Thiophosphorylated peptides

Oxidation of the intermediate phosphite triester can also be effected using elemental sulphur in CS2 (39), tetraethylthiuram disulphide in CH3CN (39), dibenzoyl tetrasulphide (40), and dithiasuccinimide (41), to yield the corresponding resin-bound thiophosphorylated peptide. 

For cleavage and side-chain deprotection of thiophosphopeptidyl resins, the use of TFA/thiophenol/water/EDTATIS (91.5 2.5 2.5 2.5 1 v/v) is recommended. 

FiguraS. HPLC of total crude phosphopeptides. (a) H-Phe-Phe-Lys-Asn-lle-Val-Thr-Pro-Arg-Thr(P03H2)-Pro-Pro-Pro-Ser-Gln-Gly-Lys-NH2. Conditions column Beckman CIS eluant A, 0.1% aq. TFA, eluant B, 0.1% TFA in acetonitrile gradient 0% to 100% B in 40 min flow rate 1 ml/min. (bl H-Gly-Asp-Phe-Glu-Glu-lle-Pro-Glu-Glu-Thr(P03H2)-Leu-NH2. Conditions column TSK-120T eluant A, 0.1% aq. TFA, eluant B, 90% acetonitrile/10% water/0.1% TFA gradient 5% B for 5 min then to 100% in 50 min flow rate 1 ml/min. Opticai density was measured at 214 nm. 

Care should be taken to avoid prolonged exposure of thiophosphoryl peptides to acidic aqueous conditions as this can result in loss of the thiophos-phate group (39, 40). Purification of these peptides is best achieved by reverse-phase HPLC using a gradient of CH3CN in 0.1 M triethylammonium acetate (pH 7.5) (39). 

Wash the resin with acetonitrile in the manner described in Protocol 1, step 13. 

---