Investigations into the biosynthesis of the polyether ionophore antibiotic, monensin-A

Holmes, Duncan Stuart (1989) Investigations into the biosynthesis of the polyether ionophore antibiotic, monensin-A. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

This thesis describes biosynthetic studies on the polyether ionophore antibiotic, monensin-A. Two approaches to this biosynthetic problem are described in this thesis. The first approach involved the synthesis of a putative triene intermediate, (2S, 3R, 4R, 5S, 6R, 7S, 12E, 16E, 18S, 20E, 22S, 24R)-21-[³H]-S-[2'-1-oxooctyl)amino-ethyl] 25-oxo-2,4,6,12,16,18,22,24-octamethyl-3,5,7-trihydroxy-12,16,20-thiopentadodecatrienoate, in a radiolabelled form, activated as a N-caprylcysteamine thioester. This material was to be fed to cultures of the monensin producers, S. cinnamonensis, in order to detect its incorporation into the antibiotic. A retrosynthetic analysis of the triene divides it into three fragments; the left hand fragment, (+)-(2S, 3R, 4R, 5S, 6S)-S-[2'-[2-oxooctyl)amino]ethyl]-3,5-isopropylidine-dihydroxy-7-oxo-2,4,6-trimethylthioheptanoate, the middle fragment, (3R, 4E, 8E, 12RS)-12-(dimethyl-t-butylsiloxy)-1-phenylsulphonyl-3,5,9-trimethyltrideca-4,8-diene, the right hand fragement, (2S^*, 4R^*)-methyl 2-methyl-4-(2-methyl-1,3-dioxolan-2-yl)-pentanoate. The last fragment was synthesised independently by another coworker at Southamtpon. The E-disubstituted double bond linking the central fragment with the right hand fragment was constructed using a modified Julia coupling while the second coupling, attaching the left hand fragment, involved a directed aldol addition. Feeding experiments were undertaken with shake flask cultures of S. cinnamonensis, A3823.5, and the mutant bacteria DMA300 which accumulates 26-deshydroxy-monensins-A and B. These experiments resulted in no significant incorporation of the labelled triene into the end products. In conjunction with feeding experiments of smaller potential chain elongation intermediates, carried out independently at Southampton, it was concluded that the triene was unable to cross the cell membrane. Using this approach it therefore remains an open question as to whether the triene is an intermediate of the monensin biosynthetic pathway. The second approach involved bioconversion studies with S. cinnamonensis. When radiolabelled E-nerolidol was added to S. cinnamonensis, two turnover products were isolated, (4E, 8RS)-4,8-dimethyl-8-hydroxy-4,9-decadieneamide and (3RS, 6E, 10RS)-3,7,11-trimethyl-1,6-dodecadiene- 3,10,11-triol in 0.4% and 0.1% turnover yields respectively. Feeding experiments with z-nerolidol revealed four turnover products, (3RS, 6Z, 10RS)-9-(3'-3'-dimethyloxiranyl)-7-hydroxy-methyl-3-methyl-1,6-noradiene, (3RS, 6S^*, 7S^*)-3,7,11-trimethyl-1,10,11-triol, and (2R^*, 5R^*)-5-(1',5'-dimethyl-1'-hydroxylhex-4'-ene)-2(ethyl-1-ene)-2-methyl-tetrahydrofuran each detected in approximately 0.05-0.1% turnover yield. These transformations demonstrate for the first time, the presence of epoxidases and hydroxylases in S. cinnamonensis. However, the enzymes involved show a preference for the oxidation of Z-trisubstituted double bonds. Therefore, if the enzymes catalysing these reactions are also the ones on the monensin pathway, then these experiments raise doubt about the stereochemistry of the putative triene intermediate of the monensin biosynthetic pathway. (DX90501)