Polyketide biosynthesis in dinoflagellates: what makes it different?

Volume 31 Number 9 September 2014 Pages 1077–1232

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REVIEW ARTICLE Jeffrey L. C. Wright et al. Polyketide biosynthesis in dinoflagellates: what makes it different?

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Polyketide biosynthesis in dinoflagellates: what makes it different? Cite this: Nat. Prod. Rep., 2014, 31, 1101

Ryan M. Van Wagoner,a Masayuki Satakeb and Jeffrey L. C. Wright*c

Covering: up to 2013 Dinoflagellates produce unique polyketides characterized by their size and complexity. The biosynthesis of a limited number of such metabolites has been reported, with studies largely hampered by the low yield of compounds and the severe scrambling of label in the isotopically-labeled precursors. Nonetheless, of the successful biosynthetic experiments that have been reported, many surprising and unique processes have been discovered. This knowledge has been accessed through a series of biochemical labeling studies, and Received 10th February 2014

while limited molecular genetic data has been amassed, it is still in the early stages of development. In an attempt to meet this challenge, this review has compared some of the biosynthetic processes with

DOI: 10.1039/c4np00016a

similar ones identified in other microbes such as bacteria and myxobacteria, with the idea that similar

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genes and enzymes are employed by dinoflagellates.

1 2 3 4 4.1 4.2 4.3 5 6 7 8 8.1 9 9.1 9.2 10 11 12 13 14

Introduction Dinoagellates and their metabolites Assembly of a typical polyketide chain Dinoagellate polyketide biosynthesis Carbon deletion b-Alkylation Pseudo a-alkylation Other dinoagellate polyketides Odd–even rule of methylation Starter groups and hybrid molecules Additional elaborations of the polyketide chain Chain termination and release Construction of polyether ring systems Basic mechanisms Examples from other organisms relevant to polyether formation Ether ring formation in okadaic acid Compounds containing isolated ether rings or spiro ring assemblies Fused-ring systems Mixed polyethers Concluding remarks

a

Sports Medicine Research and Testing Laboratory, 560 Arapeen Dr. Ste 150A, Salt Lake City, UT 84108, USA

b

Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

c Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K. Moss Ln., Wilmington, NC 28409, USA. E-mail: [email protected]; Fax: +1 910 962 2410; Tel: +1 910 962 2397

This journal is © The Royal Society of Chemistry 2014

1

Introduction

In the world of secondary metabolism, polyketides are a signicant biosynthetic group, and account for the majority of all reported secondary metabolites. These compounds are assembled by the incorporation of small acid units such as acetate, propionate, and to a lesser extent butyrate and others, though other small organic metabolites such as glycerol and glycolic acid can also be utilized. Even further variety can be added by the incorporation of amino acids along the chain to generate hybrid polyketides. They are found as metabolites of bacteria, fungi, phytoplankton, plants and invertebrates. Their distribution and structural diversity is even more remarkable when it is considered that they are all assembled from the same biosynthetic units – usually acetate. Marine dinoagellates are almost exclusively focused on the production of polyketides, and could be regarded as perfect examples for the chemotaxonomic case. To add further exclusivity, a signature feature of dinoagellate polyketides is their polyol nature, the frequent occurrence of ve- six- seven- eight- and nine-membered ether rings either fused in a ladder frame structure or as spirocyclic rings or both. Dinoagellate compounds containing nitrogen are rarer, and of the alkaloids that have been reported and studied, the carbon skeleton is usually assembled from acetate, with the inclusion of an amino acid (usually glycine) in the assembly process. Furthermore, only a few aromatic secondary metabolites, whether acetate- or shikimate-derived, have been isolated from dinoagellate cells or extracts, nor have many secondary terpenoids (where we do not include carotenoid pigments as secondary metabolites) been conclusively shown to

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arise from dinoagellates. In a sense, dinoagellates can be said to be totally invested in polyketide production as a platform for secondary metabolism. As with a specialized master crasman, this focus has led to unique, sometimes startling innovations in the process of polyketide assembly unparalleled among other polyketide producers such as bacteria and fungi. The purpose of this report is to present a detailed review of current knowledge of the biogenesis of dinoagellate polyketides, and by comparison with polyketide biosynthesis in other families of organisms, highlight the unique features of the dinoagellate process. As a result of the unique structural complexity of dinoagellate metabolites, and hence their biogenesis, the rst part of the review examines the construction of the carbon chain or backbone, while the second part follows on from this and examines the assembly processes invoked to construct the ether ring systems. Since very little is known about the genetics of dinoagellate polyketides, this review is conned to the chemical and biochemical studies that have been reported.

Ryan Van Wagoner obtained his PhD in Medicinal Chemistry from the University of Utah studying marine natural products under Chris Ireland. He did postdoctoral work with Jon Clardy at Cornell University and Harvard Medical School studying the structure and function of biosynthetic enzymes. He explored marine biotechnology applications of dinoagellates at UNC Wilmington. He returned to the Ireland group at Utah to further develop application of modern drug discovery techniques to natural products. He recently le academia and natural products to work on detection of use of performance-enhancing drugs by athletes at the Sports Medicine Research and Testing Laboratory.

Masayuki Satake obtained his PhD at Tohoku University in 1994 supervised by Prof. Takeshi Yasumoto. He spent time at the UNC Wilmington Center for Marine Science from 2005 to 2006 as a visiting research professor. Now he is an associate professor at the Department of Chemistry, of the University of Tokyo. His current interests are structural elucidation and biosynthesis of marine polyether compounds.

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2 Dinoflagellates and their metabolites Dinoagellates are eukaryotic agellated armored cells that are either benthic or pelagic. They are most famously known for the dense blooms they can form in the ocean, oen resulting in the so-called red tides,1,2 though not all blooms are red since this is determined by the predominant pigments of the plastid. Benthic species are usually found on the blades of seaweeds, or the surfaces of corals, sponges, and shellsh. Both benthic and pelagic strains can produce toxins, and it is this characteristic in particular that has to a large extent driven studies on the chemistry and biochemistry of dinoagellates. But for a few exceptions such as the saxitoxins, pinnatoxins, and symbioimine (40),3,4 the majority of dinoagellate compounds are lipophilic and display a remarkable range of potent biological activity, including ion channel modulation, phosphatase inhibition, hemolysis, mycotoxicity and cytotoxicity to name but a few.5–7 It has been suggested that the high degree of potency displayed by these marine compounds is possibly to compensate for the high dilution effect that occurs when such compounds are released into the water column.8 Apart from the massive size and structural diversity of dinoagellate metabolites,9–11 simple inspection of all the dinoagellate metabolites quickly underscores their unique structural features (Fig. 1). Perhaps the most notable is the frequent occurrence of ve-, six-, seven-, eight-, and even ninemembered ether rings. These rings can be found in isolation (e.g. amphidinolide C (3)) in spiroketal formation (e.g. okadaic acid (2)), as two fused rings (e.g. 2), or as many as fourteen ether rings continuously fused (e.g. brevetoxin B (1)). These structures pose some interesting biosynthetic questions, but in comparison with bacteria and fungi, there have been comparatively few biosynthetic studies of dinoagellate metabolites. There are several reasons for this including the low yields of microalgal metabolites requiring very large scale labeling experiments (oen >100 L), poor incorporation of precursors, and in some

Jeffrey Wright is the Carl B. Brown Distinguished Professor of Marine Science at UNC Wilmington. He obtained a BSc and PhD at the University of Glasgow, followed by a post-doctoral position at the National Research Council of Canada (NRC) and helped pioneer the use of stable isotopes and NMR spectroscopy to explore biosynthetic pathways. Aer further post-doctoral studies at University College of Wales and Oxford University, he returned to the NRC in 1972. He received the Order of Canada in 1994 in recognition of his work on marine toxins. He moved to UNCW in 2000 to continue research on marine natural products.


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Fig. 1

Structures of representative dinoflagellate metabolites.

cases extensive scrambling of the precursor label. However, from the cases that have been reported, all have been shown to be polyketide in origin, though several remarkable features have emerged that underscore notable differences from the more common polyketide assembly processes. While acetate remains a key building block, the assembly and modication of the nascent polyketide chain features several unusual steps which, coupled with other post-PKS reactions, results in the characteristic repertoire of structures that we associate with dinoagellate metabolites.

3 Assembly of a typical polyketide chain The biosynthesis of polyketides has been intensively studied for decades rst from a biochemical and later from a molecular This journal is © The Royal Society of Chemistry 2014

viewpoint. The most studied examples have been developed from bacterial and fungal organisms, and from these cases an established paradigm of polyketide biosynthesis has emerged. Collie12 rst suggested that certain natural products are assembled from activated C2 units derived from acetic acid, and he described such compounds as “polyketenes” or later as “polyketides” as they became known. Many decades later the hypothesis was further expanded by comparison of the chemical structure of various common metabolites13 and denitive biochemical proof of this hypothesis was obtained following incorporation of 14C-labeled acetate into methyl salicylic acid in a head-to-tail fashion consistent with this hypothesis.14,15 Later it was suggested that acyl-CoA was the active component in the assembly process.16 Around this time other radioisotope labeling studies quickly followed,17,18 which highlighted recurring structural features that identied a natural product as a

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polyketide and which could be used to conrm the validity of a new structure. Since those early days, an abundance of isotope labeling experiments of bacterial and fungal products, greatly facilitated by the use of magnetic isotopes in conjunction with 13 C NMR spectroscopy,19–21 have provided substantial insight into how such building blocks are arranged and processed in patterns largely consistent with Collie's original hypothesis (for a detailed historical perspective of these early years, and more recent developments consult the cited reviews17,18). In particular, the use of doubly-labeled acetate not only revealed the incorporation of intact acetate units, but also folding patterns invoked in the biosynthesis of polycyclic aromatics for example,19 and provided important quantitative information regarding the level of isotope enrichment at each labeled position.22 The generalities of polyketide biosynthesis have been extensively reviewed over the decades and will be briey described here (for a succinct review of the different polyketide assembly processes see Weissman23). Typically, the biosynthetic process employed for polyketide biosynthesis is similar to the assembly of fatty acids from acetate, in that initiation of the polyketide chain begins with acetyl CoA and is extended by a series of Claisen ester condensations with “activated acetate”

Review

units in the form of malonyl CoA until a polyketide chain of the required length and functionality is obtained. In fatty acid biosynthesis each acetate unit undergoes ketoreduction (KR), dehydration (DH), and enoyl reduction (ER), which results in a saturated acyl chain with little functionality (Fig. 2A). On the other hand, the structural variety of polyketide secondary metabolites arises when some or all of the polyketide processing steps are omitted or skipped, resulting in nascent polyketide chains containing carbonyl groups (lack of KR function), hydroxyl groups (lack of DH function) and double bonds (lack of an ER function) (Fig. 2A). It was recognized early that initiation of the polyketide chain did not necessarily require acetyl CoA, and almost any naturally occurring acyl CoA ester might be utilized to initiate chain formation of a particular metabolite. Furthermore, we know now that even the extender building blocks are not always restricted to acetate, and other simple acids can participate in this process, particularly propionate, occasionally butyrate, and others. A notable example of this is erythromycin (29) in which propionate is used as a starter unit that is extended with six additional methyl malonyl units,24,25 leading to assembly of a polyketide chain with a series of methyl branches derived from the propionate building blocks (Fig. 2B). However, in typical fungal or bacterial polyketides, such

(A) Illustrates the general outline of polyketide biosynthesis which if all the functions are employed results in a saturated fatty acid. (B) Shows two different methods employed to obtain a methyl branch in the nascent polyketide chain. The first shows a methyl introduced from S-adenosyl methionine (SAM) while the other uses propionate as a building block rather than acetate. (C) Illustrates a less frequent approach to introducing a methyl branch from a malonate unit via an HMG-CoA based enzyme.

Fig. 2

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pendant methyl groups along the chain are usually derived by addition of methyl from S-adenosyl methionine (SAM) to the nucleophilic (methyl) carbon of acetate in a process now referred to as a-alkylation (Fig. 2C). While these processes are consistent and predictable for most polyketides, sometimes referred to as polyketide logic,26–28 this is certainly not the case for dinoagellate polyketides. Of the dinoagellate examples studied to date the established rules of polyketide assembly are frequently broken, leading to the unpredictable use and processing of the basic acetate building blocks themselves, which in turn results in unexpected substitution and functionalization of the polyketide chain. There have been several reviews describing the labeling results with dinoagellate polyketides.29–38 In addition the reader is referred to reviews on the biosynthesis of cyanobacterial39–41 and myxobacterial42–45 metabolites for interesting and perhaps signicant comparisons with the dinoagellate case.

4 Dinoflagellate polyketide biosynthesis 4.1

Carbon deletion

As described above, typical polyketide chains are built up from an acetate starter unit followed by the successive addition of intact acetate units (activated as malonates) to create a chain of predetermined length and functionality. In almost every polyketide this is a consistent and reproducible pattern, to the point that the “paper biogenesis” of a putative polyketide can be reasonably proposed as originally described by Birch.15 Biosynthetic studies of brevetoxin B (PbTx-2; 1) were the rst to explore the assembly of a dinoagellate metabolite.46–48 While the fused ether system was in itself novel, some rather startling labeling data emerged following the incorporation of labeled acetate and methionine. Firstly, the polyketide chain appears to be

Fig. 3 The distribution of label following incorporation of 13C-labeled acetate. The carbon backbone contains intact acetate units as well as ones that have been cleaved leaving the methyl group (red circles) within the chain. The pendant methyl groups are either derived from SAM or from acetate by the HMG-CoA pathway.


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Fig. 4 The distribution of label in okadaic acid (2) and DTX-1 (6) following incorporation of 1-13C, 2-13C, and 1,2-13C acetate. The methyl group at C-35 in DTX-1 is also labeled from the methyl group of an acetate. The incorporation of labeled glycolate is also shown (green). Two acetate units undergo cleavage, but one of these may be the result of release of the polyketide from the synthase.

assembled from an intact malonate, much like that observed in iso-migrastatin biosynthesis,49 although in the brevetoxin case the carboxyl group is used to form a lactone ring. Secondly, although the labeling results established that brevetoxin was a polyketide, the studies showed that the polyketide chain was not assembled by successive addition of acetate units, but rather that the chain contained both intact and cleaved acetate units where the acetate carboxyl carbon had been excised or deleted (Fig. 3). Furthermore, the pendant alkyl groups found along the chain were derived from both methionine, and surprisingly, the methyl carbon of acetate. At the time it was proposed that the labeling pattern in brevetoxin could be explained by the incorporation of succinate and a-ketoglutarate precursors into the growing polyketide chain. This postulate as outlined, suffered from the fact that the nascent polyketide chain would have to detach from and then reattach to the PKS enzyme. Nonetheless, subsequent investigations of other dinoagellate metabolites including okadaic acid (2) established brevetoxin as a model child of dinoagellate polyketide biosynthesis. Early studies of okadaic acid biosynthesis50 produced by Prorocentrum lima, revealed a similar unpredictable acetate

Fig. 5 Proposed mechanism for the deletion of the carboxyl carbon from an intact acetate unit employing a flavin monooxygenase.

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incorporation which was interpreted as a mixed biogenesis from acetate and mevalonate precursors. Later biosynthetic studies of okadaic acid (2) and DTX-1 (6) including related

The cleavage and deletion of the carboxyl carbon of an acetate unit via a Favorskii rearrangement. The remaining methyl carbon of the cleaved unit is shown in red.

Fig. 6

Review

derivatives produced by the dinoagellate P. lima,51–53 revealed a similar series of carbon deletion steps, and acetate-derived pendant alkyl groups (Fig. 4). A detailed quantitative evaluation of isotope enrichment in the molecule labeled from single- and doubly-labeled acetate established that all the carbons were equally enriched.22 This indicated that all the okadaic acid acetate building blocks arose from the same biogenetic pool and that other biogenetic precursors were not involved since this would lead to variations in isotope enrichment along the polyketide chain. This result inevitably led to the proposal that carboxyl-derived carbons of intact acetate units are deleted from the polyketide chain by a Favorskii rearrangement catalyzed by a P450 or avin monooxygenase22,54 (Fig. 5). A similar labeling pattern for other related okadaic acid esters has been reported.55 A Favorskii rearrangement resulting in excision of a carbon from the polyketide chain is extremely rare in polyketides and was noted in early labeling studies of the bacterial product vulgamycin (also known as enterocin (8)) from Streptomyces hygroscopicus and S. lividans,56 the myxobacterial product ambruticin (7)42,43 and the fungal product aspyrone (9).57 In the case of 8 and 9, the carboxyl carbon is not completely excised from the molecule but is retained as a carboxyl side group, whereas in 7 and wailupemycin A (a compound closely related to enterocin),58,59 the carboxyl group is lost (Fig. 6). In spectacular contrast to this handful of natural products, this deletion step has been observed in every dinoagellate metabolite studied to date, and so can be regarded as the norm rather than the exception, hence establishing a new paradigm in dinoagellate polyketide biosynthesis. Indeed, in many cases multiple

Fig. 7 b-Alkylation in pseudomonic acid (10). (A) Distribution of label following incorporation of 13C-labeled acetate precursors and methionine. (B) Proposed scheme for the production of mupirocin H (11) the resultant product following deactivation of mupH.

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13

C


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deletion steps occur in a single dinoagellate metabolite, whereas only a single Favorskii rearrangement occurs in the bacterial and fungal examples known. In brevetoxin biosynthesis there are eleven deletion steps (though loss of the carboxyl group in the last acetate unit of the chain likely involves release from the polyketide synthase). In okadaic acid (2) there are two, with the last again likely involved in substrate release from the PKS. More recently the encM gene from S. lividans encoding for a protein involved in the Favorskii rearrangement leading to the production of enterocin (8) has been identied as an FADdependent oxygenase.60 Such proteins are involved in a number of biosynthetic oxidative processes.61 Analysis of the ambruticin gene cluster in Sorangium cellulosum uncovered several unusual biosynthetic mechanisms.62 The catalytic site responsible for the production of a putative cyclopropanone intermediate in the Favorskii rearrangement could not be identied, but a gene ambI encoding a avin monooxygenase is proposed to be involved in the Favorskii decarboxylation step. Importantly the authors note, as in the case of 8, that the Favorskii

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rearrangement occurs on the nascent chain while attached to the PKS. An alternative process requiring detachment and reattachment of the polyketide chain has been suggested.63 4.2

b-Alkylation

In the overwhelming majority of polyketides from bacteria and fungi, pendant methyl (or methylene) groups can occur by incorporation of propionate or by addition of the electrophilic methyl of S-adenosyl methionine (or formate as a C1 donor in some fungi) to a nucleophilic a-carbon in a polyketide chain (described more recently as a-alkylation, Fig. 2C) which is catalyzed by a methyl transferase enzyme located at a specic point in the polyketide synthase conglomerate. While this process is the most frequent in polyketide biosynthesis, in some bacterial metabolites a pendant alkyl group is derived from an acetate methyl which is attached to the electrophilic b-carbon of the polyketide chain. Before the studies of dinoagellate polyketide biosynthesis, such an alkylation mechanism had been observed during isotope labeling studies of pseudomonic acid (mupirocin; 10) (Fig. 7), produced by the bacterium

Other b-alkylation examples. (A) Those examples established through isotope labeling studies. (B) Those examples identified following molecular studies which identified an HMG-CoA enzyme in the biosynthetic cluster of the metabolite.

Fig. 8


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Pseudomonas aeruginosa.64 At the time it was suggested that the labeling pattern was consistent with a mixed acetate/isoprenoid pathway, and later when the mupirocin biosynthetic genes were identied, the alkylation step was shown to involve a transacting HMG-CoA synthase (HCS) cassette.65,66 Signicantly, mutation of the HCS cassette in mupirocin biosynthesis results in production of the abbreviated shunt metabolite mupirocin H (11), indicating that further elongation of the PK chain cannot proceed if the b-alkylation step is omitted. Another early b-alkylation step was observed in labeling studies of the myxovirescin A (12),67 and later genetic studies of the b-alkylation process in this myxobacterial compound identied a similar trans-acting HCS gene cluster.68–70 Molecular studies of other metabolites displaying a b-branch include the cyanobacterial products curacin (15)71 and jamaicamide (14),72 and the bacterial products pederin (16),73 bacillaene (17),74,75 difficidin (18),76 and leinamycin77,78 (Fig. 8). In all these b-alkylation examples, the data is revealing and consistent with an aldol condensation catalyzed by an HCS and several other proteins incorporated into the PKS enzyme complex. The alkylation step in these compounds and others has been reviewed in detail.79 Once again, what is an exceptional occurrence among most polyketides is in fact a regular occurrence in dinoagellate biosynthesis, and without exception the assembly of every

Review

dinoagellate metabolite studied to date contains at least one (or multiple) b-alkylation steps. As we have noted, pendant alkyl groups in brevetoxin B (PbTx-2; 1) (Fig. 3) are derived from both methionine (a-alkylation) and acetate (b-alkylation). However, in many other dinoagellate examples studied, all the alkyl groups arise exclusively from acetate. Indeed, in the biosynthesis of okadaic acid (2) and related DSP toxins, every pendant alkyl group is derived solely from acetate, though not always by a b-alkylation process (Fig. 4). Compared with okadaic acid, DTX-1 (6) contains an additional methyl group at C-35 whereas in DTX-2 (ref. 80) the common methyl group at C-31 is omitted, but the C-35 methyl is retained. A series of isotope labeling studies using 2-2H3, 2-13C-labeled acetate provided information on the mechanism of the process,22 and conrmed that the methyl groups attached at C29 and C-31 arise by a b-alkylation mechanism. As predicted these methyl groups retain only two deuterium atoms, consistent with their initial addition as malonate units. The retention of a single D at C-28 and C-32 is consistent with double bond isomerization and subsequent reduction as proposed in Fig. 9. While no molecular evidence exists at present, it seems reasonable to conclude that a similar mechanism involving an HCS-like cassette as found in various bacteria is required for this step in dinoagellates, which would be consistent with the 2H labeling data. Furthermore it is probable that the

(A) Distribution of isotope in okadaic acid (2) following incorporation of 2-2H3, 2-13C acetate. (B) Proposed mechanism for two of the balkylation steps in okadaic acid. Fig. 9

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b-alkylation step in dinoagellate biosynthesis, like those in bacterial examples, occurs during the extension of the nascent polyketide chain. Note that the retention of two deuteriums at C-18 is a result of the absence of KR and DH steps in the preceding acetate unit.


4.3

Pseudo a-alkylation

As noted above, okadaic acid (2) contains six pendant alkyl groups, ve of which are introduced by b-alkylation (Fig. 4). However, the methyl group at C-10 is added to the methyl carbon of a cleaved acetate unit in the polyketide chain. The C10 carbon is presumably in the carbonyl form following the Favorskii rearrangement, thus facilitating an aldol-based mechanism similar to that invoked in the b-alkylation process. We refer to this as pseudo a-alkylation due to the fact that alkylation occurs at the methyl carbon of acetate. Furthermore, the identical retention of deuterium at the C-10 methyl group compared with all the other methyl groups is signicant and indicates the process of alkylation likely proceeds by an aldollike mechanism. Support for a general aldol mechanism to account for the introduction of all the pendant alkyl groups in 2 was obtained following incorporation of 2-2H3, 2-13C-labeled acetate which revealed that every alkyl group (including those derived by a b-alkylation process) is equally enriched with 13C and retains two deuterium atoms (Fig. 9A).22 If alkylation resulted from nucleophilic attack by the nascent chain on an activated alkyl group, one would expect retention of three deuterium atoms. Instead, the observed retention of deuterium is consistent with an aldol-based mechanism. As a result, it is reasonable to assume that the mechanism of the process is very similar to that of b-alkylation as proposed in Fig. 9B and thus a similar suite of HMG-cassette proteins is likely involved. It is interesting to note that in brevetoxin B (1), the carbons bearing methionine-derived methyl groups (a-alkylation) are both preceded by cleaved acetate units. In a Favorskii-inspired deletion process, the remaining carbon of the cleaved acetate unit would be le as a carbonyl, thus increasing the nucleophilic nature of the methylene anked by two carbonyls.

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The timing of the b-alkylation step in the bacterial cases studied comes from the genetic data, and is linked to the location of the HMG-cassette on the PKS. However, the timing of the aldol methylation step during pseudo a-alkylation is more complex to envisage, but must occur aer the oxidative deletion step when the alkylated carbon (of the deleted unit) is present as a ketone and hence aer at least one more acetate unit has been added to the nascent polyketide chain. Such timing is also consistent with the labeling data in okadaic acid22 (2; Fig. 4 and 9A). However, since it requires that the oxidative deletion step precedes the alkylation step more constraints exist in a pseudo a-alkylation process. A proposed mechanism for this in 2 is presented in Fig. 10, which is consistent with the labeling data. If dinoagellate polyketide synthases operate by a non-iterative process, this could require that the appropriate module (or its affiliated accessory enzymes) also performs the deletion step prior to the HMG-type addition. Alternatively of

Fig. 11 Isotope distribution in goniodomin (19) following incorpora-

tion of 13C-labeled acetate and methionine.

Fig. 10 Proposed mechanism for pseudo a-alkylation at C-10 in okadaic acid (2).


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course, this addition step could occur aer formation of the entire polyketide chain, but identication of the appropriate modication sites would likely be more difficult in such a process.


5 Other dinoflagellate polyketides In contrast to most bacterial and fungal polyketides, the elaborate processing that occurs during the biosynthesis of all dinoagellate polyketides makes it extremely difficult to identify or unravel the “paper biogenesis” of these compounds. In fact this can only be determined with any condence by labeling experiments. Thus, where labeling studies have been performed, the biosynthesis of other dinoagellate metabolites continues the themes established in brevetoxin B (1) and okadaic acid (2) assembly, namely carbon deletion, a- and b-alkylation as well as pseudo a-alkylation. These examples are discussed below. A complex labeling pattern was found in goniodomin (19) following a series of labeling experiments (Fig. 11). This compound is a cyclic polyether produced by Alexandrium hiranoi possessing a spiro-ether ring system, and contains six deleted acetate units. In other respects, the labeling pattern is similar to the brevetoxin case in that pendant alkyl groups are derived from both acetate and methionine.81 One methyl is derived from methionine, while the other six arise by three b-alkylation and three pseudo a-alkylation steps. Amphidinol 4 (20), produced by Amphidinium carterae is assembled in a similar fashion to other dinoagellate metabolites and contains ve cleaved acetate units one of which is the nal acetate unit82 (Fig. 12). A more recent biosynthetic study of amphidinol 17 (21) another amphidinol congener,83 reveals seven deletion steps (Fig. 12), although the so-called hairpin

Fig. 12

Fig. 13 Labeling patterns in amphidinolides B (22) and C (3), illustrating the considerable processing of the nascent polyketide chain.

loop and polyene section of the molecule (C-25 – C-42 in AM17) displayed an identical labeling pattern to that of amphidinols 2, 3, and 4. This suggests that this portion of the amphidinol synthases is highly conserved. Of all the amphidinol

Labeling pattern in amphidinols 4 (20) and 17 (21), following incorporation of various labeled acetate precursors.

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metabolites investigated, the methyl groups are always derived from acetate. A similar biosynthetic pathway can be proposed for the karlotoxins produced by the dinoagellate Karlodinium venecum.84–86 The amphidinolides represent a large group of over 30 macrolides produced by different species of Amphidinium than those which produce the amphidinols.87,88 In every case studied, a great degree of processing of the amphidinolide polyketide chain was found to occur during their biosynthesis. While patterns of similarity can be observed among various members of this family, the frequency of carbon deletion is remarkable, even compared with other dinoagellate metabolites. For example, in amphidinolides B and C (Fig. 13), there are six and ten deletion steps respectively, and in both compounds, there is

Isotope distribution in yessotoxin (23) following incorporation of 13C-labeled acetate, methionine, and glycolate.

Fig. 14

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a run of three successive deletion steps, unseen in any other dinoagellate metabolite investigated to date. Interestingly, the polyketide chain in both molecules begins with two intact acetate units, followed by three consecutive deletion steps, then a further addition of two intact acetates. This pattern is also repeated in amphidinolide H isolated from a different Amphidinium strain.87,88 Furthermore, as in all the other dinoagellate metabolites studied, these methyl-derived carbons of deleted acetate units oen bear hallmarks of having existed as a ketone intermediate based on the frequent occurrence of oxygenation or nucleophilic alkylation at such sites in the nal product. This suggests that in addition to shortening the chain, carbon deletion by a Favorskii rearrangement process can serve as a branching point for further elaboration of the nascent polyketide chain as discussed later. Yessotoxin (23) is a ladder frame polyether toxin produced by the dinoagellate Protoceratium reticulatum. Even though it is produced by a different organism, yessotoxin resembles the brevetoxins in that it contains a series of fused 6-, 7- and 8-2membered ether rings. The recent report describing the labeling pattern of yessotoxin following incorporation of various 13C-labeled precursors89 makes the comparison between the two families of toxins even more interesting. Both toxins contain cleaved acetate units as a result of a series of Favorskii rearrangements, though yessotoxin contains more (een). Like brevetoxin, yessotoxin also contains pendant methyl groups that are derived from methionine (one) and the methyl carbon of acetate (seven). In the latter process, two of these arise by b-alkylation, and the remaining ve by pseudo-a-alkylation. In addition, labeled yessotoxin displays an interesting repeat of two intact acetate units straddling the carbon of a deleted acetate unit along the carbon chain (Fig. 14). The signicance of this is not known, but a plausible explanation is that epoxidation of the double bond between the two methyl-derived

Fig. 15 Three related spirolide derivatives identified by variations in the pattern of methyl substitution at C-13 and C-19. The revised labeling pattern for 13-desmethyl spirolide C (25) is proposed based on the results obtained following incorporation of methyl-13C methionine.


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carbons of each unit (one from a deleted acetate the other part of an intact unit) leads nicely to an arrangement that would promote ether ring formation. In such a mechanism, the double bond might result during the deletion step process. With the exception of the 8-membered ring, all the remaining 6membered rings can be formed by just such a mechanism. Brevetoxin B (1) also contains a few similar arrangements of precursor units but there is no correlation with the construction of 6-membered rings in this molecule. A later study to explore the origin of the oxygen atoms in yessotoxin determined that all the ether rings arise from O2, which is consistent with the proposed biosynthetic scheme90 and is discussed later in this report. Labeling studies have been reported for 13-desmethyl spirolide C (25) using 13C-acetate precursors.91 The study was hampered by scrambling of the label following administration of the labeled compounds to cultures of Alexandrium ostenfeldii. This is not uncommon in labeling studies of dinoagellate metabolites and proved a similar challenge in the studies of brevetoxin biosynthesis. Despite this, a labeling pattern was proposed for 25 which meets many of the anticipated results, though some anomalies do exist. One unambiguous result was the conrmation of glycine as a starter unit in the polyketide chain. However, several methyl carbons presumed to be derived from acetate are not consistent with either a pseudo a-alkylation or b-alkylation process. We (Wright, unpublished results) have recently found that both methyl groups at C-6 and C-19 are

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labeled from 13C methionine, hence a revised labeling pattern can be proposed (Fig. 15). Thus the involvement (or not) of a methionine methyl transferase at specic points in the assembly of the polyketide chain nicely accounts for the production of spirolide C (24), 13-desmethyl spirolide C (25), and 13,19-didesmethyl spirolide C (26). The structural similarity of spirolides to the pinnatoxins and gymnodimines, suggests that these compounds may share common biosynthetic genes. This notion was further supported by the discovery that 12-methyl gymnodimine and 13-desmethyl spirolide C (25) are produced simultaneously by a strain of Alexandrium peruvianum.92 Indeed, by comparison of the putative unfolded nascent chain of gymnodimine (27), spirolide, and pinnatoxin (28), it appears that spirolide is a composite of both gymnodimine and pinnatoxin biosynthetic genes. From the glycine starter unit to about 2/3rds of the chain length, the linear precursor of spirolide is identical to that for pinnatoxin. The remainder of the nascent linear spirolide is identical to gymnodimine (Fig. 16). This correspondence is tantalizingly suggestive of a genetic recombination event combining two divergent pathways to yield a third pathway. The ability of A. peruvianum to produce both spirolide and gymnodimine simultaneously provides more evidence of a potential genetic linkage between the two pathways. Furthermore, the production of gymnodimine in K. selliformis and A. peruvianum, two dinoagellates from distinct phylogenetic groups (orders

Fig. 16 Overlay of the three unfolded spiroimine nascent polyketide chains. The spirolide chain appears as a composite of gymnodimine (27) and pinnatoxin (28) biosynthesis.

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Fig. 17 Examples of common methyl substituted polyketides isolated from bacteria and fungi illustrating the common carbon separation between methyl groups.

Gymnodiniales and Peridiniales respectively) suggests possible lateral transmission of the pathway genes.

6

Odd–even rule of methylation

Although the dinoagellate metabolites described here are clearly polyketide in origin, the modications of the nascent chain and subsequent decoration to yield the nal metabolites result in specic features characteristic of dinoagellate metabolites. In particular, the cleavage of acetate units, addition of pendant methyl groups from SAM and/or the methyl of another acetate unit, inevitably lead to structural characteristics that are diagnostic of a dinoagellate polyketide. Indeed, these characteristic features coupled with structural motifs such as extended polyol chains, and multiple fused or spiro-linked

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ether rings can be used to identify the biogenetic origin of such molecules – even those isolated from a mixed assemblage of organisms. However, the most diagnostic feature of dinoagellate metabolites is perhaps the pattern of methylation along the polyketide chain. For example, typical bacterial and fungal polyketides can contain methyl groups separated by three, ve, etc., carbons, not including the starter methyl group as a consequence of SAM methylation or incorporation of propionate building blocks (Fig. 17). In other words they are separated by an odd number of carbons in the chain (incidentally this is also the case in isoprenoids such as lycopene and b-carotene). However, as a consequence of the carbon deletion steps and mixed origin of pendant methyl groups in dinoagellate metabolites (e.g. via aldol, SAM reactions), the methyl groups are frequently either vicinal or separated by an even number of carbons. This is a consistent feature of all dinoagellate polyketides and is a consequence of carbon deletion steps occurring between alkylation steps, or pendant alkyl groups derived from SAM and acetate in the same molecule. The rule is conveniently illustrated by tracing along the polyketide chain of brevetoxin B (1) or okadaic acid (2), where methyl groups are separated by an even number of carbons: for example, in 1 the SAM-derived methyl at C-8 and the methyl at C-13 are separated by six carbons as a result of a Favorskiideletion at C-10 and pseudo-a-alkylation at C-13 in the nascent chain (Fig. 18). In the same molecule, both SAM-derived methyl groups at C-22 and C-25 are separated by four carbons as a result of one Favorskii-deletion at C-24. In 2 the acetate-derived methyl groups positioned at C-10 and C-13 arise by the same aldol condensation process and might be expected to be separated by an odd number of carbons, but instead are separated by an even number of carbons as a result of a carbon deletion step (at C-10) in an acetate unit separating them (Fig. 18). The mere occurrence of just one case where two methyl groups in a molecule are separated by an even number of

Fig. 18 Examples of common dinoflagellate metabolites highlighting the carbon separation between several of the methyl groups in the carbon backbone.


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Fig. 19 Altohyrtin (34) and halichondrin (35) both isolated from sponges, but likely the products of associated dinoflagellates based on the odd–

even rule.

carbons is enough to signify that there has been a carbon deletion step or that the methyl groups arise from different sources such as methionine or acetate. In fact, every dinoagellate molecule reported to date contains methyl or vinylic methylene groups separated by an even number of carbons. Examples include goniodomin (19) where several pendant alkyl groups are separated by an even number of carbons (Fig. 10 and 18; C-33 and C-34 methyls; C-25 and C-12 vinyl groups; C-8 and C-3 vinyl groups). Once again this is a consequence of the interplay of carbon deletion and SAM/acetate alkylation. Other examples include the amphidinols (Fig. 11) and amphidinolides (Fig. 12). Indeed, of the few exceptional metabolites from non-dinoagellate sources that exhibit methyl groups separated by an even number of carbons, they do so because of the presence of SAM and acetate-derived methyl branches in the same molecule. See the review by Calderone79 for other examples in addition to those discussed in this report. In all these

non-dinoagellate examples there are no cases of pseudo a-alkylation and only myxovirescin (12)68,70 and thailandamide28,93,94 have more than one b-alkylation step. Using this rule, it is possible to conjecture the origin of a compound isolated from a mixed assemblage such as a sponge extract. For example in halichondrin (35) isolated from a sponge,95 the vicinal separation of the methyl groups (C-25 and C-26) and the even separation of other methyl groups C-26 and C-31 and between C-31 and C-42 (Fig. 10) suggests that it might plausibly arise from a dinoagellate symbiont, and other structural features including spiro ether rings support this idea. Perhaps signicantly okadaic acid (2) was isolated from the same sponge,96 perhaps suggesting a common dinoagellate source. Yet another example of this odd/even arrangement of pendant methyl groups occurs in altohyrtin (34; spongistatin 1), a compound isolated from sponge extracts97,98 which most likely arises from a dinoagellate source (Fig. 19).

Fig. 20 The labeling patterns observed in sulfated side chains of DTX-4 (36) and DTX-5b (37). Glycolate is used as a starter unit and glycine (see below) is used as an extender unit in DTX-5b. Labeling data for DTX-5c (38) is based only on the incorporation of various labeled acetate precursors (see Fig. 22).55

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The same rule also suggests that the pinnatoxins (Fig. 16), when rst isolated from shellsh tissue,99 were also of dinoagellate origin and reassuringly it has recently been discovered that these toxins are produced by a dinoagellate identied as Vulcanodinium rugosum.100


7 Starter groups and hybrid molecules All polyketides contain a starter unit, which most commonly is acetate. However in some dinoagellate polyketides it has been shown that glycolate can act as a starter unit and by “paper biogenesis” is likely to occur in other cases described below. In the early studies of okadaic acid (2) biosynthesis it was discovered that labeled acetate was not incorporated into the rst two carbons C-38 and C-37 of the polyketide chain. Instead, a subsequent study demonstrated that these two carbons arose from glycolate, and that the glycolate oxygen atom was used in the construction of the spiroketal ring system,51 which is discussed later in this report. A labeling study53 of the sulfated diester derivative DTX-4 (36) of okadaic acid showed that the diester chain also incorporated glycolate as a starter unit, and the same result was observed in the biosynthesis of the sulfated diesters DTX 5a and b54 (Fig. 20). A similar pattern of acetate incorporation was observed with DTX-5c.55 More recently, labeling studies with yessotoxin (23) conrmed that glycolate was also a starter unit for this ladder frame polyether89 (Fig. 14). Given these results, it is tempting to postulate that goniodomin (19) is also formed from a glycolate starter unit. The results of acetate labeling experiments81 showed that the entire carbon backbone is constructed from acetate except for the putative two-carbon starter unit (C-36 and C-35) which was unlabeled (Fig. 11). Similarly, the absence of labeling in the rst two carbons of the polyketide chain of amphidinol 3 and the related AM17 also leads to the idea that

Fig. 21

Various cyclic imines that may utilize glycine as a starter unit.


glycolate serves as a starter unit (Fig. 12). However, apart from glycolic acid, no other small carboxylic acids have been shown to act as starter units in dinoagellate polyketide biosynthesis, which is in marked contrast with the number of bacterial metabolic pathways which use propionate, butyrate, isobutyrate, valeric, isovaleric acid and others as starter units. The incorporation of amino acids in a polyketide chain to form hybrid polyketides is now a well-recognized feature of secondary metabolism. Once again this is a common occurrence in bacterial, myxobacterial, and cyanobacterial metabolites41,44,45 but is a less frequent occurrence in dinoagellate biogenesis, where only glycine has been utilized. Usually the process is catalyzed by a nonribosomal peptide synthase (NRPS) and the combination of an NRPS with a PKS adds considerably to the structural diversity of secondary metabolism. Amino acids can be incorporated as starter units, or as extender units

Fig. 22 Selected putative hybrid polyketides where glycine may act as a starter or extender unit.

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in the polyketide chain, and isotopic labeling data have established both modes of amino acid incorporation to occur in hybrid dinoagellate polyketides. The cyclic spiroimines represent examples where an amino acid does (or could) function as a starter unit. For example, labeling studies conrmed that glycine acts as the starter unit in 13-desmethyl spirolide C (25) biosynthesis91 (Fig. 15). In this case, the PKS/NRPS chain undergoes a Diels Alder cyclization step to form the spiro ring junction as originally proposed in the biogenesis of the pinnatoxins.99 By analogy, this amino acid is very likely the starter unit in the biogenesis of the other cyclic imines including the gymnodimines (e.g. 27), pinnatoxin (28), spirocentrimine (39), the prorocentrolides (e.g. 4) and symbioimine (40; Fig. 21), and a glycine starter and an acetate chain have been proposed for the biogenesis of zoanthamine (41).3 Amino acids can also be incorporated as extender units of the polyketide chain, again a common occurrence in bacterial, myxobacterial, and cyanobacterial metabolites but a less frequent occurrence in dinoagellate biogenesis, and to date only glycine has been shown to be incorporated as a chain extender. For example, labeled glycine was incorporated as an intact extender unit within the polyketide chain of the sulfated diester moieties of DTX-5a (not shown) and 5b (37) (Fig. 20).54 Although there is no labeling data to conrm the hypothesis, it can be proposed that several other families of dinoagellate polyethers arise via a PKS/NRPS pathway. In these cases glycine can be incorporated as a starter or extender unit (or both as in palytoxin (5)). Some examples are: DTX-5c (38),55 zooxanthellatoxin A (42; and B), palytoxin (5), and brevisamide (33)101 though it could be argued that in the latter case glycine serves as a starter unit which is subsequently acylated (Fig. 22).

8 Additional elaborations of the polyketide chain Additional modications of the dinoagellate polyketide chain have also been observed, such as insertion of oxygen in the chain via a Bayer Villigerase (BV) reaction, presumably by avoenzymes.61 To our knowledge there is only one example of this among dinoagellate metabolites which is the insertion of oxygen in the putative sulfated monoester of okadaic acid to create the sulfated diester products DTX 4, DTX-5a and b (37), and DTX-5c (38; Fig. 20). In the case of DTX-4 (36; and other related metabolites, unpublished data) the oxygen is inserted 14 carbons from the chain terminus, and separates the two carbons of an intact acetate unit22 (Fig. 20). In the case of the hybrid diesters DTX-5a, b, and c, the oxygen is introduced 15 atoms from the chain terminus (Fig. 20). This regiospecic introduction of oxygen to form these sulfated diol esters is intriguing and may be designed to generate a storage substrate that will readily undergo hydrolysis to yield a diol ester. Furthermore, the labeling results obtained with DTX-5a and b54 indicated that the oxygen atom is introduced next to a carbon of a deleted acetate unit. If BV oxidation is post-PKS, it reinforces the idea that the Favorskii elimination step is an integral part of the polyketide process. Collectively the evidence

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further supports the suggestion that these essentially inactive sulfated diol esters are the nal biosynthetic products of the dinoagellate. 8.1

Chain termination and release

Since there is a paucity of information regarding the genetics of dinoagellate polyketide biosynthesis, the methods of chain release can only be proposed. Consequently, once again, we draw on various bacterial examples. When the chain ends with an intact acetate unit, release by a thioesterase would seem to apply. This is a common occurrence among dinoagellate metabolites, and can result in formation of a lactone ring. For those cases where the chain ends with the methyl group of a cleaved acetate group – not uncommon in bacteria and fungi – a similar mechanism of release invoking decarboxylation can be proposed. Whatever the mechanism, the methyl group is either oxidized to a primary alcohol or a carboxyl group. In the latter case this can result in formation of a lactone ring as seen in the amphidinolides, goniodomin (19), prorocentrolides (4), and spiroimines to name a few. A number of dinoagellate compounds contain a terminal vinyl group and a mechanism for this process has been established in the biosynthesis of curacin (15), a cyanobacterial product. In this case it has been shown that sulfation of the penultimate carbon of the chain followed by decarboxylation and desulfation results in a terminal vinyl group.39 This mechanism could plausibly be applied to the amphidinols which all contain a terminal olen group in the lipophilic chain and is also observed among the ciguatoxins (e.g. 64). In contrast to these examples, many, if not all the metabolites of Karenia species contain an aldehyde function. An epoxide-based mechanism has been proposed to account for this.63

9 Construction of polyether ring systems A signature feature of dinoagellate polyketides is the occurrence of spiro-linked or fused ve-, six-, seven-, eight-, and ninemembered ether rings. These rings can be incorporated either in a linear chain, or in a structure containing a series of fused ether rings to form a ladder frame, or in a macrolide backbone (Fig. 1). Notwithstanding the unique biosynthetic elaborations found in the construction of the dinoagellate polyketide chain, the construction of such polyether ring systems poses another most tantalizing feature of dinoagellate biogenesis. From a

Table 1 Baldwin's rules for cyclization relevant to dinoflagellate polyethers103

Ring size

Endo/Exo

Geometry

Favored/disfavored

3–7 3–5 6–7 3–7 5–6

Exo Endo Endo Exo Endo

Trig Trig Trig Tet Tet

Favored Disfavored Favored Favored Disfavored


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mechanistic point of view, such ether rings are likely to be derived from a few basic mechanisms, and our recent understanding of the process in bacteria may shed light on the process in dinoagellates.


9.1

Basic mechanisms

Although there are a number of possible mechanisms by which ethers can be formed in natural products,102 most of the ether ring systems in dinoagellate metabolites could result from a relatively small number of transformations of nascent polyketide chains. These mechanisms can be roughly grouped as transformations involving the intramolecular addition of a hydroxyl group to an unsaturated bond and those involving the intramolecular substitution of an oxygen leaving group with a hydroxyl group nucleophile. Before describing the transformations in more detail, it is useful to consider the empirical rules of favored and disfavored cyclizations as proposed by Baldwin (Table 1).103 To apply Baldwin's rules, the proposed cyclization is described by the size of the newly formed ring (e.g. 5 or 6), whether the functional group undergoing transformation is endo or exo with respect to the newly formed ring, and whether the functional group undergoing transformation has a geometry that is digonal (dig; e.g. alkyne), trigonal (trig; e.g. alkene or ketone), or tetrahedral (tet). The rationalizations for the rules are generally stereoelectronic in nature, and thus a favored or disfavored status is primarily a kinetic phenomenon. Of course, enzymes are capable of overcoming kinetic barriers, but overall the Baldwin rules for cyclization provide a reasonable metric for understanding when enzymatic intervention might be necessary and generally how much steric guidance might need to be provided by the enzyme active site. Some of the most likely mechanisms for ether ring formation are shown in Fig. 23. Formation of hemiketals through

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intramolecular condensation is an exo-trig phenomenon and is generally favored. Similarly, intramolecular addition of an alcohol to an alkene can be either endo-trig or exo-trig with both mechanisms being favored for most of the ring sizes seen in dinoagellate metabolites (Table 1). The main exception is 5-endo-trig which is disfavored.103–105 Cyclization by the intramolecular opening of an epoxide ring with an alcohol can be either exo-tet or endo-tet, with exo-tet being favored and endo-tet being disfavored (Table 1). Cyclization by intramolecular displacement of one hydroxyl group by another is exo-tet, which is generally favored (Table 1). For the purposes of classication within this review, only the structures of the precursors and products are considered. No consideration is given as to whether the mechanism is quasi-bimolecular (e.g. SN2-like) or quasi-unimolecular (e.g. involving carbocation intermediates). In describing the stereochemistry of epoxides and their products, it is useful to adopt the nomenclature described by Gallimore and Spencer.32 This nomenclature makes apparent their insight that epoxide conguration is uniform across putative polyepoxide precursors that eventually lead to transfused polyethers. However there is one disadvantage to this nomenclature in theory, if not oen in practice. If one of the groups directly attached to the epoxide carbons is a heavy atom (i.e. chlorine) or is bound to a heavy atom, the Cahn–Ingold– Prelog priority rules106 will affect the absolute assignment for the respective center, perhaps causing some confusion. In describing the conguration of epoxides, we will use a modied nomenclature that corresponds to the Cahn–Ingold–Prelog classication in most cases. The modied priority rules are: (1) the bond to the epoxide oxygen is assigned the highest priority. (2) The bond connecting the epoxide carbons is given second priority. (3) Other bonds involved in the nascent polyketide chain are given third priority. (4) Side-chain groups (typically H or Me) are given fourth priority. To distinguish these assignments from standard congurational assignments, a prime

Fig. 23 General mechanisms of ether ring formation of relevance to dinoflagellate polyethers. (A) Exo-trig cyclization of a ketone to a hemiketal. (B) exo-tet dehydration of a 1,n-diol. (C) endo-trig and exo-trig addition to a double bond. (D) endo-tet or exo-tet opening of an epoxide. R0 and S0 nomenclature is described in the text.


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symbol is appended to the classication. For example, the trans epoxide shown in Fig. 23-D is classied as (S0 , S0 ). Applying the same rules to the ether product with the slight modication that the bonds to the oxygen atoms for each center are assigned the highest priority makes it clear in comparing the assignments for the epoxide precursor and ether product that the carbon that undergoes nucleophilic attack is inverted (Fig. 23-D). 9.2 Examples from other organisms relevant to polyether formation By far, the group of compounds that shows the strongest structural affinity to the dinoagellate polyethers are the bacterial antibiotic and ionophore polyethers, primarily from actinomycetes. A prototypical example of the latter class of compounds is monensin A (43; Fig. 24).107 The distinctive structures of polyethers such as isolasalocid A (45) led Westley to propose that formation of the rings could occur by a cascade

Monensin A (43), a polyether antibiotic from actinomycetes. Also shown is a putative polyene precursor that undergoes multiple epoxidations by MonCII followed by cyclization reactions by the epoxide hydrolases MonBI and MonBII.

Fig. 24

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of cyclization reactions acting on a polyepoxide precursor.108 Isotopic labeling studies on monensin A using [18O2, 1-13C] acetate and [18O2, 1-13C]propionate109 and later with 18O2,110 along with similar experiments with lasalocid A (44),111 supported Westley's model. Combination of the model, labeling data, and stereochemical analysis similar to that previously applied by Celmer to macrolide congurations112,113 culminated in the Cane–Celmer–Westley unied model for polyether biogenesis.114 The unied model has since proven its value as further biogenetic experiments have provided support for it.115 Studies on the biosynthesis of bacterial polyethers have undergone a recent renaissance with the cloning of multiple biosynthetic pathways and the eventual unraveling of the various enzymatic activities involved. Indeed, so much has been learned as to inspire several reviews.35,116,117 Pathways have been identied for several bacterial polyethers including monensin A (43),118,119 nanchangmycin,120 nigericin,121 lasalocid A (44),122–124 salinomycin,125,126 and tetronomycin.127 One of the key results to emerge from these studies, so far as dinoagellate polyethers are concerned, was the identication of the enzymes monBI and monBII as critical to the correct cyclization of monensin precursors and the recognition of homology of these two proteins to limonene epoxide hydrolase.128 This discovery, coupled with the apparent epoxidase monCI, strongly bolstered

Fig. 25 Cyclization of a polyepoxide precursor to the actinomycete polyether lasalocid A (44). Enzyme-catalyzed cyclization produces the disfavored endo-tet product whereas acid-catalyzed cyclization produces the favored exo-tet product.


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the Cane–Celmer–Westley model and similar models for dinoagellate polyether biosynthesis.32,129,130 Another key result came from the pathway for lasalocid A (44). In monensin A (43), nigericin, and nanchangmycin, all of the ether rings form by exo-tet epoxide ring opening, which is the favored product according to Baldwin's rules (Table 1). However, the polyepoxide cascade model for fused-ring polyether formation in dinoagellates (discussed in more detail below) requires repeated iterations of the disfavored endo-tet cyclization mechanism. The epoxide hydrolase Lsd19 from the biosynthetic pathway of lasalocid A is able to perform a 6-endotet epoxide ring opening on a synthetic intermediate (Fig. 25), whereas treatment with acid leads to the favored 5-exo-tet product.122 X-ray crystallography studies with Lsd19, which contains two highly homologous domains, in complex with synthetic lasalocid precursor analogues provided further insights into potential mechanisms.131 The substrate-binding cavity of the N-terminal domain, Lsd19A, contained a singly exotet cyclized product of a bis-epoxide precursor bound in a deeper pocket than the corresponding cavity of the C-terminal domain, Lsd19B, which was bound to a bis exo-tet cyclized product. Computational studies with the Lsd19B domain bound to the singly exo-tet cyclized epoxide precursor (formed by Lsd19A from a synthetic bis-epoxide precursor not attached

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to ACP)132 suggested that general base activation of the nucleophilic hydroxyl group coupled with general acid stabilization of the oxyanion developed at the epoxide oxygen would facilitate formation of the thermodynamically favored 6-endo-tet product. Thus, this proves in principle that epoxide hydrolases can catalyze ether ring formation via endo-tet epoxide ring openings, even when the exo-tet cyclization product is favored in the absence of enzyme. Another example of ether ring formation with possible relevance to the dinoagellate polyethers occurs in the biosynthetic pathway for spirangien from the myxobacteria Sorangium cellulosum.133 Spirangein A (46)134 contains a 6,6 spiro-ketal bicyclic system similar to those found in several dinoagellate metabolites (Fig. 26). Analysis of the biosynthetic gene cluster revealed that the PKS genes appeared to contain a functional keto reductase module responsible for the processing of the ketal carbon in spirangein A.133 Furthermore, the gene cluster contained two P450 monooxygenase enzymes (SpiC and SpiL) that the authors hypothesized to be important in cyclizing the nascent polyketide chain.133 Disruption of SpiL was found to lead to production of a partially glycosylated acyclic product bearing a hydroxyl group at the ketal carbon of spirangein A (Fig. 26). Disruption of SpiC was found to abolish spirangein production altogether. These results suggest that SpiC and SpiL act in tandem to form the spiroketal product. It has been suggested that the ketal carbon is reduced during nascent chain biosynthesis in order to allow complete enzymatic control over cyclization during the tailoring process,135 in contrast to the bacterial polyethers which appear to retain ketone groups at the corresponding ketal positions of their nascent chains. It is also interesting to note that one of the hydroxyl groups involved in spiro ketal formation is selectively glyocosylated in the acyclic shunt product (47). While glycosylation could be a means of transporting or removing an unnatural product, it could also serve the purpose of activating the oxygen atom as a leaving group for a nucleophilic reaction with a hemiketal intermediate en route to the spiro ketal (Fig. 26).

10 Ether ring formation in okadaic acid

Spirangein A (46), a spiro ketal from the myxobacterium Sorangium cellulosum. Disruption of the SpiL gene which encodes for a P450 monooxygenase causes formation of an acyclic product that is partially glycosylated at a hydroxyl group that would otherwise be involved in ether ring formation.

Fig. 26


We begin our survey of the biogenesis of ether rings in dinoagellates with the case of okadaic acid (2) and the dinophysistoxins for two reasons. First, okadaic acid contains examples of the major types of ether rings found in all dinoagellate polyethers and hence serves as an overture of sorts. Second, and more importantly, okadaic acid and DTX-4 (36) are the compounds for which the most extensive data on 18O incorporation patterns in dinoagellates are available. Such data were crucial to the formulation of the unied model of bacterial polyether antibiotic biogenesis114 that has provided the basis for some models of dinoagellate polyether biogenesis.32,129,130 Studies on the incorporation of 18O also are the sole source of direct evidence for selected carbon–oxygen connectivities in precyclization intermediates.

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Fig. 27 Isotopic labeling patterns in okadaic acid (2). Incorporation studies on [18O2]acetate, incorporation. All other studies used NMR.

The earliest 18O incorporation studies on DTX-4 (36) made use of multiply labeled [18O2, 1-13C]acetate in conjunction with 13 C NMR to identify positions at which oxygen from acetate was retained.53 The results showed that the oxygen atoms attached to C-4 and C-27 derive from acetate whereas a similar experiment with [2-13C, 2-18O]glycolate similarly showed that the oxygen atom attached to C-38 derives from glycolate (Fig. 27). Two subsequent studies were carried out by Murata and coworkers that instead used collisionally-induced dissociation (CID) MS-MS experiments to measure enrichment levels for each oxygen atom position. These experiments have advantages over NMR-based experiments in sensitivity (both in the sense of being able to detect lower percentages of labeling and in requiring much less material for the analysis) at the cost of requiring full characterization of mass spectral fragmentation

18

O2, and [18O]water used MS/MS for detecting

pathways, having a more complex analysis of the data, and in losing the ability to detect intact incorporation of C-1/O units of acetate. The rst study explored the incorporation of 18O from [18O2]acetate and from 18O2.136 The results with [18O2]acetate corroborated the earlier results of Needham et al. and further identied the oxygen atoms attached to C-12 and C-16 as having low levels of incorporation (Fig. 27). The incorporation studies with 18O2 showed that the remaining oxygen atom positions, excepting the oxygen attached to C-38, exhibited labeling. Interestingly, the oxygen attached to C-12 incorporated label from both [18O2]acetate and 18O2. The authors indicated that only one of the two oxygen atoms attached to C-1 was labeled by 18O2, but that might be explained by hydrationmediated exchange with natural abundance water during purication.

Fig. 28 Possible mechanisms for ether ring formation in okadaic acid (2). (A) Formation of rings F and G. (B) Formation of rings D, E, and F, including an endo-tet opening of an epoxide. (C) Formation of rings A and B.

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The second study by Murata and co-workers examined incorporation from [18O]water in both okadaic acid (2) and the actinomycete polyether salinomycin.137 Salinomycin exhibited partial labeling of all acetate-derived oxygen atoms, which the authors concluded as arising from reversible hydration of ketone groups in the polyketide intermediate. By contrast, incorporation of 18O in 2 from labeled water displayed a more selective pattern of isotope enrichment (Fig. 27). Of the three oxygens derived solely from [18O2]acetate (at C-4, C-16, and C27), only the oxygen at C-16 exhibited signicant incorporation from [18O]water. This suggests that the carbonyl oxygens in okadaic acid undergo less exchange through ketone hydration, possibly by exclusion of water via proteins binding to the intermediate polyketide chain, more rapid reduction of the ketone to an alcohol by the polyketide synthase, or some other means. Also worth noting, some (but not all) of the oxygen atoms labeled by 18O2 were also labeled with [18O]water, in contrast to the results with salinomycin where no such labeling took place. While some of the labeled oxygen atoms likely exist as ketones prior to cyclization (i.e. ketal oxygens like those attached to C-8, C-19, and C-34) and hence would be subject to hydrate-mediated exchange from the time of incorporation/ oxidation to the time of cyclization, this cannot explain all such

Fig. 29 Structure of belizeanic acid (48), a partially cyclized congener of okadaic acid (2). Note that the equivalent of ring E in okadaic acid is formed, but not rings C or D, presumably because C-19 is reduced to an alcohol rather than being a ketone.

Fig. 30

occurrences of labeling in okadaic acid. For example, the carbinol at C-7 exhibited labeling by both 18O2 and [18O]water. Previous studies on the incorporation of multiply labeled [2-13CD3]acetate showed retention of deuterium at C-7, which would be impossible if C-7 existed at any time as a ketone intermediate.22 One could argue that production of 2 might occur in the chloroplast where incorporation of 18O from labeled water into 18O2 via photosynthesis might lead to locally high concentrations of labeled 18O2. However, several oxygen atoms derived from 18O2 (those attached to C-2, C-22, and C-26) did not incorporate 18O from [18O]water. Overall, the results suggest that the oxidative processes in the production of 2 draw on two pools of molecular oxygen: one that is prone to labeling by [18O]water and one that is not. One possibility might involve construction of the putative polyketide chain in one location followed by translocation to another milieu where tailoring and cyclization steps are carried out. The 18O labeling results suggest several aspects of ether ring formation in okadaic acid (2). For each ring assembly in 2, the direction of ring formation could in principle proceed from right-to-le (the same direction in which the polyketide chain is built up) or le-to-right. Strong retention of 18O from [2-13C, 2-18O]glycolate favors a right-to-le direction for rings F and G (Fig. 28). The glycolate hydroxyl attacks a ketone at C-34 (derived from the methyl carbon of acetate) whose oxygen is enriched by 18 O2 and [18O]water. Although formation of ring F might also occur by cyclizing dehydration of a 1,5-diol or 6-exo-trig cyclization, it is shown here as a favored 6-endo-trig addition103 from a putative E-alkene for illustrative purposes. Observations favoring such a mechanism include the fact that the putative double bond would occur between acetate units rather than within one, as is commonly observed in dinoagellate metabolites, and that the axial disposition of the methyl group is stereochemically consistent with anti addition.

Structures of amphezonol (49) and amphidinin B (50). Neither compound contains ether rings likely to result from opening of epoxides.


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The presence of a trans-fused ether ring juncture for rings D and E suggests an intermediate epoxide derived from an Ealkene. A cyclization process that proceeds from right-to-le (i.e. the same direction as the polyketide chain is produced) is favored by multiple lines of evidence (Fig. 28).136 First, enrichment of the oxygen atom attached to C-23 by 18O2 suggests that atom is the epoxide oxygen (Fig. 27). Were the cyclization to proceed in the opposite direction the atom would derive from a ketone at C-19. Such an oxygen would necessarily derive from acetate because the retention of both deuterium atoms at C-18 indicates that C-19 cannot undergo the dehydratase-mediated reaction (Fig. 27).22 Instead, the oxygen attached to C-16 shows partial enrichment by [1-13C, 18O2]acetate.136 Further evidence supporting a right-to-le assembly comes from the isolation of belizeanic acid (48), a minor metabolite produced by Prorocentrum belizeanum that has an identical carbon skeleton to okadaic acid (2) but lacks rings C and D (Fig. 29).138 The relative congurations of C-16 and C-19 of belizeanic acid were both determined to be S* based on molecular modeling of the four possible diastereomers and comparison of the models to measured 3JH–H and ROE values. It is worth noting that SN2 displacement of a hydroxyl group with such a conguration at C-16 by a hemiketal hydroxyl at C-19 would lead to the conguration observed in 2. Thus, ring D appears to be the rst formed through 6-endo-tet opening of an (S0 ,S0 ) epoxide with the hydroxyl group at C-26, a carbon derived from the methyl carbon of a deleted acetate unit (Fig. 27). The resulting hydroxyl group at C-23 attacks the ketone at C-19, and the resulting hemiketal undergoes 5-exo-tet cyclization with the alcohol at C16, with apparent retention of the hemiketal oxygen atom (Fig. 28). In belizeanic acid, the ketone at C-19 is apparently reduced by a keto reductase before the second cyclization step can occur.138 By contrast, the formation of the spiroketal rings A and B appears to occur from le-to-right (i.e. contrary to the direction of polyketide chain elongation). Evidence for this comes from intact incorporation of the carbonyl unit of [18O2, 1-13C,]acetate at C-4 and its attached oxygen.53 Aer formation of ring A by 6exo-trig cyclization of the alcohol at C-4 with the ketone at C-8, ring B appears to be formed by a 6-exo-tet cyclization of an apparent 1,5-diol (Fig. 28). Support for such an intermediate comes from the observation that the oxygen atom attached to C12 exhibits labeling by both [18O2, 1-13C,]acetate and 18O2.136 Thus, the results indicate that in mixed polyethers like okadaic acid, which contains only one trans-fused-ring system and two spiroketal rings, there is no single uniform direction of ring assembly with respect to the direction of polyketide chain elongation. This makes inference of the mechanisms underlying ring assembly for such compounds in the absence of 18O labeling studies more difficult.

Review

In the absence of biochemical information on biosynthetic pathways, one is le to infer likely mechanisms from the structure of the nal products. Ether rings resulting from intramolecular addition of alcohols to olens will yield ether rings without constraints on the oxygenation of carbons adjacent to the ether bond. For example, rings B and C in amphezonol A (49)139 are good candidates for this type of mechanism as none of the carbons adjacent to C-13 or C-17 (in ring B) or C39 and C-43 (in ring C) bear oxygen atoms that might suggest the involvement of epoxide ring opening in ether formation. Likewise, the 5-membered ring in amphidinin B (50)140 does not appear to involve an epoxide intermediate (Fig. 30). An alternative mechanism could involve intramolecular SN2 of the corresponding 1,5- or 1,4-diol precursor or other suitably functionalized intermediate giving the corresponding tetrahydrofuran or tetrahydropyran moieties, respectively. Another mechanism of ether ring formation that appears to be common in dinoagellate metabolites is via intramolecular addition of alcohols to ketones, yielding cyclic hemiketals. Hemiketal ether rings can be seen in, for example, amphidinolide L (51),141 caribenolide I (52),142 and iriomoteolide-1a (53)143 (Fig. 31). It is interesting to note that even within this family of compounds there is no consistency in the direction of cyclization with respect to the direction of polyketide chain

11 Compounds containing isolated ether rings or spiro ring assemblies As mentioned previously, a limited number of mechanisms are likely responsible for ether ring formation in natural products.

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Fig. 31

Examples of amphidinolides that contain hemiketal rings.


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Review

Fig. 32 Cyclic imines containing spiro ether assemblies. (A) Structures of spirolides 13-desmethyl C (25), G (54), and H (55) and pinnatoxin A (28). Note that spirolide H contains only two rings. (B and C) Two possible mechanisms for formation of the spiro ring assembly in 13-desmethyl spirolide C in either of two directions.

elongation with amphidinolide L and caribenolide I being opposite in sense to iriomoteolide-1a. The spirolides are a family of cyclic imines produced by the dinoagellate Alexandrium ostenfeldii. The spirolides each contain a series of spiro-linked ether rings that suggest a small cascade of ketal formations terminated by a 5- or 6-exo-tet dehydration, depending on the direction of the cascade (Fig. 32). The three compounds 13-desmethyl spirolide C (25),144,145 spirolide G (54),146 and spirolide H (55)147 illustrate the variance seen in this family with respect to the spiroether substructure (Fig. 32). The conguration seen in 13-desmethyl spirolide C, with ring sizes of 5, 5, and 6 for rings A, B, and C, respectively, is the one that occurs most frequently in the


spirolides. In spirolide G, ring B is expanded in size by one carbon, suggesting involvement of a deletion step in conjunction with an additional acetate unit. In the dispiroketal spirolide H, ring A is absent, apparently resulting from a shortening of the putative polyketide chain by three carbons, again suggesting involvement of a deletion step. These three structures suggest some exibility in the process of ring formation with respect to the size of the rings formed. Regardless of the actual direction of cyclization, all three variants display consistency in the sizes of the rst and last rings formed. The same is not true for the structurally related cyclic imine pinnatoxin A (28)99,148 in which the equivalent of ring A is a six-membered ring. Although pinnatoxins were originally

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Review

Fig. 33 Amphidinolides containing ether rings that might arise from epoxide ring opening. (A) Amphidinolides C, E, and X. (B) Possible mechanisms for formation of ether rings in amphidinolide C (3). (C) Possible formation of ring C in amphidinolide E (56). (D) Possible formation of ring D in amphidinolide X (57).

isolated from bivalves (Pinna muricata),149 the compounds are almost certainly produced by a dinoagellate. It is interesting to note that the 6/5/6 spiro ring motif recurs in other dinoagellate polyethers such as durinskiol A150,151 and azaspiracid (68).152,153 The last major mode of ether ring formation which, as will be shown with the fused-ring polyethers, is utilized oen and effectively is through the intramolecular ring opening of epoxides by alcohols. In stark contrast to the stereochemical uniformity of apparent epoxide precursors and regioselectivity of ring closing that will shortly be described for the fused-ring polyethers, compounds containing isolated ether rings display a marked apparent exibility in the process of ether formation. This is effectively demonstrated by the amphidinolides which contain many examples of ether rings possibly resulting from

The syn–trans pattern of substitution in fused-ring polyethers. This pattern is universally observed and suggests stereochemically uniform processes for epoxide formation and fused ether ring cyclization.

Fig. 34

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ring-opening of epoxide precursors. Although the two ether rings in amphidinolide C (3)154–156 and the ether ring in amphidinolide E (56)157,158 all might result from exo-tet epoxide opening, the putative precursor epoxides would all need to have a cis conguration (Fig. 33). Also, although the ether ring in amphidinolide X (57) might result from 5-endo-tet opening of a trans epoxide, acetate incorporation studies159 indicate that the putative epoxide would span the carbons within an acetate unit rather than occurring between units as is more common.

12 Fused-ring systems The fused-ring polyethers, as exemplied by the brevetoxins, are perhaps the best known type of compounds produced by dinoagellates. The structures of the brevetoxins160 were determined around the same time that the unied model of polyether biosynthesis in actinomycetes was being developed,114 so it is not surprising that the earliest model for fused-ring polyether biogenesis in dinoagellates48,129,130 was heavily inuenced by the latter. Indeed, the typical syn–trans pattern (Fig. 34) evident in the ether ring junctures strongly suggested a stereochemically uniform cascade of cyclization reactions from a polyepoxide precursor that itself exhibited uniformity in conguration across all epoxides. As new fused-ring polyethers have been discovered, this stereochemical regularity has been maintained, prompting the observation that all epoxides within a polyether precursor might be produced by the same epoxidase acting on an all-E polyene precursor.32 Additional mechanisms


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Fig. 35

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Heptadienal polyethers from Karenia brevis.

Fig. 36 Formation of the ether ring in brevisamide (33) by opening of a

trans epoxide.

for fused-ring polyether formation in dinoagellates from cis epoxides have been proposed including a mechanism incorporating an intermediate derived from cycloaddition of an ironoxo group with an olen161 as well as one mediated by orthoester functionalization.162 However, since most instances of epoxides in other dinoagellate polyethers have been primarily trans, there is no explanatory advantage to either cis epoxide mechanism over the trans epoxide model at this time. Because of the wealth of published material on applying the bacterial polyepoxide model of ether ring formation to marine fused-ring polyethers, including recent reviews of this model and some competing models,35,116,163,164 this section of the review will focus more on particular aspects of this model that have been highlighted by the recent discovery of some aberrant polyethers from Karenia brevis, the producer of brevetoxins. The compounds brevisamide (33),101,165 brevisin (58),166 and brevenal (59)167,168 all contain a 2,3-dimethylheptadienal side chain,

suggesting relatedness in the biosynthetic pathways producing them (Fig. 35). The smallest of the three, brevisamide (33), contains only a single ether ring. This ring bears a b-hydroxyl group with a conguration that suggests that it resulted from 6-endo-tet epoxide opening of a trans epoxide (Fig. 36). Moreover, brevisamide contains a nitrogen atom, suggesting the possibility that its nascent polyketide chain has a glycine starter unit as has been seen in another nitrogenous polyether, 13-desmethyl spirolide C (25).91 The location of the b-hydroxyl group therefore suggests that the direction of cyclization is opposite the direction of polyketide chain elongation. The minimal structure of brevisamide suggests that cyclization of the rst ring can proceed in the absence of downstream epoxides from which other rings can be formed. Brevisin (58) contains a unique structural feature among fused-ring polyethers: the presence of two frames separated by a methylene bridge. The signicance of this comes when one considers a possible polyepoxide precursor to brevisin (Fig. 37). In addition to the interrupted assembly seen in brevisin, such a precursor might also form a continuous frame that incorporates a nine-membered ring. Indeed, nine-membered rings are known from other fused-ring polyethers such as brevetoxin A (PbTx-1),169 so the possibility of this alternate cyclization

Fig. 37 Ring formation in brevisin (58). (A) Brevisin's “interrupted” frame results from two separate initiation processes, each producing a sixmembered ring by 6-endo-tet cyclization. (B) A hypothetical alternative continuous cyclization process that produces a “continuous” frame version of brevisin that is not observed in extracts from K. brevis.


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product cannot be excluded on principle. The fact that so far only the interrupted frame has been observed and not the continuous frame raises the question of whether the latter is excluded by a global process (e.g. dictated by the sizes and shapes of the resulting frames) or by a local process (e.g. dictated by the patterns of functional group occurrence within the nascent polyketide chain). An analogy for a global process might be drawn from the oxidosqualene cyclases of sterol and terpenoid biosynthesis, where a linear precursor is folded and shaped by an enzyme active site cavity prior to ring formation.170 However, it is difficult to imagine how such an enzyme, were it to exist, could evolve from an ancestor that favors continuous frames such as those in the brevetoxins and brevenal (59) to one that favored the interrupted frame of brevisin over the continuous frame form. It therefore seems more likely that there are chemical cues within the nascent polyketide chain that favor the interrupted frame over the continuous frame. A simple way of achieving this is to have the initiation of polyether assemblies (i.e. formation of the rst ring) determined by the spacing between the nucleophile hydroxyl group and its nearest downstream epoxide. Then the interrupted frame would be favored if either initiation of assemblies takes place as the nascent polyketide chain is undergoing elongation or if ring frame initiation happens at a faster time scale than ring frame extension. As will be elaborated below, such a model would be consistent with the structures of all other known fused-ring polyethers to date. The third member of the family, brevenal (59), has a conventional continuous frame of ether rings, and thus connects this family to other fused-ring polyethers such as the brevetoxins. In addition, the revised absolute conguration of brevenal168 taken in conjunction with the absolute congurations of brevisamide (33) and brevisin (58),171 demonstrates an interesting consistency within all polyethers from K. brevis. If one considers the putative precursor polyepoxides for each of these polyether metabolites, one nds not only the internal stereochemical regularity for each precursor previously noted by Gallimore and Spencer,32 but also an external stereochemical regularity for all such precursors. In other words, every epoxide ring that occurs in a polyepoxide precursor in K. brevis has an absolute conguration of (S0 ,S0 ). This suggests that a single epoxidase that accepts di- and tri-substituted E-olens as substrates and that uses an acyl carrier protein as an orienting feature could in principle be sufficient for forming all epoxides in precursors to all K. brevis polyethers identied to date. One other recent development bears noting, this one coming from the world of biomimetic synthesis. Jamison and coworkers

Review

have found that formation of fused-ring polyethers by a cascade of 6-endo-tet epoxide openings proceeds spontaneously in water at neutral pH from polyepoxide precursors that contain a prebuilt initiator ring.172 Such a ring can adopt a conformation in which both the nucleophilic hydroxyl group and the chain leading to the next epoxide are located equatorially (Fig. 38). The authors postulated that a hydrogen-bonding network involving water molecules can orient the molecule in a conformation that favors 6-endo-tet attack by the hydroxyl group. The newly formed six-membered ring produces the same conguration of nucleophilic hydroxyl group and chain so that the process can be repeated. Indeed, it seems reasonable to propose that as the frame of rings is extended, the barrier to chair–chair interconversions of the terminal ring increases due to the rigidifying effects of the trans-decalin systems that are formed. The effect favoring 6-endo-tet cyclizations was found to be sufficient to overcome other directing effects due to different substitution patterns of the epoxide groups.173 Based on previous models of polyether biosynthesis32,114,129,130 and the structures of brevisamide (33), brevisin (58), and brevenal (59), we highlight several aspects of the process by which fused-ring polyethers in dinoagellates might be formed. Note that the features to be described are meant to provide a rationalizing framework for the similarities and differences observed in the architecture of such fused-ring polyethers, and not as a precise description of the physical processes underlying their assembly. Such precision is impossible without detailed knowledge of the enzymes responsible for the construction of these compounds, and there is currently nothing denite known on this matter. Nevertheless, much can be inferred from the observed structures and the limited biosynthetic studies carried out so far. (1) Fused-ring polyethers result from a series of endo-tet ring closures involving epoxide intermediates. This is the heart of the model proposed by Nakanishi.129 Further support for the involvement of polyepoxide precursors came from a labeling study showing that all ether oxygen atoms in yessotoxin (23) derive from 18O2 and not from [18O2]acetate.90 It should be noted that we propose a sequential series of closures as opposed to an unordered series of ether ring formations. We mean neither to suggest nor exclude a concerted or rapid series of ring closures, such as one might expect from terpenoid carbocation-based cyclization, where the intermediates are much less stable than those involved in epoxide-based cyclization. In particular, the proposed model is consistent both with a polyepoxide precursor undergoing a cascade of ring

The presence of a “template” ring in a synthetic intermediate might stabilize conformations favoring endo-tet cyclization in a polyepoxide intermediate.

Fig. 38

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closures as well as a process in which formation of an epoxide is followed immediately by ether ring formation, precluding the need for a polyepoxide intermediate, as proposed by Gallimore and Spencer.32 (2) The observed steric congurations of the ether rings in ladder-frame polyethers suggest that the epoxide rings of the putative intermediates are uniform in their steric congurations for a given molecule. This observation, rst made by Gallimore and Spencer,32 extends the model for antibiotic polyether biogenesis proposed by Cane, Celmer, and Westley114 to fused-ring polyethers. Nearly all of the ladder-frame polyethers have steric congurations that could result from a cascade of ring closures of the type described above in which the epoxides of the

NPR

intermediate are uniformly (R0 ,R0 )-trans epoxides or uniformly (S0 ,S0 )-trans epoxides. This is important for two reasons: rst, such an arrangement is required to make it possible for all bicycles in a given frame to have pseudo-equatorial ring junctures with respect to both rings. This is certainly true for cases involving six-membered rings, though less certain with respect to larger, more exible ring systems. Such a conguration of rings is likely to be important in overcoming the kinetic barriers to endo-tet ring closure, as noted above. Second, it is plausible for a single epoxidase or related sets of epoxidases to catalyze the formation of all epoxides, assuming a polyene intermediate in which all double bonds to be epoxidized are E in conguration. A precedent from the terrestrial world comes from the

Fig. 39 Ring formation in maitotoxin (60). A putative polyepoxide precursor is shown with each initiator ring highlighted. In addition, deviations from the usual stereochemical configuration of fused-ring polyethers are highlighted. Note also that there are no occurrences of the spacing between hydroxyl group and upstream epoxide groups noted for each initiator ring that is not within an initiator ring.


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enzyme Lsd18 which forms all of the epoxides in the bis-epoxide precursor to lasalocid A (44).174 This observation does not hold for maitotoxin (60) unless one posits that some of the ether rings are formed by a different process. The J/K ring junction in maitotoxin does not conform to the usual syn–trans conguration seen in most other ladderframe polyethers (Fig. 39). The ring juncture is trans, as is typical for these compounds, but is also anti with respect to the neighboring ring junctures in the system. Although possible misassignment of conguration for this juncture has been proposed,32 such an explanation is inconsistent with results from total synthesis, the experimental NMR evidence, and modeling studies.175–181 In addition, the steric conguration of the junctures at rings L/M and N/O would be expected from cis epoxides rather than the usual trans epoxides. Maitotoxin contains other unusual features such as cyclic systems linked together by sigma bonds (rings K/L, O/P, V/W) that could conceivably arise from either adjacency of independently formed ring systems or from 6-exo-tet ring closures rather than the usual endo-tet closures in these systems (not shown). The suggestion has been made that, even though the endo-tet cascade may dominate the formation of most ether rings in maitotoxin, it might be possible that an independent mechanism is responsible for the formation of ring J.182 Given the apparent inhomogeneity in ether-forming mechanisms within the isolated ring metabolites noted above, such a suggestion expanded to include rings L/M and N/O is reasonable. It is interesting to note that all of these deviations from canonical fused-ring formation occur in what appear to be initiator rings for the respective assemblies. (3) The cascade of ring closures is started by endo-tet epoxide opening by an alcohol in the nascent chain, and this initial ring formation is the most difficult to achieve. This observation is suggested by Jamison's studies with polyepoxides containing pre-formed initiator rings noted above.172,173,183 The enzyme most likely responsible for this is an epoxide hydrolase, as has been found in bacterial polyether biosynthesis.32,117–120,122–125,128,131,132,184 One important corollary to this observation is that the last ether ring formed by the cascade can usually be identied by the presence of an oxygen substituent b to the ether oxygen, albeit sometimes masked as part of a lactone or ketal functional group. Another important corollary is that the formation of the rst ring is more likely to impose strict requirements on the spatial conguration of an enzyme active site than any of the other rings, which can form spontaneously in water. It should be noted, however, that it is not known if seven-, eight-, or nine-membered rings can also form spontaneously in water given the presence of an initiator ring. (4) The direction of propagation of the ether-forming cascade is opposite to the direction of polyketide chain growth. In other words, the earliest formed ether ring in an assembly occurs on the last of the epoxides added by the polyketide synthase. There is precedence for such a mechanism in the world of bacterial polyethers. Studies on PKS-bound intermediates in lasalocid biosynthesis have shown that polyether ring formation occurs simultaneously with polyketide chain assembly and with a strict sequential order with the epoxides being installed from the

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starter unit towards the other end of the chain followed by ether ring formation proceeding in the opposite direction.185 It has been suggested that such a scheme is needed to prevent nonspecic ketal products.186 In the case of dinoagellate polyethers, the evidence for this is limited in that the only ladderframe polyethers for which the direction of polyketide chain assembly has been unambiguously determined are brevetoxin A (PbTx-1)47 brevetoxin B (PbTx-2; 1; Fig. 4),46,48 and yessotoxin (23; Fig. 14),89 and, due to the complexities involved in construction of nascent polyketide chains for polyethers, it is not straightforward to determine the direction of chain assembly from the structure of the nal metabolite alone. Another example is that of brevisamide (33) as noted above. In the case of the brevetoxins 33, and by extension the related compounds brevenal (59) and brevisin (58), the direction of the ring cascade appears to be opposite the direction of chain growth. Such a direction is also consistent with the 18O2 labeling patterns observed in yessotoxin.90 More studies establishing the direction of polyketide chain growth in other fused-ring polyethers are needed to further validate this observation. (5) Initiation of ether ring frames depends solely on local features of the nascent chain. The process of ring assembly initiation is temporally distinct from the process of ring assembly extension. Alternatively, ring formation occurs simultaneously with chain assembly. The presence in brevisin (58) of two separate ring assemblies (Fig. 35) suggests that multiple initiator rings can occur in a single nascent polyketide chain. The two initiator rings in brevisin (rings A and D; Fig. 35) bear some features in common with all of the other initiator rings in polyethers from K. brevis. All such initiator rings result from 6-endo-tet epoxide opening of a trans epoxide. The rings otherwise exhibit variability with respect to ring substitution patterns, whether the side chain is syn or anti with respect to the extended chain (both

Fig. 40 Occurrence of initiator ring motifs in a putative polyepoxide precursor to gambierol (61).


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Fig. 41 Occurrence of initiator ring motifs in a putative polyepoxide precursor to gambieric acid (62).

Fig. 42

groups of which are a to the ring ether), and the degree of substitution of the epoxide ring. This suggests that substrates for a possible epoxidase that forms the initiator ring might be dened merely by the spacing between the nucleophilic alcohol and the nearest upstream epoxide. What is remarkable is that if one examines the putative nascent polyepoxide precursor to all fused-ring polyethers, there is generally only one occurrence of the particular spacing between candidate hydroxyl groups and their nearest upstream epoxides that characterizes the initiator ring for each compound. The particular spacing can vary between compound families: the spacing is six for all K. brevis polyethers; six also for gambierol (61; Fig. 40),187,188 gambieric acid (62; Fig. 41),189–192 yessotoxin (23; Fig. 42),193,194 and maitotoxin (60; Fig. 39);175–178,195,196 ve for gymnocin (63; Fig. 43);197 and seven for ciguatoxins (64; Fig. 44).198–200 In all cases for which there are multiple occurrences of such a spacing between alcohol and epoxide, there are multiple initiator rings and multiple frames, as in the case of brevisin and maitotoxin. The potential nascent polyepoxide precursors for each of the families of fused-ring polyethers with initiator rings are highlighted (Fig. 39–44). The predominance of the interrupted frame in brevisin (58) over a putative continuous frame (Fig. 37) can be explained if the process of forming initiator rings is distinct in its timing

Occurrence of initiator ring motifs in a putative polyepoxide precursor to yessotoxin (23).


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Fig. 43

Review

Occurrence of initiator ring motifs in a putative polyepoxide precursor to gymnocin A (63).

Fig. 44 Occurrence of initiator ring motifs in a putative polyepoxide precursor to ciguatoxin (64).

from the extension of the ring frame (e.g. if formation of initiator rings occurs very quickly compared to the process of ring extension). For metabolites in which only one frame occurs, this is a trivial distinction. In brevisin, however, if ring initiation and ring extension were potentially co-occurring, one might expect a mixture of interrupted frame and continuous frame congeners resulting from the same putative nascent polyepoxide absent other controlling mechanisms. Another possible explanation

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would be if ring assembly construction occurs as the nascent polyketide chain is being produced. Under this scenario, rings D, E, and F might be the rst rings formed followed later by rings A, B, and C aer further extension of the nascent polyketide chain. Either mechanism has precedent in the bacterial world. For some products such as monensin and nigericin, cyclization occurs only aer the nascent chain is completely assembled and has been transferred to a specialized acyl carrier


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protein.121 For lasalocid, however, ring formation occurs simultaneous with polyketide chain assembly as most convincingly shown by the presence of fully cyclized intermediates that could be displaced directly from the PKS using synthetic chain terminators.185


13 Mixed polyethers As illustrated in this review, there are a number of polyether metabolites that contain various combinations of isolated ether rings, spiro ring systems, and fused-ring systems. One example is okadaic acid (2) discussed above. The fused-ring system in okadaic acid exhibits similar stereochemical features to those found in metabolites such as the brevetoxins. Likewise, there are isolated rings in okadaic acid that might arise from various mechanisms as outlined above. The purpose of this section is to highlight some unusual features that occur within mixed polyethers. One example is the pectenotoxins which, like okadaic acid, are members of the Diarrhetic Shellsh Poison (DSP) family of toxins. The spiro ring system A/ B in the pectenotoxins is interesting in two respects. First, in pectenotoxin 2 (PTX-2; 65) isolated from Dinophysis fortii,201,202

Fig. 45

Examples of pectenotoxin structures.


and more recently from laboratory cultures of Dinophysis spp.,203–205 the spiro ring center at C-7 is congured such that the ether oxygen of ring B is located equatorially on ring A (Fig. 45). By contrast, all tetrahydropyran rings in spiro ring systems in okadaic acid have an axial location for the ether oxygen, perhaps favored by the anomeric effect.206 Second, there have been other pectenotoxin congeners isolated from shellsh hepatopancreas in which C-7 is epimerized.206 It is not clear if formation of the alternative conguration of ring A/B occurs at the time of cyclization of the polyketide nascent chain or in the shellsh digestive system.206 Another peculiar feature of the pectenotoxins (65–67) is the bridged [3.2.1] ether bicycle at rings D/E. Based on the observation that the vast majority of ketal- or hemiketal-based ether rings in dinoagellate metabolites to date have sizes of ve or six, it appears most likely that ring E is rst formed followed by cyclization of D. Support for such a sequence comes from consideration of the structures of PTX-13 (66) and PTX-14 (67), both isolated from Dinophysis acuta (Fig. 45).207 Although the absence of detectable peaks corresponding to 67 in extracts of various cultures of Dinophysis suggested that 67 might be a dehydration artifact of 66,207 its presence suggests that such bicyclic systems can form readily given a hemiketal with a suitably positioned hydroxyl group nearby. The structure of 66 raises additional questions, particularly whether 66 arises by oxidation of PTX-2 (65) or if 65 is produced by reduction of 66. Either way, this illustrates the fact that the mechanism by which an ether ring is formed might be masked by subsequent chemical transformations of the cyclization product. Another family of compounds incorporating a bicyclic ether structure are the azaspiracids (68; Fig. 46).152 Although a likely dinoagellate origin for these compounds was remarked upon at the time of their discovery, it was another decade until a producer was found in the novel genus Azadinium.153 In addition to the [3.3.1] bicyclic system in rings F/G, azaspiracid includes a highly unusual hemiaminal ether system at rings H/I. Another unusual feature is that the fused-ring junctures at C/D and G/H have a syn conguration, suggesting that if they originated from endo-tet epoxide openings then the epoxides originally had cis geometries, or alternatively that the epoxides had trans

Fig. 46 Azaspiracid-1 (68). Note the unusual hemiaminal ether functionality (rings H/I) and the unusual geometry of fused rings C/D and G/H.

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Review

Fig. 47 Prorocentin (69) from Prorocentrum lima. (A) Prorocentin. (B) A hypothetical product (70) resulting from endo-tet epoxide opening. Note the aberrant geometry at the juncture of the newly formed ring.

geometries with subsequent inversion of conguration of the newly formed alcohol due to SN2 displacement of the hydroxyl group en route to formation of the next ether ring. A polyether incorporating a fused-ring system, albeit with a more conventional conguration, is prorocentin (69) from Prorocentrum lima.208 Prorocentin is interesting not so much for the rings that are present, which are similar to many examples given already, but more for the ring that is missing (70; Fig. 47). There is an epoxide ring that could undergo 7-endo-tet epoxide opening to form the hypothetical product shown at the bottom of Fig. 47. That this cyclization does not occur is most likely a consequence of the conguration of C-16, which is such that the hydroxyl group is axial rather than equatorial. Recall that the nucleophile hydroxyl groups generated by endo-tet epoxide openings in trans-fused polyethers are all equatorially located. This conguration at C-16 also suggests that ring B is not formed by 6-endo-tet epoxide opening of a trans epoxide. Indeed, in proposing a biogenetic scheme for ring formation in prorocentin, the authors suggested that ring B arises from a 6-exotet dehydration-like process.208 In contrast to the stereochemically uniform process by which fused-ring polyethers are formed, the formation of isolated ether rings in dinoagellate metabolites appears to be more ad hoc. Compounds that contain multiple candidate rings for formation by epoxide ring opening by virtue of the presence of a hydroxyl group b to the ether oxygen nonetheless exhibit heterogeneity within a metabolite when its rings are compared to one another on the basis of epoxide conguration, direction of cyclization, and/or whether the cyclization is endo or exo. For the cyclizations that occur by exo-tet dehydration or by endo- or exo-trig addition to olens, the question arises as to how cyclase enzymes identify which parts of the nascent chain to act on,

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particularly in cases where metabolites exhibit extensive hydroxylation and/or contain many olenic bonds. Understanding the cues for spiro ketal and hemiketal formation is more straightforward since reactive sites would be identied by the presence of ketone groups. Nonetheless, the direction of such cyclization cascades cannot be identied from the structure of the nal product alone. This is underscored by the metabolites of unrelated Amphidinium species which, regardless of the functionality that is found along the carbon chain in amphidinols or amphidinolides, no fused-ring systems are produced. A similar pattern is found among metabolites of Prorocentrum species, which despite the opportunities to form a fused-ring structure containing more than two rings, no such compounds have been found. Indeed some features of prorocentin (69) and okadaic acid (2) are remarkably similar. Similar outcomes are found with various Alexandrium ether compounds such as goniodomin (19) and the spiroimine group. Thus, among a group of organisms where it is difficult if not impossible to predict a biosynthetic pathway, this is a surprising consistency, and suggests that species belonging to the genera Amphidinium, Prorocentrum, Dinophysis, and Alexandrium lack a suitable epoxidase or epoxy hydrolase necessary for the formation of fused-ring systems. In contrast, Karenia, Gambierdiscus, and Protoceratium strains (among others), clearly possess these enzymes and are totally committed to the production of extended ladder frame ring systems that occur in the brevetoxins (1), ciguatoxins (64) and yessotoxins (23). These complexities highlight the need for more biogenetic studies exploring the incorporation of oxygen atoms from different sources to elucidate the processes by which ether rings are formed in a wide variety of compounds.


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14 Concluding remarks Despite the remarkable complexity and variety of dinoagellate chemistry, only a very limited group of precursor units are utilized to build these remarkable structures. They include only acetate, glycolate, and glycine. In contrast, the number and variety of building blocks utilized by bacteria and fungi shows more emphatically that the structural complexity of dinoagellate chemistry is quite extraordinary. Structural variety is a result of how precursor units are employed and processed, and in many cases, the intervention of polyether ring formation. It is well reported that the genetics of dinoagellates is complex, and to date this has hampered molecular studies. However, it appears that many (but not all) of the biosynthetic steps employed in the assembly of dinoagellate polyketides share similarities with those reported in a variety of bacteria and this may prove to be important as we seek to fully understand dinoagellate polyketide biosynthesis.

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