Derivatization of carboxyl groups

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

3 posts Page 1 of 1
Hi everyone,

I'm new in this forum, so I'll quickly introduce myself and my research: I'm doing my PhD in the Department of Plant Systems Biology in Ghent, Belgium. I'm working on the discovery of plant genes that are involved in the biosynthesis of so-called saponins. Those are steroidal or triterpenoidal structures with hydroxy and carboxy functions on different positions. In planta, these saponins occur with complex glycosylations.
For an experiment, I would like to specifically derivatize the carboxy functions present in a monoglycosylated analyte (of which I don't know the exact chemical sttructure) for reverse phase HPLC analysis. The nature of the derivatization is not of importance to me, as I only hope to get structural information of the analyte (i.e. how many carboxy groups are present?)
The following criteria would have to be met:
-The derivatization mustn't hydrolyze the O-glycosidic bond between sugar and triterpene aglycone,
-It should be specific for carboxy groups and musn't react with OH groups, and
-I'm working in a biochemistry lab. We don't have facilities and equipment to deal with hazardous chemical compounds. A compound which isn't too toxic and is easy to use would be nice (something like TMS derivatization for GC).

I browsed the internet a bit, but couldn't really come up with anything. Most of the alkylation reagents seem to be very toxic (for understandable reasons). Would something like boron trifluoride propanol be an option?

Thanks a lot for your help!

Greets,
Philipp
For carboxylic acids I have two derivatizing reagents I use. I use alcohol with concentrated HCl diluted to 1.2%
and a temperature of 100 deg C for 2 hours. Then I just add some water saturated with NaHCO3 and salt and take the top organic layer. I use isopropanol as the derivative of small or hydroxy acids are nonpolar enough not to tail on a db5 column. This will hydrolyze ester bonds I am uncertain about glycosidic bonds though.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817593/

The other reagent I use is the alkyl chloroformates. I prefer ethyl chloroformate ethanol for amino acids and large phenolic compounds. It wont simply derivatize caboxylic acids but amines, thiols, some phenolic hydroxyls etc but it doesn't hydrolyse ester or glycosidic bonds. They are pretty darn toxic but I use only very minute amounts (I do the reaction typically about 200ul using about 20ul of chloroformate) in a small 2ml autosampler tube. Any chemicals you need to dispose of can simply be left in the hood until it is gone.
Hey,

thank you for your reply! The esterification would be nice and easy, but I'm afraid my O-glycosidic bond will suffer from the acidic conditions. I may try it out anyway..
The ethyl chloroformate sounds interesting. Do you know whether the reaction product will be stable in water? I wouldn't be surprised if it were prone to hydrolysis.

Thank you for your help!

Philipp
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