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D. Mathew et al. / Journal of Molecular Catalysis A: Chemical 415 (2016) 65–73
2. Experimental
lized from hot ethanol containing 1% acetic acid. The substrates,
t-Boc/Nphth/Fmoc-l-phenylalanyl-p-nitroanilide and Z-l-alanyl-
p-nitroanilide were also prepared following the same procedure.
2.1. Materials and methods
Dicyclohexylcarbodiimide (DCC), ethylene glycol dimethacry-
late (EGDMA) and phenethyl boronic acid were purchased
from Sigma–Aldrich, USA. ␣-Chymotrypsin, Z/Boc/Nphth/Fmoc-l-
phenylalanine, Z-l-alanine, l-histidine, l-serine, l-aspartic acid,
benzyl carbamate, triphenyl phosphite and phenylacetaldehyde
were purchased from SRL, Mumbai. Other chemicals available from
local suppliers were purified prior to use by following the standard
procedures.
IR spectra were recorded on a Shimadzu FT-IR-8400S spec-
trophotometer. Kinetic studies were performed using Shimadzu UV
2450 spectrophotometer. JEOL JSM6390 SEM analyzer was used for
SEM analysis. 1H NMR spectra were taken using Bruker Advance
DPX-300 MHz FT-NMR spectrometer in CDCl3.
2.5. Amidolysis of Z-l-phenylalanine-p-nitroanilide
(Z-l-Phe-PNA) using TSA imprinted and non-imprinted enzyme
mimics: general procedure
A suspension of 10 mg chymotrypsin mimic C1 (0.00647 mmol)
was suspended in 5 mL acetonitrile–Tris HCl buffer (1:9 by volume,
pH 7.75) in a reagent bottle and 271 mg, 0.647 mmol of the sub-
strate Z-Phe-PNA in 50 mL acetonitrile was added. The reaction
mixture was placed in a water bath shaker at 45 ◦C and shaken
gently. Amidolysis of Z-Phe-PNA was followed by monitoring the
absorbance of released p-nitroaniline spectrophotometrically at
374 nm in the framework of Michaelis–Menten kinetics and the
reaction was monitored for two days. A blank reaction was also car-
ried out in the absence of the enzyme mimic. From the absorbance
data, the rate constants and percentage amidolysis were evaluated.
Amidase activity of the mimics C2–C7 was evaluated in a similar
manner.
2.2. Synthesis of TSA-A
(phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl
phosphonate)
2.6. Regeneration and reusability of the spent polymer
The transition state analog was synthesized by refluxing
4.10 mL, 13.20 mmol triphenyl phosphite, 2.0 g, 13.20 mmol benzyl
carbamate, 2.38 mL, 19.80 mmol phenylacetaldehyde and 2.0 mL
glacial acetic acid for 4 h at 100 ◦C in an oil bath. The diphenyl phos-
phonate formed was hydrolyzed with 0.40 N NaOH, acidified with
conc. HCl, filtered, and purified by column chromatography using
9:1 chloroform–methanol mixture. FTIR:- 1301 cm−1 (P O stretch-
The spent enzyme mimics were collected by filtration, washed
simultaneously with distilled water and Tris–HCl buffer (pH 7.75)
and dried under vacuum. In the second cycle the amidolytic reac-
tion was carried out in fresh Tris–HCl buffer solution. Catalytic
amidolysis was repeated for 6 cycles.
stretching).
O benzyl
3. Results and discussion
The transition state analog phenyl-1-benzyloxycarbonylamino-
4-methoxybenzyl phospohonate was also prepared by the same
method using 4-methoxybenzaldehyde instead of phenylacetalde-
hyde for a comparison [15].
3.1. Synthesis of TSA- A:
phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl
phosphonate
2.3. Synthesis of TSA imprinted enzyme mimics and
non-imprinted control polymers
The TSA, which has more structural resemblance with the
substrate, was synthesized using triphenyl phosphite, benzyl car-
bamate and phenylacetaldehyde. The TSA synthesized possesses Z
group of the substrate and the “specificity determinant” – C6H5CH2
group – of chymotrypsin.
The enzyme mimic polymer C1 was prepared by radical ini-
tiated bulk polymerization of 223 mg, 10 mmol, of monomer
N-methacryloyl-l-histidine (MALH) and 3.4 mL, 90 mmol, of the
crosslinking agent EGDMA in presence of 205.5 mg, 0.50 mmol TSA-
A in 40 mL DMSO for 6 h at 80 ◦C. The template was completely
leached out by washing with methanol and then subjected to Soxh-
let extraction with chloroform. The polymer obtained was collected
and dried over vacuum.
Enzyme mimics C2–C7 were also synthesized as per the same
procedure. The corresponding non-imprinted control polymers CPs
were also prepared by the same procedure in the absence of TSA.
The total amino content in the polymer mimics were estimated by
ninhydrin reagent. The morphology of the polymers was charac-
terized SEM analysis.
The TSA synthesized was characterized by FTIR and NMR spec-
troscopic techniques. FTIR of the TSA exhibited bands at 1301, 946
and 1252 cm−1 corresponding to P O stretching, P OH stretch-
ing and P
O
benzyl stretching respectively. The 1H NMR spectra
showed singlets at ␦ 1.73 and 5.21 corresponding to OH group
and CH2 of Z group respectively. The methylene protons (CH CH2)
appeared at ␦ 2.81 as doublet and the alkyl CH resonated at ␦ 4.09
as multiplet. The NH proton appeared as doublet at 6.01 ppm. The
15 aromatic protons showed a multiplet at 6.5–7.5 ppm.
3.2. Synthesis of TSA imprinted and non-imprinted polymers
The molecularly imprinted polymers were prepared by
radical initiated bulk polymerization method using the func-
tional monomers, template TSA and the cross linker EGDMA.
The monofunctional mimics (C1–C3) were synthesized using
2.4. Synthesis of the substrate Z-l-phenylalanine-p-nitroanilide
(Z-l-Phe-PNA)
The substrate Z-l-Phe-PNA was synthesized by dissolving
2.99 mg (0.01 mol) Z-l-phenylalanine and 1.38 mg (0.01 mol) p-
nitroaniline in 30 mL ethyl acetate and the solution was stirred on
a magnetic stirrer in an ice-water bath for half an hour. A solu-
tion of 2.06 mg (0.01 mol) DCC in 30 mL ethyl acetate was added
drop wise and the reaction mixture was stirred for 30 min in the
ice water bath and the stirring was continued for further 3 h at
room temperature. The DCU formed was filtered off and the filtrate
was evaporated in vacuum. The residue obtained was recrystal-
N-methacryloyl-l-histidine
acid (MALA)/N-methacryloyl-l-serine (MALS) respectively with
EGDMA and TSA-A in the molar ratio 1:9:0.5.
pared using functional monomers N-methacryloyl-l-histidine,
N-methacryloyl-l-aspartic acid and N-methacryloyl-l-serine—C4
with MALH and MALA, C5 with MALH and MALS and C6 with MALA
and MALS. The monomers, EGDMA and TSA-A were in the molar
ratio 0.5:0.5:90:0.5 (Table 1). Trifunctional mimic C7 was obtained
(MALH)/N-methacryloyl-l-aspartic