Nonactin is a polyketide antibiotic produced by Streptomyces griseus ETH A7796. It is active against a range of Gram positive organisms, acting as an ionophore that has a high selectivity for K + ions. Among the polyketides, nonactin has a unique structure. It is a 32-membered, cyclic tetraester generated from four monomers of nonactic acid, two of which are (+)-nonactic acid and two of which are (−)-nonactic acid. The monomers are arranged (+)-(−)-(+)-(−) in the macrocycle so that nonactin has S4 symmetry and is achiral. As the structure of nonactin is unique, so is the biosynthesis of the compound. There is much evidence that after an early achiral intermediate, there are two mirror image biosynthesis pathways, one for the synthesis of each enantiomer of nonactic acid. Emerging evidence suggests that the two pathways arose from gene duplication followed by divergent evolution. The central question, therefore, raised by both the structure and the biosynthesis of nonactin concerns the benefit, if any, to the producing organism in initially selecting for, and then maintaining, two independent pathways for nonactic acid synthesis and for generating only the achiral diastereoisomer of nonactin.To understand why achiral nonactin is the naturally generated diastereoisomer we prepared through synthesis two alternate diastereoisomers of nonactin, one prepared solely from (+)-nonactic acid and one prepared solely from (−)-nonactic acid, referred to here as 'all-(+)-nonactin' and 'all-(−)-nonactin' respectively. Both all-(+)-nonactin and all-(−)-nonactin were more than 500-fold less active against Gram positive organisms than nonactin confirming that the biosynthesis of both nonactic acid enantiomers, and their incorporation into nonactin, is necessary for biological activity. To understand the lack of antibacterial activity we used isothermal calorimetry to measure the association constant K a , ΔG, ΔH and ΔS of formation for the K + , Na + and NH 4 + complexes of nonactin and all-(−)-nonactin. The natural diastereoisomer had a high selectivity for K + to which it bound approximately 880-fold better than all-(−)-nonactin. We used a picrate partitioning assay to confirm that all-(−)-nonactin, unlike nonactin, could not partition K + ions into organic solvent. To complement the thermodynamic data we used a simple experimental model to mimic ion transport. Using two concentric glass tubes we arranged for two aqueous samples to be separated by a CHCl 3 layer. By following the co-transport of picrate anion from one aqueous layer to the other, through the organic phase, we were able to measure the rates of K + transport that were facilitated by the ionophores. Whereas nonactin allowed for efficient K + transport, all-(−)-nonactin was completely nigel.priestley@umontana.edu. Supporting Information Available: 1 H and 13 C NMR spectra and mass spectra for compounds 8 to 17. This material is available free of charge via the Internet at

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 ...

The polyketide nonactin, a polyketide possessing antitumor and antibacterial activity, is produced by an unusual biosynthesis pathway in Streptomyces griseus that uses both enantiomers of the nonactin precursor, nonactic acid. Despite many studies with labeled precursors, much of the biosynthesis pathway remains unconfirmed, particularly the identity of the last achiral intermediate in the pathway, which is believed to be 4,6-diketoheptanoyl-CoA. We set out to confirm the latter hypothesis with feeding studies employing [4,5-(13)C(2)]-, [5,6-(13)C(2)]-, and [6,7-(13)C(2)]-4,6-diketoheptanoate thioester derivatives. In each case the isotopic label was incorporated efficiently into nonactin; however, at positions inconsistent with the currently accepted biosynthesis pathway. To resolve the discrepancy, we conducted additional feeding studies with a [3,4-(13)C(2)]levulinate thioester derivative and again observed efficient label incorporation. The latter result was intriguing, as levulinate is not an obvious precursor to nonactin. Levulinate, however, is known to be efficiently degraded into propionate even though the pathway for the conversion is not known. On the basis of both our levulinate and diketoheptanoate isotope incorporation data we can now postulate a pathway from levulinate to propionate that can also account for the conversion of 4,6-diketoheptanoate into levulinate in S. griseus.

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We have shown that the intentional engineering of a natural product biosynthesis pathway is a useful way to generate stereochemically complex scaffolds for use in the generation of combinatorial libraries that capture the structural features of both natural products and synthetic compounds. Analysis of a prototype library based upon nonactic acid lead to the discovery of triazole-containing nonactic acid analogs, a new structural class of antibiotic that exhibits bactericidal activity against drug resistant, Gram-positive pathogens including Staphylococcus aureus and Enterococcus faecalis.