Genomics-Driven Discovery of Chlorinated Cyclic Hexapeptides Ulleungmycins A and B from a Streptomyces Species

Article abstract of DOI:10.1021/acs.jnatprod.7b00660

Analysis of the genome sequence of Streptomyces sp. KCB13F003 showed the presence of a cryptic gene cluster encoding flavin-dependent halogenase and nonribosomal peptide synthetase. Pleiotropic approaches using multiple culture media followed by LC-MS-guided isolation and spectroscopic analysis enabled the identification of two new chlorinated cyclic hexapeptides, ulleungmycins A and B (1 and 2). Their structures, including absolute configurations, were determined by 1D and 2D NMR techniques, advanced Marfey's analysis, and GITC derivatization. The new peptides, featuring unusual amino acids 5-chloro-l-tryptophan and d-homoleucine, exhibited moderate antibacterial activities against Gram-positive pathogenic bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus.

Full text of DOI:10.1021/acs.jnatprod.7b00660

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX-XXX
Genomics-Driven Discovery of Chlorinated Cyclic Hexapeptides
Ulleungmycins A and B from a Streptomyces Species
Sangkeun Son,†,‡ Young-Soo Hong,†,‡ Mina Jang,†,‡ Kyung Taek Heo,†,‡ Byeongsan Lee,† Jun-Pil Jang,†
Jong-Won Kim,† In-Ja Ryoo,† Won-Gon Kim,‡,§ Sung-Kyun Ko,†,‡ Bo Yeon Kim,†,‡ Jae-Hyuk Jang,*,†,‡
and Jong Seog Ahn*,†,‡
†Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Korea
‡Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon
34141, Korea
§Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea
S
* Supporting Information
ABSTRACT: Analysis of the genome sequence of Streptomyces sp. KCB13F003
showed the presence of a cryptic gene cluster encoding flavin-dependent
halogenase and nonribosomal peptide synthetase. Pleiotropic approaches using
multiple culture media followed by LC-MS-guided isolation and spectroscopic
analysis enabled the identification of two new chlorinated cyclic hexapeptides,
ulleungmycins A and B (1 and 2). Their structures, including absolute
configurations, were determined by 1D and 2D NMR techniques, advanced
Marfey’s analysis, and GITC derivatization. The new peptides, featuring unusual
amino acids 5-chloro-L-tryptophan and D-homoleucine, exhibited moderate
antibacterial activities against Gram-positive pathogenic bacteria including
methicillin-resistant and quinolone-resistant Staphylococcus aureus.
he genomics-driven discovery of novel bioactive natural
products. The most frequently used strategies for activation of
Tproducts has been enabled by the reduced cost of genome cryptic secondary metabolite gene clusters include alteration in
growth conditions, heterologous expression of gene clusters,
and genetic manipulation of the regulators.5 In consideration of
pleiotropic effects of growth conditions on gene expression, we
screened different culture media with LC-MS analysis to detect
the encoded compounds and consequently found conditions
for the production of two chlorinated NRPs (1 and 2) whose
structures are strongly correlated with the predicted domain
organization of the gene cluster. Structurally, they were
characterized as unusual NRPs containing 5-chloro-L-trypto-
phan (5-Cl-L-Trp) and D-homoleucine (D-Hleu). Here, we
report the isolation, structural characterization, and biological
activities as well as the biosynthetic analysis of these two new
peptides.
sequencing and by advances in bioinformatics-based predictions
of encoded compounds in biosynthetic gene clusters.1 Non-
ribosomal peptides (NRPs), among the most structurally
diverse and pharmaceutically important secondary metabolites,
are synthesized by modular multifunctional nonribosomal
peptide synthetases (NRPSs), and the core peptide structures
are often subsequently modified via the action of tailoring
enzymes including methyltransferases, glycosyltransferases, and
halogenases, further increasing structural complexity.2 Our
earlier studies on new compounds found through LC-MS
screening of bacterial culture extracts led to the discovery of
new cyclic depsipeptides, ulleungamides A and B, featuring
several novel residues from Streptomyces sp. KCB13F003.3 To
probe their biosynthetic mechanisms and further investigate the
secondary metabolic potential of the strain, we turned our
attention to analyzing the whole genome sequence and found
that the strain harbors multiple putative biosynthetic gene
clusters including polyketide synthases (natamycin), lantipep-
tide synthetases (planosporicin), and NRPSs (ulleungamide).3,4
Significantly, the genome contains diverse NRPS gene clusters
whose products were not detected in the culture extract,
indicating that the strain could be a promising NRP producer.
Among these genes, we focused on one NRPS gene cluster
adjacent to the halogenase gene capable of encoding
chlorinated hexapeptides, which are rarely reported natural
RESULTS AND DISCUSSION
■
The whole genome of Streptomyces sp. KCB13F003 was
sequenced and assembled into two contigs, with a total size
of 9.27 Mb and an overall GC content of 71.4%. Analysis of the
contigs with antiSMASH software enabled us to identify the
putative halogenase gene (ulm24) alongside an NRPS cluster
(GenBank accession number MF541667) designated ulm in
this study (Figure 1).6 The targeted halogenase was speculated
Received: July 31, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Article
NRPSpredictor software (Table S1).9 In the case of the A
domain motif (DVAMAGAV) in module 1, it did not show a
clear conserved sequence for substrate prediction. However, the
similar A domain motif (DVAMAGMV) was recently reported
to recognize L-Trp during the biosynthesis of hexapeptide
desotamides,10 thus suggesting the activation of the Trp or Trp-
associated residues by the A domain in module 1.
At this point, due to limited prediction of exact core peptides
and tailoring reactions, we prioritized the structure determi-
nation of the encoded peptides by activating the gene cluster
and performing spectroscopic analyses. As none of halogen- or
tryptophan-containing compounds were detected in a previous
culture using glycerol-based media, we then focused our efforts
to induce the expression of the gene cluster by manipulating
the culture conditions and LC-MS screening. As a result of
these tests, it was found that when one of the media used for
production of ulleungamides was diluted (2-fold dilution), the
strain produced previously unrecognized compounds exhibiting
the typical indole UV spectrum and the characteristic isotopic
pattern for the presence of one chlorine atom (Figure S2). Two
compounds, 1 and 2 (9.0 and 4.5 mg, respectively), were then
purified from the extract of the scale-up culture and subjected
to detailed structural analysis.
The molecular formula of ulleungmycin A (1) was
established as C38H58N9O8Cl by HRESIMS and NMR analyses.
The peptidic nature of 1 was evident from the presence of the
α-protons (δH 4.61−3.74), amide NH signals (δH 8.77−7.18),
and amide carbonyl signals (δC 173.2−169.4) observed in the
1D NMR spectra. A combination of HSQC, DQF-COSY, and
HMBC experiments was used to assign the amino acid residues
in the peptide. The two signals at δC/H 60.6/3.74 and 56.7/4.19
appeared to be the α-methine groups for the two amino acids
Val and Ile, which were assigned by the DQF-COSY and
HMBC correlations (Figure 2). The presence of an unusual
to be an FADH2-dependent halogenase based on the presence
of the absolutely conserved motifs (GxGxxG and GWxWxIP)
(Figure S1). Since a flavin reductase gene (ulm26) was found
very close to the halogenase gene, Ulm26 was suggested to be
involved in the halogenation reaction.7 A subsequent functional
prediction with the NCBI protein−protein BLAST search of
the targeted halogenase showed homology to many putative or
functionally established L-tryptophan halogenases, including
PrnA and PyrH.8 The gene cluster was also found to harbor
two genes involved in the NRPS assembly line consisting of a
total of six modules, implying incorporation of six amino acids
for an encoded compound. The peptide sequence was then
predicted based on the ATP-dependent adenylation (A)
domain specificities of every NRPS module using the
Figure 1. Analysis of the ulm gene cluster. (a) Proposed biosynthetic pathway for the ulleungmycins. (b) Chlorination of tryptophan by FADH2-
dependent halogenase (Ulm24) and flavin reductase (Ulm26). (c) Genetic organization of the ulm gene cluster. Genes are color-coded according to
their proposed functions.
B
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with a characteristic chemical shift of 5-NHCl‑Trp (δH 11.1),
3
suggested the presence of an indole ring. Strong JC,H HMBC
correlations of the aromatic protons (H-5/C-6; H-7/C-9, C-11;
H-8/C-6, C-10; H-10/C-4, C-6, C-8) clearly identified the
indole moiety. The nonprotonated aromatic carbon C-9Cl‑Trp
(δC 122.9), which was confirmed by no correlation in the
HSQC spectrum and meta-coupled H-10Cl‑Trp (J = 1.7 Hz), was
assigned as having a chlorine atom, establishing this residue as
5-Cl-Trp. On the other hand, the DQF-COSY correlations
from the amide 2-NHHleu to the terminal gem-dimethyls H3-
6Hleu and H3-7Hleu and the HMBC correlation of H-2Hleu to the
amide carbonyl C-1Hleu confirmed the presence of the rare
amino acid homoleucine (Hleu).
The additional spin system comprising 2-NHOH‑Asn, H-
2OH‑Asn, and the oxygenated methine H-3OH‑Asn (δH 4.41) was
obtained from DQF-COSY correlations, indicating the oxy-
genated β-carbon. A β-OH-Asn residue was then identified
through the HMBC correlations from H-3OH‑Asn and two
mutually coupled broad singlets 4-NH2OH‑Asn to the carbonyl C-
4OH‑Asn (δC 173.2). The last proton sequence starting from 2-
NHOrn through the α-proton H-2Orn and two methylenes H2-
3Orn and H2-4Orn to the aminated methylene H2-5Orn (δH 2.71)
established the ornithine residue, assigning a total of six amino
acid residues. The amino acid sequence of β-OH-Asn/5-Cl-
Figure 2. Key 2D NMR correlations used for determining the
structure of 1.
amino acid, 5-Cl-Trp, was revealed as follows. Three spin
systems could be constructed by the DQF-COSY spectra
analysis: 2-NHCl‑Trp to H2-3Cl‑Trp; H-5Cl‑Trp to 5-NHCl‑Trp; and
H-7Cl‑Trp to H-8Cl‑Trp (Figure 2). Key HMBC correlations from
H2-3Cl‑Trp to the carbonyl C-1Cl‑Trp, sp2 nonprotonated carbons
C-4Cl‑Trp and C-11Cl‑Trp, and sp2 methine C-5Cl‑Trp, together
1
Table 1. 13C (225 MHz) and H (900 MHz) NMR Spectroscopic Data for 1 and 2 in DMSO-d6
1
2
1
2
δH, mult (J
in Hz)
δH, mult (J
in Hz)
δH, mult (J
in Hz)
δH, mult (J
in Hz)
position δC, type
δC, type
position δC, type
δC, type
a
5-Cl-L-Trp
1
2
171.2, C
53.5, CH 4.61, dd
(15.1, 7.7)
27.1, CH2 3.11, dd
(14.2, 7.9)
171.5, C
7
22.2, CH3 0.80, ovl
7.82, br s
53.6, CH 4.61, dd
(14.9, 8.0)
2-NH
8.10, br s
L-allo-Ile
1
171.7, C
171.8, C
a
3a
3b
27.5, CH2 3.09, dd
(14.1, 8.3)
2
56.7, CH 4.19, t (6.9) 56.5, CH 4.25, ovl
a
3
36.0, CH 1.91, m
25.5, CH2 1.34, td
36.2, CH 1.90, ovl
25.4, CH2 1.34, m
2.83, dd
(14.2, 6.6)
2.83, dd
4a
(14.1, 6.3)
(13.9, 7.0)
4
5
6
7
8
109.9, C
109.9, C
4b
1.18, m
1.12, tt
(15.0, 7.3)
a
125.5, CH 7.13, s
134.5, C
125.4, CH 7.13, ovl
134.5, C
5
11.3, CH3 0.86, t (7.4) 11.1, CH3 0.87, t (7.4)
14.5, CH3 0.84, d (6.4) 14.4, CH3 0.83, d (6.9)
112.7, CH 7.31, d (8.5) 112.7, CH 7.32, d (8.5)
120.6, CH 7.03, dd 120.6, CH 7.03, dd
(8.5, 1.9) (8.5, 1.9)
6
2-NH
7.90, s
7.65, br d
(6.0)
9
122.9, C
122.9, C
L-Orn
1
170.5, C
170.2, C
10
117.5, CH 7.54, d (1.7) 117.5, CH 7.54, d (1.7)
2
52.9, CH 3.99, br s
53.4, CH 3.82, br s
a
a
11
128.4, C
170.9, C
128.4, C
171.1, C
3a
27.1, CH2 1.89, ovl
26.6, CH2 1.90, ovl
a
2-NH
7.90, ovl
11.1, s
7.85, d (8.0)
11.08, s
3b
1.69, m
25.1, CH2 1.51, m
1.47, m
1.70, m
a
5-NH
4a
25.3, CH2 1.51, ovl
a
D-Val
1
4b
1.47, ovl
2
60.6, CH 3.74, t (7.7) 60.8, CH 3.70, t (7.4)
29.1, CH 1.88, m 28.9, CH 1.89, m
5
38.8, CH2 2.71, br s
8.77, br s
39.0, CH2 2.72, br s
8.95, br s
3
2-NH
4
18.9, CH3 0.67, d (6.8) 18.9, CH3 0.66, d (6.8)
18.5, CH3 0.69, d (6.7) 18.6, CH3 0.68, d (6.7)
5-NH2
8.39, br s
8.39, br s
5
D-threo-β-
OH-Asn
1
2
169.4, C
169.5, C
2-NH
1
8.7, br s
8.46, br s
55.7, CH 4.54, dd
(8.3, 1.8)
55.6, CH 4.56, dd
(8.4, 1.4)
D-Hleu
172.2, C
172.1, C
a
2
52.8, CH 4.25, m
28.7, CH2 1.75, m
1.61, m
51.2, CH 4.25, ovl
39.2, CH2 1.59, m
1.54, m
3
71.3, CH 4.41, d (2.1) 71.1, CH 4.44, d (1.8)
173.2, C 173.1, C
3a
3b
4a
4b
5
4
2-NH
4-NH2
7.34, br s
7.35, br s
7.18, br s
7.21, d (5.7)
7.34, br s
34.2, CH2 1.08, m
1.06, m
24.1, CH 1.49, m
a
7.13, ovl
27.0, CH 1.45, tt
21.3, CH3 0.78, d (6.5)
22.9, CH3 0.84, d (6.6)
(13.3, 6.7)
a
a
6
22.5, CH3 0.80, ovl
Signals were overlapped with other signals.
C
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3
Trp/Val/Hleu/Ile/Orn was corroborated by the JC,H HMBC
correlations from the α-protons to the carbonyl carbons of the
adjacent residues, and the ROESY correlations between the α-
protons and the amide protons supported this result. The
ROESY cross-peak between H-2Ile (δH 4.19) and 2-NHOrn (δH
8.77) established the ring closure and completed the planar
structure of 1 as a chlorinated cyclic hexapeptide.
Table 2. Antibacterial Activities of 1 and 2
MIC (μg/mL)
a
strain
1
2
Cip
Staphylococcus aureus RN 4220
MRSA CCARM 3167
16
16
8
16
32
16
8
0.125
4
QRSA CCARM 3505
32
Bacillus subtilis KCTC 1021
Streptococcus pneumoniae KCTC 5412
Enterococcus faecalis KCTC 5191
Escherichia coli CCARM 1356
Pseudomonas aeruginosa KCTC 2004
8
0.015
0.5
The absolute configuration of the amino acids in 1 was
determined by advanced Marfey’s analysis following acid
hydrolysis.11 The standards for 5-Cl-Trp and Hleu were
obtained by acid hydrolysis of commercially available L-N-
Boc-5-chlorotryptophan and Fmoc-D-homoleucine, respec-
tively. LC-MS chromatographic comparison of the hydrolysis
products of 1, after derivatization with L- and D-FDLA (1-
fluoro-2,4-dinitrophenyl-5-leucinamide), enabled us to assign 5-
Cl-L-Trp, D-Val, D-Hleu, L-Ile, and L-Orn. Marfey’s analysis
revealed that the β-OH-Asn of 1 underwent hydrolysis to β-
OH-Asp under the acid hydrolysis conditions, which is a
frequently observed phenomenon during the preparation of the
acid hydrolysates.12 To precisely determine its absolute
configuration, the L- and D-FDLA derivatives of the authentic
standards L-threo-β-OH-Asp and L-erythro-β-OH-Asp were
prepared. The L- and D-FDLA derivatives of β-OH-Asp from
the hydrolysate of 1 were found to elute at the same time as the
D- and L-FDLA derivatives of the L-threo-β-OH-Asp, respec-
tively. This was taken as evidence for the absolute configuration
of β-OH-Asn, thus indicating the presence of D-threo-β-OH-Asn
in 1. The threo configuration was further supported by J-based
configuration analysis using vicinal coupling constants (from
HECADE and J-resolved HMBC spectra) and ROESY
correlations (Figure S3).13 In addition, chromatographic
comparisons of GITC (2,3,4,6-tetra-O-acetyl-β-D-glucopyrano-
syl isothiocyanate) derivatives of the acid hydrolysate of 1 and
those of authentic L-Ile and L-allo-Ile demonstrated that 1
contains L-allo-Ile.14
The molecular formula of ulleungmycin B (2) was
established as C38H58N9O8Cl by HRESIMS, which is 14 amu
less than the mass of 1. The 1D and HSQC NMR data of 2
closely resembled those of 1, except for a missing methylene
group. The DQF-COSY and HMBC NMR data confirmed that
2 had Leu instead of Hleu. LC-MS analyses of the FDLA and
GITC derivatives for the acid hydrolysate of 2 established the
configuration of amino acids and enabled us to conclude that
both compounds shared the same absolute configuration.
Ulleungmycins were active against multiple strains of Gram-
positive bacteria including antibiotic-resistant strains, MRSA
(methicillin-resistant Staphylococcus aureus), and QRSA (qui-
nolone-resistant Staphylococcus aureus), as well as antibiotic-
susceptible strains, with MIC values in the range of 8−32 μg/
mL (Table 2).
16
8
8
8
1
128
128
64
128
64
0.125
a
Ciprofloxacin was used as a positive control.
possessing three Hleu residues was reported recently but
without stereochemical assignment.19 Ulleungmycins A and B
are most closely related to two antibacterial cyclic hexapeptides,
desotamides and longicatenamycins.10,18a Of note, the gene
clusters for the ulleungmycins (ulm) and desotamides (dsa)
show considerable homology, but tailoring enzymes, such as
halogenase and dioxygenase, are absent in the dsa gene cluster
(Table S3). Ulleungmycins also share a similar amino acid
composition with longicatenamycins which were isolated as a
mixture of peptidic congeners. The absolute configurations of
the amino acids in longicatenamycins are opposite those of
ulleungmycins. The majority of the absolute configurations of
the amino acids in longicatenamycins were solved by ORD or
specific rotations of the pure or mixed residues.20 The total
synthesis of longicatenamycin A has reproduced its biological
activity.21
On the basis of the information from the chemical structures
and the putative biosynthetic gene cluster, we deduced a
biosynthetic model for the ulleungmycins. Although the
boundaries of the gene cluster were not established in this
study, the genes were annotated to be involved in the
biosynthesis of the ulleungmycins based on BLAST-based
functional prediction (Figure 1 and Table S2). The number of
NRPS modules of the gene cluster was in accordance with the
number of amino acid residues, and predicted incorporation of
Asn and Val based on the A domain specificities was also
consistent with the sequence of β-OH-Asn and Val in the
ulleungmycins. The presence of epimerase (E) domains
embedded in modules 2, 3, and 6 matched the R-configured
amino acids Val, Hleu/Leu, and threo-β-OH-Asn, suggesting
that compounds 1 and 2 are the products of the ulm cluster.
Furthermore, we investigated the presence of the dechlorinated
analogues by analyzing the LC-MS data, and corresponding
minor peaks were found in the extract (Figure S4). Thus,
although the stage at which L-Trp is chlorinated is not yet clear,
it is suggested that nonchlorinated L-Trp could be accepted by
the A domain of module 1. It is hypothesized that thioester
cleavage and release might be accomplished by the discrete
thioesterase (TE) Ulm7 due to the absence of a C-terminal TE
domain in the termination module 6. In addition, the gene
ulm20 codes for an enzyme with sequence similarity to TauD,
the nonheme iron enzyme taurine hydroxylase.22 Ulm20 also
shows homology to SyrP from the syringomycin biosynthetic
cluster, which catalyzes β-hydroxylation of Asp tethered to the
PCP domain.23 This fact, together with the significant degree of
the substrate prediction score for Asn, suggested that
hydroxylation might occur by Ulm20 once Asn is bound to
the PCP domain as a thioester. The gene cluster further
contains ulm31, whose predicted product is a closely related
Ulleungmycins A and B are structurally characterized by the
unusual amino acids 5-Cl-L-Trp and D-Hleu. Although an
indole ring chlorinated in the 5 position has been observed
from pyrroindomycin B as well as bisindole alkaloids, such as
cladoniamides and lynamicins,15 the unique 5-Cl-L-Trp residue
in peptide metabolites was previously described only once in
the lantibiotic NAI-107.16 The presence of Hleu is attributed to
the low specificity of biosynthetic enzymes involved in the
synthesis of branched chain amino acids valine, isoleucine, and
leucine.17 Although L-Hleu has been found in a limited number
of metabolites such as longicatenamycins and mycoplanecins,18
D-Hleu-containing metabolites have not been previously
reported. Additionally, a fungal dodecadepsipeptide decatransin
D
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carried out using a Young Lin YL9100 HPLC system equipped with a
Young Lin YL9160 PDA detector. LC-MS was performed with a
Thermo LTQ XL linear ion trap attached to an ESI source that was
connected to a Thermo Scientific Dionex Ultimate 3000 Rapid
Separation LC system using a Waters HSS T3 column (Waters, 2.1 ×
150 mm, 2.5 μm). Cosmosil 75C18 was used for ODS flash
chromatography.
Microorganism Culture and Compound Purification Meth-
ods. The strain Streptomyces sp. KCB13F003 was isolated and
identified as previously described.3 The strain maintained on an SY
agar medium was used for a large-scale culture. Two agar plugs of the
strain were directly inoculated into a 1 L baffled Erlenmeyer flask
containing 200 mL of a modified GLY medium (7.9 mL of glycerol, 5
g of lactose, 2.5 g of malt extract, 2.5 g of yeast extract, and 0.5 g of
CaCO3 per 1 L of distilled water). The cultures were shaken at 28 °C
and 125 rpm for 132 h. The cells were centrifuged for 25 min at 5000
rpm and then extracted with 1 L of acetone and filtered. Following the
removal of acetone, the aqueous solution was extracted three times
with an equal volume of ethyl acetate. The supernatant was also
extracted three times with an equal volume of ethyl acetate. The crude
extracts of the supernatant and cells were combined and fractionated
by ODS chromatography eluting with a stepwise gradient elution of
MeOH−H2O [2:8, 4:6, 6:4, 8:2, 10:0 (v/v)] to yield 10 fractions.
Fractions 8 and 9, eluted with 80% and 100% MeOH, respectively,
contained the desired materials. Fraction 9 was subjected to HPLC
purification (Cosmosil Cholester, 10 × 250 mm, 5 μm; flow rate 3
mL/min; 25−40% CH3CN−H2O containing 0.05% formic acid over
20 min) to afford compound 1 (9.0 mg, tR 14.6 min). Fraction 8 was
purified by HPLC (Cosmosil Cholester, 10 × 250 mm, 5 μm; flow rate
3 mL/min; 25−40% CH3CN−H2O containing 0.05% formic acid over
20 min) to afford compound 2 (4.5 mg, tR 12.0 min).
Ulleungmycin A (1): white powder; [α]2D2 +21.7 (c 0.05, MeOH);
UV (MeOH) λmax (log ε) 208 (3.4), 227 (3.5), 290 (2.7); for NMR
data, see Table 1; HRESIMS m/z 804.4166 [M + H]+ (calcd for
C38H59N9O8Cl, 804.4175).
Ulleungmycin B (2): white powder; [α]2D2 +38.2 (c 0.05, MeOH);
UV (MeOH) λmax (log ε) 207 (3.4), 227 (3.4), 290 (2.7); for NMR
data, see Table 1; HRESIMS m/z 790.4018 [M + H]+ (calcd for
C37H57N9O8Cl, 790.4019).
homologue to the global regulator GntR. Mutations in the
GntR family regulator of Streptomyces were shown to activate a
cryptic gene cluster or enhance the production of antibiotics.24
The regulatory role of GntR, which responds to nutritional
signals, together with the two-component regulatory system
(ulm11 and ulm12) and the LysR family transcriptional
regulator (ulm3), is a possible candidate to explain the different
production profiles of ulleungmycins depending on the culture
media.25
Screening tailoring enzymes, especially halogenases, which
have the potential to diversify the substrate structures, is
considered to be a promising genome-driven method to find
new halogen-containing metabolites, although only a limited
number of case studies have been reported.26 The discovery of
ulleungmycins provides evidence that the combination of
genome-mining approaches focusing on tailoring enzymes and
bioinformatic tools for predicting encoded compounds is an
effective strategy to access new chemical entities.
Advanced Marfey’s Analysis. Compounds 1 and 2 (0.1 mg
each) were separately hydrolyzed with 6 N HCl (0.5 mL) at 100 °C
for 1 h. The solution was dried repeatedly in vacuo and dissolved in 1
N NaHCO3 (100 μL). To a solution was added a Marfey’s solution of
1% L-FDLA (5-fluoro-2,4-dinitrophenyl-5-L-leucine amide) in acetone
(100 μL). The reaction mixture was allowed to react at 40 °C for 1 h
and quenched with 20 μL of 2 N HCl. After 2-fold dilutions in
CH3CN, the solution was analyzed by LC-MS (Waters HSS T3
column, 2.1 × 150 mm; flow rate 0.3 mL/min; 5−100% CH3CN−
H2O containing 0.05% formic acid over 15 min). D-FDLA derivatives
were prepared and analyzed in the same manner as described above.
An optimized separation condition (Waters HSS T3 column, 2.1 ×
150 mm; flow rate 0.3 mL/min; CH3CN−H2O containing 0.05%
formic acid; 5% at 0−5 min, 5−40% at 5−65 min, and 40−100% at
65−70 min) was used to resolve FDLA derivatives of β-OH-Asp.
Retention times (tR, min) of the L- and D-FDLA derivatives for
compound 1 were as follows: 5-Cl-L-Trp 13.55, 14.23; D-Val 14.01,
12.54; D-Hleu 15.33, 13.66; L-allo-Ile 12.96, 14.65; L-Orn (bis
derivative) 14.52, 14.23; D-threo-β-OH-Asp 46.41, 46.59. Retention
times (tR, min) of the L- and D-FDLA derivatives for compound 2 were
as follows: 5-Cl-L-Trp 13.54, 14.24; D-Val 13.99, 12.57; D-Leu 14.7,
13.1; L-allo-Ile 12.96, 14.65; L-Orn (bis derivative) 14.51, 14.24; D-
threo-β-OH-Asp 46.41, 46.59. Retention times (tR, min) of the L- and
D-FDLA derivatives of L-threo-β-OH-Asp and L-erythro-β-OH-Asp were
as follows: L-threo-β-OH-Asp 46.5, 46.35; L-erythro-β-OH-Asp 52.66,
51.14.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The specific rotation was
obtained in MeOH using a JASCO P-1020 polarimeter. UV spectra
were recorded in MeOH using an Optizen 2120 UV spectropho-
tometer. 1D and 2D NMR spectra were measured on a Bruker Biospin
1
Advance II 900 NMR spectrometer (900 MHz for H and 225 MHz
for 13C) at the Korea Basic Science Institute (KBSI) in Ochang, Korea.
Chemical shifts were referenced to the respective residual solvent
peaks (δH 2.50 and δC 39.51 for DMSO-d6). HRESIMS data were
obtained on a Waters SYNAPT G2 Q-TOF mass spectrometer at the
KBSI in Ochang, Korea. Semipreparative reversed-phase HPLC was
GITC Derivatization. The acid hydrolysates of 1 and 2 (0.1 mg
each) were separately dissolved in triethylamine (6% w/v in acetone,
200 μL) and a GITC solution (1% w/v in acetone, 200 μL). The
reaction mixture was stirred at room temperature for 20 min. After
adding 5% acetic acid (200 μL), the sample was directly analyzed on
E
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Journal of Natural Products
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the LC-MS (Waters HSS T3 column, 2.1 × 150 mm; flow rate 0.3
mL/min; CH3CN−H2O containing 0.05% formic acid; 5% at 0−5
min, 5−40% at 5−65 min, and 40−100% at 65−70 min). The
retention time for the GITC derivatives of 1 and 2 was 59.26 min.
GITC derivatives of authentic L-Ile and L-allo-Ile were prepared and
analyzed in the same manner (tR = 59.48 and 59.24 min, respectively).
Antibacterial Activity Assay. The whole-cell antimicrobial
activity was determined using broth microdilution as described
previously.27 Drug-resistant pathogens including methicillin-resistant
S. aureus (MRSA) CCARM 3167, quinolone-resistant S. aureus
(QRSA) CCARM 3505, and Escherichia coli CCARM 1356 were
obtained from the Culture Collection of Antimicrobial Resistant
Microbes of Korea. Most of the test strains were grown to mid log
phase in Mueller−Hinton broth and diluted 1000-fold in the same
medium. Cells (105/mL) were inoculated into Mueller−Hinton broth
and dispensed at 0.2 mL/well in 96-well microtiter plates. Streptococcus
pneumonia were grown in Todd−Hewitt medium instead of Mueller−
Hinton broth. Since test compounds and ciprofloxacin (Sigma) were
soluble in DMSO, they were prepared in DMSO, the final
concentration of which did not exceed 0.05%. Cells were treated
with either 0.05% DMSO as vehicle control or test samples. The MICs
were determined in triplicate by serial 2-fold dilutions of the test
compounds. The MIC was defined as the concentration of a test
compound that completely inhibited cell growth during a 24 h
incubation at 37 °C. Bacterial growth was determined by measuring
the absorption at 650 nm using a microtiter enzyme-linked
immunosorbent assay (ELISA) reader.
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AUTHOR INFORMATION
■
Corresponding Authors
*Tel (J.-H. Jang): +82-43-240-6164. Fax: +82-43-240-6169. E-
mail: jangjh@kribb.re.kr.
*Tel (J.-S. Ahn): +82-43-240-6160. Fax: +82-43-240-6169. E-
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Research Council of
Science & Technology (NST) granted by the International
Joint Research Project (ASIA-16-001) and KRIBB Research
Initiative Program of Korea. This work was also supported by
the National Research Foundation of Korea (NRF) grant
funded by the Korean government (MSIT; Ministry of Science
ICT) (NRF-2017R1C1B2002602). We thank the Korea Basic
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