Synthesis and applications of benzothiazole containing cyanine dyes

Monomethine cyanine dyes

Monomethine benzothiazole cyanine dyes typically absorb in the visible region (450–470 nm) of the electronic spectrum depending on the substituents attached to the benzothiazole core structure. These dyes are characterized by a narrow absorption peak and high fluorescence intensity. They are best known for their nucleic acid binding properties. The oldest method for the synthesis of the monomethine cyanine dyes involves condensation of an N-alkyl-2-(methylthio)benzothiazolium salt with another alkylated heterocycle with an activated methyl group [13]. This method was adopted for the synthesis of β-cyclodextrin functionalized benzothiazole cyanine dye 1 (Equation 1) [14]. β-Cyclodextrin possesses both a hydrophobic cavity and hydrophilic surface and is used for molecular recognition as a drug delivery agent in pharmaceutics [15]. The medicines containing vitamins are generally unstable to light, heat, and oxygen, whereas the formation of inclusion complexes of vitamins with β-cyclodextrin enhances the stability, solubility, and bioavailability of the drug [16]. Analysis of such inclusion interactions of vitamins are commonly conducted by means of spectrophotometric titration using external agents such as dyes as spectral probes. The incorporation of the dye molecule as host compound provides an option to easily recognize colorless guest molecules by direct titration. Compound 1 was used to study supramolecular interactions of β-cyclodextrin directly by visible spectroscopy, which is otherwise impossible due to an insufficient chromophore system.

The synthetic procedure shown above involves a major drawback of producing methyl mercaptan, which is a toxic pollutant. Researchers have overcome this problem by developing a new procedure that uses 2-iminobenzothiazoline instead of 2-methylthiobenzothiazolium salt [17]. This method can be used to synthesize symmetric and asymmetric cyanine dyes. In this procedure, cyanine dyes are prepared by melting 2-iminobenzothiazoline with quaternary heterocyclic salt containing 2- or 4-methyl groups, as depicted in Equation 2.

Deligeorgiev et al. described a novel procedure for the synthesis of benzothiazole monocyanines, which involves heating a sulfobetaine salt of N-alkylbenzothiazolium compound with the quaternary salt of a heterocyclic compound containing a reactive methyl group, as illustrated in Equation 3 [18]. These reactions are usually carried out without solvent by heating the mixture to approximately 150–200°C. An alternative route to less thermostable compounds involves heating a solution in polar solvent. This synthetic procedure is characterized by high yields and short reaction times.

Another approach towards the synthesis of monomethine cyanine dyes involves condensation of N-alkyl-2-methylbenzothiazolium salt with 2- or 4-chloropyridinium or quinolinium substrate in a basic medium [19]. This synthetic procedure has been used to synthesize dicationic and tricationic benzothiazole cyanine dyes (Equation 4). In this particular case, the increased cationic charge helps increase water solubility of the dye.

Several homodimeric benzothiazole cyanine dyes with polycationic charge on the overall cyanine dye have been published (Equation 5) [20].

Compounds 2–4 are structural analogs of the thiazole orange dye which is used extensively for the purpose of nucleic acid detection, flow cytometry, and gel electrophoresis [17]. Typically, monomethine cyanine dyes display almost negligible fluorescence in aqueous solutions but show multifold fluorescence enhancement upon binding to nucleic acids. This phenomenon can be used to detect nucleic acids in the solution up to certain nanomolar concentrations. The cationic monomethine cyanine dye molecules with planar aromatic rings typically intercalate within the two adjacent base pairs of DNA. It has been observed that increasing cationic charges on the dye molecule results in improved binding to the negatively charged nucleic acids. Homodimeric cyanine dye 5 interacts with nucleic acids by bis-intercalation and electrostatic interaction, which provides extra stabilization to the dye-nucleic acid complex [20]. Remarkably, cyanine dye 5 exhibits approximately a 40-nm difference between fluorescence maxima in the presence of single-stranded DNA and double-stranded DNA, which could make it useful to differentiate between both species in solution.

A novel method for the synthesis of monomeric asymmetric benzothiazole derivatives, such as 7 (Scheme 1), containing mercapto and thioacetyl substituents has been reported [21]. Compound 7 could be further modified as suggested by Ishiguro et al. by linking the mercapto group to oligonucleotides [22]. This method would facilitate in vitro transcription and gene expression studies. The synthetic methodology is depicted in Scheme 1. The preparation of the dye intermediate 6 was carried out by heating under reflux a mixture of 2-methylmercaptobenzothiazole with 1,3-dibromopropane for 2 h. Compound 6 was then allowed to react with 1,4-dimethylquinolinium iodide to furnish dye 7 in 95% overall yield.

Scheme 1

A new method for the synthesis of benzothiazole monocyanines was developed making use of solid phase resin support [23]. The schematic representation of the solid phase synthesis is presented in Scheme 2. The first step involves the attachment of 2-mercaptobenzothiazole to the Merrifield resin. This resin-attached benzothiazole is then reacted in the next step with 4-methylbenzenesulfonate to yield N-methylbenzothiazolium salt 8. In the final step, condensation of N-methylbenzothiazolium salt 8 with carboxylic acid derivative of lepidine affords cyanine dye 9. Traceless cleavage of Merrifield resin is normally carried out during the course of the synthesis of the cyanine dye. The presence of a carboxylic acid side chain attached to the quinoline ring makes the dye available for post-synthetic modification. Attaching an amino acid to the carboxylic end group and modifying it with folic acid can be used to identify cancer cells that have a folacin acceptor on the cellular surface [23]. The most important feature of the solid phase synthesis is the elimination of the lengthy purification step of the conventional liquid phase method, which allows the use of synthesized dyes directly for biological screening.

Scheme 2

Adding another vinyl unit to the central linking chain gives rise to trimethine cyanines. This class of dyes is discussed in the following section.

Trimethine cyanine dyes

Benzothiazole trimethine cyanine dyes absorb in the visible region (550–570 nm) of the electronic spectrum. The classical ortho ester method described by Koenig is the oldest known method for the synthesis of symmetric trimethine cyanine dyes [24]. The general synthetic procedure involves condensation of a quaternary benzothiazolium salt containing an activated methyl group with an ortho ester under basic conditions [25]. The ortho ester approach is illustrated in Scheme 3 by the synthesis of a dumbbell-type [60]-fullerene dimer containing a benzothiazole trimethine cyanine dye 11 [26]. The first step of the synthesis involves attachment of [60]-fullerene to the benzothiazole system, which is achieved by heating a solution of [60]-fullerene, sarcosine, and 5-formyl-2- methylbenzothiazole under a nitrogen atmosphere. The resulting compound 10 is then treated with dimethyl sulfate to generate a quaternary salt, which undergoes the coupling reaction with triethyl orthoformate in the next step to yield cyanine dye 11. Fullerene chemistry is a rapidly growing division of material science. C₆₀ dyads have been the focus of much attention in recent years due to their applicability in artificial photosynthesis and molecular electronic devices [26]. The natural photosynthesis involves multistep electron transfer reactions achieved through various electron mediators present in the matrix which accomplish long lifetime of the final charge separated state [27]. Fullerenes have been widely employed as donor/acceptor molecules in dyads to accelerate photo-induced charge separation to mimic natural photosynthesis. The presence of two or more C₆₀ units in the molecule could result in a longer lifetime of the final charge separated state, which is similar to the natural phenomenon.

Scheme 3

A new class of phosphonate labeled trimethine benzothiazole cyanine dyes has been published using the ortho ester approach [28]. Synthetic methodology for the phosphonate labeled cyanine dye starts with the synthesis of compound 12 which is obtained by heating under reflux 2-methylbenzothiazole with diethyl 3-bromopropylphosphonate in acetonitrile (Scheme 4). Phosphonate salt 12 is then converted to cyanine dye 14 using triethyl orthoformate under basic conditions. Conversion of phosphonate salt to phosphonic acid salt is achieved by acidic hydrolysis, which is used in a further step to synthesize dye 13. The presence of phosphonic acid side chain improves the water solubility of the dye. In the deprotonated form, cyanine dyes 13 and 14 are pH sensitive calcium probes. Employment of the phosphonate group on the side chain makes these dyes potent fluorescent labels in complexation with biological systems [28].

Scheme 4

Another approach to the synthesis of benzothiazole trimethine cyanine dyes involves the use of N,N-diphenylformamidine or iodoform as a condensing agent. This synthetic method also allows the synthesis of asymmetric cyanine dyes [29]. Scheme 5 shows the use of N,N-diphenylformamidine for the synthesis of benzothiazole trimethine cyanine dye 16. Alkylated benzoxazole derivative is first treated with N,N-diphenylformamidine in the presence of acetic anhydride to furnish half dye 15. Compound 15 in the next step is condensed with 2,3-dimethylbenozthiazolium iodide to yield final asymmetric dye 16. Different solvents can be used for the synthesis of asymmetric cyanine dyes using the N,N-diphenylformamidine approach. The reactions are usually carried out in acetic anhydride but can also be conducted in n-propanol or DMSO at high temperatures for several hours.

Scheme 5

Park et al. developed novel triazacarbocyanine intramolecular charge transfer dye sensor 17 (Equation 6) [30]. This dye sensor shows reversible pH induced color switching effects. Here, a methoxy group acts as a donor group, whereas the nitro group acts as an acceptor group.

Equation 7 depicts the synthesis of trimethine cyanine dye with meso substitution in the trimethine chain. The meso methyl group undergoes condensation reaction with 9-julolidine carboxaldehyde to furnish benzothiazole derivative 18 [31].

The pentamethine cyanines are prepared using a similar approach. This class of dyes is discussed next.

Pentamethine cyanine dyes

Pentamethine cyanine dyes generally absorb in the visible region (650–670 nm) but fluoresce in the near-infrared region (690–710 nm) of the electronic spectrum. The classical synthetic procedure involves use of bis(phenylimine) hydrochloride of malonaldehyde under basic conditions, as exemplified in Equation 8 [32, 33].

Modification of the trimethine chain in the malonaldehyde-derived reagent for the use in synthesis of γ-substituted pentamethine cyanine dye has been reported, as shown in Scheme 6 [34]. The new γ-substituted pentamethine cyanine dyes were synthesized using a three-step procedure. The first step involves synthesis of N-pyridinium acetic acid salt 20 by reaction between 3,5-dimethylpyridine and bromoacetic acid. In the second step, compound 20 was formylated in the presence of N,N-dimethylformamide and phosphorus oxychloride to obtain compound 21. The synthesis of cyanine dye 22 was carried out by condensation reaction between compound 21 and 2,3-dimethyl benzothiazole tetrafluoroborate in the presence of sterically hindered Hunig base. The presence of the pyridine ring in the pentamethine chain makes the dye more rigid and the cationic charge makes it water soluble. The presence of the pyridinium moiety affects electron density distribution of the dye molecule.

Scheme 6

Benzothiazole cyanine dyes with fluorine substituted pentamethine chain have been reported (Scheme 7) [35]. The Stille reaction was employed in the first step for coupling of 2-iodobenzothiazole with a fluorinated tin reagent in the presence of copper iodide and tetrakis(triphenylphosphine) palladium(0) to afford compound 23. This product was alkylated by reaction with methyl iodide in the presence of silver tetrafluoroborate to obtain compound 24, which on subsequent reaction with 2-fluoromethylbenzothiazole methylene base was transformed into cyclic compound 25. Alkylation of 25 followed by reaction with p-dimethylaminotoluidine furnished pentamethine cyanine dye 26. It is noteworthy that the electronegative fluorine atoms in the pentamethine chain act as electron donating substituents resulting in the shift of absorption maximum for compound 26 (691 nm) of 41 nm relative to the non-fluorinated dye.

Scheme 7

The synthesis of asymmetric pentamethine benzothiazole cyanine dyes can be accomplished utilizing an aldehyde analog of the benzothiazole moiety [36]. Meguellati et al. adapted the aldehyde strategy to synthesize the imino pentamethine cyanine dye, as depicted in Scheme 8. These dyes remain stable in the solid state, whereas in solution within an hour aldehyde and imine are regenerated [37]. Compound 29 was synthesized by reacting malonaldehyde bis(phenylimine) hydrochloride with a 1,2,3,3-tetramethylindolenine iodide salt. Basic hydrolysis of the activated hemicyanine derivative 27 in sodium hydroxide solution afforded compound 28 in excellent yield. In the last step, compound 28 was treated with N-methyl-2-aminobenzothiazolium salt to yield cyanine dye 29. The cyanine dye 29 is accessible through reversible and thermodynamically controlled reaction of amine and aldehyde.

Scheme 8

A series of benzothiazole fluorophores with substituted cyclohexene and cyclopentene in the pentamethine chain has been described [38]. These compounds were studied as fluorescent probes for nucleic acid and proteins. It is believed that they act as nucleic acid groove binders. Synthesis of pentamethine cyanine dyes 34 and 35 containing a cyclopentene ring in the pentmethine chain was accomplished by condensing 2-methylcylcopentane-1,3-diones 30, 31 with quaternary salts of 2-methylbenzothiazolium 32, 33 at 210°C in the presence of triethylamine (Equation 9). Compound 35 exhibits a 15-fold increase and compound 34 displays an 8-fold increase in the fluorescence intensity in the presence of DNA. Cyanine dyes 34 and 35 can be further modified to increase their binding interactions with DNA.

Synthesis of pentamethine dyes with a cyclohexene ring in the pentamethine chain was achieved by condensation of quaternary salts of benzothiazole containing an active methyl group with 1,5-dimethoxy-1,4-cyclohexadienes or with 1,3-diethoxy-5,5-dimethyl- or 1,3-diethoxy-2,5,5-trimethyl-1,3-cyclohexadienes either by melting the two substrates or by heating in benzonitrile at 120–130°C, as illustrated in Equation 10. Compound 36 displays an 8-fold increase in the fluorescence intensity in the presence of RNA, but almost negligible fluorescence changes in the presence of DNA. Remarkably, compound 36 exhibits an approximately 26-fold increase in the fluorescence intensity in the presence of bovine serum albumin [38]. It has been suggested that compound 36 could be used as a potential fluorescent probe for other proteins.

Heptamethine cyanine dyes

Heptamethine cyanine dyes typically absorb and fluoresce in the near-infrared region (750–1100 nm) of the electronic spectrum. The general synthetic method for the synthesis of benzothiazole heptacyanines with a flexible polymethine chain involves use of glutaconaldehyde dianil monohydrochloride as a precursor to the heptamethine linker [39]. As shown in Equation 11, a number of benzothiazole heptamethine derivatives with different substituents at the benzothiazole nitrogen atom have been synthesized [40, 41].

Synthesis of another class of benzothiazole heptamethine dyes containing five- or six-membered cyclic systems as a part of the heptamethine linker makes use of the Vilsmeier-Haack reagents [42, 43]. The presence of a carbocyclic ring in the heptamethine chain makes dye more rigid, which helps to increase fluorescence quantum yield and decrease aggregation of the dye in solution. Vilsmeier-Haack reagents are derived from cyclopentanone or cyclohexanone in a two-step procedure, as shown in Equation 12.

The general methodology for the synthesis of heptamethine cyanines with a rigid polymethine chain involves the condensation of Vilsmeier-Haack reagents 38 or 39 with a heterocycle containing an activated methyl group in ethanol in the presence of sodium acetate as catalyst [44]. Another approach for the synthesis of heptamethine cyanine dyes involves heating under reflux a quaternary salt of benzothiazole and bis-aldehyde in butanol/benzene solvent mixture with continuous removal of the water from the reaction mixture using a Dean-Stark apparatus [45]. The major advantage of using this method of synthesis is the elimination of a catalyst and the formation of final dye 40 in high yield (Equation 13). Benzothiazole cyanine dyes similar to compound 40 can be additionally functionalized at the meso position by reaction with various nucleophiles. The products have been examined as sensitizers for zinc oxide solar cells [43].