Organic & Biomolecular Chemistry
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using TLC. After 5 days, the reaction was cooled to room temp- washed with THF, EtOH and H2O, respectively. The product
erature, filtered off, and the filtrate was evaporated to dryness. was dried in a vacuum oven. FTIR (KBr, cm−1); 1641 (COONa),
The remaining solid was dissolved in 100 mL Et2O and washed 1621 (COONH), 1542, 1468, 1413 and 1363 (CvC), 1149, 1046,
with water to adjust to pH 7.0. The organic phase was dried 957 and 790 (Si–O), and 578 (Fe–O).
over MgSO4 and then filtered. The filtrate was evaporated
Sol–gel encapsulation of lipase with/without C[8]-C4-COOH
and magnetite nanoparticles or NP-C[8]-C4-COOH
1732 cm−1(CvO). H NMR (CDCl3): δ 1.05 (s, 72H, But), 1.11 The method of Reetz was modified for sol–gel encapsulation
under reduced pressure. The crude residue was recrystallized
from MeOH. Yield 78.2%, M.p. 165–166 °C. FTIR (ATR):
1
(t, 24H, J = 6.8 Hz, –CH3), 1.72–1.76 (m, 32H, –CH2–), 2.27 of the lipases.1 Typically, a mixture of lipase powder (lyophili-
(t, 16H, J = 6.8 Hz, –CH2–CO), 3.63 (brs, 16H, ArCH2–Ar), zate) such as CRL-type VII (245 mg) in phosphate buffer
3.99–4.04 (m, 32H, ArO–CH2– and O–CH2–), 6.92 (s, 16H, ArH). (1.56 mL, 50 mM) adjusted at pH 7.0 was vigorously shaken.
13C-NMR (100 MHz, CDCl3): δ (ppm) 14.21 (–CH3), 21.62 Either NP-C[8]-C4-COOH (0.2 g) or C[8]-C4-COOH (3) (0.1 g)
(–CH2), 29.66 (–CH2), 31.26 (–CH3 of But), 31.39 (Ar–CH2–Ar), and Fe3O4 (0.1 g) was added to the mixture, together with 400
31.59 (–C(CH3)3), 34.10 (–CH2), 60.15 (O–CH2–CH3), 73.40 µL of polyvinyl alcohol (4% w/v), 200 µL of NaF solution
(O–CH2), 125.90 (ArC), 132.95 (ArC), 145.83 (ArC), 150.58 (0.1 M) and 400 µL of isopropyl alcohol. After obtaining homo-
(ArC–O), 173.41 (CvO). Anal. Calcd for C144H208O24: C, 74.45; geneity, tetramethoxysilane (460 µL, 0.5 mmol) and octyltri-
H, 9.02. Found (%); C, 74.51; H, 8.98.
methoxysilane (3.2 mL, 2.5 mmol) were added and left to be
Synthesis of 5,11,17,23,29,35,41,47-octa-tert-butyl-49,50,51,- shaken for 10–15 s. Then, 15 mL of isopropyl alcohol was
52,53,54,55,56-octahydroxycarbonylbutoxy-calix[8]arene (C[8]- poured onto the dried white solid. The gel was washed with
C4-COOH) (3). A solution of NaOH (15%) was added to a water (10 mL) and isopropyl alcohol (10 mL), and then it was
mixture of C[8]-C4-COOEt (0.5 g, 0.43 mmol) in 50 mL EtOH. lyophilized to produce the encapsulated lipases.
The reaction mixture was refluxed for 17 h. Then, the volatile
Lipase activity and protein assay determination
component was evaporated, and the remaining solid was
treated with cold water (50 mL) and HCl (3 N, 100 mL). The p-NPP in an aqueous phosphate buffer (100 mmol sodium
collected product was neutralized with water, and dried in an phosphate, pH 7.0) was used to determine the hydrolytic activi-
oven. Yield 88.5%; m.p. 294–296 °C. FTIR: 1704 cm−1 (CvO, ties of the encapsulated lipases. A UV-visible spectrophoto-
acid). 1H-NMR (DMSO-d6): δ 0.94 (s, 72H, But), 1.60–1.65 meter was scanned at 410 nm in order to measure the
(m, 32H, –CH2–), 2.19 (t, 16H, J = 6.8 Hz, –CH2–CO), 3.36–3.61 concentration of the corresponding p-nitrophenol.29,30
(m, 16H, Ar–CH2–Ar), 3.93 (brs, 16H, ArO–CH2–), 6.83 (s, 16H,
Protein content was defined by the method of Bradford28
ArH). 13C-NMR (100 MHz, DMSO-d6): δ (ppm) 22.18 (–CH2), using a Bio-Rad protein dye reagent concentrate. Bovine serum
29.52 (–CH2), 29.88 (–CH3), 31.51 (Ar–CH2–Ar), 34.15 albumin was used as the standard.
(–C(CH3)3), 34.67 (–CH2), 72.82 (O–CH2), 125.61 (ArC), 133.03
pHs and temperatures on activity
(ArC), 145.28 (ArC), 153.24 (ArC–O), 175.30 (CvO). Anal. Calcd
for C128H176O24: C, 73.25; H, 8.45. Found (%); C, 73.17; Activity was determined between pH 4 and 9 in 50 mM phos-
H, 8.48.
phate buffer to see the changes in activity of free and immobi-
Synthesis of 5,11,17,23,29,35,41,47-octa-tert-butyl-49-mono- lized lipases.28–30 Moreover, the thermal inactivation of the
(benzotriazol-1)-oxycarbonylbutoxy-50,51,52,53,54,55,56-hepta- free and immobilized lipases was investigated at 30–60 °C.
hydroxycarbonylbutoxy-calix[8]arene (C[8]-C4-COOBaz) (4). To Both forms of enzymes were incubated in PBS (50 mM, pH 7.0)
a solution of C[8]-C4-COOH (0.3 g, 0.286 mmol) and 1-hydroxy- for 20 min at various temperatures and, after lowering the
benzotriazol (0.046 g, 0.34 mmol) in 25 mL THF was added temperature, the remaining activity was assayed under the
DCC (0.071 g, 0.34 mmol), and then allowed to be stirred at rt standard conditions and analyzed.
for 48 h. The reaction was monitored using TLC. The solvent
was then evaporated, and the residue was treated with EtOAc.
Thermal stability
The filtrate was evaporated to afford the final product. Yield Each encapsulated lipase (with or without additives) was
92%, 1H NMR (DMSO): δ 0.94–1.03 (m, 72H, But), 1.57–1.71 stored in the buffer (50 mM, pH 7.0) at 60 °C for 2 h, in order
(m, 34H, –CH2– and –CH2–CO), 2.16–2.19 (m, 14H, –CH2–CO), to estimate their activity as outlined above.
3.29–3.58 (m, 16H, ArCH2–Ar), 3.93 (brs, 16H, ArO–CH2–),
Enantioselective hydrolysis of racemic naproxen methyl ester
by encapsulated lipases
6.84–6.99 (m, 16H, ArH), 7.39 (t, 1H, J = 8.0 Hz, ArH), 7.52 (t,
1H, J = 7.2 Hz, ArH), 7.69 (d, 1H, J = 8.8 Hz, ArH), 7.96 (d, 1H,
J = 8.8 Hz, ArH). Anal. Calcd for C134H179N3O24: C, 72.63; H, A reaction system in an aqueous phase/organic solvent was
8.14; N, 1.90. Found (%); C, 72.69; H, 7.94; N, 1.92.
used for the hydrolysis reactions according to the literature
procedure.8,12,14 The conversion and enantioselectivity of
naproxen methyl ester by the encapsulated lipases in the pres-
Preparation of NP-C[8]-C4-COOH
A mixture of C[8]-C4-COOBaz (0.25 g) and Fe3O4-APTES (0.2 g) ence or absence of additives such as either C[8]-C4-COOH (3)
in 40 mL THF–DMF (v/v) was stirred at rt for 4 days. Then, and Fe3O4, or NP-C[8]-C4-COOH were expressed as the enantio-
NP-C[8]-C4-COOH was collected using a simple magnet, and meric ratio (E), which was calculated from the conversion (x)
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 6634–6642 | 6641
Sayin, Serkan
