Plausible Aggregate Architectures

Based on the cyro-TEM and FF-TEM results, as well as the fluorescence, AUC and SAXS

data, we drew plausible representations of porphyrin J-aggregates in tubular form (Figure 15)

and prior to tube formation (Figure 16). Figure 13 for tubes resembles the flat ring model of

Gandini et al.;52 in which the macrocycles lie flat to create one layer of the tube. In our simplified

representation, it is easy to see how macrocycles can experience zwitterionic and stacking

interactions with molecules in adjacent layers, which is consistent with the fluorescence

spectroscopy. Our results cannot distinguish this model from that of Vlaming et al.67 These

authors proposed partial overlap of the porphyrin molecules in two dimensions, resulting in a

planar sheet. As a result of the overlap, which provides the stabilizing interactions without

actually stacking layers, each macrocycle is tilted out of the plane by a small angle, θ ~20°. To

account for cylinder formation, the authors propose that the planar sheet is rolled; thus, the

macrocycle lies on the surface of the cylinder. By contrast, in the model of Gandini et al., the

plane of each macrocycle lies parallel to the cross-section of the cylinder. It is also not possible

to rule out a hybrid of the two models in which macrocycles join to form a ring, but with tilting

and partial overlap. This is effectively the staircase model (Figure 1b) wrapped into a ring (or

split washer). Stacking these rings (washers) could still yield a tube (helical tube). The ring

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n  f  19 Å  tube circumference  π  160 Å Equation 3-3 where f is a parameter (0 < f < 1) reflecting the degree to which one macrocycle overlaps

another. Figure 1b is drawn with f  0.5, meaning that a negatively charged sulfate group on one porphyrin group is located near the tetrapyrrole center of a neighboring macrocycle. Overlap

profoundly affects the ring occupancy number; for example, n  50 if f  0.5.

The situation is even less certain for the dilute solution aggregates. Equilibrium AUC

provides vital information on the mass of the assemblies, but not their shape. Micali et al.,68 who

studied porphyrin aggregation by light scattering while taking care to exclude fluorescent light,

reported a transition from fractal aggregates to rods. At pH 4, our AUC results instead show

assemblies made from 25  3 macrocycles. A single flat ring such as shown at the top of Figure 13 could account for the mass, but not for the fluorescence spectroscopy results, which demand

J-type stacking. The tilted ring hybrid model described in the preceding paragraph is consistent

with all our data, but structures of lower symmetry are easily imagined. In dilute solutions, the

ring motif is merely a suggestion from the SAXS observations at higher concentrations. Placing

26 macrocycles into that ring is consistent with the AUC result (25  3) but choosing 26 instead of another number in the experimental range again borrows from the nominal SAXS value at

higher concentrations. If a 26-member ring is correct, and if an overlap parameter f  0.5 is selected on the basis that such values enhance zwitterionic and stacking interactions, then a

circumference of ~250Å is expected, corresponding to a predicted diameter of ~80Å.

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Figure 3-16 One possible model (of many) for the elemental aggregate species in dilute solutions for H4TPPS2- at pH ≤ 4. The porphyrin rings are tilted out of the plane of the ring and partially overlap with their immediate neighbors.

3.4 Conclusions

A series of complementary experiments and analytical methods was used to characterize

porphyrin aggregates in aqueous solutions, as functions of concentration, solution pH, time, and

ionic strength. UV-Vis and fluorescence spectra showed that H2TPPS4- forms J-type aggregates

in dilute aqueous solutions, mainly dimers and trimers at neutral and basic solutions, and larger

aggregates with 25 ± 3 porphyrin units at low pH values (pH 4), based on AUC data. The

aggregate mass from AUC is consistent with a 26-member ring in which the macrocycles are

tilted with respect to the plane of the ring, but other structures permitting stacking interactions

are not excluded. The porphyrin is able to overcome electrostatic repulsion, even at high pH,

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In the absence of added NaCl (or at low ionic strength) and at low pH, the main aggregate

species in aqueous solution is a particularly stable 26-unit structure. As the concentration is

raised, the porphyrin molecules form hollow cylinders, as visualized by FF-TEM and modeled

using DAMMIF for the SAXS data. Our results for tubular structures are consistent with those of

Gandini et al., but we are unable to distinguish between that structure and the one suggested by

Vlaming et al. It is not yet possible to rigorously exclude helix formation. With time and/or at

high NaCl concentration, the aggregates grow, forming amorphous clusters that precipitate out of

solution. Indeed, this technique has been used for many years in porphyrin extractions from

aqueous media (using brine) and into organic phases, by decreasing their solubility in the

aqueous layer. This study provides the basis for improved understanding of the self-assembly

process of water-soluble porphyrins, and a foundation for investigation of more complex but

related systems for specific applications, such as cancer therapeutics. Especially appealing

avenues for further investigation include simulation to address the degree of overlap among the

porphyrin molecules and possible helix formation, and high-sensitivity, low-noise methods for

structure determination at very low concentrations.