Traceless reductive linker molecules for peptide purification

Abstract

This invention provides a method for purifying peptides or peptide nucleic acids produced by solid-phase peptide synthesis (SPPS), and a linker molecule used for the said purification. [Solution] Formula (1), XT b -V a The present invention provides a linker molecule of type -UYZ(1) and a method for purifying peptides using the linker molecule. The linker molecule can be coupled to a purification resin via part X and to a peptide via part Y under the release of a leaving group Z. T is an arbitrary spacer part, and V is an arbitrary electron-withdrawing part. U is an aryl or 5-membered or 6-membered heteroaryl part that is bound to at least one electron-withdrawing part V, W, or E. The linker is stable under acidic conditions and releases the peptide upon addition of a reducing agent.

Description

[Technical Field]

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

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

[0003] A widely used method for peptide purification is preparative high-performance liquid chromatography (HPLC). A drawback of this method is its low scalability in terms of desired yield; therefore, it is not possible to produce varying quantities using a single system. This results in relatively high acquisition costs for the corresponding composite devices. A further drawback is that a relatively broad range of knowledge is required to correctly analyze and evaluate individual fragments. Furthermore, HPLC purification can consume large amounts of solvent, and sometimes column material (solid phase), during operation. [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) [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Therefore, inexpensive methods with a low tendency towards defects are advantageous for reducing the cost of peptide production.

[0006] An alternative method involves using a linker molecule that can bind to the peptide, and then coupling it to a functionalized solid phase used in purification.

[0007] European Patent No. 0552368(A1) describes a linker molecule having a thiol that can be covalently bonded to a purified support. However, this method is not suitable for thiol-containing peptides such as those containing the amino acids cysteine ​​or penicillamine.

[0008] European Patent No. 2501711(B1) proposes a similar method for linking a linker to a solid phase by a 1,3-dipolar cycloaddition reaction between an azide (-N3) and an alkyne, which requires the presence of copper. However, peptides containing methionine, cysteine, arginine, and lysine may become complex with copper, making removal difficult. Due to the toxicity of copper, such peptides are not suitable for all applications, such as pharmaceutical uses.

[0009] WO2017129818(A1) discloses a linker molecule that can bind to peptides that remain bound to the synthetic resin after SPPS. Applying commonly used TFA conditions, the peptides are subsequently cleaved from synthesis. However, a drawback of the linker molecule that forms a benzyl carbamate with the peptide, as disclosed in WO2017129818(A1), is its instability to acidic treatment (TFA > 50%, pH < 0 in the presence of water). Premature degradation of the linker molecule leads to a significant decrease in the yield of purified peptides.

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

[0011] Furthermore, the sulfone linker described in WO2017129818(A1) suffers from a reactive vinyl sulfone portion that remains in the solid report and requires an additional quenching step. [Means for solving the problem]

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

[0013] [Figure 1] Figure 1 is a schematic diagram of the peptide purification of the present invention using an X-Tb-Va-U(W)-YZ type linker (wherein W is N3), the method of which is described in claim 10. SR = synthetic resin, PB = purification beads; 1.) 4 equivalents of X-Tb-Va-U(W)-YZ, 6 equivalents of oxima, 6 equivalents of DIEA, 2 hours; 3.) dissolution of crude peptide with DMSO and addition of 10 vol% sodium citrate pH 4.5, 90 minutes; 4.) washing; 5.) PPh3 in MeCN / AcOH 9:1, 15 minutes; 6.) washing with MeCN; 7.) hydrolysis with H2O / TFA; 8.) 1,6- or 1,4-elimination; 9.) final ether precipitation. [Figure 2-1]Figure 2 shows an example of peptide purification of peptide P1 (H-ARTKQTARKSTGGKA-OH) according to the present invention using linker molecules X1 and X2 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to P1, 2.) Chromatogram of P1 after purification using linker X1 and the method of claim 10, 3.) Chromatogram of P1 after purification using linker X2 and the method of claim 10. [Figure 2-2] Figure 2 shows an example of peptide purification of peptide P1 (H-ARTKQTARKSTGGKA-OH) according to the present invention using linker molecules X1 and X2 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to P1, 2.) Chromatogram of P1 after purification using linker X1 and the method of claim 10, 3.) Chromatogram of P1 after purification using linker X2 and the method of claim 10. [Figure 3-1] Figure 3 shows an example of peptide purification of peptide P2 (H-AKADEVSLHKWYG-NH2) according to the present invention using linker molecule X2 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to P2, 2.) Chromatogram of P2 after purification by the use of linker X2 and the method of claim 10. About 3-aminobenzoic acid was used as an internal standard for peptide quantification. [Figure 3-2] Figure 3 shows an example of peptide purification of peptide P2 (H-AKADEVSLHKWYG-NH2) according to the present invention using linker molecule X2 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to P2, 2.) Chromatogram of P2 after purification by the use of linker X2 and the method of claim 10. About 3-aminobenzoic acid was used as an internal standard for peptide quantification. [Figure 4-1]Figure 4 shows four examples of peptide purification according to the present invention, specifically peptides P3 (H-YFTGSEVENVSVNVH-NH2), P4 (H-PSNPFYEALST-NH2), P5 (H-DAEFRHDSGYEVHHQKLVFF-NH2), and P6 (H-CKADEVSMHKWYG-NH2), using linker molecule X1 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to the peptide, 2.) Chromatogram of the peptide after purification using linker X1 and the method described in claim 10. [Figure 4-2] Figure 4 shows four examples of peptide purification according to the present invention, specifically peptides P3 (H-YFTGSEVENVSVNVH-NH2), P4 (H-PSNPFYEALST-NH2), P5 (H-DAEFRHDSGYEVHHQKLVFF-NH2), and P6 (H-CKADEVSMHKWYG-NH2), using linker molecule X1 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to the peptide, 2.) Chromatogram of the peptide after purification using linker X1 and the method described in claim 10. [Figure 4-3] Figure 4 shows four examples of peptide purification according to the present invention, specifically peptides P3 (H-YFTGSEVENVSVNVH-NH2), P4 (H-PSNPFYEALST-NH2), P5 (H-DAEFRHDSGYEVHHQKLVFF-NH2), and P6 (H-CKADEVSMHKWYG-NH2), using linker molecule X1 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to the peptide, 2.) Chromatogram of the peptide after purification using linker X1 and the method described in claim 10. [Figure 4-4]Figure 4 shows four examples of peptide purification according to the present invention, specifically peptides P3 (H-YFTGSEVENVSVNVH-NH2), P4 (H-PSNPFYEALST-NH2), P5 (H-DAEFRHDSGYEVHHQKLVFF-NH2), and P6 (H-CKADEVSMHKWYG-NH2), using linker molecule X1 of the present invention. A = absorption (210 nm), B = time / min; 1.) Chromatogram of the crude peptide sample before linker coupling to the peptide, 2.) Chromatogram of the peptide after purification using linker X1 and the method described in claim 10. [Figure 5] 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 synthetic resin (SR) to obtain linker-modified peptide, b) incubation with aldehyde-functionalized solid support to immobilize linker-modified peptide on purified beads (PB), c) washing of 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 release electronic structure, g) spontaneous decomposition of the linker molecule by either 1,6-,1,4-elimination or nucleophilic attack. Figure 5 is an amine switch (Type 1) with an azide-reducing safety lock. The basic nitrogen atom conjugated to the cleavable aromatic core extracts electrons when protonated (E: arrow). The positive charge of structure 1 enhances solubility and provides stability of TFA during acidic cleavage of the linker-peptide structure of synthetic resin (SR). Peptide release occurs in two steps. First, the release safety lock is removed by the reduction of the azide (partial W) to -NH2. The stability of the linker is maintained even after reduction to 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 the removal of the reducing agent and washing under acidic conditions. In the second step, the purified peptide is released by raising the pH to pH > pKa relative to the pKa of the most basic nitrogen. The increase in pH induces a 1,6-elimination reaction. The linker is decomposed under the release of CO2. This 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 = arrows indicate the direction of electron withdrawal or electron donation, a) TFA cleavage of synthetic resin (SR) to obtain linker-modified peptide, b) incubation with aldehyde-functionalized solid support to immobilize linker-modified peptide on purified beads (PB), c) washing of 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 release electronic structure, 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 that has a reducing moiety other than an azide, such as a nitro, disulfide, or azo group. The basic nitrogen atom conjugated to the cleavable aromatic core extracts electrons when protonated (E: arrow). The positive charge of structure 1 enhances solubility and provides stability to TFA during acidic cleavage of the linker-peptide structure of the synthetic resin (SR). Peptide release occurs in two steps. First, the safety lock of release is removed by the reduction of nitro (partial W) to -NH2. The stability of the linker is still maintained after reduction to 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 the removal of the reducing agent and washing under acidic conditions. In the second step, the purified peptide is released by raising the pH to pH > pKa relative to the pKa of the most basic nitrogen. The increase in pH induces a 1,6-elimination reaction. The linker decomposes under the release of CO2. This peptide is 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 = arrows indicate the direction of electron withdrawal or electron donation, a) TFA cleavage of synthetic resin (SR) to obtain linker-modified peptide, b) incubation with aldehyde-functionalized solid support to immobilize linker-modified peptide on purified beads (PB), c) washing of 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 release electronic structure, 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 carbonates, the linker exhibits high stability in solution and during storage. The coupling to the peptide can be performed as a common amino acid coupling, producing an amide bond instead of a carbamate bond. Due to the absence of a carbamate bond, the linker is stable under acidic conditions such as TFA. Here, the release of the peptide occurs in two steps. First, the reducing moiety (-N3) is reduced to an amine, removing the safety lock on the release. If the pH is higher than the pKa of the most basic nitrogen, the linker remains stable after reduction. This allows for the removal of the reducing agent and washing under acidic conditions. In the second step, the purified peptide is released by increasing the pH to 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 the pH to bring about the release electron structure, g) spontaneous 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, for example - Br, and a reducing moiety W, for example - 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, for example, 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, 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 peptide purification of peptide P2 (H-AKADEVSLHKWYG-NH2) using three of the present invention linker molecules: 2.) X9 on a type 1 amine switch with a reductive safety lock of azide, 3.) X13 on a type 2 amine switch with another reductive safety lock, and 4.) X22 on a type 3 amine switch with nucleophilic release. A = absorption (210 nm), B = time / min; the identity of the isolated peptide was always confirmed by ULC-ESI / MS analysis. Identified P2 products are marked with *. 1.) Chromatogram of the crude peptide sample before linker coupling to P2(*). 2.) Chromatogram of P2(*) after purification by the use of type 1 linker X9 and the corresponding method, as described in the Examples section below. 3.) Chromatogram of P2(*) after use of type 2 linker X13 and purification by the corresponding method, as described in the Examples section below. 4.) Chromatogram of P2(*) after use of type 3 linker X22 and purification by the corresponding method, as described in the Examples section below. [Modes for carrying out the invention]

[0014] explanation According to a first aspect of the present invention, Formula 1, XT b -V a -UYZ(1) compounds are provided, During the ceremony, - X is selected from the portion of equations 2, 2a, 3, 3a, or 4, more particularly from the portion of equation 2 or 2a of equation 2, 2a, 3, or 3a, [ka] During the ceremony, - Each R 1 and R 2 These are independently selected from H or B, and at least R 1 or R 2 B is, - R 3 is selected from H or B, - R4 is selected from H, C1 - C 12 alkyl or aryl, and the aldehyde group or ketone group may be protected with an acid - labile protecting group, - B is an acid - labile amine protecting group, - T is a 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, especially - C 1-12 alkyl -, (- C2H4O -) 1~12 , - C(=O)-, - C(=O)-JR 9 -, - JR 9 - C(=O)-, - JR 9 - of at least one, especially 1 - 5, more especially 1 - 3, including a linear or branched spacer, where in the formula, J is CH or N, especially N, especially T is: - C1 - C 12 alkyl -, especially - C 1~6 alkyl -, more especially - 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)-O-R 6 -、 -C(=O)-O-R 6 -、 -R 5 -phenyl-R 6 -、 -R 5 -phenyl-、 -phenyl-R 6 -、 -phenyl-、 -R 5 -pyrrolyl、 -R 5 -pyrazolyl、 -R 5 -imidazolyl、 R 5 -piperazinyl-、 -R 5 -pyridinyl、 -R 5 -pyrimidinyl、 -R 5 -pyrazinyl、 -R 5 -pyridazinyl、 -R 5 -pyrrolyl-R 6 -、 -R 5 -pyrazolyl-R 6 -、 -R 5 -imidazolyl-R 6 -、 -R 5 -piperazinyl-R 6 -、 -R 5 -pyridinyl-R 6 -、 -R 5 -pyrimidinyl-R 6 -、 -R 5 -pyrazinyl-R 6 -、 -R 5 -pyridazinyl-R 6 -、 pyrrolyl-R 6 -、 pyrazolyl-R 6 -、 imidazolyl-R 6 -piperazinyl-R 6 -、 pyridinyl-R 6 -、 pyrimidinyl-R 6 -、 pyrazinyl-R 6-, pyridazinyl-R 6 -, pyrroyl, pyrazolyl, imidazolyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl, a spacer selected from, wherein, R 5 , R 5’ and R 6 are, independently of each other, C1-C 12 alkyl or (-C2H4O-) 1~12 selected from, especially C1-C6 alkyl, especially C1-C3 alkyl, and wherein, 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 selected from, especially H and C 1~2 alkyl, more especially R 9 is H, wherein, B is an amine protecting group labile to acids selected independently, R 15 is a blocking agent capable of reacting with the aldehyde moiety, especially R 15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, especially cysteinyl and N-methylhydroxylamine, more especially cysteinyl, wherein, the amine moiety and / or the thiol moiety of the blocking agent can be protected by an amine protecting group B labile to acids selected independently, especially Boc, and / or by a thiol protecting group labile to acids, especially trityl, - b is 0 or 1, especially 1, - V is -NR11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p -,-piperazinyl-(CH2) p -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, [ka] , From -C(=O)- and -C(=O)-O-, in particular -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -(CH2) p -,-piperazinyl-(CH2) p -, -pyridinyl-, pyrimidinyl, more specifically -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-(CH2) p -, -pyridinyl-, and pyrimidinyl are selected electron-withdrawing moieties, in the formula, R 11 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 11 H is H, R 12 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 12 It is methyl, p is 0, 1, or 2, and in particular 0 or 1. - a is either 0 or 1, and the sum of a and b is either 1 or 2. - U is a phenyl or 5-membered or 6-membered heteroaryl moiety, more particularly phenyl or 6-membered heteroaryl moiety, more particularly phenyl, which is part of V, W q and E n It is coupled to at least one of C 1~6 Alkyl, especially C 1~3 It can be optionally substituted with alkyl, in the formula, V is defined as described 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] Therefore, especially -N3, -N=NR 8 -O-CH2-N3, -SSR 8 Selected from, in the formula, R 8 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. E is an electron-withdrawing group under acidic conditions. n is an integer between 0 and 4, especially between 0 and 2, and more especially between 0 and 1, and q is an integer between 0 and 4, especially between 0 and 2, and more especially between 0 and 1, and the sum of n and q is 4 or less, in the formula, U is the phenyl moiety and Y is -(CH2) m If it is -OC(=O)-, then 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, especially 1 or 2, and more especially 1. - Z is an electron-withdrawing leaving group.

[0015] In a particular embodiment, - X is selected from the portion of equations 2, 2a, 3, 3a, or 4, more particularly from the portion of equation 2 or 2a of equation 2, 2a, 3, or 3a, [ka] During the ceremony, - Each R 1 and R 2 These are independently selected from H or B, and at least R 1 or R 2 B is, - R 3 is selected from H or B, - R 4 H, C1~C 12 Selected from alkyl or aryl groups, the aldehyde or keto group may be protected with an acid-unstable protecting group. - B is an amine protecting group that is unstable in acid, - T is a 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, in the formula, J is C or N, and in particular is N. T in particular: -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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-, A spacer selected from, in the formula, R 5 , R 5’ and R 6 These are independent of each other, C1~C 12 Alkyl or (-C2H4O-) 1~12 From, selected particularly from C1-C6 alkyl groups, and particularly from C1-C3 alkyl groups, R 9 and R 9’ H and C are independent of each other. 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 Therefore, especially H and C 1~2 Selected from alkyl groups, and more particularly R 9 H is, in the formula, B is an amine protecting group that is unstable to an independently selected acid, R 15 It is a blocking agent that can react with the aldehyde portion, and especially R 15It is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, and hydrazine, particularly from cysteinyl and N-methylhydroxylamine, and more particularly from cysteinyl, in the formula, The amine and / or thiol portions of the blocking agent may be protected by an independently selected acid-unstable amine protecting group B, particularly Boc, and / or an acid-unstable thiol protecting group, particularly trityl. - b is 0 or 1, in particular 1, - V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, [ka] , From -C(=O)- and -C(=O)-O-, in particular, -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly selected from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, in the formula, R 11 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 11 H is H, R 12 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 12 It is methyl, - a is either 0 or 1, and the sum of a and b is either 1 or 2. - U is a phenyl or 5-membered or 6-membered heteroaryl moiety, more particularly phenyl or 6-membered heteroaryl moiety, more particularly phenyl, which is part of V, W q and E n It is coupled to at least one of C 1~6 Alkyl, especially C 1~3 It can be optionally substituted with alkyl, in the formula, V is defined as described 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] Therefore, especially -N3, -N=NR 8 -O-CH2-N3, -SSR 8 Selected from, in the formula, R 8 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. E is an electron-withdrawing group under acidic conditions. n is an integer between 0 and 4, especially between 0 and 2, and more especially between 0 and 1, and q is an integer between 0 and 4, especially between 0 and 2, and more especially between 0 and 1, and the sum of n and q is 4 or less, in the formula, U is the phenyl moiety and Y is -(CH2) m If it is -OC(=O)-, then 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, especially 1 or 2, and more especially 1. - Z is an electron-withdrawing leaving group.

[0016] In a particular embodiment, - X is selected from the portion of equations 2, 2a, 3, 3a, or 4, more particularly from the portion of equation 2 or 2a of equation 2, 2a, 3, or 3a, [ka] During the ceremony, - Each R 1 and R 2 These are independently selected from H or B, and at least R 1 or R 2 B is, - R 3 is selected from H or B, - R 4 H, C1~C 12 Selected from alkyl or aryl groups, the aldehyde or keto group may be protected by an acid-unstable protecting group. - B is an amine protecting group that is unstable in acid, - T is: -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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-, A spacer selected from, in the formula, R 5 , R 5’ and R 6 These are independent of each other, C1~C 12 Selected from alkyl groups, particularly C1-C6 alkyl groups, and especially C1-C3 alkyl groups, R 9 and R 9’ H and C are independent of each other. 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 9 H is, - b is 0 or 1, in particular 1, - V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, [ka] , From -C(=O)- and -C(=O)-O-, in particular, -NR 11 -C(=O)-, -C(=O)-NR 11 -, S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl-, pyrimidinyl, more particularly selected from -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-, -pyridinyl-, pyrimidinyl, in the formula, R 11 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 11 H is, R 12 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R12 It is methyl, - a is either 0 or 1, and the sum of a and b is either 1 or 2. - U is a phenyl or 5-membered or 6-membered heteroaryl moiety, more particularly phenyl or 6-membered heteroaryl moiety, more particularly phenyl, which is part of V, W q and E n It is coupled to at least one of C 1~6 Alkyl, especially C 1~3 It can be optionally substituted with alkyl, in the formula, V is defined as described 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] Therefore, in particular, -N3, -N=NR 8 -O-CH2-N3, -SSR 8 Selected from, in the formula, R 8 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. E is an electron-withdrawing group under acidic conditions. n is an integer between 0 and 4, especially between 0 and 2, and more especially an integer between 0 and 1, and q is an integer between 0 and 4, especially between 0 and 2, and more especially an integer between 0 and 1, and the sum of n and q is 4 or less, and in the expression, U is the phenyl moiety and Y is -(CH2) m If -OC(=O)-, then the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and in the formula, 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, especially 1 or 2, and more especially 1. - Z is an electron-withdrawing leaving group.

[0017] In a particular embodiment, - X is selected from the parts of equations 2, 2a, 3, 3a, or 4, in particular from equations 2, 2a, 3, or 3a, more particularly from equation 2 or 2a, and most particularly from the part of equation 2. [ka] During the ceremony, - Each R 1 and R 2 These are independently selected from H or B, and at least R 1 or R 2 B is, - R 3 is selected from H or B, - R 4 H, C1~C 12 Selected from alkyl or aryl groups, the aldehyde or keto group may be protected with an acid-unstable protecting group. - B is an amine protecting group that is unstable in acid, - T is: -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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-, A spacer selected from, in the formula, R 5 and R 6 These are independent of each other, C1~C 12 Selected from alkyl groups, particularly C1-C6 alkyl groups, and especially C1-C3 alkyl groups, where R9 is H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 9 H is, - b is 0 or 1, in particular 1, - V is -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-, and pyrimidinyl are selected as electron-withdrawing moieties, in the formula, R 11 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 11 H is, R 12 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 12 It is methyl, - a is either 0 or 1, and the sum of a and b is either 1 or 2. - U is a phenyl or 5-membered or 6-membered heteroaryl moiety, more particularly a phenyl or 6-membered heteroaryl moiety, which is bonded to at least one of parts V, W, and E, in the formula, V is defined as described above, W is -N3, -S(=O)-R 8 -SSR8 -OCH2N3, -OC(=O)OCH2-N3, -N=N-phenyl, -N=N-pyridine, [ka] Selected from, in the formula, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1~C6 alkyl, or -(CH2) p -NMe2, where 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, and 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 in the formula, U is the phenyl moiety and Y is -(CH2) m If -OC(=O)-, then the sum of the Hammett constants of V, W, and E under acidic conditions is greater than 0.45, and in the formula, 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, especially 1 or 2, and more especially 1. - Z is an electron-withdrawing leaving group.

[0018] The linker molecule in Equation 1 is suitable for purifying peptides after solid-phase peptide synthesis (SPPS).

[0019] A common approach to purifying peptides after SPPS using a linker molecule is to couple the linker molecule to the N-terminus of the peptide in the final coupling step. In this coupling step, the N-terminus of the peptide nucleophilically attacks the linker molecule, forming a covalent carbamate or amide bond with the linker's Y portion, while releasing an electron-withdrawing leaving group Z. Subsequently, the peptide linker construct is cleaved from the synthetic resin by adding TFA.

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

[0021] Part X of the linker can be coupled to a functionalized solid phase, such as a resin used in purification. Part X can react with a suitable part of the functionalized solid phase, such as an aldehyde, ketone, aminooxy, or hydrazine, to form a hydrazone bond or an oxime bond.

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

[0023] Part T is directly bonded to part U, or bonded via part V.

[0024] Part 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 bonded to at least one of the electron-withdrawing parts E, W, and V.

[0025] When the electron-withdrawing moieties (E, W, V) are bound to the phenyl moiety, the benzyl position of the linker molecule bound to the peptide has a lower electron density, making it less susceptible to decomposition by acid catalysts. For sufficient stability of the linker molecule of the present invention under acidic conditions, a specific threshold for electron withdrawal 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 It is expressed as the sum of ( ). 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] The Hammett constant is the meta position (σ m ) and para position (σ p The Hammett values ​​are empirically determined constants for the phenyl core substituents of the benzoic acid derivatives of ), resulting in different acidity (pKa). In the context of this invention, this position is determined in relation to the bond of partial Y. For ortho substituents, the Hammett values ​​for the para substituents are a good approximation and are therefore used to calculate the sum of the Hammett values ​​of substituents V, W, and E in the context of this invention.

[0027] It should be noted that the Hammett constant is calculated for substituents V, W, and E under acidic conditions. For example, the amine moiety at neutral pH has a Hammett constant of σ. m = -0.16 and σ p = -0.66, and is therefore characterized by substituents that push out electrons. Under acidic conditions, the amine moiety is 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 the protonated form via a conjugated π system, either directly as a substituent on U or via π-conjugation of U.

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

[0029] [Table 1]

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

[0031] For the stability of linker molecules containing the heterocyclic moiety U under acidic conditions, such a surplus threshold is not required for selecting specific moieties V, W, and E. 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, if U is a nitrogen-containing heterocycle, the nitrogen is protonated during the acidic release of the peptide, resulting in a particularly low electron density of the aromatic system of U. Therefore, the benzyl cation cannot be stabilized.

[0032] Aside from mediating the stability of the linker molecule under acidic conditions, parts V, W, and E are important in relation to the peptide release mechanism. Furthermore, they can contribute to the solubility of the linker molecule under acidic conditions.

[0033] The W portion is a reducible substituent that causes the linker to break down, and thus the release of the peptide. A linker containing the reducing W portion is also called a reducing intermediate. In contrast to a stable linker molecule, the reducing intermediate is unstable. The instability of the reducing intermediate is pH-dependent. If the W portion is protonable (e.g., pyridyl), it also contributes to the solubility of the linker molecule under acidic conditions.

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

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

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

[0037] Part Y is either a -C(=O)- part or an -OC(=O)- part. When the linker molecule is coupled, an amide (-C(=O)-NH-) part or a carbamate (-OC(=O)-NH-) part is formed between the linker molecule and the N-terminus of the peptide. After purification, the peptide is released from the linker molecule and thus from the purification medium under reducing conditions, either by elimination or nucleophilic attack as described in 1.4 or 1.6. This reducing stimulus converts W to its reduced form, which then functions as an electron-donating group and nucleophile, thus enabling the release of the peptide.

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

[0039] In certain embodiments, X is selected from the portion of Equation 2.

[0040] The reaction time required for coupling portion X to the functionalized solid phase by the formation of a hydrazone or oxime bond is longer when using a linker having portion 4 of formula, and shorter when using a linker having portion 2 or 3 of formula. The formation of the hydrazone bond between the aldehyde or ketone portion of the solid support and portion 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 a linker having portion 2 of formula, almost no 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] If U is further substituted with one or more alkyl moieties, the Hammett values ​​of the alkyl moieties are taken into consideration. If U is further substituted with a phenyl moiety or heteroatom, the sum of the Hammett values ​​of V, W, E and optionally the alkyl substituents is greater than 0.45. In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N(C2H4)2NH2, -N(C2H4)2N-B, -N=N-phenyl, -N=NR 8 ,-(CH2) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 Selected from alkyl)2-, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1~C6 alkyl or -(CH2) p-NMe2, particularly pyridyl or -C1~C6 alkyl, and p is 1, 2, 3 or 4, and B is an acid-unstable amine protecting group as defined herein, particularly -C(=O)OtBu(Boc) or C(=O)CPh3, and r is 0, 1, 2, 3, or 4, and in particular 0, 1, or 2.

[0045] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridadinyl, -(CH2) 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 are selected, 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, pyridadinyl-NH-C 1~6 Alkyl, -N(C 1~6 Selected from alkyl)2-, -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 Selected from alkyl)2- or -Br.

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

[0049] In certain embodiments, E is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N=N-phenyl, -N=NR 8Selected from -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1~C6 alkyl or -(CH2) p It is -NMe2, particularly 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 Selected from -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1~C6 alkyl or -(CH2) p It is -NMe2, particularly 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, -(CH2) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 Selected from -alkyl)2-, in particular -(CH2) r -NH-C 1~3 -Alkyl, -(CH2) r -N(C 1~3 Selected from alkyl)2-, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, -C(=O)NH2, -SO2Me, -SOMe, -SO2Et, -SOEt, where r is 0, 1, or 2, and especially 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, and -SOEt.

[0053] In certain embodiments, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridadinyl, -(CH2) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 Selected from alkyl)2-, especially -(CH2) r -NH-C 1~3 Alkyl, -(CH2) r -N(C 1~3 Selected from 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, pyridadinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, -CO2H, and r is 0, 1, or 2, and in particular 0.

[0055] In certain embodiments, E is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, -F, -Cl, -Br, -I, -CN, -NO2, -N3, -CF3, -SO3H, and -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 Selected from 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, and 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, and 7) requires a protonable basic nitrogen. Such basic nitrogen can be provided by part E.

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

[0062] In certain embodiments, E is -(CH2) r -NH-C 1~6 Alkyl, -(CH2) r -N(C 1~6 Selected from alkyl)2-, especially -(CH2) r -NH-C 1~3 Alkyl, -(CH2) r -N(C 1~3 Selected from alkyl)2-, pyridyl, pyrimidinyl, and pyridazinyl, where r is 0, 1, or 2, particularly 0, and n is 1.

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

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

[0068] In a particular embodiment, 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), Benz Selected from ruamine (-CH2Ph), acetamide (-COMe), trifluoroacetamide (-COCF3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohexy-1-ylidene)-3-ethyl), and 1-(4,4-dimethyl-2,6-dioxocyclohexy-1-ylidene)-3-methylbutyl (ivDde), where B is Boc or Eei, where B is Boc, or - Selected from the following acetal or ketal protecting groups: [ka] In the formula, r is 0 to 12, especially 0 to 6, and more specifically 0, 1 or 2, and R 10 is -C1~C 12 Alkyl-, especially C 1~6 Alkyl, especially 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), Ben Dilamine (-CH2Ph), acetamide (-COMe), trifluoroacetamide (-COCF3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)-3-ethyl) and 1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)-3-methylbutyl (ivDde), in particular B being Boc or Eei, and more particularly B being Boc, or - Selected from the following acetal or ketal protecting groups: [ka] In the formula, r is 0 to 12, in particular 0 to 6, and more particularly 0, 1 or 2, and R 10 is -C1~C 12 Alkyl-, especially C 1~6 It is alkyl, and more particularly C 1~3 It is alkyl.

[0070] 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-toluenesulfone Selected from amide (-SO2C6H4Me), benzylamine (-CH2Ph), acetamide (-COMe), trifluoroacetamide (-COCF3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)-3-ethyl) and 1-(4,4-dimethyl-2,6-dioxocyclohexa-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 acetal or ketal protecting groups selected from the following: [ka] In the formula, r is 0 to 12, in particular 0 to 6, and 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 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 phenyl, a 5-membered or 6-membered heteroaryl, wherein J is CH or N, and in particular N, and R 9 H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 From alkyl-NB2, especially H and C 1~2 Selected independently of alkyl, and more particularly R 9 is H, where B is an independently selected acid-unstable 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 - A linear or branched spacer comprising 1 to 5 portions independently selected from phenyl, a 5-membered or 6-membered heteroaryl, particularly a linear spacer.

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

[0077] In certain embodiments, T is part C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 - A linear or branched spacer comprising 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 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, where J is C or N, in particular N, and where R 9 H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 From alkyl-NB2, especially H and C 1~2 Selected independently of alkyl, and more particularly R 9is H, where B is an independently selected acid-unstable amine protecting group.

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

[0081] In a particular embodiment, T is -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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 -Pyrazol, -R 5 -Imidazol, R 5 -piperazinyl-, -R 5 -pyridinyl, -R 5 -pyrimidinyl, -R 5 -pyrazinyl, -R 5 -pyridazinyl, -R 5 -Pyroyl-R 6 -, -R 5 -Pyrazol-R 6 -, -R 5 -Imidazol-R 6 -, -R 5 -Piperazinil-R 6 -, -R 5 -Pyridinyl-R 6 -, -R 5 -Pyrimidinyl-R 6 -, -R 5 -Pyrazinil-R 6 -, -R 5 -Pyridazinil-R 6 -, Pyroyl-R 6 -, Pyrazoyl-R 6 -, Imidazoil-R 6 -Piparadinil-R 6 -, pyridinyl-R 6 -, pyrimidinil-R 6 -, Pyrazinil-R 6 -, Pyridazinil-R 6 - Selected from pyrroyl, pyrazoyl, imidazoyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl.

[0082] In a particular embodiment, T is -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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 -Imidazoyl, -R 5 -Imidazol-R 6 -, Imidazoil-R 6 - Selected from imidazoyl.

[0083] In a particular embodiment, T is -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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 -Imidazoyl, -R 5 -Imidazol-R 6 -,-Imidazoi-R 6 - Selected from imidazoyl.

[0084] In a particular embodiment, T is -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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 - Select from the options.

[0085] In a particular embodiment, T is -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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, especially C 1~3 Alkyl, -R 5 -C(=O)-NR 9 -, -R 5 -NR 9 -C(=O)-R6 - from, 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 It is defined as described above.

[0086] In certain embodiments, T is part C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 - A linear or branched spacer comprising phenyl, at least one, particularly 1 to 5, 5-6 membered heteroaryls, in the formula R 9 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 Therefore, especially H and C 1~2 Selected independently of alkyl, especially R 9 is H, where B is an amine protecting group that is unstable to an independently selected acid, R 15 It is a blocking agent that can react with the aldehyde portion, and especially R 15 It is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, and hydrazine, particularly from cysteinyl and N-methylhydroxylamine, and more particularly from cysteinyl, in the formula, The amine and / or thiol portions of the blocking agent can be protected by an independently selected acid-unstable amine protecting group B, particularly Boc, and / or an acid-unstable thiol protecting group, particularly trityl.

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

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

[0089] In certain embodiments, T is part C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -A linear or branched spacer comprising at least one of the following, where R 9 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 Therefore, especially H and C 1~2 Selected independently of alkyl, and more particularly R 9is H, and in the formula, B is an amine protecting group that is unstable to an independently selected acid. R 15 It is a blocking agent that can react with the aldehyde portion, and especially R 15 This is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, and hydrazine, particularly from cysteinyl and N-methylhydroxylamine, and more particularly from cysteinyl, where, The amine and / or thiol portions of the blocking agent can be protected by an independently selected acid-unstable amine protecting group B, particularly Boc, and / or an acid-unstable thiol protecting group, particularly trityl.

[0090] As described above, spacer T is generally unreactive under commonly applied purification conditions, except for the removal of protecting groups under acidic conditions. Spacer T can enhance the solubility of linker molecules. In particular, R 9 Branched spacers containing protected or unprotected amine moieties contribute to enhanced solubility. Under acidic conditions, amine protecting group B is removed and the amine is protonated.

[0091] In certain embodiments, T is part C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -A linear or branched spacer comprising at least one of the following, where R 9 H, C 1~4 Alkyl, -C 1~6 Alkyl-NH2, -C 1~6 Alkyl-NHB, -C 1~6 From alkyl-NB2, in particular, H and C 1~2 Selected independently of alkyl, and more particularly R 9 is H, and in the formula, B is an amine protecting group that is unstable to an independently selected acid.

[0092] Spacer T may also include a blocking function. In particular, branched spacers may include a blocking agent suitable for binding to the aldehyde moiety. When the solid phase used for peptide purification contains an aldehyde moiety (e.g., agarose beads), a portion X of the linker compound can be covalently bonded to the solid phase, for example, by the formation of an oxime bond. Unreacted aldehyde moieties can cause unwanted side reactions during subsequent purification. To prevent such side reactions, the unreacted aldehyde moiety of the solid phase can be blocked by the blocking function of the spacer, for example, by a cysteinyl moiety.

[0093] In certain embodiments, T is part C 1~12 Alkyl-, -C(=O)-, -C(=O)-NR 9 -, -NR 9 -C(=O)-, -NR 9 -A linear or branched spacer comprising at least one of the following, where R 9 H, C 1~4 Alkyl, -R 15 , -C 1~6 Alkyl-R 15 , -C 1~6 Alkyl-NH-R 15 Therefore, especially H and C 1~2 Selected independently of alkyl, and more particularly R 9 is H, and in the formula, B is an amine protecting group that is unstable to an independently selected acid. R 15 It is a blocking agent that can react with the aldehyde portion, and especially R 15 This is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, and hydrazine, particularly from cysteinyl and N-methylhydroxylamine, and more particularly from cysteinyl, where, The amine and / or thiol portions of the blocking agent can be protected by an independently selected acid-unstable amine protecting group B, particularly Boc, and / or an acid-unstable thiol protecting group, particularly trityl.

[0094] In a particular embodiment, T is -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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 - From -C1~C 12 Alkyl-, especially C 1~6 From alkyl, more especially 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 -, especially -R 5 -NR 9 -C(=O)-R 6 - Selected from, R 5 , R 6 and R 9 This is defined as above.

[0095] As described above, partial U, V, W, and E contribute to the stability of the linker under acidic conditions. For example, linkers containing partial U or V that include heterocyclic elements such as amines or pyridines are stable under acidic conditions, especially at TFA > 50% and pH < 0 in the presence of water, because the amine or heterocyclic element 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 described in the last 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, pyridadinyl, [ka] , The electron-withdrawing portion is selected from -C(=O)- and -C(=O)-O-, and in the formula, R 11 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 11 H is H, R 12 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 12 It is methyl, p is 0, 1, or 2, and in particular is 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, pyridadinyl, [Chemical formula] and an electron-withdrawing moiety selected from -C(=O)-, -C(=O)-O-, wherein the pyridinyl moiety is connected 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 - selected from.

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

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

[0101] In certain embodiments, V is -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-(CH2) p - selected from.

[0102] In certain embodiments, V is -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinyl-(CH2) p - selected from, and p is 0 or 1.

[0103] In certain embodiments, V is -NH-C(=O)-, -C(=O)-NH- and -piperazinyl-(CH2) p - selected from, p is 0 or 1, and in particular p is 0.

[0104] In particular, the linker molecule that releases the peptide via the carbamate switch (Figure 8) does not contain a basic nitrogen atom, and such an atom has a pKa of less than 2 for the corresponding acid. In certain embodiments, V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, and -S(=O)-, particularly selected from -NH-C(=O)- and -C(O=)-NH-, and 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,

Chemical formula

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

[0107] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -S(=O)-, -NR 12 -, -piperazinyl-, -pyridinyl-, and pyrimidinyl are selected as electron-withdrawing moieties, where R 11 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 11 H is R 12 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 12 It is methyl.

[0108] V is -NR 11 -C(=O)-, -C(=O)-NR 11 -, -NR 12 -, -piperazinyl-, -pyridinyl-, and pyrimidinyl are selected as electron-withdrawing moieties, where R 11 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 11 H is R 12 H and C 1~4 From alkyl, especially H and C 1~2 Selected from alkyl groups, and more particularly R 12 It is methyl.

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

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

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

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

[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 amine switches require a basic nitrogen atom. The basic nitrogen atom can be provided via a partial V, E, or U (U = heteroaryl). If U is phenyl, the basic nitrogen atom can be provided via a partial V or E.

[0117] In certain embodiments, E and V are selected as described above, and if U is phenyl, then at least one part E or V is selected from the following: - 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, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -(CH2) p -Selected from NMe2, where p and B are defined as described herein, and - V:-NR 12 -(CH2) p -,-piperazinyl-(CH2) p -, -pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, [ka] And in particular, -NR 12 -(CH2) p -,-piperazinyl-(CH2) p-, -pyridinyl, pyrimidinyl, R 12 and p are defined as described herein.

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

[0119] In certain embodiments, 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] Selected from, in the formula, R 8 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 Alkyl compounds can be linear or branched, and may be, for example, butyl or tert-butyl.

[0120] In certain embodiments, 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] Selected from.

[0121] In certain embodiments, W is -N3, -S(=O)-R 8 -SSR 8 -O-CH2-N3, -N=NR 8 Therefore, especially -N3, -N=NR 8 -O-CH2-N3, -SSR8 Selected from, in the formula, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1~C6 alkyl, or -(CH2) p It is -NMe2, particularly 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 Therefore, especially -N3, -N=NR 8 -O-CH2-N3, -SSR 8 Selected from, in the formula, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -C1~C6 alkyl, or -(CH2) p It is -NMe2, particularly pyridyl or -C1~C6 alkyl, and p is 1, 2, 3, or 4.

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

[0124] Partial W=-SSR 8 Linkers containing thiols can be cleaved using thiols. Therefore, cleavage of peptide-linker complexes from synthetic resins used in SPPS under acidic conditions (e.g., TFA > 50%, pH < 0 in the presence of water) can be performed without thiols if 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 Therefore, especially -N3, -N=NR8 -O-CH2-N3, -SSR 8 Selected from, in the formula, R 8 is pyridyl, pyrimidinyl, pyrazinyl, pyridadyl, or -C1~C6 alkyl, or more particularly pyrimidinyl, pyridyl, or -C1~C6 alkyl, and more particularly pyridyl or -C1~C6 alkyl.

[0126] In certain embodiments, W is -N3, -SSR 8 (R 8 It is selected from -C1~C6 alkyl and NO2.

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

[0128] A linker molecule (Figure 6) that releases a peptide via an amine switch with an azide-free reductive safety lock contains 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 selected from pyridyl, pyrimidinyl, pyrazinyl, or pyridazyl).

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

[0130] The linker molecules that release peptides via nucleophilic release through an amine switch (Figure 7) are -N3 and -SSR. 8 (R 8 is C 1~6 Alkyl, especially tert-butyl, or R 8 It contains a reducible moiety such as pyridinyl.

[0131] As described above, linker molecules can contribute to the solubility of linker-peptide constructs under acidic conditions, particularly during the purification of hydrophobic peptides. Solubility can also be mediated by a partial W, where W is selected from groups that enhance solubility for peptides purified in the pH range of 0–7.

[0132] In certain embodiments, W is -S(=O)-R 8 -SSR 8 -N=NR 8 , [ka] Selected from, in the formula, R 8 Pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -(CH2) p -NHBoc or -(CH2) p -NMe2, especially pyridyl or -(CH2) p It is -NHBoc-, and p is 1, 2, 3, or 4.

[0133] In certain embodiments, U is selected from phenyl or a 5-membered or 6-membered heterocycle, the 5-membered or 6-membered heterocycle containing one or two heteroatoms.

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

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

[0136] In certain embodiments, U is selected from phenyl or a six-membered heterocycle, the six-membered heterocycle containing one or two heteroatoms.

[0137] In certain embodiments, the five-membered or six-membered heteroaryl portion of U comprises one or two heteroatoms, and in particular, the five-membered heteroaryl portion of partial U is selected from pyrazole and imidazole, and the six-membered heteroaryl portion of partial U is selected from pyridine, pyridazine, pyrimidine, and pyrazine, particularly from pyridine.

[0138] In certain embodiments, U is selected from phenyl or a six-membered heterocycle, the six-membered heterocycle containing one heteroatom. In certain embodiments, the five-membered heterocycle of partial U is selected from pyrazole and imidazole, and the six-membered heterocycle of partial U is selected from pyridine, pyridazine, pyrimidine, and pyrazine.

[0139] In certain embodiments, U is selected from phenyl, pyridine, pyridazine, pyrimidine, and pyrazine, particularly from phenyl or pyridine, and more particularly from phenyl.

[0140] The linker according to the present 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 at the ortho or para position relative to partial Y.

[0141] In certain embodiments, U is selected from the portion of Equation 5 or 6, [ka] During the ceremony, T, V, Y, W, and E are defined as described above. U is joined to a sub-T or V (T or V), A 1 , A 2 , A 3 , A 4 and D 1 , D2 , D 3 , D 4 These are independently selected from C, N, S, and O, particularly from C and N, and n is an integer between 0 and 3, specifically 0, 1, or 2.

[0142] In a particular embodiment, A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 Parts 2 to 4 of are C, and especially A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 Three or four parts of it are C.

[0143] In a particular embodiment, all parts 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 parts of equations 5, 6, 7, or 8. [ka] During the ceremony, T, V, Y, W, and E are defined as described above. In equations 5 and 6, U is combined with part T or V. In equations 7 and 8, U is combined with part V, A 1 , A 2 , A 3 , A 4 and D 1 , D 2 , D 3 , D4 These are selected independently from each other from C, N, S, and O, particularly from C and N, and A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 Parts 2 to 4 of are C, and especially A 1 , A 2 , A 3 and A 4 , or D 1 , D 2 , D 3 and D 4 Three or four parts of it are C, and more specifically 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 cases of equations 5 and 6, the integers are between 0 and 3. In the cases of equations 7 and 8, the integers are 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 portion of Equation 5 or 6.

[0146] In certain embodiments, U is selected from parts of formulas 5, 6, 7, or 8, where, T, V, Y, W, and E are defined as described above. In equations 5 and 6, U is combined with part T or V. In equations 7 and 8, U is combined with part V, A 1 , A 2 , A 3 , A 4 and D 1 , D 2 , D 3 , D 4These are independently selected from C, N, S, and O, particularly from C and N. n is In the cases of equations 5 and 6, the integers are between 0 and 2. In the cases of equations 7 and 8, the integers are between 0 and 2, especially between 0 and 1. q is an integer between 0 and 2, especially between 0 and 1.

[0147] In certain embodiments, U is selected from the parts of formulas 9, 10, 11, or 12, particularly from the part of formula 9 or 10. [ka] During the ceremony, T, V, Y, W, E, q, and n are defined as above. In equations 9 and 10, U is combined with part T or V. In equations 11 and 12, U is combined with part 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 of them are N, and in particular A 2 and A 3 Both are C, and D 2 is C or N, and in particular is C.

[0148] In certain embodiments, U is selected from the parts of formulas 9, 10, 11, or 12, particularly from the part of formula 9 or 10. [ka] During the ceremony, T, V, Y, W, E, q, and n are defined as above. In equations 9 and 10, U is combined with part T or V. In equations 11 and 12, U is combined with part V, A 2and A 3 Both are C, or A 2 and A 3 One of them is C, and A 2 and A 3 The other side is N, D 2 is either C or N.

[0149] If the partial U contains a nitrogen-containing heteroaryl, the nitrogen atom is protonated under acidic conditions, thus increasing the solubility of the linker molecule.

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

[0151] In certain embodiments, U is selected from parts of formulas 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. [ka] In the formula, T, V, Y, W, E, q, and n are defined as above. In the cases of equations 13, 14, 15, 19, 20, 21 and 22, U is combined with part T or V. In equations 16, 17, and 18, U is combined with part V, A 2 , A 3 and A4 is C or N, and in particular is C, D 2 is either C or N.

[0152] In certain embodiments, U is selected from parts of formulas 13, 14, 15, 16, 17, or 18. [ka] During the ceremony, T, V, Y, W, E, q, and n are defined as above. In equations 13, 14, and 15, U is combined with part T or V. In equations 16, 17, and 18, U is combined with part V, A 3 is C or N, D 2 is either C or N.

[0153] In certain embodiments, U is selected from parts of formula 13, 14, 15, 21, or 22, where, T, V, Y, W, E, and n are defined as described above. U is combined with a partial T or V, A 3 and A 4 is C or N, and in particular is C, D 2 is C or N.

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

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

[0156] Regarding amine switches (Figures 5 and 6) and carbamate switches (Figure 8) with reductive safety locks, portion Y can release CO2, particularly when a change in pH causes linker breakdown and releases a peptide with a free N-terminus -(CH2). m -OC(=O)-

[0157] In certain embodiments, U is a part of formula 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, and Y is -(CH2) m -OC(=O)-

[0158] In certain embodiments, U is a part of formula 13, 14, 15, 21, or 22, and Y is -(CH2) m -OC(=O)-

[0159] In certain embodiments, U is a part of formula 13 or 21, and Y is -(CH2) m -OC(=O)-

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

[0161] If the linker molecule has a -COOH group at its terminus, the peptide can be coupled to the linker via a general amino acid coupling. In certain embodiments, Y is -(CH2) m It is -C (=O)- and Z is -OH.

[0162] For amine switches involving nucleophilic release, U is specifically the part in Equation 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, -N3, -OH, -O(C=O)CH2(C=O)OH, -SR14 , -OCF3, -OCH2CF3, -OSO2CF3, -SO2C6H4CH3, -SO2CF3, -SO2CH3, [ka] Selected from, in particular, -OH, -Cl, [ka] Selected from, in particular -OH, [ka] Selected from, in the formula, R 14 These substituents are C1-C6 alkyl-, aryl-, or benzyl- substituents.

[0164] A type 1 linker molecule suitable for peptide release via an amine switch with an azide-reductive safety lock (Figure 5) may consist of the following parts: U is phenyl or pyrimidyl, and in particular U is 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. V'a is 1, and V is selected from -piperazinyl-, -piperazinyl-CH2-, -N(CH3)-, pyrimidinyl, pyridyl, and especially from -piperazinyl-, -piperazinyl-CH2-, -N(CH3)-, and Y is -(CH2)mOC(=O)-, where m is 1, 2, or 3, in particular 1 or 2, and more particularly 1.

[0165] The pyridyl portion is marked with a U at position 3 or 5.

[0166] In certain embodiments, U is selected from parts of formulas 13, 14, 15, 21, or 22.

[0167] A type 2 linker molecule suitable for peptide release via an amine switch without an azide (Figure 6) may consist of the following parts: U is pyridinyl or phenyl, and in particular U is formula 13, 14, or 21. W is -SS-tertbutyl, -NO2, -N=N-pyridyl, [ka] Selected from, in particular from -SS-tertbutyl,-NO2, n in E is 0, V'a is 1, and V is selected from -C(=O)-NH- and piperazinyl, and Y is -(CH2)mOC(=O)-, where m is 1, 2, or 3, in particular 1 or 2, and more particularly 1.

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

[0169] A type 3 linker molecule suitable for peptide release via an amine switch with nucleophilic release (Figure 7) may consist of the following parts: U is phenyl or pyridinyl, more particularly phenyl, and more particularly U is a part of formula 13 or 14. W is -N3, -SS-tertbutyl, -SS-pyridyl, and especially -N3. n in E is 0, V's a is 1, and V is piperazinyl, -NH-, -C(=O)-NH-, and in particular piperazinyl, and Y is -(CH2)mC(=O)-, where m is 1, 2, or 3, in particular 1 or 2, and more particularly 1.

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

[0171] A type 4 linker molecule suitable for peptide release via a carbamate switch (Figure 8) may consist of the following parts: U is phenyl, and in particular U is formula 13 or 15, and in particular part A is C. W is -N3, E's n is either 1 or 2, and E is -Br. V's a is 1, and V is -NH-C(=O)-, and Y is -(CH2)mOC(=O)-, where m is 1, 2, or 3, in particular 1 or 2, and more particularly 1.

[0172] In certain embodiments, U is selected from parts of formula 13 or 15, and in particular part 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] Selected from, in particular, -OH, -Cl, [ka] Selected from, in particular -OH, [ka] Selected from, in the formula, R 14 These substituents are C1-C6 alkyl, aryl, or benzyl substituents.

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

[0175] In a particular embodiment, the compound of formula 1 is selected from compounds 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 aims to provide a method for purifying peptides.

[0177] A second aspect of the present invention provides a method for purifying peptides. This method includes the following steps: - A step of providing a crude linker-modified peptide, wherein the crude peptide is covalently bonded to a linker molecule according to a first aspect of the present invention, - In the coupling step, the linker-modified peptide is coupled to a solid support to obtain an immobilized linker-modified peptide, - The release step, wherein a reducing agent is added under acidic conditions to release the peptide.

[0178] A linker molecule according to a first aspect of the present invention can be used in a method for purifying peptides. In the first step, a crude peptide mixture is brought into contact with a linker molecule according to a first aspect of the present invention, and the crude peptide is coupled to the linker molecule to obtain a crude linker-modified peptide. This crude peptide is coupled to the linker molecule in a standard manner commonly known to experts in the fields of chemistry, biochemistry, and pharmacy. During purification, the stability of the linker molecule can be improved by adjusting the pH. Under acidic conditions (particularly a pH lower than the pKa of the most basic heteroatom of the linker molecule), the linker molecule is stabilized due to the protonation and electron withdrawal of this heteroatom (Figures 5, 6, 7). Once the linker is bound to the peptide and immobilized on a solid support, the peptide is released via a reducing intermediate under reducing conditions, for example, by adding a reducing agent such as triphenylphosphine under acidic conditions. The reducing intermediate is characterized by a reducing linker moiety, such as an azaylide. The linker portion of the reducing intermediate decomposes over time, or triggered by factors such as an increase in pH, particularly when the pH exceeds the pKa of the most basic heteroatom in the linker portion of the reducing intermediate. Therefore, all steps prior to peptide release (coupling, any washing, and reduction of the linker) are performed under acidic conditions. When the pH rises to a level exceeding the pKa of the most basic heteroatom in the linker portion of the reducing intermediate, the linker portion of the reducing intermediate 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 as follows: 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 (-NH3 has a pKa of 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 include 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 adding 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 the 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, and the peptide is released from said reduction intermediate.

[0185] In certain embodiments, the reduction intermediate that features a reduced linker portion of the 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, and 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 - 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. Additionally, 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 0.1 M, 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 (Na2S2O4), boranedimethyl sulfide, boranetriphenylphosphine complex, NaBH4, 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 (Na2S2O4), ethanedithiol, propanedithiol, dithiothreitol, Na2S, NaSH, glutathione, 2,2'-dithiodipyridine, BH Selected from 3,4,4,5,5-tetramethyl-1,3,2-dioxaborolane, catecholborane, boranetetrahydrofuran, boranedimethylsulfide, boranedimethylamine complex, boranetriphenylphosphine complex, boranetert-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 (Na2S2O4), boranedimethyl sulfide, boranetriphenylphosphine complex, NaBH4, and ascorbic acid.

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

[0217] In certain embodiments, the reducing agent is selected from triphenylphosphine, sodium dithionite (Na2S2O4), boranedimethyl sulfide, boranetriphenylphosphine 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 a particular embodiment, the method includes the following steps: - A step of providing a crude linker-modified peptide, wherein the crude peptide is covalently bonded to a linker molecule according to a first aspect of the present invention, and the linker molecule comprises a portion W and / or E including an azide portion, - In the coupling step, the linker-modified peptide is coupled to a solid support to obtain an immobilized linker-modified peptide, - The release step, wherein the peptide is released by adding a reducing agent under acidic conditions.

[0221] In a particular embodiment, the method includes the following steps: - A step of providing a crude linker-modified peptide, wherein the crude peptide is covalently bonded to a linker molecule according to a first aspect of the present invention, and the linker molecule comprises a portion W and / or E including an azide portion, - In the coupling step, the linker-modified peptide is coupled to a solid support to obtain an immobilized linker-modified peptide, - The release step, wherein a reducing agent is added under acidic conditions to obtain a reducing intermediate, and then the pH is increased so that pH > pKa of the most basic heteroatom in the linker portion of the reducing intermediate to release the peptide. -

[0222] In a particular embodiment, the method includes the following steps: - A step of providing a crude linker-modified peptide, wherein the crude peptide is covalently bonded to a linker molecule according to a first aspect of the present invention, and the linker molecule comprises a portion W and / or E including an azide portion, - In the coupling step, the linker-modified peptide is coupled to a solid support to obtain an immobilized linker-modified peptide, - The release step, wherein triphenylphosphine, trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, particularly triphenylphosphine, is added under acidic conditions to generate a reducing intermediate, and then the pH is increased so that pH > pKa with respect to the pKa of the most basic heteroatom to release the peptide.

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

[0224] In a particular embodiment, the method for purifying a peptide includes the following steps: a) A step of providing a peptide bonded to a synthetic resin, wherein the peptide is further covalently bonded to a linker molecule according to claim 1, and the linker molecule comprises a portion W and / or E including an azide portion, b) A step of cleaving peptides from synthetic resin, c) A step of coupling the cleaved peptide mixture with a solid support, d) A step of releasing the peptide using triphenylphosphine, or trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, particularly triphenylphosphine.

[0225] The 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 ethers (Et2O, iPr2O, MeOtBu, THF / hexane (1:1)) can be used for precipitation.

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

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

[0228] The coupling product is washed with EtOH / water (7:3) containing DMSO, 6M guanidium hydrochloride, 0.1M NaCl, water, and 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 for 15 minutes.

[0230] In certain embodiments, triphenylphosphine, trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine is added to MeCN / AcOH / H2O(90:5:5), and / or the azailide formed after the addition of triphenylphosphine, particularly trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, is washed with MeCN or MeCN / H2O(9:1).

[0231] In certain embodiments, triphenylphosphine, trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine, particularly triphenylphosphine, is added to MeCN / AcOH / H2O(90:5:5). In certain embodiments, the addition is carried out for 15 minutes.

[0232] In certain embodiments, the azailides formed after the addition of triphenylphosphine, trimethylphosphine, triethylphosphine, or tris(2-carboxyethyl)phosphine are washed, in particular, with MeCN or MeCN / H2O(9:1).

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

[0234] In certain embodiments, the resulting azailide is hydrolyzed, particularly using H2O / TFA. The ratio can be 99.95% to 50% water.

[0235] If the linker, especially U, E, W, or V, is a nitrogen-containing heterocycle, the pH must exceed the pKa of the heterocycle portion, which is already the case in the hydrolysis mixture of TFA and water, or can be achieved by adding a buffer of the desired pH.

[0236] In certain embodiments, after hydrolysis, an elution step is performed using TFA / H2O in particular, in a ratio of 9:1.

[0237] In certain embodiments, after hydrolysis, an elution step is performed, particularly using TFA / H2O, in a ratio of 95:5.

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

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

[0240] In the context of this invention, the Hammett constant is the constant 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 becomes stronger as the Hammett constant increases. The Hammett constant is the constant at the meta position (σ). m ) and para position (σ p This is a constant empirically determined for the substituents of the phenyl moiety of ). In the context of the present invention, this position is determined in relation to the bonding of partial Y. For ortho substituents, the Hammett value of the para substituent is a good approximation and is therefore used to calculate the sum of the Hammett values ​​of substituents V, W, and E in the context of the present invention.

[0241] In the context of this invention, the term "under acidic conditions" refers to a pH less than 7, particularly a pH less than the linker's pKa, and more specifically, a TFA > 50%, with a pH < 0 in the presence of water.

[0242] In the context of this invention, the term alkyl means linear or branched saturated hydrocarbons. For example, C 1~12 The term alkyl refers to saturated linear or branched hydrocarbons having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Non-limiting examples of C1-C4 alkyls include methyl, ethyl, propyl, n-butyl, 2-methylpropyl, and tert-butyl. [Examples]

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

[0244] The peptide sequence was synthesized under standard solid-phase peptide synthesis conditions, thereby blocking unreacted amino groups in the synthetic resin by treating it with acetic anhydride and pyridine after each amino acid coupling. Linker X1 was coupled to P1 on the resin by using 4 equivalents of linker, 6 equivalents of oxima, 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 for 2 hours by using 4 equivalents of linker, 6 equivalents of oxima, and 6 equivalents of DIEA. Subsequently, the peptide was cleaved from the synthetic resin with a mixture of TFA / PhOH / PhSH / H2O / ethanedithiol (EDT, 82.5:5:5:2.5). The crude peptide mixture was dissolved in dimethyl sulfoxide (DMSO). Aldehyde-modified agarose beads were washed three times each with water and 0.1 M sodium citrate buffer (at pH 4.5). To a DMSO solution of the peptide, 10% by volume of sodium citrate buffer was added, and this solution was then applied to agarose beads over 90 minutes to quantitatively immobilize the target peptide onto the agarose beads. Subsequently, the beads were washed three times each with 8M urea, DMSO, EtOH / water (7:3), 0.1M NaCl, water, and MeCN to remove acetylated terminal sequences and other impurities. The immobilized linker was cut by treating the agarose resin with 50 mg of PPh3 per 1 mL of MeCN / AcOH (9:1). The support was then rinsed four times with MeCN, and a solution of H2O / 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 in the same tube with TFA / H2O (9:1). The peptide was obtained by adding 10-fold Et2O to induce precipitation, and the tube was centrifuged to process the organic supernatant.

[0245] The purity of each phase was confirmed using UPLC-MS. UPLC chromatograms of unpurified (without linker molecule) and purified peptides are shown in Figures 2 and 3, and the results are summarized in Table 2. Peptide identity was confirmed by ESI-MS. In the purification of 100 μmol of P1, using X1 with a purity of 93% yielded 26 mg of peptide P1 (62% recovery rate) (originally 41%). Using the X2 linker yielded 21 mg (50% recovery rate) with a purity of 93%. P2 was purified on a 5 μmol scale using the X2 linker sample, yielding 4 mg (73% recovery rate) with a purity of 95% (originally 55%).

[0246] [Table 2]

[0247] Linker Type 4 Example 2: Purification of Naturally Derived and Research-Grade Peptides, P3, P4, P5, and P6 In the second set, five peptides were synthesized. These are: H-YFTGSEVENVSVNVH-NH2 (SEQ ID NO: 3) (P3) fragments 81-95 of human cytomegalovirus lower matrix phosphoprotein (CMV), H-PSNPFYEALST-NH2 (SEQ ID NO: 4) (P4) fragments 510-520 of human Lemur tyrosine kinase 3 (LMTK3), H-DAEFRHDSGYEVHHQKLVFF-NH2 (SEQ ID NO: 5) (P5) fragments 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. The synthetic resin was then 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 oxima (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 synthetic resin with a mixture of TFA / PhOH / PhSH / H2O / 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 precipitated beads) were washed three times each with water and 0.1 M sodium citrate buffer at pH 4.5. A DMSO solution of the peptide was then mixed with 10 vol.% (500 μL) of sodium citrate buffer containing 8 M guanidium hydrochloride. This solution was then applied to the agarose beads for 90 minutes to quantitatively immobilize the target peptide onto the beads. Subsequently, to block unreacted aldehyde groups, a 1 wt% solution of L-cysteine ​​in 0.1 M sodium citrate buffer at pH 4.5 was directly added to the immobilized mixture for 15 minutes. The purified medium was then washed three times each with DMSO, 6 M guanidium hydrochloride, EtOH / water (7:3) containing 0.1 M NaCl, water, and MeCN to remove all acetylated terminal sequences and other impurities. The immobilized linker was cleaved by treating the agarose resin with 10 mL of 50 mg / mL PPh3 MeCN / AcOH / H2O (90:5:5). The support was then rinsed three times with MeCN / H2O (9:1), and 2 mL of H2O / TFA (60:40) solution was added over 60 minutes. 2 mL of TFA was then added to the supernatant, and the resulting mixture was filtered into a centrifuge tube. The support was then rinsed twice in the same tube with TFA / H2O (95:5). Et2O was added five times relative to the TFA-water volume to initiate precipitation, and the peptide was obtained by centrifugation of the tube and treatment of the organic supernatant.

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

[0250] [Table 3]

[0251] Implementation of Type 1 Linker 1: Purification of Research Peptide P2 The present invention's method for purifying peptides having a type 1 linker was applied to the peptide H-AKADEVSLHKWYG-NH2 (SEQ ID NO: 2) (P2) (this is the peptide sequence intended for research).

[0252] The peptides were synthesized under standard solid-phase peptide synthesis conditions, thereby blocking unreacted amino groups in the synthetic resin by treating it with acetic anhydride and pyridine after each amino acid coupling. Type 1 linker X9 was coupled to P2 on the resin for 2 hours in dimethylformamide (DMF) using 4 equivalents of linker, 6 equivalents of oxima, and 6 equivalents of diisopropylamine (DIEA). The peptides were then cleaved from the synthetic resin with a mixture of TFA / TIS / DTT / H2O (84:2:6:8) and precipitated in diethyl ether. 30 mg of linker-modified peptide was obtained. The present invention method for 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). Aldehyde-modified agarose beads were added to a cartridge containing 75 μL each of 50% bead suspension in H2O / EtOH (4:1) slurry, and the beads were then washed three times with water and 0.1 M sodium citrate buffer at pH 4.5. 10 vol.% (10 μL) of sodium citrate buffer containing 8 M guanidium hydrochloride was added to the DMSO solution of the peptide. Then, 110 μL of this solution was applied to the agarose beads over 90 minutes, thereby quantitatively immobilizing the desired peptide onto the agarose beads (UPLC analysis of the immobilized supernatant). The immobilized mixture was then interfiltration over 15 minutes to block unreacted aldehyde groups and reverse imine formation, after which 100 μL of a 2 wt% solution of L-cysteine ​​in 0.1 M sodium citrate buffer at pH 4.5 was added to the beads. Subsequently, the purified medium was washed three times with 500 μL each of DMSO containing 0.9 M guanidinium hydrochloride and EtOH / water (7:3) containing 0.1 M NaCl to remove acetylated terminal sequences and other impurities. The immobilized linker peptide was cleaved by treating the agarose resin with 200 μL of TCEP (25 mg / mL) for 30 minutes per reactor. The support was then rinsed three times with H2O containing 0.1% TFA. 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, releasing 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 present invention's method for purifying peptides having a type 2 linker was applied to the peptide H-AKADEVSLHKWYG-NH2 (SEQ ID NO: 2) (P2) (this is the peptide sequence intended for research).

[0255] The peptides were synthesized under standard solid-phase peptide synthesis conditions, thereby blocking unreacted amino groups in the synthetic resin by treating it with acetic anhydride and pyridine after each amino acid coupling. Linker X13 was coupled to P2 on the resin over 3 hours using 4 equivalents of linker, 6 equivalents of oxima, and 6 equivalents of diisopropylamine (DIEA) in dimethylformamide (DMF). This method of the present invention for linker type 2 is shown in Figure 6. Subsequently, the peptides were cleaved from the synthetic resin with a mixture of TFA / TIS / DTT / H2O (84:2:6:8). The crude peptide mixture was dissolved in dimethyl sulfoxide (DMSO). Aldehyde-modified agarose beads were washed three times each with water and 0.1 M sodium citrate buffer at pH 4.5. 10% by volume of 6 M GdmCl in sodium citrate buffer was added to the DMSO solution of the peptides. The obtained 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% by weight L-Cys in sodium citrate buffer for 15 minutes. Subsequently, the beads were washed three times each with DMSO, 6M aqueous GdmCl, EtOH / 0.1M NaCl (7:3), water, MeCN, and 0.1 vol% TFA in EtOH to remove acetylated terminal sequences and other impurities. The immobilized linker was cut by treating the agarose resin with 10 equivalents of SnCl2 in EtOH (0.5M) for 3 hours. Subsequently, the support was washed three times each with 0.1 vol% TFA in EtOH, 0.1 vol% TFA in H2O, and MeCN / H2O (9:1). A 0.2 M aqueous NH4HCO2 (pH 8.85) / MeCN (1:1) solution was added over 15 minutes. The supernatant was filtered into a centrifuge tube, and the support was rinsed twice with H2O in the same tube. The residue was freeze-dried to obtain the purified peptide.

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

[0257] [Table 5]

[0258] Implementation of Type 3 Linker 1: Purification of Research Peptide P2 The present invention's method for purifying peptides having a type 3 linker was applied to the peptide H-AKADEVSLHKWYG-NH2 (SEQ ID NO: 2) (P2) (this is the peptide sequence intended for research).

[0259] The peptides were synthesized under standard solid-phase peptide synthesis conditions, thereby blocking unreacted amino groups in the synthetic resin by treating it with acetic anhydride and pyridine after each amino acid coupling. Type 3 linker X22 was coupled to P2 on the resin over 2 hours using 4 equivalents of linker, 3.6 equivalents of 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium-hexafluorophosphat (HCTU), 4 equivalents of oxima, and 8 equivalents of diisopropylamine (DIEA) in dimethylformamide (DMF). The peptides were then cleaved from the synthetic resin with a mixture of TFA / TIS / DTT / H2O (84:2:6:8) and precipitated in diethyl ether. 27 mg of linker-modified peptide was obtained. The present invention method for this linker type 3 is shown in Figure 7. 2.1 mg of the crude peptide mixture was dissolved in 100 μL of dimethyl sulfoxide (DMSO). Aldehyde-modified agarose beads were added to a cartridge containing 75 μL each of 50% bead suspension in H2O / EtOH (4:1) slurry, and the beads were then washed three times with water and 0.1 M sodium citrate buffer at pH 4.5. 10 vol.% (10 μL) of sodium citrate buffer containing 8 M guanidium hydrochloride was added to the DMSO solution of the peptide. Then, 110 μL of this solution was applied to the agarose beads over 90 minutes, thereby quantitatively immobilizing the desired peptide onto the agarose beads (UPLC analysis of the immobilized supernatant). The immobilized mixture was then interfiltration over 15 minutes to block unreacted aldehyde groups and reverse imine formation, after which 100 μL of a 2 wt% solution of L-cysteine ​​in 0.1 M sodium citrate buffer at pH 4.5 was added to the beads. Subsequently, the purification medium was washed three times with 500 μL each of DMSO containing 0.9 M guanidia hydrochloride and EtOH / water (7:3) containing 0.1 M NaCl to remove acetylated terminal sequences and other impurities. The immobilized linker peptide was cleaved by treating the agarose resin with 200 μL of TCEP (25 mg / mL) for 30 minutes in each reactor. The support was then rinsed three times with H2O containing 0.1% TFA.By using an amine switch with nucleophilic release, the peptide was released from agarose, and therefore 200 μL of 0.2 M NEt3 in an aqueous solution at pH 7 was added to the beads for 100 hours. This released piperazinyl and aniline-NH3. + The portion was deprotonated, and then the peptide was released by nucleophilic attack with aniline-NH2. The identity of the desired peptide was confirmed by ESI / MS (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 N-bromosuccinimide (6.12 g, 34.05 mmol, 1 equivalent) was added to a cooled solution (0°C) of 6-aminophthalide (5.13 g, 34.05 mmol, 1 equivalent) in THF (80 mL). The cooling bath was removed, the solution was stirred for 1 hour, and then the solvent was removed under reduced pressure. The yellow residue was taken into 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 obtain 6-amino-7-bromophthalide as a brown solid (6.53 g, 28.63 mmol, 84%). f =0.2(cyclohexane / ethyl acetate 2:1);UPLC-MS:t R = 1.45 minutes (Gradient 10-90% B in 5 minutes); UPLC purity (210nm) = 83.1%; ESI-MS: (Calculated MH) + (227.97, 229.96, Detected: 228.01, 230.01)

[0263] 6-Azid-7-Bromophthalide 6-amino-5-bromophthalide (5.54 g, 24.17 mmol) was added to 100 mL of 0°C cold hydrochloric acid (1 M). Concentrated sulfuric acid was added dropwise to the cooled suspension with stirring until the solid was completely dissolved (25 mL), and the solution was further cooled until it reached 0°C again. A solution of sodium nitrite (3.34 g, 48.34 mmol, 2 equivalents) in water (17 mL) was slowly added (if the solution is too warm, nitrite gas will be formed). After stirring for 10 minutes, a solution of sodium azide (3.14 g, 48.34 mmol, 2 equivalents) in water (20 mL) was slowly added dropwise (Caution: hydroazic acid will be formed). After 30 minutes, 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 minutes (Gradient B with a 10-90% gradient in 5 minutes); UPLC purity (210nm) = 50.3%

[0264] N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamide)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide 6-azido-7-bromophthalide (5.54 g (94%), 24.17 mmol) was added to acetonitrile (150 mL), and the suspension was heated to 50°C with stirring. Ethylenediamine (23.4 mL, 350.47 mmol, 14.5 equivalents) was added, and the solid was completely dissolved after 10 minutes. After stirring at 50°C for 1 hour, the solvent and excess ethylenediamine were removed under reduced pressure to obtain a red oil residue (8.49 g). Saturated brine (80 mL) was added, and the resulting suspension was sonicated for 30 minutes, stirred at 40°C for 30 minutes, and filtered. The filtered cake was washed once with saturated brine (50 mL) and once with cyclohexane (100 mL). After drying the filtered 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 mins); 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)acetamide)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide Dicyclohexylcarbodiimide (DCC, 3.30 g, 15.86 mmol, 1.3 equivalents) is added to a stirred solution of bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)2AOAc-OH, 4.71 g, 15.86 mmol, 1.3 equivalents) and NHS (1.84 g, 15.86 mmol, 1.3 equivalents) in acetonitrile (40 mL). After stirring for 1 hour, the solution is separated from the white precipitate obtained by filtration, and the filtrate cake is washed with acetonitrile (40 mL). The filtrate is 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 equivalent) is added to acetonitrile (30 mL), and the suspension is sonicated for 50 minutes. Next, the filtrate containing (Boc)2AOAc-NHS is added to this suspension, and the reaction mixture is stirred for 2.5 hours. After removing the solvent under reduced pressure, ethyl acetate (150 ml) is added to the resulting orange oil (10.47 g), the suspension is sonicated for 10 minutes, and then stirred at 50°C for 10 minutes. 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 obtain 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 (measured by UV / Vis), 8.31 mmol, 68.91% yield). c=0.15(DCM / MeOH 95:5);UPLC-MS:t R = 2.60 minutes (10-90% MeCN in 3 minutes), UPLC purity (21nm) = 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 equivalents) was first added with stirring, and then a solution of p-nitrophenyl chloroformate in DCM (20 mL) (2.52 g, 12.48 mmol, 1.5 equivalents) was slowly added. The reaction mixture was warmed to room temperature and stirred for 1 hour. 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 obtain a yellow foamy solid as the crude product (8.85 g). After purification by column chromatography (silica gel, cyclohexane:ethyl acetate 2:1-1:1), the product (3.82 g) was placed in 100 mL of diethyl ether, treated with sonication for 10 minutes, stirred at 40°C for 30 minutes, and then stored overnight at -20°C. The product was filtered, washed with 100 mL of cold diethyl ether at -20°C, dried under high vacuum to obtain 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 minutes (10-90% MeCN in 5 minutes), UPLC purity (278nm) = 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 stirring 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 to the solution as a solid through a powder funnel. The ice bath was removed, and after a while the solution turned yellow. After stirring at room temperature for 2 hours, UPLC-MS and TLC showed complete conversion to dibromide. The solvent was removed under reduced pressure using 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. f =0.6 (cyclohexane / ethyl acetate 2:1), UPLC-MS:t R = 2.36 minutes (10-90% MeCN in 3 minutes), UPLC purity (254nm) = 87.0%, ESI-MS: (calculated MH) + (307.95 g / mol, detection: 307.76 m / 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. This brown solution was cooled in a large bucket of ice, and then 235 mL of 1 M HCl was slowly added, with a precipitate forming during the addition of HCl. NaNO2 (17.31 g, 248.35 mmol, 2 equivalents) was dissolved in 32 mL of water, and after it reached 5°C, it was slowly added to the suspension. The precipitate then dissolved. The solution was stirred further at 0°C for 15 minutes, and then NaN3 (16.31 g, 248.35 mmol, 2 equivalents) in 75 mL of water was added dropwise using a Pasteur pipette. Strong gas formation (N2, HN3) was observed. The foamy solution was stirred for 1 hour, and then 500 mL of water was added under ice cooling. After the bubbling stopped and the solution reached room temperature, the suspension was filtered using a Buchner funnel, while the solid was washed by transferring it to the funnel with 2 L of water. After drying the wet product under reduced pressure in a crystallization 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 minutes (10-90% MeCN in 3 minutes), UPLC purity (254nm) = 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), and insoluble impurities were filtered off. Ethylenediamine (52.73 mL, 749.32 mmol, 14 equivalents) was added at 0°C. The reaction mixture was stirred at room temperature for 1 hour, after which ULC-MS showed a quantitative transformation. The reaction mixture was transferred to a separatory funnel to which 100 mL of brine had been added. After separating the aqueous phase, the organic phase was dried over MgSO4, and the organic solvent was evaporated as an orange solid using a rotary evaporator to obtain the desired product (20.50 g, 52.16 mmol, 97.4%). f =0.25(DCM / MeOH 8:2), UPLC-MS:tR = 1.80 minutes (10-90% MeCN in 3 minutes), UPLC purity (254nm) = 84.2%, ESI-MS: (calculated MH) + (393.93 g / mol, detection: 393.87 m / z)

[0272] N-(2-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetamide)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 equivalents) and N-hydroxysuccinimide (NHS, 6.76 g, 58.18 mmol, 1.1 equivalents) were dissolved in 350 mL of acetonitrile. Dicyclohexylcarbodiimide (DCC, 12.13 g, 58.18 mmol, 1.1 equivalents) was added as a solid to this solution, and a white precipitate formed after the dissolution of DCC. The reaction mixture was stirred at room temperature for 1 hour, where (Boc)2AOAc-NHS ester was quantitatively formed according to ULC-MS. The mixture was then filtered into a 1 L flask to remove 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 subsequently added to a 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 ULC-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) / saltwater 1:1 (150 mL each). The organic phase was dried over MgSO4, thus removing the organic solvent under vacuum, and the title compound was obtained as a pale yellow oil (37.70 g, 56.58 mmol, quantitative). f =0.4(DCM / MeOH 95:5), UPLC-MS:t R = 2.97 minutes (10-90% MeCN in 3 minutes), UPLC purity (254nm) = 54.2%, ESI-MS: (calculated MH) + :667.05, MNa +(689.04 g / mol, detected at 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 CH2Cl2 (170 mL), and pyridine (4.72 mL, 58.50 mmol, 1.1 equivalents) (dried and placed on a molecular sieve) was added. Then, 4-nitrophenyl chloroformiate (12.03 g, 58.50 mmol, 1.1 equivalents) was slowly added as a solid at room temperature while maintaining a constant temperature using a water bath. The reaction may cause evaporation of the DCM in the center of the flask. Complete reaction was observed after 1 hour, as shown by LCMS and TLC. Dichloromethane was removed under vacuum to obtain 52 g of crude brown oil. The residue was dissolved in 500 mL of ethyl acetate and washed twice with 2% citric acid solution (pH 4.5) / saline solution 1:1 (250 mL each) and once with 150 mL of saline solution. The organic phase was dried using MgSO4. The suspension was then filtered through glass frit using a 50 g silica plug, but orange and reddish impurities remained in the silica. The organic solvent was removed from the filtrate under reduced pressure until a high-viscosity, slightly amber oil remained. 70 mL of Et2O was added to this oil, and the two-phase emulsion was rotated in a rotary evaporator at 45°C for 10 minutes until a single homogeneous phase was formed. A small sand cone was added to the flask as a crystallization initiator, and the flask was left in the refrigerator overnight (16 hours). A thick precipitate had formed in the flask. Next, 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 Buchner funnel filled with a filter and washed with 200 mL of cold Et2O. 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 minutes (30-95% MeCN in 3 minutes), UPLC purity (278nm) = 93.5%, ESI-MS: (calculated MH) + :832.06, MNa +(854.04 g / mol, detected at 853.87 m / 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 an amine switch with 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)piperazine-1-yl)methyl)-4-azidobenzyl(4-nitrophenyl)carbonate (X6)

[0276] 4-Nitro-3-(piperazine-1-ylmethyl)benzoic acid 5.03 g, 25.50 mmol of 4-nitro-3-methylbenzoate methyl ester was dissolved in 170 mL of dry benzene in a 500 mL round-bottom flask equipped with a stirring bar. 5.27 g, 29.33 mmol of N-bromosuccinimide and 0.62 g, 2.55 mmol of benzoyl peroxide were added, and the solution was heated under reflux. After 12 hours, ULC-UV / Visible showed only 10% conversion to the brominated starting material. Again, 3.66 g, 20.56 mmol of N-bromosuccinimide and 0.62 g, 2.55 mmol of benzoyl peroxide were added. After 36 hours, ULC-UV / Visible showed 80% conversion. The mixture was concentrated under vacuum, and 85 mL of chloroform was added. The mixture 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 stirring bar. After 2 hours, UPLC-UV / Visible showed complete conversion to the desired product. The solvent was removed using 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 evaporating, 7.3 g (not completely dried) of the desired product was obtained as a yellow oil. UPLC-MS:t R = 1.602 min (10~90% MeCN in 3 min), UPLC purity (278 nm) = 68.3%, ESI-MS: (calculated MH + (Detection: 280.12 g / mol, detection: 280.11 m / z)

[0277] (4-Nitro-3-(piperazine-1-ylmethyl)phenyl)methanol 4-nitro-3-(piperazine-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 stir bar. LiCl (0.77 g, 18.00 mmol), NaBH4 (0.69 g, 18.00 mmol), and 16 mL of ethanol were added sequentially. After 12 hours, ULC-UV / Visible showed complete conversion of the starting materials. The reaction mixture was concentrated under vacuum 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 hours, 25 mL of 1 M NaOH was slowly added and the mixture was transferred to a separation funnel. This layer was separated and the aqueous phase was extracted twice with 50 mL of ethyl acetate and twice with 50 mL of chloroform. The combined organic layers were dried over MgSO4, and after evaporation of the organic solvent, the desired product was obtained as a yellow solid (0.59 g, 2.35 mmol, 88%). UPLC-MS:t R = 1.27 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 50.4%, ESI-MS: (calculated MH) + (Detection: 252.29 g / mol, detection: 252.17 m / z)

[0278] 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazine-1-yl)ethane-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 stir bar. 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-(piperazine-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, ULC-UV / Visible showed complete conversion of the starting materials. The solvent was removed using 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 three times with 50 mL of water, three times with 50 mL of saturated NaHCO3 solution, and three times with 50 mL of brine, and then dried over MgSO4. After evaporating the organic solvent, the desired product was obtained as a yellow solid (1.8 g, not completely dried). UPLC-MS:t R = 2.24 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 19.8%, ESI-MS: (calculated MH) + (Detection: 525.14 g / mol, detection: 525.25 m / z)

[0279] 1-(4-(2-amino-5-(hydroxymethyl)benzyl)piperazine-1-yl)-2-bis-(tert-butoxycarbonyl)-(aminooxy)ethane-1-one In a round-bottom flask, 2-bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazin-1-yl)ethane-1-one was dissolved in 16 mL of water / ethanol (1:4). Then, iron powder (0.23 g, 4 mmol) and ammonium chloride (0.25 g, 4 mmol) were added to the solution. The reaction mixture was stirred overnight at room temperature. After confirming the complete consumption of the starting materials via UPLC-MS, the reaction mixture was filtered through Celite to remove excess iron. The solvent was concentrated under vacuum as much as 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 the organic solvent was removed under reduced pressure using a rotary evaporator. The final product (0.19 g, 0.4 mmol) was obtained as a brown oil. UPLC-MS:t R = 2.05 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 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)piperazine-1-yl)ethane-1-one In a round-bottom flask, 1-(4-(2-amino-5-(hydroxymethyl)benzyl)piperazin-1-yl)-2-bis-(tert-butoxycarbonyl)-(aminooxy)ethane-1-one (0.19 g, 0.4 mmol) was dissolved in dry acetonitrile. While stirring, the solution was cooled in an ice bath, and tert-butylnitrile (236 μL, 2 mmol) and then trimethylsilyl azide (351 μL, 1.6 mmol) were slowly added. This solution was further stirred in a sealed flask for 2 hours while cooling on ice. After UPLC-MS showed incomplete conversion, tert-butylnitrile (572 μL, 4 mmol) and then trimethylsilyl azide (702 μL, 3.2 mmol) were slowly added again. The reaction mixture was stirred for a further 2 hours. The solvent was then removed under reduced pressure using 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 and three times with 50 mL of aqueous salt solution, then dried on magnesium sulfate. After evaporating the organic solvent, the desired product (0.14 g, 0.20 mmol) was obtained. UPLC-MS:t R = 2.34 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 20.4%, ESI-MS: (calculated MH) + (521.27, detected: 521.30 m / z)

[0281] 3-((4-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetyl)piperazine-1-yl)methyl)-4-azidobenzyl(4-nitrophenyl)carbonate(X6) 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)benzyl)piperazin-1-yl)ethane-1-one (149 mg, 0.2 mmol) was dissolved in a round-bottom flask using 0.2 mL of dry DCM. 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 chloroformiate (32.9 mg, 0.16 mmol) in 0.2 mL of dry DCM was slowly added, and the reaction mixture was stirred overnight at room temperature. The organic solvent was removed under vacuum, and the desired product was obtained as a dark brown oil (0.21 g, not completely dried) ULC-MS:t R = 10.72 minutes (0-60% MeCN in 11 minutes), UPLC purity (278nm) = 8.39%, ESI-MS: (calculated MH) + :686.28, MNa + (708.26 g / mol, detection: 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)ethane-1-one 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethane-1-one (1.00 g, 1.96 mmol) was dissolved in 78 mL of methanol in a round-bottom flask. Magnesium powder (1.28 g, 19.6 mmol) was added to this solution. Ammonium formate (1.23 g, 19.6 mmol) was added as a solid under stirring. The solution was further stirred at room temperature for 20 minutes, during which gas formation was observed. The solution was immediately filtered to remove magnesium, and the solvent was removed under reduced pressure using a rotary evaporator. The crude product was lyophilized using a 1:1 water / acetonitrile solvent mixture. This crude product (2.20 g) was used without any further purification. UPLC-MS:t R = 2.21 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 53.2%, ESI-MS: (calculated MH) + : 481.27 g / mol, MNa + (503.25 g / mol, detection: 477.30 m / z)

[0283] 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)phenyl)piperazine-1-yl)ethane-1-one In a round-bottom flask, 1-(4-(2-amino-5-(hydroxymethyl)phenyl)piperazin-1-yl)-2-bis-(tert-butoxycarbonyl)-aminooxy)ethane-1-one (1.00 g, 2.08 mmol) was dissolved in dry acetonitrile. While stirring, the solution was cooled in an ice bath, and tert-butylnitrile (1.37 mL, 10.4 mmol) and then trimethylsilyl azide (1.16 mL, 8.32 mmol) were slowly added. This solution was further stirred in a sealed flask for 2 hours while cooling on ice. After UPLC-MS showed incomplete conversion, tert-butylnitrile (1.37 mL, 10.4 mmol) and then trimethylsilyl azide (1.16 mL, 8.32 mmol) were slowly added again. The reaction mixture was stirred further overnight. The solvent was then removed under reduced pressure using 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 and three times with 50 mL of aqueous salt solution, and dried on magnesium sulfate. After evaporating the organic solvent and purifying with a flash column (3:2 EtOAC / cyclohexane), the desired product (0.2 g, 0.39 mmol) was obtained. f =0.5(siRNA / cyclohexane 4:1), UPLC-MS:t R = 2.92 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 84.2%, ESI-MS: (calculated MH) + : 507.26 g / mol, MNa + (Detection: 529.24 g / mol, detection: 529.30 m / z)

[0284] 3-(4-(2-bis-(tert-butoxycarbonyl)-(aminooxy)acetyl)piperazine-1-yl)-4-azidobenzyl(4-nitrophenyl)carbonate(X9) 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)phenyl)piperazin-1-yl)ethane-1-one (76 mg, 0.15 mmol) was dissolved in a round-bottom flask using 1 mL of dry DCM. 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 minutes, the ice bath was removed, and the reaction mixture was stirred overnight at room temperature. The organic solvent was removed under vacuum, and the crude product was redissolved in 30 mL of ethyl acetate. 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 removed again under vacuum. The crude product was finally purified by flash column using an ethyl acetate / 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 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 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 having other reductive safety lock (type 2) linker molecules X12, X13, and X43 Synthesis of (5-(2-(2-bis-(tert-butoxycarbonyl(aminooxy)acetamide)-6-(tert-butyldisulfanyl)pyridine-3-yl)methyl(4-nitrophenyl)carbonate (X12) Synthesis of methyl 6-melacapto-5-nitropyridine-3-carboxylate To a chilled 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 gradually added. The mixture was allowed to stand for 30 minutes to 1 hour with stirring. 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 slowly adding 1 M HCl at 0°C. The resulting yellow solid material was recovered by filtration and used in further steps without purification. UPLC-MS:t R = 1.80 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 95.0%, ESI-MS: (calculated MH) + (Detection: 215.01 g / mol, detection: 215.20 m / z)

[0287] Synthesis of methyl 6-melacapto-5-aminopyridine-3-carboxylate Methyl 6-melacapto-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 over an oil bath until the starting materials were completely converted to the product. After refluxing, 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 times × 25 ml). The solvent was removed under reduced pressure, and the resulting crude residue was of sufficient purity for further analysis (3.49 mmol, 77%). UPLC-MS:t R= 1.26 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 93%, ESI-MS: (calculated MH) + (Detection: 185.04 g / mol, detection: 185.14 m / z)

[0288] Synthesis of (5-amino-6-melacaptopyridine-3-yl)methanol Methyl 6-melacapto-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 gradually added to the reaction mixture under an N2 atmosphere for 10 minutes. The resulting solution was stirred at room temperature for 24 hours. After the reaction was complete as shown in UPLC, the 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 salt was 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 times × 50 ml). The isopropanol was removed under reduced pressure to obtain the desired product in pure form (1.92 mmol, 52%). UPLC-MS:t R = 1.01 min (10-90% MeCN in 3 mins), UPLC purity (278 nm) = 96%, ESI-MS: (calculated MH) + (Detection: 157.04 g / mol, detection: 157.11 m / z)

[0289] Synthesis of (2-(2-bis-(tert-butoxycarbonyl)(aminooxy))-N-(2-(tert-butyldisulfanyl)-5-(hydroxymethyl)pyridine-3-yl)acetamide) 6-Mercapto-5-aminopyridine-3-carboxylic acid (1.3 gm, 8.03 mmol) was placed in a Schlenk flask, to which CH2Cl2 (20 mL), 2-methyl-2-propantheol (0.91 ml, 8.03 mmol), and TBHP (1.13 gm, 8.83 mmol, 70% water solution) were added under an N2 atmosphere. After 30 seconds, NIS (0.19 gm, 0.18 mmol) was added in one batch. The Schlenk flask was then allowed to react at 25°C for 1 hour. After the reaction was complete, the mixture was quenched with water (20.0 mL) and extracted with ethyl acetate (3 times × 30 mL). The organic layers were combined and evaporated under vacuum, and the compounds were used in the next step without further purification. UPLC-MS:t R = 1.68 min (10-90% MeCN in 3 mins), UPLC purity (278 nm) = 93%, ESI-MS: (calculated MH) + (Detection: 245.08 g / mol, detection: 245.18 m / z)

[0290] Synthesis of (5-(2-(2-bis-(tert-butoxycarbonyl(aminooxy)acetamide)-6-(tert-butyldisulfanyl)pyridine-3-yl)methyl(4-nitrophenyl)carbonate (X12) (5-amino-6-(tert-butyldisulfanyl)pyridine-3-yl)methanol (83.5 mg, 0.156 mmol) was added to CH2Cl2 (2 mL), and dried pyridine (0.161 mL, 0.19 mmol) was added. Then, 4-nitrophenyl chloroformiate (31.9 mg, 0.154 mmol) was added. LC-MS and TLC showed that the reaction was complete after 12 hours. Dichloromethane was removed under vacuum to obtain a pale yellow / orange crude oil. The residue was redissolved in 10 mL of ethyl acetate and washed with water and brine in a 1:1 ratio (5 mL each). Finally, EtoAc was removed under reduced pressure to obtain the desired product (0.08 mmol, 75%). UPLC-MS:t 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 + (Detection: 705.19 g / mol, detection: 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 using a stirring bar, and 3 mL of (54.02 mmol) H₂SO₄ was added. After stirring at 50°C for 42 hours, UPLC-UV / Visible showed complete conversion to methyl ester. The reaction mixture was concentrated to 10 mL using a rotary evaporator. After adding 100 mL of ethyl acetate and 100 mL of H₂O, the mixture was stirred, and then K₂CO₃ (7.00 g) was added gradually. 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 layer was dried over MgSO₄ and evaporated to obtain 4.81 g (24.15 mmol, 89%) of the desired product as an orange solid. UPLC-MS:t R = 2.53 minutes (10-90% MeCN in 3 minutes), UPLC purity (210nm) = 99.4%, ESI-MS: (calculated MH) + :200.14 g / mol, detected:-).

[0292] Methyl 4-nitro-3-(piperazine-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 using a stirring bar. Methyl 3-fluoro-4-nitrobenzoate (4.7 g, 23.60 mmol), dissolved in 50 mL of chloroform, was slowly added from a dropping funnel at room temperature over 1 hour, and the mixture was rapidly stirred. After 66 hours, ULC-UV / Visible showed that only 10% of the starting material had been converted. Piperazine (4.07 g, 47.20 mmol) was added, and after 2 hours, ULC-UV / Visible showed complete conversion. After 15 minutes, 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 with MgSO4, the organic solvent was evaporated using a rotary evaporator, and the desired product was obtained as a red solid (7.87 g, not completely dried). UPLC-MS:t R = 1.72 minutes (10-90% MeCN in 3 minutes), UPLC purity (210nm) = 87.2%, ESI-MS: (calculated MH) + (Detection: 266.27 g / mol, detection: 266.23 m / 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 successively. After 15 hours, ULC-UV / Vis showed complete conversion. The reaction mixture was concentrated under vacuum, 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 hour, 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. The combined organic layers were dried over MgSO4, and after evaporation of the organic solvent, the desired product was obtained as an orange solid (3.97 g, 16.73 mmol, 84%). UPLC-MS:t R = 1.27 minutes (10-90% MeCN in 3 minutes), UPLC purity (210nm) = 70.0%, ESI-MS: (calculated MH) + (Detection: 238.26 g / mol, detection: 238.24 m / z)

[0294] 2-Bis-(tert-butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazine-1-yl)ethane-1-one Bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)2AOAcOH, 2.96 g, 9.96 mmol, 1.2 equivalents) and N-hydroxysuccinimide (NHS, 1.15 g, 9.96 mmol, 1.2 equivalents) were dissolved in 10 mL of acetonitrile. Dicyclohexylcarbodiimide (DCC, 2.07 g, 9.96 mmol, 1.2 equivalents) was added as a solid to this solution, and a white precipitate formed after the dissolution of DCC. The reaction mixture was stirred at room temperature for 1 hour, where the (Boc)2AOAc-NHS ester was quantitatively formed according to ULC-MS. The mixture was then filtered into a round-bottom flask containing (4-nitro-3-(piperazine-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. Further precipitates were filtered off, and the organic phase was washed three times with water, three times with saturated NaHCO3 solution, and three times with aqueous salt solution. The organic phase was dried over MgSO4, thus removing the organic solvent under vacuum, and the title compound was obtained as a yellow oil (4.35 g, 8.52 mmol, quantitative, not completely dried). UPLC-MS:t R = 3.00 min (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 48.2%, ESI-MS: (calculated MH) + :511.24, MNa + :534.23 g / mol, detected:-).

[0295] 3-(4-(2-bis-(tert-butoxycarbonyl)-((aminooxy)acetyl)piperazine-1-yl)-4-nitrobenzyl(4-nitrophenyl)carbonate(X13) 2-Bis-(tert-butoxycarbonyl-aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethane-1-one (2.06 g, 4 mmol) was placed in an 8 mL round-bottom flask equipped with a stirring bar. Dry pyridine was added to this solution. Then, 4-nitrophenyl chloroformiate (0.86 g, 4.2 mmol) was slowly added as a solid at room temperature. The reaction is exothermic and may cause foaming of DCM. After stirring at room temperature for 2 hours, UPLC-MS and TLC showed complete conversion to carbonate. The solvent was removed under reduced pressure using a rotary evaporator. The product was purified by column chromatography (cyclohexane / ethyl acetate 1:1) to obtain a yellow solid (410 mg, 0.61 mmol). UPLC-MS:t R = 3.44 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 92.4%, ESI-MS: (calculated MH) + : 676.25 g / mol, MNa + (698.23 g / mol, detected at 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)ethane-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 stirring 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 successively. After x hours, UPLC-UV / Visible showed complete conversion of the starting material. The solvent was removed using 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 / cycloxane (1:1), the desired product (0.02 g, 0.03 mmol) was obtained. UPLC-MS:t R = 2.96 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 55.6%, ESI-MS: (calculated MNa + (Detection: 712.24 g / mol, detection: 712.41 m / z)

[0298] Chemical synthesis of amine switches having nucleophilic release (type 3) linker molecules X22 and X42 Synthesis of 2-(2-azido-5-(4-(2-((2-bis-(tert-butoxycarbonyl)amino)oxy)acetyl)piperazine-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 stirring 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 the reaction was complete, 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 obtain the desired product as a white solid (129.86 mmol, 98%). UPLC-MS:t R = 2.41 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 97%, ESI-MS: (calculated MH) + :214.05 g / 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 overnight at a maximum of 80°C. The progress of the reaction was monitored by UPLC-MS and TLC. After the completion of the reaction, the solution was filtered to remove solid byproducts, and the DMF was removed under reduced pressure using a rotary evaporator. Ice-cold water (25 ml) was added to the crude material, the resulting solid was filtered, washed with water (50 ml), and dried under reduced pressure to obtain the desired product (1.01 gm, 2.66 mmol, 82%) as a yellow solid UPLC: R = 3.01 min (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 96.0%, ESI-MS: (calculated MH) + :380.18 g / mol, detection: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 / H2O (25 mL, 3:1), NH4Cl (1.23 gm, 1.01 gm, 18.50 mmol) and zinc powder (1.23 gm, 18.50 mmol) were added at room temperature. The reaction mixture was stirred at the same temperature for 3 hours, and then filtered onto a Celite bed. The resulting solution was evaporated, and the crude material was compartmentalized between H2O (100 mL) and siRNA (300 mL). The organic layer was separated, dried, and concentrated to (MgSO4) to obtain the desired product as a yellow solid (1.75 mmol, 68%). UPLC-MS:t R = 2.12 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 94.0%, ESI-MS: (calculated MH) + (Detection: 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 0.35 gm, 0.99 mmol of tert-butyl 4-(4-amino-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate was dissolved in 25 mL of dry acetonitrile and cooled to 0°C. 90% tert-butyl nitrite (0.837 g, 7.94 mmol) was added dropwise to the reaction mixture, followed by the addition of TMSN3 (0.722 g, 5.95 mmol) over 10 minutes. The resulting red mixture was stirred for 3 hours. The progress of the reaction was monitored by ULC-MS. After the reaction was complete, 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 (twice in 50 mL batches). The ethyl acetate layer was dried over MgSO4 and removed under reduced pressure to obtain an orange solid (which was not further purified) (0.33 gm, 0.88 mmol). UPLC-MS:t R = 3.01 min (10-90% MeCN in 3 mins), UPLC purity (278nm) = 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)piperazine-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 H2O (0.2 mL) was stirred at room temperature for 3 hours. After the starting material was completely converted 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 ELISA (2 times × 25 mL), dried over MgSO4, and concentrated to yield the product as a yellow solid (0.65 mmol, 78%). UPLC-MS: R = 2.55 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 95.0%, ESI-MS: (calculated MH) + (362.18 g / mol, detection: 306.04 m / z)

[0304] Synthesis of 4-(4-azido-3-(carboxymethyl)phenyl)piperazine-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 the reaction was complete (monitored by UPLC-MS), cold ether (25 mL) was added, the resulting solid was filtered, and washed with cold ether. The resulting light brown solid (0.30 gm, 85%) was dried and proceeded to the next step without purification. UPLC-MS:t R = 2.80 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 87.0%, ESI-MS: (calculated MH) + (262.13 g / mol, detected at 262.11 m / z) Incorrect mass.

[0305] Synthesis of 2-(2-azido-5-(4-(2-((((2-bis-(tert-butoxycarbonyl)amino)oxy)acetyl)piperazine-1-yl)phenyl)acetic acid (X22) Bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)2AOAcOH, 88.4 mg, 0.30 mmol) and N-hydroxysuccinimide (NHS, 34.6 g, 0.30 mmol) were dissolved in 2 mL of dry acetonitrile. Dicyclohexylcarbodiimide (DCC, 62.0 mg, 0.30 mmol) was added to this solution as a solid, and a white precipitate formed after the dissolution of DCC. The reaction mixture was stirred at room temperature for 1 hour, where the (Boc)2AOAc-NHS ester was quantitatively formed according to ULC-MS. Subsequently, to remove DCC-urea, the mixture was filtered directly onto a reaction flask containing 4-(4-azido-3-(carboxymethyl)phenyl)piperazine-1-ium 2,2,2-trifluoroacetate (100 mg, 0.20 mmol) in dry DMF (2 ml) and DIEPA (155 μL, 0.89 mmol) using filter paper. The mixture was further stirred at room temperature for 1-2 hours. After the reaction was complete, the solvent was evaporated under reduced pressure, and the reaction mixture was neutralized with NH4Cl (25 ml). The compound was extracted with ELISA (twice × 25 mL), dried over MgSO4, and concentrated to yield the product as a dark brown solid (0.19 mmol, 97%). UPLC-MS:t R = 2.80 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 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)piperazine-1-yl)-2-nitrophenyl)acetic acid (X42) Synthesis of 2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-nitrophenyl)acetic acid A solution of tert-butyl 4-(3-(2-methoxy-2-oxoethyl)-4-nitrophenyl)piperazine-1-carboxylate (0.50 gm, 1.30 mmol) in MeOH:H2O (21.2 mL, 16:1) was mixed with LiOH and stirred overnight at room temperature. After completely converting 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 ELISA (4 times × 25 mL), dried over MgSO4, and concentrated under reduced pressure to yield the desired product as a yellow solid (0.78 mmol, 59%). UPLC-MS: R = 2.36 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 97.0%, ESI-MS: (calculated MH) + :380.18 g / mol, detection:310.04 m / z(2H) + -tbu m / z))

[0307] Synthesis of 4-(3-(carboxymethyl)-4-nitrophenyl)piperazine-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 the reaction was complete (monitored by UPLC-MS), cold ether (25 mL) was added, the resulting solid was filtered, and washed with cold ether. The resulting yellow solid (88%) was dried and proceeded to the next step without purification. UPLC-MS:t R = 1.35 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 88.9%, ESI-MS: (calculated MH) + (Detection: 266.11 g / mol, detection: 266.11 m / z)

[0308] Synthesis of 2-(5-(4-(2-((2-bis-)tert-butoxycarbonyl)amino)oxy)acetyl)piperazine-1-yl)-2-nitrophenyl)acetic acid (X42) Bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc)2AOAcOH, 88.4 mg, 0.30 mmol) and N-hydroxysuccinimide (NHS, 34.6 g, 0.30 mmol) were dissolved in 2 mL of dry acetonitrile. Dicyclohexylcarbodiimide (DCC, 62.0 mg, 0.30 mmol) was added to this solution as a solid, and a white precipitate formed after the dissolution of DCC. The reaction mixture was stirred at room temperature for 1 hour, where the (Boc)2AOAc-NHS ester was quantitatively formed according to ULC-MS. Subsequently, to remove DCC-urea, the mixture was filtered directly onto a reaction flask containing 4-(3-(carboxymethyl)-4-nitrophenyl)piperazine-1-ium 2,2,2-trifluoroacetate (100 mg, 0.20 mmol) in dry DMF (2 ml) and DIEPA (155 μL, 0.89 mmol) using filter paper. The mixture was stirred at room temperature for 1-2 hours, after which the completion of the reaction was confirmed by UPLC-MS. After the reaction was complete, the solvent was removed under reduced pressure, and the reaction mixture was neutralized by adding NH4Cl (25 ml). The resulting solution was extracted with siRNA (2 times × 25 mL), concentrated, and the product was obtained as a dark brown solid (0.19 mmol, 97%). UPLC-MS:t R = 2.74 minutes (10-90% MeCN in 3 minutes), UPLC purity (278nm) = 87.0%, ESI-MS: (calculated MNa + (Detection: 561.22 g / mol, detection: 561.28 m / z).

Claims

1. Formula 1, XT ｂ -V ａ A compound of the form -U-Y-Z(1), in which, - X is part of equation 2, 【Chemistry 1】 During the ceremony, - Each R １ and R ２ These are independently selected from H or B, and at least R １ or R ２ B is, - B is an amine protecting group unstable to acids, such as Boc (-C(=O)OtBu), Eei (=CMeOEt, 1-ethoxyethylidene) trityl (-C(Ph) ３ ), -C(=O)CPh ３ , Mmt (-C(Ph) ２ C ６ H ４ OMe), DMT (-C(Ph)(C ６ H ４ OMe) ２ ), Cbz (-C(=O)OCH ２ Ph), benzylideneamine (=CPh), phthalimide (= (CO) ２ C ６ H ４ ), p-toluenesulfonamide (-SO ２ C ６ H ４ Me), benzylamine (-CH ２ Ph), acetamide (-COMe), trifluoroacetamide (-COCF ３ ), 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), and is selected from - T is C １～３ Alkyl, -R ５ -C(=O)- and -R ５ -C(=O)-NR ９ -R ６ - A spacer selected from the group consisting of the following, in the formula, R ５ and R ６ They are independent of each other, C １ ~C ３ Selected from the group consisting of alkyl, and in the formula, R ９ H is, - V is -NR １１ -C(=O)-, -C(=O)-NR １１ -, -S(=O)-, -NR １２ - (CH ２ ) ｐ -,-piperazinyl-(CH ２ ) ｐ -, -pyridinyl-, and pyrimidinyl are selected electron-withdrawing moieties, in the formula, R １１ H and C １～２ Selected from alkyl groups, R １２ H and C １～２ Selected from alkyl groups, p is either 0 or 1, - U is phenyl, which is partly V, W ｑ and E ｎ Combined with at least one of the following, in the formula, V is defined as described above, W is -N ３ , -NO ２ -N = N - R ８ , -O-CH ２ -N ３ , -S-S-R ８ Selected from, in the formula, R ８ is pyridyl or -C １ ~C ６ It is alkyl, E is piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, -N(C) ２ H ４ ) ２ NH ２ , -N(C ２ H ４ ) ２ N-B, -N=N-phenyl, -N=N-R ８ ,-(CH ２ ) ｒ -NH-C １～６ Alkyl, -(CH ２ ) ｒ -N(C) １～６ Alkyl) ２ -, -F, -Cl, -Br, -I, -CN, -NO ２ , -N ３ , -CF ３ , -SO ３ H, -CO ２ H, -C(=O)NH ２ , -SO ２ Me, -SOMe, -SO ２ Selected from Et and -SOEt, R ８ is pyridyl or -C １ ~C ６ It is alkyl, and B is an amine protecting group that is unstable to the acid as defined above, and r is 0, 1, or 2. n is an integer between 0 and 2, q is 1, and in the formula, W is in the ortho or para position relative to Y. - Y is - (CH ２ ) ｍ -C (=O)- or -(CH ２ ) ｍ -O-C(=O)-, and m is 1, -Z is an electron-withdrawing leaving group, -F, -Cl, -Br, -I, -N ３ , -OH, -O(C=O)CH ２ (C=O)OH, -SR １４ , -OCF ３ , -OCH ２ CF ３ , -OSO ２ CF ３ , -SO ２ C ６ H ４ CH ３ , -SO ２ CF ３ , -SO ２ CH ３ , 【Chemistry 2】 Selected from, in the formula, R １４ C １ ~C ６ Alkyl, aryl, or benzyl substituents The aforementioned compound.

2. E stands for pyridyl, pyrimidinyl, pyrazinyl, pyridadinyl, -N=N-phenyl, -N=N-R ８ , -F, -Cl, -Br, -I, -CN, -NO ２ , -N ３ , -CF ３ , -SO ３ H, -CO ２ From H, more particularly selected from pyridyl, pyrimidinyl, pyridazinyl, or -Br, R ８ is pyridyl or -C １ ~C ６ It is alkyl. The compound according to claim 1.

3. The compound according to claim 1 or 2, wherein E is selected from pyridyl, pyrimidinyl, pyrazinyl, or -Br.

4. The compound according to any one of claims 1 to 3, wherein E is -Br.

5. - B is selected from Boc(-C(=O)OtBu) or Eei(=CMeOEt, 1-ethoxyethylidene). The compound according to any one of claims 1 to 4.

6. The compound according to any one of claims 1 to 5, wherein B is Boc(-C(=O)OtBu).

7. T is C １～３ Alkyl or -R ５ -C(=O)-NR ９ - Selected from, R ５ , R ６ and R ９ The compound according to any one of claims 1 to 6, as defined above.

8. V is -NR １１ -C(=O)-, -C(=O)-NR １１ -, and -piperazinyl-(CH ２ ) ｐ The electron-withdrawing moiety is selected from the group consisting of the following: The compound according to any one of claims 1 to 7.

9. V is -NH-C(=O)-, -C(=O)-NH-, -N(CH ３ )-,-piperazinyl-(CH ２ ) ｐ The electron-withdrawing moiety is selected from -, -pyridinyl-, and pyrimidinyl. The compound according to any one of claims 1 to 7.

10. V is -NH-C(=O)-, -C(=O)-NH-, -piperazinyl-(CH ２ ) ｐ - is an electron-withdrawing part selected from, The compound according to any one of claims 1 to 7.

11. R １１ H and C １～２ Selected from alkyl groups, The compound according to any one of claims 1 to 10.

12. R １２ H and C １～２ Selected from alkyl groups, The compound according to any one of claims 1 to 11.

13. W is -N ３ -N = N - R ８ , -O-CH ２ -N ３ , -S-S-R ８ Selected from, R ８ C １ ~C ６ It is alkyl or pyridyl. The compound according to any one of claims 1 to 12.

14. Z is -OH, -Cl, 【Transformation 3】 Selected from, The compound according to any one of claims 1 to 13.

15. Z is -OH, 【Chemistry 4】 Selected from, The compound according to any one of claims 1 to 14.

16. A method for purifying peptides, comprising the following steps: - A step of providing a crude linker-modified peptide, wherein the crude peptide is covalently bonded to the linker molecule described in claim 1, - In the coupling step, the linker-modified peptide is coupled to a solid support to obtain an immobilized linker-modified peptide, - In the release step, the peptide is released by adding a reducing agent, particularly under acidic conditions. The method, including the method described above.

17. In the release step, a reduction intermediate characterized by the reduced linker moiety of the immobilized linker-modified peptide is achieved, and the peptide is released from the reduction intermediate by a trigger, particularly by a change in temperature and / or pH, more particularly by increasing the pH to pH > pKa relative to the pKa of the most basic heteroatom of the linker moiety of the reduction intermediate in the case of an amine switch, or by decreasing the pH to pH < pKa relative to the pKa of the carbamate in the case of a carbamate switch. The method according to claim 16.

18. The method according to claim 16 or 17, wherein the linker-modified peptide is further bonded to the synthetic resin, and the synthetic resin is cleaved before the coupling step is performed.

19. The method according to any one of claims 16 to 18, wherein the linker molecule according to claim 1 comprises a portion W and / or E containing an azide portion.

20. The method according to any one of claims 16 to 19, wherein the unreacted aldehyde portion of the solid support is blocked after step (b) using a blocking agent, in particular by using a blocking agent selected from cysteine, threonine, 2-mercaptoethanol, cysteamine, ethanedithiol, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, and hydrazine, and more particularly by using a blocking agent selected from cysteine ​​and N-methylhydroxylamine.

21. The reducing agents are 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, boranetetrahydrofuran, boranedimethylsulfide, boranedimethylamine complex, boranetriphenylphosphine complex, boranetert-butylamine, LiAlH ４ LiBH ４ NaBH ４ NaBH ３ CN, NaBH (OMe) ３ , NaBH(OCCH ３ ) ３ , LiAlH(OCMe ３ ) ３ From hydroquinone, sodium ascorbate, ascorbic acid, KI-containing ascorbic acid, hydrazine, NH=NH, formaldehyde, in particular dithioerythritol, dithiothreitol, triphenylphosphine, KI-containing ascorbic acid, tributylphosphine, trimethylphosphine, tris(2-carboxyethyl)phosphine, sodium dithionite (Na ２ S ２ O ４ ), boranedimethyl sulfide, boranetriphenylphosphine complex, NaBH ４ , selected from ascorbic acid, more particularly from triphenylphosphine and trimethylphosphine, The method according to any one of claims 16 to 20.

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