ß-Glutamine-mediated self-association of transmembrane ß-peptides within lipid bilayers.

Transmembrane ß-peptide helices and their association in lipid membranes are still widely unexplored. We designed and synthesized transmembrane ß-peptides harboring different numbers of d-ß3-glutamine residues (hGln) by solid phase peptide synthesis. By means of circular dichroism spectroscopic measurements, the secondary structure of the ß-peptides reconstituted into unilamellar vesicles was determined to be similar to a right-handed 314-helix. Fluorescence spectroscopy using d-ß3-tryptophan residues strongly suggested a transmembrane orientation. Two or three hGln served as recognition units between the helices to allow helix-helix assembly driven by hydrogen bond formation. The association state of the transmembrane ß-peptides as a function of the number of hGln residues was investigated by fluorescence resonance energy transfer (FRET). Therefore, two fluorescence probes (NBD, TAMRA) were covalently attached to the side chains of the transmembrane ß-peptide helices. The results clearly demonstrate that only ß-peptides with hGln as recognition units assemble into oligomers, presumably trimers. Temperature dependent FRET experiments further show that the strength of the helix-helix association is a function of the number of hGln residues in the helix.

Variation of the intercalating proline in artificial peptides mimicking the DNA binding and bending IHF protein.

The integration host factor (IHF) is a protein which sequence specifically induces a bend of double-stranded DNA by more than 160°. Based on IHF as lead structure, a peptide mimic was introduced resembling the positively charged body of the protein by a lysine dendrimer and the minor groove recognition loop by a cyclopeptide. The proline located close to the tip of the recognition loop intercalates between the base pair plane. It was modified in order to evaluate the influence of the side chain residue with respect to size (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), aromaticity (phenylalanine), conformation of the five-membered ring [(4R)-fluoroproline, (4S)-fluoroproline, 3,4-dehydroproline], and the peptide backbone conformation (α-methylproline) on binding dsDNA and bending the double strand. Binding and bending studies were carried out by fluorescence resonance energy transfer experiments and gel electrophoresis using DNA sequences prepared by PCR with the IHF binding site in central or terminal position. Whereas aromatic residues and α-methylproline were not tolerated as proline substitute, incorporation of (4S)-fluoroproline and 3,4-dehydroproline provided enhanced binding.

Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras.

Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the alpha-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy.

Femtosecond time-resolved guanine oxidation in acridine modified alanyl peptide nucleic acids.

Alanyl peptide nucleic acids have been designed to generate linear and rigid pairing complexes. Femtosecond time resolved electron transfer dynamics studies of alanyl-PNA double strands where both strands contain an intercalated 9-amino-6-chloro-2-methoxy-acridine in its protonated state reveal a strong similarity to nearest neighbor interstrand/intrastrand guanine oxidation in the corresponding B-DNA fragment. This observation implies that the combined influence of electronic couplings and energetic parameters, driving force and reorganization energy, on electron transfer dynamics is similar in both structures. With respect to the alanyl-PNA structure, this result is consistent with the notion of stacking distances in the nucleobase staple similar to the one in B-DNA and thus provides additional structural evidence for nucleobase stacking in alanyl-PNA double strands.

Phe and Asn side chains in DNA double strands.

The contribution of amino acid side chains to the recognition of DNA by peptides or proteins is evaluated by substituting single nucleobases of a DNA double strand by amino acid side chains. C-nucleosides with the side chains of phenylalanine and asparagine were synthesized and incorporated in DNA. This modification should allow to keep the double strand conformation. Hydrogen bonds, pi-pi-interactions and solvation have an influence on the double strand stability.

Alanyl-PNA homoduplex: A-T pairing with the N7-regioisomer of adenine.

The N7-regioisomer of adeninyl alanine can be used as a building block for the synthesis of alanyl-PNA. By changing the nucleobase connectivity from N9 to N7, pairing with the Hoogsteen side is no longer possible and the order of donor/acceptor positions at the Watson-Crick side is reversed. This influences the pairing selectivity but not the stability of alanyl-PNA self-pairing complexes, as shown by UV and CD spectroscopy.