RETROBIOSYNTHETIC NMR-STUDIES WITH C-13-LABELED GLUCOSE - FORMATION OF GALLIC ACID IN PLANTS AND FUNGI

The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Funga l cultures were grown with [1-C-13]gluose or with a mixture of unlabel ed glucose and [U-C-13(6)]glucose. Young leaves of R. typhina were kep t in an incubation chamber and were supplied with a solution containin g a mixture of unlabeled glucose and [U-C-13(6)]glucose via the leaf s tem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional H-1 and C-13 NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metab olites was obtained by deconvolution of the (CC)-C-13-C-13 coupling mu ltiplets using numerical simulation methods. This approach required th e accurate analysis of heavy isotope chemical shift effects in a varie ty of different isotopomers and the analysis of long range (CC)-C-13-C -13 coupling constants. The resulting isotopomer patterns were interpr eted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show tha t both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic gr oup of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committe d precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caf feic acid, or 3,4,5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorpor ation of uniformly C-13-labeled glucose followed by quantitative NMR a nalysis of isotopomer patterns is suggested as a general method for bi osynthetic studies. As shown by the plant experiment, this approach is also applicable to systems with low incorporation rates.