Multiple-Turnover Isotopic Labeling of Fmoc- and Boc-Protected Amino Acids with Oxygen Isotopes

Seyfried, Martin S.; Lauber, Birgit S.; Luedtke, Nathan W.

doi:10.1021/ol902519g

Cited by 25 publications

(103 citation statements)

References 31 publications

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“…A promising new method for synthesizing 13 C= 18 O amino acids has recently been demonstrated [52]. This one-pot synthesis uses multiple-turnover activation chemistry to efficiently exchange 16 O with 18 O at pH ~5.…”

Section: Membrane Proteins and Amyloidsmentioning

confidence: 99%

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy

Middleton

Woys

Mukherjee

et al. 2010

Methods

123

202

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We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for 13 C= 18 O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide form type 2 diabetes.

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Section: Membrane Proteins and Amyloidsmentioning

confidence: 99%

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy

Middleton

Woys

Mukherjee

et al. 2010

Methods

123

202

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“…8-12 While the utility of such labels is continually expanding, the backbone-incorporation techniques for these labels are currently limited to solid-phase peptide synthesis (SPPS). 1-8,10-13 …”

Section: Introductionmentioning

confidence: 99%

Insights into the Mechanism of Peptide Cyclodehydrations Achieved through the Chemoenzymatic Generation of Amide Derivatives

Dunbar

Mitchell

2013

J. Am. Chem. Soc.

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Current strategies for generating peptides and proteins bearing amide carbonyl derivatives rely on solid-phase peptide synthesis for amide functionalization. Although such strategies have been successfully implemented, technical limitations restrict both the length and sequence of the synthetic fragments. Herein we report the repurposing of a thiazole/oxazole-modified microcin (TOMM) cyclodehydratase to site-specifically install amide backbone labels onto diverse peptide substrates, a method we refer to as azoline-mediated peptide backbone labeling (AMPL). This convenient chemoenzymatic strategy can generate both thioamides and amides with isotopically labeled oxygen atoms. Moreover, we demonstrate the first leader peptide-independent activity of a TOMM synthetase, circumventing the requirement that sequences of interest be fused to a leader peptide for modification. Through bioinformatics-guided site-directed mutagenesis, we also convert a strictly dehydrogenase-dependent TOMM azole synthetase into an azoline synthetase. This vastly expands the spectrum of substrates modifiable by AMPL by allowing any in vitro reconstituted TOMM synthetase to be employed. To demonstrate the utility of AMPL for mechanistic enzymology studies, an 18O-labeled substrate was generated to provide direct evidence that cyclodehydrations in TOMMs occur through the phosphorylation of the carbonyl oxygen preceding the cyclized residue. Furthermore, we demonstrate that AMPL is a useful tool for establishing the location of azolines both on in vitro modified peptides and azoline-containing natural products.

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“…Initial attempts to carry out isotope exchange reaction of Boc-(1- 13 C)Phe-OH with H 2 18 O were not fruitful when we followed a previously reported protocol for the efficient 18 O isotope exchange of N α -protected amino acids. [17] We then carried out the Boc-protection/isotope exchange reaction in the reverse order. First, we conducted the exchange reaction of H-(1- 13 C)Phe-OH with a 1:1 mixture of H 2 18 O/dioxane at 100 °C; [18] After a few cycles of the exchange reaction, satisfactory enrichment was obtained.…”

mentioning

confidence: 99%

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

et al. 2016

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Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2D 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 2D 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. 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.

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Multiple-Turnover Isotopic Labeling of Fmoc- and Boc-Protected Amino Acids with Oxygen Isotopes

Cited by 25 publications

References 31 publications

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy

Insights into the Mechanism of Peptide Cyclodehydrations Achieved through the Chemoenzymatic Generation of Amide Derivatives

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

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Multiple-Turnover Isotopic Labeling of Fmoc- and Boc-Protected Amino Acids with Oxygen Isotopes

Cited by 25 publications

References 31 publications

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy

Insights into the Mechanism of Peptide Cyclodehydrations Achieved through the Chemoenzymatic Generation of Amide Derivatives

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

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