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 journalArticle

3 Citations (Scopus)

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
JournalInternational Journal of Peptide Research and Therapeutics
Volume3
Issue number6
StatePublished - 1996

Fingerprint

Cyclization
Tandem Mass Spectrometry
Peptides
Mass spectrometry
Byproducts
High Pressure Liquid Chromatography
Ions
Lactams
Cyclic Peptides
Derivatives
Dynorphins
Guanidine
Alkylation
Mass Spectrometry

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Bioengineering
  • Molecular Medicine
  • Drug Discovery
  • Analytical Chemistry

Cite this

Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry. / Arttamangkul, Seksiri; Arbogast, Brian; Barofsky, Douglas; Aldrich, Jane V.

In: International Journal of Peptide Research and Therapeutics, Vol. 3, No. 6, 1996, p. 357-370.

Research output: Contribution to journalArticle

@article{cd97182e7f87401daa06e9ae916962b3,
title = "Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry",
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.",
keywords = "Aspartimide rearrangement, BOP, Dipyrrolidinylguanidinium, Dynorphin A derivative, HAPyU, HATU, Tetramethylguanidinium",
author = "Seksiri Arttamangkul and Brian Arbogast and Douglas Barofsky and Aldrich, {Jane V.}",
year = "1996",
language = "English (US)",
volume = "3",
pages = "357--370",
journal = "International Journal of Peptide Research and Therapeutics",
issn = "1573-3149",
publisher = "Springer Netherlands",
number = "6",

}

TY - JOUR

T1 - Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry

AU - Arttamangkul, Seksiri

AU - Arbogast, Brian

AU - Barofsky, Douglas

AU - Aldrich, Jane V.

PY - 1996

Y1 - 1996

N2 - 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.

AB - 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.

KW - Aspartimide rearrangement

KW - BOP

KW - Dipyrrolidinylguanidinium

KW - Dynorphin A derivative

KW - HAPyU

KW - HATU

KW - Tetramethylguanidinium

UR - http://www.scopus.com/inward/record.url?scp=53349146463&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=53349146463&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:53349146463

VL - 3

SP - 357

EP - 370

JO - International Journal of Peptide Research and Therapeutics

JF - International Journal of Peptide Research and Therapeutics

SN - 1573-3149

IS - 6

ER -