Synthesis and biological evaluation of pyrophosphate mimics of thiamine pyrophosphate based on a triazole scaffold

Article abstract of DOI:10.1039/b806580b

Novel triazole-based pyrophosphate analogues of thiamine pyrophosphate (TPP) have been synthesised and tested for inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis. The thiazolium ring of thiamine was replaced by a triazole in an efficient two-step procedure. Pyrophosphorylation then gave extremely potent triazole inhibitors with K_I values down to 20 pM, compared to a K_D value of 0.35 μM for TPP. This triazole scaffold was used for further investigation and six analogues containing mimics of the pyrophosphate group were synthesised and tested for inhibition of PDC. Several effective analogues were found with K_I values down to around 1 nM.

Full text of DOI:10.1039/b806580b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of pyrophosphate mimics of thiamine
pyrophosphate based on a triazole scaffold
Karl M. Erixon, Chester L. Dabalos and Finian J. Leeper*
Received 21st April 2008, Accepted 9th June 2008
First published as an Advance Article on the web 11th July 2008
DOI: 10.1039/b806580b
Novel triazole-based pyrophosphate analogues of thiamine pyrophosphate (TPP) have been synthesised
and tested for inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis. The thiazolium
ring of thiamine was replaced by a triazole in an efficient two-step procedure. Pyrophosphorylation
then gave extremely potent triazole inhibitors with KI values down to 20 pM, compared to a KD value
of 0.35 lM for TPP. This triazole scaffold was used for further investigation and six analogues
containing mimics of the pyrophosphate group were synthesised and tested for inhibition of
PDC. Several effective analogues were found with KI values down to around 1 nM.
Introduction
Thiamine pyrophosphate 2 (TPP) is an important coenzyme, made
in animals by pyrophosphorylation of thiamine 1 (vitamin B1).
TPP-dependent enzymes serve crucial roles in many metabolic
processes of carbohydrates and branched-chain amino acids.
Examples include a-keto acid decarboxylases, dehydrogenases and
oxidases, transketolase, acetohydroxyacid synthase and deoxyxy-
lulose 5-phosphate synthase.1 All TPP-dependent enzymes have
the common feature that they catalyse the cleavage and formation
of bonds adjacent to the carbon of a carbonyl group. The catalysis
occurs at the thiazolium ring of TPP. However, the pyrimidine
ring of TPP also plays an important role in the mechanism, as it
is involved in the initial activation step (as shown in Scheme 1).
The enzyme promotes the conversion of TPP 2 to its normally less
stable tautomer 2a, and it is this imino form of the amino group
of TPP that effects the deprotonation of C-2 of the thiazolium
ring to give ylid 3.2 In the case of pyruvate decarboxylase (PDC)
the ylid 3 attacks the substrate (pyruvate) to give 2-lactylTPP 4.
Scheme 1 Mechanism of pyruvate decarboxylase.
The next step is decarboxylation to give enamine 5, facilitated by
the positively charged thiazolium ring of TPP, which acts as an
electron sink. Similar bond cleavage and formation of enamine
(also called the “activated aldehyde”) intermediate, is seen in all
TPP catalysis, whatever the substrate. The fate of this enamine then
depends on the particular enzyme. In PDC 5 is protonated to give
hydroxyethylTPP 6, followed by release of acetaldehyde product
and regeneration of ylid 3. In other examples the electrophile could
be an aldehyde or a ketone of another substrate molecule, a lipoyl
group or an oxidant.1
(Fig. 1), which is an isoelectronic inactive analogue of TPP. This
analogue 7 has been studied in detail for inhibition of several TPP-
dependent enzymes, and it has been shown that 7 is an extremely
potent inhibitor, with KI <15 pM for Zymomonas mobilis PDC
(ZmPDC) compared to a KD value of approximately 0.35 lM for
TPP.4,5
There are crystal structures of many TPP-dependent enzymes
available. However, due to fast reaction of substrate bound to the
active site and instability of reaction intermediates, it has proven,
with a few exceptions,3 to be difficult to trap these natural inter-
mediates. Hence, much information regarding substrate binding
and important catalytic groups in the active site still remains to be
revealed. In light of this, we have recently synthesised deazaTPP 7
Fig. 1 DeazaTPP 7 and the triazole analogues of TPP.
All TPP-dependent enzymes are either dimers or tetramers
(dimers of dimers), e.g. PDC is a homotetramer. The two active
sites in each dimer are located at the interface of the two subunits.
The TPP 2 is anchored by the pyrimidine moiety on one side and
by the pyrophosphate group of TPP on the other side. The most
Department of Chemistry, University of Cambridge, Lensfield Road, Cam-
bridge, CB2 1EW, UK. E-mail: fjl1@cam.ac.uk; Fax: +44 1233 336362;
Tel: +44 1223 336493
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important binding interaction originates from the pyrophosphate
which is coordinated to a bivalent metal ion (usually Mg2+) in
the active site.1 However, the pyrophosphate is unsuitable for
whole cell studies or pharmaceutical development, since it is
highly charged and compounds possessing it will suffer from poor
bioavailability and cellular uptake. We therefore wanted to explore
whether the pyrophosphate group could be replaced by isosteres
without losing too much binding affinity for the enzyme. The
synthesis of deazaTPP 7 was quite difficult, however, involving 12
steps,5,6 so in this study we also sought to use a TPP analogue
which was easier to make.
In this paper we report the synthesis of eight novel triazole-
based analogues of TPP, 8–15, and inhibition studies with
ZmPDC. Preliminary results have recently been reported in a
communication7 but here we report full details of the syntheses
of all the TPP analogues and a more extensive evaluation of their
interaction with ZmPDC.
22 was identified by its NOE between the triazole methyl group
and the bridging CH2 group in a NOESY spectrum. The mixture
of isomeric tosylates was separated with difficulty to give a pure
sample of 22 (>95% isomeric purity).
Each tosylate (19 and 22) was converted to its corresponding
pyrophosphate (8 and 9) by nucleophilic displacement with
tris(tetrabutylammonium) pyrophosphate.5 Purification by anion
exchange and then cation exchange chromatography gave products
8 and 9 in moderate yields (50–60%). A cation exchange resin
was used to separate the product from the tosylate anion that
was formed in the reaction and co-eluted on the anion exchange
resin. Although successful in separating these two compounds,
the strongly acidic resin did cause a small amount (<10%) of
cleavage of the pyrophosphates, 8 and 9, to their corresponding
monophosphates.
Synthesis of methylenediphosphonate esters 10 and 11.
Difluoromethylene-diphosphonic acid 25 (Scheme 3) was syn-
thesised by a published method10 in which tetraisopropyl
methylenediphosphonate 23 was treated alternately with 10 por-
tions each of sodium hexamethyldisilazide (NaHMDS) and N-
fluorobenzenesulfonimide (NFSi). Acid 25 was then prepared
in approximately 90% yield by deprotection of the isopropyl
groups using bromotrimethylsilane and subsequent treatment with
methanol.10 Both commercially available methylenediphosphonic
acid 26 and its difluoro derivative 25 were treated with three
equivalents of tetrabutylammonium hydroxide to form the cor-
responding trianions, which were then reacted with tosylate 19 to
give pyrophosphate mimics 10 and 11. The purification and yields
were similar to those for pyrophosphates 8 and 9 described above.
Results and discussion
Synthesis of analogues
Synthesis of triazole pyrophosphates 8 and 9. Triazole ana-
logues 8 and 9 were synthesised conveniently from thiamine 1
itself (Scheme 2). In the first step the thiazolium moiety of 1
was displaced by azide in a bisulfite-catalysed process8 to give
the azidomethylpyrimidine 16 in 83% yield. Cu(I)-catalysed 1,3-
dipolar cycloaddition9 of 16 with 1-butynol or its tosylate 17
then gave triazoles 18 and 19 respectively, in good to moderate
yields (56–81%) after recrystallisation. 5-Methyltriazole 22 was
synthesised by heating 16 with 3-pentynol at reflux in butanol
for 3 days to afford the isomeric mixture of 20 and 21 (approx.
1 : 1) in 80% total yield. This mixture was partially separated by
silica gel chromatography and then tosylated. The desired isomer
Scheme 3 Synthesis of methylenediphosphonates 10 and 11. Reagents
and conditions: (i) sodium hexamethyldisilazide (NaHMDS) (3.0 eq.),
N-fluorobenzenesulfonimide (NFSI) (3.3 eq.), THF, 25 ◦C to reflux,
20 min, 48%; (ii) Me3SiBr (6.1 eq.), DCM, reflux (∼33 ◦C), 24 h, 90%;
(iii) (Bu4N)OH·30H2O (6.0 eq.), then 19 (1.0 eq.), MeCN, 0 to 25 ◦C, 12 h,
58% for 10 and 66% for 11.
Synthesis of N-(alkoxysulfonyl)phosphoramidic acid 12.
Bonnac et al. have recently described a method to synthesise
N-(alkoxysulfonyl)phosphoramidic acids as new mimics of py-
rophosphate esters.11 In their paper they report the cleavage of a
number of trimethyl phosphorimidate derivatives (as in 29) to give
trimethylsilyl esters using bromotrimethylsilane and subsequent
hydrolysis in water for seven days to give the corresponding
phosphoramidic acids. In our synthesis of phosphoramidic acid 12
Scheme 2 Synthesis of pyrophosphates 8 and 9. Reagents and conditions:
(i) NaN3 (2.5 eq.), Na2SO3 (0.1 eq.), H2O, 60–65 ◦C, 6 h, 83%; (ii) sodium
ascorbate (0.1 eq.), CuSO4·5 H2O (0.01 eq.), tBuOH–H2O (2 : 1), 25 ◦C,
16–24 h, 81% for 18 and 56% for 19; (iii) (Bu4N)3HP2O7 (2.0 eq.), MeCN,
−5 to 25 ◦C, 16 h, 50–60%; (iv) 3-pentynol (1.0 eq.), nBuOH, reflux at
130 ◦C, 96 h, 80% (isomeric mixture); (v) TsCl (2.5 eq.), pyr, −5 to 25 ◦C,
3 h, 34% (desired isomer).
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(Scheme 4), sulfamoyl chloride was prepared from chlorosulfonyl
isocyanate 27 and formic acid and then coupled in situ with 3-
butynol to give the sulfamate 28 in 67% yield (over two steps).12
Then reaction with di-tert-butyl azodicarboxylate and trimethyl
phosphite afforded trimethyl phosphorimidate 29 in 80% yield
after chromatography.
Synthesis of N-(alkoxycarbonyl)sulfamic acid 13. Analogue 13
was synthesised in a facile one-pot reaction in which alcohol 18
was added to a slight excess of chlorosulfonyl isocyanate 27 to form
intermediate 32, which was subsequently hydrolysed with water to
give carbamate 13 (Scheme 5). The mixture was purified by ion
exchange chromatography, which gave a 9 : 1 mixture of desired
analogue 13 and double reaction product 33 in approximately 55%
total yield. Attempts to avoid dimer formation, e.g. by adding
more equivalents of isocyanate, or to improve the separation, e.g.
by varying the eluant gradient, were unsuccessful. Nevertheless,
1
the purity (by H NMR spectroscopic analysis) was considered
to be good enough for biological testing, especially as 33 was not
expected to bind to the enzyme.
Scheme 4 Synthesis of N-(alkoxysulfonyl)phosphoramidic acid 12.
Reagents and conditions: (i) formic acid (1.0 eq.), then 3-butynol (0.67 eq.),
pyridine (1.0 eq.), DCM, 0 to 25 ◦C, 17 h, 67%; (ii) (MeO)3P (2.0 eq.),
di-tert-butyl azodicarboxylate (2.0 eq.), 25 ◦C, 2 h, 80%; (iii) Me3SiBr
(6.2 eq.), 25 ◦C, 3 h, 100%; (iv) MeOH, 25 ◦C, 5 min, 100%; (v) 16 (1.0 eq.),
CuI (0.6 eq.), DMF, 25 ◦C, 24 h, 57%.
Scheme 5 Synthesis of N-(alkoxycabonyl)sulfamic acid 13. Reagents and
conditions: (i) pyridine, then H2O, 0 to 25 ◦C, 2.5 h, 55% (9 : 1 molar ratio
of 13 and 33).
Following the published procedure,11 29 was treated
with bromotrimethylsilane. However, rather than forming
the tris(trimethylsilyl) ester, as described in the paper, the
bis(trimethylsilyl) ester 30 was obtained quantitatively. Hydrolysis
of 30 in water (pH 7, for seven days) was monitored in situ by
31P NMR spectroscopy and peaks were observed at similar shifts
to those reported by Bonnac et al. (i.e. −25.5, −10.5, −4.8 and
+2.5 ppm, corresponding to the starting material, different hydrol-
ysis intermediates and the final product respectively). However
isolation of the organic product of this hydrolysis gave back the
undesired sulfamate 28, product of cleavage of the N–P bond. The
conclusion that the 31P NMR peak at +2.5 ppm must be due to
inorganic phosphoric acid, rather than the phosphoramidic acid
31, was confirmed by spiking the NMR sample with commercial
phosphoric acid. We suggest that the same is probably true of the
peak at +2.5 ppm observed by Bonnac et al.
Synthesis of malonate ester 14. In our first approach, attempts
were made to simply couple malonic acid 34 with the triazole
alcohol 18 using DCC as a coupling reagent. Even though the
desired product 14 was identified, the purification of the reaction
mixture turned out to be very troublesome, since both starting
alcohol 18 and malonate 14 are highly water-soluble and 14 is
unstable on silica gel. Because of these difficulties, malonic acid 34
was first coupled with tert-butanol using DCC which gave mono-
tert-butyl malonate 35 in 66% yield (Scheme 6).13 Subsequent
coupling of 35 with alcohol 18 afforded the tert-butyl-protected
product 36 in 51% yield. Finally, deprotection of 36 using TFA
gave the malonate analogue 14 in 80% yield.
Fortunately, it was found that bis(trimethylsilyl) ester 30 could
be cleaved to give phosphoramidic acid 31 by treatment with
methanol for 5 min at room temperature. The acid 31 gave
a single peak at −5.4 ppm in its 31P NMR spectrum. Our
experience suggests that the four chemical shifts described by
Bonnac et al. are more likely due to the bis(trimethylsilyl) ester,
the mono(trimethylsilyl) ester, the phosphoramidic acid, and
inorganic phosphate, respectively.
Scheme 6 Synthesis of the malonate ester 14. Reagents and conditions:
(i) tBuOH (2.0 eq.), DCC (2.2 eq.), MeCN, 25 ◦C, 30 min, 66%; (ii) 18
(1.0 eq.), DCC (1.0 eq.), THF–pyridine, 25 ◦C, 18 h, 51%; (iii) neat TFA,
25 ◦C, 3 h, 80%.
Application of ‘click’ chemistry – cycloaddition of 31 and azide
16 in DMF with Cu(I) as catalyst – gave the triazole 12 in 57%
yield after anion exchange chromatography.
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Synthesis of iminodiacetic acid monoester 15. In our first
approach to monoester 15, iminodiacetic 37 was Boc-protected to
give 38 (Scheme 7) and then coupled with alcohol 18 via anhydride
40 using DCC.14 Unfortunately, as in the synthesis of malonate
14, it was extremely troublesome to purify the resulting mixture.
Consequently, attempts were made to couple the anhydride 40 with
tert-butanol instead to give the Boc-protected mono-tert-butyl
ester before coupling with the triazole alcohol 18. Unfortunately,
both tert-butanol and potassium tert-butoxide were completely
unreactive in this reaction. As a result, it was decided to use the
less hindered benzyl alcohol instead and, to avoid the need for two
separate deprotection steps later in the synthesis, the amine was
first protected with a Cbz-group to give 39 in 71% yield.15 In the
next step, the solid anhydride 41 was first formed (not isolated)
and then benzyl alcohol and dicyclohexylamine, as a base, were
added. The use of this base was very convenient, since it not only
deprotonated the alcohol but also formed a solid salt with the
carboxylic acid 42, which precipitated during the reaction.16 The
pure product, in its salt form, was collected and recrystallised and
the base was then removed by extraction into aqueous acid, giving
42 as an oil in 74% yield. Acid 42 was coupled with the alcohol 18
using DCC and DMAP (no reaction occurred without DMAP) to
give diester 43 in 70% yield. Final deprotection by hydrogenation
over palladium-on-carbon gave monoester 15 in 76% yield.
Scheme 8 Coupled assay of PDC.
In the binding experiments, the analogues (typically 2–10 lM)
were incubated with apo-PDC in a Mg2+-containing buffer and
small samples were taken out at timed intervals (1–15 min) and
added to the assay solution containing ADH, Mg2+, NADH and
an excess of TPP (100 lM). The assay was then started by the
addition of pyruvate. Under these conditions any apo-PDC that
does not already have the TPP analogue bound will bind TPP
when it is added and thus show activity. However, TPP analogues
that are only loosely bound may be displaced by the excess TPP
and so could also show activity.
Using this assay, the eight TPP analogues (8–15) were incubated
with apo-PDC and the decrease of activity was measured. All
analogues except for 13, 14 and 15 showed strong binding affinity
under these conditions and completely inactivated the enzyme
within 15–20 min. The iminodiacetic acid moiety of analogue 15
was intended to not only occupy the pyrophosphate binding site
but also replace the Mg2+ ion, with the protonated amine binding in
its place, and so for this analogue the binding was also performed
in the absence of Mg2+. However no inhibition was observed either
in the presence or absence of Mg2+.
In all cases of inhibition the data-points fitted poorly onto a
simple exponential curve, the initial decrease of activity being too
fast and the later decay too slow, but fitted reasonably well onto a
double exponential curve of equation:
y = (1 − x)exp(−k1t) + xexp(−k2t).
The value of x was fixed at 0.35, which gave a good fit in all
cases.
Fig. 2 shows the binding of analogue 9. The faster of the two
apparent first-order rate constants obtained from each curve was
plotted against concentration to give a straight line, which gave
the second order rate constant for the initial binding event, kon.
Similar curves were seen for the other analogues (i.e. 8 and 10–12),
and kon values are presented in Table 1.
The apparent two-stage inhibition has been seen in previous
studies,5,17 and two possible explanations are (i) normal slow-
binding inhibition, in which a more rapid initial binding is followed
by a slower irreversible step, presumably a conformational change
of the enzyme, or (ii) the two rates observed in our inhibition
experiments are for binding of the first molecule of inhibitor to
the enzyme dimer and for binding of the second molecule of
inhibitor. Many TPP-dependent enzymes show so-called half-of-
sites reactivity (see recently published review18), due to some form
of communication between the two active sites of each dimer, so
it is quite possible that the rate of binding of a TPP analogue to
one active site is dependent on whether or not the other active site
of the dimer is occupied. If this second explanation is correct, the
rate constant for the second slower step would also be dependent
on inhibitor concentration. Although the k2 values did appear
to increase with increasing inhibitor concentration, the errors in
these values were much larger than in the k1 values and, as a result,
we were not able to show a linear dependence.
Scheme 7 Synthesis of iminodiacetic acid monoester 15. Reagents and
conditions: (i) BnOCOCl (2.0 eq.), aq. NaOH (2 M), 25 ◦C, 2 h, 71%;
(ii) DCC (1.0 eq.), THF, 25 ◦C, 12 h; (iii) BnOH (3.1 eq.), dicyclohexy-
lamine (1.1 eq.), Et2O, 0 to 25 ◦C, 12 h, 74%; (iv) 18 (1.0 eq.), DCC (1.1 eq.),
DMAP (1.1 eq.), 25 ◦C, 24 h, 70%; (v) Pd/C (10%), H2 (1 atm), 25 ◦C,
20 min, 76%.
Inhibition studies with pyruvate decarboxylase
Pyruvate decarboxylase (PDC) catalyses the conversion of pyru-
vate to acetaldehyde and carbon dioxide. Its activity is measured
by a coupled enzyme assay using the NADH-dependent reduction
of acetaldehyde by alcohol dehydrogenase (ADH) (Scheme 8).4,5
Because the TPP analogues can only bind to the holo-enzyme
once TPP has dissociated and the dissociation of TPP is known to
be very slow,4 we instead studied binding of the analogues to the
apo-enzyme, prepared as described previously.5
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Fig. 3 Recovery of activity for ZmPDC fully inhibited by TPP analogue
9 (10 lM) and then incubated with TPP (10 mM). The activity is given as
a fraction of the initial activity of uninhibited enzyme.
Fig. 2 Inactivation of ZmPDC by various concentrations of TPP
analogue 9, from top to bottom: ● 2 lM, ᭺ 4 lM, ꢀ 6 lM, ꢀ 8 lM,
ꢁ 10 lM; the residual activity is given as a fraction of the initial activity
of uninhibited enzyme. Inset: Apparent first-order rate constants plotted
versus inhibitor concentrations; the slope of the graph gives kon.
interestingly it also speeds up the rate of binding quite markedly,
perhaps because of the greater hydrophobicity.
Previously we reported that we were unable to observe any
reactivation of enzyme inhibited with deazaTPP 7.5 However, in
this work, by using higher concentrations of TPP and longer time
periods, we were able to observe some reactivation in a similar way
as for 8 and 9 (3.5% activity in the presence of 10 lM of 7 and
10 mM of TPP). The KI value obtained from this experiment was
13 ( 3) pM.
Table 1 Inhibition parameters for analogues 8–15 and 18
Analogue
kon/lM−1 min−1
KI
30 3 pM
8
9
0.05 0.02
0.21 0.03
0.09 0.01
0.17 0.03
0.02 0.004
n.o.a
20 2 pM
10
11
12
13
14
15
18
0.95 0.1 nM
1.2 0.1 nM
0.14 0.02 lM
The reason why analogues such as 7, 8 and 9, bind so much
more tightly than TPP itself is thought to be because the enzyme
stabilises neutral rings at this position better than the positively
charged thiazolium ring.5,19 In this way the enzyme promotes both
the formation of the ylide and the decarboxylation reaction.
Some of the analogues containing pyrophosphate mimics also
show strong inhibitory activity. In 10 and 11, the bridging
oxygen in the pyrophosphate has been replaced by a CH2 or
CF2 group. These have recently been reported to be effective
non-hydrolysable mimics of pyrophosphate, used in e.g. antivi-
ral nucleoside analogues.10 We expected these to be effective
replacements for the pyrophosphate because the crystal structures
of PDC complexed with TPP show no hydrogen bonds to the
bridging oxygen of the pyrophosphate group.20 However, in our
case, it appears that the bridging oxygen is actually of some
importance for the binding. Although they bind faster than the
corresponding pyrophosphate 8, the KI values for both these
analogues (10 and 11) were estimated to be more than 10-fold
higher. Methylenediphosphonate esters such as 11 are known
to have higher pKa values than pyrophosphates,21 which might
explain why 11 binds faster, being less hydrophilic, but less
tightly than 8. Difluoromethylene-diphosphonate esters such as
10, however, have a similar pKa to pyrophosphates.22 Presumably
the greater size of the CF2 group compared to O reduces the
binding affinity in this instance.
8
2 lM
n.o.a
0.4 0.1 mM
0.4 0.1 mM
0.3 0.1 mM
n.o.a
n.o.a
a Not observable.
By adding a large excess of TPP (100 lM to 10 mM) to
inactivated PDC (generally <1% activity) the reversiblity of the
binding of each analogue was investigated. The recovery of activity
was followed over a period of 7–8 days and in each case some
activity was regained to reach a plateau within ca. 2 days (e.g. Fig. 3
depicts the reactivation of PDC pre-inhibited by analogue 9). As
full activity was retained in a control experiment with uninhibited
enzyme, even after 7–8 days, the plateau of activity reached with
inhibitor present can be regarded as due to establishment of the
equilibrium between binding of TPP and binding of the inhibitor.
Using the relative concentrations of TPP and inhibitor and the
previously reported KD value for TPP (0.35 lM),4 the KI values
for the analogues were calculated and are listed in Table 1.
Not surprisingly, the two analogues possessing the important
pyrophosphate group (8 and 9) were the most potent inhibitors:
only 8.0% and 5.5% respectively of the original activity was
recovered when enzyme inhibited with 10 lM of inhibitor was
reactivated with 10 mM TPP. This corresponds to KI values of
30 pM and 20 pM. The 5-methyl group of 9 slightly improves
the strength of binding compared with 8, as might be expected
because TPP also possesses a methyl group in this position, but
The assay described above is only useful for TPP analogues
which bind very tightly. This is because analogues that bind
weakly will be displaced by TPP when the enzyme activity assay
is started (TPP is added in a large excess) and consequently,
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any binding that might have occurred will not be seen. Hence,
analogues 13–15 required a different type of experiment in order
to measure their binding. In this second type of experiment, the
same coupled assay shown in Scheme 8 was used, but instead
of studying the binding of the TPP analogue to apo-PDC, the
effect of analogues 13–15 on the binding of TPP to apo-PDC was
studied. Accordingly, TPP 2 (4 lM) and analogue (13–15; various
concentrations) were incubated with apo-PDC (ca. 0.4 lM active
sites) and the activity was measured at timed intervals. As expected,
the increase of activity followed a saturation-like curve and under
these conditions the activity reached a maximum after ca. 45 min
of incubation when no analogue was present (see Fig. 4). For
comparison, alcohol 18 was also included in this study. In all cases
the presence of analogue did not have much effect on the initial
rate of activation but it did affect the maximum activity reached.
By analogy with the reactivation experiment with analogues 8–
12, it was believed that this lower level of activity was due to an
equilibrium between TPP binding and analogue binding. Hence
the corresponding KI values could be calculated. The KI values
listed in Table 1 for 13–15 and 18 are the average values obtained
from all concentrations tested for each analogue.
Using the most readily synthesised triazole 18, we set out to find
less charged and more stable mimics of the pyrophosphate group
that would still be potent inhibitors. Six pyrophosphate analogues
were synthesised and tested. The results provide a striking example
of the great importance of the pyrophosphate group, with ca.
1.0 × 107-fold higher affinity of pyrophosphate 8 for the active
site compared to its corresponding alcohol 18. However, it was
also shown that several of the analogues containing mimics of the
pyrophosphate group, particularly the methylene-diphosphonates
10 and 11 (with KI values just 30- to 40-fold greater than that of
8), still bind with high affinity.
Marked decreases of inhibition are seen in going from tri-
anionic analogues 8–11 to the dianionic phosphoramidic acid
12, to the monoanionic carbamate 13 and malonate 14. Hence,
with a magnesium ion in the binding site, the more negatively
charged analogues clearly bind tightest. With the iminodiacetic
acid analogue 15 the intention was that the protonated amino
group would replace the magnesium ion, but unfortunately this
compound did not turn out to be an effective inhibitor at all.
This study is relevant not only to TPP-dependent enzymes
but also a wide range of other enzymes and proteins that bind
pyrophosphate esters (e.g. ADP and GDP).
Experimental
General synthesis methods
Proton NMR spectra were recorded on a Bruker AM/DPX 400
(400 MHz) or a Bruker DPX 500 (500 MHz) spectrometer. The
chemical shifts (d) and coupling constants (J) are given in ppm
(downfield of TMS) and Hz, respectively. Carbon NMR spectra
were recorded on either a Bruker AC/DPX 400 (100 MHz) or
a Bruker DPX 500 (126 MHz) spectrometer. To assist in the
assignments, the numbers of attached protons were determined
using either APT (Attached Proton Test, J-resolved spin echo)
or DEPT (Distortionless Enhancement by Polarisation Transfer)
spectra. Phosphorus NMR spectra were recorded on a Bruker
DPX 400 (162 MHz) with broadband proton decoupling. IR
spectra were recorded on a Perkin-Elmer FTIR spectrometer
and only significant bands are reported. Melting points were
determined on a Reichert melting point apparatus. Mass spectra
were run on a Bruker BioApex II 4.7e FTICR or Waters LCT
Premier using electrospray ionisation (ESI). Analytical TLC
was performed using commercial Merck glass plates, coated to
thickness of 0.25 mm with Kieselgel 60 F254 silica and visualised
under UV light or by staining with vanillin, ninhydrin or KMnO4
dip solutions. Flash chromatography was performed using Merck
Kieselgel 60 (230–400 mesh) silica under a slight positive pressure
of air. Anion exchange was performed on a Pharmacia Acta Prime
chromatography system using a column packed with DEAE-
Sephacel and eluting with a gradient of 0 to 0.25 M aqueous
ammonium bicarbonate. Solvents and reagents for anhydrous
reactions were dried prior to use by conventional methods.23
Fig. 4 Inhibition of binding of TPP (4 lM) to PDC by carbamate 13,
from top to bottom: ● no inhibitor, ᭺ 20 lM, ꢀ 40 lM, ꢀ 80 lM, ꢁ
100 lM, ꢂ 160 lM; the activity is given in arbitrary units.
As the carbamate 13 had shown some activity in the first assay
for strong inhibitors, it was not surprising that 13 was most potent
among these weak binders. In fact, the KI value of 8 lM for 13
is only about 23-fold greater than the KD for TPP. Analogues 14
and 15, however, showed almost the same affinity as alcohol 18,
and thus it is likely that their interaction with the active site comes
mostly from the pyrimidine part. The iminodiacetic acid moiety
of 15 is presumably too large to fit into the pyrophosphate pocket
in the active site.
Conclusion
In conclusion, we report here the synthesis and biological evalua-
tion of eight new triazole-based analogues of TPP. The analogues
were prepared in relatively few steps using ‘click’ chemistry.
The pyrophosphate esters 8 and 9 proved to be low picomolar
inhibitors of PDC, almost as potent as deazaTPP 7.
5-Azidomethyl-2-methylpyrimidin-4-ylamine 16
To a solution of thiamine chloride (10.3 g, 30.6 mmol) and sodium
azide (4.9 g, 75.4 mmol) in water (100 ml) was added sodium sulfite
(0.38 g, 3.0 mmol) and the mixture was stirred at 65 C for 5 h.
◦
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Citric acid (21.0 g, 100 mmol, to pH ≈ 4) was added and the
aqueous solution was washed with dichloromethane. Potassium
carbonate (to pH ≈ 8) was added, upon which some precipitation
of the product occurred. The suspension was filtered and the
filtrate was extracted with ethyl acetate and the combined organic
layers were washed with brine, dried (MgSO4) and evaporated
under reduced pressure. The solid residue was pooled with the
precipitate and recrystallised from ethyl acetate–hexane to give the
azide 16 as fine needles (3.15 g, 63%), m.p. 150–153 ◦C [Found: C,
43.6; H, 4.9; N, 50.7; M + H+ (+ESI), 165.0881 C6H8N6 requires
C, 43.9; H, 4.9; N, 51.2; M + H 165.0889]; mmax/cm−1 3300 and
3094 (NH2), 2086 and 2107 (N3), 1668, 1587 and 1561 (pyrimidine
ring); dH (400 MHz, CDCl3) 2.50 (3 H, s, CH3), 4.19 (2 H, s, CH2),
5.47 (2 H, broad, NH2), 8.05 (1 H, s, CH); dC (100 MHz, DMSO-
d6) 24.5 (CH3), 46.7 (CH2), 106.9 (CCNH2), 155.1 (CH), 161.1
and 166.2 (CNCNH2).
109.3 (CCNH2), 123.3 (triazole CH), 145.4 (triazole C), 158.8
(CH), 162.2 and 167.6 (CNCNH2).
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-[1,2,3]triazol-4-
yl]ethyl toluene-4-sulfonate 19
To a stirred solution of tosylate 17 (297 mg, 1.32 mmol) and azide
16 (217 mg, 1.32 mmol) in tert-butanol–water (6 ml, 2 : 1) were
added sodium ascorbate (26 mg, 0.13 mmol) and CuSO4·5H2O
(3 mg, 0.12 mmol). The reaction mixture was stirred at room
temperature for 16 h and then evaporated under reduced pressure.
The oily residue was dissolved in 1-butanol and washed with
aqueous potassium carbonate (0.1 M) and then with brine, dried
(MgSO4) and evaporated under reduced pressure to give a white
solid. Recrystallisation from 2-propanol–petroleum ether (b.p. 60–
80 ◦C) gave◦the triazole 19 as fine yellow crystals (289 mg, 56%),
m.p. 85–88 C [Found: M + H+ (+ESI), 389.1400 C17H20N6O3S
requires M + H, 389.1396]; mmax/cm−1 3335 and 3130 (NH2), 1661
and 1568 (pyrimidine ring), 1348 and 1174 (SO2O), 905 (aromatic
C–H); dH (400 MHz, DMSO-d6) 2.28 (3 H, s, CH3), 2.38 (3
H, s, CH3), 2.92 (2 H, t, J 6.4, CH2CH2O), 4.18 (2 H, t, J 6.4,
CH2CH2O), 5.35 (2 H, s, CH2 bridge), 6.86 (2 H, br s, NH2), 7.40
(2 H, d, J 8.1, 2 × Ar-H), 7.67 (2 H, d, J 8.1, 2 × Ar-H) 7.83
(1 H, s, triazole CH), 7.93 (1 H, s, pyrimidineCH); dC (126 MHz,
DMSO-d6) 21.2 (CH3), 25.3 (CH3), 25.6 (CH2CH2O), 46.6 (CH2
bridge), 69.6 (CH2CH2O), 108.5 (CCNH2), 123.0 (triazole CH),
127.6 and 130.2 (4 × ArCH), 132.2 and 142.2 (2 × ArC), 145.0
(triazole C), 156.0 (pyrimidine CH), 161.6 and 166.9 (CNCNH2).
But-3-ynyl toluene-4-sulfonate 17
To a stirred solution of 3-butynol (0.11 ml, 1.46 mmol) in
anhydrous pyridine (15 ml) at-5 ◦C was added in portions p-
toluenesulfonyl chloride (697 mg, 3.65 mmol). The reaction
mixture was stirred at room temperature for 3 h, then quenched
with hydrochloric acid (1 M) and extracted with ethyl acetate.
The combined organic layers were washed with aqueous sodium
bicarbonate, aqueous CuSO4 and then with brine, dried (MgSO4)
and evaporated under reduced pressure to yield the tosylate 1724
as an oil (261 mg, 80%) [Found: M + Na+ (+ESI), 247.0406
C11H12O3S requires M + Na, 247.0399]; mmax/cm−1 3287 (H–CC),
1357 and 1173 (SO2O), 978 (aromatic C–H); dH (400 MHz, CDCl3)
1.95 (1 H, t, J 2.7, CH), 2.42 (3 H, s, CH3), 2.53 (2 H, dt, J 2.7 and
7.0, CH2CH2O), 4.08 (2 H, t, J 7.0, CH2CH2O), 7.33 (2 H, d, J 8.2,
2 × ArCH), 7.77 (2 H, d, J 8.2, 2 × ArCH); dC (100 MHz, CDCl3)
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-5-methyl-1H-
[1,2,3]triazol-4-yl]ethanol 21 and 2-[1-(4-amino-2-methyl-
pyrimidin-5-ylmethyl)-4-methyl-1H-
≡
17.9 (CH2CH2O), 20.1 (CH3), 66.0 (CH2CH2O), 69.3 (C CH),
[1,2,3]triazol-5-yl]ethanol 20
≡
76.9 (C C–C), 126.5 and 128.4 (4 × ArCH), 131.3 and 143.6
(2 × ArC).
A solution of azide 16 (328 mg, 2.0 mmol) and 3-pentynol (185 ll,
◦
2.0 mmol) in 1-butanol (2 ml) was heated at reflux (120 C) for
72 h. The crude mixture was concentrated under reduced pressure
and purified by silica gel chromatography eluting with DCM–
MeOH (3 : 1) to give an approximately 1 : 1 mixture of 20 and
21 (399 mg, 80%) as a light yellow solid. The two regioisomers
were partially separated by further chromatography (as above)
to give a mixture of 20 and 21 (1 : 3; 250 mg) [Found: M + H+
(+ESI), 249.1460 C11H16N6O requires M + H, 249.1464]; mmax/cm−1
3000–3450 (broad), 3395 and 3112 (NH2), 1649, 1595 and 1559
(pyrimidine ring) and 1045 (CO); dH for 21 (500 MHz, DMSO-
d6) 2.19 (3 H, s, CH3), 2.29 (3 H, s, CH3), 2.68 (2 H, t, J 7.0,
CH2CH2OH), 3.57 (2 H, q, J 5.2 and 7.0, CH2CH2OH) 4.69 (1
H, t, J 5.2, CH2CH2OH), 5.31 (2 H, s, CH2 bridge), 6.82 (2 H,
broad, NH2), 7.73 (1 H, s, pyrimidine CH); dC for 21 (126 MHz,
DMSO-d6) 10.6 (triazole CH3), 25.6 (CH3), 29.0 (CH2CH2O), 45.3
(CH2 bridge), 61.0 (CH2CH2O), 108.5 (CCNH2), 131.5 and 142.5
(triazole C), 155.2 (pyrimidine CH), 161.9 and 167.0 (CNCNH2);
dH for 20 (500 MHz, DMSO-d6) 2.17 (3 H, s, CH3), 2.29 (3
H, s, CH3), 2.79 (2 H, t, J 6.4, CH2CH2OH), 3.48 (2 H, q, J
6.4, CH2CH2OH), 5.0 (1 H, b, CH2CH2OH), 5.34 (2 H, s, CH2
bridge), 6.82 (2 H, br s, NH2), 7.73 (1 H, s, pyrimidine CH);
dC for 20 (126 MHz, DMSO-d6) 18.6 (CH3), 25.6 (CH3), 26.0
(CH2CH2O), 45.6 (CH2 bridge) 59.8 (CH2CH2O),108.8 (CCNH2),
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-[1,2,3]triazol-4-
yl]ethanol 18
To
a stirred solution of 3-butynol (907 ll, 12.0 mmol)
and aminopyrimidine azide 16 (1.968 g, 12.0 mmol) in tert-
butanol/water (12 ml; 2 : 1) were added sodium ascorbate (238 mg,
1.2 mmol) and CuSO4·5H2O (30 mg, 0.12 mmol). The reaction
mixture was stirred at room temperature for 16 h. The crude
mixture was evaporated under reduced pressure and the solid
residue was dissolved in 1-butanol. The organic layer was washed
with aqueous potassium carbonate (0.1 M) and then with brine,
dried (MgSO4) and evaporated under reduced pressure to give
a white solid. Recrystallisation from 2-propanol/hexane gave
the triazole 18 as fine needles (2.27 g, 81%), m.p. 164–165 ◦C
[Found: M + H+ (+ESI), 235.1305 C10H14N6O requires M +
H, 235.1307]; mmax/cm−1 3500–3000 (broad OH), 3340 and 3140
(NH2), 1657 and 1566 (pyrimidine ring) and 1043 (CO); dH
(400 MHz, DMSO-d6) 2.28 (3 H, s, CH3), 2.74 (2 H, t, J 6.8,
CH2CH2O), 3.58 (2 H, t, J 6.8, CH2CH2O), 4.65 (1 H, s, OH),
5.35 (2 H, s, CH2 bridge), 6.85 (2 H, s, NH2), 7.83 (1 H, s, triazole
CH), 7.93 (1 H, s, pyrimidineCH); dC (100 MHz, DMSO-d6) 26.0
(CH3), 29.9 (CH2CH2O), 47.3 (CH2 bridge), 61.0 (CH2CH2O),
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132.5 and 141.0 (triazole C), 155.2 (pyrimidine CH), 161.8 and
166.9 (CNCNH2).
to room temperature, stirred for 16 h under an atmosphere of
argon, then diluted with water (1 ml) and purified first by anion-
exchange chromatography and then by absorption onto Dowex
50 H+-form and elution with aqueous NH3 (1 M). Lyophilisation
gave the pyrophosphate 9 as a white ammonium salt (30 mg, 49%).
[Found: M + H+ (+ESI), 409.0794 C10H16N6O7 requires M +
H, 409.0785]; dH (400 MHz, D2O) 2.22 (3 H, s, CH3), 2.34 (3
H, s, CH3), 2.92 (2 H, t, J 6.5, CH2CH2O), 4.03 (2 H, dt,
J 6.9 and J 6.5, CH2CH2OP), 5.30 (2H, s, CH2 bridge), 7.78
(1H, s, pyrimidine CH); dC (100 MHz, D2O) 6.5 (triazole-CH3),
21.5 (pyrimidine-CH3), 25.1 (CH2CH2O, d, J 7.6), 44.5 (CH2-
bridge), 64.4 (CH2CH2O, d, J 5.7), 108.5 (CCNH2), 133.0 and
142.0 (triazole C), 146.9 (pyrimidine CH), and 161.6 and 164.1
(CNCNH2); dP (162 MHz, D2O) −8.8 (1P, d, J 20.4) and −9.7
(1P, d, J 20.4).
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-5-methyl-1H-
[1,2,3]triazol-4-yl]ethyl toluene-4-sulfonate 22
To a stirred solution of the mixture of alcohols 20 and 21 (230 mg,
0.93 mmol, 1 : 3 mixture) in anhydrous pyridine (10 ml) at 0 ◦C was
added in portions p-toluenesulfonyl chloride (886 mg, 4.64 mmol).
The reaction mixture was stirred at room temperature for 3 h,
then quenched with hydrochloric acid (0.1 M) and extracted with
ethyl acetate. The combined organic layers were washed with
brine, dried (MgSO4) and evaporated under reduced pressure to
yield a mixture (340 mg, 91%) of the two tosylates. This mixture
was partially separated by silica gel chromatography eluting with
DCM–MeOH (9 : 1) to give tosylate 22 (>95% isomeric purity)
as an oil (95 mg, 34%). [Found: M + H+ (+ESI), 403.1544
C18H22N6O3S requires M + H, 403.1547]; mmax/cm−1 3361 and 3139
(NH2), 1669 and 1571 (pyrimidine ring) 1358 and 1185 (SO2O),
902 (aromatic C–H); dH (400 MHz, CDCl3) 2.15 (3 H, s, CH3), 2.37
(3 H, s, CH3), 2.42 (3 H, s, CH3), 2.90 (2 H, t, J 6.7, CH2CH2O),
4.19 (2 H, t, J 6.7, CH2CH2O), 5.15 (2 H, s, CH2 bridge), 5.70 (2
H, br s, NH2), 7.23 (2 H, d, J 8, 2 × Ar-H), 7.63 (2 H, d, J 8,
2 × Ar-H), 8.08 (1 H, s, pyrimidine CH); dC (100 MHz, CDCl3)
6.7 (triazole CH3), 21.2 (CH3), 25.5 (CH3), 27.1 (CH2CH2O), 46.9
(CH2 bridge), 69.2 (CH2CH2O), 107.9 (CCNH2), 127.2 and 130.6
(4 × ArCH), 131.2 (triazole C) 132.8 and 142.7 (2 × ArC), 145.4
(triazole C), 155.6 (pyrimidine CH), 161.7 and 167.1 (CNCNH2).
Tetraisopropyl difluoromethylenediphosphonate 2410
To stirred neat tetraisopropyl methylenediphosphonate 23 (359 ll,
1.12 mmol) at room temperature were added, alternately in
10 portions each, a solution of sodium hexamethyldisilazide
(NaHMDS) in THF (1.67 M; 2 ml, 3.35 mmol) and a solution of
N-fluorobenzenesulfonimide (NFSi) (1.16 g, 3.68 mmol) in THF
(2 ml). The sequence of additions was started with NaHMDS,
and as the reaction was exothermic the temperature rose to
near reflux after each addition. After the additions, the reaction
mixture was allowed to cool to room temperature, then cooled to
−78 ◦C, quenched with saturated aqueous ammonium chloride
and diluted with diethyl ether. The mixture was warmed to room
temperature, the organic layer was separated and the aqueous
layer was extracted with diethyl ether. The combined organic
layers were washed with 5% aqueous citric acid, then saturated
aqueous sodium bicarbonate, and then brine, dried (MgSO4) and
concentrated under reduced pressure. The residual syrup was
purified by silica gel chromatography eluting with diethyl ether
to give difluoromethylenediphosphonate 24 as an oil (203 mg,
48%) dH (400 MHz, CDCl3) 1.38 (12 H, d, J 4.5, 4 × CH3), 1.40
(12 H, d, J 4.5, 4 × CH3), 4.91 (4 H, m, 4 × CH); dF (376 MHz,
CDCl3) −122.3 (2 F, t, J 87.1); all analytical data are consistent
with those previously reported.10
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-[1,2,3]triazol-4-
yl]ethyl pyrophosphate 8
To a stirred solution of tosylate 19 (20 mg, 0.05 mmol) in
anhydrous acetonitrile (150 ll) at −10 ◦C under an atmosphere
of argon was added in portions tris(tetra-n-butylammonium)
hydrogen pyrophosphate (93 mg, 0.10 mmol). The reaction
mixture was allowed to warm to room temperature, stirred for
16 h under argon, then diluted with water (1 ml) and purified
first by anion-exchange chromatography and then by absorption
onto Dowex 50 H+-form and elution with aqueous NH3 (1 M).
Lyophilisation gave the pyrophosphate 8 as a white ammonium salt
(12 mg, 60%), m.p. 209–211 ◦C [Found: M + H+ (+ESI), 395.0645
C10H16N6O7 requires M + H, 395.0628]; mmax/cm−1 3130 and 3048
({2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-[1,2,3]triazol-
4-yl]ethoxy}hydroxyphosphoryldifluoromethyl)phosphonic acid 10
=
(broad NH2), 1654 and 1547 (pyrimidine ring), 1194 (P O), 1057
(P–OR), 905 (P–O–P); dH (400 MHz, D2O) 2.31 (3 H, s, CH3),
2.94 (2 H, t, J 6.6, CH2CH2O), 4.02 (2 H, dt, J 6.7 and J 6.6,
CH2CH2OP), 5.37 (2 H, s, CH2 bridge), 7.85 (1 H, s, triazole CH),
7.97 (1 H, s, pyrimidine CH); dC (126 MHz, D2O) 23.5 (CH3),
26.3 (CH2CH2O, d, J 7.6), 47.5 (CH2 bridge), 64.3 (CH2CH2O,
d, J 5.5), 108.4 (CCNH2), 124.2 (triazole CH), 145.3 (triazole C),
154.6 (pyrimidine CH), 161.8 and 167.3 (CNCNH2); dP (162 MHz,
D2O) −6.54 and −9.95 (each 1 P, br s, OPOPO).
To a stirred solution of 24 (66 mg, 0.17 mmol) in anhydrous
CH2Cl2 (2 ml) under an atmosphere of argon was added dropwise
bromotrimethylsilane (137 ll, 1.04 mmol). The reaction mixture
was stirred and heated at gentle reflux (∼33 ◦C) for 24 h and
then evaporated under reduced pressure. CH2Cl2 (5 ml) was twice
added and evaporated again. The residual oil was cooled on ice and
methanol (5 ml) was added and then evaporated under reduced
pressure. Methanol (5 ml) was twice more added and evaporated
again. The residual oil was dissolved in water (8 ml) and the
aqueous solution was washed with ethyl acetate. Lyophilisation
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-5-methyl-1H-
[1,2,3]triazol-4-yl]ethyl pyrophosphate 9
1
gave acid 25 as a thick oil (33 mg, 90%); H NMR spectroscopy
To a stirred solution of tosylate 22 (60 mg, 0.15 mmol) in
acetonitrile (anhydrous, 350 ll) at −10 ◦C under argon was added
in portions tris(tetra-n-butylammonium) hydrogen pyrophosphate
(271 mg, 0.30 mmol). The reaction mixture was allowed to warm
showed no peaks, consistent with the desired product. The product
was used in the next step without further purification.
A solution of tetrabutylammonium hydroxide·30H2O (376 mg,
0.47 mmol) and difluoromethylenediphosphonic acid 25 (33 mg,
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0.154 mmol) in water (5 ml) was stirred at room temperature for
15 min and then lyophilised to give the tris(tetrabutylammonium)
salt. The salt was dissolved in anhydrous acetonitrile (300 ll),
and tosylate 19 (31 mg, 0.08 mmol) was added at 0 ◦C under
an atmosphere of argon. The reaction mixture was allowed to
warm to room temperature and stirred for 12 h. The mixture was
evaporated under reduced pressure, dissolved in water (1 ml) and
purified by anion-exchange chromatography. Lyophilisation gave
a mixture of 10 and tosylate anion. This mixture was dissolved in
water (0.5 ml), separated by absorption onto Dowex 50 H+-form
and elution with aqueous NH3 (1 M), and then lyophilised to
give phosphonic acid 10 as a white ammonium salt (20 mg, 58%)
[Found: M + H+ (+ESI), 429.0645; C11H18N6O6P2 requires M + H,
429.0647]; dH (400 MHz, D2O) 2.34 (3 H, s, CH3), 2.96 (2 H, t, J 6.5,
CH2CH2O), 4.14 (2 H, dt, J 7 and 6.5, CH2CH2OP), 5.40 (2 H, s,
CH2 bridge), 7.90 (1 H, s, triazole CH), 8.02 (1 H, s, pyrimidine
CH); dC (126 MHz, D2O) 22.8 (CH3), 27.5 (CH2CH2O, d, J 6.0),
49.8 (CH2-bridge), 66.1 (CH2CH2O, d, J 5.3), 110.1 (CCNH2),
128.4 (triazole CH), 141.3 (triazole C), 145.2 (pyrimidine CH),
164.7 and 165.2 (CNCNH2); dP (162 MHz, D2O) 5.0 (1 P, dt, J
51.5 and J 73.0) and 7.9 (1 P, dt, J 51.5 and J 87.0); dF (376 MHz,
D2O) −117.3 (2 F, dd, J 87.0 and 73.0).
mixture of 3-butynol (227 ll, 3.0 mmol) and pyridine (371 ll,
4.6 mmol) at 0 ◦C. The reaction mixture was allowed to warm
to room temperature, stirred for 12 h and then was evaporated
under reduced pressure. The residue was dissolved in ethyl acetate,
washed with hydrochloric acid (1 M) and then with brine, dried
(MgSO4) and concentrated under reduced pressure to give the
sulfamate 28 as an oil (300 mg, 67% over two steps) [Found: M +
Na+ (+ESI), 172.0042; C4H7NO3S requires M + Na, 172.0039];
mmax/cm−1 3287 (H–C C), 1357 and 1173 (SO2O); dH (400 MHz,
≡
≡
CDCl3) 2.06 (1 H, t, J 2.7, C CH), 2.66 (2 H, dt, J 2.7 and 6.8,
CH2CH2O), 4.29 (2 H, t, J 6.8, CH2CH2O), 4.80 (2 H, s, NH2);
dC (100 MHz, CDCl3) 19.5 (CH2CH2O), 68.5 (CH2CH2O), 71.1
≡
≡
(C CH), 79.0 (C CH); all analytical data are consistent with
those previously reported.25
N-(But-3-ynyloxysulfonyl)trimethoxyphosphazene 29
To a stirred solution of sulfamate 28 (300 mg, 2.01 mmol) and
trimethyl phosphite (475 ll, 4.03 mmol) in anhydrous THF (2 ml)
was added in portions di-tert-butyl azodicarboxylate (928 mg,
4.03 mmol). The reaction mixture was stirred at room temperature
for 2 h and then concentrated under reduced pressure. The
residue was purified by silica gel chromatography eluting with
dichloromethane–methanol (98 : 2) to give the phosphazene 29
as an oil (436 mg, 80%). [Found: M + H+ (+ESI), 272.0362;
C7H14NO6PS requires M + H, 272.0358]; mmax/cm−1 3287 (H–
({2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-[1,2,3]triazol-
4-yl]ethoxy}hydroxyphosphorylmethyl) phosphonic acid 11
≡
C C), 1379 and 1183 (SO2O), 1033 (P–O–C); dH (400 MHz,
A solution of tetrabutylammonium hydroxide·30H2O (370 mg,
0.463 mmol) and methylenediphosphonic acid 26 (27 mg,
0.154 mmol) in anhydrous acetonitrile (5 ml) was stirred at room
temperature for 1 h. The mixture was concentrated under high
vacuum to give the tris(tetrabutylammonium) salt. The salt was
dissolved in anhydrous acetonitrile (300 ll) and tosylate 19 (30 mg,
≡
CDCl3) 1.99 (1 H, t, J 2.7, C CH), 2.64 (2 H, dt, J 2.7 and
7.1, CH2CH2O), 3.93 (9 H, d, J 11.9, 3 × POCH3), 4.24 (2 H, t,
J 7.1, CH2CH2O); dC (100 MHz, CDCl3) 18.0 (CH2CH2O), 55.0
≡
≡
(POCH3, d, J 6.0), 69.7 (CH2CH2O), 71.3 (C CH), 77.2 (C CH);
dP (162 MHz, CDCl3) 3.7 (s).
◦
0.077 mmol) was added at 0 C under an atmosphere of argon.
O,O-Bis(trimethylsilyl) N-(but-3-
ynyloxysulfonyl)phosphoramidate 30
The reaction mixture was allowed to warm to room temperature
and stirred for 12 h. The mixture was evaporated under reduced
pressure, dissolved in water (1 ml) and purified by anion-exchange
chromatography and then by absorption onto Dowex 50 H+-form
and elution with aqueous NH3 (1 M) to give phosphonic acid
11 as a white ammonium salt (20 mg, 66%), m.p. 160–162 ◦C;
[Found: M − H+ (−ESI), 391.0685; C11H18N6O6P2 requires M −
H, 391.0685]; mmax/cm−1 3044 (broad NH2), 1652, 1601 and 1563
Bromotrimethylsilane (950 ll, 7.2 mmol) was added to 29 (314 mg,
1.16 mmol) at room temperature with stirring under an argon
atmosphere. The reaction mixture was stirred at room temperature
for 3 h and then concentrated under reduced pressure to give the
phosphoramidate ester 30 as a thick oil (432 mg, 100%). mmax/cm−1
≡
3287 (H–C C), 1358 and 1173 (SO2O); dH (400 MHz, CDCl3)
=
(pyrimidine ring), 1167 (P O), 1020 (P–OR); dH (400 MHz, D2O)
≡
0.35 (18 H, s, 2 × OSiMe3), 2.03 (1 H, t, J 2.7, C CH), 2.65
2.00 (2 H, t, J 19.8, PCH2P), 2.36 (3 H, s, CH3), 2.95 (2 H, t,
J 6.4, CH2CH2O), 4.00 (2 H, dt, J 6.5 and J 6.7, CH2CH2OP),
5.41 (2 H, s, CH2 bridge), 7.88 (1 H, s, triazole CH), 8.01 (1 H, s,
pyrimidine CH); dC (126 MHz, D2O) 23.4 (CH3), 26.6 (CH2CH2O,
d, J 6.8), 27.3 (PCH2P, dd, J 123 and 125), 47.4 (CH2 bridge), 63.1
(CH2CH2O, d, J 5.5), 108.5 (CCNH2), 124.1 (triazole CH), 145.3
(triazole C), 153.9 (pyrimidine CH), 161.8 and 167.0 (CNCNH2);
dP (162 MHz, D2O) 14.8 (1 P, d, J 9.3) and 18.4 (1 P, d, J 9.3).
(2 H, dt, J 2.7 and J 7.1, CH2CH2O), 4.30 (2 H, t, J 7.1,
CH2CH2O); dC (100 MHz, CDCl3) 1.4 (SiMe3), 18.9 (CH2CH2O),
≡
≡
67.9 (CH2CH2O), 70.4 (C CH), 78.4 (C CH); dP (162 MHz,
CDCl3) −23.5 (s).
N-(But-3-ynyloxysulfonyl)phosphoramidic acid 31
A mixture of anhydrous methanol (1 ml) and phosphoramidate
ester 30 (163 mg, 0.44 mmol) was stirred at room temperature
for 5 min and then evaporated under reduced pressure to give
the acid 31 as an oil (101 mg, 100%) [Found: M − H+ (−ESI),
227.9737 C6H8NO6PS requires M − H, 227.9732]; mmax/cm−1 3290
O-But-3-ynyl sulfamate 28
Formic acid (180 ll, 4.6 mmol) was added dropwise to neat
chlorosulfonyl isocyanate 27 (400 ll, 4.6 mmol) at 0 ◦C, whereupon
vigorous gas evolution was observed. The resulting viscous
suspension was stirred at 0 ◦C for 5 min. Dichloromethane (5 ml)
≡
(H–C C), 2587 (PO–H), 1358 and 1173 (SO2O); dH (400 MHz,
≡
MeOD) 2.35 (1 H, t, J 2.7, C CH), 2.64 (2 H, dt, J 2.7 and J
6.9, CH2CH2O), 4.28 (2H, t, J 6.9, CH2CH2O); dC (100 MHz,
◦
≡
was added and the solution was stirred at 0 C for 1 h and then
CD3OD) 20.2 (CH2CH2O), 68.7 (CH2CH2O), 71.8 (C CH), 80.5
at 25 ◦C for 4 h. To the stirred solution was added slowly a
(C CH); dP (162 MHz, CDCl3) −5.4 (s).
≡
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=
(2 × C O); all analytical data are identical to those previously
N-({2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-
[1,2,3]triazol-4-yl]ethoxy}sulfonyl)phosphoramidic acid 12
reported.13
To a stirred solution of phoshoramidic acid 31 (46 mg, 0.20 mmol)
and azide 16 (33 mg, 0.20 mmol) in anhydrous DMF (0.5 ml) was
added CuI (6 mg, 0.12 mmol). The reaction mixture was stirred
at room temperature for 24 h and then evaporated under reduced
pressure, dissolved in water (1 ml), filtered and purified by anion-
exchange chromatography. Lyophilisation gave the triazole 12 as a
white ammonium salt (45 mg, 57%) m.p. 138–140 ◦C [Found: M +
H+ (+ESI), 394.0702 C10H16N7O6PS requires M + H, 394.0699];
mmax/cm−1 3055 (NH2), 1651 and 1566 (pyrimidine ring), 1347 and
1170 (SO2O); dH (400 MHz, D2O) 2.32 (3 H, s, CH3), 3.04 (2 H,
t, J 6.2, CH2CH2O), 4.28 (2 H, t, J 6.2, CH2CH2O), 5.38 (2 H, s,
CH2 bridge), 7.85 (1 H, s, triazole CH), 7.98 (1 H, s, pyrimidine
CH); dC (126 MHz, D2O) 23.3 (CH3), 24.4 (CH2CH2O), 47.1
(CH2 bridge), 67.7 (CH2CH2O), 107.9 (CCNH2), 123.7 (triazole
CH), 144.1 (triazole C), 155.0 (pyrimidine CH), 161.2 and 167.3
(CNCNH2); dP (162 MHz, D2O) −3.5 (s).
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-[1,2,3]triazol-4-
yl]ethyl tert-butyl malonate 36
To a stirred solution of tert-butyl malonate 35 (150 mg, 0.94 mmol)
andDCC (194mg, 0.94mmol) inanhydrous THF (6 ml) was added
a solution of alcohol 18 (136 mg, 0.94 mmol) in anhydrous pyridine
(2 ml). The reaction mixture was stirred at room temperature
for 18 h, then filtered and evaporated under reduced pressure.
The residue was dissolved in ethyl acetate, washed with saturated
aqueous sodium bicarbonate, dried (MgSO4) and evaporated
under reduced pressure to give the diester 36 as an oil (180 mg,
51%) [Found: M + H+ (+ESI), 377.1936 C17H24N6O4 requires M +
H, 377.1937]; mmax/cm−1 3329 and 3120 (NH2), 1723 (C O), 1661,
=
1596 and 1568 (pyrimidine ring), 1141 (C–O); dH (400 MHz,
DMSO-d6) 1.34 (9 H, s, tBu), 2.27 (3 H, s, CH3), 2.92 (2 H, t, J 6.8
CH2CH2O), 3.33 (2 H, s, CH2), 4.26 (2 H, t, J 6.8, CH2CH2O),
5.36 (2 H, s, CH2 bridge), 6.84 (2 H, s, NH2), 7.90 (1 H, s, triazole
CH), 7.92 (1 H, s, pyrimidine CH); dC (100 MHz, CD3OD) 24.5
(CH3), 25.5 (CH2CH2O), 27.7 (CMe3), 34.2 (CH2), 49.0 (CH2
bridge), 64.4 (CH2CH2O), 82.5 (CMe3), 109.4 (CCNH2), 123.6
(triazole CH), 145.0 (triazole C), 155.9 (pyrimidine CH), 163.0,
O-2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-
[1,2,3]triazol-4-yl]ethyl sulfamate 13
To a solution of chlorosulfonyl isocyanate 27 (32.7 ll, 0.38 mmol)
in anhydrous dioxane (4 ml) at 0 ◦C was added dropwise a solution
of alcohol 18 (58 mg, 0.25 mmol) in anhydrous pyridine (1 ml).
The reaction mixture was stirred at 0 ◦C for 30 min. Water (1 ml)
was added slowly at 0 ◦C and the mixture was stirred at room
temperature for 2 h and then evaporated under reduced pressure.
The residue was diluted with water (to 1 ml) and purified by anion-
exchange chromatography. Lyophilisation gave a mixture of the
carbamate 13 and the dimer 33 (9 : 1 molecular ratio) as their white
ammonium salts (50 mg, 55%), m.p. 178–180 ◦C [Found: M +
H+ (+ESI), 358.0920 C11H15N7O5S requires M + H, 358.0934];
=
167.1, 167.9 and 168.4 (2 × C O and CNCNH2).
Mono{2-[1-(4-amino-2-methylpyrimidin-5-ylmethyl)-1H-
[1,2,3]triazol-4-yl]ethyl} malonate 14
A mixture of trifluoroacetic acid (4 ml) and diester 36 (173 mg,
0.46 mmol) was stirred at room temperature for 3 h, then evapo-
rated under high vacuum, dissolved in water (5 ml) and washed
with dichloromethane. One equivalent of sodium bicarbonate
(39 mg, 0.46 mmol) was added and the aqueous solution was
lyophilised to give the malonate 14 as its sodium salt as an oil
(126 mg, 80%) [Found: M + H+ (+ESI), 321.1297 C13H16N6O4
requires M + H, 321.1311]; mmax/cm−1 3342 and 3143 (NH2), 1723
mmax/cm−1 3137 (NH2), 1714 (C O), 1651 and 1601 (pyrimidine
=
ring); dH for 13 (400 MHz, D2O) 2.34 (3 H, s, CH3), 2.98 (2 H, t,
J 6.1, CH2CH2O), 4.27 (2 H, t, J 6.1, CH2CH2O), 5.39 (2 H, s,
CH2 bridge), 7.79 (1 H, s, triazole CH), 8.00 (1 H, s, pyrimidine
CH); dC (100 MHz, D2O) 22.3 (CH3), 23.8 (CH2CH2O), 46.4
(CH2 bridge), 63.5 (CH2CH2O), 107.6 (CCNH2), 123.1 (triazole
CH), 143.8 (triazole C), 152.2 (pyrimidine CH), 160.9 and 165.8
(CNCNH2).
=
and 1651 (C O), 1543 (pyrimidine ring), 1183 and 1132 (C–O);
dH (400 MHz, CD3OD) 2.54 (3 H, s, CH3), 3.07 (2 H, t, J 6.4,
CH2CH2O), 3.35 (2 H, s, CH2), 4.38 (2 H, t, J 6.4, CH2CH2O),
5.53 (2 H, s, CH2 bridge), 7.92 (1 H, s, triazole CH), 8.01 (1
H, s, pyrimidine CH); dC (100 MHz, CD3OD) 24.4 (CH3), 28.8
(CH2CH2O), 32.5 (CH2), 50.2 (CH2 bridge), 67.6 (CH2CH2O),
113.8 (CCNH2), 127.6 (triazole CH), 147.4 (pyrimidine CH),
Mono-tert-butyl malonate 3513
=
148.6 (triazole C), 166.1, 167.8, 171.2 and 172.9 (2 × C O and
To a stirred solution of malonic acid 34 (208 mg, 2.0 mmol)
and tert-butanol (296 mg, 4.0 mmol) in anhydrous acetonitrile
(10 ml) was added a solution of DCC (454 mg, 2.2 mmol) in
anhydrous acetonitrile (2 ml). The reaction mixture was stirred
at room temperature for 30 min, then filtered and evaporated
under reduced pressure. The residue was dissolved in diethyl
ether and extracted with saturated aqueous sodium bicarbonate.
The aqueous phase was acidified to pH ≈ 1 by addition of
hydrochloric acid (1 M) and extracted with ethyl acetate. The
combined organic layers were dried (MgSO4) and concentrated
under reduced pressure to give the malonate 35 as an oil (219 mg,
66%). [Found: M − H+ (−ESI), 159.0651 C7H12O4 requires M–
CNCNH2).
N-Benzyloxycarbonyliminodiacetic acid 3915
To a stirred solution of iminodiacetic acid 37 (878 mg, 6.6 mmol)
in aqueous sodium hydroxide (2 M; 15 ml) was added dropwise at
5 ◦C benzyl chloroformate (2.25 g, 13.2 mmol). A further volume
of aqueous sodium hydroxide (2 M; 8 ml) was added and the
mixture was stirred at room temperature for 2 h. The reaction
mixture was washed with diethyl ether and acidified to pH ≈ 2
with hydrochloric acid (1 M). The aqueous layer was extracted
with diethyl ether and the combined organic layers were dried
(MgSO4) and concentrated under reduced pressure to give the Cbz-
protected amine 39 as an oil (1.25 g, 71%) [Found: M − H+ (−ESI),
266.0675 C12H13NO6 requires M − H, 266.0665]; mmax/cm−1 3035
H, 159.0657]; mmax/cm−1 1713 (C O), 1142 (C–O); dH (400 MHz,
=
CDCl3) 1.50 (9 H, s, 3 × CH3), 3.34 (2 H, s, CH2); dC (100 MHz,
CDCl3) 28.3 (3 × CH3), 42.1 (CH2) 83.4 (C), 167.0 and 171.4
3570 | Org. Biomol. Chem., 2008, 6, 3561–3572
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=
(C–H), 1689 (C O); dH (400 MHz, CDCl3) 4.12 (2 H, s, CH2), 4.19
64.90 (CH2CH2O), 68.29, 68.35, 69.17 and 69.23 (2 × OCH2),
109.19 and 109.28 (CCNH2), 123.05 and 123.33 (triazole CH),
128.85, 129.02, 129.38, 129.45, 129.50, 129.54, 129.73 and 129.85
(ArCH), 136.35, 136.37, 137.10 and 137.13 (ArC), 145.90 and
145.94 (triazole C), 157.19 and 157.23 (pyrimidine CH), 163.19,
169.91 and 170.00 (CNCNH2), 170.21, 170.37 and 170.46 (2 ×
=
(2 H, s, CH2), 5.17 (2 H, s, OCH2), 7.30–7.37 (5 H, m, ArH); dC
(100 MHz, CDCl3) 48.4 and 48.5 (2 × CH2), 66.8 (OCH2), 126.0
(ArCH), 126.2 (2 × ArCH), 126.9 (2 × ArCH), 135.4 (ArC), 154.4,
=
171.4 and 172.9 (3 × C O); all analytical data are consistent with
those previously reported.15
C
O).
Monobenzyl N-benzyloxycarbonyliminodiacetate 42
Mono({2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-
A solution of 39 (1.25 g, 4.68 mmol) and DCC (964 mg,
4.68 mmol) in anhydrous THF (10 ml) was stirred at room
temperature for 12 h. The mixture was filtered and evaporated
under reduced pressure to give the solid anhydride 41, which
was used in the next step without further purification. To a
stirred suspension of 41 (944 mg, 3.79 mmol) and benzyl alcohol
(1.2 ml, 11.6 mmol) in anhydrous diethyl ether (8 ml) was added
[1,2,3]triazol-4-yl]ethyl) iminodiacetate 15
A mixture of 43 (150 mg, 0.26 mmol) and palladium-on-carbon
(10%; 40 mg) in methanol (10 ml) was stirred under hydrogen
(1 atm) at room temperature for 20 min. The suspension was
filtered through Celite and the residue was washed several times
with methanol. The filtrate was evaporated under reduced pressure
to give the acid 15 as a white solid (69 mg, 76%), m.p. 195–200 ◦C;
[Found: M + H+ (+ESI), 350.1582; C14H19N7O4 requires M + H,
◦
at 0 C dicyclohexylamine (829 ll, 4.17 mmol) and the reaction
mixture was allowed to warm to room temperature and stirred
for 12 h. The solid was collected, washed several times with
diethyl ether and recrystallised from ethyl acetate–hexane to yield
the dicyclohexylammonium salt. The solid was dissolved in ethyl
acetate, washed with hydrochloric acid (1 M) and concentrated
under reduced pressure to give the ester 4216 as an oil (1.24 g,
74%). [Found: M − H+ (−ESI), 356.1146; C19H19NO6 requires M −
350.1571]; mmax/cm−1 3134 (NH2), 1749 (C O), 1681 and 1577
=
(pyrimidine ring); dH (400 MHz, D2O) 2.33 (3 H, s, CH3), 3.00 (2
H, t, J 6.2, CH2CH2O), 3.49 (2 H, s, CH2), 3.83 (2 H, s, CH2),
4.40 (2 H, t, J 6.2, CH2CH2O), 5.39 (2 H, s, CH2 bridge), 7.78 (1
H, s, triazole CH), 7.95 (1 H, s, pyrimidine CH); dC (100 MHz,
D2O) 23.9 (CH3), 24.7 (CH2CH2O), 47.6 and 47.9 (2 × CH2), 49.7
(CH2 bridge), 65.5 (CH2CH2O), 108.9 (CCNH2), 124.2 (triazole
CH), 145.0 (triazole C), 154.8 (pyrimidine CH), 162.2 and 167.8
H, 356.1134]; mmax/cm−1 3034 (C–H), 1706 (C O); dH (400 MHz,
=
CDCl3; many of the protons show two sets of signals due to slowly
=
interconverting rotamers about the N–C O bond) 4.12 (2 H, 2 × s
=
(CNCNH2), 168.2 and 172.0 (2 × C O).
due to rotamers, CH2), 4.20 (2 H, 2 × s due to rotamers, CH2), 5.12
(2H, s, OCH2), 5.19 (2 H, 2 × s due to rotamers, OCH2), 7.26–7.37
(10 H, m, ArH); dC (100 MHz, CDCl3, many carbons show two
peaks due to the rotamers) 49.9, 50.13, 50.29 and 50.32 (2 × CH2),
67.61, 67.96, 68.48 and 68.57 (2 × OCH2), 128.09, 128.47, 128.59,
128.67, 128.76, 128.83 and 128.88 (ArCH), 135.00, 135.14, 135.86
and 135.92 (2 × ArC), 155.98, 156.16, 169.93, 170.92, 172.91 and
General methods for enzymic assays
All assays were performed on a Cary 100 Bio UV-visible spec-
trophotometer, using disposable plastic cuvettes (Fisherbrandꢀ
R
semi-micro polystyrene). Buffer and assay solutions were prepared
on the day of use. Pyruvate decarboxylase from Zymomonas
mobilis was overexpressed in E. coli and purified as described
previously.26 Protein concentrations were determined by the
Bradford assay.27
=
173.48 (3 × C O).
2-[1-(4-Amino-2-methylpyrimidin-5-ylmethyl)-1H-[1,2,3]triazol-4-
yl]ethyl benzyl N-(benzyloxycarbonyl)iminodiacetate 43
Assay for PDC activity4,5
To a suspension of 42 (715 mg, 2.0 mmol), alcohol 18 (468 mg,
2.0 mmol) and DCC (453 mg, 2.2 mmol) in anhydrous THF (15 ml)
was added DMAP (269 mg, 2.2 mmol). The reaction mixture was
stirred at room temperature for 24 h, then filtered and evaporated
under reduced pressure. The residual oil was purified by silica
gel chromatography eluting with DCM–MeOH (9 : 1) to give
the diester 43 as an oil (800 mg, 70%) [Found: M + H+ (+ESI),
574.2390 C29H31N7O6 requires M + H, 574.2414]; mmax/cm−1 3341
The assay solution used in all experiments contained alcohol
dehydrogenase (ADH; 10 units per ml), NADH (0.15 mM), TPP
(0.1 mM) and MgCl2 (5 mM) in MES–KOH buffer (50 mM;
pH 6.5). Pyruvate decarboxylase (ca. 8 nM of◦active sites) was
incubated in the assay solution for 3 min at 25 C and the assay
was then started by addition of the substrate pyruvate to give
5 mM. The consumption of NADH was followed spectrophoto-
metrically at 340 nm (e340 = 5700 M−1 cm−1).
=
and 3200 (NH2), 1744 and 1703 (C O), 1631, 1594 and 1561
(pyrimidine ring); dH (400 MHz, CDCl3, many of the protons
show two sets of signals due to slowly interconverting rotamers
=
about the N–C O bond) 2.47 (3 H, s, CH3), 2.93 and 3.04 (2
Preparation of apoPDC5
H, 2 × t, J 6.3 and 6.5, CH2CH2O), 4.07, 4.11, 4.12 and 4.14
(4 H, 4 × s, 2 × CH2), 4.31 and 4.38 (2 H, t, J 6.3 and 6.5,
CH2CH2O), 5.09, 5.16, 5.23 and 5.26 (4 H, 4 × s, 2 × OCH2),
5.52 and 5.54 (2 H, 2 × s, CH2 bridge), 7.22 and 7.35 (1 H, s,
triazole CH), 7.26–7.34 (10 H, m, ArH), 8.16 and 8.18 (1 H, 2 × s,
pyrimidine CH); dC (100 MHz, CDCl3, many carbons show two
peaks due to the rotamers) 26.46 and 26.59 (CH2CH2O), 26.70
(CH3), 49.70 (CH2 bridge), 50.70, 50.81 and 50.90 (2 × CH2),
The holoPDC was dissolved in HEPES-KOH buffer (50 mM;
pH 8.2) containing dipicolinic acid (1 mM). After incubation at
5
repeated ultrafiltration. The buffer was finally replaced by MES–
KOH buffer (50 mM; pH 6.5). The preparation of the apoenzyme
was confirmed by assays of activity with and without TPP
present.
◦C for 30 min the apoPDC was separated from cofactors by
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Time-dependent inactivation and reactivation of PDC
L. Meshalkina, Y. Lindqvist and G. Schneider, J. Biol. Chem., 1997,
272, 1864.
ApoPDC (ca. 0.3 lM of active sites) was incubated at 25 ◦C with
MgCl2 (5 mM) and inhibitor (various concentrations, e.g. from
2 lM to 10 lM) in MES–KOH buffer (50 mM; pH 6.5). At timed
intervals, aliquots (20 ll) were added to the assay solution (778 ll)
and after a further incubation at 25 ◦C for 3 min a solution of
sodium pyruvate (2 M; 2 ll, to give 5 mM) in water was added and
the decrease of A340 with time was followed.
In order to ensure the stability of the enzyme, control experi-
ments were carried out without inhibitor present. In every case
the percentage activity is expressed as the ratio of the activity with
inhibitor to the corresponding activity obtained without inhibitor.
In order to study the rate of unbinding, apoenzyme was first
incubated with inhibitor (10–20 lM) until low (or no) activity
was observed. An excess of TPP (to give 100 lM to 10 mM) was
then added and the recovery of activity was followed for several
(7–8) days.
4 R. J. Diefenbach and R. G. Duggleby, Biochem. J., 1991, 276, 439.
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837.
Time-dependent reactivation/inactivation of PDC
ApoPDC (ca. 0.4 lM of active sites) was incubated at 25 ◦C
with TPP (4 lM), MgCl2 (10 mM) and analogue (concentrations
ranging from 20 lM to 6 mM) in MES–KOH buffer (50 mM;
pH 6.5). At timed intervals, aliquots (20 ll) were added to the assay
solution containing NADH (0.15 mM), ADH (10 units ml−1) and
pyruvate (5 mM) in MES–KOH buffer (50 mM; pH 6.5) and the
decrease of A340 with time was followed.
15 A. C. Benniston, P. Gunnung and R. D. Peacock, J. Org. Chem., 2005,
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Acknowledgements
We thank the European Commission under the Marie Curie Early
Stage Research Training Programme for funding (KME).
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