Synthesis and spectroscopic characterization of fluorescent boron dipyrromethene-derived hydrazones

Abstract

Derivatives of 4,4-difluoro-4-bora-3a,4a,diaza-s-indacene (BODIPY® or BDP) that possess a hydrazine substituent on position 5 are potential "turn-on" fluorophores for labeling aldehydes The unnatural amino acid L-3-formyltyrosine can be incorporated into a protein or peptide; thus, these hydrazines are potentially site specific labels for such polymers. In this work, model compounds were synthesized to assess whether the photochemical properties of the BDP-hydrazone would be suitable for protein labeling. Hydrazones were synthesized from the fluorophore 3-chloro-5-hydrazino-BDP and different aldehydes, and the absorption and emission spectra of the products were compared. The hydrazone of an unsubstituted aromatic aldehyde displays absorption and emission maxima (531 nm and 559 nm, respectively in dioxane) that are red shifted relative to those of a hydrazone from an aliphatic aldehyde (513 nm and 543 nm, respectively, in dioxane) and an increased quantum yield (0.21 vs. 0.11, respectively, in dioxane). The presence of a hydroxyl group ortho- to the aldehyde produces a hydrazone in which the absorption and emission maxima are slightly red shifted (528 nm and 564 nm, respectively in dioxane) from the unsubstituted aromatic hydrazone, but the quantum yields of the two hydrazones are equivalent. Thus, an ortho-hydroxy substituted aromatic aldehyde is a suitable electrophile for "turn on" protein labeling using the hydrazino-BDP. The specificity of this labeling reaction for the unnatural amino acid was demonstrated through fluorescent labeling of just the 3-formyltyrosine-containing α-subunit of α,β-tubulin.

Fig. 1

Structures of BDP derivatives

Fig. 2

Structures of aromatic aldehydes and hydrazone tautomers

Fig. 3

Normalized absorption spectra of BDP hydrazones 5 (panel A) and 6 (panel B) in dioxane (black) and methanol (red)

Fig. 4

Normalized fluorescence emission spectra of 2–6 in dioxane. From left to right, compound 2 (black), compound 3 (red), compound 4 (blue), compound 5 (green), compound 6 (light blue). Molecules were excited at their absorption maxima at room temperature (23°C)

Fig. 5

a Absorption difference spectra for the reaction of 650 μM salicylaldehyde with 65 μM BDP 2 in 0.1 M phosphate buffer at pH 4 at room temperature. Spectra shown were collected immediately after mixing and at 30 s intervals thereafter. b Plot of the change in absorbance at 550 nm as a function of time. Solid line: fit of the data as a pseudo-first order reaction

Fig. 6

a Absorption difference spectra for the reaction of 2 mM propanal with BDP 2 in 0.1 M phosphate buffer at pH 4 at room temperature. Spectra shown were collected immediately after mixing and at 30 s intervals thereafter. b Change in absorption at 550 nm as a function of time. Solid line: Data fit as a pseudo-first order reaction. c Change in absorption at 482 nm as a function of time. d Data from panel C subtracted from data in panel B. Solid line: Data fit as a pseudo-first order reaction

Fig. 7

SDS-PAGE of tyrosinated tubulin (Lane 1) and 3-formyltyrosinated tubulin (Lane 2) after treatment with BDP 2. The gel was photographed in Panel A under irradiation with long wavelength UV light and in Panel B after staining with Coumassie blue