Anchor molecules for peptide synthesis in aqueous solutions

Abstract

Anchor molecules for peptide synthesis in aqueous solutions are disclosed. Wherein the anchor molecules are independently present and may be covalently coupled to a variety of hydrophilic solid supports, such as CM agarose and carboxymethyl cellulose, by amido bonds under aqueous phase conditions using a coupling agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) prior to the start of peptide synthesis. The anchor molecule has a general structure of Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n-NH2, wherein Fmoc-AA represents an amino acid protected by Fmoc, and the general structure of the anchor molecule is shown as Fmoc-AA-NH-(CH2) n-NH2. The anchoring strategy includes an acid labile ester bond located between the carboxyl terminal of Fmoc-AA and the benzyl alcohol group of the solid support, which ester bond remains stable during peptide extension and is cleavable under strongly acidic conditions, achieving final peptide release. The anchor molecule may be functionalized to interact with the ion exchange resin. The present invention enables efficient solid phase peptide synthesis (SPPS) in an aqueous phase system, enhances compatibility with hydrophilic resins, and provides improved environmental sustainability by reducing dependence on organic solvents.

Description

Technical Field

[0001] This invention generally relates to peptide synthesis. More specifically, this invention provides a method for synthesizing anchored molecules, involving the use of separable anchored molecules for linking peptides to a solid resin during solid-phase peptide synthesis in an aqueous solution. Background Technology

[0002] In solid-phase peptide synthesis (SPPS), Wang resin is one of the most widely used solid supports. In Wang resin, linking molecules connect the solid phase to the first amino acid of the peptide chain. These linking molecules contain a cleavable structure that breaks under strongly acidic conditions to release the peptide, and they are chemically bonded to the solid support.

[0003] SPPS is a widely used peptide assembly technique that assembles peptides by anchoring the C-terminus of a peptide to an insoluble polymer and then sequentially adding protected amino acids. Traditionally, SPPS relies on toxic organic solvents such as dimethylformamide (DMF) and dichloromethane (CH2Cl2), which pose environmental and health risks. To mitigate these concerns, recent research has focused on developing more environmentally friendly aqueous peptide synthesis methods. These methods utilize water-soluble protected amino acids, coupling agents, and additives. Notable innovations include: the use of propylene carbonate as an environmentally friendly alternative to DMF and CH2Cl2; the application of micellar catalysis (e.g., TPGS-750-M) to facilitate continuous deprotection and coupling in water; the use of crystallization-based techniques that leverage solvent variations; the use of water-compatible protecting groups and water-soluble coupling agents; and the use of surfactants to improve the solubility of Fmoc-amino acids and linker molecules. Summary of the Invention

[0004] The purpose of this invention is to provide an anchoring molecule for establishing a bond between a peptide and a hydrophilic solid phase in an aqueous phase.

[0005] This invention relates generally to, and more specifically to, solid-phase peptide synthesis (SPPS) in an aqueous environment, using specially designed linker molecules to link peptides to hydrophilic resins, while using Fmoc-protected amino acids (Fmoc-AA) as building blocks to facilitate the assembly of peptide sequences in aqueous solution.

[0006] To achieve the above objectives, embodiments of the present invention provide an anchoring molecule for solid-phase peptide synthesis, comprising:

[0007] a. Has the following chemical structure:

[0008] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2,

[0009] Where n is an integer from 1 to 5;

[0010] b. From -NH-(CH2) n The first terminal primary amino group provided by the -NH2 portion, in the presence of a coupling agent selected from 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and its functional equivalents, allows the free-terminal amino group to form an amide bond with the carboxyl group bound to the resin.

[0011] c. An Fmoc-protected amino acid residue (Fmoc-AA) located at the molecular end opposite to the primary amino group, the amino acid being linked to an adjacent benzyl ester bond -O-CH2-Ph- at the C-terminus of the amino acid;

[0012] d. The benzyl ester bond thereon is a bond that is easily broken under acidic conditions (pH 1-2), thereby enabling the release of the synthesized peptide from the anchoring molecule after chain assembly is complete;

[0013] e. The anchoring molecule is configured to be attached to a solid support having a carboxyl functional group, including but not limited to carboxymethyl cellulose and CM agarose;

[0014] f. Thus, the anchoring molecule (i) immobilizes Fmoc-protected amino acids during solid-phase peptide synthesis for repeated coupling and deprotection cycles, and (ii) releases the fully assembled peptide under acidic cleavage conditions without destroying the peptide backbone.

[0015] According to one embodiment of the present invention, the anchoring molecule is synthesized through the following steps:

[0016] a. Preparation of compound intermediates

[0017] An intermediate compound was synthesized by reacting a Fmoc-protected amino acid (Fmoc-AA) with bis(chloromethyl)diphenylmethane (Cl-CH2-Ph-CH2-Ph-CH2-Cl): bis(chloromethyl)diphenylmethane was dissolved in a water-miscible organic solvent selected from dimethyl sulfoxide (DMSO), acetonitrile, dimethylformamide (DMF), and tetrahydrofuran (THF) to form a solution; Fmoc-AA was dissolved in a water-miscible organic solvent; then, Fmoc-AA dissolved in the water-miscible organic solvent was added to the bis(chloromethyl)diphenylmethane solution at a molar ratio of about 1:1 to 1:2 (Fmoc-AA: bis(chloromethyl)diphenylmethane); and the reaction was carried out under anhydrous conditions at a temperature of 20°C to 60°C for 2 to 20 hours to produce the intermediate Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl.

[0018] b. Reacts with diamines to form anchoring molecules.

[0019] A diamine compound selected from ethylenediamine, 1,3-propanediamine and 1,4-butanediamine is dissolved in a water-miscible organic solvent at a concentration of 3 to 5 molar equivalents relative to the intermediate product of step 1. The intermediate product is added to the diamine solution and stirred at a temperature of 20°C to 60°C for 1 to 10 hours to produce the anchoring molecule.

[0020] c. Removal of water-soluble byproducts and unreacted diamines

[0021] The anchoring molecule, which is poorly soluble in water, precipitates from the reaction mixture, while the excess diamine, which is highly water-soluble, remains in the aqueous solution. The precipitate is washed several times with water to remove residual diamine and water-soluble byproducts, and then dried to produce purified anchoring molecules in a yield of approximately 30-60%.

[0022] According to one embodiment of the present invention, the anchoring molecule is connected to a resin having a carboxyl functional group, the resin being selected from carboxymethyl cellulose and carboxymethyl agarose beads;

[0023] The anchoring molecule is:

[0024] a. Dissolved in an aqueous medium containing a nonionic surfactant selected from polysorbate 80 (Tween 80) and its functional equivalents;

[0025] b. Contacting the resin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) at a pH of about 5 for about 4 hours promotes covalent coupling between the anchoring molecule and the resin; and

[0026] c. Subsequently, a capping step is performed, wherein any unreacted carboxyl groups on the resin are reacted with ethanolamine (NH2CH2OH) with the assistance of coupling agent EDC.

[0027] According to one embodiment of the present invention, the anchoring molecule can be fixed on an anion exchange resin via an intermediate linker sulfoacetic acid (HO3S-CH2-COOH), wherein the sulfoacetic acid comprises a sulfonic acid group and a carboxylic acid group, the sulfonic acid group being able to form an ion interaction with the anion exchange resin, and the carboxylic acid group providing a functional site for covalent coupling with the anchoring molecule containing a free amino group.

[0028] The connection process includes the following steps:

[0029] a. The anion exchange resin and sulfoacetic acid are allowed to stand at room temperature for about 2 to 4 hours in a pH range of about 6 to 7, so that some sulfonic acid ions are bound to the surface of the resin;

[0030] Washing: The resin is washed with deionized water to remove unbound sulfoacetic acid;

[0031] b. Coupling reaction: Using a carbodiimide coupling agent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), the anchoring molecule is coupled to the carboxyl group of the immobilized sulfoacetic acid at a pH of about 5 and a temperature range of about 2°C to 50°C to form a covalent amide bond.

[0032] Post-coupling washing: Remove excess reagents and byproducts by washing with an appropriate aqueous solution.

[0033] According to one embodiment of the invention, the anchoring molecule is configured to be connected to a cation exchange resin;

[0034] Carnitine (N(CH3)3-CH2-CH(OH)-CH2-COOH), comprising a quaternary ammonium moiety and a carboxylic acid moiety, is used as an intermediate for immobilizing the anchoring molecule to the cation exchange resin. The quaternary ammonium group promotes ionic interactions with the negatively charged functional groups of the cation exchange resin in an aqueous medium, while the carboxyl group provides a reaction site for forming an amide bond with the amino group of the anchoring molecule in the presence of a carbodiimide coupling agent.

[0035] The connection process may include the following steps:

[0036] a. Let the carnitine and the cation exchange resin stand at room temperature for about 2 to 4 hours at a pH of about 6 to 7;

[0037] b. Remove unbound carnitine by rinsing the resin with deionized water;

[0038] c. Using a carbodiimide coupling agent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), at a pH of about 5 and a temperature range of about 2°C to 50°C, coupling the anchoring molecule to the carboxyl group of the immobilized carnitine; and

[0039] d. Excess reagent is removed by washing, thereby producing a functionalized resin that can be used for Fmoc deprotection and subsequent peptide synthesis.

[0040] Additional aspects and advantages of the invention will be set forth in the description which follows, and in some respects will be obvious from the description or may be learned by practice of the invention.

[0041] Compared with the prior art, the beneficial effects of the embodiments of this application are:

[0042] This invention provides an anchoring molecule for peptide synthesis in aqueous solution, facilitating the transition of SPPS from an organic solvent to an aqueous system while preserving as much of the existing SPPS method as possible. This transition involves minimal changes, with no significant differences compared to organic solvent systems, except for the linker molecule and the hydrophilic solid resin. This method aims to make the transition of SPPS to aqueous solutions more readily accepted by the industry.

[0043] Although the anchoring molecule provided by the present invention functions similarly to the linker in Wang resin, unlike conventional systems such as Wang resin where the linker is pre-attached to the resin, the anchoring molecule disclosed in the present invention exists independently. This anchoring molecule can be attached to various solid resins by forming amide bonds in aqueous conditions with the aid of a coupling agent before peptide synthesis begins.

[0044] The anchoring molecule of this invention has the following characteristics: Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2). n The general structure of -NH2, where Fmoc-AA represents the Fmoc-protected amino acid. The anchoring strategy involves an acid-instable ester bond between the carboxyl terminus of Fmoc-AA and the benzyl alcohol group of the solid support. This ester bond remains stable during peptide elongation and can be cleaved under strongly acidic conditions to achieve the final peptide release.

[0045] The anchoring molecules of this invention can be functionalized to interact with ion exchange resins. For anion exchange resins, sulfoacetic acid is used to provide sulfonate groups for ion interaction and carboxyl groups for covalent bonding. For cation exchange resins, carnitine is used to provide quaternary ammonium groups for ion interaction and carboxyl groups for forming amide bonds with the anchoring molecules.

[0046] This invention enables efficient solid-phase peptide synthesis (SPPS) in aqueous systems, enhances compatibility with hydrophilic resins, and provides improved environmental sustainability by reducing dependence on organic solvents. Detailed Implementation

[0047] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0048] In solid-phase peptide synthesis (SPPS), praseodymium resin is one of the most commonly used solid supports. Peptide resin contains linker molecules that attach the solid support to the first amino acid of the peptide chain. These linkers contain a cleavable portion that breaks under strong acid conditions to release the synthesized peptide, and these linker molecules are covalently bonded to the solid support.

[0049] This invention provides an anchoring molecule that functions similarly to the linker in royal jelly resin. However, unlike the permanently linked linker in royal jelly resin, the anchoring molecule of this invention exists as a separate entity. This anchoring molecule can be linked to various solid resins in an aqueous phase by forming amide bonds with a coupling agent before peptide synthesis begins. It links the hydrophilic resin to the first amino acid of the peptide, enabling peptide assembly in an aqueous solution. After synthesis is complete, the anchoring molecule can be cleaved under strong acid, releasing the finished peptide.

[0050] Specifically, the anchoring molecule of the present invention has the following general structure:

[0051] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2,

[0052] Fmoc-AA refers to an amino acid (AA) that is protected by the Fmoc group at its N-terminus.

[0053] In the anchoring molecule of this invention, the carboxyl group of the Fmoc-protected amino acid is linked to a methylphenyl group on the resin via an acid-labile ester bond. This ester bond, formed between the carboxyl group of the amino acid and the benzyl hydroxyl group of the anchoring molecule, remains stable under standard peptide synthesis conditions but can be selectively cleaved under strongly acidic conditions. After peptide chain assembly, the ester bond is cleaved by treatment with a strong acid, thereby releasing the full-length peptide from the solid support.

[0054] The free amino group at the end of the anchoring molecule can react with the carboxyl group of the solid resin to form an amide bond, facilitated by a coupling agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). For example, CM agarose containing free carboxyl groups is suitable for binding such anchoring molecules via carbodiimide-mediated coupling. Another suitable resin is carboxymethyl cellulose, whose carboxyl groups can also react with the amino group of the anchoring molecule via EDC coupling.

[0055] The reaction scheme is as follows:

[0056] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2+HOOC- resin+EDC→Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH-COO- resin

[0057] The anchoring molecules of the present invention can be attached to anion exchange resin and cation exchange resin respectively via sulfoacetic acid and carnitine.

[0058] 1. Anchoring molecules are linked to anion exchange resins via sulfoacetic acid modification:

[0059] To immobilize the anchoring molecule onto the anion exchange resin, it is first derivatized with sulfoacetic acid, forming an amide bond between the amine group of the anchoring molecule and the carboxyl group of the sulfoacetic acid. The sulfonate group (-SO3) of the sulfoacetic acid... - It can bind to positively charged sites on the resin (e.g., quaternary ammonium, -NR3). + ).

[0060] First, sulfoacetic acid is adsorbed onto the surface of the anion exchange resin:

[0061] HO3S-CH2-COOH + anion exchange resin → resin -O3S-CH2-COOH

[0062] Then, the carboxyl groups of sulfoacetic acid on the resin surface are coupled to the anchoring molecules (EDC-mediated).

[0063] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2+ resin-O3S-CH2-COOH+EDC→Fmoc-AA-CH2-Ph-CH2-Ph-CH2-CO-NH-(CH2) n -COO-CH2-SO3 - - Resin.

[0064] 2. Anchored molecules are linked to the cation exchange resin via carnitine:

[0065] To enable the anchoring molecules to bind to the cation exchange resin, this invention uses carnitine. Carnitine is a quaternary ammonium compound containing both carboxyl and hydroxyl groups.

[0066] First, the positively charged quaternary ammonium groups of carnitine are combined with the cation exchange resin, as shown in the following reaction:

[0067] N(CH3)3-CH2-CH(OH)-CH2-COOH (carnitine) + cation exchange resin → carnitine-cation exchange resin

[0068] Subsequently, using a coupling agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), the amino group of the anchoring molecule is coupled to the carboxyl group of carnitine via a carbodiimide-mediated reaction:

[0069] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NHC-(CH2) n -NH2+COOH-carnitine-cationic resin+EDC→Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NHC-(CH2) n -NH-COO-Carnitine-Cation Resin

[0070] In one embodiment of the present invention, the synthesis process of the anchoring molecule includes the following steps:

[0071] Anchored molecules can be synthesized using the following starting materials: starting materials A, B, and C:

[0072] A = Cl-CH2-Ph-CH2-Ph-CH2-Cl (bis(chloromethyl)diphenylmethane or 4,4'-bis(chloromethyl)diphenylmethane, abbreviated as BCM-DPM)

[0073] B = Fmoc-AA, where Fmoc-AA represents an amino acid (AA) protected at the N-terminus by a fluorenylmethoxycarbonyl group (Fmoc). The type of AA depends on the first residue required by the target peptide.

[0074] C = ethylenediamine (abbreviated as EDA), or alternative diamines such as 1,3-propanediamine or 1,4-butanediamine can be used.

[0075] Step 1: Connect Fmoc-AA to A to form the intermediate product Fmoc-AA-(bisbenzylmethyl) chloride.

[0076] A water-miscible organic solvent such as dimethyl sulfoxide (DMSO), acetonitrile, dimethylformamide (DMF), or tetrahydrofuran (THF) is used as the reaction medium. The Fmoc-AA solution is slowly added to the solution of A to form an intermediate. The molar ratio of A to B is approximately 2:1 to 3:1. The reaction proceeds as follows:

[0077] A+B→Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl

[0078] (Fmoc-AA-(bisbenzylmethyl) chloride; intermediate for the next step)

[0079] The reaction mixture may also contain unwanted byproducts and unreacted starting materials, including:

[0080] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-AA-Fmoc (byproduct, small amount)

[0081] Cl-AA-CH2-Ph-CH2-Ph-CH2-Cl (Unreacted compound A)

[0082] Fmoc-AA (unreacted compound B, small amount)

[0083] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl is an intermediate compound used in the synthesis of the next step.

[0084] Step 2: Connect amino groups to form anchor molecules.

[0085] The intermediate from step 1 reacts with a diamine compound to introduce an amino group. Diamine molecules such as ethylenediamine (EDA) are soluble in organic solvents and water; this unique solubility advantage is used to prepare anchoring molecules.

[0086] The reaction is carried out by adding the intermediate product from step 1 dropwise to a solution of diamine molecules EDA. The molar ratio of A to diamine is approximately 1:3 to 1:5. The reaction of the intermediate molecules proceeds as follows:

[0087] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl+H2N-(CH2) n -NH2→Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2 (anchored molecule)

[0088] Among them, possible byproducts and unreacted materials include:

[0089] Cl-CH2-Ph-CH2-Ph-CH2-Cl+H2N-(CH2)n-NH2→NH2-(CH2)n-NH-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2

[0090] (A byproduct of step 2)

[0091] The reaction mixture after step 2 may contain:

[0092] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2 (anchored molecule)

[0093] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-AA-Fmoc (a byproduct from step 1, in small quantities)

[0094] Fmoc-AA (from unreacted compound B in step 1, trace amount)

[0095] NH2-(CH2) n -NH-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2 (a byproduct of step 2)

[0096] H2N-(CH2) n -NH2 (from the unreacted diamine in step 2)

[0097] Step 3: Purify the reaction mixture.

[0098] Purify the reaction mixture by removing water-soluble molecules (including byproducts and unreacted diamines):

[0099] NH2-(CH2) n -NH-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2 (a byproduct of step 2)

[0100] Excessive diamine:

[0101] The anchoring molecule has poor solubility in water, while the diamine and the byproduct of step 2 are highly soluble and slightly soluble in water, respectively. The reaction mixture is thoroughly washed with deionized (DI) water to remove the diamine and the byproduct of step 2. Water washing causes the anchoring molecule to precipitate, which constitutes the majority of the precipitate. The yield of the anchoring molecule is approximately 30-60%.

[0102] After washing away excess diamine, a small amount of water-insoluble byproducts and unreacted starting materials may remain. However, these compounds do not contain free amine groups and therefore do not bind to solid resins.

[0103] In one embodiment of the present invention, attaching the anchoring molecule to the solid resin includes the following steps:

[0104] Step 1: Dissolve the anchoring molecules in water.

[0105] Because the anchored molecules have poor water solubility, this invention employs a technique that utilizes surfactants to improve the solubility of hydrophobic molecules in an aqueous phase to facilitate their attachment to solid resins.

[0106] 1. Dissolve the anchoring molecules in DMSO.

[0107] 2. Add Tween 80 surfactant to the DMSO solution at a weight ratio of approximately 2-6:1 (surfactant to anchoring molecule).

[0108] 3. Add the obtained DMSO solution (containing anchoring molecules and surfactants) to water so that DMSO accounts for 20-40% of the total volume of the aqueous solution.

[0109] Step 2: Attach the anchoring molecules to a hydrophilic solid resin containing carboxyl groups.

[0110] The anchoring molecule is covalently attached to the solid resin through an amide bond formed between its amino group and the carboxyl group of the resin. The reaction proceeds as follows:

[0111] reaction:

[0112] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2)n-NH2+COOH-resin+EDC→Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NHCO-(CH2) n -NH-COO- resin

[0113] An excess of EDC is used to drive the reaction. After the reaction is complete, unreacted EDC and byproducts are washed away. The anchoring molecule is thus fixed to the solid support by stable amide bonds.

[0114] Step 3: Cap unreacted functional groups before removing Fmoc.

[0115] Before removing the Fmoc protecting group from the anchoring molecule used for peptide synthesis, unreacted functional groups on the solid resin should be capped to prevent undesirable side reactions.

[0116] Unreacted carboxyl groups on solid resins should be capped using ethanolamine (NH2CH2OH) and a coupling agent such as EDC.

[0117] Any unreacted amino groups that may originate from byproducts should also be deactivated by reacting with acetic acid and EDC.

[0118] In one embodiment of the present invention, the process of attaching anchoring molecules to anion exchange solid resin includes the following steps:

[0119] To enable the anchoring molecule to bind to the anion exchange solid resin, this invention uses sulfoacetic acid as a linker. The sulfonic acid (-SO3H) group forms an ionic bond with the positively charged ammonium group on the resin, while the carboxyl (-COOH) group of the sulfoacetic acid serves as a site for covalent coupling with the anchoring molecule via an amide bond.

[0120] Step 1: Reconstitute sulfoacetic acid in an aqueous solution with a functional group containing a positively charged group, such as a quaternary ammonium group (-NR3). + The anion exchange resin was left to stand at room temperature. The sulfonyl groups of sulfoacetic acid formed ionic bonds with these groups, fixing them to the resin surface.

[0121] Step 2: Use a coupling agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to react the anchoring molecule with the carboxyl group of the surface-bound sulfoacetic acid, thereby forming an amide bond between the anchoring molecule and the sulfoacetic acid.

[0122] Step 3: Thoroughly wash the resin to remove unreacted anchoring molecules and excess coupling agent.

[0123] Step 4: In the presence of a coupling agent, any remaining unreacted carboxyl groups of sulfoacetic acid are capped by reacting with ethanolamine (NH2CH2OH) to prevent non-specific interactions in subsequent peptide synthesis steps.

[0124] In one embodiment of the present invention, the process of attaching anchoring molecules to a cation exchange solid resin includes the following steps:

[0125] Carnitine is used as a linker to enable the anchoring molecule to bind to the cation exchange solid resin. An amide bond is formed between the carboxyl group of carnitine and the amino group of the anchoring molecule.

[0126] Carnitine structure:

[0127] N(CH3)3 + -CH2-CH(OH)-CH2-COOH

[0128] Step 1: Resin Preparation

[0129] Carnitine was incubated with an aqueous solution of a cation exchange resin at room temperature. This resin contains negatively charged functional groups (such as sulfonic acid groups, -SO3). - These functional groups are designed to attract positively charged substances, such as the quaternary ammonium groups present on carnitine (e.g., -N(CH3)3). + This step ensures that carnitine is fixed to the resin through ionic interactions.

[0130] Step 2: Coupling reaction

[0131] The anchoring molecule is then covalently linked to the carboxyl group of the immobilized carnitine via its amino group. This coupling reaction is promoted by the carbodiimide coupling agent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), thereby facilitating the formation of a stable amide bond.

[0132] Step 3: Washing

[0133] Unreacted anchoring molecules and residual EDC are removed by thoroughly washing the resin with an appropriate buffer solution.

[0134] Step 4: End sealing

[0135] To block any remaining unreacted carboxyl groups on the immobilized carnitine, a capping step is performed using ethanolamine (NH2-CH2OH) and EDC. This prevents nonspecific reactions in subsequent steps.

[0136] To facilitate a better understanding of the present invention, the following examples further illustrate the solution of the present invention.

[0137] Example 1: Anchored Molecular Synthesis

[0138] Starting materials:

[0139] A: 4,4′-bis(chloromethyl)diphenylmethane (Cl-CH2-Ph-CH2-Ph-CH2-Cl), molecular weight: 265 g / mol

[0140] B: Fmoc-AA (an amino acid protected by the Fmoc group at the N-terminus; AA represents the first amino acid in the target peptide sequence; for example, Fmoc-Gly-OH, MW: 297.3 g / mol)

[0141] C: Ethylenediamine (EDA), molar mass: 60.1 g / mol

[0142] Step 1: Preparation of intermediate molecule (Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl)

[0143] To synthesize the intermediate anchoring molecule, the following procedures were performed in this example:

[0144] 1.8 g of compound A was dissolved in dimethyl sulfoxide (DMSO) to prepare solution A.

[0145] 1.0 g of compound B was dissolved in DMSO to prepare solution B.

[0146] reaction:

[0147] Solution B is slowly added to solution A while continuously stirring.

[0148] Keep the reaction mixture at room temperature for 2 to 6 hours.

[0149] The molar ratio of chlorine group (from A) to Fmoc-AA(B) is approximately 4:1.

[0150] Expected response:

[0151] A+B→

[0152] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl (required intermediate)

[0153] +Fmoc-AA-CH2-Ph-CH2-Ph-CH2-AA-Fmoc (small amount of byproducts)

[0154] +Cl-AA-CH2-Ph-CH2-Ph-CH2-Cl (unreacted A)

[0155] +Fmoc-AA (unreacted B)

[0156] It is important to note that:

[0157] Small amounts of byproducts and unreacted starting materials (A and B) may remain in the reaction mixture. However, these will not interfere with the anchoring function, as none of the resulting molecules have free amine groups.

[0158] Step 2: Link the amino group to the intermediate molecule.

[0159] The intermediate molecule obtained in step 1 is reacted with a diamine to introduce a terminal amino group, thereby forming an anchored molecule.

[0160] Reaction conditions:

[0161] Using an intermediate in a molar ratio of 1:8-16: diamine inhibits the formation of disubstituted (dimer) byproducts derived from bis(chloromethyl)diphenylmethane.

[0162] Excess diamine ensures preferential formation of monosubstituted anchor molecules.

[0163] Preparation of diamine solution (solution C): Dissolve 2 g of ethylenediamine (EDA) in DMSO.

[0164] process:

[0165] The reaction mixture obtained in step 1 was added dropwise to solution C (a DMSO solution of diamine) with stirring.

[0166] The following reactions mainly occur:

[0167] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl+NH2-(CH2)2-NH2→Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2)2-NH2 (anchored molecule)

[0168] Possible side effects:

[0169] C1-CH2-Ph-CH2-Ph-CH2-Cl+NH2-(CH2)2-NH2→NH2-(CH2)2-NH-CH2-Ph-CH2-Ph-CH2-NH-(CH2)2-NH2

[0170] Composition of the reaction mixture after step 2:

[0171] Target product (a):

[0172] Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2)2-NH2

[0173] (Anchoring molecules; hydrophobic, poor water solubility)

[0174] Byproduct (b):

[0175] NH2-(CH2)2-NH-CH2-Ph-CH2-Ph-CH2-NH-(CH2)2-NH2

[0176] (A symmetrical bis-diamine product; slightly soluble in water)

[0177] Excess reagent (c):

[0178] NH2-(CH2)2-NH2(EDA)

[0179] (Extremely soluble in water)

[0180] Step 3: Purify the anchoring molecule.

[0181] To remove water-soluble byproducts and excess diamine, while separating poorly water-soluble anchoring molecules.

[0182] Purification process:

[0183] The crude reaction mixture was thoroughly washed with deionized (DI) water.

[0184] This step removes:

[0185] Unreacted EDA (extremely soluble in water)

[0186] Bis-diamine byproduct (slightly soluble in water)

[0187] The anchoring molecules precipitate by gradually adding water to the mixture.

[0188] Poorly water-soluble anchoring molecules will precipitate.

[0189] Collect the precipitate and redissolve it in DMSO, then precipitate it again with DI water.

[0190] Repeat the dissolution-precipitation cycle several times to improve purity.

[0191] Finally, the precipitate was washed with DI water and dried under vacuum.

[0192] result:

[0193] The final product is an anchored molecule, which is suitable for direct use in aqueous solid-phase peptide synthesis (SPPS).

[0194] Based on the starting Fmoc-AA, the typical yield is about 50%.

[0195] Example 2: Attaching anchoring molecules to carboxyl-containing solid resins

[0196] To covalently link the synthesized anchoring molecule to a carboxyl-containing solid support (such as carboxymethyl cellulose (CMC)), the reaction scheme is as follows:

[0197] Anchoring molecule + carboxyl resin + EDC coupling agent → resin-bonded anchoring molecule

[0198] Material:

[0199] Anchored molecule: 1 gram

[0200] Carboxymethyl cellulose (CMC): 5 grams

[0201] DMSO: 20mL

[0202] Tween 80 (T80): 4 grams (4 × the weight of the anchored molecule)

[0203] Deionized (DI) water: 40mL

[0204] EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide): as a coupling agent

[0205] process:

[0206] Dissolving and anchoring molecules:

[0207] Dissolve 1g of the anchoring molecule in 20mL of DMSO.

[0208] Add 4g of Tween 80 to the solution and mix thoroughly. (Solution A)

[0209] Preparation of resin suspension:

[0210] Disperse 5g of carboxymethyl cellulose (CMC) in 40mL of dispersing solution (DI) with stirring. (Solution B)

[0211] Coupling reaction:

[0212] Add solution A slowly to solution B while stirring.

[0213] EDC is added to initiate amide bond formation.

[0214] The pH was maintained between 5.0 and 6.0 during the reaction.

[0215] Stir the mixture at room temperature for 2 hours.

[0216] The solid resin is thoroughly washed with deionized (DI) water to remove excess reagents and byproducts. Any unreacted amino and acetic acid groups on the resin are then capped, and the Fmoc protecting group is removed under standard deprotection conditions. The resin is then ready for peptide synthesis.

[0217] Example 3: Connection of anchoring molecules to anion exchange resin

[0218] First, anion exchange resins are modified by introducing carboxyl groups onto the resin surface using sulfoacetic acid as a linker molecule, so as to covalently link anchor molecules.

[0219] Step 1: Surface modification of anion exchange resin

[0220] Reagents:

[0221] Sulfonylic acid (HO3S-CH2-COOH) is a bifunctional molecule containing both sulfonic acid and carboxylic acid functional groups.

[0222] process:

[0223] 1 gram of sulfoacetic acid and 5 grams of anion exchange resin were placed together in an aqueous medium and allowed to stand at room temperature.

[0224] During the period of standing at room temperature, the pH was maintained between 6 and 7 to promote effective adsorption through ion interactions.

[0225] Stir the mixture at room temperature for 2 hours.

[0226] After standing at room temperature, the resin is thoroughly washed with deionized (DI) water to remove unbound sulfoacetic acid.

[0227] The sulfonate group binds to the positively charged anion exchange resin, while the carboxylic acid group remains exposed. This modification transforms the resin surface into a carboxyl-functionalized resin, suitable for covalent coupling with anchoring molecules.

[0228] Step 2: Connection of anchoring molecules

[0229] Once modified, anion exchange resins can be considered as carboxyl-containing solid resins and can be functionalized with anchor molecules using the process outlined in "Exemplary Example: Attaching Anchor Molecules to Carboxyl-Containing Solid Resins".

[0230] Anchored molecules can also be attached to cation exchange resins. To achieve this, carnitine (N(CH3)3-CH2-CH(OH)-CH2-COOH) is used to modify the cation exchange resin by introducing a carboxyl group. This is achieved by adsorbing carnitine onto the resin surface under aqueous conditions at pH 6-7. Typically, 1 gram of carnitine is placed together with 5 grams of cation exchange resin in water and allowed to stand at room temperature. Once the carnitine is bound to the resin, the resulting carboxyl-containing resin can be functionalized with the anchored molecule using the process described in "Exemplary Example: Attaching Anchored Molecules to Carboxyl-Containing Solid Resins".

Claims

1. An anchoring molecule for solid-phase peptide synthesis, characterized in that, Include: a. Has the following chemical structure: Fmoc-AA-CH2-Ph-CH2-Ph-CH2-NH-(CH2) n -NH2, Where n is an integer from 1 to 5; b. From -NH-(CH2) n The first terminal primary amino group provided by the -NH2 portion, in the presence of a coupling agent selected from 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and its functional equivalents, can form an amide bond with the carboxyl group bound to the resin by the free-terminal amino group. c. An Fmoc-protected amino acid residue (Fmoc-AA) located at the molecular end opposite to the primary amino group, the amino acid being linked to an adjacent benzyl ester bond -O-CH2-Ph- at the C-terminus of the amino acid; d. The benzyl ester bond thereon is a bond that is easily broken under acidic conditions (pH 1-2), thereby enabling the release of the synthesized peptide from the anchoring molecule after chain assembly is complete; e. The anchoring molecule is configured to be attached to a solid support having a carboxyl functional group, including but not limited to carboxymethyl cellulose and CM agarose; f. Thus, the anchoring molecule (i) immobilizes Fmoc-protected amino acids during solid-phase peptide synthesis for repeated coupling and deprotection cycles, and (ii) releases the fully assembled peptide under acidic cleavage conditions without destroying the peptide backbone.

2. The anchoring molecule according to claim 1, characterized in that, The anchoring molecule is synthesized through the following steps: a. Formation of intermediate compounds An intermediate compound was synthesized by reacting a Fmoc-protected amino acid (Fmoc-AA) with bis(chloromethyl)diphenylmethane (Cl-CH2-Ph-CH2-Ph-CH2-Cl): bis(chloromethyl)diphenylmethane was dissolved in a water-miscible organic solvent selected from dimethyl sulfoxide (DMSO), acetonitrile, dimethylformamide (DMF), and tetrahydrofuran (THF) to form a solution; Fmoc-AA was dissolved in a water-miscible organic solvent; then, Fmoc-AA dissolved in the water-miscible organic solvent was added to the bis(chloromethyl)diphenylmethane solution at a molar ratio of about 1:1 to 1:2 (Fmoc-AA: bis(chloromethyl)diphenylmethane); and the reaction was carried out under anhydrous conditions at a temperature of 20°C to 60°C for 2 to 20 hours to produce the intermediate Fmoc-AA-CH2-Ph-CH2-Ph-CH2-Cl. b. Reacts with diamines to form anchoring molecules. A diamine compound selected from ethylenediamine, 1,3-propanediamine and 1,4-butanediamine is dissolved in a water-miscible organic solvent at a concentration of 3 to 5 molar equivalents relative to the intermediate product of step 1. The intermediate product is added to the diamine solution and stirred at a temperature of 20°C to 60°C for 1 to 10 hours to produce the anchoring molecule. c. Removal of water-soluble byproducts and unreacted diamines The anchoring molecule, which is poorly soluble in water, precipitates from the reaction mixture, while the excess diamine, which is highly water-soluble, remains in the aqueous solution. The precipitate is washed several times with water to remove residual diamine and water-soluble byproducts, and then dried to produce purified anchoring molecules in a yield of approximately 30-60%.

3. The anchoring molecule according to claim 1, characterized in that, The anchoring molecule is attached to a resin having a carboxyl functional group, the resin being selected from carboxymethyl cellulose and carboxymethyl agarose beads; The anchoring molecule is: a. Dissolved in an aqueous medium containing a nonionic surfactant selected from polysorbate 80 (Tween 80) and its functional equivalents; b. In the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), the resin is contacted at a pH of about 5 for about 4 hours to promote covalent coupling between the anchoring molecule and the resin; as well as c. Subsequently, a capping step is performed, wherein any unreacted carboxyl groups on the resin are reacted with ethanolamine (NH2CH2OH) with the assistance of coupling agent EDC.

4. The anchoring molecule according to claim 1, characterized in that, The anchoring molecule can be fixed on the anion exchange resin via the intermediate linker sulfoacetic acid (HO3S-CH2-COOH). The sulfoacetic acid contains sulfonic acid groups and carboxylic acid groups. The sulfonic acid groups can form ion interactions with the anion exchange resin, while the carboxylic acid groups provide functional sites for covalent coupling with the anchoring molecule containing free amino groups. The connection process includes the following steps: a. The anion exchange resin and sulfoacetic acid are allowed to stand at room temperature for about 2 to 4 hours in a pH range of about 6 to 7, so that some sulfonic acid ions are bound to the surface of the resin; Washing: The resin is washed with deionized water to remove unbound sulfoacetic acid; b. Coupling reaction: Using a carbodiimide coupling agent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), the anchoring molecule is coupled to the carboxyl group of the immobilized sulfoacetic acid at a pH of about 5 and a temperature range of about 2°C to 50°C to form a covalent amide bond. Post-coupling washing: Remove excess reagents and byproducts by washing with an appropriate aqueous solution.

5. The anchoring molecule according to claim 1, The anchoring molecule is configured to be attached to a cation exchange resin; Carnitine (N(CH3)3-CH2-CH(OH)-CH2-COOH), comprising a quaternary ammonium moiety and a carboxylic acid moiety, is used as an intermediate for immobilizing the anchoring molecule to the cation exchange resin. The quaternary ammonium group promotes ionic interactions with the negatively charged functional groups of the cation exchange resin in an aqueous medium, while the carboxyl group provides a reaction site for forming an amide bond with the amino group of the anchoring molecule in the presence of a carbodiimide coupling agent. The connection process may include the following steps: a. Let the carnitine and the cation exchange resin stand at room temperature for about 2 to 4 hours at a pH of about 6 to 7; b. Remove unbound carnitine by rinsing the resin with deionized water; c. Using a carbodiimide coupling agent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), the anchoring molecule is coupled to the carboxyl group of the immobilized carnitine at a pH of about 5 and a temperature range of about 2°C to 50°C. as well as d. Excess reagent is removed by washing, thereby producing a functionalized resin that can be used for Fmoc deprotection and subsequent peptide synthesis.