Metabolic alkene labeling and in vitro detection of histone acylation via the aqueous oxidative Heck reaction

Abstract

The detection of protein lysine acylations remains a challenge due to lack of specific antibodies for acylations with various chain lengths. This problem can be addressed by metabolic labeling techniques using carboxylates with reactive functionalities. Subsequent chemoselective reactions with a complementary moiety connected to a detection tag enable the visualization and quantification of the protein lysine acylome. In this study, we present EDTA-Pd(II) as a novel catalyst for the oxidative Heck reaction on protein-bound alkenes, which allows employment of fully aqueous reaction conditions. We used this reaction to monitor histone lysine acylation in vitro after metabolic incorporation of olefinic carboxylates as chemical reporters.

Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine-Thiol Displacement Reaction (FTDR)

“…We then investigated substitution effects on benzenethiol in order to efficiently convert fluoroacetamide under the mild physiological reaction condition. [31][32] Exploration of a series of benzenethiol derivatives (5)(6)(7)(8)(9)(10)(11)(12)(13) found that electron donating groups at the ortho-and para-positions facilitate the reaction by increasing the nucleophilicity (Figure 2, S7). Given their theoretical pKa's (< 7.0) ( Figure S8), almost all of these derivatives should be fully deprotonated.…”

“…5 For instance, chemical reporters on acetylation have revealed many new protein substrates of p300, [6][7] providing a more robust substrate recovery from proteome in comparison to anti-acetyl lysine antibodies. [8][9] Yet, current chemical reporters are bulky in length and size, thereby impeding their general applications. Even alkyne or azide-based minimalist reporters for copper-catalyzed azide-alkyne cycloaddition (CuAAC) are still significantly larger than the inherent carbon-hydrogen bond ( Figure 1A).…”

Activity‐Based Probes for 15‐Lipoxygenase‐1

“…Because of the observed fast inactivation t 1/2 , the labeling experiments were done in a relatively short time (2 min). The ABPP labeled enzyme was detected using covalent attachment of biotinyl phenyl boronic acid to the terminal alkene chemical reporter using the oxidative Heck reaction [42,48] and subsequent visualization by on blot luminescence imaging using HRP-conjugated Streptavidin. In the oxidative Heck reaction it proved to be important to use 10% DMF as co-solvent in the reaction mixture to avoid non-specific binding of biotinyl phenyl boronic acid to the protein.…”

“…Application of a terminal alkene as chemical reporter and not the more commonly used terminal alkyne enables straightforward synthesis of the ABPP probe using methods shown in Figure 2 without the need for protection and deprotection of the reporter functionality. As demonstrated recently, terminal alkenes can be linked to biotinylated phenylboronic acid by application of the recently developed bioorthogonal oxidative Heck reaction, [41,42] which proceeds under mild conditions. Using these methods we demonstrate for the first time activity-based labeling of lipoxygenase activity, which paves the way for exploration of this novel area.…”

“…[39,40] After modeling studies,i nc ontrast with the previous inhibitors,w es hifted the position of the bis(alkyne) moiety from the 9,12 to the 5,8 position owing to structural differences in the active sites between the two enzymes (see Figure S9 in the Supporting Information) but also with the aim to synthesize less lipophilic compounds.Next, we developed ABPP probes that included both ab is(alkyne) functionality for covalent linkage to the active enzyme and at erminal alkene as chemical reporter for the bioorthogonal linkage of ad etectable functionality (Figure 1). Thea pplication of at erminal alkene as ac hemical reporter and not the more commonly used terminal alkyne enabled straightforward synthesis of the ABPP probe by the methods shown in Scheme 1without the need for protection and deprotection of the reporter functionality.A sd emonstrated recently,t erminal alkenes can be linked to biotinylated phenylboronic acid by abioorthogonal oxidative Heck reaction, [41,42] which proceeds under mild conditions.B yu sing these methods,w ed emonstrate the activity-based labeling of lipoxygenase activity.…”