Investigation of the Nucleophilic Attack of Dichlorvos by Reduced Sulfur Species Using 1H NMR

Article abstract of DOI:10.1021/acs.jafc.7b04749

The mechanism of the reaction of dichlorvos through hydrolysis reactions and through the reaction with polysulfide (S_n^2-) and thiophenolate (PhS^-) was investigated by proton nuclear magnetic resonance (¹H NMR). The study confirmed product identities of an organophosphorus insecticide reacting with reduced sulfur species using ¹H NMR in oxygen sensitive solutions. The experiments of dichlorvos with polysulfide led to the detection of a previously undetected product. The thiophenolate experiments were further advanced to investigate second-order rate kinetics using an internal standard. The experiments provide new evidence for a nucleophilic attack by the reduced sulfur species at the methoxy carbon of dichlorvos. In addition, the observation of in situ reaction dynamics illustrates the applicability of ¹H NMR spectroscopy toward kinetic investigations in environmental science.

Full text of DOI:10.1021/acs.jafc.7b04749

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Article
Investigation of the Nucleophilic Attack of
1
Dichlorvos by Reduced Sulfur Species using H NMR
Saumil Christian, Padmanava Pradhan, and Urs Jans
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04749 • Publication Date (Web): 09 Dec 2017
Downloaded from http://pubs.acs.org on December 9, 2017
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Journal of Agricultural and Food Chemistry
Investigation of the Nucleophilic Attack of Dichlorvos by Reduced
1
Sulfur Species using H NMR
†
†
,*,†
#
Saumil Christian , Padmanava Pradhan and Urs Jans
Department of Chemistry and Biochemistry, The City College of New York
60 Convent Avenue, New York, NY 10031
1
*
Corresponding author: Urs Jans
Department of Chemistry and Biochemistry, The City College of New York
60 Convent Avenue, New York, NY 10031
1
Tel (212) 650ꢀ8369; Fax (212) 650ꢀ6107; eꢀmail: ujans@ccny.cuny.edu
†
Department of Chemistry and Biochemistry, The City College of New York, New York,
NY 10031
#
Chemistry Program, The Graduate Center of the City University, New York, NY, 10016
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The mechanism of the reaction of dichlorvos through hydrolysis reactions and through
2
ꢀ
ꢀ
the reaction with polysulfide (S ) and thiophenolate (PhS ) were investigated by proton
n
1
nuclear magnetic resonance ( H NMR). The study confirmed product identities of an
1
organophosphorus insecticide reacting with reduced sulfur species using H NMR in
oxygen sensitive solutions. The experiments of dichlorvos with polysulfide led to the
detection of a previously undetected product. The thiophenolate experiments were further
advanced to investigate secondꢀorder rate kinetics using an internal standard. The
experiments provide new evidence for a nucleophilic attack by the reduced sulfur species
at the methoxy carbon of dichlorvos. In addition, the observation of inꢀsitu reaction
1
1
1
dynamics illustrates the applicability of H NMR spectroscopy toward kinetic
investigations in environmental science.
1
1
1
1
2
3
4
KEYWORDS: Dichlorvos, H NMR, thiophenolate, methyl polysulfide, desmethyl
dichlorvos
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Introduction
Organophosphorus compounds (i.e., esters and thioesters of phosphoric acids)
1
are among the most common insecticides encountered in surface waters in the U.S.
Dichlorvos (for structure see Fig. 2) is an organophosphorus pesticide that has been in
2
widespread use for over 45 years. It is reported to be genotoxic in vitro. It causes tumors
3
,4
in rats and mice in some studies but not others. Apart from having actively been used as
a pesticide, it has been found to be the breakdown product of the pesticide trichlorfon. It
is also generated in many plants through metabolism of the pesticide naled – of which it
3
,5ꢀ8
is postulated as the chief “insecticidal” principle.
Application of dichlorvos for
various pest control approaches have resulted in its release into sensitive environments
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such as salt marshes and estuaries where anoxic conditions are known to be prevalent.
Anaerobic conditions in these waters can result in the formation of reduced sulfur species
2
ꢀ
such as bisulfide, polysulfide (S ), thiosulfate, and organosulfur species (e.g.,
n
1
0ꢀ12
glutathione and cysteine).
In order to determine dichlorvos’ fate in these
environments, hydrolysis at environmentally relevant pH and nucleophilic substitution by
reduced sulfur species were considered to be the primary reaction pathways.
Phosphate triesters are known to hydrolyze via a nucleophilic substitution with
ꢀ
the attack of OH on the phosphorus atom resulting in the cleavage of a PꢀO bond, while
1
3,14
ꢀ
H O might also attack at the carbon of a methoxy or ethoxy group.
2
When OH attacks
the phosphorus of dichlorvos, the vinyl phosphate bond is most susceptible to breakage
1
5
due to electron withdrawing chlorines on the vinylꢀgroup. This has been supported by
past investigations showing that dichlorvos hydrolysis resulted in the formation of
dichloroacetaldehyde that is in equilibrium with dichlorovinyl alcohol
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(dichloroethenol). However, the formation of dimethyl phosphate, the other expected
1
product of this reaction was not analyzed. In the current work, H NMR was employed to
1
further support the previously proposed reaction mechanism. Since H NMR has the
potential to provide insight into all hydrogen containing products formed, it can deliver
valuable information on reaction pathways, as well as potential subsequent reactions of
primary products.
Reduced sulfur species have been known to react with a wide array of organic
17ꢀ20
2ꢀ
pollutants that undergo nucleophilic substitution.
Concentrations of Sn as high as
2
1,22
0.33 mM have been reported in porewaters of marine sediment.
While concentrations
of thiophenol have not been reported, thiophenolꢀcontaining compounds are likely to
form through reactions of dissolved natural organic matter with bisulfide. Previous work
2
ꢀ
in our lab with Sn and organophosphorus pesticide (e.g., chlorpyrifosꢀmethyl) support a
2
3
2ꢀ
n
substitution reaction at the carbon of the methoxy group of the pesticide. S and
thiophenolate are expected to react analogously with dichlorvos, resulting in the
formation of Oꢀdesmethyl dichlorvos and methylated reduced sulfur species – methylated
2
ꢀ
polysulfides for S (Figure 2A) and thioanisol for thiophenolate. The attack of the
n
reduced sulfur species at the phosphorus atom resulting in the formation of sulfurꢀ
substituted dimethyl phosphate and dichloroacetaldehyde (Figure 2C) was also
considered, although less likely to occur due to unfavorable hardꢀsoft interactions.
Previous investigation in our lab determined the second order reaction rate
constant of dichlorvos with thiophenolate (pH≥9.0) by analyzing samples of aqueous
9
reaction solutions periodically by gas chromatography. In this work, reactions of
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dichlorvos in D O were monitored inꢀsitu and reaction rate constants were derived
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directly from NMR resonance changes over time.
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1
P NMR has been used to study kinetics and to identify degradation products of
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reactions of organophosphorus insecticides.
Previously, P NMR had been utilized to
investigate the hydrolysis of dichlorvos at different pH values and the collected data
26
supported expected product identities. However, the reported kinetics conflicted with
expectations, the measured reaction rate decreased with increase in basicity. It also
reported the formation of dimethyl phosphate as the product of hydrolysis (at pH 5.6, 7,
2
6
1
and 9). Our work extended toward H NMR utilization because of more structural
3
1
elucidation possibilities than P NMR. Minor chemical changes in structure that might
3
1
1
remain undetected by P NMR are more likely to be identified by H NMR. Also,
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products lacking P would effectively remain ‘hidden’ in P NMR. However, the
formation of new products can only be monitored inꢀsitu, if the product protons are in a
magnetic environment that is distinctly different from the environment of reactant
1
protons. If this condition is satisfactorily met, following kinetic experiments by H NMR
has the advantage of revealing all products.
In this work, the hydrolysis of dichlorvos was monitored in D O at pH* 6.7, 7.7,
2
and 9.1. In addition, reactions of dichlorvos with polysulfide and thiophenolate as
nucleophiles were conducted in D O to elucidate the identity of the products formed and
2
1
to test the suitability of H NMR to measure reaction rate constants in oxygen sensitive
solutions.
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Materials and Methods
Chemicals. All chemicals were used as received. Dichlorvos (2,2ꢀdichlorovinyl dimethyl
phosphate, 99%) was obtained from Chem Service (West Chester, PA). D O (99.9 atom
2
% D) and methanolꢀD4 (D, 99.8%) were received from Cambridge Isotope Laboratories,
Inc (Andover, MA). Iodomethane (CH I, 99%), sodium sulfide (Na S ), anhydrous
3
2 4
sodium tetraborate (Na B O 99.998 %) and anhydrous potassium phosphate (K PO ,
2
4
7,
3
4
97%) were received from Alfa Aesar (Ward Hill, MA). D PO (Dꢀenrichment 99%,
3
4
nitrogen flushed) was received from ACROS Organics (New Jersey, USA). Thiophenol
was received from Lancaster Synthesis Inc (99%, Pelham, NH). Thioanisole was obtained
from TCI (Portland, OR). 4,4ꢀDimethylꢀ4ꢀsilapentaneꢀ1ꢀsulfonic acid (DSS) was obtained
from SigmaꢀAldrich (St. Louis, MO). All solvents and reagents that were used were
analytical grade or equivalent.
Instrument. 500 MHz Varian Inova NMR Spectrometer was utilized. The spectra were
acquired in D O and at 25 ˚C. In general spectra were obtained as 32 scans with a
2
relaxation delay (d1) of 5 seconds. This allowed the acquisition of spectra in just under 5
minutes. It is of primary importance to verify beforehand the nuclear relaxation times of
all compounds of interest. For DDS as an internal standard we noticed a lack of
quantitative correlation between various DSS peaks applying the previously mentioned
parameters. Therefore, the relaxation delay (d1) was increased to 30 seconds while
decreasing the number of scans to 8 per spectrum in experiments with DDS. This still
produced very acceptable spectra with each spectrum still acquired in less than 5 minutes.
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1
1
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00
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Preparation of Solutions: Buffer Stock Solution: All solutions were prepared in an
anoxic glovebox (5% H , 95% N ) with argonꢀpurged solutions. Borate buffer stock
2
2
solutions were prepared in argonꢀpurged D O. 5 mM deuterated phosphoric
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acid/potassium phosphate buffer was prepared for hydrolysis experiments conducted at
pHs 6.7 and 7.7. For reduced sulfur experiments and hydrolysis experiments at pH 9,
borate buffer was utilized. 100 mg of anhydrous sodium tetraborate was dissolved in 5
mL deoxygenated D O resulting in a borate stock solution of 400 mM. The buffer
2
solution was stored in the anoxic glove box.
Dichlorvos Stock Solution: Various dichlorvos stock solutions ranging in concentrations
from 40 mM to 70 mM were prepared in deoxygenated D O or deoxygenated CD OD in
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a 1.0 mL volumetric flask and stored in the glove box.
1
H NMR Internal Standard Preparation: DSS stock solution was prepared in 10 mL
deoxygenated D O and stored in the anoxic glovebox. Stock solution concentrations
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ranged from 15 mM to 30 mM for all solutions prepared throughout the study.
Polysulfide Stock Solution Preparation: Na S powder was used to prepare 5 mL
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deoxygenated D O solutions in 100 mM borate buffer and 100 mM NaCl. The final
2
polysulfide solution concentration was determined through iodometric titration using a
starch endpoint.
Thiophenolate Stock Solution: Thiophenolate stock solutions, ranging in concentrations
from 30 mM to 60 mM, were prepared by diluting pure thiophenol in 100 mM tetraborate
buffer in the anoxic glovebox. The final thiophenol stock solution concentrations were
determined through iodometric titration using a starch endpoint.
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Polysulfide and Thiophenolate Reaction Solution Preparation. Reduced sulfur species
reaction solutions were prepared by adding an appropriate amount of the respective
reduced sulfur species stock solution to a 5ꢀmL volumetric flask and filled up with D O.
2
0.5 mL of this reaction solution was used for the 5 mm NMR glass tube to which an
appropriate amount of dichlorvos with/without DSS was added prior to it. For selected
experiments, dichlorvos and DSS were added to the thiophenolate reaction solution (in 5
mL volumetric flask) before transferring approximately 0.5 mL to the NMR glass tube.
Three time one milliliter of the remaining reaction solution were used for iodometric
titration with a starch endpoint to determine the concentrations of total reduced sulfur
concentration [S(ꢀII)] . [S(ꢀII)] of polysulfide solutions represents the sum of [H S] and
T
T
2
T
[H S ] , where the latter represents the total concentration of polysulfides, hydrogenꢀ
2
n T
2
ꢀ
ꢀ
polysulfides, and sulfanes numerically equal to ∑([S ]+[HS ]+[H S ]) for n = 2ꢀ5. The
n
n
2 n
polysulfide concentrations were calculated on the basis of the measurement of S(ꢀII) and
T
2
7
the equilibrium constants reported by Giggenbach with excess S(0) as described in
1
8
detail by Lippa and Roberts. In case of thiophenolate reaction solutions, the
concentrations determined by iodometric titration were adjusted for speciation using the
2
8
pK of thiophenol, as reported to be 6.50.
a
1
H NMR Reaction Solution Preparation: All steps to prepare the reaction solutions were
performed in the anoxic glovebox with thoroughly washed glassware that was dried
overnight at 200 ºC. The NMR tube was deoxygenated prior to its transfer to the
glovebox by flushing it with argon to ensure that all air was removed. Appropriate
amount of dichlorvos that would maintain pseudoꢀfirst order kinetics (dichlorvos reaction
concentrations ranging from 10 µM to 50 µM) was withdrawn from the dichlorvos stock
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solution and transferred to a 5 mm NMR glass tube. Appropriate amount of DSS
(concentrations maintained at values in par with the reduced sulfur concentrations) was
added to the NMR tube for selected experiments where DSS was used as an internal
standard and internal reference. All transfers were carried out using either microsyringes
(excluding reduced sulfur solutions) or micropipettes. Finally, 0.5 mL of the reduced
sulfur reaction solution was transferred to the NMR tube. The tube was closed with a
pressure cap and sealed with parafilm. The contents were mixed by shaking the NMR
tube vigorously prior to probing with a 500 MHz NMR for at least three halfꢀlives of
dichlorvos degradation. The amount of time from mixing the reaction contents to
obtaining the initial NMR spectrum was about 10 minutes. All experiments were
conducted at 25 °C. Pseudoꢀfirstꢀorder rate constants were determined by regression of
the natural logarithm of applicable dichlorvos NMR chemical signal versus time.
pH measurement. The remaining reaction solution that was not transferred to the NMR
tube was used for pH* determination using an Accumet pH meter (Fisher Scientific,
Pittsburg, PA) with a Ross combination pH electrode (ThermoOrion, Beverly, MA). The
measured values are reported as pH*, which is an uncorrected reading in a D O solution
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of the H O calibrated pHꢀmeter, where pD = pH* + 0.4.
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Results and Discussion
Degradation of dichlorvos through hydrolysis
A) Qualitative determination. Hydrolysis experiments were conducted at pH* 6.7, 7.7
1
and 9.1. H NMR peaks that were changing in intensity with reaction time were
1
scrutinized. Figure 1 shows the H NMR spectra of dichlorvos hydrolysis at pH* 9.1
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showing all relevant peaks. The signal centered at 3.47 ppm was identified as dimethyl
phosphate (the expected products when hydroxide attacks at the phosphorus atom). The
signal further downfield at 3.54 ppm was identified to be desmethyl dichlorvos, that
resulted from water attacking dichlorvos at one of the methoxy carbons. The two
equivalent dichlorvos methyl groups produces a phosphorusꢀsplit doublet at 3.81 ppm.
Two more signals are observed at 3.71 ppm and 3.22 ppm. But their lack of change in
intensity over the duration of the experiment led to the conclusion that these peaks
correspond to compounds that are either impurities or breakdown products that formed in
the dichlorvos stock solution prior to the experiment. A similar lack in intensity change
was observed for the desmethyl dichlorvos signal at 3.54 ppm. Overall, these three peaks
had no significant contribution to the dichlorvos degradation under the given conditions.
These observations indicate that the hydrolysis reaction at pH* 9.1 proceeded with an
attack of the nucleophile (e.g., hydroxide ion) at the phosphorus atom as published in
previous work. The hydrolysis experiment at pH* 7.7 also shows dimethyl phosphate as
the major product, however it also showed a minor increase in the integral for the
desmethyl dichlorvos and methanol peak (signal at 3.20 ppm). This observation seems to
indicate that at pH* 7.7 the nucleophilic attack at the phosphorus in no longer the only
reaction pathway. There also seems to be nucleophilic attack of water at the methoxy
carbon leading to the formation of desmethyl dichlorvos and methanol. In our previous
work investigating the hydrolysis of dichlorvos, the formation of dichloroacetaldehyde
9
was characterized by GCꢀMS after derivation with PFBHAꢀHCl. In general, an aldehyde
is expected to be the more stable than the corresponding enol form (tautomer). However,
the expected aldehyde peak centered around 10 ppm was completely absent in this study.
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This lack of an aldehyde proton can be explained by the enol – aldehyde and the aldehyde
– diol equilibria. The SPARC online calculator (developed at the University of Georgia
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using the computational approach outlined by Carreira et al. ) was used to calculate the
equilibrium constants of these two equilibria. Between the enol (dichloroethenol) and the
respective aldehyde (dichloroacetaldehyde), the enol is expected to be the major tautomer
by approximately 95%. In addition, the aldehyde can also react with water and form a
diol (dichloroethanediol). According to SPARC, the diol of dichloroacetaldehyde
(dichloroethanediol) occurs at more than 99% when water is the solvent at pH 7.
Therefore, if we consider all species at pH 9.0, the dichloroethanediol occurs at about
90%, while the deprotonated dichloroethanediol and the deprotonated enolate occur at
1% and 9%, respectively. Thus, the most likely structure responsible for the resonating
proton at approximately 6.8 ppm in Figure 1 is dichloroethanediol. While ChemDraw
(version 7.0.1) NMR calculations assign an estimated shift of 5.58 ppm to the
dichloroethanediol, it assigns an almost identical shift (5.60 ppm) to the dichloroethenol.
Thus, it is possible that the vinylic proton of the 9% of the dichloroethenol present may
be resonating at the same frequency as the proton of the dichloroethanediol.
B) Kinetic treatment. No internal standard was used for hydrolysis experiments. The
NMR reaction tube was removed from the instrument between acquisitions of
consecutive spectra since slow hydrolysis rate even at pH 9.1 required a considerable
wait time before significant changes in dichlorvos concentration could occur. As a result,
spectral shift assignments could not be compared directly as NMR parameters were reset
every time the reaction tube was reintroduced into the instrumental probe. This
shortcoming was overcome by normalization of all relevant peak integrals (shown
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below), which in this case were the methyoxy protons in dichlorvos and their eventual
translation to methoxy protons in dimethyl phosphate.
ꢐꢑꢒꢓꢔꢕꢖꢗꢇꢘꢙꢇꢚꢛꢜꢝꢗꢘꢕꢞꢘꢟ
ꢀꢁꢂꢃꢄꢅꢆꢁꢇ%ꢇꢅꢈꢄꢁꢉꢀꢃꢂꢇꢊꢋꢇꢌꢅꢍℎꢂꢊꢀꢆꢊꢎ = ꢇ100 ∙ ꢏ
ꢣ
ꢐꢑꢒꢓꢔꢕꢖꢗꢇꢘꢙꢇꢚꢛꢜꢝꢗꢘꢕꢞꢘꢟꢠꢐꢑꢒꢓꢔꢕꢖꢗꢇꢘꢙꢇꢡꢕꢘꢚꢢꢜꢒꢟ
(1)
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However, this step requires that all products of all competing reaction pathways are
included. To ensure that all products were included a carefully comparison of ratios of all
observed signals with one another over the entire spectrum was undertaken. This resulted
in identification of those signals that were increasing or decreasing with time as opposed
to those staying constant. The signals that remained constant with time indicates nonꢀ
reactive compounds. Pseudo firstꢀorder rate constants were obtained from the slope of the
semiꢀlogarithmic plots of the normalized peak integral of dichlorvos versus time. The
peak integral was normalized by dividing a peak integral by the sum of all integrals of
peaks that change in area over time, Eq. 1. Rate of degradation increased with pH
8
ꢀ
confirming our previous observation that OD anion is a stronger nucleophile than D O,
2
which is the dominant nucleophile in acidic solutions. The so determined reaction rate
ꢀ1
constants for the hydrolysis in D O at pH* 6.7, 7.7, and 9.1 were found to be 0.055 d ,
2
ꢀ1
ꢀ1
0.086 d , and 0.19 d , respectively. Hydrolysis experiment in water and analyzed with
gas chromatography performed in our lab at pH 7.12 and 25 °C showed a reaction rate
ꢀ1
9
constant of 0.32 d which is considerably faster than the rate observed here in D O.
2
Solvent effects (H O vs D O) are reported for the nucleophilic attack at phosphate
2
2
3
1,32
esters
and might explain the slower reaction rates in D O versus H O. The results of
2 2
the hydrolysis experiments allowed us to confirm the products identities from our
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previous work and locate the associated peaks in the H NMR spectrum. The knowledge
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of the spectral peaks of dimethyl phosphate and dichloroaldehyde/dichloroethenol made
the interpretation of the NMR spectra of the experiments with polysulfide and
thiophenolate significantly easier.
Reaction of dichlorvos with polysulfide. It has been proposed that polysulfides react
3
3
with structurally related organophosphorus insecticides by attacking a methoxy carbon.
This potential attack of polysulfide at a methoxy carbon of dichlorvos would result in the
formation of Oꢀdesmethyl dichlorvos and methylated polysulfides (Figure 2A). It might
also be possible that polysulfide attacks the phosphorus atom or the carbon in the vinyl
group (Figure 2B). Attack by polysulfide at the phosphorous atom (Figure 2C) would
result in the formation of dimethyl phosphate and
1
dichloroacetaldehyde/dichloroethanediol. H NMR was used again to monitor the
reaction with the intend to identify the forming products. Based on the analysis of the
product peaks increasing in intensity over time, we conclude that polysulfide attacks at a
1
methoxy carbon of dichlorvos. Figure 3 shows three consecutive H NMR spectra of the
reaction of dichlorvos in 2.0 mM polysulfide at pH* 8.3. (For this experiment the NMR
tube was not removed from the instrument thus no resetting the measurement parameters
was necessary). While there is a dimethyl phosphate peak centered at 3.43 ppm, a peak
centered at 6.88 ppm could signify the presence of dichloroethandiol/dichloroethenol.
This could result in erroneous conclusion that the alternative mechanism (Figure 2C) is
occurring. However, the dimethyl phosphate peak maintained a constant intensity
throughout the course of the experiment, therefore the formation of
dichloroethandiol/dichloroethanol is unlikely. The peak at 6.88 ppm is much more likely
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attributed to the lone proton on the dichloroethenoxy moiety of desmethyl dichlorvos,
which would resonate at an almost identical frequency as the dichloroethandiol.
Additional support for the polysulfide attack at the methoxy carbon is obtained by the
observation of increase in area of the signal for multiple singlets between 2.0 ppm and 2.7
ppm. These singlets agree with the expected signals resulting from various methylated
polysulfide molecules that would form as products along with desmethyl dichlorvos. Four
such peaks are observed at 2.16 ppm, 2.36 ppm, 2.48 ppm and 2.61 ppm. The sum of
those peak intensities agreed with the signal intensity resulting from desmethyl
dichlorvos at 3.54 ppm. Although these two products were not individually probed for
their respective relaxation times, a near agreement between the integral of the two
supports the mechanism postulated in Figure 2A wherein the three methoxy protons in
desmethyl dichlorvos and the three methyl protons of methylated polysulfide should
produce similar intensities due to the same number of resonating protons. To verify the
identity of the signals between 2.16 ppm and 2.61 ppm, a spectrum was obtained of 0.5
mL of 50 mM polysulfide in excess iodomethane. Figure 3 shows this spectrum; the
highest peak is assigned to the unreacted excess of iodomethane. The inset provides
further verification when a secondary spectrum is obtained after adding more polysulfide
to the NMR tube. It should be noted that any given polysulfide molecule is methylated
twice resulting in six protons per molecule when the electrophile is in excess (as in the
case for iodomethane here). But even in the experimental case where dichlorvos is the
limiting electrophile, for any dimethylated polysulfide, two molecules of desmethyl
dichlorvos would be produced resulting in six methoxy protons resonating at 3.54 ppm.
This would preserve linearity between the two signal intensities, which is what is
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ꢀ
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observed. The reaction rate for dichlorvos in 2.0 mM Sn at pH* 8.3 was determined to
ꢀ1
ꢀ
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be 5.2 d while the reaction rate of dichlorvos at pH* 9.1 through hydrolysis was 0.19 d
1
. This indicates that the contribution of hydrolysis in this experiment can be neglected.
Reaction of dichlorvos with thiophenolate. Reaction of dichlorvos with thiophenol was
previously examined showing that nucleophilic attack by thiophenol/thiophenolate at the
9
methoxy carbon resulted in the formation of thioanisol. The observed degradation rate of
dichlorvos in a thiophenol solution is the sum of three individual reaction rates resulting
from hydrolysis, reaction with thiophenol and reaction with thiophenolate:
ꢤꢘꢥꢟ = ꢤ + ꢤꢡꢝꢦꢧ[ꢨℎꢩꢪ] + ꢤꢡꢝꢦꢫ[ꢨℎꢩꢫ]
(2)
ꢝ
If the thiophenol/thiophenolate concentration is large enough, the contribution of
hydrolysis (k ) can be neglected. The mole fraction of thiophenolate (α ꢀ) at a given
h
PhS
pH is given by the equation below.
ꢫꢱ
ꢣ
[
ꢡꢝꢦꢭ]
ꢡꢝꢦꢧ]ꢠ[ꢡꢝꢦꢭ]
[ꢧ ]
ꢮ
3
00 ꢬꢡꢝꢦꢫ = [
= ꢏ1 + ꢯꢰ
(3)
301
302
303
304
305
306
307
308
309
All thiophenol experiments were carried out at pH* 9.0 where the pK of thiophenol (pK
a a
2
8
6.50 at 25 °) favors the deprotonated form (thiophenolate) by more than 99%. Hence
the observed degradation of dichlorvos in these experiments can solely be attributed to
thiophenolate.
1
H NMR spectra for the reaction the dichlorvos with thiophenolate indicate a
nucleophilic substitution pathway through an attack of thiophenolate at a methoxy carbon
resulting in the formation of Oꢀdesmethyl dichlorvos and thioanisol. Spectral results
showed three separate aromatic peaks centered at 6.8 ppm, 7.0 ppm, and 7.3 ppm that
were assigned to para-, meta-, and orthoꢀprotons on thiophenolate, respectively.
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Aromatic peaks from the forming thioanisol are overlapping with the peaks of
thiophenolate. The mechanistic pathway whereby dichlorvos undergoes nucleophilic
attack by thiophenolate at the methoxy carbon was supported by observation of a singlet
at 2.4 ppm, which was assigned to the resonating methyl group on thioanisol. This peak
was confirmed by observing a singlet at 2.4 ppm in a spectrum obtained from commercial
thioanisol. The peak at 3.2 ppm remained constant throughout all experiments. It was
attributed to resonance of protons of the solvent methanol. No dimethyl phosphate peak
growth was observed. Unlike spectra of dichlorvos in polysulfide where 1:1 signal
intensities were observed between desmethyl dichlorvos and methylated polysulfides,
signal intensities between desmethyl dichlorvos and thioanisol showed a slightly slower
growth for the methyl group of thioansiol compared to the growth of the methyl group in
desmethyl dichlorvos. Differential relaxation time between the two products is not
expected to affect the relative signal intensities of these two products. At the same time,
thioanisol solubility in water is 7.0 mM which was well above the expected concentration
of thiosanisol formed in the experiment. However, the cause of the slower peak growth of
thioanisol versus desmethyl dichlorvos was hypothesized to be the adsorption of
thioanisol to the surface of thiophenolate dimers (diphenyl disulfide) particles which can
form by (photo)ꢀoxidation of thiophenolate (see Figure 4). Diphenyl disulfide has a very
low solubility in water and therefore precipitates and forms colloids. It is possible that
thioanisol may adsorb to those diphenyl disulfide colloids. (It was observed that the
thiophenol reaction solution turned slowly turbid over the course of several hours). In
order to address this issue an internal standard (DSS) was added to the reaction solutions.
Two consecutive spectra of commercial thioanisol in borate buffer (pH* 9.0) with
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internal standard DSS were obtained within an interval of three days. It was observed that
all thioanisol resonance signals (aromatic and methyl) had decreased significantly
indicating a decrease in concentration of the compound. Except for a weak signal from
methanol, the ‘missing’ protons from thioanisol were not observed elsewhere in the
spectra, which indicated some removal of thioanisol from solution. This observation
supports the hypothesis that thioanisol might be removed from solution by adsorption. In
particular, since thioanisol is slowly hydrolyzed at elevated pH to thiophenolate and then
can oxidize to diphenyl disulfide.
34
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Internal Standard and Kinetic Measurements. The hydrolysis experiments were
conducted over serval days and the NMR reaction tube was removed frequently.
Therefore, the signal integral had to be normalized. The experiments with polysulfide had
no such requirement since they were much faster. In those cases, three halfꢀlives of
dichlorvos degradation were observed with a dedicated NMR instrument. In either case,
decreasing dichlorvos signal strength was plotted against time to obtain firstꢀorder
exponential decay plots. Semiꢀlogarithmic translation of these plots provided pseudoꢀfirst
order rate constants for the respective nucleophile concentrations. However, at low
thiophenolate concentration, due to a slower reaction rate, the NMR tube was removed
1
from the NMR magnet in between measurements. In such a case, the H NMR signals
1
could not directly be assigned a concentration value. Normalization of the H NMR
signals was not possible, since we noticed that the signal loss due to dichlorvos reacting
did not correspond to the signal increase due to thioanisol and desmethyl dichlorvos
formation. Therefore, 2,2ꢀdimethylꢀ2ꢀsilapentaneꢀ5ꢀsulfonic acid (DDS) was used as an
internal standard for experiments where thiophenolate was the nucleophile. DSS was
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chosen as the internal standard after assessing its nonꢀinterference in terms of H NMR
resonance at frequencies of interest and its lack of reactivity towards the electrophile or
the nucleophile. The singlet arising from its nine triꢀmethyl protons was chosen to
determine concentrations of all reaction participants. The internal standard was also used
to determine the thiophenolate concentration. Since thiophenolate might undergo
photooxidation, it is stored in a dark vial to minimize this process. However, during
transfer to the NMR tube and the transport to the NMR some exposure to light is difficult
to avoid completely. The possibility of inꢀsitu oxidation could effectively reduce the
reaction concentration of thiophenolate in the reaction (in the NMR tube). Since the
concentrations determined by iodometric titration were measured with an amount of the
reaction solution that was not transferred to an NMR tube, the concentration determined
by iodometric titration could potentially be higher than the concentration in the NMR
tube. The proton in paraꢀposition of thiophenolate which resonates at a relatively
‘uncrowded’ distance from the other aromatic protons was used to obtain inꢀsitu
thiophenolate concentrations. These concentrations of thiophenolate obtained for
experiments where DSS was utilized were compared to the concentration values obtained
through iodometric titration. For the lower thiophenolate concentrations, the values
obtained through the internal standard tend to be lower than those obtained through
iodometric titration. However, values for higher concentrations of thiophenolate showed
that spectral values were slightly larger than iodometric determinations. This can be
explained by significant resonance overlap that was observed between various aromatic
peaks (including the lone para-proton) when experiments were designed with very high
thiophenolate concentrations. Figure 5 shows a plot of pseudoꢀfirst order rate constants
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against thiophenolate concentrations determined through iodometric titrations. The
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00
01
second order rate constant for the reaction of thiophenolate with dichlorvos in D O was
2
ꢀ1
ꢀ1
derived from this plot. Its value of 0.80 ± 0.16 min M , when compared to the reported
ꢀ1
ꢀ1
value determined in H O and with GCꢀFID of 1.30 ± 0.09 min M might be indicative
2
of a deuterium effect.
The use of an internal standard allowed the determination of reaction rate
constants even when some of the products were not stable over time and therefore the
total area of protons considered was decreasing with time.
Acknowledgements
This work was supported by a PSCꢀCUNY grant. We thank Prof. M.K. Lakshman for his
willingness to engage in numerous discussions involving this project.
Supporting Information
Supporting Information is available free of charge on the ACD Publication website:
Figure S1: calculated equilibrium constants determining the speciation of
dichloroacetaldeyde, dichloroethandiol, and dichloroethenol. Figure S2: plot of natural
logarithm of relative signal integral at 3.8 ppm versus time for the hydrolysis of
1
dichlorvos at pH* 7.7. Figure S3: H NMR spectra (500 MHz) of mixtures of polysulfide
1
and iodomethane in D O at 25 ˚C. Figure S4: H NMR spectrum (500 MHz) of
2
dichlorvos reacting with thiophenolate in D O, pH* 9.0 at 25 ˚C. Table S1: normalized
2
signal integral of dichlorvos and its products for the hydrolysis in D O at pH* 7.7 at 25
2
˚C. Table S2: signal integrals for the reaction of 2.0 mM polysulfides with dichlorvos in
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D O, pH* 8.25. Table S3: signal integrals of the various methoxy groups for the reaction
2
of dichlorvos with 39 mM thiophenolate at pH* 9.0. Table S4: concentrations for
thiophenolate determined through iodometric titration and spectral assignment with
internal standard.
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References
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Figure Captions:
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4
96
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Figure 1. H NMR of dichlorvos and its degradation product in D O buffer and 25 ˚C at
2
4
500 MHz. The residual water signal was suppressed before signal acquisition.
498
499
500
501
502
503
504
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506
507
508
509
510
511
Figure 2. Three potential reaction pathways of dichlorvos with polysulfides that were
considered
1
Figure 3. Three H NMR spectra of the reaction of dichlorvos with 7.6 mM polysulfide
in D O at pH* 8.3 and 25 ˚C acquired after 10 min (bottom), 26 min (middle), and 175
2
min (top).
Figure 4. Oxidation of thiophenolate to diphenyl disulfide.
Figure 5. Plot of thiophenolate concentration (iodometric titration) versus observed
reaction rate constants of dichlorvos with thiophenolate in D O, 100 mM borate buffer at
2
pH* 9.0 and 25 ˚C.
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Figure 1. H NMR of dichlorvos and its degradation product in D
2
O buffer and 25 ˚C at 500 MHz. The residual
water signal was suppressed before signal acquisition.
1
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Figure 2. Three potential reaction pathways of dichlorvos with polysulfides that were considered
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Figure 3.Three H NMR spectra of the reaction of dichlorvos with 7.6 mM polysulfide in D
2
O at pH* 8.3 and
2
5 ˚C acquired after 10 min (bottom), 26 min (middle), and 175 min (top).
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Figure 4. Oxidation of thiophenolate to diphenyl disulfide.
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Figure 5. Plot of thiophenolate concentration (iodometric titration) versus observed reaction rate constants
of dichlorvos with thiophenolate in D
2
O, 100 mM borate buffer at pH* 9.0 and 25 ˚C.
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