Method for modifying peptides immobilized on solid supports by traceless reductively cleavable linker molecules - Patent Application 20070123333

Abstract

The present invention relates to a method for modifying and purifying peptides, which includes an immobilization step, a modification step, and a release step. In the immobilization step, a linker-tagged crude peptide LP is coupled to a solid support to obtain an immobilized linker-tagged peptide SLP. The immobilized linker-tagged peptide SLP is then modified with one or more organic molecules to obtain an immobilized linker-tagged peptide SL-mP. Finally, the modified peptide is released via a reduced intermediate RI. The linker molecule is represented by XT of Formula 1: b -V a -UYZ(1), which can be coupled to a purification resin via moiety X and to a peptide via moiety Y upon release of leaving group Z. T is an optional spacer moiety, and V is an optional electron-withdrawing moiety. U is an aryl or 5- or 6-membered heteroaryl moiety bound to at least one electron-withdrawing moiety V, W, or E. The linker is stable under acidic conditions and releases the peptide upon addition of a reducing agent.

Description

[Technical field]

[0001] FIELD OF THEINVENTION Modified peptides are of increasing interest as pharmaceutical-related compounds. Modifications, such as fatty acids or bridging molecules to induce peptide macrocycles, enhance the pharmacokinetics of desired peptide sequences while preventing in vivo protease degradation. Thus, peptide modifications are an important feature in modern peptide therapeutics. Nevertheless, the introduction of these modifications is often difficult, making the handling and purification of modified peptides very difficult or impossible, especially since state-of-the-art purification methods have been developed and established for unmodified peptides. [Background technology]

[0002] 2. Background of the Invention Peptide production by solid phase peptide synthesis (SPPS) is a powerful and fast method to obtain many different peptides or large quantities of a single peptide in a predictable time frame. It is therefore widely used to produce many or large quantities of peptides. The procedure consists of repeated cycles of coupling a first N-terminal protected amino acid to a synthesis resin, followed by deprotection of this N-terminus, coupling of the next N-terminal protected amino acid, and capping of the unreacted peptide sequence. Finally, the synthesized peptide is cleaved from the synthesis resin and purified. These linear peptides may be further modified to improve their properties.

[0003] Linear unmodified peptides have low stability in vivo due to rapid degradation by proteases and peptidases. Moreover, they do not show fast renal clearance rate and cell permeability in mammals (Witt et al. (2001), Peptides (22), 2329-43). Two main modifications are used to improve peptide stability and efficacy in vivo: lipidation with fatty acids to form macrocycles and cyclization of linear peptides.

[0004] Lipidation is one of the most important modifications of peptide drugs. Fatty acids attached to peptides affect the hydrophobicity, secondary structure, and aggregation behavior of peptides while retaining specificity for protein targets. The fatty acids increase metabolic stability, membrane permeability, and bioavailability, and change the pharmacokinetic and pharmacodynamic properties of modified peptides (L. Zhang, G. Bulaj (2012) Current Medicinal Chemistry 19 (11), 1602-18). Examples of drugs include GLP-1 receptor agonists such as liraglutide (Victoza®), semaglutide (Ozempic®), and insulin derivatives such as detemir (Levemir®), and degludec (Tresiba®), all of which have fatty acids on the lysine side chain. The fatty acids of lipidated drugs can interact with and bind to albumin through three high-affinity binding domains. Bound albumin has a protective effect for these lipidated peptides in addition to different circulation times in vivo.

[0005] Large-scale methods for producing lipidated drugs currently rely on two main steps. First, a biological process in which precursor peptides are expressed by suitable cell lines, mainly yeast. Second, the precursor peptides are recovered and purified, followed by acylation with fatty acids or fatty acid spacer building blocks by chemical means in a higher pH solution, followed by a final purification step. In the early stages of drug development, solid-phase peptide synthesis is utilized to synthesize a large number of lipidated drug candidates. Acylation with lipids is primarily achieved by the use of orthogonal protecting group strategies. Traditionally, orthogonally protected lysine residues are incorporated into the peptide sequence during SPPS. This protecting group is selectively removed, and lipids are introduced by adding pre-activated or in-situ activated lipids to otherwise protected peptides immobilized on a synthetic resin. Alternatively, peptides may be lipidated in solution after production via chemical or biological means. Either selective acylation techniques need to be used or the N-terminus needs to be orthogonally protected and deprotected after lipidation is achieved.

[0006] Cyclic peptides have many advantages over linear peptides. Cyclic peptides are significantly more resistant to both exo- and endo-proteases. Cyclic peptides are also most likely to exhibit superior binding affinity to the desired protein target due to a more rigid and arched binding attraction to the target receptor. Successful cyclic peptide drugs are the anticancer drug octreotide (Sandostatin®), the immunosuppressant cyclosporine (Sandimmun®, Sandimmun®, Neoral®), or the neuropeptide hormone oxytocin (Syntocinon®, etc.). Methods for forming peptide rings can be divided into two main categories: a) peptides can be cyclized by utilizing endogenous functional groups, forming rings linked, for example, by amides or disulfides. Other covalent bonds can be formed by incorporating non-natural amino acids. b) Two-component systems, where functional groups of the peptide are used to react with the crosslinking scaffold. Organic molecules can be used, which are formed with monocyclic, bicyclic, or polycyclic peptides. The most common method is to take advantage of the nucleophilic functional groups of the peptide, especially thiols, and react it with a scaffold that has an electrophilic reactive site. When using SPPS, the peptide is either cyclized on the synthesis resin or cyclized after the peptide is separated from the synthesis resin in solution. Both methods have flaws. Modification on the solid support requires orthogonally protected amino acids, which are expensive and require an additional step to deprotect before modification. Once deprotected on the solid support, pseudo-ultra-high dilution helps prevent undesired reactions of one scaffold with two or more linear peptides. In addition, excess reagents can be used because of the ease of washing out. Cyclization in solution has notorious drawbacks for side reactions, such as crosslinking of two linear peptides by one scaffold, and excess reagents are laborious to remove. As a result, the modification is performed in highly dilute solution, whereby one equivalent of scaffold is used.

[0007] Many methods have been published for peptide modification in solution, and in the context of the present invention, only disclosures that use peptides bound to a solid support to enable modification of the peptide are discussed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2004 / 077062 [Patent Document 2] International Publication No. 2009 / 098450 [Non-patent literature]

[0009] [Non-Patent Document 1] Witt et al. (2001), Peptides (22), 2329-43 [Non-Patent Document 2] L. Zhang, G. Bulaj (2012) Current Medicinal Chemistry 19(11), 1602-18 [Non-Patent Document 3] Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 [Non-Patent Document 4] Bellmann-Sickert et al. (2011), J Med Chem 54(8), 2658-67 [Non-Patent Document 5] Vila et al. (2013), Org Biomol Chem 11, 3365-74 Summary of the Invention [Problem to be solved by the invention]

[0010] In WO 2004 / 077062, membranes are used as solid supports to synthesize many desired peptides as drug candidates. After standard SPPS of the polypeptide and removal of protecting groups, the peptides are cyclized on the support by adding a molecular scaffold, e.g. tris(bromomethyl)benzene. The cyclized peptides are then used to screen for binding to proteins. The generated peptides remain covalently attached to the synthetic support on which they were generated, making this method unusable for applications other than drug discovery. Therefore, it is ultimately not possible to analyze the peptides before binding tests. Therefore, the researcher must trust that every step taken results in the desired cyclic peptide, which can lead to false positive or even false negative results.

[0011] WO 2009 / 098450 and Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 disclose a method for modifying peptides synthesized by phages and displayed on the phage surface. In this way, a large peptide library with three cysteine ​​residues in the sequence was generated and the phage was treated with a solution of tris-(bromomethyl)benzene (TBMB) to cyclize the displayed peptides into bicycles. The phage was then incubated with a biotinylated target protein and subjected to a further round of selection. After the phage titer was increased, the DNA of the phages was sequenced to deduce the selected sequences. A huge number of bicyclic peptide variants (4.4 × 10 9 In addition to the drawbacks of the method for synthetic purposes, it cannot be used to generate peptides, which are only used for diagnostics. In the diagnostic context, it also has the drawback that the peptides subjected to binding experiments cannot be analyzed, which may lead to false positive or even false negative results.

[0012] Bellmann-Sickert et al. (2011), J Med Chem 54(8), 2658-67 and Vila et al. (2013), Org Biomol Chem 11, 3365-74 disclose a method for synthesizing peptides using orthogonal protection of lysine residues on a synthetic resin, followed by selective deprotection and subsequent conjugation of fatty acids to the protected peptides. However, this requires the use of expensive building blocks, and the crude peptides are obtained already modified with fatty acids, which usually makes them difficult to handle and purify due to their low solubility. [Means for solving the problem]

[0013] To overcome the drawbacks presented in the current state of the art, namely the lack of isolating and analyzing modified peptides and the lack of using standard amino acid building blocks without orthogonal protection, the present invention utilizes linker molecules that allow the immobilization of peptide precursors, followed by subsequent modification of the peptides while they are immobilized on a solid support for purification / modification. In contrast to other methods, particularly those that use modifications in solution, the present invention simultaneously removes excess organic molecules for perturbative peptide cleavage and modification. Thus, it is a method for modifying and simultaneously purifying desired peptides. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 is a schematic diagram of the peptide modification of the present invention on a solid support: SPPS = solid phase peptide synthesis, SR = SPPS synthesis resin, S = support for modification and purification, P = peptide requiring modification, L = reductively cleavable linker, Pg = protecting group, iPg = intermediate protecting group, B = reactive scaffold for forming cyclic peptides, mP = modified peptide, FA = fatty acid, Sp = spacer, A = activation of FA or FA-Sp, a) linker coupling, b) TFA cleavage, c) immobilization, d1) stapling modification, d2) fatty acid modification, d3) disulfide formation, e) release of modified peptide, i) deprotection of intermediate protecting group. [Diagram 2]FIG. 2 shows the synthesis of peptide P1 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:18) 3 shows examples of peptide modification / purification according to the invention. A=absorption (210 nm), B=time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before linker coupling to P1. 2.) Chromatogram of P1 after coupling of linker (X1) and final TFA cleavage. 3.) Chromatogram of P1 after modification by Palm-γGlu(OH)-OtBu coupling and purification (as described in claims 1-9). [Diagram 3] FIG. 3 shows the synthesis of peptide P2 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:2) Figure 1 shows examples of peptide modification / purification according to the invention. A = Absorption (210 nm), B = Time / min; 1.) Chromatograms of Trail cleavage of crude peptide sample before coupling of linker (X1) to P1. 2.) Chromatograms of P2 after modification by Palm-γGlu(OH)-OtBu coupling and purification (as described in claims 1-9). [Figure 4] FIG. 4 shows the synthesis of peptide P3 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:3) Figure 1 shows examples of peptide modification / purification according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker (X1) to P1. 2.) Chromatogram of P3 after modification by coupling of m-xylylene dibromide and purification as described in claims 1-5 and 10-12. [Diagram 5] FIG. 5 shows the synthesis of peptide P4 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:4) Figure 1 shows examples of peptide modification / purification according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker (X1) to P1. 2.) Chromatogram of P4 after modification by coupling of m-xylylene dibromide and purification as described in claims 1-5 and 10-12. [Figure 6] FIG. 6 shows the synthesis of peptide P5 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:5) Figure 1 shows examples of peptide modification / purification according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker (X1) to P1. 2.) Chromatogram of P5 after modification by coupling of m-xylylene dibromide and purification as described in claims 1-5 and 10-12. [Figure 7] FIG. 7 shows the synthesis of peptide P6 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:6) Figure 1 shows examples of peptide modification / purification according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker (X1) to P1. 2.) Chromatogram of P6 after modification by coupling of 1,3,5-tris(bromomethyl)benzene and purification as described in claims 1-5 and 10-12. [Figure 8] FIG. 8 shows the synthesis of peptide P7 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:7)Figure 1 shows examples of peptide modification / purification according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker (X1) to P1. 2.) Chromatogram of P7 after modification by coupling of 1,3,5-tris(bromomethyl)benzene and purification as described in claims 1-5 and 10-12. [Figure 9] FIG. 9 shows the synthesis of peptide P8 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:8) Figure 1 shows examples of peptide modification / purification according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker (X1) to P1. 2.) Chromatogram of P8 after modification by coupling of 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) and purification as described in claims 1-5 and 10-12. [Figure 10] FIG. 10 shows the synthesis of peptide P8 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:8) Figure 2 shows examples of peptide modification / purification according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatograms of trail cleavage of crude peptide sample before coupling of linker (X1) to P8. 2.) Chromatograms of P8 after modification with 1,3,5-triacryloyl-1,3,5-triazinane and purification (as described in claims 1-5 and 10-12). [Figure 11] FIG. 11 shows the synthesis of peptide P3 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:3)Figure 1 shows an example of the modification / purification of a peptide according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of a crude peptide sample before coupling of the linker (X1) to P3. 2.) Chromatogram of P3 after modification by air oxidation in DMSO / (NH4)2CO3 pH 8.7 and purification (as described in claims 1-5 and claim 13). [Figure 12] FIG. 12 shows the modified peptide P12a of the present invention by using 1-bromopropyl-2,3-dipalmitate (Pam2-Br). [ka] (SEQ ID NO:22) 1 shows the ESI-MS spectrum of A=intensity, B=m / z. [Figure 13] FIG. 13 shows the modified peptide P12b of the present invention by using 1-iodopropyl-2,3-dipalmitate (Pam2-I). [ka] (SEQ ID NO:22) 1 shows the ESI-MS spectrum of A=intensity, B=m / z. [Figure 14] Figure 14 shows the UV chromatogram of modified peptide P12d (H-KKKKSC(C2H4O-Palm)-NH2) (SEQ ID NO: 23) of the present invention using Palm-OC2H4I (Pam-Et-I). A = Absorption (210 nm), B = Time / min. [Figure 15] FIG. 15 shows the modified peptide P13a of the present invention by using 1-bromopropyl-2,3-dipalmitate (Pam2-OTs). [ka] (SEQ ID NO:24) 1 shows the ESI-MS spectrum of A=intensity, B=m / z. [Figure 16] FIG. 16 shows the modified peptide P13b of the present invention by using 1-iodopropyl-2,3-dipalmitate (Pam2-I). [ka] (SEQ ID NO:24) 1 shows the ESI-MS spectrum of A=intensity, B=m / z. [Figure 17] FIG. 17 shows the UV chromatogram of modified peptide P14d (H-KKKKSC(C2H4O-Palm)-NH2) (SEQ ID NO:23) of the present invention using Palm-OC2H4I (Pam-Et-I): retention time=2.25 min, A=absorbance (210 nm), B=time / min. [Figure 18] FIG. 18 shows the modified peptide P2 / c of ​​the present invention using 1-tosylpropyl-2,3-dipalmitate (X3). [ka] (SEQ ID NO:32) 1 shows the ESI-MS spectrum of A = intensity, B = m / z [Figure 19] FIG. 19 shows the modified peptide P14b of the present invention by using 1-iodopropyl-2,3-dipalmitate (Pam2-I). [ka] (SEQ ID NO:26) 1 shows the ESI-MS spectrum of A = intensity, B = m / z [Figure 20] Figure 20 shows the ESI-MS spectrum of the modified peptide P14c (HC(C3H5O2-diPalm)RG-NH2) (SEQ ID NO: 26) of the invention using 1-tosylpropyl-2,3-dipalmitate (Pam2-OTs). A=intensity, B=m / z [Figure 21] FIG. 21 shows the UV chromatogram of modified peptide P14d (HC(C2H4O-Palm)RG-NH2) (SEQ ID NO:27) of the present invention using Palm-OC2H4I (Pam-Et-I): retention time=1.61 min, A=absorbance (210 nm), B=time / min. [Figure 22]Figure 22 shows an example of the peptide modification / purification of the invention of peptide P2b (H-HAEGTFTSDVSSYLEGQAAK(Palm-Glu-OH)EFIAWLVRGRG-OH) (SEQ ID NO: 21) by the use of the linker molecule X1 of the invention. A = Absorption (210 nm), B = Time / min; 1.) Chromatogram after TFA cleavage of the crude peptide sample after linker coupling to P2b. 2.) Chromatogram of P2b after lipidation on solid support as described in example 8 using Palm-Glu(OSu)-OtBu as the conjugate of the -OSu activated fatty acid spacer. [Figure 23] FIG. 23 shows the synthesis of peptide P15 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:28) Figure 1 shows an example of peptide modification / purification of the present invention. A = Absorption (210 nm), B = Time / min; 1.) Chromatogram after TFA cleavage of crude peptide sample after linker coupling to P15. 2.) Chromatogram of P15 after lipidation on solid support as described in Example 9 using tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu as -OSu activated fatty acid spacer conjugate. [Figure 24] Figure 24 shows an example of the peptide modification / purification of the invention of peptide P16 (Palm-SGGK(Palm)WSK(Palm)SSVIGWPAVRER-NH2) (SEQ ID NO: 29) by using the linker molecule X1 of the invention. A = Absorption (210 nm), B = Time / min; 1.) Chromatogram of trail cleavage of crude peptide sample after coupling of linker to P16. 2.) Chromatogram of P16 after lipidation on solid support as described in example 10 using Palm-S as activating lipid. [Diagram 25]Figure 25 shows an example of peptide modification / purification of the invention for peptides P17a (Palm-SGGKWSK(Palm)SSVIGWPAVRER-NH2) (SEQ ID NO: 33), P17b (Myr-SGGKWSK(Myr)SSVIGWPAVRER-NH2) (SEQ ID NO: 34)) using the linker molecule X1 of the invention. A = Absorption (210 nm), B = Time / min; 1.) Chromatogram of trail cleavage of crude peptide sample after coupling of linker to P17. 2.) Chromatogram of P17a after lipidation on solid support as described in Example 10 using Palm-S as activating lipid with spacer. 2.) Chromatogram of P17a after lipidation on solid support as described in Example 11 using Myr-OSu as activating lipid. [Figure 26] FIG. 26 shows the synthesis of peptide P6 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:6)1 shows an example of peptide modification / purification of the present invention. A=absorption (210 nm), B=time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker to P6. 2.) Chromatogram of P6 after coupling of linker (LuS1) and TFA cleavage from SPPS resin. 3.) Chromatogram of P6 after intermolecular bicyclization and purification with TATB as described in Example 12 using aldehyde-modified agarose as solid phase and DMSO+10% by volume immobilization buffer 2 as peptide solvent for immobilization. 4.) Chromatogram of P6 after intermolecular bicyclization and purification with TATB as described in Example 13 using aldehyde-modified poly(methyl acrylate) beads as solid phase and pure HFIP as peptide solvent for immobilization. 5.) Chromatogram of P6 after intermolecular bicyclization and purification with TATB as described in Example 14 using aldehyde-modified agarose as the solid phase and 1 M SDS in DMSO+10% by volume immobilization buffer 2 as the peptide solvent for immobilization. 6.) Chromatogram of P6 after intermolecular bicyclization and purification with TATB at 1° C. as described in Example 16 using aldehyde-modified poly(methyl acrylate) beads as the solid phase and pure HFIP as the peptide solvent for immobilization. [Figure 27] FIG. 27 shows the synthesis of peptide P8 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:8)1 shows an example of peptide modification / purification of the present invention. A=absorption (210 nm), B=time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before coupling of linker to P8. 2.) Chromatogram of P8 after coupling of linker (LuS1) and TFA cleavage from SPPS resin. 3.) Chromatogram of P8 after intermolecular bicyclization and purification with TATB as described in Example 12 using aldehyde-modified agarose as solid phase and DMSO+10% by volume immobilization buffer 2 as peptide solvent for immobilization. 4.) Chromatogram of P8 after intermolecular bicyclization and purification with TATB as described in Example 13 using aldehyde-modified poly(methyl acrylate) beads as solid phase and pure HFIP as peptide solvent for immobilization. 5.) Chromatogram of P8 after intermolecular bicyclization with TATB and purification as described in Example 14 using aldehyde-modified agarose as the solid phase and 1 M SDS in DMSO + 10 vol.% immobilization buffer 2 as the peptide solvent for immobilization. 6.) Chromatogram of P8 after intermolecular bicyclization with TATB and purification at 1 °C as described in Example 16 using aldehyde-modified poly(methyl acrylate) beads as the solid phase and pure HFIP as the peptide solvent for immobilization. [Figure 28] FIG. 28 shows the synthesis of peptide P3 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:3) 1 shows an example of peptide modification / purification according to the invention. A=absorption (210 nm), B=time / min; 1.) Chromatogram of P3 after coupling of the linker (X1) and final TFA cleavage. 2.) Chromatogram of P3 after disulfide formation and purification as described in claims 1-9. [Figure 29] FIG. 29 shows the synthesis of peptide P4 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:4)1 shows an example of peptide modification / purification according to the invention. A=absorption (210 nm), B=time / min; 1.) Chromatogram of trail cleavage of crude peptide sample before linker coupling to P3. 2.) Chromatogram of P4 after coupling of linker (X1) and final TFA cleavage. 3.) Chromatogram of P4 after disulfide formation and purification as described in claims 1-9. [Diagram 30] FIG. 30 shows the synthesis of peptide P9 using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:9) 1 shows an example of peptide modification / purification according to the invention. A=absorption (210 nm), B=time / min; 1.) Chromatogram of P9 after coupling of the linker (X1) and final TFA cleavage. 2.) Chromatogram of P9 after disulfide formation and purification as described in claims 1-9. [Diagram 31] FIG. 31 shows the synthesis of peptide PY using the linker molecule X1 of the present invention. [ka] (SEQ ID NO:10) 1 shows an example of peptide modification / purification according to the invention. A=absorption (210 nm), B=time / min; 1.) Chromatogram of PY after coupling of the linker (X1) and final TFA cleavage. 2.) Chromatogram of PY after disulfide formation and purification as described in claims 1-9. [Diagram 32] Figure 32 shows an example of the inventive peptide modification / purification of peptide PZ (H-CCEYCCNPACTGCY-NH2) (SEQ ID NO: 11) by use of the inventive linker molecule X1. A = absorption (210 nm), B = time / min; 1.) Chromatogram of PZ after coupling of the linker (X1) and final TFA cleavage. 2.) Chromatogram of PZ after disulfide formation and purification as described in claims 1-9. [Diagram 33]Figure 33 shows the formation of lipidated peptide P. Peptide P is immobilized on a solid support (grey spheres) and contains a lysine. In a first step, the organic molecule Palm-Glu-OtBu is coupled to the side chain of the lysine. Palmitoyl (Palm) corresponds to the moiety FA and the glutamic acid moiety protected by tert-butyl (Glu-OtBu) corresponds to the spacer Sp-OH. In a second step, the protecting tert-butyl protecting group is removed under acidic conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] explanation A first aspect of the present invention relates to a method for modifying and purifying a peptide, said method comprising the following steps: - providing a linker-tagged crude peptide LP, wherein said crude peptide is covalently bound to a linker molecule L, - in an immobilization step, said linker-tagged peptide LP is coupled to a solid support to obtain an immobilized linker-tagged peptide SLP, - in a modification step, said immobilized linker-tagged peptide SLP is modified with one or more organic molecules to obtain an immobilized linker-tagged peptide SL-mP, - in a releasing step, a reducing agent is added to obtain a reduced intermediate RI, and then the reduced intermediate RI is decomposed to obtain a modified peptide mP, Including, The linker L is XT b -V a -UYZ(1), wherein X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular from the moieties of formula 2, 2a, 3 or 3a, more in particular from the moieties of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2are independently selected from H or B, where at least R 1 or R 2 is B, - R 3 is selected from H or B; - R 4 H, C1~C 12 alkyl or aryl, wherein the aldehyde or keto group is optionally protected with an acid labile protecting group; B is an acid labile amine protecting group, - T is a linear or branched spacer and b is 0 or 1, V is an electron-withdrawing moiety; a is 0 or 1, where the sum of a and b is 1 or 2; - U is a phenyl or 5- or 6-membered heteroaryl moiety which is a moiety V, W q and E n and optionally C 1~6 and optionally substituted with alkyl, where - W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] is selected from the group consisting of R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, q is 0, 1, 2, 3 or 4; E is an electron-withdrawing group under acidic conditions and n is 0, 1, 2, 3 or 4; - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, m is 1, 2 or 3; - Z is an electron-withdrawing leaving group.

[0016] In certain embodiments, the method for modifying and purifying a peptide comprises the following steps: - providing a linker-tagged crude peptide LP, wherein said crude peptide is covalently bound to a linker molecule L, - in an immobilization step, said linker-tagged peptide LP is coupled to a solid support to obtain an immobilized linker-tagged peptide SLP, - in a modification step, said immobilized linker-tagged peptide SLP is modified with one or more organic molecules to obtain an immobilized linker-tagged peptide SL-mP, - in a releasing step, a reducing agent is added to obtain a reduced intermediate RI, and then the reduced intermediate RI is decomposed to obtain a modified peptide mP, wherein the linker L is of formula 1, b -V a -UY-(1), wherein X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular from the moieties of formula 2, 2a, 3 or 3a, more in particular from the moieties of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, where at least R 1 or R 2 is B, - R 3 is selected from H or B; - R 4 H, C1~C 12 alkyl or aryl, wherein the aldehyde or keto group is optionally protected with an acid labile protecting group; B is an acid labile amine protecting group, - T is a linear or branched spacer and b is 0 or 1, V is an electron-withdrawing moiety; a is 0 or 1, where the sum of a and b is 1 or 2; - U is a phenyl or 5- or 6-membered heteroaryl moiety which is a moiety V, W q and E n and optionally C 1~6 and optionally substituted with alkyl, where - W is -N3, -NO2, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] is selected from the group consisting of R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, q is 0, 1, 2, 3 or 4, in particular q is 1, 2, 3 or 4, E is an electron-withdrawing group under acidic conditions and n is 0, 1, 2, 3 or 4; U is a phenyl moiety and Y is -(CH2) m When -OC(=O)-, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and - W is in the ortho or para position to Y, and Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, and m is 1, 2 or 3.

[0017] In certain embodiments, X is selected from a moiety of formula 2, 3, or 4, particularly selected from a moiety of formula 2 or 3, more particularly X is formula 2.

[0018] Peptides obtained by solid-phase peptide synthesis are usually purified after cleavage of the synthetic resin. Depending on the intended use, for example as a peptide drug or drug candidate, the peptide may be further modified. As described above, the present invention does not require the use of an orthogonal protection strategy for amino acid modification, and peptide modification and purification can be performed on the same resin. An overview of the present invention is shown in Figure 1.

[0019] In the context of the present invention, the term "amino acid" relates to a molecule comprising at least one amine moiety and at least one carboxyl moiety. In particular, when used in the context of peptides, the term "amino acid" relates to alpha-amino acids (also called α-amino acids or 2-amino acids) of the general formula H2NCHRCOOH (free amino acids) or -HNCHRCOO- (amino acids in peptides), i.e. the amine moiety and the carboxylic acid moiety are attached to the first (alpha) carbon atom. R denotes the side chain of the amino acid. The term "amino acid" relates to the D- or L-isomers of α-amino acids, in particular the L-isomers. In particular, moieties which are not α-amino acids but are nevertheless amino acids in the broad sense described above, are described herein with specific formulae.

[0020] After solid phase peptide synthesis on a synthesis support such as a synthesis resin, a crude peptide P is obtained. While still attached to the synthesis resin, the crude peptide may be coupled to a tracelessly cleavable linker molecule L of formula 1.

[0021] The linker molecule of formula 1 is suitable for purification of peptides after solid phase peptide synthesis (SPPS).

[0022] A common approach to purifying peptides after SPPS using a linker molecule is to couple the linker molecule to the N-terminus of the peptide in a final coupling step, in which the N-terminus of the peptide nucleophilically attacks the linker molecule to form a covalent carbamate or amide bond with the linker moiety Y while releasing the electron-withdrawing leaving group Z. Alternatively, the linker molecule can be coupled to the peptide using a suitable moiety of the peptide's amino acid side chain, such as -NH2 or -OH. This option can be used when the N-terminus is modified by the methods described herein. Suitable amino acids for coupling the linker to the amino acid side chain are lysine or serine.

[0023] The peptide linker construct is then cleaved from the synthesis resin by adding TFA, which, if moiety X contains an acid-labile amine protecting group B, simultaneously removes moiety B.

[0024] The linkers according to the invention are stable under acidic conditions when the peptide linker construct is cleaved, for example by using TFA.

[0025] The linker moiety X may be coupled to a functionalized solid phase, such as a resin used during purification. Moiety X may form a hydrazone or oxime bond by reacting with an appropriate moiety of the functionalized solid phase, such as an aldehyde, ketone, aminooxy, or hydrazine.

[0026] The moiety T represents a spacer that is unreactive under commonly applied purification conditions.

[0027] The moiety T is attached to the moiety U either directly or via the moiety V.

[0028] The moiety U contributes to the stability of the linker molecule under acidic conditions, particularly in the presence of TFA>50%, water, and pH<0. This is achieved by using either a heterocyclic or phenyl moiety attached to at least one of the electron-withdrawing moieties E, W, and V.

[0029] When an electron-withdrawing moiety (E, W, V) is attached to the phenyl moiety, the benzylic position of the linker molecule connecting it to the peptide becomes less electron dense and therefore less susceptible to acid-catalyzed decomposition. In order for the linker molecules of the invention to be sufficiently stable under acidic conditions, a certain threshold of electron withdrawal must be met. This threshold is determined by the Hammett constants (σ) of V, W, and E under acidic conditions. m and σ p ) greater than 0.45. Hammett constants are calculated according to Hansch and Taft (1991), Chem. Rev. 91:165-195. A positive Hammett constant indicates that the substituent has an electron-withdrawing effect on the phenyl moiety, whereas a negative value indicates that the substituent has an electron-donating effect.

[0030] Hammett constant is the meta position (σ m ) and para-position (σ p pKa is an empirically determined constant resulting in different acidities (pKa) of the substituents of the phenyl core of the benzoic acid derivative at position 100. In the context of the present invention, this position is determined relative to the attachment of the moiety Y. For substituents at the ortho position, the Hammett value at the para position is a good approximation and is therefore used in the context of the present invention to calculate the sum of the Hammett values ​​of the substituents V, W, and E.

[0031] It should be noted that the Hammett constants are calculated for the substituents V, W, and E under acidic conditions. For example, the amine moiety at neutral pH has a Hammett constant σ m =-0.16 and σ p =-0.66 and is therefore characterized as an electron-pushing substituent. Under acidic conditions, the amine moiety becomes protonated. For protonated amines, the Hammett constant is σ m = +0.86 and σp = +0.60, indicating that the protonated amine is an electron-withdrawing substituent, including aromatic amines that can withdraw electrons from U through a conjugated π system, either directly as a substituent on U or in their protonated form as a π-conjugate of U.

[0032] The threshold for the sum of the Hammett constants to be greater than 0.45 is the sum of the Hammett constants when U is a phenyl moiety and Y is -(CH2) m -OC(=O)-, which applies when m=1, because -OC(=O)- contains a good leaving group at the benzylic position, thus facilitating acid-catalyzed decomposition. Therefore, the electron density of the aromatic ring needs to be low to prevent stabilization of the cation at the benzylic position of Y.

[0033] The moiety Y is -(CH2) m In the case of -C(=O)-, this threshold is not necessary since -C(=O)- is not a good leaving group at the benzylic position.

[0034] For the stability of linker molecules containing heterocyclic moieties U under acidic conditions, no such extra threshold for selecting specific moieties V, W, and E is necessary. Since the heterocyclic moiety is itself more electron deficient than the phenyl moiety, any combination of V, W, and E is believed to be sufficient for the stability of the linker molecule under acidic conditions. In particular, when U is a nitrogen-containing heterocycle, the nitrogen is protonated when the peptide is released under acidic conditions, making the electron density of the aromatic system of U particularly low and therefore unable to stabilize the benzylic cation.

[0035] The moiety Y is either a -C(=O)- or -OC(=O)- moiety. When the linker molecule is coupled, an amide (-C(=O)-NH-) or carbamate (-OC(=O)-NH-) moiety is formed between the linker molecule and the N-terminus of the peptide. After purification, the peptide is released from the linker molecule, and hence the purification medium, under reductive conditions, either by elimination as in 1.4 or 1.6 or by nucleophilic attack. The reductive stimulus converts W to a reduced form, which then functions as an electron donating group and nucleophile, thus allowing release of the peptide.

[0036] After cleavage from the resin, the linker-tagged peptide LP is provided.

[0037] In certain embodiments, the linker-tagged peptide LP is provided by linking a crude peptide P, synthesized on a synthetic support SR, with a linker L to obtain a linker-tagged supported crude peptide LP-SR, followed by cleaving the linker-tagged peptide LP from the synthetic support SR to obtain the linker-tagged peptide LP.

[0038] In certain embodiments, the linker-tagged peptide LP is prepared by tethering a crude peptide P synthesized on a synthesis support SR to a free linker L f and releasing the electron-withdrawing leaving group Z to give the linker-tagged supported crude peptide LP-SR, where L f Linker XT b -V a -UYZ, where X, T, b, V, a, U and Y are as defined above and Z is an electron-withdrawing leaving group), followed by cleaving the linker-tagged peptide LP from the synthesis support SR to obtain the linker-tagged peptide LP.

[0039] In certain embodiments, L f is the linker XT b -V a-UYZ, where X is selected from formula 2, 2a, 3, 3a or 4, and T, b, V, a, U and Y are as defined above.

[0040] In certain embodiments, the free linker L f may be coupled to the N-terminus of crude peptide P or to a free amine or hydroxyl of the side chain of an amino acid of peptide P.

[0041] In certain embodiments, the free linker L f may be coupled to the N-terminus of crude peptide P or to the side chain amine of an amino acid selected from lysine, ornithine, α-aminoglycine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, 2,6-diamino-4-hexynoic acid, β-(1-piperazinyl)alanine, 4,5-dehydrolysine or d-hydroxylysine, in particular to the side chain amine of an amino acid selected from lysine and ornithine, or to the side chain hydroxyl moiety of an amino acid selected from serine, tyrosine or threonine.

[0042] In certain embodiments, the free linker L f may be coupled to the N-terminus of crude peptide P, or to the amine of the side chain of a lysine, or to the hydroxyl moiety of the side chain of a serine.

[0043] In certain embodiments, the free linker L f may be coupled to the N-terminus of crude peptide P.

[0044] The linker-tagged supported crude peptide is a moiety, XT, since coupling of the linker L to the N-terminus of the crude peptide releases a leaving group Z. b -V a -UYP-SR, and the released linker-tagged peptide is the moiety XT b -V a -Including UYP.

[0045] The release of the linker-tagged peptide from the synthesis support is carried out under commonly used acidic conditions. In contrast to prior art linker molecules, the linker of formula 1 is stable under acidic conditions, such as TFA conditions. During this process, only the acid-labile amine protecting group B of the moiety X is removed.

[0046] In certain embodiments, cleavage of the linker-tagged peptide LP- is achieved under acidic conditions.

[0047] In certain embodiments, cleavage of the linker-tagged peptide LP- is accomplished using TFA, HBr, HCl, or acetic acid.

[0048] The linker-tagged peptide is immobilized on a solid support, such as a purification resin, applying standard methods known to those skilled in the art. Suitable solid supports contain functional groups, such as aldehyde, ketone, aminooxy or hydrazine moieties, which form hydrazine or oxime bonds with moiety X of the linker.

[0049] As described above, the peptide is usually synthesized on a synthesis resin and the linker is coupled to the peptide while still attached to the synthesis resin. The linker-tagged peptide is then cleaved from the synthesis resin by adding TFA, which simultaneously removes the acid-labile protecting group B of the moiety X. If the peptide is synthesized in solution or if the peptide is provided not attached to the synthesis support SR, the acid-labile protecting group B is removed with an acid as described above before the linker-tagged peptide LP is coupled to the solid support in the immobilization step.

[0050] In certain embodiments, the acid-labile protecting group B is removed under acidic conditions and the linker-tagged peptide LP is coupled to a solid support in an immobilization step to obtain the immobilized linker-tagged peptide SLP.

[0051] For immobilization of the linker-tagged peptides, suitable aldehyde-modified beads such as agarose or hydroxylated poly(methyl acrylate) beads can be used, especially for modifications such as disulfide formation or thioether stapling, hydroxylated poly(methyl acrylate) beads or agarose, especially hydroxylated poly(methyl acrylate) beads are used.

[0052] In certain embodiments, the solid support is aldehyde-modified.

[0053] In certain embodiments, the solid support is aldehyde-modified and selected from agarose, hydroxylated poly(methyl acrylate), poly(glycidyl acrylate), poly(glycidyl methacrylate), polylysine, polyethylene glycol, polyamide, polyacrylamide, polystyrene, or copolymers thereof.

[0054] In certain embodiments, the solid support is aldehyde-modified and selected from agarose or hydroxylated poly(methyl acrylate) beads.

[0055] In a particular embodiment, the immobilization step is carried out at a pH <5, in particular at a pH of 3-4, more in particular at a pH of 3.5.

[0056] In certain embodiments, the immobilization step is carried out in DMSO / sodium citrate / guanium chloride, TFA / water, hexafluoroisopropanol (HFIP), or trifluoroethanol, in particular in DMSO / sodium citrate / guanium chloride, TFA / water, or HFIP.

[0057] In certain embodiments, the immobilization step is carried out in DMSO / sodium citrate with 8 M guanium chloride at pH 3.5, in TFA with a content of 40-95% TFA and water, in hexafluoroisopropanol (HFIP), and in trifluoroethanol.

[0058] In certain embodiments, the immobilization step is carried out in DMSO / sodium citrate (9:1), TFA / water (1:1), TFA / water (83:27) and HFIP containing 8 M guanium chloride at pH 3.5.

[0059] The peptide can then be modified in a modification step (see below).

[0060] Finally, the modified peptide mP is released at the free N-terminus via the reduced intermediate RI.

[0061] In a particular embodiment, the reducing agent of the releasing step is selected from triphenylphosphine, dithiothreitol (DTT), dithioerythritol (DTE), trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, in particular dithiothreitol, dithioerythritol or triphenylphosphine.

[0062] In certain embodiments, the reducing agent of the releasing step is selected from triphenylphosphine, dithiothreitol (DTT), trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine.

[0063] In certain embodiments, the reducing agent in the releasing step is triphenylphosphine or dithiothreitol.

[0064] The reduced intermediate RI decomposes to release the modified peptide mP with a free N-terminus.

[0065] As mentioned above, the moieties U, V, W, and E contribute to the stability of the linker under acidic conditions. For example, linkers containing moieties U or V that contain an amine or heterocycle such as a pyridine are stable under acidic conditions, especially in the presence of water with TFA>50% and pH<0, since the amine or heterocycle is protonated. Furthermore, protonated linkers improve the solubility of the linker-peptide complex. If the pH is higher than the pKa of the linker, the linker will degrade rapidly upon the desired release of the peptide in the last step.

[0066] In certain embodiments, the reduced intermediate RI is decomposed by a pH change.

[0067] In certain embodiments, the reduced intermediate RI is decomposed by changing the pH to a pH higher than the pKa of the linker.

[0068] In certain embodiments, the reduced intermediate RI is decomposed by changing the pH to a lower pH than that used during reduction of the linker. For example, the reduced intermediate RI is obtained by adding a reducing agent at pH 8. The reduced intermediate is subsequently decomposed at a pH < 8, in particular at a pH between -0.7 and 0.7.

[0069] As mentioned above, two main modifications are used to improve peptide stability and potency in vivo: lipidation with fatty acids and cyclization of linear peptides to form macrocycles.

[0070] Lipidation refers to the coupling of fatty acids to peptides. This modification affects the hydrophobicity, secondary structure, and aggregation behavior of the peptide. Furthermore, it can increase the metabolic stability and membrane permeability of the peptide.

[0071] Peptides may be composed of natural and / or unnatural amino acids. If an amino acid of a peptide contains a nucleophilic amine in its side chain, it can form an amide bond with a carboxylic acid moiety of an organic molecule, such as the carboxylic acid moiety of a fatty acid.

[0072] In certain embodiments, the peptide comprises an amino acid that contains at least one nucleophilic amine.

[0073] In certain embodiments, the peptide comprises an amino acid that contains at least one nucleophilic amine in the side chain of the amino acid, or the nucleophilic amine is at the N-terminus of the peptide, i.e., the first residue in the peptide sequence. The amino acid that forms the N-terminus can be any amino acid.

[0074] In certain embodiments, the peptide comprises an amino acid selected from lysine, ornithine, α-aminoglycine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, 2,6-diamino-4-hexynoic acid, β-(1-piperazinyl)alanine, 4,5-dehydrolysine, or d-hydroxylysine.

[0075] In certain embodiments, the peptide comprises an amino acid comprising at least one nucleophilic amine in the amino acid side chain, said amino acid being selected from lysine, ornithine, α-aminoglycine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, 2,6-diamino-4 hexynoic acid, β-(1-piperazinyl)alanine, 4,5-dehydrolysine, or d-hydroxylysine.

[0076] In certain embodiments, the peptide comprises an amino acid selected from lysine or ornithine.

[0077] The organic molecule used for lipidation of peptides can be a fatty acid (FA-OH) or an activated fatty acid (FA-A). The fatty acid or activated fatty acid is coupled to the nucleophilic amine by forming an amide. It is also possible that the fatty acid is linked to the nucleophilic amine via one or more additional linker / spacer moieties Sp. Also here, the starting material is a fatty acid linker / spacer compound FA-Sp n -OH, or activated fatty acid linker / spacer compound FA-Sp n-A. The moieties FA and Sp are linked by an amide bond. The linker / spacer Sp-OH or the activated linker / spacer Sp-A is also coupled to the nucleophile of the peptide in an amidation reaction. In the case of multiple linker / spacer moieties Sp, the linker / spacer moieties are linked to each other by amide bonds.

[0078] In certain embodiments, the organic molecule is a compound of formula 5a, 5b, 5c or 5d, in particular 5a or 5c, FA-Sp n -A(5a), FA-Sp n -OH(5b), FA-A(5c), or FA-OH(5d), During the ceremony, - FA is -C(=O)-C 1~24 Alkyl or -C(=O)-C 2~24 Alkenyl, especially -C(=O)-C 5~17 Alkyl or -C(=O)-C 5~17 Alkenyl, more particularly -C(=O)-C 5~17 alkyl, where the alkyl or alkenyl portion is optionally unsubstituted or substituted with one or more substituents independently selected from -F and -COOH; - Sp is a spacer having a length of 2 to 50 atoms, comprising a terminal -C(=O)- moiety and an -NH- moiety, said spacer being connected to FA via the -NH- moiety; - n is an integer between 1 and 10; - A is -F, -Cl, -Br, -I, -N3, -O(C=O)CH2(C=O)OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3, [ka] is selected from In particular, -Cl, [ka] and more particularly selected from [ka] is selected from - In the formula, R 14 is a C1-C6 alkyl substituent, an aryl substituent, or a benzyl substituent.

[0079] In certain embodiments, the organic molecule is a compound of formula 5a, 5b, 5c or 5d, in particular 5a or 5c, FA x -Sp n -A(5a), FA x -Sp n -OH (5b), FA-A (5c), or FA-OH (5d), - FA is -C(=O)-C 1~24 Alkyl or -C(=O)-C 2~24 Alkenyl, especially -C(=O)-C 5~17 Alkyl or -C(=O)-C 5~17 Alkenyl, more particularly -C(=O)-C 5~17 alkyl, where the alkyl or alkenyl portion is optionally unsubstituted or substituted with one or more substituents independently selected from -F or -COOH; - Sp is a spacer having a length of 2 to 50 atoms, optionally including a terminal -C(=O)- moiety, and x linking moieties independently selected from -NH-, -O-, -S-, said spacer being connected to FA via said linking moieties; - x is an integer between 1 and 5, in particular between 1 and 3, - n is an integer between 1 and 10, in particular between 1 and 4, more in particular between 1 and 2, - A is -F, -Cl, -Br, -I, -N3, -O(C=O)CH2(C=O)OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3, [ka] is selected from In particular, -Cl, -Br, -I, [ka] is selected from More particularly, [ka] -Cl, -Br, -I; - In the formula, R 14 is a C1-C6 alkyl substituent, an aryl substituent, or a benzyl substituent.

[0080] In certain embodiments, A is [ka] It is.

[0081] The terminal -C(=O) moiety of the spacer Sp is connected to A or -OH.

[0082] C 1~24 Alkyl moiety and C 2~24 The alkenyl moiety may be linear or branched. 2~24 Alkenyl moieties contain one or more carbon-carbon double bonds, in particular one carbon-carbon double bond.

[0083] In certain embodiments, the alkyl or alkenyl moiety of FA is linear or branched.

[0084] In certain embodiments, the alkyl or alkenyl moiety of FA is linear.

[0085] In certain embodiments, the FA is derived from palmitic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, myristic acid, stearic acid, stearic diacid, perfluorooctanoic acid, perfluorodecanoic acid, perfluorolauric acid, perfluoromyristic acid, and more particularly from palmitic acid. As mentioned above, the moiety FA comprises a carbonyl moiety, which is derived from the carboxylic acid moiety of the acids described herein.

[0086] The FA moiety can contain one or more substituents. For example, the FA moiety can be derived from a perfluoro fatty acid. In this case, the alkyl or alkenyl moiety of the FA is fully substituted with -F. The FA moiety can also be derived from a fatty diacid. In this case, the terminal methyl moiety (omega carbon) of the alkyl or alkenyl moiety of the FA is substituted with one -COOH moiety.

[0087] In certain embodiments, the moiety FA is unsubstituted, the moiety FA is fully substituted with -F, or the moiety FA contains one substituent -COOH.

[0088] In certain embodiments, the moiety FA is unsubstituted, the moiety FA is fully substituted with -F, or the moiety FA contains one substituent, -COOH, at the omega carbon.

[0089] In certain embodiments, the moiety FA is unsubstituted or the moiety FA is fully substituted by -F.

[0090] In certain embodiments, the moiety FA is unsubstituted or the moiety FA comprises one substituent -COOH, in particular one substituent -COOH at the omega carbon.

[0091] In certain embodiments, the moiety FA is unsubstituted or the moiety FA comprises the substituents -COOH or -COOtBu, in particular -COOtBu.

[0092] After purification / modification, this -COOtBu is removed and the moiety FA is replaced by -COOH.

[0093] In certain embodiments, the moiety FA is unsubstituted or the moiety FA comprises the substituent -COOH or -COOtBu at the omega carbon, in particular -COOtBu.

[0094] In certain embodiments, the moiety FA comprises the substituent -COOH or -COOtBu at the omega carbon, in particular -COOtBu.

[0095] In certain embodiments, the moiety FA is unsubstituted.

[0096] The spacer Sp may be derived from a natural or unnatural amino acid. As described above, the spacer is connected to the moiety FA by an amide bond and to a further spacer moiety Sp or to a nucleophilic amine of a peptide by an amide bond. The amide bond to the moiety FA is formed by reacting the N-terminal amine of the amino acid or the amine of the side chain of the amino acid with the carboxylic acid moiety of the fatty acid FA-OH or the activated carboxylic acid moiety of FA-A. The amide bond to the further spacer moiety Sp or to a nucleophilic amine of a peptide is formed by reacting the carboxylic acid moiety of the C-terminus or the carboxylic acid moiety of the side chain of the amino acid with the amine moiety of the further spacer moiety Sp or to a nucleophilic amine of a peptide. The functional groups of the side chain, N-terminus, C-terminus of the amino acid may be protected, for example the carboxylic acid moiety may be protected with a tert-butyl protecting group.

[0097] In certain embodiments, Sp is independently selected from: - linear or branched alkyl substituted with 1 to 5, in particular 1 to 3, linking moieties, in particular said linking moieties being -O-, - -NH-[C r Alkyl-O] s -C talkyl-C(=O)-, where r is independently selected from 1, 2 or 3, s is an integer between 1 and 10, in particular 1, 2, 3 or 4, and t is 1, 2 or 3; and - an amino acid, which may be protected or unprotected, in which the N-terminus and / or side chain amine is replaced by -NH- and the C-terminus and / or side chain carboxylic acid moiety is replaced by -C(=O)-.

[0098] When n is greater than 1, identical or different moieties Sp may be combined.

[0099] In certain embodiments, the linear or branched alkyl is 1~12 Alkyl, especially C 1~6 Alkyl, more particularly C 1~3 It is an alkyl.

[0100] In certain embodiments, Sp is independently selected from: - linear alkyl substituted with 1 to x linking moieties, in particular where the linking moiety is -O-; - [ka] Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, Glu-tBu, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0101] In certain embodiments, the linear alkyl is 1~12 Alkyl, especially C 1~6 Alkyl, more particularly C 1~3 It is an alkyl.

[0102] In certain embodiments, Sp is independently -O-ethyl-, [ka] It is selected from cysteine, glutamic acid, Glu-tBu, or 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0103] In certain embodiments, Sp is an amino acid, which may be protected or unprotected, in which the N-terminus or side chain amine is replaced by -NH- and the C-terminus or side chain carboxylic acid moiety is replaced by -C(=O)-.

[0104] In certain embodiments, Sp are, independently of each other, an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, Glu-tBu, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine or 2-(2-(2-aminoethoxy)ethoxy)acetic acid, in particular an amino acid selected from glutamic acid, Glu-tBu or 2-(2-(2-aminoethoxy)ethoxy)acetic acid, in which the N-terminus or side chain amine is replaced by -NH- and the C-terminus or side chain carboxylic acid moiety is replaced by -C(=O)-.

[0105] The amino acid glutamic acid may be protected (Glu-tBu), for example the C-terminal carboxylic acid moiety may be protected with a tert-butyl protecting group.

[0106] In certain embodiments, n is 1 or 2.

[0107] In certain embodiments, n is 1.

[0108] In certain embodiments, the organic molecule is selected from the group consisting of Pam2, Pam-Et, Pam3Cys, and Pam2-Et-Cys, [ka] is selected from.

[0109] In certain embodiments, the organic molecule of formula 5a or 5c is coupled to at least one nucleophilic amine in the presence of a non-nucleophilic base in the modification step.

[0110] In a particular embodiment, the organic molecule of formula 5b or 5d is coupled in a modification step to at least one nucleophilic amine in the presence of an activating reagent, in particular an activating reagent selected from the following: tetramethylaminium tetra uoroborate (TBTU), (3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazin-4(3H)-one) (DEPBT), 2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate (C6H-Tetramethylaminium hexafluorophosphate), The reaction is coupled in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), dicyclohexylcarbodiimide (DCC), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-1H-benzotriazole (HOAt), NHS, and diisopropylcarbodiimide (DIC).

[0111] In a particular embodiment, the organic molecule of formula 5b or 5d is coupled in a modification step to at least one nucleophilic amine in the presence of an activating reagent, in particular in the presence of an activating reagent selected from the following: diisopropylcarbodiimide (DIC), (2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate) (HCTU), and tetramethylaminium hexafluorophosphate (HATU).

[0112] Besides lipidation, cyclization is often used to improve peptide stability and efficacy in vivo: cyclic peptides are more resistant to exo- and endoproteases. Due to their more rigid conformation, they show better binding affinity to the desired target. Cyclization can be achieved either by intermolecular disulfide formation or in two-component systems using scaffold molecules (intramolecular cyclization).

[0113] For both cyclization methods, a functional group on the peptide, such as a thiol moiety, is utilized.

[0114] In certain embodiments, the peptide comprises at least two amino acids that contain a nucleophilic thiol.

[0115] In certain embodiments, the peptide comprises 2-6 acids that contain a nucleophilic thiol.

[0116] In certain embodiments, the peptide comprises two amino acids that contain a nucleophilic thiol.

[0117] In certain embodiments, the peptide comprises at least two amino acids independently selected from cysteine, homocysteine, or penicillamine.

[0118] In certain embodiments, the peptide comprises 2 to 6 amino acids independently selected from cysteine, homocysteine, or penicillamine.

[0119] In certain embodiments, the peptide comprises two amino acids independently selected from cysteine, homocysteine, or penicillamine.

[0120] In two-component systems (intermolecular cyclization), functional groups of the peptide are used to react with an organic molecule, such as a crosslinking scaffold. Depending on the number of functional groups on the peptide and the scaffold molecule, the peptide and the scaffold molecule can form monocyclic, bicyclic, or polycyclic rings. The most common method is to utilize nucleophilic functional groups of the peptide, especially thiols, to react with a scaffold that has electrophilic reactive sites. Such electrophilic centers can be nucleophilically attacked by thiols.

[0121] In certain embodiments, the organic molecule is an organic scaffold molecule that comprises a scaffold having at least two electrophilic centers.

[0122] The scaffold can be a linear, branched, monocyclic or polycyclic system, which can be aliphatic or aromatic. More rigid scaffolds, especially cyclic systems, are suitable for cyclization to maintain the peptide in a desired conformation. The scaffold can also contain heteroatoms, such as O, S or N. The scaffold can further contain a substituent that forms an electrophilic center. For example, a phenyl substituted with two ethyl substituents can form the scaffold, and the Br substituent of the ethyl moiety can form the electrophilic center. This Br substituent is called benzyl Br.

[0123] In certain embodiments, the organic molecule is an organic scaffold molecule comprising a scaffold having at least two electrophilic centers, said electrophilic centers being formed by halogen substituents and / or Michael acceptors.

[0124] In certain embodiments, the electrophilic center is formed by at least two benzylic halogen substituents, at least two acrylamides, or at least two acrylic esters.

[0125] In certain embodiments, the electrophilic center is formed by at least two acrylamide or at least two benzyl halogen substituents.

[0126] In certain embodiments, the organic molecule is m-Xylylene dibromide (mDBX), 1,4-bis(bromomethyl)benzene, 1,3,5-Tris(bromomethyl)benzene (TBMB), N,N'-bis(chloroacetyl)-1,2-ethylenediamine, N,N'-bis(chloroacetyl)-1,3-propylenediamine, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-chloroacetamide), N,N'-bis(bromo ... Diamines, N,N'-bis(bromoacetyl)-1,3-propylenediamine, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-bromoacetamide), 1,3,5-triacryloyl-1,3,5-triazinan (TATA), 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (1,1',1"-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanone) (1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoacetamide ... 5-triyl)tris(2-bromoethanon)(TATB), N,N',N"-(benzene-1,3,5-triyl)-tris(2-bromoacetamide)(TBAB), N,N',N"-benzene-1,3,5-triyltrisprop-2-enamide(TAAB), 2,4,6-tris( 2,4,6-tris(bromomethyl)-s-triazine (TBMT), N,N',N"-(nitrilotris(ethane-2,1-diyl))triacrylamide, N,N',N"-(nitrilotris(ethane-2,1-diyl))tris(2-chloroacetamide), N,N',N"-(nitrilotris(ethane-2,1-diyl))tris(2-bromoacetamide), N,N',N"-(nitrilotris(ethane-2,1-diyl))triethenesulfonamide, N,N',N”-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))triacrylamide, N,N',N”-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N”-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))triethenesulfonamide, N,N',N”-(benzene N,N',N"-(benzene-1,3,5-triyltris(methylene))triacrylamide, N,N',N"-(benzene-1,3,5-triyltris(methylene))tris(2-chloroacetamide), N,N',N"-(benzene-1,3,5-triyltris(methylene))triethenesulfonamide, tri(2-hydroxyethyl)isocyanurate triacrylate, 1,3,5-tris(oxiran-2-ylmethyl)-1,3,5-triazinane-2,4,6-trione.

[0127] In certain embodiments, the organic molecule is selected from m-xylylene dibromide (mDBX), 1,3,5-tris(bromomethyl)benzene (TBMB), triazan, 1,3,5-triacryloyl-1,3,5-triazinane (TATA), 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB).

[0128] In certain embodiments, the organic molecule is selected from α,α'-dibromo-m-xylene (mDBX), 1,3,5-tris(bromomethyl)benzene (TBMB), triazan, 1,3,5-triacryloyl-1,3,5-triazinane (TATA), 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB).

[0129] In the two-component system (intermolecular cyclization), a nucleophilic thiol is obtained in a reduction step, which then attacks the electrophilic center of the organic scaffold molecule in a cyclization step.

[0130] In certain embodiments, the modification step comprises a reduction step followed by a cyclization step, wherein: - the immobilized linker-tagged peptide SLPs are treated with a reducing agent, in particular dithiothreitol (DTT) or tris-(2-carboxyethyl)-phosphine (TCEP), in a reduction step; and - An organic scaffold molecule is coupled to at least two nucleophilic thiols under basic conditions in a cyclization step.

[0131] The cyclization step is carried out under basic conditions to deprotonate the nucleophilic thiol.

[0132] In certain embodiments, the cyclization step is carried out at a pH >8.

[0133] In certain embodiments, the cyclization step is carried out in the presence of Na2CO3, NaHCO3, NH4HCO3, guanidine hydrochloride (GdmCl), diisopropylethylamine (DIPEA), triethylamine (NEt3), or N-methylmorpholine (NMM).

[0134] In certain embodiments, the cyclization step is carried out in the presence of NH4HCO3, guanidine hydrochloride (GdmCl), diisopropylethylamine (DIPEA), triethylamine (NEt3) or N-methyl-morpholine (NMM).

[0135] Cyclization can also be achieved by the formation of an intermolecular disulfide bond. For example, the thiol moieties of two cysteine ​​residues of a peptide may form a disulfide.

[0136] In certain embodiments, the number of nucleophilic thiols is even, and the nucleophilic thiols are oxidized in the modification step by a basic aqueous solution with pH>7 to obtain the macrocyclic cross-linked peptide.

[0137] In certain embodiments, the nucleophilic thiols are oxidized by an aqueous basic solution at pH>7 and / or in the presence of air (particularly oxygen) and / or in the presence of an oxidizing additive, in particular DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, Tl(CF3COO)3, CH3SiCI3-Ph(SO)Ph, [Pt(ethylenediamine)2Cl2]Cl2, 2,2'-dithiobis(5-nitropyridine), 5,5'-dithiobis-(2-nitrobenzoic acid), trans-[Pt-(CN)4Cl2] 2- , glutathione-glutathione disulfide, K3Fe(CN)6, or an oxidizing additive selected from the group consisting of .

[0138] The oxidation additives can be used as is or dissolved in a suitable solvent. The oxidation can be carried out in organic, organic-aqueous, acidic or basic solutions.

[0139] In certain embodiments, the nucleophilic thiol is oxidized in the presence of air (particularly oxygen) or in the presence of an oxidizing additive, particularly DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, Tl(CF3COO)3, CH3SiCI3-Ph(SO)Ph, [Pt(ethylenediamine)2Cl2]Cl2, 2,2'-dithiobis(5-nitropyridine), 5,5'-dithiobis-(2-nitrobenzoic acid), trans-[Pt-(CN)4Cl2] 2- , glutathione-glutathione disulfide, K3Fe(CN)6, or an oxidizing additive selected from the group consisting of .

[0140] In certain embodiments, the nucleophilic thiol is selected from the group consisting of DMSO, iodine, N-chlorosuccinimide, Tl(OAc) 3 , [Pt(ethylenediamine) 2 Cl 2 ]Cl 2 , trans-[Pt-(CN) 4 Cl 2 ] 2- , which is oxidized in the presence of glutathione.

[0141] In certain embodiments, the nucleophilic thiol is selected from the group consisting of air (oxygen), DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, Tl(CF3COO)3, CH3SiCI3-Ph(SO)Ph, [Pt(ethylenediamine))2Cl2]Cl2, 2,2'-dithiobis(5-nitropyridine), 5,5'-dithiobis-(2-nitrobenzoic acid), trans-[Pt-(CN)4Cl2] 2- , which is oxidized in the presence of glutathione-glutathione disulfide, K3Fe(CN)6.

[0142] In certain embodiments, the nucleophilic thiol is selected from the group consisting of DMSO, iodine, N-chlorosuccinimide, Tl(OAc) 3 , [Pt(ethylenediamine) 2 Cl 2 ]Cl 2 , trans-[Pt-(CN) 4 Cl 2 ] 2- , which is oxidized in the presence of glutathione.

[0143] In certain embodiments, the nucleophilic thiol is oxidized in the presence of N-chlorosuccinimide, DMSO and glutathione-glutathione disulfide.

[0144] In certain embodiments, the nucleophilic thiol is oxidized in the presence of N-chlorosuccinimide, DMSO and glutathione.

[0145] In certain embodiments, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular from the moieties of formula 2, 2a, 3 or 3a, more in particular from the moieties of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, where at least R 1 or R 2 is B, - R 3 is selected from H or B; - R 4 H, C1~C 12alkyl or aryl, wherein the aldehyde or keto group is optionally protected with an acid labile protecting group; B is an acid labile amine protecting group, - T is the part -C 1~12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9 a linear or branched spacer containing at least one of -, phenyl, and 5- or 6-membered heteroaryl, in particular a linear or branched spacer containing 1 to 5, J is C or N, in particular N; In particular, T. -C1~C 12 alkyl-, in particular C 1~6 Alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -C(=O)-R 6 -, -R 5 -NR 9 -, -R 5 -NR 9 -R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ -R 6 -, -R5 -C(=O)-NR 9 -R 5’ -NR 9’ -R 6 -, -R 5 -C(=O)-OR 6 -, -C(=O)-OR 6 -, -R 5 -phenyl-R 6 -, -R 5 -phenyl-, -phenyl-R 6 -, -phenyl-, -R 5 -Pyroyl (-R 5 -pyrroyl), -R 5 -Pyrazoyl, -R 5 -Imidazolyl, R 5 -piperazinyl-, -R 5 -pyridinyl, -R 5 -pyrimidinyl, -R 5 -pyrazinyl, -R 5 -pyridazinyl, -R 5 -Pyroyl-R 6 -, -R 5 -Pyrazoyl-R 6 -, -R 5 -Imidazolyl-R 6 -, -R 5 -Piperazinyl-R 6 -, -R 5 -Pyridinyl-R 6 -, -R 5 -Pyrimidinyl-R 6 -, -R 5 -Pyrazinyl-R 6 -, -R 5 -Pyridazinyl-R 6 -, pyrroyl-R 6 -, Pyrazoyl-R 6 -, Imidazoyl-R 6 -Piparazinyl-R 6 -, pyridinyl-R 6 -, pyrimidinyl-R 6 -, pyrazinyl-R 6 -, pyridazinyl-R 6 a spacer selected from -, pyrroyl, pyrazoyl, imidazoyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, or pyridazinyl, R 5 , R 5’ and R 6 are independent of each other, C1 to C 12 Alkyl or (-C2H4O-) 1~12 , in particular C1-C6 alkyl, in particular C1-C3 alkyl, and R 9 and R 9’ are, independently of each other, H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 In particular, H or C 1~2 alkyl, more particularly R 9 is H, where B is an independently selected acid-labile amine protecting group; R 15 is a blocking agent capable of reacting with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular selected from cysteinyl or N-methylhydroxylamine, more in particular selected from cysteinyl, wherein the amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid labile amine protecting group B, in particular by Boc, and / or by an acid labile thiol protecting group, in particular by trityl; b is 0 or 1, in particular 1, - V, -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p-,-Piperazinyl-(CH2) p -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, in particular -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p -,-Piperazinyl-(CH2) p -, -pyridinyl-, pyrimidinyl, more particularly -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-(CH2) p an electron withdrawing moiety selected from -, -pyridinyl, pyrimidinyl, R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H, R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl, p is 0, 1 or 2, in particular 0 or 1; a is 0 or 1, where the sum of a and b is 1 or 2; - U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, more in particular phenyl, which is a moiety that can be joined to the moieties V, W q and E n is bound to at least one of C 1~6 By alkyl, especially C 1~3 and optionally substituted with alkyl, V is as defined above, W is -N3, -NO2, -S(=O)-R 8 , -SSR8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] In particular, -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 is selected from the group consisting of R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, p is 1, 2, 3 or 4; E is an electron-withdrawing group under acidic conditions; n is an integer between 0 and 4, in particular between 0 and 2, more in particular between 0 and 1, and q is an integer between 0 and 4, in particular between 0 and 2, more in particular between 0 and 1, where the sum of n and q is 4 or less, and where U is a phenyl moiety and Y is -(CH2) m When -OC(=O)-, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and W is in the ortho or para position relative to Y; - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; Z is an electron-withdrawing leaving group.

[0146] In certain embodiments, q is an integer between 1 and 4, particularly an integer between 1 and 2, and more particularly q is 1.

[0147] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N(C2H4)2NH2, -N(C2H4)2N-B, -N=N-phenyl, -N=NR 8 , -(CH2)r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 alkyl)2-, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, in particular pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N=N-phenyl, -N=NR 8 , -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, more particularly pyridyl, pyrimidinyl, pyridazinyl, or -Br; R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6 alkyl or -(CH2) p -NMe2, especially pyridyl or -C1-C6 alkyl; p is 1, 2, 3 or 4; B is an acid labile amine protecting group; and r is 0, 1, 2, 3 or 4, especially 0, 1 or 2.

[0148] In certain embodiments, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular from the moieties of formula 2, 2a, 3 or 3a, more in particular from the moieties of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, where at least R 1 or R 2 is B, - R 3 is selected from H or B; - R 4 H, C1~C 12 alkyl or aryl, wherein the aldehyde or keto group is optionally protected with an acid labile protecting group; B is an acid labile amine protecting group, - T is the part -C 1~12Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9 -, wherein J is C or N, in particular N; In particular, T. -C1~C 12 A spacer selected from alkyl-, particularly -C 1~6 Alkyl-, more particularly -C 1~3 Alkyl-, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -C(=O)-R 6 -, -R 5 -NR 9 -, -R 5 -NR 9 -R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ -R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -R 6 -, -R 5 -C(=O)-OR 6 -, -C(=O)-OR 6 -, -R 5-phenyl-R 6 -, -R 5 -phenyl-, -phenyl-R 6 -, -phenyl-, R 5 , R 5’ and R 6 are independent of each other, C1 to C 12 Alkyl or (-C2H4O-) 1~12 , in particular C1-C6 alkyl, in particular C1-C3 alkyl, and R 9 and R 9’ are, independently of each other, H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 In particular, H or C 1~2 alkyl, more particularly R 9 is H, where B is an independently selected acid-labile amine protecting group; R 15 is a blocking agent capable of reacting with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular selected from cysteinyl or N-methylhydroxylamine, more in particular selected from cysteinyl, wherein the amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid labile amine protecting group B, in particular by Boc, and / or by an acid labile thiol protecting group, in particular by trityl; b is 0 or 1, in particular 1, - V, -NR11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, in particular -NR 11 -C(=O)-, -C(=O)-NR 11 -, S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H, R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl, a is 0 or 1, where the sum of a and b is 1 or 2; - U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, more in particular phenyl, which is q and E n is bound to at least one of C 1~6 By alkyl, especially C 1~3 and optionally substituted with alkyl, V is as defined above, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR8 , [ka] In particular, -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 where: R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4; E is an electron-withdrawing group under acidic conditions; n is an integer between 0 and 4, in particular between 0 and 2, more in particular between 0 and 1, and q is an integer between 0 and 4, in particular between 0 and 2, more in particular between 0 and 1, where the sum of n and q is 4 or less, and where U is a phenyl moiety and Y is -(CH2) m When -OC(=O)-, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; - Z is an electron-withdrawing leaving group.

[0149] In certain embodiments, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular from the moieties of formula 2, 2a, 3 or 3a, more in particular from the moieties of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, where at least R 1 or R 2 is B, - R3 is selected from H or B; - R 4 H, C1~C 12 alkyl or aryl, wherein the aldehyde or keto group is optionally protected with an acid labile protecting group; B is an acid labile amine protecting group, - T -C1~C 12 alkyl-, in particular C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -C(=O)-R 6 -, -R 5 -C(=O)-OR 6 -, -C(=O)-OR 6 -, -R 5 -phenyl-R 6 -, -R 5 -phenyl-, -phenyl-R 6 -, -phenyl-, where R 5 , R 5’ and R 6 are independent of each other, C1 to C 12 alkyl, in particular C1-C6 alkyl, in particular C1-C3 alkyl, and wherein R 9 and R 9’are each independently H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 9 is H, b is 0 or 1, in particular 1, - V, -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, in particular -NR 11 -C(=O)-, -C(=O)-NR 11 -, S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H, R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl, a is 0 or 1, where the sum of a and b is 1 or 2; - U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, more in particular phenyl, which is q and E n is bound to at least one of C 1~6 By alkyl, especially C 1~3and optionally substituted with alkyl, V is as defined above, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] In particular, -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 is selected from the group consisting of R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4; E is an electron-withdrawing group under acidic conditions; n is an integer between 0 and 4, in particular between 0 and 2, more in particular between 0 or 1, and q is an integer between 0 and 4, in particular between 0 and 2, more in particular between 0 and 1, where the sum of n and q is 4 or less, and where U is a phenyl moiety and Y is -(CH2) m When -OC(=O)-, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45; - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; - Z is an electron-withdrawing leaving group.

[0150] In certain embodiments, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular from the moieties of formula 2, 2a, 3 or 3a, more in particular from the moieties of formula 2 or 2a, most in particular from the moieties of formula 2, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, where at least R 1 or R 2 is B, - R 3 is selected from H or B; - R 4 H, C1~C 12 alkyl or aryl, wherein the aldehyde or keto group is optionally protected with an acid labile protecting group; B is an acid labile amine protecting group, - T -C1~C 12 alkyl-, in particular C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-NR 9 -R 6 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -R 5 -NR 9 C(=O)-R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -C(=O)-OR 6 -, -C(=O)-OR 6 -, -R 5 -phenyl-R 6 -, -R 5 -phenyl-, -phenyl-R 6 -, -phenyl-, R 5 and R 6 are independent of each other, C1 to C 12 alkyl, in particular C1-C6 alkyl, in particular C1-C3 alkyl, and wherein R 9 is H or C 1~4alkyl, in particular H or C 1~2 alkyl, more particularly R 9 is H, b is 0 or 1, in particular 1, - V, -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazyl, -C(=O)-, -C(=O)-O-, in particular -NR 11 -C(=O)-, S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -pyridinyl-, pyrimidinyl, R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H, R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl, a is 0 or 1, where the sum of a and b is 1 or 2; - U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, which is bonded to at least one of the moieties V, W and E, in which V is as defined above, W is -N3, -S(=O)-R 8 , -SSR 8 , -OCH2N3, -OC(=O)OCH2-N3, -N=N-phenyl, -N=N-pyridine, [ka] is selected from, where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p-NMe2, p is 1, 2, 3 or 4; E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, in particular selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, R 13 is selected from -F, -Cl, -Br, -I, -PF6, and U is a phenyl moiety and Y is -(CH2) m When -OC(=O)-, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45; - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; - Z is an electron-withdrawing leaving group.

[0151] In certain embodiments, X is selected from the moiety of formula 2 or 3.

[0152] In certain embodiments, X is selected from the moieties of formula 2.

[0153] The reaction time required for coupling of moiety X to a functionalized solid phase by formation of a hydrazone or oxime bond is longer when using linkers with a moiety of formula 4 and shorter when using linkers with a moiety of formula 2 or 3. The formation of the hydrazone bond between an aldehyde or ketone moiety of the solid support and moiety X of formula 3 is reversible. Due to this reversibility, the inventors observed a loss of up to about 10% of the peptide material after each washing step during purification. In contrast, almost no loss of peptide material was observed when using linkers with a moiety of formula 2.

[0154] In certain embodiments, U is C1~6 It is substituted by alkyl.

[0155] In certain embodiments, U is C 1~3 It is substituted by alkyl.

[0156] In certain embodiments, U is substituted with methyl.

[0157] When U is further substituted with one or more alkyl moieties, the Hammett values ​​of the alkyl moieties are considered. When U is further substituted with a phenyl moiety or a heteroatom, the sum of the Hammett values ​​of V, W, E and the optional alkyl substituents is greater than 0.45.

[0158] In certain embodiments, E is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N=N-phenyl, -N=NR 8 , -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt (where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2), in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4.

[0159] In certain embodiments, E is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N=N-phenyl, -N=NR 8 , -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H; R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4.

[0160] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt.

[0161] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, and -CO2H.

[0162] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyridazinyl, or -Br.

[0163] In certain embodiments, B is - Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylidene), trityl (-C(Ph)3), Mmt (-C(Ph)2C6H4OMe), DMT (-C(Ph)(C6H4OMe)2), Cbz (-C(=O)OCH2Ph), benzylideneamine (=CPh), phthalimide (=(CO)2C6H4), p-toluenesulfonamide (-SO2C6H4Me), benzylideneamine (=CPh), amine (-CH2Ph), acetamide (-COMe), trifluoroacetamide (-COCF3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), in particular B is Boc or Eei, more in particular B is Boc, or - an acetal or ketal protecting group, [ka] is selected from where r is 0 to 12, in particular 0 to 6, more in particular 0, 1 or 2; and R 10 -C1~C 12 Alkyl-, especially C 1~6Alkyl, more particularly C 1~3 It is an alkyl.

[0164] In certain embodiments, B is selected from the group consisting of Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylidene), trityl (-C(Ph)3), Mmt (-C(Ph)2C6H4OMe), DMT (-C(Ph)(C6H4OMe)2), Cbz (-C(=O)OCH2Ph), benzylideneamine (=CPh), phthalimide (=(CO)2C6H4), p-toluenesulfone amide (-SO2C6H4Me), benzylamine (-CH2Ph), acetamide (-COMe), trifluoroacetamide (-COCF3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl), or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde).

[0165] In certain embodiments, B is Boc or Eei (=CMeOEt, 1-ethoxyethylidene).

[0166] In certain embodiments, B is Boc.

[0167] In certain embodiments, B is [ka] is selected from an acetal or ketal protecting group selected from In the formula, r is 0 to 12, in particular 0 to 6, more particularly 0, 1 or 2, and R 10 -C1~C 12 Alkyl-, especially C 1~6 It is an alkyl.

[0168] In certain embodiments, T is the moiety -C 1~12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9-, where J is C or N, in particular N, and where R 9 are independently H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 alkyl-NB2, in particular H or C 1~2 alkyl, more particularly R 9 is H, where B is an independently selected acid-labile amine protecting group.

[0169] In certain embodiments, the total length of the spacer T is between 0.5 and 100 nm.

[0170] In certain embodiments, T is -C 12 alkyl-, in particular C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ C(=O)-R 6 - selected from, in particular C 1~3 Alkyl, -R 5 -C(=O)-NR 9 -, -R 5 -NR 9 -C(=O)-R 6 -From, more particularly, C 1~3 Alkyl or -R 5 -C(=O)-NR 9 - selected from R 5 , R 5’、 R 6 , R 9’ and R 9is as defined above.

[0171] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -, wherein R 9 are independently H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 In particular, H or C 1~2 alkyl, more particularly R 9 is H, where B is an independently selected acid labile amine protecting group.

[0172] R 15 is a blocking agent capable of reacting with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular selected from cysteinyl and N-methylhydroxylamine, more in particular selected from cysteinyl, wherein the amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid labile amine protecting group B, in particular by Boc, and / or by an acid labile thiol protecting group, in particular by trityl.

[0173] As mentioned above, the spacer T is generally unreactive under commonly applied purification conditions, except for removal of the protecting group under acidic conditions. The spacer T can increase the solubility of the linker molecule. In particular, R9 The branched spacer containing a protected or unprotected amine moiety at 20 contributes to improved solubility. Under acidic conditions, the amine protecting group B is removed and the amine is protonated.

[0174] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -, wherein R 9 are independently H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 alkyl-NB2, in particular H or C 1~2 alkyl, more particularly R 9 is H, where B is an independently selected acid labile amine protecting group.

[0175] The spacer T may also comprise a blocking function. In particular, the branched spacer may comprise a blocking agent suitable for binding to the aldehyde moiety. If the solid phase used for the purification of the peptide comprises an aldehyde moiety, e.g. agarose beads, the moiety X of the linker compound may be covalently attached to the solid phase, e.g. by formation of an oxime bond. Unreacted aldehyde moieties may cause unwanted side reactions during subsequent purification. To prevent such side reactions, the unreacted aldehyde moieties of the solid phase may be blocked by a blocking function of the spacer, e.g. a cysteinyl moiety.

[0176] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -, where R 9 are independently H, C1~4 Alkyl, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 In particular, H or C 1~2 alkyl, more particularly R 9 is H, where B is an independently selected acid-labile amine protecting group, R 15 is a blocking agent capable of reacting with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular selected from cysteinyl or N-methylhydroxylamine, more in particular selected from cysteinyl, wherein the amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid labile amine protecting group B, in particular by Boc, and / or protected by an acid labile thiol protecting group, in particular by trityl.

[0177] In certain embodiments, T is -C 12 alkyl-, in particular C 1~6 alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-NR 9 -R 6 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -C(=O)-OR 6 -, -C(=O)-OR 6 -, -R 5 -NR 9 -R 5 -NR 9 C(=O)-R 6 -, in particular -C1 to -C 12 alkyl-, in particular C1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -R 5 -NR 9 C(=O)-R 6 -From, most especially -R 5 -NR 9 -C(=O)-R 6 - selected from R 5 , R 6 and R 9 is as defined above.

[0178] As mentioned above, the moieties U, V, W and E contribute to the stability of the linker under acidic conditions. For example, linkers containing moieties U or V containing an amine or a heterocycle such as pyridine are stable under acidic conditions, especially in TFA>50%, in the presence of water at pH<0, since the amine or heterocycle is protonated. Furthermore, protonated linkers improve the solubility of the linker-peptide complex. If the pH is higher than the pKa of the linker, the linker will degrade rapidly upon the desired release of the peptide as the final step.

[0179] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, where R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H and R12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl.

[0180] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, where R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H and R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl.

[0181] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, pyrimidinyl, where R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H and R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl.

[0182] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, pyrimidinyl, where R11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H and R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl.

[0183] V is an electron-withdrawing moiety selected from -NH-C(=O)-, -C(=O)-HN-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, pyrazinyl, pyridazinyl.

[0184] V is an electron-withdrawing moiety selected from -NH-C(=O)-, -C(=O)-HN-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl.

[0185] In certain embodiments, V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazyl, -C(=O)-, -C(=O)-O-.

[0186] In certain embodiments, V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, where R 11 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 11 is H and R 12 is H or C 1~4 alkyl, in particular H or C 1~2 alkyl, more particularly R 12 is methyl.

[0187] In certain embodiments, V is -NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, where R 11 is H or C 1~4 alkyl, and R 12 is H or C 1~4 is selected from alkyl.

[0188] In certain embodiments, V is -NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, where R 11 is H or C 1~2 alkyl, and R 12 is H or C 1~2 is selected from alkyl.

[0189] In certain embodiments, V is selected from -NH-C(=O)-, -N-(CH3)-, -pyridinyl-, and pyrimidinyl.

[0190] In certain embodiments, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -N=NR 8 In particular, -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1 to C6 alkyl, or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4.

[0191] R 8 C 1~6 In the case of alkyl, the alkyl moiety may be linear or branched, for example tert-butyl. In particular, W is -SSR 8 In the case of R 8may be pyridyl or -C1-C4 alkyl, in particular pyridyl or tert-butyl.

[0192] Partial W=-SSR 8 Linkers containing the can be cleaved with thiols. Thus, cleavage of peptide-linker conjugates from the synthesis resins used during SPPS under acidic conditions (e.g., TFA>50%, pH<0 in the presence of water) can be performed without thiols when such linkers are used for peptide purification.

[0193] In certain embodiments, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -N=NR 8 In particular, -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 is selected from, where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl or -C1-C6 alkyl, or in particular pyrimidinyl, pyridyl or -C1-C6 alkyl, more in particular pyridyl or -C1-C6 alkyl.

[0194] In certain embodiments, U is selected from phenyl or a 5- or 6-membered heterocycle, the 5- or 6-membered heterocycle containing 1 or 2 heteroatoms.

[0195] In certain embodiments, U is selected from phenyl or a 5- or 6-membered heterocycle, the 5- or 6-membered heterocycle containing one heteroatom.

[0196] In certain embodiments, U is selected from phenyl or a 6-membered heterocycle.

[0197] In certain embodiments, U is selected from phenyl or a 6-membered heterocycle, wherein the 6-membered heterocycle contains 1 or 2 heteroatoms.

[0198] In certain embodiments, the 5- or 6-membered heteroaryl moiety of U contains one or two heteroatoms, in particular the 5-membered heteroaryl moiety of the moiety U is selected from pyrazole, imidazole and the 6-membered heteroaryl moiety of the moiety U is selected from pyridine, pyridazine, pyrimidine, pyrazine, in particular pyridine.

[0199] In certain embodiments, U is selected from phenyl or a 6-membered heterocycle, where the 6-membered heterocycle contains one heteroatom. In certain embodiments, the 5-membered heterocycle of the moiety U is selected from pyrazole, imidazole, and the 6-membered heterocycle of the moiety U is selected from pyridine, pyridazine, pyrimidine, pyrazine.

[0200] In a particular embodiment, U is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, in particular phenyl or pyridine, more in particular phenyl.

[0201] In certain embodiments, U is selected from a moiety of formula 5, 6, 7, or 8: [ka] During the ceremony, T, V, Y, W, and E are as defined above; In formulas 5 and 6, U is attached to the moiety T or V; In formulas 7 and 8, U is attached to the moiety V, A 1 , A 2 , A 3 , A 4 and D. 1 , D 2 , D 3 , D 4 are independently selected from C, N, S or O, in particular from C or N, where A 1 , A 2 , A 3 and A 4 of or D 1 , D 2 , D 3 and D. 4The 2-4 parts of are C, especially A 1 , A 2 , A 3 and A 4 of or D 1 , D 2 , D 3 and D. 4 Three or four moieties of are C, more particularly A 1 , A 2 , A 3 and A 4 of or D 1 , D 2 , D 3 and D. 4 All parts of are C, n is In the case of formulas 5 and 6, it is an integer between 0 and 3, In the case of formulas 7 and 8, it is an integer between 0 and 4, q is an integer between 0 and 4, where the sum of n and q is 4 or less.

[0202] In certain embodiments, U is selected from the moiety of formula 5 or 6.

[0203] In certain embodiments, U is selected from a moiety of formula 5, 6, 7, or 8, wherein: T, V, Y, W, and E are as defined above; In formulas 5 and 6, U is attached to the moiety T or V; In formulas 7 and 8, U is attached to the moiety V, A 1 , A 2 , A 3 , A 4 and D. 1 , D 2 , D 3 , D 4 are selected independently of one another from C, N, S or O, in particular from C or N, n is In the case of formulas 5 and 6, it is an integer between 0 and 2, In the case of formulas 7 and 8, it is an integer between 0 and 2, in particular between 0 and 1, q is an integer between 0 and 2, particularly between 0 and 1.

[0204] In certain embodiments, U is selected from a moiety of formula 9, 10, 11 or 12, in particular a moiety of formula 9 or 10, [ka] During the ceremony, T, V, Y, W, E, q, and n are as defined above; In the case of formulas 9 and 10, U is attached to a moiety T or V; In formulas 11 and 12, U is attached to the moiety V, All parts A 2 , A 3 and A 4 is C or A 2 , A 3 and A 4 Two of them are C and A 2 , A 3 and A 4 The other two are N, especially A 2 and A 3 are both C, and D 2 is C or N, in particular C.

[0205] In certain embodiments, U is selected from a moiety of formula 9, 10, 11, or 12: [ka] During the ceremony, T, V, Y, W, E, q, and n are as defined above; In the case of formulas 9 and 10, U is attached to the moiety T or V; In formulas 11 and 12, U is attached to the moiety V, A 2 and A 3 are both C or A 2 and A 3 One of them is C and the other is A 2 and A 3 The other is N, D 2 is C or N.

[0206] When the moiety U comprises an N-containing heteroaryl, the N atom becomes protonated under acidic conditions, thus increasing the solubility of the linker molecule.

[0207] In certain embodiments, U is selected from the moieties of formula 13, 14, 15, 16, 17, 18, 19, 20 or 21, particularly from the moieties of formula 13 to 19, more particularly from the moieties of formula 15 or 19; [ka] During the ceremony, T, V, Y, W, E, q, and n are as defined above; In the case of formulas 13, 14 and 15, U is bonded to the moiety T or V; In the case of formulas 16, 17 and 18, U is attached to the moiety V, A 2 , A 3 and A 4 is C or N, in particular C, D 2 is C or N.

[0208] In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 16, 17, or 18: [ka] During the ceremony, T, V, Y, W, E, q, and n are as defined above; In the case of formulas 13, 14 and 15, U is bonded to the moiety T or V; In the case of formulas 16, 17 and 18, U is attached to the moiety V, A 3 is C or N, D 2 is C or N.

[0209] In certain embodiments, Z is -F, -Cl, -Br, -I, -N, -OH, -O(C=O)CH(C=O)OH-SR 14, -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3, [ka] In particular, -OH, -Cl, [ka] is selected from In particular, -OH, [ka] is selected from In the formula, R 14 is a C1-C6 alkyl substituent, an aryl substituent, or a benzyl substituent.

[0210] In certain embodiments, Z is -F, -Cl, -Br, -I, -N3, -OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH 3、 [ka] In particular, -OH, -Cl, [ka] In particular, -OH, [ka] is selected from In the formula, R 14 is a C1-C6 alkyl substituent, an aryl substituent, or a benzyl substituent.

[0211] In certain embodiments, the compound of formula 1 is selected from compounds of formula X1, X2, X3, X4, X5, X7, X8, X9, X10, X11, X12, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, or X26. [ka] JPEG0007660555000064.jpg254156JPEG0007660555000065.jpg254152JPEG0007660555000066.jpg193166

[0212] In certain embodiments, the compound of formula 1 is a compound of formula X9, X12, X19, X22, X8, X18, X1, X14, X15, X20, X7, X11, X17, X21, X2, X16, X23, or X24.

[0213] In certain embodiments, the compound of formula 1 is a compound of formula X9, X12, X19, X22, X8, X18, X1, X14, X15, or X20.

[0214] In certain embodiments, the compound of formula 1 is a compound of formula X9, X12, X19, X22, or X8.

[0215] In certain embodiments, the compound of formula 1 is a compound of formula X1 or X2. EXAMPLES

[0216] Common methods Solid Phase Peptide Synthesis (SPPS) linker coupling and TFA cleavage: Peptide sequences P1-P8 were synthesized on an Intavis MultiPep RSi under standard solid-phase peptide synthesis conditions on a 100 μmol scale, where the synthetic resin was treated with acetic anhydride and pyridine after each amino acid coupling to block unreacted amino groups. The resin was pre-swollen for 15 min before the coupling of the linker. Linker X1 was coupled to P1-P17 on the resin by using 4 equivalents of linker X1 (301 mg), 6 equivalents of oxymer (86 mg), and 6 equivalents of diisopropylamine (DIEA, 105 μL) in 1.3 mL of DMF for 2 h. The amounts used were based on the loading of the SPPS resin (Rinkamide RAM). The reaction mixture was shaken for 120 min, filtered, washed (3 times with DMF and 3 times with DCM), and dried.

[0217] The dried resin was incubated for 120 min with TFA cleavage cocktail (TFA / HO / PhOH / PHSH / ethanedithiol, 82.5:5:5:52.5, v / v / w / v / v, 10 mL per 100 μmol synthesis scale). The cleavage solution was separated from the synthesis resin by filtration and collected in a Falcon tube with 10 times the volume of the solution. The peptide was precipitated by adding 9 times the volume of chilled diethyl ether. The vessel was centrifuged at 5000 rpm for 3.5 min, the supernatant solution was decanted, and the peptide pellet was washed again with diethyl ether and then decanted. The peptide was then dissolved in HO / MeCN / TFA, (99.95:99.95:0.1, v / v / v), frozen in liquid nitrogen, and lyophilized under high vacuum.

[0218] General procedure for modifying / purifying peptides according to the method of the invention Solubilization and immobilization of linker-tagged crude peptides The linker-tagged crude peptide mixture was dissolved in pure DMSO (4.5 mL for a 100 μmol synthesis scale). After complete dissolution, 10% by volume of immobilization buffer 2 (0.1 M citric acid / Na2CO3 pH 3.5 containing 8 M guanidine chloride, 0.45 mL for a 100 μmol synthesis scale) was added.

[0219] The linker-tagged peptides were immobilized on 1.5x amount of aldehyde-modified agarose loaded with 100μmol / 1mL of sedimentation beads (100μmol / 2mL of slurry beads) or hydroxylated poly(methyl acrylate) beads. After dispensing, the bead material was washed with 3x milli-Q water and 3x immobilization buffer 1 (0.1M citric acid / Na2CO3 pH 4.5). Then, the dissolved linker-tagged crude peptide mixture was added to the agarose and shaken. It should be noted that the beads should be able to float freely and well in the immobilization solution. After 90 minutes of reaction time, the immobilization solution was filtered off by suction.

[0220] Washing After immobilization, the supernatant was removed and the peptide-linked beads were washed three times with 5 mL each (for 100 μmol scale) with DMSO. Then, a mixture of L-cysteine ​​in 0.1 M citric acid / Na2CO3 pH 4.5 was added and the reactor was shaken for 15 minutes or more. This mixture was removed and the beads were washed three times with 5 mL (for 100 μmol scale) of the following solvents and solutions: 1) DMSO containing 0.9M guanidinium chloride 3) 70% ethanol in Milli-Q water with 0.1M NaCl

[0221] Modification of peptides immobilized on solid supports In general, the linker-tagged peptide was immobilized on the modification / purification support and the modification of the target peptide was carried out after washing of the peptide-support conjugate.

[0222] In the case of "modifying peptides with fatty acids", after immobilization and washing of the target peptide, the imines that may be constructed on the lysine side chains by excess aldehydes and said aldehydes are converted to oximes by adding O-methylhydroxylamine hydrochloride or L-cysteine. Then, the fatty acid is added together with the coupling reagent and, after a period of preactivation, an organic base is added to the peptide on the support. The excess reagent is then washed away and the peptide is released as described below.

[0223] In the case of "Formation of macrocycles via electrophilic scaffolds", after immobilization and washing of the target peptide, any disulfides that may be constructed are reduced by the addition of a small amount of DTT. After washing away the DTT, the electrophilic scaffold is added to the support. If necessary, this step can be repeated to increase the conversion to the macrocycle product. The excess reagent is then washed away and the peptide is released as described below.

[0224] For "disulfide formation", after immobilization and washing of the target peptide, imines of lysine side chains and thioacetals of cysteine ​​side chains and aldehydes that may be constructed with excess aldehydes were converted to thiazolidines by addition of guanidinium chloride. Disulfides were formed in a 1:1 mixture of DMSO, 0.4 M (NH4)2CO3, 6 M GdmCl. The oxidation buffer was then washed away and the peptides were released as described below.

[0225] Release of modified peptides 500 mg of dithiothreitol (DTT) was dissolved in 5 ml of 5 wt% NaHCO3 pH 8, added to the agarose or poly(methyl acrylate) material (5 mL of MeCN was already on the beads) and shaken for 15 min. The supernatant was discarded and the agarose material was washed three times with water and twice with MeCN to remove excess and oxidized DTT. Subsequently, 2 mL of TFA / H2O (2:3) was added and shaken for another 60 min. Then, another 2 mL of pure TFA was added to the solution and the mixture was eluted and collected in a Falcon tube. Elution was performed two more times using 2 mL each of TFA / H2O (95:5). The linker peptide was precipitated by adding 9 volumes of flash-frozen diethyl ether, followed by centrifugation at 5000 rpm for 3.5 min and decanting off the ether phase. The peptide was then present as a pellet and could be lyophilized if necessary.

[0226] Experimental explanation of the organic synthesis of FA-A and FA-Sp-A Synthesis of 1-bromopropyl-2,3-dipalmitate (Pam2-Br) 1,2-Dipalmitoyl-sn-glycerol (0.4 g, 0.7 mmol, 1 equiv.) was dissolved in 9 mL of DCM and tetrabromomethane (0.5 g, 1.5 mmol, 2.1 equiv.) was added. The solution was cooled to 0 °C and triphenylphosphine (0.4 g, 1.5 mmol, 2.1 equiv.) was added in portions within 5 min. After stirring at 0 °C for 30 min, the ice bath was removed and stirring was continued at room temperature for 3 h and the solvent was removed under reduced pressure. Cyclohexane was added and the mixture was sonicated for about 10 min. After centrifugation, the supernatant was transferred to a clean tube and the solvent was removed under reduced pressure to obtain the crude product. To remove residual O=PPh3, the product was treated with MeOH in an ultrasonic bath. After centrifugation, the solvent was decanted. Washing with methanol was repeated twice to give 1-bromopropyl-2,3-dipalmitate (Pam2-Br) as an off-white solid (0.17 g, 0.27 mmol, 39%).

[0227] R f= 0.68 (chloroform / ethyl acetate 96:4); UPLC-MS: t R =5.69 min (gradient 0-3 min C (0.1% TFA in 8:1:1 H2O / MeCN / iPrOH) 40-100% D (0.1% TFA in 9:1 MeCN / iPrOH), 3-6 min 100% D); UPLC-purity (210 nm) ≥ 98%; ESI-MS: (calculated [M+Na] + : 653.41, 655.41, Detected value: 653.21, 655.68; Calculated value [M-OCOC 15 H 31 ] + : 375.19, 377.19, Detected value: 375.27, 377.26)

[0228] 1 H NMR(400MHz,CDCl3)δ5.21(qd,J=5.5,4.4Hz,1H),4.29(ddd,J=17.5,11.9,5.0Hz,2H),3. 50(qd,J=10.9,5.5Hz,2H),2.34(m,4H),1.62(m,4H),1.28(m,48H),0.88(t,J=6.9Hz,6H)

[0229] Synthesis of 1-iodopropyl-2,3-dipalmitate (Pam2-I) 1,2-Dipalmitoyl-sn-glycerol (0.5 g, 0.88 mmol, 1 equiv.), imidazole (0.12 g, 1.76 mmol, 2 equiv.), and triphenylphosphine (0.5 g, 1.99 mmol, 2.3 equiv.) were dissolved in 7.5 mL of THF. I2 (0.32 g, 1.67 mmol, 2.0 equiv.) was slowly added to the solution. After stirring for 4 h at room temperature, the reaction was quenched by adding 250 mg of NaHCO3 and 1 mL of water. The color of the reaction mixture disappeared. The solvent was evaporated and the residue was washed with 4 mL of water. The suspension was centrifuged and the supernatant was decanted. The crude product was sonicated with 6 mL of MeOH for a few minutes. After centrifugation, the supernatant was decanted. The precipitate was washed four more times with 6 mL of MeOH. The product was dried in vacuum to give 0.47 g (0.69 mmol, 78%) of 1-iodopropyl-2,3-dipalmitate as an off-white solid.

[0230] R f = 0.66 (chloroform / ethyl acetate 96:4); UPLC-MS: t R = 5.83 min (gradient 0-3 min C 40-100% D, 3-6 min 100% D); UPLC-purity (210 nm) ≥ 90%; ESI-MS: (calculated [M+Na] + :701.40, Detected value:701.50; Calculated value [M-OCOC 15 H 31 ] + : 423.18, detected value: 423.28)

[0231] 1 H NMR(400MHz,CDCl3)δ5.00(m,1H),4.26(ddd,J=17.6,11.8,5.0Hz,2H),3.31(ddd,J=2 7.4,10.7,5.8Hz,2H),2.33(m,4H),1.63(m,4H),1.28(m,48H),0.88(t,J=6.8Hz,6H).

[0232] Synthesis of 1-tosylpropyl-2,3-dipalmitate (Pam2-OTs) 4-Toluenesulfonyl chloride (2 g, 10.5 mmol, 10 equiv.) was dissolved in 50 mL of THF. The solution was cooled to 0 °C in an ice bath. 1,2-Dipalmitoyl-sn-glycerol (0.6 g, 1.05 mmol, 1 equiv.) was dissolved in 14.4 mL of THF at room temperature, and 2.84 mL of 15 wt.% aqueous NaOH (10.7 mmol, 10 equiv.) was added. Slowly the 4-toluenesulfonyl chloride solution was added, and the reaction mixture was stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure, and the residue was taken up in chloroform. The organic phase was washed three times with brine and three times with water, then dried over Na2SO4. After filtration, the solvent was removed under reduced pressure to give the crude product as a yellow liquid, which turned into a solid after drying in vacuum. 5 mL of chilled MeOH was added, and the resulting mixture was sonicated for 10 min. After centrifugation, the supernatant was decanted. The residue was treated with MeOH five more times to remove excess tosyl chloride. The product was dried in vacuum to give 0.47 g (0.64 mmol, 61%) of an off-white solid.

[0233] R f = 0.64 (chloroform / ethyl acetate 96:4); UPLC-MS: t R = 4.99 min (gradient 0-3 min C 40-100% D, 3-6 min 100% D); UPLC-purity (210 nm) = 93%; ESI-MS: (calculated [M+Na] + : 745.51, detected value: 745.68)

[0234] 1 H NMR(400MHz,CDCl3)δ7.80(t,J=8.0Hz,2H),7.35(dd,J=8.2,3.1Hz,2H),4.88(m,1H),4 .18(m,4H),2.45(s,3H),2.23(m,4H),1.56(m,4H),1.27(m,48H),0.88(t,J=7.0Hz,6H)

[0235] Synthetic steps for synthesizing 2-iodoethyl palmitate (Pam-Et-I) Palmitoyl Chloride Thionyl chloride (7.2 mL, 100 mmol, 10 equiv.) was slowly added dropwise to palmitic acid (2.6 g, 9.9 mmol, 1 equiv.) in a round-bottom flask. After stirring at room temperature for 30 min, the reaction mixture was stirred at 75° C. for 2 h. The solution was diluted with DCM and washed three times with water. The organic layer was dried over Na2SO4. The salts were removed by filtration and the solvent was removed under reduced pressure. The resulting solution was dried under vacuum to give the product as a colorless solid (2.47 g, 9 mmol, 90%).

[0236] R f =0.52 (chloroform / ethyl acetate 96:4).

[0237] 2-Iodoethyl palmitate (Pam-Et-I) Triethylamine (0.5 mL, 3.6 mmol, 3 equiv.) was added to a solution of 2-iodoethanol (0.71 mL, 9.1 mmol, 5 equiv.) in 18 mL of DCM under nitrogen atmosphere. The solution was stirred at 0° C. for 30 min. Palmitoyl chloride (0.5 g, 1.8 mmol, 1 equiv.) was added at 0° C. Stirring was continued at room temperature for 4 h. The solution was washed three times successively with water and saturated aqueous NaHCO3. After drying over Na2SO4 and filtration, the solvent was removed under vacuum. The crude product obtained was treated with MeOH in an ultrasonic bath and the suspension was centrifuged. The supernatant was decanted and the residue was washed two more times with MeOH. The MeOH solutions from the second and third washes were combined and left at room temperature overnight, whereupon the product crystallized as colorless needles. The solvent was removed and the crystals were dried in vacuum to give 0.15 g (0.36 mmol, 20%) of product.

[0238] R f =0.43 (chloroform / ethyl acetate 96:4).

[0239] 1H NMR(400MHz,CDCl3)δ4.33(t,J=6.8Hz,2H),3.29(t,J=6.8Hz,2H),2.33(t,J=7.5Hz,2H),1.64(p,J=7.4Hz,2H),1.26(m,24H),0.88(t,J=6.9Hz,3H)

[0240] Synthesis of myristic acid N-hydroxysuccinimide ester (Myr-OSu) To a stirred solution of 1 g myristic acid (4.33 mmol, 1 equiv.) and 554 mg N-hydroxysuccinimide (4.77 mmol, 1.1 equiv.) in 44 mL acetonitrile, 994 mg dicyclohexylcarbodiimide (DCC, 4.77 mmol, 1.1 equiv.) was added and the mixture was stirred for 1.5 h. The precipitate formed was removed by filtration and the filtrate was washed with 50 mL acetonitrile. The solvent was then evaporated and dried under reduced pressure to give 1.430 g (4.33 mmol, quantitative yield) of the desired white crystalline title compound. ESI-MS: (calculated [M+Na] + : 348.22, detected value: 348.28)

[0241] Synthesis of 22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetracontanoic acid N-hydroxysuccinimide ester (tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu) To a stirred solution of 260 g of 22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontanoic acid (0.3 mmol, 1 equiv.) and 39 mg of N-hydroxysuccinimide (0.33 mmol, 1.1 equiv.) in 6 mL of acetonitrile, 67 mg of DCC (0.33 mmol, 1.1 equiv.) was added and the mixture was stirred for 1.5 h. The precipitate formed was removed by filtration and the filtrate was washed with 10 mL of acetonitrile. Then, after evaporation of the solvent and drying under reduced pressure, 0.305 g (0.30 mmol, quantitative yield) of the desired title compound was obtained as a colorless oil. ESI-MS: (calculated [M+H] + :943.59, detected value:943.72).

[0242] Example 1: Formation of purified solid support-bound lipidated peptides P1-P2a with palmitoyl-L-Glu(OH)-tBu-building blocks The method of the present invention for peptide modification and purification was applied to two peptides of different length and polarity. These are: [ka] and fragment 25-37 (K34R) of glucagon-like peptide-1 (GLP-1); [ka] and the longer sequence 7–37 (K34R) of glucagon-like peptide-1 (GLP-1).

[0243] The peptide was immobilized and washed on a 5 μmol scale according to the "General Method", with the following deviation from the General Method: after immobilization and removal of the immobilization supernatant, O-methylhydroxylamine hydrochloride solution (4Aq, 0.1M) in immobilization buffer 1 (0.1M citric acid / Na2CO3 pH 4.5) was added and shaken for 20 min. After washing according to the "General Method", it was washed three times with a 10 vol.% pyridine solution in DMF and three times with DMF. This was followed by the addition of a 0.3 M coupling solution of 2-(6-Chlor-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium-hexafluorophosphat (HCTU) or diisopropylcarbodiimide (DIC), 10 equivalents of oxime, and 12 equivalents of palmitoyl-L-Glu(OH)-tBu building block in DMF. This solution was preactivated for 20 min before being added to the agarose beads. After addition, the beads were shaken for 2 h, after which the supernatant was filtered, the solid support was washed 3 times with DMF, 3 times with HO, and 3 times with MeCN, and the modified peptide was released under the conditions shown in "General Methods\Release of Modified Peptides" using PPh3 in MeCN / AcOH / HO (9:0.5:0.5, v / v / v) instead of DTT. To remove the tBu protection of the introduced palmitoyl-L-Glu-tBu group, the released peptide pellet was dissolved in TFA / TIS / HO (96:2:2, v / v / v) and precipitated with 10 volumes of Et2O after 2 h.

[0244] [ka] (SEQ ID NO:18): The peptide precursor P1 was synthesized as described in the SPPS section above. It was immobilized and modified with the palmitic acid (Palm) building block Palm-γGlu(OH)-OtBu (purified according to the method described above). The results are shown in Figure 2 and Table 1.

[0245] [ka] (SEQ ID NO:19): The peptide precursor P2 was synthesized as described in the SPPS section above. It was immobilized and modified with the palmitic acid (Palm) building block Palm-γGlu(OH)-OtBu (purified according to the method described above). The results are shown in Figure 3 and Table 1.

[0246] Example 2: Macrocycle formation of purified solid support-bound peptides P3-P5 with m-xylylene dibromide (intramolecular cyclization) The method of the present invention for peptide modification and purification was applied to three peptides differing in cysteine ​​position and polarity. These were: [ka] It was.

[0247] Peptides were immobilized and washed according to the general method on a 25 μmol scale. To the immobilized peptide, DTT (4 eq., 0.3 M) in H2O was added and the reactor was shaken for 15 min. After removing the DTT solution by filtration, the bead material was washed 3 times with H2O and 3 times with MeCN. Then, m-xylylene dibromide (mDBX) (4 eq., 0.6 M) in acetonitrile was added, followed by an equal volume of cyclization buffer (200 mM NH4HCO3, pH 7.9). The syringe reactor was shaken for 45 min, the reaction mixture was filtered off and the bead material was washed 3 times with MeCN and 3 times with H2O. Then, this procedure was repeated twice after DTT treatment and finally, the cyclized peptide was released under the conditions indicated in "General Methods\Release of Modified Peptides" using PPh3 in MeCN / AcOH / H2O (9:0.5:0.5, v / v / v) instead of DTT.

[0248] [ka] (SEQ ID NO:3)(The underlined C residues are connected by the scaffold): Peptide precursor P3 was synthesized as described in the SPPS section above. It was immobilized and modified with m-xylylene dibromide (mDBX) scaffold and purified according to the method described above. The results are shown in Figure 4 and Table 1.

[0249] [ka] (SEQ ID NO:4) (The underlined C residues are connected by the scaffold): Peptide precursor P4 was synthesized as described in the SPPS section above. It was immobilized and modified with the mDBX scaffold and purified according to the methods described above. The results are shown in Figure 5 and Table 1.

[0250] [ka] (SEQ ID NO:5) (The underlined C residues are connected by the scaffold): Peptide precursor P4 was synthesized as described in the SPPS section above. It was immobilized and modified with the mDBX scaffold and purified according to the methods described above. The results are shown in Figure 5 and Table 1.

[0251] Example 3: Formation of the bicyclic macrocycle of peptides P6-P7 bound to purified solid support with 1,3,5-tris(bromomethyl)benzene (intramolecular cyclization) The method of the invention for peptide modification and purification was applied to two different peptides, which are: [ka] It was.

[0252] The peptide was immobilized and washed on a 5 μmol scale according to the General Methods. To the immobilized peptide, DTT (4 eq., 0.3 M) in H2O was added and the reactor was shaken for 15 min. After filtering off the DTT solution, the bead material was washed 3 times with H2O and 3 times with DMF. Then, 1,3,5-tris(bromomethyl)benzene (TBMB) (4 eq., 0.45 M) in dimethylformamide was added, followed by half the volume of cyclization buffer (400 mM NH4HCO3, 8 M GdmCl, pH 8.0). After shaking the syringe reactor for 45 min, the reaction mixture was filtered and the bead material was washed 3 times with DMF and 3 times with H2O. This procedure was then repeated twice after DTT treatment, and finally the cyclized peptide was released under the conditions indicated in “General Methods\Release of Modified Peptides” using PPh3 in MeCN / AcOH / H2O (9:0.5:0.5, v / v / v) instead of DTT.

[0253] [ka] (SEQ ID NO:6) (The underlined C residues are connected by the scaffold): Peptide precursor P6 was synthesized as described in the SPPS section above. It was immobilized and modified with 1,3,5-tris(bromomethyl)benzene (TBMT) scaffold and purified according to the method described above. The results are shown in Figure 7 and Table 1.

[0254] [ka] (SEQ ID NO:7) (The underlined C residues are connected by the scaffold): Peptide precursor P7 was synthesized as described in the SPPS section above. It was immobilized and modified with 1,3,5-tris(bromomethyl)benzene (TBMT) scaffold and purified according to the methods described above. The results are shown in Figure 8 and Table 1.

[0255] Example 4: Bicyclic macrocycle formation of purified solid support-bound peptide P8a with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (intramolecular cyclization) The method of the present invention for the modification and purification of peptides can be carried out by [ka] was applied to.

[0256] The peptide was immobilized and washed on a 5 μmol scale according to the General Methods. To the immobilized peptide, DTT (4 eq., 0.3 M) in HO was added and the reactor was shaken for 15 min. After removing the DTT solution by filtration, the bead material was washed 3 times with HO and 3 times with MeCN. Afterwards, 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB) in MeCN / cyclization buffer (400 mM NH4HCO3, 8 M GdmCl, pH 7.1, 9:1) was added. After shaking the syringe reactor for 120 min, the reaction mixture was filtered off and the bead material was washed 3 times with HO and 3 times with MeCN. This procedure was then repeated twice after DTT treatment and finally the cyclized peptide was released under the conditions indicated in "General Methods\Release of Modified Peptides" using PPh3 in MeCN / AcOH / HO (9:0.5:0.5, v / v / v) instead of DTT.

[0257] [ka] (SEQ ID NO:8) (The underlined C residues are connected by the scaffold): Peptide precursor P8 was synthesized as described in the SPPS section above. It was immobilized and modified with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TBMT) scaffold and purified according to the methods described above. The results are shown in Figure 9 and Table 1.

[0258] Example 5: Formation of the bicyclic macrocycle of peptide P8b bound to a solid support for purification with 1,3,5-triacryloyl-1,3,5-triazinane (intramolecular cyclization)

[0259] The method of the present invention for the modification and purification of peptides can be carried out by [ka] was applied to.

[0260] The peptide was immobilized and washed on a 5 μmol scale according to the General Methods. To the immobilized peptide, DTT (4 eq., 0.3 M) in HO was added and the reactor was shaken for 15 min. After removing the DTT solution by filtration, the bead material was washed 3 times with HO and 3 times with MeCN. Then, 1,3,5-triacryloyl-1,3,5-triazinane (TATA) in MeCN / cyclization buffer (400 mM NH4HCO3, 8 M GdmCl, pH 7.1, 9:1) was added. After shaking the syringe reactor for 120 min, the reaction mixture was filtered off and the bead material was washed 3 times with HO and 3 times with MeCN. This procedure was then repeated twice after DTT treatment, and finally the cyclized peptide was released under the conditions indicated in “General Methods\Release of Modified Peptides” using PPh3 in MeCN / AcOH / H2O (9:0.5:0.5, v / v / v) instead of DTT.

[0261] [ka] (SEQ ID NO:8) (The underlined C residues are connected by the scaffold): Peptide precursor P8 was synthesized as described in the SPPS section above. It was immobilized and modified with the 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TBMT) scaffold and purified according to the methods described above. The results are shown in Figure 10 and Table 1.

[0262] Example 6: Macrocycle formation of peptide P3b bound to a solid support for purification via disulfides (intramolecular cyclization) The method of the present invention for the modification and purification of peptides can be carried out by [ka] was applied to.

[0263] The peptide was immobilized and washed according to General Methods on a 10 μmol scale. A 1:1 mixture of DMSO and 0.4 M (NH4)2CO3, 6 M GdnCl pH 8.7 was then added and the syringe reactor was shaken for 120 min, after which the reaction mixture was filtered off and the bead material was washed three times with H2O. The cyclized peptide was released using PPh3 in MeCN / AcOH / H2O (9:0.5:0.5, v / v / v) instead of DTT under the conditions given in General Methods / Release of Modified Peptides to give 22% disulfide cyclized peptide.

[0264] [ka] (SEQ ID NO:3) (The underlined C residues are connected by the scaffold): Peptide precursor P3 was synthesized as described in the SPPS section above. It was immobilized and modified by air oxidation according to the method described above, and purified. The results are shown in Figure 11 and Table 1.

[0265] Example 7: Formation of lipidated peptides P1-P3 bound to purified solid support with building blocks 1-bromopropyl-2,3-dipalmitate (a), 1-iodopropyl-2,3-dipalmitate (b), 1-tosyl-2,3-dipalmitate (c), or palm-OC2H4I (d) The method of the present invention for peptide modification and purification was applied to two peptides of different length and polarity. These are model peptides: [ka] It was.

[0266] Linker-tagged peptides were immobilized on a 5 μmol scale according to the General Methods. After removal of the immobilization solution, the agarose beads were washed three times with DMSO. 250 μL of a solution of 2 w% L-cysteine ​​(5 mg) in buffer 1 was added and shaken for 15 min. This solution was aspirated and filtered off, and washing of the peptide-linker-bound beads was carried out as described in the General Methods. For reduction of the linker and removal of the StBu protecting group of the cysteine ​​residue of the peptide, a reducing solution was prepared by dissolving 25 mg (162 μmol, 32 equivalents) of DTT in 250 μL of 5 w% aqueous NaHCO3. 250 μL of MeCN was added to the agarose beads, followed by the addition of this reducing solution. The mixture of peptides with StBu protecting groups on cysteines (P1-P2) was shaken for 60 min, and the mixture of peptides without StBu (P3) was shaken for 15 min, after which the solution was aspirated and filtered off. The agarose beads with the linker-bound peptide were washed three times with water, two times with MeCN, and three times with DMF. A sonicated suspension of 3.2 mg (10 μmol, 2 equiv.) Cs2CO3 in 200 μL of DMF was added. A solution of 20 μmol of palmitoyl building blocks (4 equiv., a) Palm2-Propyl-Br = 12.6 mg, b) Palm2-Propyl-I = 13.6 mg, c) Palm2-Propyl-OTs = 14.5 mg, d) Palm-OC2H4I) in 300 μL of DMF was added. The mixture was shaken for 18–76 h. If any precipitate formed over time, an additional 200–400 μL of DMF was added. The supernatant was removed and the beads were washed five times with DMF, three times with water, and three times with MeCN. 200 μL of TFA / water (40:60) was added and shaken for 60 min to release the peptide. 200 μL of TFA was added to the cartridge and the solution was collected in a 15 mL centrifuge tube. The agarose beads were eluted twice with 200 μL of TFA / water (95:5) in the same tube. 10 mL of chilled Et2O was added to the TFA / water solution with lipidated peptide P1 or P2 to precipitate the peptide. The precipitate was collected and washed once with ether. The peptide was dissolved in water / MeCN (1:1) and lyophilized. For the sample with lipidated peptide P3, the TFA / water solution was concentrated under airflow.Water / MeCN (1:1) was added and the sample was lyophilized.

[0267] H-KKKKSC(C3H5O2-diPalm)-NH2(P12a) (SEQ ID NO:22) : Peptide precursor P12a was synthesized as described in the SPPS section above. It was immobilized and modified with 1-bromopropyl-2,3-dipalmitate, a building block of palmitic acid (Palm), according to the method described above (palmitoylation time = 18 hours). The results are shown in Table 1 and Figure 12.

[0268] H-KKKKSC(C3H5O2-diPalm)-NH2(P12b) (SEQ ID NO:22) : Peptide precursor P12b was synthesized as described in the SPPS section above. It was immobilized and modified with 1-iodopropyl-2,3-dipalmitate, a building block of palmitic acid (Palm), according to the method described above (palmitoylation time = 18 h). The results are shown in Table 1, Figure 1, and Figure 13.

[0269] H-KKKKSC(C2H4O-Palm)-NH2(P12d) (SEQ ID NO:23) : The peptide precursor P12d was synthesized as described in the SPPS section above. It was immobilized and modified with the palmitic acid (Palm) building block Palm-OC2H4I according to the method described above (palmitoylation time = 18 h). The results are shown in Table 1 and Figure 14.

[0270] H-KEKFSINSEDNNGC(C3H5O2-diPalm)-NH2(P13a) (SEQ ID NO:24) : Peptide precursor P13a was synthesized as described in the SPPS section above. It was immobilized and modified with 1-bromopropyl-2,3-dipalmitate, a building block of palmitic acid (Palm), according to the method described above (palmitoylation time = 18 h). The results are shown in Table 1 and Figure 15.

[0271] H-KEKFSINSEDNNGC(C3H5O2-diPalm)-NH2(P13b) (SEQ ID NO:24): Peptide precursor P13b was synthesized as described in the SPPS section above. It was immobilized and modified with 1-iodopropyl-2,3-dipalmitate, a building block of palmitic acid (Palm), according to the method described above (palmitoylation time = 18 h). The results are shown in Table 1 and Figure 16.

[0272] H-KEKFSINSEDNNGC(C2H4O-Palm)-NH2(P13d) (SEQ ID NO:25) : The peptide precursor P13d was synthesized as described in the SPPS section above. It was immobilized and modified with the palmitic acid (Palm) building block Palm-OC2H4I according to the method described above (palmitoylation time = 18 h). The results are shown in Table 1 and Figure 17.

[0273] HC(C3H5O2-DiPalm)RG-NH2(P14b) (SEQ ID NO:26) : Peptide precursor P14b was synthesized as described in the SPPS section above. It was immobilized and modified with 1-iodopropyl-2,3-dipalmitate, a building block of palmitic acid (Palm), according to the method described above (palmitoylation time = 76 h). The results are shown in Table 1 and Figure 18.

[0274] HC(C3H5O2-DiPalm)RG-NH2(P14b) (SEQ ID NO:26) : Peptide precursor P14c was synthesized as described in the SPPS section above. It was immobilized and modified with 1-tosylpropyl-2,3-dipalmitate, a building block of palmitic acid (Palm), according to the method described above (palmitoylation time = 76 h). The results are shown in Table 1 and Figure 19.

[0275] HC(C2H4O-Palm)RG-NH2(P14d) (SEQ ID NO:27): Peptide precursor P13c was synthesized as described in the SPPS section above. It was immobilized and modified with Palm-OC2H4I, a building block of palmitic acid (Palm) according to the method described above (time for palmitoylation = 18 h). The results are shown in Table 1 and Figure 12. Modification of peptide P3 with Palm-OC2H4I was also performed with 10 μmol (2 equiv.) triethylamine in DMF instead of Cs2CO3 in DMF, with similar results.

[0276] Example 8: Formation of purified solid support-bound lipidated peptide P2b with palmitoyl-L-Glu(OSu)-tBu-building block The method of the present invention for the modification and purification of a peptide comprises: [ka] This applies to the longer sequence 7–37 (K34R) of glucagon-like peptide-1 (GLP-1), which, when Palm-Glu-OH is attached to the Lys side chain, is known as liraglutide, a peptide drug.

[0277] The peptide was immobilized and washed on a 25 μmol scale according to "General Methods". After washing according to "General Methods", it was washed three times with DMF. Then, 2 equivalents of DIPEA and 8 equivalents of palmitoyl-L-Glu(OSu)-tBu building block in 600 μL of N-methylpyrrolidone were added. After the addition of this solution, the beads were shaken for 1 h, after which the supernatant was filtered off and the solid support was washed three times with DMSO, three times with DMF and three times with DMSO, and the modified peptide was released under the conditions indicated in "General Methods\Release of Modified Peptides". To remove the tBu protection of the introduced palmitoyl-L-Glu-tBu group, the released peptide pellet was redissolved in TFA / DTT / H2O (94:4:2, v / v / v) and precipitated after 1 h with 10 volumes of Et2O.

[0278] [ka] (SEQ ID NO:21): The peptide precursor P2 was synthesized as described in the SPPS section above. It was immobilized and modified with the palmitic acid (Palm) building block Palm-Glu(OSu)-OtBu (purified according to the method described above). The results are shown in Figure 22 and Table 1.

[0279] Example 9: Formation of purified solid support-bound lipidated peptide P15 by tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu building block The method of the present invention for the modification and purification of a peptide comprises: [ka] This applies to the longer sequence 7–37 (K34R, Aib8A) of glucagon-like peptide-1 (GLP-1), which, when CO2H-Ste-Glu(aeea-aeea-OH)-OH is attached to the Lys side chain, is known as the peptide drug semaglutide.

[0280] The peptide was immobilized and washed on a 25 μmol scale according to the "General Methods". After washing according to the "General Methods", it was washed three times with DMF. Then, 2 equivalents of DIPEA and 8 equivalents of tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu building block in 600 μL of N-methylpyrrolidone were added. After the addition of this solution, the beads were shaken for 1 h, after which the supernatant was filtered off and the solid support was washed three times with DMSO, three times with DMF and three times with DMSO, and the modified peptide was released under the conditions indicated in "General Methods\Release of Modified Peptides". To remove the tBu protection of the introduced tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu group, the released peptide pellet was redissolved in TFA / DTT / H2O (94:4:2, v / v / v) and precipitated after 1 h with 10 volumes of Et2O.

[0281] H-HAibEGTFTSDVSSYLEGQAAK(CO2H-Ste-Glu(aeea-aeea-OH)-OH)EFIAWLVRGRG-OH (SEQ ID NO:28) The peptide precursor of P15 was synthesized as described in the SPPS section above. It was immobilized, modified with the building block tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu, and purified according to the methods described above. The results are shown in Figure 23 and Table 1.

[0282] Example 10: Formation of purified solid support-bound lipidated peptides P16 and P17a with Palm-S building blocks The method of the present invention for the modification and purification of a peptide comprises: The following applies: Both of these are Nef proteins with an N-terminal myristoyl group. The sequence is 1 to 19 (S1M); [ka] and The sequences are 2 to 19. [ka] It was.

[0283] [ka] (SEQ ID NO:17) During SPPS, the building block N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine was added to Lys 3 and Boc-Gly-OH was converted to Gly. 1 After peptide assembly, the N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protecting group was removed by treatment with 2% N2H4*H2O in DMF three times for 3 min. The linker molecule X1 was then coupled as described in General Methods.

[0284] [ka] (SEQ ID NO:16) During SPPS, the building block N-α-(9-Fmoc)-O-(t-butyl-dimethyl-silyl)-L-serine was added to Ser 1 and Boc-Gly-OH was converted to Gly. 1 After peptide assembly, the t-butyl-dimethyl-silyl protecting group was removed by treatment with AcOH / THF / H2O 3:1:1 for 2 h. Then, Fmoc was removed by treatment with piperidine / DMF 1:4 twice for 5 min, the resin was washed, and the linker molecule X1 was coupled as described in General Methods.

[0285] The peptide was immobilized and washed on a 25 μmol scale according to "General Methods". After washing according to "General Methods", it was washed three times with DMF. Then, 2 equivalents of DIPEA and 8 equivalents of Palm-S-(4,6-dimethyl-2-pyrimidinyl)thioate (Palm-S) building block in 600 μL of N-methylpyrrolidone were added. After the addition of this solution, the beads were shaken for 1 h, after which the supernatant was filtered off and the solid support was washed three times with DMSO, three times with DMF and three times with DMSO, and the modified peptide was released under the conditions indicated in "General Methods\Release of Modified Peptides".

[0286] Palm-SGGK(Palm)WSK(Palm)SSVIGWPAVRER-NH2 (SEQ ID NO:29) The peptide precursor of P16 was synthesized as described in the SPPS section above. It was immobilized, modified with Palm-S building blocks, and purified according to the methods described above. The results are shown in Figure 24 and Table 1.

[0287] Palm-GGKWSK(Palm)SSVIGWPAVRER-NH2 (SEQ ID NO:30) The peptide precursor of P17a was synthesized as described in the SPPS section above. It was immobilized, modified with Palm-S building blocks, and purified according to the methods described above. The results are shown in Figure 24 and Table 1.

[0288] Example 11: Formation of purified solid support-bound lipidated peptide P17b with Palm-S building blocks The method of the present invention for the modification and purification of a peptide comprises: [ka] was applied, which is the sequence 2–19 of the Nef protein that has a myristoyl group at the N-terminus.

[0289] [ka] (SEQ ID NO:17) During SPPS, the building block N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine was added to Lys 3 and Boc-Gly-OH was converted to Gly. 1 After peptide assembly, the N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protecting group was removed by treatment with 2% N2H4*H2O in DMF three times for 3 min. The linker molecule X1 was then coupled as described in General Methods.

[0290] The peptide was immobilized and washed on a 25 μmol scale according to "General Methods". After washing according to "General Methods", it was washed three times with DMF. Then, 2 equivalents of DIPEA and 8 equivalents of Myr-OSu building block in 600 μL of N-methylpyrrolidone were added. After the addition of this solution, the beads were shaken for 1 h, after which the supernatant was filtered off and the solid support was washed three times with DMSO, three times with DMF, and three times with DMSO, and the modified peptide was released under the conditions indicated in "General Methods\Release of modified peptides".

[0291] Myr-GGKWSK(Myr)SSVIGWPAVRER-NH2 (SEQ ID NO:31)The peptide precursor of P17b was synthesized as described in the SPPS section above. It was immobilized, modified with the Myr-OSu building block, and purified according to the methods described above. The results are shown in Figure 24 and Table 1.

[0292] Example 12: Macrocycle formation of peptides P6 and P8 bound to purified solid support (aldehyde-modified agarose) with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB) (intramolecular bicyclization) The method of the present invention for peptide modification and purification was applied to two peptides of different length, cysteine ​​position and polarity. These were: [ka] It was.

[0293] The linker-tagged crude peptide was immobilized for 16 h and washed according to the "General Method", except that, as a deviation from the "General Method", after immobilization and removal of the immobilization supernatant, O-methylhydroxylamine hydrochloride solution (5 w% in H2O, 250 μL) was added and the immobilized peptide was shaken for 15 min. After washing according to the "General Method", the linker was reduced and the StBu protecting group was removed by adding 12.5 mg of DTT in 250 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) and shaking for 60 min. Then, 1.9 mg of TCEP dissolved in 30 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) was added and shaking was continued for 15 min. The supernatant was then filtered off and the solid support was washed three times with H2O, two times with MeCN and three times with 5 w% aqueous NaHCO3 (0.6 M; pH 8). 125 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) was added to the solid support, followed by 125 μL of TATB (1-2 equiv.) in MeCN. The beads were shaken for 60 min and then washed 3 times with HO and 3 times with MeCN. The modified peptide was released according to the conditions in General Methods.

[0294] [ka] (SEQ ID NO:6): The peptide precursor P6 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.3 μmol scale. The results are shown in Table 1 and Figure 26. 3.) 1.1 mg of the modified peptide with 69% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 941.1 m / z, detected value: 940.7 m / z).

[0295] [ka] (SEQ ID NO:8): Peptide precursor P8 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.0 μmol scale. The results are shown in Table 1 and Figure 27. 3.) 3.0 mg of the modified peptide with 45% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 1035.6 m / z, detected value: 1035.5 m / z).

[0296] Example 13: Macrocycle formation (intramolecular bicyclization) of peptides P6 and P8 bound to purified solid support (aldehyde-modified poly(methyl acrylate)) with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB) The method of the present invention for peptide modification and purification was applied to two peptides of different length, cysteine ​​position and polarity. These were: [ka] It was.

[0297] The linker-tagged crude peptide was dissolved in pure HFIP and immobilized for 16 h, after which the solid support, aldehyde-modified poly(methyl acrylate), was washed. After immobilization and removal of the immobilization supernatant, O-methylhydroxylamine hydrochloride solution (5 w% in H2O, 250 μL) was added and the immobilized peptide was shaken for 15 min. After washing according to the "General Method", the linker was reduced and the StBu protecting group was removed by adding 12.5 mg of DTT in 250 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) and shaking for 60 min. Then, 1.9 mg of TCEP dissolved in 30 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) was added and shaking was continued for 15 min. The supernatant was then filtered off and the solid support was washed three times with H2O, two times with MeCN, and three times with 5 w% aqueous NaHCO3 (0.6 M; pH 8). 125 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) was added to the solid support, followed by 125 μL of TATB (1-2 equiv.) in MeCN. The beads were shaken for 60 min and then washed three times with H2O and three times with MeCN. The modified peptide was released by treating the solid support with 0.4 mL of TFA / H2O (95:5) for 1 h, followed by the addition of 0.4 mL of TFA and elution of the peptide twice with 0.4 mL of TFA / H2O (95:5). The released modified peptide was precipitated with 10 mL of cold Et2O and the mixture was cooled in a freezer for 15-30 min before being centrifuged and the supernatant decanted.

[0298] [ka] (SEQ ID NO:6): The peptide precursor P6 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.3 μmol scale. The results are shown in Table 1 and Figure 26. 4.) 1.5 mg of the modified peptide with 70% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 941.1 m / z, detected value: 941.0 m / z).

[0299] [ka] (SEQ ID NO:8): Peptide precursor P8 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.0 μmol scale. The results are shown in Table 1 and Figure 27. 4.7 mg of the modified peptide with 44% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 1035.6 m / z, detected value: 1035.3 m / z).

[0300] Example 14: Macrocycle formation of peptides P6 and P8 bound to purified solid support (aldehyde-modified agarose) with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB) using sodium dodecyl sulfate as an additive for peptide dissolution (intramolecular bicyclization) The method of the present invention for peptide modification and purification was applied to two peptides of different length, cysteine ​​position and polarity. These were: [ka] It was.

[0301] The linker-tagged crude peptide was immobilized for 16 h and washed according to the "general method", except that the peptide was dissolved in 1 M SDS in DMSO + 10 vol% immobilization buffer 2, and after immobilization and removal of the immobilization supernatant, O-methylhydroxylamine hydrochloride solution (5 w% in H2O, 250 μL) was added and the immobilized peptide was shaken for 15 min. After washing according to the "general method" and three additional washes with 8 M urea in H2O / MeOH (9:1), the linker was reduced and the StBu protecting group was removed by adding 12.5 mg DTT in 250 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) and shaking for 60 min. Then, 1.9 mg TCEP dissolved in 30 μL of 5 w% aqueous NaHCO3 (0.6 M, pH 8) was added and shaking was continued for 15 min. The supernatant was then filtered off and the solid support was washed three times with HO, two times with MeCN, and three times with 5 w% aqueous NaHCO3 (0.6 M, pH 8). 125 μL of 5 w% aqueous NaHCO3 (0.6 M; pH 8) was added to the solid support, followed by 125 μL of TATB (1-2 equiv.) in MeCN. The beads were shaken for 60 min and then washed three times with HO and three times with MeCN. The modified peptide was released according to the conditions in "General Methods".

[0302] [ka] (SEQ ID NO:6): The peptide precursor P6 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.3 μmol scale. The results are shown in Table 1 and Figure 26. 5.) 5.8 mg of the modified peptide with 61% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 941.1 m / z, detected value: 940.7 m / z).

[0303] [ka] (SEQ ID NO:8):Peptide precursor P8 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.0 μmol scale. The results are shown in Table 1 and Figure 27. 5.) 6.3 mg of the modified peptide with 43% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 1035.6 m / z, detected value: 1034.9 m / z).

[0304] Example 15: Macrocycle formation (intramolecular bicyclization) of peptides P6 and P8 bound to purified solid support (aldehyde-modified poly(methyl acrylate)) with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB) using sodium dodecyl sulfate as an additive for peptide dissolution

[0305] Crude peptides with linker tags were immobilized on the solid support aldehyde-modified poly(methyl acrylate) as described in Example 13, but instead of pure HFIP, 1 M SDS in DMSO+10 vol.% immobilization buffer 2 was used to dissolve the peptide as described in Example 14. What was different was that 40 vol.% of the bead volume of aniline buffer solution (0.46 vol.% aniline dissolved in 0.79 w% NHOAc in H2O / AcOH (9:1)) was added to the solid support before adding the dissolved peptide. The immobilized peptide was washed, modified with TATB, and released as described in Example 13. What was different was that after peptide immobilization and washing, three additional washes were performed with 8 M urea in H2O / MeOH (9:1).

[0306] [ka] (SEQ ID NO:6): The peptide precursor P6 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.3 μmol scale. Results similar to those in Example 3 were obtained and are shown in Table 1. 3.9 mg of the modified peptide with 55% purity was obtained as a white powder. ESI-MS: (calculated MH2+ : 941.1 m / z, detected value: 940.7 m / z).

[0307] [ka] (SEQ ID NO:8): Peptide precursor P8 was synthesized as described in the SPPS section above. It was immobilized and modified with TATB according to the method described above on a 1.0 μmol scale. Results similar to those in Example 3 were obtained and are shown in Table 1. 3.7 mg of the modified peptide with 48% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 1035.6 m / z, detected value: 1035.3 m / z).

[0308] Example 16: Macrocycle formation (intramolecular bicyclization) of peptides P6 and P8 bound to purified solid supports (aldehyde-modified agarose or poly(methyl acrylate)) with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB) with cooling The method of the present invention for peptide modification and purification was applied to two peptides of different length, cysteine ​​position and polarity. These were: [ka] It was.

[0309] The linker-tagged crude peptides were immobilized, washed, modified with TATB, and released onto the solid support aldehyde-modified agarose or poly(methyl acrylate) as described in Examples LuS1 or LuS2, respectively, except that the modification with TATB was carried out at a temperature of 1° C. by placing the reaction vessel in an ice-water bath.

[0310] [ka] (SEQ ID NO:6):The peptide precursor P6 was synthesized as described in the SPPS section above. It was immobilized on aldehyde-modified poly(methyl acrylate) and modified with TATB according to the method described above on a 1.3 μmol scale. The results are shown in Table 1 and Figure 26. 6.) 6.2 mg of the modified peptide with 69% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 941.1 m / z, detected value: 940.5 m / z).

[0311] [ka] (SEQ ID NO:8): Peptide precursor P8 was synthesized as described in the SPPS section above. It was immobilized on aldehyde-modified poly(methyl acrylate) and modified with TATB according to the method described above on a 1.0 μmol scale. The results are shown in Table 1 and Figure 27. 6.) 7.1 mg of modified peptide with 44% purity was obtained as a white powder. ESI-MS: (calculated MH 2+ : 1035.6 m / z, detected value: 1035.2 m / z).

[0312] Example 17: Macrocycle formation of peptide P3 bound to a solid support for purification via disulfides (intramolecular cyclization) The method of the invention for peptide immobilization, modification and purification was applied to the following peptides: [ka]

[0313] [ka] (SEQ ID NO:3)(Underlined C residues are connected by the scaffold): Linker-tagged peptides are immobilized on 1.5 times the amount of aldehyde-modified polymethacrylate loaded with 152.6 μmol / g of beads. After transfer, the bead material is washed 3 times with milli-Q water and 3 times with immobilization buffer 1 (0.1 M citric acid / Na2CO3 pH 4.5). 113 μl of DMSO is added to the beads and soaked for 5 minutes. Linker-tagged crude peptide is dissolved in 223 μl of DMSO and 26 μl of buffer 2, and the mixture is added to the polymethacrylic beads and shaken. It should be noted that the beads should be able to move freely and well in the immobilization solution. After a reaction time of 72 hours, the immobilization solution is filtered off by suction. The immobilization was equally efficient with different amounts of beads. At loadings of 1.5-15x (relative to the linker-tagged peptide) of 152.6 μmol / gm of aldehyde-modified polymethacrylate, the beads were successful in quantitatively immobilizing the linker-tagged peptide (1.5x required 72 hours, 3x required 48 hours, 7.5x required 300 minutes, while 15x required 180 minutes).

[0314] Washing: After immobilization, the beads to which the peptide linker was bound were washed three times with 5 mL (in the case of a 2.5 μmol scale) of the following solvent and solution. 1) 0.9M guanidinium chloride in DMSO 3) 70% ethanol containing 0.1M NaCl Milli-Q water

[0315] Reduction: 5 ml of acetonitrile was added to the immobilized peptide. A fresh solution of DTT in 5 wt% NaHCO3 solution was added (33.6 equiv., 0.26 M) and the reactor was shaken for 60 min. After removing the DTT solution by filtration, the bead material was washed 3 times with HO and 3 times with MeCN.

[0316] Oxidation: Then, 500 μL of HO / MeCN (1:1) was added to the beads, followed by 20 μL of iodine (10 mg / mL) several times until the solution turned yellow, and the cartridge was shaken for a total of 10 min. The beads were washed three times with an aqueous solution of ascorbic acid (15 mg / mL), three times with water, and twice with MeCN.

[0317] Release of peptide: 166.6 μl of 95% TFA was added to the vial and after 30 min, the peptide was eluted twice with 166.6 μl of 95% TFA in water in pre-weighed cold ether vials, the peptide was precipitated by centrifugation, and the ether was decanted to obtain the pure peptide.

[0318] Release was also achieved equally efficiently by treating the oxidized beads with 166 μl of 80:10:10 (HO:MeCN:TFA) or 90:05:05 (HO:MeCN:TFA) for 18 h, followed by elution with 2.5 volumes of HO / MeCN (7:3) + 0.1% TFA and 1.5 volumes of HO / MeCN (3:7) + 0.1% TFA. The reconstituted solution was lyophilized to give the desired peptide in 95% purity. ESI-MS: (calculated MH 2+ : 774.13 g / mol, detected value: 773.91 m / z).

[0319] [ka] (SEQ ID NO:4)(Underlined C residues are connected by the scaffold): Linker-tagged peptides are immobilized on 3x amount of aldehyde-modified polymethacrylate loaded with 152.6 μmol / g of beads. After transfer, the bead material is washed 3 times with milli-Q water and 3 times with immobilization buffer 1 (0.1 M citric acid / Na2CO3 pH 4.5). 226 μl of 83% TFA was added to the beads. The linker-tagged crude peptide was dissolved in 498 μl of 83% TFA, and the mixture was then added to the polymethacrylate beads and shaken. It should be noted that the beads should fluctuate freely and well in the immobilization solution. After 150 min of reaction time, the immobilization solution was filtered off by suction. Immobilization was also performed efficiently using solvents such as HFIP (150 min). Immobilization was also performed in DMSO as described for P3 peptide, using different amounts of beads corresponding to P3 peptide (1.5, 3, 5 aldehyde groups per peptide). After 16 h of immobilization, 55% of the peptide was immobilized with 1.5x aldehyde, 70% of the peptide with 3x aldehyde, and no peptide was quantitatively immobilized with 5x aldehyde. Addition of a catalyst (50 mM aniline) to 3x aldehyde increased the immobilization rate by 4-fold.

[0320] Washing: When TFA was used as the immobilization solvent, after immobilization, the beads to which the linker was bound to the peptide were washed three times with 3 mL (in the case of a 2.5 μmol scale) of each of the following solvents and solutions. 1) Milli-Q water 2) 0.9M guanidinium chloride in DMSO 3) 70% ethanol in 0.1M NaCl Milli-Q water

[0321] When HFIP was used as the immobilization solvent, after immobilization, the beads to which the linker was bound to the peptide were washed three times with 3 mL (in the case of a 2.5 μmol scale) of each of the following solvents and solutions. 1) HFIP 2) Milli-Q water

[0322] In all other cases, the peptides were washed as described for the P3 peptide.

[0323] Reduction: The reduction procedure followed the method described for the P3 peptide.

[0324] Oxidation: Then, 1000 μL of DMSO was added to the beads, followed by purging air into the bead solution (5 min), shaking for 24 h, filtering, washing (3 times with H2O, 1 time with CH3CN), and drying.

[0325] Peptide release: The peptide release procedure was carried out as described for the P3 peptide, yielding the desired peptide with a purity of 79%. ESI-MS: (calculated MH 2+ : 717.43 g / mol, detected value: 717.71 m / z).

[0326] [ka] (SEQ ID NO:9) (The underlined C residues are connected by the scaffold): The procedures for immobilization, washing, and reduction of TFA followed the method described for the P4 peptide.

[0327] Oxidation: Then, 1000 μL of 20% DMSO in 0.1 M solution of disodium hydrogen phosphate was added to the beads, followed by purging the bead solution with air (5 min), shaking for 4 h, filtering, washing (3 times with HO and 1 time with CHCN) and drying.

[0328] Peptide release: The peptide release procedure was carried out according to the method described for the P3 peptide, and the desired peptide was obtained with a purity of 85% (calculated MH 2+ : 542.72 g / mol, detected value: 543.12 m / z).

[0329] [ka] (SEQ ID NO:10)(The underlined C residues are connected by the scaffold): TFA immobilization, washing and reduction, oxidation, release of the peptide were performed according to the method described for P9 peptide, yielding the desired peptide with a purity of 10% (calculated MH 4+ calc. : 959.48m / z, detected value: MH 4+ found. :959.84m / z)

[0330] [ka] (SEQ ID NO:11) (The underlined C residues are connected by the scaffold): TFA immobilization, washing and reduction, oxidation, release of the peptide were performed according to the method described for P9 peptide, yielding the desired peptide with a purity of 10% (calculated MH 2+ calc. : 763.21m / z, detected value: MH 2+ found. :763.85m / z)

[0331] [Table 1] JPEG0007660555000115.jpg254148JPEG0007660555000116.jpg25477

Claims

1. A method for modifying and purifying a peptide, comprising the steps of: providing a linker-tagged crude peptide L-P, said crude peptide being covalently bound to a linker L, - in an immobilization step, said linker-tagged peptide L-P is coupled to a solid support to obtain an immobilized linker-tagged peptide S-L-P, - in a modification step, said immobilized linker-tagged peptide S-L-P is modified with one or more organic molecules to obtain the immobilized linker-tagged peptide S-L-mP, - in a releasing step, a reducing agent is added to obtain a reduced intermediate RI, which is then decomposed to obtain a modified peptide mP, Including, The linker L is an X-T of formula 1. ｂ -V ａ -U-Y-(1), During the ceremony, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, 【Chemistry 1】 During the ceremony, - Each R １ and R ２ are independently selected from H or B, where at least R １ Or R ２ is B, - R ３ is selected from H or B; - R ４ is H, C １ ~C １２ alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group; B is an acid labile amine protecting group, T is a linear or branched spacer, b is 0 or 1 and is attached directly to the moiety U or via the moiety V, V is an electron-withdrawing moiety and a is 0 or 1, where the sum of a and b is 1 or 2; U is a phenyl or 5- or 6-membered heteroaryl moiety which is ｑ and E ｎ and optionally C １～６ and optionally substituted with alkyl, where -W is -N ３ , -NO ２ , -S(=O)-R ８ , -S-S-R ８ , —O—CH ２ -N ３ , -OC(=O)-O-CH ２ -N ３ , -N=N-phenyl, -N=N-R ８ , 【Chemistry 2】 is selected from In the formula, R ８ is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C １ ~C ６ Alkyl or -(CH ２ ) ｐ -NMe ２ wherein p is 1, 2, 3 or 4 and q is 0, 1, 2, 3 or 4; E is an electron-withdrawing group under acidic conditions and n is 0, 1, 2, 3 or 4; U is a phenyl moiety and Y is -(CH ２ ) ｍ When —O—C(═O)—, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and - W is in the ortho or para position to Y, and Y is -(CH ２ ) ｍ -C(=O)- or -(CH ２ ) ｍ -O-C(=O)-, and m is 1, 2 or 3; The method.

2. The linker-tagged peptide L-P is - The crude peptide P synthesized on the synthesis support SR is fused with a free linker L ｆ and by releasing the electron-withdrawing leaving group Z to give the linker-tagged supported crude peptide L-P-SR, where L ｆ is the linker X-T ｂ -V ａ -U-Y-Z, where X, T, b, V, a, U and Y are as defined by claim 1 and Z is an electron-withdrawing leaving group; - cleaving said linker-tagged peptide L-P from said synthesis support S-R to obtain said linker-tagged peptide L-P; The method of claim 1 provided by

3. The method of claim 2, wherein cleavage of the linker-tagged peptide L-P is achieved by TFA, HBr, HCl or acetic acid under acidic conditions.

4. The method of claim 1, wherein the solid support is aldehyde-modified and selected from agarose, hydroxylated poly(methyl acrylate), poly(glycidyl acrylate), poly(glycidyl methacrylate), polylysine, polyethylene glycol, polyamide, polyacrylamide, polystyrene, or copolymers thereof.

5. The method according to any one of claims 1 to 4, wherein the immobilization step is carried out at a pH < 5.

6. The method according to any one of claims 1 to 5, wherein the reducing agent in the releasing step is selected from triphenylphosphine, dithiothreitol (DTT), dithioerythritol (DTE), trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine.

7. The method according to any one of claims 1 to 6, wherein the reduced intermediate RI is decomposed by changing the pH to a lower pH than that used during reduction of the linker.

8. 8. The method of any one of claims 1 to 7, wherein the peptide comprises an amino acid containing at least one nucleophilic amine in the amino acid side chain, the amino acid being selected from lysine, ornithine, α-aminoglycine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, 2,6-diamino-4-hexynoic acid, β-(1-piperazinyl)alanine, 4,5-dehydrolysine, or d-hydroxylysine, or the nucleophilic amine is at the N-terminus of the peptide.

9. The organic molecule is a compound of formula 5a, 5b, 5c or 5d, FA ｘ -Sp ｎ -A(5a), FA ｘ -Sp ｎ -OH(5b), FA-A(5c), or FA-OH(5d), During the ceremony, - FA is -C(=O)-C １～２４ Alkyl or -C(=O)-C ２～２４ alkenyl, where the alkyl or alkenyl portion may be unsubstituted or substituted with one or more substituents independently selected from: -F or -COOH; - Sp is a spacer comprising a length of 2 to 50 atoms, optionally a terminal -C(=O)- moiety, and x linking moieties independently selected from -NH-, -O-, -S-, said spacer being connected to FA via said linking moieties; - x is an integer between 1 and 5, n is an integer between 1 and 10, - Aは、-F、-Cl、-Br、-I、-N ３ 、-O(C=O)CH ２ (C=O)OH、-SR １４ 、-OCF ３ 、-OCH ２ CF ３ 、-OSO ２ CF ３ 、-SO ２ C ６ H ４ CH ３ 、-SO ２ CF ３ 、-SO ２ CH ３ 、 【Chemistry 3】 is selected from - Here R １４ is C １ ~C ６ is an alkyl, aryl or benzyl substituent; The method according to claim 8.

10. Sp is independently - linear or branched alkyl substituted with 1 to 5 linking moieties; - -NH-[C ｒ Alkyl-O] ｓ -C ｔ alkyl-C(═O)—, where r is independently selected from 1, 2, or 3, s is an integer between 1 and 10, and t is 1, 2, or 3; or - amino acids, which may be protected or unprotected, in which the N-terminus and / or the side chain amine is replaced by -NH- and the C-terminus and / or the side chain carboxylic acid moiety is replaced by -C(=O)-; Selected from:

10. The method of claim 9.

11. The organic molecule of formula 5b or 5d is coupled to the at least one nucleophilic amine in the modification step in the presence of an activating reagent, which may be selected from the group consisting of tetramethylaminium tetrathioborate (TBTU), (3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazin-4(3H)-one) (DEPBT), (2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate) (HCTU), tetramethylaminium hexafluorophosphate (TCT ...

11. The method of any one of claims 8 to 10, wherein the aryl group is selected from thylaminium hexafluorophosphate (HATU), (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), dicyclohexylcarbodiimide (DCC), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-1H-benzotriazole (HOAt), NHS, or diisopropylcarbodiimide (DIC).

12. 2. The method of claim 1, wherein the peptide comprises at least two amino acids containing a nucleophilic thiol, and wherein the at least two amino acids are independently selected from cysteine, homocysteine, or penicillamine.

13. The organic molecule is an organic scaffold molecule comprising a scaffold having at least two electrophilic centers, the electrophilic centers being formed by at least two benzyl halogen substituents, at least two acrylamides, or at least two acrylic esters, and the organic scaffold molecule is selected from the group consisting of 1,4-bis(bromomethyl)benzene, m-xylylene dibromide (mDBX), 1,3,5-tris(bromomethyl)benzene (TBMB), N,N'-bis(chloroacetyl)-1,2-ethylenediamine, and N,N'-bis(chloroacetyl)-1,3-propylene. diamine, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-chloroacetamide), N,N'-bis(bromoacetyl)-1,2-ethylenediamine, N,N'-bis(bromoacetyl)-1,3-propylenediamine, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-bromoacetamide), 1,3,5-triacryloyl-1,3,5-triazinane (TATA), 1,1',1"-(1,3,5-triazinane-1,3,5-tri N,N',N"-(benzene-1,3,5-triyl)tris(2-bromoethanone) (TATB), N,N',N"-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB), N,N',N"-benzene-1,3,5-triyltrisprop-2-enamide (TAAB), 2,4,6-tris(bromomethyl)-s-triazine (TBMT), N,N',N"-(nitrilotris(ethane-2,1-diyl))triacrylamide, N,N',N"-(nitrilotris(ethane-2,1-diyl))tris(2-chloroacetamide), N,N',N"-(nitrilotris(ethane-2,1-diyl))tris(2-chloroacetamide), N,N',N"-(trilotris(ethane-2,1-diyl))triethenesulfonamide, N,N',N"-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))triacrylamide, N,N',N"-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N"-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N',N"-((1,3,5-triazinane-1,3,The method according to claim 12, wherein the aryl group is selected from N,N',N"-(benzene-1,3,5-triyltris(methylene))triethenesulfonamide, N,N',N"-(benzene-1,3,5-triyltris(methylene))triacrylamide, N,N',N"-(benzene-1,3,5-triyltris(methylene))tris(2-chloroacetamide), N,N',N"-(benzene-1,3,5-triyltris(methylene))triethenesulfonamide, tri(2-hydroxyethyl)isocyanurate triacrylate, and 1,3,5-tris(oxiran-2-ylmethyl)-1,3,5-triazinane-2,4,6-trione.

14. The modification step comprises a reduction step followed by a cyclization step, - in the reduction step, the immobilized linker-tagged peptide S-LP is treated with a reducing agent, and - in the cyclization step, the organic scaffold molecule is coupled to at least two nucleophilic thiols under basic conditions; The method of claim 13.

15. 13. The method of claim 12, wherein the number of said nucleophilic thiols is an even number, and said nucleophilic thiols are oxidized in the modification step by a basic aqueous solution having a pH>7 to obtain a macrocyclic cross-linked peptide.

16. The number of the nucleophilic thiols is an even number, and the nucleophilic thiols are, in the modification step, in the presence of oxygen, and / or - DMSO, iodine, N-chlorosuccinimide, Tl(OAc) ３ , Tl(CF ３ COO) ３ , C.H. ３ SiCI ３ -Ph(SO)Ph[Pt(ethylenediamine) ２ C ２ ]Cl ２ , 2,2'-dithiobis(5-nitropyridine), 5,5'-dithiobis-(2-nitrobenzoic acid), trans-[Pt-(CN) ４ C ２ ] ２－ , glutathione-glutathione disulfide, K ３ Fe(CN) ６ In the presence of an oxidizing additive selected from 16. The method of claim 12 or 15, wherein the macrocyclic bridged peptide is obtained by oxidation.

17. X is selected from the moieties of formula 2, 2a, 3, 3a or 4, 【Chemistry 4】 During the ceremony, - Each R １ and R ２ are each independently selected from H or B, where at least R １ Or R ２ is B, - R ３ is selected from H or B; - R ４ is H, C １ ~C １２ alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group; B is an acid labile amine protecting group, -T is -C １～１２ Alkyl-, (-C ２ H ４ O-) １～１２ , -C(=O)-, -C(=O)-JR ９ --JR ９ -C(=O)-, -JR ９ -, phenyl, a linear or branched spacer containing 1 to 5 moieties of 5- or 6-membered heteroaryl, J is C or N; R ９ are, independently of each other, H, C １～４ Alkyl, -C １～６ Alkyl-NH ２ , -C １～６ Alkyl-NHB, -C １～６ Alkyl-NB ２ , -R １５ , -C １～６ Alkyl-R １５ , -C １～６ Alkyl-NH-R １５ where: B is an independently selected acid labile amine protecting group; R １５ is a blocking agent capable of reacting with an aldehyde moiety and is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, wherein The amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid-labile amine protecting group B; b is 0 or 1, - V is -NR １１ -C(=O)-, -C(=O)-NR １１ -, -S(=O)-, -NR １２ - (CH ２ ) ｐ -,-piperazinyl-(CH ２ ) ｐ -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, 【Chemistry 5】 an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, where R １１ is H or C １～４ alkyl, R １２ is H or C １～４ alkyl, p is 0, 1 or 2; a is 0 or 1 and the sum of a and b is 1 or 2; U is a phenyl or 5- or 6-membered heteroaryl moiety which is related to the moieties V, W ｑ and E ｎ and which is linked to at least one of C １～６ and optionally substituted with alkyl, V is as described above, W is -N ３ , -NO ２ , -S(=O)-R ８ , -S-S-R ８ , —O—CH ２ -N ３ , -OC(=O)-O-CH ２ -N ３ , -N=N-phenyl, -N=N-R ８ , 【Chemistry 6】 where: R ８ is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C １ ~C ６ Alkyl or -(CH ２ ) ｐ -NMe ２ and p is 1, 2, 3 or 4; E is an electron-withdrawing group under acidic conditions; n is an integer between 0 and 4, and q is an integer between 0 and 4, where the sum of n and q is 4 or less, and where: U is a phenyl moiety and Y is -(CH ２ ) ｍ When —O—C(═O)—, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and W is in the ortho or para position relative to Y; - Y is -(CH ２ ) ｍ -C(=O)- or -(CH ２ ) ｍ -O-C(=O)-, m is 1, 2 or 3; Z is an electron-withdrawing leaving group; The method according to any one of claims 1 to 16.

18. E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N(C ２ H ４ ) ２ N.H. ２ , -N(C ２ H ４ ) ２ N—B, —N═N-phenyl, —N═N—R ８ , -(CH ２ ) ｒ -NH-C １～６ Alkyl, -(CH ２ ) ｒ -N(C １～６ Alkyl) ２ -, -F, -Cl, -Br, -I, -CN, -NO ２ , -N ３ , -CF ３ , -SO ３ H, -CO ２ H, -C(=O)NH ２ , -SO ２ Me, -SOMe, -SO ２ -Et, -SOEt, where R ８ is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C １ ~C ６ Alkyl or -(CH ２ ) ｐ -NMe ２ and p is 1, 2, 3 or 4; B is an acid labile amine protecting group, and r is 0, 1, 2, 3 or 4; The method according to any one of claims 1 to 17.

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