Efficient Total Chemical Synthesis of 13C= 18O Isotopomers of Human Insulin for Isotope-Edited FTIR

Dhayalan, Balamurugan; Fitzpatrick, Ann; Mandal, Kalyaneswar; Whittaker, Jonathan; Weiss, Michael A.; Tokmakoff, Andrei; Kent, Stephen B. H.

doi:10.1002/cbic.201500601

Efficient Total Chemical Synthesis of ¹³C= ¹⁸O Isotopomers of Human Insulin for Isotope-Edited FTIR

Journal Article · Wed Dec 30 00:00:00 EST 2015 · ChemBioChem: a European journal of chemical biology

DOI:https://doi.org/10.1002/cbic.201500601· OSTI ID:1241077

Dhayalan, Balamurugan ^[1]; Fitzpatrick, Ann ^[1]; Mandal, Kalyaneswar ^[1]; Whittaker, Jonathan ^[2]; Weiss, Michael A. ^[2]; Tokmakoff, Andrei ^[1]; Kent, Stephen B. H. ^[1]

Univ. of Chicago, IL (United States)
Case Western Reserve Univ., Cleveland, OH (United States)

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 stable¹³C=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)Phe^B24] 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)Phe^B24] 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)Phe^B24 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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: NIH; National Science Foundation; USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1241077

Journal Information:: ChemBioChem: a European journal of chemical biology, Journal Name: ChemBioChem: a European journal of chemical biology Journal Issue: 5 Vol. 17; ISSN 1439-4227

Publisher:: ChemPubSoc EuropeCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited By (3)

Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin Dhayalan, Balamurugan; Mandal, Kalyaneswar; Rege, Nischay Chemistry - A European Journal, Vol. 23, Issue 7 https://doi.org/10.1002/chem.201605578	journal	January 2017
Novel protein science enabled by total chemical synthesis Kent, Stephen B. H. Protein Science, Vol. 28, Issue 2 https://doi.org/10.1002/pro.3533	journal	December 2018
A molecular engineering toolbox for the structural biologist Debelouchina, Galia T.; Muir, Tom W. Quarterly Reviews of Biophysics, Vol. 50 https://doi.org/10.1017/s0033583517000051	journal	January 2017

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Related Subjects

2D IR spectra
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
chemical protein synthesis
human insulin
isotope labeling
native chemical ligation