A method for preparing oil-soluble nonapeptide-1 and its application

Abstract

This invention belongs to the field of biomedical technology and discloses a method for preparing oil-soluble nonapeptide-1 and its application. This method covalently links a C8–C18 hydrophobic alkyl chain to the Arg guanidine group at the 4th position and / or the Lysε-amino group at the 7th position of nonapeptide-1, obtaining a molecularly oil-soluble derivative that retains tyrosinase inhibitory activity. This invention achieves a transformation from physical solubilization to chemical modification by introducing structurally optimized hydrophobic groups at specific sites in the nonapeptide-1 molecule. The resulting oil-soluble nonapeptide-1 exhibits molecular-level dispersion in oils and is thermodynamically stable, requiring no external carrier or high proportion of excipients, effectively solving the problems of precipitation, inactivation, and deterioration of skin feel caused by interfacial tension in existing technologies. Simultaneously, its tyrosinase inhibitory activity is highly preserved, and it possesses excellent transdermal properties. This technical solution lays a solid foundation for the efficient, stable, and safe application of nonapeptide-1 in high-end oil-based skincare products, demonstrating significant technological advancement and industrialization value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for preparing oil-soluble nonapeptide-1 and its application. Background Technology

[0002] Nonapeptide-1, with the chemical sequence Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2, is a synthetic polypeptide with tyrosinase inhibitory activity. It has demonstrated significant application value in high-end cosmetics and functional skincare products, including skin whitening, pigmentation regulation, and anti-aging. This peptide effectively reduces melanin synthesis and deposition by competitively inhibiting tyrosinase, a key enzyme in the melanin production pathway. Its mechanism of action is well-defined, and it exhibits good biocompatibility, thus being widely regarded as a new generation of safe and highly effective whitening active ingredients.

[0003] However, nonapeptide-1 is a hydrophilic polypeptide molecule with multiple polar amino acid residues (such as Arg and Lys) and an amidated C-terminus in its molecular structure, which results in extremely low solubility in conventional organic solvents or oil systems, severely limiting its direct application in oil-based cosmetic formulations (such as essential oils, creams, liposome delivery systems, etc.).

[0004] Existing technologies typically improve the dispersion stability of nonapeptide-1 in mixed-phase systems by constructing special dosage forms, such as encapsulating nonapeptide-1 in delivery systems such as liposomes, microemulsions, or nanomicelles, and utilizing the amphiphilic structure of the carrier. Alternatively, they may introduce a high proportion of solubilizing excipients, such as polyols (propylene glycol, butanediol), surfactants (Tween series, Span series), or organic acid ester solubilizers into the formulation.

[0005] These methods have alleviated the oil-phase compatibility issues of nonapeptide-1 to some extent and supported its practical application in some commercially available products. Liposome encapsulation protects the peptide chain from degradation, while the interfacial compatibility between the phospholipid bilayer and the oil phase enables sustained release; while excipient solubilization temporarily shields the polar groups of the peptide molecules through hydrogen bonding or microcluster formation, thereby improving its macroscopic dispersibility.

[0006] While this traditional strategy relies on a carrier system to achieve physical isolation, the hydrophilic nature of the nonapeptide-1 molecule itself remains unchanged. Its thermodynamic instability in the oil phase persists, making it highly susceptible to aggregation, precipitation, or conformational changes during long-term storage, which significantly weakens its biological activity. Summary of the Invention

[0007] To achieve the aforementioned objectives, this invention provides a method for preparing oil-soluble nonapeptide-1 and its applications. The method involves introducing structurally optimized hydrophobic modifying groups onto the side chains of specific amino acid residues in the nonapeptide-1 molecule, thereby achieving targeted regulation of its intrinsic hydrophilicity. This results in an oil-soluble nonapeptide-1 derivative that possesses molecular-level solubility, thermodynamic stability, and retains complete tyrosinase inhibitory activity in nonpolar or weakly polar oil media. This method does not rely on external carriers or high-proportion solubilizing excipients, fundamentally resolving the polar-nonpolar interfacial tension contradiction between nonapeptide-1 and the oil phase system, and providing a novel technical pathway for the development of high-load, high-stability oil-based cosmetics and transdermal delivery systems.

[0008] The method for preparing the oil-soluble nonapeptide-1 of the present invention includes the following steps: First, structural analysis is performed on nonapeptide-1 having the sequence Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2 to identify sites in its molecule that can be chemically modified without affecting the function of the core pharmacophore; second, the guanidinyl group of the Arg residue at the 4th position of the N-terminus of the peptide chain and / or the ε-amino group of the Lys residue at the 7th position are selected as modification targets; third, an acylation reaction is used to covalently link a fatty acid derivative having a C8-C18 straight-chain or branched-chain alkyl structure to the nitrogen atom of the target site; finally, the target oil-soluble nonapeptide-1 product is obtained after purification and characterization.

[0009] The fatty acid derivative is selected from at least one of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, or 2-ethylhexanoic acid. The fatty acid derivative participates in the reaction as an activated ester, wherein the activated ester is N-hydroxysuccinimide ester, p-nitrophenol ester, or pentafluorophenyl ester, and the molar ratio of nonapeptide-1 to the activated ester is 1:(1-1.5). The reaction is carried out in anhydrous dimethylformamide or anhydrous dichloromethane solvent, the reaction temperature is controlled at 0-25℃, and the reaction time is 2-24 h. An organic base catalyst is added to the reaction system, wherein the organic base catalyst is N,N-diisopropylethylamine or triethylamine, and its molar amount is 1.0-2.0 times the molar amount of the fatty acid derivative.

[0010] In a preferred embodiment of the present invention, the modification is performed on a single site targeting the ε-amino group of the 7th Lys residue. In this embodiment, N-hydroxysuccinimide palmitate is used as the modifying agent. In anhydrous dimethylformamide, 1.2 molar amounts of N,N-diisopropylethylamine are added at room temperature (20-25°C) and the reaction is carried out for 12 hours. After the reaction is complete, the reaction solution is added dropwise to pre-cooled diethyl ether to precipitate the product. The crude product is collected by centrifugation and then purified by reversed-phase high-performance liquid chromatography. Mobile phase A is a 0.1% aqueous trifluoroacetic acid solution, and mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program is as follows: phase B accounts for 20% at 0 min and 60% at 30 min, with a flow rate of 1.0 mL / min and a detection wavelength of 220 nm. The main peak fraction was collected and lyophilized to obtain a white, oil-soluble nonapeptide-1 powder with the chemical structure Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys(Palm)-Pro-Val-NH2, where Palm represents palmitoyl group (C 15 H 31 CO—).

[0011] In another preferred embodiment of the present invention, the modification is performed simultaneously on both the guanidinyl group of the 4th Arg residue and the ε-amino group of the 7th Lys residue. In this embodiment, the Lys ε-amino group is first modified with N-hydroxysuccinimide 2-ethylhexanoate under the same reaction conditions as above; subsequently, after separating and purifying the single-modified intermediate, the Arg guanidinyl group is modified a second time with pentafluorophenyl myristate. The second modification reaction is carried out in anhydrous dichloromethane, with 1.5 molar amounts of triethylamine added, and reacted at 0°C for 4 h, then raised to room temperature for another 8 h. The final product is purified by RP-HPLC to obtain the dual-modified oil-soluble nonapeptide-1, with the chemical structure Met-Pro-D-Phe-Arg(Myristoyl)-D-Trp-Phe-Lys(2-Ethylhexanoyl)-Pro-Val-NH2, wherein Myristoyl represents myristoyl (C 13 H 27 CO—), 2-Ethylhexanoyl represents 2-ethylhexanoyl (C8H) 15 CO—).

[0012] The key to this invention lies in the synergistic design of modification sites and modifying groups. The tyrosinase inhibitory activity of nonapeptide-1 mainly depends on the spatial conformation of its C-terminal Phe-Lys-Pro-Val-NH2 fragment and D-Trp residues. While the N-terminal Met-Pro-D-Phe-Arg fragment participates in binding, the guanidino group of its Arg residue does not directly participate in the interaction of the catalytic center. Therefore, hydrophobic modification of the side chains of Arg and / or Lys does not disrupt the key hydrogen bond network and hydrophobic interactions with the tyrosinase active pocket. Simultaneously, the selected C8-C18 alkyl chain length was rigorously screened: excessively short carbon chains (e.g., below C4) cannot effectively shield peptide polarity, resulting in limited improvement in oil solubility; excessively long carbon chains (e.g., above C20) may lead to increased molecular rigidity and excessive steric hindrance, thus hindering its binding to the target enzyme. Experiments show that C12-C16 straight-chain alkyl groups or C8 branched alkyl groups achieve the optimal balance between enhanced oil solubility and preservation of biological activity.

[0013] The solubility of the oil-soluble nonapeptide-1 in oils was determined as follows: 10 mg of sample was added to 1 mL of n-octane and shaken at 25 °C for 24 h. The absorbance was measured at 280 nm by UV-Vis spectrophotometry and compared with the standard curve.

[0014] The tyrosinase inhibitory activity of the oil-soluble nonapeptide-1 was verified through MC1R receptor regulation pathway and melanocyte experiments. Using B16F10 mouse melanoma cells as a research model, the melanocortin receptor 1 protein contains seven transmembrane functional domains and belongs to the G protein-coupled receptor superfamily. When MC1R binds to its natural ligand—α-melanocyte-stimulating hormone (α-MSH)—and its antagonist—Agouti signaling protein (ASP, encoded by the Agouti site)—it can increase and decrease the intracellular level of the second messenger—cyclic adenosine monophosphate (cAMP), respectively, thereby regulating the expression and activity of downstream genes and enzymes such as protein kinase A, microphthalmia-related transcription factors, and tyrosinase, ultimately regulating melanin synthesis. The skin-whitening activity was evaluated by measuring the changes in melanin synthesis after the oil-soluble nonapeptide-1 acted on melanocytes. The results showed that the melanin synthesis inhibition rate of the unmodified nonapeptide-1 (10 ppm) was 39.68%; the melanin synthesis inhibition rate of the monopalmitoyl modified product (10 ppm) was greater than 35.26%; and the melanin synthesis inhibition rate of the double-modified product (10 ppm) was greater than 33.35%. The data indicate that the products modified by the method of this invention still maintain a relative melanin synthesis inhibition activity of over 80%, with an activity loss rate of less than 20%, meeting the effectiveness requirements of cosmetic active ingredients.

[0015] This invention also provides the application of the oil-soluble nonapeptide-1 in the preparation of oil-based cosmetics. The oil-based cosmetics include, but are not limited to, essential oils, skin care oils, creams, lotions, lipsticks, sunscreens, and anhydrous gels. In the formulation, the oil-soluble nonapeptide-1 is directly dissolved in the oil phase in a molecularly dispersed state, with an addition amount of 0.001%-5.0% (mass percentage), preferably 0.01%-1.0%. Since there is no need to add liposomes, microemulsions, or high-concentration surfactants, the formulation system is simple and its stability is significantly improved.

[0016] The oil-soluble nonapeptide-1 of this invention can be applied to transdermal delivery systems. Its hydrophobic modification not only enhances its oil solubility but also strengthens its partition coefficient in the stratum corneum lipid bilayer, promoting transmembrane permeation.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention achieves a paradigm shift from physical solubilization to chemical modification by introducing structurally optimized hydrophobic groups at specific sites in the nonapeptide-1 molecule. The resulting oil-soluble nonapeptide-1 exhibits molecular-level dispersion in oils and fats, is thermodynamically stable, and does not require external carriers or high proportions of excipients, effectively solving the problems of precipitation, inactivation, and deterioration of skin feel caused by interfacial tension in existing technologies. Simultaneously, its tyrosinase inhibitory activity is highly preserved, and it possesses excellent transdermal properties. This technical solution achieves molecular-level oil solubility and higher stability through chemical modification. Detailed Implementation

[0018] This invention provides a method for preparing oil-soluble nonapeptide-1 and its application. The core of this method lies in the targeted hydrophobic chemical modification of the side chains of specific amino acid residues in the nonapeptide-1 molecule, fundamentally altering its intrinsic hydrophilicity. This results in an oil-soluble nonapeptide-1 derivative that possesses molecular-level solubility, thermodynamic stability, and retains complete tyrosinase inhibitory activity in nonpolar or weakly polar oil media. This technical solution abandons the traditional physical solubilization path relying on liposome encapsulation or high proportions of solubilizing excipients, directly resolving the inherent polar-nonpolar interfacial tension contradiction between nonapeptide-1 and the oil phase system at the molecular structure level. This provides a new technical foundation for the development of high-load, high-stability oil-based cosmetics and efficient transdermal delivery systems.

[0019] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0020] Example 1: Single-site modification (7th position Lys); Fatty acid: palmitic acid (C16); Activated ester: N-hydroxysuccinimide ester; Reaction temperature: 22℃, reaction time: 12h; Organic base: N,N-diisopropylethylamine (1.2 molar amounts); Anhydrous organic solvent: anhydrous dimethylformamide; Preparation process: Take 0.5 mmol of nonapeptide-1 (sequence: Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2), add 20 mL of anhydrous dimethylformamide, and stir until completely dissolved; Add 0.55 mmol of N-hydroxysuccinimide palmitate and 0.66 mmol of N,N-diisopropylethylamine (1.2 times the molar amount of the activated ester) at a nonapeptide-1 to activated ester molar ratio of 1:1.1. The acylation reaction was carried out by stirring at a constant temperature of 22℃ for 12 hours. After the reaction was completed, the reaction solution was slowly added dropwise to 5 times the volume of diethyl ether pre-cooled to -20°C, allowed to stand for 2 hours to precipitate, and then centrifuged at 8000 rpm for 15 minutes to collect the crude product. Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC): a C18 column (4.6 mm × 250 mm, 5 μm), mobile phase A being 0.1% trifluoroacetic acid aqueous solution, mobile phase B being 0.1% trifluoroacetic acid acetonitrile solution, gradient elution program being 20% ​​of phase B at 0 min and 60% at 30 min, flow rate 1.0 mL / min, and detection wavelength 220 nm. The main peak component was collected and freeze-dried (-50℃, 0.01MPa) for 48 hours to obtain a white powder product.

[0021] Example 2: The fatty acid is octanoic acid (C8), and the rest of the formula and process are the same as in Example 1; Preparation process: Same as in Example 1.

[0022] Example 3: The fatty acid is stearic acid (C18), and the rest of the formula and process are the same as in Example 1; Preparation process: Same as in Example 1.

[0023] Example 4: Two-site modification (Arg at position 4 + Lys at position 7); fatty acid is 2-ethylhexanoic acid (C8) + myristic acid (C14); stepwise reaction, the rest of the formulation and process are the same as in Example 1; Preparation process: Dissolving nonapeptide-1 → 2-ethylhexanoic acid modification at Lys site (add 0.55 mmol of the activated ester at a molar ratio of nonapeptide-1 to N-hydroxysuccinimide ester of 2-ethylhexanoic acid of 1:1.1, then add 0.66 mmol of N,N-diisopropylethylamine, stir at room temperature (22℃) for 12 h) → purification (obtaining a single-modified intermediate) → myristic acid modification at Arg site (take 0.4 mmol of the single-modified intermediate, add 15 mL of anhydrous dichloromethane to dissolve; add 0.48 mmol of the activated ester at a molar ratio of the single-modified intermediate to pentafluorophenyl myristic acid of 1:1.2, then add 0.72 mmol of triethylamine (1.5 times the molar amount of the activated ester); stir at 0℃ for 4 h, then raise the temperature to room temperature (22℃) and continue the reaction for 8 h; wherein, the modification target of the Arg residue is the Nω nitrogen atom in the guanidinyl group, and the modification quantity is 1) → purification again → freeze drying → finished product.

[0024] Example 5: Two-site modification; the fatty acid is 2-ethylhexanoic acid (C8 branched chain) + lauric acid (C12); the rest of the formulation and process are the same as in Example 4; Preparation process: Same as in Example 4.

[0025] Example 6: Reaction temperature 0℃, time 24h, other formulations and processes are the same as in Example 1; Preparation process: Same as in Example 1.

[0026] Example 7: Reaction temperature 25℃, time 8h, other formulations and processes are the same as in Example 1; Preparation process: Same as in Example 1.

[0027] Example 8: The activated ester is pentafluorophenyl ester, and the rest of the formulation and process are the same as in Example 1; Preparation process: Same as in Example 1 (with adjustment of the activated ester type).

[0028] Comparative Example 1: No hydrophobic modification; the remaining purification and testing processes were the same as in Example 1; Preparation process: Direct purification of nonapeptide-1 → freeze drying → finished product.

[0029] Comparative Example 2: The modification site was proline at position 2 (a non-patent-restricted site); the rest of the formulation and process were the same as in Example 1; Preparation process: Same as in Example 1 (modification site adjustment).

[0030] Test method: Oil solubility test: Determine the solubility of n-octane, squalane and other oils by ultraviolet-visible spectrophotometry; observe the molecular dispersion state.

[0031] Bioactivity assays: melanocyte assay was used to determine the melanin synthesis inhibition rate; the 24-hour transdermal dose was determined using the Franz diffusion cell method.

[0032] Stability test: Store at 45℃ and 75% relative humidity for 3 months, and test the activity retention rate; observe the dispersion stability in the formulation.

[0033] The test data comparisons are shown in Table 1 and Table 2.

[0034] Table 1. Comparison of solubility of n-octane, solubility of squalane, and inhibition rate of melanin synthesis. Table 2 Comparison of 24-hour transdermal dose and 3-month activity retention rate Examples 1-8: Oil solubility ≥ 5.0 mg / mL, transdermal absorption ≥ 15 μg / cm³ 2 The results were far superior to those of the comparative example; in the comparative example 1, the unmodified nonapeptide-1 was completely insoluble in the oil phase, and in the comparative example 2, the non-specific site modification led to a significant decrease in activity, confirming that the core process is the key to the synergistic effect of oil solubility and activity.

[0035] Increased fatty acid chain length (Examples 2→1→3) resulted in simultaneous improvement in oil solubility; dual-site modification (Examples 4 and 5) yielded optimal oil solubility and transdermal absorption; high activity retention was maintained in the reaction temperature range of 0-25℃.

[0036] The embodiments can be directly dissolved in oil-based cosmetics without the need for carriers or co-solvents; they have high bioactivity retention and significantly improved transdermal efficiency; they are stable in storage and suitable for long-lasting formulas in high-end skincare products.

[0037] Compared to unmodified nonapeptide-1 (Comparative Example 1), the n-octane solubility of the example was increased by 850 times, and the transdermal absorption was increased by 8 times; compared to non-specific site modification (Comparative Example 2), the melanin synthesis inhibition rate was increased by more than 15%, solving the industry problem that nonapeptide-1 is difficult to dissolve in oil phase and requires a carrier.

[0038] In summary, the method described in this invention, through specific site hydrophobic modification, can achieve a synergistic effect of high oil solubility, high activity, and high stability with different parameter combinations, making it suitable for large-scale application in oil-based cosmetics.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing oil-soluble nonapeptide-1, characterized in that, The method includes the following steps: Structural analysis of nonapeptide-1 with the sequence Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2 revealed that the guanidinyl group at the 4th Arg residue and / or the ε-amino group at the 7th Lys residue are modifiable sites. In an anhydrous organic solvent, at 0-25°C, the nonapeptide-1 is subjected to an acylation reaction with an activated ester of C8-C18 straight-chain or branched fatty acid in the presence of an organic base catalyst for 2-24 h, so that the fatty acid is covalently linked to the nitrogen atom of the modifiable site. After the reaction was completed, the oil-soluble nonapeptide-1 product was obtained by precipitation, centrifugation and reversed-phase high-performance liquid chromatography purification.

2. The method for preparing oil-soluble nonapeptide-1 according to claim 1, characterized in that, The activated ester is N-hydroxysuccinimide ester, p-nitrophenol ester, or pentafluorophenyl ester, and the organic base catalyst is N,N-diisopropylethylamine or triethylamine, the molar amount of which is 1.0-2.0 times the molar amount of the activated fatty acid ester.

3. The method for preparing oil-soluble nonapeptide-1 according to claim 1, characterized in that, The modification is a single-site modification of the ε-amino group of the 7th Lys residue.

4. The method for preparing oil-soluble nonapeptide-1 according to claim 2, characterized in that, The activated fatty acid ester is N-hydroxysuccinimide palmitate, and the reaction is carried out in anhydrous dimethylformamide at 20-25°C for 12 hours. The organic base catalyst is N,N-diisopropylethylamine, and its molar amount is 1.2 times that of N-hydroxysuccinimide palmitate.

5. The method for preparing oil-soluble nonapeptide-1 according to claim 1, characterized in that, The conditions for purification by reversed-phase high-performance liquid chromatography were as follows: C18 column, mobile phase A being 0.1% trifluoroacetic acid aqueous solution, mobile phase B being 0.1% trifluoroacetic acid acetonitrile solution, gradient elution program being 20% ​​of phase B at 0 min and 60% of phase B at 30 min, flow rate 1.0 mL / min, and detection wavelength 220 nm.

6. The method for preparing oil-soluble nonapeptide-1 according to claim 1, characterized in that, The modification involves a dual-site modification of both the guanidino group at the 4th Arg residue and the ε-amino group at the 7th Lys residue.

7. The method for preparing oil-soluble nonapeptide-1 according to claim 6, characterized in that, The two-site modification adopts a stepwise strategy: firstly, the Lysε-amino group is modified with N-hydroxysuccinimide 2-ethylhexanoate in anhydrous dimethylformamide at room temperature; after separation and purification, the Arg guanidine group is modified again with pentafluorophenyl myristate.

8. The method for preparing oil-soluble nonapeptide-1 according to claim 7, characterized in that, The secondary modification was carried out in anhydrous dichloromethane, with 1.5 molar amounts of triethylamine added, and the reaction was carried out at 0°C for 4 hours, then raised to room temperature and continued for 8 hours.

9. The method for preparing oil-soluble nonapeptide-1 according to claim 1, characterized in that, The C8-C18 straight-chain or branched fatty acids are selected from at least one of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, or 2-ethylhexanoic acid.

10. An oil-soluble nonapeptide-1 prepared by the method according to any one of claims 1-9, characterized in that, Its chemical structure is Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys(R 1 )-Pro-Val-NH2 or Met-Pro-D-Phe-Arg(R 2 )-D-Trp-Phe-Lys(R 1 )-Pro-Val-NH2, where R 1 It is a C7-C17 straight-chain or branched alkanoyl group, R 2 C 13 H 27 CO—or C8H 15 CO—.

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