Spirolaxine

Spirolaxine is a natural product isolated from Sporotrichum laxum ATCC 15155, which has shown a variety of biological activities including promising anti-Helicobacter pylori property. To understand how this compound is biosynthesized, the genome of S. laxum was sequenced. Analysis of the genome sequence revealed two putative type III polyketide synthase (PKS) genes in this strain, Sl-pks1 and Sl-pks2, which are located adjacent to each other (~2.0 kb apart) in a tail-to-tail arrangement. Disruption of these two genes revealed that Sl-PKS2 is the dedicated PKS involved in the biosynthesis of spirolaxine. The intron-free Sl-pks2 gene was amplified from the cDNA of S. laxum and ligated into the expression vector pET28a for expression in Escherichia coli BL21-CodonPlus (DE3)-RIL. The major products of Sl-PKS2 in E. coli were characterized as alkylresorcinols that contain a C13-C17 saturated or unsaturated hydrocarbon side chain based on the spectral data. This enzyme was purified and reacted with malonyl-CoA and a series of fatty acyl-SNACs (C6-C10). Corresponding alkylresorcinols were formed from the decarboxylation of the synthesized tetraketide resorcylic acids, together with fatty acyl-primed triketide and tetraketide pyrones as byproducts. This work provides important information about the PKS involved in the biosynthesis of spirolaxine, which will facilitate further understanding and engineering of the biosynthetic pathway of this medicinally important molecule.

Incubation of (+)-spirolaxine (= (3R)-5-hydroxy-7-methoxy-3-{5-[(2R,5R,7R)-2-methyl-1,6-dioxaspiro[4.5]dec-7-yl]pentyl}-2-benzofuran-1(3H)-one; 1a) with Bacillus megaterium afforded two new mono- and one new dihydroxylated metabolite(s), all OH groups being introduced on the non-activated six-membered ring. In contrast, exposure of 1a to Cunninghamella echinulata gave rise to hydroxylation on the five-membered ring of the parent structure. The structures and absolute configurations of the new products 1b-e were deduced on the basis of MS and NMR data. The metabolite 1b was investigated, in comparison to 1a, for its cytotoxicity (sulforhodamin-B test) and for its antiproliferative activity towards bovine microvascular endothelial cells (BMEC).

The first enantioselective synthesis of the anti-Heliocbacter pylori agent (+)-spirolaxine methyl ether 2b has been carried out in a convergent fashion establishing that the absolute stereochemistry of the natural product is in fact (3R, 2"R, 5"R, 7"R) after initial synthesis of the unnatural (2"S)-diastereomer 2a. The key step in the synthesis of (+)-spirolaxine methyl ether 2b involved a heterocycle-activated Julia-Kocienski olefination between benzothiazole-based spiroacetal sulfone 4b and phthalide aldehyde 3a. (2"R, 5"S, 7"S)-Spiroacetal sulfone 4b was prepared via cyclisation of protected dihydroxyketone 6b, which in turn was derived from the coupling of the acetylide derived from (R)-acetylene 24b with aldehyde 3a. Phthalide aldehyde 3a was prepared via intramolecular acylation of bromocarbamate 15, which was available via titanium tetrafluoride-(+)-BINOL-mediated allylation of 3,5-dimethoxybenzaldehyde 13. Union of the sulfone 4b and aldehyde 3a fragments successfully completed the enantioselective synthesis of (+)-spirolaxine methyl ether 2b. The synthesis of the unnatural (3R, 2"S, 5"R, 7"R)-diastereomer of spirolaxine methyl ether 2a was also undertaken in a similar manner by union of phthalide aldehyde 3a with (2"S, 5"S, 7"S)-spiroacetal sulfone 4a derived from (S)-acetylene 24a.