Peptide‐Based 2‐Aminophenylamide Probes for Targeting Endogenous Class I Histone Deacetylase Complexes

Abstract

Lysine deacetylases or histone deacetylases (HDACs) remove acetylation markers from numerous cellular proteins, thereby regulating their function and activity. Recently established peptide probes containing the HDAC‐trapping amino acid α‐aminosuberic acid ω‐hydroxamate (AsuHd) have been used to investigate the compositions of HDAC complexes in a site‐specific manner. Here we report the new HDAC‐trapping amino acid 2‐amino‐8‐[(2‐aminophenyl)amino]‐8‐oxooctanoic acid (AsuApa) and the utility of AsuApa‐containing probes for HDAC complex profiling on a proteome‐wide scale. Unlike AsuHd‐containing probes, AsuApa enriched only HDACs 1, 2, and 3 efficiently and was the most potent probe tested for capturing the last of these. These findings indicate that the inherent specificity of reported small‐molecule pimelic diphenylamide HDAC inhibitors is preserved in AsuApa and that this HDAC‐trapping amino acid represents a potent tool for investigating class I HDAC complexes.

Synthesis of Nε‐acetyl‐L‐homolysine by the Lossen rearrangement and its application for probing deacetylases and binding modules of acetyl‐lysine

“…29,30,32 In order to synthesize Fmoc-hLys(Ac)-OH and Fmoc-hLys(Alloc)-OH from L-Asu (1), we adapted our synthesis for AsuHd to the Lossen rearrangement (Figure 2). 33 In a first step, we protected the α-carboxylate and amino group with 9-borabicyclo[3.3.1]nonane (9-BBN). 34 Upon activation of the side chain carboxylate in 2 with HBTU, O-(tert-butyldimethylsilyl)hydroxylamine (TBDMS-ONH 2 ) was coupled.…”

Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases

“…2a ) 31 , 32 . Consequently, the turnover of Kac-containing peptides by Zn 2+ -dependent HDACs can be inhibited by entities where the Kac has been substituted with zinc-binding groups, and peptides containing the hydroxamic acid 32 – 34 or the o -aminoanilide 35 functionalities have been shown to bind and/or capture HDAC enzymes. Here, we explored the use of these moieties in histone tail peptide microarrays to interrogate the binding of class I HDACs.…”

Investigating Peptide‐Coenzyme A Conjugates as Chemical Probes for Proteomic Profiling of N‐Terminal and Lysine Acetyltransferases

“…Peptide immobilization: SulfoLink Coupling Resin suspension (300 μL for each peptide) (Thermo Fisher Scientific, Rockford, USA) was drained and washed with coupling buffer (50 mM Tris • HCl, 5 mM EDTA-Na, pH 8.5) (5 × 800 μL). [31] 300 μL of the peptide solution (1 mM in coupling buffer) were added and the resin was incubated at room temperature for 1 h. After washing with coupling buffer (3 × 500 μL), 500 μL of blocking buffer (50 mM β mercaptoethanol in coupling buffer) were added to each resin and incubated for 1 h. Afterwards, the resin was washed with 1 M NaCl solution (6 × 1 mL), water (2 × 1 mL) and 50 % ACN in water (4 × 1 mL). The dry resin was mixed with 450 μL of 50 % ACN in water, aliquots of 40 μL were prepared and stored at À 20 °C.…”