Weight-losing peptide with high activity as well as preparation method and application of weight-losing peptide

Abstract

The invention relates to the technical field of biological peptides, and discloses a weight-losing peptide with high activity as well as a preparation method and application thereof, and the weight-losing peptide is prepared from an Arg34-GLP-1 (7-37) main chain, an acylating agent, an N-acetyl-L-arginine chaotropic regulator, a sodium benzoate interface regulator, a zinc salt masking agent and a reaction solvent. The preparation method comprises the following steps: carrying out in-situ coordination masking on the N-terminal of polypeptide by using zinc ions in a water phase, then feeding a reaction solvent to implement solvent polarity switching so as to construct a homogeneous reaction system, then carrying out high-selectivity acylation reaction, and finally carrying out metal decomplexation and deprotection treatment. According to the invention, the technical problems of serious N-terminal competitive side reaction, easy racemization of histidine and easy aggregation and precipitation of polypeptide in a mixed solvent in the modification process are effectively solved by utilizing the synergistic effect of metal coordination locking and a chaotropic regulator, and the obtained product is high in regioselectivity and complete in biological activity.

Description

Technical Field

[0001] This invention relates to the field of biopeptide technology, specifically to a highly active weight-loss peptide, its preparation method, and its applications. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1) analogs have become important drugs for treating obesity and type 2 diabetes due to their significant blood glucose-lowering and weight-loss effects. To overcome the short half-life of natural GLP-1, long-chain fatty acid side chains are typically modified onto specific lysine residues of the polypeptide backbone to increase its binding affinity to plasma albumin. However, the chemical modification of polypeptide backbones, exemplified by Arg34-GLP-1 (7-37), faces significant synthetic chemical challenges.

[0003] The Arg34-GLP-1(7-37) polypeptide structure contains multiple nucleophilic active sites. The α-amino and imidazole groups of the N-terminal histidine (His7) exhibit similar nucleophilic reactivity to the ε-amino group of the target modification site, lysine. In conventional acylation reaction systems, acylating agents struggle to distinguish these sites, leading to highly competitive acylation reactions at the N-terminus, generating N-terminal monoacylated or N,ε-diacylated impurities. These positional isomers are highly similar in physicochemical properties to the target product, making them extremely difficult to separate using conventional chromatographic methods, severely impacting the yield and purity of the final product. Furthermore, to enhance the nucleophilicity of the lysine side chain, the reaction typically requires alkaline conditions. However, this introduces a new risk: the chiral center of the N-terminal histidine is thermodynamically unstable in alkaline environments, readily undergoing proton extraction leading to configurational inversion—that is, a transformation from the biologically active L-form to the inactive D-form. This racemic side reaction directly results in the loss of drug bioactivity.

[0004] To address the aforementioned regioselectivity issue, existing technologies attempt to utilize transition metal ions (such as zinc ions) to form complexes with N-terminal histidine residues for protection. However, this strategy faces significant solvent compatibility challenges in practical applications. Since the long-chain fatty acid derivatives required for modification are typically highly hydrophobic, a high proportion of organic solvents (such as N-methylpyrrolidone) is necessary for dissolution. However, metal-peptide complexes are hydrophilic salt structures; as the proportion of organic solvent in the system increases, the dielectric constant of the solution decreases, and the metal-peptide complex readily undergoes desolvation, resulting in hydrophobic aggregation or even precipitation. This phase separation not only disrupts the homogeneity of the reaction system and hinders mass transfer but may also lead to the failure of metal ion protection due to precipitation, thereby triggering N-terminal side reactions or physical damage to the peptide backbone. Therefore, maintaining the colloidal stability of the metal-peptide coordination structure and inhibiting N-terminal racemization while ensuring the solubility of the long-chain acylating agent is a critical technical challenge that urgently needs to be addressed in the preparation of such highly active weight-loss peptides. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a highly active weight-loss peptide, its preparation method, and its application. It solves the technical problems of severe competitive side reactions at the N-terminus during peptide side chain modification, easy racemization of histidine, and poor solvent compatibility in the reaction system leading to peptide aggregation and precipitation.

[0006] To achieve the above objectives, the first aspect of the present invention provides a highly active weight-loss peptide, which is prepared from the following components: an Arg34-GLP-1(7-37) polypeptide backbone, an acylating agent, a non-nucleophilic liquid ionization regulator, an interface regulator, a metal masking agent, and a reaction solvent. In this case, based on the molar amount of the Arg34-GLP-1(7-37) polypeptide backbone as 1 part, the amount of the metal masking agent is 1.8 to 2.2 molar parts, and the amount of the acylation agent is 1.15 to 1.3 molar parts; The concentration of the non-nucleophilic liquid ionizer in the reaction composition is 0.3–0.5 mol / L, and the concentration of the interface regulator in the reaction composition is 50–100 mmol / L.

[0007] By employing the above technical solution, this invention constructs a reaction system with specific ionic strength and coordination environment, achieving highly selective modification through the synergistic chemical effect of each component. The specific principle is as follows: The metal masking agent added to the system dissociates into metal ions. These metal ions preferentially form thermodynamically stable chelates with the imidazole group of histidine and the α-amino group at the N-terminus of the peptide, utilizing the principles of coordination chemistry. This in-situ coordination structure masks the N-terminal active site through steric hindrance and reduces the electron cloud density of the α-amino group, thereby significantly reducing its nucleophilic reactivity and blocking the N-terminal side reaction pathway. Strict control of the molar fraction of metal ions is crucial to ensure complete N-terminal coordination while avoiding excessively high concentrations of free metal ions that could trigger peptide salting out or catalytic hydrolysis of active esters.

[0008] Meanwhile, the non-nucleophilic liquid-leaching regulator introduced into the system can alter the microstructure of the solution. Its charged or polar side-chain groups form a solvation layer with the polypeptide backbone, shielding the hydrophobic interactions between polypeptide molecules and preventing aggregation during metal coordination or the addition of organic solvents. The interface regulator is distributed at the microscopic interface between the polypeptide and the solvent, reducing the interfacial tension of the mixed solvent system, assisting the dispersion and mass transfer of long-chain hydrophobic acylating agents in the aqueous phase, and ensuring the reaction proceeds under homogeneous conditions.

[0009] Preferably, the non-nucleophilic liquid ionizer is N-acetyl-L-arginine; the interface regulator is sodium benzoate; the metal masking agent is zinc acetate or zinc acetate dihydrate; and the acylating agent is octadecanoic acid monotert-butyl ester-N-hydroxysuccinimide ester.

[0010] By adopting the above technical solution, N-acetyl-L-arginine provides the necessary ionic strength to stabilize the conformation of the metal complex, and its N-terminal acetylation eliminates its own nucleophilic interference; sodium benzoate improves the reaction rate by solubilizing long-chain fatty acid acylating agents through its amphiphilic structure.

[0011] Preferably, the reaction solvent is a mixture of water and N-methylpyrrolidone, wherein the volume ratio of N-methylpyrrolidone to water is 50:50 to 60:40.

[0012] By employing the above technical solution, the solvent ratio balances the relationship between peptide solubility, metal coordination stability, and acylation agent reactivity. The aqueous phase maintains the coordination balance of metal ions, preventing metal ion detachment due to an excessively high proportion of organic solvent; an appropriate amount of N-methylpyrrolidone fully extends the hydrophobic region of the peptide, fully exposing the Lys side chain reaction sites.

[0013] Secondly, the present invention provides a method for preparing a highly active weight-loss peptide, comprising the following steps: S1. Substrate dissolution and interface regulation: Dissolve the Arg34-GLP-1(7-37) polypeptide backbone in water, add a non-nucleophilic liquid ionizer and an interface regulator, adjust the pH to alkaline, and obtain the precursor solution; S2, In-situ Metal Masking: A metal masking agent is added to the precursor solution, and the reaction forms a metal-peptide complex; S3, Solvent polarity switching: Organic solvent is added to the metal-peptide complex to construct a homogeneous reaction system; S4, Highly selective acylation reaction: An acylation agent is added to the S3 system to carry out the reaction; S5. Quenching and decomplexing: After the reaction is completed, a quenching agent is added to terminate the reaction, and a metal decomplexing agent is added to remove metal ions; S6. Post-processing and deprotection: The product obtained in S5 is filtered, concentrated, and the side chain protecting groups are removed to purify and obtain the target weight loss peptide.

[0014] By employing the above technical solution, this method precisely controls the selectivity of chemical reactions through step-by-step regulation of the reaction microenvironment. The innovative principles of each step are explained below: Steps S1 and S2 employ a strategy of first regulating and then masking. Pre-establishing a buffer environment containing a liquid-dissociating agent in the pure aqueous phase maintains the unfolded conformation of the peptide, facilitating the rapid and accurate binding of subsequently added metal ions to the N-terminal sites to form a protective layer.

[0015] The solvent polarity switching implemented in step S3 solves the problems of poor solubility of metal salts in organic solvents and easy precipitation when directly mixed. After the aqueous phase is protected, the organic solvent is introduced by a feeding method, so that the reaction system smoothly transitions to a polar environment suitable for fatty acid acylation reactions, avoiding peptide denaturation and precipitation caused by sudden changes in solvent polarity.

[0016] Step S4 utilizes the pre-constructed homogeneous system with the N-terminus masked, allowing the acylating agent to undergo nucleophilic substitution reactions primarily with the exposed Lys side chain ε-amino group, thus kinetically and thermodynamically favoring the generation of the target monoacylated product.

[0017] Steps S5 and S6 competitively bind metal ions with strong chelating agents, destroying the metal-peptide complex and reversibly releasing the N-terminal structure of the polypeptide. High-purity products are then obtained through subsequent processing.

[0018] Preferably, in step S1, the concentration of the Arg34-GLP-1(7-37) polypeptide backbone is 10.0 to 15.0 g / L; and the pH range is adjusted to 7.8 to 8.2.

[0019] By adopting the above technical solution, this pH range takes into account both the reactivity (deprotonated state) of the amino group on the lysine side chain and the hydrolytic stability of the acylating agent, while maintaining the weakly alkaline environment required for zinc ion coordination.

[0020] Preferably, in step S2, the molar ratio of zinc ions to peptides in the metal masking agent is 1.8:1 to 2.2:1; the addition method is dropwise addition, followed by stirring at 20 to 25°C for 30 to 45 minutes.

[0021] By adopting the above technical solution, the dropwise addition method can control the local ion concentration and prevent non-specific aggregation caused by local supersaturation; the incubation at specific temperature and time ensures that the metal coordination reaction reaches thermodynamic equilibrium, so that the N-terminus of all polypeptide molecules is effectively masked.

[0022] Preferably, in step S3, the organic solvent is added until the volume ratio of N-methylpyrrolidone to the accumulated aqueous phase in the system reaches 50:50 to 60:40; and the system temperature is controlled at 20 to 25°C during the addition process.

[0023] By adopting the above technical solution, the fed-batch operation causes a gradual change in the polarity of the system, providing space for the conformational adjustment of peptide molecules and maintaining the colloidal stability of the solution.

[0024] Preferably, in step S4, the acylating agent is added dropwise during the reaction, and the pH value is maintained at 7.8-8.2 after dropwise addition, and the reaction time is 45-90 min.

[0025] By adopting the above technical solution, the instantaneous concentration of the acylating agent was controlled by dropwise addition, reducing the probability of non-specific multi-substitution reactions; constant pH operation compensated for the protons generated during the reaction, maintaining a stable reaction rate.

[0026] Preferably, in step S5, the quenching agent is glycine; the metal decomplexing agent is disodium edetate, and the molar ratio of disodium edetate to the added metal masking agent is 1.5:1 to 2.0:1; in step S6, the method for removing the side-chain protecting group is as follows: trifluoroacetic acid is added to the concentrated intermediate solution to make the volume percentage of trifluoroacetic acid reach 90%, and the reaction is carried out at room temperature for 30 minutes to remove the side-chain tert-butyl ester.

[0027] By adopting the above technical solutions, disodium edetate can completely remove zinc ions from peptides using its higher stability constant, eliminating the risk of heavy metal residues; high-concentration trifluoroacetic acid treatment can efficiently remove side-chain acid-sensitive protective groups and release the final active molecules.

[0028] Thirdly, the present invention provides an application of the highly active weight-loss peptide as described in the first aspect above, employing the following technical solution: The use of a highly active weight-loss peptide as described in the first aspect above in the preparation of a medicament for the prevention or treatment of obesity.

[0029] By adopting the above technical solution, the weight-loss peptide prepared by this method has an intact N-terminal structure and specific modification sites, which can maintain excellent receptor binding activity and prolong the in vivo half-life through side chain modification, thus showing good efficacy in weight loss indications.

[0030] This invention provides a highly active weight-loss peptide, its preparation method, and its applications. It possesses the following beneficial effects: 1. This invention utilizes the steric hindrance effect to block the attack of the acylation agent on the N-terminal α-amino group by forming a stable coordination structure between zinc ions and N-terminal histidine. This technical solution makes the acylation reaction highly concentrated on the lysine side chain, significantly reducing the generation of N-terminal monoacylation and N,ε-diaacylation impurities, thereby solving the technical problem of low product purity and low subsequent purification yield caused by poor site selectivity.

[0031] 2. This invention improves the solubility of peptides in organic solvent / water mixtures by introducing the non-nucleophilic liquid-dissociating regulator N-acetyl-L-arginine and the interface regulator sodium benzoate, along with a specific solvent switching strategy. This system disrupts the hydrophobic interactions between peptide molecules, constructing a stable homogeneous reaction environment and effectively avoiding mass transfer obstacles and metal protection failure caused by precipitation.

[0032] 3. This invention restricts the conformational freedom of the histidine side chain by utilizing metal coordination and strictly controls the reaction pH within a mild range of 7.8–8.2. This synergistic effect of coordination locking and pH control maintains the reactivity of lysine while improving the thermodynamic stability of the N-terminal chiral center, effectively inhibiting the conversion of L-histidine to the inactive D-isomer, and ensuring the high biological activity of the final drug. Attached Figure Description

[0033] Figure 1 This is the transmittance change curve of the reaction system during the solvent polarity switching process of the present invention; Figure 2 This is a schematic diagram of the kinetic comparison curves of the acylation reaction mediated by sodium benzoate according to the present invention; Figure 3 The figures are HPLC chromatograms of the reaction solution of the present invention with and without zinc ion masking. Figure (a) is a schematic diagram of the HPLC chromatogram of the reaction solution of Example 1, and Figure (b) is a schematic diagram of the HPLC chromatogram of the reaction solution of Comparative Example 1. Figure 4 The above are comparative diagrams of competing byproducts in the reaction system of the present invention, wherein Figure (a) is a schematic diagram of byproducts in Example 1 and Figure (b) is a schematic diagram of byproducts in Comparative Example 3. Figure 5 This is a radar chart for evaluating the sensitivity and amplification effect of the process parameters of the present invention. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0036] Arg34-GLP-1(7-37) polypeptide is a glucagon-like peptide-1 analog backbone prepared through gene recombination technology, with the molecular formula C7-37.151 H 228 N 42 O 47 The purity is greater than 98.0%.

[0037] Acylating agent: 1-tert-butyl octadecanoate-N-hydroxysuccinimide ester is a fatty acid active ester used to introduce side chains. Its chemical name is 1-tert-butyl-18-(2,5-dioxopyrrolidone-1-yl)octadecanoate, and its molecular formula is C2. 26 H 45 NO6, CAS number 843666-34-2, is a white crystalline powder with a purity greater than 97.0%.

[0038] Non-nucleophilic liquid dissociation regulator: N-acetyl-L-arginine, molecular formula: C8H 16 N4O3, CAS number 155-84-0, purity greater than 99.0%.

[0039] Metal masking agent: zinc acetate dihydrate, CAS number 5970-45-6, zinc content approximately 29.5%, analytical grade.

[0040] Organic reaction solvent: N-methylpyrrolidone (NMP), CAS No. 872-50-4, water content less than 0.1%, chromatographic grade or high-purity industrial grade.

[0041] Metal decomplexing agent: Disodium edetate (EDTA-2Na), CAS No. 6381-92-6, analytical grade.

[0042] Example 1: This embodiment provides a method for preparing a highly active weight-loss peptide, comprising the following steps: (1) Substrate dissolution and interface control: Purified water was added to a stainless steel reactor equipped with an anchor stirrer and jacket temperature control. Stirring was started and the speed was set to 80 rpm. Arg34-GLP-1(7-37) peptide backbone was added to prepare a solution with a peptide concentration of 12.0 g / L. Then, N-acetyl-L-arginine and sodium benzoate were added sequentially to make the molar concentration of N-acetyl-L-arginine 0.4 mol / L and the molar concentration of sodium benzoate 80 mmol / L. The solution was stirred at 20 °C for 20 min until it became clear. The pH was adjusted to 8.0 using 1 M NaOH aqueous solution (NaOH concentration of 1.0 mol / L) to obtain the peptide solution.

[0043] (2) In-situ metal masking: Prepare a 1.0 M zinc acetate dihydrate aqueous solution (concentration 1.0 mol / L), and slowly (dropping rate 0.5 mL / min) add it to the peptide solution in step (1) to control the zinc ion concentration (Zn). 2+The molar ratio of zinc to peptide is 2.0:1. Stir at 20°C for 30 minutes to form a clear, transparent zinc-peptide complex solution.

[0044] (3) Solvent polarity switching: N-methylpyrrolidone (NMP) is added to the reactor at a flow rate of 15.0 mL / min until the volume ratio of NMP to the accumulated aqueous phase in the system (including the purified water added in step (1) and the aqueous solution added in step (2)) reaches 55:45. At this time, the system temperature is controlled at 20℃, the solution remains clear and homogeneous, and no precipitate is formed.

[0045] (4) Highly selective acylation reaction: Octadecyl tert-butyl mono-N-hydroxysuccinimide ester was dissolved in NMP to prepare an acylation agent of 100 mg / mL (the mass-volume ratio of octadecyl tert-butyl mono-N-hydroxysuccinimide ester to NMP was 1 g:10 mL), and added to the reaction vessel. The molar ratio of acylation agent to peptide was controlled at 1.2:1. During the reaction, triethylamine was added by titration (dropping rate of 0.2 mL / min) to maintain the pH at 8.0±0.1, the temperature at 20℃, and the reaction time at 60 min.

[0046] (5) Quenching and decomplexing: After the HPLC control showed that the raw material conversion rate was >98%, glycine aqueous solution (concentration of 1.0 mol / L) was added to bring the final concentration of glycine to 15 mM, and the mixture was stirred for 10 min (80 rpm). Then, disodium edetate (EDTA-2Na) aqueous solution (concentration of 0.5 mol / L) was added, with a molar ratio of EDTA to zinc acetate of 1.8:1. The pH was adjusted to 7.2, and the mixture was stirred for 30 min to perform zinc removal treatment.

[0047] (6) Post-processing and deprotection: The reaction solution from step (5) was filtered through a 0.22 μm filter membrane and then tangentially filtered through a 3.0 kDa ultrafiltration membrane. The solvent was replaced with purified water at a volume of 5. The retentate was concentrated, and trifluoroacetic acid (TFA) was added. The amount added was controlled so that the volume percentage of TFA in the final mixed solution reached 90% (v / v). The mixture was stirred at room temperature for 30 min to remove the side-chain tert-butyl ester protecting group. After neutralization, the reaction solution was loaded onto a C18 reversed-phase high-performance liquid chromatography column and eluted with a gradient of 0.1% TFA / acetonitrile. The target main peak was collected, converted to salt, and lyophilized to obtain the high-purity target product.

[0048] Example 2: This embodiment provides a method for preparing a highly active weight-loss peptide, comprising the following steps: (1) Substrate dissolution and interface control: Purified water was added to a stainless steel reactor equipped with an anchor stirrer and jacket temperature control. Stirring was started and the speed was set to 80 rpm. Arg34-GLP-1(7-37) peptide backbone was added to prepare a solution with a peptide concentration of 10.0 g / L (based on 500 mL of purified water). N-acetyl-L-arginine and sodium benzoate were added sequentially to make the molar concentration of N-acetyl-L-arginine 0.3 mol / L and the molar concentration of sodium benzoate 50 mmol / L. The solution was stirred at 20 °C for 20 min until it became clear. The pH was adjusted to 7.8 using 1 M NaOH aqueous solution to obtain the peptide solution.

[0049] (2) In-situ metal masking: Prepare a 1.0 M zinc acetate dihydrate aqueous solution (concentration 1.0 mol / L), and slowly (dropping rate 0.5 mL / min) add it to the peptide solution in step (1) to control the zinc ion concentration (Zn). 2+ The molar ratio of zinc to peptide is 1.8:1. Stir at 20°C for 30 minutes to form a clear, transparent zinc-peptide complex solution.

[0050] (3) Solvent polarity switching: N-methylpyrrolidone (NMP) is added to the reactor at a flow rate of 12.5 mL / min until the volume ratio of NMP to the accumulated aqueous phase in the system (including the purified water added in step (1) and the aqueous solution added in step (2)) reaches 50:50. At this time, the system temperature is controlled at 20°C and the solution remains clear and homogeneous.

[0051] (4) Highly selective acylation reaction: Octadecyl tert-butyl mono-N-hydroxysuccinimide ester was dissolved in NMP to prepare an acylation agent of 100 mg / mL (mass-volume ratio 1 g: 10 mL), which was then added to the reaction vessel. The molar ratio of the acylation agent to the peptide was controlled at 1.15:1. During the reaction, triethylamine was added by titration (dropping rate of 0.2 mL / min) to maintain the pH at 7.8 ± 0.1, the temperature at 20 °C, and the reaction time at 90 min.

[0052] (5) Quenching and decomplexing: After the HPLC control showed that the raw material conversion rate was qualified, glycine aqueous solution (concentration of 1.0 mol / L) was added to make the final concentration of glycine reach 10 mM, and stirred for 10 min (80 rpm). Then, disodium edetate (EDTA-2Na) aqueous solution (concentration of 0.5 mol / L) was added, and the molar ratio of EDTA to zinc acetate was 1.5:1. The pH was adjusted to 7.0, and the stirring speed was maintained for 30 min to carry out zinc removal treatment.

[0053] (6) Post-processing and deprotection: The reaction solution from step (5) was filtered through a 0.22 μm filter membrane and then tangentially filtered through a 3.0 kDa ultrafiltration membrane. The solvent was replaced with purified water at a volume of 5. The retentate was concentrated, and trifluoroacetic acid (TFA) was added to a final concentration of 90%. The mixture was stirred at room temperature for 30 min to remove the tert-butyl ester protecting group. After neutralization, the reaction solution was loaded onto a C18 reversed-phase high-performance liquid chromatography column and eluted using a gradient elution system of 0.1% TFA / acetonitrile. The target main peak was collected, converted to salt, and lyophilized to obtain the high-purity target product.

[0054] Example 3: This embodiment provides a method for preparing a highly active weight-loss peptide, comprising the following steps: (1) Substrate dissolution and interface control: Purified water was added to a stainless steel reactor equipped with an anchor stirrer and jacket temperature control. Stirring was started and the speed was set to 100 rpm. Arg34-GLP-1(7-37) peptide backbone was added to prepare a solution with a peptide concentration of 15.0 g / L (based on 500 mL of purified water). N-acetyl-L-arginine and sodium benzoate were added sequentially to make the molar concentration of N-acetyl-L-arginine 0.5 mol / L and the molar concentration of sodium benzoate 100 mmol / L. The solution was stirred at 25 °C for 20 min until it became clear. The pH was adjusted to 8.2 using 1 M NaOH aqueous solution to obtain the peptide solution.

[0055] (2) In-situ metal masking: Prepare a 1.0 M zinc acetate dihydrate aqueous solution (concentration 1.0 mol / L), and slowly (dropping rate 0.5 mL / min) add it to the peptide solution in step (1) to control the zinc ion concentration (Zn). 2+ The molar ratio of the molecule to the polypeptide is 2.2:1. Stir at 25°C for 45 minutes.

[0056] (3) Solvent polarity switching: N-methylpyrrolidone (NMP) was added to the reactor at a flow rate of 18.0 mL / min until the volume ratio of NMP to the accumulated aqueous phase in the system (including the purified water added in step (1) and the aqueous solution added in step (2)) reached 60:40. Thanks to the liquid separation effect of the high concentration of N-acetyl-L-arginine, the system remained clear at 25°C.

[0057] (4) Highly selective acylation reaction: Octadecyl tert-butyl mono-N-hydroxysuccinimide ester was dissolved in NMP to prepare an acylation agent of 100 mg / mL (mass-volume ratio 1 g: 10 mL), which was then added to the reaction vessel. The molar ratio of the acylation agent to the peptide was controlled at 1.3:1. During the reaction, triethylamine was added by titration (dropping rate of 0.3 mL / min) to maintain the pH at 8.2 ± 0.1, the temperature at 25 °C, and the reaction time at 45 min.

[0058] (5) Quenching and decomplexing: Add glycine aqueous solution (concentration of 1.0 mol / L) to achieve a final glycine concentration of 20 mM, and stir for 10 min (100 rpm). Then add EDTA-2Na aqueous solution (concentration of 0.5 mol / L), with a molar ratio of EDTA to zinc acetate of 2.0:1. Adjust the pH to 7.5 and maintain stirring speed for 30 min.

[0059] (6) Post-processing and deprotection: The steps are the same as step (6) in Example 1.

[0060] Example 4: This embodiment provides a method for preparing a highly active weight-loss peptide, comprising the following steps: (1) Substrate dissolution and interface control: Purified water was added to a stainless steel reactor equipped with an anchor stirrer and jacket temperature control. Stirring was started and the speed was set to 80 rpm. Arg34-GLP-1(7-37) polypeptide backbone was added to prepare a solution with a peptide concentration of 12.0 g / L (based on 500 mL of purified water). N-acetyl-L-arginine and sodium benzoate were added sequentially to make the molar concentration of N-acetyl-L-arginine 0.4 mol / L and the molar concentration of sodium benzoate 80 mmol / L. The mixture was stirred at 20 °C for 20 min, and the pH was adjusted to 8.0 using 1 M NaOH aqueous solution.

[0061] (2) In-situ metal masking: Prepare a 1.0 M zinc acetate dihydrate aqueous solution (concentration 1.0 mol / L), and slowly (dropping rate 0.5 mL / min) add it to the peptide solution in step (1) to control the zinc ion concentration (Zn). 2+ The molar ratio of the molecule to the polypeptide is 2.0:1. Stir at 20°C for 30 minutes.

[0062] (3) Solvent polarity switching: N-methylpyrrolidone (NMP) is added to the reactor at a flow rate of 15.0 mL / min until the volume ratio of NMP to the accumulated aqueous phase in the system (including the purified water added in step (1) and the aqueous solution added in step (2)) reaches 55:45. At this time, the system temperature is controlled at 20℃ to maintain homogeneity.

[0063] (4) Highly selective acylation reaction: Octadecyl tert-butyl mono-N-hydroxysuccinimide ester was dissolved in NMP to prepare an acylation agent of 100 mg / mL, which was then added to the reaction vessel. The molar ratio of the acylation agent to the peptide was controlled at 1.2:1. During the reaction, triethylamine was added by titration (dropping rate of 0.2 mL / min) to maintain the pH at 8.0 ± 0.1, the temperature at 20 °C, and the reaction time at 60 min.

[0064] (5) Quenching and decomplexing: Add glycine aqueous solution (concentration of 1.0 mol / L) to achieve a final glycine concentration of 15 mM, and stir for 10 min. Then add edetate disodium (EDTA-2Na) aqueous solution (concentration of 0.5 mol / L), with a molar ratio of EDTA to zinc acetate of 1.8:1. Adjust the pH to 7.2 and stir for 30 min.

[0065] (6) Post-processing and deprotection: The steps are the same as step (6) in Example 1.

[0066] Example 5: This embodiment provides a method for preparing a highly active weight-loss peptide, comprising the following steps: (1) Substrate dissolution and interface control: Purified water (8.3L) was added to a 30L stainless steel reactor, and stirring was started at 150 rpm. 100g of Arg34-GLP-1(7-37) polypeptide backbone was added, with an initial peptide concentration of approximately 12.0g / L. N-acetyl-L-arginine (approximately 718g) and sodium benzoate (approximately 96g) were added sequentially to achieve an N-acetyl-L-arginine concentration of 0.4mol / L and a sodium benzoate concentration of 80mmol / L. The mixture was stirred at 20°C until clear, and the pH was adjusted to 8.0 using 4M NaOH aqueous solution.

[0067] (2) In-situ metal masking: Prepare a 1.0M zinc acetate dihydrate aqueous solution and slowly add it to the reactor through a metering pump (flow rate of 20mL / min) to control the molar ratio of zinc ions to peptides to be 2.0:1. Maintain stirring at 20℃ for 40min.

[0068] (3) Solvent polarity switching: Start the precision metering pump to add NMP to the reactor (flow rate of 300 mL / min) until the volume ratio of NMP to the accumulated aqueous phase in the system (including the purified water added in step (1) and the aqueous solution added in step (2)) reaches 55:45. No local precipitation was found during the entire process.

[0069] (4) Highly selective acylation reaction: The calculated amount of octadecanoic acid monotert-butyl ester-N-hydroxysuccinimide ester was dissolved in NMP and then added. The molar ratio of acylation agent to peptide was controlled at 1.2:1. During the reaction, triethylamine was added dropwise through an automatic liquid addition system to maintain the pH at 8.0±0.1, the temperature at 20℃, and the reaction time at 60 min.

[0070] (5) Quenching and decomplexing: After passing the HPLC control, glycine aqueous solution (concentration of 2.0 mol / L) was added for quenching, followed by EDTA-2Na aqueous solution (concentration of 1.0 mol / L) for decomplexing. The dosage was scaled up proportionally, and the operation logic was the same as in Example 1.

[0071] (6) Post-treatment and deprotection: The reaction solution is desalted and replaced by an industrial-grade tangential flow filtration system, followed by TFA deprotection and preparative liquid chromatography purification, and then collected and freeze-dried to obtain the finished product.

[0072] Comparative Example 1: Compared with Example 1, the difference is that in step (2), instead of adding an aqueous solution of zinc acetate dihydrate, an equal volume of purified water is added, and zinc ion complexation is not performed. All other steps are the same.

[0073] Comparative Example 2: Compared with Example 1, the difference is that N-acetyl-L-arginine is not added in step (1), but all other steps are the same.

[0074] Comparative Example 3: Compared with Example 1, the difference is that in step (1), L-arginine (without acetylation capping) of equal molar concentration is added instead of N-acetyl-L-arginine, and the rest are the same.

[0075] Comparative Example 4: Compared with Example 1, the difference is that sodium benzoate is not added in step (1), but the rest are the same.

[0076] Test Example 1: Compatibility and Solution Stability Test of the Reaction System (Turbidity Method) 1. Experimental steps: Following the methods of Example 1 and Comparative Example 2, substrate dissolution and interface regulation as well as in-situ metal masking steps were performed to obtain two sets of initial zinc-peptide complex aqueous solutions.

[0077] Start stirring and maintain a constant temperature of 20°C. Use a precision peristaltic pump to slowly add N-methylpyrrolidone (NMP) to both sets of reactors at a consistent flow rate.

[0078] Using the volume percentage of NMP in the total liquid volume of the system as the x-axis, the flow was paused at nodes of 0%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, and 55%, and 2.0 mL of sample was taken.

[0079] Immediately place the sample in a 1cm quartz cuvette, using a NMP-water mixed solvent of the corresponding ratio as a blank control, and measure the transmittance (T%) at 600nm using a UV-Vis spectrophotometer, while visually observing the appearance of the solution.

[0080] After the test, the sample is poured back into the reactor to continue the subsequent feeding operations until the endpoint ratio is reached.

[0081] 2. Experimental data are shown in Table 1: Table 1: Transmittance variation data of the reaction system under different NMP volume fractions

[0082] 3. Conclusion: According to Table 1 and Figure 1 The data were controllable. In the low organic solvent ratio (0%-20%) stage, the transmittance of Example 1 and Comparative Example 2 showed no significant difference, both remaining above 96%, indicating that the zinc-peptide complex has a certain intrinsic solubility in a water-based system. When the NMP ratio exceeded 30%, the colloidal behavior of the two systems diverged. The transmittance of Comparative Example 2 decreased sharply and non-linearly with increasing NMP ratio, dropping to 44.1% at the 40% NMP node accompanied by flocculent precipitation, and reaching a final transmittance of only 12.4% at 55% NMP. This indicates that the unmodified zinc-peptide complex, in an environment with a decreased dielectric constant, could not maintain its solvation state due to the shielding of surface charge and enhanced hydrophobicity, resulting in irreversible aggregation and precipitation.

[0083] In Example 1, the transmittance remained consistently stable within the range of 99.2%–99.9% across the entire testing range (0%–55% NMP), without any abrupt changes in optical properties. The data demonstrate that the introduced N-acetyl-L-arginine exerted the expected colloidal stabilizing effect. The guanidinium group on the side chain of this regulator, in high-concentration organic phases, disrupts the intermolecular hydrogen bond network and provides cation-π interactions, constructing an artificial solvation layer on the surface of the zinc-peptide complex, effectively resisting the phase separation tendency caused by solvent polarity switching. This homogeneous system with high transmittance ensures sufficient contact between the substrate and reagent in subsequent acylation reactions, forming the physical basis for achieving high conversion rates.

[0084] Test Example 2: Reaction Kinetics and Feed Conversion Monitoring 1. Experimental steps: Following the methods of Example 1 and Comparative Example 4, the pretreatment steps were completed respectively. After the reaction system was in a homogeneous state after the solvent polarity switching was completed, the temperature was adjusted to stabilize at 20°C.

[0085] Add an equal amount of tert-butyl octadecanoate-N-hydroxysuccinimide ester to initiate the acylation reaction, and start timing immediately.

[0086] At 5 min, 15 min, 30 min, 45 min, 60 min and 90 min of reaction, 50 μL of reaction solution was taken from the center of the reaction vessel using a micropipette.

[0087] The sample was quickly injected into a sample vial containing glycine quenching solution, vortexed for 10 seconds to terminate the reaction, and then diluted with acetonitrile to a suitable concentration.

[0088] The samples at each time point were analyzed by C18 reversed-phase high-performance liquid chromatography (HPLC). The peak area of ​​the Arg34-GLP-1 main chain (starting material) and the sum of the peak areas of each acylated product were recorded, and the conversion rate of the starting material was calculated.

[0089] 2. Experimental data are shown in Table 2: Table 2: Data on the change of feed conversion rate over time under different reaction systems

[0090] 3. Conclusion: According to Table 2 and Figure 2 The data show that Example 1 and Comparative Example 4 exhibit significantly different reaction kinetic characteristics. In the initial stage of the reaction (0–15 min), the conversion rate of Example 1 rapidly reached 61.8%, while that of Comparative Example 4 was only 18.7%, indicating that the initial reaction rate of Example 1 was approximately three times that of Comparative Example 4. As the reaction proceeded, the conversion rate of Example 1 reached 96.2% at 45 min, meeting the process endpoint requirement; in contrast, the conversion rate of Comparative Example 4 was less than 50% at the same time point, and even after extending the reaction to 90 min, the conversion rate remained at 74.2%, showing a significant reaction lag.

[0091] The data discrepancies confirm the interfacial regulation mechanism of sodium benzoate in this reaction system. In Comparative Example 4, where sodium benzoate was absent, although the solvent polarity had switched, the lysine (Lys) side chain of Arg34-GLP-1 was still partially buried in the hydrophobic core or affected by the steric hindrance of surrounding residues, making it difficult for the acylating agent to approach the attack site, resulting in a low macroscopic reaction rate. The sodium benzoate introduced in Example 1, acting as a water-soluble growth promoter, accumulated at the peptide-solvent interface, reducing local surface tension and inducing a fine-tuning of the peptide chain conformation, flipping the lysine side chain to the solvent side for exposure. This conformational change lowered the reaction activation energy and significantly increased the effective collision probability of nucleophilic attack, thereby achieving efficient substrate conversion in a shorter time.

[0092] Test Example 3: Product regioselectivity and impurity profile analysis (HPLC method) 1. Experimental steps: Take 5.0 mL of the reaction mixture after quenching and decomplexing in step (5) of Example 1 and Comparative Example 1, and add mobile phase A (0.1% trifluoroacetic acid aqueous solution) to dilute to a peptide concentration of about 1.0 mg / mL.

[0093] The diluted sample was filtered through a 0.22 μm nylon filter membrane and placed in a sample vial.

[0094] Set the parameters for the high performance liquid chromatograph (equipped with a diode array detector): use a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm), column temperature 45 ℃, detection wavelength 214 nm, and flow rate 1.0 mL / min.

[0095] Run a gradient elution program: mobile phase A is 0.1% trifluoroacetic acid / water, and mobile phase B is 0.1% trifluoroacetic acid / acetonitrile; within 0-30 min, phase B linearly increases from 25% to 55%.

[0096] Inject 20 μL of each sample and record the chromatogram. Based on the retention time, identify the main peak (Lys side chain acylated product), the pre-major impurity peak (N-terminal α-amino acylated product), and the post-major impurity peak (diacylated product). Calculate the percentage content of each component using the area normalization method.

[0097] 2. The experimental data are shown in Table 3: Table 3: Comparison of product distribution and key impurity content in the reaction system

[0098] 3. Conclusion: According to Table 3 and Figure 3 The data show a qualitative difference in product distribution between Example 1 and Comparative Example 1. In the system of Example 1 with zinc acetate, the normalized purity of the target product (Lys-acylated) reached 93.4%, while the content of the main competing byproduct N-terminal isomer was only 0.8%, and the diacylation impurity was controlled at 1.7%. This indicates that the acylation reagent accurately attacked the lysine side chain most of the time, achieving a high degree of regioselectivity.

[0099] Conversely, in Comparative Example 1 without added zinc ions, the target product content was only 44.2%, while the N-terminal isomer content was as high as 38.6%, with the two contents being close, indicating that the reaction system lost its ability to recognize the reaction site. Simultaneously, because the N-terminus and the side-chain lysine both participated in the reaction, the content of diacylated impurities surged to 12.9%.

[0100] The data validated the effectiveness of the in-situ thermodynamic masking mechanism of zinc ions. In Example 1, zinc ions formed a stable coordination structure with the histidine imidazole group and α-amino group at the N-terminus of the polypeptide. This chelation effect created significant steric hindrance and reduced the electron cloud density of the N-terminal nitrogen atom, thereby passivating its nucleophilic activity. Even under the highly active acylation reaction conditions in step (4), the locked N-terminus could not participate in the reaction, forcing the acylating agent to selectively react with the only exposed and active lysine side chain. In Comparative Example 1, due to the lack of this masking mechanism, the N-terminal α-amino group maintained high nucleophilicity and competed with the lysine side chain indiscriminately, resulting in a significant decrease in product purity.

[0101] Test Example 4: Investigation of Acylating Agent Utilization Efficiency and Competitive Side Reactions 1. Experimental steps: The reaction systems were established according to the feeding ratios and operating procedures of Example 1 and Comparative Example 3, respectively. The system of Example 1 contained 0.4 mol / L N-acetyl-L-arginine, and the system of Comparative Example 3 contained 0.4 mol / L L-arginine.

[0102] When the reaction reaches the endpoint of 60 minutes, without quenching, 1.0 mL samples are taken directly from each of the two reaction vessels.

[0103] The sample was diluted 100 times with acetonitrile / water (1:1 v / v), filtered through a 0.22 μm microporous membrane, and immediately injected into a liquid chromatography-mass spectrometry (LC-MS) instrument.

[0104] Set the scanning mode at the mass spectrometer to positive ion mode (ESI). + The mass scan range is 100–2000 m / z, with a focus on monitoring the characteristic ion peak at a relative molecular mass of 473.6 Da (corresponding to the byproduct [M+H] generated by the acylation reaction of L-arginine with tert-butyl octadecanoate). + ).

[0105] Chromatograms were recorded at the liquid chromatography stage, and the molar yield of the target peptide was quantitatively calculated using the external standard method. The peak area ratio of the regulator-acylated adduct in the low molecular weight region was also statistically analyzed.

[0106] 2. The experimental data are shown in Table 4: Table 4: Data on the influence of regulator structure on acylation reaction efficiency and competitive consumption

[0107] 3. Conclusion: According to Table 4 and Figure 4 The data show that Example 1 and Comparative Example 3 exhibit diametrically opposed results in terms of target peptide yield and side reaction control. Example 1 achieved a target peptide molar yield of 91.8%, and no adducts combining the regulator and acylating agent were detected in the system, indicating that the acylating agent was almost entirely used in the main reaction. Comparative Example 3, however, only achieved a target yield of 38.4%, while a high concentration (up to 56.7%) of arginine-octadecanoic acid adduct was detected in the low molecular weight region, and an extremely strong m / z signal of 473.6 was detected in the extracted ion chromatogram, confirming the structure of this byproduct.

[0108] The data confirms the crucial role of the non-nucleophilic design of the regulator. While L-arginine used in Comparative Example 3 possessed the same ionization ability and solubilizing effect, its unblocked α-amino group exhibited strong nucleophilicity. In the reaction system, a high concentration (0.4 mol / L) of L-arginine acted as a competitive substrate, competing with a relatively low concentration of peptide (approximately 12 g / L) for the acylation agent. Due to the rapid diffusion rate and absolute concentration advantage of small molecules, a large amount of the acylation agent was ineffectively consumed, resulting in incomplete peptide conversion. The N-acetyl-L-arginine used in Example 1 eliminated the nucleophilic activity of the α-amino group through acetylation capping. While retaining the ionization effect (provided by the side-chain guanidinium group) to stabilize the colloidal system, it remained chemically inert and did not participate in the acylation reaction, thus ensuring efficient utilization of the acylation agent and a high yield of the target product.

[0109] Test Example 5: Evaluation of process robustness and scale-up effect of different embodiments 1. Experimental steps: Collect the final product powder obtained after freeze-drying treatment in step (6) of Examples 1-5.

[0110] Accurately weigh an appropriate amount of each group of samples and dissolve them in ultrapure water to prepare a detection solution with a concentration of 1.0 mg / mL.

[0111] Analysis was performed using a high-resolution reversed-phase high-performance liquid chromatography (RP-HPLC) system. A C18 column (3.0 μm, 4.6 × 150 mm) was used, with a column temperature of 40 °C and a flow rate of 1.0 mL / min. The mobile phase consisted of 0.1% TFA / water (phase A) and 0.1% TFA / acetonitrile (phase B).

[0112] Perform a gradient elution program: 0–40 min, increasing phase B from 30% to 60%. Set the detection wavelength to 214 nm.

[0113] Record the chromatogram and calculate the main peak purity (Area%), maximum single impurity content, N-terminal acylated impurity content, and diacylated impurity content by integration.

[0114] The total molar yield was calculated based on the amount of feed and the final weight of the freeze-dried powder in each embodiment, combined with the purity data.

[0115] 2. Experimental data are shown in Table 5: Table 5: Comparison of Product Quality Attributes (CQA) under Different Process Conditions and Scale-up

[0116] 3. Conclusion: According to Table 5 and Figure 5 The data shows that the preparation process of the present invention exhibits excellent robustness under different operating parameters and production scales.

[0117] First, comparing Examples 1 with Examples 2 and 3, the purity of the main peak remained above 98.4%, and the critical N-terminal acylation impurities were controlled below 0.21%. This indicates that the in-situ thermodynamic masking mechanism of zinc ions has a wide operating window. Even with a reduced solvent ratio (Example 2) or an increased reaction concentration (Example 3), the binding constant between zinc ions and the N-terminus remains sufficiently large to effectively shield N-terminal activity. Although the yield in Example 2 was slightly reduced to 76.9% due to incomplete exposure of the lysine side chain caused by weaker solvent polarity adjustment, it was still within the industrially acceptable range and did not sacrifice selectivity.

[0118] Secondly, the data from Example 4 (purity 99.05%, N-terminal impurities 0.09%) are highly consistent with those from Example 1, demonstrating the versatility of the method. Because GLP-1 peptides exhibit high conservation in their N-terminal sequence (His7) and helical structure, the interface engineering strategy (zinc chelation + sodium benzoate induction) designed based on molecular structural features of this invention can be seamlessly transferred to the modification of similar drugs.

[0119] Finally, the data from Example 5 confirmed the scalability of the process. The peak purity (99.01%) and yield (81.9%) of the pilot batch were almost identical to those of the laboratory-scale test (Example 1). This is mainly attributed to the stable colloidal system constructed by the non-nucleophilic liquid-leaching regulator. In the 30L reactor, the hydrodynamic environment changed significantly compared to the scale-up test, which usually leads to precipitation due to local overconcentration. However, the data showed that N-acetyl-L-arginine effectively maintained the dispersion stability of the Zn-peptide complex in the macroscopic flow field, avoiding side reactions or precipitation encapsulation caused by mass transfer limitations, confirming the potential for direct industrial scale-up of this technical route.

[0120] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly active weight-loss peptide, characterized in that, The weight-loss peptide is prepared from the following components: Arg34-GLP-1(7-37) polypeptide backbone, acylating agent, non-nucleophilic liquid ionization regulator, interface regulator, metal masking agent and reaction solvent; Based on 1 molar amount of the Arg34-GLP-1(7-37) polypeptide backbone, the amount of the metal masking agent is 1.8 to 2.2 molar amounts, and the amount of the acylation agent is 1.15 to 1.3 molar amounts. The concentration of the non-nucleophilic liquid ionizer in the reaction composition is 0.3–0.5 mol / L, and the concentration of the interface regulator in the reaction composition is 50–100 mmol / L.

2. The weight-loss peptide according to claim 1, characterized in that, The non-nucleophilic liquid separation regulator is N-acetyl-L-arginine; The interface regulator is sodium benzoate; The metal masking agent is zinc acetate or zinc acetate dihydrate; The acylating agent is octadecanoic acid monotert-butyl ester-N-hydroxysuccinimide ester.

3. The weight-loss peptide according to claim 1, characterized in that, The reaction solvent is a mixture of water and N-methylpyrrolidone, wherein the volume ratio of N-methylpyrrolidone to water is 50:50 to 60:

40.

4. A method for preparing a highly active weight-loss peptide, characterized in that, The preparation of the active peptide as described in any one of claims 1-3 comprises the following steps: S1. Substrate dissolution and interface regulation: Dissolve the Arg34-GLP-1(7-37) polypeptide backbone in water, add a non-nucleophilic liquid ionizer and an interface regulator, adjust the pH to alkaline, and obtain the precursor solution; S2, In-situ Metal Masking: A metal masking agent is added to the precursor solution, and the reaction forms a metal-peptide complex; S3, Solvent polarity switching: Organic solvent is added to the metal-peptide complex to construct a homogeneous reaction system; S4, Highly selective acylation reaction: An acylation agent is added to the S3 system to carry out the reaction; S5. Quenching and decomplexing: After the reaction is completed, a quenching agent is added to terminate the reaction, and a metal decomplexing agent is added to remove metal ions; S6. Post-processing and deprotection: The product obtained in S5 is filtered, concentrated, and the side chain protecting groups are removed to purify and obtain the target weight loss peptide.

5. The method for preparing the weight-loss peptide according to claim 4, characterized in that, In step S1, the concentration of the Arg34-GLP-1(7-37) polypeptide backbone is 10.0-15.0 g / L; the pH range is adjusted to 7.8-8.

2.

6. The method for preparing the weight-loss peptide according to claim 4, characterized in that, In step S2, the molar ratio of zinc ions to peptides in the metal masking agent is 1.8:1 to 2.2:1; it is added dropwise, and then stirred at 20 to 25°C for 30 to 45 minutes.

7. The method for preparing the weight-loss peptide according to claim 4, characterized in that, In step S3, the organic solvent is added until the volume ratio of N-methylpyrrolidone to the accumulated aqueous phase in the system reaches 50:50 to 60:40; and the system temperature is controlled at 20 to 25°C during the addition process.

8. The method for preparing the weight-loss peptide according to claim 4, characterized in that, In step S4, the acylating agent is added dropwise during the reaction, and the pH value is maintained at 7.8-8.2 after the dropwise addition, and the reaction time is 45-90 min.

9. The method for preparing the weight-loss peptide according to claim 4, characterized in that, In step S5, the quenching agent is glycine; the metal decomplexing agent is disodium edetate, and the molar ratio of disodium edetate to the added metal masking agent is 1.5:1 to 2.0:1; In step S6, the method for removing the side-chain protecting group is as follows: trifluoroacetic acid is added to the concentrated intermediate solution to make the volume percentage of TFA reach 90%, and the reaction is carried out at room temperature for 30 minutes to remove the side-chain tert-butyl ester.

10. The use of a highly active weight-loss peptide as described in any one of claims 1-3 in the preparation of a medicament for the prevention or treatment of obesity.

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