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Biological and Carbon Disulfide: The Formation and Decay of Trithiocarbonates under Physiologically Relevant Conditions Maykon Lima Souza,†,§ Anthony W. DeMartino,†,‡ and Peter C. Ford*

Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, California 93106-9510, United States

*S Supporting Information

ABSTRACT: Carbon disulfide is an environmental toxin, but there are suggestions in the literature that it may also have regulatory and/or therapeutic roles in mammalian physiology. Thiols or thiolates would be likely biological targets for an electrophile, such as CS2, and in this context, the present study examines the dynamics of CS2 reactions with various thiols (RSH) in physiologically relevant near-neutral aqueous media to form the respective trithiocarbonate anions (TTC−,alsoknownas “thioxanthate anions”). The rates of TTC− formation are markedly pH-dependent, indicating that the reactive form of RSH is the conjugate base RS−. The rates of the reverse reaction, that is, decay − of TTC anions to release CS2, is pH-independent, with rates roughly antiparallel to the basicities of the RS− conjugate base. These observations indicate that the rate-limiting step of decay is − − simple CS2 dissociation from RS , and according to microscopic reversibility, the transition state of TTC formation would be − ° simple addition of the RS nucleophile to the CS2 electrophile. At pH 7.4 and 37 C, cysteine and glutathione react with CS2 at a similar rate but the trithiocarbonate product undergoes a slow cyclization to give 2-thiothiazolidine-4-carboxylic acid. The potential biological relevance of these observations is briefly discussed.

■ INTRODUCTION physiologically relevant conditions have not been previously reported. This laboratory has recently compiled literature information on the known and suggested physiological properties of carbon fi fi disul de (CS2) and identi ed certain analogies to the small- molecule bioregulators (SMBs) nitric oxide, carbon monoxide, and hydrogen sulfide (sometimes called “gasotransmitters”).1 The common properties of these SMBs include partial Elucidating prospective biological roles of carbon disulfide in aqueous and lipid systems, the ability to diffuse will depend on having vehicles for controlled CS2 release under experimental biological conditions. Certain TTCs are unstable readily in physiological structures, and known toxicity at higher 6 2 ff toward the slow release of carbon disulfide, and such reactivity concentrations. Similarly, CS2 is a nonpolar, readily di usible molecule considered to be an environmental toxin.3 In addition, may be relevant to the biological activity of CS2 as well as a desirable property for CS delivery. In this context, we describe there are indications that CS is formed endogenously or in the 2 − 2 the kinetics of CS dissociation from several prepared TTC associated gut microbiome of mammals.4 Biological sulfhydryls 2 salts in aqueous solution. The latter studies complement earlier (R−SH) would be likely targets, and the dynamics of the “on” “ ff” investigations of CS2 generation by photosensitized oxidation and o reactions of this electrophile with such nucleophiles of 1,1-dithiooxalate7 and by the thermal decay of dithiocarba- should be crucial to any physiological roles. Thus, the present 8 mate anions. The CS2 release rates from the latter precursors study is focused on exploring the reactivity of CS2 with thiols, vary considerably, thereby providing a wide range of activities such as cysteine (CysSH) and glutathione (GSH), as well as for physiological experiments. The TTC derivatives of CysSH with several model thiols to form trithiocarbonate anions and GSH also undergo a slow cyclization reaction to give 2- − (TTC , also known as “thioxanthate anions”) in near-neutral thiothiazolidine-4-carboxylic acid (TTCA), a product that, aqueous media (eq 1). From the biomedical perspective, the when found in the urine, is considered diagnostic of exposure − trithiocarbonate anion (PhCH2SCS2 ) has been studied as an inhibitor to carbonic anhydrases and as a possible therapeutic in Received: August 18, 2017 5 glaucoma treatment. However, to our knowledge, the Accepted: September 26, 2017 dynamics of the formation and decay of TTC salts under Published: October 9, 2017

© 2017 American Chemical Society 6535 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543 ACS Omega Article to carbon disulfide.9,10 Notably, this cyclization also releases an experiment, the temporal spectral data can be fit numerically equivalent of hydrogen sulfide. using a nonlinear least-squares regression program (see Experimental Section and SI Figure S2); however, the kon ■ RESULTS AND DISCUSSION values so obtained are inherently less accurate than are the Trithiocarbonate Decay. As noted above, we have corresponding koff values. recently described the kinetics for a set of dithiocarbamate −− d[TTC ]/dtk=−off [TTC ] k on [CS 2 ][RSH] (3) salts that decay by releasing CS2 with lifetimes ranging from seconds to days in near-neutral, aerobic aqueous media at 37 Figure 1 illustrates temporal spectral changes observed when a °C.8 In this section, we describe analogous decays of several sample of the sodium salt of 3-trithiocarbonatopropionate − RSCS anions under similar conditions. (Na2[PTTC]) undergoes decay in pH 7.4 aqueous solution at 2 − ° The TTC salts used here were prepared by the reaction of 37 C(eq 2). Table 1 summarizes the rate constants koff and kon the corresponding precursor with CS2 in strongly alkaline k k solution. The electronic spectrum of each displays intense Table 1. off and on Values Determined for the Decay of absorption bands at approximately 310 and 330−350 nm with PTTC− in Aqueous Phosphate Buffer Solution at 37 °C ffi ∼ × 3 −1 −1 extinction coe cient of 8 10 M s (e.g., Figure 1) that −3 −1 −1 −1 pH koff (in 10 s ) kon (M s ) 6.5a 2.43 ± 0.13 0.06 ± 0.03 7.4b 2.36 ± 0.05 0.08 ± 0.04 7.8 2.36 ± 0.02 0.22 ± 0.01 a50 mM phosphate buffer and μ = 0.154 M. bAverage of values determined at 50 and 100 mM phosphate and μ = 0.154 and 0.308 M.

calculated as described. There was no significant effect of pH − − on koff over the range 6.5 7.8; thus, the decay of PTTC is not catalyzed by acid, an observation that is in direct contrast to the decay under analogous conditions of the dithiocarbamate ions.

The kon values for the reverse reaction appear to increase with pH, as would be expected, if this step involves the reaction of

the electrophile CS2 with the thiolate group of the product. This type of reactivity is discussed in greater detail below. No Figure 1. Temporal solution spectra at 49 s intervals tracking the significant effects were seen for increasing ionic strength from ° decay of Na2[PTTC] (initially about 0.11 mM) in pH 7.4, 37 C 0.154 to 0.308 M or buffer concentration from 50 to 100 mM. aqueous solution (phosphate buffer at 100 mM). The increasing A linear Eyring plot of the koff values determined for the absorbance at 206 nm is consistent with the strong absorbance of CS2 − ff at this wavelength. decay of PTTC in pH 7.4 aqueous bu er solution over the temperature range of 5−55 °C (SI Figure S3) gave the Δ ‡ ± −1 Δ ‡ π → π* − − activation parameter values H = 70.3 0.7 kJ mol and S we assign to transitions largely localized on the SCS2 = −69 ± 2JK−1 mol−1. . Time-dependent density functional theory − − calculations (Supporting Information (SI) Figure S1) support MPTTC , the methyl ester of PTTC , shows similar this assignment. Similar but somewhat higher-energy absorp- behavior (eq 4,SIFigure S4). At 37 °C, the rate constant for tions are seen in the spectra of analogous dithiocarbamate decay was (2.40 ± 0.01) × 10−3 s−1. Kinetics data collected at (R NCS −) and xanthate (ROCS −) anions.8,11 2 2 2 27.1, 37.0, and 46.4 °C at pH 7.4 gave the activation parameters Decays of the TTC anions in aqueous media are readily ⧧ − ⧧ − followed by the temporal decreases of these two UV bands ΔH = 75.7 ± 0.1 kJ mol 1 and ΔS = −51.4 ± 0.4 J K 1 (Figure 1). These spectral changes were accompanied by the mol−1. release of at least 90% of the CS2 predicted, for example, by the stoichiometry of eq 2, using the CS2 analysis method described in detail elsewhere7,8 and briefly in the Experimental Section.

N-Acetylcysteine trithiocarbonato anion (NacTTC−, eq 5) ± As discussed below, the decays of these TTC− anions are behaved similarly but gave larger values for both koff (0.0118 −1 ± −1 −1 reversible; therefore, it is necessary to take the back-reaction 0.004 s ) and kon (0.7 0.2 M s ) in pH 7.4 aqueous into account as indicated in eq 3 when evaluating the kinetics of phosphate buffer solution (100 mM) at 37 °C. An Eyring plot the temporal spectral changes. These reactions were conducted − ° of koff values measured over the temperature range of 5 55 C in buffered solutions at specific pH values, and the effects of ‡ (SI Figure S3) gave the activation parameter values ΔH = 66.8 pH, buffer, and other factors are incorporated into the apparent ± −1 Δ ‡ − ± −1 −1 rate constants k and k for the forward reaction and back- 0.9 kJ mol and S = 67 3JK mol quite similar to off on − reaction. Although [CS2] is not constant during an individual those determined for the decay of PTTC .

6536 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543 ACS Omega Article

Scheme 1. Proposed Sequence of Steps Leading to TTC− Decay in Aqueous Media

Analogous kinetics studies for decay of the benzyl trithiocarbonate anion (BnTTC−, eq 6) displayed decreases in the 302 and 332 nm absorption band characteristic of such − pH 7.4 is roughly antiparallel to the increasing basicity of the TTC anions (SI Figure S5). The reactions are somewhat − fl − thiolate anion RS as re ected in pKa values of the respective 12−16 faster than seen for PTTC , but again the rate constants koff for thiols (RSH) (SI Table S1). the forward reaction showed essentially no sensitivity to the In other words, the more basic thiolate ions are slower to solution pH over the range of 6.5−10.1 (Table 2). In contrast, dissociate from the CS2 electrophile. However, one might expect a positive value of ΔS‡ for unimolecular dissociation of k k Table 2. off and on Values Determined for the Decay of − fi − the RS CS2 bond illustrated in the rst step of Scheme 1, but BnTTC in Aqueous Phosphate Buffer Solution at 37 °C that was not the case for PTTC−, MPTTC−, or NacTTC−. One Δ ‡ pHa k (in 10−3 s−1) k (M−1 s−1) possible explanation for the observed negative S values off on would be a pathway involving concerted protonation of the 6.5 8.8 ± 0.1 ff ± ± exiting thiolate group by a general acid (solvent or bu er 7.4 8.5 0.1 0.7 0.1 conjugate acid). However, no general acid catalysis was 7.8 8.5 ± 0.1 1.8 ± 0.1 ± ± observed and protonation by H2O itself would generate 10.1 7.3 0.1 58 1 hydroxide ion, which would be unfavorable. Thus, a more a ff μ − 100 mM phosphate bu er and = 0.18 0.29 M. likely explanation for a negative entropy of activation draws from solvent reorganization as the negative charge delocalized − − the kon value calculated was markedly larger at the highest pH, over the CS2 functional group becomes localized on the and this was reflected by the reaction not going to completion thiolate ion at the transition state (Scheme 2). but reaching an equilibrium or steady state (Figure 2).

Scheme 2. Solvation Reorganization upon CS2 Dissociation from a TTC− Anion

Formation of Trithiocarbonates. Physiologically, thiols

are likely targets in the action of CS2 either as a toxin or in potential bioregulatory or therapeutic roles.1 For example, modification of a key protein thiol by the formation of trithiocarbonate would be expected to have profound effects on that protein’s activity. The “on” reaction noted above is, of course, the formation of the TTC− adduct from the parent thiol

plus CS2. The goal in this section is to examine the dynamics of trithiocarbonate formation with cysteine and several cysteine derivatives, including glutathione, in greater detail. ff − ° Figure 3 illustrates the temporal spectrum changes that occur Figure 2. E ect of pH on the decay kinetics of BnTTC at 37 C and fl in 0.1 M phosphate buffer, except for pH 10.1, which is in 0.1 M rapidly after stopped- ow mixing of a solution containing carbonate buffer (all experiments done in duplicate). excess cysteine [CysSH] with a second solution containing CS2 in pH 7.4 aqueous buffer at 37 °C. Very similar spectral changes The absence of a pH effect on the k values in 37 °C off− − were shown to result from the reactions of CS2 with aqueous media for the decays of PTTC and BnTTC is (respectively) N-acetylcysteine (NacSH), glutathione (GSH, consistent with the rate-limiting step dissociation of CS from − 2 SI Figure S6), and cysteine methyl ester (MecSH, SI Figure the conjugate thiolate anion, RS , followed by protonation of the latter, as illustrated in Scheme 1. In accord with this S7). In each case, the appearance of the spectrum characteristic fi − sequence where the rst step is rate limiting, that is koff = k1, the of a TTC anion (eq 7) followed an exponential rise as seen in reactivity order NacTTC− > BnTTC− > PTTC− ∼ MPTTC at the figure inset.

6537 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543 ACS Omega Article

relaxing to equilibrium and CS2 is the limiting reactant, the relationship described by eq 8 holds true.17

kkkobs=+ off on[RSH] (8)

Thus, when RSH = CysSH, a plot of kobs versus [CysSH] should be linear with a slope equal to kon and an intercept equal to koff. The inset of Figure 4 is such a plot for the reaction of ° ff CS2 with excess CysSH in 37 C, pH 7.4 bu ered aqueous ± −1 −1 solutions from which the values kon = 2.9 0.1 M s and koff = 0.103 ± 0.002 s−1 were determined. Plots similar to Figure 4 were generated for the reactions of glutathione (GSH), cysteine methyl ester (MecSH), and N-acetylcysteine (NacSH) (SI − Figures S6 S8), and the kon and koff values so determined are summarized in Table 3. Notably, the values of kon and koff determined in 37 °C, pH 7.4 solution for NacSH (0.606 ± 0.008 M−1 s−1 and 0.0138 ± 0.001 s−1, respectively) can be compared to those (0.7 ± 0.2 M−1 s−1 and 0.018 ± 0.004 s−1) Figure 3. Temporal absorption spectra recorded over a period of 60 s − of a solution prepared by 1:1 stopped-flow mixing of aqueous solutions obtained above by following the decay of NacTTC under ff of cysteine and CS2 with spectra recorded every 0.6 s. Concentrations similar conditions. Given the di erent procedures and after mixing: [CysSH] = 5 mM, [CS2] = 0.2 mM. Conditions: T =37 apparatus used in these two experiments, the agreement is °C, pH 7.4, [phosphate buffer] = 50 mM, μ = 154 mM. Inset: quite good. absorbance change at 332 nm. As noted above for several other TTC anions, the koff values reported in Table 3 for the cysteine derivatives decrease as the basicity of the RS− increases owing presumably to the higher − − RS CS2 bond strength through this series. The values of kon at pH 7.4 show a similar decrease (hence, the ratio kon/koff varies only modestly over this series). The behavior of kon would be consistent with the mechanism suggested by the Figure 4 illustrates the absorption changes at 332 nm − microscopic reverse of Scheme 1.Ifkoff = k1, then kon = k2 characteristic of formation of CysTTC (eq 7) when a pH 7.4 f(H+), where f(H+)=K /(K +[H+]). As the acidity of RSH a a − (Ka) increases, more of the conjugate base thiolate RS is available at pH 7.4. Thus, one would expect kon to increase. However, the nucleophilicity of RS− is likewise also decreasing over this series, so one might expect the rate constant k for the − 2 nucleophilic attack of RS on CS2 to correspondingly decrease in going from NacS− to MeCysS−, the two trends therefore countering each other. This may help account for a reactivity difference on only an order of magnitude between NacSH and ff MeCysSH at pH 7.4 despite the much greater di erence in Ka values. SI Figure S9 displays Eyring plots of the kon and koff values determined in a similar manner for the reaction of CysSH with ff CS2 in pH 7.4 bu ered aqueous solution over the temperature − ° range of 25 45 C. The apparent activation parameters for kon are ΔH⧧ = +84.3 kJ mol−1 and ΔS⧧ = +37.4 J K−1 mol−1, and Δ ⧧ −1 Δ ⧧ − those for koff are Hoff = +75.1 kJ mol and Soff = 21.7 J −1 −1 Δ ⧧ K mol . Notably, H for koff in this case are quite similar to that recorded above at pH 7.4 for the decays of PTTC−, MPTTC−, and NacTTC−, but because ΔS⧧ remains negative in Figure 4. Exponential fit (red curve) of absorption changes at 332 nm this case, it is less so than for the other three (SI Table S2). fl According to the proposed mechanism described in Scheme 1, (black dots) upon stopped- ow mixing of cysteine (1 mM) with CS2 ⧧ ⧧ ⧧ ° ff μ k = k , so the apparent ΔH and ΔS for this step equal ΔH (0.1 mM) in 37 C, pH 7.4, aq phosphate bu er (50 mM, tot =154 off 1 1 fi Δ ⧧ mM). Inset: plot of kobs values calculated from similar ts at various and S1 , respectively. The relationship is more complex for + [CysSH]. Conditions after mixing: pH 7.4, [buffer] = 100 mM, T =37 kon because it equals k2 f(H ). If one were to make the rough ° − −1 −1 + ≫ ≅ C, [cysteine] = 5 30 mM, [CS2] = 0.5 mM. kon = 2.9 M s approximation that, at pH 7.4, [H ] Ka, then kon −1 + −1 Δ ⧧ ≅ Δ ⧧ Δ 0 Δ ⧧ ≅ (slope), koff = 0.103 s (intercept). k2Ka[H ] and then Hon H2 + Ha and Son Δ ⧧ Δ 0 Δ 0 Δ 0 S2 + Sa . The values of Ha and Sa can be calculated as buffered solution of excess CysSH was mixed with a solution +30.9 kJ/mol and −52 J K−1 mol−1, respectively, from the pH fl 19 containing CS2 in the stopped- ow spectrometer. Under such dependence of the pKa of cysteine (SI Figure S10). Thus, on “ fi ” ≫ Δ ⧧ ≅ −1 Δ ⧧ ≅ pseudo- rst-order conditions where [CysSH] [CS2], the the basis of this model, H2 53 kJ mol and S2 +89 J fi −1 −1 temporal absorption changes can be t to a simple exponential K mol for the reaction of CysSH with CS2. fi fl function from which the observed rst-order rate constant kobs Table 4 reports the results of analogous stopped- ow kinetics was obtained. Under these conditions where the system is studies of the reactions of CysSH and GSH with CS2 to form

6538 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543 ACS Omega Article k k Table 3. Values of on and off Determined Using a Stopped-Flow Kinetics Spectrophotometer for the Reactions of CysSH, ° ff a NacSH, MecSH, and GSH with CS2 in 37 C, pH 7.4 Bu ered Aqueous Solution

+ −1 −1 −1 −1 RSH pKa (SH) pKa (NH3 ) kon (M s ) koff (s ) kon/koff (M ) MecSH 6.56b 8.99b 4.3 ± 0.1 0.116 ± 0.002 37 CysSH 8.33c 10.78c 2.9 ± 0.1 0.103 ± 0.002 28 GSH 8.66b 9.12b 2.5 ± 0.1 0.036 ± 0.001 69 NacSH 9.52b 0.606 ± 0.004 0.0138 ± 0.001 44 a ff μ b c [CS2] = 0.5 mM, 100 mM phosphate bu er solution, = 0.308 M. Ref 18. Ref 19.

Table 4. Rate Constants Measured for Reaction between CS2 (0.1 mM) and CysSH (1−8 mM) or GSH (1−8 mM) as a Function of pH at 37 °CinBuffered Aqueous Solution

CysSH GSH

kon koff kon/koff kon koff kon/koff pH (M−1 s−1) (s−1) (M−1) (M−1 s−1) (s−1) (M−1) 7.4 2.9 0.103 28 2.5 0.036 69 7.6 6.4 0.099 65 8.0 11.7 0.086 136 8.7 0.039 224 8.4 14.9 0.075 198 13.7 0.036 380 8.8 18.4 0.054 340 23.5 0.034 692 9.2 22.0 0.039 563 32.5 0.034 956 9.6 26.0 0.023 1129 46.9 0.034 1340

CysTTC− and GTTC− (eqs 7 and 9, respectively) at different − pH values over the range of 7.4 9.6. The kobs values were determined for a range of different initial CysSH concentrations Figure 5. Spectral changes upon mixing CS2 (1 mM) with CysSH (1 mM) in pH 7.4 aq phosphate buffer (100 mM) at 37 °C in a sealed at each pH, and linear plots of kobs versus [CysSH] similar to − those shown in Figure 4 and SI Figures S6−S8 gave the pH- cell indicating the slow transformation of CysTTC (red spectrum) to dependent values of k and k . Both systems show dramatic TTCA (blue spectrum). Total time = 17 h. Spectra recorded at 600 s on off intervals. increases in kon at higher pH as anticipated from the simple mechanism proposed in Scheme 1. Consistent with the − − experiments for PTTC and BnTTC reported in Tables 1 assigned to the cyclized compound thiazolidine-2-thione- and 2, the k values for GTTC− are pH-independent; however, 9 fi 20,21 off − carboxylate (TTCA, eq 10), which has been identi ed as this was not the case for CysTTC , for which koff decreases by a urinary excretion product from humans who have been about a factor of four from pH 7.4 to 9.6. We attribute this exposed to CS2. The temporal decay at 332 nm and rise at 270 difference to the relative proximity of the protonated amine nm could be fit to exponential functions to give the respective group (NH +) to the trithiocarbonate functionality in fi × −5 × −5 −1 − 3 rst-order rate constants 5.2 10 and 4.9 10 s in pH CysTTC . The resulting change in the inductive effect upon 7.4 aq phosphate buffer at 37 °C (SI Figure S11). However, deprotonation of this group at higher pH should enhance the inspection of the spectral changes shows the absence of basicity of the thiolate, thus leading to slower dissociation of − isosbestic points, as well as an absorbance increase followed by CS2. Although GTTC has a similar amine, it is positioned a decrease of a shoulder at ∼230 nm, which are clear further from the TTC functionality and its deprotonation indications that a transient intermediate species is formed. would have a much smaller impact. When the reaction was run with a very large excess of CysSH ([CS2] = 0.2 mM; [CysSH] = 10 mM), the kobs value measured was nearly the same (5.8 × 10−5 s−1). Thus, it appears that this slow cyclization process is unimolecular and does not involve the reaction of free CysSH.

Subsequent Reactions of CysTTC− and GTTC−. Although the reaction of CS2 with cysteine initially showed the rapid appearance of new absorption bands at ∼295 and 332 nm consistent with the formation of the trithiocarbonate The reaction of cysteine (10 mM) and CS2 (10 mM) was CysTTC− (eq 7), a much slower subsequent reaction was further studied in a buffered phosphate deuterium oxide evidenced by further spectral changes (Figure 5) involving the solution (prepared with anhydrous Na3PO4 plus D2O, pD 7.5) disappearance of the bands at ∼295 and 332 nm and the to characterize more thoroughly the formation of TTCA. DCl appearance of a new band at 270 nm. The latter band can be solution (35 wt % in D2O) was used to correct the pD. The

6539 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543 ACS Omega Article reaction was run for 24 h at 37 °C. A small aliquot of the product solution was used to check the electronic spectrum, which showed the presence of a single and intense absorption band at 271 nm (SI Figure S12), indicating the formation of the cyclic compound because unreacted cysteine and oxidized cysteine products, such as cystine, have no strong absorption bands in this wavelength range. The 1H NMR spectrum of the product solution (SI Figure S13) showed not only resonances belonging to unreacted cysteine but also two groups of proton − − resonances at 3.67 3.63 ppm (S CH2, doublet of doublets) and 3.92−3.89 (CH, broad doublet of doublets) attributed by van Doorn et al. to the hydrogens of TTCA.22 GTTC− formed by the relatively rapid reaction of GSH with CS2 also goes through a very slow transformation to a new species reported to be TTCA (SI Figure S14). However, similar spectral changes/secondary reaction was not seen with 3- trithiocarbonatopropionate (PTTC−) but was observed with 3- trithiocarbonatopropylamine and thus an amine functionality appears to be required. This is not simply the transfer of CS2 from the thiolate to the amine to form a dithiocarbamate Figure 6. Qualitative reaction coordinate diagram for the formation − − (kon) and decay (koff) of trithiocarbonate anions (RSCS2 ) from the (DTC ) analogue because the spectrum formed by the reaction − of aqueous base and glycine displays bands at 253 and 284 nm respective thiols plus carbon disulfide. According to Scheme 1, TTC ff decay is independent of pH (koff = k1), whereas the forward reaction is (data not shown) typical of DTC anions and very di erent ⇌ − + from the characteristic of TTCA. The mechanism(s) of these strongly pH-dependent owing to the RSH RS +H equilibrium. transformations are the subject of continuing investigation. reported for metal−sulfur bonds25,26 as well as with alkoxide,27 ■ SUMMARY amine,28,29 and (even) phosphine30 ligands. Given the relatively 31 We have described the kinetics of the formation and decay of high cytosolic concentrations of GSH and plasma-based 32 trithiocarbonate derivatives formed by the reactions of carbon protein sulfhydryls, the reversible formation of unstabilized disulfide and various thiols (RSH) under physiologically TTCs may play a role in CS2 transport. relevant conditions (near-neutral pH, 37 °C). Rates of TTC− formation are strongly dependent on solution pH, indicating ■ EXPERIMENTAL SECTION that the rate-limiting step involves the reaction of the thiolate Materials. Except where otherwise noted, all materials were − − anion RS with the electrophilic CS2 substrate. Rates of TTC of analytical or reagent grade and were used without further decay are pH-independent, consistent with microscopic purification. N-Acetyl-L-cysteine (≥99%, TLC), 3-mercapto- reversibility. The relative decay rates have a reverse correlation propionic acid (≥99%), L-cysteine (97%), glycine (≥98.5%), − to the basicity of the RS anions as expected if simple and reduced L-glutathione (98%) were purchased from Sigma- fi ≥ dissociation of CS2 is rate determining, as illustrated by the Aldrich. Carbon disul de (ACS Reagent Grade, 99.9%), qualitative reaction coordinate diagram shown in Figure 6.At benzyl mercaptan (Alfa Aesar, 99%), potassium hydroxide, pH 7.4, glutathione and cysteine react with CS2 at similar rates, sodium hydroxide, and mono- and dibasic sodium phosphate and in both cases, the resulting TTC− anions undergo slow and sodium chloride used to prepare buffers were purchased cyclization reactions to a cyclized species identified previously from Fisher Scientific. as 2-thiothiazolidine-4-carboxylic acid. As noted above, the Synthesis of TTCs. Sodium 3-(Trithiocarbonato)- latter has been found to be urinary excretion products from propionate (Na2[PTTC]). This species had been generated as individuals exposed to toxic levels of carbon disulfide. Similar an intermediate in the preparation of a corresponding reactions with protein thiols could lead to irreversible trithiocarbonate ester.33 A round-bottom flask was charged fi modi cations of such targets as well as releasing an equivalent with pentane and CS2 (30 mL each) plus a stir bar and then − ° of H2S. was cooled to approximately 0 5 C with an ice bath and fi The present data do not resolve the question whether CS2 purged with inert gas. During rapid stirring, nely ground might serve some type of bioregulatory role. Although thiols NaOH (3.09 g, 77.4 mmol) was added to the flask under inert would appear to be logical targets for this electrophile, it is clear gas. Sufficient water was added to solubilize the hydroxide (1−3 that the rates and equilibrium constants for TTC− adduct mL), and to the resulting base solution, 3-mercaptopropionic formation are many orders of magnitude smaller than those for acid (4.11 g, 38.7 mmol) in (10 mL) was added reactions of NO with heme proteins, the best characterized dropwise over the course of a few minutes. A deep yellow targets of that SMB.23,24 It appears likely that any such powder immediately began to precipitate. The reaction was bioregulatory relevant sites for CS2 would have higher binding allowed to warm to room temperature, and stirring was constants, for example, an activated thiolate protein, where the continued for 6−12 h. The solid was collected via filtration and − TTC anion is stabilized by other intramolecular interactions. rinsed with cold Et2O. The yield was 3.68 g (37%). The Another could be a metal center because it is well established product proved to be soluble only in methanol or water; that transition-metal ions activate CS2 toward reaction with however, attempts to recrystallize from these solvents resulted coordinated nucleophiles and insertion into metal−ligand in decomposition. The optical spectrum of the initial product in λ bonds, owing to the strong bonding of the 1,1-dithiolate pH 7.4 water displayed bands at max = 303 and 332 nm with an − − ffi −1 −1 ( CS2 ) functionality as a ligand. Such reactivity has been extinction coe cient at 332 nm (8650 M cm ) close to

6540 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543 ACS Omega Article those previously observed for alkyl TTCs.21 The infrared reaction cell was a septum-capped, sealable quartz cuvette spectrum and CHN analysis are consistent with a hydrated with a 1.0 cm path length and an approximate volume of 4.80 sample. The solid is rather odorous. The compounds were kept mL. The cells were thermostated (±0.2 °C) at the desired in a freezer for long-term storage. Infrared spectrum: reaction temperature, and the solutions were stirred with a − (attenuated total reflection in cm 1) selected bands 989, 879 Starna “Spinnette” stirrer. Septum-sealed cells containing the (ν,C−S), 1148 (CS), 1395 (δ,C−O), 1555 (ν,C−O), 1655 buffer solution of interest temperature equilibrated (7−10 min) (δ,O−H), 3300 (ν,O−H). Anal.: (calculated values for under the desired conditions (typically 37 °C). A small amount · C4H4Na2S3 2H2O in parentheses): C, 18.1 (18.32); H, 2.8 of the solid TTC salt (5−10 mg) was then added to a vial (3.07). containing 3−5mLofbuffer to prepare a stock solution. A 100 Triethylammonium (N-Acetylcysteine)trithiocarbonate μL aliquot of this stock solution was then syringed into the − ° fl ([HNEt3][NacTTC]). To an ice-cold (0 5 C) ask containing thermostated cuvette. The volumes were chosen to minimize diethyl ether (30 mL) and triethylamine (3.72 g, 36.8 mmol) the headspace in the cuvette. Data acquisition was initiated after under inert atmosphere, a 3 g portion of N-acetylcysteine (18.4 an estimated dead time of 60−90 s. − mmol) was added. Dimethylformamide (2 3 drops) was added Kinetics of the reactions of CS2 with various thiols to to solubilize the N-acetylcysteine, after which CS2 (1.90 g, 25 examine the rates of TTC formation were generally performed mM) was added dropwise, resulting in rapid separation of an using an Applied Photophysics SX.19MV stopped-flow UV− insoluble, viscous, translucent, golden oil. This oil was visible spectrophotometer with photodiode array (temporal separated from the solvent by decanting and was dried in spectra) or a photomultiplier tube (single wavelength) detector. ∼ ° ff vacuo for several days at elevated temperature ( 40 C). The Aqueous bu ered solutions of the thiol (RSH) and CS2 were oil was then dissolved in , heated gently, and the loaded into Hamilton salt syringes (#1725 AD-SL), which were resulting solution stirred vigorously. Vacuum removal of the attached to the mixing block of the spectrophotometer and ethanol resulted again in a golden oil. This product was stored sealed from the outside atmosphere by a three-way valve. These λ in a freezer. Optical spectrum: max = 302 and 333 nm in pH 7.0 syringes were maintained at the desired temperature (generally ffi ∼ −1 −1 water with a molar extinction coe cient of 8400 M cm at 37 °C unless otherwise noted) by a circulating water bath. The the latter wavelength. ESI-MS (neg. mode) (75% ACN, 25% connector tubing and observation cell were purged with the + H2O): Anion predicted (+H ), 237.96 m/z; found, 237.94 m/z, sample solution by filling and emptying the drive syringes 3× + + 259.89 m/z (+Na ), 339.04 m/z (+TEAH ). Also observed, prior to data acquisition. Stopped-flow mixing of the two 162.17 m/z (N-acetylcysteine, impurity/decomposition). Anal. solutions initiated the reaction. The entire unit was controlled (calculated values for C18H39N3O3S3 in parentheses): C, 48.0 with “Applied Photophysics Pro-Data SX” software. Only (48.95); H, 8.22 (8.90); N, 9.39 (9.51). simultaneous symmetric mixing was used (1:1 dilution). Potassium Benzyl Trithiocarbonate (K[BnTTC]). The 5 Kinetics data were processed using Igor Pro 6.37 software by preparation was adapted from a known procedure. A round- WaveMetrics, Inc., and Prism 7 by GraphPad Software, Inc. For fl bottom ask was charged with a magnetic stirring bar, diethyl global fittings of equilibrium analyses and determination of ether (30 mL), and carbon disulfide (10 mL). The solution was − ° back-reaction values, a DynaFit (BioKin Ltd) nonlinear least- cooled to 0 5 C with an ice bath and then purged with squares regression program was used. nitrogen. Finely ground KOH (2.00 g, 35.6 mmol) was added Computational Studies. All density functional theory and then benzyl mercaptan (5 mL, 5.29 g, 42.6 mmol) was (DFT) computations were performed using Gaussian’09 added to this cloudy mixture over a period of 2 min. The software packages. Optimizations were performed using resulting solution rapidly became yellow and cloudy to give a Kohn−Sham DFT with the hybrid M06-2X exchange- chalky precipitate. This mixture was stirred for an additional 3 fi correlation functional and the 6-311+G(d,p) basis set. No h. The solid product was collected by ltration and washed with symmetry restriction was imposed, and implicit solvent effects diethyl ether and cold ethanol (4 × 10 mL). The solid was then fl were included using PCM (solvent = water) methods, as added to a ask containing 150 mL of Et2O and collected by fi implemented in Gaussian 09. Vibrational frequency calculations ltration again to remove excess dimethylformamide. Recrystal- were performed at the same level of theory to verify that no lization from a 50/50 mixture of ethanol and acetonitrile gave a imaginary frequencies were present and to ensure the true local deep yellow, mildly odorous powder that was dried in vacuo minima energies. Single-point energy and time-dependent DFT and stored in a freezer. The yield was 4.58 g (54%). K[BnTTC] calculations were performed using the M062X/6-311+G- decomposes when dissolved in water or water/alcohol mixtures (3df,3pd) level of theory. to give highly odorous product(s). CHN anal. (calculated Analysis of CS2 Release. This procedure was similar to values for C8H7KS3 in parentheses): C, 39.8 (40.3); H, 2.84 34 λ that described by Schwach and Nyanzi and to that used in (2.96). Optical spectrum: max = 302 and 332 nm in pH 7.4 −1 −1 this laboratory to detect CS2 released by the photolysis of CdSe water (extinction coefficient of ∼9300 M cm at the latter 7 quantum dots surface decorated with 1,1-dithiooxalate and for wavelength). (Note: Benzyl mercaptan has a particularly the CS released in the thermal reactions of dithiocarbamate unpleasant odor and is toxic. Proper environmental controls must 2 salts.8 be observed when handling this liquid.) Kinetics Methods. Phosphate buffers were prepared using mono- and dibasic sodium phosphate and sodium chloride (to ■ ASSOCIATED CONTENT ≥ maintain ionic strength). Nanopure water ( 18 megohm) was *S Supporting Information obtained from a Barnstead Nanopure II system and used in The Supporting Information is available free of charge on the solution preparations. ACS Publications website at DOI: 10.1021/acsomega.7b01206. The rates of the TTC decompositions were determined by fi monitoring temporal spectral changes on a Shimadzu UV-2401 Additional documentation (2 tables and 11 gures) of spectrophotometer with UVProbe kinetics software. The the studies (PDF)

6541 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543 ACS Omega Article ■ AUTHOR INFORMATION (9) van Doorn, R.; Henderson, P. T.; Vanhoorne, M.; Vertin, P. G.; Leijdekkers, J. M. Determination of thio compounds in urine of Corresponding Author * workers exposed to carbon disulfide. Arch. Environ. Health 1981, 36, E-mail: [email protected]. 289−297. ORCID (10) Domergue, J.; Lison, D.; Haufroid, V. No evidence of Peter C. Ford: 0000-0002-5509-9912 cardiovascular toxicity in workers exposed below 5 ppm carbon disulfide. Int. Arch. Occup. Environ. Health 2016, 89, 835−845. Present Addresses ‡ (11) Shankaranarayana, M. L.; et al. The Electronic Spectra of some Division of Pulmonary, Allergy, and Critical Care Medicine, Derivatives of Xanthic, Dithiocarbamic and trithiocarbonic Acids. Acta Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, Chem. Scand. 1965, 19, 1113−1119. ’ University of Pittsburgh, Pennsylvania 15213, United States (12) The SH group pKs sofN-acetylcysteine (NacSH) and 3- (A.W.D.). mercaptoproprionic acid (3-MPA) are 9.5 and 10.3, respectively (refs § CAPES Foundation, Ministry of Education of Brazil, Brasilia,́ 13 and 14). Benzyl mercaptan is not water soluble, but a value of 9.46 DF 70040-020, Brazil (M.L.S.). has been reported (ref 15) although since mercaptans are typically ∼5.5 pK units more acidic than the corresponding alcohols and Author Contributions a † benzyl alcohol has a pKa of 15.4 (ref 16), we would have estimated a ∼ M.L.S. and A.W.D. contributed equally. slightly higher pKa of 9.9. Notes (13) Burner, U.; Obinger, C. Transient-State and Steady-State fi Kinetics of the Oxidation of Aliphatic and Aromatic Thiols by The authors declare no competing nancial interest. − Carbon disulfide is toxic at high exposures, and long periods of Horseradish Peroxidase. FEBS Lett. 1997, 411, 269 274. modest exposure have been associated with a number of health (14) Srinivasan, U.; Mieyal, P. A.; Mieyal, J. J. pH Profiles Indicative of Rate-Limiting Nucleophilic Displacement in Thioltransferase conditions. CS2 and CS2-generating compounds should be Catalysis. Biochemistry 1997, 36, 3199−3206. handled and stored with care. (15) Kreevoy, M. M.; Harper, E. T.; Duvall, R. E.; Wilgus, H. S., III; Ditsch, L. T. Inductive effects on the acid dissociation constants of ■ ACKNOWLEDGMENTS mercaptans. J. Am. Chem. Soc. 1960, 82, 4899−4902. This work was supported by grants to PCF from the Chemistry (16) Cao, Z.-C.; Luo, F.-X.; Shi, W.-J.; Shi, Z.-J. Direct Borylation of Benzyl Alcohol and Its Analogues in the Absence of Bases. Org. Chem. Division of the United States National Science Foundation − (CHE-1405062 and CHE-1565702). The authors acknowledge Front. 2015, 2, 1505 1510. (17) Atkins, P. W.; DePaula, J. Physical Chemistry, 9th ed.; W. H. the UCSB Academic Senate Research Committee for seed Freeman & Co: New York, 2010; p 797. money in support of this research. They also acknowledge ’ fi (18) Dean, J. A. Lange s Handbook of Chemistry, 15th ed.; McGraw- support from the Center for Scienti c Computing from the Hill Companies: New York, 1998. CNSI, MRL: NSF MRSEC (DMR-1121053) and NSF CNS- (19) Sharma, V. K.; Casteran, F.; Millero, F. J.; De Stefano, C. 0960316. M.L.S. thanks CAPES (Coordenaca̧õ de Aperfeicoa-̧ Dissociation Constants of Protonated Cysteine Species in NaCl mento de Pessoal de niveĺ Superior), process 99999.000902/ Media. J. Solution Chem. 2002, 31, 783−792. 2015-02, for a postdoctoral fellowship. (20) Weiss, T.; Hardt, J.; Angerer, J. Determination of Urinary 2- Thiazolidinethione-4-Carboxylic Acid after Exposure to Alkylene ■ REFERENCES Bisdithiocarbamates Using Gas Chromatography−Mass Spectrometry. J. Chromatogr. B: Biomed. Sci. Appl. 1999, 726,85−94. (1) DeMartino, A. W.; Zigler, D. F.; Fukuto, J. 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6543 DOI: 10.1021/acsomega.7b01206 ACS Omega 2017, 2, 6535−6543