Environ. Sci. Technol. 1997, 31, 1973-1978
Chloramines VI: Chlorination of Glycylphenylalanine in Model Solutions and in a Wastewater DANIEL J. KEEFE, T. CHRISTOPHER FOX, BARBARA CONYERS, AND FRANK E. SCULLY, JR.* Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529-0126
Model solutions of the dipeptide glycylphenylalanine were chlorinated at pH 7.0 to five different chlorine-to-peptide (Cl2/peptide) mole ratios and analyzed after 30 min by highperformance liquid chromatography. At Cl2/peptide mole ratios e 1, N-chloroglycylphenylalanine (I) appears to be the only product. At mole ratios g 2, N,N-dichloroglycylphenylalanine (II) was the only product. II decomposes in model solutions (t1/2 ) 6.4 h) at pH 7.0 to form a compound tentatively identified as N-[2-(N′-chloroimino)ethanoyl]phenylalanine (III), an N-chloroaldimine. III, in turn, decomposes (t1/2 ) 36 h) to IV. From 13C- and 1H-NMR, GC/MS, and IR, IV was identified as N-cyanoacylphenylalanine. Glycylp-[3H]-phenylalanine (VI) was synthesized in order to monitor the reaction at low concentrations of the compound in a wastewater. A secondary wastewater (TKN ) 1.29 mg of N/L; [NH3]) 0.074 mg of N/L) was inoculated with VI and chlorinated to nine different chlorine concentrations. The stabilities of the tritiated analogs of II and III in the wastewater were comparable to those determined in model solutions.
Introduction Wastewater disinfection is practiced in the United States to ensure that pathogenic organisms do not affect humans and aquatic organisms in and around receiving waters (1). Because it is inexpensive and readily available, an estimated 100 000 t of chlorine is used annually for disinfection purposes. As a result, as much as 5000 t of chlorine-containing organic compounds is discharged into the environment (2). Most of the byproducts of wastewater chlorination are still poorly characterized, and better information is needed to determine the environmental impact of this practice (3). One of the most rapid processes to occur on the addition of chlorine to a wastewater is its reaction with ammonia and organic amino nitrogen compounds to form N-chloramines. As a class of compounds, chloramines are believed to be toxic to some species of fish (4, 5), to maintain their toxicity longer, and to generate stronger fish avoidance reactions than free residual chlorine (6-10). The formation of organic N-chloramines is believed to interfere with the accurate measurement of disinfectant concentration in wastewaters treated with aqueous chlorine (11-19). Recent work in our laboratory has focused on the chlorination reactions of a series of amino acids (20-24). However, studies of the nature of organic nitrogen in wastewaters suggest that polypeptides, polymers of the amino acids, are more abundant components of wastewaters than the free amino acids themselves. For this reason, the reactions of aqueous chlorine with polypeptides in a wastewater are potentially among the more important reactions in these * Corresponding author e-mail address: FES100U@hydrogen. chem.odu.edu.
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1997 American Chemical Society
waters. The work of others has suggested that the Nchlorinated derivatives of polypeptides are much more stable than those of the amino acids (25). Therefore, it was the objective of this paper and the following one to examine the reactions of polypeptides with aqueous chlorine to determine the identity and stabilty of their chlorination products. Apart from any side chains containing amino groups, polypeptides contain two types of nitrogen: one terminal amino nitrogen and the amide or peptide nitrogens. Model studies suggest that the reaction of the more basic terminal amino group of a polypeptide is much faster than that of amide nitrogens. Therefore, the N-chlorination of polypeptides is most likely to take place at the terminal amino group. Model studies have suggested a factor of about 1010 difference in the second-order rate constant for the chlorination of the two different nitrogens. Upon chlorination, polypeptides decompose in an entirely different fashion than do the amino acids we have previously studied (20-24) since they cannot readily undergo decarboxylation. Little work has been carried out on the reactions of HOCl with this class of amino nitrogen (26, 27). In this paper, the chlorination reactions of glycylphenylalanine are discussed. The reactions of an N,N-dichlorinated polypeptide containing an N-terminal glycyl residue would be expected to differ from those containing any other N-terminal residue, because the Cl2N-CH2-CO- moiety can lose up to 2 mol of HCl to form a nitrile residue. Chlorinated polypeptides containing any other N-terminal amino acid, such as alanylphenylalanine discussed in the accompanying paper, can only lose 1 equiv of HCl from the Cl2N-CH(CH3)CO- unit.
Experimental Methods General. All chemicals were reagent grade or better. Isobutyl chloroformate and all amino acids and dipeptides were obtained from Sigma Chemical Co. The preparation of all buffers, standard solutions, and chlorine demand free (CDF) water (28); the preparation and standardization of hypochlorite solutions; and the analysis of residual chlorine concentrations (29) were carried out as previously described. Elemental analysis was performed by Atlantic Microlabs, Inc. Glycyl-p-bromophenylalanine was converted to glycyl-p-[3H]phenylalanine by Moravek Biochemicals, Inc. by metalcatalyzed halogen reduction in the presence of tritium gas. A solution of the tritiated dipeptide (500 µL; 1 µCi/µL) was obtained with a specific activity of 25 Ci/mmol. Instrumentation. Infrared spectra and nuclear magnetic resonance spectra (13C and 1H) were recorded as previously described (20). Low-resolution GC/MS analyses were carried out on a Hewlett Packard 5880A GC interfaced with a Hewlett Packard 5970 Series quadrapole mass selective detector operated in the EI mode as previously described (24). Ethyl acetate extracts were analyzed by GC (30:1 split injection) with an injector temperature of 200 °C, and a column temperature program from 100 °C to 250 °C at 10 °C/min. A DB-5 fused silica capillary column (25 mm i.d. × 30 m) with a 0.25-µm film thickness (J&W Scientific) was used. The scan acquisition rate was 1.71 scans/s from m/z 50 to m/z 300 with an electron multiplier voltage of 1800. High-resolution mass spectra were recorded by the University of Maryland Mass Spectrometry Center. Exact mass measurements were obtained from the average of three-five representative scans. A Waters Associates liquid chromatography system (HPLC) has been described previously (21, 22, 24). Separation of chlorination products was carried out on a Whatman 5 µm Partisil ODS-3 RAC column with a dual-solvent system described previously (20). The solvent program (1 mL/min) consisted of a 10-min isocratic elution with 95% A/5% B,
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followed by a linear gradient over 15 min to 65% A/35% B where it was maintained throughout the remainder of the chromatogram. Model Solutions of Glycylphenylalanine. The breakpoint curve of model solutions of glycylphenylalanine (1.43 × 10-3 M in 0.025 M NaH2PO4, adjusted to pH 7.0) was determined by chlorinating with rapid stirring five 100-mL aliquots of the solution to each of five different chlorine-to-dipeptide mole ratios (0.5, 1.0, 1.5, 2.0, and 2.5). The solutions were then incubated in the dark at room temperature for 30 min, and the aliquots were analyzed for free and total residual chlorine by the DPD-FAS titrimetric method (29). Chlorination products were determined by chlorinating aliquots (15 mL) of model solutions to five different chlorineto-dipeptide mole ratios (0.5, 1.0, 1.5, 2.0, and 2.5), incubating them in the dark at room temperature (22 °C) for 30 min, and analyzing them by HPLC (UV detection, 254 nm). The rates of decomposition of the chlorination products were determined by analyzing the chlorinated solutions over time periods of 45-65 h. Determining the amount of each chlorination product formed was made possible by mixing a 15-mL aliquot of the model solution with 1.0 mL of a stock solution of glycyl-p[3H]-phenylalanine (0.7 µCi/mL). Each aliquot was chlorinated to the five chlorine levels listed above. Each was handled, fractionated, and assayed in a manner similar to what has been described previously (20, 21). Radiochromatograms were generated as previously reported (20, 21) with an average recovery from the column of 90 ((4)% of the radioactivity applied. The rate of decomposition of N-chloroglycylphenylalanine was determined by chlorinating with 1 equiv a model solution that was inoculated with glycyl-p-[3H]-phenylalanine. This solution was analyzed by HPLC with liquid scintillation counting of chromatographic fractions after 1, 13, 24, and 45 h. Radiochromatograms were obtained, and changes in the amount of radioactivity associated with the N-chloroglycylphenylalanine peak were measured to determine the firstorder half-life. A similar experiment was conducted to determine the stability of N,N-dichloroglycylphenylalanine (II) and N-[2(N′-chloroimino)ethanoyl]phenylalanine (III). An aliquot of the model solution was inoculated with the tritiated dipeptide as described above and chlorinated to a chlorine-to-peptide mole ratio of 2.0. Products were analyzed eight times over 65 h. The retention times observed in the UV chromatograms agreed with those observed in the radiochromatograms. The presence of chlorine in the chlorination products was determined by reacting glycylphenylalanine model solutions with 2 equiv of radioactive chlorine. A working solution of HO36Cl was prepared in a 10-mL volumetric flask by mixing 8 µL (4.4 µCi) of commercial Na36Cl with 204 µL of 14 mmol of aqueous chlorine. The solution was diluted to approximately 8 mL with 0.025 M phosphate buffer. One milliliter of the model dipeptide solution was added, and the solution was diluted to the mark (10 mL) with additional buffer. The reaction mixture was analyzed several times over 24 h by collecting HPLC fractions and assaying them as previously described (20) to determine the presence of chlorine-containing products. The detection efficiency for 36Cl was approximately 95%. Description of Wastewater. A secondary wastewater was obtained from the Williamsburg treatment facility located in Williamsburg, VA, at a sampling point prior to ammonia addition but after nitrification. Handling and storage of the wastewater has been described previously (30). The total Kjeldahl nitrogen (TKN) and ammonia concentrations were determined by the Hampton Roads Sanitation District according to published procedures (29). Amino Acid Analysis. HPLC analysis of amino acids was performed as described previously (30). A Whatman 5µm
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Partisil ODS-3 analytical column was used for this analysis. Due to co-elution of the o-phthalaldehyde derivatives of glycine and threonine, two different elution systems were employed. The solvent system used for the phenylalanine determination has been described previously (21, 22). Free dissolved phenylalanine was concentrated 10:1 by lyophilizing a 20-mL aliquot of wastewater and reconstituting the residue with 2 mL of Milli-Q water. The standard curve for phenylalanine had a slope of 3.32 × 104, an intercept of 1.24 × 104, and a correlation coefficient of 0.999. The standard curve for glycine had a slope of 1.98 × 104, an intercept of 3.18 × 104, and a correlation coefficient of 0.998. The solvent system used for glycine determinations has also been described previously (31). Additionally, the concentrations of total hydrolyzable glycine and phenylalanine in the wastewater were determined by hydrolysis and HPLC analysis as previously described (32). Analysis of Chlorination Products in Wastewater. NaH2PO4 (3.45 g) was dissolved in 1.0 L of wastewater, and the pH was adjusted to 7.0 with sodium hydroxide. The breakpoint curve was determined by chlorinating 100-mL aliquots to concentrations of 2, 4, 6, 8, 10, 12, and 14 mg of Cl2/L, incubating them in the dark at room temperature (22 °C) for 30 min, and analyzing them for free and combined residual chlorine (29). Aliquots (15.0 mL) of wastewater were inoculated with 1.0 mL of a stock solution of glycyl-p-[3H]-phenylalanine (7.0 µCi/10 mL; specific activity 25 Ci/mmol; 2.8 × 10-8 M), chlorinated to each of nine levels between 1 and 9 mg of Cl2/L, and their volumes normalized to 17.0 mL. Reaction mixtures were incubated in the dark for 30 min at room temperature (22 °C), fractionated, and assayed as described previously (20). Similarly, the rate of decomposition of N,Ndichloroglycylphenylalanine (II) and the N-chloroaldimine III were determined over a 65-h time period by HPLC analysis of buffered wastewater chlorinated to 8 mg of Cl2/L. Synthesis of N,N-Dichloroglycylphenylalanine (II). NaH2PO4 (2.07 g, 15.0 mmol) was dissolved in 100 mL of Milli-Q water, and the pH was adjusted to 7.0 with NaOH. To this solution was added glycylphenylalanine (1.11 g, 5.0 mmol) with continuous stirring. Two equivalents (10 mmol) of a standardized solution of aqueous chlorine (Clorox) were added with stirring. After 5 min the product was precipitated by the rapid dropwise addition of concentrated HCl to obtain a pH < 2. The solution was chilled in ice prior to filtration. The product was dried in a vacuum desiccator and weighed. Recrystallization from cold aqueous acetonitrile yielded pure II in 75% yield, mp 115-116 °C (lit. mp 113-115 °C) (26). Anal. Calcd. for C11H12N2O3Cl2: C, 45.38; H, 4.15; N, 9.63; Cl, 24.36. Found: C, 45.43; H, 4.18; N, 9.53; Cl, 24.26; IR (KBr) 3360 (s, N-H), 2920 (m, C-H), 1710 (m, CdO), 1640 (s, CdO), 1540 (s, C6H5) cm-1; 1H NMR (acetone-d6) (δ): 7.3 (s, 5, C6H5); 4.5-4.8 (t, 1, NH-CH); 4.4 (s, 2, N-CH2-CO): 3.1-3.4 (q, 2, C6H5-CH2). Synthesis of N-Cyanoacylphenylalanine (IV). II (0.586 g, 2.0 mmol) was added with stirring to 100 mL of Milli-Q water to form a suspension. The initial pH was approximately 3.0. The solution was slowly titrated with 1.0 N NaOH. It remained cloudy and gradually cleared as additions of base were added. Product distribution was monitored by HPLC. After 2.75 equiv of base had been added, complete conversion of starting material to IV was observed. The product was obtained by ethyl acetate extraction of the aqueous solution after lowering the pH to approximately 2.0. The product was a thick oil. IR (thin film) 3600-2800 (m, OH), 2220 (m, CtN), 1730 (m, CdO), 1680 (m, CdO), 1575 (m, C6H5) cm-1; 1H NMR (acetone-d6) (δ) 7.3 (s, 5, C6H5); 4.7-4.9 (q, 1, NH-CH), 3.1-3.4 (m, 2, C6H5-CH2); 13C-NMR (acetone-d6) (δ): 173.2 (s, CdO); 145.5 (s, CdO); 129-139 (aromatic); 114.1 (s, CtN); 56.7 (d, NH-CH); 39.0 (t, C6H5-CH2).
was added, and the sample was allowed to react for 1 h before it was purged with dry nitrogen gas. The resulting clear solution was analyzed by GC/MS and found to contain a single peak in addition to solvent, m/z (relative abundance): 205 (0.21, M - HCN), 162 (25, M - HCN - HNCO), 131 (6, m/z 162 -CH3O), 91 (100). The chemical ionization mass spectrum revealed a weak parent ion at m/z 232. Exact mass measurements on the parent ion found m/z 232.08378 (calcd mass 232.08479).
Results and Discussion
FIGURE 1. HPLC chromatograms of a model solution (1.43 × 10-3 M, pH 7.0) of glycylphenylalanine chlorinated with 2 equiv and analyzed after 0.5, 6, and 30 h. IV was methylated with diazomethane in methyl tert-butyl ether by the Fales procedure (33). An excess of diazomethane
Because wastewater treatment plants typically provide a 30min contact time for chlorinated effluent before they discharge it, the work described here examined the reactions of glycylphenylalanine within the first 30 min following chlorination. In addition, decomposition of primary chlorination products over longer time periods was studied to estimate the potential long-term environmental impact. Identification of Glycylphenylalanine Chlorination Products in Model Solutions. The 30-min breakpoint curve of a model solution of glycylphenylalanine (1.43 × 10-3 M) does not exhibit a chloramine maximum or an irreducible minimum. The combined residual chlorine concentration measured after 30 min increases proportionally to the amount of aqueous chlorine added without any apparent loss of oxidant power up to a chlorine-to-peptide ratio of 1.0. At ratios between 1.0 and 2.0, no significant increase in combined chlorine residual is observed with increasing dose. Above chlorine-to-peptide ratios of 2.0, the total chlorine residual increases linearly due to the presence of free residual chlorine. When model solutions of glycylphenylalanine were chlorinated (pH 7.0) to chlorine-to-peptide mole ratios less than 1.0, only a single, very stable chlorination product was detected by HPLC in 30 min. The column effluent containing the one chromatographic peak was found to oxidize iodide to iodine, indicating the presence of a chlorine-based oxidant such as a chloramine. Because the concentration of the one product reached a maximum as the chlorine-to-peptide ratio approached 1.0 and decreased at higher ratios, the compound was believed to be N-chloroglycylphenylalanine. At ratios greater than 1.0, a different compound was formed that also oxidized iodide to iodine. Its concentration reached a maximum at a chlorine-to-peptide mole ratio of 2.0. It could be precipitated from concentrated solution by the addition of acid, and elemental and spectral analysis identified it as N,N-dichloroglycylphenylalanine (II). No other products were observed at higher chlorination levels. II decomposed at pH 7.0 and formed a new compound, III, which decomposed over longer periods of time (>10 h) to a third compound, IV. Over very long time periods a fourth
FIGURE 2. Proposed mechanism for the decomposition of N,N-dichloroglycylphenylalanine.
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FIGURE 3. Outline of the synthesis of glycyl-p-[3H]-phenylalanine. product, V, became evident. Figure 1, panels a-c, shows HPLC chromatograms that reflect the decomposition of compound II over a 30-h period and the corresponding formation of III, IV, and V. When glycylphenylalanine was reacted with 36Cl-labeled hypochlorous acid, II and III were found to contain radioactivity. The amount of increase in 36Cl activity of the chromatographic peak associated with III was only about half (48%) of the amount of decrease in the 36Cl activity of the chromatographic peak associated with II, indicating that III contained only half as much chlorine as 1 equiv of II. Except for [36Cl]chloride, which eluted in the void volume of the column, no other chromatographic peaks contained 36Cl activity. Because of its instability, III could not be isolated sufficiently pure for spectral analysis. Based on the fact that N,N-dichlorinated R-amino acids decompose to N-chloroaldimines (20-24) and that III contains only half as much chlorine as II, the structure of III
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is believed to be N-[2-(N′-chloroimino)ethanoyl]phenylalanine (III) (Figure 2). Pereira et al. (26) synthesized and characterized a series of N,N-dichlorinated dipeptides and dehydrohalogenated them with ethanolic NaOH to produce N-chloroimines. However, none contained N,N-dichlorinated glycine residues that are capable of undergoing two successive dehydrohalogenations to form a nitrile moiety. Attempts to isolate compound III by Pereira’s method resulted in formation not only of III but also of another product, which has been identified as cyanoacylphenylalanine (IV). IV was isolated by acid precipitation and characterized by 1H- and 13C-NMR, GC/MS, and IR. The absorbance at 2220 cm-1 in the IR spectrum indicates the presence of a cyano group. This assignment is supported by a 13C-NMR spectrum that contains a singlet (off-resonance) at 114 ppm. The 1H-NMR and GC/ MS further support the assignment of the structure of IV as N-cyanoacylphenylalanine (IV) (see Figure 2). Although
FIGURE 4. Product distribution over a 65-h time period of a model solution (1.43 × 10-3 M, pH 7.0) chlorinated with 2 equiv. Stelmaszynska et al. (27) have proposed the intermediacy of compounds like IV in the breakdown of N,N-dichlorinated dipeptides, this is the first confirmation that compounds of this unusual class form on chlorination and that they are remarkably stable. Compound IV slowly decomposes to V. V has been identified as phenylalanine by its retention time and by derivatization of the product mixture with o-phthalaldehyde (OPA) and correlation of the retention time of the OPA derivative formed with the OPA derivative of phenylalanine. In the accompanying study of the chlorination reactions of alanylphenylalanine, a distinct chromatographic peak was detected that could not be isolated and identified (unknown A), but which appeared to correspond to a steadystate precursor of phenylalanine. It was tentatively identified as an isocyanate or a carbamic acid. It was estimated that the isocyanate or carbamic acid intermediate in this study would have co-chromatographed with cyanoacylphenylalanine (IV). Mechanism. The overall mechanism for the degradation of N,N-dichloroglycylphenylalanine to phenylalanine is given in Figure 2. In addition to the sequential dehydrohalogenations of II to form III and of III to form IV, IV is believed to undergo a dehydrocyanation to form an isocyanate that hydrolyzes to a carbamic acid which, in turn, readily loses CO2 to form phenylalanine. Synthesis of Glycyl-p-[3H]-phenylalanine. In order to observe the reactions of glycylphenylalanine in a wastewater and to measure the quantities of each chlorination product formed, glycyl-p-[3H]-phenylalanine was synthesized. Figure 3 outlines the method used to synthesize the precursor glycylp-bromophenylalanine (30). 1H- and 13C-NMR and IR spectra for all intermediates were consistent with proposed structures. Glycyl-p-bromophenylalanine also gave correct elemental analysis. It was converted to glycyl-p-[3H]-phenylalanine by metal-catalyzed halogen reduction of glycyl-p-bromophenylalanine with tritium gas. Decomposition of Primary Chlorination Products in Model Solutions. Changes in the concentrations of the various chlorination products with time are plotted in Figure 4. It is obvious that the N-chloroaldimine III becomes the major chlorination product within the first 10 h. As III decomposes, the significance of cyanoacylphenylalanine (IV) increases. After about 45 h, phenylalanine becomes the major chlorination product. All of the primary chlorination products of glycylphenylalanine are significantly more stable than the products of the chlorination of the simple amino acids. N-Chloroglycylphenylalanine (I) decomposes slowly at 22 °C with a half-life of 134 ( 5 h. By contrast, monochlorinated amino acids are much less stable with half-lives ranging from 52 min for N-chlorophenylalanine (pH 6.9; 25 °C) to 21.6 h for N-
FIGURE 5. Distribution of glycyl-p-[3H]-phenylalanine chlorination products in a secondary wastewater (pH 7.0, 8 mg of Cl2/L) determined over a 65-h time period. chloroglycine (pH 6.2; 21 °C) (25). The half-life of the chlorinated dipeptide N-chloroglycylglycine has been determined by others to be 233 h (pH 8.54; 22 °C) (25). These data suggest that monochlorinated dipeptides are far more stable than monochlorinated amino acids. The half-life of N,N-dichloroglycylphenylalanine (II) in a model solution at 22 °C (6.4 ( 0.3 h) suggests that it is also far more stable than the N,N-dichlorinated amino acids, which are not isolable because they decompose rapidly to nitriles and N-chloroaldimines (20-24). The half-life of the Nchloroaldimine III in a model solution (38 ( 4 h at 22 °C) is comparable to those of the N-chloroaldimines derived from the N,N-dichlorinated amino acids. The half-life of IV would be difficult to determine since it decomposes at the same time it is forming. Characteristics of a Secondary Wastewater. The secondary wastewater utilized in these experiments had a TKN concentration of 1.29 mg of N/L and an ammonia concentration of 0.074 mg of N/L. Its breakpoint curve did not contain a distinct chloramine maximum and appeared very similar to that of glycylphenylalanine discussed above. A measureable amount of free residual chlorine was not observed below a dose of 6 mg of Cl2/L. The total residual chlorine concentration increased linearly above a dose of 8 mg of Cl2/L of aqueous chlorine. The wastewater had a free dissolved phenylalanine concentration of 3.5 × 10-8 M (5.8 ppb) and total hydrolyzable phenylalanine concentration of 1.3 × 10-6 M (210 ppb). The free dissolved glycine concentration was below the detection limit (
