Anal. Chem. 1999, 71, 4178-4182
Screening of a Parallel Combinatorial Library for Selectors for Chiral Chromatography Yan Wang and Tingyu Li*
Department of Chemistry, Box 1822-B, Vanderbilt University, Nashville, Tennessee 37235
A method to screen parallel combinatorial libraries for chiral selectors is described. Crucial elements to such an approach are the efficient syntheses of potential chiral selectors on synthesis resins and the rapid screening of selectors attached to these resins with circular dichroism measurement. The method does not require any immobilization of the analyte and could complement the mixture combinatorial library method developed earlier in this laboratory. The feasibility of this parallel library screening procedure is demonstrated with a model study of the chiral HPLC resolution of (1-naphthyl)leucine ester (1) using a 16-member small library. Recently, we reported a method to screen mixture combinatorial libraries for chiral selectors based on the application of two enantiomeric libraries.1 While a large number of compounds can be synthesized and screened efficiently using this mixture library approach, analytes of interest need to be immobilized. Since the immobilization of analytes can be difficult to achieve, we became interested in exploring alternative library screening methods which avoid the necessity for such analyte immobilization. In this article, we would like to report a method for the development of chiral selectors from parallel combinatorial libraries that does not require the immobilization of analyte. In contrast to a mixture library approach in which the library is synthesized as a mixture of compounds and the entire mixture is evaluated for a desired property, library components are synthesized and screened separately in a parallel library approach.2 The parallel library approach is similar to a traditional trial and error approach except that a large number of compounds is designed to be studied quickly. In this context, the reciprocal development of chiral selectors3 and the rapid solution screening of chiral selectors by NMR or by CE4 could be considered as early examples of the application of parallel libraries in chiral chromatography. * To whom correspondence should be addressed. Phone/Fax: 615 343 8466. E-mail:
[email protected]. (1) Wu, Y.; Wang, Y.; Yang, A.; Li, T. Anal. Chem. 1999, 71, 1688-1691. (2) 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) (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. (4) (a) Armstrong, D. W. Pittcon ’98, New Orleans, 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.
4178 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Of the more recent 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 chiral HPLC.5 This method appears to be well-suited for the development of chiral selectors, except that reactions on silica gel may not work well.6 Chiral HPLC is also a relatively slow technique to screen a parallel library. The other parallel example is a reciprocal chromatographic assay of racemic library components.7 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. This method requires the pre-immobilization of the analyte. EXPERIMENTAL SECTION8 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 either 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) (Emerck, 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, USA). Preparation of Abu-AmPS Resin (Abu: 4-Aminobutyric Acid. AmPS: Aminomethylated Polystyrene). A mixture of Fmoc-AbuOH (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 (5) Welch, C. J.; Protopopova, M. N.; Bhat, G. Enantiomer 1998, 3, 471-476. (6) Our efforts to synthesize peptides onto silica gel directly have been discouraging. Yang, A.; Gehring, A. P.; Li, T., to be submitted for publication. (7) Lewandowski, K.; Murer, P.; Svec, F.; Frechet, J. M. Chem. Commun. 1998, 2237. (8) All experiments were performed at RT (about 22 °C) unless otherwise noted. 10.1021/ac9905017 CCC: $18.00
© 1999 American Chemical Society Published on Web 09/03/1999
amino concentration, 0.40 mmol/g) of AmPS resin that was swelled first in DCM (10 min). After agitating at room temperature for 2 h, the resin (Fmoc-Abu-AmPS) was collected 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 0.44 mmol/g on the basis of a Fmoc cleavage method.15,16 Preparation of Parallel 4 × 4 Library: [Anth, Bz, Dnb, Naph] -L-[Ala, Gly, Leu, Pro]-Abu-AmPS. The library was synthesized using the polyfiltronic HI-TOP manual synthesizer. Specifically, 960 mg of Abu-AmPS resin prepared above was equally distributed into 16 wells of a 96-well unifilter microplate (0.026 mmol in Abu in each well). Four identical mixtures of L-Fmoc-Ala-OH (16.4 mg, 0.0528 mmol), PyBOP (28.0 mg, 0.0528 mmol), and DIPEA (7.0 mg, 0.053 mmol) in 0.50 mL of DMF were added to four of the 16 wells. Similarly, four mixtures containing L-Fmoc-Leu-OH (18.6 mg, 0.0528 mmol), four containing Fmoc-Gly-OH (15.7 mg, 0.0528 mmol), and four containing L-Fmoc-Pro-OH (17.8 mg, 0.0528 mmol) along with their corresponding coupling reagents in 0.50 mL of DMF were added to the remaining 12 wells. 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, DCM, IPA and DCM. The acid module (Anth-OH, Bz-OH, Dnb-OH and Naph-OH) was then coupled to the deprotected R-amino acid-Abu-AmPS resin in the combination necessary to produce a 4 × 4 library, following the procedures described above for the attachment of amino acids to the Abu-AmPS resin. (9) 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. (10) For other examples of peptide libraries in chiral separation, see: (a) Jung, G.; Hofstetter, H.; Feiertag, S.; Stoll, D.; Hofstetter, O.; Wiesmuller, K.-H.; Schurig, V. Angew. Chem., Int. Ed. Engl. 1996, 35, 2148-2150. (b) Weingarten, M. D.; Sekanina, K.; Still, W. C. J. Am. Chem. Soc. 1998, 120, 9112-9113. (c) Chiari, M.; Desperati, V.; Manera, E.; Longhi, R. Anal. Chem. 1998, 70, 4967-4973. (d) Murer, P.; Lewandowski, K.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1999, 71, 1278-1284. See also ref 5. (11) The Hi-top system, Polyfiltronics, 100 Weymouth Street, Rockland, MA 02370. (12) Poole, C. F.; Poole, S. K. Chromatography today; Elsevier: New York, 1991; pp 350-353. (13) Retention factor (k′) equals (tr - t0)/t0 in which tr is the retention time and t0 is the dead time. The separation factor (R) equals k′2/k′1. Dead time t0 was measured with 1,3,5-tri-tert-butylbenzene as the void volume marker according to Pirkle, W. H.; Welch, C. J. J. Liq. Chromatogr. A 1991, 14, 1-8. (14) R-1 contains about 4% S-1, S-1 contains about 4% R-1. See ref 1. (15) This yield is calculated on the basis of the manufacturer’s certification of the AmPS resin. 110% yield indicates that the actual surface amino concentration is higher than manufacturer’s suggested value. Therefore, this coupling yield may not reflect accurately the efficiency of the coupling reaction. However, the subsequent coupling of Fmoc-Leu to Abu-AmPS, in which the Abu amount is determined accurately by the Fmoc cleavage method (see ref 16), should be a very reliable measurement of the quality of the coupling reaction. Similar argument could be made concerning the AmTG resin. (16) Method 12: Estimation of level of first residue. NovaBiochem Catalog & Peptide Synthesis Handbook; Nova Biochem: San Diego, CA, 1999; p S43. See also Experimental Section. (17) 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.
Synthesis of the 4 × 4 Library on the AmTG (Aminomethylated Tentagel) Resin. The parallel 4 × 4 library on Am-TG was prepared by following the procedure described above for the preparation of the L 4 × 4 library on AmPS resin. The only difference is that the amount of the AmTG resin used is increased from 60 to 100 mg in each well, as the surface amino group concentration (0.25 mmol/g) of the AmTG is lower than that of the corresponding AmPS resin. Screening of the Parallel 4 × 4 Library with Circular Dichroism Measurement. The resins synthesized above, each containing 0.026 mmol of selector, were transferred to 16 wells of a regular 96well plate. To each well that contains the resin was added racemic naphthylleucine ester (1.2 mg, 0.0030 mmol) in IPA-Hex (2:8, 0.6 mL). After incubating for 24 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 DNB-L-Ala-Abu-Silica Gel Stationary Phase 2 and DNB-L-Leu-Abu-Silica Gel Stationary Phase 3. Both stationary phases were prepared following similar procedures as reported for the preparation of DNB-L-Ala-Gly silica gel.1 The ligand surface concentrations were estimated to be 0.24 mmol per gram silica gel for stationary phase 2 and 0.11 mmol per gram silica gel for stationary phase 3, as determined by elemental analysis of nitrogen. Determination of the Amount of Fmoc Group Present on Resins. To about 20 mg of a 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 Our approach is based on the synthesis of potential chiral selectors on synthesis resins and the rapid screening of these selectors on the resin using circular dichroism measurement. Library syntheses on synthesis resins are expected to be much more reliable than a similar synthesis on silica gel, as synthesis resins are optimized for and used routinely in solid-phase synthesis, while silica gel is best suited for separations. The screening procedure developed is a convenient batch equilibration assay based on the measurement of circular dichroism of the analyte. In this procedure, a potential chiral selector (one parallel library member) is synthesized onto a solid-phase synthesis resin. Racemic analyte in the proper solvent is then allowed to equilibrate with this potential selector on the resin. The enantiomeric ratio of the analyte in the supernatant is analyzed after the equilibration period. A selective adsorption of one of the two enantiomers to the resin is indicative of a chiral selector. The chiral selector will then be resynthesized onto a chromatographic support, and chiral resolution of the racemic analyte will be evaluated. To demonstrate the general principle, we studied the chiral resolution of naphthyl leucine ester 19 (Figure 1). A small parallel L-(4 × 4) pseudo-peptide library consisting of 16 members (Figure Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
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Figure 1. The R and S enantiomers of racemic (1-naphthyl)leucine ester 1.
Figure 3. The circular dichroism spectra of R- and S-(1-Naphthyl)leucine ester 1. Solute concentration: 2 mg/mL. Cell pass length: 1 mm. Figure 2. The L 4 × 4 or the [Anth, Bz, Dnb, Naph]-L-[Ala, Gly, Leu, Pro] Library. The Gly in module 2 serves as a negative internal control for screening. Abu: 4-aminobutyric acid. AmPS: aminomethylated polystyrene resin.
Scheme 1. Preparation of Dnb-L-Leu Member of the Parallel 4 × 4 Librarya
Table 1. Ellipticities (mdeg) Measured at 260 nm for Each Member of the Parallel 4 × 4 Library Anth-Ala -0.28a, 0.32b Anth-Gly -0.27, -0.39 Anth-Leu 0.28, 0.20 Anth-Pro -0.56, 0.26
Bz-Ala -0.44, -0.49 Bz-Gly 0.12, -0.04 Bz-Leu -0.02, -0.30 Bz-Pro -0.19, -0.26
DNB-Ala 8.26, 4.53 DNB-Gly -0.06, -0.18 DNB-Leu 13.5, 7.84 DNB-Pro 0.02, -0.02
Naph-Ala -0.12,0.50 Naph-Gly -0.10, -0.11 Naph-Leu 0.02, 0.15 Naph-Pro 0.17, -0.28
a Data on Am-PS resin. b Data on Am-TG resin. See Experimental Section for equilibration conditions. All the amino acids are of L-configuration.
2) was chosen for this purpose.10 The Gly in module 2 served as a negative internal control. One resin chosen for the synthesis of this library is the aminomethylated polystyrene (AmPS) resin, a derivative of the widely used Merrifield resin. The synthesis of this 16-member library on this resin was performed conveniently using a Hi-top filter plate manual synthesizer.11 In this synthesizer, resin can be added to the wells of a 96-well filter plate. By adding individual amino acids into each well, as many as 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 resins 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 1 with the synthesis of the Dnb-LLeu member of the library. Other library members were synthesized following identical reactions. For the screening purpose, library components on the resins were transferred into 16 wells of a regular 96-well plate. To each of the 16 wells that contained 0.026 mmol of selector was added an established amount of the racemic analyte (1.2 mg, 0.0030 mmol) in a mixture of 2-propanol and hexanes (2:8, 0.60 mL). After equilibration for 24 h, the ellipticity (mdeg) of the supernatant in each well was measured at 260 nm, the maximum CD 4180 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Scheme 2. The Immobilization of Potential Chiral Selectors onto Silica Gel. (a) 3-Aminopropyltriethoxysilane. (b) Silica Gel, 120 °C
adsorption wavelength of the enantiomerically pure naphthylleucine ester (Figure 3). The data obtained for all 16 wells are summarized in Table 1. As seen from Table 1, the measured ellipticities of the internal negative controls (all the Gly data) on AmPS resins ranged from -0.27 to 0.12. Since no enantiomeric selectivity is expected from these Gly negative controls, -0.27 to 0.12 should be a good measure of the “noise level” of the system. The ellipticities of two wells were well above the noise level; therefore, two chiral selectors, Dnb-L-Ala and Dnb-L-Leu, were identified from this library. The two selectors were then immobilized onto silica gel (Scheme 2), and the resulting stationary phases were packed into columns using a standard slurry packing method.12 Both columns were found to resolve racemic naphthyl leucine ester 1 well; the separation factor13 with the Dnb-L-Ala column 2 was 4.7, while that of the Dnb-L-Leu column 3 was 12 (Figure 4).
Figure 4. Chromatogram of racemic (1-Naphthyl)leucine ester 1 on Dnb-L-Leu stationary phase 3. Column size: 50 × 4.6 mm. Mobile phase: 20% IPA in hexanes. Flow rate: 1.2 mL/min. UV detector (254 nm). t0 ) 0.48 min.
On the basis of its ellipticity, an enantiomeric ratio (R-1/S-1) of 59/41 was estimated for the supernatant in the well that contained selector Dnb-L-Leu. From the circular dichroism spectrum (Figure 3), a specific ellipticity of + 50 mdeg•mL•mg-1•mm-1 [+92/(2 × 1)/0.92] was calculated for enantiomerically pure R-1, after adjustment of the enantiomerical purity of the sample14 used for the measurement. The total amount of the analyte in the supernatant after equilibration was determined to be 0.95 mg (1.2 mg × 0.834/1.050), by comparing its UV adsorption (A ) 0.834 of a solution diluted 100 times) at 245 nm with that (A ) 1.050 of a solution diluted 100 times) of the analyte solution before equilibration. With the total amount of analyte in the supernatant and the specific ellipticity of enantiomerically pure R-1, the enantiomeric ratio of the supernatant was calculated. Screening experiments were also performed using aminomethylated tentagel (AmTG) resin, another popular peptide synthesis resin, as the solid support. In contrast to the AmPS resin whose surface is hydrophobic, the surface of AmTG is hydrophilic as it is coated with a layer of poly(ethylene glycol). The library on AmTG resins was synthesized following identical procedures as described above for the synthesis on AmPS resin, and the ellipticities were measured in the same manner. As can be seen from Table 1, the same two compounds were identified as chiral selectors. The magnitude of the ellipticities measured with the same selector concentration, however, is smaller when compared with the values on the AmPS resin. The difference in ellipticities observed between the AmPS and AmTG resins could be attributed either to the different swelling properties or to the different surface nonspecific interactions. In these screening experiments, roughly 24 h were needed to reach equilibration with both resins (Figure 5). This equilibration time is most likely related to the swelling of the resins in the equilibration solvents. While it is possible that the lower ellipticities observed on AmTG resins could result from its possibly lower swelling capacity, the equilibration solvent does contain a component (IPA) capable of swelling the hydrophilic AmTG resin well. The surface polarity of the AmTG resin could be another reason for the lower ellipticities observed, as polar, nonspecific interactions can cause lower chiral selectivity. Moreover, the lower loading capacity of the Tenta-gel could amplify such an effect.
Figure 5. Time dependence of the resin equilibration experiments. For conditions, see Experimental Section on the screening of the library.
As expected, the peptide-based library can be synthesized in high efficiency on both the AmPS and the AmTG resins. For example, the coupling yields of Fmoc-Abu-OH to both resins were close to 100% (110% for AmPS resin,15 92% for AmTentagel resin), as determined by a Fmoc cleavage reaction.16 Couplings of FmocL-Leu-OH to Abu-AmPS and Abu-AmTG were achieved in 98% yields in both cases, also determined by the same Fmoc cleavage reaction. In contrast, coupling of Fmoc-Abu-OH to aminopropyl silica gel can be achieved in only 64% yield. A 64% coupling yield indicates that a significant amount of aminopropyl groups still remained on the silica gel after the coupling reaction. Further coupling of Fmoc-L-Leu-OH to the Abu-silica gel prepared above also could not be completed in high yield (
