Inhibition of Streptococcus suis adhesion by dendritic galabiose compounds at low nanomolar concentration

Article abstract of DOI:10.1021/jm049476+

A series of mono-, di-, and tetravalent galabiose (Galα1-4Gal) compounds were synthesized in good yields by coupling of a general carboxylic acid-bearing sugar building block to dendritic scaffolds based on the 3,5-di-(2-aminoethoxy)benzoic acid branching

Full text of DOI:10.1021/jm049476+

J. Med. Chem. 2004, 47, 6499-6508
6499
Inhibition of Streptococcus suis Adhesion by Dendritic Galabiose Compounds at
Low Nanomolar Concentration
John A. F. Joosten,† Vuokko Loimaranta,‡ Chantal C. M. Appeldoorn,† Sauli Haataja,‡ Fatna Ait El Maate,†
Rob M. J. Liskamp,† Jukka Finne,‡ and Roland J. Pieters*,†
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Post Office Box 80082,
NL-3508 TB Utrecht, The Netherlands, and Department of Medical Biochemistry and Molecular Biology, University of Turku,
FI-20520 Turku, Finland
Received July 1, 2004
A series of mono-, di-, and tetravalent galabiose (GalR1-4Gal) compounds were synthesized
in good yields by coupling of a general carboxylic acid-bearing sugar building block to dendritic
scaffolds based on the 3,5-di-(2-aminoethoxy)benzoic acid branching unit. Furthermore, a poly-
(amidoamine)- (PAMAM-) based dendritic galabioside was synthesized containing eight
galabiose units. All galabiosides were tested in a hemagglutination assay and a surface plasmon
resonance (SPR) competition assay in order to establish their potency in the binding to the
bacterial Gram-positive pathogen Streptococcus suis. A monovalent galabioside containing a
short spacer was used as a reference compound in all the assays. Variations in the scaffold as
well as in the spacer arms were introduced to determine their influence on the inhibition. The
best inhibitor of hemagglutination was an octavalent galabioside with a minimal inhibitory
concentration (MIC) of 0.3 nM, to the best of our knowledge the first example of inhibition of
bacterial binding by a soluble carbohydrate at a subnanomolar concentration.
Introduction
taching these spacered mannose moieties to proteins or
dendrimers only led to additional enhancements of up
to 1 order of magnitude, when compared on a per sugar
basis.8 Autar et al.9 previously reported studies
involving multivalent GalNAcâ1-4Gal-containing com-
pounds targeting the pathogens F1C fimbriated E. coli
and Pseudomonas aeruginosa. As in the case of E. coli,
spacer effects (3-fold) combined with a modest multi-
valency effect (6.3-fold) led to a maximum overall en-
hancement of 19-fold per sugar for a divalent system.
Furthermore, with Streptococcus suis, Hansen et al.10
observed a relative increase in inhibitory potency of 50-
fold per carbohydrate by using small di-, tri-, and tetra-
valent galabiose-containing molecules in hemaggluti-
nation inhibition experiments. The Gram-positive bac-
terium S. suis can cause meningitis, septicemia, and
pneumonia in pigs and also meningitis in humans.11,12
S. suis recognizes the galabiose epitope (GalR1-4Gal)
on the terminal and internal positions of various gly-
colipids, wherein the disaccharide acts as the ligand for
pathogen adhesion to cells, as the first step of an in-
fective process. Other pathogenic microbes that bind to
the galabiose epitope are the uropathogenic E. coli via
its PapG adhesin13 and the bacterium Pseudomonas
aeruginosa via its PA-IL lectin.14 Furthermore, glycolip-
ids bearing the galabiose epitope can bind to bacterial
enterotoxins, for example, the Shiga-like toxins (SLTs)15
and verotoxin from E. coli.16 For S. suis, extensive
specificity studies have been performed which showed
that galabiose is the best disaccharide ligand.17 There
are two galabiose-binding subtypes of S. suis designated
as PN and PO, based on the nature of their adhesin
specificity.18 Galabiose was shown to form hydrogen
bonds to both adhesins via its HO-2, HO-3, HO-4′, and
Bacterial resistance toward traditional antibiotics is
still increasing at an alarming rate.1 For this reason the
development of new and effective preventive methods
of bacterial infections is a major priority. The first step
toward an infection is the adherence of the bacterial
pathogens to the epithelial surfaces of host tissues.
Many of these bacterial pathogens adhere to the host
via protein-carbohydrate interactions.2 Inhibitors of the
binding of bacterial lectin-like receptors (adhesins) to
oligosaccharides present on the cell surface are potential
novel antibacterial agents.3,4 It is likely that, because
the bacteria are not killed, bacterial resistance toward
these inhibitors will evolve more slowly. Moreover,
resistant mutants are likely to be less infective since
they have also lost their ability to bind to the tissue
cells.
The adhesin-carbohydrate interaction is normally
rather weak (millimolar concentration),3 but this is
amplified by the fact that both binding partners are
present in multivalent arrays.5 Several groups have
shown that, with synthetic multivalent inhibitors, these
weak interactions can also be amplified, which has
already resulted in high-affinity inhibitors, especially
for AB5 toxins.6 In the area of bacterial anti-adhesion,
relatively few studies using multivalent inhibitor design
have been reported to date. Most of them used type I
fimbriated E. coli as their target.7 In a recent study,
good inhibition was observed. It was found that a
lipophilic attachment to mannose ligands already led
to a 135-fold enhancement of the inhibitory power over
R-D-mannopyranoside. However, multivalency by at-
* Corresponding author: phone +31-30-253-6944; fax +31-30-253-
6655; e-mail R.J.Pieters@pharm.uu.nl.
† Utrecht University.
‡ University of Turku.
10.1021/jm049476+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/11/2004

6500 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Scheme 1. Synthesis of Galactose Acceptor 7a
Joosten et al.
attachment of the carboxylic acid-containing galabioside
13 (Scheme 2). Compound 13 was synthesized by
coupling of 3-azidopropyl 2,3,6-tri-O-benzyl-â-D-galac-
topyranoside acceptor 7 (Scheme 1) with 2,3,4,6-tetra-
O-benzyl-R-D-galactopyranoside trichloroacetimidate do-
nor 8 (Scheme 2).21,22 In our strategy, benzyl protective
groups were used to “arm” the O4 position of the
galactose acceptor and to favor the R-glycosylated
disaccharide. Key building block 13 was coupled to the
amine-bearing scaffolds by peptide coupling methods to
give the target compounds after sugar deprotection.
The first target in the synthesis was the benzyl-
protected acceptor 7, which contains a free HO-4 for the
glycosidic coupling and an anomeric propyl azide group
for further elaboration. As a first step in the synthesis
of 7, 3-bromopropanol was coupled to the anomeric
center of 2,3,4,6-tetra-O-acetyl-â-D-galactose 1, in the
presence of boron trifluoride ethyl etherate (BF3‚Et2O)
as a catalyst, to give 2 in 84% yield. An azido function
was introduced via SN2 displacement of the bromine,
using sodium azide in N,N-dimethylformamide (DMF).
Deacetylation of 3 by use of sodium methoxide in MeOH,
followed by benzylidenation with R,R-dimethoxytoluene
and p-toluenesulfonic acid in DMF, gave 5 in 80% yield.
Benzylation of the two remaining free hydroxyl func-
tions of 5 by use of benzylbromide, sodium hydride, and
tetrabutylammonium iodide (TBAI) in DMF yielded 6
in 75% yield. Selective opening of the benzylidene ring
to the O6 position by use of triethylsilane (TES) and
trifluoroacetic acid (TFA)23 gave the desired acceptor 7
in 76% yield. This reaction was also performed with
sodium cyanoborohydride and HCl, which gave slightly
lower yields. Coupling of the known “armed” imidate
donor 821,22 to acceptor 7 in Et2O and 15% trimethylsilyl
trifluoromethanesulfonate (TMSOTf) as a catalyst gave
disaccharide 9 in 73% yield (Scheme 2). Due to the
solvent effect of Et2O21,24 and the low temperature, no
â-linked disaccharide was isolated. To facilitate depro-
tection at the final multivalent stage, the benzyl pro-
tecting groups were replaced by acetyl groups. Selective
reduction of the azido function, by Pd-CaCO3 (Lindlar
catalyst) and H2, followed by the introduction of the
N-tert-butyloxycarbonyl (Boc) protecting group on the
free amino group, gave 10 in 92% yield.25 Debenzylation
of 10 with 10% Pd-C and H2 and subsequent acetyla-
a (a) 3-Bromopropanol, 0 °C, BF3 Et2O, 84%; (b) NaN3, DMF,
100 °C, 87%; (c) NaOMe, MeOH, CH2Cl2, 97%; (d) R,R-dimethoxy-
toluene, p-TsOH, DMF, 50 °C, 80%; (e) NaH, TBAI, BnBr, DMF,
75%; (f) TES, TFA, CH2Cl2, 0 °C, 76%.
HO-6′, while for the PO subtype additional weak hydro-
gen-bonding interactions were detected to HO-6 and
HO-3′.18,19
Though the specificities of the carbohydrate binding
proteins on bacterial surface are characterized in detail,
their relative geometry, in contrast to the structurally
well-characterized AB5 toxins,20 is mostly unknown. To
succeed in inhibitor design, it is likely important to
make a structurally diverse set of inhibitors. We here
report the synthesis of a diverse set of multivalent
galabiose derivatives and their evaluation as inhibitors
of S. suis. The length and chemical composition of the
spacer arms, as well as the nature of the scaffold
molecules themselves, were aspects of structural varia-
tion. The spacer arms for the compounds described here
were considerably longer than those previously reported,
since the longer spacers were shown to be more effec-
tive.10 Furthermore studies on AB5 toxin inhibition
reported longer spacers to be generally advantageous.20
The compounds were evaluated by a hemagglutination
assay and a surface plasmon resonance competition
assay.
Results and Discussion
Synthesis. All multivalent galabiose derivatives and
monovalent reference compounds were synthesized by
Scheme 2. Synthesis of Galabiose Building block 13a
a (a) 15% TMSOTf, Et2O, 0 °C, 73%; (b) Lindlar catalyst, H2, Boc2O, NEt3, MeOH, EtOAc, 92%; (c) Pd-C, HOAc, H2, MeOH, EtOAc;
(d) pyridine, Ac2O, 88% over two steps; (e) TFA, H2O, CH2Cl2, 0 °C, quant; (f) diglycolic anhydride, pyridine, dioxane, 90 °C, quant.

Bacterial Adhesion Inhibition by Galabiose Compounds
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6501
Scheme 3. Synthesis of Rigid Divalent Scaffoldsa
Table 1. Inhibition of the S. suis Mediated Hemagglutination
of Human Erythrocytes by Galabiose Derivatives
S. suis 628 (PN)
MIC
rel. pot.a
[nM] (per sugar)b [nM] (per sugar)b
S. suis 836 (PO)
MIC
rel. pot.a
inhibitor
22 monovalent
900
6
1 (1) 1600
1 (1)
22 (11)
16 (8)
25 divalent
150 (75)
70
100
90
50
40
2
32 divalent, long arms
29 divalent rigid 1,4
27 divalent rigid 1,3
35 tetravalent
9
100 (50)
128 (64)
225 (112)
450 (112)
7
18 (9)
4
32 (16)
40 (10)
800 (100)
2
38 octavalent, PAMAM
0.3 3000 (375)
a Relative potency ) IC50(monovalent 22)/IC50(multivalent com-
a (a) Pd(0)(PPh3)4, CuI, Et3N, CH3CN, 76% (16) or 87% (18); (b)
TFA, CH2Cl2, quant (18) or quant (19).
pound). b Relative potency per sugar ) relative potency/valency.
tion by a 1:1 mixture of pyridine and acetic anhydride
gave 11 (88% yield over two steps). Removal of the Boc-
protecting group of 11 with trifluoroacetic acid gave
compound 12, which was converted to the key building
block 13 by use of diglycolic anhydride in pyridine and
dioxane at 90 °C (quantitative over two steps).9
zotriazol-1-yloxy)-N,N,N′,N′-tetramethyluronium tet-
rafluoroborate] and DIPEA in dimethyl sulfoxide
(DMSO).33 Compound 37 was purified on an LH20
column with CH2Cl2-MeOH (1:1) as the eluent. No free
amino functions could be detected by the ninhydrin
coloring reagent. Deacetylation by sodium methoxide in
MeOH gave target compound 38 (89%) isolated after
freeze-drying.
To increase the hydrophobicity and rigidity of the
spacer arms, in addition to the 3,5-di-(2-aminoethoxy)-
benzoic acid-based compounds, two scaffolds 17 and 19
were prepared that included an acetylene moiety (Scheme
3). To fix distinct geometries, 1,3- and 1,4-disubstituted
benzene rings were used as scaffold elements. Boc-
protected propargylamine was coupled to two com-
mercially available scaffolds, 1,4-diiodobenzene 14 and
1,3-diiodobenzene 15, in good yields by a Sonogashira
coupling with Pd0(PPh3)4, CuI, and Et3N in acetonitrile.
During the purification of 16, the monosubstituted
compound was also isolated as a minor side product
(16% yield). Removal of the Boc-protecting groups of
compounds 16 and 18, with TFA in CH2Cl2, yielded
acetylene-containing scaffolds 17 and 19, both in quan-
titative yield.
Galabiose derivative 13 was attached to mono-, di-,
and tetravalent dendritic scaffolds based on the 3,5-di-
(2-aminoethoxy)benzoic acid branching unit.26,27 These
dendrimers have been used as scaffolds for the synthesis
of multivalent carbohydrate compounds.9,28 A general
coupling and deprotection route was used to produce
mono-, di-, and tetravalent galabiosides 22, 25, 32, and
35 (Scheme 4). Compound 13 was coupled in good yields
to the respective (poly)amino compounds by use of BOP
[(Benzotriazolo-1-yloxy)tris(dimethylamino)phospho-
nium hexafluorophosphate] as a coupling reagent and
DIPEA (N,N-diisopropylethylamine) as a base. Finally,
deacetylation with sodium methoxide in MeOH and
purification on a C8-cartridge column gave target
compounds 22 (80%), 25 (99%), 32 (77%), and 35 (79%).
Characterization by 1D and 2D NMR spectroscopy and
high-resolution mass spectrometry (HRMS) confirmed
the identity of the expected products. By the same
protocol, the acetylenic scaffolds 17 and 19 were con-
verted to their corresponding divalent galabiosides 27
and 29. According to their 13C NMR spectra, the
acetylene carbon signals were still present (82 and 86
ppm), indicating that no cyclization to heterocyclic
structures took place.29,30 Poly(amidoamine) (PAMAM)
dendrimer scaffolds31 have extensively been used in the
synthesis of dendritic carbohydrate compounds.32 Gala-
bioside 13 was attached to the commercially available
PAMAM dendrimer scaffold 36 containing eight end
groups by use of the TBTU coupling reagent [O-(ben-
Inhibition Studies. The synthesized compounds
were evaluated in two different assays. The first assay
method was a hemagglutination of human erythrocytes,
which express glycolipids with the GalR1-4Gal disac-
charide on their surface.18 The S. suis bacteria induce
agglutination that can be inhibited by galabiosides. The
minimal inhibitory concentrations (MIC) required for
complete inhibition of hemagglutination were deter-
mined for S. suis type PN and PO and are depicted in
Table 1. MIC values in the low nanomolar range were
obtained for both bacterial subtypes, indicating the
great inhibitory activity of these compounds. For com-
parison of the inhibition strength of each of the multi-
valent compounds, the monovalent compound 22 was
chosen as a reference compound for determining the
multivalency effects. The monovalent compound had
MIC values of 900 and 1600 nM for subtypes PN and
PO, respectively. The MICs for the multivalent com-
pounds were lower than for the monovalent reference
compound, also when corrected for valency, indicating
a multivalent or cluster effect.34 Inhibition of the PO
subtype required higher concentrations of inhibitors
than for the PN subtype, and also the response to
multivalency effects was smaller for the PO subtype.
Both of these effects may originate from the more strict
carbohydrate specificity of its adhesin.18 The MIC of
divalent compound 25 for the PN subtype was 6 nM, that
is, a 150-fold potency increase compared to reference
compound 22. Elongation of the spacer arms (compound
32) and partial preorganization of the conformation of
the divalent compounds by introducing a rigid core
structure (compounds 27 and 29) was hoped to shed
light on the favorable geometry of effective divalent
binding. The compounds were, however, almost equally
as effective as compound 25. By increasing the valency
of the inhibitor molecule from two to four galabiose
moieties (compound 35), a 3-fold increase in relative
potency per galabioside was observed. However, the
strongest multivalency effects were seen with the oc-
tavalent PAMAM compound 38. For this compound MIC
with the PN subtype of 0.3 nM was determined, which
made it 3000-fold more potent (375-fold per sugar) than
the reference compound 22. To the best of our

6502 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Scheme 4. Synthesis of Multivalent Galabiosidesa
Joosten et al.
a (a) BOP, DIPEA, CH2Cl2, DMF [however, TBTU, DIPEA, DMSO for 36]; (b) NaOMe, MeOH.
knowledge, this is the first example of a soluble carbo-
hydrate inhibiting bacterial binding at a subnanomolar
concentration.
MIC values of the most potent compounds were again
approaching the low nanomolar range, confirming their
great inhibitory potency. The monovalent compound 22
proved to be a good inhibitor (IC50 of 1800 nM), but
increasing the valency to two galabiose moieties (25) led
to an 8-fold more potent inhibitor per galabiose unit
(Figure 1, Table 2). Again, increasing the length of
spacer arms or partial preorganization of the conforma-
tion had no major effects in inhibition compared to
compound 25. By increasing the valency of the inhibitor
from two to four galabiose moieties (compound 35), a
strong increase in inhibition potency was observed. An
IC50 of 7 nM was determined, which made it 256 times
more potent than reference compound 22 and 33 times
In the surface plasmon resonance (SPR) assay, pigeon
ovomucoid, a glycoprotein rich in galabiose sequences,
was immobilized on the sensor chip surface. Whole
bacteria (S. suis 628, PN) were added, and the SPR
signal (in resonance units, RU) was detected. The whole
bacteria showed a clear SPR signal, indicative of their
association with the glycoprotein on the chip, and the
signal could be inhibited in a dose-dependent manner
by the synthesized galabiosides (Figure 1A). An inhibi-
tion curve was calculated for each compound and IC50
values were determined (Figure 1B; Table 2).

Bacterial Adhesion Inhibition by Galabiose Compounds
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6503
Figure 1. Inhibition of whole bacteria (S. suis 628, PN) binding to pigeon ovomucoid coated sensor chip in a surface plasmon
resonance assay. (A) Binding of S. suis in the presence of various concentrations of monovalent galabiose compound (RU, resonance
units). (B) Inhibition curves for monovalent 22 (9), divalent 25 ([), and tetravalent 35 (×) galabiose compound (nRU, normalized
resonance units).
Table 2. Inhibition of S. suis Binding to the Pigeon Ovomucoid
Immobilized on a Surface Plasmon Resonance Chip by
Galabiose Derivatives
and IC50 values. In one case a MIC of 0.3 nM, for
octavalent 38, was determined, representing an increase
in potency of 3000-fold, or 375-fold per galabiose unit.
This enhancement was much larger than the previously
reported most effective compounds, a divalent and a
tetravalant galabioside, which both showed a 25-fold
enhancement on a per sugar basis for the same S. suis
subtype and the same assay.10 Despite good agreement
between the data from the two highly different assay
methods, some differences were also observed. While the
octavalent 38 was the most potent in the hemaggluti-
nation assay, the tetravalent 35 showed a higher
relative potency per galabiose unit in the SPR assay.
When the performance of the four divalent compounds
was compared in the two assays, there was no strong
correlation to spacer length, rigidity, and orientation (27
vs 29). Valency seems to be a stronger determining
factor. This result has implications on the orientation
and mobility of the different binding sites determining
the multivalent enhancement. Our results may bring
the anti-adhesion approach against bacterial infections
closer to its potential, since strong inhibition was
observed as caused by multivalent presentation of the
binding galabiose moiety. Further improvements will
be pursued by evaluating complementary orientations
and valencies. Gratifyingly, we note that multivalent
carbohydrates have successfully been used in vivo, as
was recently shown in two separate studies focused on
the neutralization of the Shiga toxin of E. coli.35
S. suis 628 (PN)
inhibitor
IC50 [nM]
rel. pot.a (per sugar)b
22 monovalent
1800
235
156
309
293
7
1 (1)
8 (4)
12 (6)
6 (3)
6 (3)
256 (64)
256 (32)
25 divalent
32 divalent, long arms
29 divalent rigid 1,4
27 divalent rigid 1,3
35 tetravalent
38 octavalent, PAMAM
7
a Relative potency ) IC50(monovalent 22)/IC50(multivalent com-
pound). b Relative potency per sugar ) relative potency/valency.
more potent than divalent compound 25, indicating a
strong multivalency effect (Figure 1, Table 2). The
octavalent PAMAM compound 38 had inhibitory power
at similar levels as compound 35.
To further confirm the results of the SPR experi-
ments, similar inhibition experiments were also per-
formed by use of a previously reported enzyme-linked
immunosorbent-type assay (ELISA) with pigeon ovo-
mucoid coated plates (data not shown). The IC50 values
found in the ELISA assay were higher (IC50 compound
22, 45 µM) than the MIC values of the hemagglutination
and also higher than the IC50 values of the SPR assay.
It was also found that the differences between the
compounds were smaller than in the previously men-
tioned assay. However, in agreement with the SPR
assay, the tetravalent 35 was the most potent with a
relative potency of 12.
Experimental Section
General Methods. All chemicals were of reagent grade and
were used without further purification. Reactions were moni-
tored by thin-layer chromatography (TLC) on silica gel 60 F254
(Merck); after examination under UV light, compounds were
visualized by heating with 10% (v/v) methanolic H2SO4 or with
ninhydrin (3 mg mL-1). In the workup procedures of reaction
mixtures, organic solutions were washed with appropriate
amounts of the indicated aqueous solutions, then dried, and
concentrated under reduced pressure at 40 °C. Column chro-
matography was performed on silica gel 60 (Merck, 0.063-
0.200 mm). Final compounds were purified on an Isolute SPE
column [IST, 291-1000-F C8(EC)]. 1H NMR spectra were
recorded at 300 K with a Varian Gemini-300 (300 MHz) or a
Varian Unity INOVA 500 (500 MHz) spectrometer; the δH
values are given in parts per million (ppm) relative to the
signal for internal Me4Si (δH ) 0, CDCl3 and CD3OD) or
internal acetone (δH ) 2.225, D2O). 13C NMR spectra (APT,
75 MHz) were recorded at 300 K with a Varian Gemini-300
spectrometer; δC values are given in ppm relative to the signal
of CDCl3 (δC ) 76.9, CDCl3) or for internal acetone (δC ) 30.89,
D2O) or internal MeOH (δC ) 49.50, D2O). Two-dimensional
Conclusions
A series of mono- and multivalent galabiosides were
synthesized in high yields by coupling of one general
galabiose building block to different scaffolds. The
synthesized galabiosides were tested for their inhibition
of the binding of the bacterial pathogen S. suis. Two
different assays, hemagglutination and SPR, were used
to determine the inhibitory concentrations of the com-
pounds. For comparison of the inhibition strength of
each multivalent compound, the monovalent compound
22 was used as a reference compound. In the hemag-
glutination assay, inhibition of two different subtypes
of the adhesin of S. suis (PN and PO) were tested. All
synthesized compounds proved to be excellent inhibitors
of the binding of bacteria to galabiosylated surfaces.
Strong multivalency effects could be clearly observed,
with compounds of higher valency having lower MIC

6504 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Joosten et al.
1H-1H correlation and total correlation spectroscopy (COSY
and TOCSY) and 1H-13C correlated heteronuclear single
quantum coherence (HSQC) NMR spectra (500 MHz) were
recorded at 300 K with a Varian Unity INOVA 500 spectrom-
eter. Exact masses were measured by nanoelectrospray time-
of-flight mass spectrometry on a Micromass LCToF mass
spectrometer at a resolution of 5000 fwhm. Gold-coated
capillaries were loaded with 1 µL of sample (concentration 20
µM) dissolved in a 1:1 (v/v) mixture of CH3CN-H2O with 0.1%
formic acid. NaI or poly(ethylene glycol) (PEG) was added as
internal standard. The capillary voltage was set between 1100
and 1350 V, and the cone voltage was set at 30 V. Matrix-
assisted laser desorption ionization time-of-flight (MALDI-
TOF) spectra were recorded on a Shimadzu Axima-CFR with
R-cyano-4-hydroxycinnamic acid (CHCA) as a matrix and
adrenocorticotropin (ACTH) fragment 18-39 for calibration.
(OCHC6H5); HRMS for C16H21N3O6 (M, 351.1430) M + H found
352.152, calcd 352.1508.
3-Azidopropyl 2,3-Di-O-benzyl-4,6-di-O-benzylidene-â-
D-galactopyranoside (6). To a solution of 5 (2.1 g, 5.97 mmol)
in dry DMF (40 mL) was added NaH (0.76 g, 19.1 mmol), and
the mixture was stirred for 0.5 h. After the addition of TBAI
(0.44 g, 1.19 mmol) and BnBr (1.8 mL, 15.5 mmol), the reaction
was stirred for 5 h, and then MeOH was added and the
mixture was coconcentrated with toluene. The residue was
dissolved in EtOAc, washed with H2O, aqueous saturated
NaHCO3, and H2O, dried (Na2SO4), filtered, and concentrated.
Reduced-pressure column chromatography (toluene-EtOAc,
40:1 f 10:1) of the residue gave 6 (2.3 g, 75%), isolated as a
1
white solid. H NMR (300 MHz, CDCl3) δ ) 1.80-1.95 (m, 2
H, OCH2CH2CH2N3), 3.41 (t, 2 H, OCH2CH2CH2N3), 3.55 (dd,
J2,3 ) 9.6 Hz, J3,4 ) 3.6 Hz, 1 H, H-3), 3.58-3.64 (m, 2 H, H-5
and OCHHCH2CH2N3), 3.82 (dd, J1,2 ) 8.1 Hz, 1 H, H-2), 3.97-
4.02 (m, 2 H, H-6b and OCHHCH2CH2N3), 4.10 (d, 1 H, H-4),
4.27 (dd, J5,6a < 1 Hz, J6a,6b ) 12.3 Hz, 1 H, H-6a), 4.36 (d, 1
H, H-1), 4.75 and 4.83 (s and dd, each 2 H, 2 OCH2C6H5), 5.48
(s, 1 H, OCHC6H5), 7.28-7.38 and 7.53-7.56 (2 m, 15 H,
OCHC6H5 and 2 OCH2C6H5); 13C NMR (75.5 MHz, CDCl3) δ
) 31.0 (OCH2CH2CH2N3), 50.1 (OCH2CH2CH2N3), 68.2, 71.0,
73.7, and 77.1 (C-6, OCH2CH2CH2N3, and OCH2C6H5), 68.2,
75.5, 80.2, and 81.0 (C-2, C-3, C-4, and C-5), 103.0 and 105.3
(C-1 and OCHC6H5), 128.3, 129.4-130.7, and 139.7-140.6
(OCHC6H5); HRMS for C30H33N3O6 (M, 531.2369) M + Na
found 554.2196, calcd 554.2267.
3-Azidopropyl 2,3,4,6-Tetra-O-acetyl-â-D-galactopyra-
noside (3). A solution of 1,2,3,4,6-penta-O-acetyl-â-D-galac-
topyranoside 1 (9.2 g, 23.7 mmol) and 3-bromopropanol (4.9
g, 35.5 mmol) in dry CH2Cl2 (100 mL), containing molecular
sieve 4 Å (1.5 g), was cooled to 0 °C, and BF3‚Et2O (15 mL,
118.5 mmol) was added in 2 min. The mixture was stirred
overnight, neutralized with NEt3, washed with aqueous satu-
rated NaHCO3, H2O, and aqueous saturated NaCl, dried (Na2-
SO4), filtered, and concentrated. Reduced-pressure column
chromatography (toluene-EtOAc 8:1) of the residue gave 2
(9.36 g, 84%), isolated as a slightly yellow syrup. To a solution
of 2 (0.81 g, 1.72 mmol) in dry DMF (5 mL) was added NaN3
(0.56 g, 8.63 mmol), and the mixture was stirred at 100 °C
overnight, then filtered over Hyflo, and coconcentrated with
toluene. The residue was dissolved in dichloromethane and
washed with aqueous saturated NaCl, dried (Na2SO4), filtered,
and concentrated to give 3 (0.65 g, 87%), isolated as a slightly
yellow syrup. 1H NMR (300 MHz, CDCl3): δ ) 1.84-1.89 (m,
2 H, OCH2CH2CH2N3), 1.98, 2.05, 2.07, and 2.15 (4 s, each 3
H, 4 COCH3), 3.38 (t, 2 H, OCH2CH2CH2N3), 3.58-3.65 (m, 1
H, OCHHCH2CH2N3), 3.93-4.00 (m, 2 H, H-5 and OCHHCH2-
3-Azidopropyl 2,3,6-Tri-O-benzyl-â-D-galactopyrano-
side (7). To a solution of 6 (0.61 g, 1.15 mmol) and TES (1.1
mL, 6.9 mmol) in dry CH2Cl2 (10 mL) was added, in 1 min at
0 °C, TFA (0.5 mL, 6.9 mmol). The mixture was stirred for 5
h, during which the temperature was allowed to reach room
temperature and then neutralized with aqueous saturated
NaHCO3. The organic layer was separated, dried (Na2SO4),
filtered, and concentrated. Reduced pressure column chroma-
tography (toluene-EtOAc 10:1) of the residue yielded 7 (0.47
g, 76%), isolated as a colorless syrup. 1H NMR (300 MHz,
CDCl3) δ ) 1.70-1.75 (m, 2 H, OCH2CH2CH2N3), 3.22 (t, 2 H,
OCH2CH2CH2N3), 3.75 (dd, J2,3 ) 9.3 Hz, J3,4 ) 3.3 Hz, 1 H,
H-3), 3.86 (d, J4,5 < 1 Hz, 1 H, H-4), 4.23 (d, J1,2 ) 7.5 Hz, 1 H,
H-1), 4.44 and 4.55 (2 s, each 2 H, 2 OCH2C6H5), 4.68 (2 d,
CH2N3), 4.10-4.21 (m, 2 H, H-6a and H-6b), 4.50 (d, J1,2
)
8.1 Hz, 1 H, H-1), 5.03 (dd, J2,3 ) 10.5 Hz, J3,4 ) 3.6 Hz, 1 H,
H-3), 5.18 (dd, 1 H, H-2), 5.39 (d, J4,5 < 1 Hz, 1 H, H-4); 13C
NMR (75.5 MHz, CDCl3) δ ) 23.2, 23.3 (2 C), and 23.4
(COCH3), 31.6 (OCH2CH2CH2N3), 50.5 (OCH2CH2CH2N3), 63.9
(C-6), 69.1 (OCH2CH2CH2N3), 69.7, 71.5, 73.3, and 73.5 (C-2,
C-3, C-4, and C-5), 103.9 (C-1), 172.1, 172.8, 172.9, and 173.1
(COCH3); HRMS for C17H25N3O10 (M, 431.154) M + NH4 found
449.189, calcd 449.1883.
each 1 H, OCH2C6H5), 7.16-7.22 (m, 15 H, 3 OCH2C6H5); 13
C
NMR (75.5 MHz, CDCl3) δ ) 28.8 (OCH2CH2CH2N3), 47.8
(OCH2CH2CH2N3), 66.0, 68.8, 71.8, 73.2, and 74.8 (C-6, OCH2-
CH2CH2N3, and OCH2C6H5), 66.3, 72.8, 78.5, and 80.2 (C-2,
C-3, C-4, C-5), 103.2 (C-1), 127.2-128.0 and 137.4-138.1
(OCHC6H5); HRMS for C30H35N3O6 (M, 533.2526) M + Na
found 556.2400, calcd 556.2424.
3-Azidopropyl 4,6-Di-O-benzylidene-â-D-galactopyra-
noside (5). To a solution of 3 (2.6 g, 6.1 mmol) in MeOH (20
mL) and CH2Cl2 (6 mL) was added 30% methanolic NaOMe
(0.5 mL). The mixture was stirred for 1 h, then neutralized
with Dowex 50X8 (H+), filtered, and concentrated. Reduced-
pressure column chromatography (CH2Cl2-MeOH 10:1) of the
residue gave 4 (1.56 g, 97%), isolated as a colorless syrup.
High-resolution MS data for C9H17N3O6 (M, 263.1117): M +
H found 264.121, calcd 264.1195. To a solution of 4 (4.2 g, 15.9
mmol) in dry DMF (20 mL) were added R,R-dimethoxytoluene
(19 mL, 127.6 mmol) and a catalytic amount of p-TSOH. The
mixture was stirred for 2.5 h at 50 °C and neutralized with
NEt3. After the addition of EtOAc, the mixture was washed
with H2O and aqueous saturated NaCl, dried (MgSO4), filtered,
and coconcentrated with toluene. Reduced-pressure column
chromatography (toluene-EtOAc, 10:1 f 1:4) of the residue
3-Azidopropyl (2,3,4,6-Tetra-O-benzyl-r-D-galactopy-
ranosyl)-2,3,6-tri-O-benzyl-â-D-galactopyranoside (9). To
a solution of 7 (1.10 g, 2.05 mmol) and 2,3,4,6-tetra-O-benzyl-
R-D-galactopyranoside trichloroacetimidate 8 (2.11 g, 3.08
mmol) in dry Et2O (50 mL) was added, at 0 °C, TMSOTf (8
µL, 0.46 mmol). The mixture was stirred for 1.5 h, during
which the temperature was allowed to reach room tempera-
ture, then neutralized with NEt3, and concentrated. Reduced-
pressure column chromatography (toluene-EtOAc 40:1) of the
residue gave 9 (1.60 g, 73%), isolated as a slightly yellow syrup.
1H NMR (300 MHz, CDCl3) δ ) 1.81-1.89 (m, 2 H, OCH2CH2-
CH2N3), 3.38 (t, 2 H, OCH2CH2CH2N3), 3.66 (dd, J1,2 ) 7.8 Hz,
J2,3 ) 9.9 Hz, 1 H, H-2), 4.30 (d, 1 H, H-1), 5.04 (d, J1,2 ) 2.1
Hz, 1 H, H-1′), 7.14-7.37 (m, 35 H, 7 OCH2C6H5); 13C NMR
(75.5 MHz, CDCl3) δ ) 28.9 (OCH2CH2CH2N3), 48.0 (OCH2-
CH2CH2N3), 66.2, 67.6, 71.9, 72.0, 72.4, 72.7, 72.9, 73.4, 74.6,
and 74.9 (C-6, C-6′, OCH2CH2CH2N3, and OCH2C6H5), 69.0,
73.3, 74.3, 74.4, 76.3, 78.6, 78.7, and 80.6 (C-2, C-3, C-4, C-5,
C-2′, C-3′, C-4′, and C-5′), 100.2 and 103.5 (C-1 and C-1′),
127.0-128.1 and 137.7-138.6 (OCHC6H5); HRMS for C64H69-
N3O11 (M, 1055.4932) M + Na found 1078.477, calcd 1078.491.
1
gave 5 (4.5 g, 80%), isolated as a white solid. H NMR (300
MHz, CDCl3) δ ) 1.91-1.95 (m, 2 H, OCH2CH2CH2N3), 3.43-
3.50 (m, 3 H, H-5 and OCH2CH2CH2N3), 3.62-3.79 (m, 3 H,
H-2, H-3, and OCHHCH2CH2N3), 4.00-4.06 (m, 1 H, OCHH-
CH2CH2N3), 4.09 (dd, J5,6b ) 2.1 Hz, J6a,6b ) 12.3 Hz, 1 H,
H-6b), 4.22 (d, J3,4 ) 3.3, J4,5 < 1 Hz, 1 H, H-4), 4.30 (d, J1,2
)
7.5 Hz, 1 H, H-1), 4.34 (dd, J5,6a < 1 Hz, 1 H, H-6a), 5.56 (s, 1
H, OCHC6H5), 7.36-7.38 and 7.49-7.52 (2 m, 5 H, OCHC6H5);
13C NMR (75.5 MHz, CDCl3) δ ) 30.8 (OCH2CH2CH2N3), 50.1
(OCH2CH2CH2N3), 68.4 and 70.9 (C-6 and OCH2CH2CH2N3),
68.3, 73.1, 74.3, and 77.2 (C-2, C-3, C-4, and C-5), 103.0 and
104.7 (C-1 and OCHC6H5), 128.3, 130.0, 131.0, and 139.5
3-(N-tert-Butyloxycarbonyl)aminopropyl (2,3,4,6-Tetra-
O-benzyl-r-D-galactopyranosyl)-2,3,6-tri-O-benzyl-â-D-ga-
lactopyranoside (10). A suspension of 9 (1.2 g, 1.13 mmol),
Lindlar catalyst (0.24 g), Boc2O (0.27 g, 1.25 mmol), and 3

Bacterial Adhesion Inhibition by Galabiose Compounds
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6505
drops of NEt3 in 1:1 MeOH-EtOAc (12 mL) was stirred
overnight under an H2 atmosphere. After the addition of
EtOAc, the suspension was filtered over Hyflo and concen-
trated. Reduced-pressure column chromatography (toluene-
EtOAc 15:1) of the residue yielded 10 (1.2 g, 92%), isolated as
a colorless syrup. 1H NMR (300 MHz, CDCl3) δ ) 1.39 [s, 9 H,
OC(CH3)3], 1.78-1.82 (m, 2 H, OCH2CH2CH2NH), 3.38 (dd,
J2,3 ) 9.9 Hz, J3,4 ) 2.7 Hz, 1 H, H-3), 3.66 (dd, J1,2 ) 7.5 Hz,
1 H, H-2), 4.00 (d, 1 H, H-4), 4.10 (d, J1,2 ) 2.1 Hz, 1 H, H-2′),
4.30 (d, 1 H, H-1), 5.01 (d, 1 H, H-1′), 7.17-7.37 (m, 35 H, 7
OCH2C6H5); 13C NMR (75.5 MHz, CDCl3) δ ) 28.2 [OC(CH3)3],
29.6 (OCH2CH2CH2NH), 37.5 (OCH2CH2CH2NH), 67.4, 67.7,
71.9, 72.0, 72.7, 72.9, 73.4, 74.6, 74.9, and 78.6 (C-6, C-6′,
OCH2CH2CH2NH, and OCH2C6H5), 69.0, 73.3, 74.3, 74.4, 76.3,
78.5, 78.7, and 80.7 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and
C-5′), 100.2 and 103.6 (C-1 and C-1′), 127.1-128.4 and 137.8-
138.7 (OCHC6H5), 155.7 [OC(CH3)3]; HRMS for C69H79NO13 (M,
1129.5551) M + Na found 1152.542, calcd 1152.553.
3-(N-tert-Butyloxycarbonyl)aminopropyl (2,3,4,6-Tetra-
O-acetyl-r-D-galactopyranosyl)-2,3,6-tri-O-acetyl-â-D-ga-
lactopyranoside (11). A solution of 10 (1.68 g, 1.48 mmol)
and 3 drops of HOAc in 1:1 MeOH-EtOAc (12 mL) was stirred
overnight under a H2 atmosphere, then filtered over Hyflo, and
concentrated. The residue was dissolved in 1:1 pyridine-acetic
anhydride (100 mL) and stirred for 4 h, and then coconcen-
trated with toluene. Reduced-pressure column chromatography
(toluene-EtOAc, 4:1 f 1:1) of the residue gave 11 (1.0 g, 88%),
isolated as a white foam. 1H NMR (300 MHz, CDCl3) δ ) 1.43
[s, 9 H, OC(CH3)3], 1.75-1.81 (m, 2 H, OCH2CH2CH2NH), 1.98,
2.03, 2.06, 2.08, 2.10, and 2.13 (6 s, 3, 3, 3, 6, 3, and 3 H, 7
COCH3), 3.26 (t, 2 H, OCH2CH2CH2NH), 3.56 (m, 1 H,
OCHHCH2CH2NH), 3.93 (m, 1 H, OCHHCH2CH2NH), 4.07 (d,
J3,4 ) 3.0 Hz, J4,5 < 1 Hz, 1 H, H-4), 4.57 (d, J1,2 ) 8.1 Hz, 1
H, H-1), 4.80 (dd, J2,3 ) 10.8 Hz, 1 H, H-3), 5.00 (d, J1,2 ) 3.6
Hz, 1 H, H-1′), 5.18 (dd, 1 H, H-2), 5.20 (dd, J2,3 ) 11.4 Hz, 1
H, H-2′), 5.39 (dd, J3,4 ) 3.0 Hz, 1 H, H-3′), 5.57 (d, J4,5 < 1
Hz, 1 H, H-4′); 13C NMR (75.5 MHz, CDCl3) δ ) 20.4-20.7
(COCH3), 28.1 [OC(CH3)3], 29.4 (OCH2CH2CH2NH), 37.4 (OCH2-
CH2CH2NH), 60.2 and 61.7 (C-6 and C-6′), 67.4 (OCH2CH2-
CH2NH), 66.7, 67.1, 67.5, 68.2, 68.3, 71.6, 72.4, and 76.8 (C-2,
C-3, C-4, C-5, C-2′, C-3′, C-4′, and C-5′), 99.1 and 100.8 (C-1
and C-1′), 155.7 [OC(CH3)3], 168.9, 169.6, 169.9, 170.1, 170.2,
170.3, and 170.4 (COCH3); HRMS for C34H51NO20 (M, 793.3004)
M + Na found 816.286, calcd 816.298.
3-Aminopropyl (2,3,4,6-Tetra-O-acetyl-r-D-galactopy-
ranosyl)-2,3,6-tri-O-acetyl-â-D-galactopyranoside Triflu-
oroacetic Acid Salt (12). To a solution of 11 (222 mg, 0.279
mmol) in CH2Cl2 (8 mL) was added, at 0 °C, 90% aqueous TFA
(1 mL), and the mixture was stirred for 4 h and then
coconcentrated with ethanol to give 12 (225 mg, quantitative),
isolated as a white foam. 13C NMR (75.5 MHz, CDCl3) δ )
20.4-20.7 (COCH3), 28.1 [OC(CH3)3], 29.4 (OCH2CH2CH2NH),
37.4 (OCH2CH2CH2NH), 60.2 and 61.7 (C-6 and C-6′), 67.4
(OCH2CH2CH2NH), 66.7, 67.1, 67.5, 68.2, 68.3, 71.6, 72.4, and
76.8 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and C-5′), 99.1 and
100.8 (C-1 and C-1′), 155.7 [OC(CH3)3], 168.9, 169.6, 169.9,
170.1, 170.2, 170.3, and 170.4 (COCH3).
Short Spacer (2,3,4,6-Tetra-O-acetyl-r-D-galactopyra-
nosyl)-2,3,6-tri-O-acetyl-â-D-galactopyranoside (13). To a
solution of 12 (225 mg, 0.279 mmol) in pyridine (3 mL) and
dry dioxane (1 mL) was added a solution of diglycolic anhydride
(36 mg, 0.307 mmol) in dry dioxane (1 mL). The mixture was
stirred overnight at 90 °C and then coconcentrated with
toluene. The residue was dissolved in CH2Cl2 and washed with
aqueous 1 M KHSO4 and aqueous saturated NaCl, dried (Na2-
SO4), filtered, and concentrated, yielding 13 (226 mg, quan-
titative), isolated as a white foam. 1H NMR (300 MHz, CDCl3)
δ ) 1.82-1.89 (m, 2 H, OCH2CH2CH2NH), 2.00, 2.04, 2.06,
2.08, 2.09, 2.10, and 2.14 (7 s, each 3 H, 7 COCH3), 3.81 (t, 2
H, OCH2CH2CH2NH), 4.83 (dd, J2,3 ) 10.8 Hz, J3,4 ) 2.7 Hz,
1 H, H-3), 5.00 (d, J1,2 ) 3.6 Hz, 1 H, H-1′), 5.18 (dd, 1 H,
H-2), 5.20 (dd, J2,3 ) 10.8 Hz, 1 H, H-2′), 5.40 (dd, J3,4 ) 3.3
Hz, 1 H, H-3′), 5.57 (d, J4,5 < 1 Hz, 1 H, H-4′); 13C NMR (75.5
MHz, CDCl3) δ ) 20.2-20.6 (COCH3), 28.6 (OCH2CH2CH2NH),
36.1 (OCH2CH2CH2NH), 60.2 and 61.6 (C-6 and C-6′), 67.4
(OCH2CH2CH2NH), 68.4 and 70.6 (COCH2OCH2COOH), 66.6,
67.1, 67.5, 68.1, 68.3, 71.5, 72.3, and 76.8 (C-2, C-3, C-4, C-5,
C-2′, C-3′, C-4′, and C-5′), 99.0 and 100.7 (C-1 and C-1′), 155.7
[OC(CH3)3], 169.2, 169.8, 169.9, 170.0, 170.1, 170.2, 170.3,
170.4, and 171.4 (COCH3 and COCH2OCH2COOH); HRMS for
C33H47NO22 (M, 809.2590) M + Na found 832.2479, calcd
832.2488.
1,4-Bis[3-(tert-butoxycarbonyl)aminoprop-1-ynyl]ben-
zene (16). To a solution of 14 (2.0 g, 6.06 mmol), CuI (0.11 g,
0.6 mmol), and Pd(0)(PPh3)4 (0.34 g, 0.30 mmol) in dry CH3CN
(10 mL) was added, under N2, a solution of Boc-protected
propargylamine (2.8 g, 18.1 mmol) in dry NEt3 (1.5 mL) and
dry CH3CN (1 mL). The reaction mixture was stirred under
N2 for 2 days, then diluted with CH2Cl2, filtrated over Hyflo,
and concentrated. Reduced-pressure column chromatography
(hexanes-EtOAc 15:1) of the residue gave 16 (1.77 g, 76%),
isolated as a slightly brown solid. 1H NMR (300 MHz, CDCl3)
δ ) 1.46 [s, 18 H, 2 OC(CH3)3], 4.15 (d, J ) 5.7 Hz, 4 H, 2
CH2), 4.75 (br s, 2 H, 2 NH), 7.33 (s, 4 H, C6H4); 13C NMR
(75.5 MHz, CDCl3) δ ) 28.2 [OC(CH3)3], 31.8 (CH2), 79.9
[OC(CH3)3], 82.4 (CCH2), 87.0 (CC6H4), 122.5 and 131.4 (C6H4),
155.1 (CO); HRMS for C22H28N2O4 (M, 384.2049) M + Na found
407.2028, calcd 407.1947.
Trifluoroacetic Acid Salt of 1,4-Bis(3-aminoprop-1-
ynyl)benzene (17). To a solution of 16 (0.178 g, 0.463 mmol)
in CH2Cl2 (10 mL) was added, at 0 °C, TFA (0.35 mL). The
solution was stirred for 5 h, during which the temperature
was allowed to reach room temperature, and then concentrated
to give 17 (0.176 g, quantitative), isolated as a slightly brown
solid. HRMS for C12H12N2 (M, 184.1000) M + H found
185.1089, calcd 185.1078.
1,3-Bis[3-(tert-Butoxycarbonyl)aminoprop-1-ynyl]ben-
zene (18). To a solution of 15 (1.0 g, 3.03 mmol), CuI (6 mg,
0.30 mmol), and Pd(0)(PPh3)4 (0.17 g, 0.151 mmol) in dry CH3-
CN (5 mL) was added, under N2, a solution of Boc-protected
propargylamine (1.41 g, 9.09 mmol) in dry NEt3 (0.75 mL) and
dry CH3CN (1 mL). The reaction mixture was stirred under
N2 for 20 h, then diluted with CH2Cl2, filtrated over Hyflo,
and concentrated. Reduced-pressure column chromatography
(hexanes-EtOAc 15:1) of the residue gave 18 (1.02 g, 87%),
isolated as a slightly yellow solid. 1H NMR (300 MHz, CDCl3)
δ ) 1.47 [s, 18 H, 2 OC(CH3)3], 4.15 (d, J ) 5.7 Hz, 4 H, 2
CH2), 4.75 (br s, 2 H, 2 NH); 13C NMR (75.5 MHz, CDCl3) δ )
28.2 [OC(CH3)3], 30.9 (CH2), 79.9 [OC(CH3)3], 82.0 (CCH2), 85.9
(CC6H4), 122.8, 128.2, 131.3, and 134.6 (C6H4), 155.1 (CO);
HRMS for C22H28N2O4 (M, 384.2049) M + Na found 407.1951,
calcd 407.1947.
Trifluoroacetic Acid Salt of 1,3-Bis(3-aminoprop-1-
ynyl)benzene (19). To a solution of 18 (0.148 g, 0.385 mmol)
in CH2Cl2 (8 mL) was added, at 0 °C, TFA (0.3 mL). The
solution was stirred for 4 h, during which the temperature
was allowed to reach room temperature, and then concentrated
to give 19 (0.146 g, quantitative), isolated as a slightly yellow
solid. 1H NMR (300 MHz, CDCl3) δ ) 3.96 (s, 4 H, 2 CH2),
4.55 (br s, 2 H, 2 NH), 7.32-7.54 (m, 4 H, C6H4); 13C NMR
(75.5 MHz, CDCl3) δ ) 29.1 (CH2), 80.2 (CCH2), 85.2 (CC6H4),
121.4, 128.2, 131.8 and 134.4 (C6H4); HRMS for C12H12N2 (M,
184.1000) M + Na found 185.1019, calcd 185.1078.
General Procedure for Preparation of Acetyl-Protect-
ed Galabiose Compounds 21, 24, 26, 28, 31, and 34. To a
solution of 17, 19, 20, 23, 30, or 33, 13 (1.1 equiv per NH2
group), and BOP (1.2 equiv per NH2 group) in dry CH2Cl2 and
dry DMF was added dropwise DIPEA (1.1 equiv per NH2
group). The mixture was stirred and then concentrated. The
residue was dissolved in EtOAc, washed with 1 M KHSO4 and
aqueous saturated NaCl, dried (Na2SO4), filtered, and concen-
trated. The residue was purified by reduced-pressure column
chromatography (CH2Cl2-MeOH, 15:1 f 1:1) to give 21, 24,
26, 28, 31, or 34, isolated as white glass.
General Procedure for Preparation of Galabiose Com-
pounds 22, 25, 27, 29, 32, and 35. To a solution of 21, 24,
26, 28, 31, or 34 in MeOH (1 mL) was added methanolic

6506 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Joosten et al.
NaOMe (pH 9). The mixture was stirred for 1 h, then
neutralized with Dowex 50X8 (H+), filtered, and concentrated.
The residue was applied on a SPE-column, which was eluted
with H2O-AcCN (100:0 f 85:15) to give 22, 25, 27, 29, 32, or
35, isolated as a white solid after freeze-drying.
Galabiose-Specific 1H NMR data (300 MHz, CDCl3) for
Compounds 21, 24, 26, 28, 31, 34, and 37. δ ) 4.48 (d, H-1),
4.83 (dd, H-3), 5.00 (d, H-1′), 5.17 (dd, 1H, H-2), 5.20 (dd, H-2′),
5.38 (dd, H-3′), 5.56 (d, H-4′)
Monovalent Per-O-acetyl-Protected Galabiose Short
Spacer (21). Reaction time 1 h; CH2Cl2/DMF 14:1; yield 66%
(38 mg); 13C NMR (75.5 MHz, CDCl3) δ ) 20.4-20.7 (COCH3),
29.0 (OCH2CH2CH2NH), 36.7 (OCH2CH2CH2NH), 38.2 (OC-
H2CH2NH), 52.0 (C6H4COCH3), 60.3 and 61.6 (C-6 and C-6′),
66.5 and 68.0 (OCH2CH2CH2NH and OCH2CH2NH), 70.9 and
71.0 (COCH2OCH2CO), 66.8, 67.1, 67.5, 68.3, 68.4, 71.8, 72.3,
and 76.7 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and C-5′), 99.2
and 101.0 (C-1 and C-1′), 114.6, 119.5, 122.2, 129.4, 131.2, and
158.2 (C6H4COCH3), 166.6 (C6H4COCH3), 168.6-170.5 (COCH3
and CONH); HRMS for C43H58N2O24 (M, 986.3380) M + Na
found 1009.3435, calcd 1009.3278.
Divalent Per-O-acetyl-Protected Short Spacer 1,3-
Rigid Galabioside (26). Reaction time 2 h; CH2Cl2/DMF 7:1;
yield 68% (101 mg); 13C NMR (75.5 MHz, CDCl3) δ ) 20.4-
20.7 (COCH3), 28.9 (OCH2CH2CH2NH), 29.2 (CCH2), 36.6
(OCH2CH2CH2NH), 60.2 and 61.5 (C-6 and C-6′), 67.9 (OCH2-
CH2CH2NH), 70.8 (2 C, COCH2OCH2CO), 66.8, 67.1, 67.5, 68.2,
68.4, 71.7, 72.2, and 76.7 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′,
and C-5′), 81.7 (CCH2), 85.3 (CC6H4), 99.0 and 100.8 (C-1 and
C-1′), 122.6, 128.1, 131.3, and 134.5 (CC6H4), 168.6-170.4
(COCH3 and CONH); HRMS for C78H102N4O42 (M, 1766.5969)
M + Na found 1789.6312, calcd 1789.5867.
Divalent Short Spacer 1,3-Rigid Galabioside (27). Yield
1
76% (51 mg); H NMR (300 MHz, D2O) δ ) 1.74 (m, 4 H, 2
OCH2CH2CH2NH), 3.25 (m, 4 H, 2 OCH2CH2CH2NH), 4.02 and
4.05 (2 s, each 4 H, 2 COCH2OCH2CO), 4.14 (s, 4 H, 2 CCH2),
4.82 (d, J1,2 ) 2.7 Hz, 2 H, 2 H-1′), 7.28, 7.35, and 7.45 (3 m,
4 H, C6H4);13C NMR (75.5 MHz, D2O) δ ) 29.2 (OCH2CH2-
CH2NH), 29.6 (CCH2), 36.6 (OCH2CH2CH2NH), 60.5 and 61.1
(C-6 and C-6′), 68.1 (OCH2CH2CH2NH), 69.4, 69.6, 69.9, 71.4,
71.6, 73.0, 75.6, 77.7 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and
C-5′), 70.5 (2 C, COCH2OCH2CO), 82.2 (CCH2), 86.2 (CC6H4),
100.9 (C-1′), 103.5 (C-1), 172.1 (CONH); HRMS for C50H74N4O28
(M, 1178.4490) M + Na found 1201.4894, calcd 1201.4388.
Monovalent Galabiose Short Spacer (22). Yield 80% (22
1
mg); H NMR (300 MHz, D2O) δ ) 1.76 (m, 2 H, OCH2CH2-
Divalent Per-O-acetyl-Protected Short Spacer 1,4-
Rigid Galabioside (28). Reaction time 1 h; CH2Cl2/DMF 5:1;
yield 97% (87 mg); 13C NMR (75.5 MHz, CDCl3) δ ) 20.5-
20.8 (COCH3), 28.9 (OCH2CH2CH2NH), 29.2 (CCH2), 36.9
(OCH2CH2CH2NH), 60.3 and 61.6 (C-6 and C-6′), 68.1 (OCH2-
CH2CH2NH), 70.9 (2 C, COCH2OCH2CO), 66.9, 67.2, 67.5, 68.2,
68.5, 71.8, 72.3, and 76.8 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′,
and C-5′), 82.3 (CCH2), 86.3 (CC6H4), 99.1 and 100.9 (C-1 and
C-1′), 168.8-170.4 (COCH3 and CONH); HRMS for C78H102N4O42
(M, 1766.5969) M + Na found 1789.6218, calcd 1789.5867.
Divalent Short Spacer 1,4-Rigid Galabioside (29). Yield
93% (50 mg); 1H NMR (300 MHz, D2O) δ ) 1.73 (m, 4 H, 2
OCH2CH2CH2NH), 3.24 (m, 4 H, 2 OCH2CH2CH2NH), 3.54 (dd,
J1,2 ) 7.8 Hz, J2,3 ) 10.5 Hz, 2 H, 2 H-2), 3.53-3.59 (m, 10 H,
2 each H-3, H-5, H-6a′, H-6b′, and OCHHCH2CH2NH), 3.66-
3.79 (m, 10 H, 2 each H-2′, H-3′, H-6a, H-6b, and OCHHCH2-
CH2NH), 3.78 (s, 4 H, 2 H-4 and 2 H-4′), 3.90 and 3.93 (2 s,
each 4 H, 2 COCH2OCH2CO), 4.05 (s, 4 H, 2 CCH2), 4.12-
4.18 (m, 4 H, 2 H-1 and 2 H-5′), 4.82 (d, J1,2 ) 2.7 Hz, 2 H, 2
H-1′), 7.26 (s, 4 H, C6H4); 13C NMR (75.5 MHz, D2O) δ ) 29.2
(OCH2CH2CH2NH), 29.6 (CCH2), 36.6 (OCH2CH2CH2NH), 60.5
and 61.1 (C-6 and C-6′), 68.1 (OCH2CH2CH2NH), 69.4, 69.6,
69.9, 71.4, 71.6, 73.0, 75.6, 77.7 (C-2, C-3, C-4, C-5, C-2′, C-3′,
C-4′, and C-5′), 70.5 (2 C, COCH2OCH2CO), 82.5 (CCH2), 87.1
(CC6H4), 101.0 (C-1′), 103.6 (C-1), 172.1 (CONH); HRMS for
C50H74N4O28 (M, 1178.4490) M + Na found 1201.4495, calcd
1201.4388.
Divalent Per-O-acetyl-Protected Galabiose Long Spac-
er (31). Reaction time 1 h; CH2Cl2/DMF 15:1; yield 61% (125
mg); 13C NMR (75.5 MHz, CDCl3) δ ) 20.4-20.6 (COCH3), 28.5
(2 C) and 28.9 (OCH2CH2CH2NH), 36.3 and 36.8 (2 C, OCH2-
CH2CH2NH), 38.3 (OCH2CH2NH), 52.0 (C6H3COCH3), 60.4 and
61.6 (C-6 and C-6′), 66.8, 67.1, 67.5, 68.1, 68.5, 71.7, 72.2, and
76.8 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and C-5′), 99.1 and
100.9 (C-1 and C-1′), 106.0, 107.8, 133.6, and 159.2 (C6H3-
COCH3), 166.3 (C6H3COCH3), 169.4-170.3 (COCH3 and CONH);
HRMS for C106H160N8O58 (M, 2474.42, average mass) M + Na
found 2495.6, calcd 2497.4.
CH2NH), 3.27 (m, 2 H, OCH2CH2CH2NH), 3.48 (dd, J1,2 ) 7.8
Hz, J2,3 ) 10.2 Hz, 1 H, H-2), 3.60-3.68 (m, 7 H, H-3, H-5,
H-6a′, H-6b′, OCHHCH2CH2NH, and OCH2CH2NH), 3.73-
3.79 (m, 3 H, H-2′, H-6a, and H-6b), 3.83-3.87 (m, 5 H, H-3′,
OCHHCH2CH2NH, and C6H3COCH3), 3.97 (s, 4 H, H-4, H-4′,
and COCH2OCH2CO), 4.03 (s, 2 H, COCH2OCH2CO), 4.17 (m,
2 H, OCH2CH2NH), 4.29-4.36 (m, 2 H, H-1 and H-5′), 4.90
(d, J1,2 ) 3.9 Hz, 1 H, H-1′), 7.17 (dd, 1 H, CH5), 7.38 (dd, 1 H,
CH4), 7.46 (dd, 1 H, CH2), 7.56 (dd, 1 H, CH6), 8.16 and 8.37
(2 t, each 1 H, 2 NH); 13C NMR (75.5 MHz, D2O) δ ) 29.3
(OCH2CH2CH2NH), 36.6 (OCH2CH2CH2NH), 39.2 (OCH2CH2-
NH), 53.3 (C6H4COCH3), 60.7 and 61.1 (C-6 and C-6′), 67.2
(OCH2CH2NH), 68.2 (OCH2CH2CH2NH), 69.4, 69.5, 69.7, 71.2,
71.4, 73.0, 75.6, 77.7 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and
C-5′), 70.3 (2 C) (COCH2OCH2CO), 100.6 (C-1′), 103.5 (C-1),
115.6, 121.1, 122.9, and 130.5 (C6H4COCH3), 168.7 (C6H4CO-
CH3), 172.0 and 172.4 (CONH); HRMS for C29H44N2O17 (M,
692.2640) M + Na found 715.2514, calcd 715.2538.
Divalent Per-O-acetyl-Protected Galabiose Short
Spacer (24). Reaction time 1 h; CH2Cl2/DMF 8:1; yield 88%
(135 mg); 13C NMR (75.5 MHz, CDCl3) δ ) 20.3-20.6 (COCH3),
28.9 (OCH2CH2CH2NH), 36.5 (OCH2CH2CH2NH), 38.1 (OC-
H2CH2NH), 52.0 (C6H3COCH3), 60.2 and 61.5 (C-6 and C-6′),
66.4 and 67.9 (OCH2CH2CH2NH and OCH2CH2NH), 70.7 and
70.8 (COCH2OCH2CO), 66.7, 67.0, 67.4, 68.1, 68.3, 71.6, 72.2,
and 76.7 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and C-5′), 99.0
and 100.8 (C-1 and C-1′), 106.3, 107.8, 133.8, and 159.2 (C6H3-
COCH3), 166.1 (C6H3COCH3), 168.6-170.3 (COCH3 and CONH);
HRMS forC78H109N4O46 (M, 1836.6235) M + Na found 1859.687,
calcd 1859.621.
Divalent Galabiose Short Spacer (25). Yield 99% (74
mg); 1H NMR (300 MHz, D2O) δ ) 1.79-1.83 (m, 4 H, 2
OCH2CH2CH2NH), 3.30 (m, 4 H, 2 OCH2CH2CH2NH), 3.35 (s,
3 H, C6H3COCH3), 3.54 (dd, J1,2 ) 7.8 Hz, J2,3 ) 10.2 Hz, 2 H,
2 H-2), 3.63-3.71 (m, 14 H, 2 each H-3, H-5, H-6a′, H-6b′,
OCHHCH2CH2NH, and OCH2CH2NH), 3.78-3.93 (m, 10 H,
H-2′, H-3′, H-6a, H-6b, and OCHHCH2CH2NH), 4.02 (d, J3,4
) 3.3 Hz, J4,5 < 1 Hz, 2 H, 2 H-4), 4.03 (d, J3,4 ) 3.6 Hz, J4,5
< 1 Hz, 2 H, 2 H-4′), 4.04 and 4.10 (2 s, each 4 H, 2 COCH2-
OCH2CO), 4.13 (m, 4 H, 2 OCH2CH2NH), 4.36 (m, 2 H, 2 H-5′),
4.38 (d, 2 H, 2 H-1), 4.95 (d, J1,2 ) 3.6 Hz, 2 H, 2 H-1′), 6.70
and 7.05 (2 s, 1 and 2 H, C6H3COCH3); 13C NMR (75.5 MHz,
D2O): δ ) 29.2 (OCH2CH2CH2NH), 36.6 (OCH2CH2CH2NH),
39.1 (OCH2CH2NH), 53.4 (C6H3COCH3), 60.7 and 61.1 (C-6 and
C-6′), 67.2 (OCH2CH2NH), 68.2 (OCH2CH2CH2NH), 69.3, 69.6,
69.8, 71.4, 71.6, 73.0, 75.7, 77.7 (C-2, C-3, C-4, C-5, C-2′, C-3′,
C-4′, and C-5′), 70.5 (2 C) (COCH2OCH2CO), 100.8 (C-1′), 103.5
(C-1), 107.5, 108.9, 132.0, and 159.9 (C6H3COCH3), 168.7
(C6H3COCH3), 172.0 (2 C) and 172.5 (2 C) (CONH); HRMS for
C50H80N4O32 (M, 1248.4756) M + Na found 1271.4650, calcd
1271.4654.
Divalent Galabiose Long Spacer (32). Yield 77% (74 mg);
1H NMR (300 MHz, D2O) δ ) 1.64-1.78 (m, 12 H, 6 OCH2CH2-
CH2NH), 3.18-3.26 (m, 12 H, 6 OCH2CH2CH2NH), 4.28 (m, 2
H, 2 H-5′), 4.34 (d, J1,2 ) 7.5 Hz, 2 H, 2 H-1), 4.95 (d, J1,2
)
3.0 Hz, 2 H, 2 H-1′), 6.62 and 6.99 (2 s, 1 and 2 H,
C6H3COCH3); 13C NMR (75.5 MHz, D2O) δ ) 28.8 (2 C), 29.1
(OCH2CH2CH2NH), 36.6, 36.7 (2 C, 2 OCH2CH2CH2NH), 39.1
(OCH2CH2NH), 53.4 (C6H3COCH3), 60.6 and 61.0 (C-6 and
C-6′), 67.2 (OCH2CH2NH), 68.2, 69.0 (2 C, 2 OCH2CH2CH2-
NH), 69.3, 69.5, 69.7, 71.3, 71.5, 72.9, 75.6, 77.6 (C-2, C-3, C-4,
C-5, C-2′, C-3′, C-4′, and C-5′), 69.9 and 70.1 (each 2 C, OCH2-
CH2OCH2CH2O), 70.4 (2 C, COCH2OCH2CO), 100.8 (C-1′),
103.4 (C-1), 107.3, 108.8, 132.0, and 159.9 (C6H3COCH3), 168.7

Bacterial Adhesion Inhibition by Galabiose Compounds
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6507
(C6H3COCH3), 171.8, 172.0, 172.1, and 172.4 (CONH); HRMS
for C78H132N8O44 (M, 1885.91, average mass) M + Na found
1908.9, calcd 1908.9
The bacteria, suspended in HBS-P buffer [10 mM N-(2-
hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES),
pH 7.4, 0.15 M NaCl, and 0.005% surfactant P20] to an OD600
of 0.3, were injected over the sensor chip at a flow rate of 10
µL/min and the binding to immobilized pigeon ovomucoid was
recorded. For inhibition assays, dilutions of the galabiose
compounds were made in HBS-P buffer and incubated with
the bacteria for 5 min before injection over the sensor chip.
Between the injections, the sensor chip surface was regener-
ated with 10 mM NaOH. For inhibition curve calculations the
data from repeated experiments were normalized from 0 to
100 by use of the BIAevaluation program (version 3.0, Biacore).
The IC50 values, that is, the concentration of the compound
that elicited half the maximal response, were defined for each
compound.
Tetravalent Per-O-acetyl-Protected Galabiose Short
Spacer (34). Reaction time 4 h; CH2Cl2/DMF 15:1; yield 92%
(224 mg); 13C NMR (75.5 MHz, CDCl3) δ ) 19.9-20.2 (COCH3),
28.4 (OCH2CH2CH2NH), 35.9 (OCH2CH2CH2NH), 37.9 (OC-
H2CH2NH), 53.0 (C6H3COCH3), 60.3 and 61.6 (C-6 and C-6′),
65.6, 67.0, and 69.2 (OCH2CH2CH2NH, OCH2CH2NH, and
COCH2OCH2CO), 66.5, 67.0, 67.4, 67.9, 68.4, 71.5, 72.0, and
76.7 (C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, and C-5′), 98.8 and
100.6 (C-1 and C-1′), 166.5 (C6H3COCH3), 169.6-170.4 (COCH3,
CONH); HRMS for C166H226N10O94 (M, 3865.58, average mass)
M + Na found 3888.6, calcd 3888.6.
Tetravalent Galabiose Short Spacer (35). Yield 79%
(121 mg); 1H NMR (300 MHz, D2O) δ ) 1.73-1.88 (m, 8 H, 4
OCH2CH2CH2NH), 3.23-3.37 (m, 8 H, 4 OCH2CH2CH2NH),
4.30-4.38 (m, 8 H, 4 H-1 and 4 H-5′), 4.95 (s, 4 H, 4 H-1′),
6.64 and 6.77 (2 s, 3 and 6 H, 3 C6H3COCH3); 13C NMR (75.5
MHz, D2O) δ ) 29.1 (OCH2CH2CH2NH), 36.6 (OCH2CH2CH2-
NH), 38.9 (OCH2CH2NH), 53.1 (C6H3COCH3), 60.6 and 61.1
(C-6 and C-6′), 66.9 (OCH2CH2NH), 68.1 (OCH2CH2CH2NH),
69.3, 69.5, 69.7, 71.3, 71.5, 73.0, 75.6, 77.6 (C-2, C-3, C-4, C-5,
C-2′, C-3′, C-4′, and C-5′), 70.4 (COCH2OCH2CO), 100.8 (C-
1′), 103.5 (C-1), HRMS for C110H170N10O66 (M, 2688.55, average
mass) M + Na found 2711.9, calcd 2711.6.
Octavalent Per-O-acetyl-Protected Short Spacer PAM-
AM Generation 1 Galabioside (37). PAMAM generation 1
36 (20% weight solution in MeOH; 7.69 µmol) was evaporated
to dryness and dissolved in DMSO (5 mL). Compound 13 (54.8
mg, 67.7 µmol), TBTU (23.7 mg, 73.8 µmol), and DIPEA (62.4
µL, 369 µmol) were added and the mixture was stirred for 16
h and then concentrated. The residue was purified on a LH20
column eluted with CH2Cl2-MeOH (1:1) to give 37 (34.7 mg,
58%), isolated as a white foam. MALDI-MS for C326H488N34O180
(M, 7763.48) M + Na found 7787.0, calcd 7786.5.
Octavalent Short Spacer PAMAM Generation 1 Gala-
bioside (38). To a solution of 37 (40.7 mg, 5.24 µmol) in MeOH
(10 mL) was added a 3% solution of NaOMe in MeOH (500
µL) and the mixture was stirred for 2 h, then neutralized with
Dowex 50X8 (H+), filtered, and concentrated. The residue was
freeze-dried to give 38 (25.2 mg, 89%), isolated as a white solid.
13C NMR (75.5 MHz, D2O) δ ) 29.2 (OCH2CH2CH2NH), 30.0
(COCH2CH2N), 36.7, 38.8, 39.3 (OCH2CH2CH2NH and NC-
H2CH2NHCO), 49.1, 50.3, and 52.7 [N(CH2)3], 60.7 and 61.1
(C-6 and C-6′), 68.2, 69.3, 69.6, 69.8, 71.4, 71.6, 72.9, 75.7, 77.7
(C-2, C-3, C-4, C-5, C-2′, C-3′, C-4′, C-5′, and OCH2CH2CH2-
NH), 70.5 (COCH2OCH2CO), 100.8 (C-1′), 103.5 (C-1), 172.1-
174.4 (CONH).
Acknowledgment. This study has been carried with
financial support from the Commission of the European
Communities, specific RTD program Quality of Life and
Management of Living Resources, QLK2-CT-2002-
01852, POLYCARB, and also with support from the
Academy of Finland. We thank Johan Kemmink (De-
partment of Medicinal Chemistry, Utrecht University)
for recording 500 MHz NMR spectra and Ronald D. van
Ooijen (Department of Biomolecular Mass Spectrom-
etry, Utrecht University) for recording high-resolution
mass spectra. The Royal Netherlands Academy of Arts
and Sciences (KNAW) is acknowledged for a fellowship
to R.J.P.
Supporting Information Available: NMR and mass
spectra of the prepared compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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6508 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
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