Identification of Chiral Selectors from a 200-Member Parallel

Selection of chiral selectors for the resolution of racemic. N-(1-naphthyl)leucine ester 1 was studied with a 200- member parallel library prepared on...
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Anal. Chem. 2000, 72, 5459-5465

Identification of Chiral Selectors from a 200-Member Parallel Combinatorial Library Yan Wang, Louis H. Bluhm, and Tingyu Li*

Department of Chemistry, Vanderbilt University, Box 1822-B, Nashville, Tennessee 37235

Selection of chiral selectors for the resolution of racemic N-(1-naphthyl)leucine ester 1 was studied with a 200member parallel library prepared on polymeric synthesis resin. Through this study, the library screening procedure developed previously is improved and other pertinent issues concerning the screening process are also addressed. The equilibration time required for screening is reduced from 24 h to ∼3 h. Excellent correlation of the outcome between the resin batch equilibration experiment and chromatographic separation is further demonstrated. It is also demonstrated that selectors with separation factors as low as 1.4 could be identified by this batch screening process. In addition, a great deal of information regarding enantioselective interactions was obtained in this parallel library study. Such information should prove useful in improving future library designs. In recent years, combinatorial libraries have evolved considerably for the development of selective binders for a given target molecule.2 In these techniques, a large number of compounds (the library) can be screened for a desired property. In a short period of time, combinatorial libraries have found widespread applications in both pharmaceutical research and material sciences.3 Applications of combinatorial libraries in enantioselective separations have also been reported.4 In fact, some of the early works in chromatographic enantioseparations, such as the rapid solution screening of chiral selectors by NMR1 or by CE,5 and the reciprocal development of chiral selectors,6 are combinatorial in nature. * To whom correspondence should be addressed: (phone/fax) 615 343 8466; (e-mail) [email protected]. (1) Abbreviations: NMM, N-methylmorpholine; PyBop, benzotriazolylox tris(pyrrolidino)phosphonium hexafluorophosphate; Fmoc, 9-fluorenylmethoxycarbonyl; DIC, diisopropylcarbodiimide; HOBt, 1-hydroxybenzotriazole; TFA, trifluoroacetic acid; DMAP, 4-(dimethylamino)pyridine; DIPEA, N,N-diisopropylethylamine; DCM, dichloromethane; IPA, 2-propanol; Fmoc-Osu, 9-Fluorenylmethyloxycarbonylhydroxysuccinimide. Z, benzyloxycarbonyl. (2) For some examples, see: (a) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84. (b) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84-86. (c) Janda, K. D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10779-10785. (d) Bunin, B. A. The Combinatorial Index; Academic Press: New York, 1998; pp 5-8. See also: Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555-600. (3) For a general review, see: Borman, S. Chem. Eng. News 1998, 76 (14), 47-67. (4) Examples in chiral separation are cited in refs 7 and 8. For examples in protein purification, see: (a) Huang, P. Y.; Carbonell, R. G. Biotechnol Bioeng. 1999, 63, 633-641. (b) Palombo, G.; De Falco, S.; Tortora, M.; Cassani, G.; Fassina, G. J. Mol. Recognit. 1998, 11, 243-246. 10.1021/ac000529e CCC: $19.00 Published on Web 10/03/2000

© 2000 American Chemical Society

Some of the more recent reports of the application of combinatorial libraries in enantioselective separations use mixture libraries,7 while others use libraries of pure components (parallel libraries).8 Certain advantages encompass each technique: Parallel libraries often offer a more straightforward screening process, while a mixture library approach allows a larger number of compounds to be analyzed more readily. Of the published examples of the application of parallel libraries to the development of chiral selectors, one involves the parallel microscale synthesis of potential chiral selectors onto silica gel, followed by screening of the parallel library with HPLC.8a Another recently reported method also involves the screening of chiral selectors on silica gel.8b The third parallel example is a reciprocal chromatographic assay of racemic library components.8c In this method, one enantiomer of the racemic analyte was immobilized onto a chromatographic support, and the resolution of individual racemic library components was tested using this analyte stationary phase by HPLC. We have also published a method to screen parallel combinatorial libraries for chiral selectors based on a batch assay of potential selectors on synthesis resin.9 In this procedure, a potential chiral selector (one parallel library member) is attached to a resin suitable for solid-phase synthesis. Racemic analyte in the proper solvent is then allowed to equilibrate with this resinbound potential selector. After an equilibration time, the enantiomeric ratio of the analyte in the supernatant is analyzed by circular dichroism. An enantiomeric excess in the supernatant as indicated by CD would suggest a selective adsorption of one of the two enantiomers to the resin, which in turn is indicative of a chiral selector. The suspected chiral selector would then be resynthe(5) (a) Armstrong, D. W. Pittcon′98, New Orleans, LA, March 1-5, 1998. (b) Armstrong, D. W.; Tang, Y.; Chen, S.; Zhou, Y.; Bagwill, C.; Chen, J.-R. Anal. Chem. 1994, 66, 1473-1484. (c) Armstrong, D. W.; Rundlett, K. L.; Chen, J.-R. Chirality 1994, 6, 496-509. (6) (a) Pirkle, W. H.; Welch, C. J.; Lamm, B. J. Org. Chem. 1992, 57, 38543860. (b) Welch, C. J. J. Chromatogr., A 1994, 666, 3-26. (7) (a) Murer, P.; Lewandowski, K.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1999, 71, 1278-1284. (b) Weingarten, M. D.; Sekanina, K.; Still, W. C. J. Am. Chem. Soc. 1998, 120, 9112-9113. (c) Vries, T.; Wynberg, H.; Echten, E. V.; Koek, J.; Hoeve, W. T.; Kellogg, R. M.; Broxterman, Q. B.; Minnaard, A.; Kaptein, B.; Sluis, S. V. D.; Hulshof, L.; Kooistra, J. Angew. Chem., Int. Ed. Engl. 1998, 37, 2349. (d) M. Chiari, V. Desperati, E. Manera, R. Longhi Anal. Chem. 1998, 70, 4967-4973. (e) Jung, G.; Hofstetter, H.; Feiertag, S.; Stoll, D.; Hofstetter, O.; Wiesmuller, K.-H.; Schurig, V. Angew. Chem., Int. Ed. Engl. 1996, 35, 2148-2150. (8) (a) Welch, C. J.; Protopopova, M. N.; Bhat, G. Enantiomer 1998, 3, 471476. (b) Tobler, E.; Lammerhofer, M.; Oberleitner, W. R.; Maier, N. M.; Lindner, W. Chromatographia 2000, 51, 65-70. (c) Lewandowski, K.; Murer, P.; Svec, F.; Frechet, J. M. J. Chem. Commun. 1998, 2237. (9) Wang, Y.; Li, T. Anal. Chem. 1999, 71, 4178-4182.

Analytical Chemistry, Vol. 72, No. 21, November 1, 2000 5459

Scheme 1. Preparation of Fmoc-Protected Diaminopropionic Acid (Dp)

Figure 1. The R and S enantiomers of racemic N-(1-naphthyl)eucine ester 1.

sized onto a chromatographic support, and resolution of the racemic analyte will be evaluated. Since well-established synthesis resin is used to prepare library members, libraries with good quality could be obtained. On the other hand, library synthesis on silica gel can be more challenging.10 Although the feasibility of this approach is demonstrated with our earlier model study,9 several important questions remain unanswered. For example, the initial screening procedure required 24 h to reach equilibrium. Could this equilibration time be reduced in order to increase the overall screening efficiency? The extent of correlation between the chiral resolution in a chromatographic experiment and the selective adsorption observed in a batch equilibration experiment also needs further investigation. In the previous model study, both selectors have relatively high separation factors (R > 4.7). Can this technique identify selectors with lower separation factors? Moreover, the library used in the model study contains only 16 members. Can more efficient selectors be readily developed using a larger library? Last, besides developing effective chiral selectors, what other information can be learned from a combinatorial library study? In an effort to address these issues, we studied the resolution of racemic N-(1-naphthyl)leucine ester 111 (Figure 1) with a 200member parallel library. EXPERIMENTAL SECTION1 General Supplies and Equipment. Solid-phase synthesis resins and amino acid derivatives were purchased from NovaBiochem (San Diego, CA). All other chemicals and solvents were purchased from Aldrich (Milwaukee, WI), Fluka (Ronkonkoma, NY), or Fisher Scientific (Pittsburgh, PA). HPLC grade Allsphere silica gel (particle size 5 µm, pore size 80 Å, and surface area 220 m2/g) was purchased from Alltech (Deerfield, IL). Selecto silica gel (32-63 µm) from Fisher Scientific was used for flash column chromatographic purification of target compounds. Thin-layer chromatography was completed using EM silica gel 60 F-254 TLC plates (0.25 mm; E.Merck, Merck KGaA, 64271 Darmstadt, Germany). Elemental analyses were conducted by Atlantic Microlab, Inc. (Norcross, GA). HPLC analyses were completed with a Beckman analytical gradient system (System Gold). Circular dichroism was measured with a Jasco J-720 spectropolarimeter (cell volume 0.40 mL; cell pass length 1 mm), while UV spectra were obtained with a Shimadzu UV 201 spectrometer (cell volume 3 mL; cell pass length 10 mm). The HI-TOP manual synthesizer required for parallel library synthesis is from Whatman Polyfiltronic (Rockland, MA). (10) For example, our efforts to synthesize peptides onto silica gel directly have been discouraging. See: Yang, A.; Gehring, A. P.; Li, T. J. Chromatogr., A 2000, 878, 165-170. (11) For the synthesis of this compound and its related chiral studies, see: (a) Pirkle, W. H.; Pochapsky, T. C. J. Am. Chem. Soc. 1986, 108, 352-354. (b) Pirkle, W. H.; Deming, K. C.; Burke, J. A. Chirality 1991, 3, 183-187.

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Chromatographic Measurements. Retention factor (k) equals (tr - t0)/t0, in which tr is the retention time and t0 is the dead time. Dead time t0 was measured with 1,3,5-tri-tert-butylbenzene as the void volume marker according to a literature procedure:12 column size 50 × 4.6 mm; flow rate 1.2 mL/min. In IPA/hexanes (2/8), t0 equals 0.42 min. In CHCl3/heptane (2/8), t0 equals 0.43 min. Preparation of Abu-AmPS Resin (Abu, 4-Aminobutyric Acid; AmPS, Aminomethylated Polystyrene). A mixture of Fmoc-Abu-OH (390 mg, 1.20 mmol), PyBop (625 mg, 1.20 mmol), and DIPEA (155 mg, 1.20 mmol) in DMF (10 mL) was added to 1 g (surface amino concentration 0.40 mmol/g) of AmPS resin that was swelled first in CH2Cl2 (10 min). After agitating at room temperature for 2 h, the resin (Fmoc-Abu-AmPS) was collected by filtration and washed with DMF, DCM, IPA, and DCM (10 mL × 2). The Fmoc protecting group was then removed by treatment of the resin with 10 mL of 20% piperidine in DMF for 20 min. The deprotected resin (Abu-AmPS) was collected by filtration and washed with DMF, DCM, IPA, and DCM (10 mL × 2). The surface Abu concentration was determined to be 0.40 mmol/g based on the Fmoc cleavage method.19 Preparation of Building Block Amino Acid Fmoc-Dp-OH (5, Scheme 1). (1) N2-Z-N3-Dn-diaminopropanoic Acid (3). To a solution of 213 (3.0 g,12.5 mmol) and DIPEA (1.61 g,12.5 mmol) in THF (60 mL), dinitrobenzoyl chloride (11.5 g, 50 mmol) in THF (40 mL) was added slowly. After stirring for 30 min, THF was evaporated under vacuum and the residue was dissolved in water (100 mL). The aqueous solution was extracted with EtOAc (30 mL × 3). The organic phase was combined and washed with water. After being dried over anhydrous Na2SO4, EtOAc was evaporated to give pure 3 as a white solid (4.4 g, 80%); mp 212213 °C. (12) Pirkle, W. H.; Welch, C. J. J. Liq. Chromatogr., A 1991, 14, 1-8. (13) Prepared according to: Waki, M.; Kitajima, Y.; Izumiya, N. Synthesis 1981, 266-267. (14) (a) Pirkle, W. H. Chirality 1997, 9, 103-104. (b) Wainer, I. W.; Caldwell, J. Chirality 1997, 9, 95-6. (15) Non-π-π interactions are more important in other type of chiral selectors. See: Berthod, A.; Liu, Y.; Bagwill, C.; Armstrong, D. W. J. Chromatogr., A 1996, 731, 123-137. (16) The Hi-top system, Polyfiltronics, 100 Weymouth St., Rockland, MA 02370. (17) Poole, C. F.; Poole, S. K. Chromatography today; Elsevier: New York, 1991; pp 350-353. (18) Yang, A.; Li, T. Anal. Chem. 1998, 70, 2827-2830. (19) NovaBiochem Catalog & Peptide Synthesis Handbook, 1999, S43. Method 12: Estimation of level of first residue. See also the last paragraph of this Experimental Section.

(2) N2-Fmoc-N3-Dn-diaminopropanoic Acid (5). To a solution of 3 (4.32 g, 10 mmol) in CH3CN (50 mL), trimethylsilyl iodide (3 g, 15 mmol) was added with stirring. After 10 min, MeOH (2 mL) was added to quench the reaction. The crude amino acid 4 was obtained by evaporating the solvents in a vacuum and used directly without further purification. The crude amino acid 4 dissolved in aqueous solution of Na2CO3 (9%, 25 mL) was then added to a solution of Fmoc-OSu (5.1 g, 15 mmol) in DMF (20 mL). The mixture was stirred for 20 min and then poured into water (500 mL). After the resulting aqueous solution was extracted with ether and EtOAc (100 mL × 2), its pH was adjusted to 1-2 with concentrated hydrochloric acid. The aqueous phase was then extracted with EtOAc (80 mL × 3). The combined EtOAc extract was washed with water. The crude product after evaporation of EtOAc was recrystallized from CH2Cl2-hexane (1:1) to yield pure Fmoc-Dp-OH (5) as a pale yellow solid (3.80 g, 73% based on 3): mp 210-211 °C; 1H NMR (DMSO-d6) δ 3.5 (m, 2H), 4.0-4.2 (m, 4H), 7.2-7.8 (m, 9H), 8.99.0 (d, 3H), 9.3 (s, 1H). Preparation of the Parallel 200-Member Library. The library was synthesized using the polyfiltronic HI-TOP manual synthesizer as described before.9 The experimental procedure for the synthesis of the DnLT member of the library is shown below. Other library members were prepared following a similar sequence. To 75 mg (0.030 mmol in Abu) of Abu-AmPS resin prepared above in one well of the 96-well unifilter microplate were added mixtures of Fmoc-T(tBu)-OH (36 mg, 0.090 mmol), PyBOP (47 mg, 0.090 mmol), and DIPEA (12 mg, 0.090 mmol) in 0.50 mL of DMF. After agitating for 2 h, the resins were filtered and washed with DMF, DCM, IPA, and DCM. The Fmoc protecting group was then removed by treatment with 0.60 mL of 20% piperidine in DMF for 20 min, followed by washing with DMF. Fmoc-L-OH and dinitrobenzoic acid were then coupled to the resin following an identical reaction sequence. After that, the side-chain protecting group of T was removed by reacting with 0.6 mL of 95% TFA (2.5% water and 2.5% triisopropylsilane in TFA) for 1 h. The resin was then washed with DMF, DCM, IPA, and DCM to yield the desired library member on the solid resin. Screening of the Parallel Library with Circular Dichroism Measurement. The resins synthesized above, each containing 0.030 mmol of selector, were transferred to wells of regular 96well plates. To each well that contained resin was added racemic N-(1-naphthyl)leucine ester 1 (1.2 mg, 0.0030 mmol) in CHCl3heptane (2:8, 0.6 mL). After incubating the mixture for 3 h, the supernatant in each well was transferred into a sample cell (volume 0.40 mL) of a Jasco J-720 CD spectropolarimeter, and the ellipticity at 260 nm was recorded. Preparation of DnLT (Dn-L-T-Abu-Silica Gel) Stationary Phase. To Rink acid resin (5 g, 0.6 mmol/g) preswelled with DCM (30 mL, 30 min) were added Fmoc-Abu-OH (3.90 g, 12 mmol), DIC (1.51 g, 12 mmol), DMAP (122 mg, 1 mmol), and NMM (606 mg, 6 mmol) in DCM-DMF (2:1, 30 mL). After the mixture was agitated for 5 h, the resin was collected by filtration and washed with DMF, IPA, and DCM (20 mL × 2). The surface concentration for Fmoc-Abu-ORink resin was determined to be 0.57 mmol/g by the Fmoc cleavage method.19 The Fmoc group was then removed by treatment with 20% piperidine in DMF (30 mL) for 30 min. The deprotected Abu-

ORink resin was collected and washed with DMF, IPA, and DCM (20 mL × 2). To Abu-ORink resin (2 g, 1.14 mmol) prepared above were added Fmoc-T(tBu)-OH (1.42 g, 3.6 mmol), PyBOP (1.25 g, 2.4 mmol), HOBt (320 mg, 2.4 mmol), and DIPEA (620 mg,4.8 mmol) in DMF (10 mL). After agitating for 2 h, the resin was filtered and washed with DMF, IPA, and DCM (10 mL × 2). Surface concentration of Fmoc-T(tBu)-Abu-ORink was determined to be 0.55 mmol/g using the Fmoc cleavage method. The Fmoc group was then removed by the same procedures discussed in the previous paragraph. The T (tBu)-Abu-ORink resin was then coupled with Fmoc-LOH, by following exactly the same procedure as described above. The surface concentration of Fmoc-L-T(tBu)-Abu-ORink was 0.54 mmol/g. After removing the Fmoc group, dinitrobenzoic acid was coupled to L-T(tBu)-Abu-ORink resin. This coupling reaction was repeated one more time to ensure complete reaction. The reaction conditions are identical to those used for the coupling of FmocT(tBu)-OH to the resin. The Dn-L-T(tBu)-Abu-ORink resin was then treated with 1% TFA in DCM (10 mL, 5 min) to release Dn-L-T(tBu)-Abu-OH from the resin. The crude product obtained was purified by flash column chromatography on silica gel (mobile phase 10%MeOH in DCM) to yield the desired Dn-L-T(tBu)-Abu-OH as a white solid (460 mg, 67%): 1H NMR (DMSO-d6) δ 0.9-1.2 (m, 18H), 1.581.89 (m, 5H), 2.1 (t, 2H), 3.0 (m, 2H), 4.0 (m, 1H), 4.2 (m, 1H), 4.7 (m, 1H), 7.9 (br, 1H), 8.4 (br, 1H), 9.0-9.2 (m, 3H), 10.2 (br, 1H). A mixture of Dn-L-T(tBu)-Abu-OH (330 mg, 0.58 mmol), PyBOP (302 mg, 0.58 mmol), HOBt (40 mg, 0.29 mmol), and DIPEA (115 mg, 0.89 mmol) in DMF (5 mL) was then added to aminopropyl silica gel (0.70 g, 0.29 mmol in amino group). After agitating for 4 h, the silica gel was collected by filtration and washed with DMF, IPA, and DCM (5 mL × 2). Acetyl chloride (100 mg, 1.2 mmol) and DIPEA (155 mg, 1.2 mmol) in 5 mL of DMF (5 mL) were subsequently added to end-cap unreacted amino groups. After 20 min, the silica gel was collected by filtration. The t-Bu protecting group of Thr(T) was then removed by reacting with 5 mL of 95% TFA (2.5% water and 2.5% triisopropylsilane in TFA) for 1 h. The desired CSP was collected and washed with DCM, DMF, IPA, and methanol and then dried at 80 °C for 5 h. The surface ligand concentration was determined to be 0.22 mmol/g based on elemental analysis.18 Determination of the Amount of Fmoc Group Present on Resins (Fmoc Cleavage Method). To ∼20 mg of resin, was added 3 mL of 20% piperidine in DMF in a quartz UV cuvette. After the mixture was gently agitated for 3-5 min, the resin was allowed to settle to the bottom of the cuvette. The cuvette was then placed into the UV spectrophotometer, and the absorbance of the sample at 290 nm was recorded with a solution of 20% piperidine in DMF as the reference cell. The amount of the Fmoc group on the resin was then determined by comparing the UV absorbance with a calibration curve generated by cleaving known amounts of Fmoc-Gly-OH following similar procedures. RESULTS AND DISCUSSION The library is a dipeptide library consisting of three modules: an end-capping module (module 1) and two amino acid modules Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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Scheme 2. Preparationof Dn-L-T Member of the Parallet Librarya

a Conditions: (a) Fmoc-Abu-OH, PyBop. (b) (1) Piperidine; (2) Fmoc-T(tBu)-OH, PyBop. (c) (1) Piperidine; (2) Fmoc-L-OH, PyBop. (d) Piperidine; (2) Dnb-OH. (e) TFA.

Figure 2. The 200-member Library. Abu, 4-aminobutyric acid (NH2(CH2)3CO2H); AmPS, aminomethylated polystyrene resin.

(modules 2 and 3) (Figure 2). Module 1 consists of only two components, the acetyl group (Ac) and the electron-deficient dinitrobenzoyl group (Dn). Modules 2 and 3 have identical components: both contain the same 10 amino acids. All the possible combinations of three modules yield a library containing a total of 200 members. These modules are chosen based upon consideration of the molecular features of the target analyte. The analyte contains an electron-rich, N-substituted naphthalene ring in conjunction with some hydrogen bond donor and hydrogen bond acceptor groups. According to the chiral interaction model, a minimum of three interaction sites between the chiral selector and the analyte is needed to achieve enantioselective recognition.14 Some of these three interaction sites must be attractive, so that the analyte and the chiral selector can interact closely with each other. The electron-deficient Dn group in module 1 and also in diaminopropionic acid (Dp) of modules 2 and 3 could interact with the electron-rich, N-substituted naphthalene ring through attractive π-π interaction. Various attractive hydrogen-bonding and repulsive steric interactions are possible between the analyte and the individual library components.15 All the library building blocks in this study are commercially available, with the exception of Dp. This amino acid introduces an electron-deficient aromatic ring that is absent from natural amino acids, and its Fmoc-protected form (the one needed for library synthesis) was prepared from known amino acid 2 (Scheme 1). The resin chosen for the synthesis of this library is aminomethylated polystyrene (AmPS) resin, a derivative of the widely used Merrifield resin. The synthesis of this library was performed conveniently using a Hi-top filter plate manual synthesizer.16 In this synthesizer, resin can be added to the wells of a 96-well filter plate. By adding individual amino acids into each well, 96 different peptides can be synthesized in one run. This Hi-top system allows for quick filtration between each synthetic step without the need to remove any resin from the plate, and the overall efficiency of this synthetic process is high. In terms of the chemistry involved, Fmoc solid-phase synthesis was chosen, and the detailed chemistry is illustrated in Scheme 2 with the synthesis of the Dn-Leu5462

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Thr (DnLT) member of the library. In this synthesis, commercially available, side-chain protected form of Thr, the N-R-Fmoc-O-tertbutyl-L-threonine (Fmoc-T(tBu)-OH), is needed. Other library members were synthesized following similar reactions. For screening purposes, library components on the resins were transferred into wells of regular 96-well plates. To each of the 200 wells that contained 0.03 mmol of selector was added an equal amount of the racemic analyte (1.2 mg, 0.0030 mmol) in a mixture of chloroform and heptane (2:8, 0.60 mL). After equilibration, the ellipticity (mdeg) of the supernatant in each well was measured at 260 nm, the maximum CD adsorption wavelength of the enantiomerically pure N-(1-naphthyl)leucine ester 1. The data obtained for all 200 wells are summarized in Table 1. In contrast to the previous experiment in which the equilibration assay is performed using IPA-hexane, the equilibration time is reduced from 24 to 3 h using 2:8 CHCl3-heptane (Figure 3). The significant decrease in equilibration time increases the overall efficiency of screening process and may be attributed to the swelling property of the polymeric resin. It is known that chlorinated solvents are generally excellent solvents for swelling organic polymers. By including chloroform in the equilibration system, the swelling of the organic polymer, which is essential for the thorough interaction of analyte with immobilized library members, could be achieved more quickly. As seen from Table 1, high ellipiticity is observed with many library members, especially when Dn is used in module 1. Generally speaking, in the absence of significant amount of adsorption on the resin, which is the case in this study as evidenced by the UV absorbance measurements reported in Table 2, high ellipiticity of the supernatant implies high enantioselectivity of adsorption to the solid resin. Therefore, many chiral selectors are observed in this library. In module 2, Trp (W), Asn (N), and Tyr (Y) have a higher tendency to provide effective selectors (refer to rows 4, 7, and 9 of Table 1). In module 3, Trp (W), Ser (S), Thr (T), and Tyr (Y) are effective in providing chiral selectors (see columns 4, 5, 6, and 9 of Table 1). Interestingly, side chains of all these amino acids are capable of forming hydrogen-bonding interactions. The behavior of Thr (T) is somewhat surprising; several highly efficient selectors were observed when Thr was used in module 3 but not in module 2. Another surprising observation is that Dp in both modules 2 and 3 failed to provide effective chiral selectors, although Dn proved crucial in module 1. It is also apparent that, in general, Ser (S) is less effective than Thr (T), and Gln (Q) is less effective than Asn (N) in inducing enatioselectivity.

Table 1. Ellipticities (mdeg) Measured at 260 nm for Each Member of the Librarya DnLL +4.0 DnFL +5.8 DnPL +1.5 DnWL +10.0 DnSL +5.7 DnTL +3.9 DnNL +22.6 DnQL +4.1 DnYL +8.6 DnDpL +3.5 AcLL +2.4 AcFL 0 AcPL +0.3 AcWL +0.2 AcSL +1.0 AcTL -0.11 AcNL +1.5 AcQL +0.5 AcYL -0.1 AcDpL +0.1

DnLF +3.2 DnFF +5.5 DnPF +1.2 DNWF +4.8 DnSF +1.9 DnTF +3.9 DnNF +14.1 DnQF +2.4 DnYF +5.2 DnDpF +0.75 AcLF -0.5 AcFF 0 AcPF -0.36 ACWF -0.3 AcSF 0 AcTF +1.1 AcNF +0.7 AcQF +1.6 AcYF +0.5 AcDpF +0.4

DnLP +10.2 DnFP +9.1 DnPP -0.25 DnWP +5.4 DnSP +3.1 DnTP +2.5 DnNP +1.0 DnQP +0.7 DnYP +2.8 DnDpP +3.0 AcLP +0.6 AcFP 0 AcPP +0.6 AcWP +0.6 AcSP +0.3 AcTP +0.4 AcNP -0.25 AcQP +0.4 AcYP +1.2 AcDpP +0.6

DnLW +11 DnFW +4.8 DnPW +1.5 DnWW +11 DnSW +4.7 DnTW +2.0 DnNW +10.0 DnQW +2.7 DnYW +14.4 DnDpW +3.7 AcLW +0.2 AcFW -0.4 AcPW -0.1 AcWW -0.1 AcSW -0.9 AcTW +0.7 AcNW -0.23 AcQW -0.4 AcYW +0.3 AcDpW +1.1

DnLS +6.5 DnFS +6.0 DnPS 0 DnWS +12.4 DnSS +3.7 DnTS +2.4 DnNS +15.0 DnQS +4.0 DnYS +13.8 DnDpS +3.2 AcLS +0 AcFS +0.4 AcPS +0.8 AcWS +0.3 AcSS +0.4 AcTS +1.4 AcNS -0.3 AcQS -0.4 AcYS +0.5 AcDpS -0.8

DnLT +16.8 DnFT +6.0 DnPT +1.3 DnWT +24.0 DnST +7.5 DnTT +6.1 DnNT +19.0 DnQT +6.7 DnYT +23 DnDpT +5.0 AcLT -0.4 AcFT -0.4 AcPT -0.2 AcWT +0.4 AcST 0 AcTT +1.2 AcNT +0.2 AcQT +0.6 AcYT +1.0 AcDpT +1.5

DnLN +6.8 DnFN +2.7 DnPN +0.1 DnWN +10.3 DnSN +3.5 DnTN +3.3 DnNN +11.0 DnQN +2.9 DnYN +11.9 DnDpN +1.6 AcLN -0.1 AcFN +0.25 AcPN -0.15 AcWN +0.5 AcSN -0.1 AcTN +0.3 AcNN 0 AcQN +2.4 AcYN -0.2 AcDpN +0.1

DnLQ +2.4 DnFQ +6.7 DnPQ -0.4 DnWQ +9.0 DnSQ +3.0 DnTQ +3.1 DnNQ +5.2 DnQQ +1.9 DnYQ +9.1 DnDpQ +2.0 AcLQ -0.5 AcFQ -0.4 AcPQ -0.4 AcWQ +0.6 AcSQ +0.5 AcTQ 0 AcNQ +0.4 AcQQ 0 AcYQ +0.3 AcDp +0.7

DnLY +11.2 DnFY +11 DnPY 0 DnWY +14 DnSY +5.5 DnTY ‘+8.5 DnNY +8.5 DnQY +5.4 DnYY +18.6 DnDpY +3.0 AcLY +0.6 AcFY -0.3 AcPY +0.15 AcWY +0.3 AcSY -0.8 AcT 0 AcNY +0.6 AcQY 0 AcYY +0.7 AcDpY +1.0

DnLDp +4.0 DnFDp +5.1 DnPDp +1.3 DnWDp +5.6 DnSDp +2.4 DnTDp +3.0 DnNDp +6.3 DnQDp +2.9 DnYDp +9.6 DnDpDp +2.3 AcLDp 0 AcFDp -0.6 AcPDp +1.5 AcWDp +0.4 AcSDp -0.14 AcTDp +0.7 AcNDp 0 AcQDp +0.5 AcYDp -0.25 AcDpD p-0.3

a Equilibration solvents, CHCl -heptane (2:8). The noise level of the ellipticity measurement as detemined with previously designed Gly negative 3 controls9 is about (0.5 mdeg.

Table 2. Chiral Resolution of Racemic N-(1-Naphthyl)leucine Ester 1a mobile phase (CHCl3-heptane 2:8)

mobile phase (IPA-hexanes 2:8) CSPs

kr

R

ellipticity

R/S

A254

kr

R

ellipticity

R/S

A254

DnLT DnYT DnWT DnNT DnLY DnSY AcLL

0.68 0.83 0.64 0.78 0.56 0.84 0.07

15 7.8 9.7 4.4 6.2 2.3 1

+18 +9.4 +12 +9.1 +6.9 +3.7 +1.4

1.56 1.28 1.38 1.25 1.16 1.08 1.03

0.69 0.63 0.62 0.68 0.75 0.78 0.81

1.7 1.5 2.8 2.6 2.1 2.4 0.32

17 11 18 6.3 9.4 2.2 1.4

+17 +23 +24 +19 +11 +5.5 +2.2

1.43 1.72 1.73 1.46 1.23 1.12 1.04

0.64 0.58 0.60 0.68 0.70 0.67 0.76

a k is the retention factor of the least retained R enantiomer. Separation factor R ) k /k . R/S, A r s r 254 refer to the R/S ratio, the UV absorbance of the supernatant in the screening experiment. A254 of the stock solution used for the screening experiment is 0.82 in IPA-hexanes, 0.80 in CHCl3-heptane.

The fact that many more effective selectors are found when the dinitrobenzoyl group is used in module 1 indicates that the π-π interaction between the electron-rich naphthalene group and the electron-deficient dinitrobenzoyl group is important for enantioselective recognition. Following a model proposed for a similar system,6b one can formulate an interaction model of the analyte and library members when the Dn group is in module 1 (Figure 4). In this model, the π-π interaction between the electron-rich aromatic and the electron-deficient Dn provides one attractive interaction. Additional interactions could be generated by the

attractive hydrogen-bonding and/or the repulsive steric interactions. On the basis of this π-π interaction model, one would also expect that many effective chiral selectors would be developed when Dp is in module 2 or 3. The failure to observe such selectors may result from the fact that, in this amino acid, the Dn group is separated from the chiral center not only by the amide NH, as in the case when it is used in module 1, but also by an additional methylene unit (Figure 5). The additional methylene unit introduces more conformational flexibility, which generally has an Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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Scheme 3. Synthesis of DnLT Chiral Stationay Phasea

Figure 3. Solvent and time dependence of the resin equilibration experiments. Selector DnYT. Solvents: chloroform-heptane 2:8; IPA-hexanes 2:8.

Figure 4. Possible interactions of some library members with the analyte: (a) π-π interaction; (b) hydrogen-bonding and/or steric interactions.

Figure 5. Comparison of dinitrobenzoyl group in module 1 versus in amino acid DP, Thr versus Ser, and Asn versus Gln in chiral recognition. The arrow refers to the additional methylene unit between the chiral center and the potential attractive interaction site. See text.

undesirable effect in enantioselective separation. From this observation, it seems that when a chiral selector is being designed, it would be advantageous to keep the attractive interaction site and the chiral center in close proximity. The relative efficiency of Ser as compared to structurally similar Thr and Gln as compared to structurally similar Asn (N) could be explained in a similar manner. In Thr, the potential attractive interaction site (the OH group) is directly attached to a chiral center, whereas the OH group of Ser is attached to an achiral methylene unit. In Gln, one extra methylene unit between the potential attractive interaction site (the CONH2 group) and the chiral center exists, as compared to Asn (Figure 5). 5464

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a Conditions: Fmoc-Abu-OH, DIC. (b) (1) Piperidine; (2) FmocT(tBu)-OH, PyBop. (c) (1) Piperidine; (2) Fmoc-L-OH, PyBop. (d) (1) Piperidine; (2) dintrobenzoyl acid, PyBop. (e) AcOH. (f) Aminopropyl silica gel, PyBop. (g) TFA.

The observation that Thr is more effective when used in module 3 than in module 2 presents a more complicated situation. According to the interaction model outlined above, amino acids in module 3 may be too far away to interact effectively with the analyte if the chiral selector adopts an extended conformation. However, the chiral selector could adopt a more compact conformation that may interact effectively with the analyte. A detailed interaction model warrants a careful computational study, which is to be performed. It should be noted that some of the library members with the acetyl group in module 1 also demonstrate enantioselectivity, albeit with lower ellipiticities. For example, AcLL has a modest ellipiticity of +2.4. In this case, the chiral recognition model could be quite different from those discussed above. Several representative selectors were subsequently immobilized onto silica gel and the resulting stationary phases packed into columns using a standard slurry packing method.17 For immobilization, the peptide selector needed to be synthesized on a larger scale and in a form that can be immobilized onto a solid resin readily. The chemistry necessary to immobilize one selector is shown in Scheme 3. Other chiral selectors were immobilized following similar reactions. The key in this synthetic scheme was the use of an acidsensitive resin (Rink acid resin) to prepare a peptide acid fragment in which the side chain (OH) of Thr was still protected. The protected peptide acid fragment was then coupled to aminopropyl silica gel. After coupling to the solid support, the OH protecting group of Thr was removed by treatment with TFA, yielding the desired stationary phase on the solid support. The surface ligand concentration was determined to be 0.22 mmol/g, by following procedure published previously.18 Resolution of racemic naphthyl leucine ester 1 was studied with these columns, and separation factors obtained are tabulated (Table 2). As seen from Table 2, when chloroform-heptane was used as the mobile phase, all the selectors could resolve the racemic analyte. Separation factors up to 18 have been observed. When IPA-hexanes (2:8), which is different from the equilibration solvent used for library screening, was used as the mobile phase, all except AcLL resolved the racemic analyte. With chiral selector AcLL, the resolution experiment was also studied using 1:9 IPAhexanes. Even in this less polar solvent system, the racemic

mixture still could not be resolved, although the retention factor (0.58) is higher. For the selectors in Table 2, the screening experiments were repeated using IPA-hexanes as the equilibration solvent. The R/S ratio of analyte in each supernatant in the screening experiments as calculated from the measured ellipticity is shown in Table 2. For this calculation, the total amount of the analyte in the supernatant and the specific ellipiticity of analyte 1 are needed. The total amount of the analyte in the supernatant after equilibration can be determined by UV measurement, as described earlier.9 The specific ellipticity of (R)-1 is + 60 mdeg‚mL‚mg-1‚mm-1 in CHCl3-heptane (2:8) and + 50 mdeg‚mL‚mg-1‚mm-1 in IPAhexanes (2:8), based on the CD spectra of enantiomerically pure (R)-1. The data in Table 2 demonstrate an excellent correlation between the batch screening experiments and the chromatographic experiments. Library members with high ellipiticities could resolve racemic mixtures with excellent separation factors, while library members with low ellipiticites may fail to resolve the racemic analyte. These results also demonstrate that separation factors as low as 1.4 could be identified with these batch screening experiments. Synthetic efficiency of the library on PS resin is also studied. In the synthesis of DnLT on polymeric resin, the coupling yields of Fmoc-Abu to AmPS resin, Fmoc-T(tBu) to Abu-AmPS, and Fmoc-L to T(tBu)-Abu-AmPS are all very close to 100% (>99.5%), as determined by the Fmoc cleavage method.19 This example, along with experiments reported previously,9 demonstrates that library synthesis could indeed be achieved in high efficiency on a polystyrene resin. It should be pointed that the entire library synthesis, library screening, stationary-phase preparation, and evaluation could be repeated in about three weeks by an experienced researcher without any automation. With proper automation, development of a new selector could be accomplished in one week if all experiments were to proceed as planned. As a result, the highthroughput potential of this library screening process is enormous. CONCLUSIONS Results of this study demonstrated that this parallel library screening procedure has been improved; in addition, other important issues concerning this screening method were also answered. The equilibration time was reduced from 24 to 3 h by including chloroform in the equilibrating solvent. Excellent cor(20) Berthod, A.; Chang, S.-C.; Armstrong, D. W. Anal. Chem. 1992, 64, 395404. (21) Pancoska, P.; Bitto, E.; Janota, V.; Keiderling, T. A. In Vibrational Optical Activity: From Fundamentals to Biological Applications; The Faraday Division of the Royal Society of Chemistry: London, 1994; pp 287-311.

relation of the outcome of the resin batch equilibration experiment and chromatographic separation was further demonstrated. It was also shown that selectors with separation factors as low as 1.4 could be identified by this batch screening process. Even with this rather simple library, several selectors with high separation factors were identified. In addition to ascertaining useful chiral selectors, a large amount of data regarding enantioselective interactions was also obtained. These data should also prove useful in understanding the forces governing enantioselective separation, which in turn could lead to improved library design for such a purpose. It should also be pointed out that this kind of information regarding enantioselective interaction could also be obtained by studying the resolution of a large number of racemic analytes on a fixed chiral stationary phase, as demonstrated in a previous study.20 One, however, should exercise caution when interpreting data obtained from combinatorial library studies. The trend observed in one library study may not be applicable to other library studies and should be treated only as a “rule of thumb”. For example, it is entirely possible that, for a particular system, further separation between the attractive site and the chiral center might actually prove beneficial. Nonetheless, for the majority of analytes, a closer proximity between the attractive site and the chiral center of the chiral selector should prove more desirable. Other limitations exist to this parallel library screening approach. First, circular dichroism measurement is not applicable to compounds with low or no molar ellipticity at UV or visible wavelength. However, in these cases, one still can screen the libraries using other relatively slower techniques such as chiral LC equipped with an evaporative laser scattering detector or vibrational CD.21 The second limitation is the possible discrepancy between the outcomes of the screening experiment and the chromatographic experiments. The consolation to this limitation is that one is probably only interested in large separation factors when investigating selectors with a combinatorial library approach. With significant chiral separation, chiral selectivity observed in the batch process on the synthesis resin is very likely to be reproduced in a chromatographic experiment, as our results have demonstrated. ACKNOWLEDGMENT The financial support from Vanderbilt University, in the form of a start-up fund, research council, and natural science fund is appreciated.

Received for review May 11, 2000. Accepted August 22, 2000. AC000529E

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