The aminolysis of N-aroyl β-lactams occurs by a concerted mechanism

Article abstract of DOI:10.1039/b616420j

N-Aroyl β-lactams are imides with exo- and endocyclic acyl centres which react with amines in aqueous solution to give the ring opened β-lactam aminolysis product. Unlike the strongly base catalysed aminolysis of β-lactam antiobiotics, such as penicillins

Full text of DOI:10.1039/b616420j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
The aminolysis of N-aroyl b-lactams occurs by a concerted mechanism
Wing Y. Tsang, Naveed Ahmed and Michael I. Page*
Received 9th November 2006, Accepted 30th November 2006
First published as an Advance Article on the web 21st December 2006
DOI: 10.1039/b616420j
N-Aroyl b-lactams are imides with exo- and endocyclic acyl centres which react with amines in aqueous
solution to give the ring opened b-lactam aminolysis product. Unlike the strongly base catalysed
aminolysis of b-lactam antiobiotics, such as penicillins and cephaloridines, the rate law for the
aminolysis of N-aroyl b-lactams is dominated by a term with a first-order dependence on amine
concentration in its free base form, indicative of an uncatalysed aminolysis reaction. The second-order
rate constants for this uncatalysed aminolysis of N-p-methoxybenzoyl b-lactam with a series of
substituted amines generates a Brønsted bnuc value of +0.90. This is indicative of a large development of
positive effective charge on the amine nucleophile in the transition state. Similarly, the rate constants for
the reaction of 2-cyanoethylamine with substituted N-aroyl b-lactams gives a Brønsted blg value of
−1.03 for different amide leaving groups and is indicative of considerable change in effective charge on
the leaving group in the transition state. These observations are compatible with either a late transition
state for the formation of the tetrahedral intermediate of a stepwise mechanism or a concerted
mechanism with simultaneous bond formation and fission in which the amide leaving group is expelled
as an anion. Amide anion expulsion is also indicated by an insignificant solvent kinetic isotope effect,
O
O
kH2
2 /kD2
2 , of 1.01 for the aminolysis of N-benzoyl b-lactam with 2-methoxyethylamine. The
RNH
RNH
Brønsted blg value decreases from −1.03 to −0.71 as the amine nucleophile is changed from
2-cyanoethylamine to propylamine. The Brønsted bnuc value is more invariant although it changes from
+0.90 to +0.85 on changing the amide leaving group from p-methoxy to p-chloro substituted. The
sensitivity of the Brønsted bnuc and blg values to the nucleofugality of the amide leaving group and the
nucleophilicity of the amine nucleophiles, respectively, indicate coupled bond formation and bond
fission processes.
removal from the attacking amine and proton addition to the
leaving amino group.
Introduction
b-Lactam antibiotics such as penicillins (1) and cephalosporins
are often considered unusually reactive compared with normal
amides because most of their biologically important reactions
involve the opening of the highly strained four-membered ring.1
However, the rate of alkaline hydrolysis of b-lactams is, at most,
a hundred-fold greater than that of an analogous acyclic amide
and is, in fact, often very similar.2 In water the b-lactam ring of b-
lactam antibiotics undergoes attack by nucleophilic reagents such
as amines and alcohols in competition with that by hydroxide-
ions. Nucleophilic substitution at the carbonyl centre of b-lactams
is an acyl transfer process involving covalent bond formation
between the carbonyl carbon and the nucleophile as well as C–
N bond fission of the b-lactam. Previous studies with nitrogen
and oxygen nucleophiles have shown that the mechamism of
substitution is a stepwise process, covalent bond formation to the
incoming nucleophile occurs before C–N bond fission, resulting
in the reversible formation of a tetrahedral intermediate.3–5 The
rate-limiting step in these reactions is normally the breakdown of
the tetrahedral intermediate which may involve ring opening.5–7
The reaction of amines with penicillins gives the corresponding
penicilloyl amide and requires at least two proton transfers, proton
The aminolysis of penicillins to give penicilloyl amides is
strongly catalysed by bases (Scheme 1) which in itself is indicative
of a stepwise process and reversible formation of a zwitterionic
tetrahedral intermediate and is supported by a non-linear
dependence of the rate of aminolysis upon hydroxide-ion
concentration due to a change of rate-limiting step. In a stepwise
process the neutral amine nucleophile becomes positively charged
after forming a covalent bond with the carbonyl carbon which
also significantly changes its pKa. Proton transfer from the
protonated amine in the tetrahedral intermediate to a catalytic
base becomes thermodynamically favourable and general base
catalysed aminolysis is the dominant reaction pathway.8 In fact,
it is experimentally difficult to determine the rate constant for the
uncatalysed aminolysis9 but the evidence suggests that the rate-
limiting step is thought to be the b-lactam C–N bond fission.7,9–10
Department of Chemical and Biological Sciences, The University
of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK. E-mail:
m.i.page@hud.ac.uk; Fax: + 44 (0) 1484 473075; Tel: + 44 (0) 1484 472531
Scheme 1
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Incorporating a benzamide as a potential leaving group into
a simple azetidin-2-one gives the imide N-benzoyl b-lactam (2),
which contains an endocyclic and an exocyclic acyl centre, both
of which are potential sites for nucleophilic attack. Contrary to
expectations and despite the belief that ring strain should increase
the reactivity of b-lactams, there is competition between the two
sites for hydroxide-ion attack and between endo- and exocyclic C–
N fission as a function of substituents in the aroyl group.11 Herein
we report our studies of the aminolysis of N-aroyl b-lactams
(2) which, unlike the aminolysis of natural cephalosporins12 and
penicillins,9 does not require general base catalysis. Linear free-
energy relationships for the aminolysis of N-aroyl b-lactams were
investigated by determining the effect on the rate of reaction of
varying the basicity of the amine nucleophiles and substituents in
the aryl residue of the amide leaving group.
Fig. 1 Dependence of the observed pseudo-first-order rate constants, kobs
,
for the reaction of N-benzoyl b-lactam with 2-cyanoethylamine against
total amine concentration at various pHs in 1% acetonitrile-water (v/v) at
30 ◦C with 1.0 M ionic strength (KCl).
to zero buffer concentration corresponds to that calculated for the
background hydrolysis but is indistinguishable from zero (Fig. 1).
The slopes of these plots give the second-order rate constants, kcat,
for the aminolysis by both the protonated and the deprotonated
form of the amine. The reaction was studied using different
fractions of free base, a, of the amine and a plot of kcat against
a gives a straight line that passes through the origin (Fig. 2),
indicating that the reactive form of the amine is the free base form
and there is no significant reaction with the protonated form.
Results and discussion
The rates of the aminolysis of N-aroyl b-lactams were studied by
UV-spectrophotometry in 1% acetonitrile-water (v/v) at 30 ◦C
with 1.0 M ionic strength (I) maintained by KCl and using the
amine as both a nucleophile and a buffer. The site of nucleophilic
attack was determined by a product analysis of the reaction of
1.0 M 2-cyanoethylamine at pH 8.07 (0.6 fraction of free base) with
N-p-nitrobenzoyl b-lactam (100 mg, 45 mmol) in 20% acetonitrile-
water (v/v). NMR and ESI-MS (electrospray ionisation mass
spectrometry) analysis of the product shows that at least 90%
of nucleophilic attack occurs at the b-lactam carbonyl. This is
1
demonstrated by H, 13C HMBC (heteronuclear multiple bond
correlation) NMR which shows the coupling of the a-methylene
protons of 2-cyanoethylamine with a carbonyl carbon of the
substrate which is, in its turn, coupled to the methylene protons of
the b-lactam. 1H, 13C HMBC NMR also shows the two carbonyl
carbons of the product were coupled to the methylene protons of
the b-lactam ring indicating that there was insignificant attack at
the exocyclic amide. ESI-MS (positive mode) shows an observed
mass peak of 291 corresponding to the mass of the b-lactam ring
opened aminolysis product (Scheme 2).
The kinetics of aminolysis were studied spectrophotometrically
measuring the change in absorbance due to product formation
using the amine as both buffer and reactant and, with excess
amine over N-aroyl b-lactam, obey simple first-order kinetics.
The dependence of the observed first-order rate constants, kobs
,
Fig. 2 Dependence of the second-order rate constant, kcat, on the fraction
of free base, a, of 2-cyanoethylamine in the aminolysis of N-benzoyl
b-lactam in 1% acetonitrile-water (v/v) at 30 ◦C with 1.0 M ionic strength
(KCl).
for the reaction of N-benzoyl b-lactam in aqueous solution
buffered with 2-cyanoethylamine is linearly dependent on total
amine concentration (Fig. 1). Extrapolation of the reaction rate
Scheme 2
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Table 1 Second-order rate constants for the aminolysis of N-aroyl b-
lactams in water at 30 ◦C, I = 1.0 M (KCl)
The fact that the pseudo-first-order rate constant, kobs, for the
aminolysis of N-benzoyl b-lactam is linearly dependent on the
amine concentration is in sharp contrast to the aminolysis of
penicillin which shows a rate law with a large term which is
second-order in amine concentration. The rate law that adequately
describes the reaction is thus given as eqn 1:
2 O
pKa
kH
/M−1 s−1
RNH2
N-p-Nitrobenzoyl b-lactam
2-cyanoethylamine
N-p-Chlorobenzoyl b-lactam
2-Cyanoethylamine
2-Methoxyethylamine
Propylamine
7.91a
(1.44 0.04) × 10−1
Rate
7.91a
9.66b
(3.96 0.06) × 10−2
= kobs = ko + kRNH a[RNH2]tot
(1a)
(1b)
2
1.24 0.01
[b − lactam]
10.79b
10.9 0.1
N-Benzoyl b-lactam
Ethylenediamine monocation
2-Cyanoethylamine
2-Methoxyethylamine
Ethylenediamine
= ko + kRNH [RNH2]
2
7.43a
7.91a
(1.71 0.04) × 10−2
(2.55 0.04) × 10−2
(7.95 0.09) × 10−1
4.25 0.09
where ko is the first-order rate constant for the background
9.66b
hydrolysis and kRNH is the second-order rate constant for the
10.07b
10.79b
2
aminolysis of the b-lactam.
Propylamine
8.30 0.22
N-p-Methoxybenzoyl b-lactam
2-Cyanoethylamine
2-Methoxyethylamine
Propylamine
General base catalysis is observed in the aminolysis of
penicillin,9 cephalosporin12 and N-aryl b-lactams13 and accounts
for the major contribution to the observed rate for all of these three
substrates. Interestingly, no such catalysis is observed with the
aminolysis of N-benzoyl b-lactams reported here. The observation
of general base catalysed aminolysis seen with other b-lactams
has been taken to indicate a stepwise process and reversible
formation of a tetrahedral intermediate1–4 (Scheme 3). Formation
of this unstable intermediate changes proton transfer from the
amine nucleophile to the base from being thermodynamically
unfavourable in the reactants to favourable in this adduct. This
route, kB, is more favourable than the uncatalysed breakdown
of the intermediate, ko (Scheme 3). Although the aminolysis of
N-benzoyl b-lactam is not general base catalysed, the reaction
could be solvent catalysed with water acting as a general base,
7.91a
9.66b
(1.23 0.01) × 10−2
(4.21 0.06) × 10−1
5.02 0.20
10.79b
a Ionisation constants measured potentiometrically at 30 ◦C with 1.0 M
ionic strength in water. b Ionisation constants obtained from Ref. 21.
tetrahedral intermediate (Scheme 4) and the corresponding overall
rate equation (eqn 2).
Rate
k1k2[RNH2][base]
k−1 + k2[base]
= kobs
=
(2)
[b − lactam]
The rate-limiting step is determined by the relative rates of
partitioning of the tetrahedral intermediate to reactants and
products, k2[base]/k−1. For the aminolysis of penicillins9 and N-
aryl b-lactams,13 normally k−1 ꢀ k2[base] and the reaction rate
is dependent on base concentration. The rate-limiting step is the
diffusion controlled encounter of the base and the tetrahedral
intermediate, the k2 step.9 A change in rate-limiting step from the
diffusion step to the formation of the tetrahedral intermediate
can be observed if k−1 ꢁ k2[base] due to a high concentration
of a strong base.14 Under these conditions the rate becomes
independent of base concentration and simply first-order in
amine concentration; the rate-limiting step then becomes the
formation of the tetrahedral intermediate, k1. This pathway is
very different from the small amount of uncatalysed aminolysis
observed under normal conditions which corresponds to rate-
limiting proton transfer from the tetrahedral intermediate to water,
kH in Scheme 3. However, it is difficult to see why this should
O
2
be the exclusive pathway and more favourable than to more basic
O
O
bases. The solvent kinetic isotope effect, kH2
2 /kD2
2 , for the
RNH
RNH
reaction of N-benzoyl b-lactam with 2-methoxyethylamine is 1.01
(Table 1).
The insignificant isotope effect indicates that the reaction does
not involve a large degree of proton transfer in the transition
state of the rate-limiting step and is incompatible with a solvent
catalysed mechanism. Therefore, the simplest conclusion is that
the rate-limiting step is formation or breakdown of the tetrahedral
intermediate or that the reaction mechanism is concerted.
The base catalysed aminolysis reaction can be expressed in terms
of the rate constants, k1, for the formation of the tetrahedral
intermediate, k−1 for its breakdown to the unreacted b-lactam,
and k2 for the diffusion controlled encounter of the base and the
kH (Scheme 3).
O
2
Scheme 3
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Scheme 4
The observation seen here for the uncatalysed aminolysis of
N-aroyl b-lactams step could also be due to the normal pathway
shown in Scheme 4 but with proton transfer to base occurring
after the rate-limiting step, i.e. if k−1 ꢁ k2[base] arising from a
faster k2 process and/or a slower k−1 step. However, the rate of
breakdown of the zwitterionic tetrahedral intermediates, formed
from N-benzoyl b-lactam and other b-lactams such as N-aryl b-
lactams, to reactants by expulsion of the attacking amine (the k−1
step) is unlikely to vary significantly between different types of b-
lactams. Also, the rate of encounter of the base and the tetrahedral
intermediate (the k2 step) is diffusion controlled and should have
a similar rate in the aminolysis of N-benzoyl b-lactam and other
b-lactams under similar experimental conditions. Therefore, the
uncatalysed aminolysis of N-benzoyl b-lactam must undergo a
different pathway involving a different process for the breakdown
of the tetrahedral intermediate in the forward direction.
To further investigate the mechanism of the uncatalysed aminol-
ysis of N-aroyl b-lactams, linear free-energy relationships were
studied by varying the basicity of the amine nucleophile and also
by varying the amide leaving group with different substituents in
the aryl residue.
Fig. 3 Dependence of the second-order rate constant, kcat, on the fraction
of free base, a, of ethylenediamine monocation in the aminolysis of
N-benzoyl b-lactam in 1% acetonitrile-water (v/v) at 30 ◦C with 1.0 M
ionic strength (KCl), using a mixture of mono- and dication as the buffer.
The theoretical line was generated by using eqn 4.
The Brønsted bnuc value for the attacking amine nucleophile
where k1 is the second-order rate constant for the aminolysis of N-
benzoyl b-lactam with the ethylenediamine monocation, EDAH+,
and k2 is the apparent third-order rate constant for hydroxide-ion
catalysed aminolysis. The apparent second-order rate constant,
kcat, is (eqn 4 and 5):
The second-order rate constants for the aminolysis of N-benzoyl
b-lactam with a series of amines of varying basicity are given
in Table 1. There is a 500-fold increase in the second-order rate
constant as the basicity of the attacking amine is increased by
3.4 pKa units. As with other amines, the pseudo-first-order rate
constant, kobs, for the aminolysis in solutions of ethylenediamine
mono- and dications as buffer, is linearly dependent on the total
amine concentration indicating the insignificance of the term
which is second-order in amine concentration in the rate law.
However, in this case the second-order rate constants, kcat, exhibit
a sharp upward curvature in the plot against the fraction of its
monocation, basic form (Fig. 3). The second-order rate constant,
kcat, is indistinguishable from zero as the fraction of monocation
approaches zero, showing that the ethylenediamine dication is not
catalytically important. The non-linear dependence of the second-
order rate constants, kcat, on a is kinetically compatible with a
hydroxide-ion catalysed aminolysis mechanism. The rate law can
be described as eqn 3:
kobs
kcat
=
= k1a + kꢂ a[OH−]
(4)
[EDAH+]tot
2
kcat
a
= k1 + kꢂ [OH−]
(5)
2
A plot of kcat/a against the hydroxide-ion concentration gives a
straight line of slope kꢂ−1 (Fig. 4). Although the kꢂ−1 term formally
represents hydroxide-ion catalysed aminolysis by EDAH+ there is
no catalysis observed with other bases. This suggests that the sharp
upward curvature in the plot of kcat against a in the aminolysis with
EDAH+ is due to the aminolysis with unprotonated EDA because
this term is kinetically indistinguishable from the aminolysis with
neutral diamine, as shown in eqn 6a–b.
Rate
= kobs
[b − lactam]
[EDA][H+]
[EDAH+]
[EDA]Kw
Ka =
=
(6a)
= ko + k1a[EDAH+]tot + kꢂ a[EDAH+]tot[OH−] (3)
[EDAH+][OH−]
2
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Fig. 4 Dependence of the rate constant, kcat/a, on the concentration of
hydroxide-ion in the aminolysis of N-benzoyl b-lactam. The rate constants
were determined in 1% acetonitrile-water (v/v) at 30 ◦C with 1.0 M ionic
strength (KCl).
Fig. 5 Dependence of the statistically corrected second-order rate
constants for the reaction of amines with N-benzoyl b-lactam as a function
of the basicity of the amine in 1% acetonitrile-water (v/v) at 30 ◦C with
1.0 M ionic strength (KCl), where EDAH+ = ethylenediamine monocation;
CEA = 2-cyanoethylamine; MEA = methoxyethylamine; EDA = neutral
ethylenediamine; PA = propylamine.
Hence,
[EDA]Kw
[EDAH+][OH−] =
(6b)
(6c)
Ka
rate-limiting breakdown with little bond fission to the leaving
group or a concerted substitution mechanism.
kꢂ2[EDAH+][OH−] = k2[EDA]
Interestingly, the second-order rate constant for the aminolysis
of N-benzoyl b-lactam with neutral ethylenediamine shows no de-
viation from the Brønsted plot (Fig. 5) when statistically corrected
for the two amine groups. This is in contrast to the 30-fold rate
enhancement observed with the aminolysis of benzyl penicillin
by ethylenediamine,9 attributed to intramolecular general base
catalysed aminolysis in which the terminal amino group of the
diamine acts as a general base abstracting a proton from the amine
nucleophile in the tetrahedral intermediate. Given the absence
of a general base catalysed aminolysis pathway for N-aroyl b-
lactams, it is not surprising to see that ethylenediamine falls on the
Brønsted plot and acts as a simple monoamine. Similarly, although
the rate constant for the reaction of ethylenediamine monocation
with benzyl penicillin shows a 100-fold rate enhancement, as a
result of intramolecular general acid catalysis by the terminal
protonated amine facilitating the breakdown of the tetrahedral
intermediate by donating a proton to the b-lactam nitrogen,9 that
with N-benzoyl b-lactam shows no significant deviation from the
Brønsted plot. The importance of general acid catalysis in the
breakdown of the tetrahedral intermediate formed during the
aminolysis of penicillin and cephalosporin has been demonstrated
recently with hydroxylamine which showed a 1 × 106-fold rate
enhancement compared with that predicted from a Brønsted plot
for other primary amines.16 There is thus clear evidence that the
aminolysis of penicillin involves a rate-limiting step that occurs
after formation of the tetrahedral intermediate. The fact that
the rate constants for the aminolysis of N-benzoyl b-lactam with
ethylenediamine and its monocation are those expected for their
respective basicities suggests that the reaction does not involve
either general base or acid catalysis. This is compatible with the
solvent kinetic isotope effect of 1.01 for the aminolysis of N-
benzoyl b-lactam with methoxyethylamine (Table 1).
K
K
where k2 = kꢂ2
w
a
The second-order rate constant for the aminolysis of N-benzoyl
b-lactam with neutral ethylenediamine, EDA, k2 can thus be
obtained even in buffers of mono- and dication even when the
concentration of the unprotonated EDA is very small. The rate
law for the aminolysis with ethylenediamine thus becomes (eqn 7):
Rate
= kobs = ko + k1[EDAH+] + k [EDA]
(7)
2
[b − lactam]
The second-order rate constants, kRNH2 , for the aminolysis of
N-benzoyl b-lactam are linearly dependent on the pKa of the
amines, including EDA, and give a Brønsted bnuc value of +0.87
(Fig. 5). This indicates that approximately an effective unit positive
charge is developed on the nitrogen of the amine nucleophile in
the transition state of the rate-limiting step. There are two classes
of Brønsted bnuc values seen for the uncatalysed aminolysis of
penicillins. At high pH, where k2[OH−] ꢀ k−1 (Scheme 4) the
Brønsted bnuc value is +0.3 for rate-limiting formation of the
tetrahedral intermediate.14 There is also a Brønsted bnuc value
of +1.0 for the uncatalysed aminolysis which corresponds to
rate-limiting breakdown of the tetrahedral intermediate.9 An
unusually large sensitivity to the basicity of the attacking amine
was observed in the uncatalysed aminolysis of acetylimidazole
with a Brønsted bnuc value of +1.6, which was interpreted as rate-
limiting breakdown of the tetrahedral intermediate with complete
C–N bond formation between the amine and the acyl centre in the
transition state in which there was a large degree of bond fission
to the leaving group.15 The Brønsted bnuc value of +0.87 seen here
for the aminolysis of N-benzoyl b-lactam is compatible with a late
transition state in the formation of the tetrahedral intermediate
followed by fast expulsion of the amide leaving group, possibly
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The aminolysis of N-benzoyl b-lactam may thus occur with
an earlier rate-limiting step compared with that of other b-
lactams, i.e. formation rather than breakdown of the tetrahedral
intermediate. The leaving group of penicillins and cephalosporins
is an amine and for N-benzoyl b-lactam is an amide. Amide anions
are undoubtedly better leaving groups than amine anions and may
even be better than neutral amines giving rise to an earlier rate-
limiting step because ko ꢀ k−1 (Scheme 3). At first sight it may
appear unusual that the Brønsted bnuc of +0.87 for the aminolysis of
N-benzoyl b-lactam is greater than that observed for rate-limiting
formation of the tetrahedral intermediate with other b-lactams
(bnuc = +0.3).14 This suggests nucleophilic attack by the amine
occurs with a later transition state along the reaction coordinate
for N-benzoyl b-lactams compared with N-alkyl b-lactams.
charge on the amide nitrogen will be +0.7. The observed Brønsted
blg value of −1.03 thus indicates that the amide nitrogen has lost
considerable positive charge and has an effective charge of only
+0.1 in the transition state. It is therefore likely that the amide
leaving group is expelled as its anion and there is significant C–N
bond fission in the transition state. The rate of expulsion of the
amide anion must be faster than that of the diffusion controlled
encounter of the base and the tetrahedral intermediate so that base
catalysis is not observed and the rate of aminolysis is first-order in
amine concentration. If the reasonable assumption19 is made that
the diffusion-controlled step, k2, has a value of ca. 1 × 1010 M−1 s−1,
with the highest amine total concentration of 0.1 M used in this
study, the pseudo-first-order rate constant for this diffusion step
would be ca. 1 × 109 s−1. The rate of expulsion of the amide anion
must then be at least ca. 1 × 1010 s−1 in order to be faster than the
general base catalysed pathway which is not observed (Scheme 3).
Such a high value is not unreasonable.14,15
The Brønsted blg value for the amide leaving group
It is interesting that the aminolysis of N-aroyl b-lactams has a
high sensitivity to both the basicity of the attacking amide and
that of the amine leaving group. This could be the result of a rate-
limiting breakdown of the tetrahedral intermediate as discussed
earlier for acetylimidazole,15 but the bnuc of +0.87 is not sufficiently
large to be fully compatible with significant ring opening and
reforming the carbonyl double bond. Therefore the aminolysis
of N-aroyl b-lactams appears to occur by a concerted pathway
in which bond formation and fission occurs simultaneously
(Scheme 5). A high dependence on both the basicity of the
attacking nucleophile and that of the leaving group was also seen in
the reactions of substituted phenolate anions with phenyl formate,
with a Brønsted bnuc and blg value of +0.90 and −0.90, respectively,
and was interpreted as a concerted reaction.20
The second-order rate constants, kRNH2 , for the aminolysis of N-p-
chlorobenzoyl b-lactam and N-p-methoxybenzoyl b-lactam were
determined with a series of amines and are given in Table 1.
There is a 10-fold increase in the second-order rate constant for
the aminolysis with 2-cyanoethylamine as the substituent in the
arylamide leaving group is changed from p-methoxy to p-nitro.
As there are no literature values of the ionisation constants for
substituted benzamides, the ionisation constants for substituted
benzoic acids were used for the correlation. The rate constants
are linearly dependent on the pKa of the corresponding benzoic
acid and give a Brønsted blg value of −1.03 for the aminolysis of
N-aroyl b-lactams with 2-cyanoethylamine (Fig. 6). This indicates
that there is a large change in effective charge on the leaving group
going from the reactant state to the transition state. The amide
nitrogen, in its reactant state, is made positive by resonance and
its effective charge is 0.7+.17 As discussed elsewhere11,18 we assume
that the effective charge in imides is greater than that in amides and
use a value of +1.1. In the tetrahedral intermediate the effective
Scheme 5
As a function of attacking amine basicity, the Brønsted blg
value decreases from −1.03 for 2-cyanoethylamine to −0.71
for propylamine (Table 2), i.e. the Brønsted blg decreases with
increasing basicity of the attacking amine. This indicates less
charge development on the leaving group amide (less C–N bond
fission) in the transition state with more basic amine nucleophiles.
However, there is little variation in Brønsted bnuc for the attacking
amine as a function of substituents in the leaving group. Based
on the limited data, it appears that there is a small decrease in
bnuc from +0.90 to +0.85 when the leaving groups change from
Table 2 The dependence of the Brønsted blg values for the aminolysis of
N-aroyl b-lactams on the pKa of the amine nucleophiles
pKa
blg
2-Cyanoethylamine
Methoxyethylamine
Propylamine
7.91a
−1.03
−0.97
−0.71
9.66b
Fig. 6 Brønsted plots of the second-order rate constants, kRNH , for the
2
10.79b
aminolysis of N-aroyl b-lactams against the basicity of the benzamide
leaving group with different amines indicated. The rate constants were
determined in 1% acetonitrile-water (v/v) at 30 ◦C with 1.0 M ionic
strength (KCl).
a Ionisation constants measured potentiometrically at 30 ◦C with 1.0 M
ionic strength in water. b Ionisation constants obtained from Ref. 21.
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Table 3 The dependence of the Brønsted bnuc values for the aminolysis
of N-aroyl b-lactams on the basicity of the substituted benzamide leaving
group. The pKa of the carboxylic acid corresponding to exocyclic aryl
carboxamide is given as an indicator of the inductive effect of the
substituent in the aromatic ring. pKa values are taken from Ref. 21
measured by the Brønsted bnuc or blg values or both as indicated
(Scheme 6). In the concerted mechanism, the bond making to
the attacking nucleophile and the bond breaking to the leaving
group takes place simultaneously, does not involve a reaction
intermediate and its trajectory is indicated by the dashed diagonal
line in Scheme 6. Increasing the acidity of the amide leaving group
with electron withdrawing groups would stabilise the amide anion
product (5) and the transition state will move away from the
product corner to that of the reactants [arrow (a)], as predicted
by the ‘parallel’ effect and the Hammond postulate.17 Electron
withdrawing groups in the amide leaving group would also stabilise
the amide anion (4) of the dissociative pathway and destabilise the
tetrahedral intermediate (3) in the associative pathway. In both
cases, the transition state will tend to move towards the amide
anion (4) corner [arrow (b)] as a Thornton or ‘perpendicular’
effect. The net result is predicted to be a reduced Brønsted bnuc
value as the acidity of the leaving group is increased, as observed.
Increasing the basicity of the amine nucleophile would stabilise
the amide product (5), which causes a movement of the transition
state away from the product corner (5) towards the reactants
[arrow (c)]. A more basic amine nucleophile would also stabilise
the tetrahedral intermediate (3), which will move the transition
state towards this corner [arrow (d)] by a ‘perpendicular’ effect. A
reduced Brønsted blg is then predicted by increasing the basicity
of the amine nucleophiles.
pKa
bnuc
p-Cl
3.99
4.20
4.47
0.85
0.87
0.90
p-H
p-OMe
p-methoxy to p-chloro-benzamide (Table 3). This may not be
meaningful, but would suggest there is less charge development on
the attacking amine nucleophile with a better leaving group. The
changes in the Brønsted blg and bnuc values observed by changing
the nucleophilicity of the nucleophile and the nucleofugacity of
the leaving group are consistent with a concerted mechanism.
Bond making to the attacking nucleophile and bond breaking
to the leaving group are coupled in the transition state of a
concerted reaction. Changing either the nucleophilicity of the
nucleophile or the nucleofugacity of the leaving group will affect
the transition state position along the reaction coordinate of a
concerted pathway. This may be monitored by changes in the
Brønsted bnuc and blg values of the reaction and the effect may be
illustrated by the Jencks–More O’Ferrall diagram.17 In principle,
the expulsion of the amide anion could result from a stepwise
associative mechanism, a stepwise dissociative mechanism, or a
concerted pathway (Scheme 6). In the associative pathway, the
reaction involves the formation of a tetrahedral intermediate
(3) formed between the amine nucleophile and the b-lactam
which subsequently expels the amide anion leaving group. In the
dissociative pathway, an acylium centre and an amide anion would
be produced in the intermediate (4) and the acylium centre is
then trapped by the amine nucleophile. The axes of Scheme 6 are
The aminolysis of N-aroyl b-lactams thus appears to occur
by a concerted mechanism with significant coupling between
bond formation and bond fission and involves a transition state
with a large amount of charge development on the incoming
nucleophile as well as on the leaving group amide anion. Acyl
transfer reactions predominantly occur by a stepwise process17 and
this is true for nucleophilic substitution of b-lactams. In the case
of N-aroyl b-lactams the concerted mechanism is presumably en-
forced17, 23 by the instability of the normal tetrahedral intermediate
Scheme 6
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Table 4 A comparison of the second-order rate constants, kOH, for the
the leaving group is the b-lactam amide anion, which is less
stable than the amide anion of p-nitrobenzamide. The rate of
reaction with weaker nucleophiles, such as amines, compared with
hydroxide-ions has a greater dependence on the basicity of the
leaving group as shown by the Brønsted blg values, −0.55 for
hydroxide-ion catalysed hydrolysis11 and −0.97 for aminolysis with
methoxyethylamine (Table 2) for reactions at the b-lactam centre.
A similar situation exists in the inability of an intramolecular
carboxylate to displace the b-lactam amide anion in competition
with the alkaline hydrolysis of the b-lactam in N-o-carboxybenzoyl
b-lactam. There is no evidence of anhydride formation with N-o-
carboxybenzoyl b-lactam at neutral pH.11,18
alkaline hydrolysis and, kRNH , for the aminolysis of some b-lactams with
2
2-propylamine
kOH
kRNH
k
OH/M−1 s−1
kRNH /M−1 s−1
2
2
N-Benzoyl
9.07a
8.30a
1.09
177
11
b-lactam (6)
N-p-Nitrophenyl
b-lactam (7)
Benzylpenicillin (1)
4.42 × 10−2b
1.54 × 10−1ae
2.5 × 10−4c
1.32 × 10−2d
a At 30 ◦C with 1.0 M ionic strength. b At 25 ◦C with 1.0 M ionic streng◦th,
from Ref. 22. c At 25 ◦C with 0.1 M ionic strength, from Ref. 12. d At 30 C
with 0.25 M ionic strength. e From Ref. 1.
Experimental section
(3)—expulsion of the good leaving group amide anion is facilitated
further by ring opening of the b-lactam and occurs with a faster
rate than expulsion of the amine to regenerate the reactants.
It is of interest to compare the reactivity of N-aroyl b-lactams
with that of other b-lactams. The second-order rate constants,
(i) Synthesis
N-Aroyl b-lactams were prepared according to the general pro-
cedure: to a −78 ◦C stirred solution of 2-azetidinone (0.5 g, 7.03
mmol) in dry dichloromethane (DCM) (20 ml) was added 4,4-
dimethylaminopyridine (0.1 g, 0.82 mmol) and a solution of aroyl
chloride (1.57 g, 8.46 mmol) in dichloromethane (10 ml) dropwise
over 5 minutes. Triethylamine (0.98 ml, 7.02 mmol) was added
dropwise over 10 minutes forming a white precipitate. The reaction
mixture was stirred at −78 ◦C for 1 hour and for a further 24 hours
at ambient temperature. DCM (10 ml) was added to the reaction
mixture and the solution washed with water (15 ml) and saturated
brine (2 × 15 ml). The organic layer was dried over anhydrous
Na2SO4 and the solvent was removed under reduced pressure by
rotary evaporation at 30 ◦C to yield a pale yellow oil, which was
purified by column chromatography.
kOH, for the alkaline hydrolysis and, kRNH2 , for the uncatalysed
aminolysis of penicillin (1),9 N-benzoyl b-lactam (6), and N-p-
nitrophenyl b-lactam (7)13 with propylamine are given in Table 4.
Although the uncatalysed aminolysis of penicillin (1) represents
the very minor pathway, a reasonable estimate of the second-order
rate constant has been measured.9 It is interesting that the rate of
the uncatalysed aminolysis varies by more than 1 × 104 while
the rate of alkaline hydrolysis varies more moderately by 1 × 102
between the substrates. For penicillin (1) the rate constant for
hydrolysis is at least 10-fold greater than that of the uncatalysed
aminolysis, and is 100-fold greater for N-p-nitrophenyl b-lactam
(7) but they are similar for N-benzoyl b-lactam (6). The ratio,
k
OH/kRNH2 , indicates the relative reactivity towards hydroxide-ions
1-(4ꢀ-Methoxybenzoyl)-1-azetidin-2-one. Yield, 0.6 g (42%);
mp 125–127 ◦C; IR tmax (cm−1) (CHCl3): 3020, 3009, 2975, 2912,
2842, 1784, 1668, 1606, 1325, 1259, 1195, 1108, 1028; 1H NMR: d
(CDCl3) 7.99 (2H, d, J 8.88), 6.92 (2H, d, J 8.97), 3.85 (3H, s, CH3),
3.77 (2H, t, J 5.44, CH2N), 3.02 (2H, t, J 5.45, CH2CO); 13C NMR:
and amine nucleophiles; it is much smaller for N-benzoyl b-lactam
(6) than that of the other two b-lactams by 10–1 × 102. Since
the rates of alkaline hydrolysis of the b-lactams are similar, the
significantly reduced kOH/kRNH ratio for N-benzoyl b-lactam (6)
may also be indicative of a different mechanism for the aminolysis
2
=
=
d (CDCl3) 165.23 (C O), 163.92 (C O), 132.57 (quaternary
carbon), 131.92 (ArCH), 123.76 (quaternary carbon), 113.948
(ArCH), 55.21 (CH3), 36.45 (CH2N), 34.44 (CH2CO); HREI-MS
(high resolution electron ionisation mass spectroscopy) [M + H]+
for C11H11NO3 calculated 206.0812 measured 206.0812.
of N-benzoyl b-lactam (6) compared with the other two b-lactams.
1-Benzoyl-1-azetidin-2-one. IR tmax (cm−1) (CHCl3): 3020,
1
1786, 1673, 1327, 1298, 1216, 1192; H NMR: d (CDCl3) 8.00
(2H, d, J 7.43), 7.61 (1H, t, J 7.29), 7.49 (2H, t, 7.83), 3.81
(2H, t, J 5.49), 3.14 (2H, t, J 5.49); 13C NMR: d (CDCl3)
=
=
It is worth noting that although the alkaline hydrolysis of N-p-
nitrobenzoyl b-lactam occurs with competitive endo- and exocyclic
C–N bond fission,11 the aminolysis reaction occurs exclusively
with b-lactam ring opening. Although hydroxide-ion attack at
the exocyclic carbonyl group is competitive with that at the b-
lactam centre, amines prefer the b-lactam carbonyl group. The
second-order rate constant for aminolysis at the endocyclic b-
lactam carbonyl is at least 100-fold greater than that at the
exocyclic carbonyl centre. In the aminolysis of the b-lactam of N-
aroyl b-lactams, the zwitterionic tetrahedral intermediate appears
to breakdown by expelling the benzamide leaving group as its
anion. However, for the reaction of the exocyclic carbonyl centre,
166.26 (C O), 163.95 (C O), 133.19 (PhCH), 131.83 (quaternary
carbon), 129.69 (PhCH), 128.12 (PhCH), 36.77 (CH2), 35.05
(CH2).
1-(4ꢀ-Chlorobenzoyl)-1-azetidin-2-one. Yield, 0.85 g (58%); IR
tmax (cm−1) (CHCl3) 3020, 1788, 1673, 1593, 1404, 1324, 1284,
1217, 1093; H NMR: d (CDCl3) 7.95 (2H, d, J 6.68), 7.44 (2H,
1
d, J 8.66), 3.77 (2H, t, J 5.5, CH2N), 3.12 (2H, t, J 5.61, CH2CO);
13
=
=
C NMR: d (CDCl3) 165.01 (C O), 163.84 (C O), 139.51
(quaternary carbon), 131.14 (CH), 130.1 (quaternary carbon),
128.44 (ArCH), 36.73 (CH2N), 34.99 (CH2CO); HREI-MS [M +
H]+ for C10H8NO2Cl calculated 210.0316 measured 210.0316.
492 | Org. Biomol. Chem., 2007, 5, 485–493
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1-(4ꢀ-Nitrobenzoyl)-1-azetidin-2-one. Yield, 1.23 g (80%); mp
130–131 ◦C; IR tmax (cm−1) (CHCl3) 3020, 1787, 1686, 1599, 1523,
1397, 1309, 1252, 1204, 992; H NMR: d (CDCl3) 8.33 (2H, d, J
of 2 × 10−5 M to 2 × 10−4 M. Pseudo first-order rate constants
from exponential plots of absorbance against time or gradients
of initial slopes were obtained using the Cary Win UV kinetics
application (Version 02.00(26)). pH–Rate profiles were modelled
to theoretical equations using the Scientist program (V2.02,
Micromath Software Ltd, USA).
1
8.75), 8.14 (2H, d, J 8.74), 3.85 (2H, t, J 5.58, CH2N), 3.2 (2H,
13
=
t, J 5.57, CH2CO); C NMR: d (CDCl3) 164.16 (C O), 163.79
=
(C O), 150.29 (quaternary carbon), 137.32 (quaternary carbon),
130.76 (CH), 123.28 (CH), 37.0 (CH2N), 35.5 (CH2CO).
References
(ii) Kinectic Procedures
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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.
pH measurements were made with a ␾40 pH meter (Beckman,
Fullerton, USA) using a semi-micro calomel electrode (Beckman).
A calibration of the pH meter was carried out at 30 ◦C using
pH 6.99 0.01, pH 4.01 0.02 or pH 9.95 0.02 calibration
buffers. For solution pHs ≥3 and ≤11 the pH was controlled by the
use of ≤0.2 M buffer solutions of the amine. Buffer solutions were
prepared by partial neutralisation of solutions of their sodium salts
to the required pH. Hydroxide-i◦on concentrations were calculated
using pKw (H2O) = 13.83 at 30 C.24
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In all experiments temperatures were maintained at 30 ◦C and
ionic strength at 1.0 M with AnalaR grade KCl unless otherwise
stated. AnalaR grade reagents and deionised water were used
throughout. Organic solvents were glass distilled prior to use and
stored under nitrogen.
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Reactions studied by UV spectrophotometry were commenced
by injections (20 ll) of acetonitrile stock solutions 1 × 10−2
M
of the substrate into the cells containing pre-incubated buffer
(2.0 ml). Final reaction cells contained ≤1% acetonitrile 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 was
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. Reaction concentrations were generally within the range
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Org. Biomol. Chem., 2007, 5, 485–493 | 493
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