Investigating the interaction between merocyanine and glutathione through a comprehensive NMR analysis of three GSH-stabilized merocyanine species

Spiropyrans belong to a class of photochromic materials that are known to undergo reversible structural changes from ring-closed spiropyran to ring-open merocyanine isomer in response to different external stimuli, such as redox changes. Sensing redox active molecules such as the potent antioxidant glutathione (GSH) is of interest because of the observation that changes in GSH levels are indicative of oxidative stress and correlated with a number of pathological conditions.^1-3 Several GSH-responsive spiropyrans have been reported^4-10; however, these spiropyrans exhibited modest sensitivity and selectivity towards the antioxidant. In addition, the specificity of recognition of these photoswitches to GSH remains imperfect. Understanding the structural details of the spiropyran isomers using nuclear magnetic resonance (NMR) spectroscopy may provide insights to how these photoswitches sense GSH. While ¹H NMR spectra of different spiropyrans are readily available in the literature,^11-22 the availability of ¹H NMR data of merocyanine species is rising, but still limited.^{9, 23-33} Thiele et al.³⁴ provided nearly complete ¹³C chemical shift assignment of the merocyanine species from a light-irradiated spiropyran featuring a nitro substituent in the chromene group and a carboxylic acid attached to the indolic nitrogen (Figure 1a). However, some ¹H and ¹³C assignments, specifically the olefinic protons and carbons (shown in red asterisks in Figure 1a) at the bridging part of the molecule, were inconclusive. NMR characterization of these protons and carbons are important for determining spatial isomers (e.g., cis or trans), an important piece of structural information for understanding the mechanism governing GSH sensing using spiropyrans. Therefore, we report a complete NMR (¹H and ¹³C NMR) characterization of three GSH-stabilized merocyanines: (E)-2-(2-[5-methoxy-1,3,3-trimethyl-3H-indol-1-ium-2-yl]vinyl)phenolate (MC-1), (E)-4-methoxy-2-(2-[1,3,3-trimethyl-3H-indol-1-ium-2-yl]vinyl)phenolate (MC-2), and (E)-4-methoxy-2-(2-[5-methoxy-1,3,3-trimethyl-3H-indol-1-ium-2-yl]vinyl)phenolate (MC-3) from a series of three spiropyrans: 5′-methoxy-1′,3′,3′-trimethylspiro[chromene-2,2′-indoline] (SP-1), 6-methoxy-1′,3′,3′-trimethylspiro[chromene-2,2′-indoline] (SP-2), and 5′,6-dimethoxy-1′,3′,3′-trimethylspiro[chromene-2,2′-indoline] (SP-3) (Figure 1b). These three structures differ in the number of methoxy groups they contain: MC-1 bearing a methoxy on the para position of the indoline unit, MC-2 bearing a methoxy on the para position of the phenolic oxygen, and MC-3 bearing a methoxy on both sides. Comprehensive NMR characterization of GSH-stabilized merocyanines could aid in the identification of stereochemistry of the GSH-stabilized merocyanine species and may help in providing improved understanding of the sensing mechanism of these photoswitches for GSH.

This paper focuses on the NMR characterization of GSH-stabilized merocyanine species, MC-1, MC-2, MC-3, using techniques such as ¹H, ¹³C, COSY, NOESY, HSQC and HMBC to better understand the interaction between spiropyran and GSH. The number of methoxy groups in each compound ranged from one to two, and there were three methyl groups for all the reported spiropyrans. The proposed ¹H and ¹³C NMR chemical shifts, assignments, and general structure of merocyanines MC-1, MC-2, and MC-3 are presented in Table 1. Additionally, the proposed ¹H and ¹³C NMR chemical shifts, assignments, and general structure of spiropyrans SP-1, SP-2, and SP-3 are available in the supporting information Table S1. All protons are numbered by their attached carbons. The respective proton peaks corresponding to the closed spiro form of SP-1, SP-2, and SP-3 alone (Figures S2, S8, and S17) and with the addition of GSH (Figures 2, S9, and S18) are provided in the supporting information. The carbon peaks for the GSH stabilized merocyanines MC-1, MC-2, and MC-3 can also be found in the supporting information Figures S3, S10, and S19

The NMR spectral assignment strategy for all three merocyanine compounds was as follows: (1) identify all ¹H resonances associated with each of the three chemical species (i.e., closed-form spiropyran, open-form merocyanine, and GSH) in the sample; (2) assign all ¹H resonances by analyzing chemical shifts, peak areas, homonuclear couplings and multiplicities, and NOE cross peaks; (3) assign all ¹³C resonances with attached protons using HSQC data; and (4) assign the remaining quaternary carbons using HMBC data and chemical shift analysis. NMR data of merocyanine species MC-2 and MC-3 were determined using a similar procedure as MC-1 described here: The sample used for NMR analysis contained three different chemical species; thus, the first step was to assign all ¹H resonances to one of three compounds: GSH (grey), SP-1 (closed-form, blue), or MC-1 (open-form, black) and CD₃CN reference (red) (Figure 2).

This was easily accomplished by comparing integration areas; nine conjugated and four methyl-group resonances were identified for MC-1 and SP-1 with a ratio of 1:1.15 MC:SP. The ¹H signals at 1.69 ppm (H-19, H-20) were identified based on chemical shift and peak area (6H). The protons H-19 and H-20 are indistinguishable and appeared as a singlet in the ¹H NMR spectrum, suggesting two magnetically equivalent methyl groups in the indole moiety. The similar magnetic environment experienced by these methyl protons is a result of being in a planar merocyanine structure. However, it is interesting to note that these two methyl groups give rise to two unique ¹³C resonances in the HSQC spectrum at 26.63 and 26.58 ppm (C-19 and C-20), as shown in Figure S5, suggesting the existence of two slightly different methyl environments. This indicates either a slight bend in the planar merocyanine structure or possibly a change in environment caused by the presence of GSH on either side of the molecule. This was observed for the MC-2 and MC-3 species as well, with a singlet for the H-19 and H-20 protons in MC-2 (1.71 ppm) and MC-3 (1.69 ppm) for the ¹H spectrum x-axis of the HSQC. Two unique ¹³C peaks for MC-2 (25.38 and 25.36 ppm) and MC-3 (25.38 and 25.43 ppm) were also noticed for the ¹³C spectrum y-axis of the HSQC spectrum (Figures S11 and S20). Further insight into the interaction between GSH and spiropyrans can be gained through additional chemical shift interpretation and computational studies.

The remaining proton and carbon resonances were assigned as follows: NOESY NMR spectrum of MC-1 allowed us to examine the spatial proximity of protons H-19 and H-20 (1.69 ppm) to protons H-6 and H-11 at 7.23 and 8.40 ppm, respectively. Clear NOE cross peaks with H-19 and H-20 with H-6 and H-11 were observed in the NOESY spectrum (Figure 3).

Once the H-6 and H-11 proton chemical shifts were identified by NOESY, the direct correlation of these protons to the bound carbon was found to be C-6 (109.29 ppm) and C-11 (148.89 ppm) through HSQC NMR spectrum (Figure S6). Based on the identification of H-11, the H-10 proton could be assigned using COSY with a ¹H assignment of 7.47 ppm (Figure 4).

Additionally, H-10 and H-11 could be identified based on chemical shift, multiplicity (doublets), and the characteristic J-coupling constant of 16 Hz for olefinic protons in a trans conformation. It is noted that H-11 is the most deshielded among the merocyanine ¹H peaks because of the ring-current effects and additionally because of the delocalization of π electrons towards the positively charged indolic nitrogen, causing the carbon, to which this proton is attached, to carry a partial positive charge. The two protons H-11 and H-10 are correlated to ¹³C peaks at 148.89 (C-11) and 112.66 ppm (C-10), respectively in the HSQC spectrum (Figure S6).

The protons in the methoxy group (H-23, 3.84 ppm) and the methyl attached to the indolic nitrogen (H-21, 3.91 ppm) were close in chemical shift but could be differentiated through observed NOE cross peaks shown in Figure 3: H-21 with resonances H-3 and H-10, and H-23 with resonances H-2 and H-6. Further elucidation of their associated ¹³C chemical shifts was observed in the HSQC spectrum (Figure S6). The signals of the remaining ¹H resonances on the indoline fragment (H-2 and H-3) were assigned by analyzing homonuclear COSY and NOESY correlations. As aforementioned, an NOE cross peak was observed between H-21 and H-3, a doublet at 7.57 ppm (Figure 3). In the COSY spectrum of Figure 4, H-3 showed J-coupling to the doublet of doublets at 7.10 ppm (H-2). ¹³C resonances C-2, C-3, and C-6 were identified from the HSQC spectrum (Figure S6).

The remaining four aromatic ¹H resonances from the ring-open chromene moiety (H-14, H-15, H-16, H-17) were assigned based on COSY, HSQC, and NOESY spectra. A NOE cross peak was observed between a doublet of doublets at 7.80 ppm and both H-11 and H-10; thus, this was assigned to H-17 (Figure 3). Interestingly, the NOE cross peak intensity was much stronger between H-17 and H-10 than between H-17 and H-11, strongly suggesting a trans conformation TTT. (This is discussed further with respect to the last figure). After assigning H-17, the triplet at 6.96 ppm could then be assigned to H-16 from the COSY spectrum in Figure 4. Furthermore, the triplet H-15 (7.40 ppm) and the doublet at 6.98 ppm could then be assigned to H-14, through observed COSY cross peaks (Figure 4). These assignments are consistent with chemical shielding concepts; H-14 and H-16 were more shielded compared with H-15 and H-17 because of the partial negative charge to the carbon atoms to which H-14 and H-16 are attached because of electron delocalization. Again, associated ¹³C resonances were assigned using HSQC data by observed cross-peaks with attached protons (Figure S6).

Five quaternary carbons were identified in the HMBC spectrum and assigned to C-1, C-8, C-9, C-12, and C-13 through careful analysis (Figure 5). Basic chemical shift prediction was used to aid in quaternary carbon assignments. For example, the three most downfield quaternary carbons at 181.35 ppm (HMBC to H-11 and H-10), 161.95 ppm (HMBC to H-3 and H-6), and 159.72 ppm (HMBC to H-11, H-17, and H-15) were assigned to C-8, C-1, and C-13, respectively. This was based on HMBC correlations and by the expectation that their chemical shifts would be the most downfield of all seven quaternary carbons. Similarly, the quaternary carbon C-9 at 52.50 ppm (HMBC to H-10 and H-6) was easily identified as it is the most shielded among the seven. C-12 at 122.34 ppm was assigned from a strong HMBC correlation to H-10 and a weak coupling to H-16. The last two quaternary carbons, C-4 and C-5 were assigned at 136.08 ppm and 146.32 ppm, respectively. Both of these resonances could be assigned to either C-4 or C-5 based on ambiguous HMBC cross-peaks, so chemical-shift prediction was used to assign C-4 as the more upfield and C-5 as the more downfield. All identified HMBC correlations (solid grey lines), as well as NOESY correlations (dotted blue lines), are indicated in Figure 6.

The high amount of GSH-induced merocyanine species enabled access to complex structural information that can be obtained from 2D NMR experiments such as COSY, HSQC, HMBC, and NOESY. Whereas the closed spiro form locks the olefinic fragment in a cis configuration, computational studies show that the ring-open merocyanine isomer can assume four different conformations for each of the trans (TTC, TTT, CTC, and CTT), shown in Figure 7a, and cis isomers (CCC, CCT, TCC, and TCT).^36-38 2D NMR revealed that the trans TTC and TTT were the predominant species for MC-1, MC-2, and MC-3. NOESY showed that the olefinic protons H-10 and H-11 are in trans configuration for all MC species because of the presence of NOE cross peaks between H-11 and H-19/20 and cross peaks between H-10 and H-21 (Figures 3, S13, and S22). These correlations are illustrated using grey dotted lines in Figure 7a. Moreover, the absence of NOE cross peaks between H-10 and H-19/20 and cross peaks H-11 and H-21 further supported the trans TTC and TTT being the two prevalent conformations for all three GSH-stabilized MC species. In order to differentiate between conformation TTC and TTT, NOE peaks H-10 and H-17 were analyzed against NOE peaks H-11 and H-17 for MC-1, MC-2 and MC-3. These correlations are illustrated using blue dotted lines in Figure 7a. Visual inspection of the NOE cross peaks suggest H-10 and H-17 is stronger than the NOE cross peaks H-11 and H-17 for MC-1 and MC-2 (Figure 7b,c). This is not completely apparent for MC-3 (Figure 7d). Therefore, NOE intensity values for these cross peaks were examined for all MC species. After analyzing these intensity values for the three MC species, it was found that conformation TTT was likely to be the thermodynamically favored conformation over TTC. This contrasts with the majority of merocyanine structures in the literature that show the cis conformation, with the negatively charged oxygen species on the same side as the positively charged indole. It is only possible to observe NOE between H-10 and H-17 when these protons are in trans to each other, supporting the TTT conformation.

Complete NMR (¹H and ¹³C) assignments for three GSH-stabilized MC species were achieved by applying 2D NMR techniques such as HSQC, HMBC, COSY, and NOESY. To obtain these stabilized species, GSH was introduced to a sample of the respective spiropyran SP-1, SP-2, or SP-3. This resulted in the isomerization of spiropyran to merocyanine, which ultimately gave rise to the fixed GSH-stabilized MC-1, MC-2, or MC-3. Identification of the methyl environments in the merocyanine forms was the first action taken during spectral analysis. From there, 2D NMR was utilized to locate neighboring environments. Once all NMR assignments were appointed, the investigation of the stereochemistry for the three GSH-stabilized MC species was examined. The presence (H-11 and H-19/20, H-10 and H-21) and absence (H-10 and H-19/20, H-11 and H-21) of NOE peaks supported the trans forms TTC and TTT being the two predominant species. Visual inspection and evaluation of the intensity values for NOE peaks between H-10 and H-17 versus H-11 and H-17 suggest trans TTT being the most favorable conformation for MC-1, MC-2, and MC-3. By studying the stereochemistry of these GSH-stabilized MC species we were able to provide a full characterization of the merocyanine forms in hopes of aiding in the structural understanding of new merocyanine species that are stabilized by external chemical stimuli.