4-Azidocinnoline-Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe

Abstract

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide-amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.

Keywords: AIE and ESPT fluorescence mechanisms; amines; azides; cinnolines; environment-sensitive probe; fluorescence; fluorochromic probe; fluorogenic probe; richer cyclization.

Figure 1

Known examples of fluorogenic azide probes based on their conversion to 1,2,3-triazoles (A), amines (B) and the target structure of the current research (C).

Scheme 1

Synthesis cinnoline-4-amines.

Figure 2

Absorption spectra of the solutions of azide 5 and amine 6 in THF (c = 1 × 10⁻⁴ mol/L) (left); absorption spectra of the amine 6 solutions in DMSO, H₂O, MeCN, i-PrOH and THF, (c = 1 × 10⁻⁵ mol/L) (right).

Figure 3

Emission spectra of solutions of azide 5 and amine 6 in THF, c = 1 × 10⁻⁵ mol/L (left); emission spectra of the solutions of amine 6 in different solvents, c = 1 × 10⁻⁵ mol/L (right).

Scheme 2

The proposed emission mechanism involving intramolecular charge transfer (ICT) and excited state intermolecular proton transfer (ESPT).

Figure 4

Emission spectra of solutions of azide 5 (left) and amine 6 (right) c = 1 × 10⁻⁵ mol/L in corresponding solvents and in water with 1% v/v of the corresponding solvent.

Figure 5

Emission spectra of solutions of amine 6 in DMSO (1%)/H₂O, c = 1 × 10⁻⁵ mol/L (left) and in i-PrOH (1%)/H₂O, c = 1 × 10⁻⁵ mol/L (right) before and after the filtration through nylon and PTFE membrane filters.

Figure 6

Emission spectra of solutions of amine 6 in i-PrOH/H₂O, c = 1 × 10⁻⁵ mol/L with different water fraction (f_w) (in vol%) (left); plot of maximum intensity versus f_w (right). Water fraction (f_w) is the fraction of water in vol% in the solvent used for the preparation of corresponding solutions of amine 6, c = 1 × 10⁻⁵ mol/L from the stock solution of amine 6, c = 1 × 10⁻³ mol/L in i-PrOH.

Figure 7

Illustration of the ability of amine 6 to enter HepG2 cells and the possibility of the conversion of azide 5 into amine 6 in vitro: (A) visible light and fluorescence microscopy of control probe (HepG2 cells), HepG2 cells treated with amine 6 (10 μM) and HepG2 cells treated with azide 5 (10 μM); (B) FACS analysis of control, amine 6- and azide 5-treated probes.

Scheme 3

The proposed tautomeric forms (A and Q) of amine 6 for the associates of amine 6 with water molecules responsible for the fluorogenic properties of amine 6 through the cooperation of AIE and ESPT mechanisms.