An efficient synthesis of the precursor of AI-2, the signalling molecule for inter-species quorum sensing

Article abstract of DOI:10.1016/j.bmc.2010.12.036

Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. A synthesis of (S)-4,5-dihydroxypentane-2,3-dione (DPD), the precursor of AI-2, is described starting from methyl glycolate. The key step was an asymmetric reduction of a ketone with (S)-Alpine borane. This new method was highly reproducible affording DPD for biological tests without contaminants. The biological activity was tested with the previously available assays and compared with a new method using an Escherichia coli reporter strain thus avoiding the use of the pathogenic Salmonella reporter.

Full text of DOI:10.1016/j.bmc.2010.12.036

Bioorganic & Medicinal Chemistry 19 (2011) 1236–1241
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
An efficient synthesis of the precursor of AI-2, the signalling molecule
for inter-species quorum sensing
Osvaldo S. Ascenso a,b, João C. Marques b, Ana Rita Santos a,b, Karina B. Xavier a,b, M. Rita Ventura a,
Christopher D. Maycock a,c,
⇑
a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2780-901 Oeiras, Portugal
b Instituto Gulbenkian de Ciência, 2781-901 Oeiras, Portugal
c Faculdade de Ciências da Universidade de Lisboa, Departamento de Química e Bioquímica, 1749-016 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. A synthesis of
(S)-4,5-dihydroxypentane-2,3-dione (DPD), the precursor of AI-2, is described starting from methyl
glycolate. The key step was an asymmetric reduction of a ketone with (S)-Alpine borane. This new
method was highly reproducible affording DPD for biological tests without contaminants. The biological
activity was tested with the previously available assays and compared with a new method using an
Escherichia coli reporter strain thus avoiding the use of the pathogenic Salmonella reporter.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 20 August 2010
Revised 14 December 2010
Accepted 14 December 2010
Available online 21 December 2010
Keywords:
DPD
AI-2
Quorum sensing
Enantioselective reduction
13C-DPD
1. Introduction
Thus, using AI-2 bacteria are able to detect the presence of other
bacterial species in their vicinity and regulate gene expression
(S)-4,5-Dihydroxypentane-2,3-dione (DPD) 1 is the uncyclized
precursor of AI-2, a signalling molecule for bacterial inter-species
communication.1,2 DPD is a reactive 1,2-diketone which in aqueous
medium forms an equilibrium mixture of the linear form and two
anomeric cyclic forms 2 and 3, their hydrated versions 4 and 5
(Scheme 1).3 The hydrated isomer 4 can exist also as its 2,3-borate
diester 6. It has been shown that distinct bacteria can detect differ-
ent forms of this molecule and thus all these forms are known
collectively as AI-2. Specifically, many Vibrio have the LuxP-type
of AI-2 receptor which recognizes 6,4 whereas members belonging
to phylogenetically distinct families such as the human pathogens
Salmonella typhimurium and Bacillus anthracis as well as the plant
symbiont Sinorhizobium meliloti contain the LsrB-type of receptors
and recognize the non-borated diastereoisomer 5.3,5
Bacterial populations use cell–cell communication in order to
coordinate their behaviour and function in such a way that they
can adapt to changing environments. Chemical communication
among bacteria is called ‘quorum sensing’. Examples of quorum
sensing behaviours are biofilm formation, virulence-factor expres-
sion, antibiotic production and bioluminescence.6 AI-2 is unique in
that it is produced and detected by a wide variety of bacteria.2,7
according to the species composition in the environment.8
Ultimately, the understanding of the molecular mechanisms that
bacteria use to regulate their behaviours can lead to the develop-
ment of new therapies to control bacterial infections, and also to
develop biotechnological applications for the control of industrial
scale production of beneficial bacterial products, such as antibiot-
ics or recombinant proteins.
Although
a small molecule, DPD is highly functionalised,
optically active and highly reactive. This presents a significant
synthetic challenge and the production of reasonable quantities
presents many problems. The absence of an accessible synthesis
of DPD has been a major drawback in studies aimed at the
understanding of AI-2 quorum sensing.7 In this work we describe
an efficient and economic method to synthesise DPD on a reason-
able scale. We have also developed a simple procedure to remove
the last protective group thus furnishing uncontaminated DPD
solutions.
2. Results and discussion
Currently, there are five published9–13 syntheses of DPD but all
have problems and hence were considered unsuitable for the rou-
tine production of larger quantities required for biological studies.
These routes generally use optically active starting materials and in
⇑
Corresponding author.
E-mail address: maycock@itqb.unl.pt (C.D. Maycock).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.036

O. S. Ascenso et al. / Bioorg. Med. Chem. 19 (2011) 1236–1241
1237
OH
HO OH
O
HO O
B
HO
HO
OH
HO
H2O
OH
O
OH
O
O
4
O
6
O
HO
O
2
HO
O
HO OH
1
HO
HO
H2O
OH
OH
O
O
5
3
Scheme 1. Forms of DPD/AI-2.
most cases glyceraldehyde, derived from sugars. Each presented
chemical or practical problems and we reasoned that a route in
which the asymmetric centre was produced by the asymmetric
reduction of a ketone could resolve many of them and would allow
us to build up the carbon skeleton from two simple units
(Scheme 2), making the synthesis efficient in terms of atom econ-
omy, and flexible for the production of analogues and isomers.
Protection of the hydroxyl group of methyl glycolate with
tert-butyldiphenylsilyl chloride (TBDPSCl),14 followed by hydroly-
sis of the methyl ester with LiOH in THF/H2O afforded carboxylic
acid 914 in 94% yield. Without purification 9 was converted into
amide 1014 using N,O-dimethylhydroxylamine hydrochloride and
DCC in dichloromethane, in 85% yield. Direct formation of the
Weinreb amide 10 from the glycolate ester afforded low yields of
the product. Conversion of amide 10 to the acetylenic ketone 11
was easily accomplished by using 1-propynyl lithium (85%
yield).15All of these intermediates could be prepared on a large
scale and were stable compounds.
of the two diastereomers formed. Diol 13 was obtained as a
crystalline solid in 86% yield after cleavage of the silyl ether with
TBAF in THF. Recrystallisation from hexane/ethyl acetate increased
the ee of the product to >98%, ½a D20
ꢁ18.4 (c 0.62, CH2Cl2), mp
ꢀ
73.4–74.6 °C, as determined by chiral HPLC analysis of the
dibenzoate 16 (Scheme 2).
Reprotection of diol 13 with a cyclohexylidene group afforded
alkyne 14, ½a 2D0
ꢀ
ꢁ41.4 (c 1.16, CHCl3), Lit.10
½
a 2D0
ꢀ
ꢁ39.2 (c 0.767,
CHCl3). Oxidation of 14 with RuO2/NaIO4 as described by
Semmelhack10 afforded protected DPD 15 in 81% yield, ½a 2D0
ꢀ
ꢁ11.2 (c 1.45, CHCl3). Lit.10
½
a 2D0
ꢀ
ꢁ11.8 (c 0.900, CHCl3). This
method had the very important advantage that the acetylenic com-
pound 14 was obtained very cleanly. In our hands, methylation of
the terminal acetylene with MeI (Semmelhack)10 was not always
complete, particularly when scaling up, and the desired product
was contaminated by the unreacted terminal alkyne, which had
the same Rf and was not separable by normal chromatography.
Furthermore, oxidation of this mixture afforded a product contain-
ing acidic material and after purification low yields of the required
product 14 were obtained. Ruthenium mediated oxidation of the
pure alkyne 14, after a flash filtration through a small pad of silica
gel to remove ruthenium residues, cleanly afforded dione 15.
Treatment of 11 with (S)-Alpine borane16 afforded alcohol 12
with the required (R) configuration in 67% yield. The enantiomeric
excess (ee) of 12 was 86%, ½a D20
ꢀ
+8.85 (c 1.57, CH2Cl2), as deter-
mined by esterification with (S)-MTPCl and proton NMR analysis
a
b
TBDPSO
CO2H
e
TBDPSO
Me
CO2Me
d
HO
CO2Me
9
8
7
c
N
TBDPSO
TBDPSO
OMe
O
O
11
10
h
f
O
RO
TBDPSO
O
OR
OH
O
12
14
13: R=H
g
16: R=Bz
O
O
HO
O
HO
i
j
O
OH
HO
O
O
15
O
1
+ hydrated isomers
Scheme 2. Synthesis of DPD. Reagents and conditions: (a) TBDPSCl, Pyr, DMAP, rt, 97%; (b) LiOH, THF/H2O, rt, 94%; (c) HNMeOMeꢂCl, DCC, CH2Cl2, rt, 4 85%; (d) BuLi, propyne,
THF, ꢁ78 °C/0 °C, 95%; (e) (S)-Alpine borane, THF, rt, 67%; (f) TBAF, THF, rt, 86%; (g) BzCl, (iPr)2NEt, DMAP, CH2Cl2, 93%; (h) 1,1-dimethoxycyclohexanone, H2SO4, DMF, 91%; (i)
NaIO4, RuO2, CCl4/MeCN, rt, 86%; (j) Dowex 50WX8, H2O, pH 3, rt.

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O. S. Ascenso et al. / Bioorg. Med. Chem. 19 (2011) 1236–1241
ent-DPD was obtained by the same route using (R)-Alpine bor-
3. Conclusion
ane to reduce the acetylenic ketone 11. Oxidation of Ent-14: ½a D20
ꢀ
+40.5 (c 1.68, CHCl3), Lit.10
½
a 2D0
+39.4 (c 0.796, CHCl3) afforded
ꢀ
In conclusion, (R)- and (S)-DPD were obtained in 8 steps with a
33% overall yield and >98% ee using a common strategy. Synthetic
(S)-DPD proved to have the same biological activity as that of the
enzymatically produced DPD. All intermediates were easy to han-
dle and presented low volatility and excellent stability, the reac-
tions were very reproducible and afforded high yields. Liberation
of DPD from acetal 15 with Dowex resin and washing with chloro-
form allowed us to obtain a final DPD preparation devoid of inor-
ganic salts and organic impurities. With slight modification to
this scheme 5-[13C]-labelled DPD could be produced. This synthesis
also allows the production of novel DPD analogues.
Ent-15: ½a 2D0
ꢀ
+9.7 (c 1.83, CHCl3), Lit.10
½
a 2D0
+11.5 (c 0.733, CHCl3).
ꢀ
A rotation lower than that previously reported is explained by
partial hydration of the dione. The preparation of 13C-labelled
DPD by this method using 3-13C propyne17 is also envisioned.
Up to 0.5 g batches of 15 could be obtained by this method and
stored neat in this form. Scale up problems were not encountered
and larger batches should be possible.
A hydrolysis protocol to prepare aqueous solutions of DPD 1
from the acetal 15 was developed for the generation of small quan-
tities for NMR and biological studies. This consisted of exposing an
aqueous solution of acetal 15 to an aqueous suspension of acidic
Dowex 50WX8 resin (pH 3) for a short period followed by a simple
filtration to remove the resin. Washing the resulting aqueous solu-
tion of DPD 1 with chloroform removed the cyclohexanone. This
important step facilitates enzyme studies and NMR analysis of
DPD 1, DPD was thus obtained uncontaminated by inorganic salts
and the only salts present during the biological studies were
10 mM phosphate buffer.
The biological activity of the synthetic DPD was compared to
that of the enzymatically produced compound (Figs. 1 and 2).
Two methods that detect the borated form of AI-2 6 were used:
the in vivo method using the Vibrio harveyi reporter strain which is
the most commonly used method, and an in vitro method with
LuxP-FRET.18 By both methods activation by DPD synthesized by
the new synthesis was the same as for enzymatic DPD.
To test the biological activity of DPD with an assay for the
un-borated isomer of AI-2 5 we engineered anEscherichia coli strain
to be used as reporter because this bacterium contains the LsrB
receptor which recognizes 55 and induction of the expression of
the Lsr system is AI-2-dependent.19,20 As shown in Figure 2A using
this assay the synthetic and enzymatic DPD had the same activi-
ties. The use of E. coli for assaying this activity in vivo instead of
the S. typhimurium reporter strain used in previous studies3 has
the advantage of not requiring the use of a pathogen.
Previous studies have shown that the unnatural ent-DPD is a
weak agonist in the assays for the borated-AI-2 form 6 (Fig. 1),10,21
we have observed that the same is true for the assay for the isomer
5 (Fig. 2B). To induce the E. coli reporter, six times more ent-DPD than
DPD was necessary in order to obtain the same activity.
4. Experimental
4.1. General
1H NMR spectra were obtained at 400 MHz in CDCl3 or D2O with
chemical shift values (d) in ppm downfield from tetramethylsilane
in the case of CDCl3, and 13C NMR spectra were obtained at
100.61 MHz in CDCl3 or D2O. Assignments are supported by 2D
correlation NMR studies. Flash column chromatography: silica gel
Merck 60, 0.040–0.063 mm (230–400 mesh ASTM). Analytical
TLC: Aluminium-backed silica gel Merck 60 F254. Specific rotations
(½a 2D0
ꢀ
) were measured using an automatic polarimeter. Reagents
and solvents were purified and dried according to Ref. 22. All the
reactions were carried out in an inert atmosphere (argon), except
when the solvents were undried. The enantiomeric excesses were
determined by HPLC on a Waters 600E/U6K instrument using a
Daicel Chiralpack AD-H column.
4.1.1. Methyl 2-(tert-butyldiphenylsilyloxy)acetate 8
To a solution of methyl glycolate (2.5 g, 27.7 mmol) in pyridine
(10 mL) was added tert-butyldiphenylsilyl chloride (8.4 g,
30.5 mmol). A catalytic amount of DMAP was added at 0 °C, and
the reaction was stirred overnight at room temperature. H2O was
then added and the resulting mixture was extracted with CH2Cl2
(3 ꢃ 10 mL), the organic phase was dried with MgSO4, concen-
trated under vacuum and purified by flash chromatography (5:95
EtOAc/hexane) to give 8 as a colourless oil (8.86 g, 97% yield).
Figure 1. Dose–response curves for the borated form of AI-2 6 mediated by LuxP. Bioluminescence produced by V. harveyi strain MM32 (A) and FRET ratio of the LuxP-FRET
protein (B) were assayed following the addition of DPD produced by the new synthesis (squares), DPD produced enzymatically (triangles), and the ent-DPD (circles). Error
bars represent the standard deviation of three independent trials.

O. S. Ascenso et al. / Bioorg. Med. Chem. 19 (2011) 1236–1241
1239
Figure 2. Bioactivity of the non-borated form of AI-2 5 mediated by LsrB. Expression of the lsr-lacZ promoter fusion in E. coli strain KX1446 was assayed by determining the
b-galactosidase activity following the addition of synthetic or enzymatically produced DPD, the ent-DPD (sample concentration of 40 M was used), and no ligand as negative
l
control (A). A dose–response curve with DPD (squares) and ent-DPD (circles) for the synthetic compound is shown in (B). Error bars represent the standard deviation of three
independent cultures.
1H NMR (400 MHz, CDCl3): d 7.69–7.68 (m, 4H), 7.43–7.37 (m, 6H),
4.25 (s, 2H), 3.68 (s, 3H), 1.09 (s, 9H). 13C NMR (100 MHz, CDCl3): d
171.7, 135.6, 132.8, 129.9, 127.8, 62.1, 26.7, 19.3. FT-IR (film): 1760
(C@O). M/z 271.0 (M+ꢁtBu), 251.1 (M+ꢁPh). Elemental Anal. Calcd:
C, 69.47; H, 7.36. Obtained: C, 69.50; H, 7.08.
At ꢁ78 °C, Weinreb amide 10 (6.5 g, 18.2 mmol), previously dis-
solved in THF (10 mL), was added. The mixture was stirred for
20 min at ꢁ78 °C and then at 0 °C until all starting material had
been consumed (TLC). Saturated NH4Cl solution (3 ꢃ 30 mL) was
added and the resulting mixture was extracted with dichlorometh-
ane, dried with anhydrous MgSO4, concentrated under vacuum and
purified by flash chromatography (30:70 EtOAc/hexanes) to afford
11 as white crystals (5.79 g, 95% yield). Mp = 50–51 °C. 1H NMR
(400 MHz, CDCl3): d 7.68–7.67 (m, 4H), 7.44–7.37 (m, 6H), 4.30
(s, 2H), 1.99 (s, 3H), 1.10 (s, 9H). 13C NMR (100 MHz, CDCl3): d
186.0, 135.6, 132.8, 129.9, 127.9, 93.2, 78.3, 70.5, 26.7, 19.3, 4.2.
FT-IR (film); 1695 (C@O), 2219 (C„C). M/z 279.0 (M+ꢁtBu), 259.1
(M+ꢁPh). Elemental Anal. Calcd: C, 74.96; H, 7.19. Obtained: C,
74.90; H, 7.45.
4.1.2. 2-(tert-Butyldiphenylsilyloxy)acetic acid 9
To ester 8 (8.86 g, 27.0 mmol) in THF/H2O (4:1) (33 ml), was
added LiOH 0.75 M (97 mL, 72.9 mmol) and the mixture was stir-
red until all starting material was consumed (TLC). H2O was added
and the resulting mixture was extracted with ethyl ether
(3 ꢃ 40 mL). The aqueous phase was acidified with HCl 10%
(pH P 3), extracted with ethyl acetate (3 ꢃ 50 mL), the combined
organic phases dried with MgSO4, filtered and concentrated under
vacuum to give 9 which was pure by NMR (7.97 g, 94% yield) as a
colourless oil. 1H NMR (400 MHz, CDCl3): d 7.68–7.65 (m, 4H),
7.46–7.38 (m, 6H), 4.25 (s, 2H), 1.10 (s, 9H). 13C NMR (100 MHz,
CDCl3): d 174.9, 135.5, 132.9, 130.2, 128.0, 61.9, 26.7 19.2. M/z
237.1 (M+ꢁPh). FT-IR (film): 1737 (C@O). Elemental Anal. Calcd:
C, 68.75; H, 7.05. Obtained: C, 68.70; H, 6.91.
4.1.5. (R)-1-(tert-Butyldiphenylsilyloxy)pent-3-yn-2-ol 12
Ketone 11 (3 g, 2.97 mmol) was dissolved in THF (15 mL) at
room temperature. (S)-Alpine Borane 0.5 M in THF (26.7 mL,
4.45 mmol) was added and the mixture was stirred for 48 h.
Saturated NH4Cl solution (14 mL) was added and the resulting
mixture was extracted once with ethyl ether (15 mL) and then
with dichloromethane (2 ꢃ 15 mL), dried with anhydrous MgSO4,
concentrated under vacuum and purified by flash chromatography
4.1.3. N-Methoxy-N-methyl-2-(tert-
butyldiphenylsilyloxy)acetamide 10
Acid 9 (7.34 g, 23.3 mmol) and N,O-dimethylhydroxylamine
hydrochloride (6.83 g, 69.9 mmol) were dissolved in CH2Cl2
(20 mL) at room temperature. To this mixture was added, slowly,
DCC (14.4 g, 69.9 mmol) in CH2Cl2 (23 mL) and then refluxed until
all the starting material had been consumed (TLC). After cooling to
room temperature, H2O was added, the resulting mixture was
extracted with ethyl acetate (3 ꢃ 30 mL), the extracts dried with
anhydrous MgSO4, concentrated under vacuum and purified by
flash chromatography (30:70 EtOAc/hexanes) to afford 10 as white
crystals (7.10 g, 85% yield). Mp = 53.4–54.8 °C. 1H NMR (400 MHz,
CDCl3): d 7.74–7.72 (m, 4H), 7.42–7.37 (m, 6H), 4.43 (s, 2H), 3.43
(s, 3H), 3.13 (s, 3H), 1.10 (s, 9H). 13C NMR (100 MHz, CDCl3): d
180.4, 135.5, 133.1, 129.8, 127.7, 62.0, 61.2, 32.5, 26.7, 19.4. FT-IR
(KBr): 1691 (C@O). M/z 300.4 (M+ꢁtBu). Elemental Anal. Calcd: C,
67.19; H, 7.61; N, 3.92. Obtained: C, 67.30; H, 7.23; N, 4.30.
(5:95 AcOEt/hexane) to afford 12 (2.02 g, 67% yield, 86% ee). ½a D20
ꢀ
+8.85 (c 1.57, CH2Cl2). 1H NMR (400 MHz, CDCl3): d 7.70–7.65
(m, 4H), 7.44–7.36 (m, 6H), 4.45–4.42 (m, 1H), 3.77 (dd, J = 3.7,
10.1 Hz, 1H), 3.68 (dd, J = 7.5, 10.3 Hz, 1H), 1.81 (d, J = 2.2, 3H),
1.07 (s, 9H). 13C NMR (100 MHz. CDCl3): d 135.6, 135.5, 133.0,
132.9, 129.9, 129.8, 127.8, 127.7, 82.0, 76.9, 67.9, 63.4, 41.9, 26.8,
19.3, 3.6. FT-IR (film): 3571 (OH), 2244 (C„C). Elemental Anal.
Calcd: C, 74.51; H, 7.74. Obtained: C, 74.70; H, 7.69.
To determine the enantioselectivity of the reaction, a small
sample of alcohol 12 was treated with (R)-MTPA-Cl (3 equiv) and
DMAP (3 equiv) in CH2Cl2 at room temperature. Integration of 1H
NMR (400 MHz, CDCl3) peaks at d 1.79 (d, J = 2.1 Hz, major) and
1.75 (d, J = 1.9 Hz, minor) ppm indicated a dr of 93:7 corresponding
to 86% ee.
The same procedure using (R)-Alpine Borane afforded ent-12:
½
a 2D0
ꢀ
ꢁ8.55 (c 1.38, CH2Cl2).
4.1.4. 1-(tert-Butyldiphenylsilyloxy)pent-3-yne-2-one 11
To a solution of 1.45 M propyne in THF (16 mL, 22.5 mmol) was
added Buli 1.6 M in hexanes (13.3 mL, 21.2 mmol) dropwise from a
syringe at ꢁ78 °C and the mixture stirred at 0 °C over 30 min.
4.1.6. (R)-Pent-3-yne-1,2-diol 13
To the alcohol 12 (2 g, 5.91 mmol) in THF (10 mL) was added
TBAF 1 M in THF (7.09 mL, 7.09 mmol) at room temperature.

1240
O. S. Ascenso et al. / Bioorg. Med. Chem. 19 (2011) 1236–1241
The reaction mixture was stirred for 1 h after which all the starting
material had been consumed (TLC). The resulting mixture was
extracted with ethyl acetate (3 ꢃ 10 mL), concentrated under vac-
uum and purified by flash chromatography (70:30 AcOEt/hexane)
to give 13 as white crystals, after recrystallisation from AcOEt/
filtration. Unprotected DPD was then neutralized with 10 mM
potassium phosphate buffer, pH 7.0. The same procedure was used
to obtain ent-DPD ent-1. To determine the concentration of these
samples DPD and ent-DPD were quantified by 1H NMR using formate
as an internal reference. Spectra were acquired with full relaxation.
To remove the released cyclohexanone liquid–liquid extraction
with an equal volume of deuterated chloroform was carried out.
DPD obtained enzymatically was produced and quantified as
previously described.23
hexane 10 mL/5 mL (0.509 g, 86% yield, 98% ee). ½a D20
ꢁ18.4 (c
ꢀ
0.62, CH2Cl2). Mp = 73.4–74.6 °C. 1H NMR (400 MHz, CDCl3): d
4.44–4.40 (m, 1H), 3.71 (dd, J = 3.8, 11.3, 1H), 3.64 (dd, J = 6.5,
11.3, 1H), 1.80 (d, J = 2.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): d
82.4, 76.9, 66.7, 63.4, 3.5. FT-IR (KBr): 3603 (OH), 2305 (C„C). Ele-
mental Anal. Calcd: C, 59.98; H, 8.05. Obtained: C, 59.80; H, 8.15.
4.2. Bacterial strains and media
The same procedure afforded ent-13: ½a D20
ꢀ
+17.7 (c 1.37, CH2Cl2).
In order to determine the improved ee of the crystallised diol 13
(0.008 g, 0.08 mmol) it was converted to its dibenzoate 16 by treat-
ment with N-ethyldiisopropylamine (0.055 mL, 0.32 mmol) and
benzoyl chloride (0.027 mL, 0.24 mmol), in the presence of a cata-
lytic amount of DMAP, in CH2Cl2 (0.5 mL). The optical purity was
determined by HPLC chromatography, using a Chiralcel AD-H
column (0.5 mL/min flow in 5:95 isopropanol/hexane) to be 98%
V. harveyi MM32 (luxN::Cm, luxS::Tn5Kan) was obtained from
the laboratory of Bonnie Bassler.3 E. coli strain KX1446 (lsr-lacZ,
D
luxS, lsrFG::Cm) was constructed by replacing the lsrFG genes in
KX129017 by chloramphenicol resistance cassettes as described
previously24 using primers with 50 bp of homology to the flanking
regions of lsrF and lsrG.
(ent-13 Rf = 19.0 min, 13 Rf = 20.2 min). Dibenzoate 16:
½
a 2D0
ꢀ
4.3. DPD biological activity
ꢁ49.3 (c 0.98, CH2Cl2). 1H NMR (400 MHz, CDCl3): d 8.07–8.00
(m, 4H), 7.56–7.52 (m, 2H), 7.45–7.39 (m, 4H), 5.96–5.94 (m,
1H), 4.64 (d, J = 5.8 Hz, 2H), 1.88 (d, J = 2.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3): d 166.0, 165.5, 133.3, 133.2, 129.9, 129.7,
129.63, 129.61, 128.4, 83.9, 73.1, 65.4, 62.9, 3.7. Dibenzoate
V. harveyi in vivo response was measured using MM32 V. har-
veyi reporter strain grown in AB (autoinducer bioassay medium)
as described previously.3 Light emission was measured in a Wallac
Model 1450 Microbeta scintillation counter after 4 h incubation at
30 °C. Bioluminescence is reported as the light emitted by the cul-
tures as counts per minute (cpm). In vitro response of LuxP-FRET
protein was measured based on the method described previously17
optimized for 96 well plate reading using a multilabel counter
(1420 Victor 3, Perkin Elmer). Serial dilution of sample was added
ent-16: ½a 2D0
ꢀ
+48.9 (c 1.55, CH2Cl2).
4.1.7. (R)-1,2-Cyclohexylidenedioxypent-3-yne 14
To the diol 13 (189 mg, 1.89 mmol) in DMF (2.5 mL) at room tem-
perature, was added cyclohexanone dimethyl ketal (0.567 mL,
3.78 mmol), and two drops of H2SO4 and the reaction mixture was
stirred overnight. A saturated aqueous solution of NaHCO3 (3 mL)
was added (pH P 8). The mixture was extracted with ethyl ether
(3 ꢃ 3 mL), concentrated under vacuum and purified by flash chro-
matography (5:95 AcOEt/hexane) to afford colorless oil 14 (310 mg,
to 12.5
Phosphate buffer (pH 8), 35 mM NaCl, and 1 mM borate. Samples
(2.5 l) were added to 280 l reaction volume and AI-2 concentra-
lg/ml CFP-LuxP-YFP chimeric protein in 25 mM Sodium
l
l
tions of the samples was calculated from FRET ratio (527/485 nm).
Binding of AI-2 to the CFP-LuxP-YFP protein causes a dose-depen-
dent decrease in the FRET signal. E. coli in vivo response was
measured with the new reporter strain KX1446 grown in LB
(Luria-Bertani) using the methodology previously described for
the S. typhimurium reporter strain.3 b-Galactosidase activity of
the lsr-lacZ promoter fusion in E. coli was measured with a Bio-
Rad 3550-UV microplate Reader. b-Galactosidase units are defined
as [(OD420 minꢁ1 ꢃ dilution factor)/OD600)]. All dose–response
curves were fit to variable-slope sigmoidal curves using non-linear
least squares analysis.
91% yield). ½a 2D0
ꢀ
ꢁ41.4 (c 1.16, CHCl3); Lit.10
½
a 2D0
ꢀ
ꢁ39.2 (c 0.767,
CHCl3). 1H NMR (400 MHz, CDCl3): d 4.70–4.65 (m, 1H), 4.11 (dd,
J = 6.1, 7.8 Hz, 1H), 3.82 (dd, J = 7.8, 7.0 Hz, 1H), 1.86 (d, J = 2.1,
3H), 1.74–1.58 (m, 10H). 13C NMR (100 MHz, CDCl3): d 110.5, 82.3,
76.4, 69.7, 65.5, 35.8, 35.4, 25.1, 23.9, 23.8, 3.7. FT-IR (film): 2243
(C„C). M/z 181.0 (M++H), 180.2 (M+). The same procedure afforded
ent-14: ½a 2D0
ꢀ
+40.5 (c 1.68, CHCl3); Lit.10
½
a 2D0
+39.4 (c 0.796, CHCl3).
ꢀ
4.1.8. (S)-4,5-Cyclohexylidenedioxy-2,3-pentadione 15
To compound 14 (306 mg, 1.7 mmol) dissolved in CCl4
(9.18 mL) and MeCN (9.18 mL) was added a solution of NaIO4
(826 mg, 3.86 mmol) in H2O (14 mL) and RuO2ꢂH2O (5.6 mg,
0.042 mmol) and the reaction mixture was stirred vigorously until
all starting material had been consumed (TLC). The mixture was
extracted with CH2Cl2 (3 ꢃ 15 mL), filtered by a very short pad of
silica for medium pressure chromatography and concentrated un-
der vacuum to give the bright yellow oil 15 that crystallised as a
Acknowledgements
We acknowledge the generous financial support provided by
Fundação para a Ciência e Tecnologia (PPCDT/DG/BIA/82010/2006
Portugal). We thank CERMAX for the use of the NMR spectrome-
ters, which were purchased within the framework of the National
Programme for Scientific Re-equipment, contract REDE/1517/
RMN/2005, with funds from POCI 2010 (FEDER) and Fundação para
a Ciência e a Tecnologia (FCT). We thank Pedro Lamosa for help in
quantifying the DPD samples by NMR and Paula Chicau for help
with HPLC analysis.
low melting yellow solid (310 mg, 86% yield). ½a D20
ꢁ11.2 (c 1.45,
ꢀ
CHCl3). Lit.10
½
a 2D0
ꢀ
ꢁ11.8 (c 0.90, CHCl3). 1H NMR (400 MHz, CDCl3):
d 5.14 (dd, J = 5.3, 7.9 Hz, 1H), 4.35 (dd, J = 8.0, 8.9 Hz, 1H), 4.00 (dd,
J = 5.3, 8.9 Hz, 1H), 2.39 (s, 3H), 1.75–1.56 (m, 10H). 13C NMR
(100 MHz, CDCl3): d 198.2, 194.9, 111.9, 76.5, 65.6, 35.4, 34.7,
25.0, 24.5, 23.9, 23.8. FT-IR (film): 1795 (C@O), 1725 (C@O). The
Supplementary data
same procedure afforded ent-15: ½a D20
ꢀ
+9.7 (c 1.83, CHCl3); Lit.10
½
a 2D0
+11.5 (c 0.733, CHCl3).
ꢀ
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.036.
4.1.9. (S)-4,5-Dihydroxy-2,3-pentadione 1. Preparation of DPD
and ent-DPD samples for bio-assays
References and notes
Cyclohexylidene DPD 15 was dissolved in water to 10 mM and
the pH lowered with acidic Dowex 50WX8 resin (100 mg per mL
of sample). After stirring overnight at rt the resin was removed by
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