Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

Abstract

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 m ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5′-phosphate was 70–75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of >99%-pure 5′-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4′-phosphate, riboflavin 3′-phosphate, riboflavin 4′,5′-diphosphate; riboflavin 3′,4′-diphosphate, and riboflavin 3′,5′-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii ( K D = 9.7 × 10 −9 m). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.