Mechanisms of Alkyl and Aryl Thiol Addition to N-Methylmaleimide

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Cite This: J. Org. Chem. 2018, 83, 11674−11685

Mechanisms of Alkyl and Aryl Thiol Addition to N‑Methylmaleimide Mark A. R. Raycroft,† Karl É . Racine,† Christopher N. Rowley,‡ and Jeffrey W. Keillor*,† †

Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, Canada Department of Chemistry, Memorial University of Newfoundland, St. John’s, NL A1B 3X7, Canada

‡

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S Supporting Information *

ABSTRACT: A mechanistic study was undertaken to elucidate the reaction pathways for thiol addition to Nmethylmaleimide in water. We used linear free energy relationships, solvent kinetic isotope effects (SKIEs), activation parameters, and ionic strength effects to probe the nature of the rate-limiting transition states. Calculations were also employed and assisted in illuminating three possible mechanistic pathways: (1) stepwise addition with rate-limiting nucleophilic attack, (2) stepwise addition with rate-limiting proton transfer, and (3) concerted addition with nucleophilic attack and proton transfer occurring concurrently. Alkyl − thiolate addition exhibits βnucRS = 0.4, small negative ΔS‡ values, prominent ionic strength effects, and no evidence of − general acid catalysis, consistent with pathway 1. Aryl thiolate addition exhibited βnucArS = 1.0, large negative ΔS‡ values, normal primary SKIEs, general acid catalysis, and negligible sensitivity to ionic strength, consistent with pathways 2 and 3. The experimental and computational data depict an energy surface where ground state effects, namely the energy of the alkyl/aryl thiolate, play a major role in shaping the governing pathway. Application of these findings to bioconjugation chemistry is also discussed.

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INTRODUCTION Thiol addition to maleimide is one of the most well-known conjugate addition reactions,1−6 and it has been applied particularly broadly in protein modification. In this context, the thiol group of one or more Cys residues is subject to covalent modification, in bioconjugation,7,8 enzyme inhibition,9,10 and protein labeling11,12 applications, to name but a few. All of these applications would benefit from a thorough comprehension of the thiol addition mechanism, which would allow the reactivity of the thiol and the maleimide to be tuned accordingly. We have recently shown how substituents on the maleimide ring can attenuate its reactivity,13 and have applied this information to the design of selective protein labeling agents.14 However, the mechanism of the addition reaction has still not been completely elucidated. Although early work9,15−21 showed that this reaction is faster at higher pH, we still had questions regarding whether the addition reaction occurred in a stepwise or a concerted fashion, and whether the reaction at a given pH would be faster with a more nucleophilic thiol (having a higher pKa) or a higher proportion of thiolate from a more acidic thiol (having a lower pKa). Regarding the latter question, previous Brønsted plots9,19,20 have provided preliminary results, but over a very narrow range of thiol pKa values. In this work, we turned our attention to the thiol reactants, studying the importance of thiol pKa for a large series of both alkyl and aryl thiols, in their reaction with N-methylmaleimide (NMM) in water (Scheme 1). We studied pH−rate profiles and © 2018 American Chemical Society

Scheme 1. Thiol Addition Reaction Studied Herein, Where R Refers to Alkyl and Aryl Substituents

buffer catalysis to inform on the ionization state of the reactive species, and used broad Brønsted plots in conjunction with temperature dependence and solvent kinetic isotope effect (SKIE) studies to determine the nature of the transition states. Ionic strength experiments were also used to characterize the charge of the transition state relative to the ground state. Finally, we performed preliminary DFT calculations to aid in our interpretation of the experimental data as well as to probe nuances in the mechanism that would be experimentally challenging to investigate. Together, these data illustrate a detailed mechanistic picture and allow the prediction of rate constants for the reaction of virtually any thiol with N-alkyl maleimides, in hydroxylic medium, over a range of temperatures. Received: June 29, 2018 Published: September 5, 2018 11674

DOI: 10.1021/acs.joc.8b01638 J. Org. Chem. 2018, 83, 11674−11685

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The Journal of Organic Chemistry

Figure 1. This work studied the addition of aryl thiols (1a−g) and alkyl thiols (2a−g) to N-methylmaleimide (3).

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Scheme 2. Base-Catalyzed Hydrolysis (kHO−) and Thiol Addition (k2max) Reaction Pathways for NMM, Where R Refers to Alkyl or Aryl Substituents and HA Refers to a General Acid

RESULTS AND DISCUSSION

Experimental Design. As shown in Figure 1, a total of 14 thiolsincluding seven aryl thiols (1a−g) and seven alkyl thiols (2a−g)were selected for this study, in order to cover a broad range of pKa values from 2.6 (pentafluorothiophenol) to 10.6 (propanethiol). The aryl thiols chosen do not present any bulky ortho substituents, thereby minimizing steric effects on the nucleophilic attack. Similarly, the alkyl thiols were selected to have a two-carbon spacer between the thiol and the pKa-tuning substituent. A small amount of organic cosolvent was necessary to maintain solubility of the maleimide substrates, the addition product, and some of the aryl thiols. Acetonitrile was chosen for its miscibility with water, its capacity to solubilize all components at the desired concentrations (using only 10%), its inertness (non-nucleophilic, nonoxidizing), and its minimal perturbation of pH. Rate constants were measured at constant pH under pseudofirst-order conditions, in the presence of limiting thiol and excess maleimide. For the ArSH series, reaction progress was monitored by following the disappearance of aryl thiolate. For the RSH series, the reaction was monitored by following the decrease in maleimide. All reactions were monitored spectrophotometrically at their corresponding λmax values (see Experimental Section). Absorbance data were fitted to an equation for monoexponential decay over 3−5 half-lives (see Experimental Section), providing clean pseudo-first-order rate constants (see Figure S1). These in turn were plotted against the concentration of maleimide, exhibiting excellent linear relationships (of which an exemplary plot for 1c is shown in Figure 2) indicating the reaction is first order in maleimide, and affording the second-order rate constants (k2) tabulated in Tables S1− S14. An adventitious parallel reaction that has the potential to compete with thiolate addition to NMM under these conditions is base-catalyzed maleimide hydrolysis (refer to Scheme 2). The hydrolysis of N-alkylmaleimides has been studied extensively in

the past.8,13,15,22−24 Comparison of the rate constants for hydrolysis and thiol addition shows that thiol addition is several orders of magnitude faster, under most reaction conditions. This correlates with the empirical observation that we13 and others8,15,24 have madenamely, under the reaction conditions and time frame used to study thiol addition, the thiol addition reaction proceeds to completion and consumption of the maleimide due to hydrolysis is negligible. In this study, the kobs for addition is anticipated to converge with that of hydrolysis at pH values much greater than pKaRSH because k2 for addition will have plateaued with respect to its dependence on pH (k2max) whereas kHO− maintains a positive linear dependence on pH. Because our kinetic measurements were conducted at pH values close to pKaRSH, the effect of hydrolysis on the determination of kobs for addition (and therefore of k2 and k2max) is expected to be negligible. Furthermore, we confirmed that the disappearance of NMM at the highest pH values under the experimental conditions is relatively slow and linear ( 0.5). For example, for high-pKa thiol addition to p-nitrophenyl acetate,29 acetaldehyde,27 and acrylonitrile,30 respective βnuc values of 0.38, 0, and 0.45 were measured. For low-pKa thiol addition to acetaldehyde,27 cyanamide,28 trans-4-phenyl-3-buten-2-one,25 and aryl vinyl sulphones,31 βnuc values of 1.0, 0.55, 0.78, and 0.507 were measured. In the more relevant context of high-pKa thiol addition to maleimides, one study reported βnuc = 0.43 ± 0.03 for addition to N-ethylmaleimide (NEM),9 but this Brønsted plot was constructed from only three data points within 2.3 pKa units. Another study reported βnuc = 0.42 for addition to N-(4-(2benzimidizolyl)phenyl)maleimide,19 but this Brønsted plot exhibits considerable scatter among the alkyl thiol series Table 2. Summary of Activation Parameters and SKIE Data RSH

pKa

ΔH‡ (kcal·mol−1)

ΔS‡ (cal·mol−1·K−1)

ΔG‡ (25 °C) (kcal·mol−1)

k2H2O/k2D2O

1a 1c 1d 2a 2d 2f

2.6 4.5 6.3 6.5 8.6 10.3

4.04 ± 0.09 2.51 ± 0.07 1.35 ± 0.03 11.3 ± 0.1 7.92 ± 0.05 5.4 ± 0.1

−45.4 ± 0.3 −40.0 ± 0.2 −38.2 ± 0.1 −4.0 ± 0.4 −9.0 ± 0.2 −15.3 ± 0.3

17.6 ± 0.1 14.42 ± 0.09 12.73 ± 0.04 12.5 ± 0.2 10.60 ± 0.08 10.0 ± 0.1

2.22 ± 0.06 2.12 ± 0.06 2.05 ± 0.03 − − −

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The Journal of Organic Chemistry log k2 vs pH profiles (Figures S39, S41, and S43). The resulting Eyring plots are shown in Figures S40, S42, and S44, and the corresponding computed activation parameters are given in Table 2. Experiments involving 2a,d,f were conducted at four temperatures in the ascending wing of the log k2 vs pH profiles (Figures S45−S50). The k2max values at each temperature were predicted using the thermodynamic pKa values of 2a,d,f and a single ionization model relating log k2 and pH. In the ArSH series, the ΔS‡ values are negative and very large, suggesting considerable restriction in the degrees of freedom in the rate-limiting TS compared to the GS. In moving from nucleophiles with low pKa values to ones with higher values, the negative ΔS‡ values decrease in magnitude, as do the positive ΔH‡ values. Both of these effects lead to smaller ΔG‡ values, representing enthalpy−entropy reinforcement (see Figure 5) and

aryl thiol increases, the magnitude of the primary SKIE decreaseswhich may suggest a smaller degree of proton transfer coincides with increasing nucleophile basicity. This trend is discussed from a mechanistic perspective below. In the case of the RSH series, the absence of buffer catalysis suggests general catalysis is not operative, so it is unlikely that a proton is transferred during the rate-limiting step. While it would be helpful to confirm this assertion, similar SKIE experiments were not carried out with 2a,d,f because k2 values could not be determined reliably or at all in the plateau region of the pH−log k2 profiles. Conducting such experiments in the experimentally accessible pH-dependent region would be unreliable due to the numerous effects that changing solvents (H2O to D2O) would have on the pKa of the thiol (and that of the substituent tuning the pKaRSH), the pKa of the buffer, and the pH.33 The ratio of kH2O/kD2O in that case would be a complex composite of the actual SKIE combined with these effects. SKIE experiments have also been used to study the nature of proton transfer in the addition of thiolates to acrylonitriles26 and fumaronitrile26 as well as cyanamide28 (cued by the observation of general acid catalysis, discussed above). For fumaronitrile, which most resembles maleimides, lower-pKa thiols were found to exhibit much larger SKIEs than higher-pKa thiols; for acrylonitrile, the difference is negligible. These observations may point to substantial differences in the energies of their respective intermediates. SKIE experiments were also used to study the reverse (elimination) reaction, enabling the elucidation of a stepwise mechanism with a carbanion intermediate. In the case of cyanamide, the investigators found a central transition state − (βnucArS = 0.55) and a SKIE of kH2O/kD2O = 1.6 ± 0.2. While a stepwise mechanism is proposed where the SKIE originates from H-bonding effects in the rate-limiting addition step, the authors point out that an enforced concerted mechanism involving proton transfer in the rate-limiting step cannot be ruled out. Ionic Strength. In the extended plateau region of the pH− log k2 profile for 1c (considered to be representative of the ArSH series), the ionic strength was varied from 0.050 to 0.200 M (Figure S60). The k2 values are relatively invariant over this ionic strength range (Figure S61) and may point to little difference in overall charge in moving from the ground state to the ratelimiting transition state. The slight attenuation in k2 with increasing ionic strength may be a consequence of a rate-limiting transition state where the anionic charge remains largely on the sulfur. For the RSH series, there is a considerable attenuation of k2 with increasing ionic strength in the case of 2d (Figure S63), suggesting significant charge dissipation in attaining the TS. These data are consistent with a rate-limiting TS in which the charge on the thiolate is dispersed over multiple centers. Searching for a point of comparison, we found one report of the effect of ionic strength on the addition of alkyl (and none for aryl) thiols to maleimides. In that case, the initial rate of Lcysteine addition to eosin-5-maleimide (E5M) was found to increase with increasing ionic strength (up to 0.4 M NaCl).21 These data show the opposite trend of those found here; however, the charged nature of their maleimide differs considerably (mono- or dianionic for E5M vs neutral for NMM) and may not be an appropriate comparison. In a study of − alkyl thiolate addition to acrylonitrile (βnucRS = 0.450), the (rate-limiting) addition of homocysteine exhibited no effect due to changes in ionic strength when μ ranged from 0.15 to 1.55.30

Figure 5. Plot of −TΔS‡ vs ΔH‡ showing the markedly differing trends in the activation parameters for the reaction of NMM with either alkyl thiols (RSH, □) or aryl thiols (ArSH, ■). The solid arrows depict how these values change as pKaArSH increases; moving from top-right to bottom-left (or vice versa) represents enthalpy−entropy reinforcement. The dotted arrows depict how these values change as pKaRSH increases; moving from bottom-right to top-left (or vice versa) represents enthalpy−entropy compensation.

to ΔG‡ values that depend heavily on pKaArSH. These reinforcing effects are consistent with differences in pKaArSH being fully − realized in log k2max, leading to a large βnucArS value of 1.0. On the other hand, in the RSH series, the ΔS‡ values are negative but smaller in magnitude than those in the ArSH series, suggesting a smaller restriction in the degrees of freedom in attaining the rate-limiting TS. In moving from nucleophiles with lower pKa values to ones with higher values, the negative ΔS‡ values increase in magnitude while the positive ΔH‡ values decrease. This represents enthalpy−entropy compensation (see Figure 5) and leads to ΔG‡ values that depend to a lesser degree on pKaRSH. These compensatory effects shed light on the − energetic origins of the smaller βnucRS of 0.40. Solvent Kinetic Isotope Effects. Given the highly ordered nature of the rate-limiting transition state and the observation of general acid catalysis for the ArSH series, solvent kinetic isotope effects (SKIEs) were measured to investigate the possibility of a proton-in-flight. Experiments involving 1a,c,d were conducted in H2O and D2O in the plateau region of the log k2 vs pH profiles. The SKIE values are also given in Table 2. The magnitude of the SKIE values is normal and primary, consistent with a proton-in-flight leading up to or included in the ratelimiting transition state. These data support the observation of general acid catalysis as well as the highly ordered transition state(s) leading up to or included in the rate-limiting step. One may also perceive a trend in the SKIE valuesas the pKa of the 11678


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For the alkyl thiols, the βnucRS of 0.40 suggests only partial bond formation has occurred between RS− and NMM at the ratelimiting transition state. Unfortunately, plateau regions in the pH−log k2 profiles contained rate constants that could not be obtained with a high degree of reliability, so tests to determine the effect of deuterated solvent on k2 were not conducted. However, the independence of k2 on buffer concentration suggests only specific base catalysis is operative and that a proton-in-flight is unlikely. The entropies of activation are negative, which is consistent with the thiolate and maleimide coming together in the rate-limiting step. The small magnitude is likely due to bond formation progressing less than halfway, in addition to the release of solvent in going from a charge localized on one center to one delocalized over three centers. Charge dissipation upon attainment of the TS is also supported by the observed attenuation of k2 with increasing ionic strength. Overall, the addition reaction for the alkyl RSH series likely occurs by a stepwise mechanism, where the first step (heavy atom bond formation) is rate-limiting and probably exergonic34 (Figure 6). The proposed mechanism may be considered in light

For the addition of alkyl thiolates to p-nitrophenyl acetate −

(βnucRS = 0.38), ionic strength was varied for four thiols from 0.2 to 0.7 M with no significant changes detected.29 The difference in behavior between these systems and that of alkyl thiol addition to maleimides may be due to the development of negative charge in their transition states being more advanced and less delocalized, leading to a smaller difference in charge between the ground state and rate-limiting transition state. Solvent Effects. Also in the extended plateau region of the pH−log k2 profile for 1c, the volume fraction of MeCN was varied from 10% to 30% (Figure S68). Increasing the % MeCN over this range leads to attenuation of k2 values for the ArSH series (Figure S69). Mechanistically, this result is somewhat inconsistent with the above-mentioned data showing little change in charge in the steps leading up to the rate-limiting transition state. One explanation for the steep dependence on % MeCN could be the effect of high proportions of MeCN on thiol pKa; perhaps as the pKa increases with increasing % MeCN, the kobs descends along the pH-dependent region. Another possible source involves MeCN associating with the alkene portion of the maleimide via π−π interactions and inhibiting nucleophilic attack at those sites. A similar effect is found for 2d of the RSH series (Figures S70−S71). Previous Thiol Addition Studies. Prior to proposing a mechanism consistent with our experimental observations, it is instructive to consider what mechanisms have been proposed for related thiol addition reactions. Notably, only a small number of studies point to a mechanistic difference between high-pKa and low-pKa thiols with respect to their thiolate addition to unsaturated carbon centers. Two of them, emanating from Jencks’ research laboratories, provide a relevant context in which to interpret data from our current study. The first is a study of thiol addition to acetaldehyde where lower-pKa thiolates exhibit βnuc = 1.0 whereas higher-pKa thiolates exhibit βnuc = 0.27 Several features of this study, such as buffer catalysis, SKIE data, and Brønsted βnuc values, are similar to what we observed in the current work. The authors also studied the reverse reaction elimination of thiols from hemithioacetalswhich experimentally probes the rate-limiting step from both directions. The break in the Brønsted plots arises near the transition from aryl to alkyl thiols; however, only ∼3 thiols define each line and there is no overlap in pKa values, so it is unknown what trend would be followed by higher-pKa aryl thiols or lower-pKa alkyl thiols, if those series were extended. The data were interpreted to signify a change in rate-limiting step from diffusion-controlled separation of hydroxide ion from the neutral hemithioacetal (for low-pKa thiols) to nucleophilic attack (for high-pKa thiols). The second study pertains to mechanistic data on the addition of thiols to (and elimination of thiols from adducts of) acrylonitrile (βlg = −0.54), 1-chloroacrylonitrile (βlg = −0.52), and fumaronitrile (βlg = −0.25).26 As mentioned above, fumaronitrile, which bears resemblance to the maleimides used in this study, is proposed to follow a similar addition mechanism, with a change in rate-limiting step from proton transfer (low-pKa thiols) to nucleophlic attack (high-pKa thiols). While the biphasic kinetics, SKIE data, buffer catalysis, and Brønsted βlg values taken together point to two domains with different ratelimiting transition states, the Brønsted plots contain insufficient data for high-pKa thiols to provide evidence for this change. Alkyl Thiol Addition Mechanism. For both the alkyl and aryl thiols studied, the pH−rate data indicate the thiolate form is the active nucleophile, in agreement with Bednar’s classic work.9

Figure 6. Qualitative energy diagram for the addition of alkyl thiols on NMM, illustrating a stepwise mechanism featuring rate-limiting attack of thiolate.

of a retro-E1cBrev mechanism. This proposal aligns with the study of thiol addition to acrylonitriles and fumaronitrile where the authors propose that higher-pKa thiols add to fumaronitrile in a stepwise manner where attack is rate-limiting.26 Further distinctions may be determined in the future by conducting mechanistic studies of the reverse (elimination) reaction, akin to the work by Fishbein and Jencks.26 Aryl Thiol Addition Mechanism. As mentioned above, the pH−rate data for the aryl thiols also indicate the thiolate form is the active nucleophile, as expected.9 However, the reaction of NMM with the ArSH series exhibits a different sensitivity to thiol pKa and temperature compared to the RSH series. In fact, the entropies of activation show opposing trends with increasing thiol pKa, which suggests the reactions proceed via different ratelimiting transition states. − The βnucArS of 1.0 is consistent with a substantially formed bond between ArS− and NMM, which is further corroborated by large negative entropies of activation. A Hammett plot composed of 1c−g35 exhibits ρ− = −2.47 ± 0.09, suggesting 11679


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mechanistic studies of the reverse (elimination) reaction may allow further refinement of this proposed mechanism. Regarding Brønsted Plot Continuity. None of the reported Brønsted plots relevant to this study contains sufficient data to provide a context in which to discuss the continuous nature of these data. However, if we look more closely at the two aforementioned studies from Jencks, we see that discontinuous lines cannot be ruled out. For the addition reaction involving acetaldehyde,27 lines drawn through the aryl thiol points and alkyl thiol points would represent two intersecting, discontinuous lines. For that involving acrylonitrile,26 lines drawn separately through the aryl thiol and alkyl thiol points also indicate two distinct and discontinuous lines. In both cases, more data would be necessary to better define these relationships. If we briefly extend this discussion to consider the case of thiolate attack on the saturated centers of butyl chloride36 and methylmethanethiosulfonate,37 it was shown that alkyl and aryl

considerable reduction in electron density at the sulfur and therefore advanced bond formation to NMM. Minimal perturbation in k2 was observed upon changing ionic strength, suggesting little charge alteration occurs in the transformation of the reactants into the rate-limiting transition state. This is also consistent with advanced bond formation between ArS− and NMM, leading to a strong negative charge residing on the maleimide portion of the transition state structure or on the conjugate base of a proton donor. The normal SKIE points to considerable partial bond formation between the transforming substrate and a proton, suggesting that the strong negative charge probably resides on the conjugate base involved in proton transfer. Taken together, a rate-limiting transition state involving the restriction in degrees of freedom of the nucleophile, substrate, and a proton source such as solvent would exhibit very large negative entropies of activation, such as those shown in Table 2 for the aryl thiols. These activation parameters are very different from those measured for the alkyl thiols, both in the magnitude of the parameters and in the direction of change with increasing pKaNuc. This clearly suggests the two reactions proceed through different rate-limiting transition states, due to either a change in rate-limiting step or a change in mechanism. This result illustrates that it is probably inappropriate to fit a single Brønsted slope19 through a combined set of both aryl and alkyl thiol data. Overall, the addition reaction for the ArSH series likely occurs by a stepwise mechanism, where the second step is rate-limiting (Figure 7). According to this mechanism, the high Brønsted

−

thiols fall on different, staggered Brønsted lines (βnucArS = 0.409 −

−

−

± 0.003, βnucRS = 0.19 ± 0.03; βnucArS = 0.635, βnucRS = 0.309). Importantly, both of these studies included data gathered for both alkyl and aryl thiols beyond the break point, providing evidence for the discontinuous nature of the lines. Although the Brønsted slopes of Figure 4 appear to be staggered and discontinuous, two experimental sources should be discussed that may exaggerate this discontinuity, although they do not account for it completely. First, the ArSH pathway is general acid catalyzed whereas the RSH pathway is not. Under the experimental conditions chosen for this study (0.200 M buffer), the buffer-dependence (Figure S55) reveals the −

measured k2ArS values are ∼2.7-fold larger than the value extrapolated to zero buffer concentration. Second, the RSH pathway involves considerably more charge dissipation in attaining the TS compared with the ArSH pathway. Under the experimental conditions chosen for this study (μ = 0.100 M), the −

k2RS values are ∼1.2-fold smaller than the value extrapolated to μ = 0. Together, these effects would lower the Brønsted line for the aryl thiols and raise the Brønsted line for the alkyl thiols, bringing the two lines closer to alignment. If this continuity is real, it implies that the breaking point of the Brønsted line depends only on thiolate basicity, and the fact that it occurs exactly at the transition from the series of aryl thiols to the series of alkyl thiols is merely a coincidence. Under this interpretation, the downward break in Figure 4 may represent a change in mechanism related primarily to thiolate basicity. Starting with the strongly nucleophilic thiolates formed from the high-pKa alkyl thiols (Figure 6), as the pKa is lowered, the thiolate (ground state) is stabilized and the nucleophile is weakened, meaning the resulting adduct formation would be less exergonic. This increases the activation barrier and lowers the rate constant. As we continue to stabilize the ground state to a greater extent than the intermediate, ultimately this would lead to a case where, at a certain thiol pKa, the transition state for the formation of the intermediate would be lower in energy than that of enolate protonation (Figure 7), and a change in rate-limiting step would occur. If the pKa of the thiol were lowered even further, one could envision a case where the intermediate adduct enolate would be too unstable to exist, because the back reaction to expel an excellent thiolate leaving group would be too rapid. Presumably for very low-pKa thiols, a concerted reaction mechanism would be enforced, featuring a

Figure 7. Qualitative energy diagram for the addition of aryl thiols on NMM, illustrating a stepwise mechanism whose rate-limiting transition state involves enolate protonation to an intermediate featuring a fully formed C−S bond.

slope is reflected in the fully formed C−S bond of the second TS. The lower pKa aryl thiols react more slowly because their thiolate forms are more stable (GS stabilization). The observed primary SKIEs are due to proton transfer from a general acid in the rate-limiting step, and the entropies of activation are large and negative because they include orientation of NMM with thiolate in the first step (preassociation) and with a general acid in the second TS (partial association). This mechanism may be considered in light of a retro-E1cBirr mechanism. Again, future 11680


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The Journal of Organic Chemistry transition state that includes both nucleophilic attack and protonationthe microscopic reverse of an E2 elimination. Calculations. As a note added in proof, we have also performed preliminary DFT calculations to determine the relative energies of the reactants, intermediates, and products as well as those of TS1 and TS2 of the proposed sequential addition mechanism. TS1 was modeled as the direct attack of the thiolate with the malemide. TS2 was modeled as the transfer of a proton from a water molecule to the enolate. The calculated energies and activation energies for a select set of aryl and alkyl thiols are presented in Tables S33 and S34. The Gibbs energies of activation of these reactions are generally higher than those determined experimentally, although these calculated energies represent the solvent as a continuum. As a result, the stabilization of the transition states by the solvent may be underestimated, particularly in TS2 where the hydrogen bonding network of the aqueous solvent could stabilize the transfer of a proton from the solvent. Nevertheless, these calculations are helpful in interpreting the experimental data regarding the consequence of changing thiol pKa. The free energy of TS1 varies only within a 2-kcal·mol−1 range across the set of thiols that were modeled, despite the stability of the thiolate varying by 8.5 kcal·mol−1 in this set. The increased stability of the thiolate ground state is largely canceled by an increased barrier corresponding to the partial desolvation of the thiolate in TS1 and may contribute the enthalpy−entropy compensation noted in the experimental activation parameters. More specifically, the trend in the ΔSTS1 values may be predominantly influenced by the degree of desolvation required to attain TS1. In contrast, the activation energy of TS2 varies considerably with thiol pKa. For example, the TS2 barrier of propanethiol is predicted to be 7.2 kcal·mol−1, but the TS2 barrier for pentafluorothiophenol is predicted to be 26.3 kcal· mol−1. In this step, the resonance stabilization of the thiolate by the aryl group is lost; the atomic charges and C−S bond lengths of TS2 (Table S32) indicate it largely has thioether character. No potential energy minimum could be identified for the enolate adduct of the most acidic thiols (pentafluorothiophenol and 4-nitrothiophenol), as the complex reverts to a noncovalent complex between the maleimide and the thiolate. In this regime, TS1 ceases to exist as a discrete barrier, meaning TS2 would represent the only maximum on the free energy surface (i.e., in the retro-reaction, abstraction of a proton from the thiosuccinimide would result in a concerted formation of the maleimide and thiolate reactants, in an E2 elimination mechanism). Together, these observations point to three limiting mechanisms (2 stepwise, 1 concerted): stepwise with ratelimiting nucleophilic attack (TS1), stepwise with rate-limiting proton transfer (TS2), and concerted with nucleophilic attack and proton transfer occurring concurrently. A free energy diagram constructed using calculated free energy values is shown in Figure 8; it contains representative examples of these three mechanisms. To probe the sensitivity of each TS to the pKa of the conjugate acid of the active nucleophile, we used ΔGTS2 and ΔGTS1 to predict two sets of second-order rate constants at 25 °C (Table S34), from which two Brønsted plots (Figure S74) were constructed, exhibiting slopes of βnucTS2 = 1.7 ± 0.1 and βnucTS1 = 0.85 ± 0.09. It is clear from these computed plots that the two transition states have vastly different sensitivities to thiol pKa, and while the magnitudes differ from the experimental ones, the relative difference between the two sensitivities parallels that of the experimental gradients. We attribute the larger βnuc values to

Figure 8. Calculated free energy diagram for the addition of thiophenolates 1a and 1c and alkyl thiolate 2g to NMM.

the differences in solvation accounted for in each axis of the Brønsted plot. The horizontal axis, namely the experimentally determined solution phase pKa values, inherently accounts for explicit solvation whereas the vertical axis, namely the logarithm of the second-order rate constants determined from computed ΔGTS values, neglects explicit solvation. Without extrinsic stabilization by discrete solvent molecules, the computed pathways are expected to exhibit free energies that are more sensitive to the nature of the substituent on the nucleophile. To test this hypothesis, we plotted the computed log k2max values against computed pKa values from the literature38 that use the same functional and apply zero explicit waters of solvation. These Brønsted plots (Figure S75) exhibit βnucTS2 = 1.0 ± 0.1 and βnucTS1 = 0.4 ± 0.1, both within error of our experimental values. All together, we interpret the parity of these results to support the idea that each Brønsted slope corresponds to a different rate-limiting transition state. Tracking changes in the critical S−CNMM and H−CNMM bonds (Table S32) along the reaction coordinate establishes further parallels with, and provides additional insight into, the experimental data. In TS1, the S−CNMM bond length increases as thiol pKa increases; this is consistent with GS destabilization effects leading to an early TS in the Hammond sense. We can see by the large difference in S−CNMM bond lengths in TS1 relative to the corresponding enolate adducts that the TS is early with − respect to addition and reflects βnucRS = 0.40. In TS2, the S− CNMM bond lengths are relatively invariant as a function of thiol pKa and are intermediate in magnitude between the correspond− ing enolates and products, consistent with βnucArS = 1.0. Also of note in TS2, the H−CNMM bond length is large in magnitude relative to the corresponding product and increases as thiol pKa increases, confirming that TS2 is early with respect to proton transfer and supporting the trend in the experimental SKIE values. The lack of an enolate intermediate for 1a and 1b is a nuance that would not be easily detected experimentally, as the addition of these species would still require the complexation of the maleimide and thiolate to form the rate-limiting TS2, albeit as a concerted process rather than one with a discrete enolate intermediate. As such, we would not expect to see any clear breaks in the Brønsted plot or any drastic changes in the activation parameters or SKIE data. More sophisticated 11681


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measured using the effluent from the stopped-flow apparatus at the end of the reactions. The sspH values in water−acetonitrile solutions were determined by subtracting a correction constant of −0.03 from the electrode readings. The sspH values are referred to as pH values.43 UV−vis Kinetics. The addition of 1a−g or 2a−g to 3 was followed spectrophotometrically using an OLIS RSM stopped-flow UV−vis spectrophotometer with the cell thermostated at (25.0 ± 0.1) °C by a Julabo CF41 water bath. Reaction progress for the addition of 1a−g to 3 was monitored at the λmax values for their respective thiophenolates (1a, 252 nm; 1b, 415 nm; 1c, 277 nm; 1d, 272 nm; 1e, 265 nm; 1f, 260 nm; 1g, 265 nm). Reaction progress for the addition of 2a−g to 3 was monitored at the λmax for 3 (300 nm). Reactions were conducted in the presence of buffers composed of various ratios of acid (formic acid, acetic acid, MES, MOPS, HEPES, Tris, CHES, CAPS) and KOH. Typical kinetic experiments involved the preparation of two sets of solutions. The first contained a solution of buffer (0.200 M), KCl (to achieve μ = 0.200 M), TCEP (2 × 10−4 M to convert any disulfide to thiol), acetonitrile (10% v/v), and RSH (4 × 10−4 M) and brought to final volume with water (18.2 MΩ·cm), added in the order listed. The second set of solutions consisted of NMM (4−20 × 10−3 M) and acetonitrile (10% v/v) and brought to final volume with water (18.2 MΩ·cm), added in the order listed. All solutions were sonicated briefly to ensure complete mixing. The final concentrations of components in the 4 mm path length reaction cell were 0.100 M buffer, μ = 0.100 M (KCl), 1 × 10−4 M TCEP, 2 × 10−4 M RSH, (2−10) × 10−3 M maleimide, and 10% v/v acetonitrile. In the case of 1g, the concentration of 1g and NMM were reduced by 5-fold in order to lower kobs sufficiently to acquire reliable kinetic data. Kinetic experiments were carried out in triplicate, and the resulting Abs vs time traces were fitted to a standard single exponential equation to obtain the observed pseudo-first-order rate constants (kobs). For slower reactions, initial rates of the reactions were obtained by fitting the first 5−10% of the Abs vs time traces to a linear regression; the initial rates were then divided by the expected absorbance change (ΔAbs) had the reaction reached 100% completion (assessed by following several of the most rapid experiments to completion). Kinetic Analyses. OLIS GlobalWorks software was used to analyze the raw Abs vs λ and Abs vs time data; Microsoft Excel (2016) and GraphPad Prism 4 were used to perform further analyses. Second-order rate constants (k2) were determined by plotting kobs vs [maleimide] and computing a linear regression.26 For the ArSH series, the maximum second-order rate constants (k2max) were determined by plotting k2 vs pH and fitting the data to eq 1. For the RSH series, the linear portion of the pH−log k2 data was fitted to eq 1 with the kinetic pKa fixed to the corresponding aqueous literature32 value. The Brønsted plots were constructed using a combination of aqueous literature values and the kinetic pKa values determined from the pH−log k2 plots. To validate the use of these values, the experimentally determined kinetic pKa values were plotted against the corresponding (thermodynamic) aqueous literature values. They were found to have a very good correlation with a gradient close to 1.0 (Figure S34) suggesting the pKa values in 90% H2O−10% MeCN v/v are very close to those in neat H2O. Further, the log k2max values for NMM, log k2max values for NPM, kinetic pKa values, literature aqueous thermodynamic pKa values, and literature 95% EtOH thermodynamic pKa values were plotted against the Hammett substituent constant, σ−, exhibiting ρ− = −2.47 ± 0.09 (Figure S36), −2.4 ± 0.1 (Figure S38), −2.7 ± 0.2, −2.3 ± 0.2, and −2.4 ± 0.1,20 respectively. The similarity of these values suggests that the kinetically determined pKa values accurately represent the thermodynamic constants. Solvent Kinetic Isotope Effect (SKIE) Experiments. The solvent kinetic isotope effect (SKIE) experiments required the preparation of the above-mentioned solutions in D2O. The kinetic experiments for 1a,c,d were carried out in triplicate, and the stopped flow analyzer was rinsed with anhydrous acetonitrile followed by L2O (L = H or D) between experiments. The pH and pD values were measured after the reactions were complete. (The pD values were recorded as the pH meter readings corrected for the effect of the deuterated solvent on the reading. The exact pD value is less important since the experiments are carried out in the extensive plateau regions of the pH−log k2 profiles.)

calculations that take account of explicit solvation would be necessary to determine free energy values that better match the experimentally determined values as well as to detect the exact point of the anticipated break in the Brønsted plot. The limitations of these calculations also prohibit further speculation about the staggered nature of the discontinuity.

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CONCLUSION In this work, we present a detailed mechanistic study of the thiolmaleimide addition reaction, commonly used in a variety of bioconjugation applications. Our proposed mechanisms for the addition of alkyl and aryl thiols to NMM (Figures 6 and 7) are consistent with all of our experimental and computational data. More specifically, the mechanistic variations are primarily governed by ground state stabilization effects that have the potential to access mechanisms that are stepwise with ratelimiting nucleophilic attack, stepwise with rate-limiting proton transfer, and concerted with nucleophilic attack and proton transfer occurring concurrently. Our broad data set, considered alongside other related studies,36,37 makes it clear that the Brønsted plots of alkyl and aryl thiols should be considered separately, rather than combined.39 These proposed mechanisms may be further refined by studying the microscopic reverse reactions, formally considered as E1cb elimination reactions. More sophisticated calculations may also be useful for confirming the stepwise or concerted nature of the proposed mechanisms and providing more quantitative energy diagrams. Such studies have helped to unravel subtleties in Brønsted plots that may be too complex to interpret from the experimental data.36 Importantly, this study confirms that the reaction of alkyl thiols with maleimides is relatively insensitive to thiolate nucleophilicity, manifested in a Brønsted slope of 0.40, which is consistent with partial C---S bond formation at the ratelimiting transition state. Another consequence of this shallow slope is particularly noteworthy for protein labeling applications. For example, we have designed peptide tags40 that can be genetically fused to any protein of interest and present two Cys residues that can react with highly fluorogenic dimaleimide labeling agents.14,41 Since Cys residues present alkyl thiol functional groups, they should fall on the Brønsted slope of 0.40 (cf. thiols 1a and 1c, Figure 4). This means that for every unit drop in the pKa of a Cys residue, while its rate constant may drop 4-fold, the concentration of the Cys residue in its reactive thiolate form will increase by 10-fold. This clearly signifies that in the design of alkyl (peptide) thiols that are meant to be highly reactive at a given pH, it is more effective to increase the proportion of reactive thiolate, by designing a thiol with a lower pKa, than it is to increase the nucleophilicity of the thiolate, by designing a higher thiol pKa.42 More specifically, for a thiol addition reaction at physiological pH 7.4, the Brønsted slope of 0.40 indicates the maximum rate of reaction would be realized with a thiol whose pKa is ∼7.2 (Figure S77). We have included a spreadsheet in the Supporting Information that facilitates the visualization of the design principles based on our mechanistic conclusions.

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EXPERIMENTAL SECTION

General Methods. The concentration of H3O+ was determined potentiometrically using a combination Fisher Scientific Accumet electrode model no. 13-620-290 calibrated with certified standard aqueous buffers (pH 4.00, 7.00, and 10.00). The pH was determined as −log[H3O+]. The swpH values for the kinetic experiments were 11682



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Activation Parameters. Kinetic experiments with 1a,c,d and 2a,d,f were performed at different temperatures using a thermostattable stopped-flow analyzer. Solutions in the drive syringes were allowed to equilibrate for 10 min after the water bath reached the desired temperature. First-order rate constants were measured in at least triplicate at four different temperatures ranging from 25 to 55 °C. Buffer, HA, Ionic Strength, MeCN, TCEP Dependences. Additives such as buffer, TCEP, and MeCN as well as the maintenance of ionic strength were assessed for their effect on k2. The k2 values were determined at three different [buffer], [HA], μ, % MeCN v/v, and [TCEP] for 1c and 2d; the results are shown in Figures S54−S71. Product Analyses. The major organic products of the addition reactions were characterized using NMR and MS, either by extracting the product from a concentrated (protic) aqueous reaction mixture or by conducting the reaction in deuterated solvents. Typical product analysis experiments involved the preparation of two sets of solutions. The first contained a solution of buffer (0.200 M HEPES, 0.100 M KOH), KCl (to achieve μ = 0.200 M), TCEP (1.0 × 10−3 M), acetonitrile (10% v/v), and thiol (10.0 × 10−3 M), added in the order listed and brought to its final volume (2.5 mL) with 18.2 MΩ·cm water. The second solution consisted of NMM (10.0 × 10−3 M), and acetonitrile (10% v/v), added in the order listed and brought to its final volume (2.5 mL) with 18.2 MΩ·cm water. All solutions were sonicated briefly to ensure complete mixing. The final concentrations of components in the reaction vessel were 0.100 M buffer, μ = 0.100 M (KCl), 0.50 × 10−3 M TCEP, 5.0 × 10−3 M thiol, 5.0 × 10−3 M maleimide, and 10% v/v acetonitrile in a 5 mL (total) reaction volume. The solution was mixed thoroughly for ∼1 min and extracted with DCM (2 × 5 mL). The organic phase was dried over MgSO4 followed by removal of volatile components in vacuo. Characterization of the reaction products by NMR and MS confirmed that the corresponding thiosuccinimide is the predominant organic product of the addition reaction under these conditions. Exemplary 1H NMR spectra (Figures S72 and S73) and MS data characterizing the predominant organic product for addition of aryl thiol 1d and alkyl thiol 2e to NMM under the above-mentioned conditions are shown in the Supporting Information. Calculations. DFT calculations were performed using Gaussian 16 A03.44 The ωB97X-D exchange-correlation functional45 was used to incorporate the effects of dispersion and mitigate delocalization error.46 The TZVP basis set was used in all calculations.47 The energies of TS1, enolate, and TS2 were calculated relative to the thiolate. The effects of solvation were included using the SMD model with the parameters for an aqueous solvent.48 The stability of the thiolate was calculated based on the Gibbs energy for a 1 M standard state of the thiolate, which was calculated from the experimental thiol pKa values.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jeffrey W. Keillor: 0000-0002-8133-6862 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to J.W.K. and to C.N.R. Computational resources were provided to C.N.R. by Compute Canada.

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DEDICATION This article is dedicated to Prof. R. Stan Brown, the PhD supervisor of both M.A.R.R. and J.W.K. Along with many others, they are grateful to Prof. Brown for his legacy of rigorous physical organic chemistry and dedicated training of generations of chemists.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications Web site at DOI: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b01638. Data and plots related to Abs vs λ, Abs vs time, kobs vs [maleimide], log k2 vs pH, kinetic pKa vs thermodynamic pKa, log k2max vs pKaRSH (Brønsted plots), log k2max vs σ− (Hammett plots), ln(k2/T) vs 1/T (activation parameters), SKIE data (kH/kD), control experiments (buffer, HA, ionic strength, MeCN, TCEP dependences), product analyses, calculated bond lengths and LFERs (Brønsted and Hammett plots), fraction of thiolate, relative rate constant (k2), predicted reaction rate for addition of alkyl thiolate on NMM as a function of thiol pKa at pH 7.4, atomic Cartesian coordinates of DFT-optimized structures (PDF) Spreadsheet of relative rates (XLSX) 11683


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