Bioconjugate Chem. 1995, 6,524-528
524
Molecular Imprinting Approach for the Recognition of Adenine in Aqueous Medium and Hydrolysis of Adenosine 5'-Triphosphatet J a i n a m m a Mathew" a n d Ole Buchardt Center for Medical Biotechnology, Chemical Laboratory 11, The H. C. 0rsted Institute, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark. Received October 12, 1994@
Polymers capable of recognizing adenine in aqueous medium were developed by the molecular imprinting technique using methacrylic acid and 4(5)-vinylimidazole as comonomers with ethylene glycol dimethacrylate as the cross-linking agent under different polymerization conditions. The affinity of these polymers for adenine and other nucleotide bases was compared. The association constant for the binding of adenine to the polymer is calculated to be 4.3 x lo3 M-l. Furthermore, binding of adenosine 5'-triphosphate (ATP) to the polymers was evaluated, and a n enhanced binding compared to adenine was observed. The binding of ATP is pH dependent, with the maximum around pH 3. Results have been explained based on the hydrogen bonding and ionic interactions between ATP and the ligands on the polymer matrix. The catalytic effect of these polymers for the hydrolysis of ATP is briefly discussed.
INTRODUCTION
EXPERIMENTAL PROCEDURES
The recognition and subsequent complementary binding between a receptor and a target molecule is the first step in many vital supramolecular processes. The design of functional polymers that can selectively recognize molecules and catalyze reactions has become a n active area of research in recent years ( I -5). This technique, commonly referred to a s molecular imprinting, is based on creating cavities which correspond to the shape of the target molecule in a highly cross-linked polymer matrix. Briefly, the method involves preorganization of the target molecule with functional monomers followed by polymerization in the presence of a large excess of the crosslinking agent. Removal of the target molecule by extraction leaves behind functional groups in a rigid polymer matrix a t defined positions in a spatial arrangement that is complementary to the target molecule. Intermolecular interactions like hydrogen bonding, dipole-dipole interactions, and ionic interactions between the target molecule and the functional groups of the polymer matrix drive the molecular recognition phenomena. The design of receptors that can selectively recognize nucleotide bases has gained importance from the theoretical as well as the application point of view. Adenine has been well studied in this regard, especially in organic solvents (6, 7). K. J. Shea et al., by the molecular imprinting method, have made polymers that can selectively recognize and bind adenine and related molecules in nonaqueous media (8). Recent interest has focused on adenine receptors in aqueous media (9). The present study is aimed at developing receptor sites for adenine on a polymer matrix by the molecular imprinting technique, which in addition to recognizing adenine and adenine-containing molecules in aqueous medium is designed to have functional groups that can catalyze the ATP hydrolysis.
Ethylene glycol dimethacrylate and methacrylic acid are Aldrich products and were purified by distillation under reduced pressure. 2,2'-Azobis(isobutyronitri1e) (AIBN) (Merck) was used as such. 4(5)-Vinylimidazole was obtained by heating urocanic acid (Aldrich) as reported (10).Thymidine 5'-triphosphate, sodium salt (TTP) (Sigma), cytidine 5'-triphosphate, disodium salt hydrate (CTP) (Aldrich), guanosine 5'-triphosphate, trisodium salt hydrate (GTP) (Aldrich), ATP (Aldrich), adenosine 5'-diphosphate, sodium salt hydrate (ADP) (Aldrich), and adenosine 5'-monophosphate monohydrate (AMP) (Aldrich) were used as received. All solvents used were of HPLC grade. UV measurements were carried out on a Hewlett Packard 8452A diode array spectrophotometer. Fourier transform infrared (FTIR) spectra were recorded on a Perkin Elmer 1760 FT-IR spectrometer. Preparation of the Polymers. The polymers P1, P2, P3, P4, and RO were prepared using adenine as the template molecule. The following feed composition was used for P1-P3: methacrylic acid (2.3 mmol), vinylimidazole (1.7 mmol), adenine (0.2 mmol), and ethylene glycol dimethacrylate (10.6 mmol). The composition of P4 was the same except for a higher ethylene glycol dimethacrylate content (21.2 mmol). The reference polymer PO, without adenine, was prepared with the same monomer composition as Pl-P3. Another reference polymer RO was synthesized to study the role of imidazole groups in the polymer. The initial feed composition of RO was methacrylic acid (2.3 mmol), adenine (0.2 mmol), and ethylene glycol dimethacrylate (10.6 mmol). The solvent employed was a mixture of methanol and water (955 v/v). AIBN (0.3 mol % of the total monomer) was used as initiator. In a typical experiment, adenine (25 mg) was taken in a glass vessel and methacrylic acid (200 pL) was added followed by water (100 pL),which resulted in the gradual formation of a clear solution. After the addition of a solution of vinylimidazole (0.166 g in 1mL of methanol), ethylene glycol dimethacrylate (2 mL), AIBN (6.6 mg), and methanol (2 mL), the clear homogeneous solution was deaerated and polymerized a t 65 "C for 24 h. The polymerization mixture was deaerated by bubbling nitrogen (PO, RO, P1, and P2) or by freeze-thawing thrice
' Dr. Mathew expresses deep regret over the untimely demise of Prof. Ole Buchardt on September 5 , 1994. * Present address: Dr. Jainamma Mathew, Department of Chemistry, University of California, Irvine, CA 92717. Abstract published in Advance ACS Abstracts, June 15, 1995. @
1043-1802/95/2906-0524$09.00/0
0 1995 American Chemical Society
Roconjugate Chem., Vol. 6, No. 5, 1995 525
Molecular Imprinting: Adenine Recognition
Table 1. Binding of Various Nucleic Acid Bases to Polymersa substrate 10.07 mM) adenine adenosine cytosine thymine a
PO 0.40 f 0.25 0.36 i 0.1 0.25 0.15
RO 2.30 ic 0.05 0.80 i 0.1 0.23 0.23
substrate bound in 10 min GmoVg of polymer) P1 P2 0.90 f 0.05 0.45 f 0.07 0.02 0.05
1.20 f 0.04 0.65 0.21 0.13
P3
P4
1.40 i 0.03 1.05 f 0.05 0.35 0.22
1.20 i 0.04 1.03 0.22 f 0.01 0.12
Temp = 22 "C,pH = 6.0, in aqueous medium.
(P3 and P4). PO, RO, P1, and P2 were polymerized in a n atmosphere of nitrogen. During polymerization of PO, RO, and P2, nitrogen was continously bubbled through the reaction mixture, while for P1 the reaction vessel above the monomer mixture was deaerated by passing nitrogen. In the case of P3 and P4, the monomer mixture was taken in a glass tube, degassed by freeze-thawing, and sealed under vacuum. The polymers obtained were wet-ground and extracted (Soxhlet) with methanol (72 h) and water (24 h), resulting in 85-90% recovery of adenine. The polymers were then washed with methanol and dried under vacuum a t room temperature. The dry polymer particles were separated according to their size. Binding Studies. Binding studies were carried out by a continuous circulation (flow) method as well a s by a batch procedure. In a typical flow system, 100 mg of the polymer particles were taken in a glass column connected to a peristaltic pump. A solution of the substrate in water (20 mL) was continuously circulated through the column a t a constant flow rate. Depletion in concentration of the substrate was monitored by U V spectrophotometry. In a typical experiment, upon passing through P1 (100 mg) for 10 min, the absorbance of a 0.07 mM solution of adenine decreased from 0.99 to 0.92, leading to 0.9 pmol of adenine bindinglg of the polymer. In the case of very low binding, the experiment was carried out with 500 mg of polymer. For the binding of cytosine on P1, the difference in absorbance was very low (absorbance decreased from 0.430 to 0.427) due to its poor binding on P1 (0.02 pmollg of polymer). Batch Binding. The polymer (20 mg) was incubated with 1 mL of a n aqueous solution of adenine of known concentration in a n Eppendorf tube a t room temperature for 24 h with constant shaking. Subsequently, the solution was filtered and centrifuged and the concentration of adenine in solution determined by UV measurements a t 260 nm. Experiments were carried out with P3 and PO a t various concentrations (0.1-0.5 mM) of adenine. Solutions of adenine (0.1-0.5 mM) without polymers were examined under identical conditions as blanks. ATP Hydrolysis. Hydrolysis of ATP using the polymer was carried out a t 22 "C. A solution of ATP in water (pH adjusted to 3 with dilute HC1) was circulated through the polymer in a glass column. After definite time intervals, a known volume of the solution was analyzed by HPLC. Concentrations of ADP and AMP formed were determined by integration of the peaks on HPLC (CIS column: reversed phase (VYDAC), eluent: water containing 0.2% heptafluorobutyric acid, isocratic, flow rate = 1 mumin, detection wavelength 2 = 258 nm). The adsorbed ATP or ADPIAMP could be desorbed from the polymer by increasing the pH of the solution to 11(with dilute NaOH) and circulating it through the polymer. The products of the hydrolysis, viz., ADP and AMP, were quantitated using HPLC. In a n experiment to evaluate the selectivity of the polymers for the hydrolysis of ATP, a typical polymer (P3, 50 mg) was weighed into a conical flask containing 12 mL of a n aqueous solution of ATP (0.6 mM, pH 2.8) and
incubated a t room temperature with constant shaking for 17 h. Thereafter, (a) 2 mL of the solution was centrifuged and concentrated to 200 pL and (b) the pH of the solution was adjusted to 11by adding dilute NaOH, incubated for another 40 min. Samples (a) and (b) were analyzed by HPLC. A solution of ATP without polymer was used as a control. A similar procedure and identical conditions were employed for TTP, GTP, and CTP. RESULTS AND DISCUSSION
The hydrogen bonding involved in host-guest interactions is most effective in aprotic organic solvents and is limited in aqueous medium. However, cooperative binding of several weak noncovalent forces governs the antigen-antibody and protein-nucleic acid interactions. In the protein-nucleic acid interactions, the most selective binding contacts were realized to involve a t least two hydrogen bonds, which require the carboxylic acid group of aspartic or glutamic acid, the amide group of asparagine or glutamine, or the guanidinium group of arginine (7). Since the major interest concerned with organic host molecules is to imitate the biological molecular recognition, we have designed polymers that can recognize adenine and adenine-containing molecules and have investigated the binding of these molecules onto the polymer matrix in aqueous medium. The template molecule, adenine, has been preorganized with the functional monomer methacrylic acid through electrostatic interactions or H-bonding. Multiple hydrogen bond formation between carboxylic acids and adenine has been reported (7, 8, 11). Zimmerman et al. have reported the effect of protic solvents on the complexation between a receptor having a carboxylic acid moiety and adenine in chloroform; addition of 10% and 50% CD30D has been found to reduce the stability of the complex by ca. 2.3 kcal mol-' as compared to pure CDCl3 (7). In our study, the nature of the complex methacrylic acidadenine, whether it is formed via electrostatic interactions or H-bonding, is not clear. The complex is insoluble in water; however, it is soluble in methanol. Vinylimidazole is also expected to participate in the complex formation with adenine or with methacrylic acid. Polymerization of this organized assembly with an excess of a cross-linking agent results in a very rigid polymer matrix. Removal of the template adenine from the polymer by extraction leaves behind functional groups in a spatial arrangement that is complementary to adenine. Polymer particles of size 80-150 pm were employed for these studies. Substrate Binding and Selectivity. Rebinding of adenine from a n aqueous solution was carried out under flow conditions. Equilibrium binding was achieved in about 3 min. The amount of adenine bound per gram of polymer in 10 min for different polymers is summarized in Table 1. Binding to the reference polymer PO was carried out to account for the nonspecific interactions. Both the reference as well as the imprinted polymers P1P3 were made with the same feed composition of the monomers, under identical conditions. In the adenineimprinted polymers some of the functional groups are
Mathew and Buchardt
526 Bioconjugafe Chem., Vol. 6,No. 5, 1995
located in microcavities complementary to the shape of adenine while remaining groups are located on the surface of these polymer particles, the latter leading to nonspecific interactions. Thus, the imprinted polymers have both specific as well as nonspecific interactions. In the reference polymer where no template was used, all the functional groups are available for nonspecific interaction. The reference polymer (PO) exhibits a lower degree of adenine binding compared to the imprinted polymers P1, P2, and P3. This indicates that, in addition to the hydrogen bonding or electrostatic interaction between the functional groups of the polymers and adenine, microcavities corresponding to the shape of adenine are also necessary for effective binding. Dunkin et al., in their investigation of imprinted polymers with 2,6-diaminoanthraquinone a s the template molecule, have observed that the polymers in addition to recognizing 2,6-diaminoanthraquinone were also effective in recognizing anthracene, suggesting that the shape of the microcavity is a dominant factor (12).Within the experimental error, the binding capacity of P2 for adenine (1.2 pmoYg) is higher than that of P1 (0.9 pmollg). During the preparation of P2, nitrogen was continously bubbled through the solution; the resulting polymer has an amorphous appearance and exhibits higher adenine uptake than P1, which has a glassy appearance. Even though P2 and P3 had the same initial monomer composition, the latter exhibits higher binding capacity compared to the former. The difference may be due to the different polymerization conditions employed and the consequent changes in the morphology of the polymers. P3 and P4 were prepared with different ratios of methacrylic acid to cross-linking agent (ethylene glycol dimethacrylate). The mol % of methacrylic acid in P3 is 16% while that in P4 is 9%. The former, having a lower cross-link density, exhibits a higher binding capacity for adenine (1.4 pmollg) than P4 (1.2 pmollg). A similar observation has been made earlier by Sellergren in studies on separation of D,LPheNHPh using imprinted polymers having different methacrylic acid content (13). An increase in the concentration of methacrylic acid up to 50 mol % has been reported to increase the capacity factor and separation factor for D,L-PheNHPh. Binding of other substrates to these polymers was also carried out under identical conditions, and the results are shown in Table 1. The binding capacity of P1 and P2 for adenosine is about 50% of that for adenine, and it lies in the range 70-80% for P3 and P4. Reduced binding implies the absence of any favorable interaction between the polymer and hydroxyl groups of adenosine. Among the different nucleic acid bases, polymers Pl-P4 exhibit 7-10 times enhanced binding of adenine compared to thymine and 4-6 times compared to cytosine. Binding of adenine to PO is only about twice as high as that of cytosine and thymine. It can be suggested that, within the experimental error, the imprinted polymers exhibit selectivity toward adenine compared to other nucleotide bases. Binding Capacity. Binding curves showing the amount of adenine bound in 24 h (in a batch method) as a function of concentration of adenine in solution for PO and P3 are presented in Figure 1. The initial increase in binding is followed by a saturatiodeveling off, indicating that the available receptor sites have been saturated with adenine. The association constant (K,,,,,) for the binding of adenine to the polymer and the number of accessible sites (N) on the polymer have been calculated using the modified Scatchard equation (13):
1/K = C,/N
+ (l/K,,,,,)N
u.0
0.1
0.2
0.3
0.4
0.5
Conc. of adenine in solution (mM) Figure 1. Concentration dependence of adenine binding in a batch process at 22 "C. Polymer (20 mg) was incubated with a
solution of adenine (1mL) for 24 h. Amount of adenine bound to the polymer was evaluated from the difference in absorbance of the solution before and after incubation. Each value is given as the average of 4 independent experiments.
u.0
0.1
0.2
0.3
Cf (mM)
Figure 2. Modified Scatchard plot for the binding of adenine (data compiled from the values in Figure 1).
where K (=CdCd is the distribution constant for the solute between the polymer and solution, Cb is the concentration of solute bound to the polymer, and Cf is that of free solute in solution. In order to account for the nonspecific interactions, the amount of adenine bound to the reference polymer (PO) was subtracted from that bound to P3, and the resulting 1/K (=C$ACb) is plotted versus Cf. A linear plot is obtained with a n intercept (Figure 2). The number of accessible sites has been calculated to be 1.86 pmollg of polymer. The association constant K,,,,, calculated from the slope and intercept is 4.3 x lo3 M-l. This value is significantly lower than that calculated by K. J. Shea et al., for a similar system in nonaqueous medium (8). However, considering the fact that these experiments have been carried out in aqueous medium where water effectively competes for the hydrogen bonding interactions, the observed association constant indicates an appreciable degree of receptor-ligand interaction. The binding strength calculated (-AGO = RT In K ) is about 4.9 kcal mol-l. A recent study on the binding of adenosine derivatives to water-soluble adenine receptors has shown the association constants of 9-ethyladenine, adenosine, and 2',3'-CAMP to be 200, 150, and 660 M-l, respectively (9). A relatively higher degree of binding in the present system may be attributed to
Molecular Imprinting: Adenine Recognition
Bioconjugate Chem., Vol. 6,No. 5, 1995 527 Table 2. Influence of pH on the Binding of Nucleoside Triphosphatesa
100 L
Ye
-g
T
substrate binding (pmoYg of polymer)
P2
h
PO
80
substrate (0.08 mM) ATP GTP CTP
0
5
TTP
PO pHx 0.8 0 0.8 0
P1 PH 3 9.4 9.6 3.5 13.7
PH x 1.7 0
1.7 0.2
PH 3 18.2 10.1 6.2 10.7
a x is the pH of a solution of the substrate in water: ATP, x = 4.8; GTP, x = 5.6; CTP, x = 4.8; TTP, x = 6.2.
Table 3. pH-Dependent Variation in Binding of Adenine
"
1
2.5
3
4.5
5
12
PH Figure 3. Variation in binding of ATP as a function of pH. A solution of ATP (20 mL, 0.5 mM), after adjusting the pH with dilute HC1, was circulated through a column containing 0.1 g of polymer for 30 min. The amount bound is calculated from the depletion in concentration of ATP in solution as measured from the difference in W absorbance. Data are the average of 6 separate experiments.
simultaneous multiple interactions of adenine with the functional groups of the polymer. Unlike a homogeneous receptor or a small molecular nonhomogeneous receptor, a polymeric receptor offers additional stability to the receptor-ligand complex through its unique macromolecular properties and the cooperative functional group interactions. ATP Binding and Hydrolysis. These polymers consisting of binding sites for adenine and nucleophilic catalytic groups (imidazole) can be visualized as enzyme mimicking polymers, a s the imidazole group, similar to the histidine moieties in a n enzyme, may participate in the catalytic reactions. Esterolytic activity of polymerbound imidazoles toward activated esters such as p nitrophenyl acetate, hexanoate, etc., has been reported (14-16). However, in the present study, the imidazole groups present in the polymer matrix did not exhibit any measurable hydrolysis of the diester of the polymer as evidenced by the virtually identical FT-IR spectra of the dry polymers before and after incubation with water a t pH 2.5 for 48 h. This may be due to the relatively low imidazole content of the polymer. Even though the feed ratio of vinylimidazole to ethylene glycol dimethacrylate was 1 5 , due to the lower reactivity of the former toward radical polymerization, the extent of incorporation in the polymer chain is very low compared to that of ethylene glycol dimethacrylate. Furthermore, control experiments carried out with a solution of ethylene glycol dimethacrylate and vinylimidazole in methanol-water (4:l and 20:l v/v) did not exhibit ester hydrolysis as followed by HPLC, excluding the possibility of any hydrolysis during polymerization. Here, we report on the catalytic effect of these polymers on the hydrolysis of ATP. Two of the phosphate bonds of ATP are extremely labile and on hydrolysis release about 7-9 kcal/mol (17).Binding of ATP to the polymer has been carried out under flow conditions. Compared to the reference polymer PO, the adenine-imprinted polymers show a higher ATP uptake. The binding of ATP by the polymer was pH dependent, with the maximim a t about pH 3 (Figure 3). The ATP uptake a t pH 2.5-3 is 8-10 times higher than that at pH 5 (pH of a 0.5 mM solution of ATP in water). A similar pH dependence has been observed for TTP, GTP, and CTP (Table 2). Consistently, the uptake a t pH 3 is
time (mid 1 3 5 10 15 20 a
pH6 0.42 0.81 0.75 0.73 0.78 0.78
adenine uptake bm0Y.g of polymer) PO P1 P3 pH3 pH6 pH3 p ~ 6 0.52 0.29 0.43 1.50 0.90 0.50 1.40 0.12 0.95 0.13 1.33 0.04 0.01 0.90 0.08 1.39 0.98 0.06 0.01 0.01 0.95 0.04
p ~ 0.52 1.08 0.80 0.16
[Adenine] = 0.07 mM, temp = 22 "C, in aqueous medium.
about 10 times more for TTP, GTP, and CTP than that a t the pH of each substrate in water. Such a pHdependent variation in binding is indicative of ionic interactions between the polymer and the substrate (e.g., ATP). The functional groups of the polymer, viz., carboxyl and imidazole, may contribute to the ionic interaction. The influence of pH on the binding of adenine has also been studied by following adenine uptake as a function of time (continuous flow system) a t pH 3 and 6; the results are presented in Table 3. In addition to relatively low maximum binding, the bound adenine desorbs a t a faster rate a t pH 3. This observation excludes any positive contribution of ionic interactions between the functional groups of the polymer and adenine to the enhanced ATP uptake a t pH 3. Furthermore, other substrates such as TTP, GTP, and CTP also exhibit a n enhanced binding a t pH 3. Hence, the ionic interactions between phosphate groups and the carboxyl andlor imidazole groups of the polymer matrix may be the driving force. The role of imidazole in such interactions was investigated by following the pH-dependent binding of ATP to the reference polymer RO, which was made with only methacrylic acid a s the comonomer and no vinylimidazole. As expected, no enhancement in binding occurred a t low pH, suggesting that the ionic interactions between the imidazole groups of the polymer and phosphate groups are major contributing factors for the enhanced ATP uptake a t pH 3. Even though a satisfactory explanation is difficult a t this stage, it may be due to the pH-dependent protonatioddeprotonation of ATP and its influence on the ionic interaction and hydrogen bonding. At around pH 3, the negative charges on phosphate groups are partially neutralized by protonation; in addition, the adenine ring is also protonated (18).Hence, a t this pH, in addition to binding through specific recognition of adenine, there may be an enhanced complex formation via ionic interaction between the partially ionized phosphate groups of ATP and the imidazolium cation on the polymer. Further decrease in pH affects the ATP binding adversely. At low pH, all negative charges on phosphate groups are neutralized by protonation, decreasing the extent of ionic interactions (18).Y.&to et al., in their studies on the interaction of cyclic AMP with water-soluble adenosine receptors, have observed significantly higher binding of cyclic AMP to
3
528 Bioconjugate Chem., Vol. 6, No. 5, 1995
Mathew and Buchardt
appreciated. J.M. would like to thank Prof. Peter E. Nielsen for valuable discussions.
t
V
0
/
T
2
20
10
LITERATURE CITED
x
3
30
Time (h)
Figure 4. Concentration of AMP produced during the hydrolysis of ATP with P2 (0.1 g) as a function of time. (Initial concn of ATP: 1, 45 pM; 2, 90 pM; and 3, 180 pM.1 Initial pH of the solution = 3.0. Each value is the average of 3 experiments.
the receptor having a guanidinium moiety, which was attributed to the phosphate-guanidinium interaction resulting from both hydrogen bonding and electrostatic interactions (9). Under the present experimental conditions, during the initial 2 h of reaction, the product of hydrolysis was mainly ADP. Prolonged reaction resulted in formation of both ADP and AMP. (Separate experiments carried out under similar conditions have further shown that these polymers catalyzed the hydrolysis of ADP.) Hydrolysis has been carried out a t different ATP concentrations, and the hydrolytic pattern is shown in Figure 4. In the HPLC analysis of the reaction mixture, peaks due to ATP and ADP were not well resolved. Hence, an accurate quantitation of the ADP content was difficult. The amount of AMP produced as a function of reaction time is shown in Figure 4. The extent of hydrolysis increased with a n increase in the initial ATP concentration. Furthermore, increase in reaction time enhanced the degree of hydrolysis (AMP content) up to about 10 h, followed by the reaction reaching saturation, within experimental error. The reference polymer PO exhibited hydrolysis only to a significantly lower extent (at [ATP] = 90 pM, the concentration of AMP obtained in 10 h was only 7 pM). RO, which was designed to have no imidazole groups, did not cause ATP hydrolysis. Selectivity toward ATP hydrolysis was determined in comparison with other triphosphates, viz., TTP, GTP, and CTP. Hydrolysis of TTP was less than 5%, and within experimental error, the hydrolysis of GTP and CTP was negligible. Procedures (a) and (b) employed for product analysis gave agreeable results. Suzuki et al. have investigated the mechanism of nonenzymatic hydrolysis of ATP by polyamines and polyimines (17).The maximum rate of hydrolysis was observed a t around pH 3.0 and was attributed to the protonated imino nitrogens, forming a complex with ATP and favoring the hydrolysis of the latter. Similarly, the present study reflects that the imidazole groups of the polymer favor the hydrolysis of ATP either by a nucleophilic mechanism or by activating the attack by a water molecule. We have extended this approach to the recognition of deoxyoligonucleicacids containing adenine, and the results will be presented in due course. ACKNOWLEDGMENT
Financial support from Bionebraska, Inc., Lincoln, NE, and the Danish Biotechnology Programme is greatly
(1) Wulff, G., and Schauhoff, S. (1991) Racemic resolution of free sugars with macroporous polymers prepared by molecular imprinting. Selectivity dependence on the arrangement of functional groups versus spatial requirements. J . Org. Chem. 56, 395-400. (2) Vlatakis, G., Andersson, L. I., Muller, R., and Mosbach, K. (1993) Drug assay using antibody mimics made by molecular imprinting. Nature 361, 645-647. (3) Robinson, D. K., and Mosbach, K. (1989) Molecular imprinting of a transition state analogue leads to a polymer exhibiting esterolytic activity. J . Chem. SOC.,Chem. Commun., 969970. (4) Beach, J. V., and Shea, K. J. (1994) Designed catalysts. A synthetic network polymer that catalyzes the dehydrofluorination of 4-fluoro-4-(p-nitrophenyl)butan-2-one. J . Am. Chem. SOC.116, 379-380. (5) Dhal, P. K., and Arnold, F. H. (1992) Metal-coordination interactions in the template-mediated synthesis of substrateselective polymers: Recognition of bis(imidazo1e) substrates by copper(I1) iminodiacetate containing polymers. Macromolecules 25, 7051-7059. (6) Williams, K., Askew, B., Ballester, P., Buhr, C., Jeong, K. S., Jones, S., and Rebek, J., Jr. (1989) Molecular recognition with convergent functional groups. 7. Energetics of adenine binding with model receptors. J . Am. Chem. SOC.111,10901094. (7) Zimmerman, S. C., Wu, W., and Zeng, Z. (1991) Complexation of nucleotide bases by molecular tweezers with active site carboxylic acids: Effects of microenvironment. J. Am. Chem. SOC.113, 196-201 and references therein, (8) Shea, K. J., Spivak, D. A., and Sellergren, B. (1993) Polymer complements to nucleotide bases. Selective binding of adenine derivatives to imprinted polymers. J . Am. Chem. SOC.115, 3368-3369. (9) Kato, Y., Conn, M. M., and Rebek, J., Jr. (1994) Watersoluble receptors for cyclic-AMP and their use for evaluating 116, phosphate-guanidinium interactions. J. Am. Chem. SOC. 3279-3284. (10) Overberger, C. G., and Vorchheimer, N. (1963) Imidazolecontaining polymers. Synthesis and polymerization of the monomer 4(5)-vinylimidazole. J . Am. Chem. SOC.85, 951955. 1) Lancelot, G. (1977) Hydrogen bonding between nucleic acid 99,7037-7042. bases and carboxylic acids. J . Am. Chem. SOC. 2) Dunkin, I. R., Lenfeld, J., and Sherrington, D. C. (1993) Molecular imprinting of flat polycondensed aromatic molecules in macroporous polymers. Polymer 34, 77-84. 3) Sellergren, B. (1989) Molecular imprinting by noncovalent interactions. Enantioselectivity and binding capacity of polymers prepared under conditions favoring the formation of template complexes. Makromol. Chem. 190, 2703-2711. 4) Overberger, C. G., and Moritomo, M. (1971) Conformational effects and nonpolar interactions in poly[4(5)-vinylimidazole]catalyzed solvolyses of neutral substrates. J. Am. Chem. SOC. 93, 3222-3228. (15) Guthrie, J. P. (1972) Aggregation of p-nitrophenyl alkanoates in aqueous solution: A caution concerning their use in Chem. Commun., 897enzyme model studies. J . Chem. SOC., 899. (16) Leonhardt, A., and Mosbach, K. (1987) Enzyme-mimicking polymers exhibiting specific substrate binding and catalytic functions. React. Polym. 6, 285-290. 7) Suzuki, S., Higashiyama, T., and Nakahara, A. (1973) Nonenzymatic hydrolysis reactions of adenosine 5’-triphosphate and its related compounds. 1. Hydrolysis reactions of ATP with some continous-chain polyamines. Bioorg. Chem. 2 , 145-154. 8) Saenger, W. (1984) Princzples of Nucleic Acid Structure (Charles R. Cantor, Ed.) Springer Advanced Texts in Chemistry, Springer-Verlag. BC950033H
