Structure-reactivity relationships in the inactivation of elastase by β-sultams

Article abstract of DOI:10.1039/b208079f

N-Acyl-β-sultams are time dependent irreversible active site directed inhibitors of elastase. The rate of inactivation is first order with respect to β-sultam concentration and the second order rate constants show a similar dependence on pH to that for the hydrolysis of a peptide substrate. Inactivation is due to the formation of a stable 1:1 enzyme inhibitor complex as a result of the active site serine being sulfonylated by the β-sultam. Ring opening of the β-sultam occurs by S-N fission in contrast to the C-N fission observed in the acylation of elastase by N-acylsulfonamides. Structure-activity effects are compared between sulfonylation of the enzyme and alkaline hydrolysis. Variation in 4-alkyl and N-substituted β-sultams causes differences in the rates of inactivation by 4 orders of magnitude.

Full text of DOI:10.1039/b208079f

View Article Online / Journal Homepage / Table of Contents for this issue
Structure–reactivity relationships in the inactivation of elastase by
ꢀ-sultams
Paul S. Hinchliffe,a J. Matthew Wood,a Andrew M. Davis,b Rupert P. Austin,b R. Paul Beckettc
and Michael I. Page*a
a Department of Chemical and Biological Sciences, University of Huddersfield, Queensgate,
Huddersfield, UK HD1 3DH
b AstraZeneca R&D, Charnwood, Bakewell Road, Loughborough, UK LE11 5RH
c British Biotech Pharmaceuticals Limited, Wellington Road, Oxford, UK OX4 5LY
Received 20th August 2002, Accepted 30th September 2002
First published as an Advance Article on the web 27th November 2002
N-Acyl-β-sultams are time dependent irreversible active site directed inhibitors of elastase. The rate of inactivation
is first order with respect to β-sultam concentration and the second order rate constants show a similar dependence
on pH to that for the hydrolysis of a peptide substrate. Inactivation is due to the formation of a stable l : l enzyme
inhibitor complex as a result of the active site serine being sulfonylated by the β-sultam. Ring opening of
the β-sultam occurs by S–N fission in contrast to the C–N fission observed in the acylation of elastase by
N-acylsulfonamides. Structure–activity effects are compared between sulfonylation of the enzyme and alkaline
hydrolysis. Variation in 4-alkyl and N-substituted β-sultams causes differences in the rates of inactivation by 4 orders
of magnitude.
Proteolytic enzymes are common targets for potential thera-
peutic application and their inhibition by mechanism based and
active site directed agents is a fruitful area of study.1 In general,
this involves the covalent modification of an active site residue
which is not then readily regenerated. For example, many
inhibitors are acylating agents of the active site serine residue
of serine proteases.2 The mechanism of inhibition involves the
displacement of a leaving group from the acylating agent to
generate a relatively stable acyl enzyme which only reacts slowly
with nucleophiles, such as water, to regenerate the enzyme and
so this process leads to effective inhibition.
Human neutrophil elastase (HNE) is a serine enzyme which
is one of the most destructive proteolytic enzymes, being able to
catalyse the hydrolysis of the components of connective tissue.
HNE is released in response to inflammatory stimuli and has a
major role in protein digestion following phagacytosis. It has
been implicated in the development of diseases such as emphy-
sema, cystic fibrosis and rheumatoid arthritis and there have
been numerous studies attempting to find small molecule
inhibitors of HNE.3 The enzyme belongs to the trypsin class
and the structure of HNE has been determined by X-ray
crystallography,4 but many structural and inhibition studies
have been conducted with the related, but more readily
available, porcine pancreatic elastase (PPE).5
The majority of elastase inhibitors are based an other serine
protease inhibitors. They are, for example, peptidyl fluoro-
ketones6 or ketones attached to a strongly electron with-
drawing group, such as 2-benzoxazole, with suitable elements of
molecular recognition.7 An alternative strategy has been to use
acylating agents that generate an acyl enzyme which does not
turnover, or rearranges to a more stable adduct or even gener-
ates a second electrophilic ‘trap’ which subsequently reacts with
a nucleophilic group on the enzyme. For example, bicyclic
[3.3.0] systems containing a γ-lactone or γ-lactam which is
trans-fused to the other 5-membered ring inhibit HNE by
acylating the nucleophilic hydroxy group of serine-195 in the
active site of the enzyme.8 Interestingly, the classical β-lactams,
traditionally used as anti-bacterial agents by inhibiting serine
transpeptidases,9 have also been shown to be mechanism
based inhibitors of elastase when used as neutral derivatives.10
ESI-MS and NMR studies have shown that the first step is an
acylation process in which the four membered β-lactam ring
is opened.11 The requirements for inhibition of elastase by
monocyclic lactams have been recently described.12 They
involve the initially formed acyl enzyme intermediate undergo-
ing a conformational change to displace the hydrolytic water
molecule, effectively generating a more stable complex.
In principle, sulfonation of serine proteases offers an interest-
ing but largely unexplored strategy for inhibition as an altern-
ative to the traditional mechanism-based acylation process. In
addition to their normal acyl substrates, serine proteases are
known to react with other electrophilic centres such as phos-
phonyl derivatives.1 The main reason why sulfonation of serine
enzymes is not a well studied process is because sulfonyl
derivatives are much less reactive than their acyl counterparts.13
For example, sulfonamides are extremely resistant to alkaline
and acidic hydrolysis and, in general, sulfonyl transfer reactions
are 102 to 104 fold slower than the corresponding acyl transfer
process. However, we have recently shown that the rates of
alkaline and acid hydrolysis of N-alkyl and N-aryl β-sultams
are 102 to 103 fold greater than those for the corresponding
β-lactams.14 β-Sultams (1) show extraordinary rate enhance-
ments of 109 and 107, respectively, compared with the acid
and base catalysed hydrolysis of the corresponding acyclic
sulfonamides.15 In principle, therefore, β-sultams are excellent
candidates to explore the mechanism of sulfonation and pos-
sible inhibition of serine protease enzymes. We recently demon-
strated that ring opening of the β-sultam by elastase gives the
sulfonate ester,16 by analogy with the acyl enzyme intermediate
formed during the hydrolysis of normal substrates (Scheme 1).
Scheme 1
Herein, we report further studies on this process and the effect
of structural changes in the β-sultam on the rate of inactivation
of elastase.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
67

View Article Online
Alkaline hydrolysis of the PPelastase substrate, N-suc-(L-Ala)3-
p-nitroanilide (2)
Results and discussion
One of the major requirements for successful inhibition of an
enzyme by covalent modification is that the rate of the inacti-
vation must be faster than, or at least competitive with, the
turnover of the natural substrate and the inhibitor itself
must be relatively stable under in vivo conditions. The rate of
inactivation is controlled by two important factors:
An analysis of a particular enzyme’s selectivity for various
substrates or covalent inhibitors requires a knowledge of their
reactivity in the absence of the enzyme. Although N-suc-(-Ala)-
p-nitroanilide (2) is an established and convenient substrate for
porcine pancreatic elastase (PPelastase),19 the magnitude and
nature of its hydroxide ion catalysed hydrolysis has not been
reported.
(i) the “chemical” reactivity of the inactivator
(ii) molecular recognition by the target enzyme.
Below pH 13, N-suc-(-Ala)3-p-nitroanilide (2) undergoes
hydroxide ion catalysed hydrolysis to give p-nitroaniline, for
which the second-order rate constant, kOH, is 7.19 × 10Ϫ3 MϪ1
sϪ1 at 30 ЊC. At pHs above ca. 13 there is a pH-independent
reaction presumably due to a reversible ionisation of the
Given that most modifications involve displacement of a
leaving group by a nucleophile on the enzyme, chemical reac-
tivity is usually considered in terms of the electrophilic nature
of the inhibitor and the leaving group ability (nucleofugacity)
of the group displaced. In addition to these electronic effects,
the intrinsic chemical reactivity of a compound may be modi-
fied by steric and strain effects. Increasing intrinsic chemical
reactivity may lead to a faster rate of reaction with the target
enzyme but may also lead to greater hydrolytic and metabolic
instability and lower specificity.
Obtaining maximum activity by molecular recognition
involves using the full catalytic apparatus of the enzyme, as
used for the natural substrate, and utilising the favourable
non-covalent interactions at the various sub-sites of the enzyme
and inhibitor.17 The most desirable inhibitor may be one
where molecular recognition is maximised using these favour-
able non-covalent interactions and where covalent modification
of the enzyme, due to the inherent reactivity of the inhibitor,
is minimised.
aryl-NH group of (2) consistent with the pKa of simple
p-nitroanilides.20 The overall pathway for the hydroxide
ion hydrolysis of N-suc-(-Ala)3 p-nitroanilide (2) is shown
in Scheme 2.
Only the neutral compound undergoes hydroxide ion
catalysed hydrolysis whilst the anion is relatively stable. The
rate law for this reaction was determined based upon the
fraction of the undissociated species present in solution
[eqn. (1)], where [S] is the reactant concentration, kobs is the
observed first order rate constant for hydrolysis, kOH is the
second-order rate constant of hydroxide ion hydrolysis and
[Hϩ] / (Ka ϩ [Hϩ]) is the fraction of the anilide present in
its undissociated form.
Improving the inhibition properties of a compound may
therefore be achieved by modifying the structure to enhance
either chemical reactivity or molecular recognition, or a com-
bination of both. However, analysing these separate effects on
the rate of inactivation when the inhibitor is modified is not
straightforward. Modification of the inhibitor structure can
affect the free energies of both the initial reactant state and the
transition state of the reaction with the enzyme, whereas the
observed variation in rate constants for various inhibitors reac-
ting with the enzyme only reflects the difference in energies
between these two states. Different substituents can affect the
ease of bond-making and -breaking by classical electronic
factors such as inductive, resonance and steric effects. However,
the free energy of activation of an enzyme-catalysed reaction is
also affected by the favourable binding energies between the
protein and substrate substituents not directly involved with the
reaction site. It is, therefore, important to separate these two
effects before conclusions about the efficiency of inhibition can
be made. For substrates we have suggested18 that an ‘enzyme
rate-enhancement factor’ (EREF) can be evaluated by dividing
the second-order rate constant for the enzyme catalysed reac-
tion, kcat/Km, by that for hydrolysis of the same substrate
catalysed by hydroxide ion, kOH. For inhibitors acting by
covalent modification of the enzyme, a similar ratio of ki/kOH
can be used where ki is the rate constant for inactivation.
Rate / [S] = kobs = kOH[HOϪ]([Hϩ] / (Ka ϩ [Hϩ])) (1)
When the solution pH is less than 13, [Hϩ] ӷ Ka, eqn. (1)
simplifies to kobs = kOH[HOϪ] and a first order dependence on
hydroxide ion concentration is observed. At solution pHs
greater than 13, Ka ӷ [Hϩ], eqn. (1) becomes kobs = kOH (Kw / Ka)
resulting in a pH independent rate. Modelling the experimental
data to this equation using the Scientist software package gave
the following parameters: kOH = 7.19 × 10Ϫ3 MϪ1 sϪ1 and Ka =
9.82 × 10Ϫ14 M (pKa 13.01). Direct titration using the absorb-
ance change at 390 nm as a function of pH, immediately after
injection of (2) into sodium hydroxide solutions, gave a reason-
able sigmoidal plot from which was obtained a calculated pKa,
of 12.63 0.07, in fairly good agreement with that determined
kinetically.
Scheme 2
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
68

View Article Online
PPelastase catalysed hydrolysis of substrate, N-suc-(L-Ala)3-p-
nitroanilide (2)
incubation cell. Aliquots of the incubation solution containing
enzyme and potential inhibitor were then removed from the
incubation cell after an incubation time, ti, and assayed for
PPelastase activity against the anilide substrate (2) in separate
assay cells at pH 8.5. 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 ti. If the enzyme was incubated alone under exactly
the same conditions but in the absence of β-sultam, the
observed assay slope remained constant throughout the
experimental time frame. By taking the slope of these control
experiments as being equal to 100 % activity, the percentage
activity of PPelastase remaining in the inhibitor experiments
could be calculated and plotted against ti. This gave apparent
first-order rate constants for inactivation (Fig. 2) which were
first order with respect to inhibitor concentration (Fig. 3). The
(i) Initial rate data. Initial rate data for the hydrolysis of the
anilide substrate (2) catalysed by PPelastase were collected at
390 nm using a molar extinction coefficient change, ∆ε390, of
1.24 × 104 MϪ1 cmϪ1. The kinetic analysis was undertaken at
30 ЊC in 6% acetonitrile v/v, 1.5% methanol v/v and I = 1.0 M
(KCl). The anilide substrate (2) was dissolved in 0.1 M pH 8.5
TAPS buffer at concentrations varying from 0.08 mM to 2 mM.
Initial rates were collected up to a maximum time of 4 min.
Rates of the enzyme reaction in units M sϪ1 were obtained by
dividing the slope of the initial absorbance increase by the total
change in molar extinction coefficient for the complete reac-
tion. No effective saturation of the enzyme catalysed reaction
was observed at substrate concentrations up to 2 mM. Hence it
was not possible to obtain a reliable measurement for kcat and
Km. However, using the initial rate data, the apparent values of
kcat and kcat /Kmwere obtained via a Lineweaver–Burke plot
which gave an intercept realistically indistinguishable from zero.
Nonetheless, this plot generated kcat = 7.82 sϪ1, Km = 4.92 × 10Ϫ3
M and kcat/Km= 1.59 × 103 MϪ1 sϪ1. It is evident from these
figures that although the anilide (2) is convenient for UV assays
of PPelastase, it is only a moderately good substrate. The
second-order rate constant kcat/Km is relatively low for an
‘activated’ substrate and the EREF is 2 × 105. The relatively
high Kmvalue indicates a poor degree of enzyme-substrate
binding.
(ii) Variation of pH. The activity of PPelastase was also
determined in buffer solutions of pH 5 to pH 11. Enzyme
activity was monitored using initial rates and the N-suc-(-Ala)3-
p-nitroanilide (2) assay. The pH rate profile (Fig. 1) shows a
typical bell shaped profile but, interestingly, there appears to
Fig. 2 Plot of % PPelastase activity against incubation time for the
inactivation of PPelastase by N-benzoyl-β-sultam (3) at pH 6.75 and
30 ЊC in 0.04 M buffer, 20 % ACN v/v, 8 × 10Ϫ5 M PPelastase and
1.2 × 10Ϫ3 β-sultam.
Fig. 1 Plot of PPelastase activity (kcat/Km) towards the substrate N-
suc-(-Ala)3-p-nitroanilide (2) against pH. Initial rates measured at 1.52
× 10Ϫ6 M PPelastase, 6.0 × 10Ϫ5 M substrate, 390 nm and 30 ЊC in 0.1 M
buffer, 6% MeCN v/v.
be more than a single ionisation occurring beyond the pH
optimum. The profile is complex above pH 8.5 and either
multiple ionisations or partial denaturation of the enzyme may
be occurring. Modelling of the sigmoidal increase in enzyme
activity up to pH 8.5 from pH 5 leads to a pKa of the active-
Fig. 3 Plot of the first order rate constant for the inactivation of
PPelastase by N-benzoyl-β-sultam (3) corrected for hydrolysis at pH 6.1
against β-sultam concentration.
site’s ionisable group of 6.88
0.02. The enzyme is only
active when this group is deprotonated, which is likely to be
histidine-57 in the enzyme’s catalytic triad and is reported to be
responsible for similar increases in activity in many serine
proteases.2a,21
concentration of the enzyme is much lower than that of the
inhibitor at time zero, and so the rate of inactivation is effect-
ively pseudo-first order and the rate constant does not depend
on the enzyme concentration. Second-order rate constants for
inactivation, ki, were obtained either by dividing the observed
rate of inactivation by inhibitor concentration, kobs/[I], when
less than two inactivations were carried out or graphically from
slopes of the plots of the observed rate of inactivation against
several inhibitor concentrations (Fig. 3).
Inhibition of PPelastase by N-acyl-ꢀ-sultams
Kinetics of inhibition. In order to test the inhibitory pro-
perties of N-acyl-β-sultams with respect to porcine pancreatic
elastase (PPelastase), enzyme and β-sultam were incubated
together in a buffered solution that was referred to as the
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
69

View Article Online
Table 1 Second-order rate constants for the inactivation of PPelastase by β-sultams (ki) at pH 6, 7 and 8.5 and those for the alkaline hydrolysis, kOH
of the β-sultam at 30 ЊC
,
β-Sultam
ki at pH 6.1/MϪ1 sϪ1
ki at pH 7.0/MϪ1 sϪ1
ki at pH 8.5/MϪ1 sϪ1
kOH/MϪ1 sϪ1
EREF at pH 8.5a
N-Bz (3)
0.85
0.23
—
—
—
—
8.65
2.70
2.34
0.39
3.64
4.06
1.63
6.33
1.46 × 104
300
6.1 × 10
1.2 × 103
5.4 × 102
3.1 × 10
9.1 × 102
3.8 × 103
3.2 × 102
2.3 × 104
4-iPr-N-Bz (14)
(E)-4-Ethylidene-N-Bz (17)
N-Carbamoyl (4)
4-iPr-N-carbamoyl (15)
4-Et-N-carbamoyl (18)
N-Cbz (5)
2.55 × 103
265
2.13 × 10Ϫ2
9.14 × 10Ϫ3
0.26
3.71 × 10Ϫ2
1.59 × 10Ϫ2
0.46
3.80
26.8
38
23.6
67
106
4.48 × 104
1.0 × 103
4-iPr-N-Cbz (16)
a EREF is ki (inactivation)/kOH at pH 8.5
With some N-acyl β-sultams and at high pH there were
problems 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 normalised with respect to the decreasing
β-sultam concentration using eqn. (2).
Normalised rate at time t =
(observed rate/ [Sultam]o) × [Sultam]t (2)
The second-order rate constants ki for the inactivation of
PPelastase by various substituted β-sultams are given in Table
1.
(ii) ESIMS and X-ray crystallography data. Sulfonylation
of the enzyme by β-sultams as shown in Scheme 1 was con-
firmed by ESIMS. Samples of PPelastase inactivated by N-ben-
zoyl-β-sultam (3) were subjected to ESIMS analysis. Native
PPelastase gave MW 25,904 Da (and 25943 (M ϩ K)) which
compares well to the literature ESIMS value of 25,898.22
Incubation of the enzyme with N-benzoyl-β-sultam (3) (MW
211 Da) with PPelastase showed formation of the sulfonylated
adduct at 26115 Da (and 26153 (M ϩ K)).
Crystals of native PPelastase were soaked for 24 h at pH
5 in a saturated solution of N-benzoyl-β-sultam (3). After
soaking, X-ray crystallography revealed that the crystals were
isomorphous to native PPelastase and confirmed that ring
opening of the β-sultam was part of the inactivation process
(Fig. 5).16 A sulfonate ester formed between Ser-195 and the
inhibitor is observed along with the ethyl amino group project-
ing into the active site’s P1/P2 region. Whilst one sulfonate
oxygen atom was observed in the upper part of the S1 pocket
the other was located in the oxyanion hole within hydrogen
bonding distance of the amido-nitrogen atoms of Ser-195 (3.02
Å) and Gly-193 (3.23 Å). Crucially, the side chain of His-57 has
been displaced by approximately 90Њ from its native position,
which is now occupied by two water molecules (Wat-342 and
Wat-355). This effect is almost identical to that observed in the
inhibition of PPelastase by γ-lactams.22
Evidence for active-site sulfonation
(i) pH Dependence of inactivation. The three N-acyl-β-
sultams, N-benzoyl (3), N-(N,N-methylphenyl)carbamoyl (4)
and N-benzyloxycarbonyl-β-sultam (5) are all time dependent
inhibitors of PPelastase and their second-order rate constants
for inactivation are given in Table 1. Above pH 8, these rate
constants are unreliable because of competing hydrolysis.
However, below this pH the variation of the second-order rate
constants for inactivation (ki) with pH is very similar to that
of the second-order rate constants for the hydrolysis of the
anilide substrate (kcat/Km) catalysed by PPelastase (Fig. 4). This
N-Acylsulfonamides have been used previously to inactivate
serine enzymes.8 However, the mechanism invariably involves
acylation and C–N bond fission with the serine hydroxy group
attacking the amide to displace the sulfonamide as the leaving
group. Although the β-sultams reported here are also formally
N-acylsulfonamides inactivation occurs by sulfonylation as a
result of serine nucleophilic attack on the sulfonyl centre and
displacement of the amide as a leaving group. This appears to
be the first case of preferential S–N over C–N fission in the
reaction of a N-acylsulfonamide with a serine protease.
Fig.
4
Plot of kcat/Km for the hydrolysis of N-suc-(-Ala)3-p-
nitroanilide by PPelastase (᭜) and ki for N-benzoyl-β-sultam (3) with
PPelastase (ϩ) against pH. 30 ЊC, I = 1.0 M (KCl), 6 % MeCN v/v and
1.5% MeOH v/v.
indicates that the rate of inactivation of PPelastase by these
compounds is controlled by the same catalytic groups in the
active-site that are used for substrate hydrolysis, i.e. active-site
directed inhibition is occurring. The increase in enzyme activity
towards inactivation with increasing pH shows an apparent
pKa of about 7 and is probably due to the dissociation of the
protonated His-57 residue, crucial for effective catalysis of the
substrate residue. The hydrolysis reaction shows a pKa of 6.88,
as described earlier.
(iii) Catalysis of sulfonyl transfer. As active-site directed
inhibition is occurring in the inactivation of PPelastase by N-
benzoyl β-sultam (3) it appears that the catalytic machinery
of PPelastase is catalysing the sulfonyl transfer process. This
indicates a significant degree of flexibility within the enzyme as
the stereochemical requirements for catalysis of a reaction
involving sulfonyl transfer with
a trigonal bipyramidal
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
70

View Article Online
Fig. 5 Stereo views of the active site of PPelastase (in green) showing N-benzoyl- β-sultam (3) (in beige) covalently linked via a sulfonate ester to
Ser-195 and the ‘native’ position of His-57 in thin lines. The top picture shows the 2mFo Ϫ DFc electron density map contoured at 1 σ. Wat-342 has
not been included so that the ‘extra’ density extending from Nε2 of His-57 can be seen more clearly. The bottom picture shows the ‘native’ position of
His-57 in thin lines.
arrangement in the transition state are significantly different to
acyl transfer reactions involving a tetrahedral intermediate. For
example, elastases possess an oxyanion hole used to stabilise the
oxyanion in the tetrahedral intermediate (TI) formed after
attack of the serine oxygen at a carbonyl centre. This oxyanion
will lie at ca. 109Њ to the newly formed serine O–C bond. In
contrast, the oxyanion formed by attack at the β-sultam
sulfonyl centre is likely to occupy an equatorial position in a
trigonal bipyramidal intermediate (TBPI) or transition state
and will therefore lie at ca. 90Њ to the serine O–S bond.
Although C–N bond fission in the hydrolysis of amides requires
general acid catalysis to facilitate amine expulsion, the reac-
tivity of N-acyl-β-sultams and their amide leaving groups may
allow ring opening to occur without N-protonation. An outline
possible mechanism for sulfonyl transfer from the β-sultam to
the active site serine is shown in Scheme 3.
The preferred direction of nucleophilic attack at acyl centres
is presumed to be at an approximately tetrahedral angle at
either face. As this differs to that at sulfonyl centres, which is
along the apical position bisecting the sulfonyl oxygens, attack
of the serine group at the sulfonyl centre will alter the position
of the other catalytic group, the histidine residue, relative to the
rest of the molecule. In acyl transfer, the histidine residue has to
Scheme 3
be correctly positioned to protonate the leaving group nitrogen
in the TI in its function as a general acid catalyst. The catalysis
of sulfonyl transfer in N-acyl-β-sultams by elastase may there-
fore only be possible because ring opening occurs in a single
step without general acid catalysis by the histidine residue. The
relative position of this group in the active-site would therefore
be less important. This apparent flexibility of enzymes has also
been observed in the β-lactamase P99 which has the ability to
catalyse phosphoryl transfer reactions, via trigonal bipyramidal
intermediates.23
(iv) Reactivation. The inactivation of the enzyme is irrevers-
ible; the resulting sulfonyl enzymes are stable for up to four
weeks in the incubation solution at 30 ЊC. For some serine
proteases such as α-chymotrypsin, inactivated by sulfonyl
fluorides, it is possible to reactivate the enzyme above pH 8.5 or
by initially unfolding the enzyme and then removing from 8 M
urea.24 The sulfonyl enzyme formed from N-benzoyl-β-sultam
(3) and PPelastase could not be reactivated using a 500-fold
excess of hydroxylamine or hydrogen peroxide. Human leuco-
cyte elastase inhibited by monocyclic β-lactams via acylation
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
71

View Article Online
of the serine residue can be reactivated by hydroxylamine.25
However, this reactivation can only occur if the hydroxylamine
is added one minute after inhibitor. If added after ten minutes
no reactivation occurs due to a postulated second reaction
between the inhibitor molecule and His-57, generally involving
expulsion of a leaving group, inactivating the enzyme perman-
ently. A similar reaction in N-acyl-β-sultams is ruled out by the
ESIMS results giving no evidence for mass loss and the fact that
roughly equimolar mixtures of enzyme and inhibitor result
in complete inactivation. This indicates that a single enzyme
catalysed reaction results in inactivation. Inhibitors whose
mechanism involves a second reaction exhibit partitioning
between hydrolysis products and inactivated enzyme and charac-
teristically require a specific molar excess of inhibitor for
complete inactivation.26
indicates that there is no significant difference in binding
between these two leaving groups and the enzyme in the inacti-
vation of PPelastase. However, replacement of the N-carbamoyl
residue by the N-benzyloxycarbonyl substituent increases the
rate of inactivation by 103–104. The enzyme rate enhancement
factors (EREFs) for N-benzoyl (3), N-carbamoyl (4) and
N-benzyloxycarbonyl-β-sultam (5) are 61, 31 and 320 respect-
ively. This suggests that of all the N-substituents the benzyl-
oxycarbonyl moiety binds most favourably in the active-site of
PPelastase, possibly via π–π interactions with the His-57 residue
in the active-site or another aromatic residue in its vicinity. Such
interactions have been observed between the p-toluene ring
of N-arylsulfonyl-β-lactams and His-57.30 Apparently the
N-carbamoyl group of (4) does not reach sufficiently deeply
into the S’ sites of PPelastase’s active-site and cannot interact
as effectively with His-57. It both lacks one carbon atom
on the side chain compared to that of (5) and free rotation
is restricted by resonance of the urea moiety. There is also
cis–trans isomerism of this urea moiety resulting in the
N-carbamoyl compound (4) having two stereoisomers.
Structure–activity relationships
The leaving group. The fact that the β-sultam has ring opened
upon inactivation of the enzyme confirms the necessity of S–N
fission for activity. However, it is important to separate the
effects of substituents on chemical reactivity and molecular
recognition. For example, N-substituents not only influence the
rate of S–N bond fission and reaction through inductive effects,
altering the electrophilicity of the sulfonyl centre, N-basicity
and amine nucleofugacity, but also change binding energies
through molecular recognition effects such as interactions
between the substituent and a binding pocket in the enzyme.
In our experiments, less reactive β-sultams such as N-phenyl-
β-sultam (6) or N-alkyl-β-sultams such as N-carboxybenzyl-
β-sultam (7) and N-methoxycarbonylbenzyl-β-sultam (8) do
not inactivate PPelastase, nor do acyclic sulfonamides such as
N-benzylsulfonyl-N-methylethanamide (9) and N-methyl-
sulfonylisoleucinemethylamide (10). These acyclic compounds
show no inhibitory activity against PPelastase at concentrations
of 4 mM and 2 mM, respectively. The N-acylsulfonamide (9)
could, in principle, act as either a sulfonylating or acylating
agent. The absence of the four-membered ring enormously
reduces the reactivity of the sulfonyl centres of both of these
compounds, their second order rate constants for alkaline
hydrolysis, kOH, of ca. 1 MϪ1 sϪ1 (9) (for C–N fission) and ca.
1 × 10Ϫ8 MϪ1 sϪ1(10), are smaller than those for analogous
β-sultams by a factor of greater than 106. The four-membered
ring is required for inactivation to ensure the sulfonyl centre
is reactive enough. Acyclic sulfonyl fluorides are effective
inhibitors of serine enzymes27 as are sulfonate esters in their
more reactive cyclic form.28 There appears to be a threshold
of chemical reactivity that must be reached for sulfonyl
compounds to display effective enzyme inhibition. The least
effective PPelastase inhibitor in the simple N-acyl-β-sultams is
also the least reactive, the N-carbamoyl compound (4) with a
kOH of 265 MϪ1 sϪ1. The N-acyl sulfonamide (9) probably does
not inactivate PPelastase due to a combination of its low
reactivity and its sterically bulky benzyl substituent. Although
phenylmethanesulfonyl fluoride (PMSF) is an inhibitor of
α-chymotrypsin, the S1 pocket in α-chymotrypsin is much
larger than in PPelastase. PMSF is also likely to be more
reactive than the N-acyl sulfonamide (9) although direct kOH
comparisons are complicated by PMSF undergoing E1cB-type
elimination reactions. However, the kOH value of benzene-
sulfonyl fluoride is similar to that of (9) and N-phenyl-β-sultam
(6) at 3.4 MϪ1 sϪ1.29
The ring substituents. The rates of enzyme inactivation
observed could be increased if recognition elements are built
into the structure of the β-sultams. β-Sultams (3), (4) and (5)
are far from ideal as PPelastase inhibitors as they are relatively
hydrolytically labile. The N-benzyloxycarbonyl compound (5) is
the most potent of the simple N-acyl-β-sultams but the com-
pound is too reactive to be an effective inhibitor above neutral
pH. Effective substituent changes in the lead β-sultam
structures which are similar to those that have been previously
shown to increase recognition by PPelastase would serve two
purposes. Firstly, they could improve binding to PPelastase
and hence increase the rate of inactivation of the serine
residue. Secondly, they may reduce the hydrolytic lability of
the inhibitor, thus making it more stable in aqueous solution
and in a biological environment.
It is known that the α-chymotrypsin family of enzymes has a
well-established binding pocket adjacent to the active serine
residue, in the S1 position. In the elastase enzymes this binding
pocket is relatively small and has a preference for small hydro-
phobic substituents. The inhibition of PPelastase by mono-
cyclic β-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.30,31
Alkyl chains have therefore been added to the simple β-sul-
tam ring at the 4-position with the intention of improving bind-
ing at the active-site of PPelastase. 4-Isopropylidene β-sultam
(11) is unusual because the substituent causes nucleophiles to
react at the acyl rather than the sulfonyl centre.32 This switch of
reactive centre is accompanied by a 104-fold reduction in
reactivity towards hydroxide ion. Although N-acylsulfona-
mides may act as inhibitors of elastase by acylation and expul-
sion of the sulfonamide, 4-isopropylidene-N-benzoyl-β-sultam
(11) and 4-isopropylidene-N-benzyloxycarbonyl-β-sultam (12)
are both inactive towards PPelastase at concentrations up to 2
mM. Interestingly, 4-isopropylidene-N-(N,N-methyl phenyl)-
A comparison of the ki values at pH 7 with the second-order
rate constants for hydroxide ion hydrolysis, kOH values (Table 1),
for the N-benzoyl compound (3) and the N-carbamoyl com-
pound (4) indicates that their relative effectiveness as PPelastase
inhibitors is largely due to variations in their chemical reac-
tivity. At pH 7, the N-benzoyl derivative (3) is a better inactiva-
tor by two orders of magnitude than the N-carbamoyl β-sultam
(4) which is similar to the ratio of their kOH values, 55. This
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
72

View Article Online
carbamoyl-β-sultam (13) displayed a small amount of inacti-
vation of the enzyme with an estimated ki value of 2 × 10Ϫ3 MϪ1
sϪ1 at pH 8.5. This is a reduction of approximately 10-fold
compared to the parent compound (4) lacking the 4-isopropyl-
idene substituent and is unexpected given the extremely low
reactivity of this compound and the fact that it reacts with
hydroxide ion at the acyl centre. It is not known whether (13)
acts as a sulfonylating or acylating agent of PPelastase.
4-isopropyl and N-benzyloxycarbonyl groups creates favour-
able interactions for active-site binding. If the two isopropyl
substituted compounds, 4-isopropyl-N-carbamoyl-β-sultam
(15) and the chemically more reactive 4 isopropyl-N-benzyloxy-
carbonyl-β-sultam (16) are compared, there is an increase in
EREF of 25-fold on going from the carbamoyl to the benzyl-
oxycarbonyl N-substituent. As discussed for the simple
N-benzyloxycarbonyl compound (5), this again suggests that
of all the N-substituents the benzyloxycarbonyl moiety binds
most favourably with PPelastase. This is possibly due to its
longer length permitting more favourable π–π interactions with
His-57 or another aromatic residue.
The introduction of the 4-isopropyl substituent has increased
the effectiveness of the lead compounds as inhibitors of PPe-
lastase. ESIMS analysis of 4-isopropyl-N-benzoyl-β-sultam
(14) incubated with PPelastase in non-buffered aqueous solu-
tion confirmed that only l : l enzyme : inhibitor (EI) complexes
were being formed, indicative of a good degree of selectivity.
Native PPelastase was observed at MW 25890
2 Da and
incubation with (14) gave a time dependent emergence of a
second peak consistent with the formation of a mono-sulfo-
nylated adduct (MW 26148 7 Da) up to a relative intensity of
80%. The observed mass difference of 258 is in good agreement
with a calculated one of 254, consistent with sulfonylation of
the enzyme. Only one equivalent of the inhibitor is observed to
bind, compatible with the formation of a 1 : 1 EI complex. The
conditions of the ionisation, 1 % formic acid, should result in
protein unfolding as a result of the multiple side chain positive
charges. As a result, bound complexes can probably be regarded
as covalent because EI complexes formed from reversible
inhibitors should dissociate under these conditions. The 4-iso-
propyl-N-benzoyl compound (14) has therefore irreversibly
sulfonylated the enzyme. However, the precise location of the
sulfonylation cannot be determined from these results. An
X-ray crystal structure or further MS analysis of tryptic digests
of the inactivated enzyme are required to determine whether or
not Ser-195 is indeed sulfonylated as was observed with the
simple N-benzoyl compound (3).16
The introduction of the isopropyl substituent at the 4-pos-
ition of the thiazetidine ring lowers the reactivity of β-sultams
towards hydroxide ion by 50-fold—compare (14) with (3) (Table
1) and maintains the mechanism of S–N fission. In addition,
unlike the 4-isopropylidene derivatives, the 4-isopropyl com-
pounds are effective inhibitors. The second-order rate constants
for inhibition obtained for 4-isopropyl-N-benzoyl-β-sultam
(14), 4-isopropyl-N-(N,N-methylphenyl)carbamoyl-β-sultam
(15) and 4-isopropyl-N-benzyloxycarbonyl-β-sultam (16) are
summarised in Table 1.
In an attempt to take advantage of the smaller S1 pocket
present in PPelastase the following compounds were tested
as inhibitors: (E)-4-ethylidene-N-benzoyl-β-sultam (17) and
4-ethyl N-(N,N-methylphenyl)carbamoyl-β-sultam (18). The
kinetic results obtained are given in Table 1.
Whilst the 4-isopropyl substituent has decreased the rate of
alkaline hydrolysis of N-benzoyl β-sultam (3) by 50-fold in (14),
enzyme inhibitory activity was only decreased by 3-fold. If the
EREF values are compared between the 4-isopropyl compound
(14) and the simple N-benzoyl compound (3) then an increase
of 20-fold is observed. This matches the increase in EREF of
30-fold observed between the 4-isopropyl-N-carbamoyl com-
pound (15) and the simple N-carbamoyl compound (4). There-
fore the 4-isopropyl substituent has increased the level of active
site binding and made the β-sultams more reactive towards
the enzyme in the presence of hydroxide ion. This is possibly a
result of favourable interactions between the alkyl substituent
and the hydrophobic S1 binding pocket.
The ki value of (E)-4-ethylidene-N-benzoyl-β-sultam (17) is
similar to that for the unsubstituted N-benzoyl-β-sultam (3)
despite a 5-fold decrease in reactivity towards hydroxide ion
catalysed hydrolysis. The EREF values for the two compounds
are 540 and 61 (Table 1) respectively. These values can also be
compared to the EREF value for the 4-isopropyl-N-benzoyl
compound (14) of 1.2 × 103 which is the least reactive of the
three N-benzoyl β-sultams.
This suggests that the 4-isopropyl substituent binds slightly
more favourably into the S1 pocket of PPelastase than the
4-(E)-ethylidene substituent in the N-benzoyl series. This is
surprising given the preference of the enzyme for alanine
residues and may be due to the restricted rotation resulting
from unsaturation.
The best inactivator in this series is 4-isopropyl-N-benzyloxy-
carbonyl-β-sultam (16) with an EREF of 2.3 × 104, 70-fold
greater than that of the N-benzyloxycarbonyl compound
lacking the 4-isopropyl substituent (5). The combination of the
The effectiveness of the ethyl substituent in increasing recog-
nition was observed in 4-ethyl-N-(N,N-methylphenylcarb-
amoyl)-β-sultam (18). This compound shows relatively low ki
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
73

View Article Online
values but is by far the least reactive β-sultam to display enzyme
inhibition, being 10-fold less reactive than the simple N-carb-
amoyl compound (4). It therefore has the second highest select-
ivity towards PPelastase in the presence of hydroxide ion with
an EREF of 3.8 × 103. This suggests that the additional methyl
group in the 4-isopropyl compounds is unfavourable, possibly
due to steric interactions in the active site.
In the N-carbamoyl series the presence of the alkyl substitu-
ent in the 4-position of the β-sultam ring increases the EREF
value by an overall factor of 100-fold. The 4-ethyl substituted
compound (18) is 4-fold more potent as an elastase inhibitor
than the 4-isopropyl compound (15). This improvement is
probably due to both the increased rotational mobility of the
4-ethyl substituent as a result of its lower bulk and improved
binding in the S1 active-site pocket. Based on a structural com-
parison to an alanine-containing peptide an N-methyl-β-sultam
may show even greater binding.
It is notable, despite differing N-substituents, that the EREF
value for the conformationally restricted 4-(E)-ethylidene-
N-benzoyl-β-sultam (17) is nearly 10-fold lower than for the
4-ethyl compound (18). This suggests that the binding of
β-sultams in the S1 pocket requires the compound to have a
degree of flexibility. This direct comparison has some justifica-
tion despite differing leaving groups because the relative ki
values of the simple N-benzoyl (3) and N-carbamoyl (4)
compounds was proportional to their reactivity, which may be
indicative of similar binding of the N-substituent.
The EREF value for the 4-ethyl compound (18) is still 6-fold
lower than that of 4-isopropyl-N-benzyloxycarbonyl-β-sultam
(16)which has the highest EREF value of the series presented
here. The inhibitor EREFs have been increased by ca. 400-fold
from that of the original lead compound, N-benzoyl-β-sultam
(3). This increase represents a combination of changes in chem-
ical reactivity and molecular recognition.
rupole mass spectrometer. 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.34a Starting materials were purchased from Aldrich,
Avocado and Lancaster and were used without further
purification.
1,2-Thiazetidine 1,1-dioxide (ethane ꢀ-sultam)
Taurinesulfonyl chloride (30.4 g, 169 mmol) was added to finely
ground sodium carbonate (35.9 g, 339 mmol) in ethyl acetate
(950 ml) and stirred at ambient temperature for 48 hours. The
reaction mixture was filtered through celite and the solvent
removed by reduced pressure rotary evaporation at 30 ЊC,
giving a fine white powder (15.9 g, 89%). Mp 50–51 ЊC (Lit. 51–
52 ЊC)35 1H NMR: δ (CDCl3 3.39 (2H, dt, J 4 and 7, CH2N),
4.32 (2H, dt, J 2 and 7, CH2SO2), 5.53 (1H, br s, NH). 13C
NMR: δ (CDCl3 60.6, 26.8. IR: νmax(cmϪ1): 3307, 3048, 3022,
2991, 2918, 1416, 1336, 1299, 1249, 1212, 1171, 1156, 1107, 966,
803, 760, 668, 615. m/z (GC-MS) (MϩH): 108, 77, 54, 42.
(1,1-Dioxo-1ꢀ6,2-thiazetidin-2-yl)(phenyl)methanone (3)
To a Ϫ5 ЊC stirred solution of 1,2-thiazetidine 1,1-dioxide
(0.6 g, 5.6 mmol) in dry DCM (20 ml) was added DMAP (0.11 g,
catalytic), and benzoyl chloride (1.18 ml, 10.2 mmol) dropwise.
Triethylamine (1.88 ml, 13.5 mmol) was added dropwise over 10
minutes forming a pale yellow solution. The reaction mixture
was stirred overnight before the addition of DCM (10 ml) and
was then washed with water (15 ml) and brine (2 × 15 ml) before
drying over Na2SO4. Reduced pressure rotary evaporation at
30 ЊC gave a pale orange solid (1.6 g) which was purified by
column chromatography (75 g silica) (6 : 4 diethyl ether : DCM,
Rf = .48) yielding a white powder (0.59 g, 49.9%). Mp 103–105
ЊC. 1H NMR: δ (CDCl3 8.13 (2H br d, Ph), 7.62 (1H, br tt, Ph),
7.53 (2H, br t, Ph), 4.3 (2H, t, J 7.74, CH2SO2), 3.92 (2H, t, J
7.7, CH2N). 13C NMR: δ (CDCl3) 167.4, 133.6, 132.1, 128.9,
128.4, 56.9, 30.9 IR; νmax(cmϪ1) 2997, 2924, 2854, 1670, 1448,
1337, 1197, 1165, 778, 715.9. HREI-MS [MϩH] for C9H9SO3N
calc. 212.0381 found 212.0385.
The structure of an enzyme : inhibitor complex formed
between a 3-ethyl-N-(N-benzyl)carbamoyl-β-lactam (19)com-
pound and HLE has been modelled.33 As expected the 4-ethyl
group lies in the S1 pocket but the β-lactam ring remains intact
and the resulting oxyanion formed by attack of Ser-195 at the
carbonyl is stabilised by the oxyanion hole. It is notable that the
binding of the benzyl urea group in the so-called hydrophobic
prime site (P1Ј) is improved by alkylation of the benzylic
carbon. This is perhaps a further possible modification to the
β-sultam structure although the hydrophobic pocket exploited
may not be present in PPelastase.
1,1-Dioxo-1ꢀ62-thiazetidine-2-carboxylic acid benzyl ester (5)
To a Ϫ5 ЊC stirred solution of 1,2-thiazetidine 1,1-dioxide
(0.6 g, 5.6 mmol) in dry DCM (20 ml) was added DMAP (0.11 g,
catalytic), and benzyl chloroformate (1.21 ml, 8.5 mmol) drop-
wise. Triethylamine (1.9 ml, 13.8 mmol) was added dropwise
over 10 minutes forming a pale yellow solution. The reaction
mixture was stirred overnight before the addition of DCM (10
ml) and was then washed with water (15 ml) and brine (2 × 15
ml) before drying over Na2SO4. Reduced pressure rotary evap-
oration at 30 ЊC gave a pale yellow oil/solid (1.1 g) which was
purified by column chromatography (50 g silica) (1 : 1 DCM :
diethyl ether, Rf = 0.6) yielding a white powder (0.23 g, 17%).
Mp 99–101 ЊC. 1H NMR: δ (CDCl3) 7.4 (5H, br, Ph), 5.3 (2H, s,
CH2Ph), 4.2 (2H, t, J 7.21, CH2SO2), 3.75 (2H, t, J 7.33, CH2N)
13C NMR: δ (CDCl3) 151, 134.7, 128.6, 128.5, 128, 68.8, 57.9,
32. IR: νmax (cmϪ1) 3046, 2984, 1732, 1456, 1391, 1326, 1204,
1173, 1135, 1056, 952, 911, 780, 767, 699. HREI-MS [MϩH]
for C10H11SO4N calc. 259.0753 found 259.0749.
In conclusion, N-acyl-β-sultams are a novel class of inhibitor
compounds that inactivate serine enzymes by irreversible sul-
fonylation of the active site serine residue. They are inactive
towards enzymes lacking active serine residues, indicating that
specific sulfonylation of active site residues takes place. Vari-
ation in 4-alkyl and N-substituted β-sultams causes differences
in the rates of inactivation by 4 orders of magnitude. Such
structure–activity relationships highlight the possibilities for
further development of this compound series. Improved
enzyme binding will allow the application of more stable, and
hence more selective, enzyme inhibitors.
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 spectro-
meter. 400 MHz 1H and 67 MHz 13C NMR spectra were deter-
mined on a Bruker Advance 400 MHz spectrometer. 500 MHz
1H and 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 was performed on a Fisons Quattro VG quad-
1,1-Dioxo-1ꢀ6,2-thiazetidine-2-carboxylic acid N-methyl-N-
phenylamide (4)
To a Ϫ5 ЊC stirred solution of 1,2-thiazetidine 1,1-dioxide
(0.6 g, 5.6 mmol) in dry DCM (20 ml) was added DMAP (0.11 g,
catalytic), and methyl(phenyl)carbamoyl chloride (1.43 g, 8.4
mmol) dropwise. Triethylamine (1.88 ml, 13.5 mmol) was added
dropwise over 10 minutes forming a pale yellow solution. The
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
74

View Article Online
reaction mixture was stirred overnight before the addition of
DCM (60 ml) and was then washed with water (3 × 20 ml) and
brine (2 × 20 ml) before drying over Na2SO4. Reduced pressure
rotary evaporation at 30 ЊC gave a pale yellow solid/oil (1.5 g)
which was purified by column chromatography (75 g silica)
(1 : DCM : diethyl ether, Rf = 0.55) yielding a white powder
(0.35 g, 26%). Mp 111–112 ЊC. 1H NMR: δ (CDCl3) 7.4 (5H, m,
Ph), 4.02 (2H, t, J 7.75, CH2SO2), 3.58 (2H, dd, J 6.63 and 7.72,
CH2N), 3.38 (3H, s CH3N). 13C NMR: δ (CDCl3) 152.6, 141.2,
129.6, 128.3, 128.1, 56.5, 39.3, 32.2. IR: νmax (cmϪ1) 2954.3,
2923.5, 2853.8, 1670.3, 1458.1, 1373.3, 1346.2, 1299.8, 1279,
1212.4, 1158, 1136. HREI-MS [MϩH] for C10H12SO3N2 calc.
241.0647 found 241.0650.
Na2SO4. Reduced pressure rotary evaporation at 30 ЊC gave a
pale yellow oil (0.4 g) which was purified by column chromo-
tography (25 g silica) (2 : 3 isohexane : ethyl acetate, Rf = 0.32)
yielding a white powder (77mg, 67%). Mp 66–68 ЊC. 1H NMR:
δ (CDCl3)5.40 (1H, br s, NH), 3.72 (2H, s, CH2N), 2.08 (3H, s,
CH3), 1.70 (3H, s, CH3). 13C NMR: δ (CDCl3)142.8, 140.8, 37.6,
21.4, 20.5. IR: νmax(cmϪ1) 3274.6, 1701.5, 1444.6, 1373.9,
1326.1, 1283.0, 1245.6, 1149.1, 1130.0, 1018.1, 940.7, 920.5,
869.1, 680.9.
(4-Isopropylidene-1,1-dioxo-1ꢀ6,2-thiazetidin-2-yl)phenylmeth-
anone (11)
To a Ϫ5 ЊC stirred solution of 4-isopropylidene-1,2-thiazetidine
1,1-dioxide (77 mg, 0.523 mmol) in dry DCM (6 ml) was added
DMAP (0.1 g, catalytic), and benzoyl chloride (0.091 ml, 0.78
mmol) dropwise. Triethylamine (0.18 ml, 1.26 mmol) was added
dropwise over 10 minutes forming a pale yellow solution. The
reaction mixture was stirred overnight before the addition of
DCM (10 ml) and was then washed with water (15 ml) and
brine (2 × 15 ml)before drying over Na2SO4. Reduced pressure
rotary evaporation at 30 ЊC gave a pale yellow oil (0.4 g) which
was purified by column chromotography (25 g silica) (3 : 2
isohexane : ethyl acetate, Rf = 0.36) yielding a white powder
2,4-Bis(tert-butyldimethylsilyl)-1,2-thiazetidine 1,1-dioxide
To a Ϫ78 ЊC stirred solution of 1,2-thiazetidine 1,1-dioxide
(2.5 g, 23.3 mmol) in dry THF (190 ml) was added n-BuLi (1.6
M, 33 ml, 52.8 mmol) dropwise over 20 minutes. The mixture
was stirred for 10 minutes before the addition of TBDMSCl
(17.2 g, 114 mmol) in THF (10 ml). After a period of 30
minutes, the reaction mixture was allowed to warm to ambient
over a further 30 minutes and was then quenched with glacial
acetic acid (3.9 g, 65 mmol) in THF (4 ml). The organics were
diluted with ethyl acetate (200 ml) and saturated NH4Cl (200
ml). The aqueous layer was extracted a further 2 times with
ethyl acetate (100 ml) before the combined organic extracts
were washed with water (2 × 100 ml), brine (2 × 100 ml) and
dried over anhydrous Na2SO4. Reduced pressure rotary evapor-
ation at 30 ЊC gave a pale yellow oil (11.1 g) which was purified
by column chromatography (400 g silica) (3:2 isohexane : ethyl
acetate, Rf = 0.6) yielding a pale cream solid (6.14 g, 78%). Mp
1
(93mg, 71%). Mp 82–84 ЊC. H NMR: δ (CDCl3) 8.01 (2H, d,
J7.5, Ph), 7.61 (1H, t, J 7.4, Ph),7.51 (2H, d, J 7.6, Ph), 4.32
(2H, s, CH2), 2.10 (3H, s, CH3), 1.86 (3H, s, CH3). 13C NMR:
δ (CDCl3) 168, 143.3, 140.8, 133.9, 133.2, 129.2, 128.9, 40.9,
22.1, 20.8. IR: νmax (cmϪ1) 2923.8, 2854, 1670.1, 1600.1, 1376.8,
1337.2, 1283.5, 1199.7, 1165.4, 1125.5, 1032.1, 778, 715.9.
HREI-MS [MϩH] for C12H13SO3N calc. 252.0694 found
252.0690.
1
89–91 ЊC. H NMR: δ (CDCl3) 4.20 (1H dd, J 9.0 and 5.5,
4-Isopropylidene-1,1-dioxo-1ꢀ6,2-thiazetidine-2-carboxylic acid
benzyl ester (12)
CHSO2), 3.45 (1H dd, J 9 and 4.5, N–CH2), 3.17 (1H dd, J 4.5
and 5.3, NCH2), 1.02 (9H, s, SiC(CH3)3), 0.96 (9H, s,
SiC(CH3)3), 0.33 (3H, s, SiCH3), 0.32 (3H, s, SiCH3), 0.31 (3H,
s, SiCH3), 0.25 (3H, s, SiCH3).
To a Ϫ10 ЊC stirred solution of 4-isopropylidene-1,2-thiazeti-
dine 1,1-dioxide (78.5 mg, 0.533 mmol) in dry DCM (6 ml) was
added DMAP (0.1 g, catalytic), and benzyl chloroformate (0.12
ml, 0.795 mmol) dropwise. Triethylamine (0.19 ml, 1.33 mmol)
was added dropwise over 10 minutes forming a pale yellow
solution. The reaction mixture was stirred overnight before the
addition of DCM (10 ml) and was then washed with water (15
ml) and brine (2 × 15 ml) before drying over Na2SO4. Reduced
pressure rotary evaporation at 30 ЊC gave a pale yellow oil (0.2
g) which was purified by column chromatography (25 g silica)
(3 : 2 isohexane : ethyl acetate, Rf = 0.32) yielding a white powder
2-(tert-Butyldimethylsilyl)-4-isopropylidene-1,2-thiazetidine
1,1-dioxide
To a Ϫ78 ЊC stirred solution of diisopropylamine (2.79 ml,
19.9 mmol) in dry THF (130 ml) was added n-BuLi (1.6 M,
12.25 ml, 19.6 mmol) dropwise. The reaction mixture was
stirred for 10 minutes before the addition of 2,4-bis(tert-butyl-
dimethylsilyl)-12,-thiazetidine 1,1-dioxide (3.28 g, 9.79 mmol)
in THF (15 ml) over 20 minutes. After stirring for a further 10
minutes, acetone (7.2 ml, 98.2 mmol) was added over 5 minutes
and the mixture left to stir for 50 minutes. The reaction was
quenched with saturated NH4Cl (220 ml) and then extracted
with ethyl acetate (200 ml ϩ (2 × 100 ml)) before the combined
organics were washed with water (2 × 100 ml), brine (2 × 100
ml) and dried over Na2SO4. Reduced pressure rotary evapor-
ation at 30 ЊC yielded a pale yellow oil (5.3 g) which was puri-
fied by cloumn chromatography (200 g silica) (7 : 3 isohexane :
ethyl acetate, Rf = 0.27) yielding a pale cream powder (0.5 g,
20%). Mp 100–101 ЊC. 1H NMR: δ (CDCl3) 3.78 (2H, s, CH2N),
2.05 (3H, s, CH3), 1.78 (3H, s, CH3), 0.98 (9H, s, SiC(CH3)3),
0.28 (6H, s, SiCH3). 13C NMR : δ (CDCl3) 142.5, 137.8, 40.7,
26.3, 21.2, 20.2, Ϫ0.55. IR: νmax (cmϪ1) 2965.2, 1672.7, 1672.7,
1449.6, 1314, 1282.1, 1162.8, 1037.4, 904.2, 841, 723.7, 647.8.
m/z (EI-MS): (MϩH) 261.8.
1
(108 mg, 73%. Mp 86–88 ЊC. H NMR:δ (CDCl3) 7.35 (5H, m
Ph), 5.28 (2H, s, CH2Ph), 4.10 (2H, s, CH2N), 2.09(3H, s, CH3),
1.80 (3H, s, CH3). 13C NMR: δ (CDCl3), 142.8, 138.1, 135.3,
129, 128.92, 128.9, 128.4, 69, 41.3, 22.1, 20.8. IR: νmax (cmϪ1)
1731.2, 1445.2, 1386.2, 1315.1, 1248.0, 1179.3, 1153.0, 699.8.
HREI-MS [MϩNH4] for C13H15SO3N calc. 299.1066 found
299.1069.
4-Isopropylidene-1,1-dioxo-1ꢀ6,2-thiazetidine-2-carboxylic acid
N-methyl-N-phenylamide (13)
To a Ϫ5 ЊC stirred solution of 4-isopropylidene-1,2-thiazetidine
1,1-dioxide (120 mg 0.816 mmol) in dry DCM (10 ml) was
added DMAP (0.1 g, catalytic) and N-methyl-N-phenylcarb-
amoyl chloride (0.21 g, 1.24 mmol) in THF (2 ml) dropwise.
Triethylamine (0.27 ml, 1.89 mmol) was added dropwise over 10
minutes forming a pale yellow solution. The reaction mixture
was stirred overnight before the addition of DCM (10 ml) and
was then washed with water (15 ml) and brine (2 × 15 ml) before
drying over Na2SO4. Reduced pressure rotary evaporation at
20 ЊC gave a pale yellow oil (0.45 g) which was purified by column
chromatography (25 g silica) (DCM 7 : 3 DCM : diethyl ether,
gradient elution) and preparative HPLC (water : ACN : MeOH,
gradient elution) yielding a white powder (96 mg, 42%). Mp
4-Isopropylidene-1,2-thiazetidine 1,1-dioxide
To a stirred solution of 2-(tert-buty1dimethylsilyl)-4-isopro-
pylidene-1,2-thiazetidine 1,1-dioxide (0.204 g, 0.78 mmol) in
THF (20 ml) was added TBAF (1 M, 0.78 ml, 78 mmol) in one
portion. The reaction mixture was stirred for 3 minutes before
quenching with saturated NaCl (30 ml), extracting with ethyl
acetate (15 ml) and then the combined organics were dried over
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
75

View Article Online
102–104 ЊC. 1H NMR: δ (CDCl3): 7.35 (5H, m, Ph), 4.08 (2H, s,
NCH2), 3.39 (3H, s, NCH3), 1.95 (3H, s, CH3), 1.75 (3H, s,
CH3). 13C NMR: δ (CDCl3) 153.4, 142, 141.6, 137.4, 129.4,
128.6, 128.5, 41.9, 39.8, 21.7, 20.6. IR: νmax(cmϪ1) 2945.8,
1670.0, 1490.4, 1442.2, 1390.9, 1315.1, 1250.0, 1201.0, 1161.6,
1128.1, 1066.4, 954.8, 883.7, 762.1, 672.1. HREI-MS [MϩH]
for C13H16SO3N2 calc. 281.0960 found 281.0965.
4-Isopropyl-1,1-dioxo-1ꢀ6,2-thiazetidine-2-carboxylic acid
benzyl ester (16)
To a Ϫ10 ЊC stirred solution of 4-isopropyl-1,2-thiazetidine 1,1-
dioxide (22.2mg, 0.149 mmol) in dry DCM (2.5 ml) was added
DMAP (0.02 g, catalytic) and benzyl chloroformate (0.04 ml,
0.265 mmol) dropwise. Triethylamine (0.05 ml, 0.35 mmol) was
added dropwise over 5 minutes forming a pale yellow solution,
The reaction mixture was stirred overnight before the addition
of DCM (3 ml) and was then washed with water (2.5 ml) and
brine (2 × 2.5 ml) before drying over Na2SO4. Reduced pressure
rotary evaporation at 30 ЊC gave a pale yellow oil (70 mg) which
was purified by column chromatography (8 g silica) (3 : 2
hexane : ethy1 acetate, Rf = 0.33) yielding a white powder (28.4
mg, 67%). Mp 93–94 ЊC. 1H NMR: δ (CDCl3) 7.35 (5H, m, Ph),
5.27 (2H, s, CH2Ph), 4.09 (1H, ddd, J 6.0, 8.35 and 11.02,
CHSO2), 3.75 (1H, dd, J 6.94 and 8.49, CH2N), 3.34 (1H, dd,
2-(tert-Butyldimethylsilyl)-4-isopropyl-1,2-thiazetidine
1,1-dioxide
Palladium (0.35 g, 10% on C,) in ethyl acetate (1 ml) was added
to a solution of 2-(tert-butyldimethylsilyl)-4-isopropylidene-
1,2-thiazetidine 1,1-dioxide (0.45 g, 1.71 mmol) in dry ethanol
(12 ml) under argon. The mixture was hydrogenated at 100 psi
H2for 24 hours before being filtered through a celite plug and
rotary evaporated at 30 ЊC, yielding a white powder (278 mg,
J 6.4, CH2N), 2.31 (1H, ds, J 6.68 and 11.05, CH(CH3)2), 1.19
1
62%). Mp 101–104 ЊC. H NMR: δ (CDCl3) 4.05 (1H, ddd,
13
(3H, d, J 6.42, CH3), 1.0 (3H, d, J 6.72, CH3).
C NMR:
J 6.67, 8.59 and 11.04, CHSO2), 3.29 (1H, dd, J 7.33 and 8.36,
CH2N), 2.87 (1H, dd, J 6.86 and 7.17, CH2N), 2.20 (1H, d
septet, J 6.55 and 10.97, CH (CH3)2), 1.07 (3H, d, J 6.43,
CH3CH), 0.86 (3H, d, J 6.8, CH3CH), 0.85 (9H, s, C(CH3)3),
0.16 (6H, s, Si(CH3)2). m/z (EI-MS) (MϩH): 262.
δ (CDCl3) 153.2, 128.9, 128.4, 128.7, 128.4, 79.1, 69.1, 38.7,
29.4, 21.6, 19.4. IR: νmax (cmϪ1) 1732.0, 1455.8, 1386.3, 1341.4,
1319.7, 1207.9, 1185.5, 1170.5, 1127.9, 787.7, 769.5. HREI-MS
[MϩNH4] for C13H18SO4N calc. 301.1222 found 301.1221.
4-Isopropyl-1,1-dioxo-1ꢀ6,2-thiazetidine-2-carboxylic acid
N-methyl-N-phenylamide (15)
4-Isopropyl-1,2-thiazetidine 1,1-dioxide
To a stirred solution of 2-(tert-butyldimethylsilyl)-4-isopropyl-
1,2-thiazetidine 1,1-dioxide (0.278 g, 0.107 mmol) in THF
(20 ml) was added TBAF (1 M, 1.7 ml, 1.7 mmol) in one portion,
The reaction mixture was stirred for 3.5 minutes before quench-
ing with a saturated aqueous NaCl solution (30 ml), extracting
with ethyl acetate (15 ml) and then the combined organics were
dried over Na2SO4. Reduced pressure rotary evaporation at
30 ЊC gave a pale yellow oil (0.8 g) which was purified by column
chromatography (35 g silica) (2 : 3 isohexane : ethyl acetate,
Rf = 0.35) yielding a white powder (123 mg, 77%). Mp 64–66 ЊC.
1H NMR: δ (CDCl3) 5.60 (1H, br s, NH), 4.22 (1H, ddd, J 6.79,
8.03 and 11.14, CHSO2), 3.44 (1H, m, CH2N), 3.03 (1H, dd,
J 6.38 and 11.75, CH2N), 2.32 (1H, ds, J 6.6 and 11.01,
CH(CH3)3), 1.16 (3H, d, J 6.63, CH3), 0.98 (3H, d, J 6.87, CH3).
13C NMR: δ (CDCl3) 81.8, 35.2, 29.4, 20.5, 19.4. IR: νmax (cmϪ1)
3268.7, 2969.1, 2898.6, 2872.2, 1467.2, 1366.0, 1332.9, 1292.3,
1271.1, 1239.2, 1211.8, 1153.9, 1147.6, 1126.7, 986.0, 910.4,
842.2, 733.0, 645.7.
To a Ϫ10Њ stirred solution of 4-isopropyl-1,2-thiazetidine 1,1-
dioxide (62.3mg 0.418 mmol) in dry DCM (5 ml) was added
DMAP (0.05 g, catalytic) and N-methyl-N-phenylcarbamoyl
chloride (0.11 g, 0.65 mmol) dropwise. Triethylamine (0.14 ml,
0.98 mmol) was added dropwise over 10 minutes forming a pale
purple solution. The reaction mixture was stirred overnight at
ambient temperature before the addition of DCM (10 ml) and
was then washed with water (12 ml) and brine (2 × 12 ml) before
drying over Na2SO4. Reduced pressure rotary evaporation at
30 ЊC gave a pale purple oil (0.12 g) which was purified by
column chromatography (10 g silica) (3 : 2 hexane : ethy1
acetate, Rf = 0.31) yielding a white powder (87 mg, 74%). Mp
1
101–103 ЊC. H NMR: δ (CDCl3) 7.4 (2H, d, J 7.9, Ph), 7.36
(1H, t, J 7.37, Ph), 7.29 (2H, d, J 7.24, Ph), 3.85 (1H, ddd,
J 6.67, 8.75 and 11.15, CHSO2), 3.59 (1H, dd, J 7.31 and 8.35,
CH2N), 3.36 (3H, s, NCH3), 3.31 (1H, dd, J 6.86, CH2N), 2.23
(1H, ds, J 6.55 and 11.03 CH (CH3)2), 1.06 (3H, d, J 6.43, CH3),
0.93(3H, d, J 6.67, CH3). 13C NMR: δ (CDCl3) 153.2, 141.9,
129.9, 128.6, 77.1, 39.7, 38.9, 29.5, 21.5, 19.2. IR: νmax (cmϪ1)
2965.0, 1668.7, 1594.1, 1494.0, 1419.3, 1367.7, 1333.4, 1299.8,
1183.1, 1152.9, 1009.2, 829.0, 772.5, 697.0. HREI-MS [MϩH]
for C13H18SO3N2 calc. 283.1116 found 283.1113.
(4-Isopropyl-1,1-dioxo-1ꢀ6,2-thiazetidin-2-yl)phenylmethanone
(14)
To a Ϫ10 ЊC stirred solution of 4-isopropyl-1,2-thiazetidine 1,1-
dioxide (117 mg 0.785 mmol) in dry DCM (15 ml) was added
DMAP (0.05 g, catalytic) and benzoyl chloride (0.14 ml, 1.18
mmol) dropwise. Triethylamine (0.27 ml, 1.89 mmol) was added
dropwise over 10 minutes forming a pale yellow solution. The
reaction mixture was stirred overnight before the addition of
DCM (5 ml) and was then washed with water (12 ml) and brine
(2 × 15 ml) before drying over Na2SO4. Reduced pressure rotary
evaporation at 30 ЊC gave a pale yellow oil (0.4 g) which was
purified by column chromatography (25 g silica) (chloroform
column, eluting with 3 : 2 hexane : ethyl acetate, Rf = 0.3) yield-
2-(tert-Butyldimethylsilyl)-4-(E/Z )-ethylidene-1,2-thiazetidine
1,1-dioxide
To a Ϫ78 ЊC stirred solution of diisopropylamine (4.5 ml, 32
mmol) in dry THF (170 ml) was added n-BuLi (1.6 M, 19.4 ml,
12.1 mmol) dropwise. The reaction mixture was stirred for 10
minutes before the addition of 2,4-bis(tert-butyldimethylsilyl)-
1,2-thiazetidine 1,1-dioxide (5.2 g, 15.5 mmol) in THF (20 ml)
over 20 minutes. After stirring for a further 10 minutes,
acetaldehyde (8.7 ml, 142 mmol) was added over 10 minutes
and the mixture left to stir for 1 hour, before allowing to warm
to ambient over 30 minutes. The reaction was quenched with
saturated NaCl (90 ml) before the addition of sodium bisulfite
(400 ml, 40%) and was then stirred for a further 1 hour. The
aqueous layer was extracted with ethyl acetate (2 × 200 ml)
before the combined organics were washed with water (100 ml),
brine (2 × 100 ml) and dried over Na2SO4. Reduced pressure
rotary evaporation at 30 ЊC yielded a pale yellow oil (5.2 g)
which was purified by column chromatography (200 g silica)
(7 : 3 isohexane : ethyl acetate, Rf = 0.38 and 0.36) yielding a
1
ing a white powder (144 mg, 72%). Mp 84–86 ЊC. H NMR:
δ (CDCl3) 7.9 (2H, d, J 7.5, Ph), 7.50 (1H, t, J 7.5, Ph), 7.40 (2H,
d, J 7.85, Ph), 4.06 (1H, ddd, J 6.34, 8.7 and 11.0 CHSO2), 3.84
(1H, dd, J 8.35 and 8.5, CH2N), 3.45 (1H, dd, J 6.4 and 8.0,
CH2N), 2.23 (1H, ds, J 6.6 and 11.2, CH(CH3)2), 1.09 (3H, d,
J 6.6, CH3), 0.93 (3H, d, J 6.7, CH3). 13C NMR: δ (CDCl3)
167.9, 133.9, 132.9, 129.2, 128.9. 37.9, 29.7, 21.6, 19.2. IR:
νmax(cmϪ1) 2961.0, 1665.4, 1581.7, 1468.1, 1449.6, 1391.8,
1327.6, 1289.4, 1209.6, 1181.7, 1164.4, 1028.6, 844.8, 795.7,
705.7, 657.7. HREI-MS [MϩNH4] for C12H15SO3N calc.
271.1116 found 271.1113.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
76

View Article Online
pale yellow oil as a mixture of the two isomers E and Z (1.9 g,
50%). 1H NMR: δ (CDCl3) 6.38 (1H, tq, J 2.34 and 7.21, CH),
3.86 (2H, dq, 1.36 and 2.7, CH2N), 1.8 (3H, dt, 1.36 and 7.2,
CH3), 0.98 (9H, s, SiC(CH3)3), 0.3 (6H, s, Si(CH3)2). 13C NMR:
δ (CDCl3) 149.2, 125.6, 65.3, 39.9, 25.9, 18.3, 13.6. IR: νmax
(cmϪ1) 3518.3, 2955.5, 2931, 2859.5, 1706.6, 1471.4, 1297.4,
1255.7, 1170.8, 1126.1, 1073.3, 1005.7, 905.6, 839.9, 825.4,
785.2, 686.2, 657.9. m/z (EI-MS) (M ϩ H) 248.
1255.7, 1170.8, 1126.1, 1073.3, 1005.7, 905.6, 839.9, 825.4,
785.2, 686.2, 657.9.
4-(E )-Ethylidene-1,2-thiazetidine 1,1-dioxide
To a stirred solution of 2-(tert-butyldimethylsilyl)-4-(E)-ethyli-
dene-1,2-thiazetidine 1,1-dioxide (0.4 g, 1.6 mmol) in THF
(20 ml) was added TBAF (1 M, 1.7 ml, 1.7 mmol) in one
portion. The reaction mixture was stirred for 3 minutes, before
quenching with saturated NaCl (40 ml), extracting with ethyl
acetate (2 × 25 ml), washing with brine (25 ml) and then dried
over Na2SO4. Reduced pressure rotary evaporation at 30 ЊC
gave a pale yellow oil (0.55 g) which was purified by column
chromatography (20 g silica) (2 : 3 isohexane : ethyl acetate,
Rf = 0.3) yielding a clear colourless oil (0.15 g, 71%). 1H NMR:
δ (CDCl3) 6.44 (0.5 H, qt, J 2.52 and 7.23, CH), 5.65 (1H, br s,
NH), 3.86 (1H, m, CH2N,), 1.8 (1.5 H, dt, 1.59 and 7.22, CH3).
13C NMR: δ (CDCl3) 149.1, 128.5, 36.8, 13.9. IR: νmax (cmϪ1)
328.4, 2957.2, 2931.1, 2860.7, 2797.0, 1703.4, 1467, 1377.1,
1304.4, 1166.9, 1084, 872.3, 728.2.
4-(E/Z )-Ethylidene-1,2-thiazetidine 1,1-dioxide
To a stirred solution of 2-(tert-butyldimethylsilyl)-4-(E/Z)ethyl-
idene-1,2-thiazetidine 1,1-dioxide (1.9 g, 7.7 mmol) in THF (80
ml) was added TBAF (1 M, 8.7 ml, 8.7 mmol) in one portion.
The reaction mixture was stirred for 3 minutes, before quench-
ing with saturated NaCl (100 ml), extracting with ethyl acetate
(2 × 50 ml), washing with brine (50 ml) and then dried over
Na2SO4. Reduced pressure rotary evaporation at 30 ЊC gave a
pale yellow oil (2.5 g) which was purified by column chromato-
graphy (100 g silica) (2 : 3 isohexane : ethyl acetate, Rf = 0.3)
yielding a clear colourless oil (0.92 g, 90%). 1H NMR:
δ (CDCl3) 6.45 (0.5 H, qt, J 2.51 and 7.23, CH, E), 6.0 (0.5 H,
qt, J 2.01 and 7.26, CH, Z), 5.66 (1H, br s NH), 3.86 (1H, m,
CH2N, E),3.83 (1H, m, CH2N, Z), 2.03 (1.5 H, dt, 2.01 and
7.24, CH3, Z), 1.8 (1.5 H, dt, 1.58 and 7.23, CH3, E). 13C
NMR: δ (CDCl3) 149.148.9, 129.1, 128.4, 37.6, 36.8, 15.1, 13.9
(Z isomer in bold). IR: νmax (cmϪ1) 3287.6, 2957.3, 2931.6,
2862.1, 2797.1, 1703.5, 1467.1, 1377.2, 1304.4, 1167.5, 1084.8,
944.5, 872.3, 728.
[4-(E/Z )-Ethylidene-1,1-dioxo-1ꢀ6,2-thiazetidin-2-yl](phenyl)-
methanone (17)
To a Ϫ10 ЊC stirred solution of 4-(E/Z)-ethylidene-1,2-thiaze-
tidine 1,1-dioxide (0.32 g 2.4 mmol) in dry DCM (35 ml) was
added DMAP (0.1 g, catalytic) and benzoyl chloride (0.63 g, 3.7
mmol) dropwise. Triethylamine (0.85 ml, 6.1 mmol) was added
dropwise over 10 minutes forming a pale yellow solution. The
reaction mixture was stirred overnight at ambient temperature
before the addition of DCM (25 ml) and was then washed with
water (25 ml) and brine (2 × 25 ml) before drying over Na2SO4.
Reduced pressure rotary evaporation at 30 ЊC gave a pale yellow
oil (1 g) which was purified by column chromatography (35 g
silica) (3 : 2 hexane : ethyl acetate, Rf = 0.28 and 0.22) yielding
two fractions of white powders, E and Z isomers respectively
[120 mg (E). Mp 92–94 ЊC] and [160 mg (Z). Mp 63–65 ЊC].
Overall 49% yield. E Isomer: 1H NMR: δ (CDCl3) 8.03 (2H, m,
Ph), 7.62 (1H, m, Ph), 7.52 (2H, m, Ph), 6.65 (1H, qt, J 2.54 and
7.22 CH), 4.43 (2H, dq, 1.62 and 2.53, CH2N), 1.93 (3H, dt,
J 1.65 and 7.25, CH3). 13C NMR: δ (CDCl3) 167.5, 143.8, 133.6,
132.6, 130.7, 128.9, 128.4, 40.1, 14.4. IR: νmax (cmϪ1) 2954.7,
2924.2, 2854, 1670.4, 1464.5, 1449.3, 1376.9, 1334.4, 1311.8,
1267.1, 1187, 1158.1, 946.6. HREI-MS [MϩNH4] for
C11H11SO3N calc. 255.0803 found 255.0799.
4-Ethyl-1,2-thiazetidine 1,1-dioxide
Palladium (0.3 g, 10% on C) in ethyl acetate (1 ml) was added to
a solution of 4-(E/Z)-ethylidene-1,2-thiazetidine 1,1-dioxide
(0.32 g, 2.4 mmol) in dry ethanol (25 ml) under argon. The
mixture was hydrogenated for 72 hours before being filtered
through a celite plug and rotary evaporated at 30 ЊC, yielding a
pale yellow oil (98 mg, 30%).
1H NMR: δ (CDCl3) 5.8 (1H, br s, NH), 4.4 (1H, m, CHSO2),
3.42 (1H, dd, J 6.27 and 8.05, CH2N), 2.94 (1H, dd, J 6.12 and
6.12, CH2N), 2.09 (1H, m, CH2CH3), 1.88 (1H, m, CH2CH3),
1.04 (3H, t, J 7.39, CH3). 13 C NMR: δ (CDCl3) 75.7, 35.2, 22.3,
11.1. IR: νmax (cmϪ1) 3583.8, 3291.7, 2971.7, 2880.4, 1637,
1461.4, 1383.6, 1307, 1241.2, 1196.6, 1161.4, 1040, 978.6, 850.6,
701.3.
Z Isomer: 1H NMR: δ (CDCl3) 8.03 (2H, m, Ph), 7.65 (1 H,
m, Ph), 7.55 (2H, m, Ph), 6.27 (1H, qt, J 2.02 and 7.24, CH),
4.39 (2H, dq, 2.1 and 2.03, CH2N), 2.15 (3H, dt, J 2.14 and
7.26, CH3). 13C NMR: 167.5, 143.2, 133.6, 132.6, 131.5, 128.9,
128.4, 40.9, 159. IR: νmax (cmϪ1) 3060.2, 2962.6, 1679.9, 1599.8,
1581.5, 1451.8, 1425.6, 1378.5, 1324.9 1256.2, 1216.6, 1162.3,
1136.2, 1126.7, 1028, 1017.6, 992.1, 840.8, 755.8, 710.9, 691.6,
635.4. HREI-MS [MϩNH4] for C11H11SO3N calc. 255.0803
found 255.0801.
2-(tert-Butyldimethylsilyl)-4-(E )-ethylidene-1,2-thiazetidine
1,1-dioxide
To a Ϫ78 ЊC stirred solution of diisopropylamine (4.28 ml, 30.5
mmol) in dry THF (180 ml) was added n-BuLi (1.6 M,19 ml,
30.4 mmol) dropwise. The reaction mixture was stirred for 10
minutes before the addition of 2,4-bis(tert-butyldimethylsilyl)-
1,2-thiazetidine 1,1-dioxide (5 g, 14.9 mmol) in THF (20 ml)
over 20 minutes. After stirring for a further 10 minutes,
acetaldehyde (8.3 ml, mmol) was added over 10 minutes and the
mixture left to stir for 1 hour before quenching with glacial
acetic acid (15 ml) and stiring for a further 30 minutes. The
aqueous layer was extracted with ethyl acetate (2 × 100 ml)
before the combined organics were washed with brine (100 ml)
and dried over Na2SO4. Reduced pressure rotary evaporation at
30 ЊC yielded a pale yellow oil (8.5 g) which was purified by
column chromatography (200 g silica) (7 : 3 6 : 4 isohexane :
ethyl acetate, Rf = 0.38) yielding a pale yellow oil as the product
(0.4 g, 10.9%) and a second fraction determined to be starting
material (Rf = 0.65, 3.8 g). Yield based upon total conversion,
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 Spectra-
kinetic monochromator (Applied Photophysics, Leatherhead,
England) equipped with an absorbance photomultiplier. The
reagent syringes were thermostatted with a Grant thermo-
statted water circulator. pH-stat experiments were performed
1
45%. H NMR: δ (CDCl3) 6.38 (1H, tq, J 2.34 and 7.21, CH),
3.86 (2H, dq, 1.36 and 2.7, CH2N), 1.8 (3H, dt, 1.36 and 7.2,
CH3), 0.98 (9H, s, SiC(CH3)3), 0.3 (6H, s, Si(CH3)2). 13C NMR:
δ (CDCl3) 149.2, 125.6, 65.3, 39.9, 25.9, 18.3, 13.6. IR: νmax
(cmϪ1) 3518.3, 2955.5, 2931, 2859.5, 1706.6, 1471.4, 1297.4,
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
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
77

View Article Online
circulator. pH was measured by a pHG200–8 Glass pH
electrode and a REF200 ‘Red Rod’ reference electrode (Radio-
meter). 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
(Russel, Fife, Scotland and Beckman respectively). A calibra-
tion of the pH meter was carried out at 30 ЊC 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).
from exponential plots of absorbance against time were
obtained using the supplied fitting software (Applied
Photophysics).
For reactions studied by pH-stat standardised 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 pro-
gram (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 standardised 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 deionised water were used
throughout. 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 phenol-
phthalein as an indicator.
Organic solvents were glass distilled prior to use and stored
under nitrogen. For solution pHs ≥3 and ≤11 the pH was con-
trolled 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-amino-
propanesulfonic acid (TAPS) (pKa 8.4), 3-(cyclohexylamino)-
2-hydroxypropane-1-sulfonic acid (CAPSO) (pKa 9.6), and
3-(cyclohexylamino)propane-1-sulfonic acid (CAPS) (pKa 10.4).
For general pH work, buffers were prepared by partial neutral-
isation of solutions of their sodium salts to the required pH.
For the alcoholysis reactions, buffers were prepared by the addi-
tion of 0.25, 0.50 or 0.75 aliquots of 1 M NaOH to solutions of
the alcohol. In all experiments temperatures were maintained at
30 ЊC and ionic strength at 1.0 M with AnalaR grade KCl
unless otherwise stated. Reaction concentrations were generally
within the range ≥2×10Ϫ5 M to ≤2 × 10Ϫ4M to ensure pseudo-
first order conditions.
Hydroxide ion concentrations were calculated using pKw
(H2O) = 13.883 at 30 ЊC.34b Reactions studied by UV spectro-
photometry were usually commenced by injections of
acetonitrile or dioxan stock solutions of the substrate (5–50 µl)
into the cells containing pre-incubated buffer (2.5 ml). Final
reaction cells contained ≤5% acetonitrile or dioxan v/v. The pH
of the reaction cells was measured before and after each kinetic
run at 30 ЊC, 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 correspond-
ing 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 soft-
ware (Varian). pH-rate profiles were modelled to theoretical
equations using the Scientist program (V2.02, Micromath
Software Ltd, USA).
Enzyme inhibition studies
Assays were performed at 30 ЊC in 0.1 M pH 8.5 TAPS buffer
with I = 1.0 M (KCl). The substrate used was N-suc-(-Ala)3-p-
nitroanilide (2) and stock solutions were made up in distilled
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 ×
10Ϫ5 M was used resulting in a constant methanol concen-
tration of 1.5% v/v. Incubations of enzyme and inhibitor (500
µl) were carried out at 30 ЊC in 0.04 M buffer, 20% MeCN v/v,
8 × 10Ϫ5 M PPelastase and up to 5 × 10Ϫ3 M β-sultam. These
conditions were created in a separate UV cell containing
stock PPelastase suspension (200 µl), 0.1 M buffer (200 µ) and
inhibitor stock solution in MeCN or MeCN alone (100 µ).
Aliquots of this solution (50 µl) were assayed for PPelastase
activity at various time intervals by injection into 0.1 M pH 8.5
TAPS (2.36 ml) containing MeCN (150 µl) and substrate
solution (40 µl). This gave final assay conditions of 6.0 × 10Ϫ5
M substrate, 1.5 × 10Ϫ6 M PPelastase, 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 β-sultam solution
but then matched the remaining methodology in all respects.
Initial rates were monitored over up to eight minutes and the
gradients of the slopes obtained used as a measure of enzyme
activity. Gradients from inhibitor runs were normalised with
respect to the first order exponential decrease in β-sultam
concentration due to hydroxide ion catalysed hydrolysis at the
pH of the incubation. This was done by dividing the observed
gradient by the β-sultam concentration at the start of the
experiments, time = 0, and multiplying by the β-sultam concen-
tration at the time of the assay, time = t:
Normalised gradient at time
[Sultam]o) × [Sultam]t
The average control gradient was taken as 100% activity.
This value and the normalised gradients were used to calculate
% activity:
t = (observed gradient/
Reactions studied by stopped flow UV spectrophotometry
used stock solutions prepared at twice the standard UV concen-
tration 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 ЊC before pneumatic injection into the reaction into the reac-
tion 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 deionised
water and absorbance wavelengths common to the standard
UV experiments were used. Pseudo-first order rate constants
% Activity = (Normalised gradient/Average control gradient)
× 100
Pseudo-first order rate constants of inactivation, Kobs values,
were obtained from plots of % activity against time using the
Enzfitter program (Elsevier Biosoft). The second order rate
constants of inactivation, Ki values, were obtained by dividing
the first order Kobs values by the concentration of β-sultam in
the inactivation.
pKa determinations
A solution of the compound was placed in a UV cell at 30 ЊC
and typically titrated by means of additions of NaOH or HCl
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
78

View Article Online
(≤50 µl). Absorbance values at the wavelength of maximum
change and the cell solution pH were measured after each
injection of the 0.01, 0.05, 0.1 or 1.0 M standardised NaOH or
HCl. Near the end point injections of the 0.01 M solution (5 µl)
were made to ensure that sufficient pH values were covered in
the proximity of the pKa.
Acknowledgements
For CASE awards we thank the EPSRC and AstraZeneca
(JMW) and British Biotech (PSH). We also thank C J Schofield
and R C Wilmouth for the X-ray crystal structure.
The results were analysed using the Enzfitter software
package (Elsevier Biosoft), pKa1 determination equation, A =
References
Ϫ
Ϫ
[(Amin ϩ Amax) × (10(pH pKa))]/[(10(pH pKa)) ϩ 1] where A =
absorbance of solution at a particular pH and wavelength,
Amin = minimum absorbance where 100% of dissociating species
is in form with lowest absorption, Amax = maximum absorbance
where 100% of dissociating species is in form with highest
absorption, pH = solution pH and pKa = pKa of dissociating
species.
1 M. I. Page, Enzyme Inhibition in Comprehensive Medicinal
Chemistry, ed. P. G. Sammes, Pergamon Oxford, 1990, Vol. 2,
pp. 61–87.
2 (a) C. W. Wharton, The Serine Proteinases in Comprehensive
Biological Catalysis, ed. M. Sinnott, Academic Press, London, 1997,
Vol. 1, Ch. 11, pp. 345–379; (b) D. J. Underwood, B. G. Green,
R. Chabin, S. Mills, J. B. Doherty, P. E. Finke, M. MacCoss,
S. K. Shah, C. S. Burgey, T. A. Dickinson, P. R. Griffin, T. E. Lee,
K. M. Swiderek, T. Covey, W. M. Westler and W, B. Knight,
Biochemistry, 1995, 34, 14344–14355; (c) J. A. Katzenellenbogen,
R. Rai and W. Dai W, Bioorg. Med. Chem. Lett. 2, 1992, 11, 1399–
1404; (d ) W. B. Knight, A. L. Maycock, B. G. Green, B. M, Ashe,
P. Gale, H. Weston, P. E. Finke, W. K. Hagmann, S. K. Shah and
J. B. Doherty, Biochemistry, 1992, 31, 4980–4986; (e) C-M Kam,
K. Fujikawa and J. C. Powers, Biochemistry, 1988, 27, 2547–2557;
( f ) M. A. Navia, J. P. Springer, T. Y. Lin, H. R. Williams,
R. A. Firestone, J. M. Pisano, J. B. Doherty, P. E. Finke and
K. Hoogsteen, Nature, 1982, 327, 79–82; (g) M. H. Gelb and
R. H. Abeles, J. Med. Chem., 1986, 29, 585–589; (h) M. H. Gelb and
R. H. Abeles, Biochemistry, 1984, 23, 6569–6604.
1H-NMR experiments
The β-sultam (15 mg) was dissolved in 50 : 50 v/v CD3CN : D2O
1
(ca. 3 ml) and the H-NMR spectra acquired at 400 MHz and
300 K. One drop additions of 4% v/v NaOD in D2O were made
to the NMR tube until complete conversion of reactants had
occurred with spectra being acquired after each addition. In
buffer solution, the β-sultam (15 mg) was dissolved in 50 : 50
v/v CD3CN : 0.4 M buffer solution in D2O (ca. 3 ml), the pD of
which was measured before and after the experiment. The
solutions were also routinely submitted for both positive and
negative mode ESIMS-MS.
3 W. A. Metz and N. P. Peet, Prog. Inflammation Res., 1999, 9,
853–868.
4 (a) W, Bode, A. Z. Wei, R. Huber, E. Meyer, J. Travis and
S. Neuman, EMBO J., 1986, 5, 2453–2458; (b) M. A. Navia,
B. M. McKeever, J. P. Springer, T. Y. Lin, H. R. Williams,
E. M. Fluder, C. P. Dorn and K. Hoogsteen, Proc. Natl. Acad. Sci.
USA, 1989, 86, 7–11.
5 W. Bode, E. Meyer Jr. and J. C. Powers, Biochemistry, 1989, 28,
1951–1962.
6 (a) L. H. Takahashi, R. Radhakrishan, R. E. Rosenfeld Jr.,
E. F. Meyer Jr. and D. A. Trainer, J. Am. Chem. Soc., 1989, 111,
3368–3374; (b) A. Renaud, P. Lestienne, D. L. Hughes, J. G. Bieth
and J-L. Dimicoli, J. Biol. Chem., 1983, 258, 8312–8316.
X-Ray crystal preparation, data collection, processing and
structure refinement
PPE crystals were prepared as previously described.36 A satur-
ated solution of the N-benzoyl β-sultam (3) in sodium acetate
buffer (pH 5.0, 50 mM), sodium sulfate (25 mM) and DMSO
(10% v/v) was prepared. The mother liquor containing the
crystals was exchanged with this solution over a 1 h period.
After 24 h soaking in the inhibitor solution, the crystals were
placed in a cryoprotectant solution containing 20% (v/v)
glycerol and flash-cooled in liquid nitrogen.
Data were collected at 100 K using a Rigaku rotating anode
generator (CuKα radiation) and 345 mm Mar Research image
plate detector to a resolution limit of 1.67 Å. The crystal
belonged to the space group P212121 with a = 50.77 Å,
b = 57.61 Å, c = 74.48 Å. Raw data were processed with the
programs MOSFLM and SCALA (CCP4 suite.37 Rmerge was
4.3% for all data (19.3% in the highest resolution shell,
1.76–1.67 Å). Data were 99.3% complete (96.6% in the highest
resolution shell) with 92 216 reflections of which 25 847 were
unique after reduction. The overall I/σ1 was 20.1 and 4.8 in the
highest resolution shell (1.68–1.67 Å).
The PPE:BCM7 structure (excluding the inhibitor) (PDB
entry 1QIX) was used the starting model. The structure was
refined using REFMAC (CCP4 suite) with a total of 4% of the
reflections in the entire dataset randomly selected in order
provide a test set for the Rfree calculations. Models and electron
density maps were displayed using the program O.38 At the
stage in refinement with the Fo Ϫ Fc map showed clear and
unambiguous density for the β-sultam derived atoms, they were
included in the model and an appropriate PROTIN dictionary
file created. The sulfonate ester bond was simulated solely with
bond length restraints. Only the six atoms derived from the
β-sultam ring were visible in the structure with the benzoyl
group apparently disordered or missing. The side chains of
three residues of PPE (Arg-61, Arg-125, Arg-223) were also
disordered and were included as alanines in the model. During
the latter stages of refinement, 141 water molecules were added
along with the bound calcium and sulfate ions,36 giving a final
R-factor of 19.0% and free R-factor of 21.3%. The average
B-factor for the atoms in the β-sultam inhibitor molecule was
20.3 Å2.
7 (a) P. D. Edwards, E. F. Meyer Jr., J. Vijayalakshmi, P. A. Tuthill,
D. A. Andisk, B. Gomes and A. Strimpler, J Am. Chem. Soc., 1992,
114, 1854–1863; (b) M. J. Costanzo, B. E. Maryanoff, L. R. Hecker,
M. R. Schott, S. C. Yabut, H-C Zhang, P. Andrade-Gordon,
J. A. Kauffman, J. M. Lewis, R. Krishnan and A. Tulinsky, J. Med.
Chem., 1996, 39, 3039–3043; (c) P. R. Bernstein, B. C. Gomes,
B. J. Kosmider, E. P. Vacek and J. C. Williams, J. Med. Chem., 1995,
38, 212–215; (d ) P. D. Edwards, D. W. Andisik, A. M. Strimpler,
B. C. Gomes and P. A. Tuthill, J. Med. Chem., 1996, 39, 1112–1124;
(e) M. R. Angelastro, P. Bey, J. P. Burhart, T-M Chen, R. J. Cregge,
S. L. Durham, R. A. Farr, S. L. Gallion, C. M. Hare,
R. V. Hoffmann, E. W. Huber, M. L. Janusz, H-O. Kim, J. R. Koehl,
A. L. Marquart, S. Mehdi, W. A. Metz, N. P. Peet, H. A. Schreuder,
S. Sunder and C. Tardif, J. Med. Chem., 1998, 41, 2461–2480.
8 (a) S. J. F. Macdonald, D. B. Belton, D. M. Buckley, J. E. Spooner,
M. S. Anson, L. A. Harrison, K. Mills, R. J. Upton, M. D. Dowle,
R. A. Smith, C. Molloy and C. Risley, J. Med. Chem., 1998, 41,
3919–3922; (b) S. J. F. Macdonald, M. D. Dowle, L. A. Harrison,
J. E. Spooner, P. Shah, M. R. Johnson, G. G. A. Inglis,
G. D. E. Clarke, D. J. Belton, R. A. Smith, C. R. Molloy, M. Dixon,
G. Murkitt, R. E. Godward, T. Skarzynski, O. M. P. Singh,
K. A. Kumar, S. T. Hodgson, E. McDonald, G. W. Hardy, H. Finch,
G. Fleetwood and D. C. Humphreys, Bioorg. Med. Chem. Lett.,
2001, 11, 243–6; (c) S. J. F. Macdonald, M. D. Dowle,
L. A. Harrison, P. Shah, M. R. Johnson, G. G. A. Inglis,
G. D. E. Clarke, R. A. Smith, D. Humphreys, C. R. Molloy,
A. Amour, M. Dixon, G. Murkitt, R. E. Godward, T. Padfield,
T. Skarzynski, O. M. P. Singh, K. A. Kumar, G. Fleetwood,
S. T. Hodgson, G. W. Hardy and H. Finch, Bioorg. Med. Chem.
Lett., 2001, 11, 895–8.
9 The Chemistry of β-Lactam Antibiotics, ed. M. I. Page, Blackie,
Glasgow, 1992.
10 (a) R. Chabin, B. G. Green, P. Gale, A. L. Maycock, H. Weston,
C. P. Dorn, P. E. Finke, W. K. Hagmann, J. J. Hale, M. MacCross,
S. K. Shah, D. J. Underwood, J. B. Doherty and W. B. Knight,
Biochemistry, 1993, 32, 8970; (b) C. Beauve, M. Bouchet,
R. Touillaux, J. Fastrez and J- Marchand-Brynaert, Tetrahedron,
1999, 55, 13301.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
79

View Article Online
11 D. J. Underwood, B. G. Green, R. Chabin, S. Mills, J. B. Doherty,
P. E. Finke, M. MacCoss, S. K. Shah, C. S. Burgey, T. A. Dickinson,
P. R. Griffin, T. E. Lee, K. M. Swiderek, T. Covey, W. M. Westler
and W. B. Knight, Biochemistry, 1995, 34, 143344.
12 R. C. Wilmouth, S. Kassamally, N. J. Westwood, R. J. Sheppard,
T. D. W. Claridge, R. T. Aplin, P. A. Wright, G. J. Pritchard and
C. J. Schofield, Biochemistry, 1999, 38, 7989.
13 (a) J. F. King, R. Rathore, J. Y. L. Lam, L. E. R. Gao and
D. F. Klassen, J. Am. Chem. Soc., 1992, 114, 3028; (b) J. M. Wood
and M. I. Page, Trends Heterocycl. Chem., 2002, 8, 19.
14 N. J. Baxter, A. P. Laws, L. J. M. Rigoreau and M. I. Page, J. Am.
Chem. Soc., 2000, 112, 3375.
15 N. J. Baxter, A. P. Laws, L. J. M. Rigoreau and M. I. Page, J. Chem.
Soc., Perkin Trans. 2, 1996, 2245.
16 M. Beardsell, P. S. Hinchliffe, J. M. Wood, R. C. Wilmouth,
C. J. Schofield and M. I. Page, β-Sultams – a novel class of serine
protease inhibitors, Chem. Commun., 2001, 497–498.
17 M. I. Page, Enzyme Catalysis in Comprehensive Medicinal
Chemistry, 1990, Vol. 2, ed. P. G. Sammes, Pergamon, Oxford,
pp. 45–60.
18 (a) A. P. Laws, and M. I. Page, J. Chem. Soc., Perkin Trans. 2, 1989,
1577–1581; (b) A. P. Laws, N. J. Layland, D. G. Proctor and
M. I. Page, J. Chem. Soc., Perkin Trans 2., 1993, 17–21;
(c) N. O. Sykes, S. J. F. McDonald and M. I. Page, J. Med. Chem.,
2002, 45, 2850.
24 A. M. Gold and D. E. Fahrney, Biochemistry, 1964, 3, 783.
25 R. A. Firestone, P. L. Barker, J. M. Pisano, B. M. Ashe and
M. E. Dahlgren, Tetrahedron, 1990, 46, 2255.
26 W. B. Knight, A. L. Maycock, B. G. Green, B. M. Ashe, P. Gale,
H. Weston, P. E. Finke, W. K. Hagmann, S. K. Shah, D. Underwood
and J. B. Doherty, Biochemistry, 1992, 31, 4980.
27 (a) M. O. Lively and J. C. Powers, Biochim. Biophys. Acta, 1978, 525,
171; (b) T. Yoshimura, L. N. Barker and J. C. Powers, J. Biol. Chem.,
1982, 257, 5077; (c) D. E. Fahrney and A. M. Gold, J. Am. Chem.
Soc., 1963, 85, 997.
28 (a) O. R. Zaborsky and E. T. Kaiser, J. Am. Chem. Soc., 1970, 92,
860; (b) E. T. Kaiser, Intra-Sci. Chem. Rep., 1972, 6, 5.
29 J. L. Kice, Adv. Phys. Org. Chem., 1980, 17, 65.
30 R. C. Wilmouth, N. J. Westwood, K. Anderson, W. Brownlee,
T. D. W. Claridge, I. J. Clifton, G. J. Pritchard, R. T. Aplin and
C. J. Schofield, Biochemistry, 1998, 37, 17506.
31 R. A. Firestone, P. L. Barker, J. M. Pisano, B. M. Ashe and
M. Dahlgren, Tetrahedron, 1990, 46, 2255.
32 P. S. Hinchcliffe, J. M. Wood, A. M. Davis, R. P. Austin,
R. P. Beckett and M. I. Page, J. Chem. Soc., Perkin Trans. 2, 2001,
1503.
33 P. E. Finke, S. K. Shah, D. S. Fletcher, B. M. Ashe, K. A. Brause,
G. O. Chandler, P. S. Dellea, K. M. Hand, A. L. Maycock,
D. G. Osinga, D. J. Underwood, H. Weston, P. Davies and
J. B. Doherty, J. Med. Chem., 1995, 38, 2449.
19 J. Bieth, B. Spiess and C. G. Wermuth, Biochem. Med., 1974, 11, 350.
20 I. B. Blagoeva, A. J. Kirby, I. I. Kochiyashki, A. H. Koedjikov,
I. G. Pojarlieff and M. M. Toteva, J. Chem. Soc., Perkin Trans. 2,
2000, 1953.
21 A. R. Fersht, Enzyme Structure, Mechanism, W. H. Freeman, USA,
2nd edn., 1985, p. 405.
34 (a) D. D. Perrin and W. L. F. Armarego, Purification of Laboratory
Chemicals, 3rd edn., Pergamon, Oxford, 1988; (b) Lange’s Handbook
of Chemistry, ed. J. A. Dean, McGraw-Hill, 11th edn., 1973, pp. 5–7.
35 A. Champseix, J. Chanet-Ray, A. Etienne, A. Le Berre, J. Masson,
C. Napierale and R. Vessiere, Bull. Soc. Chim. Fr., 1985, 483.
36 E. Meyer, G. Cole, R. Radhakrishnan and O. Epp, Acta.
Crystallogr., Sect. B, 1985, B44, 26–38.
37 S. Bailey, Acta Crystallogr., Sect D, 1994, D50, 760–3.
38 A. T. Jones, J. Y. Zou, S. W. Cowan and M. Kjeldgaard, Acta
Crystallogr., Sect A., 1991, A47, 110–9.
22 R. C. Wilmouth, S. Kassamally, N. J. Westwood, R. J. Sheppard,
T. D. W. Claridge, R. T. Aplin, P. A. Wright, G. J. Pritchard and
C. J. Schofield, Biochemistry, 1999, 38, 7989.
23 M. J. Slater, A. P. Laws and M. I. Page, Bioorg. Chem., 2001, 29, 7795.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 6 7 – 8 0
80