Generalizable synthesis of bioresponsive near-infrared fluorescent probes: sulfonated heptamethine cyanine prototype for imaging cell hypoxia

Abstract

A new synthetic method produces a bioresponsive near-infrared molecular probe that undergoes “turn-on” fluorescence for microscopic imaging of hypoxia.

Structure‐Activity Studies of Nitroreductase‐Responsive Near‐Infrared Heptamethine Cyanine Fluorescent Probes

“…This work builds on our recent discovery that heptamethine cyanine probes 1 and 3 are cleaved efficiently by the overexpressed NTR within hypoxic cells to generate products 2 and 4, respectively, with "turn on" NIR fluorescence at ~780 nm when excited by 750 nm light. [31] The previous study produced strong evidence supporting the self-immolative cleavage mechanism in Scheme 1 that is triggered by NTR reduction of the nitro group. Importantly, the 4'-ester group in these molecular probes is highly resistant to direct cleavage by esterase enzymes (especially when the probe structures include sulfonate groups).…”

“…In addition, a significant strategic advantage with this synthetic method is the introduction of water-solubilizing sulfonate groups after the 4-nitrobenzyl ester trigger group has been installed on the Zincke precursor. [18,31] The benzoindolinene derivatives 5 and 7 were the first pair of molecular probes to be made and evaluated. While these fluorescent probes exhibited good photophysical properties in methanol solution, the hydrophobicity of the benzoindolinene terminal groups promoted extensive probe self-aggregation (Hand J-aggregates) in aqueous solutions.…”

“…Strong evidence that the "turn on" fluorescence is due to the catalytic action of NTR was gained by observing a decreased rate when the known NTR inhibitor dicoumarol was included in the mixture (Figure S9). [31] The selectivity of probe 9 was further assessed by treating separate solutions of 9 with common biomolecular analytes and only NTR produced the "turn on" fluorescence (Figure 3d). The inability of esterases to activate probe 9 by direct cleavage of the 4'-ester bond is especially notable.…”

“…Our previous work showed that the hypoxic conditions increased intracellular NTR activity in the A549 cellline by about 50 %. [31] Separate cell populations were treated with probes 5, 7 or 9 for 2 hours and fluorescence cell microscopy was used to quantify the level of intracellular NIR fluorescence. The data in Figures S13-S15 show representative fluorescence micrographs for cells treated with probes 5, 7 or 9 and the bar graph in Figure 4 provides the quantification gained by analyzing multiple separate microscopic fields.…”

“…The Smith group exploited this discovery to develop a new class of NTR-activated fluorescent probes based on the self-immolative fragmentation shown in Scheme 1. [31] They found that NTR catalyzed reduction of the nitro group within probe 1 or 3 triggered self-immolative cleavage to produce compounds 2 or 4, respectively. The NTR catalyzed conversion of a 4'-ester group on the heptamethine chain to a 4'-carboxyl group produced a ~40 nm blue shift in emission wavelength and a very large enhancement of fluorescence emission at ~780 nm when excited by 750 nm light.…”