Preparation and characterization of an immunoaffinity column for the selective extraction of azaspiracids.

The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC-MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26-C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC-MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90-92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.

A Practical ELISA for Azaspiracids in Shellfish via Development of a New Plate-Coating Antigen.

Azaspiracids (AZAs) are a group of biotoxins that appear periodically in shellfish and can cause food poisoning in humans. Current methods for quantifying the regulated AZAs are restricted to LC-MS but are not well suited to detecting novel and unregulated AZAs. An ELISA method for total AZAs in shellfish was reported recently, but unfortunately, it used relatively large amounts of the AZA-1-containing plate-coating conjugate, consuming significant amounts of pure AZA-1 per assay. Therefore, a new plate-coater, OVA-cdiAZA1 was produced, resulting in an ELISA with a working range of 0.30-4.1 ng/mL and a limit of quantification of 37 µg/kg for AZA-1 in shellfish. This ELISA was nearly twice as sensitive as the previous ELISA while using 5-fold less plate-coater. The new ELISA displayed broad cross-reactivity toward AZAs, detecting all available quantitative AZA reference materials as well as the precursors to AZA-3 and AZA-6, and results from shellfish analyzed with the new ELISA showed excellent correlation ( R2 = 0.99) with total AZA-1-10 by LC-MS. The results suggest that the new ELISA is suitable for screening samples for total AZAs, even in cases where novel AZAs are present and regulated AZAs are absent, such as was reported recently from Puget Sound and the Bay of Naples.

Synthesis of the C1-C19 Domain of Azaspiracid-34.

Azaspiracid-34 (AZA34) is a recently described structurally unique member of the azaspiracid class of marine neurotoxins. Its novel structure, tentatively assigned on the basis of MS and 1H NMR spectroscopy, is accompanied by a 5.5-fold higher level of toxicity against Jurkat T lymphocytes than AZA1. To completely assign the structure of AZA34 and provide material for in-depth biological evaluation and detection, synthetic access to AZA34 was targeted. This began with the convergent and stereoselective assembly of the C1-C19 domain of AZA34 designed to dovetail with the recent total synthesis approach to AZA3.

Stereochemical Definition of the Natural Product (6R,10R,13R, 14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 by Total Synthesis and Comparative Analyses.

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R, 33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned 20R epimer did not.

Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S, 25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals Non-Identity with the Natural Product.

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved by a late-stage Nozaki-Hiyama-Kishi coupling to form the C21-C22 bond with the C20 configuration unambiguously established from l-(+)-tartaric acid. Postcoupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the resulting C1 primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

Stereoselective Synthesis of the C1-C9 and C11-C25 Fragments of Amphidinolides C, C2, C3, and F.

An efficient synthesis of the C1-C9 and the C11-C25 fragments of amphidinolides C, C2, C3, and F from a common intermediate is reported. The construction of the C1-C9 fragment involves an intramolecular hetero-Michael cyclization to form the 3,5-disubstituted trans-tetrahydrofuran moiety. The approach to prepare the C11-C25 fragment utilizes a highly stereoselective aerobic cobalt-catalyzed alkenol cyclization and a chelated Mukaiyama aldol reaction to form the C13-C14 bond and to concomitantly install the C13 hydroxyl group.

Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.

Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure-activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2-epi-DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 >> 2-epi-DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs' activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors.

Total Synthesis of (-)-Salvinorin†A.

Salvinorin A (1) is natural hallucinogen that binds the human κ-opioid receptor. A total synthesis has been developed that parlays the stereochemistry of l-(+)-tartaric acid into that of (-)-1 via an unprecedented allylic dithiane intramolecular Diels-Alder reaction to obtain the trans-decalin scaffold. Tsuji allylation set the C9 quaternary center and a late-stage stereoselective chiral ligand-assisted addition of a 3-titanium furan upon a C12 aldehyde/C17 methyl ester established the furanyl lactone moiety. The tartrate diol was finally converted into the C1,C2 keto-acetate.

Synthesis of the C22-C40 Domain of the Azaspiracids.

An efficient synthesis of the C22-C40 domain of the azaspiracids is described. The synthetic route features a Nozaki-Hiyama-Kishi (NHK) coupling and chelation controlled Mukaiyama aldol reaction to access an acyclic intermediate and a double-intramolecular-hetero-Michael addition (DIHMA) to provide the FG-ring system bridged ketal.

Development of an ELISA for the Detection of Azaspiracids.

Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium spinosum and accumulate in shellfish. Ovine polyclonal antibodies were produced and used to develop an ELISA for quantitating AZAs in shellfish, algal cells, and culture supernatants. Immunizing antigens were prepared from synthetic fragments of the constant region of AZAs, while plate coating antigen was prepared from AZA-1. The ELISA provides a sensitive and rapid analytical method for screening large numbers of samples. It has a working range of 0.45-8.6 ng/mL and a limit of quantitation for total AZAs in whole shellfish at 57 µg/kg, well below the maximum permitted level set by the European Commission. The ELISA has good cross-reactivity to AZA-1-10, -33, and -34 and 37-epi-AZA-1. Naturally contaminated Irish mussels gave similar results whether they were cooked or uncooked, indicating that the ELISA also detects 22-carboxy-AZA metabolites (e.g., AZA-17 and AZA-19). ELISA results showed excellent correlation with LC-MS/MS analysis, both for mussel extract spiked with AZA-1 and for naturally contaminated Irish mussels. The assay is therefore well suited to screening for AZAs in shellfish samples intended for human consumption, as well as for studies on AZA metabolism.

Synthesis of the C1-C21 domain of azaspiracids-1 and -3.

An efficient synthesis of the C1-C21 fragment of azaspiracids-1 and -3 is described. This features a Nozaki-Hiyama-Kishi reaction to couple the AB and CD ring precursors and formation of the THF-fused ABCD trioxadispiroketal system under thermodynamic conditions.

A stereoconvergent intramolecular Diels-Alder cycloaddition related to the construction of the decalin core of neo-clerodane diterpenoids.

Several examples of a highly specific, stereoconvergent intramolecular Diels-Alder cycloaddition that led to the trans-decalin core of neo-clerodane diterpenoids are described. The relative configuration adjacent to the dienophile, which led to C4 of the decalin system, as well as the electron-withdrawing effects of various substituents and conformational rigidity of the Diels-Alder precursors were explored.

Syntheses of the C1-C14 and C15-C25 fragments of amphidinolide C.

Divergent syntheses of the C1-C14 and C15-C25 fragments of amphidinolide C have been achieved. The synthesis of the C15-C25 fragment featured cobalt-catalyzed modified Mukaiyama aerobic alkenol cyclization and sulfur-directed regiocontrolled Wacker oxidation of an internal alkene. The C1-C14 fragment was established by alkenyllithium addition to an aldehyde followed by a challenging olefination of a highly inert C9 ketone.

Comparison of splicing factor 3b inhibitors in human cells.

Name your splice: FR901464 analogues and herboxidiene inhibit constitutive splicing, most likely by inhibiting spliceosomal subunit SF3b. A parallel comparison of these compounds in a cell-based assay system showed meayamycin B as the most potent splicing inhibitor among these small molecules.

The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine.

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts containing non-α-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.

Total syntheses of the histone deacetylase inhibitors largazole and 2-epi-largazole: application of N-heterocyclic carbene mediated acylations in complex molecule synthesis.

Details of the evolution of strategies toward convergent assembly of the histone deacetylase inhibiting natural product largazole exploiting γ,δ-unsaturated-α,ß-epoxy-aldehydes and a thiazole-thiazoline containing ω-amino-acid are described. The initial N-heterocyclic carbene mediated redox amidation exploying these two types of building blocks representing largazole's structural domains of distinct biosynthetic origin directly afforded the seco-acid of largazole. This was accomplished without any protecting groups resident upon either thioester bearing epoxy-aldehyde or the tetrapeptide. However, the ineffective production of largazole via the final macrolactonization led to an alternative intramolecular esterification/macrolactamization strategy employing the established two building blocks. This provided largazole along with its C2-epimer via an unexpected inversion of the α-stereocenter at the valine residue. The biological evaluation demonstrated that both largazole and 2-epi-largazole led to dose-dependent increases of acetylation of histone H3, indicating their potencies as class I histone deacetylase selective inhibitiors. Enhanced p21 expression was also induced by largazole and its C2 epimer. In addition, 2-epi-largazole displayed more potent activity than largazole in cell viability assays against PC-3 and LNCaP prostate cancer cell lines.

Total synthesis of phorboxazole A via de novo oxazole formation: convergent total synthesis.

The phorboxazoles are mixed non-ribosomal peptide synthase/polyketide synthase biosynthetic products that embody polyketide domains joined via two serine-derived oxazole moieties. Total syntheses of phorboxazole A and analogues have been developed that rely upon the convergent coupling of three fragments via biomimetically inspired de novo oxazole formation. First, the macrolide-containing domain of phorboxazole A was assembled from C3-C17 and C18-C30 building blocks via formation of the C16-C18 oxazole, followed by macrolide ring closure involving an intramolecular Still-Genarri olefination at C2-C3. Alternatively, a ring-closing metathesis process was optimized to deliver the natural product's (2Z)-acrylate with remarkable geometrical selectivity. The C31-C46 side-chain domain was then appended to the macrolide by a second serine amide-derived oxazole assembly. Minimal deprotection then afforded phorboxazole A. This generally effective strategy was then dramatically abbreviated by employing a total synthesis approach wherein both of the natural product's oxazole moieties were installed simultaneously. A key bis-amide precursor to the bis-oxazole was formed in a chemoselective one-pot, bis-amidation sequence without the use of amino or carboxyl protecting groups. Thereafter, both oxazoles were formed from the key C18 and C31 bis-N-(1-hydroxyalkan-2-yl)amide in a simultaneous fashion, involving oxidation-cyclodehydrations. This synthetic strategy provides a total synthesis of phorboxazole A in 18% yield over nine steps from C3-C17 and C18-C30 synthetic fragments. It illustrates the utility of a synthetic design to form a mixed non-ribosomal peptide synthase/polyketide synthase biosynthetic product based upon biomimetic oxazole formation initiated by amide bond formation to join synthetic building blocks.

Total synthesis of phorboxazole A via de novo oxazole formation: strategy and component assembly.

The phorboxazole natural products are among the most potent inhibitors of cancer cell division, but they are essentially unavailable from natural sources at present. Laboratory syntheses based upon tri-component fragment coupling strategies have been developed that provide phorboxazole A and analogues in a reliable manner and with unprecedented efficiency. This has been orchestrated to occur via the sequential or simultaneous formation of both of the natural product's oxazole moieties from two serine-derived amides, involving oxidation-cyclodehydrations. The optimized preparation of three pre-assembled components, representing carbons 3-17, 18-30, and 31-46, has been developed. This article details the design and syntheses of these three essential building blocks. The convergent coupling approach is designed to facilitate the incorporation of structural changes within each component to generate unnatural analogues, targeting those with enhanced therapeutic potential and efficacy.