Method for preparing peptides

Abstract

The present invention relates to a method for preparing a peptide, the method comprising a step of forming a peptide bond in which a carboxyl group of a first amino acid or a first peptide is activated, an amino group of the activated first amino acid or first peptide is protected by a protecting group having a water-solubility enhancing group, and the activated carboxyl group of the first amino acid or the first peptide reacts with the amino group of the second amino acid or the second peptide, wherein the carboxyl group of the first amino acid or the first peptide is activated in the absence of the second amino acid or the second peptide. The invention also relates to a peptide comprising a protecting group wherein the protecting group has a water solubility enhancing group bonded to an amino group and an activated or free carboxyl group.

Description

[0001] Divisional application

[0002] This application is a divisional application of application number 201880086678.2, filing date November 23, 2018, and titled “Method for Preparing Peptides”. Technical Field

[0003] The present invention relates to an improved method for preparing peptides, and to peptides obtained by this method. Background Art

[0004] Peptides are chains of linked amino acids that represent the precursors of proteins. Peptides and proteins are fundamental building blocks of all living systems and are involved in various life processes. They have many applications in medicine and biological sciences. Therefore, the ability to synthesize peptides and proteins is of great significance to human life.

[0005] Therefore, the synthetic production of peptides is of great significance. Peptides are usually synthesized by coupling the carboxyl group (C-terminus) of one amino acid with the amino group or N-terminus of another amino acid.

[0006] In general, chemical synthesis starts with the C-terminal amino acid of the chain. In addition, it requires additional chemical groups to protect the C-terminus of the first amino acid. The coupling process of each amino acid can be completed by using different chemical coupling reagents to produce activated carboxyl groups. After the formation of the amide bond, the Nα-protecting group is removed; the conditions depend on the protecting group used. However, liquid phase chemistry is time-consuming and labor-intensive because the product must be manually separated from the reaction solution after each step through purification steps (extraction, washing or crystallization). Despite the shortcomings of this method, liquid phase chemistry remains an alternative method for the production of short peptides such as dipeptides, tripeptides or tetrapeptides, which can be obtained using fragment condensation methods to obtain longer sequences. This is because Boc- or CBz-protected amino acids are cheaper raw materials than Fmoc-amino acids, and liquid phase chemistry has almost unlimited synthetic capabilities.

[0007] Merrifield et al. adopted solid phase peptide synthesis (SPPS) in 1963 to overcome the intermediate purification problems associated with peptide assembly in solution (see Stewart and Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984), Chan and White, Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford University Press, 2000)). In solid phase synthesis, the C-terminus is anchored to an insoluble polymer support (solid phase), and amino acids are continuously coupled to produce peptide segments with the desired sequence. Once the desired sequence is assembled, the peptide is cleaved from the solid phase support.

[0008] This synthetic scheme requires protection of the α-amino group of the input 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, which react under neutral conditions in the presence of PhSiH as an allyl system scavenger. 3 In the presence of , it is removed by Pd catalysis.

[0009] Solid phase peptide synthesis methods following any of the three α-amino group protection schemes described above usually require additional protection of the reactive side chains of the constituent amino acids to avoid undesired chemical transformations. Therefore, these protecting groups need to be resistant to the reagents used in the coupling cycle. In addition, the connection between the growing peptide chain and the solid support must be stable to the conditions of α-amino group deprotection and chain assembly.

[0010] 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. Side chain protecting groups are usually removed by weak acidic reagents after the peptide chain is assembled. These side chain protecting groups are usually removed by anhydrous HF, trifluoromethanesulfonic acid or trifluoroacetic acid (TFA) after the desired peptide chain is assembled.

[0011] Since the α-amino protecting groups Fmoc and Boc (which are insoluble in water and often used in SPPS) are highly hydrophobic, the peptide assembly process is usually carried out in polar aprotic organic solvents such as dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO) and dichloromethane (DCM), or mixtures of these organic solvents. In addition, the side chain protecting groups commonly used in SPPS are usually hydrophobic, rendering the amino acids insoluble in water.

[0012] SPPS methods using Fmoc and Boc protection have been widely used but are hampered by the need for the aforementioned expensive and toxic organic solvents. For example, DMF is associated with considerable health and environmental risks; it has been linked to human cancer and is suspected of causing birth defects. Therefore, the use of these toxic solvents requires special technical equipment and precautions, such as performing the reactions under a fume hood and being handled by highly qualified personnel. In addition, disposal of the used solvents is difficult and expensive. Therefore, SPPS using organic solvents is expensive and limited to specialized laboratories with specialized equipment for organic chemical synthesis.

[0013] As 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.). They developed several protecting groups for this purpose, including 2-(phenyl(methyl)sulfonyl)ethoxycarbonyltetrathioborate (Pms), ethylsulfonylethoxycarbonyl (Esc) and 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps). In WO 2013115813 A1, CEM claims the deprotection of α,β-unsaturated sulfone in water or aqueous systems, and its application in water-based SPPS.

[0014] Furthermore, WO 2016 / 050764 A1 describes the synthesis of water-based peptides.

[0015] The above methods can be used to synthesize peptides. However, there is a permanent desire to improve these methods. Summary of the invention

[0016] Therefore, it is an object of the present invention to provide an improved method for peptide synthesis in general. The method is applicable to both liquid phase and solid phase peptide synthesis.

[0017] According to a specific purpose of the present invention, the above method can provide high yield. Further, the synthesis of peptides can be carried out at low cost. Another object of the present invention is a method for incorporating low racemic amino acid units in a peptide chain. In addition, the method for preparing peptides can be carried out in a low waste and high environmental sustainability manner.

[0018] Further, in order to obtain high acceptability, the method can be implemented using known equipment. Moreover, the impact of this method on the environment can be very small. Preferably, only environmentally acceptable solvents and compounds can be used, and / or the solvent can be easily recovered and is not expected to have a significant impact on the environment.

[0019] These objects, as well as other objects which are not explicitly stated but which can be immediately inferred or recognized by reference from the context described herein, are solved by a process for the preparation of peptides having all the features of claim 1, including a step for forming peptide bonds.

[0020] Therefore, the present invention provides a method for preparing a peptide, which includes the step of forming a peptide bond, characterized in that the carboxyl group of a first amino acid or a first peptide is activated, and the amino group of the activated first amino acid or first peptide is protected, preferably by a protecting group having a water-solubility enhancing group or by a solid phase; the activated carboxyl group of the first amino acid or the first peptide reacts with the amino group of the second amino acid or the second peptide; wherein the carboxyl group of the first amino acid or the first peptide is activated in the absence of the second amino acid or the second peptide.

[0021] Thus, the above-mentioned prior art methods for preparing peptides can be improved in ways that are difficult to predict.

[0022] The peptides can be obtained very cheaply. Surprisingly, the peptides obtained contain only very small amounts of by-products, in particular, a low racemization of the amino acid units incorporated into the peptide chain can be achieved by the process.

[0023] Furthermore, the method according to the invention enables a particularly selective preparation of peptides.Furthermore, the method according to the invention can be carried out in a simple and inexpensive manner, allows high yields of product to be obtained and, overall, has low energy consumption.

[0024] Furthermore, the method of preparing the peptides can be carried out in a low-waste and highly environmentally sustainable manner.

[0025] Further, in order to obtain high acceptability, the method can be implemented using known equipment. Moreover, the method has a small impact on the environment. Preferably, only environmentally acceptable solvents and compounds are used, and / or the solvent can be easily recovered, so that it is expected that there will be no significant impact on the environment.

[0026] According to a first embodiment of the present invention, the amino group of the activated first amino acid or first peptide is protected by a protecting group having a water-soluble enhancing group. In addition, the first amino acid or first peptide preferably includes an additional protecting group. Preferably, the functional group to be protected is preferably selected from amine, alcohol, sulfhydryl, carboxyl, phosphono and / or seleno.

[0027] In a preferred embodiment, the water solubility enhancing functional group is selected from the charged functional group SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 32- Esters and N(CH 3 ) 3 + The use of charged functional groups as water solubility enhancing functional groups has been shown to be more effective in improving solubility in environmentally friendly solvents, such as water solubility, than uncharged functional groups.

[0028] According to a second embodiment of the present invention, the amino group of the activated first amino acid or the first peptide is protected by a solid phase. Solid phase protection is preferably achieved by covalent bonding of the amino group of the activated first amino acid or the first peptide to the solid phase. Preferably, the covalent bond of the amino group of the activated first amino acid or the first peptide to the solid phase is cleavable. Therefore, the peptide or protein obtained by the method can be released from the solid phase by a cleavage reaction as described in more detail below. In addition, the first amino acid or the first peptide preferably includes other protecting groups. Preferably, the functional group to be protected is preferably selected from amine, alcohol, sulfhydryl, carboxyl, phosphono and / or seleno.

[0029] Considering sustainability, cost and efficiency, it is more preferred to protect the amino group of the activated first amino acid or the first peptide by a protecting group having a water solubility enhancing group than using solid phase protection. On the other hand, protecting the amino group of the activated first amino acid or the first peptide by solid phase provides an improvement in the production of peptides with a large number of amino acid units.

[0030] Preferably, an environmentally friendly solvent is used to achieve the formation of peptide bonds. The formation of peptide bonds includes the activation of the carboxyl group of the first amino acid or the first peptide, the reaction of the activated carboxyl group of the first amino acid or the first peptide with the amino group of the second amino acid or the second peptide, or these two steps. Usually, the synthesis of peptides is achieved in a solvent, wherein the solvent is essential for the environment or health. These solvents include dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP). Although these solvents can be used to implement this method, the present invention is not limited thereto, and environmentally friendly solvents can also be used, which provides further cost advantages and fewer precautions based on environmental standards. Therefore, one of the advantages of the present invention is to utilize amino acids and / or peptides that can be dissolved in a protected form in an environmentally friendly solvent.

[0031] As used herein, the term "soluble in an environmentally friendly solvent in a protected form" means that the composition has the solubility necessary to carry out the desired reaction in the solvent system. As with any composition, the term "soluble" does not imply unlimited solubility in any or all amounts.

[0032] Preferably, the environmental friendliness of the solvent is based on its impact on health or the environment. Substances listed as substances of very high concern (SVHC) by the European Chemicals Agency (ECHA) or included in the candidate list of substances of very high concern are not environmentally friendly solvents. In order to avoid the use of harmful organic solvents that may have serious and often irreversible effects on human health and the environment, all solvents listed as SVHC by ECHA should be avoided (2017.11.14 statement).

[0033] According to a preferred embodiment, the DNEL value of the environmentally friendly solvent is preferably at least 5 mg / m 3 , more preferably at least 10 mg / m 3 , more preferably at least 20 mg / m 3 , more preferably at least 40 mg / m 3 , more preferably at least 45 mg / m 3 , more preferably at least 60 mg / m 3 , more preferably at least 100 mg / m 3 , more preferably at least 120 mg / m 3 , even more preferably at least 200 mg / m 3 (Inhalation, systemic, as provided in ECHA statement 2017.11.14).

[0034] In a specific embodiment, the lethal dose LD50 of the environmentally friendly solvent is preferably at least 0.1 g / kg, preferably at least 0.5 g / kg, more preferably at least 1.0 g / kg, more preferably at least 1.5 g / kg, more preferably at least 2 g / kg, even more preferably at least 2.5 g / kg (oral, rat).

[0035] Preferred solvents, more preferably environmentally friendly solvents include: protic solvents, such as water, primary alcohols, secondary alcohols and / or tertiary alcohols; aprotic solvents, such as ketones, nitriles, lactones, lactams, carbonic acid amides, carbonic acid esters, ethers, urea derivatives, sulfoxides, sulfones, carbonate esters. Preferred examples of aprotic solvents are, for example, dimethylacetamide, ethyl methyl ketone, acetone, methyl isopropyl ketone, methyl isobutyl ketone, methyl n-butyl ketone, methyl tert-butyl ketone, methyl isoamyl ketone, dimethyl sulfoxide, hexamethylphosphoric triamide, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol, dimethyl ether, ethyl acetate, tert-butyl acetate or a mixture thereof.

[0036] A protic solvent is a solvent that has hydrogen atoms bonded to oxygen (such as in a hydroxyl group) or nitrogen (such as in an amine group). An aprotic solvent is a solvent that does not include hydrogen atoms bonded to oxygen (such as in a hydroxyl group) or nitrogen (such as in an amine group). Thus, ketones are aprotic solvents, but some isomers (enol isomers) may contain such hydrogen atoms. However, based on the lower percentages of these isomers formed, these isomers must be ignored for the present invention.

[0037] The present invention can be realized using protic solvents such as water, primary alcohol, secondary alcohol and / or tertiary alcohol. Based on the coupling reaction forming peptide bonds, primary amine and secondary amine cannot be used as solvents. Preferred alcohols include methanol, ethanol, isopropanol, 2-propanol, n-propanol, n-butanol, isobutanol, sec-butanol, tert-butanol or their mixtures. Protic solvents provide advantages about cost and less precautions based on environmental standards. For protic solvents, water is particularly preferred. In addition to cost advantage and high environmental acceptance, water provides improvements in safety, particularly by solvents containing water, low flammability and combustibility can be achieved. Further, compared with other protic solvents, such as alcohol, particularly primary alcohol, water provides higher productivity.

[0038] In one embodiment of the present invention, the solvent, preferably the environmentally friendly solvent, preferably comprises at least 5% by weight, more preferably at least 10% by weight, even more preferably at least 25% by weight of a protic solvent, preferably water, based on the total weight of the solvent, preferably the environmentally friendly solvent.

[0039] In another embodiment of the present invention, the solvent, preferably the environmentally friendly solvent, preferably comprises at least 5 wt.%, more preferably at least 10 wt.%, even more preferably at least 25 wt.% of a protic solvent, preferably water, based on the total weight of the solvent, preferably the environmentally friendly solvent.

[0040] Surprising improvements can be achieved by solvents, preferably environmentally friendly solvents, wherein the environmentally friendly solvents include aprotic organic solvents and / or secondary alcohols and / or tertiary alcohols, preferably aprotic organic solvents and / or tertiary alcohols, more preferably aprotic organic solvents. The use of aprotic organic solvents and / or secondary alcohols and / or tertiary alcohols provides surprising improvements in yield. Further, the use of aprotic organic solvents provides a higher conversion rate. Therefore, longer peptide chains can be obtained with considerable yields. Further, the activation of the above-mentioned and following amino acids or peptides is easier, and the activated amino acids or peptides are more stable. Therefore, a smaller amount of activating agent can be used. In addition, if the method is carried out in the above-mentioned or following ion exchangers, greater advantages can be achieved. Preferably, the ion exchanger is used in a method in which the amino group of the activated first amino acid or the first peptide is protected by a protecting group having a water-soluble enhancing group. Aprotic organic solvents and / or secondary alcohols and / or tertiary alcohols, used in combination with ion exchangers, can improve the yield of the prepared peptide. These improvements can be increased by increasing the amount of aprotic organic solvent. These improvements are achieved by using a solvent, preferably an environmentally friendly solvent, wherein the environmentally friendly solvent includes an aprotic organic solvent and / or a secondary alcohol and / or a tertiary alcohol, preferably an aprotic organic solvent and / or a tertiary alcohol, more preferably an aprotic organic solvent, for activating the carboxyl group of the first amino acid or the first peptide. Further, these improvements are achieved by using a solvent, preferably an environmentally friendly solvent, wherein the environmentally friendly solvent includes an aprotic organic solvent and / or a secondary alcohol and / or a tertiary alcohol, preferably an aprotic organic solvent and / or a tertiary alcohol, more preferably an aprotic organic solvent, for causing the activated carboxyl group of the first amino acid or the first peptide to react with the amino group of the second amino acid or the second peptide as described above or as follows. In a very preferred embodiment, the activation of the carboxyl group of the first amino acid or the first peptide, and the above or below reaction of the activated carboxyl group of the first amino acid or the first peptide with the amino group of the second amino acid or the second peptide bond, are achieved by using a solvent, preferably an environmentally friendly solvent, wherein the environmentally friendly solvent comprises an aprotic organic solvent and / or a secondary alcohol and / or a tertiary alcohol, preferably an aprotic organic solvent and / or a tertiary alcohol, more preferably an aprotic organic solvent.

[0041] In one embodiment of the present invention, the solvent, preferably the environmentally friendly solvent, preferably comprises at least 5% by weight, more preferably at least 10% by weight, even more preferably at least 25% by weight of aprotic organic solvents and / or secondary and / or tertiary alcohols, preferably aprotic organic solvents, based on the total weight of the solvent, preferably the environmentally friendly solvent.

[0042] In a particularly suitable variant, the solvent, preferably the environmentally friendly solvent, comprises an aprotic organic solvent as a first solvent, and water and / or an alcohol as a second solvent, preferably an aprotic organic solvent and water.

[0043] In a preferred embodiment, based on the total weight of the solvent, preferably the environmentally friendly solvent, the solvent, preferably the environmentally friendly solvent comprises 5% by weight to 95% by weight, preferably 10% by weight to 70% by weight of the second solvent. Further, based on the total weight of the solvent, preferably the environmentally friendly solvent, the solvent, preferably the environmentally friendly solvent comprises 5% by weight to 95% by weight, more preferably 10% by weight to 90% by weight, even more preferably 30% by weight to 70% by weight of the first solvent.

[0044] Surprisingly, the use of aprotic organic solvents with low polarity, such as dimethylacetamide, ethyl methyl ketone, acetone, methyl isopropyl ketone, methyl isobutyl ketone, methyl n-butyl ketone, methyl tert-butyl ketone, methyl isoamyl ketone, dimethyl sulfoxide, hexamethylphosphoric triamide, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol, dimethyl ether, ethyl acetate, tert-butyl acetate or mixtures thereof, and / or protic solvents, such as water and alcohols, provides a surprising improvement in the racemization of the formed peptides. In contrast, the use of polar aprotic organic solvents, such as dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP), results in a peptide having a higher racemization. Preferably, polarity is defined as the dipole moment of the molecule. Preferably, the aprotic organic solvent has a dipole of at most 12.6 10 preferably by measuring the dielectric constant using the Debye equation and / or measuring the Starr effect at 25°C and 1023 mbar. -30 Cm, more preferably at most 12.5 10 -30 In a preferred embodiment, the racemization of the peptide formed can be avoided or reduced by adding and / or using a protic solvent, more preferably water. If a polar aprotic organic solvent is used, a protic solvent, preferably water, is added to reduce the racemization of the peptide formed.

[0045] Preferably, the formation of the peptide bond is achieved in a solution without strong alkaline conditions, preferably at a pH below 12, more preferably at a pH below 10, wherein the pH is measured according to known professional methods, such as a pH electrode, by adding water to the solution at 25°C. The sample to be measured preferably contains at least 80% water by weight. Preferably, two, three or more buffer solutions with specified pH values ​​are used to calibrate the pH electrode. Carrying out the reaction in a solution without strong alkaline conditions provides a surprising improvement in the racemization of the peptide formed.

[0046] According to the present invention, the carboxyl group of the first amino acid or the first peptide is activated, and the activated carboxyl group of the first amino acid or the first peptide reacts with the amino group of the second amino acid or the second peptide.

[0047] In a preferred embodiment, the carboxyl group of the first amino acid or the first peptide is activated by the coupling agent.

[0048] Preferably, the coupling agent is a combination of carbodiimides, preferably diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 2-((ethylimino)methylene)amino)-2-methylpropane-1-sulfonate (ESC) and 2,2'-(methanediylidenebis(azidoylidene)bis(2-methylpropane-1-sulfonate) (DSC), and a compound that forms an active ester, preferably 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (NHS), ethyl cyano(hydroxyimino)acetate (Oxyma), hydroxy-3,4-dihydrobenzotriazole (HBO ... Hydrogen-4-oxo-1,2,3-benzotriazine (HOOBt), 4-dimethylaminopyridine (DMAP), 2-hydroxypyridine-N-oxide (HOPO). Coupling agents can also be based on phosphonium- and amine-(imine-) type reagents, such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate (PyBOP), bromotris(pyrrolidinyl)phosphonium hexafluorophosphate (PyBrOP), acetic acid-(hydroxyimino)acetic acid-O2)-tris-(1-pyrrolidinyl)-phosphonium hexafluorophosphate (PyOxim), (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one) (DEPBT ), (1H-benzotriazol-1-yloxy)-N,N-dimethylformimine hexachloroantimonate (BOMI), 5-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl2H-pyrrolium hexachloroantimonate (BDMP), 5-(7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (AOMP), 5-(pentafluorophenyloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (FOMP), 5-(3',4'-dihydro-4'-oxy-1',2',3'-benzotriazine-3'-yloxy)-3,4-dihydro-1-methyl2H-pyrrolium hexachloroantimonate (DOMP), 1-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl2H-pyrrolium hexachloroantimonate (DOMP), 1-(7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (AOMP), 5-(pentafluorophenyloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (FOMP), 5-(3',4'-dihydro-4'-oxy-1',2',3'-benzotriazine-3'-yloxy)-3,4-dihydro-1-methyl2H-pyrrolium hexachloroantimonate (DOMP), 1-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl triazol-1-yloxy)phenylmethylenepyrrolidinium hexachloroantimonate (BPMP), 5-(succinimidyloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (SOMP), tetramethylfluoroformamide hexafluorophosphate (TFFH), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU), (2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,33-Tetramethylamine) hexafluorophosphate (HCTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), 1-((dimethylamino)(dimethylimino)methoxy)-2-hydroxypyridinium tetrafluoroborate (TPTU), O-(cyano(ethoxycarbonyl)methyleneamino)-1,1,3,3-tetramethyluronium tetrafluoroborate (TOTU), 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate Uronium tetrafluoroborate (TDBTU), 1-[1-(cyano-2-ethoxy-2-oxyethylideneaminooxy)-dimethylamino-morpholinium] uronium hexafluorophosphate (COMU), 4-{[1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)-ylideneaminooxy](dimethylamino)-methylene}morpholin-4-ium hexafluorophosphate (COMBU), N-{[1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)-ylideneaminooxy](dimethylamino)methylene}-N-methylformamide hexafluorophosphate (TOMBU), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). It is also possible to mix coupling agents such as EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) and DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine salt), as well as other condensation products of substituted cyanuric chloride and base.

[0049] In a preferred embodiment, the carboxyl group of the first amino acid or the first peptide is activated by a coupling agent. Preferably, an excess of coupling agent is used, preferably at least two times the molar amount of coupling agent is used relative to the first amino acid or the first peptide, and more preferably at least three times the molar amount of coupling agent is used. Preferably, before adding the second amino acid or the second peptide to the activated first amino acid or the first peptide, the excess coupling agent is removed from the reaction mixture. In an optional embodiment, an equimolar amount or less of coupling agent is used to achieve that all of the added coupling agent reacts with the first amino acid or the first peptide. In this case, no excess coupling agent remains in the reaction mixture.

[0050] In one embodiment of the present invention, it can be provided that the first amino acid or the first peptide is preferably contacted with an ion exchanger ion. The contacting of the first amino acid or the first peptide can be achieved in any step of the above-mentioned and following present methods. Preferably, the first amino acid or the first peptide contacted with the ion exchanger ion comprises at least one protecting group having a water solubility enhancing group, more preferably an ionic water solubility enhancing group.

[0051] In a preferred embodiment, preferably while the first amino acid or first peptide is ionically bonded to the ion exchanger, the carboxyl group of the first amino acid or first peptide is activated by a coupling agent. Preferably, the first amino acid or first peptide ionically bonded to the ion exchanger comprises at least one protecting group having a water solubility enhancing group, more preferably an ionic water solubility enhancing group. In a variant of this method, preferably before the second amino acid or second peptide is added to the ion exchanger, excess coupling agent is removed from the ion exchanger.

[0052] In a further preferred embodiment, the formation of the peptide bond is achieved when the amino acid or peptide is bonded to an ion exchanger. Preferably, the first amino acid or first peptide ionically bonded to the ion exchanger comprises at least one protecting group having a water solubility enhancing group, more preferably an ionic water solubility enhancing group. Ionic bonding to the ion exchanger may include any steps to form a peptide bond, wherein the formation of the peptide bond comprises: activation of a carboxyl group of the amino acid or peptide, reaction of the activated carboxyl group of the amino acid or peptide with an amino group of another amino acid or another peptide, or both of these steps. Preferably, the formation of the peptide bond is achieved when the amino acid or peptide is not covalently bonded to the ion exchanger.

[0053] According to a second embodiment of the present invention, the amino group of the activated first amino acid or the first peptide is protected by a solid phase. The solid phase (solid carrier) is preferably an insoluble resin, preferably based on cross-linked polystyrene, polypropylene, polyphenols, polysaccharides, polyamides or polylysine. Preferably, by a linking group, the covalent bonding of the amino group of the activated first amino acid or the first peptide to the solid carrier is realized. The linking group preferably provides the cleavable connection of the growing peptide to the solid phase. The cracking is preferably realized using the following cracking composition. The use of the solid carrier in preparing peptides is well known in synthesis, wherein the C-terminal of the first amino acid is anchored via the N-terminal, and other amino acids are coupled to the N-terminal of the peptide anchored on the solid carrier. Solid carriers with reactive groups can be purchased on the market, wherein the reactive group can form a covalent bond with the amino group of the activated first amino acid or the first peptide. The reactive group of the solid phase can be used to form a connection with the amino group of the activated first amino acid or the first peptide, which is well known in the art. Those skilled in the art will select the group according to the reaction conditions, and the stability and cleavage required for bonding.

[0054] Preferably, the carboxyl group of the second amino acid or second peptide reacted with the activated first amino acid or first peptide is not protected. In a preferred embodiment, the carboxyl group of the second amino acid or second peptide can be avoided to protect. This embodiment provides a surprising cost advantage. Protection can be achieved by blocking groups, or by bonding to a solid phase.

[0055] In a preferred embodiment, the synthesis of the peptide is established via the carboxyl group of the amino acid or peptide, and the carboxyl group of the second amino acid or second peptide that reacts with the first activated amino acid or first peptide is not protected, wherein the method comprises the following steps: activating the first amino acid or first peptide, removing excess coupling agent, adding the second amino acid or second peptide to the activated first amino acid or first peptide, reacting the second amino acid or second peptide with the activated first amino acid or first peptide and forming a peptide bond, and removing the reaction residue by a washing step. These steps can be repeated to obtain the desired peptide.

[0056] In an optional embodiment, the synthesis of the peptide is established via the carboxyl group of the amino acid or peptide, and the carboxyl group of the second amino acid or second peptide that reacts with the first activated amino acid or first peptide is protected, and the method comprises the following steps: activating the first amino acid or first peptide, adding the second amino acid or second peptide having a protected carboxyl group to the activated first amino acid or first peptide, reacting the second amino acid or second peptide with the activated first amino acid or first peptide and forming a peptide bond, optionally blocking the unreacted carboxyl group of the first amino acid or first peptide, and deprotecting the carboxyl group of the second amino acid or second peptide. Optionally, the reaction residue is removed by a washing step implemented between the above steps. These steps can be repeated to obtain the desired peptide.

[0057] Preferably, the second amino acid or second peptide is preferably a peptide having at least two amino acid units, more preferably a peptide having at least four amino acid units, and even more preferably a peptide having at least six amino acid units. This embodiment provides an unforeseen improvement in the yield of the prepared peptide and provides a cost improvement.

[0058] According to a preferred embodiment, the amino group of the activated first amino acid or first peptide is protected by a protecting group, wherein the protecting group has a water-solubility enhancing group ionically bonded to an ion exchanger, preferably, the water-solubility enhancing group includes an ionic group ionically bonded to an ion exchanger.

[0059] The type of protecting group used to achieve ionic bonding to the ion exchanger is not critical.

[0060] Preferably, the ion exchanger is an ion exchange resin, preferably based on crosslinked polystyrene, polypropylene, polyphenol or polysaccharide and having functional groups, or the ion exchanger is a mineral ion exchanger, preferably based on silica.

[0061] Preferably, the functional group of the ion exchange resin is a tetraalkylammonium group (-NR 3 + ), primary, secondary or tertiary amine (-NH 2 , -NHR, -NR 2 ), carboxyl (-COO- ), sulfonic acid group (-SO 3 - ), wherein R is an alkyl group having 1 to 10 carbon atoms.

[0062] In this specification, conventional terms are used to describe ion exchangers. Thus, basic ion exchangers are solids that have the ability to exchange anions. Acidic ion exchangers are solids that have the ability to exchange cations.

[0063] In a specific embodiment, a basic exchange resin is used, and the amino acid or peptide contains an anionic water-solubility enhancing group, preferably a weakly basic exchange resin containing primary, secondary and / or tertiary amine groups. Preferably, an anion exchanger is used.

[0064] In another embodiment of the present invention, a cationic ion exchanger is used. Preferably, an acid exchange resin is used, and the amino acid or peptide contains a cationic water-solubility enhancing group.

[0065] The amino acid or peptide of the present invention preferably includes a protecting group with a water-soluble enhancing group. The expression "water-soluble enhancing group" describes a group that provides improved water solubility to the protecting group. In principle, each conventional protecting group used in peptide synthesis can be used, which has a group or modification that provides improved water solubility. These protecting groups with water-soluble enhancing groups include compounds such as those mentioned in Hojo et al. (Chem. Pharm. Bull. 2004, 52, 422-427 and Tetrahedron Lett. 2004, 45, 9293.) and WO 2013 115813A1, such as 2-(phenyl (methyl) sulfonyl) ethoxycarbonyl tetrathioborate (Pms), ethylsulfonyl ethoxycarbonyl (Esc) and 2-(4-sulfophenylsulfonyl) ethoxycarbonyl (Sps).

[0066] In a preferred embodiment, the protecting group having a water-solubility enhancing group comprises a main chain structure and a linking group derived from a reactive group.

[0067] In a specific embodiment, the protection of the amino acid or peptide is preferably achieved by reacting the amino acid or peptide with a protecting agent, wherein the protecting agent comprises

[0068] I. Main chain structure,

[0069] II. at least one water solubility enhancing group, and

[0070] III. at least one reactive group,

[0071] wherein the main chain structure comprises a portion selected from the group consisting of 9-methylfluorene, tert-butane and / or monophenylmethane, diphenylmethane or triphenylmethane,

[0072] Wherein the water solubility enhancing group is selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、CN、OSO 3 - Ester, OPO 3 2- esters and combinations thereof, and

[0073] The water-solubility enhancing group and the reactive group are attached to the main chain structure via at least one covalent bond.

[0074] The protective agent may contain one or more water-solubility enhancing functional groups. 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 it may be useful to contain more than one water-solubility enhancing functional group in the protective agent molecule because the water solubility is increased to a greater extent. In an embodiment where the main chain structure is tert-butane or phenylmethane, one water-solubility enhancing functional group may be sufficient.

[0075] In a preferred embodiment of the present invention, the protective agents are all of the same type, especially all SO 3 - In an alternative embodiment, the protective agent includes different types of water-solubility enhancing functional groups. As long as the water-solubility enhancing functional group is SO 3 - , then the protective agent preferably comprises at least two such functional groups. If the water-solubility enhancing functional groups are all of the same kind, the synthesis of the protective agent can be more efficient and easier.

[0076] The main chain structure comprises a moiety selected from the group consisting of 9-methylfluorene, tert-butane and / or monophenylmethane, diphenylmethane or triphenylmethane. In a preferred embodiment, the main chain structure is selected from 9-methylfluorene and tert-butane.

[0077] 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 (oxycarbonylOxyma ester), oxycarbonyl anhydride, halide, oxymethyl halide, hydroxide 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 hydroxide, halide, sulfhydryl, oxycarbonyl O-succinimide and oxycarbonyl anhydride. When the main chain structure is selected from monophenylmethane, diphenylmethane and triphenylmethane, the reactive group is preferably selected from halide, oxymethyl halide and oxycarbonyl halide.

[0078] In the present invention, the preferred protective agent having a 9-methylfluorene main chain structure can be exemplified by the following general formula 1:

[0079]

[0080] Wherein, R1 to R8 are independently selected from hydrogen, SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- ester and CN, provided that at least one, preferably at least two, of R1 to R8 are selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- In a preferred embodiment, R1 to R8 are not SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 +、OSO 3 - Ester, OPO 3 2- Esters or CN, all hydrogen.

[0081] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl O-succinimide, oxycarbonyloxy esters, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxides and mercapto groups, wherein R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyloxy esters and oxycarbonyl O-succinimide.

[0082] In a preferred embodiment, R2 and R7 are selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- ester and CN, in particular, R2 and R7 are SO 3 - Preferably, R1, R3 to R6 and R8 are hydrogen.

[0083] In a preferred embodiment, R3 and R6 are selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- ester and CN, in particular, R3 and R6 are SO 3 - Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

[0084] In a preferred embodiment, R2 and R6 are selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3- Ester, OPO 3 2- ester and CN, especially, R2 and R6 are SO 3 - Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

[0085] 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 do not limit the scope of the present invention in any way. According to the present invention, they constitute exemplary and preferred protective agents.

[0086] Table 1

[0087]

[0088]

[0089]

[0090] In the present invention, the preferred protective agent having a tert-butane main chain structure can be exemplified by the following general formula 2:

[0091]

[0092] Where R1 is selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- In a preferred embodiment, R1 is SO 3 - R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl O-succinimide, oxycarbonyl anhydride, halide, oxymethyl halide, hydroxide and mercapto groups, wherein more preferably, R99 is selected from hydroxide, halide, mercapto, oxycarbonyl O-succinimide and oxycarbonyl anhydride.

[0093] In the present invention, 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 exemplary and preferred protective agents.

[0094] Table 2

[0095]

[0096]

[0097] In the present invention, the preferred protective agent having a monophenylmethane, diphenylmethane or triphenylmethane main chain structure can be exemplified by the following general formula 3, 4 or 5, respectively:

[0098]

[0099] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- ester and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- In a preferred embodiment, R1 to R5 are not SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- Esters or CN, all hydrogen.

[0100] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxides and thiol groups, wherein more preferably, R99 is selected from halides, oxymethyl halides and oxycarbonyl halides.

[0101] In a preferred embodiment, R3 is selected from SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + 、OSO 3 - Ester, OPO 3 2- Esters and CN, especially, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0102]

[0103]

[0104] Wherein, R1 to R10 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R10 are selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R10 are not SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3) 3 + or CN, all of which are hydrogen.

[0105] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxides and mercapto groups, wherein more preferably, R99 is selected from halides, oxymethyl halides and oxycarbonyl halides.

[0106] In a preferred embodiment, R3 and R8 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 and R8 are selected from SO 3 - Preferably, R1, R2, R4 to R7, R9 and R10 are hydrogen.

[0107]

[0108] Wherein, R1 to R15 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 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 SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 +In a preferred embodiment, R1 to R15 are not SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0109] R99 is preferably selected from the group consisting of oxycarbonyl halides, oxycarbonyl oxyesters, oxycarbonyl O-succinimide, oxycarbonyl anhydrides, halides, oxymethyl halides, hydroxides 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 SO 3 - In another particularly preferred embodiment, when R3, R8 and R13 are SO 3 - 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, hydroxides and mercapto groups. In a particularly preferred embodiment, R99 is not a halide, especially not a chloride.

[0110] In a preferred embodiment, R3, R8 and R13 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3, R8 and R13 are SO 3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0111] In the present invention, preferred protective agents having a monophenylmethane, 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 exemplary and preferred protective agents.

[0112] Table 3

[0113]

[0114]

[0115]

[0116] Preferably, the functional groups are present on the amino acids, peptides or proteins and the chemical reactions are peptide or protein synthesis in a solvent as described above or below.

[0117] The above or below protecting agents provide a protecting group for an amino acid or peptide. In a preferred embodiment, the α-amine protecting group is at least one of the following formulas 6 to 10. In each case, the nitrogen shown in the following general formula belongs to the protected amino group:

[0118]

[0119] Wherein, R1 to R15 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 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 SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 3 + In a preferred embodiment, R1 to R15 are not SO 3 - ,PO 3 2-、N(CH 3 ) 2 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0120] In a preferred embodiment, R3, R8 and R13 are selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3, R8 and R13 are SO 3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0121]

[0122] Wherein, R1 to R8 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R8 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 - 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R8 are not SO3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0123] In a preferred embodiment, R2 and R7 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R2 and R7 are SO 3 - Preferably, R1, R3 to R6 and R8 are hydrogen.

[0124] In a preferred embodiment, R3 and R6 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 and R6 are SO 3 - Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

[0125] In a preferred embodiment, R2 and R6 are selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2、N(CH 3 ) 3 + and CN, in particular, R2 and R6 are SO 3 - Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

[0126]

[0127] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - ,PO 3 2- 、N(CH 3 ) 2 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0128] In a preferred embodiment, R3 is selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2-Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0129]

[0130] Wherein, R1 to R10 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R10 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R10 are not SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0131] In a preferred embodiment, R3 and R8 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2-Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 and R8 are selected from SO 3 - Preferably, R1, R2, R4 to R7, R9 and R10 are hydrogen.

[0132]

[0133] Wherein R1 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 is SO 3 - .

[0134] In a particularly preferred embodiment, the α-amine protecting group is 9-(2-sulfo)fluorenylmethoxycarbonyl (Sulfmoc), 9-(3,6-disulfo)fluorenylmethoxy-carbonyl, 9-(2,7-disulfo)fluorenylmethoxy-carbonyl or tert-butyl-(2-sulfonic acid)oxycarbonyl (Sboc). As used herein, the term "Smoc" means 9-(3,6-disulfo)fluorenylmethoxy-carbonyl or 9-(2,7-disulfo)fluorenylmethoxy-carbonyl.

[0135] In a preferred embodiment of the present invention, any reactive side chain functional groups of the first amino acid or the first peptide and / or the second amino acid or the second peptide are protected by side chain protecting groups, wherein the side chain protecting groups 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 solubility for the amino acid or peptide whose side chains are protected. Suitable side chain protecting groups are the reaction products of one of the above-mentioned protecting agents and each side chain functional group of the amino acid or peptide.

[0136] As for the side chain protecting groups, preferred amine protecting groups are those shown in the following formulas 11 to 15, wherein the nitrogen belongs to the protected amino group.

[0137]

[0138] Wherein, R1 to R15 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 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 SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R15 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0139] In a preferred embodiment, R3, R8 and R13 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3, R8 and R13 are SO 3 -Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0140]

[0141] Wherein, R1 to R8 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R8 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R8 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0142] In a preferred embodiment, R2 and R7 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 +and CN, in particular, R2 and R7 are SO 3 - Preferably, R1, R3 to R6 and R8 are hydrogen.

[0143] In a preferred embodiment, R3 and R6 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 and R6 are SO 3 - Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

[0144] In a preferred embodiment, R2 and R6 are selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R2 and R6 are SO 3 - Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

[0145]

[0146] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3- 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0147] In a preferred embodiment, R3 is selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0148]

[0149] Wherein, R1 to R10 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 +and CN, provided that at least one, preferably at least two, of R1 to R10 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R10 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0150] In a preferred embodiment, R3 and R8 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 and R8 are selected from SO 3 - Preferably, R1, R2, R4 to R7, R9 and R10 are hydrogen.

[0151]

[0152] Wherein R1 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH3 ) 3 + In a preferred embodiment, R1 is SO 3 - .

[0153] In a particularly preferred embodiment, the side chain amine protecting group is selected from 9-(2-sulfo)fluorenylmethoxycarbonyl (Sulfmoc), 9-(3,6-disulfo)fluorenylmethoxy-carbonyl, 9-(2,7-disulfo)fluorenylmethoxy-carbonyl, tris(4-sulfophenyl)methyl (Sulfo Trt), tert-butyl-(2-sulfonic acid)oxycarbonyl (Sboc) and 4-sulfo-benzyloxycarbonyl (SulfoCBz).

[0154] Preferred alcohol protecting groups are shown below in Formulas 16 to 19. In each case, the oxygen shown in the following general formula is a protected alcohol group:

[0155]

[0156] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0157] In a preferred embodiment, R3 is selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0158]

[0159] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0160] In a preferred embodiment, R3 is selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0161]

[0162] Wherein R1 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 is SO 3 - .

[0163]

[0164] Wherein, R1 to R15 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 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 SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R15 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0165] In a preferred embodiment, R3, R8 and R13 are selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3, R8 and R13 are SO 3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0166] In a particularly preferred embodiment, the alcohol protecting group is selected from 4-sulfobenzyl (BzS), 4-sulfo-benzyloxymethyl (BOMS), tris(4-sulfophenyl)methyl (Sulfo Trt) and tert-butyl-1-sulfonate (tBuS).

[0167] Preferred thiol protecting groups are shown below in Formulas 20 and 21. In each case, the sulfur shown in the following general formula is the protected thiol group:

[0168]

[0169] Wherein, R1 to R15 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 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 SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R15 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0170] In a preferred embodiment, R3, R8 and R13 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3, R8 and R13 are SO 3 -Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0171]

[0172] Wherein R1 is selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 is SO 3 - .

[0173] In a particularly preferred embodiment, the thiol protecting group is selected from tris(4-sulfophenyl)methyl (Sulfo Trt) and 1-sulfo-2-methyl-2-propanethiol (StBuS).

[0174] Preferred carboxyl protecting groups are those shown below in Formulae 22 to 25. In each case, the oxygen shown in the following general formula is the protected carboxyl group:

[0175]

[0176] Wherein, R1 to R15 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 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 SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 )3 + In a preferred embodiment, R1 to R15 are not SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0177] In a preferred embodiment, R3, R8 and R13 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3, R8 and R13 are SO 3 - Preferably, R1, R2, R4 to R7, R9 to R12, R14 and R15 are hydrogen.

[0178]

[0179] Wherein R1 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 is SO 3 - .

[0180]

[0181] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 -、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0182] In a preferred embodiment, R3 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0183]

[0184] Wherein, R1 to R5 are independently selected from hydrogen, SO3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - ,PO 3 2- 、OSO 3 - Ester, OPO 3 2- Esters, N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0185] In a preferred embodiment, R3 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0186] In a particularly preferred embodiment, the carboxyl protecting group is selected from 4-sulfobenzyl (BzS), 4-sulfo-benzyloxymethyl (BOMS), tris(4-sulfophenyl)methyl (Sulfo Trt) and tert-butyl-1-sulfonate (tBuS).

[0187] Preferred seleno protecting groups are those shown below in Formula 26. In each case, selenium shown in the following general formula is the protected seleno group:

[0188]

[0189] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0190] In a preferred embodiment, R3 is selected from SO 3 - 、OSO 3 - Ester, OPO3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0191] Preferred phosphono protecting groups are those shown below in Formulae 27 and 28. In each case, the oxygen shown in the following general formula is part of the protected phosphono group:

[0192]

[0193] Wherein R1 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 is SO 3 - .

[0194]

[0195] Wherein, R1 to R5 are independently selected from hydrogen, SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, provided that at least one, preferably at least two, of R1 to R5 are selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2-、N(CH 3 ) 2 、N(CH 3 ) 3 + In a preferred embodiment, R1 to R5 are not SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + or CN, all of which are hydrogen.

[0196] In a preferred embodiment, R3 is selected from SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- 、N(CH 3 ) 2 、N(CH 3 ) 3 + and CN, in particular, R3 is SO 3 - Preferably, R1, R2, R4 and R5 are hydrogen.

[0197] The following is a summary of the most preferred protecting groups.

[0198] Amine Protecting Groups

[0199]

[0200] Alcohol protecting group

[0201]

[0202] Thiol Protecting Group

[0203] Carboxyl protecting group

[0204] Phosphonyl protecting group

[0205]

[0206] Preferably, if an ion exchanger is used, the above-mentioned amine protecting groups are used to protect the backbone amino groups and provide an anchoring point for the amino acid or peptide to the solid phase.

[0207] In a particular embodiment, the protecting group having a water solubility enhancing group preferably comprises at least two water solubility enhancing groups, preferably ionic groups. This embodiment provides the advantage that a higher bonding with the ion exchanger can be achieved.

[0208] In another embodiment, the protecting group with water solubility enhancing group preferably comprises exactly one water solubility enhancing group, preferably an ionic group. This embodiment provides the advantage that lower hydrolysis of the protecting group can be achieved, so that reactions in water or other protic solvents provide higher yields.

[0209] Preferably, the protecting group having a water-solubility enhancing group comprises a fluorenylmethoxycarbonyl residue (Fmoc) modified with a water-solubility enhancing group, preferably an ionic group.

[0210] According to one embodiment of the present invention, it is preferred to provide any reactive side chain functional groups of the amino acid or peptide, preferably the first amino acid or the first peptide, and / or the second amino acid or the second peptide, protected by side chain protecting groups. By this embodiment, a surprising improvement in the solubility of the first amino acid or the first peptide and / or the second amino acid or the second peptide can be achieved. In addition, it is preferred to improve the bonding of the first amino acid or the first peptide to the ion exchanger.

[0211] In one embodiment, the side chain protecting group includes at least one water solubility enhancing functional group, preferably an ionic group.

[0212] In another embodiment, the side chain protecting groups do not include any water solubility enhancing functional groups.

[0213] In the method according to the present invention, it can also be provided that the protected functional groups of the first amino acid or first peptide and the second amino acid or second peptide are selected from α-amino, side chain amino, thiol, carboxyl, hydroxyl, phosphono and seleno.

[0214] In a preferred embodiment of the present invention, except the group protecting the primary amine group and the group protecting the carboxyl group, the second amino acid or the second peptide preferably do not include any blocking group. More preferably, except the group protecting the primary amine group, the second amino acid or the second peptide preferably do not include any blocking group. The present embodiment provides amazing cost improvement and easier processing. In order to obtain the higher solubility of the amino acid coupled with the growing peptide chain, blocking groups can be used in some cases.

[0215] Further, the solubility of the second amino acid or the second peptide is preferably improved by using a salt of the second amino acid or the second peptide, and the salt comprises a soluble organic ion, such as a quaternary ammonium cation. Further, preferably a compound that imparts solubility of the salt in an organic solvent, such as a crown ether, is used. In addition, the second amino acid or the second peptide can be added to the reaction mixture of the solid phase, and via a solvent, the added second amino acid or the second peptide is transferred to the first amino acid or the first peptide. This embodiment includes a cost advantage. However, the reaction time is longer than other embodiments.

[0216] The solubility of the second amino acid or second peptide is preferably sufficient to obtain a sufficient concentration of the second amino acid or second peptide in the mixture and / or the growing chain to be obtained, wherein the mixture is added to the activated first amino acid or first peptide. As mentioned above, if useful, the solubility of the second amino acid or second peptide can be increased. However, in many cases, it is not necessary to increase the solubility of the second amino acid or second peptide.

[0217] Preferably, in the absence of any protecting group, the following amino acids are used as the second amino acid or second peptide: alanine (Ala), glycine (Gly), isoleucine (Ile), leucine (Leu), proline (Pro), valine (Val), phenylalanine (Phe), arginine (Arg), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), tryptophan (Trp), histidine (His), methionine (Met), more preferably Ala, Gly, Ile, Leu, Pro, Val, Phe. These amino acids generally have sufficient solubility to obtain a sufficient concentration of the second amino acid or second peptide in a mixture added to the activated first amino acid or first peptide, and / or in the growing chain to be obtained.

[0218] The amino acids glutamic acid (Glu), aspartic acid (Asp), cysteine ​​(Cys), lysine (Lys), serine (Ser), and threonine (Thr) preferably contain a protecting group, more preferably a side chain protecting group as described above or below.

[0219] Preferably, the amino acid or peptide comprising a protecting group having a water solubility enhancing group is added to the ion exchanger in an amount of at least 10%, more preferably at least 80% of the capacity of the ion exchanger.

[0220] Preferably, the amino acid or peptide bound to the solid phase is added to the solid phase in an amount of at least 10%, more preferably at least 80% of the capacity of the solid phase.

[0221] According to the present invention, it is preferably provided that the formation of the peptide bond is achieved using a solution of a second amino acid or a peptide, wherein the concentration of the second amino acid or the second peptide is in the range of 0.01% by weight to 90% by weight, preferably in the range of 0.1% by weight to 50% by weight, and more preferably in the range of 0.5% by weight to 20% by weight, based on the total weight of the solution.

[0222] In one embodiment of the present invention, the formation of peptide bonds is preferably achieved in a temperature range of -20 to 100°C, more preferably 0 to 50°C, even more preferably 10 to 30°C.

[0223] In another embodiment of the present invention, the formation of peptide bonds is preferably achieved at a pH value in the range of 4 to 12, preferably in the range of 6 to 10, more preferably in the range of 7 to 9.5, even more preferably in the range of 7 to 9.0, even more preferably in the range of 7 to 8.5.

[0224] According to the present invention, it is preferably provided that the peptide obtained by peptide bond formation comprises 2 to 150 amino acid units, more preferably 2 to 6 amino acid units, even more preferably 2 to 4 amino acid units.

[0225] According to another embodiment of the present invention, it is preferably provided that the peptide obtained by peptide bond formation comprises 4 to 300 amino acid units, more preferably 10 to 100 amino acid units, even more preferably 15 to 45 amino acid units. Peptides with a large number of amino acid units are preferably obtained by a method in which the first amino acid or the first peptide is covalently bonded to a solid phase.

[0226] As described above and below, some embodiments of the present invention include a deprotection step. Traditionally, deprotection of a peptide or a specific amino acid unit of a peptide is achieved using a deprotection solution.

[0227] Deprotection solutions suitable for use in the present invention preferably include an acid or a base, preferably an aqueous acid or base, i.e., the acid and / or base are 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 base is used in the amount and degree 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. Suitable bases are those that have a solubility that allows dissolution of sufficient amounts to carry out deprotection in the selected solvent.

[0228] The deprotection solution used to deprotect the Sboc group preferably comprises an aqueous acid, such as phosphoric acid, hydrochloric acid or trifluoroacetic acid.

[0229] The deprotection solution for deprotecting Smoc groups, Sulfmoc groups and / or similar groups, such as fluorenylmethoxy-carbonyl groups derived from compounds or groups of formula 1, 7, 12, etc., preferably includes a water-soluble base, such as an amine, ammonia or an inorganic hydroxide. The water-soluble base is used in an amount and degree necessary to deprotect the peptide. The solubility of certain organic bases may limit the amount that can be dissolved in water, alcohol, or a mixture of water and alcohol. Suitable bases are those that have a solubility that allows dissolution of a sufficient amount to carry out deprotection in a selected solvent.

[0230] Surprisingly improved deprotection of Smoc groups, Sulfmoc groups and / or similar groups can be achieved by using at least 5% of an organic base (e.g. a primary, secondary and / or tertiary amine, preferably piperazine, piperidine, ethanolamine and / or ethylenediamine) in water or in a mixture of water and an environmentally friendly solvent (including protic solvents such as water, primary, secondary and / or tertiary alcohols, aprotic solvents such as ketones, nitriles, lactones, lactams, carbonic acid amides, carbonates, ethers, urea derivatives, sulfoxides, sulfones, carbonates). Preferred examples of aprotic solvents are, for example, dimethylacetamide, ethyl methyl ketone, acetone, methyl isopropyl ketone, methyl isobutyl ketone, methyl n-butyl ketone, methyl tert-butyl ketone, methyl isoamyl ketone, dimethyl sulfoxide, hexamethylphosphoric triamide, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol, dimethyl ether, ethyl acetate, tert-butyl acetate, or mixtures thereof. Compared with the use of ammonia or inorganic hydroxides, this reduces base-catalyzed side reactions (e.g., deprotection of OtBu-based side chain protecting groups, cleavage of esters and SPPS linker molecules) during the deprotection of Smoc groups, Sulfmoc groups and / or similar groups. This results in higher purity and product yields.

[0231] It should be understood that after deprotection, other amino acids or other peptides can be used, and the reaction and deprotection steps can be repeated until the desired target peptide is obtained, wherein the α-amino or carboxyl group of the other amino acids and / or other peptides is preferably protected by a protecting group according to the present invention, especially a Smoc group or a Sboc group.

[0232] In one embodiment, the method further comprises the step of, when the peptide is complete, releasing the resulting peptide from the polymer carrier using a release composition.

[0233] In the case where the peptide is ionically bonded to the ion exchanger via a protecting group having a water solubility enhancing group, the releasing composition is preferably a composition that deprotects the peptide from the corresponding protecting group, or a composition that can release the peptide from the ion exchanger and maintain the protection of the peptide. The deprotecting agent used to release the peptide from the solid support depends on the protecting group used. The Sboc group is conventionally released by an acidic composition, while a basic reagent is used to remove the Smoc portion.

[0234] In a particular embodiment of the invention, preferably when the amino acid or peptide is ionically bonded to the ion exchanger, the formation of a peptide bond is achieved, and after the formation of the peptide bond, the obtained peptide is removed from the ion exchanger by deprotecting the obtained peptide. In a preferred embodiment, at least one, preferably at least 50%, more preferably at least 90%, of the side chain protecting groups attached to the amino acid in the target peptide are removed by the release composition.

[0235] In another embodiment of the present invention, it is preferred that the formation of a peptide bond is achieved when an amino acid or a peptide is ionically bonded to an ion exchanger, and after the formation of the peptide bond, the obtained peptide is removed from the ion exchanger by adding an ionic solution while maintaining protection of the obtained peptide.

[0236] Preferably, the release is carried out in the presence of a scavenger composition, such as 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 depending on the protecting groups present.

[0237] The released peptide can be separated from the ion exchanger by filtration and the peptide recovered from the filtrate by conventional procedures such as evaporation or solvent driven precipitation.

[0238] In the case where the peptide is covalently bonded to a solid support, it is preferred to use an appropriate cleavage composition. Appropriate cleavage compositions and methods are well known to the skilled person. Typically, an acid, such as trifluoroacetic acid and hydrofluoric acid (HF), is used to carry out the cleavage step. Preferably, an acid suitable for cleaving the desired peptide from a polymer support while removing the side chain protecting groups of the amino acids attached to the target peptide.

[0239] Preferably, the cleavage or release is carried out 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 depending on the protecting groups present.

[0240] The cleaved or released peptide can be separated from the cleaved support (eg, resin) by filtration and the peptide recovered from the filtrate by conventional procedures such as evaporation or solvent driven precipitation.

[0241] In another preferred embodiment of the present invention, the method further comprises the steps of: washing at least once with a solvent, wherein the solvent comprises a protic and / or aprotic solvent as described above or below with respect to peptide bond formation; and, 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 waste compound, which contains a water-solubility enhancing functional group, particularly a functional group such as SO3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- and N(CH 3 ) 3 + The waste compounds can be collected and processed by affinity chromatography columns, preferably regenerated solid anion and / or cation exchange carriers. Alternatively, the affinity chromatography columns, preferably anion and / or cation exchange carrier materials, containing the waste compounds can be processed.

[0242] The washing 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 (e.g., unreacted or cleaved protecting groups) and reagents (such as coupling agents and end-capping agents described herein) from the washing solution. The waste compounds are retained on the anion and cation exchange column, and a purified solvent is obtained. The retained compounds can be treated with anion or cation exchange materials, or treated after elution, and only a minimum amount of chemical waste needs to be treated.

[0243] In another preferred embodiment of the present invention, the method further comprises a purification step after the release step, wherein the solution containing the deprotected and released target protein or peptide is contacted with an affinity chromatography column, preferably a solid anion and / or cation exchange carrier, thereby retaining waste compounds such as sulfonic acid groups, and collecting the purified target protein. In a preferred embodiment, the waste compound comprises at least one sulfonic acid group as a part of a protecting group and other reagents. Due to the sulfonic acid protecting group and the sulfonic acid containing reagent, the excess chemicals and by-products produced during the coupling, deprotection and cleavage reactions in the above or below protic and / or aprotic solvents can be substantially removed by an affinity chromatography column, preferably an ion exchange method. Of course, this also applies to other charged functional groups. Only the target peptide can pass through the ion exchange column, while the 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 needs to be handled. This is the use of charged functional groups, such as SO 3 - 、OSO 3 - Ester, OPO 3 2- Ester, PO 3 2- and N(CH 3 ) 3+ However, even if one or more uncharged functional groups are used, the method can still be carried out in water or another environmentally friendly solvent, thereby reducing the amount of waste chemicals compared to prior art methods.

[0244] A further subject of the present invention are peptides obtainable by the process according to the invention.

[0245] Another subject of the present invention is an amino acid or peptide comprising a protecting group with a water solubility enhancing group, wherein the water solubility enhancing group is bonded to the amino group and the activated or free carboxyl group, preferably the activated carboxyl group. Preferably, the peptide is ionically bonded to an ion exchanger.

[0246] It is preferably provided that the amino acid or peptide of the invention preferably has 1 to 150 amino acid units, preferably 1 to 50 amino acid units, more preferably 1 or 2 amino acid units.

[0247] 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 represented by the above general formula 6 to general formula 25, and more preferably, selected from the group consisting of those protecting groups represented by the above general formula 6 to general formula 10. In a particularly preferred embodiment, these protecting groups include 9-(2-sulfo)fluorenylmethoxycarbonyl (Sulfmoc), 9-(3,6-disulfo)fluorenylmethoxy-carbonyl, 9-(2,7-disulfo)fluorenylmethoxy-carbonyl, tris(4-sulfophenyl)methyl (Sulfo Trt), 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). DETAILED DESCRIPTION

[0248] The present invention is illustrated by the following examples, but is not limited thereto.

[0249] Example

[0250] Example 1: Synthesis of 9-(3,6-disulfonyl)fluorenylmethoxycarbonyl chloride (Smoc-Cl)

[0251] 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.

[0252]

[0253] Analytical data of Smoc-Chlorine:

[0254] 1 H NMR (500 MHz, D 2 O)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).

[0255] 13 C NMR (126MHz, D 2 O)δ=145.57,142.54,141.65,125.16,121.61,120.90,62.54,49.65.

[0256] Example 2: Synthesis of 9-(2,7-disulfonyl)fluorenylmethoxycarbonyl chloride (Smoc-Cl)

[0257] 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 in 25 ml of DCM was cooled to 0°C 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.

[0258]

[0259] NMR intermediates:

[0260] 1 H NMR (300 MHz, D 2 O)δ:6.09(s,2H),7.23–7.40(m,2H),7.72(s,2H),7.95(d,J=6.2Hz,2H). 13 C NMR (75MHz, D 2O)δ:142.61,132.99,131.74,130.23,129.28,127.22,125.57,124.69.

[0261] LC-APCI-MS of 9-(2,7-disulfo)fluorenylmethoxycarbonyl chloride:

[0262] LC-APCI-MS calculated C 15 H 9 C1O 22 m / z: 256.03. Measured m / z: 256.94 [MH-2xSO 3 ] - .

[0263] Example 3: Synthesis of Smoc-βAla-OH (Smoc-β-alanine)

[0264] 8.41 mmol of Fmoc-β-alanine was treated with 30 mL of concentrated sulfuric acid. After the reaction mixture was stopped, 8.09 mmol (96.2%) of crude Smoc-β-alanine was obtained.

[0265]

[0266] Example 4: Synthesis of L-carnosine

[0267] Smoc-β-alanine was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-β-alanine was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in water.

[0268] Afterwards, the ion exchanger loaded with activated Smoc-β-alanine was rinsed twice with water. After the washing step, 1.5 equivalents of L-histidine dissolved in water were added to the activated Smoc-β-alanine and stirred for 12 minutes at 4°C and pH 7.5. Excess L-histidine and released NHS were removed by washing with water.

[0269] The sera were isolated by using 0.25 mM NaOH (pH 10, 20 min) or Na 2 CO 3 (pH 10, 20 min) for deprotection to release the formed L-carnosine.

[0270] Example 5: Synthesis of L-carnosine

[0271] Smoc-β-alanine was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-proline was obtained in a similar manner as disclosed above for Smoc-β-alanine (see Example 3). Smoc-β-alanine was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and acetonitrile (MeCN) in a ratio of 4:1 (volume). The degree of activation was much higher than in Example 4.

[0272] Afterwards, the ion exchanger loaded with activated Smoc-β-alanine was rinsed twice with water. After the washing step, 1.5 equivalents of L-histidine dissolved in water were added to the activated Smoc-β-alanine and stirred for 12 minutes at 4°C and pH 7.5. Excess L-histidine and released NHS were removed by washing with water.

[0273] The yield of Example 5 is higher than that of Example 4.

[0274] The sera were isolated by using 0.25 mM NaOH (pH 10, 20 min) or Na 2 CO 3 (pH 10, 20 min) for deprotection to release the formed L-carnosine.

[0275] Example 6: Synthesis of Pro-Tyr-OMe

[0276] Smoc-proline was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-proline was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and tetrahydrofuran (THF) in a ratio of 3:1 (volume).

[0277] Afterwards, the ion exchanger loaded with activated Smoc-proline was rinsed twice with water. After the washing step, 1.5 equivalents of L-tyrosine methyl ester dissolved in water were added to the activated Smoc-proline and stirred for 12 minutes at 24°C and pH 8. Excess L-tyrosine methyl ester and released NHS were removed by washing with water.

[0278] The precipitate was removed by using 0.5 M NaOH (pH 9, 25 min) or Na 2 CO 3 (pH 10, 20 min) to release the formed Pro-Tyr-OMe.

[0279] Example 7: Synthesis of Pro-Tyr-OMe

[0280] Smoc-proline was added to an ion exchange resin (Amberlite IRA-900; SigmaAldrich) at maximum loading. Smoc-proline was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and tetrahydrofuran (THF) in a ratio of 3:1 (volume).

[0281] Afterwards, the ion exchanger loaded with activated Smoc-proline was rinsed twice with water. After the washing step, 1.5 equivalents of L-tyrosine methyl ester dissolved in water were added to the activated Smoc-proline and stirred for 12 minutes at 24°C and pH 8. Excess L-tyrosine methyl ester and released NHS were removed by washing with water.

[0282] The yield of Example 7 is lower than that of Example 6.

[0283] The precipitate was removed by using 0.5 mM NaOH (pH 10, 15 min) or Na 2 CO 3 (pH 10, 20 min) to release the formed Pro-Tyr-OMe.

[0284] Example 8: Synthesis of Pro-Tyr-OMe

[0285] Smoc-proline was added to an ion exchange resin (Amberlite IRA-96; SigmaAldrich) at maximum loading. Smoc-proline was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and tetrahydrofuran (THF) in a ratio of 4:1 (volume).

[0286] Afterwards, the ion exchanger loaded with activated Smoc-proline was rinsed three times with water. After the washing step, 2 equivalents of L-tyrosine methyl ester dissolved in water were added to the activated Smoc-proline and stirred for 25 minutes at 15°C and pH 8.2. Excess L-tyrosine methyl ester and released NHS were removed by washing with water.

[0287] The yield of Example 8 is similar to that of Example 7.

[0288] By using 0.25 M CaOH 2(pH 10, 15 min) or CaCO 3 (pH 10, 30 min) to release the formed Pro-Tyr-OMe.

[0289] Example 9: Synthesis of Pro-Tyr-OMe

[0290] Smoc-proline was added to an ion exchange resin (Amberlite IRA-410; SigmaAldrich) at maximum loading. Smoc-proline was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and tetrahydrofuran (THF) in a ratio of 4:1 (volume).

[0291] Afterwards, the ion exchanger loaded with activated Smoc-proline was rinsed three times with water. After the washing step, 2 equivalents of L-tyrosine methyl ester dissolved in water were added to the activated Smoc-proline and stirred for 25 minutes at 15°C and pH 8.2. Excess L-tyrosine methyl ester and released NHS were removed by washing with water.

[0292] The yield of Example 9 is lower than that of Example 6.

[0293] The precipitate was removed by using 1 M NaOH (pH 10, 10 min) or Na 2 CO 3 (pH 10, 20 min) to release the formed Pro-Tyr-OMe.

[0294] Example 10: Synthesis of Pro-Tyr-OMe

[0295] Smoc-proline was added to an ion exchange resin (Amberlite IRA-958; SigmaAldrich) at maximum loading. Smoc-proline was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and tetrahydrofuran (THF) in a ratio of 4:1 (volume).

[0296] Afterwards, the ion exchanger loaded with activated Smoc-proline was rinsed three times with water. After the washing step, 2 equivalents of L-tyrosine methyl ester dissolved in water were added to the activated Smoc-proline and stirred for 25 minutes at 15°C and pH 8.2. Excess L-tyrosine methyl ester and released NHS were removed by washing with water.

[0297] The yield of Example 10 is higher than that of Example 10.

[0298] By using 25% NH 3 (aq) (pH 10, 20 min) or (NH 4 ) 2 CO 3 (pH 10, 20 min) to release the formed Pro-Tyr-OMe.

[0299] Example 11: Synthesis of L-carnosine

[0300] Smoc-β-alanine was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-β-alanine was activated by 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) in a mixture of water and isopropanol in a ratio of 3:1 (volume).

[0301] Afterwards, the ion exchanger loaded with activated Smoc-β-alanine was rinsed once with water. After the washing step, 1.5 equivalents of L-histidine dissolved in water were added to the activated Smoc-β-alanine and stirred for 15 minutes at 24°C and pH 7.8. Excess L-histidine and released NHS were removed by washing with water.

[0302] The yield of Example 11 is similar to that of Example 4.

[0303] The precipitate was removed by using 0.5 M KOH (pH 9.8, 12 min) or K 2 CO 3 (pH 10, 20 min) for deprotection to release the formed L-carnosine.

[0304] Example 12: Synthesis of Smoc-Gly-His-OH

[0305] Smoc-glycine was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-glycine was obtained in a similar manner as disclosed above for Smoc-β-alanine (see Example 3). Smoc-glycine was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and tetrahydrofuran (THF) in a ratio of 3:1 (volume).

[0306] Afterwards, the ion exchanger loaded with activated Smoc-glycine was rinsed three times with water. After the washing step, 1.5 equivalents of L-histidine dissolved in water were added to the activated Smoc-glycine and stirred for 20 minutes at 18°C ​​and pH 8.5. Excess L-histidine and released NHS were removed by washing with water.

[0307] The formed Smoc-Gly-His-OH was released by elution with 1 M NaCl solution.

[0308] Example 13: Synthesis of Asn-Asn-OH

[0309] Smoc-Asn was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Asn was obtained in a similar manner as disclosed above for Smoc-β-alanine (see Example 3). Smoc-Asn was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and isopropanol in a ratio of 4:1 (volume).

[0310] Afterwards, the ion exchanger loaded with activated Smoc-Asn was rinsed three times with water. After the washing step, 1.5 equivalents of L-Asn dissolved in water were added to the activated Smoc-Asn and stirred for 25 minutes at 8°C and pH 8.7. Excess L-Asn and released NHS were removed by washing with water.

[0311] The precipitate was removed by using 0.5 M NaOH (pH 9.5, 12 min) or K 2 CO 3 (pH 10, 25 min) to release the formed Asn-Asn-OH.

[0312] Example 14: Synthesis of Asn-Gln-OH

[0313] Smoc-Asn was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Asn was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and isopropanol in a ratio of 4:1 (volume).

[0314] Afterwards, the ion exchanger loaded with activated Smoc-Asn was rinsed twice with water. After the washing step, 1.5 equivalents of L-Gln dissolved in water were added to the activated Smoc-Asn and stirred for 20 minutes at 24°C and pH 9. Excess L-Gln and released NHS were removed by washing with water.

[0315] The precipitate was removed by using 1 M NaOH (pH 9.5, 12 min) or K 2 CO 3 (pH 10, 25 min) to release the formed Asn-Gln-OH.

[0316] Example 15: Synthesis of Asp(OtBu)-Glu-OH

[0317] Smoc-Asp(OtBu)-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Asp(OtBu)-OH was obtained in a similar manner as disclosed above for Smoc-β-alanine (see Example 3). Smoc-Asp(OtBu)-OH was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and 2-methyltetrahydrofuran in a ratio of 4:1 (volume).

[0318] Afterwards, the ion exchanger loaded with activated Smoc-Asp(OtBu) was rinsed twice with water. After the washing step, 1.5 equivalents of L-Glu dissolved in water were added to the activated Smoc-Asp(OtBu) and stirred for 20 minutes at 24°C and pH 9. Excess L-Glu and released NHS were removed by washing with water.

[0319] The precipitate was removed by using 1 M NaOH (pH 9.5, 12 min) or K 2 CO 3 (pH 10, 25 min) to release the formed Asp(OtBu)-Glu-OH.

[0320] Example 16: Synthesis of Ile-D-Val-OH

[0321] Smoc-Ile-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Ile-OH was obtained in a similar manner as disclosed above for Smoc-β-alanine (see Example 3). Smoc-Ile-OH was activated by 4 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 3 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and dimethyl sulfoxide (DMSO) in a ratio of 3:1 (volume).

[0322] Afterwards, the ion exchanger loaded with activated Smoc-Ile was rinsed twice with water. After the washing step, 1.5 equivalents of D-Val dissolved in water were added to the activated Smoc-Ile and stirred for 17 minutes at 25°C and pH 8.5. Excess D-Val and released NHS were removed by washing with water.

[0323] The precipitate was removed by using 1 M NaOH (pH 9.5, 12 min) or Na 2 CO 3 (pH 10, 25 min) to release the formed Ile-D-Val-OH.

[0324] Example 17: Synthesis of Sulfmoc-Gly-Val-OH

[0325] 9-(2-sulfo)fluorenylmethoxycarbonylglycine (Sulfmoc-Gly-OH) was obtained in a similar manner as disclosed above for Smoc-β-alanine (see Example 3). 9-(2-sulfo)fluorenylmethoxycarbonylglycine (Sulfmoc-Gly-OH) was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in water. The preactivated Sulfmoc-Gly was then loaded onto an ion exchange resin (DEAE Sephadex A-25; GE Healthcare).

[0326] Afterwards, the ion exchanger loaded with activated Sulfmoc-Gly was rinsed twice with water. After the washing step, 1.5 equivalents of L-Val dissolved in water were added to the activated Sulfmoc-Gly and stirred for 30 minutes at 25°C and pH 9. Excess L-Val and released NHS were removed by washing with water.

[0327] The formed Sulfmoc-Gly-Val-OH was released by elution with 1 M NaCl solution.

[0328] Example 18: Synthesis of Ile-Pro-Phe-OH

[0329] Smoc-Ile-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Ile-OH was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume).

[0330] Afterwards, the ion exchanger loaded with activated Smoc-Ile was rinsed twice with water. After the washing step, 1.5 equivalents of L-Pro dissolved in water were added to the activated Smoc-Ile, and the reaction was stirred at 25°C and pH 8.5 for 12 minutes. Excess L-Pro and released NHS were removed by washing with water. In a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume), Smoc-Ile-Pro-OH was activated by 2.5 equivalents of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.2 equivalents of N-hydroxysuccinimide (NHS). Afterwards, the ion exchanger loaded with activated Smoc-Ile-Pro was rinsed twice with water. After the washing step, 1.5 equivalents of L-Phe dissolved in water were added to the activated Smoc-Ile-Pro, and the reaction was stirred at 25°C and pH 8.6 for 12 minutes. Excess L-Phe and released NHS were removed by washing with water. 2 CO 3 (pH 10, 25 min) to release the formed Ile-Pro-Phe-OH.

[0331] Example 19: Synthesis of Ile-Val-Phe-OH

[0332] Smoc-Ile-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Ile-OH was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume).

[0333] Afterwards, the ion exchanger loaded with activated Smoc-Ile was rinsed twice with water. After the washing step, 1.5 equivalents of L-Val dissolved in water were added to the activated Smoc-Ile and stirred for 12 minutes at 25°C and pH 8.5. Excess L-Val and released NHS were removed by washing with water. In a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume), Smoc-Ile-Val-OH was activated by 2.5 equivalents of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.2 equivalents of N-hydroxysuccinimide (NHS). Afterwards, the ion exchanger loaded with activated Smoc-Ile-Val was rinsed twice with water. After the washing step, 1.5 equivalents of L-Phe dissolved in water were added to the activated Smoc-Ile-Val and stirred at 25°C, pH 8.6 for 12 minutes. Excess L-Phe and released NHS were removed by washing with water. The reaction was continued for 12 minutes using 1 M NaOH (pH 9.5, 12 minutes) or Na 2 CO 3 (pH 10, 25 min) to release the formed Ile-Val-Phe-OH.

[0334] Example 20: Synthesis of Ile-His-Ile-OH

[0335] Smoc-Ile-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Ile-OH was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume).

[0336] Afterwards, the ion exchanger loaded with activated Smoc-Ile was rinsed twice with water. After the washing step, 1.5 equivalents of L-His dissolved in water were added to the activated Smoc-Ile and stirred for 10 minutes at 25°C and pH 8.2. Excess L-His and released NHS were removed by washing with water. Smoc-Ile-His-OH was activated by 2.5 equivalents of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline in a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume). Afterwards, the ion exchanger loaded with activated Smoc-Ile-His was rinsed twice with water. After the washing step, 1.5 equivalents of L-Ile dissolved in water were added to the activated Smoc-Ile-His and stirred for 10 minutes at 25°C and pH 8.5. Excess L-Ile and released 1,2-dihydroquinoline and ethanol were removed by washing with water. 2 CO 3 (pH 10, 25 minutes) to carry out deprotection and release the formed Ile-His-Ile-OH.

[0337] Example 21: Synthesis of Leu-Ile-His-OH

[0338] Smoc-Ile-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Ile-OH was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume).

[0339] By using an organic base such as ethanolamine, N,N-diisopropylethylamine, triethylamine or 5% N-methyl-morpholine (aq) Deprotection is performed to release the formed Ile-His–OH. Ile-His is precipitated by adding an aprotic solvent and washed until the base residue is removed.

[0340] Afterwards, the ion exchanger was regenerated with a 1M NaCl solution and washed twice with water. Smoc-Ile-OH was added to the ion exchange resin at the maximum loading and activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and acetonitrile (MeCN) in a ratio of 3:1 (volume). Afterwards, the ion exchanger loaded with activated Smoc-Leu was rinsed with water twice. After the washing step, 1 equivalent of L-Ile-His dissolved in water was added to the activated Smoc-Leu and stirred for 10 minutes at 25°C and pH 8.5. The released NHS was removed by washing with water. The reaction was carried out by using 1M NaOH (pH 9.5, 12 minutes) or Na 2 CO 3 (pH 10, 25 min) to release the formed Leu-Ile-His-OH.

[0341] Example 22: Synthesis of LAGV-OH

[0342] Smoc-Ile-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Leu-OH was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and isopropanol in a ratio of 2:1 (volume).

[0343] Afterwards, the ion exchanger loaded with activated Smoc-Ile was rinsed with water. After the washing step, 1.5 equivalents of L-Ala dissolved in water were added to the activated Smoc-Leu, and the reaction was stirred for 10 minutes at 25°C and pH 7. Excess L-Ala and released NHS were removed by washing with water. In a mixture of water and isopropanol in a ratio of 2:1 (volume), Smoc-Leu-Ala-OH was activated by 2.5 equivalents of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 2 equivalents of N-hydroxysuccinimide. Afterwards, the ion exchanger loaded with activated Smoc-Leu-Ala was rinsed with water twice. After the washing step, 1.5 equivalents of Gly dissolved in water were added to the activated Smoc-Leu-Ala, and the reaction was stirred for 10 minutes at 25°C and pH 7. Excess Gly and released NHS were removed by washing with water. Smoc-LAG-OH was activated by 2.5 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2 equivalents of N-hydroxysuccinimide in a mixture of water and isopropanol in a ratio of 2:1 (volume). Afterwards, the ion exchanger loaded with activated Smoc-LAG was rinsed twice with water. After the washing step, 1.5 equivalents of Val dissolved in water were added to the activated Smoc-LAG and reacted at 25°C, pH 7 with stirring for 15 minutes. Excess Val and released NHS were removed by washing with water.

[0344] By using NaOH (pH 10, 20 min) or Na 2 CO 3 (pH 10, 25 min) to release the formed LAGV-OH. Yield: 20%.

[0345] Example 23: Synthesis of Leu-enkephalin (YGGFL)

[0346] Smoc-Tyr-OH was obtained in a similar manner as disclosed above for Smoc-β-alanine (see Example 3). Smoc-Tyr-OH was added to an ion exchange resin (DEAE Sephadex A-25; GE Healthcare) at maximum loading. Smoc-Tyr-OH was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and MeCN in a ratio of 2:1 (volume).

[0347] Afterwards, the ion exchanger loaded with activated Smoc-Tyr was rinsed with water. After the washing step, 1.5 equivalents of Gly dissolved in water were added to the activated Smoc-Tyr and stirred for 10 minutes at 25°C and pH 7. Excess Gly and released NHS were removed by washing with water. In a mixture of water and MeCN in a ratio of 2:1 (volume), Smoc-YG-OH was activated by 2.5 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2 equivalents of N-hydroxysuccinimide. Afterwards, the ion exchanger loaded with activated Smoc-YG was rinsed with water. After the washing step, 1.5 equivalents of Gly dissolved in water were added to the activated Smoc-YG and stirred for 10 minutes at 25°C and pH 7. Excess Gly and released NHS were removed by washing with water. This process was repeated for each step until the complete sequence of Smoc-YGGFL-OH was synthesized.

[0348] By using NaOH (pH 10, 20 min) or Na 2 CO 3 (pH 10, 25 minutes) to release the formed YGGFL-OH. Yield: 19%.

[0349] Example 24: Synthesis of Leu-enkephalin (YGGFL) on SPPS

[0350] Tyr(tBu)-OH was added to the solid phase peptide resin ( S TRTCl resin; 0.2-0.3 mmol / g). Solid S-Tyr(tBu)-OH was activated by 4 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 equivalents of ethyl cyano(hydroxyimino)acetate (Oxyma) in a mixture of water and MeCN in a ratio of 2:1 (volume).

[0351] Afterwards, the solid support loaded with activated Tyr(tBu) was rinsed with water. After the washing step, 3 equivalents of Gly dissolved in water were added to the activated SolidS-Tyr(tBu) and stirred for 30 minutes at 25°C and pH 8. Excess Gly and released Oxyma were removed by washing with water. SolidS-YG-OH was activated by 4 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 equivalents of cyano(hydroxyimino)ethyl acetate (Oxyma) in a mixture of water and MeCN in a ratio of 2:1 (volume). Afterwards, the solid support loaded with activated Smoc-YG was rinsed with water. After the washing step, 3 equivalents of Gly dissolved in water were added to the activated SolidS-YG and stirred for 30 minutes at 25°C and pH 8. Excess Gly and released Oxyma were removed by washing with water. This process was repeated for each step until the complete sequence of SolidS-YGGFL-OH was synthesized.

[0352] The formed YGGFL-OH was cleaved from the solid support by using 95% trifluoroacetic acid (TFA) for 1.5 h. Yield: 30.5%.

[0353] Example 25: Synthesis of Acetyl Hexapeptide-3 (EEMQRR-OH) on SPPS

[0354] Glu(OtBu)-OH was added to the solid phase peptide resin ( S TRTCl resin; 0.2-0.3mmol / g). Solid S-Glu(OtBu)-OH was activated by 4 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 equivalents of ethyl cyano(hydroxyimino)acetate (Oxyma) in a mixture of water and MeCN in a ratio of 2:1 (volume).

[0355] Afterwards, the solid support loaded with activated Glu(OtBu) was rinsed with water. After the washing step, 3 equivalents of Glu(OtBu) dissolved in water were added to the activated SolidS-Glu(OtBu) and stirred for 30 minutes at 25°C and pH 8. Excess Glu(OtBu) and released Oxyma were removed by washing with water. SolidS-EE-OH was activated by 4 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 equivalents of cyano(hydroxyimino)ethyl acetate (Oxyma) in a mixture of water and MeCN in a ratio of 2:1 (volume). Afterwards, the solid support loaded with activated SolidS-EE was rinsed with water. After the washing step, 3 equivalents of Met dissolved in water were added to the activated SolidS-EE and stirred for 30 minutes at 25°C and pH 8. Excess Met and released Oxyma were removed by washing with water. This process was repeated for each step until the complete sequence of SolidS-EEMQRR-OH was synthesized. The arginine used had no side chain protecting group.

[0356] The formed EEMQRR-OH was cleaved from the solid support by using 95% trifluoroacetic acid (TFA) for 1.5 h and the side chain was deprotected. Yield: 32%.

[0357] Example 26: Synthesis of deca-Ala on SPPS

[0358] Ala-OH was added to the solid phase peptide resin ( Solid S-Ala-OH was activated by 4 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 equivalents of ethyl cyano(hydroxyimino)acetate (Oxyma) in a mixture of water and MeCN in a ratio of 2:1 (volume).

[0359] Afterwards, the solid support loaded with activated SolidS-Al was rinsed with water. After the washing step, 3 equivalents of Ala dissolved in water were added to the activated SolidS-Ala-OH, and the reaction was stirred at 25°C and pH 8 for 30 minutes. Excess Ala and released Oxyma were removed by washing with water. In a mixture of water and MeCN in a ratio of 2:1 (volume), SolidS-AA-OH was activated by 4 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 equivalents of cyano(hydroxyimino)ethyl acetate (Oxyma). Afterwards, the solid support loaded with activated SolidS-AA was rinsed with water. After the washing step, 3 equivalents of Ala dissolved in water were added to the activated SolidS-AA, and the reaction was stirred at 25°C and pH 8 for 30 minutes. Excess Ala and released Oxyma were removed by washing with water. This process was repeated for each step until the complete sequence of SolidS-decaAla-OH was synthesized.

[0360] The formed Deca-Ala-OH was cleaved from the solid support by using 95% trifluoroacetic acid (TFA) for 1.5 h. Yield: 17%.

[0361] Example 27: Synthesis of L-carnosine

[0362] 4-(((L-histidyl)oxy)methyl)-N,N,N-trimethylbenzylamine was added to an ion exchange resin (SP Sephadex C-25; GE Healthcare) at maximum loading. Smoc-β-alanine was activated by 3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 equivalents of N-hydroxysuccinimide (NHS) in a mixture of water and isopropanol in a ratio of 4:1 (volume). Thereafter, the activated Smoc-β-alanine-NHS ester was added to the histidine on the ion exchange column. The mixture was stirred and reacted at pH 7.5 for 15 minutes. Thereafter, the ion exchanger loaded with Smoc-β-Ala-His was rinsed twice with water to remove excess Smoc-β-alanine and released NHS. Thereafter, the Smoc-protecting group was removed by 5% piperazine in water and washed twice with water. The formed L-carnosine was released by deprotection using 98% TFA.

Claims

1. A method for preparing a peptide, comprising the step of forming a peptide bond, characterized in that: The carboxyl group of the first amino acid or the first peptide is activated, and the amino group of the activated first amino acid or the first peptide is protected; the activated carboxyl group of the first amino acid or the first peptide reacts with the amino group of the second amino acid or the second peptide; wherein, in the absence of the second amino acid or the second peptide, the carboxyl group of the first amino acid or the first peptide is activated; wherein the first amino acid or the first peptide is in contact with ion exchanger ions; or, the amino group of the activated first amino acid or the first peptide is protected by a solid phase; The carboxyl group of the second amino acid or second peptide that reacts with the activated first amino acid or first peptide is not protected.

2. The method according to claim 1, characterized in that The amino group of the activated first amino acid or first peptide is protected by a protecting group having a water-solubility enhancing group.

3. The method according to claim 1 or 2, characterized in that: An environmentally friendly solvent is used to activate the first amino group or the carboxyl group of the first peptide, and / or an environmentally friendly solvent is used to react the activated carboxyl group of the first amino acid or the first peptide with the amino group of the second amino acid or the second peptide.

4. The method according to claim 3, characterized in that The environmentally friendly solvent includes an aprotic organic solvent and / or a secondary alcohol and / or a tertiary alcohol.

5. The method according to claim 4, characterized in that The environmentally friendly solvent includes an aprotic organic solvent.

6. The method according to claim 1 or 2, characterized in that: The peptide bond formation is achieved in a solution without strong alkaline conditions.

7. The method according to claim 6, characterized in that The peptide bond formation is achieved in solution at a pH below 12, where the pH is measured by adding water to the solution at 25°C.

8. The method according to claim 7, characterized in that The peptide bond formation is achieved in solution at a pH in the range of 6 to 10, wherein the pH is measured by adding water to the solution at 25°C.

9. The method according to claim 1 or 2, characterized in that: The carboxyl group of the first amino acid or first peptide is activated by a coupling agent.

10. The method according to claim 1 or 2, characterized in that: When amino acids or peptides bind ions to the ion exchanger, peptide bond formation is achieved.

11. The method according to claim 1, characterized in that Peptide bond formation is achieved when amino acids or peptides are covalently bound to a solid phase.

12. The method according to claim 1 or 2, characterized in that: When the first amino acid or the first peptide is ionically bound to the ion exchanger or covalently bound to the solid phase, the carboxyl group of the first amino acid or the first peptide is activated by the coupling agent.

13. The method according to claim 1 or 2, characterized in that: Protection of the amino acid or peptide is achieved by reacting the amino acid or peptide with a protecting agent, which includes: I. Main chain structure, II. at least one water solubility enhancing 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 monophenylmethane, diphenylmethane or triphenylmethane, Wherein, the water-soluble enhancing 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 group and the reactive group are attached to the main chain structure via at least one covalent bond.

14. The method according to claim 2, characterized in that The protecting group has an ionic group having a water solubility enhancing group.

15. The method according to claim 2, characterized in that The protecting group having a water-solubility enhancing group comprises precisely one ionic group.

16. The method according to claim 2, characterized in that The protecting group having a water-solubility enhancing group includes at least two ionic groups.

17. The method according to claim 1 or 2, characterized in that: The second amino acid or second peptide does not include any protecting groups other than those protecting the primary amine group.

18. The method according to claim 1 or 2, characterized in that: 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 by side chain protecting groups.

19. The method according to claim 18, characterized in that The protected side chain functional group is selected from the group consisting of α-amino, side chain amino, thiol, carboxyl, hydroxyl, phosphono and seleno.

20. The method according to claim 9, characterized in that The coupling agent is a combination of a carbodiimide and a compound that forms an active ester.

21. The method according to claim 20, characterized in that The carbodiimide is selected from diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 2-(((ethylimino)methylene)amino)-2-methylpropane-1-sulfonate (ESC) and 2,2'-(methanediylidenebis(azaalkylene))bis(2-methylpropane-1-sulfonate) (DSC).

22. The method according to claim 20, characterized in that The active ester forming compound is selected from 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (NHS), ethyl cyano(hydroxyimino)acetate (Oxyma), hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt), 4-dimethylaminopyridine (DMAP), and 2-hydroxypyridine-N-oxide (HOPO).

23. The method according to claim 9, characterized in that The coupling agents are based on phosphonium- and amine-(imine-) type reagents.

24. The method according to claim 23, characterized in that The phosphonium- and amine-(imine-)-based reagents are selected from (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluorophosphate (PyBOP), bromotris(pyrrolidinyl)phosphonium hexafluorophosphate (PyBrOP), cyano(hydroxyimino)acetate-O2)-tris-(1-pyrrolidinyl)-phosphonium hexafluorophosphate (PyOxim), (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one) (DEPBT), (1H-benzotriazol-1-yloxy)-N,N-dimethylformimine hexachloroantimonate (BOMI), 5-(1H-benzotriazol-1-yloxy)-3,4-dihydro -1-methyl 2H-pyrrolium hexachloroantimonate (BDMP), 5-(7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (AOMP), 5-(pentafluorophenyloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate (FOMP), 5-(3',4'-dihydro-4'-oxy-1',2',3'-benzotriazin-3'-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate (DOMP), 1-(1H-benzotriazol-1-yloxy)phenylmethylenepyrrolidinium hexachloroantimonate (BPMP), 5-(succinimidyloxy)-3,4-dihydro-1-methyl-2H-pyrrolium hexachloroantimonate salt (SOMP), tetramethylfluoroformamide hexafluorophosphate (TFFH), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU), (2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylamine) hexafluorophosphate (HCTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU), 2-( 5-Norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), 1-((dimethylamino)(dimethylimino)methoxy)-2-hydroxypyridinium tetrafluoroborate (TPTU), O-(cyano(ethoxycarbonyl)methyleneamino)-1,1,3,3-tetramethyluronium tetrafluoroborate (TOTU), 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TDBTU), 1-[1-(cyano-2-ethoxy-2-oxyethylenaminooxy)-dimethylamino-morpholinium]uronium hexafluorophosphate (COMU), 4-{[1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)-ylamino](dimethylamino)-methylene}morpholin-4-ium hexafluorophosphate (COMBU), N-{[1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)-ylamino](dimethylamino)methylene}-N-methylmethanamine hexafluorophosphate (TOMBU), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). , 25. The method according to claim 9, characterized in that The coupling agents are based on EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) and DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salt), as well as other condensation products of substituted cyanuric chlorides with bases.

26. The method according to claim 9, characterized in that An excess of coupling agent was used.

27. The method according to claim 9, characterized in that At least a two-fold molar amount of coupling agent relative to the first amino acid or first peptide is used.

28. The method according to claim 26 or 27, characterized in that Prior to adding the second amino acid or second peptide to the activated first amino acid or first peptide, excess coupling agent is removed from the reaction mixture.

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