Article pubs.acs.org/jnp
Isolation of 6‑Deoxytetrodotoxin from the Pufferfish, Takif ugu pardalis, and a Comparison of the Effects of the C‑6 and C‑11 Hydroxy Groups of Tetrodotoxin on Its Activity Yuta Kudo,† Julian Finn,‡ Kohei Fukushima,§ Satsuki Sakugawa,⊥ Yuko Cho,† Keiichi Konoki,† and Mari Yotsu-Yamashita*,† †
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi, 981-8555, Japan ‡ Sciences, Museum Victoria, GPO Box 666, Melbourne, Victoria 3001, Australia § Kumamoto Prefectural Government, 6-18-1 Suizenji, Cyuo-ku, Kumamoto, Kumamoto, 862-8570, Japan ⊥ Department of Biological Sciences, Okinawa Prefectural Institute of Health and Environment, 2085 Ozato, Ozato, Nanjo, Okinawa, 901-1202, Japan S Supporting Information *
ABSTRACT: Identification of new tetrodotoxin (TTX, 1) analogues would be significant in the elucidation of its biosynthetic pathway and a study of its structure−activity relationships. In this study, a new TTX analogue, 6-deoxyTTX (2), was isolated from the ovary of the pufferfish, Takif ugu pardalis, and the structure was determined using spectroscopic methods. Compound 2 was also identified in other marine animals, Nassarius snail and blue-ringed octopuses, using LC-MS. Furthermore, we investigated the voltage-gated sodium channel blocking activity of 2 by examination of the inhibitory activities to cytotoxicity induced by ouabain and veratridine in mouse neuroblastoma cells (Neuro-2a). The activities were then compared with those of 1, 11-deoxyTTX (3), and 6,11-dideoxyTTX (4). The EC50 value for 2 was estimated to be 6.5 ± 2.2 nM, approximately 3-fold larger than that of 1 (2.1 ± 0.6 nM) and approximately 20-fold smaller than that of 3. These results suggested that contribution of the C-6 hydroxy group to the activity is less than that of the C-11 hydroxy group.
T
5,6,11-trideoxyTTX and 1-N-hydroxy-8-epi-5,6,11-trideoxyTTX, have been detected only in newts.23 Such considerable differences in the TTX analogue composition indicate different biosynthesis, metabolism, and accumulation systems or different origins of the TTX analogues among these animals.23 Therefore, we expect that the discovery of new TTX analogues would shed light on the biosynthesis or metabolism of TTX, which remains unclear. TTX blocks voltage-gated sodium channels (Nav) with high affinity and specificity.37,38 Because some types of Nav are implicated in a type of chronic pain, TTX is expected to exhibit analgesic properties.39 We and other groups have proposed that all six hydroxy groups at C-4, C-6, C-8, C-9, C-10, and C-11 and the guanidinium group in TTX contribute to Nav blocking activity due to the formation of hydrogen bonds and an ion pair, respectively.40−42 The C-10 hemilactal group would predominantly exist as the hydroxy form under physiological conditions since the pKa of the C-10 acidic hemilactal is 8.7.43
etrodotoxin (TTX, 1), a well-known potent neurotoxin, was first isolated from pufferfish.1−3 Subsequently, TTX and its analogues have been found in a wide range of marine and terrestrial animals, including crabs,4,5 snails,6−8 blue-ringed octopuses,9,10 sea slugs,11 flatworms,12,13 newts,14−17 and frogs.18−20 However, little is known about the biosynthetic pathway toward TTX. Various TTX analogues were isolated or detected from these animals by our group, also by Shimizu et al: 6-epiTTX, 11-deoxyTTX (3), and 8-epi-type and 1-Nhydroxy-type TTX analogues from newts,21−23 5-deoxyTTX, 6,11-dideoxyTTX (4), 5,6,11-trideoxyTTX, 11-norTTX-6(S)ol, and 4-S-cysteinylTTX from pufferfish,24−28 and chiriquitoxin from frogs.29 Moreover, 5,11-dideoxyTTX was recently identified in pufferfish and flatworms.30 To examine whether these analogues are species specific or not, we compared the composition of TTX analogues among various TTX-possessing organisms using hydrophilic interaction chromatography− electrospray ionization-mass spectrometry (HILIC-ESIMS).30−33 5,6,11-TrideoxyTTX is the major analogue in the ovary of pufferfish and has also been identified in other marine animals,30,34−36 but it has not been detected in newts.23 In contrast, 6-epiTTX and 8-epi-type analogues, such as 8-epi© XXXX American Chemical Society and American Society of Pharmacognosy
Received: December 30, 2013
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dx.doi.org/10.1021/np401097n | J. Nat. Prod. XXXX, XXX, XXX−XXX
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by the molecular formula, chromatographic properties, and chemical shifts of C-4 (δC 75.1 ppm) and C-8a (δC 60.5 ppm) in 2a, which are similar to C-4 (δC 75.1 ppm) and C-8a (δC 59.7 ppm), respectively, of TTX (1a, hemilactal).21 The COSY spectrum of 2a exhibited couplings between H-4/H-4a, H-5/H6, H-6/H2-11, and H-7/H-8 (Figure S1). The W-type coupling between H-5/H-7 was also observed in 2a, which is similar to TTX (1a). The presence of H-6 in 2a was deduced from the coupling shown in the COSY between H-5/H-6. The HMBC spectrum of 2 clarified the connectivities around the quaternary carbons at C-8a and C-10 by exhibiting cross-peaks due to C-4/ H-4a, C-5/H-7, C-7/H-5, C-8a/H-4a, C-8a/H-7, C-8a/H-8, C9/H-8, C-10/H-5, C-10/H-7, C-11/H-5, and C-11/H-6 in 2a. Comparing the 13C and 1H NMR signals of TTX (1a, hemilactal)21 to those of 2a (Table 1), upfield shifts (ppm) of H-4a (−0.44) and H-8 (−0.23) and a large upfield shift of C-6 (−28.1) were observed. These shifts suggested the absence of the C-6 hydroxy group in 2. The NOE measurements from the NOESY1D spectra of 2a confirmed the axial substitution of H6; irradiation at δH 1.86 ppm (H-6) enhanced the signal intensity of H-4a (δH 1.91 ppm), H-5 (δH 4.50 ppm), and H-8 (δH 4.07 ppm) of 2a (Figures S2 and S6). The value of 3 JH‑4/H‑4a (9.1 Hz in 2a) confirmed the diaxial configuration of H-4 and H-4a. The configuration of C-9 in 2 was the same as that in TTX because the chemical shifts of H-9 (δH 3.96 ppm) and C-9 (δC 70.9 ppm) in 2a are the same as H-9 (δH 3.96 ppm) and C-9 (δC 70.9 ppm) of 1a.21 In addition, partial conversion of 2 to the 4,9-anhydro form of 2 was observed using LC-MS after incubation of 2 in 5% trifluoroacetic acid− H2O (v/v) at 37 °C for 20 h, similar to TTX.1−3,50 All of these data led to the new TTX analogue being assigned as 6deoxyTTX (2). To discuss the biosynthetic significance of 2, the extracts of the Japanese snail, Nassarius glans, the Japanese blue-ringed octopus, Hapalochlaena sp., and the Australian blue-ringed octopus, H. maculosa, were analyzed using HRLCMS for TTX analogues. The toxin content per gram of these animals is listed in Table 2. In these animals, TTX was the major component among the TTX analogues, and 2 was a minor analogue that was commonly present in all of the tested animals (Figures S9− 11). However, in H. maculosa, the content of 2 was the highest for the known deoxy analogues. We have found a series of deoxy analogues, such as 5,6,11trideoxyTTX,26 6,11-dideoxyTTX,25 5,11-dideoxyTTX,30 5deoxyTTX,24 and 11-deoxyTTX,21 in pufferfish and other marine animals and hypothesized that TTX might be derived from 5,6,11-trideoxyTTX via stepwise oxidation in TTXproducing marine bacteria and, then, accumulated in these marine animals.30,51,52 In the current study, we isolated and determined the structure of a new TTX analogue, 6-deoxyTTX (2), from the ovary of the pufferfish, T. pardalis. The distribution of 2 in other marine animals supports the above hypothesis, and 2 could be a direct precursor of TTX, even though 5-deoxyTTX and 11-deoxyTTX are also potential candidates for this precursor. We speculate that these deoxy analogues of TTX would be precursors rather than its metabolites because oxidative reactions are usually more common in biosynthetic pathways than reductive ones. This issue will be experimentally examined during our future work. To investigate the contribution of C-6 and C-11 hydroxy groups to the activity, the Nav blocking activity of 2 was examined for comparison with those of TTX (1), 11deoxyTTX (3), and 6,11-dideoxyTTX (4) using the previously
Some TTX analogues have been synthesized44−48 and have provided a structure−activity relationship study of TTX.49 However, the biological activity of a TTX analogue that lacks only the C-6 hydroxy group has never been investigated. In this study, we report the isolation and structure determination of a new TTX analogue, 6-deoxyTTX (2), and the distribution of this analogue in some marine animals to elucidate the biosynthetic significance of 2. In addition, we investigated the Nav blocking activity of 6-deoxyTTX (2) for comparison with those of TTX (1), 11-deoxyTTX (3), and 6,11-dideoxyTTX (4). The effects of the C-6 and C-11 hydroxy groups were also compared.
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RESULTS AND DISCUSSION The extract of the ovary of Takif ugu pardalis showed an unknown peak corresponding to 2 on the HRLCMS extracted ion chromatogram (EIC) detecting for m/z 304.1139 ± 0.01 at the retention time that is different from those of known deoxy analogues (i.e., 11-deoxyTTX and 5-deoxyTTX) (Figure S8). Compound 2 (approximately 40 μg, by 1H NMR) was isolated by continuous column chromatography. Compound 2 was suggested to be a new monodeoxy analogue of TTX based on its molecular formula (C11H17N3O7). The structure determination of 2 was primarily achieved via NMR measurements. The 1H NMR spectrum revealed double sets of signals, suggesting that 2 exists as tautomers of hemilactal (2a) and 10,7-lactone (2b) forms similar to other TTX analogues (Figures 1 and S3).1−3
Figure 1. Hemilactal (2a) and 10,7-lactone (2b) forms of the new TTX analogue 6-deoxyTTX.
The 2a:2b ratio in CD3COOD−D2O (4:96, v/v) at 20 °C was estimated to be approximately 3:1 (mol/mol) by 1H NMR spectroscopy. The assignment of all of the 1H and 13C signals except for C-2 of 2a was derived from COSY, TOCSY, HSQC, and HMBC (Table 1, Figures S4 and S5), and the assignment of the signals from 2b was partially accomplished, but was hampered by signal intensity (Table S1). Although the HMBC correlation between C-2/H-4 was not observed in 2a, probably because of the small sample size, the presence of a guanidinium group in 2a was strongly suggested B
dx.doi.org/10.1021/np401097n | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Spectroscopic Data for the Hemilactal Forms of TTX (1a) and 6-DeoxyTTX (2a)a and Their Comparison TTX (hemilactal) (1a)21
6-deoxyTTX (hemilactal) (2a) δC, type
position 2 4 4a 5 6 7 8 8a 9 10 11
b
ND 75.1, 45.9, 71.6, 43.4, 78.6, 76.2, 60.5, 70.9, 110.0, 61.7,
δH (J in Hz) C CH CH CH CH CH CH C CH C CH2
5.51, 1.91, 4.50, 1.86, 4.26, 4.07,
δC, type
d (9.1) d (9.7) br s dd (7.3, 7.1) br s br s
3.96, s 3.95, dd (10.6, 6.9) 4.00, dd (11.4, 7.6)
156.6, 75.1, 40.7, 73.8, 71.5, 79.7, 72.8, 59.7, 70.9, 110.8, 65.5,
C CH CH CH C CH CH C CH C CH2
Δδ (2a − 1a)
δH (J in Hz)
ΔδC
ΔδH
5.50, d (9.4) 2.35, d (9.5) 4.25, br s
0.0 5.2 −2.2 −28.1 −1.1 3.4 0.8 0.0 −0.8 −3.8
0.01 −0.44 0.25
4.08, t (1.8) 4.30, d (1.5) 3.96, s 4.02, d (12.6) 4.04, d (12.6)
0.18 −0.23 0.00 −0.07 −0.04
a
The 1H (600 MHz) and 13C (150 MHz) NMR spectra were recorded using CD3COOD−D2O (4:96, v/v) as the solvent. The signal of CHD2COOD (2.06 ppm) in the 1H NMR and that of 13CD3COOD (22.4 ppm) in the 13C NMR were used as the internal references. bND denotes not determined.
reported cell-based assay.49 All of these TTX analogues exhibited a concentration-dependent inhibitory effect on the cytotoxicity induced by ouabain and veratridine in the mouse neuroblastoma cell line, Neuro-2a (Figure 2). The estimated EC50 values are shown in Figure 2. The EC50 value for 2 was only approximately 3-fold larger than that of 1, which was approximately 20-fold smaller than that of 3, indicating that 2 maintains a much higher activity than 3. A slight decrease in the activity due to the absence of the C-6 hydroxy group of TTX was observed. Similarly, the decrease in the activity due to removal of the C-6 hydroxy group of 3, which was estimated by comparing the EC50 values between 3 and 4, was also small (approximately 3-fold). On the other hand, the decrease in the activity due to loss of the C-11 hydroxy group of 1 and 2 was significant in both analogues. The EC50 values for 1 and 2 were both approximately 60-fold smaller than those of 3 and 4, respectively. These results agreed well with our previous results, where the EC50 value of 3 was 58-fold larger than that of 1.49 It was previously proposed that the hydroxy groups of TTX at C-4, C-6, C-8, C-9, C-10, and C-11 contributed to the Nav blocking activity due to the formation of hydrogen bonds.40,41,49 The result in the current study is the first experimental evidence to confirm the direct contribution of the C-6 hydroxy group of TTX to its activity. The C-6 and C-11
Table 2. Contents of TTX Analogues in the Marine Animals Japanese Takif ugu pardalis, Nassarius glans, Hapalochlaena sp., and Australian H. maculosa contents (μg/g) T. pardalis
N. glans
N. glans
H. sp.
H. maculosa posterior salivary gland
compound
ovary
viscera
muscle
salivary gland
TTX (1) 4-epiTTX 4,9-anhydroTTX 5-deoxyTTX 6-deoxyTTX (2) 11-deoxyTTX (3) 5,11-dideoxyTTX 6,11-dideoxyTTX (4) 5,6,11trideoxyTTX 11-norTTX-6(S)ol 11-oxoTTX
25 3.1 11 0.87 0.39 2.3 0.33 5.8
42 7.2 30 0.89 4.2 4.9 0.58
