Pssm 8

Isomers of αA51–60 and αB61–67 composed of L-α- and D-β-Asp residues were synthesized by Fmoc solid-phase chemistry using an automated solid-phase peptide synthesizer (Shimadzu PSSM-8). Fmoc-amino acids from Watanabe Chemical Industries (Hiroshima, Japan) were used. Crude peptides were purified by RP-HPLC using a C18 column (Capcellpak C18 ACR, 10 × 250 mm; Shiseido) with a linear gradient of 10–60% (for αA51–60) and 5–55% acetonitrile (for αB61–67) for 60 min in the presence of 0.1% TFA at a flow rate of 3.0 mL/min with monitoring at 215 nm. HPLC grade solvent was used to confirm the purity of the peptide. The purity of each peptide was confirmed to be >98% by RP-HPLC and MALDI-TOF MS or ESI-MS. The masses ([M+H]⁺) observed for the protonated precursor ions of αA51–60 and αB61–67 were 1094.6 and 696.1. These values were consistent with the theoretical ones, 1094.58 and 696.32, respectively.

The purified venom peptides were sequenced by automated Edman degradation using ABI model 477A (Applied Biosystems, USA). Peptides were synthesized by Fmoc chemistry using a Shimadzu PSSM-8 automated peptide synthesizer (Shimadzu, Japan), and purified by reverse-phase HPLC. The identity and purity of the peptides were confirmed by MALDI-TOF MS. The synthetic Xac-1 and Xac-2 were employed for circular dichroism (CD) analysis, liposome leakage assay, antimicrobial and hemolytic activity tests.

Tat-μCL (amino acid sequence, GRKKRRQRRRPPQPDALKSRTLR) was synthesized in accordance with our previously reported method [6 (link), 7 ]. Briefly, the peptide was synthesized by the fluorenylmethyloxycarbonyl method using an automated peptide synthesizer (Shimadzu PSSM-8; Shimadzu, Kyoto, Japan). The synthesized peptide was then purified by reverse-phase high performance liquid chromatography using a C18 column (Jupiter 250 mm × 10 mm; Phenomenex, Torrance, CA). The molecular weight and purity (>95%) of the peptide were confirmed by MALDI-TOF mass spectrometry using an AXIMA Confidence device (Shimadzu, Kyoto, Japan).

Fibrinopeptide B (¹QGVNDNEEGFFSAR¹⁴) was synthesized by 9-fluorenylmethyloxycarbonyl group (Fmoc)-based solid-phase peptide synthesis using an automated solid-phase peptide synthesizer (PSSM-8; Shimadzu, Japan). The coupling reaction was carried out by mixing each Fmoc amino acid (10 eq), (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (10 eq), 1-hydroxybenzotriazole hydrate (10 eq), and N-methylmorpholine (7.5 eq) in N,N-dimethylformamide (DMF). The N-terminal Fmoc group was deblocked with 30% piperidine in DMF. Spontaneous cleavage of the peptide from the resin and removal of the protective groups were achieved by treatment with a mixture containing 82.5% TFA, 5% water, 5% thioanisole, 3% ethylmethylsulfide, 2.5% 1,2-ethanedithiol, and 2% thiophenol for 6 h. The crude peptides were purified by reversed-phase high-performance liquid chromatography using a C18 column (Capcell Pak C18 ACR, 10 × 250 mm2; Shiseido, Japan) with a linear gradient of 0%−50% acetonitrile in the presence of 0.1% TFA at a flow rate of 3.0 mL/min and detection at 230 nm.

We previously designed seven PI polyamides that bind to human TGF-β1 promoter [21 (link)]. In this study, we evaluated the effect of one of the PI polyamides, GB1101, that targets sequences adjacent to the FSE2 regulatory element in the hTGF-β1 promoter. The structure of GB1101 and a mismatch PI polyamide is shown in Figure S1 [21 (link)]. Machine-assisted automatic synthesis of hairpin-type PI polyamides was carried out using a continuous-flow peptide synthesizer (PSSM-8; Shimadzu, Kyoto, Japan) at 0.1 mmol scale (200 mg of Fmoc-b-alanine CLEAR Acid Resin at 0.50 meq/g; Peptide Institute, Osaka, Japan). The automatic solid phase synthesis was performed as described previously [15 (link)].