Research

Synthesis and function of modified biooligomers
Improvement in synthesis of oligonucleotides and peptides/proteins offers bright prospects to access biooligomers with all kind of modifications incorporated in sequences of considerable length. Together with advanced analytical tools, biochemical mechanisms can be addressed next to the identification of artificial binders and inhibitors, the design of model systems, reduction of biochemical complexity, recognition between biooligomers, conformational control and signalling, aggregation, and dynamics like folding or protein motion in a membrane. Artificial nucleotides, amino acids, ligands or reporter groups are prepared by organic synthesis and incorporated in the respective oligomer; different kinds of oligomers are fused to obtain chimera. Creating biooligomers with new properties and recognition motifs in order to elucidate biochemical or pharmacological mechanisms are the main focus of our research.

DNA binding motifs and bending of double-stranded DNA:
The design and preparation of small peptides that specifically interact with double-stranded DNA is of interest with respect to translation and regulative processes. In this regard the natural product triostin A offers an interesting, structurally well-defined scaffold allowing proper orientation of two recognition units (Fig. 1). Triostin A analogues can be synthesized with a broad variety of recognition motifs that are perfectly preorganized regarding DNA binding as bisintercalator, major groove binder or as inhibitor of an abasic site.[1] Abasic sites derived from loss of a nucleobase are known as DNA lesions and intermediates in DNA repair. They are a proper diagnostic target and might be inhibited interfering with the DNA-repair process. In our approach, the recognition motifs and the rigidity of the preorganizing bicyclic peptide were synthetically varied in order to establish new selective and efficient DNA binders.

Figure 1. Natural product triostin A (right) offers a well-defined scaffold preorganizing the recognition
units like acridine and guanine in the mimetic (left).

Figure 2. Synthesis of a peptide mimicking the IHF protein bending DNA by 180° (insert).[8]

Protein modifications:
Recent improvement of solid-phase peptide synthesis using new coupling reagents, resins, amino acid derivatives like pseudoprolines, microwave-assisted synthesis as well as various ligation approaches allow the synthesis of protein domains including synthetic modifications. In some cases a combination of expression and synthesis is especially beneficial. Protein synthesis is applied to the preparation of small knotted proteins, DNA binding of zinc fingers with additional metal binding sites, to the synthesis of artificial SNARE protein domains with transmembrane helices, and to proteins with the potential to induce a conformational switch based on β-sheet formation. Knotted proteins are well-defined by a rigid secondary structure and have a protease resistant scaffold resulting from three interpenetrating disulfide bridges and macrocyclization (Fig. 3). These cyclotides are synthetically accessible with artificial units in a recognition loop, with correct folding by oxidation, with macrocyclization, and the possibility for dimerization.[3] The potential of these artificial cyclotides is derived from the possible use as orally available inhibitors with proper fitting, and perfect presentation of a broad variety of pharmacophors in the inhibitor loop. Synthesis of modified zinc fingers as well-known major groove recognition motif is motivated by introduction of a second, this time functional metal binding position that might function in DNA cleavage by cooperation with the structural zinc.

Figure 3. Cystine-knot microprotein McoTI-II (Momordica cochinchinensis).

Peptid/protein-lipid-interaction:
The arrangement, interaction, organization, and dynamics of peptide helices or barrels in the environment of a lipid bilayer being essential for transmembrane proteins, membrane channel formation and functioning, as well as signalling processes. Investigation on the molecular level is performed using well-defined helix topologies as there are β-peptide helices or helices resulting from antiparallel oriented peptides with alternating amino acid configuration (Fig. 4).[4] Attachment of various recognition elements within the membrane or on the outside of the membrane allow for specific understanding of membrane incorporation, aggregation, organization, lipid-protein interaction, and dynamics. Functional units like membrane pores might be obtained and regulated; transmembrane signalling controlled by conformational or organizational reorganization can be investigated. Membrane fusion mediated by peptides or proteins is investigated by synthesis of chimera derived from the SNARE proteins combining the transmembrane helix with artificial recognition motifs and various linkers. The membrane fusion process is investigated mechanistically based on SNARE analogues on the molecular level. The artificial recognition topologies used for organization were investigated in our laboratory in great detail with respect to topology, recognition and aggregation. Examples are the linear pairing complexes of alanyl peptide nucleic acids[5] or the nucleobase substituted β-peptides (Fig. 5).[6]

Figure 4. Peptides with alternating amino acid configuration incorporated in lipid bilayers as indicated by
X-ray scattering.

Figure 5. Specific organization of two β-peptide helices by covalently attached
nucleobases.

Synthesis of nucleotide derivatives:
Artificial, conformationally restricted nucleotides were prepared and incorporated in oligonucleotides to stabilize DNA conformations or bent DNA.[7] Nucleotides favoring the syn-conformation of purine nucleotides are designed to provide stable DNA in the less favored Z-form. Stabilized Z-DNA will facilitate the search for proteins that specifically recognize this less-favored DNA conformation. In another approach, nucleotide phosphates are synthesized as substrate mimetic or inhibitors of the enzyme orotidine decarboxylase in order to investigate the mechanism of the final step of the pyrimidine nucleoside biosynthesis, the orotidine monophosphate decarboxylation to UMP (Fig. 6).

Figure 6. The orotidine-5'-monophosphate decarboxylase
active site cavity with nucleotide 6-cyano-UMP bound..

References
[1] K. Lorenz, U. Diederichsen, A Solution-Phase Synthesis of Nucleobase-Substituted Analogues of Triostin A, J. Org. Chem., 2004, 69, 3917.
[2] E. Liebler, U. Diederichsen, From the IHF protein to design and synthesis of a sequence-specific DNA bending peptide, Org. Lett., 2004, 6, 2893.
[3] O. Avrutina, H.-U. Schmoldt, D. Gabrijelcic-Geiger, A. Wentzel, H. Frauendorf, C. P. Sommerhoff, U Diederichsen, H. Kolmar, Head-to-tail cyclized cystine-knot peptides by a combined recombinant and chemical route of synthesis, ChemBioChem., 2008, 9, 33.
[4] A. Küsel, Z. Khattari, P. E. Schneggenburger, A. Banerjee, T. Salditt, U. Diederichsen, Conformation and interaction of a d,l-alternating peptide with bilayer membrane: X-ray reflectivity, CD and FTIR spectroscopy, ChemPhysChem., 2007, 8, 2336.
[5] U. Diederichsen, D. Weicherding, N. Diezemann, Side chain homologation of alanyl peptide nucleic acids: pairing selectivity and stacking, Org. Biomol. Chem. 2005, 3, 1058.
[6] A. M. Brückner, P. Chakraborty, S. H. Gellman, U. Diederichsen, Molecular Architecture with Functionalized β-Peptide Helices, Angew. Chem. Int. Ed. 2003, 42, 4395.
[7] D. Heinrich, T. Wagner, U. Diederichsen, Synthesis and DNA incorporation of an ethynyl-bridged cytosine C-nucleoside as guanosine surrogate, Org. Lett., 2007, 5311.
[8] P. A. Rice, S. Yang, K. Mituuchi, H. A. Nash, Crystal Structure of an IHF-DNA Complex: A Protein-Induced DNA U-Turn, Cell 1996, 87, 1295.