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Arginine-rich cell-penetrating peptides (CPPs) including Tat, Penetratin and oligoarginine peptides are a series of short peptides that can be efficiently internalized into cells and have been widely used as carriers for intracellular delivery of bioactive molecules. In the early phase of the study, CPPs, as well as their conjugates, were thought to rapidly enter cells by direct penetration through membranes, which was later found to be an experimental artifact that was concluded from observations in fixed cells. Although re-evaluation using living unfixed cells revealed that endocytosis has a major role in internalization of these peptides, there are a number of studies reporting that, even if fixation is avoided, direct translocation across plasma membranes and cytosolic distribution of arginine-rich CPPs are still observed in cells without membrane perturbation. In addition, amphiphilic counteranions such as pyrenebutyrate dramatically accelerate direct translocation of these peptides into cells. These results suggest that there are at least two pathways, i.e., endocytosis and direct translocation, both of which would contribute to cellular internalization of arginine-rich CPPs. In this review, we first introduce the story of fixation artifact, which indeed led to the critical progress in CPP study, and then summarize the current understanding for direct translocation of arginine-rich CPPs. Comprehensive understanding of direct translocation of these peptides and its mechanistic elucidation would provide useful knowledge for developing methodologies that would enable efficient intracellular delivery.

A new system for the facile reduction of Met(O) in protected peptides (dimethylformamide-sulfur trioxide complex/iodide ion) was introduced. Met(O) in protected Met-enkephalins and protected leucosulfakinins were reduced to Met within 1h by this system, and the reducing ability of this new system seemed comparable to or greater than that of the formerly reported dimethylformamide-sulfur trioxide complex/ethanedithiol system. The methylsulfinylbenzyl protecting group of Tyr was also reduced to a methylthiobenzyl group by this system.

Acidolytic deprotection and cleavage conditions for an acid-labile Tyr(SO3H)-containing peptide were systematically examined with respect to acid, temperature, and scavenger. The 90% aqueous trifluoroacetic acid (TFA)-based reagent systems (90% aqueous TFA/m-cresol and 90% aqueous TFA/m-cresol/2-methylindole) at 4 degrees C were found to minimize the deterioration of Tyr(SO3Na) in the peptide. The latter deprotection/cleavage system was applied to the 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase synthesis of cholecystokinin (CCK)-12 on an acid-labile PAL-linked support (PAL = peptide amide linker), with Fmoc-Tyr(SO3Na)-OH as a building block.

To create artificial ion channels using synthetic peptides is one of the challenges of the de novo design of artificial proteins not only to obtain highly functional artificial molecules but also to understand the basic mechanisms of natural ion channel proteins. Amphiphilic helical peptides of 20 amino acid residues were reported to assemble with each other in lipid bilayers to form ion channels. To obtain more organized channels, efforts have been directed toward controlling the assembly by template molecules. Not only α-helical peptides, but also peptide nanotubes and gramicidin-like molecules were developed as channel molecules where the assembly was stabilized by hydrogen bonding between the molecules. We have developed three approaches to construct artificial helical proteins comprising helices with individual amino acid sequences. Using one of these approaches, four helices corresponding to the voltage sensor (S 4 in repeat I-IV) of the sodium channel were tethered on a peptide template to afford a protein having ion channel activity with rectification.

Artificial transcription factors targeting any desired genes are very attractive from the standpoint of regulating biological functions for life science studies and clinical applications. In order to generate such transcription factors, specific DNA binding domains are required to address a single site for each gene promoter. C(2)H(2) type zinc finger motif is one of the best frameworks to create new artificial DNA binding proteins for the following features: the zinc finger motif can recognize three bases DNA, be tandemly repeated by covalent linkage, and work as a monomer. Taking advantage of these features, manifold zinc finger proteins targeting various DNA sequences have been created so far. For application to a target in sequences as complex as the human genome, the significantly strict specificity in DNA binding must be required. Conjugating multiple fingers (multi-zinc fingers) enables to recognize longer sequences which are sufficient for addressing a single site in the human genome, whereas it has become known that as the number of finger motifs increases, the equilibrium time with the target sequence is significantly longer by in vitro experiments. Our recent study showed that the multi-zinc finger type artificial transcription factor could activate the reporter gene promptly. There is much interest in creating gene regulators, and the artificial transcription factors based on multi-zinc finger motifs could be a superior scaffold.