Abstract:

A series of small-molecular fluorescent probes based on nitrogen-fused pyridazinone scaffold were developed in this report. The design strategy involved two steps: 1) enhancing the electron-withdrawing ability of the acceptor by incorporatingan N-heterocyclic aromatic ring (pyridine or pyrazine) at the C₄ and C₅ positions of the pyridazinone skeleton and 2) anchoring a triphenylphosphine or morpholine tail as the subcellular targeting group. These fluorescent probes displayed excellent properties in live cell and brain tissue imaging.

Key words: Nitrogen-fused pyridazinone, Fluorescent probe, Biological imaging, Design, Synthesis

CLC Number: 

• R916

Supporting:

Content

1. Density functional theory calculations

2. Photophysical properties

3. Live cell imaging

4. NMR data of isolated compounds

5. HRMS data of isolated compounds

6. Reference

 

 

1. Density functional theory calculations

Table S1. Calculated total energy of optimized structures for compounds PH, PY and 4a-4c.

 

 

Table S2. Atom coordinates for the optimized geometry of PH.

  

 

Table S3. Atom coordinates for the optimized geometry of PY.

 

 

Table S4. Atom coordinates for the optimized geometry of 4a.

 

 

Table S5. Atom coordinates for the optimized geometry of 4b.

 

 

Table S6. Atom coordinates for the optimized geometry of 4c.

 

 

 

2. Photophysical properties

Table S7. Structure and photophysical properties of PH, PY and 4a-4c in different solvents. ^[a]

 

[a] All data were measured at 298K. [b] λ_max values of the one-photon absorption and emission spectra in nm. [c] Stokes shift.  

 

Determination of the absorption spectra of PH, PY and 4a-4c in DMSO

 

Figure S1. Normalized absorption spectra of compounds in DMSO. 

 

Determination of the fluorescence properties of PH, PY and 4a-4c in different solvents

 

Figure S2. Normalized fluorescence excitation and emission spectra of compounds in DMSO.

 

 

Figure S3. Normalized fluorescence excitation and emission spectra of compounds in EtOH.

 

 

Figure S4. Normalized fluorescence excitation and emission spectra of compounds in PBS.

 

Table S8. Structure and photophysical properties of probes 6a-6c and 7a-7c in different solvents. ^[a]

 

[a] All data were measured at 298K. [b] λ_max values of the one-photon absorption and emission spectra in nm. [c] Stokes shift. 

 

Determination of the absorption spectra of 6a-6c and 7a-7c in DMSO

 

Figure S5. Normalized absorption spectra of compounds in DMSO. 

 

Determination of the fluorescence properties of 6a-6c and 7a-7c in different solvents

 

Figure S6. Normalized fluorescence excitation and emission spectra of compounds in DMSO.

 

 

Figure S7. Normalized fluorescence excitation and emission spectra of compounds in EtOH.

 

 

Figure S8. Normalized fluorescence excitation and emission spectra of compounds in PBS.

 

 

3. Live cell imaging

3.1 Cytotoxicity assay

 

Figure S9. MTT assay results of nitrogen-fused pyridazinone compounds (6a-6c and 7a-7c). Cells were incubated with different concentrations (0.5/1/2/4/6 μM) of compounds.

 

 

4. NMR data of isolated compounds

¹H and ¹³C NMR spectra of compound 2a

 

 

¹H and ¹³C NMR spectra of compound 2b

 

 

¹H and ¹³C NMR spectra of compound 2c

 

 

¹H and ¹³C NMR spectra of compound 3a

 

 

¹H and ¹³C NMR spectra of compound 3b

 

 

¹H and ¹³C NMR spectra of compound 3c

 

 

¹H and ¹³C NMR spectra of compound 4a

 

 

¹H and ¹³C NMR spectra of compound 4b

 

 

¹H and ¹³C NMR spectra of compound 4c

 

 

¹H and ¹³C NMR spectra of compound 5a

 

 

¹H and ¹³C NMR spectra of compound 5b

 

 

¹H and ¹³C NMR spectra of compound 5c

 

 

¹H and ¹³C NMR spectra of compound 6a

 

 

¹H and ¹³C NMR spectra of compound 6b

 

 

¹H and ¹³C NMR spectra of compound 6c

 

 

¹H and ¹³C NMR spectra of compound 7a

 

 

¹H and ¹³C NMR spectra of compound 7b

 

 

¹H and ¹³C NMR spectra of compound 7c

 

 

 

5. HRMS data of isolated compounds

HRMS spectra of compound 2a

 

 

HR-MS spectra of compound 2b

 

 

HRMS spectra of compound 2c

 

 

HRMS spectra of compound 3a

 

 

HR-MS spectra of compound 3b

 

 

HRMS spectra of compound 3c

 

 

HRMS spectra of compound 4a

 

 

HR-MS spectra of compound 4b

 

 

HRMS spectra of compound 4c

 

 

HRMS spectra of compound 5a

 

 

HR-MS spectra of compound 5b

 

 

HRMS spectra of compound 5c

 

 

HRMS spectra of compound 6a

 

 

HRMS spectra of compound 6b

 

 

¹HRMS spectra of compound 6c

 

 

HRMS spectra of compound 7a

 

 

HRMS spectra of compound 7b

 

 

HRMS spectra of compound 7c

 

 

 

6. Reference

[1] See ref ^[20].