(Z)-N-(4-Decenoyl)homoserine lactone, a new quorum-sensing molecule, produced by endobacteria associated with Mortierella alpina A-178

Article abstract of DOI:10.1016/j.tetlet.2012.07.133

N-Acylhomoserine lactones (AHLs) are the conserved quorum-sensing signal molecules in Gram-negative bacteria. (Z)-N-(4-Decenoyl)homoserine lactone (1), a new AHL, was isolated from the culture broth of the fungus Mortierella alpina A-178 harboring bacterial endosymbionts, called endobacteria. The structure and absolute configuration of 1 were elucidated by EI-MS, chemical synthesis, and chiral GC analysis. The compound induced the expression of a QS-regulated reporter gene in Agrobacterium tumefaciens NTL4, although its activity was lower than that of N-decanoylhomoserine lactone (6).

Full text of DOI:10.1016/j.tetlet.2012.07.133

Tetrahedron Letters 53 (2012) 5441–5444
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
(
Z)-N-(4-Decenoyl)homoserine lactone, a new quorum-sensing molecule,
produced by endobacteria associated with Mortierella alpina A-178
⇑
Kenji Kai , Koji Kasamatsu, Hideo Hayashi
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Acylhomoserine lactones (AHLs) are the conserved quorum-sensing signal molecules in Gram-negative
bacteria. (Z)-N-(4-Decenoyl)homoserine lactone (1), a new AHL, was isolated from the culture broth of
the fungus Mortierella alpina A-178 harboring bacterial endosymbionts, called endobacteria. The struc-
ture and absolute configuration of 1 were elucidated by EI-MS, chemical synthesis, and chiral GC analysis.
The compound induced the expression of a QS-regulated reporter gene in Agrobacterium tumefaciens
NTL4, although its activity was lower than that of N-decanoylhomoserine lactone (6).
Ó 2012 Elsevier Ltd. All rights reserved.
Received 25 June 2012
Revised 23 July 2012
Accepted 30 July 2012
Available online 4 August 2012
Keywords:
N-Acylhomoserine lactone
Quorum sensing
Mortierella alpina
Endobacteria
Agrobacterium tumefaciens NTL4
Bacteria communicate with each other by producing and
detecting diffusible signaling molecules. This mechanism, known
as quorum-sensing (QS), allows bacteria to coordinate their activ-
O
O
N
H
4
1
ities in response to their populations. The conserved QS signaling
O
molecules of Gram-negative bacteria are N-acylhomoserine lac-
tones (AHLs), which differ in the length and substitution of their
respective acyl side chains, conferring upon them signal specific-
L-1
O
2
ity. Many important bacterial behaviors are regulated by
O
R
N
H
AHL-dependent QS, including exoenzyme production, exopolysac-
charide production, antibiotic production, motility, and biofilm
O
1
2 4 3 6
R = (CH ) CH : C -HSL (2)
formation. Such coordinated bacterial actions need to succeed in
R = (CH2)5CH3: C7-HSL (3)
R = (CH2)6CH3: C8-HSL (4)
R = (CH2)7CH3: C9-HSL (5)
R = (CH ) CH : C -HSL (6)
developing and maintaining pathogenic and symbiotic interactions
with eukaryotic hosts. Previously we discovered the production of
C
6
- to C10-homoserine lactones (HSLs) (2ꢀ6) (Fig. 1) by bacterial
2
8
3
10
endosymbionts, called endobacteria, associated with the zygomyc-
ete fungus Mortierella alpina A-178.3 Molecular biological tech-
Figure 1. Structures of AHLs produced by the endobacteria associated with M.
alpina A-178.
niques revealed that an endobacterium in the fungus has the
same 16S rRNA gene sequence as the b-proteobacterium Castellan-
iella defragrans. These findings suggested that AHL-mediated QS in
endobacteria plays important roles in this unique symbiosis. In
that study, we detected a possible new AHL in the culture extract
of M. alpina A-178 by GC/MS. In this Letter, we describe the struc-
ture elucidation of (Z)-N-(4-decenoyl)homoserine lactone (1) and
the activity for a QS-dependent reporter response in Agrobacterium
tumefaciens NTL4.
The extract of the culture broth of M. alpina A-178 was
separated by silica gel column chromatography using a gradient
4
of n-hexaneꢀEtOAc. The AHL activity was evaluated with A. tum-
efaciens NTL4 harboring a plasmid with lacZ fused to the gene traG
whose expression was induced by active AHLs supplied.5 Com-
pounds acting on A. tumefaciens NTL4 were eluted in 80% and
00% EtOAc fractions. The fractions were combined and subjected
1
to separation by HPLC with an ODS column using MeCN/H
2
O
(
10:90–70:30 over 30 min and then 70:30 for 10 min) as a mobile
phase. The active eluate collected at retention time (t
which was found to contain compound 1 by GC/MS, was further
R
) 27ꢀ30 min,
⇑
Corresponding author. Tel./fax: +81 72 254 9472.
E-mail address: kai@biochem.osakafu-u.ac.jp (K. Kai).
0
040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.133

5
442
K. Kai et al. / Tetrahedron Letters 53 (2012) 5441–5444
A 2.00 (
x10,000)
1
4.2
1
1
0
.50
.00
.50
0
5
.0
7.5
10.0
12.5
t
15.0
17.5
20.0
22.5
(min)
R
B
C
100
1
43
1
43
125
2
8
3
102
+H
−H O
2
24
168
H
8
0
6
7
196
10
−
O
4
1 55
1
25
6
4
2
0
0
0
0
O
N
H
152
150
1
156
O
102
1
82
−H +2H
2
152
1
56
68
1
10
182
1
196 210
200
253
250
1
80
224
225
5
0
75
100
125
175
m/z
275
300
100
1
68
1
31
O
8
6
4
2
0
0
0
0
0
O
N
H
7
MeS
SMe−SCH
8
7
O
4
6
1
168
5
5
216
115
115
2
52
−HSL
1
31
299
1
02
151
150
1
99
216
1
83
284
3
47
5
0
100
200
250
300
350
400
m/z
Figure 2. GC/MS analysis of compound 1 in the active HPLC eluate. (A) Selected ion monitoring chromatogram for m/z 143. (B, C) Mass spectra of 1 and its DMDS derivative
(7). The proposed fragmentations are also described in the spectra.
purified by HPLC with a C
8
2
column using MeCN/H O (40:60) as a
mobile phase to afford compound 1 (ca. 3
dard curve).
lg, estimated by stan-
a
b
OH
OTHP
9
1%
54%
R
The GC/MS analysis of compound 1 gave a peak at t 14.2 min,
8
9
+
whose EI-MS exhibited a molecular ion peak (M ) at m/z 253, and
characteristic fragment ions for AHLs such as m/z 156, 143, 125,
6
O
and 102 (Fig. 2A and B). The molecular weight was consistent with
c
that of unsaturated derivatives of C10-HSL (6). The weak fragment
ions at m/z 224, 210, 196, 182, and 168 generated by neutral loss of
the aliphatic moiety of the acyl chain suggested that the unsatura-
tion position would be C-4 in the acyl chain. To support this, com-
OTHP
OH
67%
H C(H C)
H C(H C)
3 2 4
3
2
4
10
11
pound 1 was derivatized with dimethyl disulfide (DMDS) and the
product was analyzed by GC/MS.7,8 DMDS derivative 7 provided a
d
O
e
L-1
+
spectrum with a molecular ion peak (M ) at m/z 347 and fragment
5
6%
OH
51%
ions at m/z 216 and 131, resulting from characteristic cleavage be-
tween C-4 and C-5, and also at m/z 168 and 115 originating from
the fragment at m/z 216 undergoing methylsulfide and homoserine
lactone losses (Fig. 2C). It was thus again suggested that compound
12
O
f
12
O
1
has a double bond at C-4 in the acyl chain. The double-bond
N
H
5
2%
geometry of 1 was expected to be Z because the double bond in
O
the acyl chain of AHLs was usually Z.9
–11
Therefore, we proposed
D-1
the structure of 1 to be (Z)-N-(4-decenoyl)homoserine lactone.
Scheme 1. Synthesis of compound 1. Reagents and conditions: (a) dihydropyran, p-
TsOH, 0 °C to rt; (b) 1-bromopentane, n-BuLi, HMPA, THF, ꢀ78 °C to rt; (c) CrO
SO , H O, acetone, 0 °C to rt; (d) H , Lindlar catalyst, EtOH, rt; (e) -homoserine
lactone hydrochloride, EDCꢁHCl, Et N, CH Cl , rt; (f) -homoserine lactone hydro-
chloride, EDCꢁHCl, Et N, CH Cl , rt.
To confirm the structure of 1, (Z)-N-(4-decenoyl)-L-homoserine
lactone was synthesized according to Scheme 1.12 The hydroxy
3
,
H
2
4
2
2
L
group of 4-pentyn-1-ol (8) was protected as a THP ether, giving
compound 9. Treatment of 9 with bromopentane and n-BuLi in
3
2
2
D
3
2
2

K. Kai et al. / Tetrahedron Letters 53 (2012) 5441–5444
5443
the presence of hexamethylphosphoramide (HMPA) afforded a
coupled product, 10. Accordingly, compound 10 was treated with
Jone’s reagent to achieve deprotection of the THP group and subse-
quent oxidation, yielding carboxylic acid 11. Hydrogenation of 11
over a Lindlar catalyst gave Z-olefin 12. Finally, the acid was cou-
18.7 min (minor) and 20.6 min (major) (Fig. 3).15 These retention
times were matched with those of synthetic -1 and -1, respec-
tively (Fig. 3), indicating that the -form is in excess in natural 1.
D
L
L
The identity of the peaks was also confirmed by co-injection of
synthetic standards and natural 1. The ratio of D/L in natural 1
1
6
pled with
L-homoserine lactone hydrochloride in the presence of
was calculated to be 1/9 based on standard curves.
1
-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
Finally, the ability of synthetic 1 to induce the reporter response
in A. tumefaciens NTL4 was evaluated by using paper disc diffusion
assay. We compared the activity of (Z)-N-(4-decenoyl)homoserine
13
(
EDCꢁHCl) to afford
L-1. Analysis of synthetic
L
-1 by GC/MS gave
3
the same retention time and EI-MS data as for natural 1 (Supple-
mentary data, Fig. S1). Thus, the planar structure of natural 1
was revealed to be (Z)-N-(4-decenoyl)homoserine lactone.
Next, to elucidate the absolute configuration of 1, a chiral GC
analysis of the natural and synthetic compounds was performed.
lactone (1) with that of its saturated form, C10-HSL (6). In both
AHLs,
10 times less active than
the activities between -1 and
L
-forms are more active than
-6. Such difference was also observed in
-6. Thus, it was revealed that the
D-forms (Fig. 4). L-1 was about
L
D
D
D
-1 was synthesized from 12 and
D
-homoserine lactone hydrochlo-
double bond at C-4 in acyl chain weakens the activity of compound
1 on QS in A. tumefaciens NTL4.
1
4
ride (Scheme 1). The GC/MS of natural 1 gave two peaks at t
R
In conclusion, we identified (Z)-N-(4-decenoyl)homoserine lac-
tone (1) based on EI-MS and chemical synthesis, which was previ-
ously detected in the culture extract of M. alpina A-178. This is the
first report of a novel AHL produced by endobacteria associated
with fungi. Although a quantitative analysis of 1 was not con-
ducted in this study, the production level is apparently lower than
2
0.6
1
8.7
those of C
6
- to C10-HSLs (2ꢀ6), which were found in the culture
3
2
0.5
media and mycelia of M. alpina A-178. Furthermore, the activity
of 1 for QS in A. tumefaciens NTL4 was weaker than that of C10
-
HSL (6) or any other AHL produced by the endobacteria.3 These
results seem to indicate that the contribution of 1 to QS in the
endobacteria would be small. However, there remains the possibil-
ity that compound 1 plays an important role in the development of
the Mortierella-endobacterium symbiosis. For example, recently
,5
1
8.6
Bradyrhizobium spp., plant endosymbionts, were reported to use
new and minor AHLs as their QS signaling molecules.1
7,18
The pro-
duction levels of these AHLs were low, but sufficient to invoke QS
responses in these bacteria due to their high activities. Therefore,
to evaluate the true contribution of 1 as a QS signaling molecule,
a bioassay to assess the biological activity of 1 in endobacteria
needs to be established.
5
.0
10.0
15.0
20.0
tR (min)
25.0
30.0
35.0
Figure 3. Chiral GC analysis of natural and synthetic 1. Selected ion monitoring
chromatograms (m/z 143) of natural 1 (top), synthetic -1 (middle), and synthetic
(bottom).
L
D-
1
Acknowledgments
L-1
D
-1
This work was supported by a Grant-in-Aid for Young Scientists
(
B) from Japan Society for the Promotion of Science (JSPS).
Supplementary data
10 nmol/disc
1 nmol
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.
2
012.07.133.
0
.01 nmol
0.1 nmol
References and notes
1
2
.
.
Waters, C. M.; Bassler, B. L. Annu. Rev. Cell Dev. Biol. 2005, 21, 319.
Galloway, W. R. J. D.; Hodgkinson, J. T.; Bowden, S. D.; Welch, M.; Spring, D. R.
Chem. Rev. 2011, 111, 28.
L-6
D
-6
3
4
.
.
Kai, K.; Furuyabu, K.; Tani, A.; Hayashi, H. ChemBioChem 2012, 13, 1776.
Twenty flasks, each containing 200 mL of potato dextrose broth (Difco), were
inoculated with M. alpina A-178 and incubated for 1 week at 30 °C. The
combined culture broth was extracted with equal volumes of EtOAc three
times. The EtOAc layer was dried over Na
extract (611 mg).
2 4
SO and evaporated, yielding a crude
5
6
.
.
Shaw, P. D.; Ping, G.; Daly, S. L.; Cha, C.; Cronan, J. E.; Rinehart, K. L.; Farrand, S.
K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6036.
GC/MS data were obtained with a GCMS-QP2010 Plus (Shimadzu) fitted with
an InertCap 5MS/NP capillary column (25 m ꢂ 0.25 mm i.d., 0.25 mm film, GL
Sciences). The conditions were as follows: injection, 1 lL, splitless, 60 s valve
time; injector temperature, 230 °C; carrier gas, He at 0.8 mL/min; transfer line
temperature, 280 °C; and electron energy, 70 eV. The temperature of the
column oven was programmed as follows: 150 °C for 3 min and then an
increase to 275 °C at 8.33 °C/min and kept at 275 °C for 5 min.
Buser, H. R.; Arn, H.; Guerin, P.; Rauscher, S. Anal. Chem. 1983, 55, 818.
Scribe, P.; Guezennec, J.; Dagaut, J.; Pepe, C.; Saliot, A. Anal. Chem. 1988, 60, 928.
7
8
.
.
Figure 4. Activities of compounds
expression in A. tumefaciens NTL4.
1 and 6 on AHL-responsive reporter gene

5
444
K. Kai et al. / Tetrahedron Letters 53 (2012) 5441–5444
9
.
Wagner-Döbler, I.; Thiel, V.; Eberl, L.; Allgaier, M.; Bodor, A.; Meyer, S.; Ebner,
S.; Hennig, A.; Pukall, R.; Schulz, S. ChemBioChem 2005, 6, 2195.
0. Krick, A.; Kehraus, S.; Eberl, L.; Riedel, K.; Anke, H.; Kaesler, I.; Graeber, I.;
Szewzyk, U.; König, G. M. Appl. Environ. Microbiol. 2007, 73, 3587.
1. Thiel, V.; Kunze, B.; Verma, P.; Wagner-Döbler, I.; Schulz, S. ChemBioChem 2009,
0, 479.
29.3, 30.7, 31.5, 36.1, 49.3, 66.1, 127.2, 132.0, 173.2, 175.5. Spectra of NMR and
EI-MS, see Supplementary data.
2
8
). 1H and 13
C
3
1
1
1
14. Spectroscopic data for synthetic
D
-1: ½
a
ꢃ
ꢀ11.9 ° (c 0.350, CHCl
D
NMR and EI-MS data were completely identical to those of
L
-1.
15. Chiral GC was performed with an InertCap CHIRAMIX capillary column
1
(10 m ꢂ 0.25 mm i.d., 0.25 mm film, GL Sciences). The conditions were as
1
2. H NMR data (400 MHz, CDCl
3
) for synthetic intermediates. Compound 9: d 1.50–
.61 (4H), 1.69–1.75 (2H, m), 1.82 (2H, quint, J = 7.1 Hz), 1.95 (1H, t, J = 2.6 Hz),
.32 (2H, dt, J = 7.1, 2.6 Hz), 3.46–3.56 (2H, m), 3.80–3.90 (2H, m), 4.60
follows: injection, 1 lL, splitless, 60 s valve time; injector temperature, 200 °C;
1
2
carrier gas, He at 2.0 mL/min; transfer line temperature, 200 °C; electron
energy, 70 eV; and column oven temperature, 180 °C.
(
1
1H, t, J = 3.4 Hz). Compound 10: d 0.90 (3H, t, J = 7.1 Hz), 1.26–1.39 (6H), 1.44–
.61 (6H), 1.78 (2H, quint, J = 6.8 Hz), 2.13 (2H, tt, J = 7.0, 2.3 Hz), 2.27 (2H, tt,
J = 6.8, 2.3 Hz), 3.35–3.53 (2H, m), 3.80–3.91 (2H, m,), 4.60 (1H, m). Compound
1: d 0.89 (3H, t, J = 7.1 Hz), 1.24–1.37 (4H), 1.47 (2H, quint, J = 7.1 Hz),
.12 (2H, tt, J = 7.1, 2.3 Hz), 2.44–2.64 (4H). Compound 12: d 0.89 (3H, t,
16. Other AHLs isolated or detected from the culture extract of M. alpina A-178
were also mixture of L- and D
-forms.3 The possibility of racemization of AHLs
during the isolation processes was ruled out by the model experiments with
synthetic enantiopure AHLs. We have no idea whether enantioimpure AHLs are
usual in Gram-negative bacteria, because, in most previous studies, the
1
2
J = 6.9 Hz), 1.24–1.38 (6H), 1.96–2.07 (2H, m), 2.33–2.44 (4H), 5.34 (1H, m),
.44 (1H, m).
3. Spectroscopic data for synthetic
400 MHz, CDCl ) d 0.89 (3H, t, J = 7.0 Hz), 1.24–1.38 (6H, m), 1.94–2.18 (2H,
m), 2.24–2.42 (4H, m), 2.83–2.90 (4H, m), 4.28 (1H, ddd, J = 11.6, 9.2, 5.9 Hz),
absolute configurations of natural AHLs were concluded to be
experimental supports.
17. Ahlgren, N. A.; Harwood, C. S.; Schaefer, A. L.; Giraud, E.; Greenberg, E. P. Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 7183.
18. Lindemann, A.; Pessi, G.; Schaefer, A. L.; Mattmann, M. E.; Christensen, Q. H.;
Kessler, A.; Hennecke, H.; Blackwell, H. E.; Greenberg, E. P.; Harwood, C. S. Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 16765.
L without any
5
2
8
). 1H NMR
1
L-1: ½
aꢃ
+ 13.2 ° (c 0.450, CHCl
3
D
(
3
4
.47 (1H, dt, J = 9.2, 1.0 Hz), 4.55 (1H, ddd, J = 11.7, 8.6, 5.6 Hz), 5.33 (1H, m),
13
5
3
.44 (1H, m), 6.07 (1H, br). C NMR (100 MHz, CDCl ) d 14.1, 22.6, 23.1, 27.2,