Figure 2.

Molecular platform of 9’-phenyl-3’-hydroxyfluorone-, rosamine-, and rosol-based fluorescent dyes, their analogues having a pendant benzene moiety with a charged 2-carboxylate group (-COO^-), each respective initial pK_a value, and the off-on fluorescence intensity-pH profile of the molecular platforms having a 3’- amino-6’-hydroxyxanthene-based scaffold and the maximum contrast level they would afford upon use toward visualizing tumor- draining lymphatics. A) The presence of the charged 2-carboxylate group imparts an increased pK_a value to their respective underlying xanthene core-based scaffold. B) The off-on fluorescence intensity- pH profiles of a generic rosol (X) and rhodol (Z) dye with a pK_a value of ~4.8 and ~6.3, respectively, and a proposed rosol dye (Y) designed with a pK_a value of 5.8–6.0. The dashed red lines circumscribe the pH level range that a fluorescent molecular dye having a pK_a value within them would afford optimal contrast upon use in lymphatic mapping applications. The circle identifies the pK_a value. C) Only the proposed rosol dye (Y) designed to have a pK_a value of 5.8–6.0 would afford a steady maximal response and optimal contrast. A solid or striped intensity ratio bar indicates that the dye would be either pH-insensitive or pH-sensitive when within tumor-draining lymphatic pH levels (pH 6.8–7.4), respectively.