Trifluoroethanethiol: An Additive for Efficient One-Pot Peptide Liga- tion-Desulfurization Chemistry Robert E. Thompson, Xuyu Liu, Katrina A. Jolliffe, Richard J. Payne* School of Chemistry, Building F11, The University of Sydney, NSW 2006, AUSTRALIA Supporting Information Placeholder ABSTRACT: Native chemical ligation followed by desulfuriza- tion is a powerful strategy for the assembly of proteins. Herein, we describe the development of a high yielding, one-pot ligation- desulfurization protocol that employs trifluoroethanethiol (TFET) as a novel thiol additive. The synthetic utility of this TFET- enabled methodology is demonstrated by the efficient multi-step one-pot syntheses of two tick-derived proteins, chimadanin and madanin-1, without the need for any intermediary purification. Chemical synthesis of proteins provides a means by which key structural and functional information of a given target can be elucidated. 1 Over the past two decades considerable advances in this area have been made possible owing to the development of the venerable native chemical ligation method. 2 This transfor- mation involves the chemoselective reaction between a peptide containing a C-terminal thioester and a peptide bearing an N- terminal Cys residue to afford a native peptide bond in aqueous media at neutral pH (Scheme 1). Due to the ease of preparation and stability to long term storage, alkyl thioesters are often em- ployed directly in ligation chemistry. However, this functionality is relatively inert, necessitating the inclusion of a thiol additive to generate a more reactive peptide thioester as the acyl donor in the ligation reaction. A trans-thioesterification, between the side chain of the Cys residue and the newly formed thioester moiety, then occurs followed by an irreversible intramolecular S→N acyl transfer to form a native peptide bond. Scheme 1. Native chemical ligation-desulfurization. Ligation technology has benefited greatly from the introduction of exogenous thiol additives to improve reaction rates via the in situ generation of reactive peptide thioesters. In a thorough study by Johnson and Kent, the relative reactivity of a range of com- mercially available thiols was investigated. 3 Aryl thiols with pKa values > 6 were shown to afford optimal ligation rates due to two key reactive properties: 1) the ability to rapidly exchange with alkyl thioesters to generate aryl thioesters and 2) excellent leaving group ability upon reaction with the N-terminal Cys residue. From this study the water soluble aryl thiol additive mercapto- phenylacetic acid (MPAA, pKa = 6.6) was selected as an excel- lent additive that facilitated more rapid ligations compared with the two traditionally employed thiol additives, the water soluble alkyl thiol mercaptoethane thiolate sodium salt (MESNa, pKa 9.2) and the sparingly water soluble thiophenol (pKa = 6.6). A significant advancement in ligation methodology has been the development of desulfurization chemistry which transforms Cys residues to Ala following the ligation event. 4 This methodol- ogy has sparked interest in the use of the native chemical ligation concept at a variety of unnatural mercapto- and seleno-amino acids that can subsequently be converted to native amino acids by desulfurization or deselenization. 5 Whilst desulfurization of Cys to Ala can be effected through the use of catalytic hydrogenation, 4a radical desulfurization 4b is the most widely em- ployed method and has been used in the synthesis of a number of complex protein targets. 6 Given the high yielding nature of desul- furization chemistry, the union of this transformation with effi- cient ligation chemistry into a one-pot procedure would represent a powerful addition to the toolbox of methods available for use in chemical protein synthesis. Unfortunately the necessity of aryl thiol additives in the ligation reaction prohibits this capability due to the inherent radical quenching activity (even in trace amounts) of aryl thiols. 7 As such, products produced from ligation reactions require tedious purification and lyophilization before the submis- sion of purified materials to desulfurization conditions. Solutions to this problem have therefore been sought, including the use of the alkyl thiol MESNa as the thiol additive which, despite the significantly slower ligation rates, does not interfere with the desulfurization chemistry. 6g Alternatively, methods to remove aryl thiols from the reaction mixture have been employed, includ- ing extensive liquid-liquid extraction of aryl thiols such as thio- phenol, or solid-phase extraction procedures. 5q Recently, Brik and co-workers employed a synthetic bi-functional aryl thiol catalyst which could be captured with an aldehyde-derived solid- supported reagent prior to the desulfurization reactions. 8 In an effort to streamline the two highly efficient reactions into a straightforward and operationally simple one-pot protocol, we sought to identify a novel thiol additive capable of facilitating rapid rates of ligation without disrupting the subsequent radical desulfurization. In this study we show that 2,2,2- trifluoroethanethiol (TFET) is an efficient thiol catalyst and, im- portantly, being an alkyl thiol permits in situ one-pot desulfuriza- tion reactions. To demonstrate the utility of TFET, we undertook the synthesis of two small tick-derived proteins, chimadanin and madanin-1, via the one-pot ligation-desulfurization of three pep- tide fragments either in the C- to N-terminal direction or through O H 2 N O SR 2 R N H HS + O H 2 N S O R N H O N H O R H N HS transthioesterification S N acyl shift O N H O R H N desulfurization PEPTIDE PEPTIDE PEPTIDE PEPTIDE PEPTIDE PEPTIDE PEPTIDE PEPTIDE O SR 1 R N H PEPTIDE HSR 2