Functionalized 2‐Hydroxybenzaldehyde‐PEG Modules as Portable Tags for the Engagement of Protein Lysine ϵ‐Amino Groups

Abstract

The formation of reversible‐covalent interactions between a small‐molecule ligand and its protein target is emerging as a general strategy to design binders with increased affinity. In this context, 2‐hydroxybenzaldehyde (2HB) has been recently proposed as suitable electrophilic tag to engage primary amines, such as the ϵ‐amino group of lysine residues, in remarkably stable imines. Lys residues are often expressed in high amounts on protein surfaces and in the proximity of ligand binding sites, and a fine‐tuning of the chemical connection between the ligand and 2HB is fundamental for the affinity gain. Herein we report the synthesis of four 2HB tags functionalized with a short polyethylene glycol (PEG) spacer and a suitable reactive ‘handle’ (alkyne, azide, carboxylic acid and amine, respectively) for their conjugation to virtually any type of small molecule ligand.

Synthesis of Noncoded Amino Acids Bearing a Salicylaldehyde Tag for the Design of Reversible‐Covalent Peptides

“…As in our earlier work, [15,16] we selected dihydroxybenzaldehyde derivatives (i. e., 2,5-and 2,4-dihydroxybenzaldehydes: Figure 1D, compounds 1 a and 1 b, respectively), with the aim to exploit a second phenolic OH group in the aromatic ring (5-OH or 4-OH) as anchoring unit for the SA installation in the AA side chain. In particular, as illustrated by the retrosynthetic pathway in Scheme 1A, we planned the SA tag connection to the AA scaffold through a Williamson ether synthesis, using the phenolic OH as nucleophile and a leaving group on the AA side chain.…”

“…Amide 10 a was then treated with an excess of piperidine, to remove the Fmoc protecting group at the N terminus and the resulting primary amine of compound 11 a was reacted with Fmoc‐Ser(O t Bu)‐OH, [18] previously modified as N ‐hydroxysuccinimidyl (OSu) ester. To avoid possible intra‐ and inter‐ molecular interactions between the aldehyde and the primary amino group, [15] the reaction was performed in a mixture of THF, DME and water, using sodium bicarbonate as mild base. The resulting tripeptide 12 a was obtained in good yield (42 %) and was then treated with trifluoroacetic acid to cleave the tert ‐butyl ether and ester groups.…”

“… A) Schematic binding mechanism of a SA‐bearing protein ligand, where the reaction between SA and a Lys group proximal to the ligand binding site leads to the formation of an imine adduct, stabilized by an intramolecular H bond [13] . B) Schematic representation of our previous work, consisting in the SA tag installation at either the N or C termini of a model peptide (i. e. a cyclic peptide bearing the Arg‐Gly‐Asp sequence) through a triazole connection [15] . C) Aim of this work, consisting in the installation of the SA tag at the side chain of an α‐amino acid (AA), following the synthesis of a model tripeptide.…”

“…[13] B) Schematic representation of our previous work, consisting in the SA tag installation at either the N or C termini of a model peptide (i. e. a cyclic peptide bearing the Arg-Gly-Asp sequence) through a triazole connection. [15] C) Aim of this work, consisting in the installation of the SA tag at the side chain of an α-amino acid (AA), following the synthesis of a model tripeptide. D) Molecular structure of 2,5-(1 a) and 2,4dihydroxybenzaldehyde (1 b), i. e., the SA derivatives chosen as starting material for the amino acid synthesis.…”

RGD Cyclopeptide Equipped with a Lysine‐Engaging Salicylaldehyde Showing Enhanced Integrin Affinity and Cell Detachment Potency

“…Moreover, ortho ‐hydroxy aldehydes such as pyridoxal or salicylaldehyde (SA) derivatives have been also used to form imines in aqueous media, stabilized by an intramolecular H‐bond between the imine N atom and the ortho ‐phenolic proton (Figure 1A). [6] Besides their application as site‐selective protein and peptide modifiers, [7] the use of SA fragments in the context of RC ligands was firstly described in 2012: a SA‐bearing ligand was found to engage a Lys( ϵ ‐NH 2 ) group buried in the RNAse domain of inositol‐requiring enzyme 1 endoribonuclease (IRE1) [8] . A SA fragment was also installed in the drug GBT440 (Oxbryta™, recently approved for the treatment of sickle cell disease), which prevents the polymerization by forming an imine bond between the SA handle and a N‐terminal Val( α ‐NH 2 ) group in mutant hemoglobin (HbS) [9] .…”