Über die Autorin bzw. den Autor

Thomas Schrader is Professor of Organic Chemistry at the University of Marburg. He studied chemistry and obtained his PhD in 1988 under W. Steglich at the Friedrich-Wilhelms-University of Bonn. After a postdoctorate stay at Princeton University under E.C. Taylor on total synthesis of antitumor agents, he gained his lecturing qualification at the University of Dusseldorf. In 2000 he joined the University of Marburg as associate professor. His research focuses on bioorganic aspects of supramolecular systems, optimizing and multiplying new binding motifs for characteristic structural features in biomolecules - a concept that leads to artificial receptor molecules capable of specifically interfering with biological processes. Applications include biosensors, drugs countering protein misfolding and tweezers for protein assembly. Professor Schrader is the holder of the Bredereck-symposium prize in bioorganic chemistry (2001).

Andrew D. Hamilton received his PhD from Cambridge University in 1980, and the following year carried out his postdoc research at Universite Louis Pasteur, Strasbourg. Between 1981 and 1988 he was Assistant Professor for Chemistry at Princeton University, thereafter Associate Professor until 1992, when he became Full Professor at the University of Pittsburgh, a post he held until 1997. Since 1997 he has been Irenee duPont Professor of Chemistry and, since 1998, Professor of Molecular Biophysics and Biochemistry at Yale University. Here he held the Chair of the Chemistry Department between 1999 and 2003, and has been Deputy Provost for Science and Technology since 2003. Professor Hamilton lectures at several universities in the USA.

Von der hinteren Coverseite

A timely overview of this rapidly-expanding topic, covering the most important classes of compounds and incorporating the latest literature. With its application-oriented approach, this book is the first to emphasize current and potential applications, extending to such fields as materials science, bioorganic chemistry, medicinal chemistry, and organic synthesis. In the biological context in particular, the book clarifies which receptor systems work well in water or better under physiological conditions.
From the contents:
* Amino Acid, Peptid and Protein Receptors
* Carbohydrate Receptors
* Ammonium, Amidinium and Guanidinium Receptors
* Anion Receptors
* Molecular Capsules and Self Assembly
* Dynamic Combinatorial Libraries based on Molecular Recognition
* Molecular Machines
* Self-Replication
Aimed at graduate students and specialists in the field, this is also of interest to pharmaceutical companies involved in drug design, as well as chemical companies with a polymer or nanotechnology group. In addition, analytical companies working on the advanced equipment covered here will find stimulating new applications.

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Functional Synthetic Receptors

John Wiley & Sons

Chapter One

Leonard J. Prins and Paolo Scrimin

1.1 Introduction

This chapter focuses on recognition and catalytic processes in which artificial (pseudo) peptide sequences, which can be very short, play a decisive role. The enormous amount of literature related to this topic is far beyond the scope of a single chapter, and, therefore, we intend to emphasize concepts and breakthroughs by using representative examples. Obviously, the reason for the interest in the role of (pseudo)peptides in molecular recognition and catalysis is the fact that polypeptides, e.g. proteins, play a crucial role in practically all biologically relevant processes. An incredible number of recognition events is of key importance for the occurrence of life. The origin of biological recognition is the tertiary structure of proteins, which is marvelously determined by conformationally well-defined secondary structures such as [alpha]-helices, -sheets, coiled coils, etc. These locally structured units give order to the overall system, positioning functional groups precisely in three-dimensional space, thus creating an active site where recognition takes place. Molecular recognition is especially crucial in the functioning of enzymes. To accomplish its powerful tasks an enzyme first needs to recognize the substrate and, subsequently, in the course of its chemical transformation, also the intermediate transition state that lies on the reaction pathway toward the product. These impressive results in Nature form an almost infinite source of inspiration for the chemist, not only to mimic natural functions but also to modify them and apply them in unnatural situations.

In this chapter we will discuss advances that have been made in our learning process from Nature and, more specifically, show how chemists are able to mimic natural functions using artificial synthetic molecules. First, we will focus on the biomolecular recognition of oligonucleotides (DNA/RNA) and protein surfaces by artificial oligopeptides. Next, we will show that chemists have learned to control the secondary structure of (pseudo)peptides and that specific (catalytic) functions can be introduced at will. Finally, we will conclude with a brief overview of the selection of (pseudo)peptide catalysts by a combinatorial approach.

1.2 Recognition of Biological Targets by Pseudo-peptides

1.2.1 Introduction

In all organisms, nucleic acids are responsible for the storage and transfer of genetic information. With the aim of curing gene-originated diseases, artificial molecules that can interact with DNA and RNA are of utmost interest. In this section we will discuss the current state of two major classes of pseudo-peptides that are currently under intense investigation - polyamides that bind in the minor groove of DNA and peptide nucleic acids (PNA). Both classes of compounds are inspired by naturally occurring analogs. The high synthetic accessibility and the ease with which chemical functionality can be introduced illustrate the high potential of artificial pseudo-peptides. In addition, their high biostability has enabled successful applications in both in-vitro and in-vivo studies. The limiting properties of these compounds will also be addressed.

Another way of interfering with biological processes is to obstruct the activity of proteins themselves. Pseudo-peptides that inhibit the formation of protein-protein complexes via competitive binding to the dimerization interface will be discussed. Selected examples will be given that clearly illustrate the strong increase in activity when amino acids present in a wild-type peptide sequence are replaced by artificial amino acids.

1.2.2 Polyamides as Sequence-specif ic DNA-minor-groove Binders

The discovery of the mode of interaction between the natural compounds distamycin and netropsin and the minor groove of DNA has been the impetus for the development of a set of chemical rules that determine how the minor groove of DNA can be addressed sequence-specifically. NMR and X-ray spectroscopy showed that distamycin binds to A,T-tracts 4 to 5 base pairs in length either in a 1:1 or 2:1 fashion, depending on the concentration (Fig. 1.1). It was then immediately realized by the groups of Dickerson, Lown, and Dervan that the minor groove of DNA is chemically addressable and, importantly, that chemical modifications of the natural compounds should, in theory, provide an entry to complementary molecules for each desirable sequence.

1.2.2.1 Pairing Rules

The minor groove of DNA is chemically characterized by several properties. First, the specific positions of hydrogen-bond donor and acceptor sites on each Watson-Crick base pair, as depicted schematically in Fig. 1.2. Next, the molecular shape of the minor groove in terms of specific steric size, such as the exocyclic N[H.sub.2] guanine. Finally, an important property is the curvature of the double stranded DNA helix. Having these properties as a guideline, Dervan and coworkers have developed a series of five-membered heterocycles that pairwise can recognize each of the four base pairs. These couples and their binding modes are schematically depicted in Fig. 1.3. To gain selectivity for a G,C over an A,T base pair, the pyrrole ring (Py) was substituted by an imidazole (Im), which forms an additional hydrogen-bond with the exocyclic N[H.sub.2] of guanine, as confirmed by crystal structure analysis. In addition, replacement of the pyrrole CH for an N eliminates the steric clash of pyrrole and the exocyclic N[H.sub.2] of guanine. The presence of an additional hydrogen-bond acceptor on thymine residues stimulated the synthesis of the N-methyl-3-hydroxypyrrole (Hp) monomer, which contains an additional hydrogen-bond donor. Also, in this case, the complementary molecular shape between the cleft imposed by the thymine-O2 and the adenine-C2 and the bumpy -OH are important.

The selective binding to T,A over A,T base pairs (and, similarly, G,C over C,G) originates from the antiparallel binding of two polyamide strands in the minor groove of DNA. A key NMR spectroscopy study confirmed that an ImPyPy polyamide bound antiparallel in a 2:1 fashion to a 5'-WGWCW-3' sequence (W = A or T) with the polyamide oriented N[right arrow]C with respect to the 5'[right arrow]3' direction of the adjacent DNA strand.

Recently, the repertoire of the heterocycles used (Py, Im, and Hp) has been expanded to novel structures based on pyrazole, thiophene, and furan, to increase binding specificity and stability (the Hp monomer has limited stability in the presence of free acid or radicals). In addition, benzimidazole-based monomers (Ip and Hz) were incorporated in polyamides as alternatives for the dimeric subunits PyIm and PyHp, respectively. DNase I footprinting revealed functionally similar behavior with regard to the parent compounds containing exclusively Py, Im, and Hp monomers. An important advantage is the chemical robustness of the benzimidazole monomer Hz relative to Hp.

1.2.2.2 Binding Affinity and Selectivity

The ternary complex composed of two three-ring structures, such as distamycin, and DNA is rather modest, for entropic reasons and because of the low number of hydrogen bonds involved. In an important step forward towards artificial DNA binders that can effectively compete with DNA-binding proteins, the carboxyl and amino termini of two polyamide chains were covalently connected via a [gamma]-aminobutyric acid linker (Fig. 1.4a). A so-called hairpin polyamide composed of eight heterocycles was shown to bind to the complementary six-base-pair DNA sequence with an affinity constant of the order of [10.sup.10] [M.sup.-1]. A single base-pair mismatch site induced a 10-100-fold drop in affinity. Importantly, the N[right arrow]C orientation with respect to the 5'[right arrow]3' direction of DNA is generally retained for these compounds. An additional tenfold increase in affinity was observed for a cyclic polyamide in which the two strands were covalently connected at both termini (Fig. 1.4b). The [gamma]-turn has a preference for an A,T over a G,C base pair, presumably because of a steric clash between the aliphatic turn and the exocyclic amine of guanine. New polyamide structures that are covalently bridged via the heterocycle nitrogen atoms, either at the center (H-pin, Fig. 1.4c) or terminus (U-pin, Fig. 1.4d) have recently been prepared. The U-pins resulted in a loss in affinity, because of the removal of two hydrogen bond donors, but were insensitive to the base pair adjacent to the turn. Cleverly, the H-pin polyamides were synthesized on a solid support using the Ru-catalyzed alkene metathesis reaction to connect the different polyamides. This approach enabled the rapid synthesis and screening of a series of polyamides with alkyl bridges differing in size [((C[H.sub.2]).sub.n], with n ranging from 4 to 8); the optimum affinity and specificity was obtained for n = 6.

In the gigabase-sized DNA database it is desirable to address sequences of 10-16 base pairs, because these occur much less frequently. Increasing the number of heterocycles in polyamides increases the sequence size that can be targeted, but only up to a certain limit. Studies revealed that the binding affinity is maximized at a contiguous ring number of 5. For longer systems affinity drops because the different curvatures of polyamides and B-DNA starts to give energetically strongly unfavorable interactions. These problems can be partially overcome by replacing one (or more) of the pyrrole units by a more flexible -alanine unit. In this way polyamides have been prepared that bind sequences as long as 11 base pairs with subnanomolar affinities. Alternatively, two hairpin polyamides have been covalently connected either turn-to-turn or turn-to-tail and were shown to bind ten-base-pair sequences with impressive affinities in the order of [10.sup.12] [M.sup.-1]. It should be noted, however, that these high affinities come with rather low selectivity.

The potential of these molecules in controlling gene expression is extremely important; here are examples that illustrate this point. Because excellent reviews have appeared that cover in great detail all results obtained, we will limit ourselves to recent examples that illustrate well the different concepts.

1.2.2.3 DNA Detection

The ability to detect double stranded DNA sequences, and single base-pair mismatches, is an extremely useful tool in the field of genetics. Most methods involve hybridization of single-stranded DNA by a complementary oligonucleotide probe, which carries a signaling moiety. These techniques, however, require denaturation of DNA. On the other hand, double-stranded DNA can be detected by dyes such as ethidium bromide and thiazole orange, but binding is unspecific, making these dyes useful solely as quantitative tools for DNA detection. Dervan and coworkers prepared a series of eight-ring hairpin polyamides with tetramethyl rhodamine (TMR) attached to internal pyrrole rings and studied the fluorescence in the presence and absence of 17-mer duplex DNA. In the absence of DNA, the fluorescence of the conjugates was strongly diminished compared with that of the free dye. This was hypothesized to result from the short linker separating the polyamide and the dye, which enables nonradiative decay of the excited state. The addition of increasing amounts of duplex DNA with a match sequence resulted in an increase in fluorescence until 1:1 DNA:conjugate stoichiometry was reached. Binding of the polyamide fragment to the minor groove of DNA results in forced spacing between polyamide and dye, thus diminishing any quenching effect.

In an impressive study by Laemmli and coworkers the ability of polyamide-dye conjugates to function in a genomic context was demonstrated. A series of tandem polyamides was synthesized that interact specifically with two consecutive insect-type telomeric repeat sequences (TTAGG) (Fig. 1.5). The dissociation constant for the best polyamide was 0.5 nM, as determined by DNase I footprinting. Epifluorescence microscopy studies using Texas Red-conjugated analogs of these polyamides showed a very strong staining of both insect and vertebrate telomeres of chromosomes and nuclei. Convincingly, the telomere-specific polyamide signals of HeLa chromosomes colocalize with the immunofluorescence signals of the telomere-binding protein TRF1. Studies in live Sf9 cells seem to suggest rapid uptake of the conjugates, thus enlarging the potential of these compounds as human medicine. These results should be interpreted with caution, however, because the fluorescence studies were performed after fixation of the cells, which is known to dramatically increase the membrane permeability of cells.

In a related approach, Trask et al. targeted the TTCCA motif repeated in the heterochromatic regions of human chromosomes 9, Y, and 1, using polyamides tagged with fluorescein. Staining of the targeted regions was similar to that with the conventional technique (FISH), which employs hybridization of fluorescent complementary sequences. In sharp contrast, however, polyamide-dye conjugates do not require denaturation of the chromosomes.

1.2.2.4 Gene Inhibition

Gene expression requires recruitment of the transcription machinery to the promoter region, after which transcription of the coding region into mRNA can start. Inhibition of this process by polyamides can occur in either the promoter or coding region of a gene. The latter is more difficult to achieve, because any molecule noncovalently bound to the double helix will be expelled by RNA polymerases during the transcription of DNA. To address this issue polyamides have been tagged with alkylating agents such as chlorambucil and seco-CBI. Indeed it was observed that alkylation occurs specifically at base pairs flanking the binding site of the polyamide. Whether this strategy enables effective inhibition of RNA polymerases has not yet been reported.

Most attention has been paid toward polyamides that act in the promoter region of a gene as competitors for the binding of transcription factors to DNA. Inhibition by polyamides can occur for a variety of reasons. A minor-groove-binding protein can be inhibited by a minor-groove polyamide because of steric hindrance. Binding of a protein with major-groove/minor-groove contacts can be similarly inhibited when the polyamide is crucially located in the minor groove. Alternatively, polyamides can also function as allosteric effectors that rigidify the shape of B-DNA, thus competing with a major-groove binding protein that requires helical distortion. Examples of protein-DNA complexes that have been inhibited by polyamides are TBP, LEF-1, Ets-1, and Zif268. In a key study, the viral HIV-1 gene was targeted (Fig. 1.6). The HIV-1 enhancer/promoter region contains binding sites for multiple transcription factors, among them Ets-1, TBP, and LEF-1. Two different polyamides were designed to target DNA sequences immediately adjacent to the binding sites of these transcription factors. Cell-free assays showed that these ligands specifically inhibited binding of the transcription factors to DNA and consequently repressed HIV-1 transcription. In isolated human peripheral blood cells, incubation with a combination of these two polyamides resulted in 99% inhibition of viral replication, with no obvious decrease in cell viability. RNase protection assays indicated that the transcript levels of some other genes were not affected, suggesting that the polyamides indeed affect transcription directly.

However, despite the success in inhibiting binding of a large variety of proteins to DNA, problems remain with the class of major-groove-binding proteins that are not affected by the presence of ligands in the minor groove. Recent studies have been aimed at a generic solution that would inhibit binding of any sort of transcription factor. Very promisingly, it was observed that attachment of an acridine intercalator to a polyamide locally extended and unwound the double helix and thus acted as an allosteric inhibitor for the major-groove binding of the GCN4 bZip protein.

(Continues...)

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