Inhibition of chorismate-utilising enzymes by 2-amino-4-carboxypyridine and 4-carboxypyridone and 5-carboxypyridone analogues

Article abstract of DOI:10.1039/c004062b

Several 2-amino-4-carboxypyridine, 4- and 5-carboxypyridone-based compounds were prepared and tested against three members of the chorismate-utilising enzyme family, anthranilate synthase, isochorismate synthase and salicylate synthase. Most compounds exhibited low micromolar inhibition of these three enzymes. The most potent inhibitor was a 4-carboxypyridone analogue bearing a lactate side chain on the pyridyl nitrogen which exhibited inhibition constants of 5, 91 and 54 μM against anthranilate synthase, isochorismate synthase and salicylate synthase respectively. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c004062b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Inhibition of chorismate-utilising enzymes by 2-amino-4-carboxypyridine
and 4-carboxypyridone and 5-carboxypyridone analogues†
Richard J. Payne,*a,b Esther M. M. Bulloch,a Olivier Kerbarha and Chris Abell*a
Received 9th March 2010, Accepted 19th May 2010
First published as an Advance Article on the web 9th June 2010
DOI: 10.1039/c004062b
Several 2-amino-4-carboxypyridine, 4- and 5-carboxypyridone-based compounds were prepared and
tested against three members of the chorismate-utilising enzyme family, anthranilate synthase,
isochorismate synthase and salicylate synthase. Most compounds exhibited low micromolar inhibition
of these three enzymes. The most potent inhibitor was a 4-carboxypyridone analogue bearing a lactate
side chain on the pyridyl nitrogen which exhibited inhibition constants of 5, 91 and 54 mM against
anthranilate synthase, isochorismate synthase and salicylate synthase respectively.
phoenolpyruvate and erythrose-4-phosphate into chorismate.1-4
Introduction
Chorismate serves as the branchpoint for the synthesis of a diverse
The shikimate biosynthetic pathway is utilised by algae, higher
range of aromatic compounds including the amino acids trypto-
plants, bacteria, fungi and apicomplexan parasites. The pathway
phan, tyrosine and phenylalanine, and essential vitamins including
the folate coenzymes, benzoid and naphthenoid quinones.5-7
The first committed step in the biosynthesis of these metabo-
lites is catalysed by a family of chorismate-utilising enzymes
(Scheme 1). For example, one member of this enzyme family,
isochorismate synthase catalyses the reversible conversion of
chorismate (1) into isochorismate (2).5 Escherichia coli possesses
two isochorismate synthases, EntC and MenF, which represent
the first committed steps of two distinct biosynthetic pathways.8,9
The EntC isochorismate synthase is required for the biosynthesis
consists of seven enzyme-catalysed steps which converts phos-
aDepartment 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
bSchool of Chemistry, University of Sydney, Building F11, Sydney, Australia.
E-mail: richard.payne@sydney.edu.au; Fax: +61 2 9351 3329; Tel: +61 2
9351 5877
† Electronic Supplementary Information (ESI) available: GOLD dock-
ing solutions against three chorismate-utilising enzymes. See DOI:
10.1039/c004062b/
Scheme 1 Chorismate-utilising enzymes
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of the siderophore enterobactin,10,11 whilst MenF is involved in
a separate pathway leading to the biosynthesis of menaquinone
(Vitamin K).12-14 A related enzyme, salicylate synthase catalyses the
conversion of chorismate into salicylate (3) and represents the first
commited step in the biosynthesis of iron chelating siderophores
in bacteria (Scheme 1).15 Recent evidence from HPLC and NMR
analyses suggest that this enzyme carries out the conversion
via an isochorismate (2) intermediate.16-18 Anthranilate synthase
catalyses the conversion of chorismate (1) to anthranilate (4), and
represents the first commited step in tryptophan biosynthesis.6
Anthranilate synthase is a multifunctional enzyme composed of
a glutamine amidotransferase subunit, TrpG, and a chorismate
binding subunit, TrpE.19 TrpG is responsible for the conversion
of glutamine to glutamate and delivers the ammonia generated in
the process to the chorismate binding site in the TrpE domain.
The TrpE subunit converts chorismate (1) to the intermediate
2-amino-2-deoxyisochorismate (ADIC) and then eliminates the
enol-pyruvyl side chain to form anthranilate.19,20
These three chorismate-utilising enzymes are of particular
interest due to their sequence similarity, and the similarity in the
reaction each catalyses. This has led to the suggestion that there
may be some unifying features in their mechanisms.21-23 There are
several feasible mechanisms for this initial step. One possibility
is that the reaction proceeds via an SN1 type mechanism where
the C-4 hydroxyl of chorismate (1) is first eliminated to form a
carbocation (5) at C-4 (mechanism (a), Scheme 2). The hydroxyl
is thought to be protonated by a nearby glutamate residue in the
active site of these enzymes to promote its elimination. Attack at
C-2 by either ammonia or water would then proceed to give ADIC
(7, in anthranilate synthase) and isochorismate (2, in isochorismate
synthase and salicylate synthase) respectively.11 A second possible,
andperhaps more likely, mechanism involves the direct SN2¢¢ attack
at C-2 by ammonia or water, with concomitant loss of the C-4
hydroxyl of chorismate to yield ADIC (7) or isochorismate (2)
in one step (mechanism b, Scheme 2). Indirect evidence for this
type of mechanism has been gained both in our laboratory23,24
and that of Toney22 using the structurally related 4-amino-4-
deoxychorismate (ADC) synthase, responsible for the conversion
of chorismate (1) to ADC (8) in the folate biosynthetic pathway
(Scheme 1). For anthranilate synthase and salicylate synthase there
is an additional step in which the the C-3 enol-pyruvyl side chain is
cleaved with concomitant aromatisation, to afford the enzymatic
products anthranilate (4) and salicylate (3) respectively. Recent
evidence suggests that this reaction may proceed via a pericyclic
rearrangement, a relatively rare enzymatic transformation.18,25,26
As mammals do not possess any of the chorismate-utilising
enzymes, the design of highly specific inhibitors against this
family of enzymes may lead to the development of selective
therapeutic agents including antibacterials. In particular iso-
chorismate synthase and salicylate synthase, which are critical
for the biosynthesis of siderophores, could provide effective
drug targets.7,27 Siderophores are low molecular weight organic
chelators designed to sequester ferric iron. Iron is essential for
growth of pathogenic bacteria and during infection bacteria must
obtain iron from host stores to support their normal metabolism.
For many pathogenic bacteria the efficiency of iron-uptake is
directly related to virulence.28-30
Previous inhibitor studies include the work of Kozlowski et al.
who designed several competitive inhibitors mimicking proposed
transition states for isochorismate synthase and anthranilate
synthase.21 The most potent of these (9) exhibited a KI of 0.053 and
0.62 mM against isochorismate synthase and anthranilate synthase
respectively (Fig. 1). We have recently reported the synthesis of 2,5-
dihydrochorismate-31 and benzoic acid-based32-34 inhibitors of the
chorismate-utilising enzymes. However, these compounds proved
to be less potent than 9 against the family of enzymes.
Fig. 1 Chorismate-utilising enzyme inhibitor designed by Kozlowski
et al.21
As a tool to investigate the mechanism of these enzymes and
to develop potentially useful inhibitors, a variety of 2-amino-
4-carboxypyridine (10, 11), 5-carboxypyridone (12–14) and 4-
carboxypyridone (15, 16) analogues were considered (Fig. 2). The
compounds were designed as competitive reversible inhibitors of
the chorismate-utilising enzymes. The pyridyl nitrogen (pKa
ª
7) of the 2-amino-4-carboxypyridine compounds (10, 11) would
be partially protonated under the assay conditions and, as such,
would serve as a mimic of the positive charge at C-4 of chorismate
Scheme 2 Two proposed mechanisms for anthranilate synthase, isocho-
rismate synthase and salicylate synthase, (a) two step mechanism (b) SN2¢¢
mechanism.
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Fig. 2 Proposed 2-amino-4-carboxypyridine (10, 11) and 5-carboxypyri-
done (12–14) and 4-carboxypyridone (15, 16) inhibitors.
in the transition state of one of the proposed mechanisms
(Scheme 2a). The 4- and 5-carboxypyridone compounds (12–16)
were designed as analogues of the substrate chorismate (1). A
variety of side chains were incorporated in 10–17 to probe the
enol-pyruvyl binding pocket of the enzymes.
Results and Discussion
Docking Studies
Analogues 10–16 containing glycol, lactate and acrylate side
chains were prepared for modelling using SYBYL7.1.35 These
compounds were then docked into the active site of Serratia
marcescens anthranilate synthase (AS), E. coli isochorismate syn-
thase (EntC) and Yersinia enterocolitica salicylate synthase (Irp9)
using GOLD to assess their suitability as inhibitors of the three
chorismate-utilising enzymes.36,37 Interestingly, the compounds
exhibited two distinct binding modes when docked against the
three chorismate-utilising enzymes (see ESI†). In addition, these
same two binding modes were observed when inhibitor 9 (the most
potent chorismate-utilising enzyme inhibitor prepared to date) was
docked against the three enzymes (see ESI†).
The first of these binding modes has been observed in previous
docking studies with aromatic chorismate analogues.32 In this
mode the compounds are oriented with the C-1 carboxylate bound
to the magnesium ion. The side chain carboxylate forms an ionic
interaction with a conserved arginine residue on the opposite side
of the active site, corresponding to Arg469, Arg391 and Arg347 in
AS, Irp9 and EntC, respectively. Compound (R)-11 docked in this
binding mode into the active sites of AS and Irp9 (Fig. 3a and b).
In the second binding mode, as observed when (R)-11 was docked
into the active site of EntC, the compound is flipped over so that
the side chain carboxylate interacts with the magnesium ion and
the C-1 carboxylate binds to the conserved arginine (Fig. 3c).33
For Irp9 the majority of compounds docked in the first binding
mode, while for AS and EntC the second mode was predominate
Fig. 3 Dockings of (R)-11 into the active sites of (a), S. marcescens
anthranilate synthase (AS) (b) Yersinia enterocolitica salicylate synthase
(Irp9) (c) E. coli isochorismate synthase (EntC).
(See ESI†). It was anticipated that these variations in binding
mode would be reflected in different specificities of the compounds
against the three chorismate-utilising enzymes.
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Synthesis
only occurred on the ring nitrogen. Alkylation using the above
conditions with glyoxal and pyruvic aldehyde gave the desired
2-amino alkylated products 21 and 22 in 51% and 35% yields
respectively. Finally, base hydrolysis of the methyl esters proceeded
smoothly to afford the desired dicarboxylates 10 and 11.
Synthesis of the 5-carboxypyridone analogues began from 6-
hydroxynicotinic acid 23 and involved initial methyl ester forma-
tion (Scheme 4). Alkylation with methylbromoacetate, methyl-2-
bromopropionate and methyl-(2-bromomethyl) acrylate produced
diesters 25–27 in low to moderate yields. The regiochemistry
for 25–27 was confirmed by nOe studies. Finally, methyl ester
hydrolysis with aqueous potassium hydroxide gave the desired
pyridone dicarboxylates 12–14 in excellent yields.
Synthesis of the 4-carboxypyridone analogues began from
commercially available methyl-2-amino-isonicotinic acid 17
(Scheme 3). Initial diazatisation of 17 with sodium nitrite and sul-
furic acid produced the desired 2-hydroxy functionality, however
the strongly acidic conditions also hydrolysed the methyl ester.
The methyl ester was reformed using concentrated sulfuric acid
in refluxing methanol to give 18 in 45% yield over the two steps.
Treatment of 18 with methyl-2-bromopropionate and methyl-2-
(bromomethyl)acrylate afforded the desired N-alkylated products
19 and 20 in 26% and 72% yields respectively. Their regiochemistry
was confirmed by nOe analysis. Finally, methyl ester hydrolysis
produced the pyridone dicarboxylates 15 and 16 in good yields.
Scheme 4 Synthesis of 5-carboxypyridone inhibitors 12–14.
Enzyme Assays
The 2-amino-4-carboxypyridine (10, 11), 5-carboxypyridone
(12–14) and 4-carboxypyridone (15, 16) inhibitors were tested
against three different chorismate-utilising enzymes, S. marcescens
anthranilate synthase (AS), E. coli isochorismate synthase (EntC)
and Y. enterocolitica salicylate synthase (Irp9). The assays were
carried out in 96 well plates at 25 ◦C using fluorescence
detection. AS was assayed by detecting the formation of an-
thranilate (ex. 313 nm, em. 390 nm).40 EntC and Irp9 were
assayed by detecting the formation of salicylate (ex. 305 nm, em.
440 nm).34,41,42 EntC was assayed with a coupling enzyme, Pseu-
domonas aeroginosa isochorismate pyruvate lyase (PchB), in order
to convert isochorismate into salicylate which could be detected
fluorimetrically.‡
Scheme 3 Synthesis of 2-amino-4-carboxypyridine (10, 11) and 4-car-
boxypyrididone inhibitors (15, 16).
Synthesis of the 2-amino-4-carboxypyridine analogues also
began from methyl-2-amino-isonicotinic acid 17. It is well doc-
umented that alkylation of 2-amino pyridines usually results in
alkylation at the pyridine nitrogen, giving the related imidazo[1,2-
a] derivatives through the corresponding intermediate pyridone
imines.38 A study by Alcaide et al. reported that reaction of
2-aminopyridines, 2-aminopyrimidine and 2-aminothiazole with
various glyoxal derivatives in boiling alcohol in the presence
of aqueous 60% perchloric acid, forms regiospecifically N-(2-
heteroaryl)-a-amino esters in good yields.39 This was the approach
adopted for the synthesis of the desired compounds as other
alkylation methods indeed produced adducts where alkylation had
The inhibition constants of 10–16 against the three chorismate-
utilising enzymes are shown in Table 1. All compounds proved
to be competitive reversible inhibitors of the enzymes with
respect to the substrate chorismate. Unfortunately, compounds
‡ Although the isochorismate pyruvate lyase was used in a coupling
concentration in the assay (2.4 equivalents) it was assumed that the
analogues may also inhibit this enzyme. Therefore when discussing the
inhibition results, the analogues are described as inhibiting the entire
enzymatic system rather than only isochorismate synthase
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Table 1 Inhibition constants of 2-aminopyridine and 4- and 5-pyridone
analogues against S. marcescens anthranilate synthase (AS), E. coli isocho-
rismate synthase/P.aeroginosa isochorismate pyruvate lyase (EntC/PchB)
and Y. enterocolitica salicylate synthase (Irp9)a
Conclusions
In summary, we have designed and synthesised a novel series
of chorismate-utilising enzyme inhibitors based on 2-amino-4-
carboxypyridine and 4- and 5-carboxypyridone scaffolds. These
were screened against S. marcescens anthranilate synthase (AS),
E. coli isochorismate synthase (EntC)/P. aeroginosa isochorismate
pyruvate lyase (PchB) and Y. enterocolitica salicylate synthase
(Irp9). The compounds proved to be low- to mid-micromolar
inhibitors of the enzymes and will now serve as lead structures
for the design of second generation inhibitors.
Compounds
AS
KI/mM
EntC/PchB
KI/mM
Irp9
KI/mM
10
11
12
13
14
15
16
94 19
150 18
210 17
120 29
91 10
170 17
nd
90 18
170 38
48 12
54 10
73 13
nd
50
7
100 18
5.3 1.0
120 16
41
8
59 10
nd
nd
Experimental
a Kinetic parameters of the chorismate-utilising enzymes: AS: KM = 3.7
0.5 mM, kcat = 5.6 s-1; EntC: KM = 15 2.4 mM, kcat = 13s-1; Irp9: KM
4.2 0.9 mM, kcat = 0.1s-1; nd, not determined.
=
Molecular modelling
All ligands and the receptors (TrpE, Irp9 and EntC) were prepared
using SYBYL7.1 and used as MOL2 files.35 The ligands were
prepared as carboxylate anions and their structures were energy
minimised using the Tripos force-field. Gasteiger-Huckel charges
were calculated prior to docking. Each ligand was docked using
GOLD2.1 in 25 independent genetic algorithm (GA) runs.36,37 For
each of these, a maximum number of 100 000 GA operations
was performed on a single population of 50 individuals. Operator
weights for crossover, mutation and migration in the entry box
were used as default parameters (95, 95 and 10 respectively), as
15 and 16 could not be assayed against EntC/PchB or Irp9. The
highly fluorescent nature of these compounds under the assay
conditions resulted in the spectrophotometer operating out of
the linear range of accuracy even at low inhibitor concentrations
(50 mM).
The 2-amino-4-carboxypyridine inhibitors (10 and 11) were
generally less potent than the corresponding 5-carboxypyridone
counterparts (12 and 13). This effect was especially evident in
analogue 13 containing a lactate side chain, which was significantly
more potent against all three chorismate-utilising enzymes. These
results suggest that any positive charge on the pyridyl nitrogen
does not aid in binding to the enzymes.
˚
˚
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
selected.
Compounds containing a lactyl side chain were more potent
against AS than analogues containing the alternative glycol
or acrylate side chains. This result is consistent with previous
inhibition results with aromatic analogues tested against the
chorismate-utilising enzymes.31-34 Side chain specificity does not
appear to be as obvious in the case of EntC/PchB and Irp9.
Compounds containing glycol (10 and 12) or lactate (11 and
13) side chains exhibited very similar inhibition constants against
these two enzymes. Analogue 14 containing an acrylate side chain
was two fold less potent against EntC/PchB and Irp9. Docking
studies suggest that this may be a result of the side chain being
too large to be accommodated into the enol-pyruvyl pocket in the
active site of these enzymes.
It is interesting to note that compound 16, containing an acry-
late side chain exhibited an inhibition constant of 59 mM against
AS, two fold more potent than 5-carboxypyridone analogue 14
containing the identical side chain. This result is consistent with
the molecular dockings, where an extended acrylate side chain in
16 would allow for more favourable electrostatic interactions with
the magnesium ion, if the inhibitor bound to the enzyme in this
conformation. This trend was reversed for analogues containing a
lactate side chain where 4-carboxypyridone analogue 15 was ten
fold less potent against AS than 5-carboxypyridone analogue 13.
Variation in the binding modes of these compounds, as observed
in the dockings, may explain the observed trends in the inhibition
data. Future work in our laboratory will involve crystallisation
studies of the inhibitors with the chorismate-utilising enzymes to
quantify their binding mode which should aid in the design of
more potent inhibitors of this family of enzymes.
Biological
Over-expression and purification of His6-Irp9, His6-EntC,
His6-PchB and His6-AS. Preparation of the expression vec-
tors pET-28a/irp9,16 pET-28a/entC,16 pET-28a/pchB,31and mini-
pRSETA/trpEG32 has been described previously. An overnight
culture (100 ml or 10 ml for His6-AS) of E. coli C41(DE3) cells
transformed with the appropriate expression was added to 1 l of
fresh 2YT media supplemented with 30 mg ml-1 kanamycin (or
50 mg ml-1 ampicillin for His6-AS) and divided into 2 ¥ 500 ml
cultures. Expression of His6-Irp9, His6-EntC, His6-PchB or His6-
AS was induced at an OD600 of 0.6 by the addition of isopropyl-b-
D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM.
After 4 h shaking at 37 ◦C, the IPTG-induced cells were harvested
◦
by centrifuging at 6,000 ¥ g for 15 min at 4 C. Cell pellets were
stored at -20 ◦C.
All purification steps were carried out at 4 ◦C. Harvested
cells were suspended in 0.02 M potassium phosphate, 0.5 M
NaCl, 0.02 M imidazole, pH 7.4 (buffer A) containing lysozyme
(0.35 mg ml-1) and stirred at room temperature for 30 min.
For His6-AS, cell pellets were only resuspended in buffer N1
(50 mM sodium phosphate buffer pH 8.0, 300 mM NaCl,
10 mM imidazole, 1 mM b-mercaptoethanol). The suspension was
homogenized for 20 min by sonication on ice. The crude extract
was then centrifuged for 30 min at 39,000 ¥ g. The cell-free lysate
was applied to a 5 ml Ni-NTA (nitriloacetic acid) column that
had been pre-equilibrated with buffer A or buffer N1 for His6-
AS. After washing with buffer A or buffer N2 (buffer N1 with
50 mM imidazole) for His6-AS, the protein was eluted using a
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linear gradient of 20–500 mM imidazole in 100 ml buffer A or
4 – 5 column volumes of buffer N3 (buffer N1 with 250 mM
imidazole) for His6-AS. The elution was followed via SDS-PAGE
and fractions containing the protein of interest were concentrated
using a protein centrifugal concentrator. The concentrated protein
sample was then dialyzed against 100 mM potassium phosphate,
pH 7.0 or 50 mM Tris.HCl pH 7.5, 1 mM DTT, 0.1 mM EDTA
for His6-AS using a PD-10 column containing Sephadex G-25
resin (Amersham Biosciences). The purity of the protein was
judged to be ≥ 95% by SDS-PAGE. Enzyme concentrations were
evaluated by measuring the absorbance at 280 nm and using
the conversion factor calculated using the software Vector NTI
(version 6; Invitrogen). Aliquots of pure His6-tag recombinant
proteins were frozen with liquid nitrogen and stored at -80 ◦C
until use.
High resolution mass spectrometry was carried out using a
Micromass Quadrapole-Time of Flight (Q-Tof) spectrometer.
2-Hydroxy-isonicotinic acid methyl ester (18). Methyl-2-
amino-isonicotinic acid (1.00 g, 6.57 mmol) was dissolved in
sulfuric acid (40 ml of a 2 M solution). Dropwise addition of
concentrated sulfuric acid (6 ml) was followed by cooling to 0 ◦C.
A solution of sodium nitrite (0.75 g, 0.01 mol) in water (12 ml)
was added dropwise at 0 ◦C (caution: elimination of N2). The
reaction was stirred for a further 3 h at 22 ◦C and then boiled for
one minute. The solution was neutralised with saturated aqueous
sodium bicarbonate solution at which point the carboxylic acid
precipitated and was isolated as a pale brown solid. After extensive
drying of the acid under high vacuum for 16 h, the acid was
dissolved in methanol (8 ml). Concentrated sulfuric acid (1 ml)
was added dropwise and the reaction was stirred at reflux for 15 h.
The reaction was allowed to cool to 22 ◦C before neutralising with
saturated NaHCO3 solution. The methyl ester was extracted into
ethyl acetate (2 ¥ 20 ml), the organic fractions were dried (MgSO4)
and the solvent removed under reduced pressure to afford the
desired alcohol (18) as a pale yellow solid. (0.45 g, 45%).
Enzyme and inhibition assays. Steady-state kinetic experiments
were performed spectrophotometrically on a microplate reader
by monitoring the products anthranilate 4 or salicylate 3 by
fluorescence (4: excitation 313 nm, emission 390 nm; 3: excitation
305 nm, emission 440 nm) as described previously.34,41,42 The assays
for AS containing 1 mM enzyme were performed in duplicate at
25 ◦C. The reaction of EntC with chorismate was measured using
a coupled assay with PchB, an isochorismate pyruvate lyase from
P. aeruginosa, as described previously by Gaille et al.41 Reaction
mixtures contained, in a total volume of 0.2 ml, 100 mM potassium
phosphate, pH 7.0, 10 mM MgCl2, PchB (220 nM), EntC (90
nM) and various amounts of chorismate (22.5–90 mM) and were
conducted at 25 ◦C. Irp9 assays were initiated by the addition of
1.7 mM His6-Irp9 and were monitored for 5 min by the salicylate
fluorescence assay.
nmax. (ATR): 2833 (OH stretch) 3033, 2959 (Ar C–H stretch),
-1
1
=
=
1728 (C O, str), 1656, 1618, 1540 (C C, ar) cm ; H NMR
(DMSO) d 3.81 (3H, s, CO2Me), 6.48 (1H, dd, J 6.7, 1.7 Hz, H-5),
6.78 (1H, dd, J 0.6, 1.7 Hz, H-3), 7.48 (1H, dd, J 0.6, 6.7 Hz, H-6),
11.80 (1H, br s, OH); 13C NMR (DMSO) d 53.1, 103.4, 121.5,
137.2, 141.6, 162.5, 165.2; HRMS calcd for C7H8NO3: MH+,
154.0515. Found: MH+ 154.0504.
1-(1-Methoxycarbonyl-ethyl)-2-oxo-1,2-dihydro-pyridine-4-car-
boxylic acid methyl ester (19). Methyl-2-bromopropionate
(0.85 mmol, 95 ml) was added dropwise to a solution of 18 (100 mg,
0.65 mmol) and anhydrous potassium carbonate (0.13 mmol, 18
mg) in dry DMF (5 ml) and the reaction was heated to 40 ◦C for
Initial rates for various concentrations of chorismic acid at a
number of inhibitor concentrations were measured to characterise
the mechanism of inhibition. Kinetic data were fitted to the
appropriate equations using GraFit 5.0.10. (Erithacus software).43
◦
16 h. The reaction was allowed to cool to 22 C before dilution
with ethyl acetate (20 ml). The reaction mixture was washed with
water (5 ¥ 20 ml), dried (MgSO4) and the solvent removed in
vacuo. Purification by column chromatography (eluent 6 : 1 v/v
ethyl acetate–hexane) gave 19 as a colourless oil. (40 mg, 26%).
Synthetic
General. All organic solvents were freshly distilled prior
to use. 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 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 g◦el 60. Petroleum ether refers to the fraction distilled between
RF [6 : 1 v/v ethyl acetate–hexane] = 0.50; nmax. (ATR): 3082 (Ar
-1
=
=
C–H stretch), 1730, 1665 (C O, str), 1592, 1538 (C C, ar) cm ;
1H NMR (CD3OD) d 1.66 (3H, d, J 7.2 Hz, CH3), 3.71 (3H, s,
CO2Me), 3.90 (3H, s, CO2Me), 5.24 (1H, q, J 7.2 Hz, CH), 6.80
(1H, dd, J 7.1, 1.8 Hz, H-5), 7.06 (1H, d, J 1.8 Hz, H-3), 7.76 (1H,
d, J 7.1 Hz, H-6); nOe: Irradiation of the doublet at 1.66 ppm
caused enhancement of the doublet at 7.76 ppm (H-6), thus
confirming the regiochemistry of the product; 13C NMR (CD3OD)
d 16.3, 53.6, 53.8, 58.7, 106.9, 122.4, 139.3, 143.5, 164.4, 166.4,
172.1; HRMS calcd for C11H13NO5: M+, 239.0794. Found: M+,
239.0787.
1
40–60 C. H NMR spectra were recorded on a Bruker AM-400
spectrometer or a Bruker Avance 500 spectrometer in deuterated
solvents, as indicated. 13C NMR spectra were recorded on a Bruker
AM-400 spectrometer operating at 100 MHz or a Bruker Avance
500 spectrometer operating at 125 MHz in deuterated solvents
as indicated. All chemical shifts are quoted in parts per million
(ppm). Coupling constants for 1H NMR spectroscopy are assigned
where possible and are given in Hz. Yields of final inhibitors
1-(1-Carboxy-ethyl)-2-oxo-1,2-dihydro-pyridine-4-carboxylate
(15). Potassium hydroxide (33 mg, 0.59 mmol) in milliQ water
(2 ml) was added dropwise to a solution of 19 (35 mg, 0.15 mmol)
in freshly distilled THF (2 ml) and the reaction was heated to
◦
◦
1
10–16 were calculated by using H NMR studies. Samples were
40 C for 3 h. The reaction was allowed to cool to 22 C before
dilution with water (3 ml). The aqueous fraction was washed with
dichloromethane (5 ml) before acidifying to pH 7.5 with 0.1 M
HCl. The water was lyophilised to give the desired product (15) as
a white solid. (0.13 mmol, 83%).
dissolved in D2O with a known molarity of an internal standard
(3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt). Infrared
spectra were recorded on a Perkin Elmer Spectrum One FTIR
spectrometer using attenuated transmittance reflectance (ATR).
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 3534–3542 | 3539
©

View Online
-1
1
=
=
nmax. (ATR): 3330 (br, acid OH stretch), 1610, 1558 (C O, str),
(C C, ar) cm ; H NMR (CDCl3) d 3.71 (3H, s, CO2Me), 3.85
(3H, s, CO2Me), 4.13 (2H, d, J 5.6 Hz, CH2), 5.32 (1H, t, J 5.6 Hz,
NH), 7.00 (1H, m, ArH), 7.06 (1H, dd, J 1.3, 5.3 Hz, ArH), 8.13
(1H, dd, J 0.5, 5.3 Hz, ArH); 13C NMR (CDCl3) d 45.8, 54.5, 54.8,
110.9, 114.7, 140.9, 151.0, 160.5, 168.4, 174.0; HRMS calcd for
C10H12N2O4Na: MNa+, 247.0695. Found: MNa+ 247.0696.
-1
1
=
1396, 1337 (C C, ar) cm ; H NMR (D2O) d 1.56 (3H, d, J
7.4 Hz, CH3), 5.11 (1H, q, J 7.4 Hz, CH), 6.75 (1H, dd, J 7.1,
1.6 Hz, H-5), 7.06 (1H, d, J 1.6 Hz, H-3), 7.76 (1H, d, J 7.1 Hz,
H-6); 13C NMR (CD3OD) d 16.9, 58.4, 108.1, 118.2, 136.8, 149.7,
+
164.7, 173.0, 177.8; HRMS calcd for C9H7NO5NaK2: MNaK2 ,
+
309.9496. Found: MNaK2 309.9510.
2-(Carboxymethyl-amino)-isonicotinate (10). Potassium hy-
droxide (84 mg, 1.50 mmol) in milliQ water (6 ml) was added
dropwise to a solution of 21 (112 mg, 0.50 mmol) in freshly distilled
THF (6 ml) and the reaction was heated to 40 ◦C for 3 h. The
reaction was allowed to cool to 22 ◦C before dilution with water
(7 ml). The aqueous fraction was washed with dichloromethane
(14 ml) before acidifying to pH 7.5 with 0.1 M HCl. The water was
lyophilised to give 10 as a yellow/brown solid. (0.47 mmol, 93%)
1-(2-Methoxycarbonylallyl)-2-oxo-1,2-dihydro-pyridine-4-car-
boxylic acid methyl ester (20). Methyl-(2-bromomethyl)acrylate
(81 ml, 0.68 mmol) was added dropwise to a solution of 18
(80 mg, 0.52 mmol) and anhydrous potassium carbonate (14 mg,
0.10 mmol) in dry DMF (4 ml) and the reaction was heated to 40 ◦C
for 16 h. The reaction was allowed to cool to 22 ◦C before dilution
with ethyl acetate (20 ml). The reaction mixture was washed with
water (5 ¥ 20 ml), dried (MgSO4) and the solvent removed in
vacuo. Purification by column chromatography (eluent 6 : 1 v/v
ethyl acetate–hexane) gave 20 as a colourless oil. (94 mg, 72%).
RF [6 : 1 v/v ethyl acetate–hexane] = 0.50; nmax. (ATR): 3128 (Ar
=
nmax. (ATR): 3275 (br, acid OH stretch), 1595, 1545 (C O, str),
-1
1
=
1502, 1388 (C C, ar) cm ; H NMR (D2O) d 3.80 (2H, s, CH2),
6.81 (1H, s, H-3), 6.89 (1H, d, J 5.4 Hz, ArH), 7.91 (1H, d, J
5.4 Hz, ArH); 13C NMR (D2O) d 46.1, 108.4, 112.5, 147.0, 147.3,
+
=
=
C–H stretch), 1731, 1706 (C O, str), 1667, 1639, 1581, 1540 (C C,
159.2, 174.3, 179.0; HRMS calcd for C8H6N2O4NaK2: MNaK2 ,
-1
1
+
=
ar), 1437 (C C) cm ; H NMR (CD3OD) d 3.76 (3H, s, CO2Me),
3.90 (3H, s, CO2Me), 4.83 (2H, s, CH2), 5.62 (1H, s, CHH), 6.35
(1H, s, CHH), 6.77 (1H, dd, J 7.1, 1.8 Hz, H-5), 7.08 (1H, d,
J 1.8 Hz, H-3), 7.74 (1H, d, J 7.1 Hz, H-6); 13C NMR (CD3OD)
d 51.4, 53.1, 53.8, 106.7, 122.6, 129.3, 136.5, 141.5, 143.5, 164.6,
166.4, 167.5; HRMS calcd for C12H13NO5Na: MNa+, 274.0691.
Found: MNa+ 274.0683.
294.9499. Found: MNaK2 294.9486.
2-(1-Methoxycarbonyl-ethylamino)-isonicotinic acid methyl ester
(22). A solution of pyruvic aldehyde (355 ml of a 40% wt solution
in water, 1.97 mmol) in methanol (2 ml) was added in one portion
over a slurry of methyl-2-aminopyridine-4-carboxylate (300 mg,
1.97 mmol) in 60% perchloric acid. The resulting solution was
heated to reflux for 36 h. The reaction was allowed to cool to
22 ◦C before neutralising by the dropwise addition of saturated
sodium bicarbonate solution. The solution was extracted with
ethyl acetate (2 ¥ 10 ml), the combined organic fractions washed
with brine (20 ml), dried (MgSO4) and the solvent removed in
vacuo. Purification by column chromatography (eluent 1 : 1 v/v
petroleum ether/ethyl acetate) afforded 22 as a pale yellow solid.
(160 mg, 35%).
1-(2-carboxy-allyl)-2-oxo-1,2-dihydro-pyridine-4-carboxylate
(16). Potassium hydroxide (84 mg, 1.50 mmol) in milliQ water
(2 ml) was added dropwise to a solution of 20 (94 mg, 0.38 mmol)
in freshly distilled THF (2 ml) and the reaction was heated to
◦
◦
40 C for 3 h. The reaction was allowed to cool to 22 C before
dilution with water (5 ml). The aqueous fraction was washed with
dichloromethane (5 ml) before acidifying to pH 7.5 with 0.1 M
HCl. The water was lyophilised to give the desired product (16) as
a white solid (0.34 mmol, 89%).
RF [1 : 1 v/v petroleum ether/ethyl acetate] = 0.67; nmax. (ATR):
=
3387 (N–H stretch), 1747, 1715 (C O, str), 1614, 1564, 1516
=
-1
1
=
nmax. (ATR): 3342 (br, acid OH stretch), 1661, 1567 (C O, str),
(C C, ar) cm ; H NMR (CDCl3) d 1.44 (3H, d, J 7.1 Hz, CH3),
3.70 (3H, s, CO2Me), 3.85 (3H, s, CO2Me), 4.60 (1H, dq, J 7.1,
7.6 Hz, CH), 5.23 (1H, d, J 7.6 Hz, NH), 6.99 (1H, d, J 1.3 Hz,
H-3), 7.03 (1H, dd, J 1.3, 5.2 Hz, H-5), 8.12 (1H, d, J 5.2 Hz, H-6);
13C NMR (CDCl3) d 18.8, 50.3, 52.6, 109.0, 112.6, 138.9, 149.1,
158.3, 166.4, 175.2, one undetected double-up; HRMS calcd for
C11H14N2O4Na: MNa+, 261.0851. Found: MNa+ 261.0840.
-1
1
=
1527, 1474 (C C, ar) cm ; H NMR (D2O) d 4.77 (2H, s, CH2),
5.01 (1H, s, CHH), 5.91 (1H, s, CHH), 6.77 (1H, dd, J 6.9, 1.8 Hz,
H-5), 6.90 (1H, d, J 1.8 Hz, H-3), 7.62 (1H, d, J 6.9 Hz, H-6);
13C NMR (D2O) d 51.4, 108.6, 118.7, 122.7, 139.7, 140.1, 150.4,
+
164.7, 172.7, 173.3; HRMS calcd for C10H7NO5NaK2: MNaK2 ,
+
321.9496. Found: MNaK2 321.9482.
2-(1-Methoxycarbonylmethyl-amino)-isonicotinic acid methyl es-
ter (21). A solution of glyoxal (300 ml of a 40% wt solution in
water, 1.97 mmol) in methanol (3 ml) was added in one portion
over a slurry of methyl-2-aminopyridine-4-carboxylate (300 mg,
1.97 mmol) in 60% perchloric acid (0.6 ml). The resulting solution
was heated to reflux for 48 h. The reaction was allowed to cool
to 22 ◦C before neutralising by the dropwise addition of saturated
aqueous sodium bicarbonate solution. The solution was extracted
with ethyl acetate (2 ¥ 10 ml), the combined organic fractions
washed with brine (20 ml), dried (MgSO4) and the solvent removed
in vacuo. Purification by column chromatography (eluent 1 : 1 v/v
petroleum ether/ethyl acetate) gave 21 as a pale yellow solid.
(224 mg, 51%).
2-(1-Carboxy-ethylamino)-isonicotinate (11). Potassium hy-
droxide (53 mg, 0.94 mmol) in milliQ water (4 ml) was added
dropwise to a solution of 22 (75 mg, 0.32 mmol) in THF (4 ml)
and the reaction was heated to 40 ◦C for 3 h. The reaction was
◦
allowed to cool to 22 C before dilution with water (7 ml). The
aqueous fraction was washed with dichloromethane (20 ml) before
acidifying to pH 7.5 with 0.1 M HCl. The water was lyophilised
to give 11 as a yellow solid. (0.26 mmol, 82%).
nmax. (ATR): 3274 (br, acid OH stretch), 2946 (N–H stretch),
-1
1
=
=
1586, 1549 (C O, str), 1440, 1393 (C C, ar) cm ; H NMR
(D2O) d 1.38 (3H, d, J 7.1 Hz, CH3), 4.07 (1H, q, J 7.1 Hz CH),
6.83 (1H, s, H-3), 6.92 (1H, d, J 5.0 Hz, ArH), 7.93 (1H, d, J
5.5 Hz, ArH); 13C NMR (D2O) d 18.6, 53.4, 108.7, 112.6, 147.1,
158.7, 174.2, 182.6, one undetected double-up; HRMS calcd for
RF [1 : 1 v/v petroleum ether/ethyl acetate] = 0.57; nmax. (ATR):
3390 (N–H stretch), 1748, 1726 (C O, str), 1620, 1560, 1521
C9H8N2O4NaK2: MNaK2 , 308.9656. Found: MNaK2+ 308.9632.
+
=
3540 | Org. Biomol. Chem., 2010, 8, 3534–3542
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The Royal Society of Chemistry 2010
©

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6-Hydroxy-nicotinic acid methyl ester (24). To a suspension of
6-hydroxynicotinic acid (5 g, 0.04 mol) in methanol (36 ml) was
added dropwise concentrated sulfuric acid (3.6 ml). The solution
was heated at reflux under nitrogen for 18 h. The reaction was
allowed to cool to 22 ◦C before dilution with water (20 ml).
Saturated aqueous sodium carbonate solution was added dropwise
until the solution reached pH 12. The aqueous fraction was
extracted with ethyl acetate (3 ¥ 30 ml). The combined organic
fractions were dried and the solvent removed in vacuo to afford 24
as a white crystalline solid. (4.1 g, 74%).
The reaction was allowed to cool to 22 ◦C before dilution with
ethyl acetate (50 ml). The organic fraction was washed with water
(5 ¥ 50 ml). The organic fraction was dried (MgSO4) and the
solvent removed in vacuo. Purification was achieved by column
chromatography (eluent 1 : 1 v/v ethyl acetate–hexane) to afford
the 26 as a colourless oil. (160 mg, 26%).
RF [1 : 1 ethyl acetate–hexane] = 0.35; nmax. (ATR): 3078, 2954
=
=
(Ar C–H stretch), 1744, 1717 (C O, str), 1661, 1611, 1542 (C C,
1
ar) cm-1; H NMR (MeOD) d 1.68 (3H, d, J 7.2 Hz, CH3), 3.72
(3H, s, CO2Me), 3.85 (3H, s, CO2Me), 5.26 (1H, q, J 7.2 Hz, CH),
6.51 (1H, d, J 9.5 Hz, H-3), 7.93 (1H, dd, J 2.4, 9.5 Hz, H-4), 8.42
(1H, d, J 2.4 Hz, H-6); nOe: Irradiation of the singlet at 8.42 ppm
(H-6), caused enhancement of the doublet at 1.68 ppm and the
quartet at 5.26 ppm thus confirming the regiochemistry of the
product; 13C NMR (MeOD) d 15.7, 52.3, 52.9, 58.4, 111.6, 119.7,
140.3, 143.0, 163.7, 165.6, 171.3; HRMS calcd for C11H13NO5Na:
MNa+, 262.0691. Found: MNa+, 262.0698.
nmax. (ATR): 3012 (OH stretch) 3083, 2957 (Ar C–H stretch),
-1
1
=
=
1722, 1704 (C O, str), 1677, 1639, 1590 (C C, ar) cm ; H NMR
(MeOD) d 3.82 (3H, s, CO2Me), 6.50 (1H, d, J 9.7 Hz, H-3), 7.97
(1H, dd, J 2.6, 9.7 Hz, H-4), 8.12 (1H, d, J 2.6 Hz, H-6). 13C
NMR (MeOD) d 52.4, 111.7, 120.3, 141.2, 141.8, 165.5, 165.9;
HRMS calcd for C7H7NO3Na: MNa+, 176.0324. Found: MNa+,
176.0325.
1-Methoxycarbonylmethyl-6-oxo-1,6-dihydro-pyridine-3-car-
boxylic acid methyl ester (25). Methylbromoacetate (0.34 ml,
3.59 mmol) was added dropwise to a solution of 24 (500 mg,
3.23 mmol) and potassium carbonate (90 mg, 0.65 mmol) in DMF
(20 ml) and the reaction was heated to 40 ◦C for 18 h. The reaction
was allowed to cool to 22 ◦C before dilution with ethyl acetate
(50 ml). The organic fraction was washed with water (5 ¥ 50 ml),
dried (MgSO4) and the solvent removed in vacuo. Purification
was achieved by column chromatography (eluent 4 : 1 v/v ethyl
acetate–hexane) to afford 25 as a colourless oil. (300 mg, 48%).
RF [4 : 1 ethyl acetate–hexane] = 0.36; nmax. (ATR): 3078, 2955
1-(1-Carboxy-ethyl)-6-oxo-1,6-dihydro-pyridine-3-carboxylate
(13). Potassium hydroxide (93 mg, 1.67 mol) in milliQ water
(5 ml) was added dropwise to a solution of 26 (100 mg, 0.42 mmol)
in THF (5 ml) and the reaction was heated to 40 ◦C for 3 h. The
reaction was allowed to cool to 22 ◦C before dilution with milliQ
water (10 ml). The solution was washed with ethyl acetate (15 ml)
before the aqueous phase was acidified to pH 8 by the dropwise
addition of 0.1 M HCl. The solution was lyophilised to give 13 as
a pale yellow solid (0.35 mmol, 83%).
=
nmax. (ATR): 3427 br. (acid O–H str.), 1651, 1609 (C O, str),
-1
1
=
1588, 1568 (C C, ar) cm ; H NMR (D2O) d 1.51 (3H, d, J
7.4 Hz, CH3), 4.98 (1H, q, J 7.4 Hz, CH), 6.43 (1H, d, J 9.3 Hz,
H-3), 7.84 (1H, dd, J 2.4, 9.3 Hz, H-4), 8.09 (1H, d, J 2.4 Hz,
H-6); 13C NMR (D2O) d 16.7, 58.8, 117.9, 139.7, 141.6, 164.7,
172.3, 177.8, 181.8; HRMS calcd for C9H9NO5K: MK+, 250.0112.
Found: MK+, 250.0113.
=
=
(Ar C–H stretch), 1749, 1716 (C O, str), 1659, 1612, 1542 (C C,
1
ar) cm-1; H NMR (MeOD) d 3.75 (3H, s, CO2Me), 3.82 (3H, s,
CO2Me), 4.80 (2H, s, CH2), 6.52 (1H, d, J 9.6 Hz, H-3), 7.94
(1H, dd, J 2.5, 9.6 Hz, H-4), 8.42 (1H, d, J 2.5 Hz, H-6); nOe:
Irradiation of the singlet at 4.80 ppm caused enhancement of the
doublet at 8.42 ppm (H-6), thus confirming the regiochemistry of
the product; 13C NMR (MeOD) d 51.7, 52.4, 52.9, 111.5, 119.5,
140.8, 145.4, 164.1, 165.6, 169.2; HRMS calcd for C10H11NO5Na:
MNa+, 248.0535. Found: MNa+, 248.0525.
1-(2-Methoxycarbonyl-allyl)- 6-oxo-1,6-dihydro-pyridine-4-car-
boxylic acid methyl ester (27). Methyl-(2-bromomethyl)acrylate
(0.21 ml, 1.79 mmol) was added dropwise to a solution of 24
(250 mg, 1.63 mmol) and potassium carbonate (45 mg, 0.33 mmol)
◦
1-Carboxymethyl-6-oxo-1,6-dihydro-pyridine-3-carboxylate
(12). Potassium hydroxide (79 mg, 1.41 mmol) in milliQ water
(4 ml) was added dropwise to a solution 25 (80 mg, 0.35 mmol)
in THF (4 ml) and the reaction was heated to 40 ◦C for 3 h. The
reaction was allowed to cool to 22 ◦C before dilution with milliQ
water (10 ml). The solution was washed with ethyl acetate (15 ml)
before the aqueous phase was acidified to pH 8 by the dropwise
addition of 0.1 M HCl. The solution was lyophilised to give 12 as
a white solid (0.31 mmol, 90%).
in DMF (10 ml) and the reaction was heated to 40 C for 18 h.
The reaction was allowed to cool to 22 ◦C before dilution with
ethyl acetate (25 ml). The organic fraction was washed with water
(5 ¥ 25 ml), dried (MgSO4) and the solvent removed in vacuo.
Purification was achieved by column chromatography (eluent 4 : 1
v/v ethyl acetate–hexane) to 27 as a white solid. (180 mg, 44%).
RF [4 : 1 ethyl acetate–hexane] = 0.37; nmax. (ATR): 3038, 2955
=
=
(Ar C–H stretch), 1709 (C O, str), 1661, 1644, 1612, 1538 (C C,
1
ar) cm-1; H NMR (MeOD) d 3.77 (3H, s, CO2Me), 3.83 (3H, s,
=
nmax. (ATR): 3369 br. (acid O–H str.), 1660, 1625 (C O, str),
CO2Me), 4.86 (2H, s, CH2), 5.70 (1H, s, CHH), 6.37 (1H, s, CHH),
6.51 (1H, d, J 9.5 Hz, H-3), 7.92 (1H, dd, J 2.5, 9.5 Hz, H-4),
8.48 (1H, d, J 2.5 Hz, H-6); nOe: Irradiation of the singlet at
8.48 ppm (H-6), caused enhancement of the singlet at 4.86 ppm
thus confirming the regiochemistry of the product; 13C NMR
(MeOD) d 15.7, 52.3, 52.9, 58.4, 111.6, 119.7, 140.3, 143.0, 163.7,
165.6, 171.3, one undetected double-up.
-1
1
=
1554 (C C) cm ; H NMR (D2O) d 4.42 (2H, s, CH2), 6.44 (1H,
d, J 9.3 Hz, H-5), 7.87 (1H, dd, J 2.3, 9.3 Hz, H-4), 7.99 (1H, d, J
2.3 Hz, H-2); 13C NMR (D2O) d 54.5, 118.4, 142.3, 143.0, 164.7,
172.1, 181.8, 188.2; HRMS calcd for C8H7NO5K: MK+, 235.9956.
Found: MK+, 235.9955.
1-(1-Methoxycarbonyl-ethyl)-6-oxo-1,6-dihydro-pyridine-3-car-
boxylic acid methyl ester (26). Methyl-2-bromopropionate
(0.40 ml, 3.59 mmol) was added dropwise to a solution of 24
(500 mg, 3.23 mmol) and potassium carbonate (90 mg, 0.65 mmol)
1-(2-Carboxy-allyl)-6-oxo-1,6-dihydro-pyridine-3-carboxylate
(14). Potassium hydroxide (160 mg, 2.84 mol) in milliQ water
(7 ml) was added dropwise to a solution of 27 (180 mg, 0.71 mmol)
in THF (7 ml) and the reaction was heated to 40 ◦C for 3 h. The
◦
in DMF (20 ml) and the reaction was heated to 40 C for 18 h.
This journal is The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 3534–3542 | 3541
©

View Online
reaction was allowed to cool to 22 ◦C before dilution with milliQ
water (10 ml). The solution was washed with ethyl acetate (15 ml)
before the aqueous phase was acidified to pH 8 by the dropwise
addition of 0.1 M HCl. The solution was lyophilised to give 14 as
a white solid. (0.63 mmol, 89%).
12 R. Daruwala, O. Kwon, R. Meganathan and M. E. Hudspeth, FEMS
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13 R. Muller, C. Dahm, G. Schulte and E. Leistner, FEBS Lett., 1996,
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15 C. Pelludat, D. Brem and J. Heesemann, J. Bacteriol., 2003, 185, 5648–
5653.
=
nmax. (ATR): 3328 br. (acid O–H str.), 1674, 1606 (C O, str)
-1
1
=
1558, 1563 (C C, ar) cm ; H NMR (D2O) d 4.68 (2H, s, CH2),
16 O. Kerbarh, A. Ciulli, I. Howard, N and C. Abell, J. Bacteriol., 2005,
=
=
4.91 (1H, s, C CHH), 5.80 (1H, s, C CHH), 6.46 (1H, d, J
9.4 Hz, H-3), 7.96 (1H, dd, J 2.3, 9.4 Hz, H-4), 8.02 (1H, d, J
2.3 Hz, H-6); 13C NMR (D2O) d 51.3, 117.7, 121.3, 140.0, 141.4,
141.7, 163.8, 171.1, 172.9, 181.0; HRMS calcd for C10H9NO5K:
MK+, 262.0112. Found: MK+, 262.0110.
187, 5061–5066.
17 A. J. Harrison, M. M. Yu, T. Gardenborg, M. Middleditch, R. J.
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RJP and EMMB would like to thank the Gates Cambridge
Trust for PhD funding. This work was also supported by the
Biotechnology and Biological Sciences Research Council, U.K.
We are very grateful to Professor Brendan Wren, Department
of Infectious & Tropical Diseases, London School of Hygiene
& Tropical Medicine, UK, and Professor Dieter Haas, Labo-
ratoire de Microbiologie microbienne, Universite´ de Lausanne,
Switzerland, for providing us with genomic DNA from Y.
enterocolitica (strain 8081) and plasmid pME3324, respectively.
We would also like to thank Glen Spraggon (Genomics Institute
of the Novartis Research Foundation, San Diego, California) and
Charles Yanofsky (Stanford University, Stanford, California) for
kindly supplying the ptttSmEDC vector containing the Serratia
marcescens trpEGDC operon.
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