Macrolactin N, a new peptide deformylase inhibitor produced by Bacillus subtilis

Article abstract of DOI:10.1016/j.bmcl.2006.06.058

A new 24-membered ring lactone, macrolactin N, was isolated from a culture broth of Bacillus subtilis and its structure was established by various spectral analysis. Macrolactin N inhibited Staphylococcus aureus peptide deformylase with an IC₅₀

Full text of DOI:10.1016/j.bmcl.2006.06.058

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4889–4892
Macrolactin N, a new peptide deformylase inhibitor produced
by Bacillus subtilis
a
a
a
b
a,
*
Jung-Sung Yoo, Chang-Ji Zheng, Sangku Lee, Jin-Hwan Kwak and Won-Gon Kim
a
Korea Research Institute of Bioscience and Biotechnology, PO Box 115, Yusong, Daejeon 305-600, Republic of Korea
b
School of Life and Food Sciences, Handong Global University, Pohang 791-708, Republic of Korea
Received 21 April 2006; revised 30 May 2006; accepted 16 June 2006
Available online 30 June 2006
Abstract—A new 24-membered ring lactone, macrolactin N, was isolated from a culture broth of Bacillus subtilis and its structure
was established by various spectral analysis. Macrolactin N inhibited Staphylococcus aureus peptide deformylase with an IC50 value
of 7.5 lM and also showed antibacterial activity against Escherichia coli and S. aureus.
ꢀ
2006 Elsevier Ltd. All rights reserved.
In recent years, the emergence of antibiotic-resistant
bacteria has steadily increased to become a serious
threat to the human. This emphasizes the need to dis-
cover and develop novel antibacterial drugs with new
1
modes of action. Bacterial genomics has revealed a
plethora of previously unknown targets of potential
2
use in the discovery of novel antibacterial drugs.
Among novel antibacterial targets, one target that has
received an increasing amount of attention lately is the
3
,4
bacterial peptide deformylase (PDF) (EC 3.5.1.31).
PDF, a unique subclass of metalloenzymes, catalyzes
the removal of the formyl group at the N-terminus of
bacterial proteins. PDF is essential for bacterial growth
but not required by mammalian cells, which potentially
makes it possible to identify a selective antibacterial
agent without mechanism-based toxicity. Recent studies
from several research groups have shown that PDF
inhibitors act as broad-spectrum antibacterial
Figure 1. The chemical structure of 1.
The producing strain AT29 was isolated from a soil
sample collected in Daejeon-city, Chungcheongnam-do,
Korea, and assigned to the B. subtilis AT29. Fermenta-
tion was carried out in 1-l Erlenmeyer flasks containing
soluble starch 1%, glucose 2%, soybean meal 2.5%, beef
extract 0.1%, yeast extract 0.4%, NaCl 0.2%, K HPO
3
,5,6
agents.
have been reported so far.
A few skeletons as PDF inhibitors, however,
7
–11
2
4
In the course of our screening for new PDF inhibitors
from microbial resources, we have isolated a new potent
0.025%, and CaCO 0.2% (adjusted to pH 7.2 before
3
sterilization). A piece of strain AT29 from a mature
plate culture was inoculated into a 500-ml Erlenmeyer
flask containing 80 ml of sterile seed liquid medium with
the above composition and cultured on a rotary shaker
1
2
compound, named macrolactin N (1), produced by
Bacillus subtilis AT29 (Fig. 1). In this paper, we present
the isolation, structure determination, and biological
activities of 1.
(
5
150 rpm) at 28 ꢁC for 3 days. For the production of 1,
ml of the seed culture was transferred into 1-l Erlen-
meyer flasks containing 100 ml of the above medium
and cultivated for 7 days using the same conditions.
The culture supernatant obtained from the culture broth
(9.6 l) was extracted with an equal volume of EtOAc
Keywords: Peptide deformylase; S. aureus; Macrolactin; Antibacterial.
*
Corresponding author. Tel.: +82 42 860 4298; fax: +82 42 860
595; e-mail: wgkim@kribb.re.kr
4
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.058

4890
J.-S. Yoo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4889–4892
three times and the EtOAc layer was concentrated in
vacuo. The resultant residue was subjected to SiO2
remaining carboxylic and carbonyl carbons was deter-
mined by the HMBC spectral data. The olefinic protons
at d 5.60 (2-H) and d 6.55 (3-H) were long range coupled
to the carboxylic carbon at d 166.5 (C-1). Long range cou-
plings were also observed from the methylene protons at d
1.65 and d 1.75 (13-H ) and d 2.36 (14-H ) of the first par-
(
followed by elution with CHCl /MeOH (100:1). The
Merck Art No. 7734.9025) column chromatography
3
active fractions were pooled and concentrated in vacuo
to give an oily residue. The residue was applied again
to a Sephadex LH-20 and then eluted with MeOH.
Active fraction dissolved in MeOH was further purified
by reverse-phase HPLC column (20 · 250 mm, YMC C )
2
2
tial structure to the carbonyl carbon at d 210.9 (C-15)
which was in turn long range coupled with the methylene
protons at d 2.42 (16-H ) and d 2.24 (17-H ) of the second
1
8
2
2
chromatography with a photodiode array detector. The
column was eluted with CH OH/H O (95:5) at a flow
rate of 5.5 ml/min to afford 1 (4.6 mg) at a retention time
partial structure. Together with the molecular formula,
the low-field shift of the proton at d 5.04 (23-H) suggested
the ester linkage of the proton of 23-H with the carboxylic
carbon of C-1. This linkage was confirmed by the HMBC
spectrum measured at 800 MHz in which the oxygenated
proton at 23-H was long range coupled to the carboxylic
carbon at d 66.5 (C-1) (Fig. 2). The geometric configura-
tions of the carbon–carbon double bonds were assigned
3
2
of 13 min as a white powder.
The molecular formula of 1 was determined to be
C H O on the basis of high-resolution ESI-MS
2
4
34
4
+
[
nation with H and C NMR data. The IR data suggest-
(M+Na) , 409.23355 m/z (À1.38 mmu error)] in combi-
1
13
1
on the basis of their H coupling constants together with
NOESY data. The H coupling constants between 2-H
À1
1
ed the presence of a carbonyl (1711 cm ) and a hydroxyl
(
À1
1
3449 cm ) moiety. The H and C NMR data (Table 1)
13
1
and 3-H were 11.4 Hz, while H coupling constants
with DEPT and HMQC data suggested the presence of 10
olefinic methines, two oxygenated methines, nine
methylenes, a methyl, a lactone carbonyl carbon,
between 4-H and 5-H was 15.6 Hz, indicating that the
geometries of C-2 and C-4 were Z and E configurations,
respectively. The geometry of C-8 was E configuration
1
1
1
and a ketone carbonyl carbon. The H– H COSY
spectrum indicated the presence of two partial struc-
by the H coupling constants of 15.0 Hz between 8-H
and 9-H. The geometry of C-10 was Z configuration by
2
3
4
5
6
7
1
the H coupling constants (10.8 Hz) and NOE between
tures of – CH@ CH– CH@ CH– CH – CH(OH)–
2
14
8
9
10
11
12
13
CH@ CH– CH@ CH–
CH – CH – CH – and
10-H and 11-H. The coupling constant between 18-H
and 19-H was 15.2 Hz by the decoupling experiment
2
2
21
2
22
1
6
17
18
19
20
–
CH – CH – CH@ CH–
CH – CH – CH –
2 2 2
2
2
2
3
24
CH(O–)– CH . The presence of these two partial struc-
irradiated at d 2.24 (17-H ), which indicated that the
3
2
tures was confirmed by the HMBC spectrum (Table 1).
The connectivity of these two partial structures with the
geometry of C-18 was E configuration. The absolute
stereochemistry at C-7 was determined to be 7S from
1 13
Table 1. H (600 MHz) and C (125 MHz) NMR spectral data of 1 in CDCl
3
Position
d
H
(J, Hz)
d
C
HMBC
1
2
3
4
5
6
7
8
9
166.5 C
5.60(1H, d, 11.4)
6.55(1H, t, 11.4)
7.29(1H, dd, 15.6, 11.4)
6.11(1H, m)
118.1 CH
143.3 CH
130.0 CH
139.6 CH
C-1, C-4
C-1, C-5
a
C-3 , C-6
a
C-3, C-6, C-7
2.47(2H, m)
4.30(1H, m)
41.2 CH
72.0 CH
2
C-4, C-5, C-7
C-5, C-6, C-8, C-9
C-6, C-7, C-10
C-7, C-10, C-11
5.73(1H, dd, 15.0, 6.0)
6.45(1H, dd, 15.0, 10.8)
6.05(1H, t, 10.8)
5.42(1H, m)
135.5 CH
126.1 CH
129.3 CH
132.2 CH
1
1
1
1
0
1
2
3
C-8, C-9, C-12
C-9, C-12, C-13
2.19(2H, m)
1.65(1H, m)
26.7 CH
23.0 CH
2
C-10, C-11, C-13, C-14
C-11, C-12, C-14, C-15
C-11, C-12, C-14, C-15
C-12, C-13, C-15
2
1
.75(1H, m)
1
1
1
1
1
1
2
4
5
6
7
8
9
0
2.36(2H, m)
41.2 CH
210.9 C
40.0 CH
27.2 CH
2
2.42(2H, m)
2.24(2H, m)
5.41(1H, m)
5.40(1H, m)
1.97(1H, m)
2
2
C-15, C-17, C-18
a
C-15 , C-18, C-19
129.3 CH
131.1 CH
C-21
C-17
a
a
a
32.2 CH
25.3 CH
35.7 CH
2
2
2
C-18, C-19, C-21 , C-22
a
C-18, C-19, C-21 , C-22
2
.06(1H, m)
1.42(1H, m)
.50(1H, m)
1.51(1H, m)
.68(1H, m)
a
2
1
2
C-19, C-23
a
C-19 , C-23
1
a
C-21 ,
2
a
C-21, C-20 , C-23, C-24
a
1
a
a
2
2
3
4
5.04(1H, m)
1.27(3H, d, 6.3)
70.8 CH
20.1 CH
C-1 , C-21
C-22, C-23
3
1
The assignments were aided by H– H COSY, DEPT, HMQC, and HMBC.
1
a
Detected at 800 MHz.

J.-S. Yoo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4889–4892
4891
1
Figure 2. Key H– H COSY, HMBC, and NOE correlations of
1
macrolactin N.
1
3
the (R)-and (S)-MTPA ester of 1 by modified Mosher
1
4
method, which was the same as that of C-7 of macrolac-
1
Figure 3. The mechanism of inhibition of Staphylococcus aureus PDF
by 1 with respect to f-MAS. The values are represented as means ± SD
in triplicate.
5
tin A. Thus, the structure of 1 was determined as shown
in Figure 1.
Compound 1 is a new derivative dehydroxylated at
1
6
with a MIC (lg/ml) of 100, while inhibiting weaker bac-
terial growth against S. aureus and B. subtilis with a
MIC50 (lg/ml) of 100, respectively.
C-13 of macrolactin F. A class of macrolactin is a
4-membered lactone compound and 15 macrolactin
2
compounds such as macrolactins A-M, 7-O-suc-
cinoylmacrolactin A, and 7-O-succinoylmacrolactin F
1
5–17
In summary, macrolactin N is a new 24-membered lac-
tone compound isolated from B. subtilis. Macrolactin
N strongly inhibited S. aureus PDF and also showed
antibacterial activity against E. coli, B. subtilis, and
S. aureus. Macrolactin N may serve as a new class of
PDF inhibitors for development of antibacterials.
have been reported so far.
Compound 1, however,
is the first macrolactin dehydroxylated at C-13. Macro-
lactin compounds have been isolated from an unclassifi-
able deep-sea bacterium, Actinomadura sp., or Bacillus
sp. They have been reported to exhibit weak antibacte-
rial activity against S. aureus and B. subtilis and antiviral
1
5–17
activities.
Acknowledgments
The inhibitory activity of 1 against S. aureus PDF was
1
8
evaluated according to our previously reported method
with some modifications as follows; assays contained
0 mM Hepes (pH 7.5), 10 mM NaCl, 20 lg/ml bovine
We express our thanks to Ms. Eun-Hee Kim at Korea
Basic Science Institute for NMR measurements (Avance
00, Bruker). This work was supported by the 21C
Frontier Microbial Genomics and Application Center
Program, Ministry of Science and Technology (Grant
MG05-0308-3-0), Republic of Korea.
5
8
serum albumin, 2 mM N-formylmethionine-alanine-
serine (f-MAS), 20 lM NAD, 0.00025 U formate dehy-
drogenase, and 54.3 nM S. aureus PDF in half-area,
9
6-well microtiter plates. Compound dissolved in
dimethylsulfoxide was added to each well. The rate of
increase in the amount of NADH in each reaction well
was measured at 340 nm at 30 ꢁC by a microtiter ELISA
reader using SOFTmax PRO software (Molecular
Devices, California, USA). The inhibitory activity was
calculated by the following formula: % of inhibi-
tion = 100 · [1 À (rate in the presence of compound/rate
in the untreated control)].
References and notes
1
2
3
4
. Bax, R.; Mullan, N.; Verhoef, J. Int. J. Antimicrob. Agents
000, 16, 51.
. Miesel, L.; Greene, J.; Black, T. A. Nat. Rev. Genet. 2003,
, 442.
. Yuan, Z.; Trias, J.; White, R. J. Drug Discov. Today 2001,
, 954.
. Waller, A. S.; Clements, J. M. Curr. Opin. Drug Discov.
Devel. 2002, 5, 785.
5. Jain, R.; Chen, D.; White, R. J.; Patel, D. V.; Yuan, Z.
2
4
6
Compound 1 inhibited S. aureus PDF in a dose-depen-
dent manner with an IC50 (lM) value of 7.5 lM. The
inhibition pattern of PDF by 1 with respect to the sub-
strate, f-MAS, was examined with a Lineweaver–Burk
plot analysis. As shown in Figure 3, 1 exhibited compet-
itive inhibition with f-MAS and its K and K values for
Curr. Med. Chem. 2005, 12, 1607.
6
. Chen, D.; Yuan, Z. Expert Opin. Investig. Drugs 2005, 14,
107.
7. Hu, X.; Ngujen, K. T.; Verlinde, C. L. M. J.; Hol, W. G.
1
i
m
À6
À4
PDF were 2.16 · 10 M and 1.8 · 10 M, respectively.
The antibacterial activity of 1 against S. aureus
J.; Pei, D. J. Med. Chem. 2003, 46, 3771.
8
9
. Howard, M. H.; Cenizal, T.; Gutteridge, S.; Hanna, W. S.;
Tao, Y.; Totrov, M.; Wittenbach, V. A.; Zheng, Y.
J. Med. Chem. 2004, 47, 6669.
. Davies, S. J.; Ayscough, A. P.; Beckett, R. P.; Bragg, R.
A.; Clements, J. M.; Doel, S.; Grew, C.; Launchbury, S.
B.; Perkins, G. M.; Pratt, L. M.; Smith, H. K.; Spavold, Z.
(
(
RN4220), B. subtilis (KCTC 1021), and Escherichia coli
KCTC 1924) was examined using microdilution broth
method. Compound 1 showed stronger antibacterial
activity against E. coli than S. aureus and B. subtilis.
Compound 1 inhibited bacterial growth against E. coli

4892
J.-S. Yoo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4889–4892
M.; Thomas, S. W.; Todd, R. S.; Whittaker, M. Bioorg.
Med. Chem. Lett. 2003, 13, 2709.
0. Takayama, W.; Shirasaki, Y.; Sakai, Y.; Nakajima, E.;
ester: d 5.61 (2-H), d 6.42 (3-H), d 7.25 (4-H), d 5.79 (5-H),
d 2.58 (6-H), d 4.38 (7-H), d 5.67 (8-H), d 6.57 (9-H), d 6.02
(10-H), d 5.49 (11-H), d 2.24 and 2.13 (12-H), d 1.74 and
1.66 (13-H), d 2.36 (14-H), d 2.40 (16-H), d 2.24 (17-H), d
5.39 (18-H, 19-H), d 2.05 (20-H), d 1.51 and d 1.40 (21-H),
1
Fujita, S.; Sakamoto-Mizutani, K.; Inoue, J. Bioorg. Med.
Chem. Lett. 2003, 13, 3273.
1
1. Jain, R.; Sundram, A.; Lopez, S.; Neckermann, G.; Wu,
C.; Hackbarth, C.; Chen, D.; Wang, W.; Ryder, N. S.;
Weidmann, B.; Patel, D.; Trias, J.; White, R.; Yuan, Z.
Bioorg. Med. Chem. Lett. 2003, 13, 4223.
d 1.67 and 1.52 (22-H), d 5.02 (23-H), d 1.26 (24-H). The
1
H NMR (500 MHz, CDCl ) of (S)-MTPA ester: d 5.62
3
(2-H), d 6.50 (3-H), d 7.30 (4-H), d 5.95 (5-H), d 2.648 (6-
H), d 4.38 (7-H), d 5.57 (8-H), d 6.47 (9-H), d 5.99 (10-H),
d 5.46 (11-H), d 2.16 and 2.05 (12-H), d 1.70 and 1.63 (13-
H), d 2.33 (14-H), d 2.40 (16-H), d 2.24 (17-H), d 5.39 (18-
H, 19-H), d 2.05 (20-H), d 1.51 and d 1.40 (21-H), d 1.67
and 1.52 (22-H), d 5.02 (23-H), d 1.26 (24-H).
14. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H.
J. Am. Chem. Soc. 1991, 113, 4092.
1
2. Compound 1: a white powder; kmax nm (loge) in MeOH:
2
29 (4.03), 235 (4.03), 262 (3.96); IR (KBr): 3449, 2932,
= À26.7ꢁ (c 0.2, MeOH); ESI-
À1
1711, 1273, 1190 cm ; [a]
D
+
MS: m/z 409.23355 (M+Na) , C24
34 4
H O Na requires
409.23493.
1
3. Preparation of (R)- and (S)-MTPA ester of 1: To a
solution of 4-(dimethylamino)pyridine (1.3 mg, 10.6 lmol)
and triethylamine (2.0 lL, 14.3 lmol) in chloroform
15. Gustafson, K.; Roman, M.; Fenical, W. J. Am. Chem.
Soc. 1989, 111, 7519.
(
2
0.2 mL) at room temperature were added 1 (1.0 mg,
.7 lmol) and (R)-MTPA chloride (2.0 lL, 10.7 lmol)
successively. The reaction mixture was stirred for 16 h and
-(dimethylamino)propylamine (2.0 lL, 15.9 lmol) was
added. The mixture was subjected to flash chromatogra-
phy (SiO , 25% ethyl acetate/hexane) to afford (R)-MTPA
ester of 1. The H NMR (500 MHz, CDCl
16. Jaruchoktaweechai, C.; Suwanborirux, K.; Tanasupawatt,
S.; Kittakoop, P.; Menasveta, P. J. Nat. Prod. 2000, 63,
984.
17. Nagao, T.; Adachi, K.; Sakai, M.; Nishijima, M.; Sano, H.
J. Antibiot. 2001, 54, 333.
3
2
18. Kwak, J. H.; Kim, H. J.; Seol, M. J.; Suh, B. S.; Lee, J.;
Choi, S. J. Pharm. Soc. Korea 2003, 47, 184.
1
3
) of (R)-MTPA