Linker molecule for solid phase peptide synthesis in aqueous solution and method for solid phase peptide synthesis

Abstract

Disclosed are linker molecules for solid phase peptide synthesis in aqueous solutions, including linker molecules A, linker molecules B, linker molecules C, and linker molecules D for attaching peptides to a water-compatible polymer solid phase for solid phase peptide synthesis in aqueous solutions, linker molecules A for linking peptides to anion exchange resins in aqueous solutions, linker molecules D for linking peptides to anion exchange resins in aqueous solutions, and linker molecules C for linking peptides to anion exchange resins in aqueous solutions. Linker molecule B is used to bind a peptide to a cation exchange resin in an aqueous solution, linker molecule C is used to bind a peptide to an amino resin, linker molecule D is used to bind a peptide to a carboxyl resin, in the four linker molecules, amino acids are linked to a solid phase via benzyl ester bonds, the benzyl ester linkages can cleave under acidic conditions to release the peptide chains at the end of synthesis. Also disclosed are methods of solid phase peptide synthesis in aqueous solutions for dissolving hydrophobic molecules in aqueous solutions to increase the solubility of hydrophobic compounds, in particular Fmoc-protected amino acids in aqueous solutions to promote solid phase peptide synthesis under aqueous conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of solid-phase peptide synthesis, and particularly relates to a linking molecule for solid-phase peptide synthesis in an aqueous solution and a method for solid-phase peptide synthesis. Background Art

[0002] Solid-phase peptide synthesis (SPPS) is a method for peptide synthesis. In SPPS, the peptide is linked to an insoluble polymer through its C-terminus, and the peptide is then constructed by sequential addition of protected amino acids. In the current peptide industry, solid-phase peptide synthesis (SPPS) is mainly carried out in organic solvents.

[0003] Dimethylformamide (DMF) and dichloromethane (CH2Cl2) are well-known for their serious toxicity problems and the generation of waste solvents, and are widely used in organic solvents for peptide synthesis. Therefore, there is an urgent need to develop more environmentally friendly and safer solutions in the field of peptide synthesis.

[0004] Aqueous-phase peptide synthesis generally uses protected amino acids, coupling reagents, and other essential additives that are soluble or dispersible in water. Several strategies have been conceived by researchers to promote peptide bond formation and protect the reactive functional groups of amino acids in an aqueous environment. Some researchers have proposed that propylene carbonate, which is recognized for its environmental friendliness and polarity, can effectively replace dichloromethane (CH2Cl2) and dimethylformamide (DMF) in both solution and solid-phase peptide synthesis.

[0005] Currently, a new approach has emerged in peptide synthesis, which features a tandem deprotection / coupling sequence for solution-phase peptide synthesis in water. This method is carried out under micellar catalysis conditions, using a unique surfactant, TPGS-750-M. Another new approach is a peptide synthesis method that includes the step of condensing an N-Fmoc-protected amino acid with a peptide having a C-terminal protection, and this approach uses a carrier that crystallizes in response to changes in the solvent composition.

[0006] A common method in aqueous-phase peptide synthesis is to use water-soluble coupling agents, such as water-soluble carbodiimides or peptide coupling agents that can effectively function in an aqueous medium. In addition, protecting groups that are compatible with water-based conditions are used to selectively mask functional groups that may undergo unwanted reactions during peptide bond formation.

[0007] Generally speaking, aqueous-phase peptide synthesis is an active area of research and development, with continuous efforts to improve the efficiency, scalability, and sustainability of peptide synthesis methods. Using water as the main solvent offers several advantages in terms of safety, environmental impact, and compatibility with downstream applications, making it an attractive option for peptide synthesis. Summary of the Invention

[0008] The object of the present invention is to transform the peptide synthesis method in organic solvents into a peptide synthesis method in aqueous solutions.

[0009] To this end, the first object of the present invention is to propose a linking molecule for solid-phase peptide synthesis in an aqueous solution. By changing from the use of organic solvents to an aqueous system, as much as possible of the current SPPS practice in organic solvents is retained. The present invention tries to avoid excessive changes to the peptide synthesis method in organic solvents. Except for the peptide linking molecule and the hydrophilic solid resin, the present invention has no significant difference from the current industrial use of organic solvents. This way of transformation makes it easier to be accepted by the industry.

[0010] The second object of the present invention is to propose a method for solid-phase peptide synthesis in an aqueous solution, including the processes of Fmoc deprotection, coupling, and cleavage, thereby eliminating the need for organic solvents in these stages.

[0011] The present invention more specifically focuses on using a specifically designed linking molecule to link peptides to a hydrophilic resin in solid-phase peptide synthesis (SPPS) in an aqueous environment, while Fmoc-protected amino acids (Fmoc-AA) are used as building blocks to facilitate the assembly of peptide sequences in an aqueous solution. Four specifically designed linking molecules have been developed to establish a bond between the peptide and the solid phase in the aqueous phase. A customized procedure has been conceived to improve the solubility of Fmoc-AA in the aqueous phase. This innovative approach minimizes the use of organic solvents in peptide synthesis, thus contributing to a green and sustainable solid-phase peptide synthesis.

[0012] To this end, the present invention has developed four new linking molecules for linking peptides to various water-compatible polymer solids in an aqueous solution. Linking molecule A is used to link peptides to an anion exchange resin, linking molecule B is used to bind peptides to a cation exchange resin. Both the anion and cation exchange resins can be reused after the peptide synthesis process is completed. Linking molecule C is used to link peptides to an amino resin, and linking molecule D is used to bind peptides to a carboxyl resin.

[0013] To achieve the above object, a linking molecule for solid-phase peptide synthesis in an aqueous solution according to an embodiment of the first aspect of the present invention, the linking molecule includes: linking molecule A;

[0014] The linking molecule A has a general structure of AA-CH2-Ph-Rx-SO3-. The sulfonate -SO3- provides an ionic bond to the anion exchange resin in an aqueous solution. The linking molecule A is used to link a peptide to the anion exchange resin. Once the synthesis is completed, the anion resin can be regenerated. The AA (initial amino acid) group is linked to the Ph group through the structure -COO-CH2-Ph-, which can split under strong acid conditions. Ph is a benzene ring. The Rx group provides various options, including methoxyphenyl (-O-CH2-Ph-), methylene (-CH2-), carboxylate (-OOC-), carboxymethylene (-OOC-CH2-), amino (-NH-), and ether bond group (-O-). This chemical group is used alone or in combination of two or more groups in the Rx structure. The Rx group contains two or more carbon atoms. The Rx group is a spacer that provides space between Ph and COOH. This spacer gives the linking molecule A flexibility. Such an Rx group endows the linking molecule A with a flexible structure, thus providing an easier way to establish a connection between the phenyl group and the sulfonate group.

[0015] According to another embodiment of the present invention, the linking molecule includes: a linking molecule B;

[0016] The linking molecule B is used to bind a peptide to a cation exchange resin. The linking molecule B has a general structure of AA-CH2-Ph-Rx-N(CH3)3+. The trimethylammonium group -N(CH3)3+ provides an ionic bond to the cation exchange resin in an aqueous solution. The amino acid (AA) group is linked through the structure -COO-CH2-Ph-, which can split under strong acid conditions. Once the synthesis is completed, the cation exchange resin can be regenerated. The Rx group provides various options, including methoxyphenyl (-O-CH2-Ph-), methylene (-CH2-), carboxylate group (-OOC-), carboxymethylene (-OOC-CH2-), amino (-NH-), and ether bond group (-O-). This chemical group is used alone or in combination of two or more groups in the Rx structure.

[0017] According to another embodiment of the present invention, the linking molecule includes: a linking molecule C;

[0018] The linking molecule C has the general structure of Fmoc-AA-CH2-Ph-Rx-COOH. Fmoc (fluorenylmethyloxycarbonyl) is a protecting group for AA (amino acid). The amino acid (AA) group is connected through the structure -COO-CH2-Ph-, which can split under strong acidic conditions. The carboxyl group can form a covalent bond with the amino resin: Fmoc-AA-CH2-Ph-Rx-COOH + amino resin + coupling agent → Fmoc-AA-CH2-Ph-Rx-COO-NH resin. The Rx group provides various options, including methoxyphenyl (-O-CH2-Ph-), methylene (-CH2-), carboxylate (-OOC-), carboxymethylene (-OOC-CH2-), amino (-NH-), and ether bond group (-O-). This chemical group is used alone or in combination of two or more groups in the Rx structure. The Rx group includes two or more carbon atoms. The Rx group is a spacer that provides space between Ph and COOH, and this spacer makes the linking molecule C flexible.

[0019] In addition, the linking molecule C can be used as a precursor of the linking molecule A. Coupling the linking molecule C with taurine using a coupling agent (EDC) results in the formation of the linking molecule A. Subsequently, Fmoc can be removed in an alkaline solution. Fmoc-AA-CH2-Ph-Rx-COOH + NH2-R-SO3- → Fmoc-AA-CH2-Ph-Rx-COO-NH-Rx-SO3-.

[0020] According to another embodiment of the present invention, the linking molecule includes: a linking molecule D;

[0021] The linking molecule D has the general structure of Fmoc-AA-CH2-Ph-Rx-NH2. The -NH2 group is used to form a covalent bond with the carboxyl resin (carboxymethyl cellulose): linking molecule D + carboxyl resin + coupling agent → Fmoc-AA-CH2-Ph-Rx-NH-carboxyl resin.

[0022] In addition, the linking molecule D can be used as a precursor of the linking molecule B: Fmoc-AA-CH2-Ph-R-NH2 + (3-carboxypropyl) trimethylammonium → linking molecule B. Coupling the linking molecule D with (3-carboxypropyl) trimethylammonium using a coupling agent (EDC) results in the formation of the linking molecule B. Subsequently, Fmoc can be removed in an alkaline solution.

[0023] According to one embodiment of the present invention, a protocol for solid-phase peptide synthesis (SPPS) in an aqueous solution is provided. Similar to SPPS in organic solvents, Fmoc-protected amino acids are also used as basic building blocks in the present invention. Fmoc-protected amino acids (Fmoc-AA) are applied to solid-phase peptide synthesis (SPPS) in organic solvents. However, Fmoc-AA is essentially insoluble in water. A method for dissolving Fmoc-AA in water is proposed in the present invention, enabling peptide synthesis in the aqueous phase while still using Fmoc-AA as a building block, allowing for a transition from traditional organic solvent-based solid-phase peptide synthesis (SPPS) to peptide synthesis in an aqueous environment.

[0024] According to one embodiment of the present invention, a method for enhancing the solubility of hydrophobic molecules in an aqueous solution:

[0025] The present invention provides a protocol for making some hydrophobic molecules soluble in an aqueous solution, which is applicable to dissolving Fmoc-AA in an aqueous solution. First, a non-ionic surfactant is dissolved in a small amount of water-miscible organic solvent to produce solution A; second, Fmoc-amino acid (Fmoc-AA) is dissolved in a small portion of water-miscible organic solvent to produce solution B; solution A and solution B are thoroughly mixed to form a homogeneous mixture. Finally, the resulting mixture is vigorously added to the aqueous solution. The solubility of Fmoc-AA in the aqueous solution can reach 1 - 15%, and the solubility of Fmoc-AA can vary according to the solubility of AA in the aqueous solution and temperature. For example, for AA such as glycine, Fmoc-glycine requires less surfactant to become soluble in water than Fmoc-phenylalanine.

[0026] According to one embodiment of the present invention, after coupling an initial amino acid to a solid resin via a linking molecule, peptide synthesis is achieved by introducing additional Fmoc-AA as the next amino acid to be added to the peptide chain. The additional Fmoc-AA is dissolved in the aqueous solution by using a method for enhancing the solubility of hydrophobic molecules in the aqueous solution, and the Fmoc-AA is coupled to the previously bound AA on the solid resin using a coupling agent similar to EDC.

[0027] According to one embodiment of the present invention, deprotection, coupling, and cleavage are achieved in an aqueous solution. Fmoc-deprotection is completed using a basic solution of 10% Tween 80 with a pH of 9.5 - 10.5. For example, 0.1% NaOH or 0.3% monoamine solution is used to remove Fmoc from the peptide chain, and coupling can be carried out via EDC in a 10% Tween 80 solution at pH = 4 - 5. After peptide synthesis is completed, an acidic solution such as 0.1% HCl or 0.1% H3PO4 can be used to cleave the peptide chain from the solid phase.

[0028] To achieve the above object, an embodiment of the second aspect of the present invention provides a method for solid-phase peptide synthesis in an aqueous solution, which is realized based on the linking molecule for solid-phase peptide synthesis in the aqueous solution described in any one of the above first aspects. The method includes:

[0029] Step 1: Dissolve a non-ionic surfactant in an organic solution miscible with water to produce a first solution A;

[0030] Step 2: Dissolve a hydrophobic molecule including the chemical structure Fmoc-AA in an organic solution miscible with water to produce a second solution B;

[0031] Step 3: Mix the first solution A and the second solution B to produce a mixture;

[0032] Step 4: Add the mixture to an aqueous solution to produce an aqueous solution of 1-20% Fmoc-AA.

[0033] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention.

[0034] Compared with the prior art, the beneficial effects of the embodiments of the present application are:

[0035] The present invention provides a linking molecule for solid-phase peptide synthesis in an aqueous solution. By changing from the use of organic solvents to an aqueous system, as much as possible of the current SPPS practice in organic solvents is retained. This change requires minimal alterations and, apart from the peptide linking molecule and the hydrophilic solid resin, there are no significant differences from the use of organic solvents. This way of changing makes it easier to be accepted by the industry.

[0036] The present invention provides a method for solid-phase peptide synthesis in an aqueous solution, including the processes of Fmoc deprotection, coupling, and cleavage, thus eliminating the need for organic solvents in these stages;

[0037] More specifically, it focuses on using a specifically designed linking molecule to connect peptides to a hydrophilic resin in solid-phase peptide synthesis (SPPS) in an aqueous environment, while Fmoc-protected amino acids (Fmoc-AA) are used as building blocks to facilitate the assembly of peptide sequences in an aqueous solution. Four specifically designed linking molecules have been developed to establish a bond between the peptide and the solid phase in the aqueous phase. A customized procedure has been conceived to improve the solubility of Fmoc-AA in the aqueous phase. This innovative approach minimizes the use of organic solvents in peptide synthesis, thus contributing to a green, environmentally friendly, and sustainable solid-phase peptide synthesis.

[0038] In order to more clearly understand the technical means of the present invention and be able to implement it in accordance with the content of the specification, and to make the above and other objects, features, and advantages of the present invention more obvious and understandable, the following provides preferred embodiments and, in conjunction with the accompanying drawings, details are described as follows. Other features and advantages of the present invention will be described in the subsequent specification, and, in part, will become obvious from the specification or will be embodied by implementing the present invention. The objects and other advantages of the present invention can be achieved and obtained through the structures specifically pointed out in the specification, claims, and drawings. Description of the Drawings

[0039] In order to more clearly illustrate the technical solutions in the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, a clearer understanding of the present invention can be obtained based on these drawings.

[0040] Figure 1 It is a schematic diagram of the chemical structure of linker molecule A provided according to an embodiment of the present invention;

[0041] Figure 2 It is a schematic diagram of the chemical structure of linker molecule B provided according to an embodiment of the present invention;

[0042] Figure 3 It is a schematic diagram of the chemical structure of linker molecule C provided according to an embodiment of the present invention;

[0043] Figure 4 It is a schematic diagram of the chemical structure of linker molecule D provided according to an embodiment of the present invention;

[0044] Figure 5 It is a schematic diagram of linker molecule C combined with a solid-phase amino resin provided according to an embodiment of the present invention;

[0045] Figure 6 It is a schematic diagram of linker molecule D combined with a solid-phase carboxyl resin provided according to an embodiment of the present invention;

[0046] Figure 7 It is a schematic diagram of preparing linker molecule A from linker molecule C provided according to an embodiment of the present invention;

[0047] Figure 8 It is a schematic diagram of preparing linker molecule B from linker molecule D provided according to an embodiment of the present invention;

[0048] Figure 9 It is a schematic diagram of dissolving a hydrophobic molecule in an aqueous solution provided according to an embodiment of the present invention;

[0049] Figure 10 is a flowchart of solid-phase peptide synthesis in an aqueous solution provided according to an embodiment of the present invention;

[0050] Figure 11 is a schematic diagram of the solubility test results of Fmoc-Gly and Fmoc-Phe provided according to an embodiment of the present invention. Detailed implementation manners

[0051] Embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0052] At present, solid-phase peptide synthesis (SPPS) in organic solvents is one of the most widely used methods in the industry, usually requiring a large amount of organic solvents. The main objective of the present invention is to shift from traditional SPPS in organic solvents to an aqueous solution while retaining as many of the established methods of traditional SPPS as possible, so that SPPS in an aqueous solution is easily acceptable to the peptide industry.

[0053] The present invention has developed four types of linking molecules for attaching peptides to various water-compatible solid phases. These solid phases include, but are not limited to, cationic resins, anionic resins, carboxymethyl cellulose resins, and amino PEG resins. Cationic resins, anionic resins, carboxymethyl cellulose resins, and amino PEG resins can all be easily commercially obtained, with the aim of simplifying solid-phase peptide synthesis (SPPS) in an aqueous environment.

[0054] Among them, Fmoc-protected amino acids (Fmoc-AA) are the most common building blocks in SPPS in organic solvents. In order to use Fmoc-AA in SPPS in an aqueous solution, it is necessary to make Fmoc-AA soluble in the aqueous solution. Therefore, a method for dissolving Fmoc-AA in an aqueous solution needs to be developed.

[0055] Please refer to Figure 9 , which is outlined as follows:

[0056] First, dissolve a nonionic surfactant in a small portion of a water-miscible organic solvent to produce solution A;

[0057] Second, dissolve Fmoc-AA in another small portion of a water-miscible organic solvent to produce solution B. And fully mix the two solutions (solution A and solution B) to produce a homogeneous mixture;

[0058] Finally, the mixture was vigorously added to an aqueous solvent, resulting in the successful dissolution of the Fmoc amino acid in the aqueous phase for solid-phase peptide synthesis (SPPS) in an aqueous solution.

[0059] In an embodiment of the present invention, a linker molecule for solid-phase peptide synthesis in an aqueous solution is provided, that is, four linker molecules are developed for linking peptides to various water-compatible polymer solids in an aqueous solution. The linker molecules include linker molecule A, linker molecule B, linker molecule C, and linker molecule D, respectively.

[0060] Among them, linker molecule A is used to attach peptides to anion-exchange resins, linker molecule B is used to bind peptides to cation-exchange resins, both anion and cation exchange resins can be reused after the peptide synthesis process is completed, linker molecule C is used to link peptides to amino resins, and linker molecule D is used to bind peptides to carboxyl resins.

[0061] Among them, in traditional solid-phase peptide synthesis (SPPS) in organic solvents, Wang Resin is widely favored for its effectiveness. The linker molecule in Wang Resin provides a reversible connection between the peptide chain and the solid support (resin). In Wang Resin, the linker molecule is linked to the resin via a phenyl ether bond, and amino acids (AAs) are usually linked to the linker molecule through a benzylic ester bond. Trifluoroacetic acid is used to cleave the ester bond in the linker molecule, thereby releasing the synthesized peptide at the end of the peptide synthesis process.

[0062] Next, a specific discussion will be carried out on linker molecule A, linker molecule B, linker molecule C, and linker molecule D provided by the present invention, respectively:

[0063] Linker molecule A

[0064] In an embodiment of the present invention, please refer to Figure 1 , the chemical structure of linker molecule A is AA-CH2-Ph-Rx-SO3-.

[0065] At one end of the chemical structure of linker molecule A, there is a sulfonate group, which is used to form an ionic bond with the anion-exchange resin in an aqueous solution; at the other end of the chemical structure of linker molecule A opposite to the sulfonate group, there is an amino acid, and the amino acid is connected to the chemical structure of linker molecule A through a benzylic ester bond -COO-CH2-Ph-; among them, the benzylic ester bond is a cleavable bond susceptible to acidic conditions.

[0066] Among them, the connection between the phenyl group and the sulfonate group is promoted by the Rx group, and the Rx group includes two or more carbon atoms; linker molecule A is used to link the amino acid to the anion-exchange resin in an aqueous solution to promote solid-phase peptide synthesis in an aqueous solution.

[0067] It should be noted that the amino acid (AA) is also connected to the linking molecule A through a benzylester bond, which is similar to the linking molecule in Wang resin. The main difference is that the linking molecule in Wang resin is chemically fixed to the solid phase. The linking molecule A is linked to the solid resin through an ionic bond in an aqueous solution. The linking molecule A is cleavable under acidic conditions to release the synthesized peptide when the peptide synthesis is completed.

[0068] Among them, the linking molecule A is specifically used to attach the peptide to the anion exchange resin. The linking molecule A provides a connection between the peptide and the solid phase through an ionic bond and has a general structure of AA-CH2-Ph-Rx-anionic group. The anionic group includes but is not limited to sulfonate and phosphonate groups, preferably sulfonate, and its chemical structure is as follows: AA-CH2-Ph-Rx-SO3-.

[0069] Among them, Ph represents phenyl, AA represents the initial amino acid in the peptide, and -SO3- indicates the sulfonate group. This sulfonate group is used to form an ionic bond, enabling the linking molecule A to bind to the anion exchange resin in an aqueous solution.

[0070] Among them, the sulfonate group -SO3- provides ionic binding ability in an aqueous solution to bind to the anion exchange resin. AA is the first amino acid of the peptide to be synthesized, and the carboxyl group of the amino acid (AA) is connected to the phenyl through a benzylester (-COO-CH2-Ph-). This connection can be selectively cleaved under acidic conditions at the end of the peptide synthesis process. Therefore, in such an acidic environment, the peptide will be released from the linking molecule A, and the anion exchange resin can be effectively reused through a standard anion resin regeneration procedure.

[0071] Among them, the Rx group provides various options, including but not limited to methoxyphenyl (-O-CH2-Ph-), one to ten methylenes (-CH2-), one to three carboxylate groups (-OOC-), one to three carboxymethylenes (-OOC-CH2-), one to three amino groups (-NH-), and one to ten ether bond groups (-O-).

[0072] These chemical groups are used alone or in combination of two or more groups in the Rx structure. Such a designed Rx gives a flexible structure within the linking molecule A, thus providing an easier way to establish a connection between the phenyl and the sulfonate group. The Rx group includes two or more carbon atoms, preferably 5 to 25 carbon atoms. The Rx group is a "spacer" that provides space between Ph and COOH. This spacer makes the linking molecule A flexible, and Rx can also facilitate the binding of Fmoc-AA-CH2-Ph- to the -COOH group.

[0073] Linking molecule B

[0074] In one embodiment of the present invention, please refer to Figure 2 , the chemical structure of the linking molecule B is AA-CH2-Ph-Rx-N(CH3)3+.

[0075] At one end of the chemical structure of the linking molecule B, there is a trimethylammonium group, which is used to form an ionic bond with the cation exchange resin in an aqueous solution; at the other end of the chemical structure of the linking molecule B opposite to the trimethylammonium group, there is an amino acid, and the amino acid is connected to the chemical structure of the linking molecule B through a benzylester bond -COO-CH2-Ph-; wherein the benzylester bond is a cleavable bond susceptible to acidic conditions.

[0076] Among them, the connection between the phenyl group and the trimethylammonium group is promoted by the Rx group, and the Rx group includes two or more carbon atoms; the linking molecule B is used to link the amino acid to the cation exchange resin in an aqueous solution to facilitate solid-phase peptide synthesis in the aqueous solution.

[0077] It should be noted that the linking molecule B is used to promote the connection between the peptide and the cation exchange resin in an aqueous solvent. The linking molecule B has similarity with the linking molecule A, and the key difference lies in the presence of the cationic functional group. The linking molecule B has a general structure of AA-CH2-Ph-Rx-cationic group. In the present invention, the cationic group can include but is not limited to -N(CH3)3+.

[0078] AA represents the initial amino acid in the peptide, and Ph represents the phenyl group.

[0079] Among them, similar to the linking molecule A, AA is connected to the phenyl group through a benzylester (-COO-CH2-Ph-). The linking molecule B can bind to the cation exchange resin in an aqueous solution. At the end of the peptide synthesis, the peptide can be separated from the linking molecule B under acidic conditions, and the cation exchange resin can be effectively regenerated using the standard procedures for the regeneration of the cation exchange resin.

[0080] Among them, the Rx group includes but is not limited to methoxyphenyl (-O-CH2-Ph-), one to ten methylene groups (-CH2-), one to three carboxylate groups (-OOC-), one to three carboxymethylene groups (-OOC-CH2-), one to three amino groups (-NH-), and one to ten ether bond groups (-O-).

[0081] The above chemical groups are used alone or in combination of two or more groups in the Rx structure. The Rx group contains two or more carbon atoms, preferably 5 to 25 carbon atoms. The Rx group is a "spacer" that provides space between Ph and COOH. This spacer gives flexibility to the linking molecule B, and Rx also promotes the binding of Fmoc-AA-CH2-Ph- to the -COOH group. Such Rx in the present invention endows a flexible structure within the linking molecule B, thus providing an easier way to establish a connection between the phenyl group and the trimethylammonium group (-N(CH3)3+).

[0082] Linking molecule C

[0083] In one embodiment of the present invention, referring to Figure 3 , the chemical structure of the linking molecule C is Fmoc-AA-CH2-Ph-Rx-COOH.

[0084] At one end of the chemical structure of the linking molecule C, there is a carboxyl group, which is used to form an amide bond with the amino group of the amino resin by means of a coupling agent in an aqueous solution; at the other end of the chemical structure of the linking molecule C opposite to the carboxyl group, there is an Fmoc-amino acid, and the Fmoc-amino acid is connected to the chemical structure of the linking molecule C through a benzyl ester bond -COO-CH2-Ph-; wherein the benzyl ester bond is a cleavable bond susceptible to acidic conditions.

[0085] Among them, the connection between the phenyl group and the carboxyl group is promoted by the Rx group, and the Rx group contains two or more carbon atoms; the linking molecule C is used to link an amino acid to an amino resin in an aqueous solution to promote solid-phase peptide synthesis in the aqueous solution.

[0086] It should be noted that the linking molecule C is used to promote the attachment of a peptide to the amino group of a solid resin in an aqueous solution.

[0087] Among them, the Fmoc group refers to 9-fluorenylmethoxycarbonyl and is used as a protecting group for the amino group of an amino acid. AA represents the initial amino acid in the peptide, Ph represents the phenyl group, and -COOH represents the carboxyl group. The carboxyl group of the initial amino acid is connected to the phenyl group through a benzyl ester (-COO-CH2-Ph-). This connection can be selectively cleaved under strong acidic conditions at the end of the peptide synthesis process. Therefore, in this acidic environment, the peptide will be released from the linking molecule C.

[0088] The linking molecule C can be connected to the amino group on a solid resin such as an amino polyethylene glycol resin by using a coupling agent like EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in an aqueous solution to form an amide bond.

[0089] Referring to Figure 5, in an aqueous solvent, Fmoc-AA-CH2-Ph-Rx-COOH + NH2-solid resin + coupling agent → Fmoc-AA-CH2-Ph-Rx-COO-NH2-solid resin.

[0090] The Fmoc group is attached to the N-terminus of the amino acid, and Fmoc-AA-CH2-Ph-Rx-COOH is linked to the amino group on a hydrophilic resin such as amino polyethylene glycol resin. This linking molecule C forms an amide bond between the -COOH and NH2-resin of the linking molecule C through a coupling agent DEC in an aqueous solvent, thereby acting as an intermediary between the Fmoc amino acid and the solid resin. The structure of this linking molecule C is very similar to that of linking molecule A. The main difference is that linking molecule A is attached to the solid phase via an ionic bond, while linking molecule C is linked to the amino resin through an amide bond, and the amide bond is formed between the carboxyl group in linking molecule C and the amino group of the amino resin by using a coupling agent DEC in an aqueous solvent. Similar to linking molecule A, linking molecule C can be cleaved under acidic conditions at the end of peptide synthesis, thus facilitating the release of the synthesized peptide.

[0091] Among them, the Rx group includes but is not limited to methoxyphenyl (-O-CH2-Ph-), one to ten methylenes (-CH2-), one to three carboxylic acid ester groups (-OOC-), one to three carboxymethylenes (-OOC-CH2-), one to three amino groups (-NH-), and one to ten ether bond groups (-O-). The chemical groups of the present invention are used alone or in combination of two or more groups in the Rx structure. The Rx group includes two or more carbon atoms, preferably 5 to 25 carbon atoms. The Rx group is a "spacer", providing space between Ph and COOH. This spacer makes the linking molecule C flexible, and Rx also promotes the binding of Fmoc-AA-CH2-Ph- to the -COOH group.

[0092] It should be noted that linking molecule C can also be used as a precursor of linking molecule A. Linking molecule C can be coupled with 2-aminoethanesulfonic acid (taurine) to produce linking molecule A.

[0093] Please refer to Figure 7 , first, Fmoc-AA-CH2-Ph-Rx-COOH binds to taurine through a coupling agent (EDC) in an aqueous solution, second, the Fmoc group can be removed to form linking molecule A, and finally, Fmoc-AA-CH2-Ph-Rx-COOH + NH2-CH2-CH2-SO3- (taurine) → Fmoc-AA-CH2-Ph-Rx-COO-NH-CH2-CH2-SO3-.

[0094] Linking molecule D

[0095] In one embodiment of the present invention, please refer to Figure 4 , the chemical structure of the linking molecule D is Fmoc-AA-CH2-Ph-Rx-NH2.

[0096] At one end of the chemical structure of the linking molecule D, there is an amino group, which is used to form an amide bond with the carboxyl group of the carboxyl resin by means of a coupling agent in an aqueous solution; at the other end of the chemical structure of the linking molecule D opposite to the amino group, there is an Fmoc-amino acid, and the Fmoc-amino acid is connected to the chemical structure of the linking molecule D through a benzylester bond -COO-CH2-Ph-; wherein the benzylester bond is a cleavable bond susceptible to acidic conditions.

[0097] Among them, the connection between the phenyl group and the said amino group is promoted by the Rx group, and the Rx group includes at least two carbon atoms; the linking molecule D is used to link an amino acid to the carboxyl resin in an aqueous solution to promote solid-phase peptide synthesis in the aqueous solution.

[0098] It should be noted that the linking molecule D is used to promote the attachment of the peptide in the aqueous solution to the carboxyl group on the solid resin. The structure of the linking molecule D is Fmoc-AA-CH2-Ph-Rx-NH2. The Fmoc group is the abbreviation of 9-fluorenylmethoxycarbonyl and is used as a protecting group for the amino group of an amino acid in solid-phase peptide synthesis. AA represents the initial amino acid in the peptide, Ph represents a phenyl group, and -NH2 represents an amino group. In this structure, the carboxyl group of the amino acid is connected to the phenyl group via a benzylester (-COO-CH2-Ph-), and this connection can be selectively cleaved under strong acidic conditions at the end of the peptide synthesis process, and the peptide is released from the linking molecule D in an acidic environment.

[0099] Among them, the linking molecule D can be conjugated to the carboxyl group present on a solid resin such as carboxymethyl cellulose or carboxyl agarose beads to produce an amide bond. This reaction can be promoted by using a coupling agent EDC in an aqueous solution. The -NH2 in the linking molecule D and the -COOH on the solid resin can be coupled in an aqueous solvent using a coupling agent, and this reaction results in the formation of an amide bond connecting the linking molecule D and the carboxyl resin.

[0100] Please refer to Figure 6 , in an aqueous solvent, Fmoc-AA-CH2-Ph-Rx-NH2 + HOOC-solid resin + coupling agent → Fmoc-AA-CH2-Ph-Rx-NH-OOC-solid resin.

[0101] It should be noted that linker molecule D is similar to linker molecule B, and the key difference lies in the way linker molecule D is attached to the solid phase. In this case, linker molecule D is connected to the solid phase in an aqueous solution by using a coupling agent such as EDC to directly form an amide bond on the solid phase, thereby differentiating linker molecule D from linker molecule B. Linker molecule D also has a cleavable structure -COO-CH2-Ph-, and linker molecule D can be cleaved under acidic conditions to release the peptide chain after the peptide chain reaches the desired length.

[0102] Please refer to Figure 8 , linker molecule D can also be used as a precursor of linker molecule B. Linker molecule D can react with (3-carboxypropyl)trimethylammonium in an aqueous solvent through a coupling agent (EDC) to produce linker molecule B. Subsequently, the Fmoc group is removed, resulting in the formation of linker molecule B.

[0103] The Rx group includes but is not limited to methoxyphenyl (-O-CH2-Ph-), one to ten methylenes (-CH2-), one to three carboxylic acid ester groups (-OOC-), one to three carboxymethylenes (-OOC-CH2-), one to three amino groups (-NH-), and one to ten ether bond groups (-O-). The chemical groups of the present invention are used alone or in combination of two or more groups in the Rx structure. The Rx group includes two or more carbon atoms, preferably 5 to 25 carbon atoms.

[0104] A linker molecule for solid-phase peptide synthesis in an aqueous solution provided by the present invention, by changing from the use of organic solvents to an aqueous system, retains as much as possible the current SPPS practice in organic solvents. This change requires minimal changes and has no significant difference from the use of organic solvents except for the peptide linker molecule and the hydrophilic solid resin. This way of change makes it easier to be accepted by the industry.

[0105] In another embodiment of the present invention, a method for using Fmoc-AA as a building block in solid-phase peptide synthesis in an aqueous solution is also provided, in which the hydrophobic molecule Fmoc-AA is dissolved in an aqueous solution; the method includes:

[0106] Step 1, dissolve a non-ionic surfactant in an organic solution miscible with water to produce a first solution A;

[0107] Step 2, dissolve a hydrophobic molecule including the chemical structure Fmoc-AA in an organic solution miscible with water to produce a second solution B;

[0108] Step 3, mix the first solution A and the second solution B to produce a mixture;

[0109] Step 4, adding the mixture to an aqueous solution to produce an aqueous solution of 1-20% Fmoc-AA.

[0110] A method for solid-phase peptide synthesis in an aqueous solution provided by the present invention includes the processes of Fmoc deprotection, coupling, and cleavage, thus eliminating the need for organic solvents in these stages. More specifically, it focuses on using specifically designed linking molecules to attach peptides to hydrophilic resins in solid-phase peptide synthesis (SPPS) in an aqueous environment, while Fmoc-protected amino acids (Fmoc-AA) are used as building blocks to facilitate the assembly of peptide sequences in an aqueous solution. Four specifically designed linking molecules have been developed to establish a bond between the peptide and the solid phase in the aqueous phase. A customized procedure has been conceived to improve the solubility of Fmoc-AA in the aqueous phase. This innovative approach minimizes the use of organic solvents in peptide synthesis, thus contributing to a green and sustainable solid-phase peptide synthesis.

[0111] Next, a specific discussion on the method for solid-phase peptide synthesis in an aqueous solution provided by the present invention is as follows:

[0112] Instructions for a method of dissolving hydrophobic molecules in an aqueous solution for dissolving Fmoc-AA in an aqueous solution:

[0113] In the embodiments of the present invention, Fmoc-AA is used as a building block for peptide synthesis in an aqueous solution. Fmoc-amino acids (Fmoc-AA) serve as the basic building blocks applied in solid-phase peptide synthesis (SPPS) carried out in an organic solution. In Fmoc-based solid-phase peptide synthesis (SPPS), the peptide chain is constructed sequentially, with one amino acid linked to the peptide chain one by one and fixed on an insoluble resin support. Fmoc-amino acids (Fmoc-AA) are widely used in the peptide synthesis industry.

[0114] However, it should be noted that Fmoc-AA is not easily soluble in aqueous solvents. In order to be able to utilize Fmoc-AA as a building block in an aqueous solution, a special procedure has been developed to make Fmoc-AA soluble in aqueous solvents.

[0115] Therefore, the present invention provides a method for making Fmoc-AA soluble in water. Please refer to Figure 9 , specifically:

[0116] First, a non-ionic surfactant is dissolved in a small amount of water-miscible organic solution to produce solution A; second, Fmoc-AA is dissolved in a small amount of water-miscible organic solvent to produce solution B. Solution A and solution B are fully mixed. Finally, the resulting mixture is vigorously added to an aqueous solution to obtain an aqueous solution of Fmoc-AA.

[0117] To more clearly understand the procedure of dissolving Fmoc-AA in an aqueous solution, an example is given here:

[0118] 4 grams of Tween 80 is added to 2 ml of dimethyl sulfoxide to produce solution A, 1 gram of Fmoc-AA is added to 5 ml of dimethyl sulfoxide to form solution B. Solutions A and B are thoroughly mixed, and then the resulting mixture is added to 88 ml of water to obtain a 1% concentration aqueous solution of Fmoc-AA.

[0119] To dissolve water-insoluble Fmoc-protected amino acids, such as Fmoc-phenylalanine, at room temperature, approximately eight times the amount of Tween 80 is usually required in an aqueous solution. To dissolve water-soluble Fmoc-protected amino acids, such as Fmoc-glycine, approximately four times the amount of Tween 80 is usually required in an aqueous solution.

[0120] Please refer to Figure 11 , for the listing of the solubility test results of Fmoc-Gly and Fmoc-Phe.

[0121] Among them, Figure 11 A table presenting the test results showing the surfactant to Fmoc-AA ratio (weight ratio) for two different AAs is shown. It shows that for the water-soluble amino acid Gly, the optimal ratio of surfactant to Fmoc-AAA for solubility in an aqueous solution at room temperature is 4:1 (w / w). It shows that for the insoluble amino acid Phe, the required ratio of surfactant to Fmoc-AA for solubility in an aqueous solution is 8:1 (w / w).

[0122] Although increasing the concentration of the surfactant can increase the concentration of Fmoc-AA in the aqueous solution, increasing the concentration may lead to an increase in viscosity, thereby potentially reducing the coupling efficiency. In such a case, increasing the temperature becomes very necessary to reduce the solution viscosity and improve the efficiency of the coupling process. In addition, the increase in temperature can significantly increase the solubility of Fmoc-AA in the aqueous solution.

[0123] In the embodiments of the present invention, Fmoc deprotection, amino acid coupling, and peptide cleavage are all carried out in an aqueous solution. The addition of Fmoc-AA is the only step that requires a small amount of water-miscible organic solvent, significantly reducing the overall consumption of organic solvents.

[0124] Among them, Fmoc deprotection in the aqueous solution is usually carried out under alkaline conditions with a pH range of 9.5 - 10.5 by using diluted NaOH or ethylamine in 10% Tween 80, followed by a washing step in approximately 10% Tween 80. Since the removed Fmoc is insoluble in pure aqueous solution, a surfactant aqueous solution must be used.

[0125] In the pH range of 4 - 5, EDC promotes coupling.

[0126] First, additional Fmoc - AA dissolved in an aqueous solution is introduced; second, a coupling agent such as EDC is added, and the coupling is carried out in the pH range of 4 - 5 for a duration of 20 - 30 minutes; finally, it is necessary to increase the temperature to achieve faster coupling, especially in the case of dealing with water - insoluble amino acids. In addition, the required surfactant percentage can vary according to the solubility of the amino acid.

[0127] It should be noted that a higher surfactant level may increase the viscosity, so it may be necessary to adjust the temperature to reduce the viscosity and improve the coupling efficiency.

[0128] Description of a method for synthesizing peptides in an aqueous solution using four different hydrophilic resins:

[0129] Anion - exchange resins and cation - exchange resins are commonly used in water purification, while carboxymethyl cellulose and amino resins have extensive applications in biotechnology. This embodiment utilizes these four hydrophilic resins to facilitate solid - phase peptide synthesis (SPPS) in an aqueous solution.

[0130] Among them, the synthesis of peptides in this method follows a series of steps, specifically referring to Figure 10 .

[0131] The initial amino acid is initially immobilized on the solid support through a linking molecule, and this immobilization is achieved by attaching the initial amino acid to the solid resin through the linking molecule.

[0132] Among them, linking molecule A is used to immobilize AA onto the anion - exchange resin as the solid phase, linking molecule B is used to attach AA to the cation resin, linking molecule C promotes the connection of AA with the amino resin, and linking molecule D is responsible for binding AA to the carboxyl resin. AA is the initial amino acid in peptide synthesis. Due to the benzyl ester structure in these four linking molecules, all four of these linking molecules are hydrophobic. Therefore, it is crucial to dissolve these hydrophobic molecules in an aqueous solution according to the method of dissolving the above - mentioned hydrophobic molecules in an aqueous solution before using these four linking molecules to bind the hydrophilic solid - phase resin in an aqueous solution.

[0133] Description of the procedure for applying an anion - exchange resin as the solid phase in solid - phase peptide synthesis (SPPS):

[0134] The initial step involves dissolving the linking molecule A in an aqueous solution. Subsequently, the solution of linking molecule A is co-incubated with an anion exchange resin at room temperature for several hours to attach the linking molecule A to the anion exchange resin. Then, it is washed with a 10% Tween 80 solution and DI water to prepare it for coupling with the next additional Fmoc-AA. In most cases, the amino acid (AA) in the linking molecule A is also protected by Fmoc in the following structure:

[0135] Fmoc-AA-CH2-Ph-Rx-SO3-, where AA is the initial amino acid.

[0136] If the initial amino acid in the linking molecule A is Fmoc-protected, after the linking molecule A is attached to the anion resin, the Fmoc protecting group must be removed by exposing it to a solution with a pH range of 9.5 - 10.5 in a 10% Tween 80 solution for 30 - 60 minutes, and then rinsed with DI water. At this point, it is ready to couple with the additional Fmoc-AA.

[0137] Instructions for the procedure using a cation exchange resin as the solid phase:

[0138] When using a cation exchange resin for SPPS in an aqueous solution, similar to the procedure using an anion exchange resin, the initial amino acid is attached to the solid resin using the linking molecule B.

[0139] First, dissolve the linking molecule B in an aqueous solution; second, co-incubate it with the cation exchange resin for several hours, then wash with DI water, and in the case where the amino acid is Fmoc-protected, deprotection is achieved by exposing it to a solution with a pH of 9.5 - 10.5; finally, wash with a 10% Tween 80 solution and DI water.

[0140] After these steps, the system is ready to add the additional AA,

[0141] The structure of the Fmoc-protected linking molecule B is as follows: Fmoc-AA-CH2-Ph-Rx-N(CH3)3+.

[0142] Instructions for the procedure using an amino resin as the solid phase:

[0143] The linking molecule C: Fmoc-AA-CH2-Ph-Rx-COOH is used to bind the AA to an amino resin such as the amino PEG resin commonly used in biological laboratories.

[0144] The linking molecule C contains a carboxyl group capable of binding to the amino group of an amino resin such as amino-PEG resin through a coupling agent such as EDC. The linking molecule C is dissolved in an aqueous solution, and then the resulting solution of the linking molecule C is incubated with the amino resin, followed by the addition of the coupling agent EDC to facilitate the formation of an amide bond between the carboxyl group in the linking molecule C and the amino group on the resin.

[0145] The coupling reaction occurs at pH 4 - 5 and room temperature for 30 - 60 minutes. Subsequently, any unreacted amino groups on the resin are capped by forming an amide bond with an acidic acid. Deprotection is achieved by removing the Fmoc group at pH 9.5 - 10.5, and then the system is washed with 10% T solution and DI water to prepare it for adding the next AA to the initial amino acid.

[0146] Instructions for the procedure using a carboxyl resin as the solid phase:

[0147] The linking molecule D: Fmoc-AA-CH2-Ph-Rx-NH2 is characterized in that the -NH2 group is capable of binding to the carboxyl group of the carboxyl resin through coupling with the help of a coupling agent like EDC.

[0148] For example, a carboxyl resin such as carboxymethylcellulose resin can be used. To make the linking molecule D soluble in an aqueous solution, a method for dissolving hydrophobic molecules is employed. The coupling of -NH2 and the carboxyl group occurs at pH 4 - 5 and room temperature for 30 - 60 minutes. Subsequently, any unreacted carboxyl groups on the resin are capped by forming an amide bond with a primary amine, and deprotection is achieved by removing the Fmoc group at pH 9.5 - 10.5, and then the system is washed with 10% Tween 80 solution and DI water to prepare it for adding the next amino acid (AA) to the initial amino acid.

[0149] Instructions for the procedure of peptide synthesis in an aqueous solution:

[0150] Attach the initial amino acid to the solid phase and continue with the next step according to the procedure of connecting the initial amino acid to the solid resin through the linking molecule as described above.

[0151] Deprotection includes washing the solid phase with deionized water and then removing Fmoc. Fmoc deprotection is achieved by maintaining the pH in the range of 9.5 - 10.5 using a diluted NaOH solution or a diluted monoethanolamine solution in 10% Tween 80. Subsequently, sequential washes are performed with 10% Tween 80 and deionized water.

[0152] Rinse off the excess reagent with 10% Tween 80 and deionized water to remove Fmoc from the solution.

[0153] Introduce the next Fmoc-AA. Using a method for dissolving hydrophobic molecules, first dissolve the Fmoc-AA in an aqueous solution; subsequently, combine this Fmoc-AA solution with a solid resin previously attached to a linker molecule (with an initial amino acid or as part of a growing peptide chain).

[0154] At pH 4 - 5 and room temperature, couple the Fmoc-AA to the preceding amino acid in the peptide chain using a coupling agent such as EDC in a 10% Tween 80 solution for 30 minutes. The efficiency of the coupling reaction can be increased by raising the temperature.

[0155] After the coupling reaction, eliminate the excess Fmoc-AA and EDC by washing successively with 10% Tween 80 and deionized water. Since Fmoc is insoluble in the aqueous phase, it is very necessary to include washing with 10% Tween 80.

[0156] Deprotection involves washing the solid phase with deionized water and then removing Fmoc. Fmoc deprotection is achieved by maintaining the pH in the range of 9.5 - 10.5 using a diluted NaOH solution or a diluted monoethanolamine solution in 10% Tween 80. Subsequently, perform successive washes with 10% Tween 80 and deionized water.

[0157] If additional amino acids are required, return to step 4 and repeat steps 4 to 7 until the desired peptide chain is established, then proceed to the next step.

[0158] Cleavage of the linker molecule is carried out under acidic conditions. Maintain the pH at 1 - 2 using diluted HCl or diluted H3PO4 to release the peptide from the solid phase by acid cleavage.

[0159] Instructions for preparing linker molecule A from linker molecule C:

[0160] Linker molecule A can be synthesized by bonding linker molecule C with 2 - aminoethanesulfonic acid (taurine).

[0161] Please refer to Figure 7 , first, dissolve linker molecule C in an aqueous solution. Add taurine and EDC (1 - ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide) to this solution within the pH range of 4 - 5. Keep the reaction mixture at room temperature for 60 minutes. After this coupling reaction, linker molecule A is obtained.

[0162] Instructions for preparing linker molecule B from linker molecule D:

[0163] Linker molecule B can be produced by combining linker molecule D with CTMA ((carboxypropyl)trimethylammonium).

[0164] Please refer to Figure 8, First, dissolve the linker molecule D in an aqueous solution. Introduce CTMA and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) into this solution within the pH range of 4 - 5, and keep the reaction mixture at room temperature for 60 minutes. After the coupling reaction is completed, linker molecule B is obtained.

[0165] Among them, the procedure for increasing the solubility of hydrophobic molecules is illustrated by dissolving two Fmoc-protected amino acids: Fmoc-protected glycine and Fmoc-protected phenylalanine to describe the procedure for increasing the solubility of hydrophobic molecules in an aqueous solution. Although the amino acid Gly is water-soluble, the amino acid Phe shows poor solubility in an aqueous solution. Fmoc itself has very poor solubility in an aqueous solution. It is noteworthy that both Fmoc-Gly and Fmoc-Phe show poor solubility in an aqueous solution.

[0166] The following steps outline the process of obtaining solubility of two Fmoc-protected amino acids, namely Fmoc-glycine (Fmoc-Gly) and Fmoc-phenylalanine (Fmoc-Phe) in an aqueous solution.

[0167] Instructions for dissolving Fmoc-Gly in an aqueous solution:

[0168] Please refer to Figure 11 , Step 1: Dissolve 1.08 grams of Fmoc-Gly in 6.13 milliliters of dimethyl sulfoxide solvent. Next, add 6.00 milliliters (mL) of a 70% Tween 80 DMSO solution to the Fmoc-Gly - DMSO solution;

[0169] Step 2: Pour this mixture into 87 mL of deionized (DI) water. Initially, the solution may appear slightly turbid;

[0170] Step 3: Gradually add 70% Tween 80 dropwise until the solution becomes clear. After adding 0.01 mL of 70% T, the solution becomes clear;

[0171] Step 4: This process yields a solution containing 1.07% Fmoc-Gly in an aqueous solution containing 7.85% DMSO, and the ratio of Tween 80 to Fmoc-Gly is approximately 4:1 (w / w).

[0172] Instructions for dissolving Fmoc-Phe in an aqueous solution:

[0173] Please refer to Figure 11 , Dissolve 0.18 grams of Fmoc-Phe in 1.98 dimethyl sulfoxide solvent. Next, add 2.26 milliliters (mL) of a 70% Tween 80 DMSO solution to the Fmoc-Phe solution;

[0174] Transfer this mixture into 20.38 mL of deionized water. Initially, the solution appears turbid;

[0175] Gradually add 70% T dropwise until the solution becomes clear. An additional 0.11 mL of 70% Tween 80 is required to make the solution clear;

[0176] This process yields a solution containing 0.72% Fmoc-Phe in an aqueous solution of 0.80% DMSO, with a ratio of Tween 80 to Fmoc-Phe of approximately 8:1 (w / w).

[0177] It is understood that increasing the temperature can lead to a significant increase in the solubility of Fmoc-Phe in the aqueous solution.

[0178] Instructions for solid-phase synthesis of the dipeptide His-Gly in an aqueous solution using an anion-exchange resin as the solid phase:

[0179] The linking molecule A has the structure of AA-CH2-Ph-Rx-SO3-, where AA should be Gly, i.e., Gly-CH2-Ph-Rx-SO3-.

[0180] By adopting the method of dissolving a hydrophobic molecule in an aqueous solvent, 0.2 g of the linking molecule A with Gly as the initial amino acid is dissolved in 20 mL of an aqueous solution to attach the linking molecule A to the anion-exchange resin. Then the resulting mixture is incubated with 1 g of the anion-exchange resin for 1 hour, ensuring continuous agitation by shaking or stirring. (In this case, the anion-exchange resin is characterized by quaternary ammonium cations).

[0181] To deprotect the Fmoc group, the pH is maintained in the range of 9.5 - 10.5 using a diluted NaOH solution or a diluted monoethanolamine solution in 10% Tween 80, and then washed successively with 10% Tween 80 and DI water.

[0182] Wash the mixture with 10% Tween 80 solution and deionized (DI) water respectively.

[0183] Subsequently, 0.3 g of an aqueous solution of Fmoc-histidine is introduced into the anion-exchange resin attached with the linking molecule A having Gly as the initial amino acid, ensuring continuous agitation by shaking and stirring.

[0184] Couple Fmoc-histidine with Gly in the attached linking A to the anion-exchange resin using a coupling agent such as EDC in a 10% Tween 80 solution at pH 4 - 5 and room temperature for 30 minutes.

[0185] Perform thorough washing with 10% Tween 80 solution and then deionized water.

[0186] To deprotect the Fmoc group, the pH is maintained in the range of 9.5 - 10.5 using a diluted NaOH solution or a diluted monoethanolamine solution in 10% Tween 80, and then washed successively with 10% Tween 80 and DI water.

[0187] At this time, the dipeptide His - Gly is on the anion - exchange resin. To release this dipeptide into an aqueous solution, a diluted HCl with pH = 1 can release the dipeptide from the solid - phase resin into the aqueous solution.

[0188] The anion - exchange resin can be regenerated by the standard regeneration process of the ion - exchange procedure.

[0189] Instructions for using carboxymethyl cellulose as the solid phase for peptide synthesis:

[0190] First, dissolve 1 gram of linker molecule C in 20 aqueous solution; second, add 5 grams of carboxymethyl cellulose and 1 gram of EDC, and let the mixture react at pH 4 - 5 for one hour; finally, perform a washing step using 10% Tween 80 solution and deionized (DI) water.

[0191] Before Fmoc deprotection, it is preferred to cap any unreacted carboxyl groups on the solid phase, which can be achieved by introducing 0.1 gram of monoethanolamine and an additional 0.5 EDC at pH = 4 - 5 to promote reaction with any unreacted carboxyl groups on the carboxymethyl cellulose (reminder: monoethanolamine needs to be neutralized before addition to avoid a pH jump exceeding 9), and the resulting mixture should be washed again with 10% Tween 80 solution and DI water.

[0192] After the washing step, the deprotection process can be carried out by adding a diluted NaOH or piperidine with a pH of approximately 9.5 - 10.5 and maintaining it for 30 minutes. Once this step is completed, the material is ready for further peptide synthesis processing.

[0193] Instructions for one embodiment of preparing linker molecule A from linker molecule C:

[0194] Please refer to Figure 7 , dissolve linker molecule C in an aqueous solution, add an excess of taurine, and the molar concentration ratio of taurine to C is 2:1. Subsequently, introduce EDC at a ratio of 1:1 (taurine to EDC) at room temperature in the range of pH 4 - 5 and let the reaction continue for 60 minutes.

[0195] The linking molecule A is soluble in organic solvents, while taurine has poor solubility in dimethyl sulfoxide, ethanol, ether, acetone, and methanol. To remove the excess taurine, an organic solvent such as ethanol is added to precipitate taurine from the solution. Taurine would interfere with the peptide synthesis process. After precipitating taurine, the extra organic solvent is removed under vacuum. The resulting solution contains the linking molecule A, and one end of the linking molecule A has Fmoc-AA. The resulting solution is now ready to be combined with an anion exchange resin for the synthesis process.

[0196] Although the present invention has been explained with reference to the preferred embodiments thereof, it should be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the present invention.

[0197] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included within the scope of the claims of the present invention pending approval of the application.

Claims

1. A linker molecule for solid phase peptide synthesis in aqueous solution, characterized in that The linking molecules include: linking molecules A; Wherein, the chemical structure of the linker molecule A is AA-CH2-Ph-Rx-SO3-; A sulfonate group is provided at one end of the chemical structure of the linker molecule A, and the sulfonate group is used to form an ionic bond with an anion exchange resin in an aqueous solution; an amino acid is provided at the other end of the chemical structure of the linker molecule A opposite to the sulfonate group, and the amino acid is connected to the chemical structure of the linker molecule A through a benzyl ester bond -COO-CH2-Ph-; wherein the benzyl ester bond is a cleavable bond susceptible to acidic conditions; wherein the connection between the phenyl group and the sulfonate group is facilitated by the Rx group, the Rx group comprising two or more carbon atoms; The linker molecule A is used to link the amino acid to an anion exchange resin in an aqueous solution to promote solid phase peptide synthesis in an aqueous solution.

2. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 1, characterized in that The Rx group includes methoxyphenyl-O-CH2-Ph-.

3. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 1, characterized in that The Rx group includes one to ten methylene groups -CH2-.

4. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 1, characterized in that The Rx group includes one to three carboxylate groups -OOC-.

5. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 1, characterized in that The Rx group includes one to three carboxymethylene groups -OOC-CH2-.

6. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 1, characterized in that The Rx group includes one to three amino groups -NH-.

7. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 1, characterized in that The Rx group includes one to ten ether linkage groups -O-.

8. A linker molecule for solid phase peptide synthesis in aqueous solution, characterized in that The linking molecules include: linking molecules B; Wherein, the chemical structure of the linker molecule B is AA-CH2-Ph-Rx-N(CH3)3+; A trimethylammonium group is provided at one end of the chemical structure of the linker molecule B, and the trimethylammonium group is used to form an ionic bond with a cation exchange resin in an aqueous solution; an amino acid is provided at the other end of the chemical structure of the linker molecule B opposite to the trimethylammonium group, and the amino acid is connected to the chemical structure of the linker molecule B through a benzyl ester bond -COO-CH2-Ph-; wherein the benzyl ester bond is a cleavable bond susceptible to acidic conditions; wherein the connection between the phenyl group and the trimethylammonium group is facilitated by an Rx group, wherein the Rx group includes two or more carbon atoms; The linker molecule B is used to link the amino acid to a cation exchange resin in an aqueous solution to promote solid phase peptide synthesis in an aqueous solution.

9. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 8, characterized in that The Rx group includes methoxyphenyl-O-CH2-Ph-.

10. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 8, characterized in that The Rx group includes one to ten methylene groups -CH2-.

11. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 8, characterized in that The Rx group includes one to three carboxylate groups -OOC-.

12. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 8, characterized in that The Rx group includes one to three carboxymethylene groups -OOC-CH2-.

13. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 8, characterized in that The Rx group includes one to three amino groups -NH-.

14. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 8, characterized in that The Rx group includes one to ten ether linkage groups -O-.

15. A linker molecule for solid phase peptide synthesis in aqueous solution, characterized in that The linking molecules include: linking molecules C; Wherein, the chemical structure of the linker molecule C is Fmoc-AA-CH2-Ph-Rx-COOH; A carboxyl group is provided at one end of the chemical structure of the linker molecule C, and the carboxyl group is used to form an amide bond with the amino group of the amino resin in an aqueous solution with the aid of a coupling agent; an Fmoc-amino acid is provided at the other end of the chemical structure of the linker molecule C opposite to the carboxyl group, and the Fmoc-amino acid is connected to the chemical structure of the linker molecule C via a benzyl ester bond -COO-CH2-Ph-; wherein the benzyl ester bond is a cleavable bond susceptible to acidic conditions; wherein the connection between the phenyl group and the carboxyl group is facilitated by an Rx group, wherein the Rx group includes two or more carbon atoms; The linker molecule C is used to link the amino acid to the amino resin in an aqueous solution to promote solid phase peptide synthesis in an aqueous solution.

16. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 15, characterized in that The Rx group includes methoxyphenyl-O-CH2-Ph-.

17. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 15, characterized in that The Rx group includes one to ten methylene groups -CH2-.

18. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 15, characterized in that The Rx group includes one to three carboxylate groups -OOC-.

19. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 15, characterized in that The Rx group includes one to three carboxymethylene groups -OOC-CH2-.

20. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 15, characterized in that The Rx group includes one to three amino groups -NH-.

21. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 15, characterized in that The Rx group includes one to ten ether linkage groups -O-.

22. A linker molecule for solid phase peptide synthesis in aqueous solution, characterized in that The linking molecules include: linking molecules D; Wherein, the chemical structure of the linker molecule D is Fmoc-AA-CH2-Ph-Rx-NH2; An amino group is provided at one end of the chemical structure of the linker molecule D, and the amino group is used to form an amide bond with the carboxyl group of the carboxyl resin in an aqueous solution with the aid of a coupling agent; an Fmoc-amino acid is provided at the other end of the chemical structure of the linker molecule D opposite to the amino group, and the Fmoc-amino acid is connected to the chemical structure of the linker molecule D via a benzyl ester bond -COO-CH2-Ph-; wherein the benzyl ester bond is a cleavable bond susceptible to acidic conditions; wherein the connection between the phenyl group and the amino group is facilitated by an Rx group, wherein the Rx group comprises at least two carbon atoms; The linker molecule D is used to link the amino acid to the carboxyl resin in an aqueous solution to promote solid phase peptide synthesis in an aqueous solution.

23. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 22, characterized in that The Rx group further comprises methoxyphenyl-O-CH2-Ph-.

24. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 22, characterized in that The Rx group further contains one to ten -CH2- methylene groups.

25. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 22, characterized in that The Rx group further comprises one to three carboxylate groups -OOC-.

26. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 22, characterized in that The Rx group further comprises one to three carboxymethylene groups -OOC-CH2-.

27. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 22, characterized in that The Rx group further comprises one to three amino groups -NH-.

28. The linker molecule for solid phase peptide synthesis in aqueous solution according to claim 22, characterized in that The Rx group further comprises one to ten ether linkage groups -O-.

29. A method for solid phase peptide synthesis in aqueous solution, characterized in that: Dissolving the hydrophobic molecule Fmoc-AA into an aqueous solution; the method comprises: Step 1, dissolving a nonionic surfactant in an organic solution miscible with water to produce a first solution A; Step 2, dissolving a hydrophobic molecule including a chemical structure of Fmoc-AA in an organic solution miscible with water to produce a second solution B; Step 3, mixing the first solution A and the second solution B to produce a mixture; Step 4: adding the mixture into an aqueous solution to produce an aqueous solution containing 1-20% Fmoc-AA.

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