Substructure Reactivity Affecting the Manganese Dioxide Oxidation of

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Substructure Reactivity Affecting the Manganese Dioxide Oxidation of Cephalosporin Ming-Hao Hsu, Ting-Hao Kuo, Yung-En Chen, Ching-Hua Huang, Cheng-Chih Hsu, and Angela Yu-Chen Lin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02448 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Environmental Science & Technology

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Substructure Reactivity Affecting the Manganese Dioxide Oxidation of Cephalosporins

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Ming-Hao Hsu†, Ting-Hao Kuo‡, Yung-En Chen†, Ching-Hua Huang§, Cheng-Chih

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Hsu‡, Angela Yu-Chen Lin*,†

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†

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71-Chou-shan Road, Taipei 106, Taiwan, ROC

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‡

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan, ROC

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§

School of Civil and Environmental Engineering, Georgia Institute of Technology,

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Atlanta, Georgia 30332, United States

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Graduate Institute of Environmental Engineering, National Taiwan University,

(*Corresponding author: [email protected] Tel.: ±886-2-3366-4386)

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Submitted to

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ABSTRACT

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Cefotaxime (CTX), cephalexin (CFX), cephradine (CFD), cephapirin (CFP), and

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cefazolin (CFZ), were selected as target cephalosporin antibiotics to study their

18

oxidative transformation by δ-MnO2. Although they all have the same core structure

19

(7-aminodesacetoxycephalosporanic acid), very different MnO2 oxidation rates were

20

observed at pH 4 (the initial reaction rate constant kinit ranged from 0.014 to 2.6 hr-1).

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An extensive investigation of the substructure compounds and byproduct analysis

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revealed that the oxidation mainly occurred at the following two sites on the core

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structure: (1) the sulfur atom in the cephem ring and (2) the carbon-carbon double

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bond (C=C) and its proximal carboxylic acid group. In the case of (2), when there is

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an acetyloxymethyl group at the C-3 position of the core structure, the formation of

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the keto-sulfone byproducts was inhibited. The overall results indicated that a

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substituent at the C-3 position could stabilize the core structure, which would result in

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a decrease in the oxidation rate; however, a substituent at the amine position of the

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core structure might affect the overall degradation rate of the cephalosporin,

30

depending on its reactivity with MnO2. Thus, the apparent reaction rates varied widely

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in the trend of CTX > CFP > CFD > core structure ≈ CFX > CFZ. The mechanistic

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elucidation can also help explain the degradation rates of cephalosporin antibiotics in

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other oxidation processes. 1

ACS Paragon Plus Environment


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1. INTRODUCTION

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Among emerging contaminants, antibiotics are important due to their frequent

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prescription and usage in hospitals. When antibiotics are discharged into the

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environment, several adverse impacts can occur, including increased bacterial

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resistance 1, expression of resistance genes in the environment 2, and even increased

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toxicity during transformation in the natural environment 3. Cephalosporin is the

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second most commonly used antibiotic class; for example, cefotaxime (CTX),

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cephapirin (CFP), cephradine (CFD), cephalexin (CFX) and cefazolin (CFZ)

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(Supporting Information Table S1 lists the physicochemical properties of the target

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compounds) are members of the cephalosporin class of β-lactam antibiotics that can

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act as penicillins to effectively interfere with the synthesis of gram-positive and

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gram-negative organisms. Their inhibitory mechanism involves binding to and

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inactivating enzymes that are needed for bacterial cell wall synthesis. Cephalosporin

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compounds have been frequently detected in surface waters around the world at

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concentrations of 0.076 µg/L to 1.117 µg/L 4, 5. In previous studies, the concentrations

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detected in urban wastewater treatment plant influents and effluents ranged from 2.9

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µg/L to 64 µg/L and 0.013 µg/L to 2.104 µg/L, respectively, indicating that these

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compounds are not completely removed by wastewater treatment processes 4-10. In

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addition, higher concentrations of 0.01 µg/L to 42.9 µg/L have been detected in

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hospital effluents 4, 5. Cephalosporin molecules are relatively stable (t1/2 = 10-18 d) in

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terms of hydrolysis under typical environmental conditions (pH ≈ 7, T ≈ 20°C) 11, 12.

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Another study indicated that cephalosporins could not be biodegraded in China’s

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Xuanwu Lake in 7 days 13 and exhibited low octanol-water partitioning coefficients

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(log Kow = −1.40 to −0.13) 14-16. Oxidative materials present in the environment,

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mostly sediments, are thought to be relatively important for removing cephalosporin

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antibiotics.

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Oxidative transformation by manganese dioxide (MnO2) is considered to be one

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of the most important natural attenuation processes in aqueous environments because

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MnO2 has a high reduction potential (1.23 V)

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degrade many organic compounds, and because it is ubiquitous in soils and sediments

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18, 19

17

, which enables it to effectively

. Several studies have demonstrated that MnO2 can be used as an oxidant to

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remove pharmaceuticals, such as phenol 20, aniline

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and triclosan 24, from aquatic systems.

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, aliphatic amines 22, codeine

23

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Several studies investigated the oxidative transformations of cephalosporins and

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β-lactam antibiotics, and the results indicated that these compounds can undergo

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hydrolytic cleavage or oxidation at the cephem ring during different oxidation

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processes involving ClO2, dimethyldioxirane, hydrogen peroxide and chloramine-T

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25-28

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two possible positions. Either the sulfur on the core structure is oxidized to sulfoxide

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or the amide bond in the β-lactam ring is broken. Recently, Li et al. reported the

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transformation byproducts resulting from the MnO2 oxidation of one particular

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cephalosporin, CFZ. Four major transformation byproducts of MnO2 oxidation were

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identified, and degradation pathways involving thioether and olefin oxidation,

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hydrolysis and acid-catalyzed decarboxylation were proposed 29.

. The pathways proposed in these studies suggest that oxidation mainly occurs at

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To our knowledge, no study has comprehensively investigated the reactivity and

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associated mechanism among different cephalosporin antibiotics with MnO2. In

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addition, no research has examined the oxidative sites of the core structure (the

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common structure of cephalosporins). The objective of this study was to examine the

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reactivities of the core structure (7-aminodesacetoxycephalosporanic acid) and

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substructures (substituents at the C-3 and amine positions of the core structure) of

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cephalosporin, and to identify the oxidation products of the core structure to

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determine the effects of the structure on MnO2 oxidation.

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2. MATERIALS AND METHODS

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2.1. Chemicals and Materials

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All the cephalosporin compounds (over 98% purity) were purchased from

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Sigma-Aldrich and Fluka. Individual stock solutions (100-1000 mg/L) were prepared

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with deionized water and stored at 4°C in amber glass bottles for a maximum of 14

93

days. The detailed chemicals and standards used (including the suppliers and purities)

94

in this study are given in the Supporting Information Text S1 (including Table S2).

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The δ-MnO2 synthesis method was proposed by Murray

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Supporting Information (Text S2). The stoichiometry used during δ-MnO2 preparation

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followed the equation 2 MnO4- + 3 Mn2+ + 2H2O → 5 MnO2(s) + 4H+. In brief, the

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synthesis method involved adding KMnO4 and MnCl2 into a large beaker, using 3


30

. Details are given in the


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NaOH to maintain the alkaline state, and sparging the solution with N2 gas to liberate

100

oxygen.

δ-MnO2 is consistent with natural birnessite. Detailed characterization data

101

for δ-MnO2 are given in Text S3 (including Table S4, Figure S1−S3).

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2.2. MnO2 Oxidation Experiments

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Oxidation experiments were conducted in 250 mL amber glass bottles loosely

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covered with Teflon caps. The reaction batches were continuously stirred with

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magnetic stir bars at 22±2°C. The reaction solutions were maintained at a constant pH

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with 10 mM of buffer, acetic acid and sodium acetate for pH 4 and pH 5,

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4-morpholinepropanesulfonic acid (MOPS) and its sodium salt for pH 7, and

108

2-(cyclohexylamino)ethanesulfonic acid (CHES) and its sodium salt for pH 9. Then,

109

NaCl (10 mM) was added to the solution to maintain a constant ionic strength.

110

The oxidation rate of cephalosporin apparently increased with decreasing

111

solution pH in the following order: pH 4 > pH 5 > pH 7 ≈ pH 9 (Figure S4). To ensure

112

that the degradation change could be clearly observed, the experiments investigating

113

the effects of the substructure reactivity were all conducted at pH 4 under the fixed

114

conditions of 300 nM of cephalosporin and 4 mg/L of MnO2.

115

The reaction batch volume was maintained at 100 mL. Two approaches were

116

used to quench the reaction and collect samples during the oxidation process. In the

117

first approach, aliquots were rapidly passed through 0.22 µm PVDF filter membranes

118

before analysis. In this case, the concentrations of the target compounds decreased

119

due to adsorption and oxidation by MnO2. If no obvious adsorption phenomena were

120

observed, most samples were quenched using this method. The second method was to

121

quench aliquots from the reaction by adding 5 µL of 0.5 M oxalic acid to 1 mL of

122

sample. Oxalic acid reduced MnO2 and desorbed target compounds from the MnO2

123

surface. Consequently, any loss in concentration was primarily attributed to oxidation.

124

By comparing the results of these two methods, the amount of target compounds

125

adsorbed by MnO2 was determined.

126

2.3. Analysis

127

The target compounds, substructures and byproducts were prepared at a high

128

initial concentration (10 mg/L) to meet the high detection limit of LC-MS/MS. The

129

target compounds and their substructures were analyzed using a Sciex API 4000

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LC-MS/MS with an electron-spray ionization (ESI) interface, and the byproduct

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mixtures were chromatographically separated for quantification analysis using an 4


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Agilent 1200 module equipped with a ZORBAX Eclipse XDB-C18 column (150 ×

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4.6 mm, 5 µm).

134

The target compounds and their substructures were detected through MS/MS in

135

full-scan mode, which gave their mass spectra. Then, the analytical methods were

136

modified to obtain the largest product ion signals in multiple-reaction monitoring

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transition mode (MRM) by adjusting three key parameters, the declustering potential

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(DP), the collision energy (CE), and the collision cell exit potential (CXP), and using

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established analytical methods for byproduct analysis. The parameters of the LC

140

system, including the gradient of the mobile phase, the flow rate (1.0 mL/min), and

141

the injection volume (20 µL), were adjusted to separate the analyte peaks. The mobile

142

phase contained 0.1% formic acid (v/v) in DI water or methanol in positive ion mode.

143

The byproducts were identified by comparing the chromatographic retention times

144

and ESI-MS spectral data with those of commercially available standards. The

145

detailed LC gradients and mass spectrometry conditions of all analyzed compounds

146

are described in the Supporting Information Tables S5 and S6.

147

To precisely explore the oxidation byproducts of cephalosporins, Q Exactive

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Plus hybrid quadrupole-orbitrap mass spectrometry (Thermo Fisher Scientific, USA)

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coupled with a Dionex UltiMate 3000 UHPLC system was used to analyze the

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oxidized samples. Liquid chromatographic separation was conducted by injecting 10

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L of each sample through an Acquity UPLC BEH C18 column (1.7 m, 2.1×100

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mm). The column oven temperature was controlled at 40 °C. The binary solvent

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system contained mobile phase A of water with 0.1% formic acid and mobile phase B

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of acetonitrile with 0.1% formic acid. The solvent flow rate was set at 0.25 mL/min.

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Each sample was injected with 10 L of solvent and then separated through a 10-min

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solvent gradient: 0-1 min 5% B; 1-4 min 5-95% B; 4-6.5 min 95% B; 6.5-7 min

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95-5% B; and 7-10 min 5% B. The ionization was performed through a heated ESI

158

source (Thermo HESI-II probe) using the following parameters: sheath gas flow rate

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of 46 (A.U.), auxiliary gas flow rate of 11 (A.U.), sweep gas flow rate of 2 (A.U.),

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spray voltage of 3.50 kV, capillary temperature of 253 °C, S-lens RF level of 50.0 and

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auxiliary gas heater temperature of 406 °C. MS spectra were obtained in positive

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ionization mode via top-10 data-dependent acquisition, in which full MS spectra were

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acquired with a spectral resolution of 70,000 and scan range of 100-1000 m/z. The

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data-dependent MS/MS spectra were acquired with a spectral resolution of 17,500, 5



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isolation window of 2.0 m/z, normalized CE of 20 and dynamic exclusion of 5.0

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seconds. The resulting LC-MS/MS spectra were processed with Xcalibur Qual

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Browser 2.2 (Thermo).

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3. RESULTS AND DISCUSSION

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3.1. Efficacy of Cephalosporin Removal by MnO2

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This study used batch experiments to investigate the oxidative transformation of

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five cephalosporin antibiotics (CTX, CFP, CFD, CFX, and CFZ) by δ-MnO2. In the

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absence of MnO2, all the target cephalosporins remained stable in water for 2 days.

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However, in the presence of MnO2, CTX, CFP and CFD were completely removed by

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4 mg/L of MnO2 within 12 hours. Approximately 90% of CFX and 20% of CFZ were

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removed within 30 hours (Figure 1(a)). As the reaction progressed, the amount of

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cephalosporin rapidly decreased, and the reaction deviated from pseudo-first-order

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kinetics after the first few hours (Figure S5). Similar reaction kinetics were observed

179

for reactions between organic compounds and MnO2 in previous studies

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this study, each cephalosporin had a different transformation rate, and the initial

181

reaction rate constant kinit (hr-1) was obtained by fitting the first few time data points

182

with pseudo-first-order kinetics.

20, 24, 31-35

. In

183

In this work, the reaction was quenched by filtration and oxalic acid to determine

184

the effects of the sorption and oxidation of cephalosporins by MnO2. Filtration

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removed MnO2 with chemicals adsorbed to the surface sites, thereby demonstrating

186

the effects of sorption and oxidation. In another batch, oxalic acid was added to the

187

solution to dissolve residual MnO2 and demonstrate the effect of adsorption. The

188

results indicated that the residual cephalosporin concentration quenched by the

189

filtration method was approximately equal to that quenched by oxalic acid at pH 4

190

(Figure S6), indicating that the amount of unreactive cephalosporin adsorbed on

191

MnO2 surface sites was negligible. Consequently, oxidative transformation was the

192

main mechanism for cephalosporin removal in the reaction with MnO2.

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3.2. Manganese Dioxide Oxidation Mechanism for Cephalosporins

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All the cephalosporin compounds in this study have very similar structures and

195

contain the same cephem ring, but they have very different MnO2 oxidation rates (the

196

initial reaction rate constant kinit ranged from 0.014 to 2.6 hr-1). Under the same

197

reaction conditions, the degradation rates decreased as follows, as shown in Figure 6


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1(a): CTX > CFP > CFD > core structure ≈ CFX > 7-aminocephalosporanic acid

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(abbreviated 7-ACA) > CFZ. Thus, it was hypothesized that the substructure of a

200

cephalosporin affects its overall reactivity.

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Figure 1 shows the reactivities of CFZ, CFX, CFD, CFP, CTX and their

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substructures (1H-tetrazole-5-acetic acid, 1-HTA; L-phenylglycine, LP; 2‐amino‐2‐

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(cyclohexa‐1,4‐dien‐1‐yl)acetic acid, 2-ACAA; (4-pyridylthio)acetic acid, 4-PA;

204

2-amino-α-(methoxyimino)-4-thiazoleacetic acid, 2-AMTA) at the amine position. It

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was hypothesized that (1) the substituent R1 at C-3 stabilizes the core structure, which

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leads to a decrease in the degradation rate, and (2) the substituent at the amine

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position of the core structure might affect the overall degradation rate of the

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cephalosporin, depending on its reactivity with MnO2. In the following sections, the

209

reactivities of the core structure with different substituents, the reactivities of different

210

portions of the substructures and the oxidation byproducts were examined to verify

211

these hypotheses.

212

3.2.1. Reactivity of the Core Structure with the Substituent R1 at the C-3 Position

213

As shown in Figure 1(a), when there is an acetyloxymethyl group at the C-3

214

position of the core structure (in the case of CFP, CTX, and 7-ACA), the overall

215

degradation rate is significantly changed. This is clearly demonstrated by comparing

216

the rate of 7-ACA with that of the core structure. The structure of 7-ACA is simply an

217

acetyloxymethyl group bonded to the core structure at the C-3 position, and the

218

acetyloxymethyl group was clearly shown to decrease the degradation rate of the core

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structure. This phenomenon can be potentially explained as follows: (1) the

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substituent at the C-3 position may cause steric hindrance and block the reactive sites

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of the core structure from contacting the MnO2 surface sites; and (2) the relationship

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between the free energy and the kinetics may play a role in this process. According to

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studies describing the relative reactivities of model compounds with manganese

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oxides, the relative oxidation rate decreases as the reduction potential or Hammett

225

coefficient increases

226

substituents and is primarily used to connect the substituent properties with the

227

reactivity of aromatic compounds. Generally, compounds with electron-donating

228

substituents are more susceptible to oxidation than those with electron-withdrawing

229

substituents. The slow degradation rate of 7-ACA relative to the core structure may

230

occur because the acetyloxymethyl substituent on 7-ACA has a stronger

20, 31, 36

. The Hammett constant varies depending on the specific

7



231

electron-withdrawing tendency than the methyl substituent on the core structure

232

(Table S7

233

C-3 position (5-methyl-1,3,4-thiadiazole-2-thiol, 5-M) that might also cause steric

234

hindrance; therefore, this substituent likely stabilized the compound, leading to a

235

decrease in the overall rate of CFZ oxidation. Although CFP and CTX also have

236

acetyloxymethyl substituents attached, their overall extremely fast degradation rates

237

are a result of their additional R2 substituents at the amine position (4-PA and

238

2-AMTA), which will be explained in the next section.

239

3.2.2. Effect of the Substituent R2 at the Amine Position of the Core Structure

37

). In addition, CFZ also has an electron-withdrawing substituent at the

240

To test the hypothesis that a substituent at the amine position of the core

241

structure might affect the overall degradation rate of the cephalosporin, depending on

242

its reactivity with MnO2, several experiments were performed to compare the

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substructure reactivities. The various substructures react with MnO2 at different rates,

244

which decrease in the following order: 2-AMTA > 2-ACAA > core structure > LP ≈

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1-HTA ≈ 4-PA (inert to oxidation). Rate comparisons are shown in Figure 1(b). The

246

core structure can be oxidized by MnO2; however, depending on the reactivities of the

247

substructures, substructures such as 2-AMTA and 2-ACAA will be oxidized before

248

the core structure, thereby enhancing the overall oxidation rate.

249

In the case of CTX, which has the most reactive substructure, 2-AMTA, the

250

reactivity of the 2-AMTA substructure has the same oxidation rate as CTX, as shown

251

in Figure 2(a). Since CTX (initial rate = 7.8×10-7 M/hr) degrades much faster than the

252

core structure (initial rate = 5.7×10-8 M/hr) and 7-ACA (initial rate = 1.13×10-8 M/hr),

253

this result suggests that oxidation must occur first on 2-AMTA, which is degraded

254

before the core structure.

255

Another even more obvious case is when the MnO2 oxidation rate of the core

256

structure (initial rate = 5.7×10-8 M/hr) is compared to that of CFD (initial rate =

257

2.49×10-7 M/hr). With the addition of 2-ACAA (the second most reactive substructure)

258

at the amine position in CFD, the degradation rate was significantly enhanced. Similar

259

to the case of CTX, the oxidation of CFD occurs first on the 2-ACAA substructure,

260

which determines the overall decomposition rate. The unstable dienyl moiety (part of

261

the 2-ACAA substructure) is then oxidized and transformed into a phenyl group in

262

CFX. According to the byproduct analysis, we found that more than 90% of the CFD

263

is first oxidized to CFX and then gradually degraded by MnO2 (Figure 3). Since the 8


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phenyl group (LP substructure) of CFX is nonreactive toward MnO2 oxidation (Figure

265

2(b)), CFX has a similar MnO2 oxidation rate to that of the core structure.

266

Furthermore, LP formed after 3 hours of CFX oxidation, indicating that the core

267

structure decomposes first, and then LP is released as one of the byproducts, as shown

268

in Figure 3. Moreover, several of the CFX oxidation byproducts that are similar to the

269

core structure byproducts were identified to elucidate the oxidation sites on the core

270

structure (Table S8, Figure S9), and all of the core structure byproducts (Figure 4;

271

oxidation products 1, 2, 3, 4, and 5) were also detected during CFX degradation.

272

The fast oxidation rate (initial rate = 3.45×10-7 M/hr) of CFP, for which the

273

substructure 4-PA is relatively inert, is intriguing. As shown in Figure 2(b), 4-PA was

274

minimally degraded by MnO2 at pH 4; however, it was found to be the byproduct of

275

CFP degradation by MnO2. The only possible explanation is the cleavage of the amine

276

bond between 4-PA and the core structure. In addition, another oxidation product

277

((4-pyridylthio) acetamide, 4-PA-NH) formed by cleaving the C-N bond (near the

278

amide bond, at the amine position) was detected (Figure S7). This is another case that

279

demonstrates how the amine substituent affects the overall CFP degradation rate.

280

Of the five cephalosporins studied, CFZ is the only compound that degrades

281

more slowly than the core structure (and even 7-ACA) upon exposure to MnO2, as

282

shown in Figure 1(a), and its substructure 1-HTA is relatively nonreactive toward

283

MnO2 oxidation (Figure 1(b)). Similar to the degradation of CFX and CFP, CFZ was

284

degraded by the hydrolysis of the amide bond to form a carboxylic acid byproduct,

285

and only a trace amount of 1-HTA was formed. The result indicated that the

286

degradation of CFZ was slightly associated with amine bond breaking and that the

287

core structure is first decomposed. Collectively, these results suggested that the

288

degradation rate of CFZ was mainly slower due to the 5-M substituent at the C-3

289

position, not the 1-HTA substituent at the amine position. This finding further

290

supported the hypothesis that the steric hindrance and electron-withdrawing tendency

291

of the C-3 substituent can stabilize the core structure, leading to a decrease in the

292

degradation rate.

293

3.2.3 Oxidation Sites within the Core Structure

294

The oxidative transformations of some cephalosporin antibiotics have been 25-29, 38

295

identified

296

elucidated. Herein, we investigated the oxidation sites in the core structure by

, but those of the core structure compound have not yet been

9



297

exploring its transformation byproducts during MnO2 oxidation. Five classes of

298

oxidation products, including keto-sulfone, keto-sulfoxide, sulfoxide, diketone, and

299

alcohol sulfoxide, were identified (Figure 4), and the results were further verified by

300

high-resolution MS/MS spectra (Table S8, Figure S8). In despite of the complexity of

301

these oxidation byproducts, generally oxygen addition and structural transformation

302

can occur at two sites. One site is the sulfur atom of the core structure. The lone

303

electron pair on the sulfur atom acts as a reactive nucleophile to attack the manganese

304

atom in MnO2, which is followed by an electron transfer event to form a sulfoxide

305

(S=O) or a sulfone (O=S=O)

306

formation of oxidation byproducts 1, 2, 3, and 5 in Figure 4. The second oxidation site

307

is the carbon-carbon double bond (C=C) in the core structure and its proximal

308

carboxylic acid group. The unsaturated C=C double bond could be readily attacked by

309

the nucleophilic oxygen of MnO2 to form an alcohol, diketone (through hydrolysis),

310

or ketone (through acid-catalyzed decarboxylation) (Figure 4; oxidation byproducts 1,

311

2, 4, and 5)

312

oxidation sites, such as for byproducts 1, 2, and 5.

29, 38

. This oxidation pathway was involved in the

29, 38

. Notably, the dual-oxidative transformation was observed at both

313

In addition to the core structure, the oxidation byproducts of cephalosporins

314

with one structural substituent at either the C-3 position (e.g., 7-ACA) or amine

315

position (e.g., CFX) were also investigated (Table S8, Figures S9 and S10). The

316

structural similarity among those oxidation byproducts suggests that during the

317

MnO2-based oxidation process, cephalosporins might possess similar oxidation

318

mechanisms. The proximity of the oxidation sites to the C-3 position indicates that the

319

C-3 substituent and its chemical properties, including its steric hindrance and

320

electron-withdrawing ability, might impact the oxidation process.

321

Figure 5 clearly demonstrates that the substituent R1 at the C-3 position inhibited

322

the formation of the keto-sulfone class of byproducts. The keto-sulfone class of

323

byproducts is one type of oxidation product of the core structure, and it is also found

324

in compounds such as CFX. However, when the acetyloxymethyl group was attached

325

to the C-3 position, as in 7-ACA, CFP, and CTX, the formation of keto-sulfone

326

byproducts was inhibited; only trace amounts of these byproducts were detected. This

327

result indicated that the reactivities of the oxidation sites on the core structure might

328

have been perturbed by the C-3 substituent and further supported the hypothesis that

10


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the C-3 substituent stabilizes the core structure, leading to a decrease in its

330

degradation rate.

331

In summary, this is the first study toward a comprehensive understanding of the

332

degradation of five cephalosporin antibiotics by MnO2. The analysis shows that these

333

compounds are easily oxidized at pH 4 in the presence of abundant MnO2. Moreover,

334

we elucidated the reactivities of the core structure and substituents and, consequently,

335

their overall effect on the reaction rate of the cephalosporins. The oxidation of CFX

336

mainly occurs in its core structure, but the oxidation of CFD, CFP, and CTX mainly

337

occurs in the substructure attached to the amine position of the core structure. In CFP,

338

the amide bond at the amine substituent is likely broken. In the case of CFZ, the

339

1-HTA moiety is not the oxidation site for CFZ degradation. Overall, the reactivities

340

of the amine substructures decreased in the following order: 2-AMTA > 2-ACAA >

341

core structure > LP ≈ 1-HTA ≈ 4-PA (inert to oxidation).

342

This study confirmed that a substituent at the C-3 position could stabilize the

343

reactivity of the core structure; however, the substituent at the amine position may

344

affect the overall degradation rate of the cephalosporins. This finding may help

345

predict the reactivity and rates of other cephalosporins with different types of

346

substructures. It is important to first compare the reactivity of the amine substituents

347

with that of the core structure; if the substituents are more reactive, they will be

348

oxidized first and thus determine the overall degradation rate of the cephalosporin. If

349

the substituent is less reactive than the core structure, oxidation will first occur at the

350

core structure. In addition, the presence of a substituent at C-3 position of the core

351

structure will very likely cause a further reduction in the rate of degradation.

352

The results from the substructure reactivity analysis in this study can possibly

353

help explain the degradation rates of cephalosporin antibiotics in other oxidation

354

processes.

355 356

357

Supporting Information

358

More information is available regarding the materials, the synthesis of δ-MnO2, and

359

the LC−MS/MS analysis process. This information is available free of charge via the

360

Internet at http://pubs.acs.org.

ASSOCIATED CONTENT

361 11



362

363

This research was supported by funding from the Ministry of Science and Technology

364

(MOST) through the project (MOST 103-2221-E-002-240-MY5) and from National

365

Taiwan University (NTU) through the project (NTU-CCP- 106R890905).

ACKNOWLEDGMENTS

366 367

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Environmental Science & Technology O S

H3C

OH

O

N

O

N

S

N

C-7 H NH

H

4-PA (Substructure of CFP)

1-HTA (Substructure of CFZ)

OH

R2

S

N

C-3

N

R1

O

2-AMTA (Substructure of CTX)

OH H2N

LP (Substructure of CFX)

2-ACAA (Substructure of CFD)

(a)

CH3 N

5-M (Substructure of CFZ)

O

Oxidative transformation

NH2

S

HS

Core structure

O

HO

Acetyloxymethyl group (Substructure of CFP, CTX, 7-ACA)

Core structure

(b)

Core structure

1.0

1.0 7-ACA

0.8

0.8

1-HTA

CFZ C/C0

C/C0

0.6 CFX

0.6 LP 0.4

0.4

2-ACAA

CFD 0.2

0.2

4-PA

CFP 0.0

0.0 0

10

20

30

0

CTX

10

20

30

2-AMTA

Time (h)

Time (h)

Figure 1 Oxidation of (a) core structure, 7-ACA, CFZ, CFX, CFD, CFP, and CTX, (b) core structure and cephalosporin amine substituents (1-HTA, LP, 2-ACAA,4-PA, and 2-AMTA) ([MnO2]0 = 4 mg/L, [cephalosporin and substituents]0 = 3×10-7 M, pH 4).


(a) 1.0

Page 18 of 21

(b) 1.2

70

1.0

60

0.8

50

0.6

C/C0

C/C0

0.8 CTX 2-AMTA

0.4

40 0.6 30 0.4

0.2

20

0.2

10

0.0

0.0 0

1

2

3 4 Time (h)

5

6

7

0 0

10

CFP

2-AMTA

CTX

4-PA from CFP degradation (nM)


4-PA

20 Time (h)

4-PA

30

40

4-PA from CFP degradation

CFP

Figure 2 Oxidation tendencies of the (a) CTX and (b) CFP substructures at pH 4 ([cephalosporin]0 = 3×10-7 M).


Page 19 of 21


300

1.0 250

0.8

C/C0

150

0.4

100

0.2

50

0.0

0

0

5

10

15 Time (h)

20

25

30

35

CFD CFX LP CFX formation from CFD degradation LP formation from CFX degradation

CFX L−Phenylglycine (LP) Lp formation conc. (nM)

0.6

CFD

Byproduct formation (nM)

200

8 7 6 5 4 3 2 1 0 0

5

10

15

Time (h)

Figure 3 Oxidation tendency of the CFD, CFX, LP and the byproduct at pH 4 ([MnO2]0 = 4 mg/L, [cephalosporin and substructure]0 = 3×10-7.



Figure 4 MnO2 oxidative byproducts of core structure.


Page 20 of 21

Page 21 of 21


50

0h

Extracted ion chromatogram area (105)

45

0.66 h

2h

4h

9h

22 h

40 35 30 25 20 15 10 5 0

ks-core structure

ks-CFX

ks-7-ACA

Figure 5 Formation of keto-sulfone (ks) byproducts from cephalosporins.


ks-CFP

ks-CTX

Substructure Reactivity Affecting the Manganese Dioxide Oxidation of

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