Weinberger, Lichte, Griesinger, and Kutscher 109

Small Peptide Libraries: Combinatorial Split-Mix Synthesis followed by Combinatorial Amino Acid Analysis of Selected Variants Heinz Weinbergera)'')*, Ellen Lichtea), Christian Griesingera), and Bernhard Kutscherb)

a) Institute of Organic Chemistry, J.W. Goethe-University of Frankfurt, FrankfurtMain, Germany

b, ASTA Medica AG, Department of Medicinal Chemistry, Frankfurt/Main, Germany

Key Words: Amino acid analysis; combinatorial peptide library; split-mix synthesis; TentaGel-resin

Summary

Peptides from small combinatorial libraries, covalently attached to polymeric TentaGel beads, can be directly sequenced using amino acid analysis. For libraries with restricted diversity, generated by the split-mix synthesis method, the amino acids on a selected single bead identified by pre-column derivatization with o-phthaldialde- hyde (OPA) correlate directly with the sequence of a given peptide. This is shown on a tripeptide (343 different compounds) and a tetrapeptide (4096 different compounds) library. This method allows for rapid peptide sequence determination without relying on complex encoding strategies.

Introduction

With the ongoing development of combinatorial chemistry [11, rapid and readily available analytical tools for structural determination of active compounds covalent1 attached to the bead are now of considerable importance [13]. For peptide libraries, several strategies have been devised to address this problem. Edman microsequence analysis has been widely used for this purpose [4,51, but this expensive method works only with natural amino acids and is time-consuming. Re- cently, Youngquist et al.L6] introduced a method for sequenc- ing peptides by partial capping of the growing molecule in each reaction step and finally analyzing the different peptide fragments after cleavage from the resin by matrix assisted laser desorption ionization mass spectrometry. However, this strategy is compromised by the presence of additional pep- tides on each single bead which may also interact with the soluble receptor.

Herein, we describe a method without relying on encoding strategies that enables rapid determination of the sequence of peptides from support-bound combinatorial libraries using amino acid analysis.

Results and Discussion

During the course of our work in searching for high affinity ligands to cytokine receptors, we constructed several un- coded peptide libraries by standard Fmoc/fB~-chemistry[~] on TentaGel as solid support, following the split-mix synthesis method developed by Furka [*,'I. After the corresponding

"Present address: ASTA Medica AG, Department of Medicinal Chemistry, Weismiillerstralk 45, D-603 14 FrankfudMain, Germany

bioassay, the identification of immobilized receptor binding ligands was achieved by bead staining techniques using fluo- rescein isothiocyanate (FITC) labeled antibodies. Individual staining beads were revealed by visual inspection and the determination of the peptide sequence attached to a single resin bead was accomplished by amino acid analysis using pre-column derivatization with OPA-reagent [Io1. Although yielding only information about the amino acid content of the peptide, in combination with split-mix synthesis amino acid analysis enables peptide sequencing. The repetition of the splittinghixing operations namely has an important conse- quence on the composition of the peptide. As shown in Figure 1, the 'one-bead, one-peptide' approach["] ensures that each of the resin beads carries a single peptide sequence. Moreover, every support bound peptide consists of only one amino acid of each reaction step, e.g. tripeptides which con- tain amino acid A cannot hold amino acid B or C at the same time. The same applied to the amino acids D, E, and F or G, H, and K.

Thus, if amino acid A is identified by amino acid analysis it takes position three in the corresponding tripeptide (starting from the N-terminus).

We now show that for libraries with a restricted diversity it is possible to derive the absolute position in the peptide from the result of the amino acid sequencing, resulting in the definite peptide sequence. To demonstrate the utility of this method, we have prepared a tripeptide library with 19 differ- ent L-amino acids arranged in a 7 by 3 matrix (Figure 2). Between the three coupling steps, the resin was pooled, mixed and subsequently divided in 7 reaction vessels, thus resulting in 73 = 343 trimers. Following the corresponding bioassay, the brightest beads were physically picked out under a fluo- rescence microscope with a glass syringe. After removal of the receptor complex with 5 % sodium n-dodecyl sulfate (SDS), individual stained beads were hydrolysed in 6 N HCl. The resulting mixture was derivatized with OPA-reagent and analyzed by HPLC. As shown in Figure 2, the amino acids alanine, tyrosine, isoleucine, and the linker e-aminocaproic acid (Aca) were identified by single-bead amino acid analy- sis.

In the 7 by 3 amino acid matrix Ile is contained only in the subset for AAl and therefore is AA1. Only the subset for AA2 and AA3 contains Ala and Tyr, respectively, assigning these amino acids to AA2 and AA3 unambiguously. Therefore, the peptide sequence of the trimer results in H-Tyr-Ala-Ile-OH.

Our studies established that the amount of the peptide present on any given single bead (250 pmol maximum) is more than sufficient since the detection limit in OPA-based

Arch. Phann. Phann. Med. Chem. 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1997 0365-6233/97/0404-0109 $17.50 +.50/0

110 Combinatorial Amino Acid Analysis of Selected Variants

Figure 1. The split-mix synthesis method of preparing a combinatorial library. In this illustration only three synthetic cycles with nine different amino acids (A-K) are used, giving a combinatorial library of 27 trimeric peptides.

I

Iru

L Figure 2. Amino acid analysis of a single-bead bound tripeptide after the bioassay (PEG = polyethylene glycol). The amino acids found are high- lighted in the 7 by 3 matrix on top.

amino acid analysis is 20-50 pmol. For generating larger libraries with greater diversity, it is inevitable to use amino acids several times. In this case, the above demonstrated principle of exclusion will lead to an intense reduction in the number of sequence possibilities up to the unique peptide sequence. Therefore, another tetrapeptide library attached to the above linked resin was prepared consisting of only 16 amino acids arranged in a 8 by 4 matrix resulting in 84 = 4096 different compounds (Figure 3). After the bioassay, isolation and hydrolysis, a single-bead bound tetrapeptide was ana- lyzed. Therein, glutaminic acid, tyrosine, valine, lysine, and the linker Aca were identified by amino acid analysis. The remarkably low signal intensity of the isoindole derivative of lysine was found for all lysine containing peptide hydro- lyzates.

The amino acid sequence can be derived from the amino acid analysis in the below way. Val is contained only in subset AA3. Therefore, Lys, which is contained both in subset AA3 and AAI can only be AAi. By the same taken, Tyr being in subset AAI as well as in A A 2 must represent AA2. AA4 remains for either glutamine or glutaminic acid, because Gln is hydrolyzed to Glu by treating with 6 N HCl. Consequently, amino acid analysis reduces the number of tetrapeptide se- quences to the following 2 out of 4096 possibilities: H-Glu- Val-Tyr-Lys-OH or H-Gln-Val-Tyr-Lys-OH.

Arch. P h a m P h a m Med Chem 330,109-111 (1997)

Weinberger, Lichte, Griesinger, and Kutscher 111

AA, = amino acid

mmo

F " om0

? 1 0 1 6 . 0 1 0 ~ A"

Figure 3. Amino acid analysis of a single-bead bound tetrapeptide after the bioassay. The four amino acids found are highlighted in the 8 by 4 matrix on top.

In contrast to the time consuming and expensive Edman degradation method, we have developed a strategy which is simple to use and does not require any tagging procedures. Furthermore, the identification of non-natural amino acids as well as the distinction between L- and D-amino acids and the sequencing of cyclic peptides is also possible. For small combinatorial peptide libraries, the combination of the split- mix synthesis method and amino acid analysis can be a pow- erful tool for the rapid discovery of novel biologically-active lead compounds.

I ( 0 45

Acknowledgments This work has been supported by the Bundesministerium fiir Bildung,

Wissenschaft, Forschung und Technologie (BMBF), BOM under No. 10792. The amino acid analyzer was provided by the DFG under Gr 121 111-2. We are indebted to Prof. Dr. A. Kleemann (ASTA Medica AG) and Prof. Dr. G. Quinkert (University of Frankfurt) for their active interest in establishing and running the joint postdoctoral research program.

Experimental Section TentaGel@ S-NH2 resin (0.25 meqlg, 8.9 x lo5 beaddg, 130 pm beads)

was purchased from Rapp Polymere (Tiibingen, Germany). Three Aca units

were attached to the resin serving as a linker between the polyethylene glycol (PEG) chains and the peptide by using a 4-fold excess of Fmoc-Aca in each coupling reaction. The peptide libraries were synthesized on the resin by standard Fmoc chemistry following the split-mix synthesis method. Coupling of protected amino acids was carried out with DIC in the presence of HOBt using a 20-fold excess of Fmoc-amino acid. Reactions were run on a 4.5 pM (tripeptide) and 5 pM (tetrapeptide) scale. After each coupling and mixing step, the N-a-Fmoc protecting groups of the building blocks were removed by 40 % ( v h ) piperidine in DMF. After completion of peptide synthesis, the libraries were deprotected using 4-6 ml of a mixture of TFA (8.25 ml), Hz0 (0.5 ml), thioanisole (0.5 ml), phenol (0.5 g) and 1,2-ethanedithiol(O.25 ml) per 1 0 0 mg of peptidyl resin. After 4-6 h the resin was washed five times with DMF (5 ml), three times with ether (5 ml) and finally dried under high vacuum.

For amino acid sequencing, a selected single bead was washed thoroughly with 5 % SDS and placed into a melting point glass tube. 30 pI of aqueous 6 N HCI (containing 0.1 % Phenol) was added, the glass tube was sealed and the hydrolysis were run at 110 "C for 22 h. To the resulting hydrolysate was added 30 pl of 5 N NaOH and a 30 fl aliquot of that solution was mixed with 20fl each of borate buffer and OPA-reagent, purchased from GROM (Herrenberg, Germany). After 3 min standing at room temperature, 20 pl of 1 N HCL was added and a 60 p1 aliquot of that mixture was placed directly on a GROM-SIL OPA column (5 pn, 4 x 250 nun) under the following conditions: eluant A 50 mmol Na acetate pH 7.2, eluant B: acetonitrile; for tripeptide analysis: gradient from 95% to 40% A and 5% to 60% B over 50 min, flow 1 .O d m i n ; for tetrapeptide analysis: gradient from 100% to 0% A and 0% to 80% B over 50 min, flow 1 .O ml/min; fluorescence detection at 340 nm (excitation maximum), 450 nm (emission maximum).

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