Nonactin biosynthesis: Setting limits on what can be achieved with precursor-directed biosynthesis

Abstract

Nonactin, produced by Streptomyces griseus ETH A7796, is a macrotetrolide assembled from nonactic acid. It is an effective inhibitor of drug efflux in multidrug resistant erythroleukemia K562 cells at sub-toxic concentrations and has been shown to possess both antibacterial and antitumor activity. As total synthesis is impractical for the generation of nonactin analogs we have studied precursor-directed biosynthesis as an alternative as it is known that nonactic acid can serve as a nonactin precursor in vivo. To determine the scope of the approach we prepared and evaluated a furan-based nonactic acid derivative, 11. Although no new nonactin analogs were detected when 11 was administered to S. griseus fermentative cultures, a significant inhibition of nonactin biosynthesis was noted (IC 50 ~ 100 μM). Cell mass, nonactic acid production and the generation of other secondary metabolites in the culture were unaffected by 11 demonstrating that 11 selectively inhibited the assembly of nonactin from nonactic acid. While we were unable to generate new nonactin analogs we have discovered, however, a useful inhibitor that we can use to probe the mechanism of nonactin assembly with the ultimate goal of developing more successful precursordirected biosynthesis transformations.Streptomyces griseus subsp. griseus ETH A7796 (DSM40695) makes a series of ionophore antibiotics known as the macrotetrolides (Figure 1). 1 Nonactin, the prototypical macrotetrolide (1) is assembled from two monomers of (-)-nonactic acid and two monomers of (+)-nonactic acid assembled (+)-(-)-(+)-(-) in a head-to-tail manner into a 32-membered macrocycle. Nonactin has both antibiotic and anticancer properties 2 and has been shown to be an inhibitor of drug efflux in multiple drug resistant cancers.3 The natural macrotetrolide homologues produced by S. griseus show a wide range potency with the minimum inhibitory concentration of 1 being an order of magnitude greater than that of dinactin (3) against Staphylococcus aureus and Mycobacterium bovis, a difference that is related to the stability constants of their respective Na + and K + complexes. 1,2,4 Nonactin is far too hydrophobic and insufficiently soluble to be an effective therapeutic. 5 The development of therapeutics based upon nonactin, therefore, will be dependent upon being able to make non-natural analogs in a direct and efficient manner. The total synthesis of nonactin has been achieved by a number of groups 6-9 as has the synthesis of nonactic acid. 10,11 The total synthesis of nonactin analogs is complicated as both enantiomers of nonactic acid, and its analogs, are required as is the sequential construction of a linear tetraester prior to a © 2008 Elsevier Ltd. All rights reserved. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published ...

Dereplication ofStreptomycessp. AMC 23 polyether ionophore antibiotics by accurate-mass electrospray tandem mass spectrometry

“…macrotetrolides that Streptomyces species produce. Macrotetrolides are a class of little polar compounds that exhibit ionophoric properties; they originate from nonactic acid and homononactic acid and form a large tetralactone that can insert tetrahydrofuran moieties . Linear dimer derivatives of nonactic and homononactic acids have been isolated from a Streptomyces globisporus strain .…”

Thione Esters as Substrates for the Stereoselective Alkylation of Model Compounds of Nonactic Acids

“…In this context it is worth mentioning that the biosynthesis of nonactin uses Michael addition to a linear α,β-unsaturated ester to form selectively the tetrahydrofuran ring. [36][37][38][39][40] We decided to take advantage of this inherent reactivity and to use the ring-opened products as intermediates in our quest to obtain lipophilically substituted analogues of nonactic acid. We developed a second method that proceeds via ringopened intermediates to try to ascertain the syn/anti relative configuration of the two centres at the α-and β-positions of the ester (Figure 3).…”

Pilot-plant cultivation of Streptomyces griseus producing homologues of nonactin by precursor-directed biosynthesis and their identification by LC/MS-ESI

“…A correct precursor should be used; with a wrong precursor, for example 1 mM nonactic acid having a furan ring, the production of nonactin was inhibited by more than 90%, no effect on biomass production being found up to the 10 mM concentration of added precursor. 16 Another strategy, mutasynthesis, uses mutant microorganisms deficient in a key aspect of the biosynthetic pathway; substitution of the natural precursor with a precursor analogue can again lead to the production of a new natural product. As documented, for example, by Bode et al 17 in their study of myxalamid production by myxobacterial mutant strains so manipulated, these strains may eventually exhibit the same or a weaker incorporation of suitable precursors into the Nonactins from Streptomyces griseus by LC/MS-ESI T Ř ezanka et al desirable product than the wild-type strains.…”