Doubly Caged Linker for AND‐Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

Abstract

In situ quantification of the conjugation efficiency of azide-terminated synthetic polymers/imaging probes and thiol-functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self-sorting bridging unit. Orthogonal dual "click" coupling of C1 with azide- and thiol-functionalized precursors led to highly fluorescent bioconjugates, whereas single-click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody-probe conjugates from an anti-carcinoembryonic antigen and a quinone-caged profluorescent naphthalimide derivative, the dual "click" coupling process with C1 was monitored on the basis of the emission turn-on of C1, whereas prominent changes in FRET ratios occurred for antibody-imaging-probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

Target Enzyme‐Activated Two‐Photon Fluorescent Probes: A Case Study of CYP3A4 Using a Two‐Dimensional Design Strategy

“…[15] Hydroxylation at C-4 triggers as ignificant change in the fluorescence spectra, which makes NN adesirable off-on two-photon fluorescent probe based on the mechanism of internal charge transfer (ICT). [15] Hydroxylation at C-4 triggers as ignificant change in the fluorescence spectra, which makes NN adesirable off-on two-photon fluorescent probe based on the mechanism of internal charge transfer (ICT).…”

Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers

“…To address this issue, the DDCF3 linker, containing a highly thiol‐reactive alkenyl amide/ester moiety, was synthesized as an alternative. Unfortunately, the DDCF3 linker already exhibits significant emission enhancement after a single Michael addition reaction with ME, which indicates that it loses the double‐caging feature (Figure ) …”

“…As shown in Figure , the DDCF4 linker is a coumarin derivative containing both 7‐ethynyl and highly thiol‐reactive 3‐alkenyl amide/ester functionalities. The DDCF4 linker proves to be a powerful tool for the modular and fluorogenic construction of synthetic diblock copolymers (from HS‐PDMA and PEG 45 ‐N 3 ), protein–polymer conjugates (from BSA/tris(2‐carboxyethyl)phosphine (TCEP)‐reduced‐BSA and PEG 227 ‐N 3 ), and peptide–polymer conjugates (from sCT and PEG 227 ‐N 3 ), and the coupling efficiency could be monitored in situ in real time through changes in fluorescence emission (Figure ) …”

“… The DDCF4 linker for in situ fluorescent quantification of the conjugation efficiency between azide‐terminated functional agents (molecular probes and PEG) and thiol‐functionalized (bio)macromolecules (synthetic polymers, proteins, peptides, and antibodies). Dual conjugation of DDCF4 with both azide and thiol moieties leads to highly fluorescent functional bioconjugates, whereas its emission remains caged upon single click reactions …”

“…NQO1 is overexpressed in a range of tumor cells . In combination with the targeting capability of the ACEA antibody, the detection specificity and sensitivity of antibody–probe conjugates were significantly improved to achieve cell‐type‐selective enzymatic imaging (Figure ) …”