Letter pubs.acs.org/OrgLett
Total Synthesis and Stereochemical Assignment of Gymnopeptides A and B Zhengyin Pan,† Chunlei Wu,† Wei Wang, Zhehong Cheng, Guiyang Yao, Ke Liu, Hongchang Li, Lijing Fang,* and Wu Su* Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, P. R. China S Supporting Information *
ABSTRACT: Gymnopeptides A and B are unprecedented highly N-methylated cyclic β-hairpin octadecapeptides with striking antiproliferative activities isolated from the mushroom Gymnopus f usipes. Using Fmoc-based solid-phase peptide synthesis, followed by macrolactamization of the resulting linear peptides, the first total synthesis of gymnopeptides A and B was successfully achieved in this study. The coupling methods used for the solid-phase synthesis and the cyclization were optimized, and the configuration of the Ser1/Thr1 residue in gymnopeptide A/B was determined to be L.
B
ecause of their high and specific biological activities and low toxicities, in general, peptides have gained interest as therapeutic agents in recent years. Of these peptides, cyclopeptides have shown promise as therapeutic candidates since backbone cyclization not only improves the metabolic stability but also introduces a conformational constraint to the peptide.1 Some of the cyclopeptides isolated from both marine and terrestrial sources contain N-methylated amino acids. Such structural features may further improve their pharmacological characteristics, such as lipophilicity, proteolytic stability, and receptor selectivity.2 A range of biological activities, including anticancer, antiviral, antifungal, and immunosuppressive properties, have been observed for N-methylated cyclopeptides.3 For instance, cyclosporin A isolated from the fungus Tolypocladium inf latum GAMS is a cycloundecapeptide with seven N-methylated amide bonds.4 As a successful orally available immunosuppressive drug, the development of this compound revolutionized organ transplant treatment, and cyclosporine A has also been applied for the treatment of psoriasis, rheumatoid arthritis, uveitis, etc.5 Recently, a mycochemical study of the mushroom Gymnopus f usipes by Ványolós and co-workers led to the discovery of two new cyclopeptides, gymnopeptides A and B.6 The isolated compounds exhibited striking antiproliferative activity on several human cancer cell lines with nanomolar IC50 values. Structurally, gymnopeptides A and B are unprecedented highly N-methylated cyclic octadecapeptides, where 10 of the 18 residues are N-methylated (Figure 1). These two compounds differ only in the presence of a single amino acid. Ser1 in gymnopeptide A is replaced by Thr1 in gymnopeptide B. The sequences and stereochemistries of the amino acids in these molecules were established by a combination of spectroscopic studies and Marfey’s analysis. All residues are in L © XXXX American Chemical Society
Figure 1. Structures of gymnopeptide A (1a) and gymnopeptide B (1b).
configurations except Ser1/Thr1, for which the configurations were not determined because neither the serine nor the threonine derivatives could be detected by HPLC−MS analysis. According to a preliminary molecular modeling study by Ványolós and co-workers, a geometry well agreeing with all the NMR data of gymnopeptide A was reached by calculations with L-Ser in position 1. Furthermore, an 18-mer cyclic β-hairpin structure is suggested for both peptides, wherein two antiparallel β strands stabilized by alternating natural and Nmethyl amino acids are connected with two turns, both containing a cis-amide bond (Ser1/Thr1-MeVal2 and MeVal11MeAla12). The remarkable biological activities along with their unique molecular scaffolds make them interesting targets for chemical synthesis. With the aim of identifying the absolute stereochemistry of Ser1/Thr1 and to supply samples for further pharmaceutical development, we set out to establish an efficient Received: June 9, 2017
A
DOI: 10.1021/acs.orglett.7b01742 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters synthetic strategy for the total synthesis of gymnopeptides A and B. It was envisioned that the total synthesis of gymnopeptides A or B could be realized by a head-to-tail cyclization of a linear peptide. According to the common guidelines of peptide synthesis, a proper cyclization position should avoid those sterically encumbered by the N-methylated amide bonds.7 One possible site for cyclization is at the junction of MeVal18-Ser1/ Thr1 (site B, second strategy, Figure 1). Accordingly, the linear precursor would begin from the C-terminus of MeVal18 and finish at the N-terminus of Ser1/Thr1. Thus, the linear precursors of gymnopeptides A and B share 17 amino acid residues in their sequences and differ only in the N-terminal Ser1/Thr1, which would also facilitate the stereochemical assignment of the Ser1/Thr1 residue if more than one epimer needs to be synthesized. However, a major risk of cyclization at this site is the formation of an amide bond between Ser1/Thr1 and MeVal18, two sterically hindered residues bearing bulky side chains. The other possible site for cyclization is at the junction between Ala15-Sar14 (site A, Figure 1), which is expected to cyclize readily due to minimum steric hindrance while preventing any C-terminal epimerization during the cyclization, and thus, this site was chosen in the first strategy. Consequently, the linear precursor would be assembled by solid-phase peptide synthesis (SPPS) starting from the Cterminus of Sar14 and ending at the N-terminus of Ala15. The synthesis of a peptide containing multiple dense Nmethylated amides is very challenging due to the low inherent reactivity of N-methylamino acids.8 Acylation of N-methylated residues on resin often occurs with low yields and produces racemized products. To overcome the difficulties associated with the synthesis of N-methylated amide bonds, several coupling conditions have been developed involving the use of benzotriazole-based reagents 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU)9 and N,N′-diisopropylcarbodiimide (DIC)/1-hydroxy-7-azabenzotriazole (HOAt),10 the phosphonium reagent (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP),11 and symmetric anhydride couplings utilizing N,N′-dicyclohexylcarbodiimide (DCC) as an activating agent. 12 Bis(trichloromethyl)carbonate (BTC) has also been used to improve the coupling efficiency of amidation reactions by converting Fmoc-amino acids into highly reactive and sterically unhindered acid chlorides in situ.13 In the current study, a series of different protocols were investigated to optimize the coupling conditions required for the acylation of N-methyl amino acid termini on the solid support. In view of the lability of N-alkylated peptides to acids,14 the 2-chlorotrityl chloride resin (2-CTC resin) was used as the solid support in the first strategy because it allowed the release of the linear peptide under very mild conditions. Additionally, the steric nature of 2-CTC resin would help to minimize diketopiperazine (DKP) formation at the C-terminus during the base-induced removal of an Fmoc group at the dipeptide stage as well as the coupling reaction of a third amino acid. The first amino acid, Fmoc-Sar-OH, was anchored to the resin in the presence of N,N′-diisopropylethylamine (DIEA). After removal of the Fmoc protecting group with 20% piperidine in DMF (2 × 5 min), the peptide was elongated using Fmocbased SPPS. As shown in Figure 2, BTC was found to be very efficient in activating all of the N-methylated amino acids, including MeAla, MeVal, and Sar, enabling the coupling
Figure 2. Graphical synthesis outline for the linear precursor of gymnopeptide A (1a) (1st strategy).
reactions to complete within 1 h irrespective if N-methylated or unmethylated amines were involved. Val13 and Val9 were efficiently coupled to Sar14 and Sar10, respectively, using a HATU/DIEA system. The coupling of Ala7 onto the Nmethylamino group of MeVal8 proceeded smoothly to completion using the DIC/HOAt reagents. It turned out that the reaction of Fmoc-Val-OH with the resin-bound amine of MeVal6/MeVal4 was the most difficult coupling in the sequence due to the steric hindrance of the N-methyl amino group and the bulky side chains. The various coupling protocols investigated, including the use of HATU/DIEA, DIC/HOAt, BTC/HOAt15 and PyAOP/Oxyma Pure16 systems, even under microwave-assisted coupling conditions failed to drive the reaction to completion. Finally, an acceptable yield was achieved using the symmetric anhydride formed by the reaction of Fmoc-Val-OH and DCC in a ratio of 2:1.15 The same coupling conditions were then conducted in the following coupling of Fmoc-Ala-OH and Fmoc-Ser(tBu)-OH to the resin-bound amine of MeVal. Under the optimized deprotection and coupling conditions, DKP formation was not observed in this strategy. After removal of the final Fmoc group, the linear precursor of gymnopeptide A was obtained in 8% yield after being cleaved from the resin using 1% TFA/CH2Cl2. Then the macrolactamization of the linear precursor (Ala15MeVal16-Ala17-MeVal18-Ser(tBu)1-MeVal2-Val3-MeVal4Val5-MeVal6-Ala7-MeVal8-Val9-Sar10-MeVal11-MeAla12Val13-Sar14) was performed in the presence of HATU, 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide (EDC)/HOAt, benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4methylmorpholinium chloride (DMTMM), pentafluorophenyl diphenylphophinate (FDPP), or diphenylphosphoryl azide (DPPA) under various conditions (using DMF or CH2Cl2 as a solvent; DIEA, collidine or NaHCO3 as a base; and different reaction temperatures). Unfortunately, although the cyclic dimer and trimer were detected in some cases, all of our attempts failed to provide the desired product presumably due to a conformational disposition of the linear precursor that prevented the macrocyclization. Considering the cyclic βhairpin structures of gymnopeptides A and B,6 we decided to carry out the macrolactamization at the MeVal18-Ser1/Thr1 junction (site B) in the second strategy. This site is adjacent to the Ser1/Thr1-MeVal2 turn unit containing a cis-amide bond and thus was anticipated to facilitate the head-to-tail peptide cyclization. B
DOI: 10.1021/acs.orglett.7b01742 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters As found in the first strategy, the coupling efficiencies became considerably lower after half of the linear peptide was assembled relative to those earlier in the synthetic cycle. We attribute this to the increased hydrogen bond interactions of the “hairpin turn” structure. This issue was further exacerbated when the installation of Fmoc-Ala-OH, Fmoc-Val-OH, and Fmoc-Ser(tBu)-OH to the resin-bound amines of MeVal was required in the synthesis, as the reduced reactivity of the MeVal amines resulted in extremely poor coupling efficiencies. Therefore, we prepared Fmoc-Ala-MeVal-OH (2), Fmoc-ValMeVal-OH (3), Fmoc-Ser(tBu)-MeVal-OH (4), and FmocThr(tBu)-MeVal-OH (5) in the solution phase in the second strategy. The synthesis of Fmoc-Ala-MeVal-OH (2) is shown in Scheme 1. The allylation of Fmoc-MeVal-OH with allyl
Scheme 2. Synthesis of Gymnopeptide A (1a) and Gymnopeptide B (1b) (Second Strategy)
Scheme 1. Synthesis of Fmoc-Ala-MeVal-OH (2), Fmoc-ValMeVal-OH (3), Fmoc-Ser(tBu)-MeVal-OH (4), and FmocThr(tBu)-MeVal-OH (5)
bromide and the subsequent removal of the Fmoc group with Et2NH/CH3CN allowed access to amine 6, which was coupled with Fmoc-Ala-OH in the presence of HATU/DIEA to give dipeptide 7. Fragment 2 was then afforded by removal of the allyl group in 6 with Pd(PPh3)4 and 1,3-dimethylbarbituric acid (DMBA). Fmoc-Val-MeVal-OH (3), Fmoc-Ser(tBu)-MeValOH (4), and Fmoc-Thr(tBu)-MeVal-OH (5) were synthesized using a similar procedure. Although the absolute configuration of Ser1/Thr1 was not determined by HPLC−MS analysis, the presence of L-Ser was predicted from a computational model of gymnopeptide A, constrained by NOE correlations.6 Therefore, Fmoc-L-Ser(tBu)-OH and Fmoc-L-Thr(tBu)-OH were used in the synthesis. With all of the building blocks in hand, the assembly of the linear precursors 9a and 9b on the 2-CTC resin was initiated (Scheme 2). As expected, the Fmoc-based SPPS proceeded smoothly employing BTC/collidine as the coupling reagents for the N-methylated amino acids, Sar14, MeAla12, MeVal11 and Sar10, DIC/HOAt for Val13, and Val9 and HATU/DIEA for all four building blocks 2−5. After being cleaved from the resin using 1% TFA/CH2Cl2, the linear precursors 9a and 9b were obtained in 16% and 18% yields, respectively, exhibiting 2fold improved yields compared to those in the first strategy. Macrocyclization was found to be the most challenging step. Among the various cyclization conditions investigated, only the reagent set of HATU/collidine proved to be effective, forming the desired products 10a and 10b in modest yields (ca. 40%). After removal of the tBu protecting group on the Ser/Thr residue using neat TFA at 0 °C,17 the final products 1a and 1b were purified by semipreparative RP-HPLC. To our delight, the resulting synthetic compounds and the natural products
revealed perfectly overlapped NMR spectra (see the Supporting Information). Therefore, both the absolute configuration of Ser1 in gymnopeptide A and Thr1 in gymnopeptide B are assigned as L. Furthermore, bioactivity assays were performed using the MTT method. As expected, the synthetic compounds displayed similar in vitro activity against MDA-MB-231 cells (IC50 of 44.6 and 15.8 nM, respectively) as the natural compounds (IC50 of 37.4 and 30.7 nM, respectively), corroborating the authenticity of our structural assignment. In summary, the total synthesis and stereochemical assignment of gymnopeptides A and B were achieved for the first time using Fmoc-SPPS followed by macrolactamization of the resulting linear peptides. The coupling methods used for the solid-phase synthesis and the cyclization were optimized in this study. The generation of four dipeptide building blocks in the solution phase allowed for the efficient assembly of the peptidyl chains by solid-phase peptide synthesis. A cyclization site at the MeVal18-Ser1/Thr1 junction led to the successful macrocyclization and facilitated the synthesis of both gymnopeptides A and B. The development of an efficient synthesis of gymnopeptides A and B has circumvented the issue of limited availability and paves the way for advanced in vivo evaluation of these compounds. C
DOI: 10.1021/acs.orglett.7b01742 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
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(12) Ko, S. Y.; Wenger, R. M. Helv. Chim. Acta 1997, 80, 695. (13) (a) Thern, B.; Rudolph, J.; Jung, G. Angew. Chem., Int. Ed. 2002, 41, 2307. (b) Thern, B.; Rudolph, J.; Jung, G. Tetrahedron Lett. 2002, 43, 5013. (c) Fuse, S.; Mifune, Y.; Takahashi, T. Angew. Chem., Int. Ed. 2014, 53, 851. (d) Maharani, R.; Brownlee, R. T. C.; Hughes, A. B.; Abbott, B. M. Tetrahedron 2014, 70, 2351. (e) Nabika, R.; Suyama, T. L.; Hau, A. M.; Misu, R.; Ohno, H.; Ishmael, J. E.; McPhail, K. L.; Oishi, S.; Fujii, N. Bioorg. Med. Chem. Lett. 2015, 25, 302. (14) (a) Urban, J.; Vaisar, T.; Shen, R.; Lee, M. S. Int. J. Pept. Protein Res. 1996, 47, 182. (b) Vaisar, T.; Urban, J. J. Mass Spectrom. 1998, 33, 505. (15) Yao, G.; Pan, Z.; Wu, C.; Wang, W.; Fang, L.; Su, W. J. Am. Chem. Soc. 2015, 137, 13488. (16) (a) Subiros-Funosas, R.; El-Faham, A.; Albericio, F. Org. Synth. 2014, 90, 306. (b) El-Faham, A.; Khattab, S. N.; Subirós-Funosas, R.; Albericio, F. J. Pept. Sci. 2014, 20, 1. (17) Several deprotection conditions were investigated to remove the tBu protecting group on the Ser1/Thr1 residue, such as TFA/TIPS/ H2O (95:2.5:2.5), 50% TFA/CH2Cl2, 80% TFA/CH2Cl2, and neat TFA at different reaction temperatures. The cyclized peptides proved to be unstable under all these conditions at room temperature, especially in the presence of H2O (TFA/TIPS/H2O). At 0 °C, 50% TFA/CH2Cl2 and 80% TFA/CH2Cl2 failed to remove all the tBu groups within 6 h, while some decomposed products were observed. Using neat TFA at 0 °C, synthetic compounds 1a and 1b could be obtained in acceptable yields (60−70%) within 4 h.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01742. Experimental procedures and analytical data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Wu Su: 0000-0001-9958-3434 Author Contributions †
Z.P. and C.W. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Shenzhen Sciences & Technology Innovation Council (KQCX2015033117354154, JCYJ20150316143416083, JCYJ20170413165916608, JCYJ20150401150223649, and JCYJ20160229204338907) and the National Natural Science Foundation of China (Grant Nos. 21402232, 21672254, and 21432003). We thank Dr. Zoltán Béni (Gedeon Richter Plc., Hungary) and Attila Ványolós (University of Szeged) for 1H and 13C NMR spectra of natural gymnopeptides A and B. The authors are grateful for the assistance of the Mass facility from the Peking University Shenzhen Graduate School.
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REFERENCES
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DOI: 10.1021/acs.orglett.7b01742 Org. Lett. XXXX, XXX, XXX−XXX
