Evaluation of the carbohydrate recognition domain of the bacterial adhesin FimH: Design, synthesis and binding properties of mannoside ligands

Article abstract of DOI:10.1039/b610745a

Fimbriae are proteinogeneous appendages on the surface of bacteria, which mediate bacterial adhesion to the host cell glycocalyx. The so-called type 1 fimbriae exhibit specificity for α-d-mannosides and, therefore, they are assumed to mediate bacterial adhesion via the interaction of a fimbrial lectin and α-d-mannosyl residues exposed on the host cell surface. This carbohydrate-specific adhesive protein subunit of type 1 fimbriae has been identified as a protein called FimH. The crystal structure of this lectin is known and, based on this information, the molecular details of the interaction of mannoside ligands and FimH are addressed in this paper. Computer-based docking methods were used to evaluate known ligands as well as to design new ones. Then, a series of new mannosides with extended aglycon was synthesized and tested as inhibitors of type 1 fimbriae-mediated bacterial adhesion in an ELISA. The results obtained were compared to the predictions and findings as delivered by molecular modeling. This study led to an improved understanding of the ligand-receptor interactions under investigation. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b610745a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Evaluation of the carbohydrate recognition domain of the bacterial adhesin
FimH: design, synthesis and binding properties of mannoside ligands
Oliver Sperling,† Andreas Fuchs† and Thisbe K. Lindhorst*†
Received 26th July 2006, Accepted 5th September 2006
First published as an Advance Article on the web 25th September 2006
DOI: 10.1039/b610745a
Fimbriae are proteinogeneous appendages on the surface of bacteria, which mediate bacterial adhesion
to the host cell glycocalyx. The so-called type 1 fimbriae exhibit specificity for a-D-mannosides and,
therefore, they are assumed to mediate bacterial adhesion via the interaction of a fimbrial lectin and
a-D-mannosyl residues exposed on the host cell surface. This carbohydrate-specific adhesive protein
subunit of type 1 fimbriae has been identified as a protein called FimH. The crystal structure of this
lectin is known and, based on this information, the molecular details of the interaction of mannoside
ligands and FimH are addressed in this paper. Computer-based docking methods were used to evaluate
known ligands as well as to design new ones. Then, a series of new mannosides with extended aglycon
was synthesized and tested as inhibitors of type 1 fimbriae-mediated bacterial adhesion in an
ELISA. The results obtained were compared to the predictions and findings as delivered by molecular
modeling. This study led to an improved understanding of the ligand–receptor interactions under
investigation.
carbohydrate-based antiadhesives, which might eventually be used
Introduction
as therapeutics against bacterial colonization and infection.7
Bacterial colonization of organs and cells respectively can cause
When type 1 fimbriae-mediated binding to various mannosides
severe problems for an organism. When Escherichia coli bacteria,
which are commensal residents of the intestine, enter the urogenital
and mannose-containing ligands is tested in an ELISA (enzyme-
linked immunosorbent assay), weak interactions are observed with
tract and multiply in the bladder, for example, the body reacts
IC50 values in the millimolar and low micromolar range. Attempts
with inflammation, such as in the case of cystitis.1 Colonization
of the stomach by Helicobacter pylori causes inflammation in the
stomach (gastritis) as well as ulceration of the stomach or peptic
ulcer disease.2
to improve mannose ligands as inhibitors of type 1 fimbriae-
mediated bacterial adhesion followed two different approaches: (i)
multivalent carbohydrate ligands have been designed (cf. preceding
paper8) to utilize known multivalency effects,9 and (ii) target design
of ligands has been employed to optimize the interactions of
a carbohydrate ligand to the carbohydrate recognition domain
(CRD) of the bacterial adhesin.10
In order to understand the molecular circumstances of such
processes, it is reasonable to begin with the investigation of the
molecular interactions between bacteria and their host cells. These
involve adhesion of bacteria to the glycocalyx, a complex nano-
dimensioned layer of diverse glycoconjugates, which covers every
eukaryotic cell. Bacteria enable adhesion to the glycocalyx by
adhesive organells called fimbriae or pili.3 Fimbriae are classified
according to their carbohydrate specificity. The so-called type 1
fimbriae express a specificity for terminal a-mannosyl residues,
which is mediated by a lectin called FimH forming a minor
component of the fimbrial protein complex.4 According to many
studies, type 1 fimbriae are an important and critical factor for
bacterial virulence, such as for uropathogenic E. coli.5
Rational design of carbohydrate ligands for FimH can been
guided by information obtained from the crystal structure of the
bacterial adhesin. The first X-ray structure of FimH with a ligand
bound to its carbohydrate recognition domain (CRD), in complex
with the chaperone FimC, was published in 1999,11 and was
followed by two other X-ray studies, which appeared in 200212 and
2005, respectively.13 We have employed this structural information
on FimH for our study on the design of carbohydrate ligands for
FimH, which is reported here, together with their synthesis and
biological testing.
Since more than three decades, researchers have investigated
various mannosides and oligosaccharides as inhibitors for type
1 fimbriae-mediated bacterial adhesion.6 The purpose of this
research has been twofold: (i) this work has been carried out to
learn more about the molecular details of bacterial adhesion to
the host cell glycocalyx and (ii) there has been a hope to develop
Results and discussion
Well-known ligands for FimH are depicted in Scheme 1. The
binding potency of methyl a-D-mannoside (1, MeMan) to
FimH lies in the millimolar range as reflected by inhibition
of hemagglutination or by ELISA, respectively.14 On the other
hand, a-D-mannosides carrying an aromatic aglycon such as p-
nitrophenyl a-D-mannoside (2, pNPMan) and methylumbelliferyl
a-D-mannoside (3, MeUmbMan), show a potency as inhibitors of
type 1 fimbriae-mediated bacterial adhesion, which is increased by
Otto Diels Institute of Organic Chemistry, Christiana Albertina University
of Kiel, Otto-Hahn-Platz 4, 24098, Kiel, Germany. E-mail: tklind@oc.uni-
kiel.de; Fax: (+49)431 880 7410
† These authors have contributed equally.
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the phenol moieties of TYR48 and TYR137, define a molecular
entrance to the CRD, which has been named the ‘tyrosine gate’.11,12
The aromatic aglycon of an appropriate mannoside ligand can
establish favorable p–p-interactions with this tyrosine gate, thus
leading to significantly improved affinities of mannosides such as
pNPMan (2) and MeUmbMan (3) in comparison to MeMan (1).
Based on this knowledge, we anticipated that mannosides with
an extended aromatic aglycon could satisfy further interactions
with the CRD exterior, thus improving binding to FimH. To allow
extension of the aglycon moiety of an a-D-mannoside according
to a general strategy, we have employed squaric acid diester
(diethylsquarate, DES)15 as a bifunctional linker,16 and have tested
it with 2-aminoethyl a-D-mannoside (5, Scheme 2) first. Reaction
of the amine 517 with DES in methanol gave literature-known 618
in a smooth reaction, and eventually 9 could be obtained starting
from p-aminophenyl a-D-mannoside (7)19 in analogy.
When tested in an ELISA, the potency of mannoside 6 as an
inhibitor of type 1 fimbriae-mediated bacterial adhesion was poor
as expected, whereas the inhibitory potency of the squaric acid
monoester 9 was increased 1800-fold when compared to methyl
a-D-mannoside (MeMan, 1) and some 60 times higher than that
of p-nitrophenyl a-D-mannoside (pNPMan, 2) in the same assay
(Table 1). Therefore, we concluded that the extended aglycon
moiety of 9 can establish additional, favorable interactions at the
entrance of the FimH CRD.
Consequently, we have employed computer-aided modeling to
rationalize the measured inihibitory potencies of the various FimH
ligands. We have used FlexX20 flexible docking and consensus
scoring21 as implemented in Sybyl6.822 for docking of known
Scheme 1 Standard mannoside ligands for the bacterial lectin FimH.
two orders of magnitude when compared to MeMan.6 This can
be understood on the basis of the crystal structure of the adhesive
protein FimH. All three available X-ray analyses reveal a single
carbohydrate recognition domain (CRD) on the tip of the FimH
protein, with the size of a monosaccharide.
This CRD perfectly accommodates an a-D-mannosyl residue.
It is mainly comprised of amino acids with hydrophilic side
chains, including the N-terminal amino acid of the protein, PHE1
(Fig. 1a). These amino acid side chains and, in particular, the
central aspartic acid ASP54 support complexation of the six-
membered ring of a-D-mannosides within the CRD with the a-
glycosidic linkage pointing outwards of the binding site.
Table 1 FlexX scoring values as obtained by ligand docking are com-
pared to the potencies of the different mannosides as inhibitors of
the mannose-specific adhesion of E. coli to mannan as determined by
ELISA. Lower scoring values correspond to higher affinities to the protein
FimH. Relative inhibitory potencies (RIP) are relative to the IC50 value
measured for methyl a-D-mannoside (1); thus the inhibitory potency of 1
has been defined as RIP ≡ 1. The listed RIP is an average value of the
results of at least three independently performed ELISAs and therefore
reported with its standard deviation
Mannosides investigated
Scorea
RIPb
s.d.c
1 (MeMan)
−22.5
−24.9
−20.2
−20.5
−26.5
−25.4
−29.2
−26.9
−26.6
−20.7d
−22.0e
−24.3f
1
31
380
5.7
2.7
200
1800
6900
210
340
430
330
—
13
2 (pNPMan)
3 (MeUmbMan)
240
0.32
0.43
44
920
2300
100
150
210
46
4 (BuMan)
Fig. 1 Details of the carbohydrate recognition domain (CRD) of FimH as
revealed by X-ray analysis.12 Mannose is depicted in the binding pocket as
surface representation. a) The interior of the carbohydrate binding pocket
of FimH is mainly comprised of amino acids with hydrophilic side chains,
including PHE1, the N-terminal amino acid of the protein. Especially,
the central aspartic acid forms strong interactions with the complexed
mannose or mannoside, respectively. b) The exterior of the FimH CRD
is characterized by amino acids with hydrophobic side chains, defining
a so-called “hydrophobic ridge”. The amino acids TYR48 and TYR137
flank the entrance of the binding pocket forming a gate which has been
called the “tyrosine gate”.11,12
6
8
9
10
11
12
13
14
a If not otherwise indexed, values for top scorers are listed (if the respective
conformation is reasonable, i.e. the mannosyl aglycon of the ligand is
placed correctly inside the CRD). b RIP = relative inhibitory potency.
c s.d. = standard deviation. d The highest seeded score with a reasonable
conformation out of 90 docked solutions, which were considered for
scoring, was hit number 12. e The highest seeded score with a reasonable
conformation out of 90 docked solutions, which were considered for
scoring, was hit number 60. f The highest seeded score with a reasonable
conformation out of 90 docked solutions, which were considered for
scoring, was hit number 67.
The exterior of the CRD on the other hand is characterized
by amino acids with rather lipophilic side chains, defining a so-
called ‘hydrophobic ridge’ at the entrance of the CRD11,12 (Fig. 1b).
Two aromatic amino acid side chains of this protein environment,
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Scheme 2 Synthesis of the squaric acid ethylesters 6, 9, and 10. Reagents and conditions: a) DES, MeOH, 10 h, 71%; b) from 7: DES, DMF, 63%; c)
from 8: 1. Pd/C, H2, DMF. 2. DES, DMF, 55% over two steps.
and potential carbohydrate ligands into the CRD of FimH and
for scoring of the obtained ligand conformations. Docking was
based on the published X-ray structure with D-mannose bound
in the CRD.12 This FimH CRD was held fixed during the
minimization, whereas the sugar ligand was allowed to change
its conformation freely under the influence of the force field. The
ligand conformations delivered by FlexX as docking solutions
are regarded as “unrelaxed”. To release artificial strains, which
might arise during the docking process, such conformations can
be further proceeded in an energy minimization to deliver so-called
“relaxed” conformations, which might differ from the unrelaxed
solutions.23
Either unrelaxed or relaxed (or both) solutions of the docking
have to be submitted to a scoring process to identify the most
reasonable results. In this process, FlexX produces so-called
scoring values for each docked ligand, which can be regarded
Fig. 2 Fit of top scoring conformation of pNPMan (2) depicted in the
FimH CRD. The glycon moiety of the mannoside is buried in the rather
as a rough estimate of its free binding energy. Low (more negative
respectively) scoring values correlate with high affinities, higher
deep binding pocket, whereas the phenyl aglycon sticks out of this pocket
scores reflect diminished binding potency. To validate FlexX
interacting with the hydrophobic tyrosine gate at the entrance of the CRD.
scoring, in addition to the FlexX scoring function, a number of
other scoring functions based on different algorithms were used
for scoring of docking results and the scoring results obtained
were evaluated according to the consensus scoring strategy.24 This
procedure allows a most reliable ranking of the docked solutions.
In the case of MeMan (1), docking with FlexX reproduces
both conformation and orientation of the sugar ring in the
CRD of FimH as observed by X-ray analysis of this lectin–sugar
complex.12 No difference between the unrelaxed and respective
relaxed docking solutions was found in this case and all following
ones. The mannosyl glycon is buried in the binding pocket and the
a-configured methyl aglycon points outwards of the pocket. In the
case of 2 (pNPMan), the mannosyl residue maintains its perfect fit
within the CRD and an ideal geometry of the a-positioned aglycon
allows optimal interactions of the aromatic p-nitrophenyl aglycon
with the tyrosine gate of the CRD formed by TYR48 and TYR137
(Fig. 2). These lipophilic interactions of the p-nitrophenyl ring with
the tyrosine gate lead to higher affinity to the receptor and this is
reflected in a scoring value of −24.9 which is significantly lower
than that of MeMan (−22.5, Table 1). This finding corresponds to
the measured inhibitiory potency of pNPMan, which is 31-times
higher (RIP = 31) than that of MeMan (RIP = 1).
In the case of the new squaric acid derivative 9, docking revealed
additional interactions of the extended aglycon moiety with the
FimH CRD when compared to pNPMan (2). Orientation of the
phenyl ring in 9 is as in 2 however, the squaric acid sub-structure
with its planar geometry forms further interactions with the
tyrosine gate (Fig. 3a). This moiety is also able to interact with the
THR51 hydroxyl group on the distal end of the gate by hydrogen-
bridging, and this is reflected by a diminished scoring value of
−29.2 (Table 1). The affinity to FimH is reduced dramatically
when the phenyl ring in 9 is replaced by an ethyl spacer such as in
the case of 6 (score −26.5, RIP 2.7, Table 1). Visual inspection of
the docking results for 6 reveals that the ethyl spacer is too short to
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attach various peptide side chains in the p-position of the aromatic
phenyl ring of 9 by using the squaric acid linkage chemistry.
A first ligation of DES with the amine 7 led to 9 and then a
second ligation step with an amino acid or peptide derivative,
respectively, was performed in basic media exploiting the capacity
of squaric acid amide monoesters to react with a second amine
only under basic conditions.15 This procedure yielded the amino
acid glycosides 11, 12, 13 and 14, respectively (Scheme 3) in
yields between 40 and 60%. Triglycine, tetraglycine, pentaglycine
and tyrosine methylester were used in the last ligation step. The
somewhat moderate yields are due to the reduced nucleophilicity
of the N-terminal amino functions of the employed peptides.
Docking of glycopeptides 11 to 13 reveals a complex situation.
These glycopeptide derivatives with elongated peptide aglycon
moieties carrying three, four and five glycin residues, respectively,
result in top-ranking conformations, with excellent scoring values
between −45 and −47, however the mannosyl glycon is not placed
within the binding pocket anymore. In all three cases, top-ranking
conformations refer to the molecule wound around the protein
to maximize the ligand–receptor contact area. The increased
lipophilic contact surface results in a very low scoring value
however, the underlying docking solutions cannot be considered
as reasonable, based on the initial assumption that ligand binding
of FimH depends on complexation of an a-D-mannosyl residue
within the CRD.
This finding is typical for docking of larger ligands, as rather
large molecules tend to give higher scores. In addition, the number
of rotable bonds reaches a value which is limiting for the FlexX
algorithm, thus leading to unreliable results.23 The situation in the
case of the glycopeptide mimetic 14 is similar, except that, owing to
the aromatic side chain in the aglycon moiety, none of the docked
conformations can be oriented such that the molecule receives a
convenient fit with regard to the protein surface of FimH.
To allow at least the comparison of the docking results obtained
for 11, 12, 13 and 14 with those obtained with 1–4 and 6–10, only
those conformations out of 90 docking solutions were considered,
which have the mannosyl aglycon positioned properly inside the
CRD. From this collection of solutions, the highest ranking hit
was included in Table 1. After all, the scores obtained for ligands
11–14 cannot be expected to correlate with the affinities deduced
from ELISA.
Considering the results obtained for the ligands 1, 2, 3, 4, 6, and
8–10, it can be seen from Table 1 that evaluation of ligand quality
by either docking and scoring of the docking results or by ELISA
reflected corresponding trends in many, but not all cases. In the
case of the methylumbelliferyl mannoside 3, docking places this
ligand at the last position of the ranking list of the above mentioned
eight mannosides, whereas biological testing reflects the third-best
inhibitory potency for 3 among the same eight ligands.
The rather weak scoring value of −20.2 obtained for 3 is the
result of FlexX scoring based on the X-ray structure of FimH in
complex with mannose.12 When the most recent crystal structure
for FimH, in which the lectin is complexed with butyl a-D-
mannoside (4),13 was used as the basis for docking, a different
assessment of the binding energy of 3 resulted, delivering a
score of −29.0, which correlates much better with its measured
inhibitory potency. Searching for the critical difference between
the two different X-ray structures of FimH, it can be seen that
they differ only in the conformation of the tyrosine gate at the
Fig. 3 Fit of top scoring conformations of designed mannoside ligands 9
and 10 in the CRD of FimH. a) Top scoring conformation of the squaric
acid ethylester 9. In addition to the phenyl ring, the squaric acid part also
interacts with the tyrosine gate, thus increasing the contact area between
protein and ligand. In addition, 9 interacts with the hydroxyl group of the
distal THR51 by hydrogen-bridging. These interactions sum up, resulting
in a higher affinity as reflected by lower IC50 values on the one hand and
lower scoring values on the other. Circled in yellow, a depression in the
hydrophobic ridge can be seen, which is not satisfied by ligand 9. b) Top
scoring conformation of the o-chloro-substituted squaric acid ethylester 10
depicted in the CRD of FimH as a CPK model. The chloro substituent in
the o-position of the phenyl ring fits into a depression of the hydrophobic
ridge, thus further increasing the contact area between protein and ligand,
as the favorable interaction of the aglycon moiety of 10 remains the same
as in the case of its analog 9.
allow the squaric acid moiety to properly interact with the tyrosine
gate and also H-bonding with the tyrosine OH-group is no more
possible (Figure not shown).
Upon validation of the fit of mannoside 9 within the CRD of
FimH, a lack of contact between protein and ligand in a depressed
area of the hydrophobic ridge (encircled in yellow in Fig. 3a)
became obvious. Thus, we have anticipated that the binding affinity
of 9 might be further improved by introduction of a hydrophobic
substituent in the o-position of the phenyl ring. Therefore, we
started the synthesis of o-chlorophenyl mannoside 10 employing
literature-known mannoside 8 (Scheme 2).25 Reduction of the
p-nitro group in 8 and subsequent coupling with DES could
be carried out in a one-pot procedure in good yield without
isolation of the intermediate amine. Testing results obtained with
mannoside 10 supported our initial considerations. Its inhibitory
potency was increased up to 6900-fold as compared to MeMan
(1) and almost four-fold when compared to 9 (Table 1).
Interestingly, docking of 10 and scoring of the obtained ligand
conformations delivered a relatively weak scoring value of −26.9,
thus 10 scores worse than 9 (score −29.2) though it performs better
in the ELISA. On the other hand, all top-ranking conformations
of the o-chloro-substituted mannoside 10 reflect an increased
lipophilic contact area between the ligand and the CRD (Fig. 3b).
Thus, in the case of ligand 10 the “in silico” findings are only
partly in agreement with the results obtained by biological testing.
It should be kept in mind that the potent ligand 10 was discovered
by rational concluding from the docking solutions for mannoside
9 rather than by unreflected (“blind”) docking and scoring.
Then we set out to improve the binding affinity of ligands by
further increasing the lipophilic contact surface between the ligand
aglycon and the entrance of the binding pocket. We decided to
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Scheme 3 Synthesis of the squaric acid-linked glycopeptides. Reagents and conditions: for 11 a) triglycine, DIPEA, MeOH, 10 h, acidic ion exchange
resin, 41%; for 12 b) tetraglycine, DIPEA, MeOH, 10 h, acidic ion exchange resin, 49%; for 13 c) pentaglycine, DIPEA, MeOH, 10 h, acidic ion exchange
resin, 50%; for 14 d) L-tyrosine methylester, DIPEA, MeOH, 10 h, 62%.
entrance of the binding pocket (Fig. 4). In the case of the FimH
crystal with a-mannose in the binding pocket, the conformation
of this tyrosine gate can be considered as “open”, whereas FimH
in complex with butyl a-D-mannoside has crystallized with the
tyrosine gate “closed”. This is due to a change in the torsion angle
in the TYR48 substructure CO–Ca–Cb–Caryl from 70.1◦ (“open”)
to 201.3◦ (“closed”).
To estimate the impact of this difference on scoring, all docking
studies were performed once again using the “closed” gate lectin
structure as the receptor. In Table 2 the new ranking results are
collected and compared to the older ones from Table 1, which
were obtained with the “open” gate FimH structure. In Table 3
a ranking list for the investigated compounds is provided which
was deduced from the results from three different approaches.
Table 2 Comparison of the scoring values for eight selected ligands as obtained from docking based on two different crystal structures
Ligand
Score mannosea “open” gate
Score BuMan (4)b “closed” gate
RIP based on MeMan
1 (MeMan)
−22.5
−24.9
−20.2
−20.5
−26.5
−25.4
−29.2
−26.9
−23.3
−27.4
−29.0
−21.5
−23.3
−27.9
−33.2
−32.8
1
31
2 (pNPMan)
3 (MeUmbMan)
380
5.7
2.7
200
1800
6900
4 (BuMan)
6
8
9
10
a Based on crystal structure of FimH complexed with a-mannose.12 b Based on crystal structure of FimH complexed with n-butyl a-mannoside.13
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Table 3 Ranking of compounds collected in Table 2 according to scoring
and ELISA; ranking leads from weak to better inhibitors. Significant
variations of the ranking resulting from different evaluation approaches
concern especially compounds 3 and 6
BuManFimH structure (Fig. 4b), the entire aromatic surface of
the aglycon moiety of mannoside 3 can interact with the CRD
entrance, while maintaining an optimal fit of the mannosyl glycon
within the CRD. On the other hand, less favorable interactions
are established between the same ligand and the “open-gate”
structure (Fig. 4a). This increase in contact area changing from
the “open” to the “closed” gate receptor conformation is even
more pronounced in the case of the squaric acid derivatives 9 and
10, resulting in drastically enhanced, that is lower, scoring values.
Similar considerations may explain the differing values for 6.
The docking and scoring results for the n-butyl mannoside-
based crystal structure are in good agreement with the experi-
mental ranking for the compounds with a more rigid aglycon.
Inadequate ranking occurs again for compounds 11–14 having a
very flexible aglycon. In the scoring process, the entropic penalty,
which has to be paid upon binding of this type of highly flexible
ligands outweighs the energetic gain, which is achieved through
an increased contact surface between ligand and receptor.
It seems that most of the investigated ligands can interact more
efficiently with the “closed” gate structure of FimH. However, the
tyrosine gate is a very flexible region of the protein not enclosed
or hindered by other protein parts. For the n-butyl mannoside 4
both protein conformations, “open” and “closed”, can be found.26
This might indicate a protein–ligand interaction of an “induced
fit”-type, which depends on the type of bound ligand, forcing the
protein to adopt a certain conformation.
Conclusions
We have combined our know-how in molecular modeling, carbo-
hydrate synthesis and glycobiology to shed light on the molecular
details of carbohydrate binding as mediated by bacterial type
1 fimbriae. We employed the structural information about the
protein FimH, the adhesive sub-structure of type 1 fimbriae,
which is available from, so far, three X-ray studies. These X-ray
studies are consistent in that they reveal a single carbohydrate-
binding site at the tip of FimH, which can accommodate an
a-D-mannosyl residue, with the aglycon of an a-D-mannoside
sticking out of the CRD. On the basis of this information
about the adhesin FimH, multivalency effects as measured in
various inhibition studies cannot be understood or rationalized
(cf. preceding paper8). However, the differing potencies of a-D-
mannosides with varying aglycon portions as inhibitors of type
1 fimbriae-mediated bacterial adhesion can be rationalized by
comparison of the results obtained by computer-aided docking
on one hand and ELISA on the other. In some of the investigated
cases, especially in the case of 3, the two sets of information
(obtained by docking based on the ManFimH structure12 and
ELISA, respectively) were not congruent and this prompted us to
compare the available crystal structrues of FimH. While the two X-
ray studies published first11,12 showed no significant difference with
regard to the protein structure, the most recent X-ray analysis13
revealed a different conformation of the two tyrosine residues at
the periphery of the CRD, resembling a “closed” gate rather than
an “open” one. Depending on which structure was employed as
the basis for ligand docking, scoring of the docking results for a
series of investigated ligands led to different ranking lists. This “in
silico” result, which has been discussed in this paper, also points
at the biological circumstances of ligand binding to FimH. It
Fig. 4 Comparison of two different X-ray structures available for FimH.
a) The crystal structure of FimH complexed with a-mannose is depicted
(with the tyrosine gate “open”).12 This structure allows only a suboptimal
interaction of the aromatic aglycon moiety of mannoside 3 (MeUmbMan)
with the tyrosine residues at the entrance of the CRD. Consequently FlexX
docking delivers a relatively high (less negative) scoring value (−20.2),
and this weak score is in disagreement with the relatively high inhibitory
potency of 3 as measured by ELISA. b) The crystal structure obtained of
FimH complexed with butyl a-mannoside (4) is depicted (with the tyrosine
gate “closed”).13 When this structure was used for ligand docking, the
aglycon of 3 can be favorably aligned with the surface area of the protein
CRD, leading to a more negative score of −29.0. This low score correlates
with the experimental affinity of 3 as measured in the ELISA.
It can be seen that the scores obtained based on the n-butyl
mannoside-complexed FimH structure are in fine accordance with
the ranking as measured by ELISA, other than in case of our first
study based on the X-ray analysis with FimH in complex with
mannose. The discrepancy concerns especially methylumbelliferyl
a-D-mannoside (3) and the squaric-acid modified mannoside 6.
This can be reasoned by differences regarding the exposed
lipophilic surface at the CRD entrance. In the case of the
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has to be considered that the flexibility of the protein receptor
in solution and its CRD, respectively, can significantly influence
ligand binding, and this understanding may have important
implications for the biology of carbohydrate-dependent bacterial
adhesion, which awaits further investigation. Even the finding
that fimbriae-mediated bacterial adhesion is often flow-regulated27
might be reconsidered with regard to lectin flexibility.
A second conclusion which can be drawn from this study with
regard to ligand design is that a strategy counting on additional
interactions mediated by the aglycon moiety of a-D-mannoside
ligands to improve receptor binding has its limitations. Once the
aglycon gets too large it may establish enough interactions in
the periphery of the FimH CRD so that the mannosyl glycon
gets disconnected from the binding pocket, resulting in an overall
diminished affinity. In addition, in the case of mannosides with a
highly flexible aglycon, the entropic penalty which has to be paid
for the complexation with the receptor protein FimH compensates
the enthalpic gain. Such an understanding of ligand binding to
FimH will also be of importance once binding of multivalent
carbohydrate ligands is under investigation.
from Merck. A recombinant type 1 fimbriated E. coli strain, E. coli
HB101 (pPKl4),28 was used and cultured as described earlier.29
Buffers. PBS (phosphate-buffered saline) was prepared by
dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4·2H2O and 0.2 g
KH2PO4 in 1000 ml of bidest. water (pH 7.2). PBSE was PBS
buffer + 100 mg l−1 thimerosal, PBSET was PBSE buffer + 200 ll
l−1 Tween 20. Substrate buffer was 0.1 M sodium citrate dihydrate,
adjusted to pH 4.5 with citric acid. For preparation of the ABTS
solution, ABTS (1 mg) was dissolved in substrate buffer (1 ml)
and 0.1% H2O2 (25 ll per ml) was added.
Docking
For investigation of the binding mode of various FimH ligands,
high resolution crystal structures of FimH as available in the
PDB database (FimH–FimC complex with CHEGA: 1QUN,30
FimH–FimC complex with mannose: 1KLF,31 FimH with n-butyl
a-D-mannoside: 1UWF32) were used. Docking was accomplished
applying the automated flexible docking tool FlexX 1.10.020
along with Sybyl6.822 as interface. The receptor was defined as
˚
It will be the aim of our future work to gain a better insight
into the processes of bacterial adhesion, based on the results of
this contribution and of the preceding paper8; and we will direct
our attention to the mechanistic and biological implications of our
research.
a sphere with a diameter of 10 A around the mannose-binding
pocket (specifying the carboxyl carbon atom of ASP54 as the
centre), to assure that all possible interactions are considered.
The CRD was described by the following amino acids: PHE1
ALA2 ILE13 GLY14 HIS45 ASN46 ASP47 TYR48 PRO49
GLU50 THR51 ILE52 ASP54 TYR55 ARG98 GLN133 THR134
ASN135 ASN136 TYR137 ASN138 SER139 ASP140 ASP141
PHE142 PHE144.
Experimental
General remarks
The crystal structures were used without further modification
(no minimization and side chain relaxation). Docking was per-
formed applying the FlexX standard parameters considering 30
docked solutions for scoring, unless otherwise stated. Formal
charges were used for the docking routine. To produce a more
robust evaluation of ligand–receptor interactions, a consensus of
scoring functions was calculated.21,33 The following scoring func-
tions available to the CScore module in Sybyl6.8 were considered:
FlexX original score, ChemScore,34 DockScore,35 GoldScore,36
PMFScore.37 The obtained solutions were relaxed and scored
using the above mentioned scoring functions. The FlexX original
scoring function was not available for re-scoring. Relaxation was
performed using the Tripos FF and standard parameters available
in Sybyl6.8. Rmsd values for the sugar moieties were calculated
comparing the positions of the atoms of the mannose ring (except
hydrogen) of a particular binding mode with the positions of
the respective mannose ring taken from the experimental crystal
structure using an spl-script.
All chemicals and solvents were used without further purification,
with the exception of methanol which was distilled prior to
use. Optical rotations were measured with a Perkin Elmer 241
polarimeter (10 cm cells, sodium D line: 589 nm). NMR spectra
were recorded at 300, 500 or 600 MHz on Bruker ARX 300
1
(300 MHz for H, 75.47 for 13C), Bruker DRX 500 (500 MHz
1
1
for H, 125.75 for 13C) and Bruker ARX 600 (600 MHz for H,
150.92 MHz for 13C) instruments with Me4Si (d = 0) as internal
standard. Flash column chromatography was performed on silica
gel 60 (230–400 mesh, Merck). Purifications on RP-Gel were
carried out with a Bu¨chi MPLC apparatus using Merck Licroprep
RP18 coloums. MALDI-TOF MS spectra were measured on
a Bruker Biflex apparatus at 19 kV Voltage with a sinapinic
acid matrix (saturated sinapinic acid in 9 : 1 water–acetronitrile
containing 0.1% trifluoroacetic acid). Optical densities (ODs) were
measured on an Asys DigiScan 400 ELISA reader at 405 nm with
the reference read to 492 nm. ELISA plates were incubated at
37 ◦C.
Visualization of results. The protein is presented as Connolly
surface representation38 (solvent accessible surface) and colored
according to its lipophilic potential.39 Brown colors represent
lipophilic areas, hydrophilic parts are shown in blue colors.
Molecules are presented as ball and stick models (carbon = white,
oxygen = red, nitrogen = blue, hydrogen = cyan, chlorine = green).
Reagents. Methyl a-D-mannoside (1) was purchased from
Fluka, p-nitrophenyl a-D-mannoside (2) from Senn chemicals,
methylumbelliferyl a-D-mannoside (3) and n-butyl a-D-mannoside
(4) were prepared according to the literature.13 F-shaped 96-well
microtiter plates from Sarstedt. Mannan from Saccharomyces
cerevisiae was purchased from Sigma and was used in 50 mM
aq. Na2CO3 (1 mg ml−1, pH 9.6). The peroxidase-conjugated goat
anti-rabbit antibody (IgG, H + L) was purchased from Dianova.
Skimmed milk was from Ulzena, Tween 20 from Roth, ABTS [2,2ꢀ-
azidobis-(3-ethylbenzothiazoline-6-sulfonic acid)] from Fluka and
thimerosal (2-(ethylmercuriothio) benzoic acid sodium salt) was
ELISA
To determine the potencies of the various mannoside derivatives
tested as inhibitors of type 1 fimbriae-mediated adhesion of E. coli,
an ELISA was used as published earlier.29 Polystyrene microtiter
plates were coated with mannan solution (100 ll per well) and
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3913–3922 | 3919
©

View Article Online
◦
3
3
dried overnight at 37 C. The plates were blocked once with 5%
d, aryl-H), 5.39 (1H, d, J1,2 = 1.7, H-1), 4.76 (2H, q, J = 7.0,
skimmed milk in PBSE for 30 min at 37 ◦C. The wells were washed
with PBSE (150 ll) and then PBSE (50 ll) and inhibitor solutions
(50 ll) were added. Inhibitor solutions were diluted serially two-
fold in PBSE. Bacterial suspension (50 ll per well) was added and
OCH2CH3) 3.88 (1H, dd, J2,3 = 3.2, H-2), 3.71 (1H, dd, J3,4
=
3
3
9.1, H-3), 3.40–3.63 (4H, m, H-5, H-6a, H-6b, H-4), 1.41 (3H,
t, OCH2CH3) ppm; dC (50.32 MHz, 313 K, D6-DMSO) 186.60,
183.41 (C O squaric acid, very small peaks), 178.20, 169.31
(C C squaric acid), 148.13 (man-O-Caryl), 133.53 (p-Caryl), 122.93
(m-Caryl), 121.16 (m-Caryl), 119.45 (o-Caryl-Cl), 118.27 (o-Caryl),
99.85 (C-1), 75.21 (C-5), 70.58 (C-3), 69.90 (C-2), 69.39 (CH2),
66.63 (C-4), 61.01 (C-6), 15.47 (CH3) ppm; MALDI-ToF MS:
m/z = 452.0 [M + Na]+ (calcd. 452.1) for C18H20ClNO9 (M =
429.08), HRMS (ESI), found: 452.0799 [M + Na]+.
=
◦
=
the plate was left at 37 C for 1 h to allow sedimentation of the
bacteria. Then each well was washed four times with PBSE (150 ll)
and 50 ll 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 ll). The
plates were incubated for 30 min and then washed three times with
PBSET and once with PBSE and substrate buffer. ABTS solution
(50 ll) was added, incubated for 60 min at 37 ◦C. For ELISA
controls, bacterial adhesion to blocked, uncoated microtiter plates
was checked, and the reaction of the employed antibodies with
yeast mannan was tested and found to be negligible. The low
background was substracted when calculating the IC50 values. The
percentage inhibition was calculated as OD(nI) − OD(I) × 100 ×
[OD(nI)]−1 (nI: no inhibitor, I: with inhibitor).
p-[N-(4-Triglycyl-2,3-dioxocyclobut-1-enyl)amino]phenyl a-D-
mannopyranoside 11. The squaric acid monoester 9 (100 mg,
0.25 mmol) and triglycine (50 mg, 0.26 mmol) were dissolved in
MeOH (10 ml) and DIPEA (100 ll) was added. The reaction
mixture was stirred overnight at rt and subsequently acidified
with Amberlite IR-120 ion exchange resin. The solvent was
removed in vacuo and the residue purified by MPLC on RP-18
(MeOH–H2O, 1 : 3) to furnish the title compound as a white
lyophilisate. Yield: 55 mg (0.10 mmol, 41%); [a]2D0 +66.7 (c 0.15
in DMSO); dH (500 MHz, D6-DMSO, D2O-exchange) 7.36 (2H,
IC50 values are average values from at least three independent
assays and are listed together with their standard deviations.
Relative inhibitory potencies (RIPs) are based on the IC50 value of
methyl a-D-mannopyranoside (MeMan), with RIP (MeMan) ≡ 1.
3
3
d, J = 9.0, aryl-H), 7.08 (2H, d, aryl-H), 5.29 (1H, d, J1,2
=
1.8, H-1), 4.37 (2H, d, 3J = 4.9, NH-CH2COOH), 3.73–3.83
3
(5H, m, H-2, 2 × NH-CH2-CONH), 3.67 (1H, dd, J2,3 = 3.3,
p-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl
a-D-
3J3,4 = 8.8, H-3), 3.62 (1H, dd, J5,6a = 1.7, J6a,6b = 11.3 Hz,
3
2
mannopyranoside 9. Mannoside 719 (800 mg, 2.95 mmol) was
dissolved in MeOH (10 ml), diethyl squarate (500 ll, 3.38 mmol)
was added and the reaction mixture was stirred at rt for 1 d.
Then, the solvent was removed in vacuo and the residue was
purified by two subsequent chromatographic steps, first by
flash chromatography on silica gel (MeOH–ethyl acetate, 1 : 1)
followed by chromatography on RP-18 (MeOH–H2O, 1 : 1) to
furnish the title mannoside as white lyophilisate. Yield: 1.12 g
(2.84 mmol, 63%); [a]D20 +82.3 (c 1.0, DMSO). dH (500 MHz,
D6-DMSO, D2O-exchange) 7.27 (2H, bs, aryl-H), 7.07 (2H, d,
3J = 9.0, aryl-H), 5.31 (1H, d, 3J1,2 = 1.9, H-1), 4.73 (2H, q, 3J =
H-6a), 3.41–3.50 (3H, m, H-4, H-5, H-6b) ppm; dC (125.76 MHz,
=
D6-DMSO) 183.56, 180.67 (C O squaric acid), 171.07, 171.02,
=
=
168.95 (C O), 163.84 (2 × C C squaric acid), 152.28 (i-Caryl),
133.54 (p-Caryl), 119.35 (2 × o-Caryl), 117.93 (2 × m-Caryl), 99.45
(C-1), 74.92, 70.68, 70.12, 66.76 (C-2, C-3, C-4, C-5), 61.09 (C-6),
45.93, 41.84, 40.56, 3 × Gly-CH2) ppm; MALDI-ToF MS: m/z =
561.4 [M + Na]+ (calcd. 561.1) for C22H26N4O12 (M = 538.15),
HRMS (ESI), found: 561.1424 [M + Na]+, C22H26N4O12 requires
561.1439 [M + Na]+.
p-[N-(4-Tetraglycyl-2,3-dioxocyclobut-1-enyl)amino]phenyl a-D-
mannopyranoside 12. The squaric acid monoester 9 (150 mg,
0.38 mmol) and tetraglycine (50 mg, 0.36 mmol) were dissolved in
a 1 : 1 mixture of MeOH and H2O (10 ml) and DIPEA (100 ll)
was added. The reaction mixture was stirred overnight at rt and
subsequently acidified with Amberlite IR-120 ion exchange resin.
The solvent was removed in vacuo and the residue purified by
MPLC onRP-18(MeOH–H2O, 1 : 3) to furnish the title compound
as a white lyophilisate. Yield: 104 mg (0.23 mmol, 49%); [a]2D0 +71.6
(c 0.25 in DMSO); dH (600 MHz, D6-DMSO, D2O-exchange) 7.35
3
7.1, OCH2CH3), 3.80 (1H, dd, J2,3 = 3.4, H-2), 3.65 (1H, dd,
3J3,4 = 9.0, H-3), 3.58 (1H, dd, J = 1.7, J6a,6b = 11.7, H-6a),
3
2
3.57–3.41 (3H, m, H-5, H-6b, H-4), 1.39 (3H, t, OCH2CH3) ppm;
=
dC (50.32 MHz, 313 K, D6-DMSO) 187.96, 183.26 (C O squaric
acid), 177.73, 169.32 (C C squaric acid), 153.22 (man-O-Caryl),
=
132.28 (p-Caryl), 121.05 (2 × m-Caryl), 117.39 (2 × o-Caryl), 99.31
(C-1), 74.82 (C-5), 70.67 (C-3), 70.01 (C-2), 69.30 (CH2), 66.83
(C-4), 61.10 (C-6), 15.51 (CH3) ppm; MS (MALDI-TOF): m/z:
418.5 [M + Na]+ (calcd. 418.1) for C18H21NO9 (MW 395.12),
HRMS (ESI), found: 418.1109 [M + Na]+, C18H21NO9 requires
418.1107 [M + Na]+.
(2H, d, 3J = 8.9, aryl-H), 7.07 (2H, d, aryl-H), 5.29 (1H, m, 3J1,2
=
1.5, H-1), 4.37 (2H, s NCH2COOH), 3.72–3.83 (7H, m, H-2, 3 ×
NH–CH2-CONH), 3.66 (1H, dd, 3J2,3 = 3.4, 3J3,4 = 9.1, H-3), 3.59
o-Chloro-p-[N-(2-ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl
a-D-mannopyranoside 10. Mannoside 8 (290 mg, 0.86 mmol)
was dissolved in MeOH (10 ml) and stirred for 1 h with Pd/C-
catalyst (100 mg) under a hydrogen atmosphere (1 bar) at rt. The
suspension was filtered and diethyl squarate (300 ll, 2.03 mmol)
was added. The reaction mixture was stirred overnight at rt, then
the solvent was removed in vacuo and the residue was purified
by MPLC on RP-18 (MeOH–H2O, 1 : 3) to obtain the title
compound as a white lyophilisate. Yield: 200 mg (0.47 mmol,
55%); [a]2D0 +50.0 (c 0.15 in DMSO); dH (500 MHz, D6-DMSO)
3
2
(1H, dd, J5,6a = 1.5, J6a,6b = 11.5, H-6a), 3.39–3.50 (3H, m, H-
4, H-5, H-6b) ppm; dC (150.92 MHz, D6-DMSO) 183.59, 180.77
=
=
(C O squaric acid), 171.22, 169.19, 168.98, 168.61 (4 × C O),
=
163.86 (2 × C C squaric acid), 152.33 (i-Caryl), 133.59 (p-Caryl),
119.41 (2 × o-Caryl), 117.98 (2 × m-Caryl), 99.46 (C-1), 74.99, 70.71,
70.17, 66.76 (C-2, C-3, C-4, C-5), 61.10 (C-6), 46.00, 42.11, 41.75,
40.61, 4 × Gly-CH2) ppm; MALDI-ToF MS: m/z = 617.8 [M +
Na]+ (calcd. 618.2) for C24H29N5O13 (M = 595.2), HRMS (ESI),
found: 618.1684 [M + Na]+, C24H29N5O13 requires 618.1654 [M +
Na]+.
3
7.49 (1H, bs, aryl-H), 7.33 (1H, d, Jar. = 9.0, aryl-H), 7.24 (1H,
3920 | Org. Biomol. Chem., 2006, 4, 3913–3922
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
p-[N-(4-Pentaglycyl-2,3-dioxocyclobut-1-enyl)amino]phenyl a-
D-mannopyranoside 13. The squaric acid monoester 9 (100 mg,
0.25 mmol) and pentaglycine (74 mg, 0.24 mmol) were dissolved
in a 1 : 2 mixture of MeOH and H2O (10 ml). DIPEA (100 ll)
was added. The reaction mixture was stirred overnight at rt and
subsequently acidified with Amberlite IR-120 ion exchange resin.
The solvent was removed in vacuo and the residue purified by
MPLC onRP-18 (MeOH–H2O, 1 : 3) to furnish the title compound
as a white lyophilisate. Yield: 78 mg (0.12 mmol, 50%); [a]2D0 +73.3
(c 0.25 in DMSO); dH (600 MHz, D6-DMSO, D2O-exchange) 7.35
2 (a) B. J. Marshall and R. M. Warren, Lancet, 1984, 16, 1311–1315;
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2, 161–167.
3
(2H, d, JHar. = 8.9, aryl-H), 7.07 (2H, d, aryl-H), 5.28 (1H, m,
3J1,2 = 1.6, H-1), 4.36 (2H, s NCH2COOH), 3.72–3.82 (9H, m, H-
2, 4 × NH–CH2-CONH), 3.65 (1H, dd, 3J2,3 = 3.4, 3J3,4 = 9.1, H-3),
3.59 (1H, dd, 3J5,6a = 1.6, 2J6a,6b = 11.5, H-6a), 3.39–3.52 (3H, m,
H-4, H-5, H-6b) ppm; dC (150.92 MHz, D6-DMSO) 183.60, 180.77
=
(C O squaric acid), 171.21, 169.19, 169.11, 169.08, 168.63 (5 ×
=
=
C O), 163.86 (2 × C C squaric acid), 152.33 (i-Caryl), 133.58 (p-
Caryl), 119.39 (2 × o-Caryl), 117.97 (2 × m-Caryl), 99.45 (C-1), 74.98,
70.69, 70.16, 66.75 (C-2, C-3, C-4, C-5), 61.10 (C-6), 45.99, 42.33,
42.03, 41.75, 40.61, 5 × Gly-CH2) ppm; MALDI-ToF MS: m/z =
674.6 [M + Na]+ (calcd. 675.2) for C26H32N6O14 (M = 652.2),
HRMS (ESI), found: 675.1860 [M + Na]+, C26H32N6O14 requires
675.1869 [M + Na]+.
8 M. Dubber, O. Sperling and T. K. Lindhorst, Org. Biomol. Chem., 2006,
DOI: 10.1039/b610741a.
9 (a) C. R. Bertozzi and L. L. Kiessling, Science, 2001, 291, 2357–
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Glycoconjugate J., 2000, 17, 543–551; (d) T. K. Lindhorst, Top. Curr.
Chem., 2002, 218, 201–235; (e) L. L. Kiessling, J. E. Gestwicki and
L. E. Strong, Angew. Chem., Int. Ed., 2006, 45, 2348–2368; (f) see also
ref. 8.
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F. Almqvist, J. Kihlberg and A. Linusson, J. Med. Chem., 2005, 48,
935–945.
11 D. Choudhury, A. Thompson, V. Stojanoff, S. Langerman, J.
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87–93.
p-[N-(4-L-Methyltyrosyl-2,3-dioxocyclobut-1-enyl)amino]phenyl
a-D-mannopyranoside 14. The squaric acid monoester 9 (20 mg,
50 lmol) and L-tyrosine methylester (10 mg, 51 lmol) were
dissolved in a 4 : 1 mixture of MeOH and H2O (1 ml). DIPEA
(10 ll) was added and the reaction mixture was stirred at rt
overnight. Then, the solvent was removed in vacuo and the residue
purifed by MPLC on RP-18 (MeOH–H2O, 1 : 4) to give the
title compound as a white lyophilisate. Yield: 17 mg (31 lmol,
62%); [a]2D0 +41.5 (c 0.20 in DMSO); dH (500 MHz, D6-DMSO)
3
7.32 (2H, d, J = 8.7, aryl-H), 7.06 (2H, d, aryl-H), 6.96 (2H,
3
d, J = 8.5, aryl-H, Tyr), 6.67 (2H, d, aryl-H, Tyr), 5.29 (1H, d,
3
3J1,2 = 1.7, H-1), 5.02 (1H, m, CHCH2), 3.82 (1H, dd, J2,3
=
3
3.3, H-2), 3.71 (3H, s, COOCH3), 3.67 (1H, dd, J3,4 = 9.0,
3
2
H-3), 3.62 (1H, dd, J5,6a = 1.6, J6a,6b = 11.4, H-6a), 3.39–3.51
(3H, m, H-4, H-5, H-6b), 3.08 (1H, m, CHCH2), 2.99 (1H,
m, CHCH2) ppm; dC (125.76 MHz, D6-DMSO) 183.08, 181.23
=
=
=
(C O squaric acid), 171.29 (C O), 168.04, 164.09, (C C squaric
acid), 156.34 (CAr-OH), 152.36 (Man-O-Caryl), 133.40 (p-CarylTyr),
130.39 (p-CArpAP, 125.76 (m-CarylTyr), 119.51 (m-CArpAP), 117.91
(o-CarylTyr), 115.26 (o-CArpAP), 99.44 (C-1), 74.97 (C-5), 70.68 (C-3),
70.15 (C-2), 66.73 (C-4), 61.08 (C-6), 57.39 (CH), 52.46 (CH3),
38.49 (CH2) ppm; MALDI-ToF MS: m/z = 567.4 [M + Na]+
(calcd. 567.2) for C26H28N2O11 (M = 544.2), HRMS (ESI), found:
567.1597 [M + Na]+, C26H28N2O11 requires 567.1585 [M + Na]+.
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Acknowledgements
This work was supported by the DFG (Deutsche Forschungsge-
meinschaft) in the frame of SFB 470.
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