1. Introduction
One of the main points of interrelation between chemical research on marine natural products and seafood quality control has been oriented towards chemical, structural and biosynthetic studies of secondary metabolites responsible for a variety of toxic syndromes. Within these intoxications, the toxic phenomenon known as Diarrhetic Shellfish Poisoning (DSP) occurs with frequency in northwest Europe, Canada and Japan, generating great alarm from the public health point of view and in turn within the shellfish industry. The main clinical symptoms of these intoxications consist of diarrhea, vomiting and headache, which are a result of the ingestion of shellfish contaminated by okadaic acid (OA) and dinophysistoxins (DTXs) by feeding on planktonic organisms. In addition, the scientific community has shown an enormous interest in these phenomena due to the complex structures of the toxins involved and the potent biological activities characteristic of these substances [
1,
2,
3].
In general, the toxins involved are OA and its congeners, which include several okadaates, lipophilic dinophysistoxins (DTX1–3) and the water-soluble derivatives (DTX4 and DTX5a–c) [
3]. As regards their bioactivity, these molecules stand as potent tumour promoters as well as potent and highly selective inhibitors of protein phosphatases type 1 (PP1) and 2A (PP2A), making them extremely useful tools for studying cellular processes regulated by phosphorylation, such as transduction, or cell division and memory [
4,
5,
6]. From a structural point of view, all these toxins are polyethers with a 38 carbon-atom backbone and six or seven pendant methyl groups. They are characterized by the presence of oxolane and oxane rings, which form spiro or trans-fused systems. Despite their enormous structural diversity, these polyketide metabolites are related by their common origin from highly functionalized carbon chains whose assembly is controlled by multifunctional enzyme complexes, the polyketide synthases (PKSs). Each condensation is followed by a cycle of modifying reactions: ketoreduction, dehydration and enoyl reduction. In contrast to the fatty acid biosynthesis in which reductive modifications normally follows each condensation, the PKSs can use this sequence in a highly selective and controlled manner to assemble polyketide intermediates with an enormous number of permutations in functionality along the chain. These peculiarities have encouraged intensive biosynthetic studies to determine the biosynthetic pathways of these biotoxins group [
7,
8,
9,
10]. Thus, the biosynthetic origin of the carbon backbone of DTX1 and OA was first determined and subsequently continued by the study of okadaates and water-soluble toxins DTX4, DTX5a and DTX5b [
11,
12,
13,
14,
15].
Here we report on the biosynthetic origin of the most recently determined water-soluble toxin DTX5c (
Figure 1) [
16,
17] by addition of stable isotopic precursors to a culture of the dinoflagellate
Prorocentrum belizeanum.
Figure 1.
Structure of Water-Soluble Derivative 5c (DTX5c).
Figure 1.
Structure of Water-Soluble Derivative 5c (DTX5c).
2. Results and Discussion
DSP toxins are produced in small quantities by dinoflagellates. Therefore, optimization of the culturing conditions is needed in order to maximize yield. This in turn, necessitates the development of an easy and efficient purification method. All the procedures must also be applicable to cultures involved in the feeding with labeled precursors.
The toxin studied in this work, DTX5c, was produced by an axenic culture of the dinoflagellate
Prorocentrum belizeanum [
16,
17]. Previous biosynthetic studies undertaken with similar metabolites indicated that only basic metabolic precursors could be successfully used with species of
Prorocentrum [
15]. In consequence, our biosynthetic study of DTX5c was designed as a series of feeding experiments using labeled [1-
13C] and [2-
13C] sodium acetate as the metabolic precursors that were added to artificial cultures of
P. belizeanum (PBMAO1 strain) (
Figure 2). A typical feeding experiment starts with inoculation of dinoflagellates into seven 5L Erlenmeyer flasks containing 3 L of Guillard K medium. Cells were grown for four days and at that point a mixture of two antibiotics, penicillin (40 IU/mL) and streptomycin sulphate (200 IU/mL) were added into the media. One day later, the labeled precursor was added up to a final concentration of 0.67 mM [
12]. Subsequently, three weeks later, the culture was harvested. Finally, the cells were filtered and extracted with MeOH. The extract was chromatographed using Sephadex LH-20, followed by reverse phase (C-18) chromatography and the final purification was carried out by HPLC on a XTerra column, yielding approximately 0.3 mg of DTX5c (15 μg/L) in each experiment.
Figure 2.
Selected sections of
13C NMR spectra in CD
3OD for the fragment C-8–C-10–C-43 and C-21–[C-41]–C-26 in labeled samples of DTX5c. In red, those corresponding to a sample obtained after addition of [1-
13C] sodium acetate [
], and in black those after addition of [2-
13C] sodium acetate experiment [
].
Figure 2.
Selected sections of
13C NMR spectra in CD
3OD for the fragment C-8–C-10–C-43 and C-21–[C-41]–C-26 in labeled samples of DTX5c. In red, those corresponding to a sample obtained after addition of [1-
13C] sodium acetate [
], and in black those after addition of [2-
13C] sodium acetate experiment [
].
The results obtained from the different biosynthetic experiments revealed different degrees of incorporation at all positions except for six carbon atoms located at C-37, C-38, C-1′, C-2′, C-8″ and C-9″ where no distinctive enrichment was observed using sodium acetate. Quantitative measurements indicated that
13C enrichment was to sufficient to continue the analysis, (total av.
13C 7.62% ± 0.8 SD in [2-
13C] sodium acetate experiment) although the specificity was smaller than that obtained in previous studies performed on
Prorocentrum lima cultures. Nevertheless, comparison of the
13C-NMR spectra obtained from addition of different labeled precursors showed the complementarity between the enrichment patterns, as could be observed in
Figure 2,
Figure 3,
Figure 4 and
Table 1. In fact, enrichment of 24 carbons upon addition of [1-
13C] sodium acetate and 38 carbons using [2-
13C] sodium acetate confirmed that 62 out of the 68 carbons in DTX5c derive from acetate, including all the pendant methyl groups. Furthermore, upon addition of [2-
13C] sodium acetate, it was clearly observed that, a number of signals showed the characteristic splitting due to
13C–
13C spin-spin coupling arising from the existence of adjacent
13C enriched positions that is characteristic of some marine compounds [
15]. As was already reported, slightly higher than average enrichments were observed at C-37 and C-38, however the biosynthetic origin of these carbons have been described to be deriving from glycolate [
11,
13].
Figure 3.
Selected sections of the
13C NMR spectra of the C-1′–C-10′ fragment using CD
3OD (left) and using pyridine-
d5 (right) as solvents. In red, those corresponding to a sample obtained from the [1-
13C] sodium acetate experiment [
], and in black those from the [2-
13C] sodium acetate experiment [
].
Figure 3.
Selected sections of the
13C NMR spectra of the C-1′–C-10′ fragment using CD
3OD (left) and using pyridine-
d5 (right) as solvents. In red, those corresponding to a sample obtained from the [1-
13C] sodium acetate experiment [
], and in black those from the [2-
13C] sodium acetate experiment [
].
As regards the OA moiety (C-1→C-44) of DTX5c, we found the expected incorporation pattern [
11,
12]. Thus, two alternative fragments interrupted the classic polyketide profile. The first one was found at C-9–C-10–C-43 and was detected by the existence of two weak signals that flanked C-9 and C-43, whereas the carbon signal from C-10 was flanked by four signals, with constant coupling values (
J9–10 = 72.1 Hz and
J10–43 = 42.8 Hz). The second exception was observed within the C-24→C-26 moiety. This time the coupling constants values between C-24–C-25 and C-25–C-26 were very similar (
J24–25 = 42.6 Hz;
J25–26 = 42.5 Hz), while C-25–C-41 showed a
J25–41 = 73.7 Hz. (
Figure 2).
Figure 4.
Labeling pattern observed in
13C enrichment experiments for DTX5c. [1-
13C] sodium acetate experiment is represented by [
], and the [2-
13C] sodium acetate experiment is represented by [
].
Figure 4.
Labeling pattern observed in
13C enrichment experiments for DTX5c. [1-
13C] sodium acetate experiment is represented by [
], and the [2-
13C] sodium acetate experiment is represented by [
].
Table 1.
13C NMR data for DTX5c in CD3OD (300 K; 150 MHz).
Table 1.
13C NMR data for DTX5c in CD3OD (300 K; 150 MHz).
C | δC | Origin | % Inc a [2-13C] | % Inc a [1-13C] | C | δC | Origin | % Inc a [2-13C] | % Inc a [1-13C] |
---|
1 | 176.05 | m | 7.5 | 2.0 | 34 | 96.21 | m | 8.6 | 2.9 |
2 | 74.93 | c | 2.5 | 5.1 | 35 | 36.21 | c | 1.9 | 5.7 |
3 | 45.39 | m | 8.2 | 2.0 | 36 | 19.01 | m | 7.5 | 2.4 |
4 | 67.57 | c | 1.9 | 5.1 | 37 | 25.73 | | 3.4 | 3.5 |
5 | 32.62 | m | 6.7 | 1.8 | 38 | 60.56 | | 3.4 | 3.4 |
6 | 27.21 | c | 1.4 | 4.3 | 39 | 10.27 | m | 8.1 | 2.5 |
7 | 72.26 | m | 7.3 | 1.4 | 40 | 15.89 | m | 7.4 | 2.4 |
8 | 96.86 | c | 4.0 | 6.6 | 41 | 111.70 | m | 7.9 | 2.2 |
9 | 122.61 | m | 7.6 | 2.7 | 42 | 15.79 | m | 6.6 | 2.1 |
10 | 138.87 | m | 7.3 | 2.2 | 43 | 22.41 | m | 8.2 | 2.4 |
11 | 33.19 | c | 2.3 | 4.1 | 44 | 25.34 | m | 6.1 | 2.2 |
12 | 71.46 | m | 7.7 | 2.6 | 1′ | 67.38 | | 2.6 | 2.5 |
13 | 42.20 | c | 2.3 | 4.9 | 2′, 7′ | 143.35 | | 8.0 | 3.0 |
14 | 135.78 | m | 6.5 | 1.5 | 4′ | 128.42 | c | 2.5 | 4.0 |
15 | 131.58 | c | 2.2 | 5.4 | 5′ | 128.56 | m | 7.8 | 1.9 |
16 | 79.64 | m | 6.4 | 2.1 | 3′, 6′ | 31.23 | | 7.9 | 4.3 |
17 | 30.86 | c | 2.2 | 4.7 | 8′ | 66.97 | m | 6.9 | 1.5 |
18 | 37.24 | m | 7.3 | 2.2 | 9′ | 112.45 | m | 7.9 | 1.7 |
19 | 106.34 | c | 2.8 | 6.3 | 10′ | 112.60 | m | 7.3 | 1.3 |
20 | 33.36 | m | 6.9 | 1.9 | 1″ | 172.47 | c | 2.9 | 5.5 |
21 | 26.92 | c | 1.9 | 4.0 | 2″ | 37.96 | m | 7.5 | 2.0 |
22 | 70.42 | m | 8.7 | 2.2 | 3″ | 123.29 | c | 3.1 | 5.5 |
23 | 77.56 | c | 2.4 | 5.1 | 4″ | 133.38 | m | 7.5 | 1.6 |
24 | 70.97 | m | 8.8 | 2.9 | 5″ | 29.01 | c | 1.4 | 4.0 |
25 | 146.25 | m | 8.7 | 2.7 | 6″ | 35.88 | m | 7.3 | 2.2 |
26 | 85.66 | m | 8.2 | 2.4 | 7″ | 174.99 | c | 2.5 | 6.3 |
27 | 65.27 | c | 2.2 | 4.7 | 8″ | 45.67 | | 3.8 | 3.2 |
28 | 35.97 | m | 7.6 | 2.5 | 9″ | 68.43 | | 2.1 | 3.2 |
29 | 31.51 | c | 2.6 | 5.2 | 10″ | 38.01 | m | 6.2 | 1.8 |
30 | 76.02 | m | 7.7 | 2.7 | 11″ | 69.06 | c | 1.8 | 5.1 |
31 | 27.94 | c | 1.9 | 4.7 | 12″ | 79.82 | m | 7.1 | 2.0 |
32 | 26.70 | m | 8.7 | 1.5 | 13″ | 70.76 | c | 2.0 | 4.9 |
33 | 30.46 | c | 2.1 | 4.9 | 14″ | 70.27 | m | 6.2 | 1.7 |
At first sight, the isotopic labeling pattern observed along the ester side chain of DTX5c was also similar to that reported for other related water-soluble toxins such as DTX5a and DTX5b [
14], but our observations were hindered by the structural symmetry observed in the C1′→C10′ fragment. Thus, we faced an overlapping problem with the
13C NMR signals for two pairs of carbons at C-2′, C-7′ and C-3′, C-6′ when using CD
3OD as solvent (
Figure 3). Consequently, it was impossible, under these conditions, to unambiguously assure their biogenetic origin. This problem was resolved by the alternative use of pyridine-
d5 as solvent, which shows a complementary chemical shifts profile (
Figure 5).
Figure 5.
Significant HMBC correlations for fragment C1′→C10′ in DTX5c using pyridine-d5.
Figure 5.
Significant HMBC correlations for fragment C1′→C10′ in DTX5c using pyridine-d5.
As a result the previously mentioned pairs of carbons C-2′–C-7′ and C-3′–C-6′ were now distinguishable while C-9′–C-10′ and C-4′–C-5′ were overlapped. Combining both spectra the assignment was completed and it was clear that
13C enrichment occurred at C-3′, C-5′, C-7′, C-8′, C-9′ and C-10′ upon addition of [2-
13C] sodium acetate while only C-4′ and C-6′ were enriched when [1-
13C] sodium acetate was used (
Table 2). Finally, within this fragment, C-1′ and C-2′ did not incorporate
13C distinctly from sodium acetate.
Table 2.
13C NMR data for the C-1′→C-10′ fragment in DTX5c (300 K; 150 MHz).
Table 2.
13C NMR data for the C-1′→C-10′ fragment in DTX5c (300 K; 150 MHz).
Carbon | δC CD3OD | δC Pyridine-d5 | % Inc * [2-13C] a | % Inc * [1-13C] a |
---|
1′ | 67.38 | 67.28 | 2.6 b | 2.5 b |
2′ | 143.35 | 143.73 | 1.9 c | 1.8 c |
3′ | 31.23 | 31.53 | 6.6 c | 1.5 c |
4′ | 128.42 | 128.72 | 2.5 b | 4.0 b |
5′ | 128.56 | 128.72 | 7.8 b | 1.9 b |
6′ | 31.23 | 31.56 | 1.3 c | 2.8 c |
7′ | 143.35 | 143.50 | 6.1 c | 1.2 c |
8′ | 66.97 | 66.94 | 6.9 b | 1.5 b |
9′ | 112.45 | 112.84 | 7.9 b | 1.7 b |
10′ | 112.60 | 112.84 | 7.3 b | 1.3 b |
Moreover
13C signals of C-7′, C-8′, and C-10′, appeared flanked by “satellite” signals when cultures were fed with [2-
13C] sodium acetate. Coupling constant values for these carbons,
JC7′–C10′ = 73.6 Hz and
JC7′–C8′ = 46.5 Hz, were consistent with the carbon hybridization. These data are in concordance with the existence of a “(
m)-
m-
m” sequence, similar to others previously observed in other polyketide isolated from dinoflagellates. The truncation of the carbon chain in marine polyethers to give vicinal “
m-
m” systems in the polyketide precursor has been explained in several ways. The initial proposal, which includes a biochemical process involving dicarboxylic building blocks derived from the TCA cycle [
10,
11,
15], was discarded and a second more plausible explanation given by Wright and co-workers was published. This proposal was based on the occurrence of a Favorskii or a benzyl-benzylic rearrangement in the carbon chain that would move the connectivity to the next carbon to give rise to the hypothetical intermediate 1 (
Figure 6, pathway B) [
13,
14]. The last hypothesis was published by Shimizu (
Figure 6, pathway A) and involved a terminal α,β-unsaturated acid (crotonic acid type) in the nascent polyketide chain. This would undertake epoxidation and the resultant α,β-epoxy carboxylic acid undergo facile decarboxylation [
18]. The later two proposals explains the appearance of
m-
m systems in an intermediate position of the carbon chain, as well as the fact that oxidation to a carbonyl is necessary to incorporate the pendant methyl group (C-10′), following a process similar to that suggested in the irregular moieties of okadaic acid. However, in our opinion the proposal made by Shimizu explains better the biosynthetically counterintuitive appearance of a methyl-derived aldehyde group at the terminal carbons of brevetoxins as well as the carboxylic group of okadaic acid (both enriched in feeding experiments with 2-
13C sodium acetate).
Thus, in accordance with the above comments, the
13C the incorporation pattern observed in the C-1′→C-10′ side chain of DTX5c, could be explained by the assembly of four intact acetate units to a glycolate-starting unit (C-1′–C-2′) [
14]. At this point, the necessary β-keto thioester—where the C-7′ carbonyl group derives from [2-
13C] sodium acetate—would be generated. Next, an acetate unit would attack this carbonyl. The polyketide chain would continue its growth and should be divided after a Baeyer-Villiger oxidation in a “
m-
c” bond (
Figure 6). Nevertheless, the real pathway used by dinoflagellates to achieve these particular rearrangements is still a challenge for those marine natural products research groups interested in biosynthetic studies.
Figure 6.
Biosynthesis of DTX5c according to (
A) Shimizu’s or (
B) Wright’s proposals. Enrichment from [1-
13C] or [2-
13C] sodium acetate experiments are represented by [
] and [
], respectively.
Figure 6.
Biosynthesis of DTX5c according to (
A) Shimizu’s or (
B) Wright’s proposals. Enrichment from [1-
13C] or [2-
13C] sodium acetate experiments are represented by [
] and [
], respectively.