Multivalent ligands for the mannose-specific lectin on type 1 fimbriae of Escherichia coli: Syntheses and testing of trivalent α-D-mannoside clusters

Article abstract of DOI:10.1039/a801985a

The syntheses of the triantennary cluster α-D-mannosides 16, 19, 23 and 24 and their capacities to inhibit mannose-dependent binding of E. coli HB 101 (pPK14) are described. The cluster glycosides are formed by glycosylation of tris-(3-hydroxypropyl)nitro

Full text of DOI:10.1039/a801985a

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Multivalent ligands for the mannose-specific lectin on type 1 fimbriae
of Escherichia coli: syntheses and testing of trivalent á-D-mannoside
clusters
Sven Kötter,a Ulrike Krallmann-Wenzel,b Stefan Ehlersb and
Thisbe K. Lindhorsta,*
a Department of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6,
D-20146 Hamburg, Germany
b Division of Molecular Infection Biology, Research Center Borstel, Parkallee 22,
D-23845 Borstel, Germany
The syntheses of the triantennary cluster á-D-mannosides 16, 19, 23 and 24 and their capacities to inhibit
mannose-dependent binding of E. coli HB 101 (pPKl4) are described. The cluster glycosides are formed by
glycosylation of tris-(3-hydroxypropyl)nitromethane 13, and by linking of suitable mannoside derivatives
via amide and thiourea bonds to tris-(2-carboxyethyl)nitromethane 17 and tris-(2-aminoethyl)amine 20.
Functionalized mannosides are attached to the core molecules at the 6-position of the sugar ring to allow
variation of the introduced aglycone moieties in order to compare their effects on the inhibitory potencies
of the derived mannoside clusters. The 6-peptide-bridged cluster mannoside 19 displays the highest
binding potency towards the type 1 fimbrial lectin of E. coli as tested by inhibition agglutination tests
and ELISA.
ing pocket as a model for the type 1 fimbriae agglutinin of E.
coli. Capitalizing on these findings, we have concentrated our
Introduction
Carbohydrate protein interactions play an important role in cell
adhesion1,2 such as leukocyte trafficking3,4 or microbial adhes-
ion.5 Carbohydrate derivatives have been designed to interfere
in both processes, for example to control leukocyte recruitment
in case of chronic or acute inflammatory diseases6–8 or to
inhibit the attachment of pathogens to their host cells. As firm
adhesion of the microbe to the host cell surface often is a pre-
requisite for infection, a carbohydrate-based anti-adhesion
therapy is a promising approach to the prevention of microbial
infection.9 Development of bacterial resistance as in the case of
antibiotic treatment may be less likely to occur in the case of
such treatment.
Multivalent glycomimetics have been shown to be potent
inhibitors of carbohydrate–protein interactions in many differ-
ent adhesion systems in vitro, such as in the adhesion of influ-
enza virus10 or Streptococcus suis11 with IC50-values as low as
the n range.11 In our own studies we have used multivalent
mannose derivatives to inhibit adhesion of Escherichia coli HB
101 (pPKl4) to erythrocytes and yeast mannan.12,13 E. coli
are important pathogens in urogenital and gastrointestinal
infections. Like many other bacterial strains, they carry carbo-
hydrate-recognition domains on long proteinogenous filaments
on their surfaces, called fimbriae or pili. E. coli possesses
different fimbriae, classified according to their carbohydrate
specificity. The α-mannoside-specific type 1 fimbriae have
been shown to be one of the important factors for E. coli
virulence,14,15 enabling the bacteria to adhere to the cell surface
carbohydrates of their host cells.
efforts on the synthesis of trivalent cluster mannosides.
Results and discussion
To allow for a broad variation in the structures of the glyco-
clusters, including the spatial arrangement of the mannose
residues, we have considered different attachment sites at the
sugar ring, as well as different linkage types. Mannosylation of
a trivalent core was our first approach; however, to allow for
variation of the aglycone moiety of the target mannosides, clus-
tering via the 6-position of the α-mannoside ring was the basis
for the design of the other synthesized cluster mannosides.
Thus, the effect of different aglycone moieties on the resulting
binding capacities can be explored, while the overall structure
of the glycoclusters is maintained. This is especially attractive,
as p-nitrophenyl α--mannoside binds approx. 80-fold better to
the type 1 fimbrial lectin than does the analogous methyl
glycoside.16–20 Furthermore, a decrease in binding potency upon
modification of the 6-position of the carbohydrate ring is not to
be expected as it is known from various other lectins that the 2-,
3- and 4-hydroxy groups of the carbohydrate moiety are usually
essential for binding to the lectin, whereas the 6-OH group
often plays a less important role in the recognition process.21
Syntheses of precursor molecules for cluster glycoside synthesis
Trifunctional core molecules were chosen for the synthesis of
the target mannoside clusters, which were equipped with an
additional group, suitable to be turned into a tether later in the
synthesis, for both further enlargement of the glycoclusters and
attachment of biolabels such as biotin or fluorescent markers.
Tris-(hydroxymethyl)aminomethane (Tris) has been used as a
core molecule for glycocluster synthesis in glycosylation after
protection of the amino function.22–24 The triol tris-(3-hydroxy-
propyl)nitromethane 13 appeared to be advantagous compared
with Tris for its good solubility in organic solvents, greater steri-
cal availability of the hydroxy groups, and its nitro group, which
could serve as a masked amino function. Thus, compound 13
Our recent work on testing multivalent mannose derivatives
as inhibitors of haemagglutination with E. coli revealed tri-
valent mannosyl clusters as the best inhibitors among a number
of mannose clusters with different carbohydrate valencies.12,13
This was in keeping with earlier work, mainly from Ofek and
Sharon16–20 who have suggested a trisected carbohydrate bind-
* Tel: ϩ49 40 4123-2387; Fax: ϩ49 40 4123-4325; E-mail: tklind@
chemie.uni-hamburg.de
J. Chem. Soc., Perkin Trans. 1, 1998
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was selected as an acceptor molecule for the mannosylation. Its
synthesis started from nitromethane and acrylic acid tert-butyl
ester to give the triester in a Michael addition.25 The triester was
saponified to the corresponding triacid 17, which was a suitable
core molecule in peptide-coupling reactions. The triacid 17
could be chemoselectivity reduced to the nitro-trialcohol 13 by
using BH3ؒTHF as described.25 Thus, compound 13 was
obtained in 68% overall yield starting from nitromethane.
Furthermore, tris-(2-aminoethyl)amine 20 was also used as core
molecule.
p-nitrophenyl moiety, p-nitrophenyl mannoside 9 was select-
ively tosylated at the primary hydroxy group to form compound
10, which was converted into the 6-azido-6-deoxy mannoside 11
by nucleophilic displacement reaction (Scheme 2). Subsequent
OH
HO
OTs
HO
O
O
i
HO
HO
HO
HO
OpNP
OpNP
The benzyl-protected mannosyl trichloroacetimidate 1426
was used as glycosyl donor for the complete glycosylation
of the triol 13 in order to control orthoester formation,
which regularly predominated when the analogous acetylated
mannosyl donor was used.
9
10
ii
N3
HO
NCS
HO
To gain 6-functionalized mannosides for the synthesis of
6-linked glycoclusters, the primary hydroxy groups of methyl
α--mannopyranoside and p-nitrophenyl α--mannopyrano-
side 9 were modified. Starting from the known benzoylated
6-azido-6-deoxy mannoside 1,27 the corresponding 6-amine
2 could be obtained by catalytic hydrogenation without
migration of a benzoyl group occurring (Scheme 1). The
iii
O
O
HO
HO
HO
HO
OpNP
OpNP
12
11
pNP = p-O2NC6H4
Scheme 2 Reagents, conditions and yields: i, TsCl, pyridine, 0 ЊC to rt,
57%; ii, NaN3, DMF, 70 ЊC, 8 h, 91%; iii, CS2, (EtO)3P, DME, 80 ЊC,
6 h, 76%
N3
BzO
NCS
BzO
NH2
BzO
O
O
O
i
ii
BzO
BzO
BzO
BzO
BzO
BzO
reaction of the unprotected triol 11 with triethyl phosphite and
CS2 afforded compound 12 in high yield.
Compounds 14, 2, 3, 5, 8 and 12 served as building blocks in
the subsequent synthesis of mannoside clusters.
OMe
OMe
OMe
3
1
2
iii
Syntheses of mannoside clusters
Glycosylation of the trialcohol 13 was carried out according to
the method established by Schmidt et al.31 using 3.9 mol equiv.
of the mannosyl trichloroacetimidate 14 and TMSOTf (tri-
methylsilyl trifluoromethanesulfonate) as Lewis acid catalyst.
The right choice of solvent and reaction conditions was essen-
tial for the success of this reaction. Freshly distilled, dry THF
was used and the amount of added promotor TMSOTf was
carefully controlled. Glycosylation yields were strongly depend-
ent on the reaction temperature. Performing the reaction at
Ϫ30 ЊC gave 25% product, reaction at Ϫ45 ЊC yielded 39%,
glycosylation at Ϫ50 ЊC led to 73%, and reaction at Ϫ65 ЊC
finally afforded the benzylated trivalent cluster mannoside 15
in 74% yield. Using such optimized conditions, the cluster
mannoside 15 could also be obtained on a multigram scale.
Complete debenzylation of compound 15 was achieved by
hydrogenation at 60 ЊC and 60 bar† H2 with Pd on charcoal as
catalyst and led to the unprotected triantennary O-glycosidic
α--mannoside cluster 16 in 77% yield (Scheme 3). The struc-
tures of triantennary products 15 and 16 were easily confirmed
by NMR spectroscopy, which revealed the correct integration
ratios of the core methylene protons and the multiplets for the
sugar rings in the 1H NMR spectrum.
Furthermore, the 6-modified benzoylated methyl mannoside
2 was coupled to the nitroacid 17 under standard peptide-
coupling conditions32,33 to give the 6-linked cluster 18 in good
yield (Scheme 4). This was debenzoylated by the Zemplén
method to yield the unprotected 6-peptide-bridged trimanno-
side 19 in a quantitative reaction.
The 6-deoxy-6-isothiocyanato mannosides 3, 5, 8 and 12 were
treated with tris-(2-aminoethyl)amine 20 to form thiourea-
bridged trivalent mannoside clusters, based on the thiourea-
bridging strategy which has recently been introduced (Scheme
5).34 A slight excess of the benzolyated isothiocyanate 3 and
tris-(2-aminoethyl)amine 20 led to compound 21 in over 90%
yield. However, removal of the benzoyl protecting groups was
difficult and the unprotected cluster 23 was obtained in yields
NCS
AcO
N3
AcO
O
O
iv
AcO
AcO
AcO
AcO
OMe
OMe
5
4
N3
NH2
HO
NCS
HO
HO
O
O
O
v
vi
HO
HO
HO
HO
HO
HO
OMe
OMe
OMe
6
8
7
Scheme 1 Reagents, conditions and yields: i, H2, Pd/C (10%), 1:1
EtOAc–EtOH, rt, 89%; ii, CS2, (EtO)3P, toluene, 80 ЊC, 4 h, 92%; iii (a)
NaOMe, MeOH; (b) Ac2O, pyridine, rt, 90%; iv, CS2, (EtO)3P, toluene,
80 ЊC, 6 h, 87%; v, H2, Pd/C (10%), 1:1 EtOAc–EtOH, 84%; vi, Cl2C(S),
4:1 EtOH–water, diisopropylethylamine, rt, 96%
amino-functionalized mannoside 2 later served as ‘mannoside
donor’ in the peptide-coupling reaction with the triacid 17. The
6-azido group in compound 1 was also efficiently converted into
an isothiocyanato function by the reaction with CS2 and triethyl
phosphite28 to form mannoside 3. Furthermore, mannoside
1 was converted into triacetate 4 following a deprotection–
acetylation sequence. The acetylated azide 4 was then elabor-
ated to give the 6-deoxy-6-isothiocyanato mannoside 5 by use
of CS2 and triethyl phosphite in excellent yield. Its unprotected
analogue 829 was obtained by hydrogenation of the unprotected
azide 6 to form the amine 7,30 followed by treatment of amine 7
with thiophosgene.29
The formation of the isothiocyanato function could easily be
monitored by IR [ν(N3) at 2104 cmϪ1, ν(NCS) at 2114 cmϪ1] and
by 13C NMR spectroscopy which displayed the NCS carbon at
δC 134.4. The 6-deoxy-6-isothiocyanato glycosides 3, 5 and 8
were used for the synthesis of thiourea-bridged glycoclusters.
In order to change the methyl aglycone of mannoside 8 into a
† 1 bar = 105 Pa.
2194
J. Chem. Soc., Perkin Trans. 1, 1998

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OH
NH2
NCS
R2O
OBn
BnO
O
OH
O
R2O
BnO
BnO
R2O
N
+
3
O2N
+
3
H2N
OR1
O
CCl3
NH2
OH
NH
20
3
5
8
R
1 = CH3, R2 = Bz
13
14
R1 = CH3, R2 = Ac
R1 = CH3, R2 = H
i
OR
OR
12 R1 = pNP, R2 = H
OR
O
i
OR
OR
OR
O
OR2
O
OR
OR
OR2
R2O
O
OR2
NH
OR
O2N
O
R2O
R2O
O
OR
OR1
O
R1O
NH
HN
O
S
S
OR
OR
NH
N
H
N
S
15 R = Bn
16 R = H
ii
OR2
OR2
Scheme 3 Reagents, conditions and yields: i, 0.02  TMSOTf in
CH2Cl2, THF, 4 Å, N2, Ϫ65 ЊC to rt, 74%; ii, H2, Pd/C, 3:1 EtOH–
THF, 77%
OR2
NH
O
R1O
O
OH
21 R1 = CH3, R2 = Bz
22 R1 = CH3, R2 = Ac
23 R1 = CH3, R2 = H
24 R1 = pNP, R2 = H
NH2
BzO
OH
O
O
ii
BzO
BzO
O2N
3
+
iii
OMe
OH
O
Scheme 5 Reagents, conditions and yields: i, CH2Cl2 50 ЊC, 30 min (for
3, 5 or 12), 92% (R1 = CH3, R2 = Bz), 91% (R1 = CH3, R2 = Ac), 89%
(R1 = pNP, R2 = H); 4:1 EtOH–water, 50 ЊC, 12 h, (for 8), 23%; ii, 1 
NaOMe, 5:1 MeOH–DMF, 50 ЊC, 3 days, 27%; iii, 1  NaOMe,
MeOH, rt, 82%
2
17
i
OR
was readily deprotected under Zemplén conditions to form the
thiourea-bridged target cluster 23, which was easily purified by
size-exclusion chromatography.
In the case of the OH-unprotected 6-NCS-modified pNP
mannoside 12, the thiourea-bridging reaction with tris-
(2-aminoethyl)amine leading to product 24 (Scheme 5) was
successfully performed using aprotic solvents such as CH2Cl2
(89%) or DME (86%).
RO
O
OMe
RO
RO
RO
N
O
RO
HN
H
O
OMe
OR
O2N
O
RO
RO
Testing of the inhibition properties of the mannoside clusters 16,
19, 23 and 24
N
H
O
O
Initially, the cluster mannosides were tested as inhibitors of the
haemagglutination of guinea pig erythrocytes by E. coli HB 101
(pPKl4) in classical inhibition haemagglutination tests. In these
assays, the inhibition titre (IT) is the lowest concentration of
the inhibitor at which no agglutination occurs by macroscopic
inspection. These results are highly reproducible but, however,
have to be considered semi-quantitative due to the experimental
set-up. A newly developed enzyme-linked immunosorbent assay
(ELISA) has allowed us to measure IC50-values for the inhib-
ition of E. coli adhesion to yeast mannan. IC50-Values reflect
the inhibitor concentration which causes 50% inhibition of bac-
terial binding to yeast mannan. Duplicate results were used for
the construction of the inhibition curves for each individual
ELISA experiment. Typically, the intra-assay variation of an
OMe
18 R = Bz
19 R = H
ii
Scheme 4 Reagents, conditions and yields: i, DCC, HOBT, DMF, 0 ЊC
to rt, 73%; ii, NaOMe, MeOH, 98%
not higher than 27%. Also, the analogous coupling reaction
using the OH-unprotected isothiocyanate 8 gave the glyco-
cluster 23 in only poor yields of approximately 20%, due to
solubility problems. However, when the acetylated 6-iso-
thiocyanate 5 was treated with tris-(2-aminoethyl)amine, the
trivalent cluster 22 was formed in excellent yields and, also,
J. Chem. Soc., Perkin Trans. 1, 1998
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Table 1 Inhibition titres (IT) obtained from inhibition haemagglutination tests and IC50-values measured by ELISA are listed together with the
᎐
relative values based on methyl α--mannopyranoside (RIT and RIC50 ᎐᎐ 1) as standard. IC50-Values are average values from at least three
independent ELISA experiments and the resulting standard deviations are indicated. The relative inhibition potencies based on moles of mannose
moieties present in the tested compounds (‘valency-corrected’ values) are also listed for both tests.
Methyl α--mannopyranoside
9
16
18
217
72
19
23
24
IT [µM]a
RIT
RIT based on moles of mannose
3900
42
93
93
1.1
6900
1
1
3545
1182
0.57
0.19
IC50 [µM]b
3500
800
1
49
17
71
71
39
5
90
30
11
5
318
106
5333
1892
0.66
0.22
429
140
8.2
2.7
Sc
RIC50
RIC50 based on moles of mannose
1
a Obtained from inhibition haemagglutination tests. b Obtained by ELISA. c Standard deviation.
individual ELISA is very small, whereas the IC50-values
obtained from several independently performed ELISAs differ
significantly. However, when relative inhibition potencies
(RIPs) are calculated from independently obtained data, the
results are highly reproducible. The results obtained from both
tests are listed in Table 1. IC50-Values are average values from at
least three independent ELISA experiments and are depicted
together with the resulting standard deviations.
of that shown by compound 9, the valency-corrected RIT of
the peptide-linked mannoside cluster 19 remains approximately
10-times higher than that of compound 9. This effect was not
observed in the ELISA.
As the carbohydrate-recognition domains (CRDs) are placed
at the tips and along the shafts of the fimbriae35 multivalent
carbohydrate derivatives may display enhanced inhibitory
capacities in the assays used due to simultaneous binding to
multiple CRDs. This type of enhancement effect evident with
clustering of carbohydrate substructures has been termed the
‘glycoside clustering effect’.36 Owing to the size of the tested
cluster mannosides 16, 19, 23 and 24 we can exclude the possi-
bility that their mannose moieties are simultaneously recog-
nized by more than one CRD per pilus. It is, however, possible
that they bind to adjacent CRDs on adjacent fimbriae at the
same time. On the other hand, binding could also occur to a
single CRD only, and thus different inhibitory potencies would
result from the different fit of the glycoclusters into the CRD.
This model is supported by the findings of Ofek and Sharon
who described trisaccharides as particularly suitable ligands for
the type 1 fimbrial lectin.16,20 We are currently addressing these
questions in further studies. For the design of even more potent
inhibitors of the binding of type 1 fimbriated E. coli, the
peptide-linked mannoside cluster 19 should serve as an excel-
lent prototype structure and we expect the introduction of more
hydrophobic aglycone moieties to further improve its inhibitory
capacity.
The mannoside clusters 16, 19, 23 and 24 were compared
with the known low-affinity inhibitor methyl α--manno-
pyranoside and the potent aromatic inhibitor 9. Interestingly,
the mannoside clusters greatly differed in their inhibitory
potencies. The O-glycosidic cluster 16 and the peptide-linked
cluster 19 were the best inhibitors of E. coli adhesion, clearly
surpassing the inhibitory capacity of compound 9. On the other
hand, thiourea-linked cluster 23 had a very high IT and IC50,
and therefore was a worse inhibitor than methyl α--manno-
pyranoside. The differences in the inhibition potencies of com-
pound 23 on the one hand and compounds 16 and 19 on the
other may be due to conformational differences or may be
related to the nature of the linkage. Introduction of hydro-
phobic phenyl moieties in the case of cluster 24, instead of
the methyl aglycones in cluster 23, decreased IC50-values over
100-fold, as was expected; however, the inhibitory potency of
the aromatic mannoside 9 was not reached. This result was
obtained by ELISA only, as limited water solubility of cluster
24 did not allow an inhibition haemagglutination test to be
carried out in this case.
Depending on the assay system used, the tested compounds
differed in terms of the absolute concentrations at which inhib-
ition of adhesion occurred. In the ELISA, IC50-values were
higher than titres obtained in the inhibition haemagglutination
test in the case of glycoclusters 16 and 19. This effect was
extreme for the best glycocluster 19, which showed a 10-fold
higher inhibitory concentration when determined by ELISA.
However, when relative inhibitory potencies (RIT and RIC50)
were calculated to compare different compounds, the values are
similar regardless of the assay system used. The discrepances
obtained by both methods are not surprising, as the tests reflect
very different aspects of E. coli adhesion. While in the ELISA
E. coli adhesion to yeast mannan is inhibited, in the case of
the inhibition agglutination test the bacteria bind to erythro-
cytes. Erythrocytes are likely to possess a greater variety of
oligosaccharide structures on their surface than is reflected by
yeast mannan. Furthermore, the sandwich ELISA rather has
the characteristics of a two-dimensional system in contrast to
the more complex haemagglutination assay. It is important to
keep such differences in mind when in vitro results form the
basis for predicting the usefulness of synthetic compounds in
in vivo situations.
Conclusions
A set of trivalent α--mannoside clusters was synthesized
which displayed widely different binding potencies towards the
type 1 fimbrial lectin of E. coli HB 101 (pPKL4). Our studies
using variable cluster mannoside structures provide further
insight into the putative three-dimensional structure of the
carbohydrate recognition domain. In addition, the potent
cluster 19 will be extremely useful as a lead structure for the
synthesis of even more potent inhibitors of E. coli binding with
inhibitory potencies expected in the nanomolar range.
Experimental
Materials
Methyl α--mannoside was purchased from Merck, p-nitro-
phenyl α--mannoside was from SENN Chemicals. TMSOTf
(Fluka) and tris-(2-aminoethyl)amine (Aldrich) were used
without further purification. F-shaped 96-well microtiter plates
from Sarstedt were used for ELISA and V-microtiter plates
from Nunc for inhibition haemagglutination tests. Mannan
from Saccharomyces cerevisiae was purchased from Sigma and
was used in 50 m aq. Na2CO3 (1 mg mlϪ1; pH 9.6). The poly-
clonal anti-fimA antibody was a kind gift from Prof. Dr J.
Hacker (Würzburg) and peroxidase-conjugated goat anti-
rabbit antibody (IgG, HϩL) was purchased from Dianova.
Skimmed milk was from Glücksklee, Tween 20 from Roth,
In order to estimate the net effect of clustering, the inhibitory
potencies of the glycoclusters 16 and 19 were compared based
on the number of mannose moieties they contained (RIT and
RIC50 in Table 1). Whereas the RIT of cluster 16 is in the range
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ABTS [2,2Ј-azidobis-(3-ethylbenzothiazoline-6-sulfonic acid)]
from Sigma and thimerosal {2-[(ethylmercurio)thio]benzoic
acid, sodium salt} was from Merck. A recombinant type 1 fim-
briated E. coli strain, E. coli HB101 (pPKl4),37 was used and
cultured as described.12 Light petroleum refers to the fraction
with distillation range 50–70 ЊC.
Buffers. PBS (phosphate-bufferred saline) was prepared by
dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4ؒ2H2O and 0.2 g
KH2PO4 in 1000 ml of distilled, deionized water (pH 7.2).
PBSE (PBS buffer used for ELISA) was used as 150 m solu-
tion [pH 7.2; NaCl (136.90 g lϪ1), KCl (2.68 g lϪ1), Na2HPO4
(8.0 g lϪ1), KH2PO4 (2.4 g lϪ1), 0.01% thimerosal]. PBSET was
PBSE buffer ϩ 0.05% Tween 20. Substrate buffer was 0.1 
sodium citrate dihydrate, adjusted to pH 4.5 with citric acid.
For preparation of the ABTS solution, ABTS (1 mg) was dis-
solved in substrate buffer (1 ml) by sonification and 0.1% H2O2
(25 µl per ml) was added.
µl) and inhibitor solution (50 µl) were added. Inhibitor solu-
tions were diluted serially two-fold in PBSE. Bacterial suspen-
sion (50 µl per well) was added and the plate was left at 37 ЊC
for 1 h to allow sedimentation of the bacteria. Then each well
was washed four times with PBSE (150 µl) and 50 µl of the first
antibody (anti-fimA antibody, solution as optimized prior to
the experiments) in 2% skimmed milk was added. The plates
were incubated for 30 min and then washed twice with PBSET
and the second antibody was added (50 µl). The plates were
incubated for 30 min and then subsequently washed three times
with PBSET, PBSE and substrate buffer. ABTS solution (50 µl)
was added, incubated for 30 min at 37 ЊC, and the reaction was
stopped by adding of 2% oxalic acid (50 µl).
For ELISA controls, bacterial adhesion to blocked, uncoated
microtiter plates was checked, and the reaction of antibodies
with carbohydrate derivatives as well as the reaction of the
employed antibodies with yeast mannan was tested and found
to be negligible. The low background obtained from these con-
trol experiments was subtracted when calculating the IC50
values. The percentage inhibition was calculated as [OD(nl) Ϫ
OD(l)] × 100 × [OD(nl)]Ϫ1 (nl: no inhibitor, I: with inhibitor).
General methods
Optical densities (ODs) were measured on a Dynatech 5000
ELISA reader at 405 nm with the reference read to 490 nm.
ELISA plates were incubated at 37 ЊC. For flash chroma-
tography Merck silica gel 60 (0.040–0.063 mm, 230–400 mesh)
was used. TLC was performed on Alugram Sil G/UV254
plates, purchased from Machery&Nagel, or on Kieselgel 60
F254 plates from Merck. Detection was carried out under
UV light or by spraying with 20% ethanolic sulfuric acid or
ninhydrin solution (2% in water–propan-1-ol, 1:1) with sub-
sequent heating. Size-exclusion chromatography was performed
on Sephadex G-15 (60–181 µm) and 15 m aq. NH4HCO3 as
the eluent. NMR spectra were measured on a Bruker AMX 400
(400 MHz for 1H and 100.67 MHz for 13C NMR) or a DRX 500
(500 MHz for 1H and 125.84 MHz for 13C NMR) spectrometer.
Chemical shifts are in ppm, relative to internal SiMe4 (0.00 ppm
for 1H and 13C NMR) or solvent peaks which were calibrated as
follows: CDCl3 (δ 7.26 for 1H and δC 77.00 for 13C NMR), D2O
Methyl 6-amino-2,3,4-tri-O-benzoyl-6-deoxy-á-D-manno-
pyranoside 2
Methyl
6-azido-2,3,4-tri-O-benzoyl-6-deoxy-α--manno-
pyranoside 127 (1.40 g, 2.64 mmol) was dissolved in ethyl
acetate–EtOH (1:1; 20 ml) and hydrogenated after the addition
of Pd catalyst (10% on charcoal; 200 mg) at atmospheric pres-
sure. After the reaction was complete (ca. 3.5 h), the mixture
was filtered over Celite and the filtrate was concentrated in
vacuo and purified by flash chromatography (toluene–acetone,
5:1) to yield the title compound 2 (1.19 g, 89%) as a slightly
yellow syrup; [α]D20 Ϫ51.7 (c 0.89, CHCl3); Rf (toluene–acetone,
5:1) 0.12 (UV; H2SO4); δH(400 MHz; CDCl3; Me4Si) 8.13–7.23
(15 H, m, ArH), 5.92 (1 H, dd, J3,4 10.2, 3-H), 5.83 (1 H, dd ≈ t,
J4,5 10.2, 4-H), 5.67 (1 H, dd, J2,3 3.6, 2-H), 4.97 (1 H, d, J1,2 1.5,
1-H), 4.02 (1 H, ddd, J5,6 3.6, 5-H), 3.52 (3 H, s, OCH3), 3.00–
2.90 (2 H, m, J5,6Ј 5.6, 6-H2) and 1.54 (1 H, br s, NH); δC(100.67
1
[δ 4.65 for H; for 13C NMR CD3CN (δC 1.30) or [2H6]acetone
(δC 30.60) was added], [2H4]methanol (δ 3.35 for 1H and δC 49.30
for 13C NMR), [2H6]DMSO (δ 2.49 for 1H and δC 39.70 for 13
C
MHz; CDCl ; Me Si) 165.96, 165.48, 165.42 (C᎐O), 133.48,
᎐
3
4
NMR). J Values are in Hz. Where necessary, two-dimensional
1H–1H or 1H–13C chemical-shift-correlation spectroscopy
(COSY) experiments were performed for complete signal
assignments. Mps were determined on a Leitz heating table
microscope and are uncorrected. Optical rotation values were
obtained using a Perkin-Elmer polarimeter 341 or 243 (Na-
line, 589 nm; 20 ЊC; cell length 10 cm), and are given in units of
10Ϫ1 deg cm2 gϪ1. IR spectra were recorded on an FT-IR ATI
Mattson Instruments (USA) spectrometer. Mass spectra were
measured on a VG Analytical 70-250S (FAB-MS) or a Finnigan
MAT 95 (MALDI-TOF MS) instrument.
133.43, 133.07, (aryl-CH), 129.93–128.19 (m, aryl-C), 98.58
(C-1), 71.36, 70.64, 69.84, 67.63 (C-2, -3, -4, -5), 55.35 (OCH3)
and 42.54 (C-6); m/z (FAB MS) 506.6 (Mϩ ϩ H. C28H28NO8
requires m/z, 506.18).
Methyl 2,3,4-tri-O-benzoyl-6-deoxy-6-isothiocyanato-á-D-
mannopyranoside 3
A mixture of methyl 6-azido-2,3,4-tri-O-benzoyl-6-deoxy-α--
mannopyranoside 127 (0.50 g, 0.94 mmol), CS2 (3.3 ml, 4.6
mmol) and triethyl phosphite (0.8 ml, 0.055 mmol) in dry tolu-
ene (10 ml) was stirred at 80 ЊC for 10 h. Water (10 ml) was
added and the mixture was left at room temperature (rt) over-
night. Then the aq. phase was extracted three times with
CH2Cl2 (50, 50 and 30 ml) and the combined organic phases
were subsequently washed successively with 1  H2SO4 and sat-
urated aq. NaCl, dried over MgSO4 and concentrated in vacuo.
Purification by flash chromatography (light petroleum–ethyl
acetate, 4:1) yielded the title compound 3 (474 mg, 92%) as a
solid, mp 227 ЊC (Found: C, 63.6; H, 4.6; N, 2.55; S, 5.8.
C29H25NO8S requires C, 63.61; H, 4.60; N, 2.56; S, 5.85%); [α]D20
Ϫ29.6 (c 1.0, CHCl3); νNCS 2114 cmϪ1; Rf (light petroleum–ethyl
acetate, 4:1) 0.17 (UV; H2SO4); δH(400 MHz; CDCl3; Me4Si)
8.15–7.24 (15 H, m, ArH), 5.90 (1 H, dd, J3,4 10.2, 3-H), 5.82
(1 H, dd ≈ t, J4,5 10.2, 4-H), 5.70 (1 H, dd, J2,3 3.1, 2-H), 5.02
(1 H, d, J1,2 1.5, 1-H), 4.25 (1 H, ddd, J5,6 5.6, 5-H), 3.81 (1 H,
dd, J6,6Ј 14.8, 6-H), 3.76 (1 H, dd, J5,6Ј 3.6, 6-HЈ) and 3.58 (3 H, s,
OCH3); δC(100.67 MHz; CDCl3; Me4Si) 165.55, 165.45, 165.36
Inhibition haemagglutination tests
For inhibition of haemagglutination (= erythrocyte agglutin-
ation) of guinea pig erythrocytes on microtiter plates serially
two-fold diluted carbohydrate solutions were used as
described.12 The lowest concentration that inhibited haemag-
glutination was determined visually as haemagglutination
inhibition titre (IT).
ELISA
The ELISA protocol used to determine the inhibitory potencies
of the cluster mannosides towards type 1 fimbriated E. coli was
established on the basis of published procedures.38–40 In pre-
liminary experiments, the optimal concentrations for mannan,
bacteria and antibody solutions were determined (data not
shown). Polystyrene microtiter plates were coated with mannan
solution (100 µl per well) and dried overnight at 37 ЊC. The
plates were washed once with PBS (150 µl per well) and then
blocked with 5% skimmed milk in PBSE for 30 min at 37 ЊC.
The wells were washed with PBSE (150 µl) and then PBSE (50
(C᎐O), 133.71–128.28 (m, aryl-C), 98.85 (C-1), 70.10 (C-2),
᎐
69.45 (C-3), 69.29 (C-5), 67.73 (C-4), 56.00 (OCH3) and 46.18
(C-6); m/z (EIMS) 548 (Mϩ ϩ H. C29H26NO8S requires m/z,
548.14).
J. Chem. Soc., Perkin Trans. 1, 1998
2197

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Methyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy-á-D-mannopyrano-
side 4
(aryl-Cq), 131.22, 118.06 (aryl-CH), 129.30, 126.90 (tosyl-CH),
100.22 (C-1), 73.64, 72.43, 71.64, 68.09 (C-2, -3, -4, -5), 70.65
(C-6) and 21.87 (ArCH3).
To a solution of methyl 6-azido-2,3,4-tri-O-benzoyl-6-deoxy-α-
-mannopyranoside 127 (1.591 g, 2.99 mmol) in dry MeOH (45
ml) was added a catalytic amount of NaOMe solution (1 in
MeOH; 1.5 ml) and the mixture was stirred at rt. After the
reaction was complete (TLC toluene–ethyl acetate, 25:1), the
solution was neutralized with ion-exchange resin (Levatit SP
1080 Hϩ), then filtered, and the filtrate was concentrated in
vacuo. The residual unprotected mannoside 6 was dissolved in
dry pyridine (40 ml), acetic anhydride (4.3 ml, 45 mmol) was
added, and the reaction mixture was stirred at rt until the reac-
tion was complete (TLC toluene–ethyl acetate, 25:1). Pyridine
was removed in vacuo and the residue was purified by flash
chromatography (toluene–ethyl acetate, 25:1) to give the title
compound 4 (930 mg, 90%) as a syrup; [α]D20 ϩ80.8 (c 1.50,
CHCl3) {lit.,41 [α]D ϩ63Њ (c 1, CHCl3)}; Rf (toluene–ethyl acet-
ate, 5:1) 0.30 (UV; H2SO4); δH(400 MHz; CDCl3; Me4Si) 5.33
(1 H, dd, J3,4 9.7, 3-H), 5.31 (1 H, dd ≈ t, J4,5 10.2, 4-H), 5.24
(1 H, dd, J2,3 3.6, 2-H), 4.73 (1 H, d, J1,2 1.5, 1-H), 3.94 (1 H,
ddd, J5,6 7.1, 5-H), 3.45 (3 H, s, OCH3), 3.38 (1 H, dd, J6,6Ј 13.2,
6-H), 3.29 (1 H, dd, J5,6Ј 2.6, 6-HЈ) and 2.16, 2.05 and 2.00 (each
3 H, each s, 3 × Ac); δC(100.67 MHz; CDCl3; Me4Si) 169.97,
p-Nitrophenyl 6-azido-6-deoxy-á-D-mannopyranoside 11
A mixture of the mannoside 10 (1.67 g, 3.67 mmol) and sodium
azide (1.20 g, 18.3 mmol) in dry DMF (40 ml) was stirred at
70 ЊC for 8 h. After the reaction was complete (TLC ethyl
acetate–toluene, 19:1) the solvent was removed in vacuo and the
residue was dissolved in ethyl acetate. The excess of sodium
azide and formed sodium tosylate were removed by filtration
and the filtrate was evaporated and purified by flash chrom-
atography (ethyl acetate–toluene, 19:1) to afford the title
mannoside 11 (1.09 g, 91%) as an amorphous solid (Found: C,
44.3; H, 4.3; N, 17.1. C12H14N4O7 requires C, 44.18; H, 4.33; N,
17.17%); [α]D20 ϩ63.7 (c 0.87, MeOH); Rf (ethyl acetate–toluene,
19:1) 0.21 (UV; H2SO4); δH(400 MHz; D3COD) 8.26, 7.32 (each
2 H, each d, 3J 9.15, ArH), 5.73 (1 H, d, J1,2 1.5, 1-H), 4.11 (1 H,
dd, J2,3 3.6, 2-H), 3.92 (1 H, dd, J3,4 9.2, 3-H), 3.75 (1 H, dd ≈ t,
J4,5 9.2, 4-H), 3.67 (1 H, ddd, J5,6 5.1, 5-H) and 3.47–3.46 (2 H,
m, J5,6Ј 4.1, J6,6Ј 13.7, 6-H2); δC(100.67 MHz; D3COD) 162.74
(i-aryl-Cq), 144.24 (p-aryl-Cq), 126.97, 117.93 (aryl-CH), 100.06
(C-1), 75.49 (C-5), 72.34 (C-3), 71.72 (C-2), 69.33 (C-4) and
53.02 (C-6); m/z (FAB MS) 327 (Mϩ ϩ H. C12H15N4O7 requires
327.09).
169.80, 169.77 (C᎐O), 98.36 (C-1), 69.87, 69.40, 68.75, 67.12
᎐
(C-2, -3, -4, -5), 55.37 (OCH3), 51.02 (C-6), 20.81, 20.62 and
20.59 (3 × COCH3).
p-Nitrophenyl 6-deoxy-6-isothiocyanato-á-D-mannopyranoside
12
Methyl 2,3,4-tri-O-acetyl-6-deoxy-6-isothiocyanato-á-D-manno-
pyranoside 5
A mixture of the mannoside 11 (800 mg, 2.45 mmol), CS2 (7.1
ml, 0.12 mol) and triethyl phosphite (1.65 ml, 8.51 mmol) in dry
DME (25 ml) was stirred at 80 ЊC for 5 h. Then water (100 ml)
was added and the mixture was kept at rt for 12 h. Then the aq.
phase was extracted with CH2Cl2 (100, 50 and 50 ml) and the
combined organic phases were subsequently washed with 1 
H2SO4 and saturated aq. NaCl, dried over MgSO4 and concen-
trated. Purification by flash chromatography (toluene–ethyl
acetate, 1:1) afforded the title compound 12 (395 mg, 47%, 76%
based on converted starting material) as an amorphous solid
and starting material 21 (230 mg, 0.705 mmol) was recovered
(Found: C, 45.7; H, 4.1; N, 8.2; S, 9.35. C13H14N2O7S requires
C, 45.61; H, 4.12; N, 8.18; S, 9.37%); [α]D20 ϩ85.2 (c 0.86, MeOH);
Rf (ethyl acetate–toluene, 2:1) 0.74 (UV; H2SO4); δH(400 MHz;
CDCl3; Me4Si) 8.24 (2 H, d, 3J 9.16, ArH), 7.20 (2 H, d, ArH),
5.74 (1 H, d, J1,2 1.0, 1-H), 4.56 (1 H, dd, J3,4 9.7, 3-H), 4.24
(1 H, dd, J2,3 3.1, 2-H), 4.21 (1 H, ddd, J5,6 7.1, 5-H), 4.13
(2 H, dd, J5,6Ј 2.5, J6,6Ј 10.2, 6-H2) and 3.85 (1 H, dd ≈ t, J4,5 9.7,
4-H); δC(100.67 MHz; CDCl3; Me4Si) 160.32 (i-aryl-Cq), 143.05
(p-aryl-Cq), 134.09 (Cq, NCS), 126.11 (aryl-CH), 116.36 (aryl-
CH), 97.08 (C-1), 75.16 (C-3), 70.12 (C-2), 69.85 (C-4), 65.89
(C-5) and 65.75 (C-6); m/z (FAB MS) 365 (Mϩ ϩ Na. C13H14-
SO7N2Na requires m/z, 365.04).
A mixture of compound 4 (898 mg, 2.60 mmol), CS2 (7.6 g, 6
ml, 0.10 mol) and triethyl phosphite (1.3 g, 1.4 ml, 8.06 mmol)
in dry toluene (14 ml) was heated at 80 ЊC for 6 h under nitro-
gen. Then water (50 ml) was added and the aq. phase was
extracted with CH2Cl2 three times. The combined organic
phases were subsequently washed successively with 1  H2SO4
and saturated aq. NaCl, dried over MgSO4 and concentrated.
Purification by flash chromatography (toluene–ethyl acetate,
5:1) afforded the title compound 5 (820 mg, 87%) as a solid; mp
120–121.5 ЊC (lit.,29 114–115 ЊC); [α]D20 ϩ70.1 (c 0.95, CHCl3)
[lit.,29 [α]D ϩ62 (c 1.1, CHCl3)]; Rf (toluene–ethyl acetate, 2:1)
0.47 (UV; H2SO4); δH(400 MHz; CDCl3; Me4Si) 5.35 (1 H, dd,
J3,4 10.2, 3-H), 5.23 (1 H, dd, J2,3 3.6, 2-H), 5.21, dd ≈ t, J4,5 9.7,
4-H), 4.76 (1 H, d, J1,2 1.5, 1-H), 3.95 (1 H, ddd, J4,5 9.7, 5-H),
3.62 (2 H, m, 6-H2), 3.46 (3 H, s, OCH3) and 2.17, 2.07 and 2.00
(each 3 H, each s, 3 × Ac); δC(100.67 MHz; CDCl3; Me4Si)
169.97, 169.79, 169.74 (C᎐O), 134.36 (NCS), 98.51 (C-1), 69.28
᎐
(C-2), 69.01 (C-5), 68.57 (C-3), 67.21 (C-4), 55.72 (OCH3),
46.12 (C-6) and 20.82, 20.60 and 20.58 (3 × COCH3).
p-Nitrophenyl 6-O-tosyl-á-D-mannopyranoside 10
To a solution of p-nitrophenyl α--mannopyranoside 9 (3.64 g,
12.08 mmol) in dry pyridine (60 ml) was added dropwise a solu-
tion of tosyl chloride (2.76 g, 14.5 mmol) in dry pyridine (60 ml)
within 45 min at 0 ЊC. The reaction mixture was stirred at 0 ЊC
for 1 h and then 1 h at rt. After completion of the reaction
(TLC ethyl acetate–toluene, 9:1), saturated aq. NaCl (150 ml)
was added and the aq. phase was extracted three times with
ethyl acetate (70 ml each). The combined organic phases were
dried over MgSO4, filtered and concentrated. Flash chrom-
atography (ethyl acetate–toluene, 9:1) afforded the title glyco-
side 10 (3.16 g, 57%) as an amorphous solid (Found: C, 50.2; H,
4.7; N, 3.1. C19H21NO10S requires C, 50.11; H, 4.65; N, 3.08%);
[α]D20 ϩ102.7 (c 1.30, MeOH); Rf (ethyl acetate–toluene, 9:1) 0.33
(UV; H2SO4); δH(400 MHz; D3COD) 8.19 (2 H, d, 3J 9.16,
ArH), 7.73 (2 H, d, 3J 8.65, tosyl H), 7.41 (2 H, d, tosyl H), 7.19
(2 H, d, ArH), 5.58 (1 H, d, J1,2 1.15, 1-H), 4.36 (1 H, dd, J5,6Ј
1.5, 6Ј-H), 4.24 (1 H, dd, J6,6Ј 11.2, 6-H), 4.05 (1 H, dd, J2,3 3.6,
2-H), 3.86 (1 H, dd, J3,4 8.7, 3-H), 3.69 (1 H, dd ≈ t, J4,5 10.2,
4-H), 3.64 (1 H, ddd ≈ m, J5,6 6.6, 5-H) and 2.48 (3 H, s, tosyl
CH3); δC(100.67 MHz; D3COD) 162.73, 146.78, 144.22, 134.72
Nitrotris-[3-(2,3,4,6-tetra-O-benzyl-á-D-mannopyranosyloxy)-
propyl]methane 15
The mannosyl donor 1426 (7.48 g, 10.92 mmol) and the triol 1325
(660 mg, 2.81 mmol) were dissolved in dry THF (100 ml) under
nitrogen and the solution was stirred with molecular sieves
(4 Å, 4 g) at Ϫ65 ЊC for 1 h. The reaction was started by the
addition of TMSOTf (0.02  solution in CH2Cl2; 100 µl) during
10 min and the mixture was stirred for 2 h at Ϫ65 ЊC and for 3 h
at Ϫ30 ЊC. Additional TMSOTf (0.02  solution in CH2Cl2;
150 µl) was added and the mixture was stirred at rt overnight.
Then saturated aq. NaHCO3 (100 cm3) was added and the aq.
phase was extracted with CH2Cl2 (150, 100 and 50 cm3). The
combined organic phases were concentrated, and purified by
flash chromatography (light petroleum–ethyl acetate, 3:1) to
afford the title mannoside cluster 15 (3.75 g, 74%) as a syrup
(Found: C, 74.6; H, 6.9; N, 0.8. C112H123NO20 requires C, 74.60;
H, 6.88; N, 0.78%); [α]D20 ϩ16.3 (c 1.24, in CHCl3); Rf (light
petroleum–ethyl acetate, 1:1) 0.72 (UV; H2SO4); δH(400 MHz;
2198
J. Chem. Soc., Perkin Trans. 1, 1998

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CDCl3; Me4Si) 7.36–7.23 (60 H, m, ArH), 4.84 (3 H, d, 2J 11.2,
benzyl CH2), 4.82 (3 H, d, J1,2 2.0, 1-H), 4.74–4.47 (21 H, m,
benzyl CH2), 3.96 (3 H, dd ≈ t, J4,5 9.2, 4-H), 3.84 (3 H, dd, J3,4
9.7, 3-H), 3.76–3.68 (12 H, m, J2,3 3.1, 2-, 5H and 6-H2), 3.62
(3 H, mc, CH2CH2CH2O), 3.31 (3 H, mc, CH2CH2CH2O),
1.88–1.82 (6 H, m, CH2CH2CH2O) and 1.42–1.35 (6 H, m,
CH2CH2CH2O); δC(100.67 MHz; CDCl3; Me4Si) 138.35, 138.28,
138.22, 138.19 (aryl Cq), 128.29–127.50 (aryl CH), 97.90 (C-1),
93.65 (CNO2), 80.04 (C-3), 75.12 (benzyl CH2), 74.83 (C-4),
74.75 (C-2), 73.31, 72.65 and 72.13 (benzyl CH2), 71.89 (C-5),
69.07 (C-6), 66.79 (CH2CH2CH2O), 32.08 (CH2CH2CH2O) and
23.73 (CH2CH2CH2O); m/z (FAB-MS) 1827.8 (Mϩ ϩ Na.
C112H123NNaO20 requires m/z, 1824.85).
and the filtrate was concentrated. The resulting crude material
was purified by size-exclusion chromatography on Sephadex
G-15 (with 15 m aq. NH4HCO3 as eluent) to yield the
unprotected cluster 19 (122 mg, 98%) as a amorphous solid; [α]D20
ϩ64.9 (c 1.02, water); Rf (propan-1-ol–water, 7:3, 1% NH3)
0.38 (UV; H2SO4); δH(400 MHz; D2O) 4.59 (3 H, d, J1,2 1.5,
1Ј-H), 3.78 (3 H, dd, J2Ј,3Ј 3.6, 2Ј-H), 3.60 (3 H, dd, J3Ј,4Ј 9.2,
3Ј-H), 3.50–3.45 (6 H, m, J5Ј,6Ј 7.6, 5Ј-H, 6Ј-HЈ), 3.41 (3 H,
dd ≈ t, J4Ј,5Ј 9.2, 4Ј-H), 3.29 (3 H, dd, J6Ј,6Љ 14.8, 6Ј-H) and 3.24
(9 H, s, OCH3); δC(100.67 MHz; D2O) 174.97 (NHCO), 101.23
(C-1Ј), 93.69 (CqNO2), 70.99 (C-5Ј), 70.72 (C-3Ј), 70.23 (C-2Ј),
68.40 (C-4Ј), 55.01 (OCH3), 40.42 (C-6Ј), 30.98 (CH2CH2CO)
and 30.35 (CH2CH2CO); m/z (FAB-MS) 803.7 (Mϩ ϩ H.
C31H55N4O20 requires m/z, 803.34).
Nitrotris-[3-(á-D-mannopyranosyloxy)propyl]methane 16
The benzylated cluster 15 (750 mg, 0.416 mmol) and Pd catalyst
(0.80 g; 10% on charcoal) were suspended in EtOH–THF (50
ml; 3:1) and the mixture was stirred at 60 ЊC under H2 60 bar
for 6 h. Then the catalyst was removed by filtration over a bead
of Celite, and the filtrate was concentrated, and purified by size-
exclusion chromatography on Sephadex G-15 (with 15 m aq.
NH4HCO3 as eluent). After lyophilization the unprotected clus-
ter 16 (231 mg, 77%) was obtained as a lyophilizate; [α]D20 ϩ44.2
(c 0.97, water); Rf (propan-2-ol–water, 7:3, 1% NH3) 0.38 (UV;
H2SO4); δH(400 MHz; D2O) 4.69 (3 H, d, J1,2 1.5, 1-H), 3.78
(3 H, dd, J2,3 3.6, 2-H), 3.72 (3 H, dd, J3,4 11.7, 3-H), 3.65–3.55
(9 H, m, 6-, 5- and 4-H), 3.51–3.46 (6 H, m, 6-HЈ, CH2O), 3.42–
3.37 (3 H, m, CH2O), 1.90 (6 H, mc, CH2CH2CH2O) and 1.40
(6 H, mc, CH2CH2CH2O); δC(100.67 MHz; D2O) 100.06 (C-1),
95.52 (CNO2), 73.17, 71.01, 70.45 and 67.12 (C-2, -3, -4 and
C-5), 67.43 (C-6), 61.29 (CH2CH2CH2O), 32.02 (CH2CH2-
CH2O) and 23.51 (CH2CH2CH2O); m/z (FAB-MS) 722.5
(Mϩ ϩ H. C28H52NO20 requires m/z, 722.31).
Tris-{2-[3-(2Ј,3Ј,4Ј-tri-O-benzoyl-6Ј-deoxy-1Ј-O-methyl-á-D-
mannopyranos-6Ј-yl)thioureido]ethyl}amine 21
To a solution of the mannoside 3 (394 mg, 0.72 mmol) in dry
CH2Cl2 (40 ml) was added tris-(2-aminoethyl)amine 20 (31.9
mg, 0.218 mmol) slowly at 50 ЊC. The mixture was stirred at
50 ЊC until the reaction was complete (TLC ethyl acetate–light
petroleum, 2:1). The solvent was removed in vacuo and the
remaining syrup was purified by flash chromatography (ethyl
acetate–light petroleum, 2:1) to yield the title cluster 21 (359
mg, 92%) as a slightly yellow amorphous solid (Found: C, 62.4;
H, 5.25; N, 5.5. C93H93N7O24S3 requires C, 62.44; H, 5.24; N,
5.48%); [α]D20 Ϫ92.6 (c 1.79, CHCl3); Rf (ethyl acetate–light
petroleum, 2:1) 0.20 (UV; H2SO4); δH(400 MHz; CDCl3;
Me4Si) 8.08, 7.93 and 7.80 (each 6 H, each d, ArH), 7.54–7.23
(27 H, m, ArH), 7.08 (6 H, br s, NH), 5.83 (3 H, dd, J3Ј,4Ј 9.7,
3Ј-H), 5.70 (3 H, dd ≈ t, J4Ј,5Ј 9.7, 4Ј-H), 5.61 (3 H, dd, J2Ј,3Ј 3.1,
2Ј-H), 4.85 (3 H, d, J1Ј,2Ј 2.0, 1Ј-H), 4.20 (3 H, br s, 5Ј-H), 4.11–
3.94 (3 H, br s, 6Ј-H), 3.77–3.67 (3 H, br s, 6Ј-H), 3.58 [6 H, br s,
CH2NHC(S)], 3.42 (9 H, s, OCH3) and 2.50 (6 H, s, NCH2);
Nitrotris-{2-[(2Ј,3Ј,4Ј-tri-O-benzoyl-6Ј-deoxy-1Ј-O-methyl-á-
D-mannopyranos-6Ј-yl)carbamoyl]ethyl}methane 18
δ (62.89 MHz; CDCl ) 171.11, 165.89 and 165.40 (C᎐O),
᎐
C 3
133.46, 133.37 and 133.08 (aryl-Cq), 129.93–128.18 (aryl-CH),
98.51 (C-1Ј), 70.26 (C-2Ј), 69.76 (C-3Ј), 69.51 (C-5Ј), 68.02
(C-4Ј), 60.35 (C-6Ј), 55.60 (OCH3), 53.43 (NCH2) and 45.16
[CH2NHC(S)]; m/z (FAB-MS) 1788.9 (Mϩ ϩ H. C93H94N7O24S3
requires m/z, 1788.55).
A mixture of the mannoside 2 (1.12 g, 2.22 mmol), the triacid
1725 (0.20 g, 0.72 mmol) and HOBT (0.60 g, 4.44 mmol) in dry
DMF (10 ml) was stirred at 0 ЊC for 30 min. Then DCC (0.46 g,
2.23 mmol) was added and the reaction mixture was stirred at
0 ЊC for 30 min and at rt overnight. The mixture was filtered
and the filtrate was concentrated. The resulting crude product
was diluted in ethyl acetate (80 ml) and subsequently washed
successively with hydrochloric acid (5%; twice), saturated aq.
NaHCO3 and water. The organic phase was concentrated, and
purified by flash chromatography (light petroleum–ethyl acet-
ate, 1:3) to afford the title cluster 18 (0.92 g, 73%) as a solid
(Found: C, 64.9; H, 5.2; N, 3.2. C94H90N4O29 requires C, 64.90;
H, 5.21; N, 3.22%); [α]D20 Ϫ99.6 (c 0.70, CHCl3); Rf (ethyl acetate–
light petroleum, 3:1) 0.21 (UV; H2SO4); δH(400 MHz; CDCl3;
Me4Si) 8.07–7.24 (45 H, m, ArH), 6.25 (3 H, t, JNH,6Ј 5.6, JNH,6Љ
6.6, NH), 5.85 (3 H, dd, J3Ј,4Ј 10.2, 3Ј-H), 5.70 (3 H, dd ≈ t, J4Ј,5Ј
10.2, 4Ј-H), 5.63 (3 H, dd, J2Ј,3Ј 3.6, 2Ј-H), 4.92 (3 H, d, J1Ј,2Ј
1.5, 1Ј-H), 4.12 (3 H, ddd, J5Ј,6Ј 5.6, 5Ј-H), 3.80 (3 H, ddd, J5Ј,6Љ
2.5, 6Ј-HЈ), 3.47 (9 H, s, OCH3), 3.30 (3 H, ddd, J6Ј,6Љ 14.2, 6Ј-H)
and 2.30–2.11 (12 H, m, CH2CH2CO); δC(100.67 MHz; CDCl3;
Tris-{2-[3-(2Ј,3Ј,4Ј-tri-O-acetyl-6Ј-deoxy-1Ј-O-methyl-á-D-
mannopyranos-6Ј-yl)thioureido]ethyl}amine 22
To a solution of the mannoside 5 (317 mg, 0.877 mmol) in dry
CH2Cl2 (30 ml) was added tris-(2-aminoethyl)amine 20 (39 mg,
0.266 mmol) slowly at 50 ЊC. The reaction mixture was stirred
at 50 ЊC until the reaction was complete (TLC ethyl acetate–
light petroleum, 2:1). The solvent was removed in vacuo and the
remaining syrup was purified by flash chromatography (ethyl
acetate) to yield the title cluster 22 (298 mg, 91%) as an
amorphous solid (Found: C, 46.9; H, 6.15; N, 7.95; S, 7.8.
C48H75N7O24S3 requires C, 46.86; H, 6.14; N, 7.97; S, 7.82%);
[α]D20 ϩ43.6 (c 0.84, CHCl3); Rf (ethyl acetate–EtOH, 9:1) 0.39
(UV; H2SO4); δH(400 MHz; CDCl3; Me4Si) 7.25 and 7.05 (3 H,
br s, NH), 5.32 (3 H, dd, J3Ј,4Ј 9.7, 3Ј-H), 5.23 (3 H, dd, J2Ј,3Ј 3.6,
2Ј-H), 5.12 (3 H, dd ≈ t, J4Ј,5Ј 10.2, 4Ј-H), 4.71 (3 H, d, J1Ј,2Ј 1.5,
1Ј-H), 3.90 (3 H, mc, 5Ј-H), 3.84–3.50 [12 H, br m, 6Ј-H2,
C(S)NHCH2], 3.41 (9 H, s, OCH3), 2.67 (6 H, br s, NCH2) and
2.17, 2.09 and 1.99 (each 9 H, each s, 3 × Ac); δC(100.67 MHz;
Me Si) 170.94 (NHCO), 165.98, 165.39 and 165.35 (3 × C᎐O),
᎐
4
133.69–128.27 (m, aryl-C), 98.53 (C-1Ј), 93.01 (CqNO2), 70.51
(C-2Ј), 69.62 (C-3Ј), 69.00 (C-5Ј), 67.93 (C-4Ј), 55.58 (OCH3),
39.81 (C-6Ј), 30.69 (CH2CH2CO) and 30.56 (CH2CH2CO);
m/z (FAB-MS) 1739.9 (Mϩ ϩ H. C94H91N4O29 requires m/z,
1739.58).
CDCl ; Me Si) 182.82 (NCS), 170.07, 169.82 and 169.67 (C᎐O),
᎐
3
4
98.25 (C-1Ј), 69.36 (C-2Ј), 69.25 (C-5Ј), 68.77 (C-3Ј), 67.04
(C-4Ј), 55.28 (OCH3), 52.62 (NCH2), 45.05 (C-6Ј), 42.43
[C(S)NHCH2] and 20.74, 20.73 and 20.49 [C(O)CH3].
Tris-{2-[6Јdeoxy-1Ј-O-methyl-á-D-mannopyranos-6Ј-yl)carb-
amoyl]ethyl}nitromethane 19
A solution of the protected cluster mannoside 18 (270 mg,
0.155 mmol) in diethyl ether–MeOH (1:2.5; 10 ml) was treated
with NaOMe solution (1  in MeOH; 1.5 ml) and stirred at rt
for 2 days. Then the reaction mixture was neutralized with ion-
exchange resin (Levatit SP 1080 Hϩ), the resin was filtered off,
Tris-{2-[3-(6Ј-deoxy-1Ј-O-methyl-á-D-mannopyranos-6Ј-yl)thio-
ureido]ethyl}amine 23
(a) To a solution of the unprotected mannoside 8 (136 mg,
0.578 mmol) in EtOH–ethyl acetate (4:1; 10 ml) was added tris-
(2-aminoethyl)amine 20 (25.6 mg, 0.175 mmol) slowly at 50 ЊC.
The reaction mixture was stirred at 50 ЊC until the reaction was
J. Chem. Soc., Perkin Trans. 1, 1998
2199

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complete (TLC ethyl acetate–light petroleum, 2:1). The solvent
was removed in vacuo and the remaining syrup was purified by
gel-permeation chromatography on Sephadex G-15 with 15 m
aq. NH4HCO3 as eluent to yield the title cluster 23 (35 mg, 23%)
as a lyophilizate after repeated lyophilization with distilled
deionized water until mass stability.
(b) A solution of the benzoylated cluster 21 (236 mg, 0.132
mmol) in dry 5:1 MeOH–DMF (60 ml) was treated with
NaOMe solution (1  in MeOH; 1.5 ml) and the reaction mix-
ture was stirred at rt for 1 week. It was neutralized with ion-
exchange resin (Levatit SP 1080 Hϩ), the resin was filtered off,
and the filtrate was concentrated, and purified by size-exclusion
chromatography on Sephadex G-15 (15 m aq. NH4HCO3 as
eluent). The unprotected title cluster 23 (30 mg, 27%) was
obtained as a lyophilizate after repeated lyophilization with
distilled, deionized water until mass stability.
for kindly providing the pPK14-containing strain and the
anti-fimA antibody, and to Prof. Dr J. Thiem and Prof. Dr
E. Th. Rietschel for moral support.
References
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ised water; [α]D20 ϩ14.0 (c 1.10, MeOH); Rf (propan-1-ol–water,
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J3Ј,4Ј 9.7, 3Ј-H), 4.10–3.92 [15 H, m, C(S)NHCH2, 5Ј-H and
6Ј-H2], 3.86 (3 H, dd ≈ t, J4Ј,5Ј 9.7, 4Ј-H), 3.67 (9 H, s, OCH3)
and 3.29 (6 H, br s, NCH2); δC(100.67 MHz; D2O, CD3CN)
182.76 (NCS), 101.80 (C-1Ј), 71.76, 71.26, 70.81 and 68.80
(C-2Ј, -3Ј, -4Ј and -5Ј), 55.50 (OCH3), 53.83 (NCH2), 45.54
(C-6Ј) and 42.00 [C(S)NHCH2]; m/z (FAB-MS) 852 (Mϩ ϩ H.
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Tris-(2-{3-[6Ј-deoxy-1Ј-O-(p-nitrophenyl)-á-D-mannopyranos-
6Ј-yl]thioureido}ethyl)amine 24
To a solution of the aromatic mannoside 12 (291 mg, 0.851
mmol) in dry CH2Cl2 (20 ml) was added tris-(2-aminoethyl)-
amine 20 (34 mg, 0.234 mmol) slowly at 50 ЊC. The mixture was
stirred at 50 ЊC until the reaction was complete (TLC ethyl
acetate–MeOH–water, 7:2:1). The solvent was removed in
vacuo and the remaining syrup was purified by flash chroma-
tography (ethyl acetate–MeOH, 10:1, 1% methanolic NH3;
then ethyl acetate–MeOH–water, 7:2:1, 1% methanolic NH3)
to afford the title cluster 24 (244 mg, 89%) as a lyophilizate
(Found: C, 46.2; H, 5.2; N, 11.9; S, 8.2. C45H60N10O21S3 requires
C, 46.07; H, 5.15; N, 11.94; S, 8.20%); [α]D20 ϩ89.9 (c 0.65,
MeOH); Rf (ethyl acetate–MeOH–water, 7:2:1, 1% NH3) 0.77
(UV; H2SO4); δH(400 MHz; D3COD) 8.23 (6 H, d, 3J 9.67,
3
m-ArH), 7.30 (6 H, d, J 9.16, o-ArH), 5.72 (3 H, d, J1Ј,2Ј 1.5,
1Ј-H), 4.10 (3 H, dd, J2Ј,3Ј 3.6, 2Ј-H), 3.96 (3 H, dd, J3Ј,4Ј 9.2,
3Ј-H), 3.87 (3 H, ddd, J5Ј,6Ј 5.1, 5Ј-H), 3 H, dd, J6Ј,6Ј 10.2, 6Ј-H),
3.71 (3 H, dd ≈ t, J4Ј,5Ј 9.7, 4Ј-H), 3.66 (3 H, dd, J5Ј,6Љ 3.6, 6Ј-H),
3.47 [6 H, br s, C(S)NHCH2] and 2.66 (6 H, br s, NCH2);
δC(100.67 MHz; D3COD) 162.73 (i-aryl-Cq), 144.10 (p-aryl-Cq),
127.11 and 118.19 (aryl-CH), 100.16 (C-1Ј), 74.45, 72.03,
71.78 and 69.36 (C-2Ј), -3Ј, -4Ј and -5Ј), 54.55 (NCH2), 46.39
(C-6Ј) and 43.91 [C(S)NHCH2]; m/z (MALDI-TOF) 1173.5
(Mϩ ϩ H. C45H61N10O21S3 requires m/z, 1173.32).
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Acknowledgements
This work was financed by the Deutsche Forschungsgemein-
schaft DFG (SFB 470). We are thankful to Prof. Dr J. Hacker
Paper 8/01985A
Received 11th March 1998
Accepted 8th May 1998
2200
J. Chem. Soc., Perkin Trans. 1, 1998