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.