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Chemical Synthesis of Maxadilan, a Non-mammalian Potent Vasodilatory Peptide Consisting of 61 Amino Acids with Two Disulfide Bridges, and Its Related Peptides

  • Kiyoshi Nokihara
  • Tadashi Yasuhara
  • Yoshihiro Nakata
  • Ethan A. Lerner
  • Victor Wray
Bruce Merrifield Commemorative Issue

Abstract

A potent and persistent non-mammalian derived vasodilator, maxadilan (Maxa) consists of 61 amino acids with two disulfide linkages and acts as an agonist of the type I receptor of pituitary adenylate cyclase activating polypeptide (PACAP), although there is very little sequence similarity. The total chemical syntheses of Maxa, its disulfide isomers and various fragments have been performed successfully by highly efficient solid-phase peptide synthesis (SPPS). A “difficult sequence”, envisaged in the middle region of Maxa, could be overcome by improved synthesis protocols. After assembly peptides were liberated from the resin by cleavage. Peptides having disulfide(s) were purified by two steps of preparative HPLC using cation exchange followed by reverse phase columns. Purified peptides were characterized by HPLC, Edman-sequencing, amino acid analysis and mass spectrometry in addition to disulfide form determination. The peptides obtained were used for recognition studies by the melanophore assay to confirm the native disulfide form. Peptide libraries related to Maxa, produced in the present study, will be useful for the elucidation of the structural requirements of Maxa for interaction with the PACAP type 1 receptor (PAC1).

Keywords

vasodilator maxadilan PAC1 receptor disulfide isomer highly efficient solid-phase synthesis difficult sequence melanophore assay 

Abbreviations

HATU

N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N- methylmethanaminium hexafluorophosphate N-oxide

HOBt

1-hydroxybenzotriazole

LC-IT-MS

liquid chromatograph with on-line ion-trap mass spectrometry

MALDI-TOF MS

matrix assisted laser desorption ionization time of flight mass spectrometry

Maxa

maxadilan

PACAP

pituitary adenylate cyclase activating polypeptide

PAC1

PACAP type 1 receptor

SPPS

solid-phase peptide synthesis

RP-HPLC

reverse-phase high performance liquid chromatography

PyBOP

benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate

TFA

trifluoroacetic acid

TMP

2,4,6-trimethylpyridine

Notes

Acknowledgments

The authors thank Professor T. Kasama, Tokyo Medical and Dental University for MALDI-TOF analyses. A part of the present study was supported by a grant from the Ministry of Education, Science, and Culture, Japan.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Kiyoshi Nokihara
    • 1
  • Tadashi Yasuhara
    • 2
  • Yoshihiro Nakata
    • 3
  • Ethan A. Lerner
    • 4
  • Victor Wray
    • 5
  1. 1.HiPep LaboratoriesKyotoJapan
  2. 2.Tokyo University of AgricultureTokyoJapan
  3. 3.Hiroshima UniversityHiroshimaJapan
  4. 4.Massachusetts General HospitalCharlestownUSA
  5. 5.Department of Structural BiologyHelmholtz Centre for Infection ResearchBraunschweigGermany

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