Insoluble supports for solid phase synthesis

Abstract

The present invention relates to an insoluble support comprising a distal binding site, said support comprising a homogeneous polymer matrix and a construct covalently bound to said polymer matrix, wherein the construct comprises at least one branching agent selected from aminoalkanoic acids containing at least 2 amino groups and 3 to up to 10 carbon atoms, a cleavable linker providing a distal binding site, and at least one spacer coupled to the at least one branching agent by an amide bond.

Description

Technical Field

[0001] The present invention relates to an insoluble support comprising a construct for solid phase organic synthesis such as solid phase morpholino oligomer synthesis, solid phase oligonucleotide synthesis and solid phase peptide synthesis protocols, a method for preparing the insoluble support and a method for synthesizing a polypeptide using the insoluble support.

[0002] Background and purpose of the invention

[0003] Heterogeneous liquid-solid chemical reaction schemes are attractive for the synthesis of molecules containing repeating subunits. Liquid-solid schemes enable efficient separation of the solid and liquid phases, providing suitable conditions for the application of cycles that include reaction steps for the sequential, stepwise introduction (addition) of subunits. Liquid-solid reaction schemes have been successfully applied in the field of peptide, morpholino oligomer and oligonucleotide synthesis. During the synthesis process, the nascent molecule (such as a growing peptide, morpholino oligomer or oligonucleotide) is covalently bound to an insoluble support, providing conditions for the efficient removal of by-products by washing between the reaction steps of the cycle.

[0004] Solid phase peptide, morpholine and nucleotide synthesis is an established method for producing peptides. The peptides are synthesized in a single reaction vessel, thereby reducing process complexity. In addition, isolation and purification of intermediates are avoided or significantly reduced. Since excess reactants can be used, commercial phase yields and commercially attractive purities can be obtained before post-synthesis purification.

[0005] Peptides, morpholino oligomers or oligonucleotides are gradually synthesized on an insoluble support (solid phase). The insoluble support provides binding sites for the peptide to be synthesized. Although solid phase peptide synthesis provides significant advantages that are elaborated in detail herein, the reaction occurs on an insoluble support (phase) while the key reactants are present in the liquid phase. Therefore, solid phase synthesis inherently produces phase boundaries that need to be handled. The interaction of the solid and liquid phases is important, so the swelling and solvation of the solid phase are important properties. Swelling and solvation affect, for example, the diffusion and accessibility of reagents into the solid phase and the consumption of washing solutions.

[0006] Another property of the solid phase is the number of binding sites available for peptide synthesis, often expressed as loading. An increase in the number of binding sites may not automatically increase the capacity of the insoluble support and the purity of the peptide. An increase in the capacity of the insoluble support translates into an increase in yield (in terms of peptide output). However, binding sites must be available for peptide synthesis, and high loading is known to be detrimental to the purity and yield of the synthesized peptide, especially as the number of amino acids increases.

[0007] Inadequate swelling and solvation of the solid phase often results in poor accessibility of reaction sites and reduced reaction rates. Furthermore, during peptide synthesis, swelling and solvation can be affected as the solid phase is gradually altered by the coupling of amino acids to the nascent peptide bound to the solid phase.

[0008] To synthesize a target polypeptide, a combination of a solid support and a liquid phase is selected to optimize the yield and purity of the crude target polypeptide. Typically, this optimization translates into a synthesis specification that seeks to maximize the swelling and solvation of the solid phase. An increase in swelling also equates to an increase in the volume of the reaction composition.

[0009] Sometimes, the reactor volume of a solid phase peptide synthesis process can be a limiting factor. The present invention provides a novel insoluble support obtained by transforming a standard resin with a construct comprising readily available amino acids, which significantly increases reactor throughput while showing good crude peptide yields and commercially relevant purities. Thus, by applying the novel insoluble support in a given reactor, the reactor throughput can be significantly increased. The novel insoluble support provides the opportunity to increase polypeptide yields without increasing reactor volume.

[0010] For shorter polypeptides, i.e., polypeptides with a limited number of amino acids, e.g., below 10 amino acids, an increase in the number of binding sites on the resin (often referred to as the loading capacity [mmol binding sites / g resin]) generally translates into an increase in yield while still achieving satisfactory purity. As the number of amino acids in the polypeptide increases, an increase in loading does not necessarily translate into higher yields and useful purity. Quite the contrary, polypeptides with a large number of amino acids (e.g., above 30 amino acids) provide poor yields or are even impossible to synthesize without reducing the loading on the resin. Therefore, when synthesizing long polypeptides, a resin with a low loading capacity is usually selected.

[0011] Using the insoluble support of the present invention, complex, long polypeptides can be synthesized in high yield and commercially useful purity.

[0012] Lee et al. (Tetrahedron Letters 41 (2000) 7481-7485) disclose resins having a cross-linked polystyrene (PS) core and a poly(ethylene glycol) (PEG) shell prepared by suspension polymerization for solid phase synthesis. The PEG monomer used in the suspension polymerization is formed by reacting O,O'-bis(2-aminopropyl)polyethylene glycol 500 and methacryloyl chloride to provide PEG containing ammonium functional groups. Providing a charged PEG macromer under the polymerization conditions outlined by Lee provides polymer beads having a cross-linked polystyrene core and PEG macromers arranged on the surface of the beads to provide a PEG shell. The PS core is highly cross-linked with more than 4% divinylbenzene (DVB). The loading (substitution) of the core-shell resin can be increased by coupling a shell modified with lysine. The presence of the PEG shell covering the core and the high degree of cross-linking of the core prevents polypeptide synthesis within the core. All synthesis occurs at the shell.

[0013] Lee et al. (Tetrahedron Letters 42 (2001) 7443-7445) discloses dendrimer monomers based on tris(hydroxymethyl)aminomethane (Tris) and gel-type TentaGel resins for increasing the loading capacity of core-shell resins as disclosed in Lee (2000). Lee 2001 fails to teach the use of monomers based on aminoalkanoic acids (such as diaminoalkanoic acids) as repeating units for support dendrimerization. In addition, Lee (2001) does not disclose the use of spacer molecules between the support and the dendrimer monomers or between the dendrimer monomers.

[0014] Chan et al. (Tetrahedron Letters 40 (1999) 4909-4912) disclosed the synthesis of triamino acids and the use of such molecules to generate dendrimers on resins, thereby increasing the loading capacity of the resin. Chan et al. did not disclose the use of spacer molecules between PS resins or between generations of triamino acid units.

[0015] The present invention provides an insoluble support which increases the yield of a product (polypeptide) of commercially acceptable purity at a given reaction volume, in particular the reactor throughput (the mass of the product after each swelling and cleavage of the insoluble support). Thus, a homogeneous polymer matrix, such as a modified solid phase peptide synthesis according to the present invention, i.e. by providing a construct of at least one branching agent, thereby providing a modified polymer matrix, an insoluble support, significantly increases the yield of the product (polypeptide) and the reactor throughput at a commercially acceptable purity at a given reaction volume. The insoluble support of the present invention also enables the synthesis of long (complex) polypeptides, such as polypeptides having more than, for example, 25 amino acids, with good yield and purity.

[0016] One object of the present invention is to increase the throughput of a target polypeptide in a given solid phase peptide synthesis reactor (mass of crude peptide after cleavage as a function of the volume of resin containing the target polypeptide [mass / volume g / L]).

[0017] Another object is to provide an insoluble support (polymer insoluble support) which enables an increase in capacity / productivity while reducing a disproportionate increase in swelling of the insoluble support, without affecting the purity of the synthesized peptide.

[0018] Another object is to provide an insoluble support obtained by modifying a base resin, thereby increasing the capacity / productivity for a given reactor volume and simultaneously reducing the solvent consumption in absolute terms and / or per unit of target polypeptide synthesized, especially when compared to an unmodified base resin.

[0019] Another object is to provide an insoluble support, preferably with reduced solvent consumption per unit of peptide (eg, per mmol of peptide) compared to an unmodified insoluble support.

[0020] Yet another object is to provide insoluble supports which reduce swelling and / or reduce solvent consumption (per target peptide synthesized unit) while at least maintaining or significantly increasing capacity / yield compared to unmodified insoluble supports.

[0021] Another object was to provide insoluble supports (modified resins) which significantly increase the yield of (crude) peptides without significantly decreasing the purity for a given reactor volume (compared to unmodified resins).

[0022] Another object is to provide an insoluble support for the synthesis of long polypeptides with good yield and purity, wherein the insoluble support is formed from a commercially available resin and modified using SPPS and readily available compounds such as amino acids.

[0023] Another object is to provide an insoluble support (modified resin) whose throughput (mass of crude peptide after cleavage as a function of the volume of resin containing the target polypeptide [mass / volume g / L]) is increased compared to an unmodified resin, while retaining a commercially relevant purity of the (crude) target polypeptide.

[0024] Yet another objective is to increase the throughput (mass of crude peptide after cleavage as a function of the volume of resin containing the target polypeptide) of the insoluble support (modified resin) compared to an unmodified polymer matrix / resin while maintaining a commercially relevant purity of the (crude) target polypeptide while also reducing the final volume of the polymer matrix / resin.

[0025] As mentioned above, swelling is an important characteristic of the insoluble support used in peptide synthesis. In general, the increase of swelling is conducive to the diffusion of reagents (for example, amino acids) into the insoluble support, and may also increase the availability of binding sites. However, the increase of swelling will increase required reactor volume and solvent consumption conventionally. The invention provides an insoluble support (modified insoluble support), which increases the flux under a given reactor volume and reduces solvent consumption, while increasing capacity / productivity and flux (the target peptide of cracking is a function of the final swelling volume of the insoluble support before cracking) compared with an unmodified polymer matrix.

[0026] The embodiment of the insoluble support according to the present invention is an effective measure to increase the capacity of a given reactor volume. In addition, the embodiment of the insoluble support increases the capacity of solid phase peptide synthesis without increasing the reaction volume (volume of reaction vessel) or even reducing the reaction volume, while also reducing solvent consumption while keeping the same conventional polymer matrix.

[0027] Insoluble supports are preferably used as solid phases in solid phase peptide synthesis (SPPS), such as Fmoc / tBu or Boc SPPS strategies. The use of insoluble supports in SPPS significantly increases the throughput of target peptides relative to unmodified base resins / polymer matrices.

[0028] The insoluble support of the present invention can also be successfully applied to aqueous solid phase peptide synthesis schemes, i.e., the solvent used is aqueous. Therefore, another object of the present invention is to provide SPPS conditions that can reduce organic solvents and / or replace organic solvents with aqueous-based solvents. Summary of the invention

[0029] One embodiment of the present invention relates to an insoluble support comprising a construct having an increased number of available binding sites compared to an unmodified polymer matrix. The insoluble support of the present invention is successfully applied to solid phase synthesis schemes, particularly solid phase peptide synthesis. In addition, the present invention also relates to methods for preparing insoluble supports by modified polymer matrices, and methods for synthesizing peptides using insoluble supports or constructs.

[0030] More specifically, one embodiment relates to an insoluble support (resin) in the form of particles comprising a distal binding site, the support comprising a homogeneous polymer matrix and a construct covalently bound to the polymer matrix, wherein the construct comprises at least one branching agent selected from aminoalkanoic acids containing at least 2 amino groups and 3 to up to 10 carbon atoms, a cleavable linker, and at least one spacer coupled to the at least one branching agent via an amide bond, the cleavable linker providing the distal binding site.

[0031] Another embodiment relates to a method of forming an insoluble support in the form of particles comprising secondary binding sites, the insoluble support comprising a homogeneous polymer matrix and a construct, the construct comprising a cleavable linker and at least one spacer, the method comprising providing a polymer matrix in the form of particles comprising a primary binding site, wherein the construct is formed by a solid phase synthesis scheme, the solid phase scheme comprising at least one reaction step, wherein a branching agent selected from an amino-protected aminoalkanoic acid comprising at least 2 amino groups and 3 to up to 10 carbon atoms is coupled to any of the primary binding sites, the spacer or the branching agent of the polymer matrix via an amide bond, the scheme further comprising a reaction step of coupling the cleavable linker directly to the branching agent or to it through one or more spacers.

[0032] Another embodiment relates to a method for synthesizing a polypeptide, morpholino oligomer or oligonucleotide using a solid phase peptide synthesis protocol comprising the use of an insoluble support disclosed herein.

[0033] definition

[0034] Polymer matrix: The insoluble support is formed of a polymer matrix that is modified by a construct covalently attached to the polymer matrix. The polymer matrix typically comprises binding sites, also referred to as primary binding sites. The construct is covalently attached to the binding sites of the polymer matrix. The polymer matrix may be modified to comprise any type of binding site suitable for coupling the construct. It should be noted that the primary binding sites of the polymer matrix are consumed during the formation of the insoluble support described in the present invention. Therefore, the primary binding sites of the polymer matrix are used to covalently bind the construct. Ideally, all primary binding sites of the polymer matrix are consumed by the covalent coupling of the construct, which indicates that the insoluble support of the present invention does not have a polymer matrix with primary binding sites. The polymer matrix is ​​typically a polymer network, typically comprising polymers cross-linked to a certain extent, the degree of cross-linking providing structural integrity of the polymer matrix particles while enabling significant solvation of the polymer network. The homogeneous polymer matrix is ​​preferably selected from a polymer matrix comprising binding sites distributed throughout the polymer matrix. Typically, the binding sites of the polymer matrix are substantially uniformly distributed in the matrix (on). The polymer matrix is ​​preferably selected from a homogeneous polymer matrix. The polymer matrix is ​​typically in the form of particles. The polymer matrix is ​​preferably insoluble. The size of the particles can be from about 1 μm to up to about 2000 μm, and typically from about 20 μm to up to about 500 μm. The polymer matrix can be formed by emulsion polymerization. A particularly useful homogeneous polymer matrix is ​​a styrene-based matrix crosslinked with a crosslinking agent, suitably divinylbenzene (DVB). The styrene-based homogeneous polymer matrix is ​​typically crosslinked at a crosslinking agent content of less than 4.0 wt. %, preferably less than 3.5 wt. %, preferably less than about 3.0 wt. %, suitably about 0.5 to up to 2.5 wt. %.

[0035] Insoluble Support: The insoluble support comprises the polymer matrix and the construct and constitutes the solid phase in any liquid-solid synthesis protocol, such as solid phase peptide synthesis.

[0036] Construct: A construct is a molecule that increases the number of binding sites (primary binding sites) of a polymer matrix. The construct is a branched molecule, which can be characterized as a dendrimer or a dendritic unit (dendron). According to one aspect, the construct can be called a branched polypeptide. The construct itself can also serve as an anchor molecule in liquid phase peptide synthesis (LPPS). The construct comprises a cleavable linker, at least one branching agent and at least one spacer. According to one aspect, the construct consists of a cleavable linker, at least one branching agent and at least one spacer, that is, the construct contains only compounds selected from cleavable linkers, branching agents and spacers. According to another aspect, the cleavable linker, at least one branching agent and at least one spacer are all coupled to each other through an amide bond. The simplest construct comprises one branching agent. The construct is preferably formed by repeatedly coupling a branching agent to previous generations of branching agents (repeated branching cycles), thereby obtaining a highly ordered branched macromolecule, whose binding sites continue to increase with the increase in the number of construct branching agents. Constructs can be formed by divergent reaction schemes, convergent reaction schemes or a combination of divergent and convergent reaction schemes. Preferably, insoluble constructs, i.e., the constructs are formed by divergent reaction schemes (such as solid phase organic synthesis schemes, and preferably solid phase peptide synthesis schemes), wherein all compounds constituting the constructs, especially cleavable linkers, branching agents, spacers, have functional groups such that the compounds can be coupled to each other via amide bonds.

[0037] Branching Agents: Branching agents are molecules responsible for the specific structure of the construct and the ability of the construct to increase the number of polymer matrix binding sites. Branching agents contain at least three binding sites. In its simplest form, the construct contains only one branching agent, at least one spacer coupled to the branching agent via an amide bond and a cleavable linker coupled to the branching agent.

[0038] Distal branching agent: The distal branching agent is the branching agent that is furthest from the polymer matrix. A secondary or distal binding site is provided by the distal branching agent through a cleavable linker coupled to the available binding site of the distal branching agent.

[0039] Proximal Branching Agents: Proximal branching agents are branching agents of the construct that are bound to the polymer matrix.

[0040] If a construct has only one branching agent, that one branching agent can be designated as either a proximal or distal branching agent.

[0041] Intermediate branching agents are branching agents located between distal and proximal branching agents. Intermediate branching agents require at least three generations (layers) of branching agents.

[0042] Primary, secondary, tertiary, etc. branching agents; primary branching agents, secondary branching agents, etc. refer to branching agents relative to other branching agents of the same structure. Primary branching agents or first generation branching agents (first layer branching agents) are branching agents that are coupled to the base matrix without any other branching agents in between. Secondary branching agents or second generation branching agents (second layer branching agents) are coupled to primary branching agents. Tertiary branching agents or third generation branching agents (third layer branching agents) are coupled to secondary branching agents. In other words, tertiary branching agents are coupled to the polymer matrix through the other two branching agents (secondary branching agents and primary branching agents). Primary branching agents, secondary branching agents, tertiary branching agents, etc. may also be referred to as primary (first), secondary layers or generations of branching agents.

[0043] The distal branching agent is the branching agent of the construct to which the cleavable linker is attached. The generation containing the distal branching agent is the generation with the highest integer.

[0044] The proximal branching agents are bound to the polymer matrix with or without one or more spacers.Proximal branching agents are first generation branching agents.

[0045] The presence of branching agent intermediates (one or more generations) requires at least three generations of branching agents. Intermediate generations exist between distal branching agents and proximal branching agents.

[0046] Coupling: When referring to the coupling of a molecule (e.g., branching agent, cleavable linker and spacer) to another part (e.g., branching agent, spacer or polymer matrix), such coupling can be direct coupling or coupling of the molecule to the other part through any number and type of intermediate molecules (referred to herein as spacers or spacer molecules). Coupling herein generally refers to covalent coupling. The terms 'binding' and 'coupling' are used interchangeably.

[0047] Spacer; A spacer or spacer molecule is a molecule that does not form part of the definition of a branching agent. A spacer typically contains (only) two binding sites. Thus, a spacer cannot provide a branching point. Although the binding site of the spacer can be selected from a variety of functional groups, the binding site of the spacer is preferably selected so that an amide bond is formed when the spacer is coupled to the branching agent.

[0048] Binding site: A binding site (functional group or reactive site) is a site on a molecule or polymer matrix, especially a branching agent, spacer, cleavable linker, that can be used to chemically react with a binding site on another molecule. A binding site can be considered a functional group that has the ability to form a covalent bond with a molecule other than the one containing the binding site (e.g., branching agent). Two binding sites will typically form a covalent bond.

[0049] Primary (first) binding site: A primary binding site is a binding site on a polymer matrix that can be used to covalently couple a construct to provide an insoluble support according to the invention suitable for solid phase peptide synthesis.

[0050] Secondary (second) or distal binding site: A secondary or distal binding site is a site provided by the construct, more specifically, by a cleavable linker covalently bound to the distal branching agent binding site, optionally with one or more spacers between the linker and the distal branching agent binding site. The secondary or distal binding site provides conditions for covalently anchoring the peptide during solid phase peptide synthesis.

[0051] Cleavable linker: A cleavable linker is a molecule covalently coupled to the growing polypeptide, further facilitating or enabling cleavage of the peptide from the construct. The construct comprises a cleavable linker covalently linked to the binding site of the distal branching agent directly or through one or more spacers. Preferably, the cleavable linker is coupled to the branching agent or the spacer via an amide bond.

[0052] Whenever an amino acid is mentioned, any related amino acid derivatives are also contemplated. Summary of the invention

[0054] One embodiment of the invention relates to an insoluble support. Another embodiment relates to a method of forming an insoluble support. Additional embodiments relate to solid phase peptide synthesis protocols for synthesizing polypeptides, morpholino oligomers or oligonucleotides, including the use of an insoluble support.

[0055] Insoluble support preferably serves as solid phase in solid phase peptide synthesis. According to one aspect, insoluble support is used as the solid phase in solid phase chemical synthesis scheme. Specifically, insoluble support is used as solid phase in solid phase morpholino oligomer synthesis, solid phase oligonucleotide synthesis and solid phase peptide synthesis. More specifically, insoluble support comprises binding site (also referred to as secondary binding site or distal binding site herein) and polymer matrix, wherein the distal binding site is provided by construct, and the construct comprises at least one branching agent, cleavable linker and at least one spacer. Branching agent is between secondary binding site and polymer matrix. Polymer matrix is ​​preferably a particle form representing solid phase.

[0056] Polymer matrix generally comprises binding site, also referred to herein as primary binding site.Polymer matrix preferably comprises binding site suitable for construct coupling or as binding site (anchor point) synthesized as construct.The type of polymer matrix depends on solid phase synthesis application.Polymer matrix can comprise polymer, such as comprising polymer network, for example cross-linked polymer network.Generally, polymer matrix is ​​based on commercially available polymer matrix (resin), and it is arranged to be used as solid phase in peptide or nucleotide synthesis.This construct is the entity that increases the number of binding sites of polymer matrix, makes (polymer matrix) primary binding site provide multiple secondary binding sites (at least two) by covalently bonded to the construct of primary binding site.The construct covalently bonded to polymer matrix comprises at least branching agent, and described branching agent is directly bonded to the binding site of polymer matrix or by any number and any type of molecule (also referred to as spacer or spacer molecule) between the primary binding site of branching agent and polymer matrix is ​​covalently bonded to primary binding site.

[0057] The construct can be represented as a branched molecule. According to one aspect, the construct can be referred to as a branched molecule comprising amino acids, or as a branched peptide / polypeptide. The construct comprises at least one branching agent, a cleavable linker and at least one spacer. A branching agent is a molecule comprising at least three binding sites. A spacer is a molecule that cannot act as a branching agent. Therefore, a spacer is a molecule that preferably has two binding sites or preferably has exactly two binding sites.

[0058] The insoluble support of the present invention is defined (claimed) in multiple ways (aspects) herein to fully construct the present invention. However, all aspects of the insoluble support relate to the same basic concept, providing an insoluble support comprising a construct comprising at least one branching agent, a cleavable linker and at least one spacer, increasing the number of binding sites and generally providing a specific geometric conformation.

[0059] Some aspects of the insoluble support are defined by the characterization of the final insoluble support. Some aspects of the insoluble support are defined in part by introducing features that are more related to how the insoluble support is formed.

[0060] Examples of simple, non-complex constructs include only one branching agent, such as a diaminoalkanoic acid (e.g., lysine, ornithine, etc.), a suitable cleavable linker coupled to the branching agent to provide a secondary (distal) binding site, such as glycine as a spacer, which is coupled to the branching agent via amide bonding and the binding site of the polymer matrix.

[0061] One aspect of the present invention relates to an insoluble support in particulate form for solid phase peptide synthesis, solid phase morpholino oligomer synthesis, solid phase oligonucleotide synthesis, preferably for solid phase peptide synthesis, the insoluble support providing a binding site (distal or secondary binding site), the insoluble support comprising a homogeneous polymer matrix and a construct, the construct being covalently bound to the polymer matrix, the construct comprising at least one branching agent, a cleavable linker and at least one spacer, the at least one branching agent being selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms, the at least one spacer being coupled to the at least one branching agent via an amide bond, the cleavable linker providing a distal binding site and being covalently bound to the at least one branching agent.

[0062] According to another aspect, the insoluble support can be obtained by providing a homogeneous polymer matrix comprising primary binding sites and by application of cyclic synthesis steps comprising coupling of a branching agent (such as an aminoalkanoic acid comprising at least 2 amino groups and 3 to up to 10 carbon atoms) and at least one spacer and finally coupling a cleavable linker to the binding site of a distal branching agent (or multiple distal branching agents), thereby forming a construct providing at least two secondary / distal binding sites.

[0063] According to one embodiment, the branching agent is selected from aminoalkanoic acids comprising at least 2 but not more than 3 amino groups and 3 to at most 10 carbon atoms, suitably 3 to at most 8 carbon atoms, suitably 4 to at most 8 carbon atoms.

[0064] According to another embodiment, the branching agent is selected from diaminoalkanoic acids comprising 3 to at most 10 carbon atoms, suitably 3 to at most 8 carbon atoms, suitably 4 to at most 8 carbon atoms.

[0065] The number of carbon atoms in any aminoalkanoic acid disclosed herein can range from 3, 4, 5, 6, up to 10, up to 9, up to 8, up to 7, and any combination of upper and lower limits.

[0066] According to another embodiment, the branching agent is selected from 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid, suitably, the branching agent is selected from 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid.

[0067] According to one embodiment, all branching agents of the construct are chosen from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms.

[0068] According to one embodiment, all branching agents of the construct are identical and are chosen from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms.

[0069] According to one embodiment, the branching agent of the construct may be any combination of branching agents selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms.

[0070] One embodiment relates to an insoluble support wherein the number of branching agents in the construct, d, is given by the formula d(n)=2 n -1, where n represents the generation of the branching agent and n is a positive integer.

[0071] One embodiment relates to an insoluble support wherein the number of distal binding sites l of the construct is given by the formula l(n)=2 n Given as , where n represents the generation of the branching agent and n is a positive integer.

[0072] One embodiment relates to an insoluble support wherein the number of branching agents in the construct, d, is given by the formula d(n)=2 n -1, the number of distal binding sites l of the construct is given by the formula l(n) = 2 n Given as , where n represents the generation of the branching agent and n is a positive integer.

[0073] According to one embodiment, the number n of generations of branching agents of the construct is preferably 1 to 10, suitably 1 to 5.

[0074] According to another embodiment, the number n of generations of branching agents of the construct is preferably 2 to 10, suitably 2 to 5.

[0075] The construct comprises at least one spacer. If the construct contains only one branching agent, at least one spacer may be located between the branching agent and the polymer matrix. Alternatively, the spacer may be located between each of the two or more cleavable linkers and the branching agent.

[0076] If the construct comprises multiple generations of branching agents, i.e. two or more generations, the spacer is typically located between any of the following positions: between branching agents of any number of generations, between a cleavable linker and a (distal) branching agent, between a (proximal) branching agent and the polymer matrix. Any number of spacers may be present at any position.

[0077] According to one aspect, at least one spacer is located between the branching agents. The spacer between the branching agents represents that the construct comprises at least two generations of branching agents. An example is a construct comprising three lysine branching agents, three glycine spacers and four cleavable linkers. Two glycine spacers are located between the first generation lysine and two second generation lysines. More specifically, each glycine spacer is covalently connected to the ε and α amines of the first generation lysine by an amide bond. In addition, the two second generation lysines are covalently connected to each of the two glycine spacers by an amide bond. In addition, the other third glycine spacer can be located between the first generation lysine and the homogeneous polymer matrix by two amide bonds. Four cleavable linkers are coupled to the ε and α amines of the two second generation lysines by four amide bonds.

[0078] According to one aspect, a plurality of spacers are located between the branching agents. If three or more spacers are located between the branching agents, between the branching agent and the polymer matrix, and / or between the cleavable linker and the branching agent, at least one spacer is preferably coupled to two spacers via two amide bonds.

[0079] According to one embodiment, the one or more spacers are coupled to the branching agent via an amide bond.

[0080] The polymer matrix is ​​modified with a branching agent, such as an aminoalkanoic acid containing at least 2 amino groups and 3 to up to 10 carbon atoms, to convert one binding site (primary binding site) of the polymer matrix into two or more binding sites (secondary or distal binding sites).

[0081] Distal binding sites are binding sites provided after modification of the polymer matrix.

[0082] The distal binding site is a binding site useful for synthesis in solid phase synthesis, such as solid phase peptide synthesis.

[0083] The number of additional secondary binding sites is related to the number of branching agents included in the construct. More importantly, the increase in secondary binding sites is preferably achieved by continuously coupling branching agents to other branching agents, wherein one branching agent, i.e., a proximal branching agent, is optionally coupled to a polymer matrix via a spacer, thereby generating multiple layers or generations of branching agents. Such layers may be referred to as primary (first), secondary (second), tertiary (third), quaternary (fourth) and other layers or generations. In aspects of the present invention, branching agents represent primary, secondary, tertiary, quaternary branching agents. For example, a tertiary branching agent forms a tertiary (third) layer or third generation.

[0084] One aspect of the present invention relates to an insoluble support in the form of particles, comprising a homogeneous polymer matrix and a construct, the construct being covalently bound to the polymer matrix, the construct comprising at least one branching agent, a cleavable linker and at least one spacer; the branching agent is selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms, wherein at least one branching agent is a distal branching agent comprising a binding site, the cleavable linker is covalently bound to the binding site of the distal branching agent directly or through one or more spacers, each cleavable linker providing a distal binding site; at least one spacer is located at any of the following positions: between branching agents, between the polymer matrix and the branching agent, between the cleavable linker and the binding site of the distal branching agent.

[0085] One aspect of the present invention relates to an insoluble support in the form of particles, comprising a polymer matrix and a construct, the construct being covalently bound to the polymer matrix, the construct comprising at least two branching agents, a cleavable linker and a spacer, one branching agent representing a distal branching agent, one branching agent representing a proximal branching agent, and the optional branching agent representing an intermediate branching agent; the branching agent is selected from diaminoalkanoic acids containing 3 to up to 10 carbon atoms, the distal branching agent comprising a binding site, the cleavable linker being covalently bound to the distal branching agent directly or through one or more spacers The spacer is located at any of the following positions: between the branching agents, between the polymer matrix and the branching agent, between the linker and the binding site of the distal branching agent, and the distal branching agent is only bound to one branching agent selected from the optional intermediate branching agent or the proximal branching agent; the optional intermediate branching agent is bound to three branching agents selected from the distal branching agent, the optional intermediate branching agent and the proximal branching agent, and the proximal branching agent is bound to two branching agents selected from the distal branching agent and the optional intermediate branching agent.

[0086] One aspect of the present invention relates to an insoluble support in the form of particles, comprising a polymer matrix and a construct, the construct being covalently bound to the polymer matrix, the construct comprising at least three branching agents, a cleavable linker and a spacer; the branching agent represents a distal branching agent, an intermediate branching agent and a proximal branching agent; the branching agent has three binding sites, the distal branching agent is selected from diaminoalkanoic acids containing 3 to up to 10 carbon atoms, the cleavable linker is covalently bound to the binding site of the distal branching agent directly or through one or more spacers, each cleavable linker provides a distal binding site; the spacer is located at any of the following positions: between branching agents, between the polymer matrix and the branching agent, between the linker and the binding site of the distal branching agent, and the distal branching agent is bound to only one intermediate branching agent; the intermediate branching agent is bound to three branching agents selected from the distal branching agent, the intermediate branching agent and the proximal branching agent, and the proximal branching agent is bound to two intermediate branching agents.

[0087] One aspect of the present invention relates to an insoluble support in the form of particles, comprising a homogeneous polymer matrix and a construct, the construct being covalently bound to the polymer matrix, the construct comprising a branching agent, two cleavable linkers and at least one spacer; the branching agent is selected from diaminoalkanoic acids comprising 3 to up to 10 carbon atoms, which comprises three binding sites, two cleavable linkers being covalently bound to the binding site (amino group) of the branching agent directly or through one or more spacers, each cleavable linker providing a distal binding site, the branching agent being bound to the polymer matrix; at least one spacer being located at any of the following positions: between the polymer matrix and the branching agent, between the cleavable linker and the binding site of the distal branching agent.

[0088] One aspect of the present invention relates to an insoluble support in the form of particles, which comprises a polymer matrix and a construct, wherein the construct is covalently bound to the polymer matrix, wherein the construct comprises three branching agents, four cleavable linkers and a spacer; all branching agents are selected from diaminoalkanoic acids containing binding sites and comprising 3 to at most 10 carbon atoms, two branching agents are second-generation distal branching agents containing binding sites, and one branching agent is a first-generation proximal branching agent, the cleavable linker is covalently bound to the binding site of the distal branching agent directly or by means of one or more spacers, and each cleavable linker provides a distal binding site; the first-generation proximal branching agent is bound to two second-generation distal branching agents, the first-generation proximal branching agent is bound to the matrix, and the spacer is located at any of the following positions: between the branching agents, between the polymer matrix and the branching agent, and between the linker and the binding site of the distal branching agent.

[0089] One aspect of the present invention relates to an insoluble support in the form of particles comprising a polymer matrix and a construct covalently bound to the polymer matrix, the construct comprising seven (7) branching agents, eight cleavable linkers and a spacer; all branching agents are selected from diaminoalkanoic acids containing from 3 to up to 10 carbon atoms, four (4) branching agents are third generation distal branching agents containing a binding site, two branching agents are second generation intermediate branching agents, one branching agent is a proximal branching agent, the cleavable linkers are directly or through a spacer or a plurality of spacers are covalently bound to the binding site of the distal branching agent, each cleavable linker provides a distal binding site, each second-generation intermediate branching agent is bound to two third-generation distal branching agents and to one first-generation intermediate branching agent, the first-generation intermediate branching agent is bound to two second-generation intermediate branching agents and a first-generation proximal branching agent, and the first-generation proximal branching agent is bound to the matrix; the spacer is located at any of the following positions: between the branching agents, between the polymer matrix and the branching agent, and between the linker and the binding site of the distal branching agent.

[0090] One aspect of the invention relates to an insoluble support in the form of particles comprising a polymer matrix and a construct covalently bound to the polymer matrix, the construct comprising fifteen (15) branching agents, sixteen (16) cleavable linkers and a spacer; all branching agents are selected from diaminoalkanoic acids comprising from 3 to up to 10 carbon atoms, eight (8) branching agents are fourth generation distal branching agents comprising a binding site, four (4) branching agents are third generation intermediate branching agents, two branching agents are second generation intermediate branching agents, one branching agent is a first generation proximal branching agent, the cleavable linkers are directly or through one or more A plurality of spacers are covalently bound to the binding site of the distal branching agent, each cleavable linker provides a distal binding site, each third-generation intermediate branching agent is bound to two fourth-generation distal branching agents and one second-generation intermediate branching agent, each second-generation intermediate branching agent is bound to two third-generation intermediate branching agents and one first-generation intermediate branching agent, the first-generation intermediate branching agent is bound to two second-generation intermediate branching agents and a first-generation proximal branching agent, and the first-generation proximal branching agent is bound to the matrix; the spacer is located at any of the following positions: between the branching agents, between the polymer matrix and the branching agent, and between the linker and the binding site of the distal branching agent.

[0091] One aspect of the invention relates to an insoluble support in the form of particles comprising a polymer matrix and a construct covalently bound to the polymer matrix, the construct comprising thirty-one (31) branching agents, thirty-two (32) linkers and optional spacers; all branching agents are selected from diaminoalkanoic acids comprising from 3 to up to 10 carbon atoms, sixteen (16) branching agents are fifth generation distal branching agents comprising a binding site, eight (8) branching agents are fourth generation intermediate branching agents, four (4) branching agents are third generation intermediate branching agents, two (2) branching agents are second generation intermediate branching agents, and one branching agent is a first generation proximal branching agent. A branching agent, a linker is covalently bonded to a distal branching agent binding site directly or through one or more spacers, each linker provides a distal binding site, each fourth-generation intermediate branching agent is bonded to two fifth-generation distal branching agents and one third-generation branching agent, each third-generation intermediate branching agent is bonded to two fourth-generation intermediate branching agents and one second-generation intermediate branching agent, each second-generation intermediate branching agent is bonded to two third-generation intermediate branching agents and one first-generation proximal branching agent, and the proximal branching agent is bonded to a matrix; the spacer is located at any of the following positions: between the branching agents, between the polymer matrix and the branching agent, and between the linker and the binding site of the distal branching agent.

[0092] According to another embodiment, the construct does not contain arginine.

[0093] One aspect of the invention relates to an insoluble support in the form of particles comprising a secondary binding site and a polymer matrix, wherein the secondary binding site is provided by a cleavable linker bound to a construct comprising at least one branching agent, a cleavable linker selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to up to 10 carbon atoms, and a spacer, wherein the branching agent is located between the secondary binding site and the polymer matrix, and the construct is covalently coupled to the polymer matrix.

[0094] Another aspect relates to an insoluble support in particulate form comprising a construct, a distal binding site and a polymer matrix, the construct being bound to the polymer matrix, the construct comprising a cleavable linker and at least two branching agents, the branching agents being selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to up to 10 carbon atoms, wherein (provided) the at least two branching agents comprise a different number of binding sites, one branching agent is a distal branching agent providing a distal binding site, the cleavable linker is covalently bound to the binding site of the distal branching agent directly or through one or more spacers, and each cleavable linker provides a distal binding site, and at least one spacer is located between the branching agents, between the polymer matrix and the branching agent, and between the distal branching agent and the cleavable linker.

[0095] Yet another aspect relates to an insoluble support (resin) in particulate form for solid phase peptide synthesis, solid phase morpholino oligomer synthesis and solid phase oligonucleotide synthesis, preferably solid phase peptide synthesis, comprising a distal binding site, the support comprising a homogeneous polymer matrix and a construct, the construct being covalently bound to the polymer matrix, wherein the construct comprises at least one branching agent selected from diaminoalkanoic acids comprising 3 to up to 10 carbon atoms, a cleavable linker and at least one spacer coupled to the at least one branching agent via an amide bond, the cleavable linker providing the distal binding site, and the construct being selected from branched molecules of the formula:

[0096] (A) [polymer matrix]-BA(1)-LK2;

[0097] (B) [Polymer matrix]-BA(1)-BA(2)2-LK4;

[0098] (C) [Polymer matrix]-BA(1)-BA(2)2-BA(3)4-LK8;

[0099] (D)[Polymer matrix]-BA(1)-BA(2)2-BA(3)4-BA(4)8-LK 16 ;

[0100] (E)[Polymer matrix]-BA(1)-BA(2)2-BA(3)4-BA(4)8-BA(5) 16 -LK 32 ;

[0101] wherein BA is a branching agent, an integer represents the generation of the branching agent, and LK represents a cleavable linker.

[0102] According to a further aspect, the construct is selected from the group consisting of:

[0103] (A) [Polymer matrix]-SPC-BA(1)-LK2;

[0104] (B) [Polymer matrix]-BA(1)-SPC-BA(2)2-LK4;

[0105] (C) [Polymer matrix] -BA(1)-SPC-BA(2)2-SPC-BA(3)4-LK8;

[0106] (D) [Polymer matrix] -BA (1) -SPC-BA (2) 2-SPC-BA (3) 4-SPC-BA (4) 8-LK 16 ;

[0107] (E)[Polymer matrix]-BA(1)-SPC-BA(2)2-SPC-BA(3)4-SPC-BA(4)8-SPC-BA(5) 16 -LK 32 ;

[0108] wherein BA is a branching agent and the integer represents the generation of the branching agent, LK represents a cleavable linker, and SPC represents any number of spacers (from at least one spacer) and wherein optionally any number of spacers (at least one spacer) may be located between the polymer matrix and the first generation branching agent BA (1), and between the cleavable linker and the last generation branching agent (distal branching agent).

[0109] Yet another aspect relates to an insoluble support (resin) in particulate form for solid phase morpholino oligomer synthesis, solid phase oligonucleotide synthesis and solid phase peptide synthesis, preferably solid phase peptide synthesis, comprising a distal binding site, the support comprising a homogeneous polymer matrix and a construct, the construct being covalently bound to the polymer matrix, wherein the construct comprises at least one branching agent selected from diaminoalkanoic acids comprising 3 to up to 10 carbon atoms, a cleavable linker and at least one spacer coupled to the at least one branching agent via an amide bond, the cleavable linker providing the distal binding site, and the construct being selected from the branched molecule:

[0110] A)

[0111]

[0112] B)

[0113]

[0114] C)

[0115]

[0116] D)

[0117]

[0118] E)

[0119]

[0120] wherein BA is a branching agent and the integer represents the generation of the branching agent, LK represents a cleavable linker, and wherein optionally any number of spacers (at least one spacer) may be located between the polymer matrix and the first generation branching agent BA (1), between branching agents, and between the cleavable linker and the last generation branching agent (distal branching agent).

[0121] The construct of structure A contains only one first generation proximal branching agent. The construct of structure B contains three kinds of branching agents, one first generation proximal branching agent and two second generation distal branching agents. The construct of structure B has two layers or two generations of branching agents. The construct of structure C contains seven branching agents, one first generation proximal branching agent, two second generation intermediate branching agents and four third generation distal branching agents, indicating three layers / generations of branching agents. Construct D contains fifteen branching agents, one first generation proximal branching agent, two second generation intermediate branching agents, four third generation intermediate branching agents and eight fourth generation distal branching agents, i.e., construct D contains four layers (generations) of branching agents. Construct E contains thirty-one branching agents, one first generation proximal branching agent, two second generation intermediate branching agents, four third generation intermediate branching agents, eight fourth generation intermediate branching agents and sixteen fifth generation distal branching agents, indicating that there are five layers (generations) of branching agents.

[0122] According to one aspect, the diaminoalkanoic acid (DAA) is selected from §-DAA(§) and §-DAA($), wherein DAA represents a diaminoalkanoic acid, such as a diaminoalkanoic acid containing 3 to a maximum of 10 carbon atoms, suitably selected from 3 to a maximum of 8 carbon atoms; the characters § not in brackets represent an alpha amine protecting group, and the characters § and $ in brackets represent side chain protecting groups, wherein $ represents a protecting group that is cleaved under different cleavage conditions than the protecting group § or is not cleaved in the same cleavage step as the protecting group §.

[0123] The protecting group may be selected from Fmoc (fluorenylmethoxycarbonyl), Mtt (4-methyltrityl), Mmt (4-methoxytrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene))ethyl)] or ivDde.

[0124] The protecting group § may be Fmoc and $ may be selected from Mtt (4-methyltrityl), Mmt (4-methoxytrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)] or ivDde.

[0125] According to one aspect, all branching agents are selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms, wherein both amines are protected by the same protecting group.

[0126] According to one aspect, the branching agent is exclusively selected from amine-protected diaminoalkanoic acids, preferably diaminoalkanoic acids comprising 3 to up to 10 carbon atoms, suitably 3 to up to 8 carbon atoms, wherein the amine-protected diaminoalkanoic acid is selected from diaminoalkanoic acids comprising an amine protecting group that is cleaved during the same cleavage step and / or cleaved under the same / similar cleavage conditions, and diaminoalkanoic acids comprising an amine protecting group that is retained under different cleavage conditions or is not cleaved during the same cleavage step.

[0127] The polymer matrix may also be represented by a resin. The polymer matrix may comprise a polymer network, such as a cross-linked polymer network. The insoluble support and the polymer matrix are preferably in particle form. Typically, the insoluble support is in particle form, such as in the form of beads, with a size of about 1 μm to about 1000 μm, suitably about 5 μm to about 750 μm, about 10 μm to about 300 μm or in the range of about 50 μm to about 200 μm.

[0128] According to another aspect, the polymer matrix is ​​selected from a homogeneous polymer matrix. A homogeneous polymer matrix herein means a polymer matrix in which the binding sites are distributed throughout the matrix, preferably the binding sites are uniformly (substantially uniformly) distributed throughout the polymer matrix. Homogeneous means that the important parameters of the matrix, such as the crosslinking and distribution of the (primary) binding sites, are uniformly distributed throughout the matrix.

[0129] For solid phase peptide synthesis, there is a certain type of heterogeneous resin in which the binding sites are non-uniformly distributed within the resin. In contrast, the binding sites are concentrated at or near the surface of the resin beads forming the shell, and in which the core of the bead is essentially free of binding sites.

[0130] A homogeneous polymer matrix does not include a heterogeneous resin where substantially all binding sites are located at or near the surface. Thus, a homogeneous polymer matrix in particulate form (substantially in the form of spheres / beads) does not comprise a shell located at or near the surface (phase boundary) where substantially all binding sites are located and a core having substantially no binding sites.

[0131] According to another aspect, the polymer matrix is ​​selected from polymer matrices obtained by emulsion polymerization comprising at least styrene and divinylbenzene (DVB). Preferably, DVB is present during the emulsion polymerization in an amount of less than 4.0 wt.%, preferably less than 3.5 wt.%, preferably less than 3.0 wt.%. Preferably, DVB is present in an amount of about 0.5 wt.% to at most about 2.5 wt.%.

[0132] The insoluble support may also be characterized by structural features and features associated with the formation of the insoluble support. Thus, one aspect relates to an insoluble support in the form of particles comprising a construct as defined in any one of the aspects presented herein, the insoluble support being obtained by providing a polymer matrix comprising a primary binding site, wherein the construct is covalently bound to the primary binding site, the construct provides a secondary binding site such that the primary binding site provides at least two secondary binding sites, wherein the construct comprises at least one branching agent, a linker and optionally a spacer, and wherein the construct is formed by an embodiment of a solid phase synthesis scheme comprising cyclic synthesis steps comprising coupling at least one branching agent to the polymer matrix and optionally to other branching agents.

[0133] Yet another embodiment relates to an insoluble support (resin) in particulate form for solid phase peptide synthesis, comprising a distal binding site, the support comprising a polymer matrix and a construct, the construct being covalently bound to the polymer matrix, wherein the construct comprises at least one branching agent selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to up to 10 carbon atoms, a cleavable linker and at least one spacer coupled to the at least one branching agent via an amide bond, the cleavable linker providing the distal binding site and being covalently bound to the at least one branching agent; the construct formed by synthesis comprises providing as a solid phase a polymer matrix comprising a primary binding site, a branching agent, a linker and a spacer, the construct being formed by at least one coupling step, preferably by repeated coupling steps, the one or more steps comprising at least coupling of at least one branching agent, at least one spacer and a cleavable linker to the branching agent.

[0134] Another embodiment relates to a method for forming an insoluble support in particle form for solid phase peptide synthesis, the insoluble support comprising a secondary (distal) binding site, the insoluble support comprising a polymer matrix and a construct, the construct comprising a cleavable linker and at least one spacer, the method comprising providing a polymer matrix in particle form comprising a primary binding site, wherein the construct is formed by a solid phase synthesis protocol, the solid phase protocol comprising at least one reaction step in which a branching agent selected from amino-protected aminoalkanoic acids comprising at least 2 amino groups and 3 to up to 10 carbon atoms is coupled to any one of the primary binding site, the spacer or the branching agent of the polymer matrix via an amide bond, the protocol further comprising a reaction step of coupling the cleavable linker to the branching agent.

[0135] Insoluble supports are preferably synthesized by providing a polymer matrix and by forming amide bonds and coupling related compounds (branching agents, spacers and cleavable joints) using a solid phase synthesis scheme. Preferably all compounds contain the functional groups necessary for providing amide bonds when coupled to each other. Usually, all compounds contain at least one amine / amino group and a carboxylic acid group / carboxyl group. As described in this specification, branching agents are selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms. Preferably, spacers are selected from compounds comprising two binding sites, one being an amine / amino group and the other being a carboxylic acid group. Preferably, spacers are selected from amino acids and derivatives thereof with two binding sites (i.e., an amine group and a carboxylic acid group).

[0136] It should be noted that all reactive groups of the relevant compounds (except the carboxylic acid group) require suitable protecting groups. More specifically, any amine / amino group needs to be protected with a suitable protecting group.

[0137] The reaction cycle in the solid phase peptide synthesis protocol includes at least the following steps:

[0138] a) providing a compound comprising at least one amine group and a carboxylic acid group, wherein the relevant amine group is protected by a relevant protecting group. Suitable protecting groups are covalently bonded to the amine. If the compound also comprises a side chain with a reactive group, the side chain reactive group is also protected. In the presence of a suitable coupling agent, the compound comprising at least a protected amine group and a carboxylic acid group is coupled to the primary binding site of the polymer matrix to form an amide bond. If the compound is an amino acid, the α-amine is always protected, and any reactive group of the side chain, such as the ε-amine of lysine, can be protected if necessary.

[0139] b) Separation of excess reagents and by-products after coupling of the compound of interest to the polymer matrix

[0140] c) Next, another compound comprising at least one amine group and a carboxylic acid group, wherein the corresponding amine group is protected by a relevant protecting group, is coupled to the free amine group of the previous compound coupled to the polymer resin in step.

[0141] After the last generation of branching agents is coupled, the linker can be cleaved to couple to the distal branching agent.

[0142] The process of synthesizing a polypeptide may begin by providing a polymer matrix, modifying the polymer matrix according to the invention, and continuing with the synthesis of any relevant target polypeptide once the insoluble support of the invention has been formed.

[0143] The synthesis of the insoluble support may include the following steps:

[0144] a) optionally coupling at least one spacer molecule to the primary binding site,

[0145] b) coupling a primary branching agent preferably selected from diaminoalkanoic acids (two amine groups protected by protecting groups), preferably diaminoalkanoic acids (containing 3 to up to 10 carbon atoms, suitably 3 to up to 6 carbon atoms, such as diaminopropionic acid, diaminobutyric acid, diaminopentanoic acid and diaminohexanoic acid), to the primary binding site of the polymer matrix, or optionally coupling the branching agent to a spacer molecule,

[0146] c) removing the protecting groups,

[0147] wherein steps a), b) and c) can be repeated, and

[0148] d) Coupling the linker to the unprotected amine group of the distal branching agent.

[0149] The two amines of the above diaminoalkanoic acid of step b) are suitably protected by protecting groups, which are selected from amine protecting groups that are cleaved in the same cleavage step and / or cleaved under the same / similar cleavage conditions, and diaminoalkanoic acids containing amine protecting groups that remain under different cleavage conditions or are not cleaved in the same cleavage step.

[0150] According to one embodiment, all protecting groups of the diaminoalkanoic acid are cleaved in the same deprotection step. Thus, the alpha amine and the side chain amine are suitably protected by the same protecting group.

[0151] Another embodiment of the present invention relates to a construct. The construct and the polymer matrix support form the insoluble support of the present invention.

[0152] According to one aspect, a solid phase scheme can be used to form a construct and an insoluble support. The solid phase can be a base insoluble support. The construct is an entity that increases the number of binding sites, that is, an entity that is coupled to the primary binding site of an insoluble support, ultimately providing multiple secondary binding sites. The construct comprises at least one branching agent as disclosed herein. As described above, the construct can include two or more layers / generations of branching agents. The distal or intermediate branching agents of the construct are optionally coupled to the proximal branching agent through a spacer. The preferred procedure for forming the construct and the modified insoluble support is the application of a solid phase scheme. According to one aspect, the branching agent is selected from diaminoalkanoic acid, and the construct can be synthesized using a reaction scheme established in solid phase peptide synthesis (SPPS). Once the target construct is generated, the construct can be cut off from the insoluble support. Alternatively, the construct can be synthesized by a liquid phase reaction scheme.

[0153] The construct can be considered as a branched polymer, or a branched polymer comprising an amide conjugate. The construct can also be considered as a branched peptide, or a peptide-like polymer comprising a spacer.

[0154] According to one aspect, if Φ represents the number of binding sites of the branching agent, the relationship between the number of secondary binding sites and the number of layers of the construct can be expressed as: λ = (Φ-1) n , where Φ represents the number of binding sites of the branching agent and n represents the number of layers of branching agents in the construct, provided that all branching agents in the construct have the same number of binding sites. If the construct contains only one branching agent, and the branching agent is lysine (lysine has three binding sites, then the number of secondary binding sites λ provided by the construct is λ = (3-1) 1 : Two secondary binding sites. Therefore, modification of the primary binding sites with lysine as a branching agent (and construct), providing only one layer or generation of branching agents, will provide two secondary binding sites for each primary binding site of the polymer matrix.

[0155] The following aspects of the insoluble support relate to insoluble supports comprising one, two, three, four and five layers (generations) of branching agents, which are referred to as primary, secondary, tertiary, quaternary and quinary branching agents. The primary branching agents form the first generation, the secondary branching agents form the second generation, the tertiary branching agents form the third generation, and so on. If the branching agent contains three binding sites, such as lysine, then the number of secondary binding sites provided is equal to 2", where n represents the number of generations. Thus, an insoluble support comprising lysine as a branching agent and further comprising five quinary lysine branching agents would provide 2 n , that is, each primary binding site provides 25 or 32 secondary binding sites.

[0156] The scope of the present invention also includes insoluble supports comprising more than five levels of branching agents. However, the number of secondary binding sites for each level increases exponentially. In particular, by introducing spacers (spacer molecules) between the branching agents, the availability of secondary binding sites for peptide synthesis can be enhanced.

[0157] Another aspect relates to an insoluble support / polymer matrix comprising a primary binding site, wherein a proximal branching agent is covalently bound to the primary binding site, optionally via a spacer molecule, and wherein the primary branching agent provides at least two secondary binding sites.

[0158] The construct may contain not only one type of branching agent but also different types of branching agents. The construct may contain branching agents with different numbers of binding sites and / or branching agents with the same number of binding sites but different branching agents.

[0159] Another aspect relates to any insoluble support disclosed herein for use as a solid phase in any solid phase synthesis protocol. The insoluble support is preferably used as a solid phase in solid phase peptide synthesis.

[0160] Another aspect relates to the use of an insoluble support as a solid phase for the synthesis of peptides and polypeptides in solid phase peptide synthesis.

[0161] Branching Agent

[0162] As mentioned above, the construct is a molecule that can provide a secondary / distal binding site. In its simplest form, the construct comprises a branching agent. The most general definition of a branching agent is an organic molecule comprising at least three binding / coupling sites. A binding / coupling site is a functional group that can form a covalent bond with the binding / coupling site of another molecule (e.g., two binding sites of two different molecules form couplings in the form of a coupling between a covalent bond, a spacer-spacer, a spacer-branching agent, two branching agents, e.g., a primary binding site of a secondary (middle / distal) branching agent-primary (proximal) branching agent, a primary (proximal) branching agent, and a polymer matrix).

[0163] Typically, the branching agent comprises a functional group selected from amines, carboxylic acids, alcohols, ketones and aldehydes. Alternatively, the functional group is selected so that coupling generates amides, ethers and esters. Preferably, the branching agent comprises a functional group selected from amines and carboxylic acids. The preferred binding sites of the branching agent are amines and carboxylic acids. The amine is preferably a primary amine.

[0164] The branching agent may be selected from any natural amino acid or amino acid derivative that provides at least three coupling sites.

[0165] According to a preferred aspect, the branching agent is selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms.

[0166] According to another aspect, the branching agent is selected from aminoalkanoic acids comprising at least 2 but not more than 3 amino groups and from 3 to at most 10 carbon atoms.

[0167] According to a further aspect, the branching agent is selected from diaminoalkanoic acids containing 3 to at most 10 carbon atoms, and preferably from diaminoalkanoic acids containing 3 to at most 8 carbon atoms, such as 3 to at most 6 carbon atoms.

[0168] According to another aspect, the branching agent is selected from 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid, suitably, the branching agent is selected from 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid.

[0169] According to another aspect, the branching agent is selected from (lysine analogs) such as diaminopropionic acid, diaminobutyric acid and diaminopentanoic acid, diaminohexanoic acid.

[0170] According to one aspect, the branching agent is lysine (diaminocaproic acid), more specifically 2,6-diaminocaproic acid.

[0171] The branching agent may contain one or more chiral carbon centers (asymmetric carbons). Such chiral carbons may be in S or R configuration. Branching agents containing chiral carbons may have all chiral carbons in S or R configuration, or if multiple chiral carbons are present, both S and R configurations are present.

[0172] Amino acid-based branching agents and amino acid-based spacers can be in D or L form. Most amino acids are chiral (containing chiral carbons: particularly alpha carbon). Glycine has no chiral asymmetric carbon atoms. The construct can contain branching agents and optional spacers in D and / or L form. Thus, the construct can contain amino acids in D and L form. According to one embodiment, all chiral carbons of the relevant amino acid-based branching agents and optional spacers are in L form.

[0173] Spacer

[0174] Spacers may be present between branching agents, between branching agents and the primary binding site of the polymer matrix, and between the cleavable linker and the branching agent. The spacer needs to contain two binding / coupling sites / functional groups that can be used to form a covalent coupling.

[0175] The binding / coupling / functional group of the spacer can be selected from amines, carboxylic acids, alcohols, ketones and aldehydes. Preferably, the functional group is selected from amines, carboxylic acids and alcohols. Preferably, the functional group of the spacer forms an amide and an ether. Preferably, the spacer can be selected from any molecule having two functional groups, both of which can form an amide bond. A suitable spacer is any molecule having two functional groups, both of which can form an amide bond and are used in solid phase synthesis protocols.

[0176] According to a preferred aspect, the spacer is selected from amino acids or amino acid derivatives. More specifically, the spacer is selected from amino acids having one amine.

[0177] Useful spacers may be amino hydrocarbon carboxylic acids, ie any type of hydrocarbon chain comprising an amine and a carboxylic acid. Preferably, the amine and the carboxylic acid are at the ends of the hydrocarbon chain.

[0178] The spacer may be selected from an amino acid comprising a carboxylic acid group and an amine group. Suitable amino acids may also comprise aliphatic hydrocarbons. Exemplary amino acid spacers are glycine, alanine, β-alanine, isoleucine, leucine and any derivatives thereof.

[0179] According to one aspect, the spacer is selected from aminoalkanoic acids, in particular monoaminoalkanoic acids containing 1 to up to 10 carbon atoms, such as glycine, alanine, β-alanine, valine, isoleucine, leucine, β-leucine, e-aminopropionic acid, 4-aminobutanoic acid (4-aminobutanoic acid), 5-aminovaleric acid (5-aminovaleric acid), e-aminohexanoic acid (e-aminocaproic acid), 7-aminoheptanoic acid (7-aminoenanthic acid), 8-aminooctanoic acid (8-aminocaprylic acid), 9-aminononanoic acid, 10-aminodecanoic acid.

[0180] A preferred spacer is glycine.

[0181] Any number of spacers may be applied to the construct. Spacers may be located between branching agents, between a branching agent and a primary binding site of an insoluble support, and between a construct linker and a branching agent.

[0182] The hydrophilicity / hydrophobicity of the construct and the insoluble support can be adjusted by the choice of branching agents and / or spacers.

[0183] According to one aspect, the PEG spacer is located between the last layer of branching agents (also called distal branching agents) and the linker and / or between branching agents.

[0184] The PEG spacer is preferably provided in the form of 2-[2-[2-(Fmocamino)ethoxy]ethoxy]acetic acid (AEEA). An advantage of AEEA spacers is that they are easily integrated into solid phase peptide synthesis protocols.

[0185] Cleavable linker

[0186] The cleavable linker is located between the secondary binding site and the branching agent at the far end. The secondary binding site is usually integrated with the linker. Therefore, the linker may also be referred to as the secondary binding site. The far end refers to being away from the polymer matrix. The farthest branching agent layer represents the layer farthest from the polymer matrix base. The selected linker depends on the chemical properties used to synthesize the peptide, especially the target cleavage conditions. In addition, the type of polymer matrix may have an impact on the selection of the linker. The linker can be selected from PAM, Wang, Rink (such as Rink amide and Rink acid), PAL, Ramage, Sieber, MBHA, trityl chloride, oxime, HMPA, HMPB, DHP, Weinreb aminomethyl.

[0187] According to one aspect, the cleavable linker is not a photolytically cleavable linker.

[0188] Polymer Matrix

[0189] The insoluble support comprises a polymer matrix and a construct covalently attached to the polymer matrix, the polymer matrix generally comprising binding sites, also referred to as primary binding sites. The construct is covalently bound to the binding sites of the polymer matrix. The polymer matrix can be modified to comprise any type of binding sites suitable for coupling constructs. It should be noted that the primary binding sites of the polymer matrix are consumed during the formation of the insoluble support described in the present invention. Therefore, the primary binding sites of the polymer matrix are used to covalently bind constructs. Ideally, all the major binding sites of the polymer matrix are consumed by the covalent coupling of the construct, which indicates that the insoluble support of the present invention does not have a polymer matrix with primary binding sites. The polymer matrix is ​​generally a polymer network, generally comprising polymers cross-linked to a certain degree, the degree of cross-linking providing the structural integrity of the polymer matrix particles while enabling the polymer network to be significantly solvated. The polymer matrix is ​​preferably selected from a polymer matrix comprising binding sites distributed throughout the polymer matrix. The polymer matrix can be referred to as a homogeneous polymer matrix. The polymer matrix can also be referred to as a polymer network or resin.

[0190] According to one aspect, the polymer matrix is ​​selected from a homogeneous polymer matrix. A homogeneous polymer matrix herein refers to a polymer matrix in which the binding sites are distributed throughout the matrix, preferably the binding sites are uniformly (substantially uniformly) distributed throughout the polymer matrix. Homogeneity relative to a polymer matrix refers to that certain parameters of the matrix (such as the cross-linking and distribution of (primary) binding sites) are uniformly distributed throughout the matrix.

[0191] For solid phase peptide synthesis, there is a certain type of heterogeneous resin in which the binding sites are not evenly distributed within the resin. Instead, the binding sites are concentrated (restricted) at or near the surface of the resin beads forming the shell, and in which the core of the beads is essentially free of binding sites.

[0192] A homogeneous polymer matrix does not include a heterogeneous resin in which substantially all of the binding sites are located at or near the surface.

[0193] According to another aspect, the polymer matrix is ​​selected from polymer matrices obtained by emulsion polymerization, especially homogeneous matrices, which contain at least styrene and a crosslinker, preferably divinylbenzene (DVB). Preferably, the crosslinker is present during the emulsion polymerization in an amount of less than 4.0 wt. %, preferably less than 3.5 wt. %, preferably less than 3.0 wt. %. Preferably, DVB is present in an amount of about 0.5 wt. % to at most about 2.5 wt. %.

[0194] According to one aspect, the polymer matrix is ​​selected from a homogeneously crosslinked polystyrene based matrix, preferably comprising less than 4.0 wt. % crosslinking agent, preferably less than 3.5 wt. % crosslinking agent, preferably less than 2.0 wt. % crosslinking agent.

[0195] The polystyrene-based matrix, in addition to being preferably crosslinked with DVB, can also be modified by various monomers such as hydroxyethylstyrene and polyethylene glycol.

[0196] The polymer matrix can be formed by emulsion polymerization. A particularly useful polymer matrix is ​​a styrene-based matrix crosslinked with divinylbenzene (DVB). The DVB content of the styrene-based matrix crosslinked with DVB is preferably less than 4.0 wt%, preferably less than 3.5 wt%, preferably less than about 3.0 wt%.

[0197] The polymer matrix can also be represented by a resin. The polymer matrix can be selected from polystyrene, polyacrylate, polyacrylamide, polyamide or polyethylene glycol. The polymer matrix can also be a hybrid of at least two different types of polymers. Thus, the polymer matrix can be a polystyrene modified by another type of polymer (such as ethylene glycol, polyacrylate, polyamide or polyacrylamide).

[0198] Insoluble support is preferably used as solid phase in particle form. The particle form of insoluble support is suitably spherical. Therefore, insoluble support is suitably spherical, and can be expressed as beaded or simply referred to as beads. Bead is preferably spherical. The size range of insoluble support (spherical beads) can be about 1 μm to about 2000 μm at most, such as about 1000 μm at most, suitably about 5 μm to about 750 μm at most, such as about 20 μm to about 500 μm at most, about 50 μm to about 300 μm at most, specifically about 50 to 150 μm. Not only the size of the beads is important, but also the size distribution of the beads should be considered.

[0199] The polymer matrix is ​​typically substituted. Substitution can be achieved by directly attaching the matrix to the polymer matrix, for example by copolymerization of the substituting polymer with a polymer of the polymer matrix (eg styrene) or by electrophilic aromatic substitution reactions.

[0200] Examples of polymer matrices are polystyrene-based matrices modified with aminomethyl (AM or AMS), 4-methylbenzylamine (MBHA), cross-linked polystyrene-based matrices appropriately modified with PEG (such as Tentagel) by grafting PEG onto the polystyrene-based matrix. TM Resin, and ), polyethylene glycol (PEG) based resins / matrices (such as and NovaPEG), aminomethylated polystyrene-based matrices of partially derivatized methyl-PEG-p-nitrophenyl carbonate (e.g., NovaGel TM ), polystyrene-based matrices copolymerized with hydroxyethyl polystyrene and polyethylene glycol (PEG) (e.g. NovaSyn TM ).

[0201] Constructs

[0202] The construct can be referred to as a branched molecule, and in a sense can also be referred to as a branched polymer, because the construct preferably comprises repeating units, such as a branching agent and an optional spacer. As shown, the construct comprises at least a branching agent and an optional spacer. In theory, there is no upper limit to the number of branching agents included in the construct. For most applications, the upper limit of the branching agent in the construct is about 150. Most constructs will comprise 1 to about 150 at most, 1 to about 100 at most, such as about 1 to about 80 branching agent numbers at most. Branching is induced by a branching agent. According to one aspect, the branching agent and the spacer are selected from amino acids or amino acid derivatives. The construct comprising a branching agent and a spacer selected from amino acids can be expressed as a branched peptide. Therefore, some constructs can be referred to as branched peptides or polypeptides. The molecular weight of the construct (excluding joints) can start from about 100 g / mol to about 200,000 g / mol at most, to about 100,000 g / mol at most, to about 10,000 g / mol at most. The molecular weight of an exemplary 2X (Gly) construct is about 1245 g / mol, and the molecular weight of a 16X (Gly) construct is about 3375 g / mol (all exemplary herein). The construct comprises a cleavable linker coupled to a distal branching agent suitable for targeting polypeptides and peptide chemistry, preferably taking into account an end-capping strategy.

[0203] General disclosure of the method for forming the insoluble support and construct of the present invention

[0204] The insoluble support can be formed by providing a commercially available polymer matrix and gradually synthesizing the construct (e.g., by a solid phase peptide synthesis protocol: a divergent synthesis protocol). Alternatively, the insoluble support can be formed by providing a commercially available polymer matrix and coupling the complete construct (a convergent synthesis protocol). Preferably, the insoluble support is formed by a solid phase synthesis protocol that includes the sequential addition of relevant chemical compounds, especially branching agents, linkers, and spacers.

[0205] Preferably, the construct is provided by a process scheme similar to that for providing an insoluble support, although a linker is applied between the construct and the resin to enable the construct to be cleaved from the resin. The construct cleaved from the resin can be used to modify the polymer matrix. According to one aspect, the branching agent and the spacer are selected from amino acids and amino acid derivatives, thereby providing a construct that can be referred to as a peptide, typically a branched peptide or a branched polypeptide.

[0206] Any solid phase peptide synthesis chemistry can be used for the synthesis of this construct, including various amine protection and side chain protection chemistries as well as peptide bond forming activation chemistries.

[0207] Solid phase synthesis reagents are readily available from commercial sources. Solid phase synthesis procedures typically include the following repetitive operations: deprotection of the N-terminal alpha amine protecting group and / or side chain amine protecting groups of the amino acid covalently coupled to the solid phase, coupling of the N-terminal amine protected and side chain amine protected amino acids, optional capping of unreacted amino acids (e.g., acetylation), and a washing step for displacement of by-products. In addition, the following operations may be applied: blocking interfering groups and protecting amino acids during the reaction.

[0208] According to one aspect, the branching agent is selected from aminoalkanoic acids comprising at least 2 amino groups and 3 to at most 10 carbon atoms, preferably diaminoalkanoic acids comprising 3 to at most 10 carbon atoms, such as diaminoalkanoic acids selected from lysine analogs (including diaminopropionic acid, diaminobutyric acid, diaminopentanoic acid, diaminohexanoic acid and diaminoheptanoic acid).

[0209] The amine group of the diaminoalkanoic acid can be protected with a variety of protecting groups, including fluoren-9-ylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), tert-butyl tBu, and trityl-based protecting groups, including 4-methyltrityl (Mtt) and 4-methoxytrityl (Mmt).

[0210] The advantage of the Fmoc strategy over the Boc strategy is that Fmoc can be removed under milder reaction conditions. The Fmoc strategy allows the use of base-sensitive N-terminal amine protecting groups and acid-sensitive side chain protecting groups. The Fmoc N-terminal protecting group is removed under alkaline conditions without removing the acid-sensitive side chain protecting groups.

[0211] According to one aspect, the amine group of a diaminoalkanoic acid (DAA) is protected by a group that is cleaved under similar cleavage conditions, which generally leads to the conclusion that the amine group of the diaminoalkanoic acid is protected by the same (identical) protecting group. However, the corresponding amine group, N-terminus, alpha amine group and amine group of the side chain of the diaminoalkanoic acid may also be protected by a protecting group that is cleaved under different cleavage conditions (such as different pH).

[0212] Diaminoalkanoic acid in which the amine group is protected by the same protecting group is also referred to as §-DAA(§), wherein § represents an alpha amine protecting group and (§) represents an epsilon amine protecting group.

[0213] Diaminoalkanoic acids whose amine groups are protected by different protecting groups are also called Where $ represents an α-amine protecting group, represents a side chain or orthogonal amine protecting group. DAA can be lysine (K).

[0214] The diaminoalkanoic acid protecting group may be selected from Mtt (4-methyltrityl) or Mmt (4-methoxytrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)] or ivDde.

[0215] The protecting group § may be Fmoc and $ may be selected from Mtt (4-methyltrityl) or Mmt (4-methoxytrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)] or ivDde.

[0216] According to one aspect, all aminoalkanoic acids of the construct are protected by protecting groups that are cleaved simultaneously in essentially the same cleavage step, e.g., both amine groups (of all diaminoalkanoic acids) are protected by the same protecting group (such as Fmoc), e.g., §-DAA (§). If only aminoalkanoic acids in which all amines are protected by the same protecting group are used to synthesize the construct, then, for example, both the alpha amine and the epsilon amine (if the aminoalkanoic acid is a diaminoalkanoic acid) will be coupled to the same (identical) compound (e.g., a branching agent, a spacer, or a linker).

[0217] According to one aspect, at least one aminoalkanoic acid is used as a branching agent (during the synthesis of the construct of the insoluble support), the respective amine group of which is protected by a protecting group that is cleaved under different cleavage conditions (such as different pH), e.g. By subjecting the aminoalkanoic acid of the amine protecting group to cleavage under different conditions (usually in separate cleavage steps), different compounds can be coupled to the alpha and side chain amines.

[0218] Any combination of different diaminoalkanoic acids can be used, as well as any ratio of diaminoalkanoic acids having an amine protecting group that is cleaved in the same cleavage step (and / or cleaved under substantially the same cleavage conditions) (e.g., §-DAA (§)) and diaminoalkanoic acids having an amine protecting group that is cleaved under different cleavage conditions (e.g., ) to form a construct. A construct can be formed by using a diaminoalkanoic acid specifically, wherein two amine groups (N-terminal, α amine group and side chain amine group, such as ε amine group) are all protected by the same protecting group. For example, two amine groups of all diaminoalkanoic acids are protected by Fmoc. Alternatively, a construct can be formed by using a diaminoalkanoic acid specifically, wherein each amine group is protected by different protecting groups that are cracked under different cleavage conditions (usually different pH). For example, an amine group of a diaminoalkanoic acid is protected by Fmoc, and another amine group is protected by Mtt or Mmt groups. The insoluble support 7X linear disclosed in the experimental part is synthesized by the following: first, only lysine is used as a branching agent to form the main chain, wherein the amine group of each lysine is protected by an Fmoc group and an Mtt group, and then Fmoc-Lys (Fmoc) is coupled to the side chain of the main chain lysine. For example 7X insoluble support, the main chain contains 3 lysines (for example, provided with Fmoc-Lys (Mtt)). Any odd number of binding sites (e.g., 3, 5, 7, 9, 11, etc.) of the construct can be provided by coupling any number of Fmoc-Lys(Mtt) to the polymer matrix (1, 2, 3, 4, 5, 6, etc.), thereby forming a backbone containing 1 to any number of Fmoc-Lys(Mtt). The backbone can be represented as Fmoc-Lys(Mtt)-[Lys(Mtt)] X - polymer matrix, wherein X is 0 up to an integer. X can be from 0 to 50. In this case, the backbone is a chain of consecutive lysines coupled to each other via alpha amines.

[0219] Typically, a linear construct can be formed by providing a polymer matrix and a continuous coupling branching agent and an optional spacer, wherein the branching agent is specifically selected from aminoalkanoic acid, wherein the aminoalkanoic acid has an N-terminal amine and a side chain amine, wherein the side chain amine is protected by a protecting group that is cracked under different cracking conditions (e.g., different pH), and is typically cracked in different cracking steps to form a main chain. After the main chain is formed, the side chain protecting group is cracked under suitable cracking conditions. In a subsequent step, the branching agent is specifically selected from aminoalkanoic acid, wherein the N-terminal amine and the side chain amine of these aminoalkanoic acids are protected by protecting groups, and these protecting groups are cracked under identical cracking conditions, which are actually identical protecting groups. In another reaction step, all remaining amine protecting groups are all cracked, and subsequently suitable joints are coupled to the amine groups of all deprotections.

[0220] Another aspect relates to a synthetic scheme wherein the The backbone is provided by branching agents of the form of , optionally with spacers between the branching agents and between the polymer matrix and the first generation branching agents. In a further step, the Protecting group. Subsequently, a branching agent in the form of §-DAA (§) is coupled. If no further step of coupling the branching agent is carried out, a so-called linear construct is formed. 7X linear represents a linear construct.

[0221] For certain constructs, particularly asymmetric constructs (also referred to herein as linear constructs), the side chain amine of the branching agent (eg, lysine) is protected with Mtt, Mmt, alloc, Dde, or ivDde.

[0222] According to one aspect, the construct is synthesized as a solid phase on a suitable polymer matrix by applying a solid phase synthesis protocol, which solid phase synthesis protocol includes the use of a branching agent selected from diaminoalkanoic acids containing 3 to up to 10 carbon atoms, wherein the N-terminal amine group (alpha amine group) and the amine groups of the side chains of the branching agent are both protected by groups that are cleaved under similar reaction conditions, i.e., the protecting groups are cleaved simultaneously.

[0223] Where applicable, amino acid type spacers (such as spacers selected from amino acids comprising one carboxylic acid group, one amine group and an aliphatic hydrocarbon) are Fmoc protected.

[0224] According to another aspect, constructs are synthesized on a suitable polymer matrix as a solid phase by applying a solid phase synthesis protocol, the solid phase synthesis protocol comprising the use of a branching agent selected from diaminoalkanoic acids, the diaminoalkanoic acids comprising 3 to at most 10 carbon atoms, wherein the N-terminus (alpha amine group) and the amine groups of the side chains are protected by two different protecting groups that are cleaved under different cleavage conditions (e.g., different pH). Preferably, the protecting groups are selected from acid-labile and base-labile protecting groups, suitably Fmoc, Mtt and Mmt. Different chemical moieties can be coupled to the same branching agent by coupling a base-sensitive Fmoc protecting group and an acid-sensitive Mtt or Mmt group to the corresponding amine groups of a branching agent (e.g., diaminoalkanoic acid).

[0225] When both the α chain and the side chain amine groups have protecting groups, which are cleaved substantially simultaneously in the same cleavage step, a symmetrical construct will be formed. When the corresponding amine groups of at least one branching agent (e.g., diaminoalkanoic acid) are protected by two different protecting groups and cleaved under different cleavage conditions (e.g., different pH), an asymmetric linear construct will be formed, such as Fmoc and Mtt or Mmt.

[0226] Preferably, both the α and the side chain amine groups are protected by the same protecting group, such as Boc or Fmoc, preferably Fmoc. Preferably, both the α and ε amine groups of lysine are protected by Fmoc.

[0227] 7X linear is an asymmetric linear construct. If the reaction conditions allow at least one lysine α and ε amine group to be coupled to different chemical moieties (e.g., spacers and branching agents (lysine)), an asymmetric construct is formed. In order to achieve the coupling of lysine α and ε amine groups to different chemical moieties, lysine α and ε amine groups must have protective groups that are cleaved under different reaction conditions. One method of achieving this is to have an acid-labile protective group and a base-labile protective group. Therefore, one amine group of lysine may be protected by base-labile Fmoc, while the other amine group is protected by acid-labile protective groups (such as Mtt and / or Mmt).

[0228] Lysine branching agents include Fmoc protection of the N-terminus and Mtt or Mmt protection of the side chain amine, which can be coupled to another branching agent and a spacer or linker, respectively. The synthesis of the construct can incorporate diaminoalkanoic acids containing two Fmoc groups (i.e., both the N-terminus and the side chain amine are protected by Fmoc) and / or diaminoalkanoic acids containing one Fmoc group and Mtt and / or Mmt groups.

[0229] The Fmoc group is usually removed by using a base such as a heterocyclic amine, preferably piperidine. The Mtt group can be cleaved under acidic conditions, such as by using a solution comprising dichloromethane (DCM) and trifluoroacetic acid (TFA). The Boc protecting group is unstable (sensitive) to bases and is preferably removed by using TFA.

[0230] Amino acid coupling chemistries include, Oxyma pure (ethyl cyano(hydroxyimino)acetate), diisopropylcarbodiimide (DIC), N,N-dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tris(pyrrolidinyl)phosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yl-oxytris(pyrrolidinyl)phosphonium hexafluorophosphate (PyAOP), O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)- N,N,N′N′-tetramethyluronium hexafluorophosphate (HATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HCTU), O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TDBTU), 3-(diethylphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT), ethyl-(N′,N′-dimethylamino)propylcarbodiimide hydrochloride (EDC), 4,4,4-trifluoro-N-Fmoc-O-tert-butylthreonine.

[0231] Typically, the secondary binding site provided by the insoluble support comprises a cleavable linker that is capable of cleaving the target peptide from the insoluble support. Useful linkers include Rink, PAL, Ramage, Sieber, MBHA (methylbenzhydrylamine), PAM, Wang (4-hydroxybenzyl alcohol moiety), trityl, 2-chlorotrityl, oxime, HMPA (hydroxymethylbenzoic acid), DHP, Weinreb aminomethyl.

[0232] Useful solvents for preparing insoluble supports are methyl chloride, N-methylpyrrolidone (NMP), N-methyl-2-pyrrolidone (NMP) and dimethylformamide (DMF).

[0233] Peptide synthesis using the insoluble support of the present invention

[0234] Insoluble supports of the present invention are preferably applied to the synthesis of peptides. According to one aspect, peptide synthesis starts after the insoluble supports are formed. Therefore, the peptide synthesis scheme can start with providing a polymer matrix, followed by the synthesis of constructs, thereby forming the insoluble supports of the present invention. Before the initial target peptide is synthesized, the insoluble supports can be treated with steps so that the insoluble supports are in a state suitable for the synthesis of subsequent target peptides.

[0235] Any solid phase peptide synthesis chemistry suitable for synthetically modified insoluble supports can be used for the synthesis of the target peptide, including various amine protection and side chain protection chemistries as well as peptide bond forming activation chemistries.

[0236] According to one aspect, the insoluble support is particularly suitable for the synthesis of long complex peptides, ie peptides having more than 10, 15, 20, 25, 30 amino acids, or even peptides having more than 50 amino acids.

[0237] The insoluble supports of the present invention enable the synthesis of complex peptides in commercially acceptable yields and commercially useful purities.

[0238] The insoluble supports of the present invention are typically formed using a commercially available homogeneous polymer matrix / resin that is modified by synthesizing branched constructs from the available primary binding sites of the polymer matrix.

[0239] Therefore, insoluble supports based on commercially available homogeneous polymer resins are commercially attractive options for the successful synthesis of complex peptides without the need to develop new complex resins. Commercially available homogeneous polymer resins can be converted into insoluble supports that enable the production of complex peptides in high yields, commercially relevant purities, and high throughput in SPPS by using a fairly simple, uncomplicated SPPS protocol and the application of readily available amino acids.

[0240] The present invention also relates to methods for synthesizing peptides having at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 amino acids using SPPS and the use of the insoluble support disclosed herein.

[0241] The general process of synthesizing peptides on a resin begins with attaching the first amino acid (C-terminal residue) to the resin. To prevent amino acid polymerization, the alpha amino group and reactive side chains are protected with temporary protecting groups. Once the amino acid is attached to the resin, the resin is filtered and washed to remove by-products and excess reagents. Next, the N-alpha protecting group is removed in a deprotection process, and the resin is washed again to remove by-products and excess reagents. The next amino acid is then coupled to the attached amino acid. Another washing procedure is then performed to prepare the resin-peptide for the next coupling cycle. This cycle is repeated until the peptide sequence is complete. The peptide is then typically cleaved from the resin, removing all protecting groups at the same time. Alternatively, the side chain protecting groups are removed, the peptide resin is washed, and then the peptide is cleaved from the resin.

[0242] Typically, an N-α-carbamoyl protected amino acid and the N-terminal amino acid on the growing peptide chain attached to the resin with the linker construct are coupled at room temperature in an inert solvent such as dimethylformamide in the presence of a coupling agent such as diisopropylcarbodiimide and Oxyma pure. After amide bond formation, the Nα-carbamoyl protecting group is removed from the resulting peptide resin using a reagent such as piperidine, and the coupling reaction is repeated to add the next desired Nα-protected amino acid to the peptide chain.

[0243] Suitable amine protecting groups are well known in the art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, 1991. The most commonly used examples include fluorenylmethoxycarbonyl (Fmoc) and Boc (tert-butyloxycarbonyl). After the synthesis is complete, the peptide is cleaved from the solid support using standard treatment methods, preferably under acidic conditions, with simultaneous side chain deprotection.

[0244] The crude target peptide is cleaved from the insoluble support containing the construct. The conditions for cleavage of the target peptide depend in part on the type of linker and side chain protecting groups used.

[0245] Crude peptides are typically analyzed using RP-HPLC on a C18 (CHS-Agilent UPLC) column using a water-acetonitrile gradient in 0.1% TFA. Purity can be verified by analytical RPHPLC. The identity of the peptide can be verified by mass spectrometry. Peptides can be dissolved in aqueous buffers over a wide pH range.

[0246] The insoluble support of the present invention can be regenerated after the target peptide is cleaved. The insoluble support can be regenerated after the target peptide is separated from the peptide-insoluble support solution.

[0247] Detailed disclosure of certain aspects of the invention

[0248] Insoluble supports comprising constructs are disclosed below, which demonstrate that the initial (primary) binding sites of the polymer matrix have increased by two times 2X, four times 4X, seven times 7X, eight times 8X and sixteen times 16X. Lysine (K or Lys) is always used as a branching agent. Where appropriate, glycine (G or Gly), polyethylene glycol (PEG6) or beta-alanine (bAla) are used as spacers. PEG6 represents that PEG contains six individual PEG moieties covalently bound by peptide bonds.

[0249] Aminomethyl (AMS) polystyrene resin was used as the starting polymer matrix for the synthesis of all insoluble supports described below. All AMS resins used were aminomethyl modified polystyrene resins crosslinked with 1% divinylbenzene. AMS resins were provided in the form of beads ranging from 35-150 μm (100 to 200 mesh) with degrees of substitution of 0.6, 0.93 and 1.95 mmol / gram, respectively.

[0250] Due to the use of lysine as a branching agent, the constructs of the insoluble supports disclosed in this section can be represented as branched polypeptides synthesized on a polymer matrix.

[0251] For all insoluble supports, lysine was used in both the S configuration (L amino acid) and the R configuration (D amino acid).

[0252] For all insoluble supports (2X(Gly), 4X(Gly), 8X(Gly), 16X(Gly)) except 7X Linear, the branching agent lysine carries two Fmoc groups to protect the two amines, i.e. the branching agent is provided in the form of Fmoc-Lys(Fmoc)-OH.

[0253] 7X Linear is a linear construct. In the synthesis of the linear 7X insoluble support, two types of lysine were used that differed in the amine protecting groups. One type of lysine had both amine groups protected by Fmoc: Fmoc-Lys(Fmoc)-OH. The other type of lysine had the α amine protected by Fmoc and the ε amine protected by Mtt: Fmoc-Lys(Mtt)-OH.

[0254] The structures of some reactants are shown:

[0255] The structural formula of Lys(Fmoc) (R and S configurations), where both amine groups are protected by Fmoc:

[0256]

[0257] The structural formula of Lys(Mtt)-OH (S configuration), wherein the N-terminus and the α-amine group are protected by Fmoc, and the side chain and the ε-amine group are protected by Mtt or Mmt.

[0258]

[0259] The Fmoc-Rink amide linker (Fmoc-Rink-OH) has the following chemical structure:

[0260]

[0261] Fmoc Ramage connector (MW: 505.58):

[0262]

[0263] HMPB TBDMS and HMPB connectors:

[0264]

[0265] The glycine spacer was provided as Fmoc protected glycine. PEG was provided as 2-[2-[2-(Fmocamino)ethoxy]ethoxy]acetic acid (EAAE).

[0266]

[0267] Notes on Nomenclature of Exemplary Insoluble Supports:

[0268] The number in brackets of the branching agent lysine K (or Lys) indicates a layer or generation, and the subscript indicates the total number of lysines in each layer / generation. For example, K(2)2 or Lys(2)2 indicates that the construct of the insoluble support has two secondary lysines.

[0269] Disclosure of solid supports

[0270] Solid supports 2X(Gly), 4X(Gly), and 8X(Gly) based on 0.60 mmol / g AMS polymer matrix (without linker) were synthesized using the following protocol:

[0271] The initial AMS resin (30.0 g, Table 1) with a starting loading of 0.60 mmol / g was introduced into a 1 L glass reactor equipped with a mechanical stirrer and swollen in DMF (8 mL / g initial resin, 2 x 1 h). All amino acid couplings were performed with Fmoc amino acids (4.0 equiv.), Oxyma (4.0 equiv.) and N′N′-diisopropylcarbodiimide (5.0 equiv.) in DMF (0.50 M) at a molar ratio of 1:1:1.25. Fmoc amino acids (Fmoc-Gly-OH or Fmoc-Lys(Fmoc)-OH) were dissolved in DMF (0.50 M), then Oxyma and N′N′-diisopropylcarbodiimide were added and transferred to the glass reactor after pre-activation at room temperature for 30 minutes. Couplings were performed at room temperature for 2 hours. After coupling, the resin was washed with DMF (2 x 8 mL / g), capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes, and washed with DMF (7 x 8 mL / g). Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 minutes + 1 x 20 minutes). After Fmoc deprotection, the resin was washed with DMF (7 x 8 mL / g of initial resin).

[0272] After the synthesis of 2X-Gly-AMS was completed, the resin was washed with CH2Cl2 (3x), iPrOH (3x) and MTBE (3x) and then dried overnight under vacuum. 1 / 3 of the resulting resin was retained, and the remaining 2 / 3 (28.2 g, Table 1) was used to synthesize 4X-Gly-AMS in the same manner as 2X-Gly-AMS.

[0273] After the synthesis of 4X-Gly-AMS was completed, the resin was washed with CH2Cl2 (3x), iPrOH (3x) and MTBE (3x) and then dried overnight under vacuum. 2 / 3 of the resulting resin was retained, while the remaining 1 / 3 was used to synthesize 8X-Gly-AMS in the same manner as 2X-Gly-AMS.

[0274] After the synthesis of 8X-Gly-AMS was completed, the resin was washed with CH2Cl2 (3x), iPrOH (3x), and MTBE (3x), and then dried under vacuum overnight.

[0275]

[0276] Table 1

[0277] Solid supports 2X(Gly) and 4X(Gly) based on 0.93 mmol / g AMS polymer matrix and 1.95 mmol / g AMS polymer matrix (without linker), respectively, were synthesized using the following protocol:

[0278] Initial AMS resin with a starting loading of 0.93 or 1.95 mmol / g (1.0 or 2.0, Tables 2 and 3) was introduced into a 60 mL plastic syringe and swollen in DMF (8 mL / g initial resin, 2 x 1 h). The synthesis was performed manually with the syringe shaken at 400 rpm in a horizontal position with an Activo-PLS 4x4 synthesizer purchased from Activotec. All amino acid couplings were performed with Fmoc amino acids (4.0 equiv.), Oxyma (4.0 equiv.) and N'N'-diisopropylcarbodiimide (5.0 equiv.) in DMF (0.50 M) in a molar ratio of 1:1:1.25. The Fmoc amino acid (Fmoc-Gly-OH or Fmoc-Lys(Fmoc)-OH) was dissolved in DMF (0.50 M), then Oxyma and N′N′-diisopropylcarbodiimide were added and preactivated at room temperature for 30 minutes before being transferred to a glass reactor. The coupling was performed at room temperature for 2 hours. After coupling, the resin was washed with DMF (2 x 8 mL / g), capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes, and washed again with DMF (7x 8 mL / g). Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1x 10 minutes + 1x 20 minutes). After Fmoc deprotection, the resin was washed with DMF (7x 8 mL / g initial resin).

[0279] 2X-Gly-AMS and 4X-Gly-AMS soluble supports synthesized using 0.93mmol / g AMS resin were separated from the initial AMS resin (2.0g) of the same reactor batch. After the synthesis of 2X-Gly-AMS was completed, the resin was washed with CH2Cl2 (3x), iPrOH (3x) and MTBE (3x), and then dried overnight under vacuum. 2 / 1 of the resulting resin was retained, and the remaining 1 / 2 was used to synthesize 4X-Gly-AMS in the same manner as 2X-Gly-AMS. After the synthesis of 4X-Gly-AMS was completed, the resin was washed with CH2Cl2 (3x), iPrOH (3x) and MTBE (3x), and then dried overnight under vacuum.

[0280] 2X-Gly-AMS and 4X-Gly-AMS soluble supports synthesized using 1.95 mmol / g AMS resin were isolated from 1.0 g and 2.0 g of the initial AMS resin batch, respectively. After the synthesis, the resin was washed with CH2Cl2 (3x), iPrOH (3x), and MTBE (3x), and then dried under vacuum overnight.

[0281]

[0282] Table 2

[0283]

[0284] Table 3

[0285] Synthesis schemes of different Fmoc-Rink amide insoluble supports

[0286] 2X(Gly): (Fmoc-Rink)2-Lys(1)-Gly-AMS

[0287] 4X(Gly): (Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0288] 8X(Gly): (Fmoc-Rink)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0289] The relevant soluble supports described above were modified by Rink amide coupling using the following protocol:

[0290] The initial resin (1.0 g each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2x1 h). The synthesis was performed manually with the syringe shaken at 400 rpm in a horizontal position with an Activo-PLS 4x4 synthesizer purchased from Activotec. Before the rink amide linker coupling, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1x 10 min + 1x 20 min). After Fmoc deprotection, the resin was washed with DMF (7x 6.5 mL / g initial resin). All rink amide couplings were performed using Rink amide linker, PyAOP and diisopropylethylamine (DIEA) in DMF (0.50 M) in a 1:1:2 molar ratio, which was a 5-fold molar excess compared to the synthesis scale. Rink amide was dissolved in DMF (0.50 M), then PyAOP was added and stirred for 10 minutes at room temperature, then DIEA was added. Next, the solution was introduced into the syringe containing the resin. The coupling was performed at room temperature overnight. After coupling, the resin was washed with DMF (2x 8 mL / g) and capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes. Next, the resin was washed with DMF (7x 8 mL / g), isopropanol (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0291] Table 4 shows the resin substitution before and after Rink amide coupling.

[0292]

[0293] Table 4

[0294] Synthesis schemes of different Fmoc-Ramage insoluble supports

[0295] 2X(Gly): (Fmoc-Ramage)2-Lys(1)-Gly-AMS

[0296] 4X(Gly): (Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0297] 8X(Gly): (Fmoc-Ramage)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0298] The relevant soluble supports described above were modified by Ramage coupling using the following scheme:

[0299] The initial resin (1.0 g each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2x1 h). The synthesis was performed manually with the syringe shaking at 400 rpm in a horizontal position with an Activo-PLS 4x4 synthesizer purchased from Activotec. Before the Ramage linker coupling, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1x 10 minutes + 1x 20 minutes). After Fmoc deprotection, the resin was washed with DMF (7x 6.5 mL / g initial resin). All Ramage couplings were performed using Ramage linkers (0.15M-0.40M), Oxyma and N′N′-diisopropylcarbodiimide (DIC) in DMF in an equimolar ratio of 1:1:1, which was in a 2-fold molar excess compared to the synthesis scale. Ramage was dissolved in DMF (0.15M-0.40M), then Oxyma was added and stirred at room temperature for 10 minutes, then DIC was added. Next, the solution was introduced into a syringe containing the resin. The coupling was performed overnight at room temperature. After coupling, the resin was washed with DMF (2x 8mL / g) and capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes. Next, the resin was washed with DMF (7x8mL / g), isopropanol (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0300] Table 5 shows the resin substitution before and after Ramage coupling.

[0301]

[0302]

[0303] Table 5

[0304] Synthesis schemes of different Fmoc-Leu-HMPB insoluble supports

[0305] 4X(Gly): (Fmoc-Leu-HMPB-Leu)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0306] 8X(Gly): (Fmoc-Leu-HMPB-Leu)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0307] The relevant soluble supports described above were modified by coupling Fmoc-Leu-HMPB using the following protocol:

[0308] The initial resin (1.0 g each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2x1 h). The synthesis was performed manually, with the syringe shaking at 400 rpm in a horizontal position with an Activo-PLS 4x4 synthesizer purchased from Activotec. Before TBDMS-HMPB coupling, the Fmoc group was deprotected (1x 10 minutes + 1x 20 minutes) using 20% ​​piperidine in DMF at room temperature. After Fmoc deprotection, the resin was washed with DMF (7x 6.5 mL / g initial resin). All TBDMS-HMPB couplings were performed using TBDMS-HMPB, PyOxim and diisopropylethylamine (DIEA) in DMF (0.50 M) in a 1:1:2 molar ratio, which was 2 times in molar excess compared to the synthesis scale. TBDMS-HMPB was dissolved in DMF (0.50 M), then PyOxim was added and stirred at room temperature for 10 minutes, and then DIEA was added. Next, the solution was introduced into a syringe containing the resin. The coupling was performed at room temperature overnight. After HMPB coupling, the resin was washed with DMF (2x 8 mL / g) and capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes. Next, the resin was washed with DMF (7x 8 mL / g), isopropanol (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0309] Initial HMPB-resin (each 300mg) is introduced into 12mL syringe, and swells in THF (6.5mL / g, 2x1h).Synthesis is carried out manually, and wherein syringe is shaken in horizontal position with 400rpm together with Activo-PLS 4x4 synthesizer purchased from Activotec.At room temperature, use Et3N.3HF (12 equivalents compared with synthesis scale) in THF (0.22M) to react 2 hours, after TBDMS group is deprotected, carry out Fmoc-Leu-OH coupling.After TBDMS deprotection, use DIEA (1x) to clean resin, then use DMF until pH is neutral.

[0310] After swelling the resins in DMF (6.5 mL / g, 2x 1 h), their coupling with Fmoc-Leu-OH was performed. All couplings were performed using Fmoc-Leu-OH, Oxyma, N′N′-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) in DMF (0.45 M) in a 1:1:1:0.25 molar ratio, which was a 2.4-fold molar excess compared to the synthetic scale. Fmoc-Leu-OH was dissolved in DMF (0.45 M), then Oxyma, DIC and DMAP were added and stirred at room temperature for 5 minutes before being introduced into the syringe containing the resin. The coupling was performed overnight at room temperature. After coupling, the resin was washed with DMF (2x 8 mL / g) and capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes. Next, the resin was washed with DMF (7 x 8 mL / g), isopropanol (iPrOH, 3x), and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0311] Table 6 shows the resin substitution before and after Leu-HMPB coupling.

[0312]

[0313] Table 6

[0314] Synthesis schemes of different Fmoc-Gly-HMPB insoluble supports

[0315] 4X(Gly): (Fmoc-Gly-HMPB)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0316] 8X(Gly): (Fmoc-Gly-HMPB-)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0317] The relevant soluble supports described above were modified by coupling Fmoc-Gly-HMPB using the following scheme:

[0318] The initial resin (each 1.0g) is introduced into a 25mL syringe and swelled in DMF (6.5mL / g, 2x 1h). The synthesis is carried out manually, and the syringe is shaken in a horizontal position at 400rpm together with the Activo-PLS 4x4 synthesizer purchased from Activotec. Before the TBDMS-HMPB coupling, the Fmoc group is deprotected (1x 10 minutes + 1x 20 minutes) using 20% ​​piperidine in DMF at room temperature. After Fmoc deprotection, the resin is cleaned with DMF (7x 6.5mL / g initial resin). All TBDMS-HMPB couplings are carried out using TBDMS-HMPB, PyOxim and diisopropylethylamine (DIEA) in DMF (0.50M) with a 1:1:2 molar ratio, which is 2 times more molar than the synthesis scale. TBDMS-HMPB was dissolved in DMF (0.50 M), then PyOxim was added and stirred at room temperature for 10 minutes, and then DIEA was added. Next, the solution was introduced into a syringe containing the resin. The coupling was performed overnight at room temperature. After HMPB coupling, the resin was washed with DMF (2x 8 mL / g) and capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes. Next, the resin was washed with DMF (7x 8 mL / g), isopropanol (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0319] Initial HMPB-resin (each 300mg) is introduced into 12mL syringe, and swells in THF (6.5mL / g, 2x1h).Synthesis is carried out manually, and wherein syringe is shaken in horizontal position with 400rpm together with Activo-PLS 4x4 synthesizer purchased from Activotec.At room temperature, use Et3N.3HF (12 equivalents compared with synthesis scale) in THF (0.22M) to react 2 hours, after TBDMS group is deprotected, carry out Fmoc-Gly-OH coupling.After TBDMS deprotection, use DIEA (1x) to clean resin, then use DMF until pH is neutral.

[0320] After swelling the resins in DMF (6.5 mL / g, 2x 1 h), their coupling with Fmoc-Gly-OH was performed. All couplings were performed using Fmoc-Gly-OH, Oxyma, N′N′-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) in DMF (0.45 M) in a 1:1:1:0.25 molar ratio, which was a 2.4-fold molar excess compared to the synthetic scale. Fmoc-Gly-OH was dissolved in DMF (0.45 M), then Oxyma, DIC and DMAP were added and stirred at room temperature for 5 minutes before being introduced into the syringe containing the resin. The coupling was performed overnight at room temperature. After coupling, the resin was washed with DMF (2x 8 mL / g) and capped with DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 minutes. Next, the resin was washed with DMF (7 x 8 mL / g), isopropanol (iPrOH, 3x), and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0321] Table 7 shows the resin substitution before and after Gly-HMPB coupling.

[0322]

[0323] Table 7

[0324] (Fmoc-Linker)2-Lys(1)-Gly-AMS Insoluble Support [2X(Gly)]

[0325] Connectors: Fmoc-Rink and Fmoc-Ramage

[0326] Molecular weight of the corresponding construct without support:

[0327] Fmoc-Rink: Molecular weight (g / mol): 1245.40

[0328] Fmoc-Ramage: Molecular weight (g / mol): 1291.47

[0329] The construct of the insoluble support comprises a lysine, a glycine spacer and two Fmoc-Rink-linkers or two Fmoc-Ramage-linkers coupled to the amine group of the first generation (first layer) lysine, also known as the distal lysine (distal branching agent). The glycine spacer is located between the polymer matrix and the lysine branching agent.

[0330] The (Fmoc-Rink)2-Lys(1)-Gly and (Fmoc-Ramage)2-Lys(1)-Gly constructs contain a layer (first generation) of lysines, i.e., the primary layer, i.e., the primary lysines. Each construct provides two distal binding sites that can be used for peptide synthesis. Two Rink linkers or two Ramage linkers are bound to the amine groups of each lysine (the N-terminal alpha amine and the side chain epsilon amine). Lysine is bound to glycine, and glycine is bound to the amine of the polymer matrix.

[0331] Only lysine was used, in which both amine groups were protected by Fmoc.

[0332] The polymer matrices used for modification were AMS 0.6, AMS 0.93 and AMS 1.95.

[0333] Reactants: Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH

[0334] The structural formula of (Fmoc-Rink)2-Lys(1)-Gly construct

[0335]

[0336] The structural formula of (Fmoc-Ramage)2-Lys(1)-Gly construct

[0337]

[0338] Synthesis scheme of (Fmoc-linker)2-Lys(1)-Gly-AMS:

[0339] Connectors: Rink and Ramage

[0340]

[0341] (Linker) 4-Lys(2)2-Gly-Lys(1)-Gly-AMS Insoluble Support [4X(Gly)]

[0342] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS

[0343] Molecular weight of the corresponding construct without support:

[0344] Fmoc-Rink: Molecular weight (g / mol): 2658.99

[0345] Fmoc-Ramage: Molecular weight (g / mol): 2522.93

[0346] HMPB TBDMS: molecular weight (g / mol): 1461.66

[0347] The constructs of these modified resins contain Gly (G) in addition to three lysines and four linkers coupled to the second generation lysines. Gly or β-Ala spacers are located between the first and second generation lysines and between the first generation lysines and the polymer matrix.

[0348] The (Fmoc-Rink)4-Gly(2)2-Gly2-Lys(1)-Gly, (Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly and (HMPB TBDMS)4-Lys(2)2-Gly2-Lys(1)-Gly constructs contain two layers of (alternative) lysines, a primary layer and a secondary layer, i.e., a primary lysine and a secondary lysine, the secondary lysine layer being the lysine farthest from the polymer matrix. The two secondary lysines in the second layer provide a total of four secondary binding sites that can be used for peptide synthesis. Two Fmoc-Rink linkers, two Fmoc-Ramage linkers or two HMPB (TBDMS) linkers are bound to the two amine groups (N-terminal) of each secondary lysine. α α amine and side chain ε amine). Thus, this construct has four linkers.

[0349] The polymer matrices used for modification were AMS 0.6, AMS 0.93 and AMS 1.95.

[0350] Only lysine was used, in which both amine groups were protected by Fmoc.

[0351] Reactants; Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-β-Ala-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH, HMPB TBDMS-OH

[0352] The structural formula of (Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly construct is:

[0353]

[0354] The structural formula of (Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly construct is:

[0355]

[0356] The structural formula of (TBDMS-HMPB)4-Lys(2)2-Gly2-Lys(1)-Gly construct is:

[0357]

[0358] To characterize the 4X construct, (NH2-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-NH2 and (NH2-Rink)4-Lys(2)2-βAla2-Lys(1)-βAla-NH2 were synthesized using Fmoc chemistry on a Symphony multichannel peptide synthesizer. To cleave the construct from the polymer matrix, sieber amide resin (Cas 915706-90-0) was used. Branching agents Fmoc-Lys(Fmoc)-OH, Oxyma, Dic (diisopropylcarbodiimide) and spacers (Gly, βAla = β-alanine) were used in 3 times molar amounts of the theoretical free amino group. Amide coupling was allowed to proceed for 3 hours at room temperature. The resulting constructs were characterized by LC / MC.

[0359] (NH2-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-NH2

[0360] Calculated quality: 1770.99

[0361] Calculated value: (M+2H)2+=886.49

[0362] Observed value: (M+2H)2+=885.48

[0363]

[0364] (NH2-Rink)4-Lys(2)2-βAla2-Lys(1)-βAla-NH2

[0365] Calculated mass: 1810.89

[0366] Calculated value (M+2H)2+ ​​= 906.46

[0367] Observed value: (M+2H)2+=907.36

[0368]

[0369] (Linker) 4-K(2)2-G2-K(1)-G-AMS) Synthesis Scheme:

[0370] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS

[0371]

[0372]

[0373] (Linker)8-Lys(3)4-Gly4-Lys(2)2-Gly2-K(1)-Gly-AMS Insoluble Support [8X(Gly)]

[0374] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS

[0375] Fmoc-Rink: Molecular weight (g / mol): 5486.17

[0376] Fmoc-Ramage: Molecular weight (g / mol): 4370.12

[0377] HMPB TBDMS: molecular weight (g / mol): 4005.64

[0378] The construct of this insoluble support theoretically increases each primary binding site of the basic AMS resin by 8 times. The construct contains seven lysines, seven glycines and eight linkers. The construct has three layers / three generations of lysines, one primary (proximal) lysine, two secondary (intermediate) lysines and four tertiary (distal) lysines. All secondary intermediate lysines between the primary (proximal) lysine and the distal lysine (here tertiary lysine) are bound to three different lysines, indicating that the construct is symmetrical.

[0379] The polymer matrix used for modification was AMS 0.6.

[0380] All lysines were of the type Fmoc-Lys(Fmoc)-OH.

[0381] Reactants; Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH, TBDMS-HMPB-OH

[0382] Structure of (Fmoc-Rink)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS insoluble support

[0383]

[0384] Structure of (Fmoc-Ramage)8-K(3)4-G4-K(2)2-G2-K(1)-G-AMS insoluble support

[0385]

[0386] Structure of (TBDMS-HMPB)8-K(3)4-G4-K(2)2-G2-K(1)-G-AMS insoluble derivative

[0387]

[0388] Synthesis scheme of (linker)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0389] Connector: Fmoc-Rink, Fmoc-Ramage, TBDMS-HMPB

[0390]

[0391]

[0392] (Fmoc-Rink)4-(PEG6)4-Lys(2)2-Lys(1)-AMS Insoluble Support [4X(PEG)]

[0393] The construct of this modified insoluble support contains, in addition to three lysines and four Fmoc-Rink-linkers, a PEG6 spacer, which is located between the distal lysine and the Fmoc-Rink-linker.

[0394] The PEG spacer was provided as 2-[2-[2-(Fmocamino)ethoxy]ethoxy]acetic acid (EAAE). Thus, the PEG6 spacer was formed by coupling 6 AEEA(PEG): to each amine group of the secondary (distal) lysine.

[0395]

[0396] The polymer matrix used for modification was AMS 0.6.

[0397] Only lysine was used, in which both amine groups were Fmoc-protected.

[0398] Reactants: Fmoc-Lys(Fmoc)-OH, AEEA, Fmoc-Rink-OH

[0399] Synthesis of Insoluble Support (Fmoc-Rink)4-(PEG6)4-Lys(2)2-Lys(1)-AMS

[0400] The Fmoc protected amino acid (K) and AEEA (PEG) were coupled by repeated reaction steps, including deprotection of the Fmoc group and then coupling of the relevant Fmoc protected amino acid and AEEA. After the last AEEA was coupled continuously, the coupling joint was performed. Equimolar ratios of Fmoc-Lys(Fmoc)-OH, AEEA and Fmoc-Rink-OH and Oxyma pure (Ethyl cyano(hydroxyimino)acetate: ) and diisopropylcarbodiimide (DIC) were used to form amide bonds in an excess of 3 times the theoretical free amino groups in DMF (calculated from the first AMS binding site). The Fmoc group was removed by two treatments (5 minutes and 20 minutes, respectively) with 25% (v / v) piperidine in DMF before each coupling step. The amide coupling procedure was carried out at room temperature for 2 hours.

[0401] Synthesis scheme of (Fmoc-Rink)4-(PEG6)4-LysK(2)2-LysK(1)-AMS:

[0402]

[0403]

[0404] Structure of (Fmoc-Rink)4-(PEG6)4-Lys(2)2-Lys(1)-NH2 construct:

[0405]

[0406] Molecular weight 5971.62

[0407] (Linker) 7-LysGly-Linear-Gly-AMS Insoluble Support [7X Linear]

[0408] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS

[0409] Molecular weight of the corresponding construct without support:

[0410] Fmoc-Rink: Molecular weight (g / mol): 4608.22

[0411] Fmoc-Ramage: Molecular weight (g / mol): 4370.12

[0412] TBDMS-HMPB: Molecular weight (g / mol): 3312.76

[0413] The construct of the insoluble support contains six lysines, three glycines as spacers, and seven linkers selected from Fmoc-Rink-OH, Fmoc-Ramage, HMPB TBDMS, which are coupled to four of the six lysines.

[0414] In the synthesis of the linear 7X insoluble support, two types of lysine were used that differed in terms of protecting groups: one type of lysine had both amine groups protected by Fmoc: Fmoc-Lys(Fmoc) and the ε amine protected by Mtt: Fmoc-Lys(Mtt)-OH.

[0415] The construct of 7X insoluble support is synthesized by first synthesizing the main chain only via coupling Fmoc-Lys (Mtt) -OH. The main chain is a chain in which all amide bonds are related to α amines. After the main chain comprising three lysines and three glycines is synthesized, 30% hexafluoroisopropanol HFIP solution in DCM is used to crack Mtt 1 hour at 25 ℃. In subsequent steps, Fmoc-Lys (Fmoc) is coupled to the epsilon amine of each lysine of the main chain. After this, all Fmoc groups are cracked using 25% (v / v) piperidine in DMF twice (5 minutes and 20 minutes respectively). Finally, the linker is coupled to lysine. These seven form 7 secondary (distal) binding sites.

[0416] The side chain amine of each lysine on the backbone is coupled to only one lysine each, thereby forming the construct in what is referred to herein as a "linear" conformation.

[0417] Use equal molar ratios of Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Mtt)-OH, linker and Oxymapure (ethyl cyano(hydroxyimino)acetate: ) and diisopropylcarbodiimide (DIC) were used to form amide bonds in an excess of 3 times the theoretical free amino groups in DMF (calculated from the first AMS binding site). Where applicable, the Fmoc group was removed by two treatments (5 minutes and 20 minutes, respectively) with 25% (v / v) piperidine in DMF before each coupling step. The amide coupling process lasted for 2 hours.

[0418] The polymer matrix used for modification was AMS 0.6.

[0419] 7X Linear Synthesis Scheme:

[0420]

[0421]

[0422] This modified solid resin has a construct containing lysines that bind only to one other lysine, possibly via a glycine spacer. The presence of lysines that bind only to one other lysine (lysines with other properties) provides an odd number of secondary binding sites: here there are seven secondary binding sites.

[0423] The structural formula of (Fmoc-Rink)7-LysGly-Linear-Gly-AMS insoluble support is:

[0424]

[0425] The structural formula of (Fmoc-Ramage)7-LysGly-Linear-Gly-AMS insoluble support is:

[0426]

[0427] The structural formula of (TBDMS-HMPB)7-LysGly-Linear-Gly-AMS insoluble support:

[0428]

[0429] (Fmoc-Rink) 16 -Lys(4)8-Gly8-lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS[16X (Gly)]

[0430] Molecular weight (g / mol): 1112.49

[0431] The 16X (Gly) insoluble support comprises a construct that increases the primary binding site of the basic AMS resin by 16 times. The construct comprises 15 lysines, 15 glycine spacers and 16 Fmoc-Rink-linkers. The construct is symmetrical and contains four layers of (generation) lysines, i.e., eight quaternary lysines Lys (4), four tertiary lysines Lys (3), two secondary lysines Lys (2) and one primary lysine Lys (1). In addition, all lysines (secondary Lys (2) and tertiary Lys (3)) between the lysine bound to the primary binding site of the basic resin and the quaternary lysine Lys (4) are bound to three different lysines. For example, each of the two secondary lysines Lys (2) is bound to a primary lysine Lys (1) and two tertiary lysines Lys (3). If all lysines except the distal (in this case, the distal lysine is the quaternary lysine Lys(4)) and the primary lysine are bound to three lysines, then the construct is symmetrical. In addition, the number of secondary binding sites is a function of the following equation: Y = 2 n , where n represents the number of layers of lysine in the construct. Here there are 4 layers, so there are 16 secondary binding sites.

[0432] The polymer matrix used for modification was AMS 0.6.

[0433] All lysines were Fmoc-Lys(Fmoc).

[0434] Reactants: Fmoc-Lys(Fmoc), Fmoc-Gly, Fmoc-Rink

[0435] (Fmoc-Rink) 16 -Lys(4)8-Gly8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS insoluble The structural formula of the sexual support:

[0436]

[0437] (Fmoc-Rink) 16 -Lys(4) 8 -Gly 8 -Lys(3) 4 -Gly 4 -Lys(2) 2 -Gly 2 -Lys(1)-Gly-AMS become

[0438] Peptide constructs were synthesized on a manual peptide synthesis reactor.

[0439] Fmoc-protected lysine was coupled to the amine group of AMS using equimolar ratios of Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH and Oxyma pure (ethyl cyano(hydroxyimino)acetate: ) and diisopropylcarbodiimide (DIC) were used to form amide bonds in an excess of 3 times the theoretical free amino groups in DMF (calculated from the first AMS binding site). Before each coupling step, the Fmoc group was removed by two treatments (5 minutes and 20 minutes, respectively) with 25% (v / v) piperidine in DMF. The amide coupling process lasted for 2 hours.

[0440] Synthesis scheme of 16X(Gly)

[0441]

[0442]

[0443] Peptide synthesis using the insoluble support of the present invention

[0444] The template polypeptide sequences PP1 to PP6 were synthesized using the following insoluble supports (as detailed above):

[0445] Support with Rink connector:

[0446] 1X: (Fmoc-Rink)-AMS 0.6 (resin without construct)

[0447] 1X: (Fmoc-Rink)-AMS 0.93 (resin without construct)

[0448] 1X: (Fmoc-Rink)-AMS1.95 (resin without construct)

[0449] ·2X(Gly): (Fmoc-Rink)2-Lys(1)-gly-AMS 0.6

[0450] ·2X(Gly): (Fmoc-Rink)2-Lys(1)-Gly-AMS 0.93

[0451] ·2X(Gly): (Fmoc-Rink)2-Lys(1)-Gly-AMS1.95

[0452] ·4X(Gly): (Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 0.6

[0453] ·4X(Gly): (Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 0.93

[0454] ·4X(Gly): (Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS1.95

[0455] ·8X(Gly): (Fmoc-Rink)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 0.6

[0456] Support with Ramage connector:

[0457] 1X: (Fmoc-Ramage)-AMS 0.6 (resin without construct)

[0458] 1X: (Fmoc-Ramage)-AMS 0.93 (resin without construct)

[0459] 1X: (Fmoc-Ramage)-AMS1.95 (resin without construct)

[0460] ·2X(Gly): (Fmoc-Ramage)2-Lys(1)-Gly-AMS 0.6

[0461] ·2X(Gly): (Fmoc-Ramage)2-Lys(1)-Gly-AMS 0.93

[0462] ·2X(Gly): (Fmoc-Ramage)2-Lys(1)-Gly-AMS1.95

[0463] ·4X(Gly): (Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 0.6

[0464] ·4X(Gly): (Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 0.93

[0465] ·4X(Gly): (Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS1.95

[0466] Support with HMPB TBDMS linker:

[0467] 1X: HMPB TBDMS AMS 0.6 (resin without construct)

[0468] 1X: HMPB TBDMS AMS 0.93 (resin without construct)

[0469] ·4X(Gly): (HMPB TBDMS)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 0.6

[0470] ·8X(Gly): (HMPB TBDMS)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 0.6

[0471] The reference resin is aminomethyl (AM / AMS) polystyrene resin crosslinked with 1% divinylbenzene, with a particle size of 100-200 mesh (75-150 μm) and degrees of substitution of 0.6, 0.93 and 1.95 mmol / g, respectively.

[0472] The following peptides were synthesized:

[0473] PP 1: WLFAGGPSSGAPPPS (15-mer)

[0474] PP 2: YAEEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS (39-mer)

[0475] PP 3: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (39-mer)

[0476] PP 4: fFPRPGGGGNGDFEEIPEEYL (20mer)

[0477] PP 5: FVQYLIQG (8-mer)

[0478] PP 6: HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (31mer)

[0479] Construct:

[0480] 1X: No construct, unmodified resin containing only linkers as shown below.

[0481] 2X(G1y): -Gly-Lys(1)-Linker 2

[0482] 4X(Gly):-Gly-Lys(1)-Gly2-Lys(2)2-Connector 4

[0483] 8X(Gly):-Gly-Lys(1)-Gly2-Lys(2)2-Gly4-Lys(3)4-linker 8

[0484] Tables 8 to 14 show various data related to polypeptide synthesis.

[0485] In some columns the assembly yield [%] and the cleavage yield [%] are indicated. These parameters were calculated as follows:

[0486] (1)

[0487] (2)

[0488] Synthesis scheme of PP 1 using DMF as solvent

[0489] The initial resin (400 mg each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2 x 1 h), an initial swelling measurement was performed, and then it was transferred to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 min + 1 x 20 min). All amino acid couplings were performed using equimolar ratios (1:1:1) of Fmoc amino acids (0.30 M in DMF), Oxyma (0.90 M in DMF) and N'N'-diisopropylcarbodiimide (0.90 M in DMF), which were in 5-fold molar excess compared to the synthetic scale. The coupling was performed at room temperature for 1 hour and 30 minutes without preactivation.

[0490] After the synthesis was completed, the resin was transferred to a 25 mL syringe again, its final swelling was measured, and then it was washed with isopropanol (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0491] The dried Fmoc protected peptidyl resin was re-swelled in DMF (6.5 mL / g, 2 x 1 h). The Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 min + 1 x 20 min). Next, the resin was washed with DMF (7 times), iPrOH (3 times) and MTBE (3 times) and then redried under vacuum overnight.

[0492] Use the cleavage mixture containing TFA (trifluoroacetic acid) / H2O / DTT (dithiothreitol) (13mL / g peptide-based resin, 85 / 5 / 5v / v / w) to cleave at room temperature for 2 hours, and the dry Fmoc deprotected peptide is cleaved from the resin. Then add TIS (5%v) and continue stirring for 1 hour. Filter the resin and wash with TFA (3mL / g peptide-based resin). Use MTBE (10 times the volume of TFA) to precipitate the peptide at 0°C. The resulting suspension is transferred to a 50ml conical centrifuge tube and centrifuged at 2500rcf for 10 minutes, then the supernatant is decanted. Wash the crude solid product with cold MTBE (5x) again and dry overnight under vacuum.

[0493] Molecular weight: 1426.60 Calculated mass: +2 / 2 = 714.30

[0494] Observed mass (AMS 0.932x(Gly))+2 / 2=714.05

[0495] Observed mass (AMS1.952x(Gly)): +2 / 2 = 714.43

[0496] Observed mass (AMS1.954x(Gly)): +2 / 2 = 714.64

[0497] All 15 amino acids used were Fmoc protected.

[0498] Side chain protecting groups involved: tBu, Boc: S = Ser (tBu) and W = Trp (Boc)

[0499] Molecular weight of protected PP 1: 1917,28 g / mol

[0500] Molecular weight of unprotected PP 1: 1426,60 g / mol

[0501] Table 8: Synthesis data of 15-mer polypeptide: WLFAGGPSSGAPPPS:PP 1

[0502] Solvent used for synthesis: DMF

[0503] After Rink linker coupling, substrate mass at the start of synthesis: 400 mg

[0504]

[0505]

[0506] Table 8

[0507]

[0508] Table 8 (continued)

[0509] The embodiments of the support of the present invention significantly improve the throughput while substantially maintaining or even improving the purity.

[0510] Synthesis of PP 1: DMsO / EtOAc (3:7 v / v) and DMF as solvent

[0511] The initial resin (400 mg each) was taken into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2x 1 h). The synthesis was performed manually with the syringe shaken at 400 rpm in a horizontal position with an Activo-PLS 4x4 synthesizer purchased from Activotec. Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMSO / EtOAc (3:7, v / v) or DMF at room temperature (1x 10 min + 1x 20 min). After Fmoc deprotection, the resin was washed with DMSO / EtOAc (3:7, v / v) or DMF (7x 6.5 mL / g initial resin). All amino acid couplings were performed using an equimolar ratio (1:1:1) of Fmoc amino acids, Oxyma and N'N'-diisopropylcarbodiimide in DMSO / EtOAc (3:7, v / v) or DMF (0.30 M), which was in 2-fold molar excess compared to the synthetic scale. The Fmoc amino acid was dissolved in the corresponding solvent / solvent mixture (0.30 M), then Oxyma and N'N'-diisopropylcarbodiimide were added and stirred at room temperature for 5 minutes before being introduced into the syringe containing the resin. The coupling was performed at room temperature for 1 hour and 30 minutes. After coupling, the resin was washed with DMSO / EtOAc (3:7, v / v) or DMF (2x 6.5 mL / g initial resin).

[0512] After the synthesis was completed, the final resin swelling was measured and the resin was then washed with (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x) and then dried under vacuum overnight.

[0513] The dried Fmoc-protected peptidyl resin was re-swelled in DMF (6.5 mL / g, 2 x 1 h). The Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 min + 1 x 20 min). Next, the resin was washed with DMF (7 times), iPrOH (3 times), and MTBE (3 times), and then redried under vacuum overnight.

[0514] The dried Fmoc deprotected peptide was cleaved from the resin using a cleavage mixture containing TFA / H2O / DTT (13mL / g peptide resin, 85 / 5 / 5v / v / w) for 2 hours at room temperature. TIS (5%v) was then added and stirring continued for 1 hour. The resin was filtered and washed with TFA (3mL / g peptide resin). The peptide was precipitated with MTBE (10 volumes of TFA) at 0°C. The resulting suspension was transferred to a 50ml conical centrifuge tube and centrifuged at 2500rcf for 10 minutes, then the supernatant was decanted. The crude solid product was washed again with cold MTBE (5x) and dried overnight under vacuum.

[0515] After Rink amide linker coupling, mass of resin at the start of synthesis: 400 mg

[0516] Side chain protecting groups involved: tBu, Boc: S = Ser (tBu) and W = Trp (Boc)

[0517] Molecular weight of protected PP 1: 1917,28 g / mol

[0518] Molecular weight of unprotected PP 1: 1426,60 g / mol

[0519] Table 9: Synthesis data of 15-mer polypeptide: WLFAGGPSSGAPPPS:PP1

[0520] Solvent used for synthesis: DMSO / EtOAc (3:7), except for the first row, after coupling of AMS 0.6 with DMF Rink linker, substrate mass at the beginning of the synthesis: 400 mg

[0521]

[0522] Table 9

[0523]

[0524] Table 9 (continued)

[0525] Synthesis scheme of PP 2(YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS) using DMF as solvent

[0526] The initial resin (200 mg each) was introduced into a 12 mL syringe and swelled in DMF (6.5 mL / g, 2 x 1 h), an initial swelling measurement was performed, and then it was transferred to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 min + 1 x 20 min). All amino acid couplings were performed using equimolar ratios (1:1:1) of Fmoc amino acid (0.30 M in DMF), Oxyma (0.90 M in DMF), and N'N'-diisopropylcarbodiimide (0.90 M in DMF) in a 5-fold molar excess compared to the synthetic scale. Except for Thr, 5 Coupling within 8 hours and Ile 12和17 Except for couplings within 6 hours, couplings were performed for 2 hours at room temperature without preactivation.

[0527] After the synthesis was completed, the resin was transferred again to a 12 mL syringe, its final swelling was measured, and then washed with (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0528] The peptide of dry Boc protection is cleaved from the resin using a cleavage mixture containing TFA / H2O / DTT / TIS (13mL / g peptidyl resin, 85 / 5 / 5 / 5v / v / w / v) for 3 hours at room temperature. The resin is filtered and washed with TFA (3mL / g peptidyl resin). The peptide is precipitated with MTBE (10 volumes of TFA) at 0°C. The resulting suspension is transferred to a 50ml conical centrifuge tube and centrifuged at 2500rcf for 10 minutes, then the supernatant is decanted. The crude solid product is washed with cold MTBE (5x) again and dried overnight under vacuum.

[0529] Molecular weight: 4041.54 Calculated mass: +3 / 3 = 1348.26; +4 / 4 = 1011.38

[0530] Observed mass (AMS 0.62X (Gly)): +3 / 3 = 1347.98; +4 / 4 = 1011.70

[0531] Observed mass (AMS 0.934X(Gly)): +3 / 3 = 1348.6; +4 / 4 = 1011.67

[0532] Table 10: Synthesis data of 39-mer polypeptide: YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS:PP 2

[0533] Solvent used for synthesis: DMF

[0534] After Ramage linker coupling, substrate mass at the start of synthesis: 200 mg

[0535]

[0536]

[0537] Table 10

[0538]

[0539]

[0540] Table 10 (continued)

[0541]

[0542] Table 10 (continued)

[0543] Synthesis scheme of PP 3(HGEGTFTSDLSKQMEEEAVRLFXEWLKNGGPSSGAPPPS) using DMF as solvent

[0544] The initial resin (200 mg each) was introduced into a 12 mL syringe and swelled in DMF (6.5 mL / g, 2 x 1 h), an initial swelling measurement was performed, and then it was transferred to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 min + 1 x 20 min). All amino acid couplings were performed using equimolar ratios (1:1:1) of Fmoc amino acid (0.30 M in DMF), Oxyma (0.90 M in DMF), and N'N'-diisopropylcarbodiimide (0.90 M in DMF) in a 5-fold molar excess compared to the synthetic scale. Except for Thr, 5 Couplings were performed for 2 h at room temperature without preactivation, except for coupling within 8 h and Ile 12 and 17 within 6 h.

[0545] After the synthesis was completed, the resin was transferred again to a 12 mL syringe, its final swelling was measured, and then washed with (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0546] The peptide of dry Boc protection is cleaved from the resin using a cleavage mixture containing TFA / H2O / DTT / TIS (13mL / g peptidyl resin, 85 / 5 / 5 / 5v / v / w / v) for 3 hours at room temperature. The resin is filtered and washed with TFA (3mL / g peptidyl resin). The peptide is precipitated with MTBE (10 volumes of TFA) at 0°C. The resulting suspension is transferred to a 50ml conical centrifuge tube and centrifuged at 2500rcf for 10 minutes, then the supernatant is decanted. The crude solid product is washed with cold MTBE (5x) again and dried overnight under vacuum.

[0547] Molecular weight: 4186.63

[0548] Calculated mass: +3 / 3 = 1396.54; +4 / 4 = 1047.65

[0549] Observed mass (AMS 0.62X (Gly)): +3 / 3 = 1397.12; +4 / 4 = 1048.11

[0550] Observed mass (AMS 0.64X(Gly)): +3 / 3 = 1396.89; +4 / 4 = 1047.65

[0551] All amino acids used were protected by Fmoc, except the last amino acid (Tyr) which was protected by Boc.

[0552] Side chain protecting groups involved: tBu, Boc, Trt: S = Ser (tBu), W = Trp (Boc), Q = Gln (Trt), K = Lys (Boc), D = Asp (OtBu), Y = Tyr (tBu), T = Thr (tBu), E = Glu (OtBu)

[0553] Molecular weight of protected 39-mer: 5,596.22 g / mol

[0554] Molecular weight of unprotected 39-mer: 4,041.54 g / mol

[0555] Table 11: Synthesis data of 39-mer polypeptide: HGEGTFTSDLSKQMEEEAVRLFXEWLKNGGPSSGAPPPS:PP 3

[0556] Solvent used for synthesis: DMF

[0557] After Ramage linker coupling, substrate mass at the start of synthesis: 200 mg

[0558]

[0559]

[0560] Table 11

[0561]

[0562] Table 11 (continued)

[0563]

[0564] Table 11 (continued)

[0565] Embodiments of the supports described herein provide significantly increased throughput at good / commercially relevant / useful purity.

[0566] Synthesis scheme of PP 4(fPRPGGGGNGDFEEIPEEYL) using DMF as solvent

[0567] The initial resin (200 mg each) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2 x 1 h), an initial swelling measurement was performed, and then it was transferred to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 min + 1 x 20 min). All amino acid couplings were performed using equimolar ratios (1:1:1) of Fmoc amino acids (0.30 M in DMF), Oxyma (0.90 M in DMF) and N'N'-diisopropylcarbodiimide (0.90 M in DMF), which were in 5-fold molar excess compared to the synthetic scale. The coupling was performed at room temperature for 1 hour and 30 minutes without preactivation.

[0568] After the synthesis was completed, the resin was transferred again to a 12 mL syringe, its final swelling was measured, and then washed with (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0569] The peptide of dry Boc protection is cleaved from the resin using a cleavage mixture containing TFA / H2O / DTT / TIS (13mL / g peptidyl resin, 85 / 5 / 5 / 5v / v / w / v) for 3 hours at room temperature. The resin is filtered and washed with TFA (3mL / g peptidyl resin). The peptide is precipitated with MTBE (10 volumes of TFA) at 0°C. The resulting suspension is transferred to a 50ml conical centrifuge tube and centrifuged at 2500rcf for 10 minutes, then the supernatant is decanted. The crude solid product is washed with cold MTBE (5x) again and dried overnight under vacuum.

[0570] Molecular weight: 2180.32

[0571] Calculated mass: +2 / 2 = 1091.16

[0572] Observed mass (AMS 0.64X(Gly)): +2 / 2=1091.02

[0573] Observed mass (AMS 0.68X(Gly)): +2 / 2=1091.06

[0574] All amino acids used were protected by Fmoc, except the last amino acid (D-Phe) which was protected by Boc.

[0575] Side chain protecting groups involved: tBu, Trt, Pbf: Y = Tyr (tBu), E = Glu (OtBu), D = Asp (OtBu), N = Asn (Trt), Arg (Pbf)

[0576] Molecular weight of protected 20-mer: 3233,86 g / mol

[0577] Molecular weight of unprotected 20-mer: 2,180.32 g / mol

[0578] Table 12: Synthesis data of 20-mer polypeptide: fPRPGGGGNGDFEEIPEEYL:PP 4

[0579] Solvent used for synthesis: DMF

[0580] Substrate mass at the start of synthesis after HMPB-Leucine linker coupling: 200 mg

[0581]

[0582] Table 12

[0583]

[0584] Table 12 (continued)

[0585] Weight gain [mg] Assembly yield [%] (1) Pyrolysis yield [%] (2) 62 20 57 290 53 76 330 59 67

[0586] Table 12 (continued)

[0587] The use of the support described in the present invention significantly improves the throughput while substantially maintaining the purity.

[0588] Synthesis scheme of PP 5(FVQYLIQG) using DMF as solvent

[0589] The initial resin (100 mg each Fmoc-HMPB-Gly-AMS 0.60) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2x 1 h), an initial swelling measurement was performed, and then it was transferred to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1x 10 minutes + 1x20 minutes). All amino acid couplings were performed using equimolar ratios (1:1:1) of Fmoc amino acids (0.30 M in DMF), Oxyma (0.90 M in DMF), and N'N'-diisopropylcarbodiimide (0.90 M in DMF), which were 5 times molar excess compared to the synthetic scale. Couplings were performed at room temperature for 1 hour and 30 minutes without preactivation.

[0590] After the synthesis was completed, the resin was transferred again to a 12 mL syringe, its final swelling was measured, and then washed with (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0591] The dried Fmoc protected peptide fragment was cleaved from the resin in 15 minutes using a soft cleavage mixture of dichloromethane (DCM) containing 3% TFA. This treatment was performed 3 times, filtering the resin each time and collecting the resulting solution. The combined collected solutions were evaporated under vacuum, and the concentrated oil obtained was precipitated in MTBE / heptane (1:1, v / v), then filtered under vacuum and dried overnight.

[0592] All amino acids used were Fmoc protected.

[0593] Side chain protecting groups involved: tBu, Trt: Q = Gln (Trt), Y = Tyr (tBu)

[0594] Molecular weight of protected octamer: 1,730.1 g / mol

[0595] Table 13: Synthesis of 8-mer polypeptide: FVQYLIQG:PP 5

[0596] Solvent used for synthesis: DMF

[0597] Substrate mass at the start of synthesis after HMPB-glycine linker coupling: 100 mg

[0598]

[0599] Table 13

[0600]

[0601] Table 13 (continued)

[0602]

[0603] Table 13 (continued)

[0604] The use of the support described in the present invention significantly improves the throughput while substantially maintaining the purity.

[0605] Synthesis scheme of PP 6 (HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG) using DMF as solvent

[0606] The initial resin (200 mg each) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2 x 1 h), an initial swelling measurement was performed, and then it was transferred to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% ​​piperidine in DMF at room temperature (1 x 10 min + 1 x 20 min). All amino acid couplings were performed using equimolar ratios (1:1:1) of Fmoc amino acids (0.30 M in DMF), Oxyma (0.90 M in DMF) and N'N'-diisopropylcarbodiimide (0.90 M in DMF), which were in 5-fold molar excess compared to the synthetic scale. The coupling was performed at room temperature for 1 hour and 30 minutes without preactivation.

[0607] After the synthesis was completed, the resin was transferred again to a 12 mL syringe, its final swelling was measured, and then washed with (iPrOH, 3x) and methyl tert-butyl ether (MTBE, 3x), and then dried under vacuum overnight.

[0608] The peptide of dry Boc protection is cleaved from the resin using a cleavage mixture containing TFA / H2O / DTT / TIS (13mL / g peptidyl resin, 85 / 5 / 5 / 5v / v / w / v) for 3 hours at room temperature. The resin is filtered and washed with TFA (3mL / g peptidyl resin). The peptide is precipitated with MTBE (10 volumes of TFA) at 0°C. The resulting suspension is transferred to a 50ml conical centrifuge tube and centrifuged at 2500rcf for 10 minutes, then the supernatant is decanted. The crude solid product is washed with cold MTBE (5x) again and dried overnight under vacuum.

[0609] Molecular weight: 3383.73

[0610] Calculated mass: +3 / 3 = 1128.91; +4 / 4 = 846.93

[0611] Observed mass (AMS 0.64X (Gly)): +3 / 3 = 1129.01; +4 / 4 = 847.06

[0612] Solvent used for the synthesis of PP 6 on Symphony X: DMF

[0613] Mass of resin at the start of synthesis after HMPB-glycine linker coupling: 200 mg.

[0614] All amino acids used were protected with Fmoc, except the last amino acid (His) which was protected with Boc.

[0615] Side chain protecting groups involved: tBu, Boc, Trt, Pbf: R = Arg (Pbf), W = Trp (Boc), E = Glu (OtBu), K = Lys (Boc), Q = Gln (Trt), Y = Tyr (tBu), S = Ser (tBu), D = Asp (OtBu), T = Thr (tBu), H = His (Trt)

[0616] Molecular weight of protected PP 6: 5,234.46 g / mol

[0617] Molecular weight of unprotected PP 6: 3,383.73 g / mol

[0618] Table 14 Synthesis data of PP 6 polypeptide: HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG

[0619]

[0620]

[0621] Table 14

[0622]

[0623] Table 14 (continued)

[0624]

[0625] Table 14 (continued)

Claims

1. An insoluble support (resin) in the form of particles comprising a distal binding site, the support comprising a homogeneous polymer matrix and a construct, the construct being covalently bound to the polymer matrix, wherein the construct comprises at least one branching agent selected from aminoalkanoic acids containing at least 2 amino groups and 3 to at most 10 carbon atoms, a cleavable linker and at least one spacer coupled to the at least one branching agent via an amide bond, the cleavable linker providing the distal binding site.

2. The insoluble support according to claim 1, wherein the branching agent is selected from aminoalkanoic acids comprising at least 2 but not more than 3 amino groups and 3 to at most 10 carbon atoms.

3. The insoluble support according to claim 1 or 2, wherein the branching agent is selected from diaminoalkanoic acids containing 3 to at most 10 carbon atoms.

4. An insoluble support according to any one of claims 1 to 3, wherein the branching agent is selected from diaminoalkanoic acids containing 3 to at most 8 carbon atoms, preferably 3 to 6 carbon atoms.

5. An insoluble support according to any one of claims 1 to 4, wherein the branching agent is selected from 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid, suitably, the branching agent is selected from 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid.

6. The insoluble support according to any one of claims 1 to 5, wherein the branching agent is lysine.

7. An insoluble support according to any one of claims 1 to 6, wherein all branching agents are the same.

8. The insoluble support according to claim 3, wherein the number of branching agents δ of the construct is given by the formula δ(n)=2 n -1, and the number of linkers providing distal binding sites λ is given by the formula λ(n)=2 n Given as follows, wherein n represents the generation of the branching agent, n is 1 to 10, preferably n is 1 to 5.

9. The insoluble support according to claim 8, wherein n is 2 to 10, preferably 2 to 5.

10. The insoluble support according to claim 9, wherein at least one spacer is located between all branching agents.

11. The insoluble support according to claim 3, wherein the construct is selected from (A) [polymer matrix]-BA(1)-LK2; (B) [Polymer matrix]-BA(1)-BA(2)2-LK4; (C) [Polymer matrix]-BA(1)-BA(2)2-BA(3)4-LK8; (D)[Polymer matrix]-BA(1)-BA(2)2-BA(3)4-BA(4)8-LK 16 ; (E) [Aggregate Substrate] -BA(1) -BA(2) 2-BA(3) 4-BA(4) 8-BA(5) 16 -L.K. 32 ; Wherein BA represents a branching agent, the integer in brackets represents the generation of the branching agent, and LK represents a cleavable linker.

12. An insoluble support according to any one of claims 1 to 11; and a construct according to any one of claims 2 to 11; wherein the spacer molecule is selected from an organic molecule comprising two binding sites.

13. The insoluble support according to any one of claims 1 to 12, wherein the spacer molecule is selected from an organic molecule comprising two binding sites, and the binding sites are selected from any one of carboxylic acid, amine, and hydroxyl.

14. The insoluble support according to any one of claims 1 to 13, wherein the spacer molecule is selected from organic molecules comprising two binding sites selected from amino acids and polyethylene glycol.

15. An insoluble support according to any one of claims 1 to 14, wherein the spacer is selected from amino acids comprising two binding sites, suitably glycine and alanine, preferably glycine.

16. The insoluble support according to any one of claims 1 to 15, wherein the linker is selected from the group consisting of Rink amide, Wang, 2-chlorotrityl, PAM, PAL, HMPB, Sieber and Ramage.

17. An insoluble support according to any one of claims 1 to 16; wherein the polymer matrix is ​​selected from a homogeneous polymer matrix comprising primary binding sites distributed throughout the polymer matrix.

18. The insoluble support according to any one of claims 1 to 17, wherein the polymer matrix is ​​selected from a homogeneous polymer matrix formed by emulsion polymerization comprising at least styrene and divinylbenzene (DVB).

19. An insoluble support according to any one of claims 1 to 18, wherein the polymer matrix is ​​chosen from a homogeneous polymer matrix formed by a polymeric composition comprising at least styrene and divinylbenzene (DVB), and DVB is present in an amount lower than 4.0 wt. %, preferably lower than 3.0 wt. %.

20. The insoluble support according to any one of claims 1 to 19, which is used for solid phase peptide synthesis, solid phase morpholino oligomer synthesis and solid phase oligonucleotide synthesis.

21. An insoluble support according to any one of claims 1 to 19 for use in solid phase peptide synthesis.

22. A method of forming an insoluble support as defined in any one of claims 1 to 21; said method comprising providing a polymer matrix comprising primary binding sites, and wherein said construct is formed by divergent synthesis, convergent synthesis or a combination of divergent and convergent synthesis.

23. A method of forming an insoluble support as defined in any one of claims 1 to 21, said method comprising providing a polymer matrix comprising primary binding sites, and wherein said construct is formed by a method comprising at least the steps of: a) optionally coupling at least one spacer to the primary binding site, b) coupling a branching agent to a primary binding site of a base matrix, or optionally coupling a branching agent to at least one spacer, wherein at least two amino groups are protected by protecting groups, c) removing the protecting groups, wherein steps a), b) and c) can be repeated, and d) Coupling the linker to the binding site of the distal branching agent.

24. A solid phase peptide synthesis protocol, solid phase morpholino oligomer synthesis and solid phase oligonucleotide synthesis for the synthesis of polypeptides, morpholino oligomers and oligonucleotides, said protocol comprising the use of an insoluble support as defined in any one of claims 1 to 20.

25. A solid phase peptide synthesis protocol for the synthesis of a polypeptide, said protocol comprising the use of an insoluble support as defined in any one of claims 1 to 19.

26. The solid phase peptide synthesis protocol according to claim 25, wherein the polypeptide has at least 15 amino acids, preferably at least 20 amino acids, preferably at least 25 amino acids.

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