Enzymes and chirality are intimately associated. For their mechanisms to be studied, chiral substrates are needed as probes. Here, we report a concise synthesis of (R)- and (S)-[O,O,O]phosphoenol pyruvate starting from enantiomerically pure (R)-2-chloro-1-phenylethanol, which was transformed into O-labeled 3-methyl-1-phenylbutane-1,3-diol. The diol was reacted with tris(dimethylamino)phosphane and consecutively with HO to yield a mixture of cyclic H-phosphonates labeled with O andO. They were silylated and subjected to a Perkow reaction with ethyl 3-chloropyruvate. Two protected-[O,O,O]phosphoenol pyruvates were formed and finally globally deprotected. Their configuration was reassessed by a known enzymatic test in combination with conversion of the formed d-glucose-6-phosphate into mixtures of labeled methyl d-glucose-4,6-phosphates, which were analyzed by P NMR spectroscopy. The enzymatic test supported the configuration assigned to labeled stereogenic phosphorus atoms on the basis of synthesis.

Racemic ethyl hydrogen (1-hydroxy-2-methylsulfanyl-1-phenylethyl)phosphonate was resolved with (R)-1-phenylethylamine. The (R)-configuration of the (-)-enantiomer was determined by chemical correlation. Esterification of the (-)-enantiomer with a substituted diazomethane derived from 3-hydroxy-1,3,5(10)-estratrien-17-one delivered two epimeric phosphonates separated by HPLC. Methylation with methyl fluorosulfate at the sulfur atom and treatment with a strong base induced an α-hydroxyphosphonate-phosphate rearrangement with formation of dimethyl sulphide and two enantiomerically pure enol phosphates. Their oily nature interfered with a single crystal X-ray structure analysis to determine the stereochemistry at the phosphorus atom.