Precursor-Directed Syntheses and Biological Evaluation of New Elansolid Derivatives

Article abstract of DOI:10.1002/cbic.201200228

The antibiotic elansolid C1 (8) was isolated from Chitinophaga sancti strain FxGBF13 after fermentation in the presence of anthranilic acid. Remarkably, 8 was also obtained by addition of anthranilic acid to a crude fermentation extract containing the mac

Full text of DOI:10.1002/cbic.201200228

DOI: 10.1002/cbic.201200228
Precursor-Directed Syntheses and Biological Evaluation of
New Elansolid Derivatives
Heinrich Steinmetz,[a] Wiebke Zander,[a] Muftah A. M. Shushni,[a, b] Rolf Jansen,[a]
Klaus Gerth,[a] Richard Dehn,[c] Gerald Drꢀger,[c] Andreas Kirschning,[c] and Rolf Mꢁller*[a, d]
The antibiotic elansolid C1 (8) was isolated from Chitinophaga
sancti strain FxGBF13 after fermentation in the presence of an-
thranilic acid. Remarkably, 8 was also obtained by addition of
anthranilic acid to a crude fermentation extract containing the
macrolide elansolid A2 (1*). This Michael-type conjugate addi-
tion allowed us to generate 21 new derivatives of elansolid C1
(9–29) by using various nucleophiles. Biological activities of all
derivatives were evaluated against Staphylococcus aureus, Mi-
crococcus luteus, and the mouse cell line L929.
Recently, the first polyketide antibiotic from the gliding bacteri-
um Chitinophaga sancti (formerly Flexibacter sancti) was report-
ed as elansolid A (1). Remarkably, elansolid A (1) occurs as two
distinct atropisomers, A1 and A2 (1 and 1*), with a differently
folded macrolide ring.[1] During isolation of 1/1* from the fer-
mentation extracts, three ring-opened analogues, elansolids B1
to B3 (2–4), were isolated. The particular elansolid depended
on the work-up procedure employed.[2] In parallel with our
studies, Teta et al. investigated the biosynthesis of elansolids
based on the isolation of elansolid D2 (5) from Chitinophaga
pinensis DSM 2588.[3] Compound 5 was obtained along with
the work-up artifact elansolid D1 (6) under acidic conditions
with 0.1% TFA.[2] The molecular basis of elansolid biosynthesis
was finally explored by Dehn et al., after isolation and charac-
terization of the highly reactive p-quinone methide elansolid
A3 (7).[2,4] Interconversion studies demonstrated that all elan-
solid derivatives 1–6 could be traced back to the p-quinone
methide 7, thus explaining the large number of byproducts
observed as artifacts, depending on the work-up procedure.[2]
Interestingly, atropisomer 1 was less active against Gram-
positive bacteria than 1*, while the biological activity of 7 was
identical to that of 1*. Here, we report the isolation of another
antibiotic co-metabolite from precursor-targeted biosynthesis
after feeding anthranilic acid to the culture broth. This synthet-
ic strategy was extended to 21 elansolid derivatives that like-
wise originated from 7 in one step. The minimum inhibitory
concentrations (MICs) of all new elansolid derivatives 9–29
against Staphylococcus aureus, Micrococcus luteus were deter-
mined, as well as their cytotoxicity against a mouse cell line.
elansolid C1. Thus, elansolid C1 8 was isolated from a 10 L fer-
mentation of C. sancti in a synthetic medium containing an-
thranilic acid. The adsorber resin Amberlite XAD 16 was added
on the sixth day of cultivation. After one hour of continued
stirring the resin was recovered by sieving and eluted with
acetone. Finally, 8 was isolated from the extract by RP-HPLC in
71 mg yield.
High resolution mass spectrometry of 8 revealed a molecular
formula of C44H54NO8 for the molecular ion cluster [MÀH]À at
m/z 724.3847 (calcd 724.3849). The NMR spectra recorded in
[D6]DMSO showed signals and correlations of the retained
carbon skeletons of elansolids B1–B3 (2–4) and those of an in-
corporated anthranilic acid moiety (Figure 1 and Table 1). The
bond between the anthranilic acid moiety and the elansolid
skeleton was indicated by COSY correlations between 25H and
the amino proton and by the upfield shift for C25 (56.9 ppm).
Because of the hindered rotation of the p-hydroxyl benzyl
moiety in 8, its NMR signals were very broad at room tempera-
[a] H. Steinmetz, W. Zander, M. A. M. Shushni, R. Jansen, K. Gerth, R. Mꢀller
Helmholtz Centre for Infection Research, Research Group Microbial Drugs
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
E-mail: rom@helmholtz-hzi.de
[b] M. A. M. Shushni
Department of Pharmacognosy, Faculty of Pharmacy
Garyounis University
P.O. Box 5341, Benghazi 18251 (Libya)
[c] R. Dehn, G. Drꢁger, A. Kirschning
Institut fꢀr Organische Chemie und Biomolekulares Wirkstoffzentrum
(BMWZ)
Leibniz Universitꢁt Hannover
Schneiderberg 1B, 30167 Hannover (Germany)
Results and Discussion
[d] R. Mꢀller
Initially, elansolid C1 (8) was detected as the main product in
shake-flask cultivations of C. sancti strain FxGBF13, when using
complex media containing different soy peptones, especially
the material from the Marcor Development Corporation (Hack-
ensack, USA), whereas in “synthetic medium” only elansolids A
and B were present. However, when synthetic medium was
supplemented with anthranilic acid, cultures also provided
Helmholtz Institute for Pharmaceutical Research Saarland
Helmholtz Centre for Infection Research Saarland and Saarland University
Postfach 151150, 66041 Saarbrꢀcken (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200228: Characterization of the elansolid
1
1
derivatives 9–29; H, 13C and H,13C HMQC NMR spectra of the elansolid de-
rivatives 8–29, as well as H,13C HMBC, H,1H COSY and H,1H ROESY of 8
1
1
1
measured at RT and 334 K.
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R. Mꢀller et al.
ture. However, enhanced NMR resolution was achieved when
the temperature was raised to 334 K (Table 1).
rivatives 9–29. Chosen precursors and yields of the obtained
elansolid derivatives 8–29 are listed in Scheme 2. The general
procedure as well as characterization of products 8–29 are pro-
vided in the Supporting Information.
The formation of 8 during fermentation is most likely due to
a Michael-type nucleophilic addition of the amino group of an-
thranilic acid to the p-quinone methide carbon of the primary
biosynthetic product, elansolid A3 (7; Scheme 1).[4]
Previously we have shown that formation of the proposed
reactive intermediate quinone methide 7 occurs under mild
basic conditions from 1/1*.[2] This was supported experimental-
ly by addition of triethylamine (TEA) and anthranilic acid to
a crude fermentation extract that contained elansolid A2 (1*)
in ethanol. As expected, the reaction yielded elansolid C1 (8)
as the main product (Figure S1 in the Supporting Information).
Repetition of this experiment with a number of different nucle-
ophiles allowed us to generate a small library of elansolid de-
In our previous report, we showed that 1 and 1* differ in
their biological activity.[1] Both showed the lowest MIC against
M. luteus (MIC 0.3 mgmLÀ1for 1*, 1 was less active); against me-
thicillin-resistant S. aureus MRS3, 1* had a MIC of 4.2 mgmLÀ1
while 1 showed no growth inhibition up to 33 mgmLÀ1. Note-
worthy, elansolid A2 (1*) and the quinone methide A3 (7) had
identical MICs, but differed in their cytotoxicity:[2] against the
mouse fibroblast cell line L929, 1* showed cytotoxicity at
12 mgmLÀ1, 7 was less toxic (33 mgmLÀ1), and 1 was nontoxic
up to 40 mgmLÀ1 [1,2]
.
All the new elansolid derivatives 8–29 were tested against
M. luteus, S. aureus MRS3, and in a proliferation assay with cell
line L929. The MIC values are presented in Table 2. With the
exception of thiol adduct 28 (IC90 <0.1 mgmLÀ1), all elansolid
derivatives showed no antiproliferative activity against L929. In
contrast to all other precursors, which are amino-based nucleo-
philes, 28 results from nucleophilic attack of a thiol group
onto 7. The derivatives were generally less active against
M. luteus than 1* and 7 were. Still, a few representatives of the
new elansolid C1 varients were more active than 1 (MIC
8.3 mgmLÀ1). These include the anthranilic acid derivative elan-
solid C1 (8), the sulfate containing elansolid derivative 27 and
the sulfanylbenzoic acid derivative 28 (MIC 0.5 mgmLÀ1). All
1
Figure 1. 1H,1H COSY sequences and selected H,13C HMBC correlations of
elansolid C1 (8).
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Syntheses and Evaluation of Elansolid Derivatives
Table 1. NMR data of elansolid C1 (8) in [D6]DMSO (1H 600 MHz, 13C 150 MHz).
Position
dC, mult.
dH, mult (J [Hz])
COSY[a]
HMBC[a]
ROESY[a,b]
1
2
3
167.8, C[c]
116.0, CH
148.8, CH
132.8, C
3, 2
3
30, 5
30, 2, 6 a/b, 3
5.58, d (15.8)[d]
7.15, d (15.8)
3
2
30, (3)
5, (2)
4
30
5
6 a
6 b
7
8
31
9
12.0, CH3
139.8, CH
33.2, CH2
1.64, s
3, 5
30, 3, 6 a/b
5
2
6.07, t (br) (7.8)
2.19, dt (15.5, 7.8)
2.36, m
3.55, td (7.8, 2.9)
1.54, m
0.84, d (6.6)
4.70, m
5.48, dd (11.2, 8.8)
5.89, t (11.2)
6.59, dd (14.3, 11.2)
5.96, dd (14.3, 11.4)
6.15, t (br) (11.4)
5.76, dd (10.6, 11.4)
2.74, dd (10.6, 3.7)
6 a/b> 30
5, 6 b, 7
5, 6 a, 7
6 a/b, 8,
7, 9, 31
8
3>7
(6 b, 5)
(6 b, 5)
5>10
71.2, CH
45.0, CH
10.2, CH3
66.8, CH
134.9, CH
128.0, CH
128.4, CH
129.9, CH
128.6, CH
131.9, CH
40.6, CH
134.5, C
20.8, CH3
122.5, CH
54.7, CH
74.1, C
26.2, CH3
59.9, CH2
31, 6 a, 5
31
(31)
Scheme 1. Proposed formation of 8 by
a Michael-type nucleophilic attack of
anthranilic acid on the p-quinone
moiety of 7.
>10, 8
12
(11)>7, 31
(10>12)
9, (11)
>16
(15)
23, (14), 25NH
>13
10, 8
11, 9
31, 11
10
11
12
13
14
15
16
17
32
18
19
20
33
21 a
21 b
22
34
35
23
24
25
25NH
26
27
28
29
1’
10, 12
13, 11
12, 14
13, 15
14, 16
15, 24
14, 10
11, 15, (14)
12
Experimental Section
General experimental proce-
dures: Optical rotations were de-
termined with a model 241 polar-
imeter (PerkinElmer), UV spectra
18, 32, 14, 25, 23
32
18
1.37, s (br)
5.46, s (br)
2.62, d (br) (11.8)
>18
19>32
23, 32, 18
18
32>16
32, 33, (19)
18>34
were recorded with
spectrophotometer
a
UV-2450
23, 21 b, 33, 18
33, 21 a/b>18, 23
21b, (33)
(Shimadzu,
Kyoto, Japan), and IR spectra were
measured with a Nicolet 20DXB
FTIR spectrometer (Thermo Scien-
tific). NMR spectra were recorded
with AM 300 (1H 300 MHz, 13C
75 MHz) and ARX 600 (1H 600 MHz,
1.00, s
1.66, m
1.51, d (13.9)
18
(21b)
(21a)
21b
21a
35, 33, 34
37.5, C
35, 34, 23, 21 a/b
35, 23, 21 a/b, (34)
34,23, 21 a/b, (35)
35, 34
23, 25, 25NH
25NH
24.0, CH3
30.6, CH3
44.3, CH
47.7, CH
56.9, CH
1.21, s
0.71, s
1.83, t (11.8)
2.32, m
4.66, m
25> 19
>25
15, 25NH>33
(25)>19, 16
6’, 5’, 34, (24), 35
15, 23, 25>35
13C
150 MHz)
spectrometers
19, 24
25, 23, 16
25NH, 24
25
(Bruker Daltonics). High resolution
electron stray ionization mass
spectra (HRESIMS) were obtained
with a MaXis ESI-TOF-MS (Bruker
Daltonics) and a 1200 series DAD-
UV UHPLC system (Agilent Tech-
nologies) with an Acquity UPLC
BEH C18 column (2.1ꢃ50 mm,
1.7 mm; Waters); solvent A: H2O
with formic acid (0.1%), solvent B:
acetonitrile with formic acid
(0.1%); gradient: 5% B for 0.5 min,
increasing to 100% B in 19.5 min,
8.62, d (4.8)
131.1, C
(25)
25
127.5, CH[d]
114.8, CH[d]
155.9, C
6.96, d (8.0)[c]
6.62, d (8.0)[c]
28
27
27[d]
3’
170.0, C
2’
3’
4’
5’
6’
7’
110.3, C
4’, 6’, 25NH
5’
6’
3’
4’, 25NH
3’, 5’, 25
131.5, CH
114.0, CH
134.2, CH
111.8, CH
149.9, C
7.75, dd (7.8, 1.7)
6.44, dd (7.8, 7.3)
7.11, ddd (8.6, 7.3, 1.3)
6.10, d (8.6)
4’
(4’)
3’, 5’
4’, 6’
5’
3’, 5’> 6’
4’, 25
25, 4’
[a] Signals sorted by cross peak intensity. [b] Vicinal coupling constants in parenthesis. [c] Deviations observed
between the NMR data of the carboxylic acid and the sodium salt of elansolid C1 (8). [d] NMR data measured
at 334 K.
100%
B for 5 min; flow rate
0.6 mLminÀ1; UV detection 200–
500 nm).
Fermentation of strain FxGBF13
with anthranilic acid and isolation of elansolid C1 (8): Strain
FxGBF13 (deposited at the German Collection of Microorganisms
and Cell Cultures (DSMZ), DSM 21134) was identified as C. sancti
according to 16S rDNA sequencing. The strain was cultivated in an
were less active against S. aureus MRS3 compared to 1* and 7
(MIC 4.2 mgmLÀ1), but they exerted higher activity than 1.
In conclusion, isolation of elansolid C1 (8) from precursor-di-
rected fermentation as well as by chemical reaction of 1* with
anthranilic acid proves that the different elansolid variants
result from intermolecular nucleophilic attack on the methide
carbon of elansolid A3 (7). Based on this, 22 more derivatives
were synthesized from the crude extract. The biological activity
of 1* and 7 was not reached with any of the synthetic deriva-
tives, although several showed higher biological activity than
elansolid A1 (1); all were less cytotoxic then 1*. The mode of
action of the elansolids 1–29 is currently under investigation in
our laboratories.
Erlenmeyer flask with casitone (10 gLÀ1
, Difco) MgSO4·7H2O
(1 gLÀ1), CaCl2·2H2O (1 gLÀ1), HEPES (50 mm, pH 7.0). A sterilized
10 L bioreactor (Giovanola Frꢄres SA, Monthey, Switzerland) with
the same medium supplemented with anthranilic acid (dissolved in
methanol, final concentration 100 mgLÀ1) was inoculated with pre-
culture (1 L) and stirred (100 rpm) under aeration (0.1 vvm) at
208C. After 5 days, Amberlite XAD 16 resin (150 mL) was added
and then harvested by sieving, after 1 h of stirring.
After removal of residual cells from the XAD 16 resin, the com-
pounds were eluted with acetone (3ꢃ300 mL). The organic solvent
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R. Mꢀller et al.
Fermenation of C. sancti strain FxGBF13: A 10 L bio-
reactor (Giovanola Frꢄres) with the same medium as
above was inoculated with preculture (FxGBF13, 1 L) and
stirred (100 rpm) at 208C with aeration (0.1 vvm) for
4 days. This preculture was used to inoculate 70 L fer-
mentation (blade impeller 100 rpm, aeration 0.1 vvm,
pO2 (20%), with stirring) of the same medium without
HEPES but with Amberlite XAD 16 neutral adsorber resin
(2% (v/v), Rohm and Haas) in a 100 L bioreactor (Giova-
nola Frꢄres SA). The pH was regulated with KOH (10%)
and H2SO4 (10%). Silicone antifoam (Tegosipon; Gold-
schmidt AG, Essen, Germany) was used to inhibit foam
formation. After 7 days of cultivation, the XAD 16 resin
and residual cell mass were harvested by sieving, the
resin was washed with water, and products were eluted
in an open glass chromatography column (9 L of ace-
tone/methanol (1:1)). After evaporation of the organic
solvent, the remaining water layer was extracted three
times with ethyl acetate. The combined ethyl acetate
phases were dried with sodium sulphate and evaporated
to dryness. The oily residue was partitioned between
methanol and n-heptane. The methanol layer yielded
14.0 g of extract after evaporation. This crude fraction
was directly used in the chemical syntheses with differ-
ent nucleophiles.
General aspects on mutasynthons: The nucleophilic
precursors leading to elansolid derivatives 8, 9, 15–17,
and 26–29 were commercially available. The prepara-
tions of 3-aminobenzoic acid derivatives used for the
syntheses of elansolid derivatives 10–14 were reported
in refs. [5]–[7]. The 5-arylsubstituted anthranilic acid de-
rivatives for elansoldid derivatives 18–25 were accessible
from N-Boc protected 5-bromoanthranilic acid by
Suzuki–Miyaura cross-coupling reaction with the corre-
sponding aryl boronic acids, followed by Boc-deprotec-
tion under standard conditions.
Scheme 2. New elansolid derivatives 8–29 synthesized from 20 mg of crude extract con-
taining elansolid A2 (1*) by addition of 10 mg of the corresponding nucleophiles and
TEA.
General procedure for the preparation of 5-aryl an-
thranilic acid derivatives: Anthranilic acid methyl ester (0.774 mol,
100 mL) and di-tert-butyl dicarbonate (219.6 g, 1.006 mol) was dis-
solved in tetrahydrofurane (300 mL) and heated under refluxing
conditions for 72 h. Tetrahydrofurane was removed under reduced
pressure, water (300 mL) was added, and the mixture was extract-
ed with ethyl acetate (3ꢃ300 mL). The combined extracts were
dried with anhydrous sodium sulfate and concentrated under re-
duced pressure. The crude product was dissolved in acetic acid
(600 mL), then sodium acetate (126.66 g, 1.544 mol) was added.
Bromine (0.811 mol, 41.65 mL, dissolved in acetic acid (100 mL))
was added to the stirred mixture over a period of 2 h, and stirring
was evaporated and the aqueous layer extracted with ethyl ace-
tate, dried with sodium sulfate, filtrated, and concentrated in
vacuo, to yield 489 mg. Elansolid C1 (8) was purified by preparative
RP-HPLC in 10 batches on a Nucleodur 100-7 C18 column (250ꢃ
21 mm, Macherey–Nagel, Dꢁren, Germany): eluent A: water/aceto-
nitrile (8:2) with NH4Ac (50 mm), eluent B: water/acetonitrile (3:7)
with NH4Ac (50 mm); gradient: 100% A to 100% B over 60 min,
100% B for 10 min; flow rate 15 mLminÀ1; UV detection 254 nm.
Fractions containing 8 were combined. After removal of the organ-
ic solvent in vacuo, the water layer was extracted with ethyl ace-
tate (yield 71 mg of 8).
Table 2. MICs [mgmLÀ1] of elansolid derivatives 8–29 against M. luteus and S. aureus MRS3.
No.
M. luteus
S. aureus
No.
M. luteus
S. aureus
No.
M. luteus
S. aureus
1
1*
7
8
9
10
11
12
13
8.3
0.3
0.3
0.5
4.2
2.1
2.1
4.2
1.0
>33.0
4.2
14
15
16
17
18
19
20
21
22
16.6
0.5
16.6
0.5
4.2
4.2
2.1
16.6
16.6
33.3
8.3
67.0
8.3
8.3
8.3
8.3
8.3
33.3
23
24
25
26
27
28
29
16.6
16.6
16.6
2.1
1.0
0.5
33.3
33.3
16.6
8.3
16.6
8.3
4.2
8.3
16.6
8.3
16.6
8.3
16.6
67.0
8.3
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Syntheses and Evaluation of Elansolid Derivatives
was continued 12 h. The suspension was added to a mixture of
water and ice (1:1, 2 L), and the crude product was isolated after
filtration. Recrystallization (hexane, 600 mL) yielded N-Boc-protect-
ed 5-bromo-anthranilic acid (209.56 g, 0.635 mol; yield 82%). The
corresponding 5-arylsubstituted anthranilic acids were prepared ac-
cording to the procedure of Leadbeater et al.[8] Typically, N-Boc-
protected 5-bromo anthranilic acid (2.31 g, 7 mmol), sodium car-
bonate (2.23 g, 21 mmol), tetra-n-butylammonium bromide (2.26 g,
7.0 mmol), palladium acetate (16 mg, 0.1 mmol) and the arylboron-
ic acid (7.7 mmol) were mixed with water (7 mL) and heated under
refluxing conditions in an oil bath (preheated to 2008C). After
10 min the reaction mixture was cooled to room temperature, ad-
justed to pH 4 with dilute HCl, and the crude product was isolated
by filtration. This was dissolved in tetrahydrofurane (20 mL) and
stirred with aqueous lithium hydroxide (0.88 g, 21 mmol in 20 mL
water) at room temperature overnight. Tetrahydrofurane was re-
moved under reduced pressure and the mixture was acidified with
an excess of acetic acid. The crude 5-substituted anthranilic acid
derivatives were isolated by filtration and purified by chromatogra-
phy on silica gel (yield 70–90%). Finally, the N-Boc protection was
quantitatively removed by treatment with trifluoroacetic acid in di-
chloromethane under standard conditions.
24 h. Growth inhibition was determined by visual inspection
(Table 2). The cytotoxicity tests were carried out with the mouse
fibroblast cell line L929 in Dulbecco’s modified eagle medium
(DMEM) containing fetal bovine serum (10%). IC90 was determined
after five days of incubation. Elansolid derivatives 8–29 showed no
cytotoxicity up to 16.4 mgmLÀ1, with the exception of 28 (IC90
0.1 mgmLÀ1).
<
1
Elansolid C1 (8): oil (C44H55NO8), [a]2D5 = +112.1 (c=0.7, EtOH); H
and 13C NMR (600/150 MHz, [D6]DMSO): Table 2; IR (KBr): n˜max
3374, 2966, 2926, 1684, 1616, 1578, 1512, 1234, 1024, 1002 cmÀ1
=
;
UV (EtOH): lmax (loge)=209 (4.36), 222 (4.45), 236 (4.24), 260 (4.53),
282 (4.46), 298 (shoulder), 349 nm (357); HRESIMS: m/z calcd for
C44H54NO8: 724.3849, found: 724.3847.
Acknowledgements
The work was supported by the Fonds der Chemischen Industrie
for a Ph.D. scholarship for R.D. We thank Silke Reinecke for tech-
nical support, Diana Telkemyer and Birte Trunkwalter for the
determination of the MICs and cytotoxicities, Christel Kakoschke
and Beate Jaschok-Kentner for measuring the NMR spectra and
Jꢀrgen Fischer for expert synthetic assistance.
General experimental procedure for the syntheses of elansolid
derivatives 8–29: Crude methanol extract (20 mg) was dissolved
in ethanol (5 mL), then precursor (10 mg) and TEA (20 mL) were
added. The reaction mixture was stirred at room temperature over-
night. The organic solvent was evaporated with N2. The residue
was dissolved in ethyl acetate (4 mL) and neutralized with phos-
phate buffer (pH 7, 50 mm K2HPO4·3H2O/50 mm NaH2PO4·H2O
(2:1)). The aqueous mixture was extracted twice with ethyl acetate,
and the combined organic layer was washed with water and finally
dried in vacuo. The oily residue was further purified by preparative
RP-HPLC (Nucleodur 100-7 C18 column; 250ꢃ21 mm, Macherey–
Nagel; eluent A: water/acetonitrile (8:2) with acetic acid (0.1%),
eluent B: water/acetonitrile (6:4) with acetic acid (0.1%); gradient:
100% A to 100% B over 60 min, 100% B for 60 min; flow rate
20 mLminÀ1; UV detection at 254 nm). Fractions containing elansol-
id derivatives were combined, and after evaporation of the organic
solvent the water layer was freeze dried.
Keywords: anthranilic acid · antibiotics · elansolid · liquid
chromatography · nucleophiles
[1] R. Dehn, Y. Katsuyama, A. Weber, K. Gerth, R. Jansen, H. Steinmetz, G.
Hçfle, R. Mꢁller, A. Kirschning, Angew. Chem. 2011, 123, 3968–3973;
Angew. Chem. Int. Ed. 2011, 50, 3882–3887.
[2] S. Eichner, H. G. Floss, F. Sasse, A. Kirschning, ChemBioChem 2009, 10,
1801–1805.
[3] R. Jansen, K. Gerth, H. Steinmetz, S. Reinecke, W. Kessler, A. Kirschning, R.
Mꢁller, Chem. Eur. J. 2011, 17, 7739–7744.
[4] T. Knobloch, K. Harmrolfs, F. Taft, B. Thomaszewski, F. Sasse, A. Kirschning,
ChemBioChem 2011, 12, 540–547.
[5] N. E. Leadbeater, M. Marco, J. Org. Chem. 2003, 68, 888–892.
[6] H. Steinmetz, K. Gerth, R. Jansen, N. Schlꢀger, R. Dehn, S. Reinecke, A.
Kirschning, R. Mꢁller, Angew. Chem. 2011, 123, 553–557; Angew. Chem.
Int. Ed. 2011, 50, 532–536.
[7] F. Taft, M. Brꢁnjes, H. G. Floss, N. Czempinski, S. Grond, F. Sasse, A.
Kirschning, ChemBioChem 2008, 9, 1057–1060.
Biological assays: MICs of 8–29 against M. luteus and S. aureus
MRS3 were determined in serial dilutions (1:2) from 100 mgmLÀ1 in
EBS medium (casein peptone (0.5%), protease peptone (0.5%),
meat extract (0.1%), yeast extract (0.1%), pH 7.0). The tests were
conducted in 96-well microtiter plates. On each plate a negative
control with the tested organism was inoculated, as well as a meth-
anol control. Positive controls with oxytetracyclin hydrochloride
and corallopyronin for the MRS3 showed the expected growth
inhibition. Plates were incubated on a shaker (600 rpm) at 308C for
[8] R. Teta, M. Gurgui, E. J. N. Helfrich, S. Kꢁnne, A. Schneider, G. van Echten-
Deckert, A. Mangoni, J. Piel, ChemBioChem 2010, 11, 2506–2512.
Received: April 2, 2012
Published online on && &&, 0000
ChemBioChem 0000, 00, 1 – 6
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
&
5
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
H. Steinmetz, W. Zander,
M. A. M. Shushni, R. Jansen, K. Gerth,
R. Dehn, G. Drꢁger, A. Kirschning,
R. Mꢀller*
Importent alternatives: Elansolid A is
an antibiotic secreted by Chitinophaga
sancti. We synthesized a library of deriv-
atives from the natural precursor elan-
solid C1 that was obtained by fermenta-
tion with anthranilic acid. The new com-
pounds were tested for inhibitory activi-
ty against Staphylococcus aureus and Mi-
crococcus luteus. None was as potent as
the natural antibiotic, but stability was
significantly increased.
&& – &&
Precursor-Directed Syntheses and
Biological Evaluation of New Elansolid
Derivatives
&6
&
www.chembiochem.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 6
ÝÝ
These are not the final page numbers!