The process of excimer formation was studied for a series of pyrene end-labeled polystyrenes (PS(X)-Py 2 where X is the polymer molecular weight equal to 3, 4.5, 8, 12.7, and 14.6 K) and two series of polystyrenes randomly labeled with pyrene (CoE-PS and CoA-PS) in seven different solvents. The solvent viscosities ranged from 0.41 to 1.92 mPa x s, while the solvent quality ranged from good to poor solvents for polystyrene, as determined by intrinsic viscosity measurements. Steady-state fluorescence spectra of the pyrene-labeled polymers were acquired, and the excimer to monomer ratios showed that excimer formation increased strongly with a decrease in solvent viscosity. The monomer and excimer time-resolved fluorescence decays were also acquired and fitted globally to either the Birks' scheme or the fluorescence blob model (FBM) for the end- or randomly labeled polymers, respectively. All parameters reporting on the long-range polymer chain dynamics (LRPCD) obtained from the analysis of the fluorescence data acquired with the PS(X)-Py 2, CoE-PS, and CoA-PS series yielded virtually identical trends, demonstrating that these fluorescence experiments yield results that are internally consistent with one another. Considering the substantial advantages associated with the preparation and study of randomly labeled polymers, this report presents an appealing case for the use of randomly labeled polymers in the study of LRPCD.

The "blob" model, developed to analyze the fluorescence decays of polymers randomly labeled with pyrene, has been applied to a series of pyrene-labeled poly(glutamic acid)s (PyPGA) in DMF and carbonated buffer solutions at pH 9. Poly(glutamic acid) (PGA) exists in the ionized form in the buffer solutions as poly(sodium glutamate) (PGNa). PGA adopts an alpha-helical conformation in DMF, whereas in aqueous solution PGNa is a random coil. Fluorescence, UV-vis absorption, and circular dichroism measurements indicate that in our studies pyrene pendants attached themselves along PGA in a clustered manner. Simulations were carried out to establish that the geometry of the PGA alpha-helix induces the high level of pyrene clustering. Since the level of pyrene clustering decreased with lower pyrene content, information about naked PGA was retrieved by extrapolating the trends obtained by fluorescence to zero pyrene content. Analysis of the fluorescence decays demonstrated that during its lifetime an excited pyrene probes a 32 amino acid section of the PGA alpha-helix. This result was supported by molecular mechanics optimizations. This study establishes that the blob model, originally used to monitor the encounters between pyrenes attached randomly onto a polymer adopting a random coil conformation, can also be applied to study the dynamics of the side chains of structured proteins. Since the blob model helps in monitoring the encounters between amino acids in the initial state (i.e., random coil) and in the final state (i.e., structured protein) of the folding pathway of a protein, it could be applicable to the study of protein folding.

A series of polystyrenes randomly labeled with 1-pyrenebutanol were prepared by copolymerizing styrene and 1-pyrenebutylacrylate yielding the CoBuE–PS series. Solutions of CoBuE–PS were prepared in nine organic solvents having viscosities ranging from 0.36 to 5.5 mPa·s and the fluorescence spectra and pyrene monomer and excimer fluorescence decays were acquired. Analysis of the fluorescence spectra yielded the IE/IM ratio, whereas analysis of the fluorescence decays with the fluorescence blob model (FBM) yielded the parameters N blobo , <kblob × Nblob> , and k blobo . These parameters were compared to those obtained with two other series of pyrene-labeled polystyrenes, which had been studied earlier, namely CoA–PS and CoE–PS where pyrene was attached to the polymer backbone via a methylamide and benzyl methylether linker, respectively. Although the parameters IE/IM, N blobo , <kblob × Nblob>, and k blobo took different values according to the specific nature of the linker connecting pyrene to the polystyrene backbone, they exhibited trends that were quite similar for all the pyrene-labeled polystyrene constructs. The excellent agreement between the parameters retrieved for the three different types of pyrene-labeled polystyrenes suggests that the FBM accounts satisfyingly for differences in the nature of the label used, while still retrieving information pertinent to the polymer of interest.

Two series of pyrene-labeled poly(glutamic acid) (Py-PGA) were synthesized utilizing two different linkers for pyrene attachment, namely 1-pyrenemethylamine (PMA) and 1-pyrenebutylamine (PBA). Several Py-PGAs were synthesized for each series with pyrene contents ranging from 4 to 15 mol %. Py-PGA forms a rigid alpha-helix in DMF that effectively locks the backbone in place, thus enabling only side-chain or linker motions to be monitored by time-resolved fluorescence. Time-resolved fluorescence decays were acquired for the pyrene monomer of the Py-PGA constructs and the fluorescence blob model (FBM) was used to quantify the dynamics of the different linkers connecting pyrene to the backbone. Nitromethane was used to shorten the lifetime of the pyrene monomer, in effect controlling the probing time of the pyrene group, from 50 to 155 ns for PGA-PBA and from 50 to 215 ns for PGA-PMA. The FBM analysis of the fluorescence decays led to the conclusion that excimer formation around the rigid alpha-helix backbone takes place in a compact environment. The number of glutamic acid units within a blob, N blob, decreased only slightly with decreasing probing time for both Py-PGA constructs as a result of the compact distribution of the chromophores around the alpha-helix. The PGA alpha-helix was modeled using Hyperchem software and the ability of two pyrene groups to encounter was evaluated as they were separated by increasing numbers of amino acids along the alpha-helix. The number of amino acids required for two pyrenes to lose their ability to overlap and form excimer matched closely the N blob values retrieved using the FBM.

Four types of pyrene-labeled polystyrene samples (Py−PS) were prepared and the process of excimer formation between the pyrene labels was characterized by steady-state and time-resolved fluorescence to assess the effect the mode of pyrene incorporation into a polymer has on the kinetics of excimer formation. The pyrene label was incorporated into the PS backbone by either (1) reacting 1-pyrenemethoxide with a chloromethylated polystyrene backbone to yield the GrE−PS series, (2) copolymerizing styrene with 4-(1-pyrenyl)methoxymethylstyrene to yield the CoE−PS series, (3) copolymerizing styrene with N-(1-pyrenylmethyl)acrylamide to yield the CoA−PS series, or (4) polymerizing α,ω-dicarboxyl end-capped polystyrenes with l-lysine-1-pyrenemethylamide dihydrochloride to yield the ES−PS series. Steady-state and time-resolved fluorescence experiments demonstrated that the long and flexible linker of GrE−PS and CoE−PS enabled more efficient excimer formation than the short and rigid linker of CoA−PS, and that spacing the pyrene pendants in ES−PS led to a strong reduction in excimer formation. The fluorescence blob model (FBM) was applied to analyze quantitatively the monomer and excimer fluorescence decays of the four Py−PSs. The FBM analysis confirmed that the longer ether linker of GrE−PS and CoE−PS enabled the excited pyrene label to probe a larger volume inside the polymer coil. The level of clustering of the pyrene pendants was found to be minimal for ES−PS, as expected from its structural design. Interestingly, the pyrene pendants were twice more clustered for GrE−PS than for CoE−PS, despite both polymers having an identical chemical structure. The results for the GrE−PS and CoE−PS series suggest that reacting groups distribute themselves differently in a copolymer whether they are incorporated by a grafting onto reaction or copolymerization.