Pyrene Excimer Formation: A Tool to Study Macromolecular Conformations in Solution

Abstract

The current view for fluorescence collisional quenching (FCQ) experiments is that no quantitative information can be retrieved from macromolecules containing more than a single dye-quencher pair attached at two specific positions on a macromolecule. This holds true for pyrene excimer formation (PEF), a well-established FCQ phenomenon, where an excimer is produced through the encounter between an excited and a ground-state pyrenyl labels attached onto a macromolecule. In contrast, recent studies suggest that the analysis of fluorescence decays acquired with macromolecules containing many pyrenyl labels with the model free analysis (MFA) and florescence blob model (FBM) yields quantitative information about the internal dynamics and local density of macromolecules in solution. The underlying physical principle enabling the MFA and FBM to probe macromolecules in this manner is based on the direct relationship existing between the average rate constant (<k>) for PEF and the local concentration ([Py]loc) of pyrenyl labels on the macromolecule. Yet, and despite its importance, no study has conclusively validated this relationship. This is due, in part, to the difficulty in determining [Py]loc for pyrene-labeled macromolecules (PyLM) and benchmarking this methodology against other experimental techniques. In the present thesis, this fundamental relationship was demonstrated with a series of polyamidoamine (PAMAM) dendrimers of generations GY (=0, 1, or 2) that had been labeled with pyrene derivatives having different numbers X (= 4, 8, or 12) of carbon atoms in the pyrenyl linker to yield the PyCX-PAMAM-GY samples. The fluorescence decays were acquired in N,Ndimethylformamide (DMF) and dimethylsulfoxide (DMSO) and analyzed with the MFA to retrieve <k>, which was compared to [Py]loc obtained by assuming that the internal segments of the PyCX-PAMAM-GY samples linking the pyrenyl labels obeyed Gaussian statistics. The direct relationship found between <k> and [Py]loc for the PyCX-PAMAM-GY samples provided a vi validation for this assumption and demonstrated that PEF can be employed to probe the conformation of macromolecules in solution. Subsequently, PEF was applied to probe the conformational changes induced by protonating the internal tertiary amines of the PyCX-PAMAM-GY samples, showcasing PEF's ability to study these conformational changes intramolecularly, a feat difficult to achieve by traditional methods used for characterizing macromolecular conformations in solution. Expanding beyond dendrimers, PEF was applied to study the conformation of larger macromolecules like poly(glutamic acid) (PGA) and polynorbornene (PNb) on different length scales by using 1-pyrenealkylamines with varied alkyl side chains. The fluorescence blob model (FBM) was applied to determine the number (Nblob exp) of structural units within a blob, the volume probed by an excited pyrenyl label, taken as a measure of the local macromolecular density. Comparison of Nblob exp with Nblob MMO obtained through molecular mechanic optimizations (MMOs) validated PEF's ability to probe macromolecular conformations over different length scales. The conformation of the Py-PGA constructs was found to remain unchanged when probed with 1-pyrenealkylamines having different linker lengths reflecting a homogeneous conformation over different length scales. In contrast, the Py-PNb samples appeared helical and randomly coiled for the 1-pyrenealkylamines with a shorter and longer linker, respectively, highlighting the potential of PEF at probing complex macromolecules with heterogeneous conformation across various length scales. In conclusion, this thesis further supports the applicability of PEF as a robust experimental technique for probing the conformations and internal dynamics of macromolecules in solution.