Supramolecular Chemistry Using Fluorescent Unsymmetric Squaraine Dyes

Squaraine dyes are a class of fluorescent compounds well-suited for biological applications due to their favorable photophysical properties. Squaraines exhibit moderate fluorescence quantum yields in aqueous conditions with sharp and intense peaks in the near-infrared (NIR) region, 650-900 nm, that is critical for high-performance biological imaging. Chapter 1 of this thesis provides an overview of the photophysical and supramolecular chemistry of squaraine dyes. Chapter 2 describes squaraine rotaxanes, mechanically interlocked molecules comprised of a dumbbell-shaped 3,3-dimethylindoline squaraine dye inside a tetralactam macrocycle 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. 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.

Chapter 3 describes two related fluorescent probes that are molecular conjugates of one or two zinc dipicolylamine (ZnDPA) coordination complexes with an appended solvatochromic benzothiazolium squaraine dye. The probes were designed to target the anionic phospholipid, phosphatidylserine (PS), that is exposed on the surface of dead and dying cells. A series of spectrometric and microscopy studies using liposomes and red blood cell ghosts as models showed that the probe with two ZnDPA targeting units produced higher affinity, stronger fluorescence “turn-on” effect, and better image contrast than the probe with one ZnDPA. Both fluorescent probes enabled “no-wash” time-lapse microscopic imaging of mammalian cell death within a culture. The probe with two ZnDPA units was used for non-invasive time-lapse imaging of cell death during the development of Xenopus laevis(frog) embryos. In vivofluorescence micrographs revealed probe accumulation within the embryo tail, head and spine regions that were undergoing regression and apoptosis during growth and maturation.

Chapter 4 describes four new first-generation solvatochromic squaraine dyes with unsymmetric structures. Macrocycle threading of the dyes in chloroform produces a relatively small 20 nm red-shift in absorption wavelength. In contrast, macrocycle threading in water produces ~80 nm red-shift in absorption wavelength, indicating a large change in polarity surrounding the solvatochromic squaraine as it transfers from bulk water solution to the relatively non-polar macrocycle interior and a very large 70-fold enhancement of squaraine fluorescence. Kinetic studies of macrocycle threading by a squaraine with an N-methyl substituent at the end of the structure was 5 million times faster than threading by a squaraine with an N-propyl substituent. This sensitive substituent effect permits structural fine-tuning of macrocycle/squaraine threading kinetics, which is a very useful way to pre-assemble fluorescent probes for biological imaging.

Finally, Chapters 5 and 6 explore second-generation unsymmetrical dyes and the results show a pathway that improves the association constants for macrocycle threading in water from micromolar to nanomolar affinities. These dye threading systems are likely to be useful for in situ capture applications.