Fmoc-protected amino acids and Rink resin were purchased from Iris Biotech, deuterated Fmoc-protected leucine (Fmoc-Leu-OH-5,5,5-d ) 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 ( ). The reagents HBTU (N,N,N ,N -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 DMF45 (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% purity46 (link),47 (link).
Molecular weight and peptide purity were confirmed by electrospray ionization mass spectroscopy (ESI-MS) and RP-HPLC.
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 ( ). The reagents HBTU (N,N,N ,N -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 DMF45 (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% purity46 (link),47 (link).
Molecular weight and peptide purity were confirmed by electrospray ionization mass spectroscopy (ESI-MS) and RP-HPLC.
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