Squaraine rotaxanes

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. 

Table 4 Spectral characteristics of squaraines and squaraine rotaxanes in chloroform [56, 58]...

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. 

Compared to 18, the anthrylene macrocycle 16b is a more promising building block for squaraine rotaxane fabrication and fluorescent probe development. 

An admixture of 16b and 17b self-assembles quantitatively at millimolar concentration in chloroform solution to produce an inclusion complex whose absorption and emission maxima are red-shifted by 40 nm [56]. Clicking both ends of this pseudorotaxane with two molar equivalents of stopper S2 produces the squaraine rotaxane 16b D 19b in near-quantitative yield [58]. Pseudorotaxane 16b D 17b partially dissociates at micromolar concentrations in chloroform and produces two emission peaks, one at 638 nm which corresponds to free squaraine 17b and one at 694 nm, corresponding to the pseudorotaxane. In contrast, the squaraine rotaxane 16b D 19b does not dissociate under these conditions or in more polar solvents such as pure methanol. 

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. 

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. 

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. 

Fu N, Baumes JM, Arunkumar E, Noll BC, Smith BD (2009) Squaraine rotaxanes with boat conformation macrocycles. J Org Chem 74 6462-6468... 

Johnson JR, Fu N, Arunkumar E, Leevy WM, Gammon ST, Piwnica-Worms D, Smith BD (2007) Squaraine rotaxanes superior substitutes for Cy-5 in molecular probes for near-infrared fluorescence cell imaging. Angew Chem Int Ed 46 5528-5531... 

Gassensmith JJ, Barr L, Baumes JM, Paek A, Nguyen A, Smith BD (2008) Synthesis and photophysical investigation of squaraine rotaxanes by clicked capping . Org Lett 10 3343-3346... 

Fu N, Gassensmith JJ, Smith BD (2009) Effect of stopper size on squaraine rotaxane stability. Supramol Chem 21 118-124... 

Figure 11.11 Squaraine dye is susceptible to nucleophilic attack (left), whereas the squaraine rotaxane is essentially inert (right)...

Figure 11.12 Montage from fluorescent movie showing division of bacteria cells labeled with squaraine rotaxane. Reprinted with permission from [39]. Copyright Wiley-VCH Verlag CmbH Co. KCaA...

Gassensmith JJ, Matthys S, Lee J-J, Wojcik A, Kamat PV, Smith BD (2010) Squaraine rotaxane as a reversible optical chloride sensw. Chem Eur J 16 2916-2921... 

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