Method for modifying peptides immobilized on solid supports by traceless reductively cleavable linker molecules

Abstract

The present invention relates to a method for modifying and purifying a peptide, comprising a fixation step, a modification step and a release step. In the fixation step, a crude linker-tagged peptide L-P is coupled to a solid support to produce an immobilized linker-tagged peptide S-L-P. Subsequently, the immobilized linker-tagged peptide S-L-P is modified with one or more organic molecules to produce an immobilized linker-tagged peptide S-L-mP. Finally, the modified peptide is released through a reduced intermediate RI. The linker molecule is a compound of formula 1, X-T b ‑V a ‑U‑Y‑Z (1), which can be coupled to a purification resin via moiety X and coupled to a peptide via moiety Y under release of a 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 combined with 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] Modified peptides are gaining increasing attention as pharmaceutically relevant compounds. Modifications such as fatty acids or bridging molecules to induce peptide macrocycles enhance the pharmacokinetics of the desired peptide sequence while preventing degradation by in vivo proteases. Peptide modification is therefore a key feature of modern peptide therapeutics. However, the introduction of these modifications is often difficult and makes the handling and purification of the modified peptides very difficult or impossible. Especially since the most advanced purification methods have been developed and established for unmodified peptides. Background Art

[0002] The production of peptides by solid phase peptide synthesis (SPPS) is a powerful and rapid method for obtaining a large number of different peptides or a larger number of single peptides within a foreseeable time frame. Therefore, it is widely used to make a larger number or a large number of peptides. The procedure consists of coupling the first N-terminally protected amino acid to a synthetic resin, and then repeating the following cycle: N-terminal deprotection, coupling the next N-terminally protected amino acid, and capping the unreacted peptide sequence. Finally, the synthesized peptide is cleaved from the synthetic resin and purified. These linear peptides can be further modified to improve their properties.

[0003] Due to rapid degradation by proteases and peptidases, straight chain unmodified peptides show low in vivo stability. In addition, they show no cell permeability and rapid renal clearance in mammals (Witt et al. (2001), Peptides (22), 2329-43). Two main modifications are used to improve the stability and efficacy of peptides in vivo: lipidation with fatty acids and cyclization of straight chain peptides to form macrocycles.

[0004] Lipidation is one of the most important modifications of peptide drugs. Fatty acids attached to peptides affect their hydrophobicity, secondary structure and their aggregation behavior while maintaining their specificity for protein targets. Fatty acids enhance metabolic stability, membrane permeability, bioavailability, and alter the pharmacokinetic and pharmacodynamic properties of modified peptides (L. Zhang, G. Bulaj (2012) Current Medicinal Chemistry 19 (11), 1602-18). Drug examples include GLP-1 receptor agonists such as liraglutide. Semaglutide and insulin derivatives, such as detemir and degludec All of them carry fatty acids on the lysine side chain. The fatty acids of the lipidated drugs can interact and bind to albumin through three high-affinity binding domains. In addition to the different circulation times in the organism, the attached albumin has a protective effect on these lipidated peptides.

[0005] The method of large-scale production of lipidated drugs currently depends on two main steps: first, a biological step, in which the precursor peptide is expressed by a suitable cell line (mainly yeast). Secondly, after the precursor peptide is recovered and purified, acylation with fatty acids or fatty acid spacer building blocks is achieved by chemical means in a solution with a higher pH, followed by a final purification step. In the early stages of drug development, solid phase peptide synthesis was used to synthesize a large number of candidate lipidated drugs. Lipid acylation is mainly achieved by using an orthogonal protecting group strategy. Traditionally, in the SPPS process, orthogonally protected lysine residues are incorporated into the peptide sequence. The protecting group is selectively separated and lipids are introduced by adding pre- or in-situ activated lipids to other protected peptides fixed on a synthetic resin. Alternatively, the peptide can be lipidated in solution after production via chemical or biological means. Whether it is necessary to adopt selective acylation technology or the N-terminus must be orthogonally protected and must be deprotected after successful lipidation.

[0006] Cyclic peptides have many advantages over linear peptides. They are significantly more resistant to both exo- and endo-proteases. In addition, they exhibit superior binding affinity to the desired protein target, most likely because they exhibit a more stringent and arched binding motive to the target receptor. A successful cyclic peptide drug is the anticancer drug octreotide. The immunosuppressant cyclosporine or the neuropeptide hormone oxytocin ( or others). Methods for forming peptide rings can be divided into two major categories: a) one-component systems, in which peptides can be cyclized by utilizing their intrinsic functional groups to form rings connected by, for example, amides or disulfides. Other covalent linkages can be produced by incorporating non-natural amino acids. b) two-component systems, in which the functional groups of the peptide are used for reactions with bridging scaffolds. Organic molecules can be used to form single, double or multiple rings together with peptides. The most common method is to utilize the nucleophilic functional groups of the peptide, especially thiols, to react them with scaffolds carrying electrophilic reaction sites. When SPPS is used, the peptide is cyclized on a synthetic resin or in solution after the peptide is separated from the synthetic resin. Both methods have their drawbacks. Modification on a solid support requires orthogonally protected amino acids, which are expensive and require an additional deprotection step before modification. Once deprotected, pseudo-ultra-high dilution on the solid support helps prevent undesirable reactions of one scaffold with two or more linear peptides. In addition, excess reagents can be used because they can be easily washed off. Disadvantages of cyclization in solution are that side reactions can occur, such as bridging of two linear peptides by one scaffold, and removal of excess reagents is cumbersome. Therefore, modifications are performed in highly diluted solutions using one equivalent of the scaffold.

[0007] Many methods have been published for peptide modification in solution; in the context of the present invention, only those disclosures that use peptides attached to a solid support to enable modification of the peptide will be discussed.

[0008] WO / 2004 / 077062 uses membranes as solid supports to synthesize a number of desired peptides as candidate drugs. After standard SPPS of the polypeptide and removal of the protecting groups, a molecular scaffold, such as tri(bromomethyl)benzene, is added to cyclize the peptides on the support. Therefore, the cyclized peptides are used to screen for binding to proteins. This method cannot be used in other ways than drug discovery because the peptides produced are still covalently bound to the synthetic support on which they are produced. Therefore, it is impossible to analyze the peptides before the binding test. Therefore, the research must believe that all pre-formed steps have produced the expected cyclic peptides, resulting in the possibility of false positives and even more likely false negative results.

[0009] (WO2009 / 098450 and Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7) disclose a method for modifying peptides that have been synthesized by phage and displayed on the surface of phage. A large peptide library with three cysteine ​​residues in its sequence is thus generated, and the phage is treated with a tri-(bromomethyl)benzene (TBMB) solution to cyclize the displayed peptides into a bicyclic ring. The phage is then incubated with a biotinylated target protein and further rounds of screening are performed. After the phage titer increases, the phage DNA is sequenced to deduce the selected sequence. In addition to a large number of bicyclic peptide variants (4.4x109 ) The disadvantage of this method for synthetic purposes is that it cannot be used to produce peptides. It has only diagnostic use. It also has a disadvantage in terms of diagnosis, that is, it is not possible to analyze peptides subjected to binding experiments, resulting in the possibility of false positive and even more likely false negative results.

[0010] (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 in which peptides are synthesized on a synthetic resin using orthogonal protection of lysine residues, and after selective deprotection, fatty acids are coupled to the protected peptides. However, expensive building blocks must be used, and crude peptides that have been modified with fatty acids are obtained, which are difficult to handle and purify due to their generally poor solubility.

[0011] In order to overcome the shortcomings of the prior art, namely the lack of separation and analysis of modified peptides and the use of standard amino acid building blocks without orthogonal protection, the present invention utilizes linker molecules that can immobilize peptide precursors and subsequently modify peptides when they are immobilized on a purification / modification solid support. Compared to other methods, especially those using modification in solution, the present invention simultaneously removes interfering truncated peptides and excess organic molecules used for modification, and is therefore a method for modifying and simultaneously purifying desired peptides. Summary of the invention

[0012] The first aspect of the present invention relates to a method for modifying and purifying a peptide. The method comprises the following steps:

[0013] - providing a crude linker-tagged peptide LP, wherein the crude peptide is covalently bound to a linker molecule L,

[0014] - in the immobilization step, coupling the linker-tagged peptide LP to a solid support to produce an immobilized linker-tagged peptide SLP,

[0015] - in a modification step, modifying the immobilized linker-tagged peptide SLP with one or more organic molecules to produce an immobilized linker-tagged peptide SL-mP,

[0016] - in the release step, adding a reducing agent produces a reduced intermediate RI, and subsequently decomposing the reduced intermediate RI to produce a modified peptide mP,

[0017] Wherein the linker L is a compound of formula 1, XT b -V a -UYZ(1), where

[0018] -X is selected from a moiety of formula 2, 2a, 3, 3a or 4, in particular a moiety of formula 2, 2a, 3 or 3a, more particularly a moiety of formula 2 or 2a,

[0019]

[0020] - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B.

[0021] -R 3 Selected from H or B,

[0022] -R 4 Selected from H, C1-C 12 - alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid-labile protecting group,

[0023] -B is an acid-labile amine protecting group,

[0024] -T is a linear or branched spacer, wherein b is 0 or 1,

[0025] -V is an electron withdrawing moiety, where a is 0 or 1, where the sum of a and b is 1 or 2,

[0026] -U is a phenyl or five-membered or six-membered heteroaryl moiety, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl substituted, wherein

[0027] -W is selected from -N3, -S(=O)-R 8 、-SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3,

[0028] -N=N-phenyl, -N=NR 8 ,

[0029] Where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, where q is 0, 1, 2, 3 or 4,

[0030] -E is an electron withdrawing group under acidic conditions, wherein n is 0, 1, 2, 3 or 4,

[0031] -Y is -(CH2) m -C(=O)- or -(CH2)m -OC(=O)-, wherein m is 1, 2 or 3,

[0032] -Z is an electron withdrawing leaving group.

[0033] In certain embodiments, the method for modifying and purifying a peptide comprises the following steps

[0034] - providing a crude linker-tagged peptide LP, wherein the crude peptide is covalently bound to a linker molecule L,

[0035] - in the immobilization step, coupling the linker-tagged peptide LP to a solid support to produce an immobilized linker-tagged peptide SLP,

[0036] - in a modification step, modifying the immobilized linker-tagged peptide SLP with one or more organic molecules to produce an immobilized linker-tagged peptide SL-mP,

[0037] - in the release step, adding a reducing agent produces a reduced intermediate RI, and subsequently decomposing the reduced intermediate RI to produce a modified peptide mP,

[0038] Wherein the linker L is a compound of formula 1, XT b -V a -UY-(1), wherein -X is selected from a moiety of formula 2, 2a, 3, 3a or 4, in particular a moiety of formula 2, 2a, 3 or 3a,

[0039] More particularly a moiety of formula 2 or 2a,

[0040] in,

[0041] - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B.

[0042] -R 3 Selected from H or B,

[0043] -R 4 Selected from H, C1-C 12 - alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid-labile protecting group,

[0044] -B is an acid-labile amine protecting group,

[0045] -T is a linear or branched spacer, wherein b is 0 or 1,

[0046] -V is an electron withdrawing moiety, where a is 0 or 1, where the sum of a and b is 1 or 2,

[0047] -U is a phenyl or five-membered or six-membered heteroaryl moiety, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl substituted, wherein

[0048] -W is selected from -N3, -NO2, -S(=O)-R 8 、-SSR 8 、-O-CH2-N3、

[0049] -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , Where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, wherein q is 0, 1, 2, 3 or 4, in particular wherein q is 1, 2, 3 or 4,

[0050] -E is an electron withdrawing group under acidic conditions, wherein n is 0, 1, 2, 3 or 4,

[0051] Where U is a phenyl moiety and Y is -(CH2) m -OC(=O)-, under acidic conditions, the sum of the Hammett constants of V, W, and E is greater than 0.45, and

[0052] -W is in the ortho or para position relative to Y, and Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-,

[0053] Where m is 1, 2 or 3.

[0054] In certain embodiments, X is selected from a moiety of formula 2, 3 or 4, particularly formula 2 or 3, more particularly X is formula 2,

[0055] Peptides obtained by solid phase peptide synthesis are usually purified after cleavage from the synthesis 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 allows peptide modification and purification on the same resin without the need to use orthogonal protection strategies to modify amino acids. The present invention is summarized in Figure 1 middle.

[0056] In the context of the present invention, the term "amino acid" relates to a molecule comprising at least one amine part and at least one carboxyl part. In particular, when used in the context of peptides, the term "amino acid" relates to α-amino acids, also called α-amino acids or 2-amino acids, having the general formula H2NCHRCOOH (free amino acids) or -HNCHRCOO- (amino acids in peptides), i.e., the amine and carboxylic acid parts are attached to the first (α-) carbon atom. R represents 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, parts that are not α-amino acids but amino acids in the broad sense described above are described in the present specification by specific formulas.

[0057] After solid phase peptide synthesis on a synthetic support such as a synthetic resin, a crude peptide P is obtained. While still bound to the synthetic resin, the crude peptide can be coupled to a traceless cleavable linker molecule L of formula 1.

[0058] The linker molecule of formula 1 is suitable for purifying peptides after solid phase peptide synthesis (SPPS).

[0059] A common method for purifying peptides after SPPS using a linker molecule is to couple the linker molecule to the N-terminus of the peptide in the final coupling step. In this coupling step, the N-terminus of the peptide will nucleophilically attack the linker molecule to form a covalent carbamate or amide bond with the part Y of the linker, while releasing the electron-withdrawing leaving group Z. Alternatively, a suitable part of the amino acid side chain of the peptide such as -NH2 or -OH can be used to couple the linker molecule to the peptide. If the N-terminus is modified by the method described herein, this option can be used. Suitable amino acids for coupling the linker to the amino acid side chain are lysine or serine.

[0060] Subsequently, the peptide-linker-construct is cleaved from the synthesis resin by addition of TFA. If the moiety X comprises an acid-labile amine protecting group B, the moiety B is removed simultaneously by addition of TFA.

[0061] When the peptide-linker construct is cleaved off, for example by using TFA, the linker according to the invention is stable under acidic conditions.

[0062] The portion X of the linker can be coupled to a functionalized solid phase, such as a resin, used during purification. The portion X can form a hydrazone bond or an oxime bond by reacting with a suitable portion of the functionalized solid phase, such as an aldehyde, ketone, amino-oxy group or hydrazine.

[0063] The moiety T represents a spacer which is non-reactive under the purification conditions commonly applied.

[0064] The moiety T is bonded to the moiety U directly or via the moiety V.

[0065] The moiety U contributes to the stability of the linker molecule under acidic conditions (particularly TFA>50%, in the presence of water, pH<0). This is achieved by using a heterocyclic moiety or a phenyl moiety bonded to at least one of the electron withdrawing moieties E, W and V.

[0066] If an electron withdrawing moiety (E, W, V) is attached to the phenyl moiety, the benzyl position of the linker molecule attached to the peptide gains less electron density and is therefore less susceptible to acid-catalyzed degradation. In order for the linker molecule of the present invention to have sufficient stability under acidic conditions, a certain electron withdrawing threshold must be met. This threshold is expressed as the Hammett constant (ζ) of V, W, E under acidic conditions. m and p ) is greater than 0.45. The Hammett constant is calculated according to Hansch and Taft (1991), Chem. Rev. 91: 165-195. A positive Hammett constant reflects the ability of the substituent to exert an electron-withdrawing effect on the phenyl moiety, while a negative value indicates that the substituent exerts an electron-donating effect.

[0067] The Hammett constant is the value of the meta-position (ζ) of a benzoic acid derivative. m ) and counterpoint (ζ p ) result in different acidities (pKa) of the empirically determined constants for the substituents of the phenyl core. In the context of the present invention, the position is determined relative to the bound moiety Y. For substituents in the ortho position, the Hammett value for 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 ​​for the substituents V, W and E.

[0068] It is worth noting that the Hammett constants for substituents V, W, and E are calculated under acidic conditions. For example, the amine moiety is characterized by a Hammett constant ζ at neutral pH m = -0.16 and ζ p = -0.66, and is therefore an electron-donating substituent. Under acidic conditions, the amine moiety is protonated. For a protonated amine, the Hammett constant is ζ m = +0.86 and ζ p = +0.60, indicating that the protonated amine is an electron-withdrawing substituent, which also includes aromatic amines that can withdraw electrons from U in their protonated form through the conjugated π system, whether directly as a substituent on U or in π conjugation of U.

[0069] The threshold value of the sum of Hammett constants greater than 0.45 applies when U is a phenyl moiety and Y is -(CH2) m -OC(=O)-, wherein m=1, because -OC(=O)- constitutes a good leaving group in the benzylic position, aiding acid-catalyzed degradation. Therefore, the electron density in the aromatic ring should be low enough to prevent cationic stabilization of the benzylic position in Y.

[0070] If the moiety Y is -(CH2) m For -C(=O)-, this threshold is not necessary because -C(=O)- is not a good leaving group at the benzylic position.

[0071] For the stability of the linker molecule comprising the heterocyclic moiety U under acidic conditions, such additional thresholds for the selection of specific moieties V, W and E are not necessary. Since the heterocyclic moiety itself is more electron deficient than the phenyl moiety, any combination of V, W and E seems to be sufficient for the stability of the linker molecule under acidic conditions. Especially when U is a nitrogen-containing heterocycle, the nitrogen will be protonated during the acidic release of the peptide, making the electron density of the aromatic system of U particularly low. Therefore, the benzyl cation cannot be stabilized.

[0072] The moiety Y is a -C(=O)- or -OC(=O)- moiety. Upon coupling of the linker molecule, 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 by 1.4 or 1.6 elimination or nucleophilic attack under reducing conditions and is therefore released from the purification medium. The reducing stimulus converts W to its reduced version, which now acts as an electron donating group and nucleophile, thereby enabling the release of the peptide.

[0073] After cleavage from the synthesis resin, the linker-tagged peptide LP is provided.

[0074] In certain embodiments, a linker-tagged peptide LP is provided by connecting a crude peptide P that has been synthesized on a synthetic support SR to a linker L to produce a linker-tagged, loaded crude peptide LP-SR, and then cleaving the linker-tagged peptide LP from the synthetic support SR to produce a linker-tagged peptide LP.

[0075] In certain embodiments, a linker-tagged peptide LP is provided by: combining a crude peptide P synthesized on a synthetic support SR with a free linker L f The linker-tagged, loaded crude peptide LP-SR is generated by releasing the electron-withdrawing leaving group Z, where L f Yes connector XT b -V a -UYZ, wherein X, T, b, V, a, U and Y are as defined above, and z is an electron withdrawing leaving group, and then the linker-tagged peptide LP is cleaved from the synthetic support SR to produce the linker-tagged peptide LP.

[0076] In certain embodiments, L f Yes connector XT b -V a-UYZ, wherein X is selected from Formula 2, 2a, 3, 3a and 4, and wherein T, b, V, a, U and Y are as defined above.

[0077] In certain embodiments, the free linker L f The coupling can be to the N-terminus of the crude peptide P or to the free amine or hydroxyl groups of the amino acid side chains of the peptide P.

[0078] In certain embodiments, the free linker L f It can be coupled to the N-terminus of the crude peptide P, or to an amine in the side chain 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 and d-hydroxylysine, in particular selected from lysine and ornithine, or to the hydroxyl portion in the side chain of an amino acid selected from serine, tyrosine and threonine.

[0079] In certain embodiments, the free linker L f The coupling can be to the N-terminus of the crude peptide P or to the amine in the lysine side chain or to the hydroxyl moiety in the serine side chain.

[0080] In certain embodiments, the free linker L f Can be coupled to the N-terminus of crude peptide P.

[0081] Since the leaving group Z is released when the linker L is coupled to the N-terminus of the crude peptide, the linker-tagged, loaded crude peptide contains a portion of XT b -V a -UYP-SR, and the released linker-tagged peptide contains part of XT b -V a -UYP.

[0082] The peptide labeled with the linker is released from the synthetic carrier under commonly used acidic conditions. Compared with the linker molecules of the prior art, the linker of Formula 1 is stable under acidic conditions such as TFA. In this step, only the acid-labile amine protecting group B of part X is removed.

[0083] In certain embodiments, cleavage of the linker-tagged peptide LP- is achieved under acidic conditions.

[0084] In certain embodiments, cleavage of the linker-tagged peptide LP- is achieved by using TFA, HBr, HCl, or acetic acid.

[0085] The peptide of the linker label is fixed on a solid support such as a purification resin using standard methods known to the skilled person. Suitable solid supports include functional groups such as aldehydes, ketones, amino-oxy groups or hydrazine moieties. The functional group forms a hydrazine bond or an oxime bond with the part X of the linker.

[0086] As described above, the peptide is usually synthesized on a synthesis resin and the linker is coupled to the peptide while still bound to the synthesis resin. Subsequently, the linker-tagged peptide is cleaved from the synthesis resin by adding TFA, wherein the acid-labile protecting group B of the moiety X is simultaneously removed. If the peptide is synthesized in solution, or if the peptide is provided without being bound to a synthesis support SR, the acid-labile protecting group B is removed by using an acid as described above before coupling the linker-tagged peptide LP to a solid support in an immobilization step.

[0087] 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 produce an immobilized linker-tagged peptide SLP.

[0088] In order to immobilize the peptide of linker label, suitable aldehyde modified beads, such as agarose or hydroxylated poly(methyl acrylate) beads, can be used. In particular, for modifications such as disulfide formation or thioether stapling, hydroxylated poly(methyl acrylate) beads or agarose, in particular hydroxylated poly(methyl acrylate) beads are used.

[0089] In certain embodiments, the solid support is aldehyde-modified.

[0090] In certain embodiments, the solid support is aldehyde-modified and is selected from agarose, hydroxylated poly(methyl acrylate), poly(glycidyl acrylate), poly(glycidyl methacrylate), polylysine, polyethylene glycol, polyamide, polyacrylamide, polystyrene, and copolymers of these.

[0091] In certain embodiments, the solid support is aldehyde-modified and is selected from agarose and hydroxylated poly(methyl acrylate) beads.

[0092] In certain embodiments, the fixation step is performed at pH <5, particularly at pH 3-4, more particularly at pH 3.5.

[0093] In certain embodiments, the fixation step is performed in DMSO / sodium citrate / guanidine hydrochloride, in TFA / water, in hexafluoroisopropanol (HFIP) or in trifluoroethanol, particularly in DMSO / sodium citrate / guanidine hydrochloride, in TFA / water or in HFIP.

[0094] In certain embodiments, the fixation step is performed in DMSO / sodium citrate with 8M guanidine hydrochloride at pH 3.5, in TFA and water with a TFA content of 40-95%, in hexafluoroisopropanol (HFIP), and in trifluoroethanol.

[0095] In certain embodiments, the fixation step is performed in DMSO / sodium citrate (9:1), TFA / water (1:1), TFA / water (83:27), and HFIP with 8 M guanidine hydrochloride at pH 3.5.

[0096] Subsequently, the peptide may be modified in a modification step (see below).

[0097] Finally, the modified peptide mP with a free N-terminus is released via the reduced intermediate RI.

[0098] In certain embodiments, the reducing agent in the releasing step is selected from triphenylphosphine, dithiothreitol (DTT), dithioerythritol (DTE), trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, in particular dithiothreitol, dithioerythritol or triphenylphosphine.

[0099] In certain embodiments, the reducing agent in the releasing step is selected from triphenylphosphine, dithiothreitol (DTT), trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine.

[0100] In certain embodiments, the reducing agent in the releasing step is triphenylphosphine or dithiothreitol.

[0101] The reduced intermediate RI decomposes to release the modified peptide mP with a free N-terminus.

[0102] As described above, moieties U, V, W and E contribute to the stability of the linker under acidic conditions. For example, a linker comprising moieties U or V containing an amine or heterocycle such as pyridine is stable under acidic conditions (particularly TFA>50%, in the presence of water, pH<0) because the amine or heterocycle is protonated. In addition, protonated linkers improve the solubility of the linker-peptide complex. If the pH is higher than the pKa of the linker, the linker will rapidly decompose as the last step during the desired peptide release.

[0103] In certain embodiments, the reduced intermediate RI is decomposed by a change in pH.

[0104] In certain embodiments, the reduced intermediate RI is decomposed by changing the pH to a pH above the pKa of the linker.

[0105] In certain embodiments, the reduced intermediate RI is decomposed by changing the pH to a pH lower than the pH used during the reduction of the linker. For example, the reduced intermediate RI is obtained by adding a reducing agent at pH 8. Subsequently, the reduced intermediate is decomposed at pH < 8, particularly at a pH between -0.7 and 0.7.

[0106] As mentioned above, two main types of modifications are used to improve the stability and potency of peptides in vivo: lipidation with fatty acids and cyclization of linear peptides to form macrocycles.

[0107] Lipidation involves the coupling of fatty acids to peptides. This modification affects the hydrophobicity, secondary structure, and aggregation behavior of the peptide. In addition, it can enhance the metabolic stability and membrane permeability of the peptide.

[0108] The peptides may be composed of natural and / or unnatural amino acids. If the amino acids of the peptide contain a nucleophilic amine in its side chain, it may form an amide bond with the carboxylic acid portion of an organic molecule, such as the carboxylic acid portion of a fatty acid.

[0109] In certain embodiments, the peptide comprises an amino acid containing at least one nucleophilic amine.

[0110] In certain embodiments, the peptide comprises an amino acid containing at least one nucleophilic amine in the amino acid side chain, or the nucleophilic amine is the N-terminus of the peptide, i.e., the first residue of the peptide sequence. The amino acid forming the N-terminus can be any amino acid.

[0111] In certain embodiments, the peptide comprises an amino acid selected from the group consisting of 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, and d-hydroxylysine.

[0112] In certain embodiments, the peptide comprises an amino acid containing at least one nucleophilic amine in the amino acid side chain, wherein the amino acid is 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 and d-hydroxylysine.

[0113] In certain embodiments, the peptide comprises an amino acid selected from the group consisting of lysine and ornithine.

[0114] The organic molecule used for lipidation of the peptide 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. The fatty acid can also be connected to the nucleophilic amine via one or more additional linker / spacer moieties Sp. Here too, the starting material can be 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 via an amide bond. The linker / spacer Sp-OH or activated linker / spacer Sp-A are 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 via amide bonds.

[0115] In certain embodiments, the organic molecule is a compound of formula 5a, 5b, 5c or 5d, particularly 5a or 5c,

[0116] FA-Sp n -A(5a),FA-Sp n -OH (5b), FA-A (5c), or FA-OH (5d), wherein

[0117] -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, wherein the alkyl or alkenyl portion may be unsubstituted or substituted with one or more substituents independently selected from -F and -COOH,

[0118] -Sp is a spacer comprising 2 to 50 atoms in length, a terminal -C(=O)- moiety and a -NH- moiety, wherein the spacer is attached to FA via the -NH- moiety,

[0119] -n is an integer between 1 and 10,

[0120] -A is selected from: -F, -Cl, -Br, -I, -N3, -O(C=O)CH2(C=O)OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3,

[0121]

[0122] In particular, -Cl, More specifically

[0123] -R 14 is a C1-C6-alkyl-, aryl- or benzyl substituent.

[0124] In certain embodiments, the organic molecule is a compound of formula 5a, 5b, 5c or 5d, particularly 5a or 5c,

[0125] FA x -Sp n -A(5a),FA x -Sp n -OH (5b), FA-A (5c), or FA-OH (5d), wherein

[0126] -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, wherein the alkyl or alkenyl portion may be unsubstituted or substituted with one or more substituents independently selected from -F and -COOH,

[0127] -Sp is a spacer comprising 2 to 50 atoms in length, an optional terminal -C(=O)- moiety and x linking moieties independently selected from -NH-, -O-, -S-, wherein the spacer is attached to FA via the linking moiety,

[0128] -x is an integer between 1 and 5, specifically an integer between 1 and 3,

[0129] - n is an integer between 1 and 10, in particular an integer between 1 and 4, more particularly 1 and 2,

[0130] -A is selected from: -F, -Cl, -Br, -I, -N3, -O(C=O)CH2(C=O)OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3,

[0131]

[0132] In particular, -Cl, -Br, -I, More specifically, -Cl, -Br, -I,

[0133] Where R 14 is a C1-C6-alkyl-, aryl- or benzyl substituent.

[0134] In certain embodiments, A is

[0135] The terminal -C(=O) portion of the spacer Sp is linked to A or -OH.

[0136] C1-24 -alkyl moiety and C 2-24 The -alkenyl moiety may be straight chain or branched. 2-24 The -alkenyl moiety comprises one or more carbon-carbon double bonds, in particular one carbon-carbon double bond.

[0137] In certain embodiments, the alkyl or alkenyl portion of FA is linear or branched.

[0138] In certain embodiments, the alkyl or alkenyl portion of FA is linear.

[0139] In certain embodiments, FA is derived from palmitic acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, stearic acid, stearic acid, perfluorooctanoic acid, perfluorodecanoic acid, perfluorolauric acid, perfluoromyristic acid, more particularly palmitic acid. As described above, part of FA comprises a carbonyl moiety. The carbonyl moiety is derived from the carboxylic acid moiety of the acid described herein.

[0140] Part of FA may contain one or more substituents. For example, part of FA may be derived from a perfluorinated fatty acid. In this case, the alkyl or alkenyl portion of FA is completely substituted with -F. Part of FA may also be derived from a fatty diacid. In this case, the terminal methyl portion (ω carbon) of the alkyl or alkenyl portion of FA is substituted with a -COOH portion.

[0141] In certain embodiments, the moiety FA is unsubstituted, the moiety FA is fully substituted with -F, or the moiety FA comprises one substituent -COOH.

[0142] In certain embodiments, the moiety FA is unsubstituted, the moiety FA is fully substituted with -F, or the moiety FA comprises one substituent -COOH at the ω carbon.

[0143] In certain embodiments, the FA moiety is unsubstituted or the FA moiety is fully substituted with -F.

[0144] In certain embodiments, the moiety FA is unsubstituted or the moiety FA comprises a substituent -COOH, particularly a substituent -COOH at the ω carbon.

[0145] In certain embodiments, the moiety FA is unsubstituted or the moiety FA comprises the substituent -COOH or -COOtBu, in particular -COOtBu.

[0146] After purification / modification, –COOtBu has been removed and part of the FAs are replaced by –COOH.

[0147] In certain embodiments, the moiety FA is unsubstituted or the moiety FA comprises a substituent -COOH or -COOtBu, in particular -COOtBu, at the ω carbon.

[0148] In certain embodiments, the FA moiety comprises a substituent -COOH or -COOtBu, in particular -COOtBu, at the ω carbon.

[0149] In certain embodiments, a portion of FA is unsubstituted

[0150] The spacer group Sp can be derived from natural or non-natural amino acid.As mentioned above, the spacer group is connected with part FA through amide bond, and is also connected with other spacer group parts Sp or the nucleophilic amine of peptide through amide bond.The amide bond with part FA is formed by the reaction of the activated carboxylic acid moiety of the carboxylic acid moiety of the amine in amino acidic N-terminal amine or amino acid side chain and fatty acid FA-OH or FA-A.The amide bond with other spacer group parts Sp or with the nucleophilic amine of peptide is formed by the reaction of the amine moiety of the carboxylic acid moiety in C-terminal carboxylic acid moiety or amino acid side chain with other spacer group Sp or the nucleophilic amine of peptide.The functional group in amino acidic side chain, N-terminal or C-terminal can be protected, and for example the carboxylic acid moiety can be protected by the tertiary butyl blocking group.

[0151] In certain embodiments, Sp is independently selected from

[0152] - a linear or branched alkyl group substituted by 1 to 5, in particular 1 to 3, linking moieties, wherein in particular the linking moiety is -O-,

[0153] --NH-[C r -alkyl-O] s -C t -alkyl-C(═O)-, wherein r is independently selected from 1, 2 and 3, s is an integer between 1 and 10, in particular 1, 2, 3 or 4, and t is 1, 2 or 3, and

[0154] - amino acids, which may be protected or unprotected, wherein the amine in the N-terminus and / or the side chain is replaced by -NH- and the carboxylic acid moiety in the C-terminus and / or the side chain is replaced by -C(=O)-.

[0155] In case n is greater than 1, identical or different parts Sp may be combined.

[0156] In certain embodiments, the straight chain or branched chain alkyl is C 1-12 Alkyl groups, especially C 1-6 Alkyl, more particularly C 1-3 alkyl.

[0157] In certain embodiments, Sp is independently selected from

[0158] - linear alkyl substituted by 1 to x linking moieties, wherein in particular the linking moiety is -O-,

[0159] -2-(2-(2-aminoethoxy)ethoxy)acetic acid

[0160] - 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.

[0161] In certain embodiments, the linear alkyl group is C 1-12 Alkyl groups, especially C 1-6 Alkyl, more particularly C 1-3 alkyl.

[0162] In certain embodiments, Sp is independently selected from -O-ethyl-, Cysteine, glutamic acid, Glu-tBu or 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0163] In certain embodiments, Sp is an amino acid, which may be protected or unprotected, wherein the amine in the N-terminus or in the side chain is replaced by -NH- and the carboxylic acid moiety in the C-terminus or in the side chain is replaced by -C(=O)-.

[0164] In certain embodiments, Sp is an amino acid independently 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 glutamic acid, Glu-tBu or 2-(2-(2-aminoethoxy)ethoxy)acetic acid, wherein the amine in the N-terminus or in the side chain is replaced by -NH- and the carboxylic acid moiety in the C-terminus or in the side chain is replaced by -C(=O)-.

[0165] The amino acid glutamic acid may be protected (Glu-tBu), for example the C-terminal carboxylic acid moiety may be protected by a tert-butyl protecting group.

[0166] In certain embodiments, n is 1 or 2.

[0167] In certain embodiments, n is 1.

[0168] In certain embodiments, the organic molecule is selected from Pam2, Pam-Et, Pam3Cys and Pam2-Et-Cys,

[0169]

[0170] In certain embodiments, in the modification step, the organic molecule of Formula 5a or 5c is coupled to at least one nucleophilic amine in the presence of a non-nucleophilic base.

[0171] In certain embodiments, in the modification step, the organic molecule of formula 5b or 5d is coupled to at least one nucleophilic amine in the presence of an activating reactant, particularly in the presence of an activating reactant selected from the group consisting of tetramethyluronium tetrafluoroborate (TBTU), (3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one) (DEPBT), (2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N′,N′ -tetramethyluronium hexafluorophosphate) (HCTU), tetramethyluronium 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, and diisopropylcarbodiimide (DIC).

[0172] In certain embodiments, in the modification step, the organic molecule of Formula 5b or 5d is coupled to at least one nucleophilic amine in the presence of an activating reactant, particularly in the presence of an activating reactant selected from the group consisting of diisopropylcarbodiimide (DIC), (2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) (HCTU) and tetramethyluronium hexafluorophosphate (HATU).

[0173] In addition to lipidation, cyclization is often used to improve the stability and potency of peptides in vivo: cyclic peptides are highly resistant to exo- and endoproteases. Due to their more rigid conformation, good binding affinity to the desired target is observed. Cyclization can be achieved by intramolecular disulfide formation or in a two-component system (intermolecular cyclization) using a scaffold molecule.

[0174] For both cyclization methods, functional groups of the peptides, such as thiol moieties, were used.

[0175] In certain embodiments, the peptide comprises at least two amino acids containing a nucleophilic thiol.

[0176] In certain embodiments, the peptide comprises from 2 to 6 nucleophilic thiol-containing acids.

[0177] In certain embodiments, the peptide comprises two amino acids containing nucleophilic thiols.

[0178] In certain embodiments, the peptide comprises at least two amino acids independently selected from cysteine, homocysteine, or penicillamine.

[0179] In certain embodiments, the peptide comprises 2 to 6 amino acids independently selected from cysteine, homocysteine, or penicillamine.

[0180] In certain embodiments, the peptide comprises two amino acids independently selected from cysteine, homocysteine, or penicillamine.

[0181] In a two-component system (intermolecular cyclization), the functional groups of the peptide are used to react with organic molecules such as bridging scaffolds. Depending on the number of functional groups in the peptide and scaffold molecules, single, double or multiple rings can be formed by the peptide and scaffold molecules. The most common method is to utilize the nucleophilic functional groups of the peptide, especially thiols, to react with scaffolds with electrophilic reaction sites. Such electrophilic centers can be attacked by thiols.

[0182] In certain embodiments, the organic molecule is an organic scaffold molecule comprising a scaffold having at least two electrophilic centers.

[0183] The scaffold can be a straight chain, branched, monocyclic or polycyclic system, which is aliphatic or aromatic. In particular, more rigid scaffolds such as cyclic systems are suitable for cyclization to keep the peptide in a desired conformation. The scaffold may contain heteroatoms, such as O, S or N. The scaffold further comprises substituents that form electrophilic centers. For example, a phenyl substituted with two ethyl substituents can form a scaffold and the Br substituents of the ethyl portion form an electrophilic center. The Br substituent is referred to as benzyl Br.

[0184] In certain embodiments, the organic molecule is an organic scaffold molecule comprising a scaffold having at least two electrophilic centers, wherein the electrophilic centers are formed by halogen substituents and / or Michael acceptors.

[0185] In certain embodiments, the electrophilic center is formed by at least two benzyl halogen substituents, at least two acrylamides, or at least two acrylates.

[0186] In certain embodiments, the electrophilic center is formed by at least two acrylamide or at least two benzyl halogen substituents.

[0187] In certain embodiments, the organic molecule is selected from m-dibromobenzyl (mDBX), 1,4-bis(bromomethyl)benzene, 1,3,5-tri(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(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-chloroacetamide). -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-triyl)tris(2-bromoethanone) (TATB), N,N',N"-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB), N,N',N"-benzene-1,3,5-triyltriprop-2-enamide (TAAB), 2,4,6-tris(bromomethyl)-s-triazine (TBMT), N,N',N"-(nitrilotri(ethane-2,1-diyl))triacrylamide 、N,N′,N″-(nitrilotri(ethane-2,1-diyl))tris(2-chloroacetamide), N,N′,N″-(nitrilotri(ethane-2,1-diyl))tris(2-bromoacetamide), N,N′,N″-(nitrilotri(ethane-2,1-diyl))triethylenesulfonamide, N,N′,N″-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxyethyleneethane-2,1-diyl))triacrylamide, N,N′,N″-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxyethyleneethane-2,1-diyl))tris(2-chloroacetamide), N,N′,N″-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxyethyleneethane-2,1-diyl))triethylenesulfonamide, 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))triethylenesulfonamide, tris(2-hydroxyethyl) triacrylate isocyanurate, 1,3,5-tris(oxiran-2-ylmethyl)-1,3,5-triazinane-2,4,6-trione.

[0188] In certain embodiments, the organic molecule is selected from m-dibromobenzyl (mDBX), 1,3,5-tris(bromomethyl)benzene (TBMB), triazane, 1,3,5-triacryloyl-1,3,5-triazinane (TATA), 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB).

[0189] In certain embodiments, the organic molecule is selected from α,α′-dibromo-m-xylene (mDBX), 1,3,5-tris(bromomethyl)benzene (TBMB), triazane, 1,3,5-triacryloyl-1,3,5-triazinane (TATA), 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) (TATB).

[0190] In a two-component system (intermolecular cyclization), a nucleophilic thiol is obtained in the reduction step. Subsequently, the nucleophilic thiol attacks the electrophilic center in the organic scaffold molecule in the cyclization step.

[0191] In certain embodiments, the modifying step comprises a reduction step followed by a cyclization step, wherein

[0192] - in a reduction step, the immobilized linker-tagged peptide SLP is treated with a reducing agent, in particular dithiothreitol (DTT) or tris-(2-carboxyethyl)-phosphine (TCEP), and

[0193] - In the cyclization step, the organic scaffold molecule is coupled with at least two nucleophilic thiols under basic conditions.

[0194] The cyclization step is carried out under basic conditions to deprotonate the nucleophilic thiol.

[0195] In certain embodiments, the cyclization step is performed at pH ≥8.

[0196] In certain embodiments, the cyclization step is performed in the presence of Na2CO3, NaHCO3, NH4HCO3, guanidine hydrochloride (GdmCl), diisopropylethylamine (DIPEA), triethylamine (NEt3), or N-methyl-morpholine (NMM).

[0197] In certain embodiments, the cyclization step is performed in the presence of NH4HCO3, guanidine hydrochloride (GdmCl), diisopropylethylamine (DIPEA), triethylamine (NEt3) or N-methyl-morpholine (NMM).

[0198] Cyclization can also be achieved by intramolecular disulfide bond formation. For example, the thiol moieties of two cysteine ​​residues in a peptide can form a disulfide.

[0199] In certain embodiments, the number of nucleophilic thiols is even, and in the modification step the nucleophilic thiol is oxidized by a basic aqueous solution at pH>7 to generate a macrocyclic bridged peptide.

[0200] In certain embodiments, the nucleophilic thiol is oxidized by an alkaline aqueous solution at pH>7, and / or in the presence of air (particularly in the presence of oxygen), and / or in the presence of an oxidizing additive, particularly an oxidizing additive selected from the group consisting of DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, Tl(CF3COO)3, CH3SiCl3-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.

[0201] The oxidizing additive can be used as such or dissolved in a suitable solvent. The oxidation can be carried out in an organic solution, an organic aqueous solution, an acidic or alkaline solution.

[0202] In certain embodiments, the nucleophilic thiol is oxidized in the presence of air, particularly in the presence of oxygen, or in the presence of an oxidizing additive, particularly one selected from the group consisting of DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, Tl(CF3COO)3, CH3SiCl3-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.

[0203] In certain embodiments, the nucleophilic thiol is reacted in the presence of DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, [Pt(ethylenediamine)2Cl2]Cl2, trans-[Pt-(CN)4Cl2] 2- , is oxidized in the presence of glutathione.

[0204] In certain embodiments, the nucleophilic thiol is oxidized in the presence of air (oxygen), DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, Tl(CF3COO)3, CH3SiCl3-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.

[0205] In certain embodiments, the nucleophilic thiol is reacted in the presence of DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, [Pt(ethylenediamine)2Cl2]Cl2, trans-[Pt-(CN)4Cl2] 2- , is oxidized in the presence of glutathione.

[0206] In certain embodiments, the nucleophilic thiol is oxidized in the presence of N-chlorosuccinimide, DMSO, and glutathione-glutathione disulfide.

[0207] In certain embodiments, the nucleophilic thiol is oxidized in the presence of N-chlorosuccinimide, DMSO, and glutathione.

[0208] In certain embodiments,

[0209] -X is selected from the moiety of formula 2, 2a, 3, 3a or 4, in particular the moiety of formula 2, 2a, 3 or 3a, more particularly the moiety of formula 2 or 2a,

[0210] in

[0211] - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B.

[0212] -R 3 Selected from H or B,

[0213] -R 4 Selected from H, C1-C 12 - alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid-labile protecting group,

[0214] -B is an acid-labile amine protecting group,

[0215] -T is a linear or branched spacer group comprising at least one, in particular 1 to 5, of the following moieties: -C 1-12 -alkyl-, (-C2H4O-) 1-12 、-C(=O)-、-C(=O)-JR 9 -、-JR 9 -C(=O)-、-JR 9 -, phenyl, 5-membered or 6-membered heteroaryl, wherein

[0216] J is C or N, especially N,

[0217] In particular, T is a spacer selected from:

[0218] -C1-C 12 -alkyl-, especially 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 -pyrrolyl, -R 5 -pyrazolyl, -R 5 -imidazolyl, R 5 -piperazinyl-, -R 5 -pyridyl, -R 5 -pyrimidinyl, -R 5-pyrazinyl, -R 5 -pyridazinyl, -R 5 -pyrrolyl-R 6 -、-R 5 -pyrazolyl-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 -, pyrrolyl-R 6 -, pyrazolyl-R 6 -, imidazolyl-R 6 -piperazinyl-R 6 -, pyridyl-R 6 -, pyrimidinyl-R 6 -, pyrazinyl-R 6 -, pyridazinyl-R 6 -, pyrrolyl, pyrazolyl, imidazolyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl and pyridazinyl, wherein

[0219] R 5 , R 5’ and R 6 Independently selected from C1-C 12 -alkyl or (-C2H4O-) 1-12 , especially C1-C6 alkyl, especially C1-C3 alkyl, and wherein

[0220] R 9 and R 9’ are independently selected from 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 selected from H and C 1-2 -alkyl, more particularly R 9 is H, where

[0221] B is an independently selected acid-labile amine protecting group,

[0222] R 15 It is a blocking agent that reacts with aldehyde moieties, especially R 15 is selected from cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more particularly cysteinyl, wherein

[0223] The amine and / or thiol moieties of the blocking agent may be protected by independently selected acid-labile amine protecting groups B, in particular Boc, and / or acid-labile thiol protecting groups, in particular trityl.

[0224] -b is 0 or 1, especially 1,

[0225] -V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -(CH2) p -、-Piperazinyl-(CH2) p -, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, especially -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、--NR 12 -(CH2) p -、-Piperazinyl-(CH2) p -, -pyridinyl-, pyrimidinyl, more particularly selected from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-(CH2) p -, -pyridyl-, pyrimidinyl, wherein

[0226] R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 It is H.

[0227] R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It is methyl,

[0228] p is 0, 1 or 2, in particular 0 or 1,

[0229] -a is 0 or 1, where the sum of a and b is 1 or 2,

[0230] -U is phenyl or a five-membered or six-membered heteroaryl moiety, in particular phenyl or a six-membered heteroaryl moiety, more particularly phenyl, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl, especially C 1-3 -alkyl substituted, wherein

[0231] V is defined as above,

[0232] W is selected from -N3, -NO2, -S(=O)-R 8 、-SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , Especially -N3, -N=NR 8 、-O-CH2-N3、-SSR 8 ,in

[0233] R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6-alkyl, wherein p is 1, 2, 3 or 4,

[0234] E is an electron-withdrawing group under acidic conditions.

[0235] n is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 or 1, and q is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 and 1, wherein the sum of n and q is equal to or less than 4, and wherein

[0236] Where U is a phenyl moiety and Y is -(CH2) m -OC(=O)-, under acidic conditions, the sum of the Hammett constants of V, W, and E is greater than 0.45, and

[0237] W is in the ortho or para position relative to Y,

[0238] -Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, wherein m is 1, 2 or 3, particularly 1 or 2, more particularly 1,

[0239] Z is an electron withdrawing leaving group.

[0240] In certain embodiments, q is an integer between 1 and 4, particularly 1 and 2, more particularly 1.

[0241] In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -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, wherein R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, especially pyridyl or -C1-C6-alkyl, wherein 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.

[0242] In certain embodiments,

[0243] -X is selected from the moiety of formula 2, 2a, 3, 3a or 4, in particular the moiety of formula 2, 2a, 3 or 3a, more particularly the moiety of formula 2 or 2a,

[0244] in

[0245] - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B.

[0246] -R 3 Selected from H or B,

[0247] -R 4 Selected from H, C1-C 12 - alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid-labile protecting group,

[0248] -B is an acid-labile amine protecting group,

[0249] -T is a linear or branched spacer group comprising at least one of the following moieties: -C 1-12 -alkyl-, (-C2H4O-) 1-12 、-C(=O)-、-C(=O)-JR 9 -、-JR 9 -C(=O)-、-JR 9 -,in

[0250] J is C or N, especially N,

[0251] In particular, T is a spacer selected from:

[0252] -C1-C 12 -alkyl-, especially -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 -、-R5 -C(=O)-OR 6 -、-C(=O)-OR 6 -、-R 5 -phenyl-R 6 -、-R 5 -phenyl-, -phenyl-R 6 -, -phenyl-, wherein

[0253] R 5 , R 5’ and R 6 Independently selected from C1-C 12 -alkyl or (-C2H4O-) 1-12 , especially C1-C6 alkyl, especially C1-C3 alkyl, and wherein

[0254] R 9 and R 9’ are independently selected from 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 selected from H and C 1-2 -alkyl, more particularly R 9 is H, where

[0255] B is an independently selected acid-labile amine protecting group,

[0256] R 15 It is a blocking agent that reacts with aldehyde moieties, especially R 15 is selected from cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more particularly cysteinyl, wherein

[0257] The amine and / or thiol moieties of the blocking agent may be protected by independently selected acid-labile amine protecting groups B, in particular Boc, and / or acid-labile thiol protecting groups, in particular trityl.

[0258] -b is 0 or 1, especially 1,

[0259] -V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR12 -, piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, especially -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly selected from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, wherein

[0260] R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 It is H.

[0261] R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It is methyl,

[0262] -a is 0 or 1, where the sum of a and b is 1 or 2,

[0263] -U is phenyl or a five-membered or six-membered heteroaryl moiety, in particular phenyl or a six-membered heteroaryl moiety, more particularly phenyl, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl, especially C 1-3 -alkyl substituted, wherein

[0264] V is defined as above,

[0265] W is selected from -N3, -S(=O)-R 8 、-SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , Especially -N3, -N=NR 8 、-O-CH2-N3、-SSR 8 ,in

[0266] R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p-NMe2, in particular pyridyl or -C1-C6-alkyl, wherein p is 1, 2, 3 or 4,

[0267] E is an electron-withdrawing group under acidic conditions.

[0268] n is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 or 1, and q is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 and 1, wherein the sum of n and q is equal to or less than 4, and wherein

[0269] Where U is a phenyl moiety and Y is -(CH2) m In the case of -OC(=O)-, the sum of the Hammett constants of V, W, and E is greater than 0.45 under acidic conditions.

[0270] -Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, wherein m is 1, 2 or 3, particularly 1 or 2, more particularly 1,

[0271] -Z is an electron withdrawing leaving group.

[0272] In certain embodiments,

[0273] -X is selected from the moiety of formula 2, 2a, 3, 3a or 4, in particular the moiety of formula 2, 2a, 3 or 3a, more particularly the moiety of formula 2 or 2a,

[0274] in

[0275] - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B.

[0276] -R 3 Selected from H or B,

[0277] -R 4 Selected from H, C1-C 12 - alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid-labile protecting group,

[0278] -B is an acid-labile amine protecting group,

[0279] -T is a spacer selected from the following:

[0280] -C1-C 12 -alkyl-, especially C 1-6 -alkyl, more particularly C1-3 -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-, wherein

[0281] R 5 , R 5’ and R 6 Independently selected from C1-C 12 -alkyl, especially C1-C6 alkyl, especially C1-C3 alkyl, and wherein R 9 and R 9’ are independently selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 9 It is H.

[0282] -b is 0 or 1, especially 1,

[0283] -V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, especially -NR 11-C(=O)-, -C(=O)-NR 11 -、S(=O)-、-NR 12 -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly selected from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, wherein

[0284] R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 It is H.

[0285] R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It is methyl,

[0286] -a is 0 or 1, where the sum of a and b is 1 or 2,

[0287] -U is phenyl or a five-membered or six-membered heteroaryl moiety, in particular phenyl or a six-membered heteroaryl moiety, more particularly phenyl, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl, especially C 1-3 -alkyl substituted, wherein

[0288] V is defined as above,

[0289] W is selected from -N3, -S(=O)-R 8 、-SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , Especially -N3, -N=NR 8 、-O-CH2-N3、-SSR 8 ,in

[0290] R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6-alkyl, wherein p is 1, 2, 3 or 4,

[0291] E is an electron-withdrawing group under acidic conditions.

[0292] n is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 or 1, and q is an integer between 0 and 4, in particular an integer between 0 and 2, more in particular 0 and 1, wherein the sum of n and q is equal to or less than 4, and wherein

[0293] Where U is a phenyl moiety and Y is -(CH2) m In the case of -OC(=O)-, the sum of the Hammett constants of V, W, and E is greater than 0.45 under acidic conditions.

[0294] -Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, wherein m is 1, 2 or 3, particularly 1 or 2, more particularly 1,

[0295] -Z is an electron withdrawing leaving group.

[0296] In certain embodiments,

[0297] -X is selected from a moiety of formula 2, 2a, 3, 3a or 4, particularly a moiety of formula 2, 2a, 3 or 3a, more particularly a moiety of formula 2 or 2a, most particularly a moiety of 2

[0298] in

[0299] - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B.

[0300] -R 3 Selected from H or B,

[0301] -R 4 Selected from H, C1-C 12 - alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid-labile protecting group,

[0302] -B is an acid-labile amine protecting group,

[0303] -T is a spacer selected from the following:

[0304] -C1-C 12 -alkyl-, especially C 1-6 -alkyl, more particularly C 1-3 -alkyl, -R 5 -C(=O)-NR 9 -R 6 -、-C(=O)-NR 9 -R6 -、-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-, wherein

[0305] R 5 and R 6 Independently selected from C1-C 12 -alkyl, especially C1-C6 alkyl, especially C1-C3 alkyl, and wherein R 9 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 9 It is H.

[0306] -b is 0 or 1, especially 1,

[0307] -V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, in particular -NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridyl-, pyrimidinyl, wherein

[0308] R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 It is H.

[0309] R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It is methyl,

[0310] -a is 0 or 1, where the sum of a and b is 1 or 2,

[0311] -U is a phenyl or a five-membered or six-membered heteroaryl moiety, in particular a phenyl or six-membered heteroaryl moiety, which is bound to at least one of the moieties V, W and E, wherein

[0312] V is defined as above,

[0313] W is selected from -N3, -S(=O)-R 8 、-SSR 8 , -OCH2N3, -OC(=O)OCH2-N3,

[0314] -N=N-phenyl, -N=N-pyridine, in

[0315] R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, where p is 1, 2, 3 or 4,

[0316] E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, in particular pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, wherein

[0317] R 13 is selected from -F, -Cl, -Br, -I, -PF6, and wherein

[0318] Where U is a phenyl moiety and Y is -(CH2) m In the case of -OC(=O)-, the sum of the Hammett constants of V, W, and E is greater than 0.45 under acidic conditions.

[0319] -Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, wherein m is 1, 2 or 3, particularly 1 or 2, more particularly 1,

[0320] -Z is an electron withdrawing leaving group.

[0321] In certain embodiments, X is selected from a moiety of Formula 2 or 3.

[0322] In certain embodiments, X is selected from a moiety of Formula 2.

[0323] When using a linker having a moiety of formula 4, the reaction time required to couple the moiety X to the functionalized solid phase by forming a hydrazone or oxime bond is longer, while when using a linker having a moiety of formula 2 or 3, the reaction time required is shorter. The formation of the hydrazone bond between the aldehyde or ketone moiety of the solid support and the 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 the purification process. In contrast, almost no loss of peptide material was observed when using a linker having a moiety of formula 2.

[0324] In some embodiments, U is C 1-6 -alkyl substitution.

[0325] In some embodiments, U is C 1-3 -alkyl substitution.

[0326] In certain embodiments, U is substituted with methyl.

[0327] If U is further substituted by one or more alkyl moieties, the Hammett values ​​of these alkyl moieties are taken into account. In case U is further substituted by phenyl moieties or heteroatoms, the sum of the Hammett values ​​of V, W, E and the optional alkyl substituents is greater than 0.45.

[0328] In certain embodiments, E is selected from pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -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, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6-alkyl, wherein p is 1, 2, 3 or 4.

[0329] In certain embodiments, E is selected from pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N=N-phenyl, -N=NR 8 , -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6-alkyl, wherein p is 1, 2, 3 or 4.

[0330] In certain embodiments, E is selected from pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt.

[0331] In certain embodiments, E is selected from pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H.

[0332] In certain embodiments, E is selected from pyridinyl, pyrimidinyl, pyridazinyl, or -Br.

[0333] In certain embodiments, B is selected from

[0334] -Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylene), trityl (-C(Ph)3), Mmt (-C(Ph)2C6H4OMe), DMT (-C(Ph)(C6H4OMe)2), Cbz (-C(=O)OCH2Ph), benzylamine (=CPh), phthalimide (=(CO)2C6H4), p-toluenesulfonamide (- SO2C6H4Me), benzylamine (-CH2Ph), acetamide (-COMe), trifluoroacetamide (-COCF3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), wherein in particular B is Boc or Eei, wherein more in particular B is Boc, or

[0335] -acetal- or ketal protecting group selected from

[0336]

[0337] wherein r is 0 to 12, in particular 0 to 6, more in particular 0, 1 or 2, and

[0338] R 10 Yes-C1-C 12 -alkyl-, especially C 1-6 -alkyl, more particularly C 1-3 -alkyl.

[0339] In certain embodiments, B is selected from Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylene), trityl (-C(Ph)3), Mmt (-C(Ph)2C6H4OMe), DMT (-C(Ph)(C6H4OMe)2), Cbz (-C(=O)OCH2Ph), benzylamine (=CPh), phthalimide (=(CO)2C6H4), p-toluenesulfonamide (-SO2C6H4Me), benzylamine (-CH2Ph), acetamide (-COMe), trifluoroacetamide (-COCF3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohexan-1-ylidene)-3-ethyl) and 1-(4,4-dimethyl-2,6-dioxocyclohexan-1-ylidene)-3-methylbutyl (ivDde).

[0340] In certain embodiments, B is Boc or Eei (=CMeOEt, 1-ethoxyethylene).

[0341] In certain embodiments, B is Boc.

[0342] In certain embodiments, B is selected from an acetal or ketal protecting group selected from:

[0343]

[0344] wherein r is 0 to 12, in particular 0 to 6, more in particular 0, 1 or 2, and R 10 Yes-C1-C 12 -alkyl-, especially C 1-6 -alkyl.

[0345] In certain embodiments, T is a linear or branched spacer group comprising at least one of the following moieties: -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, especially N, where R 9 Independently selected from H, C 1-4 -alkyl, -C 1-6 -alkyl-NH2, -C 1-6 -alkyl-NHB, -C 1-6 -alkyl-NB2, in particular selected from H and C 1-2 -alkyl, more particularly R 9 is H, wherein B is an independently selected acid-labile amine protecting group.

[0346] In certain embodiments, the total length of the spacer T is between 0.5 and 100 nm.

[0347] In certain embodiments, T is selected from -C1-C 12 -alkyl-(especially -C 1-6 -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 -, especially C 1-3 -alkyl, -R 5 -C(=O)-NR 9 -、-R 5 -NR 9 -C(=O)-R 6 -, more particularly C 1-3 -alkyl or -R 5 -C(=O)-NR 9 -, where R 5 , R 5’ , R 6 , R 9’ and R 9 As defined above.

[0348] In certain embodiments, T is a linear or branched spacer group comprising at least one of the following moieties: -C 1-12 -alkyl-, -C(=O)-, -C(=O)-NR 9 -、-NR 9 -C(=O)-, -NR 9 -, where R 9 Independently selected from 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-R15 , in particular selected from H and C 1-2 -alkyl, more particularly R 9 is H, wherein B is an independently selected acid-labile amine protecting group,

[0349] R 15 It is a blocking agent that reacts with aldehyde moieties, especially R 15 is selected from cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more particularly cysteinyl, wherein

[0350] The amine and / or thiol moieties of the blocking agent may be protected by independently selected acid-labile amine protecting groups B, in particular Boc, and / or acid-labile thiol protecting groups, in particular trityl.

[0351] As mentioned above, the spacer T is generally non-reactive under commonly used purification conditions, except for the removal of the protecting group under acidic conditions. The spacer T can enhance the solubility of the linker molecule. In particular, in R 9 The branched spacer containing a protected or unprotected amine moiety helps to enhance solubility. Under acidic conditions, the amine protecting group B is removed and the amine is protonated.

[0352] In certain embodiments, T is a linear or branched spacer group comprising at least one of the following moieties: -C 1-12 -alkyl-, -C(=O)-, -C(=O)-NR 9 -、-NR 9 -C(=O)-, -NR 9 -, where R 9 Independently selected from H, C 1-4 -alkyl, -C 1-6 -alkyl-NH2, -C 1-6 -alkyl-NHB, -C 1-6 -alkyl-NB2, in particular selected from H and C 1-2 -alkyl, more particularly R 9 is H, wherein B is an independently selected acid-labile amine protecting group.

[0353] The spacer T may also include a blocking function. In particular, the branched spacer may include a blocking agent suitable for binding to the aldehyde moiety. When the solid phase used for peptide purification includes the aldehyde moiety, such as agarose beads, the portion X of the linker compound may be covalently bound to the solid phase, for example, by forming 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 the blocking function of the spacer, such as the cysteinyl moiety.

[0354] In certain embodiments, T is a linear or branched spacer group comprising at least one of the following moieties: -C 1-12 -alkyl-, -C(=O)-, -C(=O)-NR 9 -、-NR 9 -C(=O)-, -NR 9 -, where R 9 Independently selected from H, C 1-4 -alkyl, -R 15 , -C 1-6 -alkyl-R 15 , -C 1-6 -alkyl-NH-R 15 , in particular selected from H and C 1-2 -alkyl, more particularly R 9 is H, wherein B is an independently selected acid-labile amine protecting group,

[0355] R 15 It is a blocking agent that reacts with aldehyde moieties, especially R 15 is selected from cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more particularly cysteinyl, wherein

[0356] The amine and / or thiol moieties of the blocking agent may be protected by independently selected acid-labile amine protecting groups B, in particular Boc, and / or acid-labile thiol protecting groups, in particular trityl.

[0357] In certain embodiments, T is selected from -C1-C 12 -alkyl-(especially 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 selected from -C1-C12 -alkyl-(especially C 1-6 -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 -, most especially -R 5 -NR 9 -C(=O)-R 6 -, where R 5 , R 6 and R 9 As defined above.

[0358] As described above, moieties U, V, W and E contribute to the stability of the linker under acidic conditions. For example, a linker comprising moieties U or V containing an amine or heterocycle such as pyridine is stable under acidic conditions (particularly TFA>50%, in the presence of water, pH<0) because the amine or heterocycle is protonated. In addition, protonated linkers improve the solubility of the linker-peptide complex. If the pH is higher than the pKa of the linker, the linker will rapidly decompose as the last step during the desired peptide release.

[0359] V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, wherein R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 Yes H, R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It's methyl.

[0360] V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12-, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, wherein R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 Yes H, R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It's methyl.

[0361] V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, wherein R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 Yes H, R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It's methyl.

[0362] V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, wherein R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 Yes H, R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It's methyl.

[0363] V is an electron withdrawing moiety selected from -NH-C(=O)-, -C(=O)-HN-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, pyrazinyl, pyridazinyl.

[0364] V is an electron withdrawing moiety selected from -NH-C(=O)-, -C(=O)-HN-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl.

[0365] In certain embodiments, V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-.

[0366] In certain embodiments, V is selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 - and -pyridyl-, pyrimidinyl, wherein R 11 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 11 is H, and R 12 Selected from H and C 1-4 -alkyl, in particular selected from H and C 1-2 -alkyl, more particularly R 12 It's methyl.

[0367] In certain embodiments, V is selected from -NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, wherein R 11 Selected from H and C 1-4 -alkyl, and R 12 Selected from H and C 1-4 -alkyl.

[0368] In certain embodiments, V is selected from -NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, wherein R 11 Selected from H and C 1-2 -alkyl, and R 12 Selected from H and C 1-2 -alkyl.

[0369] In certain embodiments, V is selected from -NH-C(=O)-, -N-(CH3)-, -pyridinyl-, pyrimidinyl.

[0370] In certain embodiments, W is selected from -N3, -S(=O)-R 8 、-SSR 8 、-O-CH2-N3、-N=NR 8 , especially -N3, -N=NR 8 、-O-CH2-N3、-SSR8 , where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or (CH2) p -NMe2, in particular pyridyl or -C1-C6-alkyl, wherein p is 1, 2, 3 or 4.

[0371] In R 8 It is C 1-6 In the case of -alkyl, the alkyl portion may be straight chain or branched, such as tert-butyl. In particular, when W is -SSR 8 In the case of R 8 It may be pyridyl or -C1-C4-alkyl, in particular pyridyl or tert-butyl.

[0372] Contains part W = -SSR 8 The linker can be cleaved with a thiol. Therefore, when such a linker is used for peptide purification, the cleavage of the peptide-linker complex from the synthesis resin used during SPPS under acidic conditions (e.g., TFA>50%, in the presence of water, pH<0) can be performed in the absence of a thiol.

[0373] In certain embodiments, W is selected from -N3, -S(=O)-R 8 、-SSR 8 、-O-CH2-N3、-N=NR 8 , especially -N3, -N=NR 8 、-O-CH2-N3、-SSR 8 , where R 8 It is pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl or -C1-C6-alkyl, or in particular pyrimidinyl, pyridinyl or -C1-C6-alkyl, more in particular pyridinyl or -C1-C6-alkyl.

[0374] In certain embodiments, U is selected from phenyl or a five-membered or six-membered heterocyclic ring, wherein the five-membered or six-membered heterocyclic ring contains 1 or 2 heteroatoms.

[0375] In certain embodiments, U is selected from phenyl or a five-membered or six-membered heterocyclic ring, wherein the five-membered or six-membered heterocyclic ring contains 1 heteroatom.

[0376] In certain embodiments, U is selected from phenyl or a six-membered heterocyclic ring.

[0377] In certain embodiments, U is selected from phenyl or a six-membered heterocyclic ring, wherein the six-membered heterocyclic ring contains 1 or 2 heteroatoms.

[0378] In certain embodiments, the five-membered or six-membered heteroaryl moiety of U contains 1 or 2 heteroatoms, in particular the five-membered heteroaryl moiety of moiety U is selected from pyrazole, imidazole, and the six-membered heteroaryl moiety of moiety U is selected from pyridine, pyridazine, pyrimidine, pyrazine, in particular pyridine.

[0379] In certain embodiments, U is selected from phenyl or a six-membered heterocyclic ring, wherein the six-membered heterocyclic ring contains 1 heteroatom. In certain embodiments, the five-membered heterocyclic ring of part U is selected from pyrazole, imidazole, and the six-membered heterocyclic ring of part U is selected from pyridine, pyridazine, pyrimidine, pyrazine.

[0380] In certain embodiments, U is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, particularly phenyl or pyridine, more particularly phenyl.

[0381] In certain embodiments, U is selected from a moiety of Formula 5, 6, 7, or 8,

[0382]

[0383] in

[0384] T, V, Y, W and E are as defined above,

[0385] In the case of formulas 5 and 6, U is combined with a part T or V,

[0386] In the case of formulas 7 and 8, U is combined with part V,

[0387] A 1 , A 2 , A 3 , A 4 and D 1 , D 2 , D 3 , D 4 are independently selected from C, N, S and O, in particular from C and N, wherein A 1 , A 2 , A 3 and A 4 or D 1 , D 2 , D 3 and D 4 2 to 4 of which are C, especially A 1 , A 2 , A 3 and A 4 or D 1 , D 2 , D 3 and D 4 3 or 4 of the parts are C, more particularly all parts A 1 , A 2 , A3 and A 4 or D 1 , D 2 , D 3 and D 4 is C, n is

[0388] In the case of equations 5 and 6, an integer between 0 and 3,

[0389] In the case of equations 7 and 8, an integer between 0 and 4,

[0390] q is an integer between 0 and 4, wherein the sum of n and q is equal to or less than 4.

[0391] In certain embodiments, U is selected from a moiety of Formula 5 or 6.

[0392] In certain embodiments, U is selected from a moiety of Formula 5, 6, 7, or 8, wherein

[0393] T, V, Y, W and E are as defined above,

[0394] In the case of formulas 5 and 6, U is combined with a part T or V,

[0395] In the case of formulas 7 and 8, U is combined with part V,

[0396] A 1 , A 2 , A 3 , A 4 and D 1 , D 2 , D 3 , D 4 are independently selected from C, N, S and O, in particular from C and N,

[0397] n is

[0398] In the case of equations 5 and 6, an integer between 0 and 2,

[0399] In the case of formulae 7 and 8, an integer between 0 and 2, in particular 0 and 1,

[0400] q is an integer between 0 and 2, in particular 0 and 1.

[0401] 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,

[0402] in

[0403] T, V, Y, W, E, q and n are as defined above,

[0404] In the case of formulas 9 and 10, U is combined with a part T or V,

[0405] In the case of formulas 11 and 12, U is combined with part V,

[0406] All parts A 2 , A 3 and A 4 It 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 all C, and

[0407] D 2 It is C or N, especially C.

[0408] In certain embodiments, U is selected from a moiety of Formula 9, 10, 11 or 12,

[0409] in

[0410] T, V, Y, W, E, q and n are as defined above,

[0411] In the case of formulas 9 and 10, U is combined with a part T or V,

[0412] In the case of formulas 11 and 12, U is combined with part V,

[0413] A 2 and A 3 All are C, or A 2 and A 3 One of them is C and A 2 and A 3 The other one is N,

[0414] D 2 It is C or N.

[0415] If the moiety U comprises a N-containing heteroaryl group, the N atom is protonated under acidic conditions and thus increases the solubility of the linker molecule.

[0416] In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20, or 21, particularly a moiety of formula 13 to 19, more particularly a moiety of formula 15 or 19,

[0417]

[0418] in

[0419] T, V, Y, W, E, q and n are as defined above,

[0420] In the case of formulas 13, 14 and 15, U is combined with a part T or V,

[0421] In the case of formulas 16, 17 and 18, U is combined with part V,

[0422] A 2 , A 3 and A 4 is C or N, especially C,

[0423] D 2 It is C or N.

[0424] In certain embodiments, U is selected from a moiety of Formula 13, 14, 15, 16, 17, or 18,

[0425] wherein T, V, Y, W, E, q and n are as defined above,

[0426] In the case of formulas 13, 14 and 15, U is combined with a part T or V,

[0427] In the case of formulas 16, 17 and 18, U is combined with part V,

[0428] A 3 is C or N,

[0429] D 2 It is C or N.

[0430] In certain embodiments, Z is selected from: -F, -Cl, -Br, -I, -N3, -OH, -O(C=O)CH2(C=O)OH-SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3

[0431]

[0432] Especially -OH, -Cl, Especially -OH,

[0433] Where R 14 is a C1-C6-alkyl-, aryl- or benzyl substituent.

[0434] In certain embodiments, Z is selected from: -F, -Cl, -Br, -I, -N3, -OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3

[0435]

[0436] Especially -OH, -Cl, Especially -OH,

[0437] Where R 14 is a C1-C6-alkyl-, aryl- or benzyl substituent.

[0438] In certain embodiments, the compound of Formula 1 is selected from a compound 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.

[0439]

[0440]

[0441]

[0442]

[0443]

[0444] 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.

[0445] In certain embodiments, the compound of Formula 1 is a compound of Formula X9, X12, X19, X22, X8, X18, X1, X14, X15, or X20.

[0446] In certain embodiments, the compound of Formula 1 is a compound of Formula X9, X12, X19, X22, or X8.

[0447] In certain embodiments, the compound of Formula 1 is a compound of Formula X1 or X2. BRIEF DESCRIPTION OF THE DRAWINGS

[0448] Figure 1 A schematic diagram of peptide modification on a solid support of the present invention is shown. SPPS = solid phase peptide synthesis, SR = synthesis resin for SPPS, S = modification and purification support, P = peptide to be modified, 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) fixation, d1) staple modification, d2) fatty acid modification, d3) disulfide formation, e) release of modified peptide, i) deprotection of intermediate protecting group.

[0449] Figure 2 The present invention shows that by using the linker molecule X1 to bind peptide P1 (H- AK(Palm-γE-OH) EFIAWLVRGRG -NH2, SEQ ID NO 18) for 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 P1. 2.) Chromatogram of P1 after coupling of linker (X1) and final TFA cleavage. 3.) Chromatogram of P1 after coupling of linker (X1) and final TFA cleavage as described in claims 1-9 by Palm -γ Chromatogram of P1 after Glu(OH)-OtBu coupling modification and purification.

[0450] Figure 3 The present invention shows that by using the linker molecule X1 to bind peptide P2 (H- HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG -NH2, SEQ ID NO 2) Examples of peptide modification / purification according to the present 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.) Preparation of peptides by Palm -γ Chromatogram of P2 after Glu(OH)-OtBu coupling modification and purification.

[0451] Figure 4 An example of peptide modification / purification of the present invention by using the linker molecule X1 of the present invention on peptide P3 (H-CRVPGDAHHADSLC-NH2, SEQ ID NO 3) is shown. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of a crude peptide sample before coupling of the linker (X1) to P1. 2.) Chromatogram of P3 after modification and purification by m-dibromobenzyl coupling as described in claims 1 to 5 and 10 to 12.

[0452] Figure 5An example of peptide modification / purification of the present invention by using the linker molecule X1 of the present invention on peptide P4 (H-VRCPGAAHHADSLC-NH2, SEQ ID NO 4) is shown. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of a crude peptide sample before coupling of the linker (X1) to P1. 2.) Chromatogram of P4 after modification and purification by m-dibromobenzyl coupling as described in claims 1 to 5 and 10 to 12.

[0453] Figure 6 An example of peptide modification / purification of the present invention by using the linker molecule X1 of the present invention on peptide P5 (H-VRVPGCAHCADSLY-NH2, SEQ ID NO 5) is shown. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of a crude peptide sample before coupling of the linker (X1) to P1. 2.) Chromatogram of P5 after modification and purification by m-dibromobenzyl coupling as described in claims 1 to 5 and 10 to 12.

[0454] Figure 7 An example of peptide modification / purification of the present invention by using the linker molecule X1 of the present invention on peptide P6 (H-ACSWPARCLHQDLCA-NH2, SEQID NO 6) is shown. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of a crude peptide sample before coupling of the linker (X1) to P1. 2.) Chromatogram of P6 after modification and purification by 1,3,5-tris(bromomethyl)benzene coupling as described in claims 1 to 5 and 10 to 12.

[0455] Figure 8 An example of peptide modification / purification of the present invention by using the linker molecule X1 of the present invention on peptide P7 (H-ACREGFLRCLHRPTVCG-NH2, SEQID NO 7) is shown. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of a crude peptide sample before coupling of the linker (X1) to P1. 2.) Chromatogram of P7 after modification and purification by 1,3,5-tris(bromomethyl)benzene coupling as described in claims 1 to 5 and 10 to 12.

[0456] Fig. 9An example of peptide modification / purification of the present invention is shown for peptide P8 (H-ACYNEFGCEDFYDICA-NH2, SEQID NO 8) by using the linker molecule X1 of the present 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 P1. 2.) Chromatogram of P8 after modification and purification by coupling with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethanone) as described in claims 1 to 5 and 10 to 12.

[0457] Fig.10 An example of peptide modification / purification of the present invention by using the linker molecule X1 of the present invention on peptide P8 (H-ACYNEFGCEDFYDICA-NH2, SEQID NO 8) is shown. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trail cleavage of a crude peptide sample before coupling of the linker (X1) to P8. 2.) Chromatogram of P8 after modification and purification by 1,3,5-triacryloyl-1,3,5-triazine coupling as described in claims 1 to 5 and 10 to 12.

[0458] Fig.11 An example of peptide modification / purification according to the invention is shown for peptide P3 (H-CRVPGDAHHADSLC-NH2, SEQ ID NO 3) by using the linker molecule X1 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 and purification by air oxidation in DMSO / (NH4)2CO3 pH 8.7 as described in claims 1 to 5 and claim 13.

[0459] Fig.12 The ESI-MS spectrum of the modified peptide P12a (H-KKKKSC (C3H5O2-diPalm) -NH2, SEQ ID NO 22) of the present invention using 1-bromopropyl-2,3-dipalmitate (Pam2-Br) is shown. A = intensity, B = m / z.

[0460] Fig.13 The ESI-MS spectrum of the modified peptide P12b (H-KKKKSC (C3H5O2-diPalm) -NH2, SEQ ID NO 22) of the present invention using 1-iodopropyl-2,3-dipalmitate (Pam2-I) is shown. A = intensity, B = m / z.

[0461] Fig.14The ESI-MS spectrum of the modified peptide P12d (H-KKKKSC(C2H4O-Palm)-NH2, SEQ ID NO 23) of the present invention using Palm-OC2H4I (Pam-Et-I) is shown. A = absorption (210 nm), B = time / min.

[0462] Fig.15 The ESI-MS spectrum of the modified peptide P13a (H-KEKFSINSEDNNGC (C3H5O2-d-Palm)-NH2, SEQ ID NO 24) of the present invention using 1-bromopropyl-2,3-dipalmitate (Pam2-OTs) is shown. A = intensity, B = m / z.

[0463] Fig.16 The ESI-MS spectrum of the modified peptide P13b (H-KEKFSINSEDNNGC (C3H5O2-diPalm)-NH2, SEQ ID NO 24) of the present invention using 1-iodopropyl-2,3-dipalmitate (Pam2-I) is shown. A = intensity, B = m / z.

[0464] Fig.17 The UV chromatogram of the modified peptide P14d (H-KKKKSC (C2H4O-Palm) -NH2, SEQ ID NO 23) of the present invention by using Palm-OC2H4I (Pam-Et-I) is shown; retention time = 2.25 min. A = absorption (210 nm), B = time / min.

[0465] Fig.18 The ESI-MS spectrum of the modified peptide P2 / c (H-KEKFSINSEDNNGC (propyl-diPalm)-NH2, SEQ ID NO 32) of the present invention using 1-tosylpropyl-2,3-dipalmitate (X3) is shown. A = intensity, B = m / z.

[0466] Fig.19 The ESI-MS spectrum of the modified peptide P14b (HC(C3H5O2-diPalm)RG-NH2, SEQ ID NO 26) of the present invention using 1-iodopropyl-2,3-dipalmitate (Pam2-I) is shown. A = intensity, B = m / z.

[0467] Fig. 20The ESI-MS spectrum of the modified peptide P14c (HC(C3H5O2-diPalm)RG-NH2, SEQ ID NO 26) of the present invention using 1-tosylpropyl-2,3-dipalmitate (Pam2-OTs) is shown. A = intensity, B = m / z.

[0468] Fig.21 The UV chromatogram of the modified peptide P14d HC(C2H4O-Palm)RG-NH2 (SEQ ID NO 27) of the present invention using Palm-OC2H4I (Pam-Et-I) is shown; retention time = 1.61 min. A = absorption (210 nm), B = time / min.

[0469] Fig. 22 An example of peptide modification / purification of the invention is shown for peptide P2b (H-HAEGTFTSDVSSYLEGQAAK(Palm-Glu-OH)EFIAWLVRGRG-OH, SEQ ID NO 21) by using the linker molecule X1 of the invention. A = absorbance (210 nm), B = time / min; 1.) Chromatogram after TFA cleavage of a crude peptide sample after coupling of the linker to P2b. 2.) Chromatogram of P2b after lipidation on a solid support as described in Example 8 using Palm-Glu(OSu)-OtBu as an -OSu activated fatty acid spacer conjugate.

[0470] Fig.23 An example of peptide modification / purification of the present invention is shown for peptide P15 (H-HAibEGTFTSDVSSYLEGQAAK(CO2H-Ste-Glu(aeea-aeea-OH)-OH)EFIAWLVRGRG-OH, SEQ ID NO 28) using the linker molecule X1 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of crude peptide sample after TFA cleavage after coupling of the linker to P15. 2.) Chromatogram of P15 after lipidation on a solid support as described in Example 9 using tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu as -OSu-activated fatty acid spacer conjugate.

[0471] Fig.24An example of peptide modification / purification of the present invention is shown for peptide P16 (Palm-SGGK(Palm)WSK(Palm)SSVIGWPAVRER-NH2, SEQ ID NO29) by using the linker molecule X1 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trial cleavage of a crude peptide sample after coupling of the linker to P16. 2.) Chromatogram of P16 after lipidation on a solid support as described in Example 10 using Palm-S as an activated lipid.

[0472] Fig.25 An example of peptide modification / purification of the present invention is shown for peptides P17a (Palm-SGGKWSK(Palm)SSVIGWPAVRER-NH2, SEQ ID NO 33) P17b (Myr-SGGKWSK(Myr)SSVIGWPAVRER-NH2, SEQ ID NO 34) by using the linker molecule X1 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of trial cleavage of crude peptide sample after coupling of the linker to P17. 2.) Chromatogram of P17a after lipidation on a solid support as described in Example 10 using Palm-S as an activated lipid with a spacer. 2.) Chromatogram of P17a after lipidation on a solid support as described in Example 11 using Myr-OSu as an activated lipid.

[0473] Fig.26 The present invention shows that by using the linker molecule X1 to bind peptide P6 (H- ACSWPARCLHQDLCA-NH2, SEQ ID NO 6) for peptide modification / purification of the present invention. A = absorbance (210 nm), B = time / min; 1.) Chromatogram of trial cleavage of crude peptide sample before linker coupling to P6. 2.) Chromatogram of P6 after linker (LuS1) coupling and TFA cleavage from SPPS resin. 3.) Chromatogram of P6 after intramolecular dicyclization with TATB and purification as described in Example 12 using aldehyde-modified agarose as solid phase and DMSO + 10 vol.% immobilization buffer 2 as peptide solvent for immobilization. 4.) Chromatogram of P6 after intramolecular dicyclization with TATB and purification 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 intramolecular bicyclization with TATB and purification as described in Example 14 as claimed, using aldehyde-modified agarose as solid phase and 1 M SDS in DMSO + 10 vol.% immobilization buffer 2 as peptide solvent for immobilization. 6.) Chromatogram of P6 after intramolecular bicyclization with TATB and purification as described in Example 16 at 1°C, using aldehyde-modified poly(methyl acrylate) beads as solid phase and neat HFIP as peptide solvent for immobilization.

[0474] Fig. 27 The present invention shows that by using the linker molecule X1 to bind peptide P8 (H- ACYNEFGCEDFYDICA -NH2, SEQ ID NO 8) for peptide modification / purification of the present invention. A = absorbance (210 nm), B = time / min; 1.) Chromatogram of trial cleavage of crude peptide sample before linker coupling to P8. 2.) Chromatogram of P8 after linker (LuS1) coupling and TFA cleavage from SPPS resin. 3.) Chromatogram of P8 after intramolecular dicyclization with TATB and purification as described in Example 12 using aldehyde-modified agarose as solid phase and DMSO + 10 vol.% immobilization buffer 2 as peptide solvent for immobilization. 4.) Chromatogram of P8 after intramolecular dicyclization with TATB and purification 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 intramolecular bicyclization with TATB and purification as described in Example 14 using aldehyde-modified agarose as solid phase and 1 M SDS in DMSO + 10 vol.% immobilization buffer 2 as peptide solvent for immobilization. 6.) Chromatogram of P8 after intramolecular bicyclization with TATB and purification as described in Example 16 at 1°C using aldehyde-modified poly(methyl acrylate) beads as solid phase and pure HFIP as peptide solvent for immobilization.

[0475] Fig.28 The present invention shows that by using the linker molecule X1 to bind peptide P3 (H- CRVPGDAHHADSLC -NH2, SEQ ID NO 3) Examples of peptide modification / purification according to the invention. A = Absorption (210 nm), B = Time / min; 1.) Chromatogram of P3 after coupling with linker (X1) and final TFA cleavage. 2.) Chromatogram of P3 after disulfide formation and purification as described in claims 1-9.

[0476] Fig.29 The present invention shows that by using the linker molecule X1 to bind peptide P4 (H-VR CPGAAHHADSLC -NH2, SEQ ID NO 4) 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 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.

[0477] Fig.30 The present invention shows that by using the linker molecule X1 to bind peptide P9 (H- QUR Example of peptide modification / purification of the invention using PRG-NH2, SEQ ID NO 9). A = Absorption (210 nm), B = Time / min; 1.) Chromatogram of P9 after linker (X1) coupling and final TFA cleavage. 2.) Chromatogram of P9 after disulfide formation and purification as described in claims 1-9.

[0478] Fig.31 The present invention shows that by using the linker molecule X1 to bind peptide PY (HK CNTATC ATQRLANFLVHSSNFGPILPPTNVGSNTY-NH2, SEQ ID NO 10) Examples of peptide modification / purification according to the invention. A = Absorption (210 nm), B = Time / min; 1.) Chromatogram of PY after linker (X1) coupling and final TFA cleavage. 2.) Chromatogram of PY after disulfide formation and purification as described in claims 1-9.

[0479] Fig.32 An example of peptide modification / purification according to the invention is shown for peptide PZ (H-CCEYCCNPACTGCY-NH2, SEQ ID NO 11) by using the linker molecule X1 according to the invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of PY after coupling with the linker (X1) and final TFA cleavage. 2.) Chromatogram of PY after disulfide formation and purification as described in claims 1-9.

[0480] Fig.33 The formation of lipidated peptide P is shown. Peptide P is immobilized on a solid support (grey balls) and the peptide contains lysine. In the first step, the organic molecule Palm-Glu-OtBu is coupled to the lysine side chain. Palmitoyl (Palm) corresponds to the part FA and the glutamic acid part protected by the tert-butyl group (Glu-OtBu) corresponds to the spacer Sp-OH. In the second step, the protective tert-butyl protecting group is removed under acidic conditions. DETAILED DESCRIPTION

[0481] Example

[0482] General approach

[0483] Solid Phase Peptide Synthesis (SPPS) Linker Coupling and TFA Cleavage:

[0484] Peptide sequences P1-P8 were synthesized on an Intavis MultiPep Rsi at a 100 μmol scale under standard solid phase peptide synthesis conditions, wherein 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. The linker X1 was coupled to P1-P17 on the resin using 4 equivalents of linker X1 (301 mg), 6 equivalents of oxyma (86 mg) and 6 equivalents of diisopropylamine (DIEA, 105 μL) in 1.3 mL DMF for 2 h. The amount used was based on the loading of the SPPS resin (Rinkamide RAM). The reaction mixture was shaken for 120 min, filtered, washed (3x DMF, 3x DCM) and dried.

[0485] The dried resin was incubated with a TFA-cleavage mixture ((TFA / H2O / PhOH / PHSH / ethanedithiol, 82.5:5:5:52.5, v / v / w / v / v, 10 mL per 100 μmol synthesis scale) for 120 min. The cleavage solution was separated from the synthesis resin by filtration and collected in a Falcon tube 10 times the volume of the solution. The peptide was precipitated by adding 9 volumes of chilled diethyl ether. The container was centrifuged at 5000 rpm for 3.5 min, the supernatant was decanted, and the peptide precipitate was washed again with diethyl ether and then decanted. The peptide was then dissolved in H2O / MeCN / TFA (99.95:99.95:0.1, v / v / v), frozen with liquid nitrogen and lyophilized under high vacuum.

[0486] General procedures for modification and purification of peptides by the methods of the invention

[0487] Solubilization and immobilization of crude linker-tagged peptides

[0488] The crude linker-tagged peptide mixture was dissolved in pure DMSO (4.5 mL for 100 μmol synthesis scale). After complete dissolution, 10 vol.% of immobilization buffer 2 (0.1 M citric acid / Na2CO3 pH 3.5, containing 8 M guanidine hydrochloride, 0.45 mL for 100 μmol synthesis scale) was added.

[0489] The peptide of the linker label is fixed on 1.5 times the amount of aldehyde-modified agarose loaded with 100 μmol / 1mL sedimentation beads (100 μmol / 2mL slurry beads), or on hydroxylated poly (methyl acrylate) beads. After equal division, the bead material is washed with 3x milli-Q water and 3x fixed buffer 1 (0.1M citric acid / Na2CO3 pH 4.5). Then the dissolved crude linker-labeled peptide mixture is added to the agarose and shaken. It is worth noting that the beads should have free and good fluctuations in the fixed solution. After a reaction time of 90 minutes, the fixed solution is filtered out by suction.

[0490] washing

[0491] After fixation, the supernatant was removed and the beads connected to the peptide with the linker were washed 3 times each with 5 mL of DMSO (for 100 μmol scale). A mixture of L-cysteine ​​in 0.1 M citric acid / Na2CO3 pH 4.5 was then added and the reactor was shaken for 15 min or longer. The mixture was removed and the beads were washed 3 times with 5 mL (for 100 μmol scale) of the following solvents and solutions:

[0492] 1) DMSO with 0.9 M guanidine hydrochloride

[0493] 3) 70% ethanol in 0.1M NaCl Milli-Q water

[0494] Modification of peptides immobilized on solid supports

[0495] Typically, modification of the target peptide is performed after immobilizing the linker-tagged peptide on the modification / purification support and washing the peptide-support conjugate.

[0496] In the case of fatty acid modification of peptides, after fixation and washing of the target peptide, the imine of the lysine side chain can be constructed with excess aldehyde and the aldehyde converted to oxime by addition of O-methylhydroxylamine hydrochloride or L-cysteine. Thereafter, after a pre-activation time, the fatty acid is added to the peptide on the support together with the coupling reagent and an organic base. Thereafter, the excess reagent is washed off and the peptide is released as described below.

[0497] In the case of macrocycle formation by electrophilic scaffolds, after immobilization and washing of the target peptide, the disulfide formation can be reduced by adding a small amount of DTT. After washing off the DTT, the electrophilic scaffold is added to the support. If necessary, this step can be repeated to increase the conversion to the macrocyclic product. Thereafter, the excess reagent is washed off and the peptide is released as described below.

[0498] In the case of disulfide formation, after immobilization and washing of the target peptide, the imine of the lysine side chain and the thioacetal of the cysteine ​​side chain can be constructed with excess aldehyde, and the aldehyde is converted to the thiazolidine by adding guanidine hydrochloride. The disulfide is formed on the support in a 1:1 mixture of DMSO and 0.4M (NH4)2CO3, 6M GdmCl. Thereafter, the oxidation buffer is washed off and the peptide is released as described below.

[0499] Release of modified peptides

[0500] 500mg dithiothreitol (DTT) is dissolved in 5ml of 5w.% NaHCO3 pH8, added to agarose or poly (methyl acrylate) material, 5mL MeCN is already on the beads and shakes for 15min. Discard the supernatant, and the agarose material is washed 3x with water and 2x with MeCN, to remove excessive DTT and oxidized DTT. Subsequently, 2mL TFA / H2O (2:3) is added and shaken for 60min again. Then another 2mL pure TFA is added to the solution, the mixture is eluted and collected in a Falcon tube. It is eluted twice more, each time with 2mL TFA / H2O (95:5). The joint peptide is precipitated by adding 9 times the volume of deep frozen diethyl ether, and then centrifuged at 5000rpm for 3.5min, and then the ether phase is decanted. The peptide is now present as a particle, and can be lyophilized if necessary.

[0501] Experimental instructions for the organic synthesis of FA-A and FA-Sp-A

[0502] Synthesis of 1-Bromopropyl-2,3-dipalmitate (Pam2-Br)

[0503] 1,2-Dipalmitoyl-sn-glycerol (0.4 g, 0.7 mmol, 1 eq) was dissolved in 9 mL DCM and tetrabromomethane (0.5 g 1.5 mmol, 2.1 eq) was added. The solution was cooled to 0 °C and triphenylphosphine (0.4 g 1.5 mmol, 2.1 eq) was added in batches over 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. 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. In order to remove residual O=PPh3, the product was treated with MeOH in an ultrasonic bath. After centrifugation, the solvent was decanted. The washing with methanol was repeated twice to obtain 1-bromopropyl-2,3-dipalmitate (Pam2-Br) (0.17 g, 0.27 mmol, 39%) as an off-white solid.

[0504] R f =0.68 (chloroform / ethyl acetate 96:4); UPLC-MS: t R =5.69 min (gradient 0-3 min 40-100% D (0.1% TFA in 9:1 MeCN / iPrOH) in C (0.1% TFA in 8:1:1 H2O / MeCN / iPrOH), 3-6 min 100% D); UPLC-purity (210 nm) ≥98%; ESI-MS: (calculated [M+Na] + :653.41,655.41, measured value:653.21,655.68; calculated value [M-OCOC 15 H 31 ] ·+ :375.19,377.19, measured values:375.27,377.26).

[0505] 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).

[0506] Synthesis of 1-iodopropyl-2,3-dipalmitate (Pam2-I)

[0507] 1,2-Dipalmitoyl-sn-glycerol (0.5 g, 0.88 mmol, 1 eq.), imidazole (0.12 g, 1.76 mmol, 2 eq.) and triphenylphosphine (0.5 g 1.99 mmol, 2.3 eq.) were dissolved in 7.5 mL THF. I2 (0.32 g 1.67 mmol, 2.0 eq.) was slowly added to the solution. After stirring at room temperature for 4 h, 250 mg NaHCO3 and 1 mL water were added to quench the reaction. The color of the reaction mixture disappeared. The solvent was evaporated and the residue was washed with 4 mL water. The suspension was centrifuged and the supernatant was decanted. The crude product was treated with 6 mL MeOH in an ultrasonic bath for several minutes. After centrifugation, the supernatant was decanted. The precipitate was washed four more times with 6 mL MeOH. The product was dried in vacuo to give 0.47 g (0.69 mmol, 78%) of 1-iodopropyl-2,3-dipalmitate as an off-white solid.

[0508] R f =0.66 (chloroform / ethyl acetate 96:4); UPLC-MS: t R =5.83min (gradient 0-3min 40-100% D in C, 3-6min 100% D); UPLC-purity (210nm) ≥90%; ESI-MS: (calculated value [M+Na] + :701.40, measured value:701.50; calculated value [M-OCOC 15 H 31 ] ·+ :423.18, measured value:423.28).

[0509] 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).

[0510] Synthesis of 1-Tosylpropyl-2,3-dipalmitate (Pam2-OTs)

[0511] 4-Toluenesulfonyl chloride (2 g, 10.5 mmol, 10 equivalents) was dissolved in 50 mL of THF. The solution was cooled to 0 ° C with an ice bath. 1,2-dipalmitoyl-sn-glycerol (0.6 g, 1.05 mmol, 1 equivalent) was dissolved in 14.4 mL of THF at room temperature, and 2.84 mL of 15w% NaOH aqueous solution (10.7 mmol, 10 equivalents) was added. 4-Toluenesulfonyl chloride solution was slowly added and the reaction mixture was stirred at room temperature for 4 h. The solvent was evaporated under reduced pressure and the residue was absorbed in chloroform. The organic phase was washed three times with brine, three times with water, and then dried with Na2SO4. After filtration, the solvent was removed under reduced pressure to obtain a crude product as a yellow liquid, which was converted into a solid after vacuum drying. 5 mL of cooled MeOH was added and the resulting mixture was ultrasonically treated for 10 min. After centrifugation, the supernatant was decanted. The residue was treated with MeOH five more times to remove excess toluenesulfonyl chloride. The product was dried in vacuo to give 0.47 g (0.64 mmol, 61%) of an off-white solid.

[0512] R f =0.64 (chloroform / ethyl acetate 96:4); UPLC-MS: t R =4.99 min (gradient 0-3 min 40-100% D in C, 3-6 min 100% D); UPLC-purity (210 nm) = 93%; ESI-MS: (calculated value [M+Na] + :745.51, measured value:745.68).

[0513] 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).

[0514] Synthetic steps for the synthesis of 2-iodoethyl palmitate (Pam-Et-I)

[0515] Palmitoyl chloride

[0516] Thionyl chloride (7.2 mL, 100 mmol, 10 eq) was slowly added dropwise to palmitic acid (2.6 g, 9.9 mmol, 1 eq) 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 with water 3 times. 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 in vacuo to give the product (2.47 g, 9 mmol, 90%) as a colorless solid.

[0517] R f =0.52 (chloroform / ethyl acetate 96:4).

[0518] 2-Iodoethyl Palmitate (Pam-Et-I)

[0519] Under nitrogen atmosphere, triethylamine (0.5 mL, 3.6 mmol, 3 eq.) was added to a solution of 2-iodoethanol (0.71 mL, 9.1 mmol, 5 eq.) in 18 mL of DCM. The solution was stirred at 0 °C for 30 min. Palmitoyl chloride (0.5 g, 1.8 mmol, 1 eq.) was added at 0 °C. Stirring was continued at room temperature for 4 h. The solution was washed three times in sequence, each time with water and saturated aqueous NaHCO3. After drying over Na2SO4 and filtering, the solvent was removed under vacuum. The resulting crude product was treated with MeOH in an ultrasonic bath and the suspension was centrifuged. The supernatant was poured off and the residue was washed twice more with MeOH. The MeOH solutions from the second and third washes were combined and kept at room temperature overnight to allow the product to crystallize as colorless needles. The solvent was removed and the crystals were dried under vacuum to give 0.15 g (0.36 mmol, 20%) of the product.

[0520] R f =0.43 (chloroform / ethyl acetate 96:4.

[0521] 1 H NMR (400MHz, CDCl3) δ4.33 (t, J = 6.8 Hz, 2H), 3.29 (t, J = 6.8 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H), 1.64 (p, J = 7.4 Hz, 2H), 1.26 (m, 24H), 0.88 (t, J = 6.9 Hz, 3H).

[0522] Synthesis of Myristic Acid N-Hydroxysuccinimide Ester (Myr-OSu)

[0523] To a stirred solution of 1 g myristic acid (4.33 mmol, 1 eq.) and 554 mg N-hydroxysuccinimide (4.77 mmol, 1.1 eq.) in 44 mL acetonitrile was added 994 mg dicyclohexylcarbodiimide (DCC, 4.77 mmol, 1.1 eq.) and the mixture was stirred for 1.5 h. The formed precipitate was removed by filtration and the filtrate was washed with 50 mL acetonitrile. Then 1.430 g (4.33 mmol, quantitative yield) of the desired white crystalline title compound was obtained by evaporating the solvent under reduced pressure and drying. ESI-MS: (calculated value [M+Na] + :348.22, measured value:348.28).

[0524] Synthesis of 22-(tert-Butyloxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxylidene-3,6,12,15,42-pentaoxa-9,18,23-triazatetradecanoic acid N-hydroxysuccinimide ester (tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu)

[0525] To a stirred solution of 260 g 22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxy-3,6,12,15,42-pentaoxa-9,18,23-triazatetradecanoic acid (0.3 mmol, 1 eq.) and 39 mg N-hydroxysuccinimide (0.33 mmol, 1.1 eq.) in 6 mL acetonitrile was added 67 mg DCC (0.33 mmol, 1.1 eq.) and the mixture was stirred for 1.5 h. The formed precipitate was removed by filtration and the filtrate was washed with 10 mL acetonitrile. Then 0.305 g (0.30 mmol, quantitative yield) of the desired title compound as a colorless oil was obtained after evaporating the solvent and drying under reduced pressure. ESI-MS: (calculated value [M+H] + :943.59, measured value:943.72).

[0526] Example 1: Lipidation using palmitoyl-L-Glu(OH)-tBu-building blocks bound to a purified solid support Formation of peptides P1-P2a

[0527] The peptide modification and purification methods of the present invention were applied to two peptides of different length and polarity, which are H- AKEFIAWLVRGRG -NH2( SEQ ID NO:1 )(P1), which is a fragment 25-37(K34R) of glucagon-like peptide-1 (GLP-1), and H- HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG -NH2( SEQ ID NO:2 )(P2a), which is a longer sequence 7-37(K34R) of glucagon-like peptide-1 (GLP-1).

[0528] Peptides were immobilized and washed at a 5 μmol scale according to the general method. As a difference from the general method, after immobilization and removal of the immobilization supernatant, an O-methylhydroxylamine hydrochloride solution (4 μmol) in immobilization buffer 1 (0.1 M citric acid / Na2CO3 pH 4.5) was added. q., 0.1M) and shake for 20min. After washing according to the general method, wash 3 times with 10vol.% pyridine solution in DMF and 3x DMF. Then add 0.3M coupling solution composed of 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) or diisopropylcarbodiimide (DIC) in DMF, 10 equivalents of Oxyma and 12 equivalents of palmitoyl-L-Glu (OH)-tBu building blocks. Before being added to the agarose beads, the solution was preactivated for 20min. After addition, the beads were shaken for 2 hours, the supernatant was then filtered, and the solid support was washed with 3x DMF, 3x H2O and 3x MeCN, and the modified 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 remove the tBu protection of the introduced palmitoyl-L-Glu-tBu group, the released peptide precipitate was dissolved in TFA / TIS / H2O (96:2:2, v / v / v) and precipitated in 10 volumes of Et2O after 2 h.

[0529] H- AK(Palm-γE-OH)EFIAWLVRGRG -NH2 (SEQ ID NO 18): The peptide precursor P1 has been synthesized as described in the SPPS section above. It has been immobilized and modified with the palmitic acid (Palm) building block Palm-γGlu(OH)-OtBu and purified according to the above method. The results are shown in Figure 2 And as shown in Table 1.

[0530] H-HAEGTFTSDVSSYLEGQAAK(Palm-γE-OH)EFIAWLVRGR G-NH2 (SEQ ID NO 19): Peptide precursor P2 has been synthesized as described in the SPPS section above. It has been immobilized and modified with the palmitic acid (Palm) building block Palm-γGlu(OH)-OtBu and purified according to the above method. The results are shown in Figure 3 And as shown in Table 1.

[0531] Example 2: Formation of a macrocycle of peptide P3-P5 bound to a purified solid support using m-dibromobenzyl (intermolecular Cyclization

[0532] The peptide modification and purification methods of the present invention were applied to peptides with three cysteine ​​positions and polarities, which are H- CRVPGDAHHADSLC -NH2( SEQ ID NO:3)(P3), H- VRCPGAAHHADSLC -NH2( SEQ ID NO:4 )(P4) and H-VRVPGCAHCADSLY-NH2( SEQ ID NO:5 )(P5).

[0533] According to the general method, peptides are fixed and washed on a 25 μmol scale. To the fixed peptide, H is added DTT (4 equivalents, 0.3M) in O, and the reactor is shaken for 15min. After removing the DTT solution by filtration, the bead material is washed with 3x H O and 3xMeCN. Subsequently, m-dibromobenzyl (mDBX) (4 equivalents, 0.6M) in acetonitrile is added, and the cyclization buffer (200mM NH4HCO3, pH 7.9) of the same volume is added subsequently. The syringe reactor is shaken for 45min, the reaction mixture is filtered out and washed with 3xMeCN and 3x H O bead material. Then the scheme is repeated twice after DTT treatment, and finally MeCN / AcOH / H is used under the conditions given in the general method\modified peptide release PPh in O (9:0.5:0.5, v / v / v) Replace DTT to release the cyclized peptide.

[0534] H- CRVPGDAHHADSLC -NH2 (SEQ ID NO 3) (underlined C residues are connected via the scaffold): Peptide precursor P3 has been synthesized as described in the SPPS section above. It has been immobilized and modified with the m-dibromobenzyl (mDBX) scaffold and purified according to the above method. The results are shown in Figure 4 And as shown in Table 1.

[0535] H-VR CPGAAHHADSLC -NH2 (SEQ ID NO 4) (underlined C residues are connected through the scaffold): Peptide precursor P4 has been synthesized as described in the SPPS section above. It has been immobilized and modified with the mDBX scaffold and purified according to the above method. The results are shown in Figure 5 And as shown in Table 1.

[0536] H-VRVPG CAHC ADSLY-NH2 (SEQ ID NO 5) (underlined C residues are connected through the scaffold): Peptide precursor P4 has been synthesized as described in the SPPS section above. It has been immobilized and modified with the mDBX scaffold and purified according to the above method. The results are shown in Figure 5 And as shown in Table 1.

[0537] Example 3: Bicyclic Macrocycles of Peptides P6-P7 Bound to Purification Solid Support Using 1,3,5-Tris(bromomethyl)benzene Formation (intermolecular cyclization)

[0538] The peptide modification and purification methods of the present invention were applied to two different peptides, which are H- ACSWPARCLHQDLCA -NH2( SEQ ID NO:6)(P6) and H-ACREGFLRCLHRPTVCG-NH2( SEQ ID NO:7 )(P7).

[0539] According to the general method, peptides are fixed and washed on a 5 μmol scale. To the fixed peptide, H is added DTT (4 equivalents, 0.3M) in O, and the reactor is shaken for 15min. After removing the DTT solution by filtration, the bead material is washed with 3x H O and 3x DMF. Subsequently, 1,3,5-tris (bromomethyl) benzene (TBMB) (4 equivalents, 0.45M) in dimethylformamide is added, and then half of the volume of cyclization buffer (400mM NH HCO , 8M GdmCl, pH 8.0) is added. The syringe reactor is shaken for 45min, the reaction mixture is filtered out and washed with 3x DMF and 3x H O bead material. Then the scheme is repeated twice after DTT treatment, and finally MeCN / AcOH / H is used under the conditions given in the general method\modified peptide release PPh in O (9:0.5:0.5, v / v / v) Replace DTT to release the cyclized peptide.

[0540] HA CSWPARCLHQDLC A-NH2 (SEQ ID NO 6) (underlined C residues are connected via a scaffold): Peptide precursor P6 has been synthesized as described in the SPPS section above. It has been immobilized and modified with a 1,3,5-tris(bromomethyl)benzene (TBMT) scaffold and purified according to the above method. The results are shown in Figure 7 And as shown in Table 1.

[0541] HA CREGFLRCLHRPTVC G-NH2 (SEQ ID NO 7) (underlined C residues are connected via the scaffold): The peptide precursor P7 has been synthesized as described in the SPPS section above. It has been immobilized and modified with 1,3,5-tris(bromomethyl)benzene (TBMT) scaffold and purified according to the above method. The results are shown in Figure 8 And as shown in Table 1.

[0542] Example 4: Purification of solid using 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tri(2-bromoethanone) Formation of the bicyclic macrocycle of carrier-bound peptide P8a (intermolecular cyclization)

[0543] The peptide modification and purification method of the present invention was applied to the peptide H-ACYNEFGCEDFYDICA-NH2( SEQ ID NO:8 )(P8a).

[0544] Peptides were fixed and washed on a 5 μmol scale according to the general method. DTT (4 equivalents, 0.3 M) in H2O was added to the fixed peptide, and the reactor was shaken for 15 min. After the DTT solution was removed by filtration, the bead material was washed with 3x H2O and 3x MeCN. Subsequently, 1,1',1"-(1,3,5-triazine-1,3,5-triyl)tris(2-bromoethylketone) (TATB) in MeCN / cyclization buffer (400 mM NH4HCO3, 8 M GdmCl, pH 7.1; 9:1) was added. The syringe reactor was shaken for 120 min, the reaction mixture was filtered out and the bead material was washed with 3x H2O and 3x MeCN. The protocol was then repeated twice after DTT treatment, and finally the PPh3 in MeCN / AcOH / H2O (9:0.5:0.5, v / v / v) was used under the conditions given in the general method\release of modified peptides to replace DTT to release the cyclized peptide.

[0545] HA CYNEFGCEDFYDIC A-NH2 (SEQ ID NO 8) (underlined C residues are connected via the scaffold): Peptide precursor P8 has been synthesized as described in the SPPS section above. It has been immobilized and modified with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylketone) (TBMT) scaffold and purified according to the above method. The results are shown in Fig. 9 And as shown in Table 1.

[0546] Example 5: Peptide P8b bound to a purified solid support using 1,3,5-triacryloyl-1,3,5-triazine Bicyclic macrocycle formation (intermolecular cyclization)

[0547] The peptide modification and purification method of the present invention was applied to the peptide H-ACYNEFGCEDFYDICA-NH2( SEQ ID NO:8 )(P8b).

[0548] Peptide was fixed and washed with 5 μmol scale according to the general method.DTT (4 equivalents, 0.3M) in H2O was added to the fixed peptide, and the reactor was shaken for 15min.After removing the DTT solution by filtration, the bead material was washed with 3x H2O and 3x MeCN. Subsequently, 1,3,5-triacryloyl-1,3,5-triazine (TATA) in MeCN / cyclization buffer (400mM NH4HCO3, 8M GdmCl, pH7.1; 9:1) was added. The syringe reactor was shaken for 120min, the reaction mixture was filtered out and the bead material was washed with 3x H2O and 3x MeCN. The scheme was then repeated twice after DTT treatment, and finally PPh in MeCN / AcOH / H2O (9:0.5:0.5, v / v / v) was used under the conditions given in the general method\modified peptide release3 to replace DTT to release the cyclized peptide.

[0549] HA CYNEFGCEDFYDIC A-NH2 (SEQ ID NO 8) (underlined C residues are connected via the scaffold): Peptide precursor P8 has been synthesized as described in the SPPS section above. It has been immobilized and modified with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylketone) (TBMT) scaffold and purified according to the above method. The results are shown in Fig.10 And as shown in Table 1.

[0550] Example 6: Macrocyclic formation of peptide P3b bound to a purified solid support via disulfide bonds (intramolecular ring change)

[0551] The peptide modification and purification method of the present invention is applied to peptide H- CRVPGDAHHADSLC -NH2( SEQ ID NO:3 )(P3b).

[0552] Peptides were fixed and washed at 10 μmol scale according to the general method. A one-to-one mixture of DMSO and 0.4M (NH4)2CO3, 6MGdnCl, pH 8.7 was then added and the syringe reactor was shaken for 120 min, the reaction mixture was filtered out and the bead material was washed with 3xH2O. The cyclic peptides were released using PPh3 in MeCN / AcOH / H2O (9:0.5:0.5, v / v / v) instead of DTT under the conditions given in the general method\release of modified peptides, resulting in 22% of disulfide cyclic peptides.

[0553] H- CRVPGDAHHADSLC -NH2 (SEQ ID NO 3) (underlined C residues are connected via the scaffold): Peptide precursor P3 has been synthesized as described in the SPPS section above. It has been immobilized and modified by air oxidation and purified according to the above method. The results are shown in Fig.11 And as shown in Table 1.

[0554] Example 7: Using building blocks 1-bromopropyl-2,3-dipalmitate (a), 1-iodopropyl-2,3-dipalmitate Lipidation of ester (b), 1-tosyl-2,3-dipalmitate (c) or palm-OC2H4I (d) bound to a purified solid support Formation of peptides P1–P3

[0555] The peptide modification and purification methods of the present invention were applied to two peptides of different lengths and polarities, which are the model peptide H- KKKKSC -NH2( SEQ ID NO:12 )(P12), H- KEKFSINSEDNNGC -NH2( SEQ ID NO:13 )(P13) and H- CRG -NH2 (SEQ ID NO: 14) (P14).

[0556] According to the general method with 5 μ mol scale fixed linker labeled peptide.After removing the fixed solution, the agarose beads are washed 3x with DMSO.Add 250 μ L of 2w% L-cysteine ​​(5mg) solution in buffer 1 and shake for 15min.The solution is filtered out by suction, and the beads connected to the peptide with a linker are washed as described in the general method.In order to reduce the linker and remove the StBu protecting group of the cysteine ​​residue in the peptide, a reducing solution of 25mg (162 μ mol, 32 equivalents) DTT in 250 μ L 5w% NaHCO3 aqueous solution is prepared.250 μ L MeCN is added to the agarose beads, and then the reducing solution is added.The mixture of the peptide (P1-P2) with StBu protecting group on cysteine ​​is shaken for 60min, and (P3) without StBu is shaken for 15min, and then the solution is filtered.The agarose beads with the peptide connected by the linker are washed 3x with water, washed 2x with MeCN, and washed 3x with DMF. Add 3.2 mg (10 μmol, 2 eq) of Cs2CO3 in a sonicated suspension in 200 μL DMF. Add 20 μmol of palmitoyl building blocks (4 eq, 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 a solution in 300 μL DMF. Shake the mixture for 18-76 h. Add another 200–400 μL DMF as some precipitate forms over time. Remove the supernatant, then wash the beads 5x with DMF, 3x with water, and 3x with MeCN. Add 200 μL TFA / water (40:60) and shake for 60 min to release the peptide. Add 200 μL TFA to the cartridge and collect the solution in a 15 mL centrifuge tube. The agarose beads were eluted 2x with 200 μL TFA / water (95:5) into the same tube. 10 L of cooled Et2O was added to the TFA / water solution containing 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 samples containing lipidated peptide P3, the TFA / water solution was concentrated under air flow. Water / MeCN (1:1) was added and the sample was lyophilized.

[0557] H-KKKKSC(C3H5O2-dipalm)-NH2(P12a) (SEQ ID NO 22): The peptide precursor P12a has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block 1-bromopropyl-2,3-dipalmitate (palmitoylation time = 18h). The results are shown in Tables 1 and Fig.12 shown.

[0558] H-KKKKSC(C3H5O2-dipalm)-NH2(P12b) (SEQ ID NO 22): The peptide precursor P12b has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block 1-iodopropyl-2,3-dipalmitate (palmitoylation time = 18h). The results are shown in Table 1, Figure 1 and Fig.13 shown.

[0559] H-KKKKSC(C2H4O-Palm)-NH2(P12d) (SEQ ID NO 23): The peptide precursor P12d has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block Palm-OC2H4I (palmitoylation time = 18h). The results are shown in Tables 1 and Fig.14 shown.

[0560] H-KEKFSINSEDNNGC(C3H5O2-dipalm)-NH2(P13a) (SEQ ID NO 24): The peptide precursor P13a has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block 1-bromopropyl-2,3-dipalmitate (palmitoylation time = 18h). The results are shown in Tables 1 and Fig.15 shown.

[0561] H-KEKFSINSEDNNGC(C3H5O2-dipalm)-NH2(P13b) (SEQ ID NO 24): The peptide precursor P13b has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block 1-iodopropyl-2,3-dipalmitate (palmitoylation time = 18h). The results are shown in Tables 1 and Fig.16 shown.

[0562] H-KEKFSINSEDNNGC(C2H4O-Palm)-NH2(P13d) (SEQ ID NO25): The peptide precursor P13d has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block Palm-OC2H4I (palmitoylation time = 18h). The results are shown in Tables 1 and Fig.17 shown.

[0563] HC(C3H5O2-dipalm)RG-NH2(P14b) (SEQ ID NO 26): The peptide precursor P14b has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block 1-iodopropyl-2,3-dipalmitate (palmitoylation time = 76h). The results are shown in Tables 1 and Fig.18 shown.

[0564] HC(C3H5O2-dipalm)RG-NH2(P14b) (SEQ ID NO 26): The peptide precursor P14c has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block 1-tosylpropyl-2,3-dipalmitate (palmitoylation time = 76h). The results are shown in Tables 1 and Fig.19 shown.

[0565] HC(C2H4O-Palm)RG-NH2(P14d) (SEQ ID NO 27): The peptide precursor P13c has been synthesized as described in the SPPS section above. It has been fixed according to the above method and modified with the palmitic acid (Palm) building block Palm-OC2H4I (palmitoylation time = 18h). The results are shown in Tables 1 and Fig.12 Modification of peptide P3 with Palm-OC2H4I was also performed using 10 μmol (2 eq.) triethylamine in DMF instead of Cs2CO3 in DMF and produced similar results.

[0566] Example 8: Lipids bound to a purified solid support using palmitoyl-L-Glu(OSu)-tBu-building blocks Formation of peptide P2b

[0567] The peptide modification and purification methods of the present invention are applied to H- HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG -OH( SEQ ID NO:20 )(P2b), which is a longer sequence 7-37(K34R) of glucagon-like peptide-1 (GLP-1), and when Palm-Glu-OH is attached to the Lys side chain, it is called the peptide drug liraglutide.

[0568] Peptide was fixed and washed on a 25 μmol scale according to the general method. After washing according to the general method, 3x DMF was used for washing. 8 equivalents of palmitoyl-L-Glu (OSu)-tBu building blocks and 2 equivalents of DIPEA were subsequently added in 600 μL N-methylpyrrolidone. After adding the solution, the beads were shaken for 1 hour, the supernatant was then filtered, and the solid support was washed with 3xDMSO, 3x DMF, 3x, and the modified peptide was released under the conditions given in the general method\release of modified peptides. In order to remove the tBu protection of the palmitoyl-L-Glu-tBu group introduced, the released peptide precipitate was redissolved in TFA / DTT / H2O (94:4:2, v / v / v), and precipitated in 10 times the volume of Et2O after 1h.

[0569] H-HAEGTFTSDVSSYLEGQAAK(Palm-Glu-OH)EFIAWLVRG RG-OH (SEQ ID NO 21): Peptide precursor P2 has been synthesized as described in the SPPS section above. It has been immobilized and modified with the palmitic acid (Palm) building block Palm-Glu(OSu)-OtBu and purified according to the above method. The results are shown in Fig. 22 And as shown in Table 1.

[0570] Example 9: Solids purified using tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu building blocks Formation of carrier-bound lipidated peptide P15

[0571] The peptide modification and purification methods of the present invention are applied to H- HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG -OH( SEQ ID NO:15 )(P15), which is a longer sequence 7-37(K34R,Aib8A) of glucagon-like peptide-1 (GLP-1), and when CO2H-Ste-Glu(aeea-aeea-OH)-OH is attached to the Lys side chain, it is called the peptide drug semaglutide.

[0572] Peptides were fixed and washed on a 25 μmol scale according to the general method. After washing according to the general method, 3x DMF was used for washing. 8 equivalents of tBuOC (O) -Ste-Glu (aeea-aeea-OSu) -OtBu building blocks and 2 equivalents of DIPEA were subsequently added in 600 μL N-methylpyrrolidone. After adding the solution, the beads were shaken for 1 hour, the supernatant was then filtered, and the solid support was washed with 3xDMSO, 3x DMF, 3x, and the modified peptide was released under the conditions given in the general method\release of modified peptides. In order to remove the tBu protection of the introduced tBuOC (O) -Ste-Glu (aeea-aeea-OSu) -OtBu group, the released peptide precipitate was redissolved in TFA / DTT / H2O (94: 4: 2, v / v / v), and precipitated in 10 volumes of Et2O after 1h.

[0573] H-HAibEGTFTSDVSSYLEGQAAK(CO2H-Ste-Glu(aeea-aeea-OH)-OH)EFIAWLVRGRG-OH (SEQ ID NO 28): The peptide precursor of P15 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu building blocks and purified according to the above method. The results are shown in Fig.23 And as shown in Table 1.

[0574] Example 10: Formation of lipidated peptides P16 and P17a bound to a purified solid support using Palm-S building blocks become

[0575] The peptide modification and purification methods of the present invention are applied to H- SGGKWSKSSVIGWPAVRER -OH( SEQ ID NO: 16 ), which is sequence 1-19 (S1M), and H- GGKWSKSSVIGWPAVRER -OH( SEQ ID NO:17 ), which are sequences 2-19, both of which are sequences of Nef protein with an N-terminal myristoyl group.

[0576] In H- GGKWSKSSVIGWPAVRER During SPPS of Lys-OH (SEQ ID NO 17), N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine building blocks were used. 3 , and Boc-Gly-OH for Gly 1After 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 for 3 x 3 min. Thereafter, the linker molecule X1 was coupled as described in the general methods.

[0577] In HS GGKWSKSSVIGWPAVRER During SPPS of Ser-OH (SEQ ID NO 16), N-α-(9-Fmoc)-O-(tert-butyl-dimethyl-silyl)-L-serine building blocks were used to 1 , and Boc-Gly-OH for Gly 1 After peptide assembly, the tert-butyl-dimethyl-silyl protecting group was removed by treatment with AcOH / THF / H2O 3:1:1 for 2 h. Thereafter, Fmoc was removed by treatment with 2x 5 min piperidine / DMF 1:4, the resin was washed and the linker molecule X1 was coupled as described in the general methods.

[0578] Peptide is fixed and washed with 25 μmol scale according to general method.After washing according to general method, wash with 3x DMF.8 equivalents of S-(4,6-dimethyl-2-pyrimidinyl) thiopalmitate (Palm-S-(4,6-Dimethyl-2-pyrimidinyl) thioate) (Palm-S) building blocks and 2 equivalents of DIPEA added in 600 μL N-methylpyrrolidone subsequently.After adding the solution, beads are shaken for 1 hour, then supernatant is filtered, and solid support is washed with 3x DMSO, 3x DMF, 3x, and modified peptide is released under the conditions given in general method\modified peptide release.

[0579] Palm-SGGK(Palm)WSK(Palm)SSVIGWPAVRER-NH2 (SEQ ID NO 29): The peptide precursor of P16 has been synthesized as described in the SPPS section above. It has been immobilized and modified with Palm-S building blocks and purified according to the above method. The results are shown in Fig.24 And as shown in Table 1.

[0580] Palm-GGKWSK(Palm)SSVIGWPAVRER-NH2 (SEQ ID NO 30): The peptide precursor of P17a has been synthesized as described in the SPPS section above. It has been immobilized and modified with Palm-S building blocks and purified according to the above method. The results are shown in Fig.24 And as shown in Table 1.

[0581] Example 11: Formation of Lipidated Peptide P17b Bound to Purification Solid Support Using Palm-S Building Blocks

[0582] The peptide modification and purification methods of the present invention are applied to H- GGKWSKSSVIGWPAVRER -OH( SEQ ID NO: 17 ), which is sequence 2-19 of the Nef protein with an N-terminal myristoyl group.

[0583] In H- GGKWSKSSVIGWPAVRER During SPPS of Lys-OH (SEQ ID NO 17), N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine building blocks were used. 3 , and Boc-Gly-OH for 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 for 3 x 3 min. Thereafter, the linker molecule X1 was coupled as described in the general methods.

[0584] Peptide was fixed and washed on a 25 μmol scale according to the general method. After washing according to the general method, 3x DMF was used for washing. 8 equivalents of Myr-OSu building blocks and 2 equivalents of DIPEA in 600 μL N-methylpyrrolidone were subsequently added. After adding the solution, the beads were shaken for 1 hour, the supernatant was then filtered, and the solid support was washed with 3x DMSO, 3x DMF, 3x, and the modified peptide was released under the conditions given in the general method\release of modified peptides.

[0585] Myr-GGKWSK(Myr)SSVIGWPAVRER-NH2 (SEQ ID NO 31): The peptide precursor of P17b has been synthesized as described in the SPPS section above. It has been immobilized and modified with the Myr-OSu building block and purified according to the above method. The results are shown in Fig.24 And as shown in Table 1.

[0586] Example 12: Utilization of 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylketone) (TATB) and Purification of macrocycle formation (intramolecular bicyclization) of peptides P6 and P8a bound to solid support (aldehyde-modified agarose)

[0587] The peptide modification and purification methods of the present invention were applied to two peptides of different lengths, cysteine ​​positions and polarities, which are H- ACSWPARCLHQDLCA -NH2( SEQ ID NO:6 )(P6) and H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO:8) (P8).

[0588] The peptide of thick joint mark is fixed 16h and washed according to general method, as different from general method, after fixing and removing fixed supernatant, add O-methylhydroxylamine hydrochloride solution (H2O in 5w%, 250 μ L), and the fixed peptide is shaken 15min.After washing according to general method, joint is reduced, and by adding 250 μ L 5w%NaHCO3 aqueous solution (0.6M; pH 8) 12.5mg DTT and shake 60min to remove StBu protecting group.Subsequently, add 30 μ L 5w%NaHCO3 aqueous solution (0.6M; pH 8) 1.9mg TCEP and continue to shake 15min.After this, filter out supernatant and use 3x H2O, 2x MeCN and 3x 5w%NaHCO3 aqueous solution (0.6M; pH 8) washing solid support. 125 μL of 5 w% NaHCO3 aqueous solution (0.6 M; pH 8) was added to the solid support, followed by 125 μL of TATB (1-2 eq.) in MeCN. The beads were shaken for 60 min and then washed with 3x H2O and 3x MeCN. The modified peptide was released according to the conditions of the general method.

[0589] H- ACSWPARCLHQDLCA -NH2 (SEQ ID NO 6): Peptide precursor P6 has been synthesized as described in the SPPS section above. It has been immobilized at 1.3 μmol scale and modified with TATB according to the above method. The results are shown in Tables LuS1 and Fig.26 3.) 1.1 mg of modified peptide with a purity of 69% was obtained as white powder. ESI-MS: (calculated MH 2+ :941.1m / z, measured value:940.7m / z).

[0590] H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO 8): Peptide precursor P8 has been synthesized as described in the SPPS section above. It has been immobilized at 1.0 μmol scale and modified with TATB according to the above method. The results are shown in Tables LuS1 and Fig. 27 3.) 3.0 mg of modified peptide with a purity of 45% was obtained as white powder. ESI-MS: (calculated MH 2+ :1035.6m / z, measured value:1035.5m / z).

[0591] Example 13: Utilization of 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoethylketone) (TATB) and Purification of macrocycle formation (intramolecular bicyclization) of solid support (aldehyde-modified poly(methyl acrylate))-bound peptides P6 and P8a

[0592] The peptide modification and purification methods of the present invention were applied to two peptides of different lengths, cysteine ​​positions and polarities, which are H- ACSWPARCLHQDLCA -NH2( SEQ ID NO:6)(P6) and H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO:8) (P8).

[0593] The peptide of thick joint labeling is dissolved in pure HFIP, and is fixed on the poly (methyl acrylate) of aldehyde modification as solid carrier for 16h and washing.After fixing and removing the fixed supernatant, add O-methylhydroxylamine hydrochloride solution (H2O in 5w%, 250 μ L), and the fixed peptide is shaken for 15min.After washing according to general method, joint is reduced, and by adding 250 μ L 5w%NaHCO3 aqueous solution (0.6M; pH 8) 12.5mg DTT and shake 60min to remove StBu protecting group. Subsequently, add 30 μ L 5w%NaHCO3 aqueous solution (0.6M; pH 8) 1.9mg TCEP and continue to shake 15min.After this, filter out supernatant and use 3x H2O, 2x MeCN and 3x 5w%NaHCO3 aqueous solution (0.6M; pH 8) washing solid carrier. 125 μ L 5w% NaHCO The aqueous solution (0.6M; pH 8) is added to the solid support, and then the TATB (1-2 equivalent) in 125 μ L MeCN is added. The beads are shaken for 60 min, then washed with 3x H o and 3x MeCN. By using 0.4 mL TFA / H o (95: 5) to process the solid support for 1 h, then add 0.4 mL TFA and elute the peptide with 2x0.4 mL TFA / H o (95: 5), thereby releasing the modified peptide. The modified peptide released is precipitated with 10 mL cold Et o, and after the mixture is cooled in a refrigerator for 15-30 min, it is centrifuged, and the supernatant is decanted.

[0594] H- ACSWPARCLHQDLCA -NH2 (SEQ ID NO 6): Peptide precursor P6 has been synthesized as described in the SPPS section above. It has been immobilized at 1.3 μmol scale and modified with TATB according to the above method. The results are shown in Tables LuS1 and Fig.26 4.) 1.5 mg of modified peptide with a purity of 70% was obtained as white powder. ESI-MS: (calculated MH 2+ :941.1m / z, measured value:941.0m / z).

[0595] H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO 8): Peptide precursor P8 has been synthesized as described in the SPPS section above. It has been immobilized at 1.0 μmol scale and modified with TATB according to the above method. The results are shown in Tables LuS1 and Fig. 27As shown. 4.7 mg of modified peptide with a purity of 44% was obtained as a white powder. ESI-MS: (calculated MH 2+ :1035.6m / z, measured value:1035.3m / z).

[0596] Example 14: Utilization of sodium dodecyl sulfate as an additive for peptide solubilization Peptide of triazinane-1,3,5-triyl)tris(2-bromoethylketone) (TATB) bound to a purification solid support (aldehyde-modified agarose) Macrocyclic formation of P6 and P8 (intramolecular bicyclization)

[0597] The peptide modification and purification methods of the present invention were applied to two peptides of different lengths, cysteine ​​positions and polarities, which are H- ACSWPARCLHQDLCA -NH2( SEQ ID NO:6 )(P6) and H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO:8) (P8).

[0598] The peptide of thick linker mark is fixed 16h and washed according to general method, as different from general method, 1M SDS in DMSO+10vol.% fixing buffer 2 is used to dissolve peptide, and after fixing and removing fixing supernatant, O-methylhydroxylamine hydrochloride solution (H2O in 5w%, 250μL) is added, and the fixed peptide is shaken for 15min.Washing according to general method and after additional washing with 3x 8M urea in H2O / MeOH (9:1), the linker is reduced, and by adding 250μL5w%NaHCO3 aqueous solution (0.6M; pH 8) 12.5mg DTT and shake 60min to remove StBu protecting group.Subsequently, add 1.9mg TCEP dissolved in 30μL 5w%NaHCO3 aqueous solution (0.6M; pH 8) and continue to shake 15min. After this, filter out the supernatant and use 3x H o, 2x MeCN and 3x 5w% NaHCO aqueous solution (0.6M; pH 8) to wash the solid support. 125 μ L 5w% NaHCO aqueous solution (0.6M; pH 8) is added to the solid support, and then TATB (1-2 equivalent) in 125 μ L MeCN is added. The beads are shaken for 60 min, then washed with 3x H o and 3x MeCN. Modified peptides are released according to the conditions of the general method.

[0599] H- ACSWPARCLHQDLCA -NH2 (SEQ ID NO 6): Peptide precursor P6 has been synthesized as described in the SPPS section above. It has been immobilized at 1.3 μmol scale and modified with TATB according to the above method. The results are shown in Tables LuS1 and Fig.26 5.) 5.8 mg of modified peptide with a purity of 61% was obtained as white powder. ESI-MS: (calculated MH 2+:941.1m / z, measured value:940.7m / z).

[0600] H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO 8): Peptide precursor P8 has been synthesized as described in the SPPS section above. It has been immobilized at 1.0 μmol scale and modified with TATB according to the above method. The results are shown in Tables LuS1 and Fig. 27 5.) 6.3 mg of modified peptide with a purity of 43% was obtained as a white powder. ESI-MS: (calculated MH 2+ :1035.6m / z, measured value:1034.9m / z).

[0601] Example 15: Utilization of sodium dodecyl sulfate as an additive for peptide solubilization Purification of triazinane-1,3,5-triyl)tris(2-bromoethanone)(TATB) with a solid support (aldehyde-modified poly(methyl acrylate)) Macrocyclic formation of bound peptides P6 and P8a (intramolecular bicyclization)

[0602] As described in Example 13, the crude linker-tagged peptides were immobilized on aldehyde-modified poly(methyl acrylate) as solid support, while as described in Example 14, DMSO + 10 vol.% 1 M SDS in immobilization buffer 2 was used instead of pure HFIP for peptide solubilization. As a difference, 40 vol.% of the bead volume of aniline buffer solution (0.46 vol.% aniline in 0.79 w% NH4OAc in H2O / AcOH (9:1)) was added to the solid support before adding the dissolved peptide. The immobilized peptides were washed, modified with TATB and released as described in Example 13. As a difference, after fixing and washing the peptides, they were additionally washed with 3x8 M urea in H2O / MeOH (9:1).

[0603] H- ACSWPARCLHQDLCA -NH2 (SEQ ID NO 6): Peptide precursor P6 has been synthesized as described in the SPPS section above. It has been immobilized at 1.3 μmol scale and modified with TATB according to the above method. Similar results as in Example 3 were obtained and are shown in Table LuS1. 3.9 mg of the modified peptide with a purity of 55% was obtained as a white powder. ESI-MS: (calculated MH 2+ :941.1m / z, measured value:940.7m / z).

[0604] H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO 8): Peptide precursor P8 has been synthesized as described in the SPPS section above. It has been immobilized at 1.0 μmol scale and modified with TATB according to the above method. Similar results as in Example 3 were obtained and are shown in Table LuS1. 3.7 mg of the modified peptide with a purity of 48% was obtained as a white powder. ESI-MS: (calculated MH 2+:1035.6m / z, measured value:1035.3m / z).

[0605] Example 16: Using 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tri(2-bromoethyl) Macrocyclic rings of peptides P6 and P8 bound to a purification solid support (aldehyde-modified agarose or poly(methyl acrylate)) with TATB Formation (intramolecular bicyclization)

[0606] The peptide modification and purification methods of the present invention were applied to two peptides of different lengths, cysteine ​​positions and polarities, which are H- ACSWPARCLHQDLCA -NH2( SEQ ID NO:6 )(P6) and H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO:8) (P8).

[0607] As described in Example LuS1 or LuS2, the crude linker-tagged peptide was immobilized on aldehyde-modified agarose or poly(methyl acrylate) as a solid support, washed, modified with TATB and released, respectively. Unlike this method, the modification with TATB was carried out by keeping the reaction vessel in an ice-water bath at a temperature of 1°C.

[0608] H- ACSWPARCLHQDLCA -NH2 (SEQ ID NO 6): Peptide precursor P6 has been synthesized as described in the SPPS section above. It has been immobilized on aldehyde-modified poly(methyl acrylate) and modified with TATB according to the above method at a 1.3 μmol scale. The results are shown in Tables LuS1 and Fig.26 6.) 6.2 mg of modified peptide with a purity of 69% was obtained as a white powder. ESI-MS: (calculated MH 2+ :941.1m / z, measured value:940.5m / z).

[0609] H- ACYNEFGCEDFYDICA -NH2 (SEQ ID NO 8): Peptide precursor P8 has been synthesized as described in the SPPS section above. It has been immobilized on aldehyde-modified poly(methyl acrylate) and modified with TATB according to the above method at a 1.0 μmol scale. The results are shown in Tables LuS1 and Fig. 27 6.) 7.1 mg of modified peptide with a purity of 44% was obtained as a white powder. ESI-MS: (calculated MH 2+ :1035.6m / z, measured value:1035.2m / z).

[0610] Example 17: Formation of the macrocycle of peptide P3 bound to a purified solid support via a disulfide (intramolecular ring change)

[0611] The peptide fixation, modification and purification methods of the present invention are applied to peptide H- CRVPGDAHHADSLC -NH2( SEQ ID NO:3 )(P3), H- VRCPGAAHHADSLC-NH2 ( SEQ ID NO:4 )(P4), H- CYFQNCPRG -NH2( SEQ ID NO:9 )(P9), H- KCNTATCATQRLANFLVHSSNFGPILPPTNVGSNTY -NH2( SEQ ID NO:10 )(P10), H- CCEYCCNPACTGCY -NH2( SEQ ID NO:11 )(P11).

[0612] H- CRVPGDAHHADSLC -NH2 (SEQ ID NO 3) (underlined C residues are connected through the scaffold): The linker-tagged peptide was immobilized on 1.5 times the amount of aldehyde-modified polymethacrylate loaded at 152.6 μmol / 1 gm beads. After transfer, the bead material was washed with 3x milli-Q water and 3x fixation buffer 1 (0.1 M citric acid / Na2CO3 pH 4.5). 113 μl DMSO was added to the beads and soaked for 5 min. The crude linker-tagged peptide was dissolved in 223 μl DMSO and 26 μl buffer 2, and the mixture was then added to the polymethacrylate beads and shaken. It is worth noting that the beads should have free and good fluctuations in the fixation solution. After a reaction time of 72 h, the fixation solution was filtered off with suction. The fixation was equally effective when using various amounts of beads. Using 1.5-fold to 15-fold (relative to linker-tagged peptide) amounts of aldehyde-modified polymethacrylate loaded at 152.6 μmol / 1 gm, the beads successfully quantitatively immobilized linker-tagged peptides (1.5-fold -72 hours, 3-fold -48 hours, 7.5-fold required 300 minutes, and 15-fold required 180 minutes).

[0613] Washing: After immobilization, the beads attached to the peptide with the linker were washed 3 times with 5 mL (for 2.5 μmol scale) of the following solvents and solutions each.

[0614] 1) 0.9 M guanidine hydrochloride in DMSO

[0615] 3) 70% ethanol in 0.1M NaCl Milli-Q water

[0616] Reduction: 5 ml of acetonitrile was added to the immobilized peptide. A fresh solution of DTT in 5 wt% NaHCO3 solution (33.6 eq., 0.26 M) was added and the reactor was shaken for 60 min. After removing the DTT solution by filtration, the bead material was washed with 3x H2O and 3xMeCN.

[0617] Oxidation: 500 μL of H2O / MeCN (1:1) was then added to the beads, followed by multiple additions of 20 μL of iodine (10 mg / mL) until the solution was light yellow, and the cartridge was shaken for a total of 10 min. The beads were washed 3x with ascorbic acid in water (15 mg / mL), 3x with water, and twice with MeCN.

[0618] Release of peptide: 166.6 μl 95% TFA was added to the vial and after 30 min and elution with 2 x 166.6 μl 95% TFA in water, the peptide was precipitated by centrifugation and decantation of the ether to obtain pure peptide in a pre-weighed vial of cold ether.

[0619] Release was also effective by treating the oxidized beads with 166 μl of 80:10:10 (H2O:MeCN:TFA) or 90:05:05 (H2O:MeCN:TFA) for 18 h and then eluting with 2.5x volume H2O / MeCN (7:3) + 0.1% TFA and 1.5x volume H2O / MeCN (3:7) + 0.1% TFA. The resulting solution was lyophilized to give the desired peptide with a purity of 95%. ESI-MS: (calculated MH 2 + :774.13 g / mol, measured value: 773.91 m / z).

[0620] H-VR CPGAAHHADSLC -NH2 (SEQ ID NO 4) (underlined C residues are connected by a scaffold): The linker-tagged peptide was fixed on 3 times the amount of aldehyde-modified polymethacrylate loaded with 152.6 μmol / 1 g beads. After transfer, the bead material was washed with 3x milli-Q water and 3x fixation buffer 1 (0.1M citric acid / Na2CO3 pH 4.5). 226 μl 83% TFA was added to the beads. The crude linker-tagged peptide was dissolved in 498 μl 83% TFA, and then the mixture was added to the polymethacrylate beads and shaken. It is worth noting that the beads should have free and good fluctuations in the fixation solution. After a reaction time of 150 minutes, the fixation solution was filtered off with suction. Fixation was equally effective using solvents such as HFIP (150 minutes). Various amounts of beads corresponding to the peptides (peptides have 1.5, 3 and 5 aldehyde groups) were also used, as described in the P3 peptide, to fix in DMSO. After 16 hours of fixation, 55% of the peptides were fixed at 1.5 times aldehyde, 70% at 3 times aldehyde, and quantitatively fixed at 5 times aldehyde. Adding a catalyst (50 mM aniline) to 3 times aldehyde increased the fixation rate 4-fold.

[0621] Washing: In case of TFA as the immobilization solvent, after immobilization, the beads attached to the peptide with the linker were washed 3 times with 3 mL (for 2.5 μmol scale) of the following solvents and solutions each.

[0622] 1) Milli-Q water

[0623] 2) 0.9 M guanidine hydrochloride in DMSO

[0624] 3) 70% ethanol in 0.1M NaCl Milli-Q water

[0625] In case of HFIP as the immobilization solvent, after immobilization, the beads connected to the peptide with the linker were washed 3 times each with 3 mL (for 2.5 μmol scale) of the following solvents and solutions.

[0626] 1) HFIP

[0627] 2) Milli-Q water

[0628] In all other cases, the peptides were washed as described for the P3 peptide.

[0629] Reduction: The reduction procedure was performed as described for the P3 peptide.

[0630] Oxidation: 1000 μL DMSO was then added to the beads, air was then purged through the bead solution (5 min) and shaken for 24 h, filtered, washed (3× H 2 O, 1× CH 3 CN) and dried.

[0631] Peptide release: The peptide release procedure was performed as described for P3 peptide, yielding the desired peptide with a purity of 79%. ESI-MS: (calculated MH 2+ :717.43g / mol, measured value: 717.71m / z).

[0632] H- QUR PRG-NH2 (SEQ ID NO 9) (underlined C residues are linked via the scaffold): TFA fixation, washing and reduction procedures were performed as described for the P4 peptide.

[0633] Oxidation: 1000 μL of 20% DMSO in 0.1 M sodium hydrogen phosphate solution was then added to the beads, and the bead solution was then purged with air (5 min) and shaken for 4 h, filtered, washed (3x H2O, 1x CH3CN) and dried.

[0634] Peptide release: The peptide release procedure was carried out as described for P3 peptide to obtain the desired peptide with a purity of 85% (calculated MH 2+ :542.72 g / mol, measured value: 543.12 m / z).

[0635] HK CNTATC ATQRLANFLVHSSNFGPILPPTNVGSNTY-NH2 (SEQ ID NO 10) (underlined C residues are connected by the scaffold): TFA fixation, washing and reduction, oxidation, peptide release were performed according to the method described for P9 peptide to obtain the desired peptide with a purity of 10% (calculated MH 4+ 计算值 :959.48m / z, measured value: MH 4+ 实测值 :959.84m / z)

[0636] H-CCEYCCNPACTGC Y-NH2 (SEQ ID NO 11) (underlined C residues are connected by scaffold): TFA fixation, washing and reduction, oxidation, peptide release were performed according to the method described for P9 peptide to obtain the desired peptide with a purity of 10% (calculated MH 2+ 计算值 :763.21m / z, measured value: MH 2+ 实测值 :763.85m / z).

[0637]

[0638]

[0639]

[0640]

Claims

1. A method for modifying and purifying a peptide, comprising the following steps - providing a crude linker-tagged peptide LP, wherein the crude peptide is covalently bound to a linker molecule L, - in the immobilization step, coupling the linker-tagged peptide LP to a solid support to produce an immobilized linker-tagged peptide SLP, - in a modification step, modifying the immobilized linker-tagged peptide SLP with one or more organic molecules to produce an immobilized linker-tagged peptide SL-mP, - in the release step, adding a reducing agent produces a reduced intermediate RI, and subsequently decomposing the reduced intermediate RI to produce a modified peptide mP, Wherein the linker L is a compound of formula 1, XT b -V a -UY-(1), where -X is selected from a moiety of formula 2, 2a, 3, 3a or 4, in, - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B. -R 3 Selected from H or B, -R 4 Selected from H, C1-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, wherein b is 0 or 1, -V is an electron withdrawing moiety, where a is 0 or 1, where the sum of a and b is 1 or 2, -U is a phenyl or five-membered or six-membered heteroaryl moiety, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl substituted, wherein -W is selected from -N3, -NO2, -S(=O)-R 8 、-SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , Where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, where q is 1, 2, 3 or 4, -E is an electron withdrawing group under acidic conditions, wherein n is 0, 1, 2, 3 or 4, U is a phenyl moiety and Y is -(CH2) m In the case of -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, and Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, wherein m is 1, 2 or 3.

2. The method according to claim 1, wherein: X is selected from a moiety of formula 2, 2a, 3 or 3a.

3. The method according to claim 2, wherein: X is selected from a moiety of formula 2 or 2a.

4. The method according to claim 1, wherein: The linker-tagged peptide LP is provided by - The crude peptide P synthesized on the synthetic support SR is combined with the free linker L f The linker-tagged, loaded crude peptide LP-SR is generated by releasing the electron-withdrawing leaving group Z, where L f Yes connector XT b -V a -UYZ, wherein X, T, b, V, a, U and Y are as defined in claim 1, and Z is an electron withdrawing leaving group, - cleaving the linker-tagged peptide LP from the synthetic support SR to produce the linker-tagged peptide LP.

5. The method according to claim 4, wherein: Cleavage of the linker-tagged peptide LP- was achieved under acidic conditions.

6. The method according to claim 4, wherein: Cleavage of the linker-tagged peptide LP- was achieved by TFA, HBr, HCl or acetic acid.

7. The method according to any one of claims 1 to 6, wherein: The solid support is aldehyde-modified.

8. The method according to any one of claims 1 to 6, wherein: The solid support is aldehyde-modified and is selected from the group consisting of agarose, hydroxylated poly(methyl acrylate), poly(glycidyl acrylate), poly(glycidyl methacrylate), polylysine, polyethylene glycol, polyamide, polyacrylamide, polystyrene, and copolymers thereof.

9. The method according to any one of claims 1 to 6, wherein: The solid support is aldehyde-modified and is selected from agarose and hydroxylated poly(methyl acrylate) beads.

10. The method according to any one of claims 1 to 6, wherein: The fixation step is at pH <5.

11. The method according to any one of claims 1 to 6, wherein: The fixation step was performed at pH 3-4.

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

13. The method according to any one of claims 1 to 6, wherein: The reducing agent in the releasing step is selected from dithiothreitol, dithioerythritol or triphenylphosphine.

14. The method according to any one of claims 1 to 6, wherein: The reduced intermediate RI is decomposed by the pH change.

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

16. The method according to any one of claims 1 to 6, wherein: The peptide comprises amino acids comprising at least one nucleophilic amine in the amino acid side chain.

17. The method of claim 16, wherein the amino acid is selected from the group consisting of 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, and d-hydroxylysine.

18. The method of claim 16, wherein the amino acid is selected from lysine and ornithine, or the nucleophilic amine is the N-terminus of the peptide.

19. The method according to claim 16, wherein: The organic molecule is 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), wherein -FA is –C(=O)-C 1-24 -alkyl or -C(=O)-C 2-24 -alkenyl, wherein the alkyl or alkenyl portion may be unsubstituted or substituted with one or more substituents independently selected from -F and -COOH, -Sp is a spacer comprising 2 to 50 atoms in length, an optional terminal -C(=O)- moiety and x linking moieties independently selected from -NH-, -O-, -S-, wherein the spacer is attached to FA via the linking moiety, -x is an integer between 1 and 5, -n is an integer between 1 and 10, -A is selected from: -F, -Cl, -Br, -I, -N3, -O(C=O)CH2(C=O)OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3, -R 14 is a C1-C6-alkyl-, aryl- or benzyl substituent.

20. The method according to claim 19, wherein: The organic molecule is a compound of formula 5a or 5c.

21. The method according to claim 19, wherein: The FA is -C(=O)-C 5-17 -alkyl or -C(=O)-C 5-17 -alkenyl.

22. The method according to claim 19, wherein: The FA is -C(=O)-C 5-17 -alkyl.

23. The method according to claim 19, wherein: The x is an integer between 1 and 3.

24. The method according to claim 19, wherein: Said n is an integer between 1 and 4.

25. The method of claim 19, wherein: The n is 1 or 2.

26. The method of claim 19, wherein: The A is selected from -Cl, -Br, -I, 27. The method of claim 19, wherein: The A is selected from -Cl, -Br, -I.

28. The method of claim 19, wherein: Sp is independently selected from - a straight or branched alkyl group substituted by 1 to 5 linking moieties, --NH-[C r -alkyl-O] s -C t -alkyl-C(═O)-, wherein r is independently selected from 1, 2 and 3, s is an integer between 1 and 10, and t is 1, 2 or 3, and - amino acids, which may be protected or unprotected, wherein the amine in the N-terminus and / or the side chain is replaced by -NH- and the carboxylic acid moiety in the C-terminus and / or the side chain is replaced by -C(=O)-.

29. The method according to claim 28, wherein: Sp is independently selected from straight or branched chain alkyl substituted with 1 to 3 linking moieties.

30. The method of claim 28, wherein: The s is 1, 2, 3 or 4.

31. The method of claim 28, wherein: Sp is independently selected from linear alkyl substituted with 1 to x linking moieties, 2-(2-(2-aminoethoxy)ethoxy)acetic acid 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.

32. The method of claim 28, wherein: Sp is independently selected from -O-ethyl-, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, cysteine, glutamic acid or Glu-tBu.

33. The method of claim 16, wherein: In the modification step, an organic molecule of formula 5b or 5d is coupled to the at least one nucleophilic amine in the presence of an activating reactant.

34. The method of claim 33, wherein: The activation reactant is selected from the following: tetramethyluronium tetrafluoroborate (TBTU), (3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazine-4(3H)-one) (DEPBT), (2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) (HCTU), tetramethyluronium 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 and diisopropylcarbodiimide (DIC).

35. The method of claim 1, wherein: The peptide comprises at least two amino acids comprising a nucleophilic thiol.

36. The method of claim 1, wherein: The peptide comprises at least two amino acids independently selected from cysteine, homocysteine ​​or penicillamine.

37. The method of claim 35, wherein: The organic molecule is an organic scaffold molecule comprising a scaffold having at least two electrophilic centers.

38. The method of claim 37, wherein: The electrophilic center is formed by a halogen substituent and / or a Michael acceptor.

39. The method of claim 37, wherein: The electrophilic center is formed by at least two benzyl halogen substituents, at least two acrylamides or at least two acrylates.

40. The method of claim 37, wherein: The electrophilic center is formed by at least two acrylamide or at least two benzyl halogen substituents.

41. The method of claim 37, wherein: The organic scaffold molecule is selected from the group consisting of: 1,4-bis(bromomethyl)benzene, m-dibromobenzyl (mDBX), 1,3,5-tri(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(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-chloroacetamide), 1-diyl))bis(2-bromoacetamide), 1,3,5-triacryloyl-1,3,5-triazinane (TATA), 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tris(2-bromoacetamide) (TATB), N,N',N"-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB), N,N',N"-benzene-1,3,5-triyltriprop-2-enamide (TAAB), 2,4,6-tris(bromomethyl)-s-triazine (TBMT), N,N',N"-(nitrilotri(ethane-2,1-diyl))triacrylamide, N , N′,N″-(nitrilotri(ethane-2,1-diyl))tris(2-chloroacetamide), N,N′,N″-(nitrilotri(ethane-2,1-diyl))tris(2-bromoacetamide), N,N′,N″-(nitrilotri(ethane-2,1-diyl))triethylenesulfonamide, N,N′,N″-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxyethyleneethane-2,1-diyl))triacrylamide, N,N′,N″-((1,3,5-triazinane-1,3,5-triyl)tris(2-oxyethyleneethane-2,1-diyl))tris(2-chloroacetamide), N ,N′,N″-((1,3,5-triazinane-1,3,5-triyl)tri(2-oxyethyleneethane-2,1-diyl))triethylenesulfonamide, N,N′,N″-(benzene-1,3,5-triyltri(methylene))triacrylamide, N,N′,N″-(benzene-1,3,5-triyltri(methylene))tri(2-chloroacetamide), N,N′,N″-(benzene-1,3,5-triyltri(methylene))triethylenesulfonamide, triacrylate tris(2-hydroxyethyl) isocyanurate, 1,3,5-tris(oxiran-2-ylmethyl)-1,3,5-triazinane-2,4,6-trione.

42. The method according to any one of claims 35 to 41, wherein: The modification step comprises a reduction step followed by a cyclization step, wherein - in the reducing step, treating the immobilized linker-tagged peptide SLP with a reducing agent, and - In the cyclization step, the organic scaffold molecule is coupled to the at least two nucleophilic thiols under basic conditions.

43. The method of claim 42, wherein: The reducing agent is DTT or TCEP.

44. The method of claim 35, wherein: The number of nucleophilic thiols is even, and in the modification step the nucleophilic thiols are oxidized by alkaline aqueous solution at pH>7 to generate a macrocyclic bridged peptide.

45. The method of claim 35 or 44, wherein: The number of nucleophilic thiols is even, and in the modification step the nucleophilic thiols are oxidized under the following conditions to produce a macrocyclic bridged peptide: - in the presence of air, and / or - In the presence of oxidizing additives.

46. ​​The method of claim 35 or 44, wherein: The number of nucleophilic thiols is even, and in the modification step the nucleophilic thiols are oxidized under the following conditions to produce a macrocyclic bridged peptide: - in the presence of oxygen, and / or - in the presence of an oxidizing additive selected from the group consisting of DMSO, iodine, N-chlorosuccinimide, Tl(OAc)3, Tl(CF3COO)3, CH3SiCl3-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.

47. The method according to any one of claims 1 to 6, wherein -X is selected from a moiety of formula 2, 2a, 3, 3a or 4, in - Each R 1 and R 2 are independently selected from H or B, wherein at least R 1 or R 2 It's B. -R 3 Selected from H or B, -R 4 Selected from H, C1-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 group comprising at least one of the following moieties: -C 1-12 -alkyl-, (-C2H4O-) 1-12 、-C(=O)-、-C(=O)-JR 9 -、-JR 9 -C(=O)-、-JR 9 -, phenyl, 5-membered or 6-membered heteroaryl, wherein J is C or N, -b is 0 or 1, -V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -(CH2) p -、-Piperazinyl-(CH2) p -, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O- wherein R 11 Selected from H and C 1-4 -alkyl, R 12 Selected from H and C 1-4 -alkyl, p is 0, 1, or 2, -a is 0 or 1, where the sum of a and b is 1 or 2, -U is a phenyl or five-membered or six-membered heteroaryl moiety, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl substituted, wherein V is an electron withdrawing moiety selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、-NR 12 -(CH2) p -、-Piperazinyl-(CH2) p -, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O- wherein R 11 Selected from H and C 1-4 -alkyl, R 12 Selected from H and C 1-4 -alkyl, p is 0, 1, or 2, W is selected from -N3, -NO2, -S(=O)-R 8 、-SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , in R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, 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, and q is an integer between 1 and 4, wherein the sum of n and q is equal to or less than 4, and wherein Where U is a phenyl moiety and Y is -(CH2) m In the case of -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)-, wherein m is 1, 2 or 3 -Z is an electron withdrawing leaving group.

48. The method of claim 47, wherein: X is selected from a moiety of formula 2, 2a, 3 or 3a.

49. The method of claim 47, wherein: X is selected from a moiety of formula 2 or 2a.

50. The method of claim 47, wherein: T is a linear or branched spacer group comprising 1 to 5 of the following moieties: -C 1-12 -alkyl-, (-C2H4O-) 1-12 、-C(=O)-、-C(=O)-JR 9 -、-JR 9 -C(=O)-、-JR 9 -, phenyl, 5-membered or 6-membered heteroaryl, wherein J is C or N.

51. The method of claim 50, wherein: J is N.

52. The method of claim 47, wherein: T is a spacer selected from the following: -C1-C 12 -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 -pyrrolyl, -R 5 -pyrazolyl, -R 5 -imidazolyl, R 5 -piperazinyl-, -R 5 -pyridyl, -R 5 -pyrimidinyl, -R 5 -pyrazinyl, -R 5 -pyridazinyl, -R 5 -pyrrolyl-R 6 -、-R 5 -pyrazolyl-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 -, pyrrolyl-R 6 -, pyrazolyl-R 6 -, imidazolyl-R 6 -piperazinyl-R 6 -, pyridyl-R 6 -, pyrimidinyl-R 6 -, pyrazinyl-R 6 -, pyridazinyl-R 6 -, pyrrolyl, pyrazolyl, imidazolyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl and pyridazinyl, wherein R 5 , R 5’ and R 6 Independently selected from C1-C 12 -alkyl or (-C2H4O-) 1-12 , and among them R 9 and R 9’ are independently selected from 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 B is an independently selected acid-labile amine protecting group, R 15 is a blocking agent that reacts with the aldehyde moiety, wherein The amine and / or thiol moieties of the blocking agent may be protected by independently selected acid-labile amine protecting groups B, and / or acid-labile thiol protecting groups.

53. The method of claim 52, wherein: The T is selected from the following spacer groups: C 1-6 -alkyl.

54. The method of claim 53, wherein: The T is selected from the following spacer groups: C 1-3 -alkyl.

55. The method of claim 52, wherein: R 5 , R 5’ and R 6 are independently selected from C1-C6 alkyl groups.

56. The method of claim 52, wherein: R 5 , R 5’ and R 6 are independently selected from C1-C3 alkyl groups.

57. The method of claim 52, wherein: The R 9 and R 9’ are independently selected from H and C 1-2 -alkyl.

58. The method of claim 57, wherein: The R 9 It's H.

59. The method of claim 52, wherein: The R 15 Selected from cysteinyl, threonyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine.

60. The method of claim 59, wherein: The R 15 Selected from cysteinyl and N-methylhydroxylamine.

61. The method of claim 60, wherein: The R 15 It is a cysteinyl group.

62. The method of claim 52, wherein: The amine protecting group B is Boc.

63. The method of claim 52, wherein: The thiol protecting group is trityl.

64. The method of claim 47, wherein: b is 1.

65. The method of claim 47, wherein: The V is selected from -NR 11 -C(=O)-, -C(=O)-NR 11 -、-S(=O)-、--NR 12 -(CH2) p -、-Piperazinyl-(CH2) p -, -pyridinyl-, pyrimidinyl.

66. The method of claim 65, wherein: The V is selected from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-(CH2) p -, -pyridinyl-, pyrimidinyl.

67. The method of claim 47, wherein: R 11 Selected from H and C 1-2 -alkyl.

68. The method of claim 47, wherein: R 11 It's H.

69. The method of claim 47, wherein: R 12 Selected from H and C 1-2 -alkyl.

70. The method of claim 47, wherein: R 12 It's methyl.

71. The method of claim 47, wherein: p is 0 or 1.

72. The method of claim 47, wherein: U is a phenyl or six-membered heteroaryl moiety, which is q and E n at least one of which is combined and may optionally be C 1-6 -alkyl substitution.

73. The method of claim 47, wherein: U is a phenyl group, which is q and E n at least one of which is combined and may optionally be C 1-3 -alkyl substitution.

74. The method of claim 47, wherein: W is selected from -N3, -N=NR 8 、-O-CH2-N3、-SSR 8 .

75. The method of claim 47, wherein: R 8 It is pyridyl or -C1-C6-alkyl.

76. The method of claim 47, wherein: n is an integer between 0 and 2.

77. The method of claim 47, wherein: n is 0 or 1.

78. The method of claim 47, wherein: q is 1 or 2.

79. The method of claim 47, wherein: q is 1.

80. The method of claim 47, wherein: m is 1 or 2.

81. The method of claim 47, wherein: m is 1.

82. The method according to any one of claims 1 to 6, wherein: E is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -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)NH 2、 -SO2Me, -SOMe, -SO2Et, -SOEt R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C1-C6-alkyl or -(CH2) p -NMe2, where p is 1, 2, 3 or 4 B is an acid-labile amine protecting group, and r is 0, 1, 2, 3 or 4.

83. The method of claim 82, wherein: E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -N=N-phenyl, -N=NR 8 , -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H.

84. The method of claim 82, wherein: E is selected from pyridinyl, pyrimidinyl, pyridazinyl or -Br.

85. The method of claim 82, wherein: R 8 It is pyridyl or -C1-C6-alkyl.

86. The method of claim 82, wherein: r is 0, 1, or 2.

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