Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

Abstract

Ar ange of isoxazole-containing amino acids was synthesized that displaced acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains.Three of these amino acids were incorporated into ah istone H4-mimicking peptide and their affinity for BRD4 (1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of ahyperacetylated histone H4-mimicking cognate peptide,a nd demonstrated ad ependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of ahistone H4-mimicking peptide,containing alysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate aK 18C mutant of histone H3.

Regioselective synthesis of methyl 5-(N-Boc-cycloaminyl)-1,2-oxazole-4-carboxylates as new amino acid-like building blocks

“…Unnatural heteroarene amino acids have also been widely used as building blocks to prepare various heterocyclic peptides [17][18][19][20][21][22]. In particular, 1,2-oxazole amino acid derivatives, such as compounds V [20], VI [21], and VII, VIII [22], can be easily synthesized and are suitable for insertion with the corresponding heterocycle into a peptide-like structure.…”

Trifluoroacetyl Lysine as a Bromodomain Binding Mimic of Lysine Acetylation

“…We considered several different acetyllysine mimetics, either because they had been incorporated previously into peptides to study bromodomain interactions , or because they had been successfully incorporated into proteins using an orthogonal synthetase/tRNA pair. , An isoxazole mimetic has the potential to provide higher-affinity binding than acetyllysine but, in addition to lack of commercial availability, might not be equally applicable to all bromodomain families. Alkylation of cysteine to yield the methylthiocarbamate analogue results in a sulfur atom at the γ position and is perhaps more useful for modification of intact proteins; in addition, the alkylating agent methylthiocarbonyl-aziridine (MTCA) is not commercially available.…”

Probing lysine posttranslational modifications by unnatural amino acids

“…These amino acids were based on well-known small molecule bromodomain binders and were shown to display binding to a panel of bromodomains. 114 These amino acids could be incorporated into full-length histone proteins by cysteine alkylation, extending the study of bromodomains via this strategy to both peptide and proteins. Finally, photoreactive probes were developed for enrichment of proteins that interact with these modifications through pull-down assays.…”