Solution phase peptide synthesis

Abstract

The invention relates to a process for the preparation of a peptide, comprising the steps of: providing a soluble synthesis support, the soluble synthesis support having attached thereto: a first peptide fragment having a first reactive group; and a second peptide fragment having a second reactive group; coupling the first reactive group of the first peptide fragment with the second reactive group of the second peptide fragment to form a bond.

Description

[0001] Solution Phase Peptide Synthesis

[0002] Introduction

[0003] The invention relates to methods for the synthesis of peptides and peptide-like compounds, and to novel compounds obtainable thereby.

[0004] Background to the Invention

[0005] Solid-phase peptide synthesis (SPPS) was introduced in 1963 by Merrifield [Reference 1]. In the standard SPPS methodology, a specific peptide is grown on solid support particles, with identical peptide chains being assembled on each bead of a polymeric solid support resin. The synthesis is a repetitive process that includes sequential deprotection, coupling and washing reaction steps to elongate these chains until the desired single peptide is assembled. The identical peptide chains are elongated simultaneously to deliver the same peptide in one synthetic sequence. At the end of the sequence, the peptide is chemically cleaved from the resin with the aid of appropriate reagents.

[0006] This protocol is straightforward, and is the conventional method used in SPPS process. Since its introduction, tremendous efforts have been invested to advance this technology, including a range of new solid supports [References 2-6], new coupling reagents [Reference 7], as well as greener technologies to meet environmental requirements.

[0007] Despite its success at both research and industrial scales, conventional SPPS often requires that some key reactions to be performed off resin. This is primarily due to undesired side reactions occurring, restricting current SPPS to the synthesis of peptides typically 15 to 20 amino acids in length; beyond this, purity and yield become problematic. Therefore, current commercial practice for manufacturing a peptide of say 45 amino acids, is to synthesise three 15 amino acid peptide fragments, and subsequently to combine these fragments in a solution condensation reaction. This approach requires detaching each 15 amino acid peptide precursor from the resin (once for each fragment, then purifying each as necessary) and then carrying out the subsequent solution assembly reaction. These additional steps impose a significant penalty on the yield and purity of the product, manufacturing time as well as the environmental footprint of the process.

[0008] SPPS suffers from a number of inherent disadvantages, the most prominent of which lies in the difficulty of scale-up: many polymer supports are expensive, and occupy the majority of the mass of the material to be worked with. Examples of economically feasible Fmoc protection schemes in solution are scarce, with few examples in the literature.

[0009] Liquid phase peptide synthesis (LPPS) is attractive for the preparation of peptides in large quantities; however, the key separation of intermediates from reaction byproducts is usually achieved by precipitation or extraction. These relatively time consuming operations must often be optimized from one cycle to the next, and from one target to another, as the physical properties of growing peptide intermediates vary unpredictably, making the overall process slow and tedious.

[0010] One approach to addressing the shortcomings of LPPS has been described by Livingstone et al. (Angew. Chem. Int. Ed. 2021 , 60, 7786-7795). This methodology involves the use of a multivalent “hub” bearing several “arms”. The arms incorporate solubilising groups (e.g. polyethylene glycol (PEG)) and are attached to the growing peptide chain. An advantage of the approach is that after each coupling step, the hub bearing the growing peptide chain can be separated from byproducts and excess reagents using diafiltration.

[0011] The present invention addresses shortcomings in the known approaches to solution phase peptide synthesis.

[0012] Prior art

[0013] US2019 / 0345636 presents methods for the generation of combinatorial libraries on supports (e.g. beads). The libraries comprise sets of molecules of different types (e.g. DNA, RNA, and peptides). WO2023 / 122698 discloses methods for the analysis of a plurality of different polypeptides in a sample.

[0014] WO2023 / 062389 relates to preparation of polymers having defined sequences, such as polynucleotides and polypeptides, in solution phase. Purification is achieved through means of membrane filtration (e.g. diafiltration).

[0015] WO2024 / 127026 relates to a membrane filtration-assisted process for the preparation of oligonucleotides by solution-phase synthesis. In one aspect, it relates to a solution-phase process for preparing oligonucleotides in which membrane filtration (e.g., diafiltration) is used to purify and / or isolate the oligonucleotide during its step-wise growth.

[0016] Summary of the Invention

[0017] According to a first aspect, the invention relates to a process for the preparation of a peptide, comprising the steps of: a) providing a soluble synthesis support, the soluble synthesis support having attached thereto: i. a first peptide fragment having a first reactive group; and ii. a second peptide fragment having a second reactive group; coupling the first reactive group of the first peptide fragment with the second reactive group of the second peptide fragment to form a bond.

[0018] Solution Phase Peptide Synthesis

[0019] Livingston et. al. (Angew. Chem. Int. Ed. 2021 , 60, 7786-7795, incorporated herein by reference) have described liquid phase peptide synthesis featuring iterative addition of amino acids to a soluble support, with organic solvent nanofiltration (OSN) for isolation of the growing peptide after each synthesis cycle.

[0020] According to the technology presented in Livingston et. al. a multivalent “hub” is provided, bearing several “arms”, each of which is covalently bound to a first amino acid AAi. Peptides are elaborated on the arms using conventional peptide synthesis methodology, to provide a soluble synthesis support having multiple copies of the same peptide.

[0021] According to the present invention, there is provided a method for the solution phase synthesis of a peptide comprising the steps of: a) providing a soluble synthesis support, the soluble synthesis support having attached thereto: i. a first peptide fragment having a first reactive group; and ii. a second peptide fragment having a second reactive group; b) coupling the first reactive group of the first peptide fragment with the second reactive group of the second peptide fragment to form a bond.

[0022] As used herein, the term “soluble synthesis support” refers to a support of general of general formula (1 ) wherein represents a central hub, Pol represents a solubilising polymer, and n is an integer from 2 to 8.

[0023] The central hub may be an atom (e.g. N or C) or an organic moiety (such as a benzene ring), onto which the one or more solubility enhancing polymer groups are attached, directly or indirectly. Suitably, the central hub of each soluble synthesis support has a molecular mass of <1500 Da. Most suitably, the central hub of each soluble synthesis support has a molecular mass of <300 Da (e.g. a carbon atom). In a particularly preferred embodiment, the central hub is a benzene ring.

[0024] Particularly suitably, the one or more solubility-enhancing polymers Pol comprise polyethylene glycol) (PEG). Polyethylene glycol) is highly soluble in acetonitrile, the solvent favoured by industry for coupling nucleotides to prepare oligonucleotides (e.g. phosphoramidite couplings to prepare oligonucleotides). In a particularly preferred embodiment, the groups Pol are selected from polyethylene glycol) oligomers having from 5 to 10 ethylene glycol repeat units. Most preferred are groups Pol consisting of 8 glycol repeat units, i.e. octaethylene glycol.

[0025] Suitable linkers L include Wang, HMP, HMPA, 2-chlorotrityl, Fmoc-PAL, Ramage amide, Rink amide, and Sieber amide, 4-sulfamylbutyryl, HMBA, Weinreb linker, and DHP. Very preferably, the linker is selected from the Wang or Rink linker.

[0026] Especially preferred soluble synthesis supports are those having the formula (2) below, wherein L and n are as defined above, and m is an integer of from 2 to 9, preferably 7.

[0027] Especially preferred soluble supports are (3) and (4) below

[0028] The synthesis of compounds (3) and (4) above is described in Angew. Chem. Int.

[0029] Ed. 2021 , 60, 7786-7795.

[0030] Suitable soluble synthesis supports can be prepared from commercially available benzene-1 ,2,4,5-tetrayltetramethanol.

[0031] In one embodiment, the soluble synthesis support is attached to two different peptide sidechains via linker L. One of the sidechains includes a free amino group, and the other a free carboxy group. Under suitable peptide coupling conditions, a new peptide bond is formed between the side chains.

[0032] As used herein, the term “first reactive group” and “second reactive group” mean that the first and second reactive groups are capable under suitable conditions of reacting to form a bond. Such a bond may be a direct chemical bond (e.g. a covalent bond), or may involve a moiety that is linked to the first and second reactive groups to form a linker. Non-limiting examples of such first and second reactive groups and the bonds formed are shown in Table 1 above.

[0033] In a preferred aspect, the first reactive group is an amine (primary or secondary), and the second reactive group is a carboxylic acid (or a reactive derivative thereof, such as an acid chloride or anhydride). In addition to formation of bonds directly between the first and second reactive groups, it is also contemplated that the first and second reactive groups may be bound via a linker. “Linker” as used herein refers to a chemical moiety capable of reacting with both the first reactive group and the second reactive group, and will usually be a bivalent entity. Suitable linkers include dicarboxylic acids (including oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid and suberic acid), diols, diamines, and amino acids.

[0034] In some embodiments, the first reactive group is an amine. Suitably, the amine group may be provided by the side chain of any amino acid present on the first peptide fragment. Lysine is a preferred example of a naturally-occurring amino acid with an amine side chain; however, it is contemplated that any non-naturally- occurring amino acid bearing an amine group on the side chain can fulfil this role.

[0035] In some embodiments, the second reactive group is a carboxylic acid. Suitably, the carboxylic acid group may be provided by the side chain of any amino acid present on the first peptide fragment. Aspartic acid and glutamic acid are preferred examples of naturally-occurring amino acids with carboxylic acid side-chains; however, it is contemplated that any non-naturally-occurring amino acid bearing a carboxylic acid on the side chain can fulfil this role.

[0036] Coupling of the first and second peptide fragments is effected whilst both fragments are bound to the soluble synthesis support. This confers advantages to the inventive methods in terms of regioselectivity and chemoselectivity; further, as both peptide fragments are bound to the same soluble synthesis support, the local relative concentration is high, and the coupling reaction generally proceeds quickly and in high yield.

[0037] The coupling reaction may be performed in a suitable organic solvent. Suitable solvents include tetrahydrofuran, N-methyl-2-pyrrolidone, and dimethyl sulfoxide. Mixtures of these solvents are preferred. In a particularly preferred embodiment, the solvent is a mixture of tetrahydrofuran and N-methyl-2-pyrrolidone preferably in the ratio of from 10:1 to 1 :10 v / v. THF-NMP 35:65 v / v is a particularly preferred solvent. This aspect of the invention also benefits from the fact that membrane filtration (e.g. diafiltration) may be performed to isolate the coupled compound obtained from step b). Membrane filtration may further be performed to separate the supported coupled compound from a reaction by-product formed as part of a coupling reaction (e.g. from an excess reagent used as part of a coupling reaction.

[0038] Membrane filtration may therefore be performed once or twice for a given coupling reaction. A first filtration may involve separating the supported coupled peptide from a reaction by-product formed as part of a coupling reaction. A second filtration may involve separating the supported coupled peptide from an excess reagent used as part of the coupling reaction (e.g. an excess of a monomeric, dimeric or oligomeric building block to be coupled). Suitably, membrane filtration is performed twice.

[0039] Membrane filtration is suitably membrane diafiltration. More suitably, membrane filtration is organic solvent nanofiltration (OSN). Three membranes are particularly suitable: a polyethyleneimine (PEI) asymmetric membrane, cross-linked with terephthalic chloride (TPC), and coated with Jeffamine® M-2005 (a polyether monoamine); and polybenzimidazole (PBI) asymmetric membrane, cross-linked with a,a’-p-dibromoxylene (DBX), and modified with a polymer brush Jeffamine U M- 2005.

[0040] Details of methods of preparation of these and other suitable membranes can be found in J. Membr. Sci. 2015, 493, 568 -579, which is incorporated by reference.

[0041] An exemplary solution-phase synthesis of a coupled peptide is illustrated in scheme (1 ) below.

[0042]

[0043] Scheme 1

[0044] Referring to Scheme 1 , “Xaa” represents an unspecified amino acid, n, m, p and q are natural numbers (including zero). Structure (5) shows soluble synthesis support bound to first (upper) and second (lower) peptide fragment. The first peptide fragment contains a lysine residue at a point along the amino acid sequence. The second peptide fragment contains an aspartic acid residue at a point along the amino acid sequence. Coupling of the lysine side chain amino group and the aspartic acid side chain carboxylic acid is achieved under amide bond forming conditions (suitably, A / ,A / '-diisopropylcarbodiimide with ethyl cyano(hydroxyimino)acetate (OxymaPure) or (1 -Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) with diisopropylethylamine)) to form (6). Compound (6) can be purified using organic solvent nanofiltration (OSN).

[0045] It will be understood that in one aspect of the invention, it is preferably that there are an even number of arms attached to the central hub, i.e. that n is an even integer. In one aspect, the invention relies on a coupling reaction between two peptide fragments both bound to a soluble synthesis support, the first peptide fragment having a free carboxy group; and the second peptide fragment having a free amino group. In contradistinction to known methodologies, both the first and second peptide fragments are chemically attached to the same soluble synthesis support.

[0046] In one embodiment, the first peptide fragment is synthesised in a conventional, stepwise fashion on the polymer support. The C-terminal amino acid in the form of an N-a-protected, if necessary side-chain protected reactive derivative, is covalently coupled soluble synthesis support. The N-a-protective group is removed, and the subsequent protected amino acids are added in a stepwise fashion.

[0047] When the desired peptide fragment chain length has been obtained, the side-chain protective groups are removed, and the peptide is cleaved from the soluble synthesis support, which might be done in separate steps or at the same time.

[0048] Any suitable peptide protection methodology may be employed. Two preferred coupling strategies are based on the use of different N-a-protective groups and matching side-chain protective groups, that using the p-Nitrobenzyloxycarbonyl (pNZ) as the N-a protective group, and that using the 9-fluorenylmethyloxycarbonyl (Fmoc) group.

[0049] The N-a-pNZ-protected peptide coupled to a soluble synthesis support is N-a- deprotected with SnCh and a catalytic amount of acid. The resulting amine salt is washed and neutralized with a base, e.g. a tertiary amine. The subsequent peptide bond is formed by reaction with an activated pNZ-amino acid, e.g. a symmetric anhydride or a hydroxybenzotriazole active ester. Deprotection is achieved with SnCh and a catalytic amount of acid. Suitably, the side-chain protection is N- Allyloxycarbonyl (Alloc) or benzyl, which are suitably deprotected with Pd(0) catalysed allyl transfer.

[0050] The N-a-Fmoc protected peptide coupled to a soluble synthesis support is N-a- deprotected by treatment with a secondary amine, preferably piperidine, in an organic solvent, e.g. N,N-dimethyl formamide (DMF) or dichloromethane (DCM). After washing, the neutral peptide soluble synthesis support is reacted with an activated Fmoc-amino acid, e.g. a hydroxybenzotriazole active ester.

[0051] The side-chain protection is preferably selected from tertbutyl, trityl and arylsulfonyl based, and for deprotection of the side chains, a strong acid such as TFA is used.

[0052] Other N-a protective groups have been proposed (Stewart, J.M. and Young, J.D., Solid phase peptide synthesis, Pierce Chemical Company (1984)) and are contemplated within the scope of the invention.

[0053] As explained above, the first peptide fragment is synthesised using a conventional solution phase sequence. Similarly, the second peptide fragment is also synthesized using a conventional solution phase sequence.

[0054] In one embodiment, the first and second peptide fragments are synthesized using the same protecting groups. In this embodiment, the first and second peptide fragments are differentiated during the synthetic sequence by the use at one or more coupling steps of a mixture of activated N-protected amino acids, such that a proportion of the growing first and second peptide fragments differ at one or more amino acid residues. Such an embodiment is illustrated in Scheme 1 below.

[0055] Coupling

[0056] F^N-AA.] ■AA-] -NH2+ FmocNH-AA2OH reageijt

[0057] (7) (8)

[0058] Piperidine

[0059] FmocNH- NHFmocDMF»- FmocNH-AA3OH Coupling reagent (11)

[0060] FmocNH-AA4OH

[0061] (10)

[0062] (12)

[0063] Piperidine DM cNH-AA3-AA2-AA1 AA1-AA2-AA4-NHFmoc -F

[0064] Fmo ►

[0065] (13)

[0066] First fragment (15) Second fragment

[0067] Scheme 1

[0068] Scheme 1 illustrates the principle of simultaneously elaborating two peptide fragments attached to the same soluble synthesis support (“hub”). Soluble synthesis support (black circle) is loaded with first amino acid residue (AA1 ) which is deprotected to reveal primary amine group ready for a subsequent coupling reaction (7). Amino acid (7) bound to the soluble synthesis support is coupled with Fmoc N-protected amino acid (8) under suitable coupling conditions; many appropriate conditions are known, and examples are given in Chem. Rev. 2011 , 1 1 1 , 1 1 , 6557-6602, which is incorporated herein by reference. N,N-diisopropylcarbodiimide (DIC) is generally suitable.

[0069] After this first coupling reaction, Fmoc protected dipeptide AA1AA2 is bound to soluble synthesis support (9), and is deprotected under usual conditions (piperidine in dimethylformamide (DMF)) to give dipeptide (10) bound to the soluble synthesis support having free amine groups. At this point, all peptides attached to the soluble synthesis support are identical.

[0070] A critical step occurs when dipeptide (10) bound to soluble synthesis support is further extended. Instead of being coupled with a single protected amino acid as usual, soluble synthesis support-bound dipeptide (10) is reacted with a mixture of two different Fmoc protected amino acids, (11 ) and (12). The exact ratio of the two Fmoc protected amino acids (1 1 ) and (12) will depend on the nature of the amino acid residues AA3 and AA4. At the completion of this step, the soluble synthesis support will now have bound two different tripeptide chains, AA1AA2AA3 and AA1AA2AA4, protected at the N terminus with Fmoc groups (13).

[0071] Although in Scheme 1 the coupling reaction of Fmoc protected amino acids (1 1 ) and

[0072] (12) occurs in a single step, it is also contemplated that these couplings could occur sequentially. For example, soluble synthesis support-bound dipeptide (10) in some embodiments is coupled with Fmoc protected amino acids (1 1 ) in a sub-stoichiometric amount, i.e. an amount insufficient to completely react with all free amine groups of soluble synthesis support-bound dipeptide (10), and then subsequently with Fmoc protected amino acid (12) to give (13).

[0073] (13) is treated under usual Fmoc deprotection conditions to give a soluble synthesis support bearing two different tripeptides (14). At this point, the soluble synthesis support is bound to two different tripeptides, namely the first and second peptide fragments. These may be elaborated further, i.e. coupled with one or more further amino acids, to provide soluble synthesis support bearing fully elaborated first peptide fragment and second peptide fragment (15).

[0074] Although in Scheme 1 above, the first peptide fragment and second peptide fragment differ at a specific amino acid residue, it will be apparent to the skilled person that the first peptide fragment and second peptide fragment could be differentiated at any point during peptide synthesis, i.e. differ at any residue in the amino acid sequence. Similarly, although in Scheme 1 above, although the first peptide fragment and second peptide fragment differ at a single amino acid residue, it will be understood that the first peptide fragment and second peptide fragment may differ at more than one amino acid residue, such as two, three, four or more residues.

[0075] Further, although Scheme 1 above shows two differentiated peptide fragments, it is within the scope of the invention that three or more peptide fragments are synthesized, attached to the same soluble synthesis support.

[0076] In an alternative embodiment, first and second peptide fragments may be synthesized sequentially using orthogonal protecting group sequence shown in Scheme 2 below.

[0077] Scheme 2

[0078] Referring to Scheme 2 above, soluble synthesis support (black circle) is initially loaded with a mixture of Fmoc protected and pNZ protected amino acids (16).

[0079] Treatment of (16) with SnCh selectively removes the pNZ group unmasks the free amine group of amino acid AA2 (17). This is coupled with pNZ protected amino acid AA3 (18) under suitable coupling conditions. At this stage, the soluble synthesis support is coupled to pNZ protected dipeptide AA2AA3 and Fmoc protected amino acid AA1 (19). Subsequent elaboration of the pNZ -protected peptide fragment via a conventional deprotection / coupling sequence gives intermediate (20). Treatment with piperidine removes the Fmoc protecting group from soluble synthesis supportbound amino acid AA1, which may be coupled in a conventional manner with Fmoc protected amino acid (22) to give (23). Subsequent elaboration of the Fmoc- protected peptide fragment via a conventional deprotection / coupling sequence gives intermediate soluble synthesis support coupled to elaborated fragments 1 and 2 (first and second peptide fragments) (24).

[0080] Coupling Reaction on Soluble Synthesis Support

[0081] A further aspect of the invention relates to a method of coupling at least two peptide fragments which are tethered to the same soluble synthesis support. Such peptide fragments may be (and in some embodiments are) prepared according to the methods hereinbefore described, they need not be, and the soluble synthesis support coupling aspect is a separate embodiment which finds utility independent of the methods used to attach such peptide fragments to the soluble synthesis support.

[0082] In this aspect, the invention relates to a process for the preparation of a peptide or peptide-like compound, comprising the steps of: a) providing a soluble synthesis support, the soluble synthesis support having attached thereto: i. a first peptide fragment having a first reactive group; and ii. a second peptide fragment having a second reactive group; b) coupling the first reactive group of the first peptide fragment with the second reactive group of the second peptide fragment to form a bond.

[0083] As used herein, the term “first reactive group” and “second reactive group” mean that the first and second reactive groups are capable under suitable conditions of reacting to form a bond. Such a bond may be a direct chemical bond (e.g. a covalent bond), or may involve a moiety that is linked to the first and second reactive groups to form a linker. Non-limiting examples of such first and second reactive groups and the bonds formed are shown in Table 1 below.

[0084] Table 1

[0085] In the examples provided in Table 1 , suitable reaction conditions will be apparent to the skilled person. For example, reaction of an amine with a carboxylic acid can be effected with any one of the many known coupling reagents, e.g. carbodiimide reagents to form an amide bond. Similarly, many appropriate conditions are known for formation of an ester bond from an alcohol and a carboxylic acid (or active derivative thereof). Coupling of thiol groups to form disulphide groups can be achieved under oxidizing conditions, and alcohols may be coupled under e.g. dehydrating conditions. A C-C bond can be formed using a metathesis crosslinking approach currently used in stapling strategy.

[0086] In addition to formation of bonds directly between the first and second reactive groups, it is also contemplated that the first and second reactive groups may be bound via a linker. “Linker” as used herein refers to a chemical moiety capable of reacting with both the first reactive group and the second reactive group, and will usually be a bivalent entity. Suitable linkers include dicarboxylic acids (including oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid and suberic acid), diols, diamines, and amino acids.

[0087] In some embodiments, the first reactive group is an amine. Suitably, the amine group may be provided by the side chain of any amino acid present on the first peptide fragment. Lysine is a preferred example of a naturally-occurring amino acid with an amine side chain; however, it is contemplated that any non-naturally- occurring amino acid bearing an amine group on the side chain can fulfil this role.

[0088] In some embodiments, the second reactive group is a carboxylic acid. Suitably, the carboxylic acid group may be provided by the side chain of any amino acid present on the first peptide fragment. Aspartic acid and glutamic acid are preferred examples of naturally-occurring amino acids with carboxylic acid side-chains; however, it is contemplated that any non-naturally-occurring amino acid bearing a carboxylic acid on the side chain can fulfil this role.

[0089] Coupling of the first and second peptide fragments is effected whilst both fragments are bound to the soluble synthesis support. This confers unique advantages to the inventive methods in terms of regioselectivity and chemoselectivity; further, as both peptide fragments are bound to the same soluble synthesis support, the local relative concentration is high due to it being an intramolecular reaction; the coupling reaction generally proceeds quickly and in high yield.

[0090] In some embodiments, the first reactive group is an amine which is the side chain amine of a lysine residue present in the first peptide fragment, and the second reactive group is a carboxylic acid group which is the side chain carboxylic acid group of an aspartic acid or glutamic residue (preferably glutamic acid) present in the second peptide fragment. It will be understood that the terms “first,” “second” etc serve merely to distinguish the peptide fragments from one another and have no further significance; e.g. the first peptide fragment could equally comprise a carboxylic acid group, and the second peptide fragment an amine group.

[0091] A generalised scheme illustrating the coupling of a lysine-containing first peptide fragment and an aspartic acid containing second peptide fragment is shown in Scheme 3.

[0092] Scheme 3

[0093] Referring to Scheme 3, “Xaa” represents an unspecified amino acid, n, m, p and q are natural numbers (including zero). Structure (25) shows soluble synthesis support (black circle) bound to first (upper) and second (lower) peptide fragment. The first peptide fragment contains a lysine residue at a point along the amino acid sequence. The second peptide fragment contains an aspartic acid residue at a point along the amino acid sequence. Coupling of the lysine side chain amino group and the aspartic acid side chain carboxylic acid is achieved under amide bond forming conditions (suitably, A / ,A / '-diisopropylcarbodiimide with ethyl cyano(hydroxyimino)acetate (OxymaPure) or (1 -Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) with diisopropylethylamine)) to form (26).

[0094] The coupled peptide may be cleaved from the soluble synthesis support under standard conditions (which will depend on the nature of the soluble synthesis support) to yield peptide

[0027] ,

[0095] During synthetic sequences according to this aspect of the invention, it may be necessary or convenient to protect amino acid termini or side chains not participating in the coupling reaction described above.

[0096] Although the structures made by means of the inventive methodology are referred to as “peptides,” they may designated “modified peptides” as they incorporate non a- amino acids in the amino acid backbone (in the case of compound (27) for example an s-amino acid link).

[0097] In a further aspect of this invention, the first and second peptide fragments having been coupled by formation of a bond between the first and second reactive groups, the first and second peptide fragments may be further extended by SPPS to include third and fourth reactive groups; these may be coupled as described above to yield soluble synthesis support-bound cyclic peptides. Such a sequence is shown in Scheme 4.

[0098]

[0099] Scheme 4

[0100] Referring to Scheme 4, soluble synthesis support-bound coupled peptide (26) is coupled at the N-terminus of the first peptide fragment (employing a suitable deprotection and coupling sequence) with an Fmoc-protected aspartic acid residue to give (28), and subsequently at the N-terminus of the second peptide with an Fmoc-protected lysine (again employing a suitable deprotection and coupling sequence) to give (29). The side chains of the introduced lysine and aspartic acid are coupled under amide bond forming conditions to form a further covalent link between first and second peptide fragments. Soluble synthesis support-bound peptide (30) is cleaved from the soluble synthesis support and the Fmoc protection removed under standard conditions to give cyclic peptide. In a further aspect, the inventive methods described herein may be used in the preparation of partial modified inverso and / or retro inverso peptides. A generalised sequence is shown in Scheme 5.

[0101] Scheme 5

[0102] Referring to Scheme 5, according to the methodology hereinbefore described, soluble synthesis support (black circle) coupled to N-terminal Fmoc protected first peptide fragment [Xaa]nand N-terminal Fmoc protected second peptide fragment [Xaa]m (31 ) is prepared. Fmoc protection is removed under suitable conditions to give (32), which is reacted with dicarboxylic acid (33) or an anhydride thereof (q representing any natural non-zero number) under amide-bond forming conditions. First and second peptide fragments are connected at the N-termini (34), which is cleaved from the soluble synthesis support to give partial modified inverso peptide (35). The sequence shown in Scheme 6 employs a dicarboxylic acid; however, any difunctional reagent could be used to form an alternative linker between the two peptide fragments, such as a dihalide etc.

[0103] This aspect of the invention (i.e. that illustrated in Scheme 5) in a preferred aspect may be used to prepare symmetrical partial modified inverso and / or retro inverso peptide in which the first and second peptide fragments are identical. However, the skilled person will appreciate that non-identical (different) peptide fragments may be linked using a similar methodology. The partial modified inverso and / or retro inverso peptides obtainable by the methodologies described form a further aspect of the invention. In some embodiments, one of the peptide fragments comprises or consists of predominantly or exclusively D-amino acids, whilst the other fragment consists of predominantly or exclusively L-amino acids.

[0104] The methodologies outlined above, i.e. side chain coupling as outlined in Scheme 3 and the N-terminal coupling as outlined in Scheme 5 may be combined. An exemplary scheme is shown below (Scheme 6).

[0105]

[0040]

[0106] Scheme 6 Referring to Scheme 7, “Xaa” represents an unspecified amino acid, n, m, p and q are natural numbers (including zero). Structure (36) shows soluble synthesis support (black circle) bound to first (upper) and second (lower) peptide fragment, both N-terminal Fmoc protected. First peptide fragment includes at least one lysine residue having unprotected amino sidechain. Second peptide fragment includes at least one aspartic acid residue having unprotected carboxylic acid sidechain. The aforementioned lysine and aspartic acid sidechains are coupled under amide bond formation conditions to give (37); the Fmoc protecting groups are removed to furnish (38).

[0107] Soluble synthesis support-bound compound (38) is reacted with divalent carboxylic acid (or an anhydride thereof such as succinic anhydride) (39) to give (40), having two links between the first and second peptide chains. Cleavage from the soluble synthesis support yields cyclic peptide (41 ).

[0108] In a further aspect of the invention, there is provided a method for the synthesis of peptides including a conventional a-amino acid backbone via the coupling of two soluble synthesis support-bound peptide fragments. This aspect is illustrated (without limitation) in Scheme 7.

[0109] Scheme 7

[0110] Referring to Scheme 7 above, the synthetic sequence commences with soluble synthesis support attached to first (lower) and second (upper) peptide fragments (42). First peptide fragment is attached to the soluble synthesis support at the C- terminus, and is protected at the N-terminus with a pNZ group. Meanwhile, second peptide fragment is attached to the soluble synthesis support via a side chain residue (for example, lysine or aspartic acid) leaving the C-terminus free for reaction. Second peptide fragment is Fmoc protected at the N-terminus.

[0111] The pNZ group is removed from the first peptide fragment with e.g. SnCl2 / H+, unmasking the terminal amino group (43). The N-terminus of first peptide fragment is coupled on the soluble synthesis support with the C-terminus of second peptide fragment to give (44), which after cleavage from the soluble synthesis support to give (45) and Fmoc group removal gives fully elaborated peptide (46).

[0112] Examples

[0113] Materials and Methods

[0114] Soluble synthesis support (47) was prepared from (1 ,3-phenylene)dimethanol using the method described in Angew. Chem. Int. Ed. 2021 , 60, 7786-7795.

[0115] Throughout the remainder of the Examples, compound () above is illustrated as Example 1 - Preparation of Liraglutide

[0116] Liraglutide is an analogue to human glucagon like-peptide (GLP-1 ) and acts as a GLP- 1 receptor agonist. Liraglutide has 31 amino acids (HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG). The peptide has been split into two fragments, where both fragments are assembled on one soluble synthesis support as pNZ and Fmoc-chains (Scheme 8) as described hereinafter. pNZ chain : GQAAKEFIAWLVRGRG

[0117] Fmoc chain: HAEGTFTSDVSSYLE

[0118] Scheme 8. Chemical structure of the designed liraglutide fragments.

[0119] The design of the second chain (Fmoc chain) fragments has considered having amino acid with active side chain (Glu). Thus, it can be anchored to the soluble synthesis support from its side chain and making the C-terminal readily available for linking it with the N-terminal of the pNZ-chain after the whole chain is constructed. This will render the common linear peptide.

[0120] Step 1. Introduction of First pNZ Residue

[0121] To a 0.1 M solution of (47) in THF-NMP 35:65 v / v mixture is added one equivalent of pNZ-Gly-OH with DIC and HOBt (1 .7 equiv).

[0122] The product (48) is purified by diafiltration using a polybenzimidazole

[0123] (PBI) asymmetric membrane, cross-linked with a,a’-p-dibromoxylene (DBX), and modified with a polymer brush JeffamineU M-2005. Step 2. Introduction of First FMOC Residue

[0124] (48) is reacted with an equimolar amount of Fmoc-Glu(OBn)-OH (49) in THF-NMP 35:65 v / v mixture with DIC and HOBt (1 .7 equiv) to attach Fmoc protected glutamic acid via the side chain.

[0125] Product (50) is purified by diafiltration as above. Step 3. Elaboration of the pNZ Chain

[0126] Residues of the pNZ chain above are elaborated by sequential pNZ deprotection (SnCl2 / H+) and coupling with pNZ amino acids (DIC and HOBt (1 .7 equiv)).

[0127] Purification between couplings is accomplished using diafiltration as above. Product (51 ) is obtained. pNZGIyArgGlyArgValLeuTrpAlallePheGluGluLysAlaAlaGInGly — NH

[0128] °\

[0129] - NH Step 4 - Elaboration of Fmoc Chain

[0130] Residues of the Fmoc chain of (51 ) above are elaborated by sequential Fmoc deprotection (HCI / ethyl acetate) and coupling with Fmoc amino acids. Purification between couplings is accomplished using diafiltration as above. Product (52) is obtained. pNZGIyArgGlyArgValLeuTrpAlallePheGluGluLysAlaAlaGInGly — NH

[0131] O

[0132] FmocHisAlaGluGlyThrPheThrSerAspValSerSerTyrLeuGlu —

[0133] OBn

[0134] (52)

[0135] Step 5 - Coupling of FMOC and pNZ Chains

[0136] The pNZ protecting group is removed from the glycine residue (HCI / ethyl acetate) of (52). The benzyl protecting group is removed from the a-carboxylic acid using catalytic hydrogenation. The fragments are coupled with DIC and HOBt (1 .7 equiv).

[0137] Product (53) is obtained.

[0138] pNZGIyArgGlyArgValLeuTrpAlallePheGluGluLysAlaAlaGInGly - NH i SnCI2 / H+ii HJPdriii DCI / HOBt

[0139] FmocHisAlaGluGlyThrPheThrSerAspValSerSerTyrLeuGlu

[0140] OBn

[0141] (52)

[0142] FmocHisAlaGluGlyThrPheThrSerAspValSerSerTyrLeuGlu

[0143] (53)

[0144] Step 6 - Release of Product The Fmoc group is removed with piperidine, and the product cleaved from the soluble synthesis support with trifluoroacetic acid to give liraglutide peptide.

[0145] References

[0146] 1 . Merrifield, R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide'. J. Am. Chem. Soc. 1963, 85: p. 2149-2154. 10.1021 / ja00897a025

[0147] 2. Zalipsky, S., J.L. Chang, F. Albericio, and G. Barany. Preparation and applications of polyethylene glycol-polystyrene graft resin supports for solid-phase peptide synthesis. React. Polym. 1994, 22: p. 243-258. 10.1016 / 0923- 1137(94)90122-8 3. Vaino, A.R. and K.D. Janda. Solid-Phase Organic Synthesis: A Critical

[0148] Understanding of the Resin. J. Comb. Chem. 2000, 2: p. 579-596. 10.1021 / cc000046o 4. Garcia-Ramos, Y., M. Paradis-Bas, J. Tulla-Puche, and F. Albericio. ChemMatrix((R)) for complex peptides and combinatorial chemistry. J. Pept. Sci.

[0149] 2010, 16: p. 675-8. 10.1002 / psc.1282

[0150] 5. Lee, T.-K., S.-J. Ryoo, and Y.-S. Lee. A new method for the preparation of 2- chlorotrityl resin and its application to solid-phase peptide synthesis. Tetrahedron Letters 2007, 48,(3): p. 389-391. 10.1016 / j.tetlet.2006.11 .071

[0151] 6. Jaradat, D.M.M. Thirteen decades of peptide synthesis: key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation. Amino Acids 2018, 50,(1 ): p. 39-68. 10.1007 / s00726-017-2516-0 7. El-Faham, A. and F. Albericio. Peptide coupling reagents, more than a letter soup. Chem. Rev. 2011 , 111 ,(11 ): p. 6557-602. 10.1021 / cr100048w

[0152] All citations are incorporated by reference.

Claims

CLAIMS1 . A process for the preparation of a peptide, comprising the steps of: a) providing a soluble synthesis support, the soluble synthesis support having attached thereto: iii. a first peptide fragment having a first reactive group; and iv. a second peptide fragment having a second reactive group; b) coupling the first reactive group of the first peptide fragment with the second reactive group of the second peptide fragment to form a bond.

2. A process according to claim 1 , wherein the soluble synthesis support comprises a plurality of solubilizing arms covalently bound at a first end to a hub, and at a second end to a peptide synthesis linker.

3. A process according to claim 1 or 2 wherein the hub is selected from a benzene ring and a carbon atom.

4. A process according to any one of claims 1 to 3 wherein the arms are selected from ethylene glycol oligomers having from 3 to 10 repeat units.

5. A process according to any one of claims 1 to 4 wherein the linkers are selected from a Rink linker and a Wang linker.

6. A process according to any one of claims 1 to 5 comprising a further step of purification by organic solvent nanofiltration.

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