BACKGROUND ART Background Solid phase synthesis of complex organic molecules such as polypeptides and nucleic acids is the overwhelming method of choice for producing many compounds used in research, and for manufacturing. The initial attachment of starting material and the final cleavage of synthesized product from the solid support are the key steps in solid phase synthesis. Consequently, many linkers and methods for derivatization of the polymer matrices have been developed for anchoring a wide range of functional groups of substrates.

An important use of solid phase supports is in the synthesis of polypeptides.

Methods of peptide synthesis in current use have a protecting group on the amino acid monomers, typically either Boc (tert-butyloxycarbonyl) or Fmoc (9- fluorenylmethoxycarbonyl). Both Boc and Fmoc chemistry routinely involve use of polymeric microspheres or crowns as supports. The support serves as the anchor for the assembly of amino acid chains to produce polypeptides. Fmoc is preferable in many ways to Boc protecting groups, because the side chain blocking groups and the peptide resin linkages are completely stable to the agents used during each step of the synthesis of peptides. However, some Fmoc syntheses employ a polyamide resin, which generally has a low loading capacity, and which tends to clump together, rendering it unsuitable for large scale production.

Geysen et al. (1984) Proc. Natl. Acad. Sci. 81: 3998-4002 has described a modification of the Merrifield solid-phase synthesis wherein the C-terminal amino acid residues are bound to solid supports in the form of polyethylene pins. The pins are treated

individually or collectively to sequentially couple additional amino acids thereto to form a plurality of chemically distinct oligopeptides. Without removing the oligomers from the supports, the synthesized oligopeptides can be individually assayed for desired pharmacological activity.

Multipin technology is a cost-effective and efficient means for the systematic investigation of peptides for use as diagnostic reagents, vaccine components, hormone analogs, agonists and antagonists of receptor mediated functions, and as ligands for affinity purification. The multipin method can also be used for the synthesis of sets of peptide sequences and multiple copies of the same peptide sequence, as well as for the synthesis of combinatorial and other libraries. The multipin system has subsequently been applied to the preparation of non-peptidic libraries and small molecule organic compounds. For example, 1,4-benzodiazepines and peptidomimetics have been prepared by this method.

There is a continuing need for resins usable in solid phase synthesis that provide for a high loading capacity, result in higher yields and higher purity, and which are applicable to the synthesis of a variety of organic molecules, including polypeptides.

RelevantLiterature Synthesis of 5- (4'-chloromethylphenyl) pentylpolystyrene resin is described by Kobayashi and Moriwaki (1997) Tetrahedron Lett. 38: 4251-4254. Friedel-Crafts acylation of the polystyrene resin with 5-phenylvaleryl chloride is followed by reduction and chloromethylation.

Various functionalized polystyrenes are used in solid phase synthesis of polypeptides. In common use is"Merrifield resin,"chloromethylpolystyrene-1%- divinylbenzene. The preparation of a hydroxamine resin via tritryl chloride resin is described in Floyd et al. (1996) Tet. Lett. 37: 8045-8048. Other resins are described in U. S.

Patent Nos. 5,563,220 to Webber et al. and 4,859,736 to Rink. Such polymers may be grafted onto a substrate, such as a polyolefin, for parallel synthesis reactions, see, e. g., U. S.

Patent No. 5,373,053 to Borg et al. Grafted polystyrene supports are commercially available, e. g. from Chiron Technologies PTY Ltd. (Melbourne, Australia).

Secondary amide forming linkers, or spacers, are described by Albericio et al.

(1990) J. Organic Chem. 55: 37301 Songster et al. (1995) Peptide Science 2: 265, and Fivush et al. (1997) Tetrahedron Lett. 38: 7151.

The use of insoluble polymer supports in general organic synthesis is reviewed by Leznoff (1978) Acct. Chem. Res. 11: 327-333. The solid phase synthesis of small organic molecules is discussed in Chen et al. (1994) J. Am. Chem. Soc. 116: 2661-2662. Specific methods for the solid phase synthesis of 1,4-benzodiazopine derivatives are described by Bunin and Ellman (1992) J. Am. Chem. Soc. 114: 10997-10998. Coupling alcohols to solid supports by employing dihydropyran-functionalized resins is described by Thompson and Ellman (1994) Tet. Lett. 35: 9333-9336.

The attachment of Fmoc-protected amino acids to 4-alkoxybenzyl alcohol polystyrene with 2,6-dichlorobenzoyl chloride is discussed by R. C. Sheppard and B. J.

Williams (1988) Int. Pep. Protein Res. 20: 451-454.

DISCLOSURE OF THE INVENTION Novel functionalized polymeric substrates are provided for use as supports in solid phase synthesis. The functionalized polymeric substrates have the generic structure (I) where P is a base polymer suitable for use in solid phase synthesis and comprises surface aromatic groups, Sp is a flexible spacer, n is 0 or 1, Ar is arylene, Rl is alkyl or benzyl, q is an optional double bond, and X is a substituent group, often based on a heteroatom or halogen, providing a reactive moiety to which a monomer can be anchored for subsequent solid phase synthesis. Different X substituents are chosen depending on the specific monomer to be anchored. The functionalized polymers of the invention may be formed by one of the synthetic reactions described herein.

In one synthetic method, the functionalized polymeric substrates are formed by reaction of a base polymer comprising phenyl groups or other aromatic moieties with an acid chloride RICOCI, where R, is defined above, to convert the aromatic groups to

substituted or unsubstituted acetophenone groups, which are then reduced to provide aromatic moieties having-CH (OH) RI substituents. The-CH (OH) RI substituents are used directly for some purposes, or undergo further reactions to provide other X substituents.

This method is illustrated in the following scheme: R,COCI Reduction 0.--ON L--i L-1 p R \OH Optional Further Conversion - ; _-J v Ri X Scheme 1 In Scheme 1, Rl and X are as defined above, i is an integer in the range of 0 to 3 inclusive, and Y is a substituent such as lower alkyl, lower alkoxy, halogen, or the like. The aromatic moieties may also be present as pendant groups, in which case the synthetic method proceeds as illustrated in Scheme 2:

M-M ¢Xf COCU Reduction (Y) i (Y) i (Y) i I \ I ici OH Pi Ri Optional Further Conversion - J X X Ri Scheme 2 In Scheme 2, M is a monomer unit typically although not necessarily formed by addition polymerization of an olefinic or vinyl monomer unit; and an exemplary base polymer herein is polystyrene.

Where it is desirable to include a spacer between the backbone of the base polymer and the reactive groups X of the functionalized polymeric substrate, an alternative synthesis is to react a hydroxyl-substituted aromatic ketone such as acetophenone with an ester having the structural formula

wherein R2 is lower alkyl, R3 is halogen, and m is an integer in the range of 1 to 10 inclusive. An example of such an ester is methyl bromovalerate. Following or during coupling, the ester group is hydrolyzed. The reaction results in the formation of a linker having an aryloxy, e. g., an acetophenoxy, moiety and a carboxylic acid moiety, which are separated by an alkylene linkage- (CH2) m. The free carboxylic acid group is then reacted with any suitable substrate having free amino groups on the surface thereof, resulting in an amide linkage. The reaction is illustrated in Scheme 3 (for purposes of illustration, the aromatic ketone is para-hydroxyl-substituted acetophenone): HO I Support NH2 (CH2) m O- z Support NH \ O 93NH o< 0 Scheme 3 In one embodiment of the invention, q is present as a double bond and X is oxygen, in which case the functionalized polymeric substrate may be used for anchoring ammonia or primary amines, including aromatic and aliphatic amines, using reductive amination. When q is absent and X is a hydroxyl, sulfhydryl, or aminooxy group, the functionalized polymer may be used for anchoring acids, particularly protected carboxylic acids such as Fmoc- protected amino acids, under mild coupling conditions. In another embodiment of the invention, q is absent and X is a halogen substituent, whereby the functionalized polymer

may be used for anchoring hydroxyl-containing compounds such as alcohols, phenols, etc.

Also provided are methods for using the polymers of the invention in solid phase synthesis.

The functionalized polymeric substrate may be provided as a graft, or crown, for multipin solid phase synthesis of peptides and non-peptides. Alternatively, the functionalized polymeric substrates are provided as microspheres suitable for use in a"tea bag,"or column format, including beaded cross-linked resins such as 1 % divinylbenzene/polystyrene.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph depicting the loading capacity of derivatized polystyrene crowns versus concentration of acylation reagent, as evaluated in Example 3.

Figure 2 is a graph depicting the cleavage of Dnp-p-Ala from hydroxyethyl polystyrene in the presence of varying concentrations of trifluoroacetic acid, as evaluated in Example 6.

Figure 3 is a graph depicting the cleavage of an acetophenone moiety from chloroethyl polystyrene in the presence of varying concentrations of trifluoroacetic acid, as evaluated in Example 6.

Figure 4 is a graph depicting the cleavage of Dnp-p-Ala from hydroxamate ethyl polystyrene in the presence of varying concentrations of trifluoroacetic acid, as evaluated in Example 6.

MODES FOR CARRYING OUT THE INVENTION Functionalized polymeric substrates are provided, which are useful as supports in solid phase synthetic reactions. Base polymers are derivatized with linkers having improved characteristics for use as a support. The functionalized polymeric substrates of the invention have the general structure (I)

wherein: P is a base polymer suitable for use in solid phase synthesis, and comprises surface aromatic groups, as either backbone moieties, pendant groups, or both; Ar is arylene, usually phenylene, optionally substituted with 1 to 3 substituents such as lower alkyl, lower alkoxy or halo, and is either 1,3-linked or 1,4-linked to adjacent moieties; Rl is alkyl or benzyl, preferably lower alkyl, and may be branched or linear, preferably linear; q is an optional double bond; n is 0 or 1; Sp is a flexible spacer of the formula -R4-Z-O- in which R4 is selected from the group consisting of-NR5C (O)-,-C (O)-NR5-,-CH20-, -CH2S-,-O-,-S-,-OC (O)-, and-C (O) O-, wherein Rs is lower alkyl or benzyl, and Z is selected from the group consisting of- (CH2) m-,- (CH2CH20) pCH2CH2-, cycloalkylene such as wherein m is an integer in the range of 1 to 12, usually 1 to 10, preferably 1 to 6, and most preferably 2 to 4, and p is an integer in the range of 1 to 50, usually 1 to 4, and wherein a particularly preferred spacer has the formula

X is a substituent group, typically heteroatom-containing or halogen-containing, providing a reactive moiety to which a monomer can be anchored for subsequent solid phase synthesis. Different X substituents are chosen depending on the specific monomer to be anchored. When q is present, X is oxygen, i. e., a carbonyl group is present. When q is absent, the linkage to"X"is through a single bond, and X may be, for example, hydroxyl, carboxyl, sulfhydryl, aminooxy, amino, substituted amino, halo, e. g., chloro, bromo or iodo, or an activated oxygen atom such as tosylate, brosylate, nosylate, mesylate, or the like.

Unsubstituted and substituted amino groups are generally of the formula-NHR'wherein R' is hydrido, alkyl, alkenyl, alkynyl, aryl, either unsubstituted or substituted at one or more available carbon atoms with a substituent selected from the group consisting of halo, amino, hydroxyl and aryl. The functionalized polymeric substrates of the invention may be formed by one of the synthetic reactions described herein.

Where the base polymer initially comprises aromatic groups, either in the polymer backbone or pendant thereto, e. g. phenyl, homoazulene, azulene, etc., the functionalized polymeric substrates of the invention may be formed by reaction of the base polymer's aromatic groups with an acid chloride of the formula Rl COCI, where Ri is as above, to yield aromatic ketones, i. e., aromatic moieties that are substituted with a- (CO) Rl group, which are then reduced to provide aromatic moieties having-CH (OH) RI substituents, such that X of formula (I) is hydroxyl. When Rl is alkyl, the substituents provided are hydroxyalkyl substituents. The-CH (OH) RI substituents are optionally further converted to provide other X substituents, i. e., substituents other than hydroxyl groups.

The reactions are illustrated in Schemes 1 and 2:

(y) i (y) i (y) R COCI I Reduction /\/\/\ _ 0.--0. p R \OH Optional Further Conversion _, -J v Ri X Scheme 1

M M M R1 COCI reduction 0 oh ii Oh Pi Ri Optional Further M Conversion (Y) i X X Ri Scheme 2 In the schemes, Rl and X are as defined earlier herein, Y is generally alkyl or lower alkoxy, i is an integer in the range of 0 to 3 inclusive, and M is a monomer unit typically although not necessarily formed by addition polymerization of an olefinic or vinyl monomer unit; an exemplary base polymer herein is polystyrene, preferably aminomethylated polystyrene.

An alternative synthesis is to react a hydroxyl-substituted aromatic ketone such as acetophenone with an ester R20- (CO)- (CH2) m-R3, wherein R2 and R3 are as defined earlier herein (e. g., methylbromovalerate), followed by hydrolysis, resulting in the formation of a linker having an aryloxy moiety (e. g., acetophenoxy) and a carboxylic acid moiety, which are separated by the alkylene linkage. The free carboxylic acid group is then reacted with a base polymer in the form of any suitable support having free-NH2 groups on its surface, e. g. aminomethyl polystyrene, via an amide linkage. This reaction is illustrated in Scheme 3:

R20 (1) coupling (1) coupling Ho-' O R1 (2) ester hydrolysis HO \ O Support NH2 (CH2) m-O ON Cari Support NH \ O CH2) m O ruz Scheme 3 In this case, the moiety X of formula (I) is O, and q is present as a double bond.

The- (CO) RI group extending from the substrate surface serves as a starting point to generate a series of novel linkers. Several examples of peptides and small molecules have been successfully made on these linkers with high yields and purities, and no side reactions were detected. Comprehensive cleavage studies showed that the desired products were cleaved under mild acidic conditions.

Definitions It is to be understood that this invention is not limited to the particular methodology, protocols, substrates, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments

only, and is not intended to limit the scope of the present invention which will be limited only by the appende claims.

As used herein the singular forms"a,""and,"and"the"include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to"a crown" includes a plurality of crowns and reference to"the substrate"includes reference to one or more substrates and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

The term"alkyl"as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Preferred alkyl groups herein have 1 to 12 carbon atoms. The term"lower alkyl"intends an alkyl group of one to six carbon atoms, preferably one to five carbon atoms.

The term"alkenyl"as used herein refers to a branched or unbranched hydrocarbon group of 2 to 24 carbon atoms containing at least one double bond, such as ethenyl, n- propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. Preferred alkenyl groups herein have 2 to 12 carbon atoms. The term"lower alkenyl"intends an alkenyl group containing two to six carbon atoms, preferably two to four carbon atoms.

The term"alkynyl"as used herein refers to a branched or unbranched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n- propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl, decynyl, and the like. Preferred alkynyl groups herein have 2 to 12 carbon atoms. The term"lower alkynyl"intends an alkynyl group containing two to six carbon atoms, preferably two to four carbon atoms.

The term"alkylene"as used herein refers to a difunctional branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methylene, ethylene, n- propylene, n-butylene, n-hexylene, decylene, tetradecylene, hexadecylene, and the like.

Preferred alkylene groups herein have 1 to 12 carbon atoms. The term"lower alkylene" refers to an alkylene group of one to six carbon atoms, preferably one to four carbon atoms.

The term"aryl"or"aromatic group"refers to an aromatic species containing 1 to 3 aromatic rings, either fused or linked, and either unsubstituted or substituted with 1 or more substituents typically selected from the group consisting of lower alkyl, lower alkoxy, halogen, and the like. Preferred aryl substituents contain 1 aromatic ring (e. g., phenyl) or 2 fused or linked aromatic rings (e. g., azulene), and particularly preferred aryl moieties are unsubstituted phenyl rings. The term"arylene"refers to a difunctional aromatic group, where"aromatic group"is as just defined, and a particularly preferred arylene moiety is phenylene.

The term"base polymer"as in a support or substrate comprised of a"base polymer" refers to a polymeric support material suitable for use in solid phase chemical synthesis, particularly solid phase synthesis in which monomers are consecutively added to a chain, or polymer, e. g. polypeptide synthesis, nucleic acid synthesis, synthesis of peptidomimetics, etc., as known in the art. The base polymer is insoluble in the solvents used in the synthetic reactions, but provides solvent accessible moieties, or linkers, that"anchor"the initial monomer. For such purposes, it is desirable to have a linker that reacts easily with the monomer, but which will form a bond that is not cleaved during the subsequent synthetic reactions. The bond must then be cleavable under conditions that do not chemically modify the synthetic product.

For the purposes of the invention, various synthetic resins can be used as the base polymer. In one embodiment of the invention, the aromatic moiety of the linker is grafted onto the base polymer, and the polymer will then comprise surface aminomethyl substituents that are suitable for derivatization. In another embodiment, the substrate will comprise aromatic groups as part of the backbone structure to which the subject linkers are added, and/or as pendant groups, as in polystyrene.

Polystyrene, which is in general use as a support for solid phase synthesis of peptides, is preferred for the base polymer. Included in the term"polystyrene"are polymers that have been substituted to some extent with substituents that are not capable of reaction under the conditions generally used for solid phase synthesis of biomolecules, including, for example, alkyl, typically lower alkyl, substituents such as methyl, ethyl, propyl, butyl, etc.; and alkoxy, typically lower alkoxy, substituents, etc. In order to increase the stability and insolubility in organic solvents, polystyrene resins that have been cross-linked by co-

polymerization with at most 5 mol%, and preferably from about 1 to 2 mol% with divinyl benzene or butadiene are also used. For some linkers, aminomethyl functionalized polystyrene may be used, as described above.

The base polymer may be fashioned in any suitable form, e. g. sheet, film, bead, pellet, disc, ring, tube, rod, net, crown, etc. In one embodiment of the invention the base polymer is provided as a graft, or crown, for multipin solid phase synthesis of peptides and non-peptides. A crown comprises a surface, such as of polyethylene or polypropylene, or other suitable surface, onto which polymer chains have been grafted, creating a derivatized surface with exposed free termini of the attached polymer chains. In another embodiment of the invention, the base polymers are provided as microspheres suitable for use in a"tea bag,"or column.

The term"functionalized polymeric substrate"refers to a solid support having a plurality of functional groups on the support surface. The base polymer is"functionalized" by the addition of a linker to the backbone, i. e., addition of a linker to an aromatic group that is present in either the backbone of the polymer that forms the substrate or as a pendant group. The aromatic group may be initially present in the base polymer or it may be subsequently introduced.

The alkyl group is introduced into the aromatic moiety by reaction of the base polymer with an acid chloride of the formula RlCOCl, where Rl is any lower alkyl, e. g. of from 1 to 6 carbon atoms. Exemplary are Friedel Craft reaction conditions, i. e., using acetyl chloride and A1C13 in dichloromethane (DCM) at room temperature. An aromatic ketone is thus formed in which the aromatic moieties of the polymer are substituted with-(CO) R groups, which can then be reduced by any convenient method to give hydroxyalkyl substituents-CH (OH) Rl, e. g., reaction with sodium borohydride.

An alternative synthesis is to react a hydroxyl-substituted aromatic ketone having the structural formula HO-Ar- (CO)-Rl with an ester having the structure

where Rl is as defined above; Ar is arylene; R2 is a lower alkyl of from 1 to 6 carbon atoms, usually methyl; R3 is halo, e. g. chloro, bromo, iodo or fluoro usually bromo; and m is a number from 1 to 10, usually from 1 to 6, preferably from 2 to 4. The reaction is conducted under conditions that promote nucleophilic coupling at R3 as well as conversion of-COOR2 to-COOH. The resulting product has the following structure, comprising an aryloxy moiety and a carboxylic acid moiety, separated by an alkylene linkage- (CH2) m-. where Ar, Rl, R2 and m are as previously defined. The free carboxylic acid group is then reacted with a base polymer having free amine groups, typically an aminomethylated resin such as aminomethyl polystyrene, to give an amide linkage. The ketone moiety is then reduced as described above, e. g., with sodium borohydride, to give the terminal substituent -CH (OH) RI.

The resulting product from either of these synthetic methods is a functionalized polymeric substrate. The hydroxyl-substituted polymeric substrate (II) can be used for anchoring acids, such as peptide acids prepared by the Fmoc strategy. The linker is used for the immobilization of Fmoc-protected amino acids and other carboxylic acid compounds under very mild coupling condition, or for anchoring alkylhalides under various reaction conditions. The linker is an alternative to the Wang or HMPA linkers (Wang et al. (1975) J.

Ors. Chem. 40: 1235; and R. C. Sheppard et al. (1988) Int. Pep. Protein Res. 20: 451-454).

The hydroxyl-substituted polymeric substrate (II) also serves as the starting material for other linkers. For example, reaction of the functionalized polymeric substrate (II) with thionyl chloride gives the chloro-substituted polymer (III). Other halogens may be similarly introduced. This linker system is designed for anchoring the hydroxyl functional group of alcohols and phenols. They can be coupled to the-CH (Cl) RI group by simple treatment of substrate with commercially available potassium or sodium alkoxide. The functionalized polymeric substrate (III) is more stable and less hindered than a chlorotrityl linker, and is more easily derivatized than Ellmans'THP linker (Thompson and Ellman (1994) Tet. Lett.

35: 9333). The use of (III) also avoids the need for prederivatization prior to attachment to the solid phase, as in the case of phenols linked to HMPA.

The chloro functional group within the linker (III) can be converted into the amino moiety, using, for example, potassium phthalimide in DMF, followed by treatment with hydrazine to afford the amino functionalized substrate (IV). This linker can be used to anchor carboxylic acids and after being cleaved with TFA it releases compounds with terminal amide groups. The general methods for conversion of chloro to amine groups is described in Tet Lett. (1972) 32: 3281-3284.

An alternative functionalized polymeric substrate derived from (II) can be prepared from (III) by reaction with nucleophiles to give products such as the aminooxy- functionalized polymeric substrate (V). Polymeric substrate (V) is designed for the solid phase synthesis of hydroxamic acids. Thus, the free amino group on this linker can be allowed to react with carboxylic acids including Fmoc-protected amino acids under mild coupling condition.

The synthesized products are cleaved from the functionalized polymeric substrate of the invention under acidic conditions. Exemplary cleavage data is provided in Figures 2,3 and 4. For example, acids are released from (II) with exceptionally high yield and purity in 10% trifluoroacetic acid. As with the linker (II), release of synthesized product can be effected by treatment of (III) with 20% trifluoroacetic acid. Cleavage of the product (IV) can be effected in 50-95% trifluoroacetic acid.

Structures of the functionalized polymers are as follows:

Linker Structure Functionality Exemplary Cleavage Resulting Function requiredfor Conditions after Cleavage attachment carboxylic acids 10% TFA in DCM acids III phenols, alcohols alcohols TFA in DCM phenols, alcohols IV carboxylic acids 50-95% TFA in primary amides DCM V carboxylic acids 50-95% TFA in hydroxamic acids DCM VI primary amines, 50% TFA in DCM secondary amides/ sulfonamides ammonia 50% TFA in DCM primary amides "Monomer"as used herein refers to a chemical entity that can be covalently linked to one or more other such entities to form an oligomer. Examples of"monomers"include amino acids, nucleotides, saccharides, peptoid monomers, and the like. In general, the monomers used in conjunction with the present invention have first and second sites, e. g. C- termini and N-termini, or 5'and 3'sites, suitable for binding to other like monomers by

means of standard chemical reactions, e. g. condensation, nucleophilic displacement of a leaving group, or the like, and typically, a diverse element that distinguishes a particular monomer from a different monomer of the same type, e. g. an amino acid side chain, a nucleotide base, etc. The initial support-bound, or anchored"ligand"is generally used as a building-block in a multi-step synthesis procedure to form an oligomer, such as in the synthesis of oligopeptides, oligopeptoids, oligonucleotides, and the like.

The term"ligand"as used herein refers to moieties that are bound to the functional groups of the substrate. For example, where the functionalized substrate is a hydroxyethyl- polystyrene, the ligand is covalently attached under esterifying conditions, to form an ester linkage. The term"ligand"in the context of the invention may or may not be an"oligomer" as defined above. However, the term"ligand"as used herein may also refer to a compound that is not synthesized on the novel substrate, but that is"pre-synthesized"or obtained commercially, and then attached to the substrate.

As used herein, the term"amino acid"is intended to include not only the L-, D-and nonchiral forms of naturally occurring amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), but also modified amino acids, amino acid analogs, and other chemical compounds that can be incorporated in conventional oligopeptide synthesis, e. g. 4-nitrophenylalanine, isoglutamic acid, isoglutamine, s-nicotinoyl-lysine, isonipecotic acid, tetrahydroisoquinoleic acid, a- aminoisobutyric acid, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, (3-alanine, 4-aminobutyric acid, and the like.

A"peptoid"is a polymer made up, at least in part, of monomer units of"amino acid substitutes"which substitutes are any molecule other than an amino acid, but which serves in the peptoid polymer to mimic an amino acid. Peptoids are produced by linking the "amino acid substitutes"into a linear chain or cyclic structure with amino acids and/or other amino acid substitutes. The links may include, without limitation, peptide bonds, esters, ethers, amines, phosphates, sulfates, sulfites, thioethers, thioesters, aliphatic bonds, and carbamates. Examples of amino acid substitutes include, without limitation, N-substituted glycine, N-alkylated glycines, N-substituted alanine, N-substituted D-alanine, urethanes,

substituted hydroxy acids, such as hydroxyacetic acid, 2-hydroxypropanoic acid, 3- hydroxypropanoic acid, 3-phenyl-2-hydroxypropanoic acid, and the like. A peptoid may comprise amino acid substitutes using more than one type of link provided the chemistry for the reaction schemes are compatible and encompassed generally by the reactions described herein. Other examples of amino acid substitutes and peptoids are described in U. S. Patent Nos. 5,811,387 to Bartlett et al., 5,877,278 to Zuckerman et al. and 5,965,695 to Simm et al., and in Zuckermann et al., PCT W094/06451.

The terms"nucleoside"and"nucleotide"are intended to include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. In addition, the terms "nucleoside"and"nucleotide"include those moieties which contain not only conventional ribose and deoxyribose sugars, but also other sugars as well. Modified nucleosides or nucleotides will also include modifications on the sugar moiety, e. g. wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like.

The term"saccharide"is intended to include not only naturally occurring mono-and di-saccharides, but also modified saccharides. Examples of monosaccharides include trioses, such as glyceraldehyde and dihydroxyacetone, tetroses, such as erythrose, erythrulose and threose, pentoses, such as ribose, ribulosem arabinose, xylose, xylulose and lyxose, hexoses, such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose, heptoses, such as sedoheptulose, and the like.

Disaccharides include dimers of the any of the above monosaccharides attached by way of a-1,2, a-1,3, a-1,4, a-1,6, ß-1,2, P-1,3, P-1,4, P-1,6 linkages, or the like. Examples of such disaccharides include maltose, lactose, sucrose, and the like. Modified saccharides include those wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, phosphates, or the like.

The term"oligomer"is used herein to indicate a chemical entity that contains a plurality of monomers. The terms"oligomer"and"polymer"may be used interchangeably.

Examples of oligomers and polymers include polypeptides, polydeoxy-ribonucleotides, polyribonucleotides, other polynucleotides which are N-or C-glycosides of a purine or

pyrimidine base, polysaccharides, and other chemical entities that contain repeating units of like chemical structure.

The terms"protection"and"deprotection"as used herein relate, respectively, to the addition and removal of chemical protecting groups using conventional materials and techniques within the skill of the art and/or described in the pertinent literature; for example, reference may be made to Greene et al., Protective Groups in Organic Synthesis, 2nd Ed., New York: John Wiley & Sons, 1991. Protecting groups prevent the site to which they are attached from participating in the chemical reaction to be carried out. Methods and conditions for the removal of protecting groups are well known in the art and described, for example, in Greene et a/., cited above.

Any number of protecting groups can be used, as will be appreciated by those skilled in the art. Again, reference may be had to Greene et al., although suitable protecting groups will be known to or easily deduced by those working in the field of synthetic organic or bio- organic chemistry. The only requirements for the protecting groups used herein are that: (1) they be"orthogonal"so as to remain in place during other chemical syntheses or procedures which are carried out on the unprotected sites, e. g., coupling of amino acids, peptide mimetics, nucleotides, and the like; and (2) they are compatible with whatever temperatures, reaction conditions and reagents are employed while they are in place, i. e., are not degraded, chemically altered, or removed from the protected site.

Frequently, although not necessarily, the protecting groups are acid-cleavable.

Examples of suitable protecting groups include, but are not limited to: (a), for diol protection, 2,2-dimethoxypropane, acetals such as benzylidene acetal andp- methoxybenzylidene acetal, bifunctional silyl ethers such as di-t-butylsilylene, and compounds which upon reaction with a 1,2-diol will form acetonides, cyclic carbonates or cyclic boronates; and (b) for protection of a single hydroxyl site, (i) protecting groups which will give rise to ethers, e. g. tetrahydropyranyl, dihydropyranyl, trimethylsilyl, substituted or unsubstituted benzyl (if substituted, typically with electron withdrawing groups such as N02), and triphenylmethyl, and (ii) protecting groups which will give rise to esters, such as acetyl, trifluoroacetyl, and trichloroacetyl.

Use of the Functionalized Polymeric Substrates The functionalized polymeric substrates of the invention are used to covalently attach a ligand, and to provide the starting point for the solid phase synthesis of a compound, which may or may not be oligomeric. For example, a ligand bound to the functionalized substrate may be divided into groups and then chemically modified by introduction of substituents to form a series of analogs of the starting ligand. Alternatively, conventional formation of an oligomer by stepwise addition of monomers to the ligand may be performed. Ligands include oligopeptides, oligonucleotides, oligosaccharides, oligomers of peptide mimetics such as oligopeptoids, and the like. Conventional reagents and methods for making oligopeptides, oligopeptoids, oligonucleotides, and the like, can be used.

In combinatorial processes, the materials and techniques now used in combinatorial chemical techniques are known in the art and discussed, see for examples, Houghten (1985) Proc. Natl. Acad. Sci. USA 82: 5131-5135); Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002; Pirrung et al. (1995), J. Am. Chem. Soc. 117: 1240-1245; Smith et al. (1994) BioMed Chem. Lett. 4: 2821-2824; Beaucage et al. (1981) Tetrahedron Lett. 22: 1859-62, and Itakura et al. (1975) J. Biol. Chem. 250 4592 (1975). (1975). Also see U. Patent Patent Nos.

5,627,210 to Valerio et al., 5,891,742 to Dollinger et al, and 5,958,792 to Desai et al.

After covalent attachment or solid phase synthesis of the ligand the protected groups are then deprotected using cleavage reagents appropriate to the selected protecting groups.

The ligand is then reacted: to add a monomer, add or delete a substituent, etc. As is common in solid phase synthesis of oligomers, after the deprotection of the anchored molecule, a reactive monomer is added to the reaction vessel. Such monomers usually comprise a protected moiety and a reactive moiety, e. g. an Fmoc protected amino acid. The reaction is allowed to proceed to completion, followed by washing steps, blocking steps, etc. as known in the art. Example 4 illustrates the use of a-hydroxyethyl-polystyrene in peptide synthesis.

After synthesis is complete, mild acidic conditions, e. g. 10-95% trifluoroacetic acid, are used to remove the completed product, as well as the protecting groups used on the individually added monomers, e. g. with amino acids, t-butyloxycarbonyl (Boc), t-butyloxy (t-Bu), and the like.

The method of using the novel functionalized substrates to prepare and use a combinatorial library involves the same general procedures outlined above. For purposes of completeness, however, the following is included as a description of the steps which would be used to carry out synthesis and use of a combinatorial library using the substrates and general methods of the invention. First, a plurality of reaction vessels is provided each containing a functionalized substrate as described herein. A different monomeric entity, each capable of binding to the substrate, is distributed into each of the reaction vessels, such that an initial support-bound monomer is provided in each vessel. Additional monomers are coupled to the growing oligomer chain, with the identity and order of monomers documented to enable synthesis of a plurality of support-bound, chemically distinct oligomers. This last step may involve a"split/mix"approach, wherein after every monomer addition, the contents of the reaction vessels are alternatively divided and mixed in a way that provides for a completely diverse set of ligands (see, Pirrung et al., supra.). The distinct oligomers in the combinatorial library so provided are then screened for activity, generally by screening individual sublibraries containing mixtures of distinct oligomers, identifying active sublibraries, and then determining the oligomeric compounds of interest by generating different sublibraries and cross-correlating the results obtained.

References describing construction of small organic molecule libraries include: Thompson et al., Chem. Rev. 96: 555-600 (1996); Gallop et al., J. Med. Chem. 37: 1233-1251 (1994); and Gordon et al., J. Med. Chem. 37: 1385-1401 (1994). A reference related to mimotopes and describing the construction of peptides on solid supports is U. S. Patent No.

4,708,871 to Geysen et al., while other references generally describing peptoids and construction of peptoid libraries include U. S. Patent Nos. 5,811,387 to Bartlett et al., 5,877,278 to Zuckerman et al. and 5,965,695 to Simm et al., and Zuckermann et al., PCT Publication No. W094/06451. References describing screening of compounds and determination of sequences include U. S. Patent Nos. 4,833,092 to Geysen et al., 5,194,392 to Geysen et al., 5,573,905 to Lerner et al., and 5,585,277 to Bowie et al.

It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples which follow are intended to illustrate and not limit the scope of the invention.

Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety.

EXPERIMENTAL The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and use the compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e. g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in C and pressure is at or near atmospheric.

Unless otherwise indicated, all starting materials and reagents were obtained commercially, e. g., from Aldrich, Sigma and ICN, and used without further purification.

Also, in these examples and throughout this specification, the abbreviations employed have their generally accepted meanings, as follows: DCM = dichloromethane DIC = diisopropylcarbodiimide DMAP = dimethylaminopyridine DMF = dimethyl formamide Dnp = dinitrophenyl ES-MS = electronspray mass spectroscopy Fmoc = fluorenylmethyl oxycarbonyl FTIR = Fourier transform infrared g = gram HOBt = 1-hydroxybenztriazole

HPLC = high performance liquid chromatography ml = milliliter PIP = pipendine PS = polystyrene RT = room temperature TFA = trifluoroacetic acid THF = tetrahydrofuran Example 1 Representative Coupling Chemistry: Reaction of Sec-Phenethyl Alcohol with Fmoc-Alanine The ester 2 (Scheme 4) was synthesized via a coupling reaction between two commercially available compounds, sec-phenethyl alcohol 1 and Fmoc-Alanine under standard coupling conditions (diisopropylcarbodiimide (DIC)/dimethylaminopyridine (DMAP)/dimethylformamide (DMF)). This ester was highly acid sensitive and readily hydrolyzed under mild acidic condition (5% TFA in DCM) to give the starting material, Fmoc-Alanine 3, in almost quantitative yield (96%) and the product 4. Surprisingly, sec- phenethyl alcohol functioned as a useful protecting group for carboxylic acids. These results prompted the development of a novel class of linker, where sec-phenethyl alcohol is incorporated into the solid support and functions as a linker system for solid phase synthesis.

Scheme 4: Preparation and Hydrolysis of Ester 2.

Reagents and Conditions: (a) 0. 1M Fmoc-Ala-OH/0. lM DIC/0. OlM DMAP/DCM/RT, 2h.

(b) 5% TFA in DCM/RT, 4h.

Example 2 Preparation of Functionalized Polymeric Substrates Synthesis of the functionalized polymeric supports 5,6,7 and 9 is illustrated in Scheme 5. The polystyrene grafted crown 8 (available from Chiron Technologies Pty, Ltd (Australia); described in Maeji et al. (1994) Reactive Polymers 22: 203;"PS"represents polystyrene and is the base polymer P of formula (I)) was subjected to Friedel Crafts acylation (acetyl chloride/AlC13/DCM). The resulting product 9 was then reduced under the standard condition of NaBH4/THF to afford compound 5. Subjection of compound 5 to a solution of 2% thionyl chloride in chloroform afforded the chlorinated product 6. The assigned structures of four compounds 5,6,8 and 9 were confirmed by FTIR analysis. The FTIR spectrum of 9 displayed a very strong stretching band of carbonyl functional group at 1678 cm-1 and is very similar with the one of acetophenone (Table 1). The synthesis of 7 began with the treatment of 6 with a commercially available N-hydroxyphthalimide in the presence of potassium tert-butoxide in dry DMF to give rise to the compound 10. This

intermediate was then refluxed with hydrazine in ethanol for 4 h. Under such a condition, the hydroxylamine was cleanly converted into the desired hydroxamate 7.

Table 1 Comparison of IR Spectral Data of 5 and Acetophenone Funtionalized Substrate 5 Acetophenone (stretching bands, cl'') (stretching bands, cl'') 1678.3 1685.2 1604.5 1599.0 1416.0 1449.3 1357.6 1359.4 1268.3 1266.3 956.4 955.4 829.8 760.3 731.4 690.5 600.4 588.3

Scheme 5: (a) AlC13/CH3COCl/CH2Cl2/RT, 16 h (b) NaBH4/THF/RT, 16h (c) SOC12/CH2C12/RT, 16 h (d) N-hydroxyphthalimide/K+t-BuO-/DMF/60°C, 16h (e) 5% hydrazine/ethanol/reflux, 4h.

Example 3 Loading Capacity of Crowns To optimize the acylation reaction condition (step a, Scheme 5), the protocol of Table 2 was used with five different concentrations of acetyl chloride and AlCl3. The reactions were performed at room temperature under gentle shaking for 16h. Functionalized substrate 5 was coupled to Fmoc-Ala-OH and the OH-loading determined by Fmoc release upon treatment with 20% piperidine in DMF (Scheme 6). The results (Table 3) indicated that for I-series crowns (Chiron Technologies Pty, Ltd. (Australia), medium size) variation of acetyl chloride concentration produced crowns with loading levels from 2 to 10 mol per crown and the optimum concentration of acetylchloride was found to be 0.6 M. The loading curve (Figure 1) appeared to be an exponential curve reaching the maximum of loading at ca. 0.6 M of acetyl chloride content, then decreasing slightly at higher concentrations of CH3COC1/A1C13 (> 0.6M). Repeated experiments with different solvents (chloroform, dichloromethane and CS2) gave similar results.

Scheme 6: (a) 0.1 M Fmoc-Ala-OH/0.1 M DIC/25% DMF in DCM/DMAP (0. 01M)/ RT, 16 h; (b) 20% piperidine in DMF/20 min. The free Fmoc level was quantitated by measuring absorbance at 301 nm. and calculated by the formula: Fmoc (nmol/crown) = [Abs (301nm) x dilution factor]/0.0078.

Table 2 Protocol for The Friedel-Crafts Reaction (Acetyl Chloride) on PS-crowns Vial # 1 2 3 4 PS-crown 20 20 20 20 20 (I-series) A1C13, M 0. 01 0. 1 0. 25 0. 5 1 AlCl3, g 0.014 0.14 0.35 0.7 1.33 CH3COC1 0.0085 0.085 0. 22 0. 42 0. 85 (ml; 1.2eq) CH2C12 10 ml 10 ml 10 ml 10 ml 10 ml Vial # 6 7 8 9 10 PS-crown 20 20 20 20 20 (1-series) A1C13, M 0. 01 0. 1 0. 25 0. 5 1 A1C13, g 0.014 0.14 0.35 0.7 1.33 Acetic anhydride 0.0113 0. 11 0. 28 0. 57 1.13 (ml; 1.2eq) CH2C12 10 ml 10 ml 10 ml 10 ml 10 ml Table 3 Derivatization of PS-crowns (with acetyl chloride/AlCl3). [acetyl nbr of pin A301nm SD Loading chloride], M (mean) (mol/crown) 0. 0 5 0. 0 0. 0 0. 00 0.012 5 0. 14277 0. 00151 2.00 0.12 5 0. 29927 0. 00354 4.22 0.3 5 0. 57593 0. 04264 8.12 0.6 5 0. 72629 0. 02649 10.24 1.2 5 0. 67980 0. 01310 9.59

Table 4 Derivatization of PS-crowns (with acetic anhydride/AIC13)- acetic nbr of pin A301nm SD Loading anhydride], M (mean) (mol/crown) 0.0 5 0. 0 0. 0 0. 00 0.012 5 0.00538 0.00593 0.04 0.12 5 0. 01907 0. 00691 0.27 0.3 5 0. 10304 0. 00665 1.45 0.6 5 0. 38778 0. 01473 5.47 1.2 5 0. 37442 0. 02472 5.28 In Figure 1, in which Table 3 results are shown represented by diamonds, and Table 4 results are shown represented by squares.

Example 4 The a-Hydroxyethyl-Polystyrene Substrate 5 for Peptide Synthesis The application of functionalized substrate 5 is demonstrated by the following protocol for the synthesis of di-peptide AA1-AA2, wherein AA1 represents a first amino acid and AA2 represents a second amino acid (see Table 5). Hydroxyethyl-PS Fmoc-AA1-OH/DIC/DMF Fmoc-AA1-Hydroxyethyl-PS 1-20% PIP/DMF 2-Fmoc-AA2-OH/DIC/HOBt Fmoc-AA2-AA1-Hydroxyethyl-PS Thus, the first Fmoc protected amino acid was attached to the substrate 5 directly via formation of an ester linkage. This initial coupling step was carried out by using the standard coupling conditions (amino acid/DIC/DMAP/25% DMF in DCM), repeating to obtain maximum loading on the crown. Unreacted hydroxyl groups were subsequently capped by acetylation. The Fmoc group was removed and the resulting product was then coupled with a second amino acid via formation of an amide linkage. Completion of the amide coupling step was monitored by the stain test (Bromophenol Blue). The Bromophenol Blue was used as an indicator for monitoring the progress of coupling in the solid phase synthesis of peptides, as in the presence of the dye, unreacted amino groups developed deep blue color (Viktor et al. (1988) Int. J. Pept. Protein Res. 32: 415).

The methodology was employed for assembling the rest of the amino acids of the desired peptide sequence. Finally, the desired peptide was cleaved off the solid support by using 20% TFA in DCM or a cocktail solution of 95% TFA, 2.5% anisole and 2.5% ethanedithiol (EDT) (for oligopeptides). The TFA was removed under a stream of N2 gas and the washing step with 50% Et2O in Petroleum spirit was employed to remove side-chain protecting groups. The sample was redissolved in 5 ml of 10% H20 in CH3CN and

submitted to HPLC and MS analysis. A model decapeptide 18 and other small molecules have been successfully prepared under these conditions, and are summarized in Table 5.

Analysis of the model peptide 18 was carried out by stepwise cleavage in which the resulting peptides were cleaved off the solid support in every single step and the peptides were examined by reverse phase HPLC, LC-MS and FAB mass spectroscopy. In each case a single major peak with correct molecular weight was observed. The result is presented in Table 6.

Table 5 Application of Functionalized Substrate 5 Compounds Retention time MS (M+1) % purity % yield (a) (Rt) (214 nm), Observed/Cald mins 14 Fmoc-Ala-OH 9.42 312.2/312.3 96 95 15 Fmoc-Phe-Ala-OH 10. 04 459. 2/ 459. 2 90 81 16 Dnp-ßAla-Ala-OH 7. 55 327.4/327.1 88 93 17 Benzylaminoacetic 2. 84 166. 3/166. 1 79 (b) acid 18Fmoc-Glu-His-Trp-9. 57 1423.6/1423.6 75 64 Ser-Tyr-Gly-Leu-Arg- Pro-Gly-OH

Notes (a) % yield was based on crude material obtained after cleavage; (b) not determined.

Retention time (Rt) of compounds 12 to 16 were obtained on an HPLC system A. (Flow rate increased from 0 to 1.5 ml/min during the first 0.5 min then remained constant for 17 min running time.) Table 6 Stepwise Analysis of Peptide 18 Peptides Observed MW Calculated MW Fmoc-Pro-Gly-OH 395. 1 395. 1 Fmoc-Arg-Pro-Gly-OH 551. 4 551.2 Fmoc-Leu-Arg-Pro-Gly-OH 664. 4 664.3 Fmoc-Gly-Leu-Arg-Pro-Gly-OH 721. 5 721.4 Fmoc-Tyr-Gly-Leu-Arg-Pro-Gly-OH 884. 6 884.4 Fmoc-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH 971. 6 971.4 Fmoc-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH 1157. 8 1157.5 Fmoc-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH 1295. 0 1294.6 Fmoc-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-1423.6 1423.6 OH

Example 5 The a-Chloroethyl-Polystyrene Substrate 6 for Reactions with Phenols and Alcohols Application of functionalized substrate 6 for anchoring phenols and alcohols is outlined in Scheme 7. In the model study, substrate 6 was coupled with 4- hydroxyacetophenone 19 under basic conditions (K+t-BuO-). The resulting product was then either cleaved off the solid support to give back the starting material 19 or subjected to further reaction. Treatment of 20 with benzylamine/NaCNBH3 and methoxylamine hydrochloride separately afforded the compounds 21 and 22 respectively. After being treated with 20% TFA/DCM the corresponding amine 23 and oxime 24 were obtained in good yield and purity. Like phenol, the substrate 6 worked very well with N- hydroxyphthalimide and subsequent treatment of the crowns with hydrazine/ethanol afforded the hydroxamate linker 7. The linker 7 was successfully reacted with Dnp-B-Ala- OH to give the compound 25. Subjection of this later compound with 95% TFA afforded 26 with high % yield and purity and this model molecule was used for later cleavage study.

HPLC and MS analysis are summarized in Table 7.

Scheme 7: (a) 0.1 M K+t-Bu0-/DMF/60 °C, 16 h; (b) 0.1M Methoxylamine/Pyridine/60 °C/16h; (c) 2M Benzylamine/DMF/60 °C, 2h then 0.01M NaBH4/THF/60 °C/16 h ; (d) 95 % TFA/RT, 2h. (e) and (f) see Scheme 5.

Table 7 Application of Functionalized Substrate 6 Products Rt (214nm) MS % Purity % Yield (Obsd/Cald) 4-hydroxyacetophenone 6. 09 137.2/137.2 (M+1) 97 (b) 3-hydroxybenzaldehyde 5.96 (a) 97 (b) N-hydroxyphthalimide 5. 94 181.2/181.2 93 (b) (M+NH4) 24 7.72 166.1/166.2 (M+l) 98 80 23 6. 03 228.1/228.3 (M+1) 84 70 26 6. 87 271.4/271.1 82 65 Notes: (a) MS-spectra of the cleavage product and the commercially available material did not show up the molecular ion peak. (b) Not available.

Example 6 Cleavage Studies Cleavage studies of functionalized substrates 5 and 7 were based on the release of the chromophoric compounds 16 and 26, respectively, which could be quantitated by measuring the absorbance at 405 nm (Bray et al. (1991) J. Org. Chem. 56: 6659-6666).

Cleavage study of 6 was based on the direct cleavage of compound 20 to give rise to 4- hydroxyacetophenone 19, which could be quantitated by HPLC. The protocol was designed to establish the % cleavage at five different TFA concentrations (5%, 10%, 20%, 50% and 95%) over a period of 2 h. Details of the study are described in Tables 8,9 and 10 and the optimal condition for cleavage is summarized in Table 11. For all linkers, the analysis result showed that the crowns were fairly stable under very mild acidic conditions (< 5% TFA) for a short period of time (30 mins). When crowns were exposed to >10% TFA, cleavage began and was completed within 2 h.

Table 8 Cleavage Study of Functionalized Substrate 5 Time (mins) % Cleavage 5% TFA 10% TFA 20% TFA 50% TFA 95% TFA 0 0 0 0 0 0 15 16 74 95 99 98 30 24 83 97 99 99 60 37 92 97 100 100 90 38 96 99 100 100 120 50 96 99 100 100

50 crowns with attachment of the Dnp-B-Ala moiety (compound 16 on substrate 5) were incubated with different concentration of TFA (from 5% to 95%). The crowns were removed at different times over a period of 2h (initial cleavage) and transferred to solutions of 95% TFA (final cleavage) to complete the cleavage. The cleavage solution was aerated with N2 gas to dryness and dissolved in 10 ml 1: 1 solution of ethanol in water. 150 1 of chromophoric solution was measured at 405nm with a Bio Tex Microplate Reader (Model MA 310). % cleavage = initial cleavage/ (initial cleavage + final cleavage). Series 1: 5% TFA; series 2: 10% TFA; series 3: 20%; series 4: 50%. Results are shown in Figure 2.

Table 9 Cleavage Study of Functionalized Substrate 6 Time (mins) % Cleavage 5% TFA 10% TFA 20% TFA 50% TFA 0 0 0 0 0 15 64 86 93 97 30 78 91 96 98 60 88 93 97 97 90 91 93 97 97 120 91 93 96 97

50 crowns 20 with attachment of the acetophenone moiety were incubated with different concentration of TFA (from 5% to 95%). The crowns were removed at different times over a period of 2h (initial cleavage) and transferred to solutions of 95% TFA (final cleavage) to complete cleavage. The cleavage solution was aerated with N2 gas to dryness and dissolved in 10 ml of acetonitrile. 10 p1 of samples were submitted to HPLC analysis. The cleavage product was determined by peak area at 214 nm. % cleavage = initial cleavage/ (initial cleavage + final cleavage). Results are shown in Figure 3, Series 1: 5% TFA; series 2: 10% TFA; series 3: 20%; series 4: 50%.

Table 10 Cleavage Study of Functionalized Substrate 7 Time (mins) % Cleavage _ » _ 5% TFA 10% TFA 20% TFA 50% TFA 95% TFA 0 0 0 0 0 0 15 11 16 15 24 20 30 14 27 34 45 55 60 21 38 48 62 63 90 19 43 62 77 82 120 19 61 77 87 90

50 crowns 25 with attachment of the Dnp-B-Ala moiety were incubated with different concentration of TFA (from 5% to 95%). The crowns were removed at different times over a period of 2h (initial cleavage) and transferred to solutions of 95% TFA (final cleavage) to complete the cleavage. The cleavage solution was aerated with N2 gas to dryness and dissolved in 10 ml 1: 1 solution of ethanol in water. 100 ul ofchromophoric solution was measured at A405nm by Bio Tex Microplate Reader (Model MA 310). % cleavage = initial cleavage/initial cleavage + final cleavage. Results are shown in Figure 4: Series 1: 5% TFA; series 2: 10% TFA; series 3: 20%; series 4: 50%.

Table 11 Cleavage Conditions for Substrates 5,6 and 7

Linker Cleavage solution (>95% cleavage) 5 10% TFA in DCM, 2h. 6 20% TFA in DCM, 30 mins. 7 95% TFA/H20,3h

Example 7 Flexible Linkers Functionalization of polystyrene with acetyl chloride (or any acid chloride RICOCI, where R is a Cl-C6 alkyl group such as methyl, ethyl, propyl, etc.) resulted in formation of a novel class of linker. Alternatively, the acetophenone moiety within the linker molecule could be attached to a side chain (methyl bromovalerate, 27) to afford the flexible linker 28 {5- (4-acetophenoxy) valeric acid}. The compound 28 with the free terminal carboxylic acid group was coupled with any commercially available aminomethylated solid supports (crown, resin, Irori tube, tentagel, etc.) via an amide linkage (Scheme 8) to afford a series of alternative functionalized substrates 30,31,32 and 33.

Scheme 8: (a) K+t-BuO-/DMF/80°C, 5 h then KOH/methanol/H20, RT, 30 min (b) DIC, HOBt, DMF/DCM, RT, 12h (c) NaBH4/THF/RT, 16h (d) SOC12/CH2Cl2/RT, 16 h (e) N- hydroxyphthalimide/K+t-BuO-/DMF/60°C, 16h then 5% hydrazine/ethanol/reflux, 4h.

The linker 28 was readily derived from commercially available methyl 5- bromovalerate 27 and acetophenone 19 as starting materials under standard coupling condition (K+t-BuO-in DMF). The aminomethylated crown 29 was derivatized with 28 to afford the functionalized substrate 30. Subjection of substrate 30 to a series of reaction conditions as described in Scheme 2 afforded 31,32 and 33, each of which is functionally identical to the previously described functionalized substrates 9,5,6 and 7, respectively.

Application of substrate 30: The functionalized substrate is equivalent to the secondary amide forming linkers described by Albericio et al. (1990) J. Organic Chem. 55: 3730; Songster et al. (1995) Peptide Science 2: 265; and Fivush et al. (1997) Tetrahedron Lett. 38: 7151.

The substrate 30 was subjected to the reductive amination reaction with a broad range of primary amines including anilines, followed by acylation with a carboxylic acid to afford a secondary amide bound solid support. Cleavage is also effected by treatment of crown with 50% TFA in DCM to release the secondary amide compound. In one example, substrate 30 was treated with benzylamine and NaBH3CN in one-step reductive amination process to afford the compound 34. Compound 34 was then acylated with Fmoc-Ala in the presence of DIC/DMAP to afford the compound 35. Subjection of compound 35 to 50% TFA in DCM liberated the model target amide 36. Scheme 9: (a) Benzylamine, NaBH3CN, AcOH/60°C, 4 h; (b) Fmoc-alanine, DIC, DMAP/ 50% DMF in DCM/RT, 12h; (c) 50% TFA in DCM.

Application of substrate 31: The functionalized substrate 31 is equivalent to the linker 5 and designed for coupling with carboxylic acids via the ester linkage. Scheme 10 outlines the synthesis of the model di-peptide 15. Briefly, the substrate 31 was allowed to react with Fmoc-alanine under standard coupling condition (DIC/DMAP). The resulting derivatized substrate 37 was deprotected with 20% PIP/DMF and followed by coupling reaction with Fmoc- phenylalanine to afford the dipeptide bound linker 38. Subjection of 38 to 20% TFA in DCM liberated the model dipeptide 15 which was identical to the one prepared from linker 5.

Characterization of the products, 36 and 15 are described in Table 12. Scheme 10: (a) 0.1 M Fmoc-Ala-OH/0.1 M DIC/25% DMF in DCM/DMAP (0.01M)/RT, 4 h; (b) 20% piperidine in DMF/20 mins then 0.1 M Fmoc-Phe-OH/0.1 M DIC/25% DMF in DCM/DMAP (0. OlM)/ R1,4 h; (c) 20% TFA/DCM, 2h, RT.

Table 12: Synthesis of the Model Molecules, 36 and 15

Compound Linker used Rt (min) MS M+H + % yield % Purity 36 30 8. 51 401. 2 76 82 15 31 8. 15 459. 2 94 93 Retention time of compounds 15 and 36 were obtained on the HPLC system B (Flow rate was constant for 20 min running time).

Example 8 Synthesis of Aminoethyl-Polystyrene linker Scheme 11 describes the detailed synthesis of aminoethyl-functionalized polystyrene 43. The substrate 43 was readily derived from the commercially available compounds 27 and 19 as starting materials under the previously described coupling condition (scheme 5).

The resulting compound 39 was reduced under standard reduction condition (NaBH4 in MeOH) to afford the alcohol 40. The compound 40 was then saponificated (aq. NaOH/MeOH) and followed by treatment with Fmoc-NH2 in acetic acid to afford 42.

The aminomethylated crown 29 was derivatized with 42 and followed by Fmoc deprotection procedure (20% PIP/DMF) to afford the functionalized substrate 43 in quantitative yield.

Scheme 11: (a) K+t-BuO-/DMF/80°C, 5h (b) NaBH4/MeOH, RT, lh (c) NaOH/ MeOH/H20, RT, Ih (d) Fmoc-NH2/AcOH/RT, 20h (e) 20% PIP/DMF/RT, lh.

Application of functionalized substrate 43: The functionalized substrate 43 was designed for solid phase synthesis of carboxamides (Scheme 12). Substrate 43 is equivalent to the Rink amide linker described by Rink, H. (1987) Tetrahedron Lett. 28: 3787. Examples are the coupling reactions with a wide range of carboxylic acids R-COOH under standard coupling condition (HOBt/DIC/DMF). Subjection of 44 to the cleavage solution (95% TFA + 5% H20) for 4-6 h afforded the target compound 45 in good yield and purity. Under the described reaction conditions, two model peptide amides (45a and 45o) have been successfully prepared via Fmoc strategy. In brief, 43 was coupled with the first Fmoc-amino acid (DIC/DMAP/50% DCM in DMF). After Fmoc deprotection the resulting product was allowed to react with the second amino acid (DIC/HOBt/DMF). At the end of the coupling cycle the desired peptide amide was cleaved off the solid support using 95% TFA (5% H20) for 4-6 h. HPLC and MS spectral data of the model peptide amides are described in Table 13.

Scheme 12: (a) RCOOH/HOBt/DIC/DMF/RT, 16h (b) 95% TFA (5% H20)/RT, 4-6h.

Table 13: Analytical Data of the Model Peptide Amides 45 (a to m) and Target molecules HPLC ES-MS b ES-MS % % R-CONH2 a M+H + [M+H] + Yield c Purity' (mins) Observed Calculated 45aFmoc-Gly-CONH2 6. 73 297. 2 297. 3 86 99 45bFmoc- (3-Ala-CONHZ 7. 06 311. 1 311. 3 67 99 45cFmoc-Val-CONH2 9. 45 339. 0 339. 4 81 99 45dFmoc-Ile-CONH2 9. 83 353. 0 353. 4 85 96 45eFmoc-Pro-CONH2 8. 98 337. 2 337. 4 86 97 45f Fmoc-Glu-CONH2 6. 26 369. 1 369. 5 85 99 45gFmoc-Asp-CONH2 6. 77 354. 9 354. 4 77 96 45hNaphthylacetamide 7. 41 186. 2 186. 2 73 93 45i 4-C6H5 (CH2) 3CONH2 7. 09 164. 2 164. 2 79 97 45k (C6H5) 2CHCONH2 6. 42 212. 2 212. 3 82 100 4513, 4- (CH30) 2C6H3CH2CONH2 4. 12 196. 2 196. 2 66 87 45m 2-ClC6H4CH2CH2CONH2 5. 96 184. 0 184. 1 73 86 45n Peptide amide-A'8. 10 614. 4 614. 7 71 96 4So Peptide amidbBe 7. 77 801. 5 801. 8 78 62

HPLC retention times were identical with the authentic compounds prepared by the Rink amide linker. b ES-MS data were identical to the authentic compounds prepared by the Rink amide linker.

Crude yield was based on the initial loading of crown. d % purity was based on the HPLC at 401 nm. ePeptide amide-A: Fmoc-Val-Gly-Phe-Ala-CONH2. Peptide amide-B: Fmoc-Tyr-Pro-Phe-Pro- Gly-CONHs

Materials and methods Mass spec. analysis (mus): Ion-spray MS was conducted with Perkin-Elmer Sciex API III using 0.1 % Acetic acid m 60% acetonitrile.

HPLC analysis: Reverse phase high performance liquid chromatography (RP- HPLC) was conducted with Rainin, Microsorb-MV Cat. # 86-200-F3,50 x 4.6 mm column using gradient mobile-phase 0-100% B over 11.5 mins. Flow rate: 1.5 ml/min. (Solvent A: 0.1% ortho-phosphoric acid in water; Solvent B: 0.1% ortho-phosphoric acid in 90% acetonitrile). Detection: 214 nm and 254 nm.

LC-MS analysis: MS: API 100 LC/MS System, Electron-spray single quadrupole mass spectrometer, model: PE SCIEX, Canada. Mass range: 40-1000 amu. Flow rate: 300 pl/min after split from column (1.5 ml/min).

LC: Column: Rainin C 18 RP 4.6 mm x 50 mm; 0-100% B over 11 mins at a flow rate of 1.5 ml/min, which is then split into the MS and UV detector at a ratio of 1: 5.; Wavelength monitored: 214 and 254 nm.; Buffer A: 100% H20 + 0. 1 % TFA; Buffer B: 90% CH3CN + 0. 1% TFA.

Compound 2 : Fmoc-Ala-OH (3.11 g, 0.01 mol) was mixed with phenethyl alcohol 1 (1.46 g, 0.012 mol) in the presence of coupling reagent (DIC, 1.52 g, 0.012 mol) and a catalytical amount of DMAP in 20% DMF in DCM at room temperature. The reaction mixture was poured into water and extracted with DCM (3 x 20 ml) and the combined organic phase was concentrated under reduced pressure to give the crude oil. Subjection of the crude oil to a flash column (Si02,20 % EtOAc in petroleum spirit) afforded a colorless oil (3.5 g, 84% yield). HPLC: Rt = 11.39 min; Ion-spray MS: 416.2 (M+1), 433.3 (M+NH4), 848.5 (2M+NH4).

Cleavage study of the model compound 2 : Compound 2 (100 mg, 0.24 mmol) was incubated with 2 ml of 5% of TFA in DCM for 4 h. The solvent was removed under a stream of N2 gas for 20 mins and the residue was dissolved in 5 ml acetonitrile and submitted to HPLC and MS. LC-MS: 7.89 {the major peak was identified as Fmoc-Ala-OH with ion-spray MS: 312.2 (M+1), 329.2 (M+NH4)}. RF-HPLC confirmed the identical Rt = 7.69 values for both compound 3 and the commercially available Fmoc-Ala-OH.

Subjection of the crude to flash chromatography afforded 3 (ca. 72 mg, 96%).

Compound 9 (Friedel-Crafts acylation of PS-Crown): Twenty I-series PS crowns (batch number: 947) were gently shaken in a glass bottle containing 10 ml of acetyl chloride (0.22 ml, 3 mmol) and Aids (0.35 g, 2.5 mmol) in dichloromethane for 16 h at RT.

The crowns were then removed and washed with CH2C12 (2x), DMF (3x) and DCM (3x) and finally dried under reduced pressure for 8 h. FTIR vmax : 2917.23,2849.55,1678.28 (strong), 1624.42,1415.95,1357.53,1268.31,1182.46,956.43,829.76,731.76 ,718.69, 701.49,600.49 cari'.

Functionalized Substrate 5 (Reduction of acylated PS-crown 9) : Twenty I-series PS-crowns 9 were incubated with 20 ml of 0.8 M NaBH4 in THF under N2 gas at RT for 16 h. The crowns were removed, washed with CH2C12 (2x), DMF (3x), DCM (3x) and then dried under reduced pressure for 4h to afford the functionalized substrate 5. Completion of reaction can be followed by FTIR. FTIR vmaX : 3338.25 (broad), 2917.81,1849.71, 1419.87,942.52,830.42,730.72,718.80 crri' Functionalized Substrate 6 (Preparation from 5) : Twenty I-series PS-crowns 5 were incubated with 10 ml of 2% SOC12 in Chloroform at RT under sonication for 4 h. This process was repeated for one more time and the crowns were removed and washed with CH2C12 (2x), DMF (3x) and DCM (3x). The crowns were finally dried under reduced pressure for 16 h before usage. FTIR V.. a,: 2917.13,2849.48,1472.93,1462.53,1421.48, 1225.03,1046.84,831.79,755.62,730.57,718.56,667.32,628.96 (strong), 569.96.

Substrate 11 and its cleavageproduct Fmoc-Ala-OH 14 : Four I-series PS-crowns 15 (loading = 5.4 gmol/crown) were incubated with Fmoc-Ala (311 mg, 1.0 mmol) and DIC (160 gel, 1.0 mmol) in 10 ml of 25% DMF in DCM at RT for 16 h. The crowns were then removed, washed with DCM (lx), DMF (2x) and DCM (2x) and finally dried in air for 30 min to afford the compound 11. One I-series crown 11 was incubated with 1 ml of 20% TFA at RT for 2 h. The crown was removed and the solution was aerated with N2 gas for 20 min. The resulting residue was redissolved in 5 ml of acetonitrile and submitted to HPLC and Ion-spray MS analysis. HPLC: Rt = 9.42 mins; Ion-spray MS: 312.2 (M+1), 329.4 (M+NH4), 640.4 (2M+NH4).

Compound 15 (Preparation of the model dipeptide) : One I-series PS-crowns 11 was incubated with 20% PIP/DMF for 20 mins and then washed with DMF (2x) and DCM (3x).

The resulting product was incubated with Fmoc-Phe-OH (66 mg, 0.2 mmol) and DIC (32

gl, 0.2 mmol) in 2 ml of 25% DMF in DCM at RT for 16 h. The crowns were then removed, washed with DCM (lx), DMF (2x) and DCM (2x) and finally dried in air for 30 min. For cleavage, the crown was incubated with 1 ml of 20% TFA in DCM at RT for 2 h.

The crown was removed and the solution was aerated with N2 gas for 20 mins. The resulting residue (15, ca. 1.8 mg crude, 81%) was redissolved in 5 ml of acetonitrile and submitted to HPLC and Ion-spray MS analysis. HPLC: Rt = 10.04 mins; Ion-spray MS: 459.2 (M+1), 476.0 (M+NH4), 934.4 (2M+NH4).

Compound 16 : The I-series PS-crowns 11 were incubated with 20% PIP/DMF for 20 mins and then washed with DMF (2x) and DCM (3x). The resulting product was incubated with Dnp-B-Ala-OH (255 mg, 1.0 mmol), HOBt (153 mg, 1 mmol) and DIC (160ul, l mmol) in 10 ml DMF for 16 h. The crowns were then removed, washed with DCM (lx), DMF (2x) and DCM (2x) and finally dried in air for 30 mins. For cleavage, the crown was incubated with 1 ml of 20% TFA in DCM at RT for 2 h. The crown was removed and the solution was aerated with N2 gas for 20 min. The resulting residue (16,1.6 mg crude, 93%) was redissolved in 5 ml of acetonitrile and submitted to HPLC and Ion- spray MS analysis. HPLC: Rt = 7.55 mins; Ion-spray MS: 327.4 (M+1), 344.3 (M+NH4).

Compound 17 : Four I-series PS-crowns 5 were incubated with bromoacetic acid (139 mg, 1 mmol), DIC (160 p1,1 mmol) and a catalytical amount of DMAP in 10 ml of 25% DMF in DCM at RT for 16 h. The crowns were then removed, washed with DCM (lx), DMF (2x) and DCM (2x) and finally dried in air for 30 mins. The crowns were then incubated with 10 ml of 2M benzylamine solution in DMF at room temperature for 16 h.

The crowns were then removed, washed with DCM (lu), DMF (2x) and DCM (2x) and finally dried in air for 15 min. For cleavage, the crown was incubated with 1 ml of 20% TFA at RT for 2 h. The crown was removed and the solution was aerated with N2 gas for 20 min. The resulting residue (15) was redissolved in 5 ml of acetonitrile and submitted to HPLC and Ion-spray MS analysis. HPLC: Rt = 2.84 mins; Ion-spray MS: 166.3 (M+1), 183.0 (M+NH4) 331.1 (2M+1).

Compound 18 : Four I-series PS-crowns 5 were incubated with Fmoc-Gly-OH (297 mg, 1 mmol), DIC (160 gl, 1 mmol) and a catalytical amount of DMAP in 10 ml of 25% DMF in DCM at RT for 16 h. The crowns were then removed, washed with DCM (lx), DMF (2x) and DCM (2x) and finally dried in air for 30 mins. The crowns were then

incubated with 10 ml of subsequent Fmoc-amino acid solution (0.1M), HOBt (0. 1M) and DIC (0.1 M) in DMF at room temperature for 16 h. The completion of reaction in each step was confirmed by bromophenol blue test. For cleavage, the crown was incubated with 1 ml solution containing 95% TFA, 2.5% anisole and 2.5% EDT at RT for 1 h. The crown was removed and the solution was aerated with N2 gas for 20 mins. The resulting residue (18) was redissolved in 1 ml of 50% Et2O in petroleum spirit. The solid was precipitated by centrifuge. The supernatant was decanted and this washing step was repeated once more time. The precipitate (18,5.1 mg crude, 64%) was dissolved in 5 ml of acetonitrile and submitted to HPLC and Ion-spray MS analysis. HPLC: Rt = 9.57 mins; Ion-spray MS: 1423.8 (M+1), 712.4 (M+2H)/2. For analysis of compound 18: at every single coupling reaction a small section of crown was removed and subjected to 1 ml of cleavage solution as described above and the resulting product was submitted for HPLC and MS analysis.

Compound 10 : One a-Chloroethyl-I-series-PS crown 6 was incubated with N hydroxyphthalimide (50 mg, 3 mmol) potassium t-butoxide (33.6 mg, 3 mmol) in 3 ml of DMF at 60 °C for 16 h. The crown was removed, washed with DMF (2x) and DCM (2x) and dried under reduced pressure for 4 h. Treatment of compound 10 with 95% TFA at RT for 2 h released N-hydroxyphthalimide. Rt = 5.94 mins. Ion-spray MS: 164.0 (M+1), 181.2 (M+ NH4), 344.0 (M+ NH4), 507.0 (3M+ NH4).

Functionalized Substrate 7 : Sixty I-series crowns 10 were refluxed with 100 ml solution of 5% hydrazine hydrate in ethanol for 4 h. The crowns were removed, washed with hot ethanol (2x), DMF (2x) and DCM (2x) and dried under reduced pressure for 4 h to afford the hydroxamate functionality attached to the ethyl linker.

Compound 26 (preparation of hydroxamic acid molecule) : Four I-series crowns 7 were incubated with 10 ml solution of Dnp-ß-Ala-OH (255 mg, 1 mmol), HOBt (153 mg, 1 mmol) and DIC (160 1 mmol) in DMF (10 ml) at room temperature for 16 h. The crowns were removed, washed with DMF (2x) and DCM (2x) and subjected to 95% TFA for 3 h. The resulting TFA solution was aerated with N2 gas for 20 min and the residue (26, 0.95 mg crude per crown, 65%) was redissolved in 5 ml of acetonitrile and submitted to HPLC and Ion-spray MS analysis. HPLC: Rt = 6.87 mins; Ion-spray MS: 271.4 (M+1), 288.4 (M+NH4) 558.2 (2M+NH4).

Substrate 20 (Attachment of phenol model to functionalized substrate 6) : One I- series PS-crown 6 (loading 0 umol/crown) was incubated with 4-hydroxyacetophenone (41 mg, 3 mmol) potassium t-butoxide (33.6 mg, 3 mmol) in 3 ml of DMF at 60 °C for 16 h. The crown was removed, washed with DMF (2x) and DCM (2x) and dried under reduced pressure for 4 h to afford 20. FTIR: Vmax 2918.33,2850.22,1674.36 (strong), 1597.39,1505.81,1418.64,1356.38,1247.03,1169.24,1066.48,1017 .79,831.11,590.22 cm-1. Treatment of 20 with 95% TFA at RT for 2 h released 4-hydroxyacetophenone. Rt = 6.09 mins. Ion-spray MS: 137.2 (M+1).

Compound 24: One I-series PS-crown 20 was incubated with methoxylamine hydrochloride (166 mg, 2.0 mmol) in 1 ml of pyridine at 60 °C for 16 h. The crown was removed, washed with DMF (2x) and DCM (2x) and dried under reduced pressure for 4 h.

The resulting substrate 22 was treated with 20% TFA to afford the oxime 24 (ca. 1.6 mg crude material, 80%). Rt = 7.72 mins. Ion-spray MS: 166.1 (M+1).

Compound 23 : One I-series PS-crown 20 was incubated with benzylamine (0.22 ml, 2.0 mmol) in 1 ml of DMF at 60 °C for 2 h. The crown was removed, quickly washed with DMF (lx), THF (2x) and subjected to NaCNBH3 solution (3.1 mg, 0.05 mmol in 1 ml THF) at 60 °C for 16 h. Finally, the crown was removed, washed with DMF (2x) and DCM (2x) and dried in air for 15 min. For cleavage, the crown was treated with 20% TFA to afford the amine 23 (1.5 mg crude, 70%). Rt = 6.03 mins. Ion-spray MS: 228.1 (M+1).

Synthesis of compound 28 {5-(4-acetophenoxy) valeric acid7 : To a suspension of potassium t-butoxide (5.6 g, 0.05 mol) in DMF (50 ml) was added dropwise 4- hydroxyacetophenone 19 (6.8 g, 0.05 mol) in 10 ml DMF at room temperature under Nitrogen atmosphere. The reaction mixture was allowed to stir at room temperature for 15 min and methyl-5-bromovalerate (9.75 g, 0.05 mol) was then injecte into the mixture via syringe. The reaction mixture was finally heated up to 80°C for 6h before being poured onto ice. The reaction mixture was extracted with ether (3 x 20 ml) and ethyl acetate (20 ml). The combined organic phase was concentrated under reduced pressure and the resulting yellowish oil (solidified after standing for few hours at room temperature) was added to an aqueous solution of NaOH (40 ml of 4N NaOH + 60 ml of methanol) at room temperature for 30 min. The reaction mixture was then poured onto ice and the aqueous phase was washed with EtOAc (30 ml) and then acidified with concentrated HC1 until pH-

3.0. The product was extracted with EtOAc (3 x 30 ml) and the combined organic phase was dried over MgS04, and finally concentrated under reduced pressure to afford the crude product. Recrystallization of this material (20% ethyl acetate in petroleum spirit 40-60) afforded compound 28 as a white powder (10. lg, 85% overal yield). Rf = 0.39 (in 50% EtOAc in petroleum spirit 40-60°), Rt = 6.16 mins. Ion spray MS m/z 237.0 [M+H] +, 254.1 [M+ NH4] +. 472.8 (55%) [2M+H] +, 490.2 [2M+NH4] +. in NMR (400 MHz): 11.80 (s, 1H), 7.90 (d, J 5Hz, 2H), 6.94 (d, 5Hz, 2H), 4.06 (t, J 4.4 Hz, 2H), 2.52 (s, 3H), 2.33 (t, J 8 Hz, 2H), 1.88-1.72 (m, 4H). l3C NMR (50 MHz): 194.8,173.4,161.5,129.1,128.7,112.8,66.4, 32.3,27.1,24.9,20.1.

Attachment of compound 28 to the crown solid support : 50 aminomethylated-PS crowns 29 (Loading = 20 p. ml/ crown) were incubated with 25 ml of the coupling solution containing compound 28 (0.71 g, 3.0 mmol), HOBt (0.46 g, 3.0 mmol) and N'N'- diisopropylcarbodiimide (0.47 ml, 3.0 mmol) in 50 ml dry DMF for 24h. The solvent was decanted and the crowns were washed with DMF (30 ml, 5 min) and DCM (2 x 50 ml, 5 min) and then allowed to dry under reduced pressure for 4h to afford 30.

Synthesis of 31,32 and 33: reaction conditions are similar to those used for compound 5,6 and 7 respectively.

Synthesis of compound 36: The functionalized substrate 30 was subjected to the following reductive amination conditions: Two I-series crowns 30 (loading = 20 pmol/ crown) were placed in 10 ml vial containing 5.15 ml of reductive amination solution which was made of 5 ml of 1M benzylamine (pH was adjusted to-6.0 with AcOH) and 0.15 ml of 0.5 M NaBH3CN in DMF: DCM (1: lv/v). The reaction vial was incubated at 60 °C for 4 h.

Crown was then removed and carefully washed with DMF, DCM (3x each) to afford 34.

For acylation, 34 was incubated in 5 ml of 100 mM Fmoc-B-alanine activated with 100 mM HOBt/DIC in 50% DMF: DCM at room temperature for 16h. Crowns were then carefully washed with DMF, DCM (3x) to afford 35. For cleavage, one crown was incubated with lml solution of 50% TFA in DCM for 2 h. The crown was then removed and washed thoroughly with DCM (3x 2ml DCM). The combined cleavage and washing solution was evaporated with a stream of N2 gas until dryness to afford 36 as a white powder (6.2 mg crude, 78%). Rt = 8.51 min. Melting point = 178°C. LC-MS m/z 401.2 [M + H] +, 418.2 [M + NH4-

Compound 15 (Preparation of the model dipeptide) : Five I-series PS-crowns 31 (loading = 20 pmol/crown) were incubated with Fmoc-ala (311 mg, 1.0 mmol), DIC (160 al, 1.0 mmol) and a catalytical amount of DMAP (5 mg) in 10 ml of 25% DMF in DCM at RT for 4 h. The crowns were then removed, washed with DCM (1 x), DMF (2x) and DCM (2x) and finally dried in air for 30 mins to afford the compound 37. One I-series PS- crowns 37 was incubated with 20% PIP/DMF for 20 min and then washed with DMF (2x) and DCM (3x). The resulting product was incubated with Fmoc-Phe-OH (66 mg, 0.2 mmol) and DIC (32 al, 0.2 mmol) in 2 ml of 25% DMF in DCM at RT for 8 h. The crowns were then removed, washed with DCM (lx), DMF (2x) and DCM (2x) and finally dried in air for 30 mins. For cleavage, the crown was incubated with 1 ml of 20% TFA in DCM at RT for 2 h. The crown was removed and the solution was aerated with N2 gas for 20 mins.

The resulting residue (15, ca. 8.6 mg crude, 94%) was redissolved in 5 ml of acetonitrile and submitted to HPLC and Ion-spray MS analysis. HPLC: Rt = 8.15 min; Ion-spray MS: 459.2 (M+1), 476.0 (M+NH4), 934.4 (2M+NH4).

Synthesis of Compound 39 (methyl 4-acetylphenoxyvalerate): To a suspension of potassium t-butoxide (8.4g, 0.075 mol) in DMF (100 mL) was added dropwise 4- hydroxyacetophenone (10.2g, 0.075 mol) in DMF (2 mL) at room temperature under nitrogen atmosphere. The reaction mixture was allowed to stir at room temperature for 15 min and methyl-5-bromovalerate (14.6g, 0.075 mol) was then injected into the mixture via syringe. The reaction mixture was finally heated up to 80°C for 5h before being poured onto ice. The reaction mixture was extracted with ether (50 ml) and ethyl acetate (50 ml). The combined organic phase was concentrated under reduced pressure to give 39 (14.5g, 77%).

Rt = 6.98 min with a 94 % purity by hplc. ES-MS m/z 251.0 [M+H] +, 501.3 [2M+H] +. in NMR (400 MHz, 8-DMSOd6): 7.92 (d, J = 0.8 Hz, 2H), 6.92 (d, J = 0.8 Hz, 2H), 4.03 (t, J = 6 Hz., 2H), 3.67 (s, 3H), 2.54 (s, 3H), 2.41 (t, J = 6.8 Hz., 2H), 1.90-1.80 (m, 4H). 13C NMR (100 MHz): 196.6,173.6,162.7,130.4,130.1,113.9,67.5,51.4,33.4,28.4,26.1 ,21.4.

Synthesis of Compound 40 (Methyl 4- (1-hydroxyethyl) phenoxyvalerate): To a solution of ketone 39 (1.25 g, 0.005 mol) in 20 ml methanol was treated with NaBH4 (0.38g, 0.01 mol). The reaction mixture was stirred at room temperature for 1 hour and the solvent was removed under reduced pressure. The resulting oil was poured into water and the product was extracted with ether (3 x 50 ml). The combined extract was concentrated

under reduced pressure to give the crude product 40 (1.2g, 95%) Rt = 6.33 min with a 92 % purity by hplc. ES-MS m/z 235.3 (M-H20) +H] +, 469.2 [2 (M-H20) +H] +, 522.5 [2M+NH4] +. lu NMR (400 MHz, 6-DMSOd6): 7.21 (d, J = 9 Hz., 2H), 6.80 (d, J = 9 Hz., 2H), 4.75 (m, 1H), 3.98-3.92 (m, 2H), 3.60 (s, 3H), 2.37 (s, broad, 1H), 2.35-2.30 (m, 2H), 1.79-1.71 (m, 3H), 1.40 (d, J = 7 Hz., 2H)."C NMR (100 MHz): 173.8,158.1,138.0, 126. 5, 114. 3, 69. 6, 67. 3, 51. 4, 38. 5, 28. 5, 24. 9, 21. 5.

Synthesis of Compound 41 (4-(1-hydroxyethyl)phenoxyvaleric acid): Methyl 4- (1- hydroxyethyl) phenoxyvalerate 40 (1.2g, 0.005 mol) was stirred in 25 ml of 40% aq. 4N NaOH in methanol at room temperature until the starting material had been consumed as determined by TLC. The reaction mixture was then poured into water and acidified by HC1 until pH = 2-3. The product was extracted with ethyl acetate (3x 50 ml) and the combined extract was dried over MgS04 and concentrated under reduced pressure to give the crude compound 41 which was used for next step without further purification.

Synthesis of Compound 42 (4- (1- (9- Fluorenylmethoxycarbonylamino) ethyl) phenoxyvaleric acid: To a solution of 41 (1.2 g, 0.005 mol) in 20 ml of acetic acid was added Fmoc-NH2 (1.2g, 0.005 mol) and few drops of concentrated H2SO4. The reaction mixture was stirred at room temperature for 20 h and then poured into water. The product was filtered to give the crude light yellow powder (l. lg, 48% yield). Chromatography of the crude (Si02,40% EtOAc in Petrleum spirit (40-60) afforded the compound 42 as a white powder. Rf = 0.3 (40% EtOAc in petroleum spirit 40- 60°C), Rt = 8.2 min with a 95 % purity by hplc. ES-MS m/z 460.2 [M+H] +, 477.1 [M+NH4] +, 919.4 2M+H +, 936.5 2M+NH4 +.'H NMR (400 MHz, 8-DMSOd6): 7.76 (d, J = 0.8 Hz., 2H), 7.64 (d, J = 0.6 Hz., 2H), 7.38 (t, J = 0.6 Hz., 2H), 7.32-7.18 (m, 4H), 6.82 (d, 0.8 Hz., 2H), 4.7 (m, 1H), 4.32 (d, J = 0.7 Hz., 2H), 4.18 (m, 1 H), 3.94 (t, J = 0.6 Hz.), 3.2 (s, broad, 1H), 2.31 (t, J = 0.7 Hz., 2H), 1.78 (m, 4H), 1.4 (d, J = 0.6,3H), 1.26 (s, 1H).

Preparation of linker 43: Five I-series crowns (loading = 20 mol/crowns) were incubated with a mixture of 42 (0.069 g, 1.5 excess), HOBt (0.023g, 1.5 mmol), DIC (0.019g, 1.5 mmol) and DMF (3 ml) at room temperature for 16 hours. Crowns were carefully washed with DMF: DCM (50: 50) and DCM and dried under reduced pressure for

several hours. Before using, crowns were treated with 20% PIP/DMF for 1 hour and then washed thoroughly with DMF, DMF: DCM (50: 50) and DCM to afford 41.

Preparation of 45 (Synthesis of the model peptide amides) : General procedure for 45b: two derivatized crowns 45 were treated with Fmoc-ß-Ala-OH (0.31 lg, 0. lmol)/HOBt (0.156g, 0.1 mol)/DIC (160 tl, 0. lmol) in 10 ml of DMF at room temperature for 16 h.

Crowns were washed with DCM: DMF (50: 50) and DCM and 20% TFA in DCM for 10 min (pre-washing). For cleavage, the crown was incubated with 95% TFA (5% H20) for 5 h and TFA was aerated under N2 gas to dryness to afford 45b (Table 13).

Synthesis ofpeptide amide-A (Fmoc-Val-Gly-Phe-Ala-CONH2): The crowns 43 were incubated with Fmoc-Ala-OH (0.31 lg, 0.1 mol), DIC (160 1,0.1 mol) and HOBt (0.156g, 0.1 mol) in 10 mL of 25% DMF in DCM at RT for 16 h. The crown was then removed, washed with DCM (lx), DMF (2x) and DCM (2x) and finally dried in air for 30 min. For further elongation steps, the crown was treated with 20% PIP/DMF for 1 h and then washed thoroughly with DCM, DMF/DCM and DCM. The crown was incubated with 10 mL of subsequent Fmoc-amino acid solution (0.1M), HOBt (0.1M) and DIC (0.1 M) in DMF at room temperature for 8 h.

For cleavage, the crown was incubated with 1 mL solution containing 95% TFA, 5% H20 at RT for 4 h. The crown was removed and the solution was aerated with N2 gas for 20 min to afford the desired peptide amide-A (45n: Fmoc-Val-Gly-Phe-Ala-CONH2). Similar procedure was applied for the synthesis of peptide amide-B (45o: Fmoc-Tyr-Pro-Phe-Pro- Gly-CONH2).

A novel class of functionalized substrates for solid phase peptide synthesis and SPOC chemistry is provided herein, which may be generated in several alternative synthetic procedures. The substrates can be prepared from inexpensive and commercially available starting materials. Reaction conditions are safe, straightforward, readily scaled up, and may be used in conjunction with a variety of different solid supports, e. g. crown, resin, Irori tube, tentagel, etc. Furthermore, the invention in actuality provides a series of linkers, or surface functionalities, that are functionally equivalent to several commercially available linkers, e. g., Wang linker, secondary amide (Barany) linker, sasrin linker, hydroxytrityl linker, chlorotrityl linker, Merrifield linker, hydroxymethyl linker, etc. The linkers are also generally stable for storage.