Acetic Acid → Boc-Gly-Oxime

Participants

Reaction Steps

Boc-Gly-Oxime-Resin was prepared by mixing oxime-resin (Novabiochem, 1.5 g 0.57 meq/g) with Boc-Gly (1.3 g, 9 eq) in the presence of N′N-dicyclohexylcarbodiimide (DCC) (9.9 ml of DCC 8%, 4.5 eq), 4-dimethylaminopyridine (DMAP) (0.3 g, 3 eq), 1-hydroxybenzotriazole hydrate (HOBt) (0.4 g, 3 eq) in dichloromethane (DCM) at room temperature for 12 hours. The resin was subjected to two washes with DCM, one wash with 2-propanol and one wash with DCM. The free oxime groups were capped by acetylation with acetic anhydride (0.4 ml, 5 eq) for 30 minutes. The peptide chain was then assembled according to the following coupling steps: (i) one wash with 25% trifluoroacetic acid in DCM (TFA-DCM); (ii) deprotection with 25% TFA-DCM (30 min); (iii) two washes with DCM; (iv) one wash with 2-propanol; (v) three washes with DCM; (vi) one wash with dimethylformamide (DMF); (viii) coupling of Boc-D-Arg(Tos)-OH (1.1 g, 3 eq) in presence of benzotriazole-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), (1.3 g, 3 eq), HOBt (0.13 g, 1 eq) and diisopropylethylamine (DIEA) (0.95 ml, 6.5 eq) in DMF (45 min.). In cycles 2 and 3, step viii is performed with Boc-Tyr(2,6-di-Cl-Bzl)—OH (1.1 g, 3 eq) and Boc-D-Gln (0.6 g, 3 eq), respectively; (ix) three washes with DMF; (x) two washes with DCM. Solvent volumes were 15 ml/g resin. Coupling efficiency was checked at each coupling cycle by the Kaiser test (Kaiser et al., Anal. Biochem., 34, 595-598, 1970, the disclosure of which is incorporated by reference). The peptide was cleaved from the resin by intrachain aminolysis in the presence of AcOH (0.097 ml, 2 eq) and DIEA (0.293 ml, 2 eq) in 30 ml DMF at room temperature for 24 hours. The product was obtained from solution phase by filtration. Protecting groups were removed with anhydrous hydrogen fluoride (HF) at 0° C. for 30 minutes. The crude product was purified by Sephadex™ G-10, then RP-HPLC (Bondapak C18 column, 10 μm×125A, 25×100 mm), with a gradient of 0-50% water-acetonitrile, 0.1% TFA over 65 minutes. The final yield was 22 mg (p5%) based on starting resin. The purity and identity of the synthetic peptide was assessed by analytical HPLC on Bondapak C18 column, 10 μm×125A, 3.9×300 mm with a gradient of 0-50% water-acetonitrile, 0.1% TFA over 50 minutes, k′:2.7, molecular mass by FAB-MS: 505 (calcd: 504.24), amino acid analysis: D-A-g (1.1), Gln (1.0), Gly (1.0), Tyr (1.0). The following cyclic peptides were synthesized based on the method given above for peptide 8.

Provenance & Evidence