Reactivity and selectivity in the inhibition of elastase by 3-oxo-β-sultams and in their hydrolysis

Article abstract of DOI:10.1039/b713899g

3-Oxo-β-sultams are both β-sultams and β-lactams and are a novel class of time-dependent inhibitors of elastase. The inhibition involves formation of a covalent enzyme-inhibitor adduct with transient stability by acylation of the active-site serine resulting from substitution at the carbonyl centre of the 3-oxo-β-sultam, C-N fission, and expulsion of the sulfonamide. The lead compound, N-benzyl-4,4-dimethyl-3-oxo-β-sultam 1 is a reasonably potent inhibitor against porcine pancreatic elastase with a second-order rate constant of 768 M^-1 s^-1 at pH 6, but also possesses high chemical reactivity with a half-life for hydrolysis of only 6 mins at the same pH in water. Interestingly, the hydrolysis of 3-oxo-β-sultams occurs at the sulfonyl centre with S-N fission and expulsion of the amide leaving group, whereas the enzyme reaction occurs at the acyl centre. Increasing selectivity between these two reactive centres was explored by examining the effect of substituents on the reactivity of 3-oxo-β-sultam towards hydrolysis and enzyme inhibition. The inhibition activity against porcine pancreatic elastase has a much higher sensitivity to substituent variation than does the rate of alkaline hydrolysis. A difference of 2000-fold is observed in the second-order rate constants, k_i, for inhibition whereas there is only a 100-fold difference in the second-order rate constants, k_OH, for alkaline hydrolysis within the series. The higher sensitivity of enzyme inhibition to substituents than that of simple chemical reactivity indicates a significant degree of molecular recognition of the 3-oxo-β-sultams by the enzyme. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b713899g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Reactivity and selectivity in the inhibition of elastase by 3-oxo-b-sultams and
in their hydrolysis†
Wing-Yin Tsang, Naveed Ahmed, Karl Hemming and Michael I. Page*
Received 10th September 2007, Accepted 11th October 2007
First published as an Advance Article on the web 30th October 2007
DOI: 10.1039/b713899g
3-Oxo-b-sultams are both b-sultams and b-lactams and are a novel class of time-dependent inhibitors
of elastase. The inhibition involves formation of a covalent enzyme–inhibitor adduct with transient
stability by acylation of the active-site serine resulting from substitution at the carbonyl centre of the
3-oxo-b-sultam, C–N fission, and expulsion of the sulfonamide. The lead compound,
N-benzyl-4,4-dimethyl-3-oxo-b-sultam 1 is a reasonably potent inhibitor against porcine pancreatic
elastase with a second-order rate constant of 768 M−1 s−1 at pH 6, but also possesses high chemical
reactivity with a half-life for hydrolysis of only 6 mins at the same pH in water. Interestingly, the
hydrolysis of 3-oxo-b-sultams occurs at the sulfonyl centre with S–N fission and expulsion of the amide
leaving group, whereas the enzyme reaction occurs at the acyl centre. Increasing selectivity between
these two reactive centres was explored by examining the effect of substituents on the reactivity of
3-oxo-b-sultam towards hydrolysis and enzyme inhibition. The inhibition activity against porcine
pancreatic elastase has a much higher sensitivity to substituent variation than does the rate of alkaline
hydrolysis. A difference of 2000-fold is observed in the second-order rate constants, ki, for inhibition
whereas there is only a 100-fold difference in the second-order rate constants, kOH, for alkaline
hydrolysis within the series. The higher sensitivity of enzyme inhibition to substituents than that of
simple chemical reactivity indicates a significant degree of molecular recognition of the 3-oxo-b-sultams
by the enzyme.
HNE.4,6,7 We have been interested in developing mechanism-based
inhibitors of HNE and the related, but more readily available,
porcine pancreatic elastase (PPE). PPE is a model enzyme that
shares ca. 40% homology and the catalytic triad consisting of Ser-
Introduction
Elastase is a proteolytic enzyme that catalyzes the hydrolysis of the
fibrous protein elastin, which comprises an appreciable percentage
195, His-57 and Asp-102 with HNE.8 Among different classes of
inhibitor of elastase, acylating agents such as b-lactams and trans-
fused c-lactones9 that inactivate elastase by formation of an acyl–
enzyme adduct appear to be one of the most promising areas for
the development of successful therapeutic agents.10 We have shown
that b-sultams, the sulfonyl analogues of b-lactams, are a novel
class of effective inhibitors of serine enzymes by sulfonylation
of the active-site serine residue.11,12 We have also shown that 3-
oxo-b-sultams, which are both b-sultams and b-lactams, are a
new class of acylating agents that inactivate the elastase from
porcine pancreas.13 The inhibition involves the formation of an
enzyme–inhibitor adduct with transient stability by acylation of
the active-site serine resulting from substitution at the carbonyl
center of the 3-oxo-b-sultam. The lead compound, N-benzyl-4,4-
dimethyl-3-oxo-b-sultam 1, is a reasonably potent inhibitor against
porcine pancreatic elastase with a second-order rate constant of
768 M−1 s−1 at pH 6. However, the 3-oxo-b-sultam is chemically
too reactive for studies towards therapeutic application – the half-
life for the degradation of the 3-oxo-b-sultam by hydrolysis is
only 6 mins at pH 6 in water. By contrast, increasing the rate of
serine acylation by enhancing the intrinsic chemical reactivity of
the b-lactams has been used as a strategy to design more potent
inhibitors.14
of all protein content in tissues such as arteries, ligaments and the
lung. Three distinct types of human elastase have been identified –
human neutrophil elastase (HNE), pancreatic elastase II (PE-
II) and macrophage metalloelastase (MME).1 The imbalance
between HNE and its endogenous inhibitors leads to excessive
elastin proteolysis and destruction of connective tissues in a
number of inflammatory diseases such as pulmonary emphysema,
adult respiratory distress syndrome, chronic bronchitis, chronic
obstructive pulmonary disease and rheumatoid arthritis.1,2 Inhi-
bition of human neutrophil elastase is therefore an important
approach to the treatment of the disease, and the search for
suitable inhibitors has been intense.3 Human neutrophil elastase is
a member of the chymotrypsin family of serine proteases and, as
such, has a rich family of classical inhibitors.4 However, there is a
need for the design of new inhibitors as currently available drugs
have shown only limited effect on the diseases.3 b-Lactams are well-
known as potent inhibitors of some serine enzymes, including the
bacterial penicillin binding proteins (PBPs) and Class A and Class
C b-lactamases.5 b-Lactams have also been structurally modified
to develop active site-directed and mechanism-based inhibitors of
Department of Chemical and Biological Sciences, The University of Hud-
dersfield, Queensgate, Huddersfield, UK HD1 3DH. E-mail: m.i.page@
hud.ac.uk; Fax: +44 (0)1484 473075; Tel: +44 (0)1484 472531
† Electronic supplementary information (ESI) available: Further experi-
mental details. See DOI: 10.1039/b713899g
Nucleophilic attack on N-benzyl-4,4-dimethyl-3-oxo-b-sultam
1 could involve either acylation or sulfonylation resulting from
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=
substitution at the carbonyl centre and expulsion of the sulfon-
amide or from substitution at the sulfonyl centre and expulsion
of the amide, respectively. Interestingly, the hydrolysis of 1 and
its enzyme reaction with elastase occur at different reaction
centres; the enzyme reaction occurs at the acyl centre whereas the
hydrolysis of 3-oxo-b-sultams occurs at the sulfonyl centre 1.13,15
Inhibition of elastase by 1 occurs by acylation of the active-site
serine and so involves C–N fission and expulsion of sulfonamide.13
By contrast, the alkaline hydrolysis of 1 occurs by hydroxide-ion
attack on the sulfonyl centre with S–N fission.15 This is a very
C O double bond of 3-oxo-b-sultam is much higher than that of
the acyclic amide and higher than the 1730–1760 cm−1 seen with
=
monocyclic b-lactams indicates more C O double bond character
and possibly weaker amide resonance due to the competing
electron withdrawal by the adjacent sulfonyl group.20 Although
reduced resonance in b-lactam amides in b-lactam antibiotics such
as penicillins, has often been stated as the reason for their chemical
reactivity and biological susceptibility, it is a fact that, contrary
to expectation, the rate of alkaline hydrolysis of b-lactams is less
than a hundred-fold greater than that of an analogous acyclic
amide.16,21 The second-order rate constant for the hydroxide-ion
catalyzed hydrolysis of N-methyl b-lactam is only 3-fold greater
than that for N,N-dimethyl acetamide.21
rapid process and occurs with a second-order rate constant, kOH
,
value of. ca. 2 × 105 M−1 s−1,15 which is about 106 times greater
than those of most clinically useful b-lactams.16 Highly reactive
inhibitors may decrease the selectivity towards the target enzyme
and reduce bioavailability.
Nucleophilic substitution at acyl centres generally proceeds
through the formation of an unstable tetrahedral intermediate 2
and it is usually assumed that the incoming nucleophile approaches
at approximately the tetrahedral angle to the carbonyl group.17 By
contrast, sulfonyl-transfer reaction involves a transition state or
intermediate with pentacoordinate trigonal bipyramidal geometry
3 and the incoming nucleophile is assumed to take the apical
position.18,19 The two processes, acyl and sulfonyl transfer, are
expected to show different sensitivity to stereochemical and
geometrical properties of the a-substituents and it is of interest to
investigate the balance between the chemical reactivity and enzy-
matic activity of 3-oxo-b-sultams by changing the a-substituents.
Scheme 1
The 3-oxo-b-sultam 1 is both a b-sultam and a b-lactam
with a second-order rate constant for alkaline hydrolysis kOH of
1.83 × 105 dm3 mol−1 s−1 (Table 1) and hydroxide-ion attack
occurs preferentially at the sulfonyl centre with expulsion of a
carboxamide leaving group, as shown by product analysis.15 That
hydroxide-ion attacks the sulfonyl centre in 1 rather than the b-
lactam carbonyl is consistent with the observation that b-sultams
are 102 to 103-fold more reactive than b-lactams towards alkaline
hydrolysis.18 Although attack at the acyl centre may be expected to
expel a better leaving group – the sulfonamide anion – the nature
of the leaving group does not have a large effect on the relative
rates of hydrolysis of imides and similar N-acyl sulfonamides.15
Increasing the steric bulk of the 4-substituent, a to the elec-
trophilic sulfonyl centre, decreases the rate of alkaline hydrolysis
(Table 1). For example, replacing the dimethyl groups by diethyl
decreases the rate 10-fold and the spiro cyclohexyl group is 30-
fold less reactive. It is known that the a-chymotrypsin family of
enzymes has a well-established binding pocket adjacent to the
active serine residue, in the S1 position.22 In the elastase enzymes
this binding pocket is relatively small and has a preference for
small hydrophobic substituents.22,23 The inhibition of elastase by
monocyclic b-lactams has also been improved by the introduction
of alkyl substituents at the 3-position, which are thought to bind
strongly in the S1 subsite of elastase.24,25 We therefore wished to
investigate any differential effects of these substituents on chemical
reactivity and enzyme binding.
In addition to varying the a-substituents, 3-oxo-b-sultams with
different nitrogen leaving groups have been synthesized in order
to study the effect of the leaving group on the reactivity of
these compounds as inhibitors of elastase. We now report that
such modifications can retain inhibitory activity while decreasing
dramatically the reactivity towards hydrolysis by hydroxide-ion.
Results and discussion
Chemistry
3-Oxo-b-sultams were prepared by a common strategy involving
ring closure of the corresponding (chlorosulfonyl)acetyl chloride
with a substituted amine (Scheme 1). The ring-closure step
was monitored conveniently by using IR spectroscopy as the
A series of 3-oxo-b-sultams with N-a-carboxylate substituents
were prepared and their rates of hydrolysis measured (Table 1).
All of these compounds undergo hydrolysis by hydroxide-ion
attack at the sulfonyl centre to give ring-opened products by
S–N fission reaction. There is no evidence of intramolecular
attack by the carboxylate oxyanion as the rate of the reaction
=
characteristic C O IR stretching frequencies of the acyclic
amide and 3-oxo-b-sultam were well-resolved and are 1690 and
1790 cm−1, respectively. That the IR stretching frequency of the
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Table 1 The second-order rate constants for alkaline hydrolysis (kOH) and
the enzyme inhibition (ki) of porcine pancreatic elastase (PPE) at pH 6.0
(0.1 M MES buffer) by 3-oxo-b-sultams in 1% acetonitrile, 1.0 M ionic
was monitored by measuring the rate of the hydrolysis of N-
succinyl-(L-ala)3-p-nitroanilide as substrate at 390 nm using a
molar extinction coefficient change, De390, of 1.24 × 104 M−1 cm−1.
The kinetic analysis was undertaken with the anilide substrate
(0.08 mM) in 0.1 M pH 8.5 TAPS buffer at 30 ◦C in 1.5% methanol
v/v and I = 1.0 M (KCl) in the assay cells. Inhibition was studied
by incubating the N-benzyl-4,4-dimethyl-3-oxo-b-sultam 1 with
PPE together in a buffered solution. Aliquots of the incubation
solution containing enzyme and potential inhibitor were then
removed after an incubation time and assayed for PPE activity
against the anilide substrate in separate assay cells. These assays
showed a time-dependent decrease in enzyme activity, given by the
slope of absorbance at 390 nm (A390) against time plots, and tended
towards zero with increasing time. Incubation of the enzyme
alone under exactly the same conditions but in the absence of
b-sultam gave constant assay slopes throughout the experimental
time frame. The enzyme activity at a given time was measured
by the initial rate of hydrolysis of the substrate and expressed as
a percentage of the initial slope relative to that for full enzyme
activity measured in a control experiment in which there is no
3-oxo-b-sultam present. The decrease of enzyme activity against
strength at 30 ◦
C
k
OH/M−1 s−1
ki/M−1 s−1 ki : kOH ratio
1
1.83 × 105
1.86 × 104
5.99 × 103
768
275
5.85
64
227
15
5
6
7
3.89 × 104
2.54
1
2
time in the inhibition of PPE with 2 × 10−5 and 4 × 10−5
M
of N-benzyl-4,4-dimethyl-3-oxo-b-sultam 1 at pH 6.0 follows an
exponential decay (Fig. 1). The concentration of the enzyme is
much lower than that of the inhibitor at time zero and therefore the
observed rate of inactivation is effectively pseudo-first order and
gives the apparent first-order rate constant, kobs. The values of kobs
show a first order dependence on the concentration of the 3-oxo-b-
sultam indicative of the simple rate laws (eqn (1) and (2)) in which
kobs is the observed pseudo-first-order rate constant for inhibition
in the presence of excess b-sultam, and ki is the corresponding
second-order rate constant for the inhibition. The second-order
rate constant, ki, was given by the slope of the linear plot of kobs
against the concentration of the 3-oxo-b-sultam (Fig. 2).
8
Not determined 5.00
9
2.42 × 104
3.27 × 104
1.74 × 103
30.1
8.30
0.37
19
4
10
11
(1)
(2)
3
occurs at similar rates to those for 3-oxo-b-sultams lacking the a-
carboxylate (Table 1). Introducing a carboxylate on the exocyclic
carbon a to the nitrogen of the amine leaving group of 9 or
11 decreases the chemical reactivity of the 3-oxo-b-sultam by
about 10-fold compared with 1 or 5, respectively (Table 1). This
decrease in chemical reactivity is of the magnitude expected due
to the electrostatic repulsion between the incoming negatively
charged nucleophile and the negative charge on the oxygens of
the dissociated carboxylate.
Fig. 1 The decrease of enzyme activity against time in the inhibition of
PPE with 2 × 10−5 (ꢀ) and 4 × 10−5 (᭹) M of N-benzyl-4,4-dimethyl-
3-oxo-b-sultam 1 at pH 6.0, I = 1 M (KCl), 25 ◦C. The solid curves were
the theoretical curve generated by fitting the data to a first-order decay
equation.
Inhibition of elastase by 3-oxo-b-sultams
N-Benzyl-4,4-dimethyl-3-oxo-b-sultam 1 is a time-dependent in-
hibitor of porcine pancreatic elastase (PPE). The activity of PPE
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4,4-diethyl-3-oxo-b-sultam 5 over a period of days, with a rate
constant of 1.39 × 10−4 s−1 at pH 8.5, whereas the enzyme remains
inactive for at least 4 weeks when inactivated by 4. Interestingly,
the rate of recovery of enzyme activity shows the same sigmoidal
dependence on pH as that for the rate of hydrolysis (kcat/Km)
of the anilide substrate (Fig. 3). Both reactions are critically
dependent on the ionization of a group with a pKa of 6.9. This
observation suggests a common residue for the two processes of
acylation and deacylation because the dependence of kcat/Km on
pH reflects the acylation process, whereas that for recovery of
activity is deacylation (Scheme 2). The much faster rate of enzyme
inactivation by the 3-oxo-b-sultam 1 compared with N-benzoyl
b-sultam 4 together with the recovery of enzyme activity suggests
different mechanisms for the two processes. It thus appears that
inactivation of elastase by the 3-oxo-b-sultam 1 is occurring by
serine attack on the b-lactam centre to form an acyl enzyme
of intermediate stability which is then slowly hydrolyzed in the
conventional manner (Scheme 2).
Fig. 2 Plot of observed first-order rate constant, kobs, for the inhibition
of PPE by N-benzyl-4,4-dimethyl-3-oxo-b-sultam 1 against the concentra-
tions of the 3-oxo-b-sultam at pH 6.0, I = 1 M (KCl), 25 ◦C.
With some 3-oxo-b-sultams and at high pH there were prob-
lems with the accurate determination of rate constants for the
inactivation process because of the competing hydrolysis of these
compounds. To correct for this, the results of the inactivations were
normalized with respect to the decreasing b-sultam concentration
using eqn (3) in which [3-oxo-b-sultam]t and [3-oxo-b-sultam]o
are the concentration of the 3-oxo-b-sultam at time = t and that
applied in the reaction, respectively. The concentration of the 3-
oxo-b-sultam at time = t was determined spectrophotometrically
in a separate experiment in which the hydrolysis of the 3-oxo-b-
sultam in the same buffer as that in the inhibition experiment was
followed.
Fig. 3 The pH dependence of the rate constants for the PPE-cat-
alyzed hydrolysis of N-succinyl-(L-ala)3-p-nitroanilide (kcat/Km, open
circles) and that for the enzyme recovery after inhibited with
N-benzyl-4,4-diethyl-3-oxo-b-sultam 5 (kr, filled circles) at different pHs.
Rate constants were determined in 0.1 M aqueous buffer solution at 30 ◦C
with 1.0 M ionic strength.
(3)
(i) Acylation or sulfonylation?
Inactivation of elastase by
3-oxo-b-sultams occurs with formation of a 1 : 1 complex. Analysis
of solutions of PPE incubated with N-benzyl-4,4-dimethyl-3-
oxo-b-sultam 1 in a 1 : 1 ratio by electrospray ionization-
mass spectrometry (ESI-MS) shows the enzyme bound to one
(ii) Variation of C-4 substituents. The rate of inhibition of
PPE by N-benzyl-3-oxo-b-sultams shows a large dependence on
the size of the C-4 substituents. The gem-dimethyl group at the
C-4 position of N-benzyl-4,4-dimethyl-3-oxo-b-sultam 1 shows
the largest rate constant, ki, for inhibition and the rate constant
decreases moderately by 3-fold in changing to gem-diethyl 5 and
decreases more significantly by 100-fold on changing to a spiro-
cyclohexyl substituent 6 (Table 1).
One of the major requirements for successful inhibition of
an enzyme by covalent modification is that the rate of the
inactivation must be faster than, or at least competitive with, the
turnover of the natural substrate and the inhibitor itself should
ideally be relatively stable under in vivo conditions. The rate of
inactivation can be considered to be controlled by two important
factors: (i) the intrinsic "chemical" reactivity of the inhibitor and
(ii) the molecular recognition reflected by the binding energy
between the inhibitor and the target enzyme. The acylation process
involves displacement of a leaving group by serine attacking the
carbonyl group of the inhibitor, so changes in chemical reactivity
brought about by substituents is usually considered in terms of
the electrophilic nature of the inhibitor and the leaving group
equivalent of b-sultam (MEI 26 133
1 Da) (98%). The native
enzyme has a mass of 25 892 and therefore a mass difference
of +241 is consistent with the formation of a covalent bond
between the enzyme and b-sultam 1. If inhibition was caused
by the hydrolysis product binding to the enzyme then a mass of
26 148 would be expected. However, ESI-MS does not distinguish
between acylation and sulfonylation of the enzyme. Previously
we have shown by X-ray crystallography that N-acyl b-sultams
inhibit elastase by sulfonylation of the active-site serine residue25
whereas b-lactams inhibit the enzyme by acylation of the same
residue.26 The second-order rate constant, ki, for the inactivation
of PPE by N-benzyl-4,4-dimethyl-3-oxo-b-sultam 1 is 768 M−1 s−1
(Table 1), which is nearly 103 greater than that by N-benzoyl
b-sultam 4 which inhibits elastase by a sulfonylation process.
This large rate difference is indicative of a different mechanism
of inhibition, possibly acylation by attack of the active-site serine
on the b-lactam of 1 giving C–N fission and acylation to form
an acyl enzyme (Scheme 2). This is supported by the recovery
of full enzyme activity when elastase is inactivated by N-benzyl-
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Scheme 2
ability (nucleofugacity) of the group displaced. In addition to these
electronic effects, the intrinsic chemical reactivity of a compound
may be modified by steric and strain effects. Although increasing
the intrinsic chemical reactivity may lead to a faster rate of reaction
with the target enzyme, it may also lead to greater hydrolytic and
metabolic instability with consequent little change in selectivity.
One method of indicating this selectivity is to use an ‘enzyme
rate-enhancement factor’ (EREF)27 which is the second-order rate
constant for the inactivation of the enzyme, ki, divided by that for
reactivity of the 3-oxo-b-sultam as 7 and 10 have similar second-
order rate constants, kOH, for their alkaline hydrolysis. Similarly,
a phenyl group does not have any significant effect on chemical
reactivity as shown by the similar second-order rate constants, kOH
,
for the alkaline hydrolysis of 7 and 9. However, the second-order
rate constant for inactivation of elastase, ki, varies 12-fold in the
series; changing iso-propyl to iso-butyl increases ki by 4-fold and
changing it to phenyl gives an 12-fold increase in the second-order
rate constant, ki (Table 1). The highest EREF (ki/kOH ratio) in
this series is shown by the phenyl substituted derivative 9. Finally,
incorporating a gem-diethyl group at C-4 and a valine nitrogen
leaving group yield the most chemically stable 3-oxo-b-sultam 11
in the series with a second order rate constant of 1.74 × 103 M−1s−1
for alkaline hydrolysis, which is still highly reactive compared with
a kOH of 10−1 M−1 s−1 for acylating agents of enzymes that are
therapeutically useful.20 However, the inhibition activity of this
compound is also the lowest in the series. The second-order rate
constants, kOH and ki, for 11 are 100- and 2000-fold less reactive,
respectively, than the most reactive 3-oxo-b-sultam 1. The higher
sensitivity of inhibition activity to substituents compared with
that of chemical activity does indicate significant recognition of
the 3-oxo-b-sultams by the enzyme.
In summary, 3-oxo-b-sultams are novel acylating agents that
inactivate elastase with reasonable specificity. These compounds
are unusual in that the enzyme reacts with the acyl centre whereas
the hydrolysis reaction occurs at the sulfonyl centre despite the
fact that b-sultams are about 103-fold more reactive than b-
lactams. Variation of the structure by simple substituents leads
to differences in rates of inactivation of up to 2000-fold. There is
a large differential effect of substituents on the relative reactivities
of hydrolysis and enzyme reaction.
hydrolysis of the inhibitor catalyzed by hydroxide ion, kOH
.
Although the 4,4-dimethyl derivative 1 has the largest rate
constant, ki, for inhibition, changing to 4,4-diethyl groups only
decreases the inhibition activity against PPE by 3-fold but by 10-
fold for chemical reactivity as measured by the rate of alkaline
hydrolysis (Table 1). Consequently the 4,4-diethyl derivative has
the largest EREF ratio of the rate constant for inhibition to that
for alkaline hydrolysis. This suggests that the 4,4-diethyl derivative
has the most favourable binding in the series. The comparison is
complicated by the fact that the enzyme reaction and the chemical
reaction involve different reaction sites on the 3-oxo-b-sultam and
the substituent effect for the hydrolysis at the two reaction centres,
acyl and sulfonyl, may not be the same. Nevertheless, the large
decrease in the rate of inhibition of PPE by N-benzyl-3-oxo-b-
sultams on increasing size of the C-4 substituents agrees with other
results that have suggested the alkyl substituents at C-3 position
of monocyclic b-lactam (which is the equivalent of the C-4 of 3-
oxo-b-sultams) binds to the S1 pocket of the enzyme,24 and for
which a small hydrophobic residue is preferred.22,23 The preference
shown by the C-4 substituents for the enzyme also confirms that
the inhibition by 3-oxo-b-sultams is active-site directed.
(iii) Variation of the amine leaving group. The inhibition
activity is severely affected by the carboxylate group; a 25-fold
decrease of the second-order rate constant, ki, for the inhibition
by 9 compared with 1 is observed (Table 1). The decrease in the
inhibition activity may be the result of a poorer binding of the
dissociated carboxylate in the hydrophobic environment of
the active site of PPE. Esterification of the N-a-carboxylate of the
3-oxo-b-sultams (7 and 9) with benzyl alcohol gave esters which
could not be studied for inhibition because of poor solubility, even
in 15% acetonitrile–water (v/v).
Experimental
Synthesis
Melting points were determined on a Gallenkamp melting point
apparatus and are uncorrected. Infrared measurements were
recorded on a Perkin Elmer 1600 series FT-IR spectrometer.
1
400 MHz H and 67 MHz 13C NMR spectra were determined
1
on a Bruker Advance 400 MHz spectrometer. 500 MHz H and
The amine leaving group was further modified by varying the
substituent at the a-carbon. Increasing the length of the alkyl
chain by one methylene unit has little or no effect on the chemical
100 MHz 13C NMR spectra were determined on a Bruker AMX
500 spectrometer. GC–MS were determined on a Varian GC–
MS with a Finnigan MAT ion trap detector. Mass spectrometry
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was performed on a Fisons Quattro VG quadrupole mass spec-
trometer. High resolution mass spectrometry was carried out by
the University of Swansea, courtesy of the EPSRC. Elemental
analyses were carried out at Medac Ltd. Fluka or Merck silica gel
60 was used for all chromatographic separations, with silica gel
60 F254 TLC plates used for initial investigations. Solvents were
purified according to Perrin and Armarego.28 Starting materials
were purchased from Aldrich, Avocado and Lancaster, and were
used without further purification.
0.56) (0.76 g, 43%), m.p. 63–64 ◦C. IR: mmax (cm−1) (CHCl3) 3020,
1772, 1339, 1216, 1182, 1122. H NMR: d (CDCl3): 7.40–7.38
(5H, m, Ph), 4.6 (2H, s, CH2), 1.8 (6H, s, 2 x CH3). 13C NMR:
1
=
d (CDCl3): 164.36 (C O), 133.17 (quaternary carbon), 128.96
(CH (Ph)), 128.52 (CH (Ph)), 128.1 (CH (Ph)), 83.03 (quaternary
carbon), 44.47 (CH2), 18.4 (CH3 × 2).
(DL)-2ꢀ -(4,4-Dimethyl-1,1-dioxido-3-oxo-1,2-thiazetidin-2-yl)
phenylacetic acid (9). Palladium (0.1 g, 10% Pd/C) was added to
dry ethyl acetate (2 ml) and then to a solution of benzyl (DL)-2ꢀ-
(4,4-dimethyl-1,1-dioxido-3-oxo-1,2-thiazetidin-2-yl) phenyl ac-
etate (S3) (0.1 g, 0.268 mmol) in dry ethyl acetate (11 ml). The
reaction vessel was flushed with nitrogen for 2 minutes followed
by hydrogen for 1 minute. The solution was hydrogenated at
atmospheric pressure for 2 hours before filtering through celite.
The solvent was removed by reduced pressure rotary evaporation
at 30 ◦C to yield a white solid (74 mg, 98%) m.p. 195–197 ◦C
(decomposes to a dark oil). IR: mmax (cm−1) (CHCl3) 3020, 2956,
Disodium 2-methyl-2-sulfonatopropanoate (S1). Concentrated
H2SO4 (5.22 ml) was added dropwise to isobutyric anhydride
(20 g, 127.0 mmol) and the mixture stirred for 30 minutes at 20–
35 ◦C. The reaction mixture was then gently heated to 90 ◦C and
stirred for approximately 7 hours. To check for the completion of
the reaction: a solution of BaCl2 (1 ml) was added to a sample of
the reaction mixture, if no precipitation appeared then the reaction
was considered complete. The hot reaction mixture was poured
into ice-cold water (50 ml) and extracted with ether (3 × 40 ml).
Ether extracts contain the isobutyric acid from the sulfonation. To
the aqueous phase, a solution of NaOH (6.16 g) in water (20 ml)
was added in small portions to adjust the pH to around pH 8.
The solution was evaporated to dryness using reduced pressure
rotary evaporation at 40 ◦C. The residue was dissolved in hot
water (10 ml) and precipitated by addition of ethanol (50 ml) and
the resulting precipitate was isolated by vacuum filtration. If the
colour of the solution was dark brown–orange, then charcoal was
added to the solution before the addition of the ethanol. A second
crop was isolated by adding ethanol to the mother liquor. The
product isolated (20.1 g, 75%) was carried through to the next step
without any further purification.
1
1776, 1732, 1354, 1337, 1216, 1181, 1124. H NMR: d (CDCl3):
7.47–7.37 (5H, m, Ph), 5.76 (1H, s, CH), 1.62 (3H, s, CH3), 1.58
(3H, s, CH3). 13C NMR: d (CDCl3): 167.98 (C O), 164.58 (C O),
134.3 (quaternary carbon), 129.06 (CH (Ph)), 128.84 (CH (Ph)),
128.45 (CH (Ph)), 82.71 (quaternary carbon), 60.15 (CH), 17.8
(CH3), 17.68 (CH3). HREI-MS [M+NH4]+ for C12H13NO5S calc.
301.0853 measured 301.0855.
=
=
4-Spiro-cyclohexyl-3-oxo-b-sultam (S17). 1-(Chlorosulfonyl)-
cyclohexane carbonyl chloride (S16) (1 g, 4.33 mmol) was
dissolved in ether (10 ml) and added dropwise over 20 minutes
(VERY SLOWLY) to liquid NH3 (5.3 ml) in ether (10 ml) at
−78 ◦C. The mixture was warmed to room temperature and stirred
until all the solvent had evaporated. The residue was dissolved
using CHCl3 (5 ml) and water (5 ml) at 0–4 ◦C, and the pH of
the solution was adjusted to pH 1 using dilute HCl. The organic
layer was separated and the aqueous layer extracted with CHCl3
(3 × 5 ml). The organic layers were combined and dried over
sodium sulfate, and then the solvent was removed by reduced
pressure rotary evaporation 30 ◦C to give a white solid (0.50 g,
65%) m.p. 61–62 ◦C. IR: mmax (cm−1) 3241, 2946, 2864, 1778, 1452,
1356, 1338, 1215, 1161, 1132, 754. 1H NMR: d (CDCl3): 8.27 (1H,
br s, NH), 2.41 (2H, m, cyclohexyl H), 2.01 (2H, m, cyclohexyl
H), 1.91 (2H, m, cyclohexyl H) 1.71 (1H, m, cyclohexyl H), 1.6
(2H, m, cyclohexyl H), 1.44 (1H, m, cyclohexyl H). 13C NMR: d
2-(Chlorosulfonyl)-2-methylpropanoyl chloride (S2). Disodium
2-methylsulfonatopropionate (S1) (4.88 g, 23 mmol) was added
to thionyl chloride (18.3 ml, 153 mmol) in small portions over
10 minutes at 0 ◦C with stirring, DMF (0.37 ml) was added
dropwise over 2 minutes and the mixture was heated to 70 ◦C. After
gas production was complete the mixture was heated for a further
◦
5 hours at 70 C. Excess thionyl chloride was evaporated under
reduced pressure on a rotary evaporator at 40 ◦C, yielding a pale
yellow residue which was dissolved in ether. The resultant NaCl
was filtered off and the solvent was removed from the filtrate by
reduced pressure rotary evaporation at 30 ◦C to yield a yellow oil
(3.48 g, 74%). IR: mmax (cm−1) (neat) 3003, 1811, 1464, 1368, 1179,
=
(CDCl3): 163.29 (C O), 86.75 (quaternary carbon), 28.11 (C1/C5
1
1127. H NMR: d (CDCl3): 2.0 (6H, s, 2 x CH3). 13C NMR: d
CH2), 24.05 (C3 CH2), 22.62 (C2/C4 CH2). HREI-MS [M–H] for
C7H11NO3S calculated 188.0376 measured 188.0379.
=
(CDCl3): 169.29 (C O), 85.44 (quaternary carbon), 22.07 (CH3).
2-Benzyl-4,4-dimethyl-1,2-thiazetidin-3-one-1,1-dioxide (1). 2-
(Chlorosulfonyl)-2-methylpropionyl chloride (S2) (1 g, 4.9 mmol)
was dissolved in dry ether (200 ml) and the mixture was cooled
to −78 ◦C. A solution of benzylamine (0.53 ml, 4.9 mmol) in
ether (10 ml) was added dropwise over 20 minutes at −78 ◦C
with stirring. The mixture was stirred for 10 minutes before
triethylamine (1.35 ml, 14.7 mmol) in ether (10 ml) was added
dropwise over 10 minutes to the mixture at −78 ◦C. The reaction
mixture was stirred at −78 ◦C for 1 hour and then for 3 hours
at room temperature and the resultant Et3N·HCl salt filtered off.
The solvent was removed by reduced pressure rotary evaporation
at 30 ◦C to yield a creamy white residue which was purified using
column chromatography (30 g silica) (3 : 2 hexane–EtOAc, Rf =
N-Benzyl-4-spiro-cyclohexyl-3-oxo-b-sultam (6). At 0 ◦C un-
der nitrogen, 4-spiro-cyclohexyl-3-oxo-b-sultam (S17) (0.1 g,
0.53 mmol) in anhydrous THF (5 ml) was added to a suspension of
NaH (0.015 g, 0.6 mmol) (pre-washed 2–3 times with petroleum
ether) in DMF (2 ml). The mixture was stirred for 15 minutes
and benzyl bromide (0.09 g, 0.526 mmol) was injected through
a septum dropwise over 5 minutes. The mixture was allowed
to warm-up to room temperature and stirred for 24 hours. The
mixture was then cooled to 0 ◦C, ether (10 ml) was added, the
solution was hydrolyzed with brine, and the pH was adjusted
to 4–5 using dilute HCl. The organic layer was separated and
the aqueous layer was extracted with ether (2 × 10 ml). The
organic layers were combined, washed with saturated brine (2 ×
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10 ml) and dried over Na2SO4. The solvent was removed by rotary
evaporation at 30 ◦C and the resulting pale yellow oil was purified
using column chromatography (silica 3 g) (1 : 8 : 1 ether–hexane–
all experiments temperatures were maintained at 30 ◦C and ionic
strength at 1.0 M with AnalaR grade KCl unless otherwise stated.
Reaction concentrations were generally within the range 2 ×
10−5 M to 2 × 10−4 M to ensure pseudo-first-order conditions.
Hydroxide ion concentrations were calculated using pKw
(H2O) = 13.883 at 30 ◦C.29 Reactions studied by UV spectropho-
tometry were usually commenced by injections of acetonitrile
or dioxane stock solutions of the substrate (5–50 ll) into the
cells containing pre-incubated buffer (2.5 ml). Final reaction
cells contained 5% acetonitrile or dioxane v/v. The pH of the
reaction cells was measured before and after each kinetic run
at 30 ◦C, kinetic runs experiencing a change >0.05 units were
rejected. Reactant disappearance or product appearance were
followed at absorbance change maxima for individual compounds.
The solubility of compounds was ensured by working within
the linear range of absorbance in corresponding Beer–Lambert
plots. If required, greater than 1% MeCN v/v was used to
aid solubility. Pseudo-first-order rate constants from exponential
plots of absorbance against time or gradients of initial slopes
were obtained using the Enzfitter package (Elsevier Biosoft,
Cambridge, England) or the CaryBio software (Varian). pH-rate
profiles were modelled to theoretical equations using the Scientist
program (V2.02, Micromath Software Ltd, USA).
Reactions studied by stopped flow UV spectrophotometry used
stock solutions prepared at twice the standard UV concentration
in 1 M KCl. Hydroxide solutions, buffer solutions or solutions
of nucleophilic reagents were prepared at twice the required
concentration. The substrate solution and the reaction mixture
were placed in separate syringes and thermostatted at 30 ◦C before
pneumatic injection into the reaction into the reaction cell. Where
applicable, the pH of solutions was measured prior to use. If
greater than 1% acetonitrile v/v was required for solubility, then
organic solvent concentration of all solutions used was fixed at the
required reaction cell amount. The photomultiplier voltage was
set to a maximum on deionized water and absorbance wavelengths
common to the standard UV experiments were used. Pseudo-first-
order rate constants from exponential plots of absorbance against
time were obtained using the supplied fitting software (Applied
Photophysics).
◦
EtOAc) Rf = 0.58 (0.082 g 53%) m.p. 73–74 C. IR: mmax (cm−1)
1
(CHCl3) 3020.6, 2944.8, 1768.8, 1335.8, 1215.9, 1168.4, 668. H
NMR: d (CDCl3): 7.40–7.36 (5H, m, Ph), 4.6 (2H, s, CH2Ph), 2.37
(2H, m, cyclohexyl H), 1.99 (2H, m, cyclohexyl H), 1.89 (2H, m,
cyclohexyl H) 1.7 (1H, m, cyclohexyl H), 1.59 (2H, m, cyclohexyl
H), 1.42 (1H, m, cyclohexyl H). 13C NMR: d (CDCl3): 163.71
=
(C O), 133.4 (quaternary carbon), 128.84 (CH (Ph)), 128.34
(CH (Ph)), 127.97 (CH (Ph)), 87.53 (quaternary carbon), 44.09
(CH2Ph), 27.91 (C1/C5 CH2), 23.99 (C3 CH2), 22.75 (C2/C4 CH2).
Kinetics
Standard UV spectroscopy was carried out on a Cary 1E UV-
visible spectrophotometer (Varian, Australia) equipped with a
twelve-compartment cell block. The instrument was used in
double-beam mode, allowing six reaction cells to be followed in a
single run. The cell block was thermostatted using a peltier system.
Stopped flow experiments used an SX 18 MV Spectrakinetic
monochromator (Applied Photophysics, Leatherhead, England)
equipped with an absorbance photomultiplier. The reagent sy-
ringes were thermostatted with a Grant thermostatted water
circulator. pH-stat experiments were performed on a ABU 91
Autoburette (Radiometer, Copenhagen, Denmark), controlled by
a VIT 90 video titrator. The SAM 90 sample station incorporated
a machined aluminium E2000 sample block rotor thermostatted
by a MGW Lauda M3 water circulator. pH was measured by a
pHG200-8 Glass pH electrode and a REF200 Red Rod reference
electrode (Radiometer). Temperature was monitored by a T101
temperature sensor.
pH measurements were made with either a 40 (Beckman,
Fullerton, USA) or 3020 (Jenway, Dunmow, England) pH meter.
Electrodes were semi-micro Ag/AgCl and calomel (Russell, Fife,
Scotland and Beckman, respectively). A calibration of the pH
meter was carried out at 30 ◦C using pH 7.00 0.01, pH 4.01
0.02 or pH 10.00 0.02 calibration buffers.
ESIMS experiments were carried out on a VG Quattro SQ II
(Micromass, Altrincham, England) and NMR experiments on a
400 MHz instrument (Bruker, Germany).
For reactions studied by pH-stat standardized NaOH was
delivered to a stirred sample solution (10 ml) held within the
thermostatted sample station. All reactions were performed under
nitrogen to prevent CO2 absorption. Data were exported to a
Windows PC via an RS232 interface and the terminal program
(Microsoft Corp., USA). Conversion into an appropriate format
was by means of an Excel (Microsoft Corp., USA) Macro
and results were fitted to first-order equations via the Enzfitter
program (Elsevier Software). The titrant used was 0.01–0.1 M
NaOH standardized prior to use by means of phenolphthalein
titration against 1.00 M HCl (volumetric reagent, D.H. Scientific).
Reactions were performed in 1 M KCl, 5% MeCN v/v, with a pH
set point of 6–7. Concentrations of sample were in the range of
1–2 mM with expected titrant added volumes of up to 1.0 ml.
AnalaR grade reagents and deionized water were used through-
out. Sodium hydroxide solutions were titrated prior to use
against a 1.00 M
0.1% hydrochloric acid volumetric reagent
(D.H. Scientific, Huddersfield, England) using phenolphthalein
as an indicator.
Organic solvents were glass-distilled prior to use and stored
under nitrogen. For solution pHs 3 and 11 the pH was controlled
by the use of 0.2
M buffer solutions of formate (pKa
3.75), ethanoate (pKa 4.72), 2-(N-morpholino)ethanesulfonic acid
(MES) (pKa 6.1), 3-(N-morpholino)propanesulfonic acid (MOPS)
(pKa 7.2), N-[tris(hydroxymethyl)methyl]-3-aminopropanesul-
fonic acid (TAPS) (pKa 8.4), 3-(cyclohexylamino)-2-hydroxy-
propane-1-sulfonic acid (CAPSO) (pKa 9.6), and 3-(cyclohexyl-
amino)propane-1-sulfonic acid (CAPS) (pKa 10.4). For general pH
work, buffers were prepared by partial neutralization of solutions
of their sodium salts to the required pH. For the alcoholysis
reactions, buffers were prepared by the addition of 0.25, 0.50
or 0.75 aliquots of 1 M NaOH to solutions of the alcohol. In
Enzyme-inhibition studies
Assays were performed at 30 ◦C in 0.1 M pH 8.5 TAPS buffer
with I = 1.0 M (KCl). The substrate used was N-suc-(L-Ala)3-
p-nitroanilide and stock solutions were made up in distilled
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methanol. Enzyme activity was monitored by following the
appearance of the p-nitroaniline product at 390 nm. During
inhibition studies a fixed assay substrate concentration of 6.0 ×
10−5 M was used resulting in a constant methanol concentration
of 1.5% v/v. Inc◦ubations of enzyme and inhibitor (500 ll) were
PPE and up to 5 × 10−3 M b-sultam. These conditions were
created in a separate UV cell containing stock PPE suspension
(200 ll), 0.1 M buffer (200 ll) and inhibitor stock solution in
MeCN or MeCN alone (100 ll). Aliquots of this solution (50 ll)
were assayed for PPE activity at various time intervals by injection
into 0.1 M pH 8.5 TAPS (2.36 ml) containing MeCN (150 ll)
and substrate solution (40 ll). This gave final assay conditions
of 6.0 × 10−5 M substrate, 1.5 × 10−6 M PPE, 6% MeCN v/v
and 1.5% MeOH v/v. In all cases assays of control incubations
were performed at the same time as inhibitor incubations. These
incubations substituted pure MeCN for the b-sultam solution but
then matched the remaining methodology in all respects.
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10 H. Ohbayashi, Expert Opin. Ther. Pat., 2002, 12, 65–84.
11 M. I. Page, Acc. Chem. Res., 2004, 37, 297–303.
12 M. I. Page, P. S. Hinchliffe, J. M. Wood, L. P. Harding and A. P. Laws,
Bioorg. Med. Chem. Lett., 2003, 13, 4489–4492.
13 W. Y. Tsang, N. Ahmed, L. P. Harding, K. Hemming, A. P. Laws and
M. I. Page, J. Am. Chem. Soc., 2005, 127, 8946–8947.
14 R. Moreira, A. B. Santana, J. Iley, J. Neres, K. T. Douglas, P. N. Horton
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17 M. I. Page, A. Williams, Organic and Bio-organic Reaction Mechanisms,
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carried out at 30 C in 0.04 M buffer, 20% MeCN v/v, 8 × 10−5
M
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Acknowledgements
We thank the University of Huddersfield for financial support
and the EPSRC National Mass Spectrometry Service Centre at
the University of Wales Swansea for accurate mass measurement.
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