Dye-rotaxanes

Reaction of 3-(9-julolidinyl)prop-2-en-l-al with N-( -adamantyl)-4-methyl-pyr-idinium chloride and a-CD in aqueous sodium hydroxide yielded styryl dye rotaxanes 6a and 7a as well as the free dye 8a (Fig. 5) [4, 27]. Analogously, the two rotaxane isomers 6b and 7b, and the free dye 8b were obtained from julolidine aldehyde and 4-methyl-2,6-diphenylpyridinium chloride. As compared to the hydrophobic dyes 8a, 8b, the rotaxanes 6a, 6b and 7a, 7b are highly soluble in water. The absorption/emission maxima of the rotaxanes 6a (525/710 nm) and 7a (535/718 nm) in DMSO are red-shifted compared to free styryl dye 8a... 

Park JS, Wilson JN, Hardcastle KI, Bunz UHF, Srinivasarao M (2006) Reduced fluorescence quenching of cyclodextrin-acetylene dye rotaxanes. J Am Chem Soc 128 7714—7715... 

Buston JEH, Marken F, Anderson HL (2001) Enhanced chemical reversibility of redox processes in cyanine dye rotaxanes. Chem Commun 11 1046—1047... 

Scheme 3 Synthesis of an azo-dye rotaxane 3 (RD) with 8-hydroxyquinoline as coupling components...

The pioneering dye rotaxane synthesis, reported by Anderson and coworkers in 1996, utilized Glaser coupling under aqueous conditions in the presence of a water soluble cyclophane macrocycle to produce a mixture of [2] and [3]rotaxanes with a conjugated phenylacetylene fiuorophore as the axle component (Figure 11.8). The purpose of the encapsulation was to insulate the conjugated tr-system from quenching processes and both rotaxanes were found to be six fold more fluorescent. 

As discussed in Section 7.3, conventional free radical polymerization is a widely used technique that is relatively easy to employ. However, it does have its limitations. It is often difficult to obtain predetermined polymer architectures with precise and narrow molecular weight distributions. Transition metal-mediated living radical polymerization is a recently developed method that has been developed to overcome these limitations [53, 54]. It permits the synthesis of polymers with varied architectures (for example, blocks, stars, and combs) and with predetermined end groups (e.g., rotaxanes, biomolecules, and dyes). 

Compared to the absorption and emission spectra of the free dye, the spectra of the rotaxane 5 C a-CD PF6 are sharper and red-shifted. The absorption maximum... 

Analysis of the available literature data on host-guest complexes based on cyanine and styryl dyes with CDs shows that rotaxane formation in general... 

References [52-54] do not include any data directly comparing squaraine rotaxanes with common cyanine dyes such as Cy5 (GE Healthcare) and Alexa 647 (Life Technologies). Nevertheless, from the available data it can be concluded that squaraine rotaxanes are remarkably resistant to chemical and photochemical degradation, and likely to be very useful as a versatile fluorescent scaffold for constructing various types of highly stable, red and near infrared (NIR) imaging probes and labels. 

Squaraine rotaxane dyes also were utilized as extremely bright and highly stable NIR fluorescent probes for in vitro and in vivo optical imaging of live and fixed cells [55]. These probes were modified for targeting of different cellular locations, namely,... 

The relatively nonpolar squaraine rotaxane 14c was found to interact with cells in a very similar way to the well-known lipophilic dye Nile Red this probe rapidly accumulates at lipophilic sites inside a living cell, such as the endoplasmic reticulum and intracellular lipid droplets [55], The red emission band for probe 14c is quite narrow and permits the acquisition of multicolor images. It displayed high chemical stability and low toxicity. 

The squaraine rotaxane tetracarboxylic acid 15a is soluble in aqueous solution at physiological pH and acts as an excellent fluorescent marker with extremely high photostability, which allows trafficking processes in cells to be monitored in realtime, with constant sample illumination, over many minutes. This type of real-time monitoring cannot be done with other available NIR fluorescent probes, such as the amphiphilic styryl dye KM4-64 and water-soluble dextran-Alexa 647 conjugate, because they are rapidly photobleached. 

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 17b and 17c have terminal acetylene residues, which allowed to convert the squaraine dyes and tetralactam macrocycles into permanently interlocked rotaxane structures using copper-catalyzed and copper-free cycloaddition reactions with bulky stopper groups [58]. 

The conversion of squaraine 19a to the rotaxane 18 D 19a causes a modest red-shift only in both absorption (10 nm) and emission (7 nm) but an approximately threefold decrease in quantum yield. The addition of two triazole rings (dye 19b) did not significantly alter the quantum yield of 17b (Table 4). A macrocycle-induced quenching effect was verified by fluorescence titration experiments adding aliquots of 18 to a solution of squaraine 17b in methylene chloride [58]. Treatment of the 18 d 17b psuedorotaxane system with the tetrabutylammonium salts of chloride, acetate, or benzoate leads to the displacement of squaraine 17b from the macrocyclic cavity and the nearly complete restoration of its fluorescence intensity. The 18-induced quenching of 17b does not support the utility of this system as a bioimaging probe however, the pseudorotaxane system 18 Z> 17b acts as an effective and selective anion sensor with NIR fluorescence. 

The squaraine rotaxane 16b z> 19c was also produced in near-quantitative yield by heating of a mixture of bis-azide squaraine dye 17d, macrocycle 16b, and stopper S3 [58]. The compound is stable enough for immediate characterization but slowly decomposes upon exposure to light. 

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. 

Some of the spectral properties of squaraine rotaxanes are given in Table 5. It can be seen that encapsulation of squaraines 22a-22d with heterocyclic end-groups in a tetralactam macrocycle results in a small blue-shift of the absorption and emission maxima while the encapsulation of squaraines 22e and 21a leads to red-shifted rotaxanes [60]. Importantly, encapsulation in a tetralactam macrocycle has a positive effect not only on the photostability (Figs. 9 and 10) of these dyes but also on the quantum yields (<2>F) and fluorescence lifetimes (Tmean)- Embedding of any type squaraines in tetralactam rotaxane system increases [Pg.175]

Table 5 Photophysical characteristics of squaraine dyes, squaraine rotaxanes, and IgG conjugates [60]...

The clipping reaction used in [52, 53, 55] to synthesize tetralactam-based squaraine rotaxanes such as 14 and 15 afforded only moderate yields (ca. 28-35%) of the rotaxanes, possibly because of the unavoidable presence of nucleophiles that react with the chemically unstable squaraines during the reaction. The slippage approach [62] minimizes the squaraine dye s contact with nucleophiles during the rotaxane formation process and therefore can be used to efficiently encapsulate a squaraine dye such as 23 in a macrocycle such as 25 [63],... 

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. 

Buston JEH, Young JR, Anderson HL (2000) Rotaxane-encapsulated cyanine dyes enhanced fluorescence efficiency and photostability. Chem Commun 11 905-906... 

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