CN100546658C - The polyoxyethylene ligand and the synthetic method thereof that contain the hepatoma-targeting peptide - Google Patents
The polyoxyethylene ligand and the synthetic method thereof that contain the hepatoma-targeting peptide Download PDFInfo
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- CN100546658C CN100546658C CNB2006100257660A CN200610025766A CN100546658C CN 100546658 C CN100546658 C CN 100546658C CN B2006100257660 A CNB2006100257660 A CN B2006100257660A CN 200610025766 A CN200610025766 A CN 200610025766A CN 100546658 C CN100546658 C CN 100546658C
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Abstract
Contain the polyoxyethylene ligand and the synthetic method thereof of hepatoma-targeting peptide, belong to medicine and chemosynthesis technical field.The present invention has following structure: measure the hepatoma-targeting polypeptide earlier: 3-maleic amide benzoic acid-N-succinimide ester MBS coupling agent: amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer: solvent=1: 0.1～0.2: 1.1～3.3: 200～4000 weight ratios, add in the reactor then, under-4 ℃～room temperature condition, reacted 2～72 hours, and made the semifinished product of the polyoxyethylene ligand that contains polypeptide; With this semifinished product dialysis, lyophilizing, obtaining the yellow solid powder is the polyoxyethylene ligand that contains the hepatoma-targeting peptide of the present invention.Synthetic route and purification of products step are simple; The productive rate height; Contrast agent with its preparation can satisfy intravenous injection; The time of staying is long in vivo; Soluble in water, but degradation in vivo toxicity is low; The contrast agent that is widely used in nuclear magnetic resonance and blood pond; Biologically active and targeting help medical diagnosis, and detect effective to hepatocarcinoma.
Description
Technical field
The present invention relates to a kind of polyoxyethylene ligand that contains the hepatoma-targeting peptide, the invention further relates to the above-mentioned synthetic method that contains the polyoxyethylene ligand of hepatoma-targeting peptide, belong to medical science and chemosynthesis technical field.
Background technology
At life science, whether target polypeptide and targeted drug have targeting to special organ or diseased region, and aspects such as the early stage safety evaluation of gene therapy method all need the more detection tracking of science.Tracking can need not to kill laboratory animal and long-time the tracking is widely adopted owing to nuclear magnetic resonance detects.Have on the one hand targeting, long half time, contrast agent that relaxation rate is high can make organ imaging to be detected clear, to clinical magnetic resonance detect and the diagnosis of the state of an illness useful and become hot research in recent years.On the other hand, biochemical druies such as most of polypeptide, protein, DNA have very fast accretion rate (plasma clearance), and can so need often this requisite crucial medicament of injection, in patient blood, be kept stable pharmacology's concentration by immunity to guarantee it.
At present, magnetic resonance imaging contrast is generally the coordination compound of paramagnetic metal ion, its part mostly is and contains the linear of polyamino and many carboxyls and ring-type micromolecular compound, so the half-life is shorter, usually increases consumption in order to obtain sharp image or repeat administration etc. in the short time.U.S. Pat 4 647 447 is with the diethylenetriamine pentaacetic acid coordination compound, with the meglumine salify, makes the Magnevist Solution contrast agent, clinically extensive use.Though it and human body have better biocompatibility, the Magnevist Solution molecular weight is little, drains too fast, eliminate about 20～100 minutes of half-life, this contrast agent is tissue and organ selectivity not, and almost completely rushes down by liver row, and is undesirable to the radiography effect of liver and gall.Because accretion rate is too fast, cranium brain and spinal cord nuclear magnetic resonance need rechallenge in 30 minutes; Judge that patient belongs to adult's pathological changes or tumor recurrence, need to increase consumption; Increase this drug dose, just increased human body toxin expelling amount, also just improved the probability of negative interaction.And the Magnevist Solution contrast agent can not pass through complete blood brain barrier, can not enter other tissue of blood capillary barrier, as spinal cord, eye and testis etc.Make when oral agents is used and to be absorbed by gastric mucosa, more can not enter erythrocyte or be attached on the hemoglobin, thereby be only limited in blood plasma and transport.Magnevist Solution is unsuitable for using in the imaging of heart and angiopathy, and usually will be with so-called blood pond image contrast reinforcing agent.In a word, up to now, also do not have a kind of part to make to make crucial medicament or contrast agent to satisfy simultaneously and have targeting, characteristics such as long half time, relaxation rate height require.
Summary of the invention
The objective of the invention is to disclose a kind of polyoxyethylene ligand that contains the hepatoma-targeting peptide, prepare targeted drug or contrast agent has targeting, long half time, relaxation rate height with this part, consumption seldom and clear image, and toxicity is low, to hepatocarcinoma detect effective.
Another object of the present invention provides the synthesis technique of above-mentioned part.
In order to achieve the above object, the present invention is through finding the polyoxyethylated performance study that has end functional group for a long time, targeted drug can with polymeric contrast agent by covalent bonds, like this macromolecule not only prolonged contrast agent half-life, prolonged half-life, the while of targeted drug not only but also can carry out tracking and monitoring.Advantages such as that polyoxyethylene and derivant thereof (PEG) have is amphipathic, nontoxic, immunogenicity is low, good biocompatibility, kind are many are usually used in protein, the preparation of polypeptide bio-pharmaceutical, Western medicine additive and preparation bio-medical material.With PEG modification contrast agent, polyoxyethylated all advantages are arranged.Having the polyoxyethylene of holding functional group has many functions, the contrast agent of the polyoxyethylene modification of both-end amido for example, and its end group is an amido, can carry out the chemical modification of medicine.And for the modification that is used for polypeptide etc., the experiment condition gentleness of requirement.
A kind of polyoxyethylene ligand that contains the hepatoma-targeting peptide provided by the invention has following structure
In the formula, R
3Represent 3-maleic amide benzoic acid-N-succinimide ester (MBS) coupling agent residue-hepatoma-targeting polypeptide, structural formula is
R in the formula
4Represent sulfydryl to participate in the hepatoma-targeting polypeptide of reaction, hepatoma-targeting polypeptide order is FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH, and wherein the FAM representative is connected to the rhodamine fluorescein C of peptamine cardinal extremity
21H
12O
7
R
2Represent polyoxyethylene-diethyl pentaacetic acid block polymer segment, structural formula is
In the formula, R
1Represent polyoxyethylene, structural formula is
-CH
2CH
2-(OCH
2CH
2)
n-
N represents 22～500 integer.
The polyoxyethylene ligand that contains the hepatoma-targeting peptide of the present invention dissolves in N, and dinethylformamide, or dichloromethane, or chloroform, or dioxane, or dimethyl sulfoxide, or distilled water, or phosphoric acid PBS buffer solvent are the yellow solid powder.
Second technical problem that the present invention will solve provides a kind of synthetic method that contains the polyoxyethylene ligand of hepatoma-targeting peptide:
First step preparation contains the semifinished product of the polyoxyethylene ligand of polypeptide
Measure the hepatoma-targeting polypeptide earlier: 3-maleic amide benzoic acid N-succinimide ester MBS coupling agent: amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer: solvent=1: 0.1～0.2: 1.1～3.3: 200～4000 weight ratios, then they are added in the reactor, under-4C °～room temperature condition, reacted 2～72 hours, and made the semifinished product of the polyoxyethylene ligand that contains polypeptide;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of polypeptide
The semifinished product dialysis of the polyoxyethylene ligand that contains polypeptide that the first step is made, lyophilizing, obtaining the yellow solid powder is the polyoxyethylene ligand that contains the hepatoma-targeting peptide of the present invention;
Above-mentioned solvent is N, dinethylformamide, or dichloromethane, or chloroform, or dioxane, or dimethyl sulfoxide, or distilled water, or phosphoric acid PBS buffer solvent;
Above-mentioned amino-contained polymer is omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, and molecular formula is NH
2-R
2-NH
2, structural formula is
In the formula, R
1Represent polyoxyethylene, structural formula is
-CH
2CH
2-(OCH
2CH
2)
n-
N represents 22～500 integer.
The above-mentioned first step prepares the method for the semifinished product of the polyoxyethylene ligand that contains polypeptide and also can be undertaken by following three steps: A, preparation earlier contain the polyoxyethylene ligand semifinished product of coupling agent; In reactor, add omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, solvent, coupling agent,-4C °～room temperature reaction 0.5～72 hour, omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer: solvent: the weight ratio of coupling agent was 1: 10～1670: 0.06～6; The purification of B, the polyoxyethylene ligand semifinished product that contains coupling agent that A is made: dialysis, lyophilizing makes the highly finished product of the polyoxyethylene ligand that contains coupling agent; C, preparation contain the part of the polyoxyethylene modification of polypeptide: go on foot adding hepatoma-targeting polypeptide and solvent in the product that obtains second, measure the highly finished product of the polyoxyethylene ligand that contains coupling agent earlier: the hepatoma-targeting polypeptide: the weight ratio of solvent is 1: 0.06～1.3: 43～1010,-4C °～room temperature reaction 1～72 hour, make the semifinished product of the polyoxyethylene ligand that contains polypeptide.
Compared with prior art, beneficial effect of the present invention is:
1. because the polyoxyethylene ligand that contains the hepatoma-targeting polypeptide of the present invention is a macromole, therefore have amphipathic, nontoxic, immunogenicity is low, good biocompatibility, kind are many, stablize, be difficult for adhering to blood vessel wall, long half time.
2. because the polyoxyethylene ligand that contains polypeptide of the present invention is the peptide bond connection by easy degraded, so this macromole can be degraded in vivo, toxicity is little.
3. because the polyoxyethylene ligand that contains polypeptide of the present invention contains diethylenetriamine pentaacetic acid, so can be as the part of synthetic contrast agent, the contrast agent long half time, the relaxation rate height that make for part with it, can be under the very little situation of metering, the time of staying prolongs in vivo, make organ imaging to be detected clear, and since contain ester group can degradation in vivo, toxicity is little.
4. because the long half time that contains the polyoxyethylene ligand of polypeptide of the present invention, can guarantee in a period of time that therefore it keeps stable pharmacology's concentration in patient blood, can be used for blood pond contrast agent.
5. owing in the polyoxyethylene ligand that contains polypeptide target polypeptide being arranged,, can adhere to hepatoma carcinoma cell, hepatocarcinoma is detected and treats by polypeptide.
6. the polyoxyethylene ligand that contains polypeptide connects gadolinium again, can be used as contrast agent, and the half-life of targeting peptide has also prolonged, and can carry out the magnetic resonance tracking monitoring simultaneously.
7. the process route of the synthetic polyoxyethylene ligand that contains polypeptide of the present invention is simple, the operating condition gentleness, and the purification of products step is simple, product productive rate height.
Description of drawings
Fig. 1 be the embodiment of the invention 4 the polyoxyethylene ligand that contains polypeptide highly finished product proton nmr spectra (
1H NMR).
The specific embodiment
Feed end amido polyoxyethylene-diethyl pentaacetic acid block polymer of the present invention needs synthetic voluntarily.Synthetic method is seen Chinese invention patent application " terminal amino oxyethylene block polymer modified Gd coordination compound and synthetic method thereof ", and application number is 200510024217.7.Chinese invention patent application " liver cancer tissue specifically binding peptide that screening method obtains in the body and uses thereof " is seen in the preparation of the raw material hepatoma-targeting polypeptide in the said method, and application number is 200410017048.X.
First step preparation contains the polyoxyethylene ligand semifinished product of coupling agent
In reactor, add omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer 28mg, solvent N, dinethylformamide 2.5ml, coupling agent 3-maleic amide benzoic acid-N-succinimide ester (MBS) 3.8mg, room temperature reaction 0.5 hour, omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, solvent N, the weight ratio of dinethylformamide, coupling agent 3-maleic amide benzoic acid-N-succinimide ester (MBS) is 1: 89: 0.136, and the structural formula of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is
In the formula, R
1Represent polyoxyethylated structural formula to be
-CH
2CH
2-(OCH
2CH
2)
n-
N represents 45;
Make the polyoxyethylene ligand semifinished product that contains coupling agent, have following structure,
In the formula, R
5Represent 3-maleic amide benzoic acid-N-succinimide ester MBS coupling agent residue
R
2Represent the not polyoxyethylene-diethyl pentaacetic acid block polymer segment of amino-contained
Second step was contained the highly finished product of the polyoxyethylene ligand of coupling agent
Lyophilizing makes the highly finished product 25mg of the polyoxyethylene ligand that contains coupling agent, product yield 79.4%;
The 3rd step made the part that contains the modification of polypeptide polyoxyethylene
In containing the highly finished product 8.2mg of polyoxyethylene ligand of coupling agent, second product that obtain of step adds hepatoma-targeting polypeptide 10mg and solvent phosphoric acid PBS buffer solvent 1ml; The weight ratio that contains highly finished product, polypeptide and the solvent phosphoric acid PBS buffer solvent of the polyoxyethylene ligand of coupling agent is 1: 1.2: 122; Room temperature reaction 1 hour, make the part of the polyoxyethylene modification that contains polypeptide, its molecular formula is
R
3Represent MBS coupling agent residue-hepatoma-targeting polypeptide, be by second step contain butanimide functional group above the polyoxyethylene ligand of coupling agent and the sulfydryl on the cysteine in the polypeptide order (SH) functional group reactions obtains, and molecular formula is,
R in the formula
4Represent hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH;
The 4th step made the highly finished product of the polyoxyethylene ligand that contains polypeptide
Dialysis, lyophilizing makes the highly finished product 7.2mg of the polyoxyethylene ligand that contains polypeptide, yellow powder, product productive rate 54.5%.
Solvent N in the first step of embodiment 1, N-dimethyl acid imide can change dimethyl sulfoxide into, or dichloromethane, or chloroform, or dioxane, or distilled water, or phosphoric acid PBS buffer solvent.
First step preparation contains the polyoxyethylene ligand semifinished product of coupling agent
In reactor, add omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer 99mg, solvent N, dinethylformamide 3ml, coupling agent 3-maleic amide benzoic acid-N-hydroxy-succinamide ester (MBS) 6.0mg, room temperature reaction 2 hours, make the polyoxyethylene ligand semifinished product that contains coupling agent; Omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, solvent N, the weight ratio of dinethylformamide, coupling agent 3-maleic amide benzoic acid-N-hydroxy-succinamide ester (MBS) is 1: 30.3: 0.061; R in the structural formula of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer
1Representative
-CH
2CH
2-(OCH
2CH
2)
n-, n represents 113;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of coupling agent
Add water, dialysis, lyophilizing makes the highly finished product 81mg of the polyoxyethylene ligand that contains coupling agent, product yield 77.1%;
The preparation of the 3rd step contains the part crude product of polypeptide polyoxyethylene modification
In containing the highly finished product 9.9mg of polyoxyethylene ligand of coupling agent, second product that obtain of step adds hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH 3mg and solvent phosphoric acid PBS buffer solvent 10ml, the weight ratio that contains highly finished product, polypeptide and the solvent phosphoric acid PBS buffer solvent of the polyoxyethylene ligand of coupling agent is 1: 0.303: 1010, room temperature reaction 24 hours, make the part of the polyoxyethylene modification that contains polypeptide;
The preparation of the 4th step contains the highly finished product of the polyoxyethylene ligand of polypeptide
Dialysis, lyophilizing makes the highly finished product 8.2mg of the polyoxyethylene ligand that contains polypeptide, yellow powder, product productive rate 63.9%;
Solvent selection in the first step of embodiment 2 is identical with embodiment's 1;
Solvent PBS buffer solvent in the 3rd step of embodiment 2 can change N into, N-dimethyl acid imide, dimethyl sulfoxide, dichloromethane, chloroform, dioxane, distilled water.
First step preparation contains the polyoxyethylene ligand semifinished product of coupling agent
In reactor, add omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer 99mg, solvent phosphoric acid PBS buffer solvent 1ml, coupling agent 3-maleic amide benzoic acid-N-hydroxy-succinamide ester (MBS) 6.0mg, room temperature reaction 24 hours, make the polyoxyethylene ligand semifinished product that contains coupling agent; The weight ratio of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, solvent phosphoric acid PBS buffer solvent, coupling agent 3-maleic amide benzoic acid-N-hydroxy-succinamide ester (MBS) is 1: 10.1: 0.061; Omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is identical with embodiment 2;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of coupling agent
Add water, dialysis, lyophilizing makes the highly finished product 90mg of the polyoxyethylene ligand that contains coupling agent, product yield 85.7%;
The preparation of the 3rd step contains the part semifinished product of polypeptide polyoxyethylene modification
In containing the highly finished product 9.9mg of polyoxyethylene ligand of coupling agent, second product that obtain of step adds hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH 3mg and solvent N, N-dimethyl acid imide 1ml, the highly finished product, polypeptide and the solvent N that contain the polyoxyethylene ligand of coupling agent, the imido weight ratio of N-dimethyl is 1: 0.303: 101, room temperature reaction 24 hours, make the part of the polyoxyethylene modification that contains polypeptide;
The preparation of the 4th step contains the highly finished product of the polyoxyethylene ligand of polypeptide
Add water, dialysis, lyophilizing makes the highly finished product 8.5mg of the polyoxyethylene ligand that contains polypeptide, yellow powder, product productive rate 65.9%.
The first step contains the polyoxyethylene ligand semifinished product of coupling agent
In reactor, add omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer 44mg, solvent N, dinethylformamide 2.5ml, coupling agent 3-maleic amide benzoic acid-N-succinimide ester (MBS) 3.9mg, room temperature reaction 2 hours, make the polyoxyethylene ligand semifinished product that contains coupling agent; Omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, solvent N, the weight ratio of dinethylformamide, coupling agent 3-maleic amide benzoic acid-N-succinimide ester (MBS) is 1: 167: 0.06, the R in the structural formula of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer
1Representative-CH
2CH
2-(OCH
2CH
2)
n-
N represents 76,
Second step was contained the highly finished product of the polyoxyethylene ligand of coupling agent
Dialysis, lyophilizing makes the highly finished product 33mg of the polyoxyethylene ligand that contains coupling agent, product yield 68.9%;
The 3rd step made the part that contains the modification of polypeptide polyoxyethylene
In containing the highly finished product 11.5mg of polyoxyethylene ligand of coupling agent, second product that obtain of step adds hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH 5mg and solvent phosphoric acid PBS buffer solvent 10ml, the weight ratio that contains highly finished product, polypeptide and the solvent phosphoric acid PBS buffer solvent of the polyoxyethylene ligand of coupling agent is 1: 0.43: 869, room temperature reaction 24 hours, make the part of the polyoxyethylene modification that contains polypeptide;
The 4th step made the highly finished product of the polyoxyethylene ligand that contains polypeptide
Dialysis, lyophilizing makes the highly finished product 12.1mg of the polyoxyethylene ligand that contains polypeptide, yellow powder, product productive rate 73.3%; The proton nmr spectra of the highly finished product of the polyoxyethylene ligand that contains polypeptide of the embodiment of the invention 4 (
1H NMR) sees accompanying drawing 1.
The first step contains the polyoxyethylene ligand semifinished product of coupling agent
In reactor, add omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer 44mg, solvent N, dinethylformamide 25ml, coupling agent 3-maleic amide benzoic acid N-succinimide ester (MBS) 390mg,-4C ° of reaction 72 hours, make the polyoxyethylene ligand semifinished product that contains coupling agent; Omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, solvent N, the weight ratio of dinethylformamide, coupling agent 3-maleic amide benzoic acid N-succinimide ester (MBS) is 1: 1670: 6, and the structure of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is identical with embodiment 4;
Second step was contained the highly finished product of the polyoxyethylene ligand of coupling agent
Dialysis, lyophilizing makes the highly finished product 35mg of the polyoxyethylene ligand that contains coupling agent, product yield 73.1%;
The 3rd step made the part that contains the modification of polypeptide polyoxyethylene
In containing the highly finished product 11.5mg of polyoxyethylene ligand of coupling agent, second product that obtain of step adds hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH 15mg and solvent phosphoric acid PBS buffer solvent 0.5ml, the weight ratio that contains highly finished product, polypeptide and the solvent phosphoric acid PBS buffer solvent of the polyoxyethylene ligand of coupling agent is 1: 1.3: 43.4,-4C ° of reaction 72 hours, make the part of the polyoxyethylene modification that contains polypeptide;
The 4th step made the highly finished product of the polyoxyethylene ligand that contains polypeptide
Dialysis, lyophilizing makes the highly finished product 12.3mg of the polyoxyethylene ligand that contains polypeptide, yellow powder, product productive rate 74.5%;
First step preparation contains the crude product of the polyoxyethylene ligand of polypeptide
In reactor, add 3mg hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH, 0.60mg MBS coupling agent and amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer 9.9mg, distilled water 1ml, room temperature reaction 24 hours, preparation contains the crude product of the polyoxyethylene ligand of polypeptide; The weight ratio of polypeptide, MBS coupling agent, amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer and distilled water is 1: 0.2: 3.3: 333, and the structure of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is identical with embodiment 2;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of polypeptide
Add water dialysis, lyophilizing obtains being connected with the yellow powder of polypeptide, 9.3mg, yield 69.0%;
Solvent distilled water in the first step of embodiment 4 can change N into, N-dimethyl acid imide, dimethyl sulfoxide, dichloromethane, chloroform, dioxane, distilled water or phosphoric acid PBS buffer solvent.
First step preparation contains the crude product of the polyoxyethylene ligand of polypeptide
In reactor, add 5mg hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH, 1.0mg MBS coupling agent and amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer 11.5mg, distilled water 1ml, room temperature reaction 24 hours, preparation contains the crude product of the polyoxyethylene ligand of polypeptide; The weight ratio of polypeptide, MBS coupling agent, amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer and distilled water is 1: 0.2: 2.3: 200, and the structural formula of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is identical with embodiment 4;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of polypeptide
Dialysis, lyophilizing obtains being connected with the yellow powder of polypeptide, 7.3mg, yield 41.7%.
First step preparation contains the crude product of the polyoxyethylene ligand of polypeptide
In reactor, add 5mg hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH, 1.0mg MBS coupling agent and amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer 11.5mg, dichloromethane 20ml,-4C ° of reaction 72 hours, preparation contained the crude product of the polyoxyethylene ligand of polypeptide; The weight ratio of polypeptide, MBS coupling agent, amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer and dichloromethane is 1: 0.2: 2.3: 4000, and the structural formula of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is identical with embodiment 4;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of polypeptide
Lyophilizing, dialysis, lyophilizing obtains being connected with the yellow powder of polypeptide, 7.1mg, yield 41.5%.
Embodiment 9
First step preparation contains the crude product of the polyoxyethylene ligand of polypeptide
In reactor, add 10mg hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH, 1.0mg MBS coupling agent and amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer 11.5mg, distilled water 20ml, room temperature reaction 2 hours, preparation contains the crude product of the polyoxyethylene ligand of polypeptide; The weight ratio of polypeptide, MBS coupling agent, amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer and distilled water is 1: 0.1: 1.1: 2000, and the structural formula of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is identical with embodiment 4;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of polypeptide
Dialysis, lyophilizing obtains being connected with the yellow powder of polypeptide, 7.5mg, yield 41.8%.
Embodiment 10
First step preparation contains the crude product of the polyoxyethylene ligand of polypeptide
In reactor, add 3mg hepatoma-targeting polypeptide FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH, 0.58mg MBS coupling agent and amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer 6.8mg, distilled water 10ml, room temperature reaction 2 hours, preparation contains the crude product of the polyoxyethylene ligand of polypeptide; The weight ratio of polypeptide, MBS coupling agent, amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer and distilled water is 1: 0.19: 2.3: 3333, and the structural formula of omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer is identical with embodiment 1;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of polypeptide
Dialysis, lyophilizing obtains being connected with the yellow powder of polypeptide, 5.5mg, yield 53.0%.
Claims (5)
1. contain the polyoxyethylene ligand of hepatoma-targeting peptide, it is characterized in that, this part has following structure:
In the formula,
R
3Representing MBS coupling agent coupling hepatoma-targeting peptide, is 3-maleic amide benzoic acid-N-succinimide ester MBS coupling agent coupling hepatoma-targeting peptide, and structural formula is
R
4Represent the hepatoma-targeting polypeptide; The order of described hepatoma-targeting polypeptide is FAM-A-G-K-G-T-P-S-L-E-T-T-P-C-COOH, and wherein the FAM representative is connected to the rhodamine fluorescein C of peptamine cardinal extremity
21H
12O
7
R
2Represent polyoxyethylene-diethyl pentaacetic acid block polymer segment, structural formula is
R
1Represent polyoxyethylene, structural formula is
-CH
2CH
2-(OCH
2CH
2)
n-
N represents 22～500 integer.
2. the described synthetic method that contains the polyoxyethylene ligand of hepatoma-targeting peptide of claim 1 is characterized in that, carries out according to following operating procedure:
First step preparation contains the semifinished product of the polyoxyethylene ligand of polypeptide
Measure the hepatoma-targeting polypeptide earlier: 3-maleic amide benzoic acid-N-succinimide ester MBS coupling agent: amino-contained polyoxyethylene-diethyl pentaacetic acid block polymer: solvent=1: 0.1～0.2: 1.1～3.3: 200～4000 weight ratios, then they are added in the reactor, under-4 ℃～room temperature condition, reacted 2～72 hours, and made the semifinished product of the polyoxyethylene ligand that contains the hepatoma-targeting polypeptide;
The preparation of second step contains the highly finished product of the polyoxyethylene ligand of polypeptide
The semifinished product dialysis of the polyoxyethylene ligand that contains polypeptide that the first step is made, lyophilizing, obtaining the yellow solid powder is the polyoxyethylene ligand that contains the hepatoma-targeting peptide of the present invention.
3. the synthetic method that contains the polyoxyethylene ligand of hepatoma-targeting peptide according to claim 2 is characterized in that, described solvent is N, dinethylformamide, or dichloromethane, or chloroform, or dioxane, or dimethyl sulfoxide, or distilled water, or phosphoric acid PBS buffer solvent.
4. the synthetic method that contains the polyoxyethylene ligand of hepatoma-targeting peptide according to claim 2 is characterized in that, the amino-contained polymer of the described first step is omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, and molecular formula is NH
2-R
2-NH
2, structural formula is
In the formula, R
1Represent polyoxyethylated structural formula to be
-CH
2CH
2-(OCH
2CH
2)
n-
N represents 22～500 integer.
5. the synthetic method that contains the polyoxyethylene ligand of hepatoma-targeting peptide according to claim 2 is characterized in that, the semifinished product that first step preparation contains the polyoxyethylene ligand of polypeptide divides following three steps to carry out:
A. preparation contains the polyoxyethylene ligand semifinished product of coupling agent: add omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer, solvent, coupling agent in reactor,-4 ℃～room temperature reaction 0.5～72 hour, omega amine polyoxyvinyl-diethylenetriamine pentaacetic acid block polymer: solvent: the weight ratio of coupling agent was 1: 10～1670: 0.06～6;
B, contain the purification of the polyoxyethylene ligand semifinished product of coupling agent: through dialysis, lyophilizing makes the highly finished product of the polyoxyethylene ligand that contains coupling agent;
C, preparation contain the part of the polyoxyethylene modification of polypeptide: go on foot adding hepatoma-targeting polypeptide and solvent in the product that obtains second, measure the highly finished product of the polyoxyethylene ligand that contains coupling agent earlier: the hepatoma-targeting polypeptide: the weight ratio of solvent is 1: 0.06～1.3: 43～1010,-4 ℃～room temperature reaction 1～72 hour, make the semifinished product of the polyoxyethylene ligand that contains polypeptide.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4647447A (en) * | 1981-07-24 | 1987-03-03 | Schering Aktiengesellschaft | Diagnostic media |
| US4789721A (en) * | 1987-07-27 | 1988-12-06 | Texaco Inc. | Curatives of epoxy resins from dicarboxylic acids, including (1) indane or (2) tert-butylisophtalic derived acids, reacted with polyetherdiamines |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4647447A (en) * | 1981-07-24 | 1987-03-03 | Schering Aktiengesellschaft | Diagnostic media |
| US4789721A (en) * | 1987-07-27 | 1988-12-06 | Texaco Inc. | Curatives of epoxy resins from dicarboxylic acids, including (1) indane or (2) tert-butylisophtalic derived acids, reacted with polyetherdiamines |
Non-Patent Citations (2)
| Title |
|---|
| 二乙三胺五乙酸吡哆醇酯钆、锰配合物的合成、弛豫率及其肝靶向性. 丁雄军等.科学通报，第46卷第18期. 2001 * |
| 靶向性磁共振成像造影剂的研究进展. 俞开潮等.化学试剂，第26卷第6期. 2004 * |
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