[Babcock_Hall]

https://global.oup.com/academic/product/spectrophotometry-and-spectrofluorimetry-9780199638123?cc=us&lang=en&
We are making two novel compounds that appear to be fluorescent.  Each one has a pyridine group, a phenolic group, and a vinyl group, and they are isomers of each other.  Based on one of the parent compounds, loss of the phenolic hydrogen as a proton will change the fluorescent properties.  I found a book chapter that was helpful in introducing me to both theory and practice.  This chapter was in the book Spectrophotometry and Spectrofluorimetry, a Practical Approach, edited by Michael G. Gore.

My question is how do we characterize the fluorescence for publication and for use of the compounds?  For example, how can we study the fluorescence as a function of pH.  By analogy to spectrophotometric studies, I envision making a series of buffers (probably Good's buffers) and using a stock solution of a compound dissolved in an organic solvent, but surely there are additional things one needs to know.  Maybe someone has a publication in mind that would serve as a model.

[rolnor]

Is there conjugation between the pyridine and the vinyl group??

[rolnor]

It would be nice to titrate a solution of the compound with base and meassure the flourescens spectrum and pH in real time and plot these as a curve

[Babcock_Hall]

Is there conjugation between the pyridine and the vinyl group??

Yes, we converted an aldehyde into a vinyl group, and the aldehyde is known to be fluorescent.

[Babcock_Hall]

It would be nice to titrate a solution of the compound with base and meassure the flourescens spectrum and pH in real time and plot these as a curve

The absorption maximum will probably shift to longer wavelengths as pH is increased.  It seems to me that even if one kept the concentration constant, the absorbance will change, leading to differences in the inner filter effect from one pH to another.

BTW the book chapter by Arthur G. Szabo that I read, "Fluorescence Principles and Measurement," is very good in introducing the novice to things like the practical difference between Rayleigh and Raman scattering.

[rolnor]

Do you think the fluorescense will affect the assay? We had an XTT assay and a group of our compounds was yellow, the biochemists missed this and the compounds looked very active because in the assay a yellow compound is formed and this is meassured in a spectrophotometer. The yellow color of the tested compounds mimiced the compound that should form in the assay. I gave the data a quick glanse and I told them,"-This lookes like physics, not biochemistry" The data were to neat, to exact and lacked the variation you see in a cellculture assay. And I was right, when tested in another assay, the compounds were inactive.

[wildfyr]

I mean... there is an instrument called a fluorimeter. if someone in your dept owns one they will know the outline of a standard assay. At each pH you can scan where the absorption and emission peaks are. That about covers it.

Remember to do it very dilute and choose your solvent thoughtfully. Both have an effect on fluorescence.

As a molecule is deprotonated or protonated its absorption and emission properties will shift significantly. Sometimes fluorescence will cease depending on pH. I wouldn't call this earth shattering info...?

The most important versions of this are that when a phenol is deprotonated its quinone becomes favorable. And a non-protonated aniline can form an iminium.

[rolnor]

I agree wildfyr, this data is not very important?

[Babcock_Hall]

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9671271/
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.

[Babcock_Hall]

Do you think the fluorescense will affect the assay? We had an XTT assay and a group of our compounds was yellow, the biochemists missed this and the compounds looked very active because in the assay a yellow compound is formed and this is meassured in a spectrophotometer. The yellow color of the tested compounds mimiced the compound that should form in the assay. I gave the data a quick glanse and I told them,"-This lookes like physics, not biochemistry" The data were to neat, to exact and lacked the variation you see in a cellculture assay. And I was right, when tested in another assay, the compounds were inactive.

We made one inhibitor a while ago that absorbed, possibly near 400 nm.  This interfered with the enzyme assay, but my collaborator figured out a work-around, which I do not recall.  Thanks for the reminder.

[rolnor]

OK, great, so my comment was not completely unnecessary then.

[rolnor]

OK Babcock, then its very clear that this data is important.