Hydrogen-Deuterium Addition and Exchange in N-Ethylmaleimide Reaction with Glutathione Detected by NMR Spectroscopy.

Glutathione (GSH) is an important and ubiquitous thiol compound abundantly present in virtually every living cell. It is a powerful antioxidant critically required to protect cells from oxidative damage and free radical injury. Its quantification in ex vivo analysis remains a major challenge because it spontaneously oxidizes to form glutathione disulfide. N-Ethylmaleimide (NEM) is a well-known Michael acceptor, which reacts rapidly and irreversibly with thiol and prevents disulfide bond formation. Based on thiol conjugation to NEM, recently, the concentration of GSH was determined in human blood using NMR spectroscopy [Anal. Chem, 2021, 93(44): 14844-14850]. It was found that hydrogen-deuterium addition and exchange occur during the thiol-maleimide reaction as well as NMR analysis, generating a series of poorly explored diastereomers/isotopomers. Here, we establish a general NMR approach to identify the thiosuccinimide diastereomers/isotopomers derived from the thiol-maleimide reaction. The thiol-Michael addition reaction was conducted for GSH and another thiol compound, cysteine, separately, using D2O and H2O. The conjugates were characterized by 1H/13C 1D/2D NMR under different solvent, buffer, and pH conditions. The Michael addition combined with the H/D exchange formed twelve unique diastereomers/isotopomers. NMR measurements allowed the distinct assignment of all structures in solutions and quantification of H/D addition and exchange. Interestingly, the deuterium exchange rate was dependent on structure, pH, and buffer. The elucidation of the thiol-maleimide reaction and H/D exchange mechanism can potentially impact areas including metabolomics, small molecule synthesis, and bioconjugation chemistry.

Introduction

Maleimides are Michael acceptors that are known to react with thiols in the pH range of 6.5–7.5.[1−3] In general, maleimides are more reactive than other Michael acceptors[4] and represent an important class of substrates for chemical and biological applications. The maleimide ring strain imposed by the alkene moiety enhances the electrophilic nature of the conjugated imide functionality.[5] Furthermore, the nature of the solvent, basic environment, and the type of thiol play important roles in the reaction kinetics and selectivity in thiol–maleimide reactions. Owing to its simplicity, efficiency, and wide utility, the thiol–maleimide reaction is characterized as a click chemistry reaction.[2,6] Hence, the reaction has implications in numerous areas including small molecule synthesis, bioconjugation chemistry, and multifunctional materials.[3]

N-Ethylmaleimide (NEM) is known to react with thiols rapidly and irreversibly. Since the first report more than 70 years ago,[7] the quantitative reaction of NEM with thiol compounds has been widely used to assay thiol-group-containing compounds including proteins, peptides, and small molecules in biological mixtures.[8−13] Glutathione (reduced form, GSH) is a highly abundant non-protein thiol in live cells. It is an important and ubiquitous cellular antioxidant critically required to protect cells from oxidative damage and free radical injury. As with other thiols, it is practically impossible to analyze GSH in its native form in biological samples because the active form (GSH) spontaneously gets converted to the oxidized form glutathione disulfide (GSSG). To block GSH oxidation, NEM is widely used such that the reacted GSH can then be analyzed using a variety of analytical techniques including spectrophotometry,[14,15] fluorometry,[16−18] and mass spectrometry.[19−24] The chemical reaction of NEM and GSH is instantaneous and it does not need any catalyst for the reaction. In addition, the product formed is stable for long periods, at least several months.[25] These characteristics offer experimental simplicity and are attractive for applications including the quantitation of thiol compounds.

Recently, we demonstrated the utility of the NEM reaction with GSH for the quantitative analysis of GSH in biological samples in human whole blood using ex vivo nuclear magnetic resonance (NMR) spectroscopy.[26] The new NMR method enables quantitation of the very unstable GSH as well as other labile metabolites including the major redox coenzymes (NAD+, NADH, NADP+, and NADPH) and energy coenzymes (ATP, ADP, and AMP) in biological mixtures, all in one step. Furthermore, interestingly, it was found that hydrogen–deuterium addition and exchange occur during the thiol NEM reaction and NMR analysis, generating a series of diastereomers/isotopomers. While the thiol-Michael addition reaction has been known for a very long time, and the formation of two diastereomers from the reaction is well known,[25,27,28] the generation of diastereomers/isotopomers has not been well explored to date. In this study, we establish a general NMR approach to correctly identify the thiosuccinimide diastereomers/isotopomers derived from the thiol NEM reaction as depicted in Figure . The thiol-Michael addition was conducted for GSH and cysteine (Cys), separately, using D2O and H2O. The conjugates were characterized by 1H/13C one-dimensional (1D)/two-dimensional (2D) NMR under different solvent, buffer, and pH conditions. These findings have implications in numerous areas including metabolomics, small molecule synthesis, isotopic enrichment, and bioconjugation chemistry.

Figure 1

Diastereomers/isotopomers formed by the Michael addition reaction in (a) H2O and (c) D2O solvents, and by H/D exchange during NMR analysis (b,d). R in the structures refers to GSH or Cys. For each diastereomer/isotopomer, the configuration for the chiral carbon centers is indicated by R or S.

Materials and Methods

Chemicals and Solvents

Monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), ammonium bicarbonate (NH4HCO3), trimethylsilyl propionic acid-d4 sodium salt (TSP), acetanilide, N-ethylmaleimide (NEM), GSH (reduced, GSH), Cys, hydrochloric acid, and sodium hydroxide were obtained from Sigma-Aldrich (St. Louis, MO) or Fisher (Waltham, MA). Deuterium oxide (D2O) was procured from Cambridge Isotope Laboratory (Tewksbury, MA). Deionized (DI) water was purified using an in-house Synergy Ultrapure Water System from Millipore (Billerica, MA). All chemicals and solvents were used without further purification.

Stock Solutions of GSH, Cys, and NEM

GSH (50 mM), Cys (50 mM), and NEM (120 mM) solutions were prepared using DI water and D2O by weighing appropriate amounts.

Buffer Solutions with Internal Standards

Phosphate buffer solution (100 mM, pH = 7.4) was prepared by dissolving 1124.0 mg of anhydrous Na2HPO4 and 249.9 mg of anhydrous NaH2PO4 in 100 g D2O or DI water. Ammonium bicarbonate buffer solution (50 mM, pH = 8.4) was prepared by dissolving 400 mg of ammonium bicarbonate in 100 g D2O. TSP (100 μM) was added to the above solutions to serve as a chemical shift reference.

NEM Reaction with the Thiols

GSH (40 μL, 50 mM) stock solution in H2O or D2O solvent was treated with six-fold excess of (NEM) (100 μL, 120 mM) solution in H2O or D2O, respectively. The solutions were mixed using a vortexer for 30 s to derivatize GSH with NEM. Then, the H2O/D2O solvents, along with the excess (unreacted) NEM, were removed by drying the solutions using a stream of nitrogen gas, as described in a recent study, where NEM was conjugated with blood GSH.[26] The dried samples were dissolved in 600 μL D2O and the pH was adjusted as needed to 6.0, 7.0, or 8.0 using hydrochloric acid or sodium hydroxide. Separately, the dried samples were dissolved in 600 μL phosphate buffer prepared in H2O or D2O (100 mM, pH = 7.5) or ammonium bicarbonate buffer prepared in D2O (50 mM, pD = 7.8). The solutions were transferred to 5 mm NMR tubes for analysis. Similarly, the NEM reaction with Cys was conducted and samples were prepared for NMR analysis. Four to ten replicates were used for each condition of the hydrogen–deuterium addition and exchange reactions.

NMR Spectroscopy

NMR experiments were performed at 298 K on a Bruker AVANCE III 800 MHz spectrometer equipped with a cryogenically cooled probe and z-gradients suitable for inverse detection. The NOESY pulse sequence with residual water suppression using presaturation with or without combining with pulsed-field gradients was used for 1H 1D NMR experiments. The spectral width of 9615 or 10,204 Hz; 3 or 55 s recycle delay; 32, 64, or 128 transients; and 32 or 64 K time-domain points were used for 1H 1D NMR experiments. The free induction decay (FID) signals were Fourier transformed after zero filling by a factor of two and multiplied using an exponential window function with a line broadening of 0.3 Hz.

Separately, for the GSH–NEM compounds obtained from the reactions in H2O as well as D2O, 1D 13C, and 2D NMR experiments were performed using H2O/D2O (90:10 v/v), D2O, or D2O buffer solvents. 1D 13C spectra were obtained using the “zgpg” pulse sequence and a spectral width of 40760 Hz, 2 s recycle delay, 1000 transients, and 128 K time-domain points. The FID signals were Fourier transformed after zero filling by a factor of two and multiplied using an exponential window function with a line broadening of 3.0 Hz. The 2D NMR experiments performed included 1H–1H COSY (correlated spectroscopy), 1H–13C heteronuclear single quantum coherence (HSQC), and 1H–13C heteronuclear multiple bond correlation (HMBC). Spectra were obtained in magnitude mode for COSY and HMBC, and phase-sensitive mode using echo-anti-echo mode for HSQC experiments. COSY experiments were performed using the “cosygpprqf” pulse sequence and a spectral width of 10,204 Hz in both dimensions. FID signals were obtained for 512 t1 increments, each with 2048 complex data points. The number of transients was 4 and the relaxation delay was 2.0 s. The obtained 2D data were zero-filled to 4096 and 1024 points in the t2 and t1 dimensions, respectively. Gaussian and unshifted sine-bell window functions were applied to the t2 and t1 dimensions, respectively, before Fourier transformation. HSQC and HMBC experiments were performed using the “hsqcetgpsisp2.2” and “hmbcgplpndprdf” pulse sequences, respectively. Spectral widths of 8802 Hz (1H) and 30,186 Hz (13C) for HSQC and 8620 Hz (1H) and 40,760 Hz (13C) for HMBC were used. FID signals were obtained with 256 or 512 t1 increments, each with 2048 and 4096 complex data points for HSQC and HMBC, respectively. The number of transients used was 8 or 16 and the relaxation delay was 1.05 s. The obtained 2D data were zero-filled to 4096 and 1024 points in t2 and t1 dimensions, respectively. A 45° shifted squared sine-bell window function was applied to both dimensions before Fourier transformation. The chemical shift scales were calibrated based on the TSP signal for 1H and 13C.

Spectral Assignment and Quantitation of Diastereomers/Isotopomers

Assignment of peaks from the products of the NEM reaction with GSH and Cys were based on the results of our recent study,[26] the comparison with NMR spectra of the reactants, and the comprehensive analyses of 1D and 2D NMR spectra of NEM reacted compounds. Quantitation of the diastereomers/isotopomers formed by H/D addition and/or exchange reactions were made based on the peak areas of methine (CH) and methylene (CH2) hydrogens of the thiosuccinimide ring under different solvent, buffer, and pH conditions. Bruker software, TopSpin version 3.6.1 or 4.1.0, was used for NMR data acquisition, processing, and analyses.

Results and Discussion

The Michael addition reaction occurred as soon as the stock solution of GSH or Cys was mixed with NEM. The formation of thiol–NEM conjugates was rapid and irreversible, promoted by thiolate ion generation in the highly polar solvent.[2] At pH ∼7, the 1,4 nucleophilic thiol addition to the C=C bond in maleimide takes place on both sides of the two sp2 carbons; only two spatial configurations are possible for the new sulfite bond because both α,β-unsaturated carbonyls are similar due to the C2 axis of symmetry. The bulkier N-substituted maleimide potentially modulates the regioselectivity of the reaction. The thiolate-Michael reaction was associated with hydrogen/deuterium addition at C4, a consequence of ketonization of the enolate and the solvent used (Figures and S1a).

Figure 2

Michael addition reaction in D2O solvent (see Figure S1a for the reaction in H2O). R in the structures refers to GSH or cysteine.

We investigated the products of the maleimide reaction in H2O or D2O using 1H and 13C 1D and 2D NMR under different solvent, buffer, and pH conditions. We first examined samples in acidic conditions using H2O at pH = 3.2 or D2O at pD = 3.3. Two diastereomers (1 and 2) were formed during the thiol–maleimide reaction in H2O (Figure a). Figures a and S2a show 1H NMR spectra for 1 and 2. The two structures show methine (C3) hydrogen peaks at ∼4.06 ppm and methylene (C4) hydrogen peaks at ∼2.69 and 3.29 ppm, with slightly different chemical shifts for the two (red and blue peaks in Figure a). The thiol-Michael addition in D2O, on the other hand, generated four diastereomers/isotopomers (5–8, Figure c). The 1H NMR spectrum showed four sets of convoluted peaks for methine and methylene hydrogens, at C3 and C4, respectively, because of partially overlapped peaks (Figures c and S2c; peaks are shown in four different colors in Figure c). Interestingly, the methylene hydrogen peaks exhibited two different intensities with a ratio ∼65:35; peak intensities for hydrogens cis to methine hydrogens (5, 6, see brown and yellow in Figure c) were higher than those that are trans to methine hydrogens (7, 8, see green and blue peaks in Figure c). This difference in the yields can be explained by the regioselective ketonization of the enolate, which favors deuteration at C4 on the opposite side of the sulfide bond because of the steric hindrance of the S-atom (Figure ).

Figure 3

Portions of 1H NMR spectra highlighting the characteristic peaks for 12 diastereomers/isotopomers formed by the Michael addition reaction of GSH with NEM and H/D exchange. In each spectrum, methine or methylene hydrogen peaks for each isotopomer are shown by the same color. Spectra (a,b) are for the reaction in H2O: (a) spectrum obtained in D2O solvent (pD = 3.3) corresponding to structures 1 and 2; (b) spectrum obtained in phosphate buffer/D2O (pD = 7.5) corresponding to structures 3 and 4; (c,d) are for the reaction in D2O: (c) spectrum obtained in D2O solvent (pD = 3.3) corresponding to structures 5 to 8; (b) spectrum obtained in phosphate buffer/D2O (pD = 7.5) corresponding to structures 9 to 12 (see also Figure S2).

When the samples were reconstituted in phosphate buffer/D2O at pD = 7.5, ammonium bicarbonate buffer/D2O at pD = 8.4, or D2O at pD = 6.0, 7.0, or 8.0 we found slow (Figure S3), and reversible (Figure S4) H/D exchange at C3. For the structures 1 and 2, the exchange generated two chiral (H/D) isotopomers, 3 and 4 (Figures b and S1b), characterized by two sets of doublets at ∼2.7 and 3.28 ppm due to 2JHH coupling between the two methylene hydrogens (Figures b and S2b). For structures 5–8, the H/D exchange led to four isotopomers, 9 to 12 (Figure d), and Figures d and S2d show peaks for all four structures. Overall, the Michael addition and H/D exchange resulted in 12 distinct isotopomers (Figure ) and all 12 were distinctly identified by NMR (Figures and S2).

For further validation of the isotopomer formation and identification, the structures were investigated using 2D NMR. Figure shows portions of typical 2D 1H–1H COSY spectra. The 2D NMR peaks from the methine and methylene hydrogens from the thiosuccinimide ring are connected by red dashed lines, vertically and horizontally. Figure a,b show spectra for the reaction in H2O. The spectrum in Figure a was obtained in D2O (pD = 3.3) and the spectrum shown in Figure b was obtained in phosphate buffer/D2O (pD = 7.5). Figure a represents structures 1 and 2 and shows peaks for the thiosuccinimide methine (at C3) and methylene (at C4) hydrogens as seen from the 2D NMR cross-peaks among all three hydrogens. The two methylene hydrogens show peaks at different chemical shifts (∼2.7 and 3.3 ppm). Figure b represents structures 3 and 4. Here, the 2D NMR cross-peaks are observed only between the two methylene hydrogens because the methine hydrogen is absent due to the exchange with D. Similarly, Figure c,d show spectra for the GSH–NEM reaction in D2O. The spectrum in Figure c was obtained in D2O (pD = 3.3) and the spectrum shown in Figure d was obtained in phosphate buffer/D2O (pD = 7.5). Figure c represents the structures 5–8; and Figure d represents the structures 9–12. The 2D NMR cross-peaks between the two methylene hydrogens are absent in Figure c because one of the hydrogens is replaced by D during the Michael addition reaction in D2O. In Figure d, none of the 2D NMR cross-peaks is seen because the methine hydrogen exchanges with D from D2O solvent and one of the methylene hydrogens is replaced by D during the Michael addition in D2O. As further evidenced for H/D addition and exchange, Figures S5–S8 show overlays of 1H–13C 2D HSQC (blue cross-peaks) and 1H–13C 2D HMBC (red cross-peaks) spectra. A few characteristic 2D NMR peaks that establish the reaction of GSH with NEM and H/D addition and exchange are enclosed within rectangular boxes. The H/D addition and exchange are indicated by the absence or broadening of 1H/13C peaks for the methine (at C3) and methylene (at C4) hydrogens/carbons. For example, in Figure S51H/13C 1D/2D NMR peaks for both C3 methine and C4 methylene groups are observed as anticipated for structures 1 and 2. In Figure S6, peaks are missing for the methine hydrogen and the C3 carbon peak is invisibly broad due to H/D exchange (structures 3 and 4); however, 2D peaks due to the long-range hydrogen/carbon couplings are still clearly seen. In Figure S7, the C4 carbon 1D NMR peak is broad due to the attached deuterium (structures 5–8), whereas in Figure S8, in accordance with the structures, 9–12, peaks for the methine hydrogens are missing and peaks for C3 and C4 carbons are broad due to deuterium exchange/addition. As anticipated, a correlation between 1H and 13C chemical shifts could be seen in all 2D NMR spectra (Figures S5–S8).

Figure 4

Portions of 2D 1H–1H COSY NMR spectra of mixtures from the reaction of GSH with NEM. For (a,b), the reaction was in H2O solvent; spectra were obtained in D2O (pD = 3.3) for (a) and phosphate buffer (100 mM, pD = 7.5) in D2O for (b); for (c,d), the reaction was in D2O; spectra were obtained in D2O (pD = 3.3) for (c) and phosphate buffer (100 mM, pD = 7.5) in D2O for (d). The characteristic thiosuccinimide methine and methylene peaks are highlighted. (a) corresponds to structures 1 and 2; (b) corresponds to structures 3 and 4; (c) corresponds to structures 5 to 8; and (d) corresponds to structures 9 to 12. Note, in (a), both methine and methylene H atoms are observed as seen by the 2D NMR cross-peaks among all the three Hs (connected by red dashed lines); in (b), 2D NMR cross-peaks are observed only between the two methylene Hs because the methine H is exchanged with D; in (c), 2D NMR cross-peak between the two methylene H is absent because one of the H atoms is substituted by D; and in (d), none of the 2D NMR cross-peaks is seen because one of the methylene H atoms is substituted by D and the methine H is exchanged with D.

The NMR peak areas of the thiosuccinimide methine and methylene hydrogens (at C3 and C4) changed dramatically due to H/D exchange (Figure and Tables S1 and S2). At higher pH/D, the keto–enol equilibrium caused the acidic α-hydrogen at C3 to exchange with deuterium. The peak area of methine hydrogens decreased with increasing pH/D, reaching ∼15% or lower at pH = 8.0 (Figure a,c). Unlike the Michael addition, where the S-atom determines the stereochemistry of the enolate ketonization, the H/D exchange proceeds with a transition state where the C3 carbon is sp2 hybridized. Interestingly, the ratio between the two methylene hydrogens changed from 65:35 at pH/D 3.3, to 55:45 at pH/D > 8.0 (Figure c). The results indicated that the methylene H/D isotopomers gradually approach the ratio 1:1 because of the H/D exchange arising from isomerization of the diastereomers.[27] Phosphate and ammonium bicarbonate buffers caused higher H/D exchange when compared to D2O or H2O alone (Figure b,d). For example, while the methine hydrogen was fully exchanged with D in phosphate buffer (pH = 7.5), the exchange was in the range of 85–90% in D2O solvent even at pH = 8.0. These results indicate that the anions of the buffers exhibit additional effects on the H/D exchange at both C3 and C4. The keto–enol tautomerism can be accelerated by both acidic and alkaline environments; however, our data support the previous observation that the base-catalyzed mechanism favors the H/D exchange, whereas the formation of an enolate intermediate was preferred over acid-catalyzed ketonization due to the sulfide bond.[29]

Figure 5

Percentage of methine and methylene Hs left after H/D addition and exchange for the NEM–GSH reaction. The reaction was performed in H2O for (a,b), and in D2O for (c,d). The black and red arrows in (b,d) indicate that the samples were in phosphate buffer or ammonium bicarbonate buffer.

The reaction between Cys and NEM also formed two diastereomers with similar H/D addition and exchange mechanisms (Figure S9). In phosphate buffer, however, the ratio between the two diastereomers changed from ∼50:50 in D2O to ∼41:59, unlike the reaction with GSH (Figure S10). Such a difference in the ratio between the diastereomers for Cys has not previously been reported to the best of our knowledge.

Taken together, the data show how the thiol NEM reaction generates a series of isotopomers under different solvent/buffer/pH conditions. Understanding how various isomers are formed and interpreting their NMR spectra are important steps to translating molecular information into biological knowledge. The identification of these isotopomers potentially has implications in areas such as metabolomics, small molecule synthesis, and bioconjugation chemistry. In the metabolomics field, for instance, the simple click chemistry reaction offers a simple route to generate deuterium-labeled internal standards for absolute quantitation of the highly unstable thiol compounds.