Nocardamin glucuronide, a new member of the ferrioxamine siderophores isolated from the ascamycin-producing strain Streptomyces sp. 80H647

Article abstract of DOI:10.1038/s41429-019-0217-5

A new siderophore glucuronide, nocardamin glucuronide (1), was isolated together with nocardamin (2) by applying the one strain-many compounds (OSMAC) approach to the ascamycin-producing strain, Streptomyces sp. 80H647, and performing multivariate analysis using mass spectral data. Structure elucidation was accomplished by a combination of NMR and MS analyses. The absolute configuration of the glucuronic acid moiety was found to be β-D-GlcA by hydrolysis using β-glucuronidase, subsequent derivatization of the hydrolysate, and comparison with standards. The siderophore activity of 1 was evaluated through the chrome azurol S assay and was comparable to that of 2 and deferoxamine (IC₅₀ 13.4, 9.5, and 6.3 μM, respectively). Nocardamin glucuronide (1) is the first example of a siderophore glucuronide.

Full text of DOI:10.1038/s41429-019-0217-5

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0217-5
SPECIAL FEATURE: BRIEF COMMUNICATION
Nocardamin glucuronide, a new member of the ferrioxamine
siderophores isolated from the ascamycin-producing strain
Streptomyces sp. 80H647
1 ●
1 ●
Yushi Futamura Takeshi Shimizu Hiroyuki Osada1
1 ●
1 ●
Julius Adam V. Lopez
Toshihiko Nogawa
Received: 21 April 2019 / Revised: 4 June 2019 / Accepted: 10 July 2019
The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
©
Abstract
A new siderophore glucuronide, nocardamin glucuronide (1), was isolated together with nocardamin (2) by applying the one
strain-many compounds (OSMAC) approach to the ascamycin-producing strain, Streptomyces sp. 80H647, and performing
multivariate analysis using mass spectral data. Structure elucidation was accomplished by a combination of NMR and MS
analyses. The absolute configuration of the glucuronic acid moiety was found to be β-D-GlcA by hydrolysis using β-
glucuronidase, subsequent derivatization of the hydrolysate, and comparison with standards. The siderophore activity of 1
was evaluated through the chrome azurol S assay and was comparable to that of 2 and deferoxamine (IC50 13.4, 9.5, and
6
.3 μM, respectively). Nocardamin glucuronide (1) is the first example of a siderophore glucuronide.
In search for new bioactive compounds, the Streptomyces
sp. 80H647 strain was subjected to the one strain-many
compounds (OSMAC) method [1]. This strain is a producer
of the nucleoside antibiotic, ascamycin [2], and we hypo-
thesized that the production of more structurally interesting
metabolites will be stimulated under different culture con-
ditions [1].
oatmeal (OA) in separate groups while the rest of the
samples were clustered with the blank (Fig. 1). This clearly
indicated that there was a difference between the metabolite
profiles of the CD and OA cultures while there was no or
reduced metabolite production in the other media; hence,
confirming the effectiveness of the OSMAC approach. In
addition, a volcano plot (p-value vs log fold change) of OA
samples against all other samples was created and it showed
that a compound with m/z 777 was unique to OA (Fig. 1). A
search of our NPPlot database [3] as well as the Dictionary
of Natural Products [4] revealed that it was possibly a new
compound and was pursued.
Six different culture media (70 mL) were prepared
(
Table S1), inoculated, and incubated with shaking at 150
rpm and 28 °C for 5 days. A small-scale extraction was
done to obtain EtOAc, BuOH, and aqueous extracts, which
were evaporated to dryness. The extracts were subjected to
bioactivity screening and LC-MS analysis. MS data were
used for the principal component analysis, which presented
samples cultured in corn steep liquor-dried yeast (CD) and
A 2.5-L OA culture was prepared and subjected to
liquid–liquid partition, HP20 Diaion resin adsorption, and a
number of medium pressure liquid chromatography runs to
obtain pure compounds, 1 and 2 (Fig. 2 and S1). Compound
1
was the target compound with m/z 777, while 2 was a
major metabolite with m/z 601. The planar structures were
deduced by interpretation of the MS and NMR data. Using
high-resolution ESI-TOFMS data (Figs. S2 and S3), the
following molecular formula were acquired: C H N O
15
This article is dedicated to Dr Kiyoshi Isono with respect and
admiration for his achievements in antibiotics research.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0217-5) contains supplementary
material, which is available to authorised users.
3
3
56
6
for 1 ([M + H]+ m/z 777.3887, calcd 777.3876), and
C H N O for
601.3556). Compound 2 had nine signals in its C NMR
2
([M + H]+ m/z 601.3570, calcd
2
7
48
6
9
1
3
*
Hiroyuki Osada
cb-secretary@ml.riken.jp
spectrum and seven spin systems plus two exchangeable
1
1
protons in its H NMR spectrum (Table 1, Fig. S4). Con-
RIKEN Center for Sustainable Resource Science, Chemical
Biology Research Group, 2-1 Hirosawa, Wako, Saitama 351-0198,
Japan
sidering its molecular formula, these NMR signals were
determined to be three repeating units of N-hydroxy-

J. A. V. Lopez et al.
Fig. 1 Principal component analysis of the MS data of Streptomyces
sp. 80H647 extracts cultured in different media and volcano plot of
OA extracts against all other samples. The composition of each
medium is listed in Table S1. Blank refers to medium only. The
numbers (3, 7, and 9) indicate pH. B and E denotes BuOH and EtOAc
extracts, respectively, and W is the corresponding aqueous layer. CD is
a modified and updated version of C4. The corresponding volcano plot
is shown in the box
to methines. COSY and TOCSY data (Figs. S11 and S12)
also showed that they form a separate spin system apart
from the methylenes of the nocardamin unit. Based on the
chemical shifts of these methines and a mass difference of
176 Da between 1 and 2, a nocardamin glucuronide struc-
ture was proposed for 1 (Fig. 2).
Connections were established by analysis of 2D NMR
data (Fig. 3 and S10–S14). The N-hydroxy-N′-succinylca-
daverine assembly was based on the following correlations:
COSY signals from H-2 through NH-7, and between H-9
and H-10; TOCSY signals from NH-7 to H-6 and H-5;
Fig. 2 Structures of nocardamin glucuronide (1), nocardamin (2), and
bisucaberin
HMBC from H-2 and H-6 to C-4 (in D O), from H-6 (in
2
D O) and H-10 to C-8, and from H-2 (in D O) and H-9 to
2
2
C-11. The glucuronic acid (GlcA) moiety was supported by
N′-succinylcadaverine, which is characteristic of nocarda-
min, also known as desferrioxamine E [5–7].
the following data: COSY signals from H-1′ though H-5′,
and HMBC from H-5′ to C-1′ and C-6′ (in D O). The
2
Although similar signals for nocardamin were evident in
proposed structure of 1 also satisfies nine degrees of unsa-
turations as calculated from the molecular formula. More-
over, MS/MS data (Fig. S15) showed fragment ions
corresponding to a loss of 176 Da (m/z 601) and 200 Da
(m/z 577; and 401, 201 from 601) consistent with the loss of
GlcA and N-hydroxy-N′-succinylcadaverine, respectively.
1
the H NMR spectrum of 1, additional smaller peaks were
observed at δ 3–5 ppm (Fig. S4). Extra peaks at δ 70–80
H
C
13
and 105.7 or 105.1 ppm were also found in the C NMR
spectrum of 1 (Figs. S7 and S8). DEPT-135 and HSQC data
(
Figs. S9 and S10) revealed that these signals corresponded

Nocardamin glucuronide, a new member of the ferrioxamine siderophores isolated from the. . .
Table 1 1H and 13C NMR data
of 1 and 2
1a
2a
Literature (bisucaberin)b,5
Position
δH (mult., J in Hz)
δC
δH (mult., J in Hz)
δC
δH (mult., J in Hz)
δC
1
2
3
4
5
6
7
8
9
1
1
, N-OH
, CH2
, CH2
, CH2
, CH2
, CH2
, NH
9.88 (br s)
9.6 (br s)
3.46 (t, 6.9)
1.51 (m)
1.20 (m)
1.37 (m)
3.00 (m)
7.72 (br t)
9.53 (s)
c
3.48, 3.69 (m)
46.8
25.9
23.2
28.6
38.3
46.8
25.8
23.1
28.6
38.3
3.48 (t, 6.3)
1.47 (m)
46.3
25.5
22.5
28.2
37.9
1.49 (m)
1.19 (m)
1.36 (m)
2.99 (m)
7.75 (br t)
1.15 (m)
1.35 (m)
3.01 (br m)
7.61 (br t)
, C=O
, CH2
0, CH2
1, C=O
171.9
30.2
172.0
30.0
171.8
30.5
2.27 (m)
2.28 (t, 6.3)
2.58 (m)
2.27 (t, 7.4)
2.57 (t, 7.4)
c
2.58, 2.86 (m)
27.7
27.5
27.8
171.5
171.4
171.5
literature (GlcA part)f,8
1
2
2
3
3
4
4
5
6
′, CH
′, CH
′-OH
′, CH
′-OH
′, CH
′-OH
′, CH
′, COOH
4.62 (d, 8.0)
3.08 (m)
105.7d
71.8
4.57 (d, 8.5)
3.04 (m)
105.7
72.2
76.6
72.0
5.38 (br d)
3.22 (m)
76.4
71.7
3.22 (m)
3.33 (m)
3.65 (m)
5.1 (br s)
3.22 (m)
5.1 (br s)
3.44 (m)
74.4
173.1e
75.9
171.3
a500 MHz for H, 125 MHz for 13C, DMSO-d6
b400 MHz for H, 100 MHz for 13C, DMSO-d6
cSignals affected by GlcA
1
1
dFrom HSQC data, DMSO-d6
eFrom HMBC data, D2O
f500 MHz for H, 125 MHz for 13C, D2O/CD3CN/DMSO-d6 (2/3.5/6)
1
eliminated on the basis of the chemical shift values on GlcA
and the weak downfield shifts on the γ (H-2) and δ (H-10)
positions from GlcA in comparison with the literature [8].
Unlike in 2, H-2 and H-10 showed nonequivalent protons in
1
as seen in the HSQC spectrum (Fig. S10) indicating
asymmetry caused by the presence of GlcA. Likewise,
1
3
paired C NMR signals (C-3, C-5, C-9, and C-10, Fig. S7)
were detected, which were not present in 2. The occurrence
of nonequivalent protons (H-2 and H-10) in 1 confirmed the
placement of GlcA on the hydroxamic acid group rather
than the amide nitrogen.
To determine the absolute configuration of GlcA,
hydrolysis, derivatization, and subsequent comparison with
standards were performed. To accomplish this, GlcA was
obtained by using β-glucuronidase (Fig. S16). The suc-
cessful enzymatic cleavage of GlcA suggested a β config-
uration of the anomeric carbon which was also supported by
chemical shift (δ 105.7, δ 4.62) and coupling constant
Fig. 3 Key 2D NMR data of 1
The chemical shifts observed for GlcA (Table 1) and its
observed cleavage from the macrolide during fragmentation
is in agreement with other reported N-O-glucuronides [8].
C
H
data (J = 8.0 Hz) [8]. Furthermore, a D configuration was
predicted since D-glycosides are usually in the β form [9].
+
In addition, the possibility of an N glucuronide was

J. A. V. Lopez et al.
respectively) demonstrated the more lipophilic property of 2
over 1.
To our knowledge, nocardamin glucuronide (1) is the
first example of a siderophore glucuronide. Glucuronides
play an important role in drug metabolism and pharmaco-
kinetics [14], such as detoxification and enterohepatic
recirculation, although majority of the studies are focused
on the mammalian system. In addition, there are only few
reports on the production of glucuronides by microbial
enzymes [12] and little is known about their function and
biosynthesis. Though a minute amount of nocardamin (2)
was also found in the CD culture, the production of 1 and 2
were particularly enhanced in the OA medium. While a
possible explanation for this outcome could not be sup-
posed, it shows that OSMAC is an efficient way of stimu-
lating cryptic and variable secondary metabolism. On the
other hand, siderophores continue to be a hot topic in the
areas of antibiotics, drug conjugates, drug resistance, and
combination therapy for the treatment of various illnesses
such as infections, cancer, and neurodegenerative diseases
Fig. 4 Dose-response curve of the siderophore activity 1, 2, and
deferoxamine (DFO)
[
15–17].
To confirm this, the hydrolysate and standards, D- and L-
GlcA, were separately reacted with L-cysteine methyl ester
followed by phenylisothiocyanate according to the method
of Tanaka et al. [10]. The derivatives were analysed by LC-
MS and results showed that the mass and retention time of
the sample matched that of D-GlcA (Fig. S17). Thus, GlcA
was unambiguously determined to have the β-D
configuration.
Since nocardamin (2) is a known siderophore [7], the
iron binding activity of 1 was evaluated via the chrome
azurol S assay [11]. Indeed, 1 exhibited iron chelating
properties comparable to 2 and deferoxamine (DFO or
desferrioxamine B) at IC50 13.4, 9.5, and 6.6 μM, respec-
tively (Fig. 4). On the other hand, the cytotoxicity test of 1
and 2 at 39 and 50 μM, respectively, was negative against
the following cell lines: HeLa (cervical cancer), HL-60
Acknowledgements This work was supported in part by JSPS
KAKENHI Grant Numbers JP16H06276, JP17H06412, and
JP18H03945. We would like to thank Ms Harumi Aono, Ms Akiko
Okano, Dr Rachael Uson-Lopez, Ms Emiko Sanada, and Dr Motoko
Uchida for technical support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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