Coordination among Bond Formation/Cleavage in a Bifunctional-Catalyzed Fast Amide Hydrolysis: Evidence for an Optimized Intramolecular N-Protonation Event

Article abstract of DOI:10.1021/acs.joc.9b03383

A density functional theory (DFT) computational analysis, using the ωB97X-D functional, of a rapid amide cleavage in 2-carboxyphthalanilic acid (2CPA), where the amide group is flanked by two catalytic carboxyls, reveals key mechanistic information: (a) General base catalysis by a carboxylate coupled to general acid catalysis by a carboxyl is not operative. (b) Nucleophilic attack by a carboxylate on the amide carbonyl coupled to general acid catalysis at the amide oxygen can also be ruled out. (c) A mechanistic pathway that remains viable involves general acid proton delivery to the amide nitrogen by a carboxyl, while the other carboxylate engages in nucleophilic attack upon the amide carbonyl; a substantially unchanged amide carbonyl in the transition state; two concurrent bond-forming events; and a spatiotemporal-base rate acceleration. This mechanism is supported by molecular dynamic simulations which confirm a persistent key intramolecular hydrogen bonding. These theoretical conclusions, although not easily verified by experiment, are consistent with a bell-shaped pH/rate profile but are at odds with hydrolysis mechanisms in the classic literature.

Full text of DOI:10.1021/acs.joc.9b03383

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Article
Coordination Among Bond Formation/Cleavage in a
Bifunctional-Catalyzed Fast Amide Hydrolysis: Evidence
for an Optimized Intramolecular N-protonation Event
Leandro Scorsin, Ricardo Ferreira Affeldt, Bruno Surdi Oliveira, Eduardo
Vieira Silveira, Matheus Santos Ferraz, Fábio Prá da Silva de Souza,
Giovanni F. Caramori, Fredric M. Menger, Bruno S. Souza, and Faruk Nome
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b03383 • Publication Date (Web): 10 Mar 2020
Downloaded from pubs.acs.org on March 13, 2020
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Coordination Among Bond Formation/Cleavage in a Bifunctional-
Catalyzed Fast Amide Hydrolysis: Evidence for an Optimized
Intramolecular N-protonation Event
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a
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Leandro Scorsin, Ricardo F. Affeldt, Bruno S. Oliveira, Eduardo V. Silveira, Matheus S. Ferraz,
a
a
,b
,a
Fábio P. S. de Souza, Giovanni F. Caramori, Fredric M. Menger,* Bruno S. Souza,* and Faruk
a,†
Nome
aDepartment of Chemistry, Federal University of Santa Catarina, Florianópolis, SC 88040-900, Brazil
bDepartment of Chemistry, Emory University, Atlanta, GA 30322, USA
†Deceased September 24, 2018.
ABSTRACT
A density functional theory (DFT) computational analysis, using the B97X-D functional, of
a rapid amide cleavage in 2-carboxyphthalanilic acid (2CPA), where the amide group is
flanked by two catalytic carboxyls, reveals key mechanistic information: (a) General base
catalysis by a carboxylate coupled to general acid catalysis by a carboxyl is not operative. (b)
Nucleophilic attack by a carboxylate on the amide carbonyl coupled to general acid catalysis
at the amide oxygen can also be ruled out. (c) A mechanistic pathway that remains viable
involves general acid proton delivery to the amide nitrogen by a carboxyl while the other
carboxylate engages in nucleophilic attack upon the amide carbonyl; a substantially
unchanged amide carbonyl in the transition state; two concurrent bond-forming events; and a
spatiotemporal-base rate acceleration. This mechanism is supported by molecular dynamic
simulations which confirm a persistent key intramolecular hydrogen bonding. These
theoretical conclusions, although not easily verified by experiment, are consistent with a bell-
shaped pH/rate profile, but are at odds with hydrolysis mechanisms in the classic literature.
INTRODUCTION
Isaac Newton recognized the presence of two types of variables: simple and
composite. Simple variables (such as time, distance, and length) each consists of a single
"pure" component. In contrast, composite variables are comprised of multiple contributors
(called "elements") as occurs with entropy, resonance stabilization, ring-strain, and solvation.
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The difference is easily appreciated when an attempt is made to compare, for example,
distance and solvation. Distance is a straightforward parameter, whereas solvation is far
more convoluted. It was for this reason that we have been interpreting organic and enzymatic
reactivity in terms of two simple variables: distance and time ("spatiotemporal theory").1
These two parameters seem preferable to "blends" of variables, especially when the goal is to
understand complex chemical behavior.
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We have previously supported experimentally the idea that rate accelerations of >108
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occur when distances of <3 Å are imposed between two reactive atoms. Stated in another
way, proper orientation at short distances, less than the diameter of water, can lead to
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enzyme-like rates. An approximate relationship was derived (e.g. E = cd where E is the
a
a
3
activation energy, c the speed of light, and d the interatomic distance) that additionally
affirms the remarkable sensitivity of rate to distance. One of our published experimental
4
examples, shown in Figure 1, serves to illustrate the concept. This amide hydrolyzes so
rapidly that, owing to its contact distances <3 Å from two carboxyls, the amide cannot be
isolated intact at 35°C and pH 4. Compare this reactivity with that of an “ordinary” amide,
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such as benzamide, having a half-life of 3 hours at 85°C in 5.9% H SO .
2
4
Et Et
- 2.75
O
2.80
Å
O
N
O
Å
H
O
O
Figure 1. 2-carboxy-N,N-diethylnaphthalamic acid, an amide which decomposes with a half-life of 20
seconds at pH 4, 35°C. The interatomic distances were determined by DFT calculations. See reference
4
for details.
Spatiotemporal notions extend to the enzymes themselves. For example, citrate
synthase holds the enolizable methyl group of acetyl-CoA a distance of ca. 2.9 Å from the
carbonyl group that the methyl carbon is destined to attack. Aspartic proteases, such as HIV-1
protease, are enzymatic systems that operate via two spatially directed carboxyl groups; a
1
010-fold acceleration ensues.6,7 A favored mechanism has an aspartate anion removing a
proton from a nucleophilic water molecule as the water adds to the amide carbonyl group.
Simultaneously, the second aspartate, in its conjugate acid form, donates a proton to the
amide's carbonyl group. The resulting tetrahedral intermediate, with a gem-diol functionality,
then collapses to the cleaved amide. Huge rates associated with this mechanism can be
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ascribed to nearly ideal contact geometries. Both experimental and computational
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observations bear this out. Among the many X-ray pictures of complexes between an
enzyme and its substrate-analog, one generalization stands out (except in cases where the
substrate analog distorts the active site): Short distances are usually imposed upon the active
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conformations.9
Carboxyl groups, the key functionalities of the discussion herein, are well known to
accelerate amide hydrolysis in intramolecular systems. For example, at pH 3 the hydrolysis of
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phthalamic acid is about 10 faster than the hydrolysis of benzamide. N-methylmaleamic
acid has a half-life of 3 h at 39°C below pH 2 where the carboxyl group is fully protonated
(the carboxylate substituent is noncatalytic).11 A Kemp's triacid derivative, which has an
enzyme-like half-life of only 8 min. at pD 7.02 and 21.5°C, shows that a protease could in
principle achieve full catalytic power by having only two properly positioned carboxyl
groups.12
We discuss herein 2-carboxyphthanilic acid (2CPA, 1 in Scheme 1) from a
spatiotemporal perspective. Decades ago Morawetz, in their classic paper, demonstrated an
intramolecular bifunctional-catalyzed amide hydrolysis, with 2CPA existing in three protopic
1
3
states (1, 1a, and 1b). By comparison, phthalanilic acid (2), 2-(benzamide)benzoic acid (3)
and 4-carboxyphthalanilic acid (4) all react slowly, verifying the bifunctional nature of the
2
CPA reaction.
1
2
Scheme 1. Protonic states of carboxyphthalanilic acid (2CPA), 1, 1a and 1b. Also shown are the
structures of much more stable related compounds.
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RESULTS AND DISCUSSION
We now report a mechanistic study on the 2-carboxyphthalanilic acid (2CPA)
hydrolysis reaction. Both proton transfer and nucleophilic attack occur in the cleavage.
Coordination and timing between the two reaction modes, the subject of prime interest, was
investigated computationally. As will be seen, in a sharp disagreement with the conclusions
of Morawetz, protonation at the N atom, but not the O of the carbonyl group, is revealed to
play a role in the amide cleavage.13
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CPA hydrolysis initially produces phthalic anhydride (PAn) and anthranilic acid
(AA) shown in Scheme 2. Under acidic conditions, the initially formed anhydride rapidly
hydrolyzes to phthalic acid (PAc).14,15 Figure 2 shows successive UV-Vis absorption spectra
at pH 3.5 for 2CPA decomposition. As can be seen, the UV-Vis spectral differences allow the
easy monitoring of the reaction progress and determination of first-order rate constants. The
sharp isosbestic point testifies to a simple reaction mechanism and purity of the reactants.
0
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30
40
50
Time / min
0.05
0
.00
2
50
275
300
325
350
375
Wavelength / nm
Figure 2. Successive UV-Vis absorption spectra for decomposition of 2CPA at 25°C, pH 3.5. The
inset shows the absorbance variation at 280 nm (left) and 330 nm (right) which are due phthalate
-
-
(
PAc ) and anthranilate (AA ), respectively.
-1
Observed rate constants kobs in s for the cleavage of 2CPA at 25.0°C as a function of
pH and pD are given in Figure 3. The bell-shaped plot supports and confirms the work of
Morawetz over fifty years ago. At pH 5 the half-life is about 100 min. For comparison,
hydrolysis of a simple amide such as benzamide is not observable at pH 5. As the pH is
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lowered below pH 5, the rate rises uniformly until its maximum is reached near pH 3.5.
Further pH reduction decreases the rate to give the left side of the bell-shaped pH/rate profile.
Full spectral data are given in the Supporting Information (SI).
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.2
.0
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
pH or pD
Figure 3. First-order rate constants kobs vs. pH or pD for the decomposition of 2CPA at 25°C. Curves
are nonlinear fittings using the equation given in the SI.
Nonlinear fitting of the pH/rate profile plot in Figure 3 according to Scheme 2
-4
-1
-3 -1
-5 -1
provides pK =3.3; pK =3.7; k =2.1x10 s ; k =4.0x10 s ; and k =1.4x10 s . The bell-
a1
a2
1
2
3
shaped curve can be understood by starting at pH 5, where 1c predominates, and moving to
the left. Lacking the possibility of intramolecular acid catalysis, the rate at pH 5 corresponds
to a slow k . As the pH is lowered, 1a, possessing both a carboxyl and a carboxylate, makes
3
its presence known kinetically. The associated rate constant, k , is fast and consistent with a
2
bifunctional catalysis where the carboxylate functions as a nucleophile and the carboxyl as a
general acid. A further pH drop (3.5 to 2.0) creates the non-ionic diacid species, 1, that is
associated with a slow k owing to the lack of a good anionic nucleophile to attack the amide
1
carbonyl. In summary, the impressive catalytic effect (i.e. an amide that cleaves with a 7
minute half-life) arises from a bifunctional catalysis contributed by the two carboxyl entities.
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Scheme 2. Species involved in the hydrolysis of 2-carboxyphthalanilic acid (2CPA).
O
O
O
Ka2
Ka1
N
N
N
H
H
H
CO -
2
CO H
2
CO H
CO2-
1a
2
CO2-
CO H
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H O
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OH
OH
O
+
H N
3
CO -
2
O
PAn
AA
Mechanistic mysteries persist. Does the nucleophilic carboxylate directly attack the
amide carbonyl, or is a carboxylate a general base that removes a proton from a water
molecule that in turn serves as the nucleophile? And do proton transfers, which are obviously
involved in the course of the mechanism, occur simultaneously with such an attack on the
amide carbonyl, or are these separate steps? To what amide atom, nitrogen or oxygen, is the
carboxyl proton delivered? Is there an intermediate, such as a tetrahedral intermediate,
involved in the reaction? What distances are involved in the transition state (or transition
states)? Such questions are addressed below with the aid of DFT calculations (see
computational details in the experimental section). Four mechanistic schemes are considered
and compared.
Direct attack by a water molecule with intramolecular catalysis was considered first
as similar reactions were reported for ester hydrolysis in related systems. 16–18. The attack is
general-base catalyzed by a well positioned carboxylate group in a transition state with a 7-
membered ring. This is accompanied by proton delivery from the general acid carboxyl group
to either (i) the carbonyl oxygen (TSw-1) or (ii) nitrogen atom (TSw-2) (Scheme 3). In both
-
cases, the final products are PAc and AA . Activation free energies, calculated by taking the
difference of the Gibbs free energy of the transition states and the reactant 1a coordinated to a
-1
-1
water molecule (see structure 1a.H O in the SI), are 29.9 kcal mol and 32.6 kcal mol ,
2
-1
respectively, more than 10 kcal mol higher than the experimental free activation energy of
-1
2
0.6 ± 2.1 kcal mol at 25 °C (see the Eyring plot in the SI). It should be noted that these
computational energies are underestimated as pointed out elsewhere.19 Therefore,
participation of a nucleophilic water can be ruled out.
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Scheme 3. Decomposition of 1a involving a nucleophilic water molecule with simultaneous general
base and general acid assistance to the oxygen (TSw-1) or nitrogen (TSw-2) atoms, respectively. Ball-
and-stick representations of transition states and respective IRC plots are given in the SI (see Figures
S1 and S2). Cartesian coordinates are given in the SI.


O
O
OH
H
O
O
O
O
O
O
H
H
O
H
1
1
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O
O
O
O
HO
HO
HO OH
r.d.s
N
N
N
H
H
H
TSw-1

O
O
H

O
O
O
O
O
H
O
O
H
HO
O
O
HO
O
OH
HO
O
H
r.d.s
+
N
H
O
N
H N
2
H
TSw-2
AA-
PAc
We then analyzed the carboxylate attack on the amide carbonyl. This attack is
accompanied by protonation of the negative charge developed on the amide oxygen by the
neighboring carboxyl group (Scheme 4). The activation free energy for TS-1, only 13.8 kcal
-1
-1
mol relative to 1a, leads to a 5-membered lactone-type structure I-1, itself 12.9 kcal mol
above reactant 1a. The 5-membered ring incorporates the classic "tetrahedral intermediate"
configuration commonly postulated on reactions of carboxylic acid derivatives. The question
now resides on decomposition of I-1 which certainly must involve activation of the leaving
group by protonation. This can occur by (i) a four membered transition state with a proton
coming from the neighboring –OH group (path a, TS-2) or (ii) through participation of a
water molecule which delivers a proton, via a six membered transition state, to the N atom
while removing a proton to restore the carbonyl group (path b, TS-3). Both transition states
-
‡´
-1
-1
lead to PAn+AA and the ΔG s are 46.9 kcal mol and 34.4 kcal mol for TS-2 and TS-3,
respectively, much too high to render path a and path b as likely mechanisms.
Scheme 4. Decomposition of 1a involving intramolecular proton transfer to carbonyl oxygen. For TS-
-
1, I-1, TS-2 and PAn+AA the reference is 1a while for TS-3 the reference is 1a.H O. For species
2
-
involved in path c the reference is I-2. The values given are DFT activation free energies in kcal mol
. The ball-and-stick models of all species are given in the SI.
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

O
H
O
O
O
H
path a
N
H
O
O
O
O
CO -
2
H
O
N
H
O

OH
O

O
TS-2
TS-1
N
46.90
O
N
H
PAn+AA-
1
3.79
H
O
O-
H
1a
I-1
H
O
O
5.76
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2.89
H
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N
H
O
CO2-
O
TS-3
H+
34.36
HO


O
O
H
 O
O

O
O

O

O
N
H
H
O
O
N
path c
OH
O
O
1
2
H2
O
O
N
TS-4
TS-5
O
N
O
H2
H2
.96
3.04
PAn+AA
I-2
I-3
O
3.00
-7.38
O
In seeking a plausible path for the collapse of I-1, we hypothesized that this species
can readily protonate via hydronium ions in solution, thereby entering path c in Scheme 4.
Nitrogen protonation of I-1 led to the tetrahedral intermediate I-2 which is then converted to
products PAn+AA through TS-4, I-3 and TS-5. The intrinsic reaction coordinate (IRC)
curves for these steps are given in Figure S3 and confirm that all species are connected. The
-1
computed activation free energy for the highest barrier (TS-5) is only 3.04 kcal mol ,
indicating that I-2 is quickly converted to products in an irreversible, barrier-less reaction.
The intermediate I-1 can be converted back to 1a or be protonated by hydronium ions
in a thermodynamically favorable diffusion controlled reaction and, therefore, does not
accumulate. Hence it is possible to apply the steady state approximation to I-1. It follows that
the apparent rate constant for conversion of 1a into PAn+AA via path c is given by Eq. 1,
where k and k are first-order rate constants for the forward and reverse reaction involving
4
-4
1
a and I-1, respectively, while k is the second order rate constant for the diffusion controlled
p
10
-1 -1
protonation of I-1 (approximately 10 M s ). Using the Eyring equation one can calculate k
4
푂
and k from the DFT activation energies. Since the observed rate constant 푘 is the product
-
4
표푏푠
푂
of 푘 and the molar fraction of 1a (Eq. 2), it is possible to simulate the pH rate profile for
푎푝푝
the mechanism involving I-1 and I-2. As can be seen in Figure 4, the simulated pH rate
profile is sigmoidal as opposed to the bell-shaped plot obtained experimentally. It is
therefore concluded that the mechanisms in Figure 4 are unlikely.
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푘4푘푝
[
H3O +
]
푂
푘
=
Eq. 1
Eq. 2
푎푝푝
H3O +
푘 ―4 +푘푝
[
]
1
푂
푂
푘
= 푘
표푏푠
푎푝푝
+
[
H O
]
퐾
a2
3
1
+
+
퐾
+
a1
[
H O
]
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.0
1
1
0
0
.5
.0
.5
.0
k
0
1
2
3
4
5
6
pH
Figure 4. Simulated pH rate profile for the stepwise decomposition of 1a involving path c in Scheme
-
4
-4
―1
10
4
푠
. The curve was simulated using Eq. 2 with K =5x10 , K =2x10 , 푘 = 477푠 , 푘 ―4 = 133푥10
a1
a2
4
―
1
10
-1 -1
, k =1x10 M s at 298 K.
p
As initial amide carbonyl protonation in Scheme 4 was not viable, we searched for 1a
decomposition mechanism involving nitrogen protonation as previously demonstrated to
4
occur with 2-carboxyl-N,N-diethylnaphthalamic (see Figure 1). Figure 5 shows the ball-and-
stick models of the calculated species found in this reaction path. In order to better visualize
the bond-forming and bond-breaking processes, the IRC plot for conversion of 1a into
-
PAn+AA is given in Figure 6.
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2
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3
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1
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1
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0
1
2
3
4
5
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8
9
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1
2
3
4
5
6
7
8
9
0
Figure 5. Ball-and-stick models of the stationary points found for the decomposition of 2CPA
involving intramolecular proton transfer to nitrogen. Values in parenthesis are the computed free
-1
energies (kcal mol ) relative to 1a. Hydrogens bound to carbon atoms were omitted for clarity
purposes. See the IRC curve of Figure 6 for a detailed description of the variation of bond lengths
along this reaction path.
The pathway for 1a decomposition shown in Figure 5 involves the stepwise C1-O3
bond formation and C1-N cleavage. The reaction starts with the reactive conformer Rconf
,
which is converted to I-3 thought transition state TS-5. As can be seen in the IRC analysis, in
TS-5 protonation of the amide nitrogen from the neighboring -CO H group is well advanced
2
(N-H1 and O1-H1 interatomic distances are 1.08 Å and 1.54 Å, respectively), the C1-N
double bond character has been considerably diminished (C1-N interatomic distances is 1.53
Å versus 1.36 Å in 1a), and an incipient bonding between the carboxylate and the amide
carbonyl carbon is observed (C1-O3 = 1.91 Å). In the high energy intermediate I-3 the C1-O3
and C1-N bonds are almost equidistant (1.55 Å and 1.60 Å, respectively). The intermediate is
decomposed through TS-6 with the main geometrical changes are the increase in the C1-N
distance and decrease in the C-O3 bond-length. The whole process occurs with a surprisingly
static amide carbonyl (C1-O1 is 1.26 Å in I-3 versus 1.22 Å in 1a).
1
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2
1
1
0
8
6
3
.0
TS-6
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
TS-7
I-4
I-4
2.8
C1-O3
2
2
2
2
1
1
.6
.4
.2
.0
.8
.6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
PAn+AA 14
1
2
N-H1
10
8
C1-N
1.4
C1-O2
6
1
conf
1.0
.2
O1-H1
4
R
-
8
-6
-4
-2
0
2
4
6
-6 -4 -2
0
2
4
6
8
10 12
IRC
Figure 6. Variation of bond lengths (dotted lines) and electronic energies (solid lines) along the IRC
for the decomposition of 1a via intramolecular N-protonation. The panels on the left and on the right
correspond to conversion of R I-4 and I-4  PAn+AA , respectively. The arrows indicate the
conf
-
conf
position of transition states, located at 0 bohr, R
taking 1a as reference. Atom numbering according to Figure 5.
and I-4. The electronic energy was computed
Proton transfer in the mechanism involving intramolecular N-protonation is consistent
with the solvent isotope effect of 1.90 observed in Figure 3. An attainable energy of 18.6 kcal
-
1
mol is involved in the first step (TS-6), while the subsequent decomposition of I-4 occurs
-1
thorough TS-7, which lies 18.9 kcal mol up in energy from 1a. This value is in good
agreement with the experimental one, the small differences in which are attributable to
explicit solvent effects which were not included in our calculations. The intermediate lies
-
1
only 0.6 and 1.0 kcal mol below TS-6 and TS-7, respectively. Therefore it is of fleeting
existence. It is either converted back to 1a or processed further into products.
The computed product in Figure 5 is an interacting complex formed by PAn+AA-.
-
1
The Gibbs free energy of this complex is 4.13 kcal mol above that of 1a. At first glance this
indicates that the equilibrium is shifted towards the reactant. However, one should bear in
mind that in solution PAn is quickly converted to phthalic acid14,15 consequently shifting the
equilibrium towards 1a decomposition.
Experimentally, the two competing paths for 1a hydrolysis, namely N- and O-
protonation, are difficult to delineate. However, a kinetic analysis using the activation
energies from the DFT results helps elucidate the question. Applying the steady state
approximation to I-3 it follows that the apparent rate constant for conversion of 1a into
1
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2
2
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2
2
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6
-
PAn+AA via I-3 is given by Eq. 3, where k and k are first-order rate constants for the
5
-5
forward and reverse reactions involving 1a and I-4, respectively, and k is the first order rate
6
constant for conversion of I-4 to products passing through TS-7. The simulated pH rate
profile of Figure 7 clearly resembles the bell-shaped experimental curve shown in Figure 3.
Moreover, at pH 3.5 the reaction involving N-protonation is approximately 50-fold faster than
O-protonation via reaction path c in Scheme 4. Thus, contrary to many speculations in the
classical literature, N-protonation in amide hydrolysis prevails.
0
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9
0
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2
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9
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0
푘5푘6
푁
푘
=
Eq. 3
푎푝푝
푘 ―5 +푘6
1
푁
푁
푘
= 푘
Eq. 4
표푏푠
푎푝푝
+
[
H O
]
퐾
a2
3
1 +
+
퐾
+
a1
[
H O
]
3
2
.5
2.0
1
.5
k
1.0
0.5
0
.0
0
1
2
3
4
5
6
7
pH
Figure 7. Simulated pH rate profile for the decomposition of 2CPA involving N-protonation
according to Figure 5. The curve was simulated using Eq. 4 with K =5x10 , K =2x10 , 푘 = 0.144
-4
-4
a1
a2
5
―1
10 ―1
10 ―1
푠
, 푘 ―5 = 181푥10 푠 , 푘 = 108푥10 푠 at 298 K.
6
The question remains as to the exact source of the observed rate acceleration. Since
the unsubstituted amide is inert to acetate, even at high concentrations, it is obvious that
intramolecularity is crucial. But this is a low-information statement, and one would prefer a
deeper understanding. In line with spatiotemporal considerations, mentioned in the beginning
of this paper, we believe that truly fast rates in solution, e.g. those associated with enzyme
8
catalysis (>10 ), arise from imposed separations among reacting atoms that approach van der
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Waals distances. We have published elsewhere numerous examples of spatiotemporal effects
1
,2
where contact distances lead to enzyme-like accelerations. It was thus incumbent on us to
examine the geometric properties of the reactive conformer Rconf shown in Figure 8. This
structure was uncovered was found by following the IRC in the reverse direction from TS-5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
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0
1
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(see Figure 6) followed by full optimization.
Figure 8. Structure of the reactive conformer Rconf involved in decomposition of 1a via intramolecular
N-protonation. Values are interatomic distances in Angstroms.
The conformer Rconf is a local minimum only 2.52 kcal mol above the 1a global
minimum. The interatomic distance between the hydrogen atom of the carboxyl group and
the amide nitrogen is 2.23 Å. More importantly, the nucleophilic carboxylate oxygen O3 is
separated from the amide carbonyl carbon electrophile by only 2.78 Å, close to the diameter
of water. In addition, the nucleophilic O3 is directly above the plane of the amide, leading to
a very significant orbital overlap (∠O3···C1=O2 is 106.45 °). Besides, the C1-N bond length
of 1.37 Å is slightly longer than the typical length observed in amides. The spatial conditions
for a fast reaction according to spatiotemporal theory are clearly in place.
-1
In order to access the life-time of reactive 2CPA conformers, we performed molecular
dynamics simulations on 200 ns time scales of Rconf with explicit solvation using the TIP3P
water model. In total, 400,000 frames were obtained. A hydrogen bond analysis revealed that
the N-H1 interatomic distance is equal or less than 3.0 Å in 14.21 % of the frames, and the
average distance is 2.69 Å. A similar analysis for O2-H1 revealed that this intramolecular
hydrogen bonding is much less frequent, being observed in only 0.41 % of the frames (the
average distance is 2.77 Å). These results indicate that 2CPA is very prone to the N-
protonation reaction path. In addition, analogously to the near attack conformer concept
20
introduced by Bruice several years ago, we analyzed the probability of finding reactive
states that simultaneously meet the following criteria: N-H1 and C1-O3 interatomic distances
1
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smaller than 3.0 Å and a ∠O3···C1=O2 angle within 107.0 ± 5.0º. These criteria are
simultaneously observed in 1.61 % of the frames, whereas the average lifetime for these
states is 1.2 ps. Hence, the temporal component of a spatiotemporal effect is not ideal. Since
reactive sites are of fleeting existence among other conformations, a proper contact distance
was present but not permanently enforced. If the rotations about the single bonds had been
restricted, as they are at active sites of enzymes, true enzyme-like accelerations would, we
feel certain, have been achieved.
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EXPERIMENTAL SECTION
Materials. All reagents were analytical grade. Deionized water was used in all
measurements. The following buffering systems were used in the kinetic studies: chloroacetic
acid (pH 2.00-3.75), acetic acid (pH 4.00-5.50) and Bis-Tris (pH 6.00-7.00). All buffered
-1
solutions had concentrations of 0.01 mol.L and the pH and pD values were adjusted with
NaOH/HCl aqueous solutions or NaOD/DCl in D O, respectively.
2
Methods. UV-Vis spectra were taken in a Cary 50 spectrophotometer coupled to a Varian
PCB 1500 thermostatic bath with Peltier water system. Kinetic runs for hydrolysis of 2-
carboxyphthalanilic acid (2CPA) were carried out under first-order conditions. The reactions
were started by adding 10 µl of a 0.01 M stock solution of 2CPA in acetonitrile to 3 ml of the
aqueous buffered solutions in a quartz stoppered cuvette (10 mm path length). The
appearance of anthranilic acid from 2CPA was followed by the increase in absorbance at 330
nm. All reactions gave strict first-order kinetics over more than five half-lives. Unless
otherwise specified, reactions were carried out at 25ºC. Activation parameters were
determined from 25°C to 55ºC using the Eyring equation with k values.
2
Computational. DFT calculations were performed using the B97X-D functional21,22 with
the 6-311++G(d,p)23–27 basis set using GAUSSIAN 09 A.02 package implemented in Linux
28
operating systems. The default parameters for convergence, were used; convergence on the
-9
density matrix was 10 atomic units, the threshold value for maximum displacement was
.0018 Å, and the maximum force 0.00045 Hartree/Bohr. Stationary points on the potential
0
energy surface were identified by frequency calculations at 1 atm and 298.15 K. All
optimizations and frequency calculations were performed using the Solvation Model Density
(
SMD) of Truhlar and coworkers.29 The transition states were identified by their single
imaginary frequencies, whereas reactants, intermediates and products show no imaginary
frequency. Reaction paths were confirmed by IRC analysis at the at the SMD/B97X-D/6-
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1+G(d,p) using the default parameters implemented in Gaussian 09 starting from transition
states. The global minimum for 1a was located on the relaxed potential energy surface
constructed by varying the dihedral angles between both aromatic rings and the amide plane
(Figure S4). Reported relative Gibbs free energies were calculated by taking the difference
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
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3
3
3
3
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0
1
2
3
4
5
6
7
8
9
0
between the sum of electronic and thermal free energies ( + G ) of stationary points.
0
corr
Molecular dynamics simulations were performed using the pmemd.cuda software from
3
0
31
AMBER 18 in a NVidia GTX 1070 graphic card. The GAFF force field and TIP3P water
model32 were used. From global minimum structure 1a, the atomic partial charges were
calculated with RESP33 procedure, Antechamber34 and Gaussian 09 softwares.28 This
structure was solvated in a periodic orthorhombic box with 1651 waters and approximately
4
0x35x35 ų (after equilibration) and more than 20 Šfrom a periodic image. Chloride
counter ions were added to neutralize the total charge of the box using the TIP3P-specific ion
35
parameters from Joung and Cheatham. The production phase was simulated in an NPT
3
6
-1
37
ensemble, Langevin thermostat 298.15 K, γ = 1 ps , Monte Carlo barostat, Lennard-Jones
3
8
cutoff 9 Å and Particle Mesh Ewald (PME) long-range eletrostatic algorithm. Because we
were interested in conformational states involving hydrogen atoms, no acceleration
algorithms involving hydrogen were used, so the time step was 0.5 fs. All the trajectory
analyses were performed using the CPPTRAJ V4.20.2 software.39
General Procedure for the Synthesis of Substrate. Preparation of 2-phthaloimidobenzoic
acid (2PIB) and 2-carboxyphthalanilic acid (2CPA). The procedure was adapted from
Wiklund et al.40
2
-phthaloimidobenzoic acid (2PIB). In a round bottom flask 6 mL of acetic acid were heated
to reflux (oil bath, 120 °C). Then phthalic anhydride (1.48 g, 10 mmol) and anthranilic acid
1.25 g, 9.09 mmol) were added. The reaction was refluxed for 15 hours. After cooling to
(
room temperature, the crude mixture was poured into 10 mL of distilled H O and the
2
precipitate formed was filtered, washed with water and dried under reduced pressure yielding
1
the pure imide (2PIB). Yield = 1.28 g (53 %), gray solid. m.p. = 216-218°C. H NMR (200
MHz, DMSO-d ) δ 13.13 (br s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 8.02-7.90 (m, 4H), 7.78 (t, J =
6
1
3
7
.5 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H). C{1H} NMR (50 MHz,
-
DMSO-d ) δ 167.1, 166.1, 134.8, 133.0, 131.8, 131.5, 131.0, 130.7, 129.3, 123.5. FTIR (cm
6
1)
: 3093 (br, w), 2752 (w), 2610 (w), 1725, 1699, 1601, 1494, 1387, 1293, 1281, 1235, 1117,
1071, 895, 756, 718.
1
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-carboxyphthalanilic acid (2CPA). In a round bottom flask 6 mL of distilled water, 2PIB
(300 mg, 1.12 mmol) and 4 equivalents of NaOH (179 mg, 4.49 mmol) were stirred at room
temperature for 18 hours. The brownish solution was acidified (pH < 2) with conc. HCl
giving a precipitate that was filtered, washed with HCl 0.2 M and dried under reduced
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pressure. Yield = 270 mg (84 %), white solid. m.p. = 165-167°C. H NMR (200 MHz,
DMSO-d ) δ 13.40 (br s, 2H), 11.56 (s, 1H), 8.64 (d, J = 8.3 Hz, 1H), 8.04 (d, J = 7.8 Hz,
6
1
3
1
H), 7.89 (d, J = 7.0 Hz, 1H), 7.71-7.59 (m, 4H), 7.22 (t, J = 7.6 Hz, 1H). C{1H} NMR (50
MHz, DMSO-d ) δ 169.7, 167.6, 167.0, 141.1, 138.1, 134.2, 131.9, 131.2, 130.6, 130.2,
6
-1
1
29.8, 127.3, 123.0, 119.9, 116.5. FTIR (cm ): 3039 (br), 2646 (w), 2598 (w), 1716, 1687,
1637, 1607, 1590, 1533, 1454, 1366, 1326, 1227, 1154, 757, 726.
ASSOCIATED CONTENT
Supporting Information
Spectroscopic data (UV-Vis analysis, H, 13C{1H} NMR spectra and FTIR), activation
parameters, relaxed potential energy surface for 2CPA monoanion, additional IRC plots,
Cartesian coordinates for all DFT structures and analysis of molecular dynamics trajectory
data.
1
AUTHOR INFORMATION
Corresponding Authors
E-mail: bruno.souza@ufsc.br
E-mail: menger@emory.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES) - Finance Code 001. We are also grateful to INCT-Catálise,
FAPESC and CNPq for financial support.
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