1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli dihydrodipicolinate synthase

Article abstract of DOI:10.1016/j.bmc.2012.01.045

Dihydrodipicolinate synthase is a key enzyme in the lysine biosynthesis pathway that catalyzes the condensation of pyruvate and aspartate semi-aldehyde. A series of phenolic ketoacid derivatives that mimic the proposed enzymatic intermediate were designed

Full text of DOI:10.1016/j.bmc.2012.01.045

Bioorganic & Medicinal Chemistry 20 (2012) 2419–2426
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Bioorganic & Medicinal Chemistry
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1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli
dihydrodipicolinate synthase
Berin A. Boughton a,b, Lilian Hor a,b, Juliet A. Gerrard c, Craig A. Hutton a,b,
⇑
a School of Chemistry, The University of Melbourne, VIC 3010, Australia
b Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC 3010, Australia
c Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Dihydrodipicolinate synthase is a key enzyme in the lysine biosynthesis pathway that catalyzes the con-
densation of pyruvate and aspartate semi-aldehyde. A series of phenolic ketoacid derivatives that mimic
the proposed enzymatic intermediate were designed as potential inhibitors of this enzyme and were syn-
thesized from simple precursors. The ketoacid derivatives were shown to act as slow and slow-tight bind-
ing inhibitors. Mass spectrometric experiments provided further evidence to support the proposed model
of inhibition, demonstrating either an encounter complex or a condensation product for the slow and
slow-tight binding inhibitors, respectively.
Received 13 December 2011
Revised 17 January 2012
Accepted 26 January 2012
Available online 10 February 2012
Keywords:
Dihydrodipicolinate synthase
Inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
Slow-binding
Slow-tight binding
Mass spectrometry
1. Introduction
angles ranging from 100 to 179°. We hypothesized that (1,3-phe-
nylene)bisketoacid 7 and (2-hydroxy-1,3-phenylene)bisketoacid 8
Lysine and its immediate precursor meso-diaminopimelate
(meso-DAP) are essential elements of bacterial peptidoglycan and
proteins.1,2 As the biosynthetic pathway to lysine is only found in
plants and bacteria, it has attracted considerable attention as a tar-
get for the design and synthesis of novel herbicides and antibiot-
ics.3–6 The first committed step in the biosynthesis of lysine is
the condensation of pyruvate 1 and (S)-aspartate semi-aldehyde
(ASA, 2), catalyzed by the enzyme dihydrodipicolinate synthase
(DHDPS) (Fig. 1).7
The DHDPS-catalyzed reaction is initiated by condensation of
pyruvate 1 with an active site lysine residue (Lys161 in Escherichia
coli DHDPS) forming a Schiff base. Subsequent tautomerization to
the enamine 5 and aldol-type reaction with ASA 2 then generates
the acyclic enzyme-bound intermediate 6 (Fig. 2). Transimination
of the acyclic intermediate 6 yields the cyclic alcohol HTPA 3, with
simultaneous release of the active site lysine residue.
would mimic the enzyme bound intermediate and would present
the ketoacid functional groups in a constrained, extended confor-
mation (Fig. 3B). Thus, we first synthesized the parent bis(keto-
acid)
7 and derivatives 11–13 (Fig. 3C) and assayed these
compounds for inhibition of DHDPS activity.8 The compounds
showed moderate inhibitory activity against DHDPS (Table 1),
and prompted further investigation into more functionalized ana-
logues that more closely mimic the structure of the enzyme-bound
intermediate 6; specifically, phenolic analogue 8 incorporating a
hydroxyl group to mimic that at C4 of the enzymatic intermediate
6. Herein we report the synthesis of phenol-containing compounds
8, 19 and 20 and detailed analysis of the inhibitory activity and
mechanism of action of these compounds and their simpler
progenitors through kinetic and mass spectrometric analysis.
We previously reported preliminary studies exploring a new
class of inhibitor based on analogy to the acyclic enzyme-bound
intermediate 6.8,9 The crystal structure of DHDPS soaked with
pyruvate and succinic semi-aldehyde (SAS) shows an enzyme-ad-
duct closely related to 6, which lacks the amino group (Fig. 3A).10
The adduct exists in an extended conformation with dihedral
⇑
Corresponding author. Tel.: +61 3 8344 2393; fax: +61 3 9347 8124.
E-mail address: chutton@unimelb.edu.au (C.A. Hutton).
Figure 1. Condensation of pyruvate 1 and ASA 2 to form HTPA 3, then dehydration
to form DHDP 4.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.045

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B. A. Boughton et al. / Bioorg. Med. Chem. 20 (2012) 2419–2426
Figure 2. Condensation of pyruvate 1 and ASA 2 to give HTPA 3 proceeds through
enamine 5 and enzyme-bound adduct 6.
Figure 4. Synthesis of bis(ketoacid)phenol 8; (i) TMS-acetylene, CuI, Et3N, 16 h; (ii)
KOH, MeOH/H2O (1:1), 20 min; (iii) NBS, acetone, 3 h; (iv) NaHCO3/MgSO4, MeOH/
H2O (2:1), KMnO4, 1 h; (v) NaSEt, DMF, 80 °C, 3 h; (vi) LiOH, THF/H2O (1:1), 3 h; (vii)
H2NOHꢀHCl, pyridine, MeOH, 16 h.
synthesis of the model compound 7 in which the ketoacid groups
were generated by oxidation of bis(acetyl)benzene 9 (Fig. 3C).8
Accordingly, we required synthesis of the corresponding 2,6-
bis(acetyl)phenol 10. However, synthetic strategies towards this
target were not successful and an alternate strategy was adopted.
Li and Liu have reported the transformation of bromoacetylenes
into a-ketoesters through treatment with potassium permanganate
in aqueous methanol.12,13 Accordingly, our modified synthetic
strategy began by double Sonogashira coupling of diiodoanisole
14 with TMS-acetylene to give bisalkyne 15 in excellent yield
(Fig. 4). Silyl-deprotection was effected upon treatment with KOH
in aqueous methanol to give the bis-acetylene 16.14 Treatment with
N-bromosuccinimide gave bis(bromoalkyne) 17 in 85% yield.15
Oxidation of the bromoacetylenes with potassium permanganate
in buffered methanol/water then gave the 2,6-bis(ketoester) 18 in
quantitative yield.12,13 Thus, a rapid and high yielding pathway to
the core scaffold of the desired bis(ketoacid)phenols was enabled.
Removal of the methyl ether group to unveil the phenol was
next investigated. Treatment of 18 with BBr3, BCl3 or AlCl3 resulted
in only recovery of starting material, or decomposition under forc-
ing conditions. However, treatment of 18 with two equivalents of
sodium ethanethiolate in DMF gave the desired phenol 19, albeit
in moderate yield (36%), with recovery of starting material in
10–25% yield.16–19 Use of one equivalent of the thiolate gave a
Figure 3. (A) Enzyme tethered intermediate 6 and crystal structure of DHDPS
soaked with SAS + pyruvate showing extended structure of analogous condensed
adduct. Dihedral bond angles along carbons bonds: A = 171°, B = 179°, C = 100°,
D = 133°11; (B) Retrosynthesis of bis(ketoacid) derivatives 7, 8 from bis(acetyl)ben-
zene synthons 9, 10; (C) Synthesis of bis(ketoacid) 7 and derivatives via oxidation of
1,3-bis-acetylbenzene 9 with SeO2.8
2. Results and discussion
2.1. Synthesis
Our initial strategy toward synthesis of the phenolic bis(keto-
acid)
8 was based on analogy to our previously developed
Table 1
Inhibition kinetics of phenolic bis(ketoacid) derivatives 8 and 18–19, compared with deoxy-analogues 7 and 11–138
Compound
Screening assay—% inhibition at 5 mMa
Time dependent inhibition
Inhibition type
Slowb
Kai pp = 2.96 mM
None
7
49%
11
12
2%
15%
N/A
Slowb
Kai pp = 0.33 mM
34% at 50 mM, t = 60 min
None
13
18
19
23%
None
10%
N/A
N/A
Kai pp = 12.0 mM
Kꢁi app = 0.04 mM
Slow-tightc
Slow-tightc
Estimated Kai pp = 11.8 mM
Kꢁi app = 0.29 mM
8
10%
a
b
c
Initial screening assays were conducted by incubating DHDPS with inhibitor for 1 min prior to assay.
See Eq. II.
See Eqs. III and IV.

B. A. Boughton et al. / Bioorg. Med. Chem. 20 (2012) 2419–2426
2421
lower yield of the deprotected product, whereas use of greater than
two equivalents resulted in the formation of complex mixtures.
Presumably, several competing reactions are taking place in which
the thiolate reacts at the aromatic ether, the methyl esters, or a
combination of both leading to a mixture of demethylated prod-
ucts. Several alternate strategies were tested where the phenol
protecting group employed was a benzyl- or para-methoxyben-
zyl-(PMB) ether (see Supplementary data). However, the PMB
ether was incompatible with the permanganate oxidation step
and poor yields were encountered in the benzyl ether deprotection
step. Nevertheless, sufficient material could be produced from
methyl ether 18 to facilitate further functional group intercon-
versions. Saponification of the bis(ketoester) 19 gave the corre-
sponding bis(ketoacid) 8 (Fig. 5). Alternatively, treatment of the
bis(ketoester) 19 with hydroxylamine hydrochloride in the pres-
ence of pyridine provided the bis(oxime)phenol 20, as a 1:1 mix-
ture of (Z,Z):(Z,E) isomers.
2.2. Enzyme inhibition assays
Kinetic assays of DHDPS activity are generally conducted using
the ‘coupled assay’, in which the product of the DHDPS-catalyzed
reaction is converted by dihydrodipicolinate reductase (DHDPR)
to THDP, with concomitant conversion of NADPH to NADP+, moni-
tored at 340 nm. Analysis of the inhibitory activity of the 2,
6-disubstituted phenols 8 and 18–20 against DHDPS was compli-
cated by their high UV absorbances at 340 nm. Accordingly,
time-dependent assays were modified to include a serial dilution
of a stock solution of DHDPS incubated with inhibitor, prior to
addition to substrates.
An initial screen was conducted in which the activity of the
enzyme was determined upon treatment with compounds 8 and
18–19 at 5 mM. The methyl ether 18 showed no inhibitory activity,
whereas the phenols 8 and 19 showed low levels of inhibition at
time t = 0 (Table 1). Due to production of only small amounts of
the separated (E,Z)- and (Z,Z)-bis(oxime)phenols 20, these com-
pounds remain to be tested in kinetic assays. Given that phenols
8 and 19 showed some inhibitory activity and are analogous to
the model series demonstrating time dependent inhibition, they
were next investigated for time-dependent inhibition.
Though only weak initial inhibition was observed, the bis(keto-
ester)phenol 19 showed time dependent inhibition, with residual
enzyme activity decreasing over time, at all concentrations of
inhibitor tested, relative to a control with no inhibitor present
(Fig. 5). Interestingly, residual activity curves display decreasing
values of v0 (i.e., y-intercept, or residual activity at t = 0, <1), sug-
gesting a slow-tight binding mechanism.20,21 Copeland describes
a simple method for estimating Kai pp—which represents the initial
fast, reversible inhibition—by plotting the values of the y-inter-
cepts on a logarithmic scale.21 A standard isotherm is then fitted
to the data and the value of Kai pp obtained from the midpoint of
the isotherm: using this method the Kai pp of 19 was determined
to be 15.3 mM. A plot of kobs (rate of inactivation) versus [19]
(concentration of inhibitor) displays a hyperbolic curve (Fig. 5C),
also typical of slow-tight binding.21 Eq. III allows for accurate
determination of Kai pp according to the slow-tight binding model.
Fitting the data for 19 to Eq. III derived Kai pp = 12.0 mM, which
correlates closely with the estimated value. From Eq. III was also
determined the reverse rate, k4 = 0.9 ꢂ 10ꢃ5 sꢃ1. This is consistent
with the observation of
a small residual rate present after
long incubation times (30–60 min). From Eq. IV was determined
Kꢁi app—which represents the slow-tight binding portion of inhibi-
tion—which was calculated for 19 to be 0.04 mM.
Analysis of the inhibitory activity of the bis(ketoacid) 8 was per-
formed in the same manner as for bis(ketoester) 19 (Fig. 6). How-
ever, the high acidity required an increase in the concentration of
buffer used to 500 mM (pH 8.0 at 4 °C HEPES buffer). Compound
8 also had the highest UV absorbance and required two twenty-
fold serial dilutions immediately prior to measurement of residual
rate. The bis(ketoacid) 8 displays time-dependent inhibition, which
was fitted to both irreversible and slow-tight binding modes, with
slow-tight binding being a better fit (Fig. 6A).20,21 Extrapolation of
the isotherm generated by plotting the y-intercepts versus [8] on a
logarithmic scale gave an estimated Kai pp = 11.8 mM (Fig. 6B). High-
er concentrations of inhibitor could not be assayed due to its high
UV absorbance. The plot of kobs versus [8] shows a linear relation-
ship from which it is difficult to differentiate slow binding from
slow-tight binding (Fig. 6C). In this case important information
from mass spectrometric experiments (see below) demonstrate
that 8 forms a covalent adduct with the enzyme, suggesting that
Figure 5. (A) Residual activity of DHDPS upon incubation with bis(ketoester) 19.
[19]: s = 0.5 mM, d = 1.0 mM, j = 2.5 mM, N = 5.0 mM, h = 10.0 mM,
D = 25.0 mM;
.
(B) Estimation of Kiapp; (C) Plot of kobs versus [19], used to determine Kapp
i

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B. A. Boughton et al. / Bioorg. Med. Chem. 20 (2012) 2419–2426
Figure 7. Substrate protection of enzyme inactivation: d = no inhibitor;
j = +bis(ketoester)19; h = +bis(ketoester)19 + 10 mM pyruvate 1; N = +bis(keto-
acid)8; = +bis(ketoacid) 8 + 10 mM pyruvate 1.
D
change in the enzyme. With identification of a series of compounds
that inhibit DHDPS with a slow-tight binding mode, we were
interested in exploring the nature of the tight-binding interaction
by mass spectrometric studies. The compounds possess
a-keto- or
a-oximo-esters or acids that could potentially condense with the
active site lysine residue in a manner similar to that of the
substrate, pyruvate.22–25 Initial binding would provide an en-
zyme–inhibitor (or encounter) complex. Nucleophilic addition of
the active site lysine e-amino group into the C@O (or C@N) bond
would then generate a tetrahedral hemi-aminal intermediate. Elim-
ination of either water or hydroxylamine from the keto or oxime
derivatives, respectively, would lead to formation of an enzyme–
inhibitor Schiff-base adduct. Either the hemi-aminal intermediate
or Schiff base could potentially represent the tightly bound form
of the enzyme–inhibitor adduct. Mass spectrometric analysis was
employed to distinguish between these species, with a simple
encounter complex expected to show a mass corresponding to
[enzyme + inhibitor], whereas a Schiff base adduct would display
a lower mass corresponding to the loss of water or hydroxylamine.
Initial investigations of the 1,3-disubstituted benzenes 7, 12 and
13 demonstrated that they formed simple [enzyme + inhibitor]
encounter complexes (Fig. 8). In each case the native enzyme
monomer [M+H]+ was the dominant peak in the spectrum. A smal-
ler peak corresponding to [M+I+H]+ was observed.
In marked contrast to the 1,3-disubstituted benzenes, the 2,6-
disubstituted phenols 8, 19 and 20 display a more complex interac-
tion with DHDPS (Fig. 9). The phenolic bis(ketoacid) 8 shows two
adducts: a minor encounter complex at m/z 31509 ([M+8+H]+)
and a predominant condensation product at m/z 31491 ([M+
8ꢃH2O]+H+). This result provides support for the assignment of
slow-tight binding kinetics to inhibitor 8. The corresponding
bis(ketoester) 19 displays only an encounter complex at m/z
31537 ([M+19+H]+), with no condensation product observed. Incu-
bation of DHDPS with bisoxime 20 displays several adducts, with
the major species being the condensation product at m/z 31534
([M+20ꢃNH2OH]+H+). Two other small peaks are present in the
mass spectrum; at m/z 31518, corresponding to DHDPS + 248 amu,
corresponding to a species where one of the oximes has condensed
with the enzyme and the second oxime group has hydrolyzed to the
ketone (i.e., [M+20ꢃ(2 ꢂ H2NOH)+H2O]+H+). The second, smaller
peak at m/z 31505 is consistent with condensation of 20 with the
enzyme and hydrolysis of both esters [M+20ꢃH2NOHꢃ(2 ꢂ
MeOH)+(2 ꢂ H2O)+H]+. Although inhibition by the bisoxime 20
was not subject to kinetic analysis, the mass spectral results suggest
that it behaves in a similar manner to the bis(ketoacid) 8 and
bis(ketoester) 19, displaying covalent attachment to the enzyme
Figure 6. (A) Residual activity of DHDPS upon incubation with bis(ketoacid) 8.
[8]: s = 1.0 mM, d = 2.5 mM, h = 5.0 mM, j = 7.5 mM,
D
= 10.0 mM; (B) Estimation
.
of Kiapp; (C) Plot of kobs versus [8], used to determine Kapp
i
the inhibitor is acting in a slow-tight binding manner rather than a
slow-binding manner. Fitting the data for 8 to Eq. IV, enables der-
ivation of Kꢁi app = 0.29 mM.
Substrate protection experiments demonstrate that pyruvate 1
protects the enzyme from inactivation by 8 and 19, confirming that
8 and 19 are competing with pyruvate for the active site (Fig. 7).
2.3. Mass spectrometric analysis
A slow-tight binding mode of enzyme inhibition generally in-
volves initial binding of the inhibitor followed by ‘transition’ to a
tighter mode of binding, for example by inducing a conformational

B. A. Boughton et al. / Bioorg. Med. Chem. 20 (2012) 2419–2426
2423
Figure 8. Deconvoluted mass spectra showing native DHDPS = 31270, (A) DHDPS + ketoacid 7 = 31492, (B) DHDPS + oxime acid 13 = 31522, (C) DHDPS + oxime ester
12 = 31550.
and presumably slow-tight binding. Interestingly, these results
demonstrate that introduction of the phenolic group promotes
condensation with the active site residue, demonstrating the
importance of the inclusion of the hydroxyl functionality in order
to mimic that at the 4-position of the carbon backbone in enzymatic
intermediate 6.
4.2. Synthetic procedures
4.2.1. 2,6-Bis((trimethylsilyl)ethynyl)anisole (15)
A 3-neck r.b.f. equipped with a nitrogen inlet and seals was
flame dried and flushed with nitrogen, then charged with 2,6-dii-
odoanisole 14 (1.00 g, 2.78 mmol), copper iodide (57.0 mg,
0.29 mmol, 10 mol %), trans-dichloro-bis(triphenylphosphine) pal-
ladium (101 mg, 0.13 mmol, 4.8 mol %) and triethylamine
(25 mL). The reaction flask was flushed with nitrogen for 15 min,
then trimethylsilylacetylene (1.9 mL, 12.9 mmol) was added via
syringe. The reaction was stirred for 16 h at rt under nitrogen, then
was suspended between EtOAc (150 mL) and HCl (1 M, 100 mL).
The organic phase was separated then washed with HCl (1 M,
2 ꢂ 75 mL), brine (100 mL), saturated NaHCO3 (100 mL), brine
(100 mL), dried (MgSO4), filtered and concentrated in vacuo to give
15 as a pale brown oil (770 mg, 92%). 1H NMR (500 MHz, CDCl3): d
7.37 (d, 2H, J = 7.9 Hz), 6.95 (t, 1H, J = 7.9 Hz), 4.02 (s, 3H), 0.25 (s,
18H). 13C NMR (125 MHz, CDCl3): d 162.8, 134.0, 123.2, 117.3,
100.5, 99.2, 60.9, ꢃ0.15. vmax (thin film)/cmꢃ1 3073 (w, br), 2959
(w, s), 2899 (w, s), 2846 (w, s), 2153 (m), 1461 (m), 1411 (m),
1248 (m). MS-ESI (m/z): [M+H]+ 301.1. MS-EI (m/z):300 [M+ꢀ],
285 [M+ꢀꢃCH3]. HRMS-ESI (m/z): [M+H]+ calcd for C17H25OSi2
301.1438; found 301.1437.
3. Conclusions
In summary, new constrained bis(ketoacid) and bis(oximino-
acid) derivatives have been synthesized and assayed for inhibition
of DHDPS. These compounds display time-dependent inhibition
and undergo condensation with the enzyme, generating an
enzyme-bound adduct that closely resembles the enzymatic inter-
mediate. Kinetic studies show these inhibitors act in a slow-tight
binding manner that has not previously been observed for inhibi-
tors of DHDPS. Inclusion of hydroxyl functionality in phenols 8
and 19 in order to better mimic the enzymatic intermediate results
in a switch of enzyme inhibition mode to a slow-tight binding
model. Mass spectrometric studies support the assignment of
slow-tight binding inhibition, through evidence of condensation
of the inhibitors with the active site lysine residue.
4. Experimental section
4.2.2. 2,6-Diethynylanisole (16)
To
a solution of 2,6-bis((trimethylsilyl)ethynyl)anisole 15
4.1. Materials and methods
(101 mg, 0.335 mmol) in MeOH (20 mL) under nitrogen was added
KOH (0.1 M, 7.0 mL, 0.7 mmol, 2.2 equiv). The reaction was stirred
for 20 min, then was concentrated in vacuo to half its volume, di-
luted with HCl (1 M, 20 mL) and extracted with CH2Cl2
Unless otherwise stated, all chemicals were purchased from Sig-
ma–Aldrich or Novabiochem. (see Supplementary data for general
experimental procedures).

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B. A. Boughton et al. / Bioorg. Med. Chem. 20 (2012) 2419–2426
Figure 9. Deconvoluted mass spectra displaying native DHDPS = 31270; (A) DHDPS + bis(ketoacid) 8—H2O = 31491, DHDPS + bis(ketoacid) 8 = 31509; (B) DHDPS + bis(keto-
ester) 19 = 31537; (C) DHDPS + bis(oxime) 20—NH2OH = 31534, and hydrolyzed condensation products at 31505 & 31518.
(3 ꢂ 15 mL). The combined organic extracts were dried (MgSO4),
filtered, then concentrated in vacuo to give the bis-acetylene 16
as a pale brown oil (48 mg, 92%). 1H NMR (500 MHz, CDCl3): d
7.46 (d, 2H, J = 7.6 Hz), 7.00 (t, 1H, J = 7.6 Hz), 4.06 (s, 3H), 3.29
(s, 2H). 13C NMR (125 MHz, CDCl3): d 163.1, 134.7, 123.4, 116.3,
81.9, 79.2, 61.4.vmax (thin film)/cmꢃ1 3289 (s), 2942 (w, br), 1463
(s), 1414 (s), 1251 (m), 1227 (m). MS-EI (m/z): 156[M+ꢀ], 141
[M+ꢀꢃCH3],126 [M+ꢀꢃOMe]. HRMS-ESI (m/z): [M+H]+ calcd for
0.39 mmol) and MgSO4 (233 mg, 0.94 mmol) in H2O (14 mL). The
reaction was cooled to 0 °C and stirred for 15 min, then KMnO4
(149.8 mg, 0.95 mmol, 4 equiv) was added in portions over 1 h.
The mixture was stirred for 1 h at 0 °C, then was diluted with
H2O (100 mL) and extracted with EtOAc (3 ꢂ 70 mL). The com-
bined organic extracts were washed with brine (5 ꢂ 60 mL), dried
(MgSO4), filtered and concentrated in vacuo to give 18 as a clear oil
(66 mg, 99%). 1H NMR (500 MHz, CDCl3): d 8.07 (d, 2H, J = 7.6 Hz),
7.41 (t, 1H, J = 7.6 Hz), 3.94 (s, 6H), 3.79 (s, 3H). 13C NMR (100 MHz,
CDCl3): d 185.4, 164.0, 162.2, 136.8, 128.3, 124.8, 66.1, 52.9. vmax
(thin film)/cmꢃ1 3457 (w, br), 2957 (w), 2926 (w), 1732 (s, br),
1693 (s), 1683 (s), 1608 (m), 1463, (m), 1438 (m), 1421 (m),
1234 (s, br), 1151 (m), 1097 (m). MS-ESI (m/z): [M+H]+ 281.1.
HRMS-ESI (m/z): [M+H]+ calcd for C13H13O7 281.0655; found
281.0654.
C11H9O 157.0647; found 157.0645.
4.2.3. 2,6-Bis(bromoethynyl)anisole (17)
To 2,6-diethynyl anisole 16 (47 mg, 0.30 mmol) in acetone
(15 mL) under argon was added N-bromosuccinimide (126 mg,
0.71 mmol), the reaction was then stirred for 3 h at rt, then diluted
with H2O (50 mL) and extracted with CH2Cl2 (3 ꢂ 30 mL). The com-
bined organic extracts were washed with saturated NaHCO3
(2 ꢂ 50 mL), dried (MgSO4), filtered and concentrated in vacuo to
give 17 as a pale brown solid (80 mg, 85%); mp 80–82 °C. Analytical
samples were prepared by column chromatography on silica gel
(25% EtOAc/Petroleum Spirits (40–60)). 1H NMR (500 MHz, CDCl3):
d 7.39 (d, 2H, J = 7.9 Hz), 6.98 (t, 1H, J = 7.9 Hz), 4.03 (s, 3H). 13C
NMR (125 MHz, CDCl3): d 163.1, 134.4, 123.3, 116.8, 75.6, 61.6,
54.2. vmax (thin film)/cmꢃ1 2940 (w, br), 1462 (s), 1414 (s), 1236
(s), 1076 (m), 999 (s). MS-EI (m/z): 312, 314, 316 [M+ꢀ], 297, 299,
301[M+ꢀꢃCH3]. HRMS-ESI (m/z): [M+H]+ calcd for C11H7Br2O
312.8858, 314.8843; found 312.8858, 314.8834.
4.2.5. Dimethyl 2,20-(2-hydroxy-1,3-phenylene)bis(2-oxoace-
tate) (19)
To a stirred solution of 18 (712 mg, 2.54 mmol) in DMF (20 mL)
was added sodium ethanethiolate (372 mg, 4.42 mmol). The reac-
tion was stirred for 3 h at 80 °C under nitrogen, then a further addi-
tion of sodium ethanethiolate (50 mg, 0.59 mmol) was added. The
reaction was stirred overnight at 80 °C under nitrogen, then diluted
with HCl solution (1 M, 100 mL) and extracted with CH2Cl2
(4 ꢂ 100 mL). The combined organic extracts were dried (MgSO4),
filtered and concentrated in vacuo. The crude yellow product
(842 mg) was purified by chromatography on silica gel (50%
EtOAc/Petroleum Spirit (40–60)) to give a yellow oil, which crystal-
lized on standing to give 19 as pale yellow crystals (242 mg, 36%
yield); mp 67–68 °C. 1H NMR (400 MHz, CDCl3): d 12.01 (s, 1H),
8.15 (d, 2H, J = 8.0 Hz), 7.14 (t, 1H, J = 8.0 Hz), 3.99 (s, 6H).
4.2.4. Dimethyl 2,20-(2-methoxy-1,3-phenylene)bis(2-oxoace-
tate) (18)
To 2,6-bis(bromoethynyl)anisole 17 (74 mg, 0.24 mmol) in
MeOH (25 mL) was added
a solution of NaHCO3 (33 mg,

B. A. Boughton et al. / Bioorg. Med. Chem. 20 (2012) 2419–2426
2425
13C NMR (100 MHz, CDCl3): d 187.3, 163.7, 163.12, 138.9, 120.1,
120.0, 53.1. vmax (thin film)/cmꢃ1 3087 (w, br), 2957 (w), 1739
(s), 1680 (m), 1645 (m), 1609 (m), 1584 (m), 1431 (m), 1318 (m,
br), 1216 (s). HRMS-ESI (m/z): [M+H]+ calcd for C12H11O7
267.0499; found 267.0499. HRMS-ESI (m/z): [MꢃH]ꢃ calcd for
would not limit the rate of reaction over the 2 min measurement.
Control assays for initial screening and time dependent inhibition
were conducted under standard modified assay conditions where
DHDPS (810
with DMSO (90
conducted by incubating a solution of DHDPS (810
200 mM pH 8.0, buffer) with inhibitor (90 L, made up in 100%
l
L, in HEPES, 200 mM pH 8.0, buffer) was incubated
L) prior to assay. Initial screening assays were
L, in HEPES,
l
C12H9O7 265.0353; found 265.0350.
l
l
4.2.6. 2,20-(2-Hydroxy-1,3-phenylene)bis(2-oxoacetic acid) (8)
DMSO) at a final concentration of 5 mM for 1 min prior to assay.
Substrate protection experiments were conducted by incubating
DHDPS with inhibitor and pyruvate (10 mM).
To a solution of dimethyl 2,20-(2-hydroxy-1,3-phenylene)bis(2-
oxoacetate) 19 (10 mg, 37.5 lmol) in dioxane (2 mL) was added a
solution of LiOH (6 mg, 0.25 mmol) in H2O (1 mL). The reaction
was stirred for 3 h then diluted with HCl solution (1 M, 20 mL)
and extracted with EtOAc (3 ꢂ 10 mL). The combined organic ex-
tracts were dried (MgSO4), filtered and concentrated in vacuo to
give 8 as a pale brown oil (6.0 mg, 67%). 1H NMR (500 MHz, CDCl3):
d 12.03 (s, 1H), 8.45 (d, 2H, J = 8.0 Hz), 7.17 (t, 1H, J = 8.0 Hz). 13C
NMR (100 MHz, d6-DMSO): d 189.5, 165.4, 161.6, 137.7, 122.1,
119.5. vmax (thin film)/cmꢃ1 2857 (w, br), 2512 (w, br), 1903 (w,
br), 1672 (s, br), 1609 (s), 1475 (w), 1436 (s), 1279 (s), 1230 (s),
1184 (m), 1156 (m), 1023 (s), 972 (s, br), 764 (m), 668 (m).
HRMS-ESI (m/z):[MꢃH]ꢃ calcd for C10H5O7 237.0040; found
237.0038.
4.4. Analysis of enzyme kinetic data
Results were analysed by the methods of Copeland,20,21 where
the fractional velocities (vt/v0) of pre-incubated solutions of DHDPS
at differing concentrations of inhibitor were determined. The ob-
served decay curve for each concentration of inhibitor was fitted
to Eq. I, to determine kobs. A plot of kobs versus [I] was fitted to
Eqs. II, III, IV to determine values koff, k3, k4, Kapp and Kiꢁapp for
i
slow-binding or slow-tight binding modes of inhibition (see
Fig. 10 for description of kon, koff, k3, k4.
vt
v0
obsꢀtÞ
¼ eðꢃk
ðIÞ
ðIIÞ
4.2.7. Dimethyl 2,20-(2-hydroxy-1,3-phenylene)bis-(2-(hydroxy-
imino)-acetate) (20)
Slow binding inhibition kinetics
To a solution of 19 (31 mg, 116
added hydroxylamine hydrochloride (33 mg, 474
l
mol) in dry MeOH (15 mL) was
mol) and pyri-
ꢀ
ꢁ
½Iꢄ
l
kobs ¼ koff 1 þ
Kai pp
dine (2 drops). The reaction was stirred under nitrogen at rt over-
night. Then the mixture was concentrated in vacuo and
resuspended between HCl solution (1 M, 20 mL) and EtOAc
(20 mL). The organic phase was separated and further washed with
HCl solution (1 M, 2 ꢂ 10 mL), then dried (MgSO4), filtered and
concentrated in vacuo to give 20 as a brown oil (29 mg, 84% yield)
as a 1:1 mixture of diastereomers (Z,Z):(E,Z). (Z,Z) Diasteromer: 1H
NMR (500 MHz, CDCl3): d 10.41 (s, 1H), 8.88 (br s, 2H), 7.36 (d,
2H, J = 7.8 Hz), 6.96 (t, 1H, J = 7.8 Hz), 3.95 (s, 6H). 13C NMR
(100 MHz, CDCl3): 162.8, 155.8, 151.1, 131.0, 119.8, 117.4, 52.8.
vmax (thin film)/cmꢃ1 3328.8 (m, br), 3037.8 (w, br), 2956.9 (w),
1728.1 (s, br), 1603.0 (w, br), 1435.9 (m), 1323.3 (m, br), 1236.3
(s), 1177.7 (w), 1111.7 (m), 1091.9 (m), 997.1 (s), 910.4 (w),
840.2, (w), 731.2 (s).(E,Z) Diasteromer: 1H NMR (500 MHz, CDCl3):
d 10.28 (s, 1H), 8.92 (br s, 1H) 8.67 (br s, 1H), 7.36 (dd, 1H,
J1 = 7.5 Hz, J2 = 2.0 Hz), 7.13 (dd, 1H, J1 = 7.5 Hz, J2 = 2.0 Hz), 6.99
(t, 1H, J = 7.5 Hz), 3.99 (s, 3H), 3.86 (s, 3H). 13C NMR (125 MHz,
CDCl3): d 163.9, 162.2, 154.8, 154.0, 148.2, 132.4, 130.1, 119.2,
118.2, 114.4, 53.1, 52.8. vmax (thin film)/cmꢃ1 3328.4 (m, br),
2958.7 (w), 1733.5 (s, br), 1630.5 (w), 1604.7 (w), 1587.4 (w),
1436.9 (s), 1322.1 (m, br), 1239.7 (s), 1193.0 (w), 1177.2 (w),
1112.2 (s), 1091.3 (m), 1021.4 (m, br), 909.4 (w), 842.3 (w),
730.1 (s). MS-ESI (m/z): [M+H]+ 297.27. HRMS-ESI (m/z): [MꢃH]ꢃ
calcd for C12H11N2O7 295.0571; found 295.0568.HRMS-ESI (m/z):
[M+Na]+ calcd for C12H11N2NaO7 319.0536; found 319.0540.
Slow-tight binding inhibition kinetics.
ꢀ
ꢁ
k3 ꢂ ½Iꢄ
kobs ¼ k4 þ
ðIIIÞ
Kai pp þ ½Iꢄ
Slow-tight binding inhibition kinetics.
2
3
½Iꢄ
i
1 þ Kapp
1 þ
4
5
kobs ¼ k4
ðIVÞ
½Iꢄ
Kꢁi app
4.5. Mass spectrometry
All analyses were performed on an Agilent 6520 ESI-qTOF LC/
MS Mass Spectrometer coupled to an Agilent 1100 LC system (Agi-
lent, Palo Alto, CA). The instrument was operated in either positive
or negative mode with: drying gas flow: 7 L minꢃ1, nebuliser:
30 psi, drying gas temp: 150–300 °C, Vcap: 3000–4000 V,
Fragmentor: 25–215 V, Skimmer: 65 V, OCT RFV: 750 V. Scan range
acquired: 100–3000 m/z. Mass spectra were deconvoluted using
Agilent MassHunter Qualitative Analysis Build 3.0 software.
DHDPS samples (10
night at rt with (1
l
L) were incubated for either 10 min or over-
l
L) solutions of each respective inhibitor (made
up in 100% DMSO)or control DMSO (1
ESI-qTOF.
lL) before injection into the
4.3. Assay for monitoring DHDPS activity
UV–vis spectra and kinetic data were collected on either a Hew-
lett Packard 8452A Diode Array spectrophotometer with a circulat-
ing water bath to maintain a constant temperature of 30 °C or a
Cary 50 Bio UV–vis Spectrophotometer equipped with a Haake
P5/DC10 circulating water bath to maintain a constant tempera-
ture of 30 °C. The concentration of (S)-ASA was determined as pre-
viously described.26 Modified assay conditions were utilized to
determine rates of inactivation of phenolic inhibitors.8,9 The con-
centration (activity) of DHDPS was optimised under control condi-
tions (see below) to ensure an optimal rate where substrates
Figure 10. Scheme of slow and slow-tight binding inhibition showing initial on/off
rates to form EI complex, with slow-tight enzyme isomerization k3 to form EꢁI
complex with slow reverse rate k4.

2426
B. A. Boughton et al. / Bioorg. Med. Chem. 20 (2012) 2419–2426
9. Boughton, B. A.; Griffin, M. D.; O’Donnell, P. A.; Dobson, R. C.; Perugini, M. A.;
Acknowledgments
Gerrard, J. A.; Hutton, C. A. Bioorg. Med. Chem. 2008, 16, 9975.
10. Blickling, S.; Renner, C.; Laber, B.; Pohlenz, H.-D.; Holak, T. A.; Huber, R.
Biochemistry 1997, 36, 24.
11. Structure provided through private correspondence with R. Huber.
12. Li, L.-S.; Wu, Y.-L. Tetrahedron Lett. 2002, 43, 2427.
13. Liu, K.-G.; Hu, S.-G.; Wu, Y.; Yao, Z.-J.; Wu, Y.-L. J. Chem. Soc., Perkin Trans. 1
2002, 1890.
14. Chimenti, F.; Bolasco, A.; Secci, D.; Chimenti, P.; Granese, A. Synth. Commun.
2004, 34, 2549.
15. Villeneuve, K.; Riddell, N.; Jordan, R. W.; Tsui, G. C.; Tam, W. Org. Lett. 2004, 6,
4543.
This work was supported by the Defense Threat Reduction
Agency (Project ID AB07CBT004) (C.A.H.) and the Royal Society of
New Zealand Marsden Fund (J.A.G. and C.A.H.). The authors thank
R.C.J. Dobson, S.R.A. Devenish, F.G. Pearce, S. Dommaraju and
M.A. Perugini for providing samples of DHDPS and DHDPR used
in enzyme assays, and for useful discussions.
16. Dodge, J. A.; Stocksdale, M. G.; Fahey, K. J.; Jones, C. D. J. Org. Chem. 1995, 60,
739.
Supplementary data
17. Feutrill, G. I.; Mirrington, R. N. Tetrahedron Lett. 1970, 1327.
18. Feutrill, G. I.; Mirrington, R. N. Aust. J. Chem. 1972, 25, 1719.
19. Feutrill, G. I.; Mirrington, R. N. Aust. J. Chem. 1972, 25, 1731.
20. Copeland, R. A. Enzymes: A Practical Introduction to Structure, Mechanism, and
Data Analysis, 2nd ed.; Wiley-VCH, Inc, 2000.
21. Copeland, R. A. Evaluation of Enzyme Inhibitors in Drug Discovery—A Guide for
Medicinal Chemists and Pharmacologists; Wiley-VCH, Inc, 2005.
22. Alexeev, D.; Baxter, R. L.; Campopiano, D. J.; Kerbarh, O.; Sawyer, L.; Tomczyk,
N.; Watt, R.; Webster, S. P. Org. Biomol. Chem. 2006, 4, 1209.
23. Aldini, G.; Granata, P.; Carini, M. J. Mass Spectrom. 2002, 37, 1219.
24. Yeboah, F. K.; Alli, I.; Yaylayan, V. A.; Konishi, Y.; Stefanowicz, P. J. Agric. Food
Chem. 2000, 48, 2766.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.045.
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