Stereo-controlled synthesis of prelasalocid, a key precursor proposed in the biosynthesis of polyether antibiotic lasalocid A

In the biosynthesis of a polyether ionophore antibiotic, lasalocid A, the cyclic ether skeleton composed of a tetrahydrofuran linked to a tetrahydropyran could be constructed by oxidative cyclization of linear dodecaketide diene precursor. Hence, we hypothesized a prelasalocid having (E,E)-trisubstituted olefins as the dodecaketide biosynthetic precursor. A stereo-controlled synthetic route to the prelasalocid has been devised in a highly convergent manner entailing installation of a variety of substituents at the trisubstituted olefins.     

SiMe3-based homologation-epoxidation-cyclization strategy for ladder THP synthesis

A trimethylsilyl (SiMe(3)) group is the basis of a strategy that emulates the three fundamental proposed processes in ladder polyether biosynthesis: chain homologation, stereoselective epoxidation (>95% ee or >95:5 dr), and endo-selective, stereospecific (inversion) hydroxyepoxide cyclization (>95:5 endo:exo, >95% dr). A tris-THP was synthesized in 18 total operations from commercial materials using this approach. [reaction: see text]     

Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry

Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High-resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M-H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs.     

The characterisation of polyether ionophore veterinary drugs by HPLC-electrospray MS.

We undertake the determination of a wide range of veterinary drug residues in a range of animal products. Various screening analyses are employed, followed by HPLC-API (atmospheric pressure ionisation)-MS for the unequivocal confirmation of significant positives. EU legislation for the use of GC-MS as a confirmatory technique requires the successful monitoring of at least four diagnostic ions and although no such requirement exists for HPLC-MS confirmation, a similar requirement would seem appropriate. Until recently, reports describing the electrospray MS confirmation of residues of the polyether ionophores have been based on monitoring one or two ions. We have found that the addition of ammonium acetate to the HPLC mobile phase, in conjunction with 'cone voltage' or 'skimmer' assisted fragmentation, is a convenient way of producing additional diagnostic ions from polyether ionophore compounds, without compromising the overall sensitivity. Results for lasalocid, the most widely used compound, are presented. Electrospray MS data and acquisition parameters for lasalocid, monensin, narasin and salinomycin are described. The advantage of this analytical approach is that it may be used to generate confirmatory data using a single quadrupole MS system, without the need for advanced MS instrumentation, e.g., MS-MS.     

Enzymatic epoxide-opening cascades catalyzed by a pair of epoxide hydrolases in the ionophore polyether biosynthesis

Our recent findings of the first epoxide hydrolase Lsd19, involved in lasalocid A biosynthesis, led us to investigate a long-standing controversial issue on the mechanism of enzymatic epoxide-opening cascades. The site-directed mutagenesis and domain dissection analysis to reveal the mechanism of the reaction catalyzed by Lsd19 is examined, especially in the role of acidic amino acid pair and catalytic domains.     

Identification of NanE as the thioesterase for polyether chain release in nanchangmycin biosynthesis

The polyketide synthase (PKS) for the biosynthesis of the polyether nanchangmycin lacks an apparent thioesterase comparable to the type I thioesterase domains of the modular PKSs responsible for macrolide biosynthesis. Three candidate polyether chain-releasing factors were examined. Both the putative CR domain and the NanE protein appeared to be genetically relevant. Among the three heterologously expressed soluble proteins (recombinant CR domain, the ACP-CR didomain, and NanE) tested, only NanE hydrolyzed the polyether-SNAC. By contrast, recombinant DEBS TE from the erythromycin pathway, and the recombinant MonAX, a type II TE associated with the polyether monensin biosynthesis for which a homolog has not been detected in the nanchangmycin cluster, hydrolyzed a diketide-SNAC but not the polyether-SNAC. We could thus conclude that NanE is a dedicated thioesterase mediating the specific release of the polyether chain during nanchangmycin biosynthesis.     

Hydrogen-bonding interactions in gas-phase polyether/ammonium ion complexes

Hydrogen bonds are among the most important interactions involved in selective complexation in host-guest chemistry. In this study a variety of hydrogen-bonded crown ether/ammonium ion complexes are generated in the gas phase by association reactions between an amine substrate and a polyether, one of which is initially protonated, and stabilized by many collisions in the chemical ionlzation source of a triple quadrupole mass spectrometer or in a quadrupole ion trap. The nature of the hydrogen-bonding interactions of the ion complexes are evaluated by comparison of their collision-activated dissociation spectra. After collisional activation, those complexes that are weakly bound dissociate to form intact protonated polyether molecules and/or ammonium ions by simple cleavages of the hydrogen-bond association interactions. In contrast, those complexes strongly bound by multiple hydrogen bonds dissociate not only to the protonated polyether and/or ammonium ions but also by extensive covalent bond cleavage of the protonated ether skeleton.This latter type of dissociation behavior suggests that the polyether/ammonium ion complexes may be sufficiently strongly bound that surpassing the high barrier to decomposition results in formation of internally excited polyether molecules that may then undergo subsequent fragmentation by skeletal cleavages. Moreover, complexes involving multiple hydrogen bonds may have slower dissociation kinetics, allowing competition from fast dissociation processes that have substantial energy barriers.     

Tetrahydropyran formation by rearrangement of an epoxy ester: a model for the biosynthesis of marine polyether toxins

Acid-catalyzed rearrangement of the (S)-epoxide derived from 2alpha-allyl cholestanyl acetate resulted in a 1:1 mixture of a steroidal tetrahydrofuran and a steroidal tetrahydropyran. Formation of a six-membered ring supports the hypothesis that epoxy ester-orthester-cyclic ether rearrangement may be involved in the biosynthesis of ladder-type marine polyether toxins. This reaction represents a new biomimetic preparation of medium ring cyclic ethers.     

Prorocentin, a new polyketide from the marine dinoflagellate Prorocentrum lima

Prorocentin (1), isolated from an okadaic acid-producing organism, Prorocentrum lima, possessed all-trans trienes, an epoxide, as well as the 6/6/6-trans-fused/spiro-linked polyether ring moieties. The unique structure supports the proposed cyclization mechanism, polyene formation, epoxidation, and cyclization, of marine polyether toxins. The relative stereostructure was determined on the basis of spectral data. [structure: see text]     

Biosynthesis of indole diterpenes, emindole, and paxilline: involvement of a common intermediate

The key step for construction of the carbon skeleton in the indole diterpenes, paxilline, and emindole DA was examined. Intact incorporation of multiply (2)H-labeled 3-geranylgeranylindole into two different fungal metabolites proves 3-geranylgeranylindole to be a biosynthetic intermediate. These results give evidence that indole diterpenes are biosynthesized via epoxidation of a common intermediate, and the subsequent cationic cyclization, analogous to those in the steroid biosynthesis. [structure: see text]     

Insights into polyether biosynthesis from analysis of the nigericin biosynthetic gene cluster in Streptomyces sp. DSM4137

Nigericin was among the first polyether ionophores to be discovered, but its biosynthesis remains obscure. The biosynthetic gene cluster for nigericin has been serendipitously cloned from Streptomyces sp. DSM4137, and deletion of this gene cluster abolished the production of both nigericin and the closely related metabolite abierixin. Detailed comparison of the nigericin biosynthetic genes with their counterparts in the biosynthetic clusters for other polyketides has prompted a significant revision of the proposed common pathway for polyether biosynthesis. In particular, we present evidence that in nigericin, nanchangmycin, and monensin, an unusual ketosynthase-like protein, KSX, transfers the initially formed linear polyketide chain to a discrete acyl carrier protein, ACPX, for oxidative cyclization. Consistent with this, deletion of either monACPX or monKSX from the monensin gene cluster effectively abolished monensin A biosynthesis.     

Chemical Degradation of Lasalocid: (A) The Mannich reaction (B) Baeyer-Villiger oxidation of the retro-aldol ketone

Under Mannich reaction conditions (diethylamine and formaldehyde in toluene under reflux) lasalocid ( 1 ) undergoes a unique transformation in which the carboxyl group is replaced by a diethylaminomethyl group. The resulting Mannich base 2 was converted back to lasalocid, proving that none of the other chemical and stereochemical features of the molecule were disturbed. Like other phenolic Mannich bases, the one derived from lasalocid readily alkylated mercaptans. The known thermal and base-induced retro-aldol degradations of lasalocid both produce a ketone fragment 9 containing the cyclic ether units. Baeyer-Villiger oxidation of this ketone afforded a carboxylic acid fragment which still contained these ether units. The normal regiochemistry involved in oxidizing this ketone (R? CH₂? CO? CHR′R″ type) was cleanly reversed by first converting it into the hydroxymethylidene derivative 10 .     

A comparison of data analysis methods for determining gas phase stabilities by cid: Alkali metal complexes of polyether ionophore antibiotics

The gas phase stabilities of Group I metal complexes of the polyether ionophore antibiotics lasalocid and monensin were investigated by collision induced dissociation mass spectrometry. Electrospray ionization was used with a triple quadrupole mass spectrometer for the determination of threshold dissociation energies upon application of increasing collision energies. Various data analysis techniques for the determination of dissociation energies are discussed to assess the most suitable method for determining the stabilities of the ionophore-metal complexes studied here. In all cases only the relative stabilities of different complexes may be obtained by the method presented in this study, which does not assess absolute gas phase dissociation energies. Correction factors have been applied, however, to account for the energy conversion during collisions of different metal complexes and the varying degrees of freedom of different sized ligands, allowing for the comparison of the stabilities of different ionophores with like-metals. The measured threshold dissociation energies were compared with respect to the ionic radius of the metal cation, revealing a maximum stability for the K+ complexes of both lasalocid and monensin. A striking decrease in the stabilities of the Rb+ and Cs+ complexes was observed and is believed to be related to a decreasing degree of coordination that the ionophores can accomplish with the larger metals.     

Functional modification of naturally occurring ionophores as a new route to chiral receptors

Abstract

New chiral receptors characterized by acyclic polyether skeletons and neutral terminal substituents were derived from naturally occurring monensin, lasalocid, salinomycin and nigericin ionophores. Their chiral recognition ability was investigated by ion-selective electrode and ¹H-NMR spectroscopic methods. Monensin ester and amide derivatives specifically formed 1:1 complexes with chiral amine salts and exhibited excellent enantiomer selectivity, while parent monensin rarely discriminated between the enantiomers of examined amine salts. This review describes how chemical modification of naturally occurring ionophores provides unique molecular recognition functions and a new synthetic strategy for chiral receptor molecules.     

An alternative synthetic approach towards erythrinan and homoerythrinan alkaloids by tandem semipinacol/intramolecular Schmidt reaction

An alternative construction of A-B-D ring-system of erythrinan and homoerythrinan alkaloids by TiCl4-mediated tandem semipinacol/intramolecular Schmidt reaction of α-siloxyepoxyazide was addressed and the unusual epoxidation stereoselectivity was observed in preparation of the substrate once again.     

Chemical Probes for the Functionalization of Polyketide Intermediates

A library of functionalized chemical probes capable of reacting with ketosynthase‐bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl‐ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology.     

A complete gene cluster from Streptomyces nanchangensis NS3226 encoding biosynthesis of the polyether ionophore nanchangmycin

The PKS genes for biosynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectively), but are separated by independent ORFs that encode an epimerase, epoxidase, and epoxide hydrolase, and, notably, an independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the process of oxidative cyclization to form the polyether structure occurs when the polyketide chain is still anchored on the independent ACP before release. 4-O-methyl-L-rhodinose biosynthesis and its transglycosylation involve four putative genes, and regulation of nanchangmycin biosynthesis seems to involve activation as well as repression. In-frame deletion of a KR6 domain generated the nanchangmycin aglycone with loss of 4-O-methyl-L-rhodinose and antibacterial activity, in agreement with the assignments of the PKS domains catalyzing specific biosynthetic steps.     

Magnetic solid‐phase extraction based on carbon nanotubes for the determination of polyether antibiotic and s‐triazine drug residues in animal food with LC–MS/MS

Carbon nanotubes‐magnetic nanoparticles, comprising ferroferric oxide nanoparticles and carbon nanotubes, were prepared through a simple one‐step synthesis method and subsequently applied to magnetic solid‐phase extraction for the determination of polyether antibiotic and s‐triazine drug residues in animal food coupled with liquid chromatography with tandem mass spectrometry. The nanocomposites were characterized by transmission electron microscopy, X‐ray diffraction, and vibrating sample magnetometry. The components within the nanocomposites endowed the material with high extraction performance and manipulative convenience. Compared with carbon nanotubes, the as‐prepared carbon nanotubes‐magnetic nanoparticles showed better extraction and separation efficiencies for polyether antibiotics and s‐triazine drugs thanks to the contribution of the iron‐containing magnetic nanoparticles. Various experimental parameters affecting the extraction efficiency had been investigated in detail. Under the optimal conditions, the good linearity ranging from 1 to 200 μg/kg for diclazuril, toltrazuril, toltrazuril sulfone, lasalocid, monensin, salinomycin, narasin, nanchangmycin, and maduramicin, low limits of detection ranging from 1 to 5 μg/kg, and satisfactory spiked recoveries (77.1–91.2%, with the inter relative standard deviation values from 4.0 to 12.2%) were shown. It was confirmed that this novel method was an efficient pretreatment and enrichment procedure and could be successfully applied for extraction and determination of polyether and s‐triazine drug residues in complex matrices.     

Liquid chromatography with ultraviolet detection of lasalocid, monensin, salinomycin and narasin in poultry feeds using pre-column derivatization

A rapid and very effective analytical procedure for the simultaneous determination of four polyether antibiotics (PEs) lasalocid, monensin, salinomycin and narasin in poultry feeds was tested. PEs were extracted from samples using methanol and without and clean-up derivatized with 2,4-dinitrophenylhydrazine (DNP) in an acidic medium at 55 degrees C. The derivatization mixture was analyzed directly on an ODS column (150 x 4.6 mm, 5 microns) with methanol--1.5% aqueous acetic acid (90:10, v/v) as eluent and UV detection was carried out at 305/392 nm. The recoveries of the PEs from spiked samples were 85-100% with RSDs of 4-10% in a concentration range of 50-150 mg/kg.     

Structural basis of regiospecificity of a mononuclear iron enzyme in antibiotic fosfomycin biosynthesis

Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that uses dioxygen to catalyze the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid (S-HPP) in the biosynthesis of the antibiotic fosfomycin. Additionally, the enzyme converts the R-enantiomer of the substrate (R-HPP) to 2-oxo-propylphosphonic acid. To probe the mechanism of HppE regiospecificity, we determined three X-ray structures: R-HPP with inert cobalt-containing enzyme (Co(II)-HppE) at 2.1 ? resolution; R-HPP with active iron-containing enzyme (Fe(II)-HppE) at 3.0 ? resolution; and S-HPP-Fe(II)-HppE in complex with dioxygen mimic NO at 2.9 ? resolution. These structures, along with previously determined structures of S-HPP-HppE, identify the dioxygen binding site on iron and elegantly illustrate how HppE is able to recognize both substrate enantiomers to catalyze two completely distinct reactions.