Site-Selective Protein Immobilization on Polymeric Supports through N-Terminal Imidazolidinone Formation

Abstract

Protein immobilization techniques on polymeric supports have enabled many applications in biotechnology and materials science. Attaching the proteins with controlled orientations has inherent advantages, but approaches for doing this have been largely limited to cysteine or noncanonical amino acid targeting. Herein, we report a method to attach the N-terminal positions of native proteins to polymer resins site-specifically through the use of 2-pyridinecarboxyaldehyde (2PCA) derivatives. For high protein loadings and practical synthesis, we initiated this work by preparing highly reactive 2PCA derivatives using Pd-catalyzed cross-coupling amination. The resulting compounds were attached to amine-containing polyethylene glycol acrylamide resin (PEGA-NH2), which subsequently reacted with the N-termini of proteins to produce linkages that were stable over the long term but could be reversed through the addition of hydroxylamine. We envision that this site-selective, 2PCA-based protein immobilization can provide a simple and generalizable immobilization protocol.

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 …”

Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

“…Site-selective post-translational modification of proteins has long been an object of study for both chemists and biologists. The augmentation of the native properties of these biomolecules to contain fluorophores, , drug molecules, and other signaling moieties has been of tremendous value to both our understanding of biology and the creation of new therapeutics. − While much work has been done in the attachment of small molecule cargoes, the facile attachment of additional whole proteins has proven more difficult. The primary challenge of protein–protein coupling lies in the steric inhibition that must be overcome in the joining of large molecules as well as the difficulty in the generation of suitable selective reaction handles due to the frequent occurrence of most native amino acids.…”

A Universal Chemical Method for Rational Design of Protein‐Based Nanoreactors**

“…Unlike other bioconjugation methods, this method is versatile and can be used to label a large number of proteins irrespective of their size, shape, surface charge, molecular weight, and secondary, tertiary, and quaternary structures. Although a terrific method, one of the severe limitations of this methodology is that it is used only for bioconjugation of “hydrophilic payloads” which includes imaging agents, drugs, and polymers to make hydrophilic protein conjugates which find applications in the area of targeted drug delivery [14] and biomaterials applications [15] . Although extremely useful, this method cannot be directly used for site‐specific bioconjugation of hydrophobic synthetic molecules or macromolecules.…”