Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins

Abstract

Chemical modifications of native proteins can facilitate production of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, we report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallography. The absence of toxic metals and biochemically incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications.

Metallvermittelte Funktionalisierung natürlicher Peptide und Proteine: Biokonjugation mit Übergangsmetallen

“…[128,129] Im Zuge der Reaktionsoptimierung entdeckten die Autoren eine signifikante Verbesserung der Reaktionseffizienz nebst der Vermeidung stark acider Bedingungen durch die Verwendung eines N-Alkylhydroxylamin-basierten Puffers, welcher eine Durchführung der Reaktion bei pH 6.5 gestattete. Modifikationen konnten an komplexen Peptiden sowie an kleinen Proteinen nachgewiesen werden.…”

Biomolecular engineering for nanobio/bionanotechnology

“…Recently, a transition metal-free method using 9-azabicyclo[3.3.1]nonane-3-one- N -oxyl (keto-ABNO) for the conjugation of Trp was reported. This new method showed novel features, such as high Trp selectivity, the formation of single conjugates with high homogeneity, facile conjugation at an ambient temperature and nearly neutral pH and a short reaction time [218]. …”

“…Inspired by natural fusion proteins, synthetic fusion proteins have been designed to achieve synergistically improved bioactivities or to generate novel functional combinations derived from each of their component moieties, which are integrated into one molecule by peptide linkers. The fusion proteins have been widely applied in various areas, including recombinant protein production by the tag-mediated enhancement of protein expression, solubility and high-throughput purification [291, 292], fluorescent protein-mediated molecular imaging [293], advanced biocatalysis [101, 108, 111, 115, 164, 290, 294–297], biosensing and bioelectronic materials [290, 298–300], pharmaceuticals, diagnostics and therapeutics [208, 290, 301, 302], reporter protein-mediated immunoassays [303–310], the chimeric receptor-mediated control of cell fate, e.g., growth, death, migration or differentiation [311–319], the library selection of antibodies [203, 320, 321] and antibody-mediated drug delivery [218, 322, 323]. …”

Synthesis and Applications of Protein/Peptide-Polymer Conjugates

“…The relatively high natural abundance of amine‐functional groups can also be disadvantageous, leading to heterogeneous products due to the addition of multiple polymer chains to target proteins/peptides, which itself is implicated in reduction or complete loss of bioactivity. Consequently, less abundant residues such as tyrosine and tryptophan have been targeted for modification through their electron‐rich aromatic side chains. However these residues are often located in poorly accessible hydrophobic domains of larger proteins and require the use of elaborate functional linkers.…”