13C-NMR Chemical Shifts in 1,3-Benzazoles as a Tautomeric Ratio Criterion

Benzimidazole is an important heterocyclic fragment, present in many biologically active compounds with a great variety of therapeutic purposes. Most of the benzimidazole activities are explained through the existence of 1,3-tautomeric equilibrium. As the binding affinity of each tautomer to a protein target depends on an established bioactive conformation, the effect of tautomers on the ligand protein binding mechanism is determinant. In this work, we searched and analyzed a series of reported 13C-NMR spectra of benzazoles and benzazolidine-2-thiones with the purpose of estimating their tautomeric equilibrium. Herein, several approaches to determine this problem are presented, which makes it a good initial introduction to the non-expert reader. This chemical shift difference and C4/C7 signals of benzimidazolidine-2-thione and 1-methyl-2-thiomethylbenzimidazole as references were used in this work to quantitatively calculate, in solution, the pyrrole–pyridine tautomeric ratio in equilibrium. The analysis will help researchers to correctly assign the chemical shifts of benzimidazoles and to calculate their intracyclic or exocyclic tautomeric ratio as well as mesomeric proportion in benzimidazoles.


Introduction
The tautomerism phenomenon is considered as a dynamic equilibrium between interconvertible structural isomers, named tautomers, with the migration of one atom or group. When a hydrogen atom migrates, the phenomenon is known as prototropy; however, other groups such as alkyl, aryl, acyl, cyano, halogens, amines, and nitro, as well as methals can migrate; Scheme 1 [1].

Introduction
The tautomerism phenomenon is considered as a dynamic equilibrium between interconvertible structural isomers, named tautomers, with the migration of one atom or group. When a hydrogen atom migrates, the phenomenon is known as prototropy; however, other groups such as alkyl, aryl, acyl, cyano, halogens, amines, and nitro, as well as methals can migrate; Scheme 1 [1]. Scheme 1. Tautomeric equilibria in organic compounds.
Nuclear magnetic resonance (NMR) spectroscopy is one of the most useful techniques to study tautomerism. The signals observed in the spectra depend on the activation energy between the tautomers, which determines their lifetimes and chemical shift difference (∆δ). When lifetimes are long, compared with 1/∆δ, a slow exchange regime gives rise to separate narrow signals for each of the tautomers. In this case, integration of the 1  In commercial databases, the presence of tautomeric duplicates has been found, and both tautomers are offered as different products [2]. On the other hand, different names for tautomers based on the IUPAC rules have been used [3]. In general, compounds with well-known tautomerism are named as the predominant tautomer at equilibrium. For example, 2-mercaptobenzimidazole (MBI) is named as benzimidazolidinethione (BIT) or 1,3-dihydro-2H-benzo[d]imidazole-2-thione (X = NH, Y = S); Figure 1.
The tautomeric interconversion with a low-energy barrier, in general, displays averaged signals in the NMR spectrum. To slow down the interconversion rate in solution, the following strategies are used: lowering temperature and/or the use of hexamethylphosphoramide-d18, which prevents the formation of hydrogen bonds. Thermodynamic and kinetic aspects of the tautomeric equilibrium have been explored using dynamic NMR studies [4][5][6] Because of the wide range of chemical shifts and high sensitivity, 15 N-NMR spectroscopy has been used to study the tautomerism of nitrogen in heteroaromatic compounds [7]. Solid-state NMR experiments [4] 4, X-ray crystallography [8- In commercial databases, the presence of tautomeric duplicates has been found, and both tautomers are offered as different products [2]. On the other hand, different names for tautomers based on the IUPAC rules have been used [3]. In general, compounds with well-known tautomerism are named as the predominant tautomer at equilibrium. For example, 2-mercaptobenzimidazole (MBI) is named as benzimidazolidinethione (BIT) or 1,3-dihydro-2H-benzo[d]imidazole-2-thione (X = NH, Y = S); Figure 1.
The tautomeric interconversion with a low-energy barrier, in general, displays averaged signals in the NMR spectrum. To slow down the interconversion rate insolution, the following strategies are used: lowering temperature and/or the use ofhexamethylphosphoramide-d 18 , which prevents the formation of hydrogen bonds. Thermodynamic and kinetic aspects of the tautomeric equilibrium have been explored using dynamic NMR studies [4][5][6] Because of the wide range of chemical shifts and high sensitivity, 15 N-NMR spectroscopy has been used to study the tautomerism of nitrogen in heteroaromatic compounds [7]. Solid-state NMR experiments [4] 4, X-ray crystallography [8][9][10], and 15 N-NMR studies in DMSOd 6 for both BIT and 1-MeBIT revealed that the thione form is the predominant tautomer [11]. Recently, Pandey et al. theoretically studied 5-MeOBIT using B3LYP methods with a 6-311++G (d, p) basis set. A comparison between the experimental and calculated structure as well as the calculated and experimental 1 H and 13 C chemical shifts showed a good correlation with the thione isomer [12].
The binding affinity of each tautomer to a protein target depends on an established bioactive conformation. Therefore, the method proposed herein to calculate in solution the prototropic ratio in BZs, on the basis of the electronic effect of pyrrole like atom (NH = N pr ) and pyridine like atom (N pd ) on C4 and C7 chemical shift resonances, is very valuable. On the other hand, the method is also helpful to correctly assign the carbon atoms on the benzene ring.

Influence of Benzene Ring Substituents in the 13 C NMR Chemical Shifts
For clarity reasons, a brief introduction about the inductive and electronic effects of heteroatoms (X = N, O, S) on the 13 C chemical shifts (δ) of the benzene ring [25] is required. Then, it is interesting to note that both NH 2 and OH groups deshield the carbon atom to which they are attached, C i (ipso), in 18.2 and 26.9 ppm, respectively, owing to a strong inductive effect. Conversely, the electronic delocalization of the free electron pairs of N and O atoms produces a considerable shielding electronic effect on C o (ortho) in 13.4 and 12.8 ppm, respectively, and C p (para) in 10.0 and 7.4 ppm, respectively. The SH group shifts C i , C o , and C m (meta) to high frequencies in only 2.1 ppm, 0.7 ppm, and 0.3 ppm, respectively, but C p to low frequency by 3.2 ppm. All comparisons are referenced to the δ of the benzene ring (128.0 ppm).
The binding affinity of each tautomer to a protein target depends on an established bioactive conformation. Therefore, the method proposed herein to calculate in solution the prototropic ratio in BZs, on the basis of the electronic effect of pyrrole like atom (NH = Npr) and pyridine like atom (Npd) on C4 and C7 chemical shift resonances, is very valuable. On the other hand, the method is also helpful to correctly assign the carbon atoms on the benzene ring.

Influence of Benzene Ring Substituents in the 13 C NMR Chemical Shifts
For clarity reasons, a brief introduction about the inductive and electronic effects of heteroatoms (X = N, O, S) on the 13 C chemical shifts () of the benzene ring [25] is required. Then, it is interesting to note that both NH2 and OH groups deshield the carbon atom to which they are attached, Ci (ipso), in 18.2 and 26.9 ppm, respectively, owing to a strong inductive effect. Conversely, the electronic delocalization of the free electron pairs of N and O atoms produces a considerable shielding electronic effect on Co (ortho) in 13.4 and 12.8 ppm, respectively, and Cp (para) in 10.0 and 7.4 ppm, respectively. The SH group shifts Ci, Co, and Cm (meta) to high frequencies in only 2.1 ppm, 0.7 ppm, and 0.3 ppm, respectively, but Cp to low frequency by 3.2 ppm. All comparisons are referenced to the  of the benzene ring (128.0 ppm).
In this sense, the C4 in the dibenzofused heteroaromatic compounds 1-3 (Table 1) appears at 111.6, 110.8, and 122.9 ppm for X = O, NH, and S, respectively [25]. As expected, the inductive effect of the heteroatom on Ci shifts  to high frequencies, in agreement with the electronegativity of the heteroatom.

Effect of Heteroatoms on 13 C-NMR of Benzazoles
From the reported 13 C NMR data, listed in Table 2, it can be seen that heteroatom (X) exerts an inductive effect, deshielding C2, C3a, and C7a; Figure 2. On the contrary, the lone electron pair (lep) of the heteroatom X exerts a protective effect, shielding C7 through the electronic delocalization of the lep into the benzofused ring. In the case of BO (1), the oxygen atom shifts C7 to 110.8 ppm, while C4 at 120.5 ppm indicates that N3 (pyridine like nitrogen) exerts a very small electronic effect on this carbon because the lep are located out of the conjugated π-electron system. In BT 2, the δ of C7 at 123.6 and δC4 at 121.8 ppm are indicative of the very small shielding effect known for sulfur and pyridine like nitrogen atoms. For BI 3, X = NH, a set of four signals appear in the 13 C NMR spectra, independently from the solvent used. The observed C2 symmetry is the result of tautomeric equilibrium, making C5 magnetically equivalent to C6, C4 to C7, and C3a to C7a. In BIs, the tautomeric species are the result of intermolecular proton transfer in solution from N1 to N3. Then, the signals of C4 and C7 appear at approximately 115.0 ppm, an intermediate δ value between a pyridine like nitrogen (N3) and pyrrole like nitrogen (N1). This observation can be interpreted as both nitrogen atoms in BIs being present in approximately a 1:1 proportion.

Effect of Heteroatoms on 13 C-NMR of Benzazoles
From the reported 13 C NMR data, listed in Table 2, it can be seen that heteroatom (X) exerts an inductive effect, deshielding C2, C3a, and C7a; Figure 2. On the contrary, the lone electron pair (lep) of the heteroatom X exerts a protective effect, shielding C7 through the electronic delocalization of the lep into the benzofused ring. In the case of BO (1), the oxygen atom shifts C7 to 110.8 ppm, while C4 at 120.5 ppm indicates that N3 (pyridine like nitrogen) exerts a very small electronic effect on this carbon because the lep are located out of the conjugated π-electron system. In BT 2, the  of C7 at 123.6 and C4 at 121.8 ppm are indicative of the very small shielding effect known for sulfur and pyridine like nitrogen atoms. For BI 3, X = NH, a set of four signals appear in the 13 C NMR spectra, independently from the solvent used. The observed C2 symmetry is the result of tautomeric equilibrium, making C5 magnetically equivalent to C6, C4 to C7, and C3a to C7a. In BIs, the tautomeric species are the result of intermolecular proton transfer in solution from N1 to N3. Then, the signals of C4 and C7 appear at approximately 115.0 ppm, an intermediate  value between a pyridine like nitrogen (N3) and pyrrole like nitrogen (N1). This observation can be interpreted as both nitrogen atoms in BIs being present in approximately a 1:1 proportion.  In general, in BIs 3, the tautomeric equilibrium is slowed down at low temperatures and/or the increasing polarity of solvents. In polar solvents, the equilibrium exchanging rate decreases and the four signals of BIs are broadened or sometimes disappear from the spectra [4]. The use of the high polar solvent HMPA-d8 makes the equilibrium exchange slow enough to observe seven signals of the most stable tautomer, in the 13 C-NMR spectrum. For instance, the spectrum of BI, recorded in HMPA-d8, shows two different broad signals for C4 and C7 in 118.7 and 110.9 ppm, respectively, in close correspondence with the observed values in the solid state (CPMAS), at 119.9 and 112.2 ppm, respectively [4].
Within the above exposed background and the analysis of reported 13 C-NMR data, herein, we expose the usefulness of C4 and C7 chemical shifts as a criterion to estimate the position of the tautomeric equilibrium in BIs.

Effect of Heteroatoms on 13 C-NMR of Benzazoles
From the reported 13 C NMR data, listed in Table 2, it can be seen that heteroatom (X exerts an inductive effect, deshielding C2, C3a, and C7a; Figure 2. On the contrary, th lone electron pair (lep) of the heteroatom X exerts a protective effect, shielding C7 throug the electronic delocalization of the lep into the benzofused ring. In the case of BO (1), th oxygen atom shifts C7 to 110.8 ppm, while C4 at 120.5 ppm indicates that N3 (pyridin like nitrogen) exerts a very small electronic effect on this carbon because the lep are locate out of the conjugated π-electron system. In BT 2, the  of C7 at 123.6 and C4 at 121.8 ppm are indicative of the very small shielding effect known for sulfur and pyridine like nitro gen atoms. For BI 3, X = NH, a set of four signals appear in the 13 C NMR spectra, inde pendently from the solvent used. The observed C2 symmetry is the result of tautomer equilibrium, making C5 magnetically equivalent to C6, C4 to C7, and C3a to C7a. In BI the tautomeric species are the result of intermolecular proton transfer in solution from N to N3. Then, the signals of C4 and C7 appear at approximately 115.0 ppm, an intermediat  value between a pyridine like nitrogen (N3) and pyrrole like nitrogen (N1). This obse vation can be interpreted as both nitrogen atoms in BIs being present in approximately 1:1 proportion.  In general, in BIs 3, the tautomeric equilibrium is slowed down at low temperature and/or the increasing polarity of solvents. In polar solvents, the equilibrium exchangin rate decreases and the four signals of BIs are broadened or sometimes disappear from th spectra [4]. The use of the high polar solvent HMPA-d8 makes the equilibrium exchang slow enough to observe seven signals of the most stable tautomer, in the 13 C-NMR spec trum. For instance, the spectrum of BI, recorded in HMPA-d8, shows two different broa signals for C4 and C7 in 118.7 and 110.9 ppm, respectively, in close correspondence wit the observed values in the solid state (CPMAS), at 119.9 and 112.2 ppm, respectively [4] Within the above exposed background and the analysis of reported 13 C-NMR dat herein, we expose the usefulness of C4 and C7 chemical shifts as a criterion to estimat the position of the tautomeric equilibrium in BIs. In general, in BIs 3, the tautomeric equilibrium is slowed down at low temperatures and/or the increasing polarity of solvents. In polar solvents, the equilibrium exchanging rate decreases and the four signals of BIs are broadened or sometimes disappear from the spectra [4]. The use of the high polar solvent HMPA-d 8 makes the equilibrium exchange slow enough to observe seven signals of the most stable tautomer, in the 13 C-NMR spectrum. For instance, the spectrum of BI, recorded in HMPA-d 8 , shows two different broad signals for C4 and C7 in 118.7 and 110.9 ppm, respectively, in close correspondence with the observed values in the solid state (CPMAS), at 119.9 and 112.2 ppm, respectively [4].
Within the above exposed background and the analysis of reported 13 C-NMR data, herein, we expose the usefulness of C4 and C7 chemical shifts as a criterion to estimate the position of the tautomeric equilibrium in BIs.

Estimation of the Anular Tautomerism on Benzimidazol
The chemical shift reference values for the estimation of the tautomeric equilibrium were set using the 1-methyl-benzimidazole (1-MeBI, 4) as a model compound. In case 4, the tautomeric equilibrium is non-existent; therefore, a set of seven narrow signals are observed in CDCl 3 . The δC4 appears at 120.4 ppm, characteristic of an N pd effect, whereas the δC7 at 109.5 ppm is characteristic of an N pr effect. In DMSOd 6 , the δC4 and δC7 values are 119.2 and 110.1, respectively. Then, a good approximation for 100% of the pyridine like character of δC4 and 100% of the pyrrole like character of δC7 is 120.0 and 110.0 ppm, respectively. Furthermore, the tautomeric proportion in BIs can be calculated on a base equal to 120.0 ppm for δC4 ref (N pd ) and 110.0 ppm for δC7 ref (N pr ) as reference values; Figure 2.
The tautomerism in BIs can be calculated considering that the observed chemical shift δ obsC4/C7 is the result of the averaged C4 and C7 chemical shifts weighted by the respective molar fraction contributions x pd (pyridine character) and x pr (pyrrolic character). Equations (1)-(3) can be used to calculate the molar fractions of each tautomer; Figure 3.

Estimation of the Anular Tautomerism on Benzimidazol
The chemical shift reference values for the estimation of the tautomeric equilibrium were set using the 1-methyl-benzimidazole (1-MeBI, 4) as a model compound. In case 4, the tautomeric equilibrium is non-existent; therefore, a set of seven narrow signals are observed in CDCl3. The C4 appears at 120.4 ppm, characteristic of an Npd effect, whereas the C7 at 109.5 ppm is characteristic of an Npr effect. In DMSOd6, the C4 and C7 values are 119.2 and 110.1, respectively. Then, a good approximation for 100% of the pyridine like character of C4 and 100% of the pyrrole like character of C7 is 120.0 and 110.0 ppm, respectively. Furthermore, the tautomeric proportion in BIs can be calculated on a base equal to 120.0 ppm for C4ref (Npd) and 110.0 ppm for C7ref (Npr) as reference values; The tautomerism in BIs can be calculated considering that the observed chemical shift obsC4/C7 is the result of the averaged C4 and C7 chemical shifts weighted by the respective molar fraction contributions xpd (pyridine character) and xpr (pyrrolic character). Equations (1)-(3) can be used to calculate the molar fractions of each tautomer; Figure 3. From the reported 13 C data for BI 3, as well as their protonated and deprotonated derivatives, in DMSOd6 as solvent, Table 3 [30], the tautomeric proportion can be calculated. For BI, the obs value is 116.3, then the estimation is 63% of the pyridine character (+N3H). In the case of obs for protonated BI (118.3 ppm), a small effect of positive charge on nitrogen atoms shifts this signal to high frequencies in approximately 2.0 ppm. On the contrary, any effect of the negative charge on nitrogen in deprotonated BI was observed.  From the reported 13 C data for BI 3, as well as their protonated and deprotonated derivatives, in DMSOd 6 as solvent, Table 3 [30], the tautomeric proportion can be calculated. For BI, the δ obs value is 116.3, then the estimation is 63% of the pyridine character (+N3H). In the case of δ obs for protonated BI (118.3 ppm), a small effect of positive charge on nitrogen atoms shifts this signal to high frequencies in approximately 2.0 ppm. On the contrary, any effect of the negative charge on nitrogen in deprotonated BI was observed.

Estimation of the Anular Tautomerism on Benzimidazol
The chemical shift reference values for the estimation of the tautomeric equilibrium were set using the 1-methyl-benzimidazole (1-MeBI, 4) as a model compound. In case 4, the tautomeric equilibrium is non-existent; therefore, a set of seven narrow signals are observed in CDCl3. The C4 appears at 120.4 ppm, characteristic of an Npd effect, whereas the C7 at 109.5 ppm is characteristic of an Npr effect. In DMSOd6, the C4 and C7 values are 119.2 and 110.1, respectively. Then, a good approximation for 100% of the pyridine like character of C4 and 100% of the pyrrole like character of C7 is 120.0 and 110.0 ppm, respectively. Furthermore, the tautomeric proportion in BIs can be calculated on a base equal to 120.0 ppm for C4ref (Npd) and 110.0 ppm for C7ref (Npr) as reference values; Figure 2.
The tautomerism in BIs can be calculated considering that the observed chemical shift obsC4/C7 is the result of the averaged C4 and C7 chemical shifts weighted by the respective molar fraction contributions xpd (pyridine character) and xpr (pyrrolic character). Equations (1)-(3) can be used to calculate the molar fractions of each tautomer; Figure 3. From the reported 13 C data for BI 3, as well as their protonated and deprotonated derivatives, in DMSOd6 as solvent, Table 3 [30], the tautomeric proportion can be calculated. For BI, the obs value is 116.3, then the estimation is 63% of the pyridine character (+N3H). In the case of obs for protonated BI (118.3 ppm), a small effect of positive charge on nitrogen atoms shifts this signal to high frequencies in approximately 2.0 ppm. On the contrary, any effect of the negative charge on nitrogen in deprotonated BI was observed. This approximation can be applied to 2-aminomethylbenzimidazole (2-AMBI); Scheme 2. In the work of Sierra-Centeno et al. [31], the acid-base equilibrium constants of 2AMBI were determined in aqueous solutions at 25 • C through 13 C NMR spectroscopy, potentiometric and spectrophotometric techniques, as well as through theoretical methods. 13 C NMR spectra were recorded for solutions of 0.1 M of 2-AMBI at pH values from 1.1 to 13.2, containing 10 % v/v of D 2 O. During the titration process, only five signals were observed. The sets of C5 and C6 signals were shifted approximately 7.0 ppm to lower frequencies and C2 and C3a/C7a in 10.0 and 15.5 ppm, respectively, to higher frequencies, whereas the set of C4/C7 signals remained almost constant at 114.5 ppm. Therefore, the BI heterocycle retains its 45% pyridine character independently from the pH. This approximation can be applied to 2-aminomethylbenzimidazole (2-AMBI); Scheme 2. In the work of Sierra-Centeno et al. [31], the acid-base equilibrium constants of 2AMBI were determined in aqueous solutions at 25 °C through 13 C NMR spectroscopy, potentiometric and spectrophotometric techniques, as well as through theoretical methods. 13 C NMR spectra were recorded for solutions of 0.1 M of 2-AMBI at pH values from 1.1 to 13.2, containing 10 % v/v of D2O. During the titration process, only five signals were observed. The sets of C5 and C6 signals were shifted approximately 7.0 ppm to lower frequencies and C2 and C3a/C7a in 10.0 and 15.5 ppm, respectively, to higher frequencies, whereas the set of C4/C7 signals remained almost constant at 114.5 ppm. Therefore, the BI heterocycle retains its 45% pyridine character independently from the pH.

Effects of the Contribution of Mesomeric Structures in 1-Substituted Benzimidazoles
This approximation can also be used to estimate the participation of mesomeric forms to the structure. Approximately the same pyrrole effect on C7 (109.5 ppm) for 1-MeBI 4 compared with C7 (110.4 ppm) of 1-phBI 5 in CDCl3 was observed; Table 4. However, in the spectrum of 5 obtained in DMSOd6, C7 appeared in 116.5 ppm, which can be explained as a solvent effect that stabilizes the electronic delocalization of the lep of N1 to the Nphenyl ring through the mesomeric form 5b, deshielding C7. Considering the C7 value of 110 ppm to the resonance form 5a, the contribution of the resonance form 5b is estimated as 35% in DMSOd6. This effect is also observed for acylBI 6 even in CDCl3, whose mesomeric structures can be represented as 6a and 6b (approximately 50%).

Effects of the Contribution of Mesomeric Structures in 1-Substituted Benzimidazoles
This approximation can also be used to estimate the participation of mesomeric forms to the structure. Approximately the same pyrrole effect on C7 (109.5 ppm) for 1-MeBI 4 compared with C7 (110.4 ppm) of 1-phBI 5 in CDCl 3 was observed; Table 4. However, in the spectrum of 5 obtained in DMSOd 6 , C7 appeared in 116.5 ppm, which can be explained as a solvent effect that stabilizes the electronic delocalization of the lep of N1 to the Nphenyl ring through the mesomeric form 5b, deshielding C7. Considering the δC7 value of 110 ppm to the resonance form 5a, the contribution of the resonance form 5b is estimated as 35% in DMSOd 6 . This effect is also observed for acylBI 6 even in CDCl 3 , whose mesomeric structures can be represented as 6a and 6b (approximately 50%). This approximation can be applied to 2-aminomethylbenzimidazole (2-AMBI); Scheme 2. In the work of Sierra-Centeno et al. [31], the acid-base equilibrium constants of 2AMBI were determined in aqueous solutions at 25 °C through 13 C NMR spectroscopy, potentiometric and spectrophotometric techniques, as well as through theoretical methods. 13 C NMR spectra were recorded for solutions of 0.1 M of 2-AMBI at pH values from 1.1 to 13.2, containing 10 % v/v of D2O. During the titration process, only five signals were observed. The sets of C5 and C6 signals were shifted approximately 7.0 ppm to lower frequencies and C2 and C3a/C7a in 10.0 and 15.5 ppm, respectively, to higher frequencies, whereas the set of C4/C7 signals remained almost constant at 114.5 ppm. Therefore, the BI heterocycle retains its 45% pyridine character independently from the pH.

Effects of the Contribution of Mesomeric Structures in 1-Substituted Benzimidazoles
This approximation can also be used to estimate the participation of mesomeric forms to the structure. Approximately the same pyrrole effect on C7 (109.5 ppm) for 1-MeBI 4 compared with C7 (110.4 ppm) of 1-phBI 5 in CDCl3 was observed; Table 4. However, in the spectrum of 5 obtained in DMSOd6, C7 appeared in 116.5 ppm, which can be explained as a solvent effect that stabilizes the electronic delocalization of the lep of N1 to the Nphenyl ring through the mesomeric form 5b, deshielding C7. Considering the C7 value of 110 ppm to the resonance form 5a, the contribution of the resonance form 5b is estimated as 35% in DMSOd6. This effect is also observed for acylBI 6 even in CDCl3, whose mesomeric structures can be represented as 6a and 6b (approximately 50%).

Effects of the Contribution of Tautomers in 2-Substituted Benzimidazoles
In general, the δC2 in 13 C NMR spectra for 2-alkyl-benzimidazoles (2-AkBIs) 7 is shifted to higher frequencies in 10-15 ppm compared with C2 in BI; Table 5. Moreover, C3a/C7a and C4/C7 appeared as broad signals with a tendency to coalesce with an increasing alkyl chain length [35].
The spectra of 2-MeBI 7 in CDCl 3 showed a set of four sharp signals for the carbons of the BI moiety, characteristic of a tautomeric equilibrium. In contrast, the spectrum of 2-MeBI 7 in DMSOd 6 showed a set of seven signals, characteristic of a non-symmetric BI ring. Because of the very slow dynamic phototropic equilibrium, δC4 and δC7 (117.8/110.5) appeared as broad separated signals, which indicate that 7a is anchoring to differentiate both pyridine and pyrrole nitrogen atoms. In these conditions, C3a and C7a are shifted 5.0 and 4.0 ppm to higher frequencies, respectively, compared with the spectra obtained in CDCl 3 .
In the case of 2-acylBIs 8, the formation of an NH···O hydrogen bond (HB) anchors the molecule and the spectra showed a set of seven signals, being δC4 and δC7 at 122.0 and 112.0 ppm, respectively, characteristic of pyridine and pyrrole nitrogen atoms.
The 2-carboxylateBI 9 showed four signals of symmetrical BI in the 13 C NMR spectra, whose tautomeric equilibrium 9a = 9b = 9c = 9d is demonstrated with the presence of the averaged value of C4/C7 signal in 116.3 ppm (about 60% pyridine nitrogen atom character 9a = 9b). This result is in accordance with the X-ray diffraction structure of this molecule found as a zwitterionic form in the solid state [36,37].

Effects of the Contribution of Tautomers in 2-Substituted Benzimidazoles
In general, the C2 in 13 C NMR spectra for 2-alkyl-benzimidazoles (2-AkBIs) 7 is shifted to higher frequencies in 10-15 ppm compared with C2 in BI; Table 5. Moreover, C3a/C7a and C4/C7 appeared as broad signals with a tendency to coalesce with an increasing alkyl chain length [35].
The spectra of 2-MeBI 7 in CDCl3 showed a set of four sharp signals for the carbons of the BI moiety, characteristic of a tautomeric equilibrium. In contrast, the spectrum of 2-MeBI 7 in DMSOd6 showed a set of seven signals, characteristic of a non-symmetric BI ring. Because of the very slow dynamic phototropic equilibrium, C4 and C7 (117.8/110.5) appeared as broad separated signals, which indicate that 7a is anchoring to differentiate both pyridine and pyrrole nitrogen atoms. In these conditions, C3a and C7a are shifted 5.0 and 4.0 ppm to higher frequencies, respectively, compared with the spectra obtained in CDCl3.
In the case of 2-acylBIs 8, the formation of an NH•••O hydrogen bond (HB) anchors the molecule and the spectra showed a set of seven signals, being C4 and C7 at 122.0 and 112.0 ppm, respectively, characteristic of pyridine and pyrrole nitrogen atoms.
The 2-carboxylateBI 9 showed four signals of symmetrical BI in the 13 C NMR spectra, whose tautomeric equilibrium 9a = 9b = 9c = 9d is demonstrated with the presence of the averaged value of C4/C7 signal in 116.3 ppm (about 60% pyridine nitrogen atom character 9a = 9b). This result is in accordance with the X-ray diffraction structure of this molecule found as a zwitterionic form in the solid state [36,37].

Tautomers and Mesomers in Benzothiazolidine Tione.
Benzazolidine-2-thiones (BZTs) 10-13 are constituted by a benzene ring fused to oxazolidine-, thiazolidine-, or imidazolidine-2-thione rings. The tautomeric equilibria between thioamide (NHC=S) and iminethiol (NCSH), thione = thiol, in compounds 10-13 are shown in the scheme on Table 6. In these tautomeric equilibria, a hydrogen atom is exchanging from the endocyclic nitrogen to the exocyclic sulfur atom, with the consequent transformation of the endocyclic nitrogen atom from the pyrrole to pyridine character. This phenomenon has been described in the solid state [8,[42][43][44][45][46]. Thermodynamic studies in solution have concluded that tautomeric equilibrium is shifted to the thione form in substituted benzene compounds [47][48][49]. The BZTs 10-13 and their corresponding 2-S-Methyl-benzazoles 14-17 (TMBZ) are interesting compounds with biological activity [50]. Table 6. 13   It has been established that compound 12 predominantly exists as the thione (BIT) form in polar solvents and in the solid state [8]. The proton is bonded to the more electronegative atom (NH) in the molecule. The formation of a strong NH hydrogen bond with the solvent or another molecule of 12 is preferred instead of the S-H bond [11]. Model compounds, whose tautomeric equilibria has been locked with an S-Me, compound 14-17, have been used to qualitatively estimate the tautomeric ratio. In addition, the formation of metallic complexes to stabilize the tautomers has also been reported [55,56]. It has been established that compound 12 predominantly exists as the thione (BIT) form in polar solvents and in the solid state [8]. The proton is bonded to the more electronegative atom (NH) in the molecule. The formation of a strong NH hydrogen bond with the solvent or another molecule of 12 is preferred instead of the S-H bond [11]. Model compounds, whose tautomeric equilibria has been locked with an S-Me, compound 14-17, have been used to qualitatively estimate the tautomeric ratio. In addition, the formation of metallic complexes to stabilize the tautomers has also been reported [55,56].
In the 13 C-NMR spectra of BIT 12, four signals were found in DMSOd 6 , in agreement with the thione tautomer 12a. The X-ray diffraction structure reported for 12a agrees with these results [42].
On these bases, the observed C4/C7 chemical shift at 109.6 ppm of BIT 12a is a good reference value for the pyrrole tautomer δC7 ref . On the other hand, δC4 of TMBI 16 at −65 • C can be used as the pyridine reference. Despite that a set of seven signals appeared in the spectra at −65 • C, Table 7, the system is not completely frozen because δC4 obs and δC7 obs were at 117.8 and 110.9 ppm, respectively [57]. However, these data can be extrapolated to determine δC4 ref ; for this, we used the δC7 ref reference value (109.6 ppm) to calculate the difference: δC7 obs − δC7 ref (110.9 − 109.6 = 1.3 ppm), which was used to determine the δC4 ref = δC4 obs + 1.3 = 117.8 + 1.3 = 119.1 ppm. the thioamide form (NHC=S): BOT 10 (100%), BTT 11 (99%), BIT 12 (92%), and 1-MeBIT 13 (98%). However, because this tautomeric ratio depends on the temperature, concentration, and solvent polarity, these values could change with the experimental conditions. The analysis of thioamide/iminothiol tautomerism has been performed on the bases of the reported 13 C-NMR spectra of BZTs 10-13 and their respective TMBZs 14-17; Table  6. In the 13 C-NMR spectra of BIT 12, four signals were found in DMSOd6, in agreement with the thione tautomer 12a. The X-ray diffraction structure reported for 12a agrees with these results [42].
On these bases, the observed C4/C7 chemical shift at 109.6 ppm of BIT 12a is a good reference value for the pyrrole tautomer C7ref. On the other hand, C4 of TMBI 16 at −65 °C can be used as the pyridine reference. Despite that a set of seven signals appeared in the spectra at −65 °C, Table 7, the system is not completely frozen because C4obs and C7obs were at 117.8 and 110.9 ppm, respectively [57]. However, these data can be extrapolated to determine C4ref; for this, we used the C7ref reference value (109.6 ppm) to calculate the difference: δC7obs − δC7ref (110.9 − 109.6 = 1.3 ppm), which was used to determine the δC4ref = δC4obs + 1.3 = 117.8 + 1.3 = 119.1 ppm. The thioamide/iminothiol tautomerism of BZTs 10-13 can be estimated with the equation proposed by Balestrero et al. [11] for 15 N-NMR data, applied to 13 C chemical shift using values of δC4ref and δC7ref, obtained from reference model compounds weighted by the respective molar fractions, as stated through previously described Equations (1)-(3).
The spectra of TMBI 16 were obtained in CDCl3 and DMSOd6 at 27 °C. In both cases, only four signals were in the spectra, indicating the presence of a symmetric structure in tautomeric equilibrium; Table 7. The use of Equation (2) to calculate each tautomeric ratio indicates that compound 16 is present in 57% in CDCl3 and 58% in DMSOd6 as the NH tautomer. The spectra of compound 16 obtained in DMF-d7 at −65 °C showed a set of six separated signals, and C4 and C7 were observed in 117. 8  The thioamide/iminothiol tautomerism of BZTs 10-13 can be estimated with the equation proposed by Balestrero et al. [11] for 15 N-NMR data, applied to 13  The spectra of TMBI 16 were obtained in CDCl 3 and DMSOd 6 at 27 • C. In both cases, only four signals were in the spectra, indicating the presence of a symmetric structure in tautomeric equilibrium; Table 7. The use of Equation (2) to calculate each tautomeric ratio indicates that compound 16 is present in 57% in CDCl 3 and 58% in DMSOd 6 as the NH tautomer. The spectra of compound 16 obtained in DMF-d 7 at −65 • C showed a set of six separated signals, and C4 and C7 were observed in 117.8 and 110.9 ppm, respectively. In this case, from δC4 obs = 117.8 ppm, the N tautomer is calculated in 86.3%, whereas from δC7 obs = 110.9 ppm, the NH tautomer is calculated in only 13.7%. The same references were used to calculate the tautomeric ratio of compounds 10-17; Table 6.  In the 13 C-NMR spectrum of BOT 10 in CDCl3, the oxygen and nitrogen ato a shielding electronic effect, shifting C7 to 110.5 ppm and C4 to 110.2 ppm. In C4ref = 120.5 referenced to BO and C7ref = 109.6 ppm referenced to BIT 12, a Equation (2) for compound 10 with C4obs = 110.2 resulted in 95% of the pyrrolic c Then, the endocyclic nitrogen atom is mainly tautomer 10a, Table 6, near to that c by Balestrero et al. in 1986 [11]. In DMSOd6, C4 appeared at higher frequencie chloroform, because of the change in the tautomeric mixture of the pyrrole 10a ( pyridine 10b (33%) character, evaluated through C4obs = 113.2 ppm.
In the same solvent, for BTT 11, no considerable shielding electronic effect atom was observed on C7 (121.6 ppm) compared with that of BT 2 (123.6 ppm). H the nitrogen atom shifted C4 to 112.3 ppm compared with 121.8 ppm in BT 2 allowing to estimate 77% of pyrrole tautomer 11a. In the case of the TMBZs 14, 1 their C4 observed values were found in 118.1, 121.7, and 117.4 ppm, respectivel into account the C4 (119.1 ppm) and C7 (109.6 ppm) reference values, the prop their mesomeric form b with pyrrole negative nitrogen atoms was calculated a proportion in 10.5%, 2.4%, and 17.9%, respectively, in DMSO-d6; Table 6.
Braun et al. [59] reported the synthesis of 1-EtBIT, analogous to compound of seven signals were found in the spectra of this compound in DMSOd6; the shifts of C3a and C7a are at 131.9 and 130.7, C4 and C7 are at 109.6/109.3, C5 a 122.2 and 122.1, and C2 = S at 167.6 ppm, respectively. We can see that the two atoms are in pyrrole form, indicating the NH tautomer to be present in 100%.
Palmer et al. [58] synthetized 1-alkylbenzimidazolidine-2-selenones (1-Ak and 19 and their corresponding diselenides 20 and 21. In the X-ray diffraction s analysis of 1-MeBISe 18 and 1t BuBISe 19, the hydrogen atom was located on th (NH), demonstrating the structure to be the selenone tautomer. The density f theory (B3LYP) calculations indicated that selenones are more stable than the se tomers. Moreover, these data indicated that the selone is best represented as a C + terion instead of as a C=Se double bond. This was confirmed in the 13 C-NMR obtained in DMSOd6 (Table 7); both N1 and N3 atoms in 1-MeBISe 18 are main role, producing the protecting effect on C4/C7 (110.0 ppm). Mesomer 18a was c in 95.8% in equilibrium with the mesomer 18b (4.2%). On the other hand, the t B In the 13 C-NMR spectrum of BOT 10 in CDCl 3 , the oxygen and nitrogen atoms exert a shielding electronic effect, shifting C7 to 110.5 ppm and C4 to 110.2 ppm. In this case, δC4 ref = 120.5 referenced to BO and δC7 ref = 109.6 ppm referenced to BIT 12, and using Equation (2) for compound 10 with δC4 obs = 110.2 resulted in 95% of the pyrrolic character. Then, the endocyclic nitrogen atom is mainly tautomer 10a, Table 6, near to that calculated by Balestrero et al. in 1986 [11]. In DMSOd 6 , δC4 appeared at higher frequencies than in chloroform, because of the change in the tautomeric mixture of the pyrrole 10a (67%) and pyridine 10b (33%) character, evaluated through δC4 obs = 113.2 ppm.
In the same solvent, for BTT 11, no considerable shielding electronic effect of sulfur atom was observed on C7 (121.6 ppm) compared with that of BT 2 (123.6 ppm). However, the nitrogen atom shifted C4 to 112.3 ppm compared with 121.8 ppm in BT 2 (δC4Nr), allowing to estimate 77% of pyrrole tautomer 11a. In the case of the TMBZs 14, 15, and 17, their C4 observed values were found in 118.1, 121.7, and 117.4 ppm, respectively. Taking into account the C4 (119.1 ppm) and C7 (109.6 ppm) reference values, the proportion of their mesomeric form b with pyrrole negative nitrogen atoms was calculated as a minor proportion in 10.5%, 2.4%, and 17.9%, respectively, in DMSO-d 6 ; Table 6.
Braun et al. [59] reported the synthesis of 1-EtBIT, analogous to compound 13. A set of seven signals were found in the spectra of this compound in DMSOd 6 ; the chemical shifts of C3a and C7a are at 131.9 and 130.7, C4 and C7 are at 109.6/109.3, C5 and C6 in 122.2 and 122.1, and C2 = S at 167.6 ppm, respectively. We can see that the two nitrogen atoms are in pyrrole form, indicating the NH tautomer to be present in 100%.
Palmer et al. [58] synthetized 1-alkylbenzimidazolidine-2-selenones (1-AkBISe) 18 and 19 and their corresponding diselenides 20 and 21. In the X-ray diffraction structural analysis of 1-MeBISe 18 and 1-t BuBISe 19, the hydrogen atom was located on the N atom (NH), demonstrating the structure to be the selenone tautomer. The density functional theory (B3LYP) calculations indicated that selenones are more stable than the selenol tautomers. Moreover, these data indicated that the selone is best represented as a C + -Sezwitterion instead of as a C=Se double bond. This was confirmed in the 13 C-NMR spectrum obtained in DMSOd 6 ( Table 7); both N1 and N3 atoms in 1-MeBISe 18 are mainly as pyrrole, producing the protecting effect on C4/C7 (110.0 ppm). Mesomer 18a was calculated in 95.8% in equilibrium with the mesomer 18b (4.2%). On the other hand, the t BuBISe 19 showed separate signals for C4/C7 (114.4/109.9 ppm), indicating, on this basis, the reference values (119.1 and 109.6 ppm) and, for C4 obs , the presence of mesomeric forms 19a/19b in a 49.5/50.5 ratio. Eventually, mesomers 18c and 19c are present in a minor proportion. In the case of diselenides 20 and 21, C7 in 120.4 and 118.8 ppm is indicative that NR atoms are principally the pyridine type. Then, their δC4 chemical shifts at 110.5 and 114.1 ppm, respectively, show the pyridine character of N3. On the basis of these observed chemical shifts, mesomers 20a/20b was calculated in a 90.5/9.5% ratio and 21a/21b in a 52.7/47.3% ratio. The presence of negative charge on N3 in tautomers 20b and 21b was confirmed because C3a is shifted approximately 13.0 ppm to higher frequencies, as the same carbon atom (~147.0 ppm) was shifted 8.0 ppm in the deprotonated BI spectrum in Table 3.

Mesomerism in N1-acyl-1,3-benzoheterazolidine-2-thiones
Some examples about N1-acyl-BZT 22-25 were found in the literature and the 13 C NMR data are listed in Table 8. In the case of compound 22, δC4 at 115.8 ppm indicates the presence of the mesomers 22a/22b in approximately a 65/35 proportion. However, compound 23 shows the presence of two separated signals for C4 and C7 in 114.3 and 108.5 ppm, respectively. The last signal corresponds to a pyrrole N1H atom (tautomer 23a). The signal in 114.3 ppm indicates that this molecule is in the mesomeric forms 23a and 23b, which contribute to the whole structure in approximately a 50/50 proportion. The same mesomers in approximately a 67/43 proportion were observed for compound 24a/24b (C4 in 115.0 ppm, C7 in 109.4 ppm). In compound 25, four signals of a symmetric BI ring were observed on the spectra C4/C7 in 113.2 ppm, indicating the mesomer 22a is present in a major proportion. In the case of diselenides 20 and 21, C7 in 120.4 and 118.8 ppm is indicative that NR ato are principally the pyridine type. Then, their C4 chemical shifts at 110.5 and 114.1 pp respectively, show the pyridine character of N3. On the basis of these observed chemi shifts, mesomers 20a/20b was calculated in a 90.5/9.5% ratio and 21a/21b in a 52.7/47. ratio. The presence of negative charge on N3 in tautomers 20b and 21b was confirm because C3a is shifted approximately 13.0 ppm to higher frequencies, as the same carb atom (~147.0 ppm) was shifted 8.0 ppm in the deprotonated BI spectrum in Table 3.

Mesomerism in N1-acyl-1,3-benzoheterazolidine-2-thiones
Some examples about N1-acyl-BZT 22-25 were found in the literature and the NMR data are listed in Table 8. In the case of compound 22, C4 at 115.8 ppm indica the presence of the mesomers 22a/22b in approximately a 65/35 proportion. Howev compound 23 shows the presence of two separated signals for C4 and C7 in 114.3 a 108.5 ppm, respectively. The last signal corresponds to a pyrrole N1H atom (tautom 23a). The signal in 114.3 ppm indicates that this molecule is in the mesomeric forms 2 and 23b, which contribute to the whole structure in approximately a 50/50 proporti The same mesomers in approximately a 67/43 proportion were observed for compou 24a/24b (C4 in 115.0 ppm, C7 in 109.4 ppm). In compound 25, four signals of a symmet BI ring were observed on the spectra C4/C7 in 113.2 ppm, indicating the mesomer 22a present in a major proportion.

Tautomeric Equilibrium in Omeprazole
In 2004, Claramunt et al. [4] reported the 1 H and 13 C NMR spectrum of Omepraz in THF-d8 at −78 °C . The integration of 1 H NMR signals led to the 37/63 ratio of the t tautomers 26a/26b; Table 9. However, on the basis of the reported 13 C NMR data, Table  we proposed the presence of two tautomeric pairs in equilibrium 26a/26b and 26c/2 which form six-membered NH•••N and five-membered NH•••O hydrogen bonding int actions, respectively. Each tautomeric pair is represented by one of the two sets of sign reported. The C7obs value at 113.1 ppm and Equation (2) allowed us to estimate that pair 26a/26b represents 63% of the four tautomers, with 26b being in a major proporti because C7 (101.1 ppm) is affected by both OMe and Npr atoms. The remaining 37% c responds to the pair 26c = 26d, where C7 in 94 ppm represents the presence of 26c as main tautomer. This was considered because, in this case, C7 in 26c suffers the electro effect of both MeO oxygen atom and NH pyrrole nitrogen. On the other hand, the C4 121.6 ppm is characteristic of a Npd electronic effect.

Tautomeric Equilibrium in Omeprazole
In 2004, Claramunt et al. [4] reported the 1 H and 13 C NMR spectrum of Omeprazole in THF-d 8 at −78 • C. The integration of 1 H NMR signals led to the 37/63 ratio of the two tautomers 26a/26b; Table 9. However, on the basis of the reported 13 C NMR data, Table 9, we proposed the presence of two tautomeric pairs in equilibrium 26a/26b and 26c/26d, which form six-membered NH···N and five-membered NH···O hydrogen bonding interactions, respectively. Each tautomeric pair is represented by one of the two sets of signals reported. The δC7 obs value at 113.1 ppm and Equation (2) allowed us to estimate that the pair 26a/26b represents 63% of the four tautomers, with 26b being in a major proportion because C7 (101.1 ppm) is affected by both OMe and N pr atoms. The remaining 37% corresponds to the pair 26c = 26d, where δC7 in 94 ppm represents the presence of 26c as the main tautomer. This was considered because, in this case, C7 in 26c suffers the electronic effect of both MeO oxygen atom and NH pyrrole nitrogen. On the other hand, the δC4 at 121.6 ppm is characteristic of a N pd electronic effect.

Conclusions
In BI derivatives, the C4/C7 chemical shift values are sensitive to the phototropic tautomeric equilibrium: N3=C2-N1-H/H-N1=C2-N3. In general, these carbon atoms are shifted from 120 to 110 ppm, which involves the change in the nitrogen atom character from pyridine N3 to pyrrole N1-H, increasing the shielding electronic effect on C3a and C7 in the whole process.
In the protonated BI (HBI + ), no electronic effect is induced by the =N-H + nitrogen atom on C4/7; however, an inductive electronic effect shifts C3a/7a to low frequencies. The N-pyridine nitrogen atom, in deprotonated benzimidazole (BI − ), exerts a protective electronic effect on C4/7 and C5/6, and deshields C2 and C3a/7a, shifting them to high frequencies.
On the other hand, any carbonyl group bonded to the pyrrolic nitrogen atom diminishes the electronic delocalization into the aromatic system, decreasing the electronic effect on C7. The N-phenyl ring was found to be perpendicular to the BI plane when the spectra is obtained in CDCl3. Meanwhile, in DMSOd6, the phenyl ring is in the same BI plane with the consequent deshielding effect on C7.
Upon increasing the alkyl chain in 2-AkBIs in CDCl3, C3a/7a and C4/7 appeared as broad signals with tendency to disappear (coalescence). An acyl group bonded to C2 in BI favors the NH tautomer owing to an N-H•••O hydrogen bonding formation.
The C4 chemical shift of BIT (δC4ref = 119.1 ppm) and the corrected C4 chemical shift of 1-MeTMBI (δC4ref = 109.6 ppm) were used as references in Equation (2) to calculate the tautomeric ratio in BI derivatives.
The concepts for BIs can be applied to the nitrogen atom in benzoxazole and benzothiazole derivatives; for instance, in the case of BZTs, C4 and C7a were found to be shifted in about 10.0 and 4.0 ppm, respectively, to low frequencies when the nitrogen atom change from the pyridine to pyrrole type. On the other hand, alkyl groups bonded to a BOTs or BTTs nitrogen atom, C4, suffer the whole protective electronic effect of N1-R pyrrole nitrogen atom, shifting C4 to low frequencies (110 ppm).

Conclusions
In BI derivatives, the C4/C7 chemical shift values are sensitive to the phototropic tautomeric equilibrium: N3=C2-N1-H/H-N1=C2-N3. In general, these carbon atoms are shifted from 120 to 110 ppm, which involves the change in the nitrogen atom character from pyridine N3 to pyrrole N1-H, increasing the shielding electronic effect on C3a and C7 in the whole process.
In the protonated BI (HBI + ), no electronic effect is induced by the =N-H + nitrogen atom on C4/7; however, an inductive electronic effect shifts C3a/7a to low frequencies.
On the other hand, any carbonyl group bonded to the pyrrolic nitrogen atom diminishes the electronic delocalization into the aromatic system, decreasing the electronic effect on C7. The N-phenyl ring was found to be perpendicular to the BI plane when the spectra is obtained in CDCl 3 . Meanwhile, in DMSOd 6 , the phenyl ring is in the same BI plane with the consequent deshielding effect on C7.
Upon increasing the alkyl chain in 2-AkBIs in CDCl 3 , C3a/7a and C4/7 appeared as broad signals with tendency to disappear (coalescence). An acyl group bonded to C2 in BI favors the NH tautomer owing to an N-H···O hydrogen bonding formation.
The C4 chemical shift of BIT (δC4 ref = 119.1 ppm) and the corrected C4 chemical shift of 1-MeTMBI (δC4 ref = 109.6 ppm) were used as references in Equation (2) to calculate the tautomeric ratio in BI derivatives.
The concepts for BIs can be applied to the nitrogen atom in benzoxazole and benzothiazole derivatives; for instance, in the case of BZTs, C4 and C7a were found to be shifted in about 10.0 and 4.0 ppm, respectively, to low frequencies when the nitrogen atom change from the pyridine to pyrrole type. On the other hand, alkyl groups bonded to a BOTs or BTTs nitrogen atom, C4, suffer the whole protective electronic effect of N1-R pyrrole nitrogen atom, shifting C4 to low frequencies (110 ppm).
The proposal criterion about the electronic effects on the 13 C chemical shifts of the heteroatoms on the aromatic ring in benzazoles can be used to the correct assignment of any substituted benzazole derivatives as well as to estimate the proportion of the tautomeric species.