In silico screening of P,N-ligands facilitates optimization of Au(III)-mediated S-arylation

Abstract

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 steric profile 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⁻¹ s⁻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⁻¹ s⁻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.

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Introduction

Effective bioconjugation reagents are chemoselective for specific residues, amenable to mild and aqueous reaction conditions, and able to perform reactions at low concentrations, which necessitates favorable reaction kinetics. Cysteine presents itself as a common target for site-selective bioconjugation due to its high nucleophilicity, relatively low abundance in the proteome, and ability to be incorporated into proteins, antibodies, and other biomolecules through molecular biology techniques.^1–3 Commonly used cysteine modifications rely on addition to maleimides or other Michael acceptors. However, these linkages are susceptible to retro-Michael reactions under reducing conditions, leading to reversible modification of the biomolecule.^4–9 Accordingly, cysteine S-arylation has emerged as a unique approach to produce C(sp²)–S bonds, which are less susceptible to reversibility.¹⁰ Many cysteine S-arylation approaches rely on nucleophilic aromatic substitution (S_NAr), which necessitates electron withdrawing groups ortho or para to a leaving group.^11–19 This limits the aryl substrate scope for potential modification. An alternative method uses metal-mediated or metal-catalyzed cysteine S-arylation, which allows for the ex situ or in situ formation of an oxidative addition complex (OAC).^20–30 These OACs are then able to permit initial thiol coordination to the metal center and subsequent reductive elimination to form the stable, C(sp²)–S bond. The use of transition metals allows for the aryl scope to be broadly expanded to contain both electron donating and withdrawing substituents as well as different substitution patterns.

The ex situ formation of the OAC can provide a set of bench stable reagents that can be used for subsequent biomolecule transformations without any additional precautions to preserve the metal reagent.^31,32 By using thiophilic metal centers (e.g. Pd and Au), exquisite chemoselectivity for thiols and rapid kinetics are observed. Notably, the mechanism for the S-arylation proceeds via a two-step process of an initial coordination of the thiol to the metal center followed by reductive elimination to form the S-aryl bond. This stands in contrast to other cysteine modification mechanisms that rely on displacement of an alkyl halide or 1,4-addition into a Michael acceptor. Our groups have reported the use of P,N-ligated Au(III) OACs for the S-arylation of cysteine (Fig. 1),^33–35 and recently, we have shown that stopped-flow/UV-vis spectroscopy is a powerful technique to characterize the rates of the two distinct elementary steps of the S-arylation. This allowed for the quantification of the bimolecular thiol coordination rate constant (k_Co) for P,N-ligated Au(III) OACs with di-1-adamantylphosphine (PAd₂) 1 and di-tert-butylphosphine (P^tBu₂) 2 P-substituents as 2460 and 4220 M⁻¹ s⁻¹, respectively. The clear steric dependence on this coordination event led to the synthesis of a dicyclohexylphosphine-based (PCy₂) P,N-ligated Au(III) OAC reagent 3 with an unprecedented k_Co of 16 600 M⁻¹ s⁻¹.³⁵

Fig. 1 P,N-ligated Au(III) complexes developed for rapid and selective cysteine S-arylation. Purple and red circles represent sites for modification on the ligand scaffold. Counterion not included for clarity.

Alternatively, by increasing the steric bulk of the aryl group, the coordination of the thiol to the Au(III) center was slowed down, which allowed for the chemoselective S-arylation of cysteine. By calculating the percent buried volume (% V_Bur) of the Au(III) OAC, a steric parameter was produced that could be used to predict the rate of coordination to the Au(III) center for both the ligand and aryl modifications. Herein, we demonstrate the extrapolation of % V_Bur as a screening metric for the expansion of Au(III) OACs for S-arylation. As the dialkylphosphine and amine could both be modified during the synthesis of the P,N-ligand, in silico screening was performed for eight dialkylphosphine substituents and five amine groups to examine their steric properties (Fig. 1, 4). Additionally, the free energy of reaction (ΔG_Rxn) as well as the free energy of activation for the reductive elimination (ΔG^‡_RE) were calculated to examine the thermodynamics of each P,N-ligated Au(III) OAC. This allowed for the rapid screening of 13 different Au(III) OACs. Three of these novel P,N-ligated Au(III) OACs were synthesized, then their kinetics and efficiency as S-arylation reagents were determined. This led to the generation of k_Co up to 20 200 M⁻¹ s⁻¹, improving upon the previously developed Au(III) OACs and demonstrating the computational and experimental synergy available in the investigation of metal-mediated bioconjugation reactions. These results also provide additional mechanistic insight into the coordination chemistry of Au(III) compounds.^36–40

Results and discussion

Computational screening of P,N-Ligated Au(III) OACs

Previously, DFT calculations for the S-arylation of methanethiol were performed with NMe₂-containing P,N-ligated Au(III) phenyl OACs with PAd₂ (5), P^tBu₂ (6), and PCy₂ (7) P-substituents.³⁵ This reaction proceeds through a reversible coordination of the thiol to the Au(III) center of SM, generating Int1. Reductive elimination (TS_RE) from Int1 irreversibly forms the C(sp²)–S bond (Int2), then product dissociation forms the free S-arylated product (P_Ar) in addition to the Au(I) chloride starting material (P_Au, Fig. 2A).⁴¹ These calculations revealed that reagents 5–7 had a low ΔG^‡_RE and a thermodynamically favorable ΔG_Rxn. Additionally, by calculating the % V_Bur of the Au(III) OAC (SM) of 5–7, % V_Bur of 70.0, 69.8, and 67.1 were obtained, respectively.⁴² This steric parameter is in agreement with the experimentally determined k_Co for each of these complexes wherein the more sterically available complexes (i.e. less % V_Bur) exhibited faster thiol coordination. We therefore hypothesized that the % V_Bur parameter could be used as a predictive metric for the rate of thiol coordination to the Au(III) center.

Fig. 2 (A) Proposed mechanism for the S-arylation of methanethiol mediated by Au(III) OACs. (B) SM geometries calculated for reagents 7, 9, 10, and 11 at the ωB97X-D/6-311+G(d,p), SDD, CPCM(water)//B3LYP-D3/6-31G(d), LANL2DZ, CPCM(water) level of theory. (C) Table of % V_Bur, ΔG^‡_RE, and ΔG_Rxn for the different dialkylphosphine substituents examined. ^aThese parameters were calculated previously.³⁵ (D) Schematic representation showing the dialkylphosphine variations in order of least to most available % V_Bur.

To further enhance this rate of coordination, we sought to screen smaller dialkylphosphines to provide more sterically available P,N-ligated Au(III) OACs using NMe₂ as the N-substituent. Accordingly, diisopropylphosphine-(PⁱPr₂) (8) and dicyclopentylphosphine-based (PCp₂) (9) P,N-ligated Au(III) SM geometries were calculated at the ωB97X-D/6-311+G(d,p), SDD, CPCM(water)//B3LYP-D3/6-31G(d), LANL2DZ, CPCM(water) level of theory. Complexes 8 and 9 had % V_Bur of 66.8 and 66.5, respectively. These values are very similar to the NMe₂-PCy₂-ligated OAC 7, so these ligands should affect a modest improvement on k_Co. For 8 and 9, the comparable angles of the dialkylphosphine alkyl groups lead to similarly sized substituents compared to the cyclohexyl ring, which contributes to the small difference in % V_Bur compared to 7. To this end, smaller ring sizes on the dialkylphosphine fragment of the P,N-ligand were examined to affect larger changes in % V_Bur. Dicyclobutylphosphine-(PCyb₂) (10) and dicylopropylphosphine-based (PCyp₂) (11) P,N-ligated OACs were determined to have 65.6 and 63.2 % V_Bur, respectively. These values demonstrate that the smaller alkyl substituents induce more steric availability around the Au(III) center (Fig. 2B). To determine the lower limit of % V_Bur for these dialkylphosphine P,N-ligated Au(III) OACs, dimethylphosphine-based (PMe₂) P,N-ligated SM (12) was examined and found to have 61.6 % V_Bur.

Inspired by recent work developing bicyclo[1.1.1]pentyl- and bicyclo[1.1.0]butyl-containing phosphines that are more strained and consequently smaller compared to their cycloalkyl counterparts,^43,44 the % V_Bur for the di(bicyclo[1.1.1]pentyl)phosphine-(PBcp₂) (13) and di(bicyclo[1.1.0]butyl)phosphine-based (PBcb₂) (14) P,N-ligated OACs were computed. Compound 13 had a % V_Bur of 65.5 while 14 had a % V_Bur of 63.5. These dialkylphosphines provide a significant decrease in % V_Bur compared to P,N-ligated SM 9 (PCp₂) and 10 (PCyb₂), which is in agreement with the additional strain in the bicyclic systems leading to smaller ring sizes and therefore less % V_Bur. The dicubylphosphine-based (PCubane₂) P,N-ligated SM (15) was also examined, which could be employed due to its similarity to 6 (P^tBu₂). Complex 15 had a % V_Bur of 65.9, indicating that formation of the cyclic scaffold does provide more steric availability to the SM. Taken together, these eight new Au(III) OACs (8–15) provide significant changes in % V_Bur compared to previously examined reagents 5–7 (Fig. 2D).

We previously determined that while the coordination to the Au(III) center is important, the subsequent, irreversible reductive elimination to form the C(sp²)–S bond (P_Ar) remains a key step in the S-arylation.³⁵ Accordingly, the ΔG^‡_RE and ΔG_Rxn were computed for each of the complexes. All of the reactions were calculated to be exergonic and irreversible. With the exception of the PMe₂ P-substituted OAC (12), the ΔG^‡_RE values are reasonable to undergo sufficiently fast reductive elimination at 25 °C (20.5–23.1 kcal mol⁻¹), but the ΔG^‡_RE of 24.8 kcal mol⁻¹ for 12 likely prevents it from performing an efficacious reductive elimination at ambient temperature (Fig. 2C).

While prior work from our group has focused on modifying the dialkylphosphine component of the P,N-ligand, there has been research varying the amino fragment of these P,N-ligands in Pd catalysis, most notably to morpholine and piperidine.^45–49 Accordingly, these Au(III) complexes with morpholine (16) and piperidine (17) were calculated with a PAd₂ P-substituent (Fig. 3A), but there was a marginal difference in % V_Bur compared to the dimethylamino reagent (5, Fig. 3B). We hypothesized that by contracting the ring size, one could generate more sterically available Au(III) OACs. Pyrrolidine (18), azetidine (19), and aziridine (20) were consequently incorporated into the PAd₂-based P,N-ligated Au(III) complex and determined to have % V_Bur of 69.5, 68.6, and 66.7 (Fig. 3B–D), respectively. These results are in line with the dialkylphosphine calculations (Fig. 2) wherein the five-membered ring is similar to the six-membered ring, but significant changes are observed in the four- and three-membered rings. To ensure that these amine modifications did not have significant effects on the thermodynamics of the reaction, the ΔG^‡_RE and ΔG_Rxn were computed for 16–20. Each reaction was calculated to be exergonic and irreversible with a low ΔG^‡_RE (12.2–18.8 kcal mol⁻¹) (Fig. 3B). Notably, the ΔG^‡_RE for the piperidine-containing OAC (16) is significantly lower than the other complexes, potentially due to relaxation of the amine fragment from the twist-boat conformation in the S-coordinated Int1 to the chair conformation in the transition state (see ESI† for details). This complex may warrant further examination to determine its ability to mediate other, more challenging reductive eliminations,⁵⁰ but the high % V_Bur indicates that it will likely not have a faster coordination than the other complexes examined herein.

Fig. 3 (A) Generalized geometry for the Au(III) SM being modified. (B) Table of SM % V_Bur, ΔG^‡_RE, and ΔG_Rxn for the different amine substituents examined. ^aThese parameters were calculated previously.³⁵ (C) SM geometry for reagent 20 calculated at the ωB97X-D/6-311+G(d,p), SDD, CPCM(water)//B3LYP-D3/6-31G(d), LANL2DZ, CPCM(water) level of theory. (D) Buried volume plot for the SM of 20.

Synthesis of P,N-ligands, Au(I) complexes, and Au(III) OACs

To validate these computational results, we sought to synthesize the compounds with both a lower % V_Bur than the NMe₂-PCy₂ ligated OAC (7) and a reasonable ΔG^‡_RE. Accordingly, the PCp₂-, PCyb₂-, and PCyp₂-based P,N-ligands with NMe₂ as the N-substituent were targeted due to their predicted rapid coordination and sufficiently fast reductive elimination. While the bicyclic systems 13 and 14 did have lower % V_Bur compared to their cyclic counterparts, they each had a similar % V_Bur to the more synthetically accessible PCyb₂- (10) and PCyp₂-based (11) systems. Due to this and their synthetic difficulty, 13 and 14 were not further examined. For the amine components, we wanted to synthesize the PAd₂ ligand with azetidine and aziridine as the amino substituent. However, the phenyl aziridine complex polymerizes in water,⁵¹ which makes it an unsuitable reagent to perform bioconjugation reactions. We also wanted to examine if modifying both the P- and the N-substituents of the ligand simultaneously generated an additive effect of the two substitutions. To do this, the azetidine-PCy₂ complex was targeted.

The NMe₂-PCp₂ ligand 21 was synthesized by in situ formation of the aryl lithiate from 2-iodo-N,N-dimethylaniline and n-BuLi then substitution with the commercially available chlorodicyclopentylphosphine (see ESI† for details). Incomplete conversion to the desired P,N-ligand necessitated purification of the ligand by preparative high performance liquid chromatography (HPLC). Attempts to generate the NMe₂-PCyb₂ and NMe₂-PCyp₂ ligands relied on formation of the aryl lithiate and in situ generation of the chlorodialkylphosphine using phosphorus trichloride and the corresponding Grignard reagent. Purification of these reagents by preparative HPLC induced significant formation of the phosphine oxide, limiting the utility of these ligands. Other synthetic efforts to obtain these ligands involved formation of the phosphine oxide and subsequent reduction,⁵² as well as attempts to generate 2-(dichlorophosphaneyl)-N,N-dimethylaniline,⁵³ which could then react with the cyclobutyl and cyclopropyl Grignard reagents. These routes provided low conversion to product and formation of multiply arylated byproducts, respectively.

However, the azetidine-PAd₂ ligand (22) and azetidine-PCy₂ ligand (23) could be readily accessed by Pd-catalyzed C(sp²)–P bond formation from the corresponding ortho-halo-substituted aniline species and the dialkylphosphine in good isolated yield (80% and 63% yield, respectively). With P,N-ligands 21–23 isolated (Fig. 4A), the Au(I) complexes (24–26) were generated using HAuCl₄·3(H₂O) and 2,2′-thiodiethanol as a water-soluble variant of the commercially available Au(I)Cl(SMe₂) (Fig. 4B, see ESI† for details). The Au(III) OACs with a p-toluene aryl substituent (27–29) were synthesized using AgSbF₆ as a halide scavenger and 4-iodotoluene to generate a model compound for kinetic investigations (Fig. 4C, see ESI† for details).

Fig. 4 Structures of P,N-ligands (A), Au(I) complexes (B), and Au(III) OACs (C) synthesized.

Thiol coordination rate monitoring

With the Au(III) OACs in hand, we wanted to test the kinetics of the thiol coordination to validate our computational predictions. To perform the coordination kinetics studies, stopped-flow/UV-vis spectroscopy experiments were performed at 5 μM of OAC with glutathione (GSH) as a model thiol under pseudo-first order conditions (500 μM–4 mM of GSH) at pH 7.4 and 20 °C (Fig. 5A, see ESI† for details). By monitoring the absorbance, one can determine the formation of Int1 and extrapolate the observed second-order rate constant, k_Co, from the experimentally observed rate constant (k_Obs). Examining 27, a k_Co of 18 700 M⁻¹ s⁻¹ was calculated, which is faster than the NMe₂-PCy₂-ligated OAC 3 (16 600 M⁻¹ s⁻¹). Also, the equilibrium of this complex is significantly shifted toward the S-coordinated intermediate (Int1) with a K_Eq (k_Co/k_Dis) of 1530 M⁻¹ (Fig. 5B and C).

Fig. 5 (A) Generalized scheme for coordination monitoring from Au(III) OACs. X⁻ is used as a generalized counterion. Experiments were performed in 1 : 1 MeCN/100 mM Tris buffer at pH 7.4 and 20 °C. (B) Kinetic plots for coordination monitoring, n = 5 – some error bars are smaller than the markers. (C) Extracted k_Co and K_Eq for reagents 27–29.

Next, rate measurements were performed for 28 to determine if modifying the amine fragment was another viable strategy to affect the k_Co. Kinetics experiments show that by utilizing the azetidine substituent, the k_Co increases to 11 600 M⁻¹ s⁻¹, which is nearly a five-fold increase compared to the NMe₂-PAd₂-ligated reagent 1 (2460 M⁻¹ s⁻¹). This increase in k_Co may also be attributed to less electron donation from azetidine compared to NMe₂, which would make the Au(III) center more electron poor and could accelerate the thiol coordination. By adding the P- and N-modifications together, we propose that 29 would provide the fastest k_Co reported. The studies were performed and a k_Co of 20 200 M⁻¹ s⁻¹ was observed for 29 (Fig. 5B and C), which represents a 25% increase in k_Co compared to 3 and is the fastest observed rate for S-coordination to an organometallic Au(III) compound to date. These results are all consistent with the % V_Bur calculations (Fig. 2C and 3B), validating the use of % V_Bur as a steric descriptor to predict the rate of bimolecular coordination for these Au(III) OACs.

S-Arylation competition experiments

With the k_Co determined for these three OACs, as well as the equilibria showing preferential thermodynamic formation of Int1, we next wanted to determine if the bimolecular coordination elementary step was still the selectivity-determining step in the reaction by performing S-arylation competition experiments. These experiments were performed by incubating one equivalent of GSH with three equivalents of two different OACs. These OACs vary by one methylene unit, which allows us to determine by liquid chromatography-mass spectrometry (LC-MS) which Au(III) reagent mediates the S-arylation more effectively (Fig. 6A). The NMe₂-PCy₂-(30) and NMe₂-PAd₂-ligated (31) Au(III) OACs with a p-ethylbenzene aryl substituent were prepared accordingly.

Fig. 6 (A) Generalized scheme and reagents used for the S-arylation competition experiments. (B) Results of the S-arylation competition experiments, n = 3. See ESI† for further details. (C) Total ion chromatogram traces from a representative replicate from each LC-MS competition experiment.

A competition experiment was performed between the NMe₂-PCp₂ (27) and NMe₂-PCy₂ (30) Au(III) OACs, which resulted in a 57 : 43 preference for the p-tolyl S-arylated product (32) over the p-ethylbenzene S-arylated product (33) (Fig. 6B and C). This preferential S-arylation from the NMe₂-PCp₂ OAC is in agreement with both the % V_Bur calculations as well as the k_Co obtained from stopped-flow kinetics. To examine the amine fragments, competition experiments were performed using the PAd₂-based P,N-ligated Au(III) OACs with azetidine (28) and dimethylamino (31) N-substitution. A 78 : 22 formation of 32 : 33 shows a preference for S-arylation from the azetidine-containing ligand (Fig. 6B and C), which is also in agreement with the % V_Bur calculations and the stopped-flow kinetics.

However, when the competition experiments were performed using the PCy₂-based P,N-ligated Au(III) OACs using the azetidine (29) and dimethylamino (30) N-substituents, a 32 : 68 ratio for 32 : 33 was observed (Fig. 6B and C). This indicates preferential S-arylation from the dimethylamino-containing ligand 30. The % V_Bur calculations and the stopped-flow kinetics indicate that coordination is faster to 29, but we hypothesize that the reductive elimination is slower, allowing for a reversible coordination. This leads to a potential switch in the selectivity-determining step for this reaction and indicates that while the two ligand permutations are additive for the thiol coordination event, both elementary steps of the S-arylation need to be sufficiently fast to induce effective conjugation.

Reductive elimination rate monitoring

To examine the effects of the azetidine compounds on the reductive elimination, stopped-flow/UV-vis spectroscopy was performed using the conditions described vide supra (Fig. 7A, see ESI† for details). From Int1 of 28, a unimolecular rate constant was observed for the reductive elimination (k_RE) of 2.66 s⁻¹ (Fig. 7B), which is approximately three times slower than k_RE of the NMe₂-PAd₂-ligated OAC 1 (Fig. 7D). This is also in line with the calculated ΔG^‡_RE for the phenyl OACs (Fig. 3B). Profiling the reductive elimination from Int1 of 29, a k_RE of 0.133 s⁻¹ was calculated (Fig. 7C), which is nearly ten times slower than the k_RE for the NMe₂-PCy₂-ligated OAC 3 (Fig. 7D). These results confirm the proposed switch in selectivity-determining step for 29 as observed in the competition experiments.

Fig. 7 (A) Generalized scheme for reductive elimination monitoring from Int1. X⁻ is used as a generalized counterion. Experiments were performed in 1 : 1 MeCN/100 mM Tris buffer at pH 7.4 and 20 °C. (B) Kinetic plot for the reductive elimination monitoring from 28-Int1, n = 5. (C) Kinetic plot for the reductive elimination monitoring from 29-Int1, n = 3 – some error bars are smaller than the markers. (D) k_RE comparison for the amine and dialkylphosphine modifications. ^aThese parameters were determined previously.³⁵

Altogether, this work demonstrates that % V_Bur can serve as a quickly calculated metric to screen the rate of bioconjugation to an Au(III) OAC, which will be valuable for the future screening of Au(III) and other metal complexes for bioconjugation. While the % V_Bur can be used to predict the rate of coordination to the Au(III) center, it can also be used to predict the rate of reductive elimination, though the trend is in the opposite direction (Fig. 8). These modifications that affect the steric bulk of the ligand may also modify the electronic properties of the complexes,^54–56 which can impact both the rate of coordination to the metal center as well as the rate of reductive elimination.^57–59 These factors should also be considered when designing new ligands for metal-catalyzed or metal-mediated transformations.

Fig. 8 (A) Ordering of the experimentally characterized Au(III) OACs from this and previous studies in order of least to most available % V_Bur.³⁵ (B) Plot of experimentally determined k_Co against % V_Bur for the Au(III) OACs shown in Fig. 8A. *% V_Bur is for the phenyl OAC and the counterion is not included. (C) Plot of experimentally determined k_RE against % V_Bur for the Au(III) OACs shown in Fig. 8A. *% V_Bur is for the phenyl OAC and the counterion is not included.

We propose that this workflow should be amenable to other metal-mediated S-arylation reactions and provide a rapid way to screen the efficacy of these reagents. This work also provides fundamental mechanistic insight into the elementary steps of Au(III) chemistry,^36,60–65 which is relatively understudied compared to Pd(II) and Pt(II) systems.

Conclusions

Thirteen Au(III) OACs were examined with eight different dialkylphosphines and five different amines in silico to determine their effects on both steps of the S-arylation reaction. Three novel Au(III) OACs were synthesized (27–29) and their bimolecular rate constants were determined to be between 11 600–20,200 M⁻¹ s⁻¹. Finally, these OACs were examined in competition experiments to elucidate which of the two elementary steps of the S-arylation controls the selectivity, and a switch in the selectivity-determining step was confirmed experimentally for one of these reagents.

Data availability

All data are available free of charge in the ESI,† including experimental details, NMR spectra, characterization, kinetic data, and coordinates of computed structures. The raw data that support the findings of this study are available at https://github.com/jtreacy01/Ultrafaster/.

Author contributions

Joseph W. Treacy: conceptualization, computation, synthesis, formal analysis, writing – original draft. James A. R. Tilden: formal analysis, writing – original draft. Elaine Y. Chao: computation, formal analysis. Zihuan Fu: synthesis. Alexander M. Spokoyny: conceptualization, supervision, writing – review and editing, funding acquisition. K. N. Houk: supervision, writing – review and editing, funding acquisition. Heather D. Maynard: supervision, writing – review and editing, funding acquisition. All authors have given approval to the final version of the manuscript.

Conflicts of interest

The authors declare the following competing financial interest(s): A. M. S. and H. D. M. are co-inventors on several patent applications from UCLA associated with the Au(III) based bioconjugation technology.

Acknowledgements

This work was funded in part by the NSF (CHE-2404202, CHE-2003946, and CHE-2153972) and the NIH (R01DK127908). A. M. S. thanks NIGMS (R35GM124746) for funding. J. W. T. thanks UCLA Dissertation Year Fellowship for support. Z. F. thanks National Institute of Health Chemistry-Biology Interface Training Grant (T32GM136614) for support. NMR spectrometers are supported in part by the S10 program of the NIH Office of Research Infrastructure Programs under grant S10OD028644. The stopped-flow/UV-vis spectrometer was supported by the NIH (1S10OD023584). This work used computational and storage services associated with the Hoffman2 Shared Cluster provided by the UCLA Institute for Digital Research and Education's Research Technology Group.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4sc05920d

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