Glycine → Glycyl-isoleucyl-tryptophyl-threonyltryptophyl-valine

655 mg of the peptide-resin was treated with 1 mL of anisole and 10 mL of HF for one hour at 0° to 4° C. The HF was removed by nitrogen stream and the resultant solids were triturated with diethyl ether (30 mL). The solids were collected by filtration, washed with diethyl ether (3 ×10 mL) and then extracted with 50% TFA/methylene chloride (3×10 mL). The extracts were concentrated by use of a nitrogen stream in a fume hood. To the residue was added diethyl ether (~75 mL) to precipitate the crude peptide. The peptide was collected via filtration, washed with ether (3×10 mL) and dried in vacuo over sodium hydroxide to yield 246 mg of crude peptide. 100 mg of the crude peptide had the Trp residues deformylated by dissolution in 1% piperidine in 50% dimethylformamide/water(10 mL) and stirring for 2 hours at room temperature. The deformylation solution was treated dropwise with acetic acid to adjust the pH to ~4.5. This solution was injected onto a Vydac 22×250 mm C18 10 μm particle sized 300 Angstrom pore packed column. Elution with a gradient of 20% to 60% B over 60 minutes at a flow rate of 10 mL/min was carried out (Solvent A=0.1% TFA; Solvent B=0.1% TFA in 80% Ethanol/Water). Fractions were collected and the appropriate ones pooled to give 53 mg of semi-pure peptide. Further purification was carried out by dissolution of the peptide in water (-20 mL) and addition of 30% ammonium hydroxide dropwise. Four prep HPLC runs were performed using the above column and elution with isocratic 40% B buffer at 10 mL/min. Fractions were collected and the appropriate ones pooled to give 120 mg of white solid. Amino Acid Analysis: Gly 1.01 (1.00), Ile 0.97 (1.00), Thr 0.94 (1.00), Val 1.01 (1.00), Trp 1.84 (2.00), 10% peptide. FAB/MS: MH+=761.3 (MW=760)

ORD record

ord-5eab5cd1c54b4e62b3c3a619465b59aa

Source dataset

uspto-grants-1995_11

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05464935