Biosynthesis and Chemical Applications of Thioamides.

Thioamidation as a posttranslational modification is exceptionally rare, with only a few reported natural products and exactly one known protein example (methyl-coenzyme M reductase from methane-metabolizing archaea). Recently, there has been significant progress in elucidating the biosynthesis and function of several thioamide-containing natural compounds. Separate developments in the chemical installation of thioamides into peptides and proteins have enabled cell biology and biophysical studies to advance the current understanding of natural thioamides. This review highlights the various strategies used by Nature to install thioamides in peptidic scaffolds and the potential functions of this rare but important modification. We also discuss synthetic methods used for the site-selective incorporation of thioamides into polypeptides with a brief discussion of the physicochemical implications. This account will serve as a foundation for the further study of thioamides in natural products and their various applications.

The effects of thioamide backbone substitution on protein stability: a study in α-helical, ß-sheet, and polyproline II helical contexts.

Thioamides are single atom substitutions of the peptide bond that serve as versatile probes of protein structure. Effective use of thioamides requires a robust understanding of the impact that the substitution has on a protein of interest. However, the thermodynamic effects of thioamide incorporation have only been studied in small structural motifs, and their influence on secondary structure in the context of full-length proteins is not known. Here we describe a comprehensive survey of thioamide substitutions in three benchmark protein systems (calmodulin, the B1 domain of protein G, and collagen) featuring the most prevalent secondary structure motifs: α-helix, ß-sheet, and polyproline type II helix. We find that in most cases, effects on thermostability can be understood in terms of the positioning and local environment of the thioamide relative to proximal structural elements and hydrogen bonding networks. These observations set the stage for the rational design of thioamide substituted proteins with predictable stabilities.

Effect of Nascent Peptide Steric Bulk on Elongation Kinetics in the Ribosome Exit Tunnel.

All proteins are synthesized by the ribosome, a macromolecular complex that accomplishes the life-sustaining tasks of faithfully decoding mRNA and catalyzing peptide bond formation at the peptidyl transferase center (PTC). The ribosome has evolved an exit tunnel to host the elongating new peptide, protect it from proteolytic digestion, and guide its emergence. It is here that the nascent chain begins to fold. This folding process depends on the rate of translation at the PTC. We report here that besides PTC events, translation kinetics depend on steric constraints on nascent peptide side chains and that confined movements of cramped side chains within and through the tunnel fine-tune elongation rates.

Thieme Chemistry Journals Awardees - Where Are They Now? Improved Fmoc Deprotection Methods for the Synthesis of Thioamide-Containing Peptides and Proteins.

Site-selective incorporation of thioamides into peptides and proteins provides a useful tool for a wide range of applications. Current incorporation methods suffer from low yields as well as epimerization. Here, we describe how the use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) rather than piperidine in fluorenylmethyloxycarbonyl (Fmoc) deprotection reduces epimerization and increases yields of thioamide-containing peptides. Furthermore, we demonstrate that the use of DBU avoids byproduct formation when synthesizing peptides containing side-chain thioamides.

Chemoselective modifications for the traceless ligation of thioamide-containing peptides and proteins.

Thioamides are single-atom substitutions of canonical amide bonds, and have been proven to be versatile and minimally perturbing probes in protein folding studies. Previously, our group showed that thioamides can be incorporated into proteins by native chemical ligation (NCL) with Cys as a ligation handle. In this study, we report the expansion of this strategy into non-Cys ligation sites, utilizing radical initiated desulfurization to "erase" the side chain thiol after ligation. The reaction exhibited high chemoselectivity against thioamides, which can be further enhanced with thioacetamide as a sacrificial scavenger. As a proof-of-concept example, we demonstrated the incorporation of a thioamide probe into a 56 amino acid protein, the B1 domain of Protein G (GB1). Finally, we showed that the method can be extended to ß-thiol amino acid analogs and selenocysteine.

Electronic interactions of i, i + 1 dithioamides: increased fluorescence quenching and evidence for n-to-π* interactions.

Thioamide residues can be effective, minimally-perturbing fluorescence quenching probes for studying protein folding and proteolysis. In order to increase the level of quenching, we have here explored the use of adjacent dithioamides. We have found that they are more effective fluorescence quenchers, as expected, but we have also observed unexpected changes in the thioamide absorption spectra that may arise from n-to-π* interactions of the thiocarbonyls. We have made use of the increased quenching to improve the fluorescence turn-on of thioamide protease sensors.

Light-controlled modulation of gene expression by chemical optoepigenetic probes.

Epigenetic gene regulation is a dynamic process orchestrated by chromatin-modifying enzymes. Many of these master regulators exert their function through covalent modification of DNA and histone proteins. Aberrant epigenetic processes have been implicated in the pathophysiology of multiple human diseases. Small-molecule inhibitors have been essential to advancing our understanding of the underlying molecular mechanisms of epigenetic processes. However, the resolution offered by small molecules is often insufficient to manipulate epigenetic processes with high spatiotemporal control. Here we present a generalizable approach, referred to as 'chemo-optical modulation of epigenetically regulated transcription' (COMET), enabling high-resolution, optical control of epigenetic mechanisms based on photochromic inhibitors of human histone deacetylases using visible light. COMET probes may be translated into new therapeutic strategies for diseases where conditional and selective epigenome modulation is required.

Semi-synthesis of thioamide containing proteins.

Our laboratory has shown that the thioamide, a single atom O-to-S substitution, can be a versatile fluorescence quenching probe that is minimally-perturbing when placed at many locations in a protein sequence. In order to make these and other thioamide experiments applicable to full-sized proteins, we have developed methods for incorporating thioamides by generating thiopeptide fragments through solid phase synthesis and ligating them to protein fragments expressed in E. coli. To install donor fluorophores, we have adapted unnatural amino acid mutagenesis methods, including the generation of new tRNA synthetases for the incorporation of small, intrinsically fluorescent amino acids. We have used a combination of these two methods, as well as chemoenzymatic protein modification, to efficiently install sidechain and backbone modifications to generate proteins labeled with fluorophore/thioamide pairs.

Monoalkoxy BODIPYs--a fluorophore class for bioimaging.

Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.