Masakazu Tanaka

Amphipathic short helix-stabilized peptides with cell-membrane penetrating ability.

We synthesized four types of arginine-based amphipathic nonapeptides, including two homochiral peptides, R-(L-Arg-L-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: FAM-1; R=Ac: Ac-1) and R-(D-Arg-D-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: ent-FAM-1; R=Ac: ent-Ac-1); a heterochiral peptide, R-(L-Arg-D-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: FAM-2; R=Ac: Ac-2); and a racemic mixture of diastereomeric peptides, R-(rac-Arg-rac-Arg-Aib)3-NH2 (R=6-FAM-β-Ala: FAM-3; R=Ac: Ac-3), and then investigated the relationship between their secondary structures and their ability to pass through cell membranes. Peptides 1 and ent-1 formed stable one-handed α-helical structures and were more effective at penetrating HeLa cells than the non-helical peptides 2 and 3.

Cell-penetrating helical peptides having l-arginines and five-membered ring α,α-disubstituted α-amino acids.

Cell-penetrating peptides are powerful tools in the delivery of drugs, proteins, and nucleic acids into cells; therefore, focus has recently been placed on their development. In this study, we synthesized seven types of peptides possessing three l-arginines (l-Arg) and six l-leucines (l-Leu) and/or 1-aminocyclopentane-1-carboxylic acids (Ac5c), and investigated their secondary structures and cell-penetrating abilities. The peptide composed of an equal number of l-Arg, l-Leu, and Ac5c formed 310/α-helical structures in TFE solution and exhibited the highest cell-penetrating ability of all the peptides examined. Additional cellular uptake studies revealed that the incorporation of Ac5c into peptides led to improved tolerability against serum. The results of the present study will help in the design of novel cell-penetrating peptides.

A Helix-Stabilized Cell-Penetrating Peptide as an Intracellular Delivery Tool

Two types of cationic cyclic α,α‐disubstituted α‐amino acids: Api C2NH2 (which possesses a lysine mimic side chain) and ApiC2Gu (which possesses an arginine mimic side chain), were developed. These amino acids were incorporated into an arginine‐based peptide sequence [(l‐Arg‐l‐Arg‐dAA)3: dAA=Api C2NH2 or ApiC2Gu], and the relationship between the secondary structures of the resulting peptides and their ability to pass through cell membranes was investigated. The peptide containing ApiC2Gu formed a stable α‐helical structure and was more effective at penetrating cells than the nonhelical Arg nonapeptide (R9). Furthermore, the peptide was able to deliver plasmid DNA into various types of cells in a highly efficient manner.

Helical-Peptide-Catalyzed Enantioselective Michael Addition Reactions and Their Mechanistic Insights

Helical peptide foldamer catalyzed Michael addition reactions of nitroalkane or dialkyl malonate to α,β-unsaturated ketones are reported along with the mechanistic considerations of the enantio-induction. A wide variety of α,β-unsaturated ketones, including β-aryl, β-alkyl enones, and cyclic enones, were found to be catalyzed by the helical peptide to give Michael adducts with high enantioselectivities (up to 99%). On the basis of X-ray crystallographic analysis and depsipeptide study, the amide protons, N(2)-H and N(3)-H, at the N terminus in the α-helical peptide catalyst were crucial for activating Michael donors, while the N-terminal primary amine activated Michael acceptors through the formation of iminium ion intermediates.

Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment

Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1–5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide 3 may be a useful candidate as a gene delivery carrier.

Helical Peptide-Foldamers Having a Chiral Five-Membered Ring Amino Acid with Two Azido Functional Groups

A chiral five-membered ring α,α-disubstituted α-amino acid (R,R)-Ac5c(dN3) having two azido functional groups has been designed and synthesized. The cyclic amino acid (R,R)-Ac5c(dN3) could be efficiently converted into several cyclic amino acids with various two 1,2,3-triazole functional groups. (R,R)-Ac5c(dN3) homochiral peptides (up to hexapeptide) and (R,R)-Ac5c(dN3)-containing l-Leu-based peptides were prepared, and their conversion of azido functional groups into triazole groups was completed. The preferred conformation of oligomers, before and after the click reaction, together with the azido gauche effect of amino acid residues were studied using FT-IR absorption, CD, (1)H NMR, and X-ray crystallographic analysis. The cyclic amino acid (R,R)-Ac5c(dN3) could be used as a helical conformation controlling residue and also has a versatile functionalizing site in its oligopeptides.

Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids

Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery.

Plasmid DNA delivery using fluorescein-labeled arginine-rich peptides

Arginine (Arg)-rich peptides exhibit an effective cell-penetrating ability and deliver membrane-impermeable compounds into cells. In the present study, three types of Arg-rich peptides, R9 containing nine Arg residues, (RRG)3 containing six Arg and three glycine (Gly) residues, and (RRU)3 containing six Arg and three α-aminoisobutyric acid (Aib) residues, were evaluated for their plasmid DNA (pDNA) delivery and cell-penetrating abilities. The transfection efficiency of R9/pDNA complexes was much higher than those of (RRG)3 and (RRU)3/pDNA complexes, and was derived from the enhanced cellular uptake of R9/pDNA complexes. The replacement of three Arg residues with the neutral amino acid Gly and hydrophobic amino acid Aib drastically changed the cell-penetrating ability and physicochemical properties of peptide/pDNA complexes, resulting in markedly reduced transfection efficiency. A comparison of the R9 peptide administration forms between a peptide alone and peptide/pDNA complex revealed that the uptake of R9 peptides was more efficient for the complex than the peptide alone, but occurred through the same internalization mechanism. The results of the present study will contribute to the design of novel Arg-rich cell-penetrating peptides for pDNA delivery.

Protein Transfection Study Using Multicellular Tumor Spheroids of Human Hepatoma Huh-7 Cells

Several protein transfection reagents are commercially available and are powerful tools for elucidating function of a protein in a cell. Here we described protein transfection studies of the commercially available reagents, Pro-DeliverIN, Xfect, and TuboFect, using Huh-7 multicellular tumor spheroid (MCTS) as a three-dimensional in vitro tumor model. A cellular uptake study using specific endocytosis inhibitors revealed that each reagent was internalized into Huh-7 MCTS by different mechanisms, which were the same as monolayer cultured Huh-7 cells. A certain amount of Pro-DeliverIN and Xfect was uptaken by Huh-7 cells through caveolae-mediated endocytosis, which may lead to transcytosis through the surface-first layered cells of MCTS. The results presented here will help in the choice and use of protein transfection reagents for evaluating anti-tumor therapeutic proteins against MCTS models.

A Cell-Penetrating Peptide with a Guanidinylethyl Amine Structure Directed to Gene Delivery

A peptide composed of lysine with a guanidinylethyl (GEt) amine structure in the side chain [Lys(GEt)] was developed as a cell-penetrating peptide directed to plasmid DNA (pDNA) delivery. The GEt amine adopted a diprotonated form at neutral pH, which may have led to the more efficient cellular uptake of a Lys(GEt)-peptide than an arginine-peptide at a low concentration. Lys(GEt)-peptide/pDNA complexes showed the highest transfection efficiency due to efficient endosomal escape without any cytotoxicity. Lys(GEt)-peptide may be a promising candidate as a gene delivery carrier.