Structure and total synthesis of fungal calpinactam, a new antimycobacterial agent

Article abstract of DOI:10.1021/ol902553z

[Chemical equation presented] A new fungal metabolite designated calpinactam (1) was isolated from the culture broth of Mortierella alpina FKI-4905, and its structure was elucidated by spectroscopic analyses including NMR experiments. Calpinactam was found to be a hexapeptide with a caprolactam ring at its C-terminal. Its absolute stereochemistry was determined by amino acid analysis and total synthesis. Calpinactam selectively inhibited the growth of mycobacteria among various microorganisms. The MIC values of calpinactam against Mycobacterium smegmatis and M. tuberculosis were 0.78 and 12.5 μg/mL, respectively.

Full text of DOI:10.1021/ol902553z

ORGANIC
LETTERS
2010
Vol. 12, No. 3
432-435
Structure and Total Synthesis of Fungal
Calpinactam, A New Antimycobacterial
Agent
Nobuhiro Koyama,† Shigenobu Kojima,† Takeo Fukuda,† Tohru Nagamitsu,†
†
Tadashi Yasuhara, Satoshi Omura, and Hiroshi Tomoda*
‡
,†
¯
Graduate School of Pharmaceutical Sciences and Kitasato Institute for Life Sciences,
Kitasato UniVersity, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
tomodah@pharm.kitasato-u.ac.jp
Received November 4, 2009
ABSTRACT
A new fungal metabolite designated calpinactam (1) was isolated from the culture broth of Mortierella alpina FKI-4905, and its structure was
elucidated by spectroscopic analyses including NMR experiments. Calpinactam was found to be a hexapeptide with a caprolactam ring at its
C-terminal. Its absolute stereochemistry was determined by amino acid analysis and total synthesis. Calpinactam selectively inhibited the
growth of mycobacteria among various microorganisms. The MIC values of calpinactam against Mycobacterium smegmatis and M. tuberculosis
were 0.78 and 12.5 µg/mL, respectively.
Our research group has focused on the discovery of anti-
infectives from microbial metabolites.1 Tuberculosis (TB)
is still the greatest single infectious cause of mortality in
the world, together with HIV and malaria.2 Moreover, the
spread of the HIV has increased the number of tuberculosis
patients.3 However, new anti-TB agents have not been
developed for over 30 years, and only 5 anti-TB drugs,
namely, isoniazid, ethambutol, rifampicin, streptomycin, and
pyrazinamide, are in clinical use. It is increasingly urgent to
discover anti-TB drugs with a new mechanism of action.
Since isoniazid and ethambutol, first-line anti-TB drugs, show
selective growth inhibition against Mycobacteria, we have
screened for microbial metabolites that selectively inhibit the
growth of M. smegmatis among 14 test microorganisms
including gram-positive and gram-negative bacteria, fungi,
and yeasts. By using this screening system,4 we discovered
and reported lariaitins, new lasso polypeptides, from the
culture broth of actinomycete Rhodococcus josti K01-
B0171.4 During the continuation of the screening program,
we have isolated calpinactam (1, Figure 1) from the culture
broth of the fungal strain Mortierella alpina FKI-4905. From
the various spectral analyses, the compound was found to
have a hexapeptide skeleton with a caprolactam ring at its
† Graduate School of Pharmaceutical Sciences.
‡ Kitasato Institute for Life Sciences.
(1) (a) Fukumoto, A.; Kim, Y. P.; Matsumoto, A.; Takahashi, Y.; Shiomi,
K.; Tomoda, H.; Omura, S. J. Antibiot. 2008, 61, 1–6. (b) Fukumoto, A.;
Kim, Y. P.; Hanaki, H.; Shiomi, K.; Tomoda, H.; Omura, S. J. Antibiot.
2008, 61, 7–10. (c) Iwatsuki, M.; Uchida, R.; Yoshijima, H.; Ui, H.; Shiomi,
K.; Matsumoto, A.; Takahashi, Y.; Abe, A.; Tomoda, H.; Omura, S. J.
Antibiot. 2008, 61, 222–229. (d) Iwatsuki, M.; Uchida, R.; Yoshijima, H.;
Ui, H.; Shiomi, K.; Kim, Y. P.; Hirose, T.; Sunazuka, T.; Abe, A.; Tomoda,
H.; Omura, S. J. Antibiot. 2008, 61, 230–236. (e) Fukuda, T.; Hasegawa,
Y.; Sakabe, Y.; Tomoda, H.; Omura, S. J. Antibiot. 2008, 61, 550–555.
(2) National Institute of Allergy and Infectious Diseases (NIAID). http://
www.niaid.nih.gov/topics/tuberculosis/ (accessed 11/3/ 2009).
(3) O’Brien, R. J. Tuberculosis 2001, 81, 1–52.
(4) (a) Iwatsuki, M.; Tomoda, H.; Uchida, R.; Gouda, H.; Hirono, S.;
Kobayashi, S.; Omura, S. J. Am. Chem. Soc. 2006, 128, 7486–7491. (b)
Iwatsuki, M.; Uchida, R.; Takakusagi, Y.; Matsumoto, A.; Jiang, C. L.;
Takahashi, Y.; Arai, M.; Kobayashi, S.; Matsumoto, M.; Inokoshi, J.;
Tomoda, H.; Omura, S. J. Antibiot. 2007, 60, 357–363. (c) Iwatsuki, M.;
Koizumi, Y.; Gouda, H.; Hirono, S.; Tomoda, H.; Omura, S. Bioorg. Med.
Chem. Lett. 2009, 19, 2888–2890.
10.1021/ol902553z  2010 American Chemical Society
Published on Web 12/23/2009

Figure 1. Structure of 1.
C-terminal, which is seen in the structures of siderophore
mycobactins.5 In this study, the structure elucidation of 1
including its absolute stereochemistry and biological activity
is described. M. alpina FKI-4905 was isolated from soil
collected in the Bonin Islands, Tokyo, Japan. This strain was
used for the production of 1. The whole broth was cultured
for 4 days (1.0 L), before being extracted with ethanol, and
then the supernatant was filtered and concentrated under
reduced pressure. The resulting aqueous layer was diluted
by adding distilled water (2.0 L) and applied to an ODS gel
column (40 g). The materials were eluted stepwise with 30,
60, and 100% CH3CN (500 mL each). The 60% CH3CN
fraction, which showed activity, was concentrated to give a
red brown material (140 mg). This material containing
enriched calpinactam was finally purified using preparative
HPLC under the following conditions: column, PEGASIL
ODS (Senshu Sci. i.d. 20 × 250 mm); solvent, a 40 min
linear gradient from 20 to 40% CH3CN containing 0.05%
trifluoroacetic acid; flow rate, 6 mL/min; detection, UV at
210 nm. The fraction eluted as a peak with the retention
time of 31 min and was concentrated and lyophilized to
dryness to yield pure 1 (13.0 mg) as a white powder.14
Calpinactam (1) showed a molecular ion peak at m/z 768
(M + H)+ in FAB-MS, and the molecular formula
C38H57O8N9 was assigned on the basis of HRFAB-MS [m/z
768.4411 (M + H)+, ∆ +2.4 mmu], requiring 15 degrees
of unsaturation. The ninhidrin reaction and IR absorptions
suggested the presence of amino and carbonyl groups in the
structure. The 13C NMR spectrum (in DMSO-d6) showed
36 resolved signals, which were classified into four methyl
carbons, 10 methylene carbons, eight sp3 methine carbons,
seven sp2 methine carbons, two sp2 quaternary carbons, and
seven carbonyl carbons by analysis of its DEPT spectra. The
1H NMR spectrum (in DMSO-d6) displayed 51 proton
signals, six of which were suggested to be NH protons (δ
7.79, 7.83, 7.92, 7.97, 8.42, and δ 8.57). Furthermore,
Figure 2. COSY, TOCSY, and HMBC correlations of 1.
partial structures (I-VII,) as shown by the bold lines in
Figure 2. Furthermore, 13C-1H long-range couplings of J
2
3
and J were measured in the HMBC spectrum (Figure 2).
First, the presence of six amino acid residues became clear
due to the following observations: (1) The cross peaks from
H2-3 (δ 1.34 and δ 1.70) and H2-6 (δ 3.02 and δ 3.14) to
C-1 (δ 174.1) and the cross peak from NH-6 (δ 7.83) to
C-2 (δ 51.4) showed a connection between the partial
structure VII and the carbonyl carbon C1, indicating the
presence of a caprolactam moiety derived from a lysine
residue. (2) The cross peak from H-9 (δ 1.80) to C-7 (δ
169.8) showed the connection between the partial structure
VI and the carbonyl carbon C-7, indicating the presence of
an isoleucine residue. (3) The cross peaks from H2-15 (δ
1.70 and δ 1.85) to C-13 (δ 171.1) and C-17 (δ 173.9)
showed the connection among the partial structure V and
the carbonyl carbons C-13 and C-17, indicating the presence
of a glutamate residue. (4) The cross peak from H-26 (δ
1.26) to C-24 (δ 171.5) showed the connection between the
partial structure III and the carbonyl carbon C-24, indicating
the presence of a leucine residue. (5) The cross peaks from
H2-32 (δ 2.96 and δ 3.00) to C-30 (δ 167.9), C-33 (δ 134.9),
C-34 (δ 129.5), and C-38 (δ 129.5) showed the connections
among the partial structures I and II and the carbonyl carbon
C-30, indicating the presence of a phenylalanine residue. (6)
The residual molecular formula and the cross peaks from
H-19 (δ 4.55) to C-21 (δ 130.5) and from H2-20 (δ 2.82
and δ3.01) to C-21 and C-22 (δ 117.2) showed that a
heterocyclic five-membered ring was formed among C-21,
C-22, N-22, C-23, and N-23 to connect C-20 and C-21. Two
1
singlet proton signals for H-22 and H-23 in the H NMR
and the chemical shifts of C-21, C-22, and C-23 (δ 134.2)
supported the presence of an imidazole ring. Next, to
determine the sequence of the six amino acid residues, the
correlation from each R-proton to the adjacent carbonyl
carbon observed in the HMBC spectrum was investigated.
The respective cross peaks from H-25 (δ 4.26) to C-30, H-19
to C-24, H-14 (δ 4.36) to C-18, H-8 (δ 4.31) to C-13, and
H-2 (δ 4.36) to C-7 indicated that the sequence from the
N-terminal was phenylalanine, leucine, histidine, glutamic
acid, isoleucine, and cyclized lysine. Taken together, the
planar structure of calpinactam was elucidated as shown in
Figure 2.
1
analysis of the H NMR spectrum in CD3OD as a solvent
revealed the presence of two new proton signals (δ 7.34 and
δ 8.79) corresponding to aromatic protons of a heterocyclic
ring. The connectivity of the proton and carbon atoms (Figure
1
2) was established by its HMQC spectrum. H-1H COSY
and TOCSY analyses revealed the presence of the seven
(5) (a) Ratledge, C. Tuberculosis 2004, 84, 110–130. (b) Vergne, A. F.;
Walz, A. J.; Miller, M. J. Nat. Prod. Rep. 2000, 17, 99–116.
(6) (a) Takano, Y.; Marumo, K.; Kobayashi, K.; Takahashi, J. Bunseki
Kagaku 2004, 53, 1507–1514. (b) Kudo, J.; Takano, Y.; Kaneko, T.;
Kobayashi, K. Bunseki Kagaku 2003, 52, 35–40.
Org. Lett., Vol. 12, No. 3, 2010
433

To elucidate the absolute stereochemistry of 1, amino acid
analysis was carried out by the established method.6 The
acid hydrolysate of 1 was derivatized with O-phthalaldehyde
(OPA) and N-acetyl-L-Cys to yield OPA derivatives, which
were analyzed by HPLC (column, TSKgel Super-ODS
(TOSOH, i.d. 4.6 × 100 mm); flow rate, 0.7 mL/min;
detection, fluorescent intensity at 335 nm with excitation at
435 nm) using two following solvent systems: (1) A 60 min
linear gradient from 0 to 20% MeOH and a subsequent 50
min linear gradient from 20 to 60% MeOH. (2) A 90 min
linear gradient from 25 to 40% MeOH. Under the conditions
of (1), the OPA derivatives prepared from authentic D/L-
Glu, -His, -Phe, -Ile, -Leu, and -Lys were detected with
retention times of 10.6/11.7, 34.4/32.7, 87.0/87.7, 91.0/88.0,
93.0/94.0, and 94.5/94.9 min, respectively. Under these
conditions, the hydrolysate of 1 was analyzed to give D-Phe,
L-Leu, L-His, D-Glu, D-Ile, and L-Lys. Regarding D-Ile, further
analysis to differentiate D-Ile and D-allo-Ile was needed.
Under the conditions of (2), authentic D-Ile and D-allo-Ile
were eluted with retention times of 46.8 and 47.6 min,
respectively, showing D-allo-Ile to be present in 1. Therefore,
the complete structure of 1 including its absolute stereo-
chemistry was elucidated to be D-phenylalanyl-L-leucyl-L-
histidyl-D-glutaminyl-D-allo-isoleucyl-L-caprolactam, as shown
in Figure 1.
1
Table 1. H (600 MHz) and 13C NMR (150 MHz) Chemical
Shifts of 1 in DMSO-d6
position
δC
δH
1
2
174.1 s
51.4 d
-
31.2 t
27.6 t
28.8 t
40.7 t
-
169.8 s
56.2 d
-
36.6 d
25.7 t
11.6 q
14.5 q
171.1 s
51.6 d
-
27.6 t
30.1 t
173.9 s
170.0 s
52.2 d
-
27.7 t
130.5
117.2
134.2
171.5 s
51.2 d
-
40.8 t
23.8 d
21.4 q
23.1 q
167.9 s
53.5 d
37.3 t
134.9 s
129.5 d
128.6 d
127.2 d
-
4.36 (1H, m)
2-NH
3
4
5
6
6-NH
7
8
8-NH
9
10
11
12
13
14
14-NH
15
16
17
18
19
19-NH
20
21
22
23
24
25
25-NH
26
27
28
29
30
31
32
33
7.79 (1H, d, J ) 6.5)
1.34 (1H, m), 1.70 (1H, m)
1.60 (1H, m), 1.84 (1H, m)
1.17 (1H, m), 1.71 (1H, m)
3.02 (1H, m), 3.14 (1H, m)
7.83 (1H, dd, J ) 5.0, 7.0)
-
4.31 (1H, dd, J ) 6.0, 9.0)
7.97 (1H, d, J ) 9.0)
1.80 (1H, m)
1.05 (1H, m), 1.27 (1H, m)
0.80 t (3H, d, J ) 7.0)
0.77 d (3H, d, J ) 6.0)
-
4.36 (1H, m)
7.92 (1H, d, J ) 8.0)
1.70 (1H, m), 1.85 (1H, m)
2.07 (2H, m)
-
-
4.55 (1H, dt, J ) 7.5, 8.0)
8.42 (1H, d, J ) 7.5)
2.82 (1H, dd, J ) 8.0, 16.0), 3.01 (1H, m)
-
To confirm the complete structure of 1, the total synthesis
of 1 was carried out (Scheme 1). First condensation between
a known ε-lactam 2, which was readily derived from L-Lys,
and Boc-D-allo-Ile-OH in the presence of EDCI, HOBt, and
Et3N provided dipeptide 3 quantitatively. The Boc depro-
tection of 3 by treatment with TFA was followed by a second
condensation reaction with Boc-D-Glu(OBn)-OH under the
same conditions as before, affording the tripeptide 4 in an
88% yield over two steps. Removal of the Boc group of 4
by TFA, a subsequent third condensation reaction with Fmoc-
L-His(Trt)-OH, and deprotection of the Fmoc group by
exposure to piperidine furnished the tetrapeptide 5 in a 91%
yield over three steps. A fourth condensation reaction
between 5 and Fmoc-L-Leu-OH followed by deprotection
of the Fmoc group by piperidine afforded pentapeptide 6 in
a 50% yield over two steps. The pentapeptide 6 was then
condensed with Boc-D-Phe-OH to give hexapeptide 7 in a
69% yield. Finally, removal of the trityl and benzyl groups
by hydrogenolysis and N-Boc deprotection by TFA furnished
crude 1, which was purified by HPLC to afford pure 1 in a
22% yield over two steps (not optimized). Synthetic 1 was
7.34a (1H, s)
8.79a (1H, s)
-
4.26 (1H, dt, J ) 7.0, 8.0)
8.57 (1H, d, J ) 8.0)
1.26 (2H, m)
1.16 (1H, m)
0.75 (3H, d, J ) 6.0)
0.72 (3H, d, J ) 6.0)
-
4.06 (1H, t, J ) 7.0)
2.96 (1H, dd, J ) 7.0, 14.0), 3.00 (1H, m)
-
7.24 (2H, m)
7.30 (2H, m)
7.26 (1H, m)
34, 38
35, 37
36
a
1H NMR was measured in CD3OD due to no signals being detected
in DMSO-d6. The value is shown here.
formobactin,9 nocobactins,10 BE-32030,11 and amamistatins12
produced by Nocardia spp. were reported to show anti-lipid
peroxidation activity or antitumor activity. All compounds
belonging to (A) and (B) have two hydroxamic acid moieties
and one 2-hydroxyphenyloxazoline moiety in their structures,
which are expected to form extremely stable hexadentate
1
identical to natural product 1 in all respects ([R]D, H and
13C NMR, IR, MS, inhibitory activity against M. smegmatis,
and retention time in HPLC). This result shows that the
foregoing results with regard to the amino acid analysis of
natural calpinactam (1) were accurate.
Several groups of caprolactam-containing peptide-like
compounds have been reported. (A) Mycobactins7 and
carboxymycobactins8 produced by Mycobacterium spp. are
well-known siderophores or iron chelators and play a role
in iron transport during mycobacteria growth. (B) The
(9) Murakami, Y.; Kato, S.; Nakajima, M.; Matsuoka, M.; Kawai, H.;
Shin-Ya, K.; Seto, H. J. Antibiot. 1996, 49, 839–845.
(10) Ratledge, C.; Snow, G. A. Biochem. J. 1974, 139, 407–413.
(11) Tsukamoto, M.; Murooka, K.; Nakajima, S.; Abe, S.; Suzuki, H.;
Hirano, K.; Kondo, H.; Kojiri, K.; Suda, H. J. Antibiot. 1997, 50, 815–
821.
(7) Snow, G. A. Biochem. J. 1965, 97, 166–175.
(12) Suenaga, K.; Kokubo, S.; Shinohara, C.; Tsuji, T.; Uemura, D.
Tetrahedron Lett. 1999, 40, 1945–1948.
(8) Ratledge, C.; Ewing, M. Microbiology 1996, 142, 2207–2212.
434
Org. Lett., Vol. 12, No. 3, 2010

Scheme 1. Total Synthesis of 1
iron(III) complexes. On the other hand, (C) VX1000,
VVD130, and ZG3000, which are produced by the plant
pathogenic fungus Periconia circinata, were reported to
inhibit plant root growth.13 These compounds have no
structural characteristics showing iron chelating activity.
Compound 1, which was also isolated from a fungal strain,
contains no hydroxamic acids or 2-hydroxyphenyloxazolines
in its structure. In this sense, calpinactam belongs to the (C)
group, although its core structures are quite different except
for having a caprolactam moiety. Furthermore, several
synthetic mycobactin analogues were reported to potently
inhibit the growth of M. tuberculosis, probably as antagonists
of mycobactin.5 From the unique structure of 1, the mech-
anism of action of 1 appears to be different from those of
compounds belonging to (A) to (C), although this remains
to be clarified.
against mycobacteria among various microorganisms includ-
ing gram-positive and gram-negative bacteria, fungi, and
yeasts. In a liquid microdilution method, 1 inhibited the
growth of M. smegmatis and M. tuberculosis with MIC
values of 0.78 and 12.5 µg/mL, respectively.
Acknowledgment. We express our thanks to Dr. Ken-
ichiro Nagai, Ms. Akiko Nakagawa, and Ms. Noriko Sato
of the School of Pharmacy, Kitasato University for their help
with the measurements of MS and NMR spectra. We also
thank Dr. Makoto Matsumoto and Mrs. Miki Matsuba of
the Microbiological Research Institute Otsuka Pharmaceutical
Co., Ltd. for the measurement of the MIC of the myco-
bacteria. This study was supported by grants from Kakenhi
16073215 (to H.T.) and 19710191 (to N.K.) from the
Ministry of Education, Culture, Sports, Science, and
Technology of Japan and the Uehara Memorial Foundation
(to H.T.).
The antimycobacterial activity of 1 was evaluated by a
paper disk method and a liquid microdilution method.4 In a
paper disk method, the compound was selectively active
Supporting Information Available: NMR spectra of
natural calpinactam (1), and experimetnal procedures and
characterization data of synthetic calpinactam (1). This
material is available free of charge via the Internet at
http://pubs.acs.org.
(13) Macko, V.; Stimmel, M. B.; Wolpert, T. J.; Dunkle, L. D.; Acklin,
W.; Ba¨nteli, R.; Jaun, B.; Arigoni, D. Proc. Natl. Acad. Sci. U.S.A. 1992,
89, 9574–9578.
(14) Calpinactam (1): white powder; [R]25 -23.0 (c 0.05, AcOH); IR
D
(KBr) υmax 3432, 3288, 1673, 1633, 1540, 1446 cm-1; UV (MeOH) λmax
1
201 (ε 11698); H and 13C NMR data (Table 1); FAB-MS m/z 768 (M +
H)+; HRFAB-MS m/z 768.4411 ((M + H)+; calcd for C38H57O8N9,
768.4387).
OL902553Z
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