Methods of chemical synthesis of peptides

Abstract

The present invention relates to methods for the synthesis of peptides and related compounds supported on a solid polymeric resin. The inventive method provides synthetic schemes that permit the coupling of two or more peptide fragments attached to the same polymeric support, in a fashion that allows access to previously unobtainable structures, and provides a more convenient and higher-yielding approach to known peptides, including those of commercial significance.

Description

[0001] Methods of Chemical Synthesis of Peptides

[0002] Summary

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

[0004] Background of 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 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, a common 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. In the present disclosure, the re-invention of the standard SPPS process is proposed in two ways.

[0008] The first method is derived from an approach currently used to synthesise a diverse range of different peptides with potential therapeutic applications. The one-bead, one-compound (OBOC) approach has been known since 1991 [Reference 10]. It is an efficient strategy that utilises the SPPS approach but prepares a huge library of peptides, thousands and even millions of different peptides. Afterwards, this peptide library is mixed with a target molecule, and the functional peptide can be identified accordingly based on which specific peptide binds to the target molecule. Here multiple different peptides are concurrently synthesised in the SPPS on a single column.

[0009] The second novel aspect of this work is a new interchain assembly reaction which allows us to synthesise a wide spectrum of valuable peptide therapeutics by chemically driving the two or three precursor peptide chains synthesised on the same beads to react with each other to form the final peptide product (Figure 1 ). The combination of these two innovations allows the creation new peptide families with potential therapeutic applications, in an easier, faster, and cheaper reaction process. For instance, in this new innovation, purification will be required once at the end of the synthesis. Therefore, in comparison to the use of multiple SPPS steps with a final solution phase coupling reaction, the present invention uses fewer steps and hence higher yields. Most importantly, in the new innovation the inventors have inhibited a serious oligomerisation reaction by having one of the termini anchored to the resin during the course of the reaction.

[0010] Prior art

[0011] 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).

[0012] WO2023 / 122698 discloses methods for the analysis of a plurality of different polypeptides in a sample. 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).

[0013] Summary of the Invention

[0014] According to a first aspect, the invention relates to a process for the preparation of a peptide, comprising the steps of: a) providing a resin, the resin having attached thereto: i. a first peptide fragment having a free carboxy group; and ii. a second peptide fragment having a free amino group; and b) coupling the free carboxy group of the first peptide fragment with the free amino group of the second peptide fragment to form an amide group connecting the first and second peptide fragments.

[0015] According to a second aspect, the invention relates to a process for the preparation of a partial modified inverso and / or retro inverso peptide, comprising the steps of: a) providing a resin, the resin having attached thereto: i. a first peptide fragment having a first free amino group; and ii. a second peptide fragment having a second free amino group; and b) reacting the resin of step a) with a divalent electrophile capable of reacting with two amino groups to form a link between the first and second peptide.

[0016] According to a third embodiment, the invention relates to a process for the solid phase synthesis of a peptide, comprising a step of coupling an amino acid bound to a resin support having a free N-terminal amino group with a mixture of at least two N- protected amino acids.

[0017] Brief Description of the Figures

[0018] Figure 1 is a schematic representation of a reaction sequence according to an embodiment of the invention.

[0019] Figures 2, 5, 6, 7, 9, 11 , 13, 16, 19, 21 , 24, 26, and 27 are chromatograms.

[0020] Figures 3, 4, 8, 10, 12, 14, 15, 17, 18, 20, 22, 23, 25 and 28 are mass spectra. Detailed description of the Invention

[0021] The inventors have improved the standard SPPS process in two ways. The first method is derived from an approach currently used to synthesise a random and diverse range of different peptides with potential therapeutic applications. The one- bead, one-compound (OBOC) approach has been known since 1991 [Reference 10]. It is an efficient strategy that utilises the SPPS approach but generates a large library of random peptides. Afterwards, this peptide library is mixed with a target molecule, and the functional peptide can be identified accordingly which binds to the target molecule. In the present invention, multiple different peptides are concurrently synthesised in the SPPS on an ensemble of single beads.

[0022] A second aspect of the invention is the provision of an interchain assembly reaction which allows the synthesis of a wide spectrum of valuable peptides by chemically driving two or more precursor peptide chains synthesised on the bead to react with each other to form the final peptide product (Figure 1).

[0023] The combination of these two aspects allows the generation new peptide families with potential therapeutic (and other) applications, in an easier, faster, and cheaper reaction scheme. For example, in the methods of this invention, only a single purification step will be required at the end of the synthesis. In comparison with solution phase coupling, fewer steps are contemplated and higher yields are achieved. Most importantly, the inventive process inhibits a serious oligomerisation reaction by having one of the termini anchored to the resin during the course of the reaction.

[0024] In the broadest aspect, the invention relies on a coupling reaction between two peptide fragments both bound to a resin, 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 polymer bead (or other particle).

[0025] Suitable polymers are known to those in the art. Crosslinked polystyrene (PS)-based resins are most commonly used for routine SPPS. Beads of 200- to 400-mesh size distribution (corresponding to a diameter of about 50 pm) and a loading of 0.5 to 0.8 mmol / g present good characteristics for polymer swelling in solvents such as DMF and DCM, diffusion of reactants into the polymer matrix, and accessibility of linker sites buried into the bead. For larger peptides (more than 25 amino acids) or more difficult sequences, a lower loading is required (0.1 -0.2 mmol / g). A non-limiting list of suitable solid supports that may be used in the preparation of peptide amides may include Merrifield polystyrene based resin, PAM resin, Wang resin, Rink amide resin, PAL resin, Siber amide resin, MBHA Resin, and trityl and 2-chlorotrityl resins. Base- labile resins may also be used, including oxime resin, HMBA resin, DHP resin, Weinreb aminomethyl resin, and polyethylene glycol-polystyrene grafted resins. Details of these resins may be found in Chemical Approaches to the Synthesis of Peptides and Proteins, Paul Lloyd-Williams, Fernando Albericio, Ernest Giralt ISBN 9780849391422.

[0026] 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 either directly or by means of a suitable linker to the support, which is swollen in an organic solvent. The N-a-protective group is removed, and the subsequent protected amino acids are added in a stepwise fashion.

[0027] When the desired peptide fragment chain length has been obtained, the side-chain protective groups are removed, and the peptide is cleaved from the resin, which might be done in separate steps or at the same time.

[0028] 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 tert butyloxycarbonyl (Boc) as the N-a protective group, and that using the 9-fluorenylmethyloxycarbonyl (Fmoc) group.

[0029] The N-a-Boc-protected peptide coupled to a resin is N-a-deprotected with a strong acid, preferably trifluoroacetic acid (TFA). 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 Boc-amino acid, e.g. a symmetric anhydride. Deprotection is achieved with HF or a sulphonic acid. Suitably, the side-chain protection is N-Allyloxycarbonyl (Alloc) or benzyl, which are suitably deprotected with Pd(0) catalyzed allyl transfer. In certain embodiments, a third protecting group may be used that is orthogonal to both Boc and Fmoc. Preferably, this is the p-Nitrobenzyloxycarbonyl (pNZ) group. The corresponding derivatives are readily synthesized solids that perform well on solid phase. The pNZ moiety is orthogonal with the most common protecting groups used in peptide chemistry, and is removed under neutral conditions in the presence of catalytic amounts of acid.

[0030] The N-a-Fmoc protected peptide coupled to a resin 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 resin is reacted with an activated Fmoc-amino acid, e.g. a hydroxybenzotriazole active ester.

[0031] 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.

[0032] 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.

[0033] As explained above, the first peptide fragment is synthesised using a conventional SPPS sequence. Similarly, the second peptide fragment is also synthesized using a conventional SPPS sequence.

[0034] In one embodiment, the first and second peptide fragments are synthesized using the same protection 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.

[0035] First fragment [9] Second fragment

[0036] Scheme 1

[0037] Scheme 1 illustrates the principle of simultaneously elaborating two peptide fragments attached to the same polymer (e.g. bead). Polymer support (black circle) is loaded with first amino acid residue (AAi) which is deprotected to reveal primary amine group ready for a subsequent coupling reaction [1]. Although two identical amino acids are shown bound to the resin, this is representative of the numerous such amino acids in fact present. Resin bound amino acid [1] is coupled with Fmoc N-protected amino acid [2] under suitable coupling conditions; many appropriate conditions are known, and examples are given in Chem. Rev. 2011 , 111 , 11 , 6557- 6602, which is incorporated herein by reference. N,N-diisopropylcarbodiimide (DIC) is generally suitable. After this first coupling reaction, Fmoc protected dipeptide AA1AA2 is bound to resin support [3], and is deprotected under usual conditions (piperidine in dimethylformamide (DMF)) to give resin-bound dipeptide [4] having free amine groups. At this point, all peptides attached to the resin are identical.

[0038] A critical step occurs when resin-bound dipeptide [4] is further extended. Instead of being coupled with a single protected amino acid as usual, resin-bound dipeptide [4] is reacted with a mixture of two different Fmoc protected amino acids, [5] and [6]. The exact ratio of the two Fmoc protected amino acids [5] and [6] will depend on the nature of the amino acid residues AA3 and AA4. At the completion of this step, the resin will now have bound two different tripeptide chains, AA1AA2AA3 and AA1AA2AA4, protected at the N terminus with Fmoc groups [7],

[0039] Although in Scheme 1 the coupling reaction of Fmoc protected amino acids [5] and [6] occurs in a single step, it is also contemplated that these couplings could occur sequentially. For example, resin-bound dipeptide [4] in some embodiments is coupled with Fmoc protected amino acids [5] in a sub-stoichiometric amount, i.e. an amount insufficient to completely react with all free amine groups of resin-bound dipeptide [4], and then subsequently with Fmoc protected amino acid [6] to give [7],

[0040] [8] is treated under usual Fmoc deprotection conditions to give a resin support bearing two different tripeptides [8]. At this point, the resin 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 resin support bearing fully elaborated first peptide fragment and second peptide fragment

[0041] [9].

[0042] 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.

[0043] 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 resin e.g. bead. In an alternative embodiment, first and second peptide fragments may be synthesized sequentially using an orthogonal protecting group sequence as shown in Scheme 2 below.

[0044] _ Trifluoroacetic Coupling

[0045] FmocNH-AA1-^-AA2-NHBocac'dFmocNH- AA1-^-AA2-NH2+ HNBoc-AA3OHreagen

[0046]

[0010]

[0011]

[0012]

[0047] FmocNH-AA1-^^-AA2-AA3-NHBoc » , FmocNH-AA1-^^-AA2-AA3-[...]-AAn-NHBoc Coupling

[0048] Scheme 2

[0049] Referring to Scheme 2 above, resin bead (black circle) is initially loaded with a mixture of Fmoc protected and Boc protected amino acids

[0010] . Again, it will be apparent to the skilled person that each bead is bound to a large number of such amino acids, rather than the two depicted.

[0050] Treatment of

[0010] with trifluoracetic acid selectively removes the Boc group unmasks the free amine group of amino acid AA2

[0011] , This is coupled with Boc protected amino acid AA3

[0012] under suitable coupling conditions. At this stage, the resin is coupled to Boc protected dipeptide AA2AA3 and Fmoc protected amino acid AA1

[0013] . Subsequent elaboration of the Boc-protected peptide fragment via a conventional deprotection / coupling sequence gives intermediate

[0014] , Treatment with piperidine removes the Fmoc protecting group from resin-bound amino acid AA1, which may be coupled in a conventional manner with Fmoc protected amino acid

[0016] to give

[0017] , Subsequent elaboration of the Fmoc-protected peptide fragment via a conventional deprotection / coupling sequence gives intermediate resin coupled to elaborated fragments 1 and 2 (first and second peptide fragments)

[0018] . On-bead Coupling Reaction

[0051] A further aspect of the invention relates to a method of coupling at least two peptide fragments which are tethered to the same resin support. Such peptide fragments may be (and in some embodiments are) prepared according to the methods hereinbefore described, they need not be, and the on-resin coupling aspect is a separate embodiment which finds utility independent of the methods used to attach such peptide fragments to the resin.

[0052] 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 resin, the resin 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.

[0053] 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.

[0054] Table 1 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Coupling of the first and second peptide fragments is effected whilst both fragments are bound to the resin. This confers unique advantages to the inventive methods in terms of regioselectivity and chemoselectivity; further, as both peptide fragments are bound to the same polymeric support, the local relative concentration is high, and the coupling reaction generally proceeds quickly and in high yield. 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.

[0059] 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.

[0060] Scheme 3

[0061] Referring to Scheme 3, “Xaa” represents an unspecified amino acid, n, m, p and q are natural numbers (including zero). Structure

[0019] shows resin (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

[0020] . The coupled peptide may be cleaved from the resin under standard conditions (which will depend on the nature of the resin) to yield peptide

[0021] . [Xaajm

[0062] 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.

[0063] Although the structures made available for the first time 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

[0021] for example an c-amino acid link.

[0064] 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 resin-bound cyclic peptides. Such a sequence is shown in Scheme 4.

[0065]

[0066]

[0024]

[0025]

[0067] Scheme 4

[0068] Referring to Scheme 4, resin-bound coupled peptide

[0022] 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

[0023] , 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

[0024] , 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. Resin-bound peptide

[0025] is cleaved from the resin and the Fmoc protection removed under standard conditions to give cyclic peptide

[0026] .

[0069]

[0070] Following this methodology, further links between the first and second peptide fragments can be incorporated into the product to give bicyclic, tricyclic and higher- order cyclic peptides.

[0071] 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.

[0072] Scheme 5 Referring to Scheme 5, according to the methodology hereinbefore described, resin support (black circle) coupled to N-terminal Fmoc protected first peptide fragment [Xaa]n and N-terminal Fmoc protected second peptide fragment [Xaa]m

[0027] is prepared. Fmoc protection is removed under suitable conditions to give

[0028] , which is reacted with dicarboxylic acid

[0029] 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

[0030] , which is cleaved from the resin support to give partial modified inverse peptide

[0031] . The sequence shown in Scheme 5 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.

[0073] This aspect of the invention (i.e. that illustrated in Scheme 5) in a preferred aspect may be used to prepare symmetrical partial modified inverse 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.

[0074] 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.

[0075] 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).

[0076]

[0077] Scheme 6

[0078] Referring to Scheme 6, “Xaa” represents an unspecified amino acid, n, m, p and q are natural numbers (including zero). Structure

[0032] shows resin (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

[0033] ; the Fmoc protecting groups are removed to furnish

[0034] , Resin-bound compound

[0034] is reacted with divalent carboxylic acid (or an anhydride thereof such as succinic anhydride)

[0035] to give

[0036] , having two links between the first and second peptide chains. Cleavage from the resin yields cyclic peptide

[0037] ,

[0079] 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 resin-bound peptide fragments. This aspect overcomes the deficiencies known with SPPS methods when applied to the synthesis of long peptide chains. This aspect is illustrated (without limitation) in Scheme 7.

[0080] Resin Piperidine

[0081] Cleavage FmOcNH— [Xaa]m-[Xaa]n— OH - ► H„N-[Xaa]n— [Xaa]rrrOH

[0082] DMF

[0083]

[0041]

[0042]

[0084] Scheme 7

[0085] Referring to Scheme 7 above, the synthetic sequence commences with resin support attached to first (lower) and second (upper) peptide fragments

[0038] . First peptide fragment is attached to the resin support at the C-terminus, and is protected at the N- terminus with a Boc group. Meanwhile, second peptide fragment is attached to the resin 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.

[0086] The Boc group is removed from the first peptide fragment with e.g. trifluoroacetic acid, unmasking the terminal amino group

[0039] , The N-terminus of first peptide fragment is coupled on the resin with the C-terminus of second peptide fragment to give

[0040] , which after cleavage from the resin to give

[0041] and Fmoc group removal gives fully elaborated peptide

[0042] , Examples

[0087] 1. Results and discussion

[0088] Using different orthogonal protecting strategies, linkers, and chemicals has allowed the concurrent synthesis of two or three of different peptide chains on one bead, rather than just one chain of a single peptide.

[0089] The study started by elongating a peptide chain to the desired length which is anchored to the bead, then using a mixture of two different amino acids, namely Fmoc-Asp- (OAII)-OH and Fmoc-Lys(Alloc)-OH, which couple to the anchor peptide, resulting in two chains that differ in their first amino acid; Asp-peptide and Lys-peptide. The allyl group was chosen as it can be independently cleaved from the side chain of both amino acids whilst the peptide is still anchored to the resin. Hence, subsequent interchain assembly reaction can be carried out between the carboxylic acid and amine groups of the Asp and Lys, respectively. 1.1 Linear peptide synthesis

[0090] An octapeptide was successfully prepared by elongating two chains comprising the same amino acid constituents except one, where one chain has Asp and the other Lys (Scheme 8).

[0091]

[0092] Scheme 8. Schematic diagram of linear peptide synthesis following interchain assembly reaction Fmoc-GDGL-OH and Fmoc-GKGL-OH. represents polymer support. 2-chlorotrityl chloride (CTC) was used in this synthesis. The mass of the peptide was confirmed by MS.

[0093] Figure 2: Chromatograms of linear octapeptide formed from Fmoc-GDGL-OH and Fmoc-GKGL-OH (tR= 22,9 min); unreacted Fmoc-GDGL-OH (tR= 17,5 min). 15-70% in 30 min gradient elution. Mobile phase A: 0.1% TFA in H2O; mobile phase B: 0.1% TFA in CH3CN; Symmetry Luna C (3.6 pm, 4.6 x 150 mm) column.

[0094] Figure 3: Mass spectrum of linear octapeptide formed from Fmoc-GDGL-OH and Fmoc-GKGL-OH. Calculated: 1159.52; found: 1160.91 [M+1]+

[0095] This work has demonstrated the key success of this new concept. Hence, the resin was loaded with equimolar amounts of each reacting species, i.e. Lys and Asp. An excess of unreacted Asp containing precursor (Fmoc-GDGL-OH) was observed and confirmed by mass spectrometry (Figure 4 - Mass spectrum of unreacted Fmoc- GDGL-OH tetrapeptide. Calculated: 582.61 ; found: 565.48 [M-18]+). Thus, a decision was made to optimise the procedure and define the optimum loading percentages of each reacting species.

[0096] 1.2 Procedure optimisation

[0097] The reaction stoichiometry is significant, and it is very important for the reacting species to be completely consumed. Otherwise, if the reacting amino acid concentration is too low, it will terminate the main synthetic route. On the other hand, excess amino acid will be involved in undesired reaction pathways, which will impact the overall purity and yield of the main product, and eventually, the whole process could fail.

[0098] Starting from the findings of linear peptide synthesis in the previous section, a preloaded Gly-CTC resins was loaded with equimolar amounts Asp and Lys. The interchain reaction was carried out and an excess of Asp was observed, which confirms the previous findings in the previous section. The amount of the final peptide product with respect to the remaining Asp was about 85%.

[0099] Figure 5. Chromatograms of: black: linear Fmoc-Lys-Gly-OH + Fmoc-Asp-Gly-OH incorporated on CTC resin following 50% loading of Asp and 50% of Lys; pink: the product after the reaction between the Asp and Lys for 22h at RT (an excess of the 2nd peak which is Asp is observed). 5-95% in 15 min gradient elution. Chromatographic conditions as for Figure 2.

[0100] Therefore, the equimolar loading is not an optimum scenario, and the amount of Asp with respect to Lys has to be reduced. Hence, this change will ensure consuming the Asp and increasing the conversion yield and enhancing the purity as a result of reducing side reactions.

[0101] Then, the resin was loaded with reduced amounts of Asp as follows: 33% Asp and 67% Lys. This seems to be an optimum amount of the reacting species. Provided that, the amount of the final peptide product with respect to the remaining Asp was about >95%

[0102] Figure 6. Chromatograms of: black: linear Fmoc-Lys-Gly-OH + Fmoc-Asp-Gly-OH incorporated on CTC resin following 34% loading of Asp and 67% of Lys; pink: the product after the reaction between the Asp and Lys for 22h at RT (only 5% of the 2ndpeak which is Asp has remained). 5-95% in 15 min gradient elution. Refer to Figure 2 for the chromatographic conditions.

[0103] According to this preliminary optimisation procedure, loading the resin with 33% Asp and 67% Lys was used in the following reported study. 1.3 Cyclic peptide analogues

[0104] Due to their conformational rigidity, cyclic peptides have been shown to infer enhanced stability against enzymatic degradation. Peptide cyclization is also commonly applied to stabilize other secondary structures, such as a-helixes and p-sheets. Furthermore, they are powerful molecules in terms of internalisation and cell penetration efficiency. Given the protein-protein interactions are increasingly considered as therapeutic targets, cyclic peptides also offer an ideal opportunity to interrogate complex structures that are needed to engage with such type of extended interactions. After the successful synthesis of the linear analogue and optimizing the procedure, a decision was made to elongate the same two chains and include another junction position. Thus, the two chains were elongated using Gly for both chains, then Asp(OAII) and Lys(Alloc) for each one of them. The same steps as explained for the linear analogue were followed which resulted in a cyclic peptide (Scheme 9) and the mass was confirmed.

[0105] Figure 7. Chromatograms of cyclic Fmoc-KD-G-KD-GL-OH peptide (tR= 18,2 min).

[0106] Refer to Figure 2 for the chromatographic conditions.

[0107] Figure 8. Mass of cyclic peptide. Calculated: 940.50; found: 471 .58 [M+2]2+ Scheme 9. Schematic representation of cyclic peptide synthesis following interchain assembly reaction. Linking from two joints.

[0108] To further examine the robustness of this approach, different solid supports were used to prepare another cyclic peptide analogue. While in the previous examples CTC resin was used, in the new synthesis, a rink amide resin was considered. The cyclic peptide shown in Scheme 10 was prepared and its mass was confirmed by MS

[0109] Figure 9. Chromatograms of cyclic Fmoc-DK-G-G-DK-NHg peptide (tp= 10,2 min). 5- 95% in 15 min gradient elution. Refer to Figure 2 for the chromatographic conditions.

[0110] Figure 10: Mass spectrum of cyclic peptide. Calculated: 1156.50; found: 1157.95 [M+1]+, 579.81 [M+2]2+

[0111] Scheme 10. Chemical structure of a cyclic peptide on rink amide resin.

[0112] 1.4 Bicyclic peptide

[0113] Diverse molecular structures can be synthesized by linking two peptide chains using three or more different linking sites. The new synthesis protocol was investigated to prepare bicyclic peptides which are difficult to prepare, and requires a solution-based reaction step. The inventors were able to prepare a bicyclic peptide by simply linking three sites between the two peptide chains (Scheme 11 ). The mass of the peptide was also confirmed by MS.

[0114] Figure 11. Chromatograms of bicyclic H-DK-L-A-F-DK-G-G-DK-NH2peptide (tR= 10,6 min). 5-95% in 15 min gradient elution. Refer to Figure 2 for the chromatographic conditions. Figure 12. Mass of bicyclic peptide. Calculated: 2044.97; found: 1021.40 [M+2]2+ Scheme 11 . Chemical structure of a bicyclic peptide on rink amide resin.

[0115] 1.5 Highly constrained analogues

[0116] Peptides of less than 7 amino acids are very difficult to cyclize, especially the head-to- tail strategy in solution or even the intrachain interaction on resin such as the head to sidechain one [Reference 11], This challenge is ascribed to the high steric hindrance of such structure and inability to bend and form the desired cyclic confirmation.

[0117] Attempts at the cyclisation of short peptides often render dimerization, trimerization and C-terminal epimerization based products.

[0118] This new strategy proved its ability to produce a very challenging cyclic peptide (Scheme 12).

[0119]

[0120] Chemical Formula: C5oH54N6Oi2 Molecular Weight: 931.01

[0121] Scheme 12. Chemical structure of a cyclic tetrapeptide on rink amide resin.

[0122] This new cyclic peptide was successfully synthesised, and its mass was confirmed by mass spectrometry.

[0123] Figure 13. Chromatograms of cyclic Fmoc-DK-DK-NH2 peptide (tp= 7,1 min). 5-95% in 15 min gradient elution.

[0124] Figure 14. Mass spectrum of cyclic tetrapeptide. Calculated: 931.01 ; found: 932.75 [M+1]+

[0125] It is noted that in such highly constrained structures, an extended reaction time was needed to achieve the complete conversion; 48 h instead of 24 h.

[0126] The inventors highlight that the new approach is also applicable to unnatural amino acids with shorter sidechains such as Orn amino acid. Orn bears one less CH2group within its structure with respect to Lys. Nevertheless, the inventors have reacted Orn- containing peptide with Asp-containing amino acid and the final peptide was successfully synthesised (data not shown).

[0127] 1.6 Partial modified inverso and / or retro inverso peptide (PMI / PMRI)

[0128] The retro-inverso (Rl) concept was initially introduced in 1970 by Professor Goodman. Various fields have embraced this approach such as immunology, diagnosis, and drug discovery [Referenced], Partial modified inverso and / or retro inverso peptide (PMI / PMRI) holds a great promise to maximise the lifetime of biologically active peptides, which is one of the serious existing drawbacks in the peptide delivery process [Reference 13]. The current method for synthesising PMI peptides requires additional reaction steps that influence the final product purity. Furthermore, some amino acids showed instability towards the chemicals used in the current synthetic process. In this new approach, no additional reagents are required other than those already considered in conventional SPPS methodology. As a proof of concept experiment, a peptide with reversed amide bond direction was synthesised. Such new peptides will be called inverso peptides. However, incorporating amino acids with other stereo- chemistries using this approach will easily render the target PMRI peptides if required.

[0129] PMI synthesis started with a short pentapeptide (H-YGFGL-NH2) immobilised on a Siber-amide resin. Figure 15 shows the mass spectrum of YGFGL-NH2pentapeptide. Calculated: 554.29; found: 555.77 [M+1]+. Succinic anhydride was used to modify about 50% of the / V-terminal using half equivalent of the succinic anhydride with respect to the scale of synthesis. The modified chains will contain carboxylic acid at their / V-terminals which will allow reaction chemistries to take place with the unmodified / V-terminals that carry amino functionality (Scheme 13).

[0130] Scheme 13. Schematic representation of PMI peptide synthesis following interchain assembly reaction.

[0131] The reaction products were analysed with the HPLC (Figure 16) and the mass of the intermediate (4-oxobutanoic acid modified peptide) and PMI has been confirmed (Fig. S15 and S16). Figure 16. HPLC Chromatograms: black: H-YGFGL-NH2(tR= 6,4 min); pink: Succinc- YGFGL-NH2(tR= 7,1 min); green: H-YGFGL-Succ-YGFGL-NH2product (tR= 8,4 min). 5-95% in 15 min gradient elution. Mobile phase A: 0.1% TFA in H2O; mobile phase B: 0.1 % TFA in CH3CN; Symmetry Luna C18 (3.6 pm, 4.6 x 150 mm) column.

[0132] Figure 17. Mass spectrum of 4-oxobutanoic-YGFGL-NH2modified pentapeptide. Calculated: 654.30; found: 655.66 [M+1]+, 1310.09 [M+2]2+

[0133] Figure 18. Mass spectrum of H-YGFGL-NH-Succinic-YGFGL-NH2decapeptide. Calculated: 1190.58; found: 1192.00 [M+1]+, 596.79 [M+2]2

[0134] The 1 :2 stoichiometry for succinic anhydride to peptide proved to be an optimum choice. Provided that an equimolar of the peptide precursors will be generated, then these will be completely consumed during the subsequent interchain assembly reaction to render the final peptide product. Possibly due to an error in the amino acids weighing, the reaction stoichiometry was confirmed by HPLC as H-YGFGL-NH2- Succinic-YGFGL-NH2(0.9:1.1 ). This represents 9% excess of the Succinic-YGFGL- NH2. Hence, this was observed in the final product mixture as unreacted species. The decapeptide was synthesised in only two reaction steps, with the first one is the modification of the AZ-terminal and the second step is the interchain assembly reaction.

[0135] Using a 1 :1 stoichiometry, a fragment (27-36) of Enfuvirtide (T-20 or Fuzeon) was also chosen to form the basis of a PMI analogue (Scheme 14). Enfuvirtide is a 36-mer membrane fusion inhibitor for the treatment of HIV, which was approved by the United States Food and Drug Administration (US-FDA) in 2003 [Reference 14].

[0136]

[0137] Scheme 14. Chemical structure of two-fragment (27-36) of Enfuvirtide (T-20 or

[0138] Fuzeon) linked via succinamide, on Sieber amide resin.

[0139] Preparing a 20 amino acid peptide using the conventional ways can be challenging in terms of time, purity and yield. On the other hand, the inventive methodology, the 20 amino acid peptide was successfully synthesised in only two steps and with a high purity; as good as the starting peptide chain precursors (Figure 19). The mass of the synthesised peptide was confirmed as well:

[0140] Figure 19. Chromatograms of: black: one-fragment (27-36) of Enfuvirtide (T-20 or Fuzeon) (T-20 or Fuzeon) (tn= 13,5 min); pink: Succinic-T-20 (tp= 15,1 min); green: two-fragment (27-36) of the same peptide (tp= 18,4 min). 15-70% in 15 min gradient elution. Refer to Figure 2 for chromatographic conditions.

[0141] Figure 20 - Mass spectrum of PMI of T-20 fragment. Calculated: 2785.28; found: 1393.42 [M+2]2+, 929.33 [M+3]3+To demonstrate the efficiency of this invention, a 70 amino acid peptide, based on a 30-mer peptide called TD2.2 was prepared using the automatic synthesiser (Scheme 15). TD2.2 has been reported to be a specific blood-brain barrier shuttle peptide, in which it targets oligodendrocytes and has been shown to not target non-glial cells, such as human neural cells and human dermal fibroblasts [Reference 15, 16]. The N- terminal was modified following the same procedure of T-20 peptide using succinic anhydride.

[0142] Scheme 15. Chemical structure of Aib modified TD2.2 peptide, two-fragment linked via succinamide, on protide resin. An alternative representation of the modified peptide is shown below. 0

[0143] SYWYRIVI SR I GRNGRI RVGRI RPVI Gl SPNH2

[0144] (H2

[0145] ' SYWYRIVI SR I GRNGRI RVGRI RPVI Gi SPNi 12

[0146] O

[0147] The peptide has been successfully synthesised with a purity that matches the purity of the starting precursor (Figure 21 ) and its mass was confirmed.

[0148] Figure 21 . Chromatograms of two-fragment of TD2.2-succinicamide-TD2.2 (tR= 8.3 min). Refer for the legend of Figure 2 for the chromatographic conditions.

[0149] Figure 22. Mass spectrum of PMI of modified TD2.2 fragment. Calculated: 7366.64; found: 1047.83 [M+7]7+", 524.54 [M+14]14+

[0150] Coupling of a-aminoisobutyric acid (Aib) amino acid has been always a challenging reaction [Reference 17], and the automatic synthesiser is considered a good alternative when the peptide contains several Aib residues [Reference 18]. Hence, it is often used to demonstrate the quality of a new coupling methodology, for example when the main procedure is modified by introducing new solvent, coupling reagent, etc. This fact is also depicted in Scheme 15, where the difficulty is clear from the purity of the Aib modified peptide, which contains 4 Aib residues within its sequence and it has been synthesised with a microwave-assisted automatic synthesiser.

[0151] However, in the inventive approach, the inventors were able to prepare a PMI 60-mer peptide that comprises 8 Aib residues in only 2 reaction steps. This is considered as a significant advancement in the field of SPPS for two reasons. Firstly, the common manual SPPS is unable to deliver a 60-mer peptide using the conventional methodology. Thus, the convergent approach is considered rather than the stepwise approach [Reference 16]. However, this means that additional steps are needed, and sometimes the hybrid approach (SPPS and LPPS) should be considered, which will impose additional cost and will influence the final yield. Second, as explained above, coupling an Aib amino acid is quite challenging.

[0152] With this advance, it is now possible to synthesise a mini protein easily. This peptide was obtained with a high purity. 1.7 Small Cyclic Peptides

[0153] Utilising this new reaction methodology, small cyclic peptides have also been prepared. To the best of the inventor’s knowledge this analogue containing 2 amino acids is the shortest cyclic peptide that has been synthesised to date. Furthermore, it is impossible to synthesise such a short cyclic analogue with a conventional SPPS procedure. Asp and Lys amino acid were incorporated onto Pam resin and their sidechains were linked together. Next, one of the peptide chains were reacted with succinic anhydride to modify its A / -terminal and convert it to carboxylic acid (as depicted in the PMI section). Subsequently, the modified Af-terminal was linked with the unmodified one to form the cyclic product shown in Scheme 16.

[0154] Chemical Formula: C14H21N3O7

[0155] Exact Mass: 343.14

[0156] Scheme 16. Chemical structure of a new 2 amino acid cyclic peptide.

[0157] The mass of this cyclic peptide has been confirmed by MS:

[0158] Figure 23 Mass spectrum of PMI of modified self-assembly peptide. Calculated: 343.14; found: 325.39 [M-18]+

[0159] 1.8 Current industrial peptide families

[0160] The invention has also been investigated for assembling current industrial peptides. This represents an advance in two main areas. Firstly, assembling two peptide chains which are completely different in their amino acid composition. Thus, by employing the orthogonality approach between the tert-butyloxycarbonyl (Boc) and Fluorenylmethyloxycarbonyl (Fmoc) chemistries, both chains were assembled independently. Given that the protecting groups for the side chain of the Boc-amino acids are stable to trifluoroacetic acid (TFA) which are required for the Boc removal during the peptide elongation process, Boc-chain has been assembled before the Fmoc one. Secondly, the interchain assembly reaction will need to take place and connect two far away residues together, namely the N- with C-termini. Liraglutide has been selected in this work to further investigate the applicability of the new approach. 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 were assembled on one resin as Boc and Fmoc- chains (Scheme 17).

[0161] Scheme 17. Chemical structure of the designed liraglutide fragments.

[0162] 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 resin from its side chain and making the C-terminal readily available for linking it with the N-terminal of the Boc-chain after the whole chain is constructed. This will render the common linear peptide.

[0163] After independently assembling both chains, the interchain assembly reaction was carried out. The desired product was obtained as the major product with a satisfactory purity (Figure 24).

[0164] In an alternative approach, rather than assembling the first and second chains completely prior to coupling, the first amino acid (Glu) of the second chain is incorporated onto the resin in the usual manner via the side-chain carboxyl group using Fmoc chemistry. The first amino acid of the first sequence (Gly) is incorporated onto the resin using Boc chemistry. The first chain is elaborated (also using Boc chemistry), the C-terminus of the second chain deprotected, and coupled to the N-terminus of the first chain. The subsequent residues of the second chain are then elaborated using Fmoc chemistry.

[0165] Figure 24. Chromatograms of Liraglutide (tp= 9.9 min). Refer for the legend of Figure 2 for the chromatographic conditions. The masses of the peptides have been confirmed by MS (Figure 25)

[0166] Figure 25 - Mass spectrum of liraglutide peptide. Calculated: 3383.73; found: 1128.7 [M+3H]3+, 846.8 [M+4H]4+

[0167] The current findings confirm that the new technology is also able to link distant residues with the new interchain assembly reaction. Thus, the technology is also able to deliver current commercial peptides free from undesired side products and with satisfactory purity.

[0168] Condensing three shorter fragments will renders a purer product than which was obtained from 2 longer fragments. Therefore, liraglutide will be assembled by splitting the peptide into 3 chains, using 3 orthogonal protecting groups. Namely, Boc, Fmoc, and p-Nitrobenzyloxycarbonyl (pNZ). The designed three fragments of liraglutide are shown in Figure 26.

[0169] Scheme 18. Chemical structure of the designed liraglutide three fragments.

[0170] The first two chains were assembled independently as shown previously. A p- nitrobenzyloxycarbonyl (pNZ) temporary protecting group has been anchored onto the resin to reserve a place for the third chain, where it can be removed on demand. Interchain assembly reaction was carried out to link the first two chains (Boc and Fmoc). H-GQAAKEFIAWLVRGRG-OH 16-mer fragment peptide was successfully obtained with a satisfactory purity (Figure 26). Figure 26 Chromatograms of H-GQAAKEFIAWLVRGRG-OH fragment (tp= 7.5 min). Refer for the legend of Figure 2 for the chromatographic conditions

[0171] Next, pNZ group was removed under reduction conditions and the third chain will be assembled following Fmoc strategy. After that, the interchain assembly reaction will be done to obtain the final liraglutide. Furthermore, the product obtained from linking the first two chains is about twice the size of fragments used in the current manufacturing processes and with competing quality. Therefore, it can serve as a fragment for subsequent condensation processes.

[0172] The current findings confirm that our new technology is also able to link far away residues with our new interchain assembly reaction. Thus, the technology is also able to deliver current industrial peptides free from undesired side products and with satisfactory purity. Furthermore, the technology has demonstrated its ability to assemble three different peptide chains, using 3 orthogonal protecting groups.

[0173] 2.9 Cross-linked peptide

[0174] After confirming the ability to assemble three different peptide chains on one bead, cross-linked peptide was synthesised using the new technology. Assembling of peptide chains was designed carefully in order to maintain the orthogonality during the course of the synthesis. The first and the second chains were linked using the active sidechains of amino acids with reactive sidechains (Lys and Glu), as well as using their N-terminals, post modifying one of them using succinic anhydride. A third bond was also assembled between the second chain with the third one, using the active sidechains of another Lys and Glu residues. The chemical structure of the novel peptide is shown in Scheme 19.

[0175]

[0176] Scheme 19. Chemical structure of novel cross-linked peptide. Green: the formed peptide bond.

[0177] The peptide was obtained in a satisfactory purity (Figure 27) and its mass has been confirmed by MS (Figure 28).

[0178] Figure 27. Chromatograms of cross-linked peptide (tR= 9.4 min). 0-50% in 15 min gradient elution. Refer for the legend of Figure 2 for the chromatographic conditions.

[0179] Figure 28. Mass of cross-linked peptide. Calculated: 1140.26; found: 1140.86 [M+H]+and 571 .23 [M+2H]2+Such structures are expected to have outstanding conformational rigidity, metabolic stability, and efficacy. Hence, circumventing the main enzymatic degradation hurdle, and assisting in bringing these valuable molecules to the market. Such classes are first-in-class and have not been reported previously. Also, this new peptide class mimics the concept of the double-stranded oligonucleotides which contain sense and antisense strands. Likewise, such cross-linked peptides can be designed where each chain will have a unique and independent functionality, thus providing the opportunity for a synergistic multifunctional peptide. An interesting advantage in these new analogues, is the opportunity to selectively modify the chains, without compromising the function of the other chain / s. This new structure could also be exploited to scaffold molecules (e.g., gelation), without the need to have self-assembling sequences. Assembling three chains will facilitate synthethesising triple helix structures (e.g., synthetic crosslinked collagen).

[0180] 2. Conclusion

[0181] The invention demonstrates a new reaction methodology which expands traditional SPPS into a new peptide synthesis reaction space. Assembling different peptide chains concurrently rather than the same single species, on the solid-phase combined with a solid-phase assembly reaction, allows access to new and potentially significant peptides families. These new peptide families are difficult to obtain, and in some cases are impossible to synthesise using conventional SPPS. For example, the inventors have successfully synthesised a new highly constrained cyclic tetrapeptide and dipeptide which are impossible to obtain using conventional SPPS procedures.

[0182] Furthermore, the inventors have demonstrated a successful approach to the synthesis of an important class of peptides known as partial-modified inverse (PMI) peptides. In only two reaction steps, a highly pure PMI was synthesised, without the need to use aggressive chemicals which are known to attack the amino acids. Intriguingly, the innovative idea proved its ability to deliver a 60-mer peptide with high efficiency and purity. It is well known that long peptides are difficult to synthesise due to various side reactions which affect the final purity and yield of the target peptide.

[0183] Interestingly, the inventors have demonstrated the ability of the new approach to synthesise the fragments needed to prepare the current therapeutic peptides in the market with satisfactory purity.

[0184] A novel class called cross-linked has been synthesised by assembling three different chains and linking them from 3 different spots including sidechains and N-termini. Such class is envisaged to have unprecedented.

[0185] 3. Materials and methods

[0186] PuroSynth CTC (1.0 mmol / g, supplier’s specification) and PuroSynth Rink Amide (0.5 mmol / g, supplier’s specification), resins were used for all syntheses. All reagents and solvents were obtained from commercial suppliers and were used without further purification unless otherwise stated. Analytical HPLC was performed on Shimadzu LC20 system using Lab Solution software for data processing. Column: Symmetry Luna C18 (3.6 pm, 4.6 x 150 mm) column, with flow rate of 1.0 mL / min and UV detection at 220 nm. Mobile phase A was 0.1 % trifluoroacetic acid (TFA) in H2O, and mobile phase B was 0.1% TFA in CH3CN. Mass analysis was done using Velos Pro mass spectrometer (ThermoFisher Scientific), a hybrid linear trap quadrupole (LTQ)- Orbitrap, using a positive electrospray ionisation mass spectrometry (ESI+-MS) by direct infusion of samples. Liberty Blue™ automated microwave-assisted peptide synthesiser (CEM).

[0187] 3.1 Incorporation procedure

[0188] 3.2 CTC resin

[0189] First amino acids were incorporated onto CTC resin using dry CH2CI2. CTC resin was swelled in CHaCMor 10-20 min. The Fmoc-amino acids (2 equiv) were dissolved in a minimum amount of the CH2CI2 (0.5 ml_ / 100 mg resin) and sonicated for 10 min. N,N- Diisopropylethylamine (DIEA) (4 equiv) was then added to the solution, which in turn was added to the previously swelled resin and allowed to react for 1 h under mechanical shaking. After this, MeOH (80 pL / 100 mg of resin) was added to endcap any unreacted chloride of the CTC resin. Finally, the resin was washed twice with CHaCh and dried over vacuum.

[0190] 3.3 Rink amide and Sieber amide resins

[0191] First amino acids were incorporated onto Rink Amide resin using dry DMF. Rink Amide resin was swelled in DMF for 10-20 min. Fmoc was removed using 20% piperidine / DMF and the mixture was allowed to shake for 2 and 7 min. The Fmoc- amino acids (3 equiv) and OxymaPure (3 equiv) were dissolved in a minimum amount of the DMF (0.5 ml_ / 100 mg resin) and sonicated for 10 min. A / , / V- diisopropylcarbodiimide (DIC) (3 equiv) was then added to the solution, which in turn was added to the previously swelled resin and allowed to react for 1 h under mechanical shaking. Finally, the resin was washed twice with CHpCh and dried over vacuum.

[0192] 3.4 Pam resin

[0193] First amino acids were incorporated onto Pam resin using dry CH2CI2. CTC resin was swelled in CFLCh for 10-20 min. 4-Dimethylaminopyridine (DMAP) (0.25 equiv) was then added to the resin. The Fmoc-amino acids (2.5 equiv) were dissolved in a minimum amount of the CH2CI2 (0.5 mL / 100 mg resin) and sonicated for 10 min. DIC (5 equiv) was then added to the solution which in turn was added to the previously swelled resin and allowed to react for 1 h under mechanical shaking. After this, acetic anhydride and DIEA (10:20) in DMF were added to endcap any unreacted OH group of the Pam resin. Finally, the resin was washed twice with CH2CI2 and dried over vacuum.

[0194] 3.5 Reactive amino acids incorporation (Asp and Lys)

[0195] When coupling Asp and Lys to the resin as key amino acids for subsequent interchain assembly reaction. 33% Asp and 67% Lys are considered as the optimum ratio that ensures a complete conversion. The incorporation of these amino acid performed done twice to ensure their full incorporation to the peptide chain.

[0196] 3.6 Peptide synthesis

[0197] Peptides were synthesised following the standard methodology performed in the Inventor’s laboratory (3 equiv. of Fmoc-AA-OH, 3 equiv. of OxymaPure, 3 equiv. of DIC) or (2 equiv. of Fmoc-AA-OH, 1.9 equiv. of (1 -Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 4 equiv. of DIEA) in DMF and then shaking for 1 h. Fmoc was then removed (see under Rink amide resin section). All Arg and the residue that comes after were double coupled to ensure complete coupling.

[0198] 3.7 Allyl protecting group cleavage

[0199] To deprotect Asp(OAII) or Lys(Alloc), tetrakis(triphenylphosphine)palladium(0) (0.1 equiv) and Triphenylsilane (10 equiv) in CH2CI2 were added to the peptidyl resin and allowed to react for 1 h under mechanical shaking. This step is done twice. The resin was washed twice with CH2CI2 and then with A / ,A / -Diethyltithiocarbamate (0.02M in DMF) three times to wash the Pd out from the resin.

[0200] 3.8 Boc, Trt, tBu protecting groups cleavage

[0201] To cleave Boc, Trt, or tBu protecting groups 50%TFA in CH2CI2 was added to the peptidyl resin and allowed to react for 30 min under mechanical shaking. The resin was washed twice with C^Cfeand then treated with 10% DIEA in CH2CI2 and allowed to react for 30 min under mechanical shaking to neutralize the protonated groups due to acid treatment. Finally, the resin was washed twice with CH2CI2 and dried over vacuum.

[0202] 3.9 Interchain assembly reaction

[0203] Once the reactive amino acids are ready to form the amide bond, (3 equiv. of Fmoc- AA-OH, 3 equiv. of OxymaPure, 3 equiv. of DIC) or (2 equiv. of Fmoc-AA-OH, 1.9 equiv. of (1 -Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium hexafluorophosphate (COMU), 4 equiv. of DIEA) in DMF and then shaking for 24 h.

[0204] 3.10 Automatic synthesis

[0205] Within the automated SPPS method, coupling of the amino acid, to the growing peptide chain was achieved through addition and heating of the Fmoc-AA-OH acid (0.25 mmol, 5 equiv, 0.2 M in DMF), (OxymaPure, 0.25 mmol, 5 equiv, 0.5 M in DMF), and DIG (0.50 mmol, 10 equiv, 0.5 M in DMF) at 90 °C for 2 min (single coupling) or 2 x 2 min (double coupling). / V-terminal deprotection of the growing peptide chains was achieved through Fmoc-cleavage via addition of piperidine (20 % v / v in DMF) and in OxymaPure (0.1 M in DMF) and heating at 90 °C for 1.5 min.

[0206] 3.11 Cleavage protocols

[0207] Rink amide and CTC resins

[0208] The final synthesised peptide was cleaved from the resin using TFA / triisopropylsilane (TIS) / H2O (95:2.5:2.5) (1 mL / 100 mg) under mechanical shaking for 1 h. Chilled diethyl ether was then added (5 times the cleavage solution volume), and the solution was kept in an ice bath for 30 min. The solution was then centrifuged for 5 min at 5000 rpm, and the supernatant was decanted. A new amount of the ether (5 times the cleavage solution volume) was added to repeat this step. Any remaining ether was dried under N2. Finally, the precipitate was dissolved in CHsCN-H2O (1 :1 ). A small amount of the solution was injected into HPLC system to check the purity of the final product.

[0209] Pam resin

[0210] The final synthesised peptide was cleaved from the resin using Triflic acid / TFA / TIS (8:3:1 ) (1 mL / 100 mg) under mechanical shaking for 1 h at 0 °C. Chilled diethyl ether was then added (5 times the cleavage solution volume), and the solution was kept in an ice bath for 30 min. The solution was then centrifuged for 5 min at 5000 rpm, and the supernatant was decanted. A new amount of the ether (5 times the cleavage solution volume) was added to repeat this step. Any remaining ether was dried under N2. Finally, the precipitate was dissolved in CH3CN-H2O (1 :1 ). A small amount of the solution was injected into HPLC system to check the purity of the final product. References

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[0218] 8. Al Musaimi, O., B.G. de la Torre, and F. Albericio. Greening Fmoc / tBu solidphase peptide synthesis. Green Chem. 2020, 22: p. 996-1018. 10.1039 / c9gc03982a

[0219] 9. Jaradat, D.M.M., O. Al Musaimi, and F. Albericio. Advances in solid-phase peptide synthesis in aqueous media (ASPPS). Green Chem. 2022, 24,(17): p. 6360- 6372. 10.1039 / D2GC02319A

[0220] 10. Lam, K.S., S.E. Salmon, E.M. Hersh, V.J. Hruby, W.M. Kazmierski, and R.J. Knapp. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991 , 354,(6348): p. 82-84. 10.1038 / 354082a0

[0221] 11. White, C.J. and A.K. Yudin. Contemporary strategies for peptide macrocyclization. Nat. Chem. 2011 , 3,(7): p. 509-524. 10.1038 / nchem.1062

[0222] 12. Chorev, M. The partial retro-inverso modification: A road traveled together. Biopolymers (Peptide Science) 2005, 80, (2-3): p. 67-84. 10.1002 / bip.20219 13. Pallai, P.V., S. Richman, R.S. Struthers, and M. Goodman. Approaches to the synthesis of retro-inverso peptides. Int. J. Pept. Protein Res. 1983, 21 ,(1): p. 84-92. 10.1111 / j.1399-3011 .1983,tb03081 .x

[0223] 14. Bray, B.L. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat. Rev. Drug Discov. 2003, 2,(7): p. 587-93. 10.1038 / nrd1133

[0224] 15. Heffernan, C., H. Sumer, G.J. Guillemin, U. Manuelpillai, and P.J. Verma. Design and screening of a glial cell-specific, cell penetrating peptide for therapeutic applications in multiple sclerosis. PLoS One 2012, 7,(9): p. e45501. 10.1371 / journal. pone.0045501

[0225] 16. Al Musaimi, O., S.V. Morse, L. Lombardi, S. Serban, A. Basso, and D.R. Williams. Successful synthesis of a glial-specific blood-brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin. J. Pept. Sci. 2022: p. e3448. 10.1002 / psc.3448

[0226] 17. Frerot, E., J. Coste, A. Pantaloni, M.N. Dufour, and P. Jouin. PyBOP® and PyBroP: Two reagents for the difficult coupling of the a,a-dialkyl amino acid, Aib. Tetrahedron 1991 , 47: p. 259-270. 10.1016 / S0040-4020(01)80922-4

[0227] 18. Zieleniewski, F., D.N. Woolfson, and J. Clayden. Automated solid-phase concatenation of Aib residues to form long, water-soluble, helical peptides. Chem,Comm, 2020, 56,(80): p. 12049-12052. 10.1039 / D0CC04698A

[0228] 19. Al Shaer, D., O. Al Musaimi, F. Albericio, and B.G. de la Torre. 2021 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals 2022, 15,(2): p. 222. 10.3390 / ph 15020222

[0229] All the references mentioned above are incorporated herein by reference.

Claims

Claims1 . A process for the preparation of a peptide, comprising the steps of: a) providing a resin, the resin having attached thereto: i. a first peptide fragment having a free carboxy group; and ii. a second peptide fragment having a free amino group; and b) coupling the free carboxy group of the first peptide fragment with the free amino group of the second peptide fragment to form an amide group connecting the first and second peptide fragments.

2. A process according to claim 1 wherein the free carboxy group of the first peptide fragment is present on the side chain of an amino acid residue.

3. A process according to claim 2 wherein the free carboxy group of the first peptide fragment is present on the side chain of an amino acid residue, wherein the amino acid is selected from aspartic acid and glutamic acid.

4. A process according to any preceding claim wherein the free amino group of the second peptide fragment is present on the side chain of an amino acid residue.

5. A process according to claim 4 wherein the free amino group of the second peptide fragment is present on the side chain of an amino acid residue, wherein the side chain is lysine.

6. A process according to any preceding claim wherein the first peptide fragment is protected at the N-terminus with a first protecting group, and the second peptide fragment is protected at the N-terminus with a second protecting group, wherein the first and second protecting groups are orthogonal.

7. A process according to claim 6 wherein the first and second protecting groups are selected from Boc and Fmoc.

8. A process according to claim 6 or 7 wherein the first peptide fragment is assembled on the resin using a stepwise solid phase peptide synthesis protocol.

9. A process according to any of claims 6 to 8 wherein the second peptide fragment is assembled on the resin using a stepwise solid phase peptide synthesis protocol.

10. A process according to any one of claims 1 to 5 wherein the first and second peptide fragments are protected at the N-terminus with the same protecting group.

11. A process according to any one of claims 1 or 4 to 10 wherein the free carboxy group of the first peptide fragment is the C-terminus.

12. A process according to claim 11 wherein the first peptide fragment is bound to the resin via a side chain residue of the first peptide fragment.

13. A process according to any one of claims 1 to 3 or 6 to 12 wherein the free amino group of the second peptide fragment is the N-terminus.

14. A process according to any one of claims 11 to 13 wherein the free carboxy group of the first peptide fragment is the C-terminus, and the free amino group of the second peptide fragment is the N-terminus.

15. A process according to any preceding claim wherein the first and / or second peptide fragment is further elaborated to include one or more further amino acid residues.

16. A process according to claim 15 wherein the first and second peptide fragments are coupled to form a further connection between the first and second peptide fragments.

17. A process according to any preceding claim comprising the further step of cleaving the linked first and second peptide fragments from the resin.

18. A process according to claim 17 comprising the further step or steps of deprotecting the linked first and second peptide fragments.

19. A process for the preparation of a partial modified inverso and / or retro inverso peptide, comprising the steps of: a) providing a resin, the resin having attached thereto:i. a first peptide fragment having a first free amino group; and ii. a second peptide fragment having a second free amino group; and b) reacting the resin of step a) with a divalent electrophile capable of reacting with two amino groups to form a link between the first and second peptide.

20. A process according to claim 19 wherein the divalent electrophile is selected from a dicarboxylic acid or an anhydride thereof.21 . A process according to claim 20 wherein the divalent electrophile is succinic anhydride.

22. A process according to any one of claims 19 to 21 wherein the first and second free amino groups are the N-termini of the first and peptide fragments.

23. A process according to any one of claims 19 to 22 wherein the first and second peptide fragments are identical.

24. A process according to any one of claims 19 to 23 including the further step of cleaving the linked first and second peptide fragments from the resin.

25. A partial modified inverse and / or retro inverso peptide obtainable by any one of claims 19 to 24.

26. A process for the solid phase synthesis of a peptide, comprising a step of coupling an amino acid bound to a resin support having a free N-terminal amino group with a mixture of at least two N-protected amino acids.

27. A process according to claim 26 wherein the at least two N-protected amino acids are selected from lysine and aspartic acid.

28. A process according to claim Z1 wherein the molar ratio of lysine to aspartic acid is in the range of 3:1 to 3:2, preferably about 2:1.

29. A process according to any one of claims 26 to 28 wherein the amino acids are side-chain protected, preferably with an allyloxycarbonyl (alloc) group.

30. A compound having the formulaor a pharmaceutically acceptable salt or ester thereof.

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