Application of Saturation Transfer Double Difference NMR to Elucidate

Jan 12, 2008 - Elucidation of mechanistic interactions of anthropogenic chemicals is critical to understanding and eventually predicting their behavio...
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Environ. Sci. Technol. 2008, 42, 1084–1090

Application of Saturation Transfer Double Difference NMR to Elucidate the Mechanistic Interactions of Pesticides with Humic Acid AZADEH SHIRZADI, MYRNA J. SIMPSON, YUNPING XU, AND ANDRÉ J. SIMPSON* Department of Chemistry, University of Toronto, Scarborough College, 1265 Military Trail, Toronto, Ontario, M1C1A4 Canada

Received September 27, 2007. Revised manuscript received November 28, 2007. Accepted November 30, 2007.

Elucidation of mechanistic interactions of anthropogenic chemicals is critical to understanding and eventually predicting their behavior in the environment. Here, a recently developed technique, saturation transfer double difference (STDD) NMR spectroscopy is employed to determine the interactions of pesticides with humic acid (HA) at the molecular level. The degree of interaction at each NMR observable nucleus in the pesticide can be quantified in the form of an epitope map, which depicts the mechanism of the pesticide-HA interaction. Our results indicate that, at pH 7, halogen atoms (F and Cl) in water-soluble pesticides (diflufenzopyr, acifluorfen, and chlorsulfuron) play a dominant role in influencing binding to HA, whereas carboxyl groups likely play a secondary role when halogen atoms are also present in the molecule, as observed with diflufenzopyr and acifluorfen. However, when present on its own, the carboxyl group dominates in binding affinity to HA (e.g., imazapyr). Electronegativity and electron density appear to play a key role in the mechanism of binding and results suggest that polar bonds are the primary points of HA contact in the water soluble pesticides investigated. Likely interactions may include hydrogen bonding and dipole–dipole interactions.

Introduction The interactions of contaminants with soil at the molecular level are central to their bioavailability, bioaccumulation, transport, and toxicity in the environment. Sorption of pesticides to soil organic matter (SOM) can significantly decrease their bioavailability (1) and thus greatly impact bioremediation efforts (2). Elucidation of the mode of interaction at a mechanistic level is critical to understanding and eventually predicting the behavior of anthropogenic chemicals in the environment (3, 4). Numerous analytical techniques are available that can potentially provide information regarding the binding of contaminants to humic acid (HA; a major component of soil organic matter), and include fluorescence spectroscopy, infrared spectroscopy, and mass spectrometry. However, because of the fact that individual nuclei are very sensitive to their chemical surroundings, NMR spectroscopy in particular is very useful for the study of binding mechanisms (3). There are a wide range of NMR accessible parameters that can be employed to study * Corresponding author phone: (416) 287-7547; fax: (416) 2877279; e-mail: [email protected]. 1084

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molecular interactions including chemical shifts (5), relaxation (6), diffusivities (7), and nuclear Overhauser effects (8). However, until now, nearly all NMR based approaches were hampered by the fact that both the free and bound forms of contaminants are observed simultaneously, which often complicates interpretation of the data. Here, a relatively new technique, saturation transfer double difference (STDD) NMR spectroscopy (9) is applied to evaluate the binding mechanisms of some common pesticides to HA. STDD is designed such that only signals from the bound forms of a ligand are observed, which permits mechanistic information to be directly extracted from the experimental data. Furthermore, because of the fact that only the bound fraction is observed, it is possible for the ligand to be present in excess. This maximizes the number of interactions occurring in solution and in turn results in increased experimental sensitivity (10). Note in this manuscript “ligand” and “epitope” are used to describe a small molecule interacting with a larger molecule (the “receptor”), these terms are used simply to be consistent with the NMR literature and are no way meant to infer the comparability of HA/pesticide to biological systems, for example, a protein/drug system. During the experiment, the receptor (in this case HA) is selectively saturated using a weak radio frequency field and any bound ligands (in this case pesticide) receive saturation from the receptor. The experiment is designed such that all other signals (e.g., water or unbound ligands) cancel and are thus not observed. In the resulting difference spectrum, only bound ligands are observed and the intensity of the various signals from these bounds ligands are proportional to their spatial proximity to the receptor (9, 11, 12). The interaction at each NMR observable nucleus in the ligand can be quantified, in the form of an epitope map, which describes the mechanism of interaction. Presently this epitope mapping technique has been most notably used to determine the mechanism of binding in medical and pharmacological studies to aid in structure-based drug design and discovery (13). Here, we describe some preliminary applications of STDD epitope mapping to environmental chemistry to elucidate pesticide-HA binding mechanisms. Specifically, interaction between four pesticides (diflufenzopyr, acifluorfen, imazapyr, and chlorsulfuron) and peat HA are investigated. Acifluorfen is a highly effective herbicide, used in the selective control of broad-leaf weeds, is persistent on soils and in aquatic environments, and is relatively mobile (14). Imazapyr is a broad spectrum herbicide for weed control in nonagricultural areas, is known to be persistent in soils and has been found to contaminate both surface and ground waters (15). Chlorsulfuron, a sulfonylurea herbicide, is used to control broadleaf weeds in small grains, has half-lives in soil ranging from 14 to 320 days and is known to be highly mobile in the environment (16). Diflufenzopyr is an auxin-transport inhibitor that has increased broadleaf weed control and it is highly water soluble thus highly mobile therefore the potential for surface runoff and leaching exists; it is not persistent in soils, however, can be persistent under aerobic aquatic conditions (17). In addition to their environmental importance, these herbicides were chosen because of their high water solubility (thus negating the need for organic solvents that are detrimental to interaction-based studies (6)), the fact that they have no proton signals that are closer than 1 ppm from the point of irradiation (allowing selective irradiation of the HA), and because they were commercially available and affordable. The mechanism of binding for each pesticide is elucidated by this method, clearly demonstrating 10.1021/es7024356 CCC: $40.75

 2008 American Chemical Society

Published on Web 01/12/2008

that the STDD technique will likely have an important role in understanding the fate and transport of anthropogenic chemicals in the environment.

Experimental Section Sample Preparation. High purity, analytical standard grade (PESTANAL) imazapyr (2-[4-methyl-4-(1-methylethyl)-5-oxo4,5-dihydro-1 H-imidazol-2-yl]pyridine-3-carboxylicacid) (99.9%), diflufenzopyr (2-[(1 E)-N-[(3,5-difluorophenyl) carbamoyl] ethanehydrazonoyl] pyridine-3-carboxylic acid) (96.5%), acifluorfen (5-[2-chloro-4-(trifluoromethyl)phenoxy]2-nitrobenzoic acid) (97.8%), and chlorsulfuron (2-chloroN-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)carbamoyl] benzenesulfonamide) (99.9%) were obtained from Sigma Aldrich (Canada). Deuterium oxide (D2O), NaOD and DCl 99.9% purity, were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA). HA and the pesticides were dissolved in D2O and the pH was adjusted to 7 using sodium deuteroxide (NaOD, 99.5% purity) and deuterium chloride, (DCl, 99% purity) accordingly, using a Metler Toledo combination NMR pH electrode (Fisher). Taking into consideration the deuterium isotope effect on pH measurements in D2O the pH of the samples was 7.4 (18). The imazapyr and diflufenzopyr samples were made at concentrations of 13.2 and 16.8 mg/mL, respectively. Because of solubility limitations, the acifluorfen and chlorsulfuron samples where prepared at lower concentrations of 2.4 and 2.6 mg/mL respectively. Pahokee (Florida) peat HA was purchased from the International Humic Substances Society (IHSS; St. Paul, MN). Peat HA was dissolved in D2O as a stock solution at a concentration of 10 mg HA/ml D2O, and the pH was adjusted to 7 using NaOD; 1 mL of this stock solution was utilized per pesticide sample. Upon addition of pesticide, the pH of the sample was adjusted accordingly using NaOD or DCl. It is important to remind the reader that because of STDD NMR detects only the bound ligand fraction, the presence of excess ligand is beneficial as this maximizes the number of ligand–receptor interactions and results in increased sensitivity in the NMR experiment (11). Note that the average molecular weight of the IHSS peat HA is at least 3000 Daltons (7) as such the pesticides will be present in molar excess in all samples. It should be noted that because of the use of deuterated solvent system, exchangeable groups are not observed in the NMR experiments. NMR Spectroscopy. All NMR spectra were acquired using a Bruker Avance 500 MHz spectrometer equipped with a 1H-BB-13C triple resonance broadband inverse (TBI) probe fitted with an actively shielded Z gradient. In all experiments excitation sculpting (19) was employed using a 2 ms selective rectangular pulse defined by 1000 points and truncated to 1% to suppress residual water signal. Saturation transfer difference (STD) experiments were carried out using the approach described by Mayer et al. (11). Selective saturation of the HA was achieved by a train of 50 ms Gauss shaped pulses, truncated at 1%, and separated by a 100 µs delay. Forty selective pulses were applied, leading to a total length of the saturation train of 2.004 s. The on-resonance irradiation of the HA was performed at a chemical shift of 0.85 ppm with the exception of imazapyr, which was irradiated at 3.4 ppm due the presence of pesticide signals close to 0.85 ppm. Offresonance irradiation was set at 114 ppm, where HA signals were not present. Selective irradiation was carried out using very carefully calibrated power levels, which depended on the distance of the nearest pesticide signal from the point of irradiation. For diflufenzopyr, chlorsulfuron and imazapyr an effective field of 26 Hz was used for irradiation (70 db attenuation on a 60 W amplifier). In the case of the acifluorfen poor signal-to-noise was encountered using Beff of 26 Hz and the field was increased to 250 Hz (∼50 db attenuation on a 60 W amplifier), which was possible as no pesticide signals

were close to the point of irradiation (0.85 ppm) for this molecule. The spectra were subtracted internally via phase cycling after every scan using different memory buffers for on- and off-resonance irradiation. 256 000 scans were accumulated for each STD experiment. Reference spectra were recorded using the identical sequence with the exception that no irradiation power was applied and that the phase cycle was changed such that each of the 256 scans were additive. All experiments were performed with 16 384 time domain points, 256 dummy scans, and additional recycle delay of 100 ms (in addition to the saturation time). Spectra were apodized through multiplication with an exponential decay corresponding to 1 Hz line broadening in the transformed spectrum, and a zero filling factor of 2. Spectral subtractions to produce the double difference spectra were performed in the interactive mode of Topspin 1.3 (Bruker BioSpin Ltd.). All contaminant assignments have been made using a combination of 2D heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), and Correlation Spectroscopy (COSY); data not shown. It is important to stress that although STD NMR is a relatively simple NMR technique, extensive calibrations of the saturation power must be carefully performed for each NMR probe used in STD studies. All experimental parameters employed here were tested on the pesticides alone and the pesticides in the presence of HA. In the case of the pesticide only samples, no signals were observed when the STD experiments were performed with the irradiation settings used here. The same is observed with the pesticide and HA mixtures when the irradiation time or irradiation power is set to zero. These tests are essential to ensure that the signals in the STD spectrum result from solely HA to pesticide saturation transfer. To further support this, a full saturation curve (11) for the acifluorfen and HA mixture is shown in the Supporting Information (Figure S1) as an example.

Results and Discussion Before proceeding to discussions pertaining to the mechanistic interactions of the pesticides, it is important to briefly introduce STDD NMR and epitope mapping. Saturation Transfer Double Difference Spectroscopy. Figure 1A shows the 1H NMR spectrum of diflufenzopyr and HA. It is clear from the spectrum that the herbicide is present in excess (when compared to Figure 1B, which shows spectrum for HA). Figure 1D shows the STD spectrum. In this case the region labeled * has been selectively saturated. At 10 mg/mL the peat HA is known to behave as a large aggregate (7). In stable aggregates and macromolecules, dipolar interactions (which are averaged for very small molecules by isotropic tumbling) become dominant providing the framework for the efficient propagation of spin diffusion which essentially results in spin population disturbance throughout the entire aggregate. In simple terms the saturation “coats” the HA aggregate. This can be seen to some extent in Figure 1E which shows an STD experiment for HA with no pesticide present. Although the signals are relatively weak; aromatics, carbohydrates, and aliphatic resonances can be observed, which arise as a result of saturation transfer throughout the HA molecular network. During the process of “saturation transfer”, any pesticide bound or interacting with the HA will also receive some saturation, with the nuclei closest to the HA receiving the most and those furthest from the HA receiving the least. Thus, after the STD experiment the proximity of the pesticide nuclei to the HA is encoded into the NMR spectrum and through careful quantification this information can be accessed. However, at this point in both the reference spectrum (Figure 1A) and the STD spectrum (Figure 1D) the pesticide signals (of interest) are situated on signals from HA VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Series of NMR spectra required for creating the “double difference spectra” from which an epitope map for the pesticide diflufenzopyr will be derived. (A) Reference 1H NMR spectrum of diflufenzopyr and HA. Arrows indicate signals from diflufenzopyr. (B) Reference 1H NMR spectrum of HA alone. Basic assignments for the HA are provided (21), for the chemical structure and assignments for diflufenzopyr see Figure 2. (C) Subtraction of B from A results in a double difference reference spectrum (DDRS). (D) 1H STD NMR spectrum of diflufenzopyr and HA. Point of irradiation is indicated by * in the STD spectrum. (E) 1 H STD NMR spectrum of HA alone. (F) Subtraction of E from D to create a saturation transfer double difference (STDD) NMR spectrum. (background and of no interest). Therefore, before accurate quantification can be performed, a method that can successfully remove the background signals from those of the ligand without affecting the quality of the spectrum is required. A common way to remove the background signals is to introduce a T1F or T2 filter into the NMR experiment, which either reduces the background signals via dephasing or preferential relaxation. However, such filters also tend to filter out the signals from ligands which are very tightly bound to the receptor (12), in turn biasing toward molecules that are in exchange with the receptor but are not permanently 1086

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bound (9). Recently, Claasen et al. introduced saturation transfer double difference spectroscopy (STDD) (9). In this approach no NMR filters are used and the background is removed by using simple spectral subtraction. Figure 1B shows the reference spectrum of HA without pesticide present. Subtraction of the spectrum shown in Figure 1B from spectrum shown in Figure 1A produces Figure 1C which is referred to as the double difference reference spectrum (DDRS). Similarly, subtraction of 1E from 1D produces Figure 1F which is called the saturation transfer double difference (STDD) spectrum. With the background signals successfully eliminated, a quantitative comparison of the DDRS (Figure

FIGURE 2. (A) Double difference reference spectrum (DDRS) for the pesticide diflufenzopyr and HA. (B) Saturation transfer double difference (STDD) spectrum of diflufenzopyr and HA. By comparing relative integrals of the diflufenzopyr signals between the DDRS and STDD spectra, we can obtain an epitope map. (C) Epitope map for diflufenzopyr. Signals which cannot be resolved in the NMR spectrum are indicated by †, see Figure 3 and text for more details. 1C) and STDD (Figure 1F) can now be performed to produce an epitope map which describes the binding mechanism of the molecule. It should be noted that in Figure 1, the contaminant signals are very intense and somewhat dwarf the signals from the HA. Furthermore, since a weak irradiation field is used (to avoid any chance of direct pesticide irradiation) the signals from HA are also relatively weak in the STD spectrum. However, this is not always the case (see Figure S2 in the Supporting Information for an example) and it is essential to employ a double difference approach to remove the background to ensure accurate integration of the peaks. Creation of an Epitope Map. Figure 2 compares the DDRS (A) and the STDD (B), in the later only the bound or interacting contaminant can be observed. In the STDD spectrum (Figure 2B) the protons most closely associated with HA, which received the greatest amount of saturation, have higher relative signals when compared to the reference (DDRS). The epitope map is created by setting the proton that has the strongest relative STD signal at 100% and all other protons are reported relative to it (11). As such the epitope map provides a quantitative description of the degrees of interaction of the various protons in the pesticide. Figure 2C shows the epitope map for diflufenzopyr as an example. Diflufenzopyr Interactions with HA. The binding epitope of diflufenzopyr is presented in Figure 3A. The protons that are neighbored by fluorine (He and Hd) show the highest binding (100% and 96% respectively). This may be explained by the fact that fluorine has the highest electronegativity of all elements based on the Pauling scale (20). If the electronegativity difference between two atoms in a bond is between 0.5 and 1.6 the bond is considered to be polar covalent. The C-F bonds in diflufenzopyr are polar covalent due to the difference in electronegativities between the F and C atoms (2.55 and 3.98 respectively). Fluorine pulls electron density away from C atoms, thus the F atoms have a slight negative charge, whereas C has a slight positive charge, which creates a dipole in the bond. This dipole would allow the fluorine atoms to interact with other polar species, and potential interactions may include the formation of hydrogen bonds, dipole–dipole interactions and various other interactions

based on partial charge. As protons adjacent to the C-F bonds show the strongest interactions with HA, it is clear that these electronegative units are likely a primary factor in determining the molecular interaction of diflufenzopyr. Interestingly, protons Hf and Hb are both near the carboxyl group and also show a high degree of interaction with HA (76 and 75%, respectively). This suggests that the carboxyl group also plays a key role in binding. In the carboxyl group, the oxygen atom is more electronegative than the C atom it is attached to (3.44 and 2.55 respectively), thus the CdO is considered polar covalent and may interact in a similar manner as the C-F groups, although it should be noted that fluorine has a higher electronegativity which may explain its apparently stronger interaction at pH 7. However, it is important to consider that at pH 7 the carboxyl group in diflufenzopyr, which dissociates at pH 3.18 (17), may repel other dissociated anions in the humic acid and thus display a considerably reduced interaction. This is reflected in the Koc for diflufenzopyr which increases from 1 at pH 5 to 46 at pH 3 (ACD laboratories Inc., ACD/Adsorption Predictor, V7.04). Thus at lower pH it is likely the COOH group will play a definitive role in the pesticide’s interaction. It is interesting to note that the IHSS peat HA does contain a considerable peptide/protein fraction (21) which theoretically may attract the COO- group in diflufenzopyr at pH 7. Similarly, it is possible that the NH groups in diflufenzopyr may play a role in the association through hydrogen bonds with the HA. However, although this is theoretically possible, it is not supported by the mechanistic interactions of imazapyr and chlorsulfuron (see later). Unfortunately, the STD NMR approaches employed here only provide information as to the pesticide interaction and do not provide evidence regarding the humic components involved in the interaction. Future studies will aim to utilize STD NMR to investigate how the binding mechanisms for common contaminants are altered by chemical/physical parameters such as temperature, electrolyte concentration and/or pH as well novel NMR techniques will be used to probe the humic constituents that are involved in binding. At this point in the discussion, it is important to clarify to the reader that, for diflufenzopyr, if the epitope map is VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Epitope maps of four water-soluble pesticides obtained using STDD NMR. (A) diflufenzopyr, (B) acifluorfen, (C) imazapyr, (D) chlorsulfuron. A and B likely associate with HA in a planar orientation, and as such they have shaded and nonshaded sides referring to their higher affinity and lower affinity, respectively. Protons that have 100–80% binding are highlighted by red circles, whereas protons with 79–65% are blue and 64% and lower are green. The lower case letter “a” next to a proton indicates it is the highest field signal; “b” refers to the second highest field signal and so on (refer to Figure 2). The percentage values for each epitope map are all relative to the strongest binding proton in each pesticide which is expressed as 100%. considered alone and without a detailed understanding of the NMR data, it could be somewhat misleading. Specifically, signals from Hd1 and Hd2 overlap in the NMR spectrum (see Figure 2A) and thus the value of 96% assigned to these protons actually describes the average interactions of Hd1 and Hd2, rather than their individual contributions. Based on the percent interactions of the other protons in this pesticide it seems likely that one side of this planar molecule (shaded) interacts to a greater degree with HA than the other (not shaded) due to the presence of the fluorine atoms and the carboxyl group on the same side. Thus, if it were possible to differentiate the degree (%) of interaction of Hd1 and Hd2, one would expect that Hd1 would preferentially interact with HA. Nevertheless, even without the ability to differentiate the extent of interaction of Hd1 and Hd2 individually, it is clear that both the C-F and COOH groups are central to the mechanism of interaction. Although it is likely that diflufenzopyr interacts in a planar fashion, as shown, an alternate explanation is also worth considering. As HA is a complex mixture, it is feasible that there are different binding sites within this mixture, some of which attract the C-F groups preferentially, whereas others attract the COOH group. The observed epitope map could thus reflect the relative distributions of these various binding sites. Evidence from the mechanisms of binding of the three other pesticides included in this study support the interaction of the molecule in a planar fashion, rather than there being separate specific binding sites for the C-F and COOH groups. At this point, however, for diflufenzopyr, this alternative cannot be completely ruled out. Acifluorfen Interactions with HA. Figure 3B shows the epitope map for acifluorfen. The DDRS and STDD NMR spectra for this and all other pesticides studied are provided in the Supporting Information (Figures S3-S5). The proton (Hb), flanked by the group of three fluorines and one chlorine molecule, has the highest affinity (100%) for HA. Both fluorine and chlorine have high degrees of electronegativity, 3.98 and 3.16, respectively, which is likely key in determining their high affinity for HA, as previously described for diflufenzopyr. Proton Hf, which is adjacent to a carboxyl group, shows the 1088

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next highest association (83%). This carboxyl has a pK a of 3.5 (22) and thus will be dissociated at pH 7. Considering that the Koc increases from 8.64 at pH 5 to 438 at pH 3 (ACD laboratories Inc., ACD/Adsorption Predictor, V7.04) it is highly probable that the carboxyl group plays a key role in the association at lower pH. Hf is on the same side of the compound as Hb (acifluorfen is relatively planar) and it is evident that one side of acifluorfen (shaded) likely binds to HA to a greater extent than does the other (unshaded), similar to that observed with diflufenzopyr (Figure 3A). It is unlikely, for example, that the COOH/NOO- functional groups of the molecule interact exclusively with many binding sites because if this were the case, one would expect the interaction at proton Ha to be similar to that of proton Hf. It is more likely that the slightly higher affinities of protons Ha and Hc when compared to Hd and He are simply their proximity to the strongly binding functionalities which on average bring them closer to the HA than protons Hd and He which are further removed. Imazapyr Interactions with HA. As the epitope map of imazapyr demonstrates (Figure 3C) the proton adjacent to a carboxyl group (Hb) shows the highest degree of interaction (100%) with HA. This again is likely explained by the polarity and electronegativity of the carboxyl group which has a pKa of 3.6 (23). The proton next to the nitrogen in the pyridine ring shows intermediate affinity with HA (44%) which, interestingly, is larger than the affinity of the aromatic proton Hc (31%). Electron density in a pyridine ring is localized around the N atom and it has been previously determined that there are at least two types of interactions that occur between pyridine and HA: bonding with the lone pair of electrons of pyridine’s nitrogen and π-π interactions between the aromatic ring of pyridine and aromatic components of HA (24). For this particular molecule, it appears that the lone pair of electrons on nitrogen plays a slightly more important role in binding to HA than aromatics alone (Ha versus Hc respectively). As demonstrated by the epitope map of imazapyr, when other functional groups are present (such as carboxyl groups) the aromatic interactions are weaker compared to those of other functional groups. At the other

end of the molecule, only very weak interactions with HA were observed for the CH3 groups (