Redox-neutral, metal-free tryptophan labeling of polypeptides in hexafluoroisopropanol (HFIP).

Despite the unmet needs for chemical tools to study biological roles of tryptophan in living systems, there has been a lack of chemical modification methods for tryptophan residues that can be used in cellular environments. Driven by a preliminary computational study of our previous research, this work experimentally examined our hypotheses to translate the metal-catalyzed tryptophan modification method in hexafluoroisopropanol (HFIP) into a metal-free process. While one of the hypotheses merely confirmed the superiority of the thiophene-ethanol reagent developed in the previous report, the second hypothesis resulted in the identification of a trifluoroborate salt and an acidic ionic liquid as alternatives for the catalysis. Labeling of lysates of a human cell line was achieved with the acidic ionic liquid catalyst, where negative impacts of the tryptophan labeling and HFIP medium on the cellular samples were apparently insignificant. Because the labeling process does not require any redox mediators and is a formal redox-neutral reaction, the metal-free approach would be of use for tryptophan biology research potentially related to their various redox roles.

Hexafluoroisopropanol as a Bioconjugation Medium of Ultrafast, Tryptophan-Selective Catalysis.

The past decade has seen a remarkable growth in the number of bioconjugation techniques in chemistry, biology, material science, and biomedical fields. A core design element in bioconjugation technology is a chemical reaction that can form a covalent bond between the protein of interest and the labeling reagent. Achieving chemoselective protein bioconjugation in aqueous media is challenging, especially for generally less reactive amino acid residues, such as tryptophan. We present here the development of tryptophan-selective bioconjugation methods through ultrafast Lewis acid-catalyzed reactions in hexafluoroisopropanol (HFIP). Structure-reactivity relationship studies have revealed a combination of thiophene and ethanol moieties to give a suitable labeling reagent for this bioconjugation process, which enables modification of peptides and proteins in an extremely rapid reaction unencumbered by noticeable side reactions. The capability of the labeling method also facilitated radiofluorination application as well as antibody functionalization. Enhancement of an α-helix by HFIP leads to its compatibility with a certain protein, and this report also demonstrates a further stabilization strategy achieved by the addition of an ionic liquid to the HFIP medium. The nonaqueous bioconjugation approaches allow access to numerous chemical reactions that are unavailable in traditional aqueous processes and will further advance the chemistry of proteins.