Journal of Protein Chemistry

, Volume 12, Issue 5, pp 533–544 | Cite as

Total synthesis, purification, and characterization of human [Phe(p-CH2SO3Na)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

  • Maria Terêsa Machini Miranda
  • A. Grey Craig
  • Charleen Miller
  • Rodger A. Liddle
  • Jean E. Rivier


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%).

Key words

CCK analogues HPLC purifications peptide characterization peptide synthesis 


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Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Maria Terêsa Machini Miranda
    • 1
  • A. Grey Craig
    • 1
  • Charleen Miller
    • 1
  • Rodger A. Liddle
    • 2
  • Jean E. Rivier
    • 1
  1. 1.The Clayton Foundation Laboratories for Peptide BiologyThe Salk Institute for Biological StudiesLa Jolla
  2. 2.Duke University and Durham VA Medical CentersDurham

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