CN117843540A - Methionine derivative, preparation method thereof and application of gene vector - Google Patents
Methionine derivative, preparation method thereof and application of gene vector Download PDFInfo
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- CN117843540A CN117843540A CN202311850711.3A CN202311850711A CN117843540A CN 117843540 A CN117843540 A CN 117843540A CN 202311850711 A CN202311850711 A CN 202311850711A CN 117843540 A CN117843540 A CN 117843540A
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- methionine
- dodecyl chloride
- chloride
- methionine dodecyl
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 239000013598 vector Substances 0.000 title claims description 11
- 150000002741 methionine derivatives Chemical class 0.000 title abstract description 5
- 150000001875 compounds Chemical class 0.000 claims abstract description 65
- 239000002114 nanocomposite Substances 0.000 claims abstract description 5
- -1 silver tetrafluoroborate Chemical compound 0.000 claims description 61
- 239000000047 product Substances 0.000 claims description 33
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 229910001494 silver tetrafluoroborate Inorganic materials 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 229930182817 methionine Natural products 0.000 claims description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 239000012043 crude product Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 claims description 5
- 239000013067 intermediate product Substances 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 150000001263 acyl chlorides Chemical class 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical group [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- 108020004459 Small interfering RNA Proteins 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 229910052786 argon Inorganic materials 0.000 claims description 2
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- 238000001035 drying Methods 0.000 claims description 2
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- 230000000171 quenching effect Effects 0.000 claims description 2
- 238000010898 silica gel chromatography Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 2
- 239000004055 small Interfering RNA Substances 0.000 claims 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims 1
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- 108091027967 Small hairpin RNA Proteins 0.000 claims 1
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- 230000000694 effects Effects 0.000 abstract description 9
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- 239000000243 solution Substances 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 22
- 239000005090 green fluorescent protein Substances 0.000 description 20
- 239000013612 plasmid Substances 0.000 description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 15
- 238000004896 high resolution mass spectrometry Methods 0.000 description 15
- 238000005481 NMR spectroscopy Methods 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 10
- 229960004452 methionine Drugs 0.000 description 10
- 238000001502 gel electrophoresis Methods 0.000 description 9
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 239000002105 nanoparticle Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- RGCKJSPKMTWLLX-UHFFFAOYSA-N 3-iodopropylbenzene Chemical compound ICCCC1=CC=CC=C1 RGCKJSPKMTWLLX-UHFFFAOYSA-N 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- KMGBZBJJOKUPIA-UHFFFAOYSA-N butyl iodide Chemical compound CCCCI KMGBZBJJOKUPIA-UHFFFAOYSA-N 0.000 description 5
- 230000000536 complexating effect Effects 0.000 description 5
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 5
- 238000013329 compounding Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- KJIFKLIQANRMOU-UHFFFAOYSA-N oxidanium;4-methylbenzenesulfonate Chemical compound O.CC1=CC=C(S(O)(=O)=O)C=C1 KJIFKLIQANRMOU-UHFFFAOYSA-N 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
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- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- RWSOTUBLDIXVET-UHFFFAOYSA-O sulfonium Chemical compound [SH3+] RWSOTUBLDIXVET-UHFFFAOYSA-O 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229940094991 herring sperm dna Drugs 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- 229930195722 L-methionine Natural products 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
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- 230000012010 growth Effects 0.000 description 1
- 230000009851 immunogenic response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- 125000005647 linker group Chemical group 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
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- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
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- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 150000003568 thioethers Chemical class 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to the technical field of chemical synthesis and application, in particular to methionine derivatives with a general formula (A), and discloses a preparation method of the compounds, wherein the compounds and genes are subjected to polycondensation to form nano-composites, the particle size of the composites is 150-300 nm, zeta potential is +8- +50, and the composites have higher uptake effect and transfection effect. The compound has the advantages of simple synthesis, higher yield, strong gene loading capacity and the application of transferring genes.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a methionine derivative, an organic synthesis preparation method thereof and application of the methionine derivative as a gene vector.
Background
The exposed exogenous gene is difficult to enter cells and is easy to be degraded by organisms, cells or environments, so that the effect of the gene is difficult to be exerted, and the gene vector is taken as a tool for introducing the gene into the cells, and carries the gene into the cells, so that the gene is difficult to be degraded, and the specific function of the target gene is exerted. Currently, there are viral vectors and nonviral vectors as genetic vectors. The clinical safety of viral vectors has been questioned because of their propensity to stimulate immunogenic responses and induce transgene insertion mutations. Non-viral vectors, lipid Nanoparticles (LNPs), cationic polymers, inorganic nanoparticles, etc., exhibit strong gene-loading ability, high safety and practicality, and are easy to prepare. Therefore, the development of the high-efficiency low-toxicity non-viral vector has wide application prospect. Cationic lipids are widely studied as gene delivery vehicles, structurally consisting of three parts, a positively charged head, a hydrophobic tail and a linking group. Most cationic lipid compounds use nitrogen atoms to form positive charge groups, and few reports of constructing gene vectors using sulfonium as a positive charge group are made. Recently, plum and the like have described a DNA nanodelivery system based on sulfonium salt center for neoantigen delivery and tumor immunotherapy (ACS Nano.2022, 16, 19509-19522), and have shown that DNA nanodevices with sulfonium center can provide an accurate, biocompatible and effective co-delivery vaccine platform for tumor immunotherapy and prevention. Li et al designed and synthesized sulfonium lipids as DNA delivery vehicles that could efficiently polycondense and transfer DNA into cells (Curr Drug Deliv.2023, 20 (7): 951-960). Sulfonium compounds as gene vectors are worthy of further development in terms of reduced cytotoxicity and increased transfection efficiency.
Disclosure of Invention
The invention takes methionine as a core, and synthesizes a series of derivatives containing sulfonium positive charges through chemical structure modification of carboxyl, amino and thioether of methionine. The compound generates electrostatic attraction through sulfonium positive ions and phosphate negative ions in nucleic acid, and the substituent group contains a hydrophobic long-chain group, so that a nano-composite is formed, and the hydrophobic chain is constructed through ester bonds and amide bonds and has degradability. The invention uses gel blocking experiment, nano particle diameter and zeta potential detection to prove that the compound can polycondense nucleic acid to form nano compound, the size of the compound is 150-300 nm and the surface potential is +8- +50mV, and the compound has higher uptake effect and transfection effect by using uptake experiment and transfection experiment. Experiments show that the compounds of the invention can be used as gene vectors.
The general formula A of the compound of the invention is as follows:
wherein the amino acid chirality is L configuration and D configuration;
R 1 ＝C 9 H 19 、C 11 H 23 、C 13 H 27 、C 15 H 31 、C 17 H 35 ；R 2 ＝CH 3 、C 4 H 9 、C 3 H 6 Ph。
the compounds of the above formula may be:
S-methyl-N-decanoyl-D-methionine dodecyl chloride (A1),
S-methyl-N-lauroyl-D-methionine dodecyl chloride (A2),
S-methyl-N-tetradecanoyl-D-methionine dodecyl chloride (A3),
S-methyl-N-hexadecyl-D-methionine dodecyl chloride (A4),
S-methyl-N-octadecanoyl-D-methionine dodecyl chloride (A5),
S-butyl-N-decanoyl-D-methionine dodecyl chloride (A6),
S-butyl-N-dodecanoyl-D-methionine dodecyl chloride (A7),
S-butyl-N-tetradecanoyl-D-methionine dodecyl chloride (A8),
S-butyl-N-hexadecyl-D-methionine dodecyl chloride (A9),
S-butyl-N-octadecanoyl-D-methionine dodecyl chloride (A10),
s- (3-phenyl-propyl) -N-decanoyl-D-methionine dodecyl chloride (A11),
s- (3-phenyl-propyl) -N-dodecanoyl-D-methionine dodecyl chloride (A12),
s (3-phenyl-propyl) -N-tetradecoyl-D-methionine dodecyl chloride (A13),
s- (3-phenyl-propyl) -N-hexadecyl-D-methionine dodecyl chloride (A14),
s- (3-phenyl-propyl) -N-octadecanoyl-D-methionine dodecyl ester chloride (A15)
The preparation method of the compounds A1-A15 is shown in Scheme 1, and corresponding reaction compounds are selected according to different substituents to generate products from methionine.
Scheme 1
A method for synthesizing a compound having the general formula a: methionine (1 eq) and n-dodecanol (1.2 eq) are dissolved in dry toluene, p-toluene sulphonic acid (1 eq) is added, reflux is carried out for 3d, cooling, the solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out by silica gel to obtain intermediate product methionine dodecyl. Under the protection of argon, methionine dodecyl (1 eq) is dissolved in dry DMF (5 mL), triethylamine (1.5 eq) is added, acyl chloride (1.2 eq) with different chain lengths is slowly dripped into the mixture at 0 ℃ for reaction for 30min, stirring is carried out at room temperature for 1h, water quenching is carried out, dichloromethane extraction is carried out, a dichloromethane layer is reserved, saturated saline water is used for washing, anhydrous sodium sulfate is used for drying, solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out by using silica gel to obtain an intermediate product X. Intermediate X (1 eq) and the different iodoalkanes (1 eq) were dissolved in dry acetonitrile solution, silver tetrafluoroborate (1.2 eq) was added, magnetically stirred in an oil bath at 65 ℃ for 24h, and then cooled to room temperature. Filtering to remove precipitate, exchanging with strong alkali chloride ion exchange resin for 2h, evaporating solvent under reduced pressure to obtain crude product, and separating with silica gel column chromatography to obtain compound with general formula A. The compounds are characterized by mass spectrum and nuclear magnetism, represented by compounds A1, A6 and A11, and are shown in figures 1-9.
The compounds A1-A15 of the invention have gene vector application, and green fluorescent protein genes (gWiz-GFP, 5757bp, alvetron company) and short-chain herring sperm DNA (hsDNA, 20bp, shanghai Yingxin laboratory equipment Co., ltd.) are selected as test genes in experiments.
The invention detects the compound capability of the compounds A1-A15 on gWiz-GFP plasmid and hsDNA by gel blocking experiments. The gel electrophoresis blocking experimental results of the compounds A1-A15 and gWiz-GF after being compounded according to different S/P ratios are shown in the accompanying drawings 10, 11 and 12, and the experiments show that the compounds A1-A15 can effectively block gWiz-GFP plasmids. The gel electrophoresis blocking experiment results of the compounds A1-A15 and hsDNA compounded according to different S/P ratios are shown in the accompanying drawings 13, 14 and 15, and the experiments show that the compounds A1-A15 can effectively block hsDNA.
The invention uses a nanometer particle size analyzer and a zeta potentiometer to detect the particle size and zeta potential of a compound formed by the compounds A1-A15, gWiz-GFP and hsDNA nucleic acid in a complete compounding proportion. The detection data of the nanocomposite formed by the compounds A1 to A15 with the two nucleic acids in the ratio of complete complexing are shown in Table 1. Particle size is 150-300 nm, and electromotive force is +8- +50 mV. The data indicate that the compounds form nanoparticles with nucleic acids that have cell penetrating ability.
TABLE 1 particle size and zeta potential results of the nanocomposites of Compounds A1-A15 with plasmid gWiz-GFP and hsDNA
The invention uses an uptake experiment to detect the cell uptake condition of the complex formed by A1-A15, cy5-gWiz-GFP and Cy5-siRNA, and uses a transfection experiment to detect the cell transfection condition of the complex formed by A1-A15 and gWiz-GFP plasmid, as shown in figures 16, 17 and 18, the complex shows better cell uptake and transfection effect.
Drawings
FIG. 1 is a mass spectrum of a compound A1 of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the compound A1 of the present invention;
FIG. 3 is a nuclear magnetic resonance carbon spectrum of the compound A1 of the present invention;
FIG. 4 is a mass spectrum of the compound A6 of the present invention;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of the compound A6 of the present invention;
FIG. 6 is a nuclear magnetic resonance carbon spectrum of the compound A6 of the present invention;
FIG. 7 is a mass spectrum of Compound A11 of the present invention;
FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of the compound A11 of the present invention;
FIG. 9 is a nuclear magnetic resonance carbon spectrum of the compound A11 of the present invention;
FIG. 10 shows gel electrophoresis patterns after compounding compounds A1-A5 with gWiz-GFP plasmid in different ratios;
FIG. 11 shows gel electrophoresis patterns of compounds A6-A10 in different ratios after complexing with gWiz-GFP plasmid;
FIG. 12 shows gel electrophoresis of compounds A11-A15 in different ratios in combination with gWiz-GFP plasmid;
FIG. 13 shows gel electrophoresis of compounds A1-A5 in different ratios with hsDNA plasmids;
FIG. 14 shows gel electrophoresis of compounds A6-A10 in different ratios with hsDNA plasmid;
FIG. 15 shows gel electrophoresis of compounds A11-A15 in different ratios after complexing with hsDNA plasmids;
FIG. 16 is a graph showing the effect of partial compounds and Cy5-gWiz-GFP plasmid complexes on uptake in HepG2 cells under serum-free conditions;
FIG. 17 is a graph showing the effect of partial compounds and Cy5-siRNA plasmid complexes on uptake in HepG2 cells under serum-free conditions;
FIG. 18 is a graph showing the effect of a portion of the compound and gWiz-GFP plasmid complex on transfection of green fluorescent protein genes in HepG2 cells under serum-free conditions;
Detailed Description
The synthesis, structural characterization and nucleic acid complex transfer ability experiments of the compounds of the present invention are described below by way of specific examples.
Experimental main materials and instruments
All chemical reagents were purchased from commercial sources and the chemical synthesis reactions were performed using GF254 TLC plates, using potassium permanganate staining, and all aqueous solutions were prepared from deionized water. The green fluorescent protein gene (gWiz-GFP) was purchased from Aldriron and cloned in E.coli-DH 5. Alpha (TIANGEN) and then extracted with the QIAGEN plasmid kit (QiagenEndoFree plasmid, sigma-Aldrich); short-chain herring sperm DNA (hsDNA, 20bp, shanghai glume laboratories equipment limited); pro-apoptotic genes (siRNA, 21bp, shanghai Biotechnology Co., ltd.); agarose (Biowest Agarose Co.); bruker Assend 600M AVANCE III HD nuclear magnetic resonance apparatus; a Thermo Finnigan mass spectrometer; thermo Nanodrop 2000C spectrophotometer; sub-Cell electrophoresis apparatus; universal Hood II gel imager (BIO-RAD Co.); nano-ZS90 nanometer particle size and Zeta potentiometer (Markov UK).
Example 1
Methionine dodecyl ester
L-methionine (4.25 g,28.5 mmol), lauryl alcohol (12.78 mL,57 mmol), P-toluenesulfonic acid monohydrate (P-TsOH) (5.42 g,28.5 mmol) and toluene (100 mL) were added sequentially to a 250mL round bottom flask, the reaction was split under reflux and monitored by TLC (dichloromethane: methanol=20:1). After 72h of reaction, the solvent is evaporated to dryness under reduced pressure to obtain a crude product, the crude product is separated by column chromatography with silica gel, and a mixed system of methanol and dichloromethane is selected for elution to obtain an intermediate product for standby. Methionine dodecyl ester (7.64 g,24.1mmol, 84.5%) was obtained.
Example 2
Methionine dodecyl ester (1.2 g,3.78mmol,1 eq.) was reacted with acyl chloride of varying chain length (4.56 mmol,1.2 eq.) triethylamine (0.633 ml,5.67mmol,1.5 eq.) to give product X as a white dry powder; r is R f =0.3 (dichloromethane-petroleum ether 1:5).
Example 3
S-methyl-N-decanoyl-D-methionine dodecyl chloride (A1),
product X (0.217 g,0.46 mmol) was reacted with methyl iodide (0.029 mL,0.46 mmol) and silver tetrafluoroborate (0.107 g,0.55 mmol) to give product A1 (0.185 g,0.38mmol, 82.9% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). 1 H NMR(CD 3 Cl 3 ，δ)：6.89(d，1H，NH)，4.58(td，1H，NHCHCH 2 )，4.12(dd，2H OCH 2 )，3.55-3.16(m，2H，SCH 2 )，2.93(d，1H，2SCH 3 )，2.36(ddd，2H，NHCHNH 2 )，2.26(dt，C0CH 2 ，2H)，1.62(ddd，1H，CH 2 )，1.28(d，5H)，0.88(d，2CH 3 ，6H). 13 C NMR(CD 3 Cl 3 ，δ)：174.63，170.65，66.39，50.79，40.50，36.03，31.91，30.05-28.88，28.44，26.44，25.81，25.52，24.93，22.69，14.11.HR-MS(ESI)m/z：Calcd for C 28 H 56 NO 3 S + {[M-Cl-]+}486.3975，found：486.4064。
Example 4
S-methyl-N-lauroyl-D-methionine dodecyl chloride (A2),
product X (0.319 g,1.2 mmol) was reacted with methyl iodide (0.075 mL,1.2 mmol) and silver tetrafluoroborate (0.272 g,1.4 mmol) to give product A2 (0.504 g,0.98mmol, 81.6% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C 30 H 60 NO 3 S + {[M-Cl-]+}514.4288，found：514.4301。
Example 5
S-methyl-N-tetradecanoyl-D-methionine dodecyl chloride (A3),
product X (0.803 g,1.5 mmol) was reacted with methyl iodide (0.093 mL,1.5 mmol) and silver tetrafluoroborate (0.350 g,1.8 mmol) to give product A3 (0.460 g,0.84mmol, 56.5% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C 32 H 64 NO 3 S + {[M-Cl-]+}542.4601，found：542.4585。
Example 6
S-methyl N-hexadecyl-D-methionine dodecyl chloride (A4),
product X (1.069 g,1.9 mmol) was reacted with methyl iodide (0.118 mL,1.9 mmol) and silver tetrafluoroborate (0.447 g,2.3 mmol) to give product A4 (0.424 g,0.74mmol, 39.2% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C 34 H 68 NO 3 S + {[M-Cl-]+}570.4914，found：570.4924。
Example 7
S-methyl-N-octadecanoyl-D-methionine dodecyl chloride (A5),
product X (0.724 g,1.2 mmol) was reacted with methyl iodide (0.075 mL,1.2 mmol) and silver tetrafluoroborate (0.292 g,1.5 mmol) to give product A5 (0.3411 g,0.57mmol, 45.9% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C 36 H 72 NO 3 S + {[M-Cl-]+}598.5227，found：598.5226。
Example 8
S-butyl N-decanoyl-D-methionine dodecyl chloride (A6),
product X (0.604 g,1.3 mmol) was reacted with n-butyl iodide (0.147 mL,1.3 mmol) and silver tetrafluoroborate (0.311 g,1.6 mmol) to give product A6 (0.313 g,0.97mmol, 74.7% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). 1 H NMR(CD 3 Cl 3 ，δ)：6.90(d，1H，NH)，4.60(d，1H，NHCH)，4.14(t，，2H，OCH 2 )，3.53(d，1H，SCH 2 )，3.35(dtd，1H，SCH 2 )，2.97-2.88(m，1H，SCH 3 )，2.42-2.22(m，，COCH 2 ，NHCHCH 2 )，1.88-1.68(m，1H，2CH 2 )，1.58(dd，1H，CH 2 )，1.43-1.17(m，5H)，1.00(t，1H，CH 3 )，0.88(td，1H，2CH 3 ). 13 C NMR(CD 3 Cl 3 ，δ)：174.73，170.61，66.32，50.76，41.47，39.00，36.02，31.91，28.43，27.12-24.40，22.50，21.56，14.12，13.31.HR-MS(ESI)m/z：Calcd for C 31 H 62 NO 3 S+{[M-Cl-]+}：528.4445，found：528.4496。
Example 9
S-butyl N-dodecanoyl-D-methionine dodecyl chloride (A7),
product X (0.6278 g,1.3 mmol) was taken with n-butyl iodide (0.147 mL,1.3 mmol), silver tetrafluoroborate (0.311 g)1.6 mmol) to give product A7 (0.390 g,0.7mmol, 54.5% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C33H66NO3S + {[M-Cl-]+}556.4758，found：556.5110。
Example 10
S-butyl-N-tetradecanoyl-D-methionine dodecyl chloride (A8),
product X (0.470 g,0.9 mmol) was reacted with n-butyl iodide (0.102 mL,0.9 mmol) and silver tetrafluoroborate (0.214 g,1.1 mmol) to give product A9 (0.255 g,0.44mmol, 48.5% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C35H70NO3S + {[M-Cl-]+}584.5071，found：584.5491。
Example 11
S-butyl-N-hexadecyl-D-methionine dodecyl chloride (A9),
product X (0.963 g,1.7 mmol) was reacted with n-butyl iodide (0.193 mL,1.7 mmol) and silver tetrafluoroborate (0.389 g,2.0 mmol) to give product A9 (0.457 g,0.74mmol, 43.5% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C37H74NO3S + {[M-Cl-]+}612.5384，found：612.5624。
Example 12
S-butyl-N-octadecanoyl-D-methionine dodecyl chloride (A10),
product X (0.503 g,0.9 mmol) was reacted with n-butyl iodide (0.102 mL,0.9 mmol) and silver tetrafluoroborate (0.195 g,1.0 mmol) to give product A10 (0.230 g,0.36mmol, 40% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C39H78NO3S + {[M-Cl-]+}640.5697，found：640.6136。
Example 13
S- (3-phenyl-propyl) N-decanoyl-D-methionine dodecyl chloride (A11),
product X (0.210 g,0.45 mmol) was taken and reacted with 1-iodo-3-phenylpropane (0.071 mL,0.45 mmol), silver tetrafluoroborate (0.105 g,0.54 mmol)Product a11 (0.256 g,0.433mmol, 96.7% yield) was obtained as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). 1 H NMR(CD 3 Cl 3 ，δ)：7.33-7.26(m，1H)，7.20(ddd，1H)，6.89(d，1H，NH)，4.58(td，1H，NHCHCH 2 )，4.12(dd，2H OCH 2 )，3.55-3.16(m，2H，SCH 2 )，2.93(d，1H，2SCH 3 )，2.36(ddd，2H，NHCHNH 2 )，2.26(dt，COCH 2 ，2H)，1.62(ddd，1H，CH 2 )，1.28(d，5H)，0.88(d，2CH 3 ，6H). 13 C NMR(CD 3 Cl 3 ，δ)：174.71，170.53，138.97，129.65-127.57，126.89，66.39，50.69，42.01-40.35，39.25，36.06，34.27-33.43，31.91，30.54-28.75，28.43，27.01-24.67，22.69，14.12.HR-MS(ESI)m/z：Calcd for C 36 H 64 NO 3 S+{[M-Cl-]+}：590.4601，found：590.4840。
Example 14
S- (3-phenyl-propyl) -N-dodecanoyl-D-methionine dodecyl chloride (A12),
product X (0.604 g,1.2 mmol) was reacted with 1-iodo-3-phenylpropane (0.189 mL,1.2 mmol) and silver tetrafluoroborate (0.280 g,1.44 mmol) to give product A12 (0.720 g,1.12mmol, 97.1% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C38H68NO3S+S + {[M-Cl-]+}618.4914，found：618.4998。
Example 15
S- (3-phenyl-propyl) -N-tetradecoyl-D-methionine dodecyl chloride (A13),
product X (0.553 g,1.0 mmol) was reacted with 1-iodo-3-phenylpropane (0.157 mL,1.0 mmol) and silver tetrafluoroborate (0.234 g,1.2 mmol) to give product A13 (0.629 g,0.97mmol, 97.3% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C40H72NO3S + {[M-Cl-]+}646.5227，found：646.5325。
Example 16
S- (3-phenyl-propyl) N-hexadecyl-D-methionine dodecyl chloride (A14),
product X (0.559 g,1.0 mmol) was reacted with 1-iodo-3-phenylpropane (0.157 mL,1.0 mmol) and silver tetrafluoroborate (0.234 g,1.2 mmol) to give product A14 (0.616 g,0.913mmol, 91.3% yield as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C42H76NO3S + {[M-Cl-]+}674.5540，found：674.5602。
Example 17
S- (3-phenyl-propyl) -N-octadecanoyl-D-methionine dodecyl ester chloride (A15)
Product X (1.0 g,1.7 mmol) was reacted with 1-iodo-3-phenylpropane (0.268 mL,1.7 mmol) and silver tetrafluoroborate (0.389 g,2.0 mmol) to give product A15 (1.128 g,1.19mmol, 94.6% yield) as a yellow oil; r is R f =0.27 (methanol-dichloromethane 1:20). HR-MS (ESI) m/z: calcd for C44H80NO3S + {[M-Cl-]+}702.5853，found：702.5992。
Gel electrophoresis blocking experiment
A1-A15 was prepared as 0.46 nmol/. Mu.L, 0.92 nmol/. Mu.L, 1.85 nmol/. Mu.L, 3.7 nmol/. Mu.L, 5.52 nmol/. Mu.L, and 7.36 nmol/. Mu.L solutions in DMSO 2. Mu.L, and each was mixed with deionized water (4. Mu.L) to prepare a total volume of 6. Mu.L solutions. gWiz-GFP is prepared into 100 ng/. Mu.L by deionized water, 3uL (300 ng) is added into prepared A1-A15 samples with different concentrations, vortex shaking is carried out for 1S, then standing culture is carried out for 15min at 37 ℃, and A/gWiz-GFP complexes with S/P ratios of 1/2, 1/1, 2/1, 4/1, 6/1 and 8/1 are prepared for standby.
A1-A15 was prepared as 0.33 nmol/. Mu.L, 0.66 nmol/. Mu.L, 1.32 nmol/. Mu.L, 2.64 nmol/. Mu.L, 5.28 nmol/. Mu.L, and 10.56 nmol/. Mu.L of solution 2. Mu.L with DMSO, and each was mixed with deionized water (4. Mu.L) to prepare a total volume of 6. Mu.L solution. The hsDNA is prepared into 300 ng/. Mu.L by deionized water, 3uL (900 ng) is added into the prepared A1-A15 samples with different concentrations, vortex shaking is carried out for 1S, then the mixture is subjected to static culture at 37 ℃ for 15min, and A/hsDNA complexes with S/P ratios of 1/8, 1/4, 1/2, 1/1, 2/1 and 4/1 are prepared for standby.
0.35g of agarose was weighed and placed in a 100mL flask, and 35mL of tris (hydroxymethyl) aminomethane acetate buffer (TAE) was added to prepare a 1% agarose gel. After the liquid is heated, 1 mu L of ethidium bromide (EB 1 mg/mL) is added, the mixture is poured into an organic glass tank, a comb is inserted, the comb is taken off when the gel is completely solidified, and agarose gel is put into an electrophoresis tank for standby.
Each of the A/gWiz-GFP and A/hsDNA complex samples was added to 1. Mu.L of bromophenol blue mixture to form a volume of 10. Mu.L, and the mixture was added to a gel well, and the gel was subjected to electrophoresis at 120V for 20min with naked DNA as a control, and the experimental results were analyzed by a gel imager.
Detection of composite nano particle size and Zeta potential
The nano-particle size and Zeta potential of the complex were measured at a ratio of complete complexing of compounds A1-A15 with gWiz-GFP. Preparing the sulfonium compound to be detected into 1 nmol/mu L solution by using DMSO respectively for standby. At the complete compounding ratio of each sample and DNA, a corresponding amount of compound is diluted to 16 mu L in volume by ionized water, mixed with 8 mu L of DNA (containing 800ng of DNA) and vibrated for 1s, and then kept stand at room temperature for 20min, the compound is diluted to 1mL by deionized water, and the particle size and Zeta potential of the compound are measured by a Nano-ZS ZEN instrument.
The size of the nanoparticle of the complex and the Zeta potential were measured at a ratio of complete complexing of the compounds A1-A15 with hsDNA. Preparing the sulfonium compound to be detected into 1 nmol/mu L solution by using DMSO respectively for standby. At the complete compounding ratio of each sample and DNA, a corresponding amount of compound is diluted to 16 mu L in volume by ionized water, mixed with 8 mu L of DNA (containing 800ng of DNA) and vibrated for 1s, and then kept stand at room temperature for 20min, the compound is diluted to 1mL by deionized water, and the particle size and Zeta potential of the compound are measured by a Nano-ZS ZEN instrument.
Uptake experiments
Plasmid labelling was performed according to the procedure using a Cy5 labelling kit, and after labelling, the plasmid was collected in an enzyme-free EP tube, diluted to 100 ng/. Mu.L with sterile water and stored at-20℃under light-shielding for further use. The compounds A1-A15 were dissolved in DMSO to prepare A1 nmol/. Mu.L solution for use.
The stably growing, logarithmic growth phase HepG2 cells were selected and 150 μl of the cell suspension was seeded in a 96-well plate at 6000 cells/well. At 37 ℃,5% CO 2 Culture in incubatorAfter 12h of culturing until the cells adhere to the wall, the culture solution is replaced by serum-free culture solution. Mixing Cy5-siRNA or Cy5-gWiz-GFP plasmid (2 μL,100 ng/. Mu.L) with compound A1-A15 solution with corresponding S/P ratio, vibrating slightly, incubating for 20min in dark place, adding the mixture into HepG2 cell, standing at 37deg.C, and placing 5% CO 2 Culturing in a concentration incubator for 4h, removing culture solution, washing with PBS solution for 5min×3 times, adding 4% paraformaldehyde solution, standing for 30min, fixing cells, washing with PBS solution for 15min×3 times, adding 0.1% triton solution, standing for 20min, perforating cell membranes, washing with PBS solution for 20min×3 times again, fixing cell climbing sheet on a glass slide with DAPI-containing sealing tablet, staining cell nuclei, observing fluorescence distribution under a Carl Zeiss lnverted Microscope (Axiovert 5 digital) fluorescence microscope, wherein Cy5 excitation and emission wavelengths are 649/670 respectively, and DAPI excitation and emission wavelengths are 364/454 respectively.
Transfection experiments
HepG2 cells in the logarithmic growth phase were selected for stable growth, and 150. Mu.L of the cell suspension was seeded at 8000 cells/well in 96-well plates. At 37 ℃,5% CO 2 After culturing in the incubator for 12 hours until the cells adhere to the wall, the culture solution is replaced with a serum-free culture solution. Mixing gWiz-GFP (2. Mu.L, 100 ng/. Mu.L) with corresponding S/P ratio compound A1-A15 solution, vibrating slightly, incubating for 20min, adding the mixture into HepG2 cells, standing at 37deg.C, 5% CO 2 Culturing in a concentration incubator for 4 hr, removing culture solution, washing with PBS solution for 2 times, adding 10% FBS culture solution, standing at 37deg.C, and 5% CO 2 After 48h incubation in incubator, the plates were removed and observed under a Carl Zeiss lnverted Microscope (Axiovert 5 digital) fluorescence microscope, with gWiz-GFP excitation and emission wavelengths of 488nm/507nm, respectively.
Claims (5)
1. A compound of formula (a):
wherein the amino acid chirality is L configuration and D configuration; r is R 1 ＝C 9 H 19 、C 11 H 23 、C 13 H 27 、C 15 H 31 、C 17 H 35 ；R 2 ＝CH 3 、C 2 H 5 、C 3 H 7 、C 4 H 9 、C 3 H 6 Ph。
2. A process for the preparation of a compound of formula (a) according to claim 1, which is synthesized by the following route:
the main operation steps are as follows: methionine (1 eq) and n-dodecanol (1 eq) are dissolved in dry toluene, p-toluene sulphonic acid (1 eq) is added, reflux is carried out for 3d, cooling, the solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out by silica gel to obtain an intermediate product methionine dodecyl. Under the protection of argon, methionine dodecyl (1 eq) is dissolved in dry DMF (5 mL), triethylamine (1.5 eq) is added, acyl chloride (1.2 eq) with different chain lengths is slowly dripped into the mixture at 0 ℃ for reaction for 30min, stirring is carried out at room temperature for 1h, water quenching is carried out, dichloromethane extraction is carried out, a dichloromethane layer is reserved, saturated saline water is used for washing, anhydrous sodium sulfate is used for drying, solvent is evaporated under reduced pressure to obtain a crude product, and column chromatography separation is carried out by using silica gel to obtain an intermediate product X. Intermediate X (1 eq) and the different iodoalkanes (1 eq) were dissolved in dry acetonitrile solution, silver tetrafluoroborate (1.2 eq) was added, magnetically stirred in an oil bath at 65 ℃ for 24h, and then cooled to room temperature. Filtering to remove precipitate, exchanging with strong alkali chloride ion exchange resin for 2h, evaporating solvent under reduced pressure to obtain crude product, and separating with silica gel column chromatography to obtain product A.
3. A compound of formula (a) according to claim 1, wherein is any one of the following compounds:
S-methyl-N-decanoyl-D-methionine dodecyl chloride (A1),
S-methyl-N-lauroyl-D-methionine dodecyl chloride (A2),
S-methyl-N-tetradecanoyl-D-methionine dodecyl chloride (A3),
S-methyl-N-hexadecyl-D-methionine dodecyl chloride (A4),
S-methyl-N-octadecanoyl-D-methionine dodecyl chloride (A5),
S-butyl-N-decanoyl-D-methionine dodecyl chloride (A6),
S-butyl-N-dodecanoyl-D-methionine dodecyl chloride (A7),
S-butyl-N-tetradecanoyl-D-methionine dodecyl chloride (A8),
S-butyl-N-hexadecyl-D-methionine dodecyl chloride (A9),
S-butyl-N-octadecanoyl-D-methionine dodecyl chloride (A10),
s- (3-phenyl-propyl) -N-decanoyl-D-methionine dodecyl chloride (A11),
s- (3-phenyl-propyl) -N-dodecanoyl-D-methionine dodecyl chloride (A12),
s- (3-phenyl-propyl) -N-tetradecoyl-D-methionine dodecyl chloride (A13),
s- (3-phenyl-propyl) -N-hexadecyl-D-methionine dodecyl chloride (A14),
s- (3-phenyl-propyl) -N-octadecanoyl-D-methionine dodecyl chloride (A15).
4. Use of a compound according to claims 1-3 for the preparation of a vector for use as a gene. Wherein the genes are: DNA selected from a reporter gene, an anti-cancer gene or a cytokine gene; and RNA selected from siRNA, miRNA, piRNA, dsRNA or shRNA.
5. The use according to claim 4, wherein the compound is useful for delivering a gene by forming a nanocomposite with the gene.
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