[Arunas77]

Hi,

I'm studying an enzymatic hydroxylation of 3-(2,4-dihydroxyphenyl)propionic acid. Hydroxylation is done in aromatic ring (conversion yeald is max ~30 %, inhibition of enzymes), we confirmed the mass of hydroxylated product by LC-MS. Also, product oxidates to chinone (red soluton), we think it's ortho-chinone (hydroxylation on C5). But it is impossible to separate product from substrate for structure determination. We tried methylation of all hydroxy groups, but we get mixtures of mono-, di-, or tri-methylated products. Also, we tried various amines which would react with chinone, but we don't get any helpful results.

Any suggestions from real organic chemists on how to chemically modificate the product, so it would be possible to separate from substrate? And modifications should not be done under hard conditions.

Thanks,
Biochemist with only little knowledge of organic chemistry

[pgk]

Indeed, it's difficult to separate because  quinones form dimers with their corresponding hydroquinones, in mixtures.
Anyway, try acetylation with acetic anhydride. Phenol hydroxyl groups will be acetylated as usual, but quinone will give the corresponding 3-acetyl-2,4,5- trihydroxyphenyl triacetate derivative (Thiele-Winter acetylation of quinones). So, you can determinate the structure.
Org. Syn. Coll. Vol. 1, p. 360, (1941)
http://www.orgsyn.org/demo.aspx?prep=CV1P0317