Quantifying boronic ester formation on nanoparticles

The binding constants of molecular species (fluorocatechols 12 and 13, and fluorosalicylic acid 14 with nanoparticle-bound boronic acids could be determined by 19_{F NMR spectroscopy. In a typical experiment to determine}

association constants, a mixture of AuNP-7b with binding partner ([AuNP-7b] = [binding partner] ≈ 5 mM) and internal standard (3-fluoro-4-bromonitrobenzene) was suspended in CD3OD. Addition of 100 eq. N-methylmorpholine led to

boronate ester formation, which was observed by 19_{F NMR spectroscopy. The}

equilibrium concentration of boronate ester and unbound binding partner was measured relative to the internal standard, and the equilibrium concentrations of boronic acid and unbound binding partner were determined from this measurement.

Table 3.2 Summary of association constants (Ka) determined for interaction of binding

partners 12, 13 and 14 with nanoparticle-bound boronic acid AuNP-7b and model boronic acid 10 in CD3OD with 100 eq. of N-methylmorpholine. Errors

quoted are the standard deviation of three repeats.

Binding partner AuNP-7b

Ka / M−1

Model boronic acid 10 Ka / M−1 a 3-Fluorocatechol 12 1260 ± 130 (10%) 1990 ± 200 (10%) 4-Fluorocatechol 13 610 ± 40 (7%) 3420 ± 720 (21%) 4-Fluorosalicylic acid 14 400 ± 60 (15%) 2500 ± 310 (12%)

a _{Values reproduced from Table 3.1.}

Two key observations can be made from these results. First, the association constants for binding with nanoparticle-bound boronic acids are lower than with model boronic acid 10 (although in the case of 3-fluorocatechol 12 the difference is relatively small). Secondly, the relative order of binding affinities for the two fluorocatechol regioisomers is swapped for the nanoparticle-bond case: 4-fluorocatechol 13 binds less strongly than 3-fluorocatechol 12. This is in opposition to the observed trends in the reversibility/cyclability experiment (Chapter 3.4), where 3-fluorocatechol 12 binds less strongly to nanoparticle- bound boronic acids than 4-fluorocatechol 13. It must be noted that the absolute values should be viewed cautiously, due to the inherent difficulty of quantifying the concentration of nanoparticle-bound species. Peak areas determined by

integrating the broad nanoparticle-bound species can vary significantly depending on phasing of the spectra and the degree of baseline correction. This variation is reduced as the peak intensity increases. Attempts to measure association constants with early batches of AuNP-7, where a high percentage of the boronic acid had oxidised to the corresponding phenol (Chapter 2.5), did not yield reproducible results. Nonetheless, utilising less oxidised AuNP-7b

(Chapter 2.5) allowed use of higher boronic acid concentrations, and gave more reproducible results. The results strongly indicate that binding is significantly weaker with nanoparticle-bound boronic acids than with the model boronic acid.

This difference could be due to limitations in the accessibility of the nanoparticle-bound boronic acids. The boronic acids form a tightly packed monolayer on the nanoparticle surface. In order to bind the binding partner, each boronic acid also requires coordination of a Lewis base, in this case N- methylmorpholine. The steric implications of trying to attach a binding partner and base at all sites may simply be too demanding, and only a certain percentage of the available boronic acids may be bound (Figure 3.15).

Figure 3.15 Schematic representation of the equilibrium binding of an exchangeable unit to

a nanoparticle. The steric bulk of the exchangeable unit makes complete binding of all boronic acids impossible. Binding saturation, therefore, is defined as binding of all accessible boronic acids, as opposed to all boronic acids.

Experiments were carried out in order to determine the total boronic ester concentration upon saturation of the nanoparticle with approximately 10 eq. of each binding partner. Preliminary titration curves (Chapter 7.4.3, Figure 7.13) indicated that this was sufficient to achieve saturation of available boronic acids.

The boronic ester concentration was once again determined by 19_{F NMR}

relative to an internal standard. The results (Table 3.3) indicate that for 4- fluorocatechol 13 and 4-fluorosalisylic acid 14, the binding partner formed boronic esters with approximately 65% of the total nanoparticle-bound boronic acids. For 3-fluorocatechol 12 this value was significantly higher, at 85%. The results show that even for these small binding partners, not all nanoparticle- bound boronic acids are accessible. This indicates a negative cooperative binding effect: the concentration of boronic ester increases, the subsequent association constants are effectively decreased (to zero once saturation binding is reached). As the single-point binding constants were measured under conditions near-saturation binding, this effective decrease in association constant is significantly reflected in the observed Ka value.

Table 3.3 Summary of the percentage of boronic esters formed with nanoparticle-boronic

acid AuNP-7b at saturation with binding partners 12, 13 and 14.

Binding partner Percentage of accessible

boronic acids

3-Fluorocatechol 12 85%

4-Fluorocatechol 13 65%

4-Fluorosalicylic acid 14 64%

At concentrations significantly lower than the saturation boronic ester concentrations, the observed Ka value may more closely match that observed

with model boronic acid 10. The inherently broad, weak spectra obtained from NMR spectra of nanoparticle-bound species make obtaining reliable data challenging at low nanoparticle concentrations; however, having now optimised access to high quality nanoparticle samples (Chapter 2.5) this could be revisited. Other techniques, such as isothermal titration calorimetry or titration and fitting to a Langmuir-type binding isotherm that has a packing coefficient as a fitting parameter may provide further details of this phenomenon.

This difference between the accessibility of boronic acids with 3-fluorocatechol

12 and 4-fluorocatechol 13 is striking, and hard to rationalise, as structurally the