Identification of the reaction products of rosmarinic acid synthase from cell cultures of Coleus blumei by ion spray mass spectrometry and tandem mass spectrometry

Abstract

AbstractDifferent substrates were used in reaction assays for rosmarinic acid synthase, an enzyme involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei. In order to introduce the cinnamoyl moiety of the ester, 4‐coumaroyl‐CoA and caffeoyl‐CoA were applied as substrates; for the hydroxyphenyllactic acid moiety, 4‐hydroxyphenyllactate and 3,4‐dihydroxyphenyllactate were used. Four different reaction products were thus produced and these were analysed by ion spray mass spectrometry and tandem mass spectrometry after purification by semi‐preparative high pressure liquid chromatography. The molecular masses and the fragmentation patterns obtained by tandem mass spectrometry of the reaction products formed by the catalysis of rosmarinic acid synthase were in agreement with the structures of rosmarinic acid (caffeoyl‐3′,4′‐dihydroxyphenyllactate) and rosmarinic acid‐like esters (4‐coumaroyl‐4′‐hydroxyphenyllactate, 4‐coumaroyl‐3′,4′‐dihydroxyphenyllactate, caffeoyl‐4′‐hydroxyphenyllactate) as expected for the substrates used in the enzyme reaction.