ortho-Methoxyphenols as Convenient Oxidative Bioconjugation Reagents with Application to Site-Selective Heterobifunctional Cross-Linkers

Abstract

The synthesis of complex protein-based bioconjugates has been facilitated greatly by recent developments in chemoselective methods for biomolecular modification. The oxidative coupling of o-aminophenols or catechols with aniline functional groups is chemoselective, mild, and rapid; however, the oxidatively sensitive nature of the electron-rich aromatics and the paucity of commercial sources pose some obstacles to the general use of these reactive strategies. Herein, we identify o-methoxyphenols as air-stable, commercially available derivatives that undergo efficient oxidative couplings with anilines in the presence of periodate as oxidant. Mechanistic considerations informed the development of a preoxidation protocol that can greatly reduce the amount of periodate necessary for effective coupling. The stability and versatility of these reagents was demonstrated through the synthesis of complex protein-protein bioconjugates using a site-selective heterobifunctional cross-linker comprising both o-methoxyphenol and 2-pyridinecarboxaldehyde moieties. This compound was used to link epidermal growth factor to genome-free MS2 viral capsids, affording nanoscale delivery vectors that can target a variety of cancer cell types.

Triazolecarbaldehyde Reagents for One‐Step N‐Terminal Protein Modification

“…It is therefore likely that N‐terminal modification will not affect the inherent function of wild‐type proteins or enzymes in most cases . Since an N‐terminal α‐amino group has a lower p K a value than the ϵ‐amino group of the Lys side chain, a reagent targeting the N‐terminal amino acid is expected to be particularly effective for modification of this site . For example, Francis and co‐workers, and other researchers have reported a site‐specific and one‐step N‐terminal α‐amino group modification Scheme using 2‐pyridinecarbaldehyde derivatives …”

“…In biological systems, pyridoxal phosphate is an important cofactor which is involved in post‐translational modification of an amino group in peptides and proteins . Specific N‐terminal modification of a protein with 2‐pyridylcarbaldehyde, a nitrogen‐containing heteroaromatic compound with a formyl group which is similar to the pyridoxal phosphate cofactor, was reported to proceed via formation of a 4‐imidazolidinone ring involving two nitrogen atoms of the N terminus and adjacent residues . On the basis of this knowledge, we began our investigation by examining a series of five‐membered nitrogen‐containing heteroaromatic aldehydes as N‐terminal specific modification reagents.…”

Considerations on Probe Design for Affinity‐Guided Protein Conjugation

“…In a recent example, we introduced an aniline reactive handle to allow selective modification by oxidative coupling with an o ‐aminophenol modified label . The oxidative coupling was developed by Francis and co‐workers and uses oxidized o ‐aminophenols or o ‐methoxyphenols for coupling with anilines …”

Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting

“…[46] To explore different medicala nd biotechnological applications,b iomolecules( such as peptides, DNA,a nd antibodies) as well as small molecules (dyes, radioisotopes, MRI contrast agents, drugs)w ere coupled in ap recise mannert op roteins of interest. [47] Tilman Hackeng (CardiovascularR esearch Institute Maastricht/Maastricht University,T he Netherlands) discussed recent applicationso fo xime ligation reactions to probe functional heteromeric chemokinep rotein-protein interactions. [48] He furthermorer eporteda no xime ligationr eaction that can be accelerated under aqueous conditions, at low concentrationsa nd at neutral pH by freezing.…”