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Prediction of Hydrolysis Pathways and Kinetics for Antibiotics under
Environmental pH Conditions: A Quantum Chemical Study on
Cephradine
Haiqin Zhang, Hongbin Xie, Jingwen Chen,* and Shushen Zhang
Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology,
Dalian University of Technology, Dalian 116024, China
S Supporting Information
*
ABSTRACT: Understanding hydrolysis pathways and kinetics of many antibiotics that have multiple hydrolyzable
functional groups is important for their fate assessment.
However, experimental determination of hydrolysis encounters
difficulties due to time and cost restraint. We employed the
density functional theory and transition state theory to predict
the hydrolysis pathways and kinetics of cephradine, a model of
cephalosporin with two hydrolyzable groups, two ionization
states, two isomers and two nucleophilic attack directions.
Results showed that the hydrolysis of cephradine at pH = 8.0
proceeds via opening of the β-lactam ring followed by
intramolecular amidation. The predicted rate constants at
different pH conditions are of the same order of magnitude as
the experimental values, and the predicted products are confirmed by experiment. This study identified a catalytic role of the
carboxyl group in the hydrolysis, and implies that the carboxyl group also plays a catalytic role in the hydrolysis of other
cephalosporin and penicillin antibiotics. This is a first attempt to quantum chemically predict hydrolysis of an antibiotic with
complex pathways, and indicates that to predict hydrolysis products under the environmental pH conditions, the variation of the
rate constants for different pathways with pH should be evaluated.
■
INTRODUCTION
Antibiotics have been frequently detected in rivers, lakes,
regional discharges and coastal waters.1 The presence of
antibiotics in the aquatic environment attracts extensive
concern, as antibiotics can induce bacterial resistance even at
environmental concentrations.2 The molecular structures of
many antibiotics contain hydrolyzable functional groups (e.g.,
ester, amide, imide, and halogen), rendering antibiotics
susceptible to hydrolytic degradation.3 Hydrolysis of some
antibiotics may lead to formation of metabolites with higher
environmental persistence and some antimicrobial properties.4
Thus, it is of great importance to investigate the hydrolysis
kinetics and products of antibiotics for their ecological risk
assessment.
According to the limited experimental data,5 hydrolysis halflives (t1/2) for organic chemicals can vary from seconds to years.
Experimental determination of the hydrolysis kinetics is
generally supposed to be time-consuming if the hydrolysis
rates are very slow. Amide and ester moieties are known as the
functional groups for many drug molecules.6 These hydrolyzable functional groups also appear frequently in many classes
of antibiotic molecules (e.g., penicillins, cephalosporins,
macrolides, and lincosamides). Thus, the experimental
determination alone cannot meet the need of ecological risk
© 2015 American Chemical Society
assessment of so many antibiotics as well as other hydrolyzable
organic pollutants.
Although linear free-energy relationships (LFERs) can be
employed to estimate hydrolysis rate constants (kH),5,7 the
construction of LFER models relies on experimental databases,
and the utility of LFERs is limited by their applicability
domains.8,9 Moreover, most LFER models can hardly provide
information on reaction pathways. As far as we know, there are
no available LFER models that can predict kH of antibiotics. In
contrast to LFERs, a combined use of the density functional
theory (DFT) and transition state theory may predict kH and
hydrolysis pathways. For example, DFT was successfully
employed to predict kH of chlorambucil and hexamethylphosphoramide.10,11 However, to the best of our knowledge, few
studies employed DFT to predict hydrolysis of antibiotics,
especially antibiotics with multiple hydrolyzable functional
groups.
It became the purpose of this study to test the feasibility of
using DFT to predict hydrolysis pathways and kinetics of
antibiotics. Cephradine (velosef), a cephalosporin antibiotic
Received:
Revised:
Accepted:
Published:
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November 4, 2014
January 7, 2015
January 15, 2015
January 15, 2015
DOI: 10.1021/es505383b
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Environmental Science & Technology
activation (ΔG‡) to distinguish favorable/unfavorable hydrolysis pathways and to predict the kH values. Then the predicted
rate constants were compared with experimental results.
with two hydrolyzable functional groups was selected as a case
for the study. The cephalosporin antibiotic has two amide
groups (the C8N5 lactam and the C14N13 amide) and an αamino group in the side chain (Figure 1). Cephalosporin
■
MATERIALS AND METHODS
Computational Details. All the DFT calculations were
performed with the Gaussian09 software package.16 All
geometries of reactants, transition states (TSs), intermediates,
and products were calculated at the B3LYP/6-311++G(d,p)
level. To evaluate the effects of functionals and basis sets on the
results, different basis sets [6-311++G(d,p), 6-311++G(2d,2p),
6-311++G(2d,2pd), 6-311++G(2df,2pd), 6-311++G(3df,2pd),
6-311++G(3df,3pd)] and functionals [M062X and B3LYP-D3,
where “D3” means dispersion correction introduced by
Grimme et al.17] were selected to calculate the important
species in the rate-determining step of the most likely pathways.
The B3LYP and M062X hybrid functionals have been
successfully used to predict kH of hexamethylphosphoramide
and chlorambucil, respectively.10,11 The integral equation
formalism of the polarized continuum model (IEFPCM)
based on the self-consistent-reaction-field method was
employed to mimic the water solvent effect.18 As definition
of solute cavity is an important factor in determining the
accuracy of the IEFPCM, we evaluated three different atomic
Figure 1. Molecular structure of cephradine (molecular weight =
349.4) and its hydrolyzable functional groups.
antibiotics (e.g., cephradine, cephalexin, and cephaloglycin)
have been frequently detected in environmental waters, with
concentrations up to μg·L−1.1,12−15 We calculated the Gibbs
free energies of reaction (ΔG) and the Gibbs free energies of
Figure 2. Potential hydrolysis pathways and the corresponding Gibbs free energies (ΔG, kJ/mol) for hydrolysis of cephradine (The pathways
marked with green are thermodynamically favorable, and those with red are unfavorable.).
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9.0). NaN3 (200 mg·L−1) was employed as a biocide to inhibit
biodegradation of the target compound. The reaction vessels
were triangular bottles wrapped with aluminum foil to prevent
photodegradation. To facilitate comparison of the experimental
kH with the predicted values, the experimental temperature was
kept at 298.15 ± 1 K by using a water bath. Periodically
throughout the experiment, 1 mL of the reaction mixture was
withdrawn for quantification purpose. A Hitachi HPLC
equipped with an Agilent Eclipse XDB−C18 column (4.6 ×
150 mm2, 5 μm) was employed for the analysis. The mobile
phase consisted of 30% methanol and 70% sodium acetate
(0.02 M), running at a flow rate of 0.8 mL·min −1. The
injection volume was 20 μL. First-order reaction kinetics was
employed to calculate the hydrolysis rate constants.
radii for defining the solute cavity, which is detailed in the
Supporting Information (SI). TSs were verified by both
intrinsic reaction coordinate (IRC)19 and frequency (i.e., only
one imaginary frequency) calculation. To facilitate comparison
of the calculated kH with the experimental values, the calculated
standard state ΔG and ΔG‡ values (at 298.15 K) were
corrected from 1 atm to 1 mol·L−1, by adding 7.9 kJ·mol −1 for
the reaction A → B + C, and reducing 7.9 kJ·mol −1 for the
reaction A + B → C and left unchanged for the reaction A →
B.20
As cephradine has two ionizable sites (pKa1 = 2.6 ± 0.1 for
the carboxyl, pKa2 = 7.3 ± 0.1 for the α-amino),21 it exists in
two ionic forms at the environmental pH range (6.0−8.5):
zwitterion (AH±) and anion (A−). As a result, cephradine has
five possible hydrolysis pathways (Figure 2): hydrolysis of the
C8N5 lactam of AH± or A−, hydrolysis of the C14N13
amide of AH± or A−, as well as intramolecular nucleophilic
attack of the α-amino group toward β-lactam of A−, i.e.,
intramolecular aminolysis.22
By taking into account the acid-catalyzed, neutral and basecatalyzed reactions, we can express kH at a given pH as kH =
kH+·[H+] + kN + kOH‑·[OH−],5 where kH+ (L·mol−1·s−1) is the
second-order rate constant for the acid-catalyzed hydrolysis of
AH±; kN (s−1) is the sum of the first-order rate constants for the
water assisted hydrolysis of AH± and A−, and intramolecular
aminolysis of A−; kOH‑ (L·mol−1·s−1) is the second-order rate
constant for the base-catalyzed hydrolysis of AH± and A−.
Yamana and Tsuji observed that cephradine is fairly acid
stable at pH < 5,22 which was also confirmed by our preliminary
experiments (SI Figure S3). Schwarzenbach et al. also noted
that for all the hydrolyzable compounds, reactions with OH−
(base catalysis) is important even at conditions of pH < 7, and
acid catalysis is relevant only for compounds showing rather
slow hydrolysis kinetics.5 Thus, it is reasonable to assume that
kH+ ≈ 0 for cephradine, and the kH expression can be simplified
to kH = kN + kOH‑·[OH−].
ΔG values for all the possible hydrolysis pathways were
calculated from the free energy differences between the
products and reactants, and employed to distinguish
thermodynamically favorable/unfavorable pathways. For the
thermodynamically favorable pathways, the ΔG‡ values were
calculated to distinguish kinetically favorable/unfavorable
pathways.
Nucleophilic attack of H2O, OH−, or −NH2 on the reaction
sites can take place from either the exo α side (where the lone
pair of electrons on the β-lactam nitrogen is located) or from
the endo β side.21 Therefore, the different directions of
nucleophilic attack were considered in the calculation (Figure
1). The rate constants for the attack from the preferential
directions were calculated by applying the pseudo steady state
approximation (detailed in the SI).
Experimental Methods. Cephradine (purity ≥99%) was
purchased from Zhejiang Anglikang Pharmaceutical Co., Ltd.
Methanol, acetic acid, phosphate, boric acid, sodium hydroxide,
sodium chloride, and hydrochloric acid were purchased from
Tianjin Kemiou Chemical Reagent Company. All the other
chemicals are of analytical or HPLC grade of purity. Ultrapure
water (18 MΩ) was obtained with an OKP ultrapure water
system of Shanghai Lakecore.
Hydrolysis of cephradine was measured in buffer solutions
with a constant ionic strength (0.06 mol·L−1) adjusted by
sodium chloride. The buffers used were acetic acid/acetate (pH
5.0), phosphate (pH 6.3, 7.0, 8.0), and boric acid/borate (pH
■
RESULTS AND DISCUSSION
Thermodynamics. As intramolecular hydrogen bonds can
be formed between the H atom of the α-amino group and the
O atom in the amide, cephradine (A− and AH±) can isomerize
between cis- (Ac− and AHc±) and trans-configurations (At− and
AHt±). If the energy difference is sufficiently large, then the less
stable isomer can isomerize to the more stable isomer.22 We
calculated the ΔG values for the isomerism (SI Figure S4). On
the basis of the ΔG values, the equilibrium constants (K) for
the interconversion were calculated. According to the calculated
K values, At− is more stable than Ac−, with At− accounting for
99.98% of the anions (A−). While for the zwitterions (AH±),
AHc± (>99.99%) is more stable. Thus, At− and AHc± were
selected for the further calculations. The calculated ΔG values
for all the hydrolysis pathways are shown in Figure 2. On the
basis of the ΔG values, we identified the thermodynamically
favorable pathways that are marked with green in Figure 2.
Hydrolysis of the C8N5 lactam. We identified two
nucleophilic attack processes from either the α or β side. One is
a direct hydrolysis process, in which H and −OH of H2O are
concertedly added to the N5 and C8 sites, respectively. The
other is an indirect hydrolysis process, in which −OH of H2O is
added to the C8 site, while H of H2O first transfers to the O12
position of the carboxyl group and then further transfers to the
N5 site. As can be seen from Figure 3(a,b), for both At− and
AHc±, the overall ΔG‡ values for the α side attack are lower
than those for the β side attack. Thus, the attack of H2O on the
C8N5 lactam occurs preferentially from the α side. More
importantly, the overall ΔG‡ values in the direct hydrolysis
processes of At− and AHc± are much higher than those in the
corresponding indirect hydrolysis processes for the nucleophilic
attack from either the α or β side, indicating that the indirect
hydrolysis process is more favorable. Thus, the carboxyl group
can accelerate water assisted hydrolysis of cephradine.
According to Pliego Jr.,23,24 explicit water molecules may
have impacts on the DFT calculated ΔG‡. We further
considered the effects of explicit water molecules on the
catalytic role of the carboxyl group. The results show that the
involvement of explicit water molecules does not have a great
impact on the process (as detailed in the SI). To the best of our
knowledge, this is the first time that the role of the carboxyl
group in the hydrolysis of cephradine is pointed out. As all the
cephalosporins and penicillins have a common parent structure,
it can be inferred that the carboxyl group also plays a catalytic
role in the hydrolysis of other cephalosporin and penicillin
antibiotics.
The base-catalyzed hydrolysis also involves the direct and
indirect processes. For both the direct and indirect processes,
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Figure 3. Schematic free energy surfaces for the nucleophilic attack of OH−/H2O to At−/AHc± species calculated at the B3LYP/6-311++G(d,p) level
(The total energy of the reactants is set as zero. The symbols RCs, IMs and TSs stand for reactant complexes, intermediates and transition states,
respectively. The corresponding geometric structures are shown in SI Figures S5−S14).
OH− attacks on the C8 site in the initial step. Subsequently, H
of OH− transfers directly to the N5 site in the direct process;
while in the indirect process, H first transfers to the carboxyl
group and then further to the N5 site. According to Figure
3(c,d), the addition of OH− to the C8 site is a rate-determining
step, and the overall ΔG‡ values for OH− attack from the α side
are lower than those from the β side. This indicates that OH−
also prefers to attack the C8N5 bond from the α side. In
addition, the ΔG‡ values of the direct processes are slightly
higher than those of the indirect processes, demonstrating that
the carboxyl group also participates in the base-catalyzed
hydrolysis processes.
Intramolecular Aminolysis. Our calculation showed that
the intramolecular aminolysis is initiated by the attack of −NH2
on the C8 site, and can occur from either the α or β side. The
schematic free energy surfaces and geometries for the
intermediates and transition states are presented in SI Figures
S15−S17. The overall ΔG‡ value for the intramolecular
aminolysis from the β side (127.8 kJ/mol) is lower than that
from the α side (219.4 kJ/mol). Llinás et al. investigated
intramolecular aminolysis of cephaloglycin by a semiempirical
AM1 method, and also found that the intramolecular
aminolysis from the β side is more favorable than from the α
side.21 However, the overall ΔG‡ value for intramolecular
aminolysis is slightly higher than that of the indirect hydrolysis
processes, although some previous studies purposely investigated the intramolecular aminolysis of cephaloglycin.21
Hydrolysis of the C14N13 Amide Bond. As shown in
Figure 2, water assisted hydrolysis of the C14N13 amide bond
is thermodynamically unfeasible (ΔG > 0). Therefore, only the
base-catalyzed hydrolysis of the C14N13 amide bond was
considered. The attack of OH− on the C14 site can also occur
from either the α or β side. According to Smith,25 the basecatalyzed hydrolysis of the amide bond follows a BAC2
mechanism. The schematic free energy surfaces and geometries
for the intermediates and transition states involved in the BAC2
mechanism are presented in SI Figures S19−S21. The overall
ΔG‡ value for the α side attack (136.5 kJ/mol) is lower than
that for the β side (152.4 kJ/mol). More importantly, the
overall ΔG‡ value for the base-catalyzed hydrolysis of the C14
N13 amide bond is much higher than that for the base-catalyzed
hydrolysis of the C8N5 lactam bond (70.7 kJ/mol), revealing
that the base-catalyzed hydrolysis most likely occurs via OH−
attacks on the C8N5 lactam bond. Thus, in the subsequent
rate constants calculation, we did not consider the hydrolysis of
the C14N13 amide bond.
Prediction of kH at Different pH Values. On the basis of
the identified pathways, the different rate constants for OH−/
H2O attack on the At−/AHc± species from the α side and -NH2
attack on At− from the β side were calculated (SI Table S1). On
the basis of these rate constants, pseudo-first-order rate
constants (kA−H2O, kAH±H2O, kA−OH−, kAH±OH−, and kA−NH2) for
the hydrolysis and intramolecular aminolysis were calculated at
different pH levels by the following formulas:
k A−H2O = kAtα ,H2O·
[A−]
[A all]
(1)
k A−OH− = kAtα ,OH·
[A−]·[OH−]
[A all]
(2)
k AH±OH− = kAHcα ,OH·
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[AH±]· [OH−]
[A all]
(3)
DOI: 10.1021/es505383b
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k AH±H2O = kAHcα ,H2O·
k A−NH2 = k tβ NH2·
−
[AH±]
[A all]
kH = kAH±H O + kAH±OH + kA −OH− + kA −H O + kA −NH
2
2
(4)
[A−]
[A all]
2
(6)
The predicted logkH values from the different DFT methods
are listed in Table 1 and SI Table S11, together with the
corresponding values determined experimentally. It can be
deduced that the calculated logkH values are highly sensitive to
the different functionals and the atomic radii for defining solute
cavity. The combined use of the B3LYP functional with the
UFF atomic radii26 yields logkH values that are the closest to
the experimental ones. The selection of basis sets also
influences the calculated logkH values, and the logkH values
from the 6-311++G(2d,2p) basis set are closer to the
experimental ones. More importantly, the predicted logkH
values at the B3LYP/6-311++G(2d,2p) level with the UFF
atomic radii 26 are in reasonable agreement with the
experimental ones.
Subsequent Reactions for the Primary Intermediates.
As shown in Figure 4, the contribution of kAH±H2O, kAH±OH− and
kA−OH− to kH is more evident than the other pathways. A main
product from these hydrolysis pathways of the C8−N5 lactam is
2H-1−3-Thiazine-2-acetic acid (HTA, pKa2 = 7.28), which has
two ionic forms in the environmental pH range: HTA− and
HTA2−. We further investigated the subsequent transformation
of HTA. As shown in Figure 2, only intramolecular amidation
of HTA− and base-catalyzed hydrolysis of HTA2− are
thermodynamically favorable. On the basis of the schematic
free energy surfaces and geometries for the intermediates and
transition states (SI Figures S22−S25), the overall ΔG‡ value
for the intramolecular amidation of HTA− to form 2H-1,3thiazine-carboxylic acid (HTCA) is 82.1 kJ/mol, implying the
amidation of HTA− is kinetically feasible. However, it is
kinetically unfeasible for the base-catalyzed hydrolysis of
HTA2− to produce 1,4-cyclohexadiene-1-acetic acid (CAA)
and 2H-1,3-thiazine-2-acetic acid (HTAA) due to the
corresponding high ΔG‡ value (157.9 kJ/mol). Thus, the
final product for hydrolysis of cephradine is HTCA. Previous
experiments on hydrolysis products identification of cephradine
have detected HTCA as the only product.27,28
Yamana and Tsuji investigated the hydrolysis of cephaloglycin in dioxane-water solutions (pH = 8.0), and observed
that the hydrolysis rate increased with the proportion of
dioxane (an aprotic solvent that can form H-bonds with water),
and concluded that water did not play a significant role in the
hydrolysis and intramolecular aminolysis.22 However, the
experimental observation cannot preclude the base-catalyzed
hydrolysis pathway of cephaloglycin. In the current study, we
found that the hydrolysis of cephradine at basic conditions
proceeds via the opening of the β-lactam ring followed by
intramolecular amidation between the α-amino and carboxylate
groups, and the process leads to HTCA that can also be formed
from intramolecular aminolysis of cephradine. As the reaction
rate of intramolecular aminolysis is much lower than that of the
(5)
where kAtα,H2O and kAHcα,H2O are the rate constants for the attack
of H2O on At− and AHc± from the α side, respectively; kAtα,OH
and kAHcα,OH are the second-order rate constants for the attack
of OH− on At− and AHc± from the α side, respectively; ktβNH2 is
the rate constant for the attack of −NH2 on At− from the β
side; and Aall stands for the total cephradine. The variations of
these pseudo-first-order rate constants with pH are shown in
Figure 4.
Figure 4. Variations of logarithms of the pseudo-first-order rate
constants for water-assisted hydrolysis of At− (kAH±H2O) and AHc±
(kAH±H2O), base-catalyzed hydrolysis of At− (kA−OH−) and AHc±
(kAH±OH−), and intramolecular aminolysis of At− (kA−NH2) at different
pH levels.
At acidic conditions, the kAH±H2O value is higher than those of
other pathways, indicating that H2O attack on the C8N5
lactam of AH± is a predominant hydrolysis pathway. Under
neutral and basic conditions, the predominant pathway is the
base-catalyzed hydrolysis, as the kAH±OH− and kA−OH− values are
greater than those of the other pathways, and the kA−NH2 value
is the smallest. This study indicates that it is necessary to
calculate the pH dependence of pseudo-first-order rate
constants corresponding with different pathways, so as to
evaluate the hydrolysis pathways of organic pollutants, as was
also pointed out by Blotevogel et al.11 As far as we know, this is
the first attempt to quantum chemically calculate the variation
of pseudo-first-order hydrolysis rate constants of an antibiotic
with pH.
The kH value was predicted by the following equation:
Table 1. Experimental Hydrolysis Rate Constants (s−1) of Cephradine at 298.15 K Compared with Those Calculated by the
B3LYP Method with the UFF Atomic Radii
a
pH
5.0
6.3
7.0
8.0
9.0
logkH (experimental)a
logkH [6-311++G(2d,2p)]
logkH [6-311++G(d,p)]
−6.96
−7.22
−6.72
−6.44
−6.63
−6.05
−6.09
−6.13
−5.53
−5.95
−5.70
−5.03
−5.92
−5.28
−4.44
According to the experimental kH values, the hydrolysis half-lives of cephradine can vary from 7 (pH = 9.0) to 72 days (pH = 5.0).
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■
ACKNOWLEDGMENTS
We thank Prof. W. Peijnenburg (Leiden University) for
constructive suggestions. This study was supported by the
National Basic Research Program (2013CB430403) and the
National Natural Science Foundation of China (21137001,
21325729).
other pathways in the considered pH range (Figure 4), the
intramolecular aminolysis does not contribute much to the
hydrolysis of cephradine. Since both cephaloglycin and
cephradine contain the α-amino groups in their side chains
and have similar molecular structures, it can be concluded that
hydrolysis of cephaloglycin may proceed via the same pathways
as cephradine, i.e., opening of the β-lactam ring followed by
intramolecular amidation between the α-amino and carboxylate
groups. It must be emphasized that a sound understanding of
the hydrolysis pathways of cephaloglycin and other cephalosporins is of great importance, as the overall rate constant kH
is mainly determined by the pseudo-first order rate constants
for the main pathways.
Environmental Implications. Herein, we employed the
DFT calculation to predict the hydrolysis pathways and kinetics
of cephradine, a model of cephalosporin that has two
hydrolyzable groups, two ionization states, two isomers, three
general-hydrolysis pathways and two directions of OH−, H2O,
and −NH2 nucleophilic attack. The predicted rate constants are
of the same order of magnitude as the experimental values, and
the predicted products were also confirmed by experimental
findings. There are no previous quantum chemical calculation
studies dealing with hydrolysis of antibiotics with so many
hydrolysis pathways. Many organic pollutants have only one
hydrolyzable functional group with a hydrolysis pathway that is
less complicated than those of cephradine. Thus, this study
implies that the DFT calculation method can be extended to
predicting hydrolysis rate constants and pathways of >100
species of cephalosporins29 as well as other hydrolyzable
organic pollutants.
This study also indicates that in order to assess hydrolysis
rates of organic pollutants, the variations of the second-order
rate constants (ki) for the different hydrolysis pathways with
pH should be evaluated, so as to identify the main hydrolysis
pathways/products under environmental pH conditions, as kH
is mainly determined by the pseudo-first order rate constants
for the main pathways. As far as we know, there are no previous
quantum chemical calculations that evaluate the contribution of
different hydrolysis pathways of antibiotics under environmental pH conditions. Only by experiment, the dominant
hydrolysis pathways can hardly be identified. Thus, the DFT
calculation in this study shows superiority in this regard. This
study also identified the role of the carboxyl group in the
hydrolysis of cephradine. We infer that the carboxyl group also
plays a catalytic role in the hydrolysis of other cephalosporin
and penicillin antibiotics.
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ASSOCIATED CONTENT
* Supporting Information
S
Details on selection of atomic radii, calculation of rate
constants, effects of explicit water molecules, experimental
data, main geometric structures and schematic free energy
surfaces; Figures S1−S25 and Tables S1−S12, as mentioned.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
Article
AUTHOR INFORMATION
Corresponding Author
*Phone/fax: +86-411-84706269; e-mail: [email protected].
Notes
The authors declare no competing financial interest.
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