Joseph William Treacy

Reader proteins that bind histone trimethyllysine (Kme3) have become active therapeutic targets in recent years. Binding of Kme3 in the conserved aromatic cages of these readers via cation-π interactions is a key contributor to these protein-protein interactions. We explored whether the replacement of one methyl group of Kme3 with an electron-withdrawing group (EWG) could improve binding by increasing the electrostatic component of the cation-π interaction. We examined these Kme3 analogs in binding studies with histone reader proteins, nuclear magnetic resonance (NMR) studies in a β-hairpin peptide model system, and computational studies to gain mechanistic insight into their binding. Kme3 analogs with both alkyl and benzyl substitution with EWGs were examined. We found that EWGs on the ligand differentially affected binding to a series of Kme3 readers but did not improve binding. This is likely due to sterics of the different analogs that preclude optimal cation-π interactions. The unique selectivity patterns of these ligands nonetheless offer promise toward selective inhibitor development within this protein class. NMR studies of the alkyl-substituted analogs in the β-hairpin model system show that EWGs strengthen the cation-π interaction enthalpically but with an entropic penalty. NMR studies demonstrate that the benzyl group makes direct contacts with Trp in the β-hairpin model, providing aromatic interactions in addition to cation-π interactions. Computational energy decomposition analysis confirms the more favorable electrostatic interactions compared to Kme3, but other factors counter the enhanced electrostatic component. This mechanistic investigation demonstrates how EWG-containing Kme3 analogs contribute to cation-π interactions and provide unique selectivity patterns relative to Kme3.

Opioids are a powerful class of medicines due to their ability to alleviate acute pain. However, the use of prescription opioids has led to an epidemic in the United States. Many efforts to combat this are ongoing, including the preparation of misuse deterrent opioid formulations, some of which can be subverted using common household chemicals. Herein, the development of an oxycodone-containing peptide prodrug is reported that contains three covalent misuse deterrent protective layers. This prodrug is resistant to degradation in the presence of acidic and basic pH conditions, common household chemicals, and enzyme supplements. After optimization of the peptide sequence, the lead prodrug is composed of a branched lysine residue coupled to a tert-butyl-protected tyrosine that is not naturally recognized by digestive enzymes. There is a necessary sequence of protecting group removal and then subsequent enzymatic peptide cleavages that trigger cyclization of a self-immolative linker to release the opioid agonist, oxycodone. The prodrug has analgesic properties in vivo in mice only after oral administration and not by intraperitoneal injection, which suggests that this prodrug may have misuse deterrent properties. The specificity for dual-enzyme release and the resulting in vivo studies warrant further examination of this prodrug scaffold for its potential as a misuse deterrent alternative to treat acute pain.

Substituted two-dimensional aromatic systems, such as arenes, exhibit well-established reactivity patterns at specific sites, largely due to the pronounced electronic directing effects of attached substituents. In contrast, the regioselectivity of three-dimensional aromatic molecules as a function of substituents remains less understood and documented. In this work, we demonstrate that a series of closo-dodecaborate ([B₁₂H₁₂]^2-) cluster isomers containing two -NMe₃⁺ moieties exhibit unprecedented regioselective reactivity at boron vertices farthest from the charged substituents. Through a combination of theoretical and experimental studies, we reveal that these boron clusters display near-perfect regioselectivity with multiple electrophiles, ultimately enabling vertex differentiation chemistry within these systems. This observed phenomenon closely parallels the reactivity patterns typically associated with icosahedral closo-carboranes, where a carbon-based vertex induces a strong electronic dipole, leading to pronounced vertex-specific reactivity differences at boron sites. Our findings suggest that these modified closo-dodecaborates serve as electronic analogs of neutral closo-carboranes, achieving similar electronic directing effects without the need for cage-based carbon atoms. Instead, exopolyhedral substituents alone govern the regioselective behavior, expanding the potential for tailored functionalization in boron cluster chemistry.

Substituted two-dimensional aromatic systems, such as arenes, exhibit well-established reactivity patterns at specific sites, largely due to the pronounced electronic directing effects of attached substituents. In contrast, the regioselectivity of three-dimensional aromatic molecules as a function of substituents remains less understood and documented. In this work, we demonstrate that a series of closo-dodecaborate ([B12H12]2-) cluster isomers containing two -NMe₃⁺ moieties exhibit unprecedented regioselective reactivity at boron vertices farthest from the charged substituents. Through a combination of theoretical and experimental studies, we reveal that these boron clusters display near-perfect regioselectivity with multiple electrophiles, ultimately enabling vertex differentiation chemistry within these systems. This observed phenomenon closely parallels the reactivity patterns typically associated with icosahedral closo-carboranes, where a carbon-based vertex induces a strong electronic dipole, leading to pronounced vertex-specific reactivity differences at boron sites. Our findings suggest that these modified closo-dodecaborates serve as electronic analogs of closo-carboranes, achieving similar electronic directing effects without the need for cage-based carbon atoms. Instead, exopolyhedral substituents alone govern the regioselective behavior, expanding the potential for tailored functionalization in boron cluster chemistry.

In silico examination of 13 P,N-ligated Au(iii) OACs determined the key mechanistic factors governing Au(iii)-mediated S-arylation. Three complexes were synthesized which exhibited bimolecular coordination rate constants as high as 20 200 M⁻¹ s⁻¹.

Oxidant-free Au(I)/Au(III)-catalyzed cross-coupling has been recently enabled by the use of bidentate P,N-ligands. To further develop these P,N-ligands, computational studies were performed to understand their effects on the oxidative addition of aryl iodide electrophiles with Au(I). Using this mechanistic understanding, six new electron-rich P,N-ligands were synthesized. The ligand exchange equilibrium and reductive elimination were then characterized by using a Au(III)-mediated S-arylation reaction. The results detailed herein provide new fundamental insights in Au(I)/Au(III) ligand design.

Organometallic oxidative addition complexes (OACs) have recently emerged as a powerful class of reagents for the rapid and chemoselective modification of biomolecules. Notably, the steric and electronic properties of the ligand and aryl group can be modified to tune the kinetic profile of the reaction and permit regioselective S-arylation. Using the recently developed dicyclohexylphosphine-based bidentate P,N-ligated Au(III) OACs, we computationally and experimentally examined the effects of sterically bulky and electron deficient aryl substrates to achieve selective S-arylation. With this mechanistic insight, aryl substrates based on 4-iodoanisole and 3,5-dimethyl-4-iodoanisole were incorporated as end groups to generate a heterotelechelic bis-Au(III) poly(ethylene glycol) (PEG). This reagent performed rapid and regioselective S-arylation with a model biomolecule, designed ankyrin repeat protein (DARPin), to form a protein-polymer OAC in situ. This OAC mediated a second S-arylation with biologically relevant thiolated small molecules (metal chelator, saccharide, and fluorophore) and macromolecules (polymer and therapeutic peptide). It is envisioned that this approach could be utilized for the rapid construction of biomacromolecular heteroconjugates with S-aryl linkages.

Oxidant-free Au(I)/Au(III) catalyzed cross-coupling has been recently enabled by the use of bidentate P,N-ligands. To further develop these P,N-ligands, computational studies were performed to understand their effects on the oxidative addition of aryl iodide electrophiles with Au(I). Using this mechanistic understanding, six new, electron rich P,N-ligands were synthesized. The ligand exchange equilibrium and reductive elimination were then characterized using an Au(III)-mediated S-arylation reaction. The results detailed herein provide new fundamental insights in Au(I)/Au(III) ligand design.

Histone serotonylation has emerged as a key post-translational modification. WDR5 preferentially binds to serotonylated histone 3 (H3), and this binding event has been associated with tumorigenesis. Herein, we utilize genetic code expansion, structure-activity relationship studies, and computation to study an edge-face aromatic interaction between WDR5 Phe149 and serotonin on H3 that is key to this protein-protein interaction. We find experimentally that this edge-face aromatic interaction is unaffected by modulating the electrostatics of the face component but is weakened by electron-withdrawing substituents on the edge component. Overall, these results elucidate that this interaction is governed by van der Waals forces as well as electrostatics of the edge ring, a result that clarifies discrepancies among previous theoretical models and model system studies of this interaction type. This is the first evaluation of the driving force of an edge-face aromatic interaction at a protein-protein interface and provides a key benchmark for the nature of these understudied interactions that are abundant in the proteome.

Metal-mediated cysteine S-arylation is an emerging bioconjugation technique due to its high chemoselectivity, rapid kinetics, and aqueous compatibility. We have previously demonstrated that by altering the sterics of the ligand and aryl groups of an Au(III) oxidative addition complex, one can modulate the kinetics of the bimolecular coordination and induce rate constants up to 16,600 M^−1s^−1. To further enhance the rate of coordination, density functional theory (DFT) calculations were performed to investigate the steric properties of the P,N-ligated Au(III) oxidative addition complex as well as the thermodynamics of the S-arylation reaction. This allowed for the accelerated screening of 13 new Au(III) oxidative addition complexes. Three of the more sterically available, synthetically accessible P,N-ligands were synthesized, incorporated into Au(I) and Au(III) complexes, and their rates determined experimentally. The comprehensive mechanistic insights from the DFT calculations led to the development of new reagents with bimolecular coordination rate constants as fast as 20,200 M^−1s^−1. Further experimental characterization of these reagents’ efficacy as S-arylation reagents led to a proposed switch in selectivity-determining step for the fastest reagent, which was further confirmed by profiling the reductive elimination kinetics. This work provides a concise workflow for the screening of metal-mediated cysteine S-arylation reagents and new fundamental insights into the coordination chemistry behavior of Au(III) systems.