Stereoisomers of 42-Hydroxy Palytoxin from Hawaiian Palythoa toxica

Feb 10, 2014 - ... to native Hawaiians that used to smear a “moss” containing the toxin on .... John W. Blunt , Brent R. Copp , Robert A. Keyzers ...
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Stereoisomers of 42-Hydroxy Palytoxin from Hawaiian Palythoa toxica and P. tuberculosa: Stereostructure Elucidation, Detection, and Biological Activities Patrizia Ciminiello,† Carmela Dell’Aversano,† Emma Dello Iacovo,† Martino Forino,*,† Luciana Tartaglione,† Marco Pelin,‡ Silvio Sosa,‡ Aurelia Tubaro,‡ O. Chaloin,§ Mark Poli,⊥ and Gary Bignami∥ †

Department of Pharmacy, University of Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy Department of Life Sciences, University of Trieste, Via A. Valerio 6, 34127 Trieste, Italy § CNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunologie et Chimie Thérapeutiques, 67000 Strasbourg, France ⊥ U.S. Army Medical Research Institute of Infectious Diseases, Ft Detrick, Maryland 21701-5011, United States ∥ Bignami Consulting, Honolulu, Hawaii, United States ‡

S Supporting Information *

ABSTRACT: Palytoxin ranks among the most potent marine biotoxins. Its lethality was well known to native Hawaiians that used to smear a “moss” containing the toxin on their spears to cause instant death to their victims. Human intoxications due to exposure to palytoxin and to its many congeners have been reported worldwide. Currently, palytoxins constitute the main threat to public health across the Mediterranean Sea. In the present work we report on the isolation and stereostructural determination of a new palytoxin analogue from a Hawaiian Palythoa tuberculosa sample. This new toxin is a stereoisomer of 42-hydroxypalytoxin isolated from Palythoa toxica. The whole absolute configuration of this latter toxin is also reported in the paper. Interestingly, the two 42-hydroxypalytoxins do not share the same biological activity. The stereoisomer from P. tuberculosa showed cytotoxicity toward skin HaCaT keratinocytes approximately 1 order of magnitude lower than that of 42hydroxypalytoxin from P. toxica and about 2 orders of magnitude lower than that of palytoxin itself. This finding holds the prospect of interesting structure−activity relationship evaluations in the future.

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depending upon their biological sources and geographical provenance. Additionally, level and content of palytoxins in toxic Palythoa spp. can vary significantly among species, among populations of the same species, and seasonally.12 In 2009, we published a paper reporting on the analysis of the toxin content of two samples of Hawaiian P. tuberculosa and P. toxica.13 These samples were collected along the Hawaiian coasts and stored at the U.S. Army Medical Research Institute of Infectious Diseases at Fort Detrick, Maryland.14 In-depth high resolution liquid chromatography−mass spectrometry (HR LC/MS) analysis allowed identification of a previously undescribed palytoxin-like compound in both samples of Palythoa spp. Interestingly, this new palytoxin analogue was found to be by far the main palytoxin derivative present in the P. toxica sample and a major component of the toxic extract of the P. tuberculosa sample, alongside palytoxin itself. By 1D- and 2D-NMR investigation, we structurally characterized the toxin

n 1961, Philip Helfrich, accompanied by John Shupe, tracked down a legendary tidepool near the village of Mu’olea on the Island of Maui, where native Hawaiians used to collect a deadly seaweed reportedly capable of causing instant death to prey or enemies.1−3 Analyses of samples collected from this tidepool revealed the organism responsible for the fabled lethality to be a new species of cnidarian zoanthid, known as Palythoa toxica, a very rare species sparingly found along the Pacific coasts.4,5 In 1971, Paul Scheuer and Richard Moore isolated the toxic molecule from samples of P. toxica and provided preliminary details about its chemical structure. The toxin was named palytoxin (1).2 It took more than 10 years to fully define the stereochemical architecture of palytoxin.6−8 In 1972, palytoxin was also isolated from Palythoa tuberculosa collected from Okinawa, Japan.4 Since then, toxins seemingly identical to palytoxin have been identified in other Palythoa species from around the world, such as P. vestitus from Hawaii,9 P. caribaeorum from the West Indian islands,10 and additional unidentified Palythoa spp. from Tahiti and Japan.11 It is known that palytoxins can differ structurally from each other © 2014 American Chemical Society and American Society of Pharmacognosy

Received: November 19, 2013 Published: February 10, 2014 351

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Figure 1. Stereostructures of palytoxin (1), 42S-hydroxy-50R-palytoxin (2), and 42S-hydroxy-50S-palytoxin (3).

contained in P. toxica as 42-hydroxypalytoxin (2). The configurations of all of the stereocenters contained in 42hydroxypalytoxin (2) were assigned, except those of C-41 and C-42.13 In the present paper, we report on the configuration assignment of the two asymmetric carbons left undetermined in 42-hydroxypalytoxin from P. toxica, as well as on the purification and stereostructure elucidation of another 42hydroxypalytoxin (3) from the P. tuberculosa sample. The latter was identified as a diastereoisomer of 2 with a configurational inversion at C-50 (Figure1). Eventually, the palytoxin analogue isolated from P. toxica (2) was characterized as 42S-hydroxy50S-palytoxin and the palytoxin analogue isolated from P. tuberculosa (3) as 42S-hydroxy-50R-palytoxin. Interestingly, the cytotoxicity of 42S-hydroxy-50R-palytoxin toward skin HaCaT keratinocytes appeared approximately 2 orders of magnitude lower than that of palytoxin and 1 order of magnitude lower than that of 42S-hydroxy-50S-palytoxin.

complemented by interpretation of dipolar couplings (ROEs).16 With the purpose of measuring 3JH−H values, a z-filtered TOCSY was run with a one-second t2 acquisition time for attaining an adequate digital resolution (Supporting Information Figure S2). Extrapolation of appropriate 1D rows from the z-TOCSY spectrum allowed us to identify the NMR multiplicities of both H-42 and H-43. However, the H-41 NMR signal could not be extrapolated, as it was overlapped with other proton resonances. In more detail, the H-42 NMR multiplicity appeared as a doublet of doublets characterized by a large coupling constant (3JH−H = 7.2 Hz) and a small one of 2.8 Hz (Figure S3). A 7.2 Hz coupling, as reported by Matsumori et al.,16 is a typical value for an H/H anti orientation in 1,2-dioxygenated methine systems. It followed that H-42 was simultaneously in a preferential anti and gauche disposition with its two vicinal protons H-41 and H-43. As the H-43 NMR multiplicity appeared as a broad singlet (Figure S3), the gauche relationship between H-42 and H-43 and, consequently, the anti relationship between H-41 and H-42 were inferred (Figure S3). The heteronuclear spin-coupling constants (2JC−H and 3 JC−H) could not be quantitatively measured because of the small amount of pure toxin. However, we were able to qualitatively classify all of the 2JC−H and 3JC−H required for a comprehensive J-based configurational analysis as either “small” or “large” on the basis of the ratio of the relative magnitude of their ps-HMBC cross-peaks with that of a common proton.16 Missing 2,3JC−H were referred to as “small”. Accordingly,

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RESULTS AND DISCUSSION 42-Hydroxypalytoxin from Palythoa toxica (2): Configuration Assignment at C-41 and C-42. Palytoxins show alternating sequences of pyranose rings and acyclic segments featuring extended 1,2 or 1,3 oxymethine systems assuming a dominant conformation as normally occurs in highly functionalized molecules.15 It follows that the configuration of the C41/C-42 diol could be defined by a J-based analysis 352

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JC41−H42 and 2JC42−H41 were classified as “large” (Figure S3). This implied that both H-41 and H-42 were in a dominant gauche relationship with the hydroxy group linked to C-42 and C-41, respectively (Figure S3).16 In such a case, the sole J-based configurational analysis is not sufficient for distinguishing between the threo and erythro stereoisomers and needs to be complemented by ROESY (or NOESY) experiments.16 As no ROE between H2-40 and H-43 was detected (Figure S4), the erythro configuration of the C-41/C-42 diol, with an anti C-40/ C-43 orientation, was suggested. Finally, the spatial coupling between H-42 and H-44 was crucial for correlating the C-42 configuration to that of the pyranose ring, as depicted in Figure 1. The absolute S configuration was thus assigned to both C-41 and C-42; consequently, the toxin was definitively identified as 42Shydroxy-50R-palytoxin. 42-Hydroxypalytoxin from Palythoa tuberculosa (3): Isolation, Stereostructure Elucidation, and Toxicological Studies. The P. tuberculosa sample containing palytoxins was obtained following the modified method of Moore and Scheuer reported by Bignami et al.17 Preliminary HR LC/MS-based analyses had ascertained that the toxin profile of the P. tuberculosa sample basically consisted of two major palytoxins: palytoxin itself and a congener with a molecular formula featuring an extra oxygen atom in comparison to palytoxin.13 Final purification of the new palytoxin analogue was achieved by HPLC on a Kinetex 2.6 μm column. The occurrence of palytoxins in the eluates was detected by HR LC/MS analysis in full MS mode (positive ions). This final step of the purification yielded 1.1 mg of 3. Extensive NMR-driven investigation allowed us to define the whole stereostructure of 3 (Figure 1). A parallel analysis of COSY (Figure S9), z-TOCSY (Figure S10), and ROESY (Figure S11) experiments was instrumental for identifying the seven spin systems of the molecule. Every proton was then associated with its carbon by means of the HSQC spectrum (Table 1, Figures S12, S15); finally, the ps-HMBC spectrum (Figure S13) allowed the identification of the quaternary carbons of the molecule and the correlation of the seven spin systems to each other. Eventually, the planar structure of 3 turned out to be identical to that of 42S-hydroxy-50R-palytoxin (2).13 The overlap of proton and carbon chemical shifts of 3 with the relevant ones of 2 suggested that the two toxins shared the same configurations at all of their stereogenic centers apart from C-49 and C-50, where significant divergences were detected between the two molecules in terms of NMR chemical shifts (Table 1, Figure S15). The configuration of the C-49/C50 methine system of 3 was investigated by a J-based configurational analysis complemented by ROEs, as our NMR data suggested this segment assumed a preferred conformation. From the appropriate z-TOCSY experiment 1D row, the NMR multiplicity of H-49 emerged as a broad singlet, thus confirming its preferential gauche orientation with both H-50 and H2-48 (Figure 2). This hypothesis was further corroborated by (1) H-49/H-50 and H2-48/H-50 ROE correlations (Figure S14) and (2) weak COSY and z-TOCSY cross-peak intensities between H-49 and both H-50 and H2-48 (Figure S14). Additionally, H-49 had dipolar coupling to Me-50 (Figure S14). So, an H-49/Me-50 gauche relationship was assessed (Figure 2). H-50 showed an ROE correlation with H-48b, while Me-50 was not involved in any dipolar coupling with H2-48 (Figure S14). Finally, a large 2JH48a‑C49 was detected in the ps-HMBC 2

Table 1. Selected NMR Spectroscopic Data (700 MHz, CD3OD) for 42-Hydroxypalytoxin from P. tuberculosa (3) and 42-Hydroxypalytoxin from P. toxica (2) 2

3

δC

δH

δC

δH

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

38.69 24.23 30.84 30.84 30.84 31.21 38.69 71.92 77.34 66.10 74.08 74.28 67.75 101.59 41.92

1.60 1.43 1.29 1.29 1.29 1.35 1.58 3.65 3.70 4.32 3.91 3.91 3.78

38.72 24.15 30.87 30.87 30.87 31.40 38.68 71.81 74.16 69.68 74.00 74.28 67.75 101.28 41.86

1.60 1.43 1.33 1.33 1.33 1.38 1.61 3.70 3.71 4.34 3.92 3.91 3.78

49 50 50-Me 51 52 53 54

72.45 44.41 16.72 134.59 134.69 74.06 35.09

position

55

27.89

56 57 58 59

73.15 72.86 74.11 33.16

1.80 1.96 3.92 2.25 1.03 5.64 5.50 4.06 1.61 1.78 1.46 1.69 3.75 3.86 3.87 1.66 2.26

72.98 42.24 16.80 133.86 134.98 74.11 35.09 26.98 73.15 72.77 74.18 33.22

1.59 1.60 3.82 2.34 1.08 5.66 5.53 4.09 1.63 1.83 1.50 1.64 3.76 3.87 3.87 1.69 2.28

spectrum, which suggested the preferential gauche orientation between H-48a and the hydroxy group linked to C-49 (Figure S14).16 The above NMR data were coherent only with the erythro diastereoisomer of the C-49/C-50 methine system, as opposed to the same methine system in 42S-hydroxy-50Rpalytoxin (2) existing as a threo diastereoisomer (Figure 2). Finally, a key ROE between H-48b and H-46 correlated the configuration of the C-49/C-50 segment to that of the C ring, thus pointing to the absolute S configuration at C-49 and the R configuration at C-50 (Figures 1 and 2). Thus, the palytoxin derivative isolated from P. tuberculosa was identified as 42Shydroxy-50S-palytoxin. The mouse anti-palytoxin monoclonal antibody (mAb), used as a capturing reagent in a recently developed immunoenzymatic assay (ELISA) for palytoxin detection,18 was evaluated for its affinity for 42S-hydroxy-50S-palytoxin by surface plasmon resonance (SPR), in comparison to that for 42Shydroxy-50R-palytoxin and palytoxin itself (Figure S16). The antipalytoxin mAb was immobilized on a sensor chip exploiting the available free cysteine residues, which were selectively allowed to react with a maleimido group previously introduced on the chip. Then, the affinity of the antibody toward the three palytoxins was measured, testing different concentrations of the toxins in the fluid phase. 42S-Hydroxy-50S-palytoxin bound to the antipalytoxin mAb with a KD in the submicromolar range, 353

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Figure 3. ELISA standard curves for 42S-hydroxy-50S-palytoxin (3), 42S-hydroxy-50R-palytoxin (2), and palytoxin (1). Each point represents the mean ± SEM of four independent experiments in triplicate. Statistical differences with respect to 42S-hydroxy-50Rpalytoxin (2): ***p < 0.001 (two-way ANOVA and Bonferroni posttest).

Considering the increasing human cases of dermotoxicity ascribed to palytoxins after handling zoanthid corals19 and the presence of 42-hydroxypalytoxins in Palythoa species, the cytotoxic effect of 42S-hydroxy-50S-palytoxin was evaluated on human skin HaCaT keratinocytes. These cells are an accepted screening method for cutaneous toxicity, as they possess morphological and functional properties similar to those of normal keratinocytes.20 MTT assays were performed on HaCaT cells after 4 h exposure, and the effects were compared to those of palytoxin and 42S-hydroxy-50R-palytoxin within the concentration range of 1.0 × 10−16 to 1.0 × 10−7 M. 42SHydroxy-50S-palytoxin exerted a concentration-dependent reduction of cell viability starting from 1.0 × 10−10 M (−25 ± 4%), which was almost totally abolished at 1.0 × 10−8 M (−94 ± 2%). The concentration of toxin that reduced cell viability by 50% (EC50) was 9.3 × 10−9 M (95% confidence limit: (7.3−11.7) × 10−10 M). Also 42S-hydroxy-50R-palytoxin induced a concentration-dependent cytotoxicity, as measured by the reduction in cell viability (EC50 = 1.0 × 10−10 M; 95% confidence limit: (0.5−2.5) × 10−10 M), with a potency approximately 1 order of magnitude higher than that of its stereoisomer (p < 0.001). In addition, the cytotoxicity of palytoxin (EC50 = 2.7 × 10−11 M, 95% confidence limits: (1.5− 4.9) × 10−11 M) was about 1 and 2 orders of magnitude higher than that of 42S-hydroxy-50R-palytoxin and 42S-hydroxy-50Spalytoxin, respectively (p = 0.023) (Figure 4).

Figure 2. (a) C-43 to C-51 segment of 42S-hydroxy-50R-palytoxin (2). Dashed arrows indicate key ROEs. (b) C-43 to C-51 segment of 42S-hydroxy-50S-palytoxin (3). Dashed arrows indicate key ROEs. In both toxins the H-49/Me-50 and H-49/H-50 gauche relationships were determined on the basis of dipolar coupling between H-49 and both Me-50 and H-50. ROEs involving H-48b suggested the C-49/C50 threo configuration for 42S-hydroxy-50R-palytoxin (2) and the C49/C-50 erythro configuration for 42S-hydroxy-50S-palytoxin (3).

while 42S-hydroxy-50R-palytoxin bound with a KD in the micromolar range. In addition, the affinity of 42S-hydroxy-50Spalytoxin for the antibody was about 1 order of magnitude lower than that of palytoxin (Table 2). Table 2. Kinetic Parameters of SPR Interactions of Palytoxin Analogues on Anti-palytoxin mAba analyte

ka (1/Ms)

3 2 1

1.27 × 10 256 1.08 × 105 5

kd (1/s) −3

1.98 × 10 1.75 × 10−3 2.26 × 10−4

KD (M) −8

1.55 × 10 6.84 × 10−6 2.09 × 10−9

Rmax (RU)

Chi2

7.57 67 9.99

0.2 5.9 0.1

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a ka, kinetic association rate; kd, kinetic dissociation rate; KD, equilibrium dissociation constant; Rmax maximal response expressed as resonance units; Chi2, chi square value.

CONCLUSIONS The first 42-hydroxypalytoxin (2) was isolated from samples of P. toxica, and its chemical structure characterized. The present paper reports on the isolation, structure elucidation, detection, and cytotoxicity evaluation of a new stereoisomer of 42hydroxypalytoxin (3) from samples of P. tuberculosa with a configurational inversion at C-50 (42S-hydroxy-50S-palytoxin) with respect to the 42-hydroxypalytoxin (2) previously isolated from P. toxica (42S-hydroxy-50R-palytoxin)13 and to palytoxin (1). Surface plasmon resonance demonstrated the affinity of the mouse anti-palytoxin mAb for 42S-hydroxy-50S-palytoxin to be about 1 order of magnitude higher than that for 42S-hydroxy50R-palytoxin (KD = 6.84 × 10−6 and 1.55 × 10−8 M, respectively) and about 2 orders of magnitude lower than that for palytoxin (KD = 2.09 × 10−9 M). An indirect sandwich ELISA, set up with the mAb as a capture reagent and rabbit polyclonal antibodies as the detection reagent, recognized both

The ability of the sandwich ELISA18 to detect 42S-hydroxy50S-palytoxin was evaluated at concentrations within the working range for 42S-hydroxy-50R-palytoxin and palytoxin (1.3−80.0 ng/mL; Figure 3). 42S-Hydroxy-50S-palytoxin was detected by ELISA with an EC50 value of 40.6 ng/mL (95% confidence limits: 38.0−43.4 ng/mL). Such detection, however, was significantly lower than that of the parent compound 42Shydroxy-50R-palytoxin (EC50 = 7.8 ng/mL; 95% confidence limits: 6.3−9.7 ng/mL; p-value equal to 5.0 × 10−14). On the other hand, the reactivity of the sandwich ELISA toward 42Shydroxy-50R-palytoxin was comparable to that of palytoxin (EC50 = 3.9 ng/mL; 95% confidence limits: 2.1−7.5 ng/mL; p = 0.05) (Figure 3). 354

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to an Agilent 1100 LC binary system including a solvent reservoir, online degasser, binary pump, and thermostated autosampler. Extraction, Isolation, and Purification of 3. Fractions containing palytoxins were obtained from Palythoa tuberculosa collected in Hawaii according to the procedure described by Moore and Scheuer and modified by Bignami et al.17 Toxins were extracted from P. tuberculosa with 70% aqueous EtOH. The extract thus obtained was concentrated in vacuo and then extracted with CH2Cl2. The toxic fraction was purified from the aqueous layer by column chromatography on Amberlite XAD-2, followed by chromatography on DEAE-Sephadex A-25 and CM-Sephadex C-25. The toxic fraction was finally desalted on Bond Elut C18 eluted with 80% aqueous EtOH. Final purification of 42-hydroxypalytoxin (3) was carried out on a Kinetex 2.6 μm HPLC column (Phenomenex) connected to an Agilent HPLC model 1100 coupled to a linear ion trap LTQ Orbitrap XL hybrid Fourier transform MS instrument equipped with an ESI ION MAX source, with gradient elution by changing ratios of H2O/ CH3CN/AcOH from 80:20:01 to 0:100:0.1 in 20 min. Eventually, the final step of the purification yielded 1.1 mg of 3. LC-MS Experiments. HR LC-MS and MS2 analyses were performed on a linear ion trap LTQ Orbitrap XL Fourier transform mass spectrometer equipped with an ESI ION MAX source and coupled to an Agilent 1100 LC binary system. A 3 μm Gemini C18 column (150 × 2.00 mm; Phenomenex) was eluted at 0.2 mL/min with H2O (eluent A) and 95% CH3CN/H2O (eluent B), both containing 30 mM acetic acid. Gradient elution (20−50% B over 20 min, 50−80% B over 10 min, 80−100% B in 1 min, and held for 5 min) was used. Injection volume was 5 μL. HR full MS experiments (positive ions) were acquired in either the range m/z 800−1400 or m/ z 2000−3000 at resolution setting 30 000 to 100 000. The following source settings were used: spray voltage 4 kV, capillary temperature 290 °C, capillary voltage 45 V, sheath gas 35 and auxiliary gas 1 (arbitrary units), and tube lens voltage 165 V (m/z 800−1400) or 250 V (m/z 2000−3000). HR collision-induced dissociation (CID) MS2 experiments were acquired on [M + H + Ca]3+ ions of 1 and 3 by using 25% collision energy. A resolving power of 60 000, activation Q of 0.250, and activation time of 30 ms were used in all cases. Calculations of elemental formulas in full MS and CID MS2 spectra were performed by using the monoisotopic ion peak of each ion cluster. A mass tolerance of 5 ppm was used, and the isotopic pattern of each ion cluster was considered. Extracted ion chromatograms were obtained from the HR full MS spectra by selecting the most abundant ion peaks of the [M + 2H − H2O]2+ and [M + H + Ca]3+ ion clusters of each compound. In particular, most abundant ion peaks at m/z 1331.7403 and 906.8157 were selected for palytoxin and ion peaks at m/z 1339.7383 and 912.1467 for 42-hydroxypalytoxin. In quantitative analysis (triplicate injections), peak areas were measured and interpolated within the calibration curve of a palytoxin standard (Wako Chemicals GmbH) at five levels of concentration (50, 25, 12.5, 6.25, and 13.13 ng/mL). Linearity of the calibration curve was indicated by a correlation coefficient (R2) of 0.9980. Because of the lack of 42-hydroxypalytoxin standards, its molar response was assumed to be the same as that of palytoxin on the basis of structural similarity. NMR Experiments. NMR spectra were run by using Shigemi 5 mm NMR tubes and CD3OD as an internal standard (δH 3.31 and δC 49.0). Standard Varian pulse sequences were employed for the respective classes of spectra; solvent signal suppression by presaturation was used when required. All NMR data reported in the text were derived from 2D COSY, Z-filtered TOCSY, ROESY, phase-sensitive HMBC, and HSQC spectra. Toxins and Other Materials. Palytoxin, isolated from P. tuberculosa, was purchased from Wako Chemicals (Neuss, Germany; lot number WKL7151). All cell culture reagents were from SigmaAldrich. Multiwell strips were from Nunc; horseradish peroxidase (HRP)-conjugated polyclonal anti-rabbit goat antibodies were from DakoCytomation; anti-palytoxin monoclonal antibody (anti-palytoxin mAb) and anti-palytoxin polyclonal antibody (anti-palytoxin pAb) were obtained as previously described.18 The Biacore 3000 system, sensor chip CM5, surfactant P20, and amine coupling kit containing

Figure 4. Effect of 42S-hydroxy-50S-palytoxin (3), 42S-hydroxy-50Rpalytoxin (2), and palytoxin (1) on HaCaT cell viability evaluated by MTT assay. Cells were exposed to increasing toxin concentrations for 4 h before performing the assay. Data are reported as % of control (untreated cells) and are the mean ± SEM of four independent experiments in triplicate. Statistical differences with respect to 42Shydroxy-50S-palytoxin (3): *p < 0.05; ***p < 0.001 (two-way ANOVA and Bonferroni post-test).

palytoxin analogues. However, the ability of the ELISA assay to detect 42S-hydroxy-50S-palytoxin (EC50 = 40.6 ng/mL) was about 5 times lower in comparison to 42S-hydroxy-50Rpalytoxin (EC50 = 7.8 ng/mL) and about 10 times lower in comparison to palytoxin (EC50 = 3.9 ng/mL), which differs from the first compound both by a configurational change at C50 and by the lack of the hydroxy group at C-42. Although the assay cannot distinguish between these palytoxin analogues, it can be used as a preliminary method for screening purposes, before confirmatory chemical analyses on ELISA positive samples. It should be noted that the detection rank of the two stereoisomers and of palytoxin by the ELISA paralleled that of their cytotoxic effect toward HaCaT keratinocytes. In fact, the cytotoxicity of 42S-hydroxy-50Spalytoxin was about 1 order of magnitude lower than that of 42S-hydroxy-50R-palytoxin (EC50 = 9.3 × 10−10 and 1.0 × 10−10 M, respectively) and 2 orders of magnitude lower than that of palytoxin (EC50 = 2.7 × 10−11 M). This suggests that the contact with P. tuberculosa would induce less severe skin lesions than those induced by P. toxica contact, although both of them can be regarded as dermotoxic corals. As previously reported, small structural changes can have large impacts on the bioactivity of palytoxins.23 This study demonstrates that even a single configurational change can play a major role in the cytotoxicity of palytoxins. In fact, the cytotoxicity of 42S-hydroxy-50S-palytoxin turned out almost 1 order of magnitude higher than that of its stereoisomer 42Shydroxy-50R-palytoxin. It can be hypothesized that, following the configurational inversion at C-50, 42S-hydroxy-50Spalytoxin undergoes some conformational changes that ultimately reduce the potency of the toxin in comparison to 42S-hydroxy-50R-palytoxin.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectroscopic data were acquired on a Spectra photometer (Tecan Italia) and on an EL 311s automated microplate reader (Bio-Tek Instruments). NMR experiments were run on a Varian Unity Inova 700 spectrometer equipped with a 13C enhanced HCN cold probe. Mass spectrometric studies were performed by using a linear ion trap LTQ Orbitrap XL hybrid Fourier transform MS instrument equipped with an ESI ION MAX source (Thermo-Fisher) and coupled 355

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N-hydroxysuccinimide (NHS) and N-ethyl-N′-dimethylaminopropyl carbodiimide (EDC) were from GE-Healthcare. All the other reagents of analytical grade were purchased from Sigma-Aldrich. 42S-Hydroxy-50S-palytoxin (3): yellowish, amorphous solid; 1H and 13C NMR data (CD3OD, 700 MHz), see Table 1; HRESIMS monoisotopic ion peak at m/z 911.8131 [M + Ca + H]3+ (calcd for C129H224O55N3Ca, 911.8144). Surface Plasmon Resonance. Anti-palytoxin mAb was immobilized through the amino groups using 35 μL of 2-(2-pyridinyldithioethaneamine) in 50 mM borate buffer pH 8.3 on the NHS/EDCactivated matrix. Then, 35 μL of anti-palytoxin mAb (100 μg/mL in formate buffer, pH 4.3) was injected until a response of 8000 RU was obtained. Finally, 20 μL of a 50 mM cysteine/1 M NaCl solution was used to saturate the unoccupied sites on the chip. All binding experiments were carried out at 25 °C with a constant flow rate (20 μL/min). Different concentrations of antibody, 1, 2, or 3 were injected for 3 min, followed by a 3 min dissociation phase. The sensor chip surface was regenerated after each experiment injecting 10 μL of 10 mM HCl. Indirect Sandwich ELISA. The ability of the indirect sandwich ELISA to detect 3 was evaluated in comparison to that of 2 and 1, as previously described.18 Briefly, multiwell strips were coated with 20 μg/mL anti-palytoxin mAb (100 μL/well) for 16 h at 4 °C. Each well was then blocked for 1 h at room temperature (rt) with 200 μL of 2% skimmed milk (w/v) dissolved in PBS containing 0.1% Tween 20 (PBS-Tw). The toxins (concentration range: 1.3−80.0 ng/mL) were then incubated for 2 h at rt in PBS-Tw. After three washes with PBSTw, followed by another three washes with PBS, 100 μL of purified pAb-palytoxin (0.17 μg/mL in blocking solution) was added for 2 h at room temperature. After washing, 100 μL of HRP-conjugated goat anti-rabbit pAbs (1:2000 in blocking solution) was incubated for 1 h at rt. After washing, 3,3′,5,5′-tetramethylbenzidine liquid substrate (60 μL) was added to each well, and the enzyme reaction was stopped after 30 min by 30 μL of 1 M H2SO4. The absorbance was read at 450 nm by a Spectra photometer (Tecan Italia). Data are reported as the mean absorbance at 450 nm ± SEM of four independent experiments performed in triplicate. Cell Line and Cell Culture. The HaCaT cell line was purchased from Cell Line Service (DKFZ). HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1.0 × 10−2 M L-glutamine, 1.0 × 10−4 g/mL penicillin, and 1.0 × 10−4 g/mL streptomycin at 37 °C in a humidified 95% air/5% CO2 atmosphere. For cytotoxicity assays, cells were plated in 96-well plates at a concentration of 3 × 103 cells/well, and the experiments performed after 3 days in culture. All the experiments were performed between passage 47 and 52. MTT Assay. The MTT assay was performed as previously described.21,22 Briefly, cells were exposed to the toxins (1.0 × 10−16 to 1.0 × 10−7 M) for 4 h at 37 °C. Cells were then washed and wells refilled with fresh culture medium containing 0.5 mg/mL MTT. After 4 h, the insoluble crystals were solubilized by 200 μL/well DMSO and the absorbance was measured by an EL 311s automated microplate reader at 540/630 nm. Data are reported as % of control (untreated cells) and are the means ± SEM of four independent experiments performed in triplicate. Statistical Analysis. The data obtained by MTT and ELISA assay were analyzed by two-way ANOVA followed by Bonferroni’s post-test (Prism GraphPad Inc.), and significant differences were considered at p < 0.05. EC50 values (effective concentration reducing cell viability by 50%, MTT assay, or giving 50% of the maximal response, ELISA) were calculated by nonlinear regression using a four-parameter curve-fitting algorithm of the Prism GraphPad software and analyzed by a twotailed paired Student’s t test. Significant differences were considered at p < 0.05.

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plasmon resonance (SPR) experimental conditions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

NMR experiments provided for 2 and 3: 1H spectrum, zfiltered TOCSY, ROESY, HSQC, and ps-HMBC. Carbon and proton NMR data (700 MHz, CD3OD) for 2 and 3. Surface 356

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Journal of Natural Products

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(22) Pelin, M.; Ponti, C.; Sosa, S.; Gibellini, D.; Florio, C.; Tubaro, A. Toxicol. Appl. Pharmacol. 2013, 266 (1), 1−8. (23) Usami, M.; Satake, M.; Ishida, S.; Inoue, A.; Kan, Y.; Yasumoto, T. J. Am. Chem. Soc. 1995, 117, 5389−.

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