In this review, we describe our approach to creating artificial receptor-channel proteins or sensor systems, using an extramembrane segment conformationally switchable by external stimuli. Alamethicin is known to self-assemble in membranes to form ion channels with various open states. Employment of an α-helical leucine-zipper segment resulted in the effective modulation of the association states of alamethicin to produce a single predominant channel-open state. A decrease in the helical content of the extramembrane segments was found to induce a channel-current increase. Therefore, conformational changes in the extramembrane segments induced by the interaction with ligands can be reflected in the current levels.

Basic peptide-mediated protein delivery into living cells is becoming recognized as a potent approach for the understanding of cellular mechanisms and drug delivery. We have prepared the conjugates of the S-peptide (1–15) derived from RNase S with membrane-permeable basic peptides, octaarginine and the human immunodeficient virus (HIV)-1 Rev (34–50). The RNase S complexes, formed among these S-peptide (1–15)–basic peptide conjugates and the S-protein and having a dissociation constant in the range of 10⁻⁵ M, efficiently penetrated into the HeLa cells. These RNase S complexes exerted an anti-HIV replication activity. The time-of-drug-addition assay suggested that the site of action for these complexes would reside in the stages between the viral entry into the cells and reverse transcription. The present study exemplified the applicability of the arginine-rich peptides to the intracellular targeting of non-covalent protein complexes and supramolecular assemblies for the research in chemical and cellular biology. Copyright © 2004 John Wiley & Sons, Ltd.

A method was developed for synthesizing α,α-disubstituted glycine residues bearing a large (more than 15-membered) hydrophobic ring. The ring-closing metathesis reactions of the dialkenylated malonate precursors proceed efficiently, particularly when long methylene chains tether both terminal olefin groups. Surprisingly, the amino groups of these α,α-disubstituted glycines are inert to conventional protective reactions (e.g., N-tert-butoxycarbonyl (Boc) protection: Boc₂O/4-dimethylaminopyridine (DMAP)/CH₂Cl₂; N-benzyloxycarbonyl (Z) protection: Z-Cl/DMAP/CH₂Cl₂). Curtius rearrangement of the carboxylic acid functionality of the malonate derivative after ring-closing metathesis leads to formation of an amine functionality and can be catalyzed by diphenylphosphoryl azide. However, only the intermediate isocyanates can be isolated, even in the presence of alcohols such as benzyl alcohol. The isocyanates obtained by Curtius rearrangement in an aprotic solvent (benzene) were isolated in high yields and treated with 9-fluorenylmethanol in a high-boiling-point solvent (toluene) under reflux to give the N-9-fluorenylmethoxycarbonyl (Fmoc)-protected aminomalonate derivatives in high yield. These hydrophobic amino acids can be incorporated into a peptide by Fmoc solid-phase peptide synthesis and the acid fluoride activation method. The stability of the monomeric α-helical structure of a 17-amino-acid peptide was enhanced by replacement of two alanine residues with two hydrophobic amino acid residues bearing a cyclic 18-membered ring. The results of sedimentation equilibrium studies suggested that the peptide assembles into hexamers in the presence of 100 mM NaCl.

Delivery of proteins and other macromolecules using membrane-permeable carrier peptides is a recently developed novel technology, which enables us to modulate cellular functions for biological studies with therapeutic potential. One of the most often used carrier peptides is the arginine-rich basic peptide derived from HIV-1 Tat protein [HIV-1 Tat (48–60)]. Using this peptide, efficient intracellular delivery of molecules including proteins, oligonucleic acids and liposomes has been achieved. We have demonstrated that these features were commonly shared among many arginine-rich peptides such as HIV-1 Rev (34–50) and octaarginine. Not only the linear peptides but also branched-chain peptides showed efficient internalization with an optimum number of arginines (approximately eight residues). The structural and mechanistic features of the translocation of these membrane-permeable arginine-rich peptides are reviewed. Copyright © 2003 John Wiley & Sons, Ltd.