Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry

Seksiri Arttamangkul, Brian Arbogast, Douglas Barofsky, Jane V. Aldrich

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

The cyclization of a linear dynorphin A (Dyn A) analogue to give the lactam derivative cyclo[D-Asp2,Dap5]Dyn A(1-13)NH2 (where Dap = α,β-diaminopropionic acid) was studied to evaluate the usefulness of different coupling reagents for side chain to side chain lactam formation. This cyclization proved to be difficult and yielded substantial byproducts that varied depending upon the activating reagent used. On-line HPLC-ion spray mass spectrometry was more practical and useful than conventional HPLC alone for characterizing the products of these cyclization reactions. Peptide byproducts could be identified from the series of multiply charged ions observed, even when some of these peptides eluted from the HPLC with similar retention times. In addition to the desired cyclic peptide, the peptide byproducts observed following the cyclization using BOP (benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate) were the linear peptide, the cyclic dimeric peptide and the linear peptide resulting from aspartimide rearrangement. The peptide byproducts obtained following cyclization using HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HAPyU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate) were predominantly linear tetramethylguanidinium (Tmg) and dipyrrolidinylguanidinium (Dpg) derivatives resulting from alkylation of the side chain of Dap by HATU and HAPyU, respectively; in addition to monomeric guanidinium derivatives, dimeric and aspartimide-containing peptides were also produced. Peptide sequencing by ion spray tandem mass spectrometry was performed to confirm the structure of both pure peptides and peptide byproducts in the crude samples. A unique fragmentation for the β,γ-bond of the Dap side chain was demonstrated and could be used to identify linear peptide byproducts. The distinctive fragment ions from this cleavage were also observed for the peptides containing the Tmg and Dpg functionalities on the Dap side chain.

Original languageEnglish (US)
Pages (from-to)357-370
Number of pages14
JournalLetters in Peptide Science
Volume3
Issue number6
DOIs
StatePublished - Jan 1997

Keywords

  • Aspartimide rearrangement
  • BOP
  • Dipyrrolidinylguanidinium
  • Dynorphin A derivative
  • HAPyU
  • HATU
  • Tetramethylguanidinium

ASJC Scopus subject areas

  • Biochemistry

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