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Abstract

This chapter discusses o-nitrophenyl-β-D-galactopyranoside 6-phosphate. The procedure for phosphorylating primary hydroxyl groups in unprotected nucleosides is applied to o-nitrophenyl- β -D-galactoside (ONPG) to synthesize the 6-phosphate. Phosphorus oxychloride in trimethyl phosphate containing a small amount of water yielded ONPG-6-P in 50% yield. The method should be applicable to the synthesis of the 6-phosphates of other ONP glycosides. The procedure given is much simpler than that employed initially to synthesize ONPG 6-P: 30 mmoles of o-nitrophenyl β-D-galactoside is dissolved in 75 ml of trimethyl phosphate containing 30 mmoles of water and 90 mmoles of phosphorus oxychloride. The mixture is stirred and kept for 3 hr in an ice bath. Crushed ice is then added, Phosphoric and hydrochloric acids are neutralized with concentrated ammonia, and the yellow solution is evaporated under diminished pressure, o-Nitrophenol is removed by evaporating water several times from the solution. The colorless solution contained 14.65 mmoles of ONPG 6-P as determined enzymically. Enzymic determination of the ONPG 6-phosphate is performed with 10 μg of a highly purified, electrophoretically homogeneous, enzyme preparation in 2.5 ml of the buffer. For high voltage paper electrophoresis, 3 mg of ONPG-6P as the dicyclohexylammonium salt, is hydrolyzed in the standard buffer with 10 μg of 6-phosphogalactosidase.