Chiral, J-Aggregate-Forming Dyes for Alternative Signal Modulation Mechanisms in Self-Immolative Enzyme-Activatable Optical Probes

Abstract

Enzyme-activatable optical probes are important for future advances in cancer imaging, but may easily suffer from low signal-to-background ratios unless not optimized. To address this shortcoming, numerous mechanisms to modulate the fluorescence signal have been explored. We report herein newly synthesized probes based on self-immolative linkers containing chiral J-aggregate-forming dyes. Signal modulation by formation of chiral J-aggregates is yet unexplored in optical enzyme probe design. The comprehensive characterization of the probes by absorption, CD, fluorescence, and time-resolved fluorescence spectroscopy revealed dye-dye interactions not observed for the free dyes in solution as well as dye-protein interactions with the enzyme. This suggested that J-aggregate formation is challenging to achieve with current probe design and that interactions of the dyes with the enzyme may interfere with achieving high signal-to-background ratios. The detailed understanding of the interactions provided herein provides valuable guidelines for the future design of similar probes.

Recent trends in molecular aggregates: An exploration of biomedicine

“…In accordance with this, Hennig et al developed a new bioprobe containing a self-immolative linker and chiral J-aggregate-forming dyes. [35] Detailed characterization of the newly identified probes using fluorescence, absorption, CD, and time-resolved fluorescence spectroscopy revealed that the dye-dye and dye-protein interactions were not detected for the free dyes in the solution and enzyme, respectively. From the experimental results, it is clear that the lifetime of the probe is longer than that of the corresponding free dyes, which is probably due to the closer proximity of the self-immolative probe system and the hydrophobic microenvironment generated by the second dye.…”

Syntheses and kinetic studies of cyclisation-based self-immolative spacers

“…The resulting solution was stirred under argon at room temperature overnight. The solvent was evaporated, and then the residue was taken up in dichloromethane; the organic phase was washed with a solution of K 2 CO 3 , water and brine; dried on MgSO 4 , filtered and concentrated in vacuo. The final carbamates were purified by HPLC using 80 to 90% gradient of CH 3 (1) and (2) tert-Butyl(2-((4,5-dimethoxy-2-nitrobenzyl)oxy)-5-methoxyphenyl) carbamate (7).…”

“…The heterogeneous mixture was allowed to warm at room temperature and stirred 1 h 30 min under argon. The excess of LiAlH 4 was neutralized with NH 4 Cl at 0°C, and then the suspension was filtered on Celite 535; the resulting filtrate was diluted with EtOAc and treated with 1 M HCl, water and brine; and finally dried on MgSO 4 , filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel, giving the alcohol 18 as an off-white powder (479 mg, 94%); rf 0.…”

“…in 10 mL of dichloromethane was added pyridinium dichromate (2.66 g, 7.08 mmol, 4 eq.). The mixture was stirred under argon for 24 h, then treated with NH 4 Cl,…”

“…The second bond is cleaved spontaneously after cleavage of the trigger (Scheme 1). A variety of self-immolative spacers have been introduced over the years in the literature [2][3][4] and for instance used in cancer chemotherapy (Adcetris®). [5][6][7][8] In this strategy, the spontaneous release introduces a second step with its own kinetic parameters.…”