Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging

Abstract

A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with twelve bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 hours in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation.

Synthetic mimics of biotin/(strept)avidin

“…The first reported example of this approach to probe pre-assembly is shown in Figure 16. 49 A tetralactam macrocycle was threaded onto a fluorescent PEGylated squaraine scaffold containing one or two squaraine docking stations. Appended to the macrocycle periphery were six iminodiacetate units that have weak affinity for bone surfaces.…”

“…( bottom ) Pre-assembly of near-infrared fluorescent molecular probes with multivalent affinity for bone surfaces. (Adapted with permission from ref 48 and 49. Copyright 2016, American Chemical Society).…”

Non‐Covalent Assembly Method that Simultaneously Endows a Liposome Surface with Targeting Ligands, Protective PEG Chains, and Deep‐Red Fluorescence Reporter Groups

“…In short, a deep‐red fluorescent squaraine dye is encapsulated by a water‐soluble tetralactam macrocycle to give an extremely stable, threaded complex that is well suited for fluorescence imaging. To date, we have pre‐assembled fluorescent molecular probes that selectively target bone, dead/dying mammalian tissue, and various microbial species . One of the major advantages of Synthavidin self‐assembly is the capability of forming threaded complexes with long PEG chains appended to the ends of the squaraine dye .…”

Squaraine‐Based Polymers: Toward Optimized Structures for Optoelectronic Devices

“…Among these materials, squaraine dyes have been the subject of many studies because of their remarkable donor–acceptor–donor (D–A–D) type structure, enabling systems with very small optical band gaps, and ultimately, unique photochemical/photophysical properties . Applications include organic solar cells, nonlinear optics, photodetectors, light‐emitting/ambipolar field‐effect transistors (FETs), and fluorescent labels/sensors for a wide range of analytes . Key attractions of squaraine dyes include facile synthetic access, a wide variety of possible structural motifs, and remarkable oxygen and moisture stability, which make it possible to avoid inert atmosphere‐based device fabrication steps.…”