Design and synthesis of a new class of membrane-permeable triazaborolopyridinium fluorescent probes

Abstract

A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.

Figure 1

Structures of Representative Fluorescent Dyes

Figure 2

Structures of 5-substituted HPY Dyes 9, 10, 12 and 14

Figure 3

Membrane permeability of 16 and 21 in living HeLa cells. HeLa cells were incubated in 20 μM 16 (A–C) or 21 (D–F) for 1 hour, washed free of the dye, and imaged live. Additionally, 100 μg/mL AlexaFluor546-labeled bovine serum albumin (BSA) was included as a marker for fluid phase endocytosis panels B and E. Bar, 20 μm.

Figure 4

Absorption and emission spectra of (a) 25 (λ_max = 400 nm, ε = 9 900, λ_emi = 489 nm, Φ_f = 0.31) and (b) 26 (λ_max = 400 nm, ε = 12 900, λ_emi = 486 nm, Φ_f = 0.29). Samples (20 μM) were prepared in 1% DMSO/PBS, pH 7.4 at 25 °C and excited at 405 nm.

Figure 5

Fluorescent cysteine conjugates retain KSP inhibitory activity. HeLa cells were incubated in the absence or presence of 10 μM 19, or conjugates 25, 26, and 28 for four hours, and then fixed and probed for tubulin (red) and DNA (blue) localization. Bar, 10 μm.

Figure 6

Imaging of fluorescent cysteine conjugates in living cells. HeLa cells were treated with fluorescent conjugates of 10 μM 25, 26, and 28, and co-incubated with fluid-phase endocytosis marker AlexaFluor546-conjugated BSA for four hours, and then imaged using wide-field epifluorescence. Fluorescence was confined to punctate structures colocalizing with the endocytosis marker (BSA) in cells treated with 28, whereas, cells treated with conjugates 25 and 26 displayed diffuse fluorescence throughout the cytoplasm. Bar, 20 μm.

Scheme 1

Synthesis of Triazaborolopyridinium HPY Dyes

Scheme 2

Synthesis of HPY Dyes 16 and 18

Scheme 3

Synthesis of HPY Dyes 21 and 22

Scheme 4

Synthesis of Oxazolidine-2,5-dione 20

Scheme 5

Synthesis of HPY-KSP Inhibitor Conjugates 25 and 26

Scheme 6

Synthesis of AlexaFluor488-KSP Inhibitor Conjugate 28

Scheme 7

Acid Stability Study of AlexaFluor488-KSP Inhibitor Conjugate 28