Immobilized DNA Aptamers as Target-Specific Chiral Stationary

Anal. Chem. 2004, 76, 1015-1020

Immobilized DNA Aptamers as Target-Specific Chiral Stationary Phases for Resolution of Nucleoside and Amino Acid Derivative Enantiomers Mickael Michaud, Eric Jourdan, Corinne Ravelet, Annick Villet, Anne Ravel, Catherine Grosset, and Eric Peyrin*

Equipe de Chimie Analytique, De´ partement de Pharmacochimie Mole´ culaire UMR 5063 CNRS-UJF, ICMG FR 2607, Universite´ Joseph Fourier, UFR de Pharmacie de Grenoble, Avenue de Verdun, 38240 Meylan, France

Recently, we described the use of a DNA aptamer as a new target-specific chiral stationary phase (CSP) for the separation of oligopeptide enantiomers (Michaud, M.; Jourdan, E.; Villet, A.; Ravel, A.; Grosset, C.; Peyrin, E. J. Am. Chem. Soc. 2003, 125, 8672). However, from a practical point of view, it was fundamental to extend the applicability of such target-specific aptamer CSP to the resolution of small (bioactive) molecule enantiomers. In this paper, immobilized DNA aptamers specifically selected against D-adenosine and L-tyrosinamide were used to resolve the enantiomers by HPLC, using microbore columns. At 20 °C, the adenosine enantioseparation was similar to that classically reported with imprinted CSPs (∼3.5) while a very high enantioselectivity was observed for the tyrosinamide enantiomers (the nontarget enantiomer was essentially nonretained on the CSP). The influence of temperature on solute binding and chiral discrimination was analyzed. The binding enthalpic contributions were determined from linear van’t Hoff plots. Very large ∆H values were obtained for the target enantiomers (-71.4 ( 0.7 kJ/mol for D-adenosine and -139.4 ( 2.0 kJ/mol for L-tyrosinamide). Such values were consistent with the formation of a tight complex between these analytes and the aptamer CSPs. This work demonstrates that target-specific aptamer CSPs constitute a powerful tool for the resolution of small (bioactive) molecule enantiomers. Chiral separation is of crucial importance in various fields such as drug and food analysis, biochemistry, or clinical pharmacology. High-performance liquid chromatography (HPLC) using chiral stationary phases (CSPs) is a popular way to perform enantiomeric separations due to the relative ease, speed, and efficiency. Two kinds of CSPs can be distinguished: (i) the conventional CSPs1 for which the selectivity is not predetermined and (ii) the targetspecific CSPs such as imprinted polymers2 or antibodies,3 which * Corresponding author. E-mail: [email protected]. (1) See Review: J. Chromatogr., A 2001, 906, 1-489. (2) Ramstrom, O.; Ansell, R. J. Chirality 1998, 10, 195. (3) Hofstetter, O.; Lindstrom, H.; Hofstetter, H. Anal. Chem. 2002, 74, 2119. 10.1021/ac035090f CCC: $27.50 Published on Web 01/10/2004

© 2004 American Chemical Society

are characterized by a predictable elution order, depending only on the enantiomer form used as imprint or antigen. Aptamers are DNA or RNA oligonucleotides capable of specifically binding a target molecule such as organic dyes, amino acids, peptides, proteins, nucleotides, or drugs. They are generated by the SELEX procedure,4,5 which involves repeated cycles of a selection round followed by an amplification round. The selectivity and affinity of aptamers have been recently used with much interest in flow cytometry,6 sensors,7 ELISA-type assays,8 capillary electrophoresis,9-11 and affinity chromatography.12,13 Aptamers present various advantages. They are produced by chemical synthesis in a short time, at low cost, with reproducibility and accuracy, and at a high degree of purity. It is also easily possible to change their sequence in order to modulate their binding selectivity. In addition, they can be modified at precise locations by molecules such as biotin in order to allow attachment to the streptavidin surface. SELEX procedure can result in sequences that are capable of enantiospecifc recognition.14-18 Recently, a DNA aptamer characterized by its stereoselective binding affinity for D-vasopressin was used, as a new class of target-specific CSPs, to separate the nonapeptide enantiomers with a very high enantioselectivity.19 (4) Tuerk, C.; Gold, L. Science 1990, 249, 505. (5) Ellington, A. D.; Szostak, J. Nature 1990, 346, 818. (6) Davis, K. A.; Abrams, B.; Lin, Y.; Jayasena, S. D. Nucleic Acids Res. 1996, 24, 702. (7) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2001, 123, 4928. (8) Drolet, D. W.; Moon-McDermott, L.; Romig, T. S. Nat. Biotechnol. 1996, 14, 1021. (9) Pavski, V.; Le, X. C. Anal. Chem. 2001, 73, 6070. (10) Rehder, M. A.; McGown, L. B. Electrophoresis 2001, 22, 3759. (11) German, I.; Buchanan, D. D.; Kennedy, R. T. Anal. Chem. 1998, 70, 4540. (12) Deng, Q.; German, I.; Buchanan, D.; Kennedy, R. T. Anal. Chem. 2001, 73, 5415. (13) Romig, T. S.; Bell, C.; Drolet, D. W. J. Chromatogr., B 1999, 731, 275. (14) Williams, K. P.; Liu, X. H.; Schumacher, T. N. M.; Lin, H. Y.; Ausiello, D. A.; Kim, P. S.; Bartel, D. P. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11285. (15) Geiger, A.; Burgstaller, P.; von der Eltz, H.; Roeder, A.; Famulok, M. Nucleic Acids Res. 1996, 24, 1029. (16) Klussman, S.; Nolte, A.; Bald, R.; Erdmann, V. A.; Furste, J. P. Nat. Biotechnol. 1996, 14, 1112. (17) Famulok, M. J. Am. Chem. Soc. 1994, 116, 1698. (18) Famulok, M.; Szostak, J. W. J. Am. Chem. Soc. 1992, 114, 3990. (19) Michaud, M.; Jourdan, E.; Villet, A.; Ravel, A.; Grosset, C.; Peyrin, E. J. Am. Chem. Soc. 2003, 125, 8672.

Analytical Chemistry, Vol. 76, No. 4, February 15, 2004 1015

Figure 1. Structures of D-adenosine and L-tyrosinamide and their respective specific sequences of single-stranded DNA (ADE and TYR). ATTATA and TAATAT sequences were inserted at the 5′ and 3′ ends of the highly conserved sequence20 of the D-adenosine aptamer.

However, from a practical point of view, it is fundamental to extend the applicability of such target-specific DNA aptamer CSPs to the resolution of small-molecule enantiomers. Here, we demonstrate that immobilized DNA aptamers, specifically selected against D-adenosine20 and L-tyrosinamide21 enantiomers (Figure 1), are able to resolve successfully the optical isomers by HPLC, using microbore affinity columns. The influence of temperature on the solute binding and chiral discrimination was analyzed in order to define the optimal utilization conditions. From the linear van’t Hoff plots, the binding and enantioselective (for D-, L-adenosine) enthalpic contributions were also determined. EXPERIMENTAL AND METHODS Reagents and Materials. D-Biotin, L-tyrosinamide, D-adenosine, D-,L-tyrosine, and D-,L-tyrosine methyl ester were obtained from Sigma Aldrich (Saint-Quentin, France). L-Adenosine was purchased from Chemgenes (Ashland, MI). D-Tyrosinamide was synthesized by Millegen (Toulouse, France) and purified by reversed-phase chromatography (C8 column: 4.6 × 30 mm with a particle diameter of 7 µm; eluent A, H2O; eluent B, H2Oacetonitrile 25:75 (v/v); gradient elution by varying the proportion of eluent B in the mobile phase from 2 to 80% in 50 min; flow rate, 1 mL/min; UV detection, 215 nm; injection volume, 20 µL; solute retention time, 10.03 min). The identity of D-tyrosinamide was confirmed by ESI-MS. Na2HPO4, NaH2PO4, KCl, and MgCl2 were supplied by Prolabo (Paris, France). Water was obtained from an Elgastat option water purification system (Odil, Talant, France) fitted with a reverse-osmosis cartridge. The DNA oligonucleotides were synthesized and 5′-biotinylated by Eurogentec (Herstal, Belgium). Biotin phosphoramidite containing a 16-atom spacer arm based on triethylene glycol was used for the aptamer biotinylation. The biotinylated oligonucleotides were purified by gel electrophoresis (Eurogentec). The streptavidin Poros bulk media (20-µm polystyrene particles) was purchased from Applied Biosystems (Courtaboeuf, France). Poros perfusion chromatography particles have large through-pores that transect the particles and short diffusive pores that branch off from the through-pores. Stationary-Phase Preparation. Prior to immobilization, the biotinylated aptamers were renaturated by heating oligonucleotides at 70 °C for 5 min in an aqueous buffer (20 mM phosphate buffer, 25 mM KCl, and 1.5 mM MgCl2 adjusted at pH 7.6; aptamer concentration, ∼80 µM) and left to stand at room temperature for 30 min. Immobilization of aptamers was attained by mixing 1 (20) Huizenga, D. E.; Szostak, J. E. Biochemistry 1995, 34, 656. (21) Vianini, E.; Palumbo, M.; Gatto, B. Biorg. Med. Chem. 2001, 9, 2543.

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(for adenosine CSP) or 0.5 mL (for tyrosinamide CSP) of the streptavidin media slurry with 0.4 or 0.2 mL of the aptamer solution during 3 h at ambient temperature. Unbound DNA was removed by washing with the same buffer. The amount of oligonucleotide coupled to the chromatographic support was estimated by subtracting the UV absorbance, at 260 nm, of the unbound DNA solution from the initial solution. It was equal to ∼26 nmol of biotinylated aptamer immobilized/mL of streptavidin media slurry. In addition, 1 mL of the streptavidin media slurry was mixed during 3 h at ambient temperature with 1 mL of ∼26 µM biotin dissolved in the aqueous buffer, corresponding to 26 nmol of biotin/mL of streptavidin media slurry. The streptavidinbiotin complex particles obtained were used as a control stationary phase since the number of streptavidin binding sites blocked by biotin was similar to the one of the DNA-modified streptavidin particles. Column Packing. The DNA modified particles were packed in-house into two microbore columns (370 × 0.76 mm for the D-adenosine aptamer and 250 × 0.76 mm for the L-tyrosinamide aptamer) using a self-pack packing device supplied by Applied Biosystems. Nonmodified streptavidin particles and streptavidinbiotin complex particles were also packed into two microbore columns (370 × 0.76 mm) used as control stationary phases. Nut and ferrule were placed on one end of the column body, which was attached to an end fitting containing a microbore frit (column outlet). The other end of the column (column inlet) was attached to the outlet of the packing device using nut and ferrule. The particle slurry was added to the packing device. The remaining volume of the packing device was filled with the aqueous buffer. The top of the packing device was connected to the HPLC pump. The pump was started at ∼0.1 mL/min and the flow rate was slowly adjusted to operate at a pressure of ∼1500 psi. Twenty milliliters of aqueous buffer was pumped through the column. The full packed column was then attached (column inlet) to another end fitting with a microbore frit. When not in use, the microbore columns were stored at 4 °C in the aqueous buffer. Apparatus. The HPLC system consisted of a LC Shimadzu pump 10AT (Sarreguemines, France), a Shimadzu SIL-10AD autoinjector, a Shimadzu SPD-10A UV-visible detector, a Shimadzu SCL-10A system controller with Class-VP software (Shimadzu), and an oven Igloocil (Interchim). Chromatographic Operating Conditions. Solute samples were prepared in the mobile phase and injected (100 nL) at least three times. The dead time was determined using sodium nitrate or potassium iodide. Measure of the retention time was assessed as previously described.19 The mobile-phase flow rate (50 µL/ min for the D-adenosine aptamer and the control streptavidin columns and 20 µL/min for the L-tyrosinamide aptamer column) was low enough to exclude any split peak effect. The linear elution conditions were tested and defined by injecting solutes at several concentrations. As an example, the sample load dependencies of the retention factor for D- and L-adenosine are presented in Figure 2. The representative curve exhibited a plateau at low sample load followed by a falloff above 140 pmol. For the thermodynamic analysis, the column temperature varied from 12 to 32 °C for the adenosine CSP and from 20 to 32 °C for the tyronisamide CSP. To assess whether the retention factor change with increasing temperature was due to a variation in the column

Figure 2. Solute amount dependencies of the retention factor (k) for D- and L-adenosine using the D-adenosine aptamer stationary phase: temperature, 20 °C; column, 370 × 0.76 mm; mobile-phase composition: phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0; flow rate, 50 µL/min. Error bars are within the experimental points.

binding capacity, the concentration dependencies of the solute retention factor were measured at different temperatures for the two CSPs. Similar ln k versus sample load curves were obtained for all the column temperatures. As previously stated by Tittelbach and Gilpin,22 this demonstrated that the number of active binding sites in the columns did not change when the column temperature varied. RESULTS AND DISCUSSION Resolution of Adenosine and Tyrosinamide Enantiomers Using Target-Specific Aptamer CSPs. The DNA aptamers used in this study were selected against the “natural” D-adenosine20 and L-tyrosinamide21 enantiomers (Figure 1). The highly conserved sequence specific of D-adenosine was expected to form a stable framework composed of two stacked G quartets20 while the secondary structure of the L-tyrosinamide specific sequence was not elucidated.21 They bind the target enantiomers with a dissociation constant in the micromolar range (Kd ) ∼6 µM for the D-adenosine-aptamer association20 and ∼45 µM for the L-tyrosinamide-aptamer association21). In both cases, no data were provided about a possible enantiorecognition of the selected oligonucleotide. The enantioseparation properties of both the adenosine and tyrosinamide aptamer CSPs were analyzed at various column temperatures using an aqueous buffer as eluent (phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0). For the two CSPs, the target (“natural”) enantiomer used for the selection procedure was significantly more retained by the column than the nontarget enantiomer (Figures 3 and 4). Retention factors, apparent enantioselectivity as well as resolution are presented in Table 1 for all the operating conditions. At the lowest temperature studied (12 °C), an apparent enantioselectivity of ∼3.7 was obtained for the adenosine enantiomers, a value similar to the typical enantioseparation factors reported with imprinted CSPs.24 For the tyrosinamide enantiomers, the apparent enantioselectivity was extremely important for a small molecule (∼80 at 20 °C). However, it is important to note that the tyrosinamide R values may have significant errors because the nontarget enantiomer is very weakly retained on the aptamer CSP. Such a chiral recognition for a small molecule (a nonsignificant retention for the first-eluted enantiomer) has been reported previously with (22) Tittelbach, V.; Gilpin, R. K. Anal. Chem. 1995, 67, 44. (23) Tanaka, Y.; Terabe, S. Chromatographia 1999, 49, 489. (24) Sellergren, B. J. Chromatogr., A 2001, 906, 227.

Figure 3. Chromatographic resolution of adenosine enantiomers using a D-adenosine target-specific DNA aptamer as CSP: column, 370 × 0.76 (i.d.) mm; mobile phase, phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0; amount of D-,L-adenosine injected, 70 pmol; injection volume, 100 nL; flow rate, 50 µL/min; detection at 260 nm.

Figure 4. Chromatographic resolution of tyrosinamide enantiomers using a L-tyrosinamide target-specific DNA aptamer as CSP: column, 250 × 0.76 (i.d.) mm; mobile phase, phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0; amount of D-,L-tyrosinamide injected, 70 pmol; injection volume, 100 nL; flow rate, 20 µL/min; detection at 224 nm.

the target-specific antibody CSPs (for amino acids; highest R ∼136)3 and some conventional CSPs such as a teicoplanin aglycon CSP (for N-acetylfluorophenylalanine; R ∼110)25 or a teicoplanin CSP (for N-acetyl amino acids; highest R f ∞).26 The high (25) Berthod, A.; Chen, X.; Kullman, J. P.; Armstrong, D. W.; Gasparrini, F.; D′Acquarica, I.; Villani, C.; Carotti, A. Anal. Chem. 2000, 72, 1767.

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Table 1. Chromatographic Resolution of the Enantiomers of Adenosine and Tyrosinamide on Their Respective Target-Specific DNA Aptamer CSPsa R

Rs

3.72 3.62 3.57 3.44 3.27 3.18

1.88 1.41 1.26 1.23 1.03 0.94

T (°C)

k

12 16 20 24 28 32

7.66 5.60 3.91 2.46 1.65 1.10

20 23 26 29 32

Tyrosinamide 2.59 78 1.72 50 1.14 31 0.51 19 0.28 11

Adenosine

3.13 1.36 1.14 0.82 nd

a Retention factor for the more retained enantiomer: k ) (retention time - dead time)/dead time. Apparent enantioselectivity: R ) (retention factor for the more retained enantiomer)/(retention factor for the less retained enantiomer). Resolution: Rs ) 2 × (distance of the two peak positions)/(sum of bandwidths of the two peaks). Relative standard deviation of the solute retention factors was typically less than 5%.

selectivity of the tyrosinamide aptamer CSP was further exemplified by testing the retention of a racemic mixture of analogues of tyrosinamide, i.e., tyrosine and tyrosine methyl ester. These racemates were not separated into their enantiomeric compounds nor did they bind to any substantial degree, i.e., the compounds eluted roughly in the void volume (data not shown). In addition, this observation emphasizes the importance of the L-tyrosinamide amido group in the specific binding to the CSP. Tanaka and Terabe23 previously studied, by capillary electrophoresis, the chiral recognition properties of avidin and streptavidin using a partial filling technique. They showed that basic avidin was useful for the chiral separation of acidic compounds while neutral streptavidin was able to discriminate the enantiomers of some acidic or basic analytes such as warfarin, dansylated amino acids, or trimipramine. To evaluate a possible contribution of immobilized streptavidin on the enantioselectivity of the aptamer CSPs, a microbore column was packed with nonmodified streptavidin Poros particles. When a racemic mixture of tyrosinamide was injected (in the same operating conditions) onto this nonmodified streptavidin column, the enantiomers were not separated and eluted roughly in the void volume whatever the column temperature (data not shown). This result indicates that the tyrosinamide enantioseparation is only dependent on the stereospecific interactions between the analytes and the L-tyrosinamide aptamer. On the other hand, the nonmodified streptavidin column was able to discriminate the enantiomers of adenosine. As an example, the D-adenosine enantiomer retention factors varied from 0.60 ( 0.003 at 20 °C to 1.09 ( 0.007 at 12 °C while the L-adenosine enantiomer was not significantly retained in both cases (Figure 5). However, when a racemic mixture of adenosine was injected onto a microbore column packed with the streptavidin-biotin complex particles (see the Experimental Section for details), the enantiomers were not separated nor did they bind to (26) Cavazzini, A.; Nadalini, G.; Dondi, F.; Gasparrini, F.; Ciogli, A.; Villani, C., J. Chromatogr., A, in press.

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Figure 5. Chromatograms obtained when a racemic mixture of adenosine was injected onto the nonmodified streptavidin (;) and streptavidin-biotin complex (‚‚‚) columns: columns, 370 × 0.76 (i.d.) mm; mobile phase, phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0; amount of D-,L-adenosine injected, 70 pmol; injection volume, 100 nL; flow rate, 50 µL/min; detection at 260 nm.

any substantial degree over the temperature range studied (Figure 5). This is in accordance with the data from Tanaka and Terabe23 who showed that the chiral recognition ability of avidin was lost when biotin was added, as it forms a strong complex with avidin in which the binding sites are blocked. So, although streptavidin possesses intrinsically chiral recognition properties for adenosine, this result demonstrates that the enantioselective ability of the D-adenosine aptamer CSP is only dependent on the aptamer specific binding sites in the operating conditions used in this study. Here, it can be noted also that, as expected, the tyrosinamide enantiomers were not separated using the streptavidin-biotin complex column (elution roughly in the void volume, data not shown). The results presented here are of great importance since they show that, although the DNA aptamers were not selected for enantioselective binding20,21 via a counter selection with the nontarget enantiomer,16 they are however able to discriminate the enantiomers. Therefore, as expected previously by Famulok and co-workers,15 it seems likely that an efficient SELEX procedure for high-affinity binding would result in the isolation of enantiospecific oligonucleotides. Of course, a necessary condition is that the target immobilization to the matrix must allow an adequate exposure of the enantiomer key functional groups to the oligonucleotide pool during the in vitro selection. Temperature Effects and Determination of the Thermodynamic Parameters. As reported by previous studies on the target-aptamer associations,19,27,28 temperature was a preponderant parameter in the solute binding to the immobilized aptamers (Table 1 and Figures 3 and 4). By increasing the column temperature by only 12 (for adenosine CSP) or 9 °C (for tyrosinamide CSP), a very significant reduction of the analysis (27) Zhai, G.; Iskandar, M.; Barilla, K.; Romaniuk, P. J. Biochemistry 2001, 40, 2032. (28) Cowan, J. A.; Ohyama, T.; Wang, D.; Natarajan, K. Nucleic Acids Res. 2000, 28, 2935.

Figure 6. Van’t Hoff plots ln k/ln R vs 1/T for D,L-adenosine (k ,retention factor; R, apparent enantioselectivity) using the D-adenosine aptamer stationary phase: temperature (T) range, 12-32 °C; column, 370 × 0.76 mm; mobile-phase composition, phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0; flow rate, 50 µL/min. Error bars are within the experimental points.

time with the baseline resolution of the racemic mixtures was attained (Figures 3 and 4). In linear conditions (see Experimental Section), the temperature dependence of retention factor (k) is given by the following relation

ln k )

mL -∆H ∆S + + ln RT R VM

(1)

where mL is the active binding site number in the column and VM the void volume of the chromatographic column. ∆H and ∆S are respectively the enthalpy and entropy of transfer of solute from the mobile to the stationary phase, T is the absolute temperature, and R is the gas constant. If the stationary phase, solute, and solvent properties are independent of temperature and ∆H and ∆S are temperature invariant, a linear van’t Hoff plot is obtained. The temperature dependence of apparent enantioselectivity (R) is given by the following relation

ln R )

ml2 -∆(∆H) ∆(∆S) + + ln RT R ml1

(2)

where ∆(∆H) and ∆(∆S) are the difference in enthalpy and entropy of transfer of the enantiomers from the eluent to the CSP, respectively. mL2 and mL1 are the active binding site number for the more and the less retained enantiomers, respectively. If the stationary phase, ∆(∆H) and ∆(∆S), solute, and solvent properties are independent of temperature, a linear van’t Hoff plot is obtained. Linear van’t Hoff plots ln k versus 1/T were obtained for the enantiomers of adenosine and L-tyrosinamide. The retention factor for D-tyrosinamide did not change substantially when the column temperature varied from 20 to 32 °C (kD-tyrosinamide ∼0.03). These van’t Hoff plots are shown in Figures 6 and 7, respectively. The regression coefficient R2 was higher than 0.9789 for the adenosine enantiomers and the L-enantiomer of tyrosinamide (R2 ) 0.4611 for the D-tyrosinamide plot). Using eq 1, the ∆H values were determined. Very large negative values were obtained varying from -65.6 ( 1.2 and -71.4 ( 0.7 kJ/mol for L- and D-adenosine enantiomers to -139.4 ( 2.0 kJ/mol for L-tyrosinamide. Such values are consistent with the formation of a very tight complex

Figure 7. Van’t Hoff plots ln k vs 1/T for D,L-tyrosinamide (k ,retention factor) using the L-tyrosinamide aptamer stationary phase: temperature (T) range, 20-32 °C; column, 250 × 0.76 mm; mobilephase composition, phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0; flow rate, 20 µL/min. Error bars are within the experimental points.

between these solutes and the aptamer CSPs, due to a large magnitude in the attractive interactions or a great number of interaction points. Using eq 2, the ∆(∆H) value was calculated for D-,L-adenosine from the linear van’t Hoff plot ln R versus 1/T (Figure 6). The regression coefficient R2 was 0.9692. ∆(∆H) was equal to -5.8 ( 1.0 kJ/mol, which is comparable to the enantioselective enthalpic contributions classically obtained on conventional29-32 or imprinted33,34 CSPs. The ∆(∆H) value for D-,L-tyrosinamide was not reported due to the inaccuracy in the estimation of the R values, as previously stated. Column Stability with Time. The stability of the columns was evaluated by comparing the solute retention factor before and after more than two months, in the same conditions. The retention factors for the adenosine enantiomers decreased by only 3.3% with no change in selectivity, indicating a good stability of the adenosine aptamer CSP. This is in accordance with the previous study using another DNA aptamer as CSP.19 In contrast, the performances of the L-tyrosinamide specific aptamer CSP decreased with time, characterized by a significant reduction in the retention factor of the target enantiomer. The decrease in the L-tyrosinamide retention factor (and then in the enantioselectivity) with time was associated with a significant deterioration of the column efficiency and peak shape (peak tailing), as expected for a reduction of the column saturation capacity.35 However, as shown in Figure 8, the partial resolution of enantiomers was still observed after more than two months at 20 °C by increasing the Stanton number,35 via a decrease in the flow rate from 20 to 8 µL/min. This diminution of the column saturation capacity was expected to be due to an oligonucleotide degradation or a loss of the aptamer active conformation. Experiments are now in process to provide satisfactory explanation about this phenomenon. (29) Castells, C. B.; Carr, P. W. Chromatographia 2000, 52, 535. (30) Peter, A.; Torok, G.; Armstrong, D. W.; Toth, G.; Tourwe´, D. J. Chromatogr., A 1998, 828, 177. (31) Yashima, E.; Yamamoto, C.; Okamoto, Y. J. Am. Chem. Soc. 1996, 118, 4036. (32) Loun, B.; Hage, D. S. Anal. Chem. 1994, 66, 3814. (33) Lin, J. M.; Nakagawa, T.; Uchiyama, K.; Hobo, T. Biomed. Chromatogr. 1997, 11, 298. (34) Sellergren, B.; Shea, K. J. J. Chromatogr., A 1995, 690, 29. (35) Gotmar, G.; Fornstedt, T.; Guiochon, G. J. Chromatogr., A 1999, 831, 17.

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Figure 8. Chromatographic resolution after more than two months of tyrosinamide enantiomers using a L-tyrosinamide target-specific DNA aptamer as CSP: column, 250 × 0.76 (i.d) mm; mobile phase, phosphate buffer 20 mM, KCl 25 mM, MgCl2 1.5 mM, pH 6.0; amount of D,L-tyrosinamide injected, 70 pmol; injection volume, 100 nL; flow rate, 8 µL/min; detection at 224 nm.

CONCLUSION In summary, the present results demonstrate that DNA aptamers, specifically selected against one enantiomer of small (bioactive) molecules, can be used successfully as target-specific

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CSPs. This is an important step toward the applicability of such an analytical method to biological and pharmaceutical fields. However, some major drawbacks could limit a broad practical use of these target-specific CSPs. A nonexhaustive list is presented here: (i) As can be seen in the present study for the tyrosinamide aptamer CSP, the oligonucleotide stability can be limited. (ii) The reversed-phase chromatographic mode is expected to be the only one that can be used. (iii) As reported previously,19 the slow kinetics of the aptamertarget complex formation and dissociation can be responsible for a low efficiency and peak tailing. (iv) Although the target specificity may be a very favorable feature, it may also cause some problems for quantification applications. As shown with the tyrosinamide CSP, the nontarget enantiomer and structurally closely related compounds elute in the void volume due to the very important selectivity of the aptamer stationary phase. Thus, it does not allow us to quantify any of these compounds as they elute as a single peak in the front. (v) The SELEX methodology requires highly sophisticated equipment, expensive reagents, and can be relatively timeconsuming. ACKNOWLEDGMENT This work was supported by the Fonds national de la science (ACI program “nouvelles me´thodologies analytiques et capteurs”). Received for review September 17, 2003. Accepted December 7, 2003. AC035090F