Synthesis and bioluminescence-inducing properties of autoinducer (S)-4,5-dihydroxypentane-2,3-dione and its enantiomer

Article abstract of DOI:10.1016/j.bmcl.2010.02.064

The autoinducer (4S)-4,5-dihydroxypentane-2,3-dione ((S)-DPD, AI-2) facilitates chemical communication, termed 'quorum sensing', amongst a wide range of bacteria, The synthesis of (S)-DPD is challenging in part due to its instability. Herein we report a novel synthesis of (S)-DPD via (2S)-2,3-O-isopropylidene glyceraldehyde, through Wittig, dihydroxylation and oxidation reactions, with a complimentary asymmetric synthesis to a key precursor. Its enantiomer (R)-DPD, was prepared from d-mannitol via (2R)-2,3-O-isopropylideneglyceraldehyde. The synthesized enantiomers of DPD have AI-2 bioluminescence-inducing properties in the Vibrio harveyi BB170 strain.

Full text of DOI:10.1016/j.bmcl.2010.02.064

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2625–2628
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and bioluminescence-inducing properties of autoinducer
(S)-4,5-dihydroxypentane-2,3-dione and its enantiomer
Manikandan Kadirvel a, William T. Stimpson b, Souad Moumene-Afifi a, Biljana Arsic a, Nicola Glynn b,
Nigel Halliday c, Paul Williams c, Peter Gilbert a, Andrew J. McBain a, Sally Freeman a, John M. Gardiner b,
*
a School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PT, UK
b School of Chemistry and Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, UK
c School of Molecular Medical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The autoinducer (4S)-4,5-dihydroxypentane-2,3-dione ((S)-DPD, AI-2) facilitates chemical communica-
tion, termed ‘quorum sensing’, amongst a wide range of bacteria, The synthesis of (S)-DPD is challenging
in part due to its instability. Herein we report a novel synthesis of (S)-DPD via (2S)-2,3-O-isopropylidene
glyceraldehyde, through Wittig, dihydroxylation and oxidation reactions, with a complimentary asym-
Received 15 December 2009
Revised 11 February 2010
Accepted 13 February 2010
Available online 19 February 2010
metric synthesis to a key precursor. Its enantiomer (R)-DPD, was prepared from D-mannitol via (2R)-
2,3-O-isopropylideneglyceraldehyde. The synthesized enantiomers of DPD have AI-2 bioluminescence-
inducing properties in the Vibrio harveyi BB170 strain.
Keywords:
Autoinducer
DPD
Ó 2010 Elsevier Ltd. All rights reserved.
AI-2
The AI-2 class of inducer, used by both Gram-positive and
Gram-negative bacteria,1 has been proposed to be an universal sig-
nal for interspecies communication (quorum sensing). In contrast,
the acyl homoserine lactones (AHLs) and autoinducing peptides
(AIPs) are believed to control intraspecies communication.2 (4S)-
4,5-Dihydroxypentane-2,3-dione (1) (DPD), an unstable diketone
biosynthesised by bacteria, is the penultimate precursor of autoin-
ducer AI-2. AI-2 coordinates bacterial behaviour and function to
enable adaptation to changing environments and possibly to facil-
itate competition with multicellular organisms.3 The receptor for
the AI-2 inducer, activates gene transcription, including those for
its biosynthesis.4 The biosynthesis of DPD involves S-adenosylme-
thionine in three enzymatic steps, the last step catalyzed by LuxS.5
DPD is proposed to exist as an equilibrium mixture of cyclic,
hydrated and borate complexes, collectively known as autoinduc-
er-2 (AI-2). Vibrio harveyi, an indicator bacterium which forms
the basis of the bioluminescence assay, recognizes the 2,3-borate
O
O
HO
OH
Me
HO
(S)
H2O
HO
(S)
HO
HO
O
Me
OH
(S)
(S)
(S)
Me
OH
O
O
1
OH
B
O
HO
HO
(S)
HO
O
(R)
HO
OH
HO
(S)
H2O
(S)
OH
Me
OH
OH
(R)
O
(S)
(R)
Me
O
O
O
Me
Scheme 1. Autoinducer AI-2: Biological forms of (S)-DPD (1).9
lene ketone.10 In addition, the di-O-acetylated derivative of (S)-
DPD was prepared as a enzyme-labile, chemically stable precursor
of DPD.11
diester of the hydrated
a
-anomer of DPD6 (Scheme 1). The role
of borate complexation in structurally-analogous sugars suggests
Here we report a novel synthesis of the unnatural enantiomer,
(R)-DPD (2), starting from D-mannitol (3). We also describe a novel
this can play a key role in signalling efficacy.7
Despite its relatively simple structure, the synthesis of DPD is
complicated by its instability at high concentrations giving rise
to oligomerisation. In the published syntheses, DPD was obtained
as dilute aqueous solutions, either from acidic hydrolysis of an
synthesis of the natural (S)-DPD (1) to provide samples of both
enantiomers to investigate bacterial coordination of gene expres-
sion, biofilm formation and other AI-2 quorum sensing-regulated
process.
orthoester8 or a ketal,9 or by reductive ozonolysis of an
a-methy-
For the synthesis of the unnatural R enantiomer, D-mannitol (3)
is an inexpensive starting material which possesses the absolute
stereochemistry at C-2/C-5 required to give an enantiospecific syn-
* Corresponding author. Tel.: +44 161 306 4530.
E-mail address: gardiner@manchester.ac.uk (J.M. Gardiner).
thesis of (R)-DPD (2).12 Using published procedures12
D-mannitol
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.064

2626
M. Kadirvel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2625–2628
deprotection of the acetonide group of (14) in D2O (to allow mon-
itoring by 1H NMR spectroscopy) led to the formation of (R)-DPD
(2), with a complex 1H NMR spectrum as described in the literature
for (S)-DPD (1).9 Further evidence for the formation of (R)-DPD (2)
was obtained by addition of 1,2-phenylenediamine, forming a sin-
gle quinoxaline derivative (15) (Scheme 4), also consistent with the
data reported for (S)-DPD (1).9
OH
O
OH OH
a
O
(R)
(S)
OH
(R)
(R)
(S)
(R)
(R)
(R)
O
HO
O
OH
OH OH
4
3
O
Whilst this synthesis establishes a simple entry to the (R)-DPD
system, storage of the acetal 14 (and certainly 2) long term may be
subject to potential degradation. Thus, the bis-O-benzoyl and bis-
O-acetyl derivatives of (R)-DPD, namely (16) and (17), were pre-
pared (Scheme 5) as stable bioprecursors of (R)-DPD, which could
then be stored for prolonged periods, prior to use in biological as-
says. Thus, acetals (6) and (7) were deprotected to give diols
(18)/(19) and then esterified with either benzoyl or acetyl chloride
to give alkene diesters (20)/(21) and (22)/(23) in 69% and 58%
yield, respectively. The alkenes were dihydroxylated as previously
with OsO4 to give the corresponding diols, which were subse-
quently oxidised with PCC to give (16) and (17) in 41% and 30%
yield, respectively.
We also report here a novel synthesis of the natural (S)-DPD (1)
from (R)-2,2-dimethyl-1,3-dioxolane-4-methanol (24) (Scheme 6),
employing the same Wittig-osmylation strategy applied above in
the synthesis of unnatural (R)-DPD. Thus, alcohol (24) was oxidised
to aldehyde (25) using PCC oxidation.20 Reaction of (25) with
(ethyl)triphenylphosphonium bromide/n-BuLi in a Wittig reaction
provided the novel alkenes (26) and (27), in the same 2:1 ratio as
observed for Wittig reaction of 5. Subsequent dihydroxylation of
these alkenes provided (28)-(31), oxidation to give (32) and depro-
tection to give (S)-DPD (1) (Scheme 6). All substrates and products
were characterised by NMR spectroscopy, IR spectrometry, mass
and high resolution mass spectroscopy, with the (R) and (S) enan-
tiomers of DPD having identical spectroscopic characterisation.
An alternative strategy to the DPD backbone was also explored in
which the 2,3-chiral centres are introduced by asymmetric synthe-
sis and these are then used to direct the establishment of the natural
product 4S centre by diastereoselective osmylation. Whilst the
asymmetrically-introduced chiral centres are then of course lost in
final oxidations, they have transferred the key chiral information
to the C4 chiral centre of the target. This route thus starts from an
achiral precursor, rather than a chiral pool source as employed in
the prior routes. Benzyl (E)-2-butenoate (33) underwent asymmet-
ric dihydroxylation to give 34, which was then elaborated via aceto-
nide protection of the new diol followed by ester reduction and
oxidation-Wittig to give 35 (Scheme 7). A second dihydroxylation
then proceeded with essentially complete diastereocontrol followed
by benzylation to give the fully-protected DPD precursor tetraol 37,
with C4 and C5 protected orthogonally to C2 and C3. This serves both
as a precursor dibenzyl analogue and natural stereochemical anti-
pode of 16, as well as precursor to 38, which is the direct dibenzyl
analogue of intermediate 28, and can thus be converted into the
dibenzyl protected variant of 32.
(E)
(Z)
b
c
(R)
(S)
(S)
+
O
O
O
O
O
O
5
6, E (major)
OH
7, Z (minor)
H
H
HO
(R)
+
(S)
(S)
(R)
d
(R)
(R)
O
9
OH
H
O
O
O
O
H
OH
O
8
6+7
OH
H
HO
H
(R)
(S)
(S)
(R)
+
(R)
(R)
d
OH
H
H
OH
O
O
11
10
Scheme 2. Synthesis of intermediate diols. Reagents and conditions: (a) 2,2-
dimethoxypropane, p-TSAÁH2O, 57%; (b) NaIO4, NaHCO3, 75%; (c) (ethyl)triphenyl
phosphonium bromide, n-BuLi, 70%; (d) 4%OsO4, NMOÁH2O, 70%.
(3) was reacted with 2,2-dimethoxypropane and catalytic p-TSA to
give 1,2:5,6-di-O-isopropylidene-D-mannitol (4), with subsequent
oxidative cleavage with NaIO4 to give known (2R)-2,3-O-isopropy-
lideneglyceraldehyde (5).13 Reaction of (5) with (ethyl)triphenyl-
phosphonium bromide/n-BuLi14 provided the novel alkenes (6)
and (7) in 70% combined yield with a geometrical isomer ratio of
>20:1 E:Z by 1H NMR spectroscopy (Scheme 2). Subsequent prepa-
rations of alkenes using n-BuLi which had been stored for even
moderate periods led to formation of alkenes with a geometrical
isomer ratio of 2:1 E:Z (1H NMR). Dihydroxylation of alkenes (6)
and (7) using catalytic OsO4 and stoichiometric N-methyl morpho-
line-N-oxide,15 afforded the diastereoisomeric diols (8)/(9) and
(10)/(11), respectively in a 70% yield.
Whilst dihydroxylation of such alkene substrates is known to
proceed with good substrate diasterecontrol or matching double
diastereocontrol using AD, in the current synthesis both the new-
ly-introduced chiral secondary alcohols are to be converted into
ketones, so that the mixture of diols (8)-(11) from achiral osmyla-
tion can be taken forwards. However, oxidation of (8)–(11) using
Swern oxidation16 led to the formation of monoketones (12) and
(13) (Scheme 3), and not the desired a-diketone (14). There are lit-
erature precedents for the difficulty of oxidising vicinal diols,17 un-
der Swern conditions, reporting mixtures of 1,2-diketone and
monoketones products.18
O
O
b
However, oxidation of (8)–(11) with PCC led to the formation of
a
(R)
(R)
8-11
HO
O
a
-diketone (14) in 30% yield.19 Subsequent acid-catalysed
O
O
OH
2
O
14
O
O
a
N
(R)
(S)
8-11
+
(R)
(R)
c
OH
H
O
O
H
OH
(S)
O
O
HO
N
OH
13
12
15
Scheme 3. Reagents and conditions: (a) Swern oxidation: oxalyl chloride, dry
Scheme 4. Reagents and conditions: (a) PCC, 30%; (b) H2O, H2SO4; (c) 1,2-
DMSO, Et3N, 49%.
phenylenediamine.

M. Kadirvel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2625–2628
2627
O
(E)
(Z)
(S)
(R)
d,e
(S)
+
RO
b
RO
RO
b
a
OR
OR
O
OR
6 / 7
18, R=H
19, R=H
16, R=Bz
17, R=Ac
20, R=Bz
21, R=Bz
c
c
22, R=Ac
23, R=Ac
Scheme 5. Synthesis of (R)-DPD esters (16) and (17). Reagents and conditions: (a) methanol, p-TSAÁH2O, 57%; (b) triethylamine, benzoyl chloride, 69%; (c) triethyamine acetyl
chloride, 53%; (d) 4%OsO4, NMO.H2O; (e) PCC, 41%.
after 2 h. As DPD was dissolved in water, sterile water served as
a negative control. Commercial (S)-DPD (OMM Scientific) was used
as the positive control. The fold bioluminescence induction is cal-
culated at the point that the blank sample gives the minimal rela-
tive light output (RLO) (described in the electronic Supplementary
data (ESI )).
OH
O
(E)
(Z)
a
b
(R)
(R)
(S)
(R)
+
O
O
O
H
O
O
O
O
O
24
26, E (major) 27, Z (minor)
OH
The inversion of stereochemistry of the hydroxyl moiety of (S)-
DPD (1) resulted in a small decrease in bioluminescent activity in
the (R)-enantiomer (Fig. 1). Recent literature reported that inversion
of stereochemistry renders the hydroxyl group inaccessible for
hydrogen bonding with that of amide side chain of Gln77 and the in-
dole of Trp82.23 (R)-DPD (2), the non-natural enantiomer exhibits
considerably lower quorum sensing activity compared with the
commercial (S)-DPD (OMM Scientific). However, lower biolumines-
cence-inducing activity of (R)-DPD was observed when compared
with the synthesised (S)-DPD (1) (Fig. 1). The apparently lower
bioluminescence-inducing activity of synthesised (S)-DPD (1), as
compared with the commercial (OMM Scientific) (S)-DPD, is in sig-
nificant part due to lower mass of materials used, and the required
25
HO
H
(S)
+
(R)
(R)
(S)
d
(S)
a
(S)
H
OH
O
O
OH
H
O
O
O
28
29
OH
(S)
d
O
a
1
26+27
O
O
HO
H
H
32
(R)
(S)
(R)
(S)
(S)
(S)
+
d
H
OH
O
O
OH
H
O
O
30
31
in situ removal of the isopropylidene protecting group from
a-dike-
tone (32), reducing the effective molarity of DPD.(Scheme 6).
Scheme 6. Synthesis of (S)-DPD (1). Reagents and conditions: (a) PCC, 30%; (b)
(ethyl)triphenylphosphonium bromide, n-BuLi, 58%; (c) 4% OsO4, NMO.H2O, 48%;
(d) H2O, H2SO4.
In conclusion, (S)-DPD (1) and its unnatural enantiomer (R)-
DPD (2) act as quorum sensing signals for the LuxP sensor protein
in Vibrio harveyi, with (S) showing higher activity than (R). Our
straightforward synthetic route compared with the previous
published procedures could be used to produce both (R)- and
(S)-DPD to investigate bacterial coordination of gene expression,
biofilm formation, and other AI-2 quorum sensing regulated
processes.
O
OH
O
O
a
b-e
O
(E)
Bn
g
Bn
(R)
O
(S)
O
(S)
O
(S)
OH
33
35
34
H
O
HO
O
f
h
(S)
O
(S)
(S)
(R)
(S)
(S)
HO
(S)
(R)
BnO
(S)
BnO
H
OH
36
OH
OBn
OBn
37
38
Scheme 7. Synthesis of (S)-DPD precursor via asymmetric dihydroxylation.
Reagents and conditions: (a) AD mix-
a
; (b) 2,2-dimethoxypropane, p-TSAÁH2O;
(c) LiBH4, 0 °C; (d) PCC; (e) (Methyl)triphenylphosphonium bromide, n-BuLi; (f) AD-
mix-b; (g) NaOH, BnBr; (h) methanol, p-TSAÁH2O.
The biological activity of (S)- and (R)-DPD were assayed using
Vibrio harveyi BB170 reporter strain that responds only to AI-2 like
molecules.21 The bioluminescence assay was performed as de-
scribed by Surette and Greenberg21,22 by testing different concen-
trations of synthetic DPD (0.5, 1 and 5 lg/ml). Briefly, Vibrio harveyi
BB170 ATCC number BAA-1117 was grown at 30 °C for 16 h with
aeration in AB medium then diluted 1:5000 in fresh AB medium.
Aliquots (180
ferent concentrations of synthetic DPD (20
l
l) of the culture were placed in a 96-well plate. Dif-
l) were added to the
l
Figure 1. AI-2 Assay of S- and R-DPD. Induction of bioluminescence ina Vibrio harveyi
strain BB170 by (S)-DPD (1) [S], (R)-DPD (2) [R] and commercial (S)-DPD (OMM
Scientific) [C]. Fold light induction was calculated at the time point that the blank
sample gave a minimal light output (t = 3 h)—see Supplementary data Figure 7.
aliquots of inoculated AB medium. Light production was measured
using a luminescence reader (Tecan Infinite 200 microplate reader)
every 30 min at 30 °C. Luminescence generally started to increase

2628
M. Kadirvel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2625–2628
Potier, N.; Van Dorsselaer, A.; Pelczer, I.; Bassler, B. L.; Hughson, F. M. Nature
Acknowledgements
2002, 415, 545.
7. Gardner, P.; Winzer, K.; Davis, B. Nat. Chem. 2009, 1, 377.
8. Meijler, M. M.; Hom, L. G.; Kaufmann, G. F.; McKenzie, K. M.; Sun, C.; Moss, J. A.;
Matsushita, M.; Janda, K. D. Angew. Chem., Int. Ed. 2004, 43, 2106.
9. Semmelhack, M. F.; Campagna, S. R.; Federle, M. J.; Bassler, B. L. Org. Lett. 2005,
7, 569.
10. (a) De Keersmaecker, S. C. J.; Varszegi, C.; van Boxel, N.; Habel, L. W.; Metzger,
K.; Daniels, R.; Marchal, K.; De Vos, D.; Vanderleyden, J. J. Biol. Chem. 2005, 280,
19563; (b) Frezza, M.; Soulere, L.; Queneau, Y.; Doutheau, A. Tetrahedron Lett.
2005, 46, 6495.
Overseas Research Scholarship and University of Manchester
award to M.K. Research funded in part by the Government of
Republic of Algeria to S.M.A. The authors acknowledge the consid-
erable contributions of Professor Peter Gilbert (1951–2008) for
ideas to the inception of this work.
Supplementary data
11. Frezza, M.; Soulere, L.; Balestrino, D.; Gohar, M.; Deshayes, C.; Queneau, Y.;
Forestier, C.; Doutheau, A. Bioorg. Med. Chem. Lett. 2007, 17, 1428.
12. (a) Ghosh, S.; Rao, R. V.; Shashidhar, J. Tetrahedron Lett. 2005, 46, 5479; (b) Jung,
M. E.; Shaw, T. J. J. Am. Chem. Soc. 1980, 102, 6304; (c) Huang, J.-M.; Xu, K.-C.;
Loh, T.-P. Synthesis 2003, 755.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.064.
13. Earle, M. J.; Abdur-Rashid, A.; Priestley, N. D. J. Org. Chem. 1996, 61, 5697.
14. Hernanz, D.; Camps, F.; Guerrero, A.; Delgado, A. Tetrahedron: Asymmetry 1995,
6, 2291.
References and notes
15. Cha, J. K.; Christ, W. J.; Kishi, Y. Tetrahedron 1984, 40, 2247.
16. Ohsugi, S.-i.; Nishide, K.; Oono, K.; Okuyama, K.; Fudesaka, M.; Kodama, S.;
Node, M. Tetrahedron 2003, 59, 8393.
17. (a) Iwahama, T.; Sakaguchi, S.; Nishiyama, Y.; Ishii, Y. Tetrahedron Lett. 1995, 36,
1523; (b) Volc, J.; Sedmera, P.; Halada, P.; Prikrylova, V.; Daniel, G. Carbohydr.
Res. 1998, 310, 151.
18. (a) Jones, T. K.; Reamer, R. A.; Desmond, R.; Mills, S. G. J. Am. Chem. Soc. 1990,
112, 2998; (b) Walker, L. F.; Bourghida, A.; Connolly, S.; Wills, M. J. Chem. Soc.,
Perkin Trans. 1 2002, 965.
19. (a) Corey, E. J.; Suggs, J. W. Tetrahedron Lett. 1975, 2647; (b) Tojo, G. Oxidation
of alcohols to aldehydes and ketones. In Basic reactions in organic synthesis;
Fernández, M., Ed.; Springer Science: Boston, 2006.
20. Ermolenko, L.; Sasaki, N. A.; Potier, P. Synlett 2001, 1565.
21. Surette, M. G.; Bassler, B. L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7046.
22. Greenberg, E. P.; Hastings, J. W.; Ulitzur, S. Arch. Microbiol. 1979, 120, 87.
23. Lowery, C. A.; McKenzie, K. M.; Qi, L.; Meijler, M. M.; Janda, K. D. Bioorg. Med.
Chem. Lett. 2005, 15, 2395.
1. Taga, M. E.; Bassler, B. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14549.
2. (a) Bassler, B. L. Cell 2002, 109, 421; (b) Schauder, S.; Bassler, B. L. Gene Dev.
2001, 15, 1468.
3. (a) Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001, 55, 165; (b) Whitehead,
N. A.; Barnard, A. M. L.; Slater, H.; Simpson, N. J. L.; Salmond, G. P. C. FEMS
Microbiol. Rev. 2001, 25, 365; (c) Waters, C. M.; Bassler, B. L. Annu. Rev. Cell Dev.
Biol. 2005, 21, 319; (d) Winans, S. C.; Bassler, B. L. J. Bacteriol. 2002, 184, 873; (e)
Hastings, J. W.; Nealson, K. H. Annu. Rev. Microbiol. 1977, 31, 549.
4. Federle, M. J.; Bassler, B. L. J. Clin. Invest. 2003, 112, 1291.
5. (a) Schauder, S.; Shokat, K.; Surette, M. G.; Bassler, B. L. Mol. Microbiol. 2001, 41,
463; (b) Winzer, K.; Hardie, K. R.; Burgess, N.; Doherty, N.; Kirke, D.; Holden, M.
T. G.; Linforth, R.; Cornell, K. A.; Taylor, A. J.; Hill, P. J.; Williams, P. Microbiology
2002, 148, 909; (c) Zhu, J.; Miller, M. B.; Vance, R. E.; Dziejman, M.; Bassler, B. L.;
Mekalanos, J. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3129; (d) Zhu, J.; Hu, X.;
Dizin, E.; Pei, D. J. Am. Chem. Soc. 2003, 125, 13379.
6. (a) Miller, S. T.; Xavier, K. B.; Campagna, S. R.; Taga, M. E.; Semmelhack, M. F.;
Bassler, B. L.; Hughson, F. M. Mol. Cell 2004, 15, 677; (b) Chen, X.; Schauder, S.;