Analytica Chimica Acta 504 (2004) 53–62

Novel stereoselective molecularly imprinted polymersvia ring-opening metathesis polymerisation

Alpesh Patel, Sandra Fouace1, Joachim H.G. Steinke∗

Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, UK

Received 6 January 2003; received in revised form 21 August 2003; accepted 29 August 2003

Abstract

Stereoselective molecularly imprinted polymers (MIPs) have been synthesised via ring-opening metathesis polymerisation, in essentially,quantitative yield. A covalent imprinting strategy was followed during the network formation of the chiral sorbent. Recognition of the substratehowever involved non-covalent interactions; a combination of hydrogen bonding and the chiral environment presented by the imprinted cavities.The enantiomeric excess achievable with these new MIPs is solvent dependent and stereoselectivities of up to 20% e.e. (separation factorα = 2.2) were found in batch equilibrations.© 2003 Elsevier B.V. All rights reserved.

Keywords: Molecularly imprinted polymers; Ring-opening metathesis polymerisation; Enantioselectivity; Non-covalent interactions

1. Introduction

There has been a considerable increase in research activ-ity on molecularly imprinted polymers (MIPs) world wide[1–8]. In particular, the application of MIPs as molecularrecognition material for separation[7–12] and detection[3,13–16] is at the forefront of academic and industrialinterest. MIPs can be used as selective chromatographicsupport including trace analysis,[17] and the separationof volatile compounds[18]. Imprinted polymers have alsobeen evaluated as active detection layers in drug assays(ELISA) [16,19–21], atmospheric trace analysis (quartzmicrobalance)[18] and as substrate specific, direct-readfluorescence sensors[12,22]. Other applications of MIPsinvolve their synthesis as novel enzyme and antibody mim-ics for the development of new, highly selective and robustsynthetic catalysts[2].

The synthesis of molecularly imprinted polymers effect-ively involves three steps (Fig. 1): firstly, the pre-organisationof a template molecule, appropriately chosen polymerisablebinding sites capable of recognising the template moleculeand crosslinker; secondly, the formation of a polymer net-

∗ Corresponding author.E-mail address: J.steinke@ic.ac.uk (J.H.G. Steinke).1 Present address: Universite de Perpignan 52, Avenue de Villeneuve,

66 860 Perpignan, France.

work and finally, the removal of the template moleculefrom the polymer in order to generate imprinted cavities formolecular recognition. Predominantly free radical polymeri-sation has been the method of choice for network formation,because of the ease of synthesis and the intrinsic functionalgroup tolerance of free radical chemistry, allowing the se-lection of a wide range of template molecules[1,2,6]. Overthe last 30 years, since the introduction of MIPs basedon the enzyme–analogue polymer approach introduced byWulff et al. [1], a significant amount of research activityhas been dedicated to optimise the process of MIP synthe-sis. Essentially, all efforts have been focussed on improvingMIP formation via free radical polymerisation chemistry.Although improvements have been made[1,23,24], one ofthe remaining issues relating to the synthesis of MIPs is theunavoidable formation of polyclonal receptor sites (cavities)[2]. In this context polyclonality means that a distribution ofcavities exist inside the MIP with a wide range of differentselectivities (binding constants) for the substrate, in starkcontrast to enzymes and antibodies that feature exclusivelymonoclonal binding sites (cavities). This leads to undesir-able peak-broadening and cross-selectivity when MIPs arebeing used in chromatographic processes. The consequenceof polyclonality on reactions catalysed by MIPs is poorsubstrate selectivity and low catalytic activity[2].

These considerations highlight the need for new syntheticapproaches for more controlled polymer network formation.

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2003.08.069

54 A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62

Fig. 1. Illustration of the synthesis of molecularly printed polymers.

Although free radical polymerisation is the method ofchoice for MIP synthesis, intrinsically network forma-tion is controlled by kinetics thereby inevitably leading topolyclonal imprinted sites. We decided to investigate thepossibility to adapt a thermodynamically controlled poly-merisation process for the synthesis of MIPs. Up to nowthere have only been very few examples in which, at leastimplicitly, MIPs were synthesised employing thermody-namically controllable network forming reactions. An earlyexample is cross-linking through disulfide bridge forma-tion as demonstrated by Takagishi and Klotz[6] and inmore recent years phase inversion precipitation processes[25–27]. The work on surface imprinted silica by Moriharaet al., (‘footprint catalysis’) stands out, as they developeda synthetic strategy where an annealing step during theimprinting process allows rearrangement of the silica net-work. This led to a substantial improvement of the catalyticperformance of the silica-based MIPs[28,29]. It is notclear why this methodology has not been taken up by otherresearchers as careful optimisation by Morihara et al., re-duced the intrinsic brittleness of the material and producedsilica surfaces that were far less prone to surface rearrange-ments. As was the case with all the other investigationsimproved recognition and catalysis was achieved. However,if the effect was the result of reduced polyclonality, i.e.a narrower distribution of binding site selectivities, or theformation of a larger proportion of cavities with higheraffinity at the expense of the simultaneous formation of alarger proportion of less selective sites was not established.Here we would like to present a new synthetic route toMIPs that employs ring-opening metathesis polymerisation,as a potential means of controlling the formation of MIPsthermodynamically.

2. Experimental

If not stated otherwise, commercially available reagentswere used as received and purchased at the highest avail-able purity.l- and d-menthyl chloroformate, bis(tricyclo)

hexylphosphine benzylidene ruthenium(IV)dichloride (Gru-bbs’ catalyst) were purchased from Lancaster. Exo-N-hyd-roxy-7-oxabicyclo(2,2,1)hept-5-ene-2,3-dicarboximide anddicyclopentene ether from Aldrich. Dicyclopentadiene(Aldrich) was purified by vacuum distillation. Dimethyl-formamide, isopropanol, dichloromethane, triethyl amine(BDH) were used after distillation from anhydrous MgSO4,CaO, P2O5 and KOH, respectively. Tetrahydrofuran and di-ethyl ether were distilled from sodium benzophenone ketyland dioxane and toluene were distilled from sodium.

Manipulation of air and oxygen-sensitive compoundswere carried out under a protective atmosphere of nitrogen.Analytical thin layer chromatography was performed usingpre-coated glass-backed plates (Merck Kieselgel 60 F254)and visualised by ultra-violet light and/or acidic potassiumpermanganate.

Unless otherwise indicated,1H and 13C spectra wererecorded in CDCl3 on Bruker AM 400 MHz, WM 250 MHzNMR or JEOL GSX 270 spectrometers, using residualprotic solvent (CHCl3, dH = 7.26 ppm) or CDCl3 (dC= 77.0 ppm, t) as internal reference. Coupling constantsare measured in Hertz. Infrared spectra were recorded ona Perkin–Elmer 1710 FTIR instrument. Mass spectra wererecorded on VG Micromass 7070E and AutoSpec-Q spec-trometers. Enantiomer analysis was performed on an Agilent4890D GC using a CP Chirasil-DEX CB column (VarianLtd.). Optical rotations were measured using a 1 dm pathlength (c given as gram per 100 ml) on a Perkin–Elmer 141polarimeter, and are reported in units of 10−1 deg cm2 g−1.Melting points were taken on a Kofler hot stage appa-ratus and are uncorrected. Column chromatography wasperformed on Merck Kieselgel 60 (230–400 mesh).

2.1. Synthesis of monomers

2.1.1. Synthesis of 4-(l-menthol)-1,6-heptadienolcarbonate, 4

To a solution ofl-menthol (1.0 g, 6.4 mmol) in dry THFwere added CDI (519 mg, 3.22 mmol) and Et3N (446�l,3.22 mmol). The reaction mixture was stirred at room tem-perature for 1 h then at 60◦C for 3 h. The reaction mix-ture was evaporated. The remaining solid was taken up inDCM/water. The aqueous layer was extracted several timeswith DCM. The combined organic layers were dried overNa2SO4, filtered and the solution concentrated in vaccuo(l-menthol-CO-Im).

NaH (81 mg, 3.2 mmol) was added at 0◦C to a solutionof 1,6-heptadiene-4-ol in dry THF. The reaction mixturewas stirred at room temperature for 3 h. Then a solutionof l-menthol-CO-Im in dry THF was slowly added. Thereaction mixture was stirred at room temperature for 18 h.Excess hydride was quenched with saturated Na2SO4 at0◦C. The reaction mixture was filtered. The filtrate was ex-tracted with DCM/water, the combined organic layers weredried over Na2SO4. The filtrate was concentrated in vaccuoand the remaining residue was purified by chromatography

A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62 55

(hexane:ethyl acetate 98:2 (v/v)).4 was obtained as colour-less oil in 81% yield.

1H NMR (CDCl3): 0.76(d, 3H, CH3–CH, J = 6.9 Hz);0.88(t, 6H, CH3–CH–CH3, J = 6.9 Hz); 0.95–1.08 (m,3H, CH2 of l-menthol, CH3–CH–CH3); 1.33–1.48 (m,2H, CH2 of l-menthol); 1.59–1.66 (m, 2H, CH2 ofl-menthol); 1.90–1.95 (m, 1H, CH3–CH); 1.97–2.04 (m,1H, CH3–CH(CH)–CH3); 2.26–2.43 (m, 4H, 2 CH2–CH=CH2); 4.43–4.53 (m, 1H, CH–O–CO of l-menthol);4.74–4.83 (m, 1H, CH–O–CO of 1,6-heptadien-4-ol);5.05–5.12 (m, 4H, 2 CH=CH2); 5.71–5.81 (m, 2H, 2CH=CH2).

IR (film) ν (cm−1): 1737 (O–CO–O); 2870 (CH2);2928–2955 (CH=CH).

2.1.2. Synthesis of 4-(l-menthol)-3-cyclopentenolcarbonate, 5

A solution of bis-(tricyclohexylphosphine)-benzylidene-ruthenium(IV)dichloride1 (48 mg, 0.058 mmol) in dry DCMwas added to a solution of 4-(l-menthol)-1,6-heptadienolcarbonate4 (685 mg; 2.33 mmol) in dry DCM. The re-action mixture was stirred at reflux for 2 h. Then a solu-tion of bis-(tricyclohexylphosphine)-benzylideneruthenium(IV)dichloride 1 (48 mg; 0.058 mmol) in dry DCM wasadded. The reaction mixture was stirred at room tempera-ture for 12 h then concentrated in vaccuo. The residue wasdiluted with hexane:ethyl acetate 9:1 (v/v) and treated withcharcoal. The reaction mixture was filtered through a plugof silica. The filtrate was concentrated in vaccuo.5 wasobtained as colourless oil in 77% yield.

1H NMR (CDCl3): 0.77(d, 3H, CH3–CH, J = 6.9 Hz);0.88(t, 6H, CH3–CH–CH3, J = 6.8 Hz); 0.95–2.02 (m, 9H,3 CH2 of l-menthol, CH3–CH, CH3–CH(CH)–CH3);2.42–2.48 (m, 2H, CH2–CH=CH2); 2.74 (dd, 2H, CH2–CH=CH2, J = 6.7;17.3 Hz); 4.49 (ddd, 1H, CH–O–CO ofl-Menthol, J = 4.4;10.9;15.3 Hz); 5.23–5.28 (m, 1H,CH–O–CO of 1,6-heptadien-4-ol); 5.69 (s, 2H, CH=CH).

13C NMR (CDCl3): 16.20(CH3); 20.75(CH3); 22.00(CH3); 23.27 (CH2); 26.02 (CH); 31.42(CH); 34.14 (CH);39.66 (2CH2–CH=CH2); 40.84 (CH2); 47.02 (CH); 78.00(CH); 128.06 (CH=CH); 154.75 (CO).

IR (film) ν (cm−1): 1727 (O–CO–O); 2872 (CH2);2930–2957 (CH=CH).

[�D]20: −55.0 (DCM,c = 0.1).

2.1.3. Synthesis of(exo–N-(l-menthol)-4-hydroxy-10-oxa-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione) carbonate, 11-L

4-Hydroxy-10-oxa-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione9 (1.00 g, 5.51 mmol and triethylamine (0.613 g,6.07 mmol) were dissolved in dry dimethylformamide(20 ml). The solution was cooled down to 0◦C andl-menthyl chloroformate10-l (1.41 g, 6.07 mmol) dis-solved in dry dimethylformamide (5 ml) was added drop-wise during 1 h. After addition was complete the solutionwas allowed to stir for a further 3 h at rt. The precipi-

tated triethylamine hydrochloride salt was filtered off andthe filtrate was poured into water (500 ml). The resultingprecipitate was combined with the precipitate obtained bydissolving the earlier isolated triethylamine hydrochloridesalt in water. The combined precipitates were washed sev-eral times with water and dried in vacuo. The crude productwas recrystallised in ethanol:acetone 1:1 (v/v).11-l wasobtained as white fluffy crystals in 92% yield.

1H NMR (CDCl3): 0.78(d, 3H, CH3–CH, J = 6.9 Hz);0.89(d, 3H, CH3–CH–CH3, J = 6.9 Hz); 0.90(d, 3H, CH3–CH–CH3, J = 6, 4 Hz); 0.95–1.19 (m, 3H, CH2 ofl-menthol, CH3–CH–CH3); 1.37–1.52 (m, 2H, CH2of l-menthol); 1.63–1.73 (m, 2H, CH2 of l-menthol);1.90–2.02 (m, 1H, CH3–CH); 2,08–2,16 (m, 1H, CH3–CH(CH)–CH3); 2,86 (s, 2H, 2 CH= CH–CH–O); 4.60 (ddd,1H, CH–O–CO,J = 4.4; 11.1; 15.3 Hz); 5.31 (m, 2H, 2CH–CO–N); 6.51 (m, 2H, CH=CH).

13C NMR (CDCl3): 16.34(CH3); 20.63 (CH3); 21.90(CH3); 23.46 (CH2); 26.15 (CH); 31.50 (CH); 33.90(CH2);40.19 (CH2); 44.34 (2 CH–CO); 46.95 (CH); 80.51 (2CH=CH–CH–O); 83.27 (CH); 136.35 (CH=CH); 148.41(O–CO–O); 168.58 (2CO–N–O). IR (film)→ (cm−1): 1729(O–CO–O); 1810 (CO–N); 2854 (CH2); 2922 (CH=CH).MS (CI, NH4): 381 (M (+18), 60%); 199 (100%); 138(60%); 80 (70%); 67 (85%). mp: 156–158◦C. [�D]20:−33.0 (DCM,c = 0.1).

11-D, the enantiomer of11-L was synthesised in an iden-tical fashion.

2.2. Synthesis of polymers

2.2.1. Synthesis of poly(2-dicyclopenteneether-co-4-(l-menthol)-3-cyclopentenol carbonate), (6)

To a solution of 4-(l-menthol)-3-cyclopentenol carbonate5 (400 mg, 1.50 mmol, 5 mol%) and dicyclopentene ether2(4.28 g, 28.5 mmol; 95 mol%) in dry DCE (3 ml) wasadded at−10◦C a solution of bis-(tricyclohexylphosphine)-benzylideneruthenium (IV)dichloride1 (25 mg; 0.034 mmol;1 mol%) in dry DCM (300�l). The cooling bath was re-moved and the solution was stirred for 5 h. A second1 mol% of1 was added and stirring continued for 36 h at rt.The catalyst was quenched with ethyl vinyl ether (388�l;4.08 mmol) in a solution of chloroform:acetonirile (1:1 v/v,20 ml) for 6 h. The polymer gel was soxhlet extracted withchloroform for 4 h.6 was obtained as a clear and transparentgel in 44% yield.

2.2.2. Synthesis ofpoly(dicyclopentadiene-co-4-(l-menthol)-3-cyclopentenolcarbonate), 8

To a solution of 4-(l-menthol)-3-cyclopentenol carbonate5 (400 mg; 1.50 mmol; 5%) and dicyclopentadiene7 (3.79 g,28.5 mmol, 95%) in dry isopropanol (3 ml) was added at−30◦C a solution of bis-(tricyclohexylphosphine)-benzyli-deneruthenium (IV)dichloride1 (25 mg; 0.03 mmol; 1%)in dry DCM (300�l). The reaction mixture was stirred

56 A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62

vigorously while the solution was cooled to−70◦C. Theampoule was sealed under vacuum. The ampoule was placedin an oven for 36 h at 80◦C. Subsequently the catalystwas quenched with ethylvinyl ether (194�l, 2.04 mmol)dissolved in chloroform:acetonirile (1:1 v/v, 80 ml) (super-natant a) for 6 h. The brittle polymer was soxhlet extractedwith toluene for 4 h and with isopropanol for 4 h (super-natant b). The combined supernatants were evaporated invacuo. The crude residue (456 mg) was purified by chro-matography (hexane:ethyl acetate: 95:5 v/v). 200 mg of4-(l-menthol)-3-cyclopentenol5 were isolated from thecolumn. A solid, brown monolith was obtained in 88%yield. 50% of5 was not incorporated within the polymer.

2.2.3. Synthesis ofpoly(exo–N-(l-menthol)-4-hydroxy-10-oxa-4-azatricyclo[5.2.1.0(2,6)] dec-8-ene-3,5-dione) carbonate, 12

To a solution of exo–N-(l-menthol)-4-hydroxy-10-oxa-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione) carbonate11-l (2 ml; 13.0 mmol) in dry DCE (1 ml) was added slowly at−30◦C a solution of bis-(tricyclohexylphosphine)-benzyli-dene ruthenium(IV)dichloride1 (11 mg; 0,013 mmol) in dryDCE (100�l). After removing the cooling bath the reactionmixture was stirred for 3 h. The catalyst was quenched byadding a solution of ethyl vinyl ether (88�l, 0.92 mmol) inchloroform:acetonitrile (1:1 (v/v), 20 ml) and stirring con-tinued for 6 h. The solvent was evaporated in vacuo. Theremaining solid was dissolved in chloroform:acetonitrile(1:1 v/v) and reprecipitated into hexane. After filtration anddrying 12 was obtained as a viscous liquid in 46% yield.

2.2.4. Synthesis ofpoly(norbornene-co-exo–N-(l-menthol)-4-hydroxy-10-oxa-4-azatri cyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione)carbonate, 13

To a solution of norbornene (104 mg, 1.10 mmol) andexo–N-(l-menthol)-4-hydroxy-10 - oxa-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione) carbonate11-l (100 mg; 0.275mmol) in dry DCE (400�l) was added slowly at−10◦C asolution of bis-(tricyclohexylphosphine)-benzylideneruthe-nium(IV)dichloride 1 (3 mg; 0.004 mmol) in dry DCE

Table 1Batch equilibration results of MIP-ROMP-l for an equimolar mixture ofd- and l-menthol

MIP-ROMP-l Supernatant (mmol) MIP d,l-Menthol Separation factorα-factor

Load. (%) Selec. cav.(%)

Solvent Mentholl (%)

d (%) Mentholl (%)

d (%) Supernatant(mmol)

MIP(mmol)

Hexane 40.1 59.9 59.2 40.8 0.015 0.036 2.2 11.2 2.1Toluene 42.5 57.5 56.1 43.9 0.021 0.030 2.0 9.4 1.3Hexane:DCM (1:1) 44.6 55.4 54.3 45.7 0.015 0.036 1.5 11.2 1.1Hexane:CHCl3 (1:1) 40.1 59.9 58.7 41.3 0.013 0.038 2.1 11.8 2.2DCM 47.2 52.6 52.1 47.9 0.013 0.038 1.2 11.8 0.52-Propanol 49.2 50.8 51.8 48.2 0.023 0.028 1.1 8.7 0.2Standarda 49.6 50.4 n/a n/a 0.051 n/a n/a n/a n/a

a 5.1 × 10−2 mmol of a 1:1 mixture ofl-and d-menthol in 10 ml of solvent.

(50�l). The reaction mixture was stirred at room temper-ature for 3 h. The catalyst was quenched with a solutionof ethylvinyl ether (7.5�l; 0.08 mmol) in chloroform:aceto-nitrile (1:1 (v/v), 15 ml) for 6 h. The crude polymer wasdissolved in chloroform and precipitated into methanol.Copolymer13 was obtained in 44% yield. Template mono-mer 11-l was incorporated at a level of 30% (by NMR).

2.2.5. Synthesis of MIP-ROMP-lTo a solution of exo–N-(l-menthol)-4-hydroxy-10-oxa-4-

azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione) carbonate11-l(300 mg; 0.825 mmol; 5 mol%) and dicyclopentadiene7(2.09 g; 15.7 mmol; 95 mol%) in dry isopropanol:DCM 1:2(v/v) (6 ml) was added at−30◦C a solution of bis (tri-cyclohexylphosphine)-benzylideneruthenium (IV)dichloride1 (14 mg; 0.016 mmol, 1 mol%) in dry DCM (200�l). Thereaction mixture was stirred vigorously while it was cooledto −70◦C. The ampoule was sealed under vacuum. The am-poule was placed in an oven at 80◦C for 36 h. The catalystwas quenched with ethylvinyl ether (106�l; 1.12 mmol) ina solution of chloroform:acetonirile (1:1 v/v, 60 ml) for 6 h.The polymer was soxhlet extracted with toluene for 4 h andthen with isopropanol for 4 h. After drying MIP-ROMP-lwas obtained as solid, beige/brown monolith in 99% yield.

IR (nujol) ν (cm−1): 1741 (O–CO–O); 1788 (CO–N);2854 (CH2); 2923 (CH=CH).

2.2.6. Synthesis of MIP-ROMP-lMIP-ROMP-d was synthesised as described for MIP-

ROMP-l using11-d instead of11-l.

2.2.7. Synthesis of MIP-ROMP-dlMIP-ROMP-dl was synthesised as described for MIP-

ROMP-l using 2.5 mol% of11-d and 2.5 mol% of11-linstead of 5 mol%11-l.

2.3. Template removal

MIP-ROMP-X (X = l, d or dl) (3.00 g, equivalentto 0.371 g, 1.03 mmol of3-l) was suspended in dioxane(20 ml) and allowed to swell for 3 h. Then triethyl amine

A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62 57

Table 2Batch equilibration results of MIP-ROMP-X for an equimolar mixture ofd-and l-menthola

Polymer Supernatant MIP d,l-Menthol Separation factorα-value

Mentholl (%)

d (%) Mentholl (%)

d (%) Supernatant(mmol)

MIP(mmol)

MIP-ROMP-l 40.1 59.9 58.7 41.3 0.013 0.038 2.1MIP-ROMP-d 58.7 41.3 40.8 59.2 0.012 0.039 2.1MIP-ROMP-dl 49.6 50.4 49.4 50.6 0.013 0.038 1.0Pre-equilibration 49.6 50.4 n/a n/a 0.051 n/a n/a

a 5.1 × 10−2 mmol of a 1:1 mixture ofl- and d-menthol in 10 ml of hexane:chloroform (1:1 (v/v)).

(1.47 ml, 10.3 mmol) and hexyl amine (1.36 ml, 10.3 mmol)were added. The suspension was stirred for 12 h beforethe polymer was filtered and washed with dioxane, thendichloromethane and finally soxhlet extracted with chloro-form for 12 h.

The organic extracts were combined and evaporated todryness in vacuo. The product, then-hexyl carbamate ofl-(d- or d/l) menthol was obtained as white crystals. Af-ter prolonged drying in vacuo the crystals were accuratelyweighed and their purity confirmed by NMR and IR spec-troscopy.

2.4. Batch equilibrations

MIP-ROMP-X (X = l, d or dl) (1.0 g) was suspendedin the incubating solvent (seeTables 1 and 2) (10 ml con-taining 5.1 mmol of an equimolar ratio of3-l and3-d). Thesuspension was stirred for 24 h. The polymer was separatedfrom the supernatant and the latter dried and weighed priorto GC analysis. The MIP was soxhlet extracted with chloro-form for 12 h. The extract was also dried and weighed priorto analysis. Each sample was dissolved in ethyl acetate toobtain concentrations of 0.2–0.3%, w/v for GC analysis.

3. Results and discussion

Metathesis is a reversible carbon–carbon bond formingprocess that in recent years has become widely used forring-closing, ring-opening and cross metathesis reactions.Depending on the reaction conditions such as, concentrationof reactants and temperature the position of the equilibrium(open chain versus. cyclised product) can be controlled(Fig. 2) [30,31]. If one could demonstrate that it is possibleto synthesise MIPs via a metathetic process, then the possi-

OH O O

O

O O

O(i, ii) (iii)

3-L 4 5

Fig. 3. Synthesis of template monomer5. Synthesis of cyclopentenol-l-menthol carbonate7: (i) CDI, NEt3, THF, 60◦C, 4 h; (ii) (1,6-heptadiene-4-ol,NaH, in THF) added, then rt, 18 h, 81%; (iii) Grubbs’ catalyst, DCM, reflux, 2 h, 77%.

RuClCl

PCy3

PCy3

Ph

ROM

RCM ROMP

n

Grubbs' catalyst

CM +

1

Fig. 2. Synthetic pathways of metathesis reactions typically employingGrubbs or Schrock alkylidene catalysts. RCM: ring closing metathesis;ROM: ring-opening metathesis; ROMP: ring-opening metathesis poly-merisation; CM: cross metathesis.

bility of forming such a network under equilibrating condi-tions could be used to prepare less polyclonal MIPs. Recentdevelopments in ring-opening metathesis polymerisation(ROMP) catalysis[30] and the advent of effective prepa-ration procedures for highly cross-linked ROMP networks[32,33], convinced us that it should be synthetically feasibleto develop network formation via ROMP into a methodol-ogy for thermodynamically controlled MIP synthesis.

Our initial model system was based on cyclopentenemoieties as slightly strained ring systems suitable forring-opening metathesis polymerisation. Rather than opt-ing for norbornene-derived monomers it was thought thatthe lower ring strain in cyclopentene rings would resultin slower polymerisation kinetics and consequentially al-lowing the polymer network to form closer to equilibriumconditions. Grubbs’ catalyst,1, was selected because of itswell-established reactivity and functional group tolerance inmetathesis reactions including ROMP (Fig. 2) [30]. In orderto maximise compatibility of the template monomer with the

58 A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62

O O

O

O O

O

O

O

O

O

O

O5 +

(iii)

5

(ii)

+

n m

n m

O O

O

5

(i)SM

Fig. 4. Homopolymerisation and copolymerisation of5: (i) 0.3 mol% 1, DCE, rt, 12 h, starting material; (ii) 20 mol%7, 80 mol% cyclopentene, 0.1 mol%1, DCE, −10◦C → rt, 3 h, 46%l; (iii) norbornene instead of cyclopentene otherwise as in (ii) 66%.

catalyst, a covalent imprinting strategy was adopted anal-ogous to Whitcombe et al.,[34]. We opted for L-menthol,3-l, as the template molecule because we expected it tosimplify the analysis of substrate recognition. The presenceof only a single hydroxyl group in3-l allowed us in only2 steps to link it to a cyclopentene moiety.3-l was reactedwith CDI to form the corresponding imidazolide3, whichwas converted in situ to diallyl ether4 [35]. Ring-closingmetathesis of4 using Grubbs’ catalyst1 afforded targettemplate monomer5 in 63% overall yield (Fig. 3).

Before the synthesis of MIPs, homo and copolymerisa-tion behaviour of2 and 5 were evaluated in the presenceof 1 to ensure successful incorporation of crosslinker andtemplate monomer during the synthesis of the correspond-

k

O

O

O

O

O(i)

n

6

O O

O

5

+

O

O

O

(ii)

n

8

(iii)

OH

k

n

k

O O

O

5

+

7

2

m

mm

Fig. 5. Synthesis of cross-linked polymer networks through copolymerisation of5 with 2 and7: (iii) 5 mol% 5, 95 mol%2, 0.1 mol%1, DCE, −10◦C →rt, 5 h, 0.1 mol%1, 36 h, 44%; (iv) 5 mol%5, 95 mol%7, 0.1 mol%1, DCM/2-propanol, 80◦C, 36 h, 88%, (iii) various conditions, 5% (see text for details).

ing MIPs (Fig. 4). Surprisingly5 did not homopolymerise,though copolymerisation with cyclopentene and norbornenewas successful (Fig. 4). Based on these results we choose2-cyclopentene ether,2, as a suitable crosslinker since itsreactivity was expected to be similar to that of5 (Fig. 5).Homopolymerisation of2 yielded a transparent gel withless than 50% conversion at room temperature. Copolymeri-sation of2 and 5 at a molar ratio of 19:1 in the presenceof catalytic amounts of1 also led to the formation of a gel,6. Despite several modifications of the synthetic protocol itwas not possible to obtain6 with conversion of more than50% (Fig. 5). Thus it was necessary to replace2 with a morereactive crosslinker, dicyclopentadiene7 [32,33]. Indeedwith 7 improved conversion was achieved by carrying out

A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62 59

the polymerisation at 80◦C for 36 h leading to polymer8 in88% yield (Fig. 5). Through model studies with the templatemonomer5 we had identified potassium trimethylsilanoatein diethyl ether as the most effective reagent leading to aclean and quantitative hydrolysis of the carbonate bond atroom temperature within 12 h. However hydrolysis of thecarbonate group functionality incorporated in8 proved tobe much more difficult than anticipated. In fact, only 5%of the total amount of3-l available on the polymer couldbe recovered despite the use of excess reagent, prolongedreaction times and elevated temperatures.

This result suggested that a template monomer wasneeded that could be hydrolysed more easily and ideallyshould be of similar reactivity to 7 to ensure a statisticalincorporation into the polymer network. A long searchled us to tricyclic norbornene derivative9 in which theN-hydroxyimide functionality was expected, based on lit-erature precedence, to accelerate the hydrolysis of theN-carbonate moiety[35–37]. Template monomer11-l wassynthesised directly from commercially available tricycle9and the chloroformate ofl-menthol10-l (Fig. 6).

It was possible to obtain homopolymer12 from 11-l andits copolymer13 with norbornene in satisfactory yield atroom temperature (Fig. 7). 11-l was also more susceptibleto hydrolysis as was evidenced by the almost quantitativehydrolysis of copolymer13 using an excess of potassiumtrimethylsilanoate in THF.

By substituting norbornene for7, we attempted again tosynthesise the corresponding MIPs. As was the case fornetwork 8, we again followed the reaction conditions es-

11-L +

(ii)

n

12

O O

O

n

N

OO

O

13

(i)

O O

ON

OO

O

O

O

O

N OO

O

O

O

O

11-L

n

N OO

O

Fig. 7. Homopolymerisation of11-l and its copolymerisation with norbornene: (i) 0.1 mol%1, DCE,−10◦C → rt, 1 h, 46%; (ii) 20 mol%11-l, 80 mol%norbornene, 0.1 mol%1, DCE, −10◦C → rt, 3 h, 44%.

O X

O

O O

ON

(i)

10-L 11-L

OO

O

Fig. 6. Synthesis of cyclopentenol-exo-N-hydroxy-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboximide carbonate11-l. (i) 9, NEt3, DMF, rt, 18 h,92%.

tablished by Mühlebach et al.[32] (Fig. 8). After 36 h at80◦C the catalyst was quenched with ethylvinyl ether. Alightweight and brittle solid was obtained in 99% yield af-ter soxhlet extraction and subsequent drying. This polymerwas shown to swell 66% in CHCl3, 90% in THF and 25% inEtOH despite its highly cross-linked nature, a behaviour typ-ically encountered for networks of this kind[33,38]. Onceagain hydrolysis on the cross-linked polymer proved moredifficult than on the linear model copolymer13. Incompletehydrolysis (only 30% of3-l was recovered) necessitatedlengthy optimisation which eventually led to the use of a10-fold excess of n-hexylamine and triethylamine resultingin the removal of more than 90% (92–94%) of all avail-ablel-menthol from the polymer network (MIP-ROMP-l)(Fig. 8).

Selectivity of MIP-ROMP-l was evaluated in a competi-tive batch mode binding assay to provide selectivities underequilibrium conditions[39]. For this purpose aliquots ofMIP-ROMP-l were suspended in a selection of solvents

60 A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62

O O

ON

OO

O

O

O

O

11-L

N OO

O

+ n

n

MIP-ROMP-L

m

(i)k

OH

m

N OO

O

k

(ii)

rebinding

(iii)

n

O

m

N OO

O

k

O

HH

7

Fig. 8. Synthesis of MIP-ROMP-l: (i) 5 mol% 11-l, 95 mol% dicyclopentadiene, 0.1 mol%1, DCM:2-propanol, 80◦C, 36 h, 99%; (ii) 10 mol eq. NEt3,10 mol eq. n-hexylamine, dioxane, rt, 12 h, 93%; (iii) for detailsTables 1 and 2.

(Table 1) containing equimolar amounts of3-l andd-menthol (3-d) and stirred for 24 h. This time frame wassufficient for the recognition process to reach equilibriumas the selectivity data for equilibrations carried out for 48 hwas found to be identical. In all cases the supernatant wascollected and the filtered polymer extracted exhaustivelyfirst with the solvent used for the batch procedure then withchloroform. The supernatant and the extract collected fromMIP-ROMP-l post equilibration were analysed by chiralGC. The results are summarised inTable 1.

As anticipated the template3-l with which MIP-ROMP-lhad been imprinted was bound by MIP-ROMP-l preferen-tially. In fact with hexane as solvent the final compositionon the polymer was almost 60% of3-l and 40% of3-d,equivalent to an enantiomeric excess (e.e.) of almost 20%.With increasing solvent polarity though discrimination be-tween the enantiomers decreased and was poorest in the caseof 2-propanol. This general trend suggests that 2-propanolcompetes quite successfully with the template molecule toform hydrogen bonds with the availableN-hydroxyimidegroups. This is also reflected in the ratio of bound to freementhol, which for most solvents is 70:30 but drops to 55:45when the equilibration is carried out in 2-propanol. Knowingthe concentration of menthol on the polymer and in solutionenabled us to calculate a separation factor (α-value). Herewe found values for MIP-ROMP-l as high as 2.2 with about10% of all available cavities being occupied by3-l and3-dat the same time (Table 1). This loading level indicates that

a substantial number of cavities available for binding are se-lective towards the template monomer. Menthol imprintedpolymers had already been prepared a few years earlier.Nedeljkovic et al., employed a racemic mixture of mentholas template for non-covalently imprinted MIPs polymerisedby �-irradiation [40]. They claim their menthol-imprintedMIP absorbs 22% more menthol when compared to theblank. Imprinting with menthol enantiomers via the sol–gelprocess resulted in MIPs with improved recognition com-pared to their blank analogues but they were not stereose-lective as shown by Pinel et al.[41]. Percival et al., preparednon-covalentlyl-menthol imprinted MIPs as sensing lay-ers for a polymer-coated quartz crystal microbalance[18].A substantial preference for the template molecule over itsenantiomer results in a sensitivity enhancement by a factorof three. Clearly these MIPs are selective towardsl-mentholthough a direct comparison with our data is not possible be-cause from the data presented it is not possible to establishthe number of occupied sites, which is required to calculateα-values. That our data is based on a competitive bindingassay is a further complicating factor because the simultane-ous binding of the template enantiomer to the polymer willinevitably lead to a different distribution of occupied cavi-ties [18] based on the difference in affinity compared to thesame assay carried out with only one enantiomer.

It is interesting to note that the chiral recognition eventseems to occur as a result of a single accurately positionedalcohol functionality within a chiral cavity. The three-point

A. Patel et al. / Analytica Chimica Acta 504 (2004) 53–62 61

O O

ON

OO

O11-D

+MIP-ROMP-D

(i,ii)

O O

ON

OO

O11-L

+MIP-ROMP-DL

(i,ii)

O O

ON

OO

O11-D

+

7

7

Fig. 9. Synthesis of MIP-ROMP-d and MIP-ROMP-dl. For (i) and (ii)seeFig. 8((i) and (ii)).

binding model requires two additional loci for chiral dis-crimination which we rationalise by assuming that the cavityitself is a chiral entity playing its part in the enantioselectiverecognition process[7,42].

For further evidence that the observed chiral discrimi-nation is the result of chiral cavities through the presenceof template11-l, we synthesised two additional MIPs re-lated to MIP-ROMP-l, MIP-ROMP-d and MIP-ROMP-dl.MIP-ROMP-d was synthesised using the enantiomer ofthe template monomer,11-d, whereas MIP-ROMP-dl wassynthesised using an equimolar mixture of11-l and 11-d(Fig. 9). Both MIPs were otherwise identical to MIP-ROMP-l, which was even reflected in essentially identical levelsof monomer conversion and amount of extracted template.MIP-ROMP-d exhibited the same level of chiral discrim-ination for its template (11-d) than had been found forMIP-ROMP-l. MIP-ROMP-dl on the other hand lacked anypreference for either11-l or 11-d (Table 1). The templaterecognition exhibited by these two polymers demonstrateunequivocally that the observed stereoselectivity is a resultof chiral cavities containing single hydroxyl groups avail-able for enantioselective hydrogen bonding interactions.

4. Conclusions

The synthetic methodology for the preparation of cova-lently imprinted MIPs has been demonstrated for the first

time. Through optimisation of monomer design, reactionand template removal conditions it was possible to preparehighly cross-linked polymer networks with >99% conver-sion and template recovery of >90%. These MIPs wereshown to be highly enantioselective with values for theenantiomeric excess-after one competitive batch equilibra-tion reaching 20%. Depending on the solvent separationfactors of up to 2.2 were obtained. We are now investigatingreaction conditions and catalysts to influence the selectivitydistribution of the cavities found in these MIPs with theaim to reduce polyclonality.

Acknowledgements

We like to thank Dr Alan Armstrong and Fred Goldbergfor access to and their assistance in the GC analyses and DrGuy Clarkson for helpful discussions. Funding by the Engi-neering and Physical Sciences Research Council (EPSRC),UK, is gratefully acknowledged.

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