UCLA

Chemical biology is an emerging field that focuses on the use of chemical tools to interrogate biological functions. One of the key points in chemical biology is the site-specific conjugation of chemical tools to biomolecules, which enables the precise use of these tools to determine the biological role for the biomolecule of interest. Site-specific conjugation methodologies are numerous, but the mechanism of these reactions often remains unclear due to the complex media in which theses reactions take place. In order to further improve these reactions and develop novel strategies, it is essential to understand the mechanism of bioconjugation. Density functional theory (DFT) is a computational tool that can be used to gain mechanistic insight into the nature of intermediates and transition states in the reaction. Throughout this thesis, I demonstrate the synergy that is available between DFT and bioconjugation methods to develop improved tools for chemical biology. Initial work focused on the mechanism of amine payload release from hydroxybenzylamines (Chapter 2). Through kinetic studies and DFT calculations, the mechanism of the reaction was determined to be reversible and proceed through a quinone methide intermediate. In silico screening of an intramolecular trapping arm led to the development of a propyl methyl ether pendant arm that prevented reversibility and accelerated the reaction five-fold. With this mechanistic understanding, aromatic substituents were examined in order to stabilize the quinone methide intermediate. This screening led to the use of a 1,4-anthracene-based hydroxybenzylamine that had a release half-life of 18 minutes, which is nearly 20 times faster than the initially reported hydroxybenzylamine containing a pendant propyl methyl ether unit. These works illustrate the use of DFT for the development of hydroxybenzylamine linkers that have applications in the synthesis of traceless protein-polymer conjugates. Cysteine is a commonly modified amino acid due to its high nucleophilicity and low abundance in the proteome, allowing for the generation of singly modified biomolecules. In order to rectify the reversibility and kinetic issues with maleimide and alkyl halide cysteine modification, respectively, cysteine S-arylation was developed as an alternative approach for cysteine bioconjugation. Metal-mediated S-arylation is a method that uses thiophilic metal centers to rapidly and chemoselective label the cysteine with a strong S-C(sp2) linkage. This chapter (Chapter 3) focuses on profiling of the bioconjugation kinetics to P,N-ligated Au(III) oxidative addition complexes (OACs) to understand the effects of the ligand on the elementary steps of thiol coordination and reductive elimination. This mechanistic knowledge led to the development of a P,N-ligated Au(III) OAC with a bimolecular thiol coordination rate constant of 16,600 M–1s–1. By understanding the effect of steric bulk on the reaction, selective S-arylation was achieved between two disparate OACs by generating sterically bulky aryl complexes that slowed down the thiol coordination. This led to the regioselective, one pot synthesis of heteroconjugates using bis-Au(III) OACs with an organic linker. In order to further accelerate the rate of bimolecular thiol coordination (Chapter 4), in silico screening of the P,N-ligand enabled the rapid examination of 13 different P,N-ligated Au(III) OACs with different diphosphine and amine modifications. Three of the synthetically accessible, more sterically available complexes were synthesized and their kinetics were determined. This led to bimolecular rate constants of 11,600–20,200 M–1s–1. Further examination of these reagents’ efficacy in S-arylation led to a proposed switch in the selectivity-determining elementary step for the fastest reagent, which was confirmed by examination of the reductive elimination. This work demonstrates the utility of DFT in the examination of metal-mediated bioconjugation reactions and provides a comprehensive workflow for the development of these reagents. To improve the scope of the available P,N-ligated Au(III) OACs, DFT calculations were performed to uncover the role of the ligand’s steric and electronic properties on the oxidative addition barrier (Chapter 5). Screening of both diphosphine and amine substituents led to the conclusion that the oxidative addition free energy of activation is controlled by the electronics of the ligand. Accordingly, six electron rich P,N-ligands were synthesized and incorporated into Au(I) complexes and Au(III) OACs. Profiling of the thiol exchange equilibrium and the reductive elimination for these novel complexes was performed using stopped-flow/UV-vis spectroscopy. These results demonstrate the effects of the ligand electronics on the elementary steps of Au(I)/Au(III) redox reactions, which has applications in both Au(I)/Au(III) catalyzed cross-coupling as well as Au(III)-mediated S-arylation.