Synthesis of 1,4-dihydropyrimidines with immobilized urease: effect of method immobilization on magnetic supports

2.1 Synthesis of supports

Fe₃O₄ nanoparticles (F) were synthesized by the co-precipitation method [23], using a molar ratio of Fe(II)/Fe(III)=0.5 at pH=11. F were encapsulated with SiO₂ using a ratio Fe₃O₄:SiO₂=1:1 [24]. Then, 0.5 g of Fe₃O₄ particles was dispersed in a mixture of ethanol (200 ml), deionized water (100 ml), and concentrated ammonia aqueous solution (6 ml). The mixture was sonicated for 1 h. Subsequently, 1.75 ml of tetraethylorthosilicate (TEOS, 99%, Sigma Aldrich) was added dropwise and stirred for 3 h. The Fe₃O₄–SiO₂ solid was filtered, washed with deionized water, and dried under vacuum at 60°C. The ratio between the magnetic particles and the silica was 1:1.

The functionalization with 3-aminopropyl-triethoxysilane (APTES, 99%, Sigma Aldrich) using the method described by Díez et al. [25] was used to obtain Fe₃O₄–SiO₂–NH₂ (FSN). Briefly, Fe₃O₄–SiO₂ was sonicated in ethanol solution (75%), and then APTES, 99% was added and vigorously stirred for 7 h under inert atmosphere. Finally, the solids were filtered, washed with ethanol solution (50%), and dried under vacuum at 343 K.

To obtain activated Fe₃O₄–SiO₂–NH₂ with glutaraldehyde (FSN-A), the Fe₃O₄–SiO₂–NH₂ support was suspended in 10 ml of glutaraldehyde solution (9.25%, pH 9.3) [26]. Then, the suspension was stirred at 70°C for 20 min. Afterwards, the mixture was filtered and washed with water. The nanoparticles were suspended in 25 ml of polyethylenimine (5%, Sigma Aldrich), and the suspension was incubated at 25°C for 3 h. Then, it was filtered and washed with water. The support was again suspended in 25 ml glutaraldehyde solution (1%), incubated at 25°C for 30 min, washed with water, and dried under vacuum.

2.2 Support characterization

X-ray diffraction (XRD) measurements were carried out with a RigakuMiniflex II using Cu Kα radiation (λ=1.54056 Å). X-ray photoelectron spectroscopy (XPS) analyses were performed in a Thermo Scientific Escalab 250 XI Photoelectron spectrometer with monochromatic Al K radiation (hυ=1486.6 eV). Core-level peak positions were determined after background subtraction according to Shirley using Avantage software.

2.3 Immobilization of urease

In a general procedure, magnetic supports were added to 0.01 m sodium phosphate buffer pH 5.8 with stirring at 4°C. Afterwards, the solids were dispersed with a urease solution (Jack beam 5 U/ml, Sigma Aldrich) for 24 h. The supernatant was collected by centrifugation. The magnetic particles with immobilized enzyme were washed several times with 0.01 m solution of phosphate buffer and stored in 1 ml of phosphate buffer. The amount of immobilized urease was calculated from the initial urease amount minus the amount remaining in the supernatant using the Bradford method [27].

2.4 Urease activity

To optimize the immobilization process, free and immobilized urease activities were determined spectrophotometrically at 488 nm. The enzymatic activity was expressed as micromoles of ammonium produced per minute. The relative activity is the ratio between the enzymatic activity of the immobilized enzyme (Ai) and the enzymatic activity of the free enzyme (Ao) expressed as:

The catalytic efficiency was used as an index for comparison of enzymes acting on the same substrate. It is also known as catalytic or constant yield potential [28], and it is defined as

2.5 Optimization of urease immobilization

The immobilization of urease was optimized by modifying only one variable in each experiment and keeping all others constant. The effect of immobilization time was studied in the range 0–18 h. The temperature was varied in the range 4°C–30°C, and the support to urease ratio (mg/g support) varied from 300 to 5000. A pH value near 6.0 was used because as previously reported, this pH was better for urease activity. The stability of immobilized enzyme was evaluated at 4°C for 30 days, and the reuse was studied for 5 recycles using the same concentration of urea to maintain urease activity constant.

2.6 Kinetic parameters of immobilized urease

The method of Lineweaver-Burk was used to determine the The Michaelis constant (k_m) value. The enzymatic activity was tested using 50 ul immobilized urease and modifying the substrate concentration from 0.02 to 0.1 mol/l. The reaction conditions were 10 min, pH 5.8, and 30°C.

2.7 Application of immobilized urease for the synthesis of 1,4-DHPs using the Biginelli synthesis

Free and immobilized urease was added to a mixture of 3 mol of benzaldehyde (99%, Sigma Aldrich), 1 mol of ethyl acetate (EtOAc) (99.5%, Sigma Aldrich), and 2 mol of urea (98%, Sigma Aldrich). The mixture was stirred at 70°C in water until the product precipitated. The reaction was monitored by thin layer chromatography using hexane:EtOAc (70:30). The reaction product was washed with cold water and filtered to give the pure 1,4-DHP product, and then the yield was calculated.

3.1 Characterization of magnetic supports

The crystalline structure of FSN and FSN-A particles was investigated by XRD. As shown in Figure 1, the diffraction peaks (2θ) at 18.2°, 30.3°, 35.6°, 43.3°, 53.8°, 57.3°, and 62.8° are ascribed to the (111), (220), (311), (400), (422), (511) and (440) planes of Fe₃O₄ (JCPDS no. 19-0629). The signal near 2θ=22° is assigned to the amorphous silica. This signal is broader in FSN-A.

Figure 1:

X-ray diffraction patterns for: (A) FSN and (B) FSN-A. (■) Fe₃O₄, (▼) SiO₂.

X-ray photoelectron spectra were obtained to compare the surface modification performance of FSN-A in the cross-linking process with glutaraldehyde and polyethylenimine. The XPS spectra collected in the region of N 1s are shown in Figure 2. The N 1s peak was deconvoluted into two spectral bands at 399.1 and 401.1 eV, which correspond to protonated and unprotonated species present in APTES [29]. By considering the relative proportions of these two species, it is concluded that unprotonated species increase in the cross-linking process.

Figure 2:

X-ray photoelectron spectra in the region of N 1s (A) FSN-A and (B) FSN.

Figure 3 displays the XPS in the Fe 2 p_3/2 and p_1/2 regions. The absence of signals associated with Fe 2 p_3/2 and p_1/2 suggests that the Fe₃O₄ particles were totally covered in the FSN-A solid.

Figure 3:

X-ray photoelectron spectra in the region Fe 2p. (A) FSN and (B) FSN-A.

3.2 Urease activity

The optimization of urease immobilization by physical adsorption was performed in FSN, while covalent immobilization was studied with the FSN-A support. Table 1 lists the results of adsorbed urease and enzymatic activity expressed as relative activity at different concentrations of urease. In both methods, the amount of coupled urease and relative activity decreased with an increase in urease concentration. This is due to the competition of enzyme molecules by surface of chemical groups [11] and possible blocking pores of the support by proteins neighborhood [30], [31]. Higher protein concentrations did not yield better immobilization.

Similar results by physical adsorption were reported by Pogorilyi et al. [14] using a poly (3-aminopropyl) siloxane matrix; they indicated 94% adsorbed urease and 73% residual activity. However, in the covalent adsorption, the values of relative activity are lower than other previously reported ones. Krishna et al. [2] found a relative urease activity near 90% on chitosan beads activated with 3% glutaraldehyde. In this work, the same result was obtained with 0.1 mg/ml and 1% glutaraldehyde concentration in activation time.

The coupling time for attachment of urease on the surface of solids was also optimized by varying it from 4 to 24 h (Figure 4). The immobilization time should be optimized due to conformational changes in the tertiary structure of the enzyme that occur during this process. Regarding physical adsorption, the highest percentage of immobilization and relative activity was found at 20 h (Figure 4A). Pogorilyi et al. [14], and Ayhan et al. [32] reported a higher percentage of enzyme adsorption and relative activity between 94% and 100%, with shorter immobilization time (1–4 h). This difference with our results is due to the immobilization temperature used by these authors (25°C).

Figure 4:

Effect of time immobilization of urease by physical immobilization and covalent bonding.

Relative activity (■) and adsorbed urease (□).

The percentage of immobilization in covalent adsorption is reached in 6 h, and the highest relative activity in 24 h (Figure 4B). However, the activity of the covalently bound urease was significantly lower than that of the adsorbed urease, in agreement with the results of Pogorilyi et al. [33]. Covalent grafting is a method that can change the enzymatic structure.

The effect of immobilization temperature on enzymatic activity is shown in Table 2. A temperature of 4°C for both immobilization methods was chosen to improve urease stability. Enzymatic deactivation occurs at room temperature [2], [34], [35] and increases in adsorbed urease by physical adsorption.

3.3 Stability, reuse, and kinetic parameters

The stability of immobilized urease is shown in Figure 5. The enzymatic activity was studied for 30 days. Using both methods of immobilization, the relative enzymatic activity is near 40% after 30 days, while the relative activity of free urease is 70% at the same time. This behavior is explained by the desorption or denaturation of the enzyme [34].

Figure 5:

Stability study in terms of relative activity (%) free urease (•) urease immobilized by physical adsorption (■) and by covalent bonding (□).

The enzyme reuse was studied in 10 cycles (Figure 6), obtaining a relative activity near 20% in 8 cycles. Other authors such as Ispirli Doğaç et al. [34] and Krishna et al. [2] reported the enzyme reuse for 11–14 cycles, respectively; this is because they did not wash out the excess dye.

Figure 6:

Immobilized urease recycles. Physical adsorption (■) covalent bonding (□).

The kinetic parameters of immobilized urease were compared to those of free urease. The V_max values and the k_m values are presented in Table 3. It can be observed that the maximum velocity (V_max) value decreases, but the k_m value considerably increases. The lowest k_m values obtained mean a loss of enzyme affinity by substrate when the enzyme is immobilized. This is because many active sites of the enzyme are buried or blocked inside the support surface. This phenomenon occurs preferentially in the covalent bonding method. However, both methods show similar catalytic efficiency.

3.4 Syntheses of 1,4-DHPs

Syntheses of 1,4-DHPs were carried out with benzaldehyde, ethyl acetoacetate, and urea catalyzed by immobilized and free urease. The products of the Biginelli/Hantzsch reactions were obtained by isolating them via dilution of the crude product with cold water. The results of yields are listed in Table 4. Product A was separated by distillation of ethanol, and the remaining solid B was isolated by filtering. The experimental control using only the supports without urease under similar conditions did not give the product even after 24 h, indicating that the presence of urease is necessary for urea dissociation.

As shown in Table 4, the most interesting result is obtained when the enzyme is immobilized onto the support Fe₃O₄–SiO₂–NH₂ due to the highest yield to product A. Tamaddon and Ghazi [5] reported selectivity to product A of 60% and 80% conversion, carrying out the reaction under the same conditions using free urease.

The experimental control using only the supports without urease under similar conditions did not give the product even after 24 h, indicating that the presence of urease is necessary for urea dissociation. The mechanism of Hantzch and Biginelli has been well described by Kappe [36]. The urease acts hydrolyzing the urea and improving the NH₃ disponibility. As the urea is hydrolyzed by the enzyme the Hantzch product is favored. The Biginelli product is formed by the reaction of aldehyde with urea through acyl imine intermediate. The urease favors preferentially the Hanztch product while the Biginelli product is formed without the presence of the enzyme. Figure 7 shows schematically the summary of the products obtained.

Figure 7:

Routes in the formation of Biginelli and Hanztch products.

Considering that some dihydropyrimidines are inhibitors of certain ureases [4] the products formed possibly cause the deactivation of the free enzyme, phenomena that does not occur when the enzyme is immobilized. It is noteworthy that urease immobilization by any of the immobilization methods enabled obtaining the product of the Hantzsch reaction. Although, the urease immobilized has a lower affinity of the enzyme towards urea (k_m values of immobilized urease is approximately 2.5 times higher than the free enzyme), the disponibility of NH₃ is sufficient for favoring the Hanztch product, and the confinement of enzyme in the support prevents the inhibition with products formed. However, this study will require a major reaction optimization