Letter pubs.acs.org/OrgLett
Total Synthesis of Theonellapeptolide Id Takefumi Kuranaga, Ayumu Enomoto, Hui Tan, Kazuto Fujita, and Toshiyuki Wakimoto* Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan S Supporting Information *
ABSTRACT: Theonellapeptolide Id is a tridecapeptide containing a 37membered lactone, originally isolated from the marine sponge Theonella swinhoei. In addition to moderate cytotoxicity, immunosuppressive activity had been reported for this natural cyclodepsipeptide. However, the synthetic material to verify its unique biological activity has not been available thus far. In this study, the first total synthesis of theonellapeptolide Id has been performed by solid phase peptide synthesis, and the biological activity has been confirmed in comparison with cyclosporin A.
T
K+ ion transport activities and cytotoxicity against several tumor cell lines. However, the reported cytotoxicity of this compound series is variable, depending on the literature.1 Considering the fact that the marine sponge Theonella is a prolific source of potent cytotoxic compounds, such as polytheonamide and swinholide, it would be difficult to exclude the possible contamination of the natural sample with trace amounts of highly potent cytotoxic metabolites.2 Thus, the total synthesis of this cyclodepsipeptide is highly desirable, to verify not only the structure but also the biological activities of theonellapeptolides. Nonetheless, the total synthesis of this class of molecules had not been reported thus far. In this study, we report the first total synthesis by solid phase peptide synthesis and the evaluation of the biological activity of 1 in comparison with that of 3. The first approach toward 1 is summarized in Scheme 1a. The depsipeptide 1 possesses several nonproteinogenic amino acids (e.g., D-α-amino acids, N-methylated α-amino acids, and β-amino acids) and an N-terminal methoxy acetic acid. Although the biosynthetic gene cluster of 1 has not been identified,2 these structural features of 1 suggest that 1 was biosynthesized by a nonribosomal peptide synthetase (NRPS). The peptide chain of 1 would be elongated from its N-terminus to C-terminus by the repeating modules consisting of condensation (C), adenylation (A), and thiolation (T) domains, and then cyclized by the action of a thioesterase (TE) domain. Theonellapeptolide IId (2) and other naturally occurring analogues would be produced as a result of the promiscuous substrate specificities of the A-domains of NRPS. In light of the putative biosynthetic pathway, the biomimetic retrosynthetic analysis3 was implemented and the depsipeptide
heonellapeptolides (1, Figure 1) are cyclic tridecadepsipeptides originally isolated from the Okinawan marine
Figure 1. Structures of theonellapeptolides and cyclosporin A.
sponge Theonella swinhoei.1a To date, 15 derivatives with various substitutions of Val for Ile and vice versa, and Nterminal modifications with a methoxyacetyl, methylsulfinylacetyl, or acetyl group, have been isolated from several marine sponges.1 The structure is composed of aliphatic amino acids and some N-methyl amide bonds and resembles the immunosuppressant cyclosporin A (3). In 2000, Higa and coworkers indeed reported that theonellapeptolide Id (1), IId (2), and Ia exhibited moderate inhibitory activities on a mixed lymphocyte reaction (MLR).1i In addition, this class of molecules shows various biological activities, such as Na+ and © XXXX American Chemical Society
Received: January 24, 2017
A
DOI: 10.1021/acs.orglett.7b00249 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 1. (a) First Retrosynthesis of 1; (b) Amino Acid Sequence of 1a
a
Fmoc =9-fluorenylmethyloxycarbonyl.
Fmoc group of 6 was removed with a base, the corresponding amine 7 cyclized immediately to form the diketopiperazine 8, even though the bulky 2-chlorotrityl resin7 was attached at the C-terminus of the dipeptide. To circumvent the uncontrollable diketopiperazine formation, we had to avoid the generation of the intermediate 12, which possesses a dipeptide on the secondary hydroxy group of a Thr residue (Scheme 3). In doing so, the depsipeptide 1 was retrosynthetically acyclized to the branched peptide 10, by the disconnection of the amide bond between the D-Leu and D-alloMeIle residues. The peptide 10 would be synthesized by the combination of SPPS and on-resin esterification from the Fmoc-D-Leu loaded resin 11. According to this strategy, the disadvantageous macrocyclization8 of the N-methyl amine moiety at the final stage is inevitable. In our preliminary study with other conceivable alternatives, the intermolecular amidation between D-allo-MeIle and the dipeptide β-Ala-D-LeuOH actually did not proceed. However, the reported intramolecular hydrogen bonding networks1e involving βAla(NH)/D-allo-MeIle(CO), D-allo-Ile(NH)/D-Leu(CO), and D-Leu(NH)/D-allo-Ile(CO) residues encouraged us to spec-
1 was retrosynthetically simplified to the resin-bound seco acid 4. Fmoc-based solid phase peptide synthesis (SPPS)4 of 4 would start from 5 (C-terminus) and proceed toward its Nterminus. However, our initial approach toward 1 revealed the formation of an undesired diketopiperazine,5 due to the cispreference of the N-methyl amide6 (Scheme 2). Once the Scheme 2. Diketopiperazine Formation
Scheme 3. (a) Second Retrosynthesis of 1; (b) Amino Acid Sequence of 1 with Hydrogen Bonding Networks
B
DOI: 10.1021/acs.orglett.7b00249 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 4. Total Synthesis of Theonellapeptolide Id (1)a
a
DMF = N,N-dimethylformamide; DIC = N,N′-diisopropylcarbodiimide; Oxyma = ethyl (hydroxyimino)cyanoacetate; DMAP = N,N-dimethyl-4aminopyridine; TFA = trifluoroacetic acid; PyBOP = benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; HOAt = 1-hydroxy-7azabenzotriazole.
PyBOP12/HOAt13 conditions delivered the cyclized peptide 1.14 After reversed-phase HPLC purification, theonellapeptolide Id (1) was obtained in a 16.8% yield over 27 steps from 11 (average yield per step: 93.6%). The NMR spectra and the HPLC retention time of synthetic 1 are identical to those of natural 1 (Figures S3−S10 in the Supporting Information). To examine the biological activity of synthetic 1 with 3 as a positive control, the osteoclast differentiation induced by the receptor activator of the nuclear factor-κB ligand (RANKL) was employed, since the inhibitory activities of immunosuppressants such as 3 and FK506 had been reported in this assay with the RAW 264.7 cell line.15,16 As the result of tartrate-resistant acid phosphatase (TRAP) staining, synthetic 1 exhibited 27% inhibition of osteoclast differentiation at 30 μM, while retaining 84% cell viability. On the other hand, 3 showed a potent inhibitory effect on osteoclast differentiation of around 59% at 500 nM. Both compounds showed weak cytotoxicity against P388 murine leukemia cells, with similar IC50 values of around 40 μM. In the original paper, the moderate inhibitory activity of 1 on MLR was detected at 20−40 μM and considered to be
ulate that the similar conformational structure of the branched peptide 10 would promote this intramolecular amidation (Scheme 3b). The total synthesis started from the loading of Fmoc-D-LeuOH onto 2-chlorotrityl resin by the action of i-Pr2NEt (Scheme 4). The loading rate of 11 (0.783 mmol/g) was determined by UV spectrophotometry.9 Treatment of the resin-bound FmocD-Leu (11) with piperidine liberated the amine 14, and then cycles of DIC/Oxyma 10 -mediated amide coupling and piperidine-promoted Nα-deprotection were applied to 14. This peptide chain elongation sequence resulted in the formation of the resin-bound dodecapeptide 15, which was subsequently coupled with the acid 16, using DIC and Oxyma as condensation reagents, to afford 17. The linear peptide 17 was converted to the branched peptide by the esterification with Fmoc-D-allo-MeIle under DIC/DMAP activation conditions,11 and then the Fmoc group was removed to generate the amine 18. The resin at the C-terminus of 18 was detached upon treatment with TFA, leading to the macrolactam precursor 19. Finally, macrolactamization of 19 under C
DOI: 10.1021/acs.orglett.7b00249 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters attributable to its cytotoxicity.1i Our data were in good agreement with the previous report in terms of the effective concentration, but suggested that the inhibitory effect on the osteoclast differentiation is not related to its cytotoxicity. Among the theonellapeptolide derivatives, the most potent immunosuppressive activity on MLR had been observed for 2.1i The total synthesis and biological evaluation of 2 are currently underway in our group and will be reported in due course.
■
(3) (a) Gaich, T.; Baran, P. S. J. Org. Chem. 2010, 75, 4657. (b) Kim, J.; Movassaghi, M. Chem. Soc. Rev. 2009, 38, 3035. (4) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis; Oxford University Press: New York, 2000. (5) (a) Ward, D. E.; Lazny, R.; Pedras, M. S. C. Tetrahedron Lett. 1997, 38, 339. (b) Bodanszky, M.; Martinez, J. Synthesis 1981, 1981 (5), 333. (6) Takeuchi, Y.; Marshall, G. R. J. Am. Chem. Soc. 1998, 120, 5363. (7) (a) Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. J. Chem. Soc., Chem. Commun. 1994, 2559. (b) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J. Pept. Protein Res. 1991, 37, 513. (8) For a review, see: White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509. (9) See the Supporting Information. (10) Subirós-Funosas, R.; Prohens, R.; Barbas, R.; El-Faham, A.; Albericio, F. Chem. - Eur. J. 2009, 15, 9394. (11) Tsakos, M.; Schaffert, E. S.; Clement, L. L.; Villadsen, N. L.; Poulsen, T. B. Nat. Prod. Rep. 2015, 32, 605. (12) Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31, 205. (13) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397. (14) For examples of these conditions, see: (a) Kuranaga, T.; Mutoh, H.; Sesoko, Y.; Goto, T.; Matsunaga, S.; Inoue, M. J. Am. Chem. Soc. 2015, 137, 9443. (b) Kuranaga, T.; Sesoko, Y.; Sakata, K.; Maeda, N.; Hayata, A.; Inoue, M. J. Am. Chem. Soc. 2013, 135, 5467. (c) Murai, M.; Kaji, T.; Kuranaga, T.; Hamamoto, H.; Sekimizu, K.; Inoue, M. Angew. Chem., Int. Ed. 2015, 54, 1556−60. (d) Yamashita, T.; Matoba, H.; Kuranaga, T.; Inoue, M. Tetrahedron 2014, 70, 7746. (15) Hirotani, H.; Tuohy, N. A.; Woo, J.-T.; Stern, P. H.; Clipstone, N. A. J. Biol. Chem. 2004, 279, 13984. (16) Guo, M.; James, A. W.; Kwak, J. H.; Shen, J.; Yokoyama, K. K.; Ting, K.; Soo, C. B.; Chiu, R. H. Sci. Rep. 2016, 6, 22378.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00249. General experimental procedures, ODS-HPLC charts, 1H and 13C NMR spectra, cytotoxicity assay, and osteoclast differentiation assay (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Toshiyuki Wakimoto: 0000-0003-2917-1797 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank emeritus Prof. J. Kobayashi, Hokkaido University for providing sponge samples. A.E. and H.T. are grateful to the Tsukushi Fellowship Foundation and the Tokyo Biochemical Research Foundation, respectively. This work was partly supported by the Takeda Science Foundation, the Astellas Foundation for Research on Metabolic Disorders, the Naito Foundation, the Akiyama Life Science Foundation, and Grantsin-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (JSPS KAKENHI Grant Numbers JP16703511, JP15547389, JP15597834, JP14544491, and JP14506930).
■
REFERENCES
(1) (a) Kitagawa, I.; Kobayashi, M.; Lee, N. K.; Shibuya, H.; Kawata, Y.; Sakiyama, F. Chem. Pharm. Bull. 1986, 34, 2664. (b) Kitagawa, I.; Lee, N. K.; Kobayashi, M.; Shibuya, H. Chem. Pharm. Bull. 1987, 35, 2129. (c) Kitagawa, I.; Lee, N. K.; Kobayashi, M.; Shibuya, H. Tetrahedron 1991, 47, 2169. (d) Kobayashi, M.; Lee, N. K.; Shibuya, H.; Momose, T.; Kitagawa, I. Chem. Pharm. Bull. 1991, 39, 1177. (e) Kobayashi, M.; Kanzaki, K.; Katayama, S.; Ohashi, K.; Okada, H.; Ikegami, S.; Kitagawa, I. Chem. Pharm. Bull. 1994, 42, 1410. (f) Li, S.; Dumdei, E. J.; Blunt, J. W.; Munro, M. H.; Robinson, W. T.; Pannell, L. K. J. Nat. Prod. 1998, 61, 724. (g) Tsuda, M.; Shimbo, K.; Kubota, T.; Mikami, Y.; Kobayashi, J. Tetrahedron 1999, 55, 10305. (h) Roy, M. C.; Ohtani, I. I.; Tanaka, J.; Higa, T.; Satari, R. Tetrahedron Lett. 1999, 40, 5373. (i) Roy, M. C.; Ohtani, I. I.; Ichiba, T.; Tanaka, J.; Satari, R.; Higa, T. Tetrahedron 2000, 56, 9079. (j) Sinisi, A.; Calcinai, B.; Cerrano, C.; Dien; Henny, A.; Zampella, A.; D’Amore, C.; Renga, B.; Fiorucci, S.; Taglialatela-Scafati, O. Beilstein J. Org. Chem. 2013, 9, 1643. (2) Wilson, M. C.; Mori, T.; Ruckert, C.; Uria, A. R.; Helf, M. J.; Takada, K.; Gernert, C.; Steffens, U. A. E.; Heycke, N.; Schmitt, S.; Rinke, C.; Helfrich, E. J. N.; Brachmann, A. O.; Gurgui, C.; Wakimoto, T.; Kracht, M.; Crusemann, M.; Hentschel, U.; Abe, I.; Matsunaga, S.; Kalinowski, J.; Takeyama, H.; Piel, J. Nature 2014, 506, 58. D
DOI: 10.1021/acs.orglett.7b00249 Org. Lett. XXXX, XXX, XXX−XXX
