Thioamide substitutions in the backbones of proteins can modulate their structure and thermostability, or serve as spectroscopic probes in fluorescence quenching experiments. Using native chemical ligation, we have produced the first examples of a protein (calmodulin) containing two thioamides. Dithioamide variants were made to explore the effects of combining stabilizing, neutral, and destabilizing single thioamide substitutions. One of the dithioamide calmodulin variants exhibited stabilization greater than any monothioamide variant, although the effect could not easily be anticipated from the results of single substitutions. Each of the calmodulin variants retained the ability to bind a target peptide, and the dithioamide proteins exhibited an increase in fluorescence quenching of tryptophan relative to their single thioamide counterparts. These results show that multiply thioamidated proteins can be synthesized, and that properly placed thioamides can be used to increase protein thermostability or enhance fluorecsence quenching in peptide binding experiments.
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Side‐chain thioamides as fluorescence quenching probes
Thioamides, single atom oxygen‐to‐sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side‐chains of amino acids. We synthesize and incorporate Fmoc‐protected, solid‐phase peptide synthesis building blocks for introducing
- NSF-PAR ID:
- 10454296
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biopolymers
- Volume:
- 112
- Issue:
- 1
- ISSN:
- 0006-3525
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Thioamides represent highly valuable isosteric in the strictest sense “single‐atom substitution” analogues of amides that have found broad applications in chemistry and biology. A long‐standing challenge is the direct transamidation of thioamides, a process which would convert one thioamide bond (R−C(S)−NR1R2) into another (R−C(S)−NR3N4). Herein, we report the first general method for the direct transamidation of thioamides by highly chemoselective N−C(S) transacylation. The method relies on site‐selective N‐
tert ‐butoxycarbonyl activation of 2° and 1° thioamides, resulting in ground‐state‐destabilization of thioamides, thus enabling to rationally manipulate nucleophilic addition to the thioamide bond. This method showcases a remarkably broad scope including late‐stage functionalization (>100 examples). We further present extensive DFT studies that provide insight into the chemoselectivity and provide guidelines for the development of transamidation methods of the thioamide bond. -
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Abstract Thioamides represent highly valuable isosteric in the strictest sense “single‐atom substitution” analogues of amides that have found broad applications in chemistry and biology. A long‐standing challenge is the direct transamidation of thioamides, a process which would convert one thioamide bond (R−C(S)−NR1R2) into another (R−C(S)−NR3N4). Herein, we report the first general method for the direct transamidation of thioamides by highly chemoselective N−C(S) transacylation. The method relies on site‐selective N‐
tert ‐butoxycarbonyl activation of 2° and 1° thioamides, resulting in ground‐state‐destabilization of thioamides, thus enabling to rationally manipulate nucleophilic addition to the thioamide bond. This method showcases a remarkably broad scope including late‐stage functionalization (>100 examples). We further present extensive DFT studies that provide insight into the chemoselectivity and provide guidelines for the development of transamidation methods of the thioamide bond. -
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Rationale N 6‐Formyl lysine is a well‐known modification of histones and other proteins. It can also be formed as a damaged product from direct formylation of free lysine and accompanied by other lysine derivatives such as acetylated or methylated forms. In relation to the activity of cellular repair enzymes in protein turnover and to lysine metabolism, it is important to accurately quantify the overall ratio of modified lysine to free lysine.Methods N 6‐Formyl lysine was quantified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data collected in a non‐targeted manner using positive mode electrospray ionization on a Q‐Exactive HF+Orbitrap mass spectrometer. Studies were performed with lysine and deuterated lysine spiked into protein digests and solvents to investigate the extent of spontaneous formation and matrix effects of formation ofN 6‐formyl lysine.Results We show that
N 6‐formyl lysine,N 2‐formyl lysine,N 6‐acetyl lysine, andN 2‐acetyl lysine are all formed spontaneously during sample preparation and LC/MS/MS analysis, which complicates quantification of these metabolites in biological samples.N 6‐Formyl lysine was spontaneously formed and correlated to the concentration of lysine. In the sample matrix of protein digests, 0.03% of lysine was spontaneously converted intoN 6‐formyl lysine, and 0.005% of lysine was converted intoN 6‐formyl lysine in pure run solvent.Conclusions Spontaneous formation of
N 6‐formyl lysine,N 6‐acetyl lysine,N 2‐formyl lysine, andN 2‐acetyl lysine needs to be subtracted from biologically formed lysine modifications when quantifying these epimetabolites in biological samples. -
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Abstract The strain irreversibility cliff (SIC), marking the abrupt change of the intrinsic irreversible strain limit
ε irr,0as a function of heat-treatment (HT) temperatureθ in Nb3Sn superconducting wires made by the restacked-rod process (RRP®), is confirmed in various wire designs. It adds to the complexity of reconciling conflicting requirements on conductors for fabricating magnets. Those intended for the high-luminosity upgrade of the Large Hardon Collider (LHC) at the European Organization for Nuclear Research (CERN) facility require maintaining the residual resistivity ratioRRR of conductors above 150 to ensure stability of magnets against quenching. This benchmark may compromise the conductors’ mechanical integrity if theirε irr,0is within or at the bottom of SIC. In this coupled investigation of strain andRRR properties to fully assess the implications of SIC, we introduce an electro-mechanical stability criterion that takes into account both aspects. For standard-Sn billets, this requires a strikingly narrow HT temperature window that is impractical. On the other hand, reduced-Sn billets offer a significantly wider choice ofθ , not only for ensuring thatε irr,0is located at the SIC plateau whileRRR ≥ 150, but also for containing the strain-induced irreversible degradation of the conductor’s critical-current beyondε irr,0. This study suggests that HT of LHC magnets, made of reduced-Sn wires having a Nb/Sn ratio of 3.6 and 108/127 restacking architecture, be operated atθ in the range of 680 to 695 °C (when the dwell time is 48 hours).
