Absolute configuration of pterocidin, a potent inhibitor of tumor cell invasion from a marine-derived Streptomyces

Article abstract of DOI:10.1016/j.tetlet.2011.11.115

During the course of screening natural products for the inhibitors of tumor cell invasion, pterocidin, a linear polyketide with a δ-lactone terminus, was rediscovered from a Streptomyces strain of a marine sediment-origin. A series of J-based configuratio

Full text of DOI:10.1016/j.tetlet.2011.11.115

Tetrahedron Letters 53 (2012) 654–656
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Tetrahedron Letters
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Absolute configuration of pterocidin, a potent inhibitor of tumor cell
invasion from a marine-derived Streptomyces
Yasuhiro Igarashi a, , Daisuke Asano a, Kazuo Furihata b, Naoya Oku a, Satoshi Miyanaga c,
⇑
Hiroaki Sakurai c, Ikuo Saiki c
a Biotechnology Research Center, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan
b Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
c Department of Bioscience, Institute of Natural Medicine, Toyama University, 2630 Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
During the course of screening natural products for the inhibitors of tumor cell invasion, pterocidin, a lin-
ear polyketide with a d-lactone terminus, was rediscovered from a Streptomyces strain of a marine sed-
iment-origin. A series of J-based configuration analyses and NOESY analysis, coupled with chemical
derivatization and chiral anisotropy analysis, established the absolute stereochemistry of five asymmetric
centers in this compound.
Received 28 October 2011
Revised 22 November 2011
Accepted 24 November 2011
Available online 28 November 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Pterocidin
Absolute configuration
Invasion inhibitor
Streptomyces
Actinomycetes, especially those belonging to the genus Strepto-
myces have been an unparalleled rich source of bioactive polyke-
tides.1,2 We recently reported the discovery of pterocidin (1), a
cytotoxic compound produced by Streptomyces hygroscopicus TP-
A0451 isolated from a stem of bracken Pteridium aquilinum.3 1 is
by silica gel and ODS column chromatographies, followed by HPLC
purification to yield 36 mg of pterocidin (1). Its spectroscopic data
were fully consistent with those previously reported.3
Stereochemical analysis of 1 was started with MTPA derivatiza-
tion.11 Esterification of 1 with (R)- and (S)-MTPA acids by DCC cou-
pling yielded (R)- and (S)-MTPA esters (2a and 2b), respectively.12,13
a linear polyketide featured by an
a,b-unsaturated d-lactone ring
on its terminus and four almost contiguous substituents in the cen-
ter of a highly unsaturated aliphatic chain. Although the former
feature is not uncommon in natural products, 1 is the first to have
a methoxy substitution on the lactone moiety in this class. In our
previous study, stereochemical assignment was hampered by the
limited availability of this material and the poor reproducibility
of production culture. During the course of our continuing effort
toward the discovery of new inhibitors of tumor cell invasion from
natural products,4–8 another Streptomyces strain obtained from a
marine sediment sample was found to produce a substantial
amount of 1 that enabled us to establish the absolute configura-
tions of all five asymmetric centers (C-4, C-5, C-10, C-12, and
C-13) present in this molecule (Fig. 1).
In the 1H NMR of 2a and 2b, positive
D
dS–R values were observed for
the protons from H-15 to H3-19, while negative dS–R values were
D
observed for the protons from H-9 to H-12 and 22-Me (Fig. 2). These
data established the absolute configuration at C-13 as S.
20
OMe
21
OMe
22
12
23
OMe
6
8
16
18
4
5
7
9
13
17
19
10
11
14
15
3
2
O
OH
1
pterocidin (1)
O
The producing strain Streptomyces sp. TP-A0879 was isolated
from a marine sediment sample collected at a depth ꢀ44.5 m in
Otsuchi Bay, Iwate, Japan.9 The strain was cultured in A11M med-
ium10 (7 L), and the whole culture broth was extracted with 1-
butanol. The crude extract (24.6 g) was consecutively fractionated
OMe
OMe
O
O
O
OH
3
⇑
Corresponding author. Tel.: +81 766 56 7500; fax: +81 766 56 2498.
E-mail address: yas@pu-toyama.ac.jp (Y. Igarashi).
Figure 1. Structures of pterocidin (1) and 14-O-demethylpterocidin (3).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.11.115

Y. Igarashi et al. / Tetrahedron Letters 53 (2012) 654–656
655
-0.05
OMe
OMe
OMe
OMe
3J (H4, H5) = 4.2 Hz
2J (C4, H5) < 2.0 Hz*
2J (C5, H4) < 2.0 Hz*
O
C6
+0.06 +0.02
+0.04
-0.04
-0.05
C5
-0.01
C3
H4
+0.13 +0.08
+0.01
-0.02
H5
O
O
OR
NOE
2a :
R = (R)-MTPA
Figure 4. Configuration of C-4 and C-5 and coupling constants for C-4–C-5 in 1.
2b : R = (S)-MTPA
⁄Absolute values are given for JCH coupling constants.
2
Figure 2.
D
dS–R values for MTPA esters (2a and 2b) of 1.
relationship between the C-10 methoxy and the C-22 methyl groups
was established, with C-10/C-13 exhibiting gauche orientation. Sim-
ilarly, the anti orientation of C-11 and C-14 around the C-12–C-13
The relative configuration for the C-12 stereocenter and the 1,3-
methine system C-10–C-12 in 1 was investigated using J-based con-
figuration analysis.14 Heteronuclear long-range coupling constants
3
bond was confirmed on the basis of large JC22,H13 and small
3
3
2JCH and JCH were determined by J-resolved HMBC experi-
2JC13,H12 values as well as the small JH12,H13 value, allowing the
ments.15–17 However, it was impossible to assign a single conformer
assignment of a syn relationship between the C-13 hydroxyl and
the C-22 methyl groups (Fig. 3C). These configurational assignments
were in good agreement with an observed NOE between H-10 and
H-13 (Fig. 3D). The 12R and 10S configurations were thus
established on the basis of the relative configuration of C-12 and
C-13.
3
around the C-11–C-12 bond because all the JCH values between
H-11 protons and C-13 and C-22 indicated intermediate values
(3.6–3.9 Hz) probably due to the presence of more than two stable
rotamers (data not shown). Next, the same method was applied to
14-O-demethylpterocidin (3)18 which was obtained as a degrada-
tion product after the long storage of 1. The diastereomeric
methylene protons H-11a (d 1.72) and H-11b (d 1.61) were stereo-
specifically assigned with respect to H-10, according to the magni-
tude of the coupling constants (Fig. 3A). The gauche orientations of
The coupling constant between H-4 and H-5 (J = 4.2 Hz) as well
as an NOE between these protons in 1 suggested their syn relation-
2
ship. This was supported by the small JCH values for H-4/C-5
(<2 Hz) and H-5/C-4 (<2 Hz) indicating anti orientations of H-4 to
O-5 and H-5 to 4-OMe (Fig. 4). In order to determine the absolute
configurations at these stereocenters, it became necessary to apply
the 1H NMR anisotropy method. 1 was subjected to Ca(BH4)2
reduction19,20 in MeOH to afford diol 4 along with the C-2–C-3-
unsaturated diol in a ratio of 5:2.21,22 4 was then treated with (R)-
or (S)-MPA (methoxyphenylacetic acid)23,24 in the presence of DIC
and DMAP, yielding tris MPA esters 5a and 5b.25,26 Analysis of 1H
3
H-11a and H-11b to C-9 were suggested by the small JC9,H11a and
3JC9,H11b values, while the anti orientations of H-11a to 10-OMe
2
and H-11b to H-10 were provided by the small JH11a,C10 and large
3JH10,H11b values. For the C-11–C-12 bond, in this case, only one rot-
amer was deduced from the observed data (Fig. 3B). The large
homonuclear coupling constant between H-11a and H-12 (9.6 Hz)
suggested an anti relationship between these protons. Small
three-bond C–H coupling constants for H-11a/C-22 and H-11b/C-
22 indicated that these protons were gauche to C-22 methyl group.
The gauche orientation of H-11a to C-13 was suggested by the small
3JC13,H11a value, while the anti orientation of H-11b to C-13 was pro-
vided by the large 3JC13,H11b value. On the basis of these data, the syn
NMR data for these MPA esters allowed the assignment of the
DdR–S
values, which were positive for the protons from H-6 to H-9, while
those for the protons from H-1 to H-4, except for one of the diaste-
reomeric H-3 methylene protons, were negative (Fig. 5). This is
sufficiently consistent to assign the absolute configuration of C-5
as R. On the basis of the relative configuration of C-4 and C-5, the
absolute configuration of C-4 was assigned as R. The absolute con-
figurations of all five asymmetric centers in 1 were determined as
4R, 5R, 10S, 12R, and 13S.
Invasion is a key feature of metastasis because it promotes
tumorigenesis by enabling tumor cells to migrate through the con-
nective tissue surrounding a tumor and enter the circulatory system
and also supporting endothelial cell migration and angiogenesis.27
In addition to antiproliferative property,3 pterocidin (1) was found
to exhibit potent antiinvasive activity at non-cytotoxic concentra-
tions. The invasion of murine colon 26-L5 carcinoma cells across
the Matrigel-fibronectin membrane28 was inhibited by 1 with an
C11-C12
C10-C11
C12
C10
C9
H12
C11
A
B
H10
OMe
H11b
C10
H11b
H11a
C13
Me22
H11a
3J (H10, H11b) = 9.0 Hz
2J (C10, H11b) = 6.2 Hz*
3J (C9, H11b) = 3.4 Hz
3J (H10, H11a) = 4.4 Hz
2J (C10, H11a) < 3.0 Hz*
3J (C9, H11a) < 3.0 Hz
3J (H12, H11b) = 5.2 Hz
3J (C13, H11b) = 5.5 Hz
3J (C22, H11b) = 2.4 Hz
3J (H12, H11a) = 9.6 Hz
3J (C22, H11a) = 3.4 Hz
3J (C13, H11a) < 3.0 Hz
IC50 value of 0.25
up to 7 M. The demethyl derivative 3 showed slightly weaker activ-
ity with an IC50 value of 1.6 M with no cytotoxic effect up to 7 M.
lM, whereas the cytotoxicity was not apparent
l
l
l
In summary, prerocidin (1), a linear polyketide with a d-lactone
terminus, was rediscovered from a marine-derived Streptomyces
C12-C13
C14
OMe
D
H12
Me22
OH
C
-0.17
O
C12
10
OMe
OMe
OMe
H
-0.57
-0.46
H
+0.15
+0.06
+0.04
H13
H
+0.11
+0.02
-0.04
-0.15
+0.02
-0.01
Hb
RO
C11
12
Me
13
-0.10
-0.35
-0.18
-0.18
-0.18
-0.03
+0.52 +0.29
+0.49
+0.14
3J (H12, H13) = 2.0 Hz
3J (C22, H13) = 5.8 Hz
3J (C11, H13) = 3.2 Hz
2J (C13, H12) < 3.0 Hz*
+0.17
Ha
NOE
OH
OR
OR
4 : R = H
5a :
R = (R)-MPA
5b : R = (S)-MPA
Figure 3. Configuration analysis for C-10–C-13 based on coupling constants and
NOE in 3. ⁄Absolute values are given for JCH coupling constants.
Figure 5. DdR–S values for MPA esters (5a and 5b) of 4.
2

656
Y. Igarashi et al. / Tetrahedron Letters 53 (2012) 654–656
2
0.25 mL; 7.9 mg of 3 in 0.25 mL). In the J-resolved HMBC spectra, JCH and 3JCH
strain as an inhibitor of tumor cell invasion, and the absolute con-
figurations of all five asymmetric centers in this molecule were
established on the basis of J-based configuration analyses and
NOESY analysis, coupled with chemical derivatization and chiral
anisotropy analysis. Linear polyketides with a d-lactone terminus
have been reported from various organisms including actinomy-
cetes,29 myxobacteria,30 fungi,31 slime molds,32 marine sponges,33
and colonial ascidians.34 Although a wide range of bioactivity
was reported for this class of natural products, pterocidin (1) rep-
resents the first example of invasion inhibitor, providing a new
template for the development of antiinvasive agents.
values are obtained as absolute values.
18. 14-O-Demethylpterocidin (3): ½a 2D9
ꢁ
ꢀ17 (c 0.18, MeOH); 1H NMR (400 MHz,
CDCl3) d 0.70 (3H, d, J = 6.9 Hz, H-22), 1.00 (3H, t, J = 7.4 Hz, H-19), 1.61 (1H,
ddd, J = 14.0, 9.0, 5.2 Hz, H-11b), 1.72 (1H, ddd, J = 14.0, 9.3, 4.4 Hz, H-11a),
2.07 (2H, m, H-18), 2.32 (1H, m, H-12), 3.19 (2H, d, J = 6.7 Hz, H-15), 3.31 (3H, s,
H-21), 3.43 (3H, s, H-20), 3.79 (1H, ddd, J = 9.0, 8.3, 4.4 Hz, H-10), 4.02 (1H, dd,
J = 4.3, 4.2 Hz, H-4), 4.29 (1H, d, J = 2.0 Hz, H-13), 4.99 (1H, dd, J = 6.7, 4.2 Hz, H-
5), 5.52 (1H, dtt, J = 15.4, 6.7, 1.3 Hz, H-16), 5.63 (1H, m, H-17), 5.65 (1H, dd,
J = 15.2, 8.3 Hz, H-9), 5.90 (1H, dd, J = 15.2, 6.7 Hz, H-6), 6.17 (1H, d, J = 9.9 Hz,
H-2), 6.28 (1H, dd, J = 15.2, 10.5 Hz, H-8), 6.46 (1H, dd, J = 15.2, 10.5 Hz, H-7),
6.95 (1H, dd, J = 9.9, 4.3 Hz, H-3); 13C NMR (100 MHz, CDCl3) d 13.6 (C-19), 13.7
(C-22), 25.8 (C-18), 31.8 (C-12), 39.6 (C-11), 42.2 (C-15), 56.6 (C-21), 57.4 (C-
20), 71.4 (C-4), 77.4 (C-13), 79.4 (C-10), 79.9 (C-5), 119.9 (C-16), 123.5 (C-2),
126.0 (C-6), 131.4 (C-8), 134.2 (C-7), 136.2 (C-9), 137.6 (C-17), 143.2 (C-3),
162.7 (C-1), 211.1 (C-14); HR-ESITOFMS m/z 391.2127 [MꢀH]ꢀ (calcd for
Acknowledgments
C
22H31O6 391.2126).
19. Mori, K.; Watanabe, H. Pure Appl. Chem. 1989, 61, 543.
20. Mori, K.; Takikawa, H.; Kido, M. J. Chem. Soc., Perkin Trans. 1 1993, 169.
We acknowledge the Atmosphere and Ocean Research Institute,
the University of Tokyo and Dr. C. Imada at Tokyo University of
Marine Science and Technology for assistance to sediment sam-
pling. This research was supported in part by a research Grant from
the Atmosphere and Ocean Research Institute, University of Tokyo
to Y.I.
21. Calcium borohydride is used to synthesize diols from a,b-unsaturated lactones
without reducing the conjugated C–C double bond.19,20 However, in case of
pterocidin, reduction with Ca(BH4)2 gave the C-2–C-3-reduced diol (4) as a major
product. The ratio of 4 to C-2–C-3-unsaturated diol varied depending on the
reaction solvent. The ratio was 5:2 in MeOH, 2:1 in 2-propanol, and 1:1 in THF.
22. Reduction of 1 to yield 4: Calcium borohydride bis(tetrahydrofuran) (1.6 mg,
7.4 lmol) was added to a solution of 1 (1.5 mg, 3.7 lmol) in MeOH (200 ll) at
room temperature, and the reaction mixture was stirred for 3 h. The reaction
was quenched by the addition of several drops of water, and the mixture was
extracted with EtOAc. The EtOAc layer was concentrated under reduced
pressure to give a mixture of 4 and C-2–C-3-unsaturated diol in a ratio of 5:2
(1.1 mg, 72% yield). This mixture was employed to the next reaction without
further purification. 4:1H NMR (500 MHz, CDCl3) d 0.90 (3H, d, J = 7.0 Hz, H-22),
1.02 (3H, t, J = 7.4 Hz , H-19), 1.57 (1H, m, H-3), 1.58 (1H, m, H-11), 1.66 (2H, m,
H-2), 1.68 (1H, m, H-11), 1.71 (1H, m, H-3), 1.96 (1H, m, H-12), 2.13 (2H, m, H-
18), 3.17 (1H, m, H-4), 3.27 (3H, s), 3.47 (3H, s), 3.67 (2H, m, H-1), 3.70 (3H, s),
3.73 (1H, m, H-10), 4.08 (1H, m, H-13), 4.13 (1H, m, H-5), 5.54 (1H, dd, J = 15.3,
8.2 Hz , H-9), 5.59 (1H, d, J = 10.9 Hz , H-15), 5.67 (1H, dt, J = 15.2, 6.6 Hz, H-17),
5.69 (1H, dd, J = 15.2, 7.2 Hz, H-6), 6.21 (1H, dd, J = 15.4, 10.5 Hz, H-8), 6.35 (1H,
dd, J = 15.2, 10.8 Hz, H-16), 6.35 (1H, dd, J = 15.2, 10.6 Hz, H-7); HR-ESITOFMS
m/z 435.2722 [M+Na]+ (calcd for C23H40O6Na 435.2717).
References and notes
1. Bérdy, J. J. Antibiot. 2005, 58, 1.
2. Goodfellow, M.; Fiedler, H.-P. Antonie van Leeuwenhoek 2010, 98, 119.
3. Igarashi, Y.; Miura, S.; Fujita, T.; Furumai, T. J. Antibiot. 2006, 59, 193.
4. Miyanaga, S.; Obata, T.; Onaka, H.; Fujita, T.; Saitoh, N.; Sakurai, H.; Saiki, I.;
Furumai, T.; Igarashi, Y. J. Antibiot. 2006, 59, 698.
5. Igarashi, Y.; Trujillo, M. E.; Martinez-Molina, E.; Yanase, S.; Miyanaga, S.; Obata,
T.; Sakurai, H.; Saiki, I.; Fujita, T.; Furumai, T. Bioorg. Med. Chem. Lett. 2007, 17,
3702.
6. Igarashi, Y.; Mogi, T.; Yanase, S.; Miyanaga, S.; Fujita, T.; Sakurai, H.; Saiki, I.;
Ohsaki, A. J. Nat. Prod. 2009, 72, 980.
7. Igarashi, Y.; Kim, Y.; In, Y.; Ishida, T.; Kan, Y.; Fujita, T.; Iwashita, T.; Tabata, H.;
Onaka, H.; Furumai, T. Org. Lett. 2010, 12, 3402.
8. Igarashi, Y.; Yu, L.; Miyanaga, S.; Fukuda, T.; Saitoh, N.; Sakurai, H.; Saiki, I.;
Alonso-Vega, P.; Trujillo, M. E. J. Nat. Prod. 2010, 73, 1943.
9. The bacterial strain TP-A0879 was isolated from a sediment sample collected at
a depth ꢀ44.5 m in Otsuchi Bay, Iwate, Japan in 2006 by using Smith–McIntyre
grab. The strain was identified as a member of the genus Streptomyces on the
basis of 98.1% 16S rRNA gene sequence (1409 nucleotides; DDBJ accession
number AB666472) identity with Streptomyces hygroscopicus subsp.
crystallogenes NBRC 16551 (accession number AB184723).
10. Igarashi, Y.; Ogura, H.; Furihata, K.; Oku, N.; Indananda, C.; Thamchaipenet, A. J.
Nat. Prod. 2011, 74, 670.
23. MTPA derivatization using MTPA-Cl or MTPA acid/DCC/DMAP was
unsuccessful due to the elimination of the MTPA acyloxy group from the
product.
24. Trost, B. M.; Belletire, J. L.; Godleski, S.; McDougal, P. G.; Balkovec, J. M.;
Baldwin, J. J.; Christy, M. E.; Ponticello, G. S.; Varga, S. L.; Springer, J. P. J. Org.
Chem. 1986, 51, 2370.
25. Tris-(R)-MPA ester of 4 (5a): To a solution of 4 (1.1 mg, 2.7
(100 l) was added (R)-MPA acid (1.8 mg, 11 mol), DIC (1.7 mg, 14
DMAP (0.1 mg, 0.8 mol) at room temperature. After standing for 3 h, the
l
mol) in dry CH2Cl2
l
l
l
mol), and
l
reaction mixture was concentrated under reduced pressure, and the residue
was purified by silica gel column chromatography (n-hexane/EtOAc = 1:0–1:1)
to give tris-(R)-MPA ester 5a (0.5 mg, 21% yield): 1H NMR (500 MHz, CDCl3) d
0.66 (3H, d, J = 6.8 Hz, H-22), 1.01 (3H, t, J = 7.5 Hz, H-19), 1.07 (1H, m, H-11),
1.07 (1H, m, H-2), 1.15 (1H, m, H-2), 1.23 (1H, m, H-11), 1.33 (1H, m, H-3), 1.52
(1H, m, H-3), 1.96 (1H, m, H-12), 2.10 (2H, m, H-18), 2.99 (1H, m, H-4), 3.44
(1H, m, H-10), 3.90 (1H, dt, J = 13.4, 6.9 Hz, H-1), 3.94 (1H, dt, J = 13.4, 6.5 Hz,
H-1), 5.24 (1H, d, J = 5.3 Hz, H-13), 5.30 (1H, dd, J = 15.3, 7.9 Hz, H-9), 5.37 (1H,
dd, J = 7.0, 5.6 Hz, H-5), 5.39 (1H, d, J = 11 Hz, H-15), 5.56 (1H, dd, J = 15.3,
7.0 Hz, H-6), 5.61 (1H, dt, J = 15.4, 6.6 Hz, H-17), 5.98 (1H, dd, J = 15.3, 10.5 Hz,
H-8), 6.16 (1H, dd, J = 15.3, 10.5 Hz, H-7), 6.27 (1H, dd, J = 15.4, 11 Hz, H-16);
HR-ESITOFMS m/z 855.4319 [MꢀH]ꢀ (calcd for C50H63O12 855.4325).
11. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092.
12. (R)-MTPA ester of 1 (2a): To a solution of 1 (5.0 mg, 12
(200 L) was add (R)-MTPA acid (5.6 mg, 24 mol), DCC (5.0 mg, 24
DMAP (0.7 mg, mol) at room temperature. After standing for 4 h, the
l
mol) in dry CH2Cl2
l
l
lmol), and
6
l
reaction mixture was concentrated under reduced pressure, and the residue
was purified by silica gel column chromatography (n-hexane/EtOAc = 1:0–1:1)
to give (R)-MTPA ester 2a (7.3 mg, 95% yield): 1H NMR (500 MHz, CDCl3) d 0.93
(3H, d, J = 7.0 Hz, H-22), 1.02 (3H, t, J = 7.5 Hz, H-19), 1.52 (1H, m, H-11), 1.61
(1H, m, H-11), 2.07 (1H, m, H-12), 2.09 (2H, m, H-18), 3.25 (3H, s), 3.43 (3H, s),
3.57 (3H, s), 3.65 (1H, m, H-10), 4.03 (1H, dd, J = 4.3, 3.8 Hz, H-4), 4.99 (1H, dd,
J = 6.7, 3.8 Hz, H-5), 5.29 (1H, d, J = 10.8 Hz, H-15), 5.36 (1H, d, J = 5.6 Hz, H-13),
5.54 (1H, dd, J = 15.4, 7.9 Hz, H-9), 5.54 (1H, dt, J = 15.5, 7.6 Hz, H-17), 5.89 (1H,
dd, J = 15.4, 6.7 Hz, H-6), 6.17 (1H, d, J = 9.9 Hz, H-2), 6.20 (1H, dd, J = 15.4,
10.7 Hz, H-8), 6.23 (1H, dd, J = 15.5, 10.8 Hz, H-16), 6.43 (1H, dd, J = 15.4,
10.7 Hz, H-7), 6.95 (1H, dd, J = 9.9, 4.3 Hz, H-3); HR-ESITOFMS m/z 645. 2640
[M+Na]+ (calcd for C33H41F3Na1O8 645.2646).
26. Tris-(S)-MPA ester of 4 (5b): In the same manner as described for 5a, 5b was
prepared from 4 and (S)-MPA acid: 1H NMR (500 MHz, CDCl3) d 0.83 (3H, d,
J = 6.8 Hz, H-22), 0.97 (3H, t, J = 7.5 Hz, H-19), 1.35 (2H, m, H-3), 1.41 (2H, m, H-
11), 1.61 (1H, m, H-2), 1.71 (1H, m, H-2), 1.97 (1H, m, H-12), 2.04 (2H, m, H-
18), 3.16 (1H, m, H-4), 3.54 (1H, m, H-10), 4.08 (1H, dt, J = 11.0, 6.8 Hz, H-1),
4.12 (1H, dt, J = 11.0, 6.6 Hz, H-1), 4.88 (1H, d, J = 10.9 Hz, H-15), 5.16 (1H, dd,
J = 15.3, 8.0 Hz, H-9), 5.22 (1H, d, J = 5.4 Hz, H-13), 5.30 (1H, dt, J = 15.4, 6.5 Hz,
H-17), 5.42 (1H, dd, J = 6.0, 5.8 Hz, H-5), 5.45 (1H, dt, J = 14.6, 6.0 Hz, H-6), 5.67
(1H, dd, J = 14.6, 10.5 Hz, H-7), 5.96 (1H, dd, J = 15.3, 10.5 Hz, H-8), 6.12 (1H, dd,
J = 15.4, 10.9 Hz, H-16); HR-ESITOFMS m/z 879.4301 [M+Na]+ (calcd for
13. (S)-MTPA ester of 1 (2b): In the same manner as described for 2a, 2b was
prepared from pterocidin and (S)-MTPA acid: 1H NMR (500 MHz, CDCl3) d 0.88
(3H, d, J = 6.8 Hz, H-22), 1.03 (3H, t, J = 7.5 Hz, H-19), 1.60 (1H, m, H-11), 1.70
(1H, m, H-11), 2.05 (1H, m, H-12), 2.12 (2H, m, H-18), 3.24 (3H, s), 3.43 (3H, s),
3.64 (1H, m, H-10), 3.67 (3H, s), 4.03 (1H, dd, J = 4.3, 4.1 Hz, H-4), 4.99 (1H, dd,
J = 6.3, 4.1 Hz, H-5), 5.40 (1H, d, J = 6.0 Hz, H-13), 5.42 (1H, d, J = 10.7 Hz, H-15),
5.52 (1H, dd, J = 15.2, 8.0 Hz, H-9), 5.62 (1H, dd, J = 15.5, 6.5 Hz, H-17), 5.89 (1H,
dd, J = 15.5, 6.3 Hz, H-6), 6.12 (1H, d, J = 9.9 Hz, H-2), 6.22 (1H, dd, J = 15.2,
10.5 Hz, H-8), 6.29 (1H, ddt, J = 15.5, 10.7, 1.6 Hz, H-16), 6.43 (1H, dd, J = 15.5,
10.5 Hz, H-7), 6.94 (1H, dd, J = 9.9, 4.3 Hz, H-3); HR-ESITOFMS m/z 645.2646
[M+Na]+ (calcd for C33H41F3O8Na 645.2646).
C
50H64O12Na 879.4290).
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at 20 °C using a microtube (Shigemi Inc., Japan) in CDCl3 (9.0 mg of 1 in