Metal-catalyzed uncaging of DNA-binding agents in living cells

Abstract

Attachment of alloc protecting groups to the amidine units of fluorogenic DNA-binding bisbenzamidines or to the amino groups of ethidium bromide leads to a significant reduction of their DNA affinity. More importantly, the active DNA-binding species can be readily regenerated by treatment with ruthenium catalysts in aqueous conditions, even in cell cultures. The catalytic chemical uncaging can be easily monitored by fluorescence microscopy, because the protected products display both different emission properties and cell distribution to the parent compounds.

Fig. 1

Structure of selected DNA binders: pentamidine (1), propamidine (2), phenyl azapentamidine (3), DAPI (4) ethidium bromide (5).

Fig. 2

Top: Structures of the alloc (©3), and allyl (6) protected derivatives of bisbenzamidine 3. Bottom: fluorescence emission of 5 μM solutions of 3 and ©3 in Tris-HCl 20 mM, 100 mM NaCl, pH 7.5 (dashed lines), and in the presence of 1 equiv. of h-A3·T3 (solid lines). h-A3·T3: 5′-GGC-AAATTT-CAGTTTTTCTG-AAATTT-GCC-3′.

Fig. 3

Top: catalytic deprotection in phosphate buffer and 10% DMSO. Bottom: HPLC trace of the uncaging reaction in phosphate buffer 100 mM pH 7.5, and 10% DMSO. (a) ©3 (75 mM); (b) ©3 + PhSH (325 mM); (c) ©3 + PhSH + [Ru] (10%), t = 1 min; (d) ©3 + PhSH + [Ru], t = 20 min.

Fig. 4

Top: Structure of ©4. Bottom: fluorescence spectra of DAPI (4) and ©4 in the absence (dashed lines) and in the presence of 1 equiv. of h-A2·T2 (solid lines). Experiments were carried out using 0.2 μM of ©4 and 4 in TrisHCl 20 mM, 100 μM NaCl, pH 7.5 at 20 °C. Sequence of: h-A2·T2: 5′-GGCG AATT CAGCTTTTTGCTG AATT GCC-3′.

Fig. 5

Top: Structure of ©5. Bottom: fluorescence emission spectra of 1 μM 5 and ©5 (dashed lines), and spectra of both compounds in the presence of 0.3 mg mL ⁻¹ of ct-DNA (solid lines). Spectra of 5 and of 5 in the presence of ct-DNA are represented multiplied by a factor of 10 for comparison. λ_exc 5 = 545 nm, λ_exc ©5 = 300 nm.

Fig. 6

HPLC trace of the deprotection reaction of ©5. (a) ©5 in buffer (50 mM); (b) ©5 + PhSH (250 mM) + ct-DNA (0.1 mg ml ⁻¹); (c) ©5 + PhSH + ct-DNA + Ru (10%) t = 0; (d) ©5 + PhSH + ct-DNA + Ru t 20 min; (e) ©5 + PhSH + ct-DNA + Ru t = 1 h. Inset: picture (left to = right) of the aliquots a, b, c and e.

Fig. 7

Ru-catalyzed deprotection of 2.5 μM ©4 in CEF cells: (a) ©4, 30 min at 37 °C, blue channel; (b) ©4, 30 min at 37 °C, brightfield; (c) ©4 treated with 2.5 μM [Ru] and 100 μM PhSH, 8 min, blue channel; (d) ©4 treated with 2.5 μM [Ru] and 100 μM PhSH, 8 min, brightfield; (e) ©4 treated 20 min with 2.5 μM [Ru] and 100 μM PhSH, 20 min, blue channel; (f) ©4 treated 20 min with 2.5 μM [Ru] and 100 μM PhSH, 20 min, brightfield. (g) control with 2.5 μM 4 after 30 min at 37 °C, blue channel; (h) control with 15 μM 4 after 30 min, 37 °C, blue channel. Blue channel fluorescence microscopy settings: excitation filter 360–370 nm, emission filter 420 nm and dichromatic mirror 400 nm.

Fig. 8

Ru-catalyzed deprotection of 2.5 μM of ©4 in Vero cells. Top row: ©4, 30 min at 37 °C (a) blue channel; (b) brightfield. Bottom row: ©4 with 2.5 μM [Ru] and 100 μM PhSH, 20 min; (c) blue channel; (d) brightfield.

Fig. 9

Ru-catalyzed deprotection of 10 μM ©5 in CEF cells. Left column: ©5, 30 min at 37 °C; (a) green channel; (b) brightfield; (c) red channel. Right column: ©5 incubated with 20 μM [Ru] and 100 μM PhSH, 20 min; (d) green channel; (e) brightfield; (f) red channel. Fluorescence microscopy settings for green channel: filter 530–550 nm, emission filter 590 nm and dichromatic mirror 570 nm; and for red channel: filter 530–550 nm, emission filter 590 nm and dichromatic mirror 570 nm.