preparation of amino acid ester-selective cavities formed by non-covalent imprinting with a...

Reactive Polymers, 9 (1988) 29-41 29 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

STATE-OF-THE-ART REPORT

PREPARATION OF A M I N O ACID ESTER-SELECTIVE CAVITIES F O R M E D BY N O N - C O V A L E N T

I M P R I N T I N G WITH A S U B S T R A T E IN HIGHLY C R O S S - L I N K E D P O L Y M E R S *

LARS ANDERSSON

Ferring Pharmaceuticals, P.O. Box 30561, S-200 62 Malm6 (Sweden)

Methods explored in order to make molecular imprints with phenylalanine ethyl esters in styrene and acrylic polymers using a non-covalent imprinting technique are described. Amino acid ester-selective polymers were prepared after ion-pair association of substrate (print molecule) and carboxyl-containing monomers in the polymerization step. A high concentration of cross-linking agents (divinylbenzene or ethylene glycol dimethacrylate) was present in the polymerization mixtures in order to produce macroporous polymers of high rigidity. After polymerization, polymers were washed free from print molecules and the substrate-selective polymers formed were examined by incubation of the polymers with racemic mixtures of doubly radiolabelled phenylalanine ethyl ester. Binding experiments in batch and chromatographic procedures have been performed. It was found that separation factors determined for polymers prepared in the presence of D- or L-phenylalanine ethyl ester were different from one, indicating that the polymers formed were chiral. It was also found that the polymers interacted preferentially with the enantiomer of the print molecule present in the polymerization mixture. Finally, a synthetic procedure has been worked out for making new amino acid-based cross-linkers suitable for preparation of substrate-selec- tire acrylic polymers. The method utilizes modification of aminoalcohols by acrvloyl chloride substitution.

INTRODUCTION

The preparation of polymers containing chiral cavities formed by imprinting with substrate (template or print molecule) present during polymerization has received great at-

* Paper presented at the Microsymposium on Macro- molecules "Polymer-supported Organic Reagents and Catalysts", Prague, Czechoslovakia, July 6-9, 1987.

tention during the last few years [1,2]. For example, substrate-selective binding sites containing functional groups stereospecifically attached to the interior of the cavities have been made in styrene [3-5] and acrylic polymers [6,7], in silica [8] and polysiloxane- coated silica [9,10], and in starch [11]. Molec- ular imprinting has been proven possible with several different compounds, and small molecules such as carbohydrates [12], dyes [6,8,13] and amino acid derivatives [7,14-16] have

0167-6989/88/$03.50 © 1988 Elsevier Science Publishers B.V.

30

been employed successfully as templates in the polymerization process. Moreover, interesting studies of applications of substrate- selective polymers, such as resolution of racemic mixtures [17], stereoselective control of organic chemical reactions [4,18] and complex formation with complementary metal ions [19,20], have been reported, demonstrat- ing the potential of this new technique in chemistry. However, for optimal utilization of the imprinting technique or template synthesis there is a need for binding sites showing improved recognition at the molecular level. Also, it would be desirable to know in more detail the factors governing the formation of asymmetric binding sites in print polymers during imprinting. Recently, studies aiming at this have been carried out, and relationships between substrate selectivity and positioning of functional groups at the polymer binding sites have been explored [10,21].

Different experimental approaches have been used for the formation of polymers showing molecular recognition. In template synthesis, usually associated with covalent imprinting, template molecules carrying vinyl groups are copolymerized with other vinyl monomers present in the reaction mixture. After polymerization template molecules are split off from the polymer, generally by hy- drolysis of covalent bonds attaching template to polymer (Fig. 1A), and the polymers thus prepared contain cavities complementary to the print structure with respect to size, shape and functional groups. This procedure, elegantly shown in several studies [3-5], re- quires that template molecules are modified by vinyl substitution before polymerization. However, sometimes it can be difficult to perform such template modifications because of the complex chemistry of the substitution reaction.

An alternative method of preparing substrate-selective polymers would be to utilize non-covalent imprinting with substrate in highly cross-linked polymers. Here we wish to

o,rme..at,on

Cleavage of covalent bonds [ between substrate and polymer

~ S u b s t r a t e ~ ? . •

® olymerlzation 4;-

Extraction T

Fig. 1. Two principal modes of imprinting with substrate in highly cross-linked polymers. (A) Covalent method. (B) Non-covalent method.

discuss such an approach. (Material describ- ing this has been taken from three short articles on molecular imprinting with amino acid derivatives [7,22,23].) Using this method, substrate and monomers are allowed to preorganize prior to polymerization and form non-covalent bindings as a result of, for example, ionic, hydrophobic, charge transfer or hydrogen bond interactions (Fig. 1B). Thus, by this procedure, exploiting the interaction of complementary binding sites of substrate and monomers during polymerization, it would be unnecessary to attach vinyl func- tionalities covalently to the template before polymerization as well as to cleave covalent bonds to remove polymer-bound template molecules after polymerization.

EXPERIMENTAL

Materials

p-Vinylbenzoic acid (PVB) was purchased from Polyscience Inc. (Warrington, PA).

Styrene, divinylbenzene (DVB), acrylic acid, acrylamide, acryloyl chloride, ethylene glycol dimethacrylate and 2,2'-azobisisobutyronitrile (AIBN) were bought from Merck (Darm- stadt). D- and L-phenylalanine, o- and L- tryptophan and benzylamine hydrochloride were purchased from Sigma (St. Louis, MO) and O-tert-butyl-L-serine methyl ester hydrochloride from Nova biochem (L~iufelfingen). L-[C-2,C-3,3H]phenylalanine (21 Ci/mmol), L-[C-2,C-3,3H]tryptophan (40-60 Ci/mmol) and benzylamine [C-l,~4C]hydrochloride (50--60 mCi/mmol) were obtained from Amersham and [C-l ,14C]ethanol (21 mCi/mmol) from NEN (Boston). All other chemicals were of reagent grade and used without further purification.

Preparation of amino acid derivatives

Amino acid ethyl esters were prepared as described by Greenstein and Winitz [24]. Ra- diolabelled esters were made using either 3H- labelled L-amino acids and "cold" ethanol or cold D-amino acids and a4C-labelled ethanol. The compounds synthesized were all at least 95% pure as judged by thin-layer chromatography (TLC), UV, 1H-NMR and optical rota- tion measurements. Prior to use in the batch experiments, radiolabelled 0- and L-phenylalanine ethyl esters were chromatographed on columns packed with CM-Sephadex C-25 (1.8 × 23 cm containing 58.5 ml gel). Elution of the esters was effected by a linear gradient of 0-0.3 M NaC1 (total volume 2 1) in 5 m M sodium phosphate, pH 7.4. Amino acid esters synthesized here were applied as their free bases in the imprinting experiments and the subsequent binding studies.

N-Acetylation of D-[14C], L-[aH]phenyl - alanine ethyl ester was performed by treating the doubly radiolabelled, racemic ester with acetic anhydride [25]. L-[3H]phenylalanine benzylamide and D-phenylalanine [14C]benzylamide were synthesized by condensing "hot" tert-butyloxycarbonyl (Boc)-protected

31

phenylalanine (BocPheOH) and cold benzylamine and vice versa using N,N'-dicyclohe- xylcarbodiimide as coupling reagent in the presence of 1-hydroxybenzotriazole [26]. The Boc protecting group was introduced after acylation of the a-amino group of phenylalanine with di-tert-butyl carbonate [27], and it was removed by brief treatment (30 min, room temperature) with trifluoracetic acid (TFA).

Specific radioactivities and R F values determined for the radiolabelled amino acid derivatives synthesized in this study are shown below. The radioactivity was measured in a liquid scintillation counter (LKB 1217 rackbeta) and the R v values were estimated after TLC on silica in chloroform/methanol (10: 1, v/v). D-Phenylalanine [C-l,14C]ethyl ester, 0.68 × 10 -2 /zCi//~mol, R v = 0.77; L- [C-2,C-3,3H]phenylalanine ethyl ester, 2.0 × 10 2/~Ci//~mol; D-tryptophan [C-1,14C]ethyl ester, 3.4 x 10 -2/~Ci/~mol, R v = 0.56; L-[C- 2,C-3,3H]tryptophan ethyl ester, 1.32 × 10 -2 ~Ci//~mol; N-acetyl-D-phenylalanine [C- 1,14C]ethyl ester, 0.45 × 1 0 - 2 ~Ci//~mol, R F = 0.85; N-acetyl-L-[C-2,C-3,3H]phenylalanine ethyl ester, 0.68 x 10 -2 #Ci//amol; D-phenylalanine [C-l,14C]benzylamide, 10.7 x 10 -2 ~Ci//zmol, R v = 0.35; L-[C-2,C- 3,3H]phenylalanine benzylamide, 1.39 × 10 -2 /aCi/~mol.

Preparation of print polymers

Styrene polymers Styrene polymers were prepared using p-

vinylbenzoic acid (acid form; 0.111 g, 0.75 mmol), D- or L-phenylalanine ethyl ester (free base; 0.145 g, 0.75 mmol), technical divinylbenzene (3.442 g, equivalent to about 14.2 mmol of cross-linking molecules as technical divinylbenzene contains only 53.9% divinylbenzene [12]) AIBN (0.035 g) and acetonitrile (4.8 ml, 3.74 g). Polymerization mixtures prepared in the absence of print molecules or in the presence of racemic phenylalanine ethyl

32

ester were also employed (to make reference polymers). Prior to polymerization, the mixtures, placed in glass tubes, were degassed. The tubes were sealed under nitrogen and then heated at 60°C, 90°C and 120°C for 24 h at each temperature. After polymerization, the polymers were ground and washed continuously with acetonitrile overnight employing a Soxhlet extractor. Finally, the polymers were dried at 60 ° C overnight and stored dry.

Acrylic polymers Polymerization mixtures were prepared by

dissolving ethylene glycol dimethacrylate (5.643 g, 28.5 mmol), acrylic acid (0.216-0.432 g, 3.0-6.0 mmol), D- or L-phenylalanine ethyl ester (0.289 g, 1.5 mmol) and AIBN (0.062 g) in acetonitrile (8.24 ml, 6.43 g). Reaction mixtures containing no amino acid ester were utilized to make reference polymers. Poly- merization and working up of the polymers formed were as described for the preparation of the styrene polymers.

Recovery of print molecules The amount of print molecules recovered

from the polymers after polymerization was estimated by analyzing polymers imprinted with tritium-labelled t-phenylalanine ethyl ester instead of the unlabelled print substrate normally used for imprinting. The regain of ester molecules was determined by analyzing samples (50 mg) of the polymers before and after the Soxhlet extraction using liquid scintillation counting following total combustion of the polymers in a Packard Tricarb oxidizer. In addition to measuring the radioactivity of the polymers per se, the radioactivity of the acetonitrile extracts containing the recovered tritium-labelled print molecules was determined. Moreover, amino acid analysis of polymer samples hydrolyzed in 6 N HC1 for 24 h at 110°C have been performed. Re- coveries determined by the latter method agreed well with those found by the radio- analyses.

Investigation of the print polymers

Binding studies were carried out to examine the substrate selectivity of the polymers formed.

Batch experiments Racemic mixtures of phenylalanine ethyl

ester containing roughly 5 ~mol each of the a4C-labelled D-form and the 3H-labelled L- form were applied to 1 g (dry weight) of print polymer in 8 ml of acetonitrile (dried over molecular sieves). Prior to incubation, the assay mixtures were sonicated and then left with gentle agitation for at least 24 h at room temperature. Following centrifugation, the radioactivities of the supernatants were measured.

Chromatographic experiments Columns for high-performance liquid chro-

matography (HPLC) were used, and in order to obtain particles of uniform size, the polymers were milled and sieved under water in a Resch sieve prior to column packing. Frac- tions containing polymer particles of 45-65 ~m were collected and stainless-steel columns (200 × 5 mm) were packed with the fractionated material (0.8 g) suspended in equal amounts of methanol and 50% (w/v) aqueous sucrose [22]. Chromatography was per formed at 50 ° C and the eluent, acetonitrile, was pumped through the column at a flow rate of 15-80 ~l /min. At each HPLC experiment roughly 0.2-0.6 /zmol of racemate were applied to the substrate-selective c o l u m n s p r e - e q u i l i b r a t e d wi th acetonitrile. In the HPLC studies, elution of substrate was followed by recording the ab- sorbance of the eluate at 257 nm or 280 nm and by measuring the 3H- and 14C-content of the eluate in a liquid scintillation counter.

Synthesis of amino acid-based acry#c-functional cross-linkers

The model synthesis of N,O-bisacryloyl-L- phenylalaninol is described here.

L-Phenylalaninol (t-2-amino-3-phenyl-l-propanol

To cold 30% (v/v) aqueous ethanol (84 ml) containing L-phenylalanine ethyl ester hydrochloride (21.6 g, 94 mmol), sodium borohydride (17.8 g, 470 mmol) was added in portions. The reaction was allowed to proceed at 4°C for 24 h. The precipitate formed was removed by filtration, and the pH of the filtrate was adjusted to about 3 with HC1. This solution was left with stirring overnight to destroy remaining sodium borohydride and then taken down to dryness. The residue obtained was dissolved in 0.2 M NaHCO 3 and the pH of the mixture was adjusted to 9.3 with NaOH. The solution was extracted with n-butanol (2 × 250 ml) and the organic phases were pooled. Butanol was evaporated and the product isolated was dried over P205 under vacuum. The dry material was mixed with diethyl ether and the mixture was refluxed for a couple of hours. Remaining non-dissolved material was removed by filtration and the ether was evaporated until the product precipitated out. L-Phenylalaninol, a white crystalline substance, was afforded by filtration and dried over P205 under vacuum. Yield: 50.9 mmol (7.7 g, 54%); R v = 0.8 on cellulose in n-butanol/acetic acid/water (5 : 2: 3, v/v); m.p. 81-83°C; [ a ] ~ = - 1 7 . 5 ° (C=0 .57 in methanol).

N,O-bisacryloyl-t-phenylalaninol (N,O-bisacryloyl-L-2-amino-3-phenyl- l-propanol)

To a solution containing L-phenylalaninol (2.5 g, 16.6 mmol) in ice-cooled N,N-dimeth- ylformamide (DMF) (15 ml), acryloyl chloride (3.0 ml, 36.5 mmol) and triethylamine (5.11 ml, 36.5 mmol) were added in five equivalent portions. After reaction overnight (room temperature) the salt formed was removed by filtration and the filtrate was di- luted with ethyl acetate (250 ml). After washing with 0.5 M NaHCO 3 (2 × 20 ml), 0.5 M citric acid (2 × 20 ml) and water (2 × 20 ml),

33

the organic phase was dried (Na2SO4) and the solvent was evaporated. The residue obtained as an oil was dissolved in methanol (10 ml) and by adding water (100 ml) to the methanol solution the product precipitated out as a slightly yellow substance. This material was isolated by filtration and dried over P205 under vacuum. Yield 3.3 mmol (0.8 g, 20%); R F = 0.73 in chloroform/methanol ( 1 0 1 , v/v); m.p. = 102°C; [~]22 = _28.1 ° (C =- 0.43 in methanol). 1H-NMR (in CDC13) 82.9 (2H, m, -CI-I2-C6H5); 64.2 (2H, d, J = 4.5 Hz, -OCH2-) ; 84.5 (1H, m, > CH-); 85.6-6.6 (7H, m, CH2= CH-CO-N__H and CH2= CH-CO-O) ; 87.3 (5H, m, C6__H5). Elemental analysis: C, 68.9 (69.5); H, 6.54 (6.56); N, 5.32 (5.41); O, 19.0 (18.5); calcu- lated values within parentheses.

Molecular imprinting in polymeric 17phenyl- alaninol

Using acrylic-based monomers derived from L-phenylalaninol two print polymers (P1 and P2) and one reference polymer (P3) were prepared. The composition of the polymerization mixtures were: P1, N,O-bisacryloyl-L- phenylalaninol (1.02 g, 3.9 mmol), acrylic acid (0.029 g, 0.41 mmol), AIBN (0.019 g), L-phenylalanine ethyl ester (0.04 g, 0.21 mmol) and acetonitrile (2.64 ml, 2.06 g); P2, N,O-bisacryloyl-L-phenylalaninol (0.31 g, 1.18 mmol), acrylic acid (0.012 g, 0.169 mmol), acrylamide (0.024 g, 0.338 mmol), AIBN (0.006 g), L- phenylalanine ethyl ester (0.016 g, 0.085 mmol), acetonitrile (0.62 ml, 0.48 g); P3, the same composition as that used for P1 but without print molecules added to the polymerization mixture. Polymerization and investigation of the polymers prepared were carried out in much the same manner as described above to make imprints with amino acid ethyl esters in styrene and acrylic polymers.

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RESULTS AND DISCUSSION

Preparation of phenylalanine ethyl ester-selective polymers by non-covalent imprinting

Figure 2 illustrates schematically the strategy used to prepare phenylalanine ethyl ester-selective polymers by the non-covalent imprinting method. A print molecule, D- or L-phenylalanine ethyl ester, is present at a low concentration in a mixture containing, in addition to print molecules, vinyl monomers, initiator and acetonitrile. During polymerization, it is envisaged that substrate molecules would interact preferentially with carboxyl- containing monomers and form ion pairs con- sisting of positively charged amino groups of substrate and oppositely charged carboxylates of carboxyl-containing monomers. After polymerization, loosely bound print molecules are

washed from the polymers, and the polymers obtained now contain imprints of added print molecules. Moreover, the so-formed cavities, shaped according to the print structure, are derivatised with carboxyl groups that can interact stereospecifically with the amino func- tion of rebound phenylalanine ethyl ester.

Two different kinds of phenylalanine ethyl ester-selective polymers have been made in this work. Polymers were prepared from either styrene- or acrylic-based monomers using a very high concentration of cross-linking agents (divinylbenzene or ethylene glycol dimethacrylate, 56 and 83-91 mole% of added monomers, respectively) to yield macroporous polymers of high rigidity. It should be mentioned that equal amounts of substrate and counter ion (p-vinylbenzoic acid) were used to produce styrene polymers, and for the preparation of acrylic polymers the concentra-

PVB

O I

H2N-CH-C--O-CH2--CH 3 I

2 L-PheOEt

ORGANIC. SOLVENT lov COPOLYMERIZATION

O O . ~ ~'~'-C-O H3N-CH-C--O-CH2--Ci-~ ~

H CONTINUOUS EXTRACTION

I INCUBATION D,L-PheOEI

C-OH

Fig. 2. Schematic il lustration showing non-covalent imprint ing with L-phenylalanine ethyl ester in polystyrene. PVB = p-vinylbenzoic acid; PheOEt = phenylalanine ethyl ester; DVB = divinylbenzene. (Reprinted with permission from Ref. [7].)

tion of acrylic acid ranged from two to four times the concentration of the interacting substrate. Print molecules added in the polymerization step were recovered under very mild conditions by extracting the polymers with acetonitrile using a Soxhlet extractor. By employing various analytical methods such as measurements of radioactivity of the extracts of acetonitrile, total combustion of the polymers in conjunction with radioanalysis of the oxidized polymer material or amino acid analysis of the polymers hydrolyzed in HC1, it could be established that roughly 55-95% of the concentration of phenylalanine ethyl ester present in the polymerization mixtures was recovered by the extraction procedure.

Studies of binding of substrate to phen),lalanine ethyl ester-imprinted polymers

In order to examine the substrate selectivity of the polymers formed, the latter were allowed to interact with racemates of the substrate and substrate analogues. To this end binding experiments were performed in batch as well as chromatographic procedures. Chro- matography on the rigid polymers formed was possible, since it is known that macroporous polymers prepared as described here show

35

sufficient mechanical stability to cope with the conditions of elution of HPLC [12].

Batch experiments A very sensitive assay procedure using dou-

bly radiolabelled phenylalanine ethyl ester was developed [7]. Mixtures containing roughly equal amounts of 14C-labelled D-form and 3H-labelled L-form were applied to the prepared polymers placed in the same solvent (acetonitrile) as that used in the polymerization step. At equilibrium the amount of substrate free in solution and bound to the polymers was determined. The results of the batch experiments are summarized in Table 1, in which the separation factor, a, indicates the substrate selectivity of the polymers obtained in this study. The separation factor was estimated by determining the ratio of the apparent distribution coefficients (KD/KL) for the partitions of the D- and L-substrate between polymer and solvent. From the table, it can be seen that a values determined for polymers prepared in the presence of print molecules were either greater or less than one, indicating that the polymers formed were chiral. Furthermore, it was found that the polymers interacted preferentially with the enantiomer of the print molecule present in

TABLE 1

Batchwise resolution of racemates of phenylalanine ethyl ester after application of the racemic mi×tures to phenylalanine ethyl ester-selective polymers

Polymer Polymer Amount of D- Amount of L- Amount of D- Amount of L- a specificity ester bound ester bound ester found ester found K D / K L b

(print mole- to polymer to polymer in supernatant in supernatant cule) (/~ mol) (/~ mol) (/~ mol) (/~ mol)

Styrene D-PheOEt a 0.537 0,550 1.756 1.875 1.061 polymers L-PheOEt 0.498 0.554 1.795 1.871 0.953

D,L-PheOEt 0.428 0.459 1.865 1.966 1

Acrylic D-PheOEt 0.637 0.578 2.246 2.119 1.088 polymers L-PheOEt 0.504 0.527 2.379 2.119 0.912

none 0.585 0.567 2.298 2.130 1

a PheOEt = phenylalanine ethyl ester, b Values of the separation factor, a, have been normalized by relating the a values of the print polymers to the a value of a reference polymer prepared in the presence of D,L-PheOEt (styrene polymers) or in the absence of print molecules (acrylic polymers).

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the polymerization mixture. Thus, a values determined for polymers prepared in the presence of D-print molecules were greater than one. The reverse situation, polymers showing L-specificity in preference to D- specificity, was found after imprinting with L-substrates (a values less than one). On the other hand, polymers prepared in the presence of racemic mixtures of the substrate or in the absence of print molecules did not show enantiomer selectivity. It was also observed that resolution powers of polymers containing L-phenylalanine ethyl ester covalently bound to the polymer after polymerization were practically zero (the polymers were prepared using N-p-vinylbenzoyl or N- acryloyl derivatives of substrate instead of unmodified substrate in the polymerization process but otherwise under identical polymerization conditions) [7,22]. This result also argues against the possible explanation that the enantiomer selectivity observed in this work could be due to interactions between substrate and print molecules en- trapped in the polymers after polymerization and subsequent extraction of the polymers with acetonitrile. In this connection, it should be added that polymeric amino acid derivatives have been used as optically active adsorbents [28].

Chromatographic experiments Chromatographic separations of D- and L-

phenylalanine ethyl ester on print polymers have been carried out as well [22]. HPLC columns packed with fractionated polymer particles (particle size 45-65 ttm) were equilibrated with acetonitrile. Using this solvent as an eluent, it was found that ap- propriate retention volumes were obtained if the chromatography was performed at 50 .o C. In the chromatographic studies, the enantiomer separation on the print columns was measured by determining the ratio of the capacity factors for the enantiomers of the applied substrate [12]. Thus, for D,L-sep-

A2~z

D

5 0 100 min

Fig. 3. Elution profiles of D- and L-phenylalanine ethyl ester (0.2 ~tmol) applied separately to a column (200 × 5 mm) packed with 0.8 g of an L-phenylalanine ethyl ester-selective acrylic polymer (83 mole% cross-linker in the polymerization mixture of monomers). Acetonitrile was used as an eluent and the chromatography was performed at 50 ° C. Flow rate: 80 /xl/min. Separation factor ( k [ / k ~ ) , c~ =1.29. (Reprinted with permission from Ref. [22]).

arations the separation factor, a, was ex- pressed as k[/kD = ( V L - Vo)/(VD- Vo)

! where k e = capacity factor for the L-form and V e = retention volume of the L-form; k D and V o are the corresponding properties for the D-form, and V 0 = dead volume of the column. Chromatographic resolution of D- and L-phenylalanine ethyl ester on a column containing acrylic polymers exhibiting selectivity for L-phenylalanine ethyl ester is shown in Fig. 3. In this experiment the enantiomers were applied separately, affording a separation factor of 1.29. A similar resolution was observed when the racemic mixture was applied to the column [22]. Thus qualitatively similar ct values were determined in the batch and chromatographic procedures. However, the resolution powers found for the columns were slightly greater than those determined for the polymers applied in the batch procedures (a = 1.08-1.10). Similar findings have been reported elsewhere [12].

Aspects of substrate binding other than enantiomer selectivity of the polymers prepared in this study have been investigated as well. For example, by changing charge, size

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TABLE 2

Chromatographic resolutions of racemates of amino acid derivatives applied to a HPLC column packed with L-phenylalanine ethyl ester-selective acrylic polymer

R l H N - C H - C O - R 3 I CH2 I R~

t a p p p

Substrate R 1 - R 2 - R 3 - k D k L ~ c~ = k L / k E~

PheOEt H - C 6H 5- CH 3CH 2 0 - 0.370 0.475 1.284 TrpOEt H - 3-indolyl- C H 3 C H 2 0 - 1.028 1.231 1.197 AcPheOEt CH 3CO- C 6 H 5 - CH 3CH 2 O - 0.222 0.222 1 PheNHBzl H - C 6 H 5 - C 6 H 5 CH 2 N H - 1.200 1.200 1

a Capacity factors were determined for ~4C-labelled D- and 3H-labelled L-forms of the compounds applied to the L-phenylalanine ethyl ester-selective acrylic polymer. PheOEt = phenylalanine ethyl ester; TrpOEt = tryptophan ethyl ester; AcPheOEt = N-c~-acetyl-phenylalanine ethyl ester; PheNHBzl = phenylalanine benzylamide. (Data reproduced with permission from Ref. [22].)

and shape of interacting molecules additional information regarding structure and chemical properties of the substrate binding sites in the print polymers might be obtained. Therefore, to study polymer-subs t ra te interaction, racemates of different analogues of phenylalanine ethyl ester have been synthesized and applied to an L-phenylalanine ethyl ester- selective column. Some binding data are listed in Table 2. It is interesting to note that the resolution power of the column was lost en- tirely on N-acetylation of phenylalanine ethyl ester. In agreement with this, low substrate affinity has been observed for polymers formed after imprinting with L-phenylalanine ethyl ester in the presence of styrene, sub- stituting p-vinylbenzoic acid in the polymerization process [7]. However, in this case some chiral binding was still found to be possible for the styrene-based print polymer. Based on this, it seems reasonable to suggest that ion- pair association of negatively charged polymer and positively charged substrate is an important factor for efficient resolution of D,L-substrates on the substrate-selective columns prepared in this work. Worth mention- ing is also the resolution (a = 1.20) observed for racemic tryptophan ethyl ester, the side

chain of which differs only slightly in size and charge from that of phenylalanine ethyl ester, as well as the lack of resolution of a D,L-mixture of phenylalanine benzylamide by the print polymers. The reason for the latter result is not obvious but steric factors due to the bulky aromatic substituent at the carboxyl group of phenylalanine may explain the non-specific interaction found in this experiment. However, further studies need to be performed to understand the mode of binding of substrate analogues to the amino acid ester-selective polymers.

Synthesis of amino acid-based cross-linkers and their use in molecular imprinting

In order to exploit fully the potential of the non-covalent imprinting approach, we feel that it is necessary to be able to utilize various interactions, not merely ionic ones, to preorganize substrate and monomers during polymerization. Such prearrangements would be possible if vinyl- and acrylic-based monomers possessing additional functional groups were available. In particular, polyfunctional monomers suitable for the preparation of highly cross-linked polymers are needed. It

38

® HCI/EtOH H2N-CH-COOH ~ H2N-CH-COOEt

! I

NaBH 4 H2N-?H-CH2-OH

CH~CH-COCI ~. H2C=CH-CO-NH-CH-CH2-O-CO-CH=CH 2

Et3N i

® NaBH 4 CH2=CH-COCI H=N-CH-GOOMe ~ H2N-CH'CH~OHt P H2C=CH-CO-NH-CH-CH2-O-CO-CH=CH21

?H= Et3N ~ " t - , N 2 cH~

I O O O

I 1 I H~C-C-CH~ H~C-C-CH~ H~C-C-CH~

C H s CH 3 CH 3

TFA

H2C=CH-CO-NH-CH-CH2-O-CO-CH=CH 2

CH 2 I

OH

Fig. 4. General scheme of synthesis of acrylic-functional cross-linkers made from amino acid derivatives. (A) Synthesis of N,O-bisacryloyl-L-phenylalaninol (N,O-bisacryloyl-L-2-amino-3-phenyl-l-propanol). (B) Synthesis of Nz,Ol-bis - acryloylserinol (Nz,Oa-bisacryloyl-L-2-amino-3-hydroxy-l-propanol). EtOH = ethanol; Me = methyl; Et3N = triethylamine; TFA = trifluoroacetic acid.

occurred to us that amino acids containing different functional groups in their side chains could be used as building blocks for the con- struction of chiral, acrylic-functional cross- linkers [23]. Methods of synthesizing amino acid derivatives carrying bisacrylic groups are shown schematically in Fig. 4.

In the first step of the synthesis of such compounds, an amino acid ester, for example methyl or ethyl ester, was converted to the corresponding aminoalcohol by reduction of the ester with sodium borohydride (yield: 60-90%). In the subsequent step, acylation of amino and hydroxyl groups took place by treating the aminoalcohol formed with a molar excess of acryloyl chloride (2.2-4 equivalents) in the presence of equal amounts of triethylamine. Eventually, the bisacylated product

dissolved in ethyl acetate was isolated after washing the organic phase with citrate and sodium bicarbonate. In this manner, homoge- neous, crystalline N,O-bisacryloyl-L-phenylalaninol (N, O-bisacryloyl-L-2-amino-3-phenyl-l-propanol) has been prepared in 20% yield [23] (Fig. 4A). A similar synthetic procedure can be applied to make a bisacrylic-functional derivative of the hydroxyl-containing amino acid serine [29]. However, in the synthesis serine was protected in the side chain as the tert-butyl ether. After acryloyla- tion, the tert-butyl protecting group was removed by brief treatment (30 min at room temperature) of the acrylic-modified serine derivative with trifluoroacetic acid (Fig. 4B). The chiral cross-linker N z,Oa-bisacryloyl-L - serinol (N2,Ol-bisacryloyl-L-2-amino-3-hy-

droxy-l-propanol) obtained as an oil was pure as judged by reversed-phase HPLC on a C18 column, 1H-NMR and TLC (R F = 0.58 on silica in cyclohexane/ethylacetate/methanol (1 :1 :1 , v/v)). Summarizing, it is fully con- ceivable that any of the naturally occurring amino acids could be modified and converted to acrylic-functional cross-linkers by using a combination of the methods described here and those of modern synthetic peptide chemistry.

The new amino acid-based cross-linkers have been tried out in radical polymeriza- tions. In addition, molecular imprinting with L-phenylalanine ethyl ester using acrylic acid as the counter ion of the substrate and N,O- bisacryloyl-L-phenylalaninol as the cross-linking agent has been carried out successfully [23]. By employing the sensitive radioactivity assay mentioned earlier, it could be shown that substrate-imprinted polymeric L-phenylalaninol bound, as postulated in the protocol, the L-enantiomer in preference to the D-enantiomer of phenylalanine ethyl ester (Table 3). Reference polymers prepared in the absence of print molecules showed hardly any resolv- ing power. Considering the results of this examination, although not complete, it is sug- gested that amino acid-based monomers as discussed here may be suitable as cross-linking agents in molecular imprinting.

39

CONCLUDING REMARKS

We have demonstrated that amino acid ester-selective polymers can be formed by non-covalent imprinting with substrate in highly cross-linked polymers. This was achieved by using ion-pair association of substrate and monomers in the polymerization step. Moreover, we feel that the polymer substrate selectivity observed in this work was indeed due to substrate binding to specific binding sites or cavities formed in the polymers after imprinting with the substrate. Specific substrate binding is supported by the following observations: (a) resolutions of applied racemates were obtained on print polymers only (not on reference polymers); (b) polymer specificity could be reversed by imprinting with D- and L-forms of the substrate; and (c) very low resolution of racemate was found on polymeric L-phenylalaninol (reference polymer). Noteworthy is also the ob- servation that a negative charge at the site of substrate interaction in the print polymers appears to improve the power of resolution of the print polymers. Similarly, it has been sug- gested that hydrophilic regions at the binding sites of macroporous polymers may account for the high substrate selectivities found in studies of molecular recognition of aromatic ketones [21]. Finally, utilization of acrylic-

TABLE 3

Racemic resolutions on polymeric L-phenylalaninol a

Polymer b Polymer specificity Amount of cross-linker Recovery d a = K t . / K D

(print molecule) applied in the poly- (% of added merization mixture print molecule) (mol% of monomers)

P1 L-PheOEt c 90.5 74 1.050_+ 0.013 e P2 L-PheOEt 70 n.d. 1.075 _+ 0.013 P3 none 90.5 - 1.017 + 0.013

a The acrylic polymers were prepared using N,O-bisacryloyl-L-phenylalaninol (N,O-bisacryloyl-L-2-amino-3-phenyl- 1-propanol) as cross-linker, b For details of polymerization, see Experimental. c PheOEt = phenylalanine ethyl ester, d The recovery of added print molecules after polymerization was determined by amino acid analysis of hydrolyzed print polymers; n.d. = not determined, e Error limits given are standard deviations.

40

func t iona l cross- l inkers der ived f r o m a m i n o acids m a y pe rmi t n o n - c o v a l e n t i m p r i n t i n g

wi th the subs t r a t e in the p resence of m o n o -

mers ca r ry ing var ious func t iona l g roups in ter -

ac t ing wi th the subs t ra t e dur ing p o l y m e r i z a -

tion. Pr int p o l y m e r s thus f o r m e d m a y al low

for mul t ip le in te rac t ions wi th the subs t r a t e at

the b ind ing sites. Th is would lead to p r in t

p o l y m e r s s h o w i n g i m p r o v e d e n a n t i o m e r

select ivi ty and eventual ly , as b i nd i ng ene rgy

seems to be imp l i ca t ed in catalysis [30], to the

des ign of ca ta ly t ic p r in t po lymers .

A C K N O W L E D G E M E N T

The s u p p o r t p r ov i ded b y Fe r r ing Pha r -

maceu t i ca l s for the p r e p a r a t i o n of this

m a n u s c r i p t is g ra te fu l ly acknowledged .

R E F E R E N C E S

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11 S. Shinkai, M. Yamada, T. Sone and O. Manabe, Template synthesis from starch as an approach to tailor-made "cyklodextrin", Tetrahedron Lett., 24 (1983) 3501.

12 G. Wulff and W. Vesper, Preparation of chromatographic sorbents with chiral cavities for racemic resolution, J. Chromatogr., 167 (1978) 171.

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14 J. Damen and D.C. Neckers, On the memory of synthesized vinyl polymers for their origins, Tetra- hedron Lett., 21 (1980) 1913.

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17 G. Wulff, H.-G. Poll and M. Minarik, Enzyme-analogue built polymers. 19. Racemic-resolution on polymers containing chiral cavities, J. Liq. Chro- matogr., 9 (1986) 385.

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20 S.N. Gupta and D.C. Neckers, Template effects in chelating polymers, J. Polym. Sci., Polym. Chem. Ed., 20 (1982) 1609.

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22 B. Sellergren, B. Ekberg and K. Mosbach, Molecular imprinting of amino acid derivatives in macroporous

polymers. Demonstration of substrate- and enantio- selectivity by chromatographic resolution of racemic mixtures of amino acid derivatives, J. Chromatogr., 347 (1985) 1.

23 L. Andersson, B. Ekberg and K. Mosbach, Synthesis of a new amino acid based cross-linker for preparation of substrate selective acrylic polymers, Tetra- hedron Lett., 26 (1985) 3623.

24 J.P. Greenstein and M. Winitz, Chemistry of the Amino Acids, Vol. 2, Wiley, New York, NY, 1961, p. 925.

25 J.P. Greenstein and M. Winitz, Chemistry of the Amino Acids, Vol. 3, Wiley, New York, NY, 1961, p. 2067.

26 M. Bodanszky and A. Bodanszky, The Practice of

41

Peptide Synthesis, Springer-Verlag, Berlin, 1984, p. 145.

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preparation of amino acid ester-selective cavities formed by non-covalent imprinting with a...

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