Synthesis and spectral characterization of a long-lifetime osmium (II) metal-ligand complex: a conjugatable red dye for applications in biophysics

Abstract

There is a need for luminescent probes, which display both long excitation and emission wavelengths and long decay times. We synthesized and characterized an osmium metal-ligand complex which displays a mean decay time of over 100 ns when bound to proteins. [Os(1,10-phenanthroline)2(5-amino-1,10-phenanthroline)[(PF6)2 can be excited at wavelengths up to 650 nm, and displays an emission maximum near 700 nm. The probe displays a modest but useful maximum fundamental anisotropy near 0.1 for 488-nm excitation, and thus convenient when using an argon ion laser. [Os(phen)2(aphen)](PF6)2 is readily activated to the isothiocyanate for coupling to proteins. When covalently linked to bovine serum albumin the intensity decay is moderately heterogeneous with a mean decay time of 145 ns. The anisotropy decay of the labeled protein displays a correlation time near 40 ns. This relatively long lifetime luminophores can be useful as a biophysical probe or in clinical applications such as fluorescence polarization immunoassays.

Fig. 1.

Spectral characteristics of [Os(phen)₂(aphen)](PF₆)₂ complex in glycerol. The absorption spectra (dotted line) and emission spectra (dashed line) were collected at 23°C. The excitation anisotropy (solid line, λ_em = 680 nm), and emission anisotropy (opened circles, λ_exc = 580 nm) spectra were measured at −65°C.

Fig. 2.

Frequency-domain intensity decay of [Os(phen)₂-(aphen)](PF₆)₂ complex in glycerol (closed circles) and acetonitrile (opened circles), room temperature, air. The solid lines show the best single decay time fit in acetonitrile, and the best two-decay time fit in glycerol.

Fig. 3.

Comparison of frequency-domain intensity decay of [Os(phen)₂(aphen)](PF₆)₂ in aerated (closed circles) and deoxygenated (opened circles) water solution in room temperature. The solid lines show the best three decay time fits to the data for the aerated sample, and the best four decay time fits for the deareated sample (Table 1).

Fig. 4.

Frequency-domain lifetime data of [Os(phen)₂-(aphen)](PF₆)₂ covalently attached to BSA, 50 mM Tris buffer (pH 9.0). The solid line shows the best three-decay time fit to the data.

Fig. 5.

Frequency-domain anisotropy decay measurements of [Os(phen)₂(aphen)](PF₆)₂ covalently attached to BSA, 50 mM Tris buffer (pH 9.0).

Scheme 1.

Chemical structure of [Os(phen)₂(aphen)]²⁺.