Isolation, structure elucidation, and total synthesis of tryptopeptins A and B, new TGF-?2 signaling modulators from streptomyces sp.

Article abstract of DOI:10.1021/ol503340k

Two new microbial metabolites, tryptopeptins A (1) and B (2), were isolated from the cultured broth of Streptomyces sp. KUSC-G11, as modulators of the transforming growth factor-?2 (TGF-?2) signaling pathway. Their chemical structures consisting of isoval

Full text of DOI:10.1021/ol503340k

Letter
pubs.acs.org/OrgLett
Isolation, Structure Elucidation, and Total Synthesis of Tryptopeptins
A and B, New TGF‑β Signaling Modulators from Streptomyces sp.
Yuta Tsunematsu,†,‡ Shinichi Nishimura, Akira Hattori, Shinya Oishi, Nobutaka Fujii,
and Hideaki Kakeya*
†
†
§
§
,†
†
§
Department of System Chemotherapy and Molecular Sciences and Department of Bioorganic Medicinal Chemistry &
Chemogenomics, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto
University, Kyoto 606-8501, Japan
*
S Supporting Information
ABSTRACT: Two new microbial metabolites, tryptopeptins A (1) and B (2), were isolated from the cultured broth of
Streptomyces sp. KUSC-G11, as modulators of the transforming growth factor-β (TGF-β) signaling pathway. Their chemical
structures consisting of isovalerate, N-Me-L-Val, L-allo-Thr, and a tryptophan-related residue were elucidated on the basis of
spectroscopic analyses, while they were unambiguously determined by total syntheses. A structure−activity relationship (SAR)
study using natural and synthesized tryptopeptins revealed the importance of the α,β-epoxyketone function located at the C
terminus. These new TGF-β signaling modulators would be highly useful for development of new drug leads targeting TGF-β-
related diseases such as fibrosis and cancer.
ransforming growth factor-β (TGF-β) is a key molecule
T
for cancer progression, since it facilitates cancer meta1-
stasis, organ fibrosis, angiogenesis and immunosuppression.
For example, epithelial−mesenchymal transition (EMT)
induced by TGF-β is a crucial event in tumor metastasis;
polarized and immotile epithelial cells differentiate into motile
mesenchymal cells to be transferred to another organ via blood
or lymph vessels. To combat cancer, small molecules inhibiting
the TGF-β signaling pathway have been developed, some of
2
which are under clinical trial. However, most chemicals target
Figure 1. Structures of tryptopeptins A (1) and B (2).
TGF-β receptors, and a variety of chemicals with unique modes
of action are requisite for exploring the therapeutic potential of
modulation of TGF-β signaling. During the course of our
screening for novel TGF-β modulators, we have found two
novel microbial metabolites from Streptomyces sp. KUSC-G11,
designated tryptopeptins A (1) and B (2). We herein describe
the isolation, structure elucidation and total synthesis of
tryptopeptins. The structure−activity relationship (SAR) is
also discussed using natural and synthetic derivatives.
We conducted a cell-based screening against thousands of
microbe culture extracts to find that the culture extract of
Streptomyces sp. KUSC-G11 exhibited potent inhibitory activity
against TGF-β signaling. Bioassay-guided fractionation by silica
gel chromatography and repetitive reversed phase HPLC
afforded novel metabolites 1 and 2 (Figure 1).
Tryptopeptin A (1) was isolated as an optically active
colorless oil ([α]D27 = −94.3, c 0.010, MeOH). The molecular
formula was determined to be C H N O with 11 degrees of
28
40
4
6
+
unsaturation, based on HR-FABMS (m/z 529.3028 [M + H] ,
Δ = +0.2 mmu, calcd. for C H N O , 529.3026) and 1D
28
41
4
6
NMR spectra. Diagnostic signals in the NMR spectra indicated
the peptidic nature of 1; two amide protons (δ
ppm) and one N-methyl signal (δ 2.87 ppm) appeared in the
7.12 and 6.78
H
H
1
13
H NMR spectrum, while the C NMR spectrum exhibited
three amide carbonyl signals (δ 174.8, 171.6, and 171.1 ppm).
C
Received: November 18, 2014
Published: January 5, 2015
©
2015 American Chemical Society
258
DOI: 10.1021/ol503340k
Org. Lett. 2015, 17, 258−261

Organic Letters
D NMR data including DQF-COSY, TOCSY, HSQC and
Letter
3
2
presence of N-Me-L-Val in 2. The configuration of Thr was
determined to be L-allo-Thr by comparing the retention times
with those of FDLA-derivatized authentic Thr and allo-Thr
(Figure S15, Supporting Information). Unfortunately, however,
we could not determine the configuration of C-4 since FDLA
derivatives of the tryptophan-related residue were not detected.
By contrast, the configuration of the Trp-EK in metabolite 1
was only speculatively deduced by the NMR analysis (Figure
S14). To determine the absolute configuration of these
tryptopeptins, we conducted the total synthesis of tryptopep-
tins.
Tryptopeptin A (1) seems to have the same configuration to
that of 2; these two molecules shared the same amino acid
backbone, suggesting that they are biosynthesized by a
common nonribosomal peptide synthetase (NRPS) machinery.
In addition, they exhibited very similar H NMR signals
especially for the backbone protons. Retrosynthetic analysis of
HMBC spectra revealed the presence of one residue each of
isovalerate, N-methylvaline (N-Me-Val) and threonine (Thr).
The remaining portion was C H N O . Five aromatic proton
1
3
13
2
2
signals (δ 7.62−7.08 ppm), one exchangeable NH signal (δ
H
H
9
.17 ppm) and eight aromatic carbon signals (δ 137.5−110.2)
C
indicated the presence of an indole ring, while a spin-system
between an amide proton (4-NH), a methine proton (H-4) and
methylene protons (H-5ab) was identified in the DQF-COSY
spectrum, suggesting that 1 includes a tryptophan-related
residue. The DQF-COSY and TOCSY spectra included
another spin system consisting of one methine (H-2) and
one methylene (H-1ab). The NMR signal that remains to be
assigned was one carbonyl (C-3). HMBC correlations were
observed from H-1, H-2, H-4 and H-5 to this carbon, indicating
that the two spin systems H-1 to H-2 and 4-NH to H-5ab were
connected through C-3 carbonyl. Finally, the unsaturation
degree indicated the presence of an epoxide group, which was
supported by characteristic chemical shift values for C-1 and C-
1
tryptopeptins 1 and 2 is shown in Scheme 1. We planned to
2
(δ 47.7 and 53.0 ppm, respectively), deducing the structure
Scheme 1. Retrosynthetic Analysis of Four and Two Possible
Diastereomers of Tryptopeptins A (1a−1d) and B (2a and
2b), Respectively
C
of an α,β-epoxyketone functionality.
The sequence of amino acid residues was determined by
interpretation of the HMBC data; the HMBC correlations from
H-4, 4-NH and H-15 to C-14, from 15-NH and H-19 to C-18,
and from H-19, H-23, H-25a and H-25b to C-24 allowed us to
determine the sequence as follows: from N-terminus,
isovalerate, N-Me-Val, Thr and C-terminally modified trypto-
phan-related residue possessing α,β-epoxyketone (Trp-EK)
(
Figure 2).
synthesize them by conjugating the corresponding N-terminal
and C-terminal fragments; 1 could be synthesized with 3 and 4,
and 2 from 3 and 5, respectively. The common segment 3
consisting of isovalerate, N-Me-L-Val and L-allo-Thr could be
obtained by a conventional peptide synthesis methodology
from 8−10. In contrast, we had to synthesize a set of
stereoisomers of the C-teminal fragment for determining the
configuration of the natural products. We expected that four
diastereomers 4a−4d could be obtained by epoxidation of an
α,β-enone 6a or 6b (6a and 6b with 4S and 4R configuration,
respectively), while another set of two diastereomers 5a and 5b
could be synthesized by reduction of 6a and 6b, respectively.
The α,β-enones 6a and 6b could be synthesized from
commercially available N-Boc-L-tryptophan 7a and its enan-
tiomer 7b, respectively.
Figure 2. 2D NMR correlations in tryptopeptin A (1).
Tryptopeptin B (2) was obtained as a colorless oil ([α] 25
96.0, c 0.010, MeOH). The molecular formula of
=
D
−
C H N O , which was deduced by HR-ESIMS (m/z
2
8
42
4
5
+
5
37.3066 [M + Na] , Δ = +1.3 mmu, calcd. for
C H N NaO , 537.3053), indicated loss of one oxygen
28
42
4
5
atom and decrease of one degree of unsaturation when
1
13
compared to the metabolite 1. The H and C NMR spectra
strongly suggested lack of an epoxide group. Instead, signals
corresponding to an ethyl group (δ 0.89 and 2.45 ppm, and δC
H
7
.6 and 33.7 ppm) were observed, indicating that the
epoxyketone function of 1 was substituted by an ethyl ketone
group. Detailed analysis of the 2D NMR data confirmed the
presence of the ethyl ketone group, and we could determine the
planar structure of 2 as depicted in Figure 1.
We began the synthetic work by preparing chiral tryptophan-
related residues (Scheme 2A). We first synthesized 6a, a
common intermediate for 4a, 4b and 5a. N-Boc-L-Trp 7a was
We next tried to determine the stereochemistry of
3
4
tryptopeptins. The advanced Marfey’s method was applied
converted to Weinreb’s amide, followed by protection of the
to determine the absolute configuration of amino acids. The
acid hydrolysate of 2 was condensed with Marfey’s reagents L-
or D-FDLA, and the products were analyzed by LC−MS. L-
FDLA derivative of N-Me-valine was eluted faster than the D-
FDLA derivative on reversed phase HPLC, which indicated the
indole nitrogen with a TBS group to afford 11. Grignard
reaction of 11 with vinylmagnesium chloride gave the
corresponding enone 6a. We next tried to synthesize
epoxyketone 4a and 4b from 6a. We first attempted Sharpless
asymmetric epoxidation or dihydroxylation to an allylic alchol
2
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Organic Letters
Letter
a
Scheme 2. Syntheses of Tryptopeptin A (1a−1d) and B (2a and 2b) Diastereomers
a
(
A) Syntheses of tryptopeptin A C-terminal fragments 4a and 4b. (B) Common N-terminal fragment synthesis. (C) Syntheses of tryptopeptin A
diastereomers (1a−1d). (D) Syntheses of tryptopeptin B diastereomers (2a and 2b).
derivative that was prepared from 6a with NaBH ; however,
with TASF to afford the four diastereomers 1a−1d,
4
6
these strategies were not satisfactory due to the low reaction
yields. We then tested a nonstereoselective route, which can
conveniently furnish diastereomers. After testing several
epoxidation conditions, we found that treatment of enone 6a
with H O /NaHCO afforded diastereomers of α,β-epoxyke-
respectively (Scheme 2C, Scheme S4). With four possible
diastereomers of natural tryptopeptin A (1) in hand, we
compared their physicochemical properties including NMR
spectra to that of the natural one. We found that only synthetic
1a exhibited similar properties to those of natural 1 (Table S3,
Figures S19−S22, S55); for example, optical rotation values of
2
2
3
tones 4a and 4b (31%, 4a:4b = 1.8:1.0). We purely separated
two products by reversed phase HPLC. The absolute
configuration of the epoxide (C2-position) was determined
by derivatization and spectroscopic analysis (Scheme S2,
Figures S16−S17). Another set of diastereomers 4c and 4d
were prepared from 7b in a similar fashion (Scheme S3). On
the other hand, hydrogenation of 6a successfully furnished the
ethyl ketone residue 5a, in addition, the enantiomer 5b was
prepared from 6b (Scheme 2D).
The N-terminal fragment was constructed by applying
conventional Fmoc chemistry (Scheme 2B). Fmoc-protected
L-allo-Thr (10) was converted to the benzyl ester and its
secondary hydroxyl group was protected by the TBDPS group
to give 12. After removal of the Fmoc group, allo-Thr was
coupled with 9, followed by Fmoc deprotection and acylation
with 8, which afforded the acylated dipeptide 13. Subsequent
removal of the benzyl ester by hydrogenation yielded the N-
terminal fragment 3.
2
7
natural 1 and synthetic 1a were close (1: [α]
= −94.3, c
D
0.010, MeOH, 1a: [α]D27 = −96.9, c 0.064, 1b: [α]
26
=
D
26
−157.2, c 0.25, MeOH, 1c: [α] = −127.1, c 0.25, MeOH, 1d:
D
25
[α] = −50.3, c 0.25, MeOH). Thus, we concluded that the
D
natural product 1 consists of isovalerate, N-Me-L-Val, L-allo-Thr
and a tryptophan-related residue (Trp-EK) with 2R,4S
configurations.
Two possible diastereomers of tryptopeptin B (2) were
synthesized from the C-terminal fragment 5a or 5b and the N-
terminal 3 in a similar way as described above; N-Boc
deprotection, peptide condensation and silyl-deprotection
afforded 2a and 2b, respectively (Scheme 2D). We compared
their physicochemical properties and found that 2a, not 2b,
exhibited similar properties to those of natural 2 (Table S4,
Figures S23−S27 and S56), concluding the configuration of 4S.
As predicted above, tryptopeptins 1 and 2 shared common
absolute configurations.
Synthesis of tryptopeptins was accomplished by conjugation
of the corresponding C-terminal and N-terminal fragments
followed by deprotection. To obtain four possible diaster-
eomers of 1, the N-terminal fragment 3 and the C-termical
fragments derived from 4 were condensed; C-termina5l
fragments (4a, 4b, 4c or 4d) were deprotected with TFA
and immediately coupled with the N-terminal fragment 3 using
HATU. Finally, we carefully deprotected the secondary alcohol
With two natural and six synthesized tryptopeptins in hand,
SAR study was conducted. Among compounds tested,
tryptopeptin A (1) showed the most potent inhibitory activity
with the IC50 of 1.0 μM against TGF-β signaling without
cytotoxicity (Figure 3 and S57). It is noteworthy that a loss of
an epoxide moiety of tryptopeptins caused a significant
decrease in the TGF-β inhibitory activities; the IC50 values
for 2 and 2b are 55 and 70 μM, respectively. We also found that
2
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Organic Letters
Letter
ASSOCIATED CONTENT
Supporting Information
■
*
S
Experimental procedures, compound data, tables of NMR data,
D and 2D NMR spectra, and results of biological assays. This
1
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*
E-mail: scseigyo-hisyo@pharm.kyoto-u.ac.jp.
Present Address
‡
Department of Pharmaceutical Sciences, University of
Figure 3. TGF-β signaling inhibitory activities of tryptopeptin A (1),
tryptopeptin B (2) and their synthetic diastereomers (1b−1d and 2b).
Shizuoka, Shizuoka 422-8526, Japan.
Notes
The authors declare no competing financial interest.
the configuration of the epoxide group affected the inhibitory
activity; the IC values of 1, 1b, 1c, and 1d were 1.0, 8.0, 10.0,
ACKNOWLEDGMENTS
We thank Dr. Kojima, S. (RIKEN Institute) and Morioka, M.
Kyoto University) for providing a p(CAGA) MLP-Luc
■
(
50
and 3.2 μM, respectively, indicating that the diastereomers with
S configuration exhibited weaker inhibition than the
9
2
reporter plasmid and supporting biological assay, respectively.
We are grateful to Dr. Takasu, Y., Dr. Sakai, Y., Hayashi, R. and
Dr. Inokuchi, E. (Kyoto University) for helpful discussions on
organic synthesis. Y.T. is grateful for assistance from JSPS
research fellowships for young scientist. This work was
supported in part by research grants from the Japan Society
for the Promotion of Science (JSPS), the Ministry of
Education, Culture, Sports, Science and Technology of Japan
compounds with 2R configuration (Figure 3). Similar tendency
was observed in the growth inhibitory activities against HeLa
cells (Figure S58). Importantly, natural tryptopeptins (1 and 2)
and synthetic tryptopeptins with the same configuration (1a
and 2a) exhibited the same biological activities (Figures S55
and S56), which also confirmed the absolute configuration of
the natural tryptopeptins. Taken together, tryptopeptin A (1)
with 2R,4S configurations exhibited the most potent biological
activities, suggesting that target molecule(s) in a cell might
recognize the configurations of the epoxyketone.
(
(
MEXT), the Ministry of Health, Labor and Welfare of Japan
MHLW), and SRF.
So far, several compounds possessing a peptidyl α,β-
epoxyketone function have been reported from microorgan-
REFERENCES
■
7
(1) Miyazono, K. Proc. Jpn. Acad., Ser. B 2009, 85, 314−323.
isms, e.g., histone deacetylase inhibitor trapoxins, and 20S
8a
8b
(2) Nagaraj, N. S.; Datta, P. K. Expert Opin. Invest. Drugs 2010, 19,
7−91.
proteasome inihibitors epoxomicin and eponemycins. The
α,β-epoxyketone group has been revealed to play a crucial role
in the enzyme inhibition, by forming a covalent bond with the
7
(
(
1
3) (a) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
b) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem.
9
catalytic residue in the enzyme. In this study, we showed the
997, 69, 3346−3352. (c) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.;
presence and the absolute configuration of epoxide in
tryptopeptins affected the potency of TGF-β signaling
inhibition. It is likely that we can fish the cellular targets taking
advantage of the reactivity of the epoxyketone group. Synthesis
and evaluation of tryptopeptin probes are under way in our
laboratory.
Harada, K. Anal. Chem. 1997, 69, 5146−5151.
(4) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815−
3818.
(5) Sin, N.; Kim, K. B.; Elofsson, M.; Meng, L. H.; Auth, H.; Kwok,
B. H. B.; Crews, C. M. Bioorg. Med. Chem. Lett. 1999, 9, 2283−2288.
6) Although we tried several desilylating conditions, only TASF gave
the desired product. In other conditions, only degradation of the
starting material was observed.
7) Itazaki, H.; Nagashima, K.; Sugita, K.; Yoshida, H.; Kawamura, Y.;
Yasuda, Y.; Matsumoto, K.; Ishii, K.; Uotani, N.; Nakai, H.; Terui, A.;
Yoshimatsu, S.; Ikenishi, Y.; Nakagawa, Y. J. Antibiot. 1990, 43, 1524−
532.
(
Tryptopeptins consist of four blocks; among them, L-allo-Thr
is a rare residue in nature, while there is no report on the C-
terminally modified tryptophan-related residue. The peptide
backbones might be constructed by NRPS and C-terminus of L-
tryptophan is likely elongated by polyketide synthase (PKS),
followed by some modifications to form α,β-epoxyketone group
in compound 1. Recently, the biosynthetic gene clusters of
epoxomicin and eponemycin were reported, whereas the
(
1
(8) (a) Hanada, M.; Sugawara, K.; Kaneta, K.; Toda, S.; Nishiyama,
Y.; Tomita, K.; Yamamoto, H.; Konishi, M.; Oki, T. J. Antibiot. 1992,
45, 1746−1752. (b) Sugawara, K.; Hatori, M.; Nishiyama, Y.; Tomita,
K.; Kamei, H.; Konishi, M.; Oki, T. J. Antibiot. 1990, 43, 8−18.
10
mechanism of the C-terminus modification is unknown.
(
9) Groll, M.; Kim, K. B.; Kairies, N.; Huber, R.; Crews, C. M. J. Am.
Genomic sequencing of the tryptopeptin producer Streptomyces
sp. KUSC-G11 would unveil the biosynthetic mechanism of
tryptopeptins including generation of the Trp-EK.
In summary, we isolated two novel natural products,
tryptopeptins A (1) and B (2), from the cultured broth of
Streptomyces sp. as TGF-β signaling inhibitors. Chemical
structures were unambiguously determined by spectroscopic
analyses and total syntheses. Since TGF-β signaling is deeply
involved in cancer progression, understanding modes of action
of tryptopeptins would open a way for cancer chemotherapy.
Chem. Soc. 2000, 122, 1237−1238.
10) Schorn, M.; Zettler, J.; Noel, J. P.; Dorrestein, P. C.; Moore, B.
S.; Kaysser, L. ACS Chem. Biol. 2013, 9, 301−309.
(
2
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