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MICROREVIEW

DOI: 10.1002/ejoc.201100407

The Bacterial Lectin FimH, a Target for Drug Discovery – Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Mirja Hartmann* [a] and Thisbe K. Lindhorst* [a]

Keywords: Lectins / Cell adhesion / Carbohydrates / Cluster effect / Inhibitors

Adhesion is a prerequisite for bacteria to colonize cell sur- faces. To accomplish cellular adhesion, many bacteria use carbohydrate-specific lectins, which are expressed as part of capillary protein appendages expanding from their surface, called fimbriae or pili. For bacteria, colonization of cell sur- faces offers advantageous conditions to persist and multiply. For the host, however, bacterial colonization can be affiliated with severe health problems such as inflammation. There- fore, to combat bacterial adhesion and inflammatory dis- eases, investigation of the molecular and biophysical details of the relevant lectin–carbohydrate interactions is important. Understanding molecular carbohydrate recognition can lead

to the development of high-affinity inhibitors of bacterial lec- tins. That way, interfering with the bacterial attachment to surfaces proves the vision of an antiadhesion therapy, among others, against uropathogenic E. coli (UPEC). One of the most important and best investigated bacterial lectins is the man- nose-specific protein FimH, which is expressed on the tips of type 1 fimbriae. During the last 30 years, many natural as well as synthetic mannosidic ligands of FimH have been de- signed and tested for their inhibitory potencies. We report key results and comment on key problems and perspectives of this research.

Contents

1. Introduction

2. Early studies on the carbohydrate specificity of type 1 fimbriae

3. Multivalent glycomimetics as inhibitors of type 1 fimbriae-medi-

ated bacterial adhesion

4. Assays to test antagonists of type 1 fimbriae-mediated bacterial

adhesion

[a] Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel Otto-Hahn-Platz 3/4, 24098 Kiel, Germany Fax: +49-431-880-7410 E-mail: mhartmann@oc.uni-kiel.de tklind@oc.uni-kiel.de

5. Structure of the type 1 fimbrial lectin FimH

6. Rational design of carbohydrate ligands for the type 1 fimbrial

lectin FimH

7. Special approaches to the inhibition of mannose-specific bacte-

rial adhesion

1. Introduction

There is a class of proteins that reversibly bind carbo- hydrates. These proteins are neither carbohydrate-specific enzymes nor antibodies. Today, the understanding of glyco- scientists is that this type of proteins has evolved to recog- nize specific carbohydrates and select them for binding to

Mirja Hartmann, M.Sc., was born in 1983. She studied Molecular Life Science at the University of Erlangen-Nuremberg with main focus on Drug Discovery, Molecular Biology and Molecular Synthesis. For her master thesis, she developed strategies for the synthesis of glycoconjugates and their biological testing with Prof. Mikael Elofsson at Umeå University in Sweden. In 2007 she received her Master of Science degree from the University of Erlangen-Nuremberg and joined the research group of Prof. Thisbe Lindhorst at Christiana Albertina University of Kiel for her Ph.D. studies. Currently, she is working on biochemical assays and biophysical setups to test type 1 fimbriae-mediated bacterial adhesion.nize specific carbohydrates and select them for binding to Thisbe Lindhorst is Full Professor at the

Thisbe Lindhorst is Full Professor at the Faculty of Mathematics and Natural Science of Christiana Albertina University of Kiel since 2000. She studied chemistry at the Universities of Munich and Münster, received her diploma in chemistry/ biochemistry in 1988 and her Ph.D. in Organic Chemistry in 1991 at the University of Hamburg in the group of Prof. J. Thiem. After a postdoctoral stay at the University of British Columbia with Prof. S. Withers, she worked on her habilita- tion and became Private Docent in 1998 at the University of Hamburg. In 1997 she was a Visiting Professor at the University of Ottawa in Canada in Prof. R. Roy’s laboratory. Since 2000, she holds a chair in Organic and Biological Chemistry in Kiel. Her scientific interests are in the field of synthetic organic chemistry and in biological chemistry, and in particular in glycochemistry and glycobiology. Current research is focussed on the study of cell adhesion to glycosylated surfaces. She is the author of the textbook “Essentials of Carbohydrate Chemistry and Biochemistry”.setups to test type 1 fimbriae-mediated bacterial adhesion. Eur. J. Org. Chem. 2011 , 3583–3609 ©

MICROREVIEW

M. Hartmann, T. K. Lindhorst

form carbohydrate–protein complexes. While, apparently, carbohydrate–protein complexation has no obvious conse- quence for the structural integrity of the complexed and subsequently released saccharide ligand, such molecular re- cognition of carbohydrates can trigger biological response of different kinds in all types of organisms, such as cell re- cognition, signalling and cell adhesion.

Lectins

The carbohydrate-complexing proteins called “lectins” comprise a remarkable variety of structures, folds, functions and occurrence. The history of lectins goes back to the end of the 19th century. [1] At that time, work with extracts from seeds of the castor tree ( Ricinus communis) led to the dis- covery that certain plant proteins have the ability to aggluti- nate erythrocytes. The agglutinating protein isolated from Ricinus communis was called “ricin”. Rapidly, many more proteins with the same feature were discovered in various plants and were called phytoagglutinins, phytohemaggluti- nins and somewhat later hemagglutinins. Soon they were employed in immunological studies, leading to the finding that specific hemagglutinins react specifically with human red blood cells of certain blood groups (A, B, or O). [2] In 1954, the term “lectin” was proposed by Boyd and Shapleigh “for these and other antibody-like substances” with blood-group-specific agglutination properties. [3] The authors quoted the name to be derived from “the Latin lego, to choose or pick out”; (in fact the Latin verb legere means to gather, choose, collect, select, pass through, read , and its perfect passive participle is lectus, meaning selected and picked out, or read out.) In the 1970s Sharon and Lis [4] suggested the term lectin as a general name for all proteins of nonimmune origin that possess the ability to agglutinate erythrocytes and other cell types. It was discovered very early that the interactions of lec- tins with cells can be inhibited by specific carbohydrates, mono- or oligosaccharides. Consequently, it was concluded that lectins are specific saccharide-binding proteins. This was first shown for the well-known plant lectin concanava- lin A. [5] As a consequence, the first attempt to classify the fast-growing class of lectins was based on their carbo- hydrate specificity. Today, lectins are being classified on the basis of their structural features and especially the re- latedness of their carbohydrate-binding sites, which are called “carbohydrate recognition domains”, in short CRDs. [6] It was Drickamer who pointed out the importance of sequence homologies in the carbohydrate recognition do- main motifs of proteins for their carbohydrate-binding properties. [7,8] The carbohydrate specificity of lectin CRDs has remained a key criterion for the assessment of lectins so far. [9]

Fimbrial Lectins of Bacteria

Early in the history of lectins, it had become evident that, besides numerous plant lectins, most, if not all, organisms express this class of proteins, for very different purposes,

however. Already during the first half of the 20th century, it was discovered that also many bacteria, in particular those of the Enterobacteriaceae family, have the ability to agglutinate erythrocytes. This hemagglutination activity of bacteria is almost always associated with the presence of multiple filamentous protein appendages projecting from their surface, called fimbriae (from the Latin word for “thread”) or pili (from the Latin word for “hair”) (Fig- ure 1). Fimbriae are adhesive organelles, comprising lectin subunits, which mediate carbohydrate-specific adhesion to cell surfaces as well as cell agglutination. Thus, bacteria uti- lize the sugar decoration of cells, the glycocalyx, to colonize the cell surface, wherever cells are in contact with the out- side environment, as for example in the case of epithelial cells.

environment, as for example in the case of epithelial cells. Figure 1. Transmission electron micrograph of

Figure 1. Transmission electron micrograph of a type 1 fimbriated E. coli cell. Some hundreds of the adhesive organelles cover the bacterial surface.

Bacterial Adhesion and Diseases

Initial bacterial adhesion to cell surfaces is in turn ampli- fied, leading to the development of a well-organized super- structure, called a biofilm. Biofilm formation is highly ad- vantageous for the colonizing microbes. It facilitates firm and irreversible adhesion to a surface, interlinking bacteria of different species, which produce a carbohydrate mucus to maintain the biofilm. [10] Through this exopolysaccharide layer, [11] bacteria can achieve chemical communication and profit from favourable coordination (quorum sensing). Biofilm formation can form the basis of a beneficial sym- biosis between a microorganism and its host such as in the gut, where Escherichia coli (E. coli) bacteria produce vita- min K in the large intestine. However, as soon as microor- ganisms invade another habitat or only slightly change their genes, disorders such as inflammation, or even apoptosis or uncontrolled cell growth can arise. [12] Typically, bacterial colonization is accompanied by infectious diseases, and this constitutes a major global health problem. [13] In addition, biofilm formation on medical devices and implants fre- quently causes complications [14] and significantly contrib- utes to the pathogenesis of implant-related infections. Among the most prevalent inflammatory diseases that are caused by pathogens are urinary tract infections. The predominant pathogens in this case are uropathogenic E.

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Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion coli (UPEC). UPEC can attach to specific niches in

coli (UPEC). UPEC can attach to specific niches in the uri- nary tract by virtue of the interaction of their surface fim- briae with carbohydrate receptors on the luminal surface of the bladder epithelium, leading to bladder infection and inflammation (cystitis). [15]

1 E , [24] but are simply referred to as type 1 fimbriae in most studies, as well as in this account. Because of the impor- tance of type 1 fimbriae for bacterial adhesion, numerous studies on their carbohydrate specificity were undertaken, in order to design effective inhibitors of bacterial adhesion to mucosa.

Carbohydrate Antagonists and Antiadhesion Therapy

Adhesion to the surface of the host cell is a prerequisite for colonization and successful reproduction conditions es- pecially for enteric, oral and respiratory bacteria. Adher- ence protects bacteria from being swept away by the normal cleansing mechanism operating on mucosal surfaces such as urinary flow. Thus, successful adhesion increases the ability of the bacteria to colonize epithelial cells, multiply and eventually also invade the host. As bacterial adhesion is mediated by interactions with cell surface carbohydrates, the intriguing idea to prevent bacterial colonization by carbohydrate inhibitors of adhesion arises. Thus, carbo- hydrates could be developed as antiadhesive drugs accord- ing to a strategy against pathogens that is an alternative to antibiotics. An antiadhesive therapy [1618] could help to fight especially those bacteria with multi-antibiotic resis- tance, constituting an increasing problem in medicine. [19] It can be considered as rather unlikely that bacteria develop resistances against antiadhesive drugs. Hence, studies on the carbohydrate-specificity of bacte- rial lectins have been largely motivated by the importance of carbohydrate-mediated bacterial adhesion in infection, and this research has led to the vision to prevent bacterial adhesion by suitable carbohydrate antagonists of natural lectin ligands. There are many different lectins of Gram- negative bacteria known with various carbohydrate specifi- cities. In E. coli, P fimbriae (specific for Gal α1,4Gal), S fim- briae (specific for Neu5Acα2,3Gal), and type G fimbriae (specific for GalNAc) are found, as well as the mannose- specific type 1 fimbriae, which are one of the most abun- dant surface structures both in pathogenic and nonpatho- genic Gram-negative bacteria. [20]

Type 1 Fimbriae

Type 1 fimbriae serve as extremely efficient adhesion tools for bacteria inhabiting diverse environments, including biotic and abiotic surfaces. [21] They are uniformly distrib- uted on Enterobacteriaceae, commonly between 100 and 400 fimbriae per cell. Their length varies between 0.1 to 2 micrometer, their width is approximately 7 nm (Figure 1). Fimbriae are present in at least 90% of all known UPEC strains, which are the main cause of urinary tract infections in humans, and they have been shown to be important viru- lence and pathogenicity factors. [22] Type 1 fimbriae mediate agglutination of guinea pig red blood cells in an α-man- nose-inhibitable manner and are responsible for bacterial binding to a wide range of glycoproteins carrying one or more N-linked high-mannose type oligosaccharide. [23] Type 1 fimbriae of the E. coli type have been classified as type

2. Early Studies on the Carbohydrate Specificity of Type 1 Fimbriae

Interest in type 1 fimbriae started relatively early. Ap- proximately since the 1970s it was known that hemaggluti- nation mediated by type 1 fimbriae can be inhibited by mannose, methyl α-d-mannoside (MeMan) and mannan. [20] Then, evidence that type 1 fimbriae are major virulence fac- tors of UPEC motivated a number of more in-depth investi- gations on the carbohydrate-specificity of type 1 fimbriae. Ofek and colleagues showed that the carbohydrate specific- ity of the E. coli type 1 fimbrial lectin can be studied quan- titatively by examination of the inhibitory potency of mono- and oligosaccharides on the agglutination of yeast cells by bacteria. In this agglutination inhibition assay, the concentration of MeMan that is required to inhibit 50 % of yeast agglutination by E. coli 364 (a strain which predomi- nately expresses type 1 fimbriae) was determined to range

between 0.15 and 0.40 mm. [25] A number of oligosaccha- rides were tested as inhibitors of yeast agglutination and compared to the inhibitory potency of MeMan, which was arbitrarily set to 1 (Figure 2). Mannobiosides Man α1,2Man (1), Manα1,3Man (2) and Manα1,6Man (3), as well as the branching trimannoside Man α1,6[Manα1,3]Manα1-OMe (4), formed a first group of tested saccharides, of which only trisaccharide 4 showed a significantly higher inhibitory po- tency than MeMan [RIP( 4) MeMan = 10.5]. In 2006, very similar saccharides were investigated as inhibitors of the ad- hesion of type 1 fimbriated E. coli to mannan in an enzyme- linked immunosorbent assay (ELISA). [26] Lindhorst and co-workers employed allyl mannobiosides Man α1,2Manα1- OAll (5), Manα1,3Manα1-OAll (6), Manα1,6Manα1-OAll (7) and Manα1,4Manα1-OAll (8), and the allyl trimanno- side Manα1,6[Manα1,3]Man1α-OAll (9), and largely found relative inhibitory potencies similar to those reported by Ofek and his colleagues. However, the α1,3-linked disaccha- ride 6 performed significantly better in the ELISA, giving

a RIP value of 11 (Figure 2). It should be noted, however,

that the standard deviation in this assay was determined as 7, which was rather high. Also the allyl trisaccharide 9

[RIP(9) MeMan = 20] was a better inhibitor than the analo- gous methyl trisaccharide 4 [RIP(4) MeMan = 10.5], though in different assays.

When Ofek and co-workers tested more complex oligo- saccharides, the strict specificity of the type 1 fimbrial lectin for α-mannosides was confirmed. However, it was also shown that the way an α-mannosyl ligand is scaffolded on

a particular oligosaccharide is crucial. While for trisaccha- ride 10, Manα1,3Manβ1,4GlcNAc, a RIP value of 21 was determined, its isomer 11, Manα1,6Manβ1,4GlcNAc, had

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MICROREVIEW M. Hartmann, T. K. Lindhorst Figure 2. Structures of oligosaccharides tested as inhibitors of mannose-specific

Figure 2. Structures of oligosaccharides tested as inhibitors of mannose-specific adhesion of E. coli together with their relative inhibitory potencies based on the reference mannoside methyl α-d-mannoside (MeMan). IP: inhibitory potency; RIP: relative inhibitory potency (with IP of MeMan 1); SD: standard deviation. [a] Values from inhibition of yeast agglutination with E. coli 346; [25] [b] values from inhibition of adhesion of type 1 fimbriated E. coli HB101 pPKL4 to the polysaccharide mannan, measured by ELISA; [26] [c] “Oligoman- nose-9 and -3” are named according to a suggestion made in the literature, [28] where they were tested as ligands for the fimbrial lectin FimH (vide infra).

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Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion a RIP of 0.7, thus performing worse than simple

a RIP of 0.7, thus performing worse than simple MeMan

as inhibitor of mannose-specific yeast agglutination. Furthermore, while the branched oligomannosides 12 and 13 were very good inhibitors, 14 and 15 were significantly weaker. The data obtained in yeast agglutination assays were confirmed with guinea pig erythrocytes a little later. [27]

Some of these results, obtained in the 1980s, can be ra- tionalized today on the basis of more modern testing results with oligosaccharides 16 and 17, which have been termed “oligomannose-9 and -3”, respectively, by Bouckaert et al.

(Figure 2). [28] At the time, however, researchers had no idea about the structure of the carbohydrate recognition domain of the type 1 fimbrial lectin and had to rely on models, which were deduced on the basis of the determined testing results. The model of the type 1 fimbrial carbohydrate com- bining site, which was finally suggested by Ofek and Sharon, was therefore based on the findings obtained with

a series of oligomannosides and, in addition, based on

structure–activity relationships obtained with a series of simple mannosides with varying aglycon moieties (Fig- ure 3).

mannosides with varying aglycon moieties (Fig- ure 3). Figure 3. The nature of the aglycon moiety

Figure 3. The nature of the aglycon moiety of simple α-d-mannos- ides is decisive for their potency as inhibitors of type 1 fimbriae- mediated bacterial adhesion. [29] RIP: relative inhibitory potency (with inhibitory potency of MeMan 1).

Strikingly, mannosides with an aromatic aglycon, such as p-nitrophenyl α-d-mannoside ( pNPMan) and 4-methylum- beliferyl α-d-mannoside (MeUmbMan), were shown to ex- ceed the inhibitory potency of MeMan significantly (Fig- ure 3), [29] performing better than all oligosaccharides that had been examined earlier. In addition, certain substitution

patterns on the aromatic ring showed a favourable effect on the inhibitory potency of the respective mannnoside. For p-nitrophenyl-o-chlorophenyl α-d-mannoside (pNoClMan), for example, a RIP value of approximately 720 was deter- mined. As mentioned earlier, this and some other testing results of the early days can today be decoded on the basis of the crystal structure of the fimbrial lectin (vide infra). Finally, Sharon and Ofek established a model for the carbohydrate combining site of the type 1 fimbrial lectin and postulated that it should correspond to the size of a trisaccharide (having three subsites) with a hydrophobic binding region adjacent to the carbohydrate-binding site, which has a strict specificity for the α-configuration of the bound mannoside. Today, crystallographic studies have par- tially approved Sharon’s model, while the three-subsite model could not be confirmed so far. Nevertheless, at pres- ent there seems to be some mystery hidden behind type 1 fimbriae-mediated binding of mannosides that has not been unravelled yet. Therefore, Sharon’ s model might contain a relevance, which is not reflected in the X-ray analysis of the type 1 fimbrial lectin, called FimH, and thus has not yet been appreciated and remains underestimated.

3. Multivalent Glycomimetics as Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

After the early investigations with natural mannosides and oligosaccharides, a series of studies has employed multivalent glycomimetics as inhibitors of type 1 fimbriae- mediated bacterial adhesion. The multivalent presentation of α-d-mannosides was anticipated as a promising approach to high-affinity (avidity, respectively) inhibitors of man- nose-specific bacterial adhesion based on multivalency ef- fects that had been observed in other carbohydrate–lectin interactions. Characteristically, lectins can contain not only one but two or more carbohydrate-binding sites, and their interaction with carbohydrates is therefore often multi- valent. Multiple lectin–carbohydrate contacts can lead to agglutination on one hand, but on the other hand, they also may lead to increased affinities as well as to a higher specificity of the interaction. When the affinity of a lectin to a carbohydrate ligand is increased by multivalent carbo- hydrate–CRD contacts, the term “avidity” is used to de- scribe the strength of the overall interaction. To achieve an avidity effect by employing multivalent glycomimetics as lectin antagonists is especially attractive, as carbohydrate binding of lectins is typically associated with only moderate affinity. Thus, the typical stabilities of many lectin–mono- saccharide complexes are low and lie in the millimolar to high micromolar range. [30] Indeed, studies on the inhibition of type 1 fimbriae-medi- ated bacterial adhesion and hemagglutination have revealed that rather high concentrations of saccharides are required for effective inhibition. In the 1970s, Y. C. Lee and co- workers approved weak binding of galactose and GalNAc binding to the asialoglycoprotein receptor (ASGPR). How- ever, when multivalent glycomimetics were employed, they

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discovered that a linear increase in the number of monosac- charide epitopes, presented in a specific glycomimetic, led to a logarithmically increased avidity to the ASGPR. Lee and co-workers, at that time, concluded that this finding strongly suggests that lectins possess more than one carbo- hydrate-binding site, hence are bi- or even multivalent. The resulting effect in binding multivalent carbohydrates they called the “cluster effect”, which today is often referred to as “multivalency effect”. [31] Inspired by Y. C. Lee’s results, chemists have strived after multivalent glycomimetics to achieve high-affinity carbohydrate ligands for lectins. Multi- valent glycomimetics have been designed and synthesized along a large variety of architectures, and this field has been extensively reviewed. [32,33] It has, however, not been easy to simply reproduce the results Y. C. Lee observed in the case of carbohydrate binding to ASGPR with other lectins. To- day it can be concluded that multivalency effects, observed in carbohydrate–protein interactions, can result from quite different biological mechanisms and according to quite dif- ferent thermodynamic and kinetic conditions. [3438] Thus, the effect of variable multivalent glycomimetics in a particu- lar testing system can not necessarily be compared in the sense of quantitative structure–activity relationships (QSARs). Multivalent glycopolymers, [3942] for example, might interact with a lectin according to fundamentally dif- ferent models than a multivalent glycocluster, which is based on a carbohydrate core, for instance. [26] Among multivalent glycomimetics, glycodendrimers are an espe- cially promising class of molecules, which have been evalu- ated as antiadhesives. Their synthesis and testing results as inhibitors of UPEC has recently been reviewed in detail. [43]

Mannose Glycoclusters with Noncarbohydrate Core

In 1998, the effect of multivalency on the inhibition of type 1 fimbriae-mediated bacterial adhesion was studied for the first time, [44] by using a hemagglutination inhibition as- say (vide infra). Branched and hyperbranched amines were employed as multivalent scaffold molecules and reacted with glycosyl isothiocyanates to achieve di-, tri-, tetra-, and hexavalent α-mannose clusters 18 to 21 by thiourea bridg- ing (Table 1). The inhibitory potencies determined with these glycoclusters and glycodendrimers [4547] turned out to be not strictly valency-dependent. [44] Whereas divalent mo- lecule 18 and tetravalent cluster 20 performed 31 and 39 times better than the reference inhibitor MeMan, respec- tively, trivalent cluster 19 had a 106-fold higher inhibitory potential. When the determined inhibitory potencies are considered as valency-corrected values (RIP vc ), trivalent glycocluster 19 was superior over all other multivalent gly- comimetics tested in this series. In valency correction, the inhibitory potency determined for a specific glycocluster is divided by the number of clustered mannosyl moieties. Thus, a RIP value of 106, determined for a trivalent gly- cocluster, results in a RIP vc of 35. The hexavalent molecule 21, for example, was shown to have the same RIP value as the trivalent cluster 19, but its valency-corrected inhibitory potency, RIP vc , is only 18.

Besides the valency of a glycocluster, the structure of the aglycon or scaffolding unit was shown to play a crucial role for lectin-binding properties. Trivalent cluster 22, which, apart from its O-glycosidic linkage, offers great structural similarity to thiourea-bridged glycocluster 19 [RIP(19) MeMan = 106], performed only 10 times better than MeMan. [44] Thus, thiourea bridging of mannosyl residues was especially effective in inhibiting mannose-specific bac- terial adhesion. This was once again confirmed in a study with tetravalent clusters 23 and 24, [48] which are based on tetraazamacrocycle scaffolds. While cyclen 23 showed a 195-fold increased inhibitory potency in a hemagglutination assay, when compared to MeMan, the more flexible cyclam analogue 24 exceeded the inhibitory potency of MeMan by

780-fold.

These findings showed that tri- and tetravalent glycoclus- ters perform particularly well as inhibitors of type 1 fim- briae-mediated bacterial adhesion, and this led to the devel- opment of a series of further small glycoclusters and cluster mannosides (Table 1). A new ELISA (vide infra) was devel- oped to determine the antiadhesive properties of such tri- and tetravalent cluster mannosides, all O-glycosidically at- tached to the respective noncarbohydrate cores. [49] In ad- dition, the idea was born to cluster mannosides via the 6- position of the sugar ring. This concept was considered in order to combine a favourable aglycon effect with the suc- cess of trivalent clustering of mannosidic ligands. Thus, in the late 1990s “mannoside donors” (instead of the classical “mannosyl donors”) were designed, each with a function- alized 6-position to achieve a library of analogous cluster mannosides with varied aglycon moiety. For clustering pep- tide coupling to branched triacids, or thiourea bridging to branched triamines was utilized. This approach to high-af- finity ligands for type 1 fimbrial lectin led to cluster glycos- ides such as 25 and 26, which were quite poor inhibitors, however. Nevertheless, direct comparison of two structur- ally similar C-6-clustered inhibitors with aliphatic [RIP(25) MeMan = 0.7] and aromatic aglycon [RIP( 26) MeMan = 8] moieties showed that the aromatic character of the ag- lycon affected binding. On the other hand, the anomerically linked trivalent cluster mannnoside 27 tested in the same assay and lacking an aromatic moiety, produced a RIP of 90. Overall, the concept of mannoside clustering via the 6- position of the sugar ring cannot be recommended. Any good testing results with this type of 6-modified cluster mannosides must be considered false positive results [49] in light of the FimH X-ray structure, [50] which indicates that complexation of mannosides within the FimH CRD is not possible once a scaffolding unit is connected to the 6-posi- tion of the sugar ring (cf. Figure 6). The first crystal structure of the type 1 fimbrial lectin FimH was published in 1999. [50] It proved that this lectin is monovalent and possesses just one CRD, thus favourable effects with multivalent ligands could not necessarily be ex- pected on the basis of this information. In spite of this, many more multivalent cluster mannosides were prepared in the following years up to the present, and they were tested as inhibitors of type 1 fimbriae-mediated bacterial

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Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion adhesion and even the isolated lectin FimH. Such research

adhesion and even the isolated lectin FimH. Such research was motivated and reasoned by numerous experimental fin-

dings, which showed that especially tri- and tetravalent clus- ter mannosides are indeed potent inhibitors of type 1 fim-

Table 1. A selection of representative mannose glycoclusters with noncarbohydrate core and their testing results. As many of the structures are rather complex, α-d-mannosyl residues have been simplified and represented as six-membered heterocycles shaded in grey, here and likewise in all other following tables.

in grey, here and likewise in all other following tables. Eur. J. Org. Chem. 2011 ,

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Table 1. (Continued)

M. Hartmann, T. K. Lindhorst

Table 1. (Continued) M. Hartmann, T. K. Lindhorst 3590 www.eurjoc.org © 2011 Wiley-VCH Verlag GmbH &

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Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Table 1. (Continued)

1 Fimbriae-Mediated Bacterial Adhesion Table 1. (Continued) [a] SD = standard deviation, included in brackets if
1 Fimbriae-Mediated Bacterial Adhesion Table 1. (Continued) [a] SD = standard deviation, included in brackets if

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan with IP MeMan 1. [c] RIP vc = valency-corrected RIP (rounded down). [d] = RIP based on d-mannose with IP Man 1.

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Table 2. A selection of representative carbohydrate dendrimers and carbohydrate-centred cluster mannosides and their testing results.

cluster mannosides and their testing results. 3592 www.eurjoc.org © 2011 Wiley-VCH Verlag GmbH & Co.

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Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Table 2. (Continued)

1 Fimbriae-Mediated Bacterial Adhesion Table 2. (Continued) [a] SD = standard deviation, included in brackets if
1 Fimbriae-Mediated Bacterial Adhesion Table 2. (Continued) [a] SD = standard deviation, included in brackets if

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan with IP MeMan 1. [c] RIP vc = valency corrected RIP (rounded down). [d] K abbreviates l-lysine as branching element.

briae-mediated bacterial adhesion and good antagonists of FimH. Thus, a collection of structurally related tri- and tetravalent cluster mannosides with a C 3 -aliphatic aglycon,

28 to 31, were investigated. [51,52] Here it was again con-

firmed that a linear increase in valency can result in a much more than linear enhancement in affinity, as observed by comparison of 28 and 31. However, the attempt to improve binding to FimH by incorporation of an aromatic moiety adjacent to the clustered mannosides, such as in the case of

28 and 29, [51] was not successful. Trivalent cluster manno-

side 30 performed approximately 6 times better than its an- alogue 29 with a phenyl ring incorporated at its focal point.

On the other hand, the importance of the nature of the scaffolding unit for the affinity of clustered mannosides was also revealed. Four clusters with ethylene glycol (EG) spa-

cers were tested (Table 1): trivalent clusters 32 and 33, [53] both yielding a high RIP of 1063, and tetravalent molecules 34 and 35, [54] which had a 100 times lower RIP in the same assay system. Thus, in spite of the fact that the same type of linkers were used for clustering in all four cases, their inhibitory behaviour differed significantly. In recent years, also additional chemistries such as “click” chemistry, [55] Sonogashira coupling [56] and squaric acid conjugation [57] were employed for the synthesis of clus- ter mannosides, leading, for example, to triazole-linked clusters 36, 37, 41 and 42, alkyne-linked multivalent man- nosides 38 to 40 and clusters such as 43, linked by squaric acid. With the isolated lectin FimH on hand, such tetra- and hexavalent cluster mannosides were tested in surface plasmon resonance (SPR) experiments (vide infra). [58] The

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obtained RIP values vary from 157 for cluster 36 to 41 for cluster 37, and even more drastically, from 4889 for tetrava- lent cluster 38 to 8 for the similar tetravalent 39. This tre- mendous discrepancy points out that the distance between the mannoside ligand and the aromatic moiety as part of the spacer is extremely critical for binding efficiency to FimH. An interesting observation was made, when the val- ency of alkyne-linked clusters was changed while the princi- pal architecture of these clusters was maintained. When tetravalent molecule 39 was expanded to hexavalent struc- ture 40, the relative inhibitory potencies remained the same. The triazole-linked cluster mannosides 41 and 42, on the other hand, led to a significant improvement, such that the RIP value of 41 was increased by a factor of 4.7 when com- pared to that of 36. The synthesis of cluster 42 [59] was in- spired by the structure of the highly potent heptyl α-d-man- noside (cf. Table 3) and indeed seems to be a very promising candidate for inhibition of bacterial adhesion. Glycocluster 42 was shown to exhibit a relative inhibitory potency of

2670 (based on mannose) when tested in a hemagglutina-

tion inhibition (HAI) assay. An impressive example for a cluster effect in FimH bind- ing was shown by applying mannosylated lysine-based den-

drimers. [60] With growing generations, the relative inhibitory potencies of the di-, tetra-, octa- and hexadecavalent den- drimers 44, 45, 46 and 47 increased from 455 over 2000 and

3571 to 11111, respectively (Table 2). Though the avidity

did not grow logarithmically, it rose to a much greater ex- tent than it would by a linear increase with respect to the increasing valency.

Carbohydrate-Centred Cluster Mannosides and Carbohydrate Dendrimers

Carbohydrate dendrimers were obtained by employing a glycosyl donor as the branching element. [61] This synthesis starts from a 3,6-di-O-allyl mannoside (Scheme 1), which can be converted into a respective diol by hydroboration or radical addition of 2-mercaptoethanol. Then, mannosyl- ation with a 3,6-di- O-allylated mannosyl donor delivers the next generation of carbohydrate dendrimers. When hydro- boration is employed to produce the diol acceptor for glyco- sylation, mannose moieties are linked by a C 3 spacer, such as in the case of 48 and 49 (Table 2). When 2-mercapto- ethanol was added upon the allylic double bonds, more flexible dendritic structures such as 50 resulted. Interest- ingly, thioethers like 50 could be oxidized to the respective sulfones (51), to increase the hydrophilicity of the spacers. Adhesion inhibition assays showed that, relative to the first generation precursor 48, a duplication of mannoside resi- dues in 49 led to a fourfold higher inhibitory potency [RIP(48) MeMan = 10; RIP(49) MeMan = 42]. Secondly, again the importance of spacer properties for protein–ligand in- teractions was apparent. Sulfone-bridged cluster 51 sur- passed the inhibitory potency of MeMan by 25-fold, whereas its less polar analogue 50 was 110 times a better inhibitor than the reference mannoside. Finally, carbohydrate-centred cluster mannosides were introduced and tested. [26,62] They were designed to closely resemble the chemical nature of natural oligosaccharides. For steric reasons, C 3 spacers were included between the

steric reasons, C 3 spacers were included between the Scheme 1. Carbohydrate dendrimers such as 50

Scheme 1. Carbohydrate dendrimers such as 50 (Table 2) can be formed in an iterative synthetic sequence employing a glycosyl donor as the branching element. [61]

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Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion glucose core and the scaffolded mannoside ligands. It was

glucose core and the scaffolded mannoside ligands. It was anticipated that, once the synthetic sequence to glucose- centred cluster glycosides is established, it can be transfer- red to others, for example, di- and trisaccharide core glyco- sides. Thus, a small library of carbohydrate-centred clusters such as 52, 53 [26] and 54 [62] (Table 2) could be produced, which gave RIP values of 180 for pentavalent cluster 52, 230 for octavalent 53 and 190 for dodecavalent mannoside 54. [62] Strikingly, these values are all in the same range, pointing out that the enhancement in molecule size and val- ency had no significant effect on inhibition properties of the respective cluster mannosides in this case.

Reasoning of Multivalency Effects in Carbohydrate Binding of Type 1 Fimbriated Bacteria

The multivalency effects observed with many different types of mannoside clusters as inhibitors of type 1 fimbriae- mediated adhesion of E. coli have not been fully under- stood. Generally, a cluster effect can be explained with the clustering of multiple CRDs, [31,34,37,63] but the type 1 fim- brial lectin FimH possesses only one CRD. Interfimbrial clustering of FimH CRDs might occur with large enough molecules. In an average glycocluster having about ten C–C single bonds between two mannoside residues, the distance between these two ligands is in the range of 1.5 nm. As the bacterial fimbriae measure about 7 nm in diameter, binding of one cluster molecule to more than one fimbrial lectin at a time is very unlikely with cluster mannosides such as those depicted in Table 1. Intramolecular binding is a much more sensible explanation of avidity enhancement observed with multivalent cluster glycosides in this adhesion system. Hence, several putative carbohydrate-binding sites on FimH, in addition to the CRDs known from X-ray studies, were suggested (vide infra). [64] Multiple binding to more than one carbohydrate-binding site on FimH could account for strengthened binding of multivalent ligands. A most obvious explanation for the observed multiva- lency effects is the elevated effective concentration of man- noside residues in the proximity of the carbohydrate re- cognition domain when a multivalent ligand is employed. Quick rebinding of a mannoside ligand is therefore statistic- ally favoured once a bound ligand is released from the CRD. Another probable explanation for multivalency effects in inhibition of type 1 fimbriae-mediated adhesion could be that cooperative effects between ligand and protein play a role. For example, intramolecular preorganization of free ligands could arise when the first ligand of a multivalent glycomimetic is bound to the lectin. This induced spatial organization could enhance binding of the other ligands (positive cooperativity) or reduce their affinity (negative cooperativity). Moreover, binding of one ligand might in- duce a change in lectin conformation, which enforces the subsequent binding event of the next mannoside moiety of a cluster mannoside. This mechanism would correspond to an allosteric activation of the protein achieved by a multi- valent ligand.

Several studies indicated that especially potent clustered inhibitors of type 1 fimbriae-mediated bacterial adhesion were tri- or tetravalent. To explain the variant potencies of these, the chemical properties of the used linkers are decis- ive. Tuning of the linker chain length and its conforma- tional flexibility makes the exposed carbohydrates more or less easily accessible for lectin binding. Furthermore, the conformational stability of the chosen linker in water (or buffer), the surrounding solvent in biological systems, is an important property. Very hydrophilic spacers may dissolve the carbohydrate cluster so well that binding to a partially hydrophobic protein would be disfavoured by enthalpy con- siderations. In contrast, hydrophobic linkers might lead to supramolecular assemblies of the clusters, in course lower- ing their accessibility for lectin binding. This is a reasonable explanation of the finding that higher valencies in many cases did not lead to drastically increased inhibitory potenc- ies of cluster mannosides and mannose glycodendrimers. Higher numbers of mannoside residues on a scaffolding core molecule of limited size does not necessarily mean bet- ter ligand availability. Individual mannoside moieties might be clustered too closely, so that enough space for the lectin to bind multiple copies is not available.

4. Assays to Test Antagonists of Type 1 Fimbriae-Mediated Bacterial Adhesion

The development of new drugs is typically based on reli- able estimation of QSAR studies with potential leads. In addition to rational ligand design, which is based on the knowledge of the structural properties of the drug target, in vitro methods to screen for potential lead compounds are indispensable tools. Therefore, any test system has to be particularly suited for the biological question that is investi- gated. In an in vitro assay, which is typically used for QSAR studies, three major components determine the situation during testing: the biological receptor or target, the native ligand and the test compound, which can be an agonist or an antagonist. If this three-compartment model is translated to the screening for potent inhibitors of type 1 fimbriae-mediated bacterial adhesion (Figure 4), the bacterial lectin FimH is the receptor, the native ligand is approximated by the glyco- sylated surface (e.g. human bladder epithelium cells, or mannan as its mimic), and the test compound is the puta- tive inhibitor of bacterial adhesion, typically an α-d-man- noside derivative. All possible mutual interactions of the three components have to be considered to influence the outcome of an assay. In this system, a number of param- eters are important, and they are discussed in the following. (1) Presentation of the lectin: How the lectin is applied in an assay plays a crucial role for the outcome. Three states of complexity of the receptor system have been employed. The lectin can be applied as recombinantly expressed, iso- lated protein or protein conjugate. Sheared-off bacterial type 1 fimbriae can be used, or an assay can be performed with whole bacteria. Even without any knowledge of the

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MICROREVIEW M. Hartmann, T. K. Lindhorst Figure 4. Three different types of interactions play key roles

Figure 4. Three different types of interactions play key roles in FimH-mediated bacterial adhesion to mammalian cells. In the development of assays to test and optimize putative adhesion inhibitors, all interactions and their mutual interplay have to be considered: the bacterial lectin FimH can either bind to a diversely glycosylated surface, a cell or cell mimetic (interaction I) or to an inhibitor in solution, a multivalent α-d-mannoside in this case (interaction II); additionally, an interplay of the carbohydrates on the surface with those in solution (interaction III) has to be considered. Each change in one of the shown interactions will influence the others, and likewise changes in the conditions of the binding process have an impact on the whole system. Thus, the results of any FimH binding assay or adhesion inhibition assay are highly dependent on the setup of the applied in vitro test system.

lectin structure, it can easily be understood that the binding conditions can differ a lot in these three different setups. (2) Glycosylated surfaces: Not only the presentation of the lec- tin FimH but also the glycosylated surface used in the assay influences the results. The ligand system can be applied in many different stages of complexity. Whole cells can be used to test adhesion at native interfaces. On the other hand, by using synthetic surfaces it is possible to tune the complexity and structural features of a glycosylation pattern in great detail. In this way, structural refinement allows to test for very specific patterns in a ligand surface, if needed. (3) Pres- entation of the inhibitor: Structural details of the test com- pound that enhance or lower its affinity are the object un- der investigation and can naturally not be varied to tune an assay. But still, they play a role in the way a FimH antago- nist is applied. As mentioned earlier, inhibitors that are clustered can be much more effective than their respective monovalent precursors. Immobilized on some surface or other rigid device, the inhibitor’s properties can change and thus lead to an inhibitory effect that differs from the prop- erties of the inhibitor in solution.

In Vitro Assay Systems to Screen for Potent FimH Inhibitors Different types of assays have been developed to test the quality of a potential new inhibitor of type 1 fimbriae-medi-

ated bacterial adhesion. The data that are generated for the same FimH ligand can vary significantly when obtained in different assay systems. To be able to understand some of the observed effects and apparent discrepancies, it is useful to categorize the tests applied according to the conditions under which inhibition of adhesion is investigated.

Assays in a Three-Dimensional Setup

The first assays that were performed to screen the inter- actions between type 1 fimbriated bacteria and eukaryotic cells were aggregation assays. [25] In the 1980s Sharon and colleagues could show that the agglutination of yeast cells by type 1 fimbriated bacteria, such as, among others, E. coli and some species of Salmonella, can be inhibited by ad- dition of α-mannosides. This agglutination assay using bac- teria and yeast cells is still used today. [65,66] The assay setup is rather close to physiological conditions regarding the gly- cosylated eukaryotic cell as well as in view of the fact that the lectin-bearing bacteria can move freely in all three di- mensions. A closely related testing system that has nearly replaced the yeast aggregation assay due to its higher sensi- tivity is the hemagglutination inhibition assay. [44,49,58,67,68] In this assay, an inhibition titre (IT) is determined, which reflects the concentration of a tested inhibitor that is needed to prevent type 1 fimbriated bacteria from agglutinating guinea pig erythrocytes. As direct IT values generally differ

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Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion much from one screen to the other even if

much from one screen to the other even if the same bacte- rial strain is used, relative IT values are commonly calcu- lated, which are referenced to a known inhibitor tested in parallel. In most cases, the reference glycoside is methyl α- d-mannoside (MeMan). In 2002, along with hemagglutina- tion inhibition data of type 1 fimbriated E. coli, Lee and co-workers detected the binding event by radioactivity as- says. [60] Mannosides coupled with 125 I-labelled human se- rum albumin (HSA) were incubated together with E. coli bacteria and different dilutions of the test substance. After centrifugation, the remaining radioactivity of the E. coli cells was an inverse measure for the inhibitory effect of the tested carbohydrate ligand. As the tested inhibitors have to compete with the mannosylated HSA surface for bacterial binding, half maximal inhibitory concentration (IC 50 ) val- ues can be derived by plotting the % inhibition against the inhibitor concentration on a logarithmic scale to obtain a sigmoidal dose response curve. The IC 50 value of a test sub- stance reflects the inhibitor concentration at which 50% of the binding is inhibited. In dose response curves the IC 50 value is the point of inflection. A second 3D approach to determine inhibitory potencies of FimH ligands was re- ported in 2001. [69] Human cells (neutrophilic granulocytes) and a dilution of potential adhesion inhibitors were added to a solution of biotinylated type 1 fimbriae. After incu- bation and washing, fluorescein-labelled streptavidin facili- tated the readout by fluorescence-activated cell sorting (FACS). In 2002, it was shown that the binding of man- nose-encapsulated gold nanoparticles (m-AuNP) to type 1 fimbriated E. coli cells is inhibited by addition of MeMan. M-AuNPs were thereby introduced as tools for the imaging of FimH mediated bacterial binding in transmission elec- tron microscopy (TEM). [70] Recently, mannosylated nano- particles, which can be used in imaging applications were introduced again. In this study, the adhesion of E. coli to mannose-functionalized hematite (iron oxide) nanoparticles was visualized by TEM. [71] Detection has been performed with 3 H-labelled mannose. After incubation of isolated FimH together with 3 H-man- nose and a competing inhibitor, the assay mixture was fil- tered, and the remaining radioactivity could be read out. When using different inhibitor dilutions, K d values could be derived for the tested substances. [72] In recent work, two additional three-dimensional assays were applied to test the binding between FimH and potential carbohydrate ligands. One of these is the investigation of the binding of fluores- cent-dye-labelled mannosides to isolated FimH in a fluores- cence polarization (FP) assay. [68] In this assay, the fluoro- phore on the FimH ligand is excited by polarized light, so it consequently emits polarized light. The more freely the molecule can move in solution the more disturbed the po- larization of the emitted light. Bound ligands partially freeze and thus move more slowly; therefore, the polariza- tion of the emitted light increases upon binding, which can be measured. As ligand binding is not competitive in this setup, the inhibitory effect of a substance is not detected directly. Nevertheless, the binding event per se can be moni- tored precisely. Thus, the characteristic measure for a tested

ligand is not an IT, but the half maximal effective concen- tration, abbreviated EC 50 . The EC 50 can again be deduced from a dose response curve, a plot of the gained effect against the corresponding ligand concentration applied. Isothermal titration calorimetry (ITC) is a different and well-known test method that was first utilized for the detec- tion of FimH binding mannosides by Bouckaert and col- leagues in 2011. [73] Mannosylated ligands can be titrated into a FimH solution to measure the temperature changes upon saccharide addition and calculate a K d value to char- acterize the binding of the ligand. Measurement of inhibi- tory effects was not performed, but binding constants for the ligand–FimH interaction were deduced.

Assays on Surfaces

Besides the three-dimensional test systems, a number of assay setups have been published that depend on the immo- bilization of one binding partner and subsequent detection of the binding event to the manipulated surface. As only one of the binding partners is in solution and can thus move freely, the setups can be categorized as two-dimen- sional. The prototype of 2D assays is probably the classical ELISA that depends on the detection of a molecule or pro- tein bound to a predefined surface by incubation with a specific antibody against the presumably surface-bound compound. Lindhorst and co-workers used microwell plates coated with mannan (mannose polymer) for ELISA to bind type 1 fimbriated E. coli cells. [49] These could be detected

with an antibody against FimA, the predominant protein of the fimbrial shaft and a peroxidase-labelled secondary antibody. Upon addition of mannoside solutions in dif- ferent concentrations, dose response curves of the inhibi- tory effect of the tested ligand against the applied concen- tration could be plotted, which led to IC 50 values as charac- teristic for each tested ligand. As these ligands compete with the mannan-coated surface for FimH binding, a direct inhibitory potency is measured in this case. To be able to compare two such ELISA measurements with one another, a standard inhibitor has to be tested on the same test plate, so that the poorly comparable IC 50 values from different experiments can be uniformly referenced. Different ELISA experiments have been performed by using whole E. coli

type 1 fimbriae [77] and the iso-

cells, [26,44,48,51–54,61,62,74–76]

lated FimCH protein complex. [78] Assays similar to the ELISA method have been devel- oped. They all share the competitive 2D binding situation, but the detection method is nonimmunological. By using type 1 fimbriated E. coli cells, two different competitive ad- hesion inhibition assay setups have been introduced. [79]

These assays basically resemble the ELISA and were per- formed on the same mannan-coated test plates, but either the bacteria were biotinylated to be detected with a strepta- vidin–peroxidase conjugate, or directly detectable self-fluo- rescing bacteria expressing the green fluorescent protein (GFP) were used. In addition to mannan-coated plates, co- valently glycosylated assay surfaces were used to screen the affinity of bacteria to a surface-bound ligand. Both assays resulted in IC 50 values for the tested substances. Covalent

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immobilization of saccharides on a test surface is also needed for current research and carbohydrate array fabrica- tion. A competitive binding assay on mannosylated chips was published in 2004. [80] Type 1 fimbriated bacteria were stained with intercalating fluorescent dyes and sub- sequently, together with a reference mannoside solution, ap- plied to a mannoside array to competitively bind to the microchip. The fluorescence readout was then used to quantify the surface-bound bacteria. A more flexible carbo- hydrate surface can be built up by self-assembled monolayer (SAM) formation on a gold surface. In 2002, an adhesion inhibition assay on mannosylated SAMs in a 96-well for- mate was performed. [81] The adhesion of type 1 fimbriated fluorescence dye-stained bacteria to the glycosylated SAM was shown to be inhibitable by addition of a mannoside solution. As this inhibition again turned out to be depend- ent on inhibitor concentration, IC 50 values for the used in- hibitors could be reported. Radioactively labelled bacteria were also used to detect binding to GP2 (a membrane gly- coprotein) in a competitive manner. [82] To test the inhibi- tory potency of a glycopeptide from soybean, Guo and co- workers [83] used an adhesion inhibition assay, in which they let E. coli and Salmonella cells adhere to eukaryotic LoVo cells (colon carcinoma cells). This adhesion was inhibited by glycopeptides. To characterize the glycoconjugate GP2 as inhibitor, the remaining bacteria cells on the LoVo cell surface were counted. A different 2D test on human cells was performed in 2008. [28] A human urothelial cell line was grown in 12-well plates. Type 1 fimbriated E. coli were pre- incubated with mannoside solutions and then transferred to the cell surface. After incubation and washing, the blad- der cells were lysed by addition of trypsin and the lysate was transferred to agar culture plates to determine the re- maining amount of colony-forming units. Turning the setup upside down, the FimH CRD can also be immobilized to microtitre wells, to which the inhibitor dilution and a biotinylated polyacrylamide glycopolymer are applied and incubated, generating a competitive binding situation. By addition of peroxidase-coupled streptavidin, the amount of bound mannoside-functionalized polymer is detected. By plotting the corresponding dose response curves, IC 50 values for the tested substances can be ob- tained. [84]

Assays under Flow

One parameter that plays an important role in physiolog- ical systems is the flow that the binding event has to with- stand. Though in some of the reported assays the samples are shaken during incubation, the shear stress that the lectin is exposed to is not regulated. This is different in flow-regu- lated SPR measurements. For SPR, two different general setups have been applied. In the first SPR measurements with FimH, either an anti-FimH antibody or a BSA–man- noside conjugate was bound to the Biacore sensor chip. When isolated FimH was coursed over the immobilized an- tibody together with a soluble adhesion inhibitor, only a small percentage of FimH could be attached to the surface, depending on the binding affinity between FimH and the

added inhibitor. [58,72] Recently, the antibody or BSA conju- gate was replaced by low-molecular-weight mannosides, which were covalently attached to the Biacore chip. [73] When a mixture of FimH and soluble inhibitors – mannos- ylated fullerenes in this case – passes the glycosylated chip surface, the lectin can either bind to the Biacore chip and be detected or stick to the glycosylated fullerenes and be washed away. Both of these setups for SPR measurements resulted in K d values for the FimH–mannoside interaction. In most cases, noncovalent binding of protein–ligand in- teractions is shorter-lived under shear stress. Such interac- tions are referred to as slip bonds. [85] Nevertheless, for some interactions of cells with surfaces, it was first proposed in 1988 [86] that a critical applied tensile force can enhance the binding strength rather than weakening the adhesion. [85] FimH was the first bacterial protein for which such catch bonds were discovered. [87] Since 2010, the way bacterial lec- tin FimH tightens the binding to its sugar ligand under ten- sile force is understood in great detail. As long as the FimH protein is in a relaxed conformation, the N-terminal lectin domain and the C-terminal pilin domain (vide infra) are in close proximity, and the lectin domain is compressed. When shear force is applied, the two domains separate, and the lectin domain is elongated by 11 Å. This elongation process stretches the conformation of the β-sheets of the lectin do- main in such a way that the CRD constricts. This, in turn, tightens the binding of the ligand to the binding site. The same mechanism is in effect when a FimH truncate is iso- lated in pure form. As the FimH tr isolates lack the attach- ment to the C-terminal pilin domain, the lectin domain is frozen in its elongated high-affinity state. [87] This finding is particularly valuable for the interpretation of the results gained in different adhesion or binding assays with type 1 fimbriated bacteria under flow. In addition, the affinity state in which the lectin FimH is applied in different assays has a great impact on the measured binding strength. Con- sidering these recent findings about the catch bond mecha- nism of lectin, many testing results with the various FimH antagonists might appear in different light and deserve an updated interpretation. The evaluation of testing results can be complex and dif- ficult. Even the interpretation of inhibitory potencies within one assay system can already be challenging. In many cases, it can be very helpful to keep in mind the three-compart- ment model of an assay (Figure 4). To be able to compare the effect of different compounds, it is essential to test a reference compound in the same assay and batch, so that all results can be referenced reliably. In FimH binding studies, MeMan (Table 3) or pNPMan (Table 4) are mostly used as reference glycosides. In particular, these relative values are needed to compare the results of different assays. As the conditions under which binding is measured vary from sys- tem to system, the same inhibitor might perform quite dif- ferently in two apparently similar assays. Illustrative exam- ples are the highly diverging values measured for pNPMan in many different test systems (Table 3). Usually, the calcu- lated relative values of the inhibitory potencies are in good agreement, though.

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Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion 5. Structure of the Type 1 Fimbrial Lectin FimH

5. Structure of the Type 1 Fimbrial Lectin FimH

Crystal structures of carbohydrate-binding proteins pro- vide valuable information about the modes of interactions that mediate carbohydrate recognition. [88,89] It has been fre- quently observed in X-ray diffraction studies with lectins that a carbohydrate ligand is complexed by a well-defined array of hydrogen bonds, involving hydrogen-bond donor and acceptor groups of the ligand and the side chains of asparagine or glutamine residues, carboxylate groups from aspartates or glutamates, OH groups in serine side chains and NH-groups from lysine, tryptophan or histidine resi- dues of the lectin CRD. Water molecules can mediate these hydrogen bonds, and in some cases divalent metal ions such as Ca 2+ or Mn 2+ are additionally involved in carbohydrate binding. Also, sandwiching of the carbohydrate ligand be- tween aromatic amino acid side chains is an important binding motif, leading to a relevant contribution of CH– π interactions in carbohydrate binding. [90,91] The type 1 fimbrial lectin is called FimH and is as- sembled at the tips of type 1 fimbrial composite structures according to the chaperone/usher pathway. [50,92] Nine fim genes are required for fimbriae assembly. The tip-fibrillar structure contains the mannose-specific adhesin FimH, whereas the fimbrial rod is mainly composed of FimA sub- units (Figure 5). Type 1 fimbriae are constructed by a “do- nor strand exchange” (DSE) process, in which the Ig fold of every subunit is completed by an amino-terminal exten- sion from the following subunit. [21]

exten- sion from the following subunit. [ 2 1 ] Figure 5. The type 1 fimbrial

Figure 5. The type 1 fimbrial rod is assembled from different Fim proteins, which attach to each other by donor strand exchange (DSE). The fimbrial lectin, FimH, terminates the fimbrial fibre at its tip, and the most abundant protein constituent, FimA, attaches the fimbriae to the bacterial outer membrane.

FimH is a 29 kDa protein with a length of 279 amino acid residues. Its exact structure has remained unknown for long, because FimH cannot be crystallized in pure form as it is missing a strand to complete its fold. In addition, it is proteolytically degraded when it is expressed alone. Knight and co-workers reasoned that, according to the two-compo- nent system that is required for fimbriae assembly (accord- ing to the DSE process), the C-terminal part of FimH re- quires interaction with another protein. For fimbriae as- sembly, FimH is complexed with the chaperone FimC, and this led to the attempt to stabilize FimH by addition of FimC. This idea delivered the first crystal structure of

FimH in 1999, [50] in a complex with the FimC chaper- one. Cyclohexylbutanoyl- N-hydroxyethyl-d-glucamide (C- HEGA), which had been added for crystallization, was found to be bound to the CRD of FimH, where it adopts a conformation that relatively closely resembles the struc- ture of a mannose pyranoside ring. Thus, it was revealed that FimH has two domains, a pilus domain, FimH P , and a lectin domain, FimH L . The N-ter- minal mannose-binding lectin domain FimH L comprises residues 1–156, and the C-terminal pilin domain, which is used to anchor the adhesin to the pilus, comprises residues 160–279. There is one single CRD to be seen in the FimH crystal structure, which is located at the tip of the lectin domain. It is capable of accommodating a mannose mono- saccharide in its α-configuration (Figure 6).

mono- saccharide in its α -configuration (Figure 6). Figure 6. A mannosidic ligand is complexed within

Figure 6. A mannosidic ligand is complexed within the FimH CRD such that the aglycon of an α-d-configured mannoside points towards the entrance of the binding pocket. This allows terminal mannose residues on complex oligosaccharides to be complexed by FimH. β-d-Mannosides cannot be complexed for steric reasons.

In 2002, the crystal structure of the FimC/FimH chap- erone–adhesin complex bound to its physiologically rel- evant ligand α-d-mannose was published, and the amino acids that are important for mannose complexation were identified in great detail. [78] Mannose was found to be bur- ied in a deep and negatively charged pocket. The mannose ring makes ten direct hydrogen bonds to the FimH binding site, and in addition indirect water-mediated hydrogen bonds are formed. All hydroxy groups of the sugar ring, other than the anomeric position, interact extensively with the CRDs of FimH, in particular with residues Phe1, Asn46, Asp47, Asp54, Gln133, Asn135, Asp140 and Phe142 (Figure 7). The mannose-binding pocket is sur- rounded by a hydrophobic ridge comprising Ile13, Tyr48, Ile52, Tyr137 and Phe142. The side chains of Tyr48 and Tyr137 are positioned such that they form a gate-like struc- ture that has been named the “tyrosine gate”. [21] Thus, man- noside ligands with an aromatic aglycon, such as pNPMan, can establish ππ interactions with the tyrosine gate flank- ing the entrance of the FimH CRD (Figures 7 and 8), lead-

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MICROREVIEW M. Hartmann, T. K. Lindhorst Figure 7. Both graphics show the amino acid residues in

Figure 7. Both graphics show the amino acid residues in an orientation revealed by X-ray diffraction analysis (PDB ID: 1KLF), [78] depicted in ball-and-stick form. Left: The bottom site of the FimH CRD with docked p-nitrophenyl α-d-mannoside ( pNPMan). The CRD contains the N-terminal Phe1 amino acid of the protein. Prominent hydrogen bonds between the carbohydrate-binding site and the ligand are depicted as grey dotted lines. Right: The amino acid residues at the entrance of the FimH CRD. The aromatic side chains of Tyr48 and Tyr137 form ππ interactions with docked pNPMan, leading to an increased affinity of the ligands with an aromatic aglycon. [93]

ing to increased affinities. Thus, the early findings with li- gands such as pNPMan or MeUmbMan (Figure 3) can now be understood on the basis of the FimH structure.

3) can now be understood on the basis of the FimH structure. Figure 8. Structures of

Figure 8. Structures of the CRD of the bacterial lectin FimH crys- tallized with the tyrosine gate (residues Tyr48 and Tyr137) in an open or closed conformation. The conformation of the tyrosine gate depends on the complexed ligand. The open gate structure (PDB ID: 1KLF) was obtained with FimH in complex with man- nose (top), [78] while the closed gate conformation (PDB ID:

1UWF) arose from complexation of BuMan (bottom). [72] The FimH receptor structure is depicted as a Connolly surface, and to highlight the tyrosine gate it is meshed in each case.

In 2005, another crystallographic study was published, which employed two FimH proteins named FimH tr1 and FimH tr2 from two different bacterial strains. [72] These FimH tr proteins were truncated to only the FimH lectin domain, built up by residues 1–158. In both cases, although no sugar was included in the crystallization setups, it turned out that butyl α-d-mannoside (BuMan) was bound in the CRD. It was shown that this mannoside originated from the LB (Luria-Bertani) medium used to grow the bacteria during expression of the protein. The butyl moiety of bound BuMan extends out of the mannose-binding pocket towards Tyr48 and Tyr137, making van der Waals contacts to both tyrosine rings and Ile52. In case of FimH tr2 the Tyr48 and Tyr137 side chains were found in an almost par- allel orientation as in the earlier FimC/FimH struc-

tures. [50,78]

In the FimH tr1 structure, on the other hand, the parallel orientation of the Tyr48 ring is prevented and instead it is packed edge-to-face with Tyr137 (Figure 8). Thus, there is a conformational flexibility of the tyrosine gate at the en- trance of the FimH CRD, which has implications on ligand design and the interpretation of testing results (vide supra). In 2008, a truncated version of FimH, FimH tr compris- ing residues 1–158, was finally crystallized in a complex with a natural ligand, “oligomannose-3” (Figure 2). [28] It was shown that the α1,3-linked mannose residue is com- plexed within the CRD and that the Man β1,4GlcNAcβ1, 4GlcNAc portion of “oligomannose-3” interacts with an extended region of the binding site. Interestingly, for the central mannose unit, a strong stacking interaction with the aromatic ring of Tyr48 can be observed, whereas Tyr137 interacts with the mannose-bound GlcNAc moiety. Thus, the FimH tyrosine gate can be likewise utilized by oligosac-

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Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion charide ligands for tighter binding as it has been

charide ligands for tighter binding as it has been observed for synthetic ligands with an aromatic aglycon moiety (Fig- ure 3, Table 4).

6. Rational Design of Carbohydrate Ligands for the Type 1 Fimbrial Lectin FimH

As the structures of the type 1 fimbrial lectin FimH and its CRD are known today from several crystallographic studies, [28,50,68,72,78] a rational, computer-aided design of high-affinity ligands to develop inhibitors of bacterial ad- hesion to mucosal surfaces should be greatly facilitated.

Only recently, a number of such more rational studies have been published, leading to three classes of FimH ligands with relatively high affinity: (i) long-chain alkyl mannos- ides, (ii) mannosides with variously substituted aromatic ag- lycon moieties, and (iii) mannosides with extended aglycon moieties.

Long-Chain Alkyl α-D -Mannosides – Hydrophobic Interactions with the Tyrosine Gate

In case of the crystallization studies with FimH tr pub- lished in 2005, [72] exogenous BuMan was found to be com-

Table 3. A selection of representative alkyl α-d-mannosides and their testing results.

alkyl α - d -mannosides and their testing results. [a] SD = standard deviation, included in

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan with IP MeMan 1.

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plexed within the CRD. As even repeated dialysis could not remove BuMan from the protein, it was concluded that this simple alkyl mannoside undergoes a stable interaction with the protein. In fact, van der Waals contacts of the butyl ag- lycon to the phenyl rings of the lectin’s Tyr48 and Tyr137 and to the side chain of Ile52 were found. Today, it can be reasoned that in its complexed conformation, BuMan can mimic the hydrophobic face of an oligosaccharide such as “oligomannose-3” (Figure 2). [28] To screen the ligand prop- erties of further mannosides with a more extended hydro- phobic alkyl aglycon, a series of alkyl α-mannosides was synthesized and tested in SPR experiments and a displace- ment assay using 3 H-labelled mannose (vide supra). [72] As indicated in Table 3, the affinity of the various alkyl α-d- mannosides for FimH increases with the length of the agly- con alkyl chain. The only irregularity in this study was a comparably high RIP value of 440 for heptyl α-d-manno- side (HeptMan), determined in SPR studies. The strong binding to FimH is assumed to result from interactions with the heptyl aglycon. These high affinities of HeptMan for FimH were approved in other studies. [68,84] However, the high value of the MeMan-based RIP(HeptMan) MeMan = 440, determined in the initial SPR study, [72] has not been confirmed in any other test system to date (cf. Table 3).

Mannosides with Aromatic Aglycon Moieties – ππ Interactions with the Tyrosine Gate

It has been known since the 1980s that aromatic aglycon moieties can enhance the affinity of the respective mannos- ides for FimH by a factor of 600 or more. [29] Later, on the basis of the FimH crystal structures, such findings could be rationalized. Thus, ππ stacking interactions of aromatic rings with the amino acid side chains of Tyr48 and Tyr137 of the FimH protein improve the affinity of a carbohydrate ligand. Also oligosaccharides, the natural ligands for FimH, can interact with this tyrosine gate by means of their hydro- phobic sites. [28] To further improve the affinity of pNPMan, extensive studies on variation of the aromatic aglycon of mannoside ligands of FimH have been performed, and some of the results are summarized in Table 4. Variation of the p-sub- stituent of phenyl mannosides had no big effect. It was shown that neither the reduction of the nitro group in pNPMan (leading to pAPMan), nor its deletion (PMan) changed the compound’s inhibitory potency to a great ex- tent. [67,68] While Sharon and co-workers had reported a dif- ference between PMan [RIP(PMan) MeMan = 40] and pNPMan [RIP( pNPMan) MeMan = 70], somewhat later this difference could not be confirmed (cf. Table 4). Only an N- acetylamino substituent in the p-position of phenyl manno- side (pNAcPMan) increased the inhibitory potency by a factor of 4 compared to that of pNPMan. On the other hand, the favourable effect of o-substitution was confirmed, as introduction of a chlorine substituent in the o-position of the phenyl ring to yield pNoClPMan increased the affin- ity for FimH significantly by a factor of 200 [74] and 720. [29]

Monochloro-substituted mannosides, oClPMan, mClPMan and pClPMan did not perform as well as pNoClPMan

when tested in a hemagglutination inhibition assay. Also in

a cyano-substituted series of mannosides, oCNPMan, mCNPMan and pCNPMan, it was confirmed that

mannnosides with o-substituted phenyl aglycon performed best as hemagglutination inhibitors, whereas the p-substi- tuted compounds were the worst inhibitors (Table 4). The high inhibitory potency of MeUmbMan, which had been reported by Sharon three decades ago, was confirmed

in three independent assay systems. [68,72,74] From the X-ray

structure of FimH it can be seen that the aromatic system

in MeUmbMan perfectly fits between the phenyl rings of

the tyrosine gate to build up strong ππ stacking interac- tions. A similar effect could also be achieved with other

fused ring systems, such as naphthalene (for 55). Manno- side 55 has a fivefold higher inhibitory potency than pNPMan. Interestingly, in the applied assay, mannosides

with an ester-substituted thiophene ring fused to the phenyl aglycon (mannoside 56) produced a RIP value of 16 (based

on pNPMan), just as MeUmbMan, though lacking the ex- tended aromatic system of MeUmbMan.

Mannosides with Extended Aromatic Aglycon Moieties – Interactions beyond the Tyrosine Gate?

When MeUmbMan was docked into the FimH binding site in its closed-gate conformation, [72] the pyrone part of the conjugated system was located exactly between the two phenyl rings of the CRD tyrosine gate. This complexation mode explains the high affinity measured for MeUmbMan (Table 4). Extension of the aromatic system by another planar substituent with the ability to establish additional

or tighter ππ interactions with the tyrosine gate seemed a

promising approach towards low-molecular-weight manno- sides with very high affinities for FimH. Thus, a series of mannosides with varied biphenyl aglycon were synthesized and tested in hemagglutination experiments, [68] a selection

of which is shown in Table 5. The biphenyl analogue of

pNPMan, 57, and its m-nitro-substituted analogue 58 sup- ported the initial assumption. The inhibitory potencies of these biphenyl mannosides exceeded that of pNPMan by a factor of 16 for 57, a value that was also found for MeUmb- Man in the same assay (Table 4). The m-nitro-substituted biphenyl mannoside 58 exceeded the inhibitory potency of pNPMan even by a factor of 62. To identify a substitution pattern on biphenyl mannosides that is optimal for FimH binding, the influence of o-, m- and p-cyano-substitution (compounds 59 to 61) was investigated. [68] Other than in the case of phenyl mannosides (cf. Table 4), the inhibitory potency could be best enhanced, namely by a factor of 31 relative to pNPMan, by the introduction of a cyano group into the m-position of the biphenyl system ( 60). CN resi- dues in the o-position led to a 16-fold inhibitory potency, while the p-substituted product 61 had a RIP value of 4 (Table 5). The same inhibitory effect could be achieved, when the biphenyl system was replaced by a benzophenone aglycon (62).

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Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion Table 4. A selection of representative mannosides with

Table 4. A selection of representative mannosides with aromatic aglycon moieties and their testing results.

with aromatic aglycon moieties and their testing results. [a] SD = standard deviation, included in brackets

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan with IP MeMan 1. [c] RIP based on pNPMan with IP p NPMan 1.

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Table 5. A selection of representative mannosides with extended aromatic aglycon moieties and their testing results.

aromatic aglycon moieties and their testing results. [a] SD = standard deviation, included in brackets if

[a] SD = standard deviation, included in brackets if literature-reported. [b] RI = relative inhibitory potency, based on MeMan with IP MeMan 1. [c] RIP based on pNPMan with IP p NPMan 1.

When pNPMan was extended by a squaric acid moiety (mannoside 63), also a very high inhibitory potency relative to pNPMan resulted, with a RIP value of 58 as determined by ELISA. Introduction of a chloro substituent in the o- position (mannoside 64) led to a RIP value of 223 (Table 5), and this could be rationalized by molecular modelling. [74] Somewhat different results for 63 and 64 were obtained later by employing a competitive binding study. [84] When the aglycon portion of mannoside 63 was further extended by short peptide chains, the affinities decreased [RIP(65) p NPMan = 7 and RIP(66) p NPMan = 14]. It has therefore been argued that the potentially reactive squaric acid monoamide moiety in 63 might block the bind- ing pocket permanently by covalent attachment to the N- terminus of the lectin, Phe1. This would imply that the af- finity for the corresponding squaric acid diamide 67 should

be lower than the one for 63. Only recently, Lindhorst et al. could show that this is not the case. [93] On the contrary, the squaric acid diamide 67 exceeded the RIP value of its corresponding monoamide derivative 63 by a factor of 3.

Conformational Flexibility of the Ligand and the Lectin

According to the induced fit model of ligand–receptor binding, it must be considered that both a carbohydrate li- gand and its lectin receptor change their conformation upon formation of a carbohydrate–lectin complex. Such conforma- tional considerations have, however, not yet been extensively investigated for carbohydrate binding of FimH. For a fo- cussed library of cluster mannosides and glycodendrimers, molecular dynamic simulations have been performed that

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Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion provide an impression of the conformational availability of

provide an impression of the conformational availability of the included mannose moieties. [94,95] Interestingly, predic- tions made in these theoretical studies can be aligned with experimental results obtained from inhibition adhesion mea- surements. When α-d-mannosides where clustered as multi- valent glycomimetics, the affinities of the respective glyco- clusters and glycodendrimers for type 1 fimbriated bacteria (cf. Tables 1 and 2) showed moderate to good improve- ment. [44,58,60,61] However, when p-nitrophenyl α-d-mannoside moieties were analogously assembled in order to increase the favourable affinity of pNPMan, testing results were disap- pointing. [44,96] In fact, clustering of pNPMan hardly led to inhibitory potencies that exceeded that of monovalent pNPMan when tested with type 1 fimbriated E. coli. This finding can be explained by results obtained in molecular modelling. [94] Molecular dynamic studies with pNPMan gly- coclusters suggest that there is a pronounced intramolecular ππ stacking of the included phenyl moieties and that the conformational space that is populated by the scaffolded pNPMan moieties is thus considerably limited. If the sugar moiety is less available for complexation with FimH in pNPMan glycoclusters than in monovalent pNPMan, conse- quently favourable multivalency effects cannot be expected in this case, and they have not been measured either. Thus, the conformational features of lectin ligands cer- tainly influence their receptor binding properties. On the other hand, the fimbrial lectin FimH will also experience conformational changes upon ligand binding. [97] Most ob- viously, speculations can be made about the meaning of dif- ferent conformations of the tyrosine gate at the entrance of the FimH CRD. In crystal structures, the tyrosine gate was in an either closed [68,72] or open conformation. [78] Conse- quently, the predictions about ligand binding to FimH, de- duced from docking studies, are different depending on which FimH conformation was taken as the basis of the modelling. [74] Some mannosides show higher affinity to the closed gate conformation of FimH, others show the oppo- site preference. It can be speculated that, depending on the ligand that is available for FimH binding in vivo, the lectin adopts an appropriate conformation in order to bind as tightly as possible. The tyrosine gate in this situation might adopt any conformation between the two extremes “closed” and “open”. Furthermore, it is intriguing to speculate that the conformational flexibility of the entrance domain of the FimH CRD (including the tyrosine gate) might have a func- tion in “selecting” and/or “preorganizing” carbohydrates for binding. Future research might address such hypotheses.

7. Special Approaches to the Inhibition of Mannose-Specific Bacterial Adhesion

From X-ray diffraction analysis and studies on the as- sembly of fimbriae, it seems to be evident that FimH is the type 1 fimbrial lectin, located at the type 1 fimbrial termini and displaying one monovalent carbohydrate recognition domain (CRD) in its lectin domain (FimH L ). Nevertheless, type 1 fimbriae-mediated bacterial adhesion is a multifac-

eted process, which is not yet understood in all of its details. Consequently, there is also room for novel conceptual ap- proaches both to better understand the mechanisms of bac- terial adhesion as well as to further improve the affinities and other advantageous features of inhibitors of bacterial adhesion. As mentioned earlier, higher-valent inhibitors effect an increased avidity compared to that of their monovalent counterparts. These results, obtained with multivalent gly- comimetics as ligands of FimH and as inhibitors of type 1 fimbriae-mediated adhesion of E. coli, [32,33,43] are not in accordance with the monovalent nature of the fimbrial lect- in, though. In addition, clustering of several FimH CRDs by typical multivalent glycomimetics is unlikely, given the size of fimbriae and their distance dimensions on the bacte- rial surface. In order to take advantage from the reported multivalency effects and combine them with the ability of carbohydrate conjugates to cluster the bacterial lectins, mannose residues can be coupled to a more extended scaf- fold. A common technique in biochemistry is to display the ligand on a protein. Some examples of serum albumins functionalized with 8 to 35 mannose residues have been tested in a competitive binding assay with type 1 fimbriated E. coli bacteria. [60] Generally, it was shown that the binding efficiency increased with the degree of mannosylation. Be- sides, as reported for glycoclusters (vide supra), the length and the hydrophilic and steric properties of the linker con- necting the mannose moiety and the protein play a crucial role for the inhibitory potency of the tested compound. The valency-corrected relative inhibitory potencies based on the values obtained for MeMan were in the range of results typically measured for glycoclusters. Most of the tested neo- glycoconjugates had a RIP vc of about 50. [60] Hence, in- creased inhibitory effects, which might arise from lectin clustering or agglutination, were not observed. Only recently, the idea of particle-bound mannosides was resumed. Fullerenes were functionalized with twelve man- noside residues, by using two different linker strategies. [73] It turned out that the valency-corrected inhibitory potenc- ies were unexpectedly low, in the range of 3 (compared to those of the monovalent counterparts). A considerable multivalency effect would have increased the avidity by a much higher factor. On the other hand, it was reported in this study that the dodecamannosylated fullerenes could bind up to seven isolated FimH proteins. Clustering of the isolated form of the lectin domain was thus achieved by this approach. An even more extended scaffold molecule was employed, when mannosylated pseudopolyrotaxanes were introduced in 2010. [98] Three, five or ten cucurbit[6]uril-based mannos- ylated “wheel structures” were threaded on polyviologen strings with approximately eleven viologen units. These pseudorotaxane inhibitors were tested in a hemagglutina- tion inhibition assay. The relative inhibitory potencies of the threaded mannose wheels were reported to be 180 to 300. This is a value typically obtained for a potent mannose cluster. Hence, the desired prominent inhibitory effects of these interesting compounds could not be measured,

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though mannosides were displayed on a voluminous scaf- fold. Another interesting approach to the development of functional FimH ligands addresses the topic of multival- ency from a different point of view. It has been speculated that multiple binding sites might exist on the FimH ad- hesin. [99] This hypothesis has been encouraged by the find- ing that certain carbohydrate ligands exhibit stimulating al- losteric effects on adhesion, [100] which might be explained by the existence of allosteric binding sites or by activation of carbohydrate complexation in an analogy to the catch bond. Although the sequence of the FimH adhesin is highly conserved, studies by Sokurenko and colleagues [101103] have indicated that allelic variation in FimH is correlated with different carbohydrate-binding profiles. However, none of the allelic variations, which gives rise to differences in mannose-binding, occurs within, or even close to the FimH mannose-binding pocket. Again, additional sugar binding sites on the FimH lectin domain could be an explanation for this finding. Multiple binding sites on FimH could aid in recognizing large and multivalent carbohydrate receptors on the host cell surface. Consequently, molecular modelling was performed to identify putative additional carbohydrate-binding pockets on FimH, and this has led to the suggestion of three loca- tions, which might constitute new potential carbohydrate- binding sites on the surface of the FimH lectin domain, in addition to the mannose pocket at the tip of the domain. [99] The hypothesis of multiple carbohydrate binding sites on FimH was tested with a bivalent carbohydrate ligand (Fig- ure 9), [75] which was shaped such that it could concomi- tantly occupy the known FimH CRD and a putative second carbohydrate-binding site with a preference for high-man- nose trisaccharides, as suggested by Knight. [99] This manno- side-trimannoside ligand was meant to clamp two carbo- hydrate-binding sites on FimH, but failed as effective inhib- itor of type 1 fimbriae-mediated bacterial adhesion, when tested by ELISA. Thus, the hypothesis of multiple carbo- hydrate-binding sites on FimH remains unapproved to date. An alternative method to prove multiple binding sites on FimH is photoaffinity labelling of the protein. According to this methodology, mannoside ligands of FimH have to be equipped with a photolabile functional group that can be activated for covalent cross-linking to the lectin upon irradiation. Proteolytic degradation and mass spectrometric analysis can then lead to the identification of receptor loci, which complex carbohydrates. To allow affinity chromatog- raphy with the proteolytic digest of a lectin labelled in this way and to thus facilitate the analysis, biotin can be incor- porated into a photolabile lectin ligand, to be selected by streptavidin-based affinity chromatography. Indeed, photoactive mannoside ligands for FimH have been made available and shown to be suited to covalently label FimH. [76] Future studies will reveal whether photoaf- finity labelling is a suitable methodology to reliably identify carbohydrate-binding sites as well as allosteric sites on FimH and other lectins.

sites as well as allosteric sites on FimH and other lectins. Figure 9. This bivalent glycopeptide

Figure 9. This bivalent glycopeptide ligand was designed to bridge two putative carbohydrate-binding sites on FimH and was synthe- sized by squaric acid conjugation of a branched trimannoside and a simple α-mannoside unit. [75]

8. Conclusions

Lectins have been defined as a class of carbohydrate- binding proteins, which are neither antibodies nor enzymes. They have been discovered in all organisms, from plants and microorganisms to vertebrates including humans. They have been allocated to manifold biochemical processes, and it must be assumed that our understanding of how lectins function is not yet comprehensive. Bacteria use lectins as part of adhesive organelles, called fimbriae or pili, to adhere to the glycocalyx of their target cells. Adherence offers a viability advantage for bacteria, as the cell surface forms an ideal site for multiplication and persistency, providing a reliable environment with stable pH and salt conditions and versatile nutrients to feed on. Pre- venting bacterial adhesion by suitable carbohydrate inhibi- tors has been envisaged as a means against bacterial coloni- zation, infection by pathogens, inflammation and other dis- eases and medical problems caused by adhesive bacteria. Antiadhesives might offer an alternative to antibiotic treat- ment, which is threatened by a growing number of multire- sistant bacterial strains. [19] The vision of an antiadhesion therapy has not yet led to pharmacological applications, but it has greatly motivated current programmes on the devel- opment of high-affinity inhibitors of bacterial lec-

tins. [66,68,93,104]

The type 1 fimbrial lectin FimH is crucial for mannose- specific adhesion of most enterobacteria including UPEC and thus forms a promising target for carbohydrate drug development. While at first various mannosides and natural oligosaccharides were investigated as FimH antagonists, mainly two approaches were taken to develop high-affinity ligands of FimH. Since the first crystal structure of FimH was solved in 1999, [50] FimH ligands have been made (i) according to rational design guided by the crystal structure of the FimH carbohydrate-binding site or (ii) more often, glycoclusters were designed for the multivalent presentation of α-d-mannoside ligands for FimH. Though the bacterial lectin FimH is a monovalent lectin with a carbohydrate-binding site that accommodates just one α-mannosyl residue, frequently favourable effects have been

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Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion observed with multivalent cluster mannosides of various

observed with multivalent cluster mannosides of various architectures. Such multivalency effects have not yet been fully understood in the case of FimH. For steric reasons, it is unlikely that the rather small glycoclusters that have been employed for the inhibition of type 1 fimbriae-mediated bac- terial adhesion can effectively cluster several FimH CRDs. It appears to be more reasonable to conclude on secondary binding sites on the lectin surface or interpret the effect of multivalent FimH ligands on the basis of statistics such as high ligand density in the close proximity of the FimH CRD. For a sensible interpretation of testing results, it is impor- tant to keep in mind the complexity of most testing systems and of the in vivo situation of bacterial adhesion to host cell surfaces. In addition to the lectin–carbohydrate interac- tion, which is “isolated” from the more complex molecular scenario to be used as the basis for rational drug design, any carbohydrate inhibitor also interacts with the surrounding carbohydrate environment. Likewise, the fimbrial lectin is embedded into a possibly favourable environment before an interaction with an added carbohydrate inhibitor occurs. The thermodynamic, enthalpic and entropic, as well as ki- netic circumstances of this multifaceted situation can hardly be sorted out in all detail. Furthermore, the biophysical pa- rameters of carbohydrate binding will be very different whether a hemagglutination assay, an ELISA or SPR mea- surements under flow, among others, are being used to de- termine the inhibitory potency of a particular FimH ligand. Moreover, it was shown that shear forces even influence the structure of the type 1 fimbrial lectin FimH, [87] leading to tighter carbohydrate binding according to a mechanistic principle that has been termed “catch bonds”. [85] All together, it cannot be taken for granted that carbo- hydrate ligands for bacterial lectins can be successfully de- veloped as antiadhesives for an antiadhesion therapy in vivo. The multidimensional in vivo scenario of cellular ad- hesion might prevent the success of the rather one-dimen- sional idea of an antiadhesion therapy. In addition, it could be a critical limitation for the concept to provide enough selectivity not to interfere with physiological cell adhesion. Given that most pathogens, bacteria and viruses, possess different kinds of lectins to use several carbohydrates for adhesion, selectivity issues will form a major challenge for any approach to an antiadhesion therapy. After all, research on lectin ligands is also highly moti- vated by fundamental questions on the mechanism of carbohydrate binding. Thus, it will continue to form an im- portant field in the glycosciences and eventually will lead to new applications in life science. The development of functional glycomimetics and lectin ligands will assist in un- ravelling the secrets of glycobiology and to understand bio- logy beyond genomics and proteomics. The key to under- standing glycobiology will be to identify so far undiscov- ered intrinsic features of carbohydrates, as displayed in the supramolecular environment of living cells.

Abbreviations

AIBN = azobis(isobutyronitrile), ASGPR = asialoglycoprotein re- ceptor, 9-BBN = 9-borabicyclo[3.3.1]nonane, BuMan = butyl α-d-

mannoside, C-HEGA = cyclohexylbutanoyl-N-hydroxyethyl-d-gluc- amide, CRD = carbohydrate recognition domain, DSE = donor strand exchange, E. coli = Escherichia coli, EC 50 = half maximal effective concentration, ELISA = enzyme-linked immunosorbent assay, FACS = fluorescence-activated cell sorting, FimH-CRD-Th- His = construct containing the FimH-CRD, thrombin cleavage site and 6His-tag; FP = fluorescence polarization, Gal = galactose, GalNAc = N-acetylgalactosamine, GFP = green fluorescent pro- tein, HAI = hemagglutination inhibition, HSA = human serum albumin, IC 50 = half maximal inhibitory concentration, IT = inhi- bition titre, ITC = isothermal titration calorimetry, LoVo cells = human colon adenocarcinoma cell line, Man = mannose, MeMan = methyl α-d-mannoside, MeUmbMan = methlyumbelliferyl α-d- mannoside, Neu5Ac = 5- N-acetylneuraminic acid, pNPMan = para-nitrophenyl α-d-mannoside, QSAR = quantitative structure– activity relationship; RIP = relative inhibitory potency, SAM = self-assembled monolayer, SD = standard deviation, SPR = surface plasmon resonance, TEM = transmission electron microscopy, TMSOTf = trimethylsilyl trifluoromethanesulfonate, UPEC = uro- pathogenic Escherichia coli.

Acknowledgments

Our own work on carbohydrate binding of type 1 fimbriated bacte- ria was financed in its first period by the Deutsche Forschungsge- meinschaft (DFG) and later by Christiana Albertina University. Most valuable over the years have been the contributions of the Lindhorst group members, in particular those of Dr. Christoffer Kieburg, Dr. Sven Kötter, Dr. Michael Dubber, Dr. Anupama Pa- tel, Dr. Oliver Sperling, Dr. Christoph Heidecke and for molecular modelling Dr. Andreas Fuchs and Jörn Schmidt-Lassen. Further- more, we are very thankful to our collaborators in this field of our research, Dr. Ulrike Krallmann-Wenzel, Prof. Dr. Stefan Ehlers and Prof. Dr. Stefan Knight.

[1] N. Sharon, H. Lis, Glycobiology 2004, 14, 53R–62R. [2] W. C. Boyd, E. Shapleigh, M. McMaster, Arch. Biochem. Bio- phys. 1955, 55, 226–234. [3] W. C. Boyd, E. Shapleigh, Science 1954, 119, 419. [4] N. Sharon, H. Lis, Science 1972, 177, 949–959. [5] J. B. Sumner, S. F. Howell, J. Bacteriol. 1936 , 32 , 227–237. [6] M. Ambrosi, N. R. Cameron, B. G. Davis, Org. Biomol. Chem. 2005, 3, 1593–1608. [7] K. Drickamer, Curr. Opin. Struct. Biol. 1993, 3, 393–400. [8] K. Drickamer, Curr. Opin. Struct. Biol. 1999, 9, 585–590. [9] H. Lis, N. Sharon, Chem. Rev. 1998, 98, 637–6749. [10] M. J. Blaser, Sci. Am. 2005 , 292 , 38–45. [11] D. Mack, A. P. Davies, L. G. Harris, J. K. M. Knobloch, H. Rohde, Top. Curr. Chem. 2009, 288, 157–182.

[12] R. J. Menaker, N. L. Jones, Microbes Infect. 2003 , 5 , 1149–

1158.

[13] J. K. Savjani, A. K. Gajjar, K. T. Savjani, Mini-Rev. Med. Chem. 2009, 9, 194–205. [14] A. B. Estrela, M. G. Heck, W. R. Abraham, Curr. Med. Chem. 2009, 16, 1512–1530. [15] H. Connell, W. Agace, P. Klemm, M. Schembri, S. Marild, C. Svanborg, Proc. Natl. Acad. Sci. USA 1996, 93, 9827–9832. [16] N. Sharon, Biochim. Biophys. Acta 2006, 1760, 527–537. [17] I. Ofek, D. L. Hasty, N. Sharon, FEMS Immunol. Med. Micro- biol. 2003, 38, 181–191. [18] R. J. Pieters, Med. Res. Rev. 2007 , 27 , 796–816. [19] S. B. Levy, Adv. Drug Delivery Rev. 2005, 57, 1446–1450.

[20] N. Sharon, FEBS Lett. 1987, 217, 145–157. [21] S. D. Knight, J. Bouckaert, Top. Curr. Chem. 2009 , 288 , 67–

107.

Eur. J. Org. Chem. 2011, 3583–3609

© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjoc.org

3607

MICROREVIEW

M. Hartmann, T. K. Lindhorst

[22] T. A. Oelschlaeger, U. Dobrindt, J. Hacker, Curr. Opin. Urol. 2002, 12, 33–38. [23] G. Zhou, W. J. Mo, P. Sebbel, G. W. Min, T. A. Neubert, R. Glockshuber, X. R. Wu, T. T. Sun, X. P. Kong, J. Cell Sci. 2001, 114, 4095–4103. [24] E. F. Boyd, D. L. Hartl, J. Bacteriol. 1999, 181, 1301–1308. [25] N. Firon, I. Ofek, N. Sharon, Carbohydr. Res. 1983, 120, 235–

249.

[26] M. Dubber, O. Sperling, T. K. Lindhorst, Org. Biomol. Chem. 2006, 4, 3901–3912. [27] J.-R. Neeser, B. Koellreutter, P. Wuersch, Infect. Immun. 1986, 52, 428–436. [28] A. Wellens, C. Garofalo, H. Nguyen, N. Van Gerven, R. Slättegård, J. P. Hernalsteens, L. Wyns, S. Oscarson, H. De Greve, S. Hu ltgren, J. Bouckaert, PLoS ONE 2008 , 3 ,

e2040.

[29] N. Firon, S. Ashkenazi, D. Mirelman, I. Ofek, N. Sharon, In- fect. Immun. 1987, 55, 472–476.

[30] J. Beuth, H. L. Ko, G. Pulverer, G. Uhlenbruck, H. Pichlmaier, Glycoconjugate J. 1995, 12, 1–6. [31] Y. C. Lee, R. T. Lee, Acc. Chem. Res. 1995 , 28 , 321–327. [32] M. Lahmann, Top. Curr. Chem. 2009, 288, 17–65. [33] Y. M. Chabre, R. Roy, Adv. Carbohydr. Chem. Biochem. 2010, 63, 165–393. [34] M. Mammen, S.-K. Choi, G. M. Whitesides, Angew. Chem. 1998, 110, 2908–2953; Angew. Chem. Int. Ed. 1998, 37, 2754–

2794.

[35] C. R. Bertozzi, L. L. Kiessling, Science 2001, 291, 2357–2364. [36] L. L. Kiessling, R. A. Splain, Annu. Rev. Biochem. 2010, 79,

619–653.

[60] N. Nagahori, R. T. Lee, S.-I. Nishimura, D. Pagé, R. Roy, Y. C. Lee, ChemBioChem 2002, 3, 836–844. [61] C. Heidecke, T. K. Lindhorst, Chem. Eur. J. 2007 , 13 , 9056–

9067.

[62] O. Sperling, M. Dubber, T. K. Lindhorst, Carbohydr. Res. 2007 , 342, 696–703. [63] A. Mulder, J. Huskens, D. N. Reinhoud, Org. Biomol. Chem. 2004, 2, 3409–3424. [64] F. G. Sauer, M. Barnhart, D. Choudhury, S. D. Knight, G. Waksman, S. J. Hultgren, Curr. Opin. Struct. Biol. 2000 , 10 ,

548–556.

[65] D. Abgottspon, G. Rölli, L. Hosch, A. Steinhuber, X. Jiang,

O. Schwardt, B. Cutting, M. Smiesko, U. Jenal, B. Ernst, A.

Trampuz, J. Microbiol. Methods 2010, 82, 249–255.

[66] T. Klein, D. Abgottspon, M. Wittwer, S. Rabbani, J. Herold,

X. Jiang, S. Kleeb, C. Lüthi, M. Scharenberg, J. Bezencon, E.

Gubler, L. Pang, M. Smiesko, B. Cutting, O. Schwardt, B. Ernst, J. Med. Chem. 2010, 53, 4779–4792. [67] T. K. Lindhorst, S. Kötter, J. Kubisch, U. Krallmann-Wenzel, S. Ehlers, V. Krén, Eur. J. Org. Chem. 1998, 1669–1674. [68] Z. Han, J. S. Pinkner, B. Ford, R. Obermann, W. Nolan, S. A. Wildman, D. Hobbs, T. Ellenberger, C. K. Cusumano, S. J. Hultgren, J. W. Janetka, J. Med. Chem. 2010 , 53 , 4779–4792.

[69] A. Horst, S. Kötter, T. K. Lindhorst, A. Ludwig, E. Brandt, C. Wagener, Med. Microbiol. Immunol. 2001, 90, 145–149. [70] C.-C. Lin, Y.-C. Yeh, C.-Y. Yang, C.-L. Chen, G.-F. Chen, C.-

C. Chen, Y.-C. Wu, J. Am. Chem. Soc. 2002, 124, 3508–3509.

[71] L.-H. Liu, H. Dietsch, P. Schurtenberger, M. Yan, Bioconjugate Chem. 2009, 20, 1349–1355.

[72] J. Bouckaert, J. Berglund, M. Schembri, E. De Genst, L. Cools,

[37] J. J. Lundquist, E. J. Toone, Chem. Rev. 2002 , 102 , 555–578.

8109–8114.

M.

Wuhrer, C.-S. Hung, J. Pinkner, R. Slättegård, A. Zavialov,

[38] E. J. Toone, Curr. Opin. Struct. Biol. 1994 , 4 , 719–728.

D.

Choudhury, S. Langermann, S. J. Hultgren, L. Wyns, P.

[39] Y. Chen, G. Chen, M. H. Stenzel, Macromolecules 2010, 43,

Klemm, S. Oscarson, S. D. Knight, H. De Greve, Mol. Micro- biol. 2005, 55, 441–455.

[40] S. G. Spain, N. R. Cameron, Polym. Chem. 2011, 2, 60–68.

[41] J. E. Gestwicki, C. W. Cairo, L. E. Strong, K. A. Oetjen, L. L. Kiessling, J. Am. Chem. Soc. 2002, 124, 14922–14933. [42] K. Godula, C. R. Bertozzi, J. Am. Chem. Soc. 2010, 132, 9963–

9965.

[43] M. Touaibia, R. Roy, Mini-Rev. Med. Chem. 2007, 7, 1270–

1283.

[44] T. K. Lindhorst, C. Kieburg, U. Krallmann-Wenzel, Glycocon- jugate J. 1998, 15, 605–613. [45] T. K. Lindhorst, Nachr. Chem. Tech. Lab. 1996 , 44 , 1073–1079.

[46] N. Röckendorf, T. K. Lindhorst, Top. Curr. Chem. 2001 , 217 ,

201–238.

[47] W. B. Turnbull, J. F. Stoddart, J. Biotechnol. 2002 , 90 , 231–255. [48] B. König, T. Fricke, A. Waßmann, U. Krallmann-Wenzel, T. K. Lindhorst, Tetrahedron Lett. 1998, 39, 2307–2310. [49] S. Kötter, U. Krallmann-Wenzel, S. Ehlers, T. K. Lindhorst, J. Chem. Soc. Perkin Trans. 1 1998, 2192–2200. [50] D. Choudhury, A. Thompson, V. Stojanoff, S. Langermann, J. Pinkner, S. J. Hultgren, S. D. Knight, Science 1999 , 285 , 1061–

1066.

[51] N. Röckendorf, O. Sperling, T. K. Lindhorst, Aust. J. Chem. 2002, 55, 87–93. [52] T. K. Lindhorst, M. Dubber, U. Krallmann-Wenzel, Eur. J. Org. Chem. 2000, 2027–2034. [53] T. K. Lindhorst, S. Kötter, U. Krallmann-Wenzel, J. Chem. Soc. Perkin Trans. 1 2001, 823–831. [54] M. M. K. Boysen, K. Elsner, T. K. Lindhorst, Eur. J. Org. Chem. 2003, 4376–4386. [55] M. Meldal, C. W. Tornoe, Chem. Rev. 2008, 108, 2952–3015. [56] R. Chinchilla, C. Najera, Chem. Rev. 2007, 107, 874–922. [57] L. F. Tietze, M. Arlt, M. Beller, K. H. Glüsenkamp, E. Jähde, M. F. Rajewsky, Chem. Ber. 1991, 124, 1215–1221.

[58] M. Touaibia, A. Wellens, T. C. Shiao, Q. Wang, S. Sirois, J. Bouckaert, R. Roy, ChemMedChem 2007, 2, 1190–1201. [59] S. G. Gouin, A. Wellens, J. Bouckaert, J. Kovensky, ChemMed- Chem 2009, 4, 749–755.

[73] M. Durka, K. Buffet, J. Iehl, M. Holler, J.-F. Nierengarten, J. Taganna, J. Bouckaert, S. P. Vincent, Chem. Commun. 2011 , 47, 1321–1323. [74] O. Sperling, A. Fuchs, T. K. Lindhorst, Org. Biomol. Chem. 2006, 4, 3913–3922. [75] T. K. Lindhorst, K. Bruegge, A. Fuchs, O. Sperling, Beilstein

J. Org. Chem. 2010, 6, 801–809.

[76] T. K. Lindhorst, M. Märten, A. Fuchs, S. D. Knight, Beilstein

J. Org. Chem. 2010, 6, 810–822.

[77] D. Chessa, C. W. Dorsey, M. Winter, A. J. Bäumler, J. Biol. Chem. 2008, 283, 8118–8124. [78] C.-S. Hung, J. Bouckaert, D. Hung, J. Pinkner, C. Widberg,

A. DeFusco, C. G. Auguste, R. Strouse, S. Langermann, G.

Waksman, S. J. Hultgren, Mol. Microbiol. 2002 , 44 , 903–915. [79] M. Hartmann, A. K. Horst, P. Klemm, T. K. Lindhorst, Chem. Commun. 2010, 46, 330–332.

[80] M. D. Disney, P. H. Seeberger, Chem. Biol. 2004, 11, 1701–

1707.

[81] X. Qian, S. J. Metallo, I. S. Choi, H. Wu, M. N. Liang, G. M. Whitesides, Anal. Chem. 2002, 74, 1805–1810. [82] S. Yu, A. W. Lowe, BMC Gastroenterol. 2009, 9, 58. [83] B. Yang, X. Zhang, X. Bao, Y. Lv, J. Zhang, S. Guo, Food Res. Int. 2008, 41, 594–599. [84] S. Rabbani, X. Jiang, O. Schwardt, B. Ernst, Anal. Biochem. 2010, 407, 188–195. [85] W. Thomas, Annu. Rev. Biomed. Eng. 2008, 10, 39–57.

[86] M. Dembo, D. C. Torney, K. Saxman, D. Hammer, Proc. R. Soc. London Ser. B 1988, 234, 55–83. [87] I. Le Trong, P. Aprikian, B. A. Kidd, M. Forero-Shelton, V. Tchesnokova, P. Rajagopal, V. Rodriguez, G. Interlandi, R. Klevit, V. Vogel, R. E. Stenkamp, E. V. Sokurenko, W. E. Thomas, Cell 2010, 141, 645–655.

[88] W. I. Weis, K. Drickamer, Annu. Rev. Biochem. 1996, 65, 441–

473.

ˇ

[89] E. P. Mitchell, C. Sabin, L. S najdrová, M. Pokorná, S. Perret,

C. Gautier, C. Hofr, N. Gilboa-Garber, J. Kocˇ a, M. Wim-

3608 www.eurjoc.org

© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Org. Chem. 2011, 3583–3609

Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion merová, A. Imberty, Proteins Struct., Funct., Bioinf.

merová, A. Imberty, Proteins Struct., Funct., Bioinf. 2005, 58,

735–746.

[90] K. Ramírez-Gualito, R. Alonso-Ríos, B. Quiroz-García, A. Rojas-Aguilar, D. Díaz, J. Jiménez-Barbero, G. Cuevas, J. Am. Chem. Soc. 2009, 131, 18129–18138. [91] D. B. Walker, G. Joshi, A. P. Davis, Cell Mol. Life Sci. 2009, 66, 3177–3191. [92] H. Remaut, C. Tang, N. S. Henderson, J. S. Pinkner, T. Wang, S. J. Hultgren, D. G. Thanassi, G. Waksman, H. Li, Cell 2008 , 133, 640–652. [93] C. Grabosch, M. Hartmann, J. Schmidt-Lassen, T. K. Lind- horst, ChemBioChem 2011, 12, 1066–1074. [94] C.-W. von der Lieth, M. Frank, T. K. Lindhorst, Rev. Mol. Biotechnol. 2002, 90, 311–337. [95] A. Fuchs, Dissertation, Kiel, 2005. [96] C. Kieburg, Dissertation, Hamburg, 1997. [97] M. Erikson, Dissertation, Kiel, 2010 ; M. Eriksson, T. K. Lind- horst, B. Hartke, to be published.

[98] J. Kim, Y. Ahn, K. M. Park, D.-W. Lee, K. Kim, Chem. Eur. J. 2010, 16, 12168–12173. [99] S. D. Knight, J. Berglund, D. Choudhury, Curr. Opin. Chem. Biol. 2000, 4, 653–660. [100] A. J. Schaeffer, S. K. Amundsen, J. M. Jones, Infect. Immun. 1980, 30, 531–537. [101] E. V. Sokurenko, V. Chesnokova, R. J. Doyle, D. L. Hasty, J. Biol. Chem. 1997, 272, 17880–17886. [102] E. V. Sokurenko, V. Chesnokova, D. E. Dykhuizen, I. Ofek, X.-R. Wu, K. A. Krogfelt, C. Struve, M. A. Schembri, D. L. Hasty, Proc. Natl. Acad. Sci. USA 1998, 95, 8922–8926. [103] M. A. Schembri, E. V. Sokurenko, P. Klemm, Infect. Immun. 2000, 68, 2638–2646. [104] B. Ernst, J. L. Magnani, Nat. Rev. Drug Discovery 2009 , 8 ,

661–677.

Received: March 24, 2011 Published Online: June 15, 2011

Eur. J. Org. Chem. 2011, 3583–3609

© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjoc.org

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