Thioamide modifications of the peptide backbone are used to perturb secondary structure, to inhibit proteolysis, as photoswitches, and as spectroscopic labels. Thus far, their incorporation has been confined to single peptides synthesized on solid phase. We have generated thioamides in C-terminal thioesters or N-terminal Cys fragments and examined their compatibility with native chemical ligation conditions. Most sequence variants can be coupled in good yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations. Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track conformational changes during aggregation of labeled α-synuclein.

Characterization of the amyloidogenic Parkinson’s Disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled αS were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.

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.