Di-, tri-, and tetravalent dendritic galabiosides that inhibit hemagglutination by Streptococcus suis at nanomolar concentration

Article abstract of DOI:10.1021/ja970859p

Galabiose (Galα1-4Gal) derivatives were coupled to carrier molecules having mono, di-, tri-, and tetrameric functionalities, thus creating mono- to tetravalent dendritic galabiosides (1-9). The di- to tetravalent galabiosides were several hundred times mo

Full text of DOI:10.1021/ja970859p

6974
J. Am. Chem. Soc. 1997, 119, 6974-6979
Di-, Tri-, and Tetravalent Dendritic Galabiosides That Inhibit
Hemagglutination by Streptococcus suis at Nanomolar
Concentration
Henrik C. Hansen,† Sauli Haataja,‡ Jukka Finne,‡ and Go1ran Magnusson*,†
Contribution from Organic Chemistry 2, Center for Chemistry and Chemical Engineering,
Lund UniVersity, P.O. Box 124, S-221 00 Lund, Sweden, and Department of Medical
Biochemistry, UniVersity of Turku, FIN-20520 Turku, Finland
ReceiVed March 17, 1997X
Abstract: Galabiose (GalR1-4Gal) derivatives were coupled to carrier molecules having mono-, di-, tri-, and tetrameric
functionalities, thus creating mono- to tetravalent dendritic galabiosides (1-9). The di- to tetravalent galabiosides
were several hundred times more efficient than the monomeric galabiosides in inhibiting hemagglutination by the
Gram-positive bacterium Streptococcus suis, resulting in complete inhibition at low nanomolar concentrations. This
is unprecedented in the field of inhibition of bacterial adhesion by soluble compounds.
Introduction
The effective inhibitory concentrations (IC50 or MIC values)
were found in the micro- to millimolar region for most of the
galabiose derivatives we have tested as inhibitors of E. coli and
S. suis hemagglutination. Such inhibitor concentrations are
typical and probably too high for practical treatment of infected
tissues. However, strong inhibitors have been prepared and
various aromatic R-mannosides inhibit the adherence of type 1
fimbriated E. coli to yeast and guinea pig intestinal epithelial
cells at low micromolar concentrations.9
Inhibition of influenza virus sialidase, as well as inhibition
of influenza virus replication, was accomplished at the low
nanomolar level by a designed guanidino derivative of N-
acetylneuraminic acid.3 Other approaches toward efficient
inhibitors of sugar-binding proteins use saccharides coupled in
a multivalent fashion to carriers, producing, for example,
polymers, dendrimers, and solid materials capped with various
sugars.10 However, it is reasonable to believe that polymers of
high molecular weight will be reluctant to cross biological
membranes and, consequently, their absorbance into animal
tissues and their systemic in ViVo antiadhesive function would
be impaired. Absorption of the inhibitors by cells is of course
not relevant when the inhibition is expected to occur by topical
administration or in the gut, but would be crucial for treatment
of, for example, S. suis infections, which cause meningitis,
septicemia, and pneumonia in pigs and also meningitis in
humans.11
Bacterial resistance against traditional antibiotics is a growing
medical problem.1 New principles for treatment of infections
are therefore highly desirable. One possibility would be to
inhibit the attachment of the infecting bacteria to the host’s cell
surfaces; such attachment is often a prerequisite for the later
stages of infection, colonization, and invasion of tissues.2 Since
the chemical structure of the adhesion inhibitors most likely
would be quite similar to the natural attachment ligands used
by the bacteria, it is unlikely that mutations (resistance) would
give them the capability to overcome the inhibitory effect of
the antiadhesive drug whithout impairing their ability to adhere
to the host cells. In the virus field, lack of formation of resistant
mutants was observed in connection with the successful
development of a potent sialidase-based inhibitor of influenza
virus replication.3
Many bacterial species carry surface-bound lectins that
recognize saccharide moieties on the surface of the host cells
and, thus, use these as anchoring points.4 However, each
saccharide-lectin interaction is normally rather weak, which
is compensated by multivalent binding;5,6 an Escherichia coli
cell carries multiple lectin molecules for this purpose.2
We are currently interested in the development of inhibitors
of different pathogens that recognize the galabiose (GalR1-4Gal)
moiety of glycolipids. The work is based on earlier determina-
tions of the galabiose epitopes used for adhesion by uropatho-
genic E. coli7 (a Gram-negative species) and the piglet pathogen
Streptococcus suis8 (a Gram-positive species).
(7) (a) Kihlberg, J.; Hultgren, S. J.; Normark, S.; Magnusson, G. J. Am.
Chem. Soc. 1989, 111, 6364-6368. (b) Striker, R.; Nilsson, U.; Stonecipher,
A.; Magnusson, G.; Hultgren, S. J. Mol. Microbiol. 1995, 16, 1021-1029.
(c) Nilsson, U.; Striker, R. T.; Hultgren, S. J.; Magnusson, G. Bioorg. Med.
Chem. 1996, 4, 1809-1817.
(8) (a) Haataja, S.; Tikkanen, K.; Liukkonen, J.; Franc¸ois-Gerard, C.;
Finne, J. J. Biol. Chem. 1993, 268, 4311-4317. (b) Haataja, S.; Tikkanen,
K.; Nilsson, U.; Magnusson, G.; Karlsson, K.-A.; Finne, J. J. Biol. Chem.
1994, 269, 27466-27472.
† Lund University.
‡ University of Turku.
X Abstract published in AdVance ACS Abstracts, July 15, 1997.
(1) Chem. Eng. News 1996, Jan. 22, 9.
(2) (a) Microbial Lectins and Agglutinins; Mirelman, D., Ed.; Wiley
Interscience: New York, 1986. (b) Zopf, D.; Roth, S. Lancet 1996, 347,
1017-1021.
(3) von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J.
C.; Jin, B.; Phan, T. V.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman,
P. M.; Varghese, J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotham,
V. J.; Cameron, J. M.; Penn, C. R. Nature 1993, 363, 418-423.
(4) Karlsson, K.-A. Curr. Opin. Struct. Biol. 1995, 5, 622-635.
(5) Bundle, D. R.; Young, N. M. Curr. Opin. Struct. Biol. 1992, 2, 666-
673.
(9) Firon, N.; Ashkenazi, S.; Mirelman, D.; Ofek, I.; Sharon, N. Infect.
Immun. 1987, 55, 472-476.
(10) (a) Lee, R. T.; Lee, Y. C. in Neoglycoconjugates: Preparation and
applications; Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego,
CA, 1994; pp 23-50. (b) Roy, R. Trends Glycosci. Glycotechnol. 1996, 8,
79-99. (c) Zanini, D.; Roy, R. J. Org. Chem. 1996, 61, 7348-7354 and
references cited therein. (d) Armstrong, G. D.; Fodor, E.; Vanmaele, R. J.
Infect. Dis. 1991, 164, 1160-1167. (e) Heerze, L. D.; Kelm, M. A.; Talbot,
J. A.; Armstrong, G. D. J. Infect. Dis. 1994, 169, 1291-1296. (f) Armstrong,
G. D.; Rowe, P. C.; Goodyer, P.; Orrbine, E.; Klassen, T. P.; Wells, G.;
MacKenzie, A.; Lior, H.; Blanchard, C.; Auclair, F.; Thompson, B.; Rafter,
D. J.; McLaine, P. N. J. Infect. Dis. 1995, 171, 1042-1045.
(6) Kiessling, L. L.; Pohl, N. L. Chem. Biol. 1996, 3, 71-77.
S0002-7863(97)00859-7 CCC: $14.00 © 1997 American Chemical Society

Dendritic Galabiosides That Inhibit Hemagglutination
J. Am. Chem. Soc., Vol. 119, No. 30, 1997 6975
Figure 1. Dendritic galabiosides used as inhibitors of hemagglutination by S. suis bacteria.
Scheme 1a
a Key: (a) CF3COOH, CH2Cl2, 22 °C, 30 min; (b) Cl3CCN, DBU, 0 °C, 50 min; (c) NaN3, 15-crown-5, 75 °C, 24 h; (d) MeONa, MeOH, then
H2, Pd/C.
We now report the synthesis and biological evaluation of the
novel mono- to tetravalent galabiosides 2-9 (Figure 1). Some
of these compounds were found to inhibit the agglutination of
human erythrocytes by S. suis bacteria, at inhibitor concentra-
tions as low as 2 nM. This result is unprecedented in the field
of bacterial antiadhesion by soluble inhibitors. The high
inhibitory power of 6-9, combined with the moderate molecular
weight (∼1-2 kD), makes these saccharides potentially useful
as starting points for the practical development of useful
antiadhesive drugs.
The 2-bromoethyl galabioside 1214 was treated with sodium
azide to give 13 (97%). Removal of the O-acetyl groups of
13, followed by hydrogenolysis of the azido group, gave the
amine 14 (82%).
Commercially available 1,3- and 1,4-benzenedimethanethiol
were glycosylated with the imidate 11 (2.5 equiv), using boron
trifluoride etherate as promoter,13 which gave 15 and 16
(Scheme 2) in 65 and 63% yield, respectively. Removal of the
O-acetyl groups of 15 and 16 gave the dimeric galabiosides 3
(94%) and 4 (92%). Treatment of 1,4-benzenedimethanethiol
with the 2-bromoethyl galabioside 12 (2.5 equiv) and cesium
carbonate15 gave 17 in 55% yield. De-O-acetylation of 17 gave
the dimeric galabioside 5 (93%).
Results and Discussion
I. Synthesis of Di- to Tetravalent Galabiosides. The
galabioside moieties 11, 12, and 14 were synthesized as
described above (Scheme 1) and coupled to the various scaffold
molecules shown in Schemes 2 and 3, thus producing the mono-
to tetradentate galabiosides 2-9, useful as inhibitors of hem-
agglutination by the S. suis bacteria. The 2-(trimethylsilyl)-
ethyl (TMSEt) galabioside 1012 was treated with trifluoroacetic
acid, which removed the TMSEt group.12 The crude hemiacetal
was then transformed into the R-trichloroacetimidate 11 by
treatment with trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]-
undec-7-ene (DBU),13 leaving 11 in an overall yield of 92%.
Alkylation of benzylthiol, 1,4-benzenedimethanethiol, and
1,3-benzenedimethanethiol with 3-bromopropionic acid (Scheme
3) gave the scaffold mono- and diacids 18 (65%), 19 (77%),
and 20 (66%), respectively. The commercially available tetra-
(bromomethyl)methane (Scheme 3) was treated with methyl
3-mercaptopropionate and cesium carbonate,15 which gave the
tetraester 22 (54%). Hydrolysis of the ester groups gave the
tetraacid 23 (96%).
The 2-aminoethyl galabioside 14 was coupled via amide
bonds to the mono- to tetraacids 18-21 and 23, which furnished
the mono- to tetravalent galabiosides 2 and 6-9, respectively.
(11) (a) Arends, J. P.; Zanen, H. C. ReV. Infect. Dis. 1988, 10, 131-
137. (b) Sihvonen, L.; Kurl, D. N.; Henrichsen, J. Acta Vet. Scand. 1988,
29, 9-13.
(12) Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnusson, G.;
Dahme´n, J.; Noori, G.; Stenvall, K. J. Org. Chem. 1988, 53, 5629-5647.
(13) Schmidt, R. R.; Kinzy, W. AdV. Carbohydr. Chem. Biochem. 1994,
50, 21-123.
(14) Dahme´n, J.; Frejd, T.; Gro¨nberg, G.; Lave, T.; Magnusson, G.;
Noori, G. Carbohydr. Res. 1983, 116, 303-307.
(15) Buter, J.; Kellogg, R. M. J. Org. Chem. 1981, 46, 4481-4485.

6976 J. Am. Chem. Soc., Vol. 119, No. 30, 1997
Hansen et al.
Scheme 2a
Scheme 3a
a Key: (a) BF3‚OEt2, CH2Cl2, 22 °C, 30-120 min; (b) MeONa,
MeOH, 22 °C, 6.5-15 h; (c) Cs2CO3, DMF, 22 °C, 15 h.
This flexible methodology permits easy variation of the
carbohydrate moieties, which would provide ligands for other
applications. The amide linkages were obtained by first
activating the carboxyl groups of the acids and isolating the
resulting active esters. Without the crucial isolation step, a
complex mixture was obtained, resulting in a low yield of the
desired amide. The acids 18-21 were activated by N-ethyl-
N′-(3-(dimethylamino)propyl)carbodiimidehydrochloride(EDC)-
mediated esterification with N-hydroxysuccinimide (NHS),16 and
the active esters were isolated and coupled with the galabio-
sylamine 14 (1.1 equiv per ester group), which gave the
galabiosyl amides 2 (98%), 6 (97%), 7 (97%), and 8 (98%).
Attempted reaction of the tetraacid 23 under the same conditions
as above and coupling with 14 left a mixture of several
compounds, and the yield of 9 was estimated by NMR to be
∼40%. Several alternative procedures were investigated, but
none of them gave 9 in high yield. The best result was obtained
by transforming 23 into the corresponding pentafluorophenyl
ester (isolated yield 32%), using pentafluorophenol and diiso-
propylcarbodiimide (DIC).17 Treatment of 14 with the penta-
fluorophenyl ester gave 9 (55%) after purification by preparative
TLC. The symmetry of compounds 3-9 simplified the
interpretation of NMR spectra; all signals from structurally
equivalent protons and carbons had the same chemical shifts,
agreeing with those of other galabiosides.18
a Key: (a) NaH, DMF, 22 °C, 15 h; (b) NHS, EDC, DMF, 22 °C,
15-18 h; (c) pyridine, 60 °C, 8 h; (d) Cs2CO3, DMF, 22 °C, 15 h; (e)
LiOH, MeOH, H2O, 22 °C, 15 h; (f) F5C6OH, DIC, DMF, 0 f 22 °C,
16 h; (g) HOBt, Et3N, DMF, 22 °C, 16 h.
Bacteria having galabiose-specific lectins on the surface will
agglutinate erythrocytes, due to a multivalent recognition of the
cell-surface sugar residues by the bacterial protein molecules.
Most cells in the body carry these glycolipids. Therefore, the
erythrocyte-bacteria agglutination system is useful for testing
the adhesion of bacteria to cell surfaces, and inhibition in Vitro
by water-soluble saccharides can be used to probe the inhibitory
efficacy and map the binding epitopes of synthetic saccharide
analogs. It is important that the same batches of erythrocytes
and bacteria are used in such investigations; experiments
performed with different batches can give rather different
absolute MIC (or IC50) values but the relative values for a series
of inhibitors are fairly constant.20 The procedure is described
in the Experimental Section, and the concentrations of the
different saccharide inhibitors needed for complete inhibition
of hemagglutination are given in Table 1.
The results show a clear connection between inhibitory
efficacy and the number of galabiose units present on the
inhibitor. We have earlier shown that the glycolipid globotriosyl
ceramide is the most efficient receptor for S. suis on the
erythrocyte surface, whereas globotetraosyl ceramide shows
much weaker inhibitory acticity. The hydrogen-bonding pattern
of the recognition of galabiosides by S. suis bacteria was
determined with the help of synthetic monodeoxygalabiosides.8
However, the protein has not yet been obtained in a form
suitable for detailed complexation studies with galabiosides and
it is at present not known in what type of structure the protein
occurs on the bacterial surface.
II. Inhibition of Hemagglutination of S. suis Bacteria.
Glycolipids of the globoseries are present on the surface of
human red blood cells (erythrocytes).19 Galabiose (GalR1-
4Galâ) is a common structural unit in these glycolipids.
(16) Kretzschmar, G.; Sprengard, U.; Kunz, H.; Bartnik, E.; Schmidt,
W.; Toepfer, A.; Ho¨rsch, B.; Krause, M.; Seiffge, D. Tetrahedron 1995,
51, 13015-13030.
(17) (a) Kisfaludy, L.; Scho¨n, I. Synthesis 1983, 325-327.
(18) (a) Bock, K.; Frejd, T.; Kihlberg, J.; Magnusson, G. Carbohydr.
Res. 1988, 176, 253-270. (b) Gro¨nberg, G.; Nilsson, U.; Bock, K.;
Magnusson, G. Carbohydr. Res. 1994, 257, 35-54.
The monomeric TMSEt galabioside 112 has been used as a
standard inhibitor in earlier investigations of bacterial adhesion
(19) (a) Marcus, D. M.; Naiki, M.; Kundu, S. K. Proc. Natl. Acad. Sci.
U.S.A. 1976, 73, 3263-3267. (b) Marcus, D. M.; Kundu, S. K.; Suzuki, A.
Semin. Hematol. 1981, 18, 63-71.
(20) Magnusson, G.; Hultgren, S. J.; Kihlberg, J. Methods Enzymol. 1995,
253, 105-114.

Dendritic Galabiosides That Inhibit Hemagglutination
J. Am. Chem. Soc., Vol. 119, No. 30, 1997 6977
Table 1. Inhibition of Agglutination of Human Erythrocytes by S.
suis Bacteria
68.9, 60.9, 60.5, 39.8, 35.7, 35.6, 27.1; HRMS calcd for C24H38O12NS
(M + H) 564.2115, found 564.2096.
inhibitory concentrationa (nM) of S. suis (strain)b
Bisgalabioside 3. Compound 15 (47 mg, 0.0334 mmol) was
dissolved in methanol (4 mL), and the mixture was treated with a
catalytic amount of sodium methoxide for 6.5 h, neutralized with
Duolite C436 (H+), and concentrated. The residue was chromato-
inhibitor
type PN (628)
type PO (836)
1
2
3
4
5
6
7
8
9
1800
300
130
90
100
6
1300
300
76
51
64
3
graphed (CH2Cl2/MeOH/H2O 5:5:1) to give 3 (25.7 mg, 94%): [R]24
D
+23 (c 0.6, H2O); 1H NMR (D2O) δ 7.17-7.31 (m, 4 H, Ar), 4.81 (d,
2 H, J ) 3.9 Hz, H-1′), 4.22 (d, 2 H, J ) 9.1 Hz, H-1), 4.21 (bt, 2 H,
J ) 6.8 Hz, H-5′), 4.05 (d, 2 H, J ) 13.5 Hz, PhCH2S), 4.00 (d, 2 H,
J ) 13.5 Hz, PhCH2S), 3.92 (bd, 2 H, J ) 2.6 Hz, H-4), 3.89 (bd, 2
H, J ) 3.1 Hz, H-4′), 3.77 (dd, 2 H, J ) 3.2, 10.5 Hz, H-3′), 3.69 (dd,
2 H, J ) 3.8, 10.5 Hz, H-2′), 3.60-3.69 (m, 4 H, H-6), 3.49-3.60 (m,
6 H, H-5,6′), 3.49 (dd, 2 H, J ) 2.7, 9.7 Hz, H-3), 3.44 (t, 2 H, J )
9.6 Hz, H-2); 13C NMR (D2O) δ 136.4, 131.3, 128.2, 100.7, 85.1, 79.1,
77.8, 74.1, 71.1, 70.0, 69.5, 69.3, 69.1, 60.8, 60.3, 31.5; HRMS calcd
for C32H50O20S2Na (M + Na) 841.2235, found 841.2252.
10
25
3
c
16
2
a Complete inhibition of hemagglutination. b Described in ref 8. c Not
determined.
(S. suis,8 E. coli,7 and the E. coli pilus protein PapG).7,21
Changing to an aromatic amide aglycon made the galabioside
2 more efficient than 1 by a factor of 6. The dimeric
galabiosides 3-5 are 14-25 times more efficient than 1. A
large increase in inhibitory power was displayed by the di- and
tetravalent galabiosides 6, 7, and 9, being several hundred times
more efficient than 1. The trimer 8 was somewhat less efficient
than 6, 7, and 9. The high efficacy of 6-9 might be due to the
rather long and flexible aromatic linkers, as compared with the
shorter linkers of 3-5 and 8, allowing effective adhesion to
the bacterial surface proteins. However, the fine molecular
details of the recognition remain to be determined.
In conclusion, we have demonstrated that very low concen-
trations of synthetic oligomeric saccharides of moderate mo-
lecular weight can inhibit the agglutination of human erythro-
cytes by S. suis bacteria. This is the first example of inhibition
of bacterial adhesion by low nanomolar concentrations of a
soluble saccharide inhibitor.
Bisgalabioside 4. Compound 16 (35 mg, 0.0249 mmol) was treated
as in the preparation of 3 to give 4 (20.4 mg, 92%): [R]26D -1 (c 0.5,
1
H2O); H NMR (D2O) δ 7.26 (s, 4 H, Ar), 4.81 (d, 2 H, J ) 3.9 Hz,
H-1′), 4.20 (bt, 2 H, J ) 6.6 Hz, H-5′), 4.16 (d, 2 H, J ) 9.3 Hz, H-1),
3.91 (bd, 2 H, J ) 2.4 Hz, H-4), 3.90 (d, 2 H, J ) 13.4 Hz, PhCH2S),
3.89 (m, 2 H, H-4′), 3.79 (d, 2 H, J ) 13.5 Hz, PhCH2S), 3.77 (dd, 2
H, J ) 3.2, 10.5 Hz, H-3′), 3.69 (dd, 2 H, J ) 3.9, 10.8 Hz, H-2′),
3.45-3.70 (m, 12 H, H-3,5,6,6′), 3.45 (t, 2 H, J ) 9.7 Hz, H-2); 13C
NMR (D2O) δ 137.4, 129.7, 100.7, 84.9, 79.2, 77.9, 74.1, 71.1, 70.0,
69.5, 69.3, 69.1, 60.8, 60.4, 33.8; HRMS calcd for C32H50O20S2Na (M
+ Na) 841.2235, found 841.2214.
Bisgalabioside 5. Compound 17 (50 mg, 0.0334 mmol) was
dissolved in dry methanol (4 mL), and the mixture was treated with a
catalytic amount of sodium methoxide overnight, neutralized with
Duolite C436 (H+), and concentrated. The residue was chromato-
graphed (CH2Cl2/MeOH/H2O 10:4:1) to give 5 (28.2 mg, 93%): [R]26
D
1
+145 (c 0.5, H2O); H NMR (D2O) δ 7.25 (s, 4 H, Ar), 4.84 (d, 2 H,
J ) 3.9 Hz, H-1′), 4.26 (d, 2 H, J ) 7.7 Hz, H-1), 4.23 (bt, 2 H, J )
6.8 Hz, H-5′), 3.90 (bd, 2 H, J ) 3.2 Hz, H-4), 3.89 (bd, 2 H, J ) 3.3
Hz, H-4′), 3.53-3.86 (m, 24 H, H-3,5,6,2′,3′,6′, OCH2CH2S, PhCH2S),
3.41 (dd, 2 H, J ) 7.7, 10.2 Hz, H-2), 2.61 (dt, 2 H, J ) 1.3, 6.5 Hz,
OCH2CH2S); 13C NMR (D2O) δ 137.9, 129.7, 103.4, 100.6, 77.4, 75.4,
72.7, 71.2, 71.1, 69.5, 69.3, 69.2, 69.1, 60.9, 60.3, 35.3, 30.5; HRMS
calcd for C36H58O22S2Na (M + Na) 929.2759, found 929.2763.
Bisgalabioside 6. Compound 19 (10 mg, 0.0318 mmol) was
dissolved in dry N,N-dimethylformamide (2 mL), and N-hydroxy-
succinimide (11 mg, 0.096 mmol) and N-ethyl-N′-(3-(dimethylamino)-
propyl)carbodiimide hydrochloride (EDC, 18.5 mg, 0.097 mmol) were
added. The mixture was stirred at 22 °C overnight, and water (10 mL)
and dichloromethane (30 mL) were added. The organic layer was dried
and concentrated. The residue was chromatographed (EtOAc/heptane
2:1) to give the N-hydroxysuccinimide ester of 19 (14.6 mg, 91%).
The diester (14.6 mg, 0.0287 mmol) was added to a solution of 14
(24.3 mg, 0.0632 mmol) in dry pyridine (7 mL). The mixture was
stirred at 60 °C for 8 h and concentrated. The residue was chromato-
graphed (Varian Mega Bond Elut Column C18; H2O/MeOH 9:1 f
Experimental Section
NMR spectra were recorded on 300 or 400 MHz instuments. 1H
NMR spectral assignments were made by the double-resonance
technique COSY. Concentrations were made using rotary evaporation
with bath temperatures at or below 40 °C. Anhydrous Na2SO4 was
used as drying agent for the organic extracts in the workup procedures.
TLC was performed on Kiselgel 60 F254 plates (Merck). Column
chromatography was performed using SiO2 (Matrex LC-gel 60 Å, 35-
70 MY, Grace). Compounds 1,7 10,12 and 1214 were synthesized as
described. Compound 21 is commercially available.
2-(5-Phenyl-4-thiapentanoylamido)ethyl 4-O-r-D-Galactopyran-
osyl-â-D-galactopyranoside (2). Compound 18 (30 mg, 0.15 mmol)
was dissolved in dry N,N-dimethylformamide (2 mL), and N-hydroxy-
succinimide (NHS, 26.5 mg, 0.229 mmol) and N-ethyl-N′-(3-(dimethyl-
amino)propyl)carbodiimide hydrochloride (EDC, 44 mg, 0.229 mmol)
were added. The mixture was stirred at 22 °C for 18 h, and water and
dichloromethane were added. The organic layer was dried and
concentrated. The residue was chromatographed (EtOAc/heptane 2:1)
to give the N-hydroxysuccinimide ester of 18 (43 mg, 98%). The ester
(13 mg, 0.044 mmol) was added to a solution of 14 (20 mg, 0.052
mmol) in dry pyridine (4 mL). The mixture was stirred at 60 °C for
8 h and concentrated. The residue was chromatographed (Varian Mega
8:2 f 7:3 f 6:4 f 5:5, 5 mL each) to give 6 (29.1 mg, 97%): [R]20
D
+67 (c 0.2, H2O); 1H NMR (D2O) δ 7.22 (s, 4 H, Ar), 4.82 (d, 2 H, J
) 3.7 Hz, H-1′), 4.33 (d, 2 H, J ) 7.7 Hz, H-1), 4.19 (t, 2 H, J ) 6.5
Hz, H-5′), 3.89 (bd, 2 H, J ) 3.0 Hz, H-4), 3.85 (bd, 2 H, J ) 3.2 Hz,
H-4′), 3.82 (m, 2 H, OCH2CH2N), 3.53-3.79 (m, 22 H, H-3,5,6,2′,3′,6′,
OCH2CH2N, PhCH2S), 3.42 (dd, 2 H, J ) 7.7, 10.2 Hz, H-2), 3.30 (m,
4 H, OCH2CH2N), 2.60 (t, 4 H, J ) 6.9 Hz, SCH2CH2CO), 2.39 (t, 4
H, J ) 6.8 Hz, SCH2CH2CO); 13C NMR (D2O) δ 174.9, 137.7, 129.6,
103.4, 100.7, 77.7, 75.4, 72.7, 71.3, 71.2, 69.5, 69.3, 69.0, 68.9, 60.9,
60.5, 39.8, 35.7, 35.2, 27.1; HRMS calcd for C42H69O24N2S2 (M + H)
1049.3682, found 1049.3684.
Bond Elut Column C18; H2O/MeOH 9:1 f 8:2 f 7:3 f 6:4 f 5:5,
5 mL each) to give 2 (24.6 mg, 98%): [R]22 +66 (c 0.7, H2O); H
1
D
NMR (D2O) δ 7.18-7.32 (m, 5H, Ar), 4.82 (d, 1 H, J ) 3.7 Hz, H-1′),
4.33 (d, 1 H, J ) 7.7 Hz, H-1), 4.19 (t, 1 H, J ) 6.7 Hz, H-5′), 3.88
(d, 1 H, J ) 3.0 Hz, H-4), 3.85 (bd, 1 H, J ) 2.8 Hz, H-4′), 3.80-3.90
(m, 1 H, OCH2CH2NH), 3.53-3.80 (m, 11 H, H-3,5,6,2′,3′,6′, CH2-
CH2NH-, PhCH2S), 3.43 (dd, 1 H, J ) 7.7, 10.2 Hz, H-2), 3.20-3.39
(m, 2 H, CH2N), 2.62 (t, 2 H, J ) 7.0 Hz, SCH2CH2CO), 2.40 (t, 2 H,
J ) 6.8 Hz, SCH2CH2CO); 13C NMR (D2O) δ 175.0, 138.8, 129.3,
129.2, 127.7, 103.4, 100.7, 77.7, 75.4, 72.7, 71.3, 71.2, 69.5, 69.3, 69.0,
Bisgalabioside 7. Compound 20 (15 mg, 0.048 mmol) was dissolved
in dry dichloromethane (2 mL), and N-hydroxysuccinimide (17 mg,
0.147 mmol) and N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide
hydrochloride (EDC, 27 mg, 0.143 mmol) were added. The mixture
was stirred at 22 °C overnight under N2. Water (10 mL) and
dichloromethane (30 mL) were added. The organic layer was dried
and concentrated. The residue was chromatographed (EtOAc/heptane
2:1) to give the N-hydroxysuccinimide ester of 20 (19 mg, 79%). The
(21) Nilsson, U.; Johansson, R.; Magnusson, G. Chem. Eur. J. 1996, 2,
295-302.

6978 J. Am. Chem. Soc., Vol. 119, No. 30, 1997
Hansen et al.
diester (8.4 mg, 0.017 mmol) was added to a solution of 14 (14 mg,
0.036 mmol) in dry pyridine (5 mL). The mixture was stirred at 60
°C for 8 h and concentrated. The residue was chromatographed (Varian
Mega Bond Elut Column C18; H2O/MeOH 9:1 f 8:2 f 7:3 f 6:4 f
5.38 (dd, 1 H, J ) 3.6, 12.5 Hz, H-2′), 5.30 (dd, 1 H, J ) 2.7, 11.2
Hz, H-3), 5.24 (dd, 1 H, J ) 3.6, 11.1 Hz, H-2), 5.02 (d, 1 H, J ) 3.6
Hz, H-1′), 4.53 (bt, 1 H, J ) 6.6 Hz, H-5 or 5′), 4.27-4.38 (m, 3 H),
4.06-4.18 (m, 3 H), 2.14, 2.12, 2.04, 2.03, 2.00 (5 s, 21 H, Ac); 13C
NMR (CDCl3) δ 170.5, 170.3, 170.1, 169.9, 169.7, 160.7, 99.0, 93.5,
90.8, 70.6, 69.4, 68.1, 67.7, 67.2, 67.1, 66.7, 61.8, 60.7, 21.0, 20.7,
20.6, 20.5; HRMS calcd for C28H36O18NCl3Na (M + Na) 802.0896,
found 802.0895.
2-Azidoethyl 2,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-r-D-
galactopyranosyl)-â-D-galactopyranoside (13). To a mixture of 1214
(495 mg, 0.666 mmol), dry N,N-dimethylformamide (10 mL), and 15-
crown-5 (0.132 mL, 0.667 mmol) was added sodium azide (130 mg,
2.00 mmol). The mixture was stirred at 75 °C for 24 h. Water (20
mL) and toluene (60 mL) were added, and the organic layer was dried
5:5, 5 mL each) to give 7 (16.8 mg, 97%): [R]20 +47 (c 0.9, H2O);
D
1H NMR (D2O) δ 7.15-7.29 (m, 4 H, Ar), 4.83 (d, 2 H, J ) 3.8 Hz,
H-1′), 4.34 (d, 2 H, J ) 7.7 Hz, H-1), 4.20 (t, 2 H, J ) 6.4 Hz, H-5′),
3.89 (bd, 2 H, J ) 3.0 Hz, H-4), 3.86 (bd, 2 H, J ) 3.4 Hz, H-4′), 3.83
(dd, 2 H, J ) 4.4, 6.5 Hz, OCH2CH2NH), 3.53-3.80 (m, 22 H,
including H-2′,3′,3), 3.43 (dd, 2 H, J ) 7.7, 10.2 Hz, H-2), 3.23-3.40
(m, 4 H, OCH2CH2NH), 2.61 (t, 4 H, J ) 7.1 Hz, SCH2CH2CO), 2.41
(t, 4 H, J ) 6.8 Hz, SCH2CH2CO); 13C NMR (H2O) δ 174.9, 139.1,
129.6, 129.4, 128.0, 103.4, 100.6, 77.6, 75.4, 72.6, 71.3, 71.1, 69.4,
69.2, 69.0, 68.8, 60.8, 60.4, 39.7, 35.7, 35.3, 26.9; HRMS calcd for
C42H68O24N2S2Na (M + Na) 1071.3501, found 1071.3506.
Trisgalabioside 8. Nitromethanetrispropionic acid (21) was con-
verted to the N-hydroxysuccinimide triester as described.16 The triester
(10.5 mg, 0.0185 mmol) was added to a solution of 14 (22.5 mg, 0.058
mmol) in dry pyridine (6 mL). The mixture was stirred at 60 °C for
8 h and concentrated. The residue was chromatographed (Varian Mega
and concentrated. The residue was chromatographed (EtOAc/heptane
1
3:1) to give 13 (454 mg, 97%): [R]23 +68 (c 0.6, CHCl3); H NMR
D
(CDCl3) δ 5.58 (dd, 1 H, J ) 1.2, 3.3 Hz, H-4′), 5.41 (dd, 1 H, J )
3.3, 11.0 Hz, H-3′), 5.24 (dd, 1 H, J ) 7.7, 10.8 Hz, H-2), 5.21 (dd 1
H, J ) 3.6, 11.0 Hz, Hz, H-2′), 5.01 (d, 1 H, J ) 3.6 Hz, H-1′), 4.82
(dd, 1 H, J ) 2.8, 10.8 Hz, H-3), 4.58 (d, 1 H, J ) 7.8 Hz, H-1), 4.54
(ddd, 1 H, J ) 1.5, 5.7, 8.4 Hz, H-5′), 4.46 (dd, 1 H, J ) 6.7, 11.2 Hz,
H-6), 4.04-4.22 (m, 5 H, OCH2CH2N3, H-4,6,6′), 3.82 (bt, 1 H, J )
6.6 Hz, H-5), 3.71 (ddd, 1 H, J ) 3.3, 8.7, 10.7 Hz, OCH2CH2N3),
3.55 (ddd, 1 H, J ) 3.4, 8.6, 13.3 Hz, OCH2CH2N3), 3.29 (ddd, 1 H,
J ) 3.3, 4.4, 13.4 Hz, OCH2CH2N3), 2.14, 2.11, 2.09, 2.08, 2.06, 2.05,
2.00 (7 s, 3 H each, Ac); 13C NMR (CDCl3) δ 171.1, 171.0, 170.88,
170.86, 170.5, 170.2, 169.6, 101.3, 99.9, 77.5, 73.1, 72.4, 69.0, 68.8,
68.6, 68.3, 67.8, 67.5, 62.4, 60.9, 50.9, 21.4, 21.2, 21.1, 21.05; HRMS
calcd for C28H39O18N3Na (M + Na) 728.2126, found 728.2128.
2-Aminoethyl 4-O-(r-D-galactopyranosyl)-â-D-galactopyranoside
(14). Compound 13 (86 mg, 0.122 mmol) was treated with methanol
(1 mL) and a catalytic amount of sodium methoxide, and the mixture
was neutralized with Duolite C436 (H+) and concentrated. The residue
was dissolved in a mixture of ethanol (3 mL) and aqueous hydrochloric
acid (1.22 mL, 0.1 M) and hydrogenated (H2, 10% Pd/C, 1 atm) for 2
h. The mixture was filtered through Celite and concentrated. The
residue was dissolved in water and passsed through a column of Duolite
A147 (OH-) and concentrated. The residue was chromatographed
(Varian Mega Bond Elut Column C18; H2O/MeOH 9:1 f 8:2 f 7:3
f 6:4 f 5:5, 5 mL each) to give 14 (38.6 mg, 82%): [R]22D + 104 (c
0.5, H2O); 1H NMR (D2O) δ 4.79 (d, 1 H, J ) 3.8 Hz, H-1′), 4.26 (d,
1 H, J ) 7.7 Hz, H-1), 4.16 (bt, J ) 6.3 Hz, H-5′), 3.86 (bs, 2 H,
H-4,4′), 3.61-3.90 (m, 5 H, H-5,6,2′,3′, OCH2CH2), 3.48-3.60 (m,
H-3,6,6′, OCH2CH2), 3.38 (dd, 1 H, J ) 7.7, 10.2 Hz, H-2), 2.65 (m,
2 H, CH2CH2N), 1.75 (s, partly exchanged, NH2); 13C NMR (D2O) δ
103.9, 101.2, 78.0, 75.6, 73.5, 72.1, 71.7, 71.6, 70.1, 69.8, 69.4, 61.2,
60.4, 40.7; HRMS calcd for C14H28O11N (M + H) 386.1662, found
386.1670.
A sample of 14 was acetylated: 1H NMR (CDCl3) δ 6.03 (bs, 1 H,
NH), 5.58 (dd, 1 H, J ) 1.3, 3.3 Hz, H-4′), 5.41 (dd, 1 H, J ) 3.3,
11.1 Hz, H-3′), 5.22 (dd, 1 H, J ) 3.6, 11.0 Hz, H-2′), 5.20 (dd, 1 H,
J ) 7.7, 10.8 Hz, H-2), 5.01 (d, 1 H, J ) 3.7 Hz, H-1′), 4.82 (dd, 1 H,
J ) 2.7, 10.8 Hz, H-3), 4.53 (bt, 1 H, J ) 7.6 Hz, H-5′), 4.49 (d, 1 H,
J ) 7.8 Hz, H-1), 4.45 (dd, 1 H, J ) 7.0, 11.2 Hz, H-6), 4.10-4.21
(m, 3 H, H-6,6′), 4.09 (bd, 1 H, J ) 2.9 Hz, H-4), 3.84-3.91 (m, 1 H,
OCH2CH2), 3.81 (bt, 1 H, J ) 6.7 Hz, H-5), 3.66-3.75 (m, 1 H, OCH2-
CH2), 3.49 (m, 2 H, OCH2CH2N), 2.15, 2.12, 2.10, 2.09, 2.08, 2.04,
2.02, 2.01 (8 s, 3 H each, Ac); HRMS calcd for C30H43O19NNa (M +
Na) 744.2327, found 744.2321.
Acetylated Bisgalabioside 15. To a solution of 11 (130 mg, 0.167
mmol) and 1,3-benzenedimethanethiol (11.3 mg, 0.067 mmol) in
dichloromethane (2 mL) was added boron trifluoride etherate (0.017
mL, 0.135 mmol) under Ar. After 2 h at 22 °C, saturated aqueous
sodium hydrogen carbonate (10 mL) and dichloromethane (20 mL) were
added. The organic layer was dried and concentrated. The residue
was chromatographed (EtOAc/heptane 2:1) to give 15 (60.9 mg,
65%): [R]24D +29 (c 0.5, CHCl3); 1H NMR (CDCl3) δ 7.2-7.3 (m, 4
H, Ar), 5.56 (bd, 2 H, J ) 3.4 Hz, H-4′), 5.36 (dd, 2 H, J ) 3.4, 11.1
Hz, H-3′), 5.22 (t, 2 H, J ) 10.0 Hz, H-2), 5.19 (dd, 2 H, J ) 3.9, 11.0
Hz, H-2′), 5.00 (d, 2 H, J ) 3.6 Hz, H-1′), 4.78 (dd, 2 H, J ) 2.7, 10.3
Hz, H-3), 4.40-4.51 (m, 4 H), 4.28 (d, 2 H, J ) 9.8 Hz, H-1), 4.00-
4.19 (m, 12 H), 3.72 (bt, 2 H, J ) 6.5 Hz, H-5), 2.13, 2.12, 2.11, 2.05,
Bond Elut Column C18; H2O/MeOH 9:1 f 8:2 f 7:3 f 6:4 f 5:5,
1
5 mL each) to give 8 (25.0 mg, 98%): [R]22 +101 (c 0.3, H2O); H
D
NMR (D2O) δ 4.83 (d, 3 H, J ) 3.9 Hz, H-1′), 4.34 (d, 3 H, J ) 7.7
Hz, H-1), 4.24 (t, 3 H, J ) 6.5 Hz, H-5′), 3.91 (d, 6 H, J ) 3.1 Hz,
H-4,4′), 3.85 (m, 3 H, -OCH2CH2N), 3.78 (dd, 3 H, J ) 3.0, 10.4 Hz,
H-3′), 3.53-3.74 (m, 24 H, H-3,5,6,2′,6′, OCH2CH2N), 3.43 (dd, 3 H,
J ) 7.7, 10.2 Hz, H-2), 3.31 (m, 6 H, OCH2CH2N), 2.17 (s, 12 H,
CH2CH2CO); 13C NMR (D2O) δ 174.9, 103.4, 100.6, 93.7, 77.5, 75.5,
72.7, 71.3, 71.2, 69.5, 69.3, 69.1, 68.8, 60.9, 60.5, 39.8, 30.8, 30.4;
HRMS calcd for C52H91O38N4 (M + H) 1379.5311, found 1379.5250.
Tetrakisgalabioside 9. Compound 23 (50 mg, 0.102 mmol) and
pentafluorophenol (226 mg, 1.23 mmol) were suspended in dry N,N-
dimethylformamide (10 mL), the mixture was cooled to 0 °C, and
diisopropylcarbodiimide (DIC, 0.095 mL, 0.614 mmol) was added. The
mixture was stirred for 1 h at 0 °C and at 22 °C overnight. Water (3
mL) was added, and the mixture was extracted with ether (100 mL).
The combined organic extracts were dried and concentrated. The
residue was chromatographed (EtOAc/heptane 1:8) to give the penta-
fluorophenyl ester of 23 (37.4 mg, 32%). The ester (12.1 mg, 0.0105
mmol) was added to a solution of 14 (43.4 mg, 0.113 mmol) in N,N-
dimethylformamide (5 mL) and triethylamine (3 mL), and 1-hydroxy-
benzotriazole (HOBt, 15 mg, 0.111 mmol) was added. The mixture
was stirred for 16 h at 22 °C and concentrated. The residue was purified
by preparative TLC (Merck silica gel 60 F254, CH2Cl2/MeOH/H2O 4:12:
3) to give 9 (11.2 mg, 55%): 1H NMR (D2O) δ 4.84 (d, 4 H, J ) 4.0
Hz, H-1′), 4.35 (d, 4 H, J ) 7.7 Hz, H-1), 4.24 (t, 4 H, J ) 6.5 Hz,
H-5′), 3.82-3.95 (m, 4 H, CH2CH2O), 3.91 (d, 8 H, J ) 3.1 Hz, H-4,4′),
3.55-3.82 (m, 36 H, H-3,2′,3′,6′, CH2CH2O), 3.44 (dd, 4 H, J ) 7.7,
10.1 Hz, H-2), 3.25-3.42 (m, 8 H, CH2N), 2.76 (t, 8 H, J ) 6.7 Hz,
SCH2CH2CO), 2.63 (s, 8 H, CCH2S), 2.48 (t, 8 H, J ) 6.6 Hz,
SCH2CH2CO); 13C NMR (D2O) δ 174.9, 103.5, 100.6, 77.6, 75.5, 72.7,
71.3, 71.2, 69.5, 69.4, 69.1, 68.9, 60.9, 60.5, 43.8, 39.8, 38.1, 36.3,
29.2; HRMS calcd for C73H128O48N4S4Na (M + Na) 1979.6479, found
1979.6489.
2,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-r-D-galactopyrano-
syl)-r-D-galactopyranosyl Trichloroacetimidate (11). 2-(Trimeth-
ylsilyl)ethyl 2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-R-D-galacto-
pyranosyl)-â-D-galactopyranoside 1012 (1.06 g, 1.44 mmol) was dis-
solved in dry dichloromethane (7.5 mL) at 22 °C under Ar. Trifluoro-
acetic acid (15 mL) was added,12 and the mixture was stirred at 22 °C.
After 30 min, n-propyl acetate and toluene were added and the mixture
was concentrated and co-concentrated with toluene to give the
corresponding hemiacetal (920 mg). The crude hemiacetal was
dissolved in dry dichloromethane (22 mL), and trichloroacetonitrile (5.5
mL, 54.6 mmol) was added. The mixture was cooled to 0 °C under
Ar. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 0.33 mL, 2.22 mmol)
was added. After 50 min of stirring at 0 °C, the mixture was washed
with ice cold saturated aqueous NaHCO3 (20 mL), dried, and
concentrated. The residue was chromatographed (heptane/EtOAc 1:1)
1
to give 11 (1030 mg, 92%): [R]24 +144 (c 1.0, CHCl3); H NMR
D
(CDCl3) δ 8.68 (s, 1 H, NH), 6.60 (d, 1 H, J ) 3.7 Hz, H-1), 5.57 (dd,
1 H, J ) 1.3, 3.2 Hz, H-4′), 5.42 (dd, 1 H, J ) 3.6, 12.5 Hz, H-3′),

Dendritic Galabiosides That Inhibit Hemagglutination
J. Am. Chem. Soc., Vol. 119, No. 30, 1997 6979
2.02, 1.98, 1.978 (7 s, 6 H each, Ac); 13C NMR (CDCl3) δ 170.7, 170.6,
170.5, 170.4, 170.1, 169.8, 169.2, 135.4, 131.1, 127.7, 99.3, 82.5, 77.2,
75.8, 73.8, 68.5, 67.8, 67.3, 67.1, 62.4, 60.6, 31.2, 20.92, 20.86, 20.8,
20.7; HRMS calcd for C60H78O34S2Na (M + Na) 1429.3714, found
1429.3711.
Acetylated Bisgalabioside 16. To a solution of 11 (258 mg, 0.33
mmol) and 1,4-benzenedimethanethiol (20.1 mg, 0.118 mmol) in
dichloromethane (2 mL) was added boron trifluoride etherate (0.031
mL, 0.247 mmol) under Ar. After 30 min at 22 °C, saturated aqueous
sodium hydrogen carbonate (10 mL) and dichloromethane (20 mL) were
added. The organic layer was dried and concentrated. The residue
Bis(4-carboxy-2-thiabutyl)-1,4-benzene (19). To a solution of 1,4-
benzenedimethanethiol (50 mg, 0.294 mmol) and 3-bromopropionic
acid (269.5 mg, 1.76 mmol) in dry N,N-dimethylformamide (4 mL)
was added sodium hydride (110 mg, 80% in oil, 3.66 mmol). The
mixture was stirred overnight at 22 °C under Ar. Methanol (1 mL),
acetic acid (5 mL), and toluene (60 mL) were added. The organic
layer was dried and concentrated. The residue was chromatographed
(EtOAc/heptane/AcOH 2:6:1) to give 19 (70.7 mg, 77%): 1H NMR
(CDCl3/CD3OD 1:1) δ 7.25 (s, 4 H, Ar), 3.70 (s, 4 H, ArCH2S), 2.64
(bt, 4 H, SCH2CH2CO), 2.50 (bt, 4 H, SCH2CH2CO); 13C NMR (CDCl3/
CD3OD 1:1) δ 174.9, 137.4, 129.3, 36.0, 34.6, 26.5; HRMS calcd for
C14H18O4S2Na (M + Na) 337.0544, found 337.0545.
Bis(4-carboxy-2-thiabutyl)-1,3-benzene (20). To a solution of 1,3-
benzenedimethanethiol (50 mg, 0.294 mmol) in dry N,N-dimethyl-
formamide (4 mL) was added sodium hydride (106 mg, 80% in oil,
3.53 mmol), followed by 3-bromopropionic acid (270 mg, 1.76 mmol).
The mixture was stirred overnight at 22 °C under N2. Aqueous
ammonium chloride (10%, 4 mL), acetic acid (3 mL), and toluene (50
mL) were added. The organic layer was dried and concentrated. The
residue was chromatographed (EtOAc/heptane/AcOH 2:6:1) to give 20
(60.5 mg, 66%): 1H NMR (CDCl3) δ 7.18-7.32 (m, 4 H, Ar), 3.73 (s,
4 H, ArCH2S), 2.67 (bt, 4 H, J ) 6.6 Hz, SCH2CH2CO), 2.58 (bt, 4 H,
J ) 6.6 Hz, SCH2CH2CO); 13C NMR (CDCl3) δ 178.6, 138.8, 129.8,
129.4, 128.1, 36.6, 34.8, 26.2; HRMS calcd for C14H18O4S2Na (M +
Na) 337.0544, found 337.0536.
Tetra(4-methoxycarbonyl-2-thiabutyl)methane (22). Tetra(bro-
momethyl)methane (100 mg, 0.258 mmol) and methyl 3-mercaptopro-
pionate (0.335 mL, 3.09 mmol) were dissolved in dry N,N-dimethyl-
formamide (4 mL), and cesium carbonate (840 mg, 2.58 mmol) was
added. The mixture was stirred at 22 °C overnight. Water (5 mL)
and dichloromethane (60 mL) were added, and the organic layer was
dried and concentrated. The residue was chromatographed (EtOAc/
heptane 2:1) to give 22 (76.5 mg, 54%): 1H NMR (CDCl3) δ 3.69 (s,
12 H, OMe), 2.83 (t, 8 H, J ) 7.3 Hz, SCH2CH2), 2.71 (s, 8 H, CCH2S),
2.63 (t, 8 H, J ) 7.3 Hz, CH2CH2CO); 13C NMR (CDCl3): δ 172.6,
52.2, 44.4, 38.7, 35.2, 29.0; HRMS calcd for C21H37O8S4 (M + H)
545.1371, found 545.1368.
Tetra(4-carboxy-2-thiabutyl)methane (23). Compound 22 (49 mg,
0.09 mmol) was dissolved in methanol/water (4 mL, 6:1), and lithium
hydroxide (25.5 mg, 1.06 mmol) was added. The mixture was stirred
at 22 °C overnight, filtered, and concentrated. The residue was
chromatographed (EtOAc/heptane/AcOH 1:3:1) to give 23 (42.2 mg,
96%): 1H NMR (CD3OD/D2O 6:1) δ 2.83 (t, 8 H, J ) 7.0 Hz, SCH2-
CH2CO), 2.74 (s, 8 H, CCH2S), 2.61 (t, 8 H, J ) 7.0 Hz, CH2CH2-
CO); 13C NMR (CD3OD/D2O 6:1) δ 177.7, 46.8, 40.7, 37.6, 31.2;
HRMS calcd for C17H28O8S4Na (M + Na) 511.0565, found 511.0574.
Inhibition of Hemagglutination. The experiments were performed
as described earlier.8 In short, 50 µL of sialidase-treated human
erythrocytes (5% hematocrit) were mixed with 25 µL of a suspension
of S. suis bacteria (∼108 cfu/mL), preincubated with 25 µL of the
serially diluted saccharide derivatives 1-9, and incubated on ice for 2
h. The lowest concentrations giving complete inhibition of the
hemagglutination were recorded (Table 1).
was chromatographed (EtOAc/heptane 2:1) to give 16 (104.2 mg,
1
63%): [R]24 +38 (c 0.9, CHCl3); H NMR (CDCl3) δ 7.23 (s, 4 H,
D
Ar), 5.53 (dd, 2 H, J ) 0.9, 3.2 Hz, H-4′), 5.35 (dd, 2 H, J ) 3.2, 11.0
Hz, H-3′), 5.25 (t, 2 H, J ) 10.2 Hz, H-2), 5.17 (dd, 2 H, J ) 3.6, 11.1
Hz, H-2′), 4.99 (d, 2 H, J ) 3.5 Hz, H-1′), 4.79 (dd, 2 H, J ) 2.7, 10.3
Hz, H-3), 4.38-4.50 (m, 4 H), 4.28 (d, 2 H, J ) 10.0 Hz, H-1), 4.01-
4.17 (m, 10 H), 3.94 (d, 2 H, J ) 12.7 Hz, PhCH2S), 3.83 (d, 2 H, J
) 12.7 Hz, PhCH2S), 3.72 (t, 2 H, J ) 6.4 Hz, H-5), 2.10, 2.08, 2.07,
2.03, 2.00, 1.99, 1.95 (7 s, 6 H each, Ac); 13C NMR (CDCl3) δ 170.6,
170.5, 170.4, 170.1, 169.8, 169.2, 136.0, 129.3, 99.1, 82.3, 77.1, 75.9,
73.7, 68.5, 67.8, 67.3, 67.1, 67.06, 62.3, 60.6, 33.1, 20.9, 20.8, 20.7,
20.6; HRMS calcd for C60H78O34S2Na (M + Na) 1429.3714, found
1429.3726.
Acetylated Bisgalabioside 17. To a solution of 1,4-benzene-
dimethanethiol (10 mg, 0.058 mmol) and 1214 (105 mg, 0.141 mmol)
in dry N,N-dimethylformamide (3 mL) was added cesium carbonate
(50 mg, 0.153 mmol), and the resulting mixture was stirred at 22 °C
overnight. Water (10 mL) and dichloromethane (30 mL) were added.
The organic layer was dried and concentrated. The residue was
chromatographed (EtOAc/heptane 2:1) to give 17 (49 mg, 55%): [R]23
D
1
+65 (c 0.6, CHCl3); H NMR (CDCl3) δ 7.28 (s, 4 H, Ar), 5.57 (dd,
2 H, J ) 1.1, 3.3 Hz, H-4′), 5.39 (dd, 2 H, J ) 3.3, 11.0 Hz, H-3′),
5.21 (dd, 2 H, J ) 3.6, 11.2 Hz, H-2′), 5.19 (dd, 2 H, J ) 7.6, 11.0
Hz, H-2), 5.01 (d, 2 H, J ) 3.7 Hz, H-1′), 4.82 (dd, 2 H, J ) 2.8, 10.8
Hz, H-3), 4.53 (bt, 2 H, J ) 6.7 Hz, H-5′), 4.48 (d, 2 H, J ) 7.7 Hz,
H-1), 4.45 (dd, 2 H, J ) 6.7, 11.1 Hz, H-6), 4.08-4.21 (m, 6 H, H-6,6′),
4.07 (bd, 2 H, J ) 2.0 Hz, H-4), 3.96-4.05 (m, 2 H, OCH2CH2S),
3.79 (t, 2 H, J ) 6.5 Hz, H-5), 3.75 (s, 4 H, PhCH2S), 3.59-3.69 (m,
2 H, OCH2CH2S), 2.60-2.73 (m, 4 H, OCH2CH2S), 2.14, 2.11, 2.09,
2.08, 2.05, 2.04, 1.99 (7 s, 6 H each, Ac); 13C NMR (CDCl3) δ 171.2,
171.0, 170.91, 170.87, 170.6, 170.2, 169.6, 137.6, 129.5, 101.6, 99.8,
77.7, 73.1, 72.4, 69.7, 69.0, 68.3, 67.8, 67.5, 62.4, 60.9, 36.7, 31.2,
21.4, 21.22, 21.21, 21.16, 21.11, 21.08; HRMS calcd for C64H86O36S2-
Na (M + Na) 1517.4238, found 1517.4249.
(4-Carboxy-2-thiabutyl)benzene (18). Benzenemethanethiol (0.050
mL, 0.42 mmol) was dissolved in dry N,N-dimethylformamide (4 mL),
and sodium hydride (89 mg, 80% in oil, 2.97 mmol) and 3-bromopro-
pionic acid (195 mg, 1.27 mmol) were added. The mixture was stirred
at 22 °C under N2 overnight. Water, aqueous ammonium chloride
(10%), acetic acid, and toluene were added. The organic layer was
dried and concentrated. The residue was chromatographed (EtOAc/
heptane/AcOH 3:12:1) to give 18 (54 mg, 65%): 1H NMR (CDCl3/
CD3OD 1:1) δ 5.85-5.99 (m, 5 H, Ar), 2.39 (s, 2 H, PhCH2S), 1.31
(bt, 2 H, SCH2CH2CO), 1.18 (bt, 2 H, SCH2CH2CO); 13C NMR (CDCl3/
CD3OD 1:1) δ 173.5, 137.2, 127.8, 127.4, 126.0, 35.0, 33.3, 25.1;
HRMS calcd for C10H11O2SNa2 (M - H + 2Na) 241.0275, found
241.0263.
Acknowledgment. This work was supported by the Swedish
Natural Science Research Council, the Finnish Academy, and
the Sigrid Juse´lius Foundation.
JA970859P