Diisopropylcarbodiimide dic

Synthesis of peptide precursors was performed manually using fritted syringes on a vacuum manifold via 9‐fluorenylmethyloxycarbonyl (Fmoc) solid‐phase peptide synthesis (SPPS). All reagents were purchased from commercial sources and used without further purification. 2‐Chlorotrityl chloride resin (sub. = 1.2 mmol/g), Wang resin (sub. = 0.73 mmol/g), Oxyma Pure and all standard Fmoc amino acids were sourced from Mimotopes Pty Ltd (Australia). Fmoc‐L‐Dap(Boc)‐OH and Fmoc‐L‐Asp‐OMe were obtained from AK Scientific, Inc. (USA). Fmoc‐Cys(StBu)‐OH was sourced from CreoSalus, Inc. (USA). (1‐Cyano‐2‐ethoxy‐2‐oxoethylidenaminooxy)dimethylamino‐morpholino‐carbenium hexafluorophosphate (COMU), 1‐[bis (dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium‐3‐oxide hexafluorophosphate (HATU) and 2,2′‐dipyridyldisulfide (DPDS) were purchased from Combi Blocks (USA). Diisopropylcarbodiimide (DIC), 4‐dimethylaminopyridine (DMAP), diphenyldiselenide, acetoacetone, triisopropylsilane (TIPS), thioanisole, 2,2′‐(ethylenedioxy)diethanethiol (DODT), trifluoroacetic acid (TFA), piperidine, diisopropylethylamine (DIEA), N‐methylmorpholine (NMM), hydrazine hydrate, tris‐carboxyethylphosphine hydrochloride (TCEP.HCl), iodine, dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, acetonitrile and trypsin were purchased from Sigma‐Merck (Australia).

The soluble peptides EEFRDYYENGEEKSNKQM (PI), SSGVRVDLGEDAEVENAK (PII), GDQRLKDGGFAFPNADDH (PIII) and KLKDGGFAFPNANDHEEFRDYYENGEEKSNKQM (tripeptide) were synthesized on a 25 μmol scale in the ResPep SL automated synthesizer (Intavis^®). Brielfy, Fmoc-amino acids were activated with a 1∶1 solution of Oxyma Pure (Merk) and diisopropylcarbodiimide (DIC, Sigma-Aldrich). The active amino acids were incorporated into TentaGel (Intavis) (Peptides I and II) or H-Rink Amide ChemMatrix (Sigma-Aldrich) (Peptide III and Tripeptide) resins. Fmoc deprotection was performed using 25% 4-methylpiperidine (25% v/v in DMF). These steps were repeated until the synthesis of each peptide was completed. The peptides were deprotected and released form the resin by treatment with a solution of 92.5% trifluoroacetic acid, 2.5% water, 2.5% triisopropylsilane and 2.5% beta-mercaptoethanol during 3 hours under agitation. Specifically, for the Tripeptide the synthesis was performed using a chaotropic agent and lithium chloride (0.8M). The time of deprotection was prolonged in order to guarantee the proper extension of the polypeptide chain. The peptides were precipitated with cold methyl tert-butyl ether and lyophilized. The molecular weight of each peptide synthetized was confirmed by mass spectrometry using Autoflex Speed MALDI/TOF equipment.