Fmoc protected amino acids

Fmoc-protected amino acids and coupling agents were purchased from Iris Biotech GmbH. α-Methoxy-ω-amino PEG (CH₃O–PEG–NH₂, M_n = 5000 g mol⁻¹), α-methoxy-ω-NHS ester (CH₃O–PEG–NHS, M_n = 5000 g mol⁻¹) were purchased from Rapp Polymere. Boc–His(Trt)–OH, 4-nitrophenyl acetate (PNPA), 4-nitrophenyl butyrate (PNPB), (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN–NHS) and other chemicals were purchased from Sigma-Aldrich. Solvents were purchased from several departmental suppliers, Honeywell, Fisher and Sigma-Aldrich.

Synthesis of carboxyamidated temporin-SHe was performed using a solid-phase FastMoc chemistry procedure on a 433A automated peptide synthesizer from Applied Biosystems, as previously described [50 (link)]. Briefly, Fmoc-Rink-Amide PEG MBHA resin and Fmoc-protected amino acids were purchased from Iris Biotech GMBH (Marktredwitz, Germany). Purification was performed by reversed-phase high-performance liquid chromatography (RP-HPLC) on a semi-preparative column (Luna C18, 10 µm, 250 × 10 mm, Phenomenex, Torrance, CA, USA) with a 40–80% linear gradient of acetonitrile (1%/min) at a flow rate of 5 mL/min. Peptide purity was assessed by analytical RP-HPLC on an Uptisphere C18 column (modulo-cart QS, 5 μm, ODS2, 250 × 4.6 mm, Interchim, Los Angeles, CA, USA) using the conditions above with a flow rate of 0.75 mL/min. The peptide mass was confirmed by MALDI-TOF-MS (Voyager DE-Pro and 4700 Proteomic analyzer, Applied Biosystems, Mass Spectrometry platform, IBPS, Sorbonne Université, France). Carboxamidated temporin-SHd and [K³]temporin-SHa, also used in the study, were synthesized using the same procedure. The figures of HPLC chromatograms (Figures S1–S3) and MS spectra (Figures S4–S6) of the synthesized peptides were provided as Supplementary Data.

Chemical reagents and solvents for the peptide synthesis were of peptide-synthesis grade; solvents for HPLC were of HPLC grade. Fmoc-protected amino acids, Rink-amide MBHA resin (100–200 mesh, loading 0.57 mmol/g), N,N-diisopropylethylamine (DIPEA), piperidine, N,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), diethyl ether, and trifluoroacetic acid (TFA) were purchased from Iris Biotech (Germany). H-Asp(OtBu)-2-chlorotrityl resin (loading 0.60 mmol/g) was purchased from Merck Schuchardt OHG (Germany). Thioanisole (TIA), acetic anhydride, α-cyano-4-hydroxycinnamic acid, triisopropylsilane (TIS), 1,2-ethanedithiol (EDT), and acetonitrile (ACN) were purchased from Sigma Aldrich (Germany). 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) were purchased from Biosolve (The Netherlands).

Peptide synthesis reagents and Fmoc-protected amino acids were purchased from Iris Biotech (Marktredwitz, Germany) or Merck Biosciences (Darmstadt, Germany). Chemicals for peptide synthesis were obtained from Merck (Darmstadt, Germany) or Biosolve (Valkenswaard, Netherlands), and solvents for HPLC purification were purchased from Fischer Scientific (Geel, Belgium). 4-Chloro-7-nitrobenzofurazan (NBD-Cl) was purchased from VWR (Bruchsal, Germany). The fluorescent probes 8-amino-naphtalene-1,3,6-trisulfonic acid sodium salt (ANTS) and p-xylene-bis-pyridinium bromide (DPX) were obtained from Invitrogen-Molecular Probes (Karlsruhe, Germany). Fluorescently labelled 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). The lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), and 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) were purchased from NOF (Grobbendonk, Belgium).

Fmoc-protected amino acids and Rink resin were purchased from Iris Biotech, deuterated Fmoc-protected leucine (Fmoc-Leu-OH-5,5,5-d ${}_3$ ) was purchased from Sigma Aldrich.
TP1 peptide was synthesized without its end-capping cysteine to prevent the formation of dimers. This did not modify its secondary structure, as it did not alter its CD spectrum, and therefore it should not modify its penetrating activity, considering that this cysteine is meant to be disulfide-bonded to a cargo.
TP1 peptide and its deuterated variations were synthesized using standard Fmoc-methodology on Rink amide resin ( $0.4\text{mmol}{\text{g}}^{-1}$ ). The reagents HBTU (N,N,N ${}^{\prime }$ ,N ${}^{\prime }$ -tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate) and HCTU (O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) were used as activators and DMF (N,N-dimethylformamide) was used as solvent. Fmoc deprotections were performed using a solution of 4-methylpiperidine in DMF^{45 (link)}. Peptide cleavage was carried out with a solution of trifluoroacetic acid, triisopropylsilane, EDT (2,2-(ethylenedioxy)diethanethiol) and water in 92.5:2.5:2.5:2.5 ratio. Reaction products were purified by reverse phase chromatography up to >90% purity^{46 (link),47 (link)}.
Molecular weight and peptide purity were confirmed by electrospray ionization mass spectroscopy (ESI-MS) and RP-HPLC.

Reagents
for peptide synthesis
(Fmoc-protected amino acids, resins, activation, and deprotection
reagents) were purchased from Iris Biotech GmbH (Waldershofer Str.
49–51, 95615); EDC/NHS, PDMS, and CRP (C- Reactive Protein)
were from Sigma-Aldrich. VEGF (Recombinant Human VEGF165) was purchased
from Peprotech. Anti-CRP (Anti-C Reactive Protein antibody (FITC)
(ab19174)) and Anti-VEGF (Anti-Recombinant Human VEGF antibody (FITC))
were from Abcam. TNF-α and Anti-TNF-α (Anti-Tumor Necrosis
Factor-α antibody (FITC)) were from Prospec. Solvents for peptide
synthesis and HPLC analyses were purchased from Sigma-Aldrich; reversed-phase
columns for peptide analysis and the LC–MS system were supplied,
respectively, from Agilent Technologies and Waters (Milan, Italy).
All SPR reagents and chips were purchased from AlfaTest (Rome, Italy).
PMMA substrates used in this study were purchased from the same batch
of the polymer supplier (Good Fellow Cambridge Limited, England);
Fluorolink S10 was from Solvay. Pooled human serum from healthy donors
was supplied by Lonza (Life Technology Ltd., Paisley, UK). All chemicals
were used as received.