Roimatacene: An antibiotic against gram-negative bacteria isolated from cystobacter ferrugineus Cb G35 (Myxobacteria)

Article abstract of DOI:10.1002/chem.201003677

Roimatacene (1) was isolated from the myxobacterium Cystobacter ferrugineus strain Cb G35 in a bioactivity-guided process, by following the antimicrobial activity against Escherichia coli. Since 1 was extremely sensitive to oxygen, a protective isolation and handling protocol was developed, by utilizing the free radical scavenger 4-ethoxyphenol. The structure of 1 was determined by HRMS, 1D and 2D NMR spectroscopy and chemical derivatization to acetonides and Mosher esters to finally establish the absolute configuration. Methionine and acetate were identified as building blocks in the biosynthesis of 1 by feeding experiments with differently ¹³C-labeled precursors. The antimicrobial activity of 1 was determined in a broad screening revealing 1 to inhibit several Gram-negative bacteria. Overcoming instability: A highly protective protocol was developed for the isolation of the oxygen-sensitive polyene roimatacene (1) from Cystobacter ferrugineus Cb G35 (Myxobacteria). The relative stereochemistry of 1 was determined by using Rychnovsky′s method and molecular modeling. In a broad screening, roimatacene (1) showed antimicrobial activity against several Gram-negative bacteria. Copyright

Full text of DOI:10.1002/chem.201003677

FULL PAPER
DOI: 10.1002/chem.201003677
Roimatacene: An Antibiotic against Gram-Negative Bacteria Isolated from
Cystobacter ferrugineus Cb G35 (Myxobacteria)
Wiebke Zander, Klaus Gerth, Kathrin I. Mohr, Wolfgang Kessler, Rolf Jansen, and
Rolf Mꢀller*[a]
Abstract: Roimatacene (1) was isolated
from the myxobacterium Cystobacter
ferrugineus strain Cb G35 in a bioactiv-
ity-guided process, by following the
antiACHTUNGTRENNUNGmicrobial activity against Escheri-
chia coli. Since 1 was extremely sensi-
tive to oxygen, a protective isolation
and handACHTUNGTRENNUNGling protocol was developed,
by utilizing the free radical scavenger
4-ethoxyphenol. The structure of 1 was
determined by HRMS, 1D and 2D
NMR spectroscopy and chemical de-
esters to finally establish the absolute
configuration. Methionine and acetate
were identified as building blocks in
the biosynthesis of 1 by feeding experi-
ments with differently 13C-labeled pre-
cursors. The antimicrobial activity of 1
was determined in a broad screening
revealing 1 to inhibit several Gram-
negative bacteria.
AHCTUNGTRENNrGUN ivatization to acetonides and Mosher
Keywords: antibiotics · Cystobacter
ferrugineus · myxobacteria · radical
scavengers · structure elucidation
Introduction
diversity of myxobacterial secondary metabolites as a rich
source for novel antibiotics.
The recent identification of multidrug-resistant “superbugs”
once again highlighted the urgency of enhanced efforts to
identify novel antibiotics.[1,3] Especially alarming is the fact
that these new superbugs did not belong to the frequently
observed multiresistant Staphylococcus aureus (MRSA)
group but to Gram-negative Enterobacteriaceae isolates re-
sistant to nearly all antibiotics.[2] During the past three de-
cades myxobacteria have been revealed as versatile produc-
ers of biologically active secondary metabolites with unique
structures and numerous different modes-of-action.[4,5] Im-
portantly, the majority of metabolites from myxobacteria
show antimicrobial activities. One example is the family of
soraphens, which selectively inhibits the fungal acetyl-CoA
carboxylase.[6–8] Other myxobacterial antibiotics are the
highly effective sorangicins (minimum inhibitory concentra-
tion (MIC) 0.004–0.1 mgmLÀ1 against various Gram-positive
bacteria)[9,10] and the a-pyranone myxopyronin,[11,12] both
targeting bacterial RNA synthesis. Whereas sorangicin binds
closely to the RNA polymerase (RNAP) active centre exact-
ly like rifampicin,[13] myxopyronin was shown to inhibit the
opening and closing of the RNAP active centre cleft, thus
establishing a novel mode of action.[14,15] Further, two myxo-
bacterial antibiotics corallopyronin[16,17] and ripostatin[18,19]
use this novel mechanism.[14] These examples highlight the
Results and Discussion
Isolation: The antibiotic activity of the extract of Cystobact-
er ferrugineus strain Cb G35 against Escherichia coli
(E.coli.) was detected in a broad antimicrobial screening.
Accordingly, roimatacene (1) was isolated in a bioactivity-
guided procedure from the Amberlite XAD-16 adsorber
resin, which was present during fermentation. To avoid de-
composition of the oxygen- and light-sensitive polyene 1,
the extracts were kept dissolved in methanol containing 4-
ethoxyphenol as free radical scavenger. Prior to use, all sol-
vents were saturated with nitrogen gas and all operations
were carried out under a nitrogen atmosphere. Ethyl acetate
was filtered over aluminium oxide to remove peroxides, and
extracts and fractions were handled in amber glassware. The
Amberlite XAD-16 resin was harvested from the fermenta-
tion by sieving and was then eluted with methanol. The anti-
biotic 1 was enriched by an acid–base partition and finally
purified by RP-chromatography providing 4-ethoxyphenol
to each HPLC fraction.
Structure elucidation: HRMS (ESI) of the molecular ion
cluster [MÀH]À at m/z: 515.3021 established the molecular
formula C30H44O7 (calcd: 515.3014) for 1, which was sup-
ported by the NMR spectroscopic data (Table 1). Interpreta-
[a] W. Zander, Dr. K. Gerth, Dr. K. I. Mohr, Dipl.-Ing. W. Kessler,
Dr. R. Jansen, Prof. Dr. R. Mꢀller
tion of the H,1H COSY spectrum furnished four structural
1
Mikrobielle Wirkstoffe, Helmholtz Zentrum
fꢀr Infektionsforschung, Inhoffenstraße 7
38124 Braunschweig (Germany)
Fax : (+49)531-61819499
E-mail: rom@helmholtz-hzi.de
elements A to D (Scheme 1). After assignment of the direct-
ly carbon-bound protons from a 1H,13C HMQC spectrum,
the carboxylic acid carbon C1 was allocated from its HMBC
correlations with 2H and 3H of subunit A. After verifica-
tion by 13C ATP NMR spectroscopy the quaternary carbon
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003677.
Chem. Eur. J. 2011, 17, 7875 – 7881
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7875

Table 1. NMR spectroscopic data of roimatacene (1) (1H: 600 MHz, 13C: 150 MHz, [D6]DMSO).[a]
C8 (dC =134.4 ppm), overlap-
ping with C13, was identified as
a connection between units A
and B from HMBC correlations
with 7H and 9H, and accessory
correlations of the methyl
group C28 at C8. Similarly, C16
and the methyl group C29 had
HMBC correlations with 15H
of subunit B and with 17H and
18Hb of subunit C. The final
connection of structural unit D
was indicated by HMBC corre-
lations of C20 with methyl
group C30 and 18H and 22H
and by correlations of the
methyl group C30 with 19H
and 21H. These were supported
by mutual HMBC correlations
of 19H and 21H, to finally
result in the 2D structure of 1
Atom dH
H
m
J [Hz]
COSY
dC
HMBC
ROESY[e]
1
2
3
4
5
–
–
1
1
2
1
–
d
ddd
m[c]
ddd
–
16.0
16.0, 7.0, 7.0
–
11.0, 6.7, 5.5
–
–
3, 4
167.1
123.6
145.6
40.0
69.9
–
3, 2
4>3
4
3, 2, 6, 5OH
7, 6, 4>3
–
–
(3)
5.79
6.80
2.35
4.18
2, 4
(2)>(4), 5
>(5), 2, (3)
7, (4,6), 3
–
3, 5[e]
4>6, 5OH
5
5OH 5.01 (1) s (br)
6
7
8
28
9
10
11
12
13
14a
14b
15
5.67
6.25
–
1
1
–
3
1
1
1
1
1
1
1
1
dd (br) 15.8, 5.9
7, 5
6
–
9
10, 12
7, 9
10, 12
11, 13
12, 14b
13, 15, 14b
132.0
133.6
7, 4
28, (7, 5), 4
9, 5, (6)
–
6, 10
7, 11
28, 12
13, 9
d
15.8
28, 6, 9, 10
–
–
134.4[d] 28, 10, 7, 9[e]
1.80
6.10
6.47
6.27
6.16
5.82
2.35
2.02
3.51
s
–
–
12.6
131.0
126.9
133.9
131.7
134.4
34.6
–
7, 9
7, 11, 28
12
m[b]
dd
14.6, 11.6
dd
14.6, 10.6
13, 9
m[b]
ddd
m[c]
dddd
dd[e]
–
10, 11>14a/b
14a/b, 11
12, 13>15OH
–
10
15.1, 7.3, 7.3
–
11, 15, (14a/b)
(14b, 15, 13), 12, 17
ACHTUNGTRENN(UNG 14a, 13)
15.4,7.7,7.7, 1.5 14 a, 15, 13
9.7, 1.7[e]
15OH, 14b
73.4
29, 18b
13, 17, (14a), 29,
18a/b
15OH 4.52 (1) s (br)
16
16OH 4.43 (1) s (br)
–
–
–
15
–
–
74.3
–
–
–
–
–
–
–
–
29, 18b
–
17>15OH
29
17
0.93
3.29
3
1
s
–
–
18.2
73.5
17OH
17, 15>12, 18a
dd[e]
10.5, 1.5[e]
18b[e]
29,[e] 18b
19, 15, (18a), 29, 30, shown in Scheme 1. The struc-
21
ture revealed an unusual accu-
mulation of reactive functional
17OH 4.08 (1) s (br)
–
17
–
–
–
18a
18b
19
1.70
1.49
4.14
1
1
1
ddd
ddd
dd
13.8, 10.2, 1.3
13.8, 10.2, 6.4
7.0, 6.4
19, 18b
19, 18a>17
18a/b>19OH 74.9
36.1
–
>18b
–
ACHTUNGTRENNUNG
groups, like a tertiary alcohol
and two a-polyunsaturated al-
cohol groups, an acrylic acid
residue and polyenes, altogeth-
er providing a well-founded jus-
tification for the observed sen-
sitivity of 1.
29, 30, 21, 18a/b 21, 17, (18a/b)
19OH 4.85 (1) s (br)
–
19
–
–
–
20
30
21
22
23
24
25
26
27
–
–
3
1
1
1
1
1
2
3
–
s
–
–
–
21
140.2
11.6
30, 18a/b>22
21, 19
30, 23, 19
21, 23
21, 25
22, 26
27, 26, 23
27, 25, 24
26, 25
–
1.67
6.00
6.37
6.14
6.14
5.74
2.08
0.97
19, 22
d (br)
dd
m[b]
m[b]
ddd
dt
11.2
14.5, 11.2
–
–
14.3,7.1, 7.1
7.1, 7.5
7.5
22, 30
23, 21
22, 24
25, 23
24, 26
27, 25
26
125.2
126.9
132.2
129.9
135.6
25.2
23, 19>(22), 17
30, 24, (23, 21)
21, (22)>25, 30
26, (23, 25)
ACHTUNGTRENNUNG
Relative configuration: The
trans configurations of the
double bonds of 1 were indicat-
ed by vicinal coupling constants
of 15 Hz and by corresponding
AHCTUNGTRENNUNG
t
13.5
[a] The compound was stabilized with 4-ethoxyphenol (1H NMR (600 MHz, [D6]DMSO): d=1.26 (t, J=
6.97 Hz, 3H) 3.89 (q, J=6.97 Hz, 2H) 6.65 (d, J=8.80 Hz, 2H) 6.72 ppm (d, J=8.80 Hz, 2H); 13C NMR
(150 MHz, [D6]DMSO): d=14.8, 63.3, 115.3, 115.7, 151.0, 151.3 ppm. [b] Multiplet of 4 protons at d=
6.15 ppm, one AA’-system consisting of 23/24 and an overlap with 9H and 12H. All proton shifts are taken
from the HMQC spectrum. [c] Overlap of protons 4H and 14Ha. Proton shift from the HMQC spectrum.
[d] 13C shift from the APT spectra after H/D exchange. [e] Data after H/D exchange.
2D
ROESY
correlations
(Table 1). To establish the rela-
tive configuration of the polyol
fragment C15 to C20, three ace-
tonides (3–5) were prepared by
reaction with 2,2-dimethoxypro-
pane (Scheme 2). 17,19-Acetonide 3 showed the typical shift
of dC =98.9 ppm for the quaternary carbon atom CMe2 and
dC =20.0 and 30.1 ppm (see Table S2 in the Supporting In-
formation) for the two methyl groups characteristic of a
chair conformation of acetonides as analyzed by Rychnov-
sky’s method.[20] Since a chair conformation is only possible
for syn-1,3-diols, 17OH and 19OH have a syn configuration.
In contrast, the 15,17-acetonide 4 showed chemical shifts of
dC =100.2 ppm for the quaternary carbon atom and similar
shifts of dC =25.2 and 23.7 ppm for the methyl groups, un-
ambiguously indicating a twisted-boat conformation and
thus the anti configuration of 15OH and 17OH.[20]
The configuration of C16 was established by taking the
relative configurations of C15, C17, and C19 into account
Scheme 1. Roimatacene (1) and selected correlations from the 2D NMR
spectra.
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Chem. Eur. J. 2011, 17, 7875 – 7881

Roimatacene
FULL PAPER
fully compatible with the distance of 2.5 ꢂ between 17H
and 14Hb in the S*,S*,S*,R* configuration (Figure 1b). Al-
though the expected NOE between methyl group 30H (dH =
1.79 ppm) and 18Ha (dH =1.71 ppm) could not be observed,
because their 1H NMR spectroscopic signals overlap. The
strong NOE between 19H and 21H requires the orientation
of the triene side chain as shown in the S*,S*,S*,R* isomer.
Thus, the NOE data are only compatible with the relative
S*,S*,S*,R* configuration of the polyol fragment shown in
the bis(acetonide) 5 in Figure 1b and the resulting relative
configuration of 1 is presented in Scheme 3.
Scheme 2. Preparation of acetonides 3 to 5 from roimatacene methyl
ester (2): a) 2,2-dimethoxypropane, pyridium-p-tolouenesulfonic acid
(PPTS), 48C, 18 h.
and the NOEs of the bis(acetonide) 5 (Figure 1b). Both pos-
sible diastereomers at C16 were modeled by applying the
“Conformational Search” module of HyperChem (Ver-
sion 8.5) by using MM+ calculations. The optimized alter-
native conformations were compared with the NMR spec-
troscopic data of 5 (see Table S4 in the Supporting Informa-
tion).
Scheme 3. The relative 15S*,16S*,17S*,19R* configuration of roimata-
cene (1).
Absolute stereochemistry: The absolute stereochemistry of
roimatacene (1) was established by applying Mosherꢃs
method.[21] Exasperatingly, the methoxy(trifluoro-methyl)-
phenylacetyl (MTPA) esters
prepared directly from
1 or
from the acetonides (3, 4) were
not stable. As an alternative ap-
proach roimatacene (1) was
completely hydrogenated under
mild conditions and then esteri-
fied with p-bromophenacyl bro-
mide. Finally, the tris-(R)- and
the tris-(S)-MTPA esters 8a/b
were derived by Yamaguchi
esterification[22] of p-bromoace-
tophenone-octahydroroimata-
cene ester 7 (Scheme 4).
Figure 1. Partial view of 5 resulting from modeling studies with HyperChem and selected NOEs of 5. a) The
relative 15S*,16R*,17S*,19R* isomer and b) the relative 15S*,16S*,17S*,19R* isomer of roimatacene (1).
The NOEs of the chair conformation of the 17,19-syn-ace-
tonide unit in 5 allowed differentiation of the equatorial
18Hb (dH =1.41 ppm) and axial 18Ha (dH =1.77 ppm) pro-
tons. The methyl group 29H at the 15,16-acetonide showed
ROESY correlations with 18Hb and 17H of the six-mem-
bered acetonide, which are feasible for both isomers
(Figure 1). However, the strongest NOE of methyl group
29H was indicated with 15H. This is only compatible with
their distance of 2.5 ꢂ in the relative 15S*,16S*,17S*,19R*
configuration in Figure 1b, but not with the distance of
3.8 ꢂ in the 15S*,16R*,17S*,19R* configuration in Figure 1a.
Besides their distance, the trans orientation of methyl group
29H and 15H at the acetonide in the S*,R*,S*,R* isomer
would not allow a strong NOE (Figure 1a). Instead, a strong
NOE between 15H and 18Ha would be expected from a
calculated distance of 2.6 ꢂ. However, this NOE is missing
1
in the H,1H ROESY spectrum. Further, an NOE between
17H and 14Hb in the side chain was observed, which is also
not possible in the S*,R*,S*,R* isomer (Figure 1a), even
after rotation of the side chain. However, the observation is
Scheme 4. Preparation of the tris-(R)- and the tris-(S)-MTPA esters of p-
bromoacetophenone-octahydroroimatacene ester 8a/b: a) Pd/C (10%),
H2, RT, 4.5 h; b) p-bromophenacyl bromide, 3 ꢂ MS, Et3N, acetone, 19 h;
c) MTPA, 2,4,6-trichlorobenzoyl chloride, 4-dimethylaminopyridine
(DMAP), Et3N, 0 8C to RT, 4 h.
Chem. Eur. J. 2011, 17, 7875 – 7881
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www.chemeurj.org
7877

R. Mꢀller et al.
Detailed 1H NMR spectroscopic signal assignment (see
the Supporting Information) of the diastereomeric MTPA-
esters 8a and 8b started from the downfield-shifted ester
protons 5H, 15H, and 19H, which were identified by their
different neighborhoods from a complete set of 2D NMR
spectra, respectively. Subsequently, comparison of the
Mosher esters by analysis of the DdSR values (Table 2) adja-
Biosynthetic precursors: To determine the biosynthetic pre-
cursors roimatacene (1) was isolated after feeding experi-
ments with [1-13C]- and [2-13C]-labeled acetate, [13CH3]-me-
thionine and [1-13C]-propionate. According to 13C NMR
spectroscopic analysis, the C3 starter unit derives from (S)-
adenosyl-l-methionine (SAM) and acetate, whereas the re-
maining carbon chain of 1 was assembled from acetate and
all methyl groups originate
from methionine (Scheme 7).
Table 2. DdSR values (1H NMR) of the tris-(S)- and tris-(R)-MTPA esters of p-bromoacetophenone-octahy-
droroimatacene ester (8a/b) in CDCl3.
Biological activity: Roimata-
cene (1) was detected in a bio-
logical screening due to its anti-
microbial activity against E.
coli. In a screening against vari-
ous Gram-positive bacteria,
Gram-negative bacteria, yeasts
and the fungus Mucor hiemalis
(see the Supporting Informa-
tion), roimtacene (1) showed
H
d(S)-MTPA
d(R)-MTPA
DdSR
H
d(S)-MTPA
d(R)-MTPA
DdSR
31
2
3a
3b
4
5.27
2.44
1.67
1.59
1.71
5.12
1.67
1.60
1.35
0.84
1.24
1.07
5.29
2.51
1.79
1,74
1.76
5.11
1.60
1.56
1.26
0.79
1.20
1.02
À0.02
À0.07
À0.12
À0.15
À0.05
0.01
0.07
0.04
0.09
0.05
14a
14b
15
29
17
18a
18b
18ꢄ
19
20
30
1.82
1.57
5.10
1.04
3.39
1.91
1.75
1.80
5.19
1.69
0.79
1.85
1.58
5.10
0.97
3.30
1.83
1.66
1.68
5.14
1.72
0.87
À0.03
À0.01
0.00
0.07
0.07
0.08
0.09
0.12
0.05
5
6a
6b
8
28
9a
9b
À0.03
0.04
0.05
À0.08
antimicrobial
activity
only
against Gram-negative bacteria
cent to C5 indicated the S configuration by negative values
for 31H to 4H and positive values for 6H, 8H, 9H, and
methyl group 28H (Scheme 5). Similarly, the 15S and 19R
configurations were identified from completely positive
DdSR values between both stereocenters C15 and C19 and
Scheme 7. Incorporation of 13C-labeled precursors in roimatacene (1).
13
13
13
&
^
*
: [1- C]-acetate; : [2- C]-acetate; : [ CH3]-methionine.
(Table 3). The MICs in the
mgmLÀ1 range are rather mod-
erate.
The
upmost
MIC
(0.1 mgmLÀ1) was achieved
against E.coli tolC, which can
be expected due to the inactiva-
tion of the major RND (resist-
ance-nodulation-cell division)
efflux pumps in this strain. One
of the mechanisms known for
multidrug resistance of Gram-
negative bacteria is the ability
to drain off drugs through the
Scheme 5. DdSR values after comparison of the 1H NMR spectroscopic data of the tris-MTPA esters 8a/b and
the evaluation of the stereocenters C5, C15, and C19; %R = side chain of the tris-MTPA esters of p-bromo-
ACHTUNGTRENNUNGacetophenone-octahydroroimatacene as numbered
efflux pump systems of the
from negative DdSR values on both sides (14H, 20H, and
30H; Table 2, Scheme 5). The Mosher-derived 15S,19R con-
figuration agrees with the relative configuration of 1 as-
signed above. Thus, the absolute configuration of roimata-
cene (1) was unambiguously derived as the all-trans-
5S,15S,16S,17S,19R configuration (Scheme 6).
outer membrane.[23] Since 1 was also active against the E.
coli 2 DC14PS mutant (MIC: 2.2 mgmLÀ1), which shows an
Table 3. MIC of roimatacene (1) (1.3 mgmLÀ1 roimatacene (1) and
0.5 mgmLÀ1 4-ethoxyphenol in DMSO; negative control of 0.5 mgmLÀ1
4-ethoxyphenol in DMSO) against various Gram-negative bacteria.
Test organism
MIC [mgmLÀ1
]
E. coli tolC
0.1
E. coli
8.6
E. coli 2 DC 14 PS
Chromobacterium violaceum
Pseudomonas stutzeri
Klebsiella pneumoniae
2.2
0.3
4.2
>9.1
Scheme 6. The absolute configuration of all-trans-5S,15S,16S,17S,19R-roi-
matacene (1).
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Chem. Eur. J. 2011, 17, 7875 – 7881

Roimatacene
FULL PAPER
overexpression of one RND efflux pump,[24] the target of
roimatacene most likely is not related to the resistance
against the tripartite efflux pump systems of the RND
family. Chromobacterium violaceum is prevalently used as a
model strain for quorum sensing. The biosynthesis of the
quorum sensing indicator violacein was inhibited by 1 with a
MIC of 0.3 mgmLÀ1. The mode-of-action against pseudomo-
nads (Pseudomonas stutzeri MIC of 4.2 mgmLÀ1) has to be
investigated further, due to the rapidly increasing clinical
multidrug resistance of Pseudomonas aeruginosa.[1,25,26] The
radical scavenger 4-ethoxyphenol showed no growth inhibi-
tion up to 3.4 mgmLÀ1. Thus, the maximum concentration of
4-ethoxyphenol used in the roimatacene-test solution was
limited to 3.0 mgmLÀ1. In the proliferation assay of the
mouse fibroblast cell line l-929, the IC50 of roimatacene is
ꢀ18 mgmLÀ1. At this dilution the IC50 (8.8 mgmLÀ1) of the
radical scavenger in the roimatacene-test solution limited
the experiment and therefore no explicit results are avail-
able.
24415) was transferred to a liquid medium consisting of soybean flour
(4 gLÀ1), glucose (2 gLÀ1), starch (8 gLÀ1), CaCl2·2H2O (1 gLÀ1),
MgSO4·7H2O (1 gLÀ1), HEPES (11.9 gLÀ1) and Na–Fe–ethylenediamine-
tetraacetic acid (EDTA) (8 mglÀ1) at pH 7.4. A 4 day-old pre-culture
(5 L) was used to inoculate a volume of 70 L of the same medium in a
100 L fermentor (Giovanola Frꢅres SA, Monthey, Switzerland) in the
presence of adsorber resin Amberlite XAD-16 (1.4 kg, Rohm & Haas).
The culture was supplemented with 1.15–1.75 gh1 of NaAc solution
(100 gkgÀ1) during seven days of cultivation (blade impeller 150 rpm, aer-
ation 7 LminÀ1, 308C, pO2 20%, pH regulated between pH 7.15 and 7.25
with 10% KOH and 5% H2SO4). For harvesting, the culture broth was
passed through a process filter collecting the absorber resin. Residual
cells were floated from the recovered XAD-16 resin with water.
Isolation: The XAD absorber resin was eluted with methanol/water
(3:7), methanol, and acetone (4 L each) saturated with N2 gas. After ad-
dition of saturated NaCl solution (250 mL), the water layer (300 mL) re-
maining after evaporation of the methanol was adjusted to pH 4.15 with
formic acid and extracted 5 times with ethyl acetate (100 mL). The ethyl
acetate was supplemented with 0.1m 4-ethoxyphenol (100 mL) and metha-
nol (100 mL). The ethyl acetate layer was evaporated and dissolved into
a methanol layer (140 mL), without drying the samples. The extract was
analyzed by HPLC to contain 504 mg of roimatacene (1) in 13.4 g of
crude extract. The extract was partitioned between methanol and n-hep-
tane to provide 12.9 g of polar raw material, containing 440 mg roimata-
cene (1) according to HPLC analyses (supplemented with 200 mL of 0.1m
4-ethoxyphenol). The methanol extract was stored in methanol (142 mL,
c=3.1 mgmLÀ1) at 48C. After 6 weeks HPLC analysis showed degrada-
tion to 256 mg (58%, c=1.8 mgmLÀ1) of 1 in the crude extract. Half of
the extract was dissolved in 2n ammonia (75 mL) and methanol was
evaporated. The pH of the water layer was adjusted to pH 9.5 with 2n
ammonia and extracted 3 times with ethyl acetate (95 mL). The water
layer was acidified to pH 4.2 with formic acid and then extracted twice
with n-heptane (55 mL) to remove fatty acids from the crude extract.
The acidic water layer was then extracted 4 times with ethyl acetate
(75 mL) supplemented with 4-ethoxyphenol (200 mL, 0.1m), methanol
and toluene before evaporation of the ethyl acetate. The methanol/tolu-
ene solution yielded 99.4 mg enriched roimatacene (1) according to
HPLC analyses. Roimatacene (1) was further purified by preparative
RP-HPLC in 7 batches (column VP250/21 Nucleodur 100–10 C-18 ec
(Macherey–Nagel), solvent methanol/water (6:4) containing 50 mmolLÀ1
of NH4Ac and 400 mLLÀ1 of acetic acid; the solvents were saturated with
N2 gas and 0.1m 4-ethoxyphenol (10 mL) were provided in each test tube
of the fraction collector). Roimatacene (1) eluted at Rt =34 min. The
fraction was evaporated and the water layer was passed through a Strata
X cartridge (Phenomenex) under N2 protection gas, washed with water
and eluted with methanol. 4-Ethoxyphenol (200 mL, 0.1m) was added to
the methanol layer before the concentration was adjusted to 50 mL con-
taining 38.5 mg of roimatacene (1). Pale-yellow solution in methanol;
[a]RDT =+58.4 (c=0.65 in methanol); for NMR spectroscopic data (1H:
600 MHz, 13C: 150 MHz, [D6]DMSO): Table 1; UV (methanol): lmax (e)=
276 (21018), 286 (21492), 308 (23385), 323 nm (20356 molÀ1 dm3 cmÀ1);
HRMS (ESI): m/z: calcd for C30H43O7: 515.3014; found: 515.3021.
Conclusion
Although roimatacene (1) easily decomposed under the
mildest conditions, for example, during cautious evaporation
of solutions, contact with air or exposure to light, a protec-
tive isolation and handling protocol could be developed,
which permitted structure elucidation, determination of the
relative and absolute stereochemistry and biological assays.
With the absolute stereochemistry of 1 in hand, structure–
activity relationship (SAR) studies now are indicated to im-
prove the stability without decreasing the activity. In the
case of other myxobacterial compounds, for example, tubu-
lysins, a high tolerance for simplification of the core struc-
ture without dramatic loss of activity was observed in SAR
studies.[27–29]
Roimatacene (1) is a new antimicrobial secondary metab-
olite from myxobacteria inhibiting selectively the growth of
Gram-negative bacteria. The biological activity against P.
stutzeri is of particular interest, because infections with mul-
tidrug resistant (ꢀthree antibiotics) pseudomonads are pro-
gressively increasing[1] and new lead structures and targets
are desperately needed, especially in the field of Gram-neg-
ative bacteria.[25] Synthesis of simplified and stabilized ana-
logues of roimatacene (1) and studies of the mode of action
will be investigated.
Roimatacene methyl ester (2): Diazald (5.3 g) was dissolved in ether
(20 mL) and ethanol (3 mL) and the mixture was stirred with aqueous
KOH (2.0 g in 3.2 mL H2O). After heating to 358C the ether/diazome-
thane solution was distilled and then added dropwise to a stirred solution
of roimatacene (1) and 4-ethoxyphenol in methanol at room temperature.
After 20 min, ether and diazomethane were evaporated under nitrogen
gas to the crude product in methanol. The product was purified by prepa-
rative RP-HPLC (column VP250/21 Nucleodur 100–10 C-18 ec (Ma-
cherey–Nagel); solvent A: methanol water (6:4) supplemented with
50 mMLÀ1 NH4Ac and 400 mLLÀ1 formic acid, solvent B: methanol same
buffer concentration; gradient: 100% A increasing to 100% B in 50 min,
continued for 15 min, FR=20 mLminÀ1, UV detection: 278 nm, Rt =
60 min). After evaporation of the methanol, the water layer was diluted
and passed through a Strata X cartridge (1.0 g, Phenomenex) under a ni-
trogen atmosphere. The cartridge was rinsed with water and roimatacene
ester (2) was eluted with methanol (supplemented with 40 mL of 0.1m 4-
ethoxyphenol). Pale-yellow solution in methanol; 1H NMR (600 MHz,
Experimental Section
General: see the Supporting Information.
Handling of roimatacene extracts and ethyl acetate: Extracts containing
roimatacene were kept in solution (methanol) supplemented with 4-
ethoxyphenol. As far as possible, N2 gas was used at all times. Ethyl ace-
tate was filtrated over aluminium oxide before use. Amber glassware was
used for handling and for storage at 48C.
Fermentation: Cystobacter ferrugineus strain Cb G35 (deposit at DSMZ-
Deutsche Sammlung fꢀr Mikrooranganismen und Zellkultur; DSM
Chem. Eur. J. 2011, 17, 7875 – 7881
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7879

R. Mꢀller et al.
[D6]DMSO): d=0.92 (s, 4H) 0.93–0.95 (m, 1H) 0.95 (t, J=7.52 Hz, 4H)
1.45–1.52 (m, 2H) 1.66 (s, 4H) 1.67–1.72 (m, 2H) 1.79 (s, 4H) 1.90 (s,
2H) 1.98–2.04 (m, 1H) 2.07 (t, J=7.34 Hz, 3H) 2.32–2.43 (m, 5H) 2.50
(dt, J=3.67, 1.83 Hz, 2H) 3.32 (d, J=10.27 Hz, 2H) 3.48–3.55 (m, 7H)
3.63 (s, 4H) 4.12–4.22 (m, 3H) 5.66 (dd, J=15.59, 6.05 Hz, 1H) 5.70–5.76
(m, 1H) 5.81 (dt, J=14.95, 7.38 Hz, 1H) 5.89 (d, J=15.41 Hz, 1H) 5.97
(d, J=11.00 Hz, 1H) 6.08–6.19 (m, 5H) 6.24 (dd, J=15.22, 8.25 Hz, 2H)
6.36 (dd, J=13.94, 11.37 Hz, 1H) 6.46 (dd, J=14.67, 11.74 Hz, 1H)
6.87 ppm (d, J=15.77 Hz, 1H); 13C NMR (150 MHz, [D6]DMSO): d=
172.4, 166.3, 146.8, 140.2, 135.9, 134.5, 134.1, 133.8, 132.4, 132.4, 132.0,
131.9, 131.2, 130.0, 127.0, 125.5, 122.4, 75.0, 74.5, 73.6, 70.0, 63.5, 51.4,
48.8, 40.0, 36.2, 34.7, 25.4, 21.2, 18.3, 13.6, 12.8, 11.7 ppm; 4-ethoxyphe-
nol: 1H NMR (600 MHz, [D6]DMSO): d=1.25 (t, J=6.97 Hz, 4H) 3.88
(q, J=6.97 Hz, 3H) 6.63–6.66 (m, 2H) 6.69–6.73 ppm (m, 2H); 13C NMR
(150 MHz, [D6]DMSO): d=151.5, 151.1, 115.9, 115.5, 15.0 ppm; HRMS
(ESI): m/z: calcd for C31H46O7Na: 553.3136; found: 553.3128.
Octahydroroimatacene (6): Yellowish oil; 1H NMR (300 MHz, MeOD):
d=0.88–0.93 (m, 10H) 1.06–1.17 (m, 5H) 1.31–1.35 (m, 24H) 1.56–1.84
(m, 10H) 2.44 (m, 4H) 3.35 (brs, 1H) 3.54 (m, 2H) 3.65 ppm (brs, 1H);
13C NMR ( 75 MHz, MeOD): d=177.8, 76.6, 76.6, 76.5, 76.4, 76.2, 76.1,
76.1, 76.0, 72.6, 72.6, 38.4, 38.2, 37.9, 37.8, 36.0, 36.0, 34.4, 34.3, 34.2, 34.1,
33.8, 33.2, 32.2, 31.3, 31.2, 31.0, 30.9, 30.7, 30.6, 28.7, 28.7, 28.3, 28.1, 23.9,
20.4, 20.2, 15.68, 15.5, 14.5 ppm; doubling of signals due to isomers at C8
and C20; 4-ethoxyphenol: 1H NMR (300 MHz, MeOD): d=3.94 (q, J=
6.97 Hz), 6.71 (m, 3H; CH3) 1.31–1.35 ppm (m, 24H); 13C NMR
(
75 MHz, MeOD): d=153.8, 152.4, 116.9, 116.8, 65.3, 14.6 ppm; HRMS
(ESI): m/z: calcd for C30H60O7Na: 555.4231; found: 555.4233.
p-Bromoacetophenone-octahydroroimatacene ester (7): Yellowish oil;
1H NMR (300 MHz, CDCl3): d=0.85–0.96 (m, 11H) 1.05 (s, 3H) 1.08–
1.18 (m, 4H) 1.27–1.33 (m, 17H) 1.39–1.51 (m, 8H) 1.63–1.81 (m, 4H)
2.55 (t, J=7.25 Hz, 2H) 3.45 (m, 1H) 3.62 (m, 1H) 3.83 (m, 1H) 3.98 (m,
1H) 5.31 (s, 2H) 7.65 (d, J=8.7 Hz, 2H) 7.79 ppm (d, J=8.7 Hz, 2H);
13C NMR (75 MHz, CDCl3): d=191.5, 173.2, 133.0, 132.3, 129.3, 129.2,
80.3, 77.3, 74.0, 74.0, 71.9, 71.8, 65.7, 39.8, 39.6, 36.9, 36.8, 36.7, 36.6, 35.1,
34.9, 33.8, 33.4, 32.9, 32.7, 32.1, 32.0, 31.9, 29.9, 29.8, 29.6, 29.3, 27.4, 27.3,
26.9, 22.7, 21.2, 21.0, 19.8, 19.7, 14.9, 14.1 ppm; doubling of signals due to
the isomers at C8 and C20; HRMS (ESI): m/z: calcd for C38H66BrO8:
729.3936; found: 729.3972.
Acetonides of roimatacene methyl esters (3–5): Roimatacene methyl
ester
2 (31.7 mg) was dried and dissolved in 2,2-dimethoxypropane
(4 mL). PPTS (12.5 mg) was added and the reaction was stirred overnight
at 48C. After quenching with 5% NaHCO3 solution the reaction was ex-
tracted 3 times with CH2Cl2 (50 mL). After evaporation of CH2Cl2 the
crude extract was further purified by RP-HPLC (column VP250/21 Nu-
cleodur 100–10 C-18 ec (Macherey–Nagel), solvent A: methanol/water
1:1; solvent B: methanol; gradient: 70% B increasing to 100% B in
50 min and maintained at 100% B for 10 min, FR=20 mLminÀ1, UV de-
tection 210–500 nm) yielding 17,19-acetonide 3 (5.5 mg, Rt =9.1 min),
Tris-(S)-MTPA ester of p-bromoacetophenone-octahydroroimatacene ester
(8a): Oil; NMR spectroscopic data (1H: 600 MHz, 13C: 150 MHz,
CDCl3): see Table S5 in the Supporting Information; HRMS (ESI): m/z:
calcd for C68H90BrF9O14N: 1394.5395; found: 1394.5404.
15,17-acetonide
4 (2.4 mg, Rt =22.9 min) and the bis(acetonide) 5
(4.2 mg, Rt =20.9 min).
Tris-(R)-MTPA ester of p-bromoacetophenone-octahydroroimatacene
ester (8b): Oil; NMR spectroscopic data (1H: 600 MHz, 13C: 150 MHz,
CDCl3): see Table S6 in the Supporting Information; HRMS (ESI): m/z:
calcd for C68H86BrF9O14Na: 1399.4949; found: 1399.4957.
Ester 3: Yellow oil; NMR spectroscopic data (1H: 600 MHz, 13C:
150 MHz, CDCl3): see Table S2 in the Supporting Information; HRMS
(ESI): m/z: calcd for C34H50O7Na: 593.3449; found: 593.3448.
Ester 4: Yellow oil; NMR spectroscopic data (1H: 600 MHz, 13C:
150 MHz, CDCl3): see Table S3 in the Supporting Information; HRMS
(ESI): m/z: calcd for C34H54O7N: 570.3789; found: 570.3801.
Ester 5: Yellow oil; NMR spectroscopic data (1H: 600 MHz, 13C:
150 MHz, CDCl3): see Table S4 in the Supporting Information; HRMS
(ESI): m/z: calcd for C37H54O7Na: 633.3762; found: 633.3768.
Biosynthesis: [13CH3]-sodium methionine (98%, 50 mg), [2-13C]-sodium
propionate (97%, 50 mg) and [1-13C]- and [2-13C]-sodium acetate
(100 mg, 99%, all Cambridge Isotope Laboratories) were fed in two por-
tions after 16 and 40 h of incubation to shaken cultures (100 mL) of
strain Cb G35. The cultures were incubated at 308C for 7 days. After
washing with water each XAD-16 resin was eluted twice with 3 bed vol-
umes of methanol and twice with acetone. The organic layers of each ex-
periment were evaporated to a small volume before adjusting their con-
centration to 100:1 under the addition of 4-ethoxyphenol (50 mL, 0.1m).
All extracts were partitioned between methanol and n-heptane. The
methanol layers were subjected to acid/base partition. They were then
dissolved in ammonia (2n, 10 mL) and the methanol was evaporated.
The aqueous layer was extracted 3 times with ethyl acetate (10 mL). The
water layers were acidified to pH 3.3 with formic acid and extracted with
n-heptane (10 mL) and then 3 times with ethyl acetate (10 mL). 4-
Tris-(S)-MTPA ester of p-bromoacetophenone-octahydroroimatacene
ester (8a): For the (S)-Mosher ester, roimatacene (1) (25 mg) was hydro-
genated in methanol in the presence of Pd/C (10%, 31.4 mg) under a H2
atmosphere at RT for 4.5 h. The reaction mixture was filtered over Celite
and dried in vacuo to yield 35.2 mg extract of octahydroroimatacene (6).
The extract was dissolved in dry acetone (2 mL) and stirred for 19 h at
RT with triethanolamine (TEA, 15 mL) and p-dibromoacetophenone
(24.1 mg) in the presence of 3 ꢂ molecular sieves. The mixture was fil-
tered over Celite and dried to give 66.9 mg. p-Bromoacetophenone-octa-
hydroroimatacene ester (7) was separated by silica gel plate chromatog-
raphy (two PSC-plates 20ꢆ20ꢆ0.1 cm, silica gel 60 F254 with concentra-
tion zone (Merck), solvent ethyl acetate/n-heptane 8:2). The UV active
zone (Rf =0.42) was eluted with ethyl acetate to give 5.3 mg of p-bromo-
EthoxyACTHNURTGNEUpGN henol (20 mL, 0.1m) was added to the ethyl acetate layers of
each experiment. Ethyl acetate was evaporated after addition of toluene
and methanol. The solvents were evaporated after addition of
[D6]DMSO and the 13C NMR spectra of all samples of the feeding ex-
periments were measured.
ACHTUNGTRENNUNGacetophenone-octahydroroimatacene ester (7). 2,4,6-Trichlorobenzoyl
chloride (12.5 mL) and 7 (5.3 mg) were dissolved in dry toluene (0.7 mL)
and added to a solution of (S)-MTPA (16.2 mg), DMAP (14.4 mg) and
TEA (12.5 mL) in dry toluene (0.4 mL) at 08C. The reaction mixture was
stirred for 15 min at 08C and 4 h at RT. The reaction was quenched with
buffer (pH 7) and extracted 3 times with ethyl acetate to yield 14.6 mg
crude product. The tris-(S)-MTPA derivative 8a was further purified by
RP-HPLC (column VP 250/10 Nucleodur 100–7 C-18 ec (Macherey–
Nagel); solvent A: methanol/water 1:1, solvent B: methanol, gradient:
85% B in 20 min to 86% B, in 3 min to 90% B, maintained at 90% B;
FR=6 mLminÀ1, UV detection at 259 nm, Rt =49 min) to yield 2.8 mg of
8a.
AHCTUNGTRENNUNG
[13CH3]-methionine-labeled roimatacene (1): 13C NMR (100 MHz,
[D6]DMSO): d (signal/noise)=18.3 (16.5:1), 13.6 (19.2:1), 12.7 (13.2:1),
11.7 ppm (14.4:1).
AHCTUNGTRENNUNG
[1-13C]-acetate-labeled roimatacene (1): 13C NMR (100 MHz, [D6]DMSO):
d (signal/noise)=167.3 (4.3:1), 145.8 (5.1:1), 135.8 (3.5:1), 134.5 (2.1:1),
134.0 (2.7:1), 133.8 (2.9:1), 132.4 (3.5:1), 131.2 (3.1:1), 125.4 (2.2:1), 75.1
(2.3:1), 73.6 (2.3:1), 70.1 ppm (3.1:1).
[2-13C]-acetate-labeled roimatacene (1): 13C NMR (100 MHz, [D6]DMSO):
AHCTUNGTRENNUNG
d (signal/noise)=140.3 (5.0:1), 133.7 (5.5:1), 132.1 (5.5:1), 131.8 (6.0:1),
130.0 (9:1), 127.0 (9.5:1), 123.7 (11:1), 74.4 (9:1), 36.1 (5.5:1), 34.7 (5.0:1),
25.3 ppm (10:1).
Tris-(R)-MTPA ester of p-bromoacetophenone-octahydroroimatacene
ester (8b): Tris-(R)-MTPA ester of p-bromoacetophenone-octahydroroi-
matacene ester (8b) was prepared analogously by starting with roimata-
cene (1) (11.6 mg) and gave 2.1 mg of tris-(R)-MTPA ester 8b for NMR
spectroscopy.
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
7880
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Chem. Eur. J. 2011, 17, 7875 – 7881

Roimatacene
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Received: December 20, 2010
Published online: May 26, 2011
Chem. Eur. J. 2011, 17, 7875 – 7881
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7881