Balance between Fluorescence Enhancement and Association Affinity in Fluorescent Heteroditopic Indicators for Imaging Zinc Ion in Living Cells

Abstract

A fluorescent heteroditopic indicator for zinc(II) ion possesses two different zinc(II) binding sites. The sequential coordination of zinc(II) at the two sites can be transmitted into distinct fluorescence changes. In the heteroditopic ligand system that our group developed, the formations of mono- and di-zinc(II) complexes along an increasing gradient of zinc(II) concentration lead to fluorescence enhancement and emission bathochromic shift, respectively. The extents of these two changes determine the sensitivity, and ultimately, the effectiveness of the heteroditopic indicator in quantifying zinc(II) ion over a large concentration range. In this work, a strategy to increase the degree of fluorescence enhancement upon the formation of the mono-zinc(II) complex of a heteroditopic ligand under simulated physiological conditions is demonstrated. Fluorination of the pyridyl groups in the pentadentate N,N,N′-tris(pyridylmethyl)ethyleneamino group reduces the apparent pKa value of the high-affinity site, which increases the degree of fluorescence enhancement as the mono-zinc(II) complex is forming. However, fluorination impairs the coordination strength of the high-affinity zinc(II) binding site, which in the triply-fluorinated ligand reduces the binding strength to the level of the low-affinity 2,2′-bipyridyl. The potential of the reported ligands in imaging zinc(II) ion in living cells was evaluated. The subcellular localization properties of two ligands in five organelles were characterized. Both benefits and deficiencies of these ligands were revealed which provides directions for the near future in this line of research.

5-Arylvinylene-2,2′-bipyridyls: Bright “push–pull” dyes as components in fluorescent indicators for zinc ions

“…To rectify this situation, the tridentate DPA is replaced by a pentadentate ligand that contains two fluorinated pyridyl groups in compound 35 (Fig. 12b) [107]. A clear-cut two-segment fluorescence change of 35 was observed in an aqueous solution buffered at neutrality.…”

Zn(ii)-coordination modulated ligand photophysical processes – the development of fluorescent indicators for imaging biological Zn(ii) ions

“…30) and the emission band shift (Δλ) when Zn(II) coordinates at the low-affinity site (LF) are the parameters for evaluating a fluorescent heteroditopic ligand. 234 The choice of the aryl ring on the fluorophore (e.g., phenyl in 31 ), affects both factors in conflicting ways. 238 Increasing the electron density of the aryl ring (e.g., from phenyl to thienyl) creates a larger emission band shift (Δλ) because the charge transfer character of the fluorophore is strengthened.…”

“…233,234 Compound 32 (Scheme 11) undergoes a more than 5-fold fluorescence enhancement followed by emission bathochromic shift over a range of 0.1 nM to 0.1 mM of free Zn(II). 234 The deficiency of this compound is still the emission band separation, which is too small (32 nm) for the two bands to be captured by two different emission filter sets, for example, blue (i.e., 420 – 480 nm) and green (i.e., 510 – 560 nm).…”

“…Occasionally, for any reason that the K d of the indicator under investigation is not fittable using any single metal-buffering ligand, a small range of the K d of the indicator between those of two metal-buffering ligands may be reported. 234 …”

“…235 (b) A modified fluorescent heteroditopic ligand 32 with a pentadentate high-affinity Zn(II) ligand. 234 …”