Molecularly imprinting of polymeric nucleophilic catalysts containing

4-alkylaminopyridine functions

Ji-Tao Huang*, Si-Hua Zheng, Jia-Qi Zhang

Department of Biochemical Engineering, Tianjin Institute of Technology, Tianjin 300191, China

Received 25 January 2004; received in revised form 17 March 2004; accepted 17 March 2004

Abstract

Imidazole group of histidine residue is the essential catalytic group in the active site of a hydrolase protein. However, the imidazole is

usually replaced with more nucleophilic 4-(N,N-dimethylamino)pyridines in organic syntheses. In order to introduce the supernucleophilic

4-dialkylaminopyridines into a catalytic site, a polymeric catalyst containing pyrrolidinopyridine moiety, is synthesized by imprinting of the

bulk polymer with the transition state analogue of a substrate, accelerates the substrate-specific hydrolysis of p-nitrophenyl acetate. The

results show that TSA-imprinted polymer containing 4-alkylaminopyridine groups is better than imidazole-appended polymer in artificial

enzyme activity.

q 2004 Elsevier Ltd. All rights reserved.

Keywords: Molecularly imprinted polymer; Supernucleophilic catalyst; 4-Dialkylaminopyridines

1. Introduction

It is well-known that nucleophilic reactions catalyzed by

pyridine or imidazole are even more effectively accelerated

by 4-(N,N-dialkylamino)pyridine derivatives and their

polymers. Among these, 4-(N,N-dimethylamino)pyridine

(DMAP) [1] and the polystyrene-supported alkylaminopyr-

idine reagents [2–8] are acylation catalysts that have

found numerous important applications since they became

commercially available nearly two decades ago. It is an

excellent catalyst for a variety of nucleophilic reactions,

being most notably useful in difficult acylation, hydrolysis

and silyation of highly hindered substrates [9–11]. 4-

Pyrrolidinopyridine (PPY) [12] and 4-(N,N-diallylamino)-

pyridine polymers (DAAP) [13–15] are superior to DMAP

as a catalyst [9], although this advantage is counterbalanced

by their higher cost and lack of availability. These

supernucleophilic catalysts are used to accelerate a wide

variety of acylations and are not specific for the substrate.

Molecular recognition plays a decisive role in enzyme

protein mechanism. Specific recognition is attributed to

binding cavities that complement molecules in size, shape

hydrogen bonding, and hydrophobic and electrostatic

profiles. A commonly employed approach to catalysis by

polymers imprinted molecularly is to use transition state

analogues (TSAs) as small bait molecules. The binding

cavity of the macromolecular matrix is designed to stabilize

the reaction transition state, thereby lowering the activation

energy requirement and resulting in reaction rate accelera-

tion [16 – 24]. The instantaneous transition state of

carboxylic ester can be mimicked by stable phosphonate

derivatives, a strategy that has been available in the

synthesis of many TSA-imprinted polymer catalysts

[25–29]. These catalysts often contain imidazole groups,

just as hydrolase proteins contain histidine residues.

We herein replaced imidazole groups with the more

efficient 4-(N,N-dialkylamino)pyridine groups. A molecule-

imprinted supernucleophilic catalyst was designed and

evaluated by the hydrolysis of p-nitrophenyl acetate.

Although 4-alkylaminopyridine supernucleophilic groups

do not exist in any proteins, the catalyst containing both

supernucleophilic moieties and imprinted cavities, from the

point of view of their biochemical nature, is enzyme mimic.

If the effect follows saturation kinetics similar to enzymes

and enabled the data to be treated by a variant of Michaelis–

Menten kinetics, the accelerations in the present of such

polymeric catalyst is due to the formation of artificial

enzyme-substrate complex.

0032-3861/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.polymer.2004.03.050

Polymer 45 (2004) 4349–4354

www.elsevier.com/locate/polymer

* Corresponding author. Tel.: þ86-2227402987; fax: þ86-2227402697.

E-mail address: jthuang@mail.tjut.edu.cn (J.T. Huang).

2. Experimental

2.1. General

4-(5)-Vinylimidazole was prepared as described by

Overberger et al. [30]. Other reagents were purchased

from Tianjin Chemical Reagent Co., Inc. (Tianjin, China).

Infrared spectra were measured on a Nicolet 205 FT-IR. 1H

NMR spectra were recorded at 200 MHz on a Bruker AP-

P200 spectrometer. Elemental analyses were run by a Carlo

Erba 1106 and HERAEUS. CHN-O elemental analysers.

The UV–visible spectra were recorded on a Shimadzu UV-

360 spectrophotometer.

2.2. Preparation of 1,4-dibromo-2,3-bis(bromomethyl)-2-

butadiene (1) [31]

To solution of 21.13 g (257 mmol) of freshly distilled

2,3-dimethyl-1,3-butadiene in 200 ml of CCl4 was added a

solution of 41.07 g (257 mmol) of bromine in 100 ml of

CCl4 over 4 h. To this solution 91.49 g (514 mmol) of N-

bromosuccinimide, 2.00 g of benzoyl peroxide and 10 ml of

CCl4 were added. The mixture was refluxed for 2 h. The hot

mixture was filtered. The filtrate was precipitated at 0 8C.

The solid was recrystallized from ethyl acetate to give

59.6 g (58%) of 1. Mp 157–159 8C; IR (KBr) 3100, 1630,

885 cm21. Anal. calcd for C6H8Br4: C, 18.02; H, 2.00; Br,

79.98; found: C, 18.17; H, 2.05; Br, 80.10.

2.3. Preparation of 2,3-bis(iodomethyl)-1,3-butadiene (2)

[32]

To solution of 54.35 g (136 mmol) of 1 in 200 ml of

acetone was added 64.50 g (408 mmol) of sodium thio-

sulfate and 67.73 g (408 mmol) of potassium iodide. The

mixture was vigorously stirred for 1 h at 45 8C. The

suspension was poured onto ice and extracted with ether.

The extracts were washed three times with 150 ml of

saturated NaCl solution, dried with MgSO4, and evaporated

to give 40.4 g (89%) of 2 as a pale yellow solid. Mp 95–

96 8C; IR (KBr) 3100, 1590, 900 cm21. Anal. calcd for

C6H8I2: C, 21.57; H, 2.40; I, 76.03; found: C, 21.54; H, 2.45;

I, 75.98.

2.4. Preparation of 4-(3,4-dimethylenepyrrolidyl)pyridine

(3) [33]

To solution of 3.86 g (41 mmol) of 4-aminopyridine in

15 ml of methanol at room temperature was added 20.03 g

(60 mmol) of newly produced 2. After the mixture was

warmed to 60 8C and stirred for 1 h under nitrogen,

potassium hydroxide (8 g) was added and the suspension

was stirred for 10 h at 60 8C under nitrogen, filtered, and

dried under vacuum. The material was recrystallized from

1:3 methanol–chloroform (v/v) to give 3.2 g (45%) of 3 as a

brown solid; IR (KBr) 2923, 1620, 1550–1500, 831 cm21;

1H NMR (DMSO-d6) 2.50 (s, 4H); 5.06–5.55 (m, 4H); 7.84

(m, 2H); 8.15 (m, 2H). Anal. calcd for C11H12N2: C, 76.74;

H, 6.98; N, 16.28; found: C, 76.79; H, 7.03; N, 16.21.

2.5. Preparation of 4,4 0-bis(3,4-

dimethylenepyrrolidyl)phenyl sulfone (4)

To solution of 4.72 g (19 mmol) of 4,40-diaminodiphenyl

sulfone in 20 ml of tetrahydrofuran (THF) at room

temperature was added 19.03 g (57 mmol) of newly

produced 2. After the mixture was warmed to 60 8C and

stirred for 1 h under nitrogen, potassium hydroxide (15 g)

was added and the suspension was stirred for 10 h at 60 8C

under nitrogen, filtered, and dried under vacuum. The

material was recrystallized from 1:4 acetone–water (v/v) to

give 2.7 g (35%) of 4 as a pale yellow solid; IR (KBr) 2990,

1640–1120, 830 cm21; 1H NMR (DMSO-d6) 2.56 (s, 4H);

5.12–5.64 (m, 4H); 7.75 (m, 2H); 8.02 (m, 2H). Anal. calcd

for C24H24N2O2S: C, 71.29; H, 5.94; N, 6.93; found: C,

71.32; H, 6.01; N, 6.95.

2.6. Synthesis of non-imprinted poly[4-(3,4-

dimethylenepyrrolidine)pyridine] (5)

To solution of 2.82 g (30 mmol) of 3 and 2.48 g

(10 mmol) of 4 in 10 ml of 15% diluted hydrochloric acid

was added 0.27 (1 mmol) g of 2,20-azo bis(2-methylpropio-

namidine) dihydrochloride (V-50) at 0 8C under nitrogen.

After nitrogen gas was bubbled into the solution for 10 min,

the mixture was stirred for 10 h at 60 8C under nitrogen. The

solid was filtrated and crushed. The powder was screened

through a sieve, 10 and 60 mm mesh size. The powder was

washed with a 5% aqueous NaOH for three times and then

with water for five times. The resulting polymer was dried

under vacuum to give 5 as puce powder; IR (KBr) 3020,

1610, 1560–1500, 850 cm21.

2.7. Substrate imprinting of poly[4-(3,4-

dimethylenepyrrolidine)pyridine] (6)

This polymer was prepared according to the procedure

described in the synthesis of 5. To reaction system was

added 0.72 g (4 mmol) of p-nitrophenyl acetate (substrate

imprint) to give 6 as brown powder; IR (KBr) 3020, 1615,

1560–1500, 856 cm21.

2.8. Preparation of p-nitrophenol methylphosphonate (TSA)

[34]

To solution of 20 g (144 mmol) of p-nitrophenol was

added 10 g (75 mmol) of methylphosphonic dichloride. The

mixture was heated slowly over 3 h to a temperature of

160 8C. Hydrogen chloride evolution began at 70 8C. The

temperature was held at 160 8C for an additional hour. The

mixture was dissolved in toluene, treated with decolorizing

charcoal, and then recrystallized twice from 1:1 toluene–ether

J.-T. Huang et al. / Polymer 45 (2004) 4349–43544350

(v/v) to give 11.3 g (49%) of bis( p-nitrophenol)

methylphosphonate.

5 g (14.7 mmol) of bis( p-nitrophenol) methylphosphonate

was placed together with 32.6 mmol of 10% NaOH in 100 ml

of water and refluxed 20 min. The solution was cooled,

acidified to pH 3.5, and extracted with three 50 ml portions of

ether. The ether solution was dried with anhydrous MgSO4 and

the crude product was allowed to crystallize in the refrigerator.

Recrystallization from refluxing ether gave 1.6 g (24%) of

TSA as white needles. Mp 113–114 8C. Anal. calcd for

C7H8NO5P: C, 38.72; H, 3.71; N, 6.45; P, 14.27; found: C,

38.81; H, 3.82; N, 6.40; P, 14.45.

2.9. TSA imprinting of poly[4-(3,4-

dimethylenepyrrolidine)pyridine] (7)

This polymer was prepared according to the procedure

described in the synthesis of 5. p-Nitrophenyl acetate was

replaced by 0.86 g (4 mmol) of p-nitrophenolmethyl-

phosphonate (TSA imprint) to give 7 as reddish brown

powder; IR (KBr) 3020, 1620, 1560–1500, 1030, 860 cm21.

2.10. TSA imprinting of poly(4-(5)-vinylimidazole) (8) [26]

To solution of 0.38 g (1.6 mmol) of cobalt chloride

hexahydrate in 5 ml of methanol–water (1:9) solution was

added 1.51 g (16 mmol) of 4-(5)-vinylimidazole (monomer).

After the solution was stirred for 1 h at room temperature,

0.35 g (1.6 mmol) of p-nitrophenol methylphodphonate (TSA

imprint), 1.35 g (8 mmol) of N,N0-ethylene bis acrylamide

(cross-linker) and 0.053 g (0.32 mmol) of azo bis(isobutyr-

onitrile) (initiator) were added. After nitrogen gas was bubbled

into the solution for 10 min, the mixture was stirred for 3 h at

60 8C under nitrogen atmosphere. The solid was filtrated and

crushed. The powder was screened through a sieve, 10 and

60 mm mesh size. The powder was washed with a 5% aqueous

NaOH for three times and then with water for five times. The

resulting polymer was dried under vacuum to give 8 as blue

powder; IR (KBr) 2920, 2840, 1470, 1460–1070, 720 cm21.

2.11. Measurements

p-Nitrophenyl acetate was added to the solution of 0.4%

(by volume) acetonitrile in 0.05 M of Tris–HCl buffer (pH

8.2) including 0.05 M of KCl and formed a

2.5 £ 1025 mol l21 solution. 0.23 g l21 of the catalyst (5,

6, 7 or 8) was added at 37 8C. The hydrolysis of p-

nitrophenyl acetate was carried out in Tris–HCl buffer at

37 ^ 0.1 8C. The catalytic activity was determined with the

spectrophotometer. Reaction temperature was kept at

37 ^ 0.1 8C using water circulation instrument. p-Nitro-

phenyl anion formation was monitored spectrophotometri-

cally by measuring the change in absorption at 400 nm as a

function of time.

3. Results and discussion

As shown in Fig. 1, as reported in earlier papers [33], we

prepared the 1,4-dibromo-2,3-bis(bromomethyl)-2-buta-

diene (1) by bromization of the 2,3-dimethyl-1,3-butadiene

according to Cope’s method [31]. Iodination of 1 with

sodium thiosulfate and potassium iodide gave the 2,3-

bis(iodomethyl)-1,3-butadiene (2) [32], and a subsequent

4-aminopyridine generated the 4-(3,4-dimethylenepyrroli-

dyl)pyridine (3). Monomer 3 and its linear homopolymer

are very similar to the DAAP homopolymer in molecular

structure (see Figs. 1 and 5). In the same way, the 4,40-

bis(3,4-dimethylenepyrrolidyl)phenyl sulfone (4) with two

conjugated-double-bonds, as a cross-linker, was prepared

by treatment of 4,40-diaminodiphenyl sulfone with com-

pound 2 according to the same approach. Non-imprinted

poly[4-(3,4-dimethylenepyrrolidine)pyridine] (5), a cross-

linked polymer with alkylaminopyridine reagent, was

synthesized as shown in Fig. 2 by co-polymerization

between monomer 3 and cross-linker 4.

As in Fig. 3, p-nitrophenol methylphosphonate, as a

template molecule, was used to orient monomer 3 prior to

polymerization [26]. The free radical polymerization was

initiated by a water–soluble initiator V-50. The formed

polymer wrapped a large number of the template molecules.

These templates were removed from the polymer matrix by

washing the crushed solid with dilute aqueous NaOH.

Examination of the filtrate showed 82% recovery of the

template molecules. Thus cavities complementary to the

templates were generated within the polymer (7). Similarly,

co-polymerization of 3 in the presence of p-nitrophenyl

acetate (substrate) followed by removal of the substrate

molecules generated substrate-imprinted polymer (6). The

Fig. 1. Preparation of monomer and cross-linker.

J.-T. Huang et al. / Polymer 45 (2004) 4349–4354 4351

high-crosslinking degrees of the polymer matrixes were

designed to stabilize the size and shape of the imprinted

cavities, thereby lowering the binding site flexibility and

leading to an enhanced recognition power.

p-Nitrophenol methylphosphonate, as a TSA, is very

similar to the transition state of p-nitrophenyl acetate

(substrate) in size and shape. Substrate transition state is

preferentially bound to the polymer 7 made in the presence of

TSA, and substrate ground state is preferentially bound to the

polymer 6 made in the presence of substrate. On the other

hand, TSA-imprinted poly(4-(5)-vinylimidazole) (8) com-

prises imidazole catalytic group and imprinted cavity. The

objectives of our work were to establish the alkylaminopyr-

idine-appended artificial enzyme model, and to compare this

model with imidazole-appended enzyme mimics.

The high-crosslinking polymers hardly swell in aqueous

reaction medium. In order to increase their surface area,

these polymers have to be pulverized to powders, 5–60 mm

in diameter. Hydrolysis of p-nitrophenyl acetate was used to

evaluate the catalytic active of these polymers. At low

concentration of substrate the reaction obeyed pseudo-first-

order rate law. The reaction rate was monitored by the

spectrophotometric determination of p-nitrophenyl anion

formation at the absorbance at 400 nm, and the rate

constants of kobs and kcontrol; obtained with and without the

catalyst, respectively, were listed in Table 1.

Table 1 shows that, even in the absence of catalyst, the

ester hydrolyzed spontaneously. TSA-imprinted alkylami-

nopyridine polymer 7 revealed 12.5-fold increase in the rate

constant, while the rate constant increases of the TSA-

imprinted imidazole polymer (8) were 8.4-fold, indicating

that the alkylaminopyridine group was better than the

corresponding imidazole group in the hydrolysis active site.

The rate constant increases of the substrate-imprinted

polymer 6 and non-imprinted polymer 5 are 3.3-fold and

1.2-fold, respectively. Transition state of p-nitrophenyl

acetate is somewhat different to p-nitrophenyl acetate in

shape [28]. The low activity of 6 is probably due to the

unfitted cavity shape within the polymer. There has been

few rate acceleration of the non-imprinted polymer 5,

because p-nitrophenyl acetate molecules are obstructed by

the macromolecular chain network and not accessible to the

catalytic sites.

We assumed that the hydrolysis followed kinetics similar to

enzymes and enabled the data to be treated by variants of

Michaelis–Menten kinetics. The rate constant ðkcatÞ and

Michaelis constants ðKmÞwere calculated by the Lineweaver–

Burk plots as shown in Fig. 4. The results given in Table 1 for

TSA-imprinted polymeric catalysts obtained from Line-

weaver-Burk plots indicate that both the alkylaminopyridine

and imidazole catalysts are comparable in their catalytic

activity. The polymer 7 hydrolyzes p-nitrophenyl acetate 1.54

and 2.54 times as fast as 8 and 6 in kcat; respectively. 7 has a

Michaelis constant Km that is 104.8% and 40.6% smaller than

that of 8 and 6, respectively. The data showed that stabilization

of catalyst-substrate complex of 7 was similar to 8 and better

than 6. The shape fitting between transition state of the

substrate and imprinted cavity in 7 are better than 6 and 8,

resulting in 1.49 and 3.25-fold larger overall reaction rate

kcat=Km than of 8 and 6, respectively. The second-order

constants ðkcat=KmÞ; the enzymatic rate constants, indicate that

the alkylaminopyridine catalyst is as efficient as the imidazole

catalyst in its enzyme mimicking.

Molecular imprinting has proven to be an effective

method that creates three-dimensional binding sites in

polymers [35]. 4-Pyrrolidinopyridine (PPY) is a very active

supernucleophilic catalyst [10,12]. The catalytic activities

Fig. 2. Polymerization of 4-(3,4-dimethylenepyrrolidyl)pyridine 3 with 4,40-bis(3,4-dimethylenepyrrolidyl)phenyl sulfone 4.

Table 1

Kinetic parameters for hydrolysis of p-nitrophenyl acetate in the presence

of non-imprinted, substrate-imprinted, TSA-imprinted poly[4-(3,4-

dimethylenepyrrolidine) pyridine] and TSA-imprinted poly[4-(5)-vinyli-

midazole] (5, 6, 7 and 8), respectively

Catalyst 105 kobs

(s21)

kobs=kcontrol 108 kcat

(s21)

104 Km

(M21)

104 ðkcat=KmÞ

(M21 s21)

None 1.32 – – – –

5 1.43 1.2 – – –

6 4.39 3.3 3.81 2.71 1.40

7 17.8 13.5 9.66 1.10 8.81

8 12.4 9.4 6.27 1.05 5.97

[ p-Nitrophenyl acetate] ¼ 2.5 £ 1025 mol l21 and [catalyst] ¼ 0.23

g l21 in 0.4% (by volume) acetonitrile in 0.05 M of Tris–HCl buffer (pH

8.2) including 0.05 M of KCl at 37 8C.

J.-T. Huang et al. / Polymer 45 (2004) 4349–43544352

of 4-(N,N-diallylamino)pyridine homopolymer (DAAP

homopolymer) and co-polymers, the polymers whose

monomer units are identical with PPY, are higher than

that of PPY in all known acylations [9] (Fig. 5). Having thus

established the systems to mimic the binding site (cavity

imprinted molecularly) and the catalytic group (PPY-like

supernucleophilic group) of hydrolase, it was logical to

combine the two essential features of enzyme action to

produce a miniature organic enzyme model. Transition state

of p-nitrophenyl acetate is known to be recognized

specifically by the TSA-imprinted cavity. Hydrolysis is

accelerated by alkylaminopyridine moiety within the cavity.

Fig. 3. Protocol for p-nitrophenol methylphosphonate imprinting of poly[4-(3,4-dimethylenepyrrolidine)pyridine]. TSA and substrate (templates) were

dissolved in monomers 3, respectively and then was carried out a co-polymerization. The templates were removed from solid polymer to retain specifically

binding cavities within the polymer matrix complementary to these template molecules. Transition state of p-nitrophenyl acetate is similar to the template

molecule in shape. p-Nitrophenyl acetate molecule was recognized by the imprinted cavity and catalyzed by alkylaminopyridine group within the cavity. The

TSA-imprinted polymer with supernucleophilic reagent is a miniature organic model of hydrolytic enzymes with catalytic activity that exhibits specificity and

large rate acceleration.

J.-T. Huang et al. / Polymer 45 (2004) 4349–4354 4353

4. Conclusions

Although the 4-(N,N-dialkylamino)pyridines as super-

nucleophilic catalysts have shown amongst the greatest rate

accelerations in acylation and hydrolysis, the catalytic

groups have not been used previously in the design an

artificial enzyme model. The TSA-imprinted polymer

catalyst with supernucleophilic moiety is synthesized; it

should be possible to build a miniature enzyme model of 4-

alkylaminopyridines, much like the functional groups used

in imidazole-appended enzyme mimics. Such model

enables us to enhance binding specificity of the polymer

catalysts as well as the catalytic activities.

Acknowledgements

The authors thank Q. Wang and A.L. Cao for stimulating

discussion. This work was supported in part by research

grant 013801311 from the Tianjin Committee of Science

and Technology and research grant 99705 from the Tianjin

Committee of Education.

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Fig. 4. The Lineweaver–Burk plots for hydrolysis of p-nitrophenyl acetate

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J.-T. Huang et al. / Polymer 45 (2004) 4349–43544354