3.3 Photophysical Evaluation
4.3.2 Changes in the Photophysical Properties of 119 and 120 Upon Enzymatic
The enzymatic activity of both probes developed above was evaluated. First, Amonafide (1 × 10-5 M) was analysed spectroscopically, by means of absorption, excitation and fluorescence emission in PBS at 25 °C (Figure A.4.2). Next, compound analysis of 119 by MS (Figure A.4.3) and UV-vis spectroscopy (Figure 4.3.3) proved that Amonafide had
Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide
112
been released, as illustrated in Scheme 4.1.4. Changes in the absorption and fluorescence spectra following enzymatic cleavage allowed the reaction to be monitored spectroscopically. As can be seen in Figure 4.3.3a, a broader absorption band (410 nm) is observed after treatment with the enzyme, which corresponds to the Amonafide absorption band (410 nm) as can be seen in Figure 4.3.3c in the normalised absorption spectra, giving good evidence of its enzymatic release. However, the absorption band found at 410 nm is broader than the one found in Amonafide, as it is a superposition of Amonafide and the released linker (compound 116).
Figure 4.3.3 Changes in the photophysical properties (a) absorption and b) emission) of 119 (1 × 10-5 M, λ exc = 347 nm) before and after the addition of the enzyme (β-galactosidase, 1 U) recorded in PBS at 30 °C and c) Normalised absorption spectra and d) normalised emission fluorescence spectra (λexc = 347 nm) of Amonafide (1 × 10-5 M) and 119 (1 × 10-5 M) after treatement with 1 U β-galactosidase, recorded in PBS at rt. Figure representative of three independent experiments.
The fluorescence emission intensity (exc = 347 nm) dropped dramatically after treatment with the 1 U of enzyme for 30 min. This decrease in the fluorescent intensity could be owed to the lower quantum yield of Amonafide. According to Qian et al.,207
a) b)
Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide
113 Amonafide’s ΦF in H2O is 7.5%, in contrast with compound 119 which is almost double at 14%. Furthermore, a new band appeared at longer wavelength (590 nm), which corresponds to the characteristic band of Amonafide. This is evidenced in the normalised fluorescence emission spectra in Figure 4.3.3d, proving that treatment of compound 119 with 1 U of enzyme releases Amonafide and this release can be monitored spectroscopically.
In order to investigate the kinetics of the enzymatic process, a time-dependant experiment was carried out with 119 (1 × 10-5 M) using 1 U of β-galactosidase from Aspergillus Oryzae in PBS (Figure 4.3.4). The experiment was also conducted at three different temperatures (25, 30 and 37 °C), in order to understand the effect of the temperature on the enzyme and the resulting kinetics.
Compound 119 was incubated in PBS at 25, 30 and 37 °C, respectively, prior to the experiment in a thermostat fitted spectrometer, to identify if the temperature has any effect in the emission that could interfere with the emission changes observed in the presence of the enzyme.
Figure 4.3.4a shows how the fluorescence emission response of compound 119 (1 × 10-5 M) increases with temperature and time, with the highest overall intensity observed at 37 °C. A plausible explanation for this could be that higher temperature disrupts the aggregation of the compounds. Naphthalimides are well-known to form aggregates in solution that self-quench the fluorescence emission. However, as shown in Figure 4.3.2, concentrations studies previously carried out indicated that at the concentration used (1 × 10-5 M) no aggregation should be occurring. As previously explained, the photophysical properties of naphthalimides are highly dependant on the environment and can be affected by pH, polarity or temperature. However, not many examples can be found in the literature where the photophysical properties of naphthalimides can be modified by the temperature.
As can be seen in Figure 4.3.4, after 5 min incubation the emission stabilises and did not increase further, reaching a plateau. Therefore, before enzyme treatment compound 119 (1 × 10-5 M) was pre-incubated at the chosen temperature for 15 min. Even though the optimal temperature of β-galactosidase from Aspergillus oryzae was 30 °C, the reaction worked successfully at the three temperatures, with 37 °C found to be the fastest time point. As seen in Figure 4.3.4b, when using 1 U of the enzyme the hydrolysis had finished after 15 min when it was carried out at 37 °C. By contrast, it took 30 min to finish when carried out at 25 °C. However, it is important to note that even at low concentration of the
Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide
114
enzyme (0.1 U), the reaction still proceeded in a relatively short period of time (two hours).
Enzymes are measured by units (U), which corresponds to the amount of enzyme that is able to catalyse the conversion of 1 μmole of substrate per minute.155 However, this activity is quantified by using a particular substrate at certain temperature and pH. Therefore, the rate of an enzymatic reaction can change depending on the substrate, pH and temperature used, even when U is kept constant.
Figure 4.3.4. Emission intensity versus time of 119 (1 × 10-5 M) at different temperatures in PBS solution when incubated with a) no enzyme, b) 1 U of β-galactosidase, c) 0.5 U of β-galactosidase and d) 0.1 U of β- galactosidase. Figure representative of two independent experiments.
Enzyme kinetics are complex as there are several species competing in the reaction. As represented in scheme 4.3.1, in an enzymatic reaction the rate constant is a combination of the rate constants of formation of enzyme-substrate complex (ES) and the formation of product (P) from this complex.
Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide
115 Scheme 4.3.1. Different species present in an enzymatic reaction.
The most common model used to study enzymatic reactions is the one developed by Michaelis and Menten. In this model, the rate constant (KM) represents the concentration of substrate at which the rate of the reaction is half of the maximum reached by the system (when saturation of the enzyme occurs).208
To spectroscopically determine the rate constant using this model, the rate of the reactions had to be determined using the absorbance, as a means of establishing the concentration of product. However, in our system both the substrate and product present similar absorption spectra, and therefore the concentration of the product formed alone could not be calculated. The values found in the literature for this enzyme shown KM values of 7.2 × 10-4 M and 1.8 × 10-2 M when ortho-nitrophenyl-β-galactoside and lactose are used as substrates, respectively, and when the enzymatic reaction took place at 30 °C and pH 4.5 and pH 4.8, respectively, as these are the specified optimal conditions for this enzyme and these substrates.209 Therefore, the rate constant depends strongly in the environment (pH and T) but also in the substrate used, therefore limiting the possibility of comparing the values obtained with these compounds and the literature.
However, it was found that all the curves in these experiments could be fitted to a mono-exponential function, which corresponds to a first-order reaction. In a first-order reaction the rate constant only depends on the concentration of one species. Therefore the experimental rate constants could be calculated by fitting the curves to a monoexponetial function. This implies that the limiting step of the reaction is the formation of product from ES (in bold in Scheme 4.3.2), omitting the reversible formation of the complex ES.
Scheme 4.3.2. Enzymatic reaction simplified to a first-order reaction and the equation to calculate the rate of a first-order reaction.
With this data, the half-life time of a first-order reaction was also calculated according to equation 4.1.
E + S K1 ES E + P
K-1
K2
Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide
116
Equ. 4.1 t½ = ln2/ k
The experimental rate constant and their corresponding half-live time are summarised in Table 4.4.
Table 4.4. Rate constants (K) and t1/2 of the enzymatic hydrolysis of compound 119 with different U of enzyme.
Units Enzyme T (°C) K (min-1)
(min -1) t1/2 (min) 1 25 0.0927 7.47 30 0.1374 5.04 37 0.1528 4.53 0.5 25 0.0424 16.34 30 0.0632 10.96 37 0.1039 6.67 0.1 25 0.0097 71.45 30 0.0130 53.31 37 0.0150 46.20
The same experiments were conducted with compound 120 (1 × 10-5 M) using β- glucuronidase from Escherichia Coli. As seen in Figure 4.3.5, both the absorption and emission spectra after 1 h treatment with 1 U of the enzyme indicated the release of Amonafide, which was also confirmed by MS.
Figure 4.3.5. a) Absorption and b) fluorescence emission spectra of 120 (1 × 10-5 M) before (black lines) and after (red line) treatment with β-glucuronidase (1 U) for 1 h at 37 °C. Figure representative of three independent experiments.
Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide
117 In contrast with compound 119, incubation of compound 120 at different temperature did not seem to have any effect on its emission intensity (Figure 4.3.5a). Therefore, only 5 min of pre-incubation were needed before adding the enzyme.
Time-dependant studies of 120 upon treatment of the enzyme indicated a much faster reaction than the one for compound 119. The enzymatic reaction appeared to conclude after only 5 min when 1 or 0.5 U of enzyme were used (Figure 4.3.6b and c). However, the reaction slowed down significantly when only 0.1 U of the enzyme were present, taking up to 40 min to conclude at 25 °C (Figure 4.3.6d, black line).
Figure 4.3.6. Emission intensity of compound 120 (1 × 10-5 M, λ
exc = 350 nm and λem = 460 nm) over time at different temperatures after a) no enzyme treatment, b) 1 U β-glucuronidase, c) 0.5 U β-glucuronidase and d) 0.1 U β-glucuronidase. Figure representative of two independent experiments.
A summary with the experimental rate constants and their half-life time are sumarised in Table 4.5.
Chapter 4. Glycosylated Naphthalimides as Prodrugs of Amonafide
118
The ICT absorption bands of compounds 136 and 141 also exhibit a shoulder at ca. 383 nm. The excitation spectra (red line) match the absorption spectra, indicating the purity of the sample. Excitation at 347 nm gives broad emission bands at 460 nm in both cases (Figure 4.3.7, black line).
Table 4.5. Rate constants (K) and t1/2 of the enzymatic hydrolysis of compound 120 when using 0.1 U of enzyme.
Units Enzyme T (ºC) K (min-1) t
1/2 (min) 0.1
25 0.0704 9.84
30 0.0727 9.53
37 0.1445 4.79
Having already studied the effect of enzymatic hydrolysis in the photophysical properties of compounds 119 and 120, and demonstrated that the enzymatic reaction takes place within minutes and that can be monitored spectroscopically, the photophysical properties of the O-acetylated derivatives 136 and 141 were investigated, as they could also present interesting in vitro properties, which will be further discussed in sections 4.4.5 – 4.4.8.