WO2020063216A1 - 类肽化合物及其制备方法、寡聚物、药物组合物和试剂盒 - Google Patents

类肽化合物及其制备方法、寡聚物、药物组合物和试剂盒 Download PDF

Info

Publication number
WO2020063216A1
WO2020063216A1 PCT/CN2019/102009 CN2019102009W WO2020063216A1 WO 2020063216 A1 WO2020063216 A1 WO 2020063216A1 CN 2019102009 W CN2019102009 W CN 2019102009W WO 2020063216 A1 WO2020063216 A1 WO 2020063216A1
Authority
WO
WIPO (PCT)
Prior art keywords
subunit
oligomer
peptoid compound
compound
peptoid
Prior art date
Application number
PCT/CN2019/102009
Other languages
English (en)
French (fr)
Inventor
赵子健
殷雨丹
Original Assignee
京东方科技集团股份有限公司
北京京东方技术开发有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 京东方科技集团股份有限公司, 北京京东方技术开发有限公司 filed Critical 京东方科技集团股份有限公司
Priority to US16/646,320 priority Critical patent/US11591370B2/en
Publication of WO2020063216A1 publication Critical patent/WO2020063216A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids

Definitions

  • Embodiments of the present disclosure relate to a peptoid compound, a method for preparing the peptoid compound, an oligomer, a pharmaceutical composition, and a use of the pharmaceutical composition in the manufacture of a medicament for detecting or diagnosing a disease related to the tyrosine kinase HER2, and the use thereof For identifying circulating tumor cells.
  • breast cancer and ovarian cancer are one of the malignant tumors that women are susceptible to, accounting for 15% of women's cancer, and gastric cancer is also a relatively high incidence disease.
  • anti-tumor drugs are often used to treat patients.
  • the anti-tumor drugs include protein drugs such as cytotoxic chemotherapy drugs and antibodies.
  • cytotoxic chemotherapeutic drugs have poor selectivity, large toxic and side effects, and easily cause adverse reactions.
  • protein drugs such as antibodies have high specificity and small toxic and side effects, due to their large molecular mass and complex structure, they can easily cause immune reactions and Drug production is dependent, and the process of preparing protein drugs such as antibodies is so complicated that it is expensive and difficult to afford for ordinary tumor patients.
  • Circulating Tumor Cells screening technology can be used to diagnose cancer.
  • the screening and detection of CTCs requires the capture of circulating tumor cells from the blood.
  • the detection process has an inseparable relationship with the probe molecules coupled to the surface of the capture device, and specific capture is achieved through the affinity of the probe molecules with the surface proteins of CTCs.
  • the probe molecule includes antibodies, polypeptides, peptoids, and aptamers such as targeting molecules for specific receptor proteins at tumor sites.
  • peptidomimetics have many advantages, such as low immunogenicity, good tissue permeability, small molecular weight, high stability, easy modification and economical.
  • At least one embodiment of the present disclosure provides a peptidic compound, the peptidic compound includes: cysteine (Cys) subunit, butylenediamine (Nlys) subunit, 3,4-methylenedioxybenzylamine (Npip ) Subunit, 3-aminopropionic acid (Nce) subunit, and 1-naphthylamine (Na) subunit.
  • cysteine Cys
  • Nlys butylenediamine
  • Npip 3,4-methylenedioxybenzylamine
  • Nce 3-aminopropionic acid
  • Na 1-naphthylamine
  • the order of the subunits contained in the peptoid compound is cysteine (Cys) subunit-butanediamine (Nlys) subunit-butanediamine (Nlys) subunit—butanediamine (Nlys) subunit—3,4-methylenedioxybenzylamine (Npip) subunit—3-aminopropionic acid (Nce) subunit—1-naphthylamine (Na) Subunit.
  • the peptoid compound has a structure represented by Formula I:
  • At least one embodiment of the present disclosure also provides a method for preparing a peptoid compound, wherein the method includes a solid-phase synthesis method to synthesize a subunit.
  • the preparation method includes the following steps:
  • step (3) reacting the donor of the second subunit of the peptoid compound with the product obtained in step (2) to replace the bromine atom to complete the connection of the second subunit;
  • the synthetic peptide-like compound is cleaved from the solid-phase carrier to obtain the peptide-like compound.
  • At least one embodiment of the present disclosure also provides an oligomer, wherein the molecular structure formula of the oligomer is:
  • the molecular structure formula of the oligomer is:
  • At least one embodiment of the present disclosure also provides a pharmaceutical composition including: any one of the above oligomers; and a pharmaceutically acceptable excipient.
  • the excipient is at least one of an excipient, a diluent, a carrier, a flavoring agent, a binder, and a filler.
  • At least one embodiment of the present disclosure also provides the use of any one of the foregoing pharmaceutical compositions in the manufacture of a medicament for detecting or diagnosing a disease related to the tyrosine kinase HER2.
  • the disease includes at least one of breast cancer, gastric cancer, and ovarian cancer.
  • At least one embodiment of the present disclosure also provides a kit for identifying circulating tumor cells, including a box body, a microfluidic chip disposed in the box body, and a fluorescent probe disposed in the box body.
  • the fluorescent probe is any one of the above oligomers with a fluorescent label.
  • FIG. 1 is a flowchart of a method for preparing a peptoid compound according to an embodiment of the present disclosure
  • FIG. 2 is a flowchart of a method for preparing an oligomer according to an embodiment of the present disclosure
  • FIG. 3 is a schematic diagram of a two-dimensional oligomer formation process provided by an embodiment of the present disclosure
  • 4A is an atomic force microscope image of an oligomer provided by an embodiment of the present disclosure.
  • 4B is an atomic force microscope image of another oligomer provided by an embodiment of the present disclosure.
  • FIG. 5 shows an oligomer and HER2 with concentrations of 2.2 ⁇ M, 4.6 ⁇ 10 -1 ⁇ M, 9.1 ⁇ 10 -2 ⁇ M, 1.8 ⁇ 10 -2 ⁇ M, and 3.6 ⁇ 10 -3 ⁇ M, according to an embodiment of the present disclosure. Results of protein-bound surface plasmon resonance detection.
  • the model of the SPRi instrument in the following examples is Plexera Kx5V2, Plexera Bioscience LLC, USA.
  • the instrument is mainly equipped with a 660nm LED light source, a CCD image collector and a sensor chip with a microfluidic channel.
  • the instrument displays the reflection on each monitoring point The change in light intensity over time is recorded as an SPR curve.
  • ⁇ M herein means “ ⁇ mol / L”
  • mM means “mmol / L”.
  • Liquid biopsy does not require the removal of tumor tissue from a patient's body. It only needs to remove the patient's blood or secretions for testing. Therefore, researchers are paying more and more attention to liquid biopsy and research.
  • Liquid biopsy techniques include the use of peptoid compounds to detect circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or exosome.
  • Circulating tumor cells are a collective name for various types of tumor cells present in peripheral blood. Due to spontaneous or diagnostic operations, they are shed from solid tumor lesions (primary, metastatic), and most CTCs enter peripheral blood After apoptosis or phagocytosis occurs, a few can escape and develop into metastases, thereby increasing the risk of death in patients with malignant tumors. The presence and quantity of CTCs is an important indicator for judging the progression and metastasis of cancer. Detecting and tracking the number of CTCs in peripheral blood is helpful for early screening, monitoring of curative effects, prognosis judgment and recurrence prediction.
  • CTCs The detection technology for CTCs can predict the occurrence of early tumors, detect tumor metastasis during the treatment of patients with drugs, and guide medications for subsequent treatments.
  • CTCs are derived from primary tumors or metastatic tumors. CTCs can enter blood vessels after they are detached from the basement membrane. Because the content of CTCs in the blood is extremely low, and its size is similar to the size of white blood cells, it makes it difficult to detect CTCs using liquid biopsy technology. However, CTCs carry cancer-specific, highly expressed proteins on their surfaces.
  • human epidermal growth factor receptors include EGFR / ErbB1 / HER1, ErbB2 / HER2, ErbB3 / HER3, and ErbB4 / HER4.
  • human epidermal growth factor receptor-2 HER2
  • High-expressing breast cancer is the most important type of breast cancer. Ovarian and gastric cancers with high HER2 expression are also common. Therefore, by specifically identifying the HER2 protein on the surface of CTCs in breast, ovarian and gastric cancer patients with high HER2 expression, Capturing the corresponding CTCs with high sensitivity provides a favorable guarantee.
  • molecular probes with high affinity and high sensitivity for the HER2 protein can be designed.
  • Peptide small molecules have the characteristics of low immunogenicity, good tissue permeability, small molecular weight, high stability, easy modification and low manufacturing cost.
  • the combination of peptoid small molecules and biosensors The ability is not strong, resulting in small peptide-like molecules that cannot be used as probe molecules; antibodies have the characteristics of tight binding to biosensors, but the arrangement of antibody molecules is disordered, and the direction of their arrangement on the sensor surface is random and difficult to control This results in lower specificity and higher cost of antibodies.
  • an oligomer formed by a peptidomimetic molecule and an antibody-like molecule can well combine the characteristics of the peptidomimetic molecule and an antibody, that is, the oligomer has the characteristics of tight binding of an antibody to a biosensor
  • small peptide-like molecules can be formed on the surface of the sensor in an orderly manner.
  • the molecular probe formed by the oligomer has a strong affinity with tumor cells, and the oligomer cannot be hydrolyzed, which can ensure natural Activity of a living sample.
  • peptides Compared with peptoids, peptides use alpha amino acids as structural units, and peptoids use N-substituted glycine as structural units.
  • Peptoid compounds have good biological activity and pharmacological properties. They can effectively detect or inhibit the deterioration in living experiments and have good cell membrane permeability. At present, the more mature peptide synthesis technology is "subunit synthesis" technology.
  • At least one embodiment of the present disclosure provides a peptidic compound, the peptidic compound includes: cysteine (Cys) subunit, butylenediamine (Nlys) subunit, 3,4-methylenedioxybenzylamine (Npip ) Subunit, 3-aminopropionic acid (Nce) subunit, and 1-naphthylamine (Na) subunit.
  • cysteine Cys
  • Nlys butylenediamine
  • Npip 3,4-methylenedioxybenzylamine
  • Nce 3-aminopropionic acid
  • Na 1-naphthylamine
  • each subunit is as follows:
  • the subunits included in the peptoid compound are arranged in the following order: cysteine (Cys) subunit-butanediamine (Nlys) subunit-butane Diamine (Nlys) subunit-butanediamine (Nlys) subunit-3,4-methylenedioxybenzylamine (Npip) subunit-3-aminopropionic acid (Nce) subunit-1-naphthylamine ( Na) subunit.
  • the peptidic compound has a structure represented by Formula I:
  • the peptide compound can be used as a probe molecule to specifically recognize the HER2 protein, that is, the peptide compound can bind to the breast cancer marker HER2 protein for detection of breast cancer.
  • At least one embodiment of the present disclosure also provides a method for preparing a peptoid compound.
  • the method includes solid phase synthesis to synthesize subunits.
  • FIG. 1 is a flowchart of a method for preparing a peptoid compound according to an embodiment of the present disclosure. As shown in FIG. 1, the method includes the following steps:
  • Step S01 connecting the first subunit of the peptoid compound to the solid-phase carrier according to the connection order of the subunits of the peptoid compound;
  • Step S02 After the bromoacetic acid is activated under the action of an activator, it reacts with the amino group of the first subunit connected to the solid-phase support to form an amide bond;
  • Step S03 reacting the donor of the second subunit of the peptoid compound with the product obtained in step S02 to replace the bromine atom to complete the connection of the second subunit;
  • Step S04 Repeat the connection of bromoacetic acid and subsequent subunits until the connection of all the subunits is completed, wherein the input order of the subunits is: Cys, t-Butyloxycarbonyl (Boc) ) Protected succinic acid (Nlys), Boc protected succinic acid (Nlys), Boc protected succinic acid (Nlys), 3,4-methylenedioxybenzylamine (Npip), 3-aminopropionic acid (Nce) and 1-naphthylamine (Na).
  • Step S05 The synthetic peptide-like compound is cleaved from the solid phase carrier to obtain the peptide-like compound.
  • the solid support is Rink® AM resin.
  • the solid phase support is swelled before the first subunit of the peptoid is attached to the solid phase support.
  • the solid phase carrier is Rink AM AM resin
  • it is swollen and deprotected with hexahydropyridine to make Rink AM AM resin barely exposed to the amino group.
  • the process of attaching the first subunit of a peptoid to a solid phase carrier is performed under the action of a condensing agent and an activator.
  • the condensing agent is 2- (3'-N-oxo-benzotriazole) -1,1 ', 3,3'-tetramethylurea hexafluorophosphate, O-benzotriazole- N, N, N ', N'-tetramethylurea tetrafluoroborate or at least one of 1-hydroxybenzotriazole.
  • the activator used in step S01 is N-methylmorpholine.
  • the activator used in step S02 is N, N'-diisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide.
  • the reaction temperature in step S02 is 20-40 ° C, such as 20 ° C, 21 ° C, 23 ° C, 24 ° C, 25 ° C, 33 ° C, 34 ° C, 36 ° C, 38 ° C, or 40 ° C.
  • the reaction time in step S02 is 10-100 min, such as 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, or 100 min.
  • the donor in step S03 refers to a peptoid subunit compound.
  • the donor for the cysteine subunit is cysteine
  • the donor for the succindiamine subunit is succindiamine
  • the donor for the 3,4-methylenedioxybenzylamine subunit is 3,4 -Methylenedioxybenzylamine
  • the donor of the 3-aminopropionic acid subunit is 3-aminopropionic acid
  • the donor of the 1-naphthylamine subunit is 1-naphthylamine.
  • the reaction temperature in step S03 is 20-40 ° C, such as 20 ° C, 21 ° C, 23 ° C, 24 ° C, 25 ° C, 33 ° C, 34 ° C, 36 ° C, 38 ° C, or 40 ° C.
  • the reaction time in step S03 is 30-180min, such as 30min, 35min, 45min, 50min, 55min, 60min, 65min, 70min, 80min, 90min, 100min, 120min, 140min, 150min, 160min, 170min, or 180min.
  • step S04 the connection of bromoacetic acid and subsequent subunits is repeated, that is, steps S04 and S03 are repeated, and the only difference is that the connected subunit is a subsequent subunit.
  • the lysing agent used in the step S05 cleavage includes the following components by mass percentage: trifluoroacetic acid with a mass percentage of 95%, ultra-pure water of 2.5%, and triisopropylsilane of 2.5%.
  • groups that do not participate in the ligation reaction can be protected by groups to ensure the accuracy of the ligation site, so that the reaction proceeds more accurately and smoothly, and then all subunits are completed. After connection, deprotection is performed to remove the protecting group.
  • the synthesis of a peptoid compound by a solid-phase subunit synthesis method includes the following steps:
  • Rink polyamide resin substitution level: 0.3mmol / g
  • cysteine and 2- (3'-N-oxo-benzotriazole) -1 , 1 ', 3,3'-tetramethylurea hexafluorophosphate are mixed in an equimolar amount, and coupled under the activation of N-methylmorpholine;
  • a step of purifying the obtained product may be further included as necessary.
  • the method of purification is not particularly limited, and methods similar to those in the art for purifying similar products can be used for purification, such as precipitation, filtration, dialysis, gel permeation chromatography, and the like.
  • At least one embodiment of the present disclosure also provides an oligomer, and the molecular structure formula of the oligomer is:
  • the oligomer includes: ⁇ -phenylethylamine subunit, biphenylethylamine subunit, cysteine (Cys) subunit, butylenediamine (Nlys) subunit, 3,4-methylenedioxy Benzylamine (Npip) subunit, 3-aminopropionic acid (Nce) subunit, 1-naphthylamine (Na) subunit, and ethylenediamine subunit.
  • the molecular structure of each subunit is as follows:
  • the subunits included in the oligomer are arranged in the following order: [ ⁇ -phenylethylamine subunit—3-aminopropionic acid subunit—biphenylethyl Amine subunit—3-aminopropionic acid subunit] n 2 — ⁇ -phenethylamine subunit—cysteine (Cys) subunit—butanediamine (Nlys) subunit—butanediamine (Nlys) subunit —Butanediamine (Nlys) subunit — 3,4-methylenedioxybenzylamine (Npip) subunit — 3-aminopropionic acid (Nce) subunit — 1-naphthylamine (Na) subunit — biphenyl Ethylamine subunit-ethylenediamine subunit-[ ⁇ -phenethylamine subunit-ethylamine subunit-biphenylethy
  • At least one embodiment of the present disclosure also provides a method for preparing an oligomer, which is synthesized by a solid-phase subunit synthesis method.
  • FIG. 2 is a flowchart of a method for preparing a peptoid compound according to an embodiment of the present disclosure. As shown in FIG. 2, the method for preparing a peptoid compound includes the following steps:
  • Step S11 Connect the first subunit of the oligomer to the solid-phase carrier according to the connection order of the subunits of the oligomer;
  • Step S12 After the bromoacetic acid is activated under the action of an activator, it reacts with the amino group of the first subunit connected to the solid support to form an amide bond;
  • Step S13 reacting the donor of the second subunit of the peptoid compound with the product obtained in step S12 to replace the bromine atom to complete the connection of the second subunit;
  • Step S14 Repeat the connection of bromoacetic acid and subsequent subunits until the connection of all subunits is completed;
  • Step S15 The synthetic oligomer is cleaved from the solid phase carrier to obtain the peptide compound.
  • the oligomer includes the peptoid compound in any one of the above embodiments, and also includes helper chains formed on the left and right sides of the peptoid compound, respectively, and the helper chains formed on the left side of the peptoid compound Both contain amino groups, and the helper chains formed on the right side of the peptide compounds contain carboxyl groups.
  • the helper chains formed on the left side and the right side of the peptide compounds help to form the oligomer into a two-dimensional layer. Structure, which can expose the intermediate peptoid compound on the surface of the sensor as a probe molecule to detect the HER2 protein.
  • the helper chains formed on the left and right side of the peptoid compound can also make the oligomerization Things are arranged more neatly.
  • n1 and n2 are less than 3, there will be a problem that the chain length is too short to assemble; when n1 and n2 are greater than 10, the formed chain is too long, and the density of the peptoid compound inserted in the middle of the oligomer Too low, there will be a problem of weakened affinity, and the problem of specific binding to HER2 protein on CTCs cannot be achieved.
  • the molecular structure is:
  • the preparation method of the oligomer specifically includes the following steps:
  • Swell Rink polyamide resin (the starting resin for peptide synthesis, with a substitution level of 0.3 mmol / g) and swell with hexahydropyridine, and equal molar amounts of ⁇ -phenylethylamine and 1-hydroxybenzotriazole Mixing and coupling under the activation of N-methylmorpholine;
  • tert-butoxycarbonyl group (t-Boc or Boc) is a protecting group for protecting an amino group.
  • DMSO dimethyl sulfoxide
  • H 2 O water
  • the molecular structure is:
  • the preparation method of the oligomer specifically includes the following steps:
  • Rink amide AM resin (the starting resin for peptide synthesis, substitution level 0.3 mmol / g) is swollen and deprotected with hexahydropyridine, and the equimolar amounts of ⁇ -phenylethylamine and 1-hydroxybenzotriazole are carried out. Mixing and coupling under the activation of N-methylmorpholine;
  • the order of subunit input is:
  • DMSO dimethyl sulfoxide
  • H 2 O water
  • the molecular structure is:
  • the preparation method of the oligomer specifically includes the following steps:
  • Rink amide AM resin (the starting resin for peptide synthesis, substitution level 0.3 mmol / g) is swollen and deprotected with hexahydropyridine, and the equimolar amounts of ⁇ -phenylethylamine and 1-hydroxybenzotriazole are carried out. Mixing and coupling under the activation of N-methylmorpholine;
  • the order of subunit input is:
  • DMSO dimethyl sulfoxide
  • H 2 O water
  • the synthesis process of the oligomer provided in the embodiments of the present disclosure is simple, and the oligomer has strong binding capacity with the HER2 protein, and can effectively screen the serum of breast cancer patients and normal people through the HER2 protein in serum
  • the HER2 protein that can specifically identify the surface of breast cancer CTCs provides a favorable guarantee for capturing the corresponding CTCs with high sensitivity.
  • molecular probes with high affinity and sensitivity to the HER2 protein can be designed.
  • FIG. 3 is a schematic diagram of the formation process of a two-dimensional oligomer provided by an embodiment of the present disclosure.
  • the process of forming a two-dimensional oligomer is: oligomerization provided by any embodiment of the present disclosure
  • the oligomer includes a hydrophilic end and a hydrophobic end.
  • the oligomers are randomly arranged at the interface between the gas and the liquid.
  • An external force is applied to the disordered oligomers, so that the oligomers are aligned at the gas-liquid interface; an external force is further applied to the ordered oligomers, so that the oligomers are squeezed to Gas and liquid interface.
  • Below the gas-liquid interface the hydrophilic end of the oligomer is exposed on the outside and the hydrophobic end is formed on the inside, forming a two-dimensional oligomer.
  • the oligomer nanosheet layer is formed as follows: the oligomer having a concentration of 2 mM obtained in any of the above examples 1 to 3 is dissolved in 10 mM 4-hydroxyethylpiperazineethanesulfonic acid and 100 mM sodium chloride, And add a buffer solution so that the pH is 8.0, so that the final concentration of the oligomer is 1-100 ⁇ M, for example, 20 ⁇ M, and then use the manual shaking method: the peptoid solution is stably stored at room temperature for 22 hours, and then gently shake 30 manually Seconds, stabilize for 1 minute, repeat the shaking-stabilizing process 5 times; or machine shaking method: slowly rotate the tube containing the oligomer solution from the horizontal direction (0.6 rpm) to the vertical direction, and rotate it every 450 seconds; in Nile red was added to the obtained oligomer nanosheet solution to the final concentration of the oligomer solution of 1 ⁇ M, and the oligomer solution was placed on agar with a mass percentage of
  • FIG. 5 is an oligomer with concentrations of 2.2 ⁇ M, 4.6 ⁇ 10 ⁇ 1 ⁇ M, 9.1 ⁇ 10 ⁇ 2 ⁇ M, 1.8 ⁇ 10 ⁇ 2 ⁇ M, and 3.6 ⁇ 10 ⁇ 3 ⁇ M in Example 1 of the present disclosure.
  • HER2 protein-bound surface plasmon resonance detection results where ⁇ RU represents the binding signal after the mobile phase passes the array minus the baseline signal of the initial PBS buffer.
  • the curve is the test result of PlexArray HT, and the fitted straight line is BIAevalution 4.1 is fitted.
  • ⁇ RU is a unit used to reflect the intensity of the combined signal in surface plasmon resonance imaging. It is a dimensionless unit.
  • the equilibrium dissociation constant KD is 3.76 ⁇ 10 -10 moles / liter. This indicates that the oligomer has a fairly high affinity level with the HER2 protein.
  • the oligomer solution was dropped on the surface of a 3D chip. Each sample was dropped three times. After being left at 4 ° C for 12 hours, 10XPBS (Phosphate Buffered Saline, phosphate buffer solution) and 1XPBS were sequentially used. Wash with ultrapure water, then block the chip with 1M hydrochloric acid aminoethanol for 30 minutes, then wash it with ultrapure water 5 times, and finally blow dry with nitrogen; install the chip on the SPRi instrument, measure the SPRi angle and adjust to Optimal optical position, select relevant detection points in the detection area, the relevant detection points include sample points and blank points, and set the experimental flow rate to 2 ⁇ L / s;
  • serum dilutions of different patients and normal persons were passed in.
  • the serum dilution concentrations of different patients and normal persons were respectively 1: 2000 and 1: 4000. , 1: 8000, 1: 16000, 1: 32000, the binding time is 300 seconds, and the dissociation time is 300 seconds.
  • the test results show that when the serum dilution ratio is less than or equal to 1: 8000, it can clearly distinguish breast cancer patients with high HER2 expression, ovarian cancer patients with high HER2 expression, patients with gastric cancer with high HER2 expression and normal people, showing that they have extremely high High sensitivity.
  • the 3D chip is a PlexArray HT 3D chip purchased from Plexera Bioscience, USA.
  • the oligomer self-assembles on the air-liquid interface to form a peptide-like nanosheet with a specific recognition of the HER2 protein on the surface.
  • the peptide-like nanosheet serves as a scaffold to display and supports the peptide-like compound as a molecular probe to the HER2 protein. For identification.
  • peptide nanosheets combined with surface plasmon resonance technology can be used to detect breast cancer.
  • the oligomer is a two-dimensional nanosheet material, so that the oligomer can be coupled to the sensor, and the peptide-like compound having an affinity effect can be displayed on the surface of the sensor.
  • Two-dimensional peptide-like nanomaterials are playing an increasingly important role in biology and electronics, such as sensing, template growth and filtration, and as protein mimics to test the molecular recognition and catalytic capabilities of proteins.
  • the Langmuir experimental device reveals that the formation of peptide-like nanosheets is a thermodynamic equilibrium process of self-assembly of peptide-like molecules and conversion of external mechanical energy into chemical energy of peptide-like molecules.
  • the pharmaceutical composition further includes: the oligomer in any of the above embodiments; and a pharmaceutically acceptable excipient.
  • the excipient includes at least one of an excipient, a diluent, a carrier, a flavoring agent, a binder, and a filler.
  • excipients are, for example, emulsions or oily suspensions, or polyalkylene glycols, such as polypropylene glycol.
  • At least one embodiment of the present disclosure also provides the use of the pharmaceutical composition in any one of the above embodiments for preparing a medicament for detecting or diagnosing a disease related to the tyrosine kinase HER2.
  • the disease includes breast, gastric, and ovarian cancer.
  • At least one embodiment of the present disclosure also provides a kit for identifying circulating tumor cells, including: a box body, a microfluidic chip provided in the box body, and a fluorescent probe and a fluorescent probe provided in the box body.
  • a kit for identifying circulating tumor cells including: a box body, a microfluidic chip provided in the box body, and a fluorescent probe and a fluorescent probe provided in the box body.
  • the microfluidic chip includes a microvalve control layer and a microvalve film layer.
  • the microvalve control layer is provided with six holes penetrating the control layer and three gas channels.
  • the three holes are sampling holes and communicate with the substrate for the inflow and outflow of samples and reagents. The remaining three holes are connected to the three gas channels. , Used to inject gas, and control the opening and closing of the micro valve.
  • the micro-valve film layer is provided with three holes penetrating the thin-film layer, which are correspondingly communicated with the three sampling holes of the micro-valve control layer.
  • the outer contour dimensions of the micro-valve control layer and the micro-valve film layer should match the dimensions of the substrate.
  • fluorescent probes are used to identify cells, and the molecular probes with fluorescence in the embodiments of the present disclosure are oligomers labeled with fluorescent molecules.
  • the embodiment of the present disclosure does not specifically limit the type of the fluorophore, as long as the modification can impart fluorescent properties to the oligomer and can also realize the basic function of the oligomer.
  • the oligomers in the embodiments according to the present disclosure may be modified with one or more fluorescent groups.
  • a single fluorescently labeled oligomer can be modified with one fluorescent group, or a double fluorescently labeled oligomer can be obtained with two fluorescent groups.
  • the fluorescent group may be selected from non-limiting blue fluorescent dyes, near-infrared fluorescent dyes, green fluorescent dyes, and the like, for example, a coumarin-containing fluorescent group, an anthracene-containing fluorescent group, and rhodamine fluorescence.
  • the kit in addition to a microfluidic chip and a fluorescent probe (Probe), can also include a fluorescence microscope (fluorescence imaging system), self-developed image analysis software (analysis counting system), and a pump to form a complete system. Processing of blood samples and isolation and counting of circulating tumor cells.
  • a fluorescence microscope fluorescence imaging system
  • image analysis software analysis counting system
  • a pump to form a complete system. Processing of blood samples and isolation and counting of circulating tumor cells.
  • a fluorescence microscope is used to detect whether cells in a micro V-shaped array have fluorescence, and a full-coverage fluorescence imaging is performed on the functional area to obtain a multi-channel fluorescence image.
  • self-developed image processing software is used to analyze the images acquired by the fluorescence microscope and obtain the corresponding CTC quantity.
  • the software accurately calculates the size, area, aspect ratio, and roundness of the cells in the image, selects the CTCs that meet the requirements, and counts the CTCs.
  • Localized cells were screened and identified according to an independently written algorithm. Circulating tumor cells that met the label's fluorescence characteristics were identified and counted. Cell locations were then reported and cell images were magnified.
  • Embodiments of the present disclosure provide a peptoid compound, a method for preparing the peptoid compound, an oligomer, a pharmaceutical composition, and a use of the pharmaceutical composition in the manufacture of a medicament for detecting or diagnosing a disease related to the tyrosine kinase HER2, and the use Kit for identifying circulating tumor cells, having at least one of the following
  • the oligomer has a strong binding ability to the HER2 protein on the surface of CTCs.
  • the equilibrium dissociation constant KD in the kinetic constant is in the order of 10 -10 moles / liter;
  • the surface plasmon resonance technique is used to detect the blood signal intensity of the oligomer to breast cancer patients and the blood signal intensity of normal people. Polymer can obviously distinguish breast cancer patients from normal people;
  • the diagnostic technology for diagnosing breast cancer based on the oligomers can realize non-invasive and label-free rapid diagnosis
  • the method for synthesizing the peptoid compound and the oligomer provided by at least one embodiment of the present disclosure is simple, the preparation efficiency is high, and the production cost is low.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Optics & Photonics (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)

Abstract

一种类肽化合物、类肽化合物的制备方法、寡聚物、药物组合物、药物组合物在制备检测或者诊断与酪氨酸激酶HER2相关的疾病的药物中的用途以及用于鉴定循环肿瘤细胞的试剂盒，该类肽化合物包括：半胱氨酸(Cys)亚单位、丁二胺(Nlys)亚单位、3,4-亚甲二氧基苄胺(Npip)亚单位、3-氨基丙酸(Nce)亚单位和1-萘胺(Na)亚单位。该类肽化合物和寡聚物与CTCs表面的HER2蛋白的结合能力较强，以该类肽化合物为基础的乳腺癌诊断技术能够实现无创无标记的快速诊断，此外，该类肽化合物和寡聚物的合成方法简单，制备效率高，且制作成本低。

Description

类肽化合物及其制备方法、寡聚物、药物组合物和试剂盒
本申请要求于2018年9月28日递交的中国专利申请第201811138563.1号的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
技术领域
本公开的实施例涉及一种类肽化合物、类肽化合物的制备方法、寡聚物、药物组合物、药物组合物在制备检测或者诊断与酪氨酸激酶HER2相关的疾病的药物中的用途以及用于鉴定循环肿瘤细胞的试剂盒。
背景技术
乳腺癌和卵巢癌是女性易患的恶性肿瘤之一，占到了女性癌症的15％，胃癌也是发病率比较高的疾病。针对乳腺癌、卵巢癌和胃癌，目前，临床上多采用抗肿瘤药物对患者进行治疗，例如，该抗肿瘤药物包括细胞毒类化疗药物和抗体等蛋白类药物。但是，细胞毒类化疗药物的选择性差、毒副作用大、容易引起不良反应；抗体等蛋白类药物虽然特异性高、毒副作用小，但由于其分子质量大、结构复杂、容易引起免疫反应和对药物产生依赖性，且制备抗体等蛋白类药物的工艺过程很复杂以至于其价格高昂，难以为普通肿瘤患者所承受。
在癌症发展的早期，可以采用循环肿瘤细胞(Circulating Tumor Cells，CTCs)筛选技术对癌症进行诊断。CTCs筛选检测需要从血液中捕获出循环肿瘤细胞，该检测过程与捕获器件表面偶联的探针分子有着密不可分的关系，通过探针分子与CTCs表面蛋白的亲和作用实现特异性捕获。例如，该探针分子包括抗体、多肽、类肽和核酸适配体等肿瘤部位特定受体蛋白的靶向分子。在各种靶向分子中，类肽小分子具备很多的优势，例如，免疫原性低、组织渗透性好、分子量小、稳定性高、易于修饰且经济等。
发明内容
本公开至少一实施例提供一种类肽化合物，该类肽化合物包括：半胱氨酸(Cys)亚单位、丁二胺(Nlys)亚单位、3,4-亚甲二氧基苄胺(Npip)亚单 位、3-氨基丙酸(Nce)亚单位和1-萘胺(Na)亚单位。
例如，在本公开至少一实施例提供的类肽化合物中，所述类肽化合物包含的亚单位的顺序为半胱氨酸(Cys)亚单位—丁二胺(Nlys)亚单位—丁二胺(Nlys)亚单位—丁二胺(Nlys)亚单位—3,4-亚甲二氧基苄胺(Npip)亚单位—3-氨基丙酸(Nce)亚单位—1-萘胺(Na)亚单位。
例如，在本公开至少一实施例提供的类肽化合物中，所述类肽化合物具有式I所示的结构：
Figure PCTCN2019102009-appb-000001
本公开至少一实施例还提供一种类肽化合物的制备方法，其中，所述制备方法包括固相合成法合成亚单位。
例如，在本公开至少一实施例提供的制备方法中，所述制备方法包括以下步骤：
(1)按照所述类肽化合物的亚单位的连接顺序，将所述类肽化合物的第一个亚单位连接至固相载体上；
(2)将溴乙酸在活化剂的活化作用下与连接至所述固相载体上的第一个亚单位的氨基进行反应形成酰胺键；
(3)将所述类肽化合物的第二个亚单位的供体与步骤(2)得到的产物进行反应，取代掉溴原子，完成第二个亚单位的连接；
(4)重复进行所述溴乙酸以及后续亚单位的连接，直至完成所有亚单位的连接；
(5)从所述固相载体上将合成得到的类肽化合物裂解下来得到该类肽化合物。
本公开至少一实施例还提供一种寡聚物，其中，所述寡聚物的分子结构式为：
Figure PCTCN2019102009-appb-000002
其中，10≥n1≥3，10≥n2≥3，n1＝n2，n1和n2均为自然数。
例如，在本公开至少一实施例提供的寡聚物中，所述寡聚物的分子结构式为：
Figure PCTCN2019102009-appb-000003
本公开至少一实施例还提供一种药物组合物，该药物组合物包括：上述任一种寡聚物；以及药学上接受的辅料。
例如，在本公开至少一实施例提供的药物组合物中，所述辅料为赋形剂、稀释剂、载体、调味剂、粘合剂和填充剂中的至少之一。
本公开至少一实施例还提供一种上述任一种药物组合物在制备检测或者诊断与酪氨酸激酶HER2相关的疾病的药物中的用途。
例如，在本公开至少一实施例提供的用途中，所述疾病包括乳腺癌、胃癌和卵巢癌中的至少之一。
本公开至少一实施例还提供一种用于鉴定循环肿瘤细胞的试剂盒，包括：盒体、设置在所述盒体内的微流控芯片，以及设置在所述盒体内的荧光探针，所述荧光探针为带有荧光标记的如上任一种寡聚物。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例的附图作简单地介绍，显而易见地，下面描述中的附图仅仅涉及本发明的一些实施例， 而非对本发明的限制。
图1为本公开一实施例提供的一种类肽化合物的制备方法的流程图；
图2为本公开一实施例提供的一种寡聚物的制备方法的流程图；
图3为本公开一实施例提供的二维寡聚物的形成过程示意图；
图4A为本公开一实施例提供的一种寡聚物的原子力显微图像；
图4B为本公开一实施例提供的另一种寡聚物的原子力显微图像；以及
图5为本公开一实施例提供的一种寡聚物与浓度为2.2μM、4.6×10 -1μM、9.1×10 -2μM、1.8×10 -2μM和3.6×10 -3μM的HER2蛋白结合的表面等离基元共振检测的结果图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
除非另外定义，本公开使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本公开中使用的“第一”、“第二”以及类似的词语并不表示任何顺序、数量或者重要性，而只是用来区分不同的组成部分。“包括”或者“包含”等类似的词语意指出现该词前面的要素涵盖出现在该词后面列举的要素及其等同，而不排除其他要素。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的SPRi仪器的型号为Plexera Kx5V2，Plexera Bioscience LLC,USA，该仪器主要装配有660nmLED光源、CCD图像采集器和带微流通道的传感芯片，仪器显示每个监测点上反射光强度随时间的变化并记录为SPR曲线。
除非特殊说明，本文中的“μM”指的是“μmol/L”，“mM”指的是“mmol/L”。
由于肿瘤具有异质性，对于癌细胞已发生转移的患者而言，仅仅取某个部位的肿瘤组织并不足以反映患者整体的状况，然而对患者体内所有的肿瘤 组织都取样又不切实际，因此，组织活检技术具有一定的局限性。液体活检技术不需要取出患者体内的肿瘤组织，只需要取出患者的血液或者分泌物即可进行检测，因此，研究者们对于液体活检技术的关注和研究越来越多。液体活检技术包括采用类肽化合物对循环肿瘤细胞(CTCs)、循环肿瘤DNA(ctDNA)或者外泌体(Exosome)等进行检测。
循环肿瘤细胞(CTCs,Circulating Tumor Cells)是存在于外周血中的各类肿瘤细胞的统称，因自发或者诊疗操作从实体瘤病灶(原发灶、转移灶)脱落，大部分的CTCs进入外周血后发生凋亡或者被吞噬，少数能够逃逸并发展成为转移灶，从而增加恶性肿瘤患者死亡风险。CTCs存在与否及数量的多少是判断癌症进展和是否转移的重要指标，检测和跟踪外周血中CTCs的数量有助于对患者进行早期筛查、疗效监控、预后判断和复发预测。
针对CTCs的检测技术能够预测早期肿瘤的发生，又能够在患者采用药物进行治疗的过程中对肿瘤转移的情况进行检测，此外，还能对后续的治疗进行指导用药。CTCs来源于原发肿瘤或者转移肿瘤，CTCs脱离基底膜之后可以进入血管中。由于CTCs在血液中的含量极低，且其尺寸与白细胞的尺寸相近，这样就导致CTCs很难采用液体活检技术进行检测。但是，CTCs的表面带有相关癌症特异性高表达的蛋白。例如，人表皮生长因子受体(Human epithelial growth factor receptor，简称HER)包括EGFR/ErbB1/HER1，ErbB2/HER2，ErbB3/HER3和ErbB4/HER4，其中，人表皮生长因子受体-2(HER2)高表达乳腺癌是最主要的一类乳腺癌，HER2高表达的卵巢癌和胃癌也很常见，因此，通过特异性地识别HER2高表达的乳腺癌、卵巢癌和胃癌患者CTCs表面的HER2蛋白为高灵敏度地捕获相应的CTCs提供了有利的保障，例如，可以设计对HER2蛋白具有高亲和、高灵敏度的分子探针。
类肽小分子具有免疫原性低、组织渗透性好、分子量小、稳定性高、易于修饰且制造成本低等特点，但是，在分子探针的应用上，类肽小分子和生物传感器的结合能力不强，从而导致类肽小分子无法作为探针分子；抗体具有与生物传感器紧密结合的特点，但是，抗体分子的排列是无序的，其在传感器表面的排布方向是随机的难以控制，从而导致其特异性较低，且抗体的成本较高。本公开的发明人发现，由类肽小分子和类抗体形成的寡聚物能够很好地结合类肽小分子和抗体的特点，即该寡聚物既具有抗体的与生物传感器结合紧密的特点，又可以将类肽小分子有序地形成在传感器的表面，此 外，该寡聚物形成的分子探针与肿瘤细胞的亲和作用强，且该寡聚物不能被酶解，能够保证天然活体样本的活性。
类肽(peptoid)与多肽相比，多肽以α氨基酸为结构单元，类肽以N-取代甘氨酸为结构单元。类肽化合物具有良好的生物活性和药理性质，它能够有效地检测或者抑制活体实验中的恶化情况并且具有良好的细胞膜穿透性。目前，比较成熟的类肽合成技术为“亚单位合成”技术。
本公开至少一实施例提供一种类肽化合物，该类肽化合物包括：半胱氨酸(Cys)亚单位、丁二胺(Nlys)亚单位、3,4-亚甲二氧基苄胺(Npip)亚单位、3-氨基丙酸(Nce)亚单位和1-萘胺(Na)亚单位。
例如，各亚单位的分子结构式如下所示：
Figure PCTCN2019102009-appb-000004
(半胱氨酸，Cys),
Figure PCTCN2019102009-appb-000005
(丁二胺，Nlys),
Figure PCTCN2019102009-appb-000006
(3-氨基丙酸，Nce),
Figure PCTCN2019102009-appb-000007
(3,4-亚甲二氧基苄胺，Npip)，
Figure PCTCN2019102009-appb-000008
(1-萘胺，Na)。
例如，在本公开至少一实施例提供的类肽化合物中，该类肽化合物包含的亚单位按照以下顺序进行排列：半胱氨酸(Cys)亚单位—丁二胺(Nlys)亚单位—丁二胺(Nlys)亚单位—丁二胺(Nlys)亚单位—3,4-亚甲二氧基苄胺(Npip)亚单位—3-氨基丙酸(Nce)亚单位—1-萘胺(Na)亚单位。
例如，该类肽化合物具有式I所示的结构：
Figure PCTCN2019102009-appb-000009
例如，该类肽化合物能作为探针分子特异性识别HER2蛋白，即该类肽化合物可以结合乳腺癌的标志物HER2蛋白，用于乳腺癌的检测。
本公开至少一实施例还提供一种类肽化合物的制备方法，该制备方法包括固相合成法合成亚单位。
例如，图1为本公开一实施例提供的类肽化合物的制备方法的流程图，如图1所示，该制备方法包括以下步骤：
步骤S01：按照类肽化合物的亚单位的连接顺序，将类肽化合物的第一个亚单位连接至固相载体上；
步骤S02：将溴乙酸在活化剂的作用下活化后与连接至固相载体上的第一个亚单位的氨基进行反应以形成酰胺键；
步骤S03：将类肽化合物的第二个亚单位的供体与步骤S02得到的产物进行反应，取代掉溴原子，以完成第二个亚单位的连接；
步骤S04：重复进行溴乙酸以及后续亚单位的连接，直至完成所有亚单位的连接，其中，亚单位的投入顺序为：半胱氨酸(Cys)、叔丁氧羰基(t-Butyloxy carbonyl，Boc)保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、3,4-亚甲二氧基苄胺(Npip)、3-氨基丙酸(Nce)和1-萘胺(Na)。
步骤S05：从固相载体上将合成得到的类肽化合物裂解下来得到该类肽化合物。
例如，固相载体为Rink amide AM树脂。
例如，将类肽的第一个亚单位连接至固相载体上之前将固相载体进行溶胀处理。
例如，当固相载体为Rink amide AM树脂时，将其溶胀，并利用六氢吡啶脱保护使得Rink amide AM树脂裸露出氨基。
例如，类肽的第一个亚单位连接至固相载体上的过程中，在缩合剂和活化剂的作用下进行。
例如，该缩合剂为2-(3'-N-氧代-苯并三唑)-1,1',3,3'-四甲基脲六氟磷酸盐、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸或1-羟基苯并三唑中的至少之一。
例如，在步骤S01中使用的活化剂为N-甲基吗啡啉。
例如，在步骤S02中所使用的活化剂为N,N'-二异丙基碳二亚胺(DIC)或二环己基碳二亚胺。
例如，步骤S02中反应的温度为20-40℃，例如20℃、21℃、23℃、24℃、25℃、33℃、34℃、36℃、38℃或者40℃。
例如，步骤S02中的反应时间为10-100min，例如10min、15min、20min、25min、30min、35min、40min、50min、60min、70min、80min、90min或者100min。
例如，步骤S03中的供体是指类肽亚单位的化合物。例如，半胱氨酸亚单位的供体为半胱氨酸，丁二胺亚单位的供体为丁二胺，3,4-亚甲二氧基苄胺亚单位的供体为3,4-亚甲二氧基苄胺，3-氨基丙酸亚单位的供体为3-氨基丙酸，以及1-萘胺亚单位的供体为1-萘胺。
例如，步骤S03中反应的温度为20-40℃，例如20℃、21℃、23℃、24℃、25℃、33℃、34℃、36℃、38℃或者40℃。
例如，步骤S03中的反应时间为30-180min，例如30min、35min、45min、50min、55min、60min、65min、70min、80min、90min、100min、120min、140min、150min、160min、170min或者180min。
例如，在步骤S04中重复进行溴乙酸以及后续亚单位的连接，即重复步骤S04和步骤S03，区别仅仅是连接的亚单位是后续亚单位。
例如，步骤S05裂解时使用的裂解剂包括质量百分含量如下的成分：质量百分含量分别为95％的三氟乙酸，2.5％的超纯水和2.5％的三异丙基硅烷。
例如，在类肽化合物的制备过程中，可以将不参与连接反应的基团进行 基团保护，以确保连接位点的准确性，使反应更加准确并且顺利地进行，而后在完成所有亚单位的连接后，再进行脱保护以脱除保护基。
例如，通过固相亚单位合成法合成类肽化合物，具体包括以下步骤：
(1)将Rink amide AM树脂(取代水平为0.3mmol/g)溶胀后用六氢吡啶脱保护，将半胱氨酸与2-(3'-N-氧代-苯并三唑)-1,1',3,3'-四甲基脲六氟磷酸盐等摩尔量进行混合，在N-甲基吗啡啉的活化作用下进行偶联；
(2)将10mL浓度为2mol/L的溴乙酸和10mL浓度为3.2mol/L的N,N’-二异丙基碳二亚胺(DIC)加入到Rink amide AM树脂(多肽合成的起始树脂，取代水平为0.3mmol/g)中，在37℃下反应30min，将树脂末端的氨基酰化；
(3)再加入10mL浓度为2mol/L伯胺在37℃下反应90min，通过亲核取代反应替换掉溴原子，完成一个亚单位的合成；
(4)重复步骤(2)和(3)直至完成其余单位的合成；
(5)待合成完毕后，侧链保护基团被除去，并用质量百分含量分别为95％的三氟乙酸，2.5％的超纯水和2.5％的三异丙基硅烷将类肽化合物从树脂上裂解下来以备用。
例如，在本公开的实施例提供的类肽化合物的制备方法中，还可以根据需要包括将所得产物进行纯化的步骤。该纯化的方法没有特殊限制，可以采用本领域中纯化相类似产物的方法进行纯化，例如沉淀、过滤、透析、凝胶渗透色谱等。
本公开至少一实施例还提供一种寡聚物，该寡聚物的分子结构式为：
Figure PCTCN2019102009-appb-000010
其中，10≥n1≥3，10≥n2≥3，n1＝n2，n1和n2均为自然数。
例如，该寡聚物包括：β-苯乙胺亚单位、联苯乙胺亚单位、半胱氨酸(Cys)亚单位、丁二胺(Nlys)亚单位、3,4-亚甲二氧基苄胺(Npip)亚单位、3-氨基丙酸(Nce)亚单位、1-萘胺(Na)亚单位和乙二胺亚单位。 例如，各亚单位的分子结构式如下所示：
Figure PCTCN2019102009-appb-000011
(β-苯乙胺),
Figure PCTCN2019102009-appb-000012
(联苯乙胺),
Figure PCTCN2019102009-appb-000013
(半胱氨酸，Cys),
Figure PCTCN2019102009-appb-000014
(丁二胺，Nlys),
Figure PCTCN2019102009-appb-000015
(3-氨基丙酸，Nce),
Figure PCTCN2019102009-appb-000016
(乙二胺)
Figure PCTCN2019102009-appb-000017
(3,4-亚甲二氧基苄胺，Npip)，
Figure PCTCN2019102009-appb-000018
(1-萘胺，Na)。
例如，在本公开至少一实施例提供的寡聚物中，该寡聚物包含的亚单位按照以下顺序进行排列：[β-苯乙胺亚单位—3-氨基丙酸亚单位—联苯乙胺亚单位—3-氨基丙酸亚单位]n 2—β-苯乙胺亚单位—半胱氨酸(Cys)亚单位—丁二胺(Nlys)亚单位—丁二胺(Nlys)亚单位—丁二胺(Nlys)亚单位—3,4-亚甲二氧基苄胺(Npip)亚单位—3-氨基丙酸(Nce)亚单位—1-萘胺(Na)亚单位—联苯乙胺亚单位—乙二胺亚单位—[β-苯乙胺亚单位—乙胺亚单位—联苯乙胺亚单位—乙胺亚单位]n 1。
本公开至少一实施例还提供一种寡聚物的制备方法，该寡聚物通过固相亚单位的合成法合成。
例如，图2为本公开一实施例提供的类肽化合物的制备方法的流程图，如图2所示，该类肽化合物的制备方法包括以下步骤：
步骤S11：按照该寡聚物的亚单位的连接顺序，将寡聚物的第一个亚单位连接至固相载体上；
步骤S12：将溴乙酸在活化剂的作用下活化后与连接至固相载体上的第一个亚单位的氨基反应形成酰胺键；
步骤S13：将类肽化合物的第二个亚单位的供体与步骤S12得到的产物进行反应，取代掉溴原子，以完成第二个亚单位的连接；
步骤S14：重复进行溴乙酸以及后续亚单位的连接，直至完成所有亚单位的连接；
步骤S15：从固相载体上将合成得到的寡聚物裂解下来得到该类肽化合物。
例如，该寡聚物包括上述任一实施例中的类肽化合物，还包括分别形成在该类肽化合物的左侧和右侧的助链，该形成在该类肽化合物左侧的助链上均含有氨基，形成在该类肽化合物右侧的助链上均含有羧基，形成在该类肽化合物左侧的助链和右侧的助链有助于使得该寡聚物形成为二维层状结构，这样可以使得中间的类肽化合物暴露在传感器的表面以作为探针分子对HER2蛋白进行检测，形成在该类肽化合物左侧的助链和右侧的助链还可以使得该寡聚物的排列更加整齐。
例如，该寡聚物的制备过程中各个步骤用到的缩合剂和活化剂可以参见上述类肽化合物的制备过程的相关描述，在此不再赘述。
例如，在该寡聚物中，n1＝n2＝3，n1＝n2＝4，n1＝n2＝6，n1＝n2＝8，或者n1＝n2＝10。
需要说明的是，当n1和n2小于3时，会出现链长太短无法组装的问题；当n1和n2大于10时，形成的链太长，该寡聚物中间插入的类肽化合物的密度太低，会出现亲和力减弱的问题，从而无法实现与CTCs上的HER2蛋白特异性结合的问题。
示例一
分子结构为：
Figure PCTCN2019102009-appb-000019
的寡聚物的制备方法具体包括以下步骤：
(1)将Rink amide AM树脂(多肽合成的起始树脂，取代水平为0.3mmol/g)溶胀后用六氢吡啶脱保护，将β-苯乙胺与1-羟基苯并三唑等摩尔量进行混合，在N-甲基吗啡啉的活化作用下进行偶联；
(2)将10mL浓度为2mol/L的溴乙酸和10mL浓度为3.2mol/L的N,N’-二异丙基碳二亚胺(DIC)加入到Rink amide AM树脂中，在38℃下反应30min，将树脂末端的氨基酰化；
(3)加入10mL浓度为2mol/L的伯胺在37℃下反应90min，通过亲核取代反应替换掉溴原子，完成一个亚单位的合成；
(4)重复上述步骤(2)和(3)直至完成其余单位的合成；
(5)待合成完毕后，侧链保护基团被除去，并用质量百分含量分别为95％的三氟乙酸，2.5％的超纯水和2.5％的三异丙基硅烷将寡聚物从树脂上裂解下来备用。
形成上述结构的寡聚物的过程中，亚单位投入的顺序为：
β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙 酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、半胱氨酸(Cys)、叔丁氧羰基(Boc)保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、3,4-亚甲二氧基苄胺(Npip)、3-氨基丙酸(Nce)、1-萘胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺。
需要说明的是，叔丁氧羰基(t-Boc或Boc)是一种用于保护氨基的保护基。
例如，该寡聚物可以溶解到物质的量之比为二甲基亚砜：水＝2:1的二甲基亚砜(DMSO)和水(H 2O)的混合溶液中，使其浓度为2mM。
示例二
分子结构为：
Figure PCTCN2019102009-appb-000020
的寡聚物的制备方法具体包括以下步骤：
(1)将Rink amide AM树脂(多肽合成的起始树脂，取代水平0.3mmol/g)溶胀后用六氢吡啶脱保护，将β-苯乙胺与1-羟基苯并三唑等摩尔量进行混合，在N-甲基吗啡啉的活化作用下进行偶联；
(2)将20mL浓度为2mol/L的溴乙酸和15mL浓度为3.2mol/L的N,N’-二异丙基碳二亚胺(DIC)加入Rink amide AM树脂中，在38℃下反应30min，将树脂末端的氨基酰化；
(3)加入10mL浓度为2mol/L的伯胺在37℃下反应90min，通过亲核取代反应替换掉溴原子，完成一个亚单位的合成；
(4)重复上述步骤(2)和(3)直至完成其余单位的合成；
(5)待合成完毕后，侧链保护基团被除去，并用质量百分含量分别为95％的三氟乙酸，2.5％的超纯水和2.5％的三异丙基硅烷将寡聚物从树脂上 裂解下来备用。
例如，形成上述结构的寡聚物的过程中，亚单位投入的顺序为：
β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、半胱氨酸(Cys)、Boc保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、3,4-亚甲二氧基苄胺(Npip)、3-氨基丙酸(Nce)、1-萘胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺。
例如，该寡聚物可以溶解到物质的量之比为二甲基亚砜：水＝2:1的二甲基亚砜(DMSO)和水(H 2O)的混合溶液中，使其浓度为2mM。
示例三
分子结构为：
Figure PCTCN2019102009-appb-000021
的寡聚物的制备方法具体包括以下步骤：
(1)将Rink amide AM树脂(多肽合成的起始树脂，取代水平0.3mmol/g)溶胀后用六氢吡啶脱保护，将β-苯乙胺与1-羟基苯并三唑等摩尔量进行混合，在N-甲基吗啡啉的活化下进行偶联；
(2)将25mL浓度为2mol/L的溴乙酸和22mL浓度为3.2mol/L的N,N’-二异丙基碳二亚胺(DIC)加入Rink amide AM树脂中，在38℃下反应30min，将树脂末端的氨基酰化；
(3)加入10mL浓度为3mol/L的伯胺在37℃下反应90min，通过亲 核取代反应替换掉溴原子，完成一个亚单位的合成；
(4)重复上述步骤(2)和(3)直至完成其余单位的合成；
(5)待合成完毕后，侧链保护基团被除去，并用质量百分含量分别为95％的三氟乙酸，2.5％的超纯水和2.5％的三异丙基硅烷将寡聚物从树脂上裂解下来备用。
例如，形成上述结构的寡聚物的过程中，亚单位投入顺序为：
β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、3-氨基丙酸、联苯乙胺、3-氨基丙酸；β-苯乙胺、半胱氨酸(Cys)、Boc保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、Boc保护的丁二胺(Nlys)、3,4-亚甲二氧基苄胺(Npip)、3-氨基丙酸(Nce)、1-萘胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺；β-苯乙胺、Boc保护的乙二胺、联苯乙胺、Boc保护的乙二胺。
例如，该寡聚物可以溶解到物质的量之比为二甲基亚砜：水＝2:1的二甲基亚砜(DMSO)和水(H 2O)的混合溶液中，使的寡聚物的浓度为2mM。
本公开的实施例提供的寡聚物的合成过程简单，并且该寡聚物与HER2蛋白的结合能力较强，能够有效地通过血清中的HER2蛋白来对乳腺癌患者和正常人的血清进行筛查，可以特异性地识别乳腺癌CTCs表面的HER2蛋白为高灵敏度地捕获相应的CTCs提供了有利的保障。例如，可以设计对HER2蛋白具有高亲和、高灵敏度的分子探针。
例如，利用表面等离子体共振成像技术对寡聚物与HER2蛋白间的结合能力进行测试的步骤如下：
(1)将寡聚物溶解到ddH 2O中至寡聚物的浓度为1-1000μM；
(2)将上述寡聚物溶液滴在一张3D芯片的表面上，每种样品重复滴3 次，在4℃下放置12小时后，依次用10XPBS缓冲液，1XPBS缓冲液和超纯水清洗干净，然后将芯片用1M的盐酸氨基乙醇封闭30分钟，接着用超纯水清洗5次，最后用氮气吹干；
(3)将芯片安装在SPRi仪器上，测定SPRi角并将该SPRi角调节至最佳光学位置，在检测区域选取相关的检测点，该相关的检测点包括样品点与空白点，设置实验流速为2μL/s；
(4)选择PBS为缓冲液通入流通池至基线稳定后依次通过浓度为2.2μM、4.6×10 -1μM、9.1×10 -2μM、1.8×10 -2μM和3.6×10 -3μM的HER2蛋白进行检测，结合时间分别为300秒，解离时间分别为300秒，在前一个浓度的HER2蛋白检测完成后通入磷酸以使得测试液回到初始状态，然后进行后一浓度的HER2蛋白的测试。
例如，图3为本公开一实施例提供的二维寡聚物的形成过程示意图，如图3所示，形成二维的寡聚物的过程为：将本公开任一实施例提供的寡聚物放置于朗格缪尔槽中，该寡聚物包括亲水的一端和疏水的一端，在无外界作用力的情况下，该寡聚物无序的排列在气体和液体的界面处；然后对该无序排列的寡聚物施加外力，使得该寡聚物在气液界面处有序地排列；进一步地对该有序排列的寡聚物施加外力，使得该寡聚物被挤压至气体和液体的界面之下。在气体和液体的界面之下，寡聚物亲水的一端暴露在外侧，疏水的一端形成在内侧，从而形成二维结构的寡聚物。
寡聚物纳米片层的形成过程如下：将以上示例一至示例三中任一示例得到的浓度为2mM的寡聚物溶于10mM 4-羟乙基哌嗪乙磺酸和100mM氯化钠中，且加入缓冲液使得pH＝8.0，使得寡聚物最终的浓度为1-100μM，例如，为20μM，然后采用手动摇晃法：将类肽溶液在室温下稳定存放22小时，然后手动轻轻摇晃30秒，再稳定1分钟，重复摇晃-稳定过程5次；或者机器摇晃法：将盛放寡聚物溶液的管从水平方向缓慢旋转(0.6rpm)至垂直方向，每隔450秒旋转一次；在得到的寡聚物纳米片层溶液中加入尼罗红，至寡聚物溶液的最终浓度为1μM，将寡聚物溶液放在质量百分含量为1％的琼脂上，使用荧光显微镜(Vert.A1,Carl Zeiss Far East,Germany)观察，结果如图4A和图4B所示，可以观察到明显的纳米片层结构。
例如，图5为本公开的示例一中的寡聚物与浓度分别为2.2μM、4.6×10 -1μM、9.1×10 -2μM、1.8×10 -2μM和3.6×10 -3μM的HER2蛋白结合的表面 等离基元共振检测的结果图，其中，△RU代表流动相通过阵列后的结合信号减去初始PBS缓冲液的基线信号，曲线是PlexArray HT的测试结果，拟合直线是BIAevalution 4.1拟合得到，△RU是在表面等离子体共振成像中用来反映结合信号强度的单位，是一种无量纲单位，经拟合，平衡解离常数KD为3.76×10 -10摩尔/升，这表明该寡聚物与HER2蛋白具有相当高的亲和力水平。
利用表面等离子体共振成像技术测试寡聚物对乳腺癌病人、胃癌病人或者卵巢癌病人的血清和正常人的血清进行检测的具体步骤如下：
(1)将寡聚物溶液滴在一张3D芯片的表面上，每种样品重复滴3次，在4℃下放置12小时后，依次用10XPBS(Phosphate Buffered Saline，磷酸盐缓冲液)，1XPBS和超纯水清洗干净，然后将芯片用1M的盐酸氨基乙醇封闭30分钟，然后用超纯水清洗5次，最后用氮气吹干；将上述芯片安装在SPRi仪器上，测定SPRi角并调节至最佳光学位置，在检测区域选取相关检测点，该相关检测点包括样品点与空白点，设置实验流速为2μL/s；
(2)选择PBS为缓冲液通入流通池至基线稳定后，分别通入不同病人与正常人的血清(1:5000)稀释，结合时间为300秒，解离时间为300秒，每个样品检测完后通入磷酸和蛋白酶K以使得测试液回到初始状态，然后进行下一种血清的测试。
根据寡聚物与表面等离子体共振成像的信号的结合强度，区分乳腺癌病人、胃癌病人或者卵巢癌病人与正常人。
例如，利用表面等离子体共振成像技术测试该乳腺癌诊断系统检测血清的灵敏度，具体步骤如下：
(1)将寡聚物溶液滴在一张3D芯片的表面上，每种样品重复滴3次，在4℃下放置12小时后，依次用10XPBS，1XPBS和超纯水清洗干净，然后将芯片用1M的盐酸氨基乙醇封闭30分钟，然后用超纯水清洗5次，最后用氮气吹干；将上述芯片安装在SPRi仪器上，测定SPRi角并调节至最佳光学位置，在检测区域选取相关检测点，包括样品点与空白点，设置实验流速为2μL/s；
(2)选择PBS为缓冲液通入流通池至基线稳定后，分别通入不同病人与正常人的血清稀释液，不同病人与正常人的血清稀释的浓度均分别为1:2000、1:4000、1:8000、1:16000、1:32000，结合时间为300秒，解离时间 为300秒，每个样品检测完后通入磷酸和蛋白酶K以使得测试液回到初始状态，然后进行下一种血清稀释液的测试。
检测结果表明，血清稀释的比例小于或等于1:8000时，可以明显区分HER2高表达的乳腺癌病人、HER2高表达的卵巢癌病人、HER2高表达的胃癌病人与正常人，展示了其具有极高的灵敏度。
例如，该3D芯片为购自美国Plexera Bioscience公司的PlexArray HT 3D芯片。
例如，该寡聚物在气-液界面上自组装形成表面带有特异性识别HER2蛋白的类肽纳米片层，类肽纳米片层作为支架展示和支撑类肽化合物作为分子探针对HER2蛋白进行识别。利用该类肽纳米片层，结合表面等离激元共振技术可用于乳腺癌的检测。
例如，该寡聚物为二维纳米片层材料，这样可以实现寡聚物耦合在传感器上，并将具有亲和作用的类肽化合物展示在传感器的表面。
二维的类肽纳米材料在生物学和电子学上扮演着越来越重要的角色，例如传感、模板的生长和过滤以及作为蛋白质的模拟物测试蛋白质的分子识别和催化能力。通过朗缪尔槽实验装置揭示了类肽纳米片层的形成是一个类肽分子的自组装和将外界机械能转换为类肽分子化学能的一个热力学平衡过程。
例如，该药物组合物还包括：上述任一实施例中的寡聚物；以及药学上接受的辅料。
例如，该辅料包括赋形剂、稀释剂、载体、调味剂、粘合剂和填充剂中的至少之一。
例如，赋形剂例如是乳剂或油性混悬剂，或者聚亚烷二醇类，例如聚丙二醇。
本公开至少一实施例还提供一种上述任一实施例中的药物组合物在制备检测或者诊断与酪氨酸激酶HER2相关的疾病的药物中的用途。
例如，该疾病包括乳腺癌、胃癌和卵巢癌。
本公开至少一实施例还提供一种用于鉴定循环肿瘤细胞的试剂盒，包括：盒体、设置在该盒体内的微流控芯片，以及设置在该盒体内的荧光探针，荧光探针为带有荧光标记的如上任一实施例中的寡聚物。
例如，该微流控芯片包括微阀控制层和微阀薄膜层。该微阀控制层上设 有六个贯通控制层的孔以及三条气体通道，该三个孔为加样孔，连通基板，用于样品和试剂流入和流出；其余三个孔分别连接三条气体通道，用于注入气体，且控制微阀的开启与闭合。微阀薄膜层上设有三个贯通薄膜层的孔，分别与上述微阀控制层的三个加样孔对应连通。
例如，该微阀控制层和微阀薄膜层的外轮廓尺寸应与基板的尺寸相匹配。
例如，荧光探针用于鉴定细胞，本公开的实施例中带有荧光的分子探针是标记了荧光分子的寡聚物。
例如，本公开的实施例对荧光基团的类型没有特别限制，只要修饰后能够赋予寡聚物荧光性能并且还能够实现寡聚物的基本功能即可。根据本公开的实施例中的寡聚物可以用一个或多个荧光基团进行修饰。例如，用一个荧光基团修饰得到单荧光标记寡聚物，或者用两个荧光基团标记得到双荧光标记寡聚物。在一些实施方式中，荧光基团可以非限制性地选自蓝色荧光染料、近红外荧光染料，绿色荧光染料等，例如，含香豆素荧光基团、含蒽荧光基团、罗丹明荧光基团、菲并咪唑荧光基团、含萘荧光基团、异硫氰酸荧光素、羧基荧光素(FAM)、硫氰酸荧光素(FITC)、丹磺酰氯(dansyl chloride)、2,4-二硝基苯(Dnp)、羧基罗丹明110(carbo-xyrhodamine110)、德克萨斯红(Texas Red)、五甲川菁染料(Cy5)和七甲川菁染料(Cy7)等。
例如，除了微流控芯片和荧光探针(Probe)之外，该试剂盒还可以包括荧光显微镜(荧光成像系统)、自主开发的图像分析软件(分析计数系统)、泵构成完整的系统，完成对血液样品的处理和循环肿瘤细胞的分离和计数。
例如，荧光显微镜用于检测微V形定位阵列中的细胞是否有荧光，并对该功能区进行全覆盖荧光成像，获取多通道荧光图像。
例如，自主开发的图像处理软件用于分析荧光显微镜获取的图像并获得相应的CTC数量。该软件通过对图像中细胞的大小、面积、长宽比、圆度等参数进行精确计算，筛选出符合要求的CTC，并对CTC进行计数。根据自主编写的算法对定位细胞进行筛选鉴定，鉴定出符合标记荧光特性的循环肿瘤细胞，并进行计数，然后报告细胞位置和放大细胞图像。
本公开的实施例提供一种类肽化合物、类肽化合物的制备方法、寡聚物、药物组合物、药物组合物在制备检测或者诊断与酪氨酸激酶HER2相关的疾病的药物中的用途以及用于鉴定循环肿瘤细胞的试剂盒，具有以下至少一项
有益效果：
(1)在本公开至少一实施例提供的寡聚物中，寡聚物与CTCs表面的HER2蛋白的结合能力较强，通过表面等离基元共振技术得到该寡聚物与HER2蛋白的结合动力学常数中的平衡解离常数KD为10 -10摩尔/升数量级；
(2)在本公开至少一实施例提供的寡聚物中，利用表面等离基元共振技术检测寡聚物对乳腺癌病人的血液信号强度与正常人的血液信号强度，可发现利用此寡聚物可以明显鉴别乳腺癌病人与正常人；
(3)在本公开至少一实施例提供的寡聚物中，以该寡聚物为基础的诊断乳腺癌的诊断技术能够实现无创无标记的快速诊断；
(4)本公开至少一实施例提供的类肽化合物和寡聚物的合成方法简单，制备的效率高，且制作成本低。
有以下几点需要说明：
(1)本发明实施例附图只涉及到与本发明实施例涉及到的结构，其他结构可参考通常设计。
(2)为了清晰起见，在用于描述本发明的实施例的附图中，层或区域的厚度被放大或缩小，即这些附图并非按照实际的比例绘制。
(3)在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合以得到新的实施例。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (12)

  1. 一种类肽化合物，包括：半胱氨酸亚单位、丁二胺亚单位、3,4-亚甲二氧基苄胺亚单位、3-氨基丙酸亚单位和1-萘胺亚单位。
  2. 根据权利要求1所述的类肽化合物，其中，所述类肽化合物包含的亚单位的顺序为半胱氨酸亚单位—丁二胺亚单位—丁二胺亚单位—丁二胺亚单位—3,4-亚甲二氧基苄胺亚单位—3-氨基丙酸亚单位—1-萘胺亚单位。
  3. 根据权利要求1或2所述的类肽化合物，其中，所述类肽化合物具有式I所示的结构：
    Figure PCTCN2019102009-appb-100001
  4. 一种如权利要求1-3中任一项所述的类肽化合物的制备方法，其中，所述制备方法包括固相合成法。
  5. 根据权利要求4所述的制备方法，其中，所述制备方法包括以下步骤：
    (1)按照所述类肽化合物的亚单位的连接顺序，将所述类肽化合物的第一个亚单位连接至固相载体上；
    (2)将溴乙酸在活化剂的活化作用下与连接至所述固相载体上的第一个亚单位的氨基进行反应形成酰胺键；
    (3)将所述类肽化合物的第二个亚单位的供体与步骤(2)得到的产物进行反应，取代掉溴原子，完成第二个亚单位的连接；
    (4)重复进行所述溴乙酸以及后续亚单位的连接，直至完成所有亚单位的连接；
    (5)从所述固相载体上将合成得到的类肽化合物裂解下来得到所述类肽化合物。
  6. 一种寡聚物，其中，所述寡聚物的分子结构式为：
    Figure PCTCN2019102009-appb-100002
    其中，10≥n 1≥3，10≥n 2≥3，n 1＝n 2，n 1和n 2均为自然数。
  7. 根据权利要求6所述的寡聚物，其中，所述寡聚物的分子结构式为：
    Figure PCTCN2019102009-appb-100003
  8. 一种药物组合物，包括：
    如权利要求7所述的寡聚物；以及
    药学上接受的辅料。
  9. 根据权利要求8所述的药物组合物，其中，所述辅料为赋形剂、稀释剂、载体、调味剂、粘合剂和填充剂中的至少之一。
  10. 一种如权利要求8或9所述的药物组合物在制备检测或者诊断与酪氨酸激酶HER2相关的疾病的药物中的用途。
  11. 根据权利要求10所述的用途，其中，所述疾病包括乳腺癌、胃癌和卵巢癌中的至少之一。
  12. 一种用于鉴定循环肿瘤细胞的试剂盒，包括：盒体、设置在所述盒体内的微流控芯片，以及设置在所述盒体内的荧光探针，所述荧光探针为带有荧光标记的如权利要求6或7所述的寡聚物。
PCT/CN2019/102009 2018-09-28 2019-08-22 类肽化合物及其制备方法、寡聚物、药物组合物和试剂盒 WO2020063216A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/646,320 US11591370B2 (en) 2018-09-28 2019-08-22 Peptoid compound and manufacturing method thereof, oligomer, pharmaceutical composition, and kit

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811138563.1A CN110981936B (zh) 2018-09-28 2018-09-28 类肽化合物及其制备方法、寡聚物、药物组合物和试剂盒
CN201811138563.1 2018-09-28

Publications (1)

Publication Number Publication Date
WO2020063216A1 true WO2020063216A1 (zh) 2020-04-02

Family

ID=69949289

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/102009 WO2020063216A1 (zh) 2018-09-28 2019-08-22 类肽化合物及其制备方法、寡聚物、药物组合物和试剂盒

Country Status (3)

Country Link
US (1) US11591370B2 (zh)
CN (1) CN110981936B (zh)
WO (1) WO2020063216A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114395126A (zh) * 2022-01-20 2022-04-26 中国药科大学 一种聚乙烯亚胺衍生物及其在制备免疫佐剂中的应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111393503B (zh) * 2019-01-03 2022-05-13 北京京东方技术开发有限公司 一种类肽及其制备方法和用途
CN112834740B (zh) * 2020-12-31 2023-10-24 北京旌准医疗科技有限公司 一种类肽寡聚物及其制备方法、药物组合物和微流控芯片
CN117642418A (zh) * 2022-06-29 2024-03-01 京东方科技集团股份有限公司 针对弥漫性大b细胞淋巴瘤早期诊断的探针和试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105753940A (zh) * 2016-02-05 2016-07-13 国家纳米科学中心 一种类肽抑制剂、其制备方法及应用
CN106581700A (zh) * 2016-12-02 2017-04-26 北京大学 一种靶向her2的新型多肽放射性药物及其制备方法和应用
CN106854233A (zh) * 2017-03-03 2017-06-16 国家纳米科学中心 一种类肽及其制备方法和应用
CN106866794A (zh) * 2017-02-15 2017-06-20 国家纳米科学中心 一种类肽及其制备方法和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005504043A (ja) * 2001-08-10 2005-02-10 パラチン テクノロジーズ インク． 生物学的に活性な金属ペプチド類のペプチド模倣体類
CN102276698B (zh) * 2010-06-10 2014-02-26 中国人民解放军军事医学科学院毒物药物研究所 一种mc4-r环肽类激动剂及其用途
CN105085631B (zh) * 2015-09-14 2019-01-25 国家纳米科学中心 一种特异靶向her2蛋白的多肽及其应用
CN105693860B (zh) * 2016-03-03 2019-08-27 国家纳米科学中心 特异性靶向her2蛋白的多肽及其应用
CN107213468A (zh) * 2017-04-14 2017-09-29 国家纳米科学中心 一种靶向纳米药物载体及其制备方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105753940A (zh) * 2016-02-05 2016-07-13 国家纳米科学中心 一种类肽抑制剂、其制备方法及应用
CN106581700A (zh) * 2016-12-02 2017-04-26 北京大学 一种靶向her2的新型多肽放射性药物及其制备方法和应用
CN106866794A (zh) * 2017-02-15 2017-06-20 国家纳米科学中心 一种类肽及其制备方法和应用
CN106854233A (zh) * 2017-03-03 2017-06-16 国家纳米科学中心 一种类肽及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZHAO ZIJIAN ET AL.: "Antiamyloidogenic Activity of Abeta2-Binding Peptoid in Modulating Amyloid Oligomerization", SMALL, vol. 13, no. 1, 13 October 2016 (2016-10-13) - 4 January 2017 (2017-01-04), pages 1 - 10, XP055697826 *
ZHU, LING ET AL.: "Antibody-Mimetic Peptoid Nanosheet for Label-Free Serum-Based Diagnosis of Alzheimer's Disease", ADVANCED MATERIALS, vol. 29, no. 30, 12 June 2017 (2017-06-12) - 11 August 2017 (2017-08-11), pages 1 - 7, XP055697828 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114395126A (zh) * 2022-01-20 2022-04-26 中国药科大学 一种聚乙烯亚胺衍生物及其在制备免疫佐剂中的应用
CN114395126B (zh) * 2022-01-20 2023-08-18 中国药科大学 一种聚乙烯亚胺衍生物及其在制备免疫佐剂中的应用

Also Published As

Publication number Publication date
US20210206808A1 (en) 2021-07-08
US11591370B2 (en) 2023-02-28
CN110981936B (zh) 2021-10-12
CN110981936A (zh) 2020-04-10

Similar Documents

Publication Publication Date Title
WO2020063216A1 (zh) 类肽化合物及其制备方法、寡聚物、药物组合物和试剂盒
CN102026671B (zh) 整联蛋白靶向试剂及使用其的体内和体外成像方法
CN108586579B (zh) 一种类肽及其衍生物、盐、制备方法和用途
CN104130315B (zh) 一种特异靶向her2蛋白的多肽
CN104955484A (zh) 用于前列腺癌成像的前列腺特异性抗原药剂及其使用方法
CN105085631B (zh) 一种特异靶向her2蛋白的多肽及其应用
US11971413B2 (en) Peptoid, and its preparation method and use
WO2021102919A1 (zh) 类肽化合物以及表面偶联有类肽化合物的检测芯片
JP5863057B2 (ja) 組織評価方法
CN110818777A (zh) 一种类肽纳米片层、其制备方法及应用
CN112834740B (zh) 一种类肽寡聚物及其制备方法、药物组合物和微流控芯片
US11370813B2 (en) Peptoid, and its preparation method and use
CN112370537A (zh) 一种双靶向磁性荧光纳米微球、其制备方法及在肝癌循环肿瘤细胞中的应用
CN106518965A (zh) 对溶血磷脂酸特异性识别的多肽探针及其制备与应用
WO2020233572A1 (zh) 多肽磁性纳米颗粒、其制备方法及应用
US9766252B2 (en) Peptide with gold binding and EGFR receptor affinity and same attached to gold nanostructure
WO2024060432A1 (zh) 一种近红外胃癌检测试剂盒及其制备方法
CN115144588A (zh) 用于高效捕获、原位培养和光学检测肺癌中CTCs的分析平台及其构建方法和临床应用
Kwak et al. Gastric cancer specific drug delivery with hydrophilic peptide probe conjugation
CN111868072B (zh) 类肽化合物及其制备方法、纳米载体以及药物组合物
CN115974966A (zh) 靶向神经丝轻链蛋白的多肽及其应用
CN115404220A (zh) 一种分离富集循环肿瘤细胞的多肽功能化微纳界面材料及其制备方法和应用
CN117164672A (zh) 一种靶向黑色素瘤的近红外二区荧光探针、制备方法及应用
CN104428674A (zh) 前列腺癌和sar-cov的人血清生物标记物
ES2315101A1 (es) Procedimiento para el diagnostico de cancer de cabeza y cuello mediante la valoracion en suero humano del receptor del factor de crecimiento epidermico y de su ligando especifico, el factor de crecimiento epidermico.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19865643

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19865643

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205N DATED 21.05.2021)

122 Ep: pct application non-entry in european phase

Ref document number: 19865643

Country of ref document: EP

Kind code of ref document: A1