Use of Molecularly Imprinted Polymers ina Biotransformation Process

Lei Ye, Olof Ramstrom,* Richard J. Ansell,† Mats-Olle Månsson,Klaus Mosbach

Pure and Applied Biochemistry, Chemical Center, Lund University, P.O.Box 124, S-22100 Lund, Sweden; telephone: +46 46 2228258; fax: +46 462224611; e-mail: Klaus.Mosbach@tbiokem.lth.se

Received 9 October 1998; accepted 18 February 1999

Abstract: Molecularly imprinted polymers are highlystable and can be sterilised, making them ideal for use inbiotransformation process. In this communication, wedescribe a novel application of molecularly imprintedpolymers in an enzymatic reaction. The enzymatic con-densation of Z-L-aspartic acid with L-phenylalanine meth-yl ester to give Z-L-Asp-L-Phe-OMe (Z-aspartame) waschosen as a model system to evaluate the applicability ofusing molecularly imprinted polymers to facilitate prod-uct formation. When the product-imprinted polymer ispresent, a considerable increase (40%) in product yield isobtained. Besides their use to enhance product yields, asdemonstrated here, we suggest that imprinted polymersmay also find use in the continuous removal of toxiccompounds during biochemical reactions. © 1999 JohnWiley & Sons, Inc. Biotechnol Bioeng 64: 650–655, 1999.Keywords: molecular imprinting; aspartame; enzymaticsynthesis; thermolysin; specific adsorbents

INTRODUCTION

Molecular imprinting is a moulding process in which a printmolecule is used as a template for the creation of a sub-strate-selective macromolecular matrix (Ansell et al., 1996;Mosbach and Ramstro¨m, 1996; Shea, 1994; Vulfson et al.,1997; Wulff, 1995). By this process, highly specific bindingsites can be introduced into synthetic polymers, which arethen able to recognise selectively the original template. Mo-lecularly imprinted polymers (MIPs) have been used asmimics of natural molecular recognition entities such asantibodies, receptors, and enzymes for a variety of applica-tions. One exceptionally important potential application liesin the separation of chiral drugs into their optically pureenantiomers, an area of utmost importance for the pharma-ceutical industry given that approximately 500 optically ac-tive drugs are presently available on the market (Nicholls etal., 1995). Other examples include the use of MIPs in drugassays (Ramstro¨m et al., 1996; Vlatakis et al., 1993), asreceptor binding mimics (Andersson et al., 1995), and in

substrate selective biosensor-like devices (Kriz and Mos-bach, 1994). All of these applications are based on the spe-cific interactions between the target molecule and the im-printed polymer, a consequence of their structural comple-mentarity that arises from the imprinting process.

MIPs are very stable, due in part to their high degree ofcross-linking. As previously demonstrated (Ansell et al.,1996), they are stable in organic solvents and at high tem-peratures without detriment to their recognition specificity,and, as shown in the course of this investigation, they can besterilised. It occurred to us that these highly specific adsor-bents could be useful in fermentation or enzymatic pro-cesses for a number of reasons: (a) Many interesting reac-tions exhibit unfavourable equilibria. In the presence of aspecific adsorbent, whether in the reaction vessel or packedinto a column and mounted onto the reaction vessel to allowrecirculation back into the vessel, these equilibria may beshifted towards product formation. Also, (b) it is known thattoxic compounds, which are harmful to the overall process,can be formed in the course of biochemical reactions. Theymay presumably be readily removed by using appropriateadsorbents. Finally, (c) it can be envisaged that MIPs car-rying the specifically adsorbed product could be relativelyeasily recovered from the fermentation/enzyme broth, eitherby applying a magnetic field for the isolation of magneti-cally susceptible MIPs (Ansell and Mosbach, 1998), or byusing expanded beds. This communication addresses in par-ticular the first application, i.e., shifting an unfavourableequilibrium towards product formation, by using product-imprinted polymeric adsorbents. Various enzymes includ-ing thermolysin have been applied in biochemical synthesesbecause of their high catalytic efficiency and specificity(Sakina et al., 1988). In a model system, we applied mo-lecularly imprinted polymers in the reaction between aspar-tic acid and phenylalanine methyl ester to give the sweet-ener aspartame, as catalysed by the thermostable enzymethermolysin (Oyama et al., 1987; Nakanishi et al., 1985).

MATERIALS AND METHODS

Materials

Z-L-Asp-L-Phe-OMe (Z-aspartame) was purchased fromIndofine Chemical Co. (Somerville, NJ). Z-L-Aspartic acid

*Present address:Laboratoire de Chimie Supramoleculaire, ISIS-UniversiteLouis Pasteur, F-67000 Strasbourg, France

†Present address:Institut fur Analytische Chemie, Chemo- und Biosen-sorik, Universita¨t Regensburg, 93053 Regensburg, Germany

Correspondence to:K. Mosbach

© 1999 John Wiley & Sons, Inc. CCC 0006-3592/99/060650-06

(Z-L-Asp), L-phenylalanine methyl ester hydrochloride (L-Phe-OMe? HCl), and thermolysin (fromBacillus thermo-proteolyticus rokko) were from Sigma (St. Louis, MO).L-Phe-OMe was prepared by treatment of the hydrochloridesalt with an equimolar quantity of Na2CO3 (Nakanishi et al.,1985). Other chemicals were from commercial sources. Allsolvents were of either analytical or HPLC grade.

Preparation of Molecularly Imprinted Polymers

In a typical preparation, the print molecule was mixed withporogenic solvent in a glass test tube. Methacrylic acid wasadded and the suspension was dissolved by sonication. Eth-ylene glycol dimethacrylate (EDMA) and azobis-isobutyronitrile (AIBN) were added, and the polymerisationsolution was subsequently purged with N2 for 5 min atice-bath temperature. Polymerisation was photolytically in-duced at 366 nm and allowed to proceed for 36 h using astandard laboratory UV source (CAMAG 23200, Buben-dorf, CH). The resulting bulk polymer monolith was groundin a mechanical mortar and wet-sieved. Particles with di-ameter smaller than 25mm were collected from which fineparticles were removed by repeated sedimentation in ac-etone (4 times). The print molecule was thoroughly ex-tracted with methanol containing 10% acetic acid (v/v),with spectrophotometric monitoring at 254 nm to ensurecomplete removal. The reference polymer was prepared us-ing exactly the same procedure except that no template wasused.

Polymer Binding Characterisation

Polymer particles were suspended in 40 mL of chloroformand packed into standard 100 × 4.6 mm stainless steelHPLC columns at 300 bar with an air driven fluid pump(Haskel Engineering Supply Co., Burbank, CA) using ac-etone as the packing solvent.

Chromatographic analyses were performed isocraticallyusing a Pharmacia LKB type 2249 solvent delivery system,a variable wavelength monitor model 2141 (PharmaciaLKB Biotechnology, Sweden), together with a softwarepackage EZChrom (Scientific Software, CA). Acetonitrilecontaining 1% acetic acid (v/v) was used as the mobilephase at a flow rate of 0.5 mL/min, and the analytes weremonitored at 254 nm. Acetone was used as void marker, and0.2mmol of each analyte was injected. Capacity factors (k8)were calculated according to standard chromatographictheory. The polymer binding characteristics were evaluatedby the normalised retention index, which was defined as(k8

MIP/k8REF)analyte/(k8MIP/k8REF)print molecule, wherek8MIP andk8REF

indicate capacity factors on columns packed with imprintedpolymer and reference polymer, respectively (Andersson etal., 1996). The column packed with polymer MIP 1 (Z-aspartame imprinted polymer) was autoclaved at 120°C for20 min and re-equilibrated with mobile phase. The retentionof Z-aspartame was determined to be identical to before theautoclave treatment.

Frontal Chromatography Analysis of Z-AspartameImprinted Polymer

Frontal chromatography analyses (Kasai and Oda, 1986;Kempe and Mosbach, 1991) were performed at room tem-perature, using a 100 × 4.6 mm HPLC column packed withpolymer MIP 1, prepared against Z-aspartame. Z-Aspartameand Z-L-aspartic acid were monitored at 240 nm. Acetoni-trile containing 1% (v/v) acetic acid was used as mobilephase at a flow rate of 0.5 mL/min. Elution fronts weregenerated by injecting 5 mL of analyte solution at differentconcentrations (range [S]0 4 0.25–2 mM) and acetone wasused as void marker. The dissociation constant (Kd) wascalculated from a plot of 1/[S]0(V − V0) versus 1/[S]0, whereV and V0 are the retention volume of the analyte and ac-etone, respectively.

Application of MIPs in Enzymatic Synthesisof Z-Aspartame

Enzymatic synthesis of Z-aspartame was performed at 37°Cin 1 mL of reaction media consisting of ethyl acetate satu-rated with aqueous MES buffer solution (50 mM MES, 5mM CaCl2, pH 6.0), with or without the presence of differ-ent MIPs and control polymer. When included, 4.0 mg ofthermolysin and specific amounts of polymer were used.After a specific incubation period, 2 mL of extraction sol-vent (MeOH containing 10% HOAc (v/v)) was added, andthe mixture was thoroughly mixed on an end-over-end rock-ing table at room temperature for 1 h. Centrifugation wasperformed and the supernatant was filtered through a 0.45-mm PTFE microfilter. The filtrate (500mL) was dried undervacuum. The residue was dissolved in 1 mL of dilutionsolvent (MeCN/water 70/30 (v/v), pH 2.5) and analysed byRP HPLC (ODS column, Nucleosil100-5C18), using thePharmacia HPLC system with monitoring at 254 nm. Amixture of acetonitrile and water (60/40, v/v) at pH 2.5 wasused as mobile phase at a flow rate of 0.2 mL/min. Thesynthetic yields were calculated from the Z-aspartame peakarea in the reverse phase chromatogram, using the authenticsample as standard, processed using the same post-reactionprocedure.

RESULTS AND DISCUSSION

Aspartame Synthesis

As seen in Fig. 1, the route to aspartame formation usingthermolysin is a straight-forward process involvingN-benzyloxycarbonyl-L-aspartic acid (Z-L-aspartic acid) (1)and L-phenylalanine methyl ester (2), leading, in a singlecondensation step, to Z-a-aspartame (3) (route I). The ac-tual sweetener,a-aspartame (4), is subsequently generatedby removal of theN-protecting group from the precursor.This enzymatic reaction, which is widely used in industry, isfully reversible, and the process proceeds until dynamicequilibrium is achieved. The use of thermolysin makes the

YE ET AL.: MOLECULARLY IMPRINTED ADSORBENTS 651

reaction very selective and only Z-a-aspartame can beformed. In contrast to this “clean” preparation, Z-b-aspartame (6) can also be formed as an impurity usingchemical peptide synthetic schemes (route II).

In the model system presented here, we employed mo-lecularly imprinted polymers in the enzymatic synthesis ofaspartame in order to facilitate product formation (route I).The Z-a-aspartame product was continuously removedfrom the enzymatic reaction via complexation with a mo-lecularly imprinted polymer, therefore product formationbecame favoured.

Preparation and characterisation of molecularlyimprinted polymers

Various molecularly imprinted polymers and a referencepolymer were prepared (Table I). The reaction product,Z-a-aspartame, as well as the reactants, Z-L-aspartic acid(Z-L-Asp) andL-phenylalanine methyl ester (L-Phe-OMe),were chosen as print molecules, and methacrylic acid wasused exclusively as the functional monomer. The referencepolymer was prepared in the same manner as the imprintedpolymers, except that no print molecule was used in thepolymerisation stage.

Table I. Preparationa and characterisation of molecularly imprinted polymers.

Polymer Print moleculeSurface areac

(m2 g−1)

Normalised retention indexd

a-L,L-ZAPM a-D,D-ZAPM b-L,L-ZAPM Z-L-Asp Z-D-Asp

MIP 1 a-L,L-ZAPM 195.2 1 0.79 0.68 0.59 0.56MIP 2 Z-L-Asp/L-Phe-OMeb 189.0 0.64 — 0.95 1 0.93REF None 337.1 1 1 1 1 1

aFor the preparation of molecularly imprinted polymers, 1.3 mmol print molecule, 13 mmol MAA, 65 mmol EDMA, and 125 mg AIBN were dissolvedin 20 mL acetonitrile.

bMolar composition of print molecule wasZ-L-Asp:L-Phe-OMe4 1:1.cSurface areas were determined by BET single point gas adsorption using a Micromeritics Flowsorb II 2400 instrument (30% N2 in He), samples were

degassed at 150°C for 16 h prior to determination.dNormalised retention index was defined as (k8MIP/k8REF)analyte/(k8MIP/k8REF)print molecule, wherek8MIP andk8REF were capacity factors on columns packed

with imprinted polymer and reference polymer, respectively.

Figure 1. Synthetic routes toN-benzyloxycarbonyl-L-aspartame (Z-aspartame).

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The recognition capabilities of the MIPs were evaluatedby chromatographic analysis. In all circumstances, the printmolecules showed the highest normalised retention index,as calculated relative to a non-imprinted reference polymer(REF) (Andersson et al., 1996), thereby demonstrating thespecific recognition of the print molecules by the imprintedpolymers. The normalised retention index is defined as theratio of the separation between the imprinted and the testsubstance, on the MIP and the reference polymer, respec-tively. The print molecule will have an index of 1, by defi-nition, and poorly recognised compounds lower than 1.Thus, for the product imprinted polymer (MIP 1), both sub-strates yielded lower retention indices than Z-aspartame, asdid both the optically active enantiomer Z-D-Asp-D-Phe-OMe (Z-D,D-aspartame) and Z-b-aspartame. The same pat-tern was observed for the substrate imprinted polymer (MIP2) prepared using a mixture of the two reactants as template,where all test compounds yielded lower retention indicesthan the print molecules.

In addition to the normalised retention index, ligandspecificity can also be verified by comparing the apparentdissociation constants (Kd) measured under the same con-dition. The Kd values of the product imprinted polymer(MIP 1) for the print molecule, Z-aspartame, and the reac-tant, Z-L-Asp, were estimated by frontal chromatographyanalysis (Kasai and Oda, 1986; Kempe and Mosbach,1991), using the same mobile phase. The dissociation con-stants were found to be 4.10 mM for Z-aspartame and 31.5mM for Z-L-Asp. Therefore the product-imprinted polymer,MIP 1, showed higher apparent affinity for the originaltemplate, Z-aspartame than for Z-L-aspartic acid. For theestimation ofKd values, acetic acid has been added in themobile phase to facilitate dissociation, this unavoidably re-sults in decreased binding strength. For practical reasons,measurements using the real enzymatic reaction solvent arefar more difficult than the determinations referred to above.The affinity for Z-aspartame for binding in ethyl acetatesaturated with aqueous buffer is expected to be considerablyhigher.

Polymer-Assisted Z-Aspartame Synthesis

The polymers were incubated at 37°C together with thereactants, Z-L-Asp andL-Phe-OMe, and the enzyme ther-molysin, using ethyl acetate saturated with aqueous buffersolution as the reaction solvent. It has been shown that whenethyl acetate containing 2–10% water (v/v) is used as thereaction solvent, the initial reaction rate increases with theconcentration ofL-Phe-OMe, but decreases with the con-centration of Z-L-Asp (Nagayasu et al., 1994). Therefore theconcentration ofL-Phe-OMe was kept twice that of Z-L-Aspin all experiments. The free enzyme reaction was evaluatedusing the same protocol but in the absence of any polymers.After a stated reaction time, the soluble products were sepa-rated from the solid materials, and monitored by reversephase HPLC.

Product formation as a function of time is displayed in

Fig. 2. As can be seen, the product-imprinted polymer (MIP1, curve 1) proved to be, as expected, the most efficient.This polymer provided the highest yield (63%) after 48 h ofreaction, while the polymer imprinted against the reactionsubstrates (MIP 2, curve 3) resulted in the lowest yieldamongst all the polymers used. Also displayed in Fig. 2 isthe result from the homogeneous, polymer-free enzyme-catalysed reaction. When no polymer was present, the yieldof Z-aspartame in the enzyme-catalysed synthesis reached amaximum after 6 h, and then gradually decreased with time(curve 4). Thus in the polymer-free enzymatic reaction onlyabout 15% of the Z-L-Asp was converted into product. Asimilar phenomenon has been reported previously when thesynthesis was performed in ethyl acetate saturated buffersolution, and the reason for this was proposed to be thenon-enzymatic decomposition ofL-Phe-OMe (Nakanishi etal., 1986). From Fig. 2 it can be deduced that after anincubation period of 48 h, all the reactions where polymershave been added have come close to completion. Prolongedincubation after 48 h was not performed, since the deacti-vation of enzyme and the decomposition ofL-Phe-OMeunder these conditions make the reaction system too com-plicated. When a polymer is introduced into the reactionsystem, partition of substrates and product amongst threedifferent environments takes place: the reaction solvent, theenzyme’s active site, and the polymer matrix. The fact thatthe reference polymer, as well as the substrate-imprinted

Figure 2. Z-Aspartame synthesis in the presence and absence of molecu-larly imprinted polymers. Thermolysin 4.0 mg, with or without 250 mg ofpolymer, was incubated in 1 mL of substrate solution (8 mM Z-L-Asp and16 mM L-Phe-OMe) at 37°C for a stated period. The reaction mixture wasthen extracted and monitored by reverse phase HPLC. Polymers preparedagainst different print molecules were tested: curve 1, polymer MIP 1,Z-aspartame as print molecule; curve 2, polymer REF, no print molecule;curve 3, polymer MIP 2, Z-L-Asp/L-Phe-OMe as print molecules; curve 4,homogeneous enzymatic reaction, no polymer added.

YE ET AL.: MOLECULARLY IMPRINTED ADSORBENTS 653

polymer, increased the product yield after a certain reactiontime (curves 2 and 3) may be explained by nonspecificbinding of the product to the polymer particles, via hydro-gen bonding to the free carboxyl groups on polymer par-ticles. For shorter reaction times, the synthetic yields of allreactions involving polymers were lower than that of thereaction in which no polymer was used, because the effec-tive substrate concentration was initially decreased by thepolymers utilised. The difference among the three polymersbecame more pronounced after approximately 6 h. Theseresults suggest that the polymers imprinted against the prod-uct enhanced the synthetic yield. Furthermore, the influenceof the product-imprinted polymers on the synthetic yieldagreed well with the recognition behaviour recorded in thechromatographic analyses.

The influence of the substrate concentration on the syn-thetic yield of Z-aspartame was further evaluated. With in-creased amounts of substrates (80 mM Z-L-Asp and 160 mML-Phe-OMe), enhancement of product yield using polymersimprinted against Z-aspartame was not considerable (datanot shown). This is probably due to the low loading capacityof these molecularly imprinted polymers, which makes theproduct adsorption effect negligible. Notably, when theproduct-imprinted polymer was incubated in enzyme-freereaction solvent under identical conditions, no Z-aspartamewas detectable amongst the components separated fromsolid materials, as examined by reverse phase chromatog-raphy. This confirms the complete absence of the print mol-

ecule in the product-imprinted MIP 1, prior to its use in theenzymatic reaction.

For the two equilibrium shifting polymers, MIP 1 andMIP 2, the enzymatic synthesis was evaluated for differentamounts of added polymers (Fig. 3). Thermolysin was in-cubated with various quantities of the two polymers in thesubstrate solution for 48 h, after which time the reactioncomponents were analysed. In the experimental range, itwas found that the difference between the product yieldsusing these two polymers was proportional to the amount ofMIPs added.

In this study we have evaluated the potential for usingmolecularly imprinted polymers to facilitate reversible en-zymatic syntheses. In view of the attractive physical fea-tures displayed by molecularly imprinted polymers, such ashigh pressure and temperature stability, which allow MIPsto withstand sterilisation conditions, we believe that thisnew methodology may find use in various synthetic appli-cations. Although the capacity of the imprinted polymer isstill relatively low, the desired shift towards product forma-tion is quite obvious. Modification of the imprinting proce-dure leading to more macroporous/perfusion type structuresis presently being studied in this laboratory, which willlikely lead to imprinted materials with increased capacities.

References

Andersson LI, Mu¨ller R, Vlatakis G, Mosbach K. 1995. Mimics of thebinding sites of opioid receptors obtained by molecular imprinting ofenkephalin and morphine. Proc Natl Acad Sci USA 92:4788–4792.

Andersson LI, Mu¨ller R, Mosbach K. 1996. Molecular imprinting of theendogenous neuropeptide Leu5-enkephalin and some derivativesthereof. Macromol Res Commun 17:65–71.

Ansell RJ, Kriz D, Mosbach K. 1996. Molecularly imprinted polymers forbioanalysis: Chromatography, binding assays and biomimetic sensors.Curr Opin Biotechnol 7:89–94.

Ansell RJ, Mosbach K. 1998.Magnetic molecularly imprinted polymerbeads for drug radioligand binding assay. Analyst. 123:1611–1616.

Kasai KI, Oda Y. 1986. Frontal affinity chromatography: Theory for itsapplication to studies on specific interactions of biomolecules. J Chro-matogr 376:33–47.

Kempe M, Mosbach K. 1991. Binding studies on substrate- and enantio-selective molecularly imprinted polymers. Anal Lett 24:1137–1145.

Kriz D, Mosbach K. 1994. Competitive amperometric morphine sensorbased on an agarose immobilised molecularly imprinted polymer. AnalChim Acta 300:71–75.

Mosbach K, Ramstro¨m O. 1996. The emerging technique of molecularimprinting and its future impact on biotechnology. Bio/Technology14:163–170.

Nagayasu T, Miyanaga M, Tanaka T, Sakiyama T, Nakanishi K. 1994.Synthesis of dipeptide precursors with an immobilised thermolysin inethyl acetate. Biotechnol Bioeng 43:1108–1117.

Nakanishi K, Kamikubo T, Matsuno R. 1985. Continuous synthesis ofN-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester withimmobilised thermolysin in an organic solvent. Bio/Technology 3:459–464.

Nakanishi K, Kimura Y, Matsuno R. 1986. Kinetics and equilibrium ofenzymatic synthesis of peptides in aqueous/organic biphasic systems,thermolysin-catalysed synthesis ofN-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester. Eur J Biochem 161:541–549.

Nicholls I, Andersson L, Mosbach K, Ekberg B. 1995. Recognition and

Figure 3. Influence of polymer amount on Z-aspartame synthetic yield.A 4-mg amount of thermolysin and stated amounts of polymer were incu-bated in 1 mL of substrate solution (8 mM Z-L-Asp and 16 mM L-Phe-OMe) at 37°C for 48 h. The reaction components were then extracted andmonitored by reverse phase HPLC. Polymers prepared against either theproduct, or the substrates were tested: (d) polymer MIP 1, Z-aspartame asprint molecule; (s) polymer MIP 2, Z-l-Asp/L-Phe-OMe as print mol-ecules.

654 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 64, NO. 6, SEPTEMBER 20, 1999

enantioselection of drugs and biochemicals using molecularly im-printed polymer technology. Trends Biotechnol 13:47–51.

Oyama K, Irino S, Hagi N. 1987. Production of aspartame by immobilizedthermoase. Methods Enzymol 136:503–516.

Ramstro¨m O, Ye L, Mosbach K. 1996. Artificial antibodies to corticoste-roids prepared by molecular imprinting. Chem Biol 3:471–477.

Sakina K, Kawazura K, Morihara K, Yajima H. 1988. Thermolysin-catalyzed synthesis of peptide amides. Chem Pharm Bull 36:4345–4354.

Shea KJ. 1994. Molecular imprinting of synthetic network polymers: The

de novo synthesis of macromolecular binding and catalytic sites.Trends Polym Sci 2:166–173.

Vlatakis G, Andersson LI, Mu¨ller R, Mosbach K. 1993. Drug assay usingantibody mimics made by molecular imprinting. Nature 361:645–647.

Vulfson E, Alexander C, Whitcombe M. 1997. Assembling the molecularcast. Chem Br Jan:23–26.

Wulff G. 1995. Molecular imprinting in cross-linked materials with the aidof molecular templates: A way towards artificial antibodies. AngewChem, Int Ed Engl 34:1812–1832.

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