Several pyrene-labeled polymers and polypeptides were synthesized and their chain dynamics were characterized using steady-state and time-resolved fluorescence techniques. Firstly, four series of pyrene-labeled polystyrenes (Py-PS) were synthesized to determine the effect that the method used for pyrene attachment, the linker connecting pyrene to the backbone, and the distribution of pyrene along the backbone all have on excimer formation. It was found that the amount of excimer formed was different in each case. The differences were described by utilizing the fluorescence blob model (FBM) analysis of the time-resolved monomer and excimer fluorescence decays. Secondly, two Py-PS series were studied in several different solvents with viscosities ranging from 0.36 to 1.19 mPa.s to demonstrate the effect of viscosity on the FBM parameters. The rate constant for excimer formation within a blob, kblob, was found to remain constant with viscosity while the number of monomer units per blob, Nblob, increased with decreasing viscosity. Thirdly, in a technical note, the inherent analogy existing between the aggregation number of surfactant micelles, Nagg, and Nblob, is taken advantage of to establish a “model-free” procedure to determine Nblob. This procedure was validated through the analysis of the time-resolved fluorescence decays of five different pyrene-labeled polymers in seven different solvents. Fourthly, the side-chain dynamics of alpha-helical pyrene-labeled poly(glutamic acid) were investigated. A longer linker connecting pyrene to the backbone resulted in an increase in Nblob which agreed quantitatively with predictions made by molecular modeling. Decreasing the lifetime of the excited pyrene from 215 ns to 50 ns with a quencher increased kblob significantly, while Nblob decreased slightly. Finally, the backbone dynamics of pyrene-labeled poly(aspartic acid) (Py-PAA) were investigated and quantified using the FBM. It was determined that excimer formation was controlled by the very rigid polypeptide backbone and not by the solvent viscosity, contrary to previous results obtained with other pyrene-labeled vinyl polymers.

Several pyrene-labeled polymers and polypeptides were synthesized and their chain dynamics were characterized using steady-state and time-resolved fluorescence techniques. Firstly, four series of pyrene-labeled polystyrenes (Py-PS) were synthesized to determine the effect that the method used for pyrene attachment, the linker connecting pyrene to the backbone, and the distribution of pyrene along the backbone all have on excimer formation. It was found that the amount of excimer formed was different in each case. The differences were described by utilizing the fluorescence blob model (FBM) analysis of the time-resolved monomer and excimer fluorescence decays. Secondly, two Py-PS series were studied in several different solvents with viscosities ranging from 0.36 to 1.19 mPa.s to demonstrate the effect of viscosity on the FBM parameters. The rate constant for excimer formation within a blob, kblob, was found to remain constant with viscosity while the number of monomer units per blob, Nblob, increased with decreasing viscosity. Thirdly, in a technical note, the inherent analogy existing between the aggregation number of surfactant micelles, Nagg, and Nblob, is taken advantage of to establish a “model-free” procedure to determine Nblob. This procedure was validated through the analysis of the time-resolved fluorescence decays of five different pyrene-labeled polymers in seven different solvents. Fourthly, the side-chain dynamics of alpha-helical pyrene-labeled poly(glutamic acid) were investigated. A longer linker connecting pyrene to the backbone resulted in an increase in Nblob which agreed quantitatively with predictions made by molecular modeling. Decreasing the lifetime of the excited pyrene from 215 ns to 50 ns with a quencher increased kblob significantly, while Nblob decreased slightly. Finally, the backbone dynamics of pyrene-labeled poly(aspartic acid) (Py-PAA) were investigated and quantified using the FBM. It was determined that excimer formation was controlled by the very rigid polypeptide backbone and not by the solvent viscosity, contrary to previous results obtained with other pyrene-labeled vinyl polymers.

The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.

Time-resolved fluorescence was applied to linear and branched pyrene-labelled macromolecules to study their internal dynamics. The linear macromolecules consisted of two series of pyrene-labelled poly(N-isopropylacrylamide)s where the polymer was either end-labelled (Py2-PNIPAM-Y where Y represents the molecular weight of the polymer and equals 6, 8, 14, 25, and 45 kDa) or randomly labelled (Py-PNIPAM-X% where X represents the pyrene content and is equal to 0.1, 2, 3, 4, 5, and 6 mol%) with pyrene. Four dendrimer generations based on a bis(hydroxymethyl)propionic acid backbone represented the branched macromolecules where the terminal sites were labelled with pyrene (PyX-GY-COOH where X represents the number of pyrene units incorporated into the Y`th generation dendrimer). A polystyrene-dendrimer hybrid was also synthesized (PyX-GY-PS). The fluorescence decays of the Py2-PNIPAM-Y and Py-PNIPAM-X% samples were acquired in solvents of varying viscosity and were analyzed with the Birks Scheme and the Fluorescence Blob Model (FBM) to yield the excimer formation rate constants and , respectively. The two parameters showed the same trends with varying viscosity, implying that the same information concerning chain dynamics is obtained from the randomly and end-labelled PNIPAM samples. The fluorescence decays of the Py2-PNIPAM-Y samples were acquired in ethanol and in water to determine how pyrene solubility affects the behavior of the polymers in solution, as probed by time-resolved fluorescence. It was found that the decreased pyrene solubility in water led to large amounts of intra- and intermolecular pyrene aggregation. Finally, the pyrene-labelled dendrimers were studied in tetrahydrofuran (THF) to probe the mobility of the chain ends as a function of generation number. The average rate of excimer formation, , obtained from the Model-Free analysis of the fluorescence decays in THF, increased linearly with generation number. This finding, combined with molecular mechanics optimizations, led to the conclusion that excimer formation was greatly enhanced due to the branched nature of the dendrimer molecule. Together, these studies illustrate three different applications of the use of time-resolved fluorescence to characterize the internal dynamics of pyrene-labelled macromolecules.

A series of pyrene end-labeled monodisperse poly(ethylene oxide)s (PEO(X)-Py2 where X represents the number average molecular weight (Mn) of the PEOs and equals 2, 5, 10 and 16.5 K) and one pyrene mono-labeled PEO (PEO(2K)-Py1) were synthesized and characterized in solution using fluorescence. First, the end-to-end cyclization (EEC) of PEO(X)-Py2 was investigated in seven organic solvents with viscosities (η) ranging from 0.32 to 1.92 mPa•s. The classical Birks scheme was used to globally fit the pyrene monomer and excimer fluorescence decays. The fraction of pyrenes that did not form excimer (ffree) was found to increase with increasing η and Mn. This result was contrary to the assumptions made by Birks’ scheme. To account for this, ffree was assumed to represent the fraction of PEO chains other than the monolabeled polymer impurities that cannot accomplish EEC. A fluorescence blob model (FBM) was applied to handle this assumption in the process of excimer formation for the PEO(X)-Py2 samples in solution. The radius of a blob, Rblob, in organic solvents was determined according to the results retrieved from the FBM. To quantitatively account for the existence of pyrene impurity in pyrene-labeled macromolecules, known amounts of PEO(2K)-Py1 were added into a PEO(2K)-Py2 solution and the fluorescence decays were fitted globally according to the Birks scheme and “model free” (MF) analysis to verify the validation of the MF analysis. The MF analysis was then applied to determine the amounts of 1-pyrenebutyric acid (PyBA) that had been added to a solution of pyrene end-labeled fourth generation dendritic hybrid (Py16-G4-PS). The results demonstrated that the contribution from unwanted fluorescent species could be isolated and quantitatively accounted for by fitting the fluorescence decays of the pyrene monomer and excimer globally with the MF analysis. Since the PEO(X)-Py2 samples form hydrophobic pyrene aggregates in aqueous solution, a sequential model (SM) was proposed to characterize the pyrene excimer formation of PEO(X)-Py2 in water at different polymer concentration (CP). The capture distance over which the pyrenyl end-groups experience hydrophobic forces in water was determined by assuming that the end-to-end distances of the PEO(X)-Py2 samples adopt a Gaussian distribution and that the fraction of pyrenes that are aggregated (fE0) determined by the sequential model corresponds to the fraction of PEO(X)-Py2 chains whose end-to-end distance is smaller than the hydrophobic capture distance. Since a surfactant can interact with a hydrophobically modified water-soluble polymer in aqueous solution, the interactions taking place between PEO(X)-Py2 and sodium dodecyl sulfate (SDS) were investigated at a low PEO(X)-Py2 concentration. The pyrene monomer and excimer fluorescence decays of the PEO(X)-Py2 and SDS solutions were acquired at various SDS concentrations and globally fitted according to the MF analysis to retrieve the parameters that described the kinetics of pyrene excimer formation. At high SDS concentrations above the critical micelle concentration (CMC), the pyrene end-groups of the short-chain samples (PEO(2K)-Py2 and PEO(5K)-Py2) were incorporated inside the same micelle and excimer was formed intramolecularly, while most pyrene groups of the long-chain samples (PEO(10K)-Py2 and PEO(16.5K)-Py2) were isolated into different micelles. Lastly, both the rheological properties and fluorescence behavior of a pyrene-labeled hydrophobically-modified alkali-swellable emulsion (Py-HASE) polymer in basic aqueous solution with SDS were studied. Furthermore, a joint experimental setup that combined a rheometer and a steady-state fluorometer was applied to investigate at the molecular level the effect that a shearing force had on the polymeric network. However, despite the dramatic decrease in solution viscosity with increasing shear rate, no change in the fluorescence spectra was detected, suggesting that changes in the polymeric network that affected the balance of intra- versus intermolecular pyrene associations did not impact the process of excimer formation. Together the experiments described in this thesis represent the broadest set of examples found in the scientific literature where information on the dynamics and level of association of pyrene-labeled polymers has been retrieved through the quantitative analysis of the fluorescence decays acquired with pyrene-labeled polymers in solution.

Time-resolved fluorescence was applied to linear and branched pyrene-labelled macromolecules to study their internal dynamics. The linear macromolecules consisted of two series of pyrene-labelled poly(N-isopropylacrylamide)s where the polymer was either end-labelled (Py2-PNIPAM-Y where Y represents the molecular weight of the polymer and equals 6, 8, 14, 25, and 45 kDa) or randomly labelled (Py-PNIPAM-X% where X represents the pyrene content and is equal to 0.1, 2, 3, 4, 5, and 6 mol%) with pyrene. Four dendrimer generations based on a bis(hydroxymethyl)propionic acid backbone represented the branched macromolecules where the terminal sites were labelled with pyrene (PyX-GY-COOH where X represents the number of pyrene units incorporated into the Y`th generation dendrimer). A polystyrene-dendrimer hybrid was also synthesized (PyX-GY-PS). The fluorescence decays of the Py2-PNIPAM-Y and Py-PNIPAM-X% samples were acquired in solvents of varying viscosity and were analyzed with the Birks Scheme and the Fluorescence Blob Model (FBM) to yield the excimer formation rate constants and , respectively. The two parameters showed the same trends with varying viscosity, implying that the same information concerning chain dynamics is obtained from the randomly and end-labelled PNIPAM samples. The fluorescence decays of the Py2-PNIPAM-Y samples were acquired in ethanol and in water to determine how pyrene solubility affects the behavior of the polymers in solution, as probed by time-resolved fluorescence. It was found that the decreased pyrene solubility in water led to large amounts of intra- and intermolecular pyrene aggregation. Finally, the pyrene-labelled dendrimers were studied in tetrahydrofuran (THF) to probe the mobility of the chain ends as a function of generation number. The average rate of excimer formation, , obtained from the Model-Free analysis of the fluorescence decays in THF, increased linearly with generation number. This finding, combined with molecular mechanics optimizations, led to the conclusion that excimer formation was greatly enhanced due to the branched nature of the dendrimer molecule. Together, these studies illustrate three different applications of the use of time-resolved fluorescence to characterize the internal dynamics of pyrene-labelled macromolecules.

A series of pyrene end-labeled monodisperse poly(ethylene oxide)s (PEO(X)-Py2 where X represents the number average molecular weight (Mn) of the PEOs and equals 2, 5, 10 and 16.5 K) and one pyrene mono-labeled PEO (PEO(2K)-Py1) were synthesized and characterized in solution using fluorescence. First, the end-to-end cyclization (EEC) of PEO(X)-Py2 was investigated in seven organic solvents with viscosities (η) ranging from 0.32 to 1.92 mPa•s. The classical Birks scheme was used to globally fit the pyrene monomer and excimer fluorescence decays. The fraction of pyrenes that did not form excimer (ffree) was found to increase with increasing η and Mn. This result was contrary to the assumptions made by Birks’ scheme. To account for this, ffree was assumed to represent the fraction of PEO chains other than the monolabeled polymer impurities that cannot accomplish EEC. A fluorescence blob model (FBM) was applied to handle this assumption in the process of excimer formation for the PEO(X)-Py2 samples in solution. The radius of a blob, Rblob, in organic solvents was determined according to the results retrieved from the FBM. To quantitatively account for the existence of pyrene impurity in pyrene-labeled macromolecules, known amounts of PEO(2K)-Py1 were added into a PEO(2K)-Py2 solution and the fluorescence decays were fitted globally according to the Birks scheme and “model free” (MF) analysis to verify the validation of the MF analysis. The MF analysis was then applied to determine the amounts of 1-pyrenebutyric acid (PyBA) that had been added to a solution of pyrene end-labeled fourth generation dendritic hybrid (Py16-G4-PS). The results demonstrated that the contribution from unwanted fluorescent species could be isolated and quantitatively accounted for by fitting the fluorescence decays of the pyrene monomer and excimer globally with the MF analysis. Since the PEO(X)-Py2 samples form hydrophobic pyrene aggregates in aqueous solution, a sequential model (SM) was proposed to characterize the pyrene excimer formation of PEO(X)-Py2 in water at different polymer concentration (CP). The capture distance over which the pyrenyl end-groups experience hydrophobic forces in water was determined by assuming that the end-to-end distances of the PEO(X)-Py2 samples adopt a Gaussian distribution and that the fraction of pyrenes that are aggregated (fE0) determined by the sequential model corresponds to the fraction of PEO(X)-Py2 chains whose end-to-end distance is smaller than the hydrophobic capture distance. Since a surfactant can interact with a hydrophobically modified water-soluble polymer in aqueous solution, the interactions taking place between PEO(X)-Py2 and sodium dodecyl sulfate (SDS) were investigated at a low PEO(X)-Py2 concentration. The pyrene monomer and excimer fluorescence decays of the PEO(X)-Py2 and SDS solutions were acquired at various SDS concentrations and globally fitted according to the MF analysis to retrieve the parameters that described the kinetics of pyrene excimer formation. At high SDS concentrations above the critical micelle concentration (CMC), the pyrene end-groups of the short-chain samples (PEO(2K)-Py2 and PEO(5K)-Py2) were incorporated inside the same micelle and excimer was formed intramolecularly, while most pyrene groups of the long-chain samples (PEO(10K)-Py2 and PEO(16.5K)-Py2) were isolated into different micelles. Lastly, both the rheological properties and fluorescence behavior of a pyrene-labeled hydrophobically-modified alkali-swellable emulsion (Py-HASE) polymer in basic aqueous solution with SDS were studied. Furthermore, a joint experimental setup that combined a rheometer and a steady-state fluorometer was applied to investigate at the molecular level the effect that a shearing force had on the polymeric network. However, despite the dramatic decrease in solution viscosity with increasing shear rate, no change in the fluorescence spectra was detected, suggesting that changes in the polymeric network that affected the balance of intra- versus intermolecular pyrene associations did not impact the process of excimer formation. Together the experiments described in this thesis represent the broadest set of examples found in the scientific literature where information on the dynamics and level of association of pyrene-labeled polymers has been retrieved through the quantitative analysis of the fluorescence decays acquired with pyrene-labeled polymers in solution.

The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.