Final Report Summary - IODONIUM-CLICK (Development of Iodonium Salts into a Versatile Class of Bioconjugation Reagents) The Fellow has made excellent progress over the course of the fellowship. As was described in Annex I, the original goal of the project during the specified period was to work towards the “development of diaryliodonium salts as a versatile reagent class for bioconjugation (click) chemistry.” (Part A, page 2) Towards that end, the original work plan (Part B, page 22) outlined the development of a series of diaryliodonium salts that contained a second reactive handle, thus making them “bifunctional” in nature. The purpose of this second reactive handle was to provide a means for the facile conjugation of an iodonium salt a target biomolecule. Following this, the second major milestone was to develop a palladium catalyzed “bio-Heck” cross coupling reaction that exploited the inherent reactivity that iodonium salts display in cross-coupling reactions, with the third major milestone being the development of collaborations to extend this reactivity into more challenging systems. However, the Fellow became aware early in the reporting period of an iodonium salt (1) which displayed exceptional reactivity characteristics (Weiss, Angew. Chem. Int. Ed. 1994, 33, 1952). 1 is a structurally remarkable iodonium salt in which an α-diazoester functions as one of the ligands at iodine. Treatment of 1 with nucleophiles results in transfer of the diazoester to the nucleophile, resulting in an equivalent electrophilic substitution at the α-carbon. 1 undergoes diazo-transfer with a number of soft nucleophiles under mild conditions, including sulfides, with rapid conversion times. The resulting sulfonium adducts are stable at room temperature and possess a remarkable array of manipulable functionality (attached document, figure S1A). It was realized that, if this transformation could be adapted to work on the amino acid methionine, it would provide a highly original and virtually unexplored retrosynthetic disconnect for bioconjugation. Furthermore, we envisioned that bioconjugation at methionine with 1 would serve as more that just a vehicle for the delivery of a pre-specified payload, but as also as an opportunity to generate high energy protein-synthons which could serve to act as a “discovery platform” for new bioorthogonal reactions (attached document, figure S1B). Preliminary attempts to functionalize a simple methionine-containing dipeptide showed that this conjugation was in fact possible and proceeds quite readily in water. With this result in hand, it was decided that pursuing this new direction would be even more ideally suited to meet the proposal’s objectives. The major results accrued during the Fellowship are summarized here. The Fellow has optimized the methionine conjugation on a simple dipeptide system resulting a conjugation that proceeds with rapid kinetics and in >95% yield (attached document, figure S2). Having established the viability of the conjugation method, the Fellow has comprehensively explored the substrate scope of the reaction. The methionine conjugation reaction demonstrates excellent substrate scope; including four large medicinal polypeptides and four proteins that contain methionine residues in varying steric environments that are converted to the sulfonium conjugate with excellent levels of conversion (attached document, figure S3). The Fellow has also examined the scope of the diazo transfer reaction. Pleasingly, a number of biologically relevant functional groups can be transferred to proteins using this chemistry (attached document figure S4A). The labeling reactions tend to display clean reaction profiles and the sulfonium conjugates, which can be readily isolated by standard purification procedures, have been shown to be stable at -20°C for up to 6 months. The met-labeling reaction can be used over a wide range of concentrations, from 5 µM-550 µM, and is also amenable to large-scale functionalization reactions. Importantly, the protein-sulfonium conjugates also display complementarity with contemporary functionalization strategies (attached document, figure S4B). This was exemplified by showing that an alkyne-containing conjugate undergoes a copper-catalyzed Huisgen cyclization with an azide in 39% conversion; a result that is quite remarkable given the propensity of Cu(I) species to react with diazo moieties (attached document, figure S4B). To test the viability of the diazo-sulfonium conjugates to function as a bioorthogonal discovery platform, the reactive properties of the sulfonium conjugates have been explored by the Fellow. The Fellow has explored the use of metal catalysis with a coworker, finding the diazo moiety undergoes reaction with Rh2(OAc)4 to generate a carbenoid species that can be trapped with nitriles, yielding 1,3-oxazole products (attached document, figure S5A). The electron deficient nature of the sulfonium-diazo conjugates allows for the use of “Staudinger-type” chemistry: triphenylphosphine can nucleophilicly add into the diazo compound, subsequently displacing the C-S bond and releasing free methionine. This provides a convenient means for controllably reversing the met-ligation (attached document, figure S5B). When we attempted a Staudinger-type ligation with the diazo moiety, we observed a remarkable shift of the entire conjugate from methionine to the N-terminus. While only a preliminary result, it is a remarkable result on several levels: with potential applications in native-chemical ligation and selective functionalization of nitrogen in a protein, which is an unsolved challenge in bioconjugation chemistry (attached document, figure S6). The results that have been discovered during the period of this fellowship represent a significant and potentially paradigm-shifting advance in the field of bioconjugation chemistry. In addition to developing a method for functionalizing an amino acid residue previously not considered for bioconjugation, the strategy with which it has been done is essentially unique. In this respect, we expect this work to have a lasting impact on the scientific community, which in turn will translate into societal impacts. Specifically, this work will contribute to the improvement of human health through by acting as a facilitator of biochemical discovery. For instance, we expect the results of this study will contribute towards the design of more advanced Antibody drug conjugates, a class of advanced therapeutic that are rapidly becoming front-line therapeutics for the treatment of cancer. Finally, IIF has provided the Fellow with the prime opportunity to develop himself both scientifically and professionally; putting him in the prime position to launch his academic career. The opportunity to participate in integration and transfer of knowledge activities within the European Union will prove invaluable to the Fellow in establishing future international collaborations and other knowledge-transfer activities. Related documents final1-final-report-supporting-figures-.pdf
