Design and synthesis of aromatic inhibitors of anthranilate synthase

Article abstract of DOI:10.1039/b510633h

Anthranilate synthase catalyses the conversion of chorismate to anthranilate, a key step in tryptophan biosynthesis. A series of 3-(1-carboxy-ethoxy) benzoic acids were synthesised as chorismate analogues, with varying functionality at C-4, the position of the departing hydroxyl group in chorismate. Most of the compounds were moderate inhibitors of anthranilate synthase, with inhibition constants between 20-30 μM. The exception was 3-(1-carboxy-ethoxy) benzoic acid, (C-4 = H), for which K₁ = 2.4 μM. These results suggest that a hydrogen bonding interaction with the active site general acid (Glu309) is less important than previously assumed for inhibition of the enzyme by these aromatic chorismate analogues. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b510633h

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A R T I C L E
Design and synthesis of aromatic inhibitors of anthranilate synthase†
Richard J. Payne,a Esther M. M. Bulloch,a Andrew D. Abellb and Chris Abell*a
a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, UK CB2
1EW. E-mail: ca26@cam.ac.uk; Fax: +44 1223 336362; Tel: +44 1223 336405
b Department of Chemistry, University of Canterbury, Christchurch, New Zealand
Received 26th July 2005, Accepted 16th August 2005
First published as an Advance Article on the web 9th September 2005
Anthranilate synthase catalyses the conversion of chorismate to anthranilate, a key step in tryptophan biosynthesis.
A series of 3-(1-carboxy-ethoxy) benzoic acids were synthesised as chorismate analogues, with varying functionality
at C-4, the position of the departing hydroxyl group in chorismate. Most of the compounds were moderate inhibitors
of anthranilate synthase, with inhibition constants between 20–30 lM. The exception was 3-(1-carboxy-ethoxy)
benzoic acid, (C-4 = H), for which KI = 2.4 lM. These results suggest that a hydrogen bonding interaction with the
active site general acid (Glu309) is less important than previously assumed for inhibition of the enzyme by these
aromatic chorismate analogues.
Anthranilate synthase represents the first committed step
Introduction
towards the biosynthesis of tryptophan.11 It is a multifunctional
The shikimate pathway is a major biosynthetic pathway utilised
enzyme composed of a small TrpG and large TrpE subunit
by plants, fungi, bacteria and protozoa to produce a range of
either as an ab dimer or a a2b2 heterotetramer. TrpG belongs to
important aromatic metabolites.1 This pathway has generated
the family of “triad” glutamine amidotransferases. The subunit
considerable interest as a target for potential herbicides2 and
hydrolyses the side chain amine of glutamine and transfers the
nascent ammonia through an intramolecular channel to the
antimicrobial agents.3,4
In E. coli the product of the pathway, chorismate (1), is a
substrate for five separate enzymes that are responsible for the
synthase active site of TrpE. The TrpE subunit catalyses the
production of anthranilate in two steps, (Scheme 1).12
production of a range of aromatic compounds, including the
aromatic amino acids phenylalanine, tyrosine and tryptophan
(Fig. 1).5 Three of the branchpoint enzymes; anthranilate
synthase, ADC synthase and isochorismate synthase, catalyse
mechanistically-related reactions6,7 and probably diverged from
a common ancestor.8–10
† Part II. For Part I see ref. 13.
Scheme 1 The two reactions catalysed by the TrpE subunit of
anthranilate synthase, (ammonia is produced by the TrpG subunit).
The first step is the reversible reaction of chorismate
(1) with ammonia to give the intermediate, 2-amino-2-
deoxyisochorismate (2, ADIC). The mechanism is thought to
proceed by attack of ammonia at C-2 of chorismate, and
concomitant loss of the C-4 hydroxyl as water via protonation by
Glu309 (Scheme 1).6 The second reaction is the elimination of
the enol-pyruvyl side chain from ADIC to produce the aromatic
product anthranilate (3).
We recently described the synthesis of a range of aromatic
inhibitors of anthranilate synthase.13 It was found that an
aromatic ring was a reasonable mimic for the cyclohexadiene
ring of the natural substrate, and that a lactyl side chain at
the C-3 position was a good replacement for the enol-pyruvyl
side chain. Racemic compounds 4–6 containing these features
exhibited inhibition constants ranging from 3–43 lM (Fig. 2).
Building on these findings we have developed a further series of
aromatic chorismate analogues, where the substituent at C-3 is
always the racemic lactyl side chain but where the substitution
Fig. 1 Chorismate-utilising enzymes.
at C-4 is varied.
T h i s j o u r n a l i s T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 2 9 – 3 6 3 5
3 6 2 9
©

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hydroxyl of the analogue is within hydrogen-bonding distance
from the carboxylate of Glu309. The hydroxyl of (R)-7 is in a
˚
˚
similar orientation but is closer to Glu309 (2.3 A cf. 2.5 A for
chorismate), suggesting a stronger hydrogen bond.
Synthesis of 4-substituted analogues
The synthesis involved initial methyl ester formation from 3-
hydroxy-4-methylbenzoic acid (13), followed by alkylation of
the 3-hydroxy functionality with methyl-2-bromopropionate to
introduce the lactyl side chain, (Scheme 2). The 4-methylbenzoic
acid analogue 11 was produced by treatment of 15 with aqueous
base.
Bromination of the 4-methyl group in 15 was achieved
using N-bromosuccinimide and AIBN in refluxing benzene, and
produced the desired bromide 16 in 64% yield. SN2 displacement
of the bromide with aqueous base occurred with concomitant
hydrolysis of the methyl esters to afford the 4-hydroxymethyl
diacid 7 in reasonable yield.
In order to synthesise mercaptomethyl analogue 9, bromide 16
was first treated with potassium thiolacetate in DMF, followed
by hydrolysis of the methyl esters and thiolacetate protecting
groups with aqueous potassium hydroxide, generating diacid 9
in 98% yield over the two steps.
The azide 10 was synthesised by SN2 displacement of the
bromide of 16 using sodium azide in DMF in 64% yield. Methyl
ester hydrolysis of 17 with aqueous potassium hydroxide gave
the 4-azidomethyl diacid 10 in 80% yield. Synthesis of the
aminomethyl analogue involved reduction of azide 17 to the
corresponding amine, and finally methyl ester deprotection.
The azide reduction was achieved by catalytic hydrogenation
using Pd black catalyst under one atmosphere of hydrogen. A
mixture of the desired 4-aminomethyl compound and the ring
closed lactam were formed. The mixture was further treated
with aqueous base to open up the lactam ring, and hydrolyse the
methyl esters to yield the desired aminomethyl dicarboxylate 8
in 54% yield over the two steps. Purification of 8 was achieved
by FPLC using a SourceQ anion exchange column.
Fig. 2 Inhibitors of S. marcescens anthranilate synthase.13
We now report a development of this study to systematically
explore the effect of changing the substituent at C-4.
Results and discussion
Design
C-4 is the site of departure of the hydroxyl group from
chorismate, and undergoes rehybridisation during the formation
of ADIC (2). It seemed probable that the enzyme would be
sensitive to the nature of the substituent at this position, and
preliminary evidence for this is seen in the relative potency of
the inhibitors 4–6, where there is a preference for a hydroxyl
group over an amino group. The design strategy was to hold the
main components (the C-1 carboxyl, the aromatic ring, the C-3
lactyl sidechain) constant, but introduce various functionality
at C-4 by making compounds 7–12 (Fig. 3).
Fig. 3 Proposed synthetic targets as anthranilate synthase inhibitors.
As the C-4 hydroxyl departs, the C–O bond will lengthen
and the hydroxyl will acquire a partial positive charge as it
is protonated by Glu309. We sought to design compounds
that would mimic these effects, e.g. by increasing the distance
between C-4 and the hydroxyl (7, R = CH2OH), and having
Synthesis of 12 began with methyl 3-hydroxybenzoate (18) and
involved alkylation with methyl 2-bromopropionate, followed
by ester hydrolysis to give the desired diacid 12 in 83% yield
(Scheme 3).
+
a positive charge near C-4 (8, R = CH2NH3 ). To probe the
space available and the importance of hydrogen bonding we
also made the mercaptomethyl (9, R = CH2SH), azidomethyl
(10, R = CH2N3), and methyl (11, R = CH3) analogues. Finally
the C-4 hydroxyl was replaced with a hydrogen (12), to allow
the binding due to the C-1 carboxylate and C-3 lactyl side chain
alone to be assessed.
Inhibition assays
The aromatic chorismate analogues were assayed against S.
marcescens anthranilate synthase. The production of anthrani-
late was detected fluorimetrically with excitation at 313 nm and
detection at 390 nm as reported previously.13 The inhibition
results are shown in Table 1. Intriguingly, analogues 7–11
exhibited very similar inhibition constants with KI values
ranging from 21–26 lM. The only difference between these
inhibitors is the substitution at C-4. This inhibition data suggests
a lack of sensitivity of the enzyme for the C-4 substituent of these
aromatic analogues.
Modelling of chorismate and analogues 7–11 into the active
site of S. marcescens anthranilate synthase using GOLD2.1,14
indicates that the C-4 substituents of the analogues are in close
proximity to Glu309 in silico. Fig. 4 shows (R)-7 docked into
the active site of anthranilate synthase and is overlayed with the
corresponding chorismate docking. It is apparent that the C-4
Table 1 Inhibition constants of C-4 functionalised aromatic inhibitors
against Serratia marcescens anthranilate synthase
Inhibitor
Substitution at C-4
KI (lM)
4
Hydroxy
Methoxy
Amino
Hydroxymethyl
Aminomethyl
Mercaptomethyl
Azidomethyl
Methyl
2.9 0.3
5
25
3
6
43 10
7
24
23
21
21
26
4
3
3
4
3
8
9
10
11
12
Hydrogen
2.4 0.3
Fig. 4 Chorismate (1) (purple) and (R)-7 (green) docked into the
active site of anthranilate synthase from Serratia marcescens (PDB code:
1I7Q).12,15
a KM (chorismate) = 3.7 0.5 lM, kcat = 5.6 s−1 @ 25 ◦C.13
3 6 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 2 9 – 3 6 3 5

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Scheme 2
In order to determine which enantiomer of 12 binds with
higher affinity to anthranilate synthase, enantiopure forms of 12
were synthesised. Treatment of commercially available (R) and
(S)-2-bromopropionic acids with diazomethane gave the corre-
sponding methyl esters (Scheme 4). Alkylation of 18 with (R)-
methyl 2-bromopropionate and (S)-methyl 2-bromopropionate,
under identical conditions to those employed for the synthesis
of racemic 12, gave the corresponding diesters. Subsequent
hydrolysis of the esters with aqueous potassium hydroxide gave
(R)-12 and (S)-12 in 50% and 72% yields respectively over the
three steps.
Scheme 3
Analogue 12, which has a hydrogen atom at C-4, is ten-
fold more potent than analogues 7–11, with a KI of 2.4 lM
and represents the most potent aromatic anthranilate synthase
analogue synthesised to date (Fig. 5). The 10-fold higher potency
of 12 compared with 7–11 may be due to a decrease in steric
bulk at C-4, or it may be that binding of 12 allows a water to
bind between C-4 and Glu309. At this stage it is not possible to
distinguish between these alternatives.
Scheme 4
Analogues (R)- and (S)-12 were assayed against S. marcescens
anthranilate synthase. (S)-12 was found to have a KI = 1.9
0.2 lM, and was slightly more potent than the (R)-enantiomer
(KI = 3.6 0.5 lM). This result indicates a relatively modest
sensitivity to the chirality of the side chain, but one that can be
incorporated in the design of future inhibitors.
Fig. 5 Least squares fitting for the reversible competitive inhibition of
anthranilate synthase by 12 (KI = 2.4 0.3 lM).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 2 9 – 3 6 3 5
3 6 3 1

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Over-expression and purification of anthranilate synthase
Conclusions
from S. marcescens was carried out as described previously.13
We have previously introduced aromatic chorismate analogues
as competitive inhibitors of anthranilate synthase. That study
established that a lactyl group bound well into the enol-pyruvyl
side chain binding site. In this paper we have explored the impor-
tance of the substituent in the position (C-4) corresponding to
the departing hydroxyl group in chorismate. It was assumed that
there would be important binding interactions to this position
and that these would be reflected in a sensitivity to the nature
of the C-4 substituent on the aromatic inhibitor. It has been
a major surprise to find that this is not the case, and that the
inhibition constants are almost invariant for a range of different
C-4 substituents. The biggest surprise was that removal of the C-
4 substituent gave the most potent inhibitor (compound 12). It
may be that having only hydrogen at C-4 allows a water molecule
to bind between C-4 and Glu309.
Gold docking
All ligands and the receptor were prepared using SYBYL6.5 and
used as MOL2 files.16 The ligands were prepared as carboxylate
anions and their structures were energy minimised using the
Tripos force-field Gasteiger-Huckel charges calculated prior
to docking. Each ligand was docked using GOLD2.1 in 25
independent genetic algorithm (GA) runs. For each of these,
a maximum number of 100 000 GA operations was performed
on a single population of 50 individuals.14 Operator weights for
crossover, mutation and migration in the entry box were used
as default parameters (95, 95 and 10 respectively), as well as the
˚
˚
hydrogen bonding (4.0 A) and van der Waals (2.5 A) parameters.
The position of the active site was introduced and the radius was
˚
set to 10 A, with the automatic active-site detection on.
There are several possible explanations for the observed lack
of sensitivity of the enzyme to the nature of the C-4 substituent.
The C-4 substituent projects out in the same plane as the
aromatic ring. This may be a poor mimic of the transition
state conformation in the conversion of chorismate to ADIC
(2) if chorismate adopts a conformation where the C-4 hydroxyl
moves to a more axial orientation prior to departure. If this is
the case, it may reflect a limitation in using aromatic analogues
as inhibitors of anthranilate synthase. It is also possible that the
inhibitor could be binding in a different orientation. Docking
studies identify another binding mode where the binding of
the C-1 and side chain carboxylates is reversed. However, this
places the C-4 substituent close to the magnesium ion, and it
would be surprising if variations of the C-4 substituent did not
modulate the inhibition constant. Whatever the origin of the
effect, the insensitivity to the C-4 substitution has important
consequences to the development of aromatic inhibitors of
anthranilate synthase.
Enzyme assays
Kinetic parameters for S. marcescens anthranilate synthase
were determined using the kinetic fluorescence assay developed
previously.13 The assay was carried out in 100 mM potassium
phosphate buffer (pH = 7); 10 mM magnesium chloride and
20 mM glutamine at 25 ◦C. Assays were initiated by the addition
of S. marcescens anthranilate synthase (final concentration =
17.3 nM). The formation of anthranilate was detected by
fluorescence (ex. 313 nm, em. 390 nm).13
Grafit17 software was used to construct Michaelis–Menton
plots of the kinetic data and carry out a least squares fitting for
the reversible competitive inhibition of anthranilate synthase.
The software was also utilised to calculate the inhibition
constants and the standard errors of these values.
3-Hydroxy-4-methyl benzoic acid methyl ester 14
Thionyl chloride (20 drops) was added dropwise to a suspension
of 3-hydroxy-4-methylbenzoic acid (2.4 g, 0.02 mol) in freshly
distilled methanol (40 ml). The reaction mixture was heated
to reflux under nitrogen for 16 h. The solution was allowed
to cool to 22 ◦C before the solvent was removed in vacuo to
give the crude product as a white solid. Purification by column
chromatography eluting with 3 : 1 v/v petroleum ether–ethyl
acetate gave 14 as a white solid. (2.66 g, quant.).
Experimental
All organic solvents were freshly distilled prior to use and milli-
Q deionised water was used for all biochemical work. Analytical
thin layer chromatography was carried out on commercial
silica gel 60 0.25 mm plates using either UV absorption or
potassium permanganate stain, (3 g potassium permanganate,
20 g potassium carbonate, 5 ml of 5% sodium hydroxide,
300 ml water), for visualisation. RF values are quoted with
respect to the solvent system used to develop the plate. Column
chromatography was carried out using 230–400 mesh silica gel
60. Unless otherwise stated petroleum ether refers to the fraction
collected between 40–60 C. H NMR spectra were recorded
on a Bruker AM-400 spectrometer in deuterated solvents, as
indicated. 13C NMR spectra were recorded on a Bruker AM-
400 spectrometer operating at 100 MHz. All chemical shifts
are quoted in parts per million (ppm) d Coupling constants
for 1H NMR spectroscopy are assigned where possible and are
given in Hz. Infrared spectra were recorded on a Perkin Elmer
Spectrum One FTIR spectrometer using attenuated transmit-
tance reflectance (ATR). High resolution mass spectrometry
was carried out using a Micromass Quadrapole-Time of Flight
(Q-Tof) spectrometer. FPLC purification was carried out on
a SourceQ anion exchange resin column using a gradient from
10 mM ammonium bicarbonate to 1 M ammonium bicarbonate.
Liquid-chromatography mass-spectrometry (LCMS) was car-
ried out using an Alliance HT Waters 2795 Separations Module
coupled to a Waters Micromass ZQ Quadrapole Mass Analyzer.
Samples were detected using a photomultiplier detection system.
Samples were run on a gradient from 10 mM ammonium acetate
containing 0.1% formic acid to 95% acetonitrile over a period of
8 min. Purified anthranilate synthase from Serratia marcescens
were stored in aliquots as concentrated solutions in buffer at
−78 ◦C.
RF (3 : 1 petroleum ether–ethyl acetate) = 0.39; mmax (ATR):
=
3266 (br OH stretch), 2962 (Ar C–H stretch), 1695 (C O, ar),
−1
1
=
=
1596, 1513 (C C, ar), 1436 (C C) cm ; H NMR (CDCl3) d
2.28 (3H, s, CH3), 3.89 (3H, s, CH3), 5.51 (1H, br s, OH), 7.17
(1H, d, J 8.0 Hz, ArH, C-5), 7.50 (2H, m, ArH, C-6 and C-
2); 13C NMR (CDCl3) d 16.5, 52.5, 116.1, 122.3, 129.3, 130.5,
131.3, 154.5, 167.7; LCMS (M− = 165.3, MH+ = 167.2), ret
time = 3.7 min; HRMS calcd for C9H10O3Na: MNa+, 189.0530.
Found: MNa+,189.0528.
◦
1
3-(1-Methoxycarbonyl-ethoxy)-4-methyl benzoic acid methyl
ester 15
Methyl-2-bromopropionate (0.87 ml, 7.83 mmol) was added
dropwise to a solution of 14 (1 g, 6.02 mmol), anhydrous
potassium carbonate (1.66 g, 0.01 mol), sodium iodide (18 mol%,
0.16 g, 1.08 mmol) in acetone (50 ml) and the reaction heated to
70 ◦C for 16 h. The reaction was allowed to cool to 22 ◦C before
the solvent was removed in vacuo. The residue was redissolved
in ethyl acetate and washed with water (50 ml), brine (50 ml),
dried (MgSO4) and the solvent removed in vacuo. The product
was purified by column chromatography eluting with 4 : 1 v/v
50/70 petroleum ether–ethyl acetate to afford 15 as a white solid
(1.19 g, 78%).
RF (4 : 1 petroleum ether/ethyl acetate) = 0.66; mmax (ATR):
−1
1
=
=
1733, 1715 (C O, str), 1610, 1584, 1507 (C C, ar) cm ; H
NMR (CDCl3) d 1.62 (3H, d, J 6.7 Hz), 2.30 (3H, s, CH3), 3.74
3 6 3 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 2 9 – 3 6 3 5

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(3H, s, CO2Me), 3.85 (3H, s, CO2Me), 4.84 (1H, q, J 6.7 Hz,
CH), 7.18 (1H, d, J 7.9 Hz, H-5), 7.39 (1H, d, J 1.2 Hz, H-2),
7.62 (1H, dd, J 7.9, 1.2 Hz, H-6); 13C NMR (CDCl3) d 16.5, 18.5,
52.0, 52.2, 72.6, 112.3, 122.7, 128.7, 130.8, 133.3, 155.6, 166.8,
172.3; HRMS calcd for C13H16O5Na: MNa+, 275.0895. Found:
MNa+, 275.0893.
3-(1-Carboxy-ethoxy)-4-mercaptomethyl benzoic acid 9
Potassium thiolacetate (99 mg, 0.87 mmol) was added to a 0 ◦C
solution of bromide 16 (200 mg, 0.61 mmol) in dry DMF (5 ml)
and the reaction stirred at 22 ◦C for 24 h. The reaction was
diluted with ethyl acetate (100 ml) and washed with brine (5 ×
50 ml). The organic fraction was dried (MgSO4) and the solvent
removed in vacuo. Purification by column chromatography
(eluent: 4 : 1 v/v petroleum ether–ethyl acetate) gave the diester
as a pale yellow oil. (194 mg, 98%).
3-(1-Carboxy-ethoxy)-4-methyl benzoic acid 11
Potassium hydroxide (0.19 g, 3.30 mmol) in milliQ water (5 ml)
was added dropwise to a solution of 15 (200 mg, 0.80 mmol) in
THF (5 ml) and the reaction was heated to 40 ◦C for 2 h. The
reaction was allowed to cool to 22 ◦C before dilution with water
(10 ml). The aqueous fraction was washed with ethyl acetate
(15 ml) before acidifying to pH 1 with 1 M HCl. The aqueous
fraction was extracted with ethyl acetate (2 × 15 ml). The organic
fractions were dried (MgSO4) before the solvent was removed in
vacuo to afford 11 as a white solid (168 mg, quant).
RF (4 : 1 v/v petroleum ether–ethyl acetate) = 0.30; mmax (ATR):
=
=
=
1756, 1719, 1687 (C O, str), 1621 (C C), 1580, 1503 (C C,
ar) cm−1; 1H NMR (CDCl3) d 1.63 (3H, d, J 6.8 Hz, CH3), 2.30
(3H, s, COCH3), 3.75 (3H, s, CO2Me), 3.86 (3H, s, CO2Me),
4.18 (1H, d, J 13.5 Hz, CHH), 4.22 (1H, d, J 13.5 Hz, CHH),
4.88 (1H, q, J 6.8 Hz, CH), 7.33 (1H, d, J 1.1 Hz, H-2), 7.39
(1H, d, J 7.9 Hz, H-5), 7.64 (1H, dd, J 7.9, 1.1 Hz, H-6); 13C
NMR (CDCl3) d 18.4, 28.0, 30.2, 52.1, 52.3, 72.7, 112.5, 122.8,
130.4, 130.6, 132.2, 155.1, 166.5,171.9, 195.2; LCMS: ret. time =
4.04 min; MH+ = 327.1; HRMS calcd for C15H22O6NS: MNH4 ,
+
mmax (ATR): 2845, 2631 (br, acid OH stretch), 1726, 1713, 1678
1
+
=
=
(C O, str), 1583, 1508 (C C, ar); H NMR (CD3OD) d 1.62
(3H, d, J 6.0 Hz, CH3), 2.30 (3H, s, CH3), 4.83 (1H, q, J 6.0 Hz,
CH), 7.21 (1H, d, J 7.8 Hz, H-5), 7.39 (1H, d, J 1.5 Hz, H-2),
7.62 (1H, dd, J 7.8, 1.5 Hz, H-6); 13C NMR (CD3OD) d 17.0,
19.2, 73.8, 113.6, 124.1, 130.8, 132.0, 134.5, 157.4, 170.0, 175.9;
HRMS calcd for C11H12O5Na: MNa+, 247.0582. Found: MNa+,
247.0578.
344.1162. Found: MNH4 , 344.1159.
Potassium hydroxide (41 mg, 0.73 mmol) in milliQ water (2 ml)
was added dropwise to a solution of the above diester (95 mg,
0.29 mmol) in THF (2 ml) and the reaction was stirred at 22 ◦C
for 3 h. The reaction was diluted with milliQ water (10 ml).
The aqueous fraction was washed with dichloromethane (10 ml)
before acidifying to pH 1 with 1 M HCl. The aqueous fraction
was extracted with ethyl acetate (2 × 15 ml), the organic fractions
were dried (MgSO4) before the solvent was removed in vacuo to
afford 9 as a white solid (75 mg, quant, 98% over the two steps).
4-Bromomethyl-3-(1-methoxycarbonyl-ethoxy) benzoic acid
methyl ester 16
=
mmax (ATR): 2987, 2901 br. (O–H acid str.), 1727, 1693 (C O,
−1
1
=
str), 1583, 1507 (C C, ar) cm ; H NMR (CD3OD) d 1.65 (3H,
d, J 6.8 Hz, CH3), 3.72 (1H, d, J 13.8 Hz, CHH), 3.78 (1H, d, J
13.8 Hz, CHH), 4.92 (1H, q, J 6.8 Hz, CH), 5.02 (1H, s, SH), 7.36
(1H, d, J 7.8 Hz, H-5), 7.42 (1H, d, J 1.4 Hz, H-2), 7.58 (1H, dd,
J 7.8, 1.4 Hz, H-6); 13C NMR (CD3OD) d 18.9, 24.0, 73.6, 113.8,
124.0, 130.6, 131.7, 137.4, 156.2, 169.3, 175.2; HRMS calcd for
A mixture of 15 (2.55 g, 0.01 mol), N-bromosuccinimide (1.78 g,
0.01 mol) and AIBN (5 mol%, 82 mg, 0.50 mmol) was dissolved
in freshly distilled benzene (65 ml). The reaction was heated to
◦
85 C under argon for 24 h. The reaction was allowed to cool
to 22 ◦C before dilution with dichloromethane (100 ml). The
organic fraction was washed with saturated sodium carbonate
solution (150 ml), brine (150 ml), dried (MgSO4) and the
solvent removed in vacuo. The product was purified by column
chromatography eluting with 9 : 1 v/v petroleum ether 50–70–
ethyl acetate to afford bromide 16 as a yellow oil (2.12 g, 64%).
RF (9 : 1 petroleum ether–ethyl acetate) = 0.12; mmax (ATR):
+
+
C11H16O5NS: MNH4 , 274.07443. Found: MNH4 , 274.0749.
4-Azidomethyl-3-(1-methoxycarbonyl-ethoxy) benzoic acid
methyl ester 17
−1
1
=
=
1736, 1717 (C O, str), 1610, 1582 1505 (C C, ar) cm ; H
NMR (CDCl3) d 1.72 (3H, d, J 6.7 Hz), 3.78 (3H, s, CO2Me),
3.91 (3H, s, CO2Me), 4.50 (1H, d, J 9.9 Hz, CHHBr), 4.75 (1H,
d, J 9.9 Hz, CHHBr), 4.95 (1H, q, J 6.7 Hz, CH), 7.42 (2H, m,
2 × ArH), 7.64 (1H, dd, J 7.9, 1.6 Hz, H-6); 13C NMR (CDCl3)
d 17.5, 26.5, 51.4, 51.5, 72.0, 112.2, 122.0, 130.1, 130.6, 131.0,
154.3, 165.2, 170.8; HRMS calcd for C13H15O5BrNa: MNa+,
353.0001. Found: MNa+, 352.9984.
Sodium azide (193 mg, 2.97 mmol) was added to a solution of
16 (490 mg, 1.48 mmol) in DMF (10 ml) and the reaction was
heated to 40 ◦C under argon for 16 h. The reaction was cooled
to 22 ◦C before dilution with ethyl acetate (40 ml). The organic
fraction was washed with water (5 × 40 ml), dried (MgSO4)
and the solvent removed in vacuo. The product was purified by
column chromatography eluting with 5 : 1 v/v petroleum ether
50–70–ethyl acetate to afford 17 as a colourless oil (0.30 g, 68%).
RF (5 : 1 v/v petroleum ether–ethyl acetate) = 0.29; mmax (ATR):
=
=
2110 (N3 stretch), 1745, 1698 (C O, str), 1612, 1583, 1502 (C C,
ar) cm−1; 1H NMR (CDCl3) d 1.67 (3H, d, J 6.8 Hz, CH3), 3.73
(3H, s, CO2Me), 3.88 (3H, s, CO2Me), 4.40 (1H, d, J 14.1 Hz,
CHH), 4.56 (1H, d, J 14.1 Hz, CHH), 4.92 (1H, q, J 6.8 Hz,
CH), 7.35 (1H, d, J 7.8 Hz, H-5), 7.41 (1H, d, J 1.4 Hz, H-
2), 7.66 (1H, dd, J 7.8, 1.4 Hz, H-6); 13C NMR (CDCl3) d
20.8, 52.2, 54.7, 75.1, 115.0, 125.4, 132.1, 132.5, 133.7, 157.7,
168.8, 174.2; HRMS calcd for C13H15N3O5Na: MNa+, 316.0909.
Found: MNa+, 316.0919.
3-(1-Carboxy-ethoxy)-4-hydroxymethyl benzoic acid 7
Potassium hydroxide (0.08 g, 1.36 mmol) in milliQ water (4 ml)
was added dropwise to a solution of 16 (150 mg, 0.45 mmol) in
◦
THF (4 ml) and the reaction was stirred at 22 C for 2 h. The
reaction was diluted with water (10 ml). The aqueous fraction
was washed with ethyl acetate (15 ml) before acidifying to pH 1
with 1 M HCl. The aqueous fraction was extracted with ethyl
acetate (2 × 15 ml), the organic fractions were dried (MgSO4)
before the solvent was removed in vacuo to afford 7 as an off
white solid (110 mg, 80%).
4-Azidomethyl-3-(1-carboxy-ethoxy) benzoic acid 10
=
mmax (ATR): 2927, 2542 (br, acid OH stretch), 1686 (C O,
1
=
str), 1581, 1506 (C C, ar); H NMR (CD3OD) d 1.62 (3H, d,
Potassium hydroxide (58 mg, 1.03 mmol) in milliQ water (2 ml)
was added dropwise to a solution of 17 (76 mg◦, 0.26 mmol) in
THF (2 ml) and the reaction was stirred at 22 C for 2 h. The
reaction was diluted with water (10 ml) and the aqueous fraction
was washed with ethyl acetate (15 ml) before acidifying to pH 1
with 1 M HCl. The aqueous fraction was extracted with ethyl
acetate (2 × 15 ml). The organic fractions were dried (MgSO4)
J 6.8 Hz, CH3), 4.68 (1H, d, J 14.6 Hz, CHH), 4.79 (1H, d,
J 14.6 Hz, CHH), 4.90 (1H, q, J 6.8 Hz, CH), 7.43 (1H, d,
J 1.3 Hz, H-2), 7.50 (1H, d, J 7.8 Hz, H-5), 7.65 (1H, dd, J
7.8, 1.3 Hz, H-6); 13C NMR (CD3OD) d 21.5, 62.8, 76.4, 116.1,
126.6, 131.2, 134.3, 139.6, 158.6, 172.1, 178.2; HRMS calcd for
C11H11O6Na: MNa+, 263.0532. Found: MNa+, 263.0537.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 2 9 – 3 6 3 5
3 6 3 3

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before the solvent was removed in vacuo to afford 10 as a white
solid (55 mg, 80%).
mmax (ATR): 2949, 2570 (br OH acid), 2093, 2068 (N3 stretch),
fractions were dried (MgSO4) before the solvent was removed in
vacuo to afford 12 as a white solid (350 mg, quant).
mmax (ATR): 2989, 2532 br. (O–H acid str.), 1682, 1610 (C O,
=
−1
1
−1
1
=
=
=
1703, 1692 (C O, str), 1584 (C C, ar) cm ; H NMR (CD3OD)
d 1.64 (3H, d, J 6.8 Hz, CH3), 4.39 (1H, d, J 14.0 Hz, CHH),
4.55 (1H, d, J 14.0 Hz, CHH), 4.92 (1H, q, J 6.8 Hz, CH), 7.37
(1H, d, J 7.8 Hz, H-5), 7.49 (1H, d, J 1.3 Hz, H-2), 7.62 (1H, dd,
J 7.8, 1.3 Hz, H-6); 13C NMR (CD3OD) d 17.2, 49.2, 72.3, 112.3,
122.2, 129.2, 129.7, 131.5, 155.4, 167.5, 173.6; HRMS calcd for
C11H11N3O5Na: MNa+, 288.0596. Found: MNa+, 288.0603.
str), 1583, 1492 (C C, ar) cm ; H NMR (CD3OD) d 1.58 (3H,
d, J 6.8 Hz, CH3), 4.82 (1H, q, J 6.8 Hz, CH), 7.08 (1H, ddd, J
0.8, 2.5, 8.0 Hz, ArH), 7.31 (1H, t, J 8.0 Hz, ArH), 7.51 (1H, dd,
J 1.3, 2.5 Hz, ArH), 7.61 (1H, dt, J 1.3, 8.0 Hz, ArH); 13C NMR
(CD3OD) d 18.7, 73.4, 116.6, 121.1, 123.7, 130.5, 133.0, 158.9,
+
169.5, 175.5; HRMS calcd for C10H14O5N: MNH4 , 228.0866.
+
Found: MNH4 , 228.0869.
4-Aminomethyl-3-(1-carboxy-ethoxy) benzoate 8
(R)-3-(1-Carboxy-ethoxy) benzoic acid (R)-12
A solution of 17 (0.15 g, 0.52 mmol) in freshly distilled methanol
(3 ml) was added to a suspension of palladium black (10 mg,
0.09 mmol) in freshly distilled methanol (2 ml) under a hydrogen
atmosphere. The reaction was stirred at 22 ◦C for 3 h. The
methanolic solution was passed through Celite and washed with
a further portion of methanol (10 ml). The solution was dried
(MgSO4) and the solvent removed in vacuo to afford the crude
product. This was dissolved in THF (2 ml) and a solution of
potassium hydroxide (87 mg, 1.55 mmol) in milliQ water (2 ml)
added dropwise. The reaction was stirred at 22 ◦C for 2 h. The
reaction was diluted with milliQ water (5 ml) and washed with
ethyl acetate (10 ml). The aqueous fraction was acidified to pH of
8 with 0.1 M HCl and the solution lyophilised. Purification was
achieved on a SourceQ anion exchange column using an FPLC
system (gradient from 10 mM ammonium bicarbonate to 1 M
ammonium bicarbonate) to afford 8 as the diammonium salt
as a white solid. (0.28 mmol, 54%, the yield was determined by
1H NMR spectroscopy using 3-(trimethylsilyl)-2,2,3,3-d4-acid
sodium salt as an internal standard).
An ethereal diazomethane (CAUTION) solution (generated
from Diazald)18 was added dropwise to a solution of (R)-(+)-
2-bromopropanoic acid (1 g, 6.54 mmol) at 0 ◦C until a bright
yellow colour persisted. The reaction was stirred at 22 ◦C for
2 h. The solvent was removed in vacuo to give an oil, which was
subsequently purified by kugelrohl distillation under reduced
pressure to afford the desired methyl ester as a colourless liquid
(1 g, 5.99 mmol). The resulting (R)-methyl 2-bromopropionate
(0.71 g, 4.28 mmol) was added dropwise to a solution of methyl
3-hydroxybenzoate (0.25 g, 3.29 mmol), anhydrous potassium
carbonate (0.45 g, 3.29 mmol) and sodium iodide (0.04 g,
0.30 mmol) in distilled acetone (7.5 ml) and the reaction was
heated at 70 ◦C under nitrogen for 24 h. The reaction was allowed
to cool to 22 ◦C before dilution with ethyl acetate (30 ml). The
organic fraction was washed with water (40 ml), brine (40 ml),
dried (MgSO4) and the solvent removed in vacuo. Purification
by column chromatography (eluent: 4 : 1 petroleum ether–ethyl
acetate) gave the diester as a colourless oil. (0.26 g, 67%).
1H NMR (CDCl3) d 1.59 (3H, d, J 6.8 Hz, CH3), 3.71 (3H, s,
CO2Me), 3.84 (3H, s, CO2Me), 4.79 (1H, q, J 6.8 Hz, CH), 7.04
(1H, ddd, J 1.3, 2.5, 8.0 Hz, ArH), 7.29 (1H, t, J 1.8 Hz, ArH),
7.48 (1H, dd, J 1.3, 2.5 Hz, ArH), 7.61 (1H, dt, J 1.3, 8.0 Hz,
ArH); 13C NMR (CDCl3) d 18.3, 52.0, 52.2, 72.5, 115.4, 120.2,
122.7, 129.5, 157.4, 166.5, 172.1.
Potassium hydroxide (188 mg, 3.03 mmol) in milliQ water
(2 ml) was added dropwise to a solution of the above diester
(200 mg, 0.84 mmol) in THF (2 ml) and the reaction was stirred
at 40 ◦C for 3 h. The reaction was diluted with water (5 ml) and
the aqueous fraction was washed with dichloromethane (8 ml)
before acidifying to pH 1 with 1 M HCl. The aqueous fraction
was extracted with ethyl acetate (2 × 8 ml). The organic fractions
were dried (MgSO4) before the solvent was removed in vacuo to
afford (R)-12 as a white solid (135 mg, 74%, 50% over the three
steps).
1
mmax (ATR): 3044, 2297, 1555, 1405 cm−1; H NMR (D2O) d
1.64 (3H, d, J 6.8 Hz, CH3), 4.07 (1H, d, J 13.3 Hz, CHH), 4.32
(1H, d, J 13.3 Hz, CHH), 4.83 (1H, q, J 6.8 Hz, CH), 7.40 (1H,
d, J 7.8 Hz, H–5), 7.49 (1H, d, J 1.4 Hz, H–2), 7.62 (1H, dd,
J 7.8, 1.4 Hz, H–6); 13C NMR (D2O) d 18.8, 39.6, 75.8, 113.3,
122.4, 124.9, 131.4, 138.4, 156.4, 173.9, 180.5; HRMS calcd for
C11H12NO5Na2: [M + Na2 + H]+, 284.0511. Found: [M + Na2 +
H]+, 284.0494.
3-(1-Carboxy-ethoxy) benzoic acid 12
Methyl 2-bromopropionate (478 ll, 4.28 mmol) was added
dropwise to a solution of methyl 3-hydroxybenzoate (0.50 g,
3.29 mmol), anhydrous potassium carbonate (0.91 g, 6.57 mmol)
and sodium iodide (0.09 g, 0.59 mmol) in distilled acetone (15 ml)
and the reaction was heated at 70 ◦C under nitrogen for 24 h. The
reaction was allowed to cool to 22 ◦C before dilution with ethyl
acetate (30 ml). The organic fraction was washed with water
(40 ml), brine (40 ml), dried (MgSO4) and the solvent removed
in vacuo. Purification by column chromatography (eluent: 4 : 1
petroleum ether–ethyl acetate) gave the diester as a colourless
oil. (0.65 g, 83%).
[a]2D5 −0.8 (c 1 in MeOH); 1H NMR (CD3OD) d 1.58 (3H, d, J
6.8 Hz, CH3), 4.82 (1H, q, J 6.8 Hz, CH), 7.08 (1H, ddd, J 0.8,
2.5, 8.0 Hz, ArH), 7.31 (1H, t, J 8.0 Hz, ArH), 7.51 (1H, dd, J
1.3, 2.5 Hz, ArH), 7.61 (1H, dt, J 1.3, 8.0 Hz, ArH); 13C NMR
(CD3OD) d 18.7, 73.4, 116.6, 121.1, 123.7, 130.5, 133.0, 158.9,
169.5, 175.5; LCMS: ret. time = 4.12 min; MH+ = 209.1.
RF (4 : 1 petroleum ether–ethyl acetate) = 0.33; mmax (ATR):
(S)-3-(1-Carboxy-ethoxy) benzoic acid (S)-12
−1
1
=
=
1756, 1719 (C O, str), 1587, 1489 (C C, ar) cm ; H NMR
(CDCl3) d 1.59 (3H, d, J 6.8 Hz, CH3), 3.71 (3H, s, CO2Me),
3.84 (3H, s, CO2Me), 4.79 (1H, q, J 6.8 Hz, CH), 7.04 (1H,
ddd, J 1.3, 2.5, 8.0 Hz, ArH), 7.29 (1H, t, J 1.8 Hz, ArH), 7.48
(1H, dd, J 1.3, 2.5 Hz, ArH), 7.61 (1H, dt, J 1.3, 8.0 Hz, ArH);
13C NMR (CDCl3) d 18.3, 52.0, 52.2, 72.5, 115.4, 120.2, 122.7,
129.5, 157.4, 166.5, 172.1; LCMS: ret. time = 3.70 min; MH+ =
(S)-12 was synthesised in an identical fashion to (R)-12 and was
produced as a white solid (150 mg, 72% over three steps).
[a]2D5 +1.0 (c 1 in MeOH); 1H NMR (CD3OD) d 1.58 (3H, d, J
6.8 Hz, CH3), 4.82 (1H, q, J 6.8 Hz, CH), 7.08 (1H, ddd, J 0.8,
2.5, 8.0 Hz, ArH), 7.31 (1H, t, J 8.0 Hz, ArH), 7.51 (1H, dd, J
1.3, 2.5 Hz, ArH), 7.61 (1H, dt, J 1.3, 8.0 Hz, ArH); 13C NMR
(CD3OD) d 18.7, 73.4, 116.6, 121.1, 123.7, 130.5, 133.0, 158.9,
169.5, 175.5; LCMS: ret. time = 4.13 min; MH+ = 209.1.
+
240.1; HRMS calcd for C12H18O5N: MNH4 , 256.1179. Found:
+
MNH4 , 256.1182.
Potassium hydroxide (375 mg, 6.05 mmol) in milliQ water
(4 ml) was added dropwise to a solution of the above diester
(400 mg, 1.67 mmol) in THF (4 ml) and the reaction was stirred
at 40 ◦C for 3 h. The reaction was diluted with water (10 ml)
and the aqueous fraction was washed with dichloromethane
(15 ml) before acidifying to pH 1 with 1 M HCl. The aqueous
fraction was extracted with ethyl acetate (2 × 15 ml). The organic
Acknowledgements
RJP and EMMB would like to thank the Gates Cambridge
Trust for PhD funding and the BBSRC for research support. We
would also like to thank Glen Spraggon (Genomics Institute of
the Novartis Research Foundation, San Diego, California) and
3 6 3 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 6 2 9 – 3 6 3 5

View Article Online
Charles Yanofsky (Stanford University, Stanford, California) for
kindly supplying the ptttSmEDC vector containing the Serratia
marcescens trpEGDC operon.
8 P. Goncharoff and B. P. Nichols, J. Bacteriol., 1984, 159, 57–
62.
9 B. A. Ozenberger, T. J. Brickman and M. A. McIntosh, J. Bacteriol.,
1989, 171, 775–783.
10 M. F. Elkins and C. F. Earhart, FEMS Microbiol. Lett., 1988, 56,
35–40.
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