Oxo-squaraines

Fig. 1 Absorption and emission spectra of Cy3, CyS, Cy7, oxo-squaraine 13b (part 3 of this chapter) and dicyanomethylene squaraine 41j (part 4 of this chapter) in water (pH 7.4)...

Symmetrical pyrrole- and phloroglucinol-based oxo-squaraines 2 and 3 were first synthesized by Treibs and Jacob in 1965 [43, 44] and aniline-based squaraines 4... 

In general, symmetrical oxo-squaraines having the same end-groups are synthesized by reacting squaric acid with two equivalents of quatemized indolenine, 2-methyl-substituted benzothiazole, benzoselenazole, pyridine, quinoline [39, 45, 46] (Fig. 4) in a mixture of 1-butanol - toluene or 1-butanol - benzene with azeotropic removal of water in presence [39, 45] or absence [47] of quinoline as a catalyst. Other reported solvent systems include 1-butanol - pyridine [48], 1-propanol - chlorobenzene, or a mixture of acetic acid with pyridine and acetic anhydride [49]. Low CH-acidic, heterocyclic compounds such as quatemized aryl-azoles and benzoxazole do not react, and the corresponding oxo-squaraines cannot be obtained using this method [23, 50]. 

The key intermediates for the synthesis of unsymmetrical, heterocyclic oxo-squaraines are the mono-squaraines (semi-squaraines) shown in Fig. 5. These intermediates can be synthesized via condensation of dialkylsquarate with an equimolar amount of methylene base [51]. The obtained alkoxy-mono-squaraines are then reacted with the second methylene base to yield unsymmetrical oxo-squaraines. These mono-squaraine intermediates display a higher reactivity compared to squaric acid or its esters they allow the synthesis of the corresponding... 

Fig. 6 Synthesis of symmetrical and unsymmetrical aniline-based oxo-squaraines...

Symmetrical, aniline-based, and aromatic oxo-squaraines are synthesized via a one-step reaction by heating two equivalents of the appropriate /V,/V-dialkylaniline or other reactive aromatic or heteroaromatic derivatives with squaric acid (Fig. 6) [38, 41]. Unsymmetrical aniline-type squaraines can be synthesized in two steps first one component is reacted with squaric acid dichloride to yield a mono-squaraine intermediate, which in a subsequent step is then reacted with the second component to yield the unsymmetrical squaraine dye [53]. 

Mixed squaraines can be synthesized via heterocyclic or aromatic mono-squaraines [49]. Symmetrical and unsymmetrical oxo-squaraines and mono-squar-aine intermediates can be obtained also by formation of squaraine ring [42]. 

The synthesis of oxo-squaraines and related compounds, including their spectral properties and applications as biomedical probes, photoconducting materials, and photosensitizers are provided in a recent review [56]. 

The synthesis, spectral properties, and applications of symmetrical as well as unsymmetrical, hydrophobic oxo-squaraine probes for noncovalent interaction with proteins, lipids, cells, and other high-molecular-weight analytes are described in numerous publications and patents [52, 57, 58]. 

A series of unsymmetrical oxo-squaraines 7, containing both heterocyclic and aniline-based end-groups, was synthesized and their absorption spectra were investigated [61]. Some of these dyes, absorbing between 680-820 nm with high molar... 

The noncovalent binding of a series of oxo-squaraine dyes 9a-e to BSA was evaluated by measurement of absorption, emission, and circular dichroism [63]. The magnitude of the association constants (Ks) for the dye-BSA complexes depended on the nature of the side chains and ranged from 34 x 103 to 1 x 107 M-1. Depending on the side chains, the Ks increase in the order [R1 = R2 = butyl-phthalimide] < R1 = R2 = cetyl] <[RJ = R2 = ethyl] <<[R = butyl-phthalimide, R2 = butyl-sulfonate] <<[RJ = R2 = butyl-sulfonate]. These dyes seem to interact mainly with a hydrophobic cavity on BSA. However, the association constants Ks increase substantially when the side chains are selected from butyl sulfonate. 

The quantum yields are 0.15-0.21 in ethanol and 0.01-0.02 in an aqueous medium, but in micelles, the quantum yields are five to tenfold increased. The aggregation of these dyes was studied in [53]. The amphiphilic squaraines 4 combine favorable photophysical properties and good solubility in aqueous media and in addition interact efficiently with micelles, and therefore have the potential to be used as NIR fluorescent sensors. However, our own investigations show that aniline-based squaraines lack chemical and photochemical stability when compared to oxo-squaraines with heterocyclic end-groups. 

Fluorescent labels based on oxo-squaraines were described in numerous articles and patents [45, 52]. Mono-reactive hydrophobic (32a) and hydrophilic (32b) squaraine labels containing one NHS ester group were synthesized by Terpetschnig et al. [62]. The initially low quantum yields and short fluorescence lifetime of 32b in aqueous solutions significantly increase after covalent binding to proteins. 

Dithio-squaraines 37 can be synthesized via thionation of the correspondent oxo-squaraines 5 with phosphor pentasulphide or Lawesson reagent (Fig. 8) [103, 104], However, dithio-squaraines containing carboxylic groups cannot be obtained by this method. Symmetrical indolenine- and benzothiazole-based monothio- (38) and dithio- (37) squaraines containing carboxy and sulfo groups are synthesized using 1,2-dithio- and tetrathiosquaric acid, respectively, while the reaction of these... 

Amino-squaraines 39 (Z1 = O, Z2 = R1R2) are synthesized by etherification of the oxo-squaraine oxygen using dimethylsulfate or methyl triflate followed by substitution of the methoxy group with amines [52] (Fig. 10). 

A series of symmetrical and unsymmetrical, hydrophobic and hydrophilic squaraine probes such as 41 (Table 1) with substituted squaraine ring oxygen was developed and compared to conventional oxo-squaraines 10a,b and 13b [18, 50, 98, 107]. The substituent on the squaraine ring have a strong influence on the spectral properties. Substitution of the squaraine oxygen by S, C(CN)2> C(CN)COOR, N(CN), N(OH), C(CN)[PO(OEt)2], indanedione, barbituric, and thiobarbituric acid causes red-shifted absorption and emission spectra [50]. 

The photostability of ring-substituted squaraines, especially dicyanomethylene-and f/no-squaraines is, in general, higher compared to oxo-squaraines and cyanines (Fig. 2). Increasing of the number of sulfo groups further improves the photostability of these dyes the photostability for hydrophilic dyes increases in the order 10b < 13b < Cy5 < 41h < 41i 41j < 41f < 41g [18]. Thio-squaraine dyes... 

Compared to oxo-squaraines or other ring-substituted squaraines, amino-squaraines 39 [45, 52, 112] have ionic character, similar to open-chain cyanine dyes, and due to the positive net charge, these dyes are to some extend water-soluble. Amino-squaraines absorb and emit at longer wavelength than the corresponding oxo-squaraines the absorption maxima are between 650-710 nm (eM = 85,000-300,000 M-1cm-1) [45, 112], The increase in solvent polarity is accompanied by a hypsochromic shift of the absorption. Amino-squaraine dyes are potentially used as fluorescent probes but because their photostability is inferior to those of oxo-squaraines and other ring substituted squaraines of similar structure, their applications are rather limited. 

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