Reaction of Lincosamide Antibiotics with Manganese Oxide in

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Environ. Sci. Technol. 2010, 44, 4486–4492

Reaction of Lincosamide Antibiotics with Manganese Oxide in Aqueous Solution WAN-RU CHEN,† YUNJIE DING,† CLIFF T. JOHNSTON,‡ BRIAN J. TEPPEN,† S T E P H E N A . B O Y D , † A N D H U I L I * ,† Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824, and Crop, Soil and Environmental Sciences, Department of Agronomy, Purdue University, West Lafayette, Indiana 47907

Received July 28, 2009. Revised manuscript received April 5, 2010. Accepted April 22, 2010.

Lincosamides are among the most frequently detected antibacterial agents in effluents from wastewater treatment plants and surface runoff at agricultural production systems. Little is known about their transformations in the environment. This study revealed that manganese oxide caused rapid and extensivedecompositionofclindamycinandlincomycininaqueous solution. The reactions occurred mainly at the pyranose ring of lincosamides, initially by formation of complexes with Mn and cleavage of the ether linkage, leading to the formation of a variety of degradation products via subsequent hydrolytic and oxidative reactions. The results of LC-MS/MS and FTIR analysis confirm cleavage of the C-O-C bond in the pyranose ring, formation of multiple carbonyl groups, and transformation of the methylthio moiety to sulfur oxide. The overall transformation was controlled by interactions of cationic species of lincosamides with MnO2 surfaces. The presence of electrolytes (i.e., NaCl, CaCl2, and MnCl2) and dissolved organic matter in aqueous solution, and increase of solution pH, diminished lincosamide binding to MnO2 hence reducing the rate and magnitude of the transformations. Results from this study indicate that manganese dioxides in soils and sediments could contribute to the decomposition of lincosamide antibiotics released into the environment.

Introduction Manganese oxides/hydroxides are common soil components that exist as colloidal particles and/or coatings on other soil minerals. They are formed in soils by both chemical and biological processes. For example, bacteria and fungi promote the conversion of Mn(II) to poorly crystalline layered Mn(IV) oxides (1, 2). In soils manganese oxides act as catalysts in the formation of humic materials via their reactions with naturally occurring polyphenols (3, 4), and as strong oxidants that transform a wide range of anthropogenic organic pollutants including phenols (5, 6) and anilines (7, 8). Similarly, several antibiotics and hormones containing phenolic and anilinic components in their structures have been shown to be highly susceptible to oxidation by Mn oxides (9–14). In contrast, few detailed studies have examined MnO2 oxidation of * Corresponding author e-mail: [email protected]. † Michigan State University. ‡ Purdue University. 4486

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antibiotic agents not containing these structural units (15), including the lincosamides. Lincosamide antibiotics are commonly used in humans and animals to control infection by gram-positive bacterial pathogens. They are also administered in livestock production to improve feeding efficiency and for disease prevention. The essential chemical structure of lincosamides consists of a pyranose ring, an amide moiety, and a pyrrolidine ring (Figure 1). Lincomycin, a member of the lincosamide family, is produced naturally by antinomycetes such as Streptomyces lincolnensis. Clindamycin, which has been utilized as an antibiotic since 1960s, was synthesized from lincomycin via modification at C-7 using thionyl chloride (Figure 1). Clindamycin is ∼20 times more effective in clinical studies for inhibiting bacterial growth compared to lincomycin. The antibacterial mode of action of lincosamides is to block formation of peptide bonds via direct binding to functional sites on ribosomes. Widespread and long-term utilization of lincosamides has resulted in the occurrence of lincosamide residues in honey (18), animal tissues, and bovine milk (19). In addition to the issues of food safety and purity, there is growing concern regarding the environmental fate and potential impacts of lincosamides on human and ecosystem health. Lincosamides are recalcitrant to conventional wastewater treatment processes and hence are released with municipal wastewater effluents. Watkinson (20) found that lincomycin and clindamycin residue concentrations in wastewater effluents were 40 to 60 ng L-1 and 2 to 5 ng L-1, respectively. Lincomycin is one of the most frequently detected antibiotics in the environment (21–23). A number of advanced treatments for removal of lincosamides have been examined such as membrane filtration (24), ozonation (25), photooxidation catalyzed with TiO2 (26), and oxidation by H2O2 (27). However, less is known about natural attenuation and transformation of lincosamides in the environment. Manganese oxides are common soil constituents, demonstrating high reactivity with many organic contaminants (5–9). The objectives of this study were to investigate the reaction mechanisms of lincosamides with Mn oxide, and relevant environmental factors influencing these reactions, viz. pH, dissolved organic matter (DOM), and background electrolytes. Our results demonstrate that such transformations occur, and a detailed reaction pathway is proposed based on the reaction intermediates and products identified. In this study, synthetic δ-MnO2 was used because its structure is similar to environmentally abundant, poorly crystalline biogenic Mn oxides (2, 28). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), and Fouriertransform infrared spectroscopy (FTIR), were utilized to elucidate the transformation products of lincosamides resulting from reactions with MnO2. The corresponding product evolution was evaluated to further reveal the transformation pathways. In addition, the reaction was evaluated to characterize the impacts of pH, DOM, and background electrolytes on the decomposition. This study documents the rapid and extensive decomposition of MnO2mediated lincosamide antibiotics and presents the first mechanistic description and detailed pathways of lincosamide degradation by Mn oxides.

Materials and Methods Chemicals. Clindamycin and lincomycin were purchased from Sigma with a reported purity >99% and used without further purification. Other reagents were purchased from Fisher Scientific, Acros, or Aldrich with purities >97%. All aqueous solutions were prepared using water obtained from 10.1021/es1000598

 2010 American Chemical Society

Published on Web 05/17/2010

FIGURE 1. Chemical structures of lincosamides and their pKa values (16, 17). a Millipore Milli-Q Water purification system followed by 0.2 µm membrane filtration. Lincosamide stock solutions were prepared in water at 100 mg L-1 and stored in aluminum foil-wrapped glass containers at 4 °C. The experiments to examine pH effects on the reaction were controlled using buffers; acetate buffer for pHs ranging from 4 to 5.9, 4-morpholinepropanesulfonic acid buffer for pHs from 6 to 8.5, and 2-(cyclohexylamino)ethanesulfonic acid buffer for pH at ∼9. The Elliott soil humic acid used to prepare DOM solutions was purchased from International Humic Substances Society. Sodium chloride (NaCl), calcium chloride (CaCl2), and MnCl2 were purchased from Sigma and used as background electrolytes. Preparation of MnO2. Manganese dioxide (δ-MnO2) was synthesized using the method of Murray (29). Briefly, deionized water was purged with N2 for 2 h prior to use, and 160 mL of 0.1 M NaMnO4 and 320 mL of 0.1 M NaOH were mixed with 3280 mL of N2-purged water. The mixture was then purged with N2 gas for another 1 h followed by the dropwise addition of 240 mL of 0.1 M MnCl2. The formed MnO2 particles were allowed to settle out by gravity, and the supernatant was decanted and then replenished with deionized water. This decanting-and-replacing process was repeated several times until the conductivity of the supernatant solution was pH 6.9 > pH 8.1 ∼pH 8.9 (Figure 3c), at which the corresponding clindamycin cationic fractions were 0.987, 0.998, 0.855, 0.271, and 0.056, respectively. At solution pH > 8, the loss of clindamycin was 95% of clindamycin disappeared from solution at pH < 5.8 within 4 h. These results again indicate that the cationic form of lincosamide is the more reactive species, consistent with the fact that the reaction was increasingly inhibited by increasing levels of metal cations in solution (Figure 3a and 3b). The relatively lower reaction rate at pH 4.9 (compared to that at pH 5.8) is likely due to fewer negatively charged sites for clindamycin binding on the mineral surfaces as pH approaches the PZC of MnO2 whereas at these two lower pH values, the cationic fractions of clindamycin are very similar. At higher pH (e.g., pH > 6.9) more negatively charged sites on MnO2 are created which would promote binding of clindamycin; however, the fraction of reactive cationic clindamycin species decreases simultaneously which would lead to less binding of lincosamide with MnO2 surfaces. Overall, the pH-induced shifts in speciation manifested lowered reaction rates with increasing pH, indicating that change in the cationic fraction of clindamyicn is the predominant speciation effect that determines the influence of pH on reactivity. This is reasonable based on the fact that the pH range tested encompasses the pKa of lincosamide, whereas it is at least 2 orders of magnitude higher than the PZC of MnO2. This leads to larger changes in speciation of lincosamides compared to MnO2. Finally, the presence of more protons in solution enhances the reduction of MnO2 to Mn2+ (e.g., MnO2(s) + 4H+ + 2e- ) Mn2+ + 2H2O). Thus, as solution pH increases, the overall corresponding reduction potential of the aqueous MnO2 system decreases. This is another factor responsible for the lower transformation of lincosamides at higher pH. Reaction Products and Pathways. The intermediates and products formed from the reaction of lincosamides with MnO2 were identified using the full scanning function of LC-MS/MS. The LC-MS/MS results, including chromatogram retention times and mass spectral data (as fragment ions) of clindamycin and its reaction products, are presented in Table S1 and Figure S6, Supporting Information. The parent compound clindamycin had the pseudomolecular ion [M + H]+ with m/z 425 yielding two major fragments m/z 377 and 126. The fragment m/z 377 was formed due to the loss of methylthio group (SCH3). The fragment m/z 126 was derived from the ionization of the pyrrolidine ring structure.

The mixture of major transformation products contained precursor ions of m/z 409 (M - 16), 439 (M + 14), 441 (M + 16), 443 (M + 18), and 455 (M + 30) in which M refers to the molecular ion of clindamycin (m/z ) 425) (Table S1 and Figure S6). Most these products contained the fragment corresponding to pyrrolidine ring structure (m/z ) 126), indicating that pyrrolidine, as a structural unit of lincosamides, is unreactive with MnO2. All compounds contained the fragments of precursor ion +2 m/z units, which is attributed to the 37Cl isotope effect. This result indicates that the C-Cl bond remained intact during the reactions. The precursor ions of products occurred within a relatively narrow range from M - 16 to M + 30, suggesting that the amide group had also remained intact. Several early studies (32, 33) reported the hydrolytic cleavage of the C-N bond in the amide moiety generating (methylthio)lincosaminide and 2-propyl-N-methylproline. Specific screens targeting these two products were conducted using selective mode of the LC-MS/MS; neither of these compounds was found, indicating that the amide functional group was unreactive with MnO2. Therefore, the major reactive site is located at the pyranose ring moiety where MnO2 promotes cleavage of the C-O-C ether bond (15, 34). A proposed pathway for the reaction of clindamycin with MnO2 is presented in Figure 4 based on the transformation products identified and the sequence of their appearance. The electrostatic attraction of the pyrrolidine ring of lincosamide to MnO2 surfaces facilitates their association. In the proposed pathway this leads to the formation of a stable six-member ring complex that activates C-1 of pyranose for hydrolysis via donating electron density from S (adjacent to C-1) to Mn(IV). Subsequently, pyranose opening occurs via MnO2-mediated hydrolytic attack of OH- at C-1 to form the product M + 18 and then oxidation to form the M + 16 product. The M + 16 structure complexes with MnO2 and is oxidized to form another carbonyl in the pyranose structure corresponding to the M + 14 product. The methylthio group of the M + 14 product undergoes two transformations; it is either substituted with OH (via hydrolysis) to form the M 16 product or further oxidized to create sulfur oxide (the M + 30 product). To further evaluate the proposed reaction pathways, the sequential evolution of major products was monitored as a function of reaction time (Figure 5). The concentration of clindamycin decreased rapidly (right scale of Figure 5). Due to the lack of authentic product standards, integrated areas of ionic chromatogram are reported on the left scale of Figure 5. The intermediate M + 18, M + 16, and M + 14 products reached their maximum amounts between 15 to 40 min of reaction time and then diminished quickly as the reaction proceeded. The M - 16 and M + 30 products approached maximum concentration after 55 to 70 min and then plateaued, indicating that the M - 16 and M + 30 products were plausibly derived from the intermediates M + 18, M + 16, and M + 14. Another lincosamide antibiotic, i.e., lincomycin was found to undergo the same transformation pathways with similar product evolution patterns (Figure S7, Supporting Information). The order of product evolution is consistent with the proposed reaction pathways described in Figure 4. Establishing the same reaction pathways for clindamycin and lincomycin indicates that the major reactive site of lincosamide antibiotics with MnO2 occurs at the pyranose ring. FTIR Measurements. The FTIR spectra of clindamycin and the reaction mixture with MnO2 (at 25 h) are shown in Figure S8, Supporting Information. For clindamycin there were two strong peaks at 1687 and 1574 cm-1, which are assigned to the amide I and II vibrational bands, respectively. The spectrum of the products also showed the absorbance at 1687 cm-1, accompanied by additional shoulder peaks. VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Proposed pathways for transformations of clindamycin by MnO2 showing possible MnO2 complexation with methyl-S and amide-N groups, facilitating the subsequent reactions with MnIV (M ) m/z 425 refers to the molecular ion of clindamycin).

FIGURE 5. Product evolution progress curves resulting from reaction of clindamycin with MnO2. Experimental conditions: [MnO2]0 ) 1 mM, [clindamycin]0 ) 13.4 µM, pH ) 4.8. To facilitate the comparison of the clindamycin spectrum with those obtained from products at different reaction periods, all collected spectra were normalized to 1687 cm-1 4490

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based on the observed lack of structural alteration involving the amide functional group (Figure 6). Additional peaks between ∼1600 and 1650 cm-1 were formed, and the absorbance increased with reaction time, indicating formation of additional CdO functional groups. This caused the appearance of broadening on the lower wavenumber of the 1687 cm-1 peak. As shown in Figure 4, clindamycin oxidation by MnO2 creates the products with one or two CdO functional groups. The appearance of the relatively broad FTIR peak patterns between 1687 and 1574 cm-1 provides additional independent evidence for the formation of multiple CdO functional groups, as described in the proposed reaction pathways (Figure 4). Environmental Implications. Lincosamide antibiotics are frequently found in the environment hence posing potential toxicological risks to human and ecological health. This study for the first time demonstrates that lincosamides undergo rapid and extensive reactions with MnO2, a class of minerals ubiquitous in soils. The results indicate that the lincosamide transformation reactions decreased with increasing pH due to less amounts of reactive cationic form of lincosamides present, and in the presence of excess inorganic cations which

FIGURE 6. FTIR spectra (1500 to 1800 cm-1) of clindamycin reaction with MnO2 as a function of reaction time. compete with lincosamides for binding sites on MnO2. Some common aqueous phase components in natural environment, such as Na+, K+, Mg2+, Ca2+, and natural organic matter, inhibit lincosamide reactions with MnO2, which may lengthen the half-lives of lincosamides in the environment. Lincosamide reactions with MnO2 occur primarily via the opening of the pyranose ring and subsequent formation of several major intermediates and terminal products via hydrolytic and oxidative reactions (Figure 4). These products manifest varying biological activities. For example, lincosamide sulfoxide (i.e., the M + 30 product) exhibited a reduced inhibitory effect on mammalian cell growth compared to the parent compound (35). The products formed from alterations of the SCH3 functional group (i.e., the M - 16 and M + 30 products) demonstrated a complete loss of cytotoxic effects (35). Taken together, the reaction mechanisms and pathways elucidated in this study contribute significantly to a more comprehensive understanding of environmental fate and natural attenuation of lincosamide antibiotics. The results indicate that naturally occurring manganese oxides could decompose lincosamides, but that reactivity might be reduced in natural environmental settings such as in soils/ sediments due to the presence of inorganic cations and, to a lesser extent, DOM.

Acknowledgments This research was funded by National Research Initiative Competitive Grant 2007-35107-18353 from the USDA Cooperative State Research, Education, and Extension Service, the Center for Water Sciences of Michigan State University, and the Michigan Agricultural Experiment Station.

Supporting Information Available The characterization of the synthesized MnO2, lincomycin reaction with MnO2 in the presence of Mn2+ and DOM, mass spectra of products from clindamycin reaction with MnO2, and product evolution results of lincomycin reaction with MnO2. These materials are available free of charge via the Internet at http://pubs.acs.org.

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