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StructuralGenomicsConsortium / CNP16-iminoquinone-SarsRdRp-mechanstic-study

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using N-acetyl cysteine as the substrate #1

Closed qxsml closed 3 months ago

qxsml commented 10 months ago

Hi anyone,

I have a concern about the reaction using free cysteine with the iminoquinone compounds. Below is a reminder of the question:

the question

I feel I should protect the amine group on the free cysteine, just in case.

Below is my thought:

I assume that the addition of the thiol to the quinone ring is the reaction that happened. Imaging I deliberately synthesised this addition product and obtained its retention time on the HPLC trace. Then I conduct the reaction under the buffer conditions, I should be able to observe this product peak. But what if the free amine also reacted with the ring, as a minor side reaction, I would get a peak that I have to explain what it is.

Also, I suppose cysteine is not the N-terminal of the RdRp protein right? So, its amine is not free.

I bought N-acetyl cysteine, just in case. But hope to consult your opinions. Please let me know what you think.

Thank you very much! @mattodd @tmw20653 @ahsgc @RebeckaIsaksson @ahsgc @alvaro-magalhaes @HadiaAmahli

tmw20653 commented 10 months ago

Xin, check the literature. I am pretty sure these cys capture experiments are typically done with free cysteine and not the protected versions. Tim

qxsml commented 10 months ago

Thank you! Yes, I read a paper that used free cysteine with o-quinone to investigate the regiochemistry. Above is my concern, thinking that the amine is also a good nucleophile.

tmw20653 commented 10 months ago

Try both cyseine and N-Ac cysteine. See if you get a single product. You should be able to tell the difference between SH or NH addition by NMR.

mattodd commented 10 months ago

@qxsml see comments coming in re use of glutathione itself as surrogate. https://twitter.com/MatToddChem/status/1724101005418041575

chriswaudby commented 10 months ago

Should be easy to work out by NMR - could even do time-resolved measurements if you want to check the relative reaction rates

mattodd commented 7 months ago

@qxsml any updates here to the question of which reagent to use to study the SH capture? Which ones have you now bought/made/used?

qxsml commented 2 months ago

After a few trial reactions, N-acetyl cysteine methyl ester was used as the proxy for the reactive SH groups.