WO2000026262A2 - Functionalized polymeric substrates for binding molecular moieties - Google Patents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
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- C08F8/04—Reduction, e.g. hydrogenation
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- C08F8/00—Chemical modification by after-treatment
- C08F8/18—Introducing halogen atoms or halogen-containing groups
- C08F8/20—Halogenation
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- C08F8/00—Chemical modification by after-treatment
- C08F8/26—Removing halogen atoms or halogen-containing groups from the molecule
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- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
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- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
- C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
Definitions
- This invention relates generally to methods and compositions for linking molecular moieties to polymeric substrates, and more particularly pertains to functionalization of a synthetic resin that contains phenyl groups to enable binding of molecular moieties thereto.
- 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.
- Solid phase supports 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.
- 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.
- 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.
- 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.
- 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.
- Novel functionalized polymeric substrates are provided for use as supports in solid phase synthesis.
- the functionalized polymeric substrates have the generic structure (I)
- 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
- Ri is alkyl or benzyl
- q is an optional double bond
- 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.
- the functionalized polymeric substrates are formed by reaction of a base polymer comprising phenyl groups or other aromatic moieties with an acid chloride RjCOCl, where Ri 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)R ⁇ substituents.
- RjCOCl acid chloride
- Ri is defined above
- Rj 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 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.
- an alternative synthesis is to react a hydroxyl-substituted aromatic ketone such as acetophenone with an ester having the structural formula
- R is lower alkyl
- R 3 is halogen
- m is an integer in the range of 1 to 10 inclusive.
- An example of such an ester is methyl bromovalerate.
- 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 -(CH 2 ) 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):
- q is present as a double bond and X is oxygen
- the functionalized polymeric substrate may be used for anchoring ammonia or primary amines, including aromatic and aliphatic amines, using reductive animation.
- the functionalized polymer may be used for anchoring acids, particularly protected carboxylic acids such as Fmoc- protected amino acids, under mild coupling conditions.
- 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.
- 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.
- 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- ⁇ -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- ⁇ -Ala from hydroxamate ethyl polystyrene in the presence of varying concentrations of trifluoroacetic acid, as evaluated in Example 6.
- 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)
- 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;
- Ri is alkyl or benzyl, preferably lower alkyl, and may be branched or linear, preferably linear; q is an optional double bond; n is O or l;
- Sp is a flexible spacer of the formula
- R 4 is selected from the group consisting of -NR 5 C(O)-, -C(O)-NR 5 -, -CH O-, -CH 2 S-, -O-, -S-, -OC(O)-, and -C(O)O-, wherein R 5 is lower alkyl or benzyl, and Z is selected from the group consisting of -(CH ) m -, -(CH 2 CH O) p CH CH 2 -, cycloalkylene such as
- 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.
- 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.
- 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 RiCOCl, where is as above, to yield aromatic ketones, i.e., aromatic moieties that are substituted with a -(CO)R ⁇ group, which are then reduced to provide aromatic moieties having -CH(OH)R ⁇ substituents, such that X of formula (I) is hydroxyl.
- Ri is alkyl
- the substituents provided are hydroxyalkyl substituents.
- the -CH(OH)R ⁇ 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:
- Ri 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 R 2 O-(CO)-(CH 2 ) m -R 3 , wherein R 2 and R 3 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 -NH groups on its surface, e.g. aminomethyl polystyrene, via an amide linkage.
- This reaction is illustrated in Scheme 3:
- 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.
- alkyl 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.
- alkenyl 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, w-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.
- alkynyl 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.
- lower alkynyl intends an alkynyl group containing two to six carbon atoms, preferably two to four carbon atoms.
- alkylene 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.
- lower alkylene refers to an alkylene group of one to six carbon atoms, preferably one to four carbon atoms.
- aryl 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.
- arylene refers to a difunctional aromatic group, where "aromatic group” is as just defined, and a particularly preferred arylene moiety is phenylene.
- 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.
- 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.
- various synthetic resins can be used as the base polymer.
- 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.
- 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.
- polystyrene 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.
- substituents such as methyl, ethyl, propyl, butyl, etc.
- alkoxy typically lower alkoxy, substituents, etc.
- 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.
- 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.
- 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.
- 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 RjCOCl, where Ri is any lower alkyl, e.g. of from 1 to 6 carbon atoms.
- RjCOCl any lower alkyl, e.g. of from 1 to 6 carbon atoms.
- exemplary are Friedel Craft reaction conditions, i.e., using acetyl chloride and A1C1 3 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)R 1 , e.g., reaction with sodium borohydride.
- An alternative synthesis is to react a hydroxyl-substituted aromatic ketone having the structural formula HO-Ar-(CO)-R] with an ester having the
- Ri is as defined above; Ar is arylene; R is a lower alkyl of from 1 to 6 carbon atoms, usually methyl; R 3 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 R 3 as well as conversion of -COOR to -COOH.
- the resulting product has the following structure, comprising an aryloxy moiety and a carboxylic acid moiety, separated by an alkylene linkage -(CH 2 ) m -.
- R ls R 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)R ⁇ .
- 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. Ore. Chem. 40:1235; and R.C. Sheppard et al. (1988) Int. Pen. Protein Res. 20:451-454).
- the hydroxyl-substituted polymeric substrate (II) also serves as the starting material for other linkers.
- 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)R ⁇ 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 (IN).
- 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.
- 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.
- acids are released from (II) with exceptionally high yield and purity in 10% trifluoroacetic acid.
- 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.
- “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.
- “monomers” include amino acids, nucleotides, saccharides, peptoid monomers, and the like.
- 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.
- 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.
- ligand refers to moieties that are bound to the functional groups of the substrate.
- 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.
- 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.
- 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.
- 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.
- 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 WO94/06451.
- 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.
- 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.
- saccharide is intended to include not only naturally occurring mono- and di-saccharides, but also modified saccharides.
- 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 ⁇ -1,2, ⁇ -1,3, ⁇ -1,4, ⁇ -1,6, ⁇ -1,2, ⁇ -1,3, ⁇ -1,4, ⁇ -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.
- oligomer is used herein to indicate a chemical entity that contains a plurality of monomers.
- 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.
- 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 al, cited above.
- 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.
- protecting groups include, but are not limited to: (a), for diol protection, 2,2-dimethoxypropane, acetals such as benzylidene acetal and p- 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.
- 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.
- 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.
- 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.
- 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.
- 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 ⁇ -hydroxy ethyl-polystyrene in peptide synthesis.
- 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 sub libraries 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.
- 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 pipe ⁇ dine
- TFA trifluoroacetic acid
- THF tetrahydrofuran
- 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.
- sec- phenethyl alcohol functioned as a useful protecting group for carboxylic acids.
- step a the protocol of Table 2 was used with five different concentrations of acetyl chloride and N1C1 .
- the reactions were performed at room temperature under gentle shaking for 16h.
- Functionalized substrate 5 was coupled to Fmoc-Nla-OH and the OH-loading determined by Fmoc release upon treatment with 20% piperidine in DMF (Scheme 6).
- the results 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 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. Pent. Protein Res. 32:415).
- the methodology was employed for assembling the rest of the amino acids of the desired peptide sequence.
- 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 N 2 gas and the washing step with 50% Et 2 O in Petroleum spirit was employed to remove side-chain protecting groups.
- the sample was redissolved in 5 ml of 10% H 2 O in CH 3 CN 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.
- Example 5 The ⁇ -Chloroethyl-Polystyrene Substrate 6 for Reactions with Phenols and Alcohols
- Scheme 7 (a) 0.1 M K + t-BuO7DMF/60 °C,16 h; (b) 0.1M Methoxylamine/Pyridine/60 °C/16h; (c) 2M Benzylamine/DMF/60 °C, 2h then 0.01M NaBH 4 /THF/ 60 °C/16 h;(d) 95 % TFA/RT, 2h. (e) and (f) see Scheme 5.
- 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.
- 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.
- 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 (T9971 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%o TFA in DCM to release the secondary amide compound.
- substrate 30 was treated with benzylamine and ⁇ aBH 3 C ⁇ 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.
- 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
- 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 (NaBELj in MeOH) to afford the alcohol 40.
- the compound 40 was then saponificated (aq.NaOH/MeOH) and followed by treatment with Fmoc-NH 2 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.
- 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).
- two model peptide amides 45a and 45o have been successfully prepared via Fmoc strategy.
- Scheme 12 (a) RCOOH/HOBt/DIC/DMF/RT, 16h (b) 95% TFA (5% H 2 O)/RT, 4-6h.
- LC Column: Rainin C18 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% H 2 O + 0.1 % TFA; Buffer B: 90% CH 3 CN + 0.1% TFA.
- 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- ⁇ -Ala-OH (255 mg, 1.0 mmol), HOBt (153 mg, 1 mmol) and DIC (160 ⁇ l,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.
- Compound 17 Four I-series PS-crowns 5 were incubated with bromoacetic acid (139 mg, 1 mmol) , DIC (160 ⁇ l, 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 (lx), DMF (2x) and DCM (2x) and finally dried in air for 15 min.
- Compound 18 Four I-series PS-crowns 5 were incubated with Fmoc-Gly-OH (297 mg, 1 mmol) , DIC (160 ⁇ l, 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.
- Substrate 20 (Attachment of phenol model to functionalized substrate 6): One I- series PS-crown 6 (loading ⁇ 12.0 ⁇ mol/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.
- 4-hydroxyacetophenone 41 mg, 3 mmol
- potassium t-butoxide 33.6 mg, 3 mmol
- 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 MgSO 4 , and finally concentrated under reduced pressure to afford the crude product.
- 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 ⁇ l, 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 incubated with 1 mL solution containing 95% TFA, 5% H 2 O at RT for 4 h. The crown was removed and the solution was aerated with N 2 gas for 20 min to afford the desired peptide amide-A (45n: Fmoc-Val-Gly-Phe-Ala-CONH 2 ). Similar procedure was applied for the synthesis of peptide amide-B (45o: Fmoc-Tyr-Pro-Phe-Pro- Gly-CONH 2 ).
- 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.
- 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.
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US09/432,294 | 1999-11-02 |
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WO2003093302A2 (en) * | 2002-05-03 | 2003-11-13 | Avecia Limited | Process for the synthesis of peptides amides by side-chain attachement to a solid phase |
US7166696B2 (en) | 2001-04-05 | 2007-01-23 | 3M Innovative Properties Company | Solid phase synthesis supports and methods |
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EP0274999A2 (en) * | 1986-12-24 | 1988-07-20 | Monsanto Company | Resin support for solid phase peptide synthesis |
EP0274998A2 (en) * | 1986-12-24 | 1988-07-20 | Monsanto Company | Resin support for solid phase peptide synthesis |
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Cited By (5)
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US7166696B2 (en) | 2001-04-05 | 2007-01-23 | 3M Innovative Properties Company | Solid phase synthesis supports and methods |
US7820785B2 (en) | 2001-04-05 | 2010-10-26 | 3M Innovative Properties Company | Solid phase synthesis supports and methods |
WO2003093302A2 (en) * | 2002-05-03 | 2003-11-13 | Avecia Limited | Process for the synthesis of peptides amides by side-chain attachement to a solid phase |
WO2003093302A3 (en) * | 2002-05-03 | 2004-01-08 | Avecia Ltd | Process for the synthesis of peptides amides by side-chain attachement to a solid phase |
US7691968B2 (en) | 2002-05-03 | 2010-04-06 | Avecia Biologics Limited | Process for the synthesis of peptides amides by side-chain attachment to a solid phase |
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