Ultrafast Au(III)-Mediated Arylation of Cysteine

Abstract

Through mechanistic work and rational design, we have developed the fastest organometallic abiotic Cys bioconjugation. As a result, the developed organometallic Au(III) bioconjugation reagents enable selective labeling of Cys moieties down to picomolar concentrations and allow for the rapid construction of complex heterostructures from peptides, proteins, and oligonucleotides. This work showcases how organometallic chemistry can be interfaced with biomolecules and lead to a range of reactivities that are largely unmatched by classical organic chemistry tools.

Figure 1.. Overview of Cys arylation and the development of kinetically tunable Au(III) Cys arylation reagents.

(A) Cartoon of the Cys bioconjugation process and key parameters for designing effective reagents. (B) Kinetic rate spectrum of several key classes of organic reagents commonly used for Cys bioconjugation and the relative placement of organometallic Cys arylation reagents on that spectrum. Copper-catalyzed Azide-Alkyne cycloaddition (CuAAC) and enzymatic labeling rates are included for benchmark purposes. (C) Decoupling concept of the catalytic cycle using stoichiometric organometallic Cys arylation reagents. The ex situ synthesis of the organometallic Cys arylation reagents effectively decouples the slow oxidative addition step from the subsequent fast transmetalation and reductive elimination components. The cyclic representation does not imply that these reactions occur in one pot like a typical catalytic reaction. (D) Overview of the use of stopped-flow kinetic experiments to quantitatively measure rates of Cys arylation kinetics and leading to the development of kinetically tunable Au(III) reagents. (E) Applications of kinetically tunable Au(III) reagents to synthesize ultra-fast reagents and construct hybrid biomolecular conjugates.

Figure 2.. Synthetic scheme and compounds used in this study.

(A) General scheme for the synthesis of Au(III) organometallic Cys arylation reagents. (B) The three ligands used to synthesize the Au(III) organometallic complexes. (C) Bioconjugation and Cys arylation reagents used in this study.

Figure 3.. Stopped flow kinetic analysis of bioconjugation reagents and the development of a mechanistic model.

(A) Initial stopped-flow kinetic analyses of a model bioconjugation reaction with GSH as the substrate and the reagents 1, 2, and 3 performing either the bioconjugation or arylation, the arrow in each absorbance trace indicates a decrease in absorbance over time. (B) The overall observed Cys arylation reaction with the Au(III) organometallic reagent 4 and the proposed two mechanistic steps, bioconjugation and arylation, observed via stopped flow kinetic analysis. (C) Kinetic data obtained for the organometallic Au(III) reagent 5.

Figure 4.. Computational analysis of several Au(III) reagents through %V_Bur and DFT calculations.

(A) %V_Bur analysis of the L1, L2, and L3 Au(III)-phenyl oxidative addition complexes varying steric bulk used in this study calculated at the ωB97X-D/6–311+G(d,p), SDD, CPCM(Water)//B3LYP-D3/6–31G(d), LANL2DZ, CPCM(Water) level of theory and their corresponding heat maps, looking down the Au-P bond. (B) Free energy diagrams for the model S-arylation of methanethiol with Au(III)-phenyl oxidative addition complexes with L1, L2, and L3. (C) %V_Bur analysis of two Au(III) reagents, 3 and 10, with different steric profiles of their aryl ligands. The steric heat maps are also shown, looking down the Au-P bond.

Figure 5.. Applications of kinetically tunable Au(III) organometallic Cys arylation reagents.

(A) Demonstration of Cys arylation at nM and μM concentrations of both Au(III) reagent and biomolecule. (B) Labeling of a DARPin protein with a fluorescent dye. SDS-PAGE gel demonstrating that labeling of the DARPin protein works down to pM concentrations. (C) Use of a bifunctional Au(III) reagent to construct biomolecular heterostructures. (D) Construction of an oligonucleotide-protein heterostructure using the bifunctional Au(III) reagent, the strong band appearing at ca. 15 kDa represents unlabeled DARPin protein, the band appearing just under 20 kDa represents the (O1)b(DARPin) conjugate product, and the band appearing at ca. 30 kDa corresponds to the linked homodimer of the DARPin protein, a (DARPin)b(DARPin) conjugate, due to an excess of DARPin being used in the conjugation experiment. Estimated conversion to (O1)b(DARPin) was 44%, determined by imageJ quantification, representing a significant improvement over previous one-pot methods.