Preparation method of medicinal 6-arm-polyethylene glycol-succinimide glutarate (amido bond)

Abstract

The invention discloses a preparation method of medicinal 6-arm-polyethylene glycol-succinimide glutaric acid ester (amido bond), which comprises the following steps: reacting 6-arm-polyethylene glycol-amino with glutaric anhydride to obtain a reaction solution 1; adding an organic solvent B into the reaction liquid 1, stirring, pulping, filtering and drying to obtain 6-arm-polyethylene glycol-glutaric acid (amido bond); mixing 6-arm-polyethylene glycol-glutaric acid (amido bond) with a condensing agent, dropwise adding organic alkali, and reacting to obtain reaction liquid 2; adding N-hydroxysuccinimide, and reacting to obtain a reaction solution 3; concentrating under reduced pressure, dropwise adding n-hexane into the filtrate to separate out solid, stirring and pulping to obtain pulp; and filtering, washing a filter cake with n-hexane, and drying to obtain the medicinal 6-arm-polyethylene glycol-succinimide glutaric acid ester (amido bond). The method has the advantages of mild reaction conditions, high reaction rate, few byproducts and high yield, the content is more than 99.0%, the acid value is less than 0.10, and the quality indexes all meet the standards of pharmaceutical enterprises.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, and in particular relates to a method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond). Background Technology

[0002] Six-arm polyethylene glycol-succinimide glutarate (amide bond) is a multi-arm polyethylene glycol derivative linked by six polyethylene glycol (PEG) chains, each terminal of which is modified with a succinimide glutarate (SG) active group, exhibiting good biocompatibility and bioactivity. The SG group can react with primary amines (-NH4+) in proteins, peptides, or drug molecules. This compound exhibits specific reactions, forming stable amide bonds. Its multi-arm structure allows it to interact with various molecules and cells in vivo. It can interact with gene molecules, encapsulating them within its structure to protect them from environmental influences, improving pharmacokinetic properties, and enhancing drug stability and efficacy. It can also achieve targeted drug delivery through coupling with drug molecules or targeting ligands. This structure can prolong the circulating half-life of drugs and reduce non-specific protein binding or cell adhesion, thereby improving drug targeting and therapeutic efficacy. Multi-arm polyethylene glycol derivatives are widely used in drug modification, novel biopharmaceuticals, and drug delivery, enabling drugs to have longer effects, longer half-lives, and better efficacy, making them of significant value in drug research and development.

[0003] Pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond), a novel multi-arm polyethylene glycol derivative, is currently lacking literature reports on its production technology. It can be used as a crosslinking agent, solubilizer, and absorption enhancer, improving drug targeting and showing significant market demand in modern pharmaceutical applications, particularly in the development of innovative drug formulations both domestically and internationally. Formulation customers have stringent requirements regarding the excipient's content, acid value, loss on drying, residue on ignition, and residual solvents; even slight deviations in quality control can adversely affect the efficacy of the formulation. Therefore, meticulous control is necessary during synthesis and purification to reduce residual impurities, improve product quality, and meet pharmaceutical requirements.

[0004] Based on the above situation, there is an urgent need for a method to prepare pharmaceutical 6-arm polyethylene glycol-succinimide glutarate (amide bond) that is of high quality, high yield, low cost, environmentally friendly, easy to operate, and suitable for industrial production. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) that is simple to process, safe to operate, low in cost, and whose product quality meets the standards of pharmaceutical enterprises.

[0006] The technical solution of this invention is summarized as follows: 1. A method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond), comprising the following steps: (1) Add 6-arm-polyethylene glycol-amino and glutaric anhydride to a reaction vessel, then add organic solvent A, stir evenly, heat to 65~110℃, react for 6~12 hours to obtain reaction solution 1. The molar ratio of 6-arm-polyethylene glycol-amino to glutaric anhydride is 1:13.8~35.7; the mass ratio of 6-arm-polyethylene glycol-amino to organic solvent A is 1:5.5~9.5; the molecular weight of 6-arm-polyethylene glycol-amino is 2K~40K. (2) The reaction solution 1 was concentrated under reduced pressure to remove organic solvent A, and a solid was obtained. The solid was washed with purified water, organic solvent B was added, and the mixture was stirred and slurried for 55-65 minutes. The mixture was filtered, and the filter cake was dried to obtain 6-arm-polyethylene glycol-glutaric acid (amide bond). The mass of organic solvent B was 0.75-1.5 times that of organic solvent A. (3) Add the 6-arm polyethylene glycol-glutaric acid (amide bond) and condensing agent to a reaction vessel, then add organic solvent C, stir evenly, add organic base dropwise at 0~10℃ with stirring, adjust the temperature to 20~45℃, react for 0.5~2 hours to obtain reaction solution 2; the molar ratio of the 6-arm polyethylene glycol-glutaric acid (amide bond), condensing agent and organic base is 1:8.4~12.6:1~2.6; the mass ratio of the 6-arm polyethylene glycol-glutaric acid (amide bond) to organic solvent C is 1:4~7; (4) Add N-hydroxysuccinimide to reaction solution 2, heat to 40~75℃, react for 2~6 hours to obtain reaction solution 3, wherein the molar number of N-hydroxysuccinimide is 6.6~10.8 times that of the 6-arm-polyethylene glycol-glutaric acid (amide bond); (5) Concentrate reaction solution 3 under reduced pressure to 10%~15% of the volume of reaction solution 3, filter to remove the filter cake, add n-hexane dropwise to the filtrate to precipitate solid, stir and slurry to obtain slurry; (6) Filter the slurry obtained in step (5), wash the filter cake with n-hexane, and dry it to obtain pharmaceutical 6-arm-polyethylene glycol-succinimide glutarate (amide bond).

[0007] Preferably, in step (1), the molar ratio of 6-arm-polyethylene glycol-amino to glutaric anhydride is 1:18.6~24.3.

[0008] Preferably, organic solvent A is tetrahydrofuran, toluene, or xylene.

[0009] Preferably, organic solvent B is ethyl acetate, diethyl ether, or n-hexane.

[0010] Preferably, the molar ratio of the 6-arm polyethylene glycol-glutaric acid (amide bond), condensing agent and organic base is 1:9.6~10.8:1.5~2.

[0011] Preferably, the condensing agent is O-(7-azabenzotriazol-1-yl)-di(dimethylamino)carbomon hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-di(dimethylamino)carbomon hexafluorophosphate (HBTU) or O-(5-chlorobenzotriazol-1-yl)-di(dimethylamino)carbomon hexafluorophosphate (HCTU).

[0012] Preferably, the organic solvent C is dichloromethane, tetrahydrofuran, or acetonitrile.

[0013] Preferably, the organic base is triethylamine or N,N-diisopropylethylamine.

[0014] Preferably, the mass of n-hexane added in step (5) is 2.5 to 4 times the mass of the 6-arm-polyethylene glycol-glutaric acid (amide bond) in step (3).

[0015] Advantages of this invention: 1. The present invention uses a condensing agent, which greatly reduces the activation energy of the acid-amine condensation reaction between the 6-arm polyethylene glycol-glutaric acid (amide bond) and N-hydroxysuccinimide, thereby improving the reaction rate and conversion rate; 2. The present invention uses a pulping method to effectively remove unreacted raw materials, by-products and other impurities from the reaction solution, and the resulting pharmaceutical 6-arm-polyethylene glycol-succinimide glutarate (amide bond) has a content of more than 99.0% and an acid value of less than 0.10. All quality indicators meet the pharmaceutical enterprise standards. 3. The production process used in this invention is simple to operate, the raw materials are readily available, the reaction conditions are mild, the yield is high, the operation is safe, the environment is friendly and low-pollution, and it is suitable for industrial production. Attached Figure Description

[0016] Figure 1 The GPC spectrum of the 6-arm polyethylene glycol-succinimide glutarate (amide bond) is shown.

[0017] Figure 2 The HPLC spectrum of the 6-arm polyethylene glycol-succinimide glutarate (amide bond) is shown. Detailed Implementation

[0018] Preparation method of pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) The synthetic route is as follows:

[0019] Among them: I--6-arm-polyethylene glycol-amino, II--6-arm-polyethylene glycol-glutaric acid (amide bond), III--6-arm-polyethylene glycol-succinimide glutarate (amide bond).

[0020] The present invention will be further described in detail below through embodiments, and should not be construed as limiting the scope of the claims of the present invention.

[0021] Example 1 A method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) includes the following steps: (1) Add 1000 g of 6-arm polyethylene glycol-amino (0.5 mol) with a molecular weight of 2K and glutaric anhydride (787.29 g, 6.90 mol) to the reaction vessel, then add organic solvent A (5500 g of xylene), stir evenly, heat to 110 °C, and react for 6 hours to obtain reaction solution 1; (2) The reaction solution 1 was concentrated under reduced pressure to remove xylene and a solid was obtained. The solid was washed twice with purified water, 5500g each time. 8250g of n-hexane was added, and the mixture was stirred and slurried for 55 minutes. The mixture was filtered, and the filter cake was dried to obtain 1274.65g of 6-arm-polyethylene glycol-glutaric acid (amide bond). (3) Add 6-arm polyethylene glycol-glutaric acid (amide bond) (1274.65g, 0.463mol) and condensing agent O-(benzotriazol-1-yl)-bis(dimethylamino)carbomony hexafluorophosphate (HBTU) (2209.96g, 5.828mol) to a reaction vessel, then add 6373.25g of dichloromethane, stir evenly, and slowly add triethylamine (121.68g, 1.203mol) dropwise at 0℃ with stirring. Adjust the temperature to 20℃ and react for 2 hours to obtain reaction solution 2; (4) Add N-hydroxysuccinimide (511.00g, 4.44mol) to reaction solution 2, heat to 60℃, and react for 3.5 hours to obtain reaction solution 3; (5) Concentrate reaction solution 3 under reduced pressure to 10% of the reaction solution volume, filter to remove the filter cake, add 5099g of n-hexane to the filtrate, precipitate solid, stir and slurry to obtain slurry mixture; (6) Filter the slurry obtained in step (5), wash the filter cake with 637g of n-hexane, dry the filter cake, and obtain pharmaceutical 6-arm-polyethylene glycol-succinimide glutarate (amide bond).

[0022] The GPC and HPLC spectra of the above 6-arm polyethylene glycol-succinimide glutarate (amide bond) are as follows: Figure 1 , Figure 2 As shown.

[0023] Sampling and testing were conducted, and the samples were aliquoted and stored. 1334.94 g of pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) was obtained, with a purification yield of 82.35%. Testing showed that all quality indicators met the pharmaceutical enterprise standards (see Table 1).

[0024] Example 2 A method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) includes the following steps: (1) Add 1000 g of 6-arm polyethylene glycol-amino (0.1 mol) and glutaric anhydride (407.34 g, 3.57 mol) with a molecular weight of 10 K to a reaction vessel, then add organic solvent A (9500 g of tetrahydrofuran), stir evenly, heat to 65 °C, and react for 12 hours to obtain reaction solution 1; (2) The reaction solution 1 was concentrated under reduced pressure to remove tetrahydrofuran and a solid was obtained. The solid was washed twice with purified water, 9500g each time. 7125g of diethyl ether was added, and the mixture was stirred and slurried for 65 minutes. The mixture was filtered, and the filter cake was dried to obtain 993.53g of 6-arm-polyethylene glycol-glutaric acid (amide bond). (3) Add 6-arm polyethylene glycol-glutaric acid (amide bond) (993.53 g, 0.0994 mol) and O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbomony hexafluorophosphate (HATU) (317.33 g, 0.835 mol) to a reaction vessel, then add 6954.71 g of acetonitrile and stir until homogeneous; at 6°C, with stirring, slowly add N,N-diisopropylethylamine (19.26 g, 0.149 mol), adjust the temperature to 40°C, and react for 1 hour to obtain reaction solution 2; (4) Add N-hydroxysuccinimide (123.49g, 1.073mol) to reaction solution 2, heat to 40℃, and react for 6 hours to obtain reaction solution 3; (5) Concentrate reaction solution 3 under reduced pressure to 15% of the volume of reaction solution 3, filter to remove the filter cake, add 2484g of n-hexane to the filtrate, precipitate solid, stir and slurry to obtain slurry; (6) Filter the slurry obtained in step (5), wash the filter cake with 497g of n-hexane, dry the filter cake, and obtain pharmaceutical 6-arm-polyethylene glycol-succinimide glutarate (amide bond).

[0025] Sampling and testing were conducted, and the samples were aliquoted and stored. 921.28 g of pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) was obtained, with a purification yield of 81.95%. Testing showed that all quality indicators met the pharmaceutical enterprise standards (see Table 1).

[0026] Example 3 A method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) includes the following steps: (1) 1000 g of 6-arm polyethylene glycol-amino (0.05 mol) with a molecular weight of 20 K and glutaric anhydride (138.63 g, 1.215 mol) were added to a reaction vessel, and organic solvent A (6000 g of toluene) was added. The mixture was stirred evenly, heated to 100 °C, and reacted for 7 hours to obtain reaction solution 1. (2) The reaction solution 1 was concentrated under reduced pressure to remove toluene and a solid was obtained. The solid was washed twice with purified water, 6000g each time. 6000g of ethyl acetate was added, the mixture was stirred and slurried for 60 minutes, filtered, and the filter cake was dried to obtain 964.22g of 6-arm-polyethylene glycol-glutaric acid (amide bond). (3) Add 6-arm polyethylene glycol-glutaric acid (amide bond) (964.22 g, 0.0482 mol) and O-(5-chlorobenzotriazol-1-yl)-di(dimethylamino)carbomony hexafluorophosphate (HCTU) (215.40 g, 0.521 mol) to a reaction vessel, then add 4821.10 g of tetrahydrofuran, stir evenly, and slowly add N,N-diisopropylethylamine (8.78 g, 0.0868 mol) dropwise at 0 °C with stirring. Adjust the temperature to 45 °C and react for 0.5 hours to obtain reaction solution 2; (4) Add N-hydroxysuccinimide (39.95g, 0.347mol) to the reaction solution obtained in step (3), heat to 65℃, and react for 3 hours to obtain reaction solution 3; (5) Concentrate reaction solution 3 under reduced pressure to 12% of the volume of reaction solution 3, filter to remove the filter cake, add 3375g of n-hexane to the filtrate, precipitate solid, stir and slurry to obtain slurry; (6) Filter the slurry obtained in step (5), wash the filter cake with 338g of n-hexane, and dry the filter cake to obtain pharmaceutical 6-arm-polyethylene glycol-succinimide glutarate (amide bond).

[0027] Sampling and testing were conducted, and the samples were aliquoted and stored. 894.39 g of pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) was obtained, with a purification yield of 84.21%. Testing showed that all quality indicators met the pharmaceutical enterprise standards (see Table 1).

[0028] Example 4 A method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) includes the following steps: (1) 1000 g of 6-arm polyethylene glycol-amino (0.033 mol) with a molecular weight of 30 K and glutaric anhydride (70.74 g, 0.62 mol) were added to a reaction vessel, followed by organic solvent A (6000 g of toluene). The mixture was stirred until homogeneous, heated to 100 °C, and reacted for 7.5 hours to obtain reaction solution 1. (2) The reaction solution 1 was concentrated under reduced pressure to remove toluene and a solid was obtained. The solid was washed twice with purified water, 6000g each time. 6600g of ethyl acetate was added, the mixture was stirred and slurried for 60 minutes, filtered, and the filter cake was dried to obtain 951.80g of 6-arm-polyethylene glycol-glutaric acid (amide bond). (3) Add 6-arm polyethylene glycol-glutaric acid (amide bond) (951.80g, 0.0317mol) and O-(benzotriazol-1-yl)-bis(dimethylamino)carbomony hexafluorophosphate (HBTU) (115.50g, 0.305mol) to a reaction vessel, then add 3807.20g of dichloromethane, stir evenly, and slowly add triethylamine (3.21g, 0.0317mol) dropwise at 5℃ with stirring. Adjust the temperature to 30℃ and react for 1.5 hours to obtain reaction solution 2; (4) Add N-hydroxysuccinimide (24.10 g, 0.209 mol) to the reaction solution obtained in step (3), heat to 50 °C, and react for 4 hours to obtain reaction solution 3; (5) Concentrate reaction solution 3 under reduced pressure to 15% of the volume of reaction solution 3, filter to remove the filter cake, add 2855g of n-hexane to the filtrate, precipitate solid, stir and slurry to obtain slurry; (6) Filter the slurry obtained in step (5), wash the filter cake with 952g of n-hexane, and dry the filter cake to obtain pharmaceutical 6-arm-polyethylene glycol-succinimide glutarate (amide bond).

[0029] Sampling and testing were conducted, and the samples were aliquoted and stored. 873.11 g of pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) was obtained, with a purification yield of 83.84%. Testing showed that all quality indicators met the pharmaceutical enterprise standards (see Table 1).

[0030] Example 5 A method for preparing pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) includes the following steps: (1) 1000 g of 6-arm polyethylene glycol-amino (0.025 mol) with a molecular weight of 40 K and glutaric anhydride (59.90 g, 0.525 mol) were added to the reaction vessel, and organic solvent A (8000 g of tetrahydrofuran) was added. The mixture was stirred evenly, heated to 70 °C, and reacted for 11 hours to obtain reaction solution 1. (2) The reaction solution 1 was concentrated under reduced pressure to remove tetrahydrofuran and a solid was obtained. The solid was washed twice with purified water, 8000g each time. 7200g of n-hexane was added, the mixture was stirred and slurried for 65 minutes, filtered, and the filter cake was dried to obtain 948.24g of 6-arm-polyethylene glycol-glutaric acid (amide bond). (3) 6-arm polyethylene glycol-glutaric acid (amide bond) (948.24 g, 0.024 mol) and O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbomony hexafluorophosphate (HATU) (91.94 g, 0.242 mol) were added to a reaction vessel, and then 5689.44 g of tetrahydrofuran was added. The mixture was stirred until homogeneous. Triethylamine (4.80 g, 0.0474 mol) was slowly added dropwise at 10 °C with stirring. The temperature was adjusted to 35 °C and the reaction was carried out for 1 hour to obtain reaction solution 2. (4) Add N-hydroxysuccinimide (21.28 g, 0.185 mol) to the reaction solution obtained in step (3), heat to 75 °C, and react for 2 hours to obtain reaction solution 3; (5) Concentrate reaction solution 3 under reduced pressure to 12% of the volume of reaction solution 3, filter to remove filter cake, add 3793g of n-hexane to the filtrate to precipitate solid, stir and slurry to obtain slurry; (6) Filter the slurry obtained in step (5), wash the filter cake with 632g of n-hexane, and dry the filter cake to obtain pharmaceutical 6-arm-polyethylene glycol-succinimide glutarate (amide bond).

[0031] Sampling and testing were conducted, and the samples were aliquoted and stored. 871.96 g of pharmaceutical-grade 6-arm polyethylene glycol-succinimide glutarate (amide bond) was obtained, with a purification yield of 84.57%. Testing showed that all quality indicators met the pharmaceutical enterprise standards (see Table 1).

[0032] Table 1. Pharmaceutical enterprise standards and test results for 6-arm polyethylene glycol-succinimide glutarate (amide bond).

[0033]

[0034] The technical solutions disclosed and proposed in this invention can be implemented by those skilled in the art by appropriately modifying the conditions and routes, etc. Although the methods and preparation techniques of this invention have been described through preferred embodiments, those skilled in the art can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit, and scope of this invention to achieve the final preparation technique. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the spirit, scope, and content of this invention.

Claims

1. Process for the preparation of pharmaceutical 6-armed-polyethylene glycol-succinimidyl glutarate (amide bond) characterized in that The method comprises the following steps: (1) adding 6-arm-polyethylene glycol-amino and glutaric anhydride into a reaction container, then adding organic solvent A, stirring uniformly, increasing temperature to 65-110 DEG C, and reacting for 6-12 hours to obtain reaction liquid 1, wherein the molar ratio of 6-arm-polyethylene glycol-amino to glutaric anhydride is 1:13.8-35.7, the mass ratio of 6-arm-polyethylene glycol-amino to organic solvent A is 1:5.5-9.5, and the molecular weight of 6-arm-polyethylene glycol-amino is 2K-40K; (2) removing organic solvent A from reaction liquid 1 by decompression concentration to obtain a solid, washing the solid with purified water, adding organic solvent B, stirring for 55-65 minutes, filtering, and drying the filter cake to obtain 6-arm-polyethylene glycol-glutaric acid (amide bond), wherein the mass of organic solvent B is 0.75-1.5 times of the mass of organic solvent A; (3) adding 6-arm-polyethylene glycol-glutaric acid (amide bond) and condensing agent into a reaction container, then adding organic solvent C, stirring uniformly, adding organic base dropwise under stirring at 0-10 DEG C, adjusting temperature to 20-45 DEG C, and reacting for 0.5-2 hours to obtain reaction liquid 2, wherein the molar ratio of 6-arm-polyethylene glycol-glutaric acid (amide bond), condensing agent and organic base is 1:8.4-12.6:1-2.6, and the mass ratio of 6-arm-polyethylene glycol-glutaric acid (amide bond) to organic solvent C is 1:4-7; (4) adding N-hydroxysuccinimide into reaction liquid 2, increasing temperature to 40-75 DEG C, and reacting for 2-6 hours to obtain reaction liquid 3, wherein the molar number of N-hydroxysuccinimide is 6.6-10.8 times of that of 6-arm-polyethylene glycol-glutaric acid (amide bond); (5) concentrating reaction liquid 3 by decompression to 10%-15% of the volume of reaction liquid 3, removing the filter cake, adding n-hexane dropwise into the filtrate to precipitate a solid, and stirring to obtain a slurry; (6) filtering the slurry obtained in step (5), washing the filter cake with n-hexane, and drying to obtain medicinal 6-arm-polyethylene glycol-succinimide glutarate (amide bond).

2. The method of claim 1 wherein In step (1), the molar ratio of 6-arm-polyethylene glycol-amino to glutaric anhydride is 1:18.6-24.

3.

3. The method of claim 1 wherein The organic solvent A is tetrahydrofuran, toluene or xylene.

4. The method of claim 1 wherein The organic solvent B is ethyl acetate, diethyl ether or n-hexane.

5. The method of claim 1 wherein The molar ratio of 6-arm-polyethylene glycol-glutaric acid (amide bond), condensing agent and organic base is 1:9.6-10.8:1.5-2.

6. The method of claim 1 or 5, wherein The condensing agent is O-(7-azabenzotriazol-1-yl)-di(dimethylamino)carbon hexafluorophosphate, O-(benzotriazol-1-yl)-di(dimethylamino)carbon hexafluorophosphate or O-(5-chlorobenzotriazol-1-yl)-di(dimethylamino)carbon hexafluorophosphate.

7. The method of claim 1 wherein The organic solvent C is dichloromethane, tetrahydrofuran or acetonitrile.

8. The method of claim 1 or 5, wherein The organic base is triethylamine or N,N-diisopropyl ethylamine.

9. The method of claim 1 wherein In step (5), the added mass of n-hexane is 2.5-4 times of the mass of 6-arm-polyethylene glycol-glutaric acid (amide bond) in step (3).

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