CN114874146A - Histidine lipid, histidine lipid nanoparticle, and preparation method and application thereof - Google Patents

Histidine lipid, histidine lipid nanoparticle, and preparation method and application thereof Download PDF

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CN114874146A
CN114874146A CN202210305388.0A CN202210305388A CN114874146A CN 114874146 A CN114874146 A CN 114874146A CN 202210305388 A CN202210305388 A CN 202210305388A CN 114874146 A CN114874146 A CN 114874146A
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compound
histidine
lipid
carbon atoms
alkyl
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CN114874146B (en
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田伟伟
文中行
徐健
秦兵彬
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王德玲
徐军
陆阳
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Suno Biomedical Technology Suzhou Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
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    • A61K9/5123—Organic compounds, e.g. fats, sugars
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Abstract

The invention relates to histidine lipid, which is compound A and/or salt of compound A, wherein the structural formula of the compound A is shown in the specification
Figure DDA0003564838210000011
Wherein R is 1 Selected from alkyl with 1-24 carbon atoms, and X is selected from H, COOR 2 Or COR 2 ，R 2 Selected from alkyl with 1-24 carbon atoms, R 1 And/or R 2 Has a branched chain. The histidine lipid has the advantages of low toxicity and small immunogenicity, can form histidine nanoparticles with high encapsulation efficiency, small and uniform particle size with mRNA, and can be used for effectively delivering the mRNA.

Description

Histidine lipid, histidine lipid nanoparticle, and preparation method and application thereof
Technical Field
The invention relates to histidine lipid, a histidine lipid nanoparticle, a preparation method and application thereof.
Background
Messenger ribonucleic acid (mRNA) is a single-stranded RNA which is transcribed from DNA and carries genetic information capable of guiding protein synthesis, and the treatment method of mRNA is widely researched in the fields of vaccines, tumor immunotherapy and the like. In the new crown epidemic situation, mRNA vaccines are greatly concerned due to the characteristics of high development speed, high protection rate and high safety. Among vectors for delivering mRNA, LNP Lipid Nanoparticles (LNP) are widely studied and used. However, the cationic or cationizable amino lipid used in the currently mainstream LNP system mainly has the problems of high biotoxicity, strong immunogenicity and low endosome escape rate, and LNP is mainly delivered to the liver and has poor selectivity on other organs.
Based on this, how to reduce the immunogenicity and biological toxicity of mRNA delivery systems and increase the escape rate of endosomes is an urgent technical problem to be solved.
Disclosure of Invention
The invention aims to provide histidine lipid, a histidine lipid nanoparticle, and a preparation method and application thereof.
Aiming at the defects of the prior art, the technical scheme adopted by the invention is as follows:
a kind ofHistidine lipid, wherein the histidine lipid is compound A and/or salt of the compound A, and the structural formula of the compound A is shown in the specification
Figure BDA0003564838190000011
Wherein R is 1 Selected from alkyl with 1-24 carbon atoms, and X is selected from H, COOR 2 Or COR 2 ，R 2 Selected from alkyl with 1-24 carbon atoms, R 1 And/or said R 2 Has a branched chain.
Preferably, said R is 1 The R is 2 Are each independently selected from R 3 (R 4 ) n (ii) a Wherein R is 3 Selected from alkyl, alkenyl or alkynyl with 1-24 carbon atoms, R 4 Selected from alkyl, alkenyl or alkynyl with 1-23 carbon atoms, R 4 R and the product of the number of carbon atoms and n 3 Is less than or equal to 24 and n is selected from 0, 1, 2 or 3.
Further preferably, said R 3 The R is 4 Are respectively selected from linear alkyl, alkenyl or alkynyl.
Even more preferably, when said R is 1 Is selected from R 3 (R 4 ) n And when n is 0, the R 2 Is selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3; or, when said R is 1 Is selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3, the R 2 Is selected from R 3 (R 4 ) n And n is selected from 0, 1, 2 or 3.
According to some preferred embodiments, when said R is 1 Is selected from R 3 (R 4 ) n And when n is 0, the R 1 R in (1) 3 Is selected from alkyl, alkenyl or alkynyl with 10-24 carbon atoms, and more preferably alkyl, alkenyl or alkynyl with 15-21 carbon atoms.
Further preferably, said R 2 R in (1) 3 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atoms.
It is further preferred that the first and second liquid crystal compositions,said R is 2 R in (1) 4 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atoms.
According to some preferred embodiments, when said R is 1 Is selected from R 3 (R 4 ) n And n is 1, 2 or 3, the R 1 R in (1) 3 And R 4 Each independently selected from an alkyl group, an alkenyl group or an alkynyl group having 1 to 10 carbon atoms, more preferably an alkyl group, an alkenyl group or an alkynyl group having 5 to 10 carbon atoms.
Further preferably, said R 2 R in (1) 3 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atoms.
Further preferably, said R 2 R in (1) 4 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atoms.
According to some specific and preferred embodiments, said R is 1 Is selected from
Figure BDA0003564838190000021
Figure BDA0003564838190000022
And/or, said R 2 Is selected from
Figure BDA0003564838190000023
Figure BDA0003564838190000024
C(CH 3 ) 3 Or CH 3 。
According to some specific and preferred embodiments, said compound a is selected from
Figure BDA0003564838190000025
Figure BDA0003564838190000031
Preferably, the salt of compound A has the structural formula
Figure BDA0003564838190000032
Wherein Y is an acid group.
Further preferably, the acid radical is selected from CF 3 COO - 、CH 3 COO - 、Cl - 、SO 4- Or Br - 。
According to some specific and preferred embodiments, said salt of compound a is selected from
Figure BDA0003564838190000033
The invention also provides a preparation method of the histidine lipid, which comprises the steps of reacting the compound B with alcohol to generate the compound A, and selectively reacting the compound A with acid to generate the salt of the compound A; wherein the structural formula of the compound B is
Figure BDA0003564838190000041
X in the compound B is the same as X in the compound A; the alcohol is R 1 OH, R in the alcohol 1 With R in said Compound A 1 The same; the acid is H n Y n- And Y in the acid is the same as Y in the salt of the compound A.
The invention also provides application of one or more of the histidine lipids or one or more of the histidine lipids prepared by the preparation method in delivery of one or more of mRNA, siRNA, miRNA or antisense oligonucleotide.
The invention also provides a histidine lipid nanoparticle, which comprises mRNA and one or more of the histidine lipids or one or more of the histidine lipids prepared by the preparation method.
The invention also provides an application of the histidine lipid nanoparticle as described above in delivery of one or more of mRNA, siRNA, miRNA or antisense oligonucleotide.
The implementation of the invention has at least the following beneficial effects:
the histidine lipid has the advantages of low toxicity and small immunogenicity, can form histidine nanoparticles with high encapsulation efficiency, small and uniform particle size with mRNA, and can be used for effectively delivering the mRNA.
Drawings
FIG. 1 is a schematic diagram of the preparation of mRNA/HLNP (histidine lipid nanoparticles) in an example of the present invention;
FIG. 2 is a diagram of agarose gel electrophoresis in an example of the invention;
FIG. 3 is a mouse fluorescence imaging picture in the example of the present invention, wherein 1-5 are mouse fluorescence imaging pictures obtained by injecting 5 mRNA/HLNP samples intramuscularly for 24 hours, respectively;
FIG. 4 is a schematic diagram of the administration and blood collection times of mice in an embodiment of the present invention;
FIG. 5 is a schematic diagram of luciferase antibody detection in an embodiment of the present invention.
Detailed Description
Among the vectors for delivering mRNA, LNP is widely used, but currently, ionizable lipids in LNP are generally synthetic amino compounds, and mainly have the problems of high toxicity and high immunogenicity. In order to solve the above problems, the inventors of the present invention have made extensive studies and extensive practices to provide a technical solution of the present invention. The technical solution, its implementation and principles, etc. will be further explained as follows.
A histidine lipid is compound A and/or salt thereof, and the structural formula of compound A is shown in the specification
Figure BDA0003564838190000051
Wherein R is 1 Selected from alkyl with 1-24 carbon atoms, and X is selected from H, COOR 2 Or COR 2 ，R 2 Selected from alkyl with 1-24 carbon atoms, R 1 And/or R 2 Has a branched chain. Histidine ester in the present inventionThe proton may be an isomer of the above structure, and may be, for example, a D configuration and/or an L configuration.
According to the invention, R 1 、R 2 Are each independently selected from R 3 (R 4 ) n (ii) a Wherein R is 3 Selected from alkyl, alkenyl or alkynyl with 1-24 carbon atoms, R 4 Selected from alkyl, alkenyl or alkynyl with 1-23 carbon atoms, R 4 R and the product of the number of carbon atoms and n 3 Is less than or equal to 24 and n is selected from 0, 1, 2 or 3.
Further, R 3 And R 4 All are straight-chain alkyl, alkenyl or alkynyl groups.
In some embodiments, when R 1 Is selected from R 3 (R 4 ) n And when n is 0, R 2 Is selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3.
Preferably, R 1 R in (1) 3 Selected from alkyl, alkenyl or alkynyl with 5-24 carbon atoms, R 3 The number of carbon atoms of (b) may be, for example, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20, 22, etc.
Preferably, R 2 R in (1) 3 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 3 The number of carbon atoms of (b) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R 3 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methylene, ethenyl, ethynyl, and propenyl. R 2 R in (1) 4 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 4 The number of carbon atoms of (b) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R 4 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methylene, ethenyl, ethynyl, and propenyl.
In some embodiments, when R 1 Is selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3, R 2 Is selected from R 3 (R 4 ) n And n is selected from 0, 1, 2 or 3.
Preferably, R 1 R in (1) 3 Selected from the group consisting of those having carbon atoms1-10 alkyl, alkenyl or alkynyl, R 3 The number of carbon atoms of (b) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R 3 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methylene, ethenyl, ethynyl, and propenyl. R 1 R in (1) 4 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 4 The number of carbon atoms of (b) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R 4 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methylene, ethenyl, ethynyl, and propenyl.
Preferably, R 2 R in (1) 3 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 3 The number of carbon atoms of (b) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R 3 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methylene, ethenyl, ethynyl, and propenyl. R 2 R in (1) 4 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 4 The number of carbon atoms of (b) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R 4 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methylene, ethenyl, ethynyl, and propenyl.
In some specific and preferred embodiments, R is a hydrogen atom, an oxygen atom, an electron, and the like 1 Is selected from
Figure BDA0003564838190000061
R 2 Is selected from
Figure BDA0003564838190000062
Figure BDA0003564838190000063
C(CH 3 ) 3 Or CH 3 。
Compound A may be, for example
Figure BDA0003564838190000064
Figure BDA0003564838190000065
Or isomers of the above structures.
According to the invention, the salts of the compounds A have the formula
Figure BDA0003564838190000071
Wherein Y is an acid group. The acid radical being selected from CF 3 COO - 、CH 3 COO - 、Cl - 、SO 4- Or Br - 。
In some specific and preferred embodiments, the salt of compound a is selected from
Figure BDA0003564838190000072
Figure BDA0003564838190000073
Or isomers of the above structures.
A method for producing a histidine lipid, comprising reacting a compound B with an alcohol to produce a compound A, and optionally reacting the compound A with an acid to produce a salt of the compound A; wherein the structural formula of the compound B is
Figure BDA0003564838190000074
X in compound B is the same as X in compound A; the alcohol is R 1 OH, R in alcohol 1 With R in Compound A 1 The same; the acid being H n Y n- Y in the acid is the same as Y in the salt of compound a.
According to the present invention, the reaction temperature of the compound B with the alcohol is controlled to 0 to 100 ℃, and may be, for example, 0 ℃,10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃ or the like.
Further, the reaction time of the compound B and the alcohol is controlled to be 1 to 50 hours, for example, 1 hour, 5 hours, 10 hours, 15 hours, 18 hours, 20 hours, 23 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, and 50 hours.
According to the invention, the molar ratio of compound B to alcohol fed is 1: (0.1 to 10) may be, for example, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.
In some embodiments, compound B and an alcohol are reacted in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 4-dimethylaminopyridine, diisopropylethylamine, and a solvent. The charging molar ratio of the compound B to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1 (1-5), and may be, for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:5, or the like. The feeding molar ratio of the compound B to the 4-dimethylaminopyridine is 1: (0.1 to 1), for example, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, etc. may be mentioned. The feeding molar ratio of the compound B to the diisopropylethylamine is 1: (1 to 10) may be, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.
In other embodiments, compound B and the alcohol are reacted in the presence of dicyclohexylcarbodiimide, 4-dimethylaminopyridine and a solvent. The feeding molar ratio of the compound B to the dicyclohexylcarbodiimide is 1: (0.5 to 5), for example, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, etc. may be mentioned. The feeding molar ratio of the compound B to the 4-dimethylaminopyridine is 1: (0.5 to 5), for example, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, etc. may be mentioned.
The solvent includes, but is not limited to, one or more of dichloromethane, chloroform, tetrahydrofuran, N-dimethylformamide.
According to the present invention, the reaction temperature of the compound A with the acid is controlled to 0 to 50 ℃ and may be, for example, 0 ℃,10 ℃, 20 ℃, 30 ℃, 40 ℃ or 50 ℃.
Further, the reaction time of the compound a and the acid is controlled to be 0.1 to 5 hours, for example, 0.1 hour, 0.5 hour, 1 hour, 1.5 hour, 2 hours, 3 hours, 4 hours, and the like.
According to the invention, the molar ratio of compound a to acid fed is 1: (0.5 to 100), for example, 1:1, 1:2, 1:3, 1:5, 1:10, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, etc. may be used.
According to the invention, compound a is reacted with an acid in the presence of a solvent. Acids include, but are not limited to, trifluoroacetic acid.
A histidine lipid nanoparticle comprising mRNA and a histidine lipid as described above.
According to the present invention, the histidine lipid nanoparticle further comprises one or more of a helper lipid, cholesterol, polyethylene glycol lipid. Wherein, the auxiliary lipid includes but is not limited to DSPC and/or DOPE, and the polyethylene glycol lipid includes but is not limited to one or more of PEG2000-DMG, PEG2000-C-DMG and PEG 2000-DSPE.
A method for preparing histidine lipid nanoparticles, comprising the following steps:
(1) dissolving histidine lipid, helper lipid, cholesterol and polyethylene glycol lipid in organic solvent;
(2) dissolving the mRNA in a buffer;
(3) mixing the solutions obtained in the step (1) and the step (2);
(4) diluting the product obtained in step (3) with PBS buffer solution, and performing ultrafiltration.
According to the invention, the feeding mass ratio of the histidine lipid to the helper lipid is 1: (0.1 to 0.5), for example, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, etc. The feeding mass ratio of the histidine lipid to the cholesterol is 1: (0.3 to 0.8), for example, 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6, 1:0.7, etc. The feeding mass ratio of the histidine lipid to the polyethylene glycol lipid is 1: (0.1 to 0.2), for example, 1:0.12, 1:0.14, 1:0.16, 1:0.18, etc. The feeding mass ratio of the histidine lipid to the mRNA is (5-15): 1 may be, for example, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, etc.
According to the present invention, the mass concentration of histidine lipids in the system of step (1) is controlled to be 1 to 10mg/mL, and may be, for example, 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL, or the like.
According to the invention, the pH of the buffer solution in the step (2) is controlled to be 3-4. The mass concentration of mRNA in the system of step (2) is controlled to 0.1 to 0.5mg/mL, and may be, for example, 0.15mg/mL, 0.2mg/mL, 0.25mg/mL, 0.3mg/mL, or the like.
According to the invention, the dilution factor in the step (4) is controlled to be 5-15 times, for example, 5, 8, 10, 13, etc.
According to the invention, the ultrafiltration in the step (4) is controlled to the original volume at 0-10 ℃.
The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and explanatory of the invention and do not restrict the invention in any way.
Unless otherwise specified, the room temperature in the following examples is 20 to 25 ℃.
The m/z values of the salts of compound a referred to in the examples below comprise only the main moiety and do not include an acid group.
The "yields" referred to in the following examples all refer to "molar yields".
Example 1
Synthesis of N-t-butyloxycarbonyl-L-histidine-9-heptadecyl ester (Compound 1) and L-histidine-9-heptadecyl bistrifluoroacetate (Compound 2)
The synthetic routes for compound 1 and compound 2 are as follows:
Figure BDA0003564838190000091
N-t-butyloxycarbonyl-L-histidine (300mg,1.18mmol,1eq.), 9-heptadecanol (840mg,3.3mmol,2.8eq.), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI,563mg,2.95mmol,2.5eq.), 4-dimethylaminopyridine (DMAP,57mg,0.47mmol,0.4eq.) were added to a 25mL reaction flask and N-times were performed with a three-way joint using a three-way joint 2 The reaction flask was replaced and heated with hot air to remove moisture in the flask. Diisopropylethylamine (DIPEA,1.3mL,7.55mmol,6.4eq.) and dichloromethane (DCM,10mL) were mixed and added to the reaction flask. Heating in water bath at 50 deg.C, tracking reaction by LC-MS, and stopping reaction after 22.5 h. The reaction mixture was poured into 40mL of DCM, and 40mL of a saturated aqueous solution of sodium hydrogencarbonate was added to conduct extraction, and the aqueous layerWashing with DCM 4 times, combining the organic layers, drying over sodium sulfate, filtering, concentrating, and purifying the crude product by column chromatography on silica gel with DCM/MeOH50:1-40:1-35:1 as eluent to give 144.5mg of Compound 1 as a pale yellow oil, 25% yield.
Characterization data for compound 1 are as follows:
ESI:m/z＝494.16[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.54(s,1H),6.81(s,1H),5.72(s,1H),4.85(p,J＝6.1Hz,1H),4.50(s,1H),3.15–2.99(m,2H),1.56–1.45(m,4H),1.42(s,9H),1.25(s,24H),0.87(dt,J＝6.9,1.8Hz,6H).
13 C NMR(100MHz,CDCl 3 )δ172.14,155.73,135.15,79.84,77.36,76.00,53.83,34.03,34.00,31.99,31.97,29.66,29.65,29.60,29.39,28.44,25.32,25.23,22.78,14.23.
a25 mL reaction flask was charged with Compound 1(124.6mg,0.25mmol), and the atmosphere in the flask was purged with N 2 After displacement, DCM (4mL) was added followed by trifluoroacetic acid (TFA,1 mL). The reaction was carried out at room temperature, followed by LC-MS, and stopped after 2 h. The reaction solution was concentrated under reduced pressure at room temperature, 10mL of ethanol was then added, ethanol was removed under reduced pressure at room temperature, 10mL of DCM was then added, DCM was removed under reduced pressure at room temperature, and the reaction solution was pumped with oil to give 156.7mg of pale yellow foamy compound 2 in 99% yield.
Characterization data for compound 2 are as follows:
ESI:m/z＝394.38[M+H]+.
1 H NMR(400MHz,DMSO-d 6 )δ9.02(s,1H),7.47(s,1H),4.83(p,J＝6.3Hz,1H),4.43(t,J＝7.4Hz,1H),3.34–3.12(m,2H),1.54–1.36(m,4H),1.22(s,24H),0.89–0.78(m,6H).
13 C NMR(100MHz,DMSO-d 6 )δ168.07,158.95,158.62,134.55,127.09,118.12,76.56,51.01,33.12,33.08,31.30,31.28,28.91,28.85,28.82,28.71,28.69,25.55,24.52,24.48,22.11,13.94.
example 2
Synthesis of N-t-butyloxycarbonyl-D-histidine-9-heptadecyl ester (Compound 3) and D-histidine-9-heptadecyl bistrifluoroacetate (Compound 4)
The synthetic routes for compound 3 and compound 4 are as follows:
Figure BDA0003564838190000111
N-tert-Butoxycarbonyl-D-histidine (300mg,1.18mmol,1eq.), 9-heptadecanol (840mg,3.3mmol,2.8eq.), EDCI (563mg,2.95mmol,2.5eq.), DMAP (57mg,0.47mmol,0.4eq.) were added to a 25mL reaction flask 20 times N with a three-way junction 2 The reaction flask was heated with hot air to remove moisture in the flask. DIPEA (1.3mL,7.55mmol,6.4eq.) and DCM (10mL) were mixed and added to the reaction flask. Heating in water bath at 50 deg.C, following the reaction by LC-MS, stopping the reaction after 17.5 h. The reaction mixture was poured into 40mL of DCM, and then 40mL of saturated aqueous sodium bicarbonate solution was added for extraction, the aqueous layer was washed with DCM 4 times, the organic layers were combined, dried over sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography with the eluent DCM/MeOH50:1-40:1-35:1 to give 107mg of Compound 3 as a pale yellow oil in 18% yield.
ESI:m/z＝494.56[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.52(s,1H),6.79(s,1H),5.78(s,1H),4.85(p,J＝6.2Hz,1H),4.49(d,J＝7.7Hz,1H),3.10(dt,J＝13.9,6.9Hz,2H),1.56–1.45(m,4H),1.41(s,9H),1.23(s,24H),0.86(t,J＝6.6Hz,6H).
13 C NMR(100MHz,CDCl 3 )δ172.13,155.73,135.13,79.85,77.36,76.01,53.82,34.03,34.00,31.99,31.98,29.89,29.67,29.65,29.60,29.39,28.44,25.32,25.23,22.78,14.23.
A25 mL reaction flask was charged with Compound 3(73.75mg,0.15mmol), and the atmosphere in the flask was purged with N 2 After displacement, DCM (4mL) was added followed by trifluoroacetic acid (1 mL). The reaction was carried out at room temperature, followed by LC-MS, and stopped after 2 h. After the reaction solution was concentrated under reduced pressure at normal temperature, 10mL of ethanol was added, after removal of ethanol under reduced pressure at normal temperature, 10mL of DCM was added, after removal of DCM under reduced pressure at normal temperature, 10mL of cyclohexane was added, after removal of cyclohexane under reduced pressure at normal temperature and about 2 hours of pumping with an oil pump, 75.17mg of the white foamy compound 4 was obtained, with a yield of 81%.
ESI:m/z＝394.34[M+H]+.
1H NMR(400MHz,DMSO-d 6 )δ8.96(s,1H),7.45(s,1H),4.83(p,J＝6.1Hz,1H),4.42(t,J＝7.4Hz,1H),3.27(dd,J＝15.4,6.6Hz,1H),3.18(dd,J＝15.4,8.1Hz,1H),1.57–1.36(m,4H),1.23(s,24H),0.85(td,J＝6.9,2.2Hz,6H).
13 C NMR(100MHz,DMSO-d 6 )δ168.10,158.78,158.46,158.15,127.38,118.50,117.94,115.53,76.54,51.05,33.11,33.07,31.28,31.26,28.88,28.83,28.80,28.69,28.67,25.65,24.49,24.47,22.09,13.94.
Example 3
Synthesis of N-tert-Butoxycarbonyl-L-histidine- (2-hexyl) -decyl ester (Compound 5) and L-histidine- (2-hexyl) -decyl ester bistrifluoroacetate (chemical 6)
The synthetic routes for compound 5 and compound 6 are as follows:
Figure BDA0003564838190000121
N-tert-Butoxycarbonyl-L-histidine (300mg,1.18mmol,1eq.), EDCI (563mg,2.95mmol,2.5eq.), DMAP (57mg,0.47mmol,0.4eq.) were added to a 25mL reaction flask and N was performed 20 times with a three-way adapter 2 The reaction flask was replaced and heated with hot air to remove moisture in the flask. 2-hexyl-decanol (1.7mL,5.9mmol,5eq.), DIPEA (1.3mL,7.55mmol,6.4eq.) and DCM (8mL) were mixed and added to the reaction flask. The reaction was carried out at room temperature, followed by LC-MS, and stopped after 26 h. The reaction was poured into 40mL of DCM, and 40mL of saturated aqueous sodium bicarbonate was added for extraction, the aqueous layer was washed with DCM 4 times, the organic layers were combined, dried over sodium sulfate, filtered, concentrated, and the crude product was purified by column chromatography using silica gel column, eluent DCM/MeOH 45:1-40:1-30:1, to give 436mg of compound 5 as a colorless oil in 77% yield.
Characterization data for compound 5 are as follows:
ESI:m/z＝480.50[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.53(d,J＝1.1Hz,1H),6.79(s,1H),5.78(s,1H),4.54(d,J＝7.9Hz,1H),3.99(d,J＝5.6Hz,2H),3.09(d,J＝5.2Hz,2H),1.64–1.55(m,1H),1.42(s,9H),1.25(s,24H),0.87(t,J＝6.7Hz,6H).
a25 mL reaction flask was charged with Compound 5(300mg,0.63mmol), and the atmosphere in the flask was purged with N 2 After displacement, DCM (6mL) was added followed by trifluoroacetic acid (1.5 mL). The reaction was carried out at room temperature, followed by LC-MS, and stopped after 2.5 h. Concentrating the reaction solution under reduced pressure at normal temperature, adding 10mL of ethanol, removing ethanol under reduced pressure at normal temperature, adding 10mL of DCM, removing DCM under reduced pressure at normal temperature, pumping with oil for 1h to obtain 448.6mg of white powdery compound 6 with a yield of 99%.
Characterization data for compound 6 are as follows:
ESI:m/z＝380.43[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ8.46(s,1H),7.29(s,1H),4.53–4.43(m,1H),4.24–4.07(m,2H),3.64–3.29(m,2H),1.68(s,1H),1.35–1.16(m,24H),0.86(t,J＝6.7Hz,6H).
13 C NMR(100MHz,DMSO-d 6 )δ168.48,158.82,158.50,134.60,127.40,118.50,117.91,115.54,68.24,51.13,36.45,31.31,31.25,30.15,29.26,29.23,28.97,28.93,28.90,28.69,26.00,25.92,25.61,22.11,22.08,13.94.
example 4
Synthesis of N-tert-Butoxycarbonyl-L-histidine-oleyl ester (Compound 7) and L-histidine-oleyl ester bistrifluoroacetate (Compound 8)
The synthetic routes for compound 7 and compound 8 are as follows:
Figure BDA0003564838190000131
N-tert-Butoxycarbonyl-L-histidine (300mg,1.18mmol,1eq.), oleyl alcohol (1.4mL,3.54mmol,3.0eq.), EDCI (676mg,3.53mmol,3.0eq.), DMAP (57.5mg,0.47mmol,0.4eq.) were added to a 25mL reaction flask for 20N times using a three-way junction 2 The reaction flask was heated with hot air to remove moisture in the flask. DIPEA (1.3mL,7.55mmol,6.4eq.) and DCM (8mL) were mixed and added to the reaction flask. The reaction was followed by LC-MS at room temperature and stopped after 22 h. The reaction mixture was poured into 40mL of DCM, and 40mL of a saturated aqueous solution of sodium hydrogencarbonate was added thereto for extractionThe aqueous layer was washed 4 times with DCM, the organic layers were combined, dried over sodium sulfate, filtered, concentrated and the product purified on a silica gel column with the eluent DCM/MeOH50:1-40:1-35: 1. 380mg of compound 7 are obtained as a colourless oil in 63.6% yield.
Characterization data for compound 7 are as follows:
ESI:m/z＝506.46[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.53(s,1H),6.79(s,1H),5.78(s,1H),5.42–5.31(m,2H),4.51(s,1H),4.07(t,J＝6.7Hz,2H),3.09(d,J＝5.3Hz,2H),2.07–1.91(m,2H),1.66–1.52(m,2H),1.42(s,9H),1.37–1.19(m,24H),0.87(t,J＝6.7Hz,3H).
13 C NMR(100MHz,CDCl 3 )δ172.27,155.64,135.14,130.01,129.78,79.85,77.25,65.54,53.63,31.91,29.77,29.75,29.73,29.71,29.67,29.53,29.44,29.33,29.25,29.23,28.56,28.33,27.23,27.20,25.84,22.69,14.12.
a25 mL reaction flask was charged with Compound 7(260mg,0.51mmol), and the gas in the flask was purged with N 2 After displacement, DCM (6mL) was added followed by trifluoroacetic acid (1.5 mL). The reaction was carried out at room temperature, followed by LC-MS, and stopped after 2.5 h. Concentrating the reaction solution under reduced pressure at normal temperature, adding 10mL of ethanol, removing ethanol under reduced pressure at normal temperature, adding 10mL of DCM, removing DCM under reduced pressure at normal temperature, and pumping with oil for 1h to obtain 310mg of white powdery compound 8 with a yield of 95%.
Characterization data for compound 8 are as follows:
ESI:m/z＝406.44[M+H]+.
example 5
Synthesis of N-acetyl-L-histidine-9-heptadecyl ester (Compound 9)
The synthetic route for compound 9 is as follows:
Figure BDA0003564838190000141
9-heptadecanol (300mg,1.17mmol,1eq.) and 4mL DCM were added to a 25mL reaction flask, and after ice-cooling for 10min, N-acetyl-L-histidine (277mg,1.29mmol,1.1eq.) dispersed in 3mL DCM was added to the reaction2mL of dicyclohexylcarbodiimide (DCC,265.48mg,1.29mmol,1.1eq.) in DCM and 2mL of DMAP (200.07mg,1.64mmol,1.4eq.) in DCM were added to the flask, the reaction temperature was naturally raised to room temperature, then the flask was heated in a water bath at 50 ℃ and followed by LC-MS, and the reaction was stopped after 24 hours. The reaction mixture was filtered to remove the resulting white precipitate, washed with 40mL of DCM, and the resulting filtrate was washed with 40mL of 1M dilute aqueous hydrochloric acid to remove DMAP. Then 40mL of saturated NaHCO was added 3 Adjusting the organic layer to weak alkaline with water solution, separating the organic layer, drying with sodium sulfate, filtering, concentrating, and purifying the obtained product by silica gel column chromatography with DCM/MeOH50:1-40: 1-15:1 as eluent. 137mg of compound 9 are obtained as a pale yellow oil, yield 26%.
Characterization data for compound 9 are as follows:
ESI:m/z＝436.55[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.53(s,1H),7.23(d,J＝7.7Hz,1H),6.78(s,1H),4.82(p,J＝6.2Hz,1H),4.75(dt,J＝7.7,5.5Hz,1H),3.14–3.00(m,2H),1.99(s,3H),1.57–1.39(m,3H),1.22(s,26H),0.85(t,J＝6.8Hz,6H).
13 C NMR(100MHz,CDCl 3 )δ171.54,170.42,135.10,77.36,76.09,52.75,33.93,33.90,31.95,29.62,29.60,29.58,29.55,29.43,29.36,25.28,25.20,23.25,22.75,14.20.
example 6
1. Preparation of mRNA-entrapped Histidine Lipid Nanoparticles (HLNPs)
a) Lipid component treatment: dissolving Histidine Lipid (Histine-Lipid) and helper Lipid (including but not limited to DSPC, DOPE, etc.), cholesterol (Chol) and PEG-Lipid (including but not limited to PEG2k-DMG, PEG2000-C-DMG, PEG2000-DSPE, etc.) in anhydrous ethanol (EtOH).
b) mRNA treatment: mRNA was dissolved in a buffer solution of pH 3-4.
c) mRNA/HLNP preparation: the solutions obtained in steps a and b are mixed by microfluidics.
d) Buffer replacement: the product obtained in step c was diluted 10-fold with PBS buffer pH 7.4, and then ultrafiltered to the original volume at 4 ℃, and repeated twice.
A schematic diagram of the histidine lipid nanoparticle is shown in FIG. 1.
2. The mRNA/HLNP recipe (mg) is shown in Table 1 below.
TABLE 1
Figure BDA0003564838190000151
3. Characterization of
The recipe was drawn up as in table 1 above, 5 mRNA/HLNP samples were prepared using the process described in 1, and the particle size, PDI and Zeta potential were measured using a malvern laser particle sizer, and the results of the relevant measurements are shown in table 2 below.
TABLE 2
Figure BDA0003564838190000152
4. Encapsulation efficiency (agarose gel electrophoresis)
The encapsulation efficiency of mRNA in the above-mentioned mRNA/HLNP sample was examined by agarose gel electrophoresis under the conditions shown in Table 3 below, and the agarose gel electrophoresis pattern is shown in FIG. 2, in which Lane a is a positive control, Lane b is a negative control, and Lane 1-5 are mRNA/HLNP samples.
TABLE 3
Agarose gel concentration 0.9％
EB concentration 5μL/100mL
Constant current/constant voltage Constant current 0.10A
mRNA quality in lanes (except NC) 600ng
As can be seen from FIG. 2, no clear bright band was observed in Lane 1-5, indicating that none or very little of the mRNA/HLNP samples were present Is provided with Free mRNA exists, and the mRNA encapsulation efficiency is higher.
5. Animal experiments
5.1 fluorescence imaging of mice in vivo
BABL/C mice intramuscular injection contains 20 u g mRNA/HLNP samples, each sample injection of 3 mice, 24 hours after each mouse intraperitoneal injection of fluorescein potassium salt solution for 5 minutes, intraperitoneal injection of Sutai 50 and Sialazine mixed drug anesthesia of mice, after waiting for 5 minutes, using a small animal imaging instrument, Luc channel selection, exposure for 1 minute and picture taking, the results are shown in the following figure 3.
As can be seen in figure 3, all mice injected intramuscularly with mRNA/HLNP samples showed fluorescent expression after 24h, indicating that histidine-based HLNPs can efficiently deliver Fluc mRNA into cells and be translated into fluorescent proteins in vivo in mice.
5.2 immunogenicity experiments (luciferase antibody detection ELISA)
Mice were dosed intramuscularly twice, 3 mice per sample, and 20 μ g mRNA/HLNP samples per mouse per injection. One week after the second administration, the mice are subjected to orbital venous plexus blood sampling of 100-200 microliter blood, placed in a refrigerator at 4 ℃ for overnight and then serum is separated (the administration and blood sampling time is shown in figure 4), luciferase is coated on an ELISA plate, the mouse serum is diluted and then added into the ELISA plate (the antibody of the luciferase is used as a positive control, and the mixed solution of phosphate and Tween 20 is used as a negative control) after overnight at room temperature, the plate is washed after full reaction, an IgG-HRP antibody is added, the plate is washed after full reaction, TMB reaction solution is added, stop solution is added after full reaction to stop the reaction, the light absorption value is read on an ELISA reader by selecting the wavelength of 450nm, and the luciferase antibody is detected.
The results show (fig. 5), where PC in fig. 5 is a positive control, PC1:250, PC1: 500 and PC1:1000 represent dilution times, NC is a negative control, and NS is physiological saline, respectively. After 2 administrations of mRNA/HLNP prepared with His-C17 and His-C16, no luciferase antibody was detected in the serum of mice, showing no immunogenicity. In contrast, the serum of mice treated with modern LNP showed some immunogenicity when antibodies were detected. According to the literature, the main reason that modern LNP has the function of causing immune response is ionizable lipid, but our His-C16LNP and His-C17LNP do not show obvious immunogenicity, which indicates that the ionizable lipid synthesized based on histidine has little immunogenicity.
While this invention has described certain examples of the compositions and methods, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that these compositions and methods are susceptible to additional examples and that certain details may be varied from the embodiments described herein without departing from the basic principles of the invention.

Claims (10)

1. A histidine lipid, characterized in that: the histidine lipid is compound A and/or salt of the compound A, and the structural formula of the compound A is shown in the specification
Figure FDA0003564838180000011
Wherein R is 1 Selected from alkyl with 1-24 carbon atoms, and X is selected from H, COOR 2 Or COR 2 ，R 2 Selected from alkyl with 1-24 carbon atoms, R 1 And/or said R 2 Has a branched chain.
2. Histidine lipids according to claim 1, characterized in that: the R is 1 The R is 2 Are each independently selected from R 3 (R 4 ) n (ii) a Wherein R is 3 Selected from alkyl, alkenyl or alkynyl with 1-24 carbon atoms, R 4 Selected from alkyl, alkenyl or alkynyl with 1-23 carbon atoms,R 4 r and the product of the number of carbon atoms and n 3 Is less than or equal to 24 and n is selected from 0, 1, 2 or 3.
3. Histidine lipids according to claim 2, characterized in that: the R is 3 The R is 4 Are respectively selected from straight-chain alkyl, alkenyl or alkynyl,
when said R is 1 Is selected from R 3 (R 4 ) n And when n is 0, the R 1 R in (1) 3 Selected from alkyl, alkenyl or alkynyl with 10-24 carbon atoms;
alternatively, the first and second electrodes may be,
when said R is 1 Is selected from R 3 (R 4 ) n And n is 1, 2 or 3, the R 1 R in (1) 3 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 1 R in (1) 4 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms.
4. Histidine lipids according to claim 1, characterized in that: the R is 1 Is selected from
Figure FDA0003564838180000012
Figure FDA0003564838180000013
The R is 2 Is selected from
Figure FDA0003564838180000014
Figure FDA0003564838180000015
C(CH 3 ) 3 Or CH 3 。
5. Histidine lipids according to claim 4, characterized in that: the compound A is selected from
Figure FDA0003564838180000021
6. Histidine lipids according to any one of claims 1 to 5, characterized in that: the structural formula of the salt of the compound A is
Figure FDA0003564838180000022
Wherein Y is an acid group.
7. Histidine lipids according to claim 6, characterized in that: the acid radical being selected from CF 3 COO - 、CH 3 COO - 、Cl - 、SO 4- Or Br - ；
And/or the presence of a gas in the atmosphere,
the salt of the compound A is selected from
Figure FDA0003564838180000023
Figure FDA0003564838180000031
8. A method of preparing histidine lipids as claimed in any one of claims 1 to 7, characterized in that: reacting compound B with an alcohol to form said compound a, and optionally reacting compound a with an acid to form a salt of said compound a; wherein the structural formula of the compound B is
Figure FDA0003564838180000032
X in the compound B is the same as X in the compound A;
the alcohol is R 1 OH, R in the alcohol 1 With R in said Compound A 1 The same;
the acid is H n Y n- And Y in the acid is the same as Y in the salt of the compound A.
9. Use of one or more of the histidine lipids of any one of claims 1 to 7 or produced by the method of claim 8 for the delivery of one or more of an mRNA, siRNA, miRNA or antisense oligonucleotide.
10. A histidine lipid nanoparticle, characterized in that: the histidine lipid nanoparticle comprises one or more of mRNA and the histidine lipid according to any one of claims 1 to 7 or the histidine lipid prepared by the preparation method according to claim 8.
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