Probing the inner local density of complex macromolecules by pyrene excimer formation

Abstract

The direct relationship existing between the average rate constant <k> for pyrene excimer formation and the local concentration [Py]loc of ground-state pyrenyl labels covalently attached to a macromolecule was established...

Accurate Determination of the Average Rate Constant of Pyrene Excimer Formation for Pyrene-Labeled Macromolecules from the Analysis of Individual Fluorescence Decays with Sums of Exponentials

“…The Pearson coefficient close to unity retrieved for ⟨τ⟩ also confirms that whereas the individual pre-exponential factors and decay times in a triexponential fit cannot be retrieved with enough accuracy to conduct a proper kinetic analysis of PEF for the Py-PEG n MA samples, ⟨τ⟩ is a reliable parameter that can be used in conjunction with ⟨k⟩ to characterize the local density and dynamics of a macromolecule in solution through, respectively, the parameters [Py] loc and k diff , as defined in eq 1. 20 As discussed earlier, the low Pearson coefficients obtained for the individual pre-exponential factors and decay times in Figure 6 retrieved from different sums of exponential analyses corroborate the challenges associated with the fit of multiexponential decays with the sums of exponentials. 7−12 Perhaps more specifically, it brings into question the value of the different models that have been introduced over the years to fit the multiexponential fluorescence decays acquired with PyLM, since the common feature of these models is that the pyrene monomer and excimer fluorescence decays acquired with PyLM can be represented by sums of exponentials.…”

“…This result is a mere consequence of the ⟨k⟩ versus [Py] loc relationship demonstrated in a previous publication. 20 However, application of the ⟨k⟩ versus [Py] loc relationship to characterize the conformation of a macromolecule requires that those pyrenyl labels form excimers by diffusive encounters so that [Py] loc truly reflects the local density of the macromolecule. Poor solubility of the pyrenyl labels, as in water, a high macromolecular density as for structured macromolecules, or pyrene clustering induced by the labeling reaction are all the effects leading to the formation of pyrene aggregates.…”

“…Since the pyrenyl labels were deemed to be unaggregated based on the A E− /A E+ ratios in Figure 2B, the ⟨k⟩ versus [Py] loc relationship should hold for the Py-PEG n MA and PyCX-PAMAM-GY samples, and ⟨k SoE-M ⟩ should increase linearly with increasing [Py] loc in the same manner as ⟨k MF ⟩ does. 20,27,28 To determine whether this was indeed the case, ⟨k SoE-M ⟩ was plotted as a function of a measure of [Py] loc for For the dendrimers, [Py] loc in Figure 3A and B was taken as the number (n Py ) of ground-state pyrenyl labels in a dendrimer divided by the cube of the average squared end-to-end distance (L Py ) between the pyrenyl labels normalized by the equivalent of a bond length (l). For the Py-PEG n MA samples, [Py] loc was taken as the ratio of the average number (⟨n⟩) of ground-state pyrenyl labels found inside a blob encompassing a polymer segment made of a number (N blob ) of structural units divided by N blob 3/2…”

“…. The expressions for [Py] loc have been derived earlier for the Py-PEG n MA 20 and PyCX-PAMAM-GY 27 samples.…”

“…63,64 Similarly, the direct relationship between ⟨k⟩ and [Py] loc in eq 1 was established with 55 pyrene-labeled macromolecules in four different organic solvents. 20 The Journal of Physical Chemistry B pubs.acs.org/JPCB Article Sums of Exponentials. To date, the PEF kinetics have been quantitatively characterized for hundreds of PyLM with the MFA and FBM.…”