Developing visible fluorogenic 'click-on' dyes for cellular imaging

Abstract

New fluorogenic dyes were designed and synthesized based on Cu(I)-catalyzed 'click' reaction. Conjugating weakly fluorescent benzothiazole derivatives with an electron-deficient alkyne group at the 2-position with azide-containing molecules in aqueous solution form 'click-on' fluorescent adducts. Model reactions and cell culture experiment indicated that the developed 'click-on' dye could be applied to labeling various biomolecules, such as nucleic acids, proteins, and other molecules, in cells.

Figure 1

Example for existing fluorogenic dyes

Figure 2

Emission spectra of benzothiazole derivatives (10μM) in 3ml pH=7 buffer solution containing: (a) Compound 10 (blue) and 18b (red) (λex=316nm); (b) Compound 16 (blue) and 19b (red) (λex=375nm).

Figure 3

Effect of pH on fluorescence intensity ratio of benzothiazole derivatives in different pH PBS buffer. Condition: (a) compound 10 (Brown) and 18b (Cyan), λex: 316nm, 10μM. (b) compound 16 (Brown) and 19b (Cyan), λex: 375nm, 10μM.

Figure 4

‘Click on’ fluorescence images of cells (A), and quantitation of fluorescent signal (B) after reacting with compound 16 (10 μM) in HeLa cells (x20). The fluorescent signal was imaged using the convention DAPI filter set.

Scheme 1

Reagents and conditions: (a) 1. p-MeC₆H₄SO₃H, MeCN, RT-10°C, 2. NaNO₂, H₂O, 10min, 10-15°C. 3 KI, RT, overnight. (b) 1.ethynyltrimethylsilane, CuI, PdCl₂(PPh₃)₂, NEt₃, DCM, 2. NaHCO₃, H₂O. (c) KF, MeOH, RT, 3h.

Scheme 2

Reagents and conditions: (a) HNO₃, H₂SO₄. (b) Fe, EtOH, AcOH, 55°C, 3h. (c) 36% formaldehyde, DCM, NaBHAc₃, HOAc, RT, 2h. (d) 1.ethynyltrimethylsilane, CuI, PdCl₂(PPh₃)₂, NEt₃, DCM, 2. NaHCO₃, H₂O. (e) KF, MeOH, RT, 3h.

Scheme 3

Model ‘Click’ reactions Reagents and conditions: CuSO₄, sodium ascorbate, TBTA, DMF/H₂O(3/1), RT, 30min