Chiral HPLC Separation of Phosphonothrixin and Phosphonate Derivatives and Electronic Circular Dichroism Characterization of the Isolated Enantiomers

Phosphonothrixin (PPX), a phosphonate natural product known for its potent herbicidal activity, has attracted renewed interest because of the need for alternative herbicides. Its biological activity is confined to the (S)-enantiomer. Although the only reported enantioselective synthesis is complex, labor-intensive, and low yielding, direct chiral liquid chromatographic separation is likewise challenging because of its high polarity and lack of aromatic moieties, which limit interactions with commonly used chiral stationary phases (CSPs). In addition, phosphonic acid-containing compounds tend to chelate metal ions and adsorb to metal surfaces, further complicating chromatographic separation by causing pronounced peak tailing. Here, we describe a reliable and reproducible analytical and preparative method for the direct chiral separation of PPX using a zwitterionic sulfohexyl quinidine and quinine carbamate CSPs, as well as two synthetic phosphonate derivatives using a cellulose tris(3,5-dichlorophenylcarbamate) stationary phase as an alternative approach. The best separation of PPX was achieved with the zwitterionic sulfohexyl quinidine CSP (Rs = 2.4). The separated enantiomers were isolated, and their chemical and stereochemical identities were confirmed by mass spectrometry, optical rotation, and electronic circular dichroism spectroscopy. This approach enabled the successful isolation of enantiomerically pure (S)-(-)- and (R)-(+)-PPX.