Fmoc protected amino acids

The Rink Amide ChemMatrix resin, used in solid phase peptide synthesis, was commercially obtained from Pcas-Biomatrix Inc. (Quebec, QC, Canada). Fmoc-protected amino acids and O-benzotriazole-N,N,N′,N′-tetramethyluraniumhexafluorophosphate (HBTU) were purchased from AAPPTec (Louisville, KY, USA), ChemPep, Inc. (Wellington, FL, USA), and Chem-Impex International (Wood Dale, IL, USA). Piperidine and diisopropylethylamine (DIEA) were procured from Alfa Aesar (Ward Hill, MA, USA). Acetonitrile, triisopropylsilane (TIPS), trifluoroacetic acid (TFA), and cobalt (II) chloride hexahydrate were all obtained from Sigma Aldrich (St. Louis, MO, USA). N,N-dimethylformamide (DMF), dichloromethane (DCM), and methanol (MeOH) were purchased from AVANTOR (Center Valley, PA, USA) and Fisher Scientific (Pittsburgh, PA, USA). Ethylenediaminetetraacetic acid disodium salt (EDTA) was purchased from Mallinckrodt JT Baker (Hazelwood, MO, USA). Scanning electron microscopy (SEM) stubs, round glass coverslips, and transmission electron microscopy (TEM) grids were obtained from Ted Pella, Inc. (Redding, CA, USA).

Novokinin was synthesized using a standard Fmoc-mediated solid-phase peptide synthesis (SPPS) using the Aapptec Focus Xi peptide synthesizer (Louisville, KY, USA). All Wang resin, Fmoc-protected amino acids, solvents, and reagents needed for peptide synthesis were purchased from Aapptec (Louisville, KY, USA). The chain elongation was performed by removing the Fmoc group from the resin-attached amino acid. Next, Fmoc-protected amino acid (based on the sequence) was added until the entire peptide was synthesized. After the completion of synthesis, the final Fmoc group was removed and cleaved from the resin. After purification of the novokinin, it was coupled with Maleimidopropionyl-PEG NHS Ester (Polypure, Oslo, Norway) in an equimolar concentration for an hour to form an intermediate compound. A 10× solution of thiol-BP (Surfactis Technologies, Angers, France) was reacted with the intermediate compound for another hour. After completion of coupling, the conjugate was dialyzed to separate it from unreacted reagents.
The resultant peptide and conjugate were characterized by Agilent 6545 LC/Q-TOF. A US patent application for this innovative Novo Conj synthesis and pharmacological effect was filed. For the animal study, a batch of Novo Conj was synthesized by AnaSpec (CA, USA) and used after structural and purity confirmation.