Cycloforskamide, a Cytotoxic Macrocyclic Peptide from the Sea Slug

Jul 12, 2013 - ABSTRACT: A macrocylic dodecapeptide, cycloforskamide, was isolated from the sea slug Pleurobranchus forskalii, collected off Ishigaki ...
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Cycloforskamide, a Cytotoxic Macrocyclic Peptide from the Sea Slug Pleurobranchus forskalii Karen Co Tan,† Toshiyuki Wakimoto,*,† Kentaro Takada,‡ Takashi Ohtsuki,§ Nahoko Uchiyama,§ Yukihiro Goda,§ and Ikuro Abe*,† †

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113-0033, Japan Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan § National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan ‡

S Supporting Information *

ABSTRACT: A macrocylic dodecapeptide, cycloforskamide, was isolated from the sea slug Pleurobranchus forskalii, collected off Ishigaki Island, Japan. Its planar structure was deduced by extensive NMR analyses and was further confirmed by MS/MS fragmentation analyses. Finally, the absolute configuration was determined by total hydrolysis and chiral-phase gas chromatographic analysis. This novel dodecapeptide contains three Damino acids and three thiazoline heterocycles and exhibits cytotoxicity against murine leukemia P388 cells, with an IC50 of 5.8 μM.

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he sea slug Pleurobranchus forskalii (class Gastropoda, order Notaspidea, family Pleurobranchidae) is widely distributed in temperate, shallow, subtidal areas around the Indo-Pacific and Mediterranean Seas. It belongs to a family of shell-less mollusks that are opportunistic carnivores, which scavenge on a variety of other invertebrates, such as sponges and ascidians.1 Although these physically defenseless mollusks are believed to secrete bioactive chemicals for their protection and are therefore a vast resource of secondary metabolites,2 there have only been a limited number of studies on the chemical components of P. forskalii. For instance, a cytotoxic cyclic peptide, keenamide A, was isolated from this gastropod collected from Manado, Indonesia,2 while lissoclimide-type diterpenes were identified from samples collected in Philippine waters.3 Furthermore, an ergot alkaloid peptide, ergosinine, which has been isolated thus far only from terrestrial higher plants and fungi, has also been found in P. forskalii.4 Likewise, a potent neurotoxin, tetrodotoxin, has been detected in the gray side-gilled sea slug, Pleurobranchaea maculata,5 which belongs to the same family as P. forskalii. The marine mollusk P. forskalii was collected by hand in the ocean near Ishigaki Island, Okinawa. The ethanolic extract of the frozen mollusk (400 g) was partitioned between H2O and CHCl3, and the organic layer was subjected to silica gel chromatography, followed by reversed-phase HPLC on ODS, to afford cycloforskamide (3.0 × 10−3%, by wet weight), as a white powder. Cycloforskamide (1) showed m/z peaks at 1175 [M + H]+ and 1197 [M + Na]+ in the FABMS spectrum, and its © 2013 American Chemical Society and American Society of Pharmacognosy

molecular formula was established as C54H86N12O11S3 by HRFABMS, which gave a pseudomolecular ion peak at 1175.5774 [M + H]+. The appearance of 1H NMR signals between 6.5 and 8.5 ppm upon changing the NMR solvent from CD3OD to CDCl3 indicated the presence of exchangeable protons, presumably amide protons, suggesting the peptide Received: May 21, 2013 Published: July 12, 2013 1388

dx.doi.org/10.1021/np400404r | J. Nat. Prod. 2013, 76, 1388−1391

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Table 1. NMR Spectroscopic Data for 1 in CDCl3 position L-Thr

1 2 3 4 NH OH L-Val 1 5 6 7 8 9 NH L-Val 2 10 11 12 13 14 NH L-Thr 2 15 16 17 18 NH OH L-Tzn 1 19 20 21 D-allo-Ile 22 23 24 25 26 27 NH a

δC, type

δH (J in Hz)

HMBC

NOESY

1

position L-Val

170.7, 57.3, 69.6, 20.2,

173.5, 59.9, 33.3, 19.5, 15.6,

172.6, 59.9, 30.8, 18.6, 12.2,

171.7, 57.9, 70.1, 20.1,

C CH CH CH3

C CH CH CH3 CH3

C CH CH CH3 CH3

C CH CH CH3

171.4, C 79.2, CH 39.2, CH2 178.7, 57.8, 39.6, 28.5,

C CH CH CH2

13.7, CH3 15.7, CH3

5.36, d (4.0) 4.28, m 1.30, d (5.6) 6.76 (br)

4.40, t (8.6) 2.08, m 0.92a 0.92a 7.35, d (9.6)

4.67, m 2.33, m 0.92a 0.87a 8.35 (br)

4.80, d (9.6) 4.04, d (2.1) 1.01, d (6.4) 7.98 (br)

1, 3

54

2, 3 5

6

5, 7, 8 7 7 10

NHVal2

10, 12, 13 11

NHVal1, 16

15, 17, 18 NHVal2

δC, type

28 29 30 31 32 NH L-Tzn 2 33 34 35 D-Pro 1 36 37 38

172.8, 59.9, 27.0, 18.8, 18.7,

C CH CH CH3 CH3

172.2, C 79.6, CH 39.8, CH2 181.7, C 62.2, CH 32.2, CH2

39

26.1, CH2

40

49.7, CH2

L-Ile 41 42 43 44

171.5, 56.6, 40.5, 26.2,

C CH CH CH2

16, 17

5.01, t (7.2) 3.71, m

19, 21, 22 19, 20, 22

4.76, d (4.6) 1.96, m 1.38, m 1.50, m 0.95, t (8.8) 0.89a 7.60 (br)

22, 24

45 46 NH L-Tzn 3 47 48 49 D-Pro 2 50 51 52 53

24 23, 24, 26, 27 24, 25 24, 25

54 29

δH (J in Hz)

HMBC

NOESY

3

13.1, CH3 17.3, CH3

171.5, C 79.2, CH 39.6, CH2 176.2, 60.4, 31.4, 25.6,

C CH CH2 CH2

48.4, CH2

4.67, 2.04, 0.83, 0.85, 7.28,

m m m m d (10.4)

28 29, 31 29 29 33

5.11, dd (10.4, 2.6) 3.53, m

30,b 33, 35, 36 33, 34, 36

4.93, 2.36, 2.08, 2.22, 1.63, 3.79, 3.69,

36, 38, 39 36, 39, 40 36 37, 38, 40 38 37,b 38, 39

d (5.6) m m m m m m

4.57, t (8.8) 1.63, m 1.40 1.03 0.84a 0.90a 7.22, d (9.6)

41, 42 43, 43, 43, 43, 47

4.98, t (7.2) 3.62, m

47, 49, 50 47, 48, 50

5.07, 2.09, 1.77, 1.96, 3.69, 4.15,

48b 50, 53 54

d (5.7) m m m m t (8.93)

43, 44, 46

NHallo‑Ile

42

40

45 45 44, 46 44

52, 53

48

NHIle

2

Overlapped. bCorrelations observed only at 800 MHz.

probably sulfur. These protons showed COSY correlations with three presumed α-protons at δH 5.11, 5.01, and 4.98, respectively. This sulfur-containing heterocycle was later inferred to be thiazoline (Tzn), by corroboration with the HMBC data. Of the three Tzn rings, two derive from the carbonyl of Pro residues, as indicated by the HMBC correlation between the proline’s β-protons and the thiazoline’s imine carbon. Incidentally, the Ile-Pro1 and Thr1-Pro2 amide bonds were both inferred to adopt a trans configuration, as indicated by the NOESY correlation between the Pro’s δ-protons and the preceding amino acid’s α-proton.6,7 The final Tzn ring derives from an Ile carbonyl, as indicated by the HMBC correlation between the Ile α-H at δH 4.76 and the Tzn imine carbon at δC 178.7. The rest of the amino acid sequence was determined by either HMBC or NOESY correlations (Figure 1), except for the Thr2−Tzn1 connection.

nature of the isolated compound. The two doublet methyl signals at δH 1.01 and 1.30 (Table 1), which showed HMBC correlations with deshielded carbons at δC 70.1 and 69.6, respectively, suggested the presence of two Thr residues. The presence of branched-chain amino acids, two Ile and three Val residues, was apparent due to the heavily overlapped upfield region and was confirmed by COSY and HMBC experiments. Among the heavily overlapping signals between δH 3.5 and 4.0, at least two protons had HMQC correlations with carbons at δC 49.7 and 48.4, reminiscent of aminomethylene groups, suggesting the presence of at least two Pro residues. As the NMR spectra recorded at 800 MHz revealed well-resolved signals in the corresponding region, these assignments were further corroborated. Some signals in the same region at δH 3.53, 3.71, and 3.62, displaying HMQC correlations with δC 39.8, 39.2, and 39.6, respectively, were assumed to be methylene protons attached to an electronegative atom, 1389

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To determine the absolute configuration of 1, it was first subjected to total acid hydrolysis, to obtain its component amino acids. The Tzn moiety is converted to Cys in this process.10 N-Trifluoroacetyl/methyl ester derivatives of the hydrolysate were subsequently subjected to a chiral-phase GC analysis, and the retention times were compared with those of standard amino acids derivatized in the same manner. Through this experiment, all three Val, both Thr, one of the two Ile, and all three Cys residues were found to be in the L-form. On the basis of the latter result, Tzn should also exist in the L-form. In contrast, the Pro and allo-Ile residues were both found to be in the D-form. On the basis of these results, the two Ile residues, one preceding Tzn and another preceding Pro, had opposite configurations at the α-position. However, the α-carbon preceding a Tzn ring is reportedly extremely labile to epimerization, due to the adjacent imine bond.11−13 Thus, it can be inferred that D-allo-Ile precedes Tzn, while L-Ile precedes Pro. In accordance with the sequence of the planar structure, it also follows that the Pro residues, which both precede a Tzn ring, exhibit the D-configuration. In order to rule out the possibility that the D-amino acids were racemized hydrolytic products, 1 was treated with ozone to cleave the imine bond of Tzn prior to hydrolysis, thus preventing racemization.10,14 The results established that the intact peptide contains D-allo-Ile and exclusively D-Pro. Wipf et al. reported that upon treatment of the unnatural [L-Val]-lissoclinamide 7 with pyridine, the α-H of Val, which neighbors Tzn, spontaneously epimerizes, yielding the naturally observed epimer, lissoclinamide 7.12 Thus, the thermodynamically stable conformation of the macrocyclic peptide would reinforce the epimerization to the D-configuration in preference to the L-isomer, by the stereochemically labile site adjacent to the thioimide functionality.15 Therefore, the complete structure of cycloforskamide was established as 1. Cycloforskamide showed moderate cytotoxicity against P388 murine leukemia cells, with an IC50 of 5.8 μM. From an ecological point of view, because patellamides are also cytotoxic and are metal chelators, they may serve as feeding deterrents and/or play a detoxification role by chelating toxic metals, such as copper and zinc.16 In addition, the biosynthetic genes for cyanobactin production have been ascribed to a symbiotic cyanobacterium, Prochloron didemni.8,9 Therefore, we envision that 1 could be a symbiont-derived peptide conferring beneficial effects to the host organism. Experiments are currently being performed to clarify whether P. forskalii hosts the cyanobacteria that produce 1 or acquires them from its diet.

Figure 1. HMBC and NOESY correlations of 1.

Despite the absence of 2D NMR correlations, tandem mass spectrometric data provided evidence that these two amino acids were indeed connected. In particular, the daughter ion at m/z 975 suggested the loss of 200 amu, which can be assigned to a Val-Thr fragment (Figure 2). Meanwhile, another daughter

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Figure 2. MS/MS fragmentation data for 1.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Jasco DIP-1000 digital polarimeter. The UV spectrum was obtained by a Gene Spec III UV/vis spectrometer. 1H and 13C NMR, COSY, HMQC, HMBC, and NOESY (mixing time, 250 μs) spectra in CDCl3 were recorded at 293 K on either a JEOL ECX-500 (1H 500 MHz, 13C 125 MHz) NMR spectrometer with a 5 mm broad band tunable probe or an ECA-800 (1H 800 MHz, 13C 200 MHz) NMR spectrometer equipped with a 5 mm C13/H1 dual cold probe with a z-axis gradient, utilizing residual solvent signals for referencing. FABMS and FABMS/MS experiments were performed on a JEOL JMS-700T spectrometer, using m-nitrobenzyl alcohol as the matrix. The geometry of the spectrometer was BEBE, with an accelerating voltage of 10 kV. The selected ion was collided with helium gas in the collision chamber, which was floated. The gas was introduced to cause the dissociation at a pressure that reduced the intensity of precursor ions to 30%.

ion appeared at m/z 862, reflecting the loss of 313 amu, which could be assigned to a Val-Thr-Tzn fragment. Hence, the planar structure of 1 was determined to be an N-to-C macrocyclic Tzn-containing dodecapeptide. The structure of 1 is similar to those of the patellamides and lissoclinamides, and 1 thus appears to belong to a class of ribosomally synthesized, posttranslationally modified peptides (RiPPs) called cyanobactins. The characteristic structural features of cyanobactins include Nto-C macrocyclization and heterocyclization to form thiazoline, methyloxazoline, and oxazoline, or their oxidized counterparts.8,9 The 36-membered ring size of 1 is comparable to that of wewakazole from Lyngbya majuscula.10 1390

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Animal Material. The Pleurobranchus forskalii sea slug was collected at a depth of 2 m in the ocean near Ishigaki Island, Japan, in May 2012. The animal was kept frozen until use. A voucher specimen (Pf-I-0512) was deposited at the Laboratory of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo. Isolation. A portion of the frozen animal (400 g) was crushed and extracted with EtOH (2.5 L). The ethanolic extract was evaporated in vacuo and partitioned between H2O (500 mL) and CHCl3 (500 mL × 5). The CHCl3-soluble material (660 mg) was subjected to flash chromatography on a silica gel column, eluted with a stepwise gradient of MeOH (0−20%) in CHCl3. The fractions eluted with around 1% MeOH in CHCl3 were then loaded on a Cosmosil MS-II column (⦶ 250 × 10 mm). A linear gradient was employed using aqueous MeOH, starting from 70%, increasing its concentration to 100% for a period of 10 min, and further washing with the organic solvent for 15 min. The flow rate was set at 3.2 mL/min, while UV detection was performed at 210 nm. The major peak, eluting between 14.5 and 15.3 min, yielded 1.2 mg of compound 1. In an effort to increase the yield, an additional 200 g portion of the mollusk was extracted and purified in the same manner as above. Furthermore, the side fractions from the silica gel chromatography and HPLC separation were analyzed for the presence of 1 and were purified by the HPLC method described above. They were subsequently combined with the original sample, after giving NMR and mass spectrometric data indistinguishable from those of 1. The final combined yield of 1 was 3.0 mg. Cycloforskamide (1): white solid; [α]22D +13 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 227 nm (2.60); 1H and 13C NMR (Table 1); HRFABMS m/z 1175.5774 [M + H]+ (calcd for C54H87N12O11S3, 1175.5779, Δ 0.6 mmu). Amino Acid Analysis by Chiral-Phase GC. Cycloforksamide (100 μg) was hydrolyzed with 6 M HCl (500 μL) at 110 °C for 24 h. The reaction mixture was treated with 5−10% HCl/MeOH (500 μL) at 100 °C for 30 min and was then treated with trifluoroacetic anhydride (TFAA)/CH2Cl2 (1:1, 500 μL) at 100 °C for 5 min. The chiral-phase GC analysis of the N-trifluoroacetyl (TFA)/methyl ester derivatives was performed using a CP-Chirasil-D-Val column (Alltech, 0.25 mm × 25 m; N2 as the carrier gas; program rate 50−200 °C at 4 °C/min), and peaks were observed at tR = 6.08, 7.03, 8.26, 8.53, 9.28, and 15.32 min. To confirm the absolute configuration of the thiazoline-based amino acids, a methanolic solution of the peptide (100 μg in 3 mL) was cooled to −78 °C, and then a stream of ozone was bubbled through this solution until the reaction mixture turned pale blue. Oxidative workup was performed by adding 7 drops of 30% hydrogen peroxide, and the reaction mixture was allowed to stand at room temperature for 1 h. After removing the solvent under nitrogen, acid hydrolysis, Me/TFAA derivatization, and chiral-phase GC analysis were conducted as above. Standard amino acids were also converted to the TFA/Me derivatives by the same procedure. Retention times (min) were as follows: L-Val (6.01), D-Val (6.58), L-Thr (6.98), D-Thr (7.77), L-allo-Thr (10.55), D-allo-Thr (11.54), L-Ile (8.24), D-Ile (8.90), L-allo-Ile (7.60), D-allo-Ile (8.43), L-Pro (8.69), D-Pro (9.28), LCys (15.88), D-Cys (16.11). Thus, the presence of L-Val, L-Thr, L-Ile, D-allo-Ile, D-Pro, and L-Cys (L-Tzn) was confirmed. Cytotoxicity Assay against P388. P388 murine leukemia cells were cultured in RPMI 1640 (Wako Chemicals) medium, supplemented with 10 μg/mL of penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (MP Biomedicals), at 37 °C under a 5% CO2 atmosphere. To each well of the 96-well microplate, containing 100 μL of 1 × 105 cells/mL tumor cell suspension, was added 100 μL of test solution (samples were dissolved in MeOH), and the plates were incubated for 3 days. After the addition of 50 μL of 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (1 mg/mL) to each well, the plates were incubated for 4 h under the same conditions. The mixtures were centrifuged, and the supernatants were removed by suction. The precipitates thus obtained were dissolved in DMSO, and the absorbance at 570 nm was measured with a microplate reader. The IC50 for the positive control, doxorubicin, was 210 nM.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental procedures and spectroscopic data. 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], [email protected]. jp. Phone: +81-3-5841-4740. Fax: +81-3-5841-4744. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. K. Tsuchiya, Tokyo University of Marine Science and Technology, for the species identification. K.C.T. is grateful to the Ajinomoto Scholarship Foundation. This work was partly supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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REFERENCES

(1) Cattaneo-Vietti, R.; Burlando, B.; Senes, L. J. Molluscan Stud. 1993, 59, 309−313. (2) Wesson, K. J.; Hamann, M. T. J. Nat. Prod. 1996, 59, 629−631. (3) Fu, X.; Palomar, A. J.; Hong, E. P.; Schmitz, F. J.; Valeriote, F. A. J. Nat. Prod. 2004, 67, 1415−1418. (4) Wakimoto, T.; Tan, K. C.; Abe, I. Toxicon 2013, 72, 1−4. (5) Wood, S. A.; Taylor, D. I.; McNabb, P.; Walker, J.; Adamson, J.; Cary, S. C. Mar. Drugs 2012, 10, 163−176. (6) Randazzo, A.; Dal Piaz, F.; Orru, S.; Debitus, C.; Roussakis, C.; Pucci, P.; Gomez-Paloma, L. Eur. J. Org. Chem. 1998, 2659−2665. (7) Wedemeyer, W.; Walker, E.; Scheraga, H. Biochemistry 2002, 41, 14637−14644. (8) Arnison, P.; Bibb, M.; Bierbaum, G.; Bowers, A.; Bugni, T.; Bulaj, G.; Camarero, J.; Campopiano, D.; Challis, G.; Clardy, J.; Cotter, P.; Craik, D.; Dawson, M.; Dittmann, E.; Donadio, S.; Dorrestein, P.; Entian, K.; Fischback, M.; Garavallie, J.; Goransson, U.; Gruber, C.; Haft, D.; Hemscheidt, T.; Hertweck, C.; Hill, C.; Horswill, A.; Jaspars, M.; Kelly, W.; Klinman, J.; Luipers, O.; Link, A.; Liu, W.; Marahiel, M.; Mitchell, D.; Moll, G.; Moore, B.; Muller, R.; Nair, S.; Nes, I.; Norris, G.; Olivera, B.; Onaka, H.; Patchett, M.; Piel, J.; Reaney, M.; Rebuffat, S.; Ross, R.; Sahl, H.; Schmidt, E.; Selsted, M.; Severinov, K.; Shen, B.; Sivonen, K.; Smith, L.; Stein, T.; Sussmuth, R.; Tagg, J.; Tang, G.; Truman, A.; Vederas, J.; Walsh, C.; Walton, J.; Wenzel, S.; Willey, J.; van der Donk, W. Nat. Prod. Rep. 2013, 30, 108−160. (9) Sivonen, K.; Leikoski, N.; Fewer, D.; Jokela, J. Appl. Microbiol. Biotechnol. 2010, 86, 1213−1225. (10) Nogle, L.; Marquez, B.; Gerwick, W. Org. Lett. 2003, 5, 3−6. (11) Donia, M.; Wang, B.; Dunbar, D.; Desai, P.; Patny, A.; Avery, M.; Hamann, M. J. Nat. Prod. 2008, 71, 941−945. (12) Wipf, P.; Fritch, S. J.; Geib, S. J.; Sefler, A. M. J. Am. Chem. Soc. 1998, 120, 4105−4112. (13) Yonetani, K.; Hirotsu, Y.; Shiba, T. Bull. Chem. Soc. Jpn. 1975, 48, 3302−3305. (14) Portmann, C.; Blom, J.; Gademann, K.; Juttner, F. J. Nat. Prod. 2008, 71, 1193−1196. (15) Lu, Z.; Harper, M.; Pond, C.; Barrows, L.; Ireland, C.; Van Wagoner, R. J. Nat. Prod. 2012, 75, 1436−1440. (16) Schmidt, E.; Nelson, J.; Rasko, D.; Sudek, S.; Eisen, J.; Haygood, M.; Ravel, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7315−7320.

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