Fluorescent labels such as fluorescein or rhodamine are sometimes incorporated at the N-terminus of peptides designed to enter a microbial cell. In a few cases fluorescent, non-naturally occurring side chains in peptides are used. A reference is given above. We synthesized two compounds with the expectation that each final product would be fluorescent. One reason to make these compounds is to facilitate measuring their uptake into microbial cells. I am not sure how our collaborators would know how to perform these measurements without some information, irrespective of whether or not the information is needed for publication. Another reason we made them is that they are similar to successful enzyme inhibitors that we previously made and might perform even better.
One reason to measure the fluorescence of the new molecules as a function of pH is that the interior of a cell does not have the same pH as its environment. Assays of our target enzyme are performed at fairly alkaline pH. It is within the universe of possibilities, that this assay (or other experiments) will be affected. Some days I wish I had made a molecule with fluorescent properties that are independent of pH in the range of interest. Today is becoming one of those days.
One biochemistry laboratory textbook (Ninfa, Ballou, and Benore) has the outline of an experiment to measure absorbance as a function of pH of 4-nitrophenol/4-nitrophenolate. This experiment was in the back of my mind as I thought how to perform a titration, with the first change being a switch to Good's buffers, as noted above. There are several fluorimeters in the department. Yet inasmuch as I know less practical spectrofluorimetry than spectrophotometry, that I decided to look into the matter. For example I learned that inelastic scattering can be accounted for by the solvent blank but elastic scattering cannot be (this is unlikely to come as a surprise to experienced workers in the area of fluorometry). I am concerned, however, that there are other problems, of which I am not yet aware. I hope that I have now explained our interest more fully.