Isolation and structure elucidation of the nucleoside antibiotic strepturidin from Streptomyces albus DSM 40763

Article abstract of DOI:10.1038/ja.2014.16

The antibiotic strepturidin (1) was isolated from the microorganism Streptomyces albus DSM 40763, and its structure elucidated by spectroscopic methods and chemical degradation studies. The determination of the relative and absolute stereocenters was partially achieved using chiral GC/EI-MS analysis and microderivatization by acetal ring formation and subsequent 2D-NMR analysis of key ¹H, ¹H-NOESY NMR correlations and extraction of ¹H, ¹³C coupling constants from ¹H, ¹³C-HMBC NMR spectra. Based on these results, a biosynthesis model was proposed.

Full text of DOI:10.1038/ja.2014.16

The Journal of Antibiotics (2014) 67, 471–477
2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14
www.nature.com/ja
&
ORIGINAL ARTICLE
Isolation and structure elucidation of the nucleoside
antibiotic strepturidin from Streptomyces albus
DSM 40763
1,4
2,4
1
1
3
¨
Alexander Pesic , Britta Steinhaus , Sebastian Kemper , Jonny Nachtigall , Hans Jurgen Kutzner ,
2
1
¨
Gerhard Hofle and Roderich D Sussmuth
¨
The antibiotic strepturidin (1) was isolated from the microorganism Streptomyces albus DSM 40763, and its structure
elucidated by spectroscopic methods and chemical degradation studies. The determination of the relative and absolute
stereocenters was partially achieved using chiral GC/EI-MS analysis and microderivatization by acetal ring formation and
subsequent 2D-NMR analysis of key 1H,1H-NOESY NMR correlations and extraction of 1H,13C coupling constants from
1H,13C-HMBC NMR spectra. Based on these results, a biosynthesis model was proposed.
The Journal of Antibiotics (2014) 67, 471–477; doi:10.1038/ja.2014.16; published online 5 March 2014
Keywords: 2D NMR; chiral GC/EI-MS analysis; nucleoside antibiotic; stereochemistry; Streptomyces; structure elucidation
INTRODUCTION
This report presents in detail the cultivation of the producing
The genus Streptomyces (Order Actinomycetales, colloquial ‘Actinomy- strain, as well as the isolation and structure elucidation of the
cetes’) is a widely studied genus of the Actinomycete family. Among all nucleoside antibiotic strepturidin (1), by means of HPLC-HR-ESI-
Actinomycetes, Streptomycetes play an important role in drug discovery ( þ )-Orbitrap-MS, chiral GC/EI-MS, extensive 2D-NMR spectro-
as from this genus 90% of the more than 3000 known antibiotics scopy including partial assignment of the relative stereochemistry and
have been isolated.1 In comparison with other Gram-positive bacteria absolute stereochemistry. Based on the investigations on the structure
like Staphylococcus, Streptococcus and Bacillus, the GC-content of elucidation and determination of the stereochemistry, we postulated a
the DNA of Streptomyces is with B70% comparatively high. Examples plausible biosynthesis model for (1).
of prominent antibiotics produced by these bacteria are the amino-
glycoside streptomycin (antituberculosis), the chromopeptide actino- RESULTS
mycin (cytostatic) and the polyketide tetracycline (antibacterial).
Producing strain and bacterium for antibiotic assay
Here we report on an antibiotic compound produced by S. albus has been extensively characterized in the past, notably by
Streptomyces albus that exhibits a specific antibacterial activity against Gordon et al.,6 Bo¨ttiger5 and Herrmann.8 This streptomycete species
the Gram-negative nitrogen-fixing soil bacterium Azotobacter chroo- can be easily isolated from its natural habitat, that is, self-heated
coccum.2 This antibacterial activity has been initially discovered in hay or compost, and can be rapidly identified by classical properties.
1956 by Kutzner3 (see also Flaig and Kutzner4), and, as shown later by The strain DSM 40763 has been selected from the numerous
5
¨
Bottiger, extends to all other members of the family Azotobacteraceae. representatives of this species, which are kept in worldwide culture
However, this antibacterial effect is independent from the nitrogen collections. For the antibacterial assay, the strain A. chroococcum DSM
status of the medium and therefore the nitrogenase system of 281 has been employed.
Azotobacter has been excluded as a molecular target.6 The first
isolation of the active compound, named strepturidin (1), and its
preliminary structure elucidation has already been described by
Steinhaus7 in 1993. Initial attempts to characterize the new
antibiotic substance with the above-mentioned features by test
reactions proved positive for guanidine-groups, sugars and amino
sugars. However, test reactions yielded negative results for aromatic
amines, organic acids, amino acids and peptides.
Cultivation and isolation of strepturidin (1)
S. albus DSM 40763 was cultivated as described by Herrmann.8 With a
seed culture of 200ml, a 10l preculture was inoculated to be used
subsequently to inoculate the production fermenter containing 80 l
of complex medium (mannitol 10 g lꢀ1, soy meal (full-fat) 10 g lꢀ1
meat extract 5 g lꢀ1, sodium chloride 1 g lꢀ1, calcium carbonate 1g lꢀ1
tegosipon 10 ml). The cultivation was carried out at 37 1C at 300r.p.m.
,
,
1
2
Institut fur Chemie, Technische Universitat Berlin, Berlin, Germany; Helmholtz Zentrum fur Infektionsforschung, Braunschweig, Germany and 3Technische Hochschule
¨
Darmstadt, Institut fur Mikrobiologie und Genetik, Darmstadt, Germany
¨
¨
¨
4These authors contributed equally to this work.
¨
¨
¨
Correspondence: Professor RD Sussmuth, Institut fur Chemie, Technische Universitat Berlin, Straꢀe des 17. Juni 124, 10623 Berlin, Germany.
E-mail: suessmuth@tu-berlin.de
Received 5 November 2013; revised 20 January 2014; accepted 27 January 2014; published online 5 March 2014

Isolation and structure elucidation of strepturidin
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Table 1 NMR spectroscopic data for strepturidin (1) and
monoacetal-strepturidin (3) (500 MHz) in d6-DMSO
Strepturidin (1)a
Monoacetal-strepturidin (3)
dH in p.p.m.
dH in p.p.m.
(multiplicity,
(multiplicity,
dC in p.p.m.,
dC in p.p.m.,
Pos.
integral, J in Hz)
multiplicity
integral, J in Hz)
multiplicity
Figure 1 Structure of strepturidin (1). Bold lines indicate 1H,1H-COSY
correlations. Blue arrows indicate 1H,13C-HMBC correlations. Asterisks mark
the five stereogenic centers. The list of chemical shifts is summarized in
Table 1.
1
2
/
ꢀ
/
ꢀ
ꢀ
/
155.9, qC
ꢀ
/
152.3, qC
3
ꢀ
ꢀ
4
ꢀ
ꢀ
165.4, qC
109.1, qC
145.4, CH
79.9, CH
71.8, CH
73.8, CH
81.3, CH
ꢀ
ꢀ
164.0, qC
110.4, qC
140.4, CH
79.4, CH
84.2, CH
82.8, CH
81.2 CH
40.6 CH2
5
and an aeration of 0.5 v.v.m. Growth and antibiotic production were
monitored by wet cell mass weight and HPLC analysis, respectively.
After 26 h, 130 g lꢀ1 wet cell mass and an antibiotic titer of 92 mg lꢀ1
were determined. The culture broth was harvested by centrifugation,
sodium heptanesulfonate was added to the supernatant and the crude
antibiotic obtained by ion-pair extraction on Amberlite XAD-16
(Sigma-Aldrich, Taufkirchen, Germany). From the eluate of the resin
an excess of sodium heptanesulfonate was extracted as an ion-pair with
methyltrioctylammonium chloride using dichloromethane as the
solvent. Further purification was carried out by preparative
6
7.34 (s, 6.4, ꢀ)
4.37 (d, 1, 5.5)
3.85 (dd, 1, 5.4)
3.64 (dd, 1, 5.0)
3.77 (ddd, 1, 4.0)
7.50 (s, 1, ꢀ)
4.60 (d, 1, 3.2)
4.83 (dd, 1, 6.3; 3.3)
4.68 (/, m, overlap)b
3.70 (dd, 1, 5.2)
3.18 (/, /, /);
3.25 (/, /, /)
/
7
8
9
10
11 3.16; 3.52 (/, 2, /)c 40.7, CH2
12
13
8.44 (s, b, 1.0)
ꢀ
ꢀ
ꢀ
169.8, qC
ꢀ
169.3, qC
14 4.77 (t, 1, 3.3, 8.6) 59.0, CH
4.70 (m, overlap, /)
59.4, CH
RP18 HPLC and desalting with Amberlite XAD-16 followed by
þ
15
16
17
18
1.99 (m, 2, /)
23.9, CH2 2.11/1.94 (m, overlap, /) 23.3, CH2
CM-Sephadex NH4 chromatography as described below. After
purification, 1.76 g of pure strepturidin (1) was obtained as a white
powder with no defined m.p.
2.07 (t, 2, 7.2)
31.8, CH2
174.1, qC
ꢀ
2.08 (m, overlap, /)
31.6, CH2
174.7, qC
ꢀ
ꢀ
ꢀ
6.77 (s, 1, ꢀ);
6.8 (br, 2, ꢀ);
7.3 (s, 1, ꢀ)
10
20
30
40
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Structure elucidation by means of MS and NMR
The exact molecular mass of (1) (m/z found 486.19193 [M þ H]þ
)
ꢀ
157.6, qC
157.9, qC
ꢀ
ꢀ
ꢀ
4.18 (m, 2, /)
ꢀ
ꢀ
derived from the HR-ESI-(þ )-Orbitrap-MS analysis (m/z calcd
486.18227 [M þ H]þ , Dm 4.883 p.p.m.) gave a molecular formula
of C17H27O9N8 [Mþ H]þ . The IR spectrum showed absorptions for
amine and hydroxyl groups (3341, 2928cmꢀ1) and amide carbonyls
(1662 cmꢀ1) but no characteristic bands for aromatic hydrocarbons
were detected below 900 cmꢀ1 (C-H deformation vibration). The UV
spectrum showed a maximum at l ¼ 262 nm (e 3.89) and displayed
characteristic absorptions for heterocycles such as nucleobases.
Subsequently to the determination of these parameters, the structure
elucidation of (1) (Figure 1) was established from extensive use
of 1H- and 2D-NMR experiments (1H,1H-COSY, 1H,13C-HMBC,
1H,13C-HSQC, 13C-NMR and 13C-DEPT) in d6-DMSO that revealed
a composition of the molecule from four distinct building blocks. The
chemical shifts are summarized in Table 1. In the following a more
detailed analysis of the NMR data is given.
4.20 (s, 2, ꢀ)
42.1, CH2
42.8 CH2
169.2, qC
28.4, CH3
113.5, qC
144.1, qC
127,7 CH
124,0 CH
124,0 CH
50
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
168.1, qC
100
200
300
400
500
600
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1.55 (s, 3, ꢀ)
ꢀ
ꢀ
7.35 (/, /, /)
7.52 (d, 2, 7.7)
7.37 (/, /, /)
The symbol ‘/’ indicates not assigned and ‘ ꢀ’ indicates not available.
aAs reported by Steinhaus.7
bOverlap in 1H-NMR, coupling constants could not be determined.
cABX-System, AB-Part with broad lines.
the subsequent spin system identified as a triol-containing carbon
chain, an open-chain aldopentose derivative named diamino aldo-
pentose. The linkage was assigned from the methylene protons of the
diamino aldopentose alkyl spin system at dH-11 3.2/3.5 p.p.m. (br, 2H;
ABX-part, AB-part with broad lines) correlating to dC-13 169.8 p.p.m.
in the 1H,13C-HMBC NMR spectrum.7 From the remaining four
aliphatic proton signals present in this spin system, three methine
The structure elucidation of (1) commenced with the inter-
1
pretation of the H-NMR (Table 1) and 13C-DEPT NMR spectra.7
The 1H-NMR spectrum of (1) reveals 14 proton NMR signals, with 8
aliphatic methylene (CH2) protons, 5 aliphatic protons and 1 olefinic
methine (CH) proton. In analogy to the 1H-NMR, the 13C-DEPT
NMR spectrum reveals the corresponding signals of four methylene,
six methine and, in addition, seven quaternary carbon signals.7
No primary carbons as represented by methyl groups were detected.
The NMR data of (1) can be formally divided into four
spin systems. The first spin system was identified as the glutamine
(Gln) Gln-Na-hydroxamate with four methylene protons at dH-16
3
protons (CH) at dH-7 4.3 p.p.m. (d, JH-7,H-8 ¼ 5.5 Hz, 1H), dH-9
3
3
3.6 p.p.m. (dd, J ¼ 5 Hz, 1H) and dH-10 3.7 p.p.m. (ddd, J ¼ 4 Hz,
1H) are bound to three carbons bearing hydroxyl groups.
One additional aliphatic methine proton at dH-8 3.8 p.p.m. (dd,
3J ¼ 5.4 Hz, 1H) is connecting a third spin system identified as an
2-aminoimidazol-5-one moiety through H,13C-HMBC correlations
1
3
0
0
to the quaternary carbons at dC-2 157.6 p.p.m. and dC-5 168.1 p.p.m.,
respectively. In addition, the 2-aminoimidazol-5-one spin system
accommodates the remaining two methylene protons of structure
(1) at dH-4 4.2 p.p.m. (s, 2H). The final spin system constitutes the
nucleobase uracil that is attached via a C-C-bond to the open-chain
2.07p.p.m. (t, JH-16,H-15 ¼ 7.2 Hz, 2H) and at dH-15 1.9 p.p.m. (m,
2H) and a single aliphatic methine proton at dH-14 4.7 p.p.m. (t,
3JH-14,H-15 ¼ 3.3 Hz, 3JH-14,NH ¼ 8.6 Hz, 1H). The Gln-Na-hydroxamate
spin system is linked through its a-carbonyl at dC-13 169.8 p.p.m. to
form an amide (dNH-12 8.4 p.p.m. (b, 1H)) with the amino group of
0
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Figure 2 (a) Scheme for preparative microreactions performed with strepturidin (1). Formation of monoacetal-strepturidin (3) with 1,1-dimethoxybenzene
and camphorsulfonic acid for 2D-NMR experiments. Reduction of strepturidin (1) to Gln-strepturidin (2) for chiral GC/EI-MS analysis. (b) HR-HPLC-ESI-
( þ )-Orbitrap-MS data of the compounds (1–3). (c) chiral GC-chromatogram on a LIPPDEX E column. Enantiomer analytics of (R/S)-N-trifluoroacetyl Glu
2-propyl esters (Rt-(S) ¼ 44.56min, Rt-(R) ¼ 44.11min) after hydrolysis and derivatization of Gln-strepturidin (2). The EI-MS spectrum of N-trifluoroacetyl
(S) Glu 2-propyl ester at Rt ¼ 44.56 min is shown in Supplementary Figure S4B. Asterisks mark the stereocenters in strepturidin (1).
diamino aldopentose moiety similar to the C-linkage of the ring the molecule (1). Therefore, the subsequent experiments focused
ribose to uracil in pseudouridine (c). This finding is corroborated by on derivatization reactions to assign a maximum number of
the presence of only one single characteristic olefinic methine proton stereocenters.
at dH-6 7.60p.p.m. (s, 1H) as a part of the uracil structure. These
Our efforts started with the carbon C-14 that is the stereocenter of
data are complemented by the quaternary carbon signals at dC-2 the Gln-Na-hydroxamate. The stereochemistry at C-14 was determined
155.9 p.p.m., dC-4 165.4 p.p.m., dC-5 109.1 p.p.m. and the olefinic by reduction of the Na-hydroxyl amine group with 2N HCl/Zn (2 h,
methine signal at dC-6 145.4 p.p.m. as part of the nucleobase uracil. room temperature) and transformation into the corresponding amino
The complete structure elucidation is further complemented by acid Gln (Figure 2a), generating Gln-strepturidin (2). The reduction was
1H,13C-HSQC NMR data7 yielding the assigned protons together followed by HPLC-HR-ESI-( þ )-Orbitrap-MS (Figure 2b) and HPLC-
with their corresponding carbon atoms and the correlations within LR-ESI-( þ )-MS2 (Supplementary Figure S3). Subsequently, reduced
the spin parts by the 1H,1H-COSY experiment.7 Figure 1 shows Gln-strepturidin (2) (Figure 2a) was hydrolyzed (6N, HCl, 24h, 1101C,
the structure of strepturidin (1) with the key 1H,13C-HMBC under vacuum) to obtain the glutamic acid (Glu) that was derivatized
contacts with blue arrows and the 1H,1H-COSY correlations in to the N-trifluoroacetyl 2-propyl ester. Amino acid analysis by chiral
¨
bold lines.
GC/EI-MS on a LIPODEX E (Macherey-Nagel, Duren, Germany)
column (Figure 2c) revealed that the N-trifluoroacetyl Glu 2-propyl
ester has (S)-configuration. Based on these results, the stereocenter at
C-14 in (1) is also (S)-configured.
Assignment of the absolute stereochemistry in the
Gln-Na-hydroxamate moiety
The above structure elucidation of (1) revealed the constitutional
formula and left the relative and absolute stereochemistry of the five Assignment of the relative stereochemistry in the aldopentose
stereocenters at C-7, C-8, C-9, C-10 and C-14 unassigned (Figure 1). moiety
Various attempts to crystalize the substance failed, most likely because The relative configuration of the remaining four stereocenters in the
of the high polarity, structural flexibility and also instability of central part of the molecule C-7 to C-10 could not be derived by the
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Figure 3 NMR analysis of monoacetal-strepturidin (3). (a) Plausible model for 1,3 acetal based on NMR analysis. Red arrows indicate 1H,1H-NOESY
3
3
contacts. Bold lines indicate JH-8,C-10 and JH-8,C-5 correlations. (b) 1H-NMR region for the three key protons H-7, H-8 and H-9 for compound
(3) and expanded 1H-NMR region for H-8. (c) Traces in F2 extracted from 1H,13C-HMBC spectra for C-10 and C-5 (d). The coupling constants
3JH-8,C-10E10–13Hz and JH-8,C-5E10–13 Hz suggest an axial orientation. (e) Traces in F2 extracted from 1H,1H-NOESY spectra for H-6. (f) Traces in F2
3
extracted from 1H,1H-NOESY spectra for the methyl group Me-100. The weak NOE between H-6 and H-8 in (e) cannot be explained with the conformation
shown in (a). Probably, a second conformer with H-8 and uracil in equatorial position exists.
13C-NMR spectroscopic database approach developed by Kishi and (3) has been unsuccessful and no product formation could be
coworkers9 as these databases are only applicable for 1,3-acetals with a detected by HPLC-HR-ESI-( þ )-Orbitrap-MS (data not shown).
methylene group (CH2) connecting the diol groups. In order to
The formation of the six-membered 1,3-acetal ring present in (3)
reduce the polarity and to increase the rigidity of the molecule for allowed the analysis of the relative configuration of the triol moiety,
additional NMR spectroscopy experiments of (1), we considered using proton–proton coupling constants directly from 1H-NMR data.
transformation of the 1,3-diol unit into the corresponding 1,3-acetal In addition, selTOCSY-NMR data (Supplementary Figure S9) and
using 1,1-dimethoxyethylbenzene and camphorsulfonic acid10 long-range couplings from 1H,1H-NOESY data (Figures 3e and f)
(Figure 2a) to give the monoacetal-strepturidin (3) (m/z 589.2 were used. Because of a lack of strong NOE contacts between the
[Mþ H]þ ). The monoacetal-strepturidin (3) was isolated by pre- methyl-group protons of dMe-1 1.55p.p.m. and the protons H-6, H-7
00
parative reversed-phase C18-HPLC (Supplementary Figure S1). and H-9 (Figure 3b and Table 1), an axial position of this group is not
The formation of the six-membered 1,3-acetal of compound (3) plausible and we assume a chair conformation model for the
1
was proven by H-NMR (Supplementary Figure S5) and 2D-NMR six-membered 1,3-acetal ring with the methyl group in equatorial
1
1
experiments (Supplementary Figures S7–S10; H,1H-COSY, H,13C- and the phenyl group in axial position (Figure 3a and Table 1).
1
1
HSQC, H,13C-HMBC and H,1H-NOESY). Application of Mosher’s Furthermore, the small homo-nuclear proton–proton coupling con-
method for assignment of the absolute configuration determination stant for JH-7,H-8 ¼ 3.3 Hz and 3JH-8,H-9 ¼ 6.3 Hz (48–621 and 30–501
3
on the free hydroxyl group at C-10 on the acetal-protected compound on the basis of the equation of Karplus11 excludes an axial–axial (3Jaa)
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Figure 4 Proposed biosynthesis pathway of strepturidin (1).
configuration between the neighboring protons (Figure 3a). The diamino aldopentose of which we partially assigned the relative
measurements of proton–carbon coupling constants by extraction of stereochemistry. Based on building blocks available from primary
3
1H,13C-HMBC traces in F2 dimension for the correlations JH-8,C-5 metabolism and biosynthetic analogies to other nucleoside antibiotics,
3
and for JH-8,C-10 (Figures 3c and d) yield, in both cases, high we propose a biosynthesis model for (1) as outlined in Figure 4, of
coupling constants of 3JH,CE10–13 Hz. Although these measurements which the sequence of intermediate biosynthetic steps may differ: the
are only guidelines, they are in line with above data suggesting biosynthetic logic suggests uridine monophosphate (I) with ribose as
an axial orientation of H-8/C-5 and of H-8/C-10, respectively precursor of the aldopentose-moiety of strepturidin (1). This is in line
(Figure 3a). Further support for the suggested conformation with the NMR data (Figure 3) that suggest a syn-configuration of C-7
(Figure 3a) is given by the analysis of 1H,1H-NOESY NMR data and C-9 and an anti-configuration of C-8 in (1). Therefore, from the
(Supplementary Figure S10) with a mixing time of 600 ms. The data four aldopentoses, arabinose and xylose are excluded as likely
showed similarly weak NOE effects from the equatorial methyl precursors. Considering that lyxose is a very rare aldopentose in
protons at dMe-1 1.55p.p.m. to the axial H-8 proton and to the nature,13 this strongly favors ribose as the most likely precursor.
00
equatorial H-7/H-9 protons (Figure 3f). In addition, a strong NOE As a subsequent step, isomerization of uridine monophosphate
effect from the uracil proton dH-6 7.60p.p.m. to H-7 and a weak NOE (UMP) (I) to pseudouridine (c-MP) (II) occurs that is catalyzed
to H-8 and H-9 only occur in the case of the postulated arrangement by a c synthase-type enzyme.14 Such a biosynthetic step has been
(Figure 3e). Based on these results, we can deduce the relative described for one of the first reported C-nucleosides, 5-b-D-
configuration for the two hydroxyl groups at C-7 and C-9 and the ribofuranosyluracil known as pseudouridine (c). Interestingly,
2-aminoimidazol-5-one group at C-8. Hence, both hydroxyl groups at C-nucleosides are known to inhibit important enzyme processes,
C-7 and C-9 are syn to each other and anti to the 2-aminoimidazol- as is shown by the recent discovery of the first C-nucleoside hepatitits
5-one substituent at C-8 (Figure 4). For the interpretation of the
stereochemistry see the discussion part below.
C
virus polymerase inhibitor GS-6620.15 Further important
C-nucleosides are ezomycin B2 (antifungal),16 showdomycin
(antibiotic, cytotoxic),17 formycin (antitumor antibiotic)18 and
pyrazofurin (antiviral).19 In this context, it is worth mentioning
that (1) shows antibiotic activity exclusively against A. chroococcum.
In subsequent steps, a nucleotidase could catalyze the hydrolysis of
c-MP (II) to c (III), which finds its analogy in the biosynthesis
of caprazamycin and tunicamycin:12 The proceeding oxidation of
the terminal 50OH-group to the 50aldehyde is followed by a
transamination to c-50-amino (IV). Subsequently, ring opening at
C-2 by formal addition of H2O occurs while the stereocenter at C-10
is maintained.20 The attachment of the unusual 2-aminoimidazol-5-
one building block in (VI) could be derived from condensation
of glycine (Gly) and carbamoyl phosphate to the 20-position of the
20,50-diamino aldopentose in (V). Therefore, we hypothesize an
enzyme with a transferase-like action to form the 20-amino group
in the aldopentose of (IV) to the intermediate c-20,50-diamino (V),
in analogy to the described 30-amino formation in the proposed
biosynthetic pathway of puromycin.21 Interestingly, the 2-amino
pentose formation in Streptomyces alboniger by enzymatic amination
is known and described to occur in cis-configuration to the
30-hydroxyl group that may also define its stereochemistry.22 Also,
a similar derivative of 2-aminoimidazol-5-one, the 4-formyl-4-
imidazolin-2-one, is found in the nikkomycin biosynthesis, derived
Biological activity
Bioactivity testing of pure strepturidin (1) showed the expected
activity against A. chroococcum but no activity against the
Gram-negative Escherichia coli and the Gram-positive Staphylococcus
aureus or Streptomyces glaucescens.6 In an agar diffusion assay on
A. chroococcum plates, 40mg/paper disc produced a 13mm inhibition
zone. Cytotoxicity against the mouse fibroblast cell line L929 was
440mg mlꢀ1, indicating very low toxicity.
DISCUSSION
With regard to its chemical structure, strepturidin (1) belongs to the
group of nucleoside antibiotics. The latter commonly target bacterial
peptidoglycan cell wall biosynthesis and fungal chitin wall biosynth-
esis.12 The group of nucleoside natural products contain a vast
number of representatives including well-known compounds, such as
tunicamycin, mureidomycin A, liposidomycin B, pacidamycin and
nikkomycin Z.12 The common core unit of such nucleoside
antibiotics is a uridine nucleotide consisting of a 10-N-glycosylation
of uridine with a (30-deoxy)-ribose unit.12 Unlike the above-
mentioned nucleoside antibiotics, strepturidin (1) is a C-nucleoside
with the C-glycosylation at the 50 position of uracil linked to a linear
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from histidine.12 Finally, the amino acid Gln, the precursor of the size 27.2ꢁ 5 cm, flow 9–10mlminꢀ1, detection at UV detection wavelength
l ¼ 254nm). The two batches (8g per batch) were dissolved in 250 ml water
Gln-Na-hydroxamate, which forms a peptide bond between its
carboxylic group and the primary 50amine group of the aldopentose
and applied to the column followed by two to three bed volumes of water with
a flow of 9–10 mlminꢀ1. The pure compound (1) eluted with 50 mM NH3
solution as a sharp peak in the chromatogram. The combined fractions were
building block in (VI). This is assumed to be performed by a non-
ribosomal peptide synthetase (NRPS) to form (VII) before or after
lyophilized to give 1.76 g of pure (1) as white powder (overall yield 27%).
constituting the Na-hydroxamate through hydroxylation with an
Analytical HPLC of (1) was performed on a Nucleosil RP-18 (7mm,
putative amino-monoxoygenase to strepturidin (1). Interestingly,
250 ꢁ 4 mm, Macherey-Nagel) column with the mobile phase acetonitrile/
Na-hydroxamate is rare in nature, whereas most hydroxamate
group modifications were found in the side chain of amino
acids.23,24 Although hydroxamates in general display siderophore-
67 mM phosphate buffer pH 5 with 10mM sodium heptanesulfonate with a
flow of 1.4 mlminꢀ1. Detection was carried out at the UV wavelength l ¼ 262
nm and the retention time of (1) at 11.7min.
like functions,25 common also among other natural products like
catecholate and carboxylate siderophores, the role of this moiety in
strepturidin is still elusive.
In summary, the structure elucidation of strepturidin (1) renders
an unprecedented structure of a novel nucleoside-type antibiotic
with an exclusive antibacterial activity against A. chroococcum.
Although the biosynthesis of strepturidin is still unknown, we
propose similarities to biosynthesis pathways of other (C)-nucleoside
antibiotics as mentioned above.
MS
HPLC-HR-ESI-( þ )-Orbitrap-MS mass spectra were recorded using
a
LTQ-Orbitrap XL (Thermo Scientific, Bremen, Germany) coupled to an
Agilent 1260 HPLC system (Agilent Technologies, Waldbronn, Germany).
The HPLC system was equipped with a Hypersil-Gold column (5mm,
50 ꢁ 2.1 mm, Thermo Scientific, Bremen, Germany). A H2O/MeOH (solvent
A/solvent B)þ 0.1% HCOOH gradient was used starting with 5% B and
increasing to 100% B in 6 min, holding 100% B for 4 min, flushing to starting
conditions 5% B for 3 min and a flow rate of 0.25mlminꢀ1
.
a QQQ-MS-6460
HPLC-LR-ESI-(þ )-MS2 spectra were measured on
mass spectrometer (Agilent Technologies) coupled to an Agilent 1290
UHPLC-system (Agilent Technologies). The HPLC system was equipped with
an Eclipse Plus C18 RRHD column (1.8mm, 2.1 ꢁ 50 mm, Agilent Technol-
ogies). A H2O/ACN (solvent A/solvent B) þ 0.1% HCOOH linear gradient was
used starting with 5% B and increasing to 100% B in 6 min, holding 100% B
for 4 min, flushing to starting conditions 5% B for 3 min and a constant flow
METHODS/EXPERIMENTAL SECTION
Producing strain
Large-scale cultivation of strain S. albus (DSM 40763) was performed by
Steinhaus7 in collaboration with the Bio-Plant of the GBF (Gesellschaft fur
¨
Biotechnologisches Forschung) in 1993. A preculture was prepared in a 15l
Giovanola bioreactor (Giovanola Freres, Monthey, Switzerland) charged with a
complex medium consisting of mannitol 10g lꢀ1, biopepton 5 g lꢀ1, yeast
extract 5 g lꢀ1, meat extract 5 g lꢀ1, calcium chloride 0.7g lꢀ1 (pH 7.2) and
tegosipon 5 ml, and inoculated with 200 ml of a seed culture. After 14 h, only
5 l of this preculture was left because of extensive foaming that, however, was
sufficient for subsequent inoculation. For the production of strepturidin (1),
80l of a complex medium consisting of mannitol 10 g lꢀ1, soy meal (full-fat)
10g lꢀ1, meat extract 5 g lꢀ1, sodium chloride 1 g lꢀ1, calcium carbonate
1 g lꢀ1 and tegosipon 10 ml in a 100-l bioreactor (MBR, Zurich, Switzerland)
was inoculated with the remaining 5 l of the preculture. The cultivation was
carried out at 371C at 300 r.p.m. and an aeration of 0.5v.v.m. (volume per
volume per min). After 26h, 130 g lꢀ1 wet cell mass and an antibiotic titer of
92mg lꢀ1 were determined. The culture broth was harvested by centrifugation,
180 g of sodium heptanesulfonate was added to the 72 l of supernatant, and the
solution passed over a column (10 cm diameter) with 4 l of Amberlite XAD-16
at a constant flow of 8 l hꢀ1. Strepturidin (1) was bound as a heptanesulfonate
ion-pair, and, after rinsing the column with water (4l), (1) eluted with MeOH/
H2O, 1:1 (v/v). The eluate was concentrated in vacuo, and the remaining
aqueous phase was lyophilized to give 106.4 g of crude extract containing 5.3g
of (1) (yield: 80% by HPLC). To reduce the sodium heptanesulfonate load,
ion-pair extraction in two batches was performed. Therefore, each batch was
dissolved in 200ml H2O with methyltrioctylammonium chloride (76.6 g) and
each batch was extracted twice with 200 ml CH2Cl2. The aqueous phase was
lyophilized to give 28.44g crude extract with 2.36 g of (1) (yield 95%).
Preparative HPLC of this material was performed on a Eurosil C-18 column
(10 mm, 250 ꢁ 50mm, Knauer GmbH, Berlin, Germany) with a MeOH/67mM
phosphate buffer (pH 5.0) (solvent A) and 10mM sodium heptanesulfonate
rate of 0.25mlminꢀ1
.
NMR spectroscopy
The 1D- and 2D-NMR spectra were recorded on a Bruker Avance III 500
spectrometer (Bruker, Karlsruhe, Germany) equipped with a broadband
inverse detection probe with Z-Gradient at 500 and 125 MHz for 1H and
13C, respectively. Sample was dissolved in 600ml d6-DMSO for NMR
experiments. Chemical shifts are given relative to TMS. For the calibration,
the residual solvent peaks of d6-DMSO (2.50 p.p.m. (1H) and 39.5p.p.m.
(13C)) were used. All experiments were performed at T¼ 298 K.
Synthesis of Gln-strepturidin (2)
To strepturidin (1) (5mg, 0.011 mmol) dissolved in 2 M HCl (5ml), Zn dust
(0.1mg) was added in small portions. The extent of reduction of the
Na-hydroxamate group of (1) was followed by HPLC-MS. After 24h, the
sample was centrifuged. The supernatant was purified by C18 solid-phase
extraction), washing with H2O and eluting with 100% MeOH. The compound
was concentrated in vacuo and then freeze-dried to obtain a yellow oily
material. Compound (2) was used for total hydrolysis, derivatization and
subsequent chiral GC/EI-MS analytics described in the section below.
Synthesis and purification of monoacetal-strepturidin (3)
The procedure was mainly adopted from the work published by Jundt et al.10
Strepturidin (1) (36 mg, 0.07 mmol), 1,1-dimethoxyethylbenzene (1.15 ml,
(solvent B) gradient starting with 4:96 (solvent A/B), and ending after 5 min 0.7mmol) and camphorsulfonic acid (1.2mg, 0.005mmol) were dissolved
with 9:91 (solvent A/B) at a flow of 100 mlminꢀ1. The detection was carried in 1 ml of d6-DMSO. After stirring overnight at 501C, saturated Na2CO3
out at the UV detection wavelength l ¼ 260 nm. For each preparative solution was added to quench the reaction. The mixture was freeze-dried
separation, 3.0–3.5 g of the crude extract was dissolved in 20ml of H2O to and the residue was dissolved in MeOH and separated by C18-HPLC
obtain in total 110g of salts, which after lyophilization of the combined peak to yield monoacetal-strepturidin (3) (5mg, 0.008mmol, 11% overall yield).
fractions contained 2.13g of (1) (yield 90%). Desalting was performed with
15–20g portions dissolved in 350 ml H2O and loaded on an Amberlite XAD- equipped with
16 column (bed volume of 370 ml). The column was rinsed with two to three A H2O/MeOH (solvent A/solvent B) gradient with a constant flow rate of
Preparative HPLC was performed on an Agilent HPLC 1100 system (Agilent)
a
Grom-Sil-120-ODS column (5mm, 250 mmꢁ 20mm).
bed volumes of H2O, and (1) eluted with MeOH/H2O, 1:1 (v:v). From the
combined fractions, MeOH was evaporated in vacuo to obtain 16.5g salts
15 mlminꢀ1 was used starting with 20% B and increasing to 80% B in 40min,
holding 100% B for 5 min, flushing to starting conditions 20% B for 3 min.
containing 1.96g (1þ) (yield 92%). Final desalting was achieved by CM- Peak collection was carried out at the UV detection wavelength l ¼ 254 nm.
Sephadex A-25 NH4 column chromatography (amount of gel 72g, column The chromatogram is shown in Supplementary Figure S1.
The Journal of Antibiotics

Isolation and structure elucidation of strepturidin
A Pesic et al
477
¨
Total hydrolysis of Gln-strepturidin (2) for chiral GC/EI-MS
analysis
3
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&
Total hydrolysis of Gln-strepturidin (2) (0.1mg) was performed at 110 1C in
aqueous 6 M hydrochloric acid solution (200ml, grade for amino acid analysis;
Sigma-Aldrich) under vacuum for 24h in glass ampoules. After 24h, the
hydrochloric acid was removed in a gentle stream of nitrogen. Compound (2)
was semi-purified by C18 solid-phase extraction before chiral GC/EI-MS
analysis.
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Chiral GC/EI-MS analysis
Chiral GC/EI-MS analysis from the total hydrolysate of Gln-strepturidin
(2) was performed on a GC800 Top Voyager (Thermo Finnigan, Waltham,
MA, USA) GC-mass spectrometer equipped with a chiral LIPODEX E column
(25 m, I.D. 0.25 mm). The scan range was 45–465amu, detection temperature
210 1C, source temperature 220 1C, ionization energy 70eV and injection
temperature 250 1C. Helium was used as a carrier gas with constant pressure at
60 kPa. Temperature program: 701C (2min isotherm), 901C, 3 1C per min
(15 min isotherm), 140 1C, 3 1C per min (10 min isotherm), 200 1C, 3 1C per
min (1min isotherm). The injection volume was 1 ml.
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Antimicrobial assay
Agar test plates inoculated with A. chroococcum were prepared according to
Reis.26 Samples to be tested were applied as 20ml solutions on paper discs of
6 mm diameter. After incubation for 20h at 30 1C, the diameter of the
inhibition zones was measured. The minimum inhibitory amount of pure
strepturidin was 1.7mg, giving an 8 mm inhibition zone.
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Cytotoxicity assay
The inhibitory activity of strepturidin (1) was tested with mouse fibroblasts
L-929 (DSMZ ACC 2). Next, 60ml of a serial dilution of the compound
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6 days. After that time, the MIC was estimated visually using an inverted
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Supporting information
MS data and 2D NMR data are free of charge available via Internet at http://
pubs.acs.org.
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ACKNOWLEDGEMENTS
We thank the members of the GBF BioPlant for the cultivation of Streptomyces
albus, H Steinmetz for his support with large-scale chromatography, B. Elxnat
and Ch Heuer for preparing test plates and Dr F Sasse for cytotoxicity assays.
We further thank Dr D Herrmann and T Reinhardt and H-H Reis (TU
Darmstadt) for valuable information. Financial support from the Cluster of
Excellence (‘Unifying concepts in catalysis’ UniCat) granted by the German
Research Council (DFG) is gratefully acknowledged.
´
23 Barona-Gomez, F., Wong, U., Giannakopulos, A. E., Derrick, P. J. & Challis, G. L.
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics