**3.1. Diastereomeric identification for the prepared LTAM molecules by 1D 1H NMR and 2D NMR experiments**

The 1H NMR spectra of LTAM molecules display characteristic signals (three groups) in the aliphatic region, namely a triplet and two doublets (group A) in the range of 4.20–5.40 ppm, two singlets (group B) between ~2.90 and ~3.30 ppm, and four identical singlets (group C) at 1.20–1.80 ppm. As a representative example, the 1H NMR spectrum of LTAM **4** in CDCl3 is shown in Fig. 8.

**Figure 8.** 1H NMR spectroscopy of LTAM **4** as a representative example.

**3. Spectroscopic Characterization** 

Newly synthesized FB analogs of LTAM molecules are not expected to be fully planar because of steric crowding, rather they can be viewed as a screw or helical, and thus may possess propeller structures. Subsequently, they can adopt a conformation where all three rings are twisted in the same direction, making a right- or left-handed propeller. As an analogy to a common screw or bolt, right- and left-handed screws are nominated as *M* and *P*, respectively, as shown in the upper line of Fig. 7. The red arrows denote the direction of bond rotation, not the helical direction (blue arrows). Two rings rotate through a

**Figure 7.** A new diastereomer can be formed via bond rotation (red arrows) of one diastereomer (blue

Theoretically, three configurational isomers, *ZE*, *EE,* and *ZZ,* can be proposed for these dyes. Since the *ZE* isomers can have *M* and *P* conformations, *ZE-*LTAM molecules can be

**3.1. Diastereomeric identification for the prepared LTAM molecules by 1D 1H** 

The 1H NMR spectra of LTAM molecules display characteristic signals (three groups) in the aliphatic region, namely a triplet and two doublets (group A) in the range of 4.20–5.40 ppm, two singlets (group B) between ~2.90 and ~3.30 ppm, and four identical singlets (group C) at 1.20–1.80 ppm. As a representative example, the 1H NMR spectrum of LTAM **4** in CDCl3 is

obtained as a racemic mixture from the synthetic reaction described previously.

perpendicular conformation while one moves in the opposite direction.

arrows denote the helical direction between *M* and *P* isomers).

**Figure 8.** 1H NMR spectroscopy of LTAM **4** as a representative example.

**NMR and 2D NMR experiments** 

shown in Fig. 8.

Judging from the chemical shifts of the signals in Fig. 8, group A may belong to *sp*2 protons, H2a and H2a' and allylic proton H1a'', and groups B and C may belong to *N*-Me (H10) and *gem*-dimethyl groups (H8 and H9), respectively. Interestingly, the *gem*-dimethyl groups show four well-separated singlets, indicating that these *gem*-dimethyl groups are diastereotopic. Thus, the *gem*-dimethyl groups are not identical and they have different chemical shifts in the NMR spectra. The most common instance of diastereotopic groups is when two similar groups are substituents on a carbon adjacent to a stereogenic center. These LTAM molecules may not be fully planar because of steric crowding and would thus be expected to exhibit chirality, having no stereogenic centers. No detailed discussions are not given for the resoncances in the range of 6 to 8 ppm, since the resonance in those ranges are of the general aromatic protons.

The 1H and 13C resonances of the LTAM molecules were assigned using COSY and onebond 1H–13C correlations obtained by both direct-detection HETCOR and indirect detection HSQC experiments. COSY was used to identify peaks from the A and B rings. The HETCOR and HSQC identified the shifts of the proton-containing carbons. HMBC was used to differentiate between the two A and B rings of one half of the molecules because these rings were not identical.

The HETCOR experiment identified the carbon shifts of those carbons with protons attached through one-bond coupling between 1H and 13C. Correlations between the protons such as H2a, H8, H9, H10 and H1a'', and their corresponding carbons are particularly useful for structure determination, as indicated in Fig. 9.

**Figure 9.** HETCOR of LTAM **9** in the range of of 0-170 ppm.

**Figure 10.** Structures and NOE correlations of the *ZE, EE*, and *ZZ* isomers of LTAM molecules.

The major chemical shift assignments within FB rings, A and B, of the molecules were mostly made using HMBC and NOE data. Since A and B rings are not identical, HMBC was needed to differentiate between the two groups of FB molecules. HMBC experiments have thus correlated C2 to H1a'', H2a, H8, H9, and H10 of ring A and correlated C2' to H1a'', H2a', H8', H9', and H10' in ring B. For the geometrical identification around the double bonds of the enamine moiety of the A and B (FB) rings, NOE experiments were carried out. Structures and NOE correlations of the *ZE, EE*, and *ZZ* isomers of the LTAM molecules are shown in Fig. 10. Determination of the configuration of the double bonds at positions 2–2a and 2'–2a' of the enamine moiety of the A and B (FB) rings was carried out by an NOE experiment. Strong NOE correlation was observed between the protons H10' (*N*-Me of B) at 2.97 ppm and H2a' (the same subunit) at 4.42 ppm. In addition, H10 (*N*-Me of A) has NOE with H2''*/*H6'', whereas the *gem*-dimethyl, H8 and H9, exhibits NOE with H2a. These observations are compatible with a *Z* arrangement around the double bond of ring A.



Novel Fischer's Base Analogous of Leuco-TAM and TAM+ Dyes – Synthesis and Spectroscopic Characterization 431

aOthers denote the phenyl ring and connecting groups of the LTAM molecules.

bCompound that has the *EE* configuration as the major isomer.

430 Advanced Aspects of Spectroscopy

Compound H or C

LTAM **4**

LTAM **5**<sup>b</sup>

**Figure 10.** Structures and NOE correlations of the *ZE, EE*, and *ZZ* isomers of LTAM molecules.

observations are compatible with a *Z* arrangement around the double bond of ring A.

2/2' - - - 155.8 2a/2a' - - 4.42 100.5 3/3' - - - 45.16 3a/3a' - - - 139.6 8/8' - - 1.39 30.6 9/9' - - 1.60 28.6 10/10' - - 2.97 29.0

*Z* Ring *E* Ring Othersa δ (H) δ (C) δ (H) δ (C) δ (H) δ (C)

2/2' - 151.6 - 151.3 - - 2a/2a' 4.36(d) 97.3 4.42(d) 101.5 - - 3/3' - 44.8 - 44.2 - - 3a/3a' - 139.4 - 139.6 - - 8/8' 1.32(s) 30.6 1.50(s) 28.6 - - 9/9' 1.41(s) 30.8 1.68(s) 29.0 - - 10/10' 3.26(s) 33.5 2.95(s) 29.2 - - 1a'' - - - - 5.22(dd) 38.7 2''/6'' - - - - 7.46(d) 127.6

1a'' - - - - 5.19(t) 38.8 2''/6'' - - - - 7.53(d) 128.8

The major chemical shift assignments within FB rings, A and B, of the molecules were mostly made using HMBC and NOE data. Since A and B rings are not identical, HMBC was needed to differentiate between the two groups of FB molecules. HMBC experiments have thus correlated C2 to H1a'', H2a, H8, H9, and H10 of ring A and correlated C2' to H1a'', H2a', H8', H9', and H10' in ring B. For the geometrical identification around the double bonds of the enamine moiety of the A and B (FB) rings, NOE experiments were carried out. Structures and NOE correlations of the *ZE, EE*, and *ZZ* isomers of the LTAM molecules are shown in Fig. 10. Determination of the configuration of the double bonds at positions 2–2a and 2'–2a' of the enamine moiety of the A and B (FB) rings was carried out by an NOE experiment. Strong NOE correlation was observed between the protons H10' (*N*-Me of B) at 2.97 ppm and H2a' (the same subunit) at 4.42 ppm. In addition, H10 (*N*-Me of A) has NOE with H2''*/*H6'', whereas the *gem*-dimethyl, H8 and H9, exhibits NOE with H2a. These

> **Table 2.** 1H and 13C NMR spectral data for LTAM molecules in CDCl3 (500 and 125 MHz, respectively).

Similarly, H2a' and H9' have NOE correlations with H10' and H2''/H6'', respectively. These NOE phenomena indicate a *ZE* geometry around the double bonds of the enamine moieties of A and B (FB) rings, respectively. Selected 1H and 13C NMR spectral data for the major diastereomer of various LTAM molecules in CDCl3 (500 and 125 MHz, respectively) are listed in Table 2.

### **3.2. Thermal diastereomerization**

#### *3.2.1. Diastereomeric mixtures in equilibrium state*

The 1H NMR spectra of LTAM **4** became complicated upon thermal treatment. After approximately 2 h they exhibited three sets of signals corresponding to two other forms (a & b) in addition to the original major set, in CDCl3, as shown in Fig. 11. Two of the double bonds in the LTAM molecules can exist as *ZE*, *EE*, and *ZZ* isomers, which may account for the complexity of the spectra. Their existence is most likely due to geometrical isomerism with respect to restricted rotation around the C=C double bond of the FB moiety and the C-CH(FB)(Ph) single bonds.

**Figure 11.** 1H NMR spectra of LTAM **4** at the thermal equilibrium state, showing three sets of analog peaks (major, a and b groups).

Detailed analysis of the 1H NMR spectra of the LTAM compounds in the thermal equilibrium state is important for determining the presence of a mixture of *ZE* and *EE* or *ZZ* isomers. After thermal equilibrium of LTAM **4** in CDCl3, 2 h after sampling in an NMR tube at room temperature, three sets of complex signals were observed, namely triplet peaks at 5.0–5.4 ppm, three doublets at 4.3–4.6 ppm, four singlets at 2.8–3.4 ppm, and eight singlets at 1.3–1.7 ppm. Among these peaks, those signals assigned to the *ZE* isomers were a triplet at 5.22 ppm, two doublets at 4.36 and 4.42 ppm, two singlets at 2.95 and 3.26 ppm, and four singlets at 1.3–1.7 ppm, as discussed previously. The residual peaks might belong to the *EE* and/or *ZZ* isomers.

For identification of each of the diastereomers of LTAM **4** in organic solvents, 2D NMR experiments such as COSY, HMBC, and NOESY, were used at the equilibrium state. As an example, a COSY of diastereomeric mixtures of LTAM **4** in the range of 4.25–5.25 ppm in CDCl3 is given in Fig. 12.

**Figure 12.** COSY of diastereomeric mixtures of LTAM **4** in the range of 4.25–5.25 ppm in CDCl3.

1H-1H COSY in the range of 4.25–5.25 ppm showed two individual sets of H1a'' and H2a/2a' protons for the diastereomeric structures *ZE* (solid and dot) and *EE* (dash-dot) isomers of LTAM **4**, respectively. The methylene doublets of the *ZZ* isomers for this compound could not be detected due to their low concentration in the equilibrium state. Three sets of *N*-Me (H10 or H10') in the range of 2.8–3.4 ppm and the germinal methyl group (H8 and H9, and H8' and H9') could be easily distinguished through the visual peak ratios of the 1H NMR.

432 Advanced Aspects of Spectroscopy

peaks (major, a and b groups).

and/or *ZZ* isomers.

CDCl3 is given in Fig. 12.

**Figure 11.** 1H NMR spectra of LTAM **4** at the thermal equilibrium state, showing three sets of analog

Detailed analysis of the 1H NMR spectra of the LTAM compounds in the thermal equilibrium state is important for determining the presence of a mixture of *ZE* and *EE* or *ZZ* isomers. After thermal equilibrium of LTAM **4** in CDCl3, 2 h after sampling in an NMR tube at room temperature, three sets of complex signals were observed, namely triplet peaks at 5.0–5.4 ppm, three doublets at 4.3–4.6 ppm, four singlets at 2.8–3.4 ppm, and eight singlets at 1.3–1.7 ppm. Among these peaks, those signals assigned to the *ZE* isomers were a triplet at 5.22 ppm, two doublets at 4.36 and 4.42 ppm, two singlets at 2.95 and 3.26 ppm, and four singlets at 1.3–1.7 ppm, as discussed previously. The residual peaks might belong to the *EE*

For identification of each of the diastereomers of LTAM **4** in organic solvents, 2D NMR experiments such as COSY, HMBC, and NOESY, were used at the equilibrium state. As an example, a COSY of diastereomeric mixtures of LTAM **4** in the range of 4.25–5.25 ppm in

**Figure 12.** COSY of diastereomeric mixtures of LTAM **4** in the range of 4.25–5.25 ppm in CDCl3.

**Figure 13.** HSQC of diastereomeric mixtures of LTAM **4**, showing one-bond correlation of C2a-H2a, C2a'-H2a' for the *ZE* isomer and C2a-H2a for the *EE* isomer in the range of 95-130ppm.

1H–13C correlations were obtained by both direct-detection HETCOR and indirect detection HSQC experiments. Fig. 13 shows some of the one-bond 1H–13C correlations. HSQC identifies the shifts of the carbons bearing protons of the major *ZE* and minor *EE* isomers.

More particularly, HMBC can identify which protons belong to which unit. For the major *ZE* isomers, HMBC experiments have correlated C2 to H1a'', H2a, H8, H9, and H10 of ring A and correlated C2' to H1a'', H2a', H8', H9', and H10' in ring B. The *gem*-dimethyls (1.50 and 1.68 ppm) are correlated with C3' at 44.2 ppm. The H2a' at 4.42 ppm is correlated to the same C3', which allow us to assign it to the same subunit (B ring). Similarly, the *N*-Me at 2.95 ppm, correlated to the same C3', is also in the same subunit (B ring). The other *gem*-dimethyl groups (1.32 and 1.41 ppm) are correlated with C3 at 44.8 ppm. The H2a at 4.36 ppm and the *N*-Me at 3.26 ppm are also correlated to the same C3, indicating that they are in the second subunit (A ring). The low-field HMBC of the diastereomeric mixtures of LTAM **4** is given in Fig. 14 and the high-field HMBC (< 95 ppm) are not given here.

**Figure 14.** HMBC of the diastereomeric mixtures of LTAM **4** in the range of 95–155 ppm.

For the minor concentration *EE* isomers, the HMBC experiments correlated C2 at 149 ppm to H1a'', H2a, H8, H9, and H10. Similar correlations of C3 were made for H2a, H4, H8, and H9. HMBC could further correlate C3a to H7, H8, and H9. Similar to the extremely minor *ZZ* isomers, HMBC correlated C2 to H1a'', H2a, H8, H9, and H10.

Similar correlations of C3 were made to H2a, H4, H8, and H9. HMBC further correlated C3a to H7, H8, and H9. These correlations provided a clear distinction between diastereomeric isomers. H2a and *N*-Me were also coupled to the same C3, which confirms that all of the protons belong to the same isomer. As H9 or *N*-Me is coupled to C2''/C6'', the protons of the aromatic ring could be identified as a substructure of each isomer.

2D NOESY showed spatial correlations for each of the diastereomeric mixtures for LTAM **4** after reaching thermal equilibrium, as in Fig. 15. Namely, the spatial correlations labeled a–f in red were detected for the *ZE* isomer, and those labeled b', d', and f' in blue are detected for the *EE* isomer. In addition, one correlation, e' in green, was detected for the *ZZ* isomer.

Unfortunately, a few of the 1H resonance peaks for the *ZZ* isomers were able to be detected, such as an *N*-methyl singlet and, very rarely, two *gem*-dimethyl peaks. This result indicates that the LTAM molecules equilibrate in a time-dependent manner, yielding a mixture of the *ZE/EE/ZZ* isomers in organic solvent (CDCl3).

**Figure 14.** HMBC of the diastereomeric mixtures of LTAM **4** in the range of 95–155 ppm.

isomers, HMBC correlated C2 to H1a'', H2a, H8, H9, and H10.

aromatic ring could be identified as a substructure of each isomer.

*ZE/EE/ZZ* isomers in organic solvent (CDCl3).

*ZZ* isomer.

For the minor concentration *EE* isomers, the HMBC experiments correlated C2 at 149 ppm to H1a'', H2a, H8, H9, and H10. Similar correlations of C3 were made for H2a, H4, H8, and H9. HMBC could further correlate C3a to H7, H8, and H9. Similar to the extremely minor *ZZ*

Similar correlations of C3 were made to H2a, H4, H8, and H9. HMBC further correlated C3a to H7, H8, and H9. These correlations provided a clear distinction between diastereomeric isomers. H2a and *N*-Me were also coupled to the same C3, which confirms that all of the protons belong to the same isomer. As H9 or *N*-Me is coupled to C2''/C6'', the protons of the

2D NOESY showed spatial correlations for each of the diastereomeric mixtures for LTAM **4** after reaching thermal equilibrium, as in Fig. 15. Namely, the spatial correlations labeled a–f in red were detected for the *ZE* isomer, and those labeled b', d', and f' in blue are detected for the *EE* isomer. In addition, one correlation, e' in green, was detected for the

Unfortunately, a few of the 1H resonance peaks for the *ZZ* isomers were able to be detected, such as an *N*-methyl singlet and, very rarely, two *gem*-dimethyl peaks. This result indicates that the LTAM molecules equilibrate in a time-dependent manner, yielding a mixture of the

**Figure 15.** 2D NOESY of the diastereomeric mixtures of LTAM **4** after reaching thermal equilibrium in CDCl3.

This NOE phenomenon indicates a *ZE* geometry around the double bonds of the enamine moieties of A and B (FB) rings of the major isomer. However, the minor isomer contains two symmetric Fischer's base units and 2D NOESY only showed the correlations of H2a'- H10' (blue mark, b'), H8'-H2''/H6'' (blue mark, d') but no correlation of H10'-H2''/H6''. This suggests that the *ZE* geometry around the double bonds of the enamine moieties does not exist for the minor isomer. Although the spatial correlations of H2a-H9, H10-H2''/H6'' and H1a''-H10 were expected for the extremely minor *ZZ* isomer, the proton peaks H2a and H2a' of the *ZZ* isomer were too small to be correlated with other protons in the 2D NOESY experiment. One spatial correlation of H1a''-H10 (green mark, e') was detected. This NOE phenomenon indicates that three diastereomers, such as *ZE*, *EE*, and *ZZ*, are in equilibrium with various ratios among the diastereomeric isomers, depending on the NMR solvent used.

The NMR data for the *Z* or *E* ring of the *ZE* isomer suggest that the signals for the geminal dimethyl group for the *EE* isomer should be shifted downfield compared to those of the *ZZ* isomer, whereas the signals of the *N*-methyl groups should be shifted upfield. The geminal dimethyl group signals for the *EE* isomer were shifted upfield compared to those of the *ZE* isomer, whereas the signals of the *N*-methyl and methylene groups were shifted downfield.


**Table 3.** Selected 1H resonances for some of the diastereomeric LTAMs.

Deshielding of the *gem*-dimethyl proton of the *EE* and the *N*-methyl proton of the *ZZ* isomer may be due to their relative proximity to the benzene ring, as indicated in the *ZE* isomers. Selected 1H resonances for the diastereomeric LTAMs are listed in Table 3.

#### *3.2.2. Dynamic behavior of LTAM molecules*

436 Advanced Aspects of Spectroscopy

LTAM **1**

LTAM **2**

LTAM **4**

LTAM **5**

LTAM **10**

LTAM **12**

Compound Ring proton Diastereomer (ppm)

*Z* 

*E* 

*Z* 

*E* 

*Z* 

*E* 

*Z* 

*E* 

*Z* 

*E* 

*Z* 

*E* 

**Table 3.** Selected 1H resonances for some of the diastereomeric LTAMs.

*ZE EE ZZ* 

8-Me 1.31 - 1.37 9-Me 1.41 - 1.57 *N*-Me 3.28 - 3.32 H2a 4.33 - N/A

8'-Me 1.51 1.44 - 9'-Me 1.68 1.64 - *N*'-Me 2.97 3.00 - H2a' 4.41 4.53 -

8-Me 1.33 - 1.38 9-Me 1.42 - 1.55 *N*-Me 3.29 - 3.33 H2a 4.31 - N/A

8'-Me 1.52 1.46 - 9'-Me 1.68 1.64 - *N*'-Me 2.99 3.01 - H2a' 4.36 4.47 -

8-Me 1.32 - N/A 9-Me 1.41 - N/A *N*-Me 3.26 - N/A H2a 4.36 - N/A

8'-Me 1.50 1.43 - 9'-Me 1.68 1.63 - *N*'-Me 2.95 2.98 - H2a' 4.42 4.55 -

8-Me 1.30 - N/A 9-Me 1.39 - N/A *N*-Me 3.22 - 3.28 H2a 4.31 - N/A

8'-Me 1.41 1.39 - 9'-Me 1.65 1.60 - *N*'-Me 2.94 2.97 - H2a' 4.32 4.42 -

8-Me 1.30 - N/A 9-Me 1.39 - N/A *N*-Me 3.25 - 3.28 H2a 4.30 - N/A

8'-Me 1.47 1.41 - 9'-Me 1.65 1.60 - *N*'-Me 2.94 2.96 - H2a' 4.37 4.49 -

8-Me 1.67 - N/A 9-Me 1.77 - N/A *N*-Me 3.44 - N/A H2a 4.47 - N/A

8'-Me 1.90 1.83 - 9'-Me 2.06 2.04 - *N*'-Me 3.08 3.11 - H2a' 4.52 4.60 - Interestingly, the stability of these molecules in solution depends upon the solvent media. Namely, they are inert in polar organic solvents such as acetone and DMSO, but they are unstable in nonpolar solvents such as benzene, THF, and chloroform. They equilibrate timedependently into a mixture of *ZE/EE* or *ZE/EE/ZZ* isomers, depending on the solvents used, as shown in Fig. 16.

**Figure 16.** Dynamic behavior of LTAM **1** in CDCl3, showing diastereomeric isomerization.

It has been reported (Keum et.al. 2008) that FB-analogs of LTAM molecules have very characteristic 1H NMR resonance patterns in the range of 1.0–5.4 ppm as a result of three consecutive protons (H2a, H2a', and H1a''), two *N*-methyl, and four diastereotopic *gem*dimethyl (8- and 9-Me) groups. Therefore, these characteristic peaks can be used to discriminate each of the diastereomers. (Ma et al., 2012) For example, 1H NMR data of the 3 pyridinyl LTAM **9** showed the expected features (A B) of resonances, *viz*. one triplet at 5.25 ppm, two doublets at 4.31 and 4.34 ppm, two singlets at 2.95 and 3.25 ppm, and four singlets at 1.30–1.65 ppm. In contrast, the spectra of 4-pyridinyl LTAM **8** showed very interesting features in the range of 2.90–5.40 ppm. This compound showed one triplet at 5.07 ppm, a doublet at 4.41 ppm, and a singlet at 2.97 ppm, as shown in C of Fig. 17.

The isomerization pattern of LTAM **8** is quite surprising because no FB-analog of MG showed a LTAM **8**-like feature (C B) in the range of 2.90–5.40 ppm. Based upon quantum mechanical calculations (Keum et al., 2010), *ZE* would be expected to predominate over *ZZ* and *EE* in all media. Experimentally, the *ZE* isomers of LTAM compounds generally predominant in all organic media examined. The relative energy differences between the minor *EE* and extremely minor *ZZ* were 0.08 and 0.26 kcal/mol in CDCl3 and DMSO-*d*6, respectively. In addition, both of the spectra, A and C, converged to that displayed by B approximately 2–3 h after mixing the LTAM molecules with CDCl3 in the NMR tube.

**Figure 17.** Characteristic proton resonance of the LTAM molecules in the range of 2.80~5.40 ppm in CDCl3 (A: *ZE*, B: mixture after reaching thermal equilibrium, and C: *EE* diastereomer); arrows show the directions of isomerization.

#### *3.2.3. Determination of diastereomeric ratios at equilibrium state*

Since the characteristic peaks can be used to discriminate each of the diastereomers, the equilibrium ratios among these diastereomeric isomers in various organic solvents can be determined by 1H NMR spectra. This is based on the intensities of either the *N*-methyl or *gem*-dimethyl signals corresponding to the three diastereomeric isomers at the equilibrium state. In some cases, the intensity of the H1a'' proton of the central carbon can be used. Since the *N*-methyl peaks show a well-separated singlet, it is more convenient to measure the ratio of the isomers. The cause of the isomerization of the LTAM compounds at room temperature is unclear. They belong to the group of conjugated enamine compounds. Enamine-imine tautomerism (C=C-NH and CH-C=N) may regulate *ZE* isomerization.

For most of the LTAM molecules examined that are listed in Table 1, the *ZE* isomers are the most stable and they at the equilibrium state for almost 100% of the time in polar solvents (*E*T(30) > 42) and 60–80% in non-polar solvents (*E*T(30) < 42) at room temperature. The minor *EE* minor and extremely minor *ZZ* isomers at the equilibrium state were 18–44% and 0–11% in nonpolar solvents, respectively, depending on the molecules examined. The percent ratios among the diastereomeric isomers of LTAM molecules in the thermal equilibrium states vary according to the molecules examined and solvents used.

However, some LTAM molecules, such as LTAMs **3**, **5**, and **8**, are exceptional. Surprisingly, the pure *EE* isomers are obtained, unlike for the LTAM molecules described previously. These exceptional compounds contain a resonance-electron withdrawing (-R) substituent, particularly on the para-position of the phenyl ring. This indicates that substituents such as *p*-NO2, *p*-CHO, or *p*-(N) on the phenyl ring make the *EE* isomer more stable than the *ZE* isomer, which is predicted to be more stable theoretically. These are summarized in Table 4.


Further detailed studies are needed to determine how the isomerization occurs and what causes the unusual stability of a certain diastereomer.

<sup>a</sup>*K*eq is the ratio of [*ZE*]/[*EE*+*ZZ*] or [*EE*]/[*ZE*+*ZZ*], depending on the identity of the major diastereomer.

bThe major isomer in the solid state.

438 Advanced Aspects of Spectroscopy

directions of isomerization.

The isomerization pattern of LTAM **8** is quite surprising because no FB-analog of MG showed a LTAM **8**-like feature (C B) in the range of 2.90–5.40 ppm. Based upon quantum mechanical calculations (Keum et al., 2010), *ZE* would be expected to predominate over *ZZ* and *EE* in all media. Experimentally, the *ZE* isomers of LTAM compounds generally predominant in all organic media examined. The relative energy differences between the minor *EE* and extremely minor *ZZ* were 0.08 and 0.26 kcal/mol in CDCl3 and DMSO-*d*6, respectively. In addition, both of the spectra, A and C, converged to that displayed by B

approximately 2–3 h after mixing the LTAM molecules with CDCl3 in the NMR tube.

**Figure 17.** Characteristic proton resonance of the LTAM molecules in the range of 2.80~5.40 ppm in CDCl3 (A: *ZE*, B: mixture after reaching thermal equilibrium, and C: *EE* diastereomer); arrows show the

Since the characteristic peaks can be used to discriminate each of the diastereomers, the equilibrium ratios among these diastereomeric isomers in various organic solvents can be determined by 1H NMR spectra. This is based on the intensities of either the *N*-methyl or *gem*-dimethyl signals corresponding to the three diastereomeric isomers at the equilibrium state. In some cases, the intensity of the H1a'' proton of the central carbon can be used. Since the *N*-methyl peaks show a well-separated singlet, it is more convenient to measure the ratio of the isomers. The cause of the isomerization of the LTAM compounds at room temperature is unclear. They belong to the group of conjugated enamine compounds.

Enamine-imine tautomerism (C=C-NH and CH-C=N) may regulate *ZE* isomerization.

For most of the LTAM molecules examined that are listed in Table 1, the *ZE* isomers are the most stable and they at the equilibrium state for almost 100% of the time in polar solvents (*E*T(30) > 42) and 60–80% in non-polar solvents (*E*T(30) < 42) at room temperature. The minor *EE* minor and extremely minor *ZZ* isomers at the equilibrium state were 18–44% and 0–11% in nonpolar solvents, respectively, depending on the molecules examined. The percent ratios among the diastereomeric isomers of LTAM molecules in the thermal equilibrium states

However, some LTAM molecules, such as LTAMs **3**, **5**, and **8**, are exceptional. Surprisingly, the pure *EE* isomers are obtained, unlike for the LTAM molecules described previously. These exceptional compounds contain a resonance-electron withdrawing (-R) substituent, particularly on the para-position of the phenyl ring. This indicates that substituents such as

*3.2.3. Determination of diastereomeric ratios at equilibrium state* 

vary according to the molecules examined and solvents used.

**Table 4.** Percent ratios among the diastereomeric isomers of LTAM molecules at thermal equilibrium states.

#### *3.2.4. Free energy change of activation, ΔG‡*

The rate constants for the formation of the *EE (*and *ZZ)* isomers were measured from a plot of ln(A−Aͦ) versus time (in min), according to the peak-intensity of the central proton at ~5.15 ppm. As an example, excellent linearity was obtained with r = 0.999 and n = 6. The *k*obs and half-life (*t*1/2) for the isomerization of LTAM **4** were 5.95 × 10−4 s−1 and 19.4 min, respectively. The first-order rate constant is a sum of the rate constants for the backward and reverse reactions. From the rate constant obtained at room temperature, the obtained onetemperature *ΔG*‡ value (Dougherty et al., 2006) for the *ZE EE* isomerization of LTAM **4** in CDCl3 was found to be 21.8 kcal/mol. Similarly, the rate constants for the diastereomeric isomerization of LTAM molecules were measured and the one-temperature *ΔG*‡ values of all of them were obtained, using the equation 1 (Dougherty et.al. 2006) given below:

$$
\Delta G^{\ddagger=4.576} \left[ 10.319 + \log \left( \text{T/k} \right) \right] \text{ kcal/mol} \tag{1}
$$

These are summarized in Table 5.


**Table 5.** Rate constants and *ΔG*‡ values for the *ZE/EE* isomerization of LTAM molecules in CDCl3.

It has been previously reported that the *ZE* isomerization of imines and their tautomeric isomers, enamines, has a very high energy barrier (*Δ*G‡ = 23 kcal/mol), unless the process is strongly accelerated by either acid/base catalysts or by push-pull substituents. (Liao & Collum, 2003). The isomerization rate was found to be slow on the NMR time-scale.

#### **3.3. UV-Vis spectroscopy of various forms of LTAM molecules**

FB analogs of TAM+ dyes were obtained from the reaction of FB analogs of LTAM molecules with DDQ in the presence of HCl, followed by separation of the deep blue form from the product mixtures by column chromatography in MC/MeOH (7:1). A reaction of TAM+ with an inorganic base such as NaOH gives the carbinol form of the LTAM molecule. Only the TAM+ cation shows deep coloration, in contrast to the LTAM and carbinol derivatives. This difference arises because only the cationic form has extended *π*-delocalization, which allows the molecule to absorb visible light.

The colored forms, TAM+, of the prepared LTAM and Un-LTAM molecules have absorption maxima at 580–705 and 350–420 nm in ethanol for the x- and y-band, respectively. The carbinol form was detected at 325–385 nm in basic media. The leuco form of these molecules decomposed in HCl-saturated EtOH to form conjugated molecules observed at 385–435 nm. UV-Vis spectral data in CDCl3 of the colored and decomposed forms LTAM **4**, and Un-LTAM **4**, as representative examples, are shown in Fig. 18.

UV-Vis spectral data for various forms of LTAM and Un-LTAM molecules, compared to those of commercial TAM+ dyes, are summarized in Table 6.

Compound *k*obs

x10-4 s-1 *<sup>t</sup>*1/2 (min)

Linearity

LTAM **4** 5.95 19.4 0.999 6 21.8 -

LTAM **5** 0.43 297 0.999 3 - 23.4

LTAM **8** 2.72 42.5 0.999 6 - 22.3

LTAM **9** 4.66 24.8 0.994 4 22.0 -

LTAM **10** 2.48 46.5 0.998 5 22.0 -

LTAM **11** 4.72 24.5 0.998 5 - 22.4

LTAM **13** 17.9 6.47 0.998 3 21.2 -

**Table 5.** Rate constants and *ΔG*‡ values for the *ZE/EE* isomerization of LTAM molecules in CDCl3.

Collum, 2003). The isomerization rate was found to be slow on the NMR time-scale.

**3.3. UV-Vis spectroscopy of various forms of LTAM molecules** 

the molecule to absorb visible light.

LTAM **4**, as representative examples, are shown in Fig. 18.

those of commercial TAM+ dyes, are summarized in Table 6.

It has been previously reported that the *ZE* isomerization of imines and their tautomeric isomers, enamines, has a very high energy barrier (*Δ*G‡ = 23 kcal/mol), unless the process is strongly accelerated by either acid/base catalysts or by push-pull substituents. (Liao &

FB analogs of TAM+ dyes were obtained from the reaction of FB analogs of LTAM molecules with DDQ in the presence of HCl, followed by separation of the deep blue form from the product mixtures by column chromatography in MC/MeOH (7:1). A reaction of TAM+ with an inorganic base such as NaOH gives the carbinol form of the LTAM molecule. Only the TAM+ cation shows deep coloration, in contrast to the LTAM and carbinol derivatives. This difference arises because only the cationic form has extended *π*-delocalization, which allows

The colored forms, TAM+, of the prepared LTAM and Un-LTAM molecules have absorption maxima at 580–705 and 350–420 nm in ethanol for the x- and y-band, respectively. The carbinol form was detected at 325–385 nm in basic media. The leuco form of these molecules decomposed in HCl-saturated EtOH to form conjugated molecules observed at 385–435 nm. UV-Vis spectral data in CDCl3 of the colored and decomposed forms LTAM **4**, and Un-

UV-Vis spectral data for various forms of LTAM and Un-LTAM molecules, compared to

r n

*ΔG*‡*ZE* 

 *EE ΔG*‡*EE* 

 *ZE*

**Figure 18.** UV-Vis spectral data of LTAM **4** (a) and Un-LTAM **4** (b) in EtOH, showing the various forms such as the TAM+ (a-1 and b-1) and decomposed forms (a-2 and b-2).


aNames of compounds are the same as in Table 1. bThe carbinol denoted a hydroxylated TAM+ dye.

c Symbols (x-, y-band) are adopted from Ref. (Ernest et al., 1989) d,eData for acetonitrile.

**Table 6.** UV-Vis spectral data for various forms of LTAM and Un-LTAM compounds.

In the UV-Vis spectral data of Table 6, MG and crystal violet dyes show absorption maxima at 620 and 430 nm for the x- and y-band, respectively, whereas the absorption maxima of the vinyl-log of MG are red-shifted for both the x- and y-bands, *i.e.*, 651 and 488 nm, respectively. This suggests that the vinyl effects of a vinyl unit may, to a large extent, behave like extended conjugation for both the x- and y-bands. Chemical skeletons for the N~N+ and C(phenyl)~N+ responsible for the x- and y-band, respectively, in the absorption spectra of various TAM+ dyes are shown in Fig. 19.

Structurally, the FB analogs of symmetric and unsymmetric TAM+ dyes in this work can be characterized as Cy3 and Cy5 dyes, respectively, as closed-chain cyanines.(Ernst et al., 1989) It was reported that Cy3 is maximally excited at 550 nm and maximally emits at 570 nm in the orange-red part of the spectrum, whereas Cy5 is maximally excited at 649 nm and maximally emits at 670 nm, which is in the red part of the spectrum. Therefore, the x-band of the Un-TAM+ are expected to be higher than 650 nm and 550 nm, for the y- and x-band, respectively.

**Figure 19.** Chemical skeleton for the N~N+ and C(phenyl)~N+ responsible for the x- and y-band, respectively, in the absorption spectra of various TAM+ dyes.

From the reaction of Un-LTAM **4** with HClO4, the decomposed product {5-chloro-1,3,3 trimethyl-2-((1E,3E)-4-(4-nitrophenyl)buta-1,3-dienyl)indolium perchlorate} was isolated, brown, yield 57%, M.p.= 257–258 °C, IR (KBr) 3072, 2984, 2934, 1707, 1596, 1340, and 1086 cm−1, 1H NMR (DMSO-*d*6) δ 1.76 (6H, s), 4.03 (3H, s), 7.37 (1H, d, *J* = 15.3 Hz), 7.66 (1H, dd, *J*  = 10.2, 15.3 Hz), 7.73 (1H, d, *J* = 9.0 Hz), 7.79 (1H, d, *J* = 15.3 Hz), 7.92 (2H, d, *J* = 6.9 Hz), 7.95 (1H, d, *J* = 9.0 Hz), 8.09 (1H, s), 8.33(1H, dd, (*J* = 10.2, 15.3 Hz), and 8.33(2H, d, *J* = 6.9 Hz).
