Method for peptide synthesis and device for implementing peptide solid phase synthesis method

Abstract

The present invention relates to a method of peptide synthesis wherein the method comprises the steps of reacting a first amino acid or a first peptide with an alpha-amine protected second amino acid in a solvent selected from the group consisting of water, alcohol, and a mixture of water and alcohol, and removing the alpha-amine protecting group with a deprotection solution. The invention also relates to protective agents, their use and devices for carrying out the solid phase synthesis process of peptides.

Description

[0001] The present invention relates to a method for synthesizing a peptide in a solvent, a compound used in the method and an apparatus for implementing a peptide solid phase synthesis method, wherein the solvent is selected from the group consisting of water, alcohol, and a mixture of water and alcohol. Background Art

[0002] Peptides are linked chains of amino acids and represent the precursors of proteins. Peptides and proteins are fundamental building blocks of all living systems and are involved in a variety of life processes. They have many applications in medicine and biological sciences. Therefore, the ability to synthesize peptides and proteins is very important for human life.

[0003] Therefore, the synthetic production of peptides is of great significance. Peptides are synthesized by coupling the carboxyl group (C-terminal) of an amino acid with the amino group or N-terminal of another amino acid. In 1963, Merrifield et al. introduced peptide solid phase synthesis (SPPS), which was intended to overcome the intermediate purification problem related to peptide assembly in solution (see Stewart and Young's article, Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed, 1984); Chan and White's books, Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford University Press, 2000)). During solid phase synthesis, amino acids are continuously coupled to produce peptides with the desired sequence, and the C-terminal is anchored to an insoluble polymer carrier (solid phase). Once the desired sequence is assembled, the peptide is cracked from the solid carrier.

[0004] This synthetic scheme requires protection of the α-amino group of the incoming amino acid to avoid self-polymerization. Standard protecting groups for the α-amino functional group are the acid-labile tert-butyloxycarbonyl (Boc) group, the base-labile fluorenylmethoxycarbonyl (Fmoc) group, and the allyloxycarbonyl (Alloc) group; under Pd-catalyzed neutral conditions, in the presence of PhSiH3 as a scavenger for the allylic system, the standard protecting groups for the α-amino functional group are removed.

[0005] Solid phase synthesis of peptides according to any of the three α-amino protection schemes described above generally requires additional protection of the reactive side chains of the constituent amino acids from unwanted chemical transformations. It is therefore necessary that these protecting groups are resistant to the reagents used during the coupling cycles. In addition, the attachment of the growing peptide to the solid support must be stable to the conditions for α-amino deprotection and chain assembly.

[0006] In the case of Fmoc-based α-amino protection, the side chain groups should be resistant to the basic reagents used to remove the Fmoc moiety. After assembling the peptide chain, mild acidic reagents are usually used to remove the side chain protecting groups. After assembling the desired peptide chain, these side chain protecting groups are usually cleaved using anhydrous HF, trifluoromethanesulfonic acid or trifluoroacetic acid (TFA).

[0007] The peptide assembly procedure is usually carried out in a polar aprotic organic solvent, such as dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO) and dichloromethane (DCM), or a mixture of these organic solvents, because the α-amino protecting groups Fmoc and Boc, which have strong hydrophobicity, are insoluble in water and are often used in SPPS. In addition, in SPPS, the side chain protecting groups commonly used are usually hydrophobic and make the amino acids insoluble in water.

[0008] The SPPS method using Fmoc and Boc protection has been widely used, but requires the expensive and toxic organic solvents mentioned above. For example, DMF has considerable health and environmental risks, it is related to human cancer, and it is suspected to cause birth defects. Therefore, the use of these toxic solvents requires special technical equipment and precautions, for example, reacting under a fume hood and being handled by highly specialized personnel. In addition, handling used solvents is problematic and expensive. Therefore, the SPPS using organic solvents is expensive and is limited to specialized laboratories with special equipment for organic chemical synthesis.

[0009] Therefore, to avoid the above-mentioned problems, water-based peptide synthesis protocols, especially SPPS, are very desirable.

[0010] In an attempt to overcome this problem, Hojo et al. proposed the use of water-soluble protecting groups (Chem. Pharm. Bull. 2004, 52, 422-427 and Tetrahedron Lett. 2004, 45, 9293). For this purpose, they developed several protecting groups, among which there are 2-(phenyl (methyl) sulfonyl) ethoxycarbonyl tetrathioborate (Pms), ethylsulfonyl ethoxycarbonyl (Esc) and 2-(4-sulfophenylsulfonyl) ethoxycarbonyl (Sps). In WO2013115813A1, CEM claims the deprotection of α, β-unsaturated sulfones in water or water systems and their use in water-based SPPS.

[0011] However, the above-mentioned protecting groups have several major disadvantages: their restricted stability (e.g., for Pms), their moderate solubility (Esc), and their expensive and complex synthesis and restricted availability, e.g., because their sulphur groups undergo auto-oxidation (Sps), excluding cysteine ​​and methionine. As a second consideration, these α-amine protecting groups do not show selective absorption, UV or fluorescent signals, which can be used to detect the amount of coupled amino acids, or to provide an indication of the degree of deprotection. Unlike traditional Fmoc groups, Pms and Esc cannot be tracked in traditional UV monitoring. Sps can be monitored by UV, but the complex and expensive synthesis of Sps often affects its use. As a result, the water solubility increase of these compounds is not enough to solve the problem of water-based SPPS as a whole.

[0012] It is therefore an object of the present invention to provide an improved method and apparatus for water-compatible reaction systems for peptide synthesis in general, and solid phase peptide synthesis in particular. Summary of the invention

[0013] In one embodiment, the present invention relates to a protecting agent suitable for forming a protecting group on a functional group of a peptide and / or an amino acid during water-based peptide synthesis, wherein the functional group to be protected is preferably selected from amine, alcohol, thiol and carboxyl, wherein the protecting agent comprises

[0014] I. A main chain structure,

[0015] II. at least one water solubility enhancing functional group and

[0016] III. at least one reactive group,

[0017] - wherein the main chain structure comprises a moiety selected from 9-methylfluorene, tert-butane and / or phenylmethane, diphenylmethane or triphenylmethane, preferably, the main chain structure consists of such moieties,

[0018] - wherein the water solubility enhancing functional group is selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、CN、OSO3 - Ester, OPO3 2- esters and combinations thereof, and

[0019] - wherein the water solubility enhancing functional group and the reactive group are connected to the main chain structure via at least one covalent bond.

[0020] The invention also relates to the use of the compounds for protecting functional groups in chemical reactions. In a preferred embodiment, during water-based peptide synthesis, the reagent is used to form a protecting group on the functional groups of peptides and / or amino acids. Preferably, the functional group to be protected is preferably selected from amines, alcohols, sulfhydryls and carboxyls.

[0021] In a preferred embodiment, the water solubility enhancing functional group is selected from the charged functional group SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters and N(CH3)3 + . It has been demonstrated that the use of charged functional groups as water-solubility enhancing functional groups is beneficial to improving water solubility, which is more effective than uncharged functional groups. The protective agent may contain one or more water-solubility enhancing functional groups, and in a preferred embodiment, the protective agent contains more than one water-solubility enhancing functional group. In a further preferred embodiment, the protective agent contains at least two water-solubility enhancing functional groups. In other preferred embodiments, the protective agent contains 1 to 8, 2 to 7 or 3 to 4 water-solubility enhancing functional groups. The inventors have found that due to the increase in water solubility to a greater extent, protective agent molecules with more than one water-solubility enhancing functional group may be beneficial. In an embodiment, a water-solubility enhancing functional group whose main chain structure is tert-butane or phenylmethane may be sufficient.

[0022] In a preferred embodiment of the present invention, the protective agents include all of the same type (especially all SO3 - In an optional embodiment, the protective agent comprises different types of water-solubility enhancing functional groups. As long as the water-solubility enhancing functional group is SO3 - , the protecting agent preferably comprises at least 2 of these functional groups. If the water-solubility enhancing functional groups are all of the same kind, the synthesis of the protecting agent can be more efficient and easier.

[0023] The main chain structure comprises a part selected from the group consisting of 9-methylfluorene, tert-butane and / or phenylmethane, diphenylmethane or triphenylmethane. In a preferred embodiment, the main chain structure is selected from 9-methylfluorene and tert-butane.

[0024] The reactive group is suitable, i.e. has the required chemical reactivity, so as to react chemically with the functional group to be protected. It is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl O-succinimide, oxycarbonyl oxyester (oxycarbonyl Oxymaester), oxycarbonyl anhydride, halide, oxymethyl halide, hydroxylate and sulfhydryl group. When the main chain structure is 9-methylfluorene, the reactive group is preferably selected from oxycarbonyl halides, oxycarbonyl oxyester and oxycarbonyl O-succinimide. When the main chain structure is tert-butane, the reactive group is preferably selected from hydroxylate, halide, sulfhydryl, oxycarbonyl O-succinimide and oxycarbonyl anhydride. When the main chain structure is selected from phenylmethane, diphenylmethane and triphenylmethane, the reactive group is preferably selected from halide, oxymethyl halide and oxycarbonyl halide.

[0025] In the present invention, the preferred protective agent having a 9-methylfluorene main chain structure can be described by the following general formula 1:

[0026]

[0027] Formula 1

[0028] Wherein, R1 to R8 are independently selected from hydrogen, SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- ester and CN, provided that at least one (preferably at least two) of R1 to R8 are selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- In a preferred embodiment, R1 to R8 are not SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- Esters or CN, all hydrogen.

[0029] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl O-succinimide, oxycarbonyloxy esters, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxyls and thiol groups, wherein R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyloxy esters and oxycarbonyl O-succinimide.

[0030] In a preferred embodiment, R2 and R7 are selected from SO3 - PO3 2-、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- Esters and CN, in particular, R2 and R7 are SO3 - Preferably, R1, R3 to R6 and R8 are all hydrogen.

[0031] In a preferred embodiment, R3 and R6 are selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- Esters and CN, in particular, R3 and R6 are SO3 - Preferably, R1, R2, R4, R5, R7 and R8 are all hydrogen.

[0032] In a preferred embodiment, R2 and R6 are selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- Esters and CN, in particular, R2 and R6 are SO3 - Preferably, R1, R3, R4, R5, R7 and R8 are all hydrogen.

[0033] In the present invention, preferred protective agents having a 9-methylfluorene main chain structure are shown in Table 1 below. The compounds listed in the table in no way limit the scope of the present invention. According to the present invention, they constitute illustrative and preferred protective agents.

[0034] Table 1

[0035]

[0036]

[0037]

[0038] In the present invention, the preferred protective agent having a tert-butane main chain structure can be described by the following general formula 2:

[0039] Formula 2

[0040] Wherein R1 is selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2-In a preferred embodiment, R1 is SO3 - R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl O-succinimide, oxycarbonyl anhydride, halide, oxymethyl halide, hydroxyl and mercapto groups, wherein more preferably, R99 is selected from the group consisting of hydroxyl, halide, mercapto, oxycarbonyl O-succinimide and oxycarbonyl anhydride.

[0041] In the present invention, the preferred protective agents having a tert-butane main chain structure are shown in Table 2 below. The compounds listed in the table do not limit the scope of the present invention in any way. According to the present invention, they constitute illustrative and preferred protective agents.

[0042] Table 2

[0043]

[0044]

[0045] In the present invention, the preferred protective agent having a phenylmethane, diphenylmethane or triphenylmethane main chain structure can be illustrated by the following general formula 3, 4 or 5, respectively:

[0046]

[0047] Formula 3

[0048] Wherein, R1 to R5 are independently selected from hydrogen, SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- ester and CN, provided that at least one (preferably at least two) of R1 to R5 are selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- In a preferred embodiment, R1 to R5 are not SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- Esters or CN, all hydrogen.

[0049] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxyls and thiols, wherein more preferably, R99 is selected from halides, oxymethyl halides and oxycarbonyl halides.

[0050] In a preferred embodiment, R3 is selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- Esters and CN, especially, R3 is SO3 - Preferably, R1, R2, R4 and R5 are all hydrogen.

[0051]

[0052] Formula 4

[0053] Wherein, R1 to R10 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one (preferably at least two) of R1 to R10 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 to R10 are not SO3 - PO3 2- 、N(CH3)2、OSO3 - Ester, OPO3 2- Esters, N(CH3)3 + or CN, all of which are hydrogen.

[0054] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxyls and mercapto groups, wherein more preferably, R99 is selected from halides, oxymethyl halides and oxycarbonyl halides.

[0055] In a preferred embodiment, R3 and R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 and R8 are selected from SO3 - Preferably, R1, R2, R4 to R7, R9 and R10 are all hydrogen.

[0056]

[0057] Formula 5

[0058] Wherein, R1 to R15 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one (preferably at least two, most preferably at least three) of R1 to R15 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 to R15 are not SO3 - PO3 2- 、N(CH3)2、OSO3 - Ester, OPO3 2- Esters, N(CH3)3 + or CN, all of which are hydrogen.

[0059] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxyls and mercapto groups, wherein more preferably, R99 is selected from halides, oxymethyl halides and oxycarbonyl halides. In a particularly preferred embodiment, when R3, R8 and R13 are SO3 - In another particularly preferred embodiment, when R3, R8 and R13 are SO3 - When R99 is not chloride. In a preferred embodiment, R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, oxymethyl halides, hydroxylates and thiol groups. In a particularly preferred embodiment, R99 is not a halide, especially not a chloride.

[0060] In a preferred embodiment, R3, R8 and R13 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3, R8 and R13 are SO3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are all hydrogen.

[0061] In the present invention, preferred protective agents having a phenylmethane, diphenylmethane or triphenylmethane main chain structure are shown in Table 3 below. The compounds listed in the table do not limit the scope of the present invention in any way. According to the present invention, they constitute illustrative and preferred protective agents.

[0062] Table 3

[0063]

[0064]

[0065] The present invention also relates to the use of a protecting agent for protecting a functional group in a chemical reaction. Preferably, the functional group is present on an amino acid, a peptide or a protein, and the chemical reaction is a peptide or protein synthesis in a solvent selected from water, an alcohol, or a mixture of water and an alcohol.

[0066] method

[0067] The present invention relates to a peptide synthesis method, which comprises the following steps: reacting a first amino acid or a first peptide with an α-amine protected second amino acid or a second peptide in a solvent selected from the group consisting of water, alcohol, and a mixture of water and alcohol, and removing the α-amine protecting group using a deprotection solution, wherein the α-amine protecting group is a reaction product of one of the protecting agents described herein and the α-amine functional group of the second amino acid or the second peptide. In a preferred embodiment, the α-amine protecting group is the following

[0068] At least one of Formulae 6 to 10. In each case, the nitrogen represented by the following general formula belongs to a protected amino group:

[0069]

[0070] Formula 6

[0071] Wherein, R1 to R15 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, and most preferably at least three of R1 to R15 are selected from SO3 - PO3 2- 、N(CH3)2、OSO3 - Ester, OPO3 2- Esters, N(CH3)3 + In a preferred embodiment, R1 to R15 are not SO3 - PO3 2-、N(CH3)2、OSO3 - Ester, OPO3 2- Esters, N(CH3)3 + or CN, all of which are hydrogen.

[0072] In a preferred embodiment, R3, R8 and R13 are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R3, R8 and R13 are SO3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0073]

[0074] Formula 7

[0075] Wherein R1 to R8 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R8 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0076] In a preferred embodiment, R2 and R7 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R2 and R7 are SO3 - Preferably, R1, R3 to R6 and R8 are hydrogen.

[0077] In a preferred embodiment, R3 and R6 are selected from SO3- 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 and R6 are SO3 - Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

[0078] In a preferred embodiment, R2 and R6 are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R2 and R6 are SO3 - Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

[0079]

[0080] Formula 8

[0081] Wherein, R1 to R5 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 to R5 are not SO3 - PO3 2- 、N(CH3)2、OSO3 - Ester, OPO3 2- Esters, N(CH3)3 + or CN, all of which are hydrogen.

[0082] In a preferred embodiment, R3 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R3 is SO3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0083]

[0084] Formula 9

[0085] Wherein, R1 to R10 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R10 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R10 are not SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + or CN, all of which are hydrogen.

[0086] In a preferred embodiment, R3 and R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 and R8 are selected from SO3 - Preferably, R1, R2, R4 to R7, R9 and R10 are hydrogen.

[0087]

[0088] Formula 10

[0089] Wherein R1 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 is SO3 - .

[0090] In a particularly preferred embodiment, the α-amine protecting group is 9-(3,6-disulfo)fluorenylmethoxy-carbonyl, 9-(2,7-disulfo)fluorenylmethoxy-carbonyl or tert-butyl-(2-sulfonate)oxycarbonyl (Sboc). As used herein, the term "Smoc" means 9-(3,6-disulfo)fluorenylmethoxy-carbonyl or 9-(2,7-disulfo)fluorenylmethoxy-carbonyl.

[0091] Compared with the protected amino acids or peptides in the prior art, the protected amino acids and peptides according to the present invention, especially the Smoc and Sboc protected amino acids, are more soluble in water, especially the Fmoc and Boc protected amino acids. The reason is that the present invention adds water-solubility enhancing functional groups to the protecting agent, especially the sulfonic acid (SO3 - ) groups. Thus, one of the advantages of the present invention is the ability to utilize amino acids and / or peptides that are soluble in water in their protected form, and to use only water, only alcohol, or only water-alcohol mixtures for peptide synthesis, i.e., no other solubility enhancing additives and no need to use toxic solvents. Peptide synthesis in non-toxic water and / or alcohol containing solvents offers various advantages for pharmaceutical and biological applications.

[0092] Preferred solvents are water and water-alcohol mixtures, with water being the most preferred solvent. Preferred alcohols are methanol, ethanol, isopropanol, 2-propanol, n-propanol, n-butanol, isobutanol, sec-butanol, tert-butanol or mixtures thereof.

[0093] As used herein, the term "soluble in water in its protected form" refers to the degree to which the composition has the necessary solubility in an aqueous solvent system to carry out the desired reaction. As in the case of any composition, the term "soluble" does not imply unlimited solubility in any or all amounts.

[0094] The deprotection solution suitable for use in the present invention preferably comprises an acid or a base, preferably a water-soluble acid or base, i.e. the acid and / or base is preferably water-soluble. Preferred acids are phosphoric acid, hydrochloric acid or trifluoroacetic acid. Preferred bases are amines and ammonia. Preferred deprotection solutions are amine and / or ammonia solutions. The amount of base used is the amount necessary to deprotect the functional group. The solubility of certain organic bases may limit the amount that can be dissolved in water, alcohol, or a mixture of water and alcohol. A suitable base is a base with a solubility that allows dissolution of a sufficient amount to carry out deprotection in the selected solvent.

[0095] The deprotection solution used to deprotect the Sboc group preferably contains a water-soluble acid such as phosphoric acid, hydrochloric acid or trifluoroacetic acid.

[0096] The deprotection solution for deprotecting the Smoc group preferably comprises a water-soluble base, such as an amine or ammonia. The amount of the water-soluble base used reaches the extent necessary for deprotecting the peptide. The solubility of some organic bases may limit the amount that can be dissolved in water, alcohol, or a mixture of water and alcohol. A suitable base is a base with a solubility that allows dissolution of a sufficient amount to carry out deprotection in a selected solvent.

[0097] It should be understood that after deprotection, the reaction and deprotection steps can be repeated using other amino acids or other peptides, wherein the α-amino groups of the other amino acids and / or other peptides are preferably protected by a protecting group according to the present invention, in particular a Smoc group or a Sboc group, until the desired target peptide is obtained.

[0098] In a preferred embodiment of the present invention, any reactive side chain functional groups of the first amino acid or first peptide and / or the second amino acid or second peptide are protected with side chain protecting groups, which preferably include at least one water solubility enhancing functional group. The side chain protecting groups may include sulfonic acid groups or sulfonic acid esters. The method preferably includes the step of removing the side chain protecting groups. The water solubility enhancing functional groups of the side chain protecting groups provide water solubility for the amino acid or peptide protected by the side chain. Suitable side chain protecting groups are the reaction products of one of the above-mentioned protecting agents and the corresponding side chain functional groups of the amino acid or peptide.

[0099] As for the side chain protecting groups, preferred amine protecting groups are those shown in the following Formulae 11 to 15, wherein the nitrogen belongs to the protected amino group.

[0100]

[0101] Formula 11

[0102] Wherein, R1 to R15 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one of R1 to R15, preferably at least two, most preferably at least three of R1 to R15, are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R15 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3+ or CN, all of which are hydrogen.

[0103] In a preferred embodiment, R3, R8 and R13 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3, R8 and R13 are SO3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0104]

[0105] Formula 12

[0106] Wherein R1 to R8 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R8 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0107] In a preferred embodiment, R2 and R7 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R2 and R7 are SO3 - Preferably, R1, R3 to R6 and R8 are hydrogen.

[0108] In a preferred embodiment, R3 and R6 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO32- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 and R6 are SO3 - Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

[0109] In a preferred embodiment, R2 and R6 are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R2 and R6 are SO3 - Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

[0110]

[0111] Formula 13

[0112] Wherein R1 to R5 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R5 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0113] In a preferred embodiment, R3 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R3 is SO3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0114]

[0115] Formula 14

[0116] Wherein R1 to R10 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R10 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R10 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0117] In a preferred embodiment, R3 and R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 and R8 are selected from SO3 - Preferably, R1, R2, R4 to R7, R9 and R10 are hydrogen.

[0118]

[0119] Formula 15

[0120] Wherein R1 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 is SO3 - .

[0121] In a particularly preferred embodiment, the side chain amine protecting group is selected from 9-(3,6-disulfo)fluorenylmethoxy-carbonyl, 9-(2,7-disulfo)fluorenylmethoxy-carbonyl, tris(4-sulfophenyl)methyl (SulfoTrt), tert-butyl-(2-sulfonic acid)oxycarbonyl (Sboc) and 4-sulfo-benzyloxycarbonyl (SulfoCBz).

[0122] Preferred alcohol protecting groups are shown in the following Formulas 16 to 19:

[0123]

[0124] Formula 16

[0125] Wherein, R1 to R5 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R5 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0126] In a preferred embodiment, R3 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R3 is SO3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0127]

[0128] Formula 17

[0129] Wherein, R1 to R5 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO32- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R5 are not SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + or CN, all of which are hydrogen.

[0130] In a preferred embodiment, R3 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R3 is SO3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0131]

[0132] Formula 18

[0133] Wherein R1 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 is SO3 - .

[0134]

[0135] Formula 19

[0136] Wherein, R1 to R15 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one of R1 to R15, preferably at least two, most preferably at least three of R1 to R15, are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 to R15 are not SO3 - PO32- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0137] In a preferred embodiment, R3, R8 and R13 are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, in particular, R3, R8 and R13 are SO3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0138] In particularly preferred embodiments, the alcohol protecting group is selected from 4-sulfobenzyl (BzS), 4-sulfo-benzyloxymethyl (BOMS), tris(4-sulfophenyl)methyl (SulfoTrt) and tert-butyl-1-sulfonate (tBuS).

[0139] Preferred thiol protecting groups are shown in Formula 20 and Formula 21:

[0140]

[0141] Formula 20

[0142] Wherein, R1 to R15 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one of R1 to R15, preferably at least two, most preferably at least three of R1 to R15, are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 to R15 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0143] In a preferred embodiment, R3, R8 and R13 are selected from SO3- 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3, R8 and R13 are SO3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0144]

[0145] Formula 21

[0146] Wherein R1 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 is SO3 - .

[0147] In a particularly preferred embodiment, the thiol protecting group is selected from tris(4-sulfophenyl)methyl (SulfoTrt) and 1-sulfo-2-methyl-2-propanethiol (StBuS).

[0148] Preferred carboxyl protecting groups are those shown in Formula 22 to Formula 25 below:

[0149]

[0150] Formula 22

[0151] Wherein, R1 to R15 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one of R1 to R15, preferably at least two, most preferably at least three of R1 to R15, are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 to R15 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2-Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0152] In a preferred embodiment, R3, R8 and R13 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3, R8 and R13 are SO3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0153]

[0154] Formula 23

[0155] Wherein R1 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 is SO3 - .

[0156]

[0157] Formula 24

[0158] Wherein, R1 to R5 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + In a preferred embodiment, R1 to R5 are not SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + or CN, all of which are hydrogen.

[0159] In a preferred embodiment, R3 is selected from SO3- 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 is SO3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0160]

[0161] Formula 25

[0162] Wherein, R1 to R5 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + In a preferred embodiment, R1 to R5 are not SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + or CN, all hydrogen.

[0163] In a preferred embodiment, R3 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 is SO3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0164] In a particularly preferred embodiment, the carboxyl protecting group is selected from 4-sulfobenzyl (BzS), 4-sulfo-benzyloxymethyl (BOMS), tris(4-sulfophenyl)methyl (SulfoTrt) and tert-butyl-1-sulfonate (tBuS).

[0165] The following is an overview of the most preferred protecting groups.

[0166]

[0167] In a further preferred embodiment of the invention, the method is an improvement of solid phase peptide synthesis, wherein the C-terminus of the first amino acid or the first peptide is anchored to an insoluble support, which is preferably polymeric. By using an insoluble support, intermediate purification work of the growing peptide is minimized. After each reaction step, purification can be easily performed by washing the anchored peptide in a solvent, which is preferably selected from water, alcohol, and a mixture of water and alcohol.

[0168] In one embodiment, the method further comprises the step of cleaving the resulting peptide from the polymer support using a cleavage composition when the peptide is intact.

[0169] Suitable cleavage compositions and methods are well known to those skilled in the art. Typically, an acid such as trifluoroacetic acid and hydrofluoric acid (HF) is used to perform the cleavage step. Preferably, an acid suitable for cleaving the desired peptide from a polymeric support will simultaneously remove the side chain protecting groups on the amino acids that are attached to the target peptide.

[0170] Preferably, the cleavage is performed in the presence of a scavenger composition (e.g., water, phenol, DTT, triethylsilane, and anisole) which protects the peptide from undesirable side reactions during and after the cleavage step. A skilled person can select a suitable scavenger for the protecting groups present.

[0171] The cleaved peptide may be separated from the cleaved support (eg, resin) by filtration and the peptide may then be recovered from the filtrate by conventional procedures such as evaporation or solvent-driven precipitation.

[0172] In a particularly preferred embodiment of the present invention, the α-amine protecting group comprises a fluorescent structure, especially a 9-methylfluorene main chain structure, and the method further comprises monitoring the extent of peptide bond formation and / or monitoring the extent of removal of the α-amine protecting group by measuring fluorescence, the fluorescence being generated by the α-amine protecting group coupled to the first amino acid or the first peptide. In a preferred embodiment, the protecting group comprising a 9-methylfluorene main chain structure is 9-(3,6-disulfo)fluorenylmethoxy-carbonyl or 9-(2,7-disulfo)fluorenylmethoxy-carbonyl.

[0173] In solid phase peptide synthesis, after irradiating the support with UV light, the extent of peptide bond formation and / or the extent of removal of the fluorescent protecting group (e.g., Smoc) coupled to the insoluble support material via the first amino acid or the first peptide is monitored by monitoring changes in fluorescence intensity and measuring the fluorescence generated by the fluorescent protecting group.

[0174] Surprisingly, it was observed that protecting groups containing 9-methylfluorene, such as 9-(3,6-disulfo)fluorenylmethoxy-carbonyl and 9-(2,7-disulfo)fluorenylmethoxy-carbonyl, have inherent fluorescent properties. The fluorescent properties of these protecting groups provide an opportunity to monitor after either or both of the deprotection and coupling reactions are completed. In some preferred embodiments of the present invention, the protecting agent and the corresponding protecting group contain two or more sulfonic groups attached to an aromatic fluorenyl ring system, such as 9-(3,6-disulfo)fluorenylmethoxy-carbonyl and 9-(2,7-disulfo)fluorenylmethoxy-carbonyl. In these compounds, the two or more sulfonic groups (SO3 - ) groups are unique. These compounds, such as Smoc, can be monitored by their intrinsic fluorescence to determine the amount of compound present at the end point of each reaction step.

[0175] Evidence of a fluorescent group (e.g., Smoc) on a support can be used to determine successful coupling of a fluorescently protected amino acid to determine whether the coupling step must be repeated to substantially complete the formation of the peptide bond. Likewise, evidence of a fluorescent group on a support can be used to determine incomplete removal of the fluorescent group at the end of a deprotection step to determine whether the deprotection step must be repeated to substantially complete the removal of the fluorescent protecting group.

[0176] Typically, the coupling step of reacting a first amino acid or a first peptide with an α-amine protected second amino acid or a second peptide comprises adding a coupling agent.

[0177] The present invention also relates to coupling agents suitable for use in the method of the present invention. Typical coupling agents are selected from the group consisting of carbodiimides and triazoles. Preferred carbodiimides are diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 2-(((ethylimino)methylene)amino)-2-methylpropane-1-sulfonate (ESC) and 2,2'-(methylenebis(azanylidene))bis(2-methylpropane-1-sulfonate) (2,2'-(methanediylidenebis(azanylylidene))bis(2-methylpropane-1-suofonate)) (DSC). Preferred triazoles are O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-]-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), Boc-histidine (tosyl); B01' and B01'-Cl, (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate (PyBOB) and 1-hydroxybenzotriazole (HOBt).

[0178] In a preferred embodiment of the present invention, the coupling agent comprises a sulfonic acid group. The sulfonic acid group increases the solubility of the coupling agent in aqueous solvents and allows the coupling agent to be subsequently separated by anion exchange.

[0179] Preferred coupling agents are those shown in the following formulas 26 to 30:

[0180]

[0181] Formula 26

[0182] Wherein R1 is preferably selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, especially, R1 is SO3 - R2 is preferably selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, especially, R2 is SO3 - R1 and R2 may be the same or different.

[0183]

[0184] Formula 27

[0185] Wherein R1 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, especially, R1 is SO3 - R66 is preferably selected from short chain alkyl groups, preferably C1 to C8 alkyl groups, more preferably C1 to C4 alkyl groups.

[0186]

[0187] Formula 28

[0188] Wherein R1 is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + and CN, especially, R1 is SO3 - .

[0189]

[0190] Formula 29

[0191] Wherein R1 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, especially, R1 is SO3 - .

[0192]

[0193] Formula 30

[0194] Wherein, R1 to R4 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one, preferably at least two, of R1 to R4 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 +In a preferred embodiment, R1 to R4 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all of which are hydrogen.

[0195] In a preferred embodiment, R3 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, in particular, R3 is SO3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0196] In a preferred embodiment, R50 is selected from one or more hydroxyl groups or one of the substituents given in the following formulas 30.1 to 30.4, wherein B represents a structure of formula 30:

[0197]

[0198] General formula 30.1

[0199]

[0200] General formula 30.2

[0201]

[0202] General formula 30.3

[0203]

[0204] General formula 30.4

[0205] Particularly preferred coupling agents are selected from the group consisting of 2-(((ethylimino)methylene)amino)-2-methylpropane-1-sulfonate (ESC), 2,2'-(methylenebis(azaylidene))bis(2-methylpropane-1-sulfonate) (DSC) and 2-((((3-(dimethylamino)propyl)imino)methylene)amino)-2-methylpropane-1-sulfonate (MPSC), as well as N-hydroxysulfosuccinimide (Sulfo-NHS) and 2-(2-cyano-2-(hydroxyimino)acetoxy)-2-methylpropane-1-sulfonate (Cyms).

[0206]

[0207] Wherein R is selected from C1 to C8 alkyl, preferably C1 to C4 alkyl.

[0208] Formula 31 (ESC)

[0209]

[0210] General formula 32 (DSC)

[0211]

[0212] General formula 33 (MPSC)

[0213]

[0214] Formula 34 (Cyms)

[0215] The coupling step may also comprise the addition of a capping agent. The described capping agents and their use in the peptide synthesis method for capping free amines form an independent part of the present invention. Capping agents can be used in the method of the present invention, but they can also be used in other methods of peptide synthesis. Capping agents are useful to facilitate simple, safe and cost-effective purification of the resulting product composition. Capping agents allow the free amines to be capped after the coupling step to prevent the formation of by-products, wherein the capping agent comprises

[0216] I. A main chain structure,

[0217] II. at least one water solubility enhancing functional group and

[0218] III. at least one reactive group,

[0219] - wherein the main chain structure comprises a moiety selected from a short chain alkyl group, preferably a C1 to C8 alkyl group, a cycloalkyl chain or an aromatic compound, more preferably a C1 to C4 alkyl group or a benzyl group,

[0220] - wherein the water solubility enhancing functional group is selected from SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + , CN and their combinations, and

[0221] - wherein the water solubility enhancing functional group and the reactive group are connected to the main chain structure via at least one covalent bond.

[0222] The reactive group is preferably selected from the group consisting of carboxylic acid, carboxylic acid halide, carboxylic acid O-succinimide, carboxylic acid oxymethyl ester, carboxylic acid anhydride, halide and mercapto group.

[0223] In one embodiment of the invention, according to the present invention, a capping agent is added to the peptide synthesis method. In an optional embodiment, the capping agent is added in another method for peptide synthesis, which comprises the following steps: reacting a first amino acid or a first peptide with an α-amine protected second amino acid or a second peptide in a solvent, and removing the α-amine protecting group using a deprotection solution, characterized in that the capping agent is added to the reaction mixture for capping the free amine. The capping agent is selected from those capping agents mentioned above. After the reaction is completed, the reaction mixture is passed through an affinity chromatography column to purify the reaction mixture. The affinity chromatography column is preferably selected from an anion or cation exchange column.

[0224] After coupling, the water solubility enhancing functional group facilitates purification. In a preferred embodiment of the present invention, the capping agent comprises a sulfonic group.

[0225] Preferred capping agents are 2-sulfoacetic acid or 4-sulfobenzoic acid.

[0226]

[0227] In another preferred embodiment of the present invention, the method further comprises at least one washing step, wherein the washing step uses a solvent selected from the group consisting of water, alcohol, and a mixture of water and alcohol, and a step of collecting the waste liquid obtained in the washing step, contacting the waste liquid with an affinity chromatography column, preferably a solid anion and / or cation exchange support, thereby retaining the water-solubility enhancing functional groups in the waste compounds, especially the charged functional groups, such as SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- and N(CH3)3 + The use of an anion exchange support is particularly effective in removing compounds containing at least one sulfonic group. The waste compounds can then be collected by regenerating an affinity chromatography column, preferably a solid anion and / or cation exchange support, and processed. Alternatively, the affinity chromatography column, preferably an anion and / or cation exchange support material containing the waste compounds can be processed.

[0228] The flushing step is usually performed after the coupling step and after the deprotection step. The anion and / or cation exchange step allows the removal of all sulfo-containing compounds, such as unreacted or cracked protecting groups and reagents, such as coupling agents and end-capping agents from the flushing solution as described herein. The waste compounds are retained on the anion or cation exchange column to obtain a purified solvent. The retained compounds can be treated with anion exchange material or cation exchange material or after elution, and only a minimum amount of chemical waste needs to be treated.

[0229] In a further preferred embodiment of the present invention, the method further comprises a purification step after the cleavage step, wherein the solution containing the deprotected and cleaved target protein or peptide is contacted with an affinity chromatography column, preferably a solid anion and / or cation exchange support, thereby retaining waste compounds such as waste compounds containing sulfonyl groups, and collecting the purified target protein. In a preferred embodiment, the waste compound contains at least one sulfonyl group as a part of a protecting group and other reagents. Due to the presence of sulfonyl protecting groups and sulfonyl reagents, excess chemicals and by-products produced during the coupling, deprotection and cleavage reactions in a solvent can be substantially removed by an affinity chromatography column (preferably an ion exchange method), and the solvent is selected from the group consisting of water, alcohol, and a mixture of water and alcohol. Of course, this also applies to other charged functional groups. Only the target peptide can pass through the ion exchange column, while peptide by-products, protecting group residues and excess reagents are retained on the column. After the column is regenerated, only a minimum amount of chemical waste must be handled. This is the use of charged functional groups, such as SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- and N(CH3)3 + However, even when one or more uncharged functional groups are used, the process can still be carried out in water, thereby reducing the amount of waste chemicals (compared to prior art processes).

[0230] A preferred aspect of the present invention relates to a protecting agent, wherein the protecting agent is selected from 9-(3,6-disulfo)fluorenylmethoxy-carbonyl halide (Smoc halide), 9-(3,6-disulfo)fluorenylmethoxy-carbonyl N-hydroxysuccinimide (Smoc-NHS), 9-(3,6-disulfo)fluorenylmethoxy-carbonyl O-succinimide (Smoc OSu), 9-(2,7-disulfo)fluorenylmethoxy-carbonyl halide (Smoc halide), 9-(2,7-disulfo)fluorenylmethoxy-carbonyl N-hydroxysuccinimide (Smoc-NHS), 9-(2,7-disulfo)fluorenylmethoxy-carbonyl O-succinimide (Smoc OSu). OSu), tris(4-sulfophenyl)methyl halide (SulfoTrt halide), tert-butyl-(2-sulfonate)oxycarbonyl anhydride (Sboc2O), tert-butyl-(2-sulfonate)oxycarbonyl O-succinimide (Sboc-OSu), 4-sulfo-carbonylbenzyloxy halide (SulfoCBz halide), 2-hydroxy-2-methylpropane-1-sulfonate (tBuS), 2-bromo-2-methylpropane-1-sulfonate (tBuS), 2-mercapto-2-methylpropane-1-sulfonate (StBuS), 4-sulfobenzyl halide (BzS halide) and 4-sulfo-benzyloxymethyl halide (BOMS halide).

[0231] According to the invention, preferred halides are chlorides and bromides. The above compounds are useful reagents for protecting functional groups, for example amine, alcohol, thiol or carboxyl groups, in chemical reactions in organic chemistry.

[0232] Another aspect of the present invention relates to modified amino acids, peptides and salts thereof, which contain a protecting group selected from the group consisting of those protecting groups shown in the above general formula 6 to general formula 25, and more preferably, selected from the group consisting of those protecting groups shown in the above general formula 6 to general formula 10. In particularly preferred embodiments, these protecting groups include 9-(3,6-disulfo)fluorenylmethoxy-carbonyl, 9-(2,7-disulfo)fluorenylmethoxy-carbonyl, tris(4-sulfophenyl)methyl (SulfoTrt), tert-butyl-(2-sulfonic acid)oxycarbonyl (Sboc), 4-sulfo-benzyloxycarbonyl (SulfoCBz), tert-butyl-1-sulfonate (tBuS), 1-sulfo-2-methyl-2-propanethiol (StBuS), 4-sulfobenzyl (BzS) and 4-sulfo-benzyloxymethyl (BOMS).

[0233] Another aspect of the present invention relates to a device for implementing a solid phase synthesis method of peptides. The device includes a reactor and a fluorometer, the reactor is used to receive an insoluble (e.g., polymer) carrier material, and the fluorometer is arranged to monitor changes in fluorescence intensity, which are produced by the protecting groups coupled to the insoluble carrier material. Due to the intrinsic fluorescence of the protective agent (e.g., Smoc) of the present invention, quantitative analysis of the fluorescence intensity of the insoluble carrier material provides information about the progress of the coupling reaction or deprotection reaction in the above-mentioned SPPS method. Integrating the fluorometer into the peptide synthesis device can accelerate automated synthesis.

[0234] In a preferred embodiment of the invention, the device further comprises at least one solid anion and / or cation exchange carrier, which is arranged to separate waste compounds. The solid anion and / or cation exchange carrier can be arranged in the product stream to separate the target protein from waste compounds (e.g., peptide byproducts containing sulfonyl groups, protective group residues and excess reagents). Alternatively or in addition, the solid anion exchange carrier can be arranged in the waste stream so that after the coupling and deprotection steps, the obtained rinse solution is separated from waste compounds (e.g., excess reagents and byproducts).

[0235] The present invention is further described with reference to the accompanying drawings, in which Figure 1 is a schematic diagram of an apparatus according to the present invention.

[0236] Figure 1The overall layout of an automatic solid phase peptide synthesizer apparatus for implementing the preferred method as described above is schematically shown. The apparatus comprises a reactor 1 containing a first amino acid or a first peptide anchored to an insoluble polymer carrier, a reagent container 2 comprising multiple reservoirs for providing coupling agents, capping agents, cleavage agents, etc., a solvent container 3, and an amino acid and / or peptide container 4 comprising multiple reservoirs for providing protected amino acids and / or peptides.

[0237] Reactor 1 and container 2, container 3, container 4 are connected by tubes and valves, and are arranged in this way so that specified amounts of specified reagents, amino acids and / or peptides can be delivered to reactor 1 in a conventional manner in a specified order under the control of a control component (not shown).

[0238] The apparatus comprises a fluorometer 5 arranged to measure changes in fluorescence intensity of the contents of the monitoring reactor 1. It will be appreciated that the reactor 1 may alternatively comprise an integrated fluorometer 5.

[0239] The reactor is equipped with a waste outlet 6 for removing a waste stream after each flushing step and a product outlet 7 for removing a product stream after the cleavage step. The waste outlet 6 is connected to a chamber 9 and a first ion exchange column 10 via a waste pipe 8, in which the pH of the waste stream is adjusted for subsequent separation. The product outlet 7 is connected to a second ion exchange column 12 via a product pipe 11.

[0240] The ion exchange columns 10, 11 are arranged to retain sulfonation waste compounds that must be subsequently disposed of, such as by-products, excess reagents and protecting group residues. The purified solvent stream from the first ion exchange column 10 can be directed to the sewer system or recycled. The purified product stream from the second ion exchange column 12 is used to obtain the target protein.

[0241] The present invention is illustrated by the following examples but is not limited thereto. Example

[0242] Example 1: Synthesis of 9-(3,6-disulfonyl)fluorenylmethoxycarbonyl chloride (Smoc-Cl)

[0243] 2 g (7.73 mmol) of Fmoc-chloride was treated with 20 mL of concentrated sulfuric acid. After the reaction mixture was quenched, 2.96 g (7.07 mmol, 91.4%) of crude Smoc-chloride was obtained as a light yellow solid.

[0244]

[0245] Analytical data of Smoc-Chlorine:

[0246] 1H NMR (500MHz, D2O) δ = 7.80 (s, 2H), 7.69 (d, J = 7.9Hz, 2H), 7.48 (d, J = 7.8Hz, 2H), 3.84 (d, J = 4.8Hz, 2H), 3.45 (t, J = 4.7Hz, 1H).

[0247] 13 C NMR (126MHz, D2O) δ = 145.57, 142.54, 141.65, 125.16, 121.61, 120.90, 62.54, 49.65.

[0248] Example 2: Synthesis of 9-(2,7-disulfonyl)fluorenylmethoxycarbonyl chloride (Smoc-Cl)

[0249] 2 g (7.73 mmol) of Fmoc-chlorine was treated with 20 mL of concentrated sulfuric acid and heated to 100°C. The sulfuric acid was neutralized with NaOH (pH 9.5), the solvent was removed under reduced pressure, and the formation of the target intermediate was confirmed by NMR analysis. The intermediate was dissolved again in a 20% aqueous sulfuric acid solution and stirred for 6 hours to form 9-(2,7-disulfonyl)fluorenylmethanol. The sulfuric acid was neutralized with NaOH (pH 6.7) and the solvent was removed under reduced pressure. A solution of 1.2 eq. of phosgene was cooled to 0°C in 25 ml of DCM, and 9-(2,7-disulfonyl)fluorenylmethanol (Carpino and Han, The Journal of Organic Chemistry 1972, 37, (22), 3404-3409) was slowly added under stirring. The solution was stirred in an ice bath for 1 hour and then allowed to stand at ice bath temperature for 4 hours. The solvent and excess phosgene were removed under reduced pressure to obtain the corresponding product.

[0250]

[0251] NMR intermediates:

[0252] 1 H NMR (300MHz, D2O) δ: 6.09 (s, 2H), 7.23-7.40 (m, 2H), 7.72 (s, 2H), 7.95 (d, J = 6.2Hz, 2H).

[0253] 13 C NMR(75MHz,D2O)6:142.61,132.99,131.74,130.23,129.28,127.22,125.57,124.69.

[0254] LC-APCI-MS of 9-(2,7-disulfo)-fluorenylmethoxycarbonyl chloride:

[0255] LC-APCI-MS was used to calculate C 15 H9ClO 22 m / z: 256.03. Measured m / z: 256.94 [MH-2xSO3]-.

[0256] Example 3: Synthesis of Smoc-Gly-OH (Smoc-Glycine)

[0257] 2.5 g (8.41 mmol) of Fmoc-glycine was treated with 30 mL of concentrated sulfuric acid. After the reaction mixture was stopped, 3.7 g (8.09 mmol, 96.2%) of crude Smoc-glycine in the form of light yellow powder was obtained.

[0258]

[0259] Analytical data of Smoc-Glycine:

[0260] LC-APCI-MS was used to calculate C 17 H 14 NO 10 S2 - m / z:456.01. Measured m / z:455.85[MH] - .

[0261] 1 H NMR (500MHz, D2O) δ = 8.01 (s, 2H), 7.87 (d, J = 7.9Hz, 2H), 7.79 (d, J = 8.0Hz, 2H), 5.95 (s, NH), 4.41 (d, J = 6.2Hz, 2H), 4.01 (t, J = 6.2Hz, 1H), 3.67 (s, 2H).

[0262] 13C NMR(126MHz,D2O)δ=158.21,144.94,142.41,141.88,125.51,122.09,121.09,65.75,46.95,44.39.

[0263] Example 4: Smoc- L -Synthesis of Ala-OH (Smoc-Alanine)

[0264] 2.5 g (8.03 mmol) of Fmoc-L-alanine was treated with 30 mL of concentrated sulfuric acid. After the reaction mixture was stopped, 3.8 g (7.64 mmol, 95.1%) of crude Smoc-L-alanine in the form of white powder was obtained.

[0265]

[0266] (dd, 2H), 5.94 (s, NH), 3.95 (m, OH+2H), 3.85 (q, J = 7.4Hz, 1H), 2.93 (t, J = 1.64Hz,

[0267] 1H), 1.01 (d, J = 7.3 Hz, 3H).

[0268] 1℃ NMR (126MHz, MeOD) δ=175.11,158.10,146.21,145.89,145.44,143.54,

[0269] 127.09,124.04,123.90,121.40,67.23,51.05,48.51,17.58.

[0270] Example 5: Synthesis of Smoc-L-I1e-OH (Smoc-isoleucine)

[0271] 2.5 g (7.07 mmol) of Fmoc-L-isoleucine was treated with 30 mL of concentrated sulfuric acid. After the reaction mixture was stopped, 3.8 g (6.82 mmol, 96.4%) of crude Smoc-L-isoleucine in the form of white powder was obtained.

[0272]

[0273] Analytical data of Smoc-isoleucine:

[0274] LC-APCI-MS was used to calculate C 21 H 22 NO7S·m / z:432.11, measured m / z:432.06[M-HSO3] - .

[0275] 1 H NMR (500MHz, MeOD) δ = 7.79 (d, J = 16.5Hz, 2H), 7.57 (d, J = 8.0Hz, 2H), 7.55 (dd, J = 8.0, 1.5Hz, 2H), 4.15-3.89 (m, OH + 2H), 3.77 (d, J = 6.2Hz, 1H), 2 .93(t,J=1.6Hz,1H),1.50(dtd,J=13.2,10.1,6.7Hz,1H),1.20-1.05(m ,1H), 0.97-0.81(m, 1H), 0.55(d, J=6.86Hz, 3H), 0.54(t, J=7.43Hz, 3H).

[0276] 13 C NMR (126MHz, MeOD) δ = 173.98, 158.49, 146.15, 145.45, 143.48, 127.09, 124.02, 123.95, 121.38, 67.33, 60.30, 48.55, 38.31, 26.30, 15.98, 11.66.

[0277] Example 6: Smoc- L Synthesis of -Leu-OH (Smoc-Leucine)

[0278] 2.5 g (7.07 mmol) of Fmoc- L -leucine. After the reaction mixture was stopped, 3.6 g (7.01 mmol, 99.1%) of crude Smoc- L -Leucine.

[0279]

[0280] Analytical data of Smoc-Leucine:

[0281] LC-APCI-MS was used to calculate C 21 H 22 NO7S·m / z: 432.11. Measured m / z: 432.06 [M-HSO3]-.

[0282] 1 H NMR(500MHz,,MeOD)δ=7.80(d,J=18.0Hz,2H),7.58(d,J=8.1Hz,2H),7.57(dd,J=8.3,4.1Hz2H),4.11-3.93(m,OH+ 2H),3.86(dd,J=9.9,5.3Hz,1H),2.94(t,1H),1.41-1.31(m,1H),1.30-1.17(m,2H),0.57(dd,J=14.7,6.5Hz,6H).

[0283] 13 C NMR(126MHz,MeOD)δ=175.08,158.41,146.18,145.95,145.38,143.52,127.1 0,124.07,123.97,121.40,67.27,53.99,48.54,41.47,25.86,23.27,21.78.

[0284] Example 7: Synthesis of Sulfo-Trt

[0285] In the round-bottomed flask of heating, under nitrogen atmosphere, 237mg (9.75mmol, 3.0eq.) magnesium is suspended in 10mL THF. Under vigorous stirring (vicious steering), 1 / 10 of 1.878g (9.75mmol, 3eq.) 4-chlorobenzenesulfonic acid solution in 15mL THF is added to the mixture. In order to start Grignard reaction, the mixture is heated to reflux and a drop of bromine is added. Then dropwise add the 4-chlorobenzenesulfonic acid solution of remainder, and the mixture is refluxed for 30 minutes again, then cooled to room temperature.

[0286] The second step uses the Kochi coupling method. For this purpose, a three-necked flask is placed under a nitrogen atmosphere and cooled to -78°C using a suspension of dry ice in methanol. Then 50 mL of THF is added, followed by 0.361 mL (3.25 mmol, 1 eq.) of carbon tetrachloride and 0.325 ml (0.03 mmol, 0.01 eq., 0.1 mol / L solution in THF) of dilithium tetrachlorocuprate (II). To this solution, the previously prepared Grignard reagent is added dropwise. After stirring at -78°C for 1 hour and at 0°C for 6 hours, the mixture is warmed to room temperature and stirred for another 18 hours. The solution is quenched by adding 20 mL of water. The volatile components were removed under reduced pressure to give 1.69 g of crude 4,4',4"-(hydroxymethanetriyl)tribenzenesulfonate (Sulfo-Trt) as a brown solid. The product was isolated by semi-preparative RP-HPLC. 4,4',4"-(hydroxymethanetriyl)tribenzenesulfonate was treated with thionyl chloride (SOCl2) to form tris(4-sulfophenyl)methyl chloride (Sulfo-Trt chloride). The solvent was removed under reduced pressure.

[0287]

[0288] Sulfo-Trt analysis data:

[0289] 1 H NMR (500MHz, D2O) δ = 7.68 (d, J = 8.6 Hz, 6H), 7.44 (d, J = 8.6 Hz, 6H).

[0290] 13 C NMR (126MHz, D2O) δ = 140.99, 136.88, 128.96, 127.02, 75.45.

[0291] Example 8: Synthesis of 2-hydroxy-2-methylpropane-1-sulfonate (tBuS-OH).

[0292] Dilute 6,00 g of 2-methyl-2-propylene-1-sulfonic acid sodium salt with 50 ml of water. Add 10 ml of sulfuric acid and stir the reaction at room temperature overnight. Remove sulfuric acid by CaCO3 precipitation. Remove the solvent under reduced pressure. Obtain a white powder product. Confirm the target compound by LC-MS and NMR analysis.

[0293]

[0294] Analytical data of tBuS-OH:

[0295] ESI-MS calculated C4H9O4S-m / z: 153.18, measured m / z: 152.9 [MH]-.

[0296] 1 H NMR (500MHz, DMSO-d6) δ: 1.18 (s, 6H), 2.67 (s, 2H), 5.20 (s, 1H).

[0297] 13 C NMR (126MHz, DMSO) δ: 67.84, 61.81, 39.52, 29.65.

[0298] Example 9: Synthesis of 2-bromo-2-methylpropane-1-sulfonate (tBuS-Br).

[0299] 6,00 g of 2-methyl-2-propene-1-sulfonic acid sodium salt was diluted with 20 ml of HBr (48% w / w). The solvent was removed under reduced pressure. A white-brown powder product was obtained. The target compound was confirmed by LC-MS and NMR analysis.

[0300]

[0301] Analytical data of tBuS-Br:

[0302] ESI-MS calculated C4H8BrO3S-m / z: 216,07, measured m / z: 216,8[MH]-.

[0303] Example 10: Synthesis of 2-mercapto-2-methylpropane-1-sulfonate (StBuS).

[0304] 3.00 g of 2-methyl-2-propylene-1-sulfonic acid sodium salt was diluted with 25 ml of concentrated HCl, and the reaction solution was stirred overnight. The acid was then neutralized with NaOH (pH = 6.8) and the solvent was removed under reduced pressure. In a heated round-bottom flask, 233.67 mg of magnesium was suspended in 20 mL of THF under a nitrogen atmosphere. 1.5 g of a tBuS-Cl suspension in 15 mL of THF was added under vigorous stirring. To start the Grignard reaction, the mixture was heated to reflux (70°C), a drop of bromine was added, the mixture was refluxed for 90 minutes, and then cooled to room temperature. 4 eq. of sulfur was added to the Grignard reagent and stirred for 8 hours. Then 200 ml of water was added. The mixture was stirred at room temperature for 2 hours. Subsequently, the solvent was removed under reduced pressure and the resulting white powder was analyzed by NMR.

[0305]

[0306] StBuS analysis data:

[0307] 1 H NMR (500MHz, Deuterium Oxide) δ: 1.56 (s, 6H), 2.18 (s, J = 1.3Hz, 1H), 3.67 (s, 2H).

[0308] 13 C NMR (126MHz, D2O) δ: 61.66, 32.13, 28.48.

[0309] Example 11: Synthesis of (9-BBN)-Lys(SBoc)

[0310] 1.68 g of lysine was suspended in anhydrous THF, and 24.28 ml of 9-boranobicyclo(3.3.1)nonane (9-BBN) in THF (0.5 mol / L) was added under inert gas. The resulting suspension was refluxed at 100°C for 18 h. The solvent was removed under reduced pressure. (9-BBN)-Lys was diluted with 20 ml of THF, and 1.97 g of 1,1'-carbonyldiimidazole and 2.54 g of BuS-OH were added under inert gas.

[0311] The resulting solution was stirred for 5 hours.The solvent was removed under reduced pressure and the crude product was washed 3 times with DCM to remove imidazole residues.

[0312]

[0313] Analytical data of (9-BBN)-Lys(SBoc):

[0314] Calculation of C using ESI-MS 19 H 33 BN2O7S2- m / z:444.35, measured m / z:443.2[MH] - .

[0315] Example 12: Synthesis of (9-BBN)-Tyr(tBus).

[0316] 2.00 g of tyrosine was suspended in anhydrous THF, and 25 ml of 9-BBN (0.5 mol / L) in THF was added under inert gas. The resulting suspension was refluxed at 100°C for 18 h. The solvent was removed under reduced pressure. (9-BBN)Tyr was suspended in 20 ml of THF, and 2.39 g of tBuS-Br was added under inert gas. The resulting solution was stirred for 2 d. The solvent was removed under reduced pressure, and the resulting crude product was washed 3 times with THF to remove bromide residues.

[0317]

[0318] Analytical data of (9-BBN)-Tyr(tBuS):

[0319] Calculation of C using ESI-MS 21 H 31 BNO6S - m / z: 436.35, measured m / z: 436.2 [M+H] + ,453.2[M+H2O] + .

[0320] Example 13: Stability test of Smoc-protected amino acids

[0321] To determine the stability of Smoc-protected amino acids in an aqueous environment, Smoc-glycine and Smoc-L-leucine were dissolved in water and incubated at ambient temperature for 8 days. The status of the amino acids was checked after 3, 6 and 8 days using analytical RP-HPLC. The compounds must be stable for a considerable period of time in an aqueous environment. To demonstrate this stability, Smoc-glycine and Smoc-leucine were dissolved in a minimum amount of water and kept at ambient temperature. The experimental results were monitored by RP-HPLC after 3, 6 and 8 days. As a result, no significant changes in the amino acid composition were observed.

[0322] Example 14: N-terminal deprotection of Smoc-protected amino acids

[0323] For use in SPPS, protecting groups must be easily and efficiently deprotected under safe conditions. To determine the deprotection conditions for Smoc-protected amino acids, a series of experiments were performed by dissolving 1 mg of Smoc-glycine in 1 mL of water or ethanol. One of the following bases was added to this solution, and the mixture was incubated at room temperature with shaking for 5 minutes:

[0324] Ammonia (10% aqueous solution),

[0325] Ethanolamine (50% aqueous solution),

[0326] Ethylenediamine (50% aqueous solution) or

[0327] Piperidine (50% in water).

[0328] The results were analyzed by analytical RP-HPLC and ESI-MS.

[0329] The results indicate that solutions of ammonia or amines in water are sufficient for Smoc deprotection at room temperature.

[0330] In addition to piperidine (used as the standard base for Fmoc-SPPS deprotection), water-soluble bases can also be used, such as ammonia, ethanolamine, and ethylenediamine, etc. This allows for a significantly cheaper and easier process compared to piperidine-based deprotection.

[0331] Example 15: Purification by affinity chromatography

[0332] Smoc-alanine was mixed with unprotected alanine as an artificial impurity and dissolved in 1 M formic acid and loaded onto a DEAE Sephadex A-25 ion exchange column. After washing the ion exchange column with additional formic acid, Smoc-alanine was eluted with a 4 M ammonium formate solution. The solution before loading, the solution after loading, and the eluted fractions were analyzed by RP-HPLC.

[0333] The results showed that unprotected alanine passed through the ion exchange column unhindered, while Smoc-protected alanine bound to the ion exchange column. With the mobile phase changed to 4M ammonium formate solution, Smoc-alanine was eluted from the column without any impurities remaining. Therefore, it has been shown that the Smoc group can be successfully used in affinity chromatography for purification purposes.

[0334] Example 16: Synthesis of test peptide HVGGVG-OH according to the Smoc method

[0335] The coupling of Smoc protected amino acids was performed as follows. Thus, 3 eq. of the corresponding Smoc protected amino acid, 2.8 eq. of the activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 3.0 eq. of N-hydroxysuccinimide (NHS) and 6 eq. of sodium bicarbonate as a general base were dissolved in a minimum amount of water and preactivated for 3 min. A typical coupling was performed for 60 min at ambient temperature. N-terminal deprotection of the peptide resin was performed as a double deprotection step with 10% aqueous ammonia solution. After cleavage from the support, the test peptides were analyzed by RP-HPLC and ESI-MS.

[0336] Analytical data of peptide HVGGVG-OH:

[0337] RP-HPLC, 10→60% MeCN, t R =19.1min.

[0338] Calculation of C using ESI-MS 16 H 29 N5O6 m / z: 387.21 Measured 387.0 [M+H] + .

[0339]

[0340] After each coupling step and the subsequent deprotection step, the fluorescence of water, solid support (PEGA resin) and PEGA resin-peptide was measured. The results are shown in Table 1. Therefore, after each coupling step, the fluorescence value rises due to the coupling of the Smoc group, and after each deprotection step, the fluorescence value decreases again, indicating that real-time monitoring of the reaction progress is possible.

Claims

1. A method for peptide synthesis, wherein the method comprises the following steps: A first amino acid or a first peptide is reacted with an α-amine protected second amino acid or a second peptide in a solvent selected from the group consisting of water, alcohol, and a mixture of water and alcohol, wherein the C-terminus of the first amino acid or the first peptide is anchored to an insoluble carrier, and the method further comprises the steps of cleaving the obtained peptide or protein from the polymer carrier using a cleavage composition, and removing the α-amine protecting group using a deprotection solution, wherein the α-amine protecting group is formed by reacting a protecting agent with a functional group on the amino acid or the peptide, - wherein the protective agent comprises I. Main chain structure, II. at least one water solubility enhancing functional group, and III. at least one reactive group, - wherein the main chain structure comprises a portion selected from the group consisting of 9-methylfluorene, tert-butane and / or phenylmethane, diphenylmethane or triphenylmethane, - wherein the water solubility enhancing functional group is selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、CN、OSO3 - Ester, OPO3 2- esters and combinations thereof, and - wherein the water-solubility enhancing functional group and the reactive group are linked to the backbone structure via at least one covalent bond.

2. The method according to claim 1, characterized in that Any reactive side chain functional groups of the first amino acid or first peptide and the second amino acid or second peptide are protected with side chain protecting groups, the side chain protecting groups comprising at least one water solubility enhancing functional group, and the method further comprises the step of removing the side chain protecting groups.

3. The method of claim 1, wherein the α-amine protecting group comprises a fluorescent structure, and the method further comprises the step of monitoring the extent of peptide bond formation and / or monitoring the extent of removal of the α-amine protecting group by measuring fluorescence generated by the α-amine protecting group coupled to the first amino acid or the first peptide. The method of claim 1 , wherein the protective agent comprises at least two water-solubility enhancing functional groups. The method according to claim 4 , wherein the protective agent comprises 2 to 7 water-solubility enhancing functional groups.

6. The method according to claim 1, wherein the water solubility enhancing functional group is SO3 - .

7. The method according to claim 6, wherein the water solubility enhancing functional group comprises at least two SO3 - Group.

8. The method of claim 1, wherein the reactive group is selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, hydroxyls, and thiol groups.

9. The method according to claim 1, wherein the protective agent has the 9-methylfluorene main chain structure of the present invention and is represented by the following general formula 1: in, R1 to R8 are independently selected from hydrogen, SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- ester and CN, provided that at least one of R1 to R8 is selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- ester and CN; R99 is selected from the group consisting of oxycarbonyl halides, oxycarbonyl O-succinimide, oxycarbonyloxy esters, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxyls and mercapto groups.

10. The method according to claim 9, wherein R99 is selected from oxycarbonyl halides, oxycarbonyl oxyesters and oxycarbonyl O-succinimide.

11. The method according to claim 9, wherein at least two of R1 to R8 are selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、OSO3 - Ester, OPO3 2- Esters and CN.

12. The method according to claim 9, wherein R2 and R7 are SO3 - , and R1, R3 to R6 and R8 are all hydrogen.

13. The method according to claim 9, wherein R3 and R6 are SO3 - , and R1, R2, R4, R5, R7 and R8 are all hydrogen.

14. The method according to claim 9, wherein R2 and R6 are SO3 - , and R1, R3, R4, R5, R7 and R8 are all hydrogen.

15. The method according to claim 1, wherein the protecting group has the following general formula 7: in, The nitrogen shown in the general formula is a protected amino group, and R1 to R8 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one of R1 to R8 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN.

16. The method according to claim 15, wherein at least two of R1 to R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN.

17. The method according to claim 15, wherein R1 to R8 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all hydrogen.

18. The method according to claim 15, wherein R2 and R7 are SO3 - , and R1, R3 to R6 and R8 are hydrogen.

19. The method according to claim 15, wherein R3 and R6 are SO3 - , and R1, R2, R4, R5, R7 and R8 are hydrogen.

20. The method according to claim 15, wherein R2 and R6 are SO3 - , and R1, R3, R4, R5, R7 and R8 are hydrogen.

21. The method of claim 1, wherein a coupling agent is used.

22. The method of claim 21, wherein the coupling agent is selected from the group consisting of carbodiimides and triazoles.

23. The method of claim 21, wherein the coupling agent is selected from the group consisting of diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 2-(((ethylimino)methylene)amino)-2-methylpropane-1-sulfonate (ESC) and 2,2'-(methylenebis(azaylidene))bis(2-methylpropane-1-sulfonate) (DSC ); preferred triazoles are O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-]-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), Boc-histidine (toluenesulfonyl); B01' and B01'-Cl, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOB) and 1-hydroxybenzotriazole (HOBt).

24. The method according to claim 1, characterized in that A capping agent is added to the reaction mixture for capping free amines, wherein after the reaction is completed, the reaction mixture is passed through an affinity chromatography column, wherein the capping agent comprises I. Main chain structure, II. at least one water solubility enhancing functional group, and III. at least one reactive group, - wherein the main chain structure comprises a part selected from C1 to C8 alkyl, cycloalkyl chain or aromatic compound, - wherein the water solubility enhancing functional group is selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、CN、OSO3 - Ester, OPO3 2- esters and combinations thereof, and - wherein the water-solubility enhancing functional group and the reactive group are linked to the backbone structure via at least one covalent bond.

25. The method of claim 24, wherein the backbone structure comprises a moiety selected from a C1 to C4 alkyl group or a benzyl group.

26. Protecting agents suitable for forming protecting groups on functional groups of peptides and / or amino acids during water-based peptide synthesis, - wherein the protective agent comprises I. Main chain structure, II. at least two water solubility enhancing functional groups, and III. at least one reactive group, - wherein the main chain structure comprises a portion selected from the group consisting of 9-methylfluorene, tert-butane and / or phenylmethane, diphenylmethane or triphenylmethane, - wherein the water solubility enhancing functional group is selected from SO3 - PO3 2- 、N(CH3)2、N(CH3)3 + 、CN、OSO3 - Ester, OPO3 2- esters and combinations thereof, and - wherein the water-solubility enhancing functional group and the reactive group are linked to the backbone structure via at least one covalent bond.

27. The protecting agent according to claim 26, wherein the reactive group is selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, hydroxyls and thiol groups.

28. The protective agent according to claim 26 or 27, wherein the water-solubility enhancing functional group is SO3 - .

29. The protective agent according to claim 26, wherein the water-solubility enhancing functional group comprises at least two SO3 - Group.

30. The protecting agent according to claim 26, wherein the protecting agent comprises 2 to 7 water-solubility enhancing functional groups.

31. A modified amino acid, peptide, protein or a salt thereof, comprising a protecting group formed by the reaction of the protecting agent according to claim 26 with a functional group of the amino acid, peptide or protein, wherein the functional group is selected from the group consisting of an α-amino group, a side chain amino group, a thiol group, a carboxyl group and a hydroxyl group.

32. The modified amino acid, peptide, protein or salt thereof according to claim 31, wherein the protecting group is the following general formula 7: in, The nitrogen shown in the general formula is a protected amino group, and R1 to R8 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one of R1 to R8 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN.

33. The modified amino acid, peptide, protein or salt thereof according to claim 32, wherein at least two of R1 to R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN.

34. The modified amino acid, peptide, protein or salt thereof according to claim 32, wherein R1 to R8 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all hydrogen.

35. The modified amino acid, peptide, protein or salt thereof according to claim 32, wherein R2 and R7 are SO3 - , and R1, R3 to R6 and R8 are hydrogen.

36. The modified amino acid, peptide, protein or salt thereof according to claim 32, wherein R3 and R6 are SO3 - , and R1, R2, R4, R5, R7 and R8 are hydrogen.

37. The modified amino acid, peptide, protein or salt thereof according to claim 32, wherein R2 and R6 are SO3 - , and R1, R3, R4, R5, R7 and R8 are hydrogen.

38. An amino acid, peptide or protein or a salt thereof containing an α-amine protecting group, wherein the protecting group has the following general formula 7: in, The nitrogen shown in the general formula is a protected amino group, and R1 to R8 are independently selected from hydrogen, SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN, provided that at least one of R1 to R8 is selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN.

39. The amino acid, peptide or protein according to claim 38, wherein at least two of R1 to R8 are selected from SO3 - 、OSO3 - Ester, OPO3 2- Ester, PO3 2- 、N(CH3)2、N(CH3)3 + and CN.

40. The amino acid, peptide or protein of claim 38, wherein R1 to R8 are not SO3 - PO3 2- 、OSO3 - Ester, OPO3 2- Esters, N(CH3)2, N(CH3)3 + or CN, all hydrogen.

41. The amino acid, peptide or protein of claim 38, wherein R2 and R7 are SO3 - , and R1, R3 to R6 and R8 are hydrogen.

42. The amino acid, peptide or protein of claim 38, wherein R3 and R6 are SO3 - , and R1, R2, R4, R5, R7 and R8 are hydrogen.

43. The amino acid, peptide or protein of claim 38, wherein R2 and R6 are SO3 - , and R1, R3, R4, R5, R7 and R8 are hydrogen.

Images