Dissecting AI-2-mediated quorum sensing through C5-analogue synthesis and biochemical analysis

Article abstract of DOI:10.1016/j.tet.2015.08.063

Autoinducer-2 (AI-2)-mediated quorum sensing (QS) is utilised for both intra- and inter-species communication by a wide variety of bacteria. An understanding of the mechanism of this communication has the potential to elucidate new targets for antibacteri

Full text of DOI:10.1016/j.tet.2015.08.063

Tetrahedron xxx (2015) 1e6
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Dissecting AI-2-mediated quorum sensing through C5-analogue
synthesis and biochemical analysis
*
Karen C. Collins, Kyoji Tsuchikama, Colin A. Lowery, Jie Zhu, Kim D. Janda
Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of Research and Medicine (WIRM), The Scripps
Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
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)-mediated quorum sensing (QS) is utilised for both intra- and inter-species com-
munication by a wide variety of bacteria. An understanding of the mechanism of this communication has
the potential to elucidate new targets for antibacterial therapeutics. Herein, we report the synthesis of
DPD analogues with modified dynamic equilibria and the evaluation of their behaviour in Gram-negative
bacteria. None of the compounds showed modulation of QS in Salmonella Typhimurium, and although no
antagonism of Vibrio harveyi was observed, chloro-analogue C5-Cl-DPD showed modest agonism in this
marine bacterium. This raises the possibility that access to a cyclic form of DPD may not be required for
AI-2-mediated QS in V. harveyi.
Received 21 July 2015
Accepted 25 August 2015
Available online xxx
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
form may not be crucial for activity in the latter species. Down-
stream biological recognition of AI-2 in this species includes
Quorum sensing (QS) is communication between bacteria
through recognition of small molecule modulators. QS is re-
sponsible for the orchestration of bacterial behaviour in a density-
dependent manner, including events that increase bacterial de-
fences such as biofilm formation and virulence factor expression,
and thus would be a important target for future therapeutics.
Autoinducer-2 (AI-2) is a prolific modulator, recognised by over 70
species of both Gram-negative and -positive bacteria.1 However, AI-
2-mediated QS has proved challenging to study given the pro-
pensity of the precursor, (4S)-DPD, to polymerisation at high con-
centration, and its existence as a complex equilibrium with
recognition of different equilibrium species by different bacteria
(Fig. 1).2 Crystallisation of the AI-2 signal with LuxP, the AI-2 sensor
protein in the marine organism Vibrio harveyi, suggests the re-
quirement of a cyclic DPD species coordinating to boron, (2S,4S)-
THMF-borate.3 The corresponding sensor protein in the enteric
bacterium Salmonella Typhimurium, LsrB, was also crystallised
with a cyclic form of DPD, albeit recognising an alternative ste-
reoisomer that lacked boron coordination, (2R,4S)-THMF.4
phosphorylation of the C5 alcohol by kinase LsrK, requiring a ring-
opened form of DPD,6 before binding of the resulting phospho-(4S)-
THP to transcriptional regulator LsrR.5
Given the effects of cyclisation on signalling activity have not
been fully elucidated, we sought to explore the importance of linear
and cyclic forms of DPD to investigate the requirements for the AI-2
structure in these systems. Analysis of the NMR spectra has shown
that DPD predominantly exists as
a
mixture of ring-closed
OH
OH
O B
O
HO OH
O
HO
-
B(OH)4
OH
H2O
active species in
V. harveyi
OH
O
HO
HO
HO
O
O
(2S, 4S)-DHMF
(2S, 4S)-THMF-borate
(2S, 4S)-THMF
O
2
OH
O
O
OH
OH
active species in
S. Typhimurium
and E. coli
LsrK
HO
5
3
1
HO
O
O
4
P
O
HO OH
(4S)-THP
OH HO OH
phospho-(4S)-THP
O
(4S)-DPD
Although AI-2 is not internalised by V. harveyi, it appears this is
not the case for all species. Binding to LsrB is the primary route
through which AI-2 can enter S. Typhimurium, but an alternative,
currently unknown, pathway is believed to exist.5 Thus the cyclic
O
O
HO OH
O
H2O
recognised by
LsrR
HO
HO
OH
OH
(2R, 4S)-DHMF
(2R, 4S)-THMF
* Corresponding author. Tel.: þ1 858 784 2516; fax: þ1 858 784 2595; e-mail
Fig. 1. Complex equilibrium of AI-2 including the known biologically recognised
species.
address: kdjanda@scripps.edu (K.D. Janda).
http://dx.doi.org/10.1016/j.tet.2015.08.063
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Collins, K. C.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.08.063

2
K.C. Collins et al. / Tetrahedron xxx (2015) 1e6
stereoisomers under physiological pH.2 We have previously in-
vestigated the use of carbocyclic analogues to mimic fixed
ring-closed conformations of DPD (DHMP and triHMP, Fig. 2).7
However, the direct analogue DHMP had no significant effect in ei-
ther V. harveyi or S. Typhimurium, and triHMP showed only limited
antagonism in V. harveyi. DFT calculations suggested limited hydra-
tion at the C3 ketone of DHMP, a requirement for formation of the
borate diester. These compounds also lack the dynamic equilibrium
that DPD utilises for multi-species recognition. We were therefore
interested in designing new analogues that would exhibit both
a predominantly ring-closed form and hydration of the C3 ketone.
We identified C5-SH-DPD as a suitable target as DFT calculations
suggested that the ring-closed form of this compound should exist
mainly as the C3-hydrate (see Table S1, Supplementary data).
C5-MeO-DPD (Scheme 1).2 Alkynyl Grignard addition to Weinreb
amide 113 and alkynylation of chloroacetyl chloride afforded ke-
tones rac-2a and rac-2b in high yield, which could be readily re-
duced by Luche reduction. Displacement of the chloride in rac-3b
allowed the introduction of a thioacetate group; acetate protection
was chosen as this was stable to the subsequent reaction conditions
but would ultimately allow for facile deprotection either under
mild conditions or in situ as was the case for diacetylated-DPD;14 in
the latter case, the absence of base should render the resulting thiol
resistant to oxidation. Subsequent TBS-protection and alkyne oxi-
dation15 proceeded smoothly to give the three DPD precursors
5aec in low to moderate yields. As with all a-diketone DPD ana-
logues, the final acid-catalysed deprotection was carried out
without purification or concentration of the resulting solution; it
has previously been shown that the tert-butyldimethylsilanol by-
product is non-toxic and inactive up to 100
mM against both V.
harveyi and S. Typhimurium.7
Fig. 2. Structures of DPD analogues. a. Existing DPD analogues. b. Targets of this
research.
In a parallel, complementary approach we proposed the in-
stallation of a C5-halide in order to mimic the (4S)-THP open-chain
form of DPD. Fluoride is considered a classical bioisostere for oxy-
gen, being of similar size and able to (weakly) participate as a hy-
drogen-bond acceptor.8 The larger, less electronegative halides
have been shown to form halogen-bonding interactions,9 potentially
mimicking the hydrogen-bond donor character of the hydroxyl
group. Indeed, this strategy has been utilised in replacement of the
hydrated ketone in C3-dihalo-DPD analogues, with both the dichloro
and dibromo analogues displaying comparable agonism to DPD
itself.10 Although non-fluoro alkyl halides have the potential to un-
dergo nucleophilic displacement and are widely considered to be
genotoxic fragments, drugs containing alkyl chloride motifs are
generally stable in vivo and are relatively common.11 There are also
examples of alkyl bromides being used in medicinal chemistry with
apparent stability (cannabinoid receptor agonists O-806 and O-
123612), although electrophilic reactivity is anticipated. However,
alkyl iodides are scarcely reported in this context due to their in-
stability; in our context, C5-I-DPD would almost certainly act as an
alkylating agent or simply hydrolyse to form DPD. We therefore
proposed the synthesis of the C5-fluoro, -chloro and -bromo ana-
logues to allow for investigation of both hydrogen-bond donors and
acceptors in place of the C5-hydroxy group. We also investigated the
biological activity of previously synthesised open-chain analogue
C5-MeO-DPD,2 for its ability to act as a hydrogen-bond acceptor.
Scheme 1. Synthesis of open- and closed-ring DPD analogues.
The final target C5-Br-DPD was synthesised in enantiopure
form via an alternative route. Tosylate 3d could be prepared in six
steps from
L-gulonic acid g
-lactone,15e17 subsequent TBS-
protection of the alcohol and oxidation of the alkyne provided
diketone 7. The bromide was then installed by displacement of the
tosylate in good yield, before acidic deprotection to give C5-Br-
DPD.
Deprotection of the resulting C5-DPD analogues was monitored
by NMR. As was previously observed, rac-C5-MeO-DPD gave
a clean spectrum with one compound, the linear hydrated form.
The same was observed for both rac-C5-F-DPD and rac-C5-Cl-
DPD; detailed analysis of the spectra of these derivatives showed
no evidence of instability. However, C5-Br-DPD was found to
slowly hydrolyse to form DPD under the deprotection conditions,
and therefore this compound was not further analysed. TBS
deprotection of thioacetate analogue rac-5c, initially formed in-
termediate rac-C5-SAc-DPD, and neutralisation subsequently ini-
tiated deprotection of the thiol (to give rac-C5-SH-DPD). As
predicted, the spectra of this compound supported the presence of
a complex equilibrium, analogous to that seen upon deprotection of
DPD.
All four stable C5-DPD analogues were then tested for both
agonism and antagonism of AI-2-mediated QS in Gram negative
bacteria S. Typhimurium and V. harveyi using previously estab-
lished reporter assays.18,19 Although one might expect that the
linear analogues cannot bind LsrB, which is believed to require
cyclisation for recognition, another pathway for DPD uptake has
2. Results and discussion
Three of our targets, rac-C5-SH-DPD, rac-C5-Cl-DPD and rac-
C5-F-DPD could be readily synthesised in a similar manner to rac-
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K.C. Collins et al. / Tetrahedron xxx (2015) 1e6
3
been postulated, albeit, much slower than LsrB-modulated trans-
port for AI-2 and analogues.5
S. Typhimurium strain Met844 (DluxS), a knockout of the gene
encoding DPD synthase luxS, was chosen for these assays. This
strain also contains a lacZ-lsr fusion, coupling activation of the AI-2-
some or all of this decrease resulted from toxicity of the compounds
at higher concentrations, as evidenced both by the decrease in
bioluminescence and cell density at higher concentrations of all
compounds, including DPD. To rule out the possibility of non-AI-2-
mediated QS being responsible for the resulting agonism, rac-C5-
dependent lsr promoter to the biosynthesis of
b
-galactosidase,
Cl-DPD was tested in V. harveyi strain BB886 (DluxPQ), wherein
enabling quantification of AI-2-based QS. The strain can be used in
the absence of DPD to identify AI-2 QS agonists, and DPD can be
added for the detection of antagonists or synergistic agonists,
which have previously been reported.20,21 All four compounds were
no agonism was observed at concentrations up to 200
m
M, likely
owing to the absence of the AI-2 receptor protein in V. harveyi.
As discussed previously, activity of DPD is believed to result
from (2S,4S)-THMF-borate, requiring cyclisation and borate com-
plexation, as observed in a crystal structure of DPD bound to the
receptor protein, LuxP. However, the observed agonism of rac-C5-
Cl-DPD, which is locked in the linear form in V. harveyi directly
counters this binding requirement.
tested at two concentrations (50 and 200
mM) in the presence or
absence of DPD (50 M), using DPD itself as an agonism control and
m
C1-Pr-DPD20 as an antagonism control (Fig. 3aeb). No significant
agonism or antagonism was observed for any of the tested com-
pounds, which aligned with our expectations for the open-chain
analogues. The lack of activity shown by rac-C5-SH-DPD is
harder to interpret due to the complexity of the AI-2 pathway in
this bacterium. There are multiple steps involved in the processing
of DPD; binding to LsrB, phosphorylation by LsrK and subsequent
binding to transcriptional regulator LsrR. Further work is required
to identify what effect if any, rac-C5-SH-DPD has on the individual
stages of AI-2 processing.
Given the instability of rac-C5-Br-DPD, it was proposed that the
chloro analogue may also form small amounts of DPD that could
conceivably account for the agonism observed in V. harveyi. Al-
though no DPD was observed by 1H or 13C NMR, the characteristic
signals of the complex DPD equilibrium are much smaller in
magnitude than those of the ca. chloride, which exists as a single,
hydrated species; the 20% rac-DPD required to account for the
observed agonism may not be detectable via this method. Direct
LCMS analysis is not possible on the DPD analogues due to their
inherent instability, but derivatisation with 1,2-phenylenediamine
allowed for analysis of the resulting quinoxaline products.23,24 Re-
action of rac-C5-Cl-DPD did in fact show evidence of DPD de-
rivative 10, suggesting that a small quantity of DPD may have been
present, potentially explaining the agonism observed in the V.
harveyi assay (Scheme 2). Analysis of the supernatant from an assay
of the compound against V. harveyi does not indicate increased
amounts of DPD after an 8 h incubation at 30 ꢀC, ruling out the
possibility of slow DPD formation at neutral pH. It is possible that
DPD is produced upon reaction of rac-C5-Cl-DPD with 1,2-
phenylenediamine, via formation of the corresponding epoxide
derivative 11, which was also detected. However, this does not rule
out the possibility that both rac-DPD and rac-epoxy-DPD formation
occurred during the acidic deprotection step or the subsequent
neutralization.
The compounds were subsequently tested in V. harveyi strain
MM32 (DluxN; DluxS) to look for agonism and strain BB170 (DluxN)
to identify antagonism; the inherent bioluminescence of this bac-
terium allows facile quantification of AI-2 mediated QS. Both strains
lack the gene encoding AHL receptor luxN, excluding the detection
of QS modulation through this pathway, and strain MM32 has also
had the AI-2 synthase, luxS, removed. No antagonism was observed
for any of the analogues, but rac-C5-Cl-DPD and rac-C5-SH-DPD
exhibited moderate and weak agonism, respectively (Fig. 3c). Direct
comparison with DPD showed a reduced response of ca. 10-fold in
the case of rac-C5-Cl-DPD and ca. 100-fold in the case of rac-C5-
SH-DPD. It should be noted that (4R)-DPD has negligible activity
compared with (4S)-DPD,22 and thus this assay likely un-
derestimates the true potency of the active enantiomer of these
racemic analogues. Furthermore, although partial agonism was
indicated by the lower comparative maximum bioluminescence,
Scheme 2. Reaction of C5-halo-DPD analogues and DPD with 1,2-phenylenediamine.
Strong evidence for the absence of a DPD impurity present in the
sample of rac-C5-Cl-DPD can be inferred from the S. Typhimurium
assays, wherein no agonism was observed at concentrations up to
200
mM compared to the DPD positive control at 50 mM. This
combination of assay results suggests that AI-2-mediated QS may
not be dependent upon access to a cyclic form of DPD, despite
analytical challenges preventing a definitive characterisation.
3. Conclusion
Fig. 3. Agonism and antagonism of C5-DPD analogues in V. harveyi and S. Typhimu-
rium. a. and b. Agonism and antagonism, respectively, of C5-DPD analogues in S. Ty-
A series of DPD analogues were synthesised and evaluated for
their ability to modulate QS in two species of gram-negative bac-
teria. Neither dynamic DPD analogue rac-C5-SH-DPD nor the ring
opened analogues showed any activity in S. Typhimurium, but
phimurium strain Met844 in the absence and presence of DPD (50
mM); Pr-DPD used
as a positive antagonism control. Bioluminescence in and -galactosidase activity in S.
b
Typhimurium were normalised to cell density. c. Agonism of C5-DPD analogues using
V. harveyi strain MM32. All data was performed in triplicate, errors represent SEM.
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4
K.C. Collins et al. / Tetrahedron xxx (2015) 1e6
linear chloride, rac-C5-Cl-DPD, was found to be a moderate agonist
of AI-2 mediated QS in V. harveyi in spite of the presumed re-
quirement for a cyclic DPD signal. As such, the activity of this
molecule may hinge on the presence of an unidentified alternative
mechanism or binding mode to the LuxP receptor protein, and ul-
timately may suggest a broader lexicon for V. harveyi communica-
tion than currently understood.
4.6. 1-Fluoropent-3-yn-2-one (2a)
1-propynylmagnesium bromide (52 mL, 0.5
M in THF,
26.0 mmol) was added to a solution of Weinreb amide 1 (2.10 g,
17.3 mmol) in THF (165 mL) at 0 ꢀC and the solution stirred at 0 ꢀC
(4.5 h). The reaction was quenched with satd aq NH4Cl (120 mL)
and H2O (30 mL) was added. The organic layer was separated and
the aqueous layer extracted with Et2O (2ꢁ100 mL), the combined
organic layers were dried (MgSO4), filtered and the solvent re-
moved in vacuo to give the ketone 2a as a yellow oil, which was
used without further purification (1.54 g, 89%), dH (500 MHz, CDCl3)
4.82 (2H, d, J 47.2, CH2), 2.02 (3H, d, J 1.2, CH3); dC (126 MHz, CDCl3)
181.5 (d, J 22.0), 95.9 (d, J 2.2), 84.83 (d, J 188.1), 76.58, 4.19; dF
(377 MHz, CDCl3) ꢂ225.0. Found: 101.0402. C5H6FO requires
101.0397.
4. Experimental section
4.1. General
Reactions were performed under an inert atmosphere at rt
using flame-dried glassware with dry solvents. Reagents were
used as commercially supplied, unless otherwise specified. TLC
was performed on glass-backed plates pre-coated with silica
(EMD 60 F254, 0.25e1 mm) and developed using standard visu-
alising agents: UV fluorescence (254 nm) and KMnO4, cerium
ammonium nitrate or ninhydrin with appropriate heating. 1H and
13C NMR were performed on Bruker spectrometers, with the ref-
erence from the residual solvent peak for 1H NMR (7.26 ppm for
CDCl3, 3.31 ppm for CD3OD), and the solvent peak for 13C NMR
(77.1 ppm for CDCl3, 49.0 ppm for CD3OD), coupling constants (J
values) are given in Hz. HRMS (ESI) were performed on an Agilent
1100 Series LC/MSD-TOF.
4.7. 1-Chloropent-3-yn-2-one (2b)
A solution of chloroacetyl chloride (478 mL, 60 mmol) and 1-
(trimethylsilyl)propyne (8.88 mL, 60 mmol) in CH2Cl2 (30 mL) was
cannulated into a suspension of AlCl3 (8.80 g, 66 mmol) in CH2Cl2
(120 mL) at 0 ꢀC over ca. 30 min. The solution was stirred at 0 ꢀC
(1 h) and rt (1 h) before being poured into ice/conc. HCl (200 mL).
The aqueous layer was extracted with CH2Cl2 (2ꢁ100 mL), the
combined organic layers were washed with brine (2ꢁ150 mL),
dried (MgSO4), filtered and the solvent removed in vacuo to give
ketone 2b as a brown liquid, which was used without further pu-
rification (6.8 g, 97%), dH (400 MHz, CDCl3) 4.17 (2H, s, CH2), 2.05
(3H, s, CH3); dC (101 MHz, CDCl3) 178.7, 95.1, 77.7, 59.7, 4.3.
4.2. General method for Luche reduction
CeCl3$7H2O (1.1 equiv) was added to a solution of crude ketone
(1.0 equiv) in MeOH (ca. 70 mM) at 0 ꢀC. After 5 min, NaBH4
(1.2 equiv) was added portionwise over 5 min, before the solution
was stirred at 0 ꢀC (1 h). The reaction was quenched with satd aq
NH4Cl, the solution extracted with CH2Cl2 (ꢁ3), the combined or-
ganic layers dried (MgSO4), filtered and the solvent removed in
vacuo. Purification by column chromatography eluting with EtOAc/
hexane (gradient, 0:1/1:2) gave the title compound.
4.8. 1-Fluoropent-3-yn-2-ol (rac-3a)
Synthesised using the general method for Luche reduction,
giving alcohol rac-3a as a colourless oil (321 mg, 37%), Rf 0.22 (1:19
EtOAc/hexane); dH (500 MHz, CDCl3) 4.62e4.52 (1H, br m, CHOH),
4.42 (1H, ddd, J 46.9, 9.3, 3.3, CHHF), 4.33 (1H, ddd, J 47.8, 9.3, 7.2,
CHHF), 2.89 (1H, br s, OH), 1.82 (3H, d, J 2.4, heCH3); dC (101 MHz,
CDCl3) 85.8 (d, J 175.5), 83.4 (d, J 1.8), 75.1 (d, J 12.6), 61.8 (d, J 23.9),
3.55.
4.3. General method for TBS protection
TBSCl (2.5 equiv) was added to a solution of alcohol (1.0 equiv)
and imidazole (5.0 equiv) in CH2Cl2 (ca. 200 mM) and the solution
stirred at rt (17 h). Satd aq NH4Cl and CH2Cl2 were added, the
aqueous layer was extracted with CH2Cl2 (ꢁ2), the combined or-
ganic layers were dried (MgSO4), filtered and the solvent removed
in vacuo. Purification by column chromatography eluting with
EtOAc/hexane (gradient, 0:1/1:19) gave the title compound.
4.9. 1-Chloropent-3-yn-2-ol (rac-3b)
Synthesised using the general method for Luche reduction,
giving alcohol rac-3b as a yellow oil (2.50 g, 66% over two steps), Rf
0.33 (1:4 EtOAc/hexane); dH (500 MHz, CDCl3) 4.57e4.53 (1H, m,
CHOH), 3.69 (1H, dd, J 11.1, 3.9, CHHCl), 3.61 (1H, dd, J 11.1, 6.8,
CHHCl), 2.27 (1H, br s, OH), 1.87 (3H, d, J 2.0, CH3); dC (101 MHz,
CDCl3) 83.2, 76.6, 62.8, 49.6, 3.7.
4.4. General method for alkyne oxidation
4.10. S-(2-Hydroxypent-3-yn-1-yl)ethanethioate (rac-3c)
A solution of NaIO4 (2.3 equiv) in H2O (ca. 700 mM) was added
to a solution of alkyne (1.0 equiv) in CCl4/MeCN (1:1, ca. 225 mM)
and stirred vigorously at rt under air for 3 min before the addition
of RuO2$xH2O (0.02 equiv) and the solution stirred vigorously
(15 min) before being filtered through a silica pad, washing with
CH2Cl2. Purification by column chromatography eluting with
EtOAc/hexane (gradient, 0:1/1:9) gave the title compound.
A solution of KSAc (21 mg, 0.186 mmol) in DMF (1 mL) was
added dropwise to
a solution of chloride rac-3b (20 mg,
0.169 mmol) in DMF (1 mL) at 0 ꢀC, and the solution allowed to
warm to rt and stirred (72 h). EtOAc (20 mL) and H2O (20 mL) were
added and the aqueous layer extracted with EtOAc (2ꢁ20 mL),
washed combined organic layers with brine (2ꢁ30 mL), dried
(MgSO4), filtered and the solvent removed in vacuo. Purification by
column chromatography eluting with EtOAc/hexane (gradient,
0:1/1:2) gave alcohol rac-3c as a pale yellow oil (16 mg, 60%), Rf
0.40 (1:2 EtOAc/hexane); dH (400 MHz, CDCl3) 4.49e4.43 (1H, m,
CHOH), 3.24 (1H, dd, J 13.8, 4.9, CHHS), 3.14 (1H, dd, J 13.8, 7.0 Hz,
CHHS), 2.37 (3H, s, COCH3), 1.84 (3H, d, 2.1, heCH3); dC (101 MHz,
CDCl3) 196.0, 82.4, 78.5, 61.9, 36.9, 30.7, 3.7. Found: MHþ, 159.0471.
C7H11O2S requires 159.0474.
4.5. General method for TBS deprotection
D2SO4 (0.82
mL) was added to a solution of silyl ether (10
mmol)
in 4:1 D2O/DMSO-d6 (500
mL) and the solution incubated at rt
(24 h). Upon NMR confirmation of complete deprotection, the
sample was carefully neutralised to pH 7 from ca. pH 1 by the ad-
dition of NaOD.
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K.C. Collins et al. / Tetrahedron xxx (2015) 1e6
5
4.11. tert-Butyl((1-fluoropent-3-yn-2-yl)oxy)dimethylsilane
(rac-4a)
(3H, s, SiCH3), 0.06 (3H, s, SiCH3); dC (126 MHz, CDCl3) 198.6, 195.9,
195.4, 71.1, 71.0, 32.7, 30.5, 25.8, 24.6, 18.3, ꢂ4.9. Also recovered
alkyne rac-4c as a colourless oil (18 mg, 21%).
Synthesised using the general method for TBS protection, giving
silyl ether rac-4a as a pale yellow oil (768 mg, 77%), Rf 0.60 (1:19
EtOAc/hexane); dH (500 MHz, CDCl3) 4.61e4.54 (1H, m, CHOSi),
4.33 (1H, ddd, J 46.8, 9.1, 3.8, CHHF), 4.28 (1H, ddd, J 47.9, 9.1, 7.5,
CHHF), 1.82 (3H, d, J 2.1, heCH3), 0.91 (9H, s, C(CH3)3), 0.13 (3H, s,
SiCH3), 0.12 (3H, s, SiCH3); dC (126 MHz, CDCl3) 86.1 (d, J 178.3), 82.4
(d, J 2.4), 76.2 (d, J 13.1), 62.7 (d, J 24.9), 28.9, 18.4, 3.60, ꢂ4.6, ꢂ4.9;
dF (377 MHz, CDCl3) ꢂ219.6.
4.17. 5-Fluoro-4-hydroxypentane-2,3-dione (rac-C5-F-DPD,
rac-6a)
Synthesised using the general method for in situ TBS-
deprotection, giving a colourless solution of rac-C5-F-DPD, rac-
6a, dH (400 MHz, 4:1 D2O/DMSO-d6) 4.64 (1H, ddd, J 46.2, 10.2, 3.3,
CHOH), 4.48 (1H, ddd, J 47.9, 10.1, 6.6, CHHF), 4.09 (1H, ddd, J 19.8,
6.6, 3.3, CHHF), 2.30 (3H, s, COCH3), 0.85 (4.5H, s, C(CH3)3), 0.03 (3H,
s, SiCH3); dC (151 MHz, 4:1 D2O/DMSO-d6) 210.8, 97.4 (d, J 7.5), 84.9
(d, J 164.8), 73.7 (d, J 18.6), 26.6 (TBS-OH), 25.8 (TBS-OH), 18.8, ꢂ3.0
(TBS-OH); dF (377 MHz, 4:1 D2O/DMSO-d6) ꢂ232.1.
4.12. tert-Butyl((1-chloropent-3-yn-2-yl)oxy)dimethylsilane
(rac-4b)
Synthesised using the general method for TBS protection, giving
silyl ether rac-4b as a pale yellow oil (73 mg, 74%), Rf 0.67 (1:19
EtOAc/hexane); dH (500 MHz, CDCl3) 4.48e4.44 (1H, m, CHOH),
3.55 (1H, ddd, J 10.8, 5.0, 0.6, CHHCl), 3.51 (1H, ddd, J 10.8, 7.3, 0.7,
CHHCl), 1.83 (3H, dd, J 2.0, 0.7, heCH3), 0.91 (9H, d, J¼0.8 Hz,
C(CH3)3), 0.14 (3H, s, SiCH3), 0.13 (3H, s, SiCH3); dC (126 MHz, CDCl3)
82.1, 77.9, 64.1, 49.1, 25.9, 18.4, 3.6, ꢂ4.5, ꢂ4.9.
4.18. 5-Chloro-4-hydroxypentane-2,3-dione (rac-C5-Cl-DPD,
rac-6b)
Synthesised using the general method for in situ TBS-
deprotection, giving a colourless solution of rac-C5-Cl-DPD, rac-
6b, dH (600 MHz, 4:1 D2O/DMSO-d6) 4.11 (1H, dd, J 9.4, 2.5, CHOH),
3.92 (1H, dd, J 11.8, 2.5, CHHCl), 3.61 (1H, dd, J 11.8, 9.4, CHHCl), 2.39
(3H, s, COCH3), 0.93 (9H, s, C(CH3)3), 0.12 (6H, s, 2ꢁ SiCH3); dC
(151 MHz, 4:1 D2O/DMSO-d6) 210.7, 97.8, 75.6, 46.0, 26.6 (TBS-OH),
25.7, 18.8 (TBS-OH), ꢂ3.0 (TBS-OH).
4.13. S-(2-((tert-Butyldimethylsilyl)oxy)pent-3-yn-1-yl) etha-
nethioate (rac-4c)
Synthesised using the general method for TBS protection, giving
silyl ether rac-4c as a pale yellow oil (84 mg, 81%), Rf 0.43 (1:9
EtOAc/hexane); dH (400 MHz, CDCl3) 4.39e4.34 (1H, m, CHOSi) 3.15
(1H, dd, 13.4, 5.9 Hz, CHHS), 3.09 (1H, dd, J 13.4, 7.0, CHHS), 2.32
(3H, s, COCH3), 1.81 (3H, d, J 2.1, heCH3), 0.89 (9H, s, C(CH3)3), 0.11
(3H, s, SiCH3), 0.09 (3H, s, SiCH3); dC (101 MHz, CDCl3) 195.4, 81.2,
79.3, 62.4, 37.5, 30.6, 25.9, 18.4, 3.6, ꢂ4.6, ꢂ4.9. Found: MHþ,
273.1345. C13H25O2SSi requires 273.1339.
4.19. 4-Hydroxy-5-mercaptopentane-2,3-dione (rac-C5-SAc-
DPD, rac-6c precursor)
Synthesised using the general method for in situ TBS-
deprotection, giving a colourless solution of rac-C5-SAc-DPD,
rac-6c precursor, dH (400 MHz, 4:1 D2O/DMSO-d6) 3.88 (1H, dd, J
10.0, 2.6, CHOH), 3.30 (1H, dd, J 14.1, 2.6, CHHS), 2.86 (1H, dd, J 14.1,
10.0, CHHS), 2.36 (3H, s, COCH3), 2.31 (3H, s, COCH3), 0.87 (9H, s,
C(CH3)3), 0.05 (6H, s, 2ꢁ SiCH3); dH (151 MHz, 4:1 D2O/DMSO-d6)
211.2, 201.6, 98.1, 73.7, 31.6, 31.5, 26.6 (TBS-OH), 25.7, 18.8 (TBS-OH),
ꢂ3.0 (TBS-OH).
4.14. 4-((tert-Butyldimethylsilyl)oxy)-5-fluoropentane-2,3-
dione (rac-5a)
Synthesised using the general method for alkyne oxidation,
giving diketone rac-5a as a yellow oil (162 mg, 40%), Rf 0.20 (1:19
EtOAc/hexane); dH (400 MHz, CDCl3) 5.02 (1H, ddd, J 22.9, 5.1, 3.5,
CHOSi), 4.67 (1H, ddd, J 47.2, 9.8, 5.1, CHHF), 4.56 (1H, ddd, J 46.9,
9.8, 3.5, CHHF), 2.36 (3H, s, COCH3), 0.89 (9H, s, C(CH3)3), 0.10 (3H, s,
SiCH3), 0.09 (3H, s, SiCH3); dC (151 MHz, CDCl3) 199.3, 196.7 (d, J
6.9), 84.3 (d, J 174.4), 73.5 (d, J 20.9), 25.7, 24.9, 18.3, ꢂ4.9, ꢂ5.0; dF
(377 MHz, CDCl3) ꢂ228.9. Also recovered alkyne rac-4a as a col-
ourless oil (126 mg, 36%).
4.20. (R)-2-((tert-Butyldimethylsilyl)oxy)pent-3-yn-1-yl 4-
methylbenzenesulfonate (7)
Synthesised using the general method for TBS protection, giving
silyl ether 7 as a colourless oil (209 mg, 81%), Rf 0.34 (1:9 EtOAc/
hexane); [
a
]
25 ꢂ46.1 (c 1.0, CHCl3); dH (400 MHz, CDCl3) 7.79 (2H, d,
D
J 7.8, AreH), 7.33 (2H, d, J 7.8, AreH), 4.55e4.49 (1H, m, CHOSi), 4.01
(1H, dd, J 9.6, 3.6, CHHOS), 3.91 (1H, dd, J 9.8, 7.9, CHHOS), 2.44 (3H,
s, CH3Ar), 1.77 (3H, s, heCH3), 0.85 (9H, s, C(CH3)2), 0.09 (3H, s,
SiCH3), 0.06 (3H, s, SiCH3); dC (101 MHz, CDCl3) 144.9, 135.1, 129.9,
128.1, 82.7, 76.3, 73.0, 61.6, 25.8, 21.8, 18.3, 3.6, ꢂ4.7, ꢂ4.9. Found:
MHþ, 369.1551. C18H29O4SSi requires 359.1556.
4.15. 4-((tert-Butyldimethylsilyl)oxy)-5-chloropentane-2,3-
dione (rac-5b)
Synthesised using the general method for alkyne oxidation,
giving diketone rac-5b as a yellow oil (364 mg, 56%), dH (400 MHz,
CDCl3) 5.06 (1H, t, J 5.0, CHOSi), 3.84e3.79 (1H, m, CHHCl),
3.79e3.74 (1H, m, CHHCl), 2.41 (3H, s, COCH3), 0.92 (9H, s, C(CH3)3),
0.13 (3H, s, SiCH3), 0.12 (3H, s, SiCH3); dC (126 MHz, CDCl3) 199.1,
196.1, 73.2, 45.0, 25.7, 24.9, 18.3, ꢂ4.8, ꢂ4.9.
4.21. (S)-2-((tert-Butyldimethylsilyl)oxy)-3,4-dioxopentyl 4-
methylbenzenesulfonate (8)
Synthesised using the general method for alkyne oxidation,
giving diketone 5d as a yellow oil (232 mg, 53%), Rf 0.54 (1:2 EtOAc/
hexane); dH (500 MHz, CDCl3) 7.76 (2H, d, J 8.3, AreH), 7.35 (2H, d, J
7.9, AreH), 5.00e4.98 (1H, m, CHOSi), 4.27 (1H, ddd, J 10.4, 5.2,
0.8 Hz, CHHOS), 4.22 (1H, ddd, J 10.5, 4.1, 0.8, CHHOS), 2.45 (3H, s,
CH3Ar), 2.33 (3H, s, COCH3), 0.83 (9H, s, C(CH3)3), 0.05 (3H, s, SiCH3),
0.04 (3H, s, SiCH3); dC (126 MHz, CDCl3) 198.8, 195.4, 145.3, 132.6,
130.1, 128.2, 71.7, 70.2, 25.7, 24.8, 21.8, 18.3, ꢂ4.9. Found: MHþ,
401.1454. C18H29O6SSi requires 401.1449. Also recovered alkyne 8 as
a colourless oil (71 mg, 18%).
4.16. S-(2-((tert-Butyldimethylsilyl)oxy)-3,4-dioxopentyl)
ethanethioate (rac-5c)
Synthesised using the general method for alkyne oxidation,
giving diketone rac-5c as a yellow oil (33 mg, 38%), Rf 0.50 (1:9
EtOAc/hexane); dH (400 MHz, CDCl3) 5.10 (1H, dd, J 6.8, 3.9, CHOSi),
3.38 (1H, dd, J 13.9, 3.9, CHHS), 3.08 (1H, dd, J 13.9, 6.8, CHHS), 2.35
(3H, s, SCOCH3), 2.29 (3H, s, OCOCH3), 0.87 (9H, s, C(CH3)3), 0.09
Please cite this article in press as: Collins, K. C.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.08.063

6
K.C. Collins et al. / Tetrahedron xxx (2015) 1e6
4.22. (R)-5-Bromo-4-((tert-butyldimethylsilyl)oxy)pentane-
2,3-dione (5d)
was added to an aliquot of the aqueous top layer (100
resulting luminescence (OD420) was recorded every 1 min for
20 min. -Galactosidase activity was calculated according to the
mL) and the
b
LiBr (6.5 mg, 0.050 mmol) was added to a solution of tosylate 8
(20 mg, 0.050 mmol) in acetone (1.5 mL) and stirred at rt (24 h),
whereupon the solution was filtered and washed through with
acetone before the solvent was removed in vacuo. Purification by
column chromatography eluting with EtOAc/hexane (gradient,
following equation: ‘activity¼Vmax/OD600’, where ‘Vmax¼maximum
slope of kinetic display of OD420/min’.
Acknowledgements
0:1/1:24) gave bromide 5d as a yellow oil (12 mg, 78%), Rf 0.24
We gratefully acknowledge Prof. Bonnie Bassler (Princeton
University) for providing us with S. Typhimurium strain Met844.
This work was funded by the National Institute of Allergy and In-
fectious Diseases grant AI077644. This is manuscript #29163 from
The Scripps Research Institute.
25
(1:19 EtOAc/hexane); [
a
]
D
þ22.3 (c 0.8, CHCl3); dH (500 MHz,
CDCl3) 5.09 (1H, t, J 5.0, CHOSi), 3.63 (1H, dd, J 10.5, 4.7, CHHBr),
3.58 (1H, dd, J 10.5, 5.3, CHHBr), 2.39 (3H, s, COCH3), 0.90 (9H, s,
C(CH3)3), 0.11 (3H, s, SiCH3), 0.09 (1H, s, SiCH3); dC (101 MHz, CDCl3)
199.0, 195.9, 72.6, 32.6, 25.7, 24.9, 18.3, ꢂ4.8, ꢂ4.8.
Supplementary data
4.23. 5-Bromo-4-hydroxypentane-2,3-dione (C5-Br-DPD, 6d)
Supplementary data (Additional tables and figures containing
DFT calculations, numerical antagonism data and 1H and 13C NMR
spectra) associated with this article can be found in the online
version, at http://dx.doi.org/10.1016/j.tet.2015.08.063.
Synthesised using the general method for in situ TBS-
deprotection, giving a colourless solution of C5-Br-DPD, 6d, dH
(600 MHz, 4:1 D2O/DMSO-d6) 4.10 (1H. d, J 9.9, CHOH), 3.74 (1H, d, J
11.0, CHHBr), 3.42e3.37 (1H, m, CHHBr), 2.33 (3H, s, CH3); dC
(151 MHz, 4:1 D2O/DMSO-d6) 210.7, 97.9, 75.6, 34.5, 26.6, 25.6, 18.8,
ꢂ3.0.
References and notes
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4.24. Modulation of bioluminescence in V. harveyi
3. Chen, X.; Schauder, S.; Potier, N.; Van Dorsselaer, A.; Pelczer, I.; Bassler, B. L.;
Hughson, F. M. Nature 2002, 415, 545e549.
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melhack, M. F.; Bassler, B. L. ACS Chem. Biol. 2007, 2, 128e136.
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Agonistic and antagonistic activity was evaluated in V. harveyi
using the procedure of Taga.19 Briefly, V. harveyi strain BB170 (ATCC
BAA-1117), MM32 (ATCC BAA-1121) or BB886 (ATCC BAA-1118) was
grown in AB medium (14 h, 30 ꢀC). The cells were diluted in fresh
AB medium (1/2500) and added (100
test compounds (previously adjusted to pH 7 using NaOD) in AB
medium (100 L/well) at a final concentration of 0.5% DMSO. The
plate was incubated (30 ꢀC, ca.
h), whereupon the bio-
mL/well) to the serially diluted
m
9. Sirimulla, S.; Bailey, J. B.; Vegesna, R.; Narayan, M. J. Chem. Inf. Model. 2013, 53,
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8
10. Guo, M.; Zheng, Y.; Terell, J. L.; Ad, M.; Opoku-Temeng, C.; Bentley, W. E.; Sintim,
H. O. Chem. Commun. 2015, 51, 2617e2620.
luminescence (OD490) and cell density (OD600) were measured.
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centration) as the concentration corresponding to 50% maximum
bioluminescence.
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ꢀ
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Agonistic and antagonistic activity was evaluated in S. Typhu-
murium using the procedure of Wolf.18 Briefly, S. Typhumurium
strain Met8445 was grown in LB medium (16 h, 37 ꢀC). The cells
were diluted in fresh LB medium (1/25) and added (50
the serially diluted test compounds (previously adjusted to pH 7
using NaOD) with or without DPD in LB medium (150 L/well) at
mL/well) to
m
a final concentration of 0.5% DMSO in deep-well polypropylene
plates. The plates were incubated (37 ꢀC, ca. 4 h), whereupon an
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aliquot (50
sured. A second aliquot (50
containing 0.35% BME), whereupon CHCl3 (20
the solution thoroughly mixed. ONPG (20 L, 4 mg/mL in Z buffer)
m
L) was diluted ¼ and the cell density (OD600) mea-
L) was added to Z buffer (500 L,
L) was added and
m
m
m
m
Please cite this article in press as: Collins, K. C.; et al., Tetrahedron (2015), http://dx.doi.org/10.1016/j.tet.2015.08.063