CA2151473A1 - Synthesis of encoded polymers - Google Patents
Synthesis of encoded polymersInfo
- Publication number
- CA2151473A1 CA2151473A1 CA 2151473 CA2151473A CA2151473A1 CA 2151473 A1 CA2151473 A1 CA 2151473A1 CA 2151473 CA2151473 CA 2151473 CA 2151473 A CA2151473 A CA 2151473A CA 2151473 A1 CA2151473 A1 CA 2151473A1
- Authority
- CA
- Canada
- Prior art keywords
- active
- polymer
- encoding
- carbon atoms
- conjugate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/0054—Means for coding or tagging the apparatus or the reagents
- B01J2219/00572—Chemical means
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Graft Or Block Polymers (AREA)
Abstract
Conjugates and methods of producing the conjugates and mixtures thereof are disclosed. Conjugates are comprised of an active polymer made up of monomer units selected from the group consisting of the monomer units of peptides and/or peptoids, and an encoding polymer comprised of encoding monomers wherein the encoding polymer corresponds to the active polymer, and a coupling moiety covalently coupled to the active polymer and the encoding polymer. In accordance with the synthesis methodology, mixtures of large numbers of conjugates are produced by providing a coupling moiety and covalent binding it to an active monomer and an encoding monomer. Additional monomer units are added to the active monomer to create an active polymer and additional monomer units are added to the encoding monomer to produce an encoding polymer until the desired length is reached for the active polymer. Mixtures of conjugates attached to support bases can be used to assay a sample. The sample is brought into contact with the conjugates and a determination is made with respect to which active proteins bind to receptor sites within the sample. When active binding proteins are determined, the encoding polymer associated with the active polymer is sequenced, and by deduction, the sequence of the active polymer is determined.
Description
WOg4/~6~ 21 ~ 14 7 3 PCT~S93/12013 ~YNl~SIS OF ENCODED POLYMERS
Field of the Invention This invention relates to the fields of biopolymer synthesis and drug design. More particularly, the invention relates to methods for synthesizing libraries of biologically active polymers in association with an included polymer which is ~noo~ to facilitate deciphering.
Back~,~ulld of the Invention Modern pharmaceutical technology has taken two divergent paths in pursuit of new therapeutic compounds. Rational drug design achieves results by intensive analysis of the molec~ r structure of b;n~ing sites, and designing compounds specifically to complement a desired b; n~; ng site. For example, one interested in preparing new antihypertensive compounds might analyze the mol~clll A~ structure of the ~-adrenergic receptor bi n~ i ng site using X-ray crystallography and/or advanced NMR techniques, and then synthesize compounds calculated to fit within the binding site and complement the charge distribution.
The other approach is to prepare an enormous library of compounds and select only those compounds which exhibit a desired activity. This approach differs from the traditional pharmaceutical cycle of design/synthesize/test/synthesize variants by conducting the scr~n; ng step in a massively parallel fashion, scree~;ng an enormous number of different compounds simultaneously. The challenge to this W094/~6~ PCT~S93/~013 ~$ approach is first to provide a group of compounds for scr~n; ng that is sufficiently numerous and diverse to insure that the activity sought is represented in the group, and c~con~ to identify the active compounds at low concentration within the group.
Rutter et al., US 5,010,175 disclosed a method of making diverse mi~L~es of peptides by adjusting the con~entration of each activated peptide in PLU~Vr Lion to its reaction rate, in order to obtain a substantially equimolar mixture of peptides. Rutter also disclosed the ~L G~e~S of providing a mixture of peptides (having at least 50 different peptides), and selecting one or more peptides having a desired property and separating them from the rest of the peptides.
Zuckermann et al., PCT WO91/17823 disclosed an alternative method for preparing diverse mixtures of oligopeptides on solid-phase resins, and a robotic device for performing the nececs~ry ma~;p~ tions. In this method, a pool of resin particles is separated into a number of groups (wherein each group is defined as one or more separate reactions), and a different amino acid coupled to the resin in each group. The groups are then mixed together, separated into a number of yLOU~ and again coupled with a different amino acid for each group. This cycle is repeated until the desired number of amino acids per oligopeptide is obt~i n~ . One advantage of this approach is that each coupling reaction occurs in isolation from other reactants, which permits one to drive each reaction to completion without carefully adjusting the initial conc~tration of each reactant.
This method also facilitates the preparation of oligopeptides wherein some positions within the peptide chain are held constant, and where some posi~
tions are restricted to less than all amino acids.
For example, one may use this method to prepare a pep-tide of the formula X1-~-X3-Glu-Ala-X4-Xs-~, where Xn wo 941~6~ 21~ 1 4 7 3 PCT~S93/~0~
~ ~. , .
can be any amino acid. If desired, one could limit, for example, X3 and X5 to l.yd~hobic residues.
Zuckermann also disclosed that this method may be applied to the synthesis of oligonucleotides, which may then be inserted into cloning and expression vectors for biological expression.
Bartlett et al., PCT WO91/19735 disclosed a variation of the Zuckermann et al. method i~ which a diverse set of non-amino acid monomers is employed to form mixtures of com~o~ C called "peptoids."
Peptoids sample a different region of physico-chemical parameter space than traditional oligopeptides, ~Pp~nA; ng on the type of linkage between monomers, and may be able to exhibit activities unavailable to peptide libraries due to the diversity (or difference) in side ch~; nc .
Houghten, US 4,631,211 disclosed a "tea-bag"
peptide synthesis method. The "tea bags" are mesh bags cont~i n; ng resin beads for peptide synthesis.
Houghten's method enables one to add the same amino acid to a number of different oligopeptides without mixing the products: a number of "tea bags~' may be reacted with an amino acid in a common pot, then separated physically.
Cook, EP 383620 described synthesis of COP-1, a random polymer of Ala, Glu, Lys, and Tyr, having an average mol~c~ r weight of 23 kDa h~ving activity in the treatment of multiple sclerosis. COP-l is made in the prior art by chemical polymerization of the amino acids. However, Cook described expression from genes made by random polymerization of oligonucleotides, and selection for those clones expressing COP-1 with the highest activity.
-Lebl et al., EP 445915 described a machine for performing multiple simultaneous peptide syntheses using a planar support surface. The planar support is, for example, paper or cotton.
W094/~ ~ PCT~S93/12013 ~S~ 4~ -4-Kauf~man et al., W086/05~03 disclosed production of peptide libraries by expression from synthetic genes which are partially or wholly "s~o~ tic." S~o~h~tic genes are prepared by polym-erizing a mixture of at least three oligonucleotides (at leaæt heptamers) to form a double-stranded sto~-hActic sequence, and ligating ~he stochastic sequence into an expression vectorO
Lam et al., WO92/00091 disclosed libraries of oligonucleotides, oli~o~e~Lides, and peptide/nucleotide chimeras, and methods for screening the librarieæ for active compollnA~. However, Lam did not disclose conjugates having an active seguence and a coA; ~ equence.
K.M. Derbyshire et al., Gene (1986) 46:145~
52 disclosed a method for "saturation mutagenesis" of a segment of DNA, by synthesizing oligonucleotides using contaminated pools of monomer. Each A, C, G, and T reservoir con~ e~ 1/54 parts of each of the other bases. The object was to prepare a DNA segment mixture having one or two mutationC per sequence.
They did not observe equal frequencies of mutation, presumably due to differences in coupling efficiency.
The authors suggested synthesizing seq~ seC using four reservoirs cont~in;n~ pure bases, and one reservoir cont~;ni~q a mixture of all four bases in the co~cPntrations npcesc~ry to h~lAnce the coupling efficiencies.
J.F. Rei~h~r-olson et al., Science (1988) 241:53-57 disclosed the generation of mutant ~
repressor proteins by replacing two co~o~c with random nucleotides (NNG/C). The resulting mutant proteins were assayed for activity to determine which amino acid positions were critical, and which positions should be conserved.
I.S. Dunn et al., Prot Enq (1988) 2:283-91 disclosed the use of random polynucleotides to gen-erate mutant ~-lactamase ~-peptides, some of which WO94/136~ ~1 S 14 7 3 PCT~S93/~013 ~ ^ , .
exhibited properties superior to the native sequence ~-peptide.
A.R. Oli~h~nt & K. Struhl, Nuc Acids Res (1988) 16:7673-83 disclosed the use of random poly-nucleotides to investigate promoter function. A
section of random polynucleotide was inserted into the -35 to -10 region of a gene conferring drug resistance in E. coli, and the transformants screened for resis-tance. Survivors were cloned and sequenced to provide a functional con~pn~lc sequence.
F.W. Studier, Proc Natl Acad Sci USA (1989) 86:6917-21 disclosed a method for sequencing large volumes of DNA by random priming of cosmid libraries.
A.R. Olirh~nt et al., Proc Natl Acad Sci USA
(1989) 86:9094-98 disclosed the generation of ~-lactamase mutants having altered properties, by cloning a random polynucleotide into the ~-lactamase gene.
D.K. Dube et al., Biochem (1989) 28:5703-07 disclosed the generation of ~-lactamase mutants having altered properties, by cloning a random polynucleotide into the ~-lactamase gene.
R.A. Owens et al., Biochem Biophys Res Comm (1991) 81:402-08 disclosed the selection of an HIV
protease inhibitor from a library of 240,000 tetrapep-tides (in 22 mixtures). The mixtures were prepared by the "mixed resin" t~h~ i ~ue.
These ~e~h~ i ques enable one to prepare libraries of diverse compounds. However, the problem of identifying the resulting compounds has ~eldom been addressed. Oligopeptides are typically se~Pnc~ by stepwise cleavage of each amino acid from tle parent comro~ln~ (which is 11~ 1 ly immobilized on a resin), with chromatographic analysis of the cleaved moiety.
Sensitive tec~n;ques are required to distinguish between twenty or more amino acids. Analysis is further complicated when uncommon amino acids are W094/136~ PCT~S93/~0~
employed (using ~ulL~l~L tec~ni~ues)~ especially when monomers are 1ink~ without using amide bonds.
SummarY of the Invention The present invention provides a method of synthesizing true mixtures of diverse oligopeptides and/or peptide-like compolln~c along with an associated enco~; ng polymer making it po sible to easily analyze those compounds exhibiting a desired activity. The invention involves synthesizing an ~nCOA i ng DNA strand simultaneously with the peptide/peptoid. Each unique peptide/peptoid sequence associated with its own unique DNA strand to provide the conjugates of the invention. These conjugates are screened to determine which peptide/
peptoid compounds exhibit a desired activity, and the active conjugates analyzed by DNA sequencing methods to determine the att~hPA peptide/peptoid sequence by deduc-tion, i.e., since each DNA sequence is associated with a known peptide/peptoid, once the DN~
sequence is deter-mined, the sequence of the peptide/peptoid can be ~ c~.
Another aspect of the invention is a conjugate comprising a peptide or peptoid coupled to and/or directly associated with a coAi~g polymer (CP), e.g. a nucleic acid (NA). The peptide/peptoid/CP
conjugate may be l;nke~ directly (i.e., covalently bound either directly or through a small organic mol-ecule), or by linkage to the same ~u~olL (e.g., bysynthesizing both peptide/peptoid and CP strand on the same particle or bead of resin).
An important object of the invention is to provide a chemical synthesis method which allows the production of libraries of peptides and/or peptoids along with a unique ~nco~ polymer such as a DNA
strand which makes it possible to readily determine the sequence of the peptide or peptoid.
WOg4/~6~ 215 14 ~ 3 PCT~S93/~013 ~ .
~ 7 An advantage of the present invention is that the methodology makes it possible to readily identify and sequence peptides and/or peptoids having desirable biological activities.
A feature of the present invention is that se~lPnc~c of peptoidæ or peptides which contain nonconventional amino acids can still be readily determined by seguencing associated polymers such as DNA se~l~nce~ which are simult~n~o~lcly synthesized with the peptoids and enco~e them.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the structure, synthesis and use as more fully set forth below, reference being made to the accompanying figures forming a part hereof.
Brief Description of the Drawinqs Figure 1 is a schematic diagram showing a specific embodiment of a conjugate of the invention which conjugate includes a "b; n~; ng~ strand or active polymer attached to a solid-~u~u~L substrate which substrate is also ho~ to an i~formation storage or "co~;ng" strand;
Figure 2 is a schematic flow diagram demonstrating how ~nco~ libraries can be synthesized on beads as the solid _U~l L substrate;
Figure 3 is a schematic diagram showing methods of the synthesis of both solid-phase and solution-phase libraries;
Figure 4 is a schematic diagram showing resin-bound libraries generated by the derivatization of non-hydrolyzable resins;
Figure 5 is an HPLC chromatogram of binding and co~;ng peptide strands simultaneously synthesized via non-hydrolyzable resin l;nk~e;
W094/~6~ PCT~S93/~013 ~ ~5~4~3 -8~
Figure 6 is an HPLC chromatogram of a coding and binding strand adduct which was synthesized via a hydrolyzable resin l;nk~;
Figure 7 iæ a plotted graph resulting from ELISA competition of bi n~ i ng se~l~nc~s versus bi n~ i ng/ enCoA; ng se~l~ec;
Figure 8 is a schematic diagram showing the analysis of a solid-phase amptide; and Figure 9 is a schematic flow diagram showing the analysis of a solution-phase amptide.
Detailed Description of Preferred Embodiments Before the present method of synthesis, conjugates and methods of using such are described, it is to be understood that this invention is not limited to the part;c~ r methodologies, conjugates, or methods of use described as such may, of course, vary.
It is also to be understood that the terminology used herein is for the ~ul~o~e of describing particular emho~;ments only and is not inten~ to be limiting since the scope of the present invention will be limited only by the apr~n~ claims.
It must be noted that as used herein and in the apr~nA~A claims, the singular forms "a," "and,"
and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes mixtures of peptides, reference to "an amino acid" includes mixtures thereof, and reference to "the reaction"
includes one or more reactions of the same type as generally understood by those skilled in the art, and so forth.
Unless defined all otherwise, all ~c-h~; cal and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or WO94/136~ 21514 7 3 PCT~S93/~013 ~ 9 testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe and disclose specific S information for which the reference was cited in ro~ection with.
In general, the invention provides a rapid method of synthesizing large numbers of conjugates which conjugates are comprised of a peptide/peptoid sequence, e.g., an amino acid sequence associated with a unique ~nco~ i ng sequence, e.g., a DNA sequence. The conjugates can be readily synthesized and thereafter screened for biological activity, and when activity is found, the particular peptide/peptoid sequence found to be active can be readily identified by its associated ~nco~;ng (DNA) strand. Each conjugate of the invention is comprised of at least two aomponents with one of the comron~nts being the peptide or peptoid sequence which binds to a receptor of interest and the other sequence being a polymer which encodes the bi n~; n~ sequence. The invention may utilize st~n~rd amino acids and DNA as encoding monomers to produce a chemically diverse library of solution-phase or solid-phase conjugates. In order to further describe the invention in detail, the following definitions are provided.
A. Definitions The terms "nucleic acid" and "NA" refer to oligomers constructed from DNA and/or RNA bases which may be se~l~nc~A using st~n~-rd DNA sequencing tech-niques. The NAs used herein may include uncommon bases so long as such bases are distingll;~hAhle from the other bases employed under the DNA sequencing methods to be used and include peptide-nucleic acids (PNAs) (disclosed by Nielsen, P.E., Egholm, M., Berg, R.H. & Rll~h~rdt, O., Science (1991) 254, 1497-lSO0).
Such PNAs could serve as co~i ng ctrands and the W094/~6~ PCT~S93/~013 ~4~3 lo detection would be by hybridi-zation. NAs will usually be constructed from monomers linke~ by phos-phodiester bonds, but other similar linkages may be substituted if desired. For example, phosphorothioates may be employed to reduce lability.
The term Upeptide'' as used herein refers to the 20 commonly oc~ ing amino acids: Al~nin~ (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylAlAni~ (F), glycine (G), histi~in~ (H), isoleucine (I), lysine (R), l~l~cine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), thr~Qnin~ (T), valine (V), tryptophan (W), and tyrosine (Y).
The term "peptoid~ as used herein refers to a non-peptide monomer of the general formula (R)~-X-(L)m~ where R is a side chain group, n is at least 1, L is a linkin~ group, m i8 at least 2, and X is a small organic radical. It is preferred to select L
radicals that may be indivi~ y protected and deprotected. Preferably n will be 1 or 2 and m will be 2. Monomers wherein m is 3 or greater may be used to form brAn~h~ active poly~ers. Presently preferred monomers are N-substituted glycine derivatives of the formula ~ x wherein R is alkyl of 2-6 carbon atoms, haloalkyl of 1-6 carbon atoms wherein halo is F, Cl, Br, or I, alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms, cyclolkyl of 3-8 carbon atoms, alkoxyalkyl of 2-8 carbon atoms, aryl of 6-10 carbon atoms, arylalkyl of 7-12 carbon atoms, arylalkyl of 7-12 carbon atoms substituted with 1-3 r~icA~ epPnApntly selected from halo and nitro and hY~LGXY~ am;~Q~1~Y1 Of 1-6 carbon atoms, ~ly~Loxyalkyl of 1-6 carbon atoms, WOg4/~6~ 21 51 4 7 3 PCT~Ss3/~013 carboxy, carboxyalkyl of 2-6 carbon atoms, carho~lk~y-alkyl of 3-10 carbon atoms, carbamyl, carbamylalkyl of 2-6 carbon atoms, imidazolyl, imid-azolylalkyl of 4-10 carbon atoms, pyridyl, pyridylalkyl of 6-10 carbon atoms, piperidyl, pip-eridylalkyl of 5-10 carbon atoms, indolyl, or indolyalkyl of 9-15 carbon atoms. Thus, active polymers composed of these monomers are equivalent to polyglycine having side ChA; nc att~ at each nitrogen. These and other monomers are described in cop~n~;~g application USSN 07/715,823, incorporated herein by reference, and PCT W091/19735.
The terms ~coA i ng~ and ~e~coAing~ indicated that one or more coAi ng monomers corresponds directly and uniquely to a given active monomer, e.g., conventional nucleic acids ~n~o~ (in y~Ou~S of three) the 20 natural amino acids. The number of co~i ng monomers used for each code ~p~nAc on the number of different coAing mono-mers and the number of different active monomers.
Typically, the number of different active monomers used will range from about 5 to about 30. A basis set of 4 coding monomers can ~ncoA~ up to 1~ active monomers taken in ~co~nc~ of 2 co~i ng monomers. By increasing the co~i ng monomer basis set to five distinct monomers, one can encode up to 25 different peptide/peptoid monomers. A basis set of 4 coA i ng monomers can ~nco~e up to 64 peptide/peptoid monomers taken in "codons" of 3 co~;~g monomers. Note that one can make the code degenerate or nonA~generate, and can insert additional co~i ng information into the sequence. For example, one may wish to begin each codon with the same base (e.g., G), using that base only in the first position, thus unambiguously identi-fying the beginning of each codon. As a practical matter, the group of monomers selected for use as coding monomers will form polymers that are easier to W094/~6~ PCT~S93/~0 sequence than the active polymers, i.e., the co~in~
monomers may be more readily identified using present day sequencing ~r~nology as compared to the monomer of the active poly~ers. With current technology, the order of preference for co~in~ monomers is nucleic acids > peptides > peptoids. Nucleic acids have the additional advantage that the roA i n~ sequence may be amplified by cloning or PCR (polymerase chain reaction) methods known in the art.
The term "active polymer" and/or "bin~in~
polymer" refers to a polymer having a desired biological activity. Suitable biological activities include bin~;n~ to natural receptors, pharmaceutical effects, immunogenicity/antigenicity, and the like.
"Immunogenicity" refers to the ability to stimulate an immune response (whole or partial serum-mediated i -unity and/or cell-mediated immunity) in a bird or mammal following administration. Antigenicity reguires only that the active polymer bind to the antigen-binAi n~ site of an antibody. Pharmaceutical activities, for the ~hL~o~es of this invention, will generally ~p~n~ on the ability of the active polymer to bind a protein, carbGl~d~ate, lipid, nucleic acid, or other com~ present in the subject. For example, an active poly~er may bind to a cell surface receptor and compete with the receptor's natural ligand, with or ~ithout activation of the receptor.
Other useful pharmaceutical activities include cleavage of endogenous molecules (e.g., protease activity, nuclease activity, and the like), catalysis of reactions either primarily or as a cofactor, donation of functional yLo~ (e.g., acyl, ATP, alkyl, and the like), pore formation, and the like. Active polymers comprise a series of monomers which are 1inkP~ sequentially. The monomers will generally be peptides, peptoids, or carbohydrates in the practice of the instant invention.
W094/~6~ 215 1~ 7 3 PCT~S93/~013 ~ .
The term "mixture" as used herein refers to a composition having a plurality of similar components in a single vessel.
The term "couple" as uæed herein refers to formation of a covalent bond.
The term "coupling moiety" refers to a soluble or insoluble ~ u~ to which can be attached one or more active monomers and the ~o~Le~pon~ing ~ncoA;~g monomers. Insoluble ~u~GLLs ("solid ~u~o~
lo means") may be any solid or semi-solid eurface which is stable to the reaction conditions required for synthesis of the active and ro~ing polymers, and is suitable for covalently att~chin~ and immobilizing both polymers, for example, most resins commonly employed in DNA and peptide synthesis, such as MBHA, Rink, and the like. The particular resin used will ~p~n~ upon the choice of coA i n~ and active polymers and their associated synthetic chemistries. Soluble coupling moieties are mol~c~ R having functional ~L Ou~A to which active and co~; ng monomers may be attached. Each soluble coupling moiety must be able to accommodate at least one coAing polymer and at least one active polymer, although the active and co~ing polymers need not be present in a 1:1 ratio.
The soluble coupling moiety may be as simple as an amino acid having an functional group in its side chain, or may be as complex as a function~ ed (soluble) polymer.
The term "conjugate" as used here n refers to the combination of any "active polymer" and its associated "co~i ng~ polymer. The conjugate may be formed using a "coupling moiety" or by b;n~in~ both the "active polymer" and "~nco~ing polymer" to the same ~lr~lJorL surface in close proximity with each other so that the two polymers are "associated" with each other. When both polymers are bound to the same support surface, æuch as a small bead, the ~nro~;ng polymer can be readily se~l~nce~ off of the bead and W094/~6~ - PCT~S93/~013 i ~5 the othér "active polymers" remaining on the bead will be identified once the enco~;ng sequence is known.
B. Related T.i hraxies and SYnthesis Mçthodologies for producin~ same.
There are many limitations with the current t~hnologies for probing the receptor-bi n~; ng properties of peptide libraries. Filamentous -bacteriophage libraries offer the largest source of peptide diversity (~107-l08 different components) of any current te~hnology to date (Scott, J. & Smith, G., Science, (1990), 249, 386-390; Devlin, J., PangAn;h~n, L. & Devlin, P., Science, (1990), 249, 404-406;
Cwirla, S., Peters, E., Barret, R. & Dower, W., Proc.
Natl. Acad. Sci. U.S.A., (1990), 87, 6378-6382).
These libraries, however, are limited to the natural set amino acids, suffer from biological biases (i.e., varying rates of growth, proteolysis, etc.) and also suffer, in practice, from high levels of backy~o~.d 2 0 bi n~ i ng . The present invention is designed to overcome these difficulties.
Multiple-peptide æynthesis te~hnology has substantially increased the ability to generate individual peptides (Geyæen, H., ~eloen, R. &
Bartel;~g, S., Proc. Natl. Acad. Sci. U.S.A., (1984), 81, 3998-4002; Houghten, R., Proc. Natl. Acad. Sci.
U.S.A., (1984), 5131-5135; !e~hn~rrenberg, G. &
Gerhardt, H., Tetrahedron, (1989), 45, 7759-7764;
Gausepohl, H., Kraft, ~., Rolll;~, C. & Frank, R. in Peptides: Chemistry. Structure and BioloqY
(Proc~;n~s of the 11th American Peptide Symposium, (1990), eds. Rivier J. & Marshall, G., (ESCOM, Leiden), pp. 1003-1004; Frank, R. & Doring, R., Tetrahedron, (1988), 44, 6031-6040; Fodor, S., Read, J., Pirrung, N., Stryer, L., Lu, A. & Solas, D., Science, (1991), 251, 767-773). The synthesis of ~104 individual peptides per cm2 of glass wafer represents the diversity limit of this technology (Fodor, S., WOg4/~6~ 215 1~ 7 3 PCT~S93/~013 Read, J., Pirrung, M., Stryer, L., Lu, A. & Solas, D., Science, (1991), 251, 767-773). A mixed-resin algorithm method (Furka, A., Sebestyén, M., Asgedom M.
& Dibo, G., Int. J. Petide Protein Res., (1991), 37, 487-493) has recently been used to generate solution-phase libraries (Houghten, R., Pi ni 1 1~ ~ C., B1O~P1 1 e, S., Appel, J., Dooley, C. & Cuervo, J., Nature, (1991), 354, 84-86) and resin-bound peptide libraries (Lam, K., Salmon, ~., Hersh, E., Hruby, V., Kazmiersky, W. & Knapp, R., Nature, (1991), 354, 82-84) that contain ~106 and ~107 comrQn~nts, respectively. The solution-phase libraries offer the advantage of providing quantitative receptor-binding information (Zuckermann, R., Kerr, J., Siani, M., Banville, S. & Santi, D.V., Proc. Natl. Acad. Sci.
U.S.A., 89, 4505-4509 (1992)). Furthermore~ these libraries allow the affinity of the solution conformation of a ligand to be determined, a quantity that is essential for rational drug design. An apparatus for the automated synthesis of equimolar peptide mixtures is disclosed in Zuckermannr R.N., Kerr, J.M., Siani, M.A. & Banville, S.C., Int. J. Pep.
Pro. Res., (1992), 40, 498-507.
The publications cited and ~ e~ above can be used in producing the active or bi n~; ng polymer which is used in producing the conjugate of the present invention. Accordingly, the disclosures of all of these publications are in~o~o~ated herein by reference in order to disclose peptide and peptoid synthesis methodology. Al~holyh the methodology ~;~c~l~ced within these references is extremely valuable with respect to the production of large amounts of different types of bin~in~ polymers, the mixtures of polymers pro~llc~ by this methodology are often so large and complex that there are many practical limitations with respect to their actual analysis and use. The present invention can be readily applied with such synthesis methodologies in wos4/~6~ PCT~S93/~013 ~$ ~ 16- i order to provide an ef~icient, commercially practical method of analyzing the proteins produced using such methodology.
Both the mixed-resin and solution-phase methods, however, do not allow incorporation of many non-st~n~rd amino acids heC~l~c~ of the limitations of peptide analysis. Resin-bound peptide libraries, in particular, suffer from a relatively slow rate of analysis (peptide se~"ci~ at 3 beads per day) and are limited in complexity to ~107 beads/ml. In order to generate a "complete" peptide library, there must be multiple copies (>lO) of any given peptide sequence. This becomes problematic at the sequenciny stage because the same "hit" sequence may ~rr~r multiple times. The alternative is to work with libraries that are not complete at the risk of losing se~l~nceC that bind.
When using the methodoloyy of the present invention, the sequence of a biologically active protein can be determined even without isolating the protein of interest. This can be done by synthesizing large numbers of different proteins on large numbers of different ~U~OL ~ ~urfaces such as small beads. An ~nco~i n~ polymer is attached to beads to identify each protein. A sample to be tested is then brought into contact with the beads and the beads are observed with respect to which proteins bind to a receptor site in the sample. The bead having the receptor bound thereon is analyzed by sequencing the coding polymer which has also been synthesized on the bead. When the encoding polymer has been se~nce~, the sequence of the active polymer, which may be a peptide, can be readily ~ ce~. Thus, the present invention makes it possible to determine the activity and seguence an active polymer, such as a biologically active peptide, without ever isolating the peptide.
W094/~6~ 21 ~1 4 7 3 PCT~Ss3/~013 C. General Methodolooy This invention describes a methodology for the synthesis and scre~ni ng of large synthetic polymer libraries that contain non-stAnA~rd amino acids and even non-amide h~C~A polymers. The strategy utilizes a modified mixed-resin peptide synthesis methodology to simult~nesll~ly ~ynthesize two polymer ~e~nceC
one polymer strand (the 'lbinAi ng~l strand) is synthesized for the in~Dn~A purpose of receptor b;nAing, and the cecon~ strand (the ~oAing~ strand) contains st~nA~rd amino acids or deoxyrihQnl~cleotides that ~ncoA~ for the binAln~ strand (Figure 1). The ability to decipher the bi nA i ng sequence by analysis of the coA i n~ strand with s~nA~rd peptide or oligonucleotide te~ni~ues allow the inclusion of a wide variety of novel h~ ing blocks and conformational constraints into a diverse ligand library.
This invention also describes a methodology to increase the size t~l8) and scr~ni n~ rate of a ligand library. The method uses two polymers as above, but specifically utilizes an oligodeoxyrihomlcleotide for the ~coA ing" strand. The use of DNA as the coA i ng strand allows for an incr~eA sensitivity of detection (fmol vs pmol for peptide analysis). This increased sensitivity allows for a larger library size since the amount of polymer ne~A for detection is reAnceA dramatically. The rate of sequence det~r~in~tion of receptor binders is increased since many samples can be analyzed in parallel.
In order to couple a polymer's sequence information with a peptide or oligonucleotide sequence, there needs to be a method that unambiguously correlates each polymer to each other.
Thus, when any particular non-st~nA~rd amino acid (or other monomer) is added to a "bi n~ i ~g~ polymer chain, the correcronAing information (amino acid or W094/~6~ PCT~S93/~013 ~S~4~ 3 -18-nucleotide monomer) must also be added to the "coding"
strand. A "genetic code" is thus establi ~h~ (Table 1) where each b;n~;n~ monomer corre~ron~c to (a multiple) of st~n~rd amino acids or nucleotides on the co~; n~ strand. For example, the use of three s~An~rd amino acids or nucleotide , in a 3:1 ratio with a novel monomer, would allow for the unambiguous representation of 27 novel ~onomers.
TABLE 1. Custom genetic code.
# of bases "codon" # of monomers length coded for The synthesis of coded libraries requires a modified mixed-resin algorithm (Figure 2). The resin beads are divided into equal portions, a unique monomer is added to the "bi nA i~g" strand, followed by the coupling of a ~ o~ g amino acid or nucleotide to the '~co~in~ strand. The resin aliquots are then combined to generate a mixture. A set of compatible protecting yL OU~S iS thus required to preferentially deprotect and extend each strand independently Two synthesis formats are possible for amptide libraries, one that generates resin-bound libraries and one that generates solution-phase libraries (Figure 3). Resin-bound libraries can be synthesized using non-hydrolyzable linkers that are derivatized with the "bin~;ng" and l'co~;ng" monomers strands. Solution-phase libraries can be synthesized W094/~6~ PCT~S93/~013 2151~73 .. ,. ,, , ;, , . . 1 9 as a 1:1 polymer:peptide/DNA conjugate via a hydrolyzable link~r attached to the resin.
Pe~tide as the "Codinq" strand The use of base-labile Fmoc-protected - monomers and acid-labile (N~-Ddz-protected ~mino acids (Birr, C., Nassal, M., Pipkorn, R., Int. J. PeDtide Protein Res., (1979), 13, 287-295), for example, allow for selective deprotection and coupling to two individual polymer strands. Resin-bound libraries can be generated by the derivatization of non hydLolyzable resins with a 1:1 ratio (or any desired ratio) of Fmoc:Ddz monomers tFigure 4). This i~lLLGl.ce-c two differently protected amino acids that an be ext~n~
;~p~n~ntly. Solution-phase libraries that contain a 1:1 ratio of bin~;ng:co~;ng strands can be synthesized by using a hydrolyzable Fmoc-Lysine(Moz)-OH l;nk~ that allowed for chain growth at both the ~-and ~-amino ~-~u~. Amino acids which do not contain functional yLOu~s are preferred for the "coding"
strand in order to minimize unwanted binding interactions.
The receptor-bi~ing ligand can be identified by bead st~i~ing ~echn;ques (Lam, K., Salmon, S., Hersh, E., Hruby, V., Kazmiersky, W.
~ rr, R-, Nature, (1991), 354, 82-84) and the sequence determined by N-terminal Edman degradation.
In order to ensure that only the '~oo~ing~ strand is se~l~nc~, it is essential that the N-terminus of the "b;n~ing" strand be acetylated or otherwise made non-seq~ nc~hle.
DNA as the "Coding" Strand The construction of libraries with DNA as the co~ing strand is similar to those with peptides but offers several advantages: the information storage and replicative properties of DNA allow for W094/~6~ - PCT~S93/~0 increased sensitivity of detection, a larger library size and an increased rate of sequence determinationO
The synthesis of DNA as ~he co~; ng polymer requires compatibility between the as~embly of Fmoc-5 h~ ~ monomers and st~n~d DNA chemistry. Thesesynthesis strategies are likely to be compatible ((a) Juby, C., Richardson, C. & Brous eau, R., ~et.
Letters, (1991), 32, 879-882. (b) Haralambidis, J., Duncan, L., Angus, B. & Tregear W.~ Nucleic Acid Res , (1990), 18, 493-499) (see Table 2)o Alternatively, allyl-based protection strategies existæ for both peptide (Lyttle, M.H.; ~l-A~on, D., Pe~tides: ChemistrY
and Biolo~y (Proc~inqs of the 12th American Peptide Symposium): Smith, J. ~nd Rivier, J.E., Eds.; ESCOM, T.~ , 1992, pp. 583-584) and oligodeoxyr;honl~cleotide (Hayakawa, y., I~~k~h~yashi, S., Kato, H. & Noyori, R., J. Am. Chem. Soc., (1990), 112, 1691-1696) synthesis. The assay of æolution-phase libraries can be facilitated by using only pyrimi~;n~c in the coAing strand, thereby avoiding the potential problem of base pairing between individual strands.
Strategies for the synthesis of co~i ng and active polymers, as well as matc~i ng the active polymer with a ~o~;ng ~equence to provide a genetic tag are described in Brenner et al. Proc. Natl. Acad.
Sci. U.S.A., 89:5381-5383 (June 1992) which is incorporated herein by reference.
wo g4/~6~ 2 1 ~ 1 4 7 3 PCT~S93/~0~
- 21 : . .~. .
Compatibility of DNA vs. Peptide Synthesis Chemistry Peptide r1lPmi.stry Oligonucleotide Chemistry ~ o ~ ~0 P~rmanent Protectin~ ~ v~
-OH t-butyl ether A,C benzoyl amide -CO2H t-butyl ester G isob~Ly~l -NH2 t-boc -P=O cyanoethyl, methyl his, cys trityl arg sulfonyl remov~d by:
85% trifluoroacetic acid conc. NE~OH 55C S hours 2 hours @ room temp T~m~orarY ~rot~ti~a ~ G~
9-fluorenylmethoxycaL~oll~l 4,4-dimethoxytrityl rQmove~ by:
20% piperidine 3% trichloroacetic acid CleavaqQ fro~ 801i~ ~UP~oXt:
85% trifluoroacetic acid conc. NE~OH 55C S hours 2 hours ~ room temp SU~;TI ~ UTE:~ S3HEElr W094/136~ PCT~S93/~OL3 ~ABLB 2 tCo~t) Compatibility of DNA vs. Peptide Symthesis Chemistry Std. conditions ~ ;ve - Problem addressed t-Butyl carbamate/ allyl amino acid ester deprotection from detritylation reagent 20% Piperidine 2% DBU substitution at exocyclic amine cyanoethyl allyl acylation of phosphate phosphate NH40H ethyl~neAi~mine/ racemization EtOH
iodine t Butyll~ o Tyr, Met, Cys peroxide oxidation controlled pore poly~yl~ne resin transferring glass resin T, C & G T, C, G & A depurination T, C T, C, G & A base pairing SUBSTITUTE Sl~,EET
WOg4/~6~ 21514 7 3 PCT~S93/~013 .--Resin-bound libraries can be synthesized by using non-hydrolyzable linkers to attach both the C-terminus of the peptide and the 3'-end of the oligonucleotide to the same bead. Solution-phase libraries can be synthesized as a 1:1 peptide-oligonucleotide conjugate, in which the C-terminus of the peptide is attached to the 3'-end of the oligonucleotide through a hydrolyzable Fmoc-Ser(O-Dmt) linker which is attached to the resin.
The identification of binders in the resin-bound peptide libraries can be detected by the bead stA;n;ng methodology (Lam, K., Salmon, S., Hersh, E., Hruby, V., Kazmiersky, W. & Knapp, R., Nature, (1991), 354, 82-84). Although the peptides are bound to a solid-phase, there does not have to be a 1:1 peptide-oligonucleotide ratio since the DNA can be amplified prior to the determination of its sequence. In fact, less DNA is preferred so that there will be less interference with the polymer's binding properties.
Once a bead is identified, the DNA sequence is determined (Stahl, S., Hultman, T., Olsson, A., Mois T., et al., Nucleic Acid Res., (1988), 16, 3025-3038) after PCR amplification or by thermal-cycle sequencing (Figure 8). This requires the inclusion of one or more primer sites neighboring the coA; ng region of the oligonucleotide. Similarly, the use of solution-phase libraries requires isolation of each sequence from each other. This can be accompl; ~h~ by restricting the DNA after PCR amplification and inserting it into M13 (or other suitable vector) for clonal isolation and sequencing (Figure 9).
EXAMPLES
The following examples will provide those skilled in the art with a complete disclosure of how to make and use the invention and are not intended to limit the scope of the invention. Efforts have been made to insure accuracy with respect to numbers used W094/~6~ PCT~S93/12013 (e.g. amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts or parts by weight, mol~c~ ~ weight is weight average molecular weight, temperature is in degrees centigrade and presæure is at or near atmospheric.
Example 1 The in~p~n~pnt synthesis of two unambiguously correlated se~lPnc~c has been sllccessfully completed. The C~lhc~uent sequence analysis of the "co~i n~l strand has also been demonstrated. For ~ol.ve~ience, two peptide se~lPnc~s were chosen. The "b; nA; ng" strand was synthesized with N~-Fmoc-protected amino acids and the "co~i ng~
strand was synthesized with N~-Ddz-protected amino acids. The simultaneous synthesis of the two peptide strands was tested in two formats, 1) resin-bound peptide library synthesis and 2) solution-phase peptide libraries using a hydrolyzable Fmoc-Lys~Moz) OH 1 ;nker (Wang, S.S.; Chen, S.T, Wang, K.T., and Merrifield, R.B., Int. J. Peptide Protein Res., (1987), 30, 662-667). These synth~c~fi were performed on single peptides (not libraries) as a demonstration of research concept and in order to allow the full characterization of the synthesis products.
Field of the Invention This invention relates to the fields of biopolymer synthesis and drug design. More particularly, the invention relates to methods for synthesizing libraries of biologically active polymers in association with an included polymer which is ~noo~ to facilitate deciphering.
Back~,~ulld of the Invention Modern pharmaceutical technology has taken two divergent paths in pursuit of new therapeutic compounds. Rational drug design achieves results by intensive analysis of the molec~ r structure of b;n~ing sites, and designing compounds specifically to complement a desired b; n~; ng site. For example, one interested in preparing new antihypertensive compounds might analyze the mol~clll A~ structure of the ~-adrenergic receptor bi n~ i ng site using X-ray crystallography and/or advanced NMR techniques, and then synthesize compounds calculated to fit within the binding site and complement the charge distribution.
The other approach is to prepare an enormous library of compounds and select only those compounds which exhibit a desired activity. This approach differs from the traditional pharmaceutical cycle of design/synthesize/test/synthesize variants by conducting the scr~n; ng step in a massively parallel fashion, scree~;ng an enormous number of different compounds simultaneously. The challenge to this W094/~6~ PCT~S93/~013 ~$ approach is first to provide a group of compounds for scr~n; ng that is sufficiently numerous and diverse to insure that the activity sought is represented in the group, and c~con~ to identify the active compounds at low concentration within the group.
Rutter et al., US 5,010,175 disclosed a method of making diverse mi~L~es of peptides by adjusting the con~entration of each activated peptide in PLU~Vr Lion to its reaction rate, in order to obtain a substantially equimolar mixture of peptides. Rutter also disclosed the ~L G~e~S of providing a mixture of peptides (having at least 50 different peptides), and selecting one or more peptides having a desired property and separating them from the rest of the peptides.
Zuckermann et al., PCT WO91/17823 disclosed an alternative method for preparing diverse mixtures of oligopeptides on solid-phase resins, and a robotic device for performing the nececs~ry ma~;p~ tions. In this method, a pool of resin particles is separated into a number of groups (wherein each group is defined as one or more separate reactions), and a different amino acid coupled to the resin in each group. The groups are then mixed together, separated into a number of yLOU~ and again coupled with a different amino acid for each group. This cycle is repeated until the desired number of amino acids per oligopeptide is obt~i n~ . One advantage of this approach is that each coupling reaction occurs in isolation from other reactants, which permits one to drive each reaction to completion without carefully adjusting the initial conc~tration of each reactant.
This method also facilitates the preparation of oligopeptides wherein some positions within the peptide chain are held constant, and where some posi~
tions are restricted to less than all amino acids.
For example, one may use this method to prepare a pep-tide of the formula X1-~-X3-Glu-Ala-X4-Xs-~, where Xn wo 941~6~ 21~ 1 4 7 3 PCT~S93/~0~
~ ~. , .
can be any amino acid. If desired, one could limit, for example, X3 and X5 to l.yd~hobic residues.
Zuckermann also disclosed that this method may be applied to the synthesis of oligonucleotides, which may then be inserted into cloning and expression vectors for biological expression.
Bartlett et al., PCT WO91/19735 disclosed a variation of the Zuckermann et al. method i~ which a diverse set of non-amino acid monomers is employed to form mixtures of com~o~ C called "peptoids."
Peptoids sample a different region of physico-chemical parameter space than traditional oligopeptides, ~Pp~nA; ng on the type of linkage between monomers, and may be able to exhibit activities unavailable to peptide libraries due to the diversity (or difference) in side ch~; nc .
Houghten, US 4,631,211 disclosed a "tea-bag"
peptide synthesis method. The "tea bags" are mesh bags cont~i n; ng resin beads for peptide synthesis.
Houghten's method enables one to add the same amino acid to a number of different oligopeptides without mixing the products: a number of "tea bags~' may be reacted with an amino acid in a common pot, then separated physically.
Cook, EP 383620 described synthesis of COP-1, a random polymer of Ala, Glu, Lys, and Tyr, having an average mol~c~ r weight of 23 kDa h~ving activity in the treatment of multiple sclerosis. COP-l is made in the prior art by chemical polymerization of the amino acids. However, Cook described expression from genes made by random polymerization of oligonucleotides, and selection for those clones expressing COP-1 with the highest activity.
-Lebl et al., EP 445915 described a machine for performing multiple simultaneous peptide syntheses using a planar support surface. The planar support is, for example, paper or cotton.
W094/~ ~ PCT~S93/12013 ~S~ 4~ -4-Kauf~man et al., W086/05~03 disclosed production of peptide libraries by expression from synthetic genes which are partially or wholly "s~o~ tic." S~o~h~tic genes are prepared by polym-erizing a mixture of at least three oligonucleotides (at leaæt heptamers) to form a double-stranded sto~-hActic sequence, and ligating ~he stochastic sequence into an expression vectorO
Lam et al., WO92/00091 disclosed libraries of oligonucleotides, oli~o~e~Lides, and peptide/nucleotide chimeras, and methods for screening the librarieæ for active compollnA~. However, Lam did not disclose conjugates having an active seguence and a coA; ~ equence.
K.M. Derbyshire et al., Gene (1986) 46:145~
52 disclosed a method for "saturation mutagenesis" of a segment of DNA, by synthesizing oligonucleotides using contaminated pools of monomer. Each A, C, G, and T reservoir con~ e~ 1/54 parts of each of the other bases. The object was to prepare a DNA segment mixture having one or two mutationC per sequence.
They did not observe equal frequencies of mutation, presumably due to differences in coupling efficiency.
The authors suggested synthesizing seq~ seC using four reservoirs cont~in;n~ pure bases, and one reservoir cont~;ni~q a mixture of all four bases in the co~cPntrations npcesc~ry to h~lAnce the coupling efficiencies.
J.F. Rei~h~r-olson et al., Science (1988) 241:53-57 disclosed the generation of mutant ~
repressor proteins by replacing two co~o~c with random nucleotides (NNG/C). The resulting mutant proteins were assayed for activity to determine which amino acid positions were critical, and which positions should be conserved.
I.S. Dunn et al., Prot Enq (1988) 2:283-91 disclosed the use of random polynucleotides to gen-erate mutant ~-lactamase ~-peptides, some of which WO94/136~ ~1 S 14 7 3 PCT~S93/~013 ~ ^ , .
exhibited properties superior to the native sequence ~-peptide.
A.R. Oli~h~nt & K. Struhl, Nuc Acids Res (1988) 16:7673-83 disclosed the use of random poly-nucleotides to investigate promoter function. A
section of random polynucleotide was inserted into the -35 to -10 region of a gene conferring drug resistance in E. coli, and the transformants screened for resis-tance. Survivors were cloned and sequenced to provide a functional con~pn~lc sequence.
F.W. Studier, Proc Natl Acad Sci USA (1989) 86:6917-21 disclosed a method for sequencing large volumes of DNA by random priming of cosmid libraries.
A.R. Olirh~nt et al., Proc Natl Acad Sci USA
(1989) 86:9094-98 disclosed the generation of ~-lactamase mutants having altered properties, by cloning a random polynucleotide into the ~-lactamase gene.
D.K. Dube et al., Biochem (1989) 28:5703-07 disclosed the generation of ~-lactamase mutants having altered properties, by cloning a random polynucleotide into the ~-lactamase gene.
R.A. Owens et al., Biochem Biophys Res Comm (1991) 81:402-08 disclosed the selection of an HIV
protease inhibitor from a library of 240,000 tetrapep-tides (in 22 mixtures). The mixtures were prepared by the "mixed resin" t~h~ i ~ue.
These ~e~h~ i ques enable one to prepare libraries of diverse compounds. However, the problem of identifying the resulting compounds has ~eldom been addressed. Oligopeptides are typically se~Pnc~ by stepwise cleavage of each amino acid from tle parent comro~ln~ (which is 11~ 1 ly immobilized on a resin), with chromatographic analysis of the cleaved moiety.
Sensitive tec~n;ques are required to distinguish between twenty or more amino acids. Analysis is further complicated when uncommon amino acids are W094/136~ PCT~S93/~0~
employed (using ~ulL~l~L tec~ni~ues)~ especially when monomers are 1ink~ without using amide bonds.
SummarY of the Invention The present invention provides a method of synthesizing true mixtures of diverse oligopeptides and/or peptide-like compolln~c along with an associated enco~; ng polymer making it po sible to easily analyze those compounds exhibiting a desired activity. The invention involves synthesizing an ~nCOA i ng DNA strand simultaneously with the peptide/peptoid. Each unique peptide/peptoid sequence associated with its own unique DNA strand to provide the conjugates of the invention. These conjugates are screened to determine which peptide/
peptoid compounds exhibit a desired activity, and the active conjugates analyzed by DNA sequencing methods to determine the att~hPA peptide/peptoid sequence by deduc-tion, i.e., since each DNA sequence is associated with a known peptide/peptoid, once the DN~
sequence is deter-mined, the sequence of the peptide/peptoid can be ~ c~.
Another aspect of the invention is a conjugate comprising a peptide or peptoid coupled to and/or directly associated with a coAi~g polymer (CP), e.g. a nucleic acid (NA). The peptide/peptoid/CP
conjugate may be l;nke~ directly (i.e., covalently bound either directly or through a small organic mol-ecule), or by linkage to the same ~u~olL (e.g., bysynthesizing both peptide/peptoid and CP strand on the same particle or bead of resin).
An important object of the invention is to provide a chemical synthesis method which allows the production of libraries of peptides and/or peptoids along with a unique ~nco~ polymer such as a DNA
strand which makes it possible to readily determine the sequence of the peptide or peptoid.
WOg4/~6~ 215 14 ~ 3 PCT~S93/~013 ~ .
~ 7 An advantage of the present invention is that the methodology makes it possible to readily identify and sequence peptides and/or peptoids having desirable biological activities.
A feature of the present invention is that se~lPnc~c of peptoidæ or peptides which contain nonconventional amino acids can still be readily determined by seguencing associated polymers such as DNA se~l~nce~ which are simult~n~o~lcly synthesized with the peptoids and enco~e them.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the structure, synthesis and use as more fully set forth below, reference being made to the accompanying figures forming a part hereof.
Brief Description of the Drawinqs Figure 1 is a schematic diagram showing a specific embodiment of a conjugate of the invention which conjugate includes a "b; n~; ng~ strand or active polymer attached to a solid-~u~u~L substrate which substrate is also ho~ to an i~formation storage or "co~;ng" strand;
Figure 2 is a schematic flow diagram demonstrating how ~nco~ libraries can be synthesized on beads as the solid _U~l L substrate;
Figure 3 is a schematic diagram showing methods of the synthesis of both solid-phase and solution-phase libraries;
Figure 4 is a schematic diagram showing resin-bound libraries generated by the derivatization of non-hydrolyzable resins;
Figure 5 is an HPLC chromatogram of binding and co~;ng peptide strands simultaneously synthesized via non-hydrolyzable resin l;nk~e;
W094/~6~ PCT~S93/~013 ~ ~5~4~3 -8~
Figure 6 is an HPLC chromatogram of a coding and binding strand adduct which was synthesized via a hydrolyzable resin l;nk~;
Figure 7 iæ a plotted graph resulting from ELISA competition of bi n~ i ng se~l~nc~s versus bi n~ i ng/ enCoA; ng se~l~ec;
Figure 8 is a schematic diagram showing the analysis of a solid-phase amptide; and Figure 9 is a schematic flow diagram showing the analysis of a solution-phase amptide.
Detailed Description of Preferred Embodiments Before the present method of synthesis, conjugates and methods of using such are described, it is to be understood that this invention is not limited to the part;c~ r methodologies, conjugates, or methods of use described as such may, of course, vary.
It is also to be understood that the terminology used herein is for the ~ul~o~e of describing particular emho~;ments only and is not inten~ to be limiting since the scope of the present invention will be limited only by the apr~n~ claims.
It must be noted that as used herein and in the apr~nA~A claims, the singular forms "a," "and,"
and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes mixtures of peptides, reference to "an amino acid" includes mixtures thereof, and reference to "the reaction"
includes one or more reactions of the same type as generally understood by those skilled in the art, and so forth.
Unless defined all otherwise, all ~c-h~; cal and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or WO94/136~ 21514 7 3 PCT~S93/~013 ~ 9 testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe and disclose specific S information for which the reference was cited in ro~ection with.
In general, the invention provides a rapid method of synthesizing large numbers of conjugates which conjugates are comprised of a peptide/peptoid sequence, e.g., an amino acid sequence associated with a unique ~nco~ i ng sequence, e.g., a DNA sequence. The conjugates can be readily synthesized and thereafter screened for biological activity, and when activity is found, the particular peptide/peptoid sequence found to be active can be readily identified by its associated ~nco~;ng (DNA) strand. Each conjugate of the invention is comprised of at least two aomponents with one of the comron~nts being the peptide or peptoid sequence which binds to a receptor of interest and the other sequence being a polymer which encodes the bi n~; n~ sequence. The invention may utilize st~n~rd amino acids and DNA as encoding monomers to produce a chemically diverse library of solution-phase or solid-phase conjugates. In order to further describe the invention in detail, the following definitions are provided.
A. Definitions The terms "nucleic acid" and "NA" refer to oligomers constructed from DNA and/or RNA bases which may be se~l~nc~A using st~n~-rd DNA sequencing tech-niques. The NAs used herein may include uncommon bases so long as such bases are distingll;~hAhle from the other bases employed under the DNA sequencing methods to be used and include peptide-nucleic acids (PNAs) (disclosed by Nielsen, P.E., Egholm, M., Berg, R.H. & Rll~h~rdt, O., Science (1991) 254, 1497-lSO0).
Such PNAs could serve as co~i ng ctrands and the W094/~6~ PCT~S93/~013 ~4~3 lo detection would be by hybridi-zation. NAs will usually be constructed from monomers linke~ by phos-phodiester bonds, but other similar linkages may be substituted if desired. For example, phosphorothioates may be employed to reduce lability.
The term Upeptide'' as used herein refers to the 20 commonly oc~ ing amino acids: Al~nin~ (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylAlAni~ (F), glycine (G), histi~in~ (H), isoleucine (I), lysine (R), l~l~cine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), thr~Qnin~ (T), valine (V), tryptophan (W), and tyrosine (Y).
The term "peptoid~ as used herein refers to a non-peptide monomer of the general formula (R)~-X-(L)m~ where R is a side chain group, n is at least 1, L is a linkin~ group, m i8 at least 2, and X is a small organic radical. It is preferred to select L
radicals that may be indivi~ y protected and deprotected. Preferably n will be 1 or 2 and m will be 2. Monomers wherein m is 3 or greater may be used to form brAn~h~ active poly~ers. Presently preferred monomers are N-substituted glycine derivatives of the formula ~ x wherein R is alkyl of 2-6 carbon atoms, haloalkyl of 1-6 carbon atoms wherein halo is F, Cl, Br, or I, alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms, cyclolkyl of 3-8 carbon atoms, alkoxyalkyl of 2-8 carbon atoms, aryl of 6-10 carbon atoms, arylalkyl of 7-12 carbon atoms, arylalkyl of 7-12 carbon atoms substituted with 1-3 r~icA~ epPnApntly selected from halo and nitro and hY~LGXY~ am;~Q~1~Y1 Of 1-6 carbon atoms, ~ly~Loxyalkyl of 1-6 carbon atoms, WOg4/~6~ 21 51 4 7 3 PCT~Ss3/~013 carboxy, carboxyalkyl of 2-6 carbon atoms, carho~lk~y-alkyl of 3-10 carbon atoms, carbamyl, carbamylalkyl of 2-6 carbon atoms, imidazolyl, imid-azolylalkyl of 4-10 carbon atoms, pyridyl, pyridylalkyl of 6-10 carbon atoms, piperidyl, pip-eridylalkyl of 5-10 carbon atoms, indolyl, or indolyalkyl of 9-15 carbon atoms. Thus, active polymers composed of these monomers are equivalent to polyglycine having side ChA; nc att~ at each nitrogen. These and other monomers are described in cop~n~;~g application USSN 07/715,823, incorporated herein by reference, and PCT W091/19735.
The terms ~coA i ng~ and ~e~coAing~ indicated that one or more coAi ng monomers corresponds directly and uniquely to a given active monomer, e.g., conventional nucleic acids ~n~o~ (in y~Ou~S of three) the 20 natural amino acids. The number of co~i ng monomers used for each code ~p~nAc on the number of different coAing mono-mers and the number of different active monomers.
Typically, the number of different active monomers used will range from about 5 to about 30. A basis set of 4 coding monomers can ~ncoA~ up to 1~ active monomers taken in ~co~nc~ of 2 co~i ng monomers. By increasing the co~i ng monomer basis set to five distinct monomers, one can encode up to 25 different peptide/peptoid monomers. A basis set of 4 coA i ng monomers can ~nco~e up to 64 peptide/peptoid monomers taken in "codons" of 3 co~;~g monomers. Note that one can make the code degenerate or nonA~generate, and can insert additional co~i ng information into the sequence. For example, one may wish to begin each codon with the same base (e.g., G), using that base only in the first position, thus unambiguously identi-fying the beginning of each codon. As a practical matter, the group of monomers selected for use as coding monomers will form polymers that are easier to W094/~6~ PCT~S93/~0 sequence than the active polymers, i.e., the co~in~
monomers may be more readily identified using present day sequencing ~r~nology as compared to the monomer of the active poly~ers. With current technology, the order of preference for co~in~ monomers is nucleic acids > peptides > peptoids. Nucleic acids have the additional advantage that the roA i n~ sequence may be amplified by cloning or PCR (polymerase chain reaction) methods known in the art.
The term "active polymer" and/or "bin~in~
polymer" refers to a polymer having a desired biological activity. Suitable biological activities include bin~;n~ to natural receptors, pharmaceutical effects, immunogenicity/antigenicity, and the like.
"Immunogenicity" refers to the ability to stimulate an immune response (whole or partial serum-mediated i -unity and/or cell-mediated immunity) in a bird or mammal following administration. Antigenicity reguires only that the active polymer bind to the antigen-binAi n~ site of an antibody. Pharmaceutical activities, for the ~hL~o~es of this invention, will generally ~p~n~ on the ability of the active polymer to bind a protein, carbGl~d~ate, lipid, nucleic acid, or other com~ present in the subject. For example, an active poly~er may bind to a cell surface receptor and compete with the receptor's natural ligand, with or ~ithout activation of the receptor.
Other useful pharmaceutical activities include cleavage of endogenous molecules (e.g., protease activity, nuclease activity, and the like), catalysis of reactions either primarily or as a cofactor, donation of functional yLo~ (e.g., acyl, ATP, alkyl, and the like), pore formation, and the like. Active polymers comprise a series of monomers which are 1inkP~ sequentially. The monomers will generally be peptides, peptoids, or carbohydrates in the practice of the instant invention.
W094/~6~ 215 1~ 7 3 PCT~S93/~013 ~ .
The term "mixture" as used herein refers to a composition having a plurality of similar components in a single vessel.
The term "couple" as uæed herein refers to formation of a covalent bond.
The term "coupling moiety" refers to a soluble or insoluble ~ u~ to which can be attached one or more active monomers and the ~o~Le~pon~ing ~ncoA;~g monomers. Insoluble ~u~GLLs ("solid ~u~o~
lo means") may be any solid or semi-solid eurface which is stable to the reaction conditions required for synthesis of the active and ro~ing polymers, and is suitable for covalently att~chin~ and immobilizing both polymers, for example, most resins commonly employed in DNA and peptide synthesis, such as MBHA, Rink, and the like. The particular resin used will ~p~n~ upon the choice of coA i n~ and active polymers and their associated synthetic chemistries. Soluble coupling moieties are mol~c~ R having functional ~L Ou~A to which active and co~; ng monomers may be attached. Each soluble coupling moiety must be able to accommodate at least one coAing polymer and at least one active polymer, although the active and co~ing polymers need not be present in a 1:1 ratio.
The soluble coupling moiety may be as simple as an amino acid having an functional group in its side chain, or may be as complex as a function~ ed (soluble) polymer.
The term "conjugate" as used here n refers to the combination of any "active polymer" and its associated "co~i ng~ polymer. The conjugate may be formed using a "coupling moiety" or by b;n~in~ both the "active polymer" and "~nco~ing polymer" to the same ~lr~lJorL surface in close proximity with each other so that the two polymers are "associated" with each other. When both polymers are bound to the same support surface, æuch as a small bead, the ~nro~;ng polymer can be readily se~l~nce~ off of the bead and W094/~6~ - PCT~S93/~013 i ~5 the othér "active polymers" remaining on the bead will be identified once the enco~;ng sequence is known.
B. Related T.i hraxies and SYnthesis Mçthodologies for producin~ same.
There are many limitations with the current t~hnologies for probing the receptor-bi n~; ng properties of peptide libraries. Filamentous -bacteriophage libraries offer the largest source of peptide diversity (~107-l08 different components) of any current te~hnology to date (Scott, J. & Smith, G., Science, (1990), 249, 386-390; Devlin, J., PangAn;h~n, L. & Devlin, P., Science, (1990), 249, 404-406;
Cwirla, S., Peters, E., Barret, R. & Dower, W., Proc.
Natl. Acad. Sci. U.S.A., (1990), 87, 6378-6382).
These libraries, however, are limited to the natural set amino acids, suffer from biological biases (i.e., varying rates of growth, proteolysis, etc.) and also suffer, in practice, from high levels of backy~o~.d 2 0 bi n~ i ng . The present invention is designed to overcome these difficulties.
Multiple-peptide æynthesis te~hnology has substantially increased the ability to generate individual peptides (Geyæen, H., ~eloen, R. &
Bartel;~g, S., Proc. Natl. Acad. Sci. U.S.A., (1984), 81, 3998-4002; Houghten, R., Proc. Natl. Acad. Sci.
U.S.A., (1984), 5131-5135; !e~hn~rrenberg, G. &
Gerhardt, H., Tetrahedron, (1989), 45, 7759-7764;
Gausepohl, H., Kraft, ~., Rolll;~, C. & Frank, R. in Peptides: Chemistry. Structure and BioloqY
(Proc~;n~s of the 11th American Peptide Symposium, (1990), eds. Rivier J. & Marshall, G., (ESCOM, Leiden), pp. 1003-1004; Frank, R. & Doring, R., Tetrahedron, (1988), 44, 6031-6040; Fodor, S., Read, J., Pirrung, N., Stryer, L., Lu, A. & Solas, D., Science, (1991), 251, 767-773). The synthesis of ~104 individual peptides per cm2 of glass wafer represents the diversity limit of this technology (Fodor, S., WOg4/~6~ 215 1~ 7 3 PCT~S93/~013 Read, J., Pirrung, M., Stryer, L., Lu, A. & Solas, D., Science, (1991), 251, 767-773). A mixed-resin algorithm method (Furka, A., Sebestyén, M., Asgedom M.
& Dibo, G., Int. J. Petide Protein Res., (1991), 37, 487-493) has recently been used to generate solution-phase libraries (Houghten, R., Pi ni 1 1~ ~ C., B1O~P1 1 e, S., Appel, J., Dooley, C. & Cuervo, J., Nature, (1991), 354, 84-86) and resin-bound peptide libraries (Lam, K., Salmon, ~., Hersh, E., Hruby, V., Kazmiersky, W. & Knapp, R., Nature, (1991), 354, 82-84) that contain ~106 and ~107 comrQn~nts, respectively. The solution-phase libraries offer the advantage of providing quantitative receptor-binding information (Zuckermann, R., Kerr, J., Siani, M., Banville, S. & Santi, D.V., Proc. Natl. Acad. Sci.
U.S.A., 89, 4505-4509 (1992)). Furthermore~ these libraries allow the affinity of the solution conformation of a ligand to be determined, a quantity that is essential for rational drug design. An apparatus for the automated synthesis of equimolar peptide mixtures is disclosed in Zuckermannr R.N., Kerr, J.M., Siani, M.A. & Banville, S.C., Int. J. Pep.
Pro. Res., (1992), 40, 498-507.
The publications cited and ~ e~ above can be used in producing the active or bi n~; ng polymer which is used in producing the conjugate of the present invention. Accordingly, the disclosures of all of these publications are in~o~o~ated herein by reference in order to disclose peptide and peptoid synthesis methodology. Al~holyh the methodology ~;~c~l~ced within these references is extremely valuable with respect to the production of large amounts of different types of bin~in~ polymers, the mixtures of polymers pro~llc~ by this methodology are often so large and complex that there are many practical limitations with respect to their actual analysis and use. The present invention can be readily applied with such synthesis methodologies in wos4/~6~ PCT~S93/~013 ~$ ~ 16- i order to provide an ef~icient, commercially practical method of analyzing the proteins produced using such methodology.
Both the mixed-resin and solution-phase methods, however, do not allow incorporation of many non-st~n~rd amino acids heC~l~c~ of the limitations of peptide analysis. Resin-bound peptide libraries, in particular, suffer from a relatively slow rate of analysis (peptide se~"ci~ at 3 beads per day) and are limited in complexity to ~107 beads/ml. In order to generate a "complete" peptide library, there must be multiple copies (>lO) of any given peptide sequence. This becomes problematic at the sequenciny stage because the same "hit" sequence may ~rr~r multiple times. The alternative is to work with libraries that are not complete at the risk of losing se~l~nceC that bind.
When using the methodoloyy of the present invention, the sequence of a biologically active protein can be determined even without isolating the protein of interest. This can be done by synthesizing large numbers of different proteins on large numbers of different ~U~OL ~ ~urfaces such as small beads. An ~nco~i n~ polymer is attached to beads to identify each protein. A sample to be tested is then brought into contact with the beads and the beads are observed with respect to which proteins bind to a receptor site in the sample. The bead having the receptor bound thereon is analyzed by sequencing the coding polymer which has also been synthesized on the bead. When the encoding polymer has been se~nce~, the sequence of the active polymer, which may be a peptide, can be readily ~ ce~. Thus, the present invention makes it possible to determine the activity and seguence an active polymer, such as a biologically active peptide, without ever isolating the peptide.
W094/~6~ 21 ~1 4 7 3 PCT~Ss3/~013 C. General Methodolooy This invention describes a methodology for the synthesis and scre~ni ng of large synthetic polymer libraries that contain non-stAnA~rd amino acids and even non-amide h~C~A polymers. The strategy utilizes a modified mixed-resin peptide synthesis methodology to simult~nesll~ly ~ynthesize two polymer ~e~nceC
one polymer strand (the 'lbinAi ng~l strand) is synthesized for the in~Dn~A purpose of receptor b;nAing, and the cecon~ strand (the ~oAing~ strand) contains st~nA~rd amino acids or deoxyrihQnl~cleotides that ~ncoA~ for the binAln~ strand (Figure 1). The ability to decipher the bi nA i ng sequence by analysis of the coA i n~ strand with s~nA~rd peptide or oligonucleotide te~ni~ues allow the inclusion of a wide variety of novel h~ ing blocks and conformational constraints into a diverse ligand library.
This invention also describes a methodology to increase the size t~l8) and scr~ni n~ rate of a ligand library. The method uses two polymers as above, but specifically utilizes an oligodeoxyrihomlcleotide for the ~coA ing" strand. The use of DNA as the coA i ng strand allows for an incr~eA sensitivity of detection (fmol vs pmol for peptide analysis). This increased sensitivity allows for a larger library size since the amount of polymer ne~A for detection is reAnceA dramatically. The rate of sequence det~r~in~tion of receptor binders is increased since many samples can be analyzed in parallel.
In order to couple a polymer's sequence information with a peptide or oligonucleotide sequence, there needs to be a method that unambiguously correlates each polymer to each other.
Thus, when any particular non-st~nA~rd amino acid (or other monomer) is added to a "bi n~ i ~g~ polymer chain, the correcronAing information (amino acid or W094/~6~ PCT~S93/~013 ~S~4~ 3 -18-nucleotide monomer) must also be added to the "coding"
strand. A "genetic code" is thus establi ~h~ (Table 1) where each b;n~;n~ monomer corre~ron~c to (a multiple) of st~n~rd amino acids or nucleotides on the co~; n~ strand. For example, the use of three s~An~rd amino acids or nucleotide , in a 3:1 ratio with a novel monomer, would allow for the unambiguous representation of 27 novel ~onomers.
TABLE 1. Custom genetic code.
# of bases "codon" # of monomers length coded for The synthesis of coded libraries requires a modified mixed-resin algorithm (Figure 2). The resin beads are divided into equal portions, a unique monomer is added to the "bi nA i~g" strand, followed by the coupling of a ~ o~ g amino acid or nucleotide to the '~co~in~ strand. The resin aliquots are then combined to generate a mixture. A set of compatible protecting yL OU~S iS thus required to preferentially deprotect and extend each strand independently Two synthesis formats are possible for amptide libraries, one that generates resin-bound libraries and one that generates solution-phase libraries (Figure 3). Resin-bound libraries can be synthesized using non-hydrolyzable linkers that are derivatized with the "bin~;ng" and l'co~;ng" monomers strands. Solution-phase libraries can be synthesized W094/~6~ PCT~S93/~013 2151~73 .. ,. ,, , ;, , . . 1 9 as a 1:1 polymer:peptide/DNA conjugate via a hydrolyzable link~r attached to the resin.
Pe~tide as the "Codinq" strand The use of base-labile Fmoc-protected - monomers and acid-labile (N~-Ddz-protected ~mino acids (Birr, C., Nassal, M., Pipkorn, R., Int. J. PeDtide Protein Res., (1979), 13, 287-295), for example, allow for selective deprotection and coupling to two individual polymer strands. Resin-bound libraries can be generated by the derivatization of non hydLolyzable resins with a 1:1 ratio (or any desired ratio) of Fmoc:Ddz monomers tFigure 4). This i~lLLGl.ce-c two differently protected amino acids that an be ext~n~
;~p~n~ntly. Solution-phase libraries that contain a 1:1 ratio of bin~;ng:co~;ng strands can be synthesized by using a hydrolyzable Fmoc-Lysine(Moz)-OH l;nk~ that allowed for chain growth at both the ~-and ~-amino ~-~u~. Amino acids which do not contain functional yLOu~s are preferred for the "coding"
strand in order to minimize unwanted binding interactions.
The receptor-bi~ing ligand can be identified by bead st~i~ing ~echn;ques (Lam, K., Salmon, S., Hersh, E., Hruby, V., Kazmiersky, W.
~ rr, R-, Nature, (1991), 354, 82-84) and the sequence determined by N-terminal Edman degradation.
In order to ensure that only the '~oo~ing~ strand is se~l~nc~, it is essential that the N-terminus of the "b;n~ing" strand be acetylated or otherwise made non-seq~ nc~hle.
DNA as the "Coding" Strand The construction of libraries with DNA as the co~ing strand is similar to those with peptides but offers several advantages: the information storage and replicative properties of DNA allow for W094/~6~ - PCT~S93/~0 increased sensitivity of detection, a larger library size and an increased rate of sequence determinationO
The synthesis of DNA as ~he co~; ng polymer requires compatibility between the as~embly of Fmoc-5 h~ ~ monomers and st~n~d DNA chemistry. Thesesynthesis strategies are likely to be compatible ((a) Juby, C., Richardson, C. & Brous eau, R., ~et.
Letters, (1991), 32, 879-882. (b) Haralambidis, J., Duncan, L., Angus, B. & Tregear W.~ Nucleic Acid Res , (1990), 18, 493-499) (see Table 2)o Alternatively, allyl-based protection strategies existæ for both peptide (Lyttle, M.H.; ~l-A~on, D., Pe~tides: ChemistrY
and Biolo~y (Proc~inqs of the 12th American Peptide Symposium): Smith, J. ~nd Rivier, J.E., Eds.; ESCOM, T.~ , 1992, pp. 583-584) and oligodeoxyr;honl~cleotide (Hayakawa, y., I~~k~h~yashi, S., Kato, H. & Noyori, R., J. Am. Chem. Soc., (1990), 112, 1691-1696) synthesis. The assay of æolution-phase libraries can be facilitated by using only pyrimi~;n~c in the coAing strand, thereby avoiding the potential problem of base pairing between individual strands.
Strategies for the synthesis of co~i ng and active polymers, as well as matc~i ng the active polymer with a ~o~;ng ~equence to provide a genetic tag are described in Brenner et al. Proc. Natl. Acad.
Sci. U.S.A., 89:5381-5383 (June 1992) which is incorporated herein by reference.
wo g4/~6~ 2 1 ~ 1 4 7 3 PCT~S93/~0~
- 21 : . .~. .
Compatibility of DNA vs. Peptide Synthesis Chemistry Peptide r1lPmi.stry Oligonucleotide Chemistry ~ o ~ ~0 P~rmanent Protectin~ ~ v~
-OH t-butyl ether A,C benzoyl amide -CO2H t-butyl ester G isob~Ly~l -NH2 t-boc -P=O cyanoethyl, methyl his, cys trityl arg sulfonyl remov~d by:
85% trifluoroacetic acid conc. NE~OH 55C S hours 2 hours @ room temp T~m~orarY ~rot~ti~a ~ G~
9-fluorenylmethoxycaL~oll~l 4,4-dimethoxytrityl rQmove~ by:
20% piperidine 3% trichloroacetic acid CleavaqQ fro~ 801i~ ~UP~oXt:
85% trifluoroacetic acid conc. NE~OH 55C S hours 2 hours ~ room temp SU~;TI ~ UTE:~ S3HEElr W094/136~ PCT~S93/~OL3 ~ABLB 2 tCo~t) Compatibility of DNA vs. Peptide Symthesis Chemistry Std. conditions ~ ;ve - Problem addressed t-Butyl carbamate/ allyl amino acid ester deprotection from detritylation reagent 20% Piperidine 2% DBU substitution at exocyclic amine cyanoethyl allyl acylation of phosphate phosphate NH40H ethyl~neAi~mine/ racemization EtOH
iodine t Butyll~ o Tyr, Met, Cys peroxide oxidation controlled pore poly~yl~ne resin transferring glass resin T, C & G T, C, G & A depurination T, C T, C, G & A base pairing SUBSTITUTE Sl~,EET
WOg4/~6~ 21514 7 3 PCT~S93/~013 .--Resin-bound libraries can be synthesized by using non-hydrolyzable linkers to attach both the C-terminus of the peptide and the 3'-end of the oligonucleotide to the same bead. Solution-phase libraries can be synthesized as a 1:1 peptide-oligonucleotide conjugate, in which the C-terminus of the peptide is attached to the 3'-end of the oligonucleotide through a hydrolyzable Fmoc-Ser(O-Dmt) linker which is attached to the resin.
The identification of binders in the resin-bound peptide libraries can be detected by the bead stA;n;ng methodology (Lam, K., Salmon, S., Hersh, E., Hruby, V., Kazmiersky, W. & Knapp, R., Nature, (1991), 354, 82-84). Although the peptides are bound to a solid-phase, there does not have to be a 1:1 peptide-oligonucleotide ratio since the DNA can be amplified prior to the determination of its sequence. In fact, less DNA is preferred so that there will be less interference with the polymer's binding properties.
Once a bead is identified, the DNA sequence is determined (Stahl, S., Hultman, T., Olsson, A., Mois T., et al., Nucleic Acid Res., (1988), 16, 3025-3038) after PCR amplification or by thermal-cycle sequencing (Figure 8). This requires the inclusion of one or more primer sites neighboring the coA; ng region of the oligonucleotide. Similarly, the use of solution-phase libraries requires isolation of each sequence from each other. This can be accompl; ~h~ by restricting the DNA after PCR amplification and inserting it into M13 (or other suitable vector) for clonal isolation and sequencing (Figure 9).
EXAMPLES
The following examples will provide those skilled in the art with a complete disclosure of how to make and use the invention and are not intended to limit the scope of the invention. Efforts have been made to insure accuracy with respect to numbers used W094/~6~ PCT~S93/12013 (e.g. amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts or parts by weight, mol~c~ ~ weight is weight average molecular weight, temperature is in degrees centigrade and presæure is at or near atmospheric.
Example 1 The in~p~n~pnt synthesis of two unambiguously correlated se~lPnc~c has been sllccessfully completed. The C~lhc~uent sequence analysis of the "co~i n~l strand has also been demonstrated. For ~ol.ve~ience, two peptide se~lPnc~s were chosen. The "b; nA; ng" strand was synthesized with N~-Fmoc-protected amino acids and the "co~i ng~
strand was synthesized with N~-Ddz-protected amino acids. The simultaneous synthesis of the two peptide strands was tested in two formats, 1) resin-bound peptide library synthesis and 2) solution-phase peptide libraries using a hydrolyzable Fmoc-Lys~Moz) OH 1 ;nker (Wang, S.S.; Chen, S.T, Wang, K.T., and Merrifield, R.B., Int. J. Peptide Protein Res., (1987), 30, 662-667). These synth~c~fi were performed on single peptides (not libraries) as a demonstration of research concept and in order to allow the full characterization of the synthesis products.
6~ 215 1~ 7 3 PCT~S93/~013 A. SYnthesis of a Resin-Bound Library Model "B;n~;ng" Sequence: Ac-Arg-Leu-Val-Thr-His (Fmoc peptide) "Coding" Sequence: H~-Ala-Ser-Gly-Glu-Phe-Ala (Ddz peptide) Synthesis Scheme:
Step # Description 1 Derivatization of MBHA resin with l:1-Ddz-Ala-OH: Fmoc-His(Trt)-OH
2 De~ection of "bin~;ng" strand with 20%
piperidine/DMF
3 Coupling of ~ConA ~b;n~;ng" amino acid, Fmoc-Thr(But)-OH
4 Deprotection of "co~;ng strand" with 7.5%
TFA/ CH2Cl2 Coupling of c~cQn~ "coding" amino acid, Ddz-Phe-OH
6 Repeat steps 2-5 with alternative Fmoc and Ddz deprotection and corresponding amino acid coupling 7 Final Deprotection of Fmoc followed by acetylation 8 TFA deprotection of side-chain yroups After the TFA deprotection, the model library bead has two in~p~n~ntly synthesized se~l~c~c and is ready for assay. Only the coding strand has a free ~-amino group and can be characterized by N-terminal Edman degradation. The binding strand is acetylated and there-fore will not interfere with the sequencing. The two peptides were cleaved from the resin with HF thereby providing both the "binding" and "ro~i ng~ se~l~nc~s as free peptides.
The amino acid composition, mass spectro-scopy and N-terminal sequencing data are consistent with thecorrect products. (See Figures 5, 6 and 7.) PCT~S93/~013 WO g4/~3623 3 ~
Mass Spectrometry:
theoretical observed Ddz peptide 580.2 580.2 Fmoc peptide 666.9 666.4 Amino Acid Composition:
Fmoc Peptide Ddz Peptide 10 Theor. Observ. Theor. Observ.
(%) (%) (%) (%) His 20 21 Ala 33 33 Thr 20 20 Glu 17 17 Val 20 19 Phe 17 17 15Leu 20 29 Gly 17 15 Arg 20 20 Ser 17 15 N-Terminal Edman Se~uencinq of Resin beads fcoding peptide only):
Cycle # amino acid pmol 1 Ala 45 2 Ser 12 3 Gly 30 254 Glu 28 Phe 28 6 Ala 29 Example 2 Synthesis of a Solution-Phase Peptide Library Model In this example, a 1:1 solution-phase adduct between a "b;n~;n~'~ and a "coA;n~" strand was synthesized and fully characterized. The "binding"
strand was assembled wi~h Fmoc-protected monomers, and the "co~ " strand was assembled with Ddz-protected monomers.
W094/~6~ 2 I 5 1 ~ 7 3 PCT~S93/~013 .
~B; nA; ng~ sequence: ~c-Glu-Ser-Thr-Arg-Pro~nLeu-Lys-B-(Fmoc peptide) Ala-NH2 ~CoA; nq~ sequence: ~N-Gly-Ala-Phe-Gly-Ala-Phe-CONH
(Ddz peptide) SYnthesis Scheme:
Step # Description 1 Derivatization of Rink Resin with Fmoc-B-Ala-OH spacer 2 Fmoc deprotection with 20% piperidine/DMF
3 Coupling of Fmoc-Lys (4-methOXYL~11ZY1OXYCa ~ LG11Y1 )-OH
4 Fmoc deprotection of '~b;n~tnq~ ~trand with 2 0~ piperidine/DMF
Coupling of first ''b;nAin~ monomer Fmoc-nLeu-OH
6 Cleavage of ~coA; nq~ strand 4-Methoxybenzyloxycarbonyl (Moz) group with 7.5% TFA/ ~ C12 7 Coupling of first ~C~A i nq~ amino acid Ddz-2 0 Phe-OH
8 Repeat steps 4-7 With ~ re~;ron~;n~ amino acids 9 Final Fmoc deprotection and acetylation Cleavage and deprotection of resin sample with TFA
Following TFA cleavage and deprotection, the model solution-phase library contains a l:1 Fmoc/Ddz conjugate peptide. One peptide sequence was synthesized and not a mixture in order to fully characterize the reaction product. The amino acid composition and mass spectroscopy data are consistent with the correct product. In addition, the "bi n~; n~
and "coA;~q" hybrid peptides were tested in a competition T'T.T,~ format. The ELSTRPnL "binding"
sequence binds to an anti-gpl20 antibody with submicromolar affinity. This value was not affected by the presence of the "coding" peptide.
W094/1~ PCT~S931~013 Mass S~ectrosco~y:
Theoretical Observed Fmoc/Ddz peptide conjugate: 1492.7 1492.6 S Amino Acid Com~osition:
Fmoc Peptide Ddz Peptide Theor. Observ. Theor. Observ.
(%) (%) (~) (%) Glu 7.1 7.0 Phe 4.2 14.7 Ser 7.1 7.1 Ala 14.2 14.4 Thr 7.1 702 Gly 14.2 13.8 Arg 7.1 7.2 Pro 7.1 7.9 Nleu 7.1 6.3 The instant invention is shown and described herein in what is considered to be the most practical, and preferred embodiments. It is reco~n;~ed~ however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure.
Step # Description 1 Derivatization of MBHA resin with l:1-Ddz-Ala-OH: Fmoc-His(Trt)-OH
2 De~ection of "bin~;ng" strand with 20%
piperidine/DMF
3 Coupling of ~ConA ~b;n~;ng" amino acid, Fmoc-Thr(But)-OH
4 Deprotection of "co~;ng strand" with 7.5%
TFA/ CH2Cl2 Coupling of c~cQn~ "coding" amino acid, Ddz-Phe-OH
6 Repeat steps 2-5 with alternative Fmoc and Ddz deprotection and corresponding amino acid coupling 7 Final Deprotection of Fmoc followed by acetylation 8 TFA deprotection of side-chain yroups After the TFA deprotection, the model library bead has two in~p~n~ntly synthesized se~l~c~c and is ready for assay. Only the coding strand has a free ~-amino group and can be characterized by N-terminal Edman degradation. The binding strand is acetylated and there-fore will not interfere with the sequencing. The two peptides were cleaved from the resin with HF thereby providing both the "binding" and "ro~i ng~ se~l~nc~s as free peptides.
The amino acid composition, mass spectro-scopy and N-terminal sequencing data are consistent with thecorrect products. (See Figures 5, 6 and 7.) PCT~S93/~013 WO g4/~3623 3 ~
Mass Spectrometry:
theoretical observed Ddz peptide 580.2 580.2 Fmoc peptide 666.9 666.4 Amino Acid Composition:
Fmoc Peptide Ddz Peptide 10 Theor. Observ. Theor. Observ.
(%) (%) (%) (%) His 20 21 Ala 33 33 Thr 20 20 Glu 17 17 Val 20 19 Phe 17 17 15Leu 20 29 Gly 17 15 Arg 20 20 Ser 17 15 N-Terminal Edman Se~uencinq of Resin beads fcoding peptide only):
Cycle # amino acid pmol 1 Ala 45 2 Ser 12 3 Gly 30 254 Glu 28 Phe 28 6 Ala 29 Example 2 Synthesis of a Solution-Phase Peptide Library Model In this example, a 1:1 solution-phase adduct between a "b;n~;n~'~ and a "coA;n~" strand was synthesized and fully characterized. The "binding"
strand was assembled wi~h Fmoc-protected monomers, and the "co~ " strand was assembled with Ddz-protected monomers.
W094/~6~ 2 I 5 1 ~ 7 3 PCT~S93/~013 .
~B; nA; ng~ sequence: ~c-Glu-Ser-Thr-Arg-Pro~nLeu-Lys-B-(Fmoc peptide) Ala-NH2 ~CoA; nq~ sequence: ~N-Gly-Ala-Phe-Gly-Ala-Phe-CONH
(Ddz peptide) SYnthesis Scheme:
Step # Description 1 Derivatization of Rink Resin with Fmoc-B-Ala-OH spacer 2 Fmoc deprotection with 20% piperidine/DMF
3 Coupling of Fmoc-Lys (4-methOXYL~11ZY1OXYCa ~ LG11Y1 )-OH
4 Fmoc deprotection of '~b;n~tnq~ ~trand with 2 0~ piperidine/DMF
Coupling of first ''b;nAin~ monomer Fmoc-nLeu-OH
6 Cleavage of ~coA; nq~ strand 4-Methoxybenzyloxycarbonyl (Moz) group with 7.5% TFA/ ~ C12 7 Coupling of first ~C~A i nq~ amino acid Ddz-2 0 Phe-OH
8 Repeat steps 4-7 With ~ re~;ron~;n~ amino acids 9 Final Fmoc deprotection and acetylation Cleavage and deprotection of resin sample with TFA
Following TFA cleavage and deprotection, the model solution-phase library contains a l:1 Fmoc/Ddz conjugate peptide. One peptide sequence was synthesized and not a mixture in order to fully characterize the reaction product. The amino acid composition and mass spectroscopy data are consistent with the correct product. In addition, the "bi n~; n~
and "coA;~q" hybrid peptides were tested in a competition T'T.T,~ format. The ELSTRPnL "binding"
sequence binds to an anti-gpl20 antibody with submicromolar affinity. This value was not affected by the presence of the "coding" peptide.
W094/1~ PCT~S931~013 Mass S~ectrosco~y:
Theoretical Observed Fmoc/Ddz peptide conjugate: 1492.7 1492.6 S Amino Acid Com~osition:
Fmoc Peptide Ddz Peptide Theor. Observ. Theor. Observ.
(%) (%) (~) (%) Glu 7.1 7.0 Phe 4.2 14.7 Ser 7.1 7.1 Ala 14.2 14.4 Thr 7.1 702 Gly 14.2 13.8 Arg 7.1 7.2 Pro 7.1 7.9 Nleu 7.1 6.3 The instant invention is shown and described herein in what is considered to be the most practical, and preferred embodiments. It is reco~n;~ed~ however, that departures may be made therefrom which are within the scope of the invention, and that obvious modifications will occur to one skilled in the art upon reading this disclosure.
Claims (20)
1. An assay conjugate, comprising:
an active polymer comprising monomers selected from the group consisting of peptide and peptoid monomers;
an encoding polymer comprising encoding monomers, wherein the encoding polymer corresponds to and allows identification of the active polymer; and a coupling moiety covalently coupled to the active peptide and the encoding polymer.
an active polymer comprising monomers selected from the group consisting of peptide and peptoid monomers;
an encoding polymer comprising encoding monomers, wherein the encoding polymer corresponds to and allows identification of the active polymer; and a coupling moiety covalently coupled to the active peptide and the encoding polymer.
2. The conjugate of claim 1, wherein the coupling moiety comprises a solid support.
3. The conjugate of claim 1, wherein the coupling moiety comprises a soluble linking group.
4. The conjugate of claim 1, wherein all active polymers coupled to a single selected solid support are identical.
5. The conjugate of claim 1, wherein the active polymer comprises a polypeptide and the encoding polymer comprises a DNA or RNA oligonucleo-tide.
6. The conjugate of claim 1, wherein the active polymer comprises a polypeptoid and the encoding polymer comprises a DNA or RNA oligonucleo-tide.
7. The conjugate of claim 6, wherein the polypeptoid comprises a polymer of monomers of the formula:
wherein R is alkyl of 2-6 carbon atoms, haloalkyl of 1-6 carbon atoms wherein halo is F, Cl, Br, or I, alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms, cyclolkyl of 3-8 carbon atoms, alkoxyalkyl of 2-8 carbon atoms, aryl of 6-10 carbon atoms, arylalkyl of 7-12 carbon atoms, arylalkyl of 7-12 carbon atoms substituted with 1-3 radicals independently selected from halo and nitro and hydroxy, aminoalkyl of 1-6 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, carboxy, carboxyalkyl of 2-6 carbon atoms, carboalkoxy-alkyl of 3-10 carbon atoms, carbamyl, carbamylalkyl of 2-6 carbon atoms, imidazolyl, imid-azolylalkyl of 4-10 carbon atoms, pyridyl, pyridylalkyl of 6-10 carbon atoms, piperidyl, pip-eridylalkyl of 5-10 carbon atoms, indolyl, or indolyalkyl of 9-15 carbon atoms.
wherein R is alkyl of 2-6 carbon atoms, haloalkyl of 1-6 carbon atoms wherein halo is F, Cl, Br, or I, alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms, cyclolkyl of 3-8 carbon atoms, alkoxyalkyl of 2-8 carbon atoms, aryl of 6-10 carbon atoms, arylalkyl of 7-12 carbon atoms, arylalkyl of 7-12 carbon atoms substituted with 1-3 radicals independently selected from halo and nitro and hydroxy, aminoalkyl of 1-6 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, carboxy, carboxyalkyl of 2-6 carbon atoms, carboalkoxy-alkyl of 3-10 carbon atoms, carbamyl, carbamylalkyl of 2-6 carbon atoms, imidazolyl, imid-azolylalkyl of 4-10 carbon atoms, pyridyl, pyridylalkyl of 6-10 carbon atoms, piperidyl, pip-eridylalkyl of 5-10 carbon atoms, indolyl, or indolyalkyl of 9-15 carbon atoms.
8. A mixture of conjugates of claim 1, wherein the mixture comprises at least two distinct active polymers, the coupling moiety is a solid support, distinct active polymers are covalently coupled to separate solid supports and a distinct encoding polymer corresponding to each active polymer is covalently coupled to the support coupled to its corresponding active polymer.
9. The mixture of claim 8, wherein the mixture comprises at least ten distinct active polymers.
10. The mixture of claim 9, wherein the mixture comprises at least 100 distinct active polymers.
11. A conjugate, comprising:
a biologically active peptide comprised of five or more amino acids;
an encoding polymer comprised of nucleic acids wherein one or more nucleic acids within the encoding polymer correspond to and can be readily identified with the amino acids of the active polymer;
and a coupling moiety covalently coupled to the active peptide and the encoding polymer.
a biologically active peptide comprised of five or more amino acids;
an encoding polymer comprised of nucleic acids wherein one or more nucleic acids within the encoding polymer correspond to and can be readily identified with the amino acids of the active polymer;
and a coupling moiety covalently coupled to the active peptide and the encoding polymer.
12. The conjugate of claim 11, wherein the amino acids are selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine.
13. The conjugate of claim 11, wherein the encoding polymer includes three nucleotides for each amino acid of the active polymer and the three nucleotides are nucleotides which naturally encode the corresponding amino acid of the active polymer.
14. The conjugate of claim 11, wherein the coupling moiety comprises a solid support.
15. The conjugate of claim 14, wherein the solid support is in the form of a spherical bead having a diameter of less than one centimeter.
16. The conjugate of claim 11, wherein the coupling moiety comprises a soluble linking group.
17. A method of synthesizing an encoded peptide or peptoid polymer, the method comprising:
a) providing a coupling moiety;
b) coupling to the coupling moiety an active monomer, and coupling to the coupling moiety an encoding monomer which corresponds to the active monomer to form a conjugate having a bound active monomer and a bound encoding monomer; and c) repeating step b) until an active polymer of the desired length is obtained.
a) providing a coupling moiety;
b) coupling to the coupling moiety an active monomer, and coupling to the coupling moiety an encoding monomer which corresponds to the active monomer to form a conjugate having a bound active monomer and a bound encoding monomer; and c) repeating step b) until an active polymer of the desired length is obtained.
18. The method of claim 17 wherein the active monomers are amino acids and the encoding monomers are nucleic acid bases.
19. A method of synthesizing a mixture of encoded polymers, the method comprising:
a) providing a plurality of coupling moieties;
b) dividing the plurality of coupling moieties into a plurality of aliquots;
c) within each aliquot, coupling to each coupling moiety an active monomer, and coupling to each said coupling moiety an encoding monomer which corresponds to the active monomer to form a plurality of conjugates having a bound active monomer and a bound encoding monomer, each in a separate aliquot, wherein different aliquots may contain different active monomers;
d) combining the aliquots of conjugates to form a mixture of conjugates; and e) repeating steps b-d) until a mixture of active polymers of the desired length is obtained.
a) providing a plurality of coupling moieties;
b) dividing the plurality of coupling moieties into a plurality of aliquots;
c) within each aliquot, coupling to each coupling moiety an active monomer, and coupling to each said coupling moiety an encoding monomer which corresponds to the active monomer to form a plurality of conjugates having a bound active monomer and a bound encoding monomer, each in a separate aliquot, wherein different aliquots may contain different active monomers;
d) combining the aliquots of conjugates to form a mixture of conjugates; and e) repeating steps b-d) until a mixture of active polymers of the desired length is obtained.
20. The method of claim 19, wherein the active monomers comprise activated amino acids and the coupling moiety comprises a solid support.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US98827892A | 1992-12-11 | 1992-12-11 | |
| US07/988,278 | 1992-12-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2151473A1 true CA2151473A1 (en) | 1994-06-23 |
Family
ID=25534002
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2151473 Abandoned CA2151473A1 (en) | 1992-12-11 | 1993-12-10 | Synthesis of encoded polymers |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0675873A4 (en) |
| JP (1) | JPH08504444A (en) |
| AU (1) | AU5827294A (en) |
| CA (1) | CA2151473A1 (en) |
| WO (1) | WO1994013623A1 (en) |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5527675A (en) * | 1993-08-20 | 1996-06-18 | Millipore Corporation | Method for degradation and sequencing of polymers which sequentially eliminate terminal residues |
| US5776737A (en) * | 1994-12-22 | 1998-07-07 | Visible Genetics Inc. | Method and composition for internal identification of samples |
| GB9502225D0 (en) * | 1995-02-04 | 1995-03-22 | Zeneca Ltd | Method |
| US5961923A (en) * | 1995-04-25 | 1999-10-05 | Irori | Matrices with memories and uses thereof |
| US6284459B1 (en) | 1995-04-25 | 2001-09-04 | Discovery Partners International | Solid support matrices with memories and combinatorial libraries therefrom |
| US6100026A (en) * | 1995-04-25 | 2000-08-08 | Irori | Matrices with memories and uses thereof |
| GB9515070D0 (en) * | 1995-07-22 | 1995-09-20 | Zeneca Ltd | Label |
| EP2177627B1 (en) * | 1999-02-23 | 2012-05-02 | Caliper Life Sciences, Inc. | Manipulation of microparticles in microfluidic systems |
| DE10041238A1 (en) * | 2000-08-22 | 2002-03-07 | Aventis Res & Tech Gmbh & Co | Process for the identification of specifically cleavable peptides and use of such peptide sequences |
| JP2005502309A (en) | 2000-12-23 | 2005-01-27 | カイロン コーポレイション | Oligonucleotide transfection screening method |
| US7727713B2 (en) | 2001-06-20 | 2010-06-01 | Nuevolution A/S | Templated molecules and methods for using such molecules |
| IL163822A0 (en) | 2002-03-15 | 2005-12-18 | Nuevolution As | An improved method for synthesising templated molecules |
| WO2004013070A2 (en) | 2002-08-01 | 2004-02-12 | Nuevolution A/S | Multi-step synthesis of templated molecules |
| EP1558744B1 (en) | 2002-10-30 | 2011-06-15 | Nuevolution A/S | Enzymatic encoding |
| US9121110B2 (en) | 2002-12-19 | 2015-09-01 | Nuevolution A/S | Quasirandom structure and function guided synthesis methods |
| EP1597395A2 (en) | 2003-02-21 | 2005-11-23 | Nuevolution A/S | Method for producing second-generation library |
| WO2004099441A2 (en) * | 2003-05-09 | 2004-11-18 | Hyscite Discovery As | Selection and evolution of chemical libraries |
| EP1670939B1 (en) | 2003-09-18 | 2009-11-04 | Nuevolution A/S | A method for obtaining structural information concerning an encoded molecule and method for selecting compounds |
| US7972994B2 (en) | 2003-12-17 | 2011-07-05 | Glaxosmithkline Llc | Methods for synthesis of encoded libraries |
| NZ547723A (en) | 2003-12-17 | 2009-09-25 | Praecis Pharm Inc | Methods for synthesis of encoded libraries |
| NZ564545A (en) | 2005-06-09 | 2011-03-31 | Praecis Pharm Inc | Methods for synthesis of encoded libraries |
| EP2368868A1 (en) | 2005-10-28 | 2011-09-28 | Praecis Pharmaceuticals Inc. | Methods for identifying compounds of interest using encoded libraries |
| SI2336315T1 (en) | 2005-12-01 | 2017-12-29 | Nuevolution A/S | Enzymatic encoding methods for efficient synthesis of large libraries |
| EP2558577B1 (en) | 2010-04-16 | 2018-12-12 | Nuevolution A/S | Bi-functional complexes and methods for making and using such complexes |
| DK3262059T3 (en) | 2015-02-24 | 2022-01-31 | Hope City | PLATFORMS FOR SCREENING CHEMICAL CODED, SPACIOUSLY ADDRESSED LIBRARIES |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4359353A (en) * | 1981-05-18 | 1982-11-16 | Hydrocarbon Research, Inc. | Polypeptides as chemical tagging materials |
| DE69217497T2 (en) * | 1991-09-18 | 1997-06-12 | Affymax Tech Nv | METHOD FOR SYNTHESISING THE DIFFERENT COLLECTIONS OF OLIGOMERS |
| WO1994008051A1 (en) * | 1992-10-01 | 1994-04-14 | The Trustees Of Columbia University In The City Of New York | Complex combinatorial chemical libraries encoded with tags |
-
1993
- 1993-12-10 JP JP6514425A patent/JPH08504444A/en not_active Withdrawn
- 1993-12-10 EP EP94904077A patent/EP0675873A4/en not_active Withdrawn
- 1993-12-10 CA CA 2151473 patent/CA2151473A1/en not_active Abandoned
- 1993-12-10 WO PCT/US1993/012013 patent/WO1994013623A1/en not_active Application Discontinuation
- 1993-12-10 AU AU58272/94A patent/AU5827294A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0675873A1 (en) | 1995-10-11 |
| JPH08504444A (en) | 1996-05-14 |
| AU5827294A (en) | 1994-07-04 |
| EP0675873A4 (en) | 2000-03-29 |
| WO1994013623A1 (en) | 1994-06-23 |
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