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Solid-Phase Synthesis of Neuropeptides by Fmoc Strategies

  • Chris Kowalczyk
  • Michael O’Shea
Part of the Methods in Molecular Biology™ book series (MIMB, volume 73)

Abstract

The aim of this chapter is to give a detailed, step-by-step description of a procedure for obtaining a batch of a desired peptide at the required level of purity. It is assumed that the initial synthesis will be done using an automated peptide synthesizer that performs solid-phase peptide synthesis using the Fmoc strategy (1). This is based on the sequential addition of amino acid residues to an insoluble polymeric support. The base-labile Fmoc group is used to protect the α-amino group of each residue. Those residues that have potentially reactive side chains are protected with acid-labile groups such as t-butyl. After removal of the Fmoc group with piperidine, the next protected amino acid is added using either a coupling reagent or preactivated amino acid derivative (2). At the end of the synthesis, the peptide is cleaved from the solid support to yield a peptide acid or amide, depending on the linking agent used, and the side-chain protecting groups are removed by treating the peptide-resin with a mixture of trifluoroacetic acid and various ion scavengers (3). Methyl t-butyl ether is then added to precipitate the peptide out of the cleavage mixture. The crude peptide is then dissolved and lyophilized, after which it can be purified by high performance liquid chromatography. The purified peptide can then be lyophilized for storage.

Keywords

High Performance Liquid Chromatography High Performance Liquid Chromatography Loop Volume Fmoc Group Cleavage Time 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Atherton, E. and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis, A Practical Approach, Oxford University Press, Oxford, UK.Google Scholar
  2. 2.
    Fields, G. B. and Noble, R. L. (1990) Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Peptide Protein Res. 35, 161–214.CrossRefGoogle Scholar
  3. 3.
    Applied Biosystems Inc., Foster City, CA (1990) Introduction to Cleavage Techniques.Google Scholar
  4. 4.
    Applied Biosystems Inc., Foster City, CA (1993) Synergy User Manual.Google Scholar
  5. 5.
    Calbiochem-Novabiochem, Nottingham, UK (1994) Catalog and Peptide Synthesis Handbook.Google Scholar

Copyright information

© Humana Press Inc. Totowa, NJ 1997

Authors and Affiliations

  • Chris Kowalczyk
    • 1
  • Michael O’Shea
    • 1
  1. 1.Sussex Centre for Neuroscience, School of BiologyUniversity of SussexBrightonUK

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