Chemistry

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Associations between Oil Additives in Base Oils Probed by Pyrene Excimer Fluorescence
(University of Waterloo, 2017-10-23) Gholami Tazeh Shahri, Kiarash; Duhamel, Jean
A recently developed method based on pyrene excimer fluorescence was used to probe the interactions of important polymeric oil additives. First, three ethylene-propylene (EP) copolymers representative of viscosity index improvers (VII) were maleated and fluorescently labeled with pyrene to yield three Py-EP samples. Each sample was dissolved in oil and toluene, an apolar substitute for oil. The fluorescence spectra of the Py-EP samples were acquired and analyzed to obtain a quantitative measure of the molar fraction (finter) of the pyrene labels that formed excimer intermolecularly for the Py-EP samples in oil and toluene as a function of solution temperature. The results demonstrated that finter remained more-or-less constant for the amorphous Py-EP sample in both solvents. However, the solutions of the semicrystalline Py-EP samples in oil and toluene showed an anomalous behavior for finter at intermediate temperatures, that was attributed to the formation of crystalline microdomains by the semicrystalline Py-EP samples and resulted in a sharp increase in the local pyrene concentration [Py]loc and thus finter. Moreover, finter and the molar fraction of aggregated pyrenes (fagg) were found to be larger in oil than in toluene for a same Py-EP sample, suggesting that oil was a worse solvent than toluene to solubilize the Py-EP samples. These experiments were repeated to determine finter for solutions of the Py-EP samples in oil in the presence of two pour point depressants (PPDs). The results suggested that PPDs slightly increased finter for the Py-EP samples, indicating some level of interactions between the two polymers. However since the succinimide group used to link the pyrene labels to the EP backbone induced intermolecular interactions that might affect the conclusions drawn from the finter values, the focus of the study was changed to investigate the interactions between a pyrene-labeled poly(alkyl methacrylate) (Py-PAMA) used as a PPD mimic and EP copolymers in oil. In this case, the pyrene label was attached to the PAMA backbone via the same ester bond that connected the alkyl chains to the polyester backbone. Consequently introduction of the pyrene label to PAMA did not induce any unwanted interactions between the Py-PAMA molecules. The finter-vs-T profiles for two Py-PAMA samples in oil were determined. They also reflected a contraction of the polymer coils due to the crystallization of the alkyl side chains as the solution temperature was lowered. The temperature where the transition occurred was found to be dependent on the side chain length as would be the case for alkanes of different lengths. Based on these results, a poly(octadecyl methacrylate) sample labeled with 6.7 mol% pyrene Py(6.7)-PC18MA was selected and its finter-versus-T profiles were obtained in the presence of different oil additives in oil and octane. The addition of EP copolymers to the Py(6.7)-PC18MA solutions resulted in an increase in finter indicative of interactions between the EP copolymers and Py(6.7)-PC18MA molecules in oil. The increase in finter was found to be more pronounced at high temperatures for both amorphous (EP(AM)) and semicrystalline (EP(SM)) EP copolymers. At low temperatures, EP(AM) led to an increase in finter for Py(6.7)-PC18MA but not EP(SM) as it had crystallized and could no longer interact with Py(6.7)-PC18MA. Engine oils naturally contain a substantial amount of wax in their formulation which can promote interactions between different oil additives like VIIs and PPDs. Therefore, octane was employed as a wax-free engine oil substituent. An amount of wax similar to that present in engine oils was added to the Py(6.7)-PC18MA solution in octane to study its effect on the finter-versus-T profile. The presence of wax increased finter for Py(6.7)-PC18MA as would be expected from a PPD mimic that is believed to bind to wax crystals and control their growth. Naked EP copolymers were then added to the mixture of Py(6.7)-PC18MA and wax in octane. This led to another increase in finter. Such an effect was observed over the entire temperature range for the amorphous EP copolymer, while the crystallization of the semicrystalline EP copolymer at low temperatures canceled the effect of this additive on finter for Py(6.7)-PC18MA in a 10 g.L wax solution in octane. The experiments conducted in this thesis expanded the applicability of the procedure originally developed by S. Pirouz to determine the level of intermolecular interactions between EP copolymers in toluene to PAMAs in engine oil and octane. This study provided quantitative information about the level of interactions between Py(6.7)-PC18MA and two EP copolymers that are representative of the interactions that would exist between PPDs and VIIs in engine oils.
Calibrating the Long-Range Backbone Dynamics of Polymers in Solution Using Two Different Models
(University of Waterloo, 2022-09-29) Little, Hunter; Duhamel, Jean
The fluorescence decays of a series of 41 pyrene-labeled poly(oligo(ethylene glycol)methyl ether methacrylate)s (Py-PEGnMA where n = 0, 1, 2, 3, 4, 5, 9, 16, 19) were prepared by free-radical polymerization of 1-pyrenebutyl methacrylate and oligo(ethylene glycol)methyl ether methacrylate. The more polar oligo(ethylene glycol) side chains increased the range of solvent polarities in which these polymers were soluble. The Py-PEGnMAs were dissolved in four solvents, namely tetrahydrofuran, toluene, dimethyl formamide, and dimethylsulfoxide which represented a broad range of solvent polarities and viscosities. The pyrene monomer and excimer decays were acquired for the different Py-PEGnMA solutions and then analyzed globally using the fluorescence blob model (FBM) and the model free analysis (MFA). The FBM analysis yielded the parameters kblob and Nblob along with the product kblob×Nblob. The parameter Nblob, which reports on the number of structural units within an imaginary volume known as a blob, describes the ability of a polymer to bend, while kblob×Nblob reports on the encounter frequency between structural units. Together, Nblob and kblob×Nblob provide information on the long-range backbone dynamics (LRBD) of the polymer. Interestingly, since a single structural parameter, the length of the side-chain, was changed in this study, it was easy to parametrize both of Nblob and kblob×Nblob in terms of the molecular weight of the structural unit and the solvent viscosity. The resulting equations were used to construct a calibration curve against which the dynamics of other polymers could be compared. This represents a great step forward as previous calibration curves were limited to a single solvent in which the benchmark polymers were soluble. Additionally, Nblob was used to extract the persistence length of the polymers from a modified version of the Kratky-Porod equation. Analysing the same decays with the MFA provided a unique opportunity to compare the two very different models. The FBM assumes the polymer has been randomly labeled with pyrene which are distributed into imaginary volumes called blobs according to a Poisson distribution and fits the monomer and excimer decays globally with an infinite sum of exponentials. Conversely, the MFA makes no assumption about the fluorophore distribution and fits the decays with a sum of 2–3 exponentials. This makes the MFA particularly apt to study any type of fluorophore-labeled macromolecules, many of which would be incompatible with the FBM. Regardless of the very different approaches, both models retrieved values representing the average rate constant of excimer formation, for the MFA and kblob× for the FBM, which were identical within experimental error. This result shows that the MFA is capable of reporting on the local pyrene concentration, which in turn provides important structural information. Therefore normalizing for pyrenyl labels incapable of forming excimer yielded the parameter blob which was also parameterized in terms of the molecular weight of the structural unit and solvent viscosity, resulting in a universal calibration curve for long-range backbone dynamics. More importantly though, the equivalency between the key parameters retrieved between the two models implied that there existed a similar parameter to Nblob within the results of the MFA which described the number of structural units probed by an excited state pyrene. This parameter highlighted a crucial difference between the two models: in the analysis of the FBM the dynamic component, represented as kblob, and the structural component, represented as , are separated in the analysis whereas in the MFA they are merged into the single parameter . Additional knowledge about the dynamics of the polymer chain is needed in order to accurately extract the structural information contained within the parameters reported by the MFA.
Characterization of Hydrophobically Modified Starch Nanoparticles by Pyrene Fluorescence
(University of Waterloo, 2017-01-25) Kim, Damin; Duhamel, Jean
Starch nanoparticles (SNPs) were hydrophobically modified with propionic (C3) and hexanoic (C6) anhydride via esterification. Different degrees of substitution (DS) of the hydroxyl groups of the SNPs were achieved by varying the reaction conditions. The relative hydrophobicity of the hydrophobically modified SNPs (HM-SNPs) was examined with pyrene fluorescence. The hydrophobes covalently attached to the SNPs conferred an amphiphilic character to the SNPs whereby hydrophobic microdomains were generated and stabilized by the hydrophilic SNPs in aqueous solution. The hydrophobic microdomains of the HM-SNPs were probed with the hydrophobic dye, pyrene. Several parameters related to the photophysical properties of pyrene, such as the (I1/I3)o ratio, the bimolecular rate constant for quenching k¬q, the natural lifetime o, and the equilibrium constant for the binding of pyrene to the HM-SNPs KB, were found to respond to the expected hydrophobicity of the HM-SNPs, and thus the level of hydrophobic modification. These four parameters, (I1/I3)o, k¬q, o, and KB, were examined as a function of the DS and the type of hydrophobic modification (propionic versus hexanoic acid). As the DS of hexanoic acid of the HM-SNPs increased, pyrene experienced a decrease in the polarity of its local environment evident from a decrease in (I1/I3)o while the microviscosity of the hydrophobic aggregates increased as indicated by an increase in o and a decrease in kq. Moreover, the interactions between SNPs and pyrene became stronger with increasing DS and were dependent on the surface area of the SNPs. All the parameters confirmed that the hydrophobicity of the HM-SNPs increased with increasing DS of hexanoic acid. The parameters also demonstrated that for a same DS, the environment generated by the SNPs modified with hexanoic acid was more hydrophobic than that of the SNPs modified with propionic acid. As it turns out, the photophysical parameters of pyrene retrieved for the SNPs modified with propionic acid were similar to those obtained with the naked SNPs. A model was proposed to account for the increase in SNP diameter due to aggregation observed at high SNP concentration. Its validity was supported by the good agreement obtained when comparing the average diameter predicted by the model with the hydrodynamic diameter of the SNPs determined by DLS. These studies led to the conclusion that hexanoic anhydride modification of SNPs appears to be more suitable than propionic acid modification to increase the hydrophobicity of SNPs. These measurements carried out with pyrene demonstrate that HM-SNPs can be generated where the level of hydrophobicity can be gauged based on the value of the (I1/I3)o, k¬q, o, and KB parameters. Finally, interactions between pyrene and SNPs seem to occur at the SNPs surface which is reduced upon aggregation of the SNPs.
Characterization of PIBSI Dispersants and Their PIBSA Components by Pyrene Excimer Fluorescence
(University of Waterloo, 2020-12-18) Frasca, Franklin; Duhamel, Jean
Methods are introduced to characterize the products resulting from the coupling of a polyisobutylene terminated at one end with a succinic anhydride (PIBSA) and a polyamine to generate PIBSI products where the polyamine is flanked by one or two PIB segments via a succinimide bond. The molar fraction of doubly modified PIBSA (PIBSA2) molecules in two PIBSA samples (PIBSA-1 and PIBSA-2) was first determined using pyrene excimer formation (PEF) to analyse both PIBSA samples after labeling fractionated and unfractionated PIBSA samples with 1-pyrenemethylamine (PyMA) to generate PIBSA-PyMA. The ratios of succinic anhydrides, succinimides, or pyrenyl labels to the number of isobutylene units NSA/NIB, NSI/NIB, and NPy/NIB determined by FTIR and UV-Vis spectroscopy established that the succinic content of the unfractionated PIBSA-PyMA samples was roughly double that of the fractionated samples, suggesting a difference in the molecular weight distribution (MWD) of the samples which was confirmed by GPC analysis. Model free analysis (MFA) of the time-resolved fluorescence (TRF) decays yielded the average rate constant of pyrene excimer formation by diffusion () which increased linearly with [Py], and the molar fraction fagg of PyMA attached on PIBSA2 forming excimer by direct excitation of aggregated pyrene dimers, and in turn the fraction (f¬doubly) of PIBSA2 in each sample. fagg was higher for PIBSA-1 and did not change between the fractionated or unfractionated samples suggesting that PIBSA2 was evenly distributed across the MWD of PIBSA. The number average degree of polymerization (Xn) was calculated for the PIBSA-PyMAs and showed a linear relationship with the rate constant (kdiff) describing PEF by diffusion. Next, it was hypothesized that the composition of the mixture of a coupling reaction between PIBSA and a polyamine could be predicted with the knowledge of the ratio of the number of amines to SA units (NAm/NSA) available for the coupling reaction, the NSA/NIB ratio, and the fraction (fdoubly) of PIBSA2 chains in the PIBSA sample. Two programs were written to simulate the products generated from the coupling of PIBSA with a diamine to form either bis-PIBSI (b-PIBSI) and higher-order PIBSI oligomers, or to only form b-PIBSI assuming that steric hindrance prevented more than one PIBSA from linking to another PIBSA molecule. A set of PIBSA to hexamethylene diamine (HMDA) coupling reactions were conducted with a fractionated PIBSA-2 sample to yield the PIBSA-2-H(X) samples, where X equals the NAm/NSA ratio used in a coupling reaction, and generate DRI traces with GPC for use with the simulated data and the validation of the simulation programs. Parameterization of the DRI traces with sums of Gaussians enabled the determination of the fractions of PIBSA-like molecules (PLMs) and non-PIBSA-like molecules (non-PLMs) and their comparison with the fractions obtained from the simulations. This along with GPC analysis suggested that PIBSA-2 contained 30 wt% of unmaleated PIB. When assuming that the PIBSA coupling reaction could only form b-PIBSI, excellent agreement was found between the experimental and simulated DRI traces supporting the validity of this assumption. Lastly, a study was conducted with a series of pyrene-labeled polyamines (Py-PAs) representative of the core of a PIBSI dispersant, to examine the relationship between the fluorescence intensity ratio of the pyrene excimer over that of the monomer (IE/IM), , and the local pyrene concentration ([Py]loc) in the Py-PAs, and ultimately assess the usefulness of for establishing a -vs-[Py]loc calibration curve to characterize the polyamine core of pyrene-labeled PIBSI dispersants. MFA of the TRF decays acquired with the Py-PAs yielded and the analysis of the fluorescence spectra yielded IE/IM. Both and IE/IM increased linearly with increasing [Py]loc, suggesting that the -vs-[Py]loc plot could be used as a calibration curve for analysis of the polyamines in the PIBSI cores. The methods detailed in this thesis expand on the use of PEF to study PIBSA-coupling procedures introduced in previous work by A. Mathew and S. Pirouz, and have resulted in a variety of interesting findings about the PIBSA samples studied herein such as the content of PIBSA2 in a PIBSA sample and accurate predictions of the PIBSI coupling products taking into account fagg determined by PEF. These experiments further emphasize the usefulness of PEF as a characterization method for macromolecules in general, and oil-additives specifically.
Characterization of Polysaccharides in Starch using Fluorescence Techniques
(University of Waterloo, 2020-09-25) Li, Lu; Duhamel, Jean
Amylose and amylopectin, which are the two main constituents of starch, were characterized using the pyrene excimer fluorescence (PEF) technique. First, an amylose sample was randomly labeled with the dye pyrene to yield Py-Amylose. Its fluorescence was compared to that of pyrene-labeled poly(methyl acrylate) (Py-PMA) as Py-PMA was expected to experience internal dynamics that were much faster than those of Py-Amylose. Analysis of the PEF signal revealed that Py-Amylose formed excimer as effectively as the more flexible Py-PMA. This result could only be rationalized if Py-Amylose generated a compact environment for the pyrene labels by adopting a helical conformation. Fluorescence blob model (FBM) analysis, where a blob is the subvolume inside a macromolecule probed by an excited pyrene, yielded a blob size (Nblob) of 11 anhydroglucose units (AGUs) for Py-Amylose. The experimental Nblob value matched the theoretical Nblob value obtained by molecular mechanism optimizations (MMOs) only if the pyrene labels were assumed to be covalently attached onto a polysaccharide helix, thus confirming the helical conformation of amylose in DMSO. Second, the FBM analysis was applied to a sample of amylopectin randomly labeled with pyrene (Py-Amylopectin). Nblob was found to increase from 11 for Py-Amylose to 20 for Py-Amylopectin in DMSO. MMOs were carried out to establish how theoretical Nblob varied with the interhelical distance (dh-h) between single and double helices of oligosaccharides laid out in a hexagonal array used as a mimic for the arrangement of the side chains of amylopectin. The dh-h value needed for Nblob to match the experimental Nblob of 20 equaled 25 or 29 Å depending on whether the side chain adopted a single or double helical conformation, respectively. This interhelical spacing suggested a density of amylopectin in DMSO which was one order of magnitude larger than the density of amylopectin determined by intrinsic viscosity ([η]). To resolve this discrepancy, the Solution-Cluster (Sol-CL) model was proposed. In this model, the interior of amylopectin was depicted as being a network of dense clusters of oligosaccharide helices connected by flexible and linear oligosaccharide segments. Third, nanosized amylopectin fragments (NAFs) prepared from waxy corn starch by high temperature extrusion were characterized by proton NMR, iodine binding test, viscometry, dynamic light scattering (DLS), and PEF. Proton NMR indicated that the NAFs shared a same chemical composition with amylopectin. The iodine binding test experiments confirmed that the NAFs were essentially amylose-free. [η] measurements suggested that their density increased from 0.04 to 0.12 g/mL in DMSO as their hydrodynamic diameter decreased from 57 nm to 8 nm. Matching the experimental Nblob value of Py-NAFs with the theoretical Nblob-vs-dh-h trends yielded dh-h values that led to densities close to that found for amylopectin. In addition, the density of the NAFs, determined from [η] approached the density values calculated from Nblob as their size decreased toward that of a cluster of helices. This result suggested that the linear segments that bridged the clusters of helices and contributed to the excluded volume of the NAFs were cleaved off as the NAFs became smaller. Poly(ethylene glycol)s (PEGs) of different lengths were then added to dilute dispersions of Py-Amylopectin and two Py-NAFs. 10K PEG was unable to penetrate the interior of the macromolecules, thus generating the osmotic pressure that compressed the polysaccharides. Shrinkage of the polysaccharides was monitored by measuring their PEF. Deformation of the polysaccharides was found to only occur in Py-Amylopectin and the larger Py-NAF. The smaller Py-NAF could not be compressed at the same 10K PEG concentration due to the absence of excluded volume. These conclusions provided further supports to the Sol-CL model. Together the results presented in this thesis suggest that PEF provides reliable structural information on amylose and amylopectin in solution, an information that would be difficult to extract from more traditional characterization methods.
Characterization of Self-Assembling Quinoline- Based Foldamers by Fluorescence Anisotropy
(University of Waterloo, 2019-09-24) Wang, Jingqi; Duhamel, Jean
Foldamers represent a family of synthetic macromolecules which, like their biological counterparts, are able to adopt a well-defined conformation in solution. Oligoquinoline-carboxamides (Qn) are a group of foldamers that adopt a helical conformation in solution. A series of Qn foldamers were prepared by chromatography-free large-scale synthesis and segment-doubling strategy. The C-terminal ester group of the Qn foldamers could be hydrolyzed to yield acid-functionalized foldamers (QnA) which could self-assemble into larger ((QnA)2-Na) complexes by metal coordination with a sodium cation. Moreover, the addition of a bis-acid functionalized tetramer (AQ2PQ2A) to a solution of (QnA)2-Na complexes resulted in insertion oligomeric products. To characterize these complexes in solution, both Qn and QnA were end-labeled with an oligo(phenylene vinylene) dye (OPV) at their N-terminus via a rigid amide bond to yield the OPV-Qn and OPV-QnA fluorescent equivalents. OPV was used to conduct time-resolved fluorescence anisotropy (TRFA) measurements on the OPV-Qn and OPV-QnA foldamers, the (OPV-QnA)2-Na complexes, and the OPV-Qn-Na-(AQ2PQ2A)n oligomers. Analysis of the TRFA of the OPV-Qn foldamers yielded the rotational time () of the fluorescent species, which was found to reflect the hydrodynamic volume (Vh) of the foldamers. The straight line obtained by plotting as a function of the number of (quinoline) units (NUs) demonstrated that the foldamers behaved in solution as rigid cylinders for all lengths examined. The linearity of the -vs-NU plot was employed as a calibration curve against which the rotational time of the QnA-complexes could be compared. Within experimental error, the rotational time of a Qn+m complex was found to equal the sum of the rotational times obtained for Qn and Qm. This result suggests that the complexation of two acid-functionalized oligoquinoline foldamers in solution generated a fully stacked foldamer with a NU equal to the sum of the NUs of its constituting elements. Hetero-complexes between OPV-Q8A and Q16A were also produced by adding a 10-fold excess of Q16A to an OPV-Q8A solution. Complexation was demonstrated by the  value of the mixture, that equaled that of an OPV-Q24 foldamer. Dilution experiments on a solution of OPV-Q8A-Na-Q16A complexes led to the dissociation of the complexes into their OPV-Q8A and Q16A constituting elements, as evidenced by the progressive decrease in  from the value obtained for OPV-Q24 to that of OPV-Q8 upon decreasing foldamer concentration. Similarly, the addition of increasing amounts of AQ2PQ2A to a solution of OPV-Q8A in chloroform resulted in an increase in  demonstrating the formation of complexes between OPV-Q8A and AQ2PQ2A until reached a plateau for large OPV-Q8A/AQ2PQ2A molar ratios. In the plateau region, the rotational time of the oligomeric complexes generated from OPV-Q8A and AQ2PQ2A stabilized by isobutyl or hexyl side chains was equal to that of an OPV-Q¬n foldamer with n equal to 24 or 30, respectively. The apparent absence of further polymerization, evidenced by the constant value reached for high OPV-Q8A/AQ2PQ2A molar ratios, was attributed to aggregation of longer complexes and their precipitation. This study represents the first example in the scientific literature where TRFA was applied to characterize the NU of helical self-assembling foldamers in solution.
Characterization of the Conformation and Internal Backbone Dynamics of a-Polyglucans by Pyrene Excimer Fluorescence
(University of Waterloo, 2021-11-23) Kim, Damin; Duhamel, Jean
The conformation and internal backbone dynamics (IBD) of several pyrene-labeled polysaccharides (Py-PSs), namely Py-Amylopectin, Py-Glycogen, Py-Dextran, and Py-Pullulan, were successfully examined with a combination of pyrene excimer formation (PEF), fluorescence blob model (FBM) analysis of fluorescence decays, and molecular mechanics optimizations (MMOs). The four Py-PSs were investigated by analysing their fluorescence response resulting from the formation of an excimer upon encounter between an excited and a ground-state pyrene. The fluorescence parameters, namely the fluorescence intensity ratio of the excimer over the monomer, the IE/IM ratio, and the number (Nblobexp) of anhydroglucose units (AGUs) found in the volume probed by an excited pyrene were determined from the analysis of the fluorescence spectra and decays of the Py-PS samples, respectively. MMOs conducted on Py-PS constructs built in silico with the program HyperChem yielded the theoretical number Nblobtheo of AGUs separating two pyrenyl labels, while still allowing for successful PEF. A satisfying match between Nblobtheo and Nblobexp supported the selection of a given conformation of the macromolecule used to conduct the MMOs. These concepts were applied as follows. At first, the compressibility of Py-Amylopectin and three pyrene-labeled Nanosized Amylopectin Fragments (Py-NAFs) with a hydrodynamic diameter (Dh) of 227, 56, 20, and 8 nm was investigated. PEF was applied to probe the changes in dh-h as the side chains of amylopectin were compressed by adding unlabeled NAF(56) (NAF with Dh = 56 nm) to the Py-PSs dispersions to increase the [NAF(56)] from the dilute to the semi-dilute regime. IE/IM and Nblobexp began to increase at a [NAF(56)] of 12 wt%, which was one order of magnitude larger than the overlap concentration. The magnitude of IE/IM and Nblobexp as a function of [NAF(56)] increased with particle size, which suggested that the magnitude of the change in PEF was related to the size of the clusters of helical side chains in the amylopectin interior. MMOs were conducted on a hexagonal close packed array of oligosaccharide helices to assess how Nblobtheo would change as a function of dh-h and the size of a cluster of helices. By matching the Nblobexp values, obtained as a function of [NAF(56)], with the Nblobtheo values, obtained as a function of dh-h and cluster size, a relationship was obtained between [NAF(56)] and dh-h, which allowed the assignment of the number of helices per cluster. Py-Amylopectin, Py-NAF(56), Py-NAF(20), and Py-NAF(8) were found to be made of clusters constituted of 37, 37, 6, and 3 helices, respectively. Considering that all models aiming to describe amylopectin focus on the clusters of side chains found in the amylopectin interior, the methodology developed to determine the size of side chain clusters in amylopectin is expected to become an important analytical tool in the characterization of amylopectin. Second, the combination of PEF, FBM, and MMOs was applied to investigate the density profile of the side chains across the interior of glycogen. Fluorescence Resonance Energy Transfer (FRET) experiments demonstrated that the pyrenyl pendants were distributed throughout the glycogen molecules. PEF experiments on two glycogen samples, that had been labeled with 1-pyrenebutyric acid (Py-Glycogen), yielded an value of 34 (±2), which was higher than the value of 18 (±1) for Py-Amylopectin. The increase in reflected the higher degree of branching of glycogen. MMOs yielded the relationship between Nblobtheo and the local density of a glycogen molecule. Two density distributions based on the Melendez-Hevia model and the Gilbert model were considered to examine the interior of glycogen. According to the Melendez-Hevia model, glycogen is constituted of oligosaccharide branches, that are arranged in concentric tiers with the local density of side chains increasing from the inner to the outer tier. On the other hand, SAXS experiments indicated that the density is highest toward the center of a glycogen particle, as predicted by the Gilbert model. Two density distributions of the local distribution of side chains in glycogen were considered based on both models. The parameters used to model these distributions were optimized to match with . Both density distributions resulted in a good agreement between and as long as the side chains located in the interior of glycogen experienced a higher density. This study successfully established the PEF-based methodology to examine the density distribution of side chains constituting the interior of glycogen. Finally, two linear polysaccharides adopting a random coil conformation in solution were examined. Dextran and pullulan were randomly labeled with 1-pyrenebutyric acid to yield Py-Dextran and Py-Pullulan, whose Nblobexp were found to equal 9.1 (±0.4) and 11 (±1), respectively. MMOs were carried out to determine Nblobtheo for dextran and pullulan. The good agreement between Nblobtheo and Nblobexp indicated that Py-Dextran and Py-Pullulan were randomly coiled in solution and that Py-Dextran was more extended than Py-Pullulan in DMSO. The IBD of these two polysaccharides were examined with the product kblob×Nblobexp×lSU, where kblob represents the rate constant for diffusive encounters between pyrene labels and lSU is the length of a structural unit (SU). The kblob×Nblobexp×lSU values obtained for Py-Dextran and Py-Pullulan were compared to those obtained for a series of pyrene-labeled poly(oligo(ethylene glycol) methyl ether methacrylate)s (Py-PEGnMAs), where n varied from 0 to 19. kblob×Nblobexp×lSU of the two polysaccharides was smaller than the kblob×Nblobexp×lSU value of a hypothetical Py-PEGnMA sample having a SU with a same molar mass per backbone atom as the Py-PSs. The smaller kblob×Nblobexp×lSU product suggested that these polysaccharides had a more rigid backbone than the PEGnMA samples, which was attributed to the cyclic nature of their SU. Comparison of the products Nblobexp×lSU and kblob×Nblobexp×lSU indicated that Py-Dextran was more extended and dynamic than Py-Pullulan in DMSO. The more dynamic backbone of dextran was attributed to the more mobile a-(1,6) glycosidic bonds constituting the backbone of dextran. In summary, this thesis represents the first example in the literature where PEF has been applied to characterize the conformation and IBD of such a wide variety of polysaccharides in solution. It has extended the realm of PEF applications to a new family of macromolecules and demonstrated the value of PEF-based methods to probe polysaccharides at the molecular level.
Characterizing Polymers with Complex Architecture using Pyrene Excimer Fluorescence
(University of Waterloo, 2021-09-29) Thoma, Janine Lydia; Duhamel, Jean
This thesis combines two different models, namely the model free analysis (MFA) and the fluorescence blob model (FBM), that were used to study the conformation and internal dynamics of two highly branched macromolecules, that were fluorescently labeled with the dye pyrene. Pyrene was selected due to its ability to form an excimer upon encounter between an excited and a ground-state pyrene. The effects of branching on the dynamics of the chain ends of 2,2-bis(hydroxymethyl)propionic acid backbone dendrons were first studied. Six generations of the dendrons, G(1)-G(6), were prepared with their terminal ends covalently labeled with a pyrenyl moiety (Pyx-G(N), x = 2N). The MFA was used to calculate the average rate constant of excimer formation, which was found to be proportional to the local concentration of pyrene [Py]loc. To account for the presence of aggregated pyrenyl groups, whose contribution was found to be significantly larger for the G5 and G6 dendrons, was divided by the fraction of pyrenyl groups forming excimer through diffusion, fdiff. was found to depend on the solvent, due to different solvent viscosity and probability (p) of pyrene excimer formation (PEF). Consequently, different trends were obtained by plotting as a function of [Py]loc in tetrahydrofuran (THF), toluene, N,N-dimethylformamide (DMF), and dimethylsulfoxide (DMSO). To account for these differences in with solvent, the model compound ethyl 4-(1-pyrene)butyrate (PyBE) was prepared. The rate constant for PEF through diffusive intermolecular encounters, kdiff, was determined for PyBE in each of the 4 solvents and used to normalize . /fdiff×kdiff–versus–[Py]loc was then plotted and the data in each of the 4 solvents collapsed onto a single master trend, which passed through the origin, thus confirming the direct relationship between and [Py]loc and suggesting that the dendron side chains obeyed Gaussian statistics. The chain end dynamics of a polymer with a different branching architecture, namely polymeric bottle brushes (PBBs), were then investigated. Four poly(oligo(ethylene glycol) methyl ether methacrylates) (P(PyEGyMA), y = 3, 5, 8, and 12) with side chain lengths containing 12, 18, 27, and 39 atoms, respectively, were end-labeled with a 1-pyrenemethoxy group. was then determined in THF, DMF, dioxane, and DMSO. [Py]loc was calculated assuming that each side chain probed a spherical blob, whose diameter was represented by the end-to-end distance of the side chains, ree,SC, assuming a Gaussian conformation in solution with ree,SC = n0.5×l. The model compound 1-pyrenemethoxy di(ethylene glycol) methyl ether (PyEG2ME) was synthesized and kdiff was determined to normalize for solvent viscosity and p. A plot of /fdiff×kdiff–versus–[Py]loc was considered again and the data obtained in THF, DMF, and dioxane were found to converge onto a single trend. Unfortunately, DMSO was found to be too polar to study the fully pyrene labeled P(PyEGyMA) PBBs, and it is believed that the hydrophobic pyrene pendant was not fully solvated and resided closer to the polymethacrylate (PMA) main chain. Excellent agreement was found when comparing the results obtained between the PBBs and the dendrons, even though the two families of macromolecules exhibited a very different branching architecture. The PBB architecture was then investigated in more detail in organic and aqueous environments. A series of poly(oligo(ethylene glycol) methyl ether methacrylate) PBBs were synthesized by randomly copolymerizing an oligo(ethylene glycol) methyl ether methacrylate with penta(ethylene glycol) 1-pyrenemethyl ether methacrylate) to yield a series of PyEG5-PEGnMA samples, where n = 0, 3, 4, 5, 7, 9, and 19). The change in main chain flexibility in organic solvents was probed as a function of increasing side chain length using the FBM. The FBM retrieves the number of structural units, Nblob, encompassed per unit volume or blob. As the side chain length of a PBB increases, there is an increase in steric repulsion between the side chains, which causes stiffening of the polymer main chain. This increase in polymer stiffness is reflected by a decrease in Nblob and an increase in the persistence length, lp, of the PBBs. In an attempt to quantify the change in Nblob with increasing side chain length, a blob-based approach was suggested, which combines the FBM and the Kratky-Porod worm like chain (KPWLC) model to obtain lp. The lp values obtained from the FBM approach were plotted as a function of the number of atoms, NS, in the side chain, where it was found that lp increased linearly with NS^2, as had been proposed theoretically. lp was also determined from conformation plots based on intrinsic viscosity using gel permeation chromatography (GPC). Using a Bohdanecky linearization, the lp was extracted from the GPC traces and found to be 1.8 times larger than the lp found using the FBM. This discrepancy was attributed to the difference in length scales probed by each technique. The PyEG5-PEGnMA PBBs were then studied in an aqueous solution to investigate the shielding effects afforded by the oligo(ethylene glycol) side chains of PBBs to the hydrophobic pyrenyl groups. The FBM was used to investigate the level of aggregation of the pyrenyl moieties as well as the effect that the side chain length had on shielding pyrene from the aqueous environment. The change in the volume probed by pyrene was studied using Nblob obtained from the FBM of the fluorescence decays and comparing the results obtained in organic solvents, where both the polymer and pyrene were fully soluble, to those obtained in an aqueous solution, where the polymer is soluble but pyrene is only slightly soluble. When compared to a series of poly(alkyl methacrylates) labeled with a shorter 1-pyrenebutyl methacrylate linker, the Nblob values obtained for PyEG5-PEGnMA PBBs in water were found to overlap perfectly. These results suggested that even though pyrene is attached onto a 16 atom-long linker, it can only probe a volume similar to that afforded by a 4 atom-long linker in water. This study also indicated that the oligo(ethylene glycol) side chains of the PBBs generate an organic-like environment around the main chain, that shields the hydrophobic pyrenyl labels from interacting with the aqueous phase.
Characterizing the Structural Arrangement of Lipoplexes by Pyrene Excimer Formation
(University of Waterloo, 2024-12-04) Lloyd, Ryan; Duhamel, Jean
Lipoplexes formed through the complexation of cationic surfactants and DNA were investigated by a combination of steady-state and time-resolved fluorescence with the fluorescent probe PyO-3-12, dynamic light scattering (DLS), and transmission electron microscopy (TEM). PyO-3-12 is an asymmetric gemini surfactant consisting of two dimethylammonium bromide headgroups linked by a propyl spacer, with one headgroup bearing a dodecyl chain and the other a 1-pyrenemethoxyhexyl derivative. Its sensitivity to the local pyrene concentration and polarity of the local environment were utilized to probe the hydrophobic microdomains generated inside lipoplexes. The overarching goal of this thesis is to demonstrate how pyrene excimer formation (PEF) between an excited and a ground-state pyrenyl labels can be applied to probe the interactions between surfactants and DNA and the resulting morphology of the lipoplexes. The first chapter of the thesis presents a review of the interactions between surfactants and water-soluble polymers such as DNA and the fundamentals of PEF with the perspective of their application for the characterization of lipoplexes. Other techniques used to probe lipoplexes such as DLS and TEM are briefly described as well. In Chapter 2, the ability of PyO-3-12 to behave as a complexing agent for the formation of lipoplexes was investigated. The interactions between PyO-3-12 and DNA were monitored as PyO-3-12 was held at a fixed concentration of 16 and 56 µM and the DNA concentration was varied between 1/10th and a 10-fold excess of the PyO-3-12 concentration. In terms of (-/+) ratio, representing the concentration of negative phosphates divided by the concentration of positive ammonium ions, the (-/+) ratio ranged from 0.1 to 10 in these experiments. Upon complexation onto DNA, PyO-3-12 showed increased PEF and indicated a more hydrophobic environment as would be expected. These results reflect the morphology of the PyO-3-12/DNA lipoplexes at the molecular level were supported by TEM and DLS experiments, which described the lipoplexes at the macroscopic level. In Chapter 3, the integrity of the PyO-3-12/DNA lipoplexes prepared with a (-/+) ratio of 1.5 and a PyO-3-12 concentration of 16 and 56 mM was investigated as they interacted with sodium dodecyl sulfate (SDS) for SDS concentrations ranging from 0 to 100 mM. The anionic surfactant generated a competing interaction for PyO-3-12 to induce the release of the negatively charged DNA. The existence of tertiary aggregates between all three species was demonstrated by fluorescence at the equicharge point between PyO-3-12 and SDS, followed by the loss of electrostatic interactions between PyO-3-12 and CT-DNA at higher SDS concentrations resulting in the release of DNA from the lipoplex. The evolution of the complexes formed between PyO-3-12, DNA, and SDS could be followed by TEM but DLS was less informative due to the polydispersity of the system that hampered the analysis of the DLS data. Chapter 4 represents the first example in the literature where PyO-3-12 was employed to probe the hydrophobic domains generated inside a lipoplex by two cationic surfactants, namely dodecyltrimethylammonium bromide (DTAB) and the gemini surfactant 12-3-12 constituted of two dimethylammonium headgroups held together by a propyl linker and bearing one dodecyl chain each. Analysis of the fluorescence data indicated that the 12-3-12/DNA lipoplexes were denser than the DTAB/DNA lipoplexes which was confirmed by conducting DLS and TEM experiments. In summary, this thesis demonstrated that PyO-3-12 is an interesting fluorescent probe to characterize the interactions between PyO-3-12 and anionic molecules such as SDS and DNA and study the interactions of other cationic surfactants such as 12-3-12 and DTAB with DNA. It opens the path for using PyO-3-12 in the molecular level characterization of soft matter generated through the interactions between surfactants and macromolecules.
Detection of Nitroaromatic Compounds through Fluorescence Quenching of Pyrene Labeled Starch Nanoparticles
(University of Waterloo, 2018-12-10) Patel, Sanjay; Duhamel, Jean
The aim of this research was to demonstrate the potential application of starch nanoparticles (SNPs) labeled with 1-pyrenebutyric acid (PBA) as the active component in optical sensors used for the detection of minute quantities of nitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT). Pyrene-labeled SNPs (Py-SNPs) with pyrene content ranging from 0.06 to 39 mol% were prepared. Quenching of the Py-SNPs by nitromethane (NM), 4-mononitrotoluene (MNT), 2,4-dinitrotoluene (DNT), and TNT was investigated in dimethyl sulfoxide (DMSO) and water. The bimolecular quenching rate constant for Py-SNPs in DMSO (kqSNP) remained constant with pyrene content and was found to equal 1.6 (± 0.1), 3.9 (± 0.2), 2.2 (± 0.2), and 1.6 (± 0.2) Mns for NM, MNT, DNT, and TNT, respectively, averaged across all Py-SNPs for a given quencher. These quenching experiments revealed that the quenching efficiency in DMSO increased according to the sequence MNT>DNT>TNT=NM, where MNT was the most efficient quencher and TNT and NM were the worst quenchers. Quenching studies conducted in water for Py-SNPs quenched by NM showed increased protective quenching with increased pyrene content, suggesting that hydrophobic aggregation of the pyrene labels induced a collapse of the Py-SNPs in water that hindered the diffusion of NM to access the pyrene labels. Hardly any diffusive quenching was observed in water for the quenching of the Py-SNPs by MNT, DNT, and TNT. Rather these quenchers targeted hydrophobic aggregates of pyrene labels, which emitted as excimer. The binding of the quenchers to the pyrene aggregates of the Py-SNPs increased with increasing pyrene content and followed the sequence TNT >> DNT > MNT. In addition to quenching studies conducted in solution, quenching studies were repeated with MNT, DNT, and TNT on solid films of Py-SNPs coated filter papers (Py-CFPs). Vapour quenching studies yielded response times of 0.48 (± 0.05) and 3.6 (± 0.3) min for air saturated with MNT and DNT, respectively. Detection limits for the quenching of Py-CFPs by MNT, DNT, and TNT deposited onto the filter paper were found to equal 30 (± 9), 11 (± 5), and 1.4 (± 0.6) ng per mm. In terms of detection limits, these results place the Py-CFPs among the best optical sensors currently available for the detection of TNT.
Enhanced Diffusion in Latex Films Induced by Oligomers and Characterized by Pyrene Excimer Fluorescence
(University of Waterloo, 2019-09-24) You, Zehou; Duhamel, Jean
Pyrene excimer fluorescence (PEF) was used to investigate the effects that the presence of low molecular weight oligo(n-butyl methacrylate) (OBMA) have on the diffusion of high molecular weight poly(n-butyl methacrylate) (PBMA) in latex films. In this project, a high molecular weight PBMA latex labeled with 1.9 mol% pyrene (Py-PBMA, Mw = 410 kg/mol, PDI = 2.0) was mixed with nine non-fluorescent latex particles which needed to be prepared. Among these nine particles, two unlabeled latexes (Mw = 360 kg/mol, PDI = 1.8 and Mw = 420 kg/mol, PDI = 1.9) were prepared without oligomers. Their molecular weight distribution (MWD) was slightly different from that of Py-PBMA. Four latex dispersions that incorporated four different weight fractions of an OBMA with an Mn of 3.0 kg/mol were prepared from a PBMA seed latex, whose MWD (Mw = 350 kg/mol, PDI = 1.9) was close to one of the unlabeled latex particles prepared without OBMA. Three more latex dispersions with three weight fractions of OBMAs with an Mn of 5.0 kg/mol were prepared from a PBMA seed latex, whose MWD (Mw = 460 kg/mol, PDI = 2.4) was similar to the other unlabeled latex polymerized without OBMA. Several blends of latex particles constituted of 5 wt% of Py-PBMA and 95 wt% of the non-fluorescent PBMA latex with or without oligomers were prepared and latex films were cast from these mixtures. The films were annealed at different temperatures and the fluorescence spectra of the films were acquired as a function of annealing time. They were analyzed to retrieve the fraction of mixing (fm), representing the molar fraction of Py-PBMA chains having diffused out of the Py-PBMA latex. In turn, the diffusion coefficients reflecting the diffusion of the polymer chains during film annealing were calculated from the fm values at each temperature and for different annealing times. Diffusion of the Py-PBMA chains was much enhanced upon mixing the Py-PBMA latex with the PBMA latex that contained a larger weight fraction of a same OBMA or a shorter OBMA at a same weight fraction. Master curves of the diffusion coefficient as a function of fm could be generated by determining the shift factors (aT). A plot of Ln(aT)-vs-1/T yielded the activation energy for the diffusion of the Py-PBMA chains, which was found to equal 163 ± 9 kJ/mol, regardless of the OBMA content or chain length. The efficiency (β) of OBMA as a plasticizer was determined as a function of oligomer length using the Fujita-Doolittle model. In addition, the temperature dependence of the efficiency was studied. The results showed that the plasticizer efficiency of the 3.0 kg/mol oligomer was larger than that of the 5.0 kg/mol at all temperatures studied, but that the difference in plasticizer efficiency between the two oligomers decreased for decreasing temperatures. The higher plasticizer efficiency of the 3.0 kg/mol oligomer was confirmed from a plot of Tg as a function of oligomer weight fraction, showing that Tg decreased more markedly with the 3.0 rather than the 5.0 kg/mol oligomer. In summary, this project further demonstrated the validity and robustness of the procedure based on pyrene excimer formation (PEF) to probe polymer chain diffusion in latex films.
Fluorescently Labeled Latex Particles to Monitor Film Formation
(University of Waterloo, 2017-10-25) Hisko, Victoria; Gauthier, Mario; Duhamel, Jean
The influence of the length of an oligo(ethylene glycol) spacer (EGn, where n is the number of ethylene glycol units) in a 1-pyrenemethoxy-EGn methacrylate (PyEG-MA) monomer on the incorporation of the monomer into a poly(n-butyl methacrylate) (PBMA) latex and on the diffusion of the pyrene-labeled PBMA (PyEGn-PBMA) in latex films was investigated. The PyEGn-MA monomers were prepared by two different methods. The anionic polymerization of ethylene oxide, using potassium 1-pyrenemethoxide as initiator, yielded ethoxylated 1-pyrenemethanol that was reacted with methacrylic anhydride to yield the PyEGn-MA monomer. Alternately, monotosylated well-defined ethylene glycol oligomers were successively coupled with 1-pyrenemethoxide and methacrylic anhydride. The monomers were copolymerized with n butyl methacrylate by emulsion polymerization. A longer EG7.4 spacer was found to enhance the incorporation of the PyEG7.4MA monomer up to 3.2 mol%, substantially higher that 1.9 mol% achieved previously with an EG3 spacer. The higher incorporation of PyEG7.4MA into the Py-PBMA latex led to a stronger pyrene excimer fluorescence (PEF) signal in the spectrum of the latex film. The goal was to use these fluorescently labeled particles to probe interparticle polymer diffusion (IPD) during film formation. Several issues arose in the copolymerization of PyEG7.4MA and n-butyl methacrylate (BMA). The aggregation of PyEG7.4MA into flower-like micelles was observed. Even though steps were taken to mitigate this effect in the emulsion polymerization process, the pyrene molecules tended to be more concentrated near the surface of the latex particles. This resulted in irreproducible annealing results, and it is recommended that these particles not be employed for the study of IPD.
Long Range Polymer Chain Dynamics Probed with Pyrene Excimer Fluorescence
(University of Waterloo, 2016-09-27) Farhangi, Shiva; Duhamel, Jean
A series of fluorescently labeled vinyl polymers bearing a C1-C18 side-chain (namely poly(methyl methacrylate), poly(butyl methacrylate), poly(octyl methacrylate), poly(lauryl methacrylate), and poly(stearyl methacrylate) were synthesized and their polymer chain dynamics (PCD) were characterized by applying the Fluorescence Blob Model (FBM). This report is the first example in the literature where the PCD of actual polymers are being compared as a function of the polymer chemical composition. The study of these polymers having large Mn values in the 170,000-810,000 g/mol range was accomplished by labeling these samples randomly with pyrene. The FBM takes advantage of the ability of the dye pyrene to form an excimer. Global FBM analysis of the pyrene monomer and excimer fluorescence decays yielded the blob size Nblob and the rate of pyrene excimer formation inside a blob from the product kblob×Nblob. In the future, the body of results found in this study is expected to become a reference for other polymer dynamics studies, including protein chain dynamics, influenced by the bulkiness of different side-chains. Secondly, we designed a functionalized pyrene derivative, namely 1-pyrenemethoxyethanol (PyMeOEtOH), that remains sensitive to solvent polarity. The 0-0 pyrene vibronic transition is symmetry-forbidden for pyrene, and in non-polar solvents the fluorescence intensity corresponding to this transition is low. Typically, the modification of pyrene with a reactive substituent destroys its symmetry, therefore functionalized pyrene derivatives lose their sensitivity to solvent polarity. In this work, the I1/I3 ratio of PyMeOEtOH was determined in 20 different solvents and the I1/I3 ratios were similar to those of the unmodified pyrene molecule. The product kblob Nblob retrieved from the FBM analysis remained the same regardless of the pyrene-labeled polymer. Therefore, PyMeOEtOH has the ability to probe the polarity of its local environment, and its use to probe polymer chain dynamics yields similar results as those obtained with PyButOH. In the third part of the study, four different pyrene-labeled polymers were prepared by radical copolymerization of n-butylmethacrylate (BMA) and 1-pyrenemethyl methacrylate, 1-pyrenemethoxyethyl methacrylate, 1-pyrenemethoxyethoxyethyl methacrylate, and 1-pyrenemethoxydiethoxyethyl methacrylate to yield PyEG0-PBMA, PyEG1-PBMA, PyEG2-PBMA, and PyEG3-PBMA, respectively. The number of atoms in the side chain of the pyrene-labeled copolymers increased from 3 in PyEG0-PBMA to 12 in PyEG3-PBMA. Steady-state fluorescence was used to monitor the efficiency of excimer formation while time-resolved fluorescence was applied to investigate and describe the kinetics of diffusive encounters between excited and ground-state pyrenes as a function of spacer length. In turn, FBM analysis of the fluorescence decays provided a means to represent how the volume probed by an excited pyrene and the kinetics of pyrene excimer formation were affected by the length of the spacer connecting pyrene to the main chain. The strong effect that the side chain length had on pyrene excimer formation in the PyEGX-PBMA samples suggests that pyrene excimer fluorescence could be an excellent technique to probe the conformation of highly branched macromolecules such as dendrimers or polymeric bottle brushes in solution. Fourthly, different pyrene-labeled constructs were studied in four different organic solvents using steady-state and time-resolved fluorescence. The Model Free Analysis (MFA) was used as a mathematical model to determine the diffusional rate constant of excimer formation for the end- and randomly labeled pyrene constructs with different architecture from linear to branched. This study established a universal calibration curve from the absolute value of IE/IM plotted as a function of , the aveage ate constant of excimer formation.
Pyrene Excimer Formation: A Tool to Study Macromolecular Conformations in Solution
(University of Waterloo, 2024-04-26) Patel, Sanjay; Duhamel, Jean
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 () 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 , 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 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.
Self-association of Oligoquinoline Foldamers Probed by Time-Resolved Fluorescence Anisotropy
(University of Waterloo, 2022-01-31) Lulic, Kristijan; Duhamel, Jean
The self-associations of oligoquinoline-based foldamers (OPV-QₙA with n = 4, 8, 17, and 33) terminated at one end with a carboxylic acid (A) and at the other end with the fluorescent dye oligo(phenylene vinylene) (OPV) with themselves, the octadecaquinoline Q₁₆A terminated with one carboxylic acid, and the AQ₂PQ₂A monomer made of a central pyridine flanked on each side by a quinoline dyad terminated with a carboxylic acid have been characterized in solution by time-resolved fluorescence anisotropy (TRFA). Complexation of the acid-terminated constructs was induced by the addition of 0.1 g of 16 M sodium hydroxide aqueous solution to the foldamer solutions in chloroform. Dimerization of the OPV-QₙA foldamers and the complexation of OPV-Q₈A with Q₁₆A followed a close association mechanism. Complexation of OPV-Q₈A with AQ₂PQ₂A occurred through an open association mechanism. Measurements were conducted on the solutions before and after addition of NaOH. Each sample was analyzed by UV-Visible absorption to determine the concentration of the species in solution and by TRFA to determine the average rotational time <ϕ> of the unassociated and associated OPV-QnA foldamers across concentrations spanning 5 orders of magnitudes in some cases. Plots of <ϕ> as a function of acid group concentration for the dimerization of the OPV-QₙA samples and complexation of OPV-Q₈A with Q₁₆A indicated that <ϕ> increased with increasing foldamer concentration, exclusively after addition of 16 M NaOH(aq). <ϕ> reached a plateau at high foldamer concentrations, indicative of a closed association mechanism. The increase in <ϕ> with increasing foldamer concentration reflected the association of the foldamers into larger objects. A plot of the <ϕ> values obtained in the high concentration plateaus, which were expected to represent foldamer dimerization, as a function of the number of quinoline units (NU) for the corresponding foldamer dimer, yielded a straight line, whose slope was the same as the slope of the straight line obtained by plotting <ϕ> of the unassociated foldamers as a function of their NU. This result was taken as evidence that complexation of the OPV-QₙA foldamers resulted in their dimerization. The <ϕ>-vs-[foldamer] profiles could be analyzed to determine the equilibrium constants K for these complexations. Large K⁻¹ values of 1.0 (±0.2)×10⁶ M⁻¹ were obtained which were similar in magnitude to those reported for other self-assembling systems, although the shortest foldamer made of four quinoline units had an equilibrium constant of 1.1 (±0.1)×10⁵ M⁻¹, that was one order of magnitude lower than its longer counterparts. The same methodology was then applied to investigate the complexation between AQ₂PQ₂A and OPV-Q₈A acting as a fluorescent stopper. Mixtures of AQ₂PQ₂A and OPV-Q₈A were analyzed by UV-Visible absorption to determine the concentration of both species in solution. These experiments led to the conclusion, that complexation of AQ₂PQ₂A under basic conditions resulted in hypochromicity due to aromatic stacking of the end-quinoline units of AQ₂PQ₂A. This observation suggested that upon complexation, AQ₂PQ₂A would form linear chains rather than random aggregates. TRFA was applied to determine the average rotational time <ϕ> of the products obtained from mixtures of either 1 µM or 10 µM OPV-Q₈A with varying concentrations of AQ₂PQ₂A. The trends obtained for <ϕ> as a function of the [AQ₂PQ₂A]/[OPV-Q₈A] ratio displayed an exponential increase at high foldamer concentration, indicative of an open association mechanism. Further analysis of these trends yielded the equilibrium constant, which was half that obtained for the dimerization of the longer OPV-QₙA with n = 8, 17, and 33, but four times larger than that of OPV-Q₄A. The complexation between AQ₂PQ₂A and OPV-Q₈A is reminiscent of a condensation polymerization, leading to the formation of many products with a broad size distribution, taking an average size similar to that of the longest oligoquinoline foldamer synthesized to date by conventional techniques. Consequently, the experiments described in this thesis demonstrate that large foldamers can be generated through the self-association of smaller specially designed molecules in solution.
The Structure and Internal Dynamics of Polypeptides Probed with Pyrene Excimer Fluorescence
(University of Waterloo, 2021-01-25) Casier, Remi; Duhamel, Jean
A combination of pyrene excimer fluorescence, fluorescence blob model (FBM), and molecular mechanics optimizations (MMOs) was applied to characterize a series of pyrene-labeled polypeptides. Analysis of the fluorescence decays with the FBM yielded information on the number (Nblobexp) of amino acids (aa’s) contained within the volume of a blob and the rate constant (kblob) at which two aa’s labeled with pyrene encountered one another inside a blob. The Nblobexp value was used as a structural parameter which was compared to the Nblobtheo value obtained from MMOs conducted on constructs of pyrene-labeled polypeptides to determine the conformation adopted by the rigid polypeptides in solution. The relationship that exists between Nblobexp and macromolecular conformation was established by using a series of pyrene-labeled homopolypeptides, namely poly(L-glutamic acid) (PLGlu), poly(D,L-glutamic acid) (PDLGlu), and poly(L-lysine) (PLL). The coiled conformations of PDLGlu and PLL yielded the smallest blob sizes, and thus the smallest Nblobexp values, due to their elongated conformation on the length-scale of a blob. Nblobexp was found to be significantly larger, when PLGlu and PLL adopted an alpha-helical conformation, indicating that the blob size was directly related to conformational density. This information was then used to identify the unknown conformation of PLGlu in DMSO and determine the interhelical distance in bundles of PLL alpha-helices. To study the effect of ionic interactions, the protonated and deprotonated forms of PDLGlu and PLGlu were compared. Nblobexp was found to remain constant, indicating that the temporal window provided by an excited pyrene was sufficient to study the rigid polypeptides even when their dynamics were slowed by the repulsion of like charges. Next, the effect that the aa sequence had on Nblobexp and the internal dynamics (kblob×Nblobexp) of a coiled polypeptide was investigated. To do this, a series of pyrene-labeled copolypeptides were synthesized through the ring opening polymerization of the N carboxyanhydride monomers obtained from racemic mixtures of glutamic acid and either glycine, alanine, or carbobenzyloxylysine (Lys(Z)) to yield PGlyGlu, PAlaGlu, and PLys(Z)Glu, respectively. Both Nblobexp and kblob×Nblobexp increased with decreasing aa’s side chain size (SCS). This indicated that the reduced steric hindrance generated by the smaller SCSs increased the internal dynamics and conformational freedom of the polypeptides. The effect, that the degree of comonomer incorporation had on the chain dynamics and internal density of polypeptides, was determined with a series of PAlaGlu samples prepared with alanine contents between 24 and 58 mol%. The constant Nblobexp value obtained with these samples suggested that the blob size was independent of the comonomer composition and instead depended only on the presence of a few small aa’s. Lastly, the relationship between Nblobexp and the side chain length (SCL) linking pyrene to the polypeptide was determined using a series of PDLGlu and PLL samples adopting a coiled conformation. This allowed for the quantitative determination of the contribution of the pyrenyl label to Nblobexp. After correcting for its contribution and taking into account the effect of SCS and SCL on Nblobexp, a program was implemented to estimate the blob size of each aa depending on the aa sequence in a heterogeneous polypeptide, such as a protein. This program was employed to determine the folding time of 145 proteins segmented into blobs. Comparison of the calculated and experimental folding times yielded a correlation coefficient of 0.73, demonstrating the ability of blob-based approaches, such as the ones described in this thesis, to predict the folding time of proteins. In summary, this thesis has introduced a novel blob-based approach to predict the folding time of proteins based on parameters that can be determined experimentally from the use of pyrene excimer fluorescence and racemic polypeptides. It opens an alternate path toward developing a better understanding for proteins on how to fold graciously (C. Levinthal).
Synthesis and Characterization of Furan-Based Non-ionic Surfactants (FBNIOS)
(University of Waterloo, 2021-01-27) Liu, Donghan; Duhamel, Jean; Gauthier, Mario
A series of furan-based non-ionic surfactants (fbnios) derived from 5-(chloromethyl)furfural (5-CMF), a feedstock prepared by Origin Materials through a carbon negative process, were prepared from commercially available 2,5-bis(hydroxymethyl) furan (2,5-bisHMF). The fbnios were synthesized by alkylating one hydroxyl of 2,5-bisHMF by Williamson ether synthesis and ethoxylating the other hydroxyl to generate an oligo(ethylene oxide) (OEO). Through systematic variations in the OEO length achieved by anionic polymerization, and the use of octyl and dodecyl groups, fbnios with different hydrophilic-lipophilic balances (HLBs) were synthesized. The number-average degree of polymerization (DPn), and purity of the fbnios samples were determined by proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), and matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-ToF-MS). The amphiphilic properties of these fbnios were characterized by surface tension and fluorescence measurements. Surface tension was applied to determine the efficiency and effectiveness of the fbnios. The critical micelle concentration (CMC) of fbnios was determined by both characterization methods. The CMC of the fbnios prepared with an octyl chain was found to decrease about 3-fold upon increasing the DPn of the OEO block from 3 to 14. The length of the OEO block had less influence on the CMC of the fbnios series prepared with a dodecyl chain. In contrast, the alkyl chain used to prepare the fbnios was found to affect their CMC, the CMC of the fbnios with an octyl chain being more than one order of magnitude larger than the CMC of the fbnios with a dodecyl chain. The range of CMC values found for the fbnios prepared in this thesis covered the range of CMCs found for well-known non-ionic surfactants (nios) such as the Triton X or Brij surfactant families. The fbnios with a dodecyl chain were found to have lower CMCs than the Brij surfactants prepared with the same alkyl chain. In summary, fbnios appear to behave as typical nios and show promising amphiphilic properties.
Synthesis and Characterization of Two Pyrene Labeled Gemini Surfactants and their Interactions with Oppositely Charged Molecules
(University of Waterloo, 2022-06-09) Ba Salem, Abdullah; Duhamel, Jean
Cationic gemini surfactants (GS) have attracted the attention of the scientific community as potential carriers of genetic cargo for gene transfection applications. These GS/polynucleotides complexes have been extensively studied in the literature, although most of the focus has been geared toward the characterization of the polynucleotide component of these complexes. This thesis utilizes steady-state (SSF) and time-resolved (TRF) fluorescence in conjunction with the model free analysis (MFA) to study and characterize two gemini surfactants, which were covalently labeled with the fluorescent dye pyrene. Pyrene was selected as the fluorophore for its ability to form an excimer upon encounter between an excited and a ground-state pyrene. First, two cationic gemini surfactants denoted as Py-3-12 and PyO-3-12 were prepared and their successful synthesis was confirmed by 1H NMR. The surfactants were constituted of two dimethylammonium bromide headgroups linked by a propyl spacer, where one headgroup bore a dodecyl chain and the other headgroup bore either a 1-pyrenehexyl or a 1-pyrenemethoxyhexyl group for Py-3-12 and PyO-3-12, respectively. The properties of Py-3-12 and PyO-3-12 were investigated by determining their critical micelle concentration (CMC) and average aggregation number (Nagg). The fluorescence from the pyrene excimer was employed for the first time to determine Nagg for the micelles generated by these pyrene-labeled gemini surfactants (PyLGS). Since PyO-3-12 was shown to be more sensitive of the polarity of its local environment, it was deemed a better fluorescent probe than Py-3-12. After the behavior of the PyLGS had been characterized in solution, their interactions with other molecules were studied. The improved properties of PyO-3-12 led to the study of its complexation with the anionic surfactant sodium dodecyl sulfate (SDS). Three series of PyO-3-12 solutions were prepared, where [PyO-3-12] was kept constant at 1, 4, and 16 M, which is below the CMC of 0.33 mM of PyO-3-12, while the SDS concentration was varied from 0 to 200 mM. The SSF and MFA results showed that complexation of PyO-3-12 with SDS was complete at the equicharge point. As more SDS molecules were added to these systems, these complexes remained stable resulting in small changes in the trends obtained from the analysis of the SSF spectra and the MFA of the TRF decays. The PyO-3-12/SDS complexes were found to break down into smaller complexes as the [SDS] increased from 2 to the 8 mM CMC of SDS as a result of the incorporation of more SDS molecules into the PyO-3-12/SDS complexes. The smaller PyO-3-12/SDS complexes that existed at the CMC of SDS retained their composition for [SDS] between the CMC and 50 mM, as SDS micelles began to form. Increasing [SDS] above 50 mM led to the complete decomposition of the PyO-3-12/SDS complexes and resulted in SDS micelles hosting a single PyO-3-12 surfactant. These findings showed that the use of fluorescence techniques like SSF and the combination of TRF with the MFA provides a powerful tool to probe the interactions between PyLGS and oppositely charged surfactants at PyLGS concentrations, that were as low as 1 M, much lower than their CMC and thus low enough to enable the study of their interactions between unassociated surfactants. Other techniques such as light scattering would require far higher concentrations of surfactants to study these aggregates. The complexation of Py-3-12 and PyO-3-12 with calf thymus DNA (CT-DNA) was investigated in the fourth research chapter of this thesis. The concentrations of Py-3-12 and PyO-3-12 were kept constant below their respective CMC, while the CT-DNA concentration was varied and the (/+) ratio, representing the ratio of the [CT-DNA] expressed in base pair concentration over [PyLGS], was used to describe changes in the [CT-DNA]. Both surfactants formed stable complexes with CT-DNA over a range of (/+) ratios around unity, as indicated by the constant parameters retrieved from the analysis of the SSF spectra and the MFA of the TRF decays. PyO-3-12 also provided some insight about the polarity of the local environment inside the PyO-3-12/CT-DNA complexes, which was found to be less hydrophobic than the interior of the PyO-3-12 micelles. The stability of the PyO-3-12/CT-DNA complexes prepared with a (/+) ratio of 1.5 was further investigated as SDS, used to represent the negatively charged amphiphilic molecules found in cell membranes, was added to the solution. At low [SDS], addition of SDS molecules led to their association with the PyO-3-12/CT-DNA complexes in a process leading to the dissociation of the PyO-3-12/CT-DNA into ter-complexes made of individual CT-DNA molecules coated with a layer of positively charged PyO-3-12 further wrapped in a layer of SDS. Above an [SDS] of 0.5 mM, the DNA molecules were released from the ter-complexes leading to the formation of PyO-3-12/SDS complexes, which had been studied earlier. The complete release of CT-DNA from the PyO-3-12/CT-DNA complexes further supports the well-accepted notion that gemini surfactants are good candidates as carriers of genetic material. In summary, this thesis has developed the use of PyLGS to characterize the interactions between unassociated gemini surfactants and oppositely charged molecules like SDS and CT-DNA. Taking advantage of the outstanding sensitivity of fluorescence, these studies can be conducted at surfactant concentrations, that are 1 to 2 orders of magnitude lower than their CMC, while the photophysical properties of the pyrenyl label affords insights into the structure and conformation adopted by the complexes generated with the PyLGS.
Using Pyrene to Probe Interparticle Polymer Diffusion in a Latex Film
(University of Waterloo, 2015-12-16) Casier, Remi; Duhamel, Jean; Gauthier, Mario
Fluorescence has been used for many years as a powerful analytical tool to probe the formation of films from aqueous latex dispersions. Currently, the most widely used method is fluorescence resonance energy transfer (FRET), which requires the use of two different fluorescently-labelled latex dispersions. As the latex particles coalesce during film formation, the amount of FRET that occurs between the particles is used to probe the level of interparticle polymer diffusion (IPD). Although FRET provides a quantitative method to probe IPD, a simpler method might only require the preparation of a single fluorescently-labelled latex using the dye pyrene. An isolated pyrene excited by a photon of light emits as a monomer. However, if the excited pyrene encounters a ground-state pyrene, it can form an excimer. The amount of excimer formed is directly proportional to the local pyrene concentration (CPy), which may be quantified by steady-state fluorescence with the ratio of the fluorescence intensity of the excimer (IE) over that of the monomer (IM), namely the IE/IM ratio. A mixture of a non-fluorescent latex with a latex consisting of polymers randomly labelled with pyrene will initially have a high CPy, generating lots of excimer and resulting in a high IE/IM ratio. As IPD occurs during film formation, the pyrene-labelled copolymer will diffuse into the surrounding non-fluorescent latex particles lowering CPy, resulting in reduced excimer formation and a decrease in the IE/IM ratio. Since variations in the IE/IM ratio reflect the extent of IPD, the IE/IM ratio was monitored over time to quantitatively describe the IPD of polymer chains between latex particles during film formation. The IE/IM ratio was used to calculate the fraction of mixing (fm) between the latex particles as a function of annealing time and temperature. The colour of the films irradiated by UV light was also monitored to determine whether a discernible colour change was apparent over the annealing process. In turn, fm was used to calculate the apparent diffusion coefficients of the pyrene-labelled copolymer. Lastly, the diffusion coefficients were used to calculate the apparent activation energy of diffusion and the c1 and c2 terms in the WLF equation.

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