|dc.description.abstract||Information about long-range polymer chain dynamics (LRPCD) can be obtained by attaching a luminophore and its quencher onto a polymer and monitoring the rate at which the luminophore is quenched. Since a quenching event represents an encounter between the luminophore and the quencher, the rate of quenching reflects the dynamics undergone by those polymer segments bearing the fluorescent probes. Following a long tradition, pyrene is considered to be the luminophore of choice for conducting these experiments due to pyrene’s ability to form an excimer upon encounter between an excited pyrene and a ground-state pyrene. In effect, pyrene acts as both the luminophore and the quencher. Unfortunately, pyrene is highly hydrophobic, which prevents its use to probe the chain dynamics of water-soluble polymers. This study is an attempt at circumventing the problems associated with the hydrophobicity of pyrene by covalently labeling a water-soluble poly(L-lysine) (PLL) with a ruthenium complex and dinitrobenzene to act as water-soluble luminophore and quencher, respectively.
A phenanthroline derivative of the ruthenium bipyridyl complex Ru(bpy)₃⁺⁺, namely ruthenium (II) bisbipyridine 5-aminophenanthroline chloride (RuNH₂), was synthesized to use as a luminophore. Phenanthroline is a ligand bearing a reactive amine, which after modification into an isothiocyanate allows the attachment of the ruthenium complex (RuNCS) onto PLL. The two positive charges of ruthenium enhance the solubility of the luminophore in aqueous solution. In addition, the observed lifetime of RuNH2 at 20 °C is ~0.6 μsec which provides a long enough time window to probe the LRPCD of PLL.
PLL was randomly labeled with the luminophore, RuNCS, and a quencher, 1-fluoro-2,4-dinitrobenzene (FDNB) to yield Ru-PLL-Q. The Ru-PLL-Q samples were characterized and the labeling level of the different Ru-PLL-Q samples was determined. Next, aqueous solutions of the Ru-PLL-Q samples were prepared at different pH’s, their time-resolved fluorescence decays were acquired and analyzed with the Fluorescence Blob Model (FBM) that accounts for the random distribution of the labels along the chain. The fluorescence quenching experiments yield a measure of the internal dynamics of PLL which appear to slow down when the pH is increased from 3 to values larger than 5. Slower internal dynamics at higher pH’s are expected since PLL adopts an α-helical conformation, as evidenced by circular dichroism experiments. Molecular mechanics optimizations suggest that the distance over which quenching of the Ru complex occurs is compatible with the geometry of the PLL construct. These preliminary results suggest that a ruthenium complex and dinitrobenzene have the potential to offer an attractive alternative to the use of pyrene to study the chain dynamics of water-soluble polymers quantitatively.||en