Analytica Chimica Acta 549 (2005) 39–44

Theoretical and experimental study of nicotinamide molecularlyimprinted polymers with different porogens

Liqing Wu, Kuaichang Zhu, Meiping Zhao, Yuanzong Li∗

Key Laboratory of Bioorganics and Molecular Engineering, College of Chemistry and Molecular Engineering,Peking University, No. 202, ChengFu Road, Beijing 100871, PR China

Received 25 April 2005; received in revised form 30 May 2005; accepted 1 June 2005Available online 11 July 2005

Abstract

Based on the computational model proposed in our previous work, a comparative study of the influence of porogens on the affinity andselectivity of MIP was carried out in this paper. Nicotinamide (NAM) was chosen as the template and methacrylic acid (MAA) was chosenas the functional monomer. The interaction energy values between NAM and MAA were calculated with methanol, acetonitrile, chloroformand toluene as the porogens, respectively. The subsequent chromatographic evaluation was carried out to give the retention factors ands tion factorso dielectricc ent and theh gy. Aproticp r, resultingi f porogenf©

K

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mteebbcnHnfi

hasutedoccu-od-late

eenity ofd

eval-and

t forper-porethepacity

0d

electivity factors of NAM and its analogues. Good correlations were found to exist between the interaction energies and the retenr selectivity factors. When the porogens had poor hydrogen bonding capacity, the interaction energy was mainly influenced byonstant of solvent. And when the porogen had strong capacity in forming hydrogen bond, both the dielectric constant of the solvydrogen bonding interference affected the forming of the template–monomer complex and the corresponding interaction enerorogen with low dielectric constant was likely to lead to large interaction energy between the template and the functional monome

n a MIP with better affinity and selectivity. The success of the predicting model provides a experiment-free way for the selection oor given template and functional monomer.

2005 Elsevier B.V. All rights reserved.

eywords:Molecular recognition; Molecularly imprinted polymer (MIP); Theoretical; Porogen; Solvent effect

. Introduction

Molecularly imprinted polymer (MIP) is a new kind ofacromolecular material, which has high affinity and selec-

ivity for its templates. Due to its advantages of selectivity,asy preparation and economical synthesis, MIP has drawnxtensive attention recently[1,2]. Now MIP technology haseen applied in chromatographic separation, sensors, sor-ent assays, membranes, solid-phase extraction (SPE) andatalysts[1–6]. In order to improve the properties of MIP,ew synthesis and evaluation methods have been developed.igh throughput screening (HTS)[7] and combinatorial tech-iques[8] have also been applied in molecular imprintingeld. With the development of computer technology and

∗ Corresponding author. Tel.: +86 10 62757954; fax: +86 10 62751708.E-mail address:yzli@pku.edu.cn (Y. Li).

quantum chemistry, the computer-aided study of MIPcome out. Wulff used electrostatic potential surface compby MolCad to represent the shapes of occupied and unpied cavities[9]. There was a report on using molecular meling software to study the functional monomer–tempconformation before polymerization[10]. Piletsky groupused a virtual library of functional monomers to scragainst the target template molecule, and the selectivMIP was greatly improved[11–13]. These results indicatethat the computer aided MIP synthesis, analysis anduation was a promising method for fast, accurate, safeeconomical study of MIP.

As reported, the polymerization porogen is importanthe recognition ability, enantioselectivities and other proties of MIP, such as surface area, swelling capacity andvolume[14]. In general, the hydrogen bonding capacity ofporogens, namely, an average of donor and acceptor ca

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.06.009

40 L. Wu et al. / Analytica Chimica Acta 549 (2005) 39–44

[15], played an important role in the recognition propertiesof the materials. Thus, the polymers synthesized with apro-tic porogens usually have higher affinity and selectivity thanthose prepared using the porogens with moderate or stronghydrogen bonding capacity.

NAM exists widely in nature and, when combined withnicotinic acid, is called Vitamin PP. NAM can be consideredto be both a nutrient and drug. It is a part of coenzymes Iand II, which participate in the biologic redox processes inthe human body. In the body, nicotinic acid is converted intoNAM, which can be used to treat vascular headache, vascularthrombus and other diseases.iso-Nicotinamide (iso-NAM),which is a positional isomer of NAM, possesses strong anti-tubercular, anti-pyretic, brinolytic and anti-bacterial proper-ties. So MIPs with NAM andiso-NAM as the template arethe potential material for NAM andiso-NAM analysis andseparation in drugs and human body samples[16–18].

In our previous work[17,18], a simplified computationalmodel was proposed to predict the affinity and selectivityof MIP with NAM and iso-NAM as the templates. Affin-ity was used to describe the retention of the template onrelated MIP packed cartridge and the selectivity to describethe retention of the analogues. The model could simulate thesynthesis of MIP, removal of the template and re-binding pro-cess of the template and its analogues. In these simulations,solvent effect was neglected in order to focus on the inter-a r, wec df ands

2

2

ry-l z-i df e( ti-n jingC phicg pain.M id( ,m inC

alyt-i tilledp edw Agi-l merw evedw G,G

Table 1Composition of the reaction mixture for MIP and blank polymer synthesis

NAM(mmol)

MAA(mmol)

EGDMA(mL)

AIBM(mg)

Solvent10 mL

P1 1.0 5.0 4.0 40 ChloroformP1′ 0 5.0 4.0 40 ChloroformP2 1.0 5.0 4.0 40 MeCNP2′ 0 5.0 4.0 40 MeCNP3 1.0 5.0 4.0 40 MethanolP3′ 0 5.0 4.0 40 MethanolP4 1.0 5.0 4.0 40 TolueneP4′ 0 5.0 4.0 40 Toluene

2.2. Polymer preparation

The procedure for the polymer synthesis was as follows:1.0 mmol NAM was dissolved in one of the four porogensin an 18 mm× 180 mm borosilicate glass test tube, to whichMAA, crosslinking agent EGDMA, and initiator AIBN wereadded. The detailed composition of the reaction mixture waslisted inTable 1. Then the mixture was sonicated for 1–2 minand bubbled with nitrogen for 10 min to remove the dissolvedoxygen before the tube was sealed under reduced pressure.The polymerization was allowed to continue in a water bathat 60◦C for 36 h. The solid polymer formed was crushedand ground, sieved and sedimented to obtain particles inthe size range of 32–50�m. Fine particles were removed byrepeated decanting in acetone for three times. Then the par-ticles were dried under vacuum at 60◦C. The blank polymer(BP) was prepared and treated identically except no templatewas present in the polymer preparation.

2.3. Chromatographic evaluation

The polymer particles were packed into 50 mm× 4.6 mmstainless steel cartridges manually. To remove templates, thecartridge was washed with methanol/acetic acid (9/1, v/v).The removal of template was confirmed by UV/vis spectrom-etry. The chromatographic evaluation was carried out on anA nm.T owr fixedap

ionk atea

2

outt u-l ory( rgyi eaki . The

ction between the template and monomer. In this papeontinued to use NAM andiso-NAM as the templates anocused on the prediction of solvent effect on the affinityelectivity of MIP.

. Experimental

.1. Reagents and instruments

Picolinamide (PAM) and ethylene glycol dimethacate (EGDMA) were obtained from Aldrich, USA. Pyranamide andiso-nicotinamide (iso-NAM) were purchaserom Acros, Pittsburgh, USA; 2,2′-azobisisobutyronitrilAIBN) was from Nankai Chemical Factory, China. Nicoamide (NAM) (chemical grade) was obtained from Beihemical Reagent Factory. Acetonitrile (chromatograrade) was obtained from Scharlau Chemie S.A., Sethacrylic acid (MAA), salicylic acid (SA), benzoic ac

BA), phenol,p-hydroxybenzoic acid (p-HBA), acetic acidethanol, toluene, chloroform were all manufacturedhina.All of the above chemicals and reagents were of an

cal grade unless stated. Monomers were freshly disrior to use to remove inhibitors. AIBN was re-crystallizith ethanol before use. HPLC was performed using an

ent 1100 Series chromatographic workstation. Solid polyas crushed and ground using MM200 ground and siith an AS200 control “g” siever (Retsch, GmbH & Co. Kermany).

gilent 1100 HPLC workstation with detector set at 260he mobile phase was MeCN:HOAc = 95:5 (v/v) and the flate was set at 0.8 mL/min. The column temperature wast 25.0◦C. The injection volume was 5�L with 10 mM tem-late or its analogues.

The capacity factork′ was calculated using the equat′ = (tr − t0)/t0, wheretr was the retention time of a substrndt0 the time to elute acetone.

.4. Computer simulation

Gaussian 03[19] was adopted as the software to carryhe simulation and B3LYP[20,21]was selected as the calcation method. B3LYP is a kind of density functional theDFT) method, which takes electronic correlation enento consideration and usually gives better results of wnteraction system compared with Hartree–Fock method

L. Wu et al. / Analytica Chimica Acta 549 (2005) 39–44 41

computer simulation was carried out on a PC with an IntelPentium 4 2.6 GHz CPU and 1 Gb memory.

3. Results and discussion

3.1. Computer simulation in porogens

The affinity and selectivity of MIP was usually influ-enced by solvent in two aspects. On one hand, the interactionbetween the template and the functional monomer was influ-enced by porogens with different dielectric constants andhydrogen bonding capacities, leading to the different affini-ties and selectivities of resultant MIPs. On the other hand,for given MIP the observed affinity and selectivity toward thesame template or analogue depended on the mobile phase inHPLC evaluation.

Considering the native affinity and selectivity of the MIPwere mainly determined by the porogen when other synthe-sis and evaluation conditions were fixed, the influence ofporogen was studied by adding the solvent model into theGaussian 03[19] input file to optimize the conformationand calculate the interaction energy between the templateand the functional monomer. Since the hydrogen bond wasweak, the potential surface of the template–monomer com-p notc onalm od-e thatt witht ed inv oro-g e andt inge

�

n)

W alm tivityo rma r pre-v .F -p wereo LYPm tiono ionsw n inp wasc CMs

Fig. 1. The relation between the interaction energies and the dielectric con-stants of porogens (a) or the retention factors (b).

3.2. The dependence of retention factors/imprintingfactors on the template–monomer interaction energies indifferent porogens

Fig. 1showed the relations between the interaction ener-gies in the porogens and the dielectric constants of the poro-gens or retention factors, respectively. Albeit a nice positivecorrelation was found for the interaction energies and dielec-tric constants, the correlation between them and the retentionfactors was not good. According to the calculation, NAM hadthe similar interaction energies with MAA in methanol andacetonitrile. But the chromatographic behaviors were signifi-cantly different. MIP synthesized with methanol as the poro-gen showed almost no imprinting effect toward NAM, whileit with MeCN as the porogen showed a moderate imprint-ing effect. This conflict was probably caused by the PCMmodel, in which only the dielectric constant and the solventmolecular size were considered.

It seems that the omitting of the hydrogen bonding inter-ference of the protic porogen (methanol) caused a significanterror to the estimation of the interaction energy betweenNAM and MAA. Taking the hydrogen bonding interferenceof methanol into consideration, the possible NAM–MeOHcomplexes were modeled and optimized in Gaussian 03[19],which were shown inFig. 2.

Clearly, both amide and pyridine N binding sites in NAMc Hm M,w lex.T twob .

as− ergy,fH .S ulesd ndMH e

lex was plain and the conformation optimization didonverge well. To avoid the problem, the template, functionomer and template–monomer complex were firstly mled and optimized in vacuum. An assumption was made

hese conformations changed little in porogen comparedhose obtained in vacuum. So the conformations obtainacuum were used for single point energy calculation in pen. Then the interaction energies between the templat

he functional monomer were calculated by the followquation with single point energy in porogen:

E = E(template–monomer complex in porogen)

−E(template in porogen)−E(monomers in poroge

(1)

ith NAM as the template and MAA as the functiononomer, the porogen influence on the affinity and selecf MIP was studied by using methanol, MeCN, chlorofond toluene as the porogens, respectively. Based on ouious work[17,18], NAM:MAA = 1:2 complex was studiedirstly, several possible NAM, MAA and NAM–MAA comlex models were set up and the related conformationsptimized in vacuum by Gaussian 03. In this step, B3ethod with 6-311G(d) basis set was used for conformaptimization and energy calculation. Then the conformatith the lowest energies were used for energy calculatioorogen environment. In this step, single point energyalculated in MP2 level with 6-311+G(d) basis set and Polvent model.

ould bind with MeOH. In amide binding site, two MeOolecules can form a hydrogen-binding ring with NAhich significantly increased the stability of the comphe interaction energies between MAA/MeOH and theinding sites in NAM were listed inTable 2for comparison

The interaction energy between NAM and MAA w0.00888 Hartree in amide binding site. However, the en

orming the hydrogen bonding ring structure was−0.01611artree, which was much higher than−0.00888 Hartreeince the hydrogen bonding between two MeOH molecid not contribute to the interaction between NAM aeOH, subtracting of it from−0.01611 gave−0.01050artree, which was still higher than−0.00888 Hartree. Th

42 L. Wu et al. / Analytica Chimica Acta 549 (2005) 39–44

Fig. 2. The possible NAM–MeOH complexes.

concentration of MeOH was much higher than that of MAAin the pre-polymerization stage, so there was little chancefor MAA to interact with NAM in amide binding site. Inpyridine N binding site the interaction energy between NAMand MAA was−0.00909 Hartree, and it between MeOH andNAM was only−0.00577 Hartree. So NAM could only inter-act with MAA through pyridine N binding site in MeOH.Thus the interaction energy between MAA and NAM at pyri-dine N site determined the complex stability and the retentionfactor of NAM on MIP synthesized in MeOH.Fig. 3showedthe correlation of the interaction energies with retention fac-tors (a) or imprinting factors (IF, b). The imprinting factorwas defined as following. Generally, a large imprinting fac-tor indicated a good imprinting effect.

Imprinting factor (IF)= retention factor on MIP

retention factor on blank polymer

Table 2The interaction energies between MAA/MeOH and NAM

�E (Hartree) Pyridine N Amide

MAA–NAM −0.00909 −0.00888MeOH–NAM −0.00577 −0.01611a

−0.01050b

a The energy to form ternary complex.ergy

b

Fig. 3. The relation between the interaction energies and retention factors(a) or imprinting factors (b) with different solvents as porogens.

Fig. 3a and b showed a highly improved positive correla-tion between the interaction energies and the retention factorsor imprinting factors when the hydrogen bonding property ofthe protic methanol was taken into consideration. The rela-tion demonstrated the influence of porogen on MIP affinitywas mainly determined by the interaction energy betweenthe template and the functional monomer. Different porogenswith different hydrogen bonding capacities and dielectricconstants resulted in different interaction energies betweenthe same template and functional monomer. Therefore, differ-ent quantities with different stabilities of template–monomercomplexes were formed in pre-polymerization stage. Largeinteraction energy meant more binding sites with high affin-ity in the resultant polymer, leading to a large retention factoror imprinting factor.

In our previous work[17,18], no solvent effect wastaken into consideration in the computer simulation, there-fore the energy was often over estimated. For example, theinteraction energy between NAM and MAA to form 1:2complex was−0.04713 Hartree without considering sol-vent effect, which was larger than those showed inFig. 3where solvent effects were taken into consideration. There-

b The energy to form ternary complex minus hydrogen bond enetween two methanol molecules.

L. Wu et al. / Analytica Chimica Acta 549 (2005) 39–44 43

fore, for aprotic porogen system, only the dielectric constantof porogen need to be taken into consideration, while forprotic porogen, both the dielectric constant and the hydro-gen bonding interference of the porogen should be consid-ered.

It was worthy to be pointed out that the retention of asubstrate on related MIP column depended not only on theaverage affinity of binding sites but also on the percent-age of binding sites. When no solvation of the template ormonomer occurred, the larger the interaction energy betweenthe template and monomer, the higher the concentration of thetemplate–monomer complex formed. In MeOH, both MAAand NAM could form solvation products with MeOH andMeOH was in large excess, thus much less NAM–MAA com-plex was formed in MeOH compared with that in MeCN,toluene and chloroform as porogens. So, it was not very strictto use only the interaction energy between NAM and MAAto reflect the retention of NAM on related MIP preparedwith methanol as the porogen. However, protic porogenswere seldom to be selected to prepare MIP. If the aver-age affinity on MIP column should be strictly predictedin protic porogen, such as methanol, not only the interac-tion energy between the template and monomer but alsothe percentage of high affinity binding sites needed to becalculated.

3aa

Thec IP’sa wasd tedb ctoro thet ten-t tionf tivityf apha dis-t

er-e

witht Eachs allert Andt nar-r d bep ivityo tives Then uldb seen

Fig. 4. The selectivity factors of analogues on MIP (a) and blank polymer(b) with different solvents as porogens.

fromFig. 4b that the native selectivity under different synthe-sis conditions were different and generally bad. Comparisonof the selectivity factors on MIP and BP showed that theselectivity factors on MIP did not necessarily have a positivecorrelation with those on BP. The selectivity factors on MIPusually had a narrower distribution range and lower valuesthan those on blank polymer.

It can be seen fromFig. 4thatiso-NAM, picolinamide andpyrazinamide could reflect the selectivity factors sensitively.So they were selected to study the relation between the selec-tivity factors and the interaction energies, which was shownin Fig. 5.

Again, a positive correlation between the interaction ener-gies and the selectivity factors was found. This could beinterpreted as following: when the interaction energy waslarge, more high affinity binding sites were formed and thecavities were regular, which showed discrimination of ana-logues. In the meantime, the non-specific binding sites wereless formed, which leaded to less non-specific interactionbetween the analogue and functional monomer. Thus the

.3. The relation between the selectivity factors of NAMnalogues and the interaction energies between NAMnd MAA in different porogens

Different porogens also affected the MIP’s selectivity.apacity of the template binding to MIP was defined as Mffinity and the capacity of an analogue binding to MIPefined as its selectivity. MIP’s selectivity could be reflecy selectivity factor, which was defined as the retention faf an analogue on MIP divided by the retention factor of

emplate on MIP. When the selectivity was good, the reion factor of the template was usually large and the retenactors of the analogues were small. So the small selecactor indicated good selectivity and in the selectivity grll the selectivity factors of the analogues had a narrow

ribution.Analogues’ selectivity factors on MIP and BP with diff

nt solvents as porogens were shown inFig. 4.Fig. 4a showed the best selectivity was achieved

oluene as the porogen and the worst with methanol.electivity factor with toluene as the porogen was smhan the relative one with other solvents as porogens.he selectivity factors with toluene as the porogen hadower distribution range compared with others. It shoulointed that the selectivity came not only from the selectf cavity produced by the template, but also from the naelectivity determined by different synthesis conditions.ative selectivity under different synthesis conditions coe reflected from the selectivity factors on BP. It can be

44 L. Wu et al. / Analytica Chimica Acta 549 (2005) 39–44

Fig. 5. The relation between selectivity factors and the interaction energiesbetween the template and monomer in different porogens.

good selectivity was achieved with large template–monomerinteraction energy.

4. Conclusion

In this paper, solvent effect was taken into considerationin the computer simulation of the interaction between tem-plate NAM and functional monomer MAA. The interactionenergy between the template and monomer varied with poro-gens. When aprotic porogens, such as chloroform, tolueneand MeCN, were used, the interaction energies were mainlyinfluenced by the dielectric constants of porogens. And whenthe porogens with strong capacity in forming hydrogen bond(like methanol) was used, the hydrogen bonding interferenceaffected the forming of the template–monomer complex andinfluenced the interaction energy between the template andthe functional monomer. The small dielectric constant andaprotic porogen was likely to lead to the large interactionenergy between the template and the functional monomerand that was in favor of forming higher concentration oftemplate–monomer complexes, which would result in MIPwith high affinity and selectivity. Both affinity and selec-tivity of MIP were determined by the interaction energybetween the template and the functional monomer and theycould be predicted by the computer simulation. The successo -freew unc-t entt ork[

Acknowledgments

This work was supported by the National Science Foun-dation of China (20275002, 20335010) and Ph.D. EducationFound of the National Ministry of Education.

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