I tried to produce TNB [5-thio-2-nitrobenzoic acid] from DTNB [5,5′-dithiobis(2- nitrobenzoic acid)] by alkaline hydrolysis following instructions of some studies : 10 mM DTNB solution made up to pH>12 by adding NaOH and then titrated back to pH 7 with HCl but I can't stabilize the pH at 7 and I have a poor product performance.
Dear Nathan,
you could try the procedure we published in this paper [Synlett 2016, 27, 2447–2450]
Dear Nathan Nicolau-Guillaumet a good description of the preparation of 5-thio-2-nitrobenzoic acid from its oxidation product DTNB [5,5′-dithiobis(2- nitrobenzoic acid)] can be found in the following article:
Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically-relevant oxidants peroxynitrite anion, hydrogen peroxide and hypochlorous acid
Article Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically...
Reference 17 in this paper refers to the following original research article:
A Spectrophotometric Assay for Allicin and Alliinase (Alliin lyase) Activity: Reaction of 2-Nitro-5-thiobenzoate with Thiosulfinates
Article A Spectrophotometric Assay for Allicin and Alliinase (Alliin...
Fortunately this paper has been posted by the authors as public full text on RG. Thus you can freely download it as pdf file. The synthetic is pretty much the same in both references. In both cases the pure acid can be isolated in the form of orange crystals.
By the way: I don't think that you can prepare 5-thio-2-nitrobenzoic acid simply by alkaline hydrolysis of DTNB. Please keep in mind that DTNB is the oxidation product of 5-thio-2-nitrobenzoic acid. Thus you formally need to reduce the S–S bond in DTNB and then add e.g. HCl to generate the free acid.
Good luck with your experiments and best wishes!
I think your approach to splitting the DTNB is fine, I've certainly seen high absorbance at 412 nm after treatment with NaOH. If you are struggling to hold the pH at 7 you may need to introduce a strong buffer with a pKa of around 7 to stop any overshoot when you add acid. However, I think you will struggle to prevent re-oxidation upon storage.
I have not actually tried this with DTNB, but a strategy commonly used to stabilise thiols is to acidify the solution (pH 2 for example) to create unreactive -SH groups (Only S- can re-oxidise). So the problem may be the fact you are trying to neutralise. You could potentially keep small aliquots frozen at pH2 and then neutralise with a powerful buffer when the material is needed.
Finally, i think you can actually purchase the material you are trying to make. Again, you will need to stabilise the thiol, so I'd take it up in acidic solution.
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