TOTAL SYNTHESIS, PURIFICATION, AND CHARACTERIZATION OF HUMAN [PHE(P-CH2SO3NA)(52), NLE(32,53,56), NAL(55)]-CCK20-58, [TYR(52), NLE(32,53,56), NAL(55)]-CCK-58, AND [PHE(P-CH2SO3NA)(52), NLE(32,53,56), NAL(55)]-CCK-58

The synthesis of [Phe(p-CH2SO3Na)(52), Nle(32,53,56) Nal(55)]-CCK20-58 , [Tyr(52), Nle(32,53,56), Nal(55)]- CCK-58 and of [Phe(p-CH2SO3Na)(52 ), Nle(32,53,56), Nal(55)]-CCK-58 using the (9-fluorenylmethyloxy)-car bonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude pep tide preparations were extremely complex when analyzed by RP-HPLC, cap illary zone electrophoresis (CZE), and ion-exchange chromatography (IE -FPLC). We found that the most effective strategy for purification inc luded cation-exchange chromatography followed by a RP-HPLC desalting s tep. The highly purified peptides (purity greater than 90%) were chara cterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spe ctrometry, amino acid analysis, and Edman sequence analysis {for [Tyr( 52), Nle(32,53,56), Nal(55)]-CCK-58}. The results demonstrate the appl icability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of lon g peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/ RP-HPLC approach for the purification of very lon g, heterogeneous crude peptides, allowing a true assessment of the bio logical properties of these analogs to be carried out. [Phe(p-CH2SO3Na )(52), Nle(32,53,56), Nal(55)]-CCK20-58 was less than 1% as potent as CCK-8 while [Tyr(52), Nle(32,53,56), Nal(55)]-CCK-58 and [Phe(p-CH2SO3 Na)(52), Nle(32,53,56), Nal(55)]-CCK-58 were inactive at the doses tes ted (< 0.01%).