The biosynthesis of chlorogenic acid and related conjugates of the hydroxycinnamic acids. Chromatographic separation and characterization.

The widespread occurrence in plant tissues of conjugates with quinic acid1 and with glucose of p-coumaric acid., caffeic acid, and ferulic acid (Fig. 1) is well established (2-6). Many of these compounds occur as only trace components in extremely complex mixtures of phenolic substances. Uncertainty exists as to the exact chemical structure of most of them, and relatively little is known about their biosynthesis. The primary concern of the present investigation is with the biosynthesis of the most familiar of these conjugates, chlorogenic acid, the structure of which was established as 3-0-caffeoylquinic acid by Fischer and Dangschat in 1932 (7,8). It has been shown previously (9) that this compound, along with possible biosynthetic intermediates, accumulates in potato tuber tissue when disks of the pulp are cultured on a variety of substrates, viz. glucose, quinic acid, phenylalanine, and cinnamic acid. However, the paper chromatographic methods employed in that work were not well suited to the purification and estimation of the minute quantities of the compounds related to chlorogenic acid that were encountered. This paper describes a procedure for the separation of conjugates of the above type by partition chromatography on silica gel. Sondheimer has shown (5) that chlorogenic acid can be separated in this way from its isomers and from caffeic acid by the procedure of Bulen, Varner, and Burrell (10). The present system differs from theirs principally in the use of a linear solvent gradient rather than of a stepwise elution regime, and is well suited to the quantitative analysis of complex mixtures of conjugates. With the aid of the analytical silica gel column, it has been possible to study the mixtures of conjugates present in the potato tuber as harvested or in slices of pulp tissue cultured on a solution containing n-phenylalanine and quinic acid. The bearing of these findings on chlorogenic acid biosynthesis is discussed. Among other things, the results establish that the potato tuber is capable of synthesizing a far greater variety of phenolic conjugates than has generally been realized. Evidence has been found for 17 or 18 such compounds, among which are previously unknown conjugates of shikimic acid.