Systematic evaluation and tuning of diazirine chemistry used in photoaffinity labeling

Abstract

Photoaffinity labeling (PAL) is one of the few biochemical techniques that can give direct evidence of biomolecular interactions in cells. Several photoactivatable functional groups have been adapted for use in PAL since its first implementation. The diversity of these chemistries has expanded the scope and fidelity of PAL experiments, but also increased the considerations during PAL probe design. Here, I use organic chemistry and chemoproteomics approaches to systematically evaluate the reactivity of the most common PAL functional group: diazirines. I use this understanding to further develop a new diazirine scaffold with reduced labeling bias for photoaffinity labeling in cells. In Chapter 1, I will review the major chemistries used in PAL and their applications for detecting small molecule–protein interactions. I discuss the reactive intermediates produced by each PAL functional groups and their major properties, namely their relative half-lives and amino acid reactivity. Following this overview of these photoactivatable functional groups, I describe how they can be incorporated into small molecules to develop PAL probes. Finally, I conclude this chapter with a series of examples that highlight the principles of PAL probe design. In Chapter 2, I detail a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. I find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while aryl-fluorodiazirines labeling patterns reflect reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, I use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive-charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome, and will facilitate design and interpretation of biomolecular labeling experiments with diazirines. iii In Chapter 3, I report the design and evaluation of PALBOX, a novel cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, designed using the analysis performed in Chapter 2. I show that PALBOX is readily derivatized and attached to small molecules to profile their binding interactions in cells. Systematic experiments with biological substrates show that the cyclobutane diazirine scaffold does not exhibit reactivity characteristic of unconstrained alkyl diazo intermediates. Using a set of small molecule fragments and ligands, I show that photoaffinity probes equipped with PALBOX can label known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, I demonstrate that ligands equipped with PALBOX can accurately map small molecule–protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule–protein interactions. In Chapter 4, I discuss the early development of oxadiazolines as pH-sensing tools for use in investigating pH in cells at the protein level. This functional group is designed to produce alkyl diazo intermediates which can label proteins in a pH dependent manner. In contrast to diazirines, oxadiazolines do not produce carbenes, suggesting all labeling from oxadiazolines in cells would proceed via a pH-dependent mechanism. I show that oxadiazoline probes react with acidic amino acids analogously to alkyl diazirine probes in solution, supporting the formation of the diazo intermediate, but that they also produce oxygen- adducts that suggest unintended carbene formation. I conclude this sections with suggestions of further experiments for evaluating this functional group. Finally, I conclude my thesis with my thoughts on future directions in PAL and small molecule target identification.