Evaluation Of Asymmetric Orthogonal Cyanine Fluorophores

Abstract Pentamethine cyanine (Cy5) fluorophores have proven to be versatile imaging agents (i.e., tracers) for a range of micro- and macroscopic imaging applications, including image-guided surgery. We studied the composition of asymmetric Cy5 fluorophores in relation to the photophysical properties. To this end, seven Cy5 analogues, bearing orthogonal N-indole substituents (H, SO3-, or benzene) were synthesised and evaluated. In-depth analysis revealed that introduction of sulfonates enhanced the fluorescence brightness and photostability, while reducing the lipophilicity, serum binding and stacking tendency. The addition of benzene moieties induced a bathochromic shift of 10–20 nm, increased the lipophilicity (LogP = -1.56–1.23) and serum binding (67.3–93.8% bound), as well as negatively impacted the brightness (0.74–42.9 · 103 M-1 · cm-1), photostability (24.4–90.6% remaining), and stacking tendency. Chemical stability was uninfluenced by the substitution pattern. Additionally, we report the generation of a c[RGDyK]-based hybrid tracer based on one of these fluorophores in combination with a diethylenetriaminepentaacetic acid (DTPA) chelate and an 111In-isotope. This compound was evaluated in vitro using αvβ3-overexpressing Geβ3 cells and in vivo using a 4T1 mouse tumour model. Overall, our results imply that alterations of the asymmetrical orthogonal Cy5 fluorophore composition have impact on the (photo)physical properties. Furthermore, the orthogonal Cy5 fluorophore framework can readily be applied in tracer development.