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
Mtm. Miranda et al., 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, Journal of protein chemistry, 12(5), 1993, pp. 533-544
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%).