Total synthesis, purification, and characterization of human [Phe(p-CH2SO 3Na)52, Nle32,53,56, Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58.
The synthesis of [Phe(p-CH2SO3Na)52, Nle32,53,56 Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58 and of [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis (for [Tyr52, Nle32,53,56, Nal55]-CCK-58). The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long 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 long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20-58 was less than 1% as potent as CCK-8 while [Tyr52, Nle32,53,56, Nal55]-CCK-58 and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 were inactive at the doses tested (< 0.01%).
Miranda
M T
MT
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California 92037.
Craig
A G
AG
Miller
C
C
Liddle
R A
RA
Rivier
J E
JE
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D002766:Cholecystokinin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010179:Pancreas / Q000187:drug effects / Q000201:enzymology; D051381:Rats
D007202:Indicators and Reagents; D002766:Cholecystokinin; D000681:Amylases