Chlorocatechelins A and B from streptomyces sp.: New siderophores containing chlorinated catecholate groups and an acylguanidine structure

Article abstract of DOI:10.1021/ol502964s

Two novel siderophores, chlorocatechelins A and B, were isolated from a culture broth of Streptomyces sp. Their structures were determined by spectroscopic analysis and degradation study. They contain chloro-substituted catecholate that has not been reported in natural products, whereas this group was conjugated to guanidine to form acylguanidine in chlorocatechelin A. This acylguanidine decomposed in weakly acidic solutions to furnish a less potent siderophore chlorocatechelin B. Chemical and biological insights into acylguanidine are also discussed.

Full text of DOI:10.1021/ol502964s

Letter
pubs.acs.org/OrgLett
Chlorocatechelins A and B from Streptomyces sp.: New Siderophores
Containing Chlorinated Catecholate Groups and an Acylguanidine
Structure
†
†
†
‡
§
Shinji Kishimoto, Shinichi Nishimura, Akira Hattori, Masafumi Tsujimoto, Masaki Hatano,
§
,†
Masayuki Igarashi, and Hideaki Kakeya*
†Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School
of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
‡
Faculty of Pharmaceutical Sciences, Teikyo-Heisei University, Nakano-ku, Tokyo 164-8530, Japan
§Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
*
S Supporting Information
ABSTRACT: Two novel siderophores, chlorocatechelins A and
B, were isolated from a culture broth of Streptomyces sp. Their
structures were determined by spectroscopic analysis and
degradation study. They contain chloro-substituted catecholate
that has not been reported in natural products, whereas this group
was conjugated to guanidine to form acylguanidine in
chlorocatechelin A. This acylguanidine decomposed in weakly
acidic solutions to furnish a less potent siderophore chloroca-
techelin B. Chemical and biological insights into acylguanidine are
also discussed.
ron is an essential element for organisms as it is requisite in
various biological processes including primary metabolism.
relatively high redox potentials such as ferrioxamine B (−454
6
I
mV) can be reduced directly with ferric reductases. However,
However, most iron exists as insoluble Fe(III) under aerobic
conditions at neutral pH and is hard to be taken up by
organisms. To overcome this problem, microbes produce and
secrete low molecular weight compounds called siderophores
when the complex exhibits much lower redox potentials such as
Fe(III)-enterobactin, the siderophore needs to be partially
9
degraded before reduction.
During the course of our screening for siderophores from
microbial extracts, we discovered novel siderophores named
chlorocatechelin A (1) and B (2) (Figure 1). The backbone
structure of metabolite 1 was a dipeptide, to which two sets of
4-chloro-2,3-dihydroxy-benzoic acid (CDB) were attached.
CDB has never been found in natural products. In addition,
one CDB unit was condensed with guanidine to form an acyl
guanidine structure, which has been reported only in a few
1
which can capture and solubilize Fe(III). Siderophores, in
many cases, possess three bidentate ligands in one molecule to
form a stable octahedral complex with Fe(III). The bidentate
groups are usually catecholate, hydroxamate, or α-hydroxy-
2
carboxylate. On the other hand, the backbone structures to
which bidentate groups are attached vary among siderophores.
Variation of the backbone and combination of the bidentate
groups give structural diversity to siderophores, which can
benefit their producers on iron availability. For example,
1
0,11
natural products, such as heterobactin (3).
Herein we
report isolation, structure elucidation, and iron-binding proper-
ties of chlorocatechelins. We also discuss the lability of
acylguanidine found in chlorocatechelin A (1).
3
amychelin, produced by Amycolatopsis sp. AA4, seems to
frustrate “siderophore piracy”: amychelin inhibited growth of
Broth extracts of microorganisms (∼1100) were screened
microbes that utilize siderophores secreted by other organ-
4
based on Fe(III)-chelating activities using chrome azurol S
isms.
12
(
CAS) assay. An extract of a culture broth of Streptomyces sp.
Once Fe(III)-siderophore complexes are formed and
incorporated into cells, microbes need to extract Fe(III) from
the stable complex. The most common way to release iron is to5
convert the coordinated Fe(III) to Fe(II) by ferric reductase.
The redox potentials (vs the normal hydrogen electrode
ML93-86F2 showed a rapid decrease of absorbance at 620 nm
that corresponds to the CAS-Fe(III) complex, indicating that
the extract contained Fe(III)-chelating substances. Addition of
a ferric or ferrous ion to the culture apparently suppressed
production of some metabolites, suggesting that the chelating
(
NHE)) of siderophore-coordinated Fe(III) range from −82
6
mV to −750 mV (enterobactin), which are reported to have
good relationships with the affinity of siderophore for Fe(III) in
Received: October 8, 2014
7
,8
physiological conditions. Fe(III)-siderophore complexes with
©
XXXX American Chemical Society
A
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Organic Letters
Letter
Figure 2. HMBC (arrow) and DQF-COSY (bold) correlations of (a)
1
and (b) 2.
correlations from H1 and amide NH of Arg to C7 of CDB1.
However, we could not determine the location of CDB2
because of the lack of HMBC correlations. Next, compound 1
+
was subjected to MS/MS analysis. The [M + H] ion of 1 was
fragmented by collision-induced dissociation (CID) to give
various product ions including fragments associated with CDB-
Arg-hfOrn (m/z 503.17), 2CDB-Arg (m/z 497.07), CDB-Arg
Figure 1. Chemical structures of chlorocatechelins A (1) and B (2),
and structurally related compounds heterobactin (3) and mirubactin
17
(4).
(m/z 327.08), and CDB-Orn (m/z 285.06) (Figure S2). The
observation of the 2CDB-Arg fragment ion excluded the
possibility that CDB2 was connected to hfOrn as reported for
18
13
substances may work as siderophores under physiological
conditions (Figure S1). For isolating the siderophores, a scale-
up culture (1400 mL) of this microorganism was prepared and
extracted with 2240 mL of EtOH. A half portion of this extract
was subjected to a series of solvent partition followed by ODS
column chromatography using stepwise elution with aqueous
MeOH (0−100% MeOH). CAS-positive fractions eluted with
mirubactin (4). We next measured the C NMR spectra of 1
in 600 μL of DMSO-d and 10 μL of H O or D O. Low-field
6
2
2
shifts due to hydrogen−deuterium exchange were observed for
signals at C2 and C3 of CDB1, suggesting that the hydroxyl
groups of CDB1 existed as free forms (Figure 3). Taken
3
0−90% MeOH were combined, which was fractionated by
reversed-phase HPLC to yield chlorocatechelins A (1) (75.6
mg) and B (2) (12.4 mg).
1
3
14
The LR-MS spectra of 1 showed a characteristic isotope
pattern; the intensity ratio of signals for m/z 673, 675, and 677
was 9:6:1, indicating that 1 contains two chlorine atoms. The
molecular formula of 1 was established as C H Cl N O on
2
6
30
2
6
11
+
the basis of HR-ESIMS (m/z 673.1432 [M + H] , calcd
73.1422). The H NMR spectrum exhibited several
1
6
Figure 3. Key deuterium shift values (Δδ = δ(+D2O) − δ(+H2O)) in 13
C
exchangeable protons from 9.5 to 8.3 ppm and two α protons
at 4.55 (1H, m) and 4.20 ppm (1H, m), indicating the presence
of a peptide moiety. Four aromatic protons at 7.48 (1H, d, J =
NMR spectra of 1 in DMSO-d .
6
8
6
.5 Hz), 7.24 (1H, d, J = 8.2 Hz), 6.86 (1H, d, J = 8.5 Hz), and
together, we deduced the connectivity of CDB2 and the
.60 ppm (1H, d, J = 8.2 Hz) were observed, whereas the 13
C
guanidino group of Arg. The absolute configuration of 1 was
1
9
NMR spectrum contained 12 aromatic carbons at 153.6, 151.1,
determined by an advanced Marfey method. An acid
hydrolysate of 1 was reacted with 1-fluoro-2,4-dinitrophenyl-
5-L/D-leucinamide (L- or D-FDLA) and analyzed by LC-MS to
reveal the presence of D-Arg and D-Orn. Hence, the structure of
1 was determined as shown in Figure 1.
1
1
43.0, 142.6, 123.5, 121.8, 119.4, 118.4, 118.2, 116.8, 116.0, and
13.6 ppm, suggesting the presence of two aromatic rings. A
detailed analysis of the 2D NMR data including DQF-COSY,
HMQC, and HMBC spectra revealed that 1 was composed of
one arginine (Arg), one N-δ-hydroxy-N-δ-formyl ornithine
The molecular formula of chlorocatechelin B (2) was
(
hfOrn), and two trisubstituted benzoic acid residues. The
established as C18
H25ClN O on the basis of HR-ESIMS
4 8
+
benzoic acids were revealed to be 4-chloro-2,3-dihydroxy
benzoic acids (CDB) on the basis of the molecular formula
and the extensive analysis of NMR and UV absorption
(m/z 461.1433 [M + H] , calcd 461.1434). Metabolite 2 gave
1
13
H and C NMR spectra that were simpler than those of 1.
The planar structure of 2 was determined by 2D NMR analysis
(Figure 2b) to be composed of one unit each of CDB, Orn, and
hfOrn. The connectivity of CDB to Orn was determined by an
HMBC correlation from H1 of Orn to C7 of CDB. No HMBC
1
5,16
spectra.
The N-hydroxy-N-formyl group in hfOrn and
two CDB residues were suggested to be putative iron-chelating
1
13
moieties (Figure 2a). Two sets of H and C signals were
observed in the hfOrn moiety due to the geometrical
isomerization of the N-hydroxy-N-formyl group.
correlation between Orn and hfOrn was observed in CD OD;
3
hence, we next recorded the NMR spectra in CD OH. Two
3
The order of the backbone in compound 1 was determined
by an HMBC correlation from H1 of hfOrn to C6 of Arg. The
connectivity of CDB1 to Arg was also determined by HMBC
amide protons at 8.67 (1H, br m) and 8.47 ppm (1H, br m)
were observed in the H NMR spectrum, and these were
assigned as 1-NH of Orn and hfOrn, respectively, based on a
1
B
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DQF-COSY experiment. An HMBC correlation from 1-NH of
hfOrn to C5 of Orn was observed, and thus hfOrn was
connected to Orn. Advanced Marfey analysis revealed the
absolute stereochemistries of both Orn and hfOrn were D, and
thus the chemical structure of 2 was determined as shown in
Figure 1.
Characteristic features of the chemical structures of
chlorocatechelins implied that they are siderophores. In fact,
chlorocatechelins A (1) and B (2) in cell culture were rarely
detected in the presence of iron (Figure S1). To investigate the
iron binding property of 1, we first examined a spectrophoto-
Fe(III)-1, indicating that compound 2 had weaker affinity for
Fe(III) than compound 1.
19
During purification procedures, chlorocatechelin A (1) was
always accompanied by a small amount of chlorocatechelin B
(2) in the LC-MS analysis. We wondered if 1 was
spontaneously converted to 2. To test this possibility, the
stability of 1 under various pH conditions (buffered with
sodium phosphate) was tested. Compound 1 was stable in
neutral and basic solutions (pH 7−11) for 24 h. On the other
hand, 1 gave three compounds when incubated in acidic
conditions (pH 3−5) (Figures S5, S6). One of them showed
the same retention time, HRMS, and MS/MS fragmentation
pattern as those of 2 in an LC-MS analysis, indicating this
newly formed compound was 2. The other two peaks had an
m/z of 213.0067 and 656.1157, respectively. Judging from the
molecular formula, the compound with an m/z of 213.0067
metric titration experiment of compound 1 with FeCl in 50
3
mM BisTris buffer (pH 7.0, I = 0.1 M, Figure 4a, S3a). An
(
corresponding to C H ClN O , Δmmu = +0.6) might be a
8
6
2
3
fragment 6 that can be produced when compound 1 is
decomposed to 2. Elimination of NH instead of 6 from
3
compound 1 should give compound 5 with m/z 656.1157
(
corresponding to C H Cl N O , Δmmu = 0.0) (Scheme 1).
26
28
2
5
11
Scheme 1. Proposed Mechanism for Decomposition of 1 and
Production of 2
Figure 4. Iron binding properties of 1. (a) Spectrophotometric
titration of 1 with FeCl (every 0.2 equiv from 0 to 1.0 equiv, black to
3
green) in 50 mM Bis-Tris buffer (pH 7.0, I = 0.1 M with NaCl). (b)
Cyclic voltammogram of Fe(III)-1 complex (100 μM of 1 and 60 μM
of FeCl ) in 5 mM BisTris buffer (pH 7.0, I = 0.1 M with NaCl).
3
increasing absorbance at 516 nm was observed until 1 equiv of
FeCl was added, indicating that 1 formed a complex with
3
Fe(III) at a 1:1 ratio. Next, the redox potential of the Fe(III)-1
complex was measured in cyclic voltammetry (CV) experi-
ments. The mixture of 1 (100 μM) and FeCl (60 μM) in 5
3
mM BisTris buffer (pH 7.0, I = 0.1 M, Figure 4b) gave a
reversible voltammogram with a half wave potential (E ) of
1/2
−
578 mV (vs NHE). Under our conditions, the well-studied
Fe(III)-enterobactin complex exhibited irreversible voltammo-
grams, whereas ferrioxamine B showed a reversible voltammo-
gram with an E1/2 of −440 mV (Figure S4a). These results
indicated that the Fe(III)-binding affinity of chlorocatechelin A
(1), possessing two CDB groups and one N-hydroxy-N-formyl
group, is higher than that of desferrioxamine B, a siderophore
with three hydroxamate groups, and lower than that of the
catecholate-type-siderophore enterobactin (reported E1
=
/2
6
−
750 mV ).
The molar ratio in the complex of chlorocatechelin B (2) and
Considering the fact that compound 2 showed much lower
affinity for Fe(III) than 1, the labile CDB-guanidine function
likely contributes to inactivation of 1. One of the well-known
inactivation systems of siderophores is hydrolysis by an
esterase; one of three ester bonds in enterobactin is hydrolyzed
by esterase when enterobactin forms a complex with Fe(III),
which facilitates reduction of Fe(III) catalyzed by ferric
reductase. In our proposed model for degradation of 1 to 2,
nucleophilic attack by 2-OH of CDB2 is requisite (Scheme 1).
Since this hydroxyl group may coordinate to Fe(III) in the
Fe(III) was also investigated by measuring the UV−vis spectral
changes, revealing that three molecules of 2 can bind one
molecule of Fe(III) (Figure S3b, S3c). To compare the redox
potential of Fe(III)-1 and that of Fe(III)-2, CV analysis of
Fe(III)-2 was conducted under various conditions. However, a
reversible voltammogram was not obtained; the cathodic peak
potential (E = −450 mV) suggested the E of Fe(III)-2 was
9
pa
1/2
higher than −450 mV (Figure S4b). These data revealed that
the redox potential of Fe(III)-2 is much higher than that of
C
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Organic Letters
Letter
−
1
+
complex of Fe(III)-1, degradation of Fe(III)-1 might not
1315, 1219 cm ; HRMS (ESI) m/z 673.1432 [M + H] calcd for
2
0
1
13
C H Cl N O , 673.1422; for H NMR and C NMR, see Table S1
occur. Therefore, degradation of 1 might occur, not for
releasing Fe(III), but for other purposes, e.g. inactivating an
excess amount of the free siderophore.
26 31
2
6
11
2
0
(
14) Chlorocatechelin B (2): brown amorphous solid: [α] −7.7 (c
.70, MeOH); UV (MeOH) λmax (log ε) 210 (4.38), 256 (4.01), 309
3.33) nm; IR (neat) 3162 (br), 3085, 2936, 1669, 1653, 1558, 1541,
437 cm ; HRMS (ESI) m/z 461.1433 [M + H] calcd for
C H ClN O , 461.1434; for H NMR and C NMR, see Table S2.
15) The molecular formula and NMR spectra suggested two
possibilities, 4-chloro-2,3-dihydroxybenzoic acid and 2-chloro-3,4-
dihydroxybenzoic acid. The UV spectra of 1 and 2 indicated that
they contained the former ones.
(16) Fox, D. T.; Hotta, K.; Kim, C.-Y.; Koppisch, A. T. Biochemistry
008, 47, 12251−12253.
17) This fragment peak might be produced by cleavage of the
guanidine carbon and 5-NH in Arg as shown in Scheme 1, in addition
to the amide bond fragmentation between Arg and hfOrn.
18) Giessen, T. M.; Franke, K. B.; Knappe, T. A.; Kraas, F. I.;
Bosello, M.; Xie, X.; Linne, U.; Marahiel, M. A. J. Nat. Prod. 2012, 75,
05−914.
19) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K.-I. Anal. Chem.
D
0
(
1
In this study, two novel siderophores chlorocatechelins A (1)
and B (2) were isolated and their structures were determined
by spectroscopic analysis and degradation study. Chlorocate-
chelins 1 and 2 were the first natural products that contain
CDB units. In addition, 1 possessed a rare acylguanidine
structure. The acylguanidine structure is essential for the potent
Fe(III)-chelating ability of chlorocatechelin A (1). Decom-
position of the acylguanidine structure in acidic conditions
furnished a lower-affinity siderophore chlorocatechelin B (2).
Producing labile compounds leads to a waste of energy for
microbes unless the decomposition plays any productive role.
Detailed analysis of the metabolism of Fe(III)-1 and Fe(III)-2
in and around the producer and surrounding organisms would
reveal new functions of siderophores.
−1
+
1
13
18
26
4
8
(
1
6
2
(
(
9
(
1
997, 69, 5146−5151. And also see Table S3.
ASSOCIATED CONTENT
(20) Although we tried to verify our hypothesis, we could not test it
■
*
S
Supporting Information
since Fe(III)-1 precipitated in acidic conditions.
Detailed experimental procedure, spectral data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: scseigyo-hisyo@pharm.kyoto-u.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Kenji Kano (Kyoto University) for supporting
cyclic voltammetry experiments and Eiko Moriyoshi (Kyoto
University) for technical assistance. This work was supported in
part by research grants from the Japan Society for the
Promotion of Science (JSPS), the Ministry of Health, Labour
and Welfare of Japan (MHLW), and the Ministry of Education,
Culture, Sports, Science and Technology of Japan (MEXT).
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13) Chlorocatechelin A (1): brown amorphous solid; [α] +2.6 (c
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