Prelandrine, the Key-Step Intermediate in the Biosynthesis of the Macrocyclic Spermine Alkaloid Aphelandrine

Abstract

Dedicated to Professor Elmar Vilsmaier on the occasion of his 60th birthday By means of the high sensitive on-line-coupled high-performance liquid chromatography and atmosphericpressure chemical-ionization mass spectrometry (HPLC/APCI-MS and HPLC/APCI-MS/MS) techniques, the new macrocyclic spermine alkaloid prelandrine (5) was detected in the roots of Aphelandra squarrosa (Acanthaceae), and its structure was elucidated as 4'-hydroxyprotoverbine ( 8-(4-hydroxyphenyl)-1,5,9,13-tetraazacycloheptadecan-6-one). It was further demonstrated that protoverbine (6) is enzymatically hydroxylated to prelandrine (5) in a reaction catalyzed by microsomes from the roots of A. squarrosa. The chemical synthesis of (À)-(S)-prelandrine is also described. The possible key role of prelandrine (5) as an intermediate in the biosynthesis of aphelandrine (1) is discussed.Introduction. ± The macrocyclic spermine alkaloid aphelandrine (1; Scheme) is a member of a rather rare class of natural products. Aphelandrine (1) has been found as the major alkaloid in the roots of several Aphelandra species [1 ± 3]. Although the structure of aphelandrine (1) displays p-coumaric acid and spermine (or spermidine and putrescine) as building blocks, little is known about the biosynthesis of this type of alkaloids. The feeding experiments with 3 H-and 14 C-labelled putrescine, spermidine, and coumaric acid confirmed the biogenetic structural units of aphelandrine; however, the question of whether mono-or dicoumaroylspermine is the precursor remained unanswered [4 ± 6]. For the further steps, there are two hypotheses: the first involves cyclization of N(1),N(5)-di(p-coumaroyl)spermine (3) leading to the species 2, which subsequently undergoes phenol coupling to give aphelandrine (1; Scheme) [7]. The second possibility is transformation of N(1)-cinnamoylspermine (4a) or N(1)(pcoumaroyl)spermine (4b) to the macrocycle 6 or 5, respectively, followed by acylation to give 2, which again undergoes phenol coupling to give aphelandrine (1; Scheme). When N(1)-cinnamoylspermine (4a) is cyclized by Michael addition, a hydroxylation step leading from 6 to 5 would be expected.So far, there exists no evidence to support one of these possibilities, presumably because of the concentration limits of the conventional analytical methods. Application of high-sensitive on-line-coupled HPLC/APCI-MS (atmospheric-pressure chemicalionization mass spectrometry) or ESI-MS (electrospray-ionization mass spectrometry) techniques allows the detection of substances, in some cases, at picomolar concentrations. Moreover, HPLC co-eluted substances can be easily differentiated by their molecular weights. The MS/MS spectra of selected quasi-molecular ions ([M H] ) very often provide substantial structural information without isolation.

Phenol oxidative coupling in the biogenesis of the macrocyclic spermine alkaloids aphelandrine and orantine in Aphelandra sp.

“…The data obtained in our foregoing studies have already shown that the biochemical pathway leading to aphelandrine involves as intermediate the monocyclic spermine alkaloid prelandrine (4). Prelandrine (4) was identi®ed in small amount in the roots of A. squarrosa and it is biosynthesized either from the monosubstituted precursor cinnamoyl spermine (6a) via Michael addition and aromatic hydroxylation, or directly from monocoumaroyl spermine (6b) via Michael addition (Nezbedova et al 2001). Monocoumaroyl spermine (6b) could be identi®ed in cell-free extracts of A. tetragona after incubation with spermine and p-coumaroyl-CoA, indicating the presence of a spermine hydroxycinnamoyl transferase in these plants (Hedberg et al 1996).…”