Design and synthesis of substrate and intermediate analogue inhibitors of S-ribosylhomocysteinase

Article abstract of DOI:10.1021/jm060047g

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage in S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2. Inhibitors of LuxS should interfere with bacterial interspecies communication and potentially provide a novel class of antibacterial agents. LuxS utilizes a divalent metal ion as a Lewis acid during catalysis. In this work, a series of structural analogues of the substrate SRH and a 2-ketone intermediate were designed and synthesized. Kinetic studies indicate that the compounds act as reversible, competitive inhibitors against LuxS, with the most potent inhibitors having K₁ values in the submicromolar range. These represent the most potent LuxS inhibitors that have been reported to date. Cocrystal structures of LuxS bound with two of the inhibitors largely confirmed the design principles, i.e., the importance of both the homocysteine and ribose moieties in high-affinity binding to the LuxS active site.

Full text of DOI:10.1021/jm060047g

J. Med. Chem. 2006, 49, 3003-3011
3003
Design and Synthesis of Substrate and Intermediate Analogue Inhibitors of
S-Ribosylhomocysteinase‡
Gang Shen,§ Rakhi Rajan, Jinge Zhu,§ Charles E. Bell, and Dehua Pei*,§
Departments of Chemistry and of Molecular and Cellular Biochemistry, Ohio State Biochemistry Program, The Ohio State UniVersity,
100 West 18th AVenue, Columbus, Ohio 43210
ReceiVed January 13, 2006
S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage in S-ribosylhomocysteine
(SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2. Inhibitors
of LuxS should interfere with bacterial interspecies communication and potentially provide a novel class of
antibacterial agents. LuxS utilizes a divalent metal ion as a Lewis acid during catalysis. In this work, a
series of structural analogues of the substrate SRH and a 2-ketone intermediate were designed and synthesized.
Kinetic studies indicate that the compounds act as reversible, competitive inhibitors against LuxS, with the
most potent inhibitors having KI values in the submicromolar range. These represent the most potent LuxS
inhibitors that have been reported to date. Cocrystal structures of LuxS bound with two of the inhibitors
largely confirmed the design principles, i.e., the importance of both the homocysteine and ribose moieties
in high-affinity binding to the LuxS active site.
Introduction
R-THMF (S. typhimurium AI-25) and (2S,4S)-2-methyl-2,3,3,4-
tetrahydroxytetrahydrofuran, which spontaneously forms a
Quorum sensing (QSa) is one type of bacterial cell-to-cell
communication, which regulates a diverse array of physiological
and pathological activities in bacteria in a cell-density-dependent
manner.1 QS is mediated by the synthesis, release, and detection
of small signaling molecules, called autoinducers (AIs). There
are two major types of QS systems. Type I QS is species
specific, with each bacterial species having a unique AI-1
molecule or in some cases a unique combination of AI-1
molecules. Gram-negative bacteria generally use acylhomoserine
lactones (AHLs) as their AI-1’s, whereas Gram-positive bacteria
employ oligopeptides or modified oligopeptides. Correspond-
ingly, each species possesses a cognate AI-1 receptor protein
to recognize and respond to the signal. Since the interactions
between AI-1’s and their cognate receptors are highly specific,
AI-1’s generally do not cause any cross talk in mixed popula-
tions of bacteria. To sense the presence of other species in a
mixed population (interspecies communication), bacteria utilize
an alternative mechanism (type II QS) in which a common
signal, AI-2, is released and detected.2,3 AI-2 has been identified
as a furanosyl borate diester in Vibrio harVeyi, by determining
the X-ray crystal structure of LuxP in complex with the active
AI-2 ligand.4 Very recently, the same team showed that active
AI-2 in Salmonella typhimurium is (2R,4S)-2-methyl-2,3,3,4-
tetrahydroxytetrahydrofuran (R-THMF).5 AI-2 is biosynthesized
from S-adenosylhomocysteine (SAH), which is hydrolyzed by
nucleosidase Pfs into adenine and S-ribosylhomocysteine (SRH).6
SRH is next converted into homocysteine (Hcys) and 4,5-
dihydroxy-2,3-pentanedione (DPD) by S-ribosylhomocysteinase
(LuxS). DPD is unstable and spontaneously cyclizes to form
complex with borate as V. harVeyi AI-2.4
Since QS controls many bacterial behaviors including biofilm
formation and bacterial virulence, proteins involved in quorum
sensing are being explored as novel targets for antibacterial drug
design.7-9 Halogenated furanone derivatives have been shown
to act as AI-1 antagonists and inhibit the expression of virulence
factors by Pseudomonas aeruginosa and increase bacterial
susceptibility to the antibiotic tobramycin.10-12 In a mouse
pulmonary infection model, the drug inhibited quorum sensing
of the infecting bacteria and promoted their clearance by the
mouse immune response.12 Suga and co-workers synthesized a
series of AHL analogues, some of which acted as antagonists
of quorum sensing and interfered with virulence expression and
biofilm formation by P. aeruginosa.13,14 LuxS/AI-2 also regu-
lates a host of bacterial behaviors including virulence, biofilm
formation, motility, toxin and antibiotic production, lumines-
cence, and ABC transporter expression.7 Further, the luxS gene
is present in the majority of Gram-positive and Gram-negative
bacteria and is highly conserved.7 Thus, AI-2 antagonists and
LuxS inhibitors have the potential as a class of unconventional,
broad-spectrum antibacterial agents. Two SRH analogues have
recently been reported as weak inhibitors of LuxS (IC50 ≈ 1
mM),15 but potent LuxS inhibitors are still lacking. In this work,
a series of substrate and intermediate analogues have been
synthesized and tested for inhibition against LuxS, and the best
compounds showed KI values in the high nanomolar range.
Chemistry
LuxS is a metalloenzyme containing an Fe2+ ion coordinated
by His-54, His-58, and Cys-126 (amino acid numbering in
Bacillus subtilis LuxS) and a water molecule.16-19 The native
enzyme is unstable under aerobic conditions, undergoing
oxidation of the Fe2+ ion and loss of enzymatic activity.16
Substitution of Co2+ for the native metal ion produces a highly
stable variant of essentially wild-type catalytic activity, whereas
the Zn2+-substituted enzyme has ∼10-fold lower activity.16 In
the proposed catalytic mechanism of LuxS (Figure 1), the metal
ion acts as a Lewis acid, facilitating two consecutive aldose-
ketose isomerization steps and a final â-elimination reaction.20
* To whom correspondence should be addressed. Phone: (614) 688-
4068. Fax: (614) 292-1532. E-mail: pei.3@osu.edu.
‡ The coordinates of the structures of Co-BsLuxS in complex with
compounds 10 and 11 have been deposited in the Protein Data Bank under
accession numbers 2FQT and 2FQO, respectively.
§ Department of Chemistry.
Department of Molecular and Cellular Biochemistry.
a Abbreviations: AHL, acylhomoserine lactone; AI, autoinducer; DPD,
4,5-dihydroxy-2,3-pentanedione; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid);
Hcys, homocysteine; QS, quorum sensing; R-THMF, (2R,4S)-2-methyl-
2,3,3,4-tetrahydroxytetrahydrofuran; SAH, S-adenosylhomocysteine; SRH,
S-ribosylhomocysteine.
10.1021/jm060047g CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/14/2006

3004 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Shen et al.
Figure 1. Proposed mechanism for LuxS-catalyzed reaction. RSH ) homocysteine.
UV-vis absorption spectroscopy16 and X-ray crystallographic
studies21 revealed that the catalytic metal ion is directly
coordinated with the C-2 carbonyl oxygen of a 2-ketone
intermediate (compound 4 in Figure 1). During the conversion
of SRH to intermediate 4 or from intermediate 4 to 3-ketone
intermediate 7, it was proposed that the metal ion plays a key
role in stabilizing the enediolate intermediates (2, 3, 5, and 6)
by binding to them in a bidentate manner (Figures 1 and 2A).
We envisioned that replacement of the unstable enediolate
moiety with a planar hydroxamate group (compound 10) should
produce a stable isostere with high affinity and specificity for
LuxS (Figure 2). Compound 11, which is an stereoisomer of
10 with opposite stereochemical configuration at the â carbon
(relative to the hydroxamate), and compound 12, which does
not contain the Hcys moiety, were synthesized for comparison.
The X-ray crystal structure of LuxS bound to intermediate 4
shows that the main interactions between the intermediate and
the protein are mediated by the two termini of the compound;
i.e., the amino and carboxyl groups of the Hcys moiety fit into
a pocket remote from the active site, formed by residues Lys-
35, Arg-65, Asp-78, Ile-79, and Ser-80, whereas the ribose is
bound to the metal center.21 There is little direct interaction
between the hydrophobic linker of compound 4 and the active
site. Therefore, we also designed compounds 13-16, in which
the amino acid moiety and a metal chelating group (thiol or
N-formylhydroxylamine) are connected with linkers of varying
Figure 2. (A) Proposed modes of interaction among the LuxS metal
center, 2-ketone intermediate 4, and inhibitor 10. (B) Structures of
inhibitors 10-18.
lengths (Figure 2).
Synthesis of compound 10 started from commercially avail-
able D-erythronic-γ-lactone, which was treated with excess
benzyl bromide and silver oxide to protect the hydroxyl groups
(Scheme 1).22 The lactone ring was then opened with O-tert-
butylhydroxylamine in the presence of AlMe3 to give alcohol
19, which was converted into the corresponding bromide 20
using CBr4 and PPh3. Coupling to homocysteine through an SN2
reaction afforded the desired compound 21. Deprotection of the
two benzyl groups proved to be problematic. A variety of
reagents/conditions including hydrogenation with Pd/C, tin(IV)
chloride,23 and boron trichloride-dimethyl sulfide complex24
were ineffective, giving either incomplete debenzylation or
unacceptable levels of side reactions. We found that the best
reaction condition was boron trichloride in cold CH2Cl2,25 which
not only completely removed the two benzyl groups but also
cleaved the Boc and the two tert-butyl ester groups in the

LuxS Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 3005
Scheme 4a
Scheme 1a
a Conditions: (a) BnBr, Ag2O, Et2O; (b) H2NOtBu, AlMe3, DCM; (c)
CBr4, PPh3, DCM; (d) BocNHCH(CH2CH2SH)CO2tBu, LDA, DMF; (e)
BCl3, DCM, -78 °C.
a Conditions: (a) MeOC6H4CHO, Na2SO4, DCM; (b) mCPBA, DCM;
(c) H2NOH, MeOH; (d) HCO2H, Ac2O, DCM; (e) TFA.
Scheme 2a
a Conditions: (a) KOH, MeOH; (b) BH3, THF; (c) CBr4, PPh3, DCM;
(d) BocHNCH(CH2CH2SH)CO2tBu, LDA, DMF; (e) H2NOH, MeOH; (f)
TFA, 1 N HCl.
Scheme 3a
Figure 3. Lineweaver-Burke plot showing the competitive inhibition
of Co-BsLuxS by inhibitor 10.
with m-chloroperbenzoic acid to furnish oxaziridine 36. Treat-
ment of 36 with hydroxylamine produced hydroxylamine 37.
Formylation of the amine followed by TFA deprotection gave
the desired compound 16. S-Ribosylcysteine (SRC and com-
pound 18 in Figure 2) was prepared enzymatically by treating
commercially available S-adenosylcysteine with nucleosidase
Pfs. δ-Ribosylornithine (compound 17) was similarly prepared
by hydrolyzing the natural product sinefungin with Pfs.
a Conditions: (a) H2O2, MeOH; (b) NR-Boc-Lys-O-tBu, DCC, DMAP;
(c) TFA; (d) TCEP.
Results and Discussion
Compounds 10-12 and 16-18 were assayed against Co(II)-
substituted B. subtilis LuxS (Co-BsLuxS) by using SRH as the
substrate and monitoring the release of Hcys with 5,5′-dithiobis-
(2-nitrobenzoic acid (DTNB).27 Compounds 10 and 11 acted
as potent, competitive inhibitors (as determined with 10), with
KI values of 0.72 and 0.37 µM, respectively (Figure 3 and Table
1). The same KI values were obtained for the native ferrous-
containing enzyme Fe-BsLuxS. To our knowledge, these are
the most potent LuxS inhibitors that have so far been reported.
They are, however, significantly weaker inhibitors against Zn-
BsLuxS (20 and 11 µM, respectively). They are also less potent
against Co(II)-substituted Escherichia coli (Co-EcLuxS) and V.
harVeyi (Co-VhLuxS) LuxS (Table 1). Interestingly, the KI
values roughly correlated with the KM values of the various
enzyme forms (against SRH); the enzymes with higher KM
values also have higher KI values, suggesting that these
compounds are good mimics of LuxS catalytic intermediate(s).
Compound 12, which contains only the ribose portion, binds to
LuxS with orders of magnitude lower affinity (KI g 150 µM),
highlighting the importance of the amino acid moiety to the
overall binding affinity. Likewise, methionine binds only weakly
to the LuxS active site (KI ) 61 µM), indicating that binding
molecule, all in one step. To prepare compound 11, com-
mercially available (-)-dimethyl 2,3-O-isopropylidene-L-tartrate
(22) was treated with potassium hydroxide to hydrolyze one of
the ester groups (Scheme 2).26 The resulting monocarboxylic
acid was reduced with borane to afford alcohol 23. The alcohol
was converted to bromide 24 with carbon tetrabromide and
triphenylphosphine and coupled to Hcys to give methyl ester
25. Incubation with excess hydroxylamine converted the ester
into hydroxamic acid 26, which was completely deprotected
by treatment with TFA and 1 N HCl to afford compound 11.
Compounds 13-15 were prepared from the corresponding
mercapto carboxylic acids (Scheme 3). The thiol group was
protected by oxidation into disulfide with hydrogen peroxide.
The carboxyl group was then reacted with a properly protected
lysine derivative to afford compounds 30-32, which were
subsequently treated with TFA to generate the corresponding
amino acids 33-35. The disulfides in 33-35 were reduced to
free thiols 13-15 by tris(carboxyethyl)phosphine prior to
inhibition assays. Compound 16 was synthesized as shown in
Scheme 4. NR-Boc-Lys-O-tBu was reacted with p-methoxyben-
zaldehyde to form an imine intermediate, which was oxidized

3006 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Table 1. Inhibition Constants against LuxS
Shen et al.
KI (µM)
KM
enzyme
BsLuxS
10
11
12
13
14
15
16
Met
(µM)
Co
Fe
Zn
Co
Co
0.72 ( 0.02
0.72 ( 0.03
19.6 ( 1.1
3.2 ( 0.3
0.37 ( 0.04
0.43 ( 0.02
10.6 ( 0.4
12.7 ( 0.4
12.8 ( 0.3
156 ( 5
147 ( 5
2400 ( 400
720 ( 120
550 ( 20
132 ( 17
NDa
ND
ND
ND
155 ( 24
ND
ND
ND
ND
473 ( 70
ND
ND
ND
ND
68 ( 8
ND
ND
ND
ND
61 ( 2
ND
2.3
1.9
58
16
39
ND
ND
ND
EcLuxS
VhLuxS
9.7 ( 0.4
a ND ) not determined.
bidentate N-formylhydroxylamine ligand, also exhibited weak
inhibition, with a KI value of 68 µM (Table 1). This KI value is
similar to that of methionine (KI ) 61 µM), suggesting that it
might bind to the enzyme via its amino acid end. We also
synthesized a series of compounds in which the thiol group of
cysteine or homocysteine is covalently linked to a hydroxamate
moiety via linkers of varying length (R. Liu and D. Pei,
unpublished results). These compounds all showed only moder-
ate inhibition of LuxS (KI values of 15-71 µM).
δ-Ribosylornithine (17) and S-ribosylcysteine (18) were also
tested as potential LuxS substrates and/or inhibitors. Compound
17 did not show any significant inhibition against LuxS in the
DTNB assay. Prolonged incubation of high concentrations of
17 with Co-BsLuxS also failed to cause any spectral changes.
Since the amino acid (ornithine) and ribose are connected
through a C-C bond, DPD formation was not expected.
Attempts to observe any 2- and/or 3-ketone intermediates by
13C NMR spectroscopy also failed. On the other hand, com-
pound 18 (SRC) acted as a slow substrate of LuxS. Although
its reactivity was too low to be quantitatively determined by
the DTNB assay, treatment of 18 with LuxS in the presence of
1,2-phenylenediamine resulted in the formation of a quinoxaline
derivative, indicating that DPD was released by the en-
zyme.16,29,32 When assayed as an inhibitor, SRC exhibited an
apparent KI value of 70 µM.
To gain insight into the structural basis of LuxS inhibition
by the above compounds, Co-BsLuxS was cocrystallized with
the inhibitors 10 and 11 from a solution of ammonium sulfate
at pH 7.0. Crystals of both LuxS complexes are isomorphous
with those of the 2-ketone intermediate 4 complex (PDB code
1YCL),21 and contain a LuxS dimer oriented along a 2-fold
crystallographic axis, with one monomer per asymmetric unit.
The active site is formed at the dimer interface. The structure
with inhibitor 10 was refined at 1.8 Å resolution to an R factor
of 19.4% and a free R factor of 22.6% (Table 2). The final
model includes residues 4-157 of LuxS, 1 Co2+ ion, 138 water
molecules, 2 sulfate ions, and 1 inhibitor molecule. The
temperature factors for the protein atoms range from 11 to 45
Å2, while those for the inhibitor range from 19 to 24 Å2,
indicating full occupancy of the inhibitor. The CR atoms of the
structures of LuxS bound to 10 and 2-ketone intermediate 4
can be superimposed to an rmsd of 0.2 Å. There are no
significant conformational changes in LuxS, even for side chains
of residues at the active site.
Figure 4. (A) Concentration-dependent spectral changes of Co-BsLuxS
in the presence of inhibitor 14. (B) Secondary plot of absorbance at
695 nm against inhibitor concentration from panel A.
to the metal center is also crucial for high affinity. On the other
hand, the similar KI values of compounds 10 and 11 suggest
that the central portion of the inhibitors (and likely substrates
and intermediates) are not engaged in specific interactions with
the enzyme, as we had anticipated.
Compounds 13-15 could not be tested with the DTNB-based
activity assay, because they contain free thiols in their structures.
Their binding affinity to the LuxS active site was determined
by taking advantage of the absorption spectral changes of the
Co(II) center upon inhibitor binding. The Co(II) ion is very
sensitive to changes to its environment and thus provides a
convenient probe for ligand changes associated with inhibitor
binding at the metal center.28 The free enzyme had a broad
absorption peak between 500 and 600 nm and a sharper peak
at 660 nm (Figure 4A). Addition of thiol 14 caused concentra-
tion-dependent spectral changes, resulting in the eventual loss
of the above two peaks and the appearance of three sharp bands
at 545, 630, and 695 nm. A plot of the absorbance at 695 nm
as a function of inhibitor concentration gave a secondary plot
as shown in Figure 4B. Curve fitting of the data gave a KD
value of 155 µM for compound 14. The KD values of compounds
13 and 15 were similarly determined to be 132 and 473 µM,
respectively. The drastic spectral changes suggest that the thiols
(and possibly the carbonyl group as well) replaced the metal-
bound water in the free enzyme and became directly coordinated
with the metal ion. Since the overall affinity is weak, we suspect
that the amino acid moiety of these compounds did not fit into
the homocysteine-binding pocket. Compound 16, which has a
Clear electron density for inhibitor 10 allowed for it to be fit
into the model unambiguously (Figure 5). Inhibitor 10 is bound
to LuxS in a manner very similar to that of 2-ketone intermediate
4 (Figure 5C,E and Table 3). A significant difference lies in
the interaction with the metal ion. While intermediate 4 binds
monodentately to the metal ion via its C2 carbonyl oxygen,
inhibitor 10 interacts with the Co2+ ion bidentately, through
the oxygen atoms of both C2 carbonyl and C3 hydroxyl groups
(metal-oxygen distances of 2.2 and 2.3 Å, respectively). The
hydroxamate oxygen atom O1 is hydrogen bonded to the side
chains of Arg-39 and His-11. The O1 atom does not however

LuxS Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 3007
Table 2. Data Collection and Refinement Statistics
data collection
space group
inhibitor 10
inhibitor 11
P6522
P6522
a, b (Å)
62.9
62.8
c (Å)
148.6
148.6
resolution (Å)
no. of reflns
no. of unique reflns
completeness (%)
redundancy
20.4-1.8
27.2-1.9
89103
97262
15675
14925
91.4 (54.5)a
5.5 (1.4)
6.2 (20.3)
17.2 (3.9)
98.6 (90.6)
5.9 (1.2)
5.2 (18.4)
22 (5.1)
b
Rmerge
I/σc
Refinement
resolution (Å)
20.4-1.8
14054/1568
91.1
21.1
0.17
19.4 (26.8)
22.6 (32.2)
138
0.004
1.2
27.2-1.9
13382/1500
98.6
19.6
0.16
19.1 (22.1)
22.1 (26.1)
130
no. of reflns (working/free)
completeness (%)
mean B factor (Å2)
estimated coordinate errord
R factor
Rfreee (%)
no. of water molecules
RMSD(bonds) (Å)
RMSD(angles) (deg)
b
a Numbers in parentheses refer to the highest shell only. Rmerge ) ∑|Ih
- I |/∑Ih, where I h is the average intensity over symmetry equivalents.
h
c I/σ is the mean of the intensity/sigma of the unique, averaged reflections.
d The estimated coordinate error is the value from the cross-validated σ
plot. e R factor ) ∑|Fobsd - Fcalcd|/∑obsd. Rfree is calculated from 10% of
the reflections that are omitted from the refinement.
interact directly with Ser-6, as was observed in the LuxS-ketone
4 complex, due to a change in the N1-C2 torsion angle imposed
by its partial double bond character. The sulfhydryl group of
catalytic residue Cys-84, which was replaced by alanine in the
complex with intermediate 4, is proximal to the N1 (3.4 Å),
C2 (3.5 Å), and C3 (4.1 Å) atoms of the inhibitor (Table 2).
This is consistent with Cys-84 serving as a proton donor and
acceptor to (and from) these atoms during the course of catalysis,
as outlined in Figure 1. The C3 hydroxyl (O3) is hydrogen
bonded to Glu-57, consistent with the role of Glu-57 as a general
base during the carbonyl migration reactions (Figure 1). The
C4 hydroxyl (O4) is hydrogen bonded to the Ser-6 side chain.
The atoms of the homocysteine portion of the inhibitor are
bound essentially the same as observed for the ketone 4
complex. Interestingly, a well-ordered water molecule, near the
O2 atom of inhibitor 10 and forming hydrogen bonds with Ser-6
and His-11, is observed in both structures. Given the proximity
of this water to the reactive groups of the protein and the
inhibitor, it could conceivably have an important role during
catalysis, presumably facilitating proton-transfer reactions.
The structure of Co-BsLuxS was also determined in complex
with inhibitor 11, and refined at 1.9 Å resolution to an R factor
of 19.1% and a free R factor of 22.2% (Table 2 and Figure
5D). The electron density allowed for all atoms of inhibitor 11
to be placed in the structure unambiguously. Inhibitor 11 differs
from inhibitor 10 only in having an inverted stereochemical
configuration at the C4 position. This change is evident in the
electron density map, although the density for the C4 and O4
atoms is slightly weaker than for the rest of the compound,
indicating a higher degree of flexibility. Due to the change in
configuration, the O4 atom of inhibitor 11 points down (as
viewed in Figure 5D), and is closer to the Co2+ atom (3.5 Å)
than is the case for the O4 atom of inhibitor 10 (5.2 Å). Thus,
the structure of the complex with inhibitor 11 demonstrates that
LuxS binds to substrate-related compounds with some degree
of conformational variation at the C4 position. In comparing
the structures of Co-BsLuxS bound to inhibitors 10, 11, and
intermediate 4, it is apparent that LuxS binds to substrate-related
Figure 5. Structure of Co-BsLuxS in complex with inhibitors 10 and
11. The two subunits of LuxS are colored gold and cyan, and the Co2+
ion is colored magenta. (A) Designation of atoms and groups in inhibitor
10 (according to atom numbering in SRH). (B) Stereoview of the
electron density for inhibitor 10. The blue cage is the 1.8 Å 2Fobsd
-
Fcalcd electron density map contoured at 1σ. The red cage is an Fobsd
-
Fcalcd map, contoured at 2.5σ, calculated before the inhibitor was added
to the model. The inhibitor is shown with green bonds. (C) Stereoview
showing hydrogen-bonding interactions between LuxS and inhibitor
10 as dotted lines. (D) Stereoview showing the hydrogen-bonding
interactions in the structure of Co-BsLuxS in complex with inhibitor
11 (gray bonds). Notice that the stereochemical configuration at the
C4 position is inverted relative to that of inhibitor 10 in panel C, causing
the O4 atom to point down, in the direction of the Co2+ atom. (E)
Stereoview of a superposition of the structures of inhibitors 10 (green),
11 (gray), and 2-ketone intermediate 4 (magenta) in complex with Co-
BsLuxS. The superposition is based on the protein atoms, which are
shown only for the structure of LuxS in complex with inhibitor 10,
but are virtually identical in the other two structures. Notice that the
O2 and O3 atoms form very similar interactions with the Co2+ ion in
the three structures.
compounds with a virtually fixed conformation for the ho-
mocysteine portion, and a more adaptable conformation for the
functional groups of the ribosyl portion, as would be predicted
from the reaction scheme in Figure 1. This suggests that, in

3008 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Table 3. Atomic Interactions between LuxS and Inhibitors 10 and 11a
Shen et al.
ppm for 1H and 39.70 ppm for 13C), CD3OD with internal CH3OH
as reference (3.30 ppm for H and 49.00 ppm for 13C), and D2O
1
inhibitor
atom
LuxS
atom
1
with H2O as reference (4.63 ppm for H). Analytical thin-layer
10
11
chromatography (TLC) was carried out on commercial silica gel
60 plates, 0.25 thickness, with a fluorescent indicator (F-254).
Visualization was accomplished by UV light, staining with 5%
phosphomolybdic acid in ethanol, or staining with KMnO4 in an
aqueous solution of sodium carbonate. Column chromatography
was performed with 32-63 µm silica gel. Solvents for chroma-
tography were reagent grade and used as received. Mass spectra
were performed with positive ion electrospray ionization or negative
ion electrospray ionization.
N1
O1
O1
O1
O1
C2
O2
O2
O3
O3
O3
O4
N
Cys-84 SG
His-11 NE2
Arg-39 NH1
Arg-39 NH2
Gly-127 N
Cys-84 SG
Co2+
3.4
2.7
3.2
2.8
3.5
3.5
2.2
3.1
2.3
2.6
3.0
2.5
2.9
2.8
3.3
3.2
3.0
3.5
2.6
3.4
3.1
3.4
3.6
2.1
3.2
2.3
2.7
3.0
3.7
2.9
2.8
3.3
3.3
3.1
water
Co2+
Glu-57 OE1
His-58 NE2
Ser-6 OG
Asp-78 OD1
Ile-79 O
Ser-80 OG
Asp78 OD1
Ile-79 N
(2R,3R)-2,3-Bis(benzyloxy)-N-tert-butoxy-4-hydroxybutyram-
ide (19). To a solution of O-tert-butylhydroxylamine hydrochloride
(300 mg, 2.39 mmol) in CH2Cl2 (17 mL) was added a 2 M solution
of AlMe3 in hexanes (1.12 mL, 2.24 mmol) at 0 °C, followed by
the addition of d-3,4-dibenzylerythronic-γ-lactone (479 mg, 1.61
mmol), which had been prepared from d-erythronic-γ-lactone
according to a literature method.22 The reaction mixture was heated
to reflux for 20 h. A saturated solution of NH4Cl in water was
added to quench the excess AlMe3, and the mixture was extracted
with CHCl3 (5 × 25 mL). The organic phase was combined, dried
over MgSO4, concentrated, and purified by silica gel chromatog-
raphy (1:1 EtOAc/hexanes) to afford 19 as a white solid (494 mg,
N
N
OXT
OXT
a Distances in angstroms. In the LuxS-10 complex, the number of water
molecules was 469, and in LuxS-11, it was 448.
designing future generations of LuxS inhibitors, one may be
able to employ a wide variety of linker structures to connect
the amino acid and metal-chelating moieties. A conformationally
constrained linker should be particularly effective.
1
79% yield, Rf ) 0.35). H NMR (400 MHz, DMSO-d6): δ 10.55
(s, 1H), 7.34-7.24 (m, 10H), 4.63 (d, J ) 12.0 Hz, 1H), 4.55 (d,
J ) 12.0 Hz, 1H), 4.49 (d, J ) 11.6 Hz, 1H), 4.42 (d, J ) 12.0
Hz, 1H), 3.96 (d, J ) 6.4 Hz, 1H), 3.74-3.70 (m, 1H), 3.68-3.65
(m, 1H), 3.54 (quintet, J ) 6.4 Hz, 1H), 1.14 (s, 9H). 13C NMR
(100 MHz, DMSO-d6): δ 167.7, 138.6, 137.9, 128.2, 127.9, 127.5
(d), 127.2, 80.8, 80.3, 77.1, 71.6, 71.3, 60.0, 26.4. HRESI-MS: m/z
calcd for C22H29NO5Na+ (M + Na+) 410.1938, found 410.1943.
It is worth noting that both inhibitors 10 and 11 coordinate
with the metal ion using the O2 and O3 atoms, instead of
bidentate interaction via the hydroxamate (O1 and O2), as we
had anticipated (Figure 2A). One possible reason for this is that
binding via O1 and O2 would eliminate stable hydrogen-bonding
interactions with Arg-39 and His-11. Alternatively, geometric
constraints exerted by locking the homocysteine moiety into
its binding pocket may prevent the planar hydroxamate from
approaching the metal ion at an optimal distance/angle. This
latter point may have important implications in LuxS catalytic
mechanism. The planar hydroxamate closely mimics the ene-
diolate intermediates, whose tighter binding to the metal ion
would slow or prevent catalytic turnover.
(2R,3S)-2,3-Bis(benzyloxy)-N-tert-butoxy-4-bromobutyram-
ide (20). To a solution of alcohol 19 (468 mg, 1.21 mmol) in CH2-
Cl2 (12 mL) were added triphenylphosphine (387 mg, 1.47 mmol)
and carbon tetrabromide (481 mg, 1.45 mmol) in an ice bath. The
solution was kept under an argon atmosphere and stirred overnight
at room temperature. The solvent was then removed by rotary
evaporation, and the residue was purified by silica gel chromatog-
raphy (1:3 EtOAc/hexanes) to give 20 as a white solid (471 mg,
1
87% yield, Rf ) 0.32). H NMR (400 MHz, CDCl3): δ 8.40 (s,
1H), 7.37-7.30 (m, 10H), 4.74 (d, J ) 11.2 Hz, 1H), 4.68-4.63
(m, 3H), 4.19 (d, J ) 3.2 Hz, 1H), 4.10-4.06 (m, 1H), 3.64-3.55
(m, 2H), 1.22 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 167.8, 137.4,
136.6, 128.7, 128.4 (d), 128.1 (d), 128.0, 82.5, 80.0, 79.2, 77.2,
73.6, 73.3, 31.0, 26.3. HRESI-MS: m/z calcd for C22H28BrNO4-
Na+ (M + Na+) 472.1094, found 472.1097.
Conclusion
A series of LuxS substrate and intermediate analogues have
been synthesized and tested. This effort has led to the first
submicromolar inhibitors against this enzyme. The cocrystal
structures of LuxS bound with inhibitors 10 and 11 revealed
that a high-affinity inhibitor should be able to bind to both the
homocysteine-binding pocket and the metal ion and suggested
possible strategies for designing future generations of LuxS
inhibitors. The structures also provided additional evidence for
the proposed roles of active-site residues (e.g., Glu-57 and Cys-
84 as general acids/bases) during catalysis.
tert-Butyl (2S)-4-[(2R,3S)-2,3-Bis(benzyloxy)-3-tert-butoxy-
carbamoylpropylmercapto]-2-tert-butoxycarbonylaminobu-
tyrate (21). Freshly prepared L-BocNHCH(CH2CH2SH)CO2tBu29
(52 mg, 0.18 mmol) was dissolved in anhydrous DMF (0.4 mL),
and the solution was purged with argon for 10 min. A 1.5 M
solution of LDA in cyclohexane (0.36 mL, 0.54 mmol) was added
to the above solution cooled in an ice-water bath. This solution
was stirred at 0 °C for 10 min and mixed with a solution of bromide
20 (84 mg, 0.19 mmol) in dry DMF (1 mL). This reaction mixture
was stirred at room temperature for 2 days. The solvent was
removed by rotary evaporation, and the yellow residue was
suspended in a saturated NH4Cl solution and extracted with EtOAc
(3 × 10 mL). The organic phase was dried over MgSO4,
concentrated, and purified by column chromatography (1:3 EtOAc/
hexanes) to provide 21 as a pale yellow gel (42 mg, 36% yield, Rf
) 0.16). 1H NMR (400 MHz, CDCl3): δ 8.64 (s, 1H), 7.38-7.27
(m, 10H), 5.22 (br d, J ) 7.6 Hz, 0.8H), 4.65 (d, J ) 11.2 Hz,
4H), 4.25-4.24 (m, 2H), 4.02-3.98 (m, 1H), 2.81 (dd, J ) 14.0
Hz, 6.8 Hz, 1H), 2.72 (dd, J ) 14.0 Hz, 5.6 Hz, 1H), 2.60-2.49
(m, 2H), 2.04-2.00 (m, 1H), 1.87-1.80 (m, 1H), 1.45 (s, 9H),
1.44 (s, 9H), 1.23 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 171.7,
168.4, 155.6, 138.0, 137.2, 128.8, 128.3 (d), 128.1 (d), 127.9, 82.5,
82.2, 80.1, 80.0 (d), 73.5 (d), 53.5, 32.9 (d), 29.1, 28.6, 28.2, 26.5.
Experimental Section
General Procedures. Pfs and Co2+-, Zn2+-, or Fe2+-substituted
LuxS from B. subtilis, E. coli, and V. harVeyi were overexpressed
in E. coli and purified to apparent homogeneity as described
previously.16 All chemicals and reagents were purchased from
Sigma-Aldrich (St. Louis, MO). Unless otherwise noted, materials
were obtained from commercial suppliers and used without further
purification. Triethylamine was dried under KOH; tetrahydrofuran
(THF), dichloromethane, and diethyl ether were treated with the
SOLV-TEK solvent purification system (Berryville, VA). DMF was
dried with molecular sieves. 1H and 13C NMR spectra were obtained
on a 400 or 250 MHz Bruker NMR spectrometer. Unless otherwise
indicated, all NMR data were collected at room temperature in
CDCl3 with internal CHCl3 as reference (7.26 ppm for 1H and 77.23
ppm for 13C), DMSO-d6 with internal DMSO as reference (2.49

LuxS Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 3009
HRESI-MS: m/z calcd for C35H52N2O8SNa+ (M + Na+) 683.3337,
found 683.3336.
2.78 (dd, J ) 14.4 Hz, 1.6 Hz, 1H), 2.72-2.59 (m, 2H), 2.09-
2.06 (m, 1H), 1.93-1.84 (m, 1H), 1.48 (s, 3H), 1.46 (s, 9H), 1.43
(s, 12H). 13C NMR (100 MHz, CDCl3): δ 171.3, 170.8, 155.3,
111.5, 82.1, 79.8, 79.0, 77.9, 53.4, 52.5, 34.4, 33.0, 28.7, 28.3, 28.0,
27.0, 25.8. HRESI-MS: m/z calcd for C21H37NO8SNa+ (M + Na+)
486.2132, found 486.2121.
(2S)-2-Amino-4-[(2R,3S)-2,3-dihydroxy-3-N-hydroxycarbam-
oylpropylmercapto]butyric Acid (10). A solution of 1 M BCl3 in
CH2Cl2 (0.50 mL, 0.50 mmol) was charged into a 10 mL round-
bottom flask and cooled to -78 °C, and compound 21 (53 mg,
0.080 mmol) in CH2Cl2 (0.72 mL) was added dropwise at -78 °C.
The yellow reaction mixture was stirred at -78 °C for 3 h. MeOH/
CH2Cl2 (2:1, 1.08 mL) was then added at -78 °C, and the mixture
was stirred for another 5 min. The solvent was removed by rotary
evaporation, and 2 mL of MeOH was added and then removed by
rotary evaporation. The residue was purified by silica gel chroma-
tography (30% H2O in CH3CN) to give product 4 as a white gel
(15 mg, 71% yield, Rf ) 0.34). The product was further purified
by affinity chromatography with Affi-Gel boronate gel (Bio-Rad),
which was eluted with 30 mM Na2HPO4-NaH2PO4 buffers of the
following pH values: 8.5, 8.1, 7.6, 7.0, 6.75, 6.5, 6.25, 6.0, 5.5,
5.0, and 4.5. Compound 4 was collected over the pH range of 6.5-
tert-Butyl (2S)-2-tert-Butoxycarbonylamino-4-{(4R,5R)-5-N-
hydroxycarbamoyl-2,2-dimethyl[1,3]dioxolan-4-ylmethylmer-
capto}butyrate (26). To a solution of ester 25 (63 mg, 0.14 mmol)
in MeOH (1 mL) cooled in icy water were added hydroxylamine
hydrochloride (40 mg, 0.55 mmol) and 85% KOH (40 mg, 0.61
mmol). This reaction mixture was stirred at rt for 36 h. The solvent
was removed under reduced pressure, and the white residue was
suspended in water. The aqueous mixture was carefully adjusted
to pH ≈ 5 with 1 N HCl and extracted with CHCl3 (3 × 10 mL).
The organic phase was concentrated and purified by silica gel
chromatography (1:1 EtOAc/hexanes) to produce 26 as a colorless
1
semisolid (44 mg, 70% yield, Rf ) 0.25). H NMR (400 MHz,
1
CDCl3): δ 9.22 (br s, 0.8H), 5.62 (br s, 0.2H), 5.18 (br m, 0.7H),
4.28-4.14 (m, 2H), 3.95 (br s, 0.3H), 3.22-3.04 (ABm, 1H), 2.80-
2.64 (ABm, 1H), 2.65-2.64 (m, 2H), 2.08-2.04 (m, 1H), 1.89-
1.88 (m, 1H), 1.46 (s, 12H), 1.44 (s, 12H). 13C NMR (100 MHz,
CDCl3): δ 171.4, 167.4, 155.5, 111.3, 82.2, 79.9, 79.5, 53.5, 34.2,
33.0, 28.7, 28.3, 28.0, 27.0, 26.0. HRESI-MS: m/z calcd for
C20H36N2O8SNa+ (M + Na+) 487.2085, found 487.2075.
6.0. H NMR (400 MHz, D2O): δ 4.09 (d, J ) 4.4 Hz, 1H), 3.86
(quintet, J ) 4.4 Hz, 1H), 3.68 (t, J ) 6.0 Hz, 1H), 2.65 (dd, J )
14.0 Hz, 4.0 Hz, 1H), 2.58-2.51 (m, 3H), 2.02-1.92 (m, 2H). 13
C
NMR (100 MHz, D2O): δ 174.2, 170.1, 72.8, 71.1, 53.9, 33.0,
30.3, 27.4. HRESI-MS: m/z calcd for C8H15N2O6S- (M - H-)
267.0651, found 267.0656.
Methyl (4R,5S)-5-Hydroymethyl-2,2-dimethyl[1,3]dioxolane-
4-carboxylate (23). To a solution of dimethyl (-)-2,3-O-isopro-
pylidene-L-tartrate (22) (0.95 mL, 5.0 mmol) in MeOH (25 mL)
was added 85% KOH pellet (0.33 g, 5.0 mmol). The solution was
stirred overnight at rt under an argon atmosphere. The solvent was
removed by rotary evaporation, and the residue was kept under
high vacuum for 3 h. The resultant white solid was mixed with
anhydrous THF (9.8 mL) and treated with 1 M BH3 in THF solution
(6.5 mL, 6.5 mmol) in an ice-water bath. The reaction mixture
was stirred for 8 h. Saturated NaHCO3 solution was slowly added
to the above mixture until bubbling stopped. The solvent was
removed by rotary evaporation, and the remaining aqueous phase
was extracted with EtOAc (100 mL). The organic phase was dried
over MgSO4 and concentrated. The residue was purified by silica
gel chromatography (1:1 EtOAc/hexanes) to provide 23 as a
(2S)-2-Amino-4-[(2R,3R)-2,3-dihydroxy-3-N-hydroxycarbam-
oylpropylmercapto]butyric Acid (11). Compound 26 (42 mg, 0.09
mmol) was dissolved in 1 mL of TFA, and the solution was stirred
at rt for 1 h. TFA was removed by rotary evaporation, and the
remaining residue was treated with 1 N HCl for 4 h at rt. The
mixture was concentrated, and the crude product was purified on
a silica gel column eluted with 30% H2O in CH3CN to produce a
1
white solid (24 mg, quantitative yield, Rf ) 0.34). H NMR (400
MHz, D2O): δ 4.25 (d, J ) 2.8 Hz, 1H), 4.02-3.98 (m, 1H), 3.79-
3.76 (m, 1H), 2.75-2.67 (m, 2H), 2.64 (t, J ) 7.6 Hz, 2H), 2.15-
2.00 (m, 2H). 13C NMR (100 MHz, D2O): δ 174.1, 170.9, 72.0,
70.6, 53.8, 33.6, 30.3, 27.2. HRESI-MS: m/z calcd for C8H15N2O6S-
(M - H+) 267.0651, found 267.0628.
D-Erythronohydroxamic Acid (12). This compound was syn-
1
colorless oil (0.34 g, 36% yield over two steps, Rf ) 0.29). H
thesized according to a literature procedure.31
NMR (400 MHz, CDCl3): δ 4.48 (d, J ) 7.6 Hz, 1H), 4.27-4.23
(m, 1H), 3.96 (dd, J ) 12.4 Hz, 3.2 Hz, 1H), 3.81 (s, 3H), 3.76
(dd, J ) 12.4 Hz, 3.2 Hz, 1H), 1.91 (br s, 0.8H), 1.50 (s, 3H), 1.48
(s, 3H).
(2S)-2-Amino-6-{2-[(5S)-5-amino-5-carboxypentylcarbamoyl-
methyldithio]acetylamino}hexanoic Acid (33). To a solution of
2-mercaptoacetic acid (14 µL, 0.20 mmol) in MeOH (0.5 mL) was
added H2O2 (20 µL, 0.18 mmol). The mixture was stirred for 1 h
at rt, and the solvent was evaporated. The white solid obtained was
kept under high vacuum for 6 h and mixed with l-NR-Boc-Lys-O-
tBu (61 mg, 0.20 mmol), followed by the addition of CH2Cl2 (1.5
mL), dicyclohexylcarbodiimide (42 mg, 0.21 mmol), and (dim-
ethylamino)pyridine (24 mg, 0.20 mmol). The reaction mixture was
stirred under argon for 18 h at rt. The mixture was filtered, and the
white precipitate was rinsed with EtOAc. The filtrate was pooled,
concentrated, and treated with TFA (0.8 mL) for 30 min. After
rotary evaporation of TFA, the residue was washed with CHCl3
and purified by silica gel chromatography (eluted with 30% H2O
in CH3CN) to afford 33 as a white solid (13 mg, 30% yield, Rf )
0.23). 1H NMR (400 MHz, D2O): δ 3.60 (t, J ) 6.4 Hz, 2H), 3.34
(s, 4H), 3.14 (t, J ) 6.8 Hz, 4H), 1.78-1.68 (m, 4H), 1.51-1.44
(m, 4H), 1.36-1.26 (m, 4H). 13C NMR (100 MHz, D2O): δ 174.8,
171.4, 54.7, 41.0, 39.5, 30.1, 28.0, 21.8. HRESI-MS: m/z calcd
for C16H30N4O6S2Na+ (M + Na+) 461.1504, found 461.1472.
(2S)-2-Amino-6-{3-[2-(5S)-5-amino-5-carboxypentylcarbamoyl-
ethyldithio]propionylamino}hexanoic Acid (34). This compound
was prepared in a manner similar to that of compound 33. 1H NMR
(400 MHz, D2O): δ 3.59 (t, J ) 6.0 Hz, 2H), 3.08 (t, J ) 6.8 Hz,
4H), 2.83 (t, J ) 6.8 Hz, 4H), 2.54 (t, J ) 6.8 Hz, 4H), 1.78-1.70
(m, 4H), 1.45-1.37 (m, 4H), 1.31-1.25 (m, 4H). 13C NMR (100
MHz, D2O): δ 174.7, 174.1, 54.7, 39.0, 35.0, 33.4, 30.1, 28.0, 27.1,
21.8. HRESI-MS: m/z calcd for C18H34N4O6S2Na+ (M + Na+)
489.1812, found 489.1801.
Methyl (4R,5R)-5-Bromomethyl-2,2-dimethyl[1,3]dioxolane-
4-carboxylate (24). To a solution of alcohol 23 (279 mg, 1.47
mmol) in CH2Cl2 (15 mL) were added triphenylphosphine (0.40 g,
1.53 mmol) and carbon tetrabromide (0.51 g, 1.53 mmol) in an
ice-water bath. The solution was stirred overnight at rt under argon.
The solvent was removed by rotary evaporation, and the residue
was purified by column chromatography (1:3 EtOAc/hexanes) to
give 24 as a colorless oil (0.23 g, 62% yield, Rf ) 0.47). 1H NMR
(250 MHz, CDCl3): δ 4.45-4.38 (m, 2H), 3.81 (s, 3H), 3.70-
3.64 (ABm, 1H), 3.60-3.54 (ABm, 1H), 1.52 (s, 3H), 1.46 (s, 3H).
Methyl (4R,5R)-5-[(3S)-3-tert-Butoxycarbonyl-3-tert-butoxy-
carbonylaminopropylsulfanylmethyl]-2,2-dimethyl[1,3]dioxolane-
4-carboxylate (25). Freshly prepared l-BocNHCH(CH2CH2SH)-
CO2tBu29 (142 mg, 0.48 mmol) was dissolved in dry DMF (2.5
mL), and the solution was purged with argon for 10 min and chilled
in an ice-water bath. A 1.5 M solution of LDA in cyclohexane
(0.6 mL, 0.9 mmol) was added, and the resulting solution was stirred
at 0 °C for 10 min, followed by the addition of bromide 24 (129
mg, 0.51 mmol) in DMF (1.5 mL). This reaction mixture was stirred
at rt for 2 days. The solvent was then removed by rotary
evaporation, and the yellow residue was suspended in saturated
NH4Cl solution and extracted with EtOAc (3 × 10 mL). The organic
layer was dried over MgSO4, concentrated, and purified by silica
gel chromatography (1:3 EtOAc/hexanes) to afford 25 as a colorless
1
semisolid (111 mg, 50% yield, Rf ) 0.32). H NMR (400 MHz,
CDCl3): δ 5.08 (br d, J ) 7.2 Hz, 0.8H), 4.37-4.32 (m, 2H), 4.25-
4.24 (m, 1H), 3.78 (s, 3H), 2.94 (dd, J ) 14.4 Hz, 3.6 Hz, 1H),
(2S)-2-Amino-6-{4-[3-(5S)-5-amino-5-carboxypentylcarbamoyl-
propyldithio]butyrylamino}hexanoic Acid (35). This compound

3010 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Shen et al.
was prepared in a manner similar to that of 33. 1H NMR (400 MHz,
D2O ): δ 3.67 (t, J ) 6.0 Hz, 2H), 3.13 (t, J ) 6.8 Hz, 4H), 2.68
(t, J ) 6.8 Hz, 4H), 2.43-2.28 (m, 4H), 2.09-1.91 (m, 4H), 1.85-
1.77 (m, 4H), 1.52-1.37 (m, 4H), 1.35-1.28 (m, 4H). 13C NMR
(100 MHz, D2O): δ 175.9, 174.8, 54.7, 38.9, 37.1, 34.3, 30.1, 28.7,
28.0, 24.7, 21.8. HRESI-MS: m/z calcd for C20H38N4O6S2Na+ (M
+ Na+) 517.2125, found 517.2105.
mM-1 cm-1).30 Release of adenine base was also indicated by the
appearance of a UV-active species on TLC (CHCl3/CH3OH, 1:1;
Rf ) 0.72).
LuxS Inhibition Assay. Assay reactions were performed in a
buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl, 150
µM DTNB, and varying concentrations of SRH and inhibitors at
room temperature. The reaction was initiated by the addition of
LuxS (final concentration of 0.2-0.4 µM) and monitored continu-
ously at 412 nm (ꢀ ) 14150 M-1 cm-1) in a Perkin-Elmer λ25
UV-vis spectrophotometer. The initial rates were calculated from
the early regions of the progress curves (∼3 min for BsLuxS
reactions and ∼30 s for VhLuxS and EcLuxS reactions). The
remaining activity as a percentage was plotted against each inhibitor
concentration to obtain IC50 values. The KI values were calculated
from the equations V/V0 ) (KM + [S])/{KM (1 + [I]/KI) + [S]}
and KI ) IC50/(1 + [S]/KM), where V and V0 are the reaction rates
in the presence and absence of inhibitors, [S] and [I] are the
concentrations of SRH and inhibitor, respectively, and KM is the
Michaelis-Menten constant.
UV-Vis Spectroscopy. Co-BsLuxS was diluted in a buffer
containing 50 mM HEPES (pH 7.0), 150 mM NaCl, and varying
amounts of inhibitor to a final concentration of 49 µM. UV-vis
spectra were recorded 5 min or 1 h after addition of the inhibitors
at room temperature. The KI values were determined by fitting the
absorbance increase ∆A at a specific wavelength between 650 and
700 nm against the inhibitor concentration [I] using the equation
∆A ) ∆Amax[I]/(KI + [I]), where ∆Amax is the maximal increase in
absorbance at saturating concentrations of the inhibitor.
Formation and Isolation of the DPD Derivative. For reactions
that involve slow substrates or weakly active LuxS mutants, the
DPD formed was converted into its quinoxaline derivative and
isolated by HPLC.16,29,32 Briefly, 1 mM SRH, 5 mg/mL LuxS, and
1 mM 1,2-phenylenediamine were mixed in a pH 7 buffer and
incubated for 24 h. Another equivalent of 1,2-phenylenediamine
was then added, and the resulting solution was incubated at pH
4-5 for another 24 h followed by ethyl acetate extraction. The
crude product was purified by reversed-phase HPLC on a semi-
preparative C18 column (Vydac) with a 30 min gradient of 10-
100% acetonitrile in water (monitored at 240 nm). The desired
compound eluted at ∼12 min (∼40% acetonitrile).
tert-Butyl (2S)-2-tert-Butoxycarbonylamino-6-[3-(4-methoxy-
phenyl)oxaziridin-2-yl]hexanoate (36). To a solution of L-NR-Boc-
Lys-tBu (0.18 g, 0.60 mmol) in CH2Cl2 (6 mL) were added
4-methoxybenzadehyde (78 µL, 0.63 mmol) and Na2SO4 (0.84 g,
5.9 mmol). The reaction mixture was stirred at rt for 16 h under an
argon atmosphere. The mixture was filtered, and the solid was
washed with EtOAc. The filtrate was concentrated under reduced
pressure to give a yellow oil. The oil was dissolved in CH2Cl2 (3
mL) and mixed with m-chloroperbenzoic acid (141 mg, 0.63 mmol)
dissolved in CH2Cl2 (3 mL) at -78 °C. The reaction mixture was
stirred overnight at rt and under argon. Saturated NaHCO3 solution
was added, and the mixture was extracted with CH2Cl2 (3 × 20
mL). The organic phase was combined, dried over MgSO4, and
concentrated. The crude product was purified by silica gel chro-
matography (15% EtOAc in hexane) to give a colorless oil (126
1
mg, 48% yield over two steps, Rf ) 0.13). H NMR (400 MHz,
CDCl3): δ 7.31 (d, J ) 8.4 Hz, 2H), 6.88 (d, J ) 8.0 Hz, 2H),
5.04 (d, J ) 7.6 Hz, 1H), 4.42 (s, 1H), 4.18-4.13 (m, 1H), 3.79
(s, 3H), 3.00-2.94 (m, 1H), 2.70-2.65 (m, 1H), 1.81-1.59 (m,
6H), 1.44 (s, 9H), 1.43 (s, 9H). 13C NMR (100 MHz, CDCl3): δ
172.1, 161.2, 155.6, 129.1, 127.0, 114.1, 82.0, 80.5 (d), 79.8, 61.9
(d), 55.5, 54.0, 33.0 (d), 28.5, 28.2, 27.8, 23.2 (d).
tert-Butyl (2S)-2-tert-Butoxycarbonylamino-6-hydroxyamino-
hexanoate (37). To a mixture of compound 36 (126 mg, 0.29 mmol)
in MeOH (3 mL) was added hydroxylamine hydrochloride (31 mg,
0.43 mmol). The reaction mixture was stirred under argon gas for
12 h at rt. After rotary evaporation of the solvent, the residue was
extracted with EtOAc/H2O. The aqueous phase was adjusted to pH
≈ 7 with a saturated NaHCO3 solution and extracted with CH2Cl2
(3 × 20 mL). The CH2Cl2 solution was combined, dried over
MgSO4, and concentrated. The crude product was purified by silica
gel chromatography with EtOAc and then 10% MeOH in EtOAc
as eluent to give a colorless oil (54 mg, 59% yield, Rf ) 0.54). 1H
NMR (400 MHz, CDCl3): δ 5.14 (d, J ) 8.0 Hz, 1H), 4.17-4.13
(m, 1H), 2.95-2.85 (m, 2H), 1.81-1.48 (m, 6H), 1.45 (s, 9H),
1.43 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 172.3, 155.7, 82.0,
79.9, 53.9, 53.7, 33.1, 28.5, 28.2, 26.7, 23.8.
Crystallization and X-ray Diffraction. Co-BsLuxS was co-
crystallized with inhibitors (compounds 10 and 11) by the hanging
drop vapor diffusion method. The well solution consisted of 0.1 M
HEPES (pH 7.0) and 2.2 M ammonium sulfate. LuxS protein
(10 mg/mL) was mixed with 5.4 mM inhibitor, 25 mM Tris-HCl
(pH 8.0), and 100 mM NaCl. The crystals were transferred to a
cryoprotectant solution consisting of 0.1 M HEPES (pH 7.0),
2.5 M ammonium sulfate, 15% sucrose, and 5.4 mM inhibitor,
mounted in nylon loops, and frozen in liquid nitrogen. X-ray
diffraction data were collected at -180 °C using a Rigaku
RUH3RHB rotating anode generator and an R-AXIS IV++ image
plate detector. The data were processed with CrystalClear software
(Molecular Structure Corp.). Crystallographic refinement using
CNS33 began with the structure of the BsLuxS 2-ketone intermediate
complex (PDB code 1YCL) after removal of all water molecules,
Co2+, and the 2-ketone intermediate. The conjugate gradient
minimization and individual temperature factor protocols were used
with a maximum likelihood target and overall anisotropic temper-
ature factor and bulk solvent corrections to the data. The inhibitors
were added to the model at the later stages of refinement after all
of the protein atoms, Co2+, and several water molecules were
positioned. Stereochemical parameters for refinement of the inhibi-
tor were generated using the Dundee PRODRG2 server.34 Model
building used the program O,35 and figures were generated with
MOLSCRIPT36 and RASTER3D.37
(2S)-2-Amino-6-(N-formyl-N-hydroxylamino)hexanoic Acid
(16). To ice-cooled Ac2O was added dropwise formic acid, and
the mixture was stirred for 10 min in the ice bath. The mixture
was then warmed to 50 °C, stirred for another 5 min, and chilled
again in the ice-water bath. A solution of compound 37 (54 mg,
0.17 mmol) in CH2Cl2 (1.5 mL) was added dropwise. The reaction
mixture was stirred for 13 h at rt under an argon atmosphere. The
solution was washed with saturated NaHCO3 and the 1 N HCl
solutions. The organic layer was dried over MgSO4 and concen-
trated. The residue was treated with TFA (1 mL) for 40 min. TFA
was removed by rotary evaporation and high vacuum. The crude
product was washed with CHCl3 six times and purified by silica
gel chromatography (30% H2O in CH3CN as eluent) to afford a
1
white solid (10 mg, 77% yield). H NMR (400 MHz, D2O): δ
7.81 (s, 1H), 3.61 (t, J ) 6.0 Hz, 1H), 3.49-3.43 (m, 2H), 1.78-
1.74 (m, 2H), 1.62-1.55 (m, 2H), 1.30-1.21 (m, 2H). 13C NMR
(100 MHz, D2O): δ 174.7, 163.6, 54.6, 50.2, 29.9, 25.8, 21.2.
HRESI-MS: m/z calcd for C7H14N2O4Na+ (M + Na+) 213.0851,
found 213.0868.
δ-Ribosylornithine (17) and S-Ribosylcysteine (18). Com-
pounds 17 and 18 were prepared by treating sinefungin and
S-adenosylcysteine (10 mM), respectively, with recombinant nucle-
osidase Pfs16 (10 µM) in a buffer containing 50 mM HEPES (pH
7.0) and 150 mM NaCl for 2 h at room temperature. The reaction
progress was monitored spectrophotometrically on the basis of the
absorption difference between adenosine and adenine (∆ꢀ276 ) -1.4
Acknowledgment. This work was supported by a grant from
the National Institutes of Health (AI 062901).
Supporting Information Available: NMR, MS, and HPLC data
of target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.

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