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Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells

Mukherjee, Kamalika; Chio, Tak Ian; Gu, Han; Banerjee, Abhijit; Sorrentino, Anthony M; Sackett, Dan L; Bane, Susan L
Detection and quantification of biomolecule carbonylation, a critical manifestation of oxidative stress, allows better understanding of associated disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomolecules in cells. Live cell detection of these species requires sensors that are nontoxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation. Presented here is a new fluorescent sensor for biomolecule carbonyl detection: a hydrazine derivative of a benzocoumarin, 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2-one (BzCH), which meets these requirements. This probe is especially well suited for live cell studies. It can be excited by a laser line common to many fluorescence microscopes. The emission maximum of BzCH undergoes a substantial red shift upon hydrazone formation (from ∼430 to ∼550 nm), which is the result of fluorophore disaggregation. Additionally, the hydrazone exhibits an exceptionally large Stokes shift (∼195 nm). The latter properties eliminate self-quenching of the probe and the need to remove unreacted fluorophore for reliable carbonyl detection. Thus, biomolecule carbonylation can be detected and quantified in cells and in cell extracts in a one-step procedure using this probe.
PMCID:7448582
PMID: 28722432
ISSN: 2379-3694
CID: 5176792

Intramolecular Catalysis of Hydrazone Formation of Aryl-Aldehydes via ortho-Phosphate Proton Exchange

Dile, Ozlem; Sorrentino, Anthony M; Bane, Susan
Bioorthogonal site-specific chemical reaction to label biomolecules in vitro and in living cells is one of the most powerful and convenient tools in chemical biology. A reactive pairs frequently used for chemical conjugation are aldehydes/ketones with hydrazines/hydrazides/hydroxylamines. Although the reaction is generally specific for the two components, even in a cellular environment, the reaction is very slow under physiological conditions. Addition of a phosphate group at the ortho-position of an aromatic aldehyde increases the reaction rate by an order of magnitude and enhances the aqueous solubility of the reagent and the product. We have synthesized phosphate-substituted aldehyde synthetic models to study kinetics of their reactions with hydrazines and hydrazides that contain a fluorophore. This rapid bioorthogonal reaction should therefore be potentially a very useful reaction for routine site-specific chemical ligations to study and image complex cellular processes in biological systems.
PMCID:7747795
PMID: 33343091
ISSN: 0936-5214
CID: 5176802