Electrophilic bioactive compounds are valuable tools for understanding biological mechanisms. Their ability to covalently modify proteins, particularly when armed with biorthogonal tags, allows for target identification and validation. The resurgence of interest in these molecules has led to FDA-approved drugs across various therapeutic areas. However, the structural diversity of screening sets is often limited by the available building blocks and the types of reactions used for functionalization. Established electrophilic screening compounds, such as α-halo and α,β unsaturated amides, are typically prepared using a narrow range of reactions. The focus on warheads targeting reactive cysteine residues further restricts diversity. To expand the available chemical space, this study explores S(VI) exchange chemistry and simple sulfonyl fluoride fragments as potential alternatives. Sulfonyl fluoride warheads are known to primarily target lysine and tyrosine, but tailored probes can target a broader range of residues. This research aims to develop a unified, diversity-oriented synthesis of sulfonyl fluorides, screen the resulting probes against *Trypanosoma brucei*, and use chemical proteomics to elucidate the mechanism of action of any identified inhibitors. *Trypanosoma brucei*, the causative agent of African trypanosomiasis (sleeping sickness), is a particularly relevant target due to the existence of specific enzymes in its glycosylphosphatidylinositol biosynthetic pathway that are inhibited by known sulfonyl fluoride compounds.

The existing literature highlights the limitations of current electrophilic screening compound libraries, which often lack structural and functional diversity due to reliance on a narrow set of reactions. This has spurred research into alternative approaches like S(VI) exchange chemistry to generate diverse fragments with complementary warheads. Simple sulfonyl fluoride fragments have also been explored, demonstrating their ability to target various residues, including cysteine, histidine, serine, threonine, and tyrosine, depending on the probe's design. Proteome-wide screens have shown a preference for lysine and tyrosine targeting by simple sulfonyl fluorides; however, tailored probes can enhance the range of targeted residues. The literature also indicates the potential of *Trypanosoma brucei* enzymes involved in glycosylphosphatidylinositol biosynthesis as therapeutic targets, as these enzymes are sensitive to inhibition by known sulfonyl fluoride compounds such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. The need for a more diverse and efficient synthesis method to create electrophilic probes, particularly sulfonyl fluorides, coupled with the biological relevance of *T. brucei* as a target organism, formed the basis of this study.

This study utilized a unified connective synthesis of skeletally-diverse sulfonyl fluorides, employing photoredox-catalyzed dehydrogenative coupling reactions between hetaryl sulfonyl fluorides and hydrogen donors (saturated nitrogen and/or oxygen heterocycles). A range of hetaryl sulfonyl fluorides (HA A-F) and hydrogen donors (HD 1-20) were selected based on their diversity and synthetic accessibility. The hetaryl sulfonyl fluorides were based on various heterocyclic rings, while the hydrogen donors were diverse saturated heterocyclic ring systems, each containing at least one C-H bond α to nitrogen or oxygen. The reaction involved the C-H functionalization of both substrates and the formation of a C-C bond between them. A screening approach identified productive substrate pairs. Typically, reactions were performed by combining a hetaryl sulfonyl fluoride, hydrogen donor (5 eq.), TFA, Ir[dF(CF3)ppy]2(dtbbpy)PF6 (1 mol%), and tert-butyl peracetate (TBPA) in acetone. However, for some substrates, higher equivalents were used. Reactions were irradiated with a 390 nm lamp for 24 hours. Crude products were analyzed by LC-MS and NMR, and promising reactions were purified by mass-directed HPLC. Initially, couplings with N-Boc pyrrolidine (HD 1) were explored. Subsequently, the most promising hetaryl sulfonyl fluorides were paired with all twenty hydrogen donors. The resulting 32 sulfonyl fluoride probes were characterized, and their molecular properties (including AlogP and the number of heavy atoms) were analyzed. The probes were then screened against *T. brucei* bloodstream forms in 96-well plates at concentrations ranging from 0.1 to 100 μM. Hits were rescreened to confirm EC50 values. Selectivity was assessed by screening against HeLa cells. Chemical proteomics experiments involved preparing an alkynylated analogue of a promising hit (C-1) and using this analogue, C-1alk, to identify target proteins via click chemistry with rhodamine-N3 and biotin-N3. The proteins pulled down were analyzed by mass spectrometry. To pinpoint the targets of the parent ligand C-1, competition experiments using C-1 to outcompete C-1alk labeling were performed. Additionally, fluorophosphonate probes (FP-Rh and FP-biotin), which are known to target serine hydrolases, were used to investigate if serine hydrolases were targeted by the anti-trypanosomal compounds. In-gel fluorescence analysis and mass spectrometry were used to identify the proteins labeled by the fluorophosphonate probes. Data analysis involved gene ontology enrichment analysis, and statistical analysis using a modified two-sample t-test with permutation-based false discovery rate (FDR).

A total of 32 diverse sulfonyl fluoride probes were successfully synthesized using a photoredox-catalyzed dehydrogenative coupling approach. The screening of these probes against *T. brucei* identified ten probes with EC50 < 10 μM, and four with EC50 < 1 μM. These four exhibited 12- to 66-fold selectivity for *T. brucei* over HeLa cells. The anti-trypanosomal activity was significantly greater than that of the parental hetaryl sulfonyl fluoride substrates, highlighting the importance of the specific substitution pattern. Chemical proteomics using the alkynylated probe C-1alk identified numerous enriched proteins, including those involved in nucleotide binding, translation machinery, and hydrolytic enzymes. Competition experiments with C-1 identified two proteins – a putative lyso-phospholipase (D6XM23) and a putative nucleoside hydrolase (Q57X73) – as potential targets. Further investigation using fluorophosphonate probes revealed that C-1 targets a subset of serine hydrolases, including the lyso-phospholipase D6XM23. However, RNAi studies suggest that lyso-phospholipase is not a crucial target for impairing parasite proliferation. In contrast, another potential target identified, inosine-adenosine-guanosine-nucleoside hydrolase (IAGNH), is involved in purine salvage and has been investigated as a drug target. The potential importance of glycosomal localization of the identified enzymes is noted. Several other abundant housekeeping proteins were also identified as enriched but exhibited no significant depletion by C-1 in C-1alk labeling, suggesting these are unlikely to be robust targets of C-1. The data suggest that the anti-trypanosomal activity of C-1 may be attributed to the covalent modification of multiple protein targets.

The unified synthetic approach described here provides straightforward access to a structurally diverse set of sulfonyl fluoride probes, many of which readily lend themselves to alkynylation for target identification via chemical proteomics. The ability to generate alkynylated analogues rapidly is a significant advantage. The chemical proteomics experiments identified multiple potential protein targets, revealing the complexity of the mechanism of action. While lyso-phospholipase and IAGNH initially emerged as strong candidates, further investigation using RNAi suggests that lyso-phospholipase is not a crucial target, whereas IAGNH's role is less clear-cut. The observed glycosomal localization of several enriched enzymes suggests that perturbations to glycosomal function may contribute to the observed anti-trypanosomal effects. The study highlights the potential of this approach, especially when scaled up, to identify high-quality tools and targets relevant to the biology of *T. brucei* and other pathogens.

This research successfully developed a high-throughput method for generating a library of diverse sulfonyl fluoride probes and successfully used these probes to identify multiple potential targets in *T. brucei*. While some initial candidates like lyso-phospholipase were subsequently ruled out by independent methods, the study demonstrates the power of the combined synthetic and chemical proteomics approach. The multi-target nature of the observed inhibition suggests a complex mechanism, which warrants further research. Future studies should focus on validating the remaining targets and investigating their individual contributions to the observed anti-parasitic effect. The expansion of the probe library and the application of this methodology to other pathogens are also promising avenues for future research.

The relatively small size of the probe library may have limited the identification of more potent and specific inhibitors. The chemical proteomics approach relies on covalent modification, which may not capture all interactions. Some identified targets such as lyso-phospholipase, despite showing initial promise, were not validated by independent studies as primary targets for inhibiting parasite proliferation. Further investigation into the roles of various identified proteins is required to confirm their contribution to the overall anti-trypanosomal activity. The reliance on in vitro assays might not fully reflect the in vivo situation.