9812
`
`J. Am. Chem. SOC. 1993,115, 98 12-98 13
`
`Synthetic Methods for the Implementation of Encoded
`Combinatorial Chemistry
`
`John Nielsen, Sydney Brenner,’ and Kim D. Janda.37
`The Scripps Research Institute
`Departments of Molecular Biology and Chemistry
`10666 North Torrey Pines Road
`La Jolla, California 92037
`Received June 2, 1993
`Screening of natural and synthetic compounds for activity as
`potential therapeutic agents has for decades been the main source
`of drug discoveries in the pharmaceutical industry. With advances
`in molecular biology, protein crystallography, and computational
`chemistry, “rational drug design” has found many advocates, but
`this approach is still difficult and slow. In recent years there has
`been a renaissance in drug screening with the advent of new
`technologies based on “combinatorial libraries”.’ These methods
`expose a large number of compounds to a target, and they allow
`compounds that bind to a target with the highest affinity to be
`filtered out from a pool of statistical sequences. Present
`technologies have some limitations; in chemically synthesized
`libraries, mostly of peptides to date, there can be intrinsic
`difficulties in the identification, selection, and enrichment of active
`compounds. In bacteriophage and nucleic acid libraries, the active
`compounds can be amplified by replication, but chemical diversity
`is inherently limited. Recently, a conceptual scheme for “encoded
`combinatorial chemistry” has been proposed as a possible solution
`to a number of these potential restri~tions.~J Such libraries would
`combine the potentially limitless diversity of synthetic libraries
`with the advantages of natural libraries based on gene technologies.
`It was proposed to link each molecule of a chemically synthesized
`entity to a particular oligonucleotide sequence constructed in
`parallel and to use this encoding genetic tag to identify and
`enrich active compounds. In this paper, we detail the chemistry
`necessary to synthesize encoded libraries, choosing peptides as
`an example.
`In essence, such libraries require alternating stepwise syntheses
`of a peptide and an oligonucleotide sequence on a common linker
`(Scheme I). Thus a linker had to be constructed and attached
`to a suitable solid support capable of housing both the oligonu-
`cleotides and the peptides synthesized. Such a spacer had to be
`compatible with both solid-phase peptide synthesis (SPPS) using
`Fmoc chemistry and oligonucleotide synthesis with phosphora-
`midites, and it needs to be functionalized in such a way that it
`is possible to deprotect and detach the oligonucleotide-tagged
`peptides in a regioselective, nondestructive manner. This latter
`process allows the encoding of molecules rather than their
`carriers. Furthermore, we wanted the linker unit to be versatile
`enough to allow a “dendritic” display of the chemical library for
`controlled multivalent ligand d i ~ p l a y . ~
`Controlled-pore glass (CPG) was used as the solid matrix,
`because this material has a long-standing reputation for efficient
`oligonucleotide synthesis and has also proved efficient in SPPS.
`* To whom reprint requests and correspondence should be addressed.
`t A. P. Sloan Fellow, 1993-1995.
`(1) For some excellent reviews, see: (a) Jung, G.; Beck-Sickinger, A. G.
`Angew. Chem., Int. Ed. Engl. 1992,31,367-383. (b) Pavia, M. R.; Sawyer,
`T. K.; Moos, W. H. Bioorg. Med. Chem. Lett. 1993, 3, 387-396.
`(2) (a) Brenner, S.; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 1992,89,
`5381-5383. (b) Amato, I. Science 1992, 257, 330-331.
`(3) Recently, two papers describing the encoding of combinatorial libraries
`have appeared: (a) Kerr, J. M.; Banville, S. C.; Zuckermann, R. N. J. Am.
`(b) Nikolaiev, V.; Stierandova, A.;
`Chem. SOC. 1993, 225, 2529-2531.
`Krchnak, V.; Seligmann, B.; Lam, K. S.; Salmon, S. E.; Lebl, M. PeptideRes.
`1993,6,161-170. However, thesemethods usepeptidesas thecoding elements,
`and enrichment by amplification is not possible.
`(4) For a discussion of multivalent peptide display, see: (a) Cull, M. G.;
`Miller, J. F.;Schatz,P. J. Proc. Natl.Acad.Sci. U.S.A. 1992,89, 1865-1869.
`(b) Lowman, H. B.; Bass, S. H.; Simpson, N.; Wells, J. A. Biochemistry 1992,
`30, 10832- 108 38.
`
`0002-7863/93/1515-9812$04.00/0
`
`Scheme I1 delineates the synthesis of four new supports, CPG-3,
`-4, -5, and -6. There are several features to be noted: (A) N-Fmoc-
`and 0-DMT-protected L-serine/lysine-branched monomers serve
`as differential orthogonal attachment points for the oligonucle-
`otides and the peptides (singular and bivalent). (B) The high
`optically purity of these CPG supports is necessary since a
`stereochemically impure linker complicates testing, analysis, and
`interpretation of results from such biopolymers. (C) Employment
`of the succinyl aminohexanol-sarcosine appendage provides a
`method for nonacidic detachment of the oligonucleotide-tagged
`peptides and serves to stabilize the succinyl ester from cleavage
`by nonaqueous bases (i.e., piperidine and 1,8-diazabicyclo[ 5.4.01-
`undec-7-ene (DBU)).S (D) Enlistment of variable-length tethers
`between the encoding genetic tag and the chemical units, combined
`with the overall general elaboration of the CPG support, provides
`a variety of “steric space”, a functionality generally agreed to be
`an important factor in efficient oligonucleotide and peptide
`synthesis.
`Initially we probed the coupling efficiencies and general overall
`capabilities of our new supports, using the simpler of the four
`matrices, CPG-3. Its ability to support oligonucleotide synthesis
`was tested by the preparation of a number of oligodeoxyribo-
`nucleotides using standard 2-cyanoethyl-protected nucleoside
`phosphoramidite chemistry. The sequences ranged from 24- to
`45-mers, and repetitive yields were typically upwards of 98%, as
`determined by dimethoxytrityl cation release. The ability of
`CPG-3 to serve as a support for SPPS was investigated by the
`synthesis of the test peptide CPG-3-Ser’(aho)-Val-Phe-Gln-Pro-
`His-H [Ser’(aho) indicates the CPG-linked serine]. The peptide
`was released from the support and analyzed by reversed-phase
`HPLC, and its identity was confirmed by ion-spray mass
`spectrometry (data not shown).
`Several additional model studies were undertaken to prepare
`for the construction of oligonucleotide-tagged peptide libraries
`on our CPG supports by alternating parallel synthesis. Appro-
`priate Fmoc-amino acid derivatives were subjected to the reagents
`used in oligonucleotide synthesis,6 and all were found to be stable.
`A study on the stability of the required protecting groups for the
`exocyclic amino moieties contained within the nucleobases
`(adenine, cytosine, and guanine) was also performed. The
`conditions used for the Fmoc deprotection do not significantly
`affect the standard exocyclic benzoyl and isobutyryl protecting
`groups, eliminating the possibility that these nucleobases could
`generate erroneous branch points. With regard to phosphate
`protection, we chose to use the methyl phosphate moiety as it has
`robust stability toward our Fmoc deprotection reagent.’
`To investigate the biochemical properties of these oligonu-
`cleotide-tagged peptides, the syntheses of several peptide ligand
`sequences containing the &endorphin epitope were performed
`on CPG-3 and -4, Chart I.* The sequences of both the monomeric
`and the dendritic peptides wereconfirmed by Edman degradation.
`All nucleotide sequences were shown to be efficiently radiolabeled
`with T4-polynucleotide kinase, and the nucleotide tags could be
`amplified using standard PCR technique^.^ Binding assays were
`initiated using both the mono- and the bivalent ligands. Our
`preliminary findings show that antibody 3-E7lO binds only the
`relevant leucine enkephalin-encoded members (7-10; Kd of 7 and
`8 = 24 nM). Notably, the potentially sterically impeding
`oligonucleotide tag does not appear to affect peptide binding.
`(5) Brown,T.; Pritchard, C. E.;Turner, G.; Salisbury, S. A. J. Chem. SOC.,
`Chem. Commun. 1989, 891-893.
`(6) 3% TCA/CH2C12 and 12/HzO/pyridine/THF.
`(7) Palom, Y.; Alazzouzi, E.; Gordillo, F.; Grandas, A.; Pedroso, E.
`Tetrahedron Lett. 1993, 34, 2195-2198.
`(8) Syntheses here were performed in a nonalternating manner.
`(9) PCR of the oligonucleotide-tagged @-endorphin peptides were suc-
`cessfully accomplished either in solution or when bound to 3-E7 coated
`DynaBeads.
`(10) Meo, T.; Gramsch, C.; Inan, R.; Hdllt, V.; Weber, E.; Herz, A.;
`Riethmuller, G. Proc. Narl. Acad. Sci. U.S.A. 1983, 80, 40844088.
`0 1993 American Chemical Society
`
`
`
`J. Am. Chem. SOC. 1993,115, 98 12-98 13
`
`Synthetic Methods for the Implementation of Encoded
`Combinatorial Chemistry
`
`John Nielsen, Sydney Brenner,’ and Kim D. Janda.37
`The Scripps Research Institute
`Departments of Molecular Biology and Chemistry
`10666 North Torrey Pines Road
`La Jolla, California 92037
`Received June 2, 1993
`Screening of natural and synthetic compounds for activity as
`potential therapeutic agents has for decades been the main source
`of drug discoveries in the pharmaceutical industry. With advances
`in molecular biology, protein crystallography, and computational
`chemistry, “rational drug design” has found many advocates, but
`this approach is still difficult and slow. In recent years there has
`been a renaissance in drug screening with the advent of new
`technologies based on “combinatorial libraries”.’ These methods
`expose a large number of compounds to a target, and they allow
`compounds that bind to a target with the highest affinity to be
`filtered out from a pool of statistical sequences. Present
`technologies have some limitations; in chemically synthesized
`libraries, mostly of peptides to date, there can be intrinsic
`difficulties in the identification, selection, and enrichment of active
`compounds. In bacteriophage and nucleic acid libraries, the active
`compounds can be amplified by replication, but chemical diversity
`is inherently limited. Recently, a conceptual scheme for “encoded
`combinatorial chemistry” has been proposed as a possible solution
`to a number of these potential restri~tions.~J Such libraries would
`combine the potentially limitless diversity of synthetic libraries
`with the advantages of natural libraries based on gene technologies.
`It was proposed to link each molecule of a chemically synthesized
`entity to a particular oligonucleotide sequence constructed in
`parallel and to use this encoding genetic tag to identify and
`enrich active compounds. In this paper, we detail the chemistry
`necessary to synthesize encoded libraries, choosing peptides as
`an example.
`In essence, such libraries require alternating stepwise syntheses
`of a peptide and an oligonucleotide sequence on a common linker
`(Scheme I). Thus a linker had to be constructed and attached
`to a suitable solid support capable of housing both the oligonu-
`cleotides and the peptides synthesized. Such a spacer had to be
`compatible with both solid-phase peptide synthesis (SPPS) using
`Fmoc chemistry and oligonucleotide synthesis with phosphora-
`midites, and it needs to be functionalized in such a way that it
`is possible to deprotect and detach the oligonucleotide-tagged
`peptides in a regioselective, nondestructive manner. This latter
`process allows the encoding of molecules rather than their
`carriers. Furthermore, we wanted the linker unit to be versatile
`enough to allow a “dendritic” display of the chemical library for
`controlled multivalent ligand d i ~ p l a y . ~
`Controlled-pore glass (CPG) was used as the solid matrix,
`because this material has a long-standing reputation for efficient
`oligonucleotide synthesis and has also proved efficient in SPPS.
`* To whom reprint requests and correspondence should be addressed.
`t A. P. Sloan Fellow, 1993-1995.
`(1) For some excellent reviews, see: (a) Jung, G.; Beck-Sickinger, A. G.
`Angew. Chem., Int. Ed. Engl. 1992,31,367-383. (b) Pavia, M. R.; Sawyer,
`T. K.; Moos, W. H. Bioorg. Med. Chem. Lett. 1993, 3, 387-396.
`(2) (a) Brenner, S.; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 1992,89,
`5381-5383. (b) Amato, I. Science 1992, 257, 330-331.
`(3) Recently, two papers describing the encoding of combinatorial libraries
`have appeared: (a) Kerr, J. M.; Banville, S. C.; Zuckermann, R. N. J. Am.
`(b) Nikolaiev, V.; Stierandova, A.;
`Chem. SOC. 1993, 225, 2529-2531.
`Krchnak, V.; Seligmann, B.; Lam, K. S.; Salmon, S. E.; Lebl, M. PeptideRes.
`1993,6,161-170. However, thesemethods usepeptidesas thecoding elements,
`and enrichment by amplification is not possible.
`(4) For a discussion of multivalent peptide display, see: (a) Cull, M. G.;
`Miller, J. F.;Schatz,P. J. Proc. Natl.Acad.Sci. U.S.A. 1992,89, 1865-1869.
`(b) Lowman, H. B.; Bass, S. H.; Simpson, N.; Wells, J. A. Biochemistry 1992,
`30, 10832- 108 38.
`
`0002-7863/93/1515-9812$04.00/0
`
`Scheme I1 delineates the synthesis of four new supports, CPG-3,
`-4, -5, and -6. There are several features to be noted: (A) N-Fmoc-
`and 0-DMT-protected L-serine/lysine-branched monomers serve
`as differential orthogonal attachment points for the oligonucle-
`otides and the peptides (singular and bivalent). (B) The high
`optically purity of these CPG supports is necessary since a
`stereochemically impure linker complicates testing, analysis, and
`interpretation of results from such biopolymers. (C) Employment
`of the succinyl aminohexanol-sarcosine appendage provides a
`method for nonacidic detachment of the oligonucleotide-tagged
`peptides and serves to stabilize the succinyl ester from cleavage
`by nonaqueous bases (i.e., piperidine and 1,8-diazabicyclo[ 5.4.01-
`undec-7-ene (DBU)).S (D) Enlistment of variable-length tethers
`between the encoding genetic tag and the chemical units, combined
`with the overall general elaboration of the CPG support, provides
`a variety of “steric space”, a functionality generally agreed to be
`an important factor in efficient oligonucleotide and peptide
`synthesis.
`Initially we probed the coupling efficiencies and general overall
`capabilities of our new supports, using the simpler of the four
`matrices, CPG-3. Its ability to support oligonucleotide synthesis
`was tested by the preparation of a number of oligodeoxyribo-
`nucleotides using standard 2-cyanoethyl-protected nucleoside
`phosphoramidite chemistry. The sequences ranged from 24- to
`45-mers, and repetitive yields were typically upwards of 98%, as
`determined by dimethoxytrityl cation release. The ability of
`CPG-3 to serve as a support for SPPS was investigated by the
`synthesis of the test peptide CPG-3-Ser’(aho)-Val-Phe-Gln-Pro-
`His-H [Ser’(aho) indicates the CPG-linked serine]. The peptide
`was released from the support and analyzed by reversed-phase
`HPLC, and its identity was confirmed by ion-spray mass
`spectrometry (data not shown).
`Several additional model studies were undertaken to prepare
`for the construction of oligonucleotide-tagged peptide libraries
`on our CPG supports by alternating parallel synthesis. Appro-
`priate Fmoc-amino acid derivatives were subjected to the reagents
`used in oligonucleotide synthesis,6 and all were found to be stable.
`A study on the stability of the required protecting groups for the
`exocyclic amino moieties contained within the nucleobases
`(adenine, cytosine, and guanine) was also performed. The
`conditions used for the Fmoc deprotection do not significantly
`affect the standard exocyclic benzoyl and isobutyryl protecting
`groups, eliminating the possibility that these nucleobases could
`generate erroneous branch points. With regard to phosphate
`protection, we chose to use the methyl phosphate moiety as it has
`robust stability toward our Fmoc deprotection reagent.’
`To investigate the biochemical properties of these oligonu-
`cleotide-tagged peptides, the syntheses of several peptide ligand
`sequences containing the &endorphin epitope were performed
`on CPG-3 and -4, Chart I.* The sequences of both the monomeric
`and the dendritic peptides wereconfirmed by Edman degradation.
`All nucleotide sequences were shown to be efficiently radiolabeled
`with T4-polynucleotide kinase, and the nucleotide tags could be
`amplified using standard PCR technique^.^ Binding assays were
`initiated using both the mono- and the bivalent ligands. Our
`preliminary findings show that antibody 3-E7lO binds only the
`relevant leucine enkephalin-encoded members (7-10; Kd of 7 and
`8 = 24 nM). Notably, the potentially sterically impeding
`oligonucleotide tag does not appear to affect peptide binding.
`(5) Brown,T.; Pritchard, C. E.;Turner, G.; Salisbury, S. A. J. Chem. SOC.,
`Chem. Commun. 1989, 891-893.
`(6) 3% TCA/CH2C12 and 12/HzO/pyridine/THF.
`(7) Palom, Y.; Alazzouzi, E.; Gordillo, F.; Grandas, A.; Pedroso, E.
`Tetrahedron Lett. 1993, 34, 2195-2198.
`(8) Syntheses here were performed in a nonalternating manner.
`(9) PCR of the oligonucleotide-tagged @-endorphin peptides were suc-
`cessfully accomplished either in solution or when bound to 3-E7 coated
`DynaBeads.
`(10) Meo, T.; Gramsch, C.; Inan, R.; Hdllt, V.; Weber, E.; Herz, A.;
`Riethmuller, G. Proc. Narl. Acad. Sci. U.S.A. 1983, 80, 40844088.
`0 1993 American Chemical Society
`
`
`
`Communications to the Editor
`
`J. Am. Chem. SOC., Vol. 115, No. 21, 1993 9813
`Scheme I. General Methodology for Obtaining Encoded Combinatorial Chemical Libraries
`
`I
`
`n
`
`3. (SC-31
`
`Chart I. Sequences (One-Letter Codes) of Compounds Used in Binding Studya
`5'-AGCTACTTCCCAAGGGAGCTGCTGCTAGTCGGGCCCTATTCTTAG-3'-S-[LFGGY-H]
`7
`5'-AGCTACTTCCCAAGGGAGCTGCTGCTAGTCGGGCCCTATTCTTAG-3'-~~G-S-[LFGGY-H]
`8
`S'-AGCTACTTCCCAAGGGAGCTGCTGCTAGTCGGGCCCTATTCTTAG-3' -5'- [ K (Ea hx-LFGGY-H) 2 ]
`9
`5'-AGCTACTTCCCAAGGGAGCTGCTGCTAGTCGGGCCCTATTCTTAG-3' -EtG-S- [ K ( € a h X-LFGGY-H) 21
`1 0
`1 1
`S'-AGCTACTTCCCAAGGATCACCACACTAGCGGGGCCCTATTCTTAG-3'-S-[VFQPH-H]
`S'-AGCTACTTCCCAAGGATCACCACACTAGCGGGGCCCTATTCTTAG-3'
`1 2
`calm is 6-aminohexanoic acid, EtG is a hexaethylene glycol spacer, and S represents the detached serine-aminohexanol appendage.
`affinity for antibody 3-E7 (Kd of 9 and 10 = 7 nM) than their
`Scheme IIa
`singular counterparts 7 and 8.
`Based on the above findings, we proceeded to make encoded
`peptide members on CPG-3 in the required alternating pattern
`depicted in Scheme I. We discovered that when CPG-3 was
`functionalized with the required primer sequence 1, deprotection
`of the Fmoc was slowed significantly, and, most importantly,
`amino acid addition was not efficient (< 10 5%). Suspecting steric
`hindrance, we tested primer 1 -functionalized CPG-5 and -6.
`CPG-5 provided a significant improvement (35-60%), while
`CPG-6 gave virtually quantitative couplings. Hence, CPG-6
`provides the elements necessary to efficiently make genetically
`encoded peptide libraries.
`In conclusion, we have designed and synthesized a set of four
`chemically pure, base-labile, orthogonally protected branched
`matrices, of which CPG-6 is capable of supporting alternating
`oligonucleotide-peptide synthesis in the required manner. This
`novel support has led to the first successful alternating bidirectional
`synthesis of encoded peptide entities. The simplicity involved in
`creating this matrix for encoded libraries, coupled with our
`controlled "dendritic" methodology, should lend itself to the rapid
`expansion of the technology. Undertakings to create tagged
`libraries based on natural and nonnatural building blocks are
`current 1 y under investigation.
`Acknowledgment. This work was supported in part through
`a fellowship from the A. P. Sloan Foundation (K.D.J.) and by
`a Markey Foundation grant (S.B.).
`Supplementary Material Available: Details of the preparation
`and characterization of encoded chemical members (7 pages).
`Ordering information is given on any current masthead page.
`
`4 ,
`
`( 8 0 - 4 1
`
`a Reagents: (i) Fmoc-sarcosine, PyBOP (benzotriazol- 1 -yloxytripyr-
`rolidinophosphonium hexafluorophosphate), DIPEA, (ii) piperidine/DMF
`(2/8,v/v); (iii) Fmoc-Lys(Fmoc)-OH, PyBOP, DIPEA; (iv) Fmoc-cahx-
`OH (Fmoc-6-aminohexanoic acid), PyBOP, DIPEA; (v) 1 1-Fmoc-amido-
`3,6,9-trioxaundecanoic acid, PyBOP, DIPEA.
`Strikingly, ligand affinity is dependent on the valency of the
`encodedpeptide. Thus the bivalent units 9 and 10 have a greater
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