Synthesis of a proposed structure for the diffusible extracellular factor of Xanthomonas campestris pv. campestris

Article abstract of DOI:10.1016/j.tetlet.2010.02.088

A proposed structure for the diffusible extracellular factor (DF) of Xanthomonas campestris pv. campestris (Xcc) has been synthesized. Its MS spectrum and biological activity, however, contradict those of the natural product previously reported, suggesting that the structure for DF of Xcc must be reexamined.

Full text of DOI:10.1016/j.tetlet.2010.02.088

Tetrahedron Letters 51 (2010) 2074–2077
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Tetrahedron Letters
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Synthesis of a proposed structure for the diffusible extracellular factor of
Xanthomonas campestris pv. campestris
a
b
a
a
Arata Yajima a, , Nagisa Imai , Alan R. Poplawsky , Tomoo Nukada , Goro Yabuta
*
a Department of Fermentation Science, Faculty of Applied Biological Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
b Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A proposed structure for the diffusible extracellular factor (DF) of Xanthomonas campestris pv. campestris
(Xcc) has been synthesized. Its MS spectrum and biological activity, however, contradict those of the nat-
ural product previously reported, suggesting that the structure for DF of Xcc must be reexamined.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 13 January 2010
Revised 12 February 2010
Accepted 16 February 2010
Available online 19 February 2010
Keywords:
Quorum sensing
Xanthomonas campestris pv. campestris
Diffusible extracellular factor
Diffusible signal factor
Pheromone
Xanthomonas campestris is a bacterial species that is responsible
for a variety of plant diseases worldwide. Xanthomonas campestris
pv. campestris (Xcc) is the causal agent of black rot, which affects
crucifers such as Brassica and Arabidopsis. The pathogen produces
extracellular polysaccharides (EPS = xanthan gums) and enzymes
including proteases, pectinases and endoglucanases which are con-
sidered important virulence determinants.1 In 1997, Barber et al.
reported that the pathogenicity of Xcc is mediated by a small dif-
fusible signal factor (DSF), which restores production of protease,
endoglucanase and polygalacturonate lyase to rpfF mutants.2 The
DSF-mediated system was not considered a mechanism for den-
sity-dependent regulation, which is a quorum-sensing system.
The complete structure of the DSF was reported as (Z)-11-
methyl-2-dodecenoic acid (1) by Wang et al. (Fig. 1).3 Structure–
activity relationship studies suggest that the cis-double bond in 1
is the most important feature for DSF activity.3 Another similar
but completely separate diffusible extracellular factor (DF) en-
coded by the pigB gene of Xcc was reported by Chun et al. in
1997.4 They called it a pheromone, and it was reported to regulate
both EPS and xanthomonadin pigment production. Furthermore,
convincing genetic evidence showed DF to be involved in epiphytic
survival and concomitant host infection.5 The structure of the
pheromone was proposed as a butyrolactone derivative (2) based
on its MS spectrum (Fig. 1). Notably the two DSF-mediated systems
independently regulate pigment and endoglucanase production,
but both regulate EPS production. In the co-regulating EPS produc-
tion system, 2 has a stronger effect than 1. This unique system of
Xcc is summarized in Figure 1. The structure of 2 is similar to acyl
homoserine lactones (AHLs), which are well-known cell density-
dependent autoregulators among Gram-negative bacteria. How-
ever, there is no published evidence that DF is an autoinducer. Re-
cently, we have reported that the stereochemistry of the acyl side
chain in AHL from the symbiotic nitrogen-fixing bacterium Rhizo-
bium leguminosarum is very important for its biological activity.6
Because pheromone 2 has two stereogenic centers whose absolute
configuration remains unknown, we attempted to clarify the ste-
reochemistry-activity relationship of the pheromone. In order to
establish the absolute structure of the natural pheromone, we syn-
thesized possible stereoisomers of 2. This communication de-
scribes the synthesis of stereoisomers of 2 and the results of a
comparison of the physical and biological properties of the syn-
thetic compounds alongside the natural pheromone.
We anticipated that it might be possible to separate diastereo-
mers of the final product or synthetic intermediates, therefore, the
synthesis began with the preparation of a diastereomeric mixture
of 2 (Scheme 1) from the known compound 3.7 Optically active
(S)-3 (>99% ee) was converted into alcohol 4 upon addition of
methyl Grignard reagent to the corresponding aldehyde (ca. 1:1
ds). The unseparated diastereomers were used directly in subse-
quent reactions. Protection of the resultant hydroxyl group of 4
as a PMB ether8 and deprotection of the TBS group gave alcohol
5. After conversion of 5 into the corresponding iodide 6, known
79 was alkylated with 6 using potassium carbonate as the base to
give 8 in 85% yield. The ester group of 8 was hydrolyzed with
methanolic KOH, followed by careful acidification (pH 4) with
* Corresponding author.
E-mail address: ayaji@nodai.ac.jp (A. Yajima).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.02.088

A. Yajima et al. / Tetrahedron Letters 51 (2010) 2074–2077
2075
O
COOH
O
O
DSF (1)
DF (2)
Endoglucanase
EPS
Pigment
Figure 1. DF and DSF autoregulatory systems in Xcc.
a, b
c, d
e, f
TBSO
g
TBSO
h
HO
I
CO2Me
OH
3
OPMB
OPMB
(S)-3
4
5
6
i, j
O
O
O
O
O
2'
3
2'
CO2Me
OPMB
OPMB
O
O
8
(3RS, 2'S)-2
(3RS,2'S)-9
TBSO
O
O
(3RS,2'R)-2
O
O
CO2Me
3
2'
CO2Me
O
(R)-3
7
Scheme 1. Synthesis of diastereomeric mixtures of 2. Reagents and conditions: (a) DIBAL, CH2Cl2, ꢀ78 °C; (b) CH3MgBr, ether, 0 °C (61% in two steps); (c) PMBOC(@NH)CCl3,
Ph3CBF4, ether (90%); (d) TBAF, THF (90%); (e) p-TsCl, pyridine, 0 °C; (f) NaI, acetone, reflux (93% in two steps); (g) 7, K2CO3, acetone/DMF = 6:1, reflux (85%); (h) KOH aq, EtOH
then AcOH, 23–80 °C (84%); (i) DDQ, CH2Cl2/H2O = 20:1, 0 °C (77%); (j) DMP, CH2Cl2, 0 °C (82%).
acetic acid. Decarboxylation by refluxing the mixture gave lactone
9 in 84% yield. The PMB group of 9 was deprotected with DDQ, and
the resulting hydroxyl group was oxidized with Dess–Martin peri-
odinane to give the desired (3RS,20S)-2 as a diastereomeric mixture.
The overall yield of the synthesis of (3RS,20S)-2 was 21% in 10 steps
from 3. The (3RS,20R)-isomer was synthesized in the same manner
as described above starting from (R)-3. Although the products con-
sisted of a mixture of diastereomers, the NMR spectrum was con-
sistent with the proposed structure of synthetic 2. Unfortunately,
diastereoisomers of 2 or any of the synthetic intermediates were
difficult to separate by typical chromatographic methods. The ratio
of the diastereomers of synthetic 2 was determined to be ca. 2:1
using NMR and GC analyses. However, the relative configuration
of each isomer could not be determined. GC analyses of synthetic
2 with a chiral stationary phase showed the C-20 methyl group
was stereochemically pure (>99%).10
In an effort to determine the relative configuration of the diaste-
reomers, stereoselective synthesis of 2 was undertaken, as shown
in Scheme 2. Dimethyl malonate was alkylated with iodide (R)-6
to give 10 in 94% yield. After the two ester groups were hydrolyzed
using excess KOH under reflux, careful acidification (pH 4) with
acetic acid and decarboxylation of the resulting di-acid under
Me2OC
a
b
c, d
I
MeO2C
HOOC
OPMB
Bn
OPMB
OPMB
(R)-6
11
10
Bn
e
f, g
h, i
O
O
N
O
N
O
3
2'
O
O
O
O
O
OPMB
OPMB
O
OPMB
(3R,2'S)-9
13
14
Bn
NH
(R)-12
I
O
O
3
2'
3
2'
O
OPMB
(S)-6
O
O
O
(3R,2'S)-2
(3S,2'S)-2
(S)-12
Scheme 2. Synthesis of (3R,20S)- and (3S,20S)-2. Reagents and conditions: (a) dimethyl malonate, K2CO3, acetone/DMF = 6:1, reflux (94%); (b) (i) 1 M KOH in MeOH, 75 °C then
acidification with AcOH (ii) toluene, reflux (88%); (c) PivCl, Et3N, toluene, 0 °C; (d) LiHMDS, (S)-12, THF (74% in two steps); (e) NaHMDS, allyl iodide, THF, ꢀ78 °C (78%); (f)
OsO4, 2,6-lutidine, NaIO4, t-BuOH/H2O = 3:1; (g) NaBH4, MeOH (59% in two steps); (h) DDQ, CH2Cl2/H2O = 20:1, 0 °C (83%); (i) DMP, CH2Cl2, 0 °C (quant).

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A. Yajima et al. / Tetrahedron Letters 51 (2010) 2074–2077
Figure 2. MS spectrum of synthetic 2.
Table 1
reflux in toluene gave acid 11 in two steps (88% yield). The PMB
group of 11 was relatively labile therefore, the decarboxylation
process was carried out under controlled conditions. Acid 11 was
condensed with Evans oxazolidinone (S)-12,11 and the resulting
13 was diastereoselectively alkylated using allyl iodide to give
14. Oxidative cleavage of the terminal olefin of 14 with OsO4 and
NaIO4, and subsequent reduction of the resulting aldehyde with
NaBH4 gave (3R,20S)-9 with spontaneous lactone formation. Treat-
ment with DDQ followed by Dess–Martin oxidation of the resulting
alcohol afforded the desired (3R,20S)-2.12 (3S,20S)-2 was synthe-
sized in the same manner from (S)-6 using (R)-12 as the chiral aux-
iliary.12 GC analyses of the synthetic samples using a chiral
stationary phase showed that the diastereoselectivity of the Evans
alkylation of (4S)- and (4R)-13 with allyl iodide was 96:4 and
>99:1, respectively.10 The difference in 1H and 13C NMR spectra
of the two isomers distinguished the major isomer in the diaste-
reomeric mixtures, (3RS,20S)- and (3RS,20R)-2, as the syn-isomer
(Scheme 1). The lactone ring of 9 was formed under thermal
conditions, therefore, the major syn-isomer is expected to be the
thermodynamically favored isomer, consistent with higher diaste-
reoselectivity in Evans alkylation giving the syn-isomer, (20R,40S)-
14.
Selected MS data for synthetic 2 and reported natural DF
Fragment ion (relative intensity, %)
Natural DF
Synthetic 2
170 (M+, 100)
155 (3.7)
140 (12.3)
128 (5.6)
170 (M+, 2.7)
a
—
—
a
128 (17.8)
a
112 (5.4)
—
a
—
99 (8.1)
86 (85.1)
86 (80.0)
72 (14.9)
a
—
69b
—
a
a,c
—
—
55 (52.4)
43 (100)
a,c
a
Not observed.
Relative intensity was not reported.
MS data below m/z 60 were not reported.
b
c
as originally proposed by Chun et al. Previous work indicates that
DF is probably not an AHL, and probably is a butyrolactone. AHLs
from several other bacterial genera were unable to restore pigB
mutant strains for pigment and EPS production,4,13 and several
Streptomyces strains which produce butyrolactones (but not AHLs)
were able to restore the pigB mutant strain for both these traits.14
Re-investigation of the structure of DF of Xcc is currently
underway.
The 1H and 13C NMR spectra of natural DF are unavailable due
to the scarcity of the natural product; only the MS spectrum of DF
has been reported.4 To compare the MS spectrum of synthetic 2
with that of the natural product, the MS spectra of the four ster-
eoisomers of 2 were recorded using GC–MS under similar condi-
tions as reported for the natural compound (Fig. 2). The MS
spectra of the four stereoisomers were identical, but none were
Acknowledgment
in agreement with that reported for the natural compound.4
A
The authors thank Dr. T. Tashiro (RIKEN) for MS measurements.
References and notes
comparison of MS data for synthetic 2 with that of the reported
natural DF is shown in Table 1. Mass spectra of 2 acquired under
various conditions, including both LC–MS and GC–MS in positive
and negative modes, also disagreed with the data reported for the
natural compound.
1. (a) Dow, J. M.; Scofield, G.; Trafford, K.; Turner, P. C.; Daniels, M. J. Physiol. Mol.
Plant Pathol. 1987, 31, 261–271; (b) Dums, F.; Dow, J. M.; Daniels, M. J. Mol. Gen.
Genet. 1991, 229, 357–364.
Next, synthetic (3RS,20S)-2 and (3RS,20R)-2 were subjected to a
biological assay according to the reported procedure.4 The com-
pounds were tested at seven different concentrations, varying from
0.64 to 10,000 mg/ml. No effects on pigment or EPS production of
the test strain were observed with either synthetic compound,
regardless of the concentration used. These results strongly sug-
gest that natural DF does not correspond to the structure of 2.
In summary, we report the synthesis of a diastereomeric mix-
ture and the stereochemically pure form of a proposed compound
for the diffusible extracellular factor of Xcc. However, the results of
the MS spectra and biological activities of the natural product and
the synthetic compounds were inconsistent. This finding clearly
shows that the structure proposed for DF of Xcc must be revised.
In this study, an intense peak at 86 was observed in the MS spectra
of both the natural product and synthetic 2 (Table 1), indicating
that the natural DF possesses at least a butyrolactone structure
2. Barber, C. E.; Tang, J. L.; Feng, J. X.; Pan, M. Q.; Wilson, T. J. G.; Slater, H.; Dow, J.
M.; Williams, P.; Daniels, M. J. Mol. Microbiol. 1997, 24, 555–566.
3. Wang, L. H.; He, Y.; Gao, Y.; Wu, J. E.; Dong, Y. H.; He, C.; Wang, S. X.; Weng,
L. X.; Xu, J. L.; Tay, L.; Fang, R. X.; Zhang, L. H. Mol. Microbiol. 2004, 51, 903–
912.
4. Chun, W.; Cui, J.; Poplawsky, A. R. Physiol. Mol. Plant Pathol. 1997, 51, 1–14.
5. Poplawsky, A. R.; Chun, W. Mol. Plant Microbe Interact. 1998, 11, 466–475.
6. Yajima, A.; van Brussel, A. N.; Schripsema, J.; Nukada, T.; Yabuta, G. Org. Lett.
2008, 10, 2047–2050.
7. Mori, K.; Koseki, K. Tetrahedron 1988, 44, 6013–6020.
8. Nakajima, N.; Horita, K.; Abe, R.; Yonemitsu, O. Tetrahedron Lett. 1988, 29,
4139–4142.
9. Quesada, M. L.; Schlessinger, R. H. J. Org. Chem. 1978, 43, 346–347.
10. Conditions of GC analysis; column: CP-Chirasil-Dex CB (0.25 mm ꢁ 25 m),
carrier gas He (50 kPa), 10–180 °C (3 °C/min), tR(3S,20R) = 23.7, tR(3R,20S) = 24.0,
tR(3R,20R) = 25.3 and tR(3S,20S) = 25.6.
11. Evans, D. A.; Weber, A. E. J. Am. Chem. Soc. 1986, 108, 6757–6761.
12. Properties of synthetic 2. (a) (3R,20S)-isomer: colorless oil; ½a 2D2
ꢀ10.4 (c 0.14,
ꢂ
CHCl3). IR mmax (film) cmꢀ1: 1711 (s), 1767 (s). 1H NMR (400 MHz, CDCl3):
d = 1.15 (d, J = 7.3 Hz, 3H, 20-CH3), 1.75 (ddd, J = 5.4, 7.3, 12.7 Hz, 1H, 10-H),

A. Yajima et al. / Tetrahedron Letters 51 (2010) 2074–2077
2077
1.85ꢀ1.97 (m, 2H, 10-H, 4-H), 2.20 (s, 3H, 40-CH3), 2.39 (m, 1H, 4-H), 2.49 (m,
1H, 3-H), 2.95 (m, 1H, 20-H), 4.17 (ddd, J = 6.5, 9.4, 16.0 Hz, 1H, 5-H), 4.33 (ddd,
J = 3.1, 8.3, 16.0 Hz, 5-CHH). 13C NMR (100 MHz, CDCl3): d = 17.2, 28.6, 29.6,
33.2, 37.2, 44.5, 66.5 (C-5), 179.2 (C-30), 212.0 (C-2). HREIMS m/z [M]+: calcd for
14.3 Hz, 1H, 10-H), 2.39 (m, 1H, 4-H), 2.52 (m, 1H, 3-H), 2.79 (sxt, J = 7.0 Hz, 20-
H), 4.17 (ddd, J = 6.5, 9.4, 16.0 Hz, 1H, 5-H), 4.39 (ddd, J = 2.8, 8.7, 16.0 Hz, 1H,
5-H). 13C NMR (100 MHz, CDCl3): d = 16.7, 27.7, 29.4, 33.1, 37.0, 44.7, 66.4 (C-
5), 178.9 (C-30), 211.6 (C-2). HREIMS m/z [M]+: calcd for C9H14O3, 170.0943;
found, 170.0947.
C9H14O3, 170.0943; found, 170.0937. (b) (3S,20S)-isomer: colorless oil; ½a 2D2
ꢂ
ꢀ23.5 (c 0.12, CHCl3). IR mmax (film) cmꢀ1
:
1712 (s), 1767 (s). 1H NMR
13. Poplawsky, A. R.; Chun, W. J. Bacteriol. 1997, 179, 439–444.
14. Poplawsky, A. R.; Walters, D. M.; Rouviere, P. E.; Chun, W. Mol. Plant Pathol.
2005, 6, 653–657.
(400 MHz, CDCl3): d = 1.19 (d, J = 7.0 Hz, 3H, 20-CH3), 1.49 (ddd, J = 7.0, 8.2,
14.3 Hz, 1H, 10-H), 1.90 (m, 1H, 4-H), 2.18 (s, 3H, 40-CH3), 2.24 (ddd, J = 7.0, 7.9,