CN113248436A - Novel cross-linked molecule for protein targeted transportation and preparation method and application thereof - Google Patents
Novel cross-linked molecule for protein targeted transportation and preparation method and application thereof Download PDFInfo
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- CN113248436A CN113248436A CN202110393686.5A CN202110393686A CN113248436A CN 113248436 A CN113248436 A CN 113248436A CN 202110393686 A CN202110393686 A CN 202110393686A CN 113248436 A CN113248436 A CN 113248436A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
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- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 7
- ZPWOOKQUDFIEIX-UHFFFAOYSA-N cyclooctyne Chemical group C1CCCC#CCC1 ZPWOOKQUDFIEIX-UHFFFAOYSA-N 0.000 claims abstract description 7
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 7
- 230000008685 targeting Effects 0.000 claims abstract description 7
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims abstract description 6
- XKJCHHZQLQNZHY-UHFFFAOYSA-N phthalimide Chemical compound C1=CC=C2C(=O)NC(=O)C2=C1 XKJCHHZQLQNZHY-UHFFFAOYSA-N 0.000 claims abstract description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 84
- 238000006243 chemical reaction Methods 0.000 claims description 81
- 239000002904 solvent Substances 0.000 claims description 50
- 150000001875 compounds Chemical class 0.000 claims description 47
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 36
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 36
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 32
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 22
- 239000000741 silica gel Substances 0.000 claims description 22
- 229910002027 silica gel Inorganic materials 0.000 claims description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000002585 base Substances 0.000 claims description 20
- 238000012544 monitoring process Methods 0.000 claims description 20
- 238000000605 extraction Methods 0.000 claims description 17
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 16
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 16
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- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
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- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 13
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- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 claims description 8
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- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 8
- HJUGFYREWKUQJT-UHFFFAOYSA-N tetrabromomethane Chemical compound BrC(Br)(Br)Br HJUGFYREWKUQJT-UHFFFAOYSA-N 0.000 claims description 8
- AQRLNPVMDITEJU-UHFFFAOYSA-N triethylsilane Chemical compound CC[SiH](CC)CC AQRLNPVMDITEJU-UHFFFAOYSA-N 0.000 claims description 8
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- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 claims description 6
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- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 claims description 6
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- CEEHFWSNEHHQPJ-UHFFFAOYSA-N ethyl 5-hydroxy-7-oxo-2-phenyl-8-prop-2-enylpyrido[2,3-d]pyrimidine-6-carboxylate Chemical compound N1=C2N(CC=C)C(=O)C(C(=O)OCC)=C(O)C2=CN=C1C1=CC=CC=C1 CEEHFWSNEHHQPJ-UHFFFAOYSA-N 0.000 claims description 4
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- ZCSHNCUQKCANBX-UHFFFAOYSA-N lithium diisopropylamide Chemical compound [Li+].CC(C)[N-]C(C)C ZCSHNCUQKCANBX-UHFFFAOYSA-N 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- NQMRYBIKMRVZLB-UHFFFAOYSA-N methylamine hydrochloride Chemical compound [Cl-].[NH3+]C NQMRYBIKMRVZLB-UHFFFAOYSA-N 0.000 claims description 4
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
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Abstract
The invention discloses a novel cross-linking molecule for protein targeted transportation and a preparation method and application thereof, belonging to the technical field of protein targeted transportation, wherein the cross-linking molecule comprises a novel cross-linking molecule which takes tetraethylene glycol as a mother chain and contains cyclooctyne derivative groups, disulfide bonds and amido bonds, and a molecule which is synthesized by reference and contains a targeting group, a PH sensitive group and an azide group of a hydrophobic carbon chain; the preparation of the novel cross-linking molecule is: firstly, one end of the tri-tetraethylene glycol is derivatized and connected with a phthalimide structure, and the other end of the tri-tetraethylene glycol is connected with a sulfydryl structure; followed by the introduction of a disulfide bond at the thiol group, followed by hydrolysis to a primary amine at one end of the phthalimide and subsequent conjugation to an activated cyclooctyne derivative-containing molecule. The novel carrier-free protein targeted delivery system can be prepared by crosslinking with protein and connecting with molecules containing structures such as targeted groups. The invention can solve the problems of low efficiency, high toxicity and the like of a protein transportation system.
Description
Technical Field
The invention relates to the technical field of protein targeted transportation, in particular to a novel cross-linked molecule for protein targeted transportation and a preparation method and application thereof.
Background
Proteins are indispensable participants in various cellular processes, and protein therapy delivers proteins into cells to replace dysfunctional proteins, thereby providing a promising method for drug development of various diseases such as cancer, inflammation, lysosomal storage diseases and the like. Intracellular delivery of proteins also represents a useful strategy for cellular imaging and diagnostics, genome engineering, and synthetic biology. However, native proteins are mostly membrane impermeable and are susceptible to degradation by proteolytic enzymes in the cell, particularly in lysosomes. A carrier system for efficient delivery of functional proteins into cells is crucial to the advancement of protein-based therapeutics.
Intracellular protein delivery is of great interest in the fields of disease therapy, genetic engineering and synthetic biology. Current protein delivery systems typically rely on the fusion of gene proteins with membrane-penetrating tags and protein-encapsulating carriers based on cationic liposomes, polymers, inorganic nanomaterials. However, gene fusion systems are not suitable for delivery of native, non-fused proteins. However, the nanocarriers often require covalent modification of proteins, and the efficiency of loading of proteins and lysosome escape is low.
Aiming at the problems of low efficiency, high toxicity and the like of the existing protein transport system, a new protein high-efficiency low-toxicity transport system based on small molecule guiding and regulating is urgently needed to be developed.
Disclosure of Invention
1. Technical problem to be solved
The invention aims to solve the technical problem of providing a novel cross-linking molecule for protein targeted transportation and a preparation method and application thereof, so as to solve the problems of low efficiency, high toxicity and the like of a protein transportation system in the prior art.
2. Technical scheme
In order to solve the problems, the invention adopts the following technical scheme:
the primary object of the present invention is to provide a cross-linking molecule for targeted protein transport that cross-links with proteins.
The invention is realized in such a way that a novel cross-linking molecule for protein targeted transportation is a molecule containing cyclooctyne derivative groups, disulfide bonds, amide bonds and polyethylene glycol groups, and the chemical structural formula of the novel cross-linking molecule is as follows:
in the formula (I), R1Is a derivative compound containing cyclooctyne structure, R2A polyethylene glycol chain of 2 to 10 carbon atoms, R3is-PhCH2OH、-CH2CH2CH2COOH, etc.:
further, said R1The structural formula of (A) is a compound with alkynyl, which comprises the following structures:
further, said R2Is- (CH)2CH2O) n-, wherein n is 1-5.
Preferably, said R is2Is- (CH)2CH2O)3-, said R3is-PhCH2OH, said R1Has the chemical structural formula of
Accordingly, the chemical structural formulas of the cross-linking molecules are respectively shown as the following (I-1) and (I-2):
it is still another object of the present invention to provide a method for preparing the novel crosslinking molecule.
A preparation method of a novel cross-linking molecule for protein targeted transportation comprises the following steps:
s1, dissolving 3 equivalents of compound 1 in 12 equivalents of solvent 1, adding 1.2 equivalents of base 1 under an ice bath condition, and carrying out ice bath reaction for 0.5-1 hour, wherein the base 1 is any one of triethylamine, N-diisopropylethylamine, 1, 8-diazabicycloundece-7-ene and potassium carbonate, and the solvent 1 is any one of dichloromethane, tetrahydrofuran, dioxane, dimethylformamide, acetonitrile and acetone; dissolving 1 equivalent of paratoluensulfonyl chloride in 10 equivalents of solvent 1, adding the solution into the system, and reacting for 1-1.5 hours in ice bath; then, the reaction is carried out overnight at room temperature, and after the TLC monitoring reaction is finished, the product is purified to obtain a compound 2;
s2, dissolving 1 equivalent of the compound 2 obtained in the step S1 in 20 equivalents of solvent 2, adding 1.2 equivalents of phthalimide, adding 1.5 equivalents of base 2, and heating for reaction overnight, wherein the solvent 2 is at least one of dimethylformamide, tetrahydrofuran and dioxane, and the base 2 is any one of triethylamine, N-diisopropylethylamine, 1, 8-diazabicycloundecen-7-ene and potassium carbonate; after TLC monitoring reaction is completed, purifying the product to obtain a compound 3;
s3, dissolving 1 equivalent of the compound 3 obtained in the step S2 in 50 equivalents of solvent 3, stirring in ice bath, sequentially adding 1.5 equivalents of triphenylphosphine and 1.5 equivalents of carbon tetrabromide, and reacting in ice bath for 30 minutes, wherein the solvent 3 is any one of dichloromethane, tetrahydrofuran and dimethylformamide; then, the reaction is carried out overnight at room temperature, and after the TLC monitoring reaction is finished, the product is purified to obtain a compound 4;
s4, dissolving 1.5 equivalents of base 3 in 20 equivalents of solvent 4, adding 1.5 equivalents of trithiol under an ice bath condition, and carrying out ice bath reaction for 0.5-1 hour, wherein the base 3 is any one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium hydride, sodium methoxide, lithium diisopropylamide and potassium tert-butoxide, and the solvent 4 is any one of acetone, acetonitrile, dimethylformamide and dimethyl sulfoxide; then, 1 equivalent of the compound 4 obtained in step S3 was dissolved in 10 equivalents of the solvent 4 and added to the above system, and reacted at room temperature overnight; after TLC monitoring reaction is completed, purifying the product to obtain a compound 5;
s5, dissolving 1 equivalent of the compound 5 obtained in the step S4 in 20 equivalents of solvent 5, adding 20 equivalents of trifluoroacetic acid under ice bath conditions, then adding 1 equivalent of triethylsilane, and reacting at room temperature overnight, wherein the solvent 5 is dichloromethane; after TLC monitoring reaction is completed, purifying the product to obtain a compound 6;
s6, dissolving 1 equivalent of the compound 6 obtained in the step S5 in 10 equivalents of the solvent 6, stirring in an ice bath, and adding 1.2 equivalents of a base 4, wherein the base 4 is any one of triethylamine, N-diisopropylethylamine and 1, 8-diazabicycloundecen-7-ene; dissolving 1.2 equivalents of 2,2' -dithiodipyridine in 10 equivalents of solvent 6, adding the solution into the system, and reacting at room temperature overnight, wherein the solvent 6 is any one of tetrahydrofuran, dichloromethane and dioxane; after TLC monitoring reaction is completed, purifying the product to obtain a compound 7;
s7, dissolving 1 equivalent of the compound 7 obtained in the step S6 in 10 equivalents of a solvent 7, stirring in an ice bath, and adding 1 equivalent of acid 1, wherein the solvent 7 is any one of methanol, ethanol and tert-butyl alcohol, and the acid 1 is any one of formic acid, acetic acid and glacial acetic acid; followed by addition of 1 equivalent of R3-SH in 10 equivalents of solvent 7 and adding the above system, and reacting overnight at room temperature, wherein R is3-SH is a substance such as 4-mercaptobenzyl alcohol; after TLC monitoring reaction is completed, purifying the product to obtain a compound 8;
s8, dissolving 1 equivalent of the compound 8 obtained in the step S7 in 10 equivalents of a solvent 8, and stirring in an ice bath, wherein the solvent 8 is any one of methanol and ethanol; dissolving 4 equivalents of methylamine hydrochloride and 4 equivalents of alkali 5 in 20 equivalents of solvent 8 in a dropping funnel, and dropping the mixture into the reaction system to react at room temperature overnight, wherein the alkali 5 is any one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate and sodium bicarbonate; after TLC monitoring reaction is completed, purifying the product to obtain a compound 9;
s9, dissolving 1 equivalent of the compound 9 obtained in the step 8 in 10 equivalents of a solvent 9, and stirring in an ice bath, wherein the solvent 9 is any one of tetrahydrofuran, dichloromethane and dioxane; dissolving 1 equivalent of a compound 10 in 10 equivalents of a solvent 9, adding the solvent into the reaction system, adding 2 equivalents of a base 6, and reacting at room temperature overnight, wherein the base 6 is any one of triethylamine, N-diisopropylethylamine and 1, 8-diazabicycloundecen-7-ene, and the chemical structural formula of the compound 10 is as follows:
after the completion of the TLC monitoring reaction, the product was purified to obtain compound 11.
Still another object of the present invention is to provide a construction of a protein targeting delivery system.
The cross-linking molecules participate in the construction of a carrier-free protein targeted transportation system, and the molecules combined with the novel cross-linking molecules are molecules containing targeting groups, PH sensitive groups and azide groups of hydrophobic carbon chains; the protein combined with the novel cross-linking molecule comprises at least any one of bovine serum albumin, acetaldehyde dehydrogenase 2 and catalase.
Specifically, the chemical structural formula of the molecule containing the targeting group, the pH sensitive group and the azide group of the hydrophobic carbon chain is as follows:
in the formula R4Is a long chain alkyl chain, R5Is an alkyl chain of 1 to 10 carbon atoms, R6A group containing a targeting moiety, having the following structure:
in the formula (I) < CHEM > -N3Reacts with alkyne in the formula (II) to generate triazole five-membered ring, so that the formula (I) and the formula (II) are connected together to form the cross-linking molecule required by carrier-free protein targeted transportation.
Preferably, the chemical structural formula of the molecule containing the targeting group, the pH sensitive group and the azide group of the hydrophobic carbon chain is as follows:
3. advantageous effects
(1) The novel cross-linking molecule provided by the invention takes a polyethylene glycol chain as a basic skeleton, and can improve the water solubility of the molecule; and then performing derivatization at two ends of the skeleton, connecting groups which are easily hydrolyzed by related enzymes in cells, such as disulfide bonds and amido bonds, and connecting a cyclooctyne derivative structure, and performing cycloaddition reaction on the derivative and molecules containing target groups, acetal groups and azide groups of hydrophobic carbon chains to generate a triazole structure, thereby constructing cross-linked molecules connected with the protein. The invention can realize direct modification of protein and construct a carrier-free transportation system, thereby having the advantages of improving efficiency, reducing toxicity and the like during application.
(2) Based on the cross-linking molecules, the invention provides a carrier-free protein targeted transportation method, and constructs a novel protein transportation system. When the molecular targeting lysosome is applied, firstly, the molecular can target a target cell, namely a specific target head is needed, and after endocytosis, a lysosome membrane is damaged through a hydrophobic carbon chain released by acetal structure hydrolysis under an acidic condition so as to realize lysosome escape; enter cells, corresponding small molecules are removed under the catalysis of glutathione, and finally natural protein can be released, so that the purpose of treating related diseases through protein targeted delivery is realized. The present invention is applicable to the delivery of native, non-fused proteins without the need for covalent modification of the protein, with greater efficiency of loading of the protein and lysosomal escape.
In conclusion, the invention can solve the problems of low efficiency, high toxicity and the like of a protein transport system in the prior art, and is suitable for delivering natural and non-fused protein.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The technical solution of the present invention is further explained by the following embodiments.
Example 1
First, a part (I-1) of a cross-linking molecule for targeted protein transport was prepared, and the synthetic route was as follows:
the method specifically comprises the following steps:
(1) 15.3g of Compound 1' (78.77mmol) was dissolved in 20ml of ultra-dry dichloromethane, 4.4ml of triethylamine (31.66mmol) was added under ice bath, and the reaction was carried out for 30 minutes in ice bath; then, 5g of p-toluenesulfonyl chloride (26.23mmol) was dissolved in 20ml of dichloromethane, slowly added to the above system (about 30 minutes), and reacted in ice bath for 1 hour; then turn to room temperature for reaction overnight. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1: 4), the product was purified by extraction and through a silica gel column to give compound 2', which was found to weigh 6.5029g, giving a yield of 71.2%;
(2) 3.005g of Compound 2' (8.62mmol) were dissolved in 30ml of ultra-dry dimethylformamide, and 1.53g of phthalimide (10.4mmol) and 1.94 ml of 1, 8-diazabicycloundec-7-ene (13.0mmol) were added and the reaction was carried out in an oil bath at 90 ℃ overnight. After TLC (pure ethyl acetate) monitoring reaction is completed, the product is extracted and purified by a silica gel column to obtain a compound 3 ', and the weight of the compound 3' is detected to be 2.0907g, and the yield is 75%;
(3) 1.4282g of compound 3' (4.42mmol) is dissolved in 30ml of ultra-dry dichloromethane, stirred in ice bath, 1.74g of triphenylphosphine (6.63mmol) is added, stirred in ice bath for 15 minutes, then 2.2g of carbon tetrabromide (6.63mmol) is added, and the mixture reacts in ice bath for 30 minutes; the reaction was allowed to warm to room temperature overnight. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1:1), the product was purified by extraction and through a silica gel column to give compound 4', which was detected to have a weight of 1.4895g, with a yield of 87.3%;
(4) 0.23g of 60% sodium hydride (5.75mmol) was dissolved in 10ml of ultra-dry dimethylformamide, and 1.6g of trityl mercaptan (5.79mmol) was added under ice-bath conditions, followed by reaction for 30 minutes in ice bath; 1.4895g of Compound 4' (3.86mmol) was dissolved in 10ml of dimethylformamide, and the mixture was allowed to react overnight at room temperature. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate 2:1), the product was purified by extraction and by silica gel column to give compound 5', which was detected to have a weight of 1.86 g, yield 85%;
(5) 1.84g of Compound 5' (3.16mmol) was dissolved in 10ml of ultra-dry dichloromethane, and 10ml of trifluoroacetic acid and 0.5ml of triethylsilane (3.13mmol) were added under ice-bath conditions, and the mixture was allowed to turn to room temperature for overnight reaction. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 2:1), the product was purified by extraction and through a silica gel column to give compound 6', which was detected to be 0.7509g in weight and 70% yield;
(6) 0.7509g of the compound 6 '(2.21 mmol) thus obtained were dissolved in 15ml of extra dry dichloromethane, stirred in an ice bath, 0.37ml of triethylamine (2.66mmol) was added, then 0.5849g of 2,2' -dithiodipyridine (2.65mmol) was dissolved in 10ml of extra dry dichloromethane and added to the above system, and the mixture was allowed to react overnight at room temperature. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1:1), the product was purified by extraction and through a silica gel column to give compound 7', which was detected to have a weight of 0.7g, and a yield of 70.5%;
(7) 0.65g of Compound 7' (1.45mmol) was dissolved in 10ml of methanol, stirred in an ice bath, and 83. mu.L of glacial acetic acid (1.45mmol) was added, followed by dissolving 0.203g of 4-mercaptobenzyl alcohol (1.45mmol) in 5ml of methanol and adding the above system, and the reaction was allowed to proceed overnight at room temperature. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1:2), the product was purified by extraction and through a silica gel column to give compound 8 ', which was detected to be 0.4g in weight of compound 8', 58% yield;
(8) 0.3536g of Compound 8' (0.74mmol) was dissolved in 10ml of methanol, stirred in an ice bath, and 0.2g of methylamine hydrochloride (2.96mmol) and 0.1185 g of sodium hydroxide (2.96mmol) were dissolved in 15ml of methanol in a dropping funnel and dropped into the above reaction system, and the mixture was allowed to react overnight at room temperature. TLC (dichloromethane: methanol 10; 1) monitored completion of the reaction, followed by extraction and purification of the product through silica gel column gave compound 9', which was detected to be 165mg by weight, 64.1% yield;
(9) 46mg of compound 9 '(0.132 mmol) are dissolved in 5ml of super-dry tetrahydrofuran, stirred in an ice bath, 53mg of compound 10' (0.132mmol) are dissolved in 5ml of super-dry tetrahydrofuran and added to the reaction system, followed by 44. mu.LN, N-diisopropylethylamine (0.266mmol) and brought to room temperature for reaction overnight. After completion of the reaction monitored by TLC (petroleum ether: acetone ═ 1:1), the product was purified by extraction and through a silica gel column to give compound 11', which was detected to have a weight of 64mg and a yield of 76.5%.
Secondly, another part (II-1) of the cross-linking molecule for targeted protein transport is prepared, and the synthetic route is as follows:
the method specifically comprises the following steps:
(1) the following compounds were synthesized first according to literature reports (In vivo delivery of transcription factors with multiple functional oligonucleotides, nat. mate., 2015,14, 701.):
(2) 100mg of compound 12(0.081mmol) was dissolved in 5mL of methanol, stirred in an ice bath, and 36mg of sodium methoxide (0.666mmol) was added and reacted at room temperature overnight. After the completion of the reaction was monitored by TLC (acetonitrile: water: triethylamine: 10:2:0.1), the product was purified by silica gel column to obtain compound 13, which was detected to have a weight of 50mg of compound 13 and a yield of 55%.
Thirdly, the prepared compound 11' and compound 13 are used for crosslinking with a template protein, so as to construct a carrier-free protein targeted delivery system, and the synthetic route is as follows:
the method specifically comprises the following steps:
(1) 50mg of compound 11' (0.079mmol) obtained in example 1 was dissolved in 5mL of dichloromethane, stirred in ice bath, followed by dissolving p-nitrophenylchloroformate (35mg, 0.174mmol) in 5mL of dichloromethane and adding the above system, and finally 26. mu.L of 1, 8-diazabicycloundec-7-ene (0.174mmol) was added to the above system, and reacted at room temperature overnight. After the completion of the reaction was monitored by TLC (petroleum ether: acetone ═ 2:1), the product was purified by silica gel column to give compound 14, which was detected to have a weight of 40mg, yield 63.5%;
(2) 104mg of bovine serum albumin was weighed out and dissolved in 2.4mL of 1 XPBS, and 10mg of Compound 14 was dissolved in 400. mu.L of dimethyl sulfoxide and added to the above system, and reacted at room temperature overnight. After the reaction is finished, washing the reaction solution for 6 times (1 XPBS solution) by using an ultrafiltration tube (10KD), paying attention to that the content of dimethyl sulfoxide in the ultrafiltration tube is not more than 5 percent, and finally freeze-drying the solution in the ultrafiltration tube to obtain a product 15, wherein the protein ration is 85mg, and the yield is 81.7 percent;
(3) 100mg of Compound 15 was dissolved in 2.4mL of 1 XPBS, and 10mg of Compound 13 was dissolved in 400. mu.L of dimethyl sulfoxide and added to the above system, followed by reaction overnight at room temperature. After completion of the reaction, the reaction mixture was washed 6 times (1 XPBS solution) with an ultrafiltration tube (10 KD). Finally, the solution in the ultrafiltration tube was freeze-dried to give product 16 in a protein quantification of 62mg and a yield of 77.5%.
Example 2
First, a part of a cross-linking molecule for targeted protein transport (I-2) was prepared, and the synthetic route was as follows:
the method specifically comprises the following steps:
(1) 15.3g of Compound 1' (78.77mmol) are dissolved in 20ml of ultra-dry dichloromethane, 5.2ml of diisopropylethylamine (31.46mmol) are added under ice bath, and the reaction is carried out in ice bath for 30 minutes; then, 5g of p-toluenesulfonyl chloride (26.23mmol) was dissolved in 20ml of dichloromethane, slowly added to the above system (about 30 minutes), and reacted in ice bath for 1 hour; then turn to room temperature for reaction overnight. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1: 4), the product was purified by extraction and through a silica gel column to give compound 2', which was found to weigh 6.102g, giving a yield of 66.8%;
(2) 3.005g of Compound 2' (8.62mmol) were dissolved in 30ml of ultra-dry dimethylformamide, and 1.53g of phthalimide (10.4mmol) and 1.94 ml of 1, 8-diazabicycloundec-7-ene (13.0mmol) were added and the reaction was carried out in an oil bath at 90 ℃ overnight. After TLC (pure ethyl acetate) monitoring reaction is completed, the product is extracted and purified by a silica gel column to obtain a compound 3 ', and the weight of the compound 3' is detected to be 2.0907g, and the yield is 75%;
(3) 1.4282g of compound 3' (4.42mmol) is dissolved in 30ml of ultra-dry dichloromethane, stirred in ice bath, 1.74g of triphenylphosphine (6.63mmol) is added, stirred in ice bath for 15 minutes, then 2.2g of carbon tetrabromide (6.63mmol) is added, and the mixture reacts in ice bath for 30 minutes; the reaction was allowed to warm to room temperature overnight. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1:1), the product was purified by extraction and through a silica gel column to give compound 4', which was detected to have a weight of 1.4895g, with a yield of 87.3%;
(4) 0.2804g of 60% sodium hydride (7.01mmol) was dissolved in 10ml of ultra-dry tetrahydrofuran, 1.9378g of trityl mercaptan (7.01mmol) was added under ice bath conditions, and the mixture was reacted for 30 minutes in ice bath; 1.8053g of Compound 4' (4.674mmol) were dissolved in 10ml of tetrahydrofuran, and the mixture was allowed to stand at room temperature overnight. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 2:1), the product was purified by extraction and through a silica gel column to give compound 5', which was detected to have a weight of 2.6g, with a yield of 95.6%;
(5) 1.84g of Compound 5' (3.16mmol) was dissolved in 10ml of ultra-dry dichloromethane, and 10ml of trifluoroacetic acid and 0.5ml of triethylsilane (3.13mmol) were added under ice-bath conditions, and the mixture was allowed to turn to room temperature for overnight reaction. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 2:1), the product was purified by extraction and through a silica gel column to give compound 6', which was detected to be 0.7509g in weight and 70% yield;
(6) 0.9226g of the compound 6 '(2.72 mmol) thus obtained were dissolved in 15ml of ultra-dry dichloromethane, stirred in an ice bath, 0.54ml of diisopropylethylamine (3.27mmol) was added, then 0.7186g of 2,2' -dithiodipyridine (3.26mmol) was dissolved in 10ml of ultra-dry dichloromethane and added to the above system, and the mixture was allowed to react overnight at room temperature. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1:1), the product was purified by extraction and through a silica gel column to give compound 7', which was detected to have a weight of 0.6482g, with a yield of 53.2%;
(7) 0.65g of Compound 7' (1.45mmol) was dissolved in 10ml of methanol, stirred in an ice bath, and 83. mu.L of glacial acetic acid (1.45mmol) was added, followed by dissolving 0.203g of 4-mercaptobenzyl alcohol (1.45mmol) in 5ml of methanol and adding the above system, and the reaction was allowed to proceed overnight at room temperature. After the completion of the reaction was monitored by TLC (petroleum ether: ethyl acetate ═ 1:2), the product was purified by extraction and through a silica gel column to give compound 8 ', which was detected to be 0.4g in weight of compound 8', 58% yield;
(8) 1.1853g of Compound 8' (2.48mmol) was dissolved in 20ml of ethanol, stirred in an ice bath, and 0.67g of methylamine hydrochloride (9.92mmol) and 0.5567 g of potassium hydroxide (9.92mmol) were dissolved in 30ml of methanol in a dropping funnel and added dropwise to the above reaction system, and the mixture was allowed to react overnight at room temperature. After completion of the reaction was monitored by TLC (dichloromethane: methanol 10; 1), the product was purified by extraction and through a silica gel column to give compound 9', which was detected to have a weight of 270mg and a yield of 31.3%;
(9) 100mg of compound 9 '(0.288 mmol) are dissolved in 5mL of extra dry tetrahydrofuran, stirred in an ice bath, 133mg of compound 10' (0.345mmol) are dissolved in 5mL of extra dry tetrahydrofuran and added to the reaction system, followed by 0.4mL of triethylamine (2.88 mmol) and allowed to warm to room temperature for overnight reaction. TLC (petroleum ether: acetone ═ 1:1) monitored the completion of the reaction, followed by extraction and purification of the product through silica gel column gave compound 11 "which was detected to be 110mg by weight and 64.4% yield.
Secondly, another part of the cross-linking molecule (II-1) for targeted protein transport was prepared, the synthetic route is shown in example 1.
Thirdly, the prepared compound 11' and compound 13 are used for crosslinking with a template protein, thereby constructing a carrier-free protein targeting transportation system, and the synthetic route is as follows:
the method specifically comprises the following steps:
(1) 100mg of compound 11' (0.168mmol) prepared in example 1 was dissolved in 5mL of dichloromethane, stirred in ice bath, followed by dissolving 68mg of p-nitrophenyl chloroformate (0.337 mmol) in 5mL of dichloromethane and adding the above system, and finally adding 50. mu.L of 1, 8-diazabicycloundecen-7-ene (0.334mmol) to the above system, and reacting at room temperature overnight. After the completion of the reaction was monitored by TLC (petroleum ether: acetone ═ 2:1), the product was purified by silica gel column to give compound 14', which was detected to have a weight of 78mg and a yield of 61%;
(2) 100mg of Catalase (CAT) was dissolved in 9mL of a mixed solution of 1 XPBS buffer and 1mL of Dimethylsulfoxide (DMSO), and stirred at 25 ℃, 16mg of Compound 14' was dissolved in 1mL of Dimethylsulfoxide (DMSO) and added dropwise to the solution, followed by reaction at 25 ℃ overnight; after the reaction was completed, 30mL of 1 XPBS buffer was added to the reaction mixture to dilute the mixture. Then putting the solution into a dialysis bag (the molecular weight cut-off is 7000), dialyzing with 1x PBS buffer solution, and freeze-drying the dialyzate to obtain a product 15', wherein the protein passing ration is 70mg, and the yield is 70%;
(3) 50mg of product 15' was dissolved in 5mL of 1 XPBS buffer, stirred at 25 ℃, and 8mg of Compound 13 was dissolved in 1mL of dimethyl sulfoxide and added dropwise to the solution, and reacted at 25 ℃ overnight. After the reaction was completed, 15mL of 1 XPBS buffer was added to the reaction mixture for dilution. The solution was then placed in dialysis bags (molecular weight cut-off 7000) and dialyzed against 1 XPBS buffer, and the dialysate was freeze-dried to give product 16' in 80% yield at 40mg protein quantification.
From the above, the present invention can solve the problems of low efficiency, high toxicity, etc. of the protein transport system in the prior art, and is suitable for the delivery of natural and non-fusion proteins.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. A novel cross-linking molecule for protein targeted transportation is characterized in that the novel cross-linking molecule is a molecule containing a cyclooctyne derivative group, a disulfide bond, an amido bond and a polyethylene glycol group, and the structural formula of the novel cross-linking molecule is as follows:
in the formula (I), R1Is a derivative compound containing cyclooctyne structure, R2A polyethylene glycol chain of 2 to 10 carbon atoms, R3is-PhCH2OH or-CH2CH2CH2COOH。
2. The novel cross-linking molecule for targeted protein transport of claim 1, wherein R is1The structural formula of (A) is a compound with alkynyl, which comprises the following structures:
3. the novel cross-linking molecule for protein targeted transportation as claimed in claim 1, wherein R is selected from the group consisting of2Is a polyethylene glycol chain having 1 to 5 carbon atoms.
4. A preparation method of a novel cross-linking molecule for protein targeted transportation is characterized by comprising the following steps:
s1, dissolving 3 equivalents of compound 1 in 12 equivalents of solvent 1, adding 1.2 equivalents of base 1 under an ice bath condition, and carrying out ice bath reaction for 0.5-1 hour, wherein the base 1 is any one of triethylamine, N-diisopropylethylamine, 1, 8-diazabicycloundece-7-ene and potassium carbonate, and the solvent 1 is any one of dichloromethane, tetrahydrofuran, dioxane, dimethylformamide, acetonitrile and acetone; dissolving 1 equivalent of paratoluensulfonyl chloride in 10 equivalents of solvent 1, adding the solution into the system, and reacting for 1-1.5 hours in ice bath; then, the reaction is carried out overnight at room temperature, and after the TLC monitoring reaction is finished, the product is purified to obtain a compound 2;
s2, dissolving 1 equivalent of the compound 2 obtained in the step S1 in 20 equivalents of solvent 2, adding 1.2 equivalents of phthalimide, adding 1.5 equivalents of base 2, and heating for reaction overnight, wherein the solvent 2 is at least one of dimethylformamide, tetrahydrofuran and dioxane, and the base 2 is any one of triethylamine, N-diisopropylethylamine, 1, 8-diazabicycloundecen-7-ene and potassium carbonate; after TLC monitoring reaction is completed, purifying the product to obtain a compound 3;
s3, dissolving 1 equivalent of the compound 3 obtained in the step S2 in 50 equivalents of solvent 3, stirring in ice bath, sequentially adding 1.5 equivalents of triphenylphosphine and 1.5 equivalents of carbon tetrabromide, and reacting in ice bath for 30 minutes, wherein the solvent 3 is any one of dichloromethane, tetrahydrofuran and dimethylformamide; then, the reaction is carried out overnight at room temperature, and after the TLC monitoring reaction is finished, the product is purified to obtain a compound 4;
s4, dissolving 1.5 equivalents of base 3 in 20 equivalents of solvent 4, adding 1.5 equivalents of trithiol under an ice bath condition, and carrying out ice bath reaction for 0.5-1 hour, wherein the base 3 is any one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium hydride, sodium methoxide, lithium diisopropylamide and potassium tert-butoxide, and the solvent 4 is any one of acetone, acetonitrile, dimethylformamide and dimethyl sulfoxide; then, 1 equivalent of the compound 4 obtained in step S3 was dissolved in 10 equivalents of the solvent 4 and added to the above system, and reacted at room temperature overnight; after TLC monitoring reaction is completed, purifying the product to obtain a compound 5;
s5, dissolving 1 equivalent of the compound 5 obtained in the step S4 in 20 equivalents of solvent 5, adding 20 equivalents of trifluoroacetic acid under ice bath conditions, then adding 1 equivalent of triethylsilane, and reacting at room temperature overnight, wherein the solvent 5 is dichloromethane; after TLC monitoring reaction is completed, purifying the product to obtain a compound 6;
s6, dissolving 1 equivalent of the compound 6 obtained in the step S5 in 10 equivalents of the solvent 6, stirring in an ice bath, and adding 1.2 equivalents of a base 4, wherein the base 4 is any one of triethylamine, N-diisopropylethylamine and 1, 8-diazabicycloundecen-7-ene; dissolving 1.2 equivalents of 2,2' -dithiodipyridine in 10 equivalents of solvent 6, adding the solution into the system, and reacting at room temperature overnight, wherein the solvent 6 is any one of tetrahydrofuran, dichloromethane and dioxane; after TLC monitoring reaction is completed, purifying the product to obtain a compound 7;
s7, dissolving 1 equivalent of the compound 7 obtained in the step S6 in 10 equivalents of a solvent 7, stirring in an ice bath, and adding 1 equivalent of acid 1, wherein the solvent 7 is any one of methanol, ethanol and tert-butyl alcohol, and the acid 1 is any one of formic acid, acetic acid and glacial acetic acid; followed by addition of 1 equivalent of R3-SH in 10 equivalents of solvent 7 and adding the above system, and reacting overnight at room temperature, wherein R is3-SH is a substance such as 4-mercaptobenzyl alcohol; after TLC monitoring reaction is completed, purifying the product to obtain a compound 8;
s8, dissolving 1 equivalent of the compound 8 obtained in the step S7 in 10 equivalents of a solvent 8, and stirring in an ice bath, wherein the solvent 8 is any one of methanol and ethanol; dissolving 4 equivalents of methylamine hydrochloride and 4 equivalents of alkali 5 in 20 equivalents of solvent 8 in a dropping funnel, and dropping the mixture into the reaction system to react at room temperature overnight, wherein the alkali 5 is any one of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate and sodium bicarbonate; after TLC monitoring reaction is completed, purifying the product to obtain a compound 9;
s9, dissolving 1 equivalent of the compound 9 obtained in the step 8 in 10 equivalents of a solvent 9, and stirring in an ice bath, wherein the solvent 9 is any one of tetrahydrofuran, dichloromethane and dioxane; dissolving 1 equivalent of a compound 10 in 10 equivalents of a solvent 9, adding the solvent into the reaction system, adding 2 equivalents of a base 6, and reacting at room temperature overnight, wherein the base 6 is any one of triethylamine, N-diisopropylethylamine and 1, 8-diazabicycloundecen-7-ene, and the chemical structural formula of the compound 10 is as follows:
after the completion of the TLC monitoring reaction, the product was purified to obtain compound 11.
5. The method for preparing a novel crosslinking molecule according to claim 4, wherein in step S2, the heating reaction is carried out overnight by turning to 90 ℃ oil bath reaction overnight.
6. The method for preparing the novel crosslinked molecule according to claim 4, wherein the purification treatment is an extraction treatment, and then the product is purified by silica gel column.
7. The application method of the novel cross-linking molecule for protein targeted transportation is characterized in that the cross-linking molecule participates in the construction of a carrier-free protein targeted transportation system.
8. The method for using the novel cross-linking molecule for protein targeted delivery according to claim 7, wherein the molecule combined with the novel cross-linking molecule is a molecule containing a targeting group, a pH sensitive group and an azide group of a hydrophobic carbon chain.
9. The method of claim 8, wherein the protein bound to the novel cross-linking molecule comprises at least one of bovine serum albumin, acetaldehyde dehydrogenase 2, and catalase.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106478718A (en) * | 2016-09-28 | 2017-03-08 | 中国药科大学 | No copper catalysis click chemistry module molecule and its application in a medicament |
| CN108271357A (en) * | 2015-09-01 | 2018-07-10 | 免疫功坊股份有限公司 | To treat the molecule construct of graft rejection |
| CN111601619A (en) * | 2017-11-07 | 2020-08-28 | 里珍纳龙药品有限公司 | Hydrophilic linkers for antibody drug conjugates |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108271357A (en) * | 2015-09-01 | 2018-07-10 | 免疫功坊股份有限公司 | To treat the molecule construct of graft rejection |
| CN106478718A (en) * | 2016-09-28 | 2017-03-08 | 中国药科大学 | No copper catalysis click chemistry module molecule and its application in a medicament |
| CN111601619A (en) * | 2017-11-07 | 2020-08-28 | 里珍纳龙药品有限公司 | Hydrophilic linkers for antibody drug conjugates |
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