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.

Organometallic Oxidative Addition Complexes for S-Arylation of Free Cysteines

“…Many biomolecules are only stable in buffered aqueous solutions at or below room temperature and deviation from these benign conditions results in protein degradation and aggregation. The rapid kinetics of these S -arylation processes allows for high levels of conversion quickly, in dilute aqueous solutions, at or below room temperature. − , These exceptionally mild reaction conditions allow for rapid and efficient bioconjugation to substrates which may otherwise be unstable to harsher reaction conditions. Additionally, these reactions are well suited for biomolecules which are only stable or soluble at nonphysiological pH, as these reactions also perform well in reaction conditions ranging from pH 0–14 (Scheme , bottom), as well as high ionic buffer strength.…”

“…In this work, it was found that modifying the Me-DalPhos ligand to contain dicyclohexylphosphine instead of diadamantylphosphine allowed for a nearly 10-fold increase in the second order rate constant of the bioconjugation. This ultrafast conjugation was employed to conjugate a fluorescent small molecule to a protein at high pM concentrations, representing an extreme example of the power of these reagents to modify proteins rapidly and under mild conditions …”

“…For example, modulation of the local steric environment around the Au­(III) center resulted in selective bioconjugation with a para -substituted OA complex in the presence of an otherwise identical ortho -substituted OA complex. To this end, bifunctional Au­(III) OA complexes have been developed to generate biomolecular heterostructures of various identities utilizing a linker containing the two, orthogonal Au­(III) centers for selective and modular bioconjugation (Scheme , d) . Examples of these structures include but are not limited to peptide–oligonucleotide, peptide–peptide, protein–polymer and protein-oligonucleotide biomolecular heterostructures.…”

“…Many bioconjugation methods attempt to circumvent this challenge by using large excess of coupling reagent to ensure high level of conversion, and while this strategy does tend to increase conversion, it can be difficult to purify the excess reagent away from the conjugate. Alternatively, optimization of these Pd­(II) and Au­(III) OA complexes resulted in standard conditions of near equimolar amounts of the reagent while still achieving complete reactions within minutes at low μM concentrations and low temperatures. − , …”

Comparison of Cyclic and Linear PEG Conjugates

“…Herein, we describe the first example of a cyclic protein–polymer conjugate and compare it to a linear protein–polymer conjugate of the same size. For our model study, we synthesized linear and cyclic 2 kDa PEG Au­(III) oxidative addition complexes and performed S -arylation of each substrate to the surface-exposed cysteine of T4 lysozyme V131C (T4L). − We compared the conformation, stability, and activity of the resulting purified conjugates. Finally, we performed molecular dynamics simulations of these conjugates to examine and quantify the effect of polymer architecture on the protein–polymer conjugate behavior.…”