Jean Duhamel

New Insights in the Study of Pyrene Excimer Fluorescence to Characterize Macromolecules and their Supramolecular Assemblies in Solution

This report highlights some of the recent developments that have been made in the quantitative analysis of fluorescence decays acquired with pyrene-labeled macromolecules. With these new analytical tools, macromolecules of different composition and architecture can now be labeled in a variety of ways with the pyrene chromophore, and the kinetics of pyrene excimer formation can be described to retrieve quantitative information about the internal dynamics of the macromolecules studied. In particular, this review presents the procedure that was followed to develop these new analytical tools and how the process of pyrene excimer formation with vinyl polymers, poly(L-glutamic acid), dendrimers, associative polymers, surfactants, and lipids labeled with pyrene has been successfully characterized thanks to these analysis programs.

Synthesis and Characterization of Novel Pyrene-Dendronized Porphyrins Exhibiting Efficient Fluorescence Resonance Energy Transfer: Optical and Photophysical Properties

A novel series of pyrene dendronized porphyrins bearing two and four pyrenyl groups (Py(2)-TMEG1 and Py(4)-TMEG2) were successfully synthesized. First and second generation Fréchet type dendrons (Py(2)-G1OH and Py(4)-G2OH) were prepared from 1-pyrenylbutanol and 3,5-dihydroxybenzyl alcohol. These compounds were further linked to a trimesitylphenylporphyrin containing a butyric acid spacer via an esterification reaction to obtain the desired products. Dendrons and dendronized porphyrins were fully characterized by FTIR and (1)H NMR spectroscopy and their molecular weights were determined by matrix-assisted laser desorption ionization time of flight mass spectrometry. Their optical and photophysical properties were studied by absorption and fluorescence spectroscopies. The formation of dynamic excimers was detected in the pyrene-labeled dendrons, with more excimer being produced in the higher generation dendron. The fluorescence spectra of the pyrene dendronized porphyrins exhibited a significant decrease in the amount of pyrene monomer and excimer emission, jointly with the appearance of a new emission band at 661 nm characteristic of porphyrin emission, an indication that fluorescence resonance energy transfer (FRET) occurred from one of the excited pyrene species to the porphyrin. The FRET efficiency was found to be almost quantitative ranging between 97% and 99% depending on the construct. Model Free analysis of the fluorescence decays acquired with the pyrene monomer, excimer, and porphyrin core established that only residual pyrene excimer formation in the dendrons could occur before FRET from the excited pyrene monomer to the ground-state porphyrin core.

Characterization of the Behavior of a Pyrene Substituted Gemini Surfactant in Water by Fluorescence

Time-resolved fluorescence was applied to characterize the behavior in solution of a gemini surfactant substituted with pyrene (Py-3-12). Upon association in water, excimer formation by Py-3-12 can be probed by acquiring pyrene monomer and excimer fluorescence decays which can be fitted globally according to the model free (MF) analysis to yield quantitative information about the internal dynamics of the Py-3-12 surfactant micelles as well as a complete description of the distribution of the different pyrene species in solution either incorporated inside the micelles or free in solution. A proof of procedure for the MF analysis was established by noting that the concentrations of free surfactant in solution, [Py-3-12](free), was found to equal the critical micelle concentration (CMC) for surfactant concentrations larger than the CMC. (I(E)/I(M))(SPC), the ratio of pyrene monomer to excimer fluorescence intensities, was calculated from parameters retrieved from the MF analysis of the fluorescence decays and was found to be independent of sample geometry. This work demonstrates how time-resolved fluorescence can be used to study the properties of pyrene-labeled macromolecules under conditions where large absorptions and inner filter effects usually distort the steady-state fluorescence signals. It was found that the pyrene excimer is formed mostly by diffusion within the Py-3-12 micelles, which suggests that the pyrene microenvironment is fluid, an important feature for future studies on the interactions of Py-3-12 with DNA.

A study of the dynamics of the branch ends of a series of pyrene-labeled dendrimers based on pyrene excimer formation.

A series of pyrene-labeled dendrimers were prepared from generation n = 1 to n = 4 where the pyrenes were attached to the end groups of the dendrimers. Pyrene excimer formation was monitored by steady-state and time-resolved fluorescence spectroscopy as a function of generation number and in terms of the I(E)/I(M) ratio and the average rate constant of excimer formation . To account for the unconventional distribution of pyrene labels which were neither randomly distributed throughout the macromolecule nor limited to just two units which are the only two pyrene-labeling schemes that can be dealt with in a straightforward manner, a Model Free (MF) analysis was applied to the global analysis of the fluorescence decays. Within experimental error, the I(E)/I(M) ratios and obtained from, respectively, the steady-state fluorescence spectra and the time-resolved fluorescence decays were found to increase linearly with increasing generation number. This result is inconsistent with the fact that both the I(E)/I(M) ratio and are proportional to the local concentration of pyrene inside the dendrimer ([Py](loc)) which is not expected to increase with generation number if the excited pyrene is assumed to diffuse freely throughout the dendrimer interior. Since the core-dense model predicts that the dendrimer terminal ends can occupy any position throughout the dendrimer interior, these results suggest that excimer formation between the pyrene-labeled ends is enhanced due to the branched nature of the dendrimer.

Molar Absorption Coefficient of Pyrene Aggregates in Water

The molar absorption coefficient of pyrene aggregates, epsilon(E0), was determined for a series of pyrene-labeled poly(N,N-dimethylacrylamide)s (Py-PDMA) having different pyrene contents. Aqueous solutions of Py-PDMA having pyrene contents ranging from 263 to 645 mumol of pyrene per gram of polymer were studied by UV-vis absorbance and time-resolved fluorescence spectroscopy. The global analysis of the monomer and excimer fluorescence decays with the fluorescence blob model yielded the fractions of the overall absorption contributed by all the pyrene species present in solution. The combined knowledge of the fractions obtained from the global analysis of the time-resolved fluorescence decays, the total absorption of the Py-PDMA solution obtained from UV-vis spectroscopy, and the total pyrene concentration in the solution obtained from the known pyrene content of each Py-PDMA sample led to the determination of the molar absorption coefficient of pyrene aggregates. Regardless of the pyrene content of the Py-PDMA samples and hence the level of association of the pyrene pendants in solution, all Py-PDMA samples yielded similar epsilon(E0) values over the range of wavelengths studied, namely, from 325 to 350 nm. The averaged epsilon(E0) was found to be red-shifted relative to unassociated pyrenes by 3 nm as well as having a value at the 0-0 peak of 21 000 M(-1).cm(-1) reduced from 34 700 M(-1).cm(-1) for unassociated pyrenes. The determination of epsilon(E0) enabled the first determination of the absolute fraction of associated pyrenes for aqueous solutions of a series of pyrene-labeled water-soluble polymers. The procedure outlined in this study is applicable to any pyrene-labeled water-soluble polymer and provides a new means to study quantitatively the effect of the hydrophilic-to-lipophilic balance on the hydrophobic associations generated by hydrophobically modified water-soluble polymers. As an application, the average number of pyrenes involved in a pyrene aggregate generated by Py-PDMA in water is determined.

Effect of polypeptide sequence on polypeptide self-assembly.

This study represents a unique example where the self-assembly of five amphiphilic polypeptides was monitored as a function of their hydrophilic-to-lipophilic balance (HLB). The HLB was tuned by preparing a series of polypeptides with aspartic acid (Asp) and phenylalanine (Phe) according to the sequence (Asp(x)Phe(y)) where x and y vary from 1 to 3. The self-assembly of the (Asp(x)Phe(y))(n) polypeptides with M(w) values ranging from 8 to 12 K was studied in solution by fluorescence quenching and nonradiative energy transfer (NRET) fluorescence experiments. Dynamic (DLS) and static (SLS) light scattering studies were used to complement the results obtained. The fluorescence quenching experiments conducted with the chromophore pyrene used as a free probe, physically bound to the polypeptides via hydrophobic interactions, demonstrated that the chromophore was less exposed to the solution for the sequences having a higher hydrophobic character. Protective quenching was also observed for those polypeptides randomly labeled with pyrene where the phenylalanine content was high, whereas pyrene was found to be fully exposed to the quencher for the polypeptides having more hydrophilic sequences. NRET used to probe interpolymeric association was not observed between a naphthalene and a pyrene labeled polypeptide for the polypeptide sequences which were richer in aspartic acid, whereas energy transfer took place with the more hydrophobic polypeptides. This observation led to the conclusion that those sequences with a higher content of aspartic acid essentially generate unimolecular polymeric micelles, whereas those with a higher content of phenylalanine generate polymeric aggregates which offer protection from the solvent to their hydrophobic cargo. The presence of these polymeric aggregates was also confirmed by DLS and SLS studies which suggest that species 100-200 nm in diameter are present in solution.

Quantifying the presence of unwanted fluorescent species in the study of pyrene-labeled macromolecules.

In order to mimic the effect that unwanted fluorescent species have on the process of excimer formation between pyrene labels covalently attached onto macromolecules, the steady-state fluorescence spectra and time-resolved fluorescence decays were acquired for mixtures of pyrene monolabeled and doubly end-labeled 2K poly(ethylene oxide) referred to as Py(1)-PEO(2K) and Py(2)-PEO(2K), respectively, and mixtures of 1-pyrenebutyric acid (PyBA) and a fourth generation dendron end-capped with pyrene (Py(16)-G4-PS). Monolabeled polymers like Py(1)-PEO(2K) and unattached fluorescent labels like PyBA are among the most typical fluorescent impurities that are encountered in the study of fluorescently labeled macromolecules. Our fluorescence experiments revealed that addition of minute amounts of Py(1)-PEO(2K) or PyBA to, respectively, Py(2)-PEO(2K) or Py(16)-G4-PS solutions induced a dramatic reduction of the ratio of the fluorescence intensity of the pyrene excimer to that of the pyrene monomer, namely the I(E)/I(M) ratio. Although the extreme sensitivity of fluorescence in general and the I(E)/I(M) ratio in particular to the presence of fluorescent impurities is a great concern, it is nevertheless reassuring that this effect can be quantitatively accounted for by analyzing the fluorescence decays of the pyrene monomer and excimer globally, according to a protocol which is described in detail in this study. The experiments presented herein demonstrate the importance of studying fluorescently labeled macromolecules that are of the highest purity when probing the rapid internal dynamics of a macromolecule by fluorescence.

Micellization and Adsorption of a Series of Succinimide Dispersants

The efficiency of a series of nonionic dispersants at stabilizing carbon-rich particles in oil was evaluated. The dispersants were synthesized by coupling a polyamine to two polyisobutylenes terminated at one end with a succinic anhydride (PIBSA). Their chemical composition was characterized by FTIR absorption. The associative strength of the dispersants was determined from their ability to self-associate in solution into reverse micelles. Their critical micelle concentration (CMC) and aggregation number (Nagg) were determined by fluorescence in an apolar solvent, namely hexane. The associative strength of the dispersants was found to increase with increasing number of secondary amines in the polyamine core. The adsorption isotherms describing the adsorption of the dispersants onto carbon black particles were determined and showed the existence of two binding regimes for the dispersants having more amines in their polar core. Much stronger binding occurred at dispersant concentration lower than the CMC. The weaker binding observed at dispersant concentrations larger than the CMC is believed to result from the dispersants having to compete between micellization and adsorption at concentrations above the CMC.

Molar Absorbance Coefficient of Pyrene Aggregates in Water Generated by a Poly(ethylene oxide) Capped at a Single End with Pyrene

The molar absorbance coefficient of ground-state pyrene aggregates (ε(E0)(λ)) in water generated by a short poly(ethylene oxide) chain labeled at a single end with a 1-pyrenemethoxide unit, namely, the polymeric construct Py₁-PEO(2.5K), was determined by a combination of UV-vis absorption and time-resolved fluorescence experiments. Since direct excitation of ground-state pyrene aggregates results in instantaneous excimer emission, whereas excimer formation by diffusive encounters between an excited- and a ground-state pyrene is delayed, acquisition of a pyrene monomer and excimer fluorescence decays with 1 nm increments of the excitation wavelength can probe the subtle effect that the excitation wavelength has on the extent of instantaneous excimer formation. These differences were taken advantage of to determine the fraction f(agg)(f)(λ) of the total absorbance of the Py₁-PEO(2.5K) solution that is due to ground-state pyrene aggregates. This was achieved by fitting the pyrene monomer and excimer fluorescence decays globally with the model free analysis. For each wavelength, an f(agg)(f)(λ) value was obtained which was then related to ε(E0)(λ) and f(agg), the molar fraction of aggregated pyrenes that is wavelength independent. These experiments were conducted for three Py₁-PEO(2.5K) concentrations yielding three similar ε(E0)(λ) spectra as expected since ε(E0)(λ) is an intrinsic property of the pyrene aggregates that should not depend on pyrene concentration. The ε(E0)(λ) spectra of the pyrene aggregates of Py₁-PEO(2.5K) in water were substantially broader and red-shifted compared to that of the pyrene monomer. Its measure provides a simple means to calculate the molar fraction of aggregated pyrene units in a Py₁-PEO(2.5K) aqueous solution from the absorption of the solution. This procedure should become widely applicable to determine f(agg) of water-soluble polymers which have been hydrophobically modified with a pyrene derivative and used as models for associative polymers.

Effect of time on the rate of long range polymer segmental intramolecular encounters.

The kinetics of encounters between the pyrene pendants randomly attached along a polystyrene chain (Py-PS) were monitored with a fluorescence blob model (FBM) as an external quencher was added to the solution to decrease the lifetime of the excited pyrene. The fluorescence decays acquired with the Py-PS samples yielded a measure of the volume Vblob probed by an excited pyrene during its lifetime in the form of N0blob, the number of monomers found within Vblob, and K0blob, which is inversely proportional to Vblob. Both N0blob and K0blob(-1) were found to increase with increasing probing time as the excited pyrene was allowed to probe a larger Vblob volume. The rate constant for pyrene-pyrene encounters was obtained from the product (KblobNblob). (KblobNblob) was found to decrease with increasing probing time, in agreement with scaling arguments suggesting that, as the probing time increases, the exited pyrene probes a larger Vblob where the local concentration of ground-state pyrenes in the polymer coil, [Py]loc, is smaller. K0blob, which is inversely proportional to Vblob, was found to scale as N0blob(alpha), where alpha equaled -1.5 and -1.2 in DMF and THF, respectively. The alpha exponents found for the Py-PS samples are in the same range as those found for other polymers exhibiting a random polymer coil conformation in solution and where much smaller than those obtained with more compact structured alpha-helical polypeptides randomly labeled with pyrene. Master curves were also constructed that describe how K0blob and the product (KblobNblob) scale as a function of solvent viscosity, probing time, and N0blob. These scaling laws illustrate the opposite effects that probing time and viscosity have on N0blob, V0blob, and the product (KblobNblob).