EP1631597A2 - Ligation chimique en phase solide effectuee avec un agent de liaison mobile - Google Patents

Ligation chimique en phase solide effectuee avec un agent de liaison mobile

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Publication number
EP1631597A2
EP1631597A2 EP04752449A EP04752449A EP1631597A2 EP 1631597 A2 EP1631597 A2 EP 1631597A2 EP 04752449 A EP04752449 A EP 04752449A EP 04752449 A EP04752449 A EP 04752449A EP 1631597 A2 EP1631597 A2 EP 1631597A2
Authority
EP
European Patent Office
Prior art keywords
linker
displaceable
ligation
support
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04752449A
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German (de)
English (en)
Other versions
EP1631597A4 (fr
Inventor
Donald Low
Ying Hu
Paolo Botti
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gryphon Therapeutics Inc
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Gryphon Therapeutics Inc
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Filing date
Publication date
Application filed by Gryphon Therapeutics Inc filed Critical Gryphon Therapeutics Inc
Publication of EP1631597A2 publication Critical patent/EP1631597A2/fr
Publication of EP1631597A4 publication Critical patent/EP1631597A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups

Definitions

  • the invention relates to methods and compositions for aqueous-compatible solid phase chemical ligation of a wide range of polymer molecules, including peptides, polypeptides, nucleic acids, lipids, carbohydrates, synthetic polymers and other molecules.
  • Chemical ligation involves the formation of a selective covalent linkage between a first chemical component and a second chemical component.
  • Unique, mutually reactive, functional groups present on the first and second components can be used to render the ligation reaction chemoselective.
  • the chemical ligation of peptides and polypeptides involves the chemoselective reaction of peptide or polypeptide segments bearing compatible unique, mutually reactive, C-terminal and N-terminal amino acid residues.
  • the ligation components can be partially or fully unprotected, rendering them useful for reactions in aqueous-based systems.
  • a first substrate is attached to the solid support through a cleavable handle or linker.
  • a second substrate is then ligated in aqueous medium to the first substrate on the solid support. Cleavage of the linker allows the reaction product to be released from the solid phase.
  • Aqueous-based chemical ligation on a solid support is discussed in the following references: Canne, et al, J. Amer. Chem. Soc. (1999) 121:8720-8727; US Pat, No. 6,326,468; and Brik, et al, J. Org. Chem. (2000) 65:3829-3835.
  • the invention is directed to methods and compositions related to aqueous- compatible solid phase chemical ligation.
  • the aqueous-compatible solid phase chemical ligation method of the invention comprises: (a) ligating under aqueous conditions a first polymer to a second polymer to form a ligation product bound to an aqueous-compatible support, the first and second polymers having mutually reactive chemical groups capable of chemoselective chemical ligation, the first polymer being attached to the support through a linker system comprising a displaceable linker; (b) releasing the ligation product from the support under conditions that displace the displaceable linker; and (c) separating the ligation product from the support.
  • the method of the invention preferably employs a multi-detachable linker system for tailoring the newly generated terminal end of the ligation product following displacement and separation from the support.
  • This method involves: (a) ligating under aqueous conditions a first polymer to a second polymer to form a ligation product bound to an aqueous- compatible support, the first and second polymers having mutually reactive chemical groups capable of chemoselective chemical ligation, the first polymer being attached to the support through a linker system having a displaceable linker and a second linker that is cleavable under conditions orthogonal to displacement of the displaceable linker, the second linker joining the first polymer to the displaceable linker; (b) displacing the displaceable linker so as to release the ligation product from the support; (c) separating the ligation product from the support; and (d) cleaving the second linker so as to remove the second linker from the ligation product.
  • compositions for aqueous-compatible solid phase chemical ligation include, in one embodiment, a support-bound polymer having (i) a first end attached to a support through a linker system that includes a displaceable linker, and (ii) a second end bearing a protected or unprotected chemoselective reactive group capable of chemoselective chemical ligation with a mutually reactive chemoselective reactive group.
  • the displaceable linkers of such compositions include chemically and/or enzymatically displaceable linkers.
  • the linker system of such compositions may include a chemically displaceable linker such as a hydrazone linker, a diol linker, a photolabile linker, a reducible linker, or a metal chelator linker.
  • the linker system of such compositions comprises an enzymatically displaceable linker that is a hydrolytic enzyme, such as an esterase, a lipase, an endoprotease, an endoglyconase, or a nucleic acid restriction enzyme.
  • the invention also is directed to a composition that includes a reaction product of a solid phase chemical ligation reaction, where the reaction product comprises a partially or fully unprotected polymer that is substantially pure and free of a solid support, and where the polymer includes a first end attached to a displaceable linker through a second linker that is cleavable under conditions orthogonal to the displaceable linker.
  • This composition may also include water and an excipient, such as a buffer, ligation catalyst, denaturant, lipid, detergent and chaotrope.
  • the composition may also be a lyophilized powder.
  • the polymer of such composition is substantially monodisperse (i.e., predominantly a single molecular species of defined covalent structure, in contrast to heterodisperse composition that contains a chemical ligation product composed of a range molecular species each having a different covalent structure).
  • Kits also are provided.
  • the kits include one or more containers having deposited therein a composition of the invention, a protocol for carrying out a method of the invention, or a combination of both.
  • the polymer components employed in the solid phase chemical ligation methods of the invention can include a wide range of chemical moieties, including amino acids, peptides, polypeptides, nucleic acids or other chemical moieties such as dyes, haptens, carbohydrates, lipids, biocompatible polymers or other polymers and the like.
  • the solid phase chemical ligation method of the invention is robust, providing faster access to polymer compounds in surprisingly high and pure yields, and increasing the average size of the polymers that can be synthesized made using standard chemical ligation approaches.
  • solid phase chemical ligation methods and compositions of the invention expand the utility of chemical ligation to multi-component ligation schemes, such as when producing a polypeptide involving multiple ligation strategies, such as a three or more segment ligation scheme or convergent ligation synthesis schemes.
  • the solid phase chemical ligation approach of the invention permits facile and improved recovery of the ligation product from the support, particularly for larger ligation products that would ordinarily adhere or otherwise bond to the support system when cleaved under harsh conditions used for non-displaceable linkers.
  • the displaceable linker can be removed or exploited for re-attachment to the same or different solid support.
  • reaction substrates also may be linked to the support through a displaceable linker to any number of second cleavable linkers for subsequent tailoring of the new terminal end of the ligation product once released in solution.
  • the second linker can be any number of moieties removable under a variety of conditions, including linkers that are cleaved under strong acid, strong base, and the like. This is particularly useful since many such linkers can be used to specifically tailor terminal end(s) of the ligated polymers.
  • Figure 1 illustrates a synthesis scheme for assembly of the h terleukin-4 (IL4) protein sequence utilizing aqueous-based solid phase chemical ligation with a displaceable oxime linker.
  • IL4 h terleukin-4
  • FIG.2A depicts RP-HPLC of purified full-length IL4 with SCAL-Oxime linker still attached.
  • Panel FIG.2B depicts RP-HPLC of purified full-length IL4 after removal of the SCAL-Oxime linker.
  • Figure 3 illustrates a synthesis scheme for assembly of the IL4 protein sequence utilizing aqueous-based solid phase chemical ligation with a displaceable metal chelator linker.
  • FIG.4A depicts RP-HPLC of purified full-length IL4 with Rink-HisTag linker still attached.
  • Panel FIG.4B depicts RP-HPLC of purified full- length IL4 after removal of the Rink-HisTag linker.
  • the invention is directed to methods and compositions related to aqueous-compatible solid phase chemical ligation.
  • the invention is directed to a method for the aqueous-compatible solid phase chemical ligation of polymers.
  • a composition is provided that includes a support-bound polymer having a first end attached to an aqueous-compatible support through a linker system having a displaceable linker, and a second end having a chemical group capable of chemoselective chemical ligation with a mutually reactive chemical group of a second polymer under aqueous conditions.
  • the support-bound polymer is then contacted, under aqueous conditions, with a second polymer having a first end bearing a chemical group that is mutually reactive with the second end of the support-bound polymer, to form a ligation product having the first and second polymers bound to the aqueous-compatible support.
  • the ligation product is then released from the support under conditions that displace or otherwise cleave the displaceable linker. Once released, the ligation product is separated from the support to yield a ligation product free of the support. This process is illustrated below in Scheme 1.
  • the composition "X ⁇ BA ⁇ Support” represents a first polymer that is bound to an aqueous-compatible support, i.e., "Support” through a displaceable linker "BA”, where the polymer portion is depicted by a wavy line.
  • the first polymer also bears a chemoselective group "X” that is capable of chemical ligation.
  • the second polymer “J ⁇ Y” is depicted as having a chemoselective group "Y” that is mutually compatible with, and capable of chemoselective chemical ligation with the X group on the first polymer.
  • first and second polymers are chemoselective for each other, they can react and form a covalent bond therein between in the presence or absence of other functional reactive groups, for instance, one or more side chain functional groups typical for unprotected amino acids.
  • first and second polymers may be partially or fully unprotected, depending on the intended end use.
  • the second polymer also is shown with pendant group J, which may be present or absent, and represents an additional group that may be converted to a group that is capable of chemical ligation, or is a group capable of a chemical ligation reaction that is orthogonal to the chemical ligation reaction employed for joining the X and Y groups of the first and second polymers, respectively.
  • Ligation of the first and second polymers results in the formation of a support-bound ligation product "J ⁇ Z ⁇ BA ⁇ Support" having ligation site "Z.”
  • the linker BA is displaced so as to release the ligation product "J ⁇ Z ⁇ B" from the support "A ⁇ Support.”
  • the displacement reaction results in a ligation product having residual linker B, whereas the support has residual linker A attached thereto.
  • the ligation product can then be separated from the solution containing the support.
  • linker moiety can be readily attached to a backbone group or side chain group (if present) on a polymer.
  • linker moiety can be readily attached to a backbone group or side chain group (if present) on a polymer.
  • Scheme 1 and other reaction schemes depicted herein illustrate attachment of such groups through or to a pendant terminal group on a polymer
  • a displaceable linker, linker system or component thereof may be attached to a backbone or side chain group on a polymer.
  • the first and second polymers may be prepared by a variety of methods, hi a preferred embodiment, the first polymer attached to the support is formed by chemical ligation, as depicted in Scheme 2.
  • the second polymer also may be the reaction product of a chemical ligation reaction.
  • the method of the invention preferably employs as part of the linker system a multi- detachable linker for tailoring the newly generated terminal end of the ligation product following its separation from the support.
  • a composition is provided that includes a support-bound polymer having a first end attached to an aqueous-compatible support through a linker system having a displaceable linker and a second linker that is cleavable under conditions orthogonal to displacement of the displaceable linker, where the second linker joins the polymer to the displaceable linker, and the displaceable linker is attached to the support.
  • the second end of the support- bound polymer includes a chemical group capable of chemoselective chemical ligation with a mutually reactive chemical group under aqueous conditions.
  • the support-bound polymer is contacted, under aqueous conditions, with a second polymer having a first end bearing a chemical group that is mutually reactive with the second end of the support-bound polymer, to form a ligation product having the support-bound polymer and the second polymer.
  • the ligation product is then released from the support under conditions that displace or otherwise cleave the displaceable linker. Once released, the ligation product is separated from the support to yield a ligation product free of the support.
  • the second linker may be removed or otherwise modified to tailor the newly generated terminal end of the ligation product free of the support. This process is depicted below in Scheme 3.
  • a multi-detachable linker system "CBA" is provided on the support-bound polymer, where BA represents a displaceable linker, and "C” represents the second linker.
  • Displacement of linker BA generates a ligation product J ⁇ Z ⁇ CB free of the support.
  • a portion of the displacement linker remains attached to the support, as is shown in Scheme 3, and represented by A ⁇ Support.
  • 'traceless' displacement linkers also may be employed, depending on the target ligation product and the terminus of the ligation product one desires to generate.
  • Second linker C may be a variety of linkers for tailoring the ligation product after separation of the product J ⁇ Z ⁇ CB from the support, particular linkers that require, or benefit from, cleavage under stringent chemical conditions, such as strong acid or base conditions. It also will be appreciated that displaceable linker BA may be directly adjacent to or separated by a divalent spacer, even a polymer, from the second linker C.
  • the spacer may be a divalent radical such as an alkyl chain (e.g., C1-C18 or longer aliphatic), a substituted alkyl chain bearing one or more side chains (e.g., C1-C18 or longer substituted with alkyl or alcohol groups etc.), an alkyl chain having one or more heteroatoms (e.g., peptide residues, ethylene oxide, etc.), or combinations thereof.
  • an alkyl chain e.g., C1-C18 or longer aliphatic
  • a substituted alkyl chain bearing one or more side chains e.g., C1-C18 or longer substituted with alkyl or alcohol groups etc.
  • an alkyl chain having one or more heteroatoms e.g., peptide residues, ethylene oxide, etc.
  • the spacer if present, maybe linear, branched, substituted or unsubstituted.
  • the spacer, if present will be stable and non-reactive under the conditions employed for
  • the displaceable linker can be cleaved as described above, and the ligation product released free of the support. Separation of the released ligated product following displacement from the support may be carried out by removing the aqueous-based solution from the solid support material, e.g., centrifugation and/or extraction, phase partition etc., and the desired target ligation product further purified from the recovered solution following standard liquid separation protocols and chromatography depending on the target ligation product (e.g., normal and reverse phase liquid chromatography, including high pressure liquid chromatography, ion exchange, size exclusion, gel filtration, affinity chromatography, affinity chromatography, electrophoresis, capillary electrophoresis, etc).
  • standard liquid separation protocols and chromatography including high pressure liquid chromatography, ion exchange, size exclusion, gel filtration, affinity chromatography, affinity chromatography, electrophoresis, capillary electrophoresis, etc).
  • the term "chemical ligation” is intended to mean the formation of a covalent linkage between chemoselective coupling partners. Chemoselectivity is achieved by the presence of unique, mutually reactive, functional groups on each of the coupling partners that render the ligation reaction chemoselective.
  • the chemical ligation of peptides and polypeptides involves the chemoselective reaction of peptide or polypeptide segments bearing compatible unique, mutually reactive, C-terminal and N-terminal amino acid residues.
  • a linker capable of being cleaved or dislodged under mild aqueous conditions.
  • mild aqueous conditions an aqueous or mixed organic-aqueous solution having a pH range from about 3 to 10.
  • Such mild aqueous conditions may include excipients, such as buffer, chaotropes, detergents, lipids, salts, reducing agents, catalysts, scavengers, redox coupling agents, and the like.
  • Linkers cleavable under mild aqueous conditions work well for coupling or linking a polymer for chemical ligation to pliable supports that swell in water, such as cellulose-, agarose- or polyethylene glycol-based supports.
  • displaceable linkers are particularly suitable for mild cleavage and release of a ligation product from the support, thereby improving separation, purification and recovery of the desired product in general.
  • Displaceable linkers employable in the invention include chemical and/or enzymatic linkers.
  • Chemically displaceable linkers are cleavable by chemical reagents or light.
  • Preferred examples of chemically displaceable linkers include oxime, hydrazone, diol, photolabile, allyl, and metal chelator linkers.
  • Enzymatically displaceable linkers are cleavable by an enzymatic reaction.
  • Preferred examples of enzymatically displaceable linkers include carbohydrate linkers containing endoglycosidase recognition sequence(s), peptide linker sequences containing an endoprotease recognition sites, as well as nucleic acid linkers containing restriction enzyme recognition or autocatalytic site(s).
  • oxime linkers are particularly useful chemically displaceable linkers in that they can be cleaved with a variety of compounds bearing an aminooxy functionality under mild aqueous conditions, typically at mildly acidic pHs around 3.5.
  • AOA aminooxyacetic acid
  • AOA can be used as a displacement reagent for cleavage of many oxime bonds, and is exemplary of a weak acid having a pH in water of about 3 to 3.5.
  • the support and first polymer are constructed to bear complementary reactive groups capable of oxime formation.
  • the first polymer is constructed to bear an aldehyde or ketone moiety (including glyoxyls), whereas the support bears an aminooxy group. Coupling of the support and the polymer forms an oxime-linked support bound first polymer.
  • the polymer can then be subjected to one or more ligation reactions to extend or otherwise elaborate the target ligation product. Once the ligation(s) are completed, the support-bound ligation product is release from the support by the addition of an aminooxy compound, such as aminooxyacetic acid.
  • R is any group compatible with aldehydes or ketones.
  • Preferred examples of R include H, carbonyl, or a variety of alkyl and aryl groups.
  • R may include -CH 3 , -CH 2 -CH 3 , -C(O)H, and -C(O)-CH 3 .
  • Substituents Ri and X are any groups compatible with aminooxy functionalities, and may be the same or different.
  • X and R ⁇ include alkyl and aryl moieties, with alkyl groups being more preferred, and may be linear, branched, substituted and unsubstituted.
  • an oxime linker may be formed as the reaction product of various aminooxy compounds with aldehyde or ketone acids; examples include oxime reaction products of an aminooxyacetyl compound with an aldehyde or ketone acid such as formyl, glyoxyl, pyruvic, levulinic, acetoacetic, acetonedicarboxyhc acid and the like.
  • the oxime linker can be provided in either orientation, depending on the intended end use.
  • Scheme 5 depicts the situation where the polymer bears the aminooxy functionality.
  • the ability to provide the displaceable bond in different orientations can be advantageous where post-ligation modifications include attachment of aldehyde or ketone bearing target molecules, such as polymers, dyes, tracers, drugs, and the like (See, e.g., EP 0 243 929 Bl; WO 94/25071; US 6,001,364; US 6,174,530; and US 6,217,873; and US 6,228,654).
  • the polymer is designed to bear the aminooxy functionality
  • the residual aminooxy component following displacement from the support can be employed as a purification handle following ligations and release from the support. It also is possible to reduce the oxime for capping or stabilization purposes, as with other Schiff-base type compounds.
  • Displaceable hydrazone linkers represent another preferred chemically displaceable linker of the invention. Hydrazone linkers are dislodged or otherwise cleavable by hydrazine compounds ' ⁇ fcN-NH-Ri" under mild conditions, and find use in the methods and compositions of the invention as illustrated in Scheme 6.
  • the hydrazone linkers may be formed with the hydrazide and aldehyde or ketone functionalities in different orientations (only one orientation shown in Scheme 6).
  • the hydrazide functionality can be provided by the support or by the first polymer for attachment to the support, hi Scheme 6,
  • R is any group compatible with aldehydes or ketones.
  • Preferred examples of R include H, carbonyl, or a variety of alkyl and aryl groups.
  • R may include -CH 3 , -CH 2 -CH 3 , -C(O)H, and -C(O)-CH 3 .
  • Substituents R v and X are any groups compatible with hydrazine / hydrazide functionalities, and may be the same or different.
  • Preferred examples of X and Ri include alkyl and aryl moieties, and may be linear, branched, substituted and unsubstituted.
  • X is an aromatic group
  • a hydrazone linker may be formed as the reaction product of various hydrazine compounds with aldehyde or ketone acids; examples include hydrazone reaction products of an hydrazide compound with an aldehyde or ketone acid such as formyl, glyoxyl, pyruvic, levulinic, acetoacetic, acetonedicarboxyhc acid and the like.
  • an hydrazide compound with an aldehyde or ketone acid such as formyl, glyoxyl, pyruvic, levulinic, acetoacetic, acetonedicarboxyhc acid and the like.
  • oximes and other Schiff-base type bonds it is possible to reduce the hydrazone linkage or resulting hydrazide for capping or stabilization purposes.
  • EP 0 243 929 Bl See, e.g., EP 0 243 929 Bl; WO 94/25071; US 6,001,364; US 6,
  • diol linkers are employed as displaceable linkers in the methods and compositions of the invention, as illustrated in Scheme 7.
  • the diol linkers are readily displaceable under mild aqueous conditions, for example, under neutral to slightly basic conditions (e.g., pH 6 to 9), and thus conditions compatible with a wide variety of supports that swell in water, such as cellulose, agarose or PEGA type resins.
  • the R group of the aldehyde or ketone bearing functionality is as described above, which is shown as part of the polymer in Scheme 7, and thus is any group compatible with aldehyde or ketone groups.
  • the group X is any group compatible with diols, and includes alkyl and aryl groups, including linear, branched, substituted and unsubstituted compounds capable of forming a suitable stable linkage with the support, where "n" is from 0 to 2.
  • Preferred diols for coupling to the support and/or as cyclic diol displacement reagents are those capable of forming a displaceable cyclic diether group linkage (i.e., a cyclic diol) with the aldehyde or ketone moiety.
  • preferred diols are those compounds of the formula l, «-diol, i.e., having two hydroxy groups present on different carbon atoms, where the hydroxy groups are usually but not necessarily adjacent. Diols may also be displaced by mild periodate oxidation.
  • preferred diols suitable as diol-formers and/or as displacement reagents for use in the methods and compositions of the invention include compounds such as HOCH 2 CH 2 OH 'ethylene glycol' (ethane-l,2-diol), HO[CH 2 ] 3 OH (1,3-propanediol), HO[CH 2 ] 4 OH butane- 1,4-diol, HOCH CH(OH)CH 3 (1,2-propanediol propylene glycol).
  • Diols suitable for coupling to the first polymer or support will bear a functional group in addition to the two hydroxy groups of the diol.
  • halogen-functionalized l,n-diols such as 3-halogenpropane-l,2-diol, meso- 1,2-diols, as well as higher alcohols, such as 1,2,3-propanetriol (glycerol, glycerin), that can be attached to a support through various techniques known in the art.
  • Vicinal 1,2-diols including functionalized pinacols such as tetra(hydrocarbyl)ethane- 1,2-diols (e.g., R 2 C(OH)C(OH)R ) of which the tetramethyl example is the simplest one and is itself commonly known as pinacol (benzpinacol is the tetraphenyl analog), and 3-chloropropane- 1,2-diol are preferred examples of diols suitable for attachment to a support.
  • the diols may be provided as a substituent(s) on aryl compounds as well.
  • the diol compounds may be purchased from commercial sources, as well as prepared de novo by numerous methods (See, e.g., Albany Molecular Research, Inc., (2002) Technical Report: Volume 6, No. 30).
  • Metal chelator linkers represent an additional type of a preferred chemically displaceable linker suitable for utilization in the methods and compositions of the invention.
  • the use of metal chelator linkers in the context of the methods and compositions of the invention are illustrated in Scheme 8.
  • the support "[Metal- — Chelator l] ⁇ Support” bears a metal chelate forming group "[Metal — Chelatorl]” that is charged with a metal ion of interest, such as copper, zinc, cobalt, iron, nickel etc., depending on the type of metal chelating ligand immobilized on the support.
  • a metal ion of interest such as copper, zinc, cobalt, iron, nickel etc.
  • the first polymer bears a metal chelator "Chelator 2" capable of binding to the metal-charged complex "[Metal- — Chelator l] ⁇ Support.”
  • the support-bound ligation product "J-[Polymer-Z] n - Polymer ⁇ [Chelator2 — Metal — Chelator l] ⁇ Support” is released from the support by addition of a competing metal, salts, pH gradients, or more preferably, ligands that can displace the coordinating metals employed for charging the initial metal coordination complex, such as pyridine and/or imidazole.
  • This aspect of the invention may employ a variety of metal chelator linkage systems, particular those utilizable for "immobilized metal chelate affinity chromatography” (IMAC).
  • IMAC employs free coordination sites of metal ions to bind a support and target molecule together by forming a compatible metal chelator complex therein between (See, e.g., Porath, et al, Nature (1975) 258:598-599; US 4,897,467; US 5,141,966; US 5,185,313; EP 0 593 417 Bl; and EP 0 437 875 Bl).
  • the principle IMAC mechanism is based on the interaction between a metal ion coordinated to (i) an aqueous compatible support bearing a covalently bound first chelating ligand and (ii) a second chelating ligand covalently bound to a target polymer of interest that also bears a chemoselective group capable of chemical ligation.
  • a target polymer capable of chemical ligation is bound to the support through the coordination bonds of a metal ion.
  • the metal chelator linkage systems employed in the methods and compositions of the invention are substantially stable to conditions employed for chemical ligation.
  • metal chelator linkage and conditions employed for subsequent chemical ligation reactions can be selected for optimal compatibility by standard manipulation of conditions for ligation, such as described below. Following chemical ligation, displacement of the metal coordination bonds, and thus the metal ion linkage releases the desired ligation product from the support.
  • iminodiacetic acid is a suitable metal chelating ligand that forms a bidentate chelating moiety after immobilization to a support, or incorporation as a metal chelating ligand of the first polymer.
  • the free bidentate chelating moiety can then be exploited for binding to a suitable a coordinating metal ion such as copper or nickel.
  • a suitable a coordinating metal ion such as copper or nickel.
  • binding of the metal ion occurs through the nitrogen atom and two carboxylate oxygens, where the metal will coordinate 4-6 ligands; the remaining coordination sites are occupied by water that can be displaced by another metal chelator ligand.
  • a target polymer bearing a group capable of chemical ligation and a group comprising a second metal chelator ligand can be attached to the Ni-IDA-Support through the remaining, free nickel coordination sites.
  • the orientation of the linker can be varied, for example, instead of the polymer bearing Chelator 2, the polymer can bear the metal charging component Chelator 1 for immobilization to a support bearing a metal Chelator 2.
  • a metal chelator such as IDA can be covalently attached to the target polymer of interest, charged with a metal ion of interest, and then attached to a support bearing a second chelator.
  • examples of preferred metal chelating ligands for attachment to the support or target polymer of interest include, but are not limited to, cyclams, penicillamine, dimercaptosuccinic acid, tartrate, thiomalic acid, crown ethers, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 3,6- dioxaoctanedithiomide, 3,6-dioxaoctanediamide, salicylaldoxime, dithio-oxamide, 8- hydroxyquinoline, cupferron, 2,2'-thiobis(ethyl acetoacetate), and 2,2'-dipyridyl.
  • NTA nitrilotriacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylenebis(oxyethylenenitrilo)t
  • metal coordinating compounds including nitrophilic, amino, carboxylates, as well as compounds with pyridine or imidazole cores (See, e.g., US 5,185,313 and EP 0 593 417 Bl), polyhistidine-tagged ("his-tag") metal chelator systems (See, e.g., US 4,877,830; US 5,047,513), polymer or lipid- based metal chelators incorporating fluorophoric sensors (See, e.g., US 5,059,654; US 5,616,790), as well as suitable metal-chelating polyamines and polyamidoamides (See., e.g., US 4,332,928) can be employed.
  • metal coordinating compounds including nitrophilic, amino, carboxylates, as well as compounds with pyridine or imidazole cores (See, e.g., US 5,185,313 and EP 0 593 417 Bl), polyhistidine-tagged (“his-tag”) metal chel
  • NTA is an aminotricarboxylic acid that generally binds bivalent metal ions in a ratio of 1 : 1. So for example, when nickel is used, every Ni 2+ ion binds to two histidine molecules in a non- conformation dependent manner, therefore it resists strong denaturants such as 6M guanidinium chloride.
  • polyamine metal chelators can be employed, and include acyclic polyamines (e.g., diethylamine, diethyltriamine, and diethyltetraamine) and macrocyclic polyamines (e.g., cyclams and cylems).
  • Desferrioxamine and its metal chelating derivatives is another metal chelating ligand of interest.
  • Desferrioxamine has multiple carbonyl and hydroxyl groups that provide electrons to coordinate with those in Fe 3+ , chelates iron in a one-to-one ratio, and has a primary amine for functional attachment to a variety of target molecules and/or supports.
  • Desferrioxamines also can bind other "hard” ions, such as Al(III), Zn(II), Ga(III), Cr(III), and, Pu(III,IV) (hard metal ions have high charge to ionic radius ratios and form strong inner sphere complexes with ligands containing "hard” donor atoms, such as oxygen).
  • Polypyridylalanine metal chelator linkers are another example, where a series of pyridylalanine residues can serve as a versatile metal affinity tag that can be used with many different immobilized metal ions.
  • Multi-dentate ligands can have extremely high metal affinities, and thus represent preferred metal chelator displaceable linkers of the invention. For instance, attachment of the normally hexadentate ligand EDTA to a target polymer such as a peptide through one of its carboxylate "arms" will result in a pentavalent ligand system that will still exhibit considerable binding affinity to a wide variety of metal ions.
  • the sixth coordination site will be open to binding to ligands immobilized onto solid supports, such as pyridine, thiol or imidazole modified aminomethyl supports. These supports may be prepared by simple reaction between halogenated derivatives of these ligands with the free base.
  • a target first polymer of interest is tagged with a string of metal-chelating amino acid residues at a terminal end opposite of the terminal end of the polymer bearing the chemoselective ligation group.
  • a string of histidines can be readily incorporated at the N- or C- terminus.
  • a sufficient number of histidine residues are provided in such a string so as to be capable of forming an electron rich metal chelator. For instance, four to six histidine residues in sequence are sufficient for this purpose.
  • sequences such as Serine-Proline-Glycine- Histidine-Histidine-Glycine, can also be employed to vary metal preference and elution parameters, as well as Histidine-Tryptophan sequences. (Smith, et al, J. Biol. Chem. (1988) 263:7211-7215).
  • the affinity of a suitable four to six long "His-tag" for Cu , Ni 2+ , Co 2+ or Zn 2+ allows a His-tagged polymer to be efficiently bound to a metal chelate affinity support, such as a nickel-agarose or a special metal-charged resin column (See, e.g., Hochuli et al, Bio/Technology (1988) 6:1321-1325).
  • the ligation product of interest that is bound to the matrix through interactions with the metal ions and can be eluted with a solution of ammonium chloride, glycine, histidine, imidizole, EDTA or high levels of competing metal ions (See, e.g., Kagedal, L. in Protein Purification: Principles, High Resolution Methods, and Applications (Janson, J.C. and Ryden, L., Eds.) (1989) pp. 227-251, VCH).
  • metal chelate supports for his-tagged or other chelator-tagged polymers of interest e.g., Novagen's HIS-BIND nickel resin (Novagen, Inc., Madison, WI, USA); Clonetech's TALON cobalt resins (Clonetech, fric, Palo Alto, CA, USA); as well as Qiagen's Ni-NTA (nickel-nitriloacetic acid) spin columns (Qiagen Inc., Chatsworth, CA, USA).
  • Novagen's HIS-BIND nickel resin Novagen, Inc., Madison, WI, USA
  • Clonetech's TALON cobalt resins Clonetech, fric, Palo Alto, CA, USA
  • Qiagen's Ni-NTA nickel-nitriloacetic acid
  • excipients are chosen so at not to significantly alter the redox state of the metal, or remove the metal completely, used for immobilizing the first polymer to the support, and in performing subsequent ligations until release of the product from the support is desired.
  • Ni-NTA supports care should be taken to limit reducing agents such as DTT, chelating agents such as EDTA and EGTA above 1 mM and certain buffers such as Tris, HEPES and MOPS, which contain secondary or tertiary amines; however, the following provides an exemplary list of preferred excipients that can be employed with Ni-NTA supports, as well as their associated higher end concentrations: 6 M guanidinium chloride, 50% glycerol, 8 M urea, 20% ethanol, 2% Triton X-100, 2 M NaCl, 2% Tween 20, 4 M MgCl 2 , 1% CHAPS, 5 mM CaCl 2 , 20 mM ⁇ -mercaptoethanol, and ⁇ 20 mM imidazole.
  • reducing agents such as DTT
  • chelating agents such as EDTA and EGTA
  • certain buffers such as Tris, HEPES and MOPS, which contain secondary or tertiary amines
  • Another consideration when a displaceable metal chelator linker system is employed is the pH of buffers and/or other solutions or conditions employed prior to a desired cleavage event of a ligation product from the support.
  • a pH that avoids lowering the metal affinity of a system prior to a desired cleavage event, including conditions employed for removal of any protecting groups during and/or after ligations.
  • the range and optimal pH conditions can be determined for any particular displaceable metal chelator linker system and ligation scheme by one of ordinary skill in the art.
  • the pH of a solution for solid phase ligations with a displaceable metal chelator linker system of the invention will be above pH 4, with a pH of around or above 4.5, 5.0, 5.5, and with a pH of 6.0 and above being most preferred. It also will be appreciated that while the reactions can be employed at higher pH's without significant unwanted cleavage, it is preferable to employ solutions that have a pH around or less than 10, with a pH around or below 9.5, 8.0 and 7.5 or below being most preferred. Again, the range and optimal pH conditions for any particular reaction and metal chelator linker system employed can be readily determined by one of ordinary skill in the art.
  • the pH of buffers or other solutions should typically be above pH 6, and preferably between pH 6 and 7.5. This pH range is sufficient for maintaining metal affinity and binding, as well as, for example, performing many chemical ligation reactions or other manipulations, such as removal of an Acm protecting group from the N-terminal cysteine of a Ni-NTA support-bound His-tag peptide for a subsequent round of on-support ligation with an incoming thioester or selenoester bearing peptide.
  • the pH such as with typical Acm removal conditions in the presence of sodium acetate at pH 4.0, the polymer may be released from the Ni-NTA support.
  • Additional examples of chemically displaceable linkers include photolabile linkers.
  • Preferred examples are supports functionalized with 3-hydroxymethyl-4-nitrophenoxymethyl (Nicolaou, et al, Angew. Chem. (1998) 110:1636), or safety-catch type photolabile linkers such as 3-(hydroxy(2-phenyl-l,3-dithian-2-yl)methyl)phenoxymethyl supports (Routledge, et al, Tet. Lett. (1997) 38:1227).
  • Additional examples include photolabile pivaloylglycol anchor / linker groups (Peukert, et al, J. Org. Chem. (1998) 63(24):9045-9051).
  • a preferred photolabile linker is the acid- and base-stable linker 3-amino-3-(2'nitrophenyl)-2,2- dimethylpropionic acid (Sternson, et al, Tet. Lett. (1998) 39:7451-7454).
  • Photolabile linkers have the advantage of being employed under a variety of chemical conditions for support- bound polymer ligations, followed by release of the ligation product via irradiation, typically by ultraviolet irradiation. However, care is taken to minimize exposure of the support and ligation products to any light until cleavage is desired.
  • Reducible linkers are those that are cleavable in the presence of a reducing agent or otherwise under reducing conditions.
  • allylic linkers such as HYCRAM (hydroxy-crotonyl-aminomethyl; Kunz and Dombo, Angew. Chem. (1988) 100:733-734) and HYCRON (Seitz and Kunz, J Org. Chem. (1997) 62:813-826), are examples of chemically displaceable linkers suitable for use with the compositions and methods of the invention.
  • the allylic linkers are particularly useful in that they are stable under a variety of solid phase synthesis conditions, and can be cleaved under mild, aqueous conditions with a hydrogen- generating catalyst, e.g., a palladium catalyst in the presence of nucleophiles.
  • a hydrogen- generating catalyst e.g., a palladium catalyst in the presence of nucleophiles.
  • Disulfide- forming linkers are additional examples of reducible linkers, which can be cleaved by reducing agents, e.g., thiols and the like.
  • Enzymatically displaceable linkers are cleavable by an enzymatic reaction.
  • Many suitable enzymatically displaceable linkers are known and can be used for this purpose, as the conditions for exploiting such enzymes are typically mild, aqueous conditions (See, e.g., Waldman, et al, Angew. Chem. (1995) 107:2425-2428; Elmore, et al, J. Chem. Soc, Chem. Commun. (1992) 14:1033-1034; Schuster, et al, J. Amer. Chem. Soc. (1994) 116:1135-1136; US Pat. No. 5,369,017; and US Pat. No. 6,271,345).
  • linkers suitable for cleavage by hydrolytic enzymes including linkages such as amide, ether, phosphoric ester or glycoside linkages, where the hydrolytic enzymes include lipases, esterases, amidases, proteases, peptidases, phosphatases, peroxidases or glycosidases.
  • suitable enzymatically displaceable linkers include these, and in particular, for instance, carbohydrate linkers containing endoglycosidase recognition sequence(s), peptide linker sequences containing an endoprotease recognition sites, as well as nucleic acid linkers containing unique restriction enzyme recognition site(s), or autocatalytic nucleic acid sequence that can be caused to self-cleave under defined conditions.
  • Glycosidases include enzymes capable of releasing N-linked oligomannose, such as PNGase F, Endo H, Endo D and Endo FI; carbohydrate linkers can be prepared from natural sources or made synthetically following standard protocols with a variety of coupling strategies (See, e.g., US Pat.
  • Nucleic acid linkers can be displaced by competition with complementary oligonucleotides, by melting, or by restriction enzymes, ribozymes, or autocatalysis, and can be attached to a support following numerous standard protocols (Jaschke, et al, Tet. Lett. (1993) 34:301-304; Ma, et al, Nucleic Acids Res. (1993) 21:2585-2589; Hendry, et al, Biochemica Biophysica Ada (1994) 1219:405-412; US Pat. Nos.
  • displaceable linkers are typically selected to cleave a unique site not present in the polymers subject to ligation. For instance, where a polymer employed in ligation includes a carbohydrate, then a displaceable linker other than an endoglucanase can be used. Where the polymer includes a peptide or polypeptide, then peptide linkers, containing a cleavage site not found in the polymer sequences, may be employed.
  • the endoprotease Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg (Nagai, et al, PNAS (1985) 82:7252-7255; Quinlan, et al, J. Cell Sci. (1989) 93:71-83; and Eaton, et al, Biochem. (1986) 25:505-512).
  • Factor Xa is particularly useful given the low occurrence of such recognition sequences in peptides and polypeptides.
  • Other enzymes that find use in the invention will be apparent to one of ordinary skill in the art, such as thrombin and enterokinase enzymes and their corresponding protease recognition sequences.
  • a preferred linker system of the invention comprises a displaceable linker and a second linker that is cleavable under conditions orthogonal to displacement of the displaceable linker, where the second linker joins the polymer to the displaceable linker, and the displaceable linker is attached to the support.
  • the second linker is chosen to be substantially stable to the conditions employed for chemical ligation. This is particularly true for multi-ligation strategies carried out on the solid support.
  • the second linker can be cleaved after the ligation product is separated from the support by cleavage of the displaceable linker, the second linker can be one that is cleaved under mild or even relatively harsh chemical or physical conditions that could otherwise damage the support.
  • the second linker can be chosen to be suitably stable under aqueous conditions for carrying out the chemical ligation reaction(s), but cleavable under more stringent chemical or physical conditions.
  • a wide range of second linkers may be employed in the methods and compositions of the invention.
  • Suitable second linkers include, for example, PAL, XAL, PAM, RINK, SCAL and Sieber-based linker systems (e.g., PAL (5-(4'-aminomethyl-3',5'- dimethoxyphenoxy)valeric acid, XAL (5-(9-aminoxanthen-2-oxy)valeric acid), 4-(alpha- aminobenzyl)phenoxyacetic acid, 4-(alpha-amino-4'-methoxybenzyl)phenoxybutyric acid, p- alkoxybenzyl (PAB) linkers, photolabile o-nitrobenzyl ester linkers, 4-(alpha-amino-4'- methoxybenzyl)-2-methylphenoxyacetic acid, 2-hydroxyethylsulfonylacetic acid, 2-(4- carboxyphenylsulfonyl) ethanol, (5-(4'-aminomethyl-3 ',5'-dimethoxyphenoxy)valeric acid) link
  • linker systems are cleavable under well known acidolysis conditions (typically trifluoroacetic acid (TFA) or hydrogen fluoride (HF)), UV photolysis ( ⁇ 350 nm) conditions, or catalytic hydrogenation conditions.
  • ester-forming linkers are examples of those typically cleavable in base, for example, HMBA (4-hydroxymethylbenzoic acid) and HMBA (4-methylbenzhydrylamine) linkers are stable to strong acids, including TFA, but cleavable in base; and carboxylic acids can be attached to this linker using the activator MSNT (l-(Mesitylene-2-sulfonyl)-3-nitro-lH-l,2,4- triazole) in the presence of 1-methylimidazole.
  • MSNT l-(Mesitylene-2-sulfonyl)-3-nitro-lH-l,2,4- triazole
  • linkers and other linker systems may also be used.
  • the most preferred linkers are cleavable in acidic conditions or light. For instance, the physical integrity of biological polymers such as peptides, polypeptides, nucleic acid, carbohydrates and the like are better maintained under acidic conditions.
  • the second linker is typically chosen depending on the type of residual group one desires to generate on the final ligation product. So for example, the following linkers can be used to generate carboxylates: PAM, DHPP (4-(l',l , -dimethyl-l'- hydropropyl)phoxyacetyl), Wang acid, SASRIN, HAL (hypersensitive Acid-Labile (HAL) tris(alkoxy)benzyl ester), Trityl, Rink acid, SAC (silyl acid), ⁇ -methylphenacyl ester, allylic linkers, and fluorene derived N-[9-hydroxymethyl-2-fluorenyl] succinamic acid (HMFS) linkers.
  • linkers that can be used for generating amides: PAL, Rink amide, XAN (xanthen-9-yl), photolabile amide linkers, SCAL and oxime linkers can be used.
  • linkers For generating alcohols and amines, the following are example of linkers that can be used: THP (tetrahydropyranyl) and Silyl chloride linkers for alcohols, as well as ketal and acetal linkers for diols and alcohols, and p-nitrophenyl carbonate for amines, and REM for the preparation of tertiary amines.
  • THP tetrahydropyranyl
  • Silyl chloride linkers for alcohols
  • ketal and acetal linkers for diols and alcohols
  • p-nitrophenyl carbonate for amines
  • REM REM for the preparation of tertiary amines.
  • Many additional linkers are known, and their preparation and use are replete in the literature
  • the second linker may be displaceable as well.
  • the displaceable linkers should be orthogonal.
  • a displaceable oxime linker may be employed in combination with a displaceable metal chelator linker as so on.
  • orthogonal linker schemes may be employed where two or more classes or groups are cleaved by differing chemical mechanisms, and therefore can be removed in any order and in the presence of the other classes. Orthogonal schemes offer the possibility of substantially milder overall conditions, because selectivity can be attained on the basis of differences in chemistry rather than reaction rates. Such orthogonal approaches are applicable when choosing a particular linker and chemical ligation system.
  • preferred supports are those that are capable of swelling in water. These include cellulose, dextran, agarose, PEGA, or other biocompatible supports. Of course, supports that do not swell in water may also be employed, provided they are not irreversibly collapsed in water or aqueous-based solutions. For instance, controlled glass pore (CGP) supports can be used, as they are not irreversibly collapsed in water or aqueous-based solutions.
  • CPG supports can be provided with a non-ionic hydrophilic coating, which is compatible with aqueous and most other solvent systems. The coating eliminates or reduces non-specific adsorption experienced with uncoated-CPG.
  • typical non-aqueous swelling supports can be modified with water-soluble polymers so that they combine both the features of a non- swelling and swelling support.
  • resins that provide a more polar environment than polystyrene can be helpful for reducing aggregation of polymers with this tendency, such as peptides.
  • TentaGel, reACTagel, PEG crosslinked, co-PEG, and pyridinyl resins are some examples.
  • TentaGel and reACTagel resins incorporate polyethylene glycol chains between the polystyrene bead and the linker.
  • Co-PEG resins are similar to corresponding polystyrene resins, but have polyethylene chains grafted to the polystyrene to provide a more polar environment.
  • PEG crosslinked resins are polystyrene crosslinked with polyethylene glycol, which not only produces a more polar environment but also creates larger pores in the swollen support.
  • the pyridyl resins incorporate pyridine groups into the resin that help to keep the attached polymers in a non-aggregated state.
  • the supports may be constructed de novo or purchased commercially.
  • the supports are prepared or otherwise provided with an initial functional group for elaboration with the displaceable linker (or linker component thereof, such as aminooxy group for forming an displaceable oxime linker) following standard coupling procedures.
  • displaceable linker or linker component thereof, such as aminooxy group for forming an displaceable oxime linker
  • linker component thereof such as aminooxy group for forming an displaceable oxime linker
  • oxime-forming and hydrazone-forming supports can be readily prepared from amine-functionalized supports. The amine is then modified to bear an aldehyde or ketone, or an aminooxy or hydrazide following standard procedures. Protocols for attaching these and other displaceable linker components are well known.
  • amino-functionalized supports are readily available from numerous vendors, and provide a universal group for immobilization of an initial displaceable linker component.
  • the amino group can be coupled under a variety of conditions to yield a variety of stable covalent bonds.
  • Various amino groups may be employed for this purpose, including primary amine-functionalized supports, as well as aminoaryl-supports.
  • the later have an arylamine that can be converted to diazonium, which is capable of reacting with imidazoles and phenolic compounds, and thus useful when a diazo linkage is required for linker immobilization.
  • carboxy-activated supports can be used.
  • the carboxyl group can be activated with carbodiimide to form a pseudourea, which reacts with an amine to form an amide bond.
  • the carboxyl-support can also be converted to N- hydroxysuccinimide by reacting with N-hydroxysuccinimide (NHS) in the presence of carbodiimide.
  • NHS N-hydroxysuccinimide
  • carbodiimide (CDI)-activated supports can be prepared or purchased and used where the immobilization occurs through the reaction of an N- nucleophile with the imidazolyl carbamate of the support to form a stable, uncharged N- alkylcarbamate linkage.
  • Halogen-functionalized supports may also be used for coupling to nucleophile-bearing linker components.
  • Glyceryl-functionalized supports have an adjacent diol group on the two terminal carbon atoms, which permits the formation of displaceable diol linkages, or oxidation to an active aldehyde by meta-sodium periodate, and thus useful for constructing displaceable oxime-forming or hydrazone-forming linkages, or for attachment of an initial displaceable linker component through an amine through reductive animation.
  • Hydrazide-functionalized supports will typically include an aliphatic arm terminating in an active hydrazide group, which can react readily with ketone or aldehyde groups to form a hydrazone bond.
  • the hydrazide supports are particularly suitable for coupling carbohydrates that have been modified to bear aldehyde or ketone groups (e.g., through mild periodate oxidation, which allows the cis-diols of the sugars to be transformed into reactive aldehyde moieties; these aldehydes then combine with hydrazide groups on the matrix to form leak-resistant linkages).
  • Epoxide chemistry is another useful way to immobilize a displaceable linker component bearing a nucleophile for attachment to the support such as an amino, thiol or hydroxy (including phenolic) functional groups.
  • Expoxide-activated supports can be produced by the immobilization of bifunctional oxiranes such as 1,4-butanediol diglycidyl ether onto agarose or other supports. These activated supports have limited stability in aqueous medium, so once activated are typically used immediately to couple the initial displaceable linker component.
  • Thiol-functionalized supports bear active thiol groups and can be used for immobilization through disulfide or thioether bonds. Thiol-functionalized supports are particularly useful for coupling halogen- or maleimide-functionalized linkers.
  • ethylene glycolbis (succinimidylsuccinate) (EGS), which creates a hydroxylamine-sensitive linkage
  • BSOCOES bis[2- succinimidooxycarbonyloxy)ethyl]sulfone
  • DST disuccinimidyl tartarate
  • DSP dithiobis(succinimidylpropionate)
  • the supports may include spacer arms and the like, and when purchased commercially these options are typically available.
  • the functional group of a support can be attached through short or longer extension arms. Longer spacer arms may help to facilitate the attachment and the approach of bulky molecules, where steric hindrance and surface repulsion can pose problems.
  • Suitable spacer arms include aliphatic or water- soluble polymers, such as polyethylene glycol and the like.
  • the solid phase chemical ligation method of the present invention can be employed with any aqueous-based ligation method, or combinations of ligation methods, for example, such as amide-forming native chemical ligation (Dawson, et al,
  • the linker system When choosing a particular ligation chemistry, the linker system, and in particular the displaceable linker is chosen such that the ligation, linker system and conditions utilized for the combined application thereof are mutually compatible, i.e., utilize orthogonal reagents and/or conditions to perform ligation versus linker cleavage.
  • safety-catch linker strategies may be employed (e.g., photolabile displaceable linker).
  • the linker system when performing oxime forming chemical ligation, the linker system may employ a displaceable linker other than an oxime. The basic considerations for maintaining orthogonality can thus be chosen accordingly.
  • the methods and compositions of the invention have many uses. They may be used in manual and/or rapid automated synthesis schemes using conventional peptide synthesis and other organic synthesis strategies. They also are particularly useful in expanding the utility of chemical ligation to multi-component ligation schemes, such as when producing a polypeptide involving orthogonal ligation strategies, such as a three or more segment ligation scheme or convergent ligation synthesis schemes.
  • the methods and compositions of the invention are exceptionally useful for ligating peptides, polypeptides and other polymers.
  • the ability to carry out solid phase chemical ligation under aqueous conditions using a variety of displaceable linkers expands the scope of chemical ligation, in general.
  • the invention can also be used to ligate polymers in addition to peptide or polypeptide segments when it is desirable to join such moieties through a displaceable linker.
  • the invention also finds use in the production of a wide range of peptide labels for expressed-protein ligation (EPL) applications.
  • EPL expressed-protein ligation
  • EPL- generated thioester polypeptides can be ligated to a wide range of peptides, depending on the intended end use, via the displaceable linker chemistries of the invention.
  • the invention can also be exploited to produce a variety of cyclic peptides and polypeptides
  • water-compatible polymer supports such as cellulose based materials are often used for protein-based affinity purifications, and are designed to support the immobilization of large polypeptides and proteins on their surfaces, these materials can be destroyed by the harsh conditions used for the cleavage of standard linking systems used in peptide chemistry (e.g., anhydrous HF or TFA). This makes it difficult to recover good material in desired amounts.
  • the displaceable linker strategy of the invention is particularly advantageous when water- compatible polymer supports, such as cellulose-based materials, are employed in aqueous- based solid phase chemical ligation reactions.
  • first linker that includes a displaceable group permits facile attachment to the support, as well as removal from the support under relatively mild conditions.
  • the displaceable linker strategy employed with solid phase chemical ligation streamlines the process of obtaining chemically synthesized proteins requiring multiple ligations.
  • the chemical synthesis of ligation intermediates and full-length products can be achieved without the need for the isolation and purification of synthetic intermediates.
  • the advantage of polymer supported peptide ligations over their solution counterparts will increase with the number of steps required to assemble the desired protein sequence. This technology should be readily applicable to the preparation of both small and large protein sequences requiring only a few to many ligation steps.
  • Convenient chemical access should facilitate further engineering of longer protein sequences of interest for research or therapeutic uses, small or large scale manufacturing, rapid analoging, on-resin modification, attachment of products to protein chips and other surfaces, as well as for specific tailoring of terminal sequences.
  • the methods and compositions also substantially increase the speed of the chemical protein synthesis approach, and * permit facile recovery and improved yields due to a reduction in the need for the isolation and purification of synthetic intermediates.
  • HATU N-[(dimethylamino)-lH-l,2,3-triazol [4,5-b] pyridiylmethylene]-N- methylmethanaminium hexafluorophosphate N-oxide.
  • HMP resin 4-hydroxymethylphenoxy resin; ?alkoxybenzyl alcohol resin; or Wang resin HOAt 1 -hydroxy-7-azabenzotriazole HOBt 1 -hydroxybenzotriazole Mbh dimethoxybenzhydryl
  • PEG-PS polyethylene glycol-polystyrene
  • Tacam Trimethylacetamidomethyl tBoc tert-butyloxycarbonyl
  • Boc-SPPS peptides were synthesized in stepwise fashion either manually or on a ABI 433 peptide synthesizer by SPPS using in situ neutralization HBTU activation protocols on an Boc-AA-O-CH 2 -PAM resin for non-thioester peptides, or on a thioester- generating resin for thioester peptides following standard protocols (Hackeng, et al, supra; Schnolzer, et al.Jnt. J. Pept. Prot. Res., (1992) 40:180-193; and Kent, S.B.H., Ann. Rev. Biochem. (1988) 57:957-984).
  • Boc SPPS protecting groups were used, namely: Arg(Tos); Asp(cHex); Asn(Xan); Cys(4MeBzl) and Cys(Acm); Glu(cHex); His(Bom); Lys(CIZ); Ser(Bzl); Thr(Bzl); Trp(formyl); Tyr(BrZ); Met, Gin were side-chain unprotected.
  • the peptides were deprotected and simultaneously cleaved by treatment with anhydrous hydrogen fluoride (HF) with 5% p- cresol and lyophilized and purified by preparative C4 reverse-phase-high pressure liquid chromatography (RP-HPLC).
  • HF hydrous hydrogen fluoride
  • peptides were synthesized in a stepwise manner either manually or on an ABI 433 peptide synthesizer by SPPS using HBTU/DIEA/DMF coupling protocols at 0.1 mmol equivalent resin scale.
  • HBTU/DIEA/DMF coupling protocols for each coupling cycle, 1 mmol N ⁇ -Fmoc-amino acid, 4 mmol DIEA and 1 mmol equivalents of HBTU were used.
  • the concentration of the activated HBTU-activated Fmoc amino acids were 0.5 M in DMF, and the couple time was 10 min.
  • Fmoc SPPS protecting groups were used, namely: Cys(Acm), Lys(Mtt), and Ser(OtBu) or Ser(OBzl).
  • the Fmoc peptides were deprotected by treatment with 20% piperidine in DMF solution for 2 x3 minutes. Deprotected peptide resin was then drained, and washed with DCM, DMF, DCM, and then dried in vacuo for 1 h. The peptide-resin was then cleaved by treatment with a TFA/TIS/H 2 O (95:2.5:2.5) solution at room temperature for 1 h.
  • HPLC fractions containing pure peptide were identified using ES-MS (electrospray ionization mass spectrometry), pooled and lyophilized for subsequent manipulation and/or ligation.
  • ES-MS electrospray ionization mass spectrometry
  • the protecting groups were retained, for instance, Acm-protected cysteines.
  • Boc-protected amino acids were obtained from Peptides International and Midwest Biotech.
  • Fmoc-protected amino acids were obtained from Midwest Biotech or Nova Biochemicals.
  • Trifluoroacetic acid (TFA) was obtained by Halocarbon. Other chemicals were from Fluka or Aldrich.
  • Analytical and preparative HPLC were performed on a Rainin HPLC system with 214 nm UV detection using Vydac C4 analytical or preparative column.
  • Peptide and protein mass spectrometry was performed on a Sciex API-I electrospray mass spectrometer.
  • AoA-Resin Aminooxy-functionalized cellulose-based affinity resin for oxime linkage was prepared as follows. Amino Spherilose affinity resin (ISCO, Lincoln, NE) was derivatized with Fmoc-aminooxy acetic acid and thoroughly washed with DMF. The Fmoc- aminooxyacetic acid was preactivated with DIG and N-hydroxysuccinamide for 45 minutes (for 9 mL of ISCO resin, 2 mmols each of NHS, DIG and Fmoc-AoA then allowed to couple to the resin for 45 minutes) and coupled to the amino Spherilose resin for 2-3 hours.
  • the resin was washed with DMF, the Fmoc group removed by treatment with 20% piperidine (2 x 3 minutes). After further washing with DMF, followed by DCM, the derivatized polymer support was thoroughly dried.
  • Ni-NTA-IMAC Resin Obtained commercially from Pharmacia or Qiagen, and used as received from the manufacturer.
  • a displaceable linker strategy was employed for solid-phase chemical ligation and its application to the synthesis of a test polypeptide, the mature full-length 129 amino acid residue sequence of human interleukin-4 (IL4).
  • the mature full-length human IL4 contains six cysteines, and a single N-linked glycosylation site at asparagine position 38.
  • the full- length IL4 polypeptide and the cysteine ligation sites employed for solid phase native chemical ligation is depicted below in Table 1 as SEQ ID NO:l.
  • Table 1 Mature full-length Interleukin 4*
  • a displaceable oxime-forming linker was employed in combination with an amide-generating SCAL ("Safety-Catch Acid Cleavable") linker.
  • the displaceable oxime-forming linker was designed to provide for attachment of the first peptide under mild conditions to a water-compatible spherical cellulose based support via a displaceable oxime linker, and the SCAL linker was designed to generate an amide capping group on the final full-length ligation product under acidic conditions following displacement of the oxime linker under mild conditions and its recovery from the support.
  • structure (1) contains the extreme C-terminal IL4 peptide segment (127- 129), and is alternatively denoted as having the amino acid sequence and linker attachment depicted below in SEQ ID NO:2.
  • C Acm SS-X SCA K lev (SEQ ID NO:2) where CSS corresponds to the C-terminal Cys-Ser-Ser residues 127-129 of human IL4 depicted in SEQ ID NO: 1;
  • C Acm is a cysteine having its side chain thiol protected with an Acm-protecting group;
  • X is an amide-generating "safety-catch acid cleavable" linker;
  • K lev is a modified lysine having its side chain epsilon nitrogen derivatized with levulinic acid.
  • a displaceable metal chelator linker was employed in combination with an amide-generating, acid labile Rink amide linker.
  • the displaceable metal chelator linker was designed to provide for attachment of the first peptide under mild conditions to a water-compatible spherical cellulose based support via a displaceable metal chelator linker, and the Rink linker was designed to generate an amide on the final full-length ligation product under acidic conditions following displacement of the metal chelator linker under mild conditions and its recovery from the support.
  • structure (2) contains the extreme C-terminal IL4 peptide segment (127- 129), and is alternatively denoted as having the amino acid sequence and linker attachment depicted below in SEQ ID NO:3 and SEQ ID NO:4.
  • IL4 ( Acm 127-129-X Rink HisTagV.
  • C Acm SS-X Rin HHHHHH (SEQ ID NO:4) where CSS corresponds to the C-terminal Cys-Ser-Ser residues 127-129 of human IL4 depicted in SEQ ID NO: 1 ;
  • C Aom is a cysteine having its side chain thiol protected with an Acm-protecting group;
  • X Rm is an amide-generating, acid cleavable Rink amide linker;
  • "HisTag” corresponds to a polyhistidine amino acid sequence (His-His-His-His) or (His-His- His-His-His-His) for binding metal ions such as nickel.
  • thioester peptides corresponding to IL4:( Acm 99-126 COSR ) (SEQ ID NO:5), IL4:( Acm 46-98 COSR ) (SEQ ID NO:6), IL4:(l-45 COSR ) a (SEQ ID NO:7) and IL4:(l-45 COSR ) b (SEQ ID NO:8) were prepared by highly optimized Boc SPPS and purified by HPLC before use in ligation reactions as described in Example 1. The amino acid sequences of the thioester peptides are depicted below.
  • C Acm Acm-protected cysteine
  • COSR thioester of a given C-terminal amino acid (e.g., a lysine-thioester in SEQ ID NO:5).
  • the peptide IL4:(l-45 COSR ) a was employed for oxime-forming linker approach, while the peptide IL4:(l-45 COSR ) (containing two internal Acm-protected cysteines) was employed for the metal chelator linker system to reduce side reactions with the free thiols.
  • EXAMPLE 3 SYNTHESIS OF PEPTIDE IL4:( AcM 127-129)-X SCA -K OXIME LINKER
  • Example 2 Structure (1) of Example 2 was accessed by Fmoc SPPS of the peptide (Fmoc)C Acm S Bzl S Bzl -X SCA K Mtt on a Leu-PAM resin (Applied Biosystems, 300 mg, 0.21 mmols), where the alpha-nitrogen of cysteine was protected with Fmoc, the thiol side chain cysteine was protected with Acm, the side chain hydroxyls of the serines were protected with benzyl (Bzl) groups, and the where epsilon nitrogen on the lysine side chain was protected with a Mtt protecting group.
  • the peptide chain was constructed using manual Fmoc cycles described in example one.
  • Structure (3) was made by reacting structure (1) dissolved in pH 3.5 sodium acetate buffer and an excess of AoA-Resin as follows. Approximately 40 mg of the ketone containing peptide of structure (1) was dissolved in 12 mL of a solution containing 20% acetonitrile and 0.1 M sodium acetate at pH 3.5, and then added to 9 ml of AoA-resin slurry from stock and allowed to react at room temperature for 16 hours. Synthesis of immobilized structure (3) was monitored by periodically removing small aliquots ( ⁇ 10 ⁇ l) of the supernatant and observing the disappearance of structure (1) from solution by HPLC. Oxime bond formation was typically found to be complete overnight.
  • EXAMPLE 6 SOLID PHASE CHEMICAL LIGATION OF IL4:( ACM 46-98 COSR ) TO IL4:(99-129)-X SCAL -K OXIME -SUPPORT
  • IL4 ( Acm 99-129)-X SCAL -K OXIME -Support was removed as described in Example 5. Following Acm removal, native chemical ligation of the thioester peptide IL4:( Acm 46-98 COSR ) with resin- bound IL4:(99-129)-X SCAL K OXIME -Support generated IL4:( Acm 46-129)-X SCAL K OXIME -Support.
  • the ligation reaction was performed by treating deprotected peptide resin (4.5 ml) with a solution of the incoming peptide (15 mg) in 0.5 ml of 6 M guanidinium chloride containing 300 mM sodium phosphate pH 7.5, 0.5 % thiophenol as described in Example 5.
  • this product When displaced from the resin with 1 M AoA as described above, this product also elutes as single peak in analytical HPLC, with an observed mass by ES-MS of 10,867 amu.
  • EXAMPLE 7 SOLID PHASE CHEMICAL LIGATION OF IL4:(l-45 COSR ) a TO IL4:(46-129)-X SCAL -K OXIME -SUPPORT
  • EXAMPLE 8 SYNTHESIS OF IL4:(FMOC)CYS ACM S ⁇ Bu S ⁇ Bu -X RlNK HISTAG LINKER
  • the resin bound peptide was treated with 20% piperidine in DMF 2 x 3 minutes. The resin was then washed with DMF.
  • 1 mmol of Fmoc- His was dissolved in 1.8 ml of 0.5 M HBTU in DMF and 0.5 ml of DIEA, and the resulting solution was added to the resin. Each coupling reaction was run for 40 minutes at room temperature. After each coupling, the resin washed with DMF, followed by removal of the Fmoc protecting group with 20% piperidine in DMF (2 X 3 minutes for each time), and finally washing the deprotected resin again with DMF.
  • the process for coupling the remaining histidine residues described above was repeated two or four more times to generate either a 4-His tag, or a 6-His tag.
  • the Rink-amide linker was added to complete assembly of the linker system as follows. 1 mmol of Fmoc-Rink amide linker was dissolved in 1.8 ml of HBTU and 0.5 ml of DIEA, and the resulting solution was added to the resin. The reaction was run for 40 minutes at room temperature. Then the resin was washed with DMF. The Fmoc protecting group was removed with 20% piperidine in DMF twice, three minutes for each time. Then the resin was washed with DMF.
  • IL4:(Fmoc)Cys Acm S tBu S tBu -X Rink HisTag from Example 8 was dissolved in 300 ⁇ L of 6 M guanidinium chloride, pH 7.5. The peptide solution was added to the Ni-NTA resin, then the resulting resin was washed with 6 M guanidinium chloride, pH 7.5 (50 mL), and the eluant was analyzed by HPLC. No IL4:(Fmoc)Cys Acm S tBu S tBu -X Rink HisTag peptide was detected, indicating that all of the peptide bound to the Ni-NTA-resin.
  • EXAMPLE 10 ESTABLISHMENT OF REVERSIBLE BINDING OF IL4:(FMOC)CYS ACM S ⁇ Bu S ⁇ Bu -X RlNK HISTAG LINKER
  • EXAMPLE 11 REMOVAL OF FMOC AND ACM PROTECTING GROUPS FROM IL4:(FMOC)CYS Ac S TBu S TBu -X RlNK HISTAG-NI-NTA-SUPPORT
  • the Acm-thiol protecting group of the product of Example 9 (structure (4)) was removed by adding 3 mL of 6.6 mg/mL Hg(OAc) 2 in 0.1 M acetate buffer, pH 6 to the peptide bound resin. The reaction was allowed to proceed for an hour.
  • the resin was washed with 0.1 M acetate buffer, pH 6.
  • the resin bound peptide was treated with 20% piperidine in DMF twice, three minutes for each time.
  • the resin was treated with the solution of 10% /?-mercaptoethanol in 6 M guanidinium chloride, pH 7.2 (2 mL) and very small amount of TCEP (0.3 mg) for 0.5 hour.
  • the deprotected peptide was then removed from the resin using the elution conditions described above in the step of reversible binding of His-Tagged peptides.
  • the different elution times were attributed to diastereomerism at the chiral center of the Rink linker.
  • the serine side chain tBu protecting groups were retained for convenience, as they were removed under the conditions for removal of the Rink linker from the final ligation product as described below.
  • EXAMPLE 12 SOLID PHASE CHEMICAL LIGATION OF IL4:( ACM 99-126 COSR ) TO IL4:(127-129)-X RlNK HISTAG-NI-NTA-SUPPORT
  • peptide segment IL4 ( Acm 99-126 COSR ) was ligated to structure (5) of Example 11 to give the first ligation product IL4:( Acm 99-129)- X Rink HisTag-Ni-NTA-Support as follows. 3 mg of peptide IL4:( Acm 99-126 COSR ) was dissolved in 200 ⁇ L of 6M guanidinium chloride, pH 7.5. The resulting solution was added to the resin and then 1 ⁇ L of thiophenol was added to the reaction solution. The reaction was run overnight and the resin was washed with of 6M guanidinium chloride, pH 7.2 (40 mL).
  • EXAMPLE 14 SOLID PHASE CHEMICAL LIGATION OF IL4:( Acm 46-98 COSR ) TO IL4:(99-129)-X Rink HisTag-Ni-NTA-Support
  • EXAMPLE 15 REMOVAL OF ACM PROTECTING GROUPS FROM IL4:( Acm 46- 129)-X Rink HisTag-Ni-NTA-Support [0109]
  • 3 mL of 6.6 mg/mL Hg(OAc) 2 in 0.1M acetate buffer, pH 6 were added to the peptide-bound resin. The reaction was allowed to proceed for an hour. The resin was washed with 0.1 M acetate buffer, pH 6. The resin was washed with 20 mL 0.1 M acetate buffer, pH 6.
  • the resin was treated with 2 mL of 10% /3-mercaptoethanol in 6 M guanidinium chloride, pH 7.2 and very small amount of TCEP (0.3 mg) for 0.5 hour and then washed with 6 M guanidinium chloride, pH 7.2.
  • Analytical HPLC found a peak of the product with a retention time of 26.3 min.
  • ES-MS analysis found a molecular weight of 10,868 amu for the product.
  • EXAMPLE 16 SOLID PHASE CHEMICAL LIGATION OF IL4:(l-45 COSR ) b TO IL4:(46-129)-X Rink HisTag-Ni-NTA-Support [0110]
  • the final on-resin peptide ligation was performed on the product of Example 15 as follows. 1.5 mg of the peptide thioester segment IL4:(l-45 COSR ) b was dissolved in 100 ⁇ L of 6M guanidinium chloride, pH 7.2. The resulting solution was added to the resin and then 0.5 ⁇ L of thiophenol was added to the reaction solution. The reaction was run for 3 hours.
  • a small analytical sample of the support bound, full-length ligation product was released from the support by displacement with imidazole-containing buffer as described above.
  • the eluant containing the released ligation product was readily separated from the resin, and HPLC analysis of the eluant showed a peak corresponding to the full-length ligation product with a retention time of 28.1 minutes and ES-MS analysis of the product found a molecular weight of 16,092 amu.
  • EXAMPLE 17 REMOVAL OF INTERNAL ACM PROTECTING GROUPS FROM IL4:(l-129)-X Rink HisTag-Ni-NTA-SUPPORT [0111] To remove two remaining internal Acm protecting groups from the last peptide ligation, 3 mL of 6.6 mg/mL Hg(OAc) 2 in 0.1 M acetate buffer, pH 6 was added to the peptide resin produced in Example 16. The reaction was allowed to proceed for one hour. The resin was washed with 20 mL 0.1 M acetate buffer, pH 6.
  • EXAMPLE 18 DISPLACEMENT OF FULL-LENGTH IL4:(l-129)-X Rink HisTag LIGATION PRODUCT FROM THE Ni-NTA-SUPPORT
  • Example 17 The full-length, product from Example 17 was released from the support by displacement with imidazole containing buffer as described above in Example 10. Specifically, the peptide resin product of Example 17 was treated with 2 mL of 10% ⁇ - mercaptoethanol in 6 M guanidinium chloride, pH 7.2 and very small amount of TCEP (0.3 mg) for a half hour and then washed with 6 M guanidinium chloride, pH 7.2, followed by release of the product using 0.5M imidazole solution in water. Analytical HPLC showed a peak with the retention time 28.2 minutes corresponding to the product (See, FIG.4A).
  • Full-length ligation product IL4 (1-129) from Example 19 was folded by dissolving 1 mg of the unfolded material in 1 ml of 6 M guanidinium chloride buffer with 100 mM Tris, 1 mM cysteine, and 0.2 mM cystine, at pH 8. The resulting solution was injected into a dialysis cassette (3500 MWCO, 0.1-0.5 ml). Then the cassette was put into 1000 ml of 0.5 M guanidinium chloride buffer with 100 mM Tris, 1 mM cysteine, and 0.2 mM cystine, at pH 8. The result was checked after overnight folding. Analytical HPLC showed a peak with the retention time 27.892 minutes for the folding product. ES-MS analysis found a molecular weight of 14,988 amu, indicative of the expected mass shift for disulfide formation. Circular dichroism (CD) also was performed following standard protocols and confirmed the formation of secondary structure, and thus folding of the protein.
  • CD Circular dichroism

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Abstract

La présente invention concerne des procédés et des compositions destinés à la ligation chimique en phase solide compatible avec un milieu aqueux. Les procédés et les compositions selon l'invention comprennent la ligation chimique de premier et deuxième polymères, ledit premier polymère étant attaché à un support au moyen d'un agent de liaison mobile. L'agent de liaison mobile peut être clivé ou bien déplacé dans des conditions aqueuses compatibles avec le retrait subséquent du produit de ligation dudit support solide. Les procédés et les compositions selon l'invention sont particulièrement utiles pour la ligation de peptides et de polypeptides sur un support solide. Le système de ligation selon l'invention peut être utilisé pour une grande diversité de molécules et peut par conséquent servir à la génération de polymères liés tels que des peptides, des polypeptides et d'autres polymères contenant des acides aminés.
EP04752449A 2003-05-22 2004-05-17 Ligation chimique en phase solide effectuee avec un agent de liaison mobile Withdrawn EP1631597A4 (fr)

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US8409526B2 (en) * 2010-12-09 2013-04-02 General Electric Company Cellulose substrates, compositions and methods for storage and analysis of biological materials
WO2014147124A1 (fr) 2013-03-21 2014-09-25 Sanofi-Aventis Deutschland Gmbh Synthèse de produits peptidiques contenant de l'hydantoïne
US10450343B2 (en) 2013-03-21 2019-10-22 Sanofi-Aventis Deutschland Gmbh Synthesis of cyclic imide containing peptide products
WO2015036481A1 (fr) 2013-09-11 2015-03-19 Danmarks Tekniske Universitet Lieur photolabile pour la synthèse en phase solide d'hydrazides et de pyranopyrazoles
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