Insoluble support for solid-phase synthesis

Abstract

The present invention relates to an insoluble support comprising a distal binding site, wherein the support comprises a homogeneous polymer matrix and a construct, the construct is covalently bonded to the polymer matrix, and the construct comprises at least one branching agent selected from at least two amino groups and an aminoalkanoic acid containing from 3 to 10 carbon atoms, a cleavable linker, and at least one spacer coupled to the at least one branching agent via an amide bond, and the cleavable linker provides the distal binding site.

Description

Technical Field

[0001] The present invention relates to insoluble supports containing constructs for use in solid-phase organic synthesis such as solid-phase morpholino oligomer synthesis, solid-phase oligonucleotide synthesis, and solid-phase peptide synthesis protocols, methods for preparing the insoluble supports, and methods for synthesizing polypeptides using the insoluble supports.

Background Art

[0002] Heterogeneous liquid-solid chemical reaction protocols are attractive for the synthesis of molecules containing repeating subunits. Liquid-solid protocols allow for the effective separation of the solid phase from the liquid phase and provide conditions suitable for the application of iterative cycles of reaction steps for the sequential stepwise introduction (addition) of subunits. Liquid-solid reaction protocols have been successfully implemented in the fields of peptide, morpholino oligomer, and oligonucleotide synthesis. During synthesis, nascent molecules (such as growing peptides, morpholino oligomers, or oligonucleotides) are covalently attached to an insoluble support, providing conditions for the efficient removal of by-products by washing between reaction steps of the iterative cycle.

[0003] Solid-phase peptide, morpholine, and nucleotide synthesis are established methodologies for generating peptides. Peptides are synthesized in a single reaction vessel, thereby reducing the complexity of the process. Additionally, the isolation and purification of intermediates are avoided or significantly reduced. The ability to use excess reactants leads to commercially reasonable yields with commercially attractive purities already before purification after synthesis.

[0004] Peptides, morpholino oligomers or oligonucleotides are gradually synthesized on an insoluble support (solid phase). The insoluble support provides a binding site for the peptide to be synthesized. Solid-phase peptide synthesis offers important advantages as detailed herein, but the reaction occurs on the insoluble support (phase) and the important reactants are present in the liquid phase. Thus, in solid-phase synthesis, by its very nature, a phase boundary is created that needs to be handled well. The interaction between the solid phase and the liquid phase is important, and the swelling and solvation of the solid phase are important properties. Swelling and solvation affect, for example, the diffusion and accessibility of reagents to the solid phase, as well as the consumption of the washing solution.

[0005] A further property of the solid phase is the number of binding sites available for peptide synthesis, often referred to as the throughput. Even as the number of binding sites increases, the capacity of the insoluble support and the purity of the peptide may not automatically increase. An increase in the capacity of the insoluble support leads to an increase in yield (with respect to peptide output). However, the binding sites must be made available for peptide synthesis, and high throughput is known to be detrimental to the purity and yield of the synthetic peptide, especially when the number of amino acids increases.

[0006] Insufficient swelling and solvation of the solid phase often lead to poor accessibility to the reaction sites and a decrease in the reaction rate. Furthermore, the solid phase is gradually modified by the coupling of amino acids to the nascent peptide bound to the solid phase, so swelling and solvation are affected during peptide synthesis.

[0007] When synthesizing a target polypeptide, a combination of a solid support and a liquid phase is selected to optimize the yield and the crude target polypeptide purity. Usually, such optimization is translated into synthetic specifications that attempt to increase the swelling and solvation of the solid phase as much as possible. An increase in swelling is also equal to an increase in the volume of the reaction composition.

[0008] The reactor volume in the solid-phase peptide synthesis process can sometimes be a limiting factor. The present invention provides a novel insoluble support obtained by converting a standard resin with a construct containing readily available amino acids, said insoluble support showing a good crude peptide yield and a commercially reasonable purity while significantly increasing the reactor throughput. Thus, by applying the novel insoluble support to a given reactor, the throughput of the reactor can be significantly increased. The novel insoluble support provides an opportunity to increase the output of polypeptides without increasing the volume of the reactor.

[0009] In the case of short polypeptides, i.e., polypeptides having a limited number of amino acids, for example less than 10 amino acids, an increase in the binding sites of the resin (often referred to as the throughput [mmol binding sites / g resin]) usually leads to an increase in yield while still achieving a satisfactory purity. As the number of amino acids in the polypeptide increases, an increase in throughput does not necessarily lead to higher yields and useful purities. On the contrary, polypeptides having a fairly large number of amino acids (e.g., more than 30 amino acids) may even be impossible to synthesize if the yield is low or the throughput of the resin is not reduced. Thus, when synthesizing long polypeptides, usually a resin with a low throughput is selected.

[0010] With the insoluble support of the present invention, it is possible to synthesize complex and long polypeptides in high yields and with commercially useful purities.

[0011] Lee et al. (Tetrahedron Letters 41(2000)7481 - 7485) disclose a resin having a crosslinked polystyrene (PS) core and a poly(ethylene glycol) (PEG) shell, prepared by suspension polymerization for use in solid-phase synthesis. The PEG monomer used in suspension polymerization is formed by reacting O,O’-bis(2-aminopropyl) polyethylene glycol 500 with methacryloyl chloride, thereby obtaining a PEG containing an ammonium functional group. By providing a charged PEG macromonomer under the polymerization conditions outlined by Lee, polymer beads are provided having a core of crosslinked polystyrene and a PEG macromonomer disposed on the surface of the beads, thereby providing a PEG shell. The PS core is highly crosslinked with more than 4% divinylbenzene (DVB). The throughput (substitution) of the core-shell resin can be increased by modifying the shell with lysine coupling. The presence of the PEG shell covering the core, along with the high degree of crosslinking of the core, hinders polypeptide synthesis within the core. All syntheses are carried out on the shell.

[0012] Lee et al. (Tetrahedron Letters 42(2001)7443 - 7445) disclose a tris(hydroxymethyl)aminomethane (Tris)-based dendrimer monomer used to increase the processing capacity of core-shell type resins and gel type TentaGel resins as disclosed by Lee (2000). Lee 2001 does not teach a monomer based on an aminoalkanoic acid such as diaminoalkanoic acid for use as a repeating unit for dendrimerization of a support. Also, Lee (2001) does not disclose the use of a spacer molecule between the support and the dendrimer monomer or between dendrimer monomers.

[0013] Chan et al. (Tetrahedron Letters 40(1999)4909 - 4912) disclose the synthesis of tri - amino acids and the use of such molecules to generate dendrimers on a resin, thereby increasing the throughput of the resin. Chan et al. do not disclose the application of spacer molecules between PS resins or between generations of tri - amino acid units.

[0014] The present invention provides an insoluble support that increases the yield of the product (polypeptide), particularly the reactor throughput (mass of the product after cleavage per swelling of the insoluble support) at a commercially acceptable purity in a given reaction volume. Thus, by providing a construct of at least one branching agent, and thereby a modified polymer matrix, an insoluble support, for example, a homogeneous polymer matrix for solid - phase peptide synthesis, significantly increases the yield of the product (polypeptide) and the reactor throughput at a commercially acceptable purity in a given reaction volume. The insoluble support of the present invention also enables the synthesis of long (complex) polypeptides, for example, polypeptides having more than 25 amino acids, with good yields and purity.

[0015] The object of the present invention is to increase the throughput of the target polypeptide (mass of the crude peptide after cleavage [mass / volume g / L] as a function of the volume of the resin containing the target polypeptide) in a given solid - phase peptide synthesis reactor.

[0016] A further object is to provide an insoluble support (polymeric insoluble support) that can increase the capacity / yield while reducing an uneven increase in the swelling of the insoluble support, without affecting the purity of the synthesized peptide.

[0017] A further object is to provide an insoluble support obtained by modifying a base resin, thereby increasing the capacity / yield per given reactor volume and at the same time reducing the solvent consumption, both absolutely and / or per unit of the target polypeptide synthesized, as compared in particular to an unmodified base resin.

[0018] A further object is to provide an insoluble support that reduces the solvent consumption per peptide unit (e.g., per 1 mmol of peptide), preferably as compared to an unmodified insoluble support.

[0019] Yet a further object is to provide an insoluble support that reduces swelling and / or reduces the solvent consumption (per synthesis unit of the target peptide) while at least maintaining or significantly increasing the capacity / yield as compared to an unmodified insoluble support.

[0020] A further object is to provide an insoluble support (modified resin) that significantly increases the yield of (crude) peptide without significantly reducing the purity at a given reactor volume (as compared to an unmodified resin).

[0021] A further object is to provide an insoluble support for synthesizing long polypeptides in good yield and purity, the insoluble support being formed from a commercially available resin and modified using readily available compounds such as SPPS and amino acids.

[0022] A further object is to provide an insoluble support (modified resin) for increasing the throughput (mass of crude peptide after cleavage [mass / volume g / L] as a function of the volume of resin containing the target polypeptide) while maintaining a commercially reasonable purity of the (crude) target polypeptide as compared to an unmodified resin.

[0023] A further objective is to increase the throughput of the insoluble support (modified resin) compared to the unmodified polymer matrix / resin (the mass of the crude peptide after cleavage as a function of the volume of the resin containing the target polypeptide), while maintaining a commercially reasonable purity of the (crude) target polypeptide and reducing the final volume of the polymer matrix / resin.

[0024] As suggested above, swelling is an important property of the insoluble support implemented in peptide synthesis. Generally, an increase in swelling promotes the diffusion of reagents (e.g., amino acids) into the insoluble support and potentially also increases the availability of binding sites. However, an increase in swelling typically increases the required reactor volume and solvent consumption. The present invention provides an insoluble support (modified insoluble support) that increases throughput at a given reactor volume and reduces solvent consumption while increasing capacity / yield and throughput (cleaved target peptide as a function of the final swollen volume of the insoluble support before cleavage) compared to an unmodified polymer matrix.

[0025] The implementation of the insoluble support according to the present invention is an efficient means for increasing the capacity of a given reactor volume. Furthermore, the implementation of the insoluble support increases the capacity of solid-phase peptide synthesis without increasing the reaction volume (the volume of the reaction vessel) or even while reducing the reaction volume, while also reducing the solvent consumption while maintaining the same conventional polymer matrix.

[0026] The insoluble support is preferably used as a solid phase in solid phase peptide synthesis (SPPS), such as the Fmoc / tBu or Boc SPPS strategy. The implementation of the insoluble support in SPPS significantly increases the throughput of the target peptide compared to an unmodified base resin / polymer matrix.

[0027] The insoluble support of the present invention can be successfully applied even in an aqueous solid-phase peptide synthesis protocol, i.e., when the solvent used is aqueous. Accordingly, a further object of the present invention is to provide SPPS conditions that enable the reduction of organic solvents and / or the replacement of organic solvents with aqueous solvents.

Summary of the Invention

[0028] One embodiment of the present invention relates to an insoluble support comprising a construct that increases the number of available binding sites compared to an unmodified polymer matrix. The insoluble support of the present invention is successfully implemented in solid-phase synthesis protocols, particularly solid-phase peptide synthesis. Further, the present invention also relates to a method for preparing an insoluble support by modifying a polymer matrix, and a method for synthesizing a peptide using the insoluble support or construct.

[0029] More specifically, one embodiment is an insoluble support (resin) in particulate form comprising a distal binding site, comprising a homogeneous polymer matrix and a construct, the construct being covalently bonded to the polymer matrix, the construct comprising at least one branching agent selected from at least two amino groups and an aminoalkanoic acid containing from 3 to 10 carbon atoms, a cleavable linker, and at least one spacer coupled to at least one branching agent via an amide bond, the cleavable linker providing a distal binding site, relating to an insoluble support (resin).

[0030] A further embodiment is a method for forming a particulate insoluble support comprising a secondary binding site, wherein the insoluble support comprises a homogeneous polymer matrix and a construct, the construct comprises a cleavable linker and at least one spacer, the method comprising providing a particulate polymer matrix comprising a primary binding site, the construct being formed by a solid-phase synthesis protocol, the solid-phase protocol comprising coupling a branching agent selected from at least two amino groups and an amino-protected amino alkanoic acid containing from 3 to 10 carbon atoms by an amide bond to any of the primary binding site, spacer or branching agent of the polymer matrix, the protocol further comprising at least one reaction step, the protocol further comprising a reaction step of coupling a cleavable linker to the branching agent, either directly or via one or more spacers.

[0031] A further embodiment relates to a method for synthesizing a polypeptide, morpholino oligomer or oligonucleotide using a solid-phase peptide synthesis protocol, including the application of the insoluble support disclosed herein.

[0032] Definitions Polymer Matrix: The insoluble support is formed from a polymer matrix modified by a construct covalently attached to the polymer matrix. The polymer matrix typically contains binding sites, also referred to as primary bonds. The construct covalently binds to the binding sites of the polymer matrix. The polymer matrix may be modified to contain 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 of the present invention. Thus, the primary binding sites of the polymer matrix are used to covalently bind the construct. Ideally, all of the 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 that usually contains a polymer crosslinked to an extent that provides structural integrity to the polymer matrix particles while allowing significant solvation of the polymer network. A homogeneous polymer matrix is preferably selected from polymer matrices containing binding sites distributed throughout the polymer matrix. Typically, the binding sites of the polymer matrix are essentially uniformly distributed throughout the matrix. The polymer matrix is preferably selected from homogeneous polymer matrices. The polymer matrix is typically in particulate form. The polymer matrix is preferably insoluble. The particle size can range from about 1 μm to about 2000 μm, typically from about 20 μm 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 with a crosslinking agent content of less than 4.0% by weight, preferably less than 3.5% by weight, preferably less than about 3.0% by weight, suitably from about 0.5 to 2.5% by weight.

[0033] Insoluble support: The insoluble support includes a polymer matrix and a construct, and constitutes the solid phase in any liquid-solid synthesis protocol such as solid-phase peptide synthesis.

[0034] Construct: A construct is a molecule that increases the number of binding sites (primary binding sites) of a polymer matrix. A construct is a branched molecule that can be characterized as a dendrimer or dendron. According to one aspect, a construct can be referred to as a branched polypeptide. The construct itself can also function as an anchor molecule in liquid phase peptide synthesis (LPPS). The construct includes 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 a cleavable linker, a branching agent, and a spacer. According to a further aspect, the cleavable linker, at least one branching agent, and at least one spacer are all coupled to each other via amide bonds. The simplest construct contains one branching agent. The construct is preferably formed by repeatedly coupling a branching agent to a previous generation branching agent (repeated branching cycle), whereby a highly ordered branched polymer is obtained in which the number of binding sites always increases as a function of the number of branching agents of the construct. The construct can be formed by a divergent reaction scheme, a convergent reaction scheme, or a combination of a divergent reaction scheme and a convergent reaction scheme. Preferably, the insoluble construct, i.e., the construct, is formed by a divergent reaction scheme such as a solid-phase organic synthesis scheme, preferably a solid-phase peptide synthesis scheme, where all the compounds constituting the construct, especially the cleavable linker, the branching agent, and the spacer, have functional groups that allow the compounds to couple to each other via amide bonds.

[0035] Branching agent: A branching agent is a molecule that is responsible for creating a specific structure of a construct and increasing the number of binding sites in the polymer matrix. The branching agent contains 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.

[0036] Distal branching agent: A distal branching agent is the branching agent that is farthest from the polymer matrix. The secondary or distal binding sites are provided by the distal branching agent via a cleavable linker coupled to the available binding sites of the distal branching agent.

[0037] Proximal branching agent: A proximal branching agent is the branching agent of the construct that is bound to the polymer matrix.

[0038] If the construct has only one branching agent, the one branching agent may be called either a proximal branching agent or a distal branching agent.

[0039] Intermediate branching agent: An intermediate branching agent is a branching agent that is arranged between the distal branching agent and the proximal branching agent. At least three generations (layers) of branching agents are required for the intermediate branching agent.

[0040] Primary, secondary, tertiary, etc. branching agents: Primary, secondary branching agents, etc. indicate branching agents relative to other branching agents of the same construct. A primary branching agent or first-generation branching agent (first-layer branching agent) is a branching agent that is coupled to the base matrix without any other branching agent intervening. A secondary branching agent or second-generation branching agent (second-layer branching agent) is coupled to the primary branching agent. A tertiary branching agent or third-generation branching agent (third-layer branching agent) is coupled to the secondary branching agent. In other words, the tertiary branching agent is coupled to the polymer matrix by two other branching agents, the secondary branching agent and the primary branching agent. Primary, secondary, tertiary, etc. branching agents may also be called primary (first), secondary layer or generation branching agents.

[0041] The distal branching agent is a branching agent of a construct to which a cleavable linker is attached. The generation containing the distal branching agent is the generation having the highest integer.

[0042] The proximal branching agent is attached to the polymer matrix, with or without one or more spacers. The proximal branching agent is the branching agent of the first generation.

[0043] The presence of an intermediate (generation) branching agent requires at least three generations of branching agents. The intermediate generation is found between the distal branching agent and the proximal branching agent.

[0044] Coupling: When referring to a molecule (e.g., a branching agent, a cleavable linker, and a spacer) being coupled to another moiety (e.g., a branching agent, a spacer, or a polymer matrix), such coupling can be direct coupling or coupling of the molecule to another moiety by any number and type of intermediate molecule herein referred to as a spacer or spacer molecule. Coupling herein typically means covalent coupling. The terms "attachment" and "coupling" are used interchangeably.

[0045] 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 branch point. The binding sites of the spacer may be selected from various functional groups, but the binding sites of the spacer are preferably selected such that an amide bond is formed when the spacer is coupled to the branching agent.

[0046] Binding site: The binding site (functional group or reactive site) is a site on a molecule or polymer matrix, specifically on a branching agent, spacer, cleavable linker, that is available for a chemical reaction with a binding site of a non-identical molecule. The binding site can be regarded as a functional group having the ability to form a covalent bond with a molecule different from the molecule containing the binding site, e.g., a branching agent. Two binding sites usually form a covalent bond.

[0047] Primary (first) binding site: The primary binding site is a binding site available for covalently coupling a construct for providing an insoluble support of the present invention suitable for solid-phase peptide synthesis on an available polymer matrix.

[0048] Secondary (second) or distal binding site: The secondary or distal binding site is a site provided by a construct, more specifically, by a cleavable linker covalently bonded to a binding site of a distal branching agent, which may have one or more spacers between the linker and the binding site of the distal branching agent in some cases. The secondary or distal binding site provides conditions for covalently immobilizing a peptide during solid-phase peptide synthesis.

[0049] Cleavable linker: A cleavable linker is a molecule to which a growing polypeptide covalently couples to facilitate or enable cleavage of the peptide from the construct. The construct includes a cleavable linker covalently connected directly or via one or more spacers to a binding site of a distal branching agent. Preferably, the cleavable linker is coupled to the branching agent or spacer via an amide bond.

[0050] Whenever an amino acid is referred to, any related amino acid derivative is also included.

[0051] Disclosure of the Invention One embodiment of the present invention relates to an insoluble support. Further embodiments relate to methods for forming an insoluble support. Further embodiments relate to solid-phase peptide synthesis protocols for synthesizing polypeptides, morpholino oligomers, or oligonucleotides, including the application of an insoluble support.

[0052] The insoluble support preferably functions as a solid phase in solid-phase peptide synthesis. According to one aspect, the insoluble support is used as a solid phase in solid-phase chemical synthesis protocols. In particular, the insoluble support is used as a solid phase in solid-phase morpholino oligomer synthesis, solid-phase oligonucleotide synthesis, and solid-phase peptide synthesis. More specifically, the insoluble support includes a binding site, also referred to herein as a secondary binding site or distal binding site, and a polymer matrix, the distal binding site being provided by a construct that includes at least one branching agent, a cleavable linker, and at least one spacer. The branching agent is disposed between the secondary binding site and the polymer matrix. The polymer matrix is preferably in particulate form, which represents that it is a solid phase.

[0053] The polymer matrix typically includes binding sites, also referred to herein as primary binding sites. The polymer matrix preferably includes binding sites suitable for coupling of the construct or binding sites (attachment points) for synthesis of the construct. The type of polymer matrix depends on the solid-phase synthesis application. The polymer matrix can include polymers such as polymer networks, e.g., cross-linked polymer networks. Typically, the polymer matrix is based on a commercially available polymer matrix (resin) configured for use as a solid phase in peptide or nucleotide synthesis. The construct is an entity that increases the number of binding sites of the polymer matrix such that one primary binding site (of the polymer matrix) provides several secondary binding sites (at least two) by the construct covalently bound to the primary binding site. The construct covalently bound to the polymer matrix includes at least a branching agent, and the branching agent is directly bound to the binding site of the polymer matrix or is covalently bound to the primary binding site by any number and any type of molecule (also called a spacer or spacer molecule) between the branching agent and the primary binding site of the polymer matrix.

[0054] The construct can be shown as a branched molecule. According to one aspect, the construct can be called a branched molecule containing amino acids or a branched peptide / polypeptide. The construct includes at least one branching agent, a cleavable linker, and at least one spacer. The branching agent is a molecule including at least three binding sites. The spacer is a molecule that cannot function as a branching agent. Thus, the spacer is preferably a molecule having two binding sites or preferably exactly two binding sites.

[0055] The insoluble support of the present invention is defined (claimed) in several ways (aspects) herein in order to properly configure the present invention. However, all aspects of the insoluble support relate to the same basic concept and are insoluble supports including a construct, the construct comprising at least one branching agent, a cleavable linker, and at least one spacer, and relate to the provision of an insoluble support that increases the number of binding sites and typically provides a specific geometric configuration.

[0056] 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 related to the method of forming the insoluble support.

[0057] An example of a simple, non-complex construct is a single branching agent, such as a single diaminoalkanoic acid, such as lysine, ornithine, etc., coupled to the branching agent, thereby providing a secondary (distal) binding site, an appropriate cleavable linker, and, for example, glycine as a spacer, the spacer being coupled to the branching agent via an amide bond and to the binding site of the polymer matrix.

[0058] One aspect of the present invention is an insoluble support in particulate form for use in solid-phase peptide synthesis, solid-phase morpholino oligomer synthesis, solid-phase oligonucleotide synthesis, preferably for use in solid-phase peptide synthesis, which provides a binding site (distal or secondary binding site), comprises a homogeneous polymer matrix and a construct, the construct being covalently bonded to the polymer matrix, the construct comprising at least one branching agent, a cleavable linker, and at least one spacer, at least one branching agent being selected from aminoalkanoic acids comprising at least two amino groups and from 3 to 10 carbon atoms, at least one spacer being coupled to at least one branching agent by an amide bond, the cleavable linker providing a distal binding site and being covalently bonded to at least one branching agent, relating to an insoluble support.

[0059] According to a further aspect, the insoluble support can be obtained by applying an iterative synthesis process that includes providing a homogeneous polymer matrix containing primary binding sites, and coupling a branching agent such as an aminoalkanoic acid containing at least two amino groups and from 3 to 10 carbon atoms, and at least one spacer, and finally coupling a cleavable linker to the binding site of the distal branching agent (or distal branching agents), thereby forming a construct that provides at least two secondary / distal binding sites.

[0060] According to one embodiment, the branching agent is selected from aminoalkanoic acids containing at least two but no more than three amino groups, and from 3 to 10 carbon atoms, suitably from 3 to 8 carbon atoms, suitably from 4 to 8 carbon atoms.

[0061] According to a further embodiment, the branching agent is selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, suitably from 3 to 8 carbon atoms, suitably from 4 to 8 carbon atoms.

[0062] The number of carbon atoms of any of the aminoalkanoic acids disclosed herein may range from 3, 4, 5, 6, up to 10, up to 9, up to 8, up to 7, and may be any combination of lower and upper numbers.

[0063] According to a further 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, and suitably the branching agent is selected from 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid.

[0064] According to one embodiment, all of the branching agents of the construct are selected from aminoalkanoic acids containing at least two amino groups and from 3 to 10 carbon atoms.

[0065] According to one embodiment, all branching agents of the construct are identical and are selected from amino alkanoic acids containing at least two amino groups and from 3 to 10 carbon atoms.

[0066] According to one embodiment, the branching agents of the construct can be any combination of branching agents selected from amino alkanoic acids containing at least two amino groups and from 3 to 10 carbon atoms.

[0067] One embodiment relates to an insoluble support in which the number δ of branching agents of the construct is given by the formula δ(n) = 2 n - 1, where n represents the generation number of the branching agent and n is a positive integer.

[0068] One embodiment relates to an insoluble support in which the number λ of distal binding sites of the construct is given by the formula λ(n) = 2 n where n represents the generation number of the branching agent and n is a positive integer.

[0069] One embodiment relates to an insoluble support in which the number δ of branching agents of the construct is given by δ(n) = 2 n - 1 and the number λ of distal binding sites of the construct is given by the formula λ(n) = 2 n where n represents the generation number of the branching agent and n is a positive integer.

[0070] According to one embodiment, the generation number n of the branching agent of the construct is preferably from 1 to 10, suitably from 1 to 5.

[0071] According to a further embodiment, the generation number n of the branching agent of the construct is preferably from 2 to 10, suitably from 2 to 5.

[0072] The construct comprises at least one spacer. If the construct contains only one branching agent, at least one spacer may be arranged between the branching agent and the polymer matrix. Alternatively, the spacer may be arranged between each of two or more cleavable linkers and the branching agent.

[0073] When the construct contains branching agents of multiple generations, i.e., two or more generations, the spacer is typically placed between any one of the following positions: between branching agents of any generation, between a cleavable linker and a (distal) branching agent, and between a (proximal) branching agent and the polymer matrix. Any number of spacers can be present at any of these positions.

[0074] According to one aspect, at least one spacer is placed between the branching agents. The spacer between the branching agents implies that the construct contains at least two generations of branching agents. One example is a construct containing three lysine branching agents, three glycine spacers, and four cleavable linkers. Two glycine spacers are placed between the first-generation lysine and the two second-generation lysines. More specifically, one glycine spacer each is covalently connected via an amide bond to the epsilon and alpha amines of the first-generation lysine. Further, the two second-generation lysines are covalently connected via an amide bond to each of the two glycine spacers. Additionally, a further third glycine spacer can be placed between the first-generation lysine and the homogeneous polymer matrix by two amide bonds. The four cleavable linkers are coupled via four amide bonds to the epsilon and alpha amines of the two second-generation lysines.

[0075] According to one aspect, multiple spacers are placed between the branching agents. When three or more spacers are placed 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 by two amide bonds.

[0076] According to one embodiment, one or more spacers are coupled to the branching agent via an amide bond.

[0077] Modification of the polymer matrix with a branching agent such as an aminoalkanoic acid containing at least two amino groups and from 3 to 10 carbon atoms converts one binding site (primary binding site) of the polymer matrix into two or more binding sites (secondary or distal binding sites).

[0078] The distal binding site is a binding site provided after modification of the polymer matrix.

[0079] The distal binding site is a binding site for synthesis in solid-phase synthesis such as solid-phase peptide synthesis.

[0080] The number of additional secondary binding sites correlates with the number of branching agents included in the construct. More importantly, the increase in secondary binding sites is preferably achieved by continuously coupling a branching agent to another branching agent, one of which, the proximal branching agent, is coupled to the polymer matrix, optionally using a spacer, thereby generating a layer or generation of branching agents. Such layers may be referred to as primary (first), secondary (second), tertiary (third), quaternary (fourth), etc. layers or generations. In aspects of the invention, the branching agents are designated as primary, secondary, tertiary, quaternary branching agents. For example, a tertiary branching agent forms a tertiary (third) layer or third generation.

[0081] One aspect of the present invention is an insoluble support in particulate form comprising a homogeneous polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, the construct comprising at least one branching agent, a cleavable linker, and at least one spacer, the branching agent being selected from at least two amino groups and an aminoalkanoic acid containing from 3 to 10 carbon atoms, at least one of which branching agents is a distal branching agent comprising a binding site, the cleavable linker being covalently bonded to the binding site of the distal branching agent either directly or via one or more spacers, each cleavable linker providing a distal binding site, and at least one spacer being located at any one of the following positions: between the branching agents, between the polymer matrix and the branching agent, or between the binding site of the distal branching agent and the cleavable linker.

[0082] One aspect of the present invention is an insoluble support in particulate form comprising a polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, the construct comprising at least two branching agents, a cleavable linker, and a spacer, one branching agent being designated as a distal branching agent, one branching agent being designated as a proximal branching agent, and optionally a branching agent being designated as an intermediate branching agent, the branching agents being selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, the distal branching agent comprising a binding site, the cleavable linker being covalently bonded to the binding site of the distal branching agent either directly or via one or more spacers, each cleavable linker providing a distal binding site, the spacer being located at any one of the following positions: between the branching agents, between the polymer matrix and the branching agent, or between the binding site of the distal branching agent and the linker, the distal branching agent being bonded to only one branching agent selected from either the optional intermediate branching agent or the proximal branching agent, the optional intermediate branching agent being bonded 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 being bonded to two branching agents selected from the distal branching agent and the optional intermediate branching agent.

[0083] One aspect of the present invention is an insoluble support in particulate form comprising a polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, the construct comprising at least three branching agents, a cleavable linker, and a spacer, the branching agents being designated as a distal branching agent, an intermediate branching agent, and a proximal branching agent, the branching agents having three binding sites, the distal branching agent being selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, the cleavable linker being covalently bonded to the binding site of the distal branching agent either directly or via one or more spacers, each cleavable linker providing a distal binding site, the spacer being disposed at any one of the following positions: between the branching agents, between the polymer matrix and the branching agent, between the binding site of the distal branching agent and the linker, the distal branching agent being bonded to only one intermediate branching agent, the intermediate branching agent being bonded to three branching agents selected from the distal branching agent, the intermediate branching agent, and the proximal branching agent, and the proximal branching agent being bonded to two intermediate branching agents.

[0084] One aspect of the present invention is an insoluble support in particulate form comprising a polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, the construct comprising one branching agent, two cleavable linkers, and at least one spacer, the branching agent comprising three binding sites and being selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, the two cleavable linkers being covalently bonded to the binding site (amino group) of the branching agent either directly or via one or more spacers, each cleavable linker providing a distal binding site, the branching agent being bonded to the polymer matrix, and at least one spacer being disposed at any one of the following positions: between the polymer matrix and the branching agent, between the binding site of the distal branching agent and the cleavable linker.

[0085] One aspect of the present invention is a particulate insoluble support comprising a polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, the construct comprising three branching agents, four cleavable linkers, and a spacer, all branching agents being selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms and including a binding site, two of the branching agents being second-generation distal branching agents including a binding site, one of the branching agents being a first-generation proximal branching agent, the cleavable linkers being covalently bonded to the binding site of the distal branching agent either directly or via one or more spacers, each cleavable linker providing a distal binding site, the first-generation proximal branching agent being bonded to two second-generation distal branching agents, the first-generation proximal branching agent being bonded to the matrix, and the spacer being disposed at any one of the following positions: between the branching agents, between the polymer matrix and the branching agent, between the binding site of the distal branching agent and the linker, relates to an insoluble support.

[0086] One aspect of the present invention is a particulate insoluble support comprising a polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, the construct comprising seven branching agents, eight cleavable linkers, and a spacer, all branching agents being selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, four of the branching agents being third-generation distal branching agents including a binding site, two of the branching agents being second-generation intermediate branching agents, one of the branching agents being a proximal branching agent, the cleavable linkers being covalently bonded to the binding site of the distal branching agent either directly or via one or more spacers, each cleavable linker providing a distal binding site, each second-generation intermediate branching agent being bonded to two third-generation distal branching agents and one first-generation intermediate branching agent, the first-generation intermediate branching agent being bonded to two second-generation intermediate branching agents and the first-generation proximal branching agent, the first-generation proximal branching agent being bonded to the matrix, and the spacer being disposed at any one of the following positions: between the branching agents, between the polymer matrix and the branching agent, between the binding site of the distal branching agent and the linker, relates to an insoluble support.

[0087] One aspect of the present invention is a particulate insoluble support comprising a polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, and the construct comprises 15 branching agents, 16 cleavable linkers, and a spacer, all branching agents being selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, 8 branching agents being fourth-generation distal branching agents containing a binding site, 4 branching agents being third-generation intermediate branching agents, 2 branching agents being second-generation intermediate branching agents, 1 branching agent being a first-generation proximal branching agent, the cleavable linkers being covalently bonded to the binding site of the distal branching agent either directly or via one or more spacers, each cleavable linker providing a distal binding site, each third-generation intermediate branching agent being bonded to 2 fourth-generation distal branching agents and 1 second-generation intermediate branching agent, each second-generation intermediate branching agent being bonded to 2 third-generation intermediate branching agents and 1 first-generation intermediate branching agent, the first-generation intermediate branching agent being bonded to 2 second-generation intermediate branching agents and the first-generation proximal branching agent, the first-generation proximal branching agent being bonded to the matrix, and the spacer being located at any one of the following positions: between branching agents, between the polymer matrix and a branching agent, between the binding site of the distal branching agent and the linker.

[0088] One aspect of the present invention is a particulate insoluble support comprising a polymer matrix and a construct, wherein the construct is covalently bound to the polymer matrix, and the construct comprises 31 branching agents, 32 linkers, and optionally a spacer, all branching agents being selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, 16 branching agents being fifth-generation distal branching agents comprising a binding site, 8 branching agents being fourth-generation intermediate branching agents, 4 branching agents being third-generation intermediate branching agents, 2 branching agents being second-generation intermediate branching agents, 1 branching agent being a first-generation proximal branching agent, the linkers being covalently bound to the binding site of the distal branching agent either directly or via one or more spacers, each linker providing a distal binding site, each fourth-generation intermediate branching agent being bound to two fifth-generation distal branching agents and one third-generation branching agent, each third-generation intermediate branching agent being bound to two fourth-generation intermediate branching agents and one second-generation intermediate branching agent, each second-generation intermediate branching agent being bound to two third-generation intermediate branching agents and one first-generation proximal branching agent, the proximal branching agent being bound to the matrix, and the spacer being located at any one of the following positions: between branching agents, between the polymer matrix and a branching agent, or between the binding site of the distal branching agent and a linker, relates to an insoluble support.

[0089] According to a further embodiment, the construct does not contain arginine.

[0090] One aspect of the present invention is a particulate insoluble support 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 selected from at least two amino groups and aminoalkanoic acids containing from 3 to 10 carbon atoms, a cleavable linker, and a spacer, the branching agent being disposed between the secondary binding site and the polymer matrix, and the construct being covalently bound to the polymer matrix, relates to an insoluble support.

[0091] A further aspect is a particulate insoluble support comprising a construct, a distal binding site, and a polymeric matrix, wherein the construct is bound to the polymeric matrix, the construct comprises a cleavable linker and at least two branching agents selected from aminoalkanoic acids having at least two amino groups and from 3 to 10 carbon atoms, provided that the at least two branching agents comprise different numbers of binding sites, one branching agent is a distal branching agent that provides the distal binding site, the cleavable linker is covalently bound to the binding site of the distal branching agent either directly or via one or more spacers, each cleavable linker provides a distal binding site, and at least one spacer is disposed between the branching agents, between the polymeric matrix and the branching agent, and between the distal branching agent and the cleavable linker.

[0092] Yet a further aspect is a particulate insoluble support (resin) for use in solid-phase peptide synthesis, solid-phase morpholino oligomer synthesis, and solid-phase oligonucleotide synthesis, preferably solid-phase peptide synthesis, comprising a distal binding site, a homogeneous polymeric matrix, and a construct, wherein the construct is covalently bound to the polymeric matrix, the construct comprises at least one branching agent selected from diaminoalkanoic acids having from 3 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 provides a distal binding site, and the construct has the formula: (A) [Polymeric matrix] - BA(1) - LK2, (B) [Polymeric matrix] - BA(1) - BA(2)2 - LK4, (C) [Polymeric matrix] - BA(1) - BA(2)2 - BA(3)4 - LK8, (D) [Polymeric matrix] - BA(1) - BA(2)2 - BA(3)4 - BA(4)8 - LK 16 , (E) [Polymeric matrix] - BA(1) - BA(2)2 - BA(3)4 - BA(4)8 - BA(5)16 -LK 32 selected from dendritic molecules of wherein BA is a branching agent, the integer indicates the generation of the branching agent, and LK indicates a cleavable linker, relating to an insoluble support (resin).

[0093] In yet a further aspect, the construct is a molecule: (A) [Polymer matrix] - SPC - BA(1) - LK2, (B) [Polymer matrix] - BA(1) - SPC - BA(2)2 - LK4, (C) [Polymer matrix] - BA(1) - SPC - BA(2)2 - SPC - BA(3)4 - LK8, (D) [Polymer matrix] - BA(1) - SPC - BA(2)2 - SPC - BA(3)4 - SPC - BA(4)8 - LK 16 , (E) [Polymer matrix] - BA(1) - SPC - BA(2)2 - SPC - BA(3)4 - SPC - BA(4)8 - SPC - BA(5) 16 -LK 32 selected from wherein BA is a branching agent, the integer indicates the generation of the branching agent, LK indicates a cleavable linker, SPC indicates any number of spacers (from at least one spacer), and optionally, any number of spacers (at least one spacer) may be disposed between the polymer matrix and the first generation branching agent BA(1) and between the cleavable linker and the final generation branching agent (distal branching agent).

[0094] Still further embodiments are particulate, insoluble supports (resins) for use in solid-phase morpholino oligomer synthesis, solid-phase oligonucleotide synthesis and solid-phase peptide synthesis, preferably solid-phase peptide synthesis, which include a distal binding site, include a homogeneous polymer matrix and a construct, the construct being covalently bonded to the polymer matrix, the construct including at least one branching agent selected from diaminoalkanoic acids containing from 3 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, the construct being a branched molecule: [Chemical formula] [Chemical formula] selected from: wherein BA is a branching agent, the integer indicates the generation of the branching agent, LK indicates a cleavable linker, and any optional number of spacers (at least one spacer) may be positioned between the polymer matrix and the first generation branching agent BA(1), between branching agents, and between the cleavable linker and the final generation branching agent (distal branching agent), relating to an insoluble support (resin).

[0095] The construct of Structure A contains only one first-generation proximal branching agent. The construct of Structure B contains three branching agents, namely, 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, namely, one first-generation proximal branching agent, two second-generation intermediate branching agents, and four third-generation distal branching agents, meaning three layers / generations of branching agents. Construct D contains 15 branching agents, namely, 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, that is, Construct D contains four layers (generations) of branching agents. Construct E contains 31 branching agents, namely, 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 16 fifth-generation distal branching agents, which means that there are five layers (generations) of branching agents.

[0096] According to one aspect, the diaminoalkanoic acid (DAA) is selected from §-DAA(§) and §-DAA($), where DAA represents a diaminoalkanoic acid, for example, a diaminoalkanoic acid containing from 3 to 10 carbon atoms, suitably from 3 to 8 carbon atoms, the character § not in parentheses represents an alpha amine protecting group, and the characters § and $ in parentheses represent side chain protecting groups, and $ represents a protecting group that cleaves under cleavage conditions different from those of protecting group § or does not cleave during the same cleavage step.

[0097] The protecting group can be selected from Fmoc (fluorenylmethoxycarbonyl), Mtt (4-methyltrityl), Mmt (4-methyloxythrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)] or ivDde.

[0098] The protecting group § may be Fmoc, and $ may be selected from Mtt (4-methyltrityl), Mmt (4-methyloxythrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)] or ivDde.

[0099] According to one embodiment, all branching agents are selected from aminoalkanoic acids containing at least two amino groups and from 3 to 10 carbon atoms, and both amines are protected by the same protecting group.

[0100] According to one aspect, the branching agent is exclusively selected from amine-protected diaminoalkanoic acids, preferably diaminoalkanoic acids containing from 3 to 10 carbon atoms, suitably from 3 to 8 carbon atoms, said amine-protected diaminoalkanoic acids being selected from diaminoalkanoic acids containing amine-protecting groups that cleave during the same cleavage step and / or under the same / similar cleavage conditions, and diaminoalkanoic acids containing amine-protecting groups that cleave under different cleavage conditions or do not cleave during the same cleavage step.

[0101] The polymer matrix may also be denoted as a resin. The polymer matrix may comprise a polymer network such as a crosslinked polymer network. Both the insoluble support and the polymer matrix are preferably in particulate form. Typically, the insoluble support is in the form of particles in the size range from about 1 μm to about 1000 μm, suitably from about 5 μm to about 750 μm, from about 10 μm to about 300 μm, or from about 50 μm to about 200 μm, for example in the form of beads.

[0102] According to a further aspect, the polymer matrix is selected from homogeneous polymer matrices. A homogeneous polymer matrix as used herein refers to a polymer matrix in which the binding sites are distributed throughout the matrix, preferably uniformly (essentially evenly) throughout the polymer matrix. Homogeneous means that important parameters of the matrix, such as the distribution of crosslinks and (primary) binding sites, are uniformly distributed throughout the matrix.

[0103] There are certain heterogeneous resins for solid-phase peptide synthesis in which the binding sites are not uniformly distributed within the resin. Rather, the binding sites are concentrated on or near the surface of the resin beads that form the shell, and the core of the beads is essentially devoid of binding sites.

[0104] The homogeneous polymer matrix does not include heterogeneous resins having essentially all of the binding sites on or near the surface. Thus, a homogeneous polymer matrix in particulate form (essentially spherical / bead form) does not include a shell at the surface (phase boundary) or near it where essentially all of the binding sites are located, nor does it include a core that is essentially devoid of binding sites.

[0105] According to a further aspect, the polymer matrix is selected from polymer matrices obtained by emulsion polymerization comprising at least styrene and divinylbenzene (DVB). Preferably, DVB is present in an amount of less than 4.0 wt%, preferably less than 3.5 wt%, preferably less than 3.0 wt% during the emulsion polymerization. Preferably, DVB is present in an amount from about 0.5 wt% to about 2.5 wt%.

[0106] The insoluble support can also be characterized by structural features and features related to the formation of the insoluble support. Thus, one aspect is a particulate insoluble support comprising a construct defined by any one of the aspects presented herein, obtained by providing a polymeric matrix comprising primary binding sites, wherein the construct is covalently bound to the primary binding sites, the construct provides secondary binding sites such that the primary binding sites provide at least two secondary binding sites, the construct comprises at least one branching agent, a linker, and optionally a spacer, the construct is formed by implementation of a solid-phase synthesis protocol, and the protocol comprises an iterative synthesis process comprising coupling at least one branching agent to the polymeric matrix and optionally other branching agents.

[0107] Still further embodiments are particulate insoluble supports (resins) for use in solid-phase peptide synthesis comprising distal binding sites, comprising a polymeric matrix and a construct, the construct being covalently bound to the polymeric matrix, the construct comprising at least one branching agent selected from at least two amino groups and aminoalkanoic acids containing from 3 to 10 carbon atoms, a cleavable linker, and at least one spacer coupled to at least one branching agent via an amide bond, the cleavable linker providing the distal binding site and being covalently bound to at least one branching agent, the construct being formed by synthesis comprising providing the polymeric matrix as a solid phase comprising primary binding sites, branching agents, linkers, and spacers, the construct being formed by at least one coupling step, preferably an iterative coupling step, the one or more steps comprising at least coupling at least one branching agent, at least one spacer, and a cleavable linker to the branching agent.

[0108] A further embodiment is a method for forming a particulate insoluble support for use in solid phase peptide synthesis, which comprises a secondary (distal) binding site, wherein the insoluble support comprises a polymeric matrix and a construct, the construct comprises a cleavable linker and at least one spacer, the method comprises providing a particulate polymeric matrix comprising a primary binding site, the construct is formed by a solid phase synthesis protocol, the solid phase protocol comprises at least one reaction step of coupling a branching agent selected from amino protected amino alkanoic acids comprising at least two amino groups and from 3 to 10 carbon atoms by an amide bond to either the primary binding site of the polymeric matrix, a spacer or a branching agent, and the protocol further comprises a reaction step of coupling a cleavable linker to the branching agent.

[0109] The insoluble support is preferably synthesized by providing a polymeric matrix and by coupling of the relevant compounds (branching agent, spacer and cleavable linker) by formation of amide bonds and use of a solid phase synthesis protocol. Preferably, all compounds contain the functional groups necessary to provide an amide bond when coupled to each other. Typically, all compounds contain at least one amine / amino group and a carboxylic acid group / carboxyl group. As presented throughout this specification, the branching agent is selected from amino alkanoic acids comprising at least two amino groups and from 3 to 10 carbon atoms. Preferably, the spacer is selected from compounds comprising two binding sites, one being an amine / amino group and the other being a carboxylic acid group. Preferably, the spacer is selected from amino acids and derivatives thereof having two binding sites, i.e. one amine group and one carboxylic acid group.

[0110] It should be noted that all reactive groups of the relevant compounds require appropriate protecting groups, except for the carboxylic acid groups. More specifically, all amine / amino groups need to be protected by appropriate protecting groups.

[0111] The reaction cycle in the solid-phase peptide synthesis protocol includes at least the following steps: 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. A suitable protecting group is covalently bonded to the amine. If the compound also includes a side chain having a reactive group, such a side chain reactive group is also protected. The compound comprising at least the protected amine group and the carboxylic acid group is coupled to the primary binding site of the polymer matrix in the presence of a suitable coupling agent, thereby forming an amide bond. If the compound is an amino acid, the α-amine is always protected, and optionally, any reactive groups of the side chain, such as the ε-amine of lysine, are protected. b) After coupling the relevant compound to the polymer matrix, separating the excess reagents and by-products c) Next, a further compound comprising at least one amine group and a carboxylic acid group, wherein the relevant amine group is protected by a relevant protecting group, is coupled to the free amine group of the previously coupled compound on the polymer resin in the process.

[0112] When the last generation of the branching agent is coupled, a cleavable linker is coupled to the distal branching agent.

[0113] The process of synthesizing a polypeptide starts with the provision of a polymer matrix, modifies the polymer matrix according to the present invention, and once the insoluble support of the present invention is formed, the synthesis of any relevant target polypeptide may be continued.

[0114] To synthesize the insoluble support, the following steps: a) Optionally, coupling at least one spacer molecule to the primary binding site; b) A primary branching agent, preferably a diaminoalkanoic acid in which two amine groups are protected by a protecting group, preferably a diaminoalkanoic acid containing from 3 to 10 carbon atoms, suitably from 3 to 6 carbon atoms, for example, a primary branching agent selected from diaminopropionic acid, diaminobutyric acid, diaminopentanoic acid and diaminocaproic acid, is coupled to the primary binding site of the polymer matrix, or optionally, the branching agent is coupled to a spacer molecule, c) A step of removing the protecting group, wherein steps a), b) and c) can be repeated, d) A step of coupling a linker to the unprotected amine group of the distal branching agent and may include.

[0115] The two amines of the diaminoalkanoic acid in step b) are suitably protected by a protecting group selected from amine protecting groups that cleave during the same cleavage step and / or under the same / similar cleavage conditions, and the diaminoalkanoic acid contains an amine protecting group that cleaves under different cleavage conditions or does not cleave during the same cleavage step.

[0116] According to one embodiment, all diaminoalkanoic acids have a protecting group that cleaves during the same deprotection step. Thus, suitably, the alpha amine and the side chain amine are protected by the same protecting group.

[0117] A further embodiment of the invention relates to a construct. The construct and the polymer matrix support form the insoluble support of the invention.

[0118] According to one aspect, the construct and the insoluble support can be formed by using a solid-phase protocol. The solid phase may be a base-insoluble support. The construct is an entity that couples to the primary binding site of the insoluble support to increase the number of binding sites, i.e., ultimately provides a plurality of secondary binding sites. The construct includes at least one branching agent disclosed herein. As described above, the construct may include two or more layers / generations of branching agents. The distal or intermediate branching agent of the construct is optionally coupled to the proximal branching agent via a spacer. A preferred procedure for forming the construct and the modified insoluble support is the application of a solid-phase protocol. According to one aspect, the branching agent is selected from diaminoalkanoic acids, and the construct can be synthesized using established reaction protocols in solid-phase peptide synthesis (SPPS). Once the target construct is generated, the construct can be cleaved from the insoluble support. Alternatively, the construct can be synthesized by a liquid-phase reaction protocol.

[0119] The construct can be regarded as a branched polymer or a branched polymer containing amide coupling. The construct may also be regarded as a branched peptide or peptidomimetic polymer containing a spacer.

[0120] According to one aspect, if φ represents the number of binding sites of the branching agent, the number of secondary binding sites of the construct as a function of the layer can be expressed as λ=(φ - 1) n and can be represented as, where φ represents the number of binding sites of the branching agent and n represents the number of layers of the branching agent in the construct, provided that all the branching agents of the construct have the same number of binding sites. If the construct contains only one branching agent and the branching agent is lysine (lysine having three binding sites), the number of secondary binding sites λ provided by this construct is λ=(3 - 1) 1 : two secondary binding sites. Therefore, modifying the primary binding site with lysine as the branching agent (and the construct) provides only one layer or one generation of branching agent and gives two secondary binding sites for all the primary binding sites of the polymer matrix.

[0121] The following embodiments of the insoluble support relate to insoluble supports containing branching agents of 1, 2, 3, 4 and 5 layers (generations), and the branching agents are referred to as primary, secondary, tertiary, quaternary and quinary branching agents. The primary branching agent forms the first layer (generation), the secondary branching agent forms the second layer, the tertiary branching agent forms the third layer, and so on. When the branching agent contains 3 binding sites, for example lysine, the number of secondary binding sites provided is 2 n equal to, where n represents the number of levels. Thus, an insoluble support containing lysine as a branching agent and further containing a quinary lysine branching agent of 5 levels provides 2 n from all primary binding sites, i.e., 2 5 or 32 secondary binding sites.

[0122] Within the scope of the present invention, there are also insoluble supports containing branching agents of more than 5 levels. However, for every level, the number of secondary binding sites increases exponentially. The availability of secondary binding sites for peptide synthesis can be enhanced, especially by introducing a spacer (spacer molecule) between the branching agents.

[0123] A further embodiment relates to an insoluble support / polymer matrix containing primary binding sites, wherein, optionally by a spacer molecule, a proximal branching agent is covalently bonded to the primary binding site and the primary branching agent provides at least two secondary binding sites.

[0124] The construct can contain not only one type of branching agent but also different types of branching agents. The construct can contain branching agents having different numbers of binding sites and / or branching agents having the same number of binding sites but different.

[0125] A further embodiment relates to any of the insoluble supports disclosed herein that are used as a solid phase in any solid phase synthesis protocol. The insoluble support is preferably implemented as a solid phase in solid phase peptide synthesis.

[0126] A further aspect relates to the use of an insoluble support as a solid phase for synthesizing peptides and polypeptides in solid phase peptide synthesis.

[0127] Branching agent As noted above, a construct is a molecule that enables the provision of a secondary / distal binding site. In its simplest embodiment, the construct comprises one branching agent. The most common definition of a branching agent is an organic molecule containing at least three binding / coupling sites. A binding / coupling site is a functional group capable of forming a covalent bond with a binding / coupling site of another molecule (e.g., two binding sites of two different molecules form a coupling in the form of a covalent bond, spacer-spacer, spacer-branching agent, coupling between two branching agents, e.g., secondary (intermediate / distal) branching agent-primary (proximal) branching agent, coupling of the primary (proximal) branching agent with the primary binding site of the polymer matrix).

[0128] Typically, the branching agent contains a functional group selected from amines, carboxylic acids, alcohols, ketones, and aldehydes. Alternatively, the functional groups are selected such that the coupling produces amides, ethers, and esters. Preferably, the branching agent contains a functional group selected from amines and carboxylic acids. Preferred binding sites for the branching agent are amines and carboxylic acids. The amine is preferably a primary amine.

[0129] The branching agent can be selected from any natural amino acid or amino acid derivative that provides at least three coupling sites.

[0130] According to a preferred embodiment, the branching agent is selected from aminoalkanoic acids containing at least two amino groups and from 3 to 10 carbon atoms.

[0131] According to a further embodiment, the branching agent is selected from aminoalkanoic acids containing at least two but no more than three amino groups and from 3 to 10 carbon atoms.

[0132] According to yet a further aspect, the branching agent is selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, preferably from diaminoalkanoic acids containing from 3 to 8 carbon atoms, for example from 3 to 6 carbon atoms.

[0133] According to a further 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, and suitably, the branching agent is selected from 2,4-diaminobutyric acid and 2,5-diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid.

[0134] According to a further aspect, the branching agent is selected from (lysine analogues) such as diaminopropionic acid, diaminobutyric acid and diaminopentanoic acid, diaminohexanoic acid.

[0135] According to one aspect, the branching agent is lysine (diaminohexanoic acid), more specifically 2,6-diaminohexanoic acid.

[0136] The branching agent may contain one or more chiral carbon centers (asymmetric carbons). Such chiral carbons can be in either the S or R configuration. The branching agent containing chiral carbons may have all chiral carbons in either the S configuration or the R configuration, or, if there are multiple chiral carbons, may have them in such a way as to exhibit both the S configuration and the R configuration.

[0137] Not only amino acid-based branching agents but also amino acid-based spacers can be in the D or L form. Most amino acids are chiral (containing chiral carbons: especially the alpha carbon). Glycine does not have a chiral asymmetric carbon atom. The construct may contain a branching agent and optionally a spacer that are in the D and / or L form. Thus, the construct may contain both D-type and L-type amino acids. According to one embodiment, all chiral carbons of the relevant amino acid-based branching agent and optionally the spacer are in their L form.

[0138] Spacer Spacers can be present between branching agents, between a branching agent and a primary binding site of the polymer matrix, and between a cleavable linker and a branching agent. The spacer needs to contain two binding / coupling sites / functional groups that can be used to form a covalent coupling.

[0139] The binding / coupling / functional groups of the spacer can be selected from amines, carboxylic acids, alcohols, ketones, and aldehydes. Preferably, the functional groups are selected from amines, carboxylic acids, and alcohols. The functional groups of the spacer preferably form amides and ethers. Preferably, the spacer can be selected from any molecule having two functional groups that can both form amide bonds. Suitable spacers are any molecule having two functional groups that form amide bonds and can be applied to solid-phase synthesis protocols.

[0140] According to a preferred embodiment, the spacer is selected from amino acids or amino acid derivatives. More specifically, the spacer is selected from amino acids having one amine.

[0141] A useful spacer can be an aminohydrocarbon carboxylic acid, i.e., any type of hydrocarbon chain containing one amine and one carboxylic acid. Preferably, the amine and the carboxylic acid are at the ends of the hydrocarbon chain.

[0142] The spacer can be selected from amino acids containing one carboxylic acid group and one amine group. Suitable amino acids can also contain aliphatic hydrocarbons. Exemplary amino acid spacers are glycine, alanine, beta-alanine, isoleucine, leucine, and any derivatives thereof.

[0143] According to one aspect, the spacer is selected from monoaminoalkanoic acids containing from 1 to 10 carbon atoms, such as aminoalkanoic acid, specifically glycine, alanine, beta-alanine, valine, isoleucine, leucine, beta-leucine, ε-aminopropionic acid, 4-aminobutanoic acid (4-aminobutyric acid), 5-aminopentanoic acid (5-aminovaleric acid), ε-aminohexanoic acid (ε-aminocaproic acid), 7-aminoheptanoic acid (7-aminoenanthic acid), 8-aminooctanoic acid (8-aminocaprylic acid), 9-aminononanoic acid, 10-aminodecanoic acid, etc.

[0144] A preferred spacer is glycine.

[0145] Any number of spacers can be applied to the construct. The spacers can be arranged between branching agents, between the branching agent and the primary binding site of the insoluble support, and between the construct linker and the branching agent.

[0146] The hydrophilicity / hydrophobicity of the construct and the insoluble support can be adjusted by the selection of the branching agent and / or the spacer.

[0147] According to one aspect, the PEG spacer is arranged between the last layer of the branching agent (also called the distal branching agent) and the linker, and / or between the branching agents.

[0148] The PEG spacer can preferably be provided as 2-[2-[2-(Fmoc amino)ethoxy]ethoxy]acetic acid (AEEA). The AEEA spacer has the advantage of being easily incorporated into the solid-phase peptide synthesis protocol.

[0149] Cleavable linker The cleavable linker is disposed between the secondary binding site and the most distal branching agent. The secondary binding site is typically integrated with the linker. Thus, the linker may also be referred to as the secondary binding site. Distal means being farthest away from the polymer matrix. The most distal layer of the branching agent means the layer farthest from the base of the polymer matrix. The linker selected depends on the chemical properties used to synthesize the peptide, particularly the cleavage conditions desired. Also, the type of polymer matrix can affect 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.

[0150] According to one aspect, the cleavable linker is not a linker cleavable by photolysis.

[0151] Polymer matrix The insoluble support includes a polymer matrix and a construct that is covalently connected to the polymer matrix and typically includes a binding site, also referred to as a primary bond. The construct covalently binds to the binding site of the polymer matrix. The polymer matrix may be modified to include 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 of the present invention. Thus, the primary binding sites of the polymer matrix are used to covalently bind to the construct. Ideally, all of the 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 that usually includes a polymer crosslinked to an extent that provides structural integrity to the polymer matrix particles while allowing significant solvation of the polymer network. The polymer matrix is preferably selected from polymer matrices that include 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.

[0152] According to one aspect, the polymer matrix is selected from homogeneous polymer matrices. A homogeneous polymer matrix as used herein refers to a polymer matrix in which the binding sites are distributed throughout the matrix, preferably uniformly (essentially evenly) throughout the polymer matrix. By homogeneous with respect to the polymer matrix is meant that specific parameters of the matrix, such as crosslinking and the distribution of (primary) binding sites, are uniformly distributed throughout the matrix.

[0153] There are certain heterogeneous resins for solid-phase peptide synthesis in which the binding sites are not uniformly distributed within the resin. Rather, the binding sites are concentrated (confined) on or near the surface of the resin beads that form a shell, and the core of the beads is essentially devoid of binding sites.

[0154] The homogeneous polymer matrix does not contain a heterogeneous resin having substantially all of its binding sites on or near the surface.

[0155] According to a further aspect, the polymer matrix is selected from polymer matrices obtained by emulsion polymerization comprising at least styrene and a crosslinking agent, preferably divinylbenzene (DVB), specifically a homogeneous matrix. Preferably, the crosslinking agent is present in an amount of less than 4.0 wt%, preferably less than 3.5 wt%, preferably less than 3.0 wt% during the emulsion polymerization. Preferably, DVB is present in an amount from about 0.5 wt% to about 2.5 wt%.

[0156] According to one aspect, the polymer matrix is selected from homogeneous crosslinked polystyrene-based matrices, preferably a homogeneous crosslinked polystyrene-based matrix comprising a crosslinking agent of less than 4.0 wt%, preferably less than 3.5 wt%, preferably less than 2.0 wt%.

[0157] The polystyrene-based matrix may be modified by various monomers such as hydroxyethylstyrene and polyethylene glycol in addition to crosslinking with DVB, preferably.

[0158] The polymer matrix can be formed by emulsion polymerization. A particularly useful polymer matrix is a styrene-based matrix crosslinked with divinylbenzene (DVB). The styrene-based matrix crosslinked with DVB preferably has a DVB content of less than 4.0 wt%, preferably less than 3.5 wt%, preferably less than about 3.0 wt%.

[0159] The polymer matrix can also be shown as a resin. The polymer matrix can be selected from polystyrene, polyacrylate, polyacrylamide, polyamide or polyethylene glycol. The polymer matrix can also be a blend of at least two different types of polymers. Thus, the polymer matrix can be polystyrene modified by another type of polymer, such as ethylene glycol, polyacrylate, polyamide or polyacrylamide.

[0160] The insoluble support is preferably in particulate form acting as a solid phase. The particulate form of the insoluble support is suitably spherical. Thus, the insoluble support is suitably spherical and can be shown in bead form or simply as beads. The beads are preferably spherical. The size of the insoluble support (spherical beads) can range from about 1 μm to about 2000 μm, for example up to about 1000 μm, suitably from about 5 μm to about 750 μm, for example from about 20 μm to about 500 μm, from about 50 μm to about 300 μm, specifically within the range of about 50 - 150 μm. Not only the bead size problem but also the bead size distribution should be considered.

[0161] The polymer matrix is usually substituted. Substitution can be achieved by directly incorporating substituents onto the polymer matrix, for example, by electrophilic aromatic substitution reactions, or by copolymerization of a substituted polymer with the polymer of the polymer matrix, such as styrene.

[0162] Examples of polymer matrices include polystyrene-based matrices modified with aminomethyl (AM or AMS), 4-methylbenzhydrylamine (MBHA), cross-linked polystyrene-based matrices modified with PEG by appropriately grafting PEG to a polystyrene-based matrix, such as Tentagel trademark resins, HypoGel registered trademark and Tentage registered trademark, polyethylene glycol (PEG)-based resins / matrices, such as ChemMatrix registered trademark and NovaPEG, aminomethylated polystyrene-based matrices partially derivatized with methyl-PEG-p-nitro-phenyl-carbonate, such as NovaGel trademark, polystyrene-based matrices copolymerized using hydroxyethyl polystyrene and polyethylene glycol (PEG), such as NovaSyn trademark.

[0163] Construct The construct preferably contains repeating units such as branching agents and optionally spacers, so the construct may be called a branched molecule and, in a sense, may also be called a branched polymer. As described above, the construct contains at least a branching agent and optionally a spacer. Theoretically, there is no upper limit to the number of branching agents contained in the construct. In most applications, the upper limit number of branching agents in the construct is about 150. Most constructs contain a plurality of branching agents from 1 to about 150, from 1 to about 100, for example from about 1 to about 80. Branching is induced by the branching agent. According to one aspect, the branching agent and the spacer are selected from amino acids or derivatives of amino acids. A construct containing a branching agent and a spacer selected from amino acids may be represented as a branched peptide. Thus, a particular construct may be called a branched peptide or polypeptide. The molecular weight of the construct excluding the linker can start from about 100 g / mol and go up to about 200,000 g / mol, up to about 100,000 g / mol, up to about 10,000 g / mol. The exemplified 2X(Gly) construct has a molecular weight of about 1245 g / mol, and the 16X(Gly) construct (both exemplified herein) has a molar weight of about 3375 g / mol. The construct contains a cleavable linker coupled to a distal branching agent that is suitable for the chemical properties of the target polypeptide and peptide, preferably taking into account the appropriate capping strategy.

[0164] General disclosure of the insoluble support of the present invention and methods for forming constructs The insoluble support can be formed by providing a commercially available polymer matrix and by the stepwise synthesis of the construct, i.e., a divergent synthesis protocol, such as a solid-phase peptide synthesis protocol. Alternatively, the insoluble support can be formed by providing a commercially available polymer matrix and coupling the complete construct, i.e., a convergent synthesis protocol. Preferably, the insoluble support is formed by a solid-phase synthesis protocol that involves the sequential addition of relevant chemical compounds, specifically a branching agent, a linker, and a spacer.

[0165] The construct is preferably provided by a process protocol similar to that for providing an insoluble support, but by applying a linker between the construct and the resin, cleavage of the construct from the resin is enabled. 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 derivatives of amino acids, providing a construct that can be called a peptide, typically a branched peptide or a branched polypeptide.

[0166] Any solid-phase peptide synthesis chemistry, including various amine protection chemistries, side-chain protection chemistries, and peptide bond formation activation chemistries, can be applied to the synthesis of the construct.

[0167] Reagents for solid-phase synthesis are readily available from commercial sources. Solid-phase synthesis procedures typically include the following iterative operations: deprotection of the N-terminal alpha-amine protecting group and / or deprotection of the side-chain amine protecting group of the amino acid covalently coupled to the solid phase, coupling of the amino acid with the N-terminal amine protected and the side-chain amine protected, a capping step in the case of unreacted amino acids, such as an acetylation step, and a washing step for the removal of by-products. Further, the following operations can be applied: blocking interfering groups during the reaction and protecting the amino acids.

[0168] According to one aspect, the branching agent is selected from diaminoalkanoic acids including at least two amino groups and an aminoalkanoic acid containing from 3 to 10 carbon atoms, preferably a diaminoalkanoic acid containing from 3 to 10 carbon atoms, such as diaminopropionic acid, diaminobutyric acid, diaminopentanoic acid, diaminohexanoic acid, and diaminoheptanoic acid, selected from lysine analogs.

[0169] The amine groups of the diaminoalkanoic acid can be protected by various protecting groups including fluoren-9-ylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), tert-butyl tBu, as well as trityl-based protecting groups including 4-methyltrityl (Mtt) and 4-methoxytrityl (Mmt).

[0170] The Fmoc strategy has the advantage over the Boc strategy that Fmoc can be removed under milder reaction conditions. The Fmoc strategy allows for 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 basic conditions without removing side-chain protecting groups that are labile to acid.

[0171] According to one aspect, the amine groups of the diaminoalkanoic acid (DAA) are protected by groups that cleave under similar cleavage conditions, and typically, it is presumed that the amine groups of the diaminoalkanoic acid are protected by the same (identical) protecting group. However, each amine group of the diaminoalkanoic acid, namely, the N-terminal alpha-amine group and the side-chain amine group, may be protected by protecting groups that cleave under different cleavage conditions such as different pH values.

[0172] Diaminoalkanoic acids in which the amines are protected by the same protecting group are also called §-DAA(§), where § represents the alpha-amine protecting group and (§) represents the epsilon-amine protecting group.

[0173] Diaminoalkanoic acids in which the amines are protected by different protecting groups are also called $-DAA (Euro symbol), where $ represents the alpha-amine protecting group and (Euro symbol) represents the side-chain or orthogonal amine protecting group. The DAA can be lysine (K).

[0174] The diaminoalkanoic acid protecting group can be selected from Mtt (4-methyltrityl) or Mmt (4-methyloxytrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)] or ivDde.

[0175] The protecting group § may be Fmoc, and $ may be selected from Mtt (4-methyltrityl) or Mmt (4-methyloxythrityl), Alloc (allyloxycarbonyl), Dde [N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)], or ivDde.

[0176] According to one aspect, all aminoalkanoic acids of the construct are protected by a protecting group that cleaves essentially all simultaneously during the same cleavage step. For example, the two amine groups (of all diaminoalkanoic acids) are all protected by the same protecting group such as Fmoc, e.g., §-DAA(§). Only aminoalkanoic acids in which all amines are protected by the same protecting group are implemented for synthesizing the construct. For example, both the alpha-amine and the epsilon-amine (when the aminoalkanoic acid is a diaminoalkanoic acid) are coupled to the same (identical) compound, e.g., a branching agent, spacer, or linker.

[0177] According to one aspect, at least one aminoalkanoic acid is used as a branching agent (during synthesis of the construct on an insoluble support), e.g., $-DAA (Euro symbol), in which each amine group is protected by a protecting group that cleaves under different cleavage conditions (e.g., different pH). By implementing aminoalkanoic acids having amine protecting groups that cleave under different conditions (typically under separate cleavage steps), it is possible to couple different compounds to the alpha-amine and the side-chain amine.

[0178] The construct can be formed using any combination of different diaminoalkanoic acids, as well as diaminoalkanoic acids having amine group protecting groups that cleave under the same cleavage step (and / or cleave under essentially the same cleavage conditions), for example §-DAA(§), and diaminoalkanoic acids having amine protecting groups that cleave under different cleavage conditions, for example $-DAA(€). The construct may be formed by using only diaminoalkanoic acids in which both amine groups (N-terminal, alpha amine group and side chain amine group, for example epsilon amine group) are protected by the same protecting group. For example, both amine groups of all diaminoalkanoic acids are Fmoc protected. Alternatively, the construct can be formed by using only diaminoalkanoic acids in which each amine group is protected by a different protecting group that cleaves under different cleavage conditions, typically at different pHs. For example, one amine group of the diaminoalkanoic acid is protected by Fmoc and the other amine group is protected by, for example, an Mtt or Mmt group. The insoluble support 7X linear disclosed in the experimental section is first synthesized by using only lysine in which the amine group of each lysine is protected by one Fmoc group and one Mtt group as a branching agent to form the main chain, and then coupling Fmoc-Lys(Fmoc) to the side chain of the lysine in the main chain. In the case of the exemplified 7X insoluble support, the main chain contains three lysines (provided, for example, as Fmoc-Lys(Mtt)). Constructs with any odd number of binding sites (e.g., 3, 5, 7, 9, 11, etc.) can be provided by coupling any number of, for example, Fmoc-Lys(Mtt) to the polymer matrix (1, 2, 3, 4, 5, 6, etc.), thereby forming a main chain containing from one to any number of Fmoc-Lys(Mtt). The main chain can be represented as Fmoc-Lys(Mtt)-[Lys(Mtt)] X - polymer matrix, where X is an integer from 0 to some value. X may be from 0 to 50. In this regard, the main chain is a chain in which consecutive lysines are coupled to each other by an alpha amine.

[0179] Generally, a linear construct can be formed by successively coupling a polymer matrix, a branching agent selected exclusively from amino alkanoic acids having an N-terminal amine and side-chain amines that are protected by protecting groups that cleave under different cleavage conditions (e.g., different pH) and typically during different cleavage steps, and optionally a spacer, thereby forming a backbone. After formation of the backbone, the side-chain protecting groups are cleaved under appropriate cleavage conditions. In subsequent steps, a branching agent selected exclusively from amino alkanoic acids having an N-terminal amine and side-chain amines that are protected by protecting groups that cleave under the same cleavage conditions, which are in effect the same protecting groups. In further reaction steps, all remaining amine protecting groups are cleaved, and then appropriate linkers are coupled to all deprotected amine groups.

[0180] A further aspect relates to a synthetic scheme in which a branching agent of format -DAA (Euro symbol) is used to provide a backbone that optionally has a spacer between branching agents and between the polymer matrix and the first-generation branching agent. In a further step, the (Euro symbol) protecting groups are removed by appropriate cleavage conditions. Subsequently, a branching agent of format §-DAA (§) is coupled. If no further steps are implemented where the branching agent is attached, a so-called linear construct is formed. 7X linear represents the linear construct.

[0181] For certain constructs, specifically for asymmetric constructs, also referred to herein as linear constructs, the side-chain amines of the branching agent, e.g., lysine, are protected by Mtt, Mmt, alloc, Dde, or ivDde.

[0182] According to one aspect, the construct is synthesized as a solid phase on a suitable polymer matrix by application of a solid-phase synthesis protocol that includes the use of a branching agent selected from diamino alkanoic acids containing from 3 to 10 carbon atoms, wherein both the N-terminal amine group (alpha amine group) and the side-chain amine groups of the branching agent are protected by groups that cleave under similar reaction conditions, i.e., the protecting groups cleave simultaneously.

[0183] When applicable, amino acid type spacers, such as spacers selected from amino acids containing one carboxylic acid group, one amine group and an aliphatic hydrocarbon, are Fmoc protected.

[0184] According to a further aspect, the construct is synthesized on a polymeric matrix suitable as a solid phase by application of a solid phase synthesis protocol involving the use of a branching agent selected from diaminoalkanoic acids containing from 3 to 10 carbon atoms, wherein the N-terminus (alpha amine group) and the amine groups of the side chains are each protected by two different protecting groups that cleave under different cleavage conditions such as different pHs. Preferably, the protecting groups are protecting groups that are labile to acid and labile to base, suitably selected from Fmoc, Mtt and Mmt. By coupling a base-sensitive Fmoc protecting group and an acid-sensitive Mtt or Mmt group to each amine group of a branching agent, for example a diaminoalkanoic acid, different chemical moieties can be coupled to the same branching agent.

[0185] When both the alpha amine group and the side chain amine group have protecting groups that cleave essentially simultaneously during the same cleavage step, a symmetric construct is formed. When each amine group of at least one branching agent, for example a diaminoalkanoic acid, is protected by two different protecting groups that cleave under different cleavage conditions such as different pHs, for example Fmoc and Mtt or Mmt, an asymmetric linear construct is formed.

[0186] Preferably, both the alpha amine group and the side chain amine group are protected with the same protecting group, such as Boc or Fmoc, preferably Fmoc. Preferably, both the alpha amine group and the epsilon amine group of lysine are Fmoc protected.

[0187] The 7X straight chain is an asymmetric straight-chain construct. When the reaction conditions are such that at least one of the alpha-amine group and the epsilon-amine group of lysine couples to different chemical moieties, such as a spacer and a branching agent (lysine), an asymmetric construct is formed. To achieve coupling of the alpha-amine group and the epsilon-amine group of lysine to different chemical moieties, the alpha-amine group and the epsilon-amine group of lysine must have protecting groups that cleave under different reaction conditions. One way to achieve this is to have a protecting group labile to one acid and a protecting group labile to a base. Thus, one amine group of lysine can be protected by Fmoc, which is labile to a base, and the other amine group can be protected by a protecting group labile to an acid, such as Mtt and / or Mmt.

[0188] Lysine branching agents containing N-terminal Fmoc protection and Mtt or Mmt protection of the side-chain amine may each be coupled to a further branching agent and to a spacer or linker. The synthesis of the construct may incorporate diaminoalkanoic acids containing two Fmoc groups (i.e., both the N-terminal amine and the side-chain amine are Fmoc-protected), and / or diaminoalkanoic acids containing one Fmoc group and Mtt and / or Mmt groups.

[0189] The Fmoc group is typically removed by application of a base, such as a heterocyclic amine, preferably piperidine. The Mtt group can cleave under acidic conditions, for example, by application of a solution containing dichloromethane (DCM) and trifluoroacetic acid (TFA). The Boc protecting group is base-labile (sensitive) and is preferably removed by application of TFA.

[0190] Examples of amino acid coupling chemistry 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(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yl-oxytris(pyrrolidino)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), and 4,4,4-trifluoro-N-Fmoc-O-tert-butyl-threonine.

[0191] Typically, the secondary binding sites provided by the insoluble support include a cleavable linker that enables cleavage of 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.

[0192] Solvents useful for the preparation of the insoluble support are methyl chloride, N-methylpyrrolidone (NMP), N-methyl-2-pyrrolidone (NMP) and dimethylformamide (DMF).

[0193] Peptide synthesis using the insoluble support of the present invention The insoluble support of the present invention is preferably applied to the synthesis of peptides. According to one aspect, peptide synthesis is initiated after the formation of the insoluble support. Thus, the peptide synthesis protocol may start with the provision of the polymeric matrix and subsequent synthesis of the construct, thereby forming the insoluble support of the present invention. Prior to the initiation of the target peptide synthesis, the insoluble support can be subjected to a processing step that provides the insoluble support with conditions suitable for subsequent target peptide synthesis.

[0194] Any solid-phase peptide synthesis chemistry compatible with the synthesis of modified insoluble supports, including various amine protection chemistries and side-chain protection chemistries, as well as peptide bond formation activation chemistries, can be applied to the synthesis of the target peptide.

[0195] According to one aspect, the insoluble support is particularly suitable for the synthesis of long and complex peptides, i.e., peptides having 10, 15, 20, 25, more than 30 amino acids, and further peptides having more than 50 amino acids.

[0196] The insoluble support of the present invention can synthesize complex peptides with commercially acceptable yields and commercially useful purities.

[0197] The insoluble support of the present invention is typically formed using a commercially available homogeneous polymer matrix / resin that is modified by synthesizing a branched construct from available primary binding sites in the polymer matrix.

[0198] Thus, insoluble supports based on commercially available homogeneous polymer resins are a commercially attractive option for successfully synthesizing complex peptides without developing new high-performance resins. Commercially available homogeneous polymer resins can be converted into insoluble supports that can generate complex peptides in high throughput in SPPS with high yields and commercially reasonable purity by applying fairly simple and uncomplicated SPPS schemes and readily available amino acids.

[0199] The present invention also relates to a method 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 to the application of the insoluble supports disclosed herein.

[0200] A general process for synthesizing a peptide on a resin begins by connecting the first amino acid, which is the C-terminal residue, to the resin. To prevent polymerization of the amino acids, the alpha amino group and reactive side chains are protected with temporary protecting groups. Once the amino acid is connected 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. Then, the next amino acid is coupled to the attached amino acid. This is followed by another washing procedure, leaving the resin peptide prepared for the next coupling cycle. This cycle is repeated until the peptide sequence is complete. Then, typically, the peptide is cleaved from the resin along with the removal of all protecting groups. Alternatively, the side chain protecting groups are removed, the peptide resin is washed, and the peptide is cleaved from the resin.

[0201] Typically, an N-α-carbamoyl protected amino acid is coupled with the N-terminal amino acid on the growing peptide chain connected to the resin by a linker construct 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 with the next desired Nα-protected amino acid to be added to the peptide chain.

[0202] Suitable amine protecting groups are well known in the art and are described, for example, in "Protecting Groups in Organic Synthesis" by Green and Wuts, John Wiley and Sons, 1991. The most commonly used examples include fluorenylmethoxycarbonyl (Fmoc) and Boc (tert-butyloxycarbonyl). After completion of the synthesis, the peptide is cleaved from the solid support, preferably using standard procedures under acidic conditions while simultaneously deprotecting the side chains.

[0203] The target crude peptide is cleaved from the insoluble support containing the construct. The target peptide cleavage conditions depend in part on the type of linker and side chain protecting groups used.

[0204] The crude peptide is typically analyzed by RP-HPLC on a C18 (CHS-Agilent UPLC) column using a water-acetonitrile gradient in 0.1% TFA. The purity can be verified by analytical RPHPLC. The identity of the peptide can be verified by mass spectrometry. The peptide can be solubilized in aqueous buffers over a wide pH range.

[0205] The insoluble support of the present invention can be regenerated after cleavage of the target peptide. The insoluble support can be regenerated after separating the target peptide from the peptide-insoluble support solution.

[0206] Detailed disclosure of specific embodiments of the present invention Disclosed below are insoluble supports comprising constructs that provide a 2-fold, 4-fold, 7-fold, 8-fold, and 16-fold increase in the initial (primary) binding sites of the polymer matrix. Lysine (K or Lys) is used throughout as the branching agent. Where applicable, glycine (G or Gly), polyethylene glycol (PEG6), or beta-alanine (βAla) is used as a spacer. PEG6 indicates that the PEG contains six individual PEG moieties covalently linked by peptide bonds.

[0207] Aminomethyl (AMS) polystyrene resin is used as the starting polymer matrix for the synthesis of all insoluble supports shown below. All AMS resins used are aminomethyl-modified polystyrene resins crosslinked with 1% divinylbenzene. The AMS resins are provided as beads in the range of 35 - 150 μm (100 - 200 mesh) with substitution degrees of 0.6, 0.93, and 1.95 mmol / gram, respectively.

[0208] Since lysine is used as the branching agent, the constructs of the insoluble supports disclosed in this section can be shown as branched polypeptides synthesized on the polymer matrix.

[0209] For all insoluble supports, lysine in the S configuration (L-amino acid) and the R configuration (D-amino acid) is used.

[0210] For all insoluble supports except 7X linear (2X(Gly), 4X(Gly), 8X(Gly), 16X(Gly)), the branching agent lysine is provided with two Fmoc groups to protect the two amines, i.e., the branching agent is provided as Fmoc-Lys(Fmoc)-OH.

[0211] The 7X straight chain is a straight-chain structure. In the synthesis of the straight-chain 7X insoluble support, two classes of lysine that differ with respect to amine protecting groups are used. For one class of lysine, both amine groups are protected by Fmoc: Fmoc-Lys(Fmoc)-OH. For the other class of lysine, the alpha amine is protected by Fmoc and the epsilon amine is protected by Mtt: Fmoc-Lys(Mtt)-OH.

[0212] Presentation of the structures of some of the reactants: Structural formula of Fmoc-Lys(Fmoc) (R and S configurations) where both amine groups are Fmoc-protected: [Chem.]

[0213] Structural formula of Fmoc-Lys(Mtt)-OH (S configuration) where the N-terminal alpha amine is Fmoc-protected and the side-chain epsilon amine group is Mtt- or Mmt-protected. [Chem.]

[0214] The Fmoc-Rink amide linker (Fmoc-Rink-OH) has the following chemical structure. [Chem.]

[0215] Fmoc Ramage linker (MW: 505.58): [Chem.]

[0216] HMPB TBDMS linker and HMPB linker: [Chem.]

[0217] The glycine spacer is provided as Fmoc - protected glycine. PEG is provided as 2 - [2 - [2 - (Fmoc - amino)ethoxy]ethoxy]acetic acid (EAAE).

Chemical formula

[0218] Comments on the naming of the exemplified insoluble supports: The numbers in parentheses for the branching agent lysine K (or Lys) indicate the 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 insoluble support construct has two secondary lysines.

[0219] Disclosure of the solid support Solid supports 2X(Gly), 4X(Gly), and 8X(Gly) (without linker) based on 0.60 mmol / g AMS polymer matrix were synthesized using the following protocol: The starting AMS resin (30.0 g, Table 1) with an initial treatment amount 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 of starting resin, 2 × 1 h). All amino acid couplings were carried out using Fmoc amino acids (0.50 M) (4.0 equivalents), Oxyma (4.0 equivalents), and N,N'-diisopropylcarbodiimide (5.0 equivalents) in DMF at 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), followed by the addition of Oxyma and N,N'-diisopropylcarbodiimide and pre-activated at room temperature for 30 min, then transferred to the glass reactor. The coupling was carried out at room temperature for 2 h. After coupling, the resin was washed with DMF (2 × 8 mL / g), capped using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min, and rewashed using DMF (7 × 8 mL / g). Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). After Fmoc deprotection, the resin was washed using DMF (7 × 8 mL / g of starting resin).

[0220] After completion of the 2X-Gly-AMS synthesis, the resin was washed with CH2Cl2 (3 times), iPrOH (3 times), and MTBE (3 times), and then dried overnight under vacuum. One-third of the obtained resin was stored, and the remaining two-thirds (28.2 g, Table 1) was used for the synthesis of 4X-Gly-AMS in the same manner as 2X-Gly-AMS.

[0221] After completion of the 4X-Gly-AMS synthesis, the resin was washed with CH2Cl2 (3 times), iPrOH (3 times), and MTBE (3 times), and then dried overnight under vacuum. Two-thirds of the obtained resin was stored, and the remaining one-third was used for the synthesis of 8X-Gly-AMS in the same manner as 2X-Gly-AMS.

[0222] After completion of the 8X-Gly-AMS synthesis, the resin was washed with CH2Cl2 (3 times), iPrOH (3 times), and MTBE (3 times), and then dried overnight under vacuum.

Table 1

[0223] Solid supports 2X(Gly) and 4X(Gly) (without linker) based on 0.93 mmol / g AMS polymer matrix and 1.95 mmol / g AMS polymer matrix respectively were synthesized using the following protocol: Starting AMS resins (1.0 or 2.0, Tables 2 and 3) with an initial loading of 0.93 mmol / g or 1.95 mmol / g were introduced into a 60 mL plastic syringe and swollen in DMF (8 mL / g starting resin, 2×1 h). Using an Activo-PLS 4×4 synthesizer purchased from Activotec, the syringe was shaken manually in a horizontal position at 400 rpm for synthesis. Coupling of all amino acids was carried out using Fmoc amino acids (0.50 M) (4.0 eq), Oxyma (4.0 eq), and N,N'-diisopropylcarbodiimide (5.0 eq) in DMF at 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), followed by the addition of Oxyma and N,N'-diisopropylcarbodiimide, pre-activated at room temperature for 30 min, and then transferred to a glass reactor. Coupling was carried out at room temperature for 2 h. After coupling, the resin was washed with DMF (2×8 mL / g), capped using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09) for 30 min, and rewashed using DMF (7×8 mL / g). Prior to each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1×10 min + 1×20 min). After Fmoc deprotection, the resin was washed using DMF (7×8 mL / g starting resin).

[0224] The 2X-Gly-AMS and 4X-Gly-AMS soluble supports synthesized using 0.93 mmol / g of AMS resin were isolated starting from the starting AMS resin (2.0 g) of the same reactor batch. After completion of the 2X-Gly-AMS synthesis, the resin was washed with CH2Cl2 (3 times), iPrOH (3 times) and MTBE (3 times), and then dried overnight under vacuum. Half of the obtained resin was stored, and the remaining half was used for the synthesis of 4X-Gly-AMS in the same manner as 2X-Gly-AMS. After completion of the 4X-Gly-AMS synthesis, the resin was washed with CH2Cl2 (3 times), iPrOH (3 times) and MTBE (3 times), and then dried overnight under vacuum.

[0225] The 2X-Gly-AMS and 4X-Gly-AMS soluble supports synthesized using 1.95 mmol / g of AMS resin were isolated starting from 1.0 g and 2.0 g starting AMS resin batches, respectively. After completion of the synthesis, the resin was washed with CH2Cl2 (3 times), iPrOH (3 times) and MTBE (3 times), and then dried overnight under vacuum.

Table 2

Table 3

[0226] Synthesis protocols for various Fmoc-Rink amide insoluble supports 2X(Gly):(Fmoc-Rink)2-Lys(1)-Gly-AMS 4X(Gly):(Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 8X(Gly):(Fmoc-Rink)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0227] The related soluble supports presented above were modified by coupling of Rink amide using the following protocol: The starting resin (1.0 g each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2 × 1 h). Using an Activo-PLS 4×4 synthesizer purchased from Activotec, the syringe was shaken manually at a horizontal position and 400 rpm for synthesis. Before rink amide linker coupling, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). After Fmoc deprotection, the resin was washed with DMF (7 × 6.5 mL / g starting resin). All rink amide couplings were performed using a 5-fold molar excess of Rink amide linker (0.50 M), PyAOP, and diisopropylethylamine (DIEA) in a 1:1:2 molar ratio compared to the synthesis scale, in DMF. After dissolving the Rink amide in DMF (0.50 M), PyAOP was added and stirred at room temperature for 10 min, then DIEA was added. Next, the solution was introduced into the syringe containing the resin. The coupling was carried out overnight at room temperature. After coupling, the resin was washed with DMF (2 × 8 mL / g) and capped for 30 min using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09). Next, the resin was washed with DMF (7 × 8 mL / g), isopropanol (iPrOH, 3 times), and methyl tert-butyl ether (MTBE, 3 times), and then dried overnight under vacuum.

[0228] Table 4 shows the resin substitution before and after rink amide coupling.

Table 4

[0229] Synthesis protocols for various Fmoc-Ramage insoluble supports 2X(Gly):(Fmoc-Ramage)2-Lys(1)-Gly-AMS 4X(Gly):(Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 8X(Gly):(Fmoc-Ramage)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0230] The related soluble support presented above was modified by Ramage coupling using the following protocol: The starting resin (1.0 g each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2×1 h). The synthesis was carried out manually by shaking the syringe at a horizontal position and 400 rpm using an Activo-PLS 4×4 synthesizer purchased from Activotec. Prior to the Ramage linker coupling, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1×10 min + 1×20 min). After Fmoc deprotection, the resin was washed with DMF (7×6.5 mL / g starting resin). All Ramage couplings were carried out using a 2-fold molar excess of the Ramage linker (0.15 M - 0.40 M) in DMF at an equimolar ratio of 1:1:1, Oxyma, and N,N'-diisopropylcarbodiimide (DIC), compared to the synthesis scale. After dissolving Ramage in DMF (0.15 M - 0.40 M), Oxyma was added and stirred at room temperature for 10 min, followed by the addition of DIC. Next, the solution was introduced into the syringe containing the resin. The coupling was carried out overnight at room temperature. After coupling, the resin was washed with DMF (2×8 mL / g) and capped for 30 min using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09). Next, the resin was washed with DMF (7×8 mL / g), isopropanol (iPrOH, 3 times), and methyl tert-butyl ether (MTBE, 3 times), and then dried overnight under vacuum.

[0231] Table 5 shows the resin substitution before and after Ramage coupling.

Table 5

[0232] Synthesis protocol for various Fmoc-Leu-HMPB insoluble supports 4X(Gly):(Fmoc-Leu-HMPB-Leu)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 8X(Gly):(Fmoc-Leu-HMPB-Leu)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0233] The relevant soluble supports presented above were modified by coupling of Fmoc-Leu-HMPB using the following protocol: The starting resins (1.0 g each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL / g, 2×1 h). Using an Activo-PLS 4×4 synthesizer purchased from Activotec, the syntheses were carried out manually with the syringes in a horizontal position and shaken at 400 rpm. Prior to the TBDMS-HMPB coupling, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1×10 min + 1×20 min). After Fmoc deprotection, the resin was washed with DMF (7×6.5 mL / g starting resin). All TBDMS-HMPB couplings were carried out using a 2-fold molar excess of TBDMS-HMPB (0.50 M) in DMF, PyOxim, and diisopropylethylamine (DIEA) in a 1:1:2 molar ratio compared to the synthesis scale. After dissolving TBDMS-HMPB in DMF (0.50 M), PyOxim was added and stirred at room temperature for 10 min, followed by the addition of DIEA. Next, the solution was introduced into the syringe containing the resin. The coupling was carried out overnight at room temperature. After HMPB coupling, the resin was washed with DMF (2×8 mL / g) and capped for 30 min using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09). Next, the resin was washed with DMF (7×8 mL / g), isopropanol (iPrOH, 3 times), and methyl tert-butyl ether (MTBE, 3 times) and then dried overnight under vacuum.

[0234] The starting HMPB resin (300 mg each) was introduced into a 12 mL syringe and swollen in THF (6.5 mL / g, 2 × 1 h). Using an Activo-PLS 4×4 synthesizer purchased from Activotec, the syringe was shaken manually at a horizontal position and 400 rpm for synthesis. Before the Fmoc-Leu-OH coupling, the TBDMS group was deprotected at room temperature for 2 h using Et3N·3HF (12 equivalents compared to the synthesis scale) (0.22 M) in THF. After TBDMS deprotection, DIEA (once) was used, and then DMF was used to wash the resin to neutral pH.

[0235] Before coupling with Fmoc-Leu-OH, the resin was swollen in DMF (6.5 mL / g, 2 × 1 h). All couplings were carried out using Fmoc-Leu-OH (0.45 M) in DMF at a 2.4-fold molar excess compared to the synthesis scale, with a 1:1:1:0.25 molar ratio of Fmoc-Leu-OH, Oxyma, N,N'-diisopropylcarbodiimide (DIC), and 4-dimethylaminopyridine (DMAP). After dissolving Fmoc-Leu-OH in DMF (0.45 M), Oxyma, DIC, and DMAP were added, stirred at room temperature for 5 min, and then introduced into the resin-containing syringe. The coupling was carried out overnight at room temperature. After coupling, the resin was washed with DMF (2 × 8 mL / g) and capped for 30 min using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09). Next, the resin was washed with DMF (7 × 8 mL / g), isopropanol (iPrOH, 3 times), and methyl tert-butyl ether (MTBE, 3 times), and then dried overnight under vacuum.

[0236] Table 6 shows the resin substitution before and after Leu-HMPB coupling.

Table 6

[0237] Synthesis protocols for various Fmoc-Gly-HMPB insoluble supports 4X(Gly):(Fmoc-Gly-HMPB)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS 8X(Gly):(Fmoc-Gly-HMPB-)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS

[0238] The relevant soluble supports presented above were modified by coupling of Fmoc-Gly-HMPB using the following protocol: The starting resins (1.0 g each) were introduced into 25 mL syringes and swollen in DMF (6.5 mL / g, 2 × 1 h). Synthesis was carried out manually by shaking the syringes in a horizontal position at 400 rpm using an Activo-PLS 4×4 synthesizer purchased from Activotec. Prior to the TBDMS-HMPB coupling, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). After Fmoc deprotection, the resin was washed with DMF (7 × 6.5 mL / g starting resin). All TBDMS-HMPB couplings were carried out using a 2-fold molar excess of TBDMS-HMPB (0.50 M) in DMF, PyOxim, and diisopropylethylamine (DIEA) in a 1:1:2 molar ratio, relative to the synthesis scale. After dissolving TBDMS-HMPB in DMF (0.50 M), PyOxim was added and stirred at room temperature for 10 min, followed by the addition of DIEA. Next, the solution was introduced into the syringe containing the resin. The coupling was carried out overnight at room temperature. After HMPB coupling, the resin was washed with DMF (2 × 8 mL / g) and capped for 30 min using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09). Next, the resin was washed with DMF (7 × 8 mL / g), isopropanol (iPrOH, 3 times), and methyl tert-butyl ether (MTBE, 3 times), and then dried overnight under vacuum.

[0239] The starting HMPB resin (300 mg each) was introduced into a 12 mL syringe and swollen in THF (6.5 mL / g, 2 × 1 h). Using an Activo-PLS 4×4 synthesizer purchased from Activotec, the synthesis was carried out manually by shaking the syringe in a horizontal position at 400 rpm. Before the Fmoc-Gly-OH coupling, the TBDMS group was deprotected at room temperature for 2 h using Et3N·3HF (12 equivalents compared to the synthesis scale) (0.22 M) in THF. After TBDMS deprotection, DIEA (once) was used, and then DMF was used to wash the resin to neutral pH.

[0240] Before the coupling with Fmoc-Gly-OH, the resin was swollen in DMF (6.5 mL / g, 2 × 1 h). All couplings were carried out using Fmoc-Gly-OH (0.45 M) in DMF at a 2.4-fold molar excess compared to the synthesis scale, in a 1:1:1:0.25 molar ratio with Oxyma, N,N'-diisopropylcarbodiimide (DIC), and 4-dimethylaminopyridine (DMAP). After dissolving Fmoc-Gly-OH in DMF (0.45 M), Oxyma, DIC, and DMAP were added, stirred at room temperature for 5 min, and then introduced into the syringe containing the resin. The coupling was carried out overnight at room temperature. After coupling, the resin was washed with DMF (2 × 8 mL / g) and capped for 30 min using DMF / acetic anhydride (Ac2O) / diisopropylethylamine (DIEA) (1:0.04:0.09). Next, the resin was washed with DMF (7 × 8 mL / g), isopropanol (iPrOH, 3 times), and methyl tert-butyl ether (MTBE, 3 times), and then dried overnight under vacuum.

[0241] Table 7 shows the resin substitution before and after the Gly-HMPB coupling.

Table 7

[0242] (Fmoc-linker)2-Lys(1)-Gly-AMS insoluble support [2X(Gly)] Linkers: Fmoc-Rink and Fmoc-Ramage Molecular weight of each construct without a support: Fmoc-Rink: Molecular weight (g / mol): 1245.40 Fmoc-Ramage: Molecular weight (g / mol): 1291.47

[0243] The insoluble support constructs contain one lysine, one glycine spacer, and two Fmoc-Rink-linkers or two Fmoc-Ramage-linkers coupled to the amine of a lysine of the first generation (first layer), also called the distal lysine (distal branching agent). The glycine spacer is placed between the polymer matrix and the lysine branching agent.

[0244] (Fmoc-Rink)2-Lys(1)-Gly and (Fmoc-Ramage)2-Lys(1)-Gly constructs contain one layer of lysine (first generation), the primary layer, i.e., the primary lysine. Each construct provides two distal binding sites available for peptide synthesis. The two Rink-linkers or two Ramage-linkers are attached to the amine groups (N-terminal alpha amine and side chain epsilon amine) of each lysine. Lysine is attached to glycine, and glycine is attached to the amine of the polymer matrix.

[0245] Only lysine with both amine groups Fmoc-protected is used.

[0246] The polymer matrices used for modification are AMS0.6, AMS0.93, and AMS1.95. Reactants: Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH

[0247] Structural formula of the (Fmoc-Rink)2-Lys(1)-Gly construct

Chemical formula

[0248] Structural formula of (Fmoc-Ramage)2-Lys(1)-Gly construct [Chemical formula]

[0249] Synthesis scheme of (Fmoc-linker)2-Lys(1)-Gly-AMS: Linker: Rink and Ramage [Table 8]

[0250] (Linker)4-Lys(2)2-Gly-Lys(1)-Gly-AMS insoluble support [4X(Gly)] Linker: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS Molecular weight of each construct without support: Fmoc-Rink: Molecular weight (g / mol): 2658.99 Fmoc-Ramage: Molecular weight (g / mol): 2522.93 HMPB TBDMS: Molecular weight (g / mol): 1461.66

[0251] These modified resin constructs contain Gly (G) in addition to three lysines and four linkers coupled to the second-generation lysine. The Gly or β-Ala spacer is placed between the first-generation lysine and the second-generation lysine, and between the first-generation lysine and the polymer matrix.

[0252] (Fmoc-Rink)4-Gly(2)2-Gly2-Lys(1)-Gly, (Fmoc-Ramage)4-Lys(2)2-Gly 2-The Lys(1)-Gly and (HMPB TBDMS)4-Lys(2)2-Gly2-Lys(1)-Gly constructs contain two layers (generations) of lysine, namely the primary and secondary layers, i.e., primary and secondary lysines, and the secondary layer of lysine is the lysine most distal to the polymer matrix. The two secondary lysines of the second layer provide a total of four secondary binding sites available for peptide synthesis. Two Fmoc-Rink-linkers, two Fmoc-Ramage-linkers, or two (HMPB TBDMS)-linkers are attached to the two amine groups (N-terminal alpha-amine and side-chain epsilon-amine) of each secondary lysine. Thus, the construct has four linkers.

[0253] The polymer matrices used for modification are AMS0.6, AMS0.93, and AMS1.95.

[0254] Only lysine with both amine groups Fmoc-protected is used. Reactants: Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-beta-Ala-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH, HMPB TBDMS-OH

[0255] Structural formula of the (Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly construct:

Chem.

[0256] Structural formula of the (Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly construct:

Chem.

[0257] Structural formula of the (TBDMS-HMPB)4-Lys(2)2-Gly2-Lys(1)-Gly construct:

Chem.

[0258] For the characterization of 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 multi-channel peptide synthesizer. Sieber amide resin (Cas 915706-90-0) was used to cleave the construct from the polymer matrix. The branching agent Fmoc-Lys(Fmoc)-OH, Oxyma, Dic (diisopropylcarbodiimide), and the spacers (Gly, βAla = beta-alanine) were used in a 3-fold molar excess relative to the theoretically free amino groups. Amide coupling was allowed to proceed for 3 h at room temperature. The resulting constructs were characterized by LC / MC.

[0259] (NH2-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-NH2 Calculated mass: 1770.99 Calculated: (M + 2H)2+ = 886.49 Observed: (M + 2H)2+ = 885.48

Chemical Structure

[0260] (NH2-Rink)4-Lys(2)2-βAla2-Lys(1)-βAla-NH2 Calculated mass: 1810.89 Calculated: (M + 2H)2+ = 906.46 Observed: (M + 2H)2+ = 907.36

Chemical Structure

[0261] (Linker)4-K(2)2-G2-K(1)-G-AMS) synthesis scheme: Linkers: Fmoc-Rink, Fmoc-Ramage, and HMPB TBDMS [Table 9]

[0262] (Linker)8-Lys(3)4-Gly4-Lys(2)2-Gly2-K(1)-Gly-AMS insoluble support [8X(Gly)] Linkers: Fmoc-Rink, Fmoc-Ramage, and HMPB TBDMS Fmoc-Rink: Molecular weight (g / mol): 5486.17 Fmoc-Ramage: Molecular weight (g / mol): 4370.12 HMPB TBDMS: Molecular weight (g / mol): 4005.64

[0263] The construct of this insoluble support theoretically increases each primary binding site of the base AMS resin eightfold. The construct contains seven lysines, seven glycines, and eight linkers. The construct has three layers / generations of lysines, namely, one primary (proximal) lysine, two secondary (intermediate) lysines, and four tertiary (distal) lysines. All secondary intermediate lysines between the primary lysine (proximal lysine) and the distal lysine (here, the tertiary lysine) are each bonded to three different lysines, indicating that the construct is symmetric.

[0264] The polymer matrix used for modification is AMS0.6.

[0265] All lysines are exclusively of the Fmoc-Lys(Fmoc)-OH type. Reactants: Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH, Fmoc-Ramage-OH, TBDMS-HMPB-OH

[0266] Structure of (Fmoc-Rink)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS insoluble support [Chemical formula]

[0267] Structure of (Fmoc-Ramage)8-K(3)4-G4-K(2)2-G2-K(1)-G-AMS insoluble support [Chemical formula]

[0268] Structure of (TBDMS-HMPB)8-K(3)4-G4-K(2)2-G2-K(1)-G-AMS insoluble support [Chemical formula]

[0269] Synthesis scheme of (Linker)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS: Linker: Fmoc-Rink, Fmoc-Ramage, TBDMS-HMPB [Table 10]

[0270] (Fmoc-Rink)4-(PEG6)4-Lys(2)2-Lys(1)-AMS insoluble support [4X(PEG)] The construct of this modified insoluble support contains a PEG6 spacer in addition to three lysines and four Fmoc-Rink-linkers. The PEG6 spacer is placed between the distal lysine and the Fmoc-Rink-linker.

[0271] The PEG spacer is provided as 2-[2-[2-(Fmoc amino)ethoxy]ethoxy]acetic acid (EAAE). Thus, the PEG6 spacer is provided by coupling six AEEA (PEG):s to each amine group of the secondary (distal) lysine. [Chemical formula]

[0272] The polymer matrix used for modification is AMS0.6.

[0273] Only lysine with both amine groups Fmoc-protected is used. Reactants: Fmoc-Lys(Fmoc)-OH, AEEA, Fmoc-Rink-OH

[0274] Synthesis of insoluble support (Fmoc-Rink)4-(PEG6)4-Lys(2)2-Lys(1)-AMS The Fmoc-protected amino acid (K) and AEEA (PEG) are coupled by an iterative reaction process involving deprotection of the Fmoc group, followed by coupling of the relevant Fmoc-protected amino acid and AEEA. After successive coupling of the last AEEA, the linker is coupled. The amide bond is formed using a 3-fold excess of each of equimolar ratios of Fmoc-Lys(Fmoc)-OH, AEEA and Fmoc-Rink-OH, Oxyma pure (ethyl cyano(hydroxyimino)acetate: Novabiochem registered trademark), and diisopropylcarbodiimide (DIC) over the theoretically free amino groups (calculated from the first AMS binding site) in DMF. Before each coupling step, the Fmoc group is removed using two treatments (5 minutes and 20 minutes each) of 25% (v / v) piperidine in DMF. The amide coupling procedure is allowed to proceed for 2 hours at room temperature RT.

[0275] Synthesis scheme of (Fmoc-Rink)4-(PEG6)4-LysK(2)2-LysK(1)-AMS:

Table 11

[0276] Structure of (Fmoc-Rink)4-(PEG6)4-Lys(2)2-Lys(1)-NH2 construct:

Chem.

[0277] (Linker)7-LysGly-linear-Gly-AMS insoluble support [7X linear] Linkers: Fmoc-Rink, Fmoc-Ramage and HMPB TBDMS Molecular weight of each construct without the support: Fmoc-Rink: Molecular weight (g / mol): 4608.22 Fmoc-Ramage: Molecular weight (g / mol): 4370.12 TBDMS-HMPB: Molecular weight (g / mol): 3312.76

[0278] This construct of the insoluble support contains six lysines, three glycines as spacers, and seven linkers selected from Fmoc-Rink-OH, Fmoc-Ramage, and HMPB TBDMS, and the linkers are coupled to four of the six lysines.

[0279] In the synthesis of the linear 7X insoluble support, two classes of lysines that differ with respect to the protecting groups are used. One class of lysine has both amine groups protected by Fmoc: Fmoc-Lys(Fmoc), and the epsilon amine is protected by Mtt: Fmoc-Lys(Mtt)-OH.

[0280] The construct of the 7X insoluble support is synthesized by first synthesizing the main chain by coupling only Fmoc-Lys(Mtt)-OH. The main chain is a chain in which all amide bonds contain an alpha amine. After the synthesis of the main chain containing three lysines and three glycines, Mtt is cleaved at 25 °C for 1 hour using a solution of 30% hexafluoroisopropanol HFIP in DCM. In the next step, Fmoc-Lys(Fmoc) is coupled to the epsilon amine of each lysine of the main chain. Then, two treatments with 25% (v / v) piperidine in DMF (5 minutes and 20 minutes each) are used to cleave all Fmoc groups. Finally, the linker is coupled to the lysine. The seven form seven secondary (distal) binding sites.

[0281] The side chain amine of each lysine of the main chain couples to only one lysine each, providing a construct having a configuration herein referred to as "linear".

[0282] The amide bond is formed using a three-fold excess over the theoretically free amino groups (calculated from the first AMS binding site) of each of equimolar ratios of Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Mtt)-OH and the linker, Oxyma pure (ethyl cyano(hydroxyimino)acetate: registered trademark of Novabiochem), and diisopropylcarbodiimide (DIC) in DMF. Where applicable, prior to each coupling step, two treatments with 25% (v / v) piperidine in DMF (5 minutes and 20 minutes each) are used to remove the Fmoc groups. The amide coupling procedure is allowed to proceed for 2 hours.

[0283] The polymer matrix used for modification is AMS0.6. Synthesis scheme of 7X linear:

Table 12-1

Table 12-2

[0284] This modified solid resin has a structure having a lysine bound to only one other lysine via a glycine spacer, where applicable. The presence of a lysine bound to only one other lysine (a lysine having a different identity) provides an odd number of secondary binding sites, here seven secondary binding sites.

[0285] (Fmoc-Rink)7-LysGly-linear-Gly-AMS insoluble support structural formula:

Chemical formula

[0286] (Fmoc-Ramage)7-LysGly-linear-Gly-AMS insoluble support structural formula:

Chemical formula

[0287] (TBDMS-HMPB)7-LysGly-linear-Gly-AMS insoluble support structural formula:

Chemical formula

[0288] (Fmoc-Rink) 16 -Lys(4)8-Gly8-lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS [16X(Gly)] Molecular weight (g / mol): 1112.49 The 16X(Gly) insoluble support contains constructs that provide a 16-fold increase in the primary binding sites of the base AMS resin. The constructs contain 15 lysines, 15 glycine spacers, and 16 Fmoc-Rink linkers. This construct is symmetric and contains four layers (generations) of lysines, namely 8 quaternary lysines Lys(4), 4 tertiary lysines Lys(3), 2 secondary lysines Lys(2), and 1 primary lysine Lys(1). Further, all lysines (secondary Lys(2) and tertiary Lys(3)) between the lysine bound to the primary binding site of the base resin and the quaternary lysine Ly(4) are bound to 3 different lysines. For example, each of the 2 secondary lysines Lys(2) is bound to the primary lysine Ly(1) and 2 tertiary lysines Lys(3). The construct is symmetric if all lysines except the distal lysine (in this case, the distal lysine is the quaternary lysine Lys(4)) and the primary lysine are bound to 3 lysines. Also, the number of secondary binding sites is a function of the following formula: Y = 2 n where n indicates the number of layers of lysines in the construct. Here, it has 4 layers and thus 16 secondary binding sites.

[0289] The polymer matrix used for modification is AMS0.6.

[0290] All lysines are exclusively Fmoc-Lys(Fmoc). Reactants: Fmoc-Lys(Fmoc), Fmoc-Gly, Fmoc-Rink

[0291] (Fmoc-Rink) 16 -Lys(4)8-Gly8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS insoluble support structural formula

Chemical Structure

[0292] (Fmoc-Rink) 16Synthesis of -Lys(4)8-Gly8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS The peptide construct is synthesized in a manual peptide synthesizer reactor.

[0293] Couple Fmoc-protected lysine to the amine of AMS. The amide bond is formed using a 3-fold excess of each of Fmoc-Lys(Fmoc)-OH, Fmoc-Gly-OH, Fmoc-Rink-OH in equimolar ratio, along with Oxyma pure (Ethyl cyano(hydroxyimino)acetate: Novabiochem registered trademark) and diisopropylcarbodiimide (DIC), relative to the theoretically free amino groups (calculated from the first AMS binding site) in DMF. Prior to each coupling step, the Fmoc group is removed using two treatments of 25% (v / v) piperidine in DMF (5 minutes and 20 minutes each). The amide coupling procedure is allowed to proceed for 2 hours.

[0294] Synthesis scheme of 16X(Gly)

Table 13-1

Table 13-2

[0295] Synthesis of polypeptides using the insoluble support of the present invention The template polypeptide sequences PP1 - PP6 were synthesized using the following insoluble supports (details presented above):

[0296] Support with Rink linker: ·1X: (Fmoc-Rink)-AMS0.6 (resin without construct) ·1X: (Fmoc-Rink)-AMS0.93 (resin without construct) ·1X: (Fmoc-Rink)-AMS1.95 (resin without construct) ·2X(Gly): (Fmoc-Rink)2-Lys(1)-gly-AMS0.6 ·2X(Gly):(Fmoc-Rink)2-Lys(1)-Gly-AMS0.93 ·2X(Gly):(Fmoc-Rink)2-Lys(1)-Gly-AMS1.95 ·4X(Gly):(Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS0.6 ·4X(Gly):(Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS0.93 ·4X(Gly):(Fmoc-Rink)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS1.95 ·8X(Gly):(Fmoc-Rink)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS0.6

[0297] Support with Ramage linker: ·1X:(Fmoc-Ramage)-AMS0.6 (resin without construct) ·1X:(Fmoc-Ramage)-AMS0.93 (resin without construct) ·1X:(Fmoc-Ramage)-AMS1.95 (resin without construct) ·2X(Gly):(Fmoc-Ramage)2-Lys(1)-Gly-AMS0.6 ·2X(Gly):(Fmoc-Ramage)2-Lys(1)-Gly-AMS0.93 ·2X(Gly):(Fmoc-Ramage)2-Lys(1)-Gly-AMS1.95 ·4X(Gly):(Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS0.6 ·4X(Gly):(Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS0.93 ·4X(Gly):(Fmoc-Ramage)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS1.95

[0298] Support with HMPB TBDMS linker: ·1X: HMPB TBDMS AMS0.6 (resin without structure) ·1X: HMPB TBDMS AMS0.93 (resin without structure) ·4X(Gly): (HMPB TBDMS)4-Lys(2)2-Gly2-Lys(1)-Gly-AMS0.6 ·8X(Gly): (HMPB TBDMS)8-Lys(3)4-Gly4-Lys(2)2-Gly2-Lys(1)-Gly-AMS0.6

[0299] The reference resins have substitution degrees of 0.6, 0.93, and 1.95 mmol / gram, respectively, and are aminomethyl (AM / AMS) polystyrene resins crosslinked with 1% divinylbenzene within the 100 - 200 mesh size range (75 - 150 μm).

[0300] The following polypeptides were synthesized: PP1: WLFAGGPSSGAPPPS (15-mer) PP2: YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS (39-mer) PP3: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (39-mer) PP4: fFPRPGGGGNGDFEEIPEEYL (20-mer) PP5: FVQYLIQG (8-mer) PP6: HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (31-mer)

[0301] Structures: 1X: Without structure, only unmodified resin with the linker shown below. 2X(Gly): -Gly-Lys(1)-linker2 4X(Gly): -Gly-Lys(1)-Gly2-Lys(2)2-linker4 8X(Gly): -Gly-Lys(1)-Gly2-Lys(2)2-Gly4-Lys(3)4-linker8

[0302] Tables 8 to 14 show various data related to polypeptide synthesis.

[0303] In some columns, the aggregate yield [%] and cleavage yield [%] are shown. These parameters are calculated as follows.

Equation

[0304] Synthesis protocol of PP1 using DMF as the solvent The starting resin (400 mg each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2 × 1 h) for initial swelling measurement before transferring to a Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). All amino acid couplings were performed using a 5-fold molar excess, equimolar ratio (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) compared to the synthesis scale. Coupling was carried out at room temperature for 1 hour 30 minutes without pre-activation.

[0305] After completion of the synthesis, the resin was transferred back to a 25 mL syringe to measure the final swelling, then washed with isopropanol (iPrOH, 3 times) and methyl tert-butyl ether (MTBE, 3 times), and dried under vacuum overnight.

[0306] The dried Fmoc-protected peptidyl resin was swollen again in DMF (6.5 mL / g, 2 × 1 h). The Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). Next, the resin was washed with DMF (7 times), iPrOH (3 times), and MTBE (3 times), and then re-dried under vacuum overnight.

[0307] The dried Fmoc-deprotected peptide was cleaved from the resin using a cleavage cocktail containing TFA (trifluoroacetic acid) / H2O / DTT (dithiothreitol) (13 mL / g of peptidyl resin, 85 / 5 / 5 v / v / w) at room temperature for 2 hours. Then, TIS (5% by volume) was added and stirring was continued for an additional 1 hour. The resin was filtered off and washed with TFA (3 mL / g of peptidyl resin). The peptide was precipitated at 0 °C using MTBE (10 times the volume of TFA). The resulting suspension was transferred to a 50 ml conical centrifuge tube and centrifuged at 2500 rcf for 10 minutes, after which the supernatant was decanted. The crude solid product was washed again with cold MTBE (5 times) and dried under vacuum overnight. Molecular weight: 1426.60 Calculated mass: +2 / 2 = 714.30 Observed mass (AMS0.93 2×(Gly)) +2 / 2 = 714.05 Observed mass (AMS1.95 2×(Gly)) +2 / 2 = 714.43 Observed mass (AMS1.95 4×(Gly)) +2 / 2 = 714.64

[0308] All 15 amino acids used were Fmoc-protected. Side chain protecting groups involved: tBu, Boc: S = Ser(tBu) and W = Trp(Boc) Molecular weight of protected PP1: 1917.28 g / mol Molecular weight of unprotected PP1: 1426.60 g / mol

[0309]

Table 14

Table 15

[0310] The implementation of the support of the present invention significantly increases throughput while essentially maintaining purity or even increasing purity.

[0311] Synthesis protocol of PP1 using DMSO / EtOAc (3:7 v / v) and DMF as solvents The starting resin (400 mg each) was introduced into a 25 mL syringe and swollen in DMF (6.5 mL / g, 2 × 1 h). Using an Activo-PLS 4 × 4 synthesizer purchased from Activotec, the synthesis was carried out manually by shaking the syringe in a horizontal position at 400 rpm. Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMSO / EtOAc (3:7, v / v) or DMF at room temperature (1 × 10 min + 1 × 20 min). After Fmoc deprotection, the resin was washed using DMSO / EtOAc (3:7, v / v) or DMF (7 × 6.5 mL / g starting resin). All amino acid couplings were carried out using a 2-fold molar excess, equimolar ratio (1:1:1), of Fmoc amino acids (0.30 M) in DMSO / EtOAc (3:7, v / v) or DMF, Oxyma, and N,N'-diisopropylcarbodiimide, relative to the synthesis scale. The Fmoc amino acids were dissolved in the corresponding solvent / solvent mixture (0.30 M), followed by the addition of Oxyma and N,N'-diisopropylcarbodiimide, and stirred at room temperature for 5 min before introducing into the resin-containing syringe. Coupling was carried out for 1 h 30 min at room temperature. After coupling, the resin was washed using DMSO / EtOAc (3:7, v / v) or DMF (2 × 6.5 mL / g starting resin).

[0312] After completion of the synthesis, the final resin swelling was measured, and then the resin was washed with (iPrOH, 3 times) and methyl tert-butyl ether (MTBE, 3 times) and then dried overnight under vacuum.

[0313] The dried Fmoc-protected peptidyl resin was swollen again in DMF (6.5 mL / g, 2 × 1 h). The Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). Next, the resin was washed with DMF (7 times), iPrOH (3 times), and MTBE (3 times) and then redried overnight under vacuum.

[0314] The dried Fmoc-deprotected peptide was cleaved from the resin using a cleavage cocktail containing TFA / H2O / DTT (13 mL / g of peptidyl resin, 85 / 5 / 5 v / v / w) at room temperature for 2 h. Then, TIS (5% by volume) was added and stirring was continued for an additional 1 h. The resin was filtered off and washed with TFA (3 mL / g of peptidyl resin). The peptide was precipitated at 0 °C using MTBE (10 times the volume of TFA). The resulting suspension was transferred to a 50 ml conical centrifuge tube and centrifuged at 2500 rcf for 10 min, after which the supernatant was decanted. The crude solid product was washed again with cold MTBE (5 times) and dried under vacuum overnight. Mass of resin at the start of synthesis after coupling of the Rink amide linker: 400 mg Side chain protecting groups involved: tBu, Boc: S = Ser(tBu) and W = Trp(Boc) Molecular weight of protected PP1: 1917.28 g / mol Molecular weight of unprotected PP1: 1426.60 g / mol

[0315]

Table 16

Table 17

[0316] Synthesis protocol of PP2 (YAEGTFTSDYSIALDKIAQKAFVQWLIAGGPSSGAPPPS) using DMF as solvent The starting resin (200 mg each) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2 × 1 h) for initial swelling measurement before transferring to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). All amino acid couplings were performed using a 5-fold molar excess, equimolar ratio (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) relative to the synthesis scale. Couplings were carried out at room temperature for 2 h without pre-activation, except for Thr 5 which was coupled for 8 h 12および17 and Ile which was coupled for 6 h

[0317] After completion of the synthesis, the resin was transferred back to a 12 mL syringe to measure the final swelling, then washed with (iPrOH, 3 times) and methyl tert-butyl ether (MTBE, 3 times), and dried under vacuum overnight.

[0318] The dried Boc-protected peptide was cleaved from the resin using a cleavage cocktail containing TFA / H2O / DTT / TIS (13 mL / g of peptidyl resin, 85 / 5 / 5 / 5 v / v / w / v) at room temperature for 3 h. The resin was filtered off and washed with TFA (3 mL / g of peptidyl resin). The peptide was precipitated at 0 °C using MTBE (10 times the volume of TFA). The resulting suspension was transferred to a 50 ml conical centrifuge tube, centrifuged at 2500 rcf for 10 min, and the supernatant was decanted. The crude solid product was washed again with cold MTBE (5 times) and dried under vacuum overnight. Molecular weight: 4041.54 Calculated mass: +3 / 3 = 1348.26; +4 / 4 = 1011.38 Observed mass (AMS0.6 2X(Gly)): +3 / 3 = 1347.98; +4 / 4 = 1011.70 Observed mass (AMS0.93 4X(Gly)): +3 / 3 = 1348.6; +4 / 4 = 1011.67

[0319]

Table 18

Table 19

Table 20

[0320] Synthesis protocol of PP3 (HGEGTFTSDLSKQMEEEAVRLFXEWLKNGGPSSGAPPPS) using DMF as a solvent The starting resin (200 mg each) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2 × 1 h) for initial swelling measurement before transferring to a Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). All amino acid couplings were performed using a 5-fold molar excess of Fmoc amino acids (0.30 M in DMF), Oxyma (0.90 M in DMF), and N’N’-diisopropylcarbodiimide (0.90 M in DMF) in an equimolar ratio (1:1:1) compared to the synthesis scale. Couplings were performed for 2 h at room temperature without pre-activation, except for Thr5 which was coupled for 8 h, and Ile12 and 17 which were coupled for 6 h.

[0321] After completion of the synthesis, the resin was transferred back to a 12 mL syringe to measure the final swelling, then washed with (iPrOH, 3 times) and methyl tert-butyl ether (MTBE, 3 times), and dried overnight under vacuum.

[0322] The dried Boc-protected peptide was cleaved from the resin using a cleavage cocktail containing TFA / H2O / DTT / TIS (13 mL / g of peptidyl resin, 85 / 5 / 5 / 5 v / v / w / v) at room temperature for 3 hours. The resin was filtered off and washed with TFA (3 mL / g of peptidyl resin). The peptide was precipitated at 0 °C using MTBE (10 times the volume of TFA). The resulting suspension was transferred to a 50 ml conical centrifuge tube and centrifuged at 2500 rcf for 10 minutes, after which the supernatant was decanted. The crude solid product was washed again with cold MTBE (5 times) and dried under vacuum overnight. Molecular weight: 4186.63 Calculated mass: +3 / 3 = 1396.54; +4 / 4 = 1047.65 Observed mass (AMS0.6 2X(Gly)): +3 / 3 = 1397.12; +4 / 4 = 1048.11 Observed mass (AMS0.6 4X(Gly)): +3 / 3 = 1396.89; +4 / 4 = 1047.65

[0323] All amino acids used were Fmoc-protected, except for the last amino acid (Tyr) which was Boc-protected. 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) Molecular weight of the protected 39-mer: 5596.22 g / mol Molecular weight of the unprotected 39-mer: 4041.54 g / mol

[0324]

Table 21

Table 22

Table 23

[0325] The implementation of the support of the present invention significantly increases throughput with good / commercially reasonable / useful purity.

[0326] Synthesis protocol of PP4 (fPRPGGGGNGDFEEIPEEYL) using DMF as solvent The starting resin (200 mg each) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2×1 h) for initial swelling measurement before transferring to a Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1×10 min + 1×20 min). All amino acid couplings were performed using a 5-fold molar excess, equimolar ratio (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) compared to the synthesis scale. Coupling was carried out for 1 hour 30 minutes at room temperature without pre-activation.

[0327] After completion of the synthesis, the resin was transferred back to a 12 mL syringe to measure the final swelling, then washed with (iPrOH, 3 times) and methyl tert-butyl ether (MTBE, 3 times), and dried overnight under vacuum.

[0328] The dried Boc-protected peptide was cleaved from the resin using a cleavage cocktail containing TFA / H2O / DTT / TIS (13 mL / g of peptidyl resin, 85 / 5 / 5 / 5 v / v / w / v) at room temperature for 3 hours. The resin was filtered off and washed with TFA (3 mL / g of peptidyl resin). The peptide was precipitated at 0 °C using MTBE (10 times the volume of TFA). The resulting suspension was transferred to a 50 ml conical centrifuge tube, centrifuged at 2500 rcf for 10 minutes, and the supernatant was decanted. The crude solid product was washed again with cold MTBE (5 times) and dried overnight under vacuum. Molecular weight: 2180.32 Calculated mass: +2 / 2 = 1091.16 Observed mass (AMS0.6 4X (Gly)): +2 / 2 = 1091.02 Observed mass (AMS0.6 8X(Gly)): +2 / 2 = 1091.06

[0329] All of the amino acids used were Fmoc-protected, except for the last amino acid (D-Phe) which was Boc-protected. Side-chain protecting groups involved: tBu, Trt, Pbf: Y = Tyr(tBu), E = Glu(OtBu), D = Asp(OtBu), N = Asn(Trt), Arg(Pbf) Molecular weight of the protected 20-mer: 3233.86 g / mol Molecular weight of the unprotected 20-mer: 2180.32 g / mol

[0330]

Table 24

Table 25

Table 26

[0331] The application of the support of the present invention significantly increases the throughput while essentially maintaining the purity.

[0332] Synthesis protocol of PP5 (FVQYLIQG) using DMF as a solvent The starting resin (0.60 of Fmoc-HMPB-Gly-AMS, 100 mg each) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2 × 1 h) for initial swelling measurement before transferring to the Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 min + 1 × 20 min). All amino acid couplings were performed using a 5-fold molar excess, equimolar ratio (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) compared to the synthesis scale. The coupling was carried out for 1 hour 30 minutes at room temperature without pre-activation.

[0333] After completion of the synthesis, the resin was transferred back to a 12 mL syringe to measure the final swelling, then washed with (iPrOH, 3 times) and methyl tert-butyl ether (MTBE, 3 times), and dried under vacuum overnight.

[0334] The dried Fmoc-protected peptide fragment was cleaved from the resin using a gentle cleavage cocktail containing 3% TFA in dichloromethane (DCM) for 15 minutes. This process was carried out 3 times, filtering the resin each time and collecting the resulting solution. The collected solutions were combined and evaporated under vacuum, and the resulting concentrated oil was precipitated in MTBE / heptane (1:1, v / v), then filtered and dried under vacuum overnight.

[0335] All amino acids used were Fmoc-protected. Side chain protecting groups involved: tBu, Trt: Q = Gln(Trt), Y = Tyr(tBu) Molecular weight of the protected octamer: 1730.1 g / mol

[0336]

Table 27

Table 28

Table 29

[0337] The application of the support of the present invention significantly increases the throughput while essentially maintaining the purity.

[0338] Synthesis protocol of PP6 (HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG) using DMF as a solvent The starting resin (200 mg each) was introduced into a 12 mL syringe and swollen in DMF (6.5 mL / g, 2 × 1 hour) for initial swelling measurement before transferring to a Symphony X reactor. Before each coupling step, the Fmoc group was deprotected using 20% piperidine in DMF at room temperature (1 × 10 minutes + 1 × 20 minutes). All amino acid couplings were performed using a 5-fold molar excess, equimolar ratio (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) compared to the synthesis scale. Coupling was carried out for 1 hour 30 minutes at room temperature without pre-activation.

[0339] After completion of the synthesis, the resin was transferred back to a 12 mL syringe to measure the final swelling, then washed with (iPrOH, 3 times) and methyl tert-butyl ether (MTBE, 3 times), and then dried under vacuum overnight.

[0340] The dried Boc-protected peptide was cleaved from the resin using a cleavage cocktail containing TFA / H2O / DTT / TIS (13 mL / g of peptidyl resin, 85 / 5 / 5 / 5 v / v / w / v) at room temperature for 3 hours. The resin was filtered off and washed with TFA (3 mL / g of peptidyl resin). The peptide was precipitated at 0 °C using MTBE (10 times the volume of TFA). The resulting suspension was transferred to a 50 ml conical centrifuge tube, centrifuged at 2500 rcf for 10 minutes, and then the supernatant was decanted. The crude solid product was washed again with cold MTBE (5 times) and dried under vacuum overnight. Molecular weight: 3383.73 Calculated mass: +3 / 3 = 1128.91; +4 / 4 = 846.93 Observed mass (AMS0.6 4X(Gly)): +3 / 3 = 1129.01; +4 / 4 = 847.06 Solvent used for the synthesis of PP6 in Symphony X: DMF

[0341] Mass of the resin at the start of synthesis after coupling of the HMPB - glycine linker: 200 mg

[0342] All amino acids used were Fmoc - protected, except for the last amino acid (His) which was Boc - protected. 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) Molecular weight of protected PP6: 5234.46 g / mol Molecular weight of unprotected PP6: 3383.73 g / mol

[0343]

Table 30

Table 31

Table 32

Claims

1. An insoluble support (resin) in particle form containing distal binding sites, comprising a homogeneous polymer matrix and a construct, wherein the construct is covalently bonded to the polymer matrix, and the construct comprises at least one branching agent selected from aminoalkanoic acids containing at least two amino groups and 3 to 10 carbon atoms, a cleavable linker, and at least one spacer coupled to the at least one branching agent via an amide bond, wherein the cleavable linker provides the distal binding sites.

2. The insoluble support according to claim 1, wherein the branching agent is selected from an aminoalkanoic acid containing at least two but three or fewer amino groups and three to ten carbon atoms.

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

4. The insoluble support according to claim 1, wherein the branching agent is selected from diaminoalkanoic acid containing 3 to 8, preferably 3 to 6, carbon atoms.

5. The insoluble support according to claim 1, wherein the branching agent is selected from 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid, and 2,5-diaminopentanoic acid (ornithine), and 2,6-diaminohexanoic acid, and preferably the branching agent is selected from 2,4-diaminobutyric acid, and 2,5-diaminopentanoic acid (ornithine), and 2,6-diaminohexanoic acid.

6. The insoluble support according to claim 1, wherein the branching agent is lysine.

7. The insoluble support film according to claim 1, wherein all branching agents are the same.

8. The number of branching agents in a structure δ is given by equation δ(n) = 2 ｎ The number of linkers λ that provide distal bonding sites, given by -1, is given by the equation λ(n) = 2 ｎ The insoluble support according to claim 3, given by the formula, where n represents the number of generations of the branching agent, and n is 1 to 10, preferably 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 positioned between all the branching agents.

11. The aforementioned structure, (A) [polymer matrix]-BA(1)-LK ２ , (B) [polymer matrix] - BA(1) - BA(2) ２ -LK ４ , (C) [polymer matrix] - BA(1) - BA(2) ２ -BA(3) ４ -LK ８ , (D) [Polymer Matrix] - BA(1) - BA(2) ２ - BA(3) ４ - BA(4) ８ - LK １６ , (E) [polymer matrix] - BA(1) - BA(2) ２ -BA(3) ４ -BA(4) ８ -BA(5) １６ -LK ３２ Selected from, The insoluble support according to claim 3, wherein BA represents a branching agent, the integer in parentheses indicates the generation of the branching agent, and LK represents a cleavable linker.

12. The insoluble support according to claim 1, wherein the spacer molecule is selected from an organic molecule having two binding sites.

13. The insoluble support according to claim 1, wherein the spacer molecule is selected from an organic molecule having two binding sites selected from one of carboxylic acid, amine, or hydroxyl.

14. The insoluble support according to claim 1, wherein the spacer molecule is selected from an organic molecule having two binding sites, selected from an amino acid and polyethylene glycol.

15. The insoluble support according to claim 1, wherein the spacer is an amino acid having two binding sites, appropriately selected from glycine and alanine, and preferably glycine.

16. The insoluble support according to claim 1, wherein the linker is selected from Rink amide, Wang, 2-chlorotrityl, PAM, PAL, HMPB, Sieber, and Ramage.

17. The insoluble support according to claim 1, wherein the polymer matrix is ​​selected from a homogeneous polymer matrix containing primary binding sites distributed throughout the polymer matrix.

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

19. The insoluble support according to claim 1, wherein the polymer matrix is ​​selected from a homogeneous polymer matrix formed from a polymerization composition comprising at least styrene and divinylbenzene (DVB), and DVB is present in an amount of less than 4.0% by weight, preferably less than 3.0% by weight.

20. An insoluble support according to claim 1, for use in solid-phase peptide synthesis, solid-phase morpholino oligomer synthesis, and solid-phase oligonucleotide synthesis.

21. An insoluble support according to claim 1, for use in solid-phase peptide synthesis.

22. A method for forming an insoluble support according to claim 1, comprising providing a polymer matrix containing primary binding sites, wherein the construct is formed by a divergent synthetic approach, a convergent synthetic approach, or a combination of a divergent synthetic approach and a convergent synthetic approach.

23. The invention includes providing a polymer matrix containing primary binding sites, wherein the construct is at least, a) Depending on the case, a step of coupling at least one spacer to the primary coupling portion, b) A coupling step of coupling a branching agent to the primary binding site of the base matrix, or optionally, coupling the branching agent to at least one spacer, wherein at least two amino groups are protected by protecting groups. c) A step of removing the protecting group, Here, steps a), b), and c) can be repeated. d) A step of coupling the linker to the binding site of the distal branching agent. A method for forming the insoluble support described in claim 1, comprising a method including the following:

24. A solid-phase peptide synthesis protocol for synthesizing polypeptides, morpholino oligomers, and oligonucleotides, wherein the protocol includes using an insoluble support as described in claim 1.

25. A solid-phase peptide synthesis protocol for synthesizing polypeptides, comprising using an insoluble support as described in claim 1.

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, and preferably at least 25 amino acids.

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