Efficient Total Chemical Synthesis of 13C= 18O Isotopomers of Human Insulin for Isotope-Edited FTIR
Abstract
Isotope–edited two–dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site–specific incorporation of stable 13C=18O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis—via a key ester insulin intermediate—of 97 % enriched [(1–13C=18O)PheB24] human insulin: stable–isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X–ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1–13C=18O)PheB24] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red–shifted amide I carbonyl band peak at 1595 cm–1 resulting from the (1–13C=18O)PheB24 backbone label. Furthermore, this work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.
- Authors:
-
- Univ. of Chicago, IL (United States)
- Case Western Reserve Univ., Cleveland, OH (United States)
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); National Science Foundation (NSF); National Institutes of Health (NIH)
- OSTI Identifier:
- 1241077
- Grant/Contract Number:
- AC02‐06CH11357; CHE‐1414486; R01K08993
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ChemBioChem: a European journal of chemical biology
- Additional Journal Information:
- Journal Volume: 17; Journal Issue: 5; Journal ID: ISSN 1439-4227
- Publisher:
- ChemPubSoc Europe
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; human insulin; chemical protein synthesis; native chemical ligation; isotope labeling; 2D IR spectra
Citation Formats
Dhayalan, Balamurugan, Fitzpatrick, Ann, Mandal, Kalyaneswar, Whittaker, Jonathan, Weiss, Michael A., Tokmakoff, Andrei, and Kent, Stephen B. H. Efficient Total Chemical Synthesis of 13C= 18O Isotopomers of Human Insulin for Isotope-Edited FTIR. United States: N. p., 2015.
Web. doi:10.1002/cbic.201500601.
Dhayalan, Balamurugan, Fitzpatrick, Ann, Mandal, Kalyaneswar, Whittaker, Jonathan, Weiss, Michael A., Tokmakoff, Andrei, & Kent, Stephen B. H. Efficient Total Chemical Synthesis of 13C= 18O Isotopomers of Human Insulin for Isotope-Edited FTIR. United States. https://doi.org/10.1002/cbic.201500601
Dhayalan, Balamurugan, Fitzpatrick, Ann, Mandal, Kalyaneswar, Whittaker, Jonathan, Weiss, Michael A., Tokmakoff, Andrei, and Kent, Stephen B. H. Wed .
"Efficient Total Chemical Synthesis of 13C= 18O Isotopomers of Human Insulin for Isotope-Edited FTIR". United States. https://doi.org/10.1002/cbic.201500601. https://www.osti.gov/servlets/purl/1241077.
@article{osti_1241077,
title = {Efficient Total Chemical Synthesis of 13C= 18O Isotopomers of Human Insulin for Isotope-Edited FTIR},
author = {Dhayalan, Balamurugan and Fitzpatrick, Ann and Mandal, Kalyaneswar and Whittaker, Jonathan and Weiss, Michael A. and Tokmakoff, Andrei and Kent, Stephen B. H.},
abstractNote = {Isotope–edited two–dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site–specific incorporation of stable 13C=18O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis—via a key ester insulin intermediate—of 97 % enriched [(1–13C=18O)PheB24] human insulin: stable–isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X–ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1–13C=18O)PheB24] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red–shifted amide I carbonyl band peak at 1595 cm–1 resulting from the (1–13C=18O)PheB24 backbone label. Furthermore, this work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.},
doi = {10.1002/cbic.201500601},
journal = {ChemBioChem: a European journal of chemical biology},
number = 5,
volume = 17,
place = {United States},
year = {Wed Dec 30 00:00:00 EST 2015},
month = {Wed Dec 30 00:00:00 EST 2015}
}
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Works referencing / citing this record:
Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin
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