A novel protected amino acid, a preparation method and application thereof

Abstract

The application provides a novel protected amino acid, a preparation method and application thereof, and belongs to the technical field of organic chemistry. A chemical structural general formula of the novel protected amino acid is shown as formula (1). When Amoc-AA is used for polypeptide synthesis, an organic base with low concentration is used to remove the Amoc group. Not only can the organic solvent be recycled and be green and environment-friendly, but also the purity of a polypeptide crude product obtained by synthesis and preparation is higher than that of a polypeptide crude product obtained by Fmoc method (1).

Description

Technical Field

[0001] This invention belongs to the field of organic chemistry technology, specifically relating to a novel protected amino acid, its preparation method, and its application. Background Technology

[0002] Polypeptides are a class of compounds formed by multiple amino acids linked by peptide bonds, similar to proteins, and have a relative molecular mass of less than 10,000. Polypeptides are ubiquitous in living organisms; tens of thousands of peptides have been discovered to date. They widely participate in and regulate the functional activities of various systems, organs, tissues, and cells within the body, playing a vital role in life processes. With the increasing maturity of biotechnology and peptide synthesis technology, more and more peptide drugs are being developed and applied clinically. Due to their high safety and significant efficacy, peptide drugs are currently widely used in the prevention, diagnosis, and treatment of diseases such as cancer, hepatitis, diabetes, and AIDS, and have broad development prospects.

[0003] Peptide synthesis primarily employs solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis offers advantages such as a wide selection of protecting groups, low cost, and easy scale-up. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group peptide solid-phase synthesis method excels in rapid, efficient, and controllable peptide synthesis. It is widely used in drug development, biomedical research, and bioengineering. Through rational design and optimization of reaction conditions, high-yield, high-purity peptide synthesis can be achieved, providing a feasible route for synthesizing complex peptide compounds and drugs. The Fmoc peptide solid-phase synthesis method is simple to operate, has mild reaction conditions, and high synthesis efficiency, making it the most commonly used method for chemical peptide synthesis. However, the Fmoc removal step typically requires approximately 20% piperidine solution, which presents two problems: first, the high concentration of piperidine solution makes subsequent solvent recovery difficult, resulting in low solvent recycling rates, especially under significant environmental pressure; second, the high concentration of piperidine solution is highly alkaline, leading to known or unknown side reactions, resulting in low purity and poor quality of the crude peptide.

[0004] Studies have shown that Fmoc can be removed by modification, such as by introducing a nitro group at the 2-position of Fmoc, which enables photolytic removal of Fmoc (NO2). However, this method cannot prepare amino acids with protecting groups on the side chains. Kolmar et al. introduced sulfonic acid groups at the 2 and 7 positions of Fmoc-Cl to obtain Smoc-Cl, which then reacted with amino acids to prepare Smoc-AA. Smoc-AA was used for aqueous peptide synthesis. In this process, the reaction of acyl chlorides with amino acids easily generates impurities such as dipeptides and peptides that are extremely difficult to separate, making commercial conversion difficult.

[0005] Therefore, it is necessary to develop a method for removing protective amino acids using low-concentration alkaline solutions, which can not only improve peptide quality and yield but also be environmentally friendly. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to provide a novel protective amino acid, its preparation method and its application.

[0007] This invention introduces electron-withdrawing groups onto Amoc, enabling the modified Amoc groups to be removed with a low-concentration alkaline solution, thereby protecting the amino acids protected by the modified Amoc.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a novel protected amino acid, the general chemical structure of which is shown in formula (1):

[0010] (1)

[0012] in,

[0013] R1 and R2 are either H or methyl groups, respectively.

[0014] R3 represents methyl, ethyl, propyl, isopropyl, tert-butyl, or C. 4-30 Any one of straight-chain or branched saturated alkyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclopentyl, cyclopentylmethyl, cyclopentylethyl and cyclopentylpropyl.

[0015] R represents a hydrogen atom, C represents a hydrogen atom. 1-6 straight-chain alkyl, C 3-6 Straight-chain alkenyl, C 3-6 Any one of the following: straight-chain alkynyl, isopropyl, isobutyl, tert-butyl, phenyl, benzyl, benzyl containing a substituent, (S)2-butyl, cyclopentylmethyl, and cyclohexylmethyl. Wherein, the carbon atom adjacent to R is in the S or R configuration or is achiral.

[0016] The C mentioned 1-6 The straight-chain alkyl group is any one of methyl, ethyl, propyl, butyl, pentyl, and hexyl; the C 3-6 The straight-chain alkenyl group is any one of 1-propenyl, 1-butenyl, 1-pentenyl, and 1-hexenyl; the C 3-6 The straight-chain alkynyl group is any one of 1-propynyl, 1-butynyl, 1-pentynyl and 1-hexynyl.

[0017] The substituent-containing benzyl group is selected from... .

[0018] Among them, Rm, Rn, Rp, Rs, and Rt are each independently any one of hydrogen atom, methyl, ethyl, isopropyl, tert-butyl, nitro, cyano, methoxy, tert-butoxy, benzyloxy, chlorine atom, and fluorine atom; except for hydrogen atom, the number of identical substituents is less than 3.

[0019] R is also selected from ;

[0020] in,

[0021] Ra is selected from tert-butyloxycarbonyl, benzyloxycarbonyl, or methyl;

[0022] Rb, Rc, Rd, and Re are each independently one of hydrogen, methyl, methoxy, benzyloxy, fluorine, and chlorine atoms; except for hydrogen atoms, the number of identical substituents is less than 3.

[0023] R is also selected from ;

[0024] Wherein, Rf is any one of methyl, triphenylmethyl, (p-tolyldiphenyl)methyl, acetamide methyl, p-methoxybenzyl, and p-methylbenzyl.

[0025] X1 is a sulfur atom or a selenium atom.

[0026] n1 is either 0 or 1.

[0027] R is also selected from or ;

[0028] Wherein, Rg is any one of tert-butyl, benzyl, and triphenylmethyl; * indicates that the chiral carbon bonded to the oxygen atom has an S or R configuration.

[0029] R is also selected from ;

[0030] Wherein, Rh is any one of methyl, benzyl, and tert-butyl; n2 is 0-5.

[0031] R is also selected from ;

[0032] Wherein, Ri is any one of hydrogen atom, triphenylmethyl and (p-tolyldiphenyl)methyl; n2 is 0-5.

[0033] R is also selected from ;

[0034] Wherein, Rj is any one of tert-butoxycarbonyl, benzyloxycarbonyl, triphenylmethyl and (p-tolyldiphenyl)methyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylmethylene)-3-methylbutyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylmethylene)ethyl; n3 is 0-4.

[0035] R is also selected from ;

[0036] Wherein, Rk is any one of tert-butoxycarbonyl, benzyloxycarbonyl, triphenylmethyl and (p-tolyldiphenyl)methyl.

[0037] R is also selected from ;

[0038] Where n4 is 0-3.

[0039] Preferably, the novel protective amino acid is selected from:

[0040]

[0041]

[0042]

[0043] This invention also provides a method for preparing the above-mentioned novel protected amino acid, the reaction route of which is as follows:

[0044]

[0045]

[0046]

[0047]

[0048] Where R0 is .

[0049] Specifically, the preparation method of the above-mentioned novel protective amino acid includes the following steps:

[0050] (1) Preparation of compound 6:

[0051] Compound 4 and pyridine were dissolved in a weakly polar solvent and cooled to 0°C. Then, compound 5 was slowly added. After compound 4 was completely consumed, the reaction solution was washed with dilute hydrochloric acid, then washed with water and saturated brine, respectively. The resulting solution was dried with a drying agent, filtered, and the filtrate was evaporated under reduced pressure to obtain the crude product. The crude product was then separated by normal phase chromatography to obtain the pure compound 6.

[0052] (2) Preparation of compound (1):

[0053] First, compound 7 is mixed with a weak inorganic base in water, then a water-soluble organic solvent is added, and finally compound 6 is added. The mixture is stirred at room temperature until compound 6 is completely consumed. After adding an equal volume of water, the reaction solution is extracted with petroleum ether to remove the impurities. After acidification, the product is precipitated as a solid or oil. After filtration, it is slurried with water or purified by chromatography to obtain the final compound (1).

[0054] The weakly polar solvent mentioned in step (1) is dichloromethane, dibromoethane, chloroform or toluene.

[0055] The weak inorganic base mentioned in step (2) is sodium carbonate, sodium bicarbonate or sodium tetraborate.

[0056] The water-soluble organic solvent mentioned in step (2) is acetone, tetrahydrofuran, acetonitrile or dioxane.

[0057] Compound 4 was prepared in accordance with patent CN112358399A.

[0058] This invention also provides the application of the above-mentioned novel protective amino acids in the preparation of polypeptide drugs.

[0059] Fmoc(NO2)-AA and Smoc-AA (AA being an amino acid residue or an amino acid residue with a protection group) are not easily synthesized. The possible principle behind the successful preparation of compound (1) is analyzed as follows:

[0060]

[0061] In the carbonates described above, both groups on either side of the carbonyl group have leaving activity. When X is a sulfonic acid group or a nitro group, which are strong electron-withdrawing groups, the Cl atom has a stronger leaving ability, resulting in a fluorene-protected product. However, the OSu group is more stable than the fluorene group, and since the fluorene group leaves first, a fluorene-protected product cannot be obtained. When X is an alkanoyl group, which is a weak electron-withdrawing group, both the OSu group and the chlorine atom leave first, resulting in a fluorene-protected product. Therefore, the synthesis of Fmoc(NO2)-AA and Smoc-AA is only feasible when M is Cl. However, the synthesis of protected amino acids via acyl chlorides can produce many impurities that are difficult to separate. Theoretically, X could also be an F or Cl atom, but the cost of synthesizing 2-halofluorene compounds would likely increase significantly.

[0062] Currently, most solid-phase or liquid-phase peptide synthesis methods utilize Fmoc-protected amino acids to achieve peptide chain growth. In the Fmoc removal step, the solid-phase method typically requires approximately 20% piperidine solution, while the liquid-phase method requires a strong base DBU or DBU / piperidine solution for removal. By replacing the traditional Fmoc-protected amino acid with compound (1) (compound (1) is named Amoc-AA, and Amoc will be used hereinafter to represent 2-acyl-9-fluorenylmethoxycarbonyl), a 1% piperidine solution can rapidly remove Amoc. Since the deprotection mechanism of Amoc is consistent with that of Fmoc, the degree of racemic side reactions occurring when synthesizing peptides using the Amoc method is the same as that of the Fmoc method.

[0063]

[0064] Chemical structure of Amoc

[0065] The principle behind Amoc-AA peptide synthesis is quite simple. However, traditional peptide synthesis techniques have been constrained by the peptide synthesis carrier and coupling reagents, leading some in the industry to believe that Amoc-AA is useless because Amoc is easily removed in an organic base environment. This is not the case. Compared to Fmoc peptide synthesis, the process of synthesizing peptides using Amoc-AA is similar to traditional Fmoc peptide synthesis, except that peptide synthesis mainly requires neutral, slightly alkaline, or slightly acidic conditions. If the coupling reaction is fast enough, Amoc-AA can also be used to synthesize peptides in a moderately alkaline environment. In this process, 0.5%-5% piperidine or piperazine solution is used for Amoc removal at room temperature. Piperazine has not been used in traditional processes mainly because large amounts of piperazine have poor solubility in DMF, but 0.5%-5% piperazine is completely soluble in DMF. Piperazine has low toxicity and a mild odor, therefore it can be used to replace carcinogenic and extremely malodorous piperidine, which is beneficial to the health and safety of synthesis personnel.

[0066] Due to the adoption of the above technologies, the present invention has the following beneficial effects compared with the prior art:

[0067] When Amoc-AA is used for peptide synthesis, the present invention uses a low concentration of organic base to remove the Amoc group. Not only is the organic solvent easy to recycle and environmentally friendly, but the purity of the crude peptide product obtained by synthesis is higher than that of the Fmoc method.

[0068] This invention can reduce the cost of peptide production, is environmentally friendly, and is beneficial to the health of personnel involved in on-site synthesis.

[0069] The abbreviations used in this invention and their corresponding Chinese names in English are shown in Table 1 below.

[0070] Table 1

[0071] Attached Figure Description

[0072] Figure 1 The NMR spectrum of Amoc-02-D-Ala-OH in Example 1;

[0073] Figure 2 The NMR spectrum of Amoc-O2-Asp(OtBu)-OH in Example 2;

[0074] Figure 3 The NMR spectrum of Amoc-01-Ala-OH in Example 3;

[0075] Figure 4 The NMR spectrum of Amoc-02-α-Me-DL-Phe-OH in Example 4;

[0076] Figure 5 The NMR spectrum of Amoc-O3-N-Me-Ala-OH in Example 5;

[0077] Figure 6 The NMR spectrum of Amoc-01-Aib-OH in Example 6;

[0078] Figure 7 The high-performance liquid chromatogram of crude leuprolide from Application Example 1;

[0079] Figure 8 The mass spectrum of crude leuprolide from Application Example 1;

[0080] Figure 9 The high-performance liquid chromatogram of crude leuprolide in Comparative Example 1 is shown.

[0081] Figure 10 The high-performance liquid chromatogram of the crude bivalirudin in Application Example 2;

[0082] Figure 11 The mass spectrum of crude bivalirudin from Application Example 2;

[0083] Figure 12 The high performance liquid chromatogram of crude bivalirudin in Comparative Example 2 is shown.

[0084] Figure 13 The high-performance liquid chromatogram of crude acetyl hexapeptide-38 from Application Example 3;

[0085] Figure 14 The mass spectrum of crude acetyl hexapeptide-38 from Application Example 3;

[0086] Figure 15The high-performance liquid chromatogram of crude acetyl hexapeptide-38 in Comparative Example 3 is shown.

[0087] Figure 16 The high-performance liquid chromatogram of the crude Fmoc-Ala-Tyr(tBu)-Aib-Pro-N-Me-Asp(OtBu)-OH from Application Example 4;

[0088] Figure 17 The mass spectrum of the crude Fmoc-Ala-Tyr(tBu)-Aib-Pro-N-Me-Asp(OtBu)-OH from Application Example 4 is shown. Detailed Implementation

[0089] This invention discloses a novel method for preparing and applying protected amino acids. The embodiments are merely some examples, not all examples. The embodiments are only for illustrating the technical concept and features of this invention, and are intended to enable those skilled in the art to understand the content of this invention and implement it accordingly. They should not be used to limit the scope of protection of this invention. All equivalent changes or modifications made according to the spirit and essence of this invention should be covered within the scope of protection of this invention. The invention is further illustrated below with reference to the embodiments.

[0090] Basic Example 1: 2-Palmitoyl-9-fluorenylmethyl-N-succinimide carbonate (hereinafter referred to as: Amoc-01-OSu)

[0091] Step 1: Synthesis of 2-palmitoylfluorene methanol acetate

[0092]

[0093] 7 g (25 mmol) of fluorenyl alcohol acetate was added to a 100 mL three-necked flask and dissolved in 25 mL of DCM. 9.2 g (69 mmol) of anhydrous aluminum chloride was added, and the mixture was cooled to -5 °C. A mixture of 7 g palmitoyl chloride and 12 mL of DCM was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to 20-25 °C and reacted for 1 hour. The reaction mixture was slowly added to a beaker containing 25 mL of concentrated hydrochloric acid and 75 g of ice water with stirring. A large amount of flocculent material was produced. After the ice melted, the mixture was filtered through a small layer of diatomaceous earth. The organic phase was washed with water (40 mL × 2 times), dried over anhydrous sodium sulfate, filtered, and concentrated into an oily substance. 70 mL of acetonitrile was added, and the mixture was crystallized at 20-25 °C for 3 hours. The result was 8.4 g obtained after filtration, with a yield of 62.3%.

[0094] Step 2: Synthesis of 2-palmitoylfluorenemethanol

[0095]

[0096] In a 100 mL three-necked flask, add 10 g of 2-palmitoylfluorene methanol acetate and 30 mL of methanol. Cool to 0-5 °C, then slowly add 30 mL of sulfuric acid dropwise, controlling the temperature below 30 °C. After the addition is complete, raise the temperature to 75-80 °C and maintain this temperature with stirring for 2 h. Slowly add the reaction solution to 100 mL of ice water, causing a solid to precipitate. Stir rapidly at room temperature for 0.5 h, filter, dissolve the solid in 40 mL of DCM, wash with 10% NaCl aqueous solution (50 mL × 2 times), dry with anhydrous sodium sulfate, and filter to obtain 7.4 g of solid.

[0097] Step 3: Synthesis of 2-palmitoyl-chloroformate-9-fluorenyl methyl ester

[0098]

[0099] Add 2g of 2-palmitoylfluorene methanol, 20mL of DCM, and 1.24g of triphosgene to a 100mL three-necked flask. Stir and cool to -2℃. Add 2.5mL of DCM solution containing 0.66g of pyridine dropwise. The reaction is exothermic. Control the temperature at 0-15℃. After the addition is complete, allow it to rise naturally to 20-25℃ and maintain the temperature for 3 hours. Concentrate the reaction solution to dryness, add 20mL of anhydrous acetonitrile, and stir at room temperature for 1 hour. Then cool to 0-5℃ and stir for another hour. Filter and dry to obtain 1.8g of solid.

[0100] Step 4: Synthesize Amoc-01-OSu

[0101]

[0102] Add 4.95 g of 2-palmitoyl-chloroformate-9-fluorenyl methyl ester, 30 mL of DCM, and 1.58 g of pyridine to a 100 mL three-necked flask. Stir and cool to -5 °C. Add HOSu in portions. The reaction is exothermic. Control the temperature between 0 and 15 °C. After the addition is complete, allow it to rise naturally to room temperature. Wash the reaction solution with water (20 mL × 3 times). Concentrate the reaction solution to dryness. Add 40 mL of anhydrous acetonitrile and stir at room temperature for 1 h. Then cool to 0-5 °C and stir for 1 h. Filter and dry to obtain 5.1 g of solid, yield 89.5%.

[0103] ESI-Ms: 573.33 [M+1];

[0104] 1H NMR(400MHz, CDCl3): δ0.86-0.89(t,3H), 1.25-1.41(m,21H), 1.75-1.78(t,3H), 3.00-3.04(t,J=8.0,3H), 4.39-4.41(t,3H), 4.54-4.65(m,2H), 7.41-7.47(m,2H), 7.66-7.68(d, J=8.0,1H), 7.82-7.84(m,2H), 8.05-8.07(d, J=8.0,1H),8.19(s,1H).

[0105] Basic Example 2: 2-Acetyl-9-fluorenylmethyl-benzotriazolyl carbonate (hereinafter referred to as: Amoc-02-OBt)

[0106]

[0107] Step 1: Synthesis of acetate of 2-acetylfluorenemethanol

[0108] 23.8 g (100 mmol) of fluorene-methanol acetate was added to a 250 mL three-necked flask and dissolved in 200 mL of DCM. 26.7 g (200 mmol) of anhydrous aluminum chloride was added, and the mixture was cooled to -5 °C. A mixture of 8 g (102 mmol) of acetyl chloride and 20 mL of DCM was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to 20-25 °C and reacted for 2 hours. The reaction solution was slowly added to a beaker containing 200 mL of concentrated hydrochloric acid and 200 g of ice water while stirring. After the ice melted, the mixture was filtered through a small amount of diatomaceous earth. The organic phase was washed with water (100 mL × 2 times), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to half its original volume. Then, 200 mL of acetonitrile was added, precipitating a solid. The solid was filtered, and the filter cake was allowed to air dry to obtain 18.4 g of the target product, with a yield of 65%.

[0109] Step 2: Synthesis of 2-acetylfluorenylmethanol

[0110] In a 250 mL three-necked flask, add 28.1 g (100 mmol) of 2-acetylfluorene methanol acetate and 100 mL of methanol. Cool to 0-5 °C, and slowly add 100 mL of concentrated sulfuric acid dropwise, controlling the temperature below 30 °C. After the addition is complete, raise the temperature to 75-80 °C and maintain this temperature with stirring for 2 hours. Slowly add the reaction solution to 200 mL of ice water, and stir rapidly at room temperature. Add 100 mL of DCM, wash with 10% NaCl aqueous solution (100 mL × 3 times), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 16.4 g of crude oil, yield 68.9%.

[0111] Step 3: Synthesis of 2-acetyl-chloroformate-9-fluorenylmethyl ester

[0112] In a 250 mL three-necked flask, add 23.8 g (100 mmol) of 2-acetylfluorene methanol, 200 mL of DCM, and 10.1 g (34 mmol) of triphosgene. Stir and cool to -2 °C. Add 30 mL of DCM solution containing 15.7 g of pyridine dropwise. The reaction is exothermic. Control the temperature at 0-10 °C. After the addition is complete, allow the temperature to rise naturally to 20-25 °C and maintain the temperature for 3 h. Wash the reaction solution with 0.1 M hydrochloric acid solution (100 mL × 3 times), then with water (100 mL × 3 times). Dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 31.2 g of crude oil, with a yield greater than 100%. This crude product was used directly in the next synthesis without purification.

[0113] Step 4: Synthesize Amoc-02-OBt

[0114] In a 100 mL three-necked flask, add 30.1 g (100 mmol) of 2-acetyl-chloroformate-9-fluorenyl methyl ester, 220 mL of DCM, and 15.8 g (200 mmol) of pyridine. Stir and cool to -5 °C. Add 16.2 g (120 mmol) of HOBt in portions. The reaction is exothermic, and the temperature is controlled between 0-15 °C. After the addition is complete, allow it to rise naturally to room temperature. Dissolve the reaction solution in water (100 mL × 3 times), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a crude oily product. Dissolve the crude product in 200 mL of DCM, then add petroleum ether to saturate. Heat to 40 °C, and continue to slowly add petroleum ether until just as solids precipitate, maintaining the temperature at 40 °C. At this point, cool to 25 °C, and a large amount of product precipitates. Filter, dry, and obtain 31.6 g of the target product, with a yield of 79.2%.

[0115] ESI-Ms: 340.33 [M+1];

[0116] 1 H NMR(400MHz, CDCl3): δ2.71(s,3H), 4.49-4.53(t,1H), 4.84-4.86 (m,2H),7.43-7.51(m,2H), 7.53-7.57(m,1H), 7.70-7.78(t,1H), 7.79-7.81(d,1H), 7.86-7.88(d,1H), 8.00-8.12(d,d,4H), 8.39(s,1H).

[0117] Basic Example 3: Synthesis of 2-acetyl-9-fluorenylmethyl-succinimide carbonate (hereinafter referred to as: Amoc-02-OSu)

[0118] Amoc-02-OSu was synthesized according to Basic Example 1:

[0119]

[0120] The final product was 36.2 g, with an overall yield of 74.3%.

[0121] ESI-Ms: 380.27 [M+1];

[0122] 1 H NMR(400MHz,DMSO-d6): δ2.51-2.59(t,4H), 2.61-2.67(s,3H),3.53-3.54(m,1H), 5.76 (m,2H), 7.43-7.50(m,2H), 7.96-8.05(m,4H), 8.47(s,1H).

[0123] Basic Example 4: Synthesis of 2-cyclohexylyl-9-fluorenylmethyl-succinimide carbonate (hereinafter referred to as: Amoc-03-OSu)

[0124] Amoc-03-OSu was synthesized according to Basic Example 1:

[0125]

[0126] The final product was 17.7g, with an overall yield of 21.3%.

[0127] ESI-Ms: 448.23 [M+1];

[0128] 1 H NMR(400MHz,DMSO-d6): δ1.23-1.49(m,5H), 1.69-1.85(m,5H), 2.51-2.60(m,4H), 3.53-3.54 (m,1H), 5.76 (m,1H), 6.41 (d,1H), 6.53 (d,1H), 7.44-7.50(m,2H), 7.95-8.05(m,4H), 8.47 (s,1H).

[0129] Basic Example 5: Synthesis of 2-cyclohexylyl-9-fluorenylmethyl-1-benzotriazole carbonate (hereinafter referred to as: Amoc-03-OBt)

[0130] Amoc-02-OBt was synthesized according to Basic Example 2:

[0131]

[0132] The final product was 19.3g, with an overall yield of 20.1%.

[0133] ESI-Ms: 468.23 [M+1];

[0134] 1 H NMR (400MHz, DMSO-d6): δ1.23-1.49(m,5H), 1.69-1.85(m,5H), 3.53-3.54(m,1H), 4.68 (m,1H), 5.01-5.21(m,2H), 7.41-7.70(m,5H), 7.95-8.09(m,5H), 8.42(s,1H).

[0135] Example 1: Preparation of Amoc-O2-D-Ala-OH

[0136]

[0137] 50 g (0.56 mol) of D-alanine and 141.4 g (1.68 mol) of sodium bicarbonate were mixed with 250 ml of water. Then, 201.3 g (0.5 mol) of acetone (Amoc-02-OBt) was added sequentially to 250 ml of acetone. The mixture was stirred overnight at room temperature until Amoc-02-OBt was completely consumed. The reaction solution was diluted with 250 ml of water, and impurities were extracted with petroleum ether (200 ml × 3 times). The pH was then adjusted to 3-4 with 2N hydrochloric acid solution, precipitating a white solid. The solid was filtered, and the filtrate was slurried with water (200 ml × 2 times), air-dried, and finally vacuum-dried to obtain 164 g of the final product, with a yield of 88.8%.

[0138] ESI-Ms: 354.43 [M+1]; NMR spectrum see Figure 1 .

[0139] Example 2: Preparation of Amoc-O2-Asp(OtBu)-OH

[0140]

[0141] 50 g (0.26 mol) of H-Asp(OtBu)-OH and 65.5 g (0.78 mol) of sodium bicarbonate were mixed with 250 ml of water. Then, 88.8 g (0.23 mol) of Amoc-O2-OSu were added sequentially to 250 ml of acetone. The mixture was stirred overnight at room temperature until Amoc-O2-OSu was completely consumed. The reaction solution was diluted with 250 ml of water, and impurities were extracted with petroleum ether (200 ml × 2 times). The pH was then adjusted to 3-4 with 2N hydrochloric acid solution, precipitating a white solid. The solid was filtered, and the filtrate was slurried with water (200 ml × 2 times), air-dried, and finally vacuum-dried to obtain 98.4 g of the final product, with a yield of 91%.

[0142] ESI-Ms: 454.56 [M+1]; NMR spectrum see Figure 2 .

[0143] Example 3: Preparation of Amoc-01-Ala-OH

[0144]

[0145] 50 g (0.56 mol) of L-alanine and 141.4 g (1.68 mol) of sodium bicarbonate were mixed with 250 ml of water. Then, 286.4 g (0.5 mol) of acetone (Amoc-01-OSu) was added sequentially to 250 ml of acetone. The mixture was stirred overnight at room temperature until Amoc-01-OSu was completely consumed. The reaction solution was diluted with 250 ml of water, and the pH was adjusted to 3-4 with 2N hydrochloric acid solution, resulting in the precipitation of a white solid. The solid was filtered, and the filtrate was slurried with water (200 ml × 2 times), then with acetonitrile (200 ml × 2 times). The mixture was air-dried and finally vacuum-dried to obtain 244.6 g of the final product, with a yield of 89%.

[0146] ESI-Ms: 550.71 [M+1]; NMR spectrum see Figure 3 .

[0147] Example 4: Preparation of Amoc-02-α-Me-DL-Phe-OH

[0148]

[0149] Following the method of Example 1, the amino acid was replaced with H-α-Me-DL-Phe-OH, and 38.8g of the finished product was obtained, with a yield of 80.8%.

[0150] ESI-Ms: 444.36 [M+1]; NMR spectrum see Figure 4 .

[0151] Example 5: Preparation of Amoc-O3-N-Me-Ala-OH

[0152]

[0153] Following the method of Example 2, the amino acid was replaced with HN-Me-Ala-OH, and 25.6g of the finished product was obtained, with a yield of 76.9%.

[0154] ESI-Ms: 436.56 [M+1]; NMR spectrum see Figure 5 .

[0155] Example 6: Synthesis of Amoc-01-Aib-OH

[0156]

[0157] Following the method in Example 3, the amino acid was replaced with H-Aib-OH, and 35.6g of the finished product was obtained, with a yield of 73.5%.

[0158] ESI-Ms: 564.61 [M+1]; NMR spectrum see Figure 6 .

[0159] Example 7: Synthesis of Amoc-01-Thr(tBu)-OH

[0160]

[0161] Following the method in Example 3, the amino acid was replaced with H-Thr(tBu)-OH, and 38.1 g of the finished product was obtained, with a yield of 83.2%.

[0162] ESI-Ms: 436.73 [M+1];

[0163] 1 H NMR(400MHz,DMSO-d6): δ0.84-0.86(t,3H), 1.12(s,12H), 1.23-1.31(m,2H),1.63-1.65(m,2H), 2.60-2.67(m,1H), 3.08(m, 2H), 3.99-4.09(m,1H), 4.21-4.32(m,3H),7.40-7.47(m,2H), 7.66-7.68 (d,1H), 7.77-7.78(d, 1H), 7.99-8.03(m,3H),8.22(s, 1H), 12.72 (1H).

[0164] Example 8: Synthesis of Amoc-01-N-Me-Ser(tBu)-OH

[0165]

[0166] Following the method in Example 3, the amino acid was replaced with HN-Me-Asp(OtBu)-OH, and 23.7g of the finished product was obtained, with a yield of 74.3%.

[0167] ESI-Ms: 436.57 [M+1];

[0168] 1H NMR (400MHz, DMSO-d6): δ0.85-0.87(t,3H), 1.12(s,12H),1.24-1.32(m,2H),1.64-1.66(m,2H), 2.86-2.88(s,3H),3.08(m, 2H), 3.66-3.73(m,2H), 4.21-4.37(m,3H), 4.58-4.67(m,1H), 7.41-7.48 (m,2H), 7.67-7.69(d,1H), 7.78-7.79(d,1H), 7.99-8.04(m, 3H),8.23(s, 1H), 12.92(1H).

[0169] From an atom economy perspective, Amoc-02 has the smallest molecular weight among all Amoc protecting groups, making it the most suitable for Amoc-based peptide synthesis.

[0170] Examples 9 to 25 all followed the method of Example 1, with the protecting group uniformly set to Amoc-02. The amino acids were replaced with H-Gly-OH, H-Pro-OH, H-Val-OH, H-Leu-OH, H-Ile-OH, H-Phe-OH, H-Met-OH, H-Cys(Trt)-OH, H-Tyr(tBu)-OH, H-Trp(Boc)-OH, H-Arg(Pbf)-OH, H-Lys(Boc)-OH, H-His(Trt)-OH, H-Glu(OtBu)-OH, H-Asn(Trt)-OH, H-Gln(Trt)-OH, and H-Ser(tBu)-OH to prepare the finished product.

[0171] Example 9: Synthesis of Amoc-02-Gly-OH

[0172]

[0173] ESI-Ms: 340.26 [M+1];

[0174] 1H NMR (400MHz, DMSO-d6): δ2.60-2.67(s,3H),3.70-3.85(d, J=6.4Hz,2H),4.07-4.12(t,1H), 4.23-4.31(d,2H), 7.41-7.50(m,2H), 7.65-7.71(d, J=7.2Hz,1H),7.78-7.81(t, J=7.2Hz,1H), 8.01-8.05(m,3H), 8.27-8.27(m,1H), 12.67(1H).

[0175] Example 10: Synthesis of Amoc-02-Pro-OH

[0176]

[0177] ESI-Ms: 380.39 [M+1];

[0178] 1 H NMR (400MHz, DMSO-d6): δ1.79-1.97(m,3H),2.11-2.32(m,1H),3.31-3.42(m,2H), 2.61-2.69 (s,3H), 4.15-4.21(m,2H), 4.29-4.35(m,2H), 7.42-7.51(m,2H),7.65-7.71(d, J=7.2Hz,1H), 7.77- 7.80(t, J=7.2Hz,1H), 8.01-8.06(m,3H), 8.27-8.29(m,1H),12.69(1H).

[0179] Example 11: Synthesis of Amoc-O2-Val-OH

[0180]

[0181] ESI-Ms: 382.71 [M+1];

[0182] 1H NMR(400MHz,DMSO-d6): δ0.86-0.92(S,6H), 2.03-2.11(t,1H), 2.60-2.67(s,3H),3.86-3.89(t,1H), 4.20-4.30(m,3H), (7.38-7.47(m,2H), 7.62-7.68(d, J=7.2Hz,1H), 7.75-7.78(t, J=7.2Hz,1H), 7.98- 8.02(m,3H), 8.24-8.27(m,1H),12.64(1H).

[0183] Example 12: Synthesis of Amoc-02-Leu-OH

[0184]

[0185] ESI-Ms: 396.44 [M+1];

[0186] 1 H NMR (400MHz, DMSO-d6): δ0.854-0.91(q,6H), 1.47-1.57(m,3H), 2.61-2.67(s,3H), 3.97-4.00 (m,1H), 4.22-4.31(m,3H), (7.40-7.49(m,2H),7.64-7.70(d,J=7.2Hz,1H), 7.77-7.80(t, J=7.2Hz,1H), 8.00-8.04(m,3H), 8.26-8.27(m,1H),12.41-12.53 (1H).

[0187] Example 13: Synthesis of Amoc-O2-Ile-OH

[0188]

[0189] ESI-Ms: 396.37 [M+1];

[0190] 1H NMR (400MHz, DMSO-d6): δ0.84-0.89(s,6H), 1.20-1.27(m,1H), 1.40-1.46(m,1H), 1.78-1.84(m,1H), 2.61-2.65(s,3H), 3.92-3.95(m,1H), 4.22-4.31(m,3H),7.41-7.49(m,2H), 7.64-7.71(d, J=7.2Hz,1H), 7.76-7.81(t, J=7.2Hz,1H), 8.01-8.05(m,3H), 8.26-8.27(m,1H), 12.43-12.57(1H).

[0191] Example 14: Synthesis of Amoc-O2-Phe-OH

[0192]

[0193] ESI-Ms: 430.72 [M+1];

[0194] 1 H NMR (400MHz, DMSO-d6): δ2.63-2.69(s,3H),2.87-2.90(m,1H), 3.06-3.10(m,1H), 4.15-4.21(m,4H), 7.20-7.31(m, 5H), 7.39-7.47(m,2H),7.63-7.68(d, J=7.2Hz,1H), 7.75-7.78(t, J=7.2Hz,1H), 7.99-8.03(m,3H), 8.24-8.27(m,1H),12.78(1H).

[0195] Example 15: Synthesis of Amoc-O2-Met-OH

[0196]

[0197] ESI-Ms: 414.45 [M+1];

[0198] 1H NMR (400MHz, DMSO-d6): δ1.86-1.99(m,2H), 2.04(s,3H), 2.46-2.55(m,2H), 2.62-2.68(s,3H), 4.07-4.13(m,1H), 4.21-4.31(m,3H), 7.40-7.49(m,2H),7.64-7.70(d, J=7.2Hz,1H), 7.77-7.80(t, J=7.2Hz,1H), 8.00-8.04(m,3H), 8.26-8.27(m,1H),12.66-12.73 (1H).

[0199] Example 16: Synthesis of Amoc-02-Cys(Trt)-OH

[0200]

[0201] ESI-Ms: 428.62 [M+1];

[0202] 1 H NMR (400MHz, DMSO-d6): δ2.42-2.43(m,1H), 2.60-2.67(m,4H), 3.83-3.84(m,1H), 4.23-4.29(m,3H), 4.23-4.29(m,3H), 7.22-7.35(m,15H), 7.64-7.70(d, J=7.2Hz,1H), 7.77-7.80(t, J=7.2Hz,1H), 8.00-8.04(m,3H), 8.26-8.27(m,1H),12.46-12.56 (1H).

[0203] Example 17: Synthesis of Amoc-O2-Tyr(tBu)-OH

[0204]

[0205] ESI-Ms: 502.47 [M+1];

[0206] 1H NMR (400MHz, DMSO-d6): δ1.19-1.25(s,9H), 2.64-2.71(s,3H), 2.79-2.86(m,1H), 3.01-3.06(d,1H), 4.13-4.23(m,4H), 6.80-6.86(d, J=8.4,Hz,2H), 7.13-7.18(d, J=8.4Hz,2H), (7.40-7.49(m,2H), 7.64-7.70(d, J=7.2Hz,1H), 7.77-7.80(t,J=7.2Hz,1H), 8.00-8.04(m,3H), 8.26-8.27(m,1H),12.77(1H).

[0207] Example 18: Synthesis of Amoc-O2-Trp(Boc)-OH

[0208]

[0209] ESI-Ms: 569.53 [M+1];

[0210] 1 H NMR (400MHz, DMSO-d6): δ1.57-1.60(s,9H), 2.64-2.71(s,3H), 3.00-3.16(m,1H), 3.21-3.33(m,1H), 4.19-4.21(m,3H), 4.30-4.32(m,1H), 7.22-7.31(m,2H), (7.40-7.49(m,2H), 7.51-7.59(m, 1H), 7.65-7.72(d, J=7.2Hz,1H), 7.78-7.81(t, J=7.2Hz,1H), 7.84-7.90(m, 2H), 8.01-8.04(m,3H), 8.25-8.27 (m,1H), 12.83(1H).

[0211] Example 19: Synthesis of Amoc-O2-Arg(Pbf)-OH

[0212]

[0213] ESI-Ms: 691.82 [M+1];

[0214] 1H NMR(400MHz,DMSO-d6): δ1.37-1.56(m,10H), 1.68-1.76(m,1H), 2.00(s,3H), 2.43(s,3H), 2.94(s,2H), 3.03-3.05(m,2H), 2.61-2.67(s,3H), 3.88-3.93(m,1H), 4.20-4.29(m,3H), 6.41(1H), 6.49(1H), 7.39-7.49(m,2H),7.63-7.69(d, J=7.2Hz,1H), 7.77-7.80(t, J=7.2Hz,1H), 7.99-8.02 (m,3H), 8.24-8.25 (m, 1H). 12.64 (1H).

[0215] Example 20: Synthesis of Amoc-O2-Lys(Boc)-OH

[0216]

[0217] ESI-Ms: 511.42 [M+1];

[0218] 1 H NMR (400MHz, DMSO-d6): δ1.38(s,9H), 1.52-1.67(m,2H), 2.61-2.66(s,3H), 2.81-2.91(m,2H), 3.33-3.39(m,1H), 3.87-3.91(m,1H), 4.21-4.29(m,3H), 6.78(1H), 7.20-7.34(m,15H), 7.65-7.70(d, J=7.2Hz,1H), 7.75-7.78(t, J=7.2Hz,1H),8.01-8.04(m,3H), 8.26-8.29(m,1H).12.47-12.57(1H).

[0219] Example 21: Synthesis of Amoc-O2-His(Trt)-OH

[0220]

[0221] ESI-Ms: 662.56 [M+1];

[0222] 1H NMR (400MHz, DMSO-d6): δ2.62-2.68(s,3H),2.71-2.81(m,1H),2.87-2.93(m,1H), 4.15-4.25 (m,4H), 7.02-7.05(m,6H), 7.31-7.49(m,11H), 7.63-7.70(d,1H),7.77-7.80(t,1H), 8.01-8.04(m,3H), 8.25-8.27(m,1H).12.69 (1H).

[0223] Example 22: Synthesis of Amoc-O2-Glu(OtBu)-OH

[0224]

[0225] ESI-Ms: 468.35 [M+1];

[0226] 1 H NMR (400MHz, DMSO-d6): δ1.40(S,9H), 1.77-1.79(m,1H), 1.96-1.99(m,1H), 2.27-2.31(m,2H), 2.62-2.69(s,3H),3.98-3.99(m,1H), 4.22-4.31(m,3H),7.40-7.49(m,2H),7.64-7.70(d, J=7.2Hz,1H), 7.77-7.80(t, J=7.2Hz,1H), 8.00-8.04(m,3H), 8.26-8.27(m,1H).12.66(1H).

[0227] Example 23: Synthesis of Amoc-O2-Asn(Trt)-OH

[0228]

[0229] ESI-Ms: 639.64 [M+1];

[0230] 1 H NMR(400MHz,DMSO-d6): δ2.61-2.69(m,5H), 4.17-4.39(m,4H), 7.11-7.32(m,16H), 7.40-7.49(m,2H), 7.64-7.70 (d,1H), 7.77-7.80(t,1H), 8.00-8.04(m,3H),8.26-8.27(m,1H), 8.61-8.63(m,1H), 12.74(1H).

[0231] Example 24: Synthesis of Amoc-O2-Gln(Trt)-OH

[0232]

[0233] ESI-Ms: 653.66 [M+1];

[0234] 1 H NMR (400MHz, DMSO-d6): δ1.62-1.71(m,1H), 1.89-1.98(m,1H), 2.40-2.42(m,2H), 2.59-2.68 (s,3H), 3.96-3.99(m,1H), 4.21-4.31(m,3H), 7.16-7.41(m,18H),7.63-7.71 (d,1H), 7.77-7.81(t,1H), 8.01-8.04(m,2H), 8.25-8.27(m,1H), 8.53(m,1H), 12.63 (1H).

[0235] Example 25: Synthesis of Amoc-O2-Ser(tBu)-OH

[0236]

[0237] ESI-Ms: 426.57 [M+1];

[0238] 1 H NMR(400MHz,DMSO-d6): δ1.12(s,9H), 2.60-2.67(s,3H), 3.54-3.61(m,2H), 4.09-4.14(m,1H), 4.21-4.28(m,3H), 7.42-7.49(m,2H),7.65-7.71(d, J=7.2Hz,1H),7.78-7.82(t,1H), 8.01-8.06(m,3H), 8.27-8.29(m,1H),12.72(1H).

[0239] Application Example 1: Synthesis of Leuprorelin

[0240] Peptide sequence: pGlu 1 -His 2 -Trp 3 -Ser 4 -Tyr 5 -DLeu 6 -Leu 7 -Arg 8-Pro 9 -NHEt

[0241] Prepared using a method similar to Example 14 of Patent CN105330726A.

[0242] The specific preparation process is as follows:

[0243]

[0244] The Fmoc amino acids used in patent CN105330726A are replaced with the following Amoc: Amoc-O2-His 2 (Trt)-OH, Amoc-O2-Trp 3 (Boc)-OH, Amoc-O3- Ser 4 (tBu)-OH, Amoc-O2-Tyr 5 (tBu)-OH, Amoc-02-DLeu 6 -OH, Fmoc-Leu 7 -OH、Amoc-02- Pro 9 -OH. (Note: The superscript number of an amino acid represents the position of the amino acid in the polypeptide sequence, and this will be followed below.)

[0245] 1. Synthesis of Fmoc-Leu-Arg(Pbf)-OH

[0246] The preparation of Fmoc-Leu-Arg(pbf)-OH was carried out exactly according to Example 14 of CN105330726A. 65.3g of product was obtained.

[0247] 2. Preparation of H-Pro-CTC Resin

[0248] 68.3 g (180 mmol) of Amoc-02-Pro-OH, 14.2 g (180 mmol) of pyridine, and 50 g of DCM-impregnated CTC resin (degree of substitution: 1.2 mmol / g) were mixed in 1000 mL of DMF and stirred at room temperature for 4 hours. The solution was dried under vacuum, and blocking reagent (anhydrous methanol / pyridine / DCM = 1 / 2 / 17 (volume ratio) was added for two blocking cycles of 400 mL each, 10 minutes each time. The resulting resin was washed with DCM (600 mL × 3 times). 800 mL of 1% piperidine DCM solution was added to the above resin, stirred for 20 minutes, filtered, and the resulting resin was washed with DCM (600 mL × 3 times) to obtain H-Pro-CTC Resin with a degree of substitution of 0.74 mmol / g.

[0249] 3. Synthesis of H-Leu-Arg(Pbf)-Pro-CTC Resin

[0250] 22.86 g (30 mmol) of Fmoc-Leu-Arg(pbf)-OH, 10.24 g (27 mmol) of HBTU, and 4.05 g (30 mmol) of HOBt were dissolved in 80 mL of DMF and stirred. The mixture was then placed in an ice bath for 5 minutes. 7.8 mL (45 mmol) of DIEA was added, and the mixture was activated in an ice bath for 15 minutes. The mixture was then added to a peptide reactor containing 13.5 g (10 mmol) of H-Pro-CTC Resin. The reactor was stirred under nitrogen for 2 hours, and the reaction mixture was dried under vacuum. The peptide resin was washed three times with 100 mL of DMF for 1 minute each time. Then, 20% piperidine / DMF solution was added to the resulting peptide resin to remove Fmoc protection twice, for 5+15 minutes each time, using 80 mL of DMF. The resin was washed with DMF (80 mL × 5 times) to obtain H-Leu-Arg(Pbf)-Pro-CTC Resin.

[0251] 4. Synthesis of H-DLeu-Leu-Arg(Pbf)-Pro-CTC Resin

[0252] 11.9 g (30 mmol) of Amoc-02-DLeu-OH, 3.8 g (30 mmol) of DIC, and 4.3 g (30 mol) of Oxyma pure were mixed in 100 ml of cold DMF and stirred for 5 minutes. This mixture was then added to the H-Leu-Arg(Pbf)-Pro-CTC Resin obtained in step 3. After the amino groups on the H-Leu-Arg(Pbf)-Pro-CTC Resin were completely consumed, the mixture was filtered. The resin was mixed with 100 ml of DMF, and 2.6 g (20 mol) of DIEA and 2 g (20 mmol) of acetic anhydride were added for end-capping for 1 hour. The mixture was then filtered and washed with DMF (100 ml × 3 times). 100 ml of 1% piperidine DCM solution was added to the resin, and the mixture was stirred at room temperature for 30 minutes. The resin was dried under vacuum and washed with DMF (100 ml × 3 times). The resulting resin was used directly in the next synthesis step.

[0253] 5. Synthesis of pGlu-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-DLeu-Leu-Arg(Pbf)-Pro-CTCResin (Napeptide-Resin)

[0254] The entire polypeptide resin was prepared by following the feeding molar ratio, coupling, and Amoc removal method in step 4 above, yielding 26.8g of nonapeptide-resin.

[0255] 6. Synthesis of leuprorelin

[0256] The 26.8g of nonapeptide-resin obtained in step 5 was processed completely according to steps 6, 8, and 11 of Example 14 in CN105330726A to obtain 8.4g of crude product with a purity of 86.6%. The HPLC analysis of the crude product is shown in [Figure 1]. Figure 7 Ms. Figure 8 .

[0257] Comparative Example 1: Synthesis of Leuprorelin

[0258] Leuprolide was prepared by strictly repeating steps 1, 2, 3, 6, 8, and 11 of Example 14 in patent CN105330726A, yielding 6.2 g of crude product with a purity of 82.4%. The HPLC analysis of the crude product is shown below. Figure 9 .

[0259] By comparison, the crude leuprolide prepared using Amoc not only had higher purity than the comparative example, but also significantly improved yield and reduced cost due to fewer side reactions and the absence of root impurities. Simultaneously, the amount of piperidine used was reduced by 95%.

[0260] Application Example 2: Synthesis of Bivalirudin

[0261] Polypeptide sequence: D-Phe 1 -Pro 2 -Arg 3 -Pro 4 -Gly 5 -Gly 6 -Gly 7 -Gly 8 -Asn 9 -Gly 10 -Asp 11 -Phe 12 -Glu 13 -Glu 14 -Ile 15 -Pro 16 -Glu 17 -Glu 18 -Tyr 19 -Leu 20 -OH

[0262] The amino acids protected by Amoc are as follows: Amoc-O1-D-Phe 1 -OH、Amoc-02-Pro 2 -OH, Amoc-02-Arg 3 (Pbf)-OH, Amoc-02-Pro 4 -OH, Amoc-03-Gly 5 -OH、Amoc-02-Gly 6 -OH、Amoc-02-Gly7 -OH、Amoc-02-Gly 8 - OH, Amoc-02- Asn 9 (Trt)-OH, Amoc-02-Gly 10 -OH, Amoc-02-Asp 11 (OtBu)-OH, Amoc-O3-Phe 12 - OH, Amoc-02-Glu 13 (OtBu)-OH, Amoc-O2-Glu 14 (OtBu)-OH, Amoc-O2-Ile 15 -OH、Amoc-02- Pro 16 -OH, Amoc-O2-Glu 17 (OtBu)-OH, Amoc-O2-Glu 18 (OtBu)-OH, Amoc-O2-Tyr 19 (tBu)-OH, Amoc-O2-Leu 20 -OH.

[0263] 1. H-Leu-Wang Resin Synthesis

[0264] 13.2 g (33.6 mol) of Amoc-02-Leu-OH, 4.2 g (33.6 mol) of DIC, 4.8 g (33.6 mol) of Oxyma pure, and 20 g of pre-wetted Wang resin (degree of substitution: 0.56 mmol / g) were mixed in 150 ml of DCM and stirred at room temperature for 5 hours. The mixture was filtered, and the resulting resin was washed with DCM (200 ml × 3 times). 150 ml of 1% piperidine DCM solution was added to the resin, stirred for 20 minutes, filtered, and the resulting resin was washed with DCM (200 ml × 3 times) to obtain H-Leu-Wang resin.

[0265] 2. Synthesis of Amoc-02-Tyr(tBu)-Leu-Wang Resin

[0266] 16.8 g (33.6 mmol) of Amoc-02-Tyr(tBu)-OH, 4.2 g (33.6 mmol) of DIC, and 4.8 g (33.6 mol) of Oxyma pure were mixed in 150 ml of DMF and stirred for 5 minutes. Then, all the H-Leu-Wang resin obtained in step 8.1 was added to the reaction mixture. After the H-Leu-Wang resin was completely consumed, the mixture was filtered. The resin was mixed with 150 ml of DMF, and 2.17 g (22.4 mol) of DIEA and 3.42 g (33.6 mmol) of acetic anhydride were added for end-capping for 1 hour. The mixture was then filtered and washed with DMF (200 ml × 3 times). The resulting resin was used directly in the next synthesis step.

[0267] 3. Synthesis of HD-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin (bivalerate resin):

[0268] The entire polypeptide was synthesized following the feeding molar ratio, coupling, and Amoc removal methods described in steps 1 and 2 above.

[0269] 4. Preparation of crude bivalirudin

[0270] 46 g of bivalirudin resin was mixed in 400 mL of peptide cleavage buffer (80% TFA: 5% anethole: 5% phenol: 5% water: 5% TIS) and stirred at room temperature for 3 hours. 1200 mL of cold diethyl ether was added to the reaction solution, precipitating a peptide solid. After multiple centrifugations and washing with methyl ether, the solid was dried under vacuum at room temperature to obtain 16.7 g of crude peptide with a purity of 83.4%. HPLC analysis of the crude product is shown below. Figure 10 Ms. Figure 11 .

[0271] Comparative Example 2: Synthesis of Bivalirudin

[0272] Following the method described in Application Example 2, synthesis began with H-Leu-Wang resin. The molar ratios of Fmoc amino acids, condensing agent, and resin, as well as the weight-to-volume ratio of materials, were consistent with those in Application Example 2. During peptide chain synthesis, the condensing agent combination was changed to HBTU / HOBt / DIEA. Fmoc removal was performed using a 20% piperidine / DMF solution at room temperature for 30 minutes. After processing with the same peptide cleavage method, 14.2 g of crude product was obtained with a purity of 31.2%. HPLC analysis of the crude product is shown below. Figure 12 .

[0273] Comparative analysis revealed that the purity of crude bivalirudin prepared by the Fmoc solid-phase synthesis method was significantly lower than that prepared by the Amoc method. This is likely due to the alternating increase or decrease in the number of Gly molecules under alkaline conditions during the coupling of four consecutive Gly molecules. This conclusion demonstrates that removing Amoc with a low concentration of organic base significantly reduces side reactions.

[0274] Application Example 3: Synthesis of Acetyl Hexapeptide-38

[0275] Peptide sequence: Ac-Ser 1 -Val 2 -Val 3 -Val 4 -Arg 5 -Thr 6 -NH2

[0276] The amino acids protected by Amoc are as follows: Ac-Ser(tBu)-OH, Amoc-O2-Val 2 -OH, Amoc-02-Val 3 -OH, Amoc-03-Val 4 -OH, Amoc-02-Arg 5 (Pbf)-OH, Amoc-O2-Thr 5 (tBu)-OH.

[0277] 1. Synthesis of H-Thr(tBu)-MBHA resin

[0278] 17.9 g (40.8 mmol) of Amoc-02-Thr(tBu)-OH, 5.1 g (40.8 mmol) of DIC, 5.8 g (40.8 mmol) of Oxyma pure, and 20 g of pre-wetted MAHA resin (degree of substitution: 0.68 mmol / g) were mixed in 150 ml of DCM and stirred at room temperature for 3 hours. The mixture was filtered, and the resulting resin was washed with DCM (200 ml × 3 times). 150 ml of 1% piperazine DCM solution was added to the resin, stirred for 20 minutes, filtered, and the resulting resin was washed with DCM (200 ml × 3 times) to obtain H-Thr(tBu)-MBHA resin.

[0279] 2. Synthesis of Amoc-02-Arg(Pbf)-Thr(tBu)-MBHA resin

[0280] 28.1 g (40.8 mmol) of Amoc-02-Arg(Pbf)-OH, 5.1 g (40.8 mmol) of DIC, and 5.8 g (40.8 mmol) of Oxyma pure were mixed in 150 ml of DMF and stirred for 5 minutes. Then, all the H-Thr(tBu)-MBHA resin obtained in step 1 was added to the reaction mixture. After the H-Thr(tBu)-MBHA resin was completely consumed, the mixture was filtered. The resin was mixed with 200 ml of DMF, and 3.5 g (27.2 mmol) of DIEA and 4.2 g (40.8 mmol) of acetic anhydride were added for end-capping for 1 hour. The mixture was filtered and washed with DMF (200 ml × 3 times). The resulting resin was used directly in the next synthesis step.

[0281] 3. Synthesis of Ac-Ser(tBu)-Val-Val-Val-Arg(Pbf)-Thr(tBu)-MBHA resin (acetyl hexapeptide-38 resin)

[0282] The entire polypeptide was synthesized following the feeding molar ratio, coupling, and Amoc removal methods described in steps 1 and 2 above.

[0283] 4. Preparation of crude acetyl hexapeptide-38

[0284] 67g of acetyl hexapeptide-38 resin was mixed in 500mL of peptide cleavage buffer (80% TFA: 5% anethole: 5% phenol: 5% water: 5% TIS) and stirred at room temperature for 3 hours. 1200mL of cold diethyl ether was added to the reaction solution, precipitating a peptide solid. After multiple centrifugations and washing with methyl ether, the solid was dried under vacuum at room temperature to obtain 28.3g of crude peptide with a purity of 90.9%. HPLC analysis of the crude product is shown below. Figure 13 Ms. Figure 14 .

[0285] Application Comparative Example 3: Synthesis of Acetyl Hexapeptide-38

[0286] Following the method described in Application Example 3, synthesis was initiated from H-Thr(tBu)-MBHA resin. The molar ratios of Fmoc amino acids, condensing agent, and resin, as well as the weight-to-volume ratio of materials, were consistent with those in Application Example 3. During peptide chain synthesis, the condensing agent combination was changed to HBTU / HOBt / DIEA. Fmoc removal was performed using a 20% piperidine / DMF solution at room temperature for 30 minutes. After treatment with the same peptide cleavage method, 25.4 g of crude product was obtained with a purity of 90.4%. HPLC analysis of the crude product is shown below. Figure 15 .

[0287] Comparative analysis revealed that using 1% piperazine to remove Amoc avoided high concentrations of piperidine, while also preventing the generation of foul odors during peptide preparation, resulting in a more environmentally friendly working environment.

[0288] Application Example 4: Synthesis of Fmoc-Ala-Tyr(tBu)-Aib-Pro-N-Me-Asp(OtBu)-OH

[0289] Peptide sequence: Fmoc-Ala 1 -Tyr 2 (tBu)-Aib 3 -Pro 4 -N-Me-Asp 5 (OtBu)-OH

[0290] The protected amino acids are as follows: Fmoc-Ala 1 -OH, Amoc-02-Tyr 2 (tBu)-OH, Amoc-02-Aib 3 -OH、Amoc-02-Pro 4 -OH、Amoc-O2- N-Me-Asp 5 (OtBu)-OH.

[0291] 1. Synthesis of HN-Me-Asp(OtBu)-2-Cl resin

[0292] 10.7 g (22.8 mmol) of Amoc-02-N-Me-Asp(OtBu)-OH, 1.8 g (22.8 mmol) of pyridine, and 10 g of pre-wetted 2-Cl resin (degree of substitution: 0.76 mmol / g) were mixed in 100 ml of DCM and stirred at room temperature for 5 hours. The mixture was filtered, and the resulting resin was washed with DCM (200 ml × 3 times). 100 ml of 1% piperidine DCM solution was added to the resin, stirred for 20 minutes, filtered, and the resulting resin was washed with DCM (200 ml × 3 times) to obtain HN-Me-Asp(OtBu)-2-Cl resin.

[0293] 2. Synthesis of Amoc-02-Pro-N-Me-Asp(OtBu)-2-Cl resin

[0294] 8.7 g (22.8 mmol) of Amoc-02-Pro-OH, 2.8 g (22.8 mmol) of DIC, and 3.2 g (22.8 mmol) of Oxyma pure were mixed in 100 ml of cold DMF and stirred for 5 minutes. The resulting reaction solution was then mixed with HN-Me-Asp(OtBu)-2-Cl resin. After the amino groups on the HN-Me-Asp(OtBu)-2-Cl resin were completely consumed, the mixture was filtered. The resin was mixed with 100 ml of DMF, and 1.9 g (15.2 mmol) of DIEA and 1.6 g (15.2 mmol) of acetic anhydride were added for end-capping for 1 hour. The mixture was then filtered and washed with DMF (100 ml × 3 times). The resulting resin was used directly in the next synthesis step.

[0295] 3. Synthesis of Fmoc-Ala-Tyr(tBu)-Aib-Pro-N-Me-Asp(OtBu)-2-Cl resin

[0296] The entire polypeptide was synthesized following the feeding molar ratio, coupling, and Amoc removal methods described in steps 1 and 2 above.

[0297] Note: The Fmoc protective group of the last amino acid does not need to be removed.

[0298] 4. Preparation of Fmoc-Ala-Tyr(tBu)-Aib-Pro-N-Me-Asp(OtBu)-OH

[0299] 17.3 g of the Fmoc-Ala-Tyr(tBu)-Aib-Pro-N-Me-Asp(OtBu)-2-Cl resin obtained in step 3 was mixed with 150 ml of 1% TFA / DCM solution and stirred at room temperature for 3 hours. The resin was removed by filtration, and the filtrate was washed with water (70 ml × 6 times), dried with anhydrous sodium sulfate, and then concentrated by rotary evaporation at 0-5℃ under negative pressure to half the original solution. 200 ml of petroleum ether was added to this concentrate, and a solid precipitated. The solid was filtered, and the filtrate was slurried twice with petroleum ether to obtain 5.6 g of the target product. The purity was 94.3%. The HPLC data of the product are shown below. Figure 16 Mass spectrometry Figure 17 .

[0300] The above embodiments are merely preferred technical solutions of the present invention and should not be regarded as limitations on the present invention. The scope of protection of the present invention should be the technical solution described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A protected amino acid, characterized in that: The general chemical structural formula of the protected amino acid is shown in formula (1): （1） in, R1 and R2 are either H or methyl groups, respectively; R3 represents methyl, ethyl, propyl, isopropyl, or C. 4-30 Any one of straight-chain or branched saturated alkyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclopentyl, cyclopentylmethyl, cyclopentylethyl and cyclopentylpropyl; R represents a hydrogen atom, C represents a hydrogen atom. 1-6 Any one of straight-chain alkyl, isopropyl, isobutyl, tert-butyl, phenyl, benzyl, benzyl containing a substituent, cyclopentylmethyl, and cyclohexylmethyl; wherein the carbon atom adjacent to R is in the S or R configuration or is achiral; The substituent-containing benzyl group is selected from... ; Among them, Rm, Rn, Rp, Rs, and Rt are each independently any one of hydrogen atom, methyl, ethyl, isopropyl, tert-butyl, nitro, cyano, methoxy, tert-butoxy, benzyloxy, chlorine atom, and fluorine atom; except for hydrogen atom, the number of identical substituents is less than 3.

2. The protected amino acid according to claim 1, characterized in that: The C mentioned 1-6 The straight-chain alkyl group is any one of methyl, ethyl, propyl, butyl, pentyl, and hexyl.

3. The protected amino acid according to claim 1, characterized in that: R is also selected from ; in, Ra is selected from tert-butyloxycarbonyl, benzyloxycarbonyl, or methyl; Rb, Rc, Rd, and Re are each independently one of hydrogen, methyl, methoxy, benzyloxy, fluorine, and chlorine atoms; except for hydrogen atoms, the number of identical substituents is less than 3.

4. The protected amino acid according to claim 1, characterized in that: R is also selected from ; Wherein, Rf is any one of methyl, triphenylmethyl, (p-tolyldiphenyl)methyl, acetamidemethyl, p-methoxybenzyl, and p-methylbenzyl; X1 is a sulfur atom or a selenium atom; n1 is either 0 or 1.

5. The protected amino acid according to claim 1, characterized in that: R is also selected from or ; Wherein, Rg is any one of tert-butyl, benzyl, and triphenylmethyl; * indicates that the chiral carbon bonded to the oxygen atom has an S or R configuration.

6. The protected amino acid according to claim 1, characterized in that: R is also selected from ; Wherein, Rh is any one of methyl, benzyl, and tert-butyl; n2 is 0-5.

7. The protected amino acid according to claim 1, characterized in that: R is also selected from ; Wherein, Ri is any one of hydrogen atom, triphenylmethyl and (p-tolyldiphenyl)methyl; n2 is 0-5.

8. The protected amino acid according to claim 1, characterized in that: R is also selected from ; Wherein, Rj is any one of tert-butoxycarbonyl, benzyloxycarbonyl, triphenylmethyl and (p-tolyldiphenyl)methyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylmethylene)-3-methylbutyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylmethylene)ethyl; n3 is 0-4.

9. The protected amino acid according to claim 1, characterized in that: R is also selected from ; Wherein, Rk is any one of tert-butoxycarbonyl, benzyloxycarbonyl, triphenylmethyl and (p-tolyldiphenyl)methyl.

10. The protected amino acid according to claim 1, characterized in that: R is also selected from PBF is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Where n4 is 0-3.

11. The protected amino acid according to any one of claims 1-10, characterized in that: The protective amino acids are selected from: 。 12. The method for preparing the protected amino acid according to any one of claims 1-11, characterized in that: The reaction route is as follows: Where R0 is R, R1, R2 and R3 are as described in any one of claims 1-11.

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