Detection and cellular imaging of human cancer enzyme using a turn-on, wavelength-shiftable, self-immolative profluorophore

Abstract

A frontier area in the development of activatable (turn-on) fluorescence-based probes is that concerned with rapid and selective stimulus triggering of probe activation so as to allow for biomarker identification and cellular imaging. The work here is concerned with a cloaked fluorophore composed of a reporter whose fluorescence is efficiently quenched by it being bound to an activatable trigger group through a novel self-immolative linker. Highly selective and rapid activation of the trigger group is achieved by chemical and enzymatic means that result in activated trigger group detachment from the self-immolative linker, with the latter subsequently cleaved from the reporter autonomously, thereby unmasking intense, red-shifted fluorescence emission. To achieve this success, we used a trimethyl-locked quinone propionic acid trigger group and an N-methyl-p-aminobenzyl alcohol self-immolative linker attached to the reporter. Delineated here are the synthesis and characterization of this cloaked fluorophore and the evaluation of its triggered turning on in the presence of an up-regulated enzyme in human cancer cells,

Nad(p)h: quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2).

Scheme 1. Turn-On Self-Immolative Profluorophore 1

Figure 1

Absorbance and emission spectra of 2 μM probe 1 and reporter 2 in pH 7.40, 0.1 M phosphate buffer. T = 25 °C.

Figure 2

Reduction-stimulated turning on of reporter 2. Time-course fluorescence (λ_ex = 432 nm, λ_emis = 540 nm) from 10 μM probe 1 (pH 7.40, 0.1 M phosphate buffer) after reduction by 16 μM sodium dithionite. The inset contains time-dependent spectra of 2 μM probe 1 upon reduction. T = 25 °C.

Figure 3

Kinetics of human NQO1 (40 μg) with probe 1 (2–60 μM) in pH 7.4, 0.1 M phosphate buffer. Values (n = 3) are the average ±1 sample standard deviation. The curve is the best fit to the average data. T = 25 °C.

Figure 4

Microscopy images of hNQO1-positive HT29 colon (A, B, C) and hNQO1-negative H596 lung (D, E, F) cancer cells after incubation at 37 °C with 20 μM probe 1. Confocal images in A, B, D, and E; differential interference contrast in C and F. DRAQ5 nuclear stain was used for B and E. Scale bar = 20 μm.