Matthew E. McCarroll

Computational studies on response and binding selectivity of fluorescence sensors.

Using a computational strategy based on density functional theory calculations, we successfully designed a fluorescent sensor for detecting Zn(2+) [J. Phys. Chem. B 2006, 110, 22991-22994]. In this work, we report our further studies on the computational design protocol for developing Photoinduced Electron Transfer (PET) fluorescence sensors. This protocol was applied to design a PET fluorescence sensor for Zn(2+) ions, which consists of anthracene as the fluorophore connected to pyridine as the receptor through dimethylethanamine as the linker. B3LYP and time-dependent B3LYP calculations were performed with the basis set 6-31G(d,p), 6-31+G(d,p), 6-311G(d,p), and 6-311+G(d,p). The calculated HOMO and LUMO energies of the fluorophore and receptor using all four basis sets show that the relative energy levels remain unchanged. This indicates that any of these basis sets can be used in calculating the relative molecular orbital (MO) energy levels. Furthermore, the relative MO energies of the independent fluorophore and receptor are not altered when they are linked together, which suggests that one can calculate the MO energies of these components separately and use them as the MO energies of the free sensor. These are promising outcomes for the computational design of sensors, though more case studies are needed to further confirm these conclusions. The binding selectivity studies indicate that the predicted sensor can be used for Zn(2+) even in the presence of the divalent cation, Ca(2+).

Anilinomethylrhodamines: pH sensitive probes with tunable photophysical properties by substituent effect.

A series of pH dependent rhodamine analogues possessing an anilino-methyl moiety was developed and shown to exhibit a unique photophysical response to pH. These anilinomethylrhodamines (AnMR) maintain a colorless, nonfluorescent spirocyclic structure at high pH. The spirocyclic structures open in mildly acidic conditions and are weakly fluorescent; however, at very low pH, the fluorescence is greatly enhanced. The equilibrium constants of these processes show a linear response to substituent effects, which was demonstrated by the Hammett equation.

DIABLA: a new screening method for the discovery of protein targets.

Dynamic isoelectric/anisotropy binding ligand assay (DIABLA) is a new method to identify proteins in a complex sample that bind to a molecule of interest. This is accomplished by first using capillary isoelectric focusing (cIEF) to separate the proteins in a capillary based on their isoelectric point. This separation is performed while the compound being tested is present in the separation buffer. When the proteins are focused, the entire capillary is scanned to identify regions of nonzero anisotropy, which are locations where the test compound is interacting with a focused protein band. DIABLA was demonstrated by observing the binding of fluorescein-tagged progesterone to an MCF-7 breast cancer cell lysate. The proteins were tagged with rhodamine to permit their observation and then focused in the presence of the tagged progesterone. Anisotropy measurements show that progesterone binds to six different proteins bands in the sample.

Detection of target proteins by fluorescence anisotropy.

Understanding molecular interactions is critical to understanding most biological mechanisms of cells and organisms. In the case of small molecule–protein interactions, many molecules have significant biological activity through interactions with unknown target proteins and by unknown modes of action. Identifying these target proteins is of significant importance and ongoing work in our laboratories is developing a technique termed Dynamic Isoelectric Anisotropy Binding Ligand Assay (DIABLA) to meet this need. Work presented in this manuscript aims to characterize the fundamental parameters affecting the use of fluorescence anisotropy to detect target proteins for a given ligand. Emphasis is placed on evaluating the use of fluorescence anisotropy as a detection mechanism, including optimization factors that affect the protein detection limit. Effects of ligand concentration, pH, and nonspecific binding are also examined.

Spectroscopic Studies of Cyclodextrin Complexes with 2,5-Bis-(4-Methylphenyl)Oxazole:

The complexation of γ-cyclodextrins (γ-CDs) with the guest molecule 2,5-bis-(4-methylphenyl)oxazole (MPPO) has been studied by use of fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and dynamic light scattering (DLS). The changes in the 1H NMR aromatic signals of MPPO in aqueous γ-CD provide evidence of complexation and inclusion. DLS was used to confirm the existence of a distribution of the aggregates in the nanometer range. Excimer fluorescence was pronounced in the presence of γ-CD. The evidence of the existence of two species was shown through fluorescence lifetime data. The emission intensities of the steady state fluorescence and the fluorescence anisotropies of the linear aggregates revealed a phase transition temperature of 60 °C.

Measurement of Chiral Recognition in Cyclodextrins by Fluorescence Anisotropy

Publisher Summary This chapter discusses the chiral recognition in CD, which can be described as the discrimination between the two enantiomers of a chiral molecule. Despite the subtle differences between enantiomers, the biological effects of chiral drugs can be radically distinct for each enantiomer. The section on measurement of chiral recognition discusses various processes in meaning accurately the phenomenon of chiral recognition. The polarization based spectroscopic technique that has been widely used to study molecular interactions, is discussed with mathematical methods and fluorescence anisotropy. The determination of thermodynamic binding parameters in CD is explained with examples and schematic illustrations. The results from experiments are provided with illustration for CD types and the determination of binaphthyl phosphate (BNP) are given in form of a table. The studies presented in this chapter clearly demonstrate the potential that exists for using fluorescent anisotropy to study chiral recognition in CD-based systems, which has ramifications for both fundamental and applied perspectives.