Profiling sirtuin activity using Copper-free click chemistry

Multifunctional activity-based chemical probes for sirtuins

“…1). 23 These biocompatible probes not only demonstrate isoform-selective labeling of an individual sirtuin isoform, but also allow the Cu-free conjugation to a fluorophore or a biotin. In the following sections, the utility of these ABPs will be further illustrated.…”

“…Probe 3B is a selective SIRT2 inhibitor with an IC 50 value of 9.14 ± 0.98 μM. 23 This ABP was applied to a mixture of partially purified recombinant sirtuins including SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6. SIRT2 was the only isoform that can be labeled (Fig.…”

“…We took a "less is more" strategy to engineer a simple Ala-Ala-Lys tripeptide as the backbone. [21][22][23] The epsilon-amino of the lysine residue was functionalized with a thioacyl group. The rst generation ABPs harbor a benzophenone photoaffinity group for its synthetic easiness (Fig.…”

“…In the previous studies, the ABPs demonstrated isoformselective labeling of individual sirtuin isoform in puried proteins as well as cell lysates. [21][22][23] The labeled proteins were visualized on a SDS-PAGE gel or by cellular imaging upon conjugation to a uorophore. The versatility of the probes can be further illustrated in affinity enrichment of a particular sirtuin isotype.…”

“…[18][19][20] We took advantage of the distinct reactivity of thioacyllysines to develop several series of activity-based chemical probes (ABPs) for human sirtuins. [21][22][23] These probes are Ala-Ala-Lys tripeptide featuring a thioacyl "warhead". A benzophenone or diazirine photoaffinity group is included for the covalent tethering of the ABP with the enzyme target upon UV irradiation.…”