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Cu(II)−Catalyzed Transformation of Benzylpenicillin Revisited: The Overlooked Oxidation Jiabin Chen,†,‡,§ Peizhe Sun,‡ Xuefei Zhou,† Yalei Zhang,*,† and Ching-Hua Huang*,‡ †

College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, P. R. China School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States § School of Environmental Science and Engineering, Suzhou University of Science and Technology, Suzhou 215001, P. R. China ‡

S Supporting Information *

ABSTRACT: Penicillins, a class of widely used β-lactam antibiotics, are known to be susceptible to catalyzed hydrolysis by metal ions such as CuII. However, new results in this study strongly indicate that the role of CuII is not merely a hydrolysis catalyst but also an oxidant. When benzylpenicillin (i.e., penicillin G (PG)) was exposed to CuII ion at an equal molar ratio and pH 7, degradation of PG occurred rapidly in the oxygen-rich solution but gradually slowed down to a halt in the oxygen-limited solution. In-depth studies revealed that CuII catalyzed hydrolysis of PG to benzylpenicilloic acid (PA) and oxidized PA to yield phenylacetamide and other products. The availability of oxygen played the role in reoxidizing CuI back to CuII, which sustained fast degradation of PG over time. The overall reaction was also influenced by pH, with CuII-catalyzed hydrolysis of PG occurring throughout pH 5, 7 and 9, while CuII oxidation of PA occurring at pH 7 and 9. Note that the potential of CuII to oxidize penicillins was largely overlooked in the previous literature, and catalyzed hydrolysis was frequently assumed as the only reaction. This study is among the first to identify the dual roles of CuII in the entire degradation process of PG and systematically investigate the overlooked oxidation reaction to elucidate the mechanism. The new mechanistic knowledge has important implications for many other β-lactam antibiotics for their interactions with CuII, and significantly improves the ability to predict the environmental fate and transformation products of PG and related penicillins in systems where CuII species are also present.

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INTRODUCTION The penicillins, such as penicillin G (PG, also referred to as benzylpenicillin), ampicillin, and amoxicillin, are used extensively in medical practice for prevention and treatment of infectious diseases due to their antibacterial activity by disrupting the synthesis of bacterial cell walls.1 Penicillins are among the most frequently utilized and least toxic antibiotics. For example, they accounted for 44% of the human use antibiotic medicine in the U.S. from 2010 to 2011,2 and 4.7% of the antimicrobial sale in animal husbandry in the U.S.3 Because a large portion of the administered doses are excreted unchanged,4 a substantial amount of penicillins are released to the environment. Consequentially, penicillins have been frequently detected in wastewater effluent (up to 1660 ng/L),5−7 surface water (up to 48 ng/L),8,9 and wastewater-impacted coastal water (0.64−76 ng/L).5 The frequent detection of antibiotics in the environment is becoming a worldwide concern, as they may foster antibioticresistant bacteria and pose potential threats to aquatic wildlife, the ecosystem and human health.10 Penicillins share the basic structure of a four-membered βlactam ring fused with a five-membered thiazolidine ring. The strained β-lactam ring causes nonplanarity of the molecule with large angle and torsional rotation,11,12 which reduces the resonance of the amide linkage and leads to β-lactam’s instability.13 The labile β-lactam ring of penicillins is susceptible © 2015 American Chemical Society

to cleavage under various conditions in water including by treatment of acid or alkali and in the presence of hydrolyases and metal ions.12 Penicillins undergo rapid degradation in alkaline conditions, in which the β-lactam ring hydrolyzes to give the sole product penicilloic acid.14−16 On the other hand, the β-lactam ring breaks open in the presence of acid to give an array of unstable intermediates (e.g., penicilloic acid, penicillenic acid and penillic acid) and end products (e.g., penilloic acid, penicillamine, and penilloaldehyde).17−20 The β-lactamase enzyme can hydrolyze penicillins to produce acidic derivatives which have no antibacterial properties.21 The promoted degradation of penicillins in the presence of metal ions such as ZnII,13,22 CuII,23,24 CdII,25,26 and HgII has been studied.27,28 The metal ion−catalyzed degradation is supposed to occur through the formation of penicillin-metal ion complex, followed by hydrolytic opening of the β-lactam ring.12 Among the metal ions, CuII appears to be special in the catalyzed degradation of penicillins. For example, CuII was reported to enhance the hydrolysis rate of PG by 8 × 107 folds, much higher than the other metal ions (ZnII 4 × 104, NiII 4 × 104, Received: Revised: Accepted: Published: 4218

October 20, 2014 March 6, 2015 March 11, 2015 March 11, 2015 DOI: 10.1021/es505114u Environ. Sci. Technol. 2015, 49, 4218−4225

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Environmental Science & Technology and CoII 3 × 104 folds).29 While it is known that CuII can catalyze hydrolysis of β-lactam, amide, phosphorus ester, carbamate, and urea moieties,30,31 CuII-facilitated oxidation was instead observed in the transformation of daminozide,30 tetracyclines,32 flavonols,33 catechol,34 1,1-dimethylhydrazine,35 and hydroquinone.36,37 The above ability of copper to participate in redox reactions is consistent with the fact that copper can be found in the +II or +I valence state in natural waters, and that copper redox chemistry is crucial in many physiological reactions of living organisms and physicochemical processes of environmental aquatic systems.37−40 Extensive literature review indicates that most of the previous studies on the metal ion-catalyzed degradation of penicillins occurred in several decades ago and there has been a significant gap of new information in recent years. Most of the previous studies were motivated by pharmacology and thus experiments were conducted in simulating human physiological conditions rather than environmental conditions. Furthermore, almost all the previous studies have attributed the CuII-promoted degradation of penicillins as catalyzed hydrolysis. It has been proposed that, after rapid complexation between CuII and intact PG, a rate-limiting hydrolysis of the complex by hydroxyl ion attack leads to the corresponding penicilloic acid (PA)−CuII complex.24 The CuII-promoted conversion of PG into PA was reported based on formation of intense blue color with an arsenomolybdic acid/mercuric chloride regent and variation in the UV absorption spectra.41,42 In contrast, Kiichiro et al. reported that penicillenic acid, instead of PA, was the product of CuII-catalyzed hydrolysis of PG.43 Gunther reported splitting of PG in the presence of CuII into penicillamine and other products via the PA intermediate,44 but a hydrolysis catalyst role of CuII was still assigned. Only in one study on the interactions between CuII and ampicillin, possible oxidation by CuII was implied but without obtaining direct evidence.45 In our investigation of the interactions of β-lactam antibiotics with CuII ions, we encountered experimental results that could not be fully explained by hydrolysis reaction. For example, we discovered that oxygen had a strong influence on the CuIIpromoted degradation of PG. This new discovery motivated us to conduct an in-depth study to elucidate the role of CuII in promoting the degradation of PG. As will be shown in detail later, this study elucidates that CuII is not only a catalyst to promote hydrolysis of PG to PA, but also an oxidant to oxidize PA to generate oxidation products. All the reactions could occur under mild environmental conditions (pH 7−9 and room temperature). To our best knowledge, this study is among the first to unambiguously identify the oxidative role of CuII in the degradation process of PG and to systematically investigate the oxidation reaction to elucidate the mechanism. The new findings of this study have important implications for the environmental fate of many other penicillin compounds as will be discussed further later.

sulfonic acid disodium salt hydrate were obtained from Fisher Scientific or Acros Organics at analytical grade. Deionized (DI) reagent water (resistivity >18 ΩM) was produced from a Millipore Milli-Q Ultrapure Gradient A10 purification system. Stock solutions of PG were prepared in DI water at 1 g/L and stored at 5 °C before use. Experimental Procedures. Batch reactions were conducted in 100 mL amber glass serum bottles with Teflon-lined caps and wrapped with aluminum foil to prevent light. The solution in the bottle was constantly mixed by magnetic stirring at room temperature (22 °C). MOPS buffer (10 mM) was used to control the solution pH at 7 in all the experiments, except in experiments investigating the influence of pH, in which 10 mM MES (pH 5) and CHES (pH 9) were used. These buffers were selected because of their very weak or negligible metal complex properties46 and were used in previous studies.32 Reaction was initiated by adding dissolved CuII (CuSO4 was dissolved in DI water at pH 3 adjusted by HCl) or other metal ions (i.e., ZnII) to the solution containing buffer and PG. The initial concentration of 0.1 mM of PG was employed to generate sufficient signal to measure. During the reaction, the serum bottle was exposed to the ambient air. To assess the role of oxygen in the reaction, experiments were also conducted in serum bottles sealed with rubber stoppers and the solution was purged by nitrogen gas for 30 min prior to the addition of CuII to initiate the reaction, and for 1 min after every sampling during the reaction, to exclude dissolved oxygen. This condition was termed “oxygen-limited”, in contrast to the “oxygen-rich” condition which was open to the air. The sample aliquots taken at the predetermined time intervals were immediately quenched by excess EDTA. The quenched samples were stored in 2 mL amber vials at 5 °C and analyzed within 24 h. For analysis of transformation products, the samples were immediately injected for analysis without the addition of EDTA. Control experiments without the addition of CuII were also performed under the same experimental conditions. All the experiments were conducted in duplicate or more. Analytical Methods. PG was analyzed by an Agilent 1100 high performance liquid chromatography (HPLC) equipped with a diode array detector. Sample injection of 20 μL was separated on a Zorbax RX-C18 column (4.6 × 250 mm, 5 μm) at the flow rate of 1 mL/min. Isocratic elution was employed by a mixture of water with 10 mM H3PO4 (A) and pure acetonitrile (B) at 35:65 v/v. The detection wavelength was 220 nm. Transformation products were analyzed by an Agilent 1100 HPLC/UV/MSD system with a Zorbax SB-C18 column (2.4 × 150 mm, 5 μm). Gradient elution was carried out using 0.1% formic acid in water (A) and pure acetonitrile (B) at a flow rate of 0.3 mL/min: 10% B was kept for 2 min, then ramped to 50% B over 8 min, kept for 5 min, and finally ramped back to 10% B. The injection volume was 40 μL. The products were analyzed by electrospray ionization at positive mode (ESI+) at the fragmentation voltage 70−220 eV with a mass scan range of m/z 50−1000. Other parameters were set as follows: drying gas 6 L/min at 350 °C, capillary voltage 4000 V, and nebulizer pressure 25 psig. Formation of CuI was determined by spectrophotometric measurement of the CuI complex with bathocuproine according to Moffett et al.47 After the sample was taken at the predetermined time, 20 mM ethylenediamine was added before the addition of bathocuproine as a masking ligand to inhibit the interference of CuII. The orange complex of CuI and bathocuproine was measured on a Backman DU520 UV/vis

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MATERIALS AND METHODS Chemicals. PG sodium salt and 2-phenylacetamide were obtained from Fisher Scientific at the highest purity. All the buffers (2-(N-morpholino)ethanesulfonic acid (MES), 4-morpholinepropanesulfonic acid (MOPS), and 2(cyclohexylamino)ethanesulfonic acid (CHES)) were from Acros Organics at 99% purity. Cupric sulfate (CuSO4·5H2O), zinc chloride (ZnCl2), hydrochloric acid (HCl), sodium hydroxide (NaOH), humic acid, ethylenediaminetetraacetic acid (EDTA), tert-butyl alcohol (TBA) and bathocuproinedi4219

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Figure 1. Hydrolysis of PG at alkaline pHs (A) and in the presence of ZnII (B) at pH 7. Reaction conditions: [PG]0 = 0.1 mM, T = 22 °C, 10 mM phosphate buffer for (A), 10 mM MOPS buffer for (B), O2 = open to air (i.e., oxygen-rich), w/o O2 = closed to air and purged by nitrogen (i.e., oxygenlimited).

Figure 2. Reaction of PG with different amounts of CuII in the oxygen-rich (A) and oxygen-limited (B) solutions ([PG]0 = 0.1 mM, pH 7, T = 22 °C); (C) Restart of degradation of PG after 240 min in oxygen-limited solution (similar to the case of (B)). For (a), 0.027 mM CuII was added into the oxygen-limited reactor; for (b), the reactor was open to the air by removing the stopper.

those at pH 10 and 11 (SI Table S1). Phosphate buffer had little effect on the hydrolysis of PG. ZnII has been reported to significantly enhance the hydrolysis of penicillins in water.29 Our experimental results agreed with previous research that, while PG (0.1 mM) alone was stable at pH 7, it underwent hydrolysis in the presence of 0.1 or 1.0 mM of ZnII (Figure 1B) and generated the same only hydrolysis product PA as the alkaline hydrolysis of PG. The ZnII-catalyzed hydrolysis of PG also conformed to the first-order kinetics, and was faster with a higher ZnII concentration (SI Table S1). Adding excess (50 mM) EDTA to the solution completely inhibited the catalytic effect of ZnII (Figure 1B); thus, the complexation of PG and ZnII was essential for the catalyzed hydrolysis. Moreover, the

spectrophotometer at 484 nm. In these experiments, the reactor was sealed by a rubber stopper and nitrogen gas was purged to exclude oxygen in the reactor before the reaction and during the sampling.

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RESULTS AND DISCUSSION Hydrolysis. Previous studies showed that PG is relatively stable at pH 5−8 and lower temperatures.14 At a higher pH, PG undergoes hydrolysis rapidly to generate PA.42 Our experiments showed that the hydrolysis rate of PG at pH 10, 11, and 12 conformed to the first-order kinetics and was highly pHdependent (Figure 1A). Almost all PG was hydrolyzed after 60 min at pH 12, with a hydrolysis rate constant much higher than 4220

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3.5, 55 °C), the authors also proposed Cu(I) complex formation with transformation products on the basis of unique fluorescent property, and implied the involvement of oxidation.45 The detection of CuI also suggested that the slowing down of the CuII-promoted degradation of PG in the oxygen-limited solution was due to decreasing CuII concentration in the solution. Only when the initial CuII concentration was higher than the initial PG concentration (i.e., [PG]0 = 0.1 mM and [CuII]0 = 0.2 mM), PG degradation did not slow down to a complete stop (Figure 2B) because CuII was not depleted in the 240 min reaction time. Additional experiments were conducted to remove the stopper from the oxygen-limited reactor after 240 min. Significant increase in PG degradation was observed after the air exposure, and almost all the remaining PG disappeared at 300 min (Figure 2C). Alternatively, while still keeping the oxygenlimited reactor closed to the ambient air, 0.027 mM of CuII (equivalent to the remaining amount of PG at 240 min) was added into the reactor. The remaining PG degraded fast in the initial few minutes, and then the reaction slowed down again in a trend similar to that during the first 240 min (Figure 2C). Overall, the above results confirmed that the degradation of PG involved oxidation by CuII, which generated CuI. In the oxygen-limited experiments, CuII concentration was decreased overtime, resulting in slowing down to stopping of PG degradation. When oxygen was available, CuII was regenerated by oxidation of CuI by oxygen, which sustained the continuing degradation of PG. Studies were also conducted to assess the effect of competing ligands (EDTA) and radical quenchers (TBA) on the CuIIcatalyzed degradation of PG. Experiments showed that excess (5 mM) EDTA completely inhibited the degradation of PG in the presence of CuII and oxygen (SI Figure S3), indicating the complexation of PG with CuII was essential for the promoted degradation. Because oxidation of CuI by oxygen could generate reactive oxygen species (ROS),40 TBA (a hydroxyl radical scavenger) was added to assess the potential role of radical species. With the addition of 50 mM TBA, the degradation trend of PG in the presence of CuII and oxygen was nearly identical to that without the addition of TBA (SI Figure S3). The negligible effect of TBA, along with the nearly identical initial rate of CuIIpromoted PG degradation in the oxygen-rich or limited solution (Figure 2), indicated that the degradation of PG in the presence of CuII and oxygen was not due to transient radical species that could be generated from oxidation of CuI intermediate by oxygen. Product Identification. The primary products with molecular weights (MW) of 135, 195, 308, 322, 352a, 352b, 368a, 368b, 368c, and 368d were observed during the CuIIpromoted degradation of PG at pH 7 (two products had the same MW of 352, and four products had the same MW of 368). The MW of PG is 334. The MWs of the above products were identified based on the appearance of the ions observed by ESI LC/MS at 70 eV, e.g., [M + H]+, [M + Na]+, [2M + H]+ and [2M + Na]+. In most cases, [M + H]+ was the predominant ion with the highest intensity (SI Table S2). These products, except for 195, were detected both in the oxygen-rich and limited reactions but at different distribution. The 352a and 352b products might be isomers because they had different LC retention times but the same MW and fragmentation pattern on MS. The 352b product was identified to be PA because its retention time and MS spectra matched with those of the PA hydrolysis product when PG was hydrolyzed in the presence of ZnII or at alkaline pH. Likewise, 368a-d were likely isomers based on their same MW and

availability of oxygen had little impact on the hydrolysis of PG in the presence of ZnII (Figure 1B). Oxidation versus Hydrolysis: Effect of CuII. The presence of CuII (0.01−0.2 mM) also significantly promoted the degradation of PG at pH 7, but with trends quite different from those of ZnII (Figure 2). Note that no visible precipitation of CuII was observed in any of the reactions except for those with the highest CuII concentration of 0.2 mM. Complexation of CuII with PG was expected; indeed, a discernible peak shift of PG’s UV spectrum at 220−300 nm was observed when an equal molar amount of CuII was added (measured immediately in the oxygenlimited solution), indicating complexation (SI Figure S1). Complexation of CuII by PG and PA has been investigated previously, with reported 1:1 complexation constant logβ = 2.61−4.8 for PG and 4.50 for PA.24,48 The complexation of CuII by PG is expected to lower the tendency of CuII to precipitate. Even though speciation calculation (by MINEQL+ 4.6) indicated that precipitation of Cu(OH)2(s) could occur at the commonly used experimental conditions where [CuII]0 = [PG]0 = 0.1 mM, pH 7 and T = 25 °C, separate experiments with similar CuII concentration and pH showed no visible precipitation or detectable absorbance in the visible enrage (700 nm), confirming little precipitation. As shown in Figure 2, CuII catalyzed the degradation of PG much more strongly than ZnII (at 0.1 mM metal concentration), the reaction kinetics were more complicated than the first-order kinetics, and the CuII-catalyzed degradation was much more rapid in the oxygen-rich than the oxygen-limited solution. Indeed, when oxygen was available, around 80% of PG was decomposed in the first 5 min, and complete disappearance of PG was observed at 240 min (Figure 2A). When oxygen was limited, CuII (0.1 mM) caused fast degradation of about 70% of PG in the initial 5 min; however, the degradation of PG slowed down after 5 min and stopped after about 60 min (Figure 2B). In either oxygen-rich or limited solution, the CuII-catalyzed degradation of PG increased with increasing CuII concentration. Evidently, oxygen played a critical role in the CuII-catalyzed degradation of PG. CuII was shown to have the potential to directly oxidize certain compounds, such as hydroquinone, daminozide and tetracyclines, and involvement of the CuII/CuI couple was verified in those reactions.30,32,40 PG possesses several O and N atoms, resulting in its strong tendency to complex with CuII, which has been well documented.48,49 The significant influence of oxygen (Figure 2) strongly suggested that an oxidative role of CuII was involved in the degradation of PG. To verify this hypothesis, the generation of CuI during the reaction was measured. CuI was found to accumulate in the oxygen-limited solution (SI Figure S2). The increase of CuI was initially fast and then slowed down during the reaction; such a slowing accumulation trend of CuI was similar to the slowing degradation trend of PG in the oxygenlimited solution (SI Figure S2 and Figure 2). In the reaction exposed to oxygen, CuI was also detected during the reaction, although the concentration was much lower than that in the oxygen-limited solution. The concentration of CuI increased in the initial 15 min and then decreased later (SI Figure S2). Normally, CuI is quite unstable with oxygen and can be quickly oxidized to CuII with a half-life of less than 1 min in freshwater.36 However, the complexation of CuI with organic ligands or chloride can slow down its oxidation rate.50 Thus, CuI might be complexed with the transformation products of PG, increasing its stability in this study. In the previous study by FernándezGonzález et al. on the interactions of CuII with ampicillin (pH 4221

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Furthermore, their fragments of m/z 114 and 160 are characteristics of the thiazolidine ring fused to the β-lactam ring (SI Table S2). On the basis of the above structural characteristics and other MS spectral information (SI Table S2), the structures of the intermediate products are proposed in SI Figure S4. Most of these products have not been reported in the literature. Note that while penicillenic acid and penicillamine were previously reported as products in CuII-promoted degradation of PG,43,44 they were not observed in this study, which might be due to different experimental conditions (the previous study used pH 5.0 phosphate buffer at 37 °C).43 In the oxygen-limited solution (SI Figure S5), PA was also a major intermediate product, and phenylacetamide and the other intermediate products were also detected. Phenylacetamide was one of the two dominant end products but at a much lower amount than in the oxygen-rich solution. The other end product 195 was found only in the oxygen-limited solution, but the MS information was insufficient to identify its structure. Even so, the 195 product was likely generated from the side chain of the βlactam, due to the presence of a major benzyl fragment (m/z 91) in its MS spectra (SI Table S2). Moreover, the 195 product appeared to be stable, and was probably generated via a different pathway from phenylacetamide (SI Text S3, Figures S6−S8). PG versus PA. According to the product evolution of PG in the presence of CuII (Figure 3 and SI Figure S5), the transformation of PG to PA might be the first step and then PA was further degraded to other products. To assess this hypothesis, the degradation of PA in the presence of CuII was investigated. PA was produced by exposing PG to 0.01 M NaOH for 1 h. After almost all of the PG was transformed to PA, the solution was adjusted to pH 5, 7, or 9 by HCl, followed by addition of 10 mM pH buffer and 0.1 mM CuII. The degradation of PG and PA by CuII was examined under the same conditions at the three different pHs (Figure 4). Control experiments confirmed that PA alone (without CuII) was stable in the experimental conditions (data not shown). As Figure 4 shows, at pH 7 in the oxygen-rich solution, the degradation of PG and PA was fastest; PA appeared to degrade faster than PG, particularly because its degradation did not slow down as much as the degradation of PG. At pH 7 in the oxygenlimited solution, slower degradation occurred but generally followed similar behavior for PG versus PA. At pH 9 in the oxygen-rich or limited solution, the degradation rates of PG and PA were slower than those at pH 7 but followed similar trends,

fragmentation pattern but different retention times. The products of 308 and 322, originally overlapped with the peaks of PA and PG, respectively, were discovered and separated after changing the mobile phase composition (SI Text S1). Negative mode of ESI-MS was also used but no additional transformation products were detected. Furthermore, an intense peak at 2.3 min was observed to increase over time in all the reactions when oxygen was limited, which was identified to be the complex of one CuI with two acetonitrile molecules from the mobile phase (SI Text S2). Figure 3 shows product evolution from the CuII-promoted degradation of PG in the oxygen-rich solution. Along with the

Figure 3. Degradation products of PG in the presence of CuII in the oxygen-rich solution. [PG]0 = 0.1 mM, [CuII]0 = 0.1 mM, pH 7, T = 22 °C. Product 135 was confirmed to be phenylacetamide.

rapid loss of PG (334), PA (352b) was the dominant product in the initial 5 min and then quickly degraded. Other intermediate products including 135, 322, and 368a−d were also observed and later decreased to disappearance except for 135, which accumulated to be the only major end product at 270 min. The 135 product was confirmed to be phenylacetamide by matching its LC retention time and MS spectra with those of an authentic standard. The LC/MS analysis at a higher fragmentation voltage (220 eV) indicated that PA and the intermediate products (322, 352a, 308, and 368a−d) all contained the phenylacetamide moiety in their structures.

Figure 4. Degradation of PG (A) and PA (B) in the presence of CuII at different pHs in the oxygen-rich (w/O2) and oxygen-limited (w/o O2) solutions. [PG or PA]0 = 0.1 mM, [CuII]0 = 0.1 mM, T = 22 °C. 4222

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Figure 5. Proposed mechanism for the CuII-catalyzed degradation of PG. Note: The redox reaction is prominent at neutral to alkaline (pH 7−9) conditions. Oxygen, if present, reoxidizes CuI generated from CuII reduction.

oxidation products. At acidic conditions (pH 5), CuII mostly catalyzes the hydrolysis of PG to PA and does not oxidize PA significantly. Although the possibility of direct oxidation of PG by CuII at pH 7 and 9 cannot be completely ruled out based on the experimental data, all the evidence suggests that hydrolysis followed by oxidation was the main degradation mechanism. Proposed Reaction Mechanism. Previous studies have reported the ability of several metal ions such as CuII, ZnII, CoII and NiII to catalyze hydrolysis of the unstable β-lactam ring in penicillin antibiotics.29 Although the CuII-catalyzed degradation was much faster than that promoted by other metal ions, the role of CuII in the degradation was assumed to be a hydrolysis catalyst only. In this study, we have revisited the role of CuII in the degradation of PG. Indeed, CuII catalyzed hydrolysis of the βlactam ring in PG, and the catalyzed hydrolysis was the dominant mechanism at pH 5. However, CuII played dual roles in the entire degradation process of PG at pH 7 and 9, that is, a hydrolysis catalyst for PG, and subsequently an oxidant for the hydrolysis product PA. Based on the study results, we proposed a revised mechanism for the CuII-catalyzed degradation of PG in Figure 5. PG first forms a strong complex with CuII, and then CuII catalyzes hydrolysis of PG within the complex to generate PA. While PA is still complexed with CuII, it can be oxidized by CuII (particularly at pH 7−9) to generate phenylacetamide and other products. CuII itself is reduced to CuI, which is accumulated in the oxygenlimited system, or reoxidized to CuII in the presence of oxygen to sustain the fast degradation of PG. Complexation of CuII with PG is essential for the degradation of PG. Fazakerley et al. applied NMR techniques to study the complexation of CuII and PG at pH 5.5 and 25 °C, and reported that the majority of CuII coordinated to the carboxyl group and tertiary nitrogen (proposed structure I in Figure 5).52 Such chelate mode was thought to stabilize the tetrahedral intermediate formed by hydroxide ion attack on the β-lactam, facilitating the OH−-catalyzed hydrolysis of PG.1,29 However, Cressman et al. postulated the sites of PG complexation with CuII (at pH 5.5 and 30 °C) were the β-lactam carbonyl group and the side-chain amide nitrogen (proposed structure II in Figure 5) on the basis of side-chain substitution effect. They proposed that such chelate mode was conducive to the production of “super acid” catalysis to accelerate the hydrolysis of PG.24 Moreover,

that is, PA degraded faster than PG and its degradation did not slow down as much. At pH 5, PA did not undergo degradation by CuII either in the oxygen-rich or limited reaction. PG’s degradation at pH 5 with oxygen was slower than those at pH 7 and 9. Notably, oxygen had little effect on PG’s degradation at pH 5, in contrast to the cases at higher pH. The product generation from the degradation of PG and PA at the three pHs was also compared. At pH 7, the degradation of PA by CuII in the oxygen-rich or limited reaction generated the same products as that of PG, with similar product evolution profiles (SI Figure S9 versus Figure 3 and SI Figure S5). At pH 9 with oxygen, the types of products from the degradation of PG were similar to those observed at pH 7. As SI Figure S10A shows, along with the degradation of PG, PA was the dominant intermediate product at first and then degraded further to a final disappearance. Other products such as 368a-d and 322 were also significant during the reaction, and they increased at first and then later decreased. Phenylacetamide was also the main end product at 240 min. SI Figure S10B shows that the degradation of PA at pH 9 was a little slower than that at pH 7, and the evolution of transformation products was similar to the degradation of PG at pH 9, with phenylacetamide as the main end product. At pH 5, most of PG was transformed to PA and PA was accumulated without significant further transformation (SI Figure S11). Although trace levels of the phenylacetamide, 322 and 368 product were also detected, their amounts were far lower than those observed at pH 7 and 9. The product evolution was essentially the same in the oxygen-rich or limited reaction, except for the product 352a. Epimerization of PA was reported in the hydrolysis of PG at pH 7.5 previously.51 Thus, the increase of the 352a isomer product might be due to epimerization of PA. The potential transformation of PA in the presence of CuII at pH 5 was also evaluated (SI Figure S12). There was a very slow degradation of PA over time, and the other transformation products were generated at small amounts similarly to the case in PG degradation (SI Figure S11A). Combining all the results above, it can be concluded that PA is the first transformation product of PG in the presence of CuII, resulted from CuII-catalyzed hydrolysis of PG. At neutral to alkaline conditions (pH 7 and 9), CuII can oxidize PA at a rate faster than the catalyzed hydrolysis of PG to generate a range of 4223

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Environmental Science & Technology potentiometric titration investigation also suggested CuII coordinating to PG’s side-chain amide nitrogen and inducing its deprotonation (pKa estimated at 4.92), with β-lactam oxygen the likely second donor atom in complexation.53 In that study, hydrolysis of PG (at pH 4.3−5.3 and 30 °C) was thought to be promoted by intramolecular attack of a Cu−OH species (one of the water molecules on CuII had ionized) on the β-lactam carbonyl carbon. In our work, the intact thiazolidine ring was present in most of the transformation products, suggesting that the reactive site of PG likely was located at the opposite side of the β-lactam ring. Moreover, bond cleavage occurred between the amide side group and the β-lactam ring to yield phenylacetamide as the main oxidation product. Hence, it is more plausible that PG’s degradation occurs through complexation of CuII to the sidechain amide nitrogen and the β-lactam carbonyl (structure II in Figure 5). Within the CuII−PG complex, PG’s hydrolysis was promoted likely by an intramolecular Cu−OH species to form the CuII−PA complex.53 Deprotonation of the side-chain amide nitrogen, which is more favorable at a higher pH, may render electron transfer to CuII easier, leading to reduction of CuII to CuI and oxidation of the organic substrate. We hypothesize that the inability of CuII to oxidize PA (and PG) at pH 5 may be due to very weak deprotonation of the side-chain amide nitrogen or complexation primarily via the carboxyl group and tertiary nitrogen.53 When CuII is complexed to PG’s carboxyl group and tertiary nitrogen, such a chelate mode may also catalyze hydrolysis of PG to yield PA (Figure 5),29 but is unlikely to undergo oxidative transformation observed in this study. As for the rate of CuII-promoted degradation of PG (and PA) being slightly slower at pH 9 than at pH 7, some CuII precipitation and other factors might play a role. Separate experiments with CuII concentration of 0.1 mM at pH 9 yielded no visible precipitation but a small increase in the absorbance at 700 nm, suggesting a small amount of CuII precipitation might have occurred. Only after more than 4 d, small amounts of CuII precipitation became visible, indicating that the oversaturation of CuII in the experimental solutions was likely due to slow rate of precipitation. Finally, based on the mechanism elucidated in this study, it is possible to rationalize why the oxidative role of CuII might not be recognized in previous studies conducted under simulated physiological conditions (i.e., acidic pH and higher temperature),29,53 which were unfavorable for the oxidation reaction. Furthermore, if the reaction was conducted under excess CuII to penicillin ratio, without limiting oxygen, or without evaluating the transformation products, the involvement of oxidation would not be apparent based on the PG degradation rate alone and thus be overlooked. Environmental Significance. Copper is not only an essential transition metal in living organisms, but also a common trace metal in natural waters and soils, participating in various physicochemical processes and physiological reactions. Copper salts are also widely used as fungicides in agricultural applications. This study shows that CuII is capable of promoting degradation of PG by first catalyzing hydrolysis and then oxidizing the main hydrolysis product PA to other oxidation products. The role of CuII is mostly a hydrolysis catalyst at acidic conditions (pH 5), but a combined hydrolysis catalyst and oxidant at neutral to alkaline conditions (pH 7−9). The new findings provide a more accurate understanding of the mechanism of CuII−promoted degradation of PG. Considering that PG is widely used as the raw material for the production of many semisynthetic penicillins

which still retain the basic structure of PG, similarly dual roles of CuII in the degradation of other β-lactams are likely and demand further research. Overall, the results of this study will significantly improve the ability to predict the environmental fate and transformation products of PG and other penicillins in the presence of CuII. Results of this work may also be useful for developing degradation strategies for penicillin contaminants. Moreover, the concept demonstrated in this work may warrant further research to explore the potential redox reactions of penicilins with other environmental metal ions and minerals.

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ASSOCIATED CONTENT

S Supporting Information *

Text S1−S3, Tables S1−S2 and Figures S1−S12. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(C.-H.H.) Phone: 404-894-7694; fax: 404-358-7087; e-mail: [email protected]. *(Y.Z.) Phone: 86-21-65980624; fax: 86-21-65989961; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was partially supported by the U.S. Department of Agriculture Grant 2009-65102-05843. J.C. gratefully acknowledges financial support from the China Scholarship Council.

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REFERENCES

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