Evaluation of asymmetric orthogonal cyanine fluorophores

Pentamethine cyanine (Cy5) fluomphores have proven to be versatile imaging agents (i.e., tracers) for a range of micro- and macroscopic imaging applications, including image-guided surgery. In this study the relationship between the structure of asymmetric Cy5 fluorophores and their photophysical properties was studied. To this end, seven Cy5 analogues, bearing orthogonal N-indole substituents (H, SC3-, 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 . 10(3) M-1 cm(-1)), photostability (24.4-90.6% remaining), and stacking tendency. Chemical stability was uninfluenced by the substitution pattern. Additionally, the generation of a c[RGDyK]-based hybrid tracer based on one of these fluomphores in combination with a diethylenetriaminepentaacetic acid (DTPA) chelate and an In-111-isotope was reported. This compound was evaluated in vitro using alpha(v)beta(3)-overexpressing Ge beta 3 cells and in vivo using a 4T1 mouse tumour model. Overall, the presented results imply that alterations of the asymmetrical orthogonal Cy5 fluomphore structure have impact on the (photo)physical properties. Furthermore, the orthogonal Cy5 fluorophore framework can readily be applied in tracer development.Radiolog