Article pubs.acs.org/jnp
Biosynthetic Studies of 13-Desmethylspirolide C Produced by Alexandrium ostenfeldii (= A. peruvianum): Rationalization of the Biosynthetic Pathway Following Incorporation of 13C‑Labeled Methionine and Application of the Odd−Even Rule of Methylation Matthew Anttila, Wendy Strangman, Robert York, Carmelo Tomas, and Jeffrey L. C. Wright* UNCW Center for Marine Science, 5600 Marvin K. Moss Lane, Wilmington, North Carolina 28409, United States S Supporting Information *
ABSTRACT: Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding 13C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd−even methylation rule, the complete biosynthetic pathway has been established. inoflagellates are a large group of unicellular flagellate protists that constitute the phylum Dinoflagellata. They are most commonly found as marine plankton and can form large blooms, but they can occur in freshwater habitats as well. Many marine strains are notorious for forming blooms that are toxic. The toxins produced by these organisms have a wide range of extremely potent bioactivities.1 The chemistry of these dinoflagellate toxins and indeed nearly all of their secondary metabolites is distinctly unique with almost no precedents in nature. This arises due to some unique features of dinoflagellate biosynthesis, which invokes many unusual or rare biosynthetic steps.2 There are many challenges to studying the biosynthesis of dinoflagellate secondary metabolites. Dinoflagellate genomes are quite large, ranging in size from approximately half the size of the human genome for the smallest (Symbiodinium minutum) to roughly 100× the size of the human genome.3 Coupled with the size issue are further challenges in that these genomes have modified DNA bases, exist in liquid-crystalline permanently condensed chromosomes that lack nucleosomes, and do not organize into the conserved motifs common to most eukaryotic species.3−7 Because of these massive hurdles, by far the most effective approach to studying dinoflagellate biosynthesis has been through 13C, 15N, and 18O labeling studies with biosynthetic precursors (Figure 1). These labeling studies have shown that the biosynthesis of dinoflagellate polyketides and hybrid polyketides does not follow the polyketide logic observed in most bacteria and fungi.
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© XXXX American Chemical Society and American Society of Pharmacognosy
While intact acetate units provide the majority of the building blocks of the dinoflagellate polyketide chain, they nevertheless always contain cleaved acetate units where the carboxyl carbon of an acetate unit has been excised or deleted.2 In addition, while the pendant methyl groups of polyketides from fungi are almost always derived from S-adenosylmethionine (or SAM) and in bacteria from SAM or methylmalonyl CoA, such groups in dinoflagellate metabolites are usually derived from the methyl carbon of acetate and in a few cases in combination with a methionine source. The unpredictable carbon deletion steps and variety of methylation processes make it difficult to foretell the biosynthesis of a dinoflagellate polyketide. This is compounded by the fact that in dinoflagellates it is not uncommon for extensive scrambling of the labeled acetate to occur, rendering interpretation of the biosynthetic data even more tenuous.15−18 Nonetheless, some notable successes have been reported (Figure 1), and while a common theme of biosynthetic manipulations has been observed, there is no clear repeatable biosynthetic logic that can be consistently applied to each case studied. However, there are some processes that are predictable and unvarying, and these involve the origin of the pendant methyl groups. Using the application of methylation Special Issue: Special Issue in Honor of John Blunt and Murray Munro Received: September 30, 2015
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Figure 1. Structures representing notable biosynthetic labeling studies of dinoflagellate compounds: amphidinolide C (1),8 amphidinolide B (2),9 amphidinol 4 (3),10 goniodomin (4),11 okadaic acid (5),12 brevetoxin B (6),15−17 yessotoxin (7).13,14
patterns following the incorporation of 13C-labeled methionine, we have reinvestigated the biosynthesis of 13-desmethylspirolide C (9), produced by Alexandrium ostenfeldii, and propose a modified assembly process that does follow known methylation patterns in dinoflagellates. The occurrence of acetate-derived methyl groups is a characteristic of all dinoflagellate polyketides and occurs by attack of the nucleophilic carbon of malonyl CoA on an electrophilic carbon in the polyketide chain by an aldol-type mechanism. Such a mechanism requires that the alkylated carbon of the polyketide chain be a carbonyl group. This can be accommodated when the carbon is the carboxyl carbon of an intact acetate unit that has not undergone reduction and is referred to as β-methylation.19−21 However, when the carboxyl carbon of an intact acetate unit is deleted from the chain by a Favorskii-style reaction, the remaining methyl carbon of the acetate unit is left in the carbonyl form, providing another site for aldol-methylation to occur.12,22 We describe this as pseudo α-alkylation.2 In some dinoflagellate metabolites such as brevetoxin B (6)15−17 and goniodomin (4),11 pendant methyl groups can also be derived via the more usual SAM pathway,
which is very common in bacterial and fungal polyketides and requires attack by a nucleophilic carbon in the polyketide chain on the electrophilic carbon of SAM. All these labeling patterns have been consistently observed in the biosynthesis of every dinoflagellate polyketide that has been reported to date, and while the pattern of incorporation of intact and cleaved acetate units is unpredictable, the mechanism for the addition of pendant methyl groups from acetate is not. Because pendant alkyl groups can be added to a dinoflagellate polyketide chain by any of the three processes, namely, by SAM addition, by β-alkylation, and by pseudo αalkylation, it results in the odd−even rule of methylation.2 These various processes are illustrated in Figure 2. This combination of alkylation processes during biosynthesis can result in the introduced methyl or alkyl groups separated by an odd or even number of carbons along the polyketide chain. Briefly, in non-dinoflagellate polyketides, methyl groups arising by SAM addition to a nucleophilic carbon of an acetate group will result in a series of alkyl groups separated by an odd number of carbons (Figure 2A). The same separation can be observed in dinoflagellates where alkyl groups are both B
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Figure 2. Methylation patterns that can occur in dinoflagellate polyketides. {Note the models illustrated are all based on the premise that there are an odd number of deletions between the alkyl groups. When there is an even number of deletions (usually two), the models are reversed.}
introduced from acetate by β-methylation (Figure 2B). Other combinations also result in methyl or alkyl groups separated by an odd number of carbons. For example this occurs when SAM addition is followed by pseudo α-methylation (Figure 2C) or pseudo α-methylation is followed by β-methylation (Figure 2D), or when SAM addition is followed by β-methylation with an intervening excision of a carbon from an intact acetate unit (Figure 2E). Note that in the last case an odd separation can occur only if there is deletion of an intermediate carbon in the polyketide chain. Alkyl groups separated by an even number of carbons can arise by the following means: SAM alkylation followed by βalkylation (Figure 2F), two SAM methylation steps with an intervening deletion (Figure 2G), two β-alkylation steps with an intervening deletion (Figure 2H), β-alkylation followed by a pseudo α-alkylation step (Figure 2I), and pseudo α-alkylation followed by SAM addition with no intervening excision (Figure 2J). In the odd−even rule all these processes are counted from the starter end of the nascent polyketide chain since pseudo αalkylation followed by β-alkylation can result in an odd carbon separation (Figure 2D), but an even separation when βalkylation precedes pseudo α-alkylation (Figure 2I). The same phenomenon is observed when pseudo α-alkylation precedes SAM (Figure 2J; even separation) compared with the case where SAM precedes pseudo α-alkylation (Figure 2C; odd
separation). While often complex, it is still possible to determine the direction of a dinoflagellate polyketide chain, and so these pendant alkyl patterns become very useful in predicting the incorporation and nature of the acetate units in the carbon backbone. Thus, if it is known whether a methyl group in a dinoflagellate arises from acetate or methionine, it becomes possible to determine whether carbon deletion of an intervening acetate unit has occurred or not, provided only a single deletion step has occurred. This approach has been applied to rationalize the biosynthesis of spirolides.
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RESULTS AND DISCUSSION Spirolide Biosynthesis. The spirolides were first isolated and characterized from the dinoflagellate Alexandrium ostenfeldii and display characteristic fast-acting toxicity in the mouse bioassay.23,24 Since then, this group of compounds has grown, and now they belong to a group of toxins known as the spiroimines, which also includes the gymnodimines and the pinnatoxins.25,26 Recent studies have indicated that the pinnatoxins and other spiroimines possess important biological activity.25,26 Spirolides have been shown to display potent antagonism of neuronal and muscarinic nicotinic acetylcholine receptors.27,28 Additionally, the most potent member of this family of these phycotoxins, 13-desmethylspirolide C, has been C
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Figure 3. Examples of selected spiroimine compounds.
Figure 4. Unraveled polyketide chain for spirolide revealing the revised labeling pattern for 13-desmethylspirolide C. The labeling pattern shown is derived from a combination of the original labeled acetate experiments and this current work using 13C-labeled methionine.
Figure 5. Comparison of the original (A) and revised (B) labeling patterns of 13-desmethylspirolide C.
Simple inspection of the structures of these compounds suggests a biogenetic relationship among this group of compounds, and this was underscored by the simultaneous isolation of 12-methylgymnodimine (8) and 13-desmethylspirolide C (9) from cultures of A. ostenfeldii.33 Furthermore, upon unraveling the putative polyketide chain of each member of the different toxin groups, it became clear that spirolides can be regarded as a biogenetic composite of the gymnodimines and pinnatoxins (Figure 4).32,33 A retro-biosynthetic analysis indicates a key step in the process is a Diels−Alder cyclization of a linear polyketide chain to create the spiroimine function as originally proposed for the biogenesis of the pinnatoxins.31 The challenges of labeling studies of dinoflagellate metabolites are well known and involve low yields of labeled
shown to decrease levels of hyperphosphorylated tau isoforms and intracellular amyloid beta pathogenic proteins within triply transgenic mouse models bred to display Alzheimer’s disease.29 The potent biological activities of 13-desmethylspirolide C may allow it to serve as an important tool in the continued development of Alzheimer’s disease therapies. Some related spiroimine derivatives are shown in Figure 3. The gymnodimines were originally isolated from another dinoflagellate Gymnodinium sp.,30 while the pinnatoxins were initially obtained from extracts of the toxic shellfish Pinna muricata.31 Later it was found that pinnatoxins H (10) and G are produced by Vulcanodinium rugosum,32 finally establishing a dinoflagellate origin for these compounds as well. D
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the case of the C-32 methyl it is added to the carbonyl group of glycine by a process analogous to β-methylation of acetate. Consequently the vicinal methyl group on C-31, which is labeled from acetate, must arise by pseudo α-methylation on an electrophilic carbon of a cleaved acetate unit. Continuing along the chain, because the methylene group on C-29 is separated by an odd number of carbons from the C-31 methyl, it follows that this group must arise by β-methylation of an intact acetate unit, as illustrated in Figure 2D. The nature of the following four carbons in the chain is uncertain in the original study, although an intact acetate unit at C-25/C-26 was observed. This is important because the C-24 methylene is separated from the C29 vinylic methyl group by an even number of carbons, thus indicating that the C-27/C-28 acetate unit is also intact (see Figure 2I). The origin of the C-24 methylene by a pseudo αalkylation process is further confirmed by its separation from the methionine-derived methyl at C-19 by an even number of carbons, as illustrated in Figure 2J, and indicates C-20 through C-23 contain two intact acetate units. The methionine origin of the methyl on C-19 immediately established that C-18/C-19 is also an intact acetate unit. Furthermore, since the original acetate labeling data revealed that C-14/C-15, C-12/C-13, and C-10/C-11 are intact units, the acetate unit C-16/C-17 must also be intact because the C-19 methyl is separated from the C9 methyl by an odd number of carbons, and hence the C-9 methyl is derived by a pseudo α-alkylation process (see Figure 2C). The origin of the C-6 methyl from methionine established not only that C-5/C-6 is an intact unit but remarkably that it undergoes both SAM and β-alkylation steps (see Figure 2E). Thus, the penultimate acetate unit (C-5/C-6) undergoes both α- and β-methylation, an extremely rare occurrence, which has only been observed in one other dinoflagellate polyketide (4).11 Furthermore, because the C-6 methyl is separated from the C-2 methyl by an odd number of carbons, which includes the intact units C-5/C-6 and C-3/C-4, it confirmed that the C-2 methyl must arise by pseudo α-alkylation (see Figure 2C) and finally that C-1 is part of a cleaved acetate unit. In conclusion, reevaluation of the original acetate labeling data and identification of methionine as the source of the methyl groups attached to C-6 and C-19 of the spirolide polyketide chain have led to a revision of a proposed biosynthetic pathway for 13-desmethylspirolide C (9) as shown in Figure 5B. It also explains the occurrence of the spirolide congeners spirolide C,24 13-desmethylspirolide C (9), and 13,19-didesmethylspirolide C,35 which must arise by the function (or absence) of a SAM methyl transferase.
compounds as well as the frequent occurrence of isotope scrambling of the labeled precursors. The original labeling study of 13-desmethylspirolide C (Figure 5A)18 established a hybrid polyketide pathway for the compound. However, despite employing several different feeding regimens, the acetate precursor was considerably scrambled, which made interpretation of precursor incorporation difficult. Nevertheless, glycine was clearly shown to be the starter unit, and many acetate units were incorporated intact, although a few were uncertain. A detailed review of the labeling patterns observed in the original study of 13-desmethylspirolide C (9), and in particular the incorporation of deuterium following the addition of 2-13CD3 acetate, clearly supports the acetate origin of the methyl groups located at C-2, C-9, C-31, and C-32 of the polyketide chain.18 The retention of two deuteriums at each of the methyl carbons is consistent with an aldol condensation involving malonate as originally observed in okadaic acid (6) biosynthesis.12,22 However, two other methyl groups at C-6 and C-19 as well as the exomethylene group at C-24 are also reported to be labeled from acetate, although they retain no deuterium. This is somewhat surprising as similar labeling experiments with other dinoflagellate metabolites would suggest retention of deuterium. However, regardless of whether they retain deuterium or not, assigning acetate-derived methyl groups at C-6 and C-19 of the polyketide chain is problematic, because these carbons are the methyl carbons of intact acetate units, and consequently their associated methyl groups would be expected to arise by a SAM pathway, rather than by an aldol condensation. In order to clarify the origin of these methyl groups in 9, cultures of the producing organism A. ostenfeldii were supplemented with 13C-methyl-methionine in two feeding cycles. After 4 weeks the cultures were harvested and the extract was processed as before to yield pure 9.34 The 13C NMR spectrum of the compound labeled from 13C-methylmethionine revealed substantial and specific enrichment of the methyl carbons at C-6 and C-19 in 9 only, hence establishing their origin by a SAM mechanism. As this process requires addition of methyl groups to the methyl carbon of an acetate unit, it confirms that C-5/C-6 and C-18/C-19 are indeed intact acetate units. Because the exomethylene carbon at C-24 is not labeled from methionine, it must indeed arise from acetate by an aldol mechanism involving the carbon of a deleted acetate unit (as there are intact units on either side of C-24), although surprisingly in the original study no deuterium is retained in the process. While the original data established that the methyl groups attached to C-9, C-31, and C-32 arise by an aldol condensation at an electrophilic carbon on the polyketide chain, the original data indicated that the acetate-derived methyl group at C-2 is attached to the methyl carbon of an intact acetate unit. As discussed above, this is not feasible and a more plausible explanation is that both C-1 and C-2 are the methyl carbons of consecutive acetate units in which the carboxyl carbons have been excised, as is seen in the biosynthesis of goniodomin.11 The labeling pattern is perhaps more clearly viewed by unraveling the putative polyketide chain as shown in Figure 4, where it becomes possible to envisage a plausible explanation for the labeling pattern observed in the original and revised studies. For example, application of the odd−even rule of methylation establishes that the adjacent acetate-derived methyl groups at C-31 and C-32 must arise by a different process. In
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EXPERIMENTAL SECTION
General Experimental Procedures. NMR spectra were acquired in CD3OD on a Bruker Avance spectrometer operating at Larmor frequencies of 500 and 125 MHz for 1H and 13C, respectively, with a 1.7 mm TXI probe. NMR data were analyzed using Topspin 2.0 (Bruker Biospin, Inc.). LC/MS data were acquired on a Waters/ Micromass ZQ with an ESI interface coupled to an Agilent 1100 HPLC system with a diode array detector. Preparative HPLC was accomplished using a system with two Waters 515 HPLC pumps, a gradient controller, and a Waters 2487 dual-wavelength UV detector. Extraction and Isolation. A clonal culture of Alexandrium ostenfeldii AP0411 (previously called A. peruvianum) collected from a bloom in Wickford Cove, RI, in 2009, and currently stored in UNCW’s Toxic Algal Culture Collection as CMSTACC Clone AP0905-1 was grown in an IKA PBR 10 photobioreactor using modified L1Medium33 at 22 °C and 65 mol quanta/m2/s or in 12 L E
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glass carboys. When culture achieved stationary phase, cells were harvested with a Sorvall RC2 continuous flow centrifuge as a moist pellet and either extracted immediately or stored at −80 °C until further processing. Batch cultures of A. ostenfeldii were used for labeling experiments described below as multiple (4) 10 L volumes. To this 40 L culture, 0.1 mM 13C-methyl-L-methionine was added. After 2 weeks, an additional 0.05 mM was added. After another week of growth, the cells were harvested and processed according to the previously described procedure.34 Briefly, the filtered cells of A. ostenfeldii were extracted with 80% aqueous MeOH (3 × 2 L). The extract was then filtered through glass microfiber filter papers (VWR, 691), and the filtrate dried in vacuo to yield a crude extract (0.6 g). The extract was resuspended in 1 L of 100% MeOH and dried onto 50 g of Dianion HP-20 resin in vacuo. Next, the extract-bound HP-20 resin was fractionated stepwise using 25% intervals of aqueous acetone. The 50% and 75% fractions were found to contain labeled 13desmethylspirolide C by LC-ESIMS monitoring, and they were combined and further fractionated on a 10 g Phenomenex Strata C18 SPE cartridge with a 20% step gradient of aqueous MeCN containing 0.1% TFA. The 40% and 60% fractions were found to contain labeled 13-desmethylspirolide C by LC-ESIMS and were combined. Final purification was performed by a reversed-phase C18 column (Phenomenex, Gemini 5u 110A, 250 × 10.0 mm, 5 μm) eluted under a gradient of aqueous MeCN containing 0.1% TFA (34% MeCN to 100% MeCN over 18 min) at a flow rate of 2.0 mL/min, while monitoring continuously at 214 and 245 nm. 13-Desmethylspirolide C (0.5 mg) was found to elute from the column at tR = 12.8 min.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00869. 1 H and 13C NMR spectra for 13C-methionine-enriched 13-desmethylspirolide C (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge funding from the UNCW MARBIONC program, as well as the UNCW Summer Undergraduate Research Stipend and the UNCW Carl B. Brown Trust to J.L.C.W.
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DEDICATION Dedicated to Professors John Blunt and Murray Munro, of the University of Canterbury, for their pioneering work on bioactive marine natural products.
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REFERENCES
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