Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α-Amino

Publication Date (Web): March 14, 2018. Copyright © 2018 American Chemical Society. *Tel: +81-95-819-2424, Fax: +81-95-819-2424, e-mail: moba@nagasak...
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Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α‑Amino Acids with Basic Functional Groups Takuma Kato,†,‡ Makoto Oba,*,† Koyo Nishida,† and Masakazu Tanaka*,† †

Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Osaka University of Pharmaceutical Sciences, 40-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

‡

S Supporting Information *

ABSTRACT: In the delivery of cell-impermeable molecules, cell-penetrating peptides (CPPs) have been attracting increasing attention as intracellular delivery tools. In the present study, we designed four types of cyclic α,αdisubstituted α-amino acids (dAAs) with basic functional groups on their five-membered rings and different chiralities at the α-position and introduced them into arginine (Arg)-rich peptides. The evaluation of cell-penetrating abilities indicated that these peptides exhibited better cell permeabilities than an Arg nonapeptide. Furthermore, peptides containing dAAs delivered plasmid DNA (pDNA) better than a commercially available transfection reagent with a longer incubation time. These results demonstrate that the introduction of cyclic dAAs with basic functional groups into Arg-rich peptides is an effective strategy for the design of CPPs as a pDNA delivery tool. KEYWORDS: unnatural amino acid, peptide, cell-penetrating peptide, helical structure, plasmid DNA delivery

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structures and cell permeabilities of peptides.21 The cellular uptake of one of the helical CPPs was significantly higher than that of the Tat (48−60) peptide. However, the introduction of hydrophobic Ac5c into Arg-rich peptides led to low solubility against aqueous solution, and thus, their cell permeabilities need to be improved for their utilization as a delivery tool. We herein designed four types of cyclic dAAs (Ac5cNH2 or Ac5cGu) with an amino or guanidino group on their fivemembered rings and different chiralities at the α-position and introduced them into Arg-rich peptides (Figure 1). We expected an additional basic functional group in the side chain of Ac5c to potentially improve the solubilities of Arg-rich peptides and enhance their cell permeabilities. The influence of chirality at the α-position and basic functional group of cyclic dAAs on the cell permeabilities of Arg-rich peptides was also evaluated. The CPPs designed here formed right-handed (P) helices, even in 100% aqueous solution, and showed markedly higher cell permeabilities than an Arg nonapeptide (R9), which is one of the most commonly used CPPs. Differences in chiralities and basic functional groups only had a limited impact on cell permeability. Furthermore, CPPs containing cyclic dAAs delivered pDNA better than a commercially available transfection reagent with a longer incubation time. The present

INTRODUCTION The efficient intracellular delivery of cell-impermeable drugs, proteins, and nucleic acids has been challenging, and many studies have been conducted to develop a delivery tool.1−4 Cellpenetrating peptides (CPPs) have been widely used as vectors to deliver various cell-impermeable compounds including plasmid DNA (pDNA)5−7 into cells since human immunodeficiency virus-1 (HIV-1) Tat protein (position 48−60) was first reported as a CPP.8,9 The Tat (48−60) peptide has six Larginines (L-Arg) and two L-lysines (L-Lys), and these basic amino acids contribute to high cell permeability.10,11 Electrostatic interactions between basic functional groups in peptides and acidic functional groups on the cell surface lead to the absorption of peptides with the cell membrane and their internalization into cells. A guanidino group in the side chain of Arg strongly interacts with a carboxylate, sulfate, and phosphate on cellular components, and thus, Arg-rich peptides and their derivatives bearing guanidino groups have been developed as CPPs.12,13 The stabilization of peptide secondary structures is considered to be one of the promising strategies for increasing the cell permeabilities of Arg-rich peptides by fixing guanidino groups spatially.14−16 Nonproteinogenic amino acids, such as α,α-disubstituted α-amino acids (dAAs),17−20 are often incorporated into relatively short peptides to stabilize secondary structures. We recently reported helical CPPs with five-membered ring dAAs (Ac5c), and revealed that the number of Ac5c in the peptide sequences affected the types of secondary © XXXX American Chemical Society

Received: February 13, 2018 Accepted: March 6, 2018

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DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 1. Structures of cell-penetrating peptides 9−13 designed in the present study. 82.4, 66.8, 64.6, 52.9, 43.4, 38.4, 35.4, 32.1; DART-MS m/z calcd for C16H22NO7S [M+ + H] 372.1117, found 372.1114. Synthesis of (1S,3R)- and (1R,3R)-3-Azido-1(benzyloxycarbonylamino)cyclopentanecarboxylic Acid Methyl Esters ((1S,3R)-3 and (1R,3R)-3). A mixture of (1S,3S)-2 (24.1 g, 64.8 mmol), sodium azide (NaN3) (8.4 g, 129.6 mmol), and 15-crown-5 ether (2.0 mL, 10.1 mmol) in DMF (150 mL) was stirred at 85 °C for 4 h. The solution was then evaporated to remove DMF, diluted with H2O, extracted with EtOAc, and dried over MgSO4. Removal of the solvent gave an oily residue, which was purified by column chromatography (SiO2; 25% EtOAc in n-hexane) to give (1S,3R)-3 (17.5 g, 85%) as a colorless oil: [α]27 D −11.8 (c 1.07, CHCl3); IR (neat) ν 3348, 2955, 2099, 1720, 1520, 1439, 1250 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.34−7.31 (m, 5H), 5.49 (s, 1H), 5.09 (s, 2H), 4.14 (m, 1H), 3.71 (s, 3H), 2.66 (m, 1H), 2.26−2.25 (m, 1H), 2.09 (m, 3H), 1.89 (m, 1H); 13C NMR (400 MHz, CDCl3) δ 174.0, 155.4, 136.2, 128.5, 128.1, 128.0, 66.8, 64.8, 61.6, 52.8, 42.8, 36.0, 31.1; DART-MS m/z calcd for C15H19N4O4 [M+ + H] 319.1406, found 319.1405. The diastereomeric (1R,3R)-3 was also obtained from (1R,3S)-2 in 95% yield by the same procedure as that described above: [α]20 D +2.38 (c 1.01, CHCl3); IR (neat) ν 3343, 2953, 2100, 1717, 1522, 1456, 1261 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.36−7.30 (m, 5H), 5.62 (s, 1H), 5.07 (s, 2H), 4.13 (m, 1H), 3.72 (s, 3H), 2.42 (m,1H), 2.36− 2.27 (m, 2H), 2.12−2.09 (m, 1H), 1.99 (m, 1H), 1.85−1.82 (m, 1H); 13 C NMR (400 MHz, CDCl3) δ 174.0, 155.4, 136.0, 128.5, 128.2, 128.0, 66.8, 64.7, 61.0, 52.9, 42.6, 35.8, 31.3; ESI-MS m/z calcd for C15H18N4O4Na [M+ + Na] 341.1226, found 341.1245. Synthesis of (1S,3R)- and (1R,3R)-3-Amino-1-benzyloxycarbonyl)aminocyclopentanecarboxylic Acid Methyl Esters ((1S,3R)-4 and (1R,3R)-4). A mixture of (1S,3R)-3 (2.2 g, 6.9 mmol), PPh3 (7.2 g, 27.5 mmol), and H2O (0.5 mL, 27.5 mmol) in THF (80 mL) was stirred at 60 °C for 8 h. The solution was then evaporated to remove THF, acidified with 1 M aqueous HCl, and washed with Et2O. The aqueous solution was then alkalified with saturated aqueous NaHCO3, extracted with CHCl3, and dried over MgSO4. Removal of the solvent gave an oily residue, which was purified by column chromatography (SiO2; 10% MeOH in CHCl3) to give (1S,3R)-4 (16.1 g, 94%) as amorphous: [α]24 D +7.63 (c 1.07, CHCl3); IR (neat) ν 3356, 3032, 2951, 1720, 1523, 1438, 1257 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.36−7.31 (m, 5H), 5.98 (br s, 1H), 5.10 (s, 2H), 3.72 (s, 3H), 3.65− 3.63 (m, 1H), 2.43−2.37 (m, 1H), 2.29−2.24 (m, 1H), 2.10 (m, 2H), 1.74 (m, 1H), 1.57 (m, 3H); 13C NMR (400 MHz, CDCl3) δ 174.4, 155.4, 136.3, 128.4, 128.0, 128.0, 66.6, 65.5, 52.6, 52.2, 46.4, 35.7, 34.3; ESI-MS m/z calcd for C15H20N2O4Na [M+ + Na] 315.1321, found 315.1295. The diastereomeric (1R,3R)-4 was also obtained from (1R,3R)-3 in 93% yield by the same procedure as that described above: [α]24 D +0.69 (c 1.08, CHCl3); IR (neat) ν = 3354, 3032, 2953, 1716, 1525, 1456, 1265 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.32−7.26 (m, 5H), 5.50 (br s, 1H), 5.06 (s, 2H), 3.70 (s, 3H), 3.58 (m, 1H), 2.38−2.31 (m, 2H), 2.08−2.01 (m, 2H), 1.96 (m, 3H), 1.54 (m, 1H); 13C NMR (400 MHz, CDCl3) δ 174.8, 155.4, 136.2, 128.4, 128.1, 128.0, 66.6, 65.2,

results demonstrate that the introduction of cyclic dAAs with basic functional groups into Arg-rich peptides is an effective strategy for the design of CPPs as a pDNA delivery tool.

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MATERIALS AND METHODS

General. Optical rotations [α]rtD were recorded on a JASCO DIP370 polarimeter (JASCO, Tokyo, Japan) using a 0.5 dm cell. Infrared (IR) spectra were measured with a Shimadzu IRAffinity-1 spectrometer (Shimadzu Corporation, Kyoto, Japan) for conventional measurements (neat or KBr). 1H NMR and 13C NMR spectra were examined with a JEOL AL 400 (JEOL Ltd., Tokyo, Japan). MALDITOF-MS spectra were taken on an Ultraflex (Bruker Daltonics, Fermont, CA). All commercial materials were used without further purification. Dulbecco’s modified Eagle’s medium (DMEM) was a product of Sigma-Aldrich Co. (St. Louis, MO). Cell lysis buffer M was a product of Wako Pure Chem. Co., Inc. (Osaka, Japan). A micro-BCA protein assay reagent kit was a product of Pierce (Rockford, IL). The plasmid pCAcc+Luc, coding for firefly luciferase under the control of the CAG promoter, was provided by the RIKEN Gene Bank (Tsukuba, Japan) and amplified and purified by GenScript (Tokyo, Japan). The luciferase assay kit was purchased from Promega (Madison, WI). Hoechst 33342 was obtained from Dojindo Laboratories (Kumamoto, Japan). LysoTracker Red was a product of Molecular Probes (Eugene, OR). pDNA was labeled with Cy5 (Cy5-pDNA) using the Label IT Tracker Intracellular Nucleic Acid Localization Kit; Cy5 was obtained from Mirus Bio LLC (Madison, WI). Synthesis of Fmoc-Protected dAAs. Synthesis of (1S,3R)- and (1R,3R)-1-(Benzyloxycarbonyl)amino-3-(methanesulfonyloxy)cyclopentanecarboxylic Acid Methyl Esters ((1S,3S)-2 and (1R,3S)2). A mixture of (1S,3S)-122 (6.4 g, 21.9 mmol), triethylamine (Et3N) (6.1 mL, 43.8 mmol), and methanesulfonyl chloride (MsCl) (3.4 mL, 43.8 mmol) in CH2Cl2 (160 mL) was stirred. After being stirred at room temperature for 21 h, the solution was washed with 1 M aqueous HCl, saturated aqueous NaHCO3, and brine and dried over MgSO4. Removal of the solvent gave an oily residue, which was purified by column chromatography (SiO2; 50% EtOAc in n-hexane) to give (1S,3S)-2 (7.18 g, 88%) as a colorless oil: [α]23 D −2.34 (c 1.04, CHCl3); IR (neat) 3363, 3030, 2954, 1732, 1519, 1352, 1257, 1170, 1112 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.39−7.35 (m, 5H), 5.39 (s, 1H), 5.30 (m, 1H), 5.09 (s, 2H), 3.74 (s, 3H), 3.02 (s, 3H), 2.65− 2.63 (m, 2H), 2.41−2.27 (m, 2H), 2.17−2.10 (m, 1H), 2.04 (m, 1H); 13 C NMR (400 MHz, CDCl3) δ 173.5, 155.4, 136.0, 128.5, 128.2, 128.0, 81.5, 66.8, 64.5, 52.9, 43.5, 38.3, 35.1, 32.3; DART-HRMS m/z calcd for C16H22NO7S [M+ + H] 372.1117, found 372.1135. The diastereomeric (1R,3S)-2 was also obtained from (1R,3S)-1 in 92% yield by the same procedure as that described above: [α]22 D +9.73 (c 1.05, CHCl3); IR (neat) ν 3367, 3032, 2954, 1765, 1519, 1357, 1249, 1166, 1130 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.35−7.31 (m, 5H), 5.49 (s, 1H), 5.25 (m, 1H), 5.09 (s, 2H), 3.72 (s, 3H), 2.95 (s, 3H), 2.80−2.75 (m, 2H), 2.41−2.37 (m, 1H), 2.28−2.16 (m, 4H); 13C NMR (400 MHz, CDCl3) δ 173.7, 155.4, 136.2, 128.5, 128.2, 128.1, B

DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering 52.7, 52.2, 46.7, 36.1, 34.9; ESI-MS m/z calcd for C15H20N2O4Na [M+ + Na] 315.1321, found 315.1290. Synthesis of (1S,3R)- and (1R,3R)-1-(Benzyloxycarbonyl)amino-3(tert-butoxycarbonyl)aminocyclopentanecarboxlyil acid methyl esters ((1S,3R)-5 and (1R,3R)-5). A solution of NaHCO3 (0.6 g, 7.1 mmol) and NaCl (1.0 g, 17.1 mmol) in H2O (20 mL) was added to a stirred solution of (1S,3R)-4 (1.9 g, 6.5 mmol) and di-tert-butyl dicarbonate (1.6 mL, 7.1 mmol) in CHCl3 (25 mL) and the solution was refluxed for 12 h. The solution was then extracted with CHCl3 and dried over MgSO4. Removal of the solvent gave a residue, which was purified by column chromatography (SiO2; 10% MeOH in CHCl3) to give (1S,3R)-5 (2.3 g, 91%) as amorphous: [α]25 D −3.21 (c 1.09, CHCl3); IR (KBr) ν 3356, 2976, 1713, 1522, 1456, 1250 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.34 (m, 5H), 5.94 (br s, 1H), 5.43 (br s, 1H), 5.09 (s, 2H), 4.19 (m, 1H), 3.71 (s, 3H), 2.61−2.56 (m,1H), 2.14 (m, 3H), 1.91 (m, 1H), 1.75 (m, 1H), 1.43 (s, 9H); 13C NMR (400 MHz, CDCl3) δ 174.6, 155.3, 136.1, 128.4, 128.0,128.0, 79.1, 66.6, 64.8, 52.7, 51.2, 43.3, 35.3, 32.6, 28.3; DART-MS m/z calcd for C20H28N2O6Na [M+ + Na] 415.1845, found 415.1829. The diastereomeric (1R,3R)-5 was also obtained from (1R,3R)-4 in 93% yield by the same procedure as that described above: [α]25 D +2.35 (c 1.08, CHCl3); IR (KBr) ν 3325, 2978, 1720, 1620, 1419, 1253 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.35−7.31 (m, 5H), 5.41 (s, 1H), 5.08 (s, 2H), 5.02 (br s, 1H), 4.25 (br s, 1H), 3.72 (s, 3H), 2.31− 2.14 (m, 4H), 1.91 (br s, 1H), 1.67−1.64 (m, 1H), 1.43 (s, 9H); 13C NMR (400 MHz, CDCl3) δ 175.0, 155.5, 155.3, 136.2, 128.4, 128.1, 128.0, 79.2, 66.7, 64.7, 52.8, 51.0, 36.1, 32.2, 28.3; DART-MS m/z calcd for C20H29N2O6 [M+ + H] 393.2026, found 393.2019. Synthesis of (1S,3R)- and (1R,3R)-3-(tert-Butoxycarbonyl)amino1-(9-fluorenylmethoxycarbonyl)aminocyclopentanecarboxylic Acids ((1S,3R)-6 and (1R,3R)-6). A solution of 0.2 M aqueous NaOH (24.8 mL, 4.96 mmol) was added to a stirred solution of (1S,3R)-5 (973 mg, 2.48 mmol) in MeOH (25 mL). After being stirred at room temperature for 12 h, the solution was acidified with 1 M aqueous HCl and evaporated to remove MeOH. The aqueous solution was extracted with CHCl3, dried over MgSO4, and evaporated to give a crude carboxylic acid (938 mg, quantitative). A mixture of the crude carboxylic acid (938 mg, 2.48 mmol) and 10% Pd−C (100 mg) in MeOH (10 mL) was vigorously stirred under a H2 atmosphere at room temperature for 5 h. The Pd−C catalyst was filtered off, and the filtrate was evaporated to give a crude amino acid (572 mg, 94%). A mixture of the crude amino acid (572 mg, 2.34 mmol) and diisopropylethylamine (DIPEA) (1.43 mL, 8.20 mmol) in CH2Cl2 (50 mL) was stirred at room temperature for 30 min. After adding TMS-Cl (0.60 mL, 4.69 mmol), the solution was refluxed for 3 h. Fmoc-Cl (606 mg, 2.34 mmol) was then added to the stirred solution at −20 °C, and the solution was stirred at room temperature for 20 h. The solution was diluted with 1% aqueous HCl, extracted with CHCl3, and dried over MgSO4. Removal of the solvent gave a white solid, which was purified by column chromatography (SiO2; 10% MeOH in CHCl3) to give (1S,3R)-6 (543 mg, 47%) as colorless crystals: mp 91−92 °C; [α]23 D +1.70 (c 1.06, CHCl3); IR (KBr) ν 3337, 2978, 1686, 1512, 1450, 1254 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.3 Hz, 2H), 7.58 (d, J = 5.4 Hz, 2H), 7.37 (t, J = 7.3 Hz, 2H), 7.28 (t, J = 7.3 Hz, 2H), 6.19 (br s, 1H), 5.45 (br s, 1H), 4.36 (m, 2H), 4.19 (m, 2H), 2.61 (m, 1H), 2.17 (m, 3H), 2.01 (m, 1H), 2.00 (br s, 1H), 1.82 (m, 1H), 1.45 (s, 9H); 13C NMR (400 MHz, CDCl3) δ 177.8, 155.8, 155.4, 143.8, 141.3, 127.7, 127.1, 125.1, 120.0, 79.7, 66.8, 64.4, 51.7, 47.1, 42.9, 35.1, 32.7, 29.7, 28.4; ESI-MS m/z calcd for C26H31N2O6 [M+ + H] 467.2182, found 467.2153. The diastereomeric (1R,3R)-6 was also obtained from (1R,3R)-5 in 27% yield by the same procedure as that described above: mp 92−93 °C; [α]24 D +1.12 (c 0.99, CHCl3); IR (KBr) ν 3414, 2976, 1697, 1508, 1450, 1252 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.3 Hz, 2H), 7.57 (d, J = 6.8 Hz, 2H), 7.38 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.3 Hz, 2H), 5.96 (br s, 1H), 5.66 (br s, 1H), 4.37 (br s, 2H), 4.25 (m, 1H), 4.19 (m, 1H), 2.35 (m, 3H), 2.17 (m, 1H), 2.00 (m, 1H), 1.74 (m, 1H), 1.45 (s, 9H); 13C NMR (400 MHz, CDCl3) δ 178.2, 157.2, 155.5, 143.8, 141.3, 127.7, 127.1, 125.1, 119.9, 79.5, 66.8, 64.7, 51.3,

47.1, 43.6, 36.1, 33.4, 32.4, 28.4; ESI-MS m/z calcd for C26H31N2O6 [M+ + H] 467.2182, found 467.2175. Synthesis of (1S,3R)- and (1R,3R)-1-(Benzyloxycarbonyl)amino-3(di-tert-butoxycarbonyl)guanidinocyclopentanecarboxylic Acid Methyl Esters ((1S,3R)-7 and (1R,3R)-7). A solution of (1S,3R)-4 (933 mg, 3.19 mmol) in CH2Cl2 (20 mL) was added to a stirred solution of N,N’-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (1.04 g, 3.36 mmol) and Et3N (467 μL, 3.36 mmol) in CH2Cl2 (20 mL), and the solution was then stirred at room temperature for 3 days. The solution was evaporated to give a residue that was purified by column chromatography (SiO2; 30% EtOAc in nhexane) to give (1S,3R)-7 (1.5 g, 89%) as colorless crystals: mp 150− 155 °C; [α]21 D +5.81 (c 1.06, CHCl3); IR (KBr) ν 3383, 2982, 1735, 1620, 1412, 1373, 1261 cm−1; 1H NMR (400 MHz, CDCl3) δ 11.47 (s, 1H), 8.59 (br s, 1H), 7.36−7.35 (m, 5H), 5.56 (br s, 1H), 5.12− 5.07 (m, 2H), 4.63 (m, 1H), 3.71 (s, 3H), 2.70−2.65 (m, 1H), 2.22− 2.13 (m, 4H), 1.84 (m, 1H), 1.49 (s, 9H), 1.47 (s, 9H); 13C NMR (400 MHz, CDCl3) δ 174.0, 163.4, 155.6, 155.3, 153.1, 136.2, 128.4, 128.0, 127.9, 83.0, 79.2, 66.7, 65.1, 52.6, 50.8, 43.1, 35.9, 31.9, 28.2, 28.0; DART-MS m/z calcd for C26H39N4O8 [M+ + H] 535.2768, found 535.2805. The diastereomeric (1R,3R)-7 was also obtained from (1R,3R)-4 in 91% yield by the same procedure as that described above: mp 149− 156 °C; [α]22 D −7.70 (c 1.07, CHCl3); IR (KBr) ν 3323, 2980, 1720, 1612, 1417, 1337, 1254 cm−1; 1H NMR (400 MHz, CDCl3) δ 11.49 (s, 1H), 8.62 (br s, 1H), 7.34 (m, 5H), 5.55 (br s, 1H), 5.08 (s, 2H), 4.75−4.74 (m, 1H), 3.76 (s, 3H), 2.43−2.23 (m, 4H), 1.97 (m, 1H), 1.72 (m, 1H), 1.49 (s, 18H); 13C NMR (400 MHz, CDCl3) δ 174.3, 163.5, 155.5, 155.5, 153.0, 136.2, 128.4, 128.0, 128.0, 83.0, 79.2, 66.7, 64.8, 52.8, 43.7, 50.7, 36.0, 31.9, 28.2, 28.0; ESI-MS m/z calcd for C26H39N4O8 [M+ + H] 535.2768, found 535.2729. Synthesis of (1S,3R)- and (1R,3R)-3-(Di-tert-butoxycarbonyl)guanidino-1-(9-fluorenylmethoxycarbonyl)aminocyclopentanecarboxylic Acids ((1S,3R)-8 and (1R,3R)-8). A solution of 0.1 M aqueous NaOH (26.7 mL, 2.67 mmol) was added to a stirred solution of (1S,3R)-7 (714 mg, 1.34 mmol) in MeOH (25 mL), and the solution was then stirred at room temperature for 11 h. The solution was acidified with 1 M aqueous HCl and evaporated to remove MeOH. The aqueous solution was extracted with CHCl3, dried over MgSO4, and evaporated to give a crude carboxylic acid (695 mg, quantitative). A mixture of the crude carboxylic acid (695 mg, 1.34 mmol) and 5% Pd−C (400 mg) in MeOH (30 mL) was vigorously stirred under a H2 atmosphere at room temperature for 7 h. The Pd− C catalyst was filtered off, and the filtrate was evaporated to give a crude amino acid (516 mg, quantitative). A mixture of the crude amino acid (516 mg, 1.34 mmol) and DIPEA (814 μL, 4.67 mmol) was stirred at room temperature for 30 min. After adding TMS-Cl (339 μL, 2.67 mmol), the solution was refluxed for 3 h. Fmoc-Cl (346 mg, 1.34 mmol) was then added to the stirred solution at −20 °C, and the solution was stirred at room temperature for 14 h. The solution was diluted with 1% aqueous HCl, extracted with CHCl3, and dried over MgSO4. Removal of the solvent gave a white solid, which was purified by column chromatography (SiO2; 5% MeOH in CHCl3) to give (1S,3R)-8 (702 mg, 86%) as colorless crystals: mp 112−114 °C; [α]27 D +8.33 (c 1.03, CHCl3); IR (KBr) ν 3321, 2982, 2554, 1709, 1609, 1412, 1339, 1254 cm−1; 1H NMR (400 MHz, CDCl3) δ 11.62 (br s, 1H), 8.80 (br s, 1H), 7.75 (d, J = 7.3 Hz, 2H), 7.60 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.3 Hz, 2H), 7.30 (t, J = 7.3 Hz, 2H), 6.66 (br s, 1H), 6.00 (s, 1H), 4.69 (m, 1H), 4.36 (m, 2H), 4.24−4.21 (m, 1H), 2.53 (m, 1H), 2.25 (m, 4H), 1.99 (m, 1H), 1.48 (s, 9H), 1.47 (s, 9H); 13C NMR (400 MHz, CDCl3) δ 176.1, 162.5, 155.9, 155.1, 153.0, 143.8, 141.3, 127.6, 127.0, 125.1, 119.9, 83.6, 80.1, 66.6, 63.8, 51.9, 47.2, 42.0, 35.3, 32.6.28.1, 28.0; DART-MS m/z calcd for C32H41N4O8 [M+ + H] 609.2924, found 609.2971. The diastereomeric (1R,3R)-8 was also obtained from (1R,3R)-7 in 60% yield by the same procedure as that described above: mp 113− 114 °C; [α]25 D −6.63 (c 1.06, CHCl3); IR (KBr) ν 3323, 2980, 2594, 1720, 1612, 1418, 1337, 1254, 1138, 760, 741 cm−1; 1H NMR (400 MHz, CDCl3) δ 11.47 (br s, 1H), 8.70 (br s, 1H), 7.69 (d, J = 7.3 Hz, 2H), 7.55 (d, J = 6.3 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.25 (t, J = 7.3 C

DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering Scheme 1. Synthesis of Fmoc-Protected dAAs 6 and 8

PBS or 0.00025 w/v% Pronase was incubated at 37 °C. After each incubation time, 100 μL of peptide solution was taken and diluted with 250 μL of 1% TFA/PBS to inactivate proteases. Fifty microliters of saturated α-cyano-4-hydroxycinnamic acid aqueous solution was added as an internal standard followed by the RP-HPLC analysis. Preparation of Peptide/pDNA Complexes. Peptide/pDNA complexes were prepared as previously reported.23 The N/P ratio used in this paper was defined as the residual molar ratio of the amino and/or guanidino groups of amino acids in the peptide to the phosphate groups of pDNA. Fluorescence Measurements. The fluorescence intensities of peptide/pDNA solutions prepared at various N/P ratios were measured using a spectrofluorometer (ND-3300). Results are presented as the mean and standard deviation of 3 measurements. Size and Zeta-Potential Measurements. The size and size distribution of the peptide/pDNA complexes in 10 mM Hepes buffer (pH 7.3) were evaluated by dynamic light scattering (DLS) using Nano ZS (ZEN3600, Malvern Instruments, Ltd., UK) with an incident light (633 nm). Measurements were carried out with a detection angle of 173° and temperature of 25 °C, and data were subsequently analyzed by the cumulant method. The zeta-potential was evaluated using the same apparatus by the laser-Doppler electrophoresis method. Results were presented as a mean and standard deviation of three measurements. Transfection. Huh-7 cells were seeded on 96-well culture plates (2,500 cells/well) and incubated for 24 h in 100 μL of DMEM containing 10% FBS. The medium was exchanged with fresh medium, and the peptide/pDNA complexes prepared at N/P ratios = 2, 4, and 8, naked pDNA, and transfection reagent TurboFect/pDNA, prepared according to the manufacturer’s protocol, were applied to each well at a concentration equivalent to 0.25 μg of pDNA per well. The cells were incubated for 24 h, and then the medium was replaced with fresh medium. Following further incubations for the indicated times (postincubation time), luciferase gene expression was assessed by photoluminescence intensity using a luminometer (Gene Light GL210A, Microtec, Co., Ltd., Chiba, Japan) and luciferase assay kit. The amount of protein in each well was concomitantly assessed by a Micro BCA protein assay kit. Results are presented as the mean and standard deviation of four samples. Cell Viability. The cell viability of Huh-7 cells treated with peptide/pDNA complexes, naked pDNA, and TurboFect/pDNA complex were evaluated as previously reported.23 The results are presented as the mean and standard deviation obtained from six

Hz, 2H), 6.43 (br s, 1H), 6.12 (s, 1H), 4.66 (m, 1H), 4.29 (m, 2H), 4.12 (t, J = 6.6 Hz, 1H), 2.47 (m, 2H), 2.25 (m, 2H), 2.05 (m, 1H), 1.70 (m, 1H), 1.49 (s, 9H), 1.46 (s, 9H); 13C NMR (400 MHz, CDCl3) δ 177.6, 163.1, 155.9, 155.7, 153.0, 143.7, 141.2, 127.6, 127.0, 125.1, 119.8, 83.2, 79.5, 66.8, 64.5, 50.7, 47.1, 43.4, 35.6, 31.8, 28.2, 28.0; ESI-MS m/z calcd for C32H40N4O8Na [M+ + Na] 631.2744, found 631.2758. Synthesis of Peptides. Peptides were synthesized on a solid support by Fmoc solid-phase methods using commercially available Rink amide resin and Fmoc-amino acids, as previously reported.21 HATU/HOAt (for peptides 9−11 and 13) and COMU (for peptide 12) were used as coupling reagents. The crude peptides were purified by reverse-phase (RP)-HPLC using COSMOSIL Packed Column 5C18-AR-II (20 ID × 250 nm) (Nacalai, Japan). The purified peptides were characterized by analytical RP-HPLC (COSMOSIL Packed Column 5C18-AR-II, 4.6 ID × 250 nm) and MALDI-TOF-MS (Bruker Daltonics Ultraflex, Fremont, CA). The RP-HPLC conditions described here were the same as previously reported.21 CD Spectrum Measurement. CD spectra were examined with a JASCO J-725N spectropolarimeter (JASCO) using a 1.0 mm path length cell. Data are expressed in terms of [θ]R, residue molar ellipticity (deg cm2 dmol−1). Phosphate buffer (20 mM; pH 7.4) was used as a solvent. Cellular Uptake of Peptides. Huh-7 cells were seeded on 24-well culture plates (40,000 cells/well) and incubated for 24 h in 400 μL of DMEM containing 10% fetal bovine serum (FBS). After a 24 h incubation, the medium was replaced with fresh medium containing 10% FBS. A peptide solution was added at a concentration of 2 μM, and the cells were incubated for the appropriate amount of time. Following each time incubation, the medium was removed, and the cells were washed with ice-cold PBS and trypsinized. After being diluted with fresh medium, the cells were moved to a microtube and centrifuged at 1,600 rpm at 4 °C for 3 min. The cell pellets obtained were suspended in ice-cold PBS, centrifuged at 1,600 rpm at 4 °C for 3 min, and treated with cell lysis buffer M. The fluorescence intensity of each lysate was measured using a spectrofluorometer (515 nm; ND3300, NanoDrop, Wilmington, DE). The amount of protein in each well was concomitantly assessed using a Micro BCA protein assay kit. The results are presented as a mean and standard deviation obtained from four samples. P values were calculated by Student’s t-test compared to the result of peptide 13 with *P < 0.05, **P < 0.01, and ***P < 0.001. Peptide Stability against Protease Digestion. Five hundred microliters of the peptide solution (10 μM) in 0.00025 w/v% trypsin/ D

DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering samples. P values were calculated by Student’s t-test, compared to the nontreated cells with ***P < 0.001. Confocal Laser Scanning Microscope (CLSM) Observations. Huh-7 cells were seeded on 8-well chambered cover glasses (Iwaki, Tokyo, Japan) (20,000 cells/well) and incubated for 24 h in 200 μL of DMEM containing 10% FBS. The medium was replaced with fresh medium, and peptide/Cy5-pDNA at an N/P ratio = 8 and TurboFect/ Cy5-pDNA solutions were applied to each well at a concentration equivalent to 0.5 μg of pDNA per well. Following each time incubation, the medium was removed, and cells were washed 3 times with PBS supplemented with heparin (20 units/mL). Intracellular distributions of Cy5-pDNA (green) were observed by CLSM, LSM 710 (Carl Zeiss, Oberkochen, Germany), after staining late endosomes/lysosomes with LysoTracker Red (red) and nuclei with Hoechst 33342 (blue), as previously reported.23 The colocalization ratio (%) of Cy5-pDNA with LysoTracker Red was quantified23 as

colocalization ratio =

Cy5‐pDNA pixelscolocalization Cy5‐pDNA pixels total

× 100

Figure 2. CD spectra of peptides 9−13 in 20 mM phosphate buffer (pH 7.4). Peptide concentration: 25 μM.

where Cy5-pDNA pixelscolocalization represents the number of Cy5pDNA pixels colocalizing with LysoTracker Red in the cell, and Cy5pDNA pixelstotal represents the total number of pixels in the cell. The results are presented as the mean and standard deviation obtained from 20 samples. P values were calculated by student’s t-test compared to the result of peptide 13 with *P < 0.05 and ***P < 0.001, respectively.

right-handed (P) helical structure, whereas a negative maximum at 195−200 nm (π → π*) and positive maximum at 217 nm (n → π*) were diagnostic of a random coil structure.25−27 The ratio of R (θ222/θ208) has been used as a parameter to distinguish α-helical structures from 310-helical structures (i.e., R ≃ 1: α-helix; R < 0.4: 310-helix).28−30 The CD spectra of peptides 9−12 showed negative maxima at 208 and 222 nm, which indicated that the dominant structure of these peptides was a right-handed (P) helical structure. On the other hand, the R9 peptide 13 formed a random-coil structure. The ratio of R suggested that the dominant secondary structure of the peptides containing (1S,3R)-cyclic dAAs, peptides 9 (R = 0.48) and 11 (R = 0.58), was a 310-helix, whereas that of the peptides containing (1R,3R)-cyclic dAAs, peptide 10 (R = 0.98) and 12 (R = 0.74), was an α-helix. Cellular Uptake. The uptake of peptides 9−13 by Huh-7 cells was evaluated with various incubation times (Figure 3).

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RESULTS Synthesis of Fmoc-Protected dAAs. The synthetic routes of Fmoc-protected dAAs (1S,3R)-6, (1R,3R)-6, (1S,3R)-8, and (1R,3R)-8 are shown in Scheme 1. The two diastereomers (1S,3S)-1 and (1R,3S)-1 were synthesized according to a previous study.22,24 The treatment of alcohols 1 with MsCl and Et3N afforded mesylates 2 ((1S,3S)-2: 88%, (1R,3S)-2: 92%), which were converted into azide compounds 3 by substitution with NaN3 ((1S,3R)-3: 85%, (1R,3R)-3: 95%). Azides 3 were then converted to amines 4 by the Staudinger reduction ((1S,3R)-4: 94%, (1R,3R)-4: 93%), and subsequent Boc protection afforded compound 5 ((1S,3R)-5: 91%, (1R,3R)-5: 93%). The hydrolysis of 5 under alkaline conditions followed by deprotection of the Cbz-protecting group by hydrogenolysis using H2 and Pd−C gave crude dAAs, the primary amines of which were protected by Fmoc to afford Fmoc-protected dAAs 6 in three steps ((1S,3R)-6: 47%, (1R,3R)-6: 27%). Guanidinylation of the amino group on the side chain of 4 was accomplished by the treatment of N,N′-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine under basic conditions to obtain dAAs 7 ((1S,3R)-7: 89%, (1R,3R)-7: 91%). Fmoc-protected dAAs 8 were synthesized from 7 in a similar manner to the synthesis of dAAs 6 from 5 ((1S,3R)-8: 86%, (1R,3R)-8: 60%). Synthesis of Cell-Penetrating Peptides with dAAs. CPPs were designed based on the Arg nonapeptide (R9) (Figure 1). Fmoc-protected amino acids 6 and 8 were replaced with Arg residues at positions 3, 6, and 9 to prepare peptides 9−12, which were labeled with carboxyfluorescein (CF) through the glycine (Gly) linker. The peptides were synthesized by standard Fmoc solid-phase methods and purified with RP-HPLC. The R9 peptide 13 was also synthesized as a control. Secondary Structures of Peptides. The CD spectra of peptides 9−13 were measured in phosphate buffer (pH 7.4) to obtain information on their secondary structures (Figure 2). A positive maximum at 192 nm (π → π*) and negative maxima at 208 nm (π → π*) and 222 nm (n → π*) were diagnostic of a

Figure 3. Cellular uptake of peptides 9−13. Incubation timedependency with a peptide concentration of 2 μM. Error bars represent the standard deviation, n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 vs peptide 13.

The cellular uptake of R9 peptide 13 gradually decreased after 4 h. On the other hand, peptides 9−12, which contained cyclic dAAs, showed similar results in that the amount of peptides uptaken reached a maximum after a 16 h incubation. The cellular uptake of peptides 9−12 was significantly higher than that of R9 peptide 13 after a 2 h incubation. Neither R or S chirality at the α-position nor a basic functional group (amino E

DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 4. Peptide stability against (a) trypsin (0.00025 w/v%) and (b) Pronase (0.00025 w/v%) with various incubation times.

Figure 5. Transfection efficiencies of peptide/pDNA complexes against Huh-7 cells (a) at various N/P ratios and (b) with various postincubation times. Error bars represent the standard deviation, n = 6.

reached 100−200 nm with a narrow PDI and approximately +15 mV, respectively, and these values were suitable for in vitro pDNA delivery.37−39 Transfection. The transfection efficiency of pDNA encoding luciferase was evaluated among peptide/pDNA complexes at N/P ratios = 2, 4, and 8, naked pDNA, and commercially available reagent TurboFect by luciferase assays (Figure 5). We used such N/P ratios (up to 8) because transfection efficiency of the R9 peptide 13/pDNA complex prepared at an N/P ratio = 16 was a similar level to that prepared at an N/P ratio = 8 (data not shown). Figure 5a shows the results of transfection experiments under the condition of a 24 h incubation with complexes followed by a 24 h postincubation without complexes. Peptide/pDNA complexes prepared at an N/P ratio = 2, in which the sizes were more than several hundred nm, showed low transfection abilities. The transfection efficiencies of all peptide/pDNA complexes increased with an increase in N/P ratios and reached maxima at an N/P ratio = 8. Peptides 9−12 containing cyclic dAAs showed significantly higher transfection abilities than the R9 peptide 13 at an N/P ratio = 8 (P < 0.05, peptides 9−12 vs peptide 13) but lower abilities than TurboFect (P < 0.05, peptides 9−12 vs TurboFect). For more information to be obtained on the transfection abilities of the peptides, peptides 9, 11, and 13/pDNA complexes at an N/P ratio = 8 and TurboFect/pDNA were transfected into Huh-7 cells with various postincubation times (Figure 5b). The transfection ability of TurboFect was significantly higher than those of all peptides 0 h postincubation (P < 0.001, peptides 9, 11, and 13 vs TurboFect) and decreased with an increase in postincubation times. On the other hand, peptides 9 and 11

or guanidino) in cyclic dAAs influenced the cell permeabilities of peptides 9−12. Peptide Stability against Protease Digestion. The stabilities of peptides 9, 11, and 13 against the proteases, trypsin and Pronase were evaluated (Figure 4). Previous studies reported the efficient cellular uptake of CPPs with nonproteinogenic amino acids because of their tolerance to proteases.31,32 The R9 peptide 13 was easily degraded by trypsin, and less than 20% of the intact peptide remained after a 4 h incubation (Figure 4a). On the other hand, 80% of intact peptides remained even after a 12 h incubation for peptides 9 and 11. Figure 4b, in which peptides were treated with Pronase, shows similar results to Figure 4a. Peptides 9 and 11 with cyclic dAAs were more stable against proteases than the R9 peptide 13. Preparation and Characterization of Peptide/pDNA Complexes. Peptides were evaluated as pDNA carriers by using peptide/pDNA complexes. The formation of peptide/ pDNA complexes was confirmed by fluorescence measurements of peptide/pDNA complex solutions at various N/P ratios (Figure S1). Fluorescence quenching was expected to occur with the formation of assembly structures through selfquenching.33−36 Marked increases in the fluorescence intensities of all peptide/pDNA complexes were observed over an N/P ratio = 1.5, suggesting a stoichiometric N/P ratio in these complexes. The size and zeta-potential of the peptide/pDNA complexes were also similar among all peptides (Table S1). At an N/P ratio = 2, their sizes were more than several hundred nm, and their zeta-potentials were slightly positive. Their sizes decreased and zeta-potentials increased with an increase in N/P ratios. At an N/P ratio = 8, their sizes and zeta-potentials F

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Figure 6. Intracellular distribution of Cy5-pDNA (green) with peptides (a) 9, (b) 11, (c) 13, and (d) TurboFect. Acidic late endosomes/lysosomes and nuclei were stained with LysoTracker Red (red) and Hoechst 33342 (blue). (e) Colocalization ratio of Cy5-pDNA with LysoTracker Red. Error bars represent the standard deviation, n = 20. *P < 0.05 and ***P < 0.001 vs peptide 13.

high right-handed (P) helicities of peptides containing cyclic dAAs with an R chiral center at the α-position. These results are consistent with our previous report of (1S,3S)-and (1R,3S)Ac5cOM peptides.40 The screw sense of helical peptides was more controllable by chiral centers at the α-position, and peptides composed of (1R,3S)-Ac5cOM adopted a right-handed (P) helical structure. As judged from the ratio of R (θ222/θ208), peptides containing (1S,3R)-cyclic dAAs preferred a 310-helical structure, whereas those containing (1R,3R)-cyclic dAAs preferred an α-helical structure. The chirality at the α-position of cyclic dAAs affected the type of helix. However, these conformational differences had almost no impact on the cell permeabilities of the peptides (Figure 3). Peptides 9−12 containing cyclic dAAs showed significantly higher cellular uptake than the R9 peptide 13 under longer incubation times; this may be due to the high stabilities of these peptides against proteases (Figure 4). In general, peptides containing dAAs exhibit tolerance to protease digestion,20,31,32,41,42 which is consistent with the results shown in Figure 4, leading to the long-term cell permeabilities of peptides 9−12. A basic functional group in the side chain of dAAs only exerted a weak effect on the secondary structures and cell permeabilities of peptides. Peptides 9−13 formed complexes with pDNA by electrostatic interactions, and sizes and zeta-potentials at an N/P ratio = 8 were approximately 150 nm and +15 mV (Table S1), respectively, which are suitable for in vitro pDNA delivery.37−39 The transfection efficiencies of peptides 9−12 were more than 1 order of magnitude higher than that of the R9 peptide 13 at an N/P ratio = 8 (Figure 5a). Peptides 9−12 efficiently delivered biomacromolecules including pDNA as well as a small compound such as fluorescein. Among peptides 9−12, peptide 11 showed higher transfection efficiency than the others at N/P ratios = 2 and 4; however, each maximum transfection efficiency was similar. Chirality at the α-position and a basic functional group in the side chain of cyclic dAAs were not related to the transfection abilities of those containing peptides. The commercially available reagent TurboFect showed higher transfection efficiency than all peptides under the condition of a 24 h incubation with complexes followed by a 24 h postincubation (Figure 5a). However, transfection experiments with various postincubation times revealed that peptides 9 and 11 showed significantly better transfection abilities than those of TurboFect 48 and 72 h postincubation (Figure 5b).

reached the highest transfection efficiencies 48 h postincubation, which were significantly higher than TurboFect with a longer postincubation (P < 0.05, peptides 9 and 11 vs TurboFect at 48 h post incubation; P < 0.01, peptides 9 and 11 vs TurboFect at 72 h post incubation). The negligible cytotoxicities of all peptide/pDNA complexes at various N/P ratios were detected after a 24 h incubation, whereas TurboFect exhibited ∼60% cell viability (Figure S2). CLSM Observations. The intracellular distribution of Cy5labeled pDNA (Cy5-pDNA) (green) complexed with peptides 9, 11, and 13 (N/P ratio = 8) and TurboFect were observed, particularly for the colocalization of pDNA with late endosomes and lysosomes (Figure 6). Late endosomes and lysosomes were stained with LysoTracker Red (Red), and nuclei were stained with Hoechst 33342 (blue). The amount of Cy5-pDNA observed in the cells after a 24 h incubation was higher for peptides 9 and 11 than for peptide 13 but was lower for peptides 9 and 11 than for TurboFect (Figure 6a−d). The colocalization ratio of Cy5-pDNA with LysoTracker Red was quantified at various incubation times as shown in Figure 6e. Peptides 9 and 11 exhibited lower colocalization ratios than that of the R9 peptide 13 and similar values to TurboFect, suggesting more effective endosomal escape of peptides 9 and 11. The intracellular distribution of Cy5-pDNA (magenta) with peptides 9 and 11 (green) was also evaluated by CLSM observations at 37 and 4 °C after staining nuclei (blue) (Figure S3). Peptides and Cy5-pDNA were observed at 37 °C but not at 4 °C, suggesting that peptide/pDNA complexes internalized into cells by an energy-dependent mechanism.

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DISCUSSION In the present study, we designed four types of cyclic dAAs with a basic functional group and replaced three L-Arg residues of R9 peptides with dAAs to improve cell permeability with high water solubility (Figure 1). All peptides synthesized herein dissolved, even in 100% aqueous solution, and their secondary structures in phosphate buffer (pH 7.4) were evaluated by CD spectra measurements (Figure 2). The dominant secondary structure of the R9 peptide 13 was a random-coil structure, whereas that of peptides 9−12 was a right-handed (P) helical structure. The introduction of cyclic dAAs into Arg-rich peptides led to a marked conformational change as previously reported.17,20,21 The stronger negative maximum at 222 nm of peptides 10 and 12 than peptides 9 and 11 demonstrated the G

DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Notes

Maximum transfection efficiency was reached 0 h postincubation for the R9 peptide 13 and 48 h postincubation for peptides 9 and 11. The prolonged transfection abilities of peptides 9 and 11 may have been due to the tolerance of peptides to protease digestion through the introduction of cyclic dAAs into the peptides (Figure 4) and consequently led to higher transfection efficiencies than TurboFect with a longer time postincubation. pDNA complexed with peptides containing cyclic dAAs was protected from protease digestion in the cultured medium and lysosomes.23 It is important to note that, although the viability of Huh-7 cells treated with the TurboFect/pDNA complex was less than 60%, those treated with peptide/pDNA complexes were approximately 100% (Figure S2). The peptides designed in the present study delivered pDNA with negligible cytotoxicity. For the transfection mechanism of peptide/pDNA complexes to be clarified, CLSM observations at a low temperature (Figure S3) and after staining late endosomes/lysosomes (Figure 6) were performed using Cy5-pDNA. CLSM observations of peptide/pDNA complexes at 4 °C demonstrated the energy-dependent cellular uptake of complexes into Huh-7 cells (Figure S3). Figure 6 revealed that pDNA delivered by peptides 9 and 11 into cells localized less in acidic late endosomes/lysosomes than the R9 peptide 13 with increasing incubation times. The effective endosomal escape of pDNA complexed with peptides 9 and 11 appeared to be one of the reasons for their efficient transfection abilities. Although the mechanisms underlying effective endosomal escape have yet to be elucidated in detail, the introduction of cyclic dAAs with a basic functional group in Arg-rich peptides may be highly advantageous for the construction of an efficient gene delivery peptide.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported in part by JSPS KAKENHI Grant Numbers JP25713008 (for M.O.), JP17H03998 (for M. T.), and JP15J05945 (for T.K.) and by a Grant from the Takeda Science Foundation (for M.O.).

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CONCLUSIONS We designed four types of dAAs with a basic functional group on their five-membered rings and synthesized dAA-containing Arg-rich peptides for CPPs. The introduction of dAAs into peptides changed the peptide secondary structure from a random coil to a helical structure in aqueous solution and elevated the stabilities of the peptides against protease digestion. The cell permeabilities and transfection efficiencies of peptides 9−12 containing cyclic dAAs were markedly higher than those of the R9 peptide 13, particularly with longer incubation times. These results may be helpful for designing novel CPPs as a drug delivery tool.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00180. 1 H NMR, 13C NMR, characterization of peptide/pDNA complexes, cell viability, and CLSM observations (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +81-95-819-2424, Fax: +81-95-819-2424, e-mail: moba@ nagasaki-u.ac.jp. *Tel: +81-95-819-2423, Fax: +81-95-819-2423, e-mail: [email protected]. ORCID

Takuma Kato: 0000-0002-2561-8582 Makoto Oba: 0000-0002-3691-3608 H

DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.8b00180 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX