Transition state analogue imprinted polymers as artificial amidases for amino acid p-nitroanilides: morphological effects of polymer network on catalytic efficiency

Figures & data

Figure 1. SEM photographs of TSA imprinted in different porogens.

Table 1. Details of polymer synthesis and characterization.

Polymer catalysts	Porogen Used	Surface area (m^2/g)	Pore volume (cm^3/g)	TFGC (mmol/g)	[TSA imprints] (mmol/g)
C1	DMSO	311.55	0.898	0.542	0.184
C2	ACN	301.47	0.876	0.564	0.196
C3	CHCl3	201.35	0.354	0.557	0.179

Table 2. Details of amino acid p-nitroanilides synthesized.

S. no.	Amino acid p-nitroanilide
S1	Z-L-Phe-PNA
S2	Z-D-Phe-PNA
S3	Boc-L-Phe-PNA
S4	Nphth-L-Phe-PNA
S5	Fmoc-L-Phe-PNA
S6	Z-L-Tyr-PNA
S7	Z-L-Trp-PNA
S8	Z-L-Ala-PNA
S9	Z-L-Leu-PNA

Table 3. Kinetic parameters for the amidolysis of S1.

Amidase MIPs	kacc	kim	St (h)
C1	75.38	4.22	22
C2	61.53	3.67	24
C3	4.36	1.01	Nd

All kinetic parameters within the limit ±5%.

Nd: not determined.

Figure 2. Schematic representation of catalytic amidolysis.

Figure 3. Effect of solvent composition on catalytic amidolysis of the amidase MIPs.

Table 4. Swelling studies.

Polymer catalysts	Solvent composition (ACN–Tris HCl buffer)
	1:9	2:8	3:7	5:5	7:3	9:1
C1	4.32	4.68	5.04	5.78	6.58	6.76
C2	4.24	4.71	4.84	5.59	6.06	6.65
C3	1.56	2.05	2.87	3.87	4.23	4.56

All kinetic parameters within the limit ±5%.

Figure 4. Percent amidolysis versus time plot for the amidases in (1:9) and (9:1) ACN–Tris HCl medium.

Figure 5. Michaelis–Menten plots.

Figure 6. Lineweaver – Burk plots.

Table 5. Kinetic parameters for the amidolysis S1 using the amidase MIPs.

Polymer catalyst	Solvent composition	Km (mmol)	$1/K_m$ (mmol^−1)	10^3 $\frac{V_{max}}{K_m}$ (min^−1)	$k_{cat}/K_m$ (mmol/min)
C1	1:9	0.273	3.663	26.608	4.908
C2		0.353	2.833	20.583	3.798
C3		8.948	0.112	0.893	0.165
C1	9:1	8.627	0.116	0.474	0.087
C2		11.077	0.090	0.411	0.076
C3		0.458	2.183	18.683	3.447

All kinetic parameters within the limit ±5%.

Figure 7. Comparison of the active sites from the natural chymotrypsin and the mimic.

Table 6. Kinetic parameters for substrate stereospecificity of the artificial amidases.

	1:9 ACN–Tris HCl buffer			9:1 ACN–Tris HCl buffer
Enantio–selectivity ratio	C1	C2	C3	C1	C2	C3
$k_{acc}^L/k_{acc}^D$	7.652	6.931	1.021	1.032	1.031	7.033
$K_m^D/K_m^L$	10.592	10.014	1.182	1.423	1.422	9.872
$\Delta (\Delta G_{binding}^{\#})$	−1.489	−1.454	−0.105	−0.223	−0.222	−1.445
$\Delta (\Delta G_{[C.S]}^{\#})$	−1.284	−1.221	−0.013	−0.020	−0.019	−1.231

All kinetic parameters within the limit ±5%.

Table 7. Porogen effect on substrate shape-selectivity of the polymer catalysts.

	1:9 ACN–Tris HCl buffer			9:1 ACN–Tris HCl buffer
Substrate–selectivity ratio	C1	C2	C3	C1	C2	C3
$K_m^{S1}/K_m^{S3}$	4.56	3.56	1.03	1.04	1.02	3.47
$K_m^{S1}/K_m^{S4}$	8.21	7.09	1.05	1.06	1.03	7.85
$K_m^{S1}/K_m^{S5}$	14.24	13.11	1.06	1.07	1.05	12.74

All kinetic parameters within the limit ±5%.

Table 8. Porogen effect on the substrate specificity of the polymer catalysts.

	1:9 ACN–Tris HCl buffer			9:1 ACN–Tris HCl buffer
Specificity ratio	C1	C2	C3	C1	C2	C3
$K_m^{S1}/K_m^{S6}$	1.12	1.01	1.01	1.01	1.03	1.09
$K_m^{S1}/K_m^{S8}$	4.53	1.02	1.02	1.01	1.02	4.26
$K_m^{S1}/K_m^{S9}$	5.31	1.04	1.04	1.03	1.02	5.14

All kinetic parameters within the limit ±5%.

Figure 8. TSA inhibition study.