Re-Engineering P(V) Chemical Warfare: Harnessing Stereogenic Phosphorus-Azoles for Protein Ligand Discovery In Vivo

P(V) electrophiles such as tabun, sarin, soman, and VX are notorious for their lethality and nefarious intent in chemical warfare. Consequently, these deadly agents have largely been abandoned except for fluorophosphonate tool compounds that were repurposed for activity-based protein profiling (ABPP). Stereogenic P(V) centers hold strong potential as enabling scaffolds for synthetic and medicinal chemistry due to their inherent chirality and favorable bioavailability but are limited principally by potent off-target toxicity. Herein, we developed phosphorus-azole exchange (PhAzE) chemistry for tuning reactivity of the stereogenic P(V) pharmacophore to increase selectivity and mitigate off-target activity in cells and animal models. We demonstrate ultrapotent (300 pM in cells, 1 mg kg -1 in mice), enantioselective, covalent inhibition of the serine hydrolases DPP8/9 with PhAzE ligand in cells and in vivo; no overt toxicity was detected in mice treated daily over the course of a week. These finding show the P(V) electrophile can potently and enantioselectively engage a target protein without a deadly outcome, charting a path towards broader adoption of these agents in laboratory and industry settings. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint

Among the chemical warfare agents employed throughout modern history, the organophosphorus compounds VX, tabun, sarin, and soman are notorious for their potent lethality. As P(V) electrophiles, these molecules can react covalently with biological targets, most notably enzymes involved in neurotransmission (Figure 1A).1 Sarin was stockpiled in large quantities in Germany during World War II, though it was never used on the European battlefield. Efforts to understand its deadly mechanism revealed that sarin, like tabun and soman, irreversibly inhibits acetylcholinesterase, the enzyme responsible for breaking down the neurotransmitter acetylcholine.2-3 Acetylcholinesterase inhibition leads to uncontrolled accumulation of acetylcholine at neuromuscular junctions and synapses, resulting in muscle paralysis, convulsions, respiratory failure, and ultimately death. The widespread condemnation of nerve agents culminated in global banning of the development, production, stockpiling, and use of chemical weapons.4- 5 This tragic legacy has largely stigmatized this compound class, precluding its further exploration for drug development despite potent in vivo activity. While ill-suited for medicine because of toxicity, the P(V) electrophile has been successfully deployed for academic investigations. Fluorophosphonates covalently modify the catalytic serine in active sites of most, if not all, metabolic serine hydrolases that when coupled to proteomic readouts can facilitate activity-based protein profiling (ABPP, Figure S1A).6-8 This functional proteomic method has enabled basic discoveries in serine hydrolase biology, which led to inhibitor and drug development (Figure S1A).9- 19 Moreover, the success of fluorophosphonates was foundational for exploring additional electrophiles for covalent targeting of reactive cysteines and less nucleophilic sites harboring, for example, a tyrosine, lysine or histidine (Figure S1A). 20-27 The adoption of covalency into mainstream drug discovery campaigns had produced precision therapeutics such as ibrutinib, sotorasib, and others that exploit strategically located or mutated residues for selective and durable target protein engagement. 28 20, 29 More recently, stereoselective labeling with electrophilic compounds offers a promising strategy to improve proteome-wide selectivity (Figure S1B).24, 30-37 However, because the electrophiles themselves are achiral, stereoselectivity relies on bulky, chiral substituents near the reactive site.35-36, 38 Rare examples like a chiral sulfonimidoyl fluoride demonstrate that chirality at the electrophilic center alone can drive enantioselective protein modification. 39,40 P(V) electrophiles are uniquely positioned to enable enantioselective covalent modification of biomolecules, offering a direct strategy to interrogate and exploit chirality at the electrophilic center (Figure S1C). 41 Only a few studies have explored stereoselective P(V)-based reactivity in complex proteomes. Notable examples include sulphostin, a chiral natural product that inhibits dipeptidyl peptidases 4, 8 and 9 (DPP4/8/9) 42-45 and the antiviral pro-drug (S)-remdesivir, which is more potent than the (R)-stereoisomer, likely due to CES1-mediated stereoselective hydrolysis (Figure S1C).46-48 Yet, both .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint molecules contain multiple stereocenters, limiting mechanistic understanding on whether the P(V) electrophile alone drives stereoselectivity. The use of P(V) chirality has been demonstrated in part through pioneering work in the development of chiral phosphorothioate “ψ -reagents” to synthesize antisense oligonucleotides (ASOs) with chiral backbones,49-54 nucleoside thioisosteres,55 and chemoselective phosphorylation reagents for alcohols56 and serine residues.57 These studies underscore the value of chiral P(V) centers in drug development. Phosphorothioate chemistry was recently extended to include tryptoline-based covalent ligands targeting various nucleophilic residues; however, these reagents were racemic at the P(V) center.58 Herein, we disclose the development of phosphorus-azole exchange (PhAzE) chemistry as a potent and safe stereogenic P(V) electrophile for enantioselective targeting of enzymes in vivo. The selection of an azole leaving group mitigated toxicity inherent to phosphoryl-fluorides while facilitating the design of picomolar inhibitors that enantioselectively engaged serine hydrolases DPP8/9 in the brain of treated mice. Importantly, daily dosing of the DPP8/9 PhAzE molecule over the course of a week did not produce overt toxicity or engagement with acetylcholinesterase as measured by quantitative chemoproteomics. PhAzE chemistry bridges the gap to therapeutics using stereogenic P(V) electrophiles and repositions this toxic pharmacophore for synthetic and therapeutic investigations. Figure 1. Development of PhAzE as a stereogenic P(V) electrophile for therapeutic investigation. Schematic of re-engineering phosphoryl-fluoride chemistry (A) to develop highly tunable enantioselective Phosphorus-Azole Exchange (PhAzE) chemistry (B).

Initially, we tested whether enantiomers of fluorophosphonate (FP)-alkyne could be utilized for enantioselective ABPP. These studies would assess the extent to which a less sterically hindered stereogenic P(V) electrophile could facilitate enantioselective recognition of enzyme active sites. THP1 cells were treated with 20 µM (-)- or (+)-FP-alkyne followed by cell lysis, copper-catalyzed azide-alkyne cycloaddition (CuAAC) 59-63 conjugation of biotin-azide, avidin enrichment, and tandem mass tag (TMT)- based quantitative ABPP (See Supporting Information).64-65 Despite detection of 40+ family members, .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint only a single serine hydrolase displayed enantioselective binding as defined by a >50% difference in enrichment signals between (-) and (+) enantiomers ((+)/(-) ratio of >1 or <0.5; Figure 2B and S2A). Next, we assessed the effects of rigidifying the electrophile using a previously reported cyclic phosphoryl-fluoride strategy 66 to incorporate recognition elements akin to ψ -reagents49. After purification of enantiomers, we treated THP1 cells with (-)- or (+)-RJG-2273 and observed substantially enhanced enantioselective ABPP labeling (9 or 4 serine hydrolases displaying enantioselective binding, respectively; Figure 2B and S2B). While the cyclic phosphoryl-fluoride strategy improved enantioselectivity, this probe retained the potent acetylcholinesterase (ACHE) inhibitory activity inherent to P(V) electrophiles (Figure 2C and S3E). 66-67 We considered alternative leaving groups (LGs) as a means to temper the electrophile and mitigate covalent binding to ACHE. Azoles are effective and tunable nucleofuges to activate carbonyl15-17 and sulfonyl electrophiles for reactions at protein sites.20, 24, 68-79 Initially, we installed a 1,2,4-triazole in place of fluoride to produce RJG-2259A for mediating protein reactions via phosphorus-azole exchange (PhAzE) chemistry. We purified enantiomers and tested activity of the enantiomeric probes in THP1 monocytes and observed covalent binding to >20 serine hydrolases (Figure 2B), but negligible enantioselectivity (Figure S2C). Replacing the triazole with a purine and purifying the resulting enantiomers produced alkynyl probes (-)- and (+)RJG-3241, the latter of which enantioselectively enriched 3 serine hydrolases in THP1 cells (Figure 2B and S2D). Next, we treated live Neuro-2A (N2a) mouse neuroblastoma cells with varying concentrations of (-)- or (+)-FP-Alkyne, -RJG-2273, -RJG-2259A, or -RJG-3241 to further assess enantioselective serine hydrolase binding and ACHE off-target activity in living cells. Importantly, PhAzE probes RJG-2259A and -3241 exhibited reduced inhibitory activity towards ACHE compared with phosphoryl-fluorides (Figure 2C and S3E). As expected, (-)- and (+)FP-Alkyne completely inhibited ACHE activity at 1 µM in live N2a cells (Figure 2C and S3E). Akin to the LC-MS/MS data, we found (-)RJG-2273 was a more potent inhibitor of ACHE biochemical activity compared to (+)RJG-2273, whereas both enantiomers of RJG-2259A and -3241 were weak inhibitors of ACHE (Figure 2B and S3E). In contrast, (+)RJG-2273 was a more potent inhibitor of FAAH biochemical activity, supporting the importance of P(V) chirality for recognition and covalent inactivation of serine hydrolases (Figure 2C and S3E). We also observed subnanomolar, enantioselective inhibition of FAAH with (-)FP-Alkyne compared to the (+)FP-Alkyne (Figure 2C). We observed comparable inhibition of FAAH biochemical activity (Figure 2C and S3E) and fluorophosphonate-rhodamine (FP-Rh) labeling (Figure S3C) with (-)- and (+)RJG-2259A, suggesting RJG-2259A is a covalent inhibitor of serine hydrolases. In contrast, (+)RJG-3241 was a ~10-fold more potent dipeptidyl peptidase (DPP) inhibitor compared to (-)RJG-3241, and we observed negligible inhibition with FP-Alkyne, RJG-2273, and RJG-2259A, thus highlighting the .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint tunability of the PhAzE scaffold for enantioselective reactivity and specificity. Notably, several proteins were enantioselectively inhibited by (-)- or (+)FP-Alkyne and -RJG-2273 at lower concentrations as observed by gel-based competitive ABPP with FP-Rh (Figure S3A,B). Figure 2. Enantioselective targeting of serine hydrolases with PhAzE. (A) Drawings of (-)- or (+)FP- Alkyne, RJG-2273, RJG-2259A, and RJG-3241 with respective specific rotation values or crystal structures. (B) Bar graph showing total and >50% enantioselectively enriched serine hydrolases from live THP1 monocytes treated with 20 µM ( -)- or (+)-enantiomers. Data is representative of three independent biological replicates See Supporting Information Figure 2 for complete graph of enriched serine hydrolases. (C) Table of IC50 values of derived from biochemical assays. Live N2a cells were treated with respective enantiomers of compounds for 4 hours at 37 °C, then lysate was used to perform acetylcholinesterase (ACHE), fatty-acid amide hydrolase (FAAH), and dipeptidyl peptidase (DPP) biochemical assays. See Supporting Information for experimental details. To initiate development of PhAzE ligands, we envisaged a structure-activity relationship (SAR) that would explore N-substitutions and azole LGs capable of potent inhibition of diverse serine hydrolase targets. The sp3-rich cyclopropyl moiety80 was installed to produced N-cyclopropyl or N- cyclopropylmethyl analogs RJG-3130-1 and -3045, respectively (Figure 3A). We treated live N2a cells with RJG-2273, RJG-3130-1, or RJG-3045 (1 µM compounds) followed by cell lysis, labeling of proteomes with FP-biotin, and TMT-based quantitative ABPP to assess inhibitory activity against the serine hydrolase family.6 FP-biotin labeled proteins were enriched by avidin chromatography, multiplexed using TMT, and quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS, see .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint Supporting Information for additional details).64-65 Phosphoryl-fluoride molecule RJG-2273 inhibited ACHE, whereas no inhibition was observed with PhAzE compounds RJG-3130-1 or RJG-3045 (Figure 3B). Both RJG-3130-1 or RJG-3045 showed comparable inhibitory activity against DPP8, APEH, and DPP9 (competition ratio or CR <0.5, 50% inactivation; Figure 3B and Figure S4). A dose response study in live N2a cells revealed RJG-3130-1 was about a 10-fold more potent inhibitor of DPPs and APEH compared to RJG-3045 (IC50 ≈ 8.5 nM for DPPs and APEH, Figure 3C, D and Figure S5). RJG-3130-1 was selected for further development given its superior activity against serine peptidases. We synthesized several PhAzE molecules based on the RJG-3130 scaffold but further diversified by various azole LGs. PhAzE ligands were tested directly in live N2a cells to assess serine hydrolase inactivation as determined by FP-Rh-mediated, gel-based competitive ABPP and blockade of biochemical activity using substrate assays (1-1000 nM, 4 hours; Figure S6 and S7). We found RJG-3130- 9 was a subnanomolar inhibitor of DPP probe labeling with apparent selectivity for DPPs over APEH compared to RJG-3130-8; neither compound showed activity towards ACHE (Figure S7). Compared with existing APEH (AA74-1 15) and DPP8/9 (talabostat81-83) inhibitors, RJG-3130-9 was clearly a scaffold more optimized for DPP inactivation (Figure 3E and S7B). Note that two isomers of RJG-3130-9 were isolated after purification; the first isomer, presumed to be the N7-regioisomer, degraded quickly and was therefore not tested (Figure S8). Widening the dose range revealed RJG-3130-9 is a picomolar inhibitor (~300 pM) of the ~100 kDa band matching the molecular weight of DPP8/9 in live cells (Figure 3E and S9A,B). Considering the existence of at least seven known DPPs 83-84, we next performed TMT-based competitive ABPP to assess DPP and serine hydrolase family-wide selectivity of compounds. Treatment of N2a cells with RJG-3130- 9 resulted in significant blockade of DPP8 and 9 probe labeling across all concentrations tested (1 – 1000 nM, Figure 3F and S10B-E). Off-target activity (e.g., APEH, ACOT2, CES) of RJG-3130-9 was only observed at higher concentrations and absent at lower concentrations that retained potent DPP8/9 inactivation (>50% blockade at 1 nM RJG-3130-9, Figure S10E). Notably, RJG-3130-9 was able to achieve a similar degree of DPP8 and 9 inhibition at a 1000X lower concentration compared to talabostat in live cells (Figure 3G). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint Figure 3. Developing PhAzE chiral ligands for ultrapotent enzyme inactivation. ( A) Phosphoryl- fluoride molecule RJG-2273 and non-alkynyl ligands RJG-3130-1 and RJG-3045. ( B) Graph of inhibited proteins quantified using Tandem Mass-Tag (TMT) LC-MS/MS. Live N2a cells were treated with 1µM RJG-2273, RJG-3130-1, or RJG-3045. The red dashed box highlights inhibition of acetylcholinesterase (ACHE) with RJG-2273, but not PhAzE ligands. Data is representative of three independent biological replicates. See Supporting Information Figure S4 for complete list of serine hydrolases. ( C) SDS-PAGE based competition experiment using FP-Rh as a probe and a dose response of PhAzE ligands in live N2a cells. See Supporting Information Figure 5 for full length gel. ( D) Graph of dose response curves derived from dividing raw fluorescence values of treatments over vehicle from Figure 3C. ( E) Drawing of lead DPP inhibitor RJG-3130-9 and a table of IC 50 values for APEH and DPPs obtained from dose response curves derived from dividing raw fluorescence values of treatments over vehicle and biochemical assays (See Supporting Information Figure 9 for full length gel and graphs). ( F) Dose response curves of inhibited proteins derived from competition ratios using TMT abundances, e.g. , competition ratio (3130- 9/V ehicle), of proteins inhibited by RJG-3130-9 in live THP1 monocytes. (G) Graph comparing inhibition of DPP8 and 9, but not DPPs 2/7, 3, PREP, or PRCP, with 1 µM talabostat or 1 nM RJG-3130-9. % .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint Inhibition was calculated using the competition ratio (CR) of (compound / vehicle). See Supporting Information Figure 9 for volcano plots. Next, we investigated chiral purification to determine whether PhAzE ligands display enantioselective binding activity. We initiated efforts with chiral purification of RJG-3130-1 to test if the RJG-3130 scaffold was advantageous compared to alkynyl probe RJG-2259A (Figure 2). Although enantiomeric excess (e.e.) was >98% after chiral purification, (-)- and (+)RJG-3130-1 began to racemize during concentration to dryness (Figure 4A and S11A,B). We attempted chiral purification of RJG-3130-3 containing an imidazole LG that is more basic and presumably more stable because of reduced electrophilicity (Figure S6B); however, almost complete racemization occurred upon concentration (Figure 4A and S11C,D). RJG-3130-9, which is modified with a bulkier purine LG, was readily purified by chiral supercritical fluid chromatography (SFC) and retained e.e. after concentrating to dryness (Figure 4A and S11E,F). We solved the crystal structure of (+)RJG-3130-9 by microcrystal electron diffraction (microED) and observed (S)-configuration at the P(V) electrophile and the N9-substituted regioisomer (Figure 4B). In addition, the N20 nitrogen is directed at the benzene ring at an angle of 124°, and N20 is ~2.8 Å from the bond between carbons C5-C6 and ~3.6 Å from C5, which is within range of a n – π * interaction and is near the summed van der Waals radii of 3.44 Å between N and C (Figure 4B).85-86 We analyzed inhibition of DPPs with (-)- or (+)RJG-3130-9 in THP1 soluble lysate initially by gel-based competitive ABPP. (+)RJG-3130-9 displayed ultrapotent, time-dependent blockade of DPP probe labeling, supporting an irreversible covalent inactivation mechanism (Figure S12A, B). We compared inhibitory activity of (-)- and (+)RJG-3130-9 at 1 nM and found initial evidence for enantioselectivity at all time points tested (Figure S12C). Next, we evaluated whether the enantioselective blockade of probe labeling translated into DPP biochemical inactivation. (+)RJG-3130-9 displayed dose and time-dependent inhibition of DPPs achieving an estimated potency value (IC 50) of 160 pM (4-hour treatment). (-)RJG-3130-9, in contrast, exhibited moderately potent inhibition with a ~7-15-fold reduction in potency across the different treatment times tested (Figure S12D-F). Next, we treated THP1 cells with (-)- or (+)RJG-3130-9 at varying concentrations to test whether the observed enantioselective activity against DPPs in vitro was recapitulated in live cells. Akin to in vitro findings, we detected >50% inhibition of DPP8/9 with (+)RJG-3130-9 compared to <25% inhibition with (-)RJG-3130-9 at 1 nM as measured by TMT-based competitive ABPP (Figure 4C and S13D, E). Additional DPPs identified were not significantly inhibited at 1 nM (Figure 4C). At higher concentrations of (-)- and (+)RJG-3130-9, we observed PREP, CES1, and CES2 off-target activity, which was not enantioselective and largely eliminated at lower concentrations where (+)RJG-3130-9 retained potent DPP8/9 blockade (Figure 4C, S13C,F,G). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint Given the promising cellular activity of PhAzE chiral ligands, we pursued assessment of in vivo activity with (-) and (+)RJG-3130-9. To the best of our knowledge, enantioselective covalent ligands published to date and stereogenic P(V) electrophiles – excluding nefarious intent with fluorophosphonates – have not been tested for enantioselective covalent modification of proteins in vivo. Mice were treated with vehicle, (-)-, or (+)RJG-3130-9 for 4 hours (1 mg/kg) or 7 days (10 or 1 mg/kg). Tissue was harvested and samples were multiplexed for quantitation of protein inhibition after 4 hours or 7 days of treatment via 6- or 16-plex TMT-based competitive ABPP, respectively. Strikingly, we discovered only DPP8/9 were significantly inhibited by >50% in the brains of (+)RJG-3130-9-treated mice, whereas negligible inhibition was observed with (-)RJG-3130-9 across the 48 serine hydrolases quantified (Figure 4D and S14). These findings support the ability of PhAzE ligands to cross the blood-brain barrier and enantioselectively engage target proteins. Importantly, minimal-to-no ACHE inhibition was observed after acute (4 hours) or chronic dosing consisting of a 7-day treatment regime with 1 or 10 mg/kg (-)- or (+)RJG-3130-9 (Figure 4D,F). We did not observe changes in body weight of mice treated with RJG-3130-9 enantiomers, providing initial evidence of a promising safety profile for PhAzE in vivo (Figure 4E and S15). Finally, we compared the competition binding profile of 10 and 1 mg/kg (-)- or (+)RJG-3130-9 after 7 days of treatment. At the higher dose (10 mg/kg), we detected only APEH and BCHE as additional competed targets of (+)RJG-3130-9 (Figure 4F and S16A). At lower concentrations (1 mg/kg), DPP8/9 remained the only significantly and enantioselectively competed targets of (+)RJG-3130-9 (>50% competition, Figure 4F and S16B). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint Figure 4. Enantioselective targeting of proteins in vivo with brain-penetrant PhAzE ligands. ( A) RJG-3130-1, -3, and -9 were synthesized as racemates and respective enantiomers purified by supercritical fluid chromatography. Enantiomers of RJG-3130-1 and -3 isomerized upon concentration, whereas RJG-3130-9 retained enantiomeric excess (e.e.). (B) Drawing of (S)-(+)RJG-3130-9 and a crystal structure characterized by microED. The distance between N 4 and C5 is 3.59 Å (black dashed line) and N4 and the bond between C5 and C6 is 2.89 Å (red dashed line). ( C) Bar graph representing DPPs identified in a 16-plex TMT LC-MS/MS experiment using samples from live THP1 monocytes treated with vehicle or 1 nM (-)- or (+)RJG-3130-9. See Supporting Information Figure 13 for full dose response graphs. Data is representative of three independent biological replicates. ( D) Bar graph of serine hydrolases enriched from brain tissue of mice treated with vehicle or 1 mg/kg of ( -)- or (+ )RJG-3130-9 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint for 4 hours. Samples were combined with TMT 6-plex and analyzed by LC-MS/MS. See Supporting Information Figure 14 for complete graph of enriched serine hydrolases. Data is representative of three independent biological replicates. ( E) Average weights of mice over a seven-day treatment with vehicle, 10 or 1 mg/kg (-)RJG-3130-9, or 10 or 1 mg/kg (+)RJG-3130-9. See Supporting Information Figure 15 for additional graphs. ( F) Bar graph of serine hydrolases enriched from brain tissue of mice treated with vehicle, 10 mg/kg, or 1 mg/kg of ( -)- or ( +)RJG-3130-9 for 7 days. Samples were combined with TMT 16-plex and analyzed by LC-MS/MS. Data is representative of three independent biological replicates. See Supporting Information Figure 16 for complete graph of enriched serine hydrolases.

Despite their utility in chemical biology, fluorophosphonates and related P(V) electrophiles remain challenging to deploy more broadly due to their inherent toxicity and origins as lethal nerve agents, leaving no clear path for translational applications. If toxicity could be mitigated, stereogenic P(V) hubs would serve as enabling starting points for synthetic organic chemistry and drug discovery because of their potent bioavailability and inherent chirality. The latter feature is highly desirable for drug development 30-31, 49 but often relies on bulky chiral substitutions adjacent or distal to achiral electrophiles,34-39, 58, 87 with only a few examples of chirality at the electrophile.40 Here, we developed a stereogenic P(V) electrophile dubbed PhAzE for safe and enantioselective protein inactivation in vivo, repositioning these agents from deadly poisons to valuable leads for therapeutic discovery. We discovered that chiral phosphoryl-fluorides including FP-based agents could achieve enantioselective targeting of serine hydrolases but largely retained potent ACHE binding activity (Figure 2). The switch from an acyclic 6 to cyclic P(V) electrophile66 improved enantioselectivity and reduced ACHE inactivation (Figure 2C and S3E). These promising findings along with the relative ease for accessing the tunable and constrained stereogenic P(V) electrophile in RJG-2273 prompted development of PhAzE analogs bearing azole LGs. Azoles are effective LGs for tunable activation of electrophilic protein modification 17-20, 24, 68, 88-93 providing a testable path to reduce toxicity of the P(V) electrophile by selectivity optimization. We tested this hypothesis by synthesizing stereogenic PhAzE probes and discovered that the purine azole mitigated ACHE off-target activity while retaining sufficient enantioselective binding activity for downstream ligand discovery efforts (Figure 2C, S2, and S3). The initial PhAzE design using a triazole LG ((-)- and (+)RJG-2259A) showed cellular activity but low enantioselectivity, prompting exploration of additional azoles. The natural metabolite purine emerged as an effective LG for enantioselective protein modification. We speculate that the bulkier nature of purines compared with the triazole could be contributing to improved enantioselectivity; future studies expanding on the SAR of PhAzE will further support this hypothesis. Nonetheless, we were encouraged to see a significant improvement in enantioselectively enriched serine hydrolases and differential, potent inhibition of DPP biochemical activity with RJG-3241 that was not observed with the phosphoryl-fluoride probes FP-Alkyne or RJG-2273 (Figure 2C and S3E). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint Given the enantioselective activity of RJG-3241, we transitioned from probe to ligand optimization to assess whether PhAzE could achieve enantioselective inactivation across the serine hydrolase superfamily. Our SAR campaign characterized RJG-3130-9 as a picomolar DPP8/9 inhibitor with good selectivity over APEH and no inhibition of ACHE in live cells (Figure 3E, S7 and S9). Comparison of RJG-3130-9 with the existing DPP8/9 inhibitor talabostat revealed the PhAzE ligand could achieve similar target engagement at ~1,000-fold lower concentrations than this clinical candidate (Figure 3G). Although chiral purification of phosphoryl-fluorides was straightforward, we had to overcome several challenges with chiral purification of PhAzE. Partial or complete racemization of RJG-3130-1 (1,2,4-triazole LG) or RJG-3130-3 (imidazole LG) was observed upon concentration of fractions after chiral purification, respectively, a surprising result given the relative ease in purifying enantiomers of PhAzE probe RJG-2259A (Figure 2). Ultimately, we observed that (-) and (+)RJG-3130-9 retained >99% e.e. after concentration; these chiral ligands exhibited time-dependent, enantioselective inhibition of DPP8/9 in THP1 monocytes and soluble lysate (Figure 4C and S12, respectively). Crystallography revealed (+)RJG-3130-9 exists in a (S)-configuration, the purine is substituted at N9, and this regioisomer is stabilized by an intramolecular n – π interaction (Figure 4B).86 Similar to enantioselective enrichment of serine hydrolases with (+)RJG-3241, (+)RJG-3130-9 enantioselectively inhibited DPP8 and 9 by >50% compared to negligible inhibition with (-)RJG-3130-9 at the same concentration (1 nM compounds, Figure 4C and S13). Finally, we demonstrated covalent and enantioselective inhibition in vivo with a chiral P(V) electrophile. The DPP8/9 selectivity and enantioselectivity of (+)RJG-3130-9 detected in cells was recapitulated in brain proteomes from treated mice. Specifically, (+)RJG-3130-9 (1 mg/kg, intraperitoneal or i.p.) inhibited FP-biotin probe labeling of DPP8/9 by >50% with minimal to no inhibition of the >40 serine hydrolases detected in brain. Notably, ACHE was not inhibited in mice treated under acute (4 hours) and more chronic exposure (7 days) at 1 or 10 mg/kg i.p. treatment (Figure 4D,F , S14, and S16). We did not detect overt changes in the weight of mice in any PhAzE treatment group (Figure 4E and S15). The lack of ACHE inhibitory activity combined with stable body weight under prolonged treatment provides early but critical evidence of in vivo safety for the PhAzE compound class. These findings represent, to the best of our knowledge, the first case of enantioselective inhibition of proteins in vivo with a P(V) electrophile that was not deadly. By extension, the improved safety profile of PhAzE chemistry paves the way for designing stereogenic PhAzE molecules that are readily available for synthetic organic chemistry. The availability of phosphoryl-chlorides is lacking compared to sulfonyl-chloride congeners used for various synthetic chemistry applications including the synthesis of sulfonyl-fluorides that are now widely used in synthetic .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint and medicinal chemistry.94 Phosphorus-fluoride exchange (PFEx) click chemistry was recently published as a versatile and efficient P(V) electrophilic hub for synthesizing phosphoryl moieties.66 Synthesis of PFEx molecules requires precautions such as harsh quenching conditions and plasticware when using fluorides that are toxic and known to etch/damage glassware, respectively.95 Similar to results we presented above, PFEx molecules are potent inhibitors of ACHE activity that require scientists to take precautions to prevent accidental exposure, 66 thus limiting their potential for commercial availability and general accessibility. We envision that PhAzE chemistry presented herein has the potential to be translated to synthetic organic chemistry applications with minimal-to-no additional requirements to achieve chemical transformations similar to PFEx.66

In summary, we re-engineered fluorophosphonates by substituting the fluoride for azole leaving groups to produce potent, enantioselective, and in vivo-active PhAzE ligands for covalent ligand and potential therapeutic discovery. We optimized PhAzE to achieve picomolar and enantioselective inhibition of serine hydrolases in live cells. Moreover, (S)-(+)RJG-3130-9 enantioselectively inhibited DPP8/9 in vivo, marking these findings as, to the best of our knowledge, the first disclosure of enantioselective and non- deadly covalent inhibition in vivo with a P(V) electrophile.

We thank all members of the Hsu lab for review of this manuscript. We thank Peimin Fan and Yonghong Li (Pharmaron Inc.) for small molecule synthesis, chiral purification, and microED. Contributions R.J.G. and K.-L.H conceived the study. R.J.G. synthesized and characterized all PhAzE compounds, as well as RJG-2273, and supervised synthesis and characterization of FP-biotin and chiral compounds. R.J.G. performed all experiments including tissue culture and cellular treatments, live animal treatments and tissue harvesting, biochemical assays, gel-ABPP, LC-MS/MS ABPP to generate proteomic data, and analysis of proteomic data. M.L.W. and O.L.M. designed the biotin enrichment protocol and LC-MS/MS methods. S.V . performed X-ray crystallography. K.-L.H. supervised the study. All authors edited and approved the paper. Competing Interests K.-L.H. is a founder and scientific advisory board member of Hyku Biosciences. A patent application has been filed on the work presented in this manuscript. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 28, 2026. ; https://doi.org/10.64898/2026.01.27.702106doi: bioRxiv preprint

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