Con A binding studies

5.3.2.1 Con A binding studies at low concentration

The changes in the absorption and fluorescence emission spectra of Man-Nap (1 × 10-5 _M) upon binding with Con A where recorded in DPBS pH 7.2 (0.1 mM CaCl2 and 0.1 mM MnCl2) at 25 °C and can be seen in Figure 5.3.3. Interestingly, even though precipitation occurs when Man-Nap binds with Con A, due to the complex formed being more insoluble, the UV-vis spectra still reflects the binding with Con A, with not much scattering being observed (Figure 5.3.3a).

Figure 5.3.3. Changes in the absorption (a) and fluorescence emission spectra (b) of Man-Nap (1 × 10-5 _M) upon the addition of Con A recorded in DPBS pH 7.2 (0.1 mM CaCl2 and 0.1 mM MnCl2) at 25 °C. c) changes in the fluorescence emission intensity (max = 545 nm) of Man-Napvs. Con A equivalents. Figures representative of three independent experiments. d) Changes in the fluorescence emission intensity (max =

545 nm) of Man-Napvs. Con A equivalents from three independent experiments.

a) b)

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The band observed at max = 280 belongs to the Con A, as previous concentrations studies with solely Con A demonstrated (see later discussion). Excitation at 433 nm showed that the fluorescence emission intensity increased upon binding with Con A, with a significant increase found between 0.2 and 0.3 equivalents, and a blue shift in the max of approximately 20 nm (Figure 5.3.3b).

This notable increase in fluorescence emission intensity can be clearly observed in Figure 5.3.3c, where the emission intensity is plotted versus Con A equivalents. Importantly, this behaviour was completely reproducible, as can be seen in Figure 5.3.3d, which shows the changes in fluorescence emission vs. Con A equivalents of three independent titrations with Con A. It is important to note that above 1 equivalent of Con A, no further increase in the fluorescence intensity of Man-Nap was observed and a plateau was reached, which could suggest a 1:1 binding.

As it was mentioned previously, the band cantered at 280 nm in the absorption spectra belongs to the Con A, and it might suggest that the changes seen in the characteristic ICT band from Man-Nap, max = 430 nm, are a result from the tail of the band at 280 nm. However, as can be seen in Figure 5.3.4, concentrations studies carried out with Con A demonstrated that although there was influence of this band at 430 nm it was not as significant as shown in Figure 5.3.3. Importantly, excitation at 433 nm gave no fluorescence emission (Figure 5.3.4b), therefore, proving that the changes previously seen with Con A correspond solely to changes in the photophysical properties of Man-Nap upon binding with Con A.

Figure 5.3.4. Changes in the absorption (a) and fluorescence emission spectra (exc = 433 nm) of Con A at different concentrations, recorded in DPBS pH 7.2 (0.1 mM CaCl2 and 0.1 mM MnCl2) at 25 °C.

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159 Unfortunately, attempts to obtain a binding constant were not successful. Recently Bernardi et al.227 have reported a glycodendrom-rhenium complex as probe for Con A, and were able to determine the binding constant, by means of Kd, using the Hill model, which can quantify the interactions between ligands and binding site and is usually employed to determine cooperativity.228 Using this method they obtained a Kd of 7.27 μM. However, attempts to fit the data obtained with Man-Nap into the Hill plot using GraphPad prism software resulted unsuccessful (Kd = 0.4 μM (± 0.1), R2 =0.7268).

When the studies were carried out using Man-Tb, similar results were obtained (Figure 5.3.5). In this case, the scattering occurring was a lot higher than for the previous compound, therefore, the changes in the absorption spectra did not follow a logical trend. An increase and decrease in the OD were observed alternatively, regardless of the addition of Con A, as a result from the scattering occurring. Interestingly, these changes were observed at lower equivalents of Con A than the ones required for Man-Nap (0.2 – 0.3 equiv). Here, the addition of only 0.1 equivalents of Con A already induced significant changes in the absorption spectra (Figure 5.3.5a).

Nevertheless, as a result of this scattering, these changes were not reproducible at concentrations of Con A above 0.3 equivalents, as can be seen in Figure 5.3.5c, which shows the changes in the absorption (max = 380 nm) vs. Con A equivalents of three independent experiments. The fluorescence emission spectra (exc = 380 nm) did not show significant changes. In Figure 5.3.5b, a sharp band can be seen at max = 420 nm, this band was most likely Raman scattering from the H2O, as a result of having a wide excitation slit (5 nm) to monitor the low fluorescence emission. Similar results have been observed in the literature.229 Apart from this band, only small changes can be observed, with a subtle decrease in the fluorescence emission intensity observed at max = 525 nm. Figure 5.3.5d shows the changes in the fluorescence emission (max = 525 nm) vs. Con A equivalents of three independent experiments, and similarly to the changes observed in the UV-vis spectra, no reproducibility was observed. As the changes observed were not reproducible, no attempts to fit the data were made.

As the aforementioned experiments did not give much information about the binding occurring between Con A and the glycosylated naphthalimides and TB-naphthalimides, further studies were performed at higher concentration, and will be discussed in the next section.

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Figure 5.3.5. Changes in the absorption (a) and fluorescence emission spectra (b) of Man-Tb (1 × 10-5 _M)

upon the addition of Con A recorded in DPBS pH 7.2 (0.1 mM CaCl2 and 0.1 mM MnCl2) at 25 °C. Figure

representative of three independent experiments. c) Changes in the absorption (max = 390 nm) of Man-Tb

vs. Con A equivalents of three independent studies and d) Changes in the absorption (max = 525 nm) of

Man-Tbvs. Con A equivalents of three independent studies.

5.3.2.2 Con A binding studies at high concentration

The binding affinity of Man-Nap and Man-Tb towards Con A at high concentration (1 × 10-4 M) was investigated. It is important to note that at this concentration both compounds were aggregating (see 5.3.1) and therefore, the emission was being quenched by aggregation (self-quenching). As Con A is a large macromolecule, it was expected that the binding of compounds Man-Nap and Man-Tb to it would prevent their aggregation, leading to a change in the fluorescence emission. Measurements of the ground state by UV-vis spectroscopy were not possible due to scattering problems (Figure A.5.2), as the binding with Con A forms aggregates that precipitate out of the solution, as previously observed.

a)

b)

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161 Figure 5.3.6. Fluorescence emission spectra of a) Man-Nap (1 × 10-4 _M, 

exc = 430 nm) and b) Man-Tb (1 × 10-4 _M, 

exc = 380 nm) before (black lines) and after the addition of 0.1 Equivalents of Con A (red lines), respectively, measured in DPBS (0.1 mM CaCl2 and 0.1 mM MnCl2) at 22 °C. Representative images of three independent experiments.

As a qualitative test, 0.1 equivalents of Con A were added to a solution of Man-Nap (1 × 10-4 _{M) and Man-Tb (1 × 10}-4 _{M), respectively, in DPBS (0.1 mM CaCl}

2 and 0.1 mM MnCl2) at 22 °C. As can be observed in Figure 5.3.6, the results varied depending on whether the Man-Nap or Man-Tb derivatives were used. In the case of Man-Nap, the fluorescence was turned on significantly upon the addition of Con A; by contrast, the fluorescence of Man-Tb was turned off (λem = 510 nm) and a new band appeared at 450 nm. These results are in agreements with the results obtained at lower concentrations.

In this case, where aggregation and self-quenching was occurring, the decrease in the fluorescence signal of Man-Tb was unexpected, but could be explained by the fact that

aggregation did not induce as much quenching as for Man-Nap. A new band appeared in

the fluorescence spectrum of Man-Tb at 450 nm and increased linearly with the concentration of Con A. This band was not observed at the experiments performed at lower concentration.

A back titration was carried out with Con A by treating compounds Man-Nap and Man-Tb with 0.1 equivalents of Con A and decreasing its concentration while keeping the

concentration of Man-Nap and Man-Tb constant. As the changes observed for Man-Tb

were not so noticeable, the deviation from the initial fluorescence (F0) respect to the fluorescence response upon binding Con A (F) was plotted (Figure 5.3.7), for Man-Tb as well as for Man-Nap for direct comparison. In the case of Man-Tb, represents the

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Figure 5.3.7. Changes in the fluorescence increase or decrease of a) Man-Nap (1 × 10-4 _M, 

exc = 430 nm) and b) Man-Tb (1 × 10-4 _M, 

exc = 380 nm) upon addition of different concentrations of Con A. Experiments carried out in DPBS (0.1 mM CaCl2 and 0.1 mM MnCl2) at 22 °C. Representative figures of three independent experiments.

increase and decrease observed at 450 and 510 nm, respectively, whereas in the case of Man-Nap only an increase was found at 535 nm.

As can be seen in Figure 5.3.7, the fluorescence intensity of Man-Nap upon treatment with Con A increased linearly up to 9 × 10-6 _{M, after which the fluorescence intensity} plateaus. Similarly, the lowest concentration of Con A at which the fluorescence emission changed was 1 × 10-6 _{M (0.1 equiv).}

For compound Man-Tb the fluorescence increased linearly at λ = 410 nm up to 8 × 10-6 M, reaching a plateau afterwards in a similar manner to Man-Nap. In addition, the lowest concentration of Con A invoking a change was 1 × 10-6 M as well (0.1 equiv).

Attempts to fit this data into a Hill plot gave a Kd = 7 × 10-6 M (± 0.1) (R2 = 0.9892). However, these values were not reproducible in further titrations (Figure A.5.3), with Kd values obtained from 9 × 10-6 M to 1 × 10-6 M, and therefore and accurate binding constant could not be given for this system. It has to be taken into account that there are three different factors taking place in this system: First, Con A binding with Man-Nap inducing a change in the fluorescence emission intensity, as demonstrated with the studies performed at lower concentration. Secondly, de-aggregation of Man-Nap caused by this binding, and therefore also modifying the fluorescence emission intensity, as aggregation of Man-Nap leads to self-quenching (as demonstrated in Figure 5.3.2). And third and last, the Con A binding form a lower water-soluble complex, which makes the solution turn turbid and can have an effect in the emission intensity due to the secondary inner filter

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163 effect. Therefore, it is not surprising that the fitting in order to get a binding constant was not reproducible, as there are many factors in play. However, the ability of these compounds to bind to Con A at high (and low) concentration was still demonstrated.

Following this, the effect of Con A binding under different conditions was subsequently investigated. Figure 5.3.8 represents the normalised emission intensity of Man-Nap and Man-Tb with Con A after different treatments.

Whereas the addition 0.1 equivalents of Con A led to a ca. 70% increase in fluorescence intensity in the case of Man-Nap and a decrease of 33% for Man-Tb, the addition of an excess of -D-mannose (0.2 equiv) to the mixture did not induce any changes. This may

suggest that the ligands bind more efficiently to Con A. However, further studies, including computational analysis, would need to be undertaken to confirm this.

Figure 5.3.8. Effects on the Con A binding of Man-Nap (1 × 10-4 _{M, blue) and Man-Tb (1 × 10}-4 _M, magenta) under different conditions measured in DPBS (0.1 mM CaCl2 and 0.1 mM MnCl2) at 22 °C.

In a separate experiment, the ligands were incubated with denatured Con A (heated at 100 °C for 30 min), and no significant change in the emission was observed, demonstrating that the initial changes observed in the fluorescence were due to binding with the -D

mannose unit, and not only due to the presence of large macromolecules in solution. Following this rationale, 0.1 equivalents of BSA were added to the ligands under identical conditions, but in this instance, no significant changes were observed, further demonstrating that the fluorescence change was due to substrate recognition and selective binding with Con A.

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In another experiment, 0.1 equivalents of Con A were added to a mixture of BSA with either Man-Nap or Man-Tb, to investigate if the presence of BSA would interfere with binding to Con A. Gratifyingly, the fluorescence changes followed an identical trend as the experiment carried out without BSA, proving that the binding of Con A is as efficient and selective in the presence of other biomacromolecules.

Finally, the binding of Man-Nap to Con A in the presence of another lectin was

investigated. When Man-Nap or Man-Tb were incubated with 0.1 equivalents of Peanut

Agglutinin (PNA), no changes in fluorescence intensity were observed. However, when Con A (0.1 equiv) was added to the mixture, the same changes in fluorescence intensity were observed to those observed previously, providing further evidence of substrate selectivity in binding Con A.

The previous experiments demonstrated that the compounds are able to bind efficiently with Con A even when other macromolecules were present. After this, a series of negative control studies were carried out, to further demonstrate that the fluorescence changes were only observed when efficient binding was taking place.

Structurally related compounds 63a (Gal-Nap) and 63d (Lac-Nap), synthesised and described in Chapter 2, containing a β-D-galactose and β-D-lactose unit, respectively, were

incubated with Con A, as well as compound 149 as it is the acetylated version of Man-Tb.

Figure 5.3.9. Changes in the normalised intensity of a series glycosylated naphthalimide (1 × 10-4 _{M) upon} addition of 0.1 equivalents of Con A in DPBS (0.1 mM CaCl2 and 0.1 mM MnCl2) at 22 °C.

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165 As shown in Figure 5.3.9, none of these exhibited any changes in their fluorescence

intensity upon addition of Con A. Thus it is clear that compounds Man-Nap and Man-Tb

bind selectively with Con A, and this binding process can be measured via fluorescence spectroscopy, up to low concentrations of the lectin Con A of 1 μM.

Following the demonstration of a successful binding between Man-Nap and Man-Tb to Con A, independently of the presence of other macromolecules, and having proven that the monovalent system (Man-Nap) binds more efficiently, the ability of other glycosylated naphthalimides towards other lectins was investigated. The next section describes the capacity of compounds 63a (Gal-Nap) and 63d (Lac-Nap) to bind to the lectin PNA.