Enzymes DOI: 10.1002/anie.201106323 Insights into Lasalocid A Ring Formation by Chemical Chain Termination In Vivo** Manuela Tosin,* Luke Smith, and PeterF. Leadlay* Polyether antibiotics are a unique class of compounds that are broadly used in veterinary medicine and in animal husbandry for their ability to complex inorganic cations and aid their transport across membrane barriers. They include monensin, nigericin, nanchangmycin, salinomycin, lasalocid A, tetrona- sin, and tetronomycin among others, [1] and all are produced by Streptomyces and related filamentous bacteria. Recent reports on their outstanding potency against a variety of critical infectious disease targets including protozoa, bacteria, and viruses, [2] as well as their ability to selectively kill cancer stem cells, [3] have led to a revived interest in the biosynthesis of these compounds for combinatorial chemistry purposes. Polyether ionophores possess two or more ether rings and a terminal carboxy group, all of which serve as ligands for cation binding. Early experiments involving the feeding of labeled precursors to whole cells showed that the carbon skeleton and specific oxygen atoms were of polyketide origin, with the additional ether oxygen atoms originating from molecular oxygen. On this basis Cane, Celmer, and Westley proposed a unified mechanism for polyether biosynthesis involving the initial formation of an all-trans unsaturated polyketide that would undergo oxidative cyclization by stereospecific epoxidation, epoxide hydrolysis, and a cascade of nucleophilic hydroxy cyclizations. [4] This hypothesis was first validated in our laboratories, where an (E,E,E)-poly- ketide triene precursor to monensin was isolated and characterized from a blocked mutant of Streptomyces cinna- monensis . [5] Since then enormous progress has been made in increasing our knowledge of polyether biosynthesis through the identification and the cloning of various polyether biosynthetic clusters from actinomycetes, [6] and functional studies of epoxidases, [7] epoxide hydrolases, [8] and putative thioesterases that are responsible for polyether release from their polyketide synthases (PKSs). [9] However many details of the biosynthesis remain undefined, in particular in relation to the timing and the mechanism of ring formation. We and others [10] have cloned and characterized the gene cluster of lasalocid A (1), a polyether produced by Strepto- myces lasaliensis . Lasalocid A is widely used as a coccidiostat but it also displays antimalarial and antischistosomiasis activity. [11] Based on early feeding experiments the biosyn- thesis of 1 involves the formation of a dodecaketide from the decarboxylative condensation of malonate, methylmalonate, and ethylmalonate units. On this basis Westley et al. sug- gested that the stereoselective bis(epoxidation) of a putative dodecaketide acid precursor, prelasalocid (3), and a subse- quent epoxide hydrolysis and cyclization cascade, would lead to 1 and its stereoisomer isolasalocid (2 ; Scheme 1). [12] We have recently demonstrated the role of the epoxide hydrolase LasB in directing the formation of the tetrahydrofuran (THF) and tetrahydropyran (THP) rings of lasalocid A, which is in contrast to the non-enzymatic formation of the two THF rings of isolasalocid. [10a] The Oikawa group has reported the stereoselective synthesis of prelasalocid (3) and its bis- (epoxide) 4, and showed that 4 can be enzymatically converted into 1, albeit inefficiently, by the recombinant epoxide hydrolase (LasB or Lsd19). [13] More recent work on the recombinant epoxide hydrolase LasB has proven the Scheme 1. Biosynthetic pathway to lasalocid A (1) proposed by West- ley: bis(epoxidation) of prelasalocid (3) [12–13] is followed by epoxide hydrolysis and cyclization controlled by the epoxide hydrolase LasB. [10a] [*] Dr. M. Tosin, L. Smith, Prof. P. F. Leadlay Department of Biochemistry, University of Cambridge 80 Tennis Court Road, Cambridge CB2 1GA (UK) E-mail: pfl10@mole.bio.cam.ac.uk Homepage: http://www.bioc.cam.ac.uk/uto/leadlay.html Dr. M. Tosin Department of Chemistry, University of Warwick Library Road, Coventry CV4 7AL (UK) E-mail: M.Tosin@warwick.ac.uk Homepage: http://www2.warwick.ac.uk/fac/sci/chemistry/ research/tosin/ [**] We thank the Herchel Smith Fund (Fellowship to M.T.) and BBSRC (project grant BB/I/002513/1 to P.F.L.) for financial support. Supporting information (including general methods, the construc- tion of S. lasaliensis mutant strains, and LC/HRMS analysis of all the isolated derivatives of the intermediates) for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201106323. Communications 11930  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2011, 50, 11930 –11933