Excited State Rotational Freedom Impacts Viscosity Sensitivity in Arylcyanoamide Fluorescent Molecular Rotor Dyes.

The microviscosity of intracellular environments plays an important role in monitoring cellular function. Thus, the capability of detecting changes in viscosity can be utilized for the detection of different disease states. Viscosity-sensitive fluorescent molecular rotors are potentially excellent probes for these applications; however, the predictable relationships between chemical structural features and viscosity sensitivity are poorly understood. Here, we investigate a set of arylcyanoamide-based fluorescent probes and the effect of small aliphatic substituents on their viscosity sensitivity. We found that the location of the substituents and the type of π-network of the fluorophore can significantly affect the viscosity sensitivity of these fluorophores. Computational analysis supported the notion that the excited state rotational energy barrier plays a dominant role in the relative viscosity sensitivity of these fluorophores. These findings provide valuable insight into the design of molecular rotor-based fluorophores for viscosity measurement.

Development of fluorophores for the detection of oligomeric aggregates of amyloidogenic proteins found in neurodegenerative diseases.

Alzheimer's disease and Parkinson's disease are the two most common neurodegenerative diseases globally. These neurodegenerative diseases have characteristic late-stage symptoms allowing for differential diagnosis; however, they both share the presence of misfolded protein aggregates which appear years before clinical manifestation. Historically, research has focused on the detection of higher-ordered aggregates (or amyloids); however, recent evidence has shown that the oligomeric state of these protein aggregates plays a greater role in disease pathology, resulting in increased efforts to detect oligomers to aid in disease diagnosis. In this review, we summarize some of the exciting new developments towards the development of fluorescent probes that can detect oligomeric aggregates of amyloidogenic proteins present in Alzheimer's and Parkinson's disease patients.

Exploring the Effect of Aliphatic Substituents on Aryl Cyano Amides on Enhancement of Fluorescence upon Binding to Amyloid-ß Aggregates.

The self-assembly of amyloid-ß (Aß) peptides into amyloid aggregates is a pathological hallmark of Alzheimer's Disease. We previously reported a fluorescent Aryl Cyano Amide (ARCAM) probe that exhibits an increase in fluorescence emission upon binding to Aß aggregates in solution and in neuronal tissue. Here, we investigate the effect of introducing small aliphatic substituents on the spectroscopic properties of ARCAM both free in solution and when bound to aggregated Aß. We found that introducing substituents designed to hinder the rotation of bonds between the electron donor and acceptor on these fluorophores can affect the overall brightness of fluorescence emission of the probes in amyloid-free solutions, but the relative fluorescence enhancement of these probes in amyloid-containing solutions is dependent on the location of the substituents on the ARCAM scaffold. We also observed the capability to tune the excitation or emission wavelength of these probes by introducing electron-donating or -withdrawing substituents that putatively affect either the energy required for photoexcitation or the stability of the photoexcited state. These studies reveal new design principles for developing ARCAM-based fluorescent Aß-binding probes with an enhanced fluorescence signal compared to background and tunable spectroscopic properties, which may lead to improved chemical tools for aiding in the diagnosis of amyloid-associated neurodegenerative diseases.

Method to discriminate amyloids using fluorescent probes.

The aggregation of misfolded proteins into amyloids is a common characteristic of many neurodegenerative and non-neurologic diseases. Fluorescent amyloid-targeting probes that discriminate amyloids based on differences in protein composition can provide rapid information to aid in disease diagnosis. In this chapter, we present protocols for the synthesis and use of ANCA-11 as an environmentally-sensitive amyloid-targeting probe that can fluorescently discriminate between amyloids with different disease origin. We also present a protocol for preparing amyloid samples of synthetic Amyloid-ß(1-42), as problems with amyloid preparations can be a large driver of time and cost for research. The methods presented here can be generalized for evaluation of other amyloid-targeting fluorescent probes with different aggregates of amyloidogenic proteins in solution or in tissue.

Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing.

The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of a general method to predict their photophysics, a problem that is especially acute when base pairing and stacking change fluorescence. To better understand these effects, a series of tricyclic cytidine (tC and tCO ) analogues ranging from electron-rich to electron-deficient was designed and synthesized. They were then incorporated into oligonucleotides, and photophysical responses to base pairing and stacking were studied. When inserted into double-stranded DNA oligonucleotides, electron-rich analogues exhibit a fluorescence turn-on effect, in contrast with the electron-deficient compounds, which show diminished fluorescence. The magnitude of these fluorescence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover, matched base pairing enhances fluorescence turn-on for the electron-rich compounds, and it causes a fluorescence decrease for the electron-deficient compounds. For the tCO compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering a potential new tool for studying nucleic acid structure and dynamics. These results, supported by DFT calculations, help to rationalize fluorescence changes in the base stack and will be useful for selecting the best fluorescent nucleoside analogues for a desired application.

Fluorescence Turn-On Sensing of DNA Duplex Formation by a Tricyclic Cytidine Analogue.

Most fluorescent nucleoside analogues are quenched when base stacked and some maintain their brightness, but there has been little progress toward developing nucleoside analogues that markedly increase their fluorescence upon duplex formation. Here, we report on the design and synthesis of a new tricyclic cytidine analogue, 8-diethylamino-tC (8-DEA-tC), that responds to DNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. These results, supported by DFT calculations, suggest a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

Mechanically induced pyrogallol[4]arene hexamer assembly in the solid state extends the scope of molecular encapsulation.

Pyrogallol[4]arene hexamers are hydrogen-bonded molecular capsules of exceptional kinetic stability that can entrap small molecule guests indefinitely, without exchange, at ambient temperatures. Here, we report on the use of a ball mill to induce self-assembly of the capsule components and the guests in the solid state. Stoichiometric amounts of pyrogallol[4]arene and a guest, which can be an arene, alkane, amine, or carboxylic acid, were milled at 30 Hz for fixed durations, dissolved, and characterization by NMR. Most of the resulting encapsulation complexes were kinetically stable but thermodynamically unstable in solution, and the yield of their formation correlates with the duration of the milling and is related to the structures of guest and host. This method extends the scope of molecular encapsulation, as demonstrated by the preparation of kinetically trapped encapsulation complexes of [2.2]paracyclophane, for which we could find no other method of preparation. To gain mechanistic insights into the solid-state assembly process, we characterized the milled powders using 13C CP-MAS NMR, we studied the effects of changing the alkane domain of the host, and we examined how dissolution conditions impact on the distribution of observed encapsulation complexes once in solution. The results support a mechanism comprising mechanically induced solid-state reorganization to produce a mixture rich in nearly or fully assembled guest-filled capsules.