Synthesis, structure determination, and biological evaluation of destruxin e

Article abstract of DOI:10.1021/ol101449x

The total synthesis of destruxin E (1) has been achieved for the first time, and the stereochemistry of its chiral center at the epoxide has been determined to be (S). The cyclization precursor 3a was synthesized by solid-phase peptide synthesis. Macrolac

Full text of DOI:10.1021/ol101449x

ORGANIC
LETTERS
2010
Vol. 12, No. 17
3792-3795
Synthesis, Structure Determination, and
Biological Evaluation of Destruxin E
Masahito Yoshida,† Hisayuki Takeuchi,† Yoshitaka Ishida,† Yoko Yashiroda,‡
Minoru Yoshida,‡ Motoki Takagi,§ Kazuo Shin-ya,| and Takayuki Doi*,†
Graduate School of Pharmaceutical Sciences, Tohoku UniVersity, 6-3 Aza-Aoba,
Aramaki, Aoba-ku, Sendai 980-8578, Japan, Chemical Genomics Research Group,
RIKEN AdVanced Science Institute, Wako, Saitama 351-0198, Japan, Biomedicinal
Information Research Center, Japan Biological Informatics Consortium, 2-4-7 Aomi,
Koto-ku, Tokyo 135-0064, Japan, and National Institute of AdVanced Industrial
Science and Technology, 2-4-7, Aomi, Koto-ku, Tokyo 135-0064, Japan
doi_taka@mail.pharm.tohoku.ac.jp
Received June 24, 2010
ABSTRACT
The total synthesis of destruxin E (1) has been achieved for the first time, and the stereochemistry of its chiral center at the epoxide has been
determined to be (S). The cyclization precursor 3a was synthesized by solid-phase peptide synthesis. Macrolactonization of 3a utilizing MNBA-
DMAPO, followed by formation of the epoxide, then furnished destruxin E. Its diastereomer, epi-destruxin E (2), was also synthesized in the
same manner. Furthermore, the biological evaluation indicated that destruxin E exhibits V-ATPase inhibitory activity 10-fold greater than that
of epi-destruxin E.
Destruxin E (1) is a 19-membered cyclic depsipeptide,
consisting of five amino acids (proline, isoleucine, NMe-
valine, NMe-alanine, and ꢀ-alanine) and epoxide-containing
R-hydroxy acid. It was first isolated from a culture medium
of the strain of Metarhizium anisopliae by Pa¨ıs et al. in
1981.1 To date, more than 35 destruxin derivatives have been
found from cultures such as Oospora destructor,2 Alternaria
brassica,3 Nigrosabulum globosum,4 BeauVeria felina,5 and
Metarhizium anisopliae.6 Among the destruxins, destruxin
E (1) has been shown to strongly inhibit V-ATPase (IC50
0.4 µM)7 that is localized on the membranes enclosing the
organelles such as the lysosomes and function to acidify a
wide array of intracellular compartments.8 V-ATPases have
also an important role for the control of tumor microenvi-
ronment by proton extrusion, and therefore V-ATPase
inhibitors can be a drug candidate for the cancer therapy.9
Moreover, destruxin E (1) can reversibly induce morphologi-
† Tohoku University.
‡ RIKEN Advanced Science Institute.
(5) Lira, S. P.; Vita-Marques, A. M.; Seleghim, M. H. R.; Bugni, T. S.;
LaBarbera, D. V.; Sette, L. D.; Sponchiado, S. R. P.; Ireland, C. M.;
Berlinck, R. G. S. J. Antibiot. 2006, 59, 553–563.
§ Japan Biological Informatics Consortium (JBIC).
| National Institute of Advanced Industrial Science and Technology
(AIST).
(6) (a) Suzuki, A.; Taguchi, H.; Tamura, S. Agric. Biol. Chem. 1970,
34, 813–816. (b) Wahlman, M.; Davidson, B. S. J. Nat. Prod. 1993, 56,
643–647.
(1) Pa¨ıs, M.; Das, B. C.; Ferron, P. Phytochemisty 1981, 20, 715–723.
(2) Kodaira, Y. Agric. Biol. Chem. 1962, 26, 36–42.
(3) Ayer, W. A.; Pen˜a-Rodriguez, L. M. J. Nat. Prod. 1987, 50, 400–
407.
(7) Va´zquez, M. J.; Albarra´n, M. I.; Espada, A.; Rivera-Sagredo, A.;
D´ıaz, E.; Hueso-Rodr´ıguez, J. A. Chem. BiodiVers. 2005, 2, 123–130.
(8) Nishi, T.; Forgac, M. Nat. ReV. Mol. Cell. Biol. 2002, 3, 94–103,
and references therein.
(4) Che, Y.; Swenson, D. C.; Gloer, J. B.; Koster, B.; Malloch, D. J.
Nat. Prod. 2001, 64, 555–558.
10.1021/ol101449x  2010 American Chemical Society
Published on Web 07/30/2010

cal changes in osteoclast-like multinucleated cells (OCLs)
in a dose-dependent manner (0.01-0.05 µM).10 It has
recently been reported that 1 can also reduce ꢀ-amyloid
generation without affecting ꢀ-amyloid-cleaving enzyme
(BACE) or PS/γ-secretase activity similar to how bafilomycin
does.11,12 Several groups have previously reported the
synthesis of destruxin analogues and evaluated their biologi-
cal activities.13 These reports demonstrated that the epoxide
in the side chain could be crucial for exhibiting the biological
activity. The absolute stereochemistry of the epoxide,
however, has not been established. To elucidate the
structure-activity relationships involving destruxin E (1),
we are interested in the total synthesis and the library
synthesis of 1 via solid-phase synthesis. We thus report herein
a solid-phase-assisted total synthesis of the two possible
diastereomers of destruxin E in order to determine the
absolute stereochemistry of the epoxide and evaluate the
biological activity of destruxin E.
coupling of five fragments, ꢀ-alanine, NMe-alanine, NMe-
valine, isoleucine, and R-hydroxy acid-proline derivative
(HA-Pro-OH) 4, by a solid-phase peptide synthesis using a
trityl linker. Both diastereomers 4 will be prepared from
chiral lactones 5a and 5b by hydrolysis of the lactones,
followed by amidation with proline. The optically active
lactones 5 would be provided utilizing Evans asymmetric
allylation of 6, followed by dihydroxylation and concomitant
cyclization.
Initially, HA-Pro-OH 4a and 4b were prepared according
to Scheme 2. Acylation of Evans chiral auxiliary 7 with
Scheme 2. Synthesis of 4a and 4b
Scheme 1. Retrosynthetic Analysis of Destruxin E (1)
benzyloxyacetyl chloride, followed by removal of the benzyl
group and protection of the resulting alcohol with a TBS
group provided 6 in 98% yield. The Evans asymmetric
alkylation15 of 6 proceeded smoothly at -30 °C (LiHMDS/
allyl bromide) to afford allylated product 8 with high
diastereoselectivity (81%, dr >98:2). Dihydroxylation of 8
with OsO4/NMO induced concomitant formation of γ-lac-
tones, which were separated by silica gel column chroma-
tography to afford the two desired diastereomers 5a16 (44%)
and 5b17 (42%). Acid 9a was formed by hydrolysis of the
Our synthetic strategy is illustrated in Scheme 1. Destruxin
E (1) and epi-destruxin E (2) can be synthesized from
cyclization precursor 3a or 3b through macrolactonization,14
followed by formation of the epoxide in the side chain. The
cyclization precursor 3 would be obtained by sequential
(13) (a) Cavelier, F.; Jacquier, R.; Mercadier, J.-L.; Verducci, J.
Tetrahedron 1996, 52, 6173–6186. (b) Cavelier, F.; Jacquier, R.; Mercadier,
J.-L.; Verducci, J.; Traris, M.; Vey, A. J. Peptide Res. 1997, 50, 94–101.
(c) Caveliar, F.; Vercci, J.; Andre´, F.; Haraux, F.; Sigalat, C.; Traris, M.;
Vey, A. Pestic. Sci. 1998, 52, 81–89. (d) Ast, T.; Barran, E.; Kinne, L.;
Schmidt, M.; Germeroth, L.; Simmons, K.; Wenschuh, H. J. Peptide Res.
2001, 58, 1–11.
(9) Pe´rez-Saya´ns, M.; Somaza-Mart´ın, J. M.; Barros-Angueria, F.; Rey,
J. M. G.; Garc´ıa-Garc´ıa, A. Cancer Treat. ReV. 2009, 35, 707–713, and
refernces therein.
(14) Synthetic studies of destruxin derivatives by macrolactonization
have been reported. (a) Total synthesis of destruxin B was achieved using
macrolactonization though the yield was not reported: Kuyama, S.; Tamura,
S. Agric. Biol. Chem. 1965, 29, 168–169. (b) Macrolactonization failed in
the synthetic study for protodestruxin: Lee, S.; Izumiya, N.; Suzuki, A.;
Tamura, S. Tetrahedron Lett. 1975, 11, 883–886.
(10) Nakagawa, H.; Takami, M.; Udagawa, N.; Sawae, Y.; Suda, K.;
Sasaki, T.; Takahashi, N.; Wachi, M.; Nagai, K.; Woo, J. T. Bone 2003,
33, 443–455.
(11) Itoh, N.; Okochi, M.; Tagami, S.; Nishitomi, K.; Nakayama, T.;
Yanagida, K.; Fukutomi, A.; Jiang, J.; Mori, K.; Hosono, M.; Kikuchi, J.;
Nakano, Y.; Takinami, Y.; Dohi, K.; Nishigaki, A.; Takemoto, H.;
Minagawa, K.; Katoh, T.; Willem, M.; Haass, C.; Morihara, T.; Tanaka,
T.; Kudo, T.; Hasegawa, H.; Nishimura, M.; Sakaguchi, G.; Kato, A.;
(15) (a) Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem. Soc.
1982, 104, 1737–1739. (b) Crimmins, M. T.; Emmitte, K. A.; Katz, J. D.
Org. Lett. 2000, 2, 2165–2167.
(16) 5a was converted into reported (2R,4S)-2-hydroxy-4-hydroxy-
methyl-4-butanolide, whose spectral data were in good agreement with those
previously reported: Uchikawa, O.; Okukado, N.; Sakata, T.; Arase, T.;
Terada, K. Bull. Chem. Soc. Jpn. 1988, 61, 2025–2029.
Takeda, M. Neurodegener. Dis. 2009, 6, 230–239
.
(12) Knops, J.; Suomensaari, S.; Lee, M.; McConlogue, L.; Seubert,
P.; Sinha, S. J. Biol. Chem. 1995, 270, 2419–2422
Org. Lett., Vol. 12, No. 17, 2010
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3793

lactone 5a with LiOH·H2O, followed by protection of the
resulting diols as an acetonide. Owing to the instability of 9
under acidic and basic conditions, the acid 9a was im-
mediately utilized in the next step without further purification.
The amidation of 9a with L-proline benzyl ester was
performed using PyBroP18/DIEA to afford 10a in 70% yield.
Finally, removal of the benzyl ester at the C-terminus of 10a
provided the desired acid 4a in quantitative yield. According
to the synthesis of 4a, 4b was also prepared from lactone
5b.
acylation of the resulting amine with Fmoc-MeAla-OH was
performed utilizing DIC/HOBt to afford dipeptide 13 with
high purity, which was monitored by reversed-phase HPLC-
MS analysis (UV 214 nm) after cleavage from the polymer
support. Removal of the Fmoc group in 13 (20% piperidine/
DMF, rt, 30 min), followed by amidation with Fmoc-MeVal-
OH utilizing PyBroP/DIEA provided tripeptide 14 (purity
99%, UV 214 nm). We next attempted the synthesis of
tetrapeptide 15. The coupling reaction of 14 with Fmoc-Ile-
OH was, however, not complete due to the difficulty of the
coupling between sterically hindered N-Me valine and
isoleucine. Therefore, we repeated the reaction under the
same conditions to obtain the tetrapeptide 15 with high
conversion (purity 92%, UV 214 nm). After removal of the
Fmoc group of 15, coupling with acids 4a and 4b using
PyBroP/DIEA in parallel, followed by removal of the TBS
group utilizing TBAF, and cleavage from the polymer
support under weakly acidic conditions yielded the desired
cyclization precursors 3a and 3b, respectively.22 Macrolac-
tonization of 3a was performed using 2-methyl-6-nitroben-
zoic anhydride (MNBA)/4-(dimethylamino)pyridine N-oxide
(DMAPO)23 under high dilution conditions (3 mM) for 3 d
to afford the desired macrocycle 16a in 65% yield; 16b was
also synthesized in the same manner.
With the desired 4a and 4b in hand, the cyclization
precursors 3a and 3b were prepared by solid-phase peptide
synthesis on SynPhase lanterns19 (Scheme 3). Attachment
Scheme 3. Synthesis of Macrocycles 3a and 3b
After synthesizing macrocycles 16a and 16b, the epoxide
was formed, leading to the synthesis of destruxin E (1).
Removal of the acetonide in 16a was performed with 3 M
HCl aq/1,4-dioxane at 10 °C to yield the diol 17a (73%).
Regioselective tosylation of the primary alcohol was carefully
achieved with TsCl/NEt3/DMAP at room temperature to
provide desired tosylate 18a (87%). Initial attempt for the
formation of epoxide using K2CO3 in MeOH resulted in the
ring opening of the macrocycle. Therefore, we used less
nucleophilic isopropyl alcohol instead of MeOH. Finally,
formation of the epoxide with K2CO3 in isopropyl alcohol/
CH2Cl2 afforded destruxin E (1). Spectral data, including
optical rotation, of synthetic destruxin E (1) are in good
agreement with those of the natural product, destruxin E.1,24
1
On the other hand, the H NMR spectrum of epi-destruxin
E (2) synthesized from macrocycle 16b was found to be
distinguishable from that of natural and synthetic 1. Specif-
ically, the chemical shift of the R-proton of the R-hydroxy
acid in 1H NMR of 2 was found to be δ 5.07 ppm, which is
obviously different from those of natural destruxin E (δ 4.91
ppm) and synthetic 1 (δ 4.99 ppm). Further drastic differ-
ences between natural destruxin E and epi-2 were observed
in C1R (+0.5 ppm), C1ꢀ (+0.6 ppm), C1γ (+0.7 ppm), and
C1δ (+0.8 ppm) positions, though the corresponding chemi-
cal shifts of synthetic 1 were identical to those of the natural
product (see Supporting Information).
of Fmoc-ꢀAla-OH onto trityl alcohol lantern 1120 provided
polymer-supported 12. The quantitative immobilization of
the Fmoc-ꢀAla-OH was observed by gravimetric analysis
after acidic cleavage from the polymer support (30%
hexafluoroisopropyl alcohol (HFIP)/CH2Cl2, rt, 1 h).21 After
removal of the Fmoc group with 20% piperidine/DMF,
(17) Spectral data of synthetic chiral lactone 5b were in good agreement
with those previously reported . (a) Enantiomer: Zhen, Z.-B.; Gao, J.; Wu,
Y. J. Org. Chem. 2008, 73, 7310–7316. (b) Liu, S. Y.; Huang, D. F.; Huang,
H. H.; Huang, L. Chin. Chem. Lett. 2000, 11, 957–960.
(18) PyBroP: bromo-tris-pyrrolidino-phosphonium hexafluorophosphate.
Fre´rot, E.; Coste, J.; Pantaloni, A.; Dufour, M.-N.; Jouin, P. Tetrahedron
1991, 47, 259–270.
(22) The linear precursors 3a and 3b have several conformational
isomers shown in 1H NMR spectra (see Supporting Information). Therefore,
we identified the compounds after macrolactonization.
(19) Rasoul, F.; Ercole, F.; Pham, Y.; Bui, C. T.; Wu, Z.; James, S. N.;
Trainor, R. W.; Wickham, G.; Maeji, N. J. Biopolymers 2000, 55, 207–
216.
(23) MNBA: 2-methyl-6-nitrobenzoic anhydride. DMAPO: 4-(dimeth-
ylamino)pyridine N-oxide. (a) Shiina, I.; Ibuka, R.; Kubota, M. Chem. Lett.
2002, 286–287. (b) Shiina, I.; Kubota, M.; Ibuka, R. Tetrahedron Lett. 2002,
43, 7535–7539. (c) Shiina, I.; Kubota, M.; Oshiumi, H.; Hashizume, M. J.
Org. Chem. 2004, 69, 1822–1830.
(20) Trityl alcohol lantern was purchased from Mimotopes Pty Ltd.,
Clayton, Vic 3168, Australia.
(21) Wade, J. D.; Bedford, J.; Sheppard, R. C.; Tregear, G. W. Pep.
Res. 1991, 4, 194–199.
(24) Gupta, S.; Roberts, D. W.; Renwick, J. A. A. J. Chem. Soc., Perkin
Trans. 1 1989, 2347–2357
.
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Org. Lett., Vol. 12, No. 17, 2010

Scheme 4
.
Total Synthesis of Destruxin E (1) and
epi-Destruxin E (2)
Table 1. V-ATPase Inhibitory Activity of Destruxin E (1) and
epi-Destruxin E (2)a
concentration [µM]
compound
40 10 2.5 0.63 0.16 0.04
0
destruxin E (isolated)
destruxin E (synthesized)
epi-destruxin E
N/D
-
-
-
-
-
-
-
+
-
-
+
+
+
+
+
+
+
+
+
+
a : Positive (+) ) V-ATPase activity detected. Negative (-) ) inhibition
of V-ATPase activity.
by solid-phase synthesis. On the basis of the synthesis, the
stereochemistry of the chiral center at the epoxide was
determined to be (S). It was found that natural destruxin E
(1) exhibits V-ATPase inhibitory activity 10-fold greater than
that of 2, which indicates that the stereochemistry of the
epoxide is crucial for exhibiting the potent V-ATPase
inhibitory activity. The library synthesis of destruxin E based
on the solid-phase synthesis and the biological evaluations
are underway in our laboratory.
Synthetic destruxin E (1) and epi-destruxin E (2) were
evaluated for their V-ATPase inhibitory activity by acridine
orange staining of V-ATPase-dependent acidic or-
ganelles.25,26 Accumulation of the dye was not detected when
V-ATPase was inhibited. Both natural and synthetic destruxin
E were found to be active at 0.63 µM, although epi-destruxin
E was not active until 10 µM. Therefore, 1 exhibited
V-ATPase inhibitory activity 10-fold greater than that of epi-
destruxin E (2) (Table 1).
Acknowledgment. This work was supported by the New
Energy and Industrial Technology Development Organization
(NEDO).
In summary, we have accomplished the first total synthesis
of destruxin E (1) and its diastereomer epi-destruxin E (2)
Supporting Information Available: Experimental details
and NMR spectra of 1-6, 8, 10, and 16-18. This material
is available free of charge via the Internet at http://pubs.acs.org.
(25) Robbins, E.; Marcus, P. I. J. Cell. Biol. 1963, 18, 237–250
(26) Muroi, M.; Shiragami, N.; Takatsuki, A. Biochem. Biophys. Res.
Commun. 1994, 205, 1358–1365
.
.
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