Practical Synthesis of Starburst PAMAM &α-Thiosialodendrimers for Probing Multivalent Carbohydrate-Lectin Binding Properties

Full text of DOI:10.1021/jo972061u

3486
J . Org. Chem. 1998, 63, 3486-3491
lactones of lactose and maltose have been conjugated
P r a ctica l Syn th esis of Sta r bu r st P AMAM
to PAMAM dendrimers.9 A TN-antigen peptide10 has
been coupled to the amine-terminated dendrimers via
amide bond formation. Last, an isothiocyanate coupling
strategy11-13 has been employed to conjugate PAMAM
dendrimers to R-11-13 and â-D-mannose,11 â-D-glucose,11
â-cellobiose,11 and â-lactose11 isothiocyanate derivatives.
Spherical Starburst PAMAM dendrimers (1-4)14 are
made of â-alanine repeating units and are commercially
available in fairly large quantities. They represent the
first successful synthesis of spherical, highly branched
dendrimers up to generation 10 with a defined number
of amine surface groups, and their structures are depicted
in Figure 1. Due to their hydrophilic backbones, they
could be used advantageously over other dendrimers for
biological investigations. PAMAM-based glycodendrim-
ers incorporating sialic acid residues have thus far not
been reported. In keeping with the design of chemically
well-defined multivalent sialosides and for purposes of
comparison to previously described R-thiosialodendrim-
ers,4,5 the synthesis and relative binding properties of
sialylated PAMAM dendrimers are presented herein.
Such novel “nanostructures” should be helpful to better
refine our understanding of multiple carbohydrate-
protein interactions over poorly defined polymers.
r-Th iosia lod en d r im er s for P r obin g
Mu ltiva len t Ca r boh yd r a te-Lectin Bin d in g
P r op er ties
Diana Zanini and Rene´ Roy*
Department of Chemistry, University of Ottawa, Ottawa,
ON, Canada K1N 6N5
Received November 10, 1997
Synergistic cluster or multivalent effects are known to
compensate for the weak binding constants of natural
oligosaccharides via the cooperative combination of mul-
tiple carbohydrate-protein interactions.1 Exploiting the
concept of multivalency, sialic acid-containing polymers2
and, more recently, sialodendrimers3-5 have been shown
to be potent inhibitors of the hemagglutination of human
erythrocytes by influenza viruses. Glycodendrimers6 are
monodispersed macromolecules with covalently attached
carbohydrate residues. Because glycodendrimers are a
chemically well-defined series of structurally similar
neoglycoconjugates differing in the number of sugar
epitopes, they lend themsleves as useful tools in a
systematic analysis of multivalency.
Amine-terminated PAMAM cores 1-4 were conjugated
to p-isothiocyanatophenyl R-sialoside 8 to give polythio-
urea derivatives. This approach to carbohydrate-den-
drimer conjugation allowed minimal manipulation of the
dendritic cores and proceeded smoothly.11-13 Several
methods for the preparation of glycosyl isothiocyanates
have been reported.15-17 While these procedures may
proceed efficiently for most simple carbohydrates, sialic
acid, with its unique anomeric configuration, does not
lend itself easily to these techniques. Instead, liquid-
liquid phase transfer catalysis (PTC)18 was used for the
synthesis of the key p-nitrophenyl 2-thio-R-sialoside 6.
Acetochloroneuraminic acid (5) was prepared according
to published procedures.19 This was then treated with
tetra-n-butylammonium hydrogen sulfate (TBAHS, 1
equiv, 25 °C) as the catalyst with p-nitrothiophenol (1.5
equiv, 25 °C) in equal volumes of ethyl acetate and 1 M
Na2CO3 to give thioglycoside 6 in 75% yield (Scheme 1).
The reaction occurred with complete stereocontrol to give
the glycoside shown (6) as judged by well-documented
data for 6 (1H NMR (CDCl3) δ 2.77 for H-3eq).20 The nitro
group in sialic acid derivative 6 was efficiently reduced
with tin(II) chloride in refluxing ethanol to give the
In fact, since the first reported synthesis of L-lysine-
based R-thiosialodendrimers,3 much scientific effort has
gone into the preparation of other glycodendrimers.
Ashton et al.7,8 have reported glycodendrimers bearing
up to 36 D-glucose residues. R-Thiosialodendrimers based
on N-(3-aminopropyl)-1,3-propanediamine [3,3′-iminobis-
(propylamine)]4 and gallic acid5 cores have been previ-
ously described. Other work has stemmed from the
attachment of various carbohydrate derivatives to
Starburst PAMAM dendrimers. To date, disaccharide
(1) For reviews on multivalency, see: (a) Zanini, D.; Roy, R.
Architectonic Neoglycoconjugates: Effects of Shapes and Valencies in
Multiple Carbohydrate-Protein Interactions. In Carbohydrate Mim-
ics: Concepts and Methods; Chapleur, Y., Ed.; Verlag Chemie: Wein-
heim, Germany, 1998; Chapter 20, pp 385-415. (b) Roy, R. Top. Curr.
Chem. 1997, 187, 241. (c) Lee, Y. C.; Lee, R. Acc. Chem. Res. 1995, 28,
321. (d) Roy, R. Sialoside Mimetics and Conjugates as Anti-inflam-
matory Agents and Inhibitors of Flu Virus Infections. In Carbohydrates
in Drug Design; Witczak, Z. J .; Nieforth K. A., Eds.; Marcel Dekker:
NY, 1997; Chapter 3, pp 83-135. (e) Kiessling, L. L.; Pohl, N. L. Chem.
Biol. 1996, 3, 71. (f) Roy, R. The Chemistry of Neoglycoconjugates. In
Carbohydrate Chemistry; Boons, G.-J ., Ed.; Blackie Academic &
Professional: London, U.K., 1998; Chapter 7, pp 243-321.
(2) (a) Roy, R.; Laferrie`re, C. A. Carbohydr. Res. 1988, 177, C1. (b)
Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M. J . Am.
Chem. Soc. 1996, 118, 3787. (c) Roy, R.; Andersson, F. O.; Harms, G.;
Kelm, S.; Schauer, R. Angew. Chem., Int. Ed. Engl. 1992, 31, 1479.
(d) Byramova, N. E.; Mochalova, L. V.; Belyanchikov, J . M.; Matroso-
vich, M. N.; Bovin, N. V. J . Carbohydr. Chem. 1991, 10, 691 and
references therein.
(9) Aoi, K.; Itoh, K.; Okada, M. Macromolecules 1995, 28, 5391.
(10) Toyokumi, T.; Singhal, K. Chem. Soc. Rev. 1995, 231.
(11) Lindhorst, T. K.; Kieburg, C. Angew. Chem., Int. Ed. Engl. 1996,
35, 1953.
(12) Page´, D.; Aravind, S.; Roy, R. Chem. Commun. 1996, 1913.
(13) Page´, D.; Roy, R. Bioconjugate Chem. 1997, 8, 714.
(14) (a) Tomalia, D. A.; Naylor, A. N.; Goddard, W. A., III. Angew.
Chem., Int. Ed. Engl. 1990, 29, 617. (b) Tomalia, D. A.; Dvornic, P. R.
Nature 1994, 617. Tomalia, D. A.; Esfand, R. Chem. Ind. 1997, 11,
416 and references therein.
(3) (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.
(4) (a) Zanini, D.; Roy, R. J . Org. Chem. 1996, 61, 7348. (b) Zanini,
D.; Roy, R. J . Am. Chem. Soc. 1997, 119, 2088.
(5) Meunier, S. J .; Wu, Q.; Wang, S.-N.; Roy, R. Can. J . Chem. 1997,
75, 1472.
(6) For recent reviews on glycodendrimers and glycopolymers, see
also: (a) Roy, R. Curr. Opin. Struct. Biol. 1996, 6, 992. (b) Roy, R.
Polym. News 1996, 21, 226. (c) Roy, R. Trends Glycosci. Glycotechnol.
1996, 8, 79-99.
(7) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; J ayaraman, N.;
Nepogodiev, S. A.; Stoddart, J . F. Chem.sEur. J . 1996, 2, 1115.
(8) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; J ayaraman, N.; Stoddart,
J . F. Angew. Chem., Int. Ed. Engl. 1997, 36, 732.
(15) Camarosa, M. J .; Fernandez-Resa, P.; Garcia-Lopez, M. T.; de
las Heras, F. G. Synthesis 1984, 504.
(16) Rothermal, J .; Weber, B.; Faillard, H. Leibigs Ann. Chem. 1992,
799.
(17) Lindhorst, T. K.; Kieburg, C. Synthesis 1995, 1228.
(18) Roy, R.; Tropper, F. D.; Cao, S.; Kim, J .-M. ACS Symp. Ser.
1997, 659, 163.
(19) Kuhn, R.; Luts, P.; MacDonald, D. L. Chem. Ber. 1966, 99, 611.
S0022-3263(97)02061-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/17/1998

Notes
J . Org. Chem., Vol. 63, No. 10, 1998 3487
F igu r e 1. G0 PAMAM core with 4 surface amines 1. G1 PAMAM core with 8 surface amines 2. G2 PAMAM core with 16 surface
amines 3. G3 PAMAM core with 32 surface amines 4.
corresponding p-aminophenyl thioglycoside 7 (2 h, 99%).
Derivative 7 was treated with thiophosgene in dichlo-
romethane (2 equiv, diisopropylethylamine) to provide
p-isothiocyanatophenyl sialoside 8 in 87% yield. Isolated
8 was used for dendrimer conjugation.
Commercially available methanolic solutions of PAM-
AM dendrimers 1-4 were concentrated in vacuo. CH2-
Cl2 was then added and coevaporated with any residual
MeOH three times under reduced pressure. The result-
ing residues were dissolved in CH2Cl2 for 1 or in DMF
for 2-4. Diisopropylethylamine (DIPEA, 1 equiv per
amine functionality) was then added to the solution.
Sialyl isothiocyanate derivative 8 (1.2 equiv per amine
moiety) in CH2Cl2 was added dropwise to the solutions
which were stirred overnight at room temperature
(Scheme 2). The reaction mixtures were concentrated in
vacuo, and the residues were dialyzed against a mixture
of DMSO and H2O in which the fully protected sialoden-
drimers were freely soluble (1:1, v/v, MW cutoff 2 kDa).
PAMAM-based R-thiosialodendrimers 9a -12a were ob-
tained in excellent yields (71-100%) after freeze-drying.
Complete deprotection of sialodendrimers 9a -12a by
sequential ester hydrolysis ((i) NaOMe/MeOH, (ii) 0.05
M NaOH) followed by dialysis against DMSO/H2O (1:1,
v/v, MW cutoff 2 kDa) afforded water-soluble dendrimers
9b-12b with 4, 8, 16, and 32 NeuAc residues, respec-
tively (59-93%), after liophylization. The high field (500
MHz) 1H NMR spectra confirmed complete incorporation
of sialic acid residues ((2-3%). Key signals included the
â-CH2 of the dendritic core at δ 2.28 ppm and the NAc,
H-3eq, and H-4 of the NeuAc moieties at δ 1.65, 2.70,
and 4.69 ppm in DMSO-d6, respectively.
(20) (a) Dabrowski, U.; Friebolin, H.; Brossman, R.; Supp, M.
Tetrahedron Lett. 1979, 4637. (b) Paulsen, H.; Tiex, H. Carbohydr. Res.
1984, 125, 47. (c) Okamoto, K.; Kodo, T.; Goto, T. Tetrahedron Lett.
1986, 27, 5229. (d) Okamoto, K.; Kodo, T.; Goto, T. Tetrahedron 1987,
43, 5919. (e) Hori, H.; Nakajima, T.; Nishida, Y.; Ohrui, H.; Maguro,
H. Tetrahedron Lett. 1988, 29, 6317.
To demonstrate the ability of these R-thiosialoden-
drimers to bind to the slug lectin from Limax flavus
(LFA), turbidimetric analysis (nephelometry) was ini-

3488 J . Org. Chem., Vol. 63, No. 10, 1998
Notes
Sch em e 1
solid-phase inhibition of the binding of human R1-acid
glycoprotein to LFA. These results confirm that an
amplification in carbohydrate-protein interactions is
related to an increase in the valency of sugar residues
in neoglycoconjugates. It is unlikely that the origin of
this effect relied on the spanning of these novel sialo-
dendrimers with two remotely positioned binding sites
of this dimeric lectin, as might be the case with some
high molecular weight glycopolymers. Alternatively, one
may speculate that the higher permanent local concen-
tration of the multivalent carbohydrate ligands is re-
sponsible for the higher avidity (not affinity) of these
molecules, presumably by affecting their kon/koff ratios.
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 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 DMSO-d6 (39.4
ppm). Assignments were based on COSY, HMQC, and DEPT
experiments. Phenyl 2-thio-R-sialoside 6a was obtained as
previously described.21 Human R1-acid glycoprotein (orosomu-
coid) was purchased from Sigma Chemical Co., and the lectins
LFA and peroxidase labeled LFAwere obtained from E-Y Labo-
ratories (San Mateo, CA).
P r ep a r a tion of Meth yl (4-Nitr op h en yl 5-a ceta m id o-
4,7,8,9-tetr a -O-a cetyl-3,5,-d id eoxy-2-th io-D-glycer o-r-D-ga -
la cto-2-n on u lop yr a n osid )on a te (6). To a solution of freshly
prepared acetochloroneuraminic acid 5 (3.30 g, 6.47 mmol) in
EtOAc (60 mL) was added a solution of p-nitrothiophenol (1.21
g, 7.73 mmol) and TBAHS (2.20 g, 6.47 mmol) in 1 M Na2CO3
(60 mL). The mixture was stirred vigorously for 1 h at room
temperature and next diluted with 125 mL each of EtOAc and
of saturated NaHCO3. The organic phase was separated and
washed with saturated NaHCO3 (2 × 100 mL) followed by
saturated NaCl (100 mL). The organic phase was dried over
Na2SO4 and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography using a
gradient of hexanes to EtOAc to give compound 6 as an off-white
foam in 75% yield (3.05 g, 4.85 mmol): mp 167.0-173.0 °C; [R]D
28.5° (c ) 1.2, CHCl3); lit.21 mp 168.0-172.0 °C, [R]D 27.6° (c )
1.0, MeOH).
tially performed. These microquantitative precipitation
experiments confirmed the direct binding and cross-
linking properties of the PAMAM-based R-thiosialoden-
drimers with LFA since nice and well-organized precipi-
tates could be directly visualized with the naked eye and
quantified as a function of time by optical density
measurements at 490 nm (results not shown, see Ex-
perimental Section).
The relative efficiency of sialylated PAMAM dendrim-
ers 9b-12b to inhibit the binding of horseradish peroxi-
dase (HRPO) labeled LFA to human R1-acid glycoprotein
(orosomucoid) was then determined by a competitive
enzyme-linked lectin assay (ELLA). In this experiment,
the IC50 of each sialodendrimer was measured, and the
results are shown in Table 1. Phenyl 2-thio-R-sialoside
(6a )21 was used as monovalent standard.
In this spherical R-sialodendrimer series, an increase
in multivalency resulted in a steady increase in inhibitory
potential. Glycodendrimers with valencies of 4 (9b), 8
(10b), 16 (11b), and 32 (12b) exhibited IC50’s of 69.2, 21.5,
2.89, and 1.13 nM, respectively (277, 172, 46.2, and 36.2
nM on a per sialoside residue basis, respectively, Table
1). This represents a 210-fold (6.7-fold/sialoside) jump
in inhibitory potential over the monomeric analogue.
Once again, the increased binding between carbohydrates
and proteins may be attributed to an increase in multi-
valency as reflected by the well-known cluster effect.1b
This is directly evident when comparing IC50 and/or
relative potency as a function of dendrimer valency.
It should be noted that when compared to the previ-
ously reported tetravalent 3,3′-iminobis(propylamine)-
based R-thiosialodendrimer,4 in the same set of experi-
ments, tetravalent sialylated PAMAM dendrimer 9b
exhibited a higher IC50 value. That is, at the second
generation, the 3,3′-iminobis(propylamine)-based diver-
gent series seems to be a better inhibitor of the carbo-
hydrate-protein interaction studied. However, at higher
generations, these spherical sialodendrimers appear to
have structural organizations and/or aglycon spacer
requirements more suitable than the 3,3′-iminobis(pro-
pylamine) R-thiosialodendrimers reported earlier4 for the
P r ep a r a tion of Meth yl (4-Am in op h en yl 5-a ceta m id o-
4,7,8,9-tetr a -O-a cetyl-3,5-d id eoxy-2-th io-D-glycer o-r-D-ga -
la cto-2-n on u lop yr a n osid )on a te (7). p-Nitrophenyl derivative
6 (40.0 mg, 0.064 mmol) was suspended in absolute ethanol (10
mL) to which was added tin(II) chloride dihydrate (SnCl2‚2H2O,
75.0 mg, 0.402 mmol). The reaction mixture was stirred at 70
°C for 2 h and then cooled and poured onto ice-water and the
final pH adjusted to ∼8 with NaHCO3. The resulting mixture
was filtered and the clear filtrate extracted with EtOAc (3 × 20
mL). The organic layers were combined, washed successively
with saturated NaHCO3 (20 mL), H2O (20 mL), and saturated
NaCl (20 mL), dried over Na2SO4, and concentrated under
reduced pressure. Title compound 7 was obtained as an off-
white powder in 99% yield (37.7 mg, 0.063 mmol) and used
directly in subsequent reactions without further purification: mp
96.0-101.0 °C; [R]D 26.0° (c ) 1.0, CHCl3); lit.21 mp 98.0-102.0
°C, [R]D 26.1° (c ) 1.270, MeOH).
P r ep a r a tion of Meth yl (4-Isoth iocya n a top h en yl 5-a c-
eta m id o-4,7,8,9-tetr a -O-a cetyl-3,5-d id eoxy-2-th io-D-glycer o-
r-D-ga la cto-2-n on u lop yr a n osid )on a te (70). p-Aminophenyl
derivative 7 (35.0 mg, 0.058 mmol) was dissolved in CH2Cl2 (20
mL) containing diisopropylethylamine (DIPEA, 18.7 mg, 0.145
mmol). Thiophosgene (16.7 mg, 0.145 mmol) was added and the
solution stirred at 25 °C for 1 h. The solvent was evaporated
under reduced pressure and the residue purified by silica gel
column chromatography using a gradient of hexanes to EtOAc
as eluent to afford 8 in 87% yield (32.6 mg, 0.051 mmol): mp
125.0-130.1 °C; [R]D 26.3° (c ) 1.0, CHCl3); 1H NMR (CDCl3) δ
1.85 (s, 3H, NAc), 2.00, 2.03, 2.04, 2.13 (4s, 12H, OAc’s), 2.02
(m, 1H, H-3ax), 2.80 (dd, 1H, J 3ax,3eq 12.8 Hz, J 3eq,4 4.7 Hz,
H-3eq), 3.57 (s, 3H, OCH3), 3.78-3.90 (m, 2H, H-5, H-6), 3.96-
(21) Cao, S.; Meunier, S. J .; Andersson, F. O.; Letellier, M.; Roy, R.
Tetrahedron: Asymmetry 1994, 5, 2303.

Notes
J . Org. Chem., Vol. 63, No. 10, 1998 3489
Sch em e 2

3490 J . Org. Chem., Vol. 63, No. 10, 1998
Notes
11a : 1H NMR (DMSO-d6) δ 1.64 (s, 48H, NAc), 1.77 (m, 16H,
H-3ax), 1.89, 1.99, 2.00, 2.01 (4s, 192H, OAc’s), 2.22 (bs, 56H,
b-CH2), 2.45-2.99 (m, 76H, a-CH2, c-CH2, H-3eq), 3.19-3.40
(2m, 56H, d-CH2), 3.52-3.60 (m, 56H, e-CH2), 3.53 (s, 48H,
OCH3), 3.59-3.79 (2m, 32H, H-5, H-6), 4.09 (m, 16H, H-9), 4.28
(m, 16H, H-9′), 4.69 (ddd, 16H, H-4), 5.13 (m, 16H, H-7), 5.19
(m, 16H, H-8), 7.38 (d, 32H, J ortho,meta 8.2 Hz, H-ortho), 7.54 (d,
32H, H-meta), 7.62 (d, 16H, J 5,NHAc 9.5 Hz, NHAc), 7.80-8.15
(3bs, 44H, NHC(S)NHPh, amide NH’s), 9.90 (bs, 16H, NHC(S)-
NHPh); 13C NMR (DMSO-d6) δ 20.6, 20.8 (OAc’s), 22.6 (NAc),
37.7 (C-3), 37.8 (C-d), 40.5 (C-a), 43.4 (C-e), 47.7 (C-5), 52.8
(OCH3), 61.7 (C-9), 67.4 (C-7), 68.9 (C-8), 69.5 (C-4), 74.1 (C-6),
87.3 (C-2), 122.0 (C-meta), 122.2 (C-para), 1366.4 (C-ortho), 141.3
(C-ipso), 167.8-170.1 (CdO’s), 180.4 (CdS).
Ta ble 1. In h ibition of Bin d in g of Hu m a n r1-Acid
Glycop r otein to Lim a x fla vu s by Sia lod en d r im er s
relative
compound
IC50 (nM)a
242
potencya
phenyl 2-thio-R-sialoside 6a
PAMAM-based 4mer 9b
PAMAM-based 8mer 10b
PAMAM-based 16mer 11b
PAMAM-based 32mer 12b
3,3′-iminobis(propylamine)-
based 4mer4,5
1
69.2 (277)
21.5 (172)
2.89 (46.2)
1.13 (36.2)
37.2 (149)
3.5 (0.87)
11 (1.4)
84 (5.2)
210 (6.7)
6.5 (1.6)
a
Values in parentheses are based on a per sialoside residue.
12a : 1H NMR (DMSO-d6) δ 1.65 (s, 96H, NAc), 1.78 (m, 32H,
H-3ax), 1.90, 2.00, 2.01, 2.02 (4s, 384H, OAc’s), 2.25 (bs, 120H,
b-CH2), 2.52-2.85 (m, 156H, a-CH2, c-CH2, H-3eq), 3.00-3.60
(m, 240H, d-CH2, e-CH2), 3.56 (s, 96H, OCH3), 3.59-3.79 (2m,
64H, H-5, H-6), 4.09 (m, 32H, H-9), 4.28 (m, 32H, H-9′), 4.69
(ddd, 32H, H-4), 5.13 (m, 32H, H-7), 5.19 (m, 32H, H-8), 7.38 (d,
64H, J ortho,meta 8.2 Hz, H-ortho), 7.54 (d, 64H, H-meta), 7.62 (d,
32H, J 5,NHAc 9.5 Hz, NHAc), 7.75-8.15 (m, 92H, NHC(S)NHPh,
amide NH’s), 9.90 (bs, 32H, NHC(S)NHPh); 13C NMR (DMSO-
d6) δ 20.6, 20.7 (OAc’s), 22.5 (NAc), 32.4 (C-b), 37.8 (C-3), 43.4
(C-e), 47.6 (C-5), 49.4 (C-c), 52.8 (OCH3), 61.7 (C-9), 67.3 (C-7),
68.9 (C-8), 69.5 (C-4), 74.1 (C-6), 87.3 (C-2), 122.0 (C-meta), 122.2
(C-para), 136.4 (C-ortho), 141.3 (C-ipso), 167.7-170.1 (CdO’s),
180.4 (CdS).
P r ep a r a tion of F u lly Dep r otected P AMAM-Ba sed r-Th i-
osia lod en d r im er s 9b, 10b, 11b, a n d 12b. Typ ica l P r oce-
d u r e. To peracetylated glycodendrimer 9b (47.2 mg, 0.015
mmol) dissolved in DMSO (0.5 mL) was added 1 M NaOMe in
MeOH (5 mL), and the solution was stirred at 25 °C. After 1 h,
MeOH was evaporated under reduced pressure and 0.05 M
NaOH (5 mL) added. After 1.5 h, the solution was neutralized
with 1 M HCl and the solvent removed by lyophilization. The
resulting residue was purified by dialysis against 1:1 DMSO/
H2O (MW cutoff 2 kDa) and then freeze-dried. Compound 9b
was isolated as a white, spongy solid in 80% yield (28.9 mg, 0.012
mmol).
Fully deprotected R-thiosialodendrimers 10b, 11b, and 12b
were obtained in the same manner from glycodendrimers 10a ,
11a , and 12a in 79, 100, and 100% yields, respectively.
9b: 1H NMR (DMSO-d6) δ 1.66 (dd, 4H, J 11.9 Hz, H-3ax),
1.84 (s, 12H, NAc), 2.25-2.59 (m, 12H, b-CH2, H-3eq), 2.63-
3.00 (m, 12H, a-CH2, c-CH2), 3.05-3.60 (m, 44H, d-CH2, e-CH2,
and NeuAc residues excluding above), 4.27, 4.59, 4.72, 5.18 (4bs,
16H, OH’s), 7.22-8.15 (4m, 28H, amide NH’s, NHC(S)NHPh,
H-ortho, H-meta, NHAc), 9.88-10.20 (4bs, 4H, NHC(S)NHPh);
13C NMR (DMSO-d6) δ 22.6 (NAc), 37.8 (C-d), 40.4 (C-3), 40.7
(C-a), 42.1 (C-c), 43.2 (C-e), 51.8 (C-5), 63.2 (C-9), 66.8 (C-7), 68.7
(C-8), 71.3 (C-4), 76.1 (C-6), 86.5 (C-2), 121.5, 122.2 (C-meta,
C-para), 136.2 (C-ortho), 141.5 (C-ipso), 169.2-172.1 (CdO’s),
180.4 (CdS).
4.11 (m, 1H, H-9), 4.31 (m, 1H, H-9′), 4.83 (ddd, 1H, J 3ax,4 11.7
Hz, J 4,5 10.3 Hz, H-4), 5.10 (d, 1H, J NH,5 9.9 Hz, NH), 5.23-5.27
(m, 2H, H-7, H-8), 7.16 (d, 2H, J ortho,meta 8.6 Hz, H-ortho), 7.45
(d, 1H, H-meta); 13C NMR (CDCl3) δ 20.7, 20.9 (OAc’s), 23.1
(NAc), 38.1 (C-3), 49.3 (C-5), 52.8 (OCH3), 60.3 (C-9), 62.0 (C-7),
67.5 (C-7), 69.2 (C-4), 69.4 (C-8), 74.5 (C-6), 126.0 (C-ortho), 127.9
(NdCdS), 132.9 (C-para), 137.1 (C-ipso), 137.2 (C-meta), 167.5-
170.8 (CdO’s); FAB-MS (pos.) calcd for C27H32N2O12S 640.14;
found 641.2 (M+ + 1, 18.0). Anal. Calcd for C27H32N2O12S: C,
50.62; H, 5.03; N, 4.37. Found: C, 50.23; H, 5.10; N, 4.52.
P r ep a r a tion of P er a cetyla ted P AMAM-Ba sed r-Th io-
sia lod en d r im er s 9a , 10a , 11a , a n d 12a . Typ ica l P r oced u r e.
A methanolic solution of amine-terminated tetravalent Starburst
PAMAM dendritic core 1 (23.0 mg of a 36.02% (w/w) solution in
MeOH, 0.016 mmol, Dendritech (Midland, MI)) was evaporated
under reduced pressure. The resulting residue was redissolved
in CH2Cl2 (5 mL) and the solution re-evaporated. This was
repeated three times. Compound 1 was then dissolved in CH2-
Cl2 (5 mL), to this was added diisopropylethylamine (DIPEA,
8.3 mg, 0.064 mmol), and the solution was stirred at room
temperature. Sialic acid isothiocyanato derivative 8 (50.0 mg,
0.078 mmol) dissolved in CH2Cl2 (5 mL) was added dropwise to
the stirred solution and the reaction left at 25 °C. After 20 h,
the reaction mixture was concentrated in vacuo and then
dialyzed against 1:1 DMSO/H2O (MW cutoff 2 kDa). The
resulting solution was lyophilized to give tetravalent PAMAM-
based R-thiosialodendrimer 9a as an off-white solid in 94% yield
(47.2 mg, 0.015 mmol).
Fully protected R-thiosialodendrimers 10a , 11a , and 12a were
obtained in the same manner from Starburst PAMAM dendritic
cores 2, 3, and 4 using DMF as the reaction solvent, in 71, 100,
and 100% yields, respectively.
9a : 1H NMR (DMSO-d6) δ 1.65 (s, 12H, NAc), 1.77 (dd, 4H, J
12.1 Hz, H-3ax), 1.90, 1.99, 2.00, 2.02 (4s, 48H, OAc’s), 2.28 (bs,
8H, b-CH2), 2.52-2.99 (3m, 16H, a-CH2, c-CH2, H-3eq), 3.25 (m,
8H, d-CH2), 3.52-3.60 (m, 8H, e-CH2), 3.54 (s, 12H, OCH3),
3.59-3.79 (2m, 8H, H-5, H-6), 4.09 (dd, 4H, J 8,9 6.0 Hz, J 9,9′ 12.2
Hz, H-9), 4.28 (dd, 4H, J 8,9′ 2.5 Hz, H-9′), 4.69 (ddd, 4H, H-4),
5.13 (m, 4H, H-7), 5.19 (m, 4H, H-8), 7.38 (d, 8H, J ortho,meta 8.2
Hz, H-ortho), 7.54 (d, 8H, H-meta), 7.62 (d, 4H, J 5,NHAc 9.5 Hz,
NHAc), 8.00 (bs, 4H, NHC(S)NHPh), 8.05 (bs, 4H, amide NH’s),
9.86 (bs, 4H, NHC(S)NHPh); 13C NMR (DMSO-d6) δ 20.6, 20.8
(OAc’s), 22.6 (NAc), 32.4 (C-b), 37.7 (C-3), 37.8 (C-d), 40.5 (C-a),
43.4 (C-e), 47.7 (C-5), 48.8 (C-c), 52.8 (OCH3), 61.7 (C-9), 67.4
(C-7), 68.9 (C-8), 69.5 (C-4), 74.1 (C-6), 87.3 (C-2), 122.0 (C-meta),
122.2 (C-para), 136.4 (C-ortho), 141.3 (C-ipso), 167.8-170.1
(CdO’s), 180.4 (CdS).
10b: 1H NMR (DMSO-d6) δ 1.66 (m, 8H, H-3ax), 1.83 (s, 24H,
NAc), 2.25-2.61 (m, 32H, b-CH2, H-3eq), 2.63-3.00 (m, 28H,
a-CH2, c-CH2), 3.05-3.68 (m, 104H, d-CH2, e-CH2, and NeuAc
residues excluding above), 4.27, 4.59, 4.98, 5.20 (4bs, 32H, OH’s),
7.22-8.15 (4m, 60H, amide NH’s, NHC(S)NHPh, H-ortho,
H-meta, NHAc), 9.88 (bs, 8H, NHC(S)NHPh); 13C NMR (DMSO-
d6) δ 22.7 (NAc), 37.8 (C-d), 40.5 (C-3), 40.7 (C-a), 43.4 (C-e),
51.9 (C-5), 63.2 (C-9), 66.8 (C-7), 68.8 (C-8), 71.3 (C-4), 76.1 (C-
6), 86.5 (C-2), 121.8 (C-meta), 136.4 (C-ortho), 169.3-171.6
(CdO’s), 180.4 (CdS).
1
10a : H NMR (DMSO-d6) δ 1.64 (s, 24H, NAc), 1.77 (dd, 8H,
J 12.1 Hz, H-3ax), 1.90, 1.99, 2.00, 2.02 (4s, 96H, OAc’s), 2.25
(bs, 12H, b-CH2), 2.55-2.95 (2m, 24H, a-CH2, b-CH2, H-3eq),
3.25-3.49 (2m, 48H, c-CH2, d-CH2), 3.52-3.60 (m, 24H, e-CH2),
3.54 (s, 24H, OCH3), 3.59-3.79 (2m, 16H, H-5, H-6), 4.09 (m,
8H, H-9), 4.28 (dd, 8H, J 8,9′ 2.5 Hz, J 9,9′ 12.2 Hz, H-9′), 4.69 (ddd,
8H, H-4), 5.13 (m, 8H, H-7), 5.19 (m, 8H, H-8), 7.38 (d, 16H,
J ortho,meta 8.2 Hz, H-ortho), 7.54 (d, 16H, H-meta), 7.62 (d, 8H,
J 5,NHAc 9.5 Hz, NHAc), 8.00-8.20 (2m, 20H, NHC(S)NHPh,
amide NH’s), 9.90 (bs, 8H, NHC(S)NHPh); 13C NMR (DMSO-
d6) δ 20.5, 20.7 (OAc’s), 22.5 (NAc), 37.6 (C-3), 37.7 (C-d), 40.4
(C-a), 43.3 (C-e), 47.6 (C-5), 49.4 (C-c), 52.7 (OCH3), 61.6 (C-9),
67.3 (C-7), 68.9 (C-8), 69.4 (C-4), 74.0 (C-6), 87.2 (C-2), 121.9
(C-meta), 122.7 (C-para), 136.3 (C-ortho), 141.2 (C-ipso), 167.7-
170.0 (CdO’s), 180.4 (CdS).
11b: 1H NMR (DMSO-d6) δ 1.66 (dd, 16H, J 12.0 Hz, H-3ax),
1.83 (s, 48H, NAc), 2.25-2.62 (m, 72H, b-CH2, H-3eq), 2.63-
3.00 (m, 60H, a-CH2, c-CH2), 3.05-3.70 (m, 224H, d-CH2, e-CH2,
and NeuAc residues excluding above), 4.27, 4.59, 4.72, 5.18 (4bs,
64H, OH’s), 7.22-8.15 (4m, 124H, amide NH’s, NHC(S)NHPh,
H-ortho, H-meta, NHAc), 9.90 (bs, 16H, NHC(S)NHPh); 13C
NMR (DMSO-d6) δ 22.7 (NAc), 37.9 (C-d), 40.5 (C-3), 40.7 (C-a),
43.3 (C-e), 51.9 (C-5), 63.2 (C-9), 66.8 (C-7), 68.7 (C-8), 71.3 (C-
4), 76.1 (C-6), 86.5 (C-2), 121.8, (C-meta), 136.4 (C-ortho), 141.2
(C-ipso), 169.3-171.6 (CdO’s), 180.4 (CdS).
12b: 1H NMR (DMSO-d6) δ 1.66 (dd, 32H, J 11.9 Hz, H-3ax),
1.84 (s, 96H, NAc), 2.25 (bs, 120H, b-CH2), 2.50-2.80 (m, 154H,
a-CH2, c-CH2, H-3eq), 3.05-3.70 (m, 464H, d-CH2, e-CH2, and

Notes
J . Org. Chem., Vol. 63, No. 10, 1998 3491
NeuAc residues excluding above), 4.27, 4.59, 5.18 (3bs, 128H,
OH’s), 7.22-8.15 (4m, 252H, amide NH’s, NHC(S)NHPh, H-
ortho, H-meta, NHAc), 9.95 (bs, 32H, NHC(S)NHPh); 13C NMR
(DMSO-d6) δ 22.7 (NAc), 32.5, 32.9 (C-b), 37.8 (C-d), 39.1 (C-3),
40.5 (C-a), 42.1 (C-c), 43.4 (C-e), 51.9 (C-5), 63.2 (C-9), 66.8 (C-
7), 68.8 (C-8), 71.3 (C-4), 76.1 (C-6), 86.5 (C-2), 121.9, 122.5 (C-
meta, C-para), 136.4 (C-ortho), 141.2 (C-ipso), 169.3-171.6
(CdO’s), 180.4 (CdS).
Tu r b id im et r ic An a lysis b et w een t h e Lect in fr om L.
fla vu s a n d P AMAM-Ba sed r-Th iosia lod en d r im er s 9b-12b.
Turbidimetry experiments were performed in Linbro (Titertek)
microtitration plates where 50 µL/well of stock lectin solutions
prepared from L. flavus (1 mg/mL in PBS) were mixed with 50
µL each of glycodendrimers 9b-12b (0.22, 0.24, 0.24, and 0.25
mg/mL in PBS, respectively, or 0.371 mM sialoside content each)
and incubated at room temperature for up to 4 h. The turbidity
of the solutions was monitored by reading the optical density
(OD) at 490 nm at regular time intervals until no noticeable
changes could be observed.
monomer, tetravalent 3,3′-iminobis(propylamine)-based sialo-
dendrimer,4,5 and PAMAM-based, spherical glycodendrimers
9b-12b. Each inhibitor was added in serial 2-fold dilutions (60
µL/well) in PBS with the appropriate lectin-enzyme conjugate
concentration (60 µL/well of 100-fold dilution of a 1 mg/mL stock
solution of L. flavus in PBS) on Nunclon (Delta) microtiter plates
and incubated at 37 °C for 1 h. These inhibitor solutions (100
µL) were transferred to the antigen-coated plates and incubated
for a second hour. The plates were washed, and 50 µL/well of
2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammo-
nium salt (ABTS) (1 mg/mL) in citrate-phosphate buffer (0.2 M,
pH 4.0 with 0.015% H2O2) was added. The reaction was stopped
after 20 min by adding 50 µL/well of 1 M H2SO4 and the optical
density measured at 410 nm relative to 570 nm. Percent
inhibition was calculated as follows (eq 1):
% inhibition )
(A(no inhibitor) - A(with inhibitor))/A(no inhibitor) × 100 (1)
Com p etitive In h ibition ELLA Usin g Hu m a n r1-Acid
Glycop r otein a n d P AMAM-Ba sed r-Th iosia lod en d r im er s
9b-12b a s In h ibtor s. Linbro (Titertek) microtitration plates
were coated with human R1-acid glycoprotein (orosomucoid) at
100 µL/well of a stock solution of 10 µg/mL in 0.01 M phosphate
buffer (pH 7.3) overnight. The wells were then washed three
times with 300 µL/well of 0.01 M phosphate buffer (pH 7.3)
containing 0.05% (v/v) Tween 20 (PBST). Similar washings with
PBST were repeated after each incubation period. Wells were
then blocked with 150 µL/well of 1% BSA/PBS for 1 h at 37 °C.
After being washed, the wells were filled with 100 µL/well of
inhibitor solutions and incubated again at 37 °C for 1 h.
Inhibitors included phenyl 2-thio-R-sialoside 6a 21 as a reference
IC50’s were reported as the concentration required for 50%
inhibition of the coating antigen. Each test was performed six
times, and average values are reported in Table 1.
Ack n ow led gm en t. We are thankful to Dr. R. Spin-
dler from Dendritech Inc. (Midland, MI) for a generous
gift of Starburst PAMAM dendrimers and to Dr. M.
Kawase from NGK Insulators Ltd. (Handa, J apan) for
sialic acid. An NSERC postgraduate scholarship to D.Z.
is also acknowledged.
J O972061U