A polypeptide compound and a method for preparing the same

Abstract

The application belongs to the field of health food and medicine, and relates to a polypeptide compound (9-fluorenylmethyloxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivative (formula I)) and a preparation method and application thereof in enhancing immunity. The polypeptide compound is 9-fluorenylmethyloxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivative shown in formula I, and the compound can significantly enhance the immunity function.

Description

Technical Field

[0001] This invention belongs to the fields of health food and pharmaceutical technology, and relates to a polypeptide compound (9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivative (Formula I)) and its preparation method and its application in enhancing immunity. Background Technology

[0002] Scientific research has discovered the existence of various physiologically active polypeptides in living organisms, each with different structures and functional activities. Proteins ingested by the human body are primarily digested and absorbed in the form of peptides after enzymatic hydrolysis. Almost all cells are regulated by polypeptides. They possess the dual function of regulating physiological functions and providing nutrition to the body. Various active polypeptides have been isolated from various organisms, including plants and animals, and extensive research has been conducted on their properties, preparation methods, separation and purification methods, identification techniques, functional activities, and applications, achieving certain results. Scientists have discovered that almost all cells are regulated by polypeptides; for example, cell differentiation, neurohormonal transduction regulation, and immune regulation are all closely related to active polypeptides. Polypeptides possess the dual function of regulating physiological functions and providing nutrition to the body. Because polypeptides have physiological activities such as regulating the autonomic nervous system, activating vesicular immune function, improving cardiovascular function, and anti-aging, this provides a theoretical basis for the development of peptide drugs and peptide health foods. Summary of the Invention

[0003] The purpose of this invention is to provide a polypeptide compound (Fmoc-4-tert-butoxy-L-prolyl-N-serine derivative (Formula I)) and its preparation method and its application in enhancing immunity.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A polypeptide compound, wherein the polypeptide compound is a 9-fluorenylmethoxycarbonyl(Fmoc)-4-tert-butoxy-L-prolyl-N-serine derivative as shown in Formula I, in, R1 is selected from H, alkyl, alkoxy, substituted alkyl, alkali metal, alkaline earth metal or transition metal; R2 is selected from H, OH, alkyl, alkoxy, or substituted alkyl.

[0005] Preferably, in the general formula of the compound, R1 is selected from H, C1-C5 alkyl, C1-C5 alkoxy, alkali metal or alkaline earth metal; R2 is selected from H, OH, C1-C5 alkoxy, unsubstituted or substituted C1-C5 alkyl groups, wherein the following groups are five- or six-membered rings containing at least one heteroatom, wherein the heteroatom is N or O.

[0006] The alkali metal is Li, Na, or K; the alkaline earth metal is Mg or Ca.

[0007] More preferably, in the general formula of the compound, R1 is selected from H, C1-C5 alkyl or C1-C5 alkoxy; R2 is selected from H, OH, C1-C5 alkoxy, unsubstituted or substituted C1-C5 alkyl groups, wherein the following groups are pentagonal groups containing 1-2 heteroatoms, wherein the heteroatoms are N or O.

[0008] A method for preparing a polypeptide compound, wherein the reaction formula of the compound shown in Formula I is: a: 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline (V) was reacted with compound IV in an organic solvent under the action of an acid-binding agent at -20~100℃ for 1-30 h to prepare III; b: React the reactant (compound III) with compound II in an organic solvent at -20~100℃ for 1-30 h to obtain the compound shown in formula I.

[0009] In step a), the molar ratio of compounds V, IV, and the acid-binding agent is 1:1-1.5:1-1.5, the organic solvent is an aprotic inert solvent, and the acid-binding agent is an organic base or an inorganic base; in the reaction formula, R3 is selected from C1-C4 alkyl or C1-C4 alkoxy.

[0010] The organic solvent is one or more of dichloromethane, dichloroethane, chloroform, tetrahydrofuran, dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, toluene, and chlorobenzene; the acid-binding agent is one or more of triethylamine, N-methylmorpholine, pyridine, sodium carbonate, sodium bicarbonate, and potassium carbonate.

[0011] In step b), the molar ratio of compound III to compound II is 1:1-1.5, and the organic solvent is an aprotic inert solvent.

[0012] The application of a compound, characterized in that: the application of the 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivative represented by general formula I in the preparation of immune-enhancing agents.

[0013] A pharmaceutical composition for enhancing immune function, comprising a 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivative as shown in general formula I of claim 1.

[0014] Advantages of this invention: The 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivative of Formula I obtained in this invention can significantly enhance immune function. In the sheep red blood cell (SRBC) induced DTH (foot thickening method) experiment in mice, compared with the negative control group, the difference in foot thickness in the high-dose group was significant, indicating that the compound of this invention has the function of enhancing immunity. Detailed Implementation

[0015] The following description is merely an overview of the technical method of the present invention. To provide a clearer understanding of the technical means of the present invention and to facilitate its implementation according to the specification, preferred embodiments of the present invention are described in detail below. These non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0016] Example 1: Preparation of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-NL serine methyl group In a 1L four-necked flask equipped with a stirrer and thermometer, add 40.9g (0.1mol) of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline, 400ml of tetrahydrofuran, and 11.1g (0.11mol) of triethylamine. Stir until completely dissolved, cool to below 0°C in an ice-water bath, and add 13.4g (0.11mol) of isopropyl chloroformate dropwise, keeping the temperature below 0°C during the addition. The addition is completed over 30 minutes, and the reaction is maintained at this temperature for 12 hours. Thin-layer chromatography analysis showed no raw material. The developing solvent was ethyl acetate:petroleum ether = 4:1 (v / v). The reaction solution was used directly for the next reaction without further treatment.

[0017] 13.2 g (0.11 mol) of L-serine methyl ester was added in portions to the above reaction solution over 10 minutes, maintaining the solution temperature at 0–5 °C during the addition process. The exothermic reaction was minimal. After addition, the reaction was allowed to proceed at room temperature for 12 hours. Thin-layer chromatography analysis showed no starting material. The developing solvent was ethyl acetate:petroleum ether = 4:1 (v / v). The reaction solution was then transferred to a 1000 ml separatory funnel, and 200 ml of distilled water was added for separation. 200 ml of 5% citric acid was added to the organic phase to separate it. The organic phase was dried over anhydrous sodium sulfate and then dissolved under reduced pressure to obtain 42 g of product, with a yield of 80.72% and a purity of 99.45% according to liquid chromatography.

[0018] 1 HNMR (600MH Z ,CDCl3)δ7.74(d, J =6.23,2H),7.56(t, J =8.32,2H),7.39(t, J=8.21,2H),7.30(t, J =4.34, 2H), 7.15 (D, J =6.34, 2H), 6.97 (d, J =4.16, 1H), 4.58 (m, 1H), 4.43 (m, 1H), 4.39 (m, 1H), 4.04 (m, 1H), 3.85 (m, 1H), 3.78 (s, 3H), 3.35 (m, 1H), 2.31 (m, J =4.56,1H), 2.20 (d, J =3.56,2H), 2.04 (m, J =8.23,1H),1.21(s,9H),1.18(s,1H).

[0019] EI-MS m / z[M+H]511.24.

[0020] Example 2 Preparation of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-NL serine In a 1L four-necked flask equipped with a stirrer and thermometer, add 40.9g (0.1mol) of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline, 400ml of tetrahydrofuran, and 11.1g (0.11mol) of triethylamine. Stir until completely dissolved, cool to below 0°C in an ice-water bath, and add 13.4g (0.11mol) of isopropyl chloroformate dropwise, keeping the temperature below 0°C during the addition. The addition is completed over 30 minutes, and the reaction is maintained at this temperature for 12 hours. Thin-layer chromatography analysis showed no raw material. The developing solvent was ethyl acetate:petroleum ether = 4:1. The reaction solution was used directly for the next reaction without further treatment.

[0021] 11.5 g (0.11 mol) of L-serine was added in portions over 10 minutes to the above reaction solution. The solution temperature was maintained at 0–5 °C during the addition process, and the exothermic reaction was not significant. After the addition was completed, the reaction was allowed to proceed at room temperature for 12 hours. Thin-layer chromatography analysis showed no starting material. The developing solvent was ethyl acetate:petroleum ether = 4:1 (v / v). The reaction solution was then transferred to a 1000 ml separatory funnel, and 200 ml of distilled water was added for separation. 200 ml of 5% citric acid was added to the organic phase to separate it. The organic phase was dried over anhydrous sodium sulfate and then dissolved under reduced pressure to obtain 40.2 g of product, with a yield of 81.04% and a purity of 99.34% according to liquid chromatography.

[0022] 1 HNMR (600MH Z ,CDCl3)δ7.74(d, J =6.23,2H),7.56(t,J =8.32,2H),7.39(t, J =8.21,2H),7.30(t, J =4.34, 2H), 7.15 (D, J =6.34, 2H), 6.97 (d, J =4.16, 1H), 4.58 (m, 1H), 4.43 (m, 1H), 4.39 (m, 1H), 4.04 (m, 1H), 3.85 (m, 1H), 3.35 (m, 1H), 2.31 (m, J =4.56,1H),2.20 (d, J =3.56,2H), 2.04 (m, J =8.23,1H),1.21(s,9H),1.18(s,1H).

[0023] EI-MS m / z[M+H]497.22.

[0024] Example 3 Preparation of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-NL isoleucine In a 1L four-necked flask equipped with a stirrer and thermometer, add 40.9g (0.1mol) of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline, 400ml of tetrahydrofuran, and 11.1g (0.11mol) of triethylamine. Stir until completely dissolved, cool to below 0°C in an ice-water bath, and add 13.4g (0.11mol) of isopropyl chloroformate dropwise, keeping the temperature below 0°C during the addition. The addition is completed over 30 minutes, and the reaction is maintained at this temperature for 12 hours. Thin-layer chromatography analysis showed no raw material. The developing solvent was ethyl acetate:petroleum ether = 4:1 (v / v). The reaction solution was used directly for the next reaction without further treatment.

[0025] 14.41 g (0.11 mol) of L-isoleucine was added in portions over 10 minutes to the above reaction solution. The solution temperature was maintained at 0–5 °C during the addition process, and the exothermic reaction was not significant. After the addition was completed, the reaction was allowed to proceed at room temperature for 12 hours. Thin-layer chromatography analysis showed no starting material. The developing solvent was ethyl acetate:petroleum ether = 4:1 (v / v). The reaction solution was then transferred to a 1000 ml separatory funnel, and 200 ml of distilled water was added for separation. 200 ml of 5% citric acid was added to the organic phase to separate it. The organic phase was dried over anhydrous sodium sulfate and then dissolved under reduced pressure to obtain 38.8 g of product, with a yield of 74.32% and a purity of 99.65% according to liquid chromatography.

[0026] 1 HNMR (600MH Z ,CDCl3)δ7.74(d,J =6.23,2H),7.56(t, J =8.32,2H),7.39(t, J =8.21,2H),7.30(t, J =4.34, 2H), 7.15 (D, J =6.34, 2H), 6.97 (d, J =4.16, 1H), 4.58 (m, 1H), 4.43 (m, 1H), 4.39 (m, 1H), 4.04 (m, 1H), 3.85 (m, 1H), 3.35 (m, 1H), 2.31 (m, J =4.56,1H),2.20 (d, J =3.56,2H), 2.04 (m, J =8.23,1H),1.21(s,9H),1.16(d, J =2.56,3H), 1.11 (t, J =2.25,3H).

[0027] EI-MS m / z[M+H] 523.27.

[0028] Example 4: Preparation of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-NL histidine In a 1L four-necked flask equipped with a stirrer and thermometer, add 40.9g (0.1mol) of 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline, 400ml of tetrahydrofuran, and 11.1g (0.11mol) of triethylamine. Stir until completely dissolved, cool to below 0°C in an ice-water bath, and add 13.4g (0.11mol) of isopropyl chloroformate dropwise, keeping the temperature below 0°C during the addition. The addition is completed over 30 minutes, and the reaction is maintained at this temperature for 12 hours. Thin-layer chromatography analysis showed no raw material. The developing solvent was ethyl acetate:petroleum ether = 4:1 (v / v). The reaction solution was used directly for the next reaction without further treatment.

[0029] 17.05 g (0.11 mol) of L-histidine was added in portions over 10 minutes to the above reaction solution. The solution temperature was maintained at 0-5°C during the addition process, and the exothermic reaction was not significant. After the addition was completed, the reaction was allowed to proceed at room temperature for 12 hours. Thin-layer chromatography analysis showed no starting material. The developing solvent was ethyl acetate:petroleum ether = 5:1 (v / v). The reaction solution was then transferred to a 1000 ml separatory funnel, and 200 ml of distilled water was added for separation. 200 ml of 5% citric acid was added to the organic phase to separate it. The organic phase was dried over anhydrous sodium sulfate and then dissolved under reduced pressure to obtain 37.5 g of product, with a yield of 68.66% and a purity of 99.85% according to liquid chromatography.

[0030] 1 HNMR (600MH Z , CDCl3) δ9.89 (s, 1H), 9.62 (s, 1H), 8.73 (s, 1H), 8.64 (s, 1H) 7.74 (d, J =6.45, 2H), 7.56 (t, J =6.32,2H),7.39(t, J =4.56,2H),7.30(t, J =4.86, 2H), 6.97 (d, J =4, 1H), 4.58 (m, 1H), 4.43 (m, 1H), 4.39 (m, 1H), 4.04 (m, 1H), 3.85 (m, 1H), 3.35 (m, 1H), 2.31 (m, J =4.13,1H),2.04 (m, J =6.34,1H),1.21(s,9H),1.18(s,1H).

[0031] EI-MS m / z[M+H] 547.25.

[0032] Example 5 Pharmacological experiments on 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivatives (1) Route of administration Mice were orally administered Fmoc-4-tert-butoxy-L-prolyl-N-serine derivative powder at a high dose of 10 g / kg BW and a low dose of 1 g / kg BW, once daily for 7 consecutive days. In a sheep erythrocyte (SRBC)-induced DTH (pedicel thickening assay) test in mice, the difference in paw thickness was significantly different in the high-dose group compared to the negative control group.

[0033] (2) Dosage setting and preparation of test substance A. Dosage setting Test substances: Compounds prepared in Examples 1-4, with high-dose and low-dose groups. The high-dose group was 10 g / kg and the low-dose group was 1 g / kg, which were equivalent to 10 times and 1 times the recommended human dose, respectively, administered orally, with a gavage volume of 40 ml / kg.

[0034] Negative control: Purified water, administered orally at a gavage volume of 40 ml / kg.

[0035] B. Preparation of the test substance High dose: Grind the test substance into a fine powder and pass it through a 200-mesh standard sieve. Weigh the test substance and add purified water to prepare a suspension solution of 0.25 mg / ml.

[0036] Low dose: The portion of the test substance that cannot pass through a 200-mesh standard sieve after grinding is added to purified water to prepare a suspension solution of 0.025 mg / ml.

[0037] Negative control: purified water.

[0038] All the test substances were freshly prepared at the time of use.

[0039] (3) Experimental methods Mice were randomly divided into a negative control group, a high-dose test substance group, and a low-dose test substance group according to their body weight. The negative control group was given purified water, while the high- and low-dose groups were orally administered the corresponding doses of the test substance. Administered once daily for 7 consecutive days, followed by the following steps: Sensitization: Mice were immunized intraperitoneally with 2% (v / v) SRBC, with each mouse injected with 0.2 mL (approximately 1 × 10⁻⁶). 8 SRBC.

[0040] DTH production and measurement: Four days after immunization, the thickness of the left hind paw metatarsal region of mice was measured and recorded. Then, 20% (v / v) SRBC was subcutaneously injected into the measurement site, 20 μL per mouse (approximately 1 × 10⁻⁶). 8 (1 SRBC), and the thickness of the left hind foot metatarsal region was measured again 24 hours after injection. The same site was measured three times, and the average value was taken.

[0041] The degree of DTH was represented by the difference in plantar thickness before and after the attack. The difference in the test group was significantly higher than that in the control group, indicating a positive result for this test.

[0042] (4) Data processing Analysis of variance was used, and the homogeneity of variance was tested first according to the procedure of analysis of variance.

[0043] If the variances are homogeneous, calculate the F-value. If the F-value < F... 0.05 Conclusion: There were no significant differences between groups; if the F-value ≥ F 0.05 For values ​​of P ≤ 0.05, statistical analysis was performed using pairwise comparisons of the means among multiple experimental groups and a control group.

[0044] If the data is not normally distributed or has unequal variances, perform appropriate variable transformations until it meets the requirements of normality or homogeneity of variance. Then, use the transformed data for statistical analysis. If the transformation still does not achieve normality or homogeneity of variance, use the rank-sum test for statistical analysis.

[0045] SPSS 22 statistical software was used.

[0046] (5) Test results Table 1 shows the results of sheep red blood cell (SRBC) induced DTH (pedisole thickening method) body weight in mice, and Table 2 shows the results of sheep red blood cell induced DTH pedisole thickness difference measurement in mice.

[0047] Table 1. Body weight results of sheep erythrocyte (SRBC)-induced DTH (foot metatarsal thickening method) in mice. )

[0048] Table 2. Measurement results of the difference in foot thickness of mice induced by sheep erythrocytes (DTH). )

[0049] As shown in Table 1, there were significant differences in the last weight and weight gain between the high-dose group and the negative control group; as shown in Table 2, there were significant differences in the difference in plantar thickness between the high-dose group and the negative control group.

[0050] (9) Conclusion Sheep red blood cells (SRBC) induced DTH (plantar thickening assay) in mice: Compared with the negative control group, the high-dose group showed a significant difference in plantar thickness, suggesting that the test substance may have the function of enhancing immunity.

Claims

1. A polypeptide compound, characterized in that: The polypeptide compound is a 9-fluorenylmethoxycarbonyl-4-tert-butoxy-L-prolyl-N-serine derivative as shown in Formula I. in, R1 is selected from H, C1-C5 alkyl groups; R2 is selected from OH, unsubstituted or substituted C1-C5 alkyl groups, wherein the following groups are five-membered rings containing at least one heteroatom, wherein the heteroatom is N; The polypeptide compound is: , , or .

2. A method for preparing the polypeptide compound as described in claim 1, characterized in that: The reaction formula for the polypeptide compound shown in Formula I is: a: Compound III was prepared by reacting 9-fluorenmethoxycarbonyl-4-tert-butoxy-L-proline V with compound IV in an organic solvent under the action of an acid-binding agent at -20~100℃ for 1-30h; b: React compound III and compound II in an organic solvent at -20~100℃ for 1-30 h to obtain the compound shown in formula I; In the reaction formula, R3 is selected from C1-C4 alkyl or C1-C4 alkoxy.

3. The method for preparing the polypeptide compound according to claim 2, characterized in that: In step a, the molar ratio of compound V, compound IV, and acid-binding agent is 1:1-1.5:1-1.5, the organic solvent is an aprotic inert solvent, and the acid-binding agent is an organic base or an inorganic base.

4. The method for preparing the polypeptide compound according to claim 3, characterized in that: The organic solvent is one or more of dichloromethane, dichloroethane, chloroform, tetrahydrofuran, dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, toluene, and chlorobenzene; the acid-binding agent is one or more of triethylamine, N-methylmorpholine, pyridine, sodium carbonate, sodium bicarbonate, and potassium carbonate.

5. The method for preparing the polypeptide compound according to claim 3, characterized in that: In step b, the molar ratio of compound III to compound II is 1:1-1.5, and the organic solvent is an aprotic inert solvent.

6. An application of the polypeptide compound according to claim 1, characterized in that: The use of the polypeptide compound of claim 1 in the preparation of immune-enhancing agents.

7. A pharmaceutical composition for enhancing immune function, characterized in that: The composition contains the polypeptide compound of claim 1.

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