Enzymatic Activity Evaluation

The changes in the absorption and emission spectra of 63a and 64 over time after the addition of -galactosidase were next examined at pH 7.2 and 30 °C (optimal conditions for this enzyme). For compound 63a, enzymatic hydrolysis did not lead to a significant change in the ICT absorption or fluorescence emission over time (Figure 2.3.8a and b). Different concentrations of the enzyme (0.01, 0.1 and 1.0 U) were added to the solution, and the same behaviour was observed, however, the treatment of 64 with 0.1 U of the enzyme led to a significant reduction in fluorescence intensity (Figure 2.3.8c and d) with no shift in the λmax = 550 nm. Importantly, no significant changes were seen in the absorption spectra of 74 and 64 upon addition of enzymes, which rules out any non- specific or strong association between the enzyme and the naphthalimide substrate.

Chapter 2. Glycosylated Naphthalimides as Enzyme-Activatable Probes

53 Figure 2.3.8. Study of -galactosidase activity with 63a (1 × 10-5 _{M) and 12 (1 × 10}-5 _{M), respectively. a)} and c) UV-vis absorption and b) and d) fluorescence spectra of 63a (λmax = 550 nm) and 64 (λmax = 440 nm), respectively. All the measurements were recorded at 37 ºC. Representative image of two independent experiments.

Since no noticeable changes were observed for the enzymatic release of compound 50, no further studies were carried out with them. However, time-dependent studies were carried out with compound 64 and -galactosidase at different temperatures, as can be seen in Figure 2.3.9. Prior to incubation with the enzyme, the changes in the fluorescence emission of compound 64 were monitored over time at 25 and 37 °C (Figure 2.3.9a). Although no changes were observed in the emission intensity at 25 °C, a significant decrease occurred when the compound was incubated at 37 °C. Temperature could cause changes in the fluorescence emission due to the disruption of aggregation, as higher temperatures would inhibit the formation of aggregates. However, the aggregation of naphthalimides leads to self-quenching, as previously discussed, therefore an increase in fluorescence emission was expected. Thus, this decrease in the fluorescence emission must be owed to the increase with the temperature of non-radiative processes (or the increase of

a) b)

Chapter 2. Glycosylated Naphthalimides as Enzyme-Activatable Probes

54

their rate-constant) affecting the excited state. Unfortunately, no examples supporting this hypothesis can be found in the literature for naphthalimides.

After 10 min, the fluorescence intensity remained stable and no further changes were observed. Therefore, compound 64 was pre-incubated at the desired temperature for 15 min before the addition of the enzyme, to ensure that the changes observed in the emission intensity were solely due to the enzymatic hydrolysis. Following this experiment, compound 64 was incubated at different temperatures with 1, 0.1 and 0.01 U of β- galactosidase enzyme (Figure 2.3.9b-d), respectively. Although the optimal temperature specified for this enzyme is 30 °C, in all cases the enzymatic reaction proceeded quicker when carried out at 37 °C.

Figure 2.3.9. Changes in the emission spectra of 64 (1 × 10-5 _{M, λ}

em = 440 nm) at different temperatures (a) and upon the addition of -galactosidase enzyme b) 1 U, c) 0.1 U and d) 0.01 U recorded in PBS.

Importantly, the reaction at low concentrations of enzyme (0.01 U, Figure 2.3.9d) also proceeded at relatively short times. As can be seen in Figure 2.3.9d the hydrolysis had

a)

c) d)

Chapter 2. Glycosylated Naphthalimides as Enzyme-Activatable Probes

55 almost completely finished in 1 h. These results demonstrated that this class of compounds could be successfully used for the enzymatic cleavage in short periods of time.

Unfortunately, attempts to fit this data in order to obtain the rate constant were not successful, indicating that the kinetics are more complex than a first order rate, which is common in enzymatic reactions as a mixture of substrate, substrate + Enzyme, Enzyme + product and product are present in the reaction mixture. Therefore, enzymatic reactions are usually studied using the Michaelis-Menten model.35 This model assumes that the product formation is the limiting reaction step and therefore the binding and dissociation occurred much faster. The Michaelis-Menten constant (KM) can be obtained experimentally by measuring the changes in the absorption spectrum and plotting them against the substrate concentration. Unfortunately, no changes were observed in our system and this method could not be employed.

A control experiment was carried out with compound 64 (1 × 10-5 M) and β-D-galactose

β-galactosidase (1 U) and β-glucosidase, respectively, to prove that only the right enzyme (β-galactosidase) was able to release compound 74, inducing a decrease in the fluorescence emission. As seen in Figure 2.3.10a, the treatment of 64 with 1 U of β-glucosidase did not provoke any changes in the fluorescence emission.

Figure 2.3.10. Changes in the emission intensity of a) compound 64 (1 × 10-5 _{M, λ}

em = 440 nm) after the addition of 1 U of -galactosidase and -glucosidase, respectively, in PBS at 37 °C and b) compound 74 (1 × 10-5 _{M, λ}

em = 440 nm) upon the addition of β-D-galactose (1 equivalent) and -galactosidase (1 U) recorded in PBS.

A second control experiment was carried out by combining the previously isolated compound 74 (1 × 10-5 _{M), with β-}

D-galactose (1 U) and β-galactosidase (1 U),

respectively. The results, shown in Figure 2.3.10b, proved that the combination of compound 74 with β-D-galactose or the enzyme did not lead to any change in the

Chapter 2. Glycosylated Naphthalimides as Enzyme-Activatable Probes

56

fluorescence, proving that the decreased in emission intensity previously observed for the enzymatic hydrolysis of compound 64 is due to the release of compound 74, which fluorescence emission is lower, and not to an interaction between compound 64 with the release unit β-D-galactose or the enzyme β-galactosidase.

After demonstrating the enzymatic hydrolysis, the capacity of compounds 50 and 63a to act as DNA intercalator was then investigated.