Jomthonic acid A, a modified amino acid from a soil-derived streptomyces

Article abstract of DOI:10.1021/np200742c

Jomthonic acid A (1), a new modified amino acid, was isolated from the culture broth of a soil-derived actinomycete of the genus Streptomyces. The structure and absolute configuration of 1 were determined by spectroscopic analyses and chemical conversion. Jomthonic acid A (1) induced differentiation of preadipocytes into mature adipocytes at 2-50 μM.

Full text of DOI:10.1021/np200742c

Note
pubs.acs.org/jnp
Jomthonic Acid A, a Modified Amino Acid from a Soil-Derived
Streptomyces
Yasuhiro Igarashi,*,† Linkai Yu,† Megumi Ikeda,‡ Tsutomu Oikawa,‡ Shigeru Kitani,§ Takuya Nihira,§,⊥
Baatar Bayanmunkh,⊥ and Watanalai Panbangred⊥,#
†Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
‡School of Nutrition and Dietetics, Kanagawa University of Human Services, 1-10-1 Heisei-cho, Yokosuka, Kanagawa 238-8522, Japan
§International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan
⊥Mahidol University and Osaka University Collaborative Research Center on Bioscience and Biotechnology, Bangkok 10400,
Thailand
#Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
S
* Supporting Information
ABSTRACT: Jomthonic acid A (1), a new modified amino acid, was isolated from the culture
broth of a soil-derived actinomycete of the genus Streptomyces. The structure and absolute
configuration of 1 were determined by spectroscopic analyses and chemical conversion.
Jomthonic acid A (1) induced differentiation of preadipocytes into mature adipocytes at 2−50
μM.
ctinomycetes are Gram-positive bacteria that have served
as an important source of drug discovery owing to their
entiation of preadipocytes into adipocytes. Herein, we report
the isolation, structure determination, and biological properties
of 1.
A
unparalleled ability to produce structurally diverse secondary
metabolites. Despite the recent decline in drug discovery efforts
from microbial metabolites, Streptomyces is still believed to be a
rich source of new and useful compounds, and up to 40% of
known microbial metabolites are derived from this group.1,2
Even after the intensive isolation and screening of this group
over several decades, it is estimated that less than 10% of its
potential in secondary metabolite biosynthesis has been
realized.3 Genome analyses have shown that Streptomyces
coelicolor contains 21 biosynthetic gene clusters, and
Streptomyces avermitilis contains 25 biosynthetic gene clusters
for secondary metabolites. The predicted number of gene
clusters in these two strains, however, exceeds the number of
reported compounds, suggesting that additional rare metabo-
lites await to be discovered.4,5
In our continuing search for structurally novel secondary
metabolites from Streptomyces,6−8 strain BB47 isolated from a
soil sample collected in Bangkok, Thailand, was chosen for
chemical investigation because of its potent antagonism against
Colletotrichum, a causative agent of anthracnose disease in
crops.9 The strain was cultured in A-3 M liquid medium, and
the whole culture broth was extracted with 1-butanol. HPLC/
UV analysis of the extract and subsequent comparison to our
in-house metabolite database suggested that the antifungal
principles were clethramycin-type hexaenes10,11 and pierici-
dins.12 In addition to these metabolites, an unknown metabolite
showing an UV absorption band at 264 nm was detected
although it lacked antifungal activity. HPLC/UV-guided
purification from the extract led to the isolation of a new
modified amino acid, jomthonic acid A (1). During diverse
biological evaluation, 1 was found to promote the differ-
Jomthonic acid A (1) was obtained as a pale yellow oil that
analyzed for a molecular formula of C22H29NO5 (9 degrees of
unsaturation) by interpretation of HR-ESITOFMS. This
1
molecular formula was corroborated by H and 13C NMR
spectral data (Table 1). Analysis of the combined 1D and 2D
NMR data established that 1 possessed eight olefinic methines,
two sp2 quaternary and three carbonyl carbons, four sp3
methines, and five methyls, in addition to two exchangeable
protons. The IR absorptions at 1717 and 1656 cm−1
demonstrated the presence of carbonyl functionalities, which
was confirmed by the presence of resonances at δ 166.9, 171.3,
and 177.5 in the 13C NMR spectrum. The nine degrees of
unsaturation inherent in the molecular formula of 1, coupled
with the data showing the presence of three carbonyl and 10
olefinic carbons, indicated that 1 must possess one ring.
Further analysis of 1H−1H COSY and HMBC spectra
provided three substructures. The first was an unsaturated fatty
acid containing two small COSY-defined fragments. The first
fragment was a two-carbon unit consisting of a terminal methyl
Received: September 15, 2011
Published: May 14, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
986
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Journal of Natural Products
Note
Table 1. 1H and 13C NMR Data for Jomthonic Acid A (1) in
CDCl3
C-8, to complete the planar structure of 1 as depicted in Figure
1.
a
b
bc
,
position
δC
δH mult (J in Hz)
HMBC
2, 3
1
14.5, CH3
135.6, CH
133.4, qC
146.8, CH
116.9, CH
166.9, qC
11.9, CH3
171.3, qC
57.8, CH
43.3, CH
141.4, qC
127.9, CH
128.4, CH
127.2, CH
128.4, CH
127.9, CH
17.8, CH3
16.1, CH3
72.5, CH
44.3, CH
177.5, qC
12.3, CH3
1.79, d (7.0)
5.93, q (7.0)
2
1, 7
3
4
7.23, d (15.4)
5.79, d (15.4)
2, 5, 6, 7
3, 6
5
6
7
1.74, s
2, 3, 4
Figure 1. 1H−1H COSY, key HMBC, and key NOESY correlations for
1.
8
9
4.88, dd (8.9, 8.0)
3.16, dq (8.0, 7.1)
6, 8, 10, 11, 17
8, 9, 11, 12, 17
10
11
12
13
14
15
16
17
18
19
20
21
22
9-NH
The absolute stereochemistry of 1 was assigned by
application of Marfey’s analysis14 and the chiral anisotropy
method.15 First, the relative configuration of the β-methyl-
phenylalanine (βMePhe) residue was determined by HPLC
analysis. A commercially available diastereomeric mixture of
βMe-DL-Phe was separated into the (2RS,3SR)-isomer (syn-
form) and the (2RS,3RS)-isomer (anti-form) by preparative
HPLC, and their relative configurations were assigned by
d
7.22
10, 14
7.28, t (7.8)
11, 15
d
7.22
12, 16
7.28, t (7.8)
11, 13
d
7.22
10, 14
1.39, d (7.1)
9, 10, 11
19, 20
0.88, d (6.4)
4.98, dq (7.1, 6.4)
2.59, dq (7.1, 7.2)
8, 20, 21
18, 19, 21, 22
16
1
comparing the H NMR data with reported values. The acid
hydrolysate of 1 was compared with these standards, and the
βMePhe residue was determined to have a (2RS,3SR)-
configuration. In a second step, the hydrolysate was derivatized
using 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) and
compared with the standard (2RS,3SR)-βMePhe that was
similarly derivatized with L-FDLA. The βMePhe residue was
assigned as a (2S, 3R)-isomer. The remaining stereocenters at
C-19 and C-20 of 1 were determined using MTPA and PGME
methods17,18 (Figure 2). Since the recovery of the 3-hydroxy-2-
methylbutanoate residue was hampered by difficulties with
detection, 1 was tagged with a fluorescent chromophore, 1-
pyrenemethylamine, to give the corresponding amide 2.
Alkaline hydrolysis of 2 gave 3, which was easily detected by
TLC. Esterification of 3 with (R)- and (S)-MTPA acid in the
presence of DCC and DMAP gave the (R)- and (S)-MTPA
esters 4 and 5, respectively. The Δδ value distribution pattern
suggested the R configuration at C-19. The C-20 stereo-
chemistry was assigned as R on the basis of the Δδ values of
(R)- and (S)-PGME amides (6 and 7), which showed negative
values for H-9, H-10, H-18, and H-19.
The biological activity of jomthonic acid A (1) is still under
investigation. Initial screening showed that 1 was active in an
assay designed to detect antidiabetic and antiatherogenic
activities using mouse ST-13 preadiopocytes.19 At 2 to 50
μM 1 displayed inducing activity of preadipocyte differentiation
into mature adipocytes. At the highest concentration about 80%
of preadipocytes were differentiated into adipocytes. Preadipo-
cyte differentiation is associated with the production of
adipocytokines, proteinogenic hormones secreted by mature
adipocytes and beneficial to relieve lifestyle deseases.20 1 was
inactive in a cancer-cell cytotoxicity assay (IC50 >100 μM
against MCF7 human breast cancer cells) and in an
antimicrobial assay (MIC >50 μg/mL against Escherichia coli,
Micrococcus luteus, and Candida albicans).
1.08, d (7.2)
6.35, d (8.9)
19, 20, 21
6
a
b
c
Recorded at 100 MHz. Recorded at 500 MHz. HMBC correlations
d
are from proton(s) stated to the indicated carbon. Overlapping
signals.
group (H3-1) attached to a vinyl methine (H-2). The second
fragment was also a two-carbon unit comprising two vinyl
protons (H-4 and H-5) mutually coupled with a large coupling
constant (15.5 Hz), indicating the E geometry at C-4 and C-5.
These two fragments were connected to the quaternary carbon
C-3 on the basis of HMBC correlations from the methyl proton
at δH 1.75 (H3-7) to C-2, C-3, and C-4. This unit was then
expanded to include the carbonyl carbon C-6 based on HMBC
correlations from H-4 and H-5 to this carbon, providing a 4-
methyl-2,4-hexadienoate substructure. This was consistent with
the UV spectrum, which displayed an absorption band at λmax
264 nm.13 The E geometry for the C-2−C-3 double bond was
assigned by the observation of NOEs between H-2 and H-4 and
between H3-7 and H-5. The second part was elucidated starting
from an NH proton at δH 6.35 that showed a series of COSY
correlations to the methyl proton H3-17. A monosubstituted
benzene ring was established by analysis of the H, 13C, and
1
HSQC spectra, showing relatively intense signals at δC 127.9
and 128.4, each accounting for two proton-bearing carbons (C-
12/C-16 and C-13/C-15, respectively), and resonances at δH
7.22 and 7.28 accounting for five protons. The aromatic part
was connected to C-10 on the basis of HMBC correlations
from H3-17 and H-9 to C-11 and from H-10 to C-12 and C-16.
HMBC correlations from H-9 and H-10 allowed the
connection of carbonyl carbon C-8 to C-9, establishing this
substructure as a phenylalanine bearing a methyl substitution at
the β-position. The third substructure was a 3-hydroxy-2-
methylbutanoic acid, which was deduced from a series of COSY
correlations from H3-18 to H3-22 and HMBC correlations from
H-19 and H3-22 to carbonyl carbon C-21. These three
substructures were combined on the basis of HMBC
correlations from 9-NH and H-9 to C-6 and from H-19 to
Jomthonic acid A (1) is an interesting modified amino acid
that contains rare structural features, and no related metabolites
have been reported. The unsaturated fatty acid moiety 4-
methyl-2E,4E-hexadienoate present in 1 has been found only in
salinamide C21 from Streptomyces and daldinin F13 from
Hypoxylon. β-Methylphenylalanine is a rare amino acid reported
987
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Journal of Natural Products
Note
1
Figure 2. Preparation of 3 and H NMR ΔδS−R values for MTPA esters (4 and 5) of 3 and PGME amides (6 and 7) of 1.
from two classes of microbial metabolites, bottromycins22 from
Streptomyces and AK toxins23 from Alternaria. While these
compounds contain the (2S,3S)-isomer of this unusual amino
acid, 1 is the first natural product that contains the (2S,3R)-
isomer. The discovery of jomthonic acid A (1) provides
additional evidence that Streptomyces still has great potential as
a source of novel secondary metabolites. Further structural
analysis of minor analogues of 1 present in the culture extract is
currently in progress.
MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v). Fraction 4 was
concentrated to provide 1.4 g of brown oil, which was further purified
by reversed-phase ODS column chromatography with a gradient of
MeCN/0.15% KH2PO4 (pH 3.5) (2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2
v/v). Fraction 6 was evaporated, the remaining aqueous solution was
extracted with EtOAc, and the organic layer was concentrated to give a
brown solid (114 mg). Final purification was achieved by repeated C18
RP HPLC using a Cosmosil 5C18-AR-II column (Nacalai Tesque Inc.,
20 × 250 mm) with MeCN/0.15% KH2PO4 (pH 3.5) (50:50),
followed by evaporation and extraction with EtOAc, yielding
jomthonic acid A (1, 64 mg).
Jomthonic acid A (1): pale yellow oil; [α]24D −3.0 (c 0.80, MeOH);
UV (MeOH) λmax (log ε) 264 (4.72) nm; IR (ATR) νmax 3321, 2981,
1717, 1656 cm−1; 1H and 13C NMR data, see Table 1; HR-
ESITOFMS [M − H]− 386.1973 (calcd for C22H28NO5 386.1973).
Stereochemical Determination of β-Methylphenylalanine
Residue. Jomthonic acid A (1, 1 mg) was hydrolyzed at 140 °C
with 6 N HCl for 20 h. The dried hydrolysate and standards of syn-
and anti-forms of racemic β-methylphenylalanine (β-MePhe)16 were
analyzed by HPLC using an XTerra RP18 column (Waters, 5 μm, 4.6 ×
250 mm), starting with 15% MeOH/85% 10 mM NH4HCO3 followed
by a gradient elution profile to 25% MeOH/75% 10 mM NH4HCO3
over 12 min at a flow rate of 1.0 mL/min, with monitoring at 254 nm.
Retention times for the standards were 6.46 min for (2RS,3SR)-β-
MePhe (syn-form) and 6.87 min for (2RS,3RS)-β-MePhe (anti-form),
while the hydrolysate gave a peak at 6.44 min.
To the remaining hydrolysate was added a 0.1 M NaHCO3 solution
(20 μL) and a solution of 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide
(L-FDLA) in acetone (1%, 25 μL). The reaction vial was sealed and
incubated at 35 °C for 1.5 h. To quench the reaction, 20 μL of 1 N
HCl was added and then diluted with 800 μL of MeCN. L-FDLA
derivatives of (2RS,3SR)-β-MePhe were prepared in the same manner
as described above. The Marfey’s derivatives of the hydrolysate and
standards were analyzed by HPLC using an XTerra RP18 column
(Waters, 5 μm, 4.6 × 250 mm). The reaction mixtures were analyzed
starting with 40% MeCN/60% 0.15% KH2PO4 (pH 3.5) followed by a
gradient elution profile to 80% MeCN/20% 0.15% KH2PO4 over 30
min at a flow rate of 1.0 mL/min, monitoring at 340 nm. Retention
times for the amino acid standards were 14.5 min for (2S,3R)-β-L-
MePhe and 15.4 min for (2R,3S)-β-D-MePhe, while the hydrolysate
gave a peak at 14.5 min.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation was
measured using a JASCO DIP-3000 polarimeter. The UV spectrum
was recorded on a Hitachi U-3210 spectrophotometer. The IR
spectrum was measured on a Perkin-Elmer Spectrum 100. NMR
spectra were obtained on a Bruker AVANCE 400 or a Bruker
AVANCE 500 spectrometer and referenced to residual solvent signals
(δH 7.27, δC 77.0). HR-ESITOFMS were recorded on a Bruker
microTOF Focus. Silica gel 60-C18 (Nacalai Tesque, 250−350 mesh)
was used for ODS column chromatography.
Microorganism. Strain BB47 was isolated from a soil sample
collected in Bangkok, Thailand. The strain was identified as a member
of the genus Streptomyces on the basis of 98.7% similarity in the 16S
rRNA gene sequence (1425 nucleotides; GenBank accession number
HM051280) to Streptomyces catenulae ISP 5258 (accession number
AY999778).
Fermentation. Strain BB47 cultured on a Bn-2 slant [soluble
starch 0.5%, glucose 0.5%, meat extract (Kyokuto Pharmaceutical
Industrial Co., Ltd.) 0.1%, yeast extract (Difco Laboratories) 0.1%,
NZ-case (Wako Chemicals USA, Inc.) 0.2%, NaCl 0.2%, CaCO3 0.1%,
agar 1.5%] was inoculated into 500 mL K-1 flasks each containing 100
mL of the V-22 seed medium consisting of soluble starch 1%, glucose
0.5%, NZ-case 0.3%, yeast extract 0.2%, Tryptone (Difco Laboratories)
0.5%, K2HPO4 0.1%, MgSO4·7H2O 0.05%, and CaCO3 0.3% (pH 7.0).
The flasks were placed on a rotary shaker (200 rpm) at 30 °C for 4
days. The seed culture (3 mL) was transferred into 500 mL K-1 flasks
each containing 100 mL of the A-3 M production medium consisting
of glucose 0.5%, glycerol 2%, soluble starch 2%, Pharmamedia
(Traders Protein) 1.5%, yeast extract 0.3%, and Diaion HP-20
(Mitsubishi Chemical Co.) 1%. The pH of the medium was adjusted
to 7.0 before sterilization. The inoculated flasks were placed on a
rotary shaker (200 rpm) at 30 °C for 6 days.
Extraction and Isolation. At the end of the fermentation period,
100 mL of 1-butanol was added to each flask, and they were allowed to
shake for 1 h. The mixture was centrifuged at 5000 rpm for 10 min,
and the organic layer was separated from the aqueous layer containing
the mycelium. Evaporation of the solvent gave approximately 12.5 g of
extract from 2 L of culture. The crude extract (12.5 g) was subjected to
silica gel column chromatography with a step gradient of CHCl3/
1-Pyrenemethylamide of 1 (2). Jomthonic acid A (1, 13 mg, 0.034
mmol) was treated with 1-pyrenemethylamine hydrochloride (25 mg,
0.093 mmol) in dry DMF (2.5 mL) containing DMAP (4.0 mg, 0.034
mmol) and EDAC (33 mg, 0.17 mmol) for 18 h at room temperature.
The reaction mixture was diluted with ice−water and extracted with
EtOAc. The organic layer was concentrated to dryness, and the residue
was purified by silica gel column chromatography (n-hexane/EtOAc,
1:0−1:1) to give 2 (5.6 mg, 27% yield): 1H NMR (500 MHz, CDCl3)
δ 0.71 (3H, d, J = 6.5 Hz, H-18), 1.08 (3H, d, J = 7.0 Hz, H-22), 1.09
(3H, d, J = 7.1 Hz, H-17), 1.69 (3H, s, H-7), 1.73 (3H, d, J = 7.1 Hz,
988
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Journal of Natural Products
Note
H-1), 2.38 (1H, dq, J = 5.4, 7.0 Hz, H-20), 2.88 (1H, dq, J = 9.0, 7.1
Hz, H-10), 4.30 (1H, dd, J = 9.0, 8.5 Hz, H-9), 4.84 (1H, dq, J = 5.4,
6.5 Hz, H-19), 4.95 (1H, dd, J = 14.5, 4.3 Hz, NHCH2), 5.38 (1H, dd,
J = 14.5, 6.9 Hz, NHCH2), 5.69 (1H, d, J = 15.3 Hz, H-5), 5.82 (1H, d,
J = 8.5 Hz, 9-NH), 5.83 (1H, q, J = 7.1 Hz, H-2), 6.56 (1H, dd, J = 6.9,
4.3 Hz, NHCH2), 6.76 (2H, d, J = 7.4 Hz, H-12 and H-16), 6.96 (2H,
dd, J = 7.4, 7.4 Hz, H-13 and H-15), 7.06 (1H, t, J = 7.4 Hz, H-14),
7.16 (1H, d, J = 15.3 Hz, H-4), 8.01−8.07 (4H, m), 8.16−8.23 (4H,
m), 8.36 (1H, d, J = 9.3 Hz); 13C NMR (100 MHz, CDCl3) δ 11.8 (C-
7), 13.8 (C-22), 14.4 (C-1), 17.0 (C-18), 17.9 (C-17), 41.6 (NHCH2),
42.6 (C-10), 46.7 (C-20), 58.3 (C-9), 72.6 (C-19), 116.6 (C-5), 123.4,
124.78, 124.81, 125.0, 125.2, 125.3, 126.0, 127.1 (C-14), 127.40,
127.43, 127.6 (C-13, C-15), 128.0, 128.2 (C-12, C-16), 128.9, 130.9,
131.0, 131.3, 132.0, 133.2 (C-3), 135.8 (C-2), 141.0 (C-11), 146.8 (C-
4), 166.5 (C-6), 171.2 (C-8), 172.4 (C-21); HR-ESITOFMS m/z
599.2921 [M − H]− (calcd for C39H39N2O4 599.2915).
Hydrolysis of 2 to Yield 3. Compound 2 (4.5 mg, 7.5 μmol) was
hydrolyzed at 0−5 °C with 1 M NaOH (450 μL) in MeOH/THF
(1:1, 900 μL) for 16 h at room temperature. To stop the reaction, 0.5
N HCl (2.25 mL) was added. The reaction mixture was then diluted
with 2.25 mL of H2O and lyophilized. The residual solid was subjected
to HPLC purification using an XTerra RP18 column (Waters, 10 μm,
19 × 300 mm) with an isocratic solvent system of MeCN/0.15%
KH2PO4 aqueous solution (pH 3.5) (40:60) at a flow rate of 10 mL/
min, yielding 3 (2.5 mg, 100% yield): 1H NMR (CDCl3, 500 MHz) δ
1.25 (3H, d, J = 6.3 Hz, H-18), 1.25 (3H, d, J = 7.1 Hz, H-22), 2.21
(1H, dq, J = 7.1, 6.4 Hz, H-20), 3.90 (1H, dq, J = 6.4, 6.3 Hz, H-19),
5.18 (1H, d, J = 15.1 Hz, NHCH2), 5.20 (1H, d, J = 15.1 Hz,
NHCH2), 6.13 (1H, br s, NHCH2), 7.98 (1H, d, J = 7.7 Hz), 8.03−
8.10 (3H, m), 8.15−8.23 (4H, m), 8.26 (1H, d, J = 9.2 Hz); HR-
ESITOFMS m/z 354.1462 [M + Na]+ (calcd for C22H21NO2Na
354.1465).
6.7, 6.4 Hz, H-19); HR-ESITOFMS m/z 557.2651 [M + Na]+ (calcd
for C31H38N2O6Na 557.2622).
(S)-PGME amide (7): 1H NMR (CDCl3, 500 MHz) δ 0.93 (3H, d, J
= 6.4 Hz, H-18), 1.09 (3H, d, J = 7.1 Hz, H-22), 1.36 (3H, d, J = 7.1
Hz, H-17), 2.45 (1H, dq, J = 6.7, 7.1 Hz, H-20), 3.15 (1H, dq, J = 7.9,
7.1 Hz, H-10), 4.84 (1H, dd, J = 8.5, 7.9 Hz, H-9), 4.91 (1H, dq, J =
6.7, 6.4 Hz, H-19); HR-ESITOFMS m/z 557.2623 [M + Na]+ (calcd
for C31H38N2O6Na 557.2622).
Biological Assays. Cytotoxicity assay,24 antimicrobial assay,24 and
preadipocyte differentiation assay17 were carried out according to the
procedures previously reported.
ASSOCIATED CONTENT
* Supporting Information
1D and 2D NMR spectra of jomthonic acid A (1) and H
■
S
1
NMR spectra of 2−7. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +81-766-56-7500. Fax: +81-766-56-2498. E-mail: yas@
pu-toyama.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study is supported in part by the joint program in the field
of biotechnology under the Japan Society for the Promotion of
Science (JSPS) and the National Research Council of Thailand
(NRCT).
Preparation of (R)-MTPA Ester and (S)-MTPA Ester of 3 (4 and 5).
To a solution of 3 (1.0 mg, 2.6 μmol) in dry CH2Cl2 (100 μL) was
added (R)-MTPA acid (1.0 mg, 4.3 μmol), DCC (1.0 mg, 4.8 μmol),
and DMAP (trace amount) at room temperature. After stirring for 18
h, the 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 4 (0.6 mg). (S)-
MTPA ester 5 was obtained by the same experimental procedure as
described for (R)-MTPA ester 4.
REFERENCES
■
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(R)-MTPA Ester (4): 1H NMR (CDCl3, 500 MHz) δ 1.20 (3H, d, J =
7.2 Hz, H-22), 1.34 (3H, d, J = 6.4 Hz, H-18), 2.50 (1H, dq, J = 7.2,
7.2 Hz, H-20), 4.85 (1H, dd, J = 14.4, 4.4 Hz, NHCH2), 5.27 (1H, dd,
J = 14.4, 6.3 Hz, NHCH2), 5.33 (1H, dq, J = 7.2, 6.4 Hz, H-19), 5.92
(1H, br t, J = 5.4 Hz, NHCH2); HR-ESITOFMS m/z 570.1864 [M +
Na]+ (calcd for C32H28F3NO4Na 570.1863).
(5) Omura, S.; Ikeda, H.; Ishikawa, J.; Hanamoto, A.; Takahashi, C.;
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(S)-MTPA Ester (5). 1H NMR (CDCl3, 500 MHz) δ 1.10 (3H, d, J =
7.1 Hz, H-22), 1.40 (3H, d, J = 6.4 Hz, H-18), 2.53 (1H, dq, J = 6.7,
7.1 Hz, H-20), 4.71 (1H, dd, J = 14.4, 4.2 Hz, NHCH2), 5.24 (1H, dd,
J = 14.4, 6.5 Hz, NHCH2), 5.32 (1H, dq, J = 6.7, 6.4 Hz, H-19), 5.85
(1H, br t, J = 5.4 Hz, NHCH2); HR-ESITOFMS m/z 570.1878 [M +
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7). To a solution of jomthonic acid A (1, 5.0 mg, 13 μmol) in dry
DMF (0.35 mL) and dry triethylamine (0.35 mL) were added (R)-
PGME (5.2 mg, 26 μmol), PyBOP (14 mg, 26 μmol), and HOAT (3.5
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mixture was passed through an ODS column cartridge with 5%
HCO2H (25 mL), and the column was eluted with MeOH. The eluent
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5C18-AR-II column, Nacalai Tesque Inc., 10 × 250 mm) with MeCN/
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