Chemical synthesis methods for peptides

Abstract

This invention relates to a method for synthesizing peptides and peptide-related compounds supported on a polymer solid-phase resin. The method of the present invention provides a synthetic scheme that enables the coupling of two or more peptide fragments attached to the same polymer support by allowing access to previously unobtainable structures, and also provides a simpler and higher yield method for known peptides, including those of commercial importance.

Description

[Technical Field]

[0001] This invention relates to methods for synthesizing peptides and peptide-like compounds, and novel compounds obtained thereby. [Background technology]

[0002] The solid-phase peptide synthesis (SPPS) method was published by Merrifield in 1963 [Reference 1]. In the standard SPPS method, a specific peptide is grown on solid-phase support particles, and the same peptide chain is assembled on individual beads of a polymer solid-phase support resin. This synthesis involves repeated deprotection, coupling, and washing reaction steps to extend these chains until the desired single peptide is assembled. The same peptide chain is extended simultaneously, producing the same peptide in a single synthesis sequence. At the end of the sequence, the peptide is chemically cleaved from the resin using appropriate reagents.

[0003] This protocol is a simple, conventional method used in the SPPS method. Since the introduction of this technology, considerable effort has been made to advance it, including the development of various new solid-phase supports [References 2-6], new coupling reagents [Reference 7], and more environmentally friendly techniques to meet environmental requirements.

[0004] Despite its success at both research and industrial scales, conventional SPPS methods often require several key reactions to be carried out outside the resin. This is primarily due to the occurrence of undesirable side reactions, and current SPPS methods are typically limited to the synthesis of peptides with a length of 15-20 amino acids. Beyond this length, purity and yield problems arise. Therefore, for example, the currently common commercial method for producing a 45-amino acid peptide involves synthesizing three 15-amino acid peptide fragments and then combining these fragments in a solution condensation reaction. This method requires cleaving each precursor of the 15-amino acid peptide from the resin (once for each fragment, followed by purification as needed) and then organizing them in solution. These additional steps impose significant constraints not only on product yield and purity, and manufacturing time, but also on the environmental footprint of the process.

[0005] This disclosure proposes two ways of reinventing the standard SPPS method.

[0006] The first method is derived from techniques currently used to synthesize a wide range of peptides that are expected to have therapeutic applications. The one-bead-one-compound (OBOC) method has been known since 1991 [Reference 10]. This method is an efficient method that utilizes the SPPS method, but it requires the preparation of a huge library of thousands or millions of different peptides. Subsequently, this peptide library can be mixed with a target molecule, and functional peptides can be identified based on which peptides bind to the target molecule. In this specification, multiple different peptides are synthesized simultaneously in a single column using the SPPS method.

[0007] A second novel aspect of this research is a new interchain assembly reaction (ACR) that enables the synthesis of a broad spectrum of useful peptide therapeutics by chemically reacting two or three precursor peptide chains synthesized on the same bead to form the final peptide product (Figure 1). The combination of these two technological innovations makes it possible to produce new peptide homologs with therapeutic applications in a simpler, faster, and less expensive reaction process. For example, this innovative technique requires only one purification step at the end of the synthesis. Therefore, compared to methods that involve multiple SPPS steps requiring a final liquid-phase coupling reaction, the present invention has fewer steps and thus higher yields. Most importantly, by immobilizing one end of the chain to a resin during the reaction, the inventors were able to suppress serious oligomerization reactions. [Prior art documents] [Patent Documents]

[0008] U.S. Patent Application Publication 2019 / 0345636 describes a method for generating a combinatorial library on a support (such as beads). This library contains a set of various molecules (e.g., DNA, RNA, peptides).

[0009] International Publication No. 2023 / 122698 discloses a method for analyzing multiple types of polypeptides in a sample.

[0010] International Publication No. 2023 / 062389 relates to a method for preparing polymers having a defined sequence, such as polynucleotides and polypeptides, in a liquid phase. Purification is carried out by membrane filtration (e.g., diafiltration). [Overview of the project]

[0011] According to a first aspect, the present invention relates to a process for preparing peptides. This process is a)i. A first peptide fragment having a free carboxyl group, ii. a second peptide fragment having a free amino group, and a step of preparing a resin to which is attached; 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 that links the first peptide fragment and the second peptide fragment.

[0012] According to a second aspect, the present invention relates to a process for preparing a partially modified inverso peptide and / or a partially modified retro-inverso peptide, the process comprising: a) i. a first peptide fragment having a first free amino group, ii. a second peptide fragment having a second free amino group, and a step of preparing a resin to which is attached; b) reacting the resin of step a) with a divalent electrophile capable of reacting with two amino groups to form a linkage between the first peptide and the second peptide.

[0013] According to a third embodiment, the present invention relates to a process for solid-phase synthesis of a peptide, the process comprising a step of coupling an amino acid bound to a resin carrier having a free N-terminal amino group with a mixture of at least two N-protected amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] FIG. 1 is a schematic diagram of a reaction sequence according to an embodiment of the present invention. [Figure 2] FIG. 2 is a diagram showing a chromatogram. [Figure 3] FIG. 3 is a diagram showing a mass spectrum. [Figure 4] FIG. 4 is a diagram showing a mass spectrum. [Figure 5] FIG. 5 is a diagram showing a chromatogram. [Figure 6] FIG. 6 is a diagram showing a chromatogram. [Figure 7] FIG. 7 is a diagram showing a chromatogram. [Figure 8]This is a diagram showing the mass spectrum. [Figure 9] This is a diagram of a chromatogram. [Figure 10] This is a diagram showing the mass spectrum. [Figure 11] This is a diagram of a chromatogram. [Figure 12] This is a diagram showing the mass spectrum. [Figure 13] This is a diagram of a chromatogram. [Figure 14] This is a diagram showing the mass spectrum. [Figure 15] This is a diagram showing the mass spectrum. [Figure 16] This is a diagram of a chromatogram. [Figure 17] This is a diagram showing the mass spectrum. [Figure 18] This is a diagram showing the mass spectrum. [Figure 19] This is a diagram of a chromatogram. [Figure 20] This is a diagram showing the mass spectrum. [Figure 21] This is a diagram of a chromatogram. [Figure 22] This is a diagram showing the mass spectrum. [Figure 23] This is a diagram showing the mass spectrum. [Figure 24] This is a diagram of a chromatogram. [Figure 25] This is a diagram showing the mass spectrum. [Figure 26] This is a diagram of a chromatogram. [Figure 27] This is a diagram of a chromatogram. [Figure 28] This is a diagram showing the mass spectrum. [Modes for carrying out the invention]

[0015] The inventors have improved the standard SPPS method in two ways. The first method is derived from a technique currently used to randomly synthesize a wide range of peptides that are expected to have therapeutic applications. The one-bead-one-compound (OBOC) method has been known since 1991 [Reference 10]. This method is an efficient method that utilizes the SPPS method, but it generates a large library of random peptides. Subsequently, this peptide library is mixed with a target molecule, and the functional peptide to be bound to the target molecule can be identified by determining which peptides bind to the target molecule. In the present invention, multiple different peptides are synthesized simultaneously on a single-bead assembly using the SPPS method.

[0016] A second aspect of the present invention provides a novel interchain organization reaction that enables the synthesis of useful peptide products with a broad spectrum by chemically reacting two or more precursor peptide chains synthesized on beads to form the final peptide product (Figure 1).

[0017] By combining these two embodiments, novel peptide homologs with potential therapeutic (and other) applications can be produced using a simpler, faster, and less expensive reaction scheme. For example, in the method of the present invention, the purification step only needs to be performed once at the end of the synthesis. Compared to liquid-phase coupling, the number of expected steps is reduced, and high yields are possible. Most importantly, in the process according to the present invention, serious oligomerization reactions can be suppressed by fixing one end to the resin during the reaction.

[0018] In its broadest embodiment, the present invention is based on a coupling reaction between two peptide fragments bonded together to a resin, wherein the first peptide fragment has a free carboxyl group and the second peptide fragment has a free amino group. Unlike known methods, both the first and second peptide fragments are chemically added to the same polymer beads (or other particles).

[0019] Suitable polymers are known to those skilled in the art. In the usual SPPS method, cross-linked polystyrene (PS) resins are most commonly used. Beads with a particle size distribution of 200-400 mesh (corresponding to a diameter of approximately 50 μm) and a load of 0.5-0.8 mmol / g exhibit good properties for polymer swelling in solvents such as DMF and DCM, diffusion of reactants into the polymer matrix, and access to linker sites embedded in the beads. For larger peptides (25 amino acids or more) or complex sequences, lower loads are required (0.1-0.2 mmol / g). Non-limiting examples of suitable solid supports that can be used for the preparation of peptide amides include Merrifield polystyrene resins, PAM resins, Wang resins, Rink amide resins, PAL resins, Siber amide resins, MBHA resins, trityl resins, and 2-chlorotrityl resins. Basic-instability resins may also be used, including oxime resins, HMBA resins, DHP resins, Weinreb aminomethyl resins, and polyethylene glycol-polystyrene graft resins. Details of these resins are described in "Chemical Approaches to Synthesis of Peptides and Proteins" by Paul Lloyd-Williams, Fernando Albericio, and Ernest Giralt, ISBN 9780849391422.

[0020] In one embodiment, a first peptide fragment is synthesized on a polymer support using a conventional stepwise method. A C-terminal amino acid in the form of a reactive derivative having an N-α protecting group and optionally a side-chain protecting group is covalently bonded directly or via an appropriate linker to a support swollen with an organic solvent. The N-α protecting group is removed, and then amino acids having protecting groups are added sequentially.

[0021] Once the desired peptide fragment chain length is obtained, the side chain protecting groups are removed, and the peptide is cleaved from the resin. These steps can be performed separately or simultaneously.

[0022] Any suitable method can be used for peptide protection. Two preferred coupling methods are those based on different combinations of N-α protecting groups and side-chain protecting groups. Specifically, these are the method using the tert-butyloxycarbonyl (Boc) group as the N-α protecting group and the method using the 9-fluorenylmethyloxycarbonyl (Fmoc) group.

[0023] The N-α-Boc protected peptide coupled to the resin is N-α-deprotected with a strong acid, preferably trifluoroacetic acid (TFA). The resulting amine salt is washed and neutralized with a base, such as a tertiary amine. The subsequent peptide bond is formed by reaction with an activated Boc-amino acid, such as a symmetric anhydride. Deprotection is carried out with HF or a sulfonic acid. Preferably, the side chain protecting group is N-allyloxycarbonyl (Alloc) or benzyl, which is preferably deprotected by Pd(0) catalyzed allyl transfer.

[0024] In certain embodiments, a third protecting group orthogonal to both the Boc and Fmoc groups can be used. Preferably, this protecting group is a p-nitrobenzyloxycarbonyl (pNZ) group. The corresponding derivatives are readily synthesized solids that function well in the solid phase. The pNZ moiety is orthogonal to the most common protecting groups used in peptide chemistry and is removed under neutral conditions with a catalytic amount of acid.

[0025] The N-α-Fmoc protected peptide coupled to the resin is N-α-deprotected by treatment with a secondary amine, preferably piperidine, in an organic solvent, such as N,N-dimethylformamide (DMF) or dichloromethane (DCM). After washing, the neutral peptide resin is reacted with an activated Fmoc amino acid, such as a hydroxybenzotriazole active ester.

[0026] Side chain protection is preferably selected from tert-butyl, trityl, and arylsulfonyl systems, and a strong acid such as TFA is used for side chain deprotection.

[0027] Other N-α protecting groups have also been proposed (Stewart, JM and Young, JD, "Solid phase peptide synthesis," Pierce Chemical Company (1984)), and are included within the scope of the present invention.

[0028] As explained above, the first peptide fragment is synthesized using a conventional SPPS sequence. Similarly, the second peptide fragment is also synthesized using a conventional SPPS sequence.

[0029] In one embodiment, the first peptide fragment and the second peptide fragment are synthesized using the same protecting group. In this embodiment, the first peptide fragment and the second peptide fragment are differentiated during the synthesis sequence by performing one or more coupling steps in which activated N-protected amino acids are mixed, such that the ratio of the growing first peptide fragment to the second peptide fragment differs by one or more amino acid residues. Such an embodiment is shown in Scheme 1 below. (Scheme 1) TIFF2026531690000002.tif154170

[0030] Scheme 1 illustrates the principle of simultaneously constructing two peptide fragments attached to the same polymer (e.g., beads). The polymer support (black circle) is supported with a first amino acid residue (AA1), which is deprotected to reveal a primary amine group, making it ready for a subsequent coupling reaction [1]. The resin is shown with two identical amino acids bound to it, which are representative of the many such amino acids that actually exist. The resin-bound amino acid [1] is coupled to the FmocN-protected amino acid [2] under appropriate coupling conditions. Many suitable conditions are known; see, for example, Chem. Rev. 2011, Vol. 111, No. 11, pp. 6557-6602, incorporated herein by reference. N,N-diisopropylcarbodiimide (DIC) is generally suitable.

[0031] Following this initial coupling reaction, the Fmoc-protected dipeptides AA1AA2 are bound to the resin support [3] and deprotected under normal conditions (piperidine in dimethylformamide (DMF)) to obtain resin-bound dipeptides with free amine groups [4]. At this point, all peptides attached to the resin are identical.

[0032] A crucial step occurs when further extending the resin-bound dipeptide[4]. Instead of coupling the resin-bound dipeptide[4] with a single protecting amino acid as is typical, it is reacted with a mixture of two different Fmoc-protecting amino acids,[5] and[6]. The exact ratio of the two Fmoc-protecting amino acids[5] and[6] depends on the properties of amino acid residues AA3 and AA4. Upon completion of this step, the resin is bound to two different tripeptide chains, AA1AA2AA3 and AA1AA2AA4, whose N-terminuses are protected with Fmoc groups[7].

[0033] In Scheme 1, the coupling reaction of Fmoc-protected amino acids [5] and [6] occurs in a single step, but it is also conceivable that these couplings may occur sequentially. For example, the resin-bound dipeptide [4] in some embodiments may be coupled with the Fmoc-protected amino acid [5] in an amount less than stoichiometric, i.e., an amount insufficient to completely react with all the free amine groups of the resin-bound dipeptide [4], and then coupled with the Fmoc-protected amino acid [6] to obtain [7].

[0034] [8] is treated under normal Fmoc deprotection conditions to obtain a resin carrier having two tripeptides [8]. At this point, the resin is bound to the two tripeptides, namely the first peptide fragment and the second peptide fragment. These can be further constructed, i.e., coupled with one or more additional amino acids, to prepare a resin carrier having the fully constructed first peptide fragment and the second peptide fragment [9].

[0035] In Scheme 1 above, the first peptide fragment and the second peptide fragment differ by a specific amino acid residue. However, as those skilled in the art will know, the first peptide fragment and the second peptide fragment can be made to differ at any point during peptide synthesis. That is, it will be obvious that they can differ at any residue in their amino acid sequences. Similarly, in Scheme 1 above, the first peptide fragment and the second peptide fragment differ by a single amino acid residue. However, it will be understood that the first peptide fragment and the second peptide fragment may differ by one or more amino acid residues, such as two, three, four, or more residues.

[0036] Furthermore, although Scheme 1 above shows two different peptide fragments, synthesizing three or more peptide fragments and attaching them to the same resin, such as beads, is within the scope of the present invention.

[0037] In an alternative embodiment, the first peptide fragment and the second peptide fragment can be synthesized sequentially using an orthogonal protecting group sequence, as shown in Scheme 2 below.

[0038] TIFF2026531690000003.tif83170

[0039] Referring to Scheme 2 above, the resin beads (black circles) are initially supported with a mixture of Fmoc-protected amino acids and Boc-protected amino acids

[10] . It will be obvious to those skilled in the art that each bead is bound to a number of such amino acids, rather than the two described above.

[0040] Treatment with trifluoroacetic acid

[10] selectively removes the Boc group, exposing the free amine group of amino acid AA2

[11] . This is then coupled with Boc-protected amino acid AA3

[12] under appropriate coupling conditions. At this stage, the resin is coupled to Boc-protected dipeptides AA2AA3 and Fmoc-protected amino acid AA1

[13] . Constructing the Boc-protected peptide fragments using a standard deprotection / coupling sequence yields an intermediate

[14] . Treatment with piperidine removes the Fmoc protecting group from resin-bound amino acid AA1, and coupling with Fmoc-protected amino acid

[16] using a standard method yields

[17] . Constructing these Fmoc-protected peptide fragments using a standard deprotection / coupling sequence yields intermediate resins coupled with these constructed fragments 1 and 2 (first and second peptide fragments)

[18] . Onbees coupling reaction Further aspects of the present invention relate to a method for coupling at least two peptide fragments attached to the same resin carrier. Such peptide fragments can be prepared by the methods described herein (and have been prepared in some embodiments), but are not necessarily required. A coupling method on a resin is another useful embodiment, independently of the method used to attach such peptide fragments to the resin.

[0041] In this embodiment, the present invention relates to a process for preparing peptides or peptide-like compounds. This process is a)i. A first peptide fragment having a first active group, ii. A second peptide fragment having a second active group and The process of preparing a resin to which the compound is added, b) The process includes the step of coupling the first active group of the first peptide fragment with the second active group of the second peptide fragment to form a bond.

[0042] In this specification, the terms “first active group” and “second active group” mean that the first and second active groups can react under appropriate conditions to form a bond. Such a bond may be a direct chemical bond (e.g., a covalent bond) or it may involve a portion that links to the first and second active groups to form a linker. Table 1 below shows non-limiting examples of such bonds formed with the first and second active groups.

[0043] [Table 1]

[0044] The appropriate reaction conditions in the examples shown in Table 1 will be obvious to those skilled in the art. For example, the reaction of an amine and a carboxylic acid can form an amide bond using any of the many known coupling reagents, such as carbodiimide reagents. Similarly, many suitable conditions are known for forming ester bonds from alcohols and carboxylic acids (or their active derivatives). The coupling of thiol groups to form disulfide groups can be carried out under oxidative conditions, and alcohols can be coupled, for example, under dehydration conditions. CC bonds can be formed using the metathesis crosslinking method currently used in stapling.

[0045] In addition to the direct formation of a bond between the first and second active groups, it is also conceivable that the first and second active groups be linked via a linker. As used herein, "linker" refers to a chemical moiety that can react with both the first and second active groups, and is usually a divalent entity. Suitable linkers include dicarboxylic acids (oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, etc.), diols, diamines, amino acids, etc.

[0046] In some embodiments, the first active group is an amine. Preferably, the amine group can be provided by the side chain of any amino acid present on the first peptide fragment. Lysine is a preferred example of a native amino acid having an amine side chain. However, it is conceivable that any non-native amino acid having an amine group in its side chain could also play this role.

[0047] In some embodiments, the second active group is a carboxylic acid. Preferably, the carboxylic acid group can 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 native amino acids having carboxylic acid side chains. However, it is conceivable that any non-native amino acid having a carboxylic acid in its side chain could also play this role.

[0048] The coupling of the first and second peptide fragments occurs while both fragments are bound to the resin. This provides the unique advantages of the present invention in terms of regioselectivity and chemoselectivity. Furthermore, because both peptide fragments are bound to the same polymer support, the local relative concentrations are high, and the coupling reaction proceeds generally rapidly and in high yield.

[0049] In some embodiments, the first active group is an amine, which is a side-chain amine of a lysine residue present in the first peptide fragment, and the second active group is a carboxylic acid group, which is a side-chain carboxylic acid group of an aspartic acid or glutamine residue (preferably glutamic acid) present in the second peptide fragment. It should be understood that the terms "first," "second," etc., are merely used to distinguish the peptide fragments from each other and have no further meaning. For example, the first peptide fragment may similarly contain a carboxylic acid group, while the second peptide fragment may contain an amine group.

[0050] A generalized scheme showing the coupling of a first peptide fragment containing lysine with a second peptide fragment containing aspartic acid is shown in Scheme 3.

[0051] (Scheme 3) TIFF2026531690000005.tif69170

[0052] Referring to Scheme 3, "Xaa" represents an unspecified amino acid, and n, m, p, and q are natural numbers (including 0). Structure

[19] shows the resin (black circle) bonded to a first peptide fragment (top) and a second peptide fragment (bottom). The first peptide fragment contains a lysine residue at a location along its amino acid sequence. The second peptide fragment contains an aspartic acid residue at a location along its amino acid sequence. The coupling of the lysine side-chain amino group to the aspartic acid side-chain carboxylic acid is formed under amide bond-forming conditions (preferably N,N′-diisopropylcarbodiimide and ethylcyano(hydroxyimino)acetate (OxymaPure) or (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) and diisopropylethylamine))

[20] .

[0053] The coupled peptide is cleaved from the resin under standard conditions (which vary depending on the properties of the resin) to obtain the peptide

[21] . TIFF2026531690000006.tif57170

[0054] In the synthetic sequence according to this embodiment of the present invention, it may be necessary or advantageous to protect amino acid terminals or side chains that do not participate in the coupling reaction described above.

[0055] The structure made available for the first time by the method of the present invention is called a "peptide," but since it incorporates a non-α-amino acid (for example, a linkage of ε-amino acids in the case of compound

[21] ) into the amino acid backbone, it may also be called a "modified peptide."

[0056] In a further embodiment of the present invention, the first and second peptide fragments are coupled by the formation of a bond between the first and second active groups, and the first and second peptide fragments may be further extended by the SPPS method to include third and fourth active groups. These can be coupled as described above to produce a resin-bound cyclic peptide. Such a sequence is shown in Scheme 4.

[0057] (Scheme 4) TIFF2026531690000007.tif128170

[0058] Referring to Scheme 4, the resin-bound coupled peptide

[22] is coupled at the N-terminus of the first peptide fragment (using appropriate deprotection and coupling sequences) to an Fmoc-protected aspartate residue

[23] , and subsequently at the N-terminus of the second peptide (also using appropriate deprotection and coupling sequences) to an Fmoc-protected lysine

[24] . The introduced lysine and aspartate side chains are coupled under amide bond formation conditions to form a further covalent link between the first and second peptide fragments. The resin-bound peptide

[25] is cleaved from the resin, and the Fmoc protection is removed under standard conditions to obtain a cyclic peptide

[26] . TIFF2026531690000008.tif70170

[0059] According to this method, by further incorporating the linkage between the first peptide fragment and the second peptide fragment, bicyclic, tricyclic, and even higher-order cyclic peptides can be generated.

[0060] In a further embodiment, the methods of the present invention described herein can be used for the preparation of partially modified inversopeptides and / or partially modified retroinversopeptides. A generalized sequence is shown in Scheme 5.

[0061] (Scheme 5) TIFF2026531690000009.tif106170

[0062] Referring to Scheme 5, a resin carrier (black circle)

[27] is prepared by coupling a first peptide fragment [Xaa]n with an N-terminus protected by Fmoc with an N-terminus protected by Fmoc with an N-terminus protected by Fmoc with an N-terminus protected by an N-terminus protected by an Fmoc with Fmoc with an N-terminus protected by an N-terminus protected by an N-terminus protected by an Fmoc with an N-terminus protected by an N-terminus protected by an Fmoc with an N-terminus protected by an N-terminus protected by an Fmoc with an N-terminus protected by an N-terminus protected by an N-termin

[0063] Preferred embodiments of the present invention (i.e., the embodiments shown in Scheme 5) can be used to prepare symmetrically modified inversopeptides and / or partially modified retroinversopeptides in which the first peptide fragment and the second peptide fragment are identical. However, it will be understood by those skilled in the art that non-identical (different) peptide fragments may be linked using a similar method.

[0064] Partially modified inversopeptides and / or partially modified retroinversopeptides obtained by the described method constitute further embodiments of the present invention. In some embodiments, one of the peptide fragments mainly or exclusively comprises or consists of D-amino acids, and the other fragment mainly or exclusively consists of L-amino acids.

[0065] The methods outlined above, namely the side-chain coupling outlined in Scheme 3 and the N-terminal coupling outlined in Scheme 5, may also be combined. An example scheme is shown below (Scheme 6).

[0066] (Scheme 6) TIFF2026531690000010.tif185170

[0067] Referring to Scheme 6, "Xaa" represents an unspecified amino acid, and n, m, p, and q are natural numbers (including 0). Structure

[32] shows the bonding of the resin (black circle) to the first (top) peptide fragment and the second (bottom) peptide fragment, both of which are protected at the N-terminus with Fmoc. The first peptide fragment contains at least one lysine residue having an unprotected amino side chain. The second peptide fragment contains at least one aspartic acid residue having an unprotected carboxylic acid side chain. Coupling the aforementioned lysine and aspartic acid side chains under amide bond formation conditions yields

[33] . Removal of the Fmoc protecting group yields

[34] .

[0068] When the resin-bound compound

[34] is reacted with a divalent carboxylic acid (or its anhydride such as succinic anhydride)

[35] , a compound having two links between the first peptide chain and the second peptide chain

[36] is obtained. When cleaved from the resin, a cyclic peptide

[37] is obtained.

[0069] In a further embodiment of the present invention, a method for synthesizing peptides containing a conventional α-amino acid backbone is provided via the coupling of two resin-bound peptide fragments. This embodiment overcomes the drawbacks known to the SPPS method when applied to the synthesis of long peptide chains. This embodiment is shown (but is not limited to) Scheme 7.

[0070] (Scheme 7) TIFF2026531690000011.tif64170

[0071] Referring to Scheme 7 above, the synthetic sequence begins with a resin carrier to which the first (lower) peptide fragment and the second (upper) peptide fragment are attached

[38] . The first peptide fragment is attached to the resin carrier at its C-terminus and protected at its N-terminus with a Boc group. The second peptide fragment, on the other hand, is attached to the resin carrier via a side-chain residue (e.g., lysine or aspartic acid) and has a free end at its C-terminus for reaction. The second peptide fragment is protected at its N-terminus with Fmoc.

[0072] The Boc group is removed from the first peptide fragment with trifluoroacetic acid or the like to expose the terminal amino group

[39] . The N-terminus of the first peptide fragment is coupled to the C-terminus of the second peptide fragment on the resin to obtain

[40] . This is then cleaved from the resin to obtain

[41] , and the Fmoc group is removed to obtain the fully constructed peptide

[42] .

[0073] Examples

[0074] 1. Results and Discussion By using different orthogonal protection measures, linkers, and chemicals, it has become possible to synthesize not just one peptide chain, but two or three different peptide chains simultaneously on a single bead.

[0075] In this study, a peptide chain immobilized on beads was first extended to a desired length. Then, using a mixture of two different amino acids coupled to this immobilized peptide, namely Fmoc-Asp-(OAll)-OH and Fmoc-Lys(Alloc)-OH, two chains with different initial amino acids, namely the Asp peptide and the Lys peptide, were generated. The allyl group was chosen because it can be cleaved independently from the side chains of both amino acids while the peptide remains immobilized on the resin. Therefore, the subsequent interchain organization reaction can be carried out between the carboxylic acid group and the amine group of Asp and Lys, respectively.

[0076] 1.1 Linear Peptide Synthesis We successfully prepared octapeptides by extending two chains containing the same amino acid components, except that one amino acid is in the Asp form in one chain and the Lys form in the other (Scheme 8).

[0077] (Scheme 8) TIFF2026531690000012.tif132170 Scheme 8. Schematic diagram of linear peptide synthesis after interchain organization reaction of Fmoc-GDGL-OH and Fmoc-GKGL-OH. TIFF2026531690000013.tif7170 represents a polymer support. 2-chlorotrityl chloride (CTC) was used in this synthesis. The peptide mass was confirmed by mass spectrometry.

[0078] Figure 2 shows Fmoc-GDGL-OH and Fmoc-GKGL-OH(t R (=22.9 minutes), as well as unreacted Fmoc-GDGL-OH(t R This figure shows the chromatogram of a linear octapeptide generated from (17.5 min). Gradient elution of 15-70% was performed for 30 minutes. Mobile phase A is 0.1% TFA in H2O, and mobile phase B is 0.1% TFA in CH3CN. Symmetry Luna C 18 A column (3.6 μm, 4.6 × 150 mm) was used.

[0079] Figure 3 shows the mass spectra of linear octapeptides generated from Fmoc-GDGL-OH and Fmoc-GKGL-OH. The calculated value is 1159.52, and the measured value is 1160.91 [M+1]. + That was the case.

[0080] This embodiment demonstrated the significant success of this new concept. Each reactant, namely Lys and Asp, was loaded onto the resin in equimolar amounts. Excess unreacted Asp-containing precursor (Fmoc-GDGL-OH) was observed and confirmed by mass spectrometry (Figure 4 shows the mass spectrum of the unreacted Fmoc-GDGL-OH tetrapeptide; the calculated value was 582.61, and the measured value was 565.48[M-18]+). Therefore, we decided to optimize the procedure and define the optimal loading rate for each reactant.

[0081] 1.2 Optimizing the Procedure Reaction stoichiometry is crucial, and it is essential that the reacting species are completely consumed. Otherwise, if the concentration of reacting amino acids is too low, the main synthetic route will be interrupted. On the other hand, excess amino acids will lead to undesirable reaction pathways, affecting the overall purity and yield of the main product, and ultimately potentially causing the entire process to fail.

[0082] Based on the findings from the linear peptide synthesis described in the previous section, equimolar amounts of Asp and Lys were pre-supported on a Gly-CTC resin. Upon performing the interchain reaction, an excess of Asp was observed, confirming the findings from the previous section. The amount of the final peptide product relative to the remaining Asp was approximately 85%.

[0083] Figure 5 shows the chromatogram. The black line represents the product obtained by loading 50% Asp and 50% Lys onto a CTC resin, followed by the incorporation of linear Fmoc-Lys-Gly-OH and Fmoc-Asp-Gly-OH. The pink line represents the product after reacting Asp and Lys at room temperature for 22 hours (the second peak shows excess Asp). Gradient elution from 5% to 95% was performed for 15 minutes. The chromatographic conditions were the same as in Figure 2.

[0084] Therefore, equimolar loading is not the optimal scenario, and the amount of Asp relative to Lys needs to be reduced. This modification reduces side reactions, ensuring that Asp is consumed, increasing the conversion yield, and improving purity.

[0085] Next, the amount of Asp was reduced to 33% Asp and 67% Lys, and this mixture was supported on the resin. This ratio appears to be the optimal amount of reactive species. However, the amount of the final peptide product relative to the remaining Asp was higher than approximately 95%.

[0086] Figure 6 shows the chromatogram. The black line represents the product after loading with 34% Asp and 67% Lys, followed by the incorporation of linear Fmoc-Lys-Gly-OH and Fmoc-Asp-Gly-OH on CTC resin. The pink line represents the product after reacting Asp and Lys at room temperature for 22 hours (only 5% of Asp remained at the second peak). Gradient elution from 5% to 95% was performed for 15 minutes. Refer to Figure 2 for chromatographic conditions.

[0087] Following this preliminary optimization procedure, the following report describes a resin with 33% Asp and 67% Lys supported.

[0088] 1.3 Cyclic Peptide Analogues Cyclic peptides have been shown to exhibit improved stability against enzymatic degradation due to their structural rigidity. Peptide cyclization is also commonly applied to stabilize other secondary structures such as α-helices and β-sheets. Furthermore, cyclic peptides are powerful molecules in terms of their efficiency in intracellular uptake and penetration. Given the increasing consideration of protein-protein interactions as therapeutic targets, cyclic peptides also offer ideal conditions for investigating the complex structures necessary to participate in such broad interactions.

[0089] After successfully synthesizing a linear analog and optimizing the procedure, we decided to extend the same two chains and use a different junction site. Therefore, we used Gly on both chains, and then extended the two chains using Asp(OAll) and Lys(Alloc) on each chain. Following the same procedure as described for the linear analog, we obtained a cyclic peptide (Scheme 9), and confirmed its mass.

[0090] FIG. 7 is a diagram showing the chromatogram of cyclic Fmoc-KD-G-KD-GL-OH peptide (t R = 18.2 min). Please refer to FIG. 2 for chromatography conditions.

[0091] FIG. 8 is a diagram showing the mass of the cyclic peptide. The calculated value is 940.50, and the actually measured value is 471.58 [M+2 2+ .

[0092] (Scheme 9) TIFF2026531690000014.tif122170 Scheme 9. Schematic diagram of cyclic peptide synthesis after interchain organization reaction. Linked from two junction positions

[0093] To further verify the robustness of this method, another cyclic peptide analog was prepared using a different solid support. While the previous example used CTC resin, this new synthesis explored Rink amide resin. The cyclic peptide shown in Scheme 10 was prepared, and its mass was confirmed by mass spectrometry.

[0094] FIG. 9 is a diagram showing the chromatogram of cyclic Fmoc-DK-G-DK-NH2 peptide (t R = 10.2 min). Gradient elution of 5-95% was carried out for 15 minutes. Please refer to FIG. 2 for chromatography conditions.

[0095] FIG. 10 is a diagram showing the mass spectrum of the cyclic peptide. The calculated value is 1156.50, and the actually measured value is 1157.95 [M+1 + , 579.81 [M+2 2+ .

[0096] (Scheme 10) TIFF2026531690000015.tif88170 Scheme 10. Chemical structure of cyclic peptide on Rink amide resin

[0097] 1.4 Bicyclic peptides By linking two peptide chains at three or more different linkage sites, a variety of molecular structures can be synthesized. This new synthesis protocol was studied for the preparation of bicyclic peptides, which are difficult to prepare and require solution-based reaction steps. The inventors were able to prepare a bicyclic peptide simply by linking two peptide chains at three sites (Scheme 11). The mass of this peptide was also confirmed by mass spectrometry.

[0098] Figure 11 shows the chromatogram of the bicyclic H-DK-LAF-DK-G-DK-NH2 peptide (t R This figure shows the result (10.6 minutes). Gradient elution was performed from 5% to 95% for 15 minutes. Please refer to Figure 2 for chromatography conditions.

[0099] Figure 12 shows the mass of the bicyclic peptide. The calculated value is 2044.97, and the measured value is 1021.40 [M+2]. 2+ That was the case.

[0100] (Scheme 11) TIFF2026531690000016.tif130170 Scheme 11. Chemical structure of bicyclic peptides on linkamide resin

[0101] 1.5 Highly constrained analogues Peptides with fewer than 7 amino acids are extremely difficult to cyclize, and intrachain interactions such as head-tail type in solution or head-side chain type on resins are particularly challenging [Reference 11]. This difficulty stems from the significant steric hindrance of such structures, which prevents them from being folded into the desired cyclic shape. Attempts to cyclize short peptides often yield products based on dimerization, trimerization, or C-terminal epimerization.

[0102] This new approach demonstrated the ability to generate extremely difficult-to-manage cyclic peptides (Scheme 12).

[0103] (Scheme 12) TIFF2026531690000017.tif75170 Scheme 12. Chemical structure of cyclic tetrapeptides on linkamide resins

[0104] We successfully synthesized this new cyclic peptide and confirmed its mass using mass spectrometry.

[0105] Figure 13 shows the chromatogram of the cyclic Fmoc-DK-DK-NH2 peptide (t R This figure shows the result (7.1 minutes). Gradient elution from 5 to 95% was performed for 15 minutes.

[0106] Figure 14 shows the mass spectrum of the cyclic tetrapeptide. The calculated value was 931.01, and the measured value was 932.75[M+1]+.

[0107] Furthermore, in such a highly constrained structure, achieving complete conversion required a long reaction time, namely 48 hours instead of 24 hours.

[0108] The inventors have noted that this new method can also be applied to unnatural amino acids with short side chains, such as Orn amino acids. Orn has one fewer CH2 group than Lys. Nevertheless, the inventors successfully synthesized a final peptide by reacting an Orn-containing peptide with an Asp-containing amino acid (data not shown).

[0109] 1.6 Partially modified inversopeptides (PMI) and / or partially modified retroinversopeptides (PMRI) The concept of retroinverso (RI) was published by Professor Goodman in 1970. This method has been adopted in various fields, including immunology, diagnostics, and drug discovery [Reference 12]. Partially modified inversopeptides (PMI) and / or partially modified retroinversopeptides (PMRI) are expected to maximize the lifespan of bioactive peptides, which is one of the serious existing problems in peptide production processes [Reference 13]. Current methods for synthesizing PMI peptides require additional reaction steps that affect the purity of the final product. Furthermore, some amino acids have shown instability with the chemicals used in current synthesis processes. This new method does not require the addition of reagents other than those already considered in the conventional SPPS method. As a proof-of-concept experiment, peptides with the amide bond in the opposite direction were synthesized. Such new peptides are called inversopeptides. However, by incorporating amino acids into other stereochemicals using this method, it should be possible to easily create the desired PMRI peptide as needed.

[0110] PMI synthesis began with a short pentapeptide (H-YGFGL-NH2) immobilized on sievertamide resin. Figure 15 shows the mass spectrum of the YGFGL-NH2 pentapeptide. The calculated value is 554.29, and the measured value is 555.77 [M+1]. + The N-terminus was modified with succinic anhydride, with approximately 50% being the modified portion. Half an equivalent of succinic anhydride was used relative to the scale of the synthesis. The modified chain contained a carboxylic acid at the N-terminus, which enabled chemical reactions with the unmodified N-terminus, which had amino functionalities (Scheme 13).

[0111] (Scheme 13) TIFF2026531690000018.tif118170 Scheme 13. Schematic diagram of PMI peptide synthesis after interchain organization reaction.

[0112] The reaction products were analyzed by high-performance liquid chromatography (HPLC) (Figure 16), and the masses of the intermediate (4-oxobutanoic acid modified peptide) and PMI were confirmed (Figures S15 and S16).

[0113] Figure 16 shows the HPLC chromatogram, where the black line represents the H-YGFGL-NH2 product (t R =6.4 min), the pink line represents succinate-YGFGL-NH2 product (t R =7.1 min), the green line represents the H-YGFGL-Succ-YGFGL-NH2 product (t R (=8.4 minutes) was shown. Gradient elution from 5 to 95% was performed for 15 minutes. Mobile phase A was 0.1% TFA in H2O, and mobile phase B was 0.1% TFA in CH3CN, and Symmetry Luna C 18 A column (3.6 μm, 4.6 × 150 mm) was used.

[0114] Figure 17 shows the mass spectrum of the 4-oxobutane-YGFGL-NH2 modified pentapeptide. The calculated value is 654.30, and the measured value is 655.66 [M+1]. + , 1310.09[M+2] 2+ That was the case.

[0115] Figure 18 shows the mass spectrum of the H-YGFGL-NH-succinate-YGFGL-NH2 decapeptide. The calculated value is 1190.58, and the measured value is 1192.00 [M+1]. + , 596.79 [M+2] 2 That was the case.

[0116] The optimal stoichiometry for succinic anhydride versus peptide was found to be 1:2. Equimolar peptide precursors are generated, which are then completely consumed in the subsequent interchain organization reaction to form the final peptide product. The reaction stoichiometry was confirmed by HPLC as H-YGFGL-NH2-succinate-YGFGL-NH2 (0.9:1.1), which is likely due to measurement errors of the amino acids. This value indicates a 9% surplus of succinate-YGFGL-NH2. Therefore, this surplus was observed as unreacted species in the final product mixture. The decapeptide is synthesized in just two steps: the first step is N-terminal modification, and the second step is interchain organization.

[0117] Assuming a 1:1 stoichiometric ratio, fragments (27-36) of enfuvirtide (T-20 or Fuzeon) were also selected for the basoformation of the PMI analog (Scheme 14). Enfuvirtide is a 36-mer membrane fusion inhibitor for the treatment of HIV and was approved by the U.S. Food and Drug Administration (US-FDA) in 2003 [Reference 14].

[0118] (Scheme 14) TIFF2026531690000019.tif123170 Scheme 14. Chemical structure of two fragments (27-36) of enfubiltide (T-20 or fusion) linked via succinamide on a sieberamide resin.

[0119] Preparing a 20-amino acid peptide using conventional methods is difficult in terms of time, purity, and yield. In contrast, the present invention successfully synthesized a 20-amino acid peptide in just two steps, and with high purity. This purity is equivalent to that of the starting peptide chain precursor (Figure 19). The mass of the synthesized peptide was also confirmed.

[0120] Figure 19 shows a chromatogram, where the black line represents one fragment (27-36) of enfubiltide (T-20 or fusion) (T-20 or fusion) (t R =13.5 minutes), the pink line is succinate-T-20(t R =15.1 min), the green line represents two fragments of the same peptide (27-36) (t R This shows the result (=18.4 minutes). Gradient elution was performed at 15-70% for 15 minutes. See Figure 2 for chromatography conditions.

[0121] Figure 20 shows the PMI mass spectrum of the T-20 fragment. The calculated value is 2785.28, and the measured value is 1393.42 [M+2]. 2+ , 929.33[M+3] 3+ That was the case.

[0122] To demonstrate the efficiency of the present invention, a 70-amino acid peptide based on a 30-mer peptide called TD2.2 was prepared using an automated synthesis apparatus (Scheme 15). TD2.2 has been reported to be a specific blood-brain barrier shuttle peptide, and it has been shown to target oligodendrocytes and not non-glial cells such as human neurons and human dermal fibroblasts [References 15, 16]. The N-terminus was modified with succinic anhydride using the same procedure as for the T-20 peptide.

[0123] (Scheme 15) TIFF2026531690000020.tif193170 Scheme 15. Chemical structure of Aib-modified TD2.2 peptide linked via succinamide on protide resin. Another representation of the modified peptide is shown below.

[0124] TIFF2026531690000021.tif31170

[0125] This peptide was successfully synthesized with a purity comparable to that of the starting precursor (Figure 21), and its mass was also confirmed.

[0126] Figure 21 shows the chromatograms of two fragments of TD2.2-succinamide-TD2.2 (t R This figure shows the result (8.3 minutes). Please refer to the legend in Figure 2 for the chromatography conditions.

[0127] Figure 22 shows the PMI mass spectrum of the TD2.2 fragment. The calculated value is 7366.64, and the measured value is 1047.83 [M+7]. 7+- , 524.54 [M+14] 14+ That was the case.

[0128] The coupling of α-aminoisobutyric acid (Aib) amino acids is always a difficult reaction [Reference 17], and when peptides contain multiple Aib residues, automated synthesizers are considered a superior alternative [Reference 18]. Therefore, they are often used to demonstrate the quality of new coupling methods, such as when the main procedure is modified by introducing new solvents or coupling reagents. This fact is also depicted in Scheme 15, and the difficulty is evident from the purity of the Aib-modified peptide containing four Aib residues in its sequence. This peptide contains four Aib residues in its sequence and was synthesized using a microwave-assisted automated synthesizer.

[0129] However, the present invention made it possible to prepare a PMI60-mer peptide containing eight Aib residues in just two reaction steps. This is considered a significant advance in the field of SPPS for two reasons. First, conventional manual SPPS methods cannot produce 60-mer peptides using conventional methods. Therefore, convergent approaches rather than stepwise approaches are considered [Reference 16]. However, this means that further steps are required, and in some cases, hybrid approaches (of SPPS and LPPS) need to be considered. However, this incurs additional costs and affects the final yield. Second, as mentioned above, coupling Aib amino acids is extremely difficult.

[0130] This advancement has made it possible to easily synthesize miniproteins. These peptides were obtained in high purity.

[0131] 1.7 Microcyclic peptides Using this new reaction method, a microcyclic peptide was also prepared. To the best of our knowledge, this analog, containing two amino acids, is the shortest cyclic peptide synthesized to date. Furthermore, it is impossible to synthesize such a short cyclic analog using the conventional SPPS method. The amino acids Asp and Lys were incorporated into a PAM resin, and their side chains were linked. Next, one of the peptide chains was reacted with succinic anhydride to modify its N-terminus and convert it to a carboxylic acid (as described in the PMI section). Subsequently, the modified N-terminus was linked to the unmodified one to form the cyclic product shown in Scheme 16.

[0132] (Scheme 16) TIFF2026531690000022.tif65170 Scheme 16. Chemical structure of a cyclic peptide formed from two new amino acids.

[0133] The mass of this cyclic peptide was determined by mass spectrometry.

[0134] Figure 23 shows the PMI mass spectrum of a modified self-assembly peptide. The calculated value is 343.14, and the measured value is 325.39 [M-18]. + That was the case.

[0135] 1.8 Current Industrial Peptide Congeners In this invention, we have also studied the organization of current industrial peptides. This research represents progress in two main areas. First, we organized two peptide chains with completely different amino acid compositions. Thus, by using an orthogonal approach of tert-butyloxycarbonyl (Boc) chemistry and fluorenylmethyloxycarbonyl (Fmoc) chemistry, we organized both chains independently. Since the protecting group of the Boc-amino acid side chain is stable to trifluoroacetic acid (TFA), which is required to remove Boc in the peptide elongation process, we organized the Boc chain before the Fmoc chain. Second, the interchain organizing reaction requires connecting two distantly located residues, namely the N-terminus and the C-terminus. To further investigate the applicability of the new method, we selected liraglutide in this study. Liraglutide is an analog of human glucagon-like peptide (GLP-1) and acts as a GLP-1 receptor agonist. Liraglutide has 31 amino acids (HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG). This peptide was split into two fragments, and both fragments were organized as a Boc chain and an Fmoc chain on a single resin (Scheme 17).

[0136] (Scheme 17) TIFF2026531690000023.tif69170 Scheme 17. Chemical structure of the designed liraglutide fragment

[0137] In designing the second chain (Fmoc chain) fragment, we considered having amino acids with an active side chain (Glu). Therefore, the side chain can be immobilized on the resin, and after the entire chain is constructed, the C-terminus can be easily used to ligate to the N-terminus of the Boc chain. This yields a typical linear peptide.

[0138] After organizing both chains independently, an interchain organizing reaction was carried out. The desired product was obtained as the main product with satisfactory purity (Figure 24).

[0139] In the alternative method, instead of completely organizing the first and second chains before coupling, the first amino acid (Glu) of the second chain is incorporated into the resin in the usual form via the side-chain carboxyl group using Fmoc chemistry. The first amino acid (Gly) of the first sequence is incorporated into the resin using Boc chemistry. The first chain is constructed (again using Boc chemistry), the C-terminus of the second chain is deprotected, and it is coupled to the N-terminus of the first chain. The residues of the second chain that subsequently form are constructed using the Fmoc reaction.

[0140] Figure 24 shows the chromatogram of liraglutide (t R This figure shows the result (=9.9 minutes). Please refer to the legend in Figure 2 for the chromatography conditions.

[0141] The mass of the peptide was confirmed by mass spectrometry (Figure 25).

[0142] Figure 25 shows the mass spectrum of liraglutide peptide. The calculated value is 3383.73, and the measured value is 1128.7 [M+3H]. 3+ , 846.8 [M+4H] 4+ That was the case.

[0143] This discovery confirms that this new technology can link even distant residues through interchain organization reactions. Therefore, this technology can supply currently available commercially available peptides with satisfactory purity and without the presence of undesirable by-products.

[0144] Condensing three short fragments yields a purer product than that obtained from two long fragments. Therefore, liraglutide is organized by splitting the peptide into three chains using three orthogonal protecting groups: Boc, Fmoc, and p-nitrobenzyloxycarbonyl (pNZ). The three designed liraglutide fragments are shown in Figure 26.

[0145] (Scheme 18) TIFF2026531690000024.tif100170 Scheme 18. Chemical structures of the three designed liraglutide fragments.

[0146] The first two chains were organized independently as previously shown. A temporary protecting group of p-nitrobenzyloxycarbonyl (pNZ) was immobilized on the resin to reserve space for the third chain. This protecting group can be removed if necessary. An interchain organization reaction was performed to link the first two chains (Boc and Fmoc). We successfully obtained the H-GQAAKEFIAWLVRGRG-OH16-mer fragment peptide with satisfactory purity (Figure 26).

[0147] Figure 26 shows the chromatogram of the H-GQAAKEFIAWLVRGRG-OH fragment (t R (7.5 minutes). For chromatography conditions, please refer to the legend in Figure 2.

[0148] Next, the pNZ group is removed under reducing conditions, and the third chain is organized according to the Fmoc procedure. Subsequently, an interchain organizing reaction is performed to obtain the final liraglutide. Furthermore, the product obtained by linking the first two chains is about twice the size of the fragments used in the current manufacturing process, and is of comparable quality. Therefore, it functions as a fragment for the subsequent condensation process.

[0149] This discovery confirms that the inventors' new technology allows for the linking of distantly separated residues through a novel interchain organizing reaction. Therefore, this technology can supply current industrial peptides with satisfactory purity and without undesirable by-products. Furthermore, this technology demonstrates the ability to organize three different peptide chains using three orthogonal protecting groups.

[0150] 2.9 Crosslinked Peptides After confirming the ability to organize three different peptide chains on a single bead, a cross-linked peptide was synthesized using this new technique. The peptide chain organization was carefully designed to maintain orthogonality during synthesis. The first and second chains were linked using the active side chains of amino acids (Lys and Glu) with reactive side chains, and their N-terminuses, one of which was modified with succinic anhydride. Furthermore, a third linkage was organized between the second and third chains using the active side chains of another Lys residue and Glu residue. The chemical structure of this novel peptide is shown in Scheme 19.

[0151] (Scheme 19) TIFF2026531690000025.tif118170 Scheme 19. Chemical structure of a novel crosslinked peptide. Green: formed peptide bond

[0152] The peptide was obtained with sufficient purity (Figure 27), and its mass was confirmed by mass spectrometry (Figure 28).

[0153] Figure 27 shows the chromatogram of the cross-linked peptide (tR = 9.4 min). Gradient elution from 0 to 50% was performed for 15 minutes. Please refer to the legend in Figure 2 for chromatographic conditions.

[0154] Figure 28 shows the mass of the cross-linked peptide. The calculated value is 1140.26, and the measured value is 1140.86 [M+H]. + and 571.23[M+2H] 2+ That was the case.

[0155] Such structures are expected to possess excellent conformational rigidity, metabolic stability, and efficacy. Therefore, they can help avoid the major obstacle of enzymatic degradation and bring these useful molecules to market. This type of structure is groundbreaking (first-in-class) and has not been reported before. Furthermore, this novel peptide species mimics the concept of a double-stranded oligonucleotide containing a sense chain and an antisense chain. Similarly, such cross-linked peptides can be designed so that each chain has its own independent functionality, thereby enabling the realization of synergistic, multifunctional peptides. An interesting advantage of these new analogues is the ability to selectively modify chains without impairing the function of other chains. This novel structure does not require a self-assembly sequence and could also be used for molecular scaffolding (e.g., gelation). Organizing the three chains facilitates the synthesis of triple helix structures (e.g., synthetic cross-linked collagen).

[0156] 2. Conclusion This invention demonstrates a novel reaction method that extends the conventional SPPS method to a new peptide synthesis reaction space. By simultaneously organizing different peptide chains on a solid phase, rather than the same single species, and combining this with a solid-phase organizing reaction, novel and potentially important peptide homologs can be obtained. Obtaining these new homologs is difficult, and in some cases, impossible using the conventional SPPS method. For example, the inventors have succeeded in synthesizing highly constrained cyclic tetrapeptides and dipeptides, which were impossible with the conventional SPPS method.

[0157] Furthermore, the inventors demonstrated the successful synthesis of an important type of peptide known as partially modified inversopeptides (PMIs). High-purity PMIs were synthesized in just two steps, without the use of aggressive chemicals known to attack amino acids. Interestingly, this innovative idea demonstrated the ability to produce 60-mer peptides with high efficiency and purity. Long peptides are well known to be difficult to synthesize due to various side reactions that affect the final purity and yield of the target peptide.

[0158] Interestingly, the inventors demonstrated the ability of a novel method to synthesize fragments necessary for preparing commercially available therapeutic peptides with satisfactory purity.

[0159] We synthesized a new species called a bridge by organizing three different chains and linking them from three different positions, including the side chains and the N-terminus. Such a species appears to be unprecedented.

[0160] 3. Materials and Methods PuroSynth CTC (1.0 mmol / g, supplier specifications) and PuroSynth Rink Amide (0.5 mmol / g, supplier specifications) resins were used for all synthesis. All reagents and solvents were obtained from suppliers and used without further purification unless otherwise specified. A Shimadzu LC20 system was used for analytical HPLC, and Lab Solution software was used for data processing. A Symmetry Luna C18 (3.6 μm, 4.6 × 150 mm) column was used at a 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 spectrometry was performed using a Velos Pro mass spectrometer (ThermoFisher Scientific), a hybrid linear trap quadrupole (LTQ)-orbitrap, with positive electrospray ionization mass spectrometry (ESI+-MS) by direct injection of the sample. The Liberty Blue™ automated microwave-assisted peptide synthesizer (CEM) was used.

[0161] 3.1 Installation Procedure 3.2 CTC resin The first amino acid was incorporated into the CTC resin using dry CH2Cl2. The CTC resin was swollen in CH2Cl2 for 10-20 minutes. Fmoc-amino acid (2 equivalents) was dissolved in the minimum amount of CH2Cl2 (0.5 mL / 100 mg resin) and sonicated for 10 minutes. Next, N,N-diisopropylethylamine (DIEA) (4 equivalents) was added to the solution and added to the previously swollen resin, and reacted under mechanical shaking for 1 hour. After this, MeOH (80 μL / 100 mg resin) was added to end-cap the unreacted chlorides of the CTC resin. Finally, the resin was washed twice with CH2Cl2 and vacuum-dried.

[0162] 3.3 Link amide resins and sieber amide resins The first amino acid was incorporated into the linkamide resin using dry DMF. The linkamide resin was swollen in DMF for 10-20 minutes. Fmoc was removed using 20% ​​piperidine / DMF, and the mixture was shaken for 2 minutes and 7 minutes. Fmoc-amino acid (3 equivalents) and Oxymapure (3 equivalents) were dissolved in a minimum amount of DMF (0.5 mL / 100 mg resin) and sonicated for 10 minutes. Next, N,N-diisopropylcarbodiimide (DIC) (3 equivalents) was added to the solution, which was then added to the previously swollen resin, and reacted under mechanical shaking for 1 hour. Finally, the resin was washed twice with CH2Cl2 and vacuum dried.

[0163] 3.4 PAM resin The first amino acid was incorporated into the PAM resin using dried CH2Cl2. The CTC resin was swollen in CH2Cl2 for 10-20 minutes. Next, 4-dimethylaminopyridine (DMAP) (0.25 equivalents) was added to the resin. Fmoc-amino acid (2.5 equivalents) was dissolved in the minimum amount of CH2Cl2 (0.5 mL / 100 mg resin) and sonicated for 10 minutes. Next, DIC (5 equivalents) was added to the solution and this was added to the previously swollen resin, and the reaction was carried out under mechanical shaking for 1 hour. Then, acetic anhydride and DIEA (10:20) in DMF were added to end-cap the unreacted OH groups of the PAM resin. Finally, the resin was washed twice with CH2Cl2 and vacuum-dried.

[0164] 3.5 Incorporation of reactive amino acids (Asp and Lys) When coupling Asp and Lys to a resin as key amino acids for subsequent interchain organization reactions, a ratio of 33% Asp and 67% Lys is considered the optimal ratio to ensure complete conversion. These amino acids were incorporated twice to ensure complete integration into the peptide chain.

[0165] 3.6 Peptide Synthesis Peptide synthesis was carried out according to standard methods used in the inventors' laboratory. (3 equivalents of Fmoc-AA-OH, 3 equivalents of Oxymapure, and 3 equivalents of DIC) or (2 equivalents of Fmoc-AA-OH, 1.9 equivalents of (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), and 4 equivalents of DIEA) were dissolved in DMF and shaken for 1 hour. Fmoc was then removed (see the section on Linkamide Resin). To ensure complete coupling, all Arg and the subsequent residue were double-coupled.

[0166] 3.7 Cleavage of the allyl protecting group To deprotect Asp(OAll) or Lys(Alloc), tetrakis(triphenylphosphine)palladium (0) (0.1 equivalents) and triphenylsilane (10 equivalents) in CH2Cl2 were added to the peptidyl resin and reacted for 1 hour under mechanical shaking. This process was repeated twice. The resin was washed twice with CH2Cl2, and then three times with N,N-diethylthiocarbamate (0.02 M in DMF) to wash away Pd from the resin.

[0167] 3.8 Cleavage of Boc, Trt, and tBu protecting groups To cleave the Boc, Trt, and tBu protecting groups, 50% TFA in CH2Cl2 was added to the peptidyl resin and reacted under mechanical shaking for 30 minutes. The resin was washed twice with CH2Cl2, then treated with 10% DIEA in CH2Cl2 and reacted under mechanical shaking for 30 minutes to neutralize the protonated groups by acid treatment. Finally, the resin was washed twice with CH2Cl2 and vacuum dried.

[0168] 3.9 Interchain organizing reaction Once the reactive amino acids were ready to form amide bonds, (3 equivalents of Fmoc-AA-OH, 3 equivalents of Oxymapure, and 3 equivalents of DIC) or (2 equivalents of Fmoc-AA-OH, 1.9 equivalents of (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), and 4 equivalents of DIEA) were dissolved in DMF and shaken for 24 hours.

[0169] 3.10 Automated Synthesis In the automated SPPS method, amino acid coupling to the grown peptide chain was achieved by adding Fmoc-AA-OH acid (0.25 mmol, 5 equivalents, 0.2 M in DMF), (Oxymapure, 0.25 mmol, 5 equivalents, 0.5 M in DMF), and DIC (0.50 mmol, 10 equivalents, 0.5 M in DMF) and heating at 90°C for 2 minutes (single coupling) or 2 × 2 minutes (double coupling). N-terminal deprotection of the peptide chain grown by Fmoc cleavage was performed by adding piperidine (20% v / v in DMF) and Oxymapure (0.1 M in DMF) and heating at 90°C for 1.5 minutes.

[0170] 3.11 Disconnection Protocol Linkamide resin and CTC resin The final synthesized 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 hour. Next, cooled diethyl ether (5 times the volume of the cleavage solution) was added, and this solution was allowed to stand in an ice bath for 30 minutes. Then, this solution was centrifuged at 5000 rpm for 5 minutes, and the supernatant was carefully poured off. Another volume of ether (5 times the volume of the cleavage solution) was added, and this process was repeated. The 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 an HPLC system to confirm the purity of the final product.

[0171] PAM resin The final synthesized peptide was cleaved from the resin using triflic acid / TFA / TIS (8:3:1) (1 mL / 100 mg) while mechanically shaking at 0°C for 1 hour. Next, cooled diethyl ether (5 times the volume of the cleavage solution) was added, and this solution was allowed to stand in an ice bath for 30 minutes. Then, this solution was centrifuged at 5000 rpm for 5 minutes, and the supernatant was carefully poured off. Another volume of ether (5 times the volume of the cleavage solution) was added, and this process was repeated. The 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 an HPLC system to confirm the purity of the final product.

[0172] References 1. Merrifield, RB. "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide." J. Am. Chem. Soc. 1963, 85: p. 2149-2154. 10.1021 / ja00897a025 2. Zalipsky, S., JL 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, AR and KD Janda. “Solid-Phase Organic Synthesis: A Critical 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. 2010, 16: p. 675-8. 10.1002 / psc.1282 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 6. Jaradat, DMM, "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 8. Al Musaimi, O., BG de la Torre, and F. Albericio. "Greening Fmoc / tBu solid-phase peptide synthesis." Green Chem. 2020, 22: p. 996-1018. 10.1039 / c9gc03982a 9. Jaradat, DMM, 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 10. Lam, KS, SE Salmon, EM Hersh, VJ Hruby, WM Kazmierski, and RJ Knapp. "A new type of synthetic peptide library for identifying ligand-binding activity." Nature 1991, 354,(6348): p. 82-84. 10.1038 / 354082a0 11. White, CJ and AK Yudin. "Contemporary strategies for peptide macrocyclization." Nat. Chem. 2011, 3,(7): p. 509-524. 10.1038 / nchem.1062 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, PV, S. Richman, RS 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 14. Bray, BL. "Large-scale manufacture of peptide therapeutics by chemical synthesis." Nat. Rev. Drug Discov. 2003, 2,(7): p. 587-93. 10.1038 / nrd1133 15. Heffernan, C., H. Sumer, GJ Guillemin, U. Manuelpillai, and PJ 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 16. Al Musaimi, O., SV Morse, L. Lombardi, S. Serban, A. Basso, and DR 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 17. Frerot, E., J. Coste, A. Pantaloni, MN Dufour, and P. Jouin. "PyBOP and PyBroP: Two reagents for the difficult coupling of the α,α-dialkyl amino acid (Aib)." Tetrahedron 1991, 47: p. 259-270. 10.1016 / S0040-4020(01)80922-4 18. Zieleniewski, F., DN 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 19. Al Shaer, D., O. Al Musaimi, F. Albericio, and BG de la Torre. "2021 FDA TIDES (Peptides and Oligonucleotides) Harvest." Pharmaceuticals 2022, 15,(2): p. 222. 10.3390 / ph15020222 All of the above references are incorporated herein by reference.

Claims

1. a) i. A first peptide fragment having a free carboxyl group, ii. A second peptide fragment having a free amino group and The process of preparing a resin to which the compound is added, b) A step of coupling the free carboxyl group of the first peptide fragment with the free amino group of the second peptide fragment to form an amide group that connects the first peptide fragment and the second peptide fragment. A process for preparing peptides, including [the specified element].

2. The process according to claim 1, wherein the free carboxyl group of the first peptide fragment is present in the side chain of an amino acid residue.

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

4. The process according to any of the preceding claims, wherein the free amino group of the second peptide fragment is present in the side chain of an amino acid residue.

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

6. The process according to any of the preceding claims, wherein the first peptide fragment is protected at its N-terminus by a first protecting group, and the second peptide fragment is protected at its N-terminus by a second protecting group, and the first protecting group and the second protecting group are orthogonal to each other.

7. The process according to claim 6, wherein the first protecting group and the second protecting group are selected from Boc and Fmoc.

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

9. The process according to any one of claims 6 to 8, wherein the second peptide fragment is organized on the resin using a stepwise peptide solid-phase synthesis protocol.

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

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

12. The process according to claim 11, wherein the first peptide fragment is bound to the resin via the side chain residues of the first peptide fragment.

13. The 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 at the N-terminus.

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

15. The process according to any of the preceding claims, wherein the first peptide fragment and / or the second peptide fragment is further constructed to include one or more further amino acid residues.

16. The process according to claim 15, wherein the first peptide fragment and the second peptide fragment are coupled to form a further link between the first peptide fragment and the second peptide fragment.

17. The process according to any of the preceding claims, further comprising the step of cleaving the linked first peptide fragment and the second peptide fragment from the resin.

18. The process according to claim 17, further comprising one or more steps of deprotecting the linked first peptide fragment and the second peptide fragment.

19. A process for preparing partially modified inversopeptides and / or partially modified retroinversopeptides, a) i. A first peptide fragment having a first free amino group, ii. A second peptide fragment having a second free amino group and The process of preparing a resin to which the compound is added, b) A step of reacting the resin from step a) with a divalent electrophile that can react with two amino groups to form a linkage between the first peptide and the second peptide. A process that includes this.

20. The process according to claim 19, wherein the divalent electrophile is selected from dicarboxylic acids or their anhydrides.

21. The process according to claim 20, wherein the divalent electrophile is succinic anhydride.

22. The process according to any one of claims 19 to 21, wherein the first free amino group and the second free amino group are the first peptide fragment and the N-terminus of the peptide fragment.

23. The process according to any one of claims 19 to 22, wherein the first peptide fragment and the second peptide fragment are identical.

24. The process according to any one of claims 19 to 23, further comprising the step of cleaving the linked first peptide fragment and the second peptide fragment from the resin.

25. A partially modified inversopeptide and / or partially modified retroinversopeptide obtained by the process described in any one of claims 19 to 24.

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

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

28. The process according to claim 27, wherein the molar ratio of lysine to aspartic acid is in the range of 3:1 to 3:2, preferably about 2:

1.

29. The process according to any one of claims 26 to 28, wherein the amino acid has a side chain protected, preferably protected with an allyloxycarbonyl (alloc) group.

30. A compound having the following formula, or a pharmaceutically acceptable salt or ester of said compound.

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