Real-time monitoring of newly acidified organelles during autophagy enabled by reaction-based BODIPY dyes

Abstract

Real-time monitoring of newly acidified organelles during autophagy in living cells is highly desirable for a better understanding of intracellular degradative processes. Herein, we describe a reaction-based boron dipyrromethene (BODIPY) dye containing strongly electron-withdrawing diethyl 2-cyanoacrylate groups at the α-positions. The probe exhibits intense red fluorescence in acidic organelles or the acidified cytosol while exhibiting negligible fluorescence in other regions of the cell. The underlying mechanism is a nucleophilic reaction at the central meso-carbon of the indacene core, resulting in the loss of π-conjugation entailed by dramatic spectroscopic changes of more than 200 nm between its colorless, non-fluorescent leuco-BODIPY form and its red and brightly emitting form. The reversible transformation between red fluorescent BODIPY and leuco-BODIPY along with negligible cytotoxicity qualifies such dyes for rapid and direct intracellular lysosome imaging and cytosolic acidosis detection simultaneously without any washing step, enabling the real-time monitoring of newly acidified organelles during autophagy.

A Finding on the Direct Regioselective Cyanation of BODIPY

“…11 There are several reported examples in which a nucleophile adds to the meso-carbon of BODIPY when both the -positions are occupied. 6,12 In our case, in which one of the -positions is unoccupied, substitution occurred preferentially at -position. The addition to the meso-carbon took place only after reaction at the vacant -position.…”

“…The latter outcome is consistent with reported findings. 6,12 Scheme 3 Reaction of 4 with CNion 11 Photophysical properties of 4 were studied in acetonitrile (see the Supporting Information). Compared to compound 3 ( abs,max = 540 nm,  ems,max = 552 nm in ACN), substitution with CNcaused small redshifts both in absorption and fluorescence maxima ( abs,max = 550 nm,  ems,max = 562 nm) and a decrease in fluorescence quantum yield from 0.62 to 0.26.…”

“…5 Later, Rurack and Shen revealed the mechanism behind the change in fluorescence of such di-substituted molecules and specified that the intro-duction of electron-withdrawing groups at various positions of a BODIPY makes the core suitable for further reactions. 6 Then, we turned our attention to the chemistry of 3-(methoxycarbonyl)ethenyl BODIPY 3 (Scheme 1). Studies on this compound have revealed intriguing results regarding the reactions at the core.…”

Synthesis, Optical Properties, Preliminary Antimycobacterial Evaluation and Docking Studies of Trifluoroacetylated 3‐Pyrrolyl Boron‐Dipyrromethene

“…The second way is often more appropriate for greater availability of initial reagents and its complementation of the first customization way. [12][13][14][15][16] For these reasons, numerous synthetic strategies of BODIPY modifications have been developed. It is possible to use nucleophiles for oxidative nucleophilic substitution (positions 3 and 5) and electrophiles to customize 2 and 6 positions.…”

Fluorescence lifetime imaging of pH along the secretory pathway

“…Fluorescent dyes that allow the measurement of intraorganellar pH exist and are commercially available [26][27][28][29][30] , but the inability of specific organellar targeting is a major drawback. The pH in the lumen of the Golgi and ER in mammalian cells have been measured using Shiga-like toxins covalently bound to fluorescent dyes 31,32 and with the biotin-avidin system 33 .…”