Fmoc-l-orn(boc)-gly-oh and preparation method and application thereof

Abstract

The application provides Fmoc-L-Orn(Boc)-Gly-OH, a preparation method and application thereof. The method comprises the following steps: activating a carboxyl group of N-fluorenylmethyloxy carbonyl-N'-tert-butyloxy carbonyl-L-ornithine Fmoc-L-Orn(Boc)-OH by using a condensing agent, and then condensing with an activating agent to generate an intermediate; condensing glycine H-Gly-OH with the intermediate to form a peptide bond, so as to generate a target product Fmoc-L-Orn(Boc)-Gly-OH. The method provided by the application can effectively prepare a short peptide fragment, does not need column chromatography, can be synthesized on a large scale, and has the advantages of low cost and high purity.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis technology, and in particular to an N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-L-ornithine glycine (Fmoc-L-Orn(Boc)-Gly-OH), its preparation method, and its applications. Background Technology

[0002] Atosiban is a uterine contraction inhibitor with advantages such as good anti-uterine contraction effect, few side effects, and long-term use. Atosiban competes with oxytocin receptors on the uterine smooth muscle cell membrane, thereby inhibiting oxytocin-induced uterine contractions and reducing prostaglandin secretion mediated by cyclic peptide oxytocin, thus achieving the purpose of maintaining pregnancy. It can be used for pregnant women over 18 years of age, with a gestational age of 24-33 weeks, and a normal fetal heart rate.

[0003] Currently, the preparation method for atosiban involves: preparing short peptide fragments of atosiban by sequentially coupling amino acids using peptide synthesis; then, coupling these short peptide fragments to obtain partially or fully protected atosiban peptides; and finally, removing the protecting groups to obtain the atosiban peptide. However, the current method for synthesizing atosiban short peptide fragments uses a solid-phase synthesis method. This involves first attaching Fmoc-Gly-OH to a resin to obtain an amino acid resin, then removing the Fmoc protecting group from the amino acid resin, and then attaching Fmoc-Orn(Boc)-OH to obtain an Fmoc-Orn(Boc)-Gly peptide resin. Finally, removing the resin yields the Fmoc-Orn(Boc)-Gly-OH fragment. While this solid-phase synthesis method offers advantages such as relatively fast synthesis speed and simpler post-processing, it requires expensive resins, a large excess of amino acids, and a large amount of solvent, resulting in high costs for preparing atosiban short peptide fragments. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies of the prior art and provide a Fmoc-L-Orn(Boc)-Gly-OH, its preparation method, and its application.

[0005] The molecular structure of Fmoc-L-Orn(Boc)-Gly-OH is as follows:

[0006]

[0007] A method for preparing Fmoc-L-Orn(Boc)-Gly-OH includes the following steps:

[0008] Step (1): Activate the carboxyl group of N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-L-ornithine Fmoc-L-Orn(Boc)-OH with a condensing agent, and then react with the activator to generate an intermediate;

[0009] Step (2): Glycine H-Gly-OH is used to condense with the intermediate to form a peptide bond and generate the target product Fmoc-L-Orn(Boc)-Gly-OH.

[0010] Furthermore, in the preparation method of Fmoc-L-Orn(Boc)-Gly-OH as described above, step (1) specifically includes the following steps:

[0011] The Fmoc-L-Orn(Boc)-OH and the activator were stirred and dissolved in dichloromethane to obtain mixture I;

[0012] The condensing agent is added dropwise to the mixture I at a temperature below 0°C. After the addition is complete, the mixture is stirred at room temperature to obtain the intermediate.

[0013] Furthermore, in the preparation method of Fmoc-L-Orn(Boc)-Gly-OH as described above, step (2) specifically includes the following steps:

[0014] Add the glycine H-Gly-OH to an alkaline aqueous solution, stir until dissolved, and obtain mixed solution II;

[0015] Two organic solvents were added to the mixed solution II and stirred until dissolved to obtain mixed solution III;

[0016] The intermediate was added dropwise to the mixed solution III to carry out the reaction at a temperature of 20–25°C. The reaction product was then extracted, dried, filtered, and concentrated to obtain the target product Fmoc-L-Orn(Boc)-Gly-OH.

[0017] Further, in the preparation method of Fmoc-L-Orn(Boc)-Gly-OH as described above, the condensing agent includes one of the following: N,N'-diisopropylcarbodiimide DIC, N,N'-dicyclohexylcarbodiimide DCC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC.HCl, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate TBTU, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate HBTU, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate PyBOP, and benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate BOP.

[0018] Furthermore, in the preparation method of Fmoc-L-Orn(Boc)-Gly-OH as described above, the activator is one of N-hydroxy-5-norbornene-2,3-dicarboximide HONB, N-hydroxysuccinimide HOSu, 1-hydroxybenzotriazole HOBt, and 1-hydroxy-7-azabenzotriazole HOAt.

[0019] Furthermore, in the preparation method of Fmoc-L-Orn(Boc)-Gly-OH as described above, the two organic solvents include: organic solvent I and organic solvent II;

[0020] The organic solvent I is one of tetrahydrofuran (THF), acetonitrile (ACN), dioxane, methyl tert-butyl ether (MTBE), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0021] The organic solvent II is one of ethanol (C2H5OH), methanol (CH3OH), isopropanol (IPA), acetone, DMF, and DMSO.

[0022] Furthermore, in the preparation method of Fmoc-L-Orn(Boc)-Gly-OH as described above, the pH of the alkaline aqueous solution is 8-9.

[0023] Application of Fmoc-L-Orn(Boc)-Gly-OH in the preparation of atosiban peptides.

[0024] The method provided by this invention prepares Fmoc-Orn(Boc)-Gly-OH via liquid-phase peptide synthesis, which has the advantages of low cost and high purity of dipeptide fragments. The prepared Fmoc-Orn(Boc)-Gly-OH dipeptide fragments can be directly linked to resin, attaching two amino acids at a time, simplifying the preparation steps of atosiban peptides, reducing raw material consumption, and further lowering the cost of atosiban peptide preparation. Attached Figure Description

[0025] Figure 1 The structural diagram of the target product Fmoc-L-Orn(Boc)-Gly-OH in Example 2 is shown.

[0026] Figure 2 The target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 2 1 H NMR spectrum;

[0027] Figure 3 The target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 2 13 C NMR spectrum;

[0028] Figure 4 The HRMS spectrum of the target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 2;

[0029] Figure 5 The HPLC chromatogram of the target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 4 is shown. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] Example 1:

[0032] (1) Preparation of intermediate N-fluorenemethoxycarbonyl-N'-tert-butoxycarbonyl-L-ornithine succinimide ester Fmoc-L-Orn(Boc)-OSu

[0033]

[0034] In a 500 mL reaction flask, 0.1 mol of N-fluorenylmethoxycarbonyl-N'-tert-butyloxycarbonyl-L-ornithine (Fmoc-L-Orn(Boc)-OH), 0.12 mol of N-hydroxysuccinimide (HOSu), and 300 mL of dichloromethane were added sequentially. The mixture was stirred until dissolved, and the temperature was maintained below 0 °C. While stirring, 0.12 mol of N,N'-diisopropylcarbodiimide (DIC, purchased from Suzhou Haofan Biotechnology Co., Ltd., analytical grade) was added at a rate of 5 drops per second. The reaction was carried out at room temperature. Thin-layer chromatography (TLC) was used to monitor the reaction process, with chloroform:methanol:acetic acid = 10:1:0.1 as the developing solvent. After the reaction was completed (approximately 4 hours), the mixture was filtered, and the filtrate was collected and concentrated to obtain an oily substance, N-fluorenylmethoxycarbonyl-N'-tert-butyloxycarbonyl-L-ornithine succinimide ester Fmoc-L-Orn(Boc)-OSu, for later use.

[0035] (2) Preparation of N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-L-ornithine glycine Fmoc-L-Orn(Boc)-Gly-OH

[0036]

[0037] In a 1000 mL reaction flask, add 0.12 mol glycine (H-Gly-OH), 0.132 mol Na2CO3, and 200 mL H2O sequentially. After stirring until dissolved, add 50 mL tetrahydrofuran (THF) and 100 mL ethanol (C2H5OH) sequentially and stir until homogeneous. Dissolve the oily substance obtained in step (1) in 50 mL THF and add it to the reaction system at a rate of 5 drops per second with stirring. Maintain the pH of the reaction system at 8-9 using Na2CO3 and control the temperature at 20-25 °C. Monitor the reaction process by TLC, with ethyl acetate:petroleum ether as the developing solvent at a ratio of 1:1. After the reaction was complete (3-4 h), impurities were extracted twice with ethyl acetate / petroleum ether (50 mL ethyl acetate / 150 mL petroleum ether each time). The aqueous layer was collected, and the pH of the system was adjusted to 2-4 with concentrated hydrochloric acid. 300 mL ethyl acetate was added to extract the product, and the ethyl acetate layer was collected. The product was washed twice with saturated brine (200 mL each time), and the ethyl acetate layer was collected. The product was dried with 30 g anhydrous sodium sulfate, filtered after 2 h, and the filtrate was collected. The product was concentrated under reduced pressure to 1 / 3 volume, and 400 mL petroleum ether was added. After stirring for 4 h, crystallization failed. 0.12 mol dicyclohexylamine (DCHA) was added and stirred, and a white solid precipitated. The solid was collected by filtration, yielding 48.1 g Fmoc-L-Orn(Boc)-Gly-OH.DCHA, with a yield of 69.6%.

[0038] The high-performance liquid chromatography (HPLC) chromatogram of the target product prepared in this embodiment shows that the purity of the target product is 88.80% and the single largest impurity is 8.13%.

[0039] Example 1 shows that the present invention uses HOSu as an activator to prepare the corresponding active ester Fmoc-L-Orn(Boc)-OSu, thereby obtaining the dipeptide fragment target molecule Fmoc-L-Orn(Boc)-Gly-OH; with reasonable post-processing, the target fragment with high purity can be obtained without column chromatography.

[0040] Liquid-phase synthesis of dipeptides, with a main raw material feed ratio close to 1, significantly reduces costs compared to solid-phase synthesis. This method saves on relatively expensive protective amino acids, eliminates the need for costly peptide resins, and requires fewer washing cycles, resulting in significantly lower costs and less wastewater generation, making it more environmentally friendly. Undoubtedly, the dipeptide fragment synthesis method of this invention has significant advantages over its corresponding solid-phase synthesis method.

[0041] Example 2:

[0042] (1) Preparation of intermediate N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-L-ornithine-5-norbornene-2,3-dicarboximide ester Fmoc-L-Orn(Boc)-ONB

[0043]

[0044] In a 500 mL reaction flask, 0.1 mol Fmoc-L-Orn(Boc)-OH (99.65% purity), 0.12 mol N-hydroxy-5-norbornene-2,3-dicarboximide (HONB, purchased from Suzhou Haofan Biotechnology Co., Ltd., analytical grade), and 200 mL dichloromethane were added sequentially. The mixture was stirred until dissolved, and the temperature was maintained below 0 °C. 0.12 mol DIC was added at a rate of 5 drops per second with stirring. The reaction was carried out at room temperature, and the reaction was monitored by TLC. The developing solvent was chloroform:methanol:acetic acid = 10:1:0.1. After the reaction was complete (approximately 4 hours), the mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the oily intermediate Fmoc-L-Orn(Boc)-ONB for later use.

[0045] (2) Preparation of Fmoc-L-Orn(Boc)-Gly-OH

[0046]

[0047] In a 1000 mL reaction flask, 0.12 mol H-Gly-OH, 0.132 mol Na2CO3, and 200 mL H2O were added sequentially and stirred until dissolved. Then, 50 mL THF and 100 mL C2H5OH were added sequentially and stirred until homogeneous. The oily intermediate Fmoc-L-Orn(Boc)-ONB obtained in step (1) was dissolved in 50 mL THF and added to the reaction system at a rate of 5 drops per second with rapid stirring. The pH of the reaction system was maintained at 8-9 with Na2CO3, and the temperature was controlled at 20-25 °C. The reaction process was monitored by TLC, with ethyl acetate:petroleum ether = 1:1 as the developing solvent. After the reaction was completed (3-4 h), impurities were extracted twice with ethyl acetate / petroleum ether (50 mL ethyl acetate / 150 mL petroleum ether each time). The aqueous layer was collected, and the pH of the system was adjusted to 2-4 with concentrated hydrochloric acid. 300 mL ethyl acetate was added to extract the product, and the ethyl acetate layer was collected. It was washed with saturated brine (300 mL × 2). The ethyl acetate layer was dried with 30 g anhydrous sodium sulfate and filtered after 2 h. The filtrate was collected, concentrated under reduced pressure to 1 / 3 volume, and 400 mL petroleum ether was added. The mixture was stirred and crystallized for 4 h. The solid was collected by filtration, yielding 43.6 g Fmoc-L-Orn(Boc)-Gly-OH, with a yield of 85.3%. The high-performance liquid chromatography (HPLC) chromatogram of the target product prepared in this example showed that the purity of the target product was 99.76%, and the single largest impurity was 0.09%.

[0048] Characterization data of Fmoc-L-Orn(Boc)-Gly-OH:

[0049] 1 H NMR (400MHz, DMSO) δ8.14(t,1H),7.88(d,J=8Hz,2H),7.75-7.72(m,2H),7.49(d,J=8Hz,1H),7.42(t,2H),7.33(t,2H),6.78(t,3H ),4.29-4.23(m,2H),4.22-4.20(m,1H),4.06-4.00(m,1H),3.83-3.70(m,2H),2.95-2.87(m,2H),1.71-1.42(m,4H),1.38(s,9H).

[0050] 13 C NMR (100MHz, DMSO) δ172.76,171.59,156.44,156.08,144.38,144.23,141.17,128.1 0,127.55,125.83,120.57,77.90,66.13,54.56,47.13,41.12,29.76,28.74,26.57.

[0051] HR MS calcd for C 27 H 33 N3O7, [M+Na] + :534.2,found:534.1.

[0052] Figure 1 The structural diagram of the target product Fmoc-L-Orn(Boc)-Gly-OH in Example 2 is shown. Figure 2 The target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 2 1 H NMR spectrum; Figure 3 The target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 2 13 C NMR spectrum; Figure 4 The HRMS spectrum of the target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 2 is shown.

[0053] from Figure 2-4 It can be seen that the target dipeptide fragment 1 H NMR, 13 The C NMR and HR MS spectra are in perfect agreement with its molecular structure, indicating that the method provided in this invention can successfully obtain Fmoc-L-Orn(Boc)-Gly-OH.

[0054] Example 2 shows that using HONB as an activator, the target molecule of Fmoc-L-Orn(Boc)-Gly-OH dipeptide fragment can be prepared efficiently; with reasonable post-processing, the target fragment with extremely high purity can be obtained without column chromatography, which is suitable for large-scale production.

[0055] Comparing Examples 1 and 2, it can be seen that Example 2 uses HONB instead of HOSu to prepare Fmoc-L-Orn(Boc)-OSu, resulting in higher reaction yield, improved product purity, and lower single-impurity ratio. Its synthesis results are significantly better than those of Example 1.

[0056] Table 2 shows the purity and yield of the target product obtained by changing the amount of each reactant and the solvent system based on Example 2. Experiment number 1 corresponds to Example 2.

[0057] Table 2. Other synthetic experimental results of Fmoc-L-Orn(Boc)-Gly-OH

[0058]

[0059] The high-performance liquid chromatography (HPLC) chromatograms of the target products corresponding to experiments 1-12 show that although the synthesis conditions of the target dipeptide fragments are not exactly the same, the HPLC purity of the obtained products is very high, with the lowest purity reaching 94.23% and the highest purity reaching 99.77%, which meets the stringent requirements of subsequent synthesis without further purification.

[0060] Furthermore, as can be seen from Table 2, when the molar ratio of the materials Fmoc-L-Orn(Boc)-OH, HONB, DIC, and H-Gly-OH is 1:1.2:1.2:1.2, and the solvent system is THF / C2H5OH / H2O, the product purity and yield are both high, and the cost is the lowest.

[0061] This invention utilizes a mixed reaction system of two miscible water-soluble solvents (THF and C2H5OH) with water to achieve higher product purity. This is because Fmoc-L-Orn(Boc)-ONB dissolves in THF, while Gly-ONa dissolves in water. The addition of ethanol facilitates the contact and reaction between the two reactants. Ethanol plays two roles: firstly, it increases the contact between H-Gly-OH and Fmoc-L-Orn(Boc)-ONB in ​​the organic solvent, thus accelerating the reaction; secondly, ethanol effectively inhibits the decomposition of Fmoc-L-Orn(Boc)-ONB in ​​the alkaline aqueous system, thereby reducing the formation of the impurity Fmoc-L-Orn(Boc)-OH.

[0062] Example 3:

[0063] (1) Preparation of intermediate Fmoc-L-Orn(Boc)-ONB

[0064] In a 1000 mL reaction flask, 0.4 mol Fmoc-L-Orn(Boc)-OH, 0.48 mol HONB, and 800 mL dichloromethane were added sequentially. The mixture was stirred until dissolved, and the temperature was maintained below 0 °C. 0.48 mol DIC was added at a rate of 5 drops per second, and the reaction was stirred at room temperature. The reaction was monitored by TLC, with chloroform:methanol:acetic acid = 10:1:0.1 as the developing solvent. After the reaction was complete (approximately 4 hours), the mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the oily substance Fmoc-L-Orn(Boc)-ONB for later use.

[0065] (2) Preparation of Fmoc-L-Orn(Boc)-Gly-OH

[0066] In a 3000 mL reaction flask, add 0.48 mol H-Gly-OH, 0.53 mol Na2CO3, and 800 mL H2O sequentially, stirring until dissolved. Then, add 200 mL THF and 400 mL C2H5OH sequentially, stirring until homogeneous. Dissolve the oily substance obtained in the previous step in 200 mL THF and add it to the reaction system at a rate of 5 drops per second while stirring rapidly. Maintain the pH of the reaction system at 8–9 using Na2CO3, control the temperature at 20–25 °C, and monitor the reaction process by TLC using ethyl acetate:petroleum ether in a 1:1 ratio as the developing solvent. After the reaction was completed (3-4 h), impurities were extracted twice with ethyl acetate / petroleum ether (200 mL ethyl acetate / 600 mL petroleum ether each time). The aqueous layer was collected, and the pH of the system was adjusted to 2-4 with concentrated hydrochloric acid. 1200 mL ethyl acetate was added to extract the product, and the ethyl acetate layer was collected. The product was washed with saturated brine (1200 mL × 2), and the ethyl acetate layer was collected. The product was dried with 120 g anhydrous sodium sulfate, filtered after 2 h, and the filtrate was collected. The product was concentrated under reduced pressure to 1 / 3 volume, and 1600 mL petroleum ether was added. The product was stirred and crystallized for 4 h. The solid was collected by filtration, yielding 180.0 g Fmoc-L-Orn(Boc)-Gly-OH, with a yield of 88.1%.

[0067] The high-performance liquid chromatography (HPLC) chromatogram of the target product prepared in this embodiment shows that the purity of the target product is 99.76% and the single largest impurity is 0.08%.

[0068] Example 4:

[0069] (1) Preparation of Fmoc-L-Orn(Boc)-ONB

[0070] 1 mol Fmoc-L-Orn(Boc)-OH, 1.2 mol HONB, and 2000 mL dichloromethane were added sequentially to a 3000 mL reaction flask. The mixture was stirred until dissolved, and the temperature was maintained below 0 °C. 1.2 mol DIC was added at a rate of 5 drops per second with stirring, and the reaction was continued at room temperature. The reaction was monitored by TLC, with chloroform:methanol:acetic acid = 10:1:0.1 as the developing solvent. After the reaction was complete (approximately 4 hours), the mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the oily substance Fmoc-L-Orn(Boc)-ONB for later use.

[0071] (2) Preparation of Fmoc-L-Orn(Boc)-Gly-OH

[0072] In a 10000 mL reaction flask, add 1.2 mol H-Gly-OH, 1.32 mol Na2CO3, and 2000 mL H2O sequentially, stirring until dissolved. Then, add 500 mL THF and 1000 mL C2H5OH sequentially, stirring until homogeneous. Dissolve the oily substance obtained in the previous step in 500 mL THF and add it to the reaction system at a rate of 5 drops per second while stirring rapidly. Maintain the pH of the reaction system at 8–9 using Na2CO3 and control the temperature at 20–25 °C. Monitor the reaction process by TLC, using ethyl acetate:petroleum ether in a 1:1 ratio as the developing solvent. After the reaction was complete (3-4 h), impurities were extracted twice with ethyl acetate / petroleum ether (500 mL ethyl acetate / 1500 mL petroleum ether each time). The aqueous layer was collected, and the pH of the system was adjusted to 2-4 with concentrated hydrochloric acid. 3000 mL ethyl acetate was added to extract the product, and the ethyl acetate layer was collected. The product was washed twice with saturated brine (3000 mL each time), and the ethyl acetate layer was collected. The product was dried with 300 g anhydrous sodium sulfate, filtered after 2 h, and the filtrate was collected. The filtrate was concentrated to 1 / 3 volume, and 4000 mL petroleum ether was added. The mixture was stirred and crystallized for 4 h. The solid was collected by filtration, yielding 442.6 g of Fmoc-L-Orn(Boc)-Gly-OH, with a yield of 86.6%.

[0073] Figure 5 The HPLC chromatogram of the target product Fmoc-L-Orn(Boc)-Gly-OH prepared in Example 4 is shown. Figure 5 HPLC showed that the purity of the target product was 99.82%, and the single largest impurity was 0.08%.

[0074] As can be seen from Examples 2-4, the synthesis of Fmoc-L-Orn(Boc)-Gly-OH using the method of the present invention has excellent scale-up effect: the reaction yield is excellent, the product purity is very good, and there is no significant increase in single impurities.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing Fmoc-L-Orn(Boc)-Gly-OH, characterized in that, Includes the following steps: Step (1): Dissolve N-fluorenylmethoxycarbonyl-N'-tert-butoxycarbonyl-L-ornithine Fmoc-L-Orn(Boc)-OH with an activator in dichloromethane to obtain mixture I; A condensing agent was added dropwise to the mixture I at a temperature below 0°C. After the addition was completed, the mixture was stirred at room temperature to obtain the intermediate Fmoc-L-Orn (Boc)-ONB. The activator was N-hydroxy-5-norbornene-2,3-dicarboximide HONB, and the condensing agent was N,N'-diisopropylcarbodiimide DIC. Step (2): Add glycine H-Gly-OH to an alkaline aqueous solution, stir until dissolved, and obtain mixed solution II; Add tetrahydrofuran and ethanol to the mixed solution II and stir until dissolved to obtain mixed solution III; The intermediate was added dropwise to the mixed solution III to carry out the reaction at a temperature of 20–25°C. The reaction product was then extracted, dried, filtered, and concentrated to obtain the target product Fmoc-L-Orn(Boc)-Gly-OH, whose structural formula is: .

2. The preparation method according to claim 1, characterized in that, The pH of the alkaline aqueous solution in step (2) is 8 to 9.

3. The preparation method according to any one of claims 1-2, characterized in that, The materials in steps (1) and (2) include Fmoc-L-Orn(Boc)-OH, activator HONB, condensing agent DIC and H-Gly-OH, with a molar ratio of 1:1.2:1.2:1.

2.

4. The application of the preparation method of Fmoc-L-Orn(Boc)-Gly-OH according to any one of claims 1-3 in the preparation of atosiban polypeptide.

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