Molecular tools for fluorescent imaging of specific compartments in cells are essential for understanding the function and activity of cells. All calculations were done using SPSS Statistics 24. Results and discussion Synthesis and optical characterization of the phospholes For the AMG706 preparation of the phosphole derivatives, two structural motifs were selected, the thienyl, and the pyridyl unit. Pyridyl-substituted phospholes have started to find their way into biological and medicinal chemistry and the idea was to use phosphole 2b (Scheme 1) as a reference system since it has shown to have a low toxicity in the human breast cancer cell line MCF-7 (Viry et al., 2008). In addition, the thienyl substituent was chosen in order to have a probe with a more red-shifted spectrum compared to the pyridyl-substituted phosphole (Hay et al., 2001). The synthesis of the phospholes is presented in Scheme 1, and was performed in accordance with established procedures (Hay et al., 2001; Fadhel et al., 2009). Firstly, octadiynes 1a and 1b were synthesized through a Sonogashira cross-coupling reaction, with Pd(PPh3)2Cl2 and CuI as catalysts in triethyl amine. Secondly, the Fagant-Nugent approach was applied in order to form the phosphole (Fagan and Nugent, 1988; Fagan et al., 1994). In this reaction, a zirconium intermediate is formed, which converts to a phosphole upon addition of P,P-dichlorophenylphosphine (Hay et al., 2001; Fadhel et al., AMG706 2009). Phospholes 2a,b were filtered on basic alumina and in contrast to earlier reports, 2a,b were purified by flash chromatography on silica. This methodology decreased the yield, compared to the earlier reported methods, but was a convenient way to isolate 2a,b as yellow solids. In the last step, phospholes 2a,b were oxidized with elemental sulfur and thioxophosphole 3a was obtained as an orange solid and 3b as an orange to yellow solid. The structure of the synthesized compounds were confirmed with 1H-NMR, 13C-NMR, and 31P NMR (Supplementary Material). Next, the optical properties of the probes in different solvents were explored. For cell imaging using phosphole probes, phosphate buffer saline pH: 7.4 (PBS) with DMSO as co-solvent have been employed earlier (Viry et al., 2008; Wang et al., 2015). Therefore, the excitation- and emission characteristics of the newly synthesized phospholes were measured both in pure DMSO and in PBS with 0.7% DMSO (PBSD) as co-solvent. In DMSO, 2b and 3b exhibited excitation maxima at 377 and 374 nm, whereas 2a and 3a displayed excitation maxima at longer wavelength, 439 and 429 nm, respectively (Figure ?(Figure22 and Table ?Table1).1). In addition, 2a and 3a, displayed a red-shift of the excitation maxima in PBSD compared to pure DMSO. The thienyl associated red-shifts were also apparent when AMG706 comparing the emission characteristic of the phospholes. In DMSO, 2a and 3a exhibited emission maxima at 554 and 550 nm, whereas the pyridyl analogs, 2b and 3b showed emission maxima at shorter wavelength, 470 and 512 nm, respectively (Figure ?(Figure22 and Table ?Table1).1). Thus, in agreement with the chemical design, introduction of the thienyl substituent to the phosphole induced more red-shifted excitation and emission characteristics compared to the previously reported pyridyl moiety (Hay et al., 2001). Figure 2 Optical characterization of the phospholes. Normalized excitation spectra (left column) and emission spectra (right column) of probes 2a,b and 3a,b in phosphate buffer saline pH: 7.4 (PBS) with 0.7% DMSO as co-solvent (top) and DMSO (bottom). Normalization … Table 1 Excitation- GDF5 and emission maxima of probes 2a,b and 3a,b in PBSDa and DMSO. Staining of living cells with the phospholes Next, the non-fused phospholes were utilized to stain normal human skin fibroblasts (AG01518) and malignant melanoma cells (SK-MEL-28). Living cells were stained with 10 M phosphole probes in complete cell culture medium for 24 h.