Novel multivalent mannose compounds and their inhibition of the adhesion of type 1 fimbriated uropathogenic E. coli

Article abstract of DOI:10.1016/j.tetasy.2004.11.014

A series of multivalent mannose containing compounds were prepared varying in size from small divalent, to 16-valent glycodenrimers and 21-valent glycopolymers. The molecules were approached via a common mannose building block. As scaffolds dendrimers and

Full text of DOI:10.1016/j.tetasy.2004.11.014

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 361–372
Novel multivalent mannose compounds and their inhibition of
the adhesion of type 1 fimbriated uropathogenic E. coli
Chantal C. M. Appeldoorn,a John A. F. Joosten,a Fatna Ait el Maate,a Ulrich Dobrindt,b
Jo¨rg Hacker,b Rob M. J. Liskamp,a A. Salam Khanb,* and Roland J. Pietersa,*
aDepartment of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
PO Box 80082, 3508 TB Utrecht, The Netherlands
bInstitute for Molecular Biology of Infectious Diseases, University of Wu¨rzburg, Ro¨ntgenring 11, D-97070 Wu¨rzburg, Germany
Received 2 November 2004; accepted 8 November 2004
Available online 24 December 2004
Abstract—A series of multivalent mannose containing compounds were prepared varying in size from small divalent, to 16-valent
glycodenrimers and 21-valent glycopolymers. The molecules were approached via a common mannose building block. As scaffolds
dendrimers and dendrons based on the 3,5-di-(2-aminoethoxy)-benzoic acid branching unit were used along with commercially
available PAMAM dendrimers. To include larger structures, linear glycopolymers with varying amounts of mannose were prepared
via radical polymerization. The compounds were tested for their biological activity using a newly developed ELISA based inhibition
assay, for their ability to inhibit the binding of recombinant type I fimbriated E. coli to a monolayer of T24 cell line derived from
human urinary bladder epithelium. All compounds showed enhanced affinity as compared to mannose with IC50Õs down to the low
micromolar range.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
sins with carbohydrate binding specificities.7 The most
common fimbrial type is known to be the mannose spe-
cific type 1, which contains the FimH adhesion protein8
and is a common adhesive organelle found in many spe-
cies of the enterobacterial family. Epidemiological stud-
ies indicated the prevalence of type 1 fimbriae in UTIs,
since about half of the E. coli strains from patients with
pyelonephritis and the majority of strains from patients
with cystitis are described to express type 1 fimbriae.9
While bacterial adhesion is often a prerequisite to infec-
tion10 the fluid sheer conditions prevalent in the human
bladder particularly necessitate such interactions. For
type I fimbriae the FimH adhesin binds to mannose res-
idues present on the bladder epithelial cell surface of
members of the glycoprotein family of the uroplakins.11
The ability of type 1 fimbriated strains to bind mannose
strongly differs between strains, yet is the crucial factor
for pathogenicity.21,12 Binding to mannotriose, an abil-
ity common to type 1 fimbriated E. coli does not corre-
late to UTI causing abilities. The residues in the
mannose binding pocket appeared to be highly con-
served, being present in over 200 strains of UPEC.
The UTI-infection leads to exfoliation of bladder epithe-
lial cells, a host defence mechanism, while bacteria es-
cape this by invading into deeper tissue, a mechanism
likely responsible for recurrent infections.13
Bacterial adhesion, a prelude to many infections, is com-
monly mediated by protein–carbohydrate interactions
where the bacteria contain lectin-like adhesion proteins
that bind to specific surface exposed carbohydrates of
glycolipids or glycoproteins on tissue cells.1–3 Inhibition
of the adhesion would be an attractive method of pre-
venting and treating infections. The emergence of anti-
biotic resistance is becoming a substantial problem in
medicine,4 creating an urgency in the design of addi-
tional anti-infectious approaches.5 As the bacterial
adhesion involves carbohydrate–protein interactions,
interference in this process provides a logical strategy.
Urinary tract infections (UTIs) rank amongst the most
frequently occurring bacterial diseases in humans.6
The vast majority of these UTIs are caused by uropath-
ogenic Escherichia coli (UPEC).6b In addition to viru-
lence factors such as O-antigens, capsules, toxins and
serum resistance, UPEC strains express fimbrial adhe-
*
Corresponding authors. Tel.: +49 0931 312130; fax: +49 0931 312578
(A.S.K.); tel.: +31 030 2536944; fax: +31 030 2536655 (R.J.P.);
e-mail addresses: s.khan@mail.uni-wuerzburg.de; r.j.pieters@pharm.
uu.nl
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.014

362
C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
For the generation of effective anti-adhesion compounds
a major hurdle is the fact that, in general, monosaccha-
rides display low affinities for their target, typically in
the millimolar range.10 The problem may be overcome
by the use of multivalent ligands.14 Synthetic multi-
valent carbohydrates have been shown highly effective
for well-defined targets such as the AB5 toxins.15 For
interference with bacterial adhesion only a few studies
involving multivalent carbohydrates have been reported.
A particularly effective inhibition was observed in two
studies involving the pig pathogen that can also cause
meningitis in humans, Streptococcus suis.16 The adhe-
sion of the Gram-positive pathogen was effectively
inhibited by multivalent galabiose (Gala1-4Gal) com-
pounds, where an octavalent compound inhibited hae-
magglutination with an MIC of 0.3nM, to the best of
our knowledge the first example of sub-nanomolar inhi-
bition of bacterial binding by a synthetic multivalent
compound.16b We previously reported on multivalent
GalNAcb1-4Gal- and GalNAcb1-4Galb1-4Glc-contain-
ing glycodendrimers as inhibitors for F1C fimbriated E.
coli and for Pseudomonas aeruginosa.17 A combination
of spacer and multivalency effects led to a 38-fold in-
crease in the potency of a divalent inhibitor as compared
to the parent sugar. However, the most prominently
studied target has been type 1 fimbriated uropathogenic
E. coli. Lindhorst et al. prepared a number of multiva-
lent mannose compounds of varying efficacy against
the type 1 E. coli.18 Their most effective system was a
trivalent system with the mannose residues linked via
C-6,18a,d which was on a per sugar basis more active
than p-nitrophenyl a-D-mannoside, a previously ob-
served19 active ligand. Lee and co-workers20 prepared
larger glycondendrimers as well as neoglycoproteins.
These studies showed a significant effect of the aglycon
part and also multivalency effects of one order of magni-
tude. Similar aglycon effects have previously been
observed19 and can be understood when examining the
crystal structure of the adhesin, which has hydrophobic
areas just outside the mannose binding pocket.21 The
multivalency effect was attributed to bridging between
separate fimbrial tips.
observations were made to suggest the presence of mul-
tiple copies of FimH at the fimbrial tip,23b but also data
consistent with a single copy exists.25 Based on all of this
information the preparation of a variety of mannose
containing systems seemed appropriate. To this end gly-
copolymers were prepared that represent a linear
arrangement of mannose moieties covering relatively
long distances. Members of this class were not previ-
ously evaluated as inhibitors for type 1 fimbriated E.
coli, although previous work suggested their potential
in this case.20 In other systems, for example, in viral
adhesion inhibition, glycopolymers have proven highly
effective.26 Furthermore, glycodendrimers were made
with the potential to simultaneously block several bind-
ing sites on the fimbrial shaft. Moreover, especially the
smaller ones, could act as mimics of mannotriose, a
20-fold stronger binding moiety than mannose,20 whose
binding site encompasses the mannose binding site.21
Possible amplification of this effect with an additional
135-fold20 benefit of hydrophobic aglycon interactions
further motivated our studies.
In order to evaluate compounds for their potential the
biological context should approximate the actual situa-
tion as much as possible. Haemagglutination experi-
ments are often used, although they are complicated
by the diversity of displayed carbohydrates on red blood
cells and also reproducibility can be problematic. Roy
and co-workers20 used a new assay based on the inhibi-
tion of mannosides on the bacterial binding to a radio-
labelled glycoprotein in solution. This assay gave
much lower IC50 values that observed by haema-
gglutination. Whitesides et al.27 have developed an
assay based on the association of fluorescently labelled
bacteria to mannose displaying self-assembled mono-
layers generated on gold coated 96-well plates. We chose
for an assay that is close to the in vivo situation and
used a solid phase assay of the ELISA type using a
bladder cell line in the well. IC50Õs resulting from the
assay were not in the nanomolar range for p-nitro-
phenyl a-D-mannoside as in the assay of Lee et al.,
and neither was there a complete absence of back-
ground binding as in the assay of Whitesides, yet in
our view the conditions used were realistic. Non-
specific binding of bacteria to cell surfaces is a real
occurrence and caused by hydrophobic, Van der Waals
and/or electrostatic interaction. In the reported studies
here the background binding, which could amount to
about 30% of the tota observed binding, was determined
by experiments with nonfimbriated bacteria and sub-
tracted consistently.
In this study we describe our efforts for the preparation
and evaluation of a significant variety of multivalent
mannosides, including small divalent systems, glycoden-
drimers and glycopolymers, as inhibitors of type 1 fim-
briated uropathogenic E. coli. They were evaluated in
a novel highly biorelevant assay involving a bladder
cell-line. Multivalency effects have previously been ob-
served in the peeling of bacteria from a mannose con-
taining self-assembled monolayer.22 The observed
effects were attributed to individual fimbriae binding
to and releasing from the surface. While the FimH adhe-
sins are known to be located at the tip of the fimbriae,23
others located in the fimbrial shaft are present as well
but deemed only active upon fragmentation and expo-
sure of the binding site.24 However, previous observa-
tions were made where glyconjugates were seen to
actively bind along the shaft of the fimbriae.8 The latter
arrangement would imply that the binding sites were
more closely spaced and thus would make the binding
more susceptible to multivalency effects. Furthermore
2. Results and discussion
2.1. Synthesis of general mannose building block
An appropriate mannose building block had to be syn-
thesized for coupling to the various scaffolds. As the
scaffolds contain an amine functionality, a carboxylic
acid moiety was introduced on the carbohydrate, en-
abling peptide coupling conditions. Hereto, 1,2,3,4,6-
penta-O-acetyl-a-D-mannopyranose 1 was converted to

C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
363
OAc
O
OAc
O
OAc
O
OAc
O
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
i
ii
iii
AcO
AcO
AcO
AcO
OAc
Br
O
Br
O
N3
1
3
4
2
OAc
O
AcO
AcO
OAc
O
AcO
AcO
AcO
iv
v
O
O
AcO
O
O
N
OH
O
NH2
H
5
6
Scheme 1. Reagents and conditions: (i) HBr, AcOH, CH2Cl2, rt (98%); (ii) IBr, HO(CH2)3Br, CH2Cl2, CH3CN, 0ꢁC to rt (55%); (iii) NaN3, DMF
(83%); (iv) H2, Pd–C, EtOAc, Et3N (98%); (v) diglycolic anhydride, pyridine, dioxane (64%).
bromide 2 using HBr, after which an anomeric 3-bromo-
propanol spacer was introduced using IBr yielding 3
(Scheme 1).28 After introduction of an azido function
using NaN3 in DMF to give 4, the compound was con-
verted to amine 5 by hydrogenation. Finally, elaboration
with diglycolic anhydride resulted in key building block 6.
using ammonium persulfate and N,N,N0,N0-tetramethyl-
ethylenediamine (TMEDA) in water,35 containing
various amounts of mannose, ranging from 0 (control)
to 40mol%. N-(2-Hydroxylpropyl) methacrylamide
(HPMA) was chosen as a co-monomer for its good
water solubility and excellent biocompatibility.36
Mannose monomer 38 was prepared, from amine 5,
which was converted to acrylamide 37 by the action of
acryloyl chloride (Scheme 2). Subsequently, this com-
pound was deacetylated with NaOMe in MeOH, yield-
ing unprotected monomer 38, which was used
immediately for the polymerization reaction to prevent
unwanted premature polymerization of the monomer it-
self. Hereto, 38 was dissolved in water together with
HPMA and TMEDA and polymerized by ammonium
persulfate. The polymer was dialyzed against water
and lyophilized. The chemical identity of the glycopoly-
mers was analyzed via proton NMR to determine the
ratio of each monomer, which was consistent with
the input. GPC was used for the determination of the
molecular weight (Mw ꢀ 10.000) and polydispersity
(1.3 < Mw/Mn < 1.8).
2.2. Synthesis of glycodendrimers
Building block 6 was coupled to various scaffolds gener-
ating compounds containing 1–16 mannose molecules
(Fig. 1). Dendrimers based on the 3,5-di-(2-aminoeth-
oxy)-benzoic acid branching unit29 were used as they
were previously shown to be successful in generating
multivalent carbohydrates.17,29d,30 Mannose 6 was cou-
pled to the amine containing scaffolds using BOP or
TBTU as the coupling reagent and DIPEA as the base
in good yields (Table 1). Deacetylation using sodium
methoxide in MeOH and subsequent neutralization with
Dowex H+ resulted in mono-, di-, tetra- and octavalent
end products 9, 12, 27 and 30. Mono- and divalent com-
pounds 21 and 24, containing a significantly longer
spacer, were synthesized to examine the effect of the
spacer on binding. All compounds were fully character-
ized by NMR and high-resolution mass spectrometry,
confirming their identity. Two other divalent com-
pounds 15 and 18 were prepared on the bis-3-amino-
prop-1-ynyl benzene scaffold16b introducing some pre-
orientation of the carbohydrates. Finally, PAMAM
dendrimers were used as an alternative dendritic scaf-
folds, as others31 have achieved good results with this
type of dendrimer. Acetylated compounds 32 and 35
were synthesized in DMSO rather than DCM due solu-
bility problems and purified by size exclusion chroma-
tography (LH20). The structures were confirmed by
NMR and MALDI mass spectrometry.
2.4. Adhesion inhibition
A biologically relevant testing system was developed:
compounds were assayed in an ELISA-based assay for
their ability to inhibit the binding of mannose binding
type I fimbriated E. coli to a monolayer of T24 cell line
derived from human urinary bladder epithelium. The
inhibitory potencies of the glycodendrimers and poly-
mers are summarized in Table 2 and exemplary inhibi-
tion curves are shown in Figure 2. Mannose itself was
a poor inhibitor of E. coli binding with an IC50 of
7.6mM. Potencies improved with the attachment of
scaffolds, as was seen for 9, which showed an IC50 of
337lM, that is, a relative potency of 23 when compared
to mannose. Increasing the number of mannoses to two
and even four, improved this affinity both in absolute
value (IC50 of 27 = 51lM) and relative when divided
by the number of attached saccharides (rel. pot. per
sugar 37-fold). For the octavalent 30, the activity was
slightly decreased again. The partially rigidified 15 and
18 displayed inhibitory potencies similar to the flexible
divalent 9. The compounds with elongated spacers,
2.3. Synthesis of glycopolymers
Linear glycopolymers were also synthesized with vari-
able numbers of mannose molecules attached to it.
The use of carbohydrate containing polymers has
been successful,26,32–34 especially for the inhibition of
selectins, and the influenza virus. The polyacrylamide
polymers were prepared by radical polymerization

364
C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
NHR
NHR
NHR
NHR
NHR
NHR
13-15
O
O
MeOOC
16-18
MeOOC
O
NHR
7-9
10-12
O
O
O
O
NHR
NHR
N
H
O
O
N
O
H
O
O
MeOOC
MeOOC
19-21
NH
O
O
O
O
NHR
NHR
MeOOC
O
O
O
O
O
O
O
O
NHR
NHR
N
H
O
O
O
N
H
NH
O
O
O
25-27
O
NHR
O
N
H
N
H
22-24
NHR
RHN
NHR
O
HN
O
NH
RHN
O
NH
O
NHR
HN
O
NH
O
O
O
N
NHR
O
O
O
N
HN
HN
O
O
N
O
NH
NH
H
O
O
NHR
N
NH
O
MeOOC
N
O
O
NH
O
O
N
NHR
O
O
N
O
NH
N
H
O
NHR
O
HN
RHN
O
HN
O
NHR
NH
O
28-30
NH
O
O
NHR
NHR
O
RHN
31-33
NHR
RHN
NHR
NHR
NH
NHR
HN
N
O
HN
O
NHR
HN
H
N
NHR
NH
N
RHN
O
O
N
O
O
NH
HN
O
NH
NH
O
O
N
NH
N
O
O
RHN
RHN
O
N
O
O
O
HN
HN
O
O
NHR
NH
NH
N
N
34-36
O
NHR
O
N
HN
N
O
NH
NH
N
O
HN
HN
O
N
HN
NH
NH
O
RHN
O
N
O
NHR
O
N
O
HN
NH
O
NH
NHR
RHN
HN
RHN
NHR
Figure 1. Chemical structures of the prepared (glyco)dendrimers.
monovalent 21 and divalent 24, showed a 3–8-fold in-
creased activity compared to their short spacered coun-
terparts. In fact the divalent 24 showed the highest
relative potency per sugar of 141-fold enhancement ver-
sus mannose. The PAMAM glycodendrimer 36 dis-
played the highest affinity towards the target, although
their relative potency per mannose was decreased. The
glycopolymers (with 3–21 mannose units per polymer)
showed enhanced activity with increasing mannose sub-
stitution to an IC50 as low as 12lM. The relative
potency per mannose for the polymer series was rela-
tively constant at around 30–40. No binding of the con-

C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
365
Table 1.
R =
R =
OAc
AcO
OH
HO
O
AcO
AcO
O
R = H.TFA or H2
HO
HO
O
O
O
O
O
O
NH
O
O
NH
3
3
7
10
8
11
9
12
1314
6 + 16
19
15
17
20
18
21
22
25
2324
26
BOP, DIPEA, DCM
or
TBTU, DIPEA, DMSO
27
30
NaOMe, MeOH
28
3
29
1
4
3
3
3
OAc
O
OAc
AcO
AcO
AcO
O
AcO
AcO
i
ii
AcO
O
O
O
37
NH2
N
H
5
OH
HO
y
x
O
HO
HO
O
iii
HN
O HN
O
HO
O
N
H
x/y = 0: Poly0-100
x/y = 19: Poly5-95
x/y = 9: Poly10-90
x/y = 4: Poly20-80
x/y = 1.5: Poly40-60
O
O
38
HO
HO
OH
HO
Scheme 2. Reagents and conditions: (i) acryloyl chloride, dioxane, water, NaHCO3, 0ꢁC to rt (76%); (ii) NaOMe, MeOH, rt (quant.); (iii) HPMA,
APS, TMEDA, H2O, 40ꢁC (40–65%).
trol polymer, containing no carbohydrate, was observed
in our assay.
3. Conclusions
In this study we successfully prepared a series of multi-
valent mannose compounds, that can be divided into the
categories: small divalent systems, larger glycodendri-
mers and linear glycopolymers. They were evaluated as
inhibitors in a novel ELISA-type assay that reports on
the adhesion of type 1 fimbriated uropathogenic E. coli
to a relevant cell line. Multivalency in the binding to
type 1 fimbriae could in principle occur at different lev-
els. Multiple binding sites on a single FimH molecule are
present, considering the enhanced affinity of mannotri-
ose over mannose itself.21 Multiple copies of FimH have
been suggested to be present at fimbrial tips.23b Multiple
binding occurrences of a glycoconjugate to the fimbrial
shaft have been observed at short and longer distance
The results indicate that a major multivalency effect, as
seen with S. suis using essentially the same scaffold
systems,16b did not occur. Affinity enhancements were
observed, but the main part of the effect can be attrib-
uted to the beneficial effect of a lipophilic aglycon part.
Nonetheless the divalent 24, combined these effects with
a moderate multivalency effect leading to a compound
with a 281-fold overall affinity improvement over man-
nose itself. The linear orientation of the glycopolymers
did not yield additional benefits over glycodendrimers
of similar size and valency, as PAMAM derivative 36
displayed a similar IC50 to Poly40–60, both in the low
micromolar range.

366
C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
Table 2.
4. Experimental
Compound Valency pIC50 SD
IC50 Relative potency
(lM) to mannose
(per mannose)
4.1. General methods
Chemicals were obtained from commercial sources and
used without further purification unless stated other-
wise. Reactions were monitored by TLC on Silica gel
60 F254 (Merck, Amsterdam, the Netherlands). After
examination under UV light, compounds were visual-
ized by heating with 10% (v/v) methanolic H2SO4 or
with ninhydrine (3mgmLÀ1). 1H NMR spectra were re-
corded at 300Kwith a Varian Gemini-300 (300MHz) or
a Varian Unity UNOVA 500 (500MHz spectrometer);
Mannose
1
1
7600
1
9
12
À3.47 0.13
À3.69 0.043
À3.43 0.035
À3.80 0.038
À3.88 0.050
À4.56 0.055
À4.29 0.10
À4.14 0.032
À4.43 0.033
À4.71 0.047
337 23 (23)
204 37 (19)
376 20 (10)
159 48 (24)
131 58 (58)
27 281 (141)
51 149 (37)
72 106 (13)
37 205 (26)
19 400 (25)
2
15
18
2
2
21
24
1
2
27
30
4
8
33
36
8
d
H values are given in ppm relative to the signal of inter-
16
0
nal Me4Si (dH = 0, CDCl3 or CD3OD). 13C NMR spec-
tra were recorded at 300Kwith a Varian Gemini-300
(75MHz); dC values are given in ppm relative to the sig-
nal of CDCl3 (dC = 77.0) or CD3OD (dC = 49.0). Exact
mass spectra were measured by nanoelectrospray time-
of-flight mass spectrometry using a Micromass LCToF
mass spectrometer at a resolution of 5000 FWHM.
Dowex 50 · 8 (H+ form; 20–50 mesh, Fluka) was used
for neutralization. Organic layers were dried with
Na2SO4. Amines 7, 10, 13, 16, 19, 22, 25 and 28 were
prepared according to the literature procedures.16b,17,29
Compounds 31 and 34 were obtained from Sigma–
Aldrich (Zwijndrecht, the Netherlands).
Poly0–100
Poly5–95
Poly10–90
n.d.a
—
3
À4.19 0.038
À4.48 0.032
À4.70 0.050
À4.92 0.050
65 117 (39)
33 230 (33)
20 380 (32)
12 633 (30)
7
Poly20–80 12
Poly40–60 21
a n.d. = no detectable inhibition (less than 10% at 0.5mM).
4.2. Synthesis of 1-bromo-2,3,4,6-O-acetyl-a-D-manno-
pyranoside 2
Peracetylated mannose (1, 2.12g, 5.43mmol) was dis-
solved in dry DCM (100mL), cooled to 0ꢁC, after which
HBr–AcOH (4.64mL, 33% solution) was added drop-
wise. After overnight stirring (TLC, EtOAc–hexane,
1:1), the solution was diluted with DCM and ice water,
and neutralized with NaHCO3. The organic layer was
dried and concentrated, yielding bromide 2 as a oil
(2.19g, 98% yield), which was used without further puri-
fication. 1H NMR (300MHz, CDCl3): d 2.01, 2.09, 2.11,
2.18 (4 · s, 12H, COCH3), 4.13 (dd, 1H, J 2.1, 12.4Hz,
H-6b), 4.20–4.26 (m, 1H, H-5), 4.34 (dd, 1H, J 4.7,
12.4Hz, H-6a), 5.38 (t, 1H, J 10.9Hz, H-4), 5.44 (dd,
1H, J 1.6, 3.3Hz, H-2), 5.70 (dd, 1H, J 3.3, 10.1Hz,
H-3), 6.33 (d, 1H, J 1.1Hz, H-1).
Figure 2. Competition of type I fimbriated E. coli binding to human
bladder cells by mannose (.), 9 (n), 12 (Æ), 24 (m) and 27 (j). Human
bladder cells were incubated with type I fimbriated E. coli with T24
human bladder cells for 15min at room temperature in the presence of
various inhibitors. Data points are means from
experiments of 9 data points.
3 independent
separations.8 Finally, multivalency as the bacteria use it
effectively in tissue binding is the simultaneous attach-
ment of multiple fimbriae. To inhibit the latter interac-
tions truly large systems would be required, larger
than we here prepared and larger than previously made
glycoconjugated proteins.20 It seems that at the smaller
end of the spectrum more can be gained, and in fact
was gained judging from the mentioned 146-fold affinity
gain by the divalent 24 and also the trivalent C6 linked
mannoside reported by Lindhorst and co-workers.18a,d
At which level these gains are made is still unclear. Over-
all further affinity improvement may also come from the
design of improved monovalent systems based on the
FimH X-ray structure,21 harnessing the full potential
of interactions, lipophilic and other, between ligand
and protein. A recent example of such an approach indi-
cates its potential.37
4.3. Synthesis of 3-bromopropyl-2,3,4,6-O-acetyl-a-D-
mannopyranoside 3
To a cooled solution of bromide 2 (1.87g, 4.55mmol) in
dry CH3CN (30mL) was added IBr (11.4mL,
11.4mmol). After stirring for 3h (TLC, EtOAc–hexane,
1:1), the solution was diluted with DCM, washed with
an 5% aqueous Na2S2O5 solution, dried and concen-
trated. Column chromatography (20–50% EtOAc–hex-
1
ane) yielded compound 3 (1.08g, 55% yield). H NMR
(300MHz, CDCl3): d 1.94–1.99 (m, 2H, OCH2CH2-
CH2Br), 2.00, 2.06, 2.11, 2.17 (4 · s, 12H, COCH3),
3.51–3.58 (m, 3H, OCHHCH2CH2Br), 3.88–3.95 (m,
1H, OCHHCH2CH2Br), 3.95–4.07 (m, 1H, H-5), 4.11
(dd, 1H, J 2.2, 12.4Hz, H-6b), 4.28 (dd, 1H, J 4.7,
12.4Hz, H-6a), 4.84 (d, 1H, J 1.4Hz, H-1), 5.21–5.29
(m, 3H, H-2, H-3, H-4). 13C NMR (75MHz, CDCl3):

C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
367
d 20.0 (COCH3), 29.6 (OCH2CH2CH2Br), 31.4 (OCH2-
CH2CH2Br), 61.7 (OCH2CH2CH2Br), 64.8, 65.3, 68.0,
68.4, 68.7 (C-2, C-3, C-4, C-5, C-6), 96.9 (C-1), 169.0,
169.1, 169.2, 169.8 (COCH3). MS for C17H25O10Br
(M, 468.06): [M+Na+] calcd 491.05, found: 491.20.
(4 · s, 12H, COCH3), 3.42–3.58 (m, 3H, OCHHCH2-
CH2NH), 3.75–3.85 (m, 1H, OCHHCH2CH2NH),
3.97–4.02 (m, 1H, H-5), 4.07–4.11 (m, H-6b), 4.15 and
4.21 (2 · s, 4H, COCH2OCH2CO), 4.28 (dd, 1H, J 4.9,
12.4Hz, H-6a), 4.83 (d, 1H, J < 1Hz, H-1), 5.23–5.34
(m, 3H, H-2, H-3, H-4). 13C NMR (75.5MHz, CDCl3):
4.4. Synthesis of 3-azidopropyl 2,3,4,6-O-acetyl-a-D-
mannopyranoside 4
d 20.7 (COCH3), 28.7 (OCH2CH2CH2NH), 36.6
(OCH2CH2CH2NH), 62.5 (OCH2CH2CH2NH), 66.1,
66.3, 68.4, 69.1, 69.4, 69.5, 71.3 (C-2, C-3, C-4, C-5,
C-6, COCH2OCH2CO), 97.7 (C-1), 169.8 170.1, 172.3,
172.9 (COCH3). HR-MS for C21H31NO14 (M,
521.175): [M+Na+] calcd 522.182, found 522.189.
To a solution of 3 (7.18g, 15.3mmol) in dry DMF
(75mL) was added NaN3 (4.67g, 71.9mmol). After
overnight stirring at 100ꢁC (TLC, toluene–EtOAc,
1:1), the solution was filtered over Hyflo and co-concen-
trated with toluene. The residue was diluted with DCM
and washed with brine, dried and concentrated. Column
chromatography (toluene–EtOAc, 15/1 ! 10/1) yielded
4 (5.47g, 83% yield) as a white solid. 1H NMR
(300MHz, CDCl3): d 1.89–1.96 (m, 2H, OCH2CH2-
CH2N3), 2.00, 2.06, 2.11, 2.17 (4 · s, 12H, COCH3),
3.45 (t, 2H, J 6.6Hz, OCH2CH2CH2N3), 3.50–3.57 (m,
1H, OCHHCH2CH2N3), 3.79–3.86 (m, 1H, OCHH-
CH2CH2N3), 3.95–4.00 (m, 1H, H-5), 4.12 (dd, 1H, J
2.5, 12.1Hz, H-6b), 4.29 (dd, 1H, J 5.4, 12.1Hz,
H-6a), 4.83 (d, J 1.4Hz, H-1), 5.23–5.35 (m, 3H, H-2,
H-3, H-4). 13C NMR (75.5MHz, CDCl3): d 20.9
(COCH3), 28.6 (OCH2CH2CH2N3), 48.0 (OCH2CH2-
CH2N3), 62.4, 64.8 (OCH2CH2CH2N3, C-6), 66.0,
68.6, 69.0, 69.4 (C-2, C-3, C-4, C-5), 97.6 (C-1), 169.7,
169.9, 170.1, 170.6 (COCH3). MS for C17H25N3O10
(M, 431.15): [M+Na+] calcd 454.14, found: 454.40.
4.7. General procedure for synthesis of protected
glycodendrimers
Amine containing scaffolds (as free amine or TFA salt)
were dissolved in dry DCM and general building block
6 (1.2equiv per amine), BOP (1.2equiv per amine) and
DIPEA (6equiv per amine) were added. After stirring
for 4–16h at rt the solution was diluted with DCM
and the organic layer washed with 1N KHSO4, 1N
NaOH, water and brine. After drying and concentra-
tion, acetylated glycodendrimers were purified by col-
umn chromatography (0–50% MeOH–DCM). For
larger dendrimers (P8 amine groups), DMSO instead
of DCM and TBTU (1.2equiv per amine) instead of
BOP was used. Purifications of these large glycodendri-
mers was performed based on size exclusion (LH20, elu-
ents DCM–MeOH, 1:1).
4.5. Synthesis of 3-aminopropyl 2,3,4,6-O-acetyl-a-D-
mannopyranoside 5
4.7.1. Monovalent O-acetyl protected mannose short
spacer, compound 8. Compound 8 was prepared
according to general procedure 4.7 from 7. 98mg, 83%
yield. 1H NMR (300MHz, CDCl3): d 1.78–1.95 (m,
2H, OCH2CH2CH2NH), 1.99, 2.06, 2.11, 2.18 (4 · s,
12H, COCH3), 3.40–3.58 (m, 3H, OCHHCH2CH2NH),
3.74–3.78 (m, 3H, OCHHCH2CH2NH, OCH2CH2NH),
3.91 (s, 3H, OCH3), 3.95–4.00 (m, 1H, H-5), 4.07 and
4.10 (2 · s, 4H, COCH2OCH2CO), 4.11–4.15 (m, 1H,
H-6b), 4.28 (dd, 1H, J 4.2, 12.4Hz, H-6a), 4.80 (d,
1H, J 1.2Hz, H-1), 5.24–5.29 (m, 3H, H-2, H-3, H-4),
7.20 (t, 1H, J 5.5Hz, CHarom), 7.36 (t, 1H, J 8.0Hz,
CHarom), 7.54 (s, 1H, CHarom), 7.64 (d, 1H, J 7.6Hz,
Azido 4 (712mg, 1.65mmol) was dissolved in dry
EtOAc, after which Pd–C (142mg) and a few drops of
Et3N were added. After stirring for 3h under H2
(TLC, DCM–MeOH, 2:1) the reaction mixture was fil-
tered off over Hyflo and concentrated, yielding 5 as a
slightly yellow oil (656mg, 98% yield). 1H NMR
(300MHz, CDCl3): d 1.94–1.98 (m, 2H, OCH2CH2-
CH2NH2), 2.00, 2.06, 2.11, 2.17 (4 · s, 12H, COCH3),
2.97–3.08 (m, 2H, OCH2CH2CH2NH2), 3.40–3.47 (m,
2H, OCH2CH2CH2NH2), 4.29 (dd, 1H, J 4.7, 12.4Hz,
H-6a), 4.83 (d, 1H, J < 1Hz, H-1), 5.23–5.28 (m, 3H,
H-2, H-3, H-4). 13C NMR (75.5MHz, CDCl3): d 20.6
(COCH3), 37.3 (OCH2CH2CH2NH2), 46.2 (OCH2CH2-
CH2NH2), 62.2, 64.8 (OCH2CH2CH2NH2, C-6), 65.6,
66.0, 70.2, 71.0 (C-2, C-3, C-4, C-5), 97.3 (C-1), 169.6,
170.0, 170.1, 170.6 (COCH3). MS for C17H27NO10 (M,
405.16): [M+H+] calcd 406.17, found 406.25.
CHarom). 13C NMR (75.5MHz, CDCl3):
d 20.6
(COCH3), 28.9 (OCH2CH2CH2NH), 36.7 (OCH2CH2-
CH2NH), 38.3 (OCH2CH2NH), 52.1 (OCH3), 62.3
(OCH2CH2CH2NH), 65.9, 66.4, 66.5, 68.3, 69.0, 69.2,
71.0 (C-2, C-3, C-4, C-5, C-6, COCH2OCH2CO,
OCH2CH2NH), 97.5 (C-1), 114.7, 119.5, 122.3, 129.4
(CHarom), 131.4 (Cq aromCOOMe), 158.2 (Cq arom),
166.6 (COOMe), 168.6, 168.9, 169.6, 169.9, 170.0,
170.6 (COCH3, COCH2OCH2CO). HR-MS for
C31H42N2O16 (M, 698.2534), calcd [M+H+] 699.2612,
found 699.2673, [M+Na+] calcd 721.2432, found
721.2371.
4.6. Synthesis of general building block 6
Amine 5 (86mg, 0.21mmol) was dissolved in pyridine
(6mL)/dioxane (3mL) and glycolic anhydride (24.5mg,
0.21mmol) was added. After stirring overnight under
N2 at 90ꢁC (TLC, DCM–MeOH, 2:1) the solution was
concentrated and co-evaporated with toluene. The resi-
due was diluted with DCM, washed with 1N KHSO4
and brine, dried and concentrated, yielding 6 (70mg,
4.7.2. Divalent O-acetyl protected mannose-short spacer,
compound 11. Compound 11 was prepared according
to general procedure 4.7 from 10. 37.4mg, 52% yield.
1H NMR (300MHz, CDCl3): d 1.82–1.92 (m, 4H,
OCH2CH2CH2NH), 1.99–2.16 (4 · s, 24H, CH3), 3.40–
3.45 (m, 4H, OCH2CH2CH2NH), 3.50–3.55 (m, 2H,
1
64% yield). H NMR (300MHz, CDCl3): d 1.87–1.93
(m, 2H, OCH2CH2CH2NH), 2.00, 2.06, 2.11, 2.17

368
C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
OCHHCH2CH2NH), 3.72 (dd, 4H, J 5.5, 10.6Hz,
OCH2CH2NH), 3.77–3.82 (m, 2H, OCHHCH2-
CH2NH), 3.90 (s, 3H, OCH3), 3.92–3.99 (m, 2H, H-5),
4.06 and 4.10 (2 · s, 8H, COCH2OCH2CO), 4.11–4.15
(m, 2H, H-6b), 4.28 (dd, 2H, J 4.1, 12.2Hz, H-6a),
4.81 (d, 2H, J 0.9Hz, H-1), 5.24–5.31 (m, 6H, H-2, H-
3, H-4), 6.92 (s, 1H, CHarom), 7.18 (s, 2H, CHarom).
13C NMR (75.5MHz, CDCl3): d 20.7 (COCH3), 29.0
(OCH2CH2CH2NH), 36.9 (OCH2CH2CH2NH), 38.4
(OCH2CH2NH), 52.3 (OCH3), 62.3 (OCH2CH2-
CH2NH), 66.0, 66.6, 66.8, 68.4, 69.1, 69.3, 71.1 (C-2,
C-3, C-4, C-5, C-6, COCH2OCH2CO, OCH2CH2NH),
97.6 (C-1), 106.6, 108.1 (CHarom), 132.2 (Cq arom-
COOMe), 159.5 (Cq arom), 168.7, 169.1, 169.7, 170.1,
170.7 (COCH3, COCH2OCH2CO). HR-MS calcd for
C54H76N4O30: 1260.4544, found 1261.4323 [M+H+],
1283.4461 [M+Na+].
39% yield. 1H NMR (300MHz, CDCl3): d 1.61–1.82
(m, 6H, OCH2CH2CH2NH), 1.97, 2.03, 2.09, 2.14
(4 · s, 12H, COCH3), 3.14–3.77 (m, 22H, OCH2CH2-
CH2NH, OCH2CH2NH, OCH2CH2O), 3.88 (s, 3H,
OCH3), 3.90–4.14 (m, 13H, COCH2OCH2CO, OCH2-
CH2NH, H-5, H-6a, H-6b), 4.77 (d, 1H, J < 1Hz, H-
1), 5.21–5.31 (m, 3H, H-2, H-3, H-4), 7.34 (s, 1H,
CHarom), 7.27–7.32 (m, 3H, CHarom). 13C NMR
(75.5MHz, CDCl3): d 20.5 (COCH3), 28.5 (OCH2-
CH2CH2NH), 36.9 (OCH2CH2CH2NH), 38.5 (OCH2-
CH2NH), 52.1 (OCH3), 62.3 (OCH2CH2CH2NH),
65.9, 68.3, 69.2, 69.3, 69.8, 70.0 (C-2, C-3, C-4,
C-5, C-6, COCH2OCH2CO, CH2OCH2CH2O, OCH2-
CH2NH), 97.1 (C-1), 114.7, 119.5, 122.1, 129.4
(CHarom), 131.2, 158.4 (Cq arom), 166.8 (COOMe),
169.6, 169.8, 170.2, 170.3, 170.7 (COCH3, CO-
CH2OCH2CO). HR-MS for C45H68N4O22 (M,
1016.4325), [M+H+] calcd 1017.4404, found: 1017.4350.
4.7.3. Divalent O-acetyl protected mannose 1–3rigid
short spacer, compound 14. Compound 14 was pre-
pared according to general procedure 4.7 from 13.
138mg, 90% yield. 1H NMR (300MHz, CDCl3): d
1.81–1.92 (m, 4H, OCH2CH2CH2NH), 2.00, 2.05, 2.10,
2.15 (4 · s, 24H, COCH3), 3.34–3.51 (m, 6H,
OCHHCH2CH2NH), 3.74–3.79 (m, 2H, OCHHCH2-
CH2NH), 3.95–3.98 (m, 2H, H-5), 4.08–4.19 (m, 10H,
COCH2OCH2CO, H-6b), 4.23–4.36 (m, 6H, C„CH2,
H-6a), 4.80 (d, 2H, J < 1Hz, H-1), 5.23–5.32 (m, 6H,
H-2, H-3, H-4), 7.20–7.39 (m, 4H, CHarom). 13C NMR
(75.5MHz, CDCl3): d 20.5 (COCH3), 28.7, 29.1, 36.6
(CH2C„, OCH2CH2CH2NH, OCH2CH2CH2NH),
62.3 (OCH2CH2CH2NH), 65.8, 66.3, 68.2, 69.0, 69.1,
70.7 (C-2, C-3, C-4, C-5, C-6, COCH2OCH2CO), 81.8
(„CCH2), 85.3 („CCH), 97.4 (C-1), 122.5 (Cq arom),
131.3 (CHarom), 168.8, 168.9, 169.5, 170.1, 170.6
(COCH3, CONH, COCH2OCH2CO). HR-MS for
4.7.6. Divalent O-acetyl protected mannose-long spacer,
compound 23. Compound 23 was prepared according
to general procedure 4.7 from 22. 60mg, 65% yield. H
1
NMR (300MHz, CDCl3):
d 1.58–1.82 (m, 12H,
OCH2CH2CH2NH), 1.98, 2.04, 2.09, 2.15 (4 · s, 24H,
COCH3), 3.11 (dd, 12H, OCH2CH2CH2NH), 3.15–
3.87 (m, 32H, OCH2CH2CH2NH, OCH2CH2NH,
OCH2CH2O), 3.87 (s, 3H, COCH3), 3.91–4.22 (m,
26H, COCH2OCH2CO, OCH2CH2NH, H-5, H-6a, H-
6b), 4.79 (d, 2H, J < 1Hz, H-1), 5.18–5.36 (m, 3H, H-
2, H-3, H-4), 7.06–7.25 (2 · s, 3H, CHarom). HR-MS
for C82H128N8O42 (M, 1896.8126), calcd [MÀ2Ac+Na+]
1835.781, found 1835.813.
4.7.7. Tetravalent O-acetyl protected mannose-short
spacer, compound 26. Compound 26 was prepared
according to general procedure 4.7 from 25. 50.4mg,
66% yield. 1H NMR (300MHz, CDCl3): d 1.78–1.92
(m, 8H, OCH2CH2CH2NH), 1.99, 2.05, 2.10, 2.15
C54H70N4O26
1191.4356, found: 1191.2833.
(M,
1190.4278),
[M+H+]
calcd
(4 · s,
48H,
COCH3),
3.16–3.83
(m,
28H,
4.7.4. Divalent O-acetyl protected mannose 1–4 rigid
short spacer, compound 17. Compound 17 was pre-
pared according to general procedure 4.7 from 16.
84mg, 89% yield. 1H NMR (300MHz, CDCl3): d
1.81–1.93 (m, 4H, OCH2CH2CH2NH), 2.00, 2.05, 2.10,
2.15 (4 · s, 24H, COCH3), 3.40–3.55 (m, 6H,
OCHHCH2CH2NH), 3.74–3.81 (m, 2H, OCHHCH2-
CH2NH), 3.94–4.02 (m, 1H, H-5), 4.06–4.19 (m, 10H,
COCH2OCH2CO, H-6b), 4.23–4.34 (m, 6H, C„CH2,
H-6a), 4.80 (d, 2H, J < 1Hz, H-1), 5.24–5.33 (m, 6H,
H-2, H-3, H-4), 7.27–7.41 (m, 4H, CHarom). 13C NMR
(75.5MHz, CDCl3): d 20.5 (COCH3), 28.6, 29.1, 36.3
(CH2C„, OCH2CH2CH2NH, OCH2CH2CH2NH),
62.2 (OCH2CH2CH2NH), 65.7, 66.3, 68.2, 68.9, 69.1,
70.7 (C-2, C-3, C-4, C-5, C-6, COCH2OCH2CO), 82.1
(„CCH2), 86.3 („CCH), 97.3 (C-1), 122.2 (Cq arom),
131.3 (CHarom), 168.7, 168.9, 169.5, 170.1, 170.6
(COCH3, CONH, COCH2OCH2CO). HR-MS for
OCH2CH2CH2NH, OCH2CH2NH), 3.89 (s, 3H,
OCH3), 4.03 and 4.07 (2 · s, 16H, COCH2OCH2CO),
4.08–4.16 (m, 12H, OCH2CH2NH), 4.17–4.31 (m,
12H, H-5, H-6a, H-6b), 4.79 (d, 4H, J < 1Hz, H-1),
5.23–5.29 (m, 12H, H-2, H-3, H-4), 6.85–6.96 (m, 3H,
CHarom), 7.13–7.20 (m, 6H, CHarom). HR-MS for
C118H162N10O62 (M, 2712.6), [M+Na+] calcd average
2735.6, found 2735.3.
4.7.8. Octavalent O-acetyl protected mannose-short
spacer, compound 29. Compound 29 was prepared
according to general procedure 4.7 from 28. 26.8mg,
54% yield. 1H NMR (300MHz, CDCl3): d 1.77–1.97
(m, 16H, OCH2CH2CH2NH), 1.98, 2.04, 2.09, 2.14
(4 · s, 96H, COCH3), 3.16–4.32 (m, 147H, CO-
CH2OCH2CO, OCH2CH2CH2NH, OCH2CH2NH,
OCH3, H-5, H-6a, H-6b), 4.79 (d, 8H, J < 1Hz, H-1),
5.22–5.26 (m, 12H, H-2, H-3, H-4), 6.21–6.58 (m, 7H,
CHarom), 6.73–7.13 (m, 14H, CHarom). HR-MS for
C246H334N22O126 (M, 5612.0), [M+H]+ average calcd
5616.4, found 5619.4
C54H70N4O26
1191.4356, found: 1191.5167.
(M,
1190.4278),
[M+H+]
calcd
4.7.5. Monovalent O-acetyl protected mannose-long
spacer, compound 20. Compound 20 was prepared
according to general procedure 4.7 from 19. 44mg,
4.7.9. Octavalent O-acetyl protected PAMAM based
mannose-short spacer, compound 32. Compound 32

C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
369
was prepared according to general procedure 4.7 from
31. 17.6mg, 48% yield. H NMR (300MHz, MeOD): d
(s, 3H, COCH3), 3.84 and 3.91 (2 · s, 4H, CO-
CH2OCH2CO), 3.93–4.03 (m, 4H, OCH2CH2NH),
4.59 (d, 2H, J 2.0Hz, H-1), 6.58 (s, 1H, CHarom), 7.35
(s, 2H, CHarom). HR-MS calcd for C38H60N4O22 (M,
924.3699) found: [M+H+] calcd 925.3777, found
925.3823, [M+Na+] calcd 947.3597, found 947.3153.
1
1.87–1.94 (m, 16H, OCH2CH2CH2NH), 1.95, 2.04,
2.06, 2.13 (4 · s, 96H, COCH3), 2.34–2.42 (m, 24H,
NCH2CH2NHCO), 2.46–2.64 (m, 12H, NCH2CH2N,
CONHCH2CH2N), 2.72–2.90 (m, 24H, NCH2CH2-
NHCO), 3.20–3.42 (m, 64H, OCHHCH2CH2NH,
CONHCH2CH2N, NHCH2CH2NH), 3.47–3.58 (m,
8H, OCHHCH2CH2NH), 3.77–3.84 (m, 8H, H-5),
3.97–4.17 (m, 40H, COCH2OCH2CO, H-6a), 4.25 (dd,
8H, J 4.8, 12.4Hz, H-6b), 4.83 (8H, H-1), 5.19–5.23
(m, 24H, H-2, H-3, H-4). MALDI-MS calcd for
C230H360N34O116: 5457.5, found 5456.8.
4.8.3. Divalent mannose 1–3 rigid-short spacer, compound
15. Compound 15 was prepared according to general
1
procedure 4.8 from 14. 43.9mg, 73% yield. H NMR
(300MHz, D2O): d 1.58–1.65 (m, 4H, OCH2CH2-
CH2NH), 3.12–3.22 (m, 4H, OCH2CH2CH2NH), 3.25–
3.78 (m, 16H, OCH2CH2CH2NH, H-2, H-3, H-4, H-5,
H-6a, H-6b), 3.97 and 4.05 (2 · s, 4H, COCH2OCH2-
CO), 4.11 (s, 4H, CCH2), 4.63 (d, 2H, H-1), 7.13–7.19
(m, 3H, CHarom), 7.29 (s, 1H, CHarom). HR-MS for
C38H54N4O18 (M, 854.3433), [M+H+] calcd 855.3511,
found: 855.6205, [M+Na+] calcd 877.3331, found
877.5468.
4.7.10. Hexadecavalent O-acetyl protected PAMAM
based mannose-short spacer, compound 35. Compound
35 was prepared according to general procedure
1
4.7 from 32. 27.4mg, 43% yield. H NMR (300MHz,
MeOD): d 1.87–1.94 (m, 32H, OCH2CH2CH2NH),
1.96, 2.05, 2.07, 2.15 (4 · s, 96H, COCH3), 2.34–2.42
(m, 56H, NCH2CH2NHCO), 2.48–2.63 (m, 28H,
NCH2CH2N, CONHCH2CH2N), 2.76–2.91 (m, 56H,
NCH2CH2NHCO), 3.20–3.42 (m, 156H, OCHHCH2-
CH2NH, CONHCH2CH2N, NHCH2CH2NH), 3.49–
3.62 (m, 16H, OCHHCH2CH2NH), 3.76–3.87 (m,
16H, H-5), 3.98–4.19 (m, 60H, OCH2CH2CH2NH, H-
6a), 4.21–4.27 (m, 16H, H-6b), 4.83 (16H, H-1), 5.21–
5.38 (m, 48H, H-2, H-3, H-4).
4.8.4. Divalent mannose 1–4 rigid-short spacer, compound
18. Compound 18 was prepared according to general
1
procedure 4.8 from 17. 76.1mg, 77% yield. H NMR
(300MHz, D2O): d 1.58–1.67 (m, 4H, OCH2CH2-
CH2NH), 3.13–3.22 (m, 4H, OCH2CH2CH2NH), 3.24–
3.77 (m, 16H, OCH2CH2CH2NH, H-2, H-3, H-4, H-5,
H-6a, H-6b), 3.95 and 4.07 (2 · s, 4H, COCH2OCH2-
CO), 4.12 (s, 2H, CCH2), 4.63 (d, 2H, H-1), 7.32–7.45
(m, 4H, CHarom). HR-MS for C38H54N4O18 (M,
854.3433), [M+H+] calcd 855.3511, found: 855.4216,
[M+Na+] calcd 877.3331, found 877.2683.
4.8. General procedure for preparation of unprotected
glycodendrimers
The protected glycodendrimers were dissolved in dry
MeOH and sodium methoxide (30% w/w, 10· diluted)
was added. TLC analysis (MeOH–CH2Cl2, 1:9) was
used to determine if the reaction was completed. If prod-
uct precipitated from the solution, a few drops of water
were added and stirring was continued for 1h. The reac-
tion mixture was neutralized with Dowex H+ and con-
centrated in vacuo. Products were purified using a
Seppack column with water/ACN as eluents and
lyophilized.
4.8.5. Monovalent mannose-long spacer, compound
21. Compound 21 was prepared according to general
procedure 4.8 from 20. 12mg, 31% yield. 1H NMR
(300MHz, D2O): d 1.68–1.81 (m, 6H, OCH2CH2-
CH2NH), 3.20–3.31 (m, 6H, OCH2CH2CH2NH), 3.45–
3.52 (m, 6H, OCH2CH2CH2NH), 3.57–3.80 (m, 16H,
H-2, H-3, H-4, H-5, H-6a, H-6b, OCH2CH2O,
OCH2CH2NH), 3.86 (s, 3H, COCH3), 3.98 4.04 (2 · s,
8H, COCH2OCH2CO), 4.14–4.18 (t, 2H, OCH2-
CH2NH), 4.63 (d, 1H, H-1), 7.14–7.18 (m, 1H, CHarom),
7.37 (t, 1H, CHarom), 7.45 (s, 1H, CHarom), 7.53–7.57 (m,
1H CHarom). MS for C37H60N4O18 (M, 848.390),
[M+H+] calcd 849.398, found: 849.375.
4.8.1. Monovalent mannose-short spacer, compound
9. Compound 9 was prepared according to general
procedure 4.8 from 8. 41.1mg, 55% yield. 1H NMR
(300MHz, D2O): d 1.58–1.62 (m, 2H, OCH2CH2-
CH2NH), 3.03–3.76 (m, 12H, OCH2CH2CH2NH,
OCH2CH2NH, H-2, H-3, H-4, H-5, H-6a, H-6b), 3.73
(s, 3H, COCH3), 3.83 and 3.89 (2 · s, 4H, CO-
CH2OCH2CO), 3.95–4.11 (m, 2H, OCH2CH2NH),
4.60 (1H, H-1), 6.96 (d, 1H, CHarom), 7.16–7.23 (m,
2H, CHarom), 7.34 (d, 1H, CHarom). HR-MS for
C23H34N2O12 (M, 530.2113), [M+H+] calcd 531.2190,
found: 531.2032, [M+Na+] calcd 553.2010, found
553.2054.
4.8.6. Divalent mannose-long spacer, compound
24. Compound 24 was prepared according to general
procedure 4.8 from 23. 32mg, 71% yield. 1H NMR
(300MHz, D2O): d 1.67–1.78 (m, 12H, OCH2CH2-
CH2NH), 3.19–3.25 (m, 12H, OCH2CH2CH2NH),
3.37–3.81 (m, 44H, OCH2CH2CH2NH, H-2, H-3, H-4,
H-5, H-6a, H-6b, OCH2CH2O, OCH2CH2NH), 3.81
(s, 3H, COCH3), 3.98 4.04 (2 · s, 16H, COCH2OCH2-
CO), 4.14–4.18 (t, 4H, OCH2CH2NH), 4.69 (d, 2H, H-
1), 6.68 (s, 1H, CHarom), 7.05 (s, 3H, CHarom). HR-
MS for C66H112N8O34 (M, 1560.728), calcd [M+H+]
1561.736, found: 1561.831, [M+Na+] calcd 1583.718,
found 1583.551.
4.8.2. Divalent mannose-short spacer, compound
12. Compound 12 was prepared according to general
1
procedure 4.8 from 11. 14.1mg, 51% yield. H NMR
(300MHz, D2O): d 1.58–1.62 (m, 4H, OCH2CH2-
CH2NH), 3.08–3.77 (m, 21H, OCH2CH2CH2NH,
OCH2CH2NH, H-2, H-3, H-4, H-5, H-6a, H-6b), 3.71
4.8.7. Tetravalent mannose-short spacer, compound
27. Compound 27 was prepared according to general
1
procedure 4.8 from 26. 37.8mg, 98% yield. H NMR

370
C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
(300MHz, D2O): d 1.53–1.63 (m, 8H, OCH2CH2-
CH2NH), 3.79–4.11 (m, 31H, COCH2OCH2CO,
OCH2CH2NH, COCH3), 4.58 (4H, H-1), 6.59 (s, 3H,
CHarom), 7.31 (s, 6H, CHarom). HR-MS for
C86H130N10O46 (M, 2038.8), [M+Na+] average 2063.0,
calcd found: 2063.0.
C-6), 65.7, 65.9, 68.9, 69.2 (C-2, C-3, C-4, C-5), 97.5
(C-1), 126.5 (C@CH2), 130.6 (C@CH), 165.7, 169.6,
170.0 (CO, COCH3). MS for C20H29NO11 (M,
459.17), [M+Na+] calcd 482.43, found 482.35.
Compound 37 was deacetylated as according to
general procedure 4.8, yielding monomer 38 in quantita-
1
4.8.8. Octavalent mannose-short spacer, compound
30. Compound 30 was prepared according to general
tive yield. H NMR (300MHz, D2O): d 1.61–1.71 (m,
2H, OCH2CH2CH2NH), 3.29 (m, 2H, OCH2-
CH2CH2NH), 3.52–3.92 (m, 8H), 4.79 (d, 1H, H-1),
5.73 (m, 1H, @CH2), 6.18 (m, 2H, HC@CH2). 13C
NMR (75.5MHz, D2O/MeOD): d 28.1 (OCH2CH2),
36.6 (CH2NH), 61.0, 65.2 (OCH2, C-6), 66.8, 70.1,
70.6, 72.8 (C-2, C-3, C-4, C-5), 99.9 (C-1), 127.2
(CH@CH2), 130.0 (CH@CH2), 168.6 (C@O). MS for
C12H21NO7 (M, 291.13), [M+Na+] calcd 314.12, found
314.25.
1
procedure 4.8 from 29. 19.3mg, 87% yield. H NMR
(300MHz, D2O): d 1.51–1.71 (m, 16H, OCH2CH2-
CH2NH), 3.79–4.11 (m, 31H, COCH2OCH2CO,
OCH2CH2NH, COCH3), 6.31 (s, 7H, CHarom), 6.60
(s, 14H, CHarom). HR-MS for C184H272N22O95 (M,
4309.7), [M+H]2+ average calcd 2157.7, found 2158.3.
4.8.9. Octavalent PAMAM based mannose-short spacer,
compound 33. Compound 33 was prepared according
to general procedure 4.8 from 32. 11.7mg, 51% yield.
1H NMR (300MHz, MeOD): d 1.52–1.67 (m, 16H,
OCH2CH2CH2NH), 2.10–2.21 (m, 24H, NCH2CH2-
NHCO), 2.34–2.44 (m, 12H, NCH2CH2N, CON-
HCH2CH2N), 2.52–2.63 (m, 24H, NCH2CH2NHCO),
3.02–3.22 (m, 64H, OCHHCH2CH2NH, CONH-
CH2CH2N, NHCH2CH2NH), 3.19–3.67 (m, 56H,
OCHHCH2CH2NH, H-2, H-3, H-4, H-5, H-6a, H-6b),
3.85 (s, 32H, COCH2OCH2CO). MALDI-MS for
C166H296N34O84 (M, 4110.0), found: 4114.0.
4.10. General procedure for preparation of glycopolymers
Monomer 38 and N-(2-hydroxypropyl) methacrylamide
monomer (HPMA) were dissolved in water in various
ratios. After degassing of the solution by addition of
N2 for 30min N,N,N0,N0-tetramethylethylenediamine
(TMEDA, 0.3equiv) and ammonium persulfate (APS,
0.05equiv) were added after which N2 was bubbled
through the solution for another 10min. After stirring
overnight at rt, the solution was dialyzed (MWCO
3500) against water for 2days and lyophilized to afford
white fluffy powder in 40–65% yield. The co-monomer
ratio in the copolymer was determined from the ratio
of the integral from the CH3CH (d = 1.00) from HPMA
to the integral from the H-1 of the mannose monomer
(d = 4.88), being consistent with the input. GPC was
used to determine the molecular weight (6760 < Mw <
11,160) and polydispersity (1.3 < Mw/Mn < 1.8). For
calibration of the column polystyrene standards of
known molecular weights and narrow molecular weight
distribution were used.
4.8.10. Hexadecavalent PAMAM based mannose-short
spacer, compound 36. Compound 36 was prepared
according to general procedure 4.8 from 35. 18.3mg,
1
55% yield. H NMR (300MHz, MeOD): d 1.51–1.65
(m, 32H, OCH2CH2CH2NH), 2.08–2.19 (m, 56H,
NCH2CH2NHCO), 2.32–2.41 (m, 28H, NCH2CH2N,
CONHCH2CH2N), 2.50–2.61 (m, 56H, NCH2CH2-
NHCO), 2.97–3.17 (m, 156H, OCHHCH2CH2NH,
CONHCH2CH2N, NHCH2CH2NH), 3.20–3.63 (m,
112H, OCHHCH2CH2NH, H-2, H-3, H-4, H-5, H-6a,
H-6b), 3.84 (s, 64H, COCH2OCH2CO).
4.11. Bacterial strains, growth conditions and plasmids
4.9. Synthesis of mannose monomer 38
The E. coli K-12 strain HB 101 used in this study
was transformed with the plasmid pPKL4 (pBR322
cloning vector) carrying the complete fim gene
cluster cloned from the uropathogenic E. coli strain.38
Recombinant bacteria expressing type 1 fimbriae were
cultivated with shaking at 37ꢁC overnight in Luria–
Bertani (LB) broth supplemented with the appropriate
antibiotic (i.e., 100lg of ampicillin per mL). The anti-
biotics were purchased from Sigma (Deisenhofen,
Germany).
To amine 5 (4.90g, 12.1mmol) in dioxane (120mL) was
added NaHCO3 (1.32g, 15.7mmol) in water (120mL).
This mixture was cooled to 0ꢁC and acryloyl chloride
(1.48mL, 18.2mmol) was added slowly, after which
the mixture was allowed to warm up. After 2h, the mix-
ture was diluted with EtOAc and the organic layer
washed with water, dried and concentrated. Com-
pound 37 was purified by column chromatography
(0–5% MeOH–DCM). 422mg, 76% yield. 1H NMR
(300MHz, CDCl3): d 1.85–1.98 (m, 2H, OCH2CH2-
CH2NH), 2.01, 2.06, 2.11, 2.17 (4 · s, 12H, COCH3),
3.43–3.58 (m, 3H, OCHHCH2CH2NH), 3.78–3.85 (m,
1H, OCHHCH2CH2NH), 3.97–4.04 (m, 1H-, H-5),
4.11 (dd, 1H, J 2.5, 12.1Hz, H-6b), 4.29 (dd, 1H, J
5.2, 12.1Hz, H-6a), 4.82 (d, 1H, J < 1Hz, H-1), 5.25–
5.30 (m, 3H, H-2, H-3, H-4), 5.66 (dd, 1H, J 1.5,
9.9Hz, CH@CHH), 6.13–6.33 (m, 2H, CH@CHH),
6.66 (br s, 1H, NH). 13C NMR (75.5MHz, CDCl3): d
20.6 (COCH3), 28.8 (OCH2CH2CH2NH), 37.1
(OCH2CH2CH2NH), 62.5, 66.5 (OCH2CH2CH2NH,
4.12. Cell lines, media and culture conditions
The cell line (T24) derived from human bladder was pur-
chased from the American Type Culture Collection
(Manassas, Va.). Cell line was grown in medium
(McCoyÕs 5A medium supplemented with 2mM gluta-
mine, nonessential amino acids, and 10% fetal calf ser-
um) without antibiotic at 37ꢁC in a 5% CO2–95% air
atmosphere with 90% humidity and were split twice a
week at a ratio of 1:5. Cell culture media was purchased

C. C. M. Appeldoorn et al. / Tetrahedron: Asymmetry 16 (2005) 361–372
371
from C.C. Pro (Neustadt, Germany) and supplements
were from Gibco (Gaithersburg, Md.).
Acknowledgements
This study has been carried out with financial support
from the Commission of the European Communities,
specific RTD program ÔQuality of Life and Management
of Living ResourcesÕ, QLKT-CT-2002-01852, ÔPOLY-
CARBÕ. The Royal Netherlands Academy of Arts and
Sciences (KNAW) is gratefully acknowledged for a fel-
lowship of R.J.P. We thank Dr. R. van Nostrum and
M. van Steenbergen (Department of Biopharmaceutics,
Utrecht University) for assistance with the preparation
and characterization of the polymers and to M. Schad
(Institut fur Molekulare Infektionsbiologie, University
of Wuerzburg, Germany) for his help in maintaining cell
line and ELISA assays. Dr. J. Kemmink (Department of
Medicinal Chemistry, Utrecht University) is acknowl-
edged for recording 500 NMR-spectra and R.D. van
Ooijen (Department of Biomolecular Mass Spectrome-
try, Utrecht University) for recording high-resolution
MS-spectra.
4.13. Adherence assay
To quantify the bacterial adhesion a cell line (T24) of
epithelial-like morphology and of human origin was
used as a model to eukaryotic cells. After harvesting
cells from confluent monolayers, epithelial cells were
seeded in 96-well flat-bottom cell culture plates (Gre-
iner) in 200lL aliquots for 24–30h. The culture medium
was removed and after washing monolayers once with
sterile phosphate-buffered saline (PBS) they were fixed
by addition of 100lL of glutaraldehyde (1.25% in
PBS) at room temperature for 30min. After washing
three times with PBS, monolayers were blocked
with 3% BSA–PBS (pH7.4) for 2h at 37ꢁC. A serial
dilution of type 1 fimbriated recombinant E. coli
HB101(pPKL4),
nonfimbriated
control
strain
HB101(pBR322) was first tested to determine the num-
bers of bacteria required to obtain 50% binding to this
cell line. The number of fimbriated and nonfimbriated
control strains was determined by measuring absor-
bance at a wavelength of 550nm, with a standardized
chart correlating absorbance with viable counts.39 After
removal of blocking solution and washing, 100lL of
serially diluted bacterial suspensions (4 · 108 bacteria
in the first well) in PBS with 1% BSA was added to each
well, and plates were incubated for 2h at 37ꢁC. To
determine the binding of bacteria to the monolayers,
wells were washed as mentioned above and incubated
with rabbit anti-E. coli polyclonal antibody (Biodesign
International, Kennebunk, Maine) in PBS–1% BSA
(1:1700) for 1.5h at 37ꢁC (100lL/well). Following an-
other washing step, peroxidase-conjugated (1:1700) goat
anti-rabbit immunoglobulin G (Dako, Hamburg, Ger-
many) was added and incubated for 1h at 37ꢁC
(100lL/well). Finally, the wells were washed as above
and the bound enzyme was detected by the addition of
100lL of substrate (Pierce ImmunoPure TMB substrate
Kit) in each well for 5–30min. To stop the reaction
100lL of 2 M H2SO4 was added to each well and the
absorbance was measured at a wavelength of 450nm
with an enzyme-linked immunosorbant assay (ELISA)
reader. The control wells were treated in the same man-
ner except that blank control wells have no bacteria.
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