert-butyl protected acid functionalities.
Trifluoroacetolysis of 6 gave acid 7 (96% yield) and
hydrogenolysis of the Cbz groups of 6 afforded diamine
8 (90%) (Scheme 2). Acid 7 and diamine 8 represent key
precursors in the synthesis of the glycodendrimers de-
scribed here.
Compounds 7 and 8 were coupled using diisopropyl-
carbodiimide (DIC) and HOBt to provide tetravalent Cbz-
protected dendrimer 10 in 82% yield. In order to avoid
tedious silica gel chromatography, it was found that the
crude mixture containing 10 could be treated with anion
exchange resin (HO-, Amberlite IRA-400) thereby elimi-
nating HOBt and excess acid used in the coupling. The
residue was then concentrated and diisopropylcarbodi-
imide urea and 10 were easily separated by chromatog-
Resu lts a n d Discu ssion
The approach described for the synthesis of these
glycodendrimers was based on the convergent assembly
of suitably multibranched dendrimers having N-chloro-
acetylated end groups to which thiolated carbohydrate
derivatives could be added at a later stage.10,12,13 This
strategy allows the incorporation of different carbohy-
drate haptens onto prebuilt dendritic structures. More-
over, the choice of thiolated glycosides stemmed from the
desire to generate glycodendrimers that would be resis-
tant to glycohydrolase activity. The method is general,
high-yielding and readily amenable to existing com-
mercially available amine-terminated dendrimers.
The R-thiosialoside functionality was introduced under
phase transfer catalysis (PTC) by treatment of acetochlo-
roneuraminic acid (1) with thioacetic acid12 to obtain
methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-
dideoxy-2-thio-D-glycero-R-D-galacto-2-nonulopyrano-
sonate 2. The thioacetate derivative 2 was chemoselec-
tively converted to thiol 3 (H2NNH2‚HOAc) using a
(11) (a) Ardoin, N.; Astruc, D. Bull. Soc. Chim. Fr. 1995, 132, 875.
(b) Issberner, J .; Moors, R.; Vo¨gtle, F. Angew. Chem., Int. Ed. Engl.
1994, 33, 2413. (c) Fre´chet, J . M. J . Science 1994, 263, 1710 and
references cited therein.
(12) (a) Roy, R.; Zanini, D.; Meunier, S. J .; Romanowska, A. J . Chem.
Soc., Chem. Commun. 1993, 1869. (b) Roy, R.; Zanini, D.; Meunier, S.
J .; Romanowska, A. ACS Symp. Ser. 1994, 560, 104.
(13) Page´, D.; Zanini, D.; Roy, R. Bioorg. Med. Chem. 1996, 4, in
press.
(15) The approach described here is also amenable to solid-phase
synthesis as described for L-lysine dendrimers (reference 12). Work is
in progress to evaluate the efficiency of this approach.
(16) Murahashi, S.-I.; Naota, T.; Nakajima, N. Chem. Lett. 1987,
879.
(14) Park, W. K. C.; Meunier, S. J .; Zanini, D.; Roy, R. Carbohydr.
Lett. 1994, 1, 179.

7350 J . Org. Chem., Vol. 61, No. 21, 1996
Zanini and Roy
Sch em e 3
of acid 11 and tetraamine 12. Acid 11 was prepared by
deprotection of tetravalent ester 10 with trifluoroacetic
acid. Using the above established HOBt/DIC coupling
techniques, 17 was isolated in 57% yield for the two step
process (Scheme 4). Deprotection of the Cbz-protected
Sch em e 4
17 provided hexadecavalent amine 18 in excellent yield
(97%).
N-Chloroacetylated di- (9), tetra- (13), octa- (16), and
hexadecavalent (19) dendrimers were obtained by treat-
ing the corresponding amines (8, 12, 15, and 18) with
chloroacetic anhydride in nonprotic solvents (CH3CN for
8, DMF for 12, and DMSO for 15 and 18). Changes of
solvent were necessary as the solubility properties of di-
(8), tetra- (12), octa- (15), and hexadecavalent (18) amines
were slightly different. Basic alumina chromatography
using 20% H2O/CH3CN as eluent was used to remove the
chloroacetate anions generated. Treatment of the crude
mixtures with HO- resin destroyed N-chloroacetyl func-
tionalities. DIPEA and similar bases could be added to
the reaction mixture to scavenge the chloroacetate an-
ions. However, these bases were not easily removed
during final purification as the multivalent chloroacetyl
moiety was far too polar to allow easy isolation of desired
compounds by standard chromatographic techniques.
Each dendrimer generation (9, 13, 16, and 19) was
then treated with a slight excess of methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-R-D-ga-
lacto-2-nonulopyranosonate (3) to provide completely
protected glycodendrimers 20, 22, 24, and 26. DMSO
was removed by lyophilization and the residue redis-
solved in the minimum amount of DMSO and precipi-
tated from ethyl acetate (Schemes 5-7) to give 20, 22,
24, and 26 in good to excellent yields (76 to 96%). The
polarity of the NeuAc end groups dismisses the possibility
of purification by silica gel chromatography for glyco-
dendrimers of higher generation. Dendrimers 22, 24, and
26 are soluble only in polar solvents, and precipitation
from these solvents must be accomplished with ethyl
acetate. Using other solvents such as ether or hexanes
also precipitated R-thiosialoside 3 and some disulfide
byproduct formed during the reaction. This phenomenon
was not envisioned for glycosides less polar than NeuAc.
Complete coupling was evident upon analysis of NMR
spectral data. The disappearance of the N-chloroacetyl
signal at 4.01 ppm (CDCl3 for the di- (9), tetra- (13), and,
octavalent (16) dendrimers, and DMSO-d6 for the hexa-
raphy to afford 10. Tetravalent dendrimer 10 was
deprotected to give tetraamine 12 (10% Pd/C, H2) which
was coupled to acid 8 using the strategy described above
to give Cbz-protected octamer 14 (66% yield). Repetition
of deprotection procedures gave octaamine 15 which was
again coupled to acid 8 providing hexadecavalent 17
(46%) (Scheme 3). All Cbz-protected dendrimers exhib-
ited consistent NMR and mass spectral data which
confirmed the purity of dendrimers 10, 15, and 17.
Dendrimer characterization was made easy by virtue of
their symmetry. 1H-NMR signals were superimposable
for each generation, and characteristic 1H NMR (CDCl3)
signals were observed at δ 2.50-2.55, 1.50-1.60, 3.00-
3.20, and 5.05 ppm for R-, â-, γ-, and Cbz CH2 residues,
respectively.
Alternatively, hexadecavalent dendrimer 17 could also
be prepared more efficiently by the convergent assembly

Novel Dendritic R-Sialosides
Sch em e 5
J . Org. Chem., Vol. 61, No. 21, 1996 7351
can be precisely integrated ((3%) relative to other
signals. Following carbohydrate attachment, the signal
was shifted upfield (3.41 and 3.81 ppm) due to the sulfur
atom and is further transformed into two doublets by
virtue of the newly created diastereotopic protons.
In conclusion, this methodology enabled the synthesis
of symmetrical sialodendrimers with sialic acid densities
corresponding to 2n, where n represents the n’th genera-
tion of the 3,3′-iminobis(propylamine) core. All yields
were fair to excellent, and the biological testing of
glycodendrimers 21, 23, 25, and 27, including enzyme
linked lectin assays (ELLA) with Limax flavus lectin to
various mucins and the inhibition of hemagglutination
of human erythrocytes by Influenza viruses, is in progress
and will be reported in due course.
Exp er im en ta l Meth od s
Gen er a l Meth od s. Proton chemical shifts (δ) are given
relative to internal CHCl3 (7.24 ppm) for CDCl3 solutions, to
internal dimethyl sulfoxide (2.49 ppm) for DMSO-d6 solutions,
and to internal HOD (4.76 ppm) for D2O solutions. Carbon
chemical shifts are given relative to deuterochloroform (77.0
ppm) and DMSO-d6 (39.4 ppm). Assignments were based on
COSY, HMQC, and DEPT experiments. Thiosialic acid de-
rivatives 2 and 3 were synthesized as previously described.12,14
P r ep a r a tion of 3,3′-Bis(ca r boben zyloxy)-3,3′-im in obis-
(p r op yla m in e) (5). To a solution of 3,3′-iminobis(propyl-
amine) (4) (2.00 g, 0.015 mol) in dry CH2Cl2 (50 mL) was added
dropwise a solution of benzyl cyanoformate (4.91 g, 0.030 mol)
in dry CH2Cl2 (25 mL) over a period of 2 h at 25 °C. Hydrogen
cyanide generated during the reaction was carefully introduced
into a solution of sodium hydroxide in water. After removal
of the organic solvent, the residue was subjected to column
chromatography and eluted with CHCl3 and MeOH (2:1) to
give compound 5 as a white solid in 72% yield (4.36 g, 0.11
mol).
5: 1H-NMR (CDCl3) δ 1.31 (bs, 1H, NH amine), 1.63 (t, 4H,
â-CH2), 2.61 (t, 4H, J R,â 7.4 Hz, R-CH2), 3.24 (m, 4H, γ-CH2),
5.06 (s, 4H, Cbz-CH2), 5.49 (s, 2H, Cbz-NH), 7.24-7.31 (m,
10H, Cbz-Ph); 13C-NMR (CDCl3) δ 29.6 (â-C), 39.5 (γ-C), 47.4
(R-C), 66.5 (Cbz-CH2), 128.0 (2×), 128.3, and 128.5 (Cbz-Ph,
ortho, meta, para), 136.7 (Cbz-Ph, C-1), 156.5 (CdO); FAB-
MS (pos) calcd for C22H29N3O4 399.22, found 400.3 (M+ + 1,
83.7% base peak).
P r ep a r a tion of Mon om er 6. To a solution of 5 (4.00 g,
0.010 mol) in acetonitrile (25 mL) was added tert-butyl
bromacetate (1.94 g, 0.010 mol). One equivalent of diisopro-
pylethylamine (DIPEA, 1.28 g, 0.010 mol) was added and the
solution stirred for 30 min at 25 °C. After solvent evaporation,
the residue was subjected to column chromatography and
eluted with a gradient of hexane/ethyl acetate to give yellow
resin 6 in 87% yield (4.42 g, 0.009 mol).
6: 1H-NMR (CDCl3) δ 1.40 (s, 9H, tBu), 1.62 (t, 4H, â-CH2),
2.51 (t, 4H, J R,â 6.3 Hz, R-CH2), 3.10 (s, 2H, NCH2C(O)), 3.24
(m, 4H, γ-CH2), 5.05 (s, 4H, Cbz-CH2), 5.72 (bs, 2H, Cbz-NH),
7.24-7.31 (m, 10H, Cbz-Ph); 13C-NMR (CDCl3) δ 27.0 (â-C),
28.1 (CH3’s), 39.4 (γ-C), 52.2 (R-C), 56.1 (NCH2C(O)), 66.3 (Cbz-
CH2), 81.4 (OCMe3), 2 × 127.9 and 128.4 (Cbz-Ph, ortho, meta,
para), 136.9 (Cbz-Ph, C-1), 156.6 (CdO); FAB-MS (pos) calcd
for C28H39N3O6 513.28, found 514.3 (M+ + 1, 29.1% base peak).
decamer (19)) and the emergence of new signals corre-
sponding to the incorporated NeuAc residues (H-4 at 4.83
ppm, H-3 equatorial at 2.70 ppm, and N-Ac at 1.83 ppm,
CDCl3 for di- (20) and tetravalent (22) R-sialosides and
DMSO-d6 for the octa- (24) and hexadecameric (26)
protected glycodendrimers) clearly established the extent
of sialoside incorporation (Figure 1).
Deprotection of glycodendrimers 20, 22, 24, and 26 by
sequential ester hydrolysis ((i) NaOMe/MeOH; (ii) 0.05
N NaOH) followed by gel permeation chromatography
(GPC, Biogel-P2, H2O as eluent) afforded fully depro-
tected glycodendrimers 21, 23, 25, and 27 with two, four,
eight, and sixteen NeuAc residues in moderate yields
(47-58%). Surprisingly, a two-step deprotection strategy
was necessary to ensure complete de-O-acetylation. No
deprotection was observed with shorter reaction times
nor when Zemple´n deprotection was avoided and NaOH
treatment performed directly.
The strategy presented here is a convenient and
efficient way of synthesizing dendritic glycosides. How-
ever, higher generation N-chloroacetylated dendrimers
and protected glycodendrimers are very polar compounds
and not readily isolable by standard chromatography
techniques (silica gel chromatography, reverse phase
chromatography). Alternative methods of carbohydrate
conjugation are currently under investigation. Still, the
distinct advantage of the N-chloroacetylated strategy
resides in the ability to determine the extent of glycoside
Syn th esis of Acid s 7 a n d 11. Typical Procedure:
A
solution of 6 (2.00 g, 3.90 mmol) in 50 mL of 30% trifluoroacetic
acid in CH2Cl2 was stirred vigorously for 3 h at 25 °C. The
solution was concentrated and dried under vacuum overnight.
The trifluoroacetate salt of 7 was recovered as a yellow resin
in 96% yield (2.14 g, 3.75 mmol) and used without further
purification.
The trifluoroacetate salt of 11 was isolated as above to
provide 11 as a yellow resin in quantitative yield.
7: 1H-NMR (CDCl3) δ 1.78 (m, 4H, â-CH2), 3.11 (m, 8H,
R-CH2, γ-CH2), 3.79 (s, 2H, NCH2C(O)), 4.99 (s, 4H, Cbz-CH2),
5.81 (bs, 2H, Cbz-NH), 7.24-7.28 (m, 10H, Cbz-Ph), 10.6-11.2
(bs, CO2H); 13C-NMR (CDCl3) δ 27.8 (â-C), 37.6 (γ-C), 53.0 (R-
C), 53.4 (NCH2C(O)), 66.9 (Cbz-CH2), 114.9 and 117.2 (tri-
1
coupling by high field H-NMR spectroscopy. As already
stated, the well resolved singlet corresponding to the
chloromethylene groups at 4.01 ppm (CDCl3, DMSO-d6)

7352 J . Org. Chem., Vol. 61, No. 21, 1996
Zanini and Roy
Sch em e 6
fluoroacetate salt), 127.9, 128.2, and 128.4 (Cbz-Ph, ortho,
meta, para), 136.3 (Cbz-Ph, C-1), 157.4 (acid CdO), 161.0 and
161.3 (Cbz CdO’s), 167.9 (trifluoracetate salt CdO); FAB-MS
(pos) calcd for C24H31N3O6 457.22, found 458.2 (M+ + 1, 17.2%
base peak).
P r ep a r a tion of Di- (8), Tetr a - (12), Octa - (15), a n d
Hexa d eca - (18) Am in es. Typical Procedure: To compound
6 (400 mg, 0.78 mmol) was added MeOH (10 mL) containing
10% Pd/C (40 mg). H2 was bubbled through the solution and
the mixture stirred for 30 min at 25 °C. The mixture was then
filtered and the Pd/C washed twice with MeOH. All filtrates
were combined and dried down giving compound 8 in 90% yield
(264 mg, 0.70 mmol). Yellow resin 8 was used as is.
Amines 12, 15, and 18 were obtained in a similar manner,
but with longer reaction times (4 h for 12, 20 h for both 15
and 18), as white solids in yields of 97, 98, and 97%,
respectively.
solvent used (DMF and DMSO for compounds 14 and 17) and
reaction times required for complete coupling as measured via
ninhydrin testing (20 h and 48 h, respectively). After chro-
matography, isolated yields were 66% for 14 and 46% for 17.
Using the same coupling procedure with tetraamine 12 and
acid 11 also gave compound 17 in a higher yield of 57%.
10: 1H-NMR (CDCl3) δ 1.40 (s, 9H, tBu), 1.52-1.59 (m, 12H,
â-CH2’s), 2.47-2.57 (m, 12H, R-CH2’s), 3.02-3.27 (m, 18H,
γ-CH2’s, NCH2C(O)’s), 5.04 (s, 8H, Cbz-CH2), 5.52 (bs, 4H, Cbz-
NH), 7.24-7.36 (m, 20H, Cbz-Ph), 7.58 (bs, 2H, amide NH);
13C-NMR (CDCl3) δ 27.2 (â-C’s), 28.1 (CH3’s), 37.3 and 38.9
(γ-C’s), 52.0 and 52.4 (R-C’s), 55.8 and 57.8 (NCH2C(O)’s), 57.8
(Cbz-CH2), 81.4 (OCMe3), 128.0 and 128.5 (Cbz-Ph, ortho,
meta, para), 136.7 (Cbz-Ph, C-1), 165.8-171.0 (CdO’s); FAB-
MS (pos) calcd for C60H85N9O12 1123.63, found 1125.1 (M+
1, 14.5% base peak).
+
14: 1H-NMR (CDCl3) δ 1.41 (s, 9H, tBu), 5.03 (s, 16H, Cbz-
CH2), 7.24-7.32 (m, 40H, Cbz-Ph); 13C-NMR (CDCl3) δ 28.1
(CH3’s), 66.6 (Cbz-CH2), 128.0, 128.1, and 128.5 (Cbz-Ph, ortho,
meta, para), 136.6 (Cbz-Ph, C-1); FAB-MS (pos) calcd for
C124H177N21O24 2344.32, found 2346.6 (M+ + 1, 0.4% base peak).
17: 1H-NMR (CDCl3) δ 1.41 (s, 9H, tBu), 5.04 (s, 32H, Cbz-
CH2), 7.24-7.30 (m, 80H, Cbz-Ph); 13C-NMR (CDCl3) δ 28.1
(CH3’s), 66.5 (Cbz-CH2), 128.0, 128.1, 128.3, and 128.5 (Cbz-
Ph, ortho, meta, para), 136.7 (Cbz-Ph, C-1).
8: 1H-NMR (CDCl3) δ 1.34 (s, 9H, tBu), 1.49 (m, 4H, â-CH2),
1.75 (bs, 4H, NH2), 2.49 (t, 4H, J R,â 7.0 Hz, R-CH2), 2.64 (t,
4H, J â,γ 6.7 Hz, γ-CH2), 3.07 (s, 2H, NCH2C(O)); 13C-NMR
(CDCl3) δ 28.1 (CH3’s), 30.9 (â-C), 40.3 (γ-C), 52.0 (R-C), 56.0
(NCH2C(O)), 170.9 (CdO); mass spectrum (CI) (rel intensity)
m/ z 246.1 (M+, 51.6%).
12: 1H-NMR (CDCl3) δ 1.43 (s, 9H, tBu), 2.44-2.78 (m, 28H,
R-CH2’s, γ-CH2NH2’s, NH2’s); FAB-MS (pos) calcd for C28H61N9O4
587.48, found 588.5 (M+ + 1, 7.3% base peak).
P r ep a r a tion of N-Ch lor oa cetyla ted Den d r im er s 9, 13,
16, 19. Typical Procedure: To a solution of amine 8 (294 mg,
0.78 mmol) in CH3CN (5 mL) was added chloroacetic anhydride
(1.2 equiv per amine functionality, 310 mg, 1.86 mmol). The
solution was stirred for 2 h at 25°C. The reaction was judged
to be complete by ninhydrin test. The solvent was removed
in vacuo, and the residue was subjected to basic alumina
chromatography using 20% H2O in CH3CN as eluent in order
to remove the chloroacetate anion of 9. Compound 9 was
isolated in 82% yield as a yellow resin (254 mg, 0.64 mmol).
N-Chloroacetylated dendrimers 13, 16, and 19 were isolated
in 82, 86, and 91% yields, respectively. The only difference in
their preparation included choice of solvent. Compound 12
was dissolved in DMF to give 13; amines 15 and 18 were each
dissolved in DMSO to prepare compounds 16 and 19, respec-
tively.
15: 1H-NMR (CDCl3) δ 1.42 (s, 9H, tBu), 1.45-1.72 (m, 44H,
â-CH2’s, NH2’s).
Syn th esis of Cbz-P r otected Den d r im er s 10, 14, 17.
Typical Procedure: To a solution of 8 (170 mg, 0.45 mmol) in
CH3CN (15 mL) were added acid 7 (1.2 equiv. per amine group,
617 mg, 1.08 mmol) already dissolved in CH3CN (2 mL) and
neutralized with DIPEA. To the stirred solution were added
diisopropylcarbodiimide (DIC, 136 mg, 1.08 mmol) and hy-
droxybenzotriazole (HOBt, 146 mg, 1.08 mmol), and the
mixture was stirred at 25 °C. The pH was kept at 9 by the
addition of DIPEA. Completion of the reaction was monitored
by ninhydrin test. After 3 h, the solution was treated with
HO- resin for 15 min in order to remove excess acid and HOBt.
The solution was concentrated and the residue subjected to
column chromatography using a slow gradient of CH3CN to
20% water in CH3CN. Tetravalent 10 was isolated as a yellow
resin in 82% yield (448 mg, 0.40 mmol).
9: 1H-NMR (CDCl3) δ 1.39 (s, 9H, tBu), 1.60 (m, 4H, â-CH2),
2.51 (t, 4H, J R,â 6.2 Hz, R-CH2), 3.11 (s, 2H, NCH2C(O)), 3.33
(m, 4H, γ-CH2), 3.95 (s, 4H, ClCH2), 7.47 (bs, 2H, amide NH);
13C-NMR (CDCl3) δ 26.4 (â-C), 28.1 (CH3’s), 38.2 (γ-C), 42.7
Compounds 14 and 17 were prepared in a similar fashion
from amines 12 and 15, respectively. Differences include

Novel Dendritic R-Sialosides
J . Org. Chem., Vol. 61, No. 21, 1996 7353
Sch em e 7
(ClCH2), 52.2 (R-C), 55.9 (NCH2C(O)), 81.5 (OCMe3), 166.2 and
171.0 (CdO’s); FAB-MS (pos) calcd for C16H29N3O4Cl2 398.11,
found 398.2 (M+ + 1, 5.8% base peak).
Peracetylated NeuAc dendrimers 22, 24, and 26 were
isolated in a similar manner. Of note, these protected gly-
codendrimers were too polar to be subjected to standard
chromatographic techniques. Instead, they were isolated
by redissolution in the minimum amount of DMSO (typically
300 µL) and precipitated with ethyl acetate. NMR spectral
data shows the incorporation of ethyl acetate in the dendrimer
core.
13: 1H-NMR (CDCl3) δ 1.42 (s, 9H, tBu), 4.01 (s, 8H, ClCH2);
13C-NMR (CDCl3) δ 28.2 (CH3’s), 42.8 (ClCH2); FAB-MS (pos)
calcd for C36H69N9O8Cl4 893.29, found 894.4 (M+ + 1, 1.3% base
peak).
1
16: H-NMR (CDCl3) δ 1.43 (s, 9H, tBu), 4.02 and 4.03 (2s,
16H, ClCH2’s); 13C-NMR (CDCl3) δ 28.2 (CH3’s), 41.0 and 42.8
(ClCH2’s).
20:
1H-NMR (CDCl3) δ 1.41 (s, 9H, tBu), 1.64 (t, 4H, J 6.7
19: 1H-NMR (DMSO-d6) δ 1.38 and 1.39 (2s, 9H, tBu), 4.03
(s, 32H, ClCH2); 13C-NMR (DMSO-d6) δ 27.8 (CH3’s), 42.7
(ClCH2).
Hz, â-CH2), 1.83 (s, 6H, NAc), 1.99, 2.01, 2.11, and 2.14 (4s,
24H, OAc’s), 2.02-2.10 (m, 2H, H-3ax), 2.61 (m, 4H, R-CH2),
2.70 (dd, 2H, J 3eq,4 12.7 Hz, J 3ax,3eq 4.7 Hz, H-3eq), 3.81 (m,
8H, γ-CH2, NCH2C(O), SCH2), 3.49 (d, 2H, J 15.9 Hz, SCH2),
3.73 (s, 6H, CO2CH3), 3.75-3.77 (m, 2H, H-6), 3.98-4.05 (m,
4H, H-5, H-9), 4.25 (dd, 2H, J 8,9, 12.4 Hz, J 9,9, 2.7 Hz, H-9′),
4.83 (ddd, 2H, H-4), 5.27 (dd, 2H, J 2.2 Hz, J 8.9 Hz, H-7),
5.35 (d, 2H, J 5,NHAc 10.0 Hz, NHAc), 5.37-5.39 (m, 2H, H-8),
6.88 (t, J R,NH 5.6 Hz, amide NH); 13C-NMR (CDCl3) δ 3 × 20.8,
21.4 (OAc’s), 23.1 (NAc), 27.2 (â-C), 28.2 (CH3’s), 32.5 (SCH2),
37.4 (C-3), 38.0 (γ-C), 49.3 (C-5), 51.8 (R-C), 53.2 (MeO), 55.2
(NCH2C(O)), 62.3 (C-9), 67.1 (C-7), 68.2 (C-8), 69.4 (C-4), 74.0
Syn th esis of P er a cetyla ted Den d r itic r-Th iosia losid es
20, 22, 24, 26. Typical Procedure: N-Chloroacetylated den-
drimer 9 (45 mg, 0.11 mmol) was dissolved in 1% Et3N/DMSO
(5 mL) and N2 bubbled through the solution. To this was
added thiol 3 (1.1 equiv per N-chloroacetyl functionality, 137
mg, 0.25 mmol) and the solution left stirring, under N2,
overnight at 25 °C. The solution was concentrated by lyo-
philization and subjected to column chromatography using a
gradient of CH3CN to 20% H2O/CH3CN giving off-white solid
20 in 96% yield (145 mg, 0.11 mmol).

7354 J . Org. Chem., Vol. 61, No. 21, 1996
Zanini and Roy
F igu r e 1. 1H NMR spectrum (CDCl3, 500 MHz) of compound 20.
(C-6), 82.3 (C-2), 168.2-171.0 (CdO’s); FAB-MS (pos) calcd for
C56H85N5O29S2 1339.48, found 1340.8 (M+ + 1, 28.0% base
peak).
(NAc), 23.1 (â-C), 32.6 (SCH2), 36.2 (γ-C), 38.6 and 40.1 (C-3),
41.0 and 51.2 (C-5), 51.8, 52.3, and 52.5 (R-C’s), 55.2 (NCH2C-
(O)), 62.2 and 62.8 (C-9), 66.8 and 67.4 (C-7), 67.7, 68.0, and
68.1 (C-8), 68.2, 71.2, and 71.4 (C-4), 74.1, 74.4, and 74.6 (C-
6), 84.5 and 85.1 (C-2), 169.7-174.6 (CdO’s); FAB-MS (pos)
calcd for C34H57N5O20S2 919.30, found 920.3 (M+ + 1, 0.6% base
peak).
23: 1H-NMR (D2O) δ 1.76-2.18 (m, 28H, â-CH2’s, H-3ax,
NAc), 2.80-2.94 (m, 4H, H-3eq), 2.55-2.68 and 3.20-3.95 (2m,
76H, R-CH2’s, γ-CH2’s, NCH2C(O), SCH2, and NeuAc residues
excluding above); 13C-NMR (D2O) δ 21.6 (NAc), 38.7 (SCH2),
84.4 (C-2).
22: 1H-NMR (CDCl3) δ 1.44 (bs, 9H, tBu), 1.84 (s, 12H, NAc),
1.99, 2.01, 2.02, and 2.11 (4s, 48H, OAc’s), 3.76 (s, 12H, CO2-
CH3), 3.76-3.83 (m, 4H, H-6); 13C-NMR (CDCl3) δ 20.9, 21.0,
and 21.5 (OAc’s), 23.1 (NAc), 28.1 (CH3’s), 32.5 (SCH2), 82.2
(C-2).
1
24: H-NMR (DMSO-d6) δ 1.40 (s, 9H, tBu), 1.65 (s, 24H,
NAc), 1.92, 1.98, 2.00, and 2.08 (4s, 96H, OAc’s), 3.75 (s, 24H,
CO2CH3), 3.76-3.95 (m, 24H, H-5, H-6, H-9); 13C-NMR (DMSO-
d6) δ 2 × 20.6, 20.8, and 21.0 (OAc’s), 22.6 (NAc), 27.8 (CH3’s),
32.5 (SCH2), 82.3 (C-2).
25: 1H-NMR (D2O) δ 1.84-2.15 (m, 60H, â-CH2’s, H-3ax,
NAc), 2.86 (dd, 8H, J 3eq,4 12.7 Hz, J 3ax,3eq 4.8 Hz, H-3eq), 3.20-
3.99 (m, 164H, R-CH2’s, γ-CH2’s, NCH2C(O), SCH2, and NeuAc
residues excluding above); 13C-NMR (D2O) (from HMQC) δ 21.5
(NAc), 32.5 (SCH2).
1
26: H-NMR (DMSO-d6) δ 1.40 (bs, 9H, tBu), 1.66 (s, 48H,
NAc), 1.92, 1.97, 2.02, and 2.08 (4s, 192H, OAc’s), 3.74 (s, 48H,
CO2CH3), 3.74-3.82 (m, 48H, H-5, H-6, H-9); 13C-NMR (DMSO-
d6) δ 2 × 20.6, 20.8, and 21.0 (OAc’s), 22.6 (NAc), 28.1 (CH3’s
by HMQC only), 32.2 (SCH2), 82.3 (C-2).
1
27: H-NMR (D2O) δ 1.80-2.11 (m, 124H, â-CH2’s, H-3ax,
P r ep a r a tion of F u lly Dep r otected Den d r itic r-Th io-
sia losid es 21, 23, 25, 27. Typical Procedure: To peracety-
lated glycodendrimer 20 (50 mg, 0.04 mmol) dissolved in
DMSO (0.5 mL) was added 1 N NaOMe in MeOH (5 mL) and
the solution stirred at 25 °C. After 8 h, MeOH was removed
in vacuo and 0.05 N NaOH added (5 mL). Again the mixture
was stirred for 8 h at 25 °C. Solvent was removed by
lyophilization, and the residue was purified by gel permeation
chromatography on Biogel-P2 column. Title compound 21 was
isolated, after freeze-drying, as a white, spongy solid in 58%
yield (21 mg, 0.02 mmol).
NAc), 2.80-2.92 (m, 16H, H-3eq), 3.14-4.00 (m, 340H, R-CH2’s,
γ-CH2’s, NCH2C(O), SCH2, and NeuAc residues excluding
above); 13C-NMR (D2O) δ 21.5 (NAc), 32.5 (SCH2), 85.2 and
86.9 (C-2).
Ack n ow led gm en t. We thank Dr. Glenn Facey and
Dr. Raj Capoor for running NMR spectral data. We are
grateful to the National Sciences and Engineering
Council of Canada (NSERC) for financial support and
for a postgraduate scholarship to D.Z.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
data of chromatographically isolated compounds 5, 6, 8-10,
14, 17, 20, 21, 23, 25, and 27 (24 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
Glycodendrimers 23, 25, and 27 were synthesized as above
in 58, 47, and 53% yields after GPC from compounds 22, 24,
and 26, respectively.
1
21: H-NMR (D2O) δ 1.86 and 1.93 (2dd, 2H, J 12.3 Hz, J
12.3 Hz, H-3ax), 1.97-2.08 (m, 4H, â-CH2), 2.10 and 2.11 (2s,
6H, NAc), 2.84-2.95 (m, 2H, H-3eq), 3.30-3.49 (m, 8H, R-CH2,
γ-CH2), 3.54-3.99 (m, 20H, NCH2C(O), SCH2, and NeuAc
residues excluding above); 13C-NMR (D2O) δ 21.6 and 21.7
J O961047Z