Toxicon 47 (2006) 920–924 www.elsevier.com/locate/toxicon
DTX5c, a new OA sulphate ester derivative from cultures of Prorocentrum belizeanum Patricia G. Cruz, Antonio H. Daranas, Jose´ J. Ferna´ndez *, Marı´a L. Souto, Manuel Norte * Instituto Universitario de Bio-Orga´nica ‘Antonio Gonza´lez’, Universidad de La Laguna, Astrofı´sico Francisco Sa´nchez 2, 38206 La Laguna, Tenerife, Spain Received 2 December 2005; accepted 10 March 2006 Available online 27 March 2006
Abstract Prorocentrum belizeanum is a dinoflagellate known for its okadaic acid (OA) and dinophysitoxins (DTXs) production, both OA and DTX are polyether toxins of the Diarrhetic Shellfish Poisoning (DSP) group. We have recently published the isolation of a new diol-ester of okadaic acid from cultures of P. belizeanum. On this occasion we present a new sulphated water-soluble derivative, DTX-5c, isolated from this microalga, whose structure was established on the basis of its spectroscopical data. q 2006 Elsevier Ltd. All rights reserved. Keywords: Marine toxins; DSP; Dinophysistoxins; Okadaic acid; DTX5c
1. Introduction Marine phytoplankton produces various types of bioactive substances (Yasumoto and Murata, 1993) that are involved in both food poisoning and massive fish kills. Furthermore, in the last decades, they have become formidable tools in pharmacology laboratories in the study of cellular processes. An important number of these toxins possess polyether structures, therefore a representative group of these structures consists of okadaic acid (OA) and the related toxins known as dinophysistoxins (DTX’s), both are involved in the red-tide DSP (Diarrhetic Shellfish Poisoning) syndrome (Daranas et al., 2001). These toxins are produced by dinoflagellates of genera Prorocentrum and Dinophysis and accumulated inside shellfish in waters containing blooms of these algae. They have been shown
* Corresponding authors. Tel.: C34 922 318586; fax: C34 922 318571. E-mail addresses:
[email protected] (J.J. Ferna´ndez), mnorte@ull. es (M. Norte).
0041-0101/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.03.005
to be potent phosphatase inhibitors and to have tumourpromoting activity (Ferna´ndez et al., 2002). For these reasons, cultures of these dinoflagellates have been studied widely (Sua´rez-Go´mez et al., 2001; Ferna´ndez et al., 2003; Britton et al., 2003) to establish their lipidsoluble polyether productions in relation to DSP outbreaks. Although the most abundant toxins in this group are OA and DTX1, several members of the so-called ‘OA diol-esters’ have been isolated recently from artificial cultures of different strains of Prorocentrum. In these compounds, the carboxyl group of OA is converted to its ester derivative with different unsaturated C4 to C10 diols to form allylic diol-esters (Hu et al., 1992; Norte et al., 1994). These organisms also produce other OA derivatives where the diol-esters are further conjugated to a highly polar side chain containing sulphate groups. Presently, only three of these sulphated derivatives of OA, named DTX4, DTX5a and DTX5b (Fig. 1), have been isolated from cultures of P. lima and P. maculosum by Hu et al. (1995a,b). These authors proposed that those types of derivatives may be a means of excreting the lipid soluble DSP acids and esters from the cell to the aqueous solution.
P.G. Cruz et al. / Toxicon 47 (2006) 920–924
921
43
OH O
4
12
O
7
OH
39
41
O
16
25
O 42
40
O
O 1
3
OH
1
O
HO
HO
1' 10'
9'
2
O
O 1'
1
8' 9'
O
OSO3H
O 2''
O
HO
OH
8'
1 Okadaic Acid (OA)
3
38
O OH
3
O
30
O
6''
1''
7''
N H
10'
8''
14''
OH
OH
OSO3H
OH
3 DTX5c
O
O
DTX5a
O
O
DTX5b
Fig. 1. OA derivatives isolated from genus Prorocentrum.
Recently, we reported the structure of a new OA diol ester 2 from artificial cultures of P. belizeanum (Sua´rez-Go´mez et al., 2005). Following on that work, we report here on the isolation of its sulphate water-soluble derivative DTX-5c 3 whose structure was established using spectroscopic methods.
2. Materials and methods 2.1. Instrumentation and general methods Optical rotation was carried out on a Perkin–Elmer 241 polarimeter. IR spectrum was measured on a Bruker IFS55 spectrometer. NMR spectra were recorded on Bruker 400 Advance and Bruker AMX 500 instruments. Chemical shifts are given relative to TMS and coupling constants are calculated in Hertz. HRMS was performed on a VG Auto Spec FISON spectrometer. HPLC analyses were performed on a Shimadzu instrument equipped with a differential diffractometer detector and an X-Terra column. TLC’s were carried out using Si gel Merck 60G, and were visualized with 10% phosphomolybdic acid in ethanol. 2.2. Strain The dinoflagellate Prorocentrum belizeanum was obtained from the IEO Vigo collection by courtesy of
Santiago Fraga. We were given 3 mL of approximately 7000 cell/mL of a clonal culture stored in L1 medium. 2.3. Preparation of batch algal cultures Large scale cultures of the dinoflagellate P. belizeanum were grown in 80 L tanks containing 40 L of sea water enriched with Guillard K medium at 21 8C under 16 light: 8 dark cycle. Cultures were incubated statically for 3 weeks up to a final volume of 1020 L. 2.4. Toxins extraction and isolation Cultured cells were harvested discarding the supernatant followed by centrifugation at 7000 rpm, sonication and extraction with acetone. The solvent was evaporated and the resulting extract was subjected to successive chromatographies: first, gel filtration on a Sephadex LH-20 column eluted with a mixture of CHCl3/MeOH/n-hexane (1:1:2) followed by mediumpressure reversed-phase Lobar LiChroprep RP-18 column with CH3CN/H2O (1:1) and MeOH/H2O (3:2). Toxin-containing fractions were pooled and subjected to a HPLC final purification on an X-Terra column using an isocratic elution MeOH/H2O (7:3) to obtain DTX-5c, 3 (4 mg).
922
P.G. Cruz et al. / Toxicon 47 (2006) 920–924
Full scan data were acquired from m/z 500 to 2000, in positive ion mode.
2.5. NMR spectroscopy NMR spectra were obtained dissolving compound 3 in CD3OD (99.8Cat.% D, Aldrich, USA) using Bruker 400 Advance and Bruker AMX 500 spectrometers. NMR assignments (Table 1) were obtained from examination of 1D and 2D experiments (1H, 13C, COSY, TOCSY, ROESY, HSQC, and HMBC). Chemical shifts are reported relative to TMS (dH 0.0 ppm) and CD3OD (dC 49.0 ppm) at 300 K. 2.6. MS–MS analysis MS–MS analysis was performed using a Finnigan LQC Deca XP Max and NanoSapray source with a solution of compound 3 in MeOH using flow rates of 50 nL/min. Table 1 13 C and 1H NMR chemical shift data (CD3OD) for compound 3 Carbon
d13C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
176.2 74.9 45.8 67.6 32.5 26.9 72.5 97.4 123.0 138.7 32.5 71.8 42.3 135.8 131.6 79.6 31.1 37.4 106.8 33.2 26.9 70.4 77.5 71.1 146.2 85.9 65.5 36.7 31.8 76.1 28.3 26.9 30.4 95.6 36.7
d1H
Carbon
d13C
d1 H
19.1 26.2 60.6 10.0 16.3 111.9 16.3 22.6 25.4 67.6 142.4 31.8 128.8 129.3 31.8 148.1 66.9 112.6 109.8 172.4 38.1 123.7 133.7 29.0 36.0 175.2 45.8 69.0 38.1 69.7 80.3 71.1 70.7
1.87/1.52 1.50/1.38 3.70/3.52 0.93 1.05 5.33/5.01 1.05 1.73 1.42 4.62/4.52
3.92 4.08 1.36/0.94 1.87 3.26 1.81 2.00/1.89 1.56, 1.36
36 37 38 39 40 41 42 43 44 10 20 30 40 50 60 70 80 90 10 0 1 00 2 00 3 00 4 00 5 00 6 00 7 00 8 00 9 00 10 00 11 00 12 00 13 00 14 00
1.59/1.43
N–H
1.89/1.81 4.00 1.73/1.36 1.79/1.66 3.34 5.26 1.99/1.89 3.69 2.35 5.78 5.54 4.57 2.21/1.57 1.99/1.85 1.92/1.87 1.98/1.85 3.64 3.36 4.10
2.86 [2H] 5.55 5.57 2.83 [2H] 4.52 [2H] 5.06/4.99 5.01/4.87 3.08 [2H] 5.61 5.63 2.33 [2H] 2.29 [2H] 3.26 [2H] 3.90 1.94/1.73 4.24 4.33 4.22 4.38/4.12 4.08
13 C NMR recorded in a Bruker 400 Avance, chemical shifts refer to CD3OD dC 49.0; 1H NMR in a Bruker 500 AMX, chemical shifts refers to TMS dH 0.
3. Results and discussion Large-scale unialgal cultures of P. belizeanum (Morton et al., 1998) were carried out up to a final volume of 1020 L using Guillard K medium at 21 8C under 16 light: 8 dark cycle in 80 L tanks. Cells were harvested by centrifuging at 7000 rpm, immediately sonicated and extracted with acetone. The solvent was evaporated in vacuo obtaining a yellow–brown oil (21 g). After that, it was successively fractionated using Sephadex LH-20, LiChroprep RP-18 and HPLC equipped with an X-Terra column, yielding the purified toxin 3 (4.0 mg). It was isolated as a yellow amorphous solid; ½a25 D C 4:5 (c 0.19, MeOH); IR (CHCl3) gmax 3417, 2931, 1731, 1644, 1454, 1235, 10711 978 and 976 cmK1; 1H and 13C NMR see Table 1 and Fig. 2; MS/MS m/z: 1499/1380/1261/977/831; FAB HRMS 1498.5851 (calcd 1498.5852 for [C68H103NO27S2Na3]C). A molecular formula of C68H 103NO27S2 Na 2 was established for DTX5c 3 based on the mass spectrum that presented an ion at m/z 1498.5851 corresponding to [C68H103NO27S2Na3]C. Comparison of the proton and carbon NMR spectra of 3 (Table 1) with those of the OA C10-diol ester 2 (Sua´rez-Go´mez et al., 2005), previously isolated from this alga, showed that all the signals from the diol-ester 2 were present in the spectra of 3 except the one from methylene H2-8 0 , which was shifted from dH 3.93 to dH 4.52 in the new compound, thus indicating the presence of a new ester group. In addition, compound 3 showed new proton and carbon signals which were connected in the COSY, TOCSY and HSQC experiments to form two structural units: fragment (A) comprising C-2 00 to C-6 00 and fragment (B) from C-8 00 to C-14 00 (Fig. 2). The structure of fragment (A) was established using COSY experiment which allowed us to connect a methylene signal centered at dH 3.08 (H2-2 00 , dC 38.1) with the olefinic signal at dH 5.61 (H-3 00 , dC 123.7), which, in turn, was connected with another olefinic signal centered at dH 5.63 (H-4 00 , dC 133.7). This was, in turn, connected with the methylene allylic signals at dH 2.33 (H2K5 00 , dC 29.0) and, which was connected with a second methylene centered dH 2.29 (H2-6 00 , dC 36.0), thus establishing the hydrocarbon nature of this fragment. The partial structure of fragment (B) was constructed starting from the methylene centered at dH 3.26 (H2K8 00 , dC 45.8) which is coupled to the methine H-9 00 (dH 3.90, dC 69.0) and in turn connected with the methylene H2-10 00 (dH 1.94 and 1.73, dC 38.1). The COSY and TOCSY experiments then clearly showed the correlations H-11 00 to H-14 00 . According to the proton and carbon chemical shift values, fragment (B) contains all the new heteroatoms present in the molecule which, in accordance with the molecular formula, were 1 nitrogen, 11 oxygen and 2 sulphur atoms. Thus, it was clear that the nitrogen was linked to carbon C-8 00 as shown by its
P.G. Cruz et al. / Toxicon 47 (2006) 920–924 A
B
5.61
O
H
O 2.33
O
3.08
O
2.29
H
5.63
4.08
123.2 38.1
N
H
O 172.4
3.26 1.94/1.73 3.90
OH
O 29.0
133.7
175.2 45.8 36.0
N H
38.1 69.0
OH
OSO3H 4.33 4.38/4.12 4.24
4.22
OSO3H
OH
OH
OSO3H 80.3 69.7
OH
71.1
70.7
OSO3H
OH
Fig. 2. Chemical shift data, and fragments (A) and (B) obtained by COSY and TOCSY are drawn in bold lines.
carbon dC 45.8 and proton dH 3.26 (2H) chemical shifts values and also with the IR absorption at 1.650 cmK1 corresponding to the presence of an amide group. With the exception of C-10 00 (dH 1.94 and 1.73, dC 38.1) therefore assigned as a methylene group, all the other carbons present in this fragment bear an oxygen atom and two of them must be sulphate groups, according to the sequential losses of 119 Da (OSO3Na) observed in the MS/MS analysis. Moreover, the differences observed for the proton and carbon chemical shifts values of the methine C-12 00 (dH 4.33, dC 80.3) and the methylene C-14 00 (dH 4.38 and 4.12, dC 70.1) related with those of C-9 00 (dH 3.90, dC 69.0), C-11 00 (dH 4.24, dC 69.7) and C-13 00 (dH 4.22, dC 71.1) suggested that the sulphate groups were located at carbons C-12 00 and C-14 00 (Fig. 2). Using the HMBC correlations for the carbonyl moieties, the new building blocks that form part of DTX5c were
923
connected. Furthemore, the connection between the C-10 diol ester of OA and fragment (A) was indicated to be through the sp2 quaternary carbon C-1 00 (dC 172.4) bearing the carbonyl group and this was revealed by the HMBC correlations for H2-8 0 and H2-2 00 with C-1 00 . A similar situation was observed for the carbonyl C-7 00 (dC 175.2) and its HMBC correlations with H2-6 00 and H2-8 00 (Fig. 3). Three-dimensional information was obtained from the analysis of ROESY data, which confirmed that the relative stereochemistry of all the common chiral centers of 3 and 2 were identical, as well as, the Z configuration of the double bound connecting carbons C-4 0 –C-5 0 of the ester side chain. In addition, the measured value J3 00 –4 00 Z15.4 Hz for the coupling constant observed in the proton H-4 00 , suggests an E configuration for the double bound at C-3 00 –C-4 00 . This metabolite is the fourth example of a sulphated ester produced by dinoflagellates of the genus Prorocentrum included in a particular class of water-soluble DSP toxin derivatives. Previously reported toxins DTX4, DTX5a and DTX5b isolated from P. maculosum showed that these microalgae biosynthesizes common structural fragments and that the differences appreciated in these series are related to the most abundant enol-ester present in the species, P. maculosum with C8 and C9 side chains giving DTX5a and DTX5b, respectively. The strain subject of the present study possesses mainly a C10 ester side chain resulting DTX5c, 3. When the volume of the unialgal cultures increases, an exhaustive study can be carried out, and the possibility of identifying new sulphate derivatives related to each of the diol esters reported previously, should be forthcoming.
Fig. 3. Significant HMBC carbonyl correlations (arrows) for the sulphate ester side chain in compound 3.
924
P.G. Cruz et al. / Toxicon 47 (2006) 920–924
Acknowledgements The authors acknowledge financial support from the Spanish MCYT (AGL2004-08241-C04-01/ALI; PPQ200204361-C04-04); MCYT-FSE (Programa Ramo´n y Cajal, MLS) and ICIC (PGC). Strain of P. belizeanum was obtained from the IEO (Vigo) courtesy of S. Fraga. Thanks also are extended to J. Franco and M. Carrera from IEO for MS/MS spectrum.
References Britton, R., Roberge, M., Brown, C., Van Soest, R., Andersen, R.J., 2003. New okadaic acid analogues from the marine sponge Meriamum oxeato and their effects on mitosis. J. Nat. Prod. 66 (6), 838–843. Daranas, A.H., Norte, M., Ferna´ndez, J.J., 2001. Toxic marine microalgae. Toxicon 39 (8), 1101–1132. Ferna´ndez, J.J., Candenas, M.L., Souto, M.L., Trujillo, M.M., Norte, M., 2002. Okadaic acid, useful tool for studying cellular processes. Curr. Med. Chem. 9 (2), 229–262. Ferna´ndez, J.J., Sua´rez-Go´mez, B., Souto, M.L., Norte, M., 2003. Identification a new okadaic acid derivative from laboratory cultures of Prorocentrum lima. J. Nat. Prod. 66 (9), 1294–1296.
Hu, T., de Freitas, A.S.W., Marr, J., Quilliam, M.A., Walter, J.A., Wright, J.L.C., 1992. New diol esters (of okadaic acid) isolated from cultures of the dinoflagellates Prorocentrum lima and Prorocentrum concavum. J. Nat. Prod. 55 (11), 1631–1637. Hu, T., Curtis, J.M., Walter, J.A., McLachlan, J.L., Wright, J.L.C., 1995a. Two new water-soluble DSP toxin derivatives from the dinoflagellate Prorocentrum maculosum: possible storage and excretion products. Tetrahedron Lett. 36 (51), 9273–9276. Hu, T., Curtis, J.M., Walter, J.A., Wright, J.L.C., 1995b. J. Chem. Soc., Chem. Commun., 597–599. Morton, S.L., Moeller, P.D.R., Young, K.A., Lanoue, B., 1998. Okadaic acid production from the marine dinoglagellate Prorocentrum beliceanum faust isolated from the Belizean coral reef ecosystem. Toxicon 36 (1), 201–206. Norte, M., Padilla, A., Ferna´ndez, J.J., Souto, M.L., 1994. Structural determination and biosynthetic origin of two ester derivatives of okadaic acid isolated from Prorocentrum lima. Tetrahedron 50 (30), 9175–9180. Sua´rez-Go´mez, B., Souto, M.L., Norte, M., Ferna´ndez, J.J., 2001. Isolation and structural determination of DTX6, a new okadaic acid derivative. J. Nat. Prod. 64 (10), 1363–1364. Sua´rez-Go´mez, B., Souto, M.L., Cruz, P.G., Ferna´ndez, J.J., Norte, M., 2005. New targets in diarrhetic shellfish poisoning control. J. Nat. Prod. 68 (4), 596–599. Yasumoto, T., Murata, M., 1993. Marine toxins. Chem. Rev. 93 (5), 1867–1909.
