Squaraines applications

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]. 

Symmetrical and unsymmetrical quinaldine-based squaraines 14 linked to cellular recognition elements that exhibit near-infrared absorption (>740 nm) could have potential biological and photodynamic therapeutical applications [68]. 

Dialkylanthracene-containing squaraine dyes 17 show intense absorption and emission in the NIR region (720-810 nm) [74]. They are compatible with aqueous environments and show substantial enhancement of quantum yields and fluorescence lifetimes in hydrophobic and micellar media, suggesting that these dyes can be potentially useful as fluorescent probes in biological applications, e.g., for imaging of hydrophobic domains such as cell membranes. 

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. 

Due to their low sensitivity toward the environment, cyanine dyes are perfect candidates as fluorescent labels. Squaraine dyes on the other hand display a highly environment-sensitive response and are therefore not only useful as fluorescent probes and labels but also, in particular, well-suited for lifetime-based applications. 

Terpetschnig E, Szmacinski H, Ozinskas A, Lakowicz JR (1994) Synthesis of squaraine-iV-hydroxysuccinimide esters and their biological application as long-wavelength fluorescent labels. Anal Biochem 217 197-204... 

Suzuki Y, Yokoyama K (2007) A protein-responsive chromophore based on squaraine and its application to visual protein detection on a gel for SDS-PAGE. Angew Chem Int Ed 46 4097 1099... 

Ros-Lis JV, Garcia B, Jimenez D, Martmez-Manez R, Sancenon F, Soto J, Gonzalvo F, Valldecabres MC (2004) Squaraines as fluoro-chromogenic probes for thiol-containing compounds and their application to the detection of biorelevant thiols. J Am Chem Soc 126 4064-4065... 

Umezawa K, Citterio D, Suzuki K (2008) Water-soluble NIR fluorescent probes based on squaraine and their application for protein labeling. Anal Sci 24 213-217... 

This chapter describes the synthesis, properties, and biomedical applications of cyanine and squaraine dyes encapsulated in CDs, CBs, Leigh-type tetralactam macrocycles, aptamers, and micro- or nano-particles. The optical and photochemical properties of supramolecular guest-host nanostructures that are based on intra-and intermolecular complexes of crown-containing styryl dyes with metal cations, and aggregates of carbocyanine dyes are discussed in a separate review [18]. 

With the example of stained E. coli cells, the squaraine rotaxane 15b containing a zinc(II)-dipicolylamine (Zn-DPA) ligand, which is known to selectively associate with the anionic surfaces of bacterial cells, was found to be almost 100 times more photostable as compared to Cy5-Zn-DPA [55]. This can be attributed to stronger cell-surface affinity of 15b, leading to a slower off rate for the probe. The remarkable stability of 15b permits fluorescence imaging experiments that are impossible with probes based on conventional NIR cyanine dyes such as Cy5. Squaraine rotaxanes are likely to be superior substitutes for conventional cyanine dyes for biomedical imaging applications that require NIR fluorescent probes. 

Squaraines 17a-17c were encapsulated in these macrocyles to form the corresponding pseudorotaxanes. Squaraine rotaxanes 14 and 15 with a phenylene tetralactam macrocycle have absorption/emission profiles (Table 3) that closely match those of Cy5, whereas squaraine rotaxanes 16 D 17 with an anthrylene macrocycle have a red-shifted absorption/emission that matches that of the homologous cyanine Cy5.5 (Table 4). These rotaxanes should be useful for fluorescence microscopy imaging applications. 

The squaraine rotaxanes based on the macrocycle 16b exhibit intense NIR absorption and emission maxima, and it should be possible to develop them into molecular probes for many types of photonic and bioimaging applications. In contrast, the squaraine fluorescence intensity is greatly diminished when the dye is encapsulated with macrocycle 18. The fluorescence is restored when a suitable anionic guest is used to displace the squaraine dye from a pseudorotaxane complex, which indicates that the multicomponent system might be applicable as a fluorescent anion sensor. 

A variety of hydrophobic and hydrophilic squaraine rotaxane probes and labels such as 21a-21e c Rp and 22a-22e c Rp, containing reactive carboxylic functionalities and hydrophilic sulfo groups, are disclosed in a recent patent application [60]. It was shown that not only aniline-based squaraines 21a-21e but also heterocyclic squaraines 22a-22e can form stable pseudorotaxane complexes. The indo-lenine-based squaraine 22a forms rotaxane 22a C Rp. Importantly, also the sulfonated squaraine 22b could be successfully encapsulated in a Leigh-type, phenylene-based, tetralactam macrocycle to yield the water-soluble rotaxane 22b C Rp. Quatemized, indolenme-based squaraines do not form pseudorotaxanes probably because of sterical hindrance caused by /V-alkyl and 3,3 -dimethyl groups. On the other hand, quatemized benzothiazole (22c) and benzoselenazole (22d) squaraines could be embedded in a Leigh-type macrocycle to yield rotaxanes 22c C Rp and 22d C Rp, respectively. The hydrophilic, mono-reactive rotaxane 22e-NHS C Rp based on asymmetric squaraine, synthesized by a cross-reaction of squaric acid with the two different indolenines, was also obtained. 

An overview of the synthesis, structure, photophysical properties, and applications of squaraine rotaxanes as fluorescent imaging probes and chemosensors is provided in a recent review [67]. Although a variety of squaraine dyes form rotaxanes with the molecular cage 25 or with a tetralactam macrocyclic system introduced by Leigh and co-workers [16, 17], there is no evidence in the literature that conventional cyanine dyes can be embedded in these macrocycles. 

Squaraines can be readily synthesized via the reaction of squaric acid (Treibs and Jacob, 1965) or its diester (Law and Bailey, 1986) with aromatic amines. For photoreceptor applications, Law and Bailey (1987) reported that the best results were obtained with squaraines prepared via the diester. This has been ascribed to the formation of a different crystal modification with lower impurity concentrations. Other purification methods have been reported by Lin and... 

A squaraine prepared from N-chlorobenzyl-N-methylaniline and squaric acid has found utility as a generation layer in a dual-layer photoreceptor with an inverted structure for positive charging applications (Yamamoto et al 1986). The photoreceptor showed full-process stability. 

Molecular complexes, such as the complex formed between poly(N-vinylcaibazole) and 2,4,7-trinitro-9-fluorenone, and dye-polymer aggregates were widely used as generation materials in many early applications. Since these materials are not infrared sensitive, there has been increasing emphasis on pigments. The more widely studied are various azo, phthalocyanine, squaraine, and peiylene diimide derivatives. A common feature of all of these materials is that they are polymorphic and exist in many different crystal forms. The properties are thus very sensitive to the conditions used in their preparation. Further, the sensitivity of these materials is strongly field dependent as well as dependent on the transport material. For a review of generation materials, see Law (1993). 

The absorption, emission, and redox properties of squaraines make them highly suited for applications as photosensitizers. In view of this, the early studies on squaraines were focused on thin photovoltaic and semiconductor photosensitization properties [1,4,5,91-97], Champ and Shattuck [98] first demonstrated that squaraines could photogenerate electron-hole (e-h) pairs in bilayer xerographic devices. Subsequently, extensive work has been carried out on the xerographic properties of squaraines [2,24,34,47,48,99,100], and these properties have been reviewed recently [11]. In an extensive smdy on the correlation s between cell performance and molecular structure in organic photovoltaic cells, squaraines were found to have much better solar energy conversion efficiencies than a variety of other merocyanine dyes [4,5]. 

Similar to other triptycene-derived macrocyclic arenes, triptycene-derived tetralactam macrocycles also had fixed conformations with large electron-rich cavities, which made them promising candidates as the host for some electron-deficient guests with comparatively large sizes. Squaraines [26] were a family of fluorescent dyes with specific near-IR photophysical properties, which had wide potential applications. However, their instability limited the utilization of them, and thus improving their chemical stability and the photophysical properties were the key to applications of squaraines [27]. Consequently, we [25, 28] found that macrocycles 35a-b could form a new kind of stable pseudorotaxane-type complexes with the squaraine in both solution and solid state. We further studied the chemical stability of squaraine in these complexes, and found that free guest 35b underwent hydrolytic decomposition to turn colorless in polar THF-water solvent in 4 days, but for squaraine 36b (Fig. 18.15) in complexation with 35a-b, its blue colors could be retained for several weeks. This observation revealed that the formation of complexes could efficiently protect the squaraine dyes from polar solvents. 

Squaraines with ring substituents which absorb and emit in the red and near-infrared regions have also been developed [72]. When compared to conventional Cy5—NHS systems (open chain), these derivatives have several advantages. For example, these compounds can be excited not only with red lasers but also with blue lasers or with luminescent diodes being useful compounds, they can be excited all over the visible range of the electromagnetic spectrum. These compounds are particularly useful in biomedical applications due to their favorable spectral and photophysical properties, such as fluorescence [72]. 

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