Absolute Configuration and Antibiotic Activity of Piceamycin

Article abstract of DOI:10.1021/acs.jnatprod.9b00678

The cultivation of a Streptomyces sp. SD53 strain isolated from the gut of the silkworm Bombyx mori produced two macrolactam natural products, piceamycin (1) and bombyxamycin C (2). The planar structures of 1 and 2 were identified by a combination of NMR, MS, and UV spectroscopic analyses. The absolute configurations were assigned based on chemical and chromatographic methods as well as ECD calculations. A new chromatography-based experimental method for determining the configurations of stereogenic centers β to nitrogen atoms in macrolactams was established and successfully applied in this report. These compounds exhibited significant bioactivities against the silkworm entomopathogen Bacillus thuringiensis and various human pathogens as well as human cancer cell lines. In particular, piceamycin potently inhibited Salmonella enterica and Proteus hauseri with MIC values of 0.083 μg/mL and 0.025 μg/mL, respectively. The biosynthetic pathway involved in the formation of the cyclopentenone moiety in piceamycin is discussed.

Full text of DOI:10.1021/acs.jnatprod.9b00678

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Absolute Configuration and Antibiotic Activity of Piceamycin
Yern-Hyerk Shin, Saeyeon Kang, Woong Sub Byun, Chang-Wook Jeon, Beomkoo Chung, Ji Yoon Beom,
Suckchang Hong, Jeeyeon Lee, Jongheon Shin, Youn-Sig Kwak, Sang Kook Lee, Ki-Bong Oh,
Yeo Joon Yoon, and Dong-Chan Oh*
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ABSTRACT: The cultivation of a Streptomyces sp. SD53 strain
isolated from the gut of the silkworm Bombyx mori produced two
macrolactam natural products, piceamycin (1) and bombyxamycin
C (2). The planar structures of 1 and 2 were identified by a
combination of NMR, MS, and UV spectroscopic analyses. The
absolute configurations were assigned based on chemical and
chromatographic methods as well as ECD calculations. A new
chromatography-based experimental method for determining the
configurations of stereogenic centers β to nitrogen atoms in
macrolactams was established and successfully applied in this
report. These compounds exhibited significant bioactivities against
the silkworm entomopathogen Bacillus thuringiensis and various human pathogens as well as human cancer cell lines. In particular,
piceamycin potently inhibited Salmonella enterica and Proteus hauseri with MIC values of 0.083 μg/mL and 0.025 μg/mL,
respectively. The biosynthetic pathway involved in the formation of the cyclopentenone moiety in piceamycin is discussed.
acrocyclic lactams are a structural class of biologically
possessing a branching methyl group β to the amide nitrogen,
Mactive natural products mainly produced by actino- which is a motif commonly found in macrolactams prepared
bacteria.1 Members of this unique class of natural products are
from a β-amino acid, and we used electronic circular dichroism
(ECD) calculations to elucidate the stereogenic center in the
cyclopentenone of 1. We also report the evaluation of 1 and 2
in antibacterial and antiproliferative assays as well as the
predicted post-PKS modification biosynthetic steps leading to
1 from 2.
biosynthesized by type I modular polyketide synthases (PKS),
which adopt a starting unit originating from an amino acid to
incorporate a nitrogen atom into the macrocyclic ring.2 The
carbon frameworks of macrolactams are diversified by
secondary cyclizations in their macrocycles as well as by the
number of linear PKS modules, which governs the elongation
of the polyketide chain during biosynthesis.3 Recently, we
discovered two new macrocyclic lactams, bombyxamycins A
and B, and their biosynthetic gene cluster, from a gut
bacterium, Streptomyces sp. SD53, from the silkworm Bombyx
mori.4 Further investigations based on chemical profiling of the
bacterial strain during cultivation in various culture media
allowed us to detect additional macrocyclic lactams from the
strain SD53. Large-scale cultivation of the strain in a specific
medium enabled the production of piceamycin (1), which was
discovered from Streptomyces sp. GB4-2 isolated from the
mycorrhizosphere of Picea abies,5 along with a new metabolite,
bombyxamycin C (2). Although the planar structure of
piceamycin was reported previously, the configurations of its
two stereogenic centers remained unassigned. One of the
centers is at the β-position of the amide nitrogen and has a
branching methyl substituent, and the other is at the junction
of the cyclopentenone and originates from a secondary
cyclization after the biosynthesis of the 26-membered macro-
cycle. In this work, we developed a convenient chromatog-
raphy-based method to determine the chirality of a carbon
RESULTS AND DISCUSSION
During the cultivation of Streptomyces sp. SD53 in YPM
medium, a metabolite with a polyene UV signature (λmax values
■
Received: July 22, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
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1
Table 1. H and 13C NMR Data for Piceamycin (1) and Bombyxamycin C (2) in DMSO-d6
a
b
piceamycin (1)
bombyxamycin C (2)
position
δC
type
δH
mult (J in Hz)
δC
type
δH
mult (J in Hz)
1
2
3
4
5
6
7
8
164.9
122.4
132.1
124.2
133.7
121.3
143.8
49.6
C
165.9
123.3
130.6
124.4
133.3
120.9
140.3
135.7
129.7
C
CH
CH
CH
CH
CH
CH
C
5.08
6.22
7.21
6.10
6.02
5.99
d (11.5)
CH
CH
CH
CH
CH
CH
C
5.58
6.59
6.82
6.24
6.49
6.50
d (11.5)
dd (11.5, 11.5)
dd (11.5, 11.5)
dd (11.5, 11.5)
dd (15.0, 11.5)
d (15.0)
dd (11.5, 11.5)
dd (11.5, 11.5)
dd (11.5, 11.5)
dd (15.0, 11.5)
d (15.0)
9a
49.2
CH2
2.43
2.39
d (19.0)
d (19.0)
CH
5.84
dd (7.0, 7.0)
9b
10
11
12a
12b
13
14
15
16
17
18
19
20
21
22
23
24
25a
25b
1′
203.4
155.2
138.9
C
C
C
33.7
67.3
43.0
CH2
CH
CH2
2.19
4.09
3.08
2.18
m
m
dd (13.0, 10.5)
dd (13.0, 4.5)
192.3
129.2
142.3
129.8
138.5
127.3
134.1
127.3
132.2
129.2
136.2
33.3
C
199.1
130.5
144.3
131.2
136.6
127.5
134.6
127.0
132.3
132.3
137.8
34.4
C
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH2
7.24
7.06
6.52
7.22
6.20
6.29
6.66
6.59
6.06
5.13
2.68
3.36
2.65
1.58
0.96
11.18
7.52
d (15.0)
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH2
5.99
7.00
6.42
6.84
6.04
6.19
6.39
6.58
5.97
5.19
2.71
3.36
2.74
1.49
0.96
d (15.5)
dd (15.0, 11.0)
dd (14.5, 11.0)
dd (14.5, 11.0)
dd (11.0, 10.5)
dd (10.5, 10.5)
dd (15.0, 10.5)
dd (15.0, 10.0)
dd (10.0, 10.0)
dd (10.0, 10.0)
m
dd (15.5, 11.0)
dd (14.5, 11.0)
dd (14.5, 11.5)
dd (11.5, 11.5)
dd (11.5, 11.5)
dd (14.5, 11.5)
dd (14.5, 11.0)
dd (11.0, 10.5)
dd (10.5, 10.5)
m
c
43.6
m
m
s
44.0
m
m
s
d (6.5)
28.4
17.7
CH3
CH3
12.3
17.7
CH3
CH3
2′
11-OH
25-NH
d (6.5)
br s
dd (10.0, 2.0)
7.71
dd (10.0, 2.5)
a
b
c
1H 500 MHz, 13C 212.5 MHz. 1H 850 MHz, 13C 212.5 MHz. Overlapped.
of 296 and 410 nm), which is unlike those of bombyxamycins
A and B, was detected by LC/MS analysis. Subsequent
chromatographic separation yielded a pure orange powder,
enabling spectroscopic analysis of the metabolite (1). The
molecular formula of 1 was identified as C27H29NO4,
corresponding to 14 double bond equivalents, by HR-FAB-
MS data. The planar structure of 1 was deduced as that of
piceamycin, which was previously reported from Streptomyces
sp. GB4-2 isolated from the mycorrhizosphere of Picea abies,
by comparing its H and 13C NMR chemical shifts in DMSO-
1
d6 (Table 1) with those in the literature.5 It was further
confirmed to be piceamycin by COSY, HSQC, and HMBC
spectroscopic analyses (Figure 1). Careful comparison of the
NMR data of 1 with the literature data of piceamycin revealed
that the chemical shifts and coupling constants of the
methylene protons at C-9 must be revised. In the original
report, H2-9 was assigned as δH 2.42 (d, J = 5.53 Hz).
However, if the methylene protons are magnetically equivalent,
these protons should appear as a singlet because this
methylene group is flanked by a ketone group and an aliphatic
quaternary carbon. In fact, these two protons are not
magnetically equivalent and should be considered separate
signals at δH 2.43 and 2.39 with 19.0 Hz geminal coupling
(Table 1), which is characteristic of a five-membered ring.6
Figure 1. Key COSY and HMBC correlations of piceamycin (1) and
bombyxamycin C (2).
The two stereogenic centers of piceamycin (1) remain
unassigned, prompting us to take on this challenge. Indeed, the
stereogenic center at the β-position of the amide nitrogen is
derived from the β-amino acid starting material (3-amino-2-
methylpropanoic acid) and is a common motif to all
macrocyclic lactams in this class, as shown in cyclamenol A,7
macrotermycins A−D,8 sceliphrolactam,9 niizalactam C,10 and
vicenistatin11 (Figure 2). However, the absolute configurations
of the chiral carbons at this position can be either R or S
depending on the natural product, indicating that L-glutamic
acid is being converted to 3-amino-2-methylpropanoic acid
stereoselectively by an unknown mechanism in the biosyn-
B
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Figure 2. Structures of macrocyclic lactams biosynthetically derived from β-amino acid units.
Scheme 1. Preparing S- and R-PGME Amide Products from 1 and 2
thesis of the starting material. The unambiguous determination
of this β-amino acid starting material-derived stereogenic
center has been time-consuming and inefficient as a total
synthesis or at least a 4-step degradation process of a
directions in the S-R (or R-S) PGME amide, resulting in
weaker interactions with the reversed-phase stationary phase
and thus faster elution (Figure 3a,b). The PGME derivatives
originating from 1 showed retention times consistent with
those of the PGME amides of 2S-3-amino-2-methylpropanoic
acid (Figure 3c), confirming the absolute configuration of 3-
amino-2-methylpropanoic acid derived from piceamycin (1) as
2S and thus 24R in 1. This convenient procedure provides an
empirical rule: if the S-PGME amide of the 3-amino-2-
methylpropanoic acid unit derived from a macrocyclic lactam
elutes slower than its R-PGME amide, this β-amino acid must
possess a 2S-configuration. Conversely, the 2R configuration
can be assigned when the R-PGME amide elutes later than its
S-PGME adduct. This method was also conducted with a small
quantity of piceamycin (1, 0.2 mg), which provided the
identical experimental results, indicating its validity on the 0.2
mg scale (Figure S30).
The other asymmetric carbon in piceamycin (1) is the
unprotonated C-8 moiety of the cyclopentenone. Because
chemical derivatization of this chiral center for stereochemical
analysis is not possible, electronic circular dichroism (ECD)
calculations were utilized.14 Three-dimensional models of two
possible diastereomers of 1 (8S/24R and 8R/24R) were
constructed, and ECD calculations were conducted for each
diastereomer. The experimental ECD spectrum of 1 showed a
positive Cotton effect at 300 nm and a negative Cotton effect
at 455 nm. The calculated spectrum of the 8S,24R
diastereomer (positive Cotton effect at 305 nm and negative
Cotton effect at 460 nm) (Figure 5) was similar to the
experimental ECD spectrum, indicating that the configuration
of C-8 of 1 is 8S.
1
macrocyclic lactam followed by product purification and H
NMR spectroscopic analysis were required.12
Therefore, we developed a new method for elucidating this
common but problematic stereogenic center by combining
simple chemical derivatization and chromatographic analysis
1
without preparative purification and H NMR spectroscopic
analysis of the products (Scheme 1). First, to obtain a β-amino
acid unit bearing the C-24 stereogenic center, piceamycin (1)
was sequentially subjected to ozonolysis and acid hydrolysis.
Then, portions of the hydrolysate were separately derivatized
with S- or R-phenylglycine methyl ester (PGME).13 The S- and
R-PGME products were analyzed via LC/MS, and their
retention times were compared with those of the PGME
amides (3−6) prepared with authentic 2S- and 2R-3-amino-2-
methylpropanoic acids (Figure 3). The S-PGME adduct (3) of
2S-3-amino-2-methylpropanoic acid eluted later (8.3 min)
than the R-PGME derivative (4) (6.4 min) (Figure 3). LC/MS
analysis of the PGME adducts of 2R-3-amino-2-methylpropa-
noic acid showed that its R-PGME adduct (6) eluted later than
the S-PGME derivative (5), as expected. The elution order
could be deduced based on the conformations determined by
density functional theory (DFT) calculations (Figure 4). The
S-S (or R-R) PGME amide has a hydrophobic methyl group
from the β-amino acid and the phenyl group of PGME on the
same side, which allows the formation of more hydrophobic
interaction with the reversed-phase stationary phase, resulting
in a higher retention time (Figure 3a,b). On the other hand,
these hydrophobic functional groups are oriented in opposite
C
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Figure 5. Comparison of the experimental ECD data of 1 with the
calculated ECD data; black line: experimental ECD data; red line:
calculated ECD data (8S, 24R); blue line: calculated ECD data (8R,
24R).
Bombyxamycin C (2), purified as a dark yellow powder, was
found to have a formula of C27H33NO3, corresponding to 12
degrees of unsaturation, by HR-FABMS. Analysis of its 1H and
HSQC NMR spectroscopic data (Table 1) indicated that 2
possessed one amide proton (δH 7.71), 17 olefinic protons (δH
7.00−5.19), and one carbinol proton (δH 4.09) in the
deshielded region. In addition, six methylene protons (δH
3.36, 3.08, 2.74, 2.19 (2H), and 2.18), one aliphatic methine
proton (δH 2.71) and two methyl groups (δH 1.49 and 0.96)
Figure 3. Chromatographic analysis for the determination of the
absolute configuration of the β-amino acid unit (3-amino-2-
methylpropanoic acid) of 1 by PGME derivatization. LC/MS
chromatograms of the S- and R-PGME derivatives of (a) 2S-3-
amino-2-methylpropanoic acid (3 and 5), (b) 2R-3-amino-2-
methylpropanoic acid (4 and 6), and (c) 3-amino-2-methylpropanoic
acid from 1 (Phenomenex, Luna, C18(2), 100 × 4.6 mm; 10−25%
CH3CN−H2O over 30 min with 0.1% formic acid; flow rate: 0.7 mL/
min; ion extraction for [M + H]+ m/z at 251).
were identified in the shielded region. Interpretation of the 13
C
and HSQC NMR spectra of 2 indicated the presence of 2
carbonyl carbons (δC 199.1 and 165.9) and 18 olefinic methine
carbons (δC 144.3, 140.3, 137.8, 136.6, 135.7, 134.6, 133.3,
132.3, 132.3, 131.2, 130.6, 130.5, 129.7, 127.5, 127.0, 124.4,
123.3, and 120.9) in the structure. In addition, one oxygen-
bound carbon (δC 67.3) and six aliphatic carbons (δC 44.0,
43.0, 34.4, 33.7, 17.7, and 12.3) were identified (Table 1).
Initial analysis of its 1D and HSQC NMR spectra suggested
that bombyxamycin C (2) should possess 2 carbonyl groups
and 9 double bonds, explaining 11 out of the 12 unsaturations
inherent in the molecular formula. Therefore, 2 must have one
ring.
The discrete spin systems were elucidated by analysis of the
COSY NMR correlations. Consecutive COSY correlations of
the olefinic methine protons, from H-2 (δH 5.58) to H-7 (δH
6.50), revealed the connectivity from C-2 (δC 123.3) to C-7
(δC 140.3). Another spin system (C-9−C-12 linkage) was
1
constructed via three-bond H−1H couplings among H-9 (δH
5.84), H2-10 (δH 2.19), H-11 (δH 4.09), and H2-12 (δH 3.08
and 2.18). A long spin system from H-14 (δH 5.99) to 25-NH
(δH 7.71) was established based on the COSY signals of the
protons belonging to this system. CH3-2′ could be assigned at
1
C-24 by the H−1H COSY correlation between H3-2′ (δH
0.96) and H-24 (δH 2.71). An HMBC correlation between H-2
and the amide carbon C-1 (δC 165.9) indicated that C-1 is
adjacent to C-2. 1H−13C HMBC signals from H3-1′ (δH 1.49)
to C-7, C-8 (δC 135.7), and C-9 (δC 129.7) connected the first
Figure 4. Energy-minimized structures of S- and R-PGME amides of
3-amino-2-methylpropanoic acids (3−6).
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and the second spin systems, forming the tetraenone
chromophore of 2. This partial structure could be combined
with the long spin system bearing the pentaene through the C-
13 ketone carbon based on the HMBC correlations from H2-
12 and H-14 to C-13 (δC 199.1). Furthermore, the 25-NH/C-
1 1H−13C correlation connected 25-NH to C-1, generating the
26-membered macrocyclic structure of 2. The geometries of
the double bonds in bombyxamycin C (2) could be assigned as
2Z, 4Z, 6E, 14E, 16E, 18Z, 20E, and 22Z from the 3JHH values
between olefinic protons (Table 1). These geometries are
identical to those of piceamycin (1). In addition, the NOESY
correlation between H3-1′ and H-9 enabled the identification
of the 8Z geometry (Figure S12).
Bombyxamycin C (2) has two stereogenic centers: C-11
bearing a secondary alcohol and C-24 at the β-position of the
amide nitrogen. The secondary hydroxy group at C-11 was
esterified with R- and S-α-methoxy-α-(trifluoromethyl)-
phenylacetyl chloride (MTPA-Cl), yielding S- and R-MTPA
esters (2a and 2b) of 2, respectively. Detailed analyses of the
1H and COSY NMR spectra of 2a and 2b confirmed that the
absolute configuration at C-11 was R based on the distribution
of the signs of the ΔδS‑R values by the modified Mosher’s
method (Figure 6).15
Piceamycin and bombyxamycin C (1 and 2) appear to be
biosynthesized through the bombyxamycin biosynthetic path-
way (GenBank accession No. MK433001).4 The post-PKS
modification pathways involved in the transformation of
bombyxamycin C into piceamycin seem to be similar to the
proposed post-PKS tailoring steps in the synthesis of
hitachimycin, which also possesses a pentacarbocyclic moiety
in its structure.16 Although the mechanism of the formation of
the cyclopentenone moiety in hitachimycin was not exper-
imentally elucidated, we could predict the post-PKS pathway
leading to piceamycin based on the presence of three highly
homologous enzymes in the bombyxamycin gene cluster
(BomK, BomL, and BomP) and hitachimycin cluster. First,
the hydroxy group at C-11 of bombyxamycin C is likely
converted to a ketone by BomP (GenBank accession No.
MN095224), a putative short-chain dehydrogenase/reductase.
Then, BomK (putative cytochrome P450) catalyzes a
hydroxylation at C-10 and further oxidation to a carbonyl.
With the catalytic action of sugar phosphate isomerase/
epimerase (BomL), this α,β-unsaturated ketone moiety in the
intermediate could be transformed into a cyclopentenone by a
Michael addition (Figure 7), as proposed in the biosynthesis of
hitachimycin.
Piceamycin (1) strongly inhibited the silkworm pathogen B.
thuringiensis KACC 10168 with an MIC value of 3.2 μg/mL,
whereas bombyxamycin C (2) displayed weak activity (MIC =
3.8 × 102 μg/mL). Bombyxamcyin A showed even weaker
inhibitory activity (MIC = 2.1 mg/mL) (Table 2). In the
evaluation of these compounds against human pathogenic
bacteria, piceamycin (1), which was previously reported
inactive against Gram-negative bacteria,5 displayed potent
activity against Gram-negative pathogens Proteus hauseri
NBRC 3851 (MIC = 0.025 μg/mL) and Salmonella enterica
ATCC 14028 (MIC = 0.083 μg/mL). Piceamycin also
inhibited Gram-positive pathogenic bacteria Staphylococcus
aureus ATCC 25923, Enterococcus faecalis ATCC 19433, and E.
faecium ATCC 19434 (MIC = 0.025−32 μg/mL). Bomb-
yxamycin C (2) exhibited only weak activity against Gram-
negative pathogens P. hauseri and S. enterica with an MIC value
of 1.3 × 102 μg/mL, and Gram-positive pathogen E. faecium
(MIC = 1.3 × 102 μg/mL) but did not suppress the growth of
S. aureus and E. faecalis. These results indicated the additional
cyclization in piceamycin (1) play a significant role in their
antibacterial activity mechanism.
Figure 6. ΔδS‑R values in ppm of the S- and R-MTPA esters of 2.
The absolute configuration of the asymmetric carbon at C-
24 of 2 was also determined as R by the same three-step
reaction followed by LC/MS analysis as used for 1, and this
process did not require NMR experiments. As proof of the
versatility of the method developed in this work, this chiral
center was successfully established using only 1 mg of 2.
Figure 7. Predicted post-PKS modification pathway for piceamycin (1).
E
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Table 2. Antibacterial Activities of 1 and 2 against Human and Silkworm Pathogenic Bacterial Strains
MIC values (μg/mL)
strains
1
2
ampicillin
tetracycline
streptomycin
S. aureus ATCC 25923
E. faecalis ATCC 19433
E. faecium ATCC 19434
P. hauseri NBRC 3851
K. pneumoniae ATCC 10031
S. enterica ATCC 14028
E. coli ATCC 25922
0.025 0.010
>1.3 × 102
0.17 0.06
0.50 0.00
1.0 0.0
0.030 0.000
>1.3 × 102
0.25 0.00
-
-
-
-
-
-
-
-
-
-
-
32
0
>1.3 × 102
5.3 1.9
1.3 × 102 0.0 × 102
1.3 × 102 0.0 × 102
>1.3 × 102
1.3 × 102 0.0 × 102
>1.3 × 102
3.8 × 102 0.1 × 102
0.025 0.010
>1.3 × 102
0.083 0.033
>1.3 × 102
3.2 0.1
0.44 0.11
-
-
-
16
-
0
B. thuringiensis KACC 10168
1.3 × 102 0.1 × 102
The antiproliferative activities of 1 and 2 against lung cancer
A549, colon cancer HCT116, stomach cancer SNU638, liver
cancer SK-HEP-1, breast cancer MDA-MB-231, and leukemia
K562 were measured (Table 3). Piceamycin (1) exhibited
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation data were
measured at 20 °C using a JASCO P-2000 polarimeter with a 1 cm
quartz cell. Ultraviolet (UV) spectral data and circular dichroism data
were recorded on an Applied Photophysics Chirascan plus circular
dichroism detector using a 1 cm quartz cell. Infrared (IR) spectra
were recorded using a Thermo N1 COLET iS10 spectrometer. 1D
and 2D NMR spectra were acquired on Bruker Avance 850 MHz and
500 MHz spectrometers at the National Center for Inter-University
Research Facilities (NCIRF) at Seoul National University. Low-
resolution electrospray ionization (LR-ESI) LC/MS data were
obtained on an Agilent Technologies 6130 quadrupole mass
spectrometer combined with an Agilent Technologies 1200-series
HPLC using a reversed-phase C18(2) column (Phenomenex Luna,
100 × 4.6 mm). High-resolution fast atom bombardment mass
spectrometry (HR-FAB-MS) data were acquired on a JEOL JMS-600
W high-resolution mass spectrometer at the NCIRF.
Table 3. Antiproliferative Activities of 1 and 2 against
Various Human Cancer Cell Lines
IC50 values (μM)
cell lines
A549
HCT116
SNU638
SK-HEP-1
MDA-MB-231
K562
1
2
etoposide
0.28 0.04
0.22 0.03
0.38 0.06
0.32 0.05
0.66 0.08
0.74 0.10
0.78 0.10
1.01 0.09
0.85 0.04
1.87 0.11
1.00 0.04
0.96 0.07
0.60 0.04
1.36 0.06
0.52 0.03
0.38 0.05
3.32 0.09
0.98 0.08
Cultivation and Extraction. Spores of Streptomyces sp. SD53
were inoculated into 125 mL of YPM liquid medium (2 g of yeast
extract, 2 g of peptone, and 4 g of mannitol per 1 L of sterilized water)
in a 500 mL Erlenmeyer flask to prepare the seed culture, and the
culture was shaken at 180 rpm at 30 °C. The YPM seed culture of the
SD53 strain (25 mL) was inoculated into 1 L of YPM liquid medium.
The mixture was cultivated for 5 days (160 rpm, 30 °C) and extracted
with ethyl acetate (EtOAc). The organic extract, which contained
piceamycin (1), was concentrated in vacuo by evaporating the EtOAc.
In total, 24 L of YPM culture was extracted, and 3.5 g of extract was
harvested. For preparing bombyxamycin C (2), the seed culture was
prepared in the same manner as described above. A portion of the 2-
day-old seed culture (5 mL) was transferred into 125 mL of K liquid
medium (25 g of starch, 15 g of soybean peptone, and 2 g of yeast
extract per 1 L of sterilized water) in a 500 mL Erlenmeyer flask. After
incubating the culture for 5 days (30 °C, 180 rpm), the whole culture
(24 L) was extracted with EtOAc, and the extract was dried under low
pressure, yielding 5 g of dry material.
Purification of Piceamycin and Bombyxamycin C (1 and 2).
The extract from the YPM culture was filtered through a syringe filter
(Advantec, 25HP045AN) and directly injected into a semipreparative
HPLC instrument (YMC C18(2) column, 5 μm, 250 × 10 mm). Pure
piceamycin (1, 30 mg) was obtained at an elution time of 24 min
under an isocratic solvent system (58% CH3CN−H2O, detection: UV
280 nm, flow rate: 2 mL/min). The K medium extract was dissolved
in MeOH, filtered through a syringe filter (Advantec, 25HP045AN),
and directly injected into an HPLC system equipped with a C18(2)
column (YMC, 5 μm, 250 × 10 mm). Under isocratic solvent
conditions (58% CH3CN−H2O, detection: UV 280 nm, flow rate: 2
mL/min), bombyxamycin C (2) eluted at 14 min. Bombyxamycin C
was further purified by HPLC with a gradient solvent system (YMC
C18(2) column, 5 μm, 250 × 10 mm, 65−90% CH3OH−H2O over 60
min with 0.1% formic acid, UV 280 nm, flow rate: 2 mL/min)
(retention time: 21 min, 5 mg).
potent cytotoxicity comparable to that of the positive control
(etoposide) against these cancer cell lines, with IC50 values
between 0.22 and 0.74 μM. Bombyxamycin C (2) also
inhibited the proliferation of the tested cancer cells (IC50
=
0.78−1.87 μM).
Piceamycin and bombyxamycin C (1 and 2) did not display
the growth inhibition activity against the human pathogenic
fungi Candida albicans ATCC 10231, Aspergillus fumigatus
HIC 6094, Trichophyton rubrum NBRC 9185, or Trichophyton
mentagrophytes IFM 40996.
In summary, a chemical study of the silkworm gut bacterial
strain, Streptomyces sp. SD53, which produced previously
reported macrocyclic lactams, bombyxamycins A and B,
allowed the identification of piceamycin (1) and a new
bombyxamycin derivative (2), which is a geometric isomer of
bombyxamycin A: bombyxamycin C (2) possesses the 16E
configuration in contrast to the 16Z geometry of bomb-
yxamycin A. In the course of determining the absolute
configuration of the center β to the amide bond of the
macrocyclic lactams (1 and 2), a new chromatography-based
method using PGME derivatization was developed. This
method provides a convenient approach for elucidating the
absolute configurations of commonly encountering macro-
lactams biosynthesized from 3-amino-2-methylpropanoic acid
as a starting material. The significant antibiotic activity of
piceamycin against the silkworm entomopathogen Bacillus
thuringiensis KACC 10168 indicates the possibility that the
silkworm Bombyx mori might utilize such antibiotic bacterial
metabolites to protect against potential entomopathogenic
threats. In addition, the potent antibacterial activities of 1
against the Gram-negative pathogens Salmonella enterica and
Proteus hauseri could provide a structural lead for the
development of new antibiotics.
Piceamycin (1). Orange powder; UV (MeOH) λmax (log ε) 296
(2.52), 410 (1.89) nm; ECD (c 0.7 × 10−4 M, MeOH) λmax (Δε) 228
(−1.25), 300 (1.31), and 455 (−2.99) nm; IR (neat) νmax 2970, 2870,
1349, 1051, 1022, and 1010 cm−1. For NMR spectral data, see Table
F
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1. HR-FAB-MS m/z 432.2174 [M + H]+ (calcd for C27H30NO4,
432.2175). [α]2D0 was not exactly measurable (−619 to −487).
Bombyxamycin C (2). Dark yellow powder; UV (MeOH) λmax (log
ε) 320 (2.42), 396 (1.95) nm; ECD (c 0.1 × 10−4 M, MeOH) λmax
(Δε) 216 (−1.01), 244 (2.72), 290 (0.44), 320 (1.74), and 376
(−1.50) nm; IR (neat) νmax 2920, 2860, 1639, 1052, 1032, and 1010
cm−1. For NMR spectral data, see Table 1. HR-FAB-MS m/z
420.2534 [M + H]+ (calcd for C27H34NO3, 420.2539). [α]2D0 was not
exactly measurable (+86 to + 140).
R-PGME Amide of 2R-3-Amino-2-methylpropanoic acid (6).
[α]2D0 −46 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (2.70) nm;
ECD (c 0.4 × 10−4 M, MeOH) λmax (Δε) 219 (−4.31) nm; IR (neat)
νmax 3703, 3671, 2972, 2868, 1058, and 1011 cm−1; 1H NMR
(CD3OD, 850 MHz) δH 7.4241−7.3442 (5H), 5.5213 (1H, s),
3.7187 (3H, s), 3.1549 (1H, dd, J = 12.8, 8.2 Hz), 3.0183 (1H, dd, J =
12.8, 4.6 Hz), 2.8745 (1H, m), and 1.1992 (3H, d, J = 7.1 Hz); 13C
NMR (CD3OD, 212.5 MHz) δC 175.4, 172.9, 137.2, 130.0, 130.0,
129.8, 128.8, 128.8, 58.2, 53.2, 42.9, 38.5, and 16.2; HR-FAB-MS m/z
251.1394 [M + H]+ (calcd for C13H19N2O3, 251.1396).
Preparing PGME Amides of 2S- and 2R-3-Amino-2-
methylpropanoic Acid (3−6). A commercial racemic mixture of
3-amino-2-methylpropanoic acid (CAS no. 144-90-1, Sigma-Aldrich)
was purchased and derivatized with S- and R-PGME. To prepare the
S-PGME amides, 30 mg of the β-amino acid was dissolved in
anhydrous dimethylformamide (DMF, 1.5 mL) and treated with S-
PGME (20 mg), hydroxybenzotriazole hydrate (HOBt, 20 mg),
benzotriazole-1-yl-oxytrispyrrolidinophosphonium hexafluorophos-
phate (PyBOP, 10 mg), and 4-methylmorpholine (300 μL). The
reaction mixture was stirred for 3 h at rt, quenched by adding 5% HCl
solution (3 mL), and concentrated in vacuo. The dried products were
injected onto a preparative HPLC column (Phenomenex Luna C18(2)
10 μm, 250 × 21.2 mm) and purified under a gradient solvent system
(10−30% CH3CN−H2O over 20 min with 0.1% formic acid,
detection: UV 210 nm, flow rate: 10 mL/min). As a result, two
different S-PGME amides (3 and 5) eluted at different retention times
(5 at 19.5 min and 3 at 20.5 min, respectively), and the molecular
formula of each product was determined to be C13H18N2O3 by HR-
FAB-MS ([M + H]+ m/z at 251). By comparing the LC/MS retention
times of the two S-PGME amides with that of the S-PGME amide
derived from authentic 2S-3-amino-2-methylpropanoic acid (CAS no.
4249-19-8, Sigma-Aldrich), S-PGME product 3 was assigned as the S-
PGME amide of 2S-3-amino-2-methylpropanoic acid, whereas 5 was
revealed as the S-PGME amide of 2R-3-amino-2-methylpropanoic
acid (Figure S15). Through the same derivatization, purification, and
analysis procedures with R-PGME and the racemic mixture of 3-
amino-2-methylpropanoic acid (CAS no. 144-90-1, Sigma-Aldrich),
R-PGME products (4 and 6) were acquired and assigned as the R-
PGME amide of 2S-3-amino-2-methylpropanoic acid and the R-
PGME amide of 2R-3-amino-2-methylpropanoic acid, respectively
(Figure S16).
Determination of the Stereogenic Center at the β-Position
of the Amide Nitrogen. Piceamycin (1, 5 mg) was dissolved in 2
mL of MeOH and frozen at −80 °C. The chilled solution was treated
with ozone gas generated from a micro-ozonizer for 5 min, and the
residual ozone was removed by purging with argon. The ozonolysis
products were dried in vacuo, redissolved in 7 mL of 1:1
CH3COOH−H2O2 (30%) and refluxed at 115 °C for 6 h. The
mixture was concentrated under low pressure, and 1.5 mL of 6 N HCl
was added into the vial for acid hydrolysis (120 °C, 15 h). After
hydrolysis, the HCl was evaporated on a rotary evaporator. To
eliminate residual HCl, the hydrolysate was dissolved in 1 mL of
deionized water, and the HCl was quickly evaporated three times. The
hydrolysate was dissolved in 2 mL of dry dimethylformamide (DMF)
and divided equally into two vials. The vials were treated with
benzotriazole-1-yl-oxytrispyrrolidinophosphonium hexafluorophos-
phate (PyBOP, 5 mg), hydroxybenzotriazole (HOBt, 3 mg), 4-
methylmorpholine (200 μL), and S- or R-phenylglycine methyl ester
(PGME, 10 mg). The reaction mixtures were stirred at rt for 3 h. The
reactions were quenched by adding 5% HCl solution (2 mL) and
analyzed by using LC/MS (Phenomenex, C18(2), 100 × 4.6 mm,
gradient solvent system: 10−25% CH3CN−H2O over 30 min with
0.1% formic acid, detection: UV 210 nm, flow rate: 0.7 mL/min). For
bombyxamycin C (2), the same procedure was performed with 1 mg
of 2.
Molecular Modeling of PGME Products (3−6) and ECD
Calculations for Piceamycin (1). The first structural energy
minimizations of 3−6 were conducted using Avogadro 1.2.0 with
the MMFF force field. Then, density functional theory (DFT)
calculations were conducted via Tmolex 3.4 with the DFT settings
(functional B3-LYP/gridsize m3), 6-31G basis set for all atoms, and
geometry optimization options (energy 10−6 hartree, gradient norm |
dE/dxyz| = 10−3 hartree/bohr). The initial energy-minimized
structures of 1 were also obtained by using Avogadro 1.2.0 with
MMFF94. The ground-state geometries were optimized via Tmolex
3.4 by using DFT with the 6-31G basis set for all atoms at the DFT
level (functional B3-LYP/gridsize m3). The theoretical ECD data
were calculated by TD-DFT with the 6-311++G** basis set for all
atoms at the DFT level (functional B3-LYP/gridsize m3). The
calculated ECD spectra were simulated by overlapping each
transition, where σ is the width of the band at 1/e height. ΔEi and
Ri are the excitation energies and rotatory strengths, respectively, for
transition i. In this study, the value of σ was fixed at 0.10 eV.
Preparing the S- and R-MTPA Esters of Bombyxamycin C
(2). Bombyxamycin C (2, 1 mg) was transferred into an amber vial
and completely dried under high vacuum. Then, the compound was
dissolved in 1 mL of distilled pyridine, and 15 μL of R-MTPA-Cl was
added into the vial. The reaction mixture was stirred at rt for 30 min
and then quenched with 30 μL of MeOH. The mixture was dried in
vacuo and resuspended in MeOH for purification by reversed-phase
HPLC (Kromasil, C18(2), 5 μm, 250 × 10 mm). The desired product,
S-MTPA-ester (2a, 0.5 mg) of 2, was observed at TR of 26.3 min
under gradient solvent conditions (0−20 min: 40−100% CH3OH−
H2O over 20 min; 20−40 min: 100% CH3OH; detection: UV 280
nm; flow rate: 2 mL/min). The R-MTPA-ester (2b) of 2 was also
prepared through the same procedure used for 2a. R-MTPA-ester
(2b, 0.4 mg) eluted at 26.7 min under the same gradient conditions.
Antibacterial Activity Assay. Three species of Gram-positive
bacteria (Staphylococcus aureus ATCC 25923, Enterococcus faecalis
ATCC 19433, and Enterococcus faecium ATCC 19434) and four
species of Gram-negative bacteria (Proteus hauseri NBRC 3851,
S-PGME Amide of 2S-3-Amino-2-methylpropanoic acid (3). [α]D20
+ 46 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (2.70) nm; ECD
(c 0.4 × 10−4 M, MeOH) λmax (Δε) 219 (3.97) nm; IR (neat) νmax
1
3703, 3671, 2972, 2867, 1058, and 1011 cm−1; H NMR (CD3OD,
850 MHz) δH 7.4025−7.3477 (5H), 5.5217 (1H, s), 3.7186 (3H, s),
3.1548 (1H, dd, J = 12.8, 8.2 Hz), 3.0158 (1H, dd, J = 12.8, 4.6 Hz),
2.8757 (1H, m), and 1.1987 (3H, d, J = 7.1 Hz); 13C NMR (CD3OD,
212.5 MHz) δC 175.4, 172.9, 137.2, 130.0, 130.0, 129.8, 128.8, 128.8,
58.2, 53.2, 42.9, 38.5, and 16.2; HR-FAB-MS m/z 251.1397 [M + H]+
(calcd for C13H19N2O3, 251.1396).
R-PGME Amide of 2S-3-Amino-2-methylpropanoic acid (4).
[α]2D0 −103 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (2.71)
nm; ECD (c 0.4 × 10−4 M, MeOH) λmax (Δε) 219 (−4.31) nm; IR
1
(neat) νmax 3703, 3671, 2972, 2868, 1058, and 1011 cm−1; H NMR
(CD3OD, 850 MHz) δH 7.4090−7.3501 (5H), 5.4939 (1H, s),
3.7078 (3H, s), 3.1472 (1H, dd, J = 12.8, 8.2 Hz), 2.9796 (1H, dd, J =
12.8, 4.6 Hz), 2.8344 (1H, m), and 1.3128 (3H, d, J = 7.1 Hz); 13C
NMR (CD3OD, 212.5 MHz) δC 175.6, 172.5, 137.0, 130.0, 130.0,
129.8, 129.0, 129.0, 58.3, 53.0, 42.7, 38.4, and 16.4; HR-FAB-MS m/z
251.1396 [M + H]+ (calcd for C13H19N2O3, 251.1396).
S-PGME Amide of 2R-3-Amino-2-methylpropanoic acid (5).
[α]2D0 + 106 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (2.71)
nm; ECD (c 0.4 × 10−4 M, MeOH) λmax (Δε) 219 (4.06) nm; IR
1
(neat) νmax 3703, 3671, 2972, 2867, 1055, and 1011 cm−1; H NMR
(CD3OD, 850 MHz) δH 7.4077−7.3494 (5H), 5.4931 (1H, s),
3.7075 (3H, s), 3.1480 (1H, dd, J = 12.8, 8.2 Hz), 2.9779 (1H, dd, J =
12.8, 4.6 Hz), 2.8363 (1H, m), and 1.3114 (3H, d, J = 7.1 Hz); 13C
NMR (CD3OD, 212.5 MHz) δC 175.6, 172.5, 137.0, 130.0, 130.0,
129.8, 129.0, 129.0, 58.3, 53.0, 42.7, 38.4, and 16.4; HR-FAB-MS m/z
251.1399 [M + H]+ (calcd for C13H19N2O3, 251.1396).
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Klebsiella pneumoniae ATCC 10031, Salmonella enterica ATCC 14028,
and Escherichia coli ATCC 25922) as human pathogens and a
silkworm pathogen (Bacillus thuringiensis KACC 10168) were selected
as test strains and prepared for the antibacterial activity assays. The
test bacterial strains were cultured overnight in Mueller−Hinton
broth (MHB) at 37 °C for human pathogens and in tryptic soy broth
(TSB) at 28 °C for B. thuringiensis. The grown strains were harvested
by using a centrifuge and washed twice with sterilized water.
Ampicillin, tetracycline, and streptomycin17 were used as positive
controls, and dimethyl sulfoxide (DMSO) was used as a negative
control. Each compound, including bombyxamycin C and piceamycin,
was dissolved in DMSO and diluted with MHB or TSB to prepare
serial 2-fold dilutions (from 128 μg/mL to 0.015 μg/mL). For the
antibacterial activity assay, 10 μL of suspensions containing 1 × 106
colony-forming units (cfu)/mL of the test bacteria and 190 μL of
MHB or TSB, which included the positive control compounds or test
compounds, were added into each well of a 96-well plate (final
concentration of test bacteria was 5 × 104 cfu/mL). Then, the plate
was incubated at 37 or 28 °C for 24 h. The MIC values of each
compounds were defined as the lowest concentration at which the
compound inhibited the growth of the test bacteria. Results are in
Table 2.
Cell Culture. Human lung cancer (A549), colorectal cancer
(HCT116), breast cancer (MDA-MB-231), liver cancer (SK-HEP-1),
and leukemia (K-562) cells were obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA), and stomach
cancer (SNU-638) cells were obtained from the Korean Cell Line
Bank (Seoul, Korea). The cells were cultured in an appropriate
medium (Roswell Park Memorial Institute 1640 for A549, HCT116,
SNU-638, and K562 cells; Dulbecco’s modified Eagle’s medium for
MDA-MB-231 and SK-HEP-1 cells) supplemented with 10% heat-
inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin,
and 0.25 μg/mL amphotericin B. The cells were incubated at 37 °C
with 5% CO2 in a humidified atmosphere. All reagents were
purchased from Gibco Invitrogen Corp. (Grand Island, NY, USA).
Cell Proliferation Assay. Cell proliferation was measured by the
sulforhodamine B (SRB) assay.18 Briefly, cells were seeded in 96-well
plates and incubated for 30 min (for zero-day controls) or treated
with 1 and 2 for the indicated times. After incubation, the cells were
fixed, dried, and stained with 0.4% SRB in 1% acetic acid. The
unbound dye was removed by washing, and the stained cells were
suspended in 10 mM Tris (pH 10.0). The absorbance was measured
at 515 nm, and the cell proliferation was determined. IC50 values were
calculated by nonlinear regression analysis using TableCurve 2D v
5.01 software (Systant Software Inc., Richmond, CA, USA). All
reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Saeyeon Kang − Department of Agricultural Biotechnology,
College of Agriculture & Life Sciences, Seoul National
University, Gwanak-gu, Seoul 08826, Republic of Korea
Woong Sub Byun − Natural Products Research Institute,
College of Pharmacy, Seoul National University, Gwanak-gu,
Seoul 08826, Republic of Korea
Chang-Wook Jeon − Department of Plant Medicine and IALS,
Gyeongsang National University, Jinju, Gyeongsang Nam-do
52828, Republic of Korea
Beomkoo Chung − Department of Agricultural Biotechnology,
College of Agriculture & Life Sciences, Seoul National
University, Gwanak-gu, Seoul 08826, Republic of Korea
Ji Yoon Beom − Department of Chemistry and Nanoscience,
Ewha Womans University, Seoul 03760, Republic of Korea
Suckchang Hong − College of Pharmacy, Seoul National
University, Gwanak-gu, Seoul 08826, Republic of Korea
Jeeyeon Lee − College of Pharmacy, Seoul National University,
Gwanak-gu, Seoul 08826, Republic of Korea; orcid.org/
0000-0003-3995-5747
Jongheon Shin − Natural Products Research Institute, College of
Pharmacy, Seoul National University, Gwanak-gu, Seoul
08826, Republic of Korea; orcid.org/0000-0002-7536-
8462
Youn-Sig Kwak − Department of Plant Medicine and IALS,
Gyeongsang National University, Jinju, Gyeongsang Nam-do
52828, Republic of Korea
Sang Kook Lee − Natural Products Research Institute, College of
Pharmacy, Seoul National University, Gwanak-gu, Seoul
08826, Republic of Korea; orcid.org/0000-0002-4306-
7024
Ki-Bong Oh − Department of Agricultural Biotechnology,
College of Agriculture & Life Sciences, Seoul National
University, Gwanak-gu, Seoul 08826, Republic of Korea
Yeo Joon Yoon − Department of Chemistry and Nanoscience,
Ewha Womans University, Seoul 03760, Republic of Korea;
orcid.org/0000-0002-3637-3103
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b00678
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
ASSOCIATED CONTENT
■
■
This work was supported by the National Research
Foundation of Korea Grants funded by the Korean Govern-
ment (Ministry of Science and ICT) (2017R1A2B2009393,
2018R1A4A1021703, and 2019R1A2B5B03069338).
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00678.
1D and 2D NMR spectra data, HR-FAB-MS data,
energy-minimized coordinates for 1−6 (PDF)
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