N-terminal dual protein functionalization by strain-promoted alkyne–nitrone cycloaddition

Abstract

Strain-promoted alkyne-nitrone cycloadditon (SPANC) was optimized as a versatile strategy for dual functionalization of peptides and proteins. The usefulness of the dual labeling protocol is first exemplified by the simultaneous introduction of a chloroquine and a stearyl moiety, two endosomal escape-improving functional groups, into the cell-penetrating peptide hLF (human lactoferrin). Additionally, we demonstrate that dual labeling of proteins is feasible by combining metal-free and copper-catalyzed click chemistry. First, SPANC is applied to enhanced green fluorescent protein to introduce both biotin and a terminal alkyne. The terminal acetylene then serves as a convenient anchor point for the CuAAC reaction with azido-containing fluorescein, thereby demonstrating the potential of combined SPANC and CuAAC for the straightforward, dual functionalization of proteins.

Dual modification of biomolecules

“…The main approaches to dually modify peptides and proteins can be classified in two separate categories (Figure 1). 44 The first one is the modification of two amino acids on the proteins using various methods that include: exploiting the difference of accessibility or reactivity of amino acids, [45][46][47] functionalisation of the C-and N-terminus of proteins, 48 use of unnatural amino acids, [49][50][51] use of specific enzymes 50 or a combination of these strategies 52 . The second category involves conjugation of a multifunctional linker to the protein of interest.…”

“…The multifunctional scaffold can then be reacted to give a dually modified conjugate. 19,31,44,53 engineered a dual cysteine mutant of Escherichia coli adenylate kinase and, by carefully selecting the position of the engineered cysteines, the reaction times and the stoichiometry of reagents, the authors could generate doubly labelled proteins. 45 This method relies on sufficient difference in reactivity of the cysteines so that the most reactive cysteine can be selectively reacted by addition of a weakly thiolreactive reagent, and the less reactive cysteine subsequently modified with a highly thiol-reactive reagent.…”

Site-Specific Functionalization of Proteins and Their Applications to Therapeutic Antibodies

“…N-terminal serine and threonine residues can selectively be oxidized by sodium periodate to form an aldehyde [11], followed by oxime ligation [12]. Besides oxime ligation, the oxidized serine was recently also used for the one step N-terminal dual protein functionalization using strain promoted alkyne–nitrone cycloaddition [13]. …”

1H‐1,2,3‐Triazole: From Structure to Function and Catalysis

“…Recently, functionalized analogues of BCN (bicyclo[6.1.0]non‐4‐yne, Fig. ) were prepared by the Rutjes group . Besides a much easier preparation, the BCN derivatives were shown to be stable, relatively non‐lipophilic, and symmetrical, which prevented the formation of regioisomers during cycloaddition.…”