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General procedure: 2.1 Loading of the 2-chlorotritylchloride resin. General procedure.The synthesis was done in a plastic syringe equipped with Teflon filters. The 2-chlorotritylchloride resin (100-300 mesh, 1.20 mmol/g) was swelled in CH2Cl2 (3 × 30 sec). A solution of the first protected amino acid (Fmoc-AA-OH, 1.0 eq for a resin load of 0.6-0.8 mmol/g) in CH2Cl2 and DIPEA (3.0 eq) were incorporated as a coupling system and were gently shaken for 10 min Then an extra 7.0 eq. of DIPEA was added and shaking was continued for 50 min. MeOH (0.8 mL/ g of resin) was added to the previous mixture in order to cap unreacted functional groups on the resin, and shaken for 10 min. After filtering, the resin was washed with CH2Cl2 (x3), DMF (x3) and CH2Cl2 (x3). The loading capacity was estimated by Fmoc determination after the first residue attachment.2.2 Fmoc removal and quantificationFmoc protecting group was removed by treating the resin with piperidine-DMF solution (1:4) for 1, 5 and 5 minutes successively.For quantification, all the solvents were collected into a 25 mL volumetric flask. DMF was added to a volume of 25 mL. For a resin loading and amount of 0.6 mmol/g and 400 mg of resin, the volumetric flask was shaken and 200 muL were transferred into a 10 mL volumetric flask. DMF was added to a volume of 10 mL. The flask was shaken and UV absorption measured at 290 nm. Fmoc loading was calculated taking into account that extinction coefficient for the dibenzofulvene-piperidine adduct at 290 nm is 5800 l/mol/cm.2.3 SPPS general procedure for elongation of the peptide chain.The Fmoc-AA-2-chlorotritylchloride resin was shaken with 20% piperidine in DMF (1 x 1 min and 2 x 5 min), in order to deprotect N-terminus. Then, the resin was washed with DMF (x3), CH2Cl2 (x3) and DMF (x3). A solution of Fmoc-AA-OH (3 eq.) and DIPEA (6 eq.) in DMF was added to the resin, followed by a solution of HBTU or HATU (2.9 eq) in DMF. The mixture was stirred for 60 min. After the coupling was completed, the resin was washed with DMF (×3) and CH2Cl2 (x3). Deprotection and coupling cycles were repeated with the appropriate amino acids to provide the desired compound. Completion of the coupling was monitored by colorimetric assays; Kaiser test in case of primary amines and Chloranil test for secondary amines. Analytical HPLC was also realized after each coupling step for further process control. Unless otherwise stated, the peptide was cleaved from the resin by treatment with 1-10% TFA in CH2Cl2 for 2-3 minutes at room temperature followed by filtration and collection of the filtrate in MeOH. The treatment was repeated three times and then the resin washed with CH2Cl2 (x5) and MeOH (x3). Solvents were removed in vacuo to obtain the crude peptide.
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