Supramolecular capture of highly polar amidosquaraine dye in water with nanomolar affinity and large turn-on fluorescence.

A supramolecular dye-capture system comprising anionic amidosquaraine guest and macrocyclic tetralactam host exhibits nanomolar affinity and "turn on" visible fluorescence. Utility is demonstrated with a new fluorescent assay for liposome leakage induced by the biomedically important enzyme phospholipase A2.

Design, Synthesis, and Evaluation of a Luminescent Cholesterol Mimic.

The development of new chemical tools with improved properties is essential to chemical and cell biology. Of particular interest is the development of mimics of small molecules with important cellular function that allow the direct observation of their trafficking in a cell. To this end, a novel 15-azasterol has been designed and synthesized as a luminescent cholesterol mimic for the monitoring of cholesterol trafficking. The brightness of this probe, which is ∼32-times greater than the widely used dehydroergosterol probe, is combined with resistance to photobleaching in solution and in human fibroblasts and an exceptionally large Stokes-like shift of ∼150-200 nm. The photophysical properties of the probe have been studied experimentally and computationally, suggesting an intersystem crossing to the triplet excited state with subsequent phosphorescent decay. Molecular dynamics simulations show a similar binding mode of cholesterol and the azasterol probe to NPC proteins, demonstrating the structural similarity of the probe to cholesterol.

Paired Agent Fluorescence Imaging of Cancer in a Living Mouse Using Preassembled Squaraine Molecular Probes with Emission Wavelengths of 690 and 830 nm.

New methods are described for the construction of targeted fluorescence probes for imaging cancer and the assessment of tumor targeting performance in a living mouse model. A novel noncovalent assembly process was used to fabricate a set of structurally related targeted fluorescent probes with modular differences in three critical assembly components: the emission wavelength of the squaraine fluorochrome, the number of cRGDfK peptide units that target the cancer cells, and the length of the polyethylene glycol chains as pharmacokinetic controllers. Selective targeting of cancer cells was proven by a series of cell microscopy experiments followed by in vivo imaging of subcutaneous tumors in living mice. The mouse imaging studies included a mock surgery that completely removed a fluorescently labeled tumor. Enhanced tumor accumulation due to probe targeting was first evaluated by conducting Single Agent Imaging (SAI) experiments that compared tumor imaging performance of a targeted probe and untargeted probe in separate mouse cohorts. Although there was imaging evidence for enhanced tumor accumulation of the targeted probe, there was moderate scatter in the data due to tumor-to-tumor variability of the vasculature structure and interstitial pressure. A subsequent Paired Agent Imaging (PAI) study coinjected a binary mixture of targeted probe (with emission at 690 nm) and untargeted probe (with emission at 830 nm) into the same tumor-burdened animal. The conclusion of the PAI experiment also indicated enhanced tumor accumulation of the targeted probe, but the statistical significance was much higher, even though the experiment required a much smaller cohort of mice. The imaging data from the PAI experiment was analyzed to determine the targeted probe's Binding Potential (BP) for available integrin receptors within the tumor tissue. In addition, pixelated maps of BP within each tumor indicated a heterogeneous spatial distribution of BP values. The results of this study show that the combination of fluorescent probe preassembly and PAI is a promising new way to rapidly develop targeted fluorescent probes for tumors with high BP and eventual use in clinical applications such as targeted therapy, image guided surgery, and personalized medicine.

High affinity threading of a new tetralactam macrocycle in water by fluorescent deep-red and near-infrared squaraine dyes.

A new tetralactam macrocycle was prepared and found to encapsulate deep-red and near-infrared squaraine and croconaine dyes in water with tunable threading kinetics. The new supramolecular paradigm of guest back-folding was used to increase macrocycle/squaraine affinity by 370-fold and achieve an association constant of 2.8 × 109 M-1.

New tetralactam hosts for squaraine dyes.

The photophysical properties of a deep-red fluorescent squaraine dye can be improved by encapsulating it within a tetralactam macrocycle. Three new tetralactams are described with different substituents (methyl, methoxy, methylenedioxy) on the macrocycle aromatic sidewalls. The capability of each tetralactam to encapsulate a squaraine dye in chloroform solution was determined experimentally using absorption, fluorescence, and NMR spectroscopy. Two of the tetralactams were found to thread a squaraine dye with association constants on the order of 106 M-1, while a third macrocycle exhibited no squaraine affinity. An X-ray crystal structure of the third tetralactam showed that the substituents sterically blocked squaraine association. Of the two tetralactams that encapsulate a squaraine, one induces an increase in squaraine fluorescence quantum yield, while the other quenches the squaraine fluorescence. The results suggest that these new squaraine binding systems will be useful for biological imaging and diagnostics applications.

Synthesis and Structure of 3,3-Dimethylindoline Squaraine Rotaxanes.

Squaraine rotaxanes are mechanically interlocked molecules comprised of a dumbbell shaped squaraine dye inside a tetralactam macrocycle. Previous squaraine rotaxanes have employed planar squaraine dyes with 4-aminophenyl, 2-aminothiophene, or N-amino units appended to the central C4O2 core. Here we describe two rotaxanes that encapsulate a 3,3-dimethylindoline squaraine inside a tetralactam with anthracene sidewalls. The rotaxanes were prepared by a templated clipping reaction and an X-ray crystal structure shows that the squaraine gem-dimethyl groups force a relatively wide separation between the macrocycle anthracene sidewalls. The decreased interaction between the encapsulated squaraine and the anthracene sidewalls leads to a smaller red shift of the squaraine absorption and emission bands. Solution-state studies show that the gem-dimethyl groups in 3,3-dimethylindoline squaraine dyes are large enough to prevent macrocycle threading or rotaxane unthreading. One of the new rotaxanes emits an orange light (560-650 nm), and there is a 10-fold enhancement in the squaraine fluorescence quantum yield upon encapsulation as a rotaxane. This orange-emitting dye completes the palette of known squaraine rotaxane fluorophores whose emission profiles span the color range from green to near-infrared.