I’ll preface my question by noting that I am strictly a synthetic organic chemist with limited knowledge of cell work. I did attend your talk at ACS.

Have you considered that attaching a large fluorescent label to a natural product before treating cells with it can significantly impact its lipophilicity and membrane permeability, thereby changing dramatically its localization within a cell? It seemed from your talk that you meant to design the labelling into a natural product synthesis (thereby making it the synthesis of a natural product analog, and not a natural product synthesis). I can only guess that this would mean attaching the label earlier than the final step (following a global deprotection that, as you deftly pointed out, leaves a number of synthetic handles with which to attach said label).

Secondly, regarding your point about “design(ing) a natural product de novo” in order to effect a desired biological response: isn’t that what medicinal chemists have been doing for years using HTS and SAR? Drug discovery has been around a long time. I don’t see how locating a molecule within a cell is more useful than the battery of assays against specific proteins that are run in high throughput screening. It may be a nice complement to those assays.

Philip

(the previous two anonymous posters were me. i’ll just go by cheese from now on.)

Hmmm… I may have missed the point of the first part of your talk. I understand the coumarin dye derivatives, that’s all fine. The mobile dye is neat, that’s fine, but I just couldn’t figure out what you were talking about with the Gambogic acid. I’m probably just missing the point so, please, what did you mean by that?

I’ll admit that my first question was a rumor, but as I heard it directly from my advisor, I thought I would ask.

As for my question #2, I think it is valid. You reported synthesizing the molecule on the gram scale. Now you have publically said you are re-making it. These seem incompatible.

I’d like nothing more than for you to come out correct (having made the compound you claimed — it would be a good lesson for the gosspimongers, me included), but the question as to why it needs to be re-made is still valid.

But back to your talk… Your cyclic/linear peptide localization work was interesting for sure. Could the peptides be used as localization tags (for macromolecules)? What is the end goal for this work (meaning: it looks very cool, but what are the applications)?

1) Sure u can use stains to determine the regions of the cell however the reason to make a natural product fluorescent is quite a different query. It is one method that one can use to determine the biological role of the natural product. By determining the localization of where a material goes in a cell allows one to determine what potential role a natural product has? For instance it is not likely that a DNA binding natural product will appear in the cell membrane rather one would see it in the nucleus.

2) There are a number of quite easy to use human cell lines. I usually end up using what the laboratory I am collaborating with has grown a use to using. When I do work myself and I am in the mood for exhaustive efforts I will use between 20-60 cell lines but for target work this is quite exhaustive. It’s best to start with a small set of cell lines that you are comfortable with culturing.

3) The idea of that presentation was to create thought not close it. If I gave u the answer you would not be able to make the question. There are a number of programs in the cell that movement would around the cell would be interesting to investigate for instance Golgi genesis.

Dear Mitch,

Thanks man…

Dear Anonymous,

1+2 I hope someday that we learn that rumors are not good for science.

3) Good question. Actually over time I screened a large set of dyes for their lack of biological activity and easy in synthetic manipulation. The DMC (coumarin) label is easy to make (at least 20+ labs have done it at the gram scale) and it can be manipulated easily. It is uncharged enters cells well and can be washed out of them.

Human cells have only modest blue fluorescence and backgrounds can always be eliminated with the proper blue filter sets (Semrock makes excellent ones). Also there are several controls (with other colors one can do when concerned). However the key to moving the cell to a fluorescent probe in my hands has been to stay with the same dye for as many studies as possible as each natural-product dye conjugate serves as a control for the next study.

1. Did the government really take your hexacyclinol product away?

2. If you made 3 grams of said product, why are you re-making it?

3. Why did you use coumarin (a blue dye) for your peptide work when the cells autofluoresce blue? Wouldn’t another dye be more appropriate? And why did you once use dimethyl amino coumarin and once diethyl amino coumarin?