Identification of Affinity Ligands for Protein Purification fromSynthetic Chemical Combinatorial Libraries

Yan Wang and Tingyu Li*

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

A method to screen combinatorial libraries for the development of selective ligandsfor protein affinity chromatographic purification is described. The method is based onthe application of parallel combinatorial libraries, and it has several potentialadvantages. The screening procedure is simple and straightforward, and it does notrequire the chemical derivatization of the target proteins or even that the target proteinbe pure. The experiment can also be designed to select binders that are less likely tocause protein denaturation. Feasibility of this approach is demonstrated with a modelstudy of the chromatographic purification of bovine albumin serum (BSA) and Avidin.

In recent years, chemical combinatorial libraries haveevolved considerably for the development of selectivebinders for a given target molecule (1-2). In thesetechniques, a large number of compounds (the library)can be screened for a desired property. In a short periodof time, combinatorial libraries have found widespreadapplications in both pharmaceutical research and mate-rial sciences (3). Applications of combinatorial librariesin protein separations have also been reported. Forexample, a two-color peptide library immunostainingchromatographic analysis (PELICAN) technique wasinvestigated for the affinity purification of Factor IX fromplasma (4). A protein A mimetic ligand has also beendeveloped from a combinatorial library and used for theaffinity purification of antibodies (5). In this method, thelibrary’s ability to interfere with the interaction betweenprotein A and a biotinylated immunoglobulin is measuredusing an enzyme-linked immunosorbent assay (ELISA).Another method to identify selective ligands for proteinpurification is based on a radiological assay (6). In thismethod, the radiolabeled target protein is allowed toequilibrate with a library synthesized using the one bead-one library member approach. If a library member bindsto the target protein, the bead that bears the particularlibrary member can be visualized with autoradiography.The structure of the library member on the bead can thenbe determined by micropeptide sequencing. A more recentreport is based on the affinity chromatographic screeningof soluble combinatorial peptide libraries (7). In thistechnique, a library of potential selective binders ispassed through a column immobilized with the targetprotein. Structures of the trapped library members (theselective binders) can then be identified by mass spec-trometry. Another recent report involved the develop-ment of an IgG binding ligand (8). In this method,individual columns of all library members are prepared,and their ability to purify IgG is evaluated.

In association with our interests in enantioselectiveseparations, we have developed efficient methods for theselection of ligands suitable for the chromatographicenantioselective separations from combinatorial libraries

(9). Recently, we expanded these studies to proteinpurification and have developed a screening method foridentification of affinity ligands for protein purificationfrom combinatorial libraries. The method is ratherstraightforward, and it does not require the chemicalderivatization of the target proteins. The experiment canalso be designed to select binders that are less likely tocause protein denaturation.

Our method is based on the application of parallelcombinatorial libraries. As opposed to a mixture libraryapproach, in which the library is synthesized as a mixtureof compounds and the entire mixture is evaluated for adesired property, a parallel library contains librarycomponents that are synthesized and screened separately(10). In other words, a parallel library is a collection ofpure compounds. The parallel library approach is similarto a traditional trial-and-error approach except that alarge number of compounds are designed to be studiedquickly. Apparently, practicality of a parallel screeningmethod depends on the synthetic efficiency of the libraryand the screening efficiency.

In this paper, we present and demonstrate the feasibil-ity of this screening procedure with a model study.

Experimental Section

General Supplies and Equipment. The UltraLinkbio-support resin with azlactone groups was purchasedfrom Pierce (Rockford, IL). Biotin, iminobiotin, bovineserum albumin (BSA), and Avidin were purchased fromSigma (St. Louis, MO). All other chemicals and solventswere purchased from either Aldrich (Milwaukee, WI),Fluka (Ronkonkoma, NY), or Fisher Scientific (Pitts-burgh, PA). An HR 5/5 empty glass column (5 mm × 50mm) was purchased from Pharmacia (Piscataway, NJ).Chromatographic analyses were completed with a Beck-man analytical gradient HPLC system (software, systemGold). Gel electrophoreses were accomplished using aMini-Protean 3 system and ready gels from Bio-Rad(Hercules, CA). The gel images of electropherograms wereobtained with the Eagle Eye system from Stratagene (LaJolla, CA). UV spectra needed to quantify the surfaceligand concentration with the Fmoc method were ob-tained with a Shimadzu UV 201 spectrometer (cellvolume, 3 mL; cell pass-length, 10 mm).

* To whom correspondence should be addressed. Phone/Fax:(615) 343-8466. E-mail: tingyu.li@vanderbilt.edu.

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10.1021/bp020033d CCC: $22.00 © 2002 American Chemical Society and American Institute of Chemical EngineersPublished on Web 05/10/2002

Synthesis of UltraLink Resin-NH(CH2)3NH-GlyGlyNH2. To a solution of N-Boc-1,3-diaminopropane(304 mg, 1.75 mmol) in 5 mL of phosphate buffer (KH2-PO4, pH 10) was added 0.70 g of UltraLink bio-supportresin (surface azlactone loading >0.25 mmoL/g). After 5h of shaking at room temperature, propylamine (206 mg,3.5 mmol) was introduced to the reaction mixture to blockthe unreacted azlactone functional group on the resin.After the shaking was continued for another 3 h, the resin(resin-NH(CH2)3NHBoc) was collected by filtration andwashed with the phosphate buffer (pH 10), water, andmethanol. The Boc protecting group was then removedby treatment with 30% TFA in DCM for 30 min. Subse-quently, the aminopropyl bound resin (resin-NH(CH2)3-NH2‚TFA) was obtained by filtration and washed withDCM, methanol, and DCM.

The above resin was then added into a solution ofFmoc-Gly-OH (312 mg, 1.05 mmol), PyBOP (546 mg, 1.05mmol), HOBt (142 mg, 1.05 mmol), and DIPEA (271 mg,2.1 mmol) in 5 mL of DMF. After shaking gently for 1 h,the resin was filtered and washed with DMF, methanol,and DCM to yield resin-NH(CH2)3NHGlyFmoc. Thesurface Gly loading for this resin was 0.30 mmol/g, asdetermined by the Fmoc cleavage method (11).

The Fmoc group was then removed by treatment with25% piperidine in DMF for 20 min to yield resin-NH-(CH2)3NHGlyNH2. The second Gly unit was then coupledto the resin following the same procedure as describedabove for the coupling of the first Gly unit to yield resin-NH(CH2)3NHGlyGlyFmoc. The surface loading of thesecond Gly unit was determined to be 0.29 mmol/g, asdetermined again by the Fmoc cleavage method. Removalof the Fmoc group with piperidine yielded resin-NH-(CH2)3NHGlyGlyNH2.

Synthesis of the Model Library. (1) Library Mem-bers 1 and 4-12. To prepare library member 1, asolution of D-(+)-biotin (88 mg, 0.36 mmol), PyBOP (187mg, 0.36 mmol), HOBt (48 mg, 0.36 mmol), and DIPEA(93 mg, 0.72 mmol) in 4 mL of HMPA was added to resin-NH(CH2)3NHGlyGlyNH2 prepared above (200 mg, 0.060mmol in amino group). After the mixture was shakengently for 4 h, library member 1 was collected byfiltration and washed with HMPA, methanol, and DCM.

Library members 4-12 were prepared following thesame procedure except that the solvent HMPA wasreplaced with the less toxic DMF.

(2) Library Member 2. After the pH of a solution ofiminobiotin (88 mg, 0.36 mmol) and the resin preparedabove (200 mg, 0.060 mmol in amino group) in water (5mL) was adjusted to 4-5 with 0.4 M HBr, the couplingreagent 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemetho-p-toluenesulfonate (761 mg, 1.80 mmol) was added.The mixture was shaken gently for 5 h. After that, thedesired library member 2 was collected by filtration andwashed with tris buffer (0.04 M Tris‚HCl, 1 M NaCl, pH) 6), water, methanol, and DCM.

(3) Library Members 3 and 13-18. To preparelibrary member 14, a solution of 3-(trifloromethyl)-benzene sulfonyl chloride (88 mg, 0.36 mmol) and TEA(73 mg, 0.72 mmol) in 4 mL of DMF was added to theresin (200 mg, 0.060 mmol in amino group) preparedabove. After the mixture was shaken gently for 4 h,library member 14 was collected by filtration and washedwith DMF, methanol, and DCM.

Library members 3, 13, and 15-18 were prepared bythe same procedure.

Screening of the Parallel Library for SelectiveProtein Binders. To each 1 mL centrifuge tube contain-ing 5 mg of one individual library member was added

100 µL of a solution of Avidin (concentration, 0.3 mg/mL)and BSA (concentration, 0.3 mg/µL) in a loading buffer(0.04 M Tris-HCl, 1 M NaCl, pH 9.0). These samples weregently shaken for 10 min. After the samples stood foranother 10 min to allow the resin to sink to the bottom,10 µL of supernatant was drawn out of each tube for gelelectrophoresis analysis.

Gel Electrophoresis Analysis. Ten milliliters of thesupernatant was mixed with 20 µL of bromophenol bluesample buffer that contained 5% â-mercaptoethanol. Themixture was then heated for 5 min at 95 °C to denaturethe protein. The gel electrophoresis was run on a 12-well,gradient (4-20%) SDS-PAGE tris-HCl ready gel. Therunning power was 200 V, and the current was 100 mA.For visualization, the gel was stained with CoomassieBlue R-250 solution for about 3 h.

Column Packing and Chromatographic Measure-ments. The column was packed using a slurry method.The outlet tubing of the column was connected to a wateraspirator pump. A slurry of 0.20 g of stationary phase in5 mL of tris-HCl buffer (0.04 M Tris-HCl, 1 M NaCl, pH9.0) was added to the column. An additional amount ofbuffer (5-10 mL) was pumped through the column withthe aid of the water pump until the column bed volumeno longer changed. The inlet cap was then replaced, andthe column was ready to be tested.

For chromatographic measurement, a flow rate of 1.0mL/min was used. The elution process was monitoredwith an UV detector at 280 nm. The solvent gradientemployed was 0-10 min, 100% loading buffer; 10-11min, 100% loading buffer to 100% eluting buffer; 11-31min 100% eluting buffer; and 31-32 min, 100% elutingbuffer to loading buffer. See the specific chromatogramsfor the composition of loading and eluting buffers.Column size was 50 mm × 5 mm.

Results and Discussion:General Protocol. The general protocol we use to

develop selective stationary phases for protein purifica-tion is as follows. First, the parallel library will beprepared by synthesizing each potential selective binder(each parallel library member) onto a proper solid sup-port. To determine whether an individual library memberis a selective protein binder, mixture containing targetprotein in a proper solvent, a loading buffer, will beallowed to equilibrate with this individual library mem-ber attached to the solid support (Figure 1). Afterequilibration, the supernatant will be analyzed by SDS-PAGE. A decrease in the concentration of the targetprotein relative to other impurities in the buffer after

Figure 1. Equilibration assay of selective binding of targetprotein to a member of the library. Decrease of the target proteinconcentration relative to other impurities is indicative of aselective binder.

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equilibration is indicative of a selective binder. Such abinder could then be immobilized to a suitable chromato-graphic support for the affinity purification of the crudeprotein mixture.

Minimized denaturation is another important goal indeveloping a new method for protein purification. Inimmunoaffinity chromatography, the buffer required toelute the target protein from the column can oftendenature the protein. With the parallel library method,the possibility of selecting binders that are less likely topresent elution problems exists through a two-stepscreening procedure. The first step is to identify all ofthe selective binders by following the method outlinedabove using a loading buffer. The second step is to repeatthe equilibration assay using only these previouslyidentified selective binders, with one exception: theprotein is dissolved in a buffer that is to be used forelution (Figure 2). The binders that yield specific adsorp-tion of the target protein at initial loading buffer yetpossess relatively weak adsorption with the eluting bufferwould probably be best suited to avoid protein denatur-ation in the eventual chromatographic purification oftarget proteins.

As pointed out correctly by one reviewer, identifyingselective binders by this two-step screening sequencecannot guarantee that the target protein will be isolatedin its native form. One possibility is that denaturation

could occur just by binding to the selective ligand, andSDS-PAGE could not distinguish native protein from itsdenatured forms. Moreover, both screenings are basedon adsorption, which in case of slow kinetics may notcorrelate well with the corresponding desorption behav-ior. Although these concerns are valid and can beaddressed in future studies, the screening procedureoutlined above should provide binders that are less likelyto cause protein denaturation.

Library Design and Synthesis. To demonstrate thefeasibility of this approach, we studied the chromato-graphic separation of bovine serum albumin (BSA) andAvidin using a small model library (Figure 3). The librarycontained immobilized biotin (1) and its analogue imi-nobiotin (2). Biotin is known to bind Avidin strongly,while binding by iminobiotin is weaker and also moresensitive to pH changes (12). Other library memberscontained relatively hydrophobic aromatic functionalgroups, which are more likely to bind to BSA morestrongly, as one of the important biological functions ofBSA is to transport hydrophobic organic molecules.

The solid support used to prepare the library was theUltraLink resin, a commercially available, hydrophilic,polyacrylamide-based resin suitable for protein chro-matographic purification. Although the UltraLink resinis commonly used as a chromatographic support, ourresults demonstrated that it is also suitable for solid-phase synthesis. The advantage of using UltraLink resinas a solid support for library synthesis is that screeningresults using ligands immobilized on this resin shouldbe closely correlated with the subsequent chromato-graphic experiments, which will use the same resin asthe stationary phases.

For library synthesis, the preactivated, commerciallyavailable UltraLink resin was first functionalized withan aminopropyl group by treatment with N-Boc-1,3-diaminopropane (Scheme 1). Subsequently, two Gly unitswere introduced as a linker group. The individual librarymember was then synthesized by attaching the properprecursor to the solid support. The synthesis of the biotinmember of the library (1) is shown in Scheme 1 and otherlibrary members were prepared following similar proce-dures.

Syntheses on the solid resin were monitored by Fmoccleavage reaction (11) and ninhydrin test (13). For allthose reactions involving free amino groups on the resin

Figure 2. Identifying selective binders that can offer mildereluting conditions. Lane a: before equlibration with the librarymember. Lane b: after equlibration with a loading buffer. Lanec: after equlibration with an eluting buffer.

Figure 3. The small model parallel library. X ) NH-Gly-Gly-NH(CH2)3-UltraLink resin.

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as the starting materials, negative ninhydrin tests wereobserved, which indicates the complete reaction of theamino group. According to the Fmoc cleavage reaction,the concentration of the first introduced Gly unit was 0.30mmol/g, which is higher than the suggested surfaceazlactone concentration (0.25 mmol/g) of the resin. Theconcentration of the second Gly unit was 0.29 mmol/g asdetermined by the same method. Adjusting for the weightincrease of the resin, the coupling yield of the second Glyto the first Gly unit was about 98%. These resultsdemonstrate that efficient library syntheses were achievedon this UltraLink resin.

Screening for Selective Binders. To screen thelibrary for selective binders, a mixture of Avidin and BSAin a loading buffer (0.04 M Tris‚HCl, 1 M NaCl, pH ) 9,100 µL) was added to the individual library membersprepared above on UltraLink resin (5 mg each in totalweight) in a 1 mL vial. After the samples were gentlymixed by shaking for 20 min, 10 µL of the supernatantwas drawn and analyzed by SDS gel electrophoresis.

The electrophoragrams obtained for all 18 librarymembers are shown in Figure 4. As seen clearly fromFigure 4, library members 1 and 2 have selective bindingtoward Avidin, while library members 12 and 14 haveselective binding toward BSA. Library member 11 hasstrong affinity toward both proteins, while others haveno significant binding toward either protein. Therefore,selective library members 1, 2, 12, and 14 are potentiallyuseful for the separation of a mixture of BSA and Avidin.

Screening for Elution Conditions. As in otherprotein affinity methods, the selective ligands identifiedabove may bind the target proteins too strongly to allowfor their effective chromatographic elution. Therefore,additional screening experiments are needed to deter-mine if a proper elution condition could be developed ifthese library members are to be used as stationaryphases for the chromatographic separation of BSA andAvidin. For this purpose, equilibration experiments in

three potential elution solvents were performed withthese four selective library members. Eluting buffer A,a pH 6 Tris buffer (0.04 M Tris‚HCl, 1 M NaCl), is rathermild and there is a high probability that proteins canpreserve their biological activity in this buffer. Elutingbuffers B (4 M urea in buffer A) and C (4 M guanidine inbuffer A) are designed to disrupt the interaction ofproteins with their selective ligands, and they are power-ful eluting solvents. However, proteins tend to be dena-tured in these solvents. It should be pointed out at pH )6, the buffering capacity of the Tris system may berelatively low. However, since this model study involvesrelatively pure proteins at low concentration, the some-what lower buffering capacity should not have materialimpact in this case.

The equilibration experiments were performed andanalyzed in the same manner as those described for thescreening of selective binders. The electropherogramsobtained for these experiments are shown in Figure 5.

As seen from Figure 5, with biotin (1) strong bindingstill existed between Avidin and this compound in all ofthese three solvents. Therefore, if a biotin-based affinitycolumn were to be used to separate BSA and Avidin, noneof the three solvents are likely to be able to elute Avidinfrom such a column. Since buffers B and C are normallyconsidered as powerful eluting solvents, the prospect offinding a proper elution condition is rather slim usingsuch a column. With iminobiotin (2), it is apparent thatthe mild buffer A should already be able to elute Avidinfrom an iminobiotin-based column. Therefore, equilibra-tion experiments with the more powerful eluting buffersB and C were not performed with this library member.As to the two selective BSA binders (library members12 and 14), library member 14 binds to BSA fairlystrongly in all three solvents. In contrast, the bindingbetween BSA and library member 12 was weakenedconsiderably in buffer C.

On the basis of these results, it can be concluded thatiminobiotin (2) should serve as an excellent affinity ligandto separate BSA from Avidin and the trapped Avidincould be eluted with a mild eluting buffer A. Chromato-graphic separation of BSA and Avidin is also possibleusing library member 12, although a powerful elutingsolvent (buffer C), which is very likely to denature theentrapped protein, has to be used. With library members1 and 14, elution could be more difficult with these threesolvent systems investigated.

Chromatographic Evaluations. Chromatographicseparation of a mixture of BSA and Avidin was investi-gated using two columns, one packed with library mem-ber 2 and the other with library member 12. As expected,excellent separation of BSA and Avidin was achieved on

Scheme 1. Preparation of the Biotin Member (1) of theParallel Librarya

a Conditions: (a) NH2(CH2)2NHBOC. (b) (1) TFA; (2) Fmoc-Gly-OH, PyBop. (c) (1) piperidine; (2) Fmoc-Gly-OH, PyBop; (3)piperidine. (d) biotin, Pybop

Figure 4. Screening outcome of the parallel library with a mixture of BSA and Avidin. Lane Std: mixture before equilibration withlibrary members. Lanes 1-18: mixture after equilibration with the respective library members 1-18.

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both columns. With the iminobiotin column (2), BSA waseluted out quickly with the loading buffer, and uponswitching to buffer A, Avidin was eluted (Figure 6). Withthe column packed with library member 12, Avidinemerged from the column quickly with loading buffer.Upon switching over to buffer C, BSA was washed out(Figure 7).

Conclusions

The model study demonstrates the feasibility of theparallel library screening method, which could provehighly efficient for the development of affinity ligandsfor protein purifications. Principle advantages of thisscreening method include its simplicity, its ability toscreen the library without protein immobilization, itsability to screen the library without pure target proteins,and the possibility of identifying ligands that are lesslikely (however, it cannot guarantee no denaturation) toinduce protein denaturation. It is hoped that throughfurther studies practical libraries could be developed forthe efficient purification of many protein molecules.

AcknowledgmentFinancial support from Vanderbilt University, in the

form of start-up fund, research council, and naturalscience fund is appreciated. The project is also supportedin part with a pilot project fund (P30 ES00267) from theNational Institute of Environmental Health Sciences,NIH. The authors would also like to thank Dr. JenniferPietenpol for helpful discussions.

References and Notes(1) PyBop: benzotriazolyloxy-tris[pyrrolidino]-phosphonium

hexafluorophosphate; Fmoc: 9-fluorenylmethoxycarbonyl;HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid;DIPEA: N,N-diisopropylethylamine; DMF: dimethyl forma-mide; DCM: dichloromethane; HMPA: hexamethylphos-phoramide.

(2) For some examples, see: (a) Lam, K. S.; Salmon, S. E.;Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J.A new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991, 354, 82-84. (b) Houghten, R.A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.;Cuervo, J. H. Generation and use of synthetic peptidecombinatorial libraries for basic research and drug discovery.Nature 1991, 354, 84-86. (c) Janda, K. D. Tagged versusuntagged libraries: methods for the generation and screening

Figure 5. Screening for eluting conditions. Lane numbers 1, 2, 12, and 14 represent the electrophoragrams of the mixture afterequilibration with library members 1, 2, 12, and 14, respectively. Buffer A; 0.04 M Tris‚HCl, 1 M NaCl, pH 6. Buffer B: 4 M ureain buffer A. Buffer C: 4 M guanidine in buffer A.

Figure 6. Chromotogram of a mixture of BSA and Avidin onthe iminobiotin column 2. Column size: 50 mm × 5 mm. Mobilephase: 0-10 min, 100% loading buffer (0.04 M Tris‚HCl, 1 MNaCl, pH ) 9); 10-11 min, 100% loading buffer to 100% elutingbuffer (0.04 M Tris‚HCl, 1 M NaCl, pH ) 6); 11-31 min, 100%eluting buffer; and 31-32 min, 100% eluting buffer to loadingbuffer. Flow rate: 1.0 mL/min. UV detector (280 nm). t0 ) 0.48min.

Figure 7. Chromotogram of a mixture of BSA and Avidin oncolumn 12. Column size: 50 mm × 5 mm. Mobile phase: 0-10min, 100% loading buffer (0.04 M Tris‚HCl, 1 M NaCl, pH ) 9);10-11 min, 100% loading buffer to 100% eluting buffer (0.04M Tris‚HCl, 4 M guanidine, pH ) 6); 11-31 min, 100% elutingbuffer; and 31-32 min, 100% eluting buffer to loading buffer.Flow rate: 1.0 mL/min. UV detector (280 nm). t0 ) 0.48 min.

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of combinatorial libraries. Proc. Natl. Acad. Sci. U.S.A. 1994,91, 10779-10785.

(3) For a general review, see: Borman, S. Combinatorialchemistry. Chem. Eng. News 1998, April, 47-67.

(4) Buettner, J. A.; Dadd, C. A.; Baumbach, G. A.; Masecar, B.L.; Hammond, D. J. Chemically derived peptide libraries: Anew resin and methodology for lead identification. Int. J. Pept.Protein Res. 1996, 47, 70-84.

(5) Palombo, G.; De Falco, S.; Tortora, M.; Cassani, G.; Fassina,G. A synthetic ligand for IgA affinity purification. J. Mol.Recognit. 1998, 11, 243-246.

(6) Mondorf, K.; Kaufman, D. B.; Carbonell, R. G. Screening ofcombinatorial peptide libraries: Identification of ligands foraffinity purification of proteins using a radiological approach.J. Peptide Res. 1998, 52, 526-536.

(7) Huang, P. Y.; Carbonell, R. G. Affinity chromatographicscreening of soluble combinatorial peptide libraries. Biotech-nol. Bioeng. 1999, 63, 633-641.

(8) Teng, S. F.; Sproule, K.; Hussain, A.; Lowe, C. R. A strategyfor the generation of biomimetic ligands for affinity chroma-tography. Combinatorial synthesis and biological evaluationof an IgG binding ligand. J. Mol. Recognit. 1999, 12, 67-75.

(9) (a) Wang, Y.; Li, T. Screening of a parallel combinatoriallibrary for selectors for chiral chromatography. Anal. Chem.1999, 71, 4178-4182. (b) Wu, Y.; Wang, Y.; Yang, A.; Li, T.Screening of mixture combinatorial libraries for chiral selec-tors: A reciprocal chromatographic approach using enantio-meric libraries. Anal. Chem. 1999, 71, 1688-1691.

(10) Bunin, B. A. The Combinatorial Index; Academic Press:New York, 1998; pp 5-8. See also: Thompson, L. A.; Ellman,J. A. Synthesis and applications of small molecule libraries.Chem. Rev. 1996, 96, 555-600.

(11) Method 12: Estimation of level of first residue. In Nova-Biochem Catalog & Peptide Synthesis Handbook; NovaBio-chem: Darmstadt, 1999; S43. See also ref 9a.

(12) Orr, G. A. The use of the 2-iminobiotin-Avidin interactionfor the selective retrieval of labeled plasma membane com-ponents. J. Biol. Chem. 1981, 256, 761-766.

(13) Method 13: Kaiser test. In NovaBiochem Catalog &Peptide Synthesis Handbook; NovaBiochem: Darmstadt,1999; S43.

Accepted for publication March 1, 2002.

BP020033D

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