Tumescenamide C, an antimicrobial cyclic lipodepsipeptide from Streptomyces sp.

Article abstract of DOI:10.1016/j.tet.2012.04.075

Tumescenamide C, a new cyclic lipodepsipeptide, was isolated from a culture broth of an actinomycete Streptomyces sp. KUSC-F05. Tumescenamide C was a congener of tumescenamides A and B, representing a sixteen-membered ring system, consisting of two proteinogenic and three non-proteinogenic amino acids, to which a methyl-branched fatty acid was attached. The planar structure was determined by spectroscopic analysis, while its absolute stereochemistry was determined by chemical degradation and asymmetric synthesis. Tumescenamide C exhibited antimicrobial activity with high selectivity against Streptomyces species.

Full text of DOI:10.1016/j.tet.2012.04.075

Tetrahedron 68 (2012) 5572e5578
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Tumescenamide C, an antimicrobial cyclic lipodepsipeptide from Streptomyces sp.
*
Shinji Kishimoto, Yuta Tsunematsu, Shinichi Nishimura, Yutaka Hayashi, Akira Hattori, Hideaki Kakeya
Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University,
Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 March 2012
Received in revised form 15 April 2012
Accepted 20 April 2012
Available online 27 April 2012
Tumescenamide C, a new cyclic lipodepsipeptide, was isolated from a culture broth of an actinomycete
Streptomyces sp. KUSC_F05. Tumescenamide C was a congener of tumescenamides A and B, representing
a sixteen-membered ring system, consisting of two proteinogenic and three non-proteinogenic amino
acids, to which a methyl-branched fatty acid was attached. The planar structure was determined by
spectroscopic analysis, while its absolute stereochemistry was determined by chemical degradation and
asymmetric synthesis. Tumescenamide C exhibited antimicrobial activity with high selectivity against
Streptomyces species.
Keywords:
Cyclic lipodepsipeptide
Streptomyces
Ó 2012 Elsevier Ltd. All rights reserved.
Methyl-branched fatty acid
Antimicrobial
1. Introduction
acid were determined by PGME method7 and synthetic work.
Herein we report the isolation, structure elucidation and bi-
Secondary metabolites produced by microbes continue to at-
tract the attention of organic chemists, biological chemists and
chemical biologists, due to their sophisticated chemical structures
and highly specific biological activities. Cyclic depsipeptide is one
of the important classes of such metabolites, e.g., daptomycin
produced by Streptomyces roseosporus disturbs the bacterial cell
membrane and is used as an antibacterial drug1; FK228 produced
by Chromobacterium violaceum is a potent histone deacetylase
inhibitor and used as a treatment for cutaneous T-cell lymphoma.2
In spite of the huge number of natural products so far reported, it
is an important task to discover novel secondary metabolites, as
they will provide new insights into natural product chemistry and
have the potential to be developed as research tools and phar-
maceuticals. In our continuing search for bioactive metabolites
from microbes, a culture broth of an actinomycete Streptomyces sp.
KUSC_F05 was found to contain antitumor trieneansamycin
compounds including cytotrienin A.3 Analysis of the constituents
in the same culture broth indicated the presence of another
abundant metabolite 1. Spectral analysis revealed that this me-
tabolite was a new congener of tumescenamides produced by
Streptomyces tumescens (Fig. 1).4 To determine the stereochemis-
try of 1, we applied advanced Marfey method for amino acid
residues,5,6 while the stereocenters in the methyl-branched fatty
ological activity of 1.
2. Results and discussion
2.1. Isolation
A culture broth of Streptomyces sp. KUSC_F05 (3 L) was extracted
with the same volume of n-BuOH. The extract was concentrated in
vacuo and separated by silica gel column chromatography. Analysis
of the obtained fractions by ODS HPLC revealed the presence of
a variety of trieneansamycin compounds including cytotrienin A.3
Meanwhile, a metabolite whose UV absorption pattern was differ-
ent from those of trieneansamycin compounds was found. The
corresponding fraction was subjected to reversed-phase HPLC to
give tumescenamide C (1, 61.5 mg) as a colorless amorphous solid.
* Corresponding author. Tel.: þ81 75 753 4524; fax: þ81 75 753 4591; e-mail
address: scseigyo-hisyo@pharm.kyoto-u.ac.jp (H. Kakeya).
Fig. 1. Structures of tumescenamide C (1) and tumescenamide A (2).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.075

S. Kishimoto et al. / Tetrahedron 68 (2012) 5572e5578
5573
2.2. Structure determination
HMBC spectra revealed the presence of one residue each of leucine
(Leu), threonine (Thr), valine (Val), tyrosine (Tyr), 2-amino-2-
butenoic acid (Abu), and 2,4-dimethylheptanoic acid (Dmh)
(Fig. 2). The connectivity of the five amino acid residues and one
acyl moiety was determined based on the HMBC and ROESY cor-
relations (Fig. 2). For example, HMBC correlations from the NH
The molecular formula of tumescenamide C (1) was established
as C37H57N5O8 on the basis of HR-FABMS (m/z 698.4129 [MꢀH]-,
D
mmu¼þ1.1) and NMR spectral data (Table 1). The 1H NMR spec-
trum exhibited signals characteristic of a peptide, including ex-
changeable NH signals at
(1H, br s),
7.75 (1H, d, J¼8.2 Hz), and 7.66 (1H, d, J¼8.6 Hz), and
four -protons at
d
8.75 (1H, br s),
d
8.44 (1H, br s),
d
8.13
signal at
the amide carbon at
connectivity of Thr to Abu was established by an HMBC correlation
d
7.75 (6-NH, Tyr) and the
a-proton at d 3.83 (H-15, Val) to
d
d
173.7 (C-14) connected Tyr and Val. The
a
d
4.72 (1H, dd, J¼8.6, 3.2 Hz), 4.50 (1H, ddd,
J¼12.3, 8.5, 3.5 Hz), 4.31 (1H, t, J¼7.6 Hz) and 3.83 (1H, br d,
from the protons at
carbonyl carbon at
d
5.15 (H-27, Thr) and d 6.79 (H-3, Abu) to Abu
165.3 (C-1). The planar structure of 1 was
J¼3.0 Hz). Additionally, nine signals corresponding to methyl
d
groups at
d
1.73 (3H, d, J¼7.0 Hz), 1.42 (3H, d, J¼6.3 Hz), 1.10 (3H, d,
revealed to be same to that of tumescenamide A (2, Fig. 1).4 Next,
we conducted chemical degradation and asymmetric synthesis to
determine the chirality for all stereogenic centers in 1.
J¼7.0 Hz), 1.00 (3H, d, J¼6.6 Hz), 0.94 (3H, d, J¼6.6 Hz), 0.81 (3H, d,
J¼6.8 Hz), 0.79 (3H, t, J¼7.3 Hz), 0.70 (3H, d, J¼6.9 Hz) and 0.61 (3H,
d, J¼6.8 Hz) were observed. Two aromatic doublets at
d 7.18 (2H, d,
J¼8.6 Hz) and 6.69 (2H, d, J¼8.6 Hz) indicated the presence of
a para-substituted phenyl ring. The 13C NMR spectrum indicated
the existence of a peptide backbone; six amide or ester carbonyl
carbon signals at
four -carbons at
d
180.1, 178.5, 173.7, 172.0, 171.4, and 165.3, and
62.4, 57.2, 56.8, and 54.5 were observed. A de-
a
d
tailed analysis of the 2D NMR data including DQF-COSY, HMQC and
Table 1
1H and 13C NMR spectra of 1 in CD3OHa
Fig. 2. Selected DQF-COSY (bold) and HMBC (arrow) correlations for 1 (left) and ROESY
correlations for 1 (right).
Position
dH
Mult.(J in Hz)
dC
(Z)-Abu
1
2
3
4
165.3
127.6
137.9
14.6
6.79
1.73
8.13
q (7.2)
d (7.0)
br s
The absolute configurations of the amino acids were determined
by advanced Marfey method.5,6 Acid hydrolysate of 1 was con-
2-NH
densed with 1-fluoro-2,4-dinitro-phenyl-5-
FDLA), and analyzed by LC-MS to reveal the presence of
and -Tyr (Table 2, Fig. S43). The stereochemistry of Thr was de-
termined to be -Thr by comparing the retention times of FDLA de-
rivatives of authentic standard amino acids -Thr and -allo-Thr
(Table 2). The geometry of the double bond in Abu was determined
to be Z, based on a ROESY correlation betweenprotons at 1.73 (H-4)
and 8.13 (2-NH) (Fig. 2). The absolute configuration of C-30 was
L/D-leucinamide (L- or
D-
D
-Tyr
5
6
7
172.0
57.2
36.6
L-Leu,
D-Val
4.50
a 3.37
b 3.07
ddd (12.3, 8.5, 3.5)
dd (13.9, 3.2)
dd (14.2, 12.3)
D
L
8
9
10
11
12
13
6-NH
11-OH
14
15
16
17
18
15-NH
19
20
129.7
131.3
116.3
157.4
116.3
131.4
L
L
7.18
6.69
d (8.6)
d (8.6)
d
6.69
7.18
7.75
d (8.6)
d (8.6)
d (8.2)
d
determined byapplying the PGME method.7 2,4-Dimethylheptanoic
acid (3) was obtained from the acid hydrolysate of 1 and condensed
with (R)- or (S)- phenylglycine methyl ester (PGME) to afford 3a and
3b, respectively. The 1H NMR spectra of the obtained diastereomers
were compared, and Dd values revealed the 30S stereochemistry
(Fig. 3a). The remaining chiral center at C-32 could not be de-
termined by NMR analysis and required the synthesis of authentic
standards for comparison purposes.
D
-Val
173.7
62.4
29.9
19.1
17.1
3.83
2.08
0.81
0.61
8.75
br d (3.0)
m
d (6.8)
d (6.8)
br s
L
-Leu
-Thr
178.5
54.5
40.9
4.31
t (7.6)
m
m
21
a 1.56
b 1.51
1.65
1.00
0.94
Table 2
Retention times of
L- and D
-FDLA derivatives of amino acidsa
22
23
24
20-NH
25
26
27
28
26-NH
29
m
25.7
22.9
22.7
tRLb (min)
tRDc (min)
m/z
Absolute
d (6.6)
d (6.6)
br s
Amino acid
configuration
8.44
Tyrd
Val
Leu
Thr
30.7
22.3
19.5
12.0
12.0
12.7
27.9
17.4
25.0
15.5
15.5
14.0
770.2740
412.1827
426.1983
414.1619
414.1619
414.1619
D
D
L
L
171.4
56.8
75.4
16.3
4.72
5.15
1.42
7.66
dd (8.6, 3.2)
qd (6.2, 3.4)
d (6.3)
L
L-Thr
L
d (8.9)
L-allo-Thr
L
2S,4S-Dmh
180.1
39.4
42.4
a
b
c
Amino acid derivatives were analyzed by LC-ESI MS.
30
31
2.60
m
m
m
m
m
m
m
t (7.3)
d (7.0)
d (6.9)
Retention times of
Retention times of
L
-FDLA derivatives.
-FDLA derivatives.
a 1.67
b 1.05
1.33
a 1.15
b 0.89
1.20
0.79
1.10
0.70
D
d
Retention times for bis derivatives are shown.
32
33
31.4
40.2
To determine the absolute configuration of C-32, we planned to
34
35
30-Me
32-Me
20.8
14.5
19.1
20.2
synthesize (2S,4R)-Dmh 4 and (2S,4S)-Dmh 5 and to compare their
spectral data with that of the natural Dmh 3. 2S configuration was
expected to be obtained by the asymmetric alkylation introduced
by Evans and co-workers,8 while 4S and 4R could be derived from
1H and 13C NMR spectra were recorded at 500 and 125 MHz, respectively.
a

5574
S. Kishimoto et al. / Tetrahedron 68 (2012) 5572e5578
and 5b, gave 1H NMR spectra that were indistinguishable from those
(a)
(b)
of 3a and 3b, respectively (Supplementary data, Fig. S42). Next, Dd
values of the 1H NMR spectra for 5a and 5b completely matched to
those obtained for 3a and 3b (Fig. 3). Finally, specific optical rotations
of 3a and 5a (½a 2D6
ꢁ ¼ꢀ143.3 and ꢀ144.4, respectively) were smaller
than 4a (½a 2D6
ꢁ ¼ꢀ134.4). In conclusion, the side chain of 1 was de-
(c)
termined to be (2S,4S)-2,4-dimethylheptanoic acid.
The chemical structure of tumescenamide
C
D
(1) was un-
-Tyr, -Thr, (Z)-
ambiguously determined, consisting of
L-Leu,
D-Val,
L
Abu and (2S,4S)-Dmh. Tumescenamide C was revealed to be an
isomer of tumescenamide A (2) since Val of 2 was reported to have
Fig. 3. Dd
4b; (c) 5a and 5b.
(
d(S)-PGME
ꢀ
d(R)-PGME) values for PGME derivatives. (a) 3a and 3b; (b) 4a and
an -configuration. Dmh moieties in 1 and 2 had same relative ste-
L
reochemistry although the absolute stereochemistry of Dmh in 2 is
not known; the stereochemistry of Dmh in 2 was deduced to be 2S*,
4S* by J-based NMR analysis. Curiously, the 1H and 13C NMR spectra
of 1 in acetone-d6 were similar to those reported for 2 (Table S1).
the commercially available starting materials (Scheme 1). We first
synthesized (R)-2-methypentyl trifluoromethanesulfonate (12)
from (S)-methyl 3-hydroxy-2-methypropanoate (6) in six steps
with 23% yield in a similar way to that reported by Organ and co-
workers.9 Then, diastereoselective alkylation of (4R)-propionylox-
azolidinone (13) with the chiral triflate 12 was carried out to obtain
(2S,4R)-2,4-dimethylsubstituted oxazolidinone 14a and its C-2 di-
astereomer 14b in a ca. 8:1 ratio. These two diastereomers were
easily separated by column chromatography to give 14a in 24%
yield. Hydrolytic cleavage of the chiral auxiliary from 14a was done
with alkaline hydrogen peroxide to yield 4. Synthesis of 5, a C-4
diastereomer of 4, was started from commercially available racemic
alcohol 15. Alcohol 15 was converted to triflate 16, followed by
a reaction with oxazolidinone 13 to yield a 2S mixture of 14a and
14c in 32% yield and a 2R mixture of 14b and 14d in 4% yield. The
major mixture was easily separated from the minor mixture by
silica gel column chromatography. Although 14a and 14c exhibited
similar elution properties on a reversed-phase HPLC column, we
could successfully separate them on a small scale sufficient for the
next two steps. Purified 14c was subjected to oxidative hydrolysis
with alkaline hydrogen peroxide to obtain 5.
2.3. Biological activities
Tumescenamide C (1) is composed of a sixteen-membered
depsipeptide portion and a methyl-branched acyl group. The ring
size is the same to those of actinomycin D and FK228, although the
constituent amino acids differed.2,10 The most characteristic feature
of 1 is the acyl group. Although several natural products possessing
fatty acids with branched methyl groups have been reported,11,12
tumescenamides A (2) and C (1) are the only examples possess-
ing 2,4-dimethylheptanoic acid. Tumescenamide A was reported to
weakly upregulate the expression of a reporter gene under the
control of the insulin degrading enzyme promoter. However, the
physiological function of 2 remains to be understood.
We conducted an antimicrobial assay and found that tumesce-
namide C (1) inhibited the growth of streptomycetes cells including
Streptomyces coelicolor A3(2) and Streptomyces lividans TK23;
tumescenamide C exhibited growth inhibitory zone at 3.0 mg/paper
disk (Table 3). Tumescenamide C also inhibited the growth of the
Synthesized carboxylic acids 4 and 5 exhibited apparently differ-
ent 1H NMR spectra, while 1H NMR spectra of the natural acid 3
closely resembled thatof5, suggesting that stereocenters in 3 were 2S
and 4S (Fig. 4). Further analysis was carried out by using non-volatile
PGME derivatives of 3, 4, and 5. First, the PGME derivatives of 5, 5a,
producing strain Streptomyces sp. KUSC_F05, although a larger
amount of the compound, 30 mg/paper disc, was required. Anti-
microbial activity against gram positive bacteria including Bacillus
subtilis and Staphylococcus aureus and gram negative bacteria in-
cluding Escherichia coli and Pseudomonas aeruginosa was not
(i)
(ii)
Scheme 1. Synthesis of (i) (2S,4R)-Dmh and (ii) (2S,4S)-Dmh.

S. Kishimoto et al. / Tetrahedron 68 (2012) 5572e5578
5575
a 500 MHz instrument. 1H and 13C chemical shifts are relative to the
solvent; dH 3.31 and dC 49.00 for CD3OD and dH 7.26 and dC 77.16 for
CDCl3. Mass spectral data were collected with FABMS or ESI MS.
5
4
3
4.2. Fermentation, extraction and isolation
A slant culture of Streptomyces sp. KUSC_F05 was inoculated into
a 100 mL of culture medium A (1% of glucose, 2.5% of soluble starch,
0.5% of fish meal, 0.3% of NZ case, 0.3% of pharma media, 0.2% of yeast
extract, and 0.2% of CaCO3) in a 500 mL flask. The flasks were shaken
on a rotary shaker (150 rpm) at 28 ꢂC for 72 h. This preculture (2 mL)
was transferred into a 100 mL of a culture medium B (2.0% of oatmeal,
1% of glucose, 2.5% of dextrin, 0.5% of fish meal, 0.5% of B. molasses,
1.0% of pharma media, 0.2% of ebios, and 0.2% of CaCO3), and the
fermentation was carried out at 28 ꢂC for 72 h. To the culture broth
(totally 3 L) was added the same volume of n-BuOH. The n-BuOH
extract (10.96g) wasfractionated on a silicagelcolumn(120ꢃ70 mm)
with a stepwise elution of CHCl3/MeOH (from 100:0 to 0:100). The
fraction eluted with CHCl3/MeOH (100:5) (1.4 g) was chromato-
graphed on a silica gel column (360ꢃ46 mm) with another solvent
system (n-hexane/EtOAc¼50:50 to 0:100). A portion of the EtOAc
fraction(110 mgof 714 mg)wassubjected to ODS HPLConSenshu Pak
PEGASIL ODS SP100 (250ꢃ20 mm) with MeOH/H2O (50:50) to afford
tumescenamide C (1, 61.9 mg, tR¼40min):colorlessamorphous solid;
3.0
2.5
2.0
1.5
1.0
0.5
/ppm
Fig. 4. Comparison of the 1H NMR spectra for 2,4-dimethylheptanoic acids 3 (natural),
4 (synthesized 2S,4R) and 5 (synthesized 2S,4S) in CDCl3 (500 MHz).
Table 3
Antimicrobial activity of tumescenamide C (1)
Organism
Relevant
characteristics
Minimal amount
for growth inhibition
(m
g/disc)a
Streptomyces coelicolor A3(2)
Streptomyces lividans TK23
Streptomyces sp. KUSC_F05
Staphylococcus aureus IFO13276
Bacillus subtilis IFO3134
Gram (þ)
Gram (þ)
Gram (þ)
Gram (þ)
Gram (þ)
Gram (-)
Gram (-)
3
3
30
>100
>100
>100
>100
mp 173e176 ꢂC; ½a D
ꢁ
25þ37.0 (c 2.1, MeOH); UV (MeOH) lmax (log ε)
Escherichia coli IFO3972
Pseudomonas aeruginosa IFO13275
223 (4.24), 278 (3.09) nm; IR (neat) 3301 (br), 2959, 2933, 2872,1651,
1517, 1257 cmꢀ1; HRMS (FAB) m/z 698.4129 [MꢀH]- calcd for
C37H56N5O8, 698.4134; 1H NMR and 13C NMR, see Table 1.
Organism
Relevant
characteristics
IC50 (m
g/mL)b
Schizosaccharomyces pombe JY3
Saccharomyces cerevisiae BY4741
Candida albicans IFO1594
Yeast
Yeast
Yeast
>100
>100
>100
4.3. Acid hydrolysis of 1 and advanced Marfey method
A portion of 1 (0.1 mg) was hydrolyzed with 6 N HCl (200
42 h at 105 ꢂC and then dried in vacuo. The obtained hydrolysate
was dissolved in H2O (100 L), to which 1 M NaHCO3 (20 L) was
added, and the solution was split into two. To a 60 L aliquot of the
-FDLA (1% w/v in acetone, 50 L) or -FDLA
L), and the mixtures were stirred for 1 h at
mL) for
a
Examined by paper disc diffusion assay.
Examined in a liquid medium.
b
m
m
detected even when 100 mg of 1 was loaded onto paper discs. Yeast
cells including Schizosaccharomyces pombe, Saccharomyces cer-
evisiae and Candida albicans were tolerant to 1 at a concentration of
m
hydrolysate was added
(1% w/v in acetone, 50
L
m
D
m
70 ꢂC. The solutions were cooled to room temperature, neutralized
100
mg/mL. Growth of mammalian cells (HeLa, HT1080, ARPE19 and
Mv1Lu cells) was not affected by 1 at a concentration of 100
mg/mL.
with 1 N HCl (10
mL), evaporated, and then dissolved in MeCN
Tumescenamide C seems to affect some cellular architecture or
signaling pathways, which are conserved in streptomycetes.
(200 L). The derivatives were analyzed by LC-MS; LC separation
m
was performed on a reversed-phase column (Shim-Pack XR-ODS,
75ꢃ2.0 mm) with a gradient elution system of H2O/MeCN con-
taining 0.1% formic acid (80:20 to 20:80 for 40 min, 0.2 mL/min). ESI
MS was performed in a positive ionization mode.
3. Conclusion
In summary, a cyclic depsipeptide tumescenamide C (1), a new
congener of tumescenamides, was isolated and its chemical struc-
4.4. PGME derivatives of the natural Dmh (3a and 3b)
ture was determined. The depsipeptide portion of 1 consists of
Leu, -Val, -Tyr, -Thr and (Z)-2-amino-2-butenoic acid, revealing
that 1 and 2 are epimeric at the carbon of the Val residue. The acyl
L-
Tumescenamide C (1, 10.4 mg, 1.49ꢃ10ꢀ2 mmol) was hydrolyzed
with 6 N HCl (2 mL) for 33.5 h at 105 ꢂC, which was subsequently
extracted with CHCl3 (5ꢃ0.8 mL). The combined extract was dried
over anhydrous Na2SO4, concentrated in vacuo, and separated with
preparative TLC (CHCl3/MeOH¼10:1) to afford 2,4-dimethylheptanoic
D
D
L
a
groups, 2,4-dimethylheptanoic acid, had same relative stereo-
chemistry. This is the first report to synthesize (2S,4S)-2,4-
dimethylheptanoic acid and its (4R)-epimer in a diastereo-specific
manner, by which we unambiguously determined the absolute
stereochemistry of the acyl group found in 1. Tumescenamide C
exhibited unique biological properties; selective growth inhibition
against streptomycetes cells was observed. The mechanism of ac-
tion and biological role of tumescenamide C is of interest.
acid (2, 0.8 mg, 5.1ꢃ10ꢀ3 mmol).
A portion of 3 (0.4 mg,
2.5ꢃ10ꢀ3 mmol) was mixed with HATU (3.4 mg, 8.9ꢃ10ꢀ3 mmol),
HOAt (2.1 mg, 1.5ꢃ10ꢀ2 mmol), (R)-PGME$HCl (1.4 mg,
6.9ꢃ10ꢀ3 mmol) and DIEA (1.5 mg, 1.2ꢃ10ꢀ2 mmol) in DMF (100
mL),
which was allowed stirred at room temperature. After 3.5 h, 1.5 mL of
EtOAc was added to the reaction mixture. The organic layer was
washed with satd aq NH4Cl (3ꢃ1.5 mL), concentrated, and subjected
to preparative TLC (CHCl3/MeOH¼10:1), followed by purification by
reversed-phase HPLC on Cosmosil 5C18-MS-II (250ꢃ10 mm) with
H2O/MeCN (40:60) to afford an (R)-PGME derivative of Dmh (3a,
0.4 mg, 1.3ꢃ10ꢀ3 mmol, tR¼21 min) as a colorless amorphous solid:
4. Experimental
4.1. General
All reagents and solvents were used as received from commercial
suppliers. IR spectra were measured using an FTIR spectrometer
equipped with a ZnSe ATR plate. Optical rotations were determined
using the sodium D line (589 nm). NMR spectra were measured on
½
a D
ꢁ
26ꢀ143.3 (c 0.03, CHCl3); IR (neat) 3298 (br), 2957, 2930, 2871,
1748, 1652, 1531, 1456, 1211 cmꢀ1 1H NMR (CDCl3, 500 MHz)
;
d
7.37e7.29 (m, 5H), 6.40 (br d, J¼6.8 Hz, 1H), 5.59 (d, J¼7.2 Hz, 1H),

5576
S. Kishimoto et al. / Tetrahedron 68 (2012) 5572e5578
3.73 (s, 3H), 2.39 (m, 1H), 1.66 (m, 1H), 1.34e1.15 (m, 4H), 1.15 (d,
J¼6.8 Hz, 3H), 1.10 (m, 1H), 1.01 (m, 1H), 0.83 (d, J¼6.6 z, 3H), 0.80 (t,
(4 mL, 49.6 mmol) and TsCl (1.94 g, 10.2 mmol) at 0 ꢂC. After being
stirred at 0 ꢂC for 10 h, satd aq NH4Cl (20 mL) was added. The organic
layer was dried over anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by flush column chromatography (SiO2, n-
hexane/MeOH¼50:1 and 20:1) to yield 9 (1.87 g, 3.86 mmol, 77.0%) as
J¼7.1 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d 176.1, 171.7, 136.9, 129.1
(2C), 128.6, 127.4 (2C), 56.3, 52.9, 42.0, 39.5, 39.1, 30.4, 20.0, 19.7, 18.6,
14.4; HRMS (ESI) m/z 328.1842 [MþNa]þ calcd for C18H27NNaO3,
328.1883.
a colorless oil: ½a D
ꢁ
26þ6.0 (c 0.74, CHCl3); IR (neat) 2960, 2931, 2858,
The remaining portion of 3 (0.4 mg, 2.5ꢃ10ꢀ3 mmol) was mixed
with HATU (3.3 mg, 8.7ꢃ10ꢀ3 mmol), HOAt (1.0 mg,
7.4ꢃ10ꢀ3 mmol), (S)-PGME$HCl (1.5 mg, 7.4ꢃ10ꢀ3 mmol) and DIEA
1472, 1428, 1362, 1189, 1177, 1112 cmꢀ1 1H NMR (CDCl3, 500 MHz)
;
d
7.79 (d, J¼8.3 Hz 2H), 7.59 (m, 4H), 7.45e7.41 (m, 2H), 7.39e7.35 (m,
4H), 7.31 (d, J¼8.2 Hz, 2H), 4.13 (dd, J¼9.2, 5.7 Hz, 1H), 4.01 (dd, J¼9.3,
6.0 Hz, 1H), 3.56 (dd, J¼10.2, 4.8 Hz, 1H), 3.47 (dd, J¼10.2, 6.6 Hz, 1H),
2.42 (s, 3H), 2.01 (m, 1H), 0.98 (s, 9H), 0.89 (d, J¼6.9 Hz, 3H); 13C NMR
(1.5 mg, 1.2ꢃ10ꢀ2 mmol) in DMF (100
mL) and allowed stirred for
3.5 h at room temperature. The reaction mixture was separated as
described above to afford (S)-PGME derivative of Dmh (3b, 0.3 mg,
(CDCl3, 125 MHz) d 144.7, 135.7 (2C), 135.6 (2C), 133.5, 133.5, 133.2,
9.8ꢃ10ꢀ4 mmol, tR¼21 min) as a colorless oil: ½a D
ꢁ
26þ147.3 (c 0.02,
129.9 (2C), 129.8 (2C), 128.1 (2C), 127.8 (4C), 72.3, 64.7, 35.8, 26.9 (3C),
21.8, 19.3, 13.4; HRMS (ESI) m/z 505.1838 [MþNa]þ calcd for
C27H34NaO4SSi, 505.1839. Spectral data were in agreement with those
reported previously.14
CHCl3); IR (neat) 3298, 2956, 2929, 2871, 1749, 1652, 1533, 1456,
1219 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d 7.37e7.30 (m, 5H), 6.37 (br
d, J¼6.9 Hz,1H), 5.58 (d, J¼7.1 Hz,1H), 3.73 (s, 3H), 2.40 (m,1H), 1.71
(m, 1H), 1.45 (m, 1H), 1.38e1.20 (m, 3H), 1.16e1.04 (m, 2H), 1.11 (d,
J¼6.9 Hz, 3H), 0.89 (d, J¼6.6 Hz, 3H), 0.86 (t, J¼7.1 Hz, 3H); 13C NMR
4.5.4. (R)-tert-Butyl(2-methylpentyloxy)diphenylsilane (10). A mix-
ture of 9 (349 mg, 0.723 mmol) and CuI (91.4 mg, 0.480 mmol) was
cooled to 0 ꢂC under nitrogen atmosphere, to which 8 mL of 1 M
EtMgBr in THF (8 mmol) was added. After being stirred for 3 h at
(CDCl3, 125 MHz)
d 176.1, 171.7, 136.9, 129.1 (2C), 128.6, 127.4 (2C),
56.3, 52.9, 41.9, 39.6, 39.1, 30.5, 20.1, 19.8, 18.7, 14.5; HRMS (ESI) m/z
328.1843 [MþNa]þ calcd for C18H27NNaO3, 328.1883.
0
ꢂC, the reaction was quenched with satd aq NH4Cl (8 mL). The
4.5. Synthesis of (2S,4R)-2,4-dimethylheptanoic acid (4)
aqueous layer was extracted with EtOAc (2ꢃ8 mL), and the com-
bined organic layers were dried over anhydrous Na2SO4 and con-
centrated in vacuo. The residue was chromatographed (SiO2, n-
hexane/EtOAc¼20:1) to give 10 (241 mg, 0.708 mmol, 98.0%) as
4.5.1. (S)-Methyl 3-(tert-butyldiphenylsilyloxy)-2-methylpropanoate
(7). To
a stirred solution of (S)-methyl 3-hydroxy-2-methylpr-
opanoate (6; 916 mg, 7.75 mmol) in DMF (7.8 mL) under nitrogen at-
mosphere was added imidazole (817 mg, 12.0 mmol) and TBDPSCl
(2.57 g, 9.34 mmol). After being stirred for 1.5 h at room temperature,
the reaction was quenched by addition of satd aq NH4Cl (40 mL). The
mixture was extracted with EtOAc, washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residuewas chromatographed
(SiO2, n-hexane/EtOAc¼50:1) to give 7 (2.26 g, 6.33 mmol, 81.7%) as
a colorless oil: ½a D
ꢁ
25þ1.5 (c 0.54, CHCl3); IR (neat) 2957, 2930, 2858,
1471, 1427, 1111 cmꢀ1 1H NMR (CDCl3, 500 MHz)
; d 7.67 (m, 4H),
7.44e7.35 (m, 6H), 3.52 (dd, J¼9.7, 5.6 Hz, 1H), 3.43 (dd, J¼9.7,
6.4 Hz, 1H), 1.66 (m, 1H), 1.45e1.17 (m, 3H), 1.09 (m, 1H), 1.06 (s, 9H),
0.91 (d, J¼6.7 Hz, 3H), 0.87 (t, J¼7.3 Hz, 3H); 13C NMR (CDCl3,
125 MHz)
d 135.8 (4C), 134.3 (2C), 129.6 (2C), 127.7 (4C), 69.2, 35.6
(2C), 27.0 (3C), 20.2, 19.5, 17.0, 14.5; HRMS (ESI) m/z 363.2091
a colorless oil: ½a D
ꢁ
25þ16.8 (c 0.34, CHCl3); IR (neat) 2956, 2934, 2857,
[MþNa]þ calcd for C22H32NaOSi, 363.2115.
1742, 1595, 1112 cmꢀ1 1H NMR (CDCl3, 500 MHz)
; d 7.66 (m, 4H),
7.46e7.37 (m, 6H), 3.84 (dd, J¼9.6, 6.9 Hz, 1H), 3.74 (dd, J¼9.6, 5.8 Hz,
4.5.5. (R)-2-Methyl-1-pentanol (11). To a stirred solution of 10
(237 mg, 0.695 mmol) in 5 mL of dry THF under nitrogen atmo-
sphere was added 1.5 mL of 1 M TBAF in THF (1.5 mmol) at 0 ꢂC. The
mixture was stirred for 20 min at 0 ꢂC, warmed to room temper-
ature, and stirred for 5 h. The reaction was quenched with satd aq
NH4Cl (5 mL). The aqueous layer was extracted with EtOAc
(2ꢃ5 mL), and the combined organic layers were dried over an-
hydrous Na2SO4 and concentrated in vacuo. The residue was
chromatographed (SiO2, n-hexane/EtOAc¼3:1) to give 11 (46.1 mg,
1H), 3.69 (s, 3H), 2.73 (m, 1H), 1.17 (d, J¼7.1 Hz, 3H), 1.04 (s, 9H); 13C
NMR (CDCl3,125MHz) d 175.5,135.7 (4C),133.7,133.6,129.8 (2C),127.8
(4C), 66.1, 51.7, 42.5, 26.9 (3C), 19.4, 13.6; HRMS (ESI) m/z 379.1695
[MþNa]þ calcd for C21H28NaO3Si, 379.1700. Spectral data were in
agreement with those reported previously.13
4.5.2. (R)-3-(tert-Butyldiphenylsilyloxy)-2-methyl-1-propanol (8). A
solution of 7 (2.21 g, 6.20 mmol) in 40 mL of dry toluene was cooled
to ꢀ78 ꢂC under nitrogen atmosphere, to which DIBAL-H (15 mL,
15 mmol, 1 M solution in toluene) was added dropwise. After being
stirred for 20 min at ꢀ78 ꢂC, the mixture was warmed to 0 ꢂC, and
stirred for 10 min. The reaction was quenched with satd aq sodium
potassium tartrate (40 mL) and the slurry was stirred at room
temperature for 10 h. The mixture was extracted with EtOAc
(2ꢃ40 mL), and the combined organic layers were dried over an-
hydrous Na2SO4 and concentrated in vacuo. The residue was
chromatographed (SiO2, n-hexane/EtOAc¼15:1 to 5:1) to yield 8
0.451 mmol, 64.9%) as a colorless liquid: ½a D
ꢁ
24þ13.1 (c 0.26, CHCl3);
IR (neat) 3318 (br), 2957, 2928, 2871, 1727, 1596 cmꢀ1 1H NMR
;
(CDCl3, 500 MHz)
d
3.75 (dd, J¼10.4, 5.7 Hz, 1H), 3.39 (dd, J¼10.5,
6.6 Hz, 1H), 1.61 (m, 2H), 1.42e1.21 (m, 3H), 1.08 (m, 1H), 0.90 (d,
J¼6.7 Hz, 3H), 0.89 (t, J¼7.0 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d
68.5, 35.6, 35.5, 20.2, 16.7, 14.4. The 1H NMR spectrum matched
with that of the commercially available racemic 2-methyl-1-
pentanol.
(1.83 g, 5.57 mmol, 89.9%) as a colorless oil: ½a D
ꢁ
25þ5.7 (c 0.98,
4.5.6. (R)-2-Methylpentyl trifluoromethanesulfonate (12). A stirred
solution of 11 (69.6 mg, 0.681 mmol) in 3 mL of CH2Cl2 under ni-
trogen atmosphere was cooled to ꢀ78 ꢂC, to which pyridine
CHCl3); IR (neat) 3395 (br), 2959, 2930, 2858, 1472, 1428,
1111 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d 7.70 (m, 4H), 7.48e7.40 (m,
6H), 3.75 (dd, J¼10.1, 4.6 Hz, 1H), 3.71e3.68 (m, 2H), 3.63 (dd,
(100 mL, 98 mg, 1.24 mmol) and Tf2O (200 mL, 344 mg, 1.22 mmol)
J¼10.2, 7.6 Hz, 1H), 2.57 (s, 1H), 2.01 (m, 1H), 1.09 (s, 9H), 0.86 (d,
were added. After being stirred for 50 min at ꢀ78 ꢂC, the reaction
was quenched with satd aq NH4Cl (3 mL). The aqueous layer was
extracted with CHCl3 (3 mL), and the combined organic layers were
dried over anhydrous Na2SO4 and concentrated in vacuo. The res-
idue was chromatographed (SiO2, n-hexane/EtOAc¼50:1) to give 12
J¼7.0 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d 135.7 (4C), 133.3, 133.3,
129.9 (2C), 127.9 (4C), 68.7, 67.7, 37.5, 27.0 (3C), 19.3, 13.3; HRMS
(ESI) m/z 351.1738 [MþNa]þ calcd for C20H28NaO2Si, 351.1751.
Spectral data were in agreement with those reported previously.13
(70.1 mg, 0.299 mmol, 43.9%) as a colorless liquid: ½a D
ꢁ
24þ0.1 (c
4.5.3. (S)-3-(tert-Butyldiphenylsilyloxy)-2-methylpropyl 4-methylben-
zenesulfonate (9). To a stirred solution of 8 (1.65 g, 5.02 mmol) in
18.5 mL of CH2Cl2 under nitrogen atmosphere were added pyridine
0.78, CHCl3); IR (neat) 2966, 2936, 2877, 1415, 1247, 1205,
1146 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
4.32 (dd, J¼9.6, 6.6 Hz, 1H), 1.97 (m, 1H), 1.45e1.27 (m, 3H), 1.22 (m,
d
4.40 (dd, J¼9.6, 5.6 Hz, 1H),

S. Kishimoto et al. / Tetrahedron 68 (2012) 5572e5578
5577
1H), 1.02 (d, J¼6.8 Hz, 3H), 0.92 (t, J¼7.1 Hz, 3H); 13C NMR (CDCl3,
20.1, 19.6, 17.8, 14.4; HRMS (ESI) m/z 328.1847 [MþNa]þ calcd for
125 MHz)
d
118.8 (q, J¼318.8 Hz), 82.1, 34.6, 33.2, 19.9, 16.2, 14.2.
C18H27NNaO3, 328.1883.
4.5.7. (R)-4-Benzyl-3-((2S,4R)-2,4-dimethylheptanoyl)-2-oxazolidinone
(14a). A stirred solution of (R)-4-benzyl-3-propionyl-2-oxazolidinone
(13; 139 mg, 0.595 mmol) in dry THF (1.5 mL) under nitrogen atmo-
sphere was cooled to ꢀ78 ꢂC, to which 0.31 mL of 1.9 M NaHMDS in
THF (0.589 mmol) was added. After being stirred for 30 min at ꢀ78 ꢂC,
12 (64.1 mg, 0.274 mmol) in 0.5 mL of dry THF was added. The re-
action mixture was stirred for 2.5 h at ꢀ78 ꢂC, warmed to 0 ꢂC and
stirred for 1.5 h, and then quenched with satd aq NH4Cl (1.5 mL). The
aqueous layer was extracted with EtOAc (2ꢃ1.5 mL), and the com-
bined organic layers were dried over anhydrous Na2SO4 and con-
centrated in vacuo. The residue was chromatographed (SiO2, n-
hexane/EtOAc¼10:1) to give 14a (20.5 mg, 0.0646 mmol, 23.6%) as
4.6.2. (S)-Methyl 2-((2S,4R)-2,4-dimethylheptanamido)-2-phenylace-
tate (4b). A solution of 4 (1.9 mg, 0.012 mmol), HATU (14.9 mg,
0.0392 mmol), HOAt (4.8 mg, 0.035 mmol), (S)-PGME$HCl (7.2 mg,
0.036 mmol) and DIEA (9.0 mL, 6.8 mg, 0.052 mmol) in 0.4 mL of
DMF was stirred for 7.5 h at room temperature. As described above
for 4a, 4b (1.8 mg, 0.0059 mmol, 49%, tR¼21 min) was purified and
obtained as a colorless amorphous solid: ½a D
ꢁ
26þ144.4 (c 0.15,
CHCl3); IR (neat) 3299 (br), 2957, 2930, 2873, 1749, 1652, 1533, 1456,
1219 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d 7.38e7.30 (m, 5H), 6.37 (br
d, J¼6.3 Hz, 1H), 5.58 (d, J¼7.0 Hz, 1H), 3.73 (s, 3H), 2.37 (ddq, J¼6.9,
6.9, 6.9 Hz, 1H), 1.53e1.44 (m, 2H), 1.40e1.22 (m, 4H), 1.12 (m, 1H),
1.10 (d, J¼6.8 Hz, 3H), 0.88 (t, J¼6.8 Hz, 3H), 0.85 (d, J¼5.8 z, 3H); 13C
a colorless oil: ½a D
ꢁ
25ꢀ43.1 (c 0.50, CHCl3); IR (neat) 2958, 2929, 2873,
NMR (CDCl3, 125 MHz) d 176.3, 171.7, 136.8, 129.1 (2C), 128.6, 127.4
1783, 1699, 1600, 1456, 1387, 1351, 1210, 1099 cmꢀ1; 1H NMR (CDCl3,
(2C), 56.3, 52.9, 41.4, 39.5, 38.9, 30.2, 20.1,19.6,17.6,14.5; HRMS (ESI)
500 MHz)
d
7.35e7.31 (m, 2H), 7.28 (m, 1H), 7.24e7.20 (m, 2H), 4.68
m/z 328.1843 [MþNa]þ calcd for C18H27NNaO3, 328.1883.
(m,1H), 4.19 (dd, J¼9.0, 7.5 Hz,1H), 4.15 (dd, J¼9.1, 3.4 Hz,1H), 3.87 (m,
1H), 3.30 (dd, J¼13.3, 3.3 Hz, 1H), 2.72 (dd, J¼13.3, 9.8 Hz, 1H), 1.60 (m,
1H), 1.53 (m, 1H), 1.40 (m, 1H), 1.38e1.25 (m, 3H), 1.16 (m, 1H), 1.15 (d,
J¼6.7 Hz, 3H), 0.91 (d, J¼6.2 Hz, 3H), 0.89 (t, J¼7.2 Hz, 3H); 13C NMR
4.7. Synthesis of (2S,4S)-2,4-dimethylheptanoic acid (5)
4.7.1. (R)-4-benzyl-3-((2S,4S)-2,4-dimethylheptanoyl)-2-oxazolidinone
(14c). A stirred solution of racemic 2-methyl-1-pentanol (15, 414 mg,
4.05 mmol) in 20 mL of CH2Cl2 was cooled to ꢀ78 ꢂC under nitrogen
(CDCl3, 125 MHz)
d 178.0, 153.2, 135.5, 129.6 (2C), 129.1 (2C), 127.5,
66.1, 55.5, 41.0, 39.8, 38.2, 35.5, 30.3, 20.2, 19.2, 16.8, 14.4; HRMS (ESI)
m/z 340.1867 [MþNa]þ calcd for C19H27NNaO3, 340.1883.
atmosphere, to which pyridine (500 mL, 490 mg, 6.19 mmol) and Tf2O
(1.00 mL, 1.72 g, 7.34 mmol) were added. The mixture was stirred for
20 min at ꢀ78 ꢂC and for 40 min at 0 ꢂC, and quenched with satd aq
NH4Cl (20 mL). The aqueous layer was extracted with CHCl3 (20 mL).
The combined organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was chromatographed (SiO2, n-
hexane/EtOAc¼50:1) to give racemic 2-methylpentyl tri-
fluoromethanesulfonate (16, 415 mg, 1.77 mmol, 43.7%) as a colorless
liquid. 1H NMR spectra of 16 was identical with that of 12. To a stirred
solution of 13 (169 mg, 0.726 mmol) in 3 mL of dry THF at ꢀ78 ꢂC
under nitrogen atmosphere was added 0.48 mL of 1.9 M NaHMDS in
THF (0.912 mmol). The mixture was stirred for 30 min at ꢀ78 ꢂC and
then 16 (100 mg, 0.427 mmol) in 1.1 mL of dry THF was added. After
being stirred for 2.5 h at ꢀ78 ꢂC, the reaction mixture was allowed to
slowly warm to 0 ꢂC for 1 h, stirred for another 1 h at 0 ꢂC and
quenched with satd aq NH4Cl (3 mL). The aqueous layer was
extracted with EtOAc (3 mL), and the combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and concentrated
in vacuo. The residue was chromatographed (SiO2, n-hexane/
EtOAc¼10:1) to give a mixture of 14a and 14c (43.2 mg, 0.136 mmol,
31.9%, the ratio of 14a and 14c was 7:5 judged from the 1H NMR
spectrum) as a colorless oil. The mixture was subjected to reversed-
phase HPLC (Cosmosil 5C18-AR-II, 250ꢃ20 mm, H2O/MeOH (25:75)),
4.5.8. (2S,4R)-2,4-Dimethylheptanoic acid (4). To a stirred solution
of 14a (10.1 mg, 0.0318 mmol) in a mixed solvent of THF (0.25 mL)
and H2O (0.15 mL) was added LiOH$H2O (7.85 mg, 0.125 mmol) and
31% aq H2O2 (28.5 m
L, 0.260 mmol) at 0 ꢂC. The mixture was stirred
for 1.5 h at 0 ꢂC and concentrated in vacuo. The residue was diluted
with H2O (0.4 mL), washed with CH2Cl2 (2ꢃ0.4 mL), acidified with
6 N HCl until pH reached under 2, and extracted with CH2Cl2
(2ꢃ1 mL). The CH2Cl2 extracts of the acidified aqueous layer were
concentrated in vacuo to give 4 (4.6 mg, 0.029 mmol, 91%) as
a colorless liquid: ½a D
ꢁ
26þ11.2 (c 0.38, CHCl3); IR (neat) 2959, 2931,
2871, 1708, 1465 cmꢀ1
;
1H NMR (CDCl3, 500 MHz)
d 11.2 (br, 1H),
2.55 (ddq, J¼6.9, 6.9, 6.9 Hz, 1H), 1.58e1.46 (m, 2H), 1.38 (m, 1H),
1.36e1.24 (m, 2H), 1.20e1.08 (m, 2H), 1.16 (d, J¼6.9 Hz, 3H), 0.88 (t,
J¼7.0 Hz, 3H), 0.86 (d, J¼6.3 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d
183.2, 40.9, 39.4, 37.2, 30.3, 20.1, 19.4, 17.0, 14.4; HRMS (ESI) m/z
203.1007 [M-Hþ2Na]þ calcd for C9H17Na2O2, 203.1018.
4.6. PGME derivatives of (2S,4R)-2,4-dimethylheptanoic acid
(4a and 4b)
4.6.1. (R)-Methyl 2-((2S,4R)-2,4-dimethylheptanamido)-2-phenylace-
tate (4a). A solution of 4 (1.7 mg, 0.011 mmol), HATU (12.3 mg,
0.0324 mmol), HOAt (4.1 mg, 0.030 mmol), (R)-PGME$HCl (6.9 mg,
to yield 14c: ½a D
ꢁ
26ꢀ33.0 (c 0.73, CHCl3); IR (neat) 2958, 2929, 2872,
1783, 1698, 1456, 1387, 1350, 1235, 1207, 1100 cmꢀ1; 1H NMR (CDCl3,
500 MHz)
d 7.31e7.35 (m, 2H), 7.28 (m, 1H), 7.24e7.20 (m, 2H), 4.69
0.034 mmol) and DIEA (8.5
m
L, 6.4 mg, 0.050 mmol) in 0.4 mL of
(m, 1H), 4.18 (dd, J¼9.0, 7.6 Hz, 1H), 4.15 (dd, J¼9.0, 3.1 Hz, 1H), 3.94
(m,1H), 3.30 (dd, J¼13.3, 3.3 Hz,1H), 2.73 (dd, J¼13.3, 9.7 Hz,1H),1.87
(m, 1H), 1.47 (m, 1H), 1.42e1.24 (m, 3H), 1.19 (m, 1H), 1.17 (d, J¼6.7 Hz,
3H),1.11 (m,1H), 0.91 (d, J¼6.6 Hz, 3H), 0.89 (t, J¼7.1 Hz, 3H); 13C NMR
DMF was allowed stirred for 7.5 h at room temperature. The reaction
was quenched with satd aq NH4Cl (1 mL) and diluted with EtOAc
(1 mL). The organic layer was washed with satd aq NH4Cl (2ꢃ1 mL),
dried over anhydrous Na2SO4 and concentrated in vacuo. The resi-
due was separated by preparative TLC with CHCl3/MeOH (10:1)
followed by purification by reversed-phase HPLC (Cosmosil 5C18-
MS-II, 250ꢃ10 mm) with H2O/MeCN (40:60) to afford 4a (1.3 mg,
0.0043 mmol, 39%, tR¼21 min) as a colorless amorphous solid:
(CDCl3, 125 MHz)
d 177.9, 153.2, 135.5, 129.6 (2C), 129.1 (2C), 127.5,
66.1, 55.5, 41.5, 39.2, 38.2, 35.4, 30.7, 20.1, 20.1, 18.1, 14.4; HRMS (ESI)
340.1871 [MþNa]þ calcd for C19H27NNaO3, 340.1883.
4.7.2. (2S,4S)-2,4-Dimethylheptanoic acid (5). To a stirred solution
of 14c (12.4 mg, 0.0391 mmol) in a mixed solvent of THF (0.3 mL)
and H2O (0.2 mL) was added LiOH$H2O (6.2 mg, 0.125 mmol) and
½
a D
ꢁ
25ꢀ134.4 (c 0.15, CHCl3); IR (neat) 3288 (br), 2955, 2928, 2872,
1746, 1650, 1520, 1455, 1200, 1158 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d
7.38e7.30 (m, 5H), 6.39 (br d, J¼6.6 Hz, 1H), 5.59 (d, J¼7.1 Hz, 1H),
31% aq H2O2 (20 m
L, 0.182 mmol) at 0 ꢂC. The mixture was stirred for
3.74 (s, 3H), 2.37 (ddq, J¼6.9, 6.9, 6.9 Hz, 1H), 1.45 (m, 1H), 1.41e1.15
(m, 5H), 1.14 (d, J¼6.8 Hz, 3H), 1.05 (m, 1H), 0.83 (t, J¼6.9 Hz, 3H),
20 min at 0 ꢂC and concentrated in vacuo. The residue was diluted
with H2O (0.5 mL), washed with CH2Cl2 (4ꢃ0.5 mL), acidified with
6 N HCl until pH reached under 2, and extracted with CH2Cl2
(3ꢃ1 mL). The CH2Cl2 extracts of the acidified aqueous layer were
0.79 (d, J¼6.2 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d 176.3, 171.8,
136.9, 129.1 (2C), 128.6, 127.4 (2C), 56.3, 52.9, 41.6, 39.4, 39.0, 30.3,

5578
S. Kishimoto et al. / Tetrahedron 68 (2012) 5572e5578
concentrated in vacuo to give 5 (4.8 mg, 0.030 mmol, 77%) as
IFO3134, E. coli IFO 0392 or P. aeruginosa IFO13275 were suspended
in 1% agar medium and overlaid on solid medium plates. Methanol
solutions of tumescenamide C were loaded onto paper disks (f
a colorless liquid: ½a D
ꢁ
25þ17.5 (c 0.08, CHCl3); IR (neat) 2958, 2930,
2872, 1707, 1464, 1428, 1111 cmꢀ1; 1H NMR (CDCl3, 500 MHz)
d
2.58
(m, 1H), 1.73 (m, 1H), 1.48 (m, 1H), 1.39e1.21 (m, 3H), 1.19 (d,
J¼7.0 Hz, 3H), 1.19e1.06 (m, 2H), 0.89 (d, J¼6.6 Hz, 3H), 0.88 (t,
6 mm), which were dried and placed on the plates. After incubating
at 30 ꢂC for 3 days, growth inhibitory zone was measured. Maltose-
Bennett’s medium (NBRC medium 231; 0.1% of yeast extract, 0.1% of
fish extract, 0.2% of NZ amine and 1% of maltose$H2O) was used to
cultivate S. coelicolor, S. lividans and Streptomyces sp. KUSC_F05.
NBRC medium 802 (1% of polypepton, 0.2% of yeast extract and 0.1%
of MgSO4$7H2O) was used to cultivate S. aureus subsp. aureus, B.
subtilis, E. coli and P. aeruginosa. Growth inhibition against yeast
cells including S. pombe JY3, S. cerevisiae BY4741 and C. albicans
IFO1594, was examined in a liquid medium as described before.15
J¼7.1 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d 182.4, 41.4, 39.4, 37.3,
30.6, 20.0, 19.7, 18.0, 14.4; HRMS (ESI) m/z 203.1004 [M-Hþ2Na]þ
calcd for C9H17Na2O2, 203.1018.
4.8. PGME derivatives of (2S,4R)-2,4-dimethylheptanoic acid
(5a and 5b)
4.8.1. (S)-Methyl 2-((2S,4R)-2,4-dimethylheptanamido)-2-phenylace-
tate (5a). A solution of 5 (1.9 mg, 0.012 mmol), HATU (14.9 mg,
0.0392 mmol), HOAt (4.8 mg, 0.035 mmol), (S)-PGME$HCl (7.2 mg,
Acknowledgements
0.036 mmol) and DIEA (9.0 mL, 6.8 mg, 0.052 mmol) in 0.4 mL of
DMF was allowed stirred for 7.5 h at room temperature. Following
the isolation procedure described above for 4a, 5a (1.8 mg,
0.0059 mmol, 49%, tR¼21 min) was purified and obtained as a col-
This work was supported in part by research grants from the
Japan Society for the Promotion of Sciences (JSPS) and the Ministry
of Education, Culture, Sports, Science and Technology (MEXT) of
Japan.
orless amorphous solid: ½a D
ꢁ
26ꢀ144.4 (c 0.15, CHCl3); IR (neat) 3299
(br), 2957, 2930, 2873, 1749, 1652, 1533, 1456, 1219 cmꢀ1 1H NMR
;
(CDCl3, 500 MHz)
d
7.36e7.29 (m, 5H), 6.39 (br d, J¼6.6 Hz,1H), 5.59
(d, J¼7.2 Hz, 1H), 3.73 (s, 3H), 2.39 (m, 1H), 1.66 (m, 1H), 1.34e1.15
(m, 4H), 1.15 (d, J¼6.9 Hz, 3H), 1.10 (m, 1H), 1.01 (m, 1H), 0.83 (d,
J¼6.6 z, 3H), 0.80 (t, J¼7.1 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
Supplementary data
1D and 2D NMR spectra for tumescenamide C, 1D NMR data for
other synthetic compounds and LC-MS chromatogram of FDLA
derivatives. Supplementary data related to this article can be found
online at doi:10.1016/j.tet.2012.04.075.
d
176.1, 171.7, 136.9, 129.1 (2C), 128.6, 127.4 (2C), 56.3, 52.9, 42.0,
39.5, 39.1, 30.4, 20.0, 19.7, 18.6, 14.4; HRMS (ESI) m/z 328.1843
[MþNa]þ calcd for C18H27NNaO3, 328.1883.
4.8.2. (S)-methyl
2-((2S,4S)-2,4-dimethylheptanamido)-2-phenyl-
acetate (5b). A solution of 5 (2.0 mg, 0.013 mmol), HATU (9.6 mg,
0.025 mmol), HOAt (4.0 mg, 0.029 mmol), (S)-PGME$HCl (11.5 mg,
References and notes
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0.057 mmol) and DIEA (15.0 mL,11.3 mg, 0.0872 mmol) in 0.25 mL of
DMF was stirred for 7.5 h at room temperature. 5b (2.0 mg,
0.0066 mmol, 51%, tR¼21 min) was purified as described above and
obtained as a colorless oil: ½a D
ꢁ
26þ155.7 (c 0.16, CHCl3); IR (neat)
3296, 2957, 2928, 2872, 1749, 1649, 1527, 1456, 1206 cmꢀ1; 1H NMR
(CDCl3, 500 MHz)
d
7.37e7.30 (m, 5H), 6.37 (br d, J¼6.8 Hz,1H), 5.59
(d, J¼7.1 Hz,1H), 3.73 (s, 3H), 2.40 (m, 1H),1.71 (m,1H), 1.45 (m, 1H),
1.38e1.20 (m, 3H), 1.16e1.04 (m, 2H), 1.11 (d, J¼6.9 Hz, 3H), 0.89 (d,
J¼6.6 Hz, 3H), 0.86 (t, J¼7.1 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d
176.0, 171.7, 136.9, 129.1 (2C), 128.6, 127.4 (2C), 56.3, 52.9, 41.9,
39.6, 39.1, 30.5, 20.1, 19.8, 18.7, 14.5; HRMS (ESI) m/z 328.1839
[MþNa]þ calcd for C18H27NNaO3, 328.1883.
4.9. Antimicrobial assay
Growth inhibitory activity of 1 against gram positive and gram
negative bacteria was examined by paper disc diffusion assay.
Spores of S. coelicolor A3(2), S. lividans TK23 or Streptomyces sp.
KUSC_F05, or cells of S. aureus subsp. aureus IFO13276, B. subtilis