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

© 2012 Keum et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Novel Fischer’s Base Analogous of Leuco-TAM and TAM Dyes – Synthesis and Spectroscopic Characterization


Introduction
Triarylmethane (TAM) dyes are organic compounds containing triphenylmethane backbones. TAM compounds are sometimes called leuco-TAMs (LTAMs) or leuco-bases. (Nair et al., 2006) LTAM molecules are the precursors of TAM + dyes since TAM + dyes are the oxidized form of LTAM molecules. Backbones of TAM molecules are also known to be an important group in intermediates in the synthesis of various organic functional compounds, including the preparation of polymers and supramolecules. (Bartholome & Klemm, 2006) TAM + dyes potentially have numerous applications in the chemical, pharmaceutical, and life science industries, including as staining agents, ink dyes, thermal imaging materials, carbonless copying materials, drugs, leather, ceramics, cotton, and as a cytochemical staining agent. (Balko & Allison, 2000) A number of TAM + dye molecules are well known, such as malachite green (MG), brilliant green, crystal violet, and pararosanilin, etc. The chemical structures of some of these well-known TAM + dyes are shown in Fig. 1. Among them, MG is one of the most commonly used chemicals in dye chemistry. MG is a common green dye but it is absorbed into the human body in its carbinol and leuco forms (see the section on UV-Vis spectroscopy). MG is very active with the fungus Saprolegnia, which infects fish eggs in commercial aquaculture, and is known to be good for Ichthyophthirius in fresh water aquaria. (Indig et al., 2000) It has been known, however, for MG to be highly toxic to mammalian cells, even at low concentrations. (Plakas et al., 1999;Cho et al., 2003) Because of its low cost, effectiveness as an antifungal agent for commercial fish hatcheries, and ready availability, many people can be exposed to this dye through the consumption of treated fish. Since MG is similar in structure to carcinogenic triphenylmethane dyes, it may be a potential human health hazard.
In addition, in their oxidized form, TAM + dyes are highly absorbing fluorophores with extinction coefficients of ~2.0 × 10 5 mol −1 cm −1 and high quantum yields. Their absorption maxima can easily be matched with the laser lines by simply changing the length of the conjugated chain and/or the heterocyclic moiety. Thus, TAM + dyes can be employed as fluorescence labels and sensors of biomolecules in vivo because their spectra reach the nearinfrared region. Özer (2002) reported efficient non-photochemical bleaching of a TAM + dye by chicken ovalbumin and human serum albumin, showing that dye-protein adducts can also form and suggesting that proteins may be primary, rather than indirect, targets of TAM + action. However, the use of this substance has been banned in many countries because of its toxicity and possible carcinogenicity. Substitutive materials for MG compounds have thus been in considerable demand. Numbers of researchers (Gessner & Mayer, 2005) have been interested in developing TAM + molecules. We developed Fischer's base (FB) analogs of LTAM molecules (Keum et al., 2008(Keum et al., , 2009(Keum et al., , 2010(Keum et al., , 2011, whose chemical structures contain a couple of heterocyclic FB rings and a substituted phenyl group on a central carbon of the molecules. The structures and numbering system for the Fischer's base (FB) analogs of LTAMs are shown in Fig. 2. In this chapter, abbreviations (LTAM and Un-LTAM) will be used to designate the Fischer's base analogs of symmetric and unsymmetric LTAMs, respectively. Note that "the general LTAM" in Section 2.2 denotes the LTAM/TAM + dyes that contain no FB moieties.

General leuco-TAM and TAM + molecules
Generally, the Friedel-Crafts-type catalytic alkylation of aromatic rings with aromatic aldehydes is an effective method for TAM + formation. (Li et al., 2008;Kraus et al., 2008) Several mild and efficient triaryl-and triheteroarylmethanes formations using [Ir(COD)Cl]2-SnCl4, AuCl3, Cu(OTf)2, and Sc(OTf)3 as catalysts have also been reported. Grignard reagents or n-butyl lithium compounds have also been used for their preparation. A brief summary of the preparation methods of general LTAM/TAM + molecules is shown in Fig. 3. Although a number of methods are available for the synthesis of triarylmethanes, most are multistep processes and/or require harsh reaction conditions.

Fischer's base analogs of Leuco-TAM
Fischer's base analogs of LTAM molecules can be obtained from a reaction of a molar excess of Fischer's base and substituted aryl aldehydes. The prepared LTAM dyes consist of two FB rings on the central carbon, where a substituted phenyl ring is located. The LTAM molecules can be symmetric or unsymmetric, depending on the identity of the two FB rings. They are the precursors of the TAM + dyes, which are structurally close to the polymethine dyes (e.g., Cy3,Cy5,etc.). (Ernst et al., 1989)

Symmetric LTAM FB analogs
The FB analogs of symmetric leuco-TAM molecules {2,2′-(2-phenylpropane-1,3diylidene)bis(1,3,3-trimethylindoline)} derivatives were obtained from the reaction of 5substituted benzaldehyde and excess (2-to 3-fold) FB in ethanol at room temperature for 2-4 h, as shown in Fig. 4. The white precipitate was filtered from the reaction mixture and washed thoroughly with cold ethyl alcohol. Purification was carried out through precipitation from acetone. TAM + dyes were then obtained from a reaction of LTAM molecule with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in the presence of HCl, followed by separation of the deep blue form from the product mixtures by column chromatography in MC/MeOH (9:1).
Melting points, yields, and other characteristic data of the prepared LTAM molecules are summarized in Table 1.

Unsymmetric LTAM FB analogs
Unsymmetric LTAMs (Un-LTAM) were obtained from a reaction of excess Fischer's base with the substituted cinnamaldehydes, as shown in Fig. 5. The Un-LTAMs have two different FB skeletons on the central carbon, 1,3,3-trimethyl-2-methyleneindoline and 2allylidene-1,3,3-trimethylindoline groups. The symmetric TAM + dyes with styryl-ring pendants are expected to possess elongated conjugation from the N + center of the FB ring to the phenyl ring. However, these LTAM molecules were not successfully obtained from the reactions of Fischer's base with the substituted cinnamaldehydes. Experimentally, Un-LTAM molecules were formed as the sole product and no symmetrical LTAM dyes were formed. This suggests that the Michael-type addition of the second molecule of FB occurs on the δ-carbon and not on the β-carbon of the extended ,βunsaturated iminium salts that were formed from the reaction of FB and cinnamaldehydes. The mechanistic processes for the formation of Un-LTAM molecules are shown in Fig. 6. Melting points, yields, and other characteristic data of the prepared LTAM and Un-LTAM molecules are summarized in Table 1

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 perpendicular conformation while one moves in the opposite direction. 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 obtained as a racemic mixture from the synthetic reaction described previously.

Diastereomeric identification for the prepared LTAM molecules by 1D 1 H NMR and 2D NMR experiments
The 1 H 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 1 H NMR spectrum of LTAM 4 in CDCl3 is 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 1 H and 13 C resonances of the LTAM molecules were assigned using COSY and onebond 1 H-13 C 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 1 H and 13 C. 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.
a Others denote the phenyl ring and connecting groups of the LTAM molecules.
b Compound that has the EE configuration as the major isomer. 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 1 H and 13 C NMR spectral data for the major diastereomer of various LTAM molecules in CDCl3 (500 and 125 MHz, respectively) are listed in Table 2.

Diastereomeric mixtures in equilibrium state
The 1 H 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.  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.   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 1 H 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). 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. 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 1 H resonances for the diastereomeric LTAMs are listed in Table 3.

Dynamic behavior of LTAM molecules
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. It has been reported (Keum et.al. 2008) that FB-analogs of LTAM molecules have very characteristic 1 H 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 gemdimethyl (8-and 9-Me) groups. Therefore, these characteristic peaks can be used to discriminate each of the diastereomers.    (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-d6, 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.

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 1 H 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 (ET(30) > 42) and 60-80% in non-polar solvents (ET(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.

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 kobs and half-life (t1/2) 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 one- It has been previously reported that the ZE isomerization of imines and their tautomeric isomers, enamines, has a very high energy barrier (ΔG ‡ = 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.

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

LTAM molecules
The X-ray crystal structure of LTAM 12, as a representative example, displays an orthorhombic crystal system with space group Pna21, with a residual factor of R1 = 0.0517. ORTEP diagrams of LTAM 12, showing atom numbering, are provided in Fig. 20(a).

Ring
Compound 12 is stacked so that a dimer is formed in the unit cell of the crystal. The packing in the unit cell of LTAM 12 is distinct, as can be seen in Fig. 21.

Un-LTAM molecules
The Un-LTAM 4 was only successfully crystallized from acetone. Unfortunately, crystal growth was unsuccessful for the remainder of the Un-LTAM molecules. Selected bond lengths and bond angles are listed in Table 9. The X-ray crystal structure of Un-LTAM 4 shows a triclinic crystal system with space group P-1. An ORTEP diagram of Un-LTAM 4, including the atom-numbering scheme, is shown in Fig. 22(a). The C9''-C10'' and C10''-C11''" distances are 1.516 and 1.509 Å, respectively, typical for C-C single bonds. The C7''-C8'' single bond distance, however, was 1.443 Å, which is shorter than a typical single bond and longer than a typical double bond. This is perhaps due to conjugation since the length (1.47 Å) of the central single bond of 1,3-butadiene is approximately 6 ppm shorter than that of the analogous single bond (1.53 Å) in butane. The two enamine C2=C7'' and C2'=C11'', and C8''=C9'' double bonds were 1.354, 1.324, and 1.328 Å, respectively, which are typical C=C bond lengths. In the crystal, the three aromatic rings of 4 are linked to three different layers, viz. a vinyl FB, a FB, and a phenyl group. The unsymmetrical molecule is a distorted version of the well-known three-bladed propeller conformation. (Keum et al., 2011). The inter-plane angles of the aromatic rings A-B, A-C, and B-C in Un-LTAM 4 are 87.4°, 67.5°, and 61.5°, respectively ( Fig. 22(b)). The EEE isomers of these LTAM dyes are formed as the sole product in all cases, even though there are three possible isomers, the two other diastereomers being the ZEE and ZEZ isomers, as shown in Fig. 23. Generally, the central carbon-carbon double bond of these LTAM dyes is expected to have an E configuration.
Although the presence of the ZEE and ZEZ diastereomers was generally expected to be found in organic solvents, none of these isomers were detected, unlike for the LTAM molecules examined previously.

Conclusion
Novel Fischer's base analogs of LTAM and Un-LTAM molecules and their corresponding TAM + dyes have been successfully developed. 1 H and 13 C NMR assignments for the prepared LTAM molecules have been completed by 1D and 2D NMR experiments, including DEPT, COSY, HSQC, HMBC, and NOESY. The geometry of the double bond was ZE in most cases, as measured directly by NOESY. The EE and ZZ isomers have C2 symmetry, and hence, the two FB rings of these isomers are identical. Therefore, the 1 H NMR spectra of the EE and ZZ isomers are expected to be relatively simple compared to those of the ZE isomer. The novel LTAM molecules exist as a single isomer (ZE or EE) in the solid phase and they are equilibrated with other isomers in organic solvents. The percent ratios among the diastereomeric isomers of LTAM derivatives in the thermal equilibrium states vary according to the molecules examined and solvents used.
UV-Vis spectral data shows various structural forms of the LTAM and Un-LTAM molecules, such as (a) leuco-, (b) colored TAM + , (c) carbinol-, and (d) decomposed-forms, similar to the commercially known TAM + dyes, such as MG, crystal violet, etc. Particularly, UV-Vis spectroscopic data for the Un-TAM + dyes showed absorptions in the near-IR region.
X-ray crystal analysis showed that the ZE isomers were generally formed with a so-called three-bladed propeller conformation. These isomers stacked to form a dimer or double dimer. However, the EE isomers were also formed specifically for the LTAMs 3, 5, 8, and 11, which have a resonance-electron withdrawing (-R) group at the para-position of the phenyl ring. Further analysis of a variety of substituted LTAM molecules is required to determine what makes the diastereomer structures change in the solid state.

Author details
Sam-Rok Keum, So-Young Ma and Se-Jung Roh Korea University at Sejong Campus, South Korea