Traceless reductively cleavable linker molecules for peptide purification

Abstract

To provide a method for purifying peptides or peptide nucleic acids produced by solid-phase peptide synthesis in a way that is inexpensive and is less prone to faults, and linker molecules for use in the purification.SOLUTION: The present invention relates to linker molecules of formula (1), X-Tb-Va-U-Y-Z (1) and a method for purifying peptides using the linker molecules. The linker molecule can be coupled to a purification resin via the moiety X and to a peptide via the moiety Y under the release of the leaving group Z. T is an optional spacer moiety and V is an optional electron withdrawing moiety. U is an aryl or 5- or 6-membered heteroaryl moiety bound to at least one electron withdrawing moiety V, W, or E. The linker is stable under acidic conditions and releases the peptide upon addition of a reducing agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for purifying peptides or peptide nucleic acids produced by solid phase peptide synthesis (SPPS), and to linker molecules for use in said purification. [Background technology]

[0002] Solid-phase peptide synthesis is an established method for synthesizing peptides. The standard procedure involves coupling the first N-terminally protected amino acid to a synthesis resin, followed by repeated cycles of N-terminal deprotection, coupling the next N-terminally protected amino acid, and capping any remaining peptide residues. Finally, the synthesized peptide is cleaved from the synthesis resin and purified.

[0003] A widely used method for purifying peptides is preparative high-performance liquid chromatography (HPLC). The drawback of this method is its low scalability with respect to the desired production volume, which means that different amounts cannot be produced on the same system. This makes the acquisition costs of the corresponding combined devices relatively high. A further drawback is that a relatively extensive knowledge of the individual fragments is required for the correct analytical characterization. Furthermore, HPLC purification can consume large amounts of solvent and sometimes column material (solid phase) during the run. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] European Patent No. 0552368(A1) [Patent Document 2] European Patent No. 2501711(B1) [Patent Document 3] WO2017129818(A1) Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, cheaper and less error-prone methods would be advantageous in reducing the costs of peptide production.

[0006] An alternative method uses a linker molecule that can be attached to the peptide, which is then coupled to a functionalized solid phase that is used during purification.

[0007] EP 0 552 368 A1 describes thiol-bearing linker molecules that can be covalently attached to a purification support, although this method is not suitable for thiol-containing peptides such as those containing the amino acids cysteine ​​or penicillamine.

[0008] A similar method is proposed in EP 2501711(B1) to attach linkers to solid phases via a 1,3-dipolar cycloaddition reaction between azides (-N3) and alkynes, which requires the presence of copper. However, peptides containing methionine, cysteine, arginine, or lysine can complex with copper, making their removal difficult. Due to the toxicity of copper, such peptides are not suitable for all applications, such as pharmaceutical use.

[0009] WO2017129818(A1) discloses a linker molecule that can be attached to a peptide that is still attached to the synthesis resin after SPPS. The peptide is then cleaved from the synthesis using commonly used TFA conditions. However, as disclosed in WO2017129818(A1), a drawback of the linker molecule that forms a benzyl carbamate with the peptide is its instability to acidic treatment (TFA>50%, pH<0 in the presence of water). Premature decomposition of the linker molecule causes a significant decrease in the yield of purified peptide.

[0010] Undesirable side reactions also occur with peptides containing Thr, Ser, or Cys at the N-terminus due to nucleophilic attack of the β-hydroxyl or β-thiol groups on the sulfoethylene carbamate moiety of the linker under the basic conditions (pH > 9) used to release the peptide. Additional side reactions of the linker molecules disclosed in WO2017129818(A1) include aspartimide formation and conversion of arginine to citrulline in Arg-Glu sequences, as well as disulfide formation and nucleophilic side reactions with internal Cys residues under basic conditions.

[0011] Furthermore, the sulfone linkers described in WO2017129818(A1) suffer from a reactive vinyl sulfone moiety that remains in the solid state and requires an additional quenching step. [Means for solving the problem]

[0012] To overcome the drawbacks of side reactions under basic conditions and premature decomposition of the linker under acidic conditions, the present invention provides a linker molecule that is stable under TFA conditions and allows peptide release under mildly acidic conditions, particularly at pH ≤ 7. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a schematic diagram of the peptide purification of the present invention by using a linker of the type X-Tb-Va-U(W)-YZ (wherein W is N3), which method is described in claim 10. SR = synthetic resin, PB = purification beads; 1.) 4 equivalents of X-Tb-Va-U(W)-YZ, 6 equivalents of Oxyma, 6 equivalents of DIEA, 2 hours, 3.) Dissolution of crude peptide in DMSO and addition of 10% by volume of Na-citrate pH 4.5, 90 minutes, 4.) Washing, 5.) PPh3 in MeCN / AcOH 9:1, 15 minutes, 6.) Washing with MeCN, 7.) Hydrolysis with HO / TFA, 8.) 1,6- or 1,4-elimination, 9.) Final ether precipitation. [Figure 2-1]2 shows an example of the inventive peptide purification of peptide P1 (H-ARTKQTARKSTGGKA-OH) by using linker molecules X1 and X2 of the invention. A = absorbance (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to P1, 2.) chromatogram of P1 after use of linker X1 and purification by the method of claim 10, 3.) chromatogram of P1 after use of linker X2 and purification by the method of claim 10. [Figure 2-2] 2 shows an example of the inventive peptide purification of peptide P1 (H-ARTKQTARKSTGGKA-OH) by using linker molecules X1 and X2 of the invention. A = absorbance (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to P1, 2.) chromatogram of P1 after use of linker X1 and purification by the method of claim 10, 3.) chromatogram of P1 after use of linker X2 and purification by the method of claim 10. [Figure 3-1] 3 shows an example of the peptide purification of the present invention for peptide P2 (H-AKADEVSLHKWYG-NH2) by using the linker molecule X2 of the present invention. A = absorbance (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to P2, 2.) chromatogram of P2 after use of linker X2 and purification by the method of claim 10. Approximately 3-aminobenzoic acid was used as an internal standard for quantification of the peptide. [Figure 3-2] 3 shows an example of the peptide purification of the present invention for peptide P2 (H-AKADEVSLHKWYG-NH2) by using the linker molecule X2 of the present invention. A = absorbance (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to P2, 2.) chromatogram of P2 after use of linker X2 and purification by the method of claim 10. Approximately 3-aminobenzoic acid was used as an internal standard for quantification of the peptide. [Figure 4-1]4 shows four examples of peptide purification of the invention: peptide P3 (H-YFTGSEVENVSVNVH-NH), peptide P4 (H-PSNPFYEALST-NH), peptide P5 (H-DAEFRHDSGYEVHHQKLVFF-NH) and peptide P6 (H-CKADEVSMHKWYG-NH) by using the linker molecule X1 of the invention. A = absorption (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to the peptide, 2.) chromatogram of the peptide after use of linker X1 and purification by the method of claim 10. [Figure 4-2] 4 shows four examples of peptide purification of the invention: peptide P3 (H-YFTGSEVENVSVNVH-NH), peptide P4 (H-PSNPFYEALST-NH), peptide P5 (H-DAEFRHDSGYEVHHQKLVFF-NH) and peptide P6 (H-CKADEVSMHKWYG-NH) by using the linker molecule X1 of the invention. A = absorption (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to the peptide, 2.) chromatogram of the peptide after use of linker X1 and purification by the method of claim 10. [Figure 4-3] 4 shows four examples of peptide purification of the invention: peptide P3 (H-YFTGSEVENVSVNVH-NH), peptide P4 (H-PSNPFYEALST-NH), peptide P5 (H-DAEFRHDSGYEVHHQKLVFF-NH) and peptide P6 (H-CKADEVSMHKWYG-NH) by using the linker molecule X1 of the invention. A = absorption (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to the peptide, 2.) chromatogram of the peptide after use of linker X1 and purification by the method of claim 10. [Figure 4-4]4 shows four examples of peptide purification of the invention: peptide P3 (H-YFTGSEVENVSVNVH-NH), peptide P4 (H-PSNPFYEALST-NH), peptide P5 (H-DAEFRHDSGYEVHHQKLVFF-NH) and peptide P6 (H-CKADEVSMHKWYG-NH) by using the linker molecule X1 of the invention. A = absorption (210 nm), B = time / min; 1.) chromatogram of crude peptide sample before linker coupling to the peptide, 2.) chromatogram of the peptide after use of linker X1 and purification by the method of claim 10. [Figure 5] Overview of Figures 5-8: In Figures 5-8, the following abbreviations are used: P = peptide, E = arrow indicates electron-withdrawing or electron-donating direction. a) TFA cleavage of the synthetic resin (SR) to obtain the linker-modified peptide, b) incubation with an aldehyde-functionalized solid support to immobilize the linker-modified peptide on purification beads (PB), c) washing of the beads to remove synthetic impurities, d) reduction by adding a reducing agent, e) washing of the reducing agent, f) adjustment of pH to bring about the electronic structure for release, and g) spontaneous decomposition of the linker molecule by either 1,6-, 1,4-elimination, or nucleophilic attack. Figure 5 shows an amine switch (Type 1) with an azide-reduced safety lock. The basic nitrogen atom conjugated to the cleavable aromatic core withdraws electrons when protonated (E: arrow). The positive charge of Structure 1 enhances solubility and provides stability during acidic cleavage of the linker-peptide structure on the synthetic resin (SR) with TFA. Peptide release occurs in two steps. First, the azide (moiety W) is reduced to -NH2, thereby removing the safety lock on release. The stability of the linker is still maintained after reduction of structure 2 due to the electron-withdrawing nature of the protonated amine group when the pH is lower than the pKa of the most basic nitrogen. This allows for removal of the reducing agent and washing under acidic conditions. In a second step, the purified peptide is released by raising the pH to a pH >pKa relative to the pKa of the most basic nitrogen. The increase in pH induces a 1,6-elimination reaction. The linker decomposes with the release of CO2. The peptide is obtained with a free N-terminus. [Figure 6] Overview of Figures 5-8: In Figures 5-8, the following abbreviations are used: P = peptide, E = arrow indicates the direction of electron withdrawal or electron donation. a) TFA cleavage of the synthesis resin (SR) to obtain the linker-modified peptide, b) incubation with an aldehyde-functionalized solid support to immobilize the linker-modified peptide on purification beads (PB), c) washing of the beads to remove synthetic impurities, d) reduction by adding a reducing agent, e) washing of the reducing agent, f) adjustment of the pH to bring about the release electronic structure, and g) spontaneous decomposition of the linker molecule by either 1,6-, 1,4-elimination, or nucleophilic attack. Figure 6 shows an amine switch (Type 2) with a reductive safety lock bearing other reducible moieties, such as nitro, disulfide, or azo groups, rather than azide. The basic nitrogen atom conjugated to the cleavable aromatic core withdraws electrons when protonated (E: arrow). The positive charge of Structure 1 enhances solubility and provides stability in TFA during acidic cleavage of the linker-peptide structure from the synthetic resin (SR). Release of the peptide occurs in two steps. First, the nitro (moiety W) is reduced to -NH2, removing the release safety lock. Linker stability is maintained after reduction of Structure 2 due to the electron-withdrawing properties of the protonated amine group when the pH is lower than the pKa of the most basic nitrogen. This allows for removal of the reducing agent and washing under acidic conditions. In the second step, the purified peptide is released by raising the pH to a pH >pKa relative to the pKa of the most basic nitrogen. The increase in pH induces a 1,6-elimination reaction. The linker decomposes with the release of CO2. The peptide is then obtained with a free N-terminus. [Figure 7]Overview of Figures 5-8: In Figures 5-8, the following abbreviations are used: P = peptide, E = arrow indicates electron-withdrawing or electron-donating direction, a) TFA cleavage of the synthesis resin (SR) to obtain the linker-modified peptide, b) incubation with an aldehyde-functionalized solid support to immobilize the linker-modified peptide on purification beads (PB), c) washing of the beads to remove synthetic impurities, d) reduction by adding a reducing agent, e) washing of the reducing agent, f) adjustment of pH to bring about the electronic structure for release, and g) spontaneous decomposition of the linker molecule by either 1,6-, 1,4-elimination, or nucleophilic attack. Figure 7 shows an amine switch (Type 3) with nucleophilic release. Linkers capable of releasing peptides via nucleophilic release contain a carboxylate (Y = C(=O), Z = OH) instead of a carbonate moiety (Y = -OC(=O), Z = OR). Without carbonate, the linker exhibits high stability in solution and during storage. Coupling to peptides can be performed as a standard amino acid coupling, resulting in an amide bond instead of a carbamate bond. Due to the lack of a carbamate bond, the linker is stable under acidic conditions, such as TFA. Here, release of the peptide is achieved in two steps. First, the reducing moiety (-N3) is reduced to an amine, thereby removing the safety lock on release. If the pH is higher than the pKa of the most basic nitrogen, the linker remains stable after reduction. This allows for removal of the reducing agent and washing under acidic conditions. In the second step, the purified peptide is released by raising the pH to a pH >pKa relative to the pKa of the most basic nitrogen. The increase in pH induces nucleophilic release, yielding a peptide with a free N-terminus. If the pH is higher than the pKa of the carbamate, the linker remains stable after reduction. [Figure 8]General discussion of FIGS. 5 - 8: In FIGS. 5 - 8, the following abbreviations are used: P = peptide, E = the arrow indicates the direction of electron withdrawal or electron donation, a) TFA cleavage of the synthetic resin (SR) to obtain the linker - modified peptide, b) incubation with an aldehyde - functionalized solid support to immobilize the linker - modified peptide on the purification beads (PB), c) washing of the beads to remove synthetic impurities, d) reduction by adding a reducing agent, e) washing of the reducing agent, f) adjustment of pH to bring about the released electronic structure, g) natural decomposition of the linker molecule by either 1,6 - or 1,4 - elimination or nucleophilic attack. FIG. 8 shows a carbamate switch (type 4). The linker that can release the peptide via the carbamate switch contains the moiety Y=-O-(=O)-. Further, the linker contains an electron - withdrawing moiety E, such as -Br, and a reducing moiety W, such as -N3. The stability of TFA is mediated by the electron - withdrawing moiety -Br during TFA cleavage, by the immobilization of the linker - peptide structure to a solid support, such as a purification resin, and by the subsequent washing step under acidic conditions. Reduction of the reducing moiety -N3 to -NH2 removes the safety lock for release. The linker molecule is stable when the pH is higher than the pKa of this carbamate. This allows for the removal and washing of the reducing agent. Finally, the purified peptide is released by 1,6 - or 1,4 - elimination and the release of CO2, and by decreasing the pH to pH < pKa relative to the pKa of the carbamate. This peptide is obtained with a free N - terminus. [Figure 9]Figure 9 shows examples of the purification of peptide P2 (H-AKADEVSLHKWYG-NH2) using three linker molecules of the invention: 2.) X9, a type 1 amine switch with an azide reductive safety lock; 3.) X13, a type 2 amine switch with another reductive safety lock; and 4.) X22, a type 3 amine switch with a nucleophilic release. A = absorbance (210 nm), B = time / min; the identity of the isolated peptides was always confirmed by UPLC-ESI / MS analysis. Identified P2 products are marked with an *. 1.) Chromatogram of the crude peptide sample before linker coupling to P2 (*). 2.) Chromatogram of P2 (*) after use of type 1 linker X9 and purification by the corresponding method, as described in the Examples section below. 3.) Chromatogram of P2(*) after use of linker X13 of type 2 and purification by the corresponding method, as described in the Examples section below. 4.) Chromatogram of P2(*) after use of linker X22 of type 3 and purification by the corresponding method, as described in the Examples section below. DETAILED DESCRIPTION OF THE INVENTION

[0014] explanation According to a first aspect of the present invention, a compound of formula 1, XT b -V a -UYZ(1) During the ceremony, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular of formula 2, 2a, 3 or 3a, more in particular of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, and at least R 1 or R 2 is B, -R 3 is selected from H or B, -R4 H, C1~C 12 alkyl or aryl, and the aldehyde or keto group may be protected with an acid-labile protecting group; B is an acid-labile amine protecting group, - T is part, - C 1~12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9 -, phenyl, 5- or 6-membered heteroaryl, especially -C 1-12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9 - is a linear or branched spacer comprising at least one, in particular 1 to 5, more in particular 1 to 3, of the formula J is CH or N, in particular N; In particular, T: -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 -, -R5 -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 -pyrazoyl, -R 5 -Imidazolyl, R 5 -piperazinyl-, -R 5 -pyridinyl, -R 5 -pyrimidinyl, -R 5 -pyrazinyl, -R 5 -pyridazinyl, -R 5 -Pyrrolyl-R 6 -, -R 5 -Pyrazoyl-R 6 -, -R 5 -Imidazol-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 -, Pyrazoyl-R 6 -, Imidazoyl-R 6 -Piparazinyl-R 6 -, pyridinyl-R 6 -, pyrimidinyl-R 6 -, pyrazinyl-R 6-, pyridazinyl-R 6 -, pyrroyl, pyrazoyl, imidazoyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl, is a spacer selected from the group consisting of: R 5 , R 5’ and R 6 are independent of each other, C1 to C 12 Alkyl or (-C2H4O-) 1~12 in particular C1-C6 alkyl, in particular C1-C3 alkyl, and R 9 and R 9’ are, independently of each other, H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 From, especially H and C 1~2 alkyl, more particularly R 9 is H, where: B is an independently selected acid-labile amine protecting group; R 15 is a blocking agent that can react with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular from cysteinyl and N-methylhydroxylamine, more in particular from cysteinyl, wherein the amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid-labile amine protecting group B, particularly Boc, and / or an acid-labile thiol protecting group, particularly trityl; b is 0 or 1, in particular 1; -V, -NR11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p -,-Piperazinyl-(CH2) p -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] , -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 -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-(CH2) p an electron-withdrawing moiety selected from -, -pyridinyl-, pyrimidinyl, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H, R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl, p is 0, 1 or 2, in particular 0 or 1; a is 0 or 1, and the sum of a and b is 1 or 2; U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, more in particular phenyl, which can be used in combination with the moieties V, W q and E n and C 1~6 By alkyl, especially C 1~3 and optionally substituted by alkyl, wherein V is defined as above, W is -N3, -NO2, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] From this, especially -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 is selected from: R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4; E is an electron withdrawing group under acidic conditions, n is an integer between 0 and 4, particularly between 0 and 2, more particularly between 0 and 1, and q is an integer between 0 and 4, particularly between 0 and 2, more particularly between 0 and 1, the sum of n and q being 4 or less, in the formula: U is a phenyl moiety and Y is -(CH2) m When -OC(=O)-, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and During the ceremony, W is in the ortho or para position relative to Y; - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; Z is an electron-withdrawing leaving group.

[0015] In certain embodiments, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular of formula 2, 2a, 3 or 3a, more in particular of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, and at least R 1 or R 2 is B, -R 3 is selected from H or B, -R 4 H, C1~C 12 alkyl or aryl, and the aldehyde or keto group may be protected with an acid-labile protecting group; B is an acid-labile amine protecting group, - T is part, - C 1~12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9 - a linear or branched spacer comprising at least one of the following: J is C or N, in particular N; In particular, T: -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -C(=O)-R 6 -, -R 5 -NR9 -, -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-, is a spacer selected from the group consisting of: R 5 , R 5’ and R 6 are independent of each other, C1 to C 12 Alkyl or (-C2H4O-) 1~12 selected from, in particular from C1-C6 alkyl, in particular from C1-C3 alkyl; and R 9 and R 9’ are, independently of each other, H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 From, especially H and C 1~2 alkyl, more particularly R 9 is H, where: B is an independently selected acid-labile amine protecting group; R 15 is a blocking agent that can react with aldehyde moieties, particularly R 15is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular from cysteinyl and N-methylhydroxylamine, more in particular from cysteinyl, wherein the amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid-labile amine protecting group B, particularly Boc, and / or an acid-labile thiol protecting group, particularly trityl; b is 0 or 1, in particular 1; -V, -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] , -C(=O)-, -C(=O)-O-, in particular -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H, R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl, a is 0 or 1, and the sum of a and b is 1 or 2; U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, more in particular phenyl, which can be used in combination with the moieties V, W q and E n and C 1~6 By alkyl, especially C 1~3 and optionally substituted by alkyl, wherein V is defined as above, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] From this, especially -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 is selected from: R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4; E is an electron withdrawing group under acidic conditions, n is an integer between 0 and 4, particularly between 0 and 2, more particularly between 0 and 1, and q is an integer between 0 and 4, particularly between 0 and 2, more particularly between 0 and 1, the sum of n and q being 4 or less, in the formula: U is a phenyl moiety and Y is -(CH2) m When the group is -OC(=O)-, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; Z is an electron-withdrawing leaving group.

[0016] In certain embodiments, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular of formula 2, 2a, 3 or 3a, more in particular of formula 2 or 2a, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, and at least R 1 or R 2 is B, -R 3 is selected from H or B, -R 4 H, C1~C 12 alkyl or aryl, and the aldehyde or keto group may be protected by an acid-labile protecting group; B is an acid-labile amine protecting group, -T: -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -C(=O)-R 6 -, -R 5 -C(=O)-OR 6 -, -C(=O)-OR 6 -, -R5 -phenyl-R 6 -, -R 5 -phenyl-, -phenyl-R 6 -, -phenyl-, is a spacer selected from the group consisting of: R 5 , R 5’ and R 6 are independent of each other, C1 to C 12 alkyl, in particular C1-C6 alkyl, in particular C1-C3 alkyl; R 9 and R 9’ are, independently of each other, H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 9 is H, b is 0 or 1, in particular 1; -V, -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] , -C(=O)-, -C(=O)-O-, in particular -NR 11 -C(=O)-, -C(=O)-NR 11 -, S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H, R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R12 is methyl, a is 0 or 1, and the sum of a and b is 1 or 2; U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, more in particular phenyl, which can be used in combination with the moieties V, W q and E n and C 1~6 By alkyl, especially C 1~3 and optionally substituted by alkyl, wherein V is defined as above, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] In particular, -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 is selected from: R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4; E is an electron withdrawing group under acidic conditions, n is an integer between 0 and 4, particularly between 0 and 2, more particularly an integer of 0 or 1, and q is an integer between 0 and 4, particularly between 0 and 2, more particularly an integer of 0 and 1, the sum of n and q being 4 or less, and in the formula: U is a phenyl moiety and Y is -(CH2) m When the formula is —OC(═O)—, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and In particular, W is in the ortho or para position relative to Y; - Y is -(CH2) m-C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; Z is an electron-withdrawing leaving group.

[0017] In certain embodiments, X is selected from the moieties of formula 2, 2a, 3, 3a or 4, in particular of formula 2, 2a, 3 or 3a, more in particular of formula 2 or 2a, most in particular of formula 2, [ka] During the ceremony, - Each R 1 and R 2 are independently selected from H or B, and at least R 1 or R 2 is B, -R 3 is selected from H or B, -R 4 H, C1~C 12 alkyl or aryl, and the aldehyde or keto group may be protected with an acid-labile protecting group; B is an acid-labile amine protecting group, -T: -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-NR 9 -R 6 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -R 5 -NR 9 C(=O)-R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -C(=O)-OR 6 -, -C(=O)-OR 6 -, -R 5-phenyl-R 6 -, -R 5 -phenyl-, -phenyl-R 6 -, -phenyl-, is a spacer selected from the group consisting of: R 5 and R 6 are independent of each other, C1 to C 12 alkyl, in particular C1-C6 alkyl, in particular C1-C3 alkyl; R9 is selected from H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 9 is H, b is 0 or 1, in particular 1; -V, -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazyl, -C(=O)-, -C(=O)-O-, especially -NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H, R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl, a is 0 or 1, and the sum of a and b is 1 or 2; U is a phenyl or a 5- or 6-membered heteroaryl moiety, in particular a phenyl or a 6-membered heteroaryl moiety, which is bonded to at least one of the moieties V, W and E, in which V is defined as above, W is -N3, -S(=O)-R 8 , -SSR8 , -OCH2N3, -OC(=O)OCH2-N3, -N=N-phenyl, -N=N-pyridine, [ka] is selected from: R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1 to C6 alkyl, or -(CH2) p -NMe2, and p is 1, 2, 3, or 4; E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, in particular from pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H; R 13 is selected from -F, -Cl, -Br, -I, -PF6, and wherein: U is a phenyl moiety and Y is -(CH2) m When the formula is —OC(═O)—, the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and In particular, W is in the ortho or para position relative to Y; - Y is -(CH2) m -C(=O)- or -(CH2) m -OC(=O)-, where m is 1, 2 or 3, in particular 1 or 2, more in particular 1; Z is an electron-withdrawing leaving group.

[0018] The linker molecule of Formula 1 is suitable for purification of peptides after solid phase peptide synthesis (SPPS).

[0019] A common approach to purifying peptides after SPPS using linker molecules is to couple the linker molecule to the N-terminus of the peptide in a final coupling step, where the N-terminus of the peptide nucleophilically attacks the linker molecule, forming a covalent carbamate or amide bond with the linker moiety Y while releasing the electron-withdrawing leaving group Z. The peptide linker construct is then cleaved from the synthesis resin by adding TFA.

[0020] The linkers according to the invention are stable under acidic conditions when the peptide linker construct is cleaved, for example, by using TFA.

[0021] The linker moiety X can be coupled to a functionalized solid phase, such as a resin used during purification. Moiety X can react with an appropriate moiety on the functionalized solid phase, such as an aldehyde, ketone, aminooxy, or hydrazine, to form a hydrazone or oxime bond.

[0022] The moiety T represents a spacer that is unreactive under commonly applied purification conditions.

[0023] The moiety T is attached to the moiety U directly or via the moiety V.

[0024] The moiety U contributes to the stability of the linker molecule under acidic conditions, particularly in the presence of TFA > 50%, water, and pH < 0. This is achieved by either using a heterocyclic or phenyl moiety attached to at least one of the electron-withdrawing moieties E, W, V.

[0025] When electron-withdrawing moieties (E, W, V) are attached to the phenyl moiety, the benzylic positions of the linker molecule attached to the peptide become less electron dense and therefore less susceptible to acid-catalyzed degradation. For sufficient stability of the linker molecules of the present invention under acidic conditions, a certain threshold of electron-withdrawing must be met. This threshold is determined by the Hammett constants (σ) of V, W, and E under acidic conditions greater than 0.45. m and σp ) 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.

[0026] Hammett constants are meta-positions (σ m ) and para-position (σ p pKa is an empirically determined constant for substituents on the phenyl core of benzoic acid derivatives of the formula (I), resulting in different acidities (pKa). In the context of the present invention, this position is determined relative to the attachment of the 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 ​​of the substituents V, W, and E.

[0027] It should be noted that the Hammett constants are calculated for the substituents V, W, and E under acidic conditions. For example, an amine moiety at neutral pH has a Hammett constant of σ m =-0.16 and σ p = -0.66 and are therefore characterized by electron-pushing substituents. Under acidic conditions, the amine moiety becomes protonated. For protonated amines, the Hammett constant is σ m = +0.86 and σ 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 its protonated form through a conjugated π system, whether directly as a substituent on U or in a π-conjugate with U.

[0028] The threshold for the sum of Hammett constants greater than 0.45 is when U is a phenyl moiety and Y is -(CH2) m This applies when -OC(=O)- and m=1, because -OC(=O)- has a good elimination at the benzylic position, facilitating acid-catalyzed decomposition. Therefore, the electron density of the aromatic ring must be low enough to prevent stabilization of the cation at the benzylic position of Y.

[0029] [Table 1]

[0030] The moiety Y is -(CH2) m In the case of -C(=O)-, this threshold is not necessary since -C(=O)- is not a good leaving group at the benzylic position.

[0031] For the stability of a linker molecule containing the heterocyclic moiety U under acidic conditions, no such threshold for selecting specific moieties V, W, and E is required. Since the heterocyclic moiety itself is electron-deficient compared to the phenyl moiety, any combination of V, W, and E appears to be sufficient for the stability of the linker molecule under acidic conditions. In particular, when U is a nitrogen-containing heterocycle, the nitrogen is protonated during the acidic release of the peptide, making the electron density of the aromatic system of U particularly low. Therefore, it cannot stabilize the benzyl cation.

[0032] Apart from mediating the stability of the linker molecule under acidic conditions, moieties V, W and E are important with respect to the release mechanism of the peptide. Furthermore, they can contribute to the solubility of the linker molecule under acidic conditions.

[0033] The moiety W is a reducible substituent that causes the linker to collapse, thus releasing the peptide. Linkers containing the reduced moiety W are also called reduced intermediates. In contrast to stable linker molecules, reduced intermediates are unstable. The instability of reduced intermediates is pH dependent. When the moiety W is protonatable (e.g., pyridyl), it also contributes to the solubility of the linker molecule under acidic conditions.

[0034] The moiety E is an electron-withdrawing substituent, which exhibits an electron-withdrawing effect under acidic conditions. For example, a moiety with a positive Hammett constant under acidic conditions is electron-withdrawing, particularly at pH 3-6, more particularly at pH 4.5. If the moiety E is protonatable, it also contributes to the solubility of the linker molecule under acidic conditions.

[0035] In addition to or alternatively to moiety E, moiety V may exhibit an electron-withdrawing effect under acidic conditions and may contribute to the stability and solubility of the linker under acidic conditions.

[0036] Linker molecules according to the present invention can release peptides via an amine switch mechanism (see Figures 5, 6 and 7) or a carbamate switch mechanism (see Figure 8).

[0037] The moiety Y is either a -C(=O)- or an -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 under reducing conditions by either elimination as in 1.4 or 1.6 or nucleophilic attack, and thus from the purification medium. This reductive stimulus converts W to its reduced form, where it can function as an electron donating group and nucleophile, thereby allowing release of the peptide.

[0038] In certain embodiments, X is selected from the moiety of formula 2 or 3.

[0039] In certain embodiments, X is selected from the moiety of Formula 2.

[0040] The reaction time required for coupling of moiety X to a functionalized solid phase via hydrazone or oxime bond formation is longer when using linkers with a moiety of Formula 4 and shorter when using linkers with a moiety of Formula 2 or 3. The formation of the hydrazone bond between an aldehyde or ketone moiety of the solid support and moiety X of Formula 3 is reversible. Due to this reversibility, the inventors observed a loss of up to approximately 10% of the peptide material after each washing step during purification. In contrast, when using linkers with a moiety of Formula 2, little loss of peptide material was observed.

[0041] In certain embodiments, U is C 1~6 It is substituted with alkyl.

[0042] In certain embodiments, U is C 1~3 It is substituted with alkyl.

[0043] In certain embodiments, U is substituted with methyl.

[0044] When U is further substituted with one or more alkyl moieties, the Hammett values ​​of the alkyl moieties are considered. When U is further substituted with a phenyl moiety or a heteroatom, the sum of the Hammett values ​​of V, W, E, and the optional alkyl substituents is greater than 0.45. In certain embodiments, E is selected from the group consisting of piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N(C2H4)2NH2, -N(C2H4)2N-B, -N=N-phenyl, and -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; R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p-NMe2, in particular pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4, and B is an acid-labile amine protecting group as defined herein, in particular -C(=O)OtBu(Boc) or C(=O)CPh3; and r is 0, 1, 2, 3 or 4, in particular 0, 1 or 2.

[0045] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -(CH) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 alkyl)2-, -N=N-phenyl, -N=NR 8 , —F, —Cl, —Br, —I, —CN, —NO2, —N3, —CF3, —SO3H, —CO2H, and r is 0, 1, 2, 3 or 4, in particular 0, 1 or 2.

[0046] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyridazinyl-NH—C 1~6 Alkyl, -N(C 1~6 -N=N-pyridinyl or -Br.

[0047] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyridazinyl-NH—C 1~6 Alkyl, -N(C 1~6 alkyl)2-, or -Br.

[0048] In certain embodiments, E is pyridyl, pyrimidinyl, pyridazinyl-NH—C 1~6 Alkyl, -N(C 1~6 alkyl)2-, or -Br.

[0049] In certain embodiments, E is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —N═N-phenyl, —N═NR 8, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, and R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, and p is 1, 2, 3 or 4.

[0050] In certain embodiments, E is pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, —N═N-phenyl, —N═NR 8 , -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, and R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, and p is 1, 2, 3 or 4.

[0051] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -(CH) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 -alkyl)2-, in particular -(CH2) r -NH-C 1~3 -Alkyl, -(CH2) r -N(C 1~3 alkyl)2-, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, and r is 0, 1 or 2, in particular 0.

[0052] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt.

[0053] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -(CH) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 alkyl)2-, in particular -(CH2) r -NH-C 1~3 Alkyl, -(CH2) r -N(C 1~3 alkyl)2-, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H.

[0054] In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, and r is 0, 1 or 2, particularly 0.

[0055] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H.

[0056] In certain embodiments, E is pyridyl, pyrimidinyl, pyridazinyl, -NH-C 1~6 Alkyl, -N(C 1~6 alkyl)2-, especially -NH-C 1-3 Alkyl, -N(C 1-3 alkyl)2-, or -Br.

[0057] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyridazinyl, or -Br.

[0058] In certain embodiments, n is 0, 1, or 2.

[0059] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyridazinyl, and n is 1; or E is —Br, and n is 1 or 2.

[0060] Peptide release via an amine switch (Figures 5, 6, 7) requires a basic nitrogen that can be protonated. Such a basic nitrogen can be provided by moiety E.

[0061] In certain embodiments, E is —(CH) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 alkyl)2-, in particular -(CH2) r -NH-C 1~3 Alkyl, -(CH2) r -N(C 1~3 alkyl), piperidinyl, piperazinyl, pyridyl, pyrimidinyl and pyridazinyl, and r is 0, 1 or 2, especially 0, where especially n is 1.

[0062] In certain embodiments, E is —(CH) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 alkyl)2-, in particular -(CH2) r -NH-C 1~3 Alkyl, -(CH2) r -N(C 1~3 alkyl), pyridyl, pyrimidinyl and pyridazinyl, r is 0, 1 or 2, in particular 0, and n is 1.

[0063] In particular, linker molecules that release peptides via a carbamate switch (Figure 8) contain an electron-withdrawing moiety that is not reduced by the reducing agent used to reduce moiety W. Substituents E suitable for a carbamate switch can be aprotic, such as halogen or nitro substituents. Substituents characterized by the pKa of the corresponding acid being less than 0 are also suitable substituents E for a carbamate switch.

[0064] In certain embodiments, E is -F, -Cl, -Br, -I, -NO2, -CF3, -CN, -NC, BF2, -PF4, -OCF3, -SOCF3, -SOR8, -SO2R8.

[0065] In certain embodiments, E is -Br.

[0066] In certain embodiments, E is —Br and n is 1 or 2.

[0067] When it is necessary to increase the solubility of the linker under acidic conditions, for example for the purification of hydrophobic peptides, E can be selected from protonatable moieties. In certain embodiments, E is selected from piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N(C2H4)2NH2, -N(C2H4)2N-B, -N=NR 8 , -(CH2)r-NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 alkyl)2-, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, and R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -(CH2) p -NMe2, where p is 1, 2, 3 or 4, in particular 1 or 2.

[0068] In certain embodiments, B is - Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylidene), trityl (-C(Ph)3), -C(=O)CPh3, Mmt (-C(Ph)2C6H4OMe), DMT (-C(Ph)(C6H4OMe)2), Cbz (-C(=O)OCH2Ph), benzylideneamine (=CPh), phthalimide (=(CO)2C6H4), p-toluenesulfonamide (-SO2C6H4Me), benzyl dimethylamine (-CHPh), acetamide (-COMe), trifluoroacetamide (-COCF), 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), in particular B is Boc or Eei, more in particular B is Boc; or - an acetal or ketal protecting group selected from: [ka] wherein r is 0 to 12, particularly 0 to 6, more particularly 0, 1 or 2, and R 10 is -C1~C 12 Alkyl-, especially C 1~6 Alkyl, more particularly C 1~3 It is alkyl.

[0069] In certain embodiments, B is - Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylidene), trityl (-C(Ph)3), Mmt (-C(Ph)2C6H4OMe), DMT (-C(Ph)(C6H4OMe)2), Cbz (-C(=O)OCH2Ph), benzylideneamine (=CPh), phthalimide (=(CO)2C6H4), p-toluenesulfonamide (-SO2C6H4Me), benzylamine (=CPh), phthalimide (=(CO)2C6H4), p-toluenesulfonamide (-SO2C6H4Me), diethylamine (-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), particularly where B is Boc or Eei, more particularly where B is Boc, or - an acetal or ketal protecting group selected from: [ka] wherein r is 0 to 12, particularly 0 to 6, more particularly 0, 1 or 2; and R 10 is -C1~C 12 Alkyl-, especially C 1~6 alkyl, more particularly C 1~3 It is alkyl.

[0070] In certain embodiments, B is selected from the group consisting of Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylidene), trityl (-C(Ph)), Mmt (-C(Ph)CHOMe), DMT (-C(Ph)(CHOMe)), Cbz (-C(=O)OCHPh), benzylideneamine (=CPh), phthalimide (=(CO)CHO), p-toluenesulfone amide (-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).

[0071] In certain embodiments, B is Boc or Eei (=CMeOEt, 1-ethoxyethylidene).

[0072] In certain embodiments, B is Boc.

[0073] In certain embodiments, B is selected from an acetal or ketal protecting group selected from: [ka] wherein r is 0 to 12, particularly 0 to 6, more particularly 0, 1 or 2; R 10 is -C1~C 12 Alkyl-, especially C 1~6 It is alkyl.

[0074] In certain embodiments, T is the moiety -C 1~12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9 -, phenyl, 5- or 6-membered heteroaryl, wherein J is CH or N, in particular N, and wherein R 9 is H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, especially H and C 1~2 independently selected from alkyl, more particularly R 9 is H, where B is an independently selected acid-labile amine protecting group.

[0075] In certain embodiments, T is -C 1~12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR9 -, phenyl, 5- or 6-membered heteroaryl.

[0076] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 and a linear or branched spacer containing at least one, particularly 1 to 5, of -, phenyl, piperazinyl, pyrroyl, pyrazoyl, imidazoyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.

[0077] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 It is a linear or branched spacer containing at least one, particularly 1 to 5, of -, phenyl, imidazoyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.

[0078] In certain embodiments, T is a linear spacer.

[0079] In certain embodiments, T is the moiety -C 1~12 Alkyl-, (-C2H4O-) 1~12 , -C(=O)-, -C(=O)-JR 9 -, -JR 9 -C(=O)-, -JR 9 -, wherein J is C or N, in particular N, and wherein R 9 is H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, especially H and C 1~2 independently selected from alkyl, more particularly R 9is H, where B is an independently selected acid-labile amine protecting group.

[0080] In certain embodiments, the total length of the spacer T is between 0.5 and 100 nm.

[0081] In certain embodiments, T is -C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -C(=O)-R 6 -, -R 5 -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-R6 -, -phenyl-, -R 5 -pyrroyl, -R 5 -pyrazoyl, -R 5 -Imidazolyl, R 5 -piperazinyl-, -R 5 -pyridinyl, -R 5 -pyrimidinyl, -R 5 -pyrazinyl, -R 5 -pyridazinyl, -R 5 -Pyroyl-R 6 -, -R 5 -Pyrazoyl-R 6 -, -R 5 -Imidazol-R 6 -, -R 5 -piperazinyl-R 6 -, -R 5 -pyridinyl-R 6 -, -R 5 -pyrimidinyl-R 6 -, -R 5 -pyrazinyl-R 6 -, -R 5 -pyridazinyl-R 6 -, pyrroyl-R 6 -, Pyrazoyl-R 6 -, Imidazoyl-R 6 -Piparazinyl-R 6 -, pyridinyl-R 6 -, pyrimidinyl-R 6 -, pyrazinyl-R 6 -, pyridazinyl-R 6 -, pyrroyl, pyrazoyl, imidazoyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.

[0082] In certain embodiments, T is -C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR9 -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 -Imidazolyl, -R 5 -Imidazol-R 6 -, Imidazoyl-R 6 -, imidazolyl.

[0083] In certain embodiments, T is -C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9-, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -NR 9 -R 5’ -NR 9’ C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ -C(=O)-R 6 -, -R 5 -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 -Imidazolyl, -R 5 -Imidazol-R 6 -,-Imidazol-R 6 -, imidazolyl.

[0084] In certain embodiments, T is -C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -C(=O)-R6 -, -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 - is selected from.

[0085] In certain embodiments, T is -C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-, -R 5 -C(=O)-NR 9 -, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -C(=O)-NR 9 -R 5’ -NR 9’ C(=O)-R 6 - selected from, in particular C 1~3 Alkyl, -R 5 -C(=O)-NR 9 -, -R 5 -NR 9 -C(=O)-R6 -From, more especially C 1~3 Alkyl or -R 5 -C(=O)-NR 9 - selected from R 5 , R 5’ , R 6 , R 9’ and R 9 is as defined above.

[0086] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -, phenyl, and 5- or 6-membered heteroaryl, and a linear or branched spacer containing at least one, particularly 1 to 5, of the following: 9 is 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 From, especially H and C 1~2 alkyl, and in particular R 9 is H, wherein B is an independently selected acid-labile amine protecting group, R 15 is a blocking agent that can react with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular from cysteinyl and N-methylhydroxylamine, more in particular from cysteinyl, wherein The amine and / or thiol moieties of the blocking agent can be protected by an independently selected acid-labile amine protecting group B, particularly Boc, and / or an acid-labile thiol protecting group, particularly trityl.

[0087] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 It is a linear or branched spacer containing at least one, particularly 1 to 5, of -, phenyl, pyrroyl, pyrazoyl, imidazoyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.

[0088] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 It is a linear or branched spacer containing at least one, particularly 1 to 5, of -, phenyl, imidazoyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.

[0089] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -, wherein R 9 is 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 From, especially H and C 1~2 independently selected from alkyl, more particularly R 9is H, where B is an independently selected acid-labile amine protecting group; R 15 is a blocking agent that can react with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular from cysteinyl and N-methylhydroxylamine, more in particular from cysteinyl, wherein The amine and / or thiol moieties of the blocking agent can be protected by an independently selected acid-labile amine protecting group B, particularly Boc, and / or an acid-labile thiol protecting group, particularly trityl.

[0090] As mentioned above, the spacer T is generally unreactive under commonly applied purification conditions, except for the removal of protecting groups under acidic conditions. The spacer T may enhance the solubility of the linker molecule. In particular, R 9 The branched spacer containing a protected or unprotected amine moiety at ##STR00002## contributes to enhanced solubility. Under acidic conditions, the amine protecting group B is removed and the amine is protonated.

[0091] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -, wherein R 9 is H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 Alkyl-NB2, in particular H and C 1~2 independently selected from alkyl, more particularly R 9 is H, where B is an independently selected acid-labile amine protecting group.

[0092] The spacer T may also contain a blocking function. In particular, a branched spacer may contain a blocking agent suitable for binding to an aldehyde moiety. If the solid phase used for peptide purification contains an aldehyde moiety (e.g., agarose beads), the portion X of the linker compound can be covalently attached 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, unreacted aldehyde moieties on the solid phase can be blocked by a blocking function of the spacer, for example, a cysteinyl moiety.

[0093] In certain embodiments, T is the moiety -C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -, wherein R 9 is H, C 1~4 Alkyl, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 From, especially H and C 1~2 independently selected from alkyl, more particularly R 9 is H, where B is an independently selected acid-labile amine protecting group; R 15 is a blocking agent that can react with aldehyde moieties, particularly R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular from cysteinyl and N-methylhydroxylamine, more in particular from cysteinyl, wherein The amine and / or thiol moieties of the blocking agent can be protected by an independently selected acid-labile amine protecting group B, particularly Boc, and / or an acid-labile thiol protecting group, particularly trityl.

[0094] In certain embodiments, T is -C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -C(=O)-NR 9 -R 6 -, -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -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 -, especially -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more particularly C 1~3 Alkyl, -R 5 -NR 9 -C(=O)-R 6 -, -R 5 -C(=O)-NR 9 -R 6 -, -R 5 -NR 9 -R 5 -NR 9 C(=O)-R 6 -, most especially -R 5 -NR 9 -C(=O)-R 6 - selected from R 5 , R 6 and R 9 is as defined above.

[0095] As noted above, the moieties U, V, W, and E contribute to the stability of the linker under acidic conditions. For example, linkers containing moieties U or V containing an amine or heterocycle such as pyridine are stable under acidic conditions, particularly in the presence of TFA >50% and water at pH <0, because the amine or heterocycle is protonated. Furthermore, protonated linkers improve the solubility of the linker-peptide complex. If the pH is higher than the pKa of the linker, the linker will rapidly decompose upon release of the desired peptide, as in the final step.

[0096] In certain embodiments, V is —NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p -,-Piperazinyl-(CH2) p -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] , an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H, R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl, p is 0, 1 or 2, in particular 0 or 1.

[0097] In certain embodiments, V is —NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p -,-Piperazinyl-(CH2) p -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] , an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, wherein the pyridinyl moiety is attached to U at the 3-position.

[0098] In certain embodiments, V is —NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p -,-Piperazinyl-(CH2) p - is selected from.

[0099] In certain embodiments, V is —NR 11 -C(=O)-, -C(=O)-NR 11 -, -NR 12 -(CH2) p -,-Piperazinyl-(CH2) p - is selected from.

[0100] In certain embodiments, V is -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -NH-, -piperazinyl-(CH2) p - is selected from.

[0101] In certain embodiments, V is -NH-C(=O)-, -C(=O)-NH-, -N-(CH)-, -piperazinyl-(CH) p - is selected from.

[0102] In certain embodiments, V is -NH-C(=O)-, -C(=O)-NH-, -N-(CH)-, -piperazinyl-(CH) p -, and p is 0 or 1.

[0103] In certain embodiments, V is selected from the group consisting of -NH-C(=O)-, -C(=O)-NH-, and -piperazinyl-(CH) p -, where p is 0 or 1, in particular p is 0.

[0104] In particular, linker molecules that release peptides via carbamate switches (Figure 8) do not contain basic nitrogen atoms, such atoms having a corresponding acid pKa of less than 2. In certain embodiments, V is -NR 11 -C(=O)-, -C(=O)-NR 11 and -S(=O)-, in particular -NH-C(=O)- and -C(O=)-NH-, more particularly V is -NH-C(=O)-.

[0105] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] , an electron-withdrawing moiety selected from —C(═O)—, —C(═O)—O—, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H and R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl.

[0106] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, -C(=O)-, -C(=O)-O-, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H and R 12is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl.

[0107] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, and pyrimidinyl, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H and R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl.

[0108] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -NR 12 an electron-withdrawing moiety selected from -, -piperazinyl-, -pyridinyl-, and pyrimidinyl, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H and R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl.

[0109] V is an electron-withdrawing moiety selected from -NH-C(=O)-, -C(=O)-HN-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, pyrazinyl, pyridazinyl.

[0110] V is an electron-withdrawing moiety selected from -NH-C(=O)-, -C(=O)-HN-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl.

[0111] In certain embodiments, V is —NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazyl, -C(=O)-, -C(=O)-O-.

[0112] In certain embodiments, V is —NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 - and -pyridinyl-, pyrimidinyl, wherein R 11 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 11 is H and R 12 is H and C 1~4 Alkyl, especially H and C 1~2 alkyl, more particularly R 12 is methyl.

[0113] In certain embodiments, V is —NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, wherein R 11 is H and C 1~4 alkyl, and R 12 is H and C 1~4 alkyl.

[0114] In certain embodiments, V is —NR 11 -C(=O)-, S(=O)-, -NR 12 -, -pyridinyl-, pyrimidinyl, wherein R 11 is H and C 1~2 alkyl, and R12 is H and C 1~2 alkyl.

[0115] In certain embodiments, V is selected from -NH-C(=O)-, -N-(CH3)-, -pyridinyl-, and pyrimidinyl.

[0116] In particular, linker molecules that release peptides via an amine switch require a basic nitrogen atom. The basic nitrogen atom can be provided via the moieties V, E, or U (U=heteroaryl). When U is phenyl, the basic nitrogen atom can be provided via the moieties V or E.

[0117] In certain embodiments, E and V are selected as described above, and when U is phenyl, at least one moiety E or V is selected from: - E: piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N(C2H4)2NH2, -N(C2H4)2N-B, -N=NR 8 , -(CH2) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 alkyl)2-, -N3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, where R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -(CH2) p -NMe2, where p and B are defined as described herein, and - V:-NR 12 -(CH2) p -,-Piperazinyl-(CH2) p -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, [ka] In particular, -NR 12 -(CH2) p -,-Piperazinyl-(CH2) p-, -pyridinyl, pyrimidinyl, and R 12 and p is defined as described herein.

[0118] As noted above, W is a reducible moiety. Upon reduction of W, the reduced intermediate further decomposes in a pH-dependent manner, releasing a peptide with a free N-terminus.

[0119] In certain embodiments, W is —N 3 , —NO 2 , —S(═O)—R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] is selected from: R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1-C6 alkyl or -(CH2) p -NMe2, particularly pyridyl or -C1-C6 alkyl, where p is 1, 2, 3 or 4. 1~6 The alkyl may be linear or branched, for example, butyl or tert-butyl.

[0120] In certain embodiments, W is —N 3 , —NO 2 , —S(═O)—R 8 , -SSR 8 , -O-CH2-N3, -OC(=O)-O-CH2-N3, -N=N-phenyl, -N=NR 8 , [ka] is selected from.

[0121] In certain embodiments, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -N=NR 8 From this, especially -N3, -N=NR 8 , -O-CH2-N3, -SSR8 wherein R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1 to C6 alkyl, or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, and p is 1, 2, 3 or 4.

[0122] In certain embodiments, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -N=NR 8 From this, especially -N3, -N=NR 8 , -O-CH2-N3, -SSR 8 wherein R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1 to C6 alkyl, or -(CH2) p -NMe2, in particular pyridyl or -C1-C6 alkyl, and p is 1, 2, 3 or 4.

[0123] R 8 C 1~6 When W is alkyl, the alkyl moiety may be linear or branched, for example, tert-butyl. In particular, W is -SSR 8 If R 8 may be pyridyl or -C1-C4 alkyl, in particular pyridyl or tert-butyl.

[0124] Partial W=-SSR 8 Linkers containing the following can be cleaved using thiols. Thus, cleavage of peptide-linker conjugates from synthesis resins used during SPPS under acidic conditions (e.g., TFA > 50%, pH < 0 in the presence of water) can be performed without thiols when such linkers are used for peptide purification.

[0125] In certain embodiments, W is -N3, -S(=O)-R 8 , -SSR 8 , -O-CH2-N3, -N=NR 8 From this, especially -N3, -N=NR8 , -O-CH2-N3, -SSR 8 wherein R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl or -C1-C6 alkyl, or in particular pyrimidinyl, pyridyl or -C1-C6 alkyl, more in particular pyridyl or -C1-C6 alkyl.

[0126] In certain embodiments, W is -N3, -SSR. 8 (R 8 is —C1-C6 alkyl), and NO2.

[0127] Linker molecules that release peptides via an amine switch (Figure 5) or a carbamate switch (Figure 8) with an azide reductive safety lock contain a reducible moiety, N3. In certain embodiments, W is -N3.

[0128] Linker molecules that release peptides via an amine switch with an azide-free reductive safety lock (Figure 6) contain a reducible moiety such as -NO2 or -S-tert-butyl. In certain embodiments, W is -SSR 8 (R8 is -C1-C6 alkyl), -NO2, [ka] -N=NR 8 (R 8 is pyridyl, pyrimidinyl, pyrazinyl or pyridazyl).

[0129] In certain embodiments, W is -SSR. 8 (R 8 is -C1-C6 alkyl), -NO2, -N=NR 8 (R 8 is pyridyl, pyrimidinyl, pyrazinyl or pyridazyl), in particular -NO2 or -SSR 8 (R 8 is —C1-C6 alkyl).

[0130] The linker molecules that release the peptide via amine switch by nucleophilic release (Figure 7) are -N3, -SSR 8 (R 8 is C 1~6 alkyl, especially tert-butyl, or R 8 is pyridinyl).

[0131] As noted above, the linker molecule can contribute to the solubility of the linker-peptide construct under acidic conditions, particularly during hydrophobic peptide purification. Solubility can also be mediated by the moiety W, i.e., W is selected from groups that confer enhanced solubility on the peptide being purified in the pH range of 0-7.

[0132] In certain embodiments, W is —S(═O)—R 8 , -SSR 8 , -N=NR 8 , [ka] is selected from: R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -(CH2) p -NHBoc or -(CH2) p -NMe2, especially pyridyl or -(CH2) p -NHBoc-, and p is 1, 2, 3, or 4.

[0133] In certain embodiments, U is selected from phenyl or a 5- or 6-membered heterocycle, wherein the 5- or 6-membered heterocycle contains 1 or 2 heteroatoms.

[0134] In certain embodiments, U is selected from phenyl or a 5- or 6-membered heterocycle, wherein the 5- or 6-membered heterocycle contains one heteroatom.

[0135] In certain embodiments, U is selected from phenyl or a 6-membered heterocycle.

[0136] In certain embodiments, U is selected from phenyl or a 6-membered heterocycle, wherein the 6-membered heterocycle contains 1 or 2 heteroatoms.

[0137] In certain embodiments, the 5- or 6-membered heteroaryl moiety of U contains 1 or 2 heteroatoms, and in particular the 5-membered heteroaryl moiety of the moiety U is selected from pyrazole, imidazole, and the 6-membered heteroaryl moiety of the moiety U is selected from pyridine, pyridazine, pyrimidine, pyrazine, in particular pyridine.

[0138] In certain embodiments, U is selected from phenyl or a 6-membered heterocycle, wherein the 6-membered heterocycle contains one heteroatom. In certain embodiments, the 5-membered heterocycle of the moiety U is selected from pyrazole, imidazole, and the 6-membered heterocycle of the moiety U is selected from pyridine, pyridazine, pyrimidine, and pyrazine.

[0139] In a particular embodiment, U is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, in particular from phenyl or pyridine, more in particular from phenyl.

[0140] Linkers according to the invention can release peptides via an amine switch (Figures 5-7) or a carbamate switch (Figure 8). These release mechanisms require a reducible substituent W located ortho or para to the moiety Y.

[0141] In certain embodiments, U is selected from a moiety of formula 5 or 6: [ka] During the ceremony, T, V, Y, W, and E are defined as above; U is attached to the moiety T or V; A 1 , A 2 , A 3 , A 4 and D 1 , D2 , D 3 , D 4 are selected independently from C, N, S and O, in particular from C and N, and n is an integer between 0 and 3, in particular 0, 1 or 2.

[0142] In certain embodiments, A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 2 to 4 parts of C, especially A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 Three or four of the moieties are C.

[0143] In certain embodiments, all moieties A 1 , A 2 , A 3 and A 4 or D 1 , D 2 , D 3 and D 4 The part is C.

[0144] In certain embodiments, U is selected from a moiety of formula 5, 6, 7, or 8: [ka] During the ceremony, T, V, Y, W, and E are defined as above; In formulas 5 and 6, U is attached to the moiety T or V; In formulas 7 and 8, U is attached to the moiety V, A 1 , A 2 , A 3 , A 4 and D 1 , D 2 , D 3 , D4 are independently selected from C, N, S and O, in particular from C and N; A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 2 to 4 parts of C, especially A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 3 or 4 moieties are C, more particularly A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 All parts of are C, n is In the case of formulas 5 and 6, it is an integer between 0 and 3, In the case of formulas 7 and 8, it is an integer between 0 and 4, q is an integer between 0 and 4, and the sum of n and q is 4 or less.

[0145] In certain embodiments, U is selected from the moieties of formula 5 or 6.

[0146] In certain embodiments, U is selected from a moiety of formula 5, 6, 7, or 8, wherein: T, V, Y, W, and E are defined as above; In formulas 5 and 6, U is attached to the moiety T or V; In formulas 7 and 8, U is attached to the moiety V, A 1 , A 2 , A 3 , A 4 and D 1 , D 2 , D 3 , D 4are selected independently from C, N, S and O, in particular from C and N, n is In the case of formulas 5 and 6, it is an integer between 0 and 2, In the case of formulas 7 and 8, it is an integer between 0 and 2, in particular between 0 and 1, q is an integer between 0 and 2, particularly between 0 and 1.

[0147] In certain embodiments, U is selected from a moiety of formula 9, 10, 11 or 12, particularly formula 9 or 10: [ka] During the ceremony, T, V, Y, W, E, q and n are as defined above; In formulas 9 and 10, U is attached to the moiety T or V; In formulas 11 and 12, U is attached to the moiety V, All parts A 2 , A 3 and A 4 is C or A 2 , A 3 and A 4 Two of them are C and A 2 , A 3 and A 4 The other two are N, especially A 2 and A 3 are both C, and D 2 is C or N, in particular C.

[0148] In certain embodiments, U is selected from a moiety of formula 9, 10, 11 or 12, particularly formula 9 or 10: [ka] During the ceremony, T, V, Y, W, E, q and n are as defined above; In formulas 9 and 10, U is attached to the moiety T or V; In formulas 11 and 12, U is attached to the moiety V, A 2and A 3 are both C, or A 2 and A 3 One of them is C and A 2 and A 3 The other is N, D 2 is C or N.

[0149] When the moiety U comprises an N-containing heteroaryl, the N atom becomes protonated under acidic conditions, thus increasing the solubility of the linker molecule.

[0150] In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20 or 21, particularly formulas 13-19, more particularly formula 15 or 19; [ka] During the ceremony, T, V, Y, W, E, q and n are as defined above; In formulas 13, 14 and 15, U is attached to the moiety T or V; In formulas 16, 17 and 18, U is attached to the moiety V; A 2 , A 3 and A 4 is C, N or N + Me, especially C, D 2 is C or N.

[0151] In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22: [ka] wherein T, V, Y, W, E, q and n are defined as above; In the case of formulas 13, 14, 15, 19, 20, 21 and 22, U is bonded to the moiety T or V; In formulas 16, 17 and 18, U is attached to the moiety V; A 2 , A 3 and A4 is C or N, in particular C, D 2 is C or N.

[0152] In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 16, 17, or 18: [ka] During the ceremony, T, V, Y, W, E, q and n are as defined above; In formulas 13, 14 and 15, U is attached to the moiety T or V; In formulas 16, 17 and 18, U is attached to the moiety V; A 3 is C or N, D 2 is C or N.

[0153] In certain embodiments, U is selected from a moiety of formula 13, 14, 15, 21, or 22, wherein: T, V, Y, W, E and n are defined as above; U is attached to the moiety T or V; A 3 and A 4 is C or N, in particular C, D 2 is C or N,

[0154] In certain embodiments, U is selected from the moieties of formula 13, 21, or 22.

[0155] In particular, for linker molecules that release peptides via carbamate switches, U is phenyl. In certain embodiments, U is selected as above and all A and all D are C.

[0156] For amine switches (Figures 5 and 6) and carbamate switches (Figure 8) with a reductive safety lock, the moiety Y can release CO2 -(CH2), especially when a change in pH causes the linker to collapse, releasing a peptide with a free N-terminus. m -OC(=O)-.

[0157] In certain embodiments, U is a moiety of formula 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, and Y is —(CH) m -OC(=O)-.

[0158] In certain embodiments, U is a moiety of formula 13, 14, 15, 21, or 22, and Y is —(CH) m -OC(=O)-.

[0159] In certain embodiments, U is a moiety of formula 13 or 21 and Y is -(CH) m -OC(=O)-.

[0160] For amine switches with nucleophilic release (Figure 7), the linker is stable only under acidic conditions if the moiety Y forms an amide bond with the peptide. In certain embodiments, Y is -(CH2) m -C(=O)-.

[0161] If the linker molecule terminates in -COOH, the peptide can be coupled to the peptide via standard amino acid coupling. In certain embodiments, Y is -(CH2) m It is -C(=O)- and Z is -OH.

[0162] For amine switches with nucleophilic release, U is specifically the moiety of formula 22 and Y is -(CH2) m It is —C(═O)— and Z is —OH.

[0163] In certain embodiments, Z is one of the following: -F, -Cl, -Br, -I, -N, -OH, -O(C=O)CH(C=O)OH, -SR14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3, [ka] In particular, -OH, -Cl, [ka] In particular, -OH, [ka] wherein R 14 is a C1-C6 alkyl-, aryl- or benzyl-substituent.

[0164] Type 1 linker molecules suitable for peptide release via an amine switch with an azide reductive safety lock (Figure 5) can be composed of the following moieties: U is phenyl or pyrimidyl, in particular U is of formula 13, 14, 15, 21 or 22, W is -N3; n in E is 1 and E is selected from pyridyl, pyrimidinyl, pyridazinyl, -N(CH3)2, -N=N-pyridyl, or n is 0, in particular n is 0; a of V is 1 and V is selected from -piperazinyl-, -piperazinyl-CH2-, -N(CH3)-, pyrimidinyl, pyridyl, in particular -piperazinyl-, -piperazinyl-CH2-, -N(CH3)-, and Y is —(CH 2 ) m OC(═O)—, where m is 1, 2 or 3, particularly 1 or 2, more particularly 1.

[0165] The pyridyl moiety is attached to U at the 3- or 5-position.

[0166] In certain embodiments, U is selected from the moieties of formula 13, 14, 15, 21, or 22.

[0167] Type 2 linker molecules (Figure 6) suitable for peptide release by azide-free amine switching can be composed of the following moieties: U is pyridinyl or phenyl, in particular U is of formula 13, 14 or 21, W is -SS-tertbutyl, -NO2, -N=N-pyridyl, [ka] selected from, in particular -SS-tertbutyl, -NO2, n of E is 0, a of V is 1 and V is selected from -C(=O)-NH- and piperazinyl; Y is —(CH 2 ) m OC(═O)—, where m is 1, 2 or 3, particularly 1 or 2, more particularly 1.

[0168] In certain embodiments, U is selected from the moieties of formula 13, 14, or 21.

[0169] Type 3 linker molecules suitable for peptide release by amine switching with nucleophilic release (Figure 7) can be composed of the following moieties: U is phenyl or pyridinyl, especially phenyl, more especially U is a moiety of formula 13 or 14, W is -N3, -SS-tertbutyl, -SS-pyridyl, especially -N3; n of E is 0, a of V is 1 and V is piperazinyl, -NH-, -C(=O)-NH-, in particular piperazinyl, and Y is —(CH 2 ) m C(═O)—, where m is 1, 2 or 3, particularly 1 or 2, more particularly 1.

[0170] In certain embodiments, U is selected from moieties of formula 13 or 14, and in particular all moieties D and A are C.

[0171] Type 4 linker molecules (Figure 8) suitable for the release of peptides via carbamate switches can be composed of the following moieties: U is phenyl, in particular U is of formula 13 or 15, in particular the moiety A is C, W is -N3; n of E is 1 or 2, E is -Br, a of V is 1 and V is —NH—C(═O)—, and Y is —(CH 2 ) m OC(═O)—, where m is 1, 2 or 3, particularly 1 or 2, more particularly 1.

[0172] In certain embodiments, U is selected from a moiety of formula 13 or 15, and in particular the moiety A is C.

[0173] In certain embodiments, Z is one of the following: -F, -Cl, -Br, -I, -N3, -OH, -SR 14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3, [ka] In particular, -OH, -Cl, [ka] In particular, -OH, [ka] wherein R 14 is a C1-C6 alkyl, aryl or benzyl substituent.

[0174] When Z is -OH, the -OH moiety is activated by coupling reagents, such as those commonly used in solid phase peptide synthesis, and functions as a leaving group.

[0175] In certain embodiments, the compound of Formula 1 is selected from a compound of formula: X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, or X47. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0176] A second aspect of the present invention is directed to a method for purifying a peptide.

[0177] According to a second aspect of the present invention, there is provided a method for purifying a peptide, the method comprising the steps of: - providing a crude linker-modified peptide, which crude peptide is covalently attached to a linker molecule according to the first aspect of the invention, - coupling the linker-modified peptide to a solid support in a coupling step to obtain an immobilized linker-modified peptide; - in a releasing step, adding a reducing agent under acidic conditions to release the peptide.

[0178] The linker molecule according to the first aspect of the present invention can be used in a method for purifying a peptide. In a first step, a crude peptide mixture is contacted with a linker molecule according to the first aspect of the present invention, and the crude peptide is coupled to the linker molecule to obtain a crude linker-modified peptide. The crude peptide is coupled to the linker molecule by standard methods generally known to those of ordinary skill in the fields of chemistry, biochemistry, and pharmacology. During purification, the stability of the linker molecule can be improved by adjusting the pH. Under acidic conditions (especially at a pH lower than the pKa of the most basic heteroatom of the linker molecule), the linker molecule is stable because this heteroatom is protonated and electron-withdrawing (Figures 5, 6, and 7). Once the linker is attached to the peptide and immobilized on a solid support, the peptide can be released via a reduced intermediate under reducing conditions, for example, by adding a reducing agent such as triphenylphosphine under acidic conditions. The reduced intermediate is characterized by a reduced linker moiety, such as an azaylide. The linker moiety of the reduced intermediate decomposes over time or upon a trigger, such as an increase in pH, particularly when the pH is greater than the pKa of the most basic heteroatom of the reduced intermediate's linker moiety. Therefore, all steps prior to releasing the peptide (coupling, optional washing, and reduction of the linker) are performed under acidic conditions. When the pH is increased to a pH above the pKa of the most basic heteroatom of the reduced intermediate's linker moiety, the reduced intermediate's linker moiety decomposes via a 1.4 / 1.6 elimination reaction or nucleophilic attack, releasing the peptide.

[0179] The most basic heteroatom of the linker molecule / linker portion of the reduction intermediate is related to the following: For example, moiety U consists of pyridine substituted with -N3. The heteroatom N of pyridine has a pKa of about 5, which is higher than the pKa of the -N3 moiety or the reduced -N3 moiety (-the pKa of -NH3 is about 4.6). Thus, the most basic heteroatom is the N of the pyridine moiety. By shifting the pH to pH > 5, the linker portion of the reduction intermediate undergoes an elimination reaction and the peptide is released. This mechanism is called an amine switch.

[0180] Alternatively, the linker molecule can be cleaved by a carbamate switch (Figure 8). Suitable linker molecules contain an electron-withdrawing moiety such as -Br and a reducing moiety such as -N3. This linker molecule is stable under TFA conditions due to this electron-withdrawing property. When reduced, the linker molecule is stable when the pH is higher than the pKa of this carbamate. Finally, by decreasing the pH such that pH < pKa with respect to the pKa of this carbamate, the peptide is released via 1,6-elimination.

[0181] In certain embodiments, at the pH in all steps prior to the addition of the reducing agent, pH < pKa with respect to the pKa of the most basic heteroatom of the linker portion.

[0182] In one embodiment, the peptide is released via an intermediate.

[0183] In certain embodiments, the peptide is released via a reduction intermediate characterized by the reduced linker portion of an immobilized linker-modified peptide by adding a reducing agent under acidic conditions.

[0184] In certain embodiments, in the release step, a reduction intermediate is achieved that features a reduced linker portion of an immobilized linker-modified peptide, and the peptide is triggered, particularly by a change in temperature and / or pH, more particularly by increasing the pH to pH > pKa with respect to the pKa of the most basic heteroatom of the linker portion of the reduction intermediate in the case of an amine switch, or by decreasing the pH to pH < pKa with respect to the pKa of the carbamate in the case of a carbamate switch, whereby the peptide is released from said reduction intermediate.

[0185] In certain embodiments, the reduction intermediate featuring a reduced linker portion of an immobilized linker-modified peptide is achieved in the release step, and the peptide is triggered, particularly by a change in temperature and / or pH, more particularly by increasing the pH to pH > pKa with respect to the pKa of the most basic heteroatom of the linker portion of the reduction intermediate, whereby the peptide is released from said reduction intermediate.

[0186] The peptide is released from the intermediate either spontaneously or upon triggering.

[0187] In certain embodiments, a change in temperature and / or pH serves as the trigger.

[0188] In certain embodiments, the change in temperature is an increase from ambient temperature (20 °C to 30 °C) to a higher temperature, and the higher temperature does not exceed 100 °C, particularly 70 °C, more particularly does not exceed 50 °C.

[0189] As described above, a linker suitable for an amine switch decomposes when the pH is increased to a pH exceeding the pKa of the most basic heteroatom when reduced.

[0190] In certain embodiments, the trigger is a pH shift, particularly an increase in pH.

[0191] In certain embodiments, the peptide is released from the reduced intermediate by raising the pH to pH > pKa relative to the pKa of the most basic heteroatom of the linker portion of the reducing intermediate.

[0192] Linkers suitable for the carbamate switch decompose when the pH decreases - when reduced - to a pH below the pKa of the carbamate.

[0193] In certain embodiments, the trigger is a pH shift, particularly a decrease in pH.

[0194] In certain embodiments, the peptide is released from the reducing intermediate by decreasing the pH to pH < pKa relative to the pKa of the carbamate of the linker portion.

[0195] When the reducing intermediate is stable under acidic conditions (amine switch) or stable at pH > pKa relative to the pKa of the carbamate (carbamate switch), an additional washing step can be performed to remove excess reducing agent. Since the reducing agent can react with not only the linker portion but also the peptide, adding the washing step reduces unwanted side reactions between the reducing agent and the peptide. Furthermore, non-volatile reducing agents or the products of their use are also impurities and will need to be removed by additional purification steps.

[0196] In certain embodiments, after the reducing intermediate is formed and before the peptide is released from the reducing intermediate, the reducing agent is removed by washing.

[0197] In certain embodiments, the reducing agent is removed using MeCN.

[0198] In certain embodiments, the linker molecule according to the first aspect of the invention comprises a moiety W and / or E comprising an azide (-N3) moiety.

[0199] In certain embodiments, the linker-modified peptide is additionally attached to a synthetic resin, and the synthetic resin is cleaved prior to performing the coupling step, particularly at a pH < pKa with respect to the pKa of the most basic heteroatom of the linker molecule.

[0200] Cleavage of the peptide from the synthetic support is achieved using TFA, and finally the peptide is precipitated from the TFA mixture (e.g., after 2 - 8 hours) to obtain a crude peptide mixture. Cold ether (Et2O, iPr2O, MeOtBu, THF / hexane (1:1)) can be used for precipitation.

[0201] The crude peptide mixture is dissolved in a suitable organic solvent, particularly DMSO, and a buffer system, particularly 10 volume% sodium citrate buffer 0.1M, pH 4.5 is added.

[0202] The solid support for the coupling step is an aldehyde-modified solid support, particularly agarose beads, polylysine, polyethylene glycol, polyamide, polystyrene, and their copolymers, to which the dissolved crude peptide mixture is added.

[0203] In certain embodiments, the solid support contains an aldehyde moiety.

[0204] In certain embodiments, after performing the coupling step, the unreacted aldehyde portion of the solid support is blocked using a blocking agent.

[0205] In certain embodiments, the blocking agent reacts with the aldehyde portion of the solid support and contains a thiol and / or amine moiety.

[0206] In certain embodiments, the blocking agent is selected from cysteine, threonine, 2 - mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O - methylhydroxylamine, N - methylhydroxylamine, dithiothreitol, hydrazine.

[0207] In certain embodiments, the blocking agent is selected from cysteine and N-methylhydroxylamine.

[0208] The coupled product is washed, in particular, with DMSO, guanidinium hydrochloride 6M, EtOH / water (7:3) with 0.1M NaCl, water, MeCN.

[0209] In certain embodiments, the release step is carried out at pH < pKa with respect to the pKa of the most basic heteroatom of the linker molecule.

[0210] In certain embodiments, the release step is carried out at pH < pKa with respect to the pKa of the carbamate of the linker molecule.

[0211] In certain embodiments, the reducing agent is selected from triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine or tris(2-carboxyethyl)phosphine, trimethylphosphite, triethylphosphite, tributylphosphine, diethylphosphite, 5,5'-dithiobis(2-nitrobenzoic acid), sodium dithionite (Na2S2O4), ethanedithiol, propanedithiol, dithioerythritol, dithiothreitol, Na2S, NaSH, glutathione, 2,2'-dithiodipyridine, BH3, 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, catecholborane, borane tetrahydrofuran, borane dimethylsulfide, borane dimethylamine complex, borane triphenylphosphine complex, borane tert-butylamine, LiAlH4, LiBH4, NaBH4, NaBH3CN, NaBH(OMe)3, NaBH(OCCH3)3, LiAlH(OCMe3)3, hydroquinone, sodium ascorbate salt, ascorbic acid, ascorbic acid containing KI, hydrazine, NH=NH, formaldehyde.

[0212] In certain embodiments, the reducing agent is selected from dithioerythritol, dithiothreitol, triphenylphosphine, KI-containing ascorbic acid, tributylphosphine, trimethylphosphine, tris(2-carboxyethyl)phosphine, sodium dithionite (NaSO), borane dimethyl sulfide, borane triphenylphosphine complex, NaBH, and ascorbic acid.

[0213] In certain embodiments, the reducing agent is selected from dithioerythritol, dithiothreitol, KI-containing ascorbic acid, triphenylphosphine, and trimethylphosphine.

[0214] In certain embodiments, the reducing agent is triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine or tris(2-carboxyethyl)phosphine, trimethylphosphite, triethylphosphite, tributylphosphine, diethylphosphite, 5,5′-dithiobis(2-nitrobenzoic acid), sodium dithionite (NaSO), ethanedithiol, propanedithiol, dithiothreitol, NaS, NaSH, glutathione, 2,2′-dithiodipyridine, BH Selected from 3,4,4,5,5-tetramethyl-1,3,2-dioxaborolane, catecholborane, borane tetrahydrofuran, borane dimethyl sulfide, borane dimethylamine complex, borane triphenylphosphine complex, borane tert-butylamine, LiAlH4, LiBH4, NaBH4, NaBH3CN, NaBH(OMe)3, NaBH(OCCH3)3, LiAlH(OCMe3)3, hydroquinone, sodium ascorbate, ascorbic acid, hydrazine, NH=NH, and formaldehyde.

[0215] In certain embodiments, the reducing agent is selected from triphenylphosphine, tributylphosphine, trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, sodium dithionite (NaSO), borane dimethyl sulfide, borane triphenylphosphine complex, NaBH, and ascorbic acid.

[0216] In certain embodiments, the reducing agent is selected from triphenylphosphine, trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, sodium dithionite (Na2S2O4), borane dimethyl sulfide, borane triphenylphosphine complex, NaBH4, ascorbic acid.

[0217] In certain embodiments, the reducing agent is selected from triphenylphosphine, sodium dithionite (Na2S2O4), borane dimethyl sulfide, borane triphenylphosphine complex, NaBH4, and ascorbic acid.

[0218] In certain embodiments, the reducing agent is selected from triphenylphosphine and trimethylphosphine.

[0219] In certain embodiments, the reducing agent is triphenylphosphine.

[0220] In certain embodiments, the method comprises the following steps: - providing a crude linker-modified peptide, which crude peptide is covalently attached to a linker molecule according to the first aspect of the invention, the linker molecule comprising moieties W and / or E which comprise an azide moiety, - coupling the linker-modified peptide to a solid support in a coupling step to obtain an immobilized linker-modified peptide; - in a releasing step, the peptide is released by adding a reducing agent under acidic conditions.

[0221] In certain embodiments, the method comprises the following steps: - providing a crude linker-modified peptide, which crude peptide is covalently attached to a linker molecule according to the first aspect of the invention, the linker molecule comprising moieties W and / or E which comprise an azide moiety, - coupling the linker-modified peptide to a solid support in a coupling step to obtain an immobilized linker-modified peptide; - in a releasing step, a reducing agent is added under acidic conditions to obtain a reduced intermediate, and then the pH is increased so that the pH is greater than the pKa of the most basic heteroatom in the linker moiety of the reduced intermediate, thereby releasing the peptide. -

[0222] In certain embodiments, the method comprises the following steps: - providing a crude linker-modified peptide, which crude peptide is covalently attached to a linker molecule according to the first aspect of the invention, the linker molecule comprising moieties W and / or E which comprise an azide moiety, - coupling the linker-modified peptide to a solid support in a coupling step to obtain an immobilized linker-modified peptide; - in a releasing step, adding triphenylphosphine, trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, in particular triphenylphosphine, under acidic conditions to generate a reduced intermediate, followed by releasing the peptide by increasing the pH so that the pH is greater than the pKa of the most basic heteroatom.

[0223] According to a sub-embodiment of the second aspect of the present invention there is provided a method for purifying a crude peptide prepared by solid phase peptide synthesis.

[0224] In certain embodiments, the method for purifying a peptide comprises the following steps: a) providing a peptide bound to a synthetic resin, said peptide being further covalently bound to a linker molecule according to claim 1, said linker molecule comprising moieties W and / or E comprising an azide moiety; b) cleaving the peptide from the synthetic resin; c) coupling the cleaved peptide mixture to a solid support; d) Releasing the peptide with triphenylphosphine, or trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, in particular triphenylphosphine.

[0225] Cleavage of the peptide from the synthesis support is achieved using TFA, and finally, the peptide is precipitated from the TFA mixture (e.g., after 2-8 hours) to obtain the crude peptide mixture. Precipitation can be performed using cold ether (EtO, iPrO, MeOtBu, THF / hexane (1:1)).

[0226] The crude peptide mixture is dissolved in a suitable organic solvent, particularly DMSO, and a buffer system is added, particularly 10% by volume of sodium citrate buffer 0.1 M, pH 4.5.

[0227] The solid support for the coupling step is an aldehyde-modified solid support, in particular agarose beads, polylysine, polyethylene glycol, polyamide, polystyrene and copolymers thereof, to which the dissolved crude peptide mixture is added.

[0228] The coupled product is washed with, inter alia, DMSO, 6 M guanidinium hydrochloride, EtOH / water (7:3) with 0.1 M NaCl, water, MeCN.

[0229] In certain embodiments, triphenylphosphine, trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, particularly triphenylphosphine, is added to MeCN / AcOH (9:1). In certain embodiments, the addition is carried out over a period of 15 minutes.

[0230] In a particular embodiment, triphenylphosphine, trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine is added to MeCN / AcOH / HO (90:5:5) and / or after addition of triphenylphosphine, trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, in particular triphenylphosphine, the formed aza-ylide is washed out, in particular with MeCN or MeCN / HO (9:1).

[0231] In certain embodiments, triphenylphosphine, trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, particularly triphenylphosphine, is added to MeCN / AcOH / HO (90:5:5). In certain embodiments, the addition is carried out over a period of 15 minutes.

[0232] In a particular embodiment, after addition of triphenylphosphine, trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, the aza-ylide formed is washed, in particular with MeCN or MeCN / H 2 O (9:1).

[0233] In certain embodiments, after addition of triphenylphosphine, trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, the aza-ylide formed is washed, in particular with MeCN.

[0234] In certain embodiments, the aza-ylide formed is hydrolyzed, in particular using H2O / TFA, the ratio of which can be 99.95% to 50% water.

[0235] If the linker, particularly U, E, W, or V, is a nitrogen-containing heterocycle, the pH must be above the pKa of the heterocyclic moiety, which may already be the case with the hydrolysis mixture of TFA and water, or may be achieved by adding a buffer at the desired pH.

[0236] In a particular embodiment, the hydrolysis is followed by an elution step, in particular with TFA / H2O, in particular in a ratio of 9:1.

[0237] In a particular embodiment, the hydrolysis is followed by an elution step, in particular with TFA / H2O, in particular in a ratio of 95:5.

[0238] In certain embodiments, the hydrolysis product is precipitated, in particular by adding cold ether, more particularly Et2O, iPr2O, MeOtBu, THF / hexane (1:1).

[0239] Terms and Definitions In the context of the present invention, an "electron withdrawing group" or "EWG" is any chemical group that is capable of withdrawing an electron from its connected atom or aryl system via the inductive or mesomeric effect.

[0240] In the context of the present invention, Hammett constants are constants as calculated and described in 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. The electron-withdrawing effect is stronger the larger the Hammett constant. The Hammett constant is the ratio of the number of electrons at the meta position (σ m ) and para-position (σ p ) is an empirically determined constant for substituents on the phenyl moiety of the formula (I). In the context of the present invention, this position is determined relative to the attachment of the 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.

[0241] In the context of the present invention, the term "under acidic conditions" relates to a pH below pH 7, in particular a pH below the pKa of the linker, more in particular a pH<0 in the presence of TFA>50%, water.

[0242] In the context of the present invention, the term alkyl means a linear or branched saturated hydrocarbon. 1~12 The term alkyl refers to a saturated straight or branched chain hydrocarbon having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Non-limiting examples of C1-C4 alkyl include methyl, ethyl, propyl, n-butyl, 2-methylpropyl, and tert-butyl. [Example]

[0243] Linker Type 4 Example 1: Purification of Naturally Occurring and Research Peptides, P1 and P2 The peptide purification method of the present invention was applied to two peptides with different polarities: H-ARTKQTARKSTGGKA-OH (SEQ ID NO: 1) (P1) fragment 2-16 of histone H3 protein, and H-AKADEVSLHKWYG-NH2 (SEQ ID NO: 2) (P2) was the peptide sequence for research.

[0244] The peptide sequences were synthesized under standard solid-phase peptide synthesis conditions, whereby the synthesis resin was treated with acetic anhydride and pyridine after each amino acid coupling to block unreacted amino groups. Linker X1 was coupled to P1 on the resin using 4 equivalents of linker, 6 equivalents of oxymer, and 6 equivalents of diisopropylamine (DIEA) in DMF for 2 hours. The method of the present invention is shown in Figure 1. Linker X2 was coupled to P1 and P2 on the resin using 4 equivalents of linker, 6 equivalents of oxymer, and 6 equivalents of DIEA for 2 hours. The peptide was then cleaved from the synthesis resin with a mixture of TFA / PhOH / PhSH / HO / ethanedithiol (EDT, 82.5:5:5:2.5). The crude peptide mixture was dissolved in dimethyl sulfoxide (DMSO). The aldehyde-modified agarose beads were washed three times each with water and 0.1 M Na-citrate buffer (pH 4.5). A DMSO solution of the peptide was added to 10% (v / v) sodium citrate buffer, and the solution was then applied to agarose beads for 90 minutes. The peptide was quantitatively immobilized on the agarose beads. To remove acetylated terminal sequences and other impurities, the beads were washed three times with 8 M urea, DMSO, EtOH / water (7:3), 0.1 M NaCl, water, and MeCN. The immobilized linker was cleaved by treating the agarose resin with 50 mg of PPh3 per mL of MeCN / AcOH (9:1). The support was then rinsed four times with MeCN and a solution of HO / MeCN / TFA (70:29:1) was added over 180 minutes. The supernatant was then filtered into a centrifuge tube, and the support was rinsed three times with TFA / HO (9:1) into the same tube. The peptide was precipitated by adding 10x EtO, centrifuged, and the organic supernatant was collected.

[0245] The purity of each phase was confirmed using UPLC-MS. UPLC chromatograms of the unpurified (no linker molecule) and purified peptides are shown in Figures 2 and 3, and the results are summarized in Table 2. The identity of the peptides was confirmed by ESI-MS. Purification of 100 μmol of P1 yielded 26 mg (62% recovery) of peptide P1 (originally 41%) using X1 with 93% purity. Use of the X2 linker yielded 21 mg (50% recovery) with 93% purity. P2 was purified on a 5 μmol scale using the X2 linker sample, yielding 4 mg (73% recovery) with 95% purity (originally 55%).

[0246] [Table 2]

[0247] Linker Type 4 Example 2: Purification of Naturally Occurring and Research Peptides, P3, P4, P5, and P6 In the second set, five peptides were synthesized: H-YFTGSEVENVSVNVH-NH2 (SEQ ID NO: 3) (P3) fragment 81-95 of human cytomegalovirus lower matrix phosphoprotein (CMV), H-PSNPFYEALST-NH2 (SEQ ID NO: 4) (P4) fragment 510-520 of human Lemur tyrosine kinase 3 (LMTK3), H-DAEFRHDSGYEVHHQKLVFF-NH2 (SEQ ID NO: 5) (P5) fragment 1-20 of human amyloid beta, and H-CKADEVSMHKWYG-NH2 (SEQ ID NO: 6) (P6) peptide sequence intended for research.

[0248] Peptide sequences P3, P4, P5, and P6 were synthesized on a 100 μmol scale under standard solid-phase peptide synthesis conditions, whereby the resin was treated with acetic anhydride and pyridine after each amino acid coupling to block unreacted amino groups. Linker X1 was coupled to P3, P4, P5, and P6 on the resin using 4 equivalents of linker X1 (301 mg), 6 equivalents of oxime (86 mg), and 6 equivalents of diisopropylamine (DIEA, 105 μL) in 1.3 mL of DMF for 2 hours. The peptides were then cleaved from the resin with a mixture of TFA / PhOH / PhSH / HO / ethanedithiol (EDT, 82.5:5:5:2.5) and precipitated in cold diethyl ether. The crude peptide mixture was dissolved in 4.5 mL of dimethyl sulfoxide (DMSO). Aldehyde-modified agarose beads (1.5 mL of sedimented beads) were washed three times with water and 0.1 M sodium citrate buffer, pH 4.5, respectively. A 10 vol.% (500 μL) solution of 8 M guanidinium chloride in 10% sodium citrate buffer was then added to the agarose beads for 90 minutes, allowing the target peptide to be quantitatively immobilized to the agarose beads. To block unreacted aldehyde groups, a 1 wt.% solution of L-cysteine ​​in 0.1 M sodium citrate buffer, pH 4.5, was then added directly to the immobilization mixture for 15 minutes. The purified medium was then washed three times with DMSO, 6 M guanidinium chloride, 0.1 M NaCl in EtOH / water (7:3), water, and MeCN, respectively, to remove any acetylated terminal sequences and other impurities. Cleavage of the immobilized linker was performed by treating the agarose resin with 10 mL of 50 mg / mL PPh3 in MeCN / AcOH / HO (90:5:5). The support was then rinsed three times with MeCN / HO (9:1) and 2 mL of HO / TFA (60:40) was added for 60 min. 2 mL of TFA was then added to the supernatant, the resulting mixture was filtered into a centrifuge tube, and the support was rinsed twice with TFA / HO (95:5) into the same tube. Precipitation was initiated by adding 5 times the volume of Et2O relative to the TFA-water volume. The peptide was isolated by centrifugation of the tube and treatment of the organic supernatant.

[0249] The purity of each phase was verified using UPLC-MS. The UPLC-chromatograms of the unpurified peptide (without linker molecules) and the purified peptide are shown in Figure 4. The identity of the peptide was confirmed by ESI-MS. The amount of peptide obtained after lyophilization, the calculated recovery, and the UV purity before and after purification are shown in Table 3.

[0250] [Table 3]

[0251] Implementation of Type 1 Linker 1: Purification of Research Peptide P2 The method of the present invention for purifying peptides with type 1 linkers was applied to the peptide H-AKADEVSLHKWYG-NH2 (SEQ ID NO: 2) (P2), which is the peptide sequence intended for study.

[0252] The peptides were synthesized under standard solid-phase peptide synthesis conditions, whereby the resin was treated with acetic anhydride and pyridine after each amino acid coupling to block any unreacted amino groups. Type 1 linker X9 was coupled to P2 on the resin using 4 equivalents of linker, 6 equivalents of oxime, and 6 equivalents of diisopropylamine (DIEA) in dimethylformamide (DMF) for 2 hours. The peptide was then cleaved from the resin with a mixture of TFA / TIS / DTT / HO (84:2:6:8) and precipitated in diethyl ether. 30 mg of the linker-modified peptide was obtained. The method for preparing this linker type 1 is shown in Figure 5. 1.9 mg of the crude peptide mixture was dissolved in 100 μL of dimethyl sulfoxide (DMSO). The aldehyde-modified agarose beads were added to a cartridge containing 75 μL of a 50% bead suspension in HO / EtOH (4:1) slurry. The beads were then washed three times with water and 0.1 M sodium citrate buffer, pH 4.5. A 10 vol.% (10 μL) solution of 8 M guanidinium hydrochloride in sodium citrate buffer was added to the peptide solution in DMSO. 110 μL of this solution was then applied to the agarose beads over 90 min, resulting in quantitative immobilization of the desired peptide to the agarose beads (UPLC analysis of the immobilization supernatant). The immobilization mixture was then filtered for 15 min to block unreacted aldehyde groups and reverse imine formation. After this, 100 μL of a 2 wt.% solution of L-cysteine ​​in 0.1 M sodium citrate buffer, pH 4.5, was added to the beads. The purification medium was then washed three times with 500 μL each of 0.9 M guanidinium hydrochloride in DMSO and 0.1 M NaCl in EtOH / water (7:3) to remove acetylated end sequences and other impurities. Cleavage of the immobilized linker peptide was performed by treating the agarose resin with 200 μL of TCEP (25 mg / mL) per reactor for 30 minutes. The support was then rinsed three times with 0.1% TFA-containing H2O. To release the peptide from the agarose using an amine switch, 200 μL of 0.2 M NH4HCO3 solution at pH 9 was added to the beads over 15 minutes. This deprotonated the piperazinyl moiety and released the peptide.The identity of the desired peptide was confirmed by ESI / MS (Figure 9, Table 4).

[0253] [Table 4]

[0254] Implementation of Type 2 Linker 1: Purification of Research Peptide P2 The method of the present invention for purifying peptides with type 2 linkers was applied to the peptide H-AKADEVSLHKWYG-NH2 (SEQ ID NO: 2) (P2), which was the peptide sequence intended for study.

[0255] The peptides were synthesized under standard solid-phase peptide synthesis conditions, whereby the resin was treated with acetic anhydride and pyridine after each amino acid coupling to block unreacted amino groups. Linker X13 was coupled to P2 on the resin using 4 equivalents of linker, 6 equivalents of oxime, and 6 equivalents of diisopropylamine (DIEA) in dimethylformamide (DMF) for 3 hours. This method for linker type 2 is shown in Figure 6. The peptide was then cleaved from the resin with a mixture of TFA / TIS / DTT / HO (84:2:6:8). The crude peptide mixture was dissolved in dimethyl sulfoxide (DMSO). The aldehyde-modified agarose beads were washed three times each with water and 0.1 M sodium citrate buffer, pH 4.5. 10% (v / v) of 6 M GdmCl in sodium citrate buffer was added to the DMSO solution of the peptide. The resulting solution was added to agarose beads, and the mixture was shaken for 90 minutes to quantitatively immobilize the desired peptide onto the agarose beads. The supernatant was removed, and the residue was treated with 1% (wt%) L-Cys in Na-citrate buffer for 15 minutes. The beads were then washed three times each with DMSO, 6 M aqueous GdmCl, EtOH / 0.1 M NaCl (7:3), water, MeCN, and 0.1% (v / v) TFA in EtOH to remove acetylated end sequences and other impurities. Cleavage of the immobilized linker was carried out by treating the agarose resin with 10 equivalents of SnCl2 in 0.5 M EtOH for 3 hours. The support was then washed three times each with 0.1% (v / v) TFA in EtOH, 0.1% (v / v) TFA in HO, and MeCN / HO (9:1). A solution of 0.2 M aqueous NH4HCO2 (pH 8.85) / MeCN (1:1) was added over 15 min. The supernatant was filtered into a centrifuge tube, and the support was rinsed twice with HO into the same tube. The residue was lyophilized to obtain the purified peptide.

[0256] The purity of each step was verified using UPLC-MS. The UPLC-chromatograms of the unpurified peptide (without linker molecules) and the purified peptide are shown in Figure 9. The identity of the peptide was confirmed by ESI-MS (Table 5). Using X13 and 50 μmol of crude P2 in the purification experiment, 36 mg (73% recovery) of peptide P2 was obtained with a final purity of 97% (originally 77%).

[0257] [Table 5]

[0258] Implementation of Type 3 Linker 1: Purification of Research Peptide P2 The method of the present invention for purifying peptides with type 3 linkers was applied to the peptide H-AKADEVSLHKWYG-NH2 (SEQ ID NO: 2) (P2), which is the peptide sequence intended for study.

[0259] The peptide was synthesized under standard solid-phase peptide synthesis conditions, whereby the resin was treated with acetic anhydride and pyridine after each amino acid coupling to block any unreacted amino groups. Type 3 linker X22 was coupled to P2 on the resin using 4 equivalents of linker, 3.6 equivalents of 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), 4 equivalents of oxime, and 8 equivalents of diisopropylamine (DIEA) in dimethylformamide (DMF) for 2 hours. The peptide was then cleaved from the resin with a mixture of TFA / TIS / DTT / HO (84:2:6:8) and precipitated in diethyl ether. 27 mg of the linker-modified peptide was obtained. The linker type 3 synthesis method of the present invention is shown in Figure 7. 2.1 mg of the crude peptide mixture was dissolved in 100 μL of dimethyl sulfoxide (DMSO). The aldehyde-modified agarose beads were added to a cartridge containing 75 μL of a 50% bead suspension in HO / EtOH (4:1) slurry. The beads were then washed three times with water and 0.1 M sodium citrate buffer, pH 4.5. A 10 vol.% (10 μL) solution of 8 M guanidinium hydrochloride in sodium citrate buffer was added to the peptide solution in DMSO. 110 μL of this solution was then applied to the agarose beads over 90 min, resulting in quantitative immobilization of the desired peptide to the agarose beads (UPLC analysis of the immobilization supernatant). The immobilization mixture was then filtered for 15 min to block unreacted aldehyde groups and reverse imine formation. After this, 100 μL of a 2 wt.% solution of L-cysteine ​​in 0.1 M sodium citrate buffer, pH 4.5, was added to the beads. The purified medium was then washed three times with 500 μL each of 0.9 M guanidinium chloride in DMSO and 0.1 M NaCl in EtOH / water (7:3) to remove acetylated end sequences and other impurities. Cleavage of the immobilized linker peptide was performed by treating the agarose resin with 200 μL of TCEP (25 mg / mL) per reactor for 30 minutes. The support was then rinsed three times with 0.1% TFA in HO.The peptide was released from the agarose by using an amine switch with nucleophilic release, thus 200 μL of 0.2 M NEt3 in aqueous solution at pH 7 was added to the beads for 100 h. This resulted in the release of piperazinyl and aniline-NH3. + The moiety was deprotonated, followed by nucleophilic attack of aniline-NH2 to release the peptide. ESI / MS confirmed the identity of the desired peptide (Figure 9, Table 6).

[0260] [Table 6]

[0261] Chemical synthesis of carbamate switch (type 4) linker molecules X1 and X2 Synthetic steps for synthesizing 2-((2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3-bromobenzyl(4-nitrophenyl)carbonate (X1)

[0262] 6-Amino-7-bromophthalide To a cooled solution (0 °C) of 6-aminophthalide (5.13 g, 34.05 mmol) in THF (80 mL) was added N-bromosuccinimide (6.12 g, 34.05 mmol, 1 equiv.). The cooling bath was removed, the solution was stirred for 1 h, and then the solvent was removed under reduced pressure. The yellow residue was taken up in ethyl acetate (400 mL) and washed three times with water (200 mL each). The organic phase was dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure to give 6-amino-7-bromophthalide as a brown solid (6.53 g, 28.63 mmol, 84%). R f = 0.2 (cyclohexane / ethyl acetate 2:1); UPLC-MS: t R = 1.45 min (gradient 10-90% B in 5 min); UPLC purity (210 nm) = 83.1%; ESI-MS: (calculated M + :227.97, 229.96,Detection:228.01, 230.01)

[0263] 6-Azido-7-bromophthalide 6-Amino-5-bromophthalide (5.54 g, 24.17 mmol) was added to cold hydrochloric acid (1 M, 100 mL) at 0 °C. Concentrated sulfuric acid was added dropwise to the cooled suspension with stirring (25 mL) until the solid was completely dissolved. The solution was then further cooled until it reached 0 °C again. A solution of sodium nitrite (3.34 g, 48.34 mmol, 2 equiv.) in water (17 mL) was added slowly (nitrous acid gas will form if the solution is too warm). After stirring for 10 min, a solution of sodium azide (3.14 g, 48.34 mmol, 2 equiv.) in water (20 mL) was added slowly dropwise (Caution: formation of hydrazoic acid). After 30 min, the suspension was extracted with ethyl acetate (200 mL). The aqueous phase was filtered, and the filter cake was washed three times with water (100 mL each) and once with cyclohexane (150 mL). 6-Azido-7-bromophthalide (6.58 g (94%), 24.17 mmol, quantitative) was obtained as a yellow solid. f = 0.3 (cyclohexane / ethyl acetate 2:1); UPLC-MS: t R = 2.24 min (gradient 10-90% B in 5 min); UPLC purity (210 nm) = 50.3%

[0264] N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide 6-Azido-7-bromophthalide (5.54 g (94%), 24.17 mmol) was taken up in acetonitrile (150 mL), and the suspension was heated to 50 °C with stirring. Ethylenediamine (23.4 mL, 350.47 mmol, 14.5 equiv.) was added, so that after 10 min the solid was completely dissolved. After stirring at 50 °C for 1 h, the solvent and excess ethylenediamine were removed under reduced pressure to give a red oil (8.49 g) as a residue. Saturated brine (80 mL) was added, and the resulting suspension was sonicated for 30 min, stirred at 40 °C for 30 min, and filtered. The filter cake was washed once with saturated brine (50 mL) and once with cyclohexane (100 mL). After drying the filter cake, the title compound was obtained as a yellow solid (3.84 g, 12.2 mmol, 50.6%). Product 3 was also obtained from the filtrate by extraction with ethyl acetate (6 times with 150 ml each time) (4.79 g, 15.22 mmol, 63.1%). f = 0.1 (DCM / MeOH 8:2); UPLC-MS: t R = 1.03 min (gradient 10-90% B in 5 min); UPLC purity (210 nm) = 83.5%; ESI-MS: (calculated MNa + : 336.01, 338.01 g / mol, detected: 335.95, 337.96 m / z)

[0265] N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide To a stirred solution of bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)AOAc-OH, 4.71 g, 15.86 mmol, 1.3 equiv.) and NHS (1.84 g, 15.86 mmol, 1.3 equiv.) in acetonitrile (40 mL) was added dicyclohexylcarbodiimide (DCC, 3.30 g, 15.86 mmol, 1.3 equiv.). After stirring for 1 h, the solution was separated from the resulting white precipitate by filtration, and the filter cake was washed with acetonitrile (40 mL). The filtrate was diluted to 120 mL with acetonitrile. N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide (3.79 g, 12.06 mmol, 1 equiv.) is taken up in acetonitrile (30 mL) and the suspension is sonicated for 50 min. The filtrate with (Boc)2AOAc-NHS is then added to this suspension and the reaction mixture is stirred for 2.5 h. After removing the solvent under reduced pressure, ethyl acetate (150 ml) is added to the resulting orange oil (10.47 g) and the suspension is sonicated for 10 min and stirred at 50 °C for 10 min. After washing the suspension (three times with 80 mL of 5 wt% NaHCO3 solution (pH 8), once with 80 mL of 2% citric acid solution (pH 4.5), and twice with 80 mL of brine), the organic phase was separated, dried over magnesium sulfate, and the solvent was removed under reduced pressure to give a yellow foam as the crude product (6.84 g). After drying the crude product under high vacuum, the product was obtained as a yellow solid (6.43 g, 75.89% purity (as determined by UV / vis), 8.31 mmol, 68.91% yield). c = 0.15 (DCM / MeOH 95:5); UPLC-MS: t R = 2.60 min (10–90% MeCN in 3 min), UPLC purity (21 nm) = 79.1%, ESI-MS: (calculated MNa + : 609.13, 611.13 g / mol, detected: 609.03, 611.06 m / z)

[0266] 2-((2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3-bromobenzyl(4-nitrophenyl)carbonate N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide (6.39 g (79%), 8.26 mmol) was dissolved in DCM (20 mL) and cooled to 0 °C. To this solution, anhydrous pyridine (1.00 mL, 12.48 mmol, 1.5 equiv.) was first added with stirring, followed by the slow addition of a solution of p-nitrophenyl chloroformate (2.52 g, 12.48 mmol, 1.5 equiv.) in DCM (20 mL). The reaction mixture was allowed to warm to room temperature and stirred for 1 h. The solvent was removed under reduced pressure, and the resulting orange oil (9.99 g) was dissolved in 150 mL of ethyl acetate. The suspension was filtered, and the solvent was removed from the filtrate under reduced pressure to give a yellow foamy solid as the crude product (8.85 g). After purification by column chromatography (silica gel, cyclohexane:ethyl acetate 2:1 to 1:1), the product (3.82 g) was taken up in 100 mL of diethyl ether, sonicated for 10 min, stirred at 40 °C for 30 min, and then stored at -20 °C overnight. The product was filtered, washed with 100 mL of cold diethyl ether at -20 °C, and dried under high vacuum to a pale yellow solid (2.47 g, 3.29 mmol, 39.5%).

[0267] R f =0.25 (ethyl acetate / cyclohexane 2:1), UPLC-MS:t R = 3.18 min (10–90% MeCN in 5 min), UPLC purity (278 nm) = 88.4%, ESI-MS: (calculated MNa + : 774.13, 776.13 g / mol, detected: 773.91, 775.88 m / z)

[0268] 1 H NMR (500MHz, DMSO) δ 8.71(s,1H),8.32(d,J=9.2 Hz,2H),7.95(s,1H),7.62(d,J=8.3Hz,1H),7.57(d,J=9.2Hz,1H),7.52(d,J=8.3Hz,1H),5.25(s,2H),4.36(s,2H),3.33(m,4H),1.46(s,18H)

[0269] 2-((2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3,5-dibromobenzyl(4-nitrophenyl)carbonate (X2) 5,7-Dibromo-6-aminophthalide 6-Aminophthalide (20.00 g, 132.75 mmol) was placed in a 1 L round-bottom flask equipped with a stir bar, and 550 mL of THF and 30 mL of MeCN were added at 0 °C. After the solution turned brown, N-bromosuccinimide was slowly added as a solid through a powder funnel. The ice bath was removed, and the solution turned yellow after a while. After stirring at room temperature for 2 h, UPLC-MS and TLC showed complete conversion to the dibromide. The solvent was removed under reduced pressure on a rotary evaporator. The remaining solid was dissolved in 600 mL of ethyl acetate and washed three times with water. The organic phase was dried over MgSO4, and after evaporation, 39.86 g (129.86 mmol, 98%) of the desired product was obtained as a pale yellow solid. R f =0.6 (cyclohexane / ethyl acetate 2:1), UPLC-MS:t R = 2.36 min (10-90% MeCN in 3 min), UPLC purity (254 nm) = 87.0%, ESI-MS: (calculated MH + : 307.95g / mol, detection: 307.76m / z)

[0270] 55,7-Dibromo-6-azidophthalide 5,7-Dibromo-6-aminophthalide (38.50 g, 124.18 mmol) was dissolved in 200 mL of concentrated H2SO4 in a 2 L flask. The brown solution was cooled in a large ice bucket, and then 235 mL of 1 M HCl was slowly added. A precipitate formed during the HCl addition. NaNO2 (17.31 g, 248.35 mmol, 2 equiv.) was dissolved in 32 mL of water and slowly added to the suspension after it reached 5 °C. The precipitate then dissolved. The solution was stirred for an additional 15 min at 0 °C, after which NaN3 (16.31 g, 248.35 mmol, 2 equiv.) in 75 mL of water was added dropwise via a Pasteur pipette. Strong formation of gas (N2, HN3) was observed. The foamy solution was stirred for 1 h, after which 500 mL of water was added under ice cooling. After the bubbling ceased and the solution reached room temperature, the suspension was filtered using a Buchner funnel, while 2 L of water was used to transfer the solid to the funnel, thereby washing the solid. After drying the wet product under reduced pressure in a crystallizing dish, the product was obtained as a slightly brownish solid (36.01 g, 108.16 mmol, 87.1%). f =0.45 (cyclohexane / ethyl acetate 2:1), UPLC-MS:t R = 2.89 min (10 to 90% MeCN in 3 min), UPLC purity (254 nm) = 95.3%,

[0271] N-(2-aminoethyl)-3-azido-2,4-dibromo-6-(hydroxymethyl)benzamide 5,7-Dibromo-6-azidophthalide (18.00 g, 53.52 mmol) was dissolved in ethyl acetate (490 mL), insoluble impurities were filtered off, and ethylenediamine (52.73 mL, 749.32 mmol, 14 equiv.) was added at 0 °C. The reaction was stirred at room temperature for 1 h, after which UPLC-MS showed quantitative conversion. The reaction mixture was transferred to a separatory funnel to which 100 mL of brine was added. After separating the aqueous phase, the organic phase was dried over MgSO4, and the desired product was obtained after evaporation of the organic solvent on a rotary evaporator as an orange solid (20.50 g, 52.16 mmol, 97.4%). f =0.25(DCM / MeOH 8:2), UPLC-MS:tR = 1.80 min (10–90% MeCN in 3 min), UPLC purity (254 nm) = 84.2%, ESI-MS: (calculated MH + : 393.93 g / mol, detection: 393.87 m / z)

[0272] N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2,4-dibromo-6-(hydroxymethyl)benzamide Bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)2AOAcOH, 17.30 g, 58.18 mmol, 1.1 equiv.) and N-hydroxysuccinimide (NHS, 6.76 g, 58.18 mmol, 1.1 equiv.) were dissolved in 350 mL of acetonitrile. To this solution, dicyclohexylcarbodiimide (DCC, 12.13 g, 58.18 mmol, 1.1 equiv.) was added as a solid, and a white precipitate formed after dissolution of DCC. The reaction mixture was stirred at room temperature for 1 h, whereupon (Boc)2AOAc-NHS ester was quantitatively formed according to UPLC-MS. The mixture was then filtered into a 1 L flask to remove the DCC-urea. N-(2-aminoethyl)-3-azido-2,4-dibromo-6-(hydroxymethyl)benzamide (20.5 g, 52.90 mmol) was dissolved in 530 mL of ethyl acetate and then added to the solution of (Boc)2AOAc-NHS. The mixture was stirred at room temperature for 1 hour, after which the completion of the reaction was confirmed by TLC and UPLC-MS. The additional precipitate formed was filtered off, and the organic phase was washed twice with 5% NaHCO3 (200 mL each), once with brine, and twice with 2% citric acid solution (pH 4.5) / brine 1:1 (150 mL each). The organic phase was dried over MgSO4, and the organic solvent was then removed in vacuo to give the title compound as a pale yellow oil (37.70 g, 56.58 mmol, quantitative). R f =0.4(DCM / MeOH 95:5), UPLC-MS:t R = 2.97 min (10–90% MeCN in 3 min), UPLC purity (254 nm) = 54.2%, ESI-MS: (calculated MH + :667.05, MNa +: 689.04 g / mol, detection: 688.95 m / z)

[0273] 2-((2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3,5-dibromobenzyl(4-nitrophenyl)carbonate N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2,4-dibromo-6-(hydroxymethyl)benzamide (37.70 g (94%), 52.90 mmol) was placed in CHCl (170 mL) and pyridine (4.72 mL, 58.50 mmol, 1.1 equiv.) (dried and placed over molecular sieves) was added. 4-Nitrophenyl chloroformiate (12.03 g, 58.50 mmol, 1.1 equiv.) was then added slowly as a solid at room temperature, keeping the temperature constant with the use of a water bath. The reaction may have caused evaporation of DCM in the center of the flask. LCMS and TLC showed complete reaction after 1 h. The dichloromethane was removed in vacuo to give 52 g of a crude brown oil. The residue was dissolved in 500 mL of ethyl acetate and washed twice with 2% citric acid solution (pH 4.5) / brine 1:1 (250 mL each) and once with 150 mL of brine. The organic phase was dried over MgSO4. The suspension was then filtered through a 50 g silica plug on a glass frit, with orange and reddish impurities remaining on the silica. The organic solvent was removed from the filtrate under reduced pressure until a thick, slightly amber oil remained. 70 mL of Et2O was added to the oil, and the two-phase emulsion was spun on a rotary evaporator at 45 °C for 10 minutes until a single homogeneous phase formed. A small sand corn was added to the flask as a crystallization initiator, and the flask was placed in the refrigerator overnight (16 hours). A thick precipitate had formed in the flask. An additional 200 mL of cold (-25°C) ether was added to the flask, and the flask was gently shaken and stirred in an ice bath. The white, star-like crystals were transferred to a filter-filled Buchner funnel and washed with an additional 200 mL of cold EtO. Thus, the title compound was obtained as a white solid (29.95 g, 36.02 mmol, 68.1%). f =0.6 (ethyl acetate / cyclohexane 2:1), UPLC-MS:t R = 2.86 min (30 to 95% MeCN in 3 min), UPLC purity (278 nm) = 93.5%, ESI-MS: (calculated MH + :832.06, MNa +: 854.04g / mol, detected: 853.87m / z)

[0274] 1 H NMR(400 MHz,DMSO) δ 8.73(s,1H),8.33(d,J=9.1Hz,2H),7.93(s,1H),7.57(d,J=9.1Hz,2H),5.24(s,2H),4.36(s,2H),3.40-3.32(m,4H),1.46(s,18H)

[0275] Chemical synthesis of amine switches bearing azide reduction safety lock (type 1) linker molecules X6 and X9 Synthetic steps for the synthesis of 3-((4-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)methyl)-4-azidobenzyl(4-nitrophenyl)carbonate (X6)

[0276] 4-Nitro-3-(piperazin-1-ylmethyl)benzoic acid 4-Nitro-3-methylbenzoic acid methyl ester (5.03 g, 25.50 mmol) was dissolved in 170 mL of dry benzene in a 500 mL round-bottom flask equipped with a stir bar. N-Bromosuccinimide (5.27 g, 29.33 mmol) and benzoyl peroxide (0.62 g, 2.55 mmol) were added, and the solution was heated to reflux. After 12 h, UPLC-UV / vis indicated only 10% conversion to the brominated starting material. Again, N-Bromosuccinimide (3.66 g, 20.56 mmol) and benzoyl peroxide (0.62 g, 2.55 mmol) were added. After 36 h, UPLC-UV / vis indicated 80% conversion. The mixture was concentrated in vacuo, and 85 mL of chloroform was added. It was slowly filtered into a mixture of piperazine (8.87 g, 102.00 mmol) and K2CO3 (4.63 g, 33.15 mmol) in 85 mL of chloroform in a 500 mL round-bottom flask equipped with a stir bar. After 2 h, UPLC-UV / vis showed complete conversion to the desired product. The solvent was removed on a rotary evaporator. 250 mL of ethyl acetate was added to the residue, and the mixture was filtered into a separatory funnel. The organic phase was washed three times with 100 mL of saturated NaHCO3 and three times with 100 mL of brine. After drying the organic phase over MgSO4 and evaporation, 7.3 g (not completely dried) of the desired product was obtained as a yellow oil. UPLC-MS: R = 1.602 min (10~90% MeCN in 3 min), UPLC purity (278 nm) = 68.3%, ESI-MS: (calculated MH + : 280.12 g / mol, detection: 280.11 m / z)

[0277] (4-nitro-3-(piperazin-1-ylmethyl)phenyl)methanol 4-Nitro-3-(piperazin-1-ylmethyl)benzoic acid (1.10 g, estimated 3.00 mmol) was dissolved in 8 mL of THF in a 250 mL round-bottom flask and stirred at room temperature using a magnetic stirrer. LiCl (0.77 g, 18.00 mmol), NaBH4 (0.69 g, 18.00 mmol), and 16 mL of ethanol were added sequentially. After 12 h, UPLC-UV / visible analysis indicated complete conversion of the starting material. The reaction mixture was concentrated in vacuo, and the residue was suspended in 60 mL of ethyl acetate. 25 mL of 1 M NH4Cl was added dropwise while the mixture was rapidly stirred. After 2 h, 25 mL of 1 M NaOH was slowly added, and the mixture was transferred to a separatory funnel. The layers were separated, and the aqueous phase was extracted twice with 50 mL of ethyl acetate and twice with 50 mL of chloroform. After drying the combined organic layers over MgSO4 and evaporating the organic solvent, the desired product was obtained as a yellow solid (0.59 g, 2.35 mmol, 88%). UPLC-MS: R = 1.27 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 50.4%, ESI-MS: (calculated MH + : 252.29 g / mol, detection: 252.17 m / z)

[0278] 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazin-1-yl)ethan-1-one In a 100 mL round-bottom flask, Bis-Boc-protected aminooxyacetic acid (0.78 g, 2.63 mmol) and N-hydroxysuccinimide (0.31 g, 2.63 mmol) were dissolved in 25 mL of acetonitrile and stirred with a magnetic stirrer. Dicyclohexylcarbodiimide (0.55 g, 2.63 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After filtering into a dropping funnel, it was slowly added to a solution of 4-nitro-3-(piperazin-1-ylmethyl)benzyl alcohol (0.59 g, 2.35 mmol) in 25 mL of chloroform in a 250 mL round-bottom flask. After 1 hour, UPLC-UV / visible analysis indicated complete conversion of the starting material. The solvent was removed on a rotary evaporator, and the residue was suspended in 100 mL of ethyl acetate and transferred to a separatory funnel. The organic phase was washed with 3 x 50 mL of water, 3 x 50 mL of saturated NaHCO3 solution, 3 x 50 mL of brine, and dried over MgSO4. After evaporation of the organic solvent, the desired product was obtained as a yellow solid (1.8 g, not completely dried). UPLC-MS: R = 2.24 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 19.8%, ESI-MS: (calculated MH + : 525.14 g / mol, detection: 525.25 m / z)

[0279] 1-(4-(2-amino-5-(hydroxymethyl)benzyl)piperazin-1-yl)-2-bis-(tert-butoxycarbonyl)-(aminooxy)ethan-1-one In a round-bottom flask, 2-bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazin-1-yl)ethan-1-one was dissolved in 16 mL of water / ethanol (1:4). Iron powder (0.23 g, 4 mmol) and ammonium chloride (0.25 g, 4 mmol) were then added to the solution. The reaction mixture was stirred overnight at room temperature. After confirming complete consumption of the starting material via UPLC-MS, the reaction mixture was filtered through Celite to remove excess iron. The solvent was concentrated in vacuo to the extent possible. After adding 50 mL of CHCl3, the organic phase was washed three times with 350 mL of saturated NaHCO3 solution and three times with 50 mL of brine, dried over MgSO4, and then the organic solvent was removed under reduced pressure on a rotary evaporator. The final product (0.19 g, 0.4 mmol) was obtained as a brown oil. UPLC-MS: R = 2.05 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 24.7%, ESI-MS: (calculated MH + :495.28, MNa + : 517.26 g / mol, detection: 517.29 m / z)

[0280] 2-Bis(tert-butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)benzyl)piperazin-1-yl)ethan-1-one In a round-bottom flask, 1-(4-(2-amino-5-(hydroxymethyl)benzyl)piperazin-1-yl)-2-bis-(tert-butoxycarbonyl)-(aminooxy)ethan-1-one (0.19 g, 0.4 mmol) was dissolved in dry acetonitrile. With stirring, the solution was cooled in an ice bath, and tert-butylnitrile (236 μL, 2 mmol) was added slowly, followed by trimethylsilyl azide (351 μL, 1.6 mmol). The solution was further stirred in a sealed flask for 2 h with ice cooling. After UPLC-MS showed incomplete conversion, tert-butylnitrile (572 μL, 4 mmol) was added slowly again, followed by trimethylsilyl azide (702 μL, 3.2 mmol). The reaction mixture was stirred for an additional 2 h. The solvent was then removed under reduced pressure on a rotary evaporator. The crude product was dissolved in 50 mL of ethyl acetate, and the organic phase was washed three times with 50 mL of NaHCO3, three times with 50 mL of brine, and dried over magnesium sulfate. After evaporation of the organic solvent, the desired product (0.14 g, 0.20 mmol) was obtained. UPLC-MS: R = 2.34 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 20.4%, ESI-MS: (calculated MH + : 521.27, detection: 521.30 m / z)

[0281] 3-((4-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)methyl)-4-azidobenzyl(4-nitrophenyl)carbonate (X6) 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)benzyl)piperazin-1-yl)ethan-1-one (149 mg, 0.2 mmol) was dissolved in 0.2 mL of dry DCM in a round-bottom flask. Pyridine (19.2 μL, 0.24 mmol) was added, and the solution was cooled using an ice bath. To this mixture, a solution of p-nitrophenyl chloroformate (32.9 mg, 0.16 mmol) in 0.2 mL of dry DCM was slowly added, and the reaction mixture was stirred at room temperature overnight. The organic solvent was removed in vacuo to give the desired product as a dark brown oil (0.21 g, not completely dried). UPLC-MS: t R = 10.72 min (0–60% MeCN in 11 min), UPLC purity (278 nm) = 8.39%, ESI-MS: (calculated MH + :686.28, MNa + : 708.26 g / mol, detected: 686.38 m / z)

[0282] Synthetic steps for the synthesis of 3-(4-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)-4-azidobenzyl(4-nitrophenyl)carbonate (X9) 1-(4-(2-amino-5-(hydroxymethyl)phenyl)piperazin-1-yl)-2-bis-(tert-butoxycarbonyl)-(aminooxy)ethan-1-one 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethan-1-one (1.00 g, 1.96 mmol) was dissolved in 78 mL of methanol in a round-bottom flask. To this solution was added magnesium powder (1.28 g, 19.6 mmol). Under stirring, ammonium formate (1.23 g, 19.6 mmol) was added as a solid. The solution was further stirred at room temperature for 20 minutes, during which gas formation was observed. The solution was immediately filtered to remove the magnesium, and the solvent was removed under reduced pressure on a rotary evaporator. The crude product was lyophilized using a 1:1 solvent mixture of water / acetonitrile. The crude product (2.20 g) was used without any further purification. UPLC-MS: R = 2.21 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 53.2%, ESI-MS: (calculated MH + : 481.27g / mol, MNa + : 503.25 g / mol, detection: 477.30 m / z)

[0283] 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)phenyl)piperazin-1-yl)ethan-1-one In a round-bottom flask, 1-(4-(2-amino-5-(hydroxymethyl)phenyl)piperazin-1-yl)-2-bis-(tert-butoxycarbonyl)-aminooxy)ethan-1-one (1.00 g, 2.08 mmol) was dissolved in dry acetonitrile. Under stirring, the solution was cooled in an ice bath, and tert-butylnitrile (1.37 mL, 10.4 mmol) was added slowly, followed by trimethylsilyl azide (1.16 mL, 8.32 mmol). The solution was further stirred in a sealed flask for 2 h with ice cooling. After UPLC-MS showed incomplete conversion, tert-butylnitrile (1.37 mL, 10.4 mmol) was added slowly again, followed by trimethylsilyl azide (1.16 mL, 8.32 mmol). The reaction mixture was further stirred overnight. The solvent was then removed under reduced pressure on a rotary evaporator. After dissolving the crude product in 50 mL of ethyl acetate, the organic phase was washed with 50 mL of NaHCO3 three times, 50 mL of brine three times, and dried over magnesium sulfate. After evaporation of the organic solvent and purification by flash column (3:2 EtOAC / cyclohexane), the desired product (0.2 g, 0.39 mmol) was obtained. f =0.5 (EtOAc / cyclohexane 4:1), UPLC-MS:t R = 2.92 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 84.2%, ESI-MS: (calculated MH + : 507.26g / mol, MNa + : 529.24 g / mol, detection: 529.30 m / z)

[0284] 3-(4-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)-4-azidobenzyl(4-nitrophenyl)carbonate (X9) 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)phenyl)piperazin-1-yl)ethan-1-one (76 mg, 0.15 mmol) was dissolved in 1 mL of dry DCM in a round-bottom flask. Pyridine (12.2 μL, 0.15 mmol) was added, and the solution was cooled using an ice bath. To this solution, p-nitrophenyl chloroformiate (20.3 mg, 0.15 mmol) was slowly added as a solid. After 10 min, the ice bath was removed, and the reaction mixture was stirred at room temperature overnight. The organic solvent was removed in vacuo, and the crude product was redissolved in 30 mL of EtOAc. The organic phase was then washed three times with saturated NaHCO3 solution and three times with brine, dried over MgSO4, and the organic solvent was again removed in vacuo. The crude product was finally purified by flash column chromatography using an EtOAc / cyclohexane (1:1) solvent mixture. The desired compound was obtained as a pale yellow oil (30 mg, 0.05 mmol). f = 0.54 (EtOAC / cyclohexane 1:1), UPLC-MS:t R = 3.53 min (10 to 90% MeCN in 3 min), UPLC purity (278 nm) = 93.6%, ESI-MS: (calculated MH + :671.66, MNa + : 694.24 g / mol, detection: 694.40 m / z)

[0285] 1 H-NMR(400MHz, CDCl3,25 °C):δ[ppm]=8.28(d,J=9.0Hz,2H),7.38(d,J=9.0Hz,2H),7.22-7.06(m,3H),5.22(s,2H),4.62(s,2H) ),3.94(t,J=3.93,2H),3.81(t,J=3.81,2H),3.12(t,J=3.12,2H),3.07(t,J=3.07,2H),1.54(s,18H)

[0286] Chemical synthesis of amine switches with other reductive safety-lock (type 2) linker molecules X12, X13, and X43 Synthesis of (5-(2-(2-bis-(tert-butoxycarbonyl(aminooxy)acetamido)-6-(tert-butyldisulfanyl)pyridin-3-yl)methyl(4-nitrophenyl)carbonate (X12) Synthesis of methyl 6-meracapto-5-nitropyridine-3-carboxylate To a cooled solution of methyl 6-chloro-5-nitropyridine-3-carboxylate (1.6 gm, 7.037 mmol) in methanol (25 ml), 70% sodium hydrogen sulfide hydrate (1.115, 14.127 mmol) was added portionwise. The mixture was left to stand with stirring for 30 minutes to 1 hour. The solid material was then filtered. The remaining solution was reduced to 5 ml using a rotary evaporator. The remaining solution was acidified to pH 2 by slow addition of 1 M HCl at 0°C. The resulting yellow solid material was collected by filtration, and the material was used in further steps without further purification. UPLC-MS: R = 1.80 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 95.0%, ESI-MS: (calculated MH + : 215.01 g / mol, detection: 215.20 m / z)

[0287] Synthesis of methyl 6-meracapto-5-aminopyridine-3-carboxylate Methyl 6-meracapto-5-nitropyridine-3-carboxylate (1.00 gm, 4.537 mmol) and 1.85 gm (32.48 mmol) of iron powder were placed in a reaction flask containing 50 mL of 75% methanol and 25% water. Calcium chloride (0.41 gm, 3.63 mmol) was then added, and the mixture was refluxed on an oil bath until complete conversion of the starting material to the product. After refluxing was complete, the mixture was filtered through Celite to remove excess iron. The filtrate was concentrated to near dryness, followed by the addition of water (25 ml), and the compound was extracted using ethyl acetate (3 x 25 ml). The solvent was removed under reduced pressure, and the resulting crude residue was sufficiently pure for further workup (3.49 mmol, 77%). UPLC-MS:t R= 1.26 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 93%, ESI-MS: (calculated MH + : 185.04 g / mol, detection: 185.14 m / z)

[0288] Synthesis of (5-amino-6-meracaptopyridin-3-yl)methanol Methyl 6-meracapto-5-aminopyridine-3-carboxylate (0.7 gm, 3.68 mmol) was dissolved in dry THF (20 mL), and the solution was cooled to 0°C. Lithium aluminum hydride (1.42 gm, 36.89 mmol) was added portionwise to the reaction mixture under a N2 atmosphere over 10 minutes. The resulting solution was stirred at room temperature for 24 hours. After the reaction was complete, as shown by UPLC, excess LiAlH4 was quenched by simultaneously adding 1.4 mL of water, 1.4 mL of 10% NaOH, and 4.2 mL of water at 0°C. The Al salts were filtered off, and the solid material was washed with water and MeOH (1:1, 100 mL). The solvent was evaporated, and the compound was extracted from the solid cake using isopropanol (3 x 50 mL). The isopropanol was removed under reduced pressure to give the desired product (1.92 mmol, 52%) in pure form. UPLC-MS: R = 1.01 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 96%, ESI-MS: (calculated MH + : 157.04 g / mol, detection: 157.11 m / z)

[0289] Synthesis of (2-(2-bis-(tert-butoxycarbonyl)(aminooxy))-N-(2-(tert-butyldisulfanyl)-5-(hydroxymethyl)pyridin-3-yl)acetamide) To a Schlenk flask containing 6-mercapto-5-aminopyridine-3-carboxylic acid (1.3 gm, 8.03 mmol), CHCl (20 mL), 2-methyl-2-propanethiol (0.91 mL, 8.03 mmol), and TBHP (1.13 gm, 8.83 mmol, 70% solution in water) were added under a N atmosphere. After 30 seconds, NIS (0.19 gm, 0.18 mmol) was added in one batch. The Schlenk flask was then incubated at 25°C for 1 hour. After completion of the reaction, the mixture was quenched with water (20 mL) and extracted with ethyl acetate (3 x 30 mL). The organic layers were combined and evaporated under vacuum, and the compound was used in the next step without further purification. UPLC-MS: R = 1.68 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 93%, ESI-MS: (calculated MH + : 245.08g / mol, detection: 245.18m / z)

[0290] Synthesis of (5-(2-(2-bis-(tert-butoxycarbonyl(aminooxy)acetamido)-6-(tert-butyldisulfanyl)pyridin-3-yl)methyl(4-nitrophenyl)carbonate (X12) (5-Amino-6-(tert-butyldisulfanyl)pyridin-3-yl)methanol (83.5 mg, 0.156 mmol) was placed in CHCl (2 mL) and dry pyridine (0.161 mL, 0.19 mmol) was added. 4-Nitrophenyl chloroformate (31.9 mg, 0.154 mmol) was then added. LCMS and TLC showed the reaction was complete after 12 h. The dichloromethane was removed in vacuo to give a pale yellow / orange crude oil, and the residue was redissolved in 10 mL of ethyl acetate and washed with 1:1 water and brine (5 mL each). Finally, EtoAc was removed under reduced pressure to give the desired product (0.08 mmol, 75%). UPLC-MS: R = 12.59 min (10-60% MeCN at 11.50 min, 60-90% at 11.51-13 min), UPLC purity (= nm) = 70%, ESI-MS: (calculated MNa + : 705.19 g / mol, detected: 705.41 m / z)

[0291] Synthetic steps for the synthesis of 3-(4-(2-bis-(tert-butoxycarbonyl)-((aminooxy)acetyl)piperazin-1-yl)-4-nitrobenzyl (4-nitrophenyl) carbonate (X13) Methyl 3-fluoro-4-nitrobenzoate 3-Fluoro-4-nitrobenzoic acid (5.00 g, 27.01 mmol) was dissolved in 100 mL of methanol in a 250 mL round-bottom flask with a stir bar, and 3 mL (54.02 mmol) of H2SO4 was added. After stirring at 50 °C for 42 h, UPLC-UV / visible indicated complete conversion to the methyl ester. The reaction mixture was concentrated to 10 mL on a rotary evaporator. After adding 100 mL of ethyl acetate and 100 mL of H2O, the mixture was stirred, followed by the portionwise addition of K2CO3 (7.00 g). The mixture was transferred to a separatory funnel, the layers were separated, and the aqueous phase was extracted three times with 100 mL of ethyl acetate. The combined organic layers were dried over MgSO4, and after evaporation, 4.81 g (24.15 mmol, 89%) of the desired product was obtained as an orange solid. UPLC-MS: R = 2.53 min (10–90% MeCN in 3 min), UPLC purity (210 nm) = 99.4%, ESI-MS: (calculated MH + :200.14g / mol, detected:-).

[0292] Methyl 4-nitro-3-(piperazin-1-yl)benzoate Piperazine (4.07 g, 47.20 mmol) and K2CO3 (4.24 g, 30.68 mmol) were suspended in 50 mL of chloroform in a 250 mL round-bottom flask with a stir bar. Methyl 3-fluoro-4-nitrobenzoate (4.7 g, 23.60 mmol) dissolved in 50 mL of chloroform was slowly added via a dropping funnel over 1 h at room temperature, and the mixture was rapidly stirred. After 66 h, UPLC-UV / vis indicated only 10% conversion of the starting material. Piperazine (4.07 g, 47.20 mmol) was added, and after 2 h, UPLC-UV / vis indicated complete conversion. After 15 min, 100 mL of chloroform and 150 mL of saturated NaHCO3 solution were added, and the mixture was transferred to a separatory funnel. The layers were separated and the organic phase was washed twice with 100 mL of saturated NaHCO3 solution and once with 100 mL of brine. After drying over MgSO4 and evaporating the organic solvent on a rotary evaporator, the desired product was obtained as a red solid (7.87 g, not completely dried). UPLC-MS: R = 1.72 min (10–90% MeCN in 3 min), UPLC purity (210 nm) = 87.2%, ESI-MS: (calculated MH + : 266.27g / mol, detected: 266.23m / z)

[0293] (4-nitro-3-(piperazin-1-yl)phenyl)methanol Methyl 4-nitro-3-(piperazin-1-yl)benzoate (5.85 g, not completely dried, estimated 20.00 mmol) was dissolved in 40 mL of THF in a 500 mL round-bottom flask and stirred with a magnetic stir bar. LiCl (5.09 g, 120.00 mmol), NaBH4 (4.54 g, 120.00 mmol), and 80 mL of ethanol were added sequentially. After 15 h, UPLC-UV / vis indicated complete conversion. The reaction mixture was concentrated in vacuo, 20 mL of chloroform was added, and the mixture was rapidly stirred while 30 mL of 2 M NH4Cl solution was added dropwise. After 1 h, 120 mL of 1 M NaOH and 80 mL of chloroform were slowly added, and the mixture was transferred to a separatory funnel. The layers were separated, and the aqueous phase was extracted three times with 100 mL of chloroform. After drying the combined organic layers over MgSO4 and evaporating the organic solvent, the desired product was obtained as an orange solid (3.97 g, 16.73 mmol, 84%). UPLC-MS: R = 1.27 min (10–90% MeCN in 3 min), UPLC purity (210 nm) = 70.0%, ESI-MS: (calculated MH + : 238.26 g / mol, detection: 238.24 m / z)

[0294] 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethan-1-one Bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)2AOAcOH, 2.96 g, 9.96 mmol, 1.2 equiv.) and N-hydroxysuccinimide (NHS, 1.15 g, 9.96 mmol, 1.2 equiv.) were dissolved in 10 mL of acetonitrile. To this solution, dicyclohexylcarbodiimide (DCC, 2.07 g, 9.96 mmol, 1.2 equiv.) was added as a solid, and a white precipitate formed after dissolution of DCC. The reaction mixture was stirred at room temperature for 1 h, whereupon (Boc)2AOAc-NHS ester was quantitatively formed according to UPLC-MS. The mixture was then filtered into a round-bottom flask containing (4-nitro-3-(piperazin-1-yl)phenyl)methanol (1.98 g, 8.30 mmol) in 83 mL of chloroform. The mixture was stirred at room temperature for 1 hour, after which the completion of the reaction was confirmed by TLC and UPLC-MS. The further precipitate formed was filtered off, and the organic phase was washed three times with water, three times with saturated NaHCO3 solution, and three times with brine solution. The organic phase was dried over MgSO4, and the organic solvent was then removed in vacuo to give the title compound as a yellow oil (4.35 g, 8.52 mmol, quantitative, not completely dried). UPLC-MS: R = 3.00 min (10 to 90% MeCN in 3 min), UPLC purity (278 nm) = 48.2%, ESI-MS: (calculated MH + :511.24, MNa + :534.23g / mol, detected:-).

[0295] 3-(4-(2-bis-(tert-butoxycarbonyl)-((aminooxy)acetyl)piperazin-1-yl)-4-nitrobenzyl(4-nitrophenyl)carbonate (X13) 2-Bis-(tert-butoxycarbonyl-aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethan-1-one (2.06 g, 4 mmol) was placed in an 8 mL round-bottom flask equipped with a stir bar. To this solution, dry pyridine was added. Then, 4-nitrophenyl chloroformiate (0.86 g, 4.2 mmol) was added slowly as a solid at room temperature. The reaction was exothermic and may cause the foaming of DCM. After stirring at room temperature for 2 hours, UPLC-MS and TLC showed complete conversion to the carbonate salt. The solvent was removed under reduced pressure on a rotary evaporator. The product was purified by column chromatography (cyclohexane / ethyl acetate 1:1) to give a yellow solid (410 mg, 0.61 mmol). UPLC-MS: R = 3.44 min (10 to 90% MeCN in 3 min), UPLC purity (278 nm) = 92.4%, ESI-MS: (calculated MH + : 676.25g / mol, MNa + : 698.23 g / mol, detection: 698.51 m / z)

[0296] 1 H-NMR(400MHz, CDCl3,25 °C):δ[ppm]=8.29(d,J=9.2Hz,2H),7.84(d,J=8.3Hz,1H),7.39(d,J=9.2Hz,2H),7.19(s,1H),7.16(d,J=9.7Hz,1H),5.2 9(s,2H),4.61(s,2H),3.89(t,J=3.90,2H),3.78(t,J=3.78,2H),3.15(t,J=3.15,2H),3.10(t,J=3.10,2H),1.54(s,18H)

[0297] Synthetic steps for synthesizing 3-((4-(2-(aminooxy)acetyl)piperazin-1-yl)methyl)-4-nitrobenzyl (4-nitrophenyl) carbonate (X43) 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazin-1-yl)ethan-1-one (1.27 g, not completely dried, estimated 1.86 mmol) was dissolved in 5 mL of chloroform in a 50 mL round-bottom flask using a magnetic stir bar and cooled to 0 °C in an ice bath. Dry pyridine (0.23 mL, 0.23 g, 2.88 mmol) and p-nitrophenyl chloroformate (0.59 g, 2.88 mmol) were added sequentially. After x h, UPLC-UV / vis indicated complete conversion of the starting material. The solvent was removed on a rotary evaporator, and the residue was dissolved in 5 mL of ethyl acetate / cyclohexane (1:1). After purification by flash column chromatography using ethyl acetate / cyclohexane (1:1), the desired product (0.02 g, 0.03 mmol) was obtained. UPLC-MS: R = 2.96 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 55.6%, ESI-MS: (calculated MNa + : 712.24 g / mol, detected: 712.41 m / z)

[0298] Chemical synthesis of amine switches bearing nucleophilic release (type 3) linker molecules X22 and X42 Synthesis of 2-(2-azido-5-(4-(2-((2-bis-(tert-butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)phenyl)acetic acid (X22)

[0299] Synthesis of methyl 2-(5-fluoro-2-nitrophenyl)acetate 5-Fluoro-2-nitrophenylacetic acid (3.00 gm, 14.92 mmol) was placed in a 100 ml round-bottom flask with a stir bar. The compound was dissolved in 40 ml of MeOH, and 1.62 ml (29.84 mmol) of H2SO4 was slowly added at room temperature. The resulting reaction mixture was refluxed for 6 hours. The progress of the reaction was monitored by UPLC-MS and TLC. After completion of the reaction, methanol was removed under reduced pressure using a rotary evaporator. 20 ml of water was added to the resulting crude product, and the solution was neutralized with saturated K2CO3 solution (50 ml). The precipitated solid was filtered, washed with water, and dried under reduced pressure to give the desired product as a white solid (129.86 mmol, 98%). UPLC-MS: R = 2.41 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 97%, ESI-MS: (calculated MH + : 214.05g / mol, detected: ...)

[0300] Synthesis of tert-butyl 4-(3-(2-methoxy-2-oxoethyl)-4-nitrophenyl)piperazine-1-carboxylate Methyl 2-(5-fluoro-2-nitrophenyl)acetate (0.70 gm, 3.25 mmol) was dissolved in dry DMF. To this solution, 1-Boc-piperazine (0.83 gm, 4.39 mmol) and Na2CO3 (0.71 gm, 6.5 mmol) were added at room temperature, and the resulting reaction mixture was heated at a maximum temperature of 80°C overnight. The progress of the reaction was monitored by UPLC-MS and TLC. After completion of the reaction, the solution was filtered to remove solid by-products, and the DMF was removed under reduced pressure using a rotary evaporator. Ice-cold water (25 ml) was added to the crude material, and the resulting solid was filtered, washed with water (50 ml), and dried under reduced pressure to give the desired product (1.01 gm, 2.66 mmol, 82%) as a yellow solid UPLC-MS: R = 3.01 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 96.0%, ESI-MS: (calculated MH + : 380.18 g / mol, Detected: 324.10 (M2H + -t-butyl (tbu)).

[0301] Synthesis of tert-butyl 4-(4-amino-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate To a solution of tert-butyl 4-(3-(2-methoxy-2-oxoethyl)-4-nitrophenyl)piperazine-1-carboxylate (1.00 gm, 2.58 mmol) in dioxane / HO (25 mL, 3:1) was added NH4Cl (1.23 gm, 1.01 gm, 18.50 mmol) and zinc dust (1.23 gm, 18.50 mmol) at room temperature. The reaction mixture was stirred at the same temperature for 3 hours, after which it was filtered through a celite bed. The resulting solution was evaporated and the crude material was partitioned between HO (100 mL) and EtOAc (300 mL). The organic layer was separated, dried and concentrated (MgSO4) to give the desired product as a yellow solid (1.75 mmol, 68%). UPLC-MS: R = 2.12 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 94.0%, ESI-MS: (calculated MH + : 350.21 g / mol, detection: 350.19 m / z)

[0302] Synthesis of tert-butyl 4-(4-azido-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate tert-Butyl 4-(4-amino-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (0.35 gm, 0.99 mmol) was dissolved in dry acetonitrile (25 mL) and cooled to 0°C. 90% tert-butyl nitrite (0.837 g, 7.94 mmol) was added dropwise to the reaction mixture, followed by TMSN3 (0.722 g, 5.95 mmol) over 10 minutes. The resulting red mixture was stirred for 3 hours. The reaction progress was monitored by UPLC-MS. After completion of the reaction, excess TMSN3, t-BuONO, and solvent were removed under reduced pressure, and the resulting red residue was dissolved in 50 mL of ethyl acetate and washed with water (2 x 50 mL). The ethyl acetate layer was dried over MgSO4 and removed under reduced pressure to give an orange solid (0.33 gm, 0.88 mmol), which was carried forward without further purification. UPLC-MS: R = 3.01 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 95.0%, ESI-MS: (calculated MH + : 376.20 g / mol, detection: 376.28 m / z)

[0303] Synthesis of 2-(2-azido-5-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)acetic acid A solution of tert-butyl 4-(4-azido-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (0.314 gm, 0.83 mmol), LiOH (0.102 gm, 4.14 mmol), MeOH (5 mL), and HO (0.2 mL) was stirred at room temperature for 3 hours. After complete conversion of the starting material to the product (monitored by UPLC-MS), the reaction mixture was evaporated, and saturated NH4Cl was added to the resulting crude material until the pH of the solution reached 6. The resulting solution was extracted with EtOAc (2 x 25 mL), dried over MgSO4, and concentrated to afford the product as a yellow solid (0.65 mmol, 78%). UPLC-MS: R = 2.55 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 95.0%, ESI-MS: (calculated MH + : 362.18 g / mol, detected: 306.04 m / z)

[0304] Synthesis of 4-(4-azido-3-(carboxymethyl)phenyl)piperazin-1-ium 2,2,2-trifluoroacetate Pure trifluoroacetic acid (0.6 ml, 6.14 mmol) was added dropwise to a round-bottom flask containing 2-(2-azido-5-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)acetic acid (0.22 gm, 0.61 mmol), and the resulting solution was stirred at room temperature for 1 hour. After completion of the reaction (monitored by UPLC-MS), cold ether (25 mL) was added, and the resulting solid was filtered and washed with cold ether. The resulting light brown solid (0.30 gm, 85%) was dried and taken forward to further steps without purification. UPLC-MS: R = 2.80 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 87.0%, ESI-MS: (calculated MH + : 262.13 g / mol, Found: 262.11 m / z) Wrong mass.

[0305] Synthesis of 2-(2-azido-5-(4-(2-((((2-bis-(tert-butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)phenyl)acetic acid (X22) Bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)AOAcOH, 88.4 mg, 0.30 mmol) and N-hydroxysuccinimide (NHS, 34.6 g, 0.30 mmol) were dissolved in 2 mL of dry acetonitrile. To this solution, dicyclohexylcarbodiimide (DCC, 62.0 mg, 0.30 mmol) was added as a solid, and a white precipitate formed after dissolution of DCC. The reaction mixture was stirred at room temperature for 1 h, whereupon the (Boc)AOAc-NHS ester was quantitatively formed according to UPLC-MS. The mixture was then filtered through filter paper directly into a reaction flask containing 4-(4-azido-3-(carboxymethyl)phenyl)piperazin-1-ium 2,2,2-trifluoroacetate (100 mg, 0.20 mmol) in dry DMF (2 mL) and DIEPA (155 μL, 0.89 mmol) to remove DCC-urea. The mixture was further stirred at room temperature for 1-2 h. After completion of the reaction, the solvent was evaporated under reduced pressure, and NH4Cl (25 mL) was added to neutralize the reaction mixture. The compound was extracted with EtOAc (2 x 25 mL), dried over MgSO4, and concentrated to give the product as a dark brown solid (0.19 mmol, 97%). UPLC-MS: R = 2.80 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 87.0%, ESI-MS: (calculated MNa + : 557.23 g / mol, detection: 557.28 m / z) 1 H NMR(400 MHz,)δ 7.06(d,J=8.7Hz,1H),6.90(d,J=8.8Hz,1H),6.83(s,1H),4.60(s,2H),3.90- 3.84(m,2H),3.77-3.72(m,2H),3.59(s,2H),3.24-3.11(m,4H),1.54(s,18H)

[0306] Synthesis of 2-(5-(4-(2-((2-bis-)tert-butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)-2-nitrophenyl)acetic acid (X42) Synthesis of 2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-nitrophenyl)acetic acid To a solution of tert-butyl 4-(3-(2-methoxy-2-oxoethyl)-4-nitrophenyl)piperazine-1-carboxylate (0.50 gm, 1.30 mmol) in MeOH:HO (21.2 mL, 16:1) was added LiOH and stirred at room temperature overnight. After complete conversion of the starting material to the product (monitored by UPLC-MS), the reaction mixture was evaporated and saturated NH4Cl (25 mL) was added to the resulting crude material until the pH of the solution reached 6. The compound was extracted with EtOAc (4 x 25 mL), dried over MgSO4, and concentrated under reduced pressure to give the desired product as a yellow solid (0.78 mmol, 59%). UPLC-MS: R = 2.36 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 97.0%, ESI-MS: (calculated MH + : 380.18 g / mol, detected: 310.04 m / z (2H + -tbu m / z))

[0307] Synthesis of 4-(3-(carboxymethyl)-4-nitrophenyl)piperazin-1-ium 2,2,2-trifluoroacetate Pure trifluoroacetic acid (0.73 ml, 7.59 mmol) was added dropwise to a round-bottom flask containing 2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-nitrophenyl)acetic acid (0.28 gm, 0.76 mmol) at room temperature, and the resulting solution was stirred for 1 hour. After completion of the reaction (monitored by UPLC-MS), cold ether (25 mL) was added and the resulting solid was filtered and washed with cold ether. The resulting yellow solid (88%) was dried and taken forward to further steps without purification. UPLC-MS: R = 1.35 min (10–90% MeCN in 3 min), UPLC purity (278 nm) = 88.9%, ESI-MS: (calculated MH + : 266.11 g / mol, detection: 266.11 m / z)

[0308] Synthesis of 2-(5-(4-(2-((2-bis-)tert-butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)-2-nitrophenyl)acetic acid (X42) Bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)AOAcOH, 88.4 mg, 0.30 mmol) and N-hydroxysuccinimide (NHS, 34.6 g, 0.30 mmol) were dissolved in 2 mL of dry acetonitrile. To this solution, dicyclohexylcarbodiimide (DCC, 62.0 mg, 0.30 mmol) was added as a solid, and a white precipitate formed after dissolution of DCC. The reaction mixture was stirred at room temperature for 1 h, whereupon the (Boc)AOAc-NHS ester was quantitatively formed according to UPLC-MS. The mixture was then filtered through filter paper directly into a reaction flask containing 4-(3-(carboxymethyl)-4-nitrophenyl)piperazin-1-ium 2,2,2-trifluoroacetate (100 mg, 0.20 mmol) in dry DMF (2 mL) and DIEPA (155 μL, 0.89 mmol) to remove DCC-urea. The mixture was stirred at room temperature for 1-2 hours, after which the completion of the reaction was confirmed by UPLC-MS. After completion of the reaction, the solvent was evaporated under reduced pressure, and NHCl (25 mL) was added to neutralize the reaction mixture. The resulting solution was extracted with EtOAc (2×25 mL) and concentrated to give the product as a dark brown solid (0.19 mmol, 97%). UPLC-MS: R = 2.74 min (10-90% MeCN in 3 min), UPLC purity (278 nm) = 87.0%, ESI-MS: (calculated MNa + : 561.22 g / mol, detected: 561.28 m / z).

Claims

1. Formula 1, XT ｂ -V ａ -U-Y-Z(1) The compound of formula 1 is selected from compounds of formulas X5 to X47: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 The compound.

2. 1. A method for purifying a peptide, comprising the steps of: - providing a crude linker-modified peptide, wherein the crude peptide is covalently attached to a linker molecule according to claim 1; - in a coupling step, the crude linker-modified peptide prepared in said step is coupled to a solid support to obtain an immobilized linker-modified peptide; - in a releasing step, the peptide is released by adding a reducing agent under acidic conditions, The method comprising:

3. In the release step, a reduced intermediate characterized by a reduced linker moiety of the immobilized linker-modified peptide is achieved, and the peptide is released from said reduced intermediate by increasing the pH to a pH > pKa relative to the pKa of the most basic heteroatom of the linker moiety of the reduced intermediate in the case of an amine switch, or by decreasing the pH to a pH < pKa relative to the pKa of the carbamate in the case of a carbamate switch. The method of claim 2.

4. The method according to claim 2 or 3, wherein the linker-modified peptide is further bound to a synthetic resin, and the synthetic resin is cleaved before the coupling step is carried out.

5. 5. The method according to any one of claims 2 to 4, wherein the unreacted aldehyde moieties of the solid support are blocked using a blocking agent after carrying out the coupling step by using a blocking agent selected from cysteine, threonine, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine.

6. The reducing agent may be triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine or tris(2-carboxyethyl)phosphine, trimethylphosphite, triethylphosphite, tributylphosphite, diethylphosphite, 5,5'-dithiobis(2-nitrobenzoic acid), sodium dithionite (Na ２ S ２ O ４ ), ethanedithiol, propanedithiol, dithioerythritol, dithiothreitol, Na ２ S, NaSH, glutathione, 2,2'-dithiodipyridine, BH ３ , 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, catecholborane, borane tetrahydrofuran, borane dimethyl sulfide, borane dimethylamine complex, borane triphenylphosphine complex, borane tert-butylamine, LiAlH ４ , LiBH ４ , NaBH ４ , NaBH ３ CN, NaBH (OMe) ３ , NaBH(OCCH ３ ) ３ , LiAlH(OCMe ３ ) ３ , hydroquinone, sodium ascorbate, ascorbic acid, ascorbic acid containing KI, hydrazine, NH═NH, formaldehyde; The method according to any one of claims 2 to 5.

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