Synthesis method and application of perfluoroalkyl chain modified amino acid derivative

Abstract

The invention discloses a synthetic method and application of a perfluoroalkyl chain modified amino acid derivative, and the synthetic method comprises the following steps: dissolving alkyl ester of perfluoroalkyl carboxylic acid, amino acid and organic alkali in an organic solvent, and reacting under closed conditions to prepare the perfluoroalkyl chain modified amino acid derivative. The synthesis method disclosed by the invention has the advantages of simple reaction system, mild conditions, high yield, cheap and easily available raw materials, economy, safety and the like, and meanwhile, accurate modification of a fluorinated group in a polypeptide or polypeptide-like sequence can also be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and particularly relates to a method for synthesizing a perfluoroalkyl chain-modified amino acid derivative and its application. Background Art

[0002] Due to their unique physicochemical properties, fluorides have attracted extensive attention in the fields of organic chemistry, biochemistry, and pharmaceutical chemistry. Since peptides and modified peptides have good biotolerance and compatibility as drugs and fluorine substitution has become a standard strategy for regulating drug properties, fluorinated amino acids and polypeptides have become increasingly prominent in new drugs (Vulpetti A, Dalvit C. Fluorine local environment: from screening to drug design[J]. Drug discovery today, 2012, 17(15-16): 890-897.). In the fields of gene delivery and protein delivery, perfluoroalkyl chain-modified amino acids have unique fluorine effects and can significantly improve the gene delivery efficiency of cationic polymer carriers. However, all the natural amino acids discovered so far do not contain fluorine atoms. Therefore, introducing fluoroalkyl groups into amino acids can construct fluorinated polypeptides or proteins. Currently, most of them introduce fluorine atoms or simple fluoroalkyl groups (such as trifluoromethyl, trifluoromethoxy, difluoromethyl, difluoromethoxy, trifluoromethylthio, difluoromethylthio, etc.) into amino acids. There is a literature report on a method for synthesizing perfluoroalkyl chain-modified amino acids using acyl chloride. Specifically, acyl chloride is slowly added dropwise to an aqueous sodium hydroxide solution of amino acids. After reacting at room temperature for two hours, the pH is adjusted to 1-2 with aqueous hydrochloric acid, and the target product is obtained by extraction with ether and recrystallization with ethyl acetate (Cong Z, Shoji O, Kasai C, et al. Activation of wild-type cytochrome P450BM3 by the next generation of decoy molecules: enhanced hydroxylation of gaseous alkanes and crystallographic evidence[J]. Acs Catalysis, 2015, 5(1): 150-156.). However, this method has high raw material prices, low yields, and the impurities generated are difficult to separate, and it is not suitable for industrial production. There is also a literature report on the preparation of perfluoroalkyl chain-modified phenylalanine through the classical Schotten-Baumann acylation reaction, that is, perfluorooctanoyl chloride is added dropwise to a dilute aqueous alkali solution of phenylalanine. After reacting at low temperature for 1 hour, the reaction is transferred to room temperature for 4 hours, the pH is adjusted to 1, and the target product is obtained by washing with water and methanol and then drying under vacuum. However, the yield of this reaction is low, only 28%, and the problem of high raw material prices has not been changed yet.(Ta HY, Perquis L, Balayssac S, Déjugnat C, Wodrinski A, Collin F, Gilard V, Couderc F. Separation of unsaturated C18 fatty acids using perfluorinated-micellar electrokinetic chromatography: I. Optimization and separation process. Electrophoresis. 2023Feb;44(3-4):431-441. doi:10.1002 / elps.202200151. Epub 2022Nov 24. PMID:36398472;PMCID:PMC10098715.). Therefore, the development of new safe, economical, environmentally friendly and efficient methods for the synthesis of perfluoroalkyl chain-modified amino acids has very important theoretical significance and potential application value.

[0003] Fluorinated drug delivery carriers often have stronger cellular uptake capabilities and show broad application prospects. The current common fluorination modification strategies are often based on the existing carrier structure in the form of perfluoroalkyl chlorides, etc., and the reaction sites and fluorination modification ratios are difficult to control. In oligomer drug delivery carriers represented by polypeptides or polypeptide-like molecules, the introduction of fluorinated groups is often carried out after the construction of the polypeptide sequence is completed, and there is also the problem of difficulty in achieving precise modification of fluorinated groups. These problems have caused inconvenience in systematically studying the effects of fluorination modification on the performance of drug delivery carriers, and further optimization is difficult. Therefore, a strategy that can achieve precise modification of fluorinated groups in polypeptide or polypeptide-like molecules needs to be explored. Summary of the invention

[0004] One of the purposes of the present invention is to provide a method for synthesizing a perfluoroalkyl chain-modified amino acid derivative, wherein an alkyl ester of a perfluoroalkyl carboxylic acid, an amino acid and an organic base are dissolved in an organic solvent and reacted under closed conditions to obtain a perfluoroalkyl chain-modified amino acid derivative;

[0005] The structural formula of the alkyl ester of the perfluoroalkyl carboxylic acid is as follows:

[0006]

[0007] Wherein, R' is a methyl group or an ethyl group, and n is an integer from 1 to 8;

[0008] The structural formula of the amino acid is as follows:

[0009]

[0010] Among them, R is hydrogen, methyl or isopropyl;

[0011] The organic base is triethylamine or pyridine.

[0012] Furthermore, the amino acid is glycine.

[0013] Furthermore, the molar ratio of the alkyl ester of perfluoroalkyl carboxylic acid, amino acid and organic base is 1:1-3:2-4, preferably 1:2:3.

[0014] Furthermore, the organic solvent is methanol or ethanol, preferably methanol.

[0015] Furthermore, the conditions of the reaction are stirring at room temperature for 1-4 days or heating to 55±10°C and stirring for 0.5-24 hours.

[0016] The reaction formula of the above reaction is as follows:

[0017]

[0018] In an embodiment of the present invention, after the reaction is completed, post-treatment is carried out on the product, and the post-treatment includes the following steps:

[0019] (1) Remove most of the solvent from the reaction solution to obtain an oily viscous substance, add an appropriate amount of water until the oily viscous substance is completely dissolved;

[0020] (2) Add hydrochloric acid with a certain concentration to adjust the pH value to 1-2, extract with an organic solvent, combine the organic solvents, and remove the organic solvent to obtain a solid crude product;

[0021] (3) Add the crude product to a mixed solution of an appropriate amount of methanol and water until it is completely dissolved;

[0022] (4) Rotate and evaporate to remove most of the solvent until a small amount of product precipitates, place it at 0°C for cooling and crystallization, after complete crystallization, filter by suction, wash the filter cake with a small amount of ice water, and dry to obtain.

[0023] The second object of the present invention is to provide a perfluoroalkyl chain-modified polypeptide or polypeptide-like oligomer, including the perfluoroalkyl chain-modified amino acid derivative prepared by the above synthesis method.

[0024] In an embodiment of the present invention, using the above perfluoroalkyl chain-modified amino acid derivative as a fluorination modification unit, through classical solid-phase synthesis reaction conditions, it is precisely connected to the sequence of the polypeptide or polypeptide-like oligomer that needs to be modified with a perfluoroalkyl chain. The obtained perfluoroalkyl chain-modified polypeptide or polypeptide-like oligomer can be used as a drug delivery carrier to construct a drug delivery system, and has better cell uptake ability than the unfluorinated modified carrier.

[0025] The synthesis method of the present invention has the advantages of simple reaction system, mild conditions, high yield, cheap and easily available raw materials, economic safety, etc. At the same time, it can also achieve precise modification of fluorinated groups in polypeptide or polypeptide-like sequences.

[0026] For the prior art, the present invention has the following beneficial technical effects:

[0027] The present invention provides a preparation method of a perfluoroalkyl chain-modified amino acid derivative. This method uses perfluoroalkyl chain-modified methyl ester, amino acid, and triethylamine, which are cheap and easily available, as raw materials to achieve the rapid large-scale preparation and synthesis of fluorinated amino acids and fluorinated polypeptides. Its synthesis method is simple, with high yield, environmental protection and safety, and is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the mass spectrum of pentafluoropropionylglycine in Example 1 of the present invention.

[0029] Figure 2 It is the mass spectrum of heptafluorobutyryl glycine in Example 2 of the present invention.

[0030] Figure 3 It is the mass spectrum of nonafluorovaleryl glycine in Example 3 of the present invention.

[0031] Figure 4 It is the mass spectrum of undecafluorohexanoyl glycine in Example 4 of the present invention.

[0032] Figure 5 It is the mass spectrum of tridecafluoroheptanoyl glycine in Example 5 of the present invention.

[0033] Figure 6 It is the mass spectrum of pentadecafluorooctanoyl glycine in Example 6 of the present invention.

[0034] Figure 7 It is the mass spectrum of heptadecafluorononanoyl glycine in Example 7 of the present invention.

[0035] Figure 8 It is the mass spectrum of nonadecafluorodecanoyl glycine in Example 8 of the present invention.

[0036] Figure 9 It is the cell uptake result of the drug delivery system prepared from perfluoroalkyl chain-modified polypeptide oligomers in Example 10 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0037] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0039] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial sources.

[0040] In the following examples, siFAM was purchased from General Biosystems (Anhui) Co., Ltd.

[0041] Example 1

[0042] Preparation of pentafluoropropionylglycine

[0043]

[0044] Take 5 g of methyl pentafluoropropionate (1 equiv, 28.08 mmol), 4.22 g of glycine (2 equiv, 56.16 mmol), and 8.52 g of triethylamine (84.24 mmol, 3 equiv) in a 250 mL eggplant-shaped flask, add 30 mL of methanol, stopper it, and stir the reaction at room temperature for 3 days. Rotate to remove most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and adjust the pH value to 1 - 2 with 6N hydrochloric acid. Extract 4 times with 50 mL of ethyl acetate. Combine the ethyl acetate and completely rotate to dryness to obtain a white solid product.

[0045] Hydrochloride removal and purification: Add the white product to an eggplant-shaped flask, add 60 mL of a mixed solution of methanol:water = 1:2 to obtain a clear solution. If the product cannot be completely dissolved, add methanol until it is completely dissolved. Rotate to remove most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant-shaped flask in an ice-water mixture to cool and crystallize. After the product has completely precipitated, filter it by suction, and wash the filter cake with a small amount of cold water. Dry it to obtain a white solid product. Titrate with silver nitrate and there is no residual chloride ion, and the pH value of the solution is 2 - 3. Finally, 4.22 g of pentafluoropropionylglycine is obtained with a yield of 68%. The product is a white solid, and the NMR data of the compound are as follows: 1 H NMR (300 MHz, DMSO-d 6 ): δ 12.93 (s, 1H, -OH), 9.87 (t, 1H, -NH-), 3.89 (d, 2H, -CH 2 -); 19 F NMR (282 MHz, DMSO-d 6 ): δ -82.49 (t, 3F), -122.18 (q, 2F); Mass spectrum: m / z 220.00 is the molecular ion peak of pentafluoropropionylglycine, i.e., [M - H] - .

[0046] Example 2

[0047] Preparation of heptafluorobutyryl glycine

[0048]

[0049] Take 5 g of methyl heptafluorobutyrate (1 equiv, 21.93 mmol), 3.29 g of glycine (2 equiv, 43.85 mmol), and 6.66 g of triethylamine (65.79 mmol, 3 equiv) in a 250 mL eggplant-shaped flask. Add 30 mL of methanol. After closing with a stopper, stir the reaction at room temperature for 3 days. Rotavapor most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and adjust the pH value to 1 - 2 with 6N hydrochloric acid. Extract with 50 mL of ethyl acetate 4 times. Combine the ethyl acetate and completely rotavapor to obtain a white solid product.

[0050] Hydrochloride removal and purification: Add the white product to an eggplant-shaped flask, add a mixed solution of 60 mL of methanol:water = 1:2 to obtain a clear solution. If the product cannot be completely dissolved, add methanol until it is completely dissolved. Rotavapor most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant-shaped flask in an ice-water mixture to cool and crystallize. After the product has completely precipitated, filter it by suction, and wash the filter cake with a small amount of cold water. Dry to obtain a white solid product. There is no chloride ion residue detected by silver nitrate titration, and the pH value of the solution is 2 - 3. Finally, 4.75 g of heptafluorobutyryl glycine is obtained with an 80% yield. The product is a white porous solid, and the NMR data of the compound is as follows: 1 H NMR (300 MHz, DMSO-d 6 ): δ 12.95 (s, 1H, -OH), 9.87 (t, 1H, -NH-), 3.89 (d, 2H, -CH 2 -); 19 F NMR (282 MHz, DMSO-d 6 ): δ -80.31 (t, 3F), -119.72~-119.88 (m, 2F), -126.92 (t, 2F); Mass spectrum: m / z 270.00 is the molecular ion peak of heptafluorobutyryl glycine, i.e., [M - H] - .

[0051] Example 3

[0052] Preparation of nonafluoropentanoyl glycine

[0053]

[0054] Take 5 g of methyl nonafluorovalerate (1 equiv, 17.98 mmol), 2.70 g of glycine (2 equiv, 35.96 mmol), and 5.45 g of triethylamine (3 equiv, 53.94 mmol) in a 250 mL eggplant-shaped flask. Add 30 mL of methanol. After closing with a stopper, stir the reaction at room temperature for 3 days. Rotavapor most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and add 6N hydrochloric acid to adjust the pH value to 1 - 2. Extract with 50 mL of ethyl acetate 4 times. Combine the ethyl acetate and completely rotavapor to obtain a white solid product.

[0055] Hydrogen chloride removal and purification: Add the white product to an eggplant-shaped flask, add 60 mL of a methanol:water = 1:2 mixed solution to obtain a clear solution. If the product cannot completely dissolve, add additional methanol until it is completely dissolved. Rotavapor most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant-shaped flask in an ice-water mixture to cool and crystallize. After the product completely precipitates, filter it by suction, and wash the filter cake with a small amount of cold water. Dry to obtain a loose white solid product. Titrate with silver nitrate and there is no chloride ion residue, and the pH value of the solution is 2 - 3. Finally, 3.98 g of nonafluorovaleryl glycine is obtained with a yield of 69%. The product is a white solid, and the NMR data of the compound is as follows: 1 H NMR (300 MHz, DMSO-d 6 ): δ 12.89 (s, 1H, -OH), δ 9.86 (t, 1H, -NH-), δ 3.89 (d, 2H, -CH 2 -); 19 F NMR (282 MHz, DMSO-d 6 ): δ -80.31 (t, 3F), -118.71~-118.99 (m, 2F), -122.30~-122.67 (m, 2F), -126.03 (t, 2F); Mass spectrum: m / z 320.00 is the molecular ion peak of nonafluorovaleryl glycine, i.e., [M - H] - 。

[0056] Example 4

[0057] Preparation of undecafluorocaproyl glycine

[0058]

[0059] Take 5 g of methyl 1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexanoate (1 equiv, 15.24 mmol), 2.29 g of glycine (2 equiv, 30.48 mmol), and 4.63 g of triethylamine (3 equiv, 45.72 mmol) in a 250 mL eggplant-shaped flask. Add 30 mL of methanol. After stoppered, stir the reaction at room temperature for 3 days. Rotate to remove most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and adjust the pH value to 1 - 2 with 6N hydrochloric acid. Extract with 50 mL of ethyl acetate 4 times. After combining the ethyl acetate, rotate to dry completely to obtain a pale yellow solid product.

[0060] Hydrochloride removal and purification: Add the pale yellow product to an eggplant-shaped flask, add 60 mL of a mixed solution of methanol:water = 1:2 to obtain a clear solution. If the product cannot dissolve completely, add methanol until it is completely dissolved. Rotate to remove most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant-shaped flask in an ice-water mixture to cool and crystallize. After the product completely precipitates, filter it by suction, and wash the filter cake with a small amount of cold water. Dry to obtain a loose pale yellow solid product. There is no chloride ion residue detected by silver nitrate titration, and the pH value of the solution is 2 - 3. Finally, 4.58 g of 1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexanoyl glycine is obtained with a yield of 81%. The product is a pale yellow solid, and the NMR data of the compound are as follows: 1 H NMR(300MHz,DMSO-d 6 ):δ12.92(s,1H,-OH),δ9.87(t,1H,-NH-),δ3.89(d,2H,-CH 2 -); 19 F NMR(282MHz,DMSO-d 6 ):δ-80.68(t,3F),-118.20~-119.05(m,2F),-121.82~-122.19(m,2F),-122.79~-122.97(m,2F),-126.22(t,2F); The mass spectrum: m / z 369.99 is the molecular ion peak of 1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexanoyl glycine, that is, [M - H] - 。

[0061] Example 5

[0062] Preparation of 1,1,2,2,3,3,4,4,5,5,6,6,7,7-tridecafluoroheptanoyl glycine

[0063]

[0064] Take 5 g of methyl tridecafluoroheptanoate (1 equiv, 13.23 mmol), 1.99 g of glycine (2 equiv, 26.46 mmol), and 4.02 g of triethylamine (3 equiv, 39.69 mmol) in a 250 mL eggplant-shaped flask. Add 30 mL of methanol. After closing with a stopper, stir the reaction at room temperature for 3 days. Rotate to remove most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and add 6N hydrochloric acid to adjust the pH value to 1 - 2. Extract with 50 mL of ethyl acetate 4 times. After combining the ethyl acetate, rotate to dry completely to obtain a pale yellow solid product.

[0065] Hydrochloride removal and purification: Add the pale yellow product to an eggplant-shaped flask, add 60 mL of a mixed solution of methanol:water = 1:2 to obtain a clear solution. If the product cannot dissolve completely, add methanol until it is completely dissolved. Rotate to remove most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant-shaped flask in an ice-water mixture to cool and crystallize. After the product has completely precipitated, filter it by suction, and wash the filter cake with a small amount of cold water. Dry to obtain a pale yellow solid product. Titrate with silver nitrate and there is no residual chloride ion, and the pH value of the solution is 2 - 3. Finally, obtain 3.23 g of tridecafluoroheptanoyl glycine with a yield of 58%. The product is a pale yellow solid, and the NMR data of the compound are as follows: 1 H NMR(300MHz,DMSO-d 6 ):δ12.94(s,1H,-OH),δ9.85(t,1H,-NH-),δ3.89(d,2H,-CH 2 -); 19 F NMR(282MHz,DMSO-d 6 ):δ-80.60(t,3F),-118.50~-119.05(m,2F),-121.86~-122.15(m,2F),-122.45~-122.93(m,2F),-122.97~-123.16(m,2F),δ-126.03(t,2F); The mass spectrum: m / z 419.99 is the molecular ion peak of tridecafluoroheptanoyl glycine, that is, [M - H] - 。

[0066] Example 6

[0067] Preparation of pentadecafluorooctanoyl glycine

[0068]

[0069] Take 5 g of methyl perfluorooctanoate (1 equiv, 11.68 mmol), 1.75 g of glycine (2 equiv, 23.36 mmol), and 3.55 g of triethylamine (3 equiv, 35.04 mmol) in a 250 mL eggplant-shaped flask. Add 30 mL of methanol. After stoppered, stir the reaction at room temperature for 3 days. Rotate to remove most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and add 6N hydrochloric acid to adjust the pH value to 1 - 2. Extract with 50 mL of ethyl acetate 4 times. After combining the ethyl acetate, rotate to dry completely to obtain a pale yellow solid product.

[0070] Hydrochloride removal and purification: Add the pale yellow product to an eggplant-shaped flask, add a mixed solution of 60 mL of methanol:water = 1:2 to obtain a clear solution. If the product cannot be completely dissolved, add methanol until it is completely dissolved. Rotate to remove most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant-shaped flask in an ice-water mixture to cool and crystallize. After the product has completely precipitated, filter by suction, and wash the filter cake with a small amount of cold water. Dry to obtain a pale yellow solid product. Titrate with silver nitrate to have no chloride ion residue, and the pH value of the solution is 2 - 3. Finally, obtain 3.30 g of perfluorooctanoyl glycine with a yield of 60%. The product is a pale yellow solid, and the NMR data of the compound are as follows: 1 H NMR(300MHz,DMSO-d 6 ):δ12.93(s,1H,-OH),δ9.87(t,1H,-NH-),δ3.89(d,2H,-CH 2 -); 19 F NMR(282MHz,DMSO-d 6 ):δ-80.63(t,3F),-118.48~-118.87(m,2F),-121.25~-122.55(m,2F),-122.00~-122.06(m,2F),-122.36~-122.69(m,2F),-122.71~-123.33(m,2F),-126.14(t,2F); The mass spectrum: m / z 469.99 is the molecular ion peak of perfluorooctanoyl glycine, i.e., [M - H] - 。

[0071] Example 7

[0072] Preparation of perfluorononanoyl glycine

[0073]

[0074] Take 5 g of methyl 1,1,2,2,3,3,4,4,5,5,6,6,7,7,7 - heptadecafluorononanoate (1 equiv, 10.46 mmol), 1.57 g of glycine (2 equiv, 20.92 mmol), and 3.18 g of triethylamine (3 equiv, 31.38 mmol) in a 250 - mL eggplant - shaped flask. Add 30 mL of methanol. After stopper - closing, stir the reaction at room temperature for 3 days. Rotavap most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and then add 6N hydrochloric acid to adjust the pH value to 1 - 2. Extract with 50 mL of ethyl acetate 4 times. Combine the ethyl acetate layers and completely rotavap to obtain a pale - yellow solid product.

[0075] Hydrochloride - removal and purification: Add the pale - yellow product to an eggplant - shaped flask, add a 60 - mL mixed solution of methanol:water = 1:2 to obtain a clear solution. If the product cannot be completely dissolved, supplement methanol until it is completely dissolved. Rotavap most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant - shaped flask in an ice - water mixture for cooling and crystallization. After the product has completely precipitated, filter it by suction, and wash the filter cake with a small amount of cold water. Dry it to obtain a pale - yellow solid product. Titrate with silver nitrate and there is no chloride ion residue, and the pH value of the solution is 2 - 3. Finally, 3.32 g of 1,1,2,2,3,3,4,4,5,5,6,6,7,7,7 - heptadecafluorononanoyl glycine is obtained with a yield of 61%. The product is a pale - yellow solid, and the NMR data of the compound are as follows: 1 H NMR(300MHz,DMSO - d 6 ):δ12.92(s,1H, - OH),δ9.77(t,1H, - NH - ),δ3.89(d,2H, - CH 2 - ); 19 F NMR(282MHz,DMSO - d 6 ):δ - 81.29(t,3F), - 118.75~ - 121.82(m,2F), - 121.93~ - 121.96(m,2F), - 122.19~ - 122.26(m,2F), - 122.36~ - 122.39(m,2F), - 122.77~ - 123.10(m,2F), - 123.18~ - 123.26(m,2F), - 126.75(t,2F); Mass spectrum: m / z 519.98 is the molecular ion peak of 1,1,2,2,3,3,4,4,5,5,6,6,7,7,7 - heptadecafluorononanoyl glycine, that is, [M - H] - 。

[0076] Example 8

[0077] Preparation of 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 - nonadecafluorodecanoyl glycine

[0078]

[0079] Take 5 g of methyl nonadecafluorodecanoate (1 equiv, 9.47 mmol), 1.42 g of glycine (2 equiv, 18.94 mmol), and 2.87 g of triethylamine (3 equiv, 28.41 mmol) in a 250 mL eggplant-shaped flask. Add 30 mL of methanol. After stoppered, stir the reaction at room temperature for 3 days. Rotavap most of the solvent from the reaction solution to obtain an oily viscous substance. Add 60 mL of water to dissolve it, and then add 6N hydrochloric acid to adjust the pH value to 1 - 2. Extract with 50 mL of ethyl acetate 4 times. Combine the ethyl acetate and completely rotavap it to obtain a pale yellow solid product.

[0080] Hydrochloride removal and purification: Add the pale yellow product into an eggplant-shaped flask, add 60 mL of a mixed solution of methanol:water = 1:2 to obtain a clear solution. If the product cannot be completely dissolved, add methanol until it is completely dissolved. Rotavap most of the solvent at 60 °C. At this time, a small amount of the product precipitates. Place the eggplant-shaped flask in an ice-water mixture to cool and crystallize. After the product completely precipitates, filter it by suction, and wash the filter cake with a small amount of cold water. Dry it to obtain a pale yellow solid product. Titrate with silver nitrate and there is no residual chloride ion, and the pH value of the solution is 2 - 3. Finally, 3.41 g of nonadecafluorodecanoyl glycine is obtained with a yield of 63%. The product is a pale yellow solid, and the NMR data of the compound are as follows: 1 H NMR(300MHz,DMSO-d 6 ):δ9.75(t,1H,-NH-),δ3.86(d,2H,-CH 2 -); 19 F NMR(282MHz,DMSO-d 6 ):δ-81.76(t,3F),-118.15~-118.97(m,2F),-122.03~-122.07(m,2F),-122.13~-122.38(m,2F),-122.41~-122.50(m,2F),-122.58~-122.77(m,2F),-122.83~-123.06(m,2F),-123.10~-123.84(m,2F),-126.88(t,2F); Mass spectrum: m / z 569.98 is the molecular ion peak of nonadecafluorodecanoyl glycine, that is, [M - H] - 。

[0081] Example 9

[0082] Solid-phase synthesis of perfluoroalkyl chain-modified polypeptide-like oligomers

[0083] Perfluoroalkyl-chain modified peptidomimetic oligomers are used as gene delivery vectors because they contain protonatable groups under normal physiological conditions at pH 7.4 and can encapsulate nucleic acid drugs through electrostatic adsorption. Existing perfluoroalkyl-chain modifications often involve reactions between perfluoroalkyl acyl chlorides and amino groups on the constructed peptide chains, making it difficult to determine the reaction sites and modification ratios. The introduction of perfluoroalkyl-chain modified amino acids enables precise positioning and quantitative modification of fluorinated groups. Taking the perfluoroalkyl-chain modified peptidomimetic oligomer HO-Lys-(Gly-F 7 )-(His-Stp) 3 -His-Cys-NH 2 (The sequence order is from C-terminus to N-terminus, and Stp is an artificial amino acid) as an example, this sequence is abbreviated as K-(G-F 7 )-(H-Stp) 3 -H-C). The synthetic raw materials for the sequence include protected amino acids HO-Lys(ivDde)-Fmoc, Fmoc-His(Trt)-OH, Boc-Cys(Trt)-OH suitable for Fmoc solid-phase synthesis, perfluoroalkyl-chain modified heptafluorobutyryl glycine Gly-F 7 , and Fmoc-protected artificial amino acid Fmoc-Stp-COOH.

[0084] Among them, the Fmoc-protected artificial amino acid Fmoc-Stp-COOH contains a protonatable tetraethylenepentamine structure and can be used for the loading of various nucleic acid drugs. Its structural formula is as follows:

[0085]

[0086] The synthesis of the peptidomimetic oligomer K-(G-F 7 )-(H-Stp) 3 -H-C starts from lysine at the C-terminus and uses the classical Fmoc-based solid-phase peptide synthesis reaction. First, HO-Lys(ivDde)-Fmoc is coupled to 2-chlorotrityl chloride resin, and amino acid coupling and Fmoc deprotection operations are sequentially carried out step by step towards the N-terminus until the coupling of the terminal amino acid Cys is completed to finish the construction of the main chain. The perfluoroalkyl-chain of this peptidomimetic oligomer is precisely modified on the side chain of lysine. First, the ivDde protecting group on lysine ε-NH 2 is removed using a 4% hydrazine hydrate-DMF solution (v:v) to expose lysine ε-NH 2 , and finally, heptafluorobutyryl glycine is condensed with this NH 2 site through classical solid-phase peptide reaction conditions to complete the site-directed modification of the heptafluorobutyryl group. After the fluorination modification is completed, an appropriate amount of TFA / TIS / H 2O / DODT 94 / 1 / 2.5 / 2.5 (v / v / v / v) mixed solution, under nitrogen protection, stir and react for 90 min to cleave the polypeptide chain from the resin and simultaneously remove the protecting groups. After the reaction, filter with a polytetrafluoroethylene (PTFE) membrane to obtain the filtrate, and wash the resin with trifluoroacetic acid and DCM in sequence. Add 20 μL TIS to the reaction solution to capture the positive ions, and rotary evaporate and concentrate the volume of the reaction solution to about 0.5 mL. Precipitate the obtained reaction solution in pre-cooled MTBE / n-hexane (1:1) (v:v), centrifuge and collect the precipitate, and dry it under nitrogen and store it frozen. Purify using a Sephadex G-10 column, with 10 mM hydrochloric acid / acetonitrile 7:3 (v:v) as the eluent, collect the samples, combine and lyophilize to obtain a white to pale yellow fluffy solid powder. The NMR data of the compound are as follows: 1 H NMR (300 MHz, D 2 O) δ (ppm) = 1.06 - 1.85 (m, 6H, β, γ, δ lysine), 2.30 - 2.56 (m, 12H, -CO-CH 2 -CH 2 -CO, Stp), 2.89 - 3.64 (m, 60H, -CH 2 -tetraethylenepentamine, -CH 2 -ε lysine, -CH 2 -β cysteine, -CH 2 -β histidine), 3.93 - 4.65 (m, 8H, -CH-α cysteine, α lysine, α histidine, -CH 2 -α glycine), 7.19 (d, 4H, =CH-NH-histidine), 8.56 (d, 4H, -NH=CH-NH-histidine). 19 F NMR (282 MHz, D 2 O): δ -80.82~-80.88 (t, 3F), -121.34~-121.43 (m, 2F), -127.42 (t, 2F).

[0087] Example 10

[0088] Preparation and Cellular Uptake of a Drug Delivery System Based on a Perfluoroalkyl Chain-Modified Peptidomimetic Oligomer

[0089] Preparation of a drug delivery system based on a perfluoroalkyl chain-modified polypeptide-like oligomer, i.e., preparation of a complex solution of the polypeptide-like oligomer and siRNA: Take 1.07 μL, 2.13 μL, and 4.26 μL of the 5 mg / mL polypeptide-like oligomer solution according to different N / P (N / P 6, 12, and 24), dilute it to 50 μL with HEPES buffer (20 mM, pH 7.4), and then mix it with an equal volume of siFAM solution (1.35 μg siFAM). Pipette and mix well, and incubate at room temperature for 40 min to obtain the product.

[0090] Preparation of the control siFAM solution: Take siFAM dry powder and dilute it to 13.5 μg / mL with HEPES buffer (20 mM, pH 7.4) to obtain the product.

[0091] Preparation of the complex solution of the control Lipofectamine 3000 and siRNA: Take 1 μL of Lipofectamine 3000, dilute it to 50 μL with HEPES, and then mix it with an equal volume of siFAM solution (1.35 μg siFAM). Pipette and mix well, and incubate at room temperature for 15 min to obtain the product.

[0092] Cell uptake: Select 4T1 cells in the logarithmic growth phase, inoculate 10 5 cells per well into a 24-well culture plate, add 0.5 mL of sample to each well, and culture for 24 h. After the cells adhere to the wall, aspirate the culture medium and rinse with PBS buffer. Set the complex solution of Lipofectamine 3000 and siRNA as the positive control group, and set naked siFAM as the negative control group. Add 400 μL of serum-free medium and 100 μL of siFAM solution or complex solution (1.35 μg siFAM / well), and use untreated cells as the blank control group. Place it in an incubator and culture for 4 h, then discard the old liquid, gently rinse with pre-cooled PBS buffer at 4 °C, and then add 200 μL of trypsin to each well to digest the cells. Incubate in an incubator for 3 min, place it under an inverted microscope to observe. When the cell edges are clear and the retracted protrusions become round, add 0.6 mL of fresh medium to terminate the digestion. Transfer the mixture to a 1.5 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add pre-cooled PBS buffer at 4 °C to resuspend, centrifuge at 1000 rpm for 5 min, discard the supernatant, repeat 2 times, add 200 μL of PBS buffer again and gently pipette to mix evenly, pass it through a 200-mesh sieve, and use a flow cytometer to measure the quantitative uptake. The results are as Figure 9 shown. The delivery system constructed based on the perfluoroalkyl chain-modified polypeptide-like oligomer has a better uptake effect than the commercial transfection reagent Lipofectamine 3000.

Claims

1. A method for synthesizing a perfluoroalkyl chain-modified amino acid derivative, characterized in that: The alkyl ester of perfluoroalkyl carboxylic acid, amino acid and organic base are dissolved in an organic solvent and reacted under closed conditions to prepare a perfluoroalkyl chain-modified amino acid derivative; The structural formula of the alkyl ester of the perfluoroalkyl carboxylic acid is as follows: Wherein, R' is a methyl group or an ethyl group, and n is an integer from 1 to 8; The organic base is triethylamine or pyridine.

2. The synthesis method according to claim 1, characterized in that The structural formula of the amino acid is as follows: Wherein, R is hydrogen, methyl or isopropyl.

3. The synthesis method according to claim 2, characterized in that The amino acid is glycine.

4. The synthesis method according to claim 1, characterized in that The molar ratio of the alkyl ester of the perfluoroalkyl carboxylic acid, the amino acid and the organic base is 1:1-3:2-4.

5. The synthesis method according to claim 4, characterized in that The molar ratio of the alkyl ester of the perfluoroalkyl carboxylic acid, the amino acid and the organic base is 1:2:

3.

6. The synthesis method according to claim 1, characterized in that The organic solvent is methanol or ethanol.

7. The synthesis method according to claim 6, characterized in that The organic solvent is methanol.

8. The synthesis method according to claim 1, characterized in that The reaction conditions are stirring at room temperature for 1-4 days or heating to 55±10° C. for 0.5-24 hours.

9. A polypeptide or polypeptide-like oligomer modified with a perfluoroalkyl chain, characterized in that: It comprises a perfluoroalkyl chain modified amino acid derivative prepared by the synthesis method described in claim 1.

10. A drug delivery system, characterized in that: A polypeptide or polypeptide-like oligomer modified with a perfluoroalkyl chain as described in claim 9.

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