The formation of benzophenanthridine alkaloids

Isoquinoline alkaloids are a major group of pharmacologically important compounds. The universal precursor to the majority of this alkaloid class is (S)-reticuline. The biosynthesis of this important intermediate starting from the primary metabolite, Ltyrosine, has been completely solved at the enzyme level. One of the most diverse structures in the class of isoquinoline alkaloids are the benzo[c]phenanthridines. The most highly oxidized member is macarpine, an alkaloid produced in considerable quantity in cell suspension cultures of Eschscholtzia californica. This plant source was used to isolate all of the enzymes involved in this pathway. Twelve steps are necessary for the transformation of (S)-reticuline to macarpine, eleven of these are enzyme catalyzed, one is spontaneous. Nature's pathways to complex secondary products, expecially alkaloids, have attracted for a long time the interest of scientists in the chemical fields (e.g. 1). However, it was not until the 1960's that a picture of the biosynthesis of these compounds began to emerge, largely speculative, based on isotopically labelled precursor feeding experiments to differentiated plants (2). It became quite clear at that time that the question of how these metabolites are truly formed was to identify and characterize the enzymes involved in the biosynthesis of these natural products (3). This aim has largely been achieved for select groups of compounds in the past two decades through the consequent use of plant cell suspension cultures to chase the biosynthetic enzymes of the plant kingdom (4). The rational behind biosynthetic studies these days is at least threefold: for one, the aesthetics of learning how nature with its protein catalysts created a repertoire of synthetic methods, far ahead of synthetic organic chemistry; Second to provide chemists with ideas for biomimetic synthesis and thirdly, and most important, to provide (catalytic) targets for gene technological manipulation. Therefore the knowledge of a biosynthetic pathway is an absolute prerequisite for the isolation of the genes and regulatory elements underlaying these reactions. No doubt, enzyme and gene technology will soon become available on a larger scale, which will have a major impact on the production of agriculturally and medicinally useful plants. It has been predicted that the second "revolution" in gene technology will concern low molecular weight compounds, including alkaloids. Within the class of alkaloids, the isoquinoline W o i d s are of particular interest, due to their enormous potential chemical variations of the basic precursor molecules and on the other hand comprises some of the most important drugs for therapy and euphoria (e.g. morphine and its chemical derivatives, papaverine, berberine, dimeric bisbenzylisoquinolines). Of about 29 alkaloids used as pure compounds in western medicine today, six are benzylisoquinoline derived compounds (5). Now what is the common precursor to benzylisoquinolines? No doubt, (S)-reticuline. This compound was predicted on theoretical grounds to be the biogentic precursor of, for instance, morphine (6) even prior to its discovery in nature (7). The next question to be asked is, how is (S)-reticuline biosynthesized in the plant. Visually dissecting the then-known benzylisoquinoline alkaloids, Winterstein and Trier (8) and later Robinson (9) came to the conclusion that all of these alkaloids were derived from a simple tetrahydroxylated benzylisoquinoline named norlaudanosoline. These authors also had already suggested that this molecule might arise by condensation of dopamine with 3,4dihydroxyphenylacetaldehyde to afford the tetraoxygenated base norlaudanosoline (8,9). This proposal was put to test when radiolabelled tracer molecules became commercially available. Satisfactory incorporation of the labelled tracer molecule, norlaudanosoline, was found to occur into different classes of benzvlisoauinoline alkaloids (10. 11) which seemindv verified the old Winterstein and Trier