CN114957356A - Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof - Google Patents

Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof Download PDF

Info

Publication number
CN114957356A
CN114957356A CN202210721691.9A CN202210721691A CN114957356A CN 114957356 A CN114957356 A CN 114957356A CN 202210721691 A CN202210721691 A CN 202210721691A CN 114957356 A CN114957356 A CN 114957356A
Authority
CN
China
Prior art keywords
derivative
glucuronidase
beta
responsive
glucuronic acid
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
CN202210721691.9A
Other languages
Chinese (zh)
Inventor
周志昉
吴志猛
李艳春
洪皓飞
龚亮
林汉
周坤
李丹
施杰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
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 Jiangnan University filed Critical Jiangnan University
Priority to CN202210721691.9A priority Critical patent/CN114957356A/en
Publication of CN114957356A publication Critical patent/CN114957356A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • 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"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Optics & Photonics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Materials Engineering (AREA)
  • Saccharide Compounds (AREA)

Abstract

The invention discloses a beta-glucuronidase responsive saccharide derivative and a preparation method and application thereof, wherein the saccharide derivative takes a saccharide precursor substrate as a raw material, and a chemical report group and a beta-glucuronidase responsive chemical structure are modified on the saccharide precursor substrate, wherein the saccharide precursor substrate comprises monosaccharide and any one of monosaccharide derivative, disaccharide and disaccharide derivative, the chemical report group is used for marking or treating, and the beta-glucuronidase responsive chemical structure takes a beta-glycosidic bond and the like as a self-breaking connecting arm and is connected with glucuronic acid/glucuronic acid derivative. Under the catalysis of beta-glucuronidase, a beta-glucuronic acid response chemical structure can be broken from the carbohydrate derivative, and a hydroxyl group on the monosaccharide or the disaccharide or the derivative thereof is exposed. The carbohydrate derivative can be taken up by tumor cells in a large amount, and metabolizes non-natural sugar on the surface of the tumor cells through a carbohydrate metabolism path, so that the carbohydrate derivative is used for marking and treating tumors.

Description

Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof
Technical Field
The invention relates to the field of biomedical research, in particular to a saccharide derivative responding to beta-glucuronidase and a preparation method and application thereof.
Background
The glycocalyx on the cell surface mainly comprises proteoglycan, glycoprotein, glycolipid and the like, is a layer of compact carbohydrate compounds on the surface of mammalian cells, and plays an important role in maintaining the cell function. The structure and properties of the glycocalyx on the surface of cancer cells (tumor cells) differ from those of normal cells, i.e., aberrant glycosylation modifications. Abnormalities of the tumor glycocalyx, such as sialic acid (sialic acid) on the outermost glycan layer on the cell surface, are closely associated with bacterial infection, tumorigenesis, and malignant tumor metastasis, among others. These tumor-associated aberrant glycosylation modifications are important targets for tumor cell labeling, tumor diagnosis, tumor imaging, and cancer therapy. Thus, a series of synthetic unnatural sugars derive from metabolic markers for cells based on the properties of carbohydrates for metabolism in the cell.
The use of artificially synthesized non-natural sugar derivatives as carbohydrate precursors, the labeling of tumor cells via sugar metabolism has become an important strategy for tumor labeling, diagnosis and tumor therapy. For example, unnatural sialic acids with bio-orthogonal functionalities (azide groups or alkynyl groups) are widely used in labeling of cells, and further in glycan analysis, proteomic analysis, cell labeling, tumor therapy, and the like. However, such sialic acid metabolism markers lack cell selectivity and it is difficult to specifically label tumor cells, and therefore, how to improve the cell selectivity of the carbohydrate metabolism markers is very important for the wider application of this strategy.
Beta-glucuronidase has strong activity in a plurality of malignant tumors, so the beta-glucuronidase is also an important biomarker of tumors, particularly cancers such as colorectal cancer and breast cancer, and the development of tumor diagnosis and treatment strategies aiming at the beta-glucuronidase is a hot direction. The invention provides design and preparation of a beta-glucuronidase response carbohydrate derivative and application of the beta-glucuronidase response carbohydrate derivative in tumor marking, tumor imaging and tumor treatment by combining carbohydrate metabolism engineering and click chemistry strategies. By modifying the beta-glucuronic acid of the monosaccharide and the disaccharide, the monosaccharide and the disaccharide can only be selectively catalyzed by the beta-glucuronic acid enzyme to release monosaccharide and disaccharide precursors, and the monosaccharide and the disaccharide precursors are accumulated in tumor cells with high beta-glucuronic acid enzyme activity, so that the specific marker of the tumor cells is enhanced, and the beta-glucuronic acid can be used for diagnosis and treatment of tumors.
Disclosure of Invention
The invention aims to provide a beta-glucuronidase response carbohydrate derivative and application thereof in tumor diagnosis and marking, which are used for carbohydrate metabolism engineering of tumor cells, and the tumor cells containing high-activity beta-glucuronidase can selectively accumulate the carbohydrate derivative and express the carbohydrate derivative on the cell surface, so that the tumor cells can be selectively marked and further used for imaging and treating tumors.
In order to solve the problems in the prior art, the invention provides the following technical scheme:
a first object of the present invention is to provide a β -glucuronidase-responsive saccharide derivative comprising:
a. the chemical structure of the response of the beta-glucuronidase comprises glucuronic acid/glucuronic acid derivatives and a self-breaking connecting arm,
b. the sugar precursor substrate comprises monosaccharide and any one of monosaccharide derivatives, disaccharide and disaccharide derivatives,
c. chemical reporter groups, including chemical groups that can be used in click chemistry reactions;
the chemical structure of the a, beta-glucuronidase response is modified on the hydroxyl group of the b.
In one embodiment of the invention, the self-cleaving linker arm comprises a β -glycosidic bond.
In one embodiment of the invention, when the self-cleaving linker arm is a β -glycosidic linkage, the β -glucuronidase responsive chemical structure is a β -glycosidic linked glucuronic acid/glucuronic acid derivative.
The invention modifies a beta-glucuronidase responsive chemical structure and a chemical report group on a sugar precursor substrate, wherein the sugar is monosaccharide and monosaccharide derivatives, disaccharide and disaccharide derivatives, and the beta-glucuronidase responsive chemical structure is a glucuronic acid/glucuronic acid derivative connected with a self-breaking connecting arm such as a beta-glycosidic bond. Under the catalysis of the beta-glucuronidase, the corresponding chemical structure of the beta-glucuronidase can be broken from the carbohydrate derivative, and hydroxyl groups on the monosaccharide/disaccharide or the derivative thereof are exposed. Therefore, tumor cells containing high-activity beta-glucuronidase can selectively accumulate the carbohydrate derivatives and express the carbohydrate derivatives on the cell surface, so that the tumor cells can be selectively marked and further used for imaging and treating tumors, as shown in figure 1.
In an embodiment of the present invention, the monosaccharide and the monosaccharide derivative include any one of mannose, mannosamine, acetamidomannose, sialic acid, glucose, glucosamine, galactose, galactosamine, or fucose, and the disaccharide derivative include any one of trehalose, lactose, maltose, and the like.
In one embodiment of the present invention, the self-cleaving linker arm comprises a β -glucosidic bond, i.e., a β -glucosidic bond.
In one embodiment of the invention, when the self-cleaving linker arm is a β -glycosidic linkage, the β -glucuronidase responsive chemical structure is a β -glycosidic linked glucuronic acid/glucuronic acid derivative.
In one embodiment of the present invention, the chemical reporter group is a group containing a bio-orthogonal functional group.
In one embodiment of the present invention, the bio-orthogonal functional group includes any one of azide-N3, alkynyl, ketone, thiol, and the like.
In one embodiment of the invention, the beta-glucuronidase-responsive chemical structure is modified at the hydroxyl group at position 6 of the sugar precursor substrate, i.e., at the hydroxyl group at position 6 of a monosaccharide or derivative thereof, or at the hydroxyl group at position 6 of a disaccharide or derivative thereof.
In one embodiment of the invention, the chemical reporter group is modified at the amino or hydroxyl group at position 2 of the monosaccharide or derivative thereof, or at the amino or hydroxyl group at position 2 of the disaccharide or derivative thereof.
In one embodiment of the present invention, the β -glucuronidase-responsive carbohydrate derivative has a structure represented by formula (I) or (II):
Figure BDA0003700763930000031
the second purpose of the invention is to provide a preparation method of a saccharide derivative responding to beta-glucuronidase, which comprises the following steps:
s1, preparing a carbonate compound, specifically preparing a sugar precursor substrate carbonate activated ester derivative activated by 4-nitrophenyl chloroformate by using a sugar precursor substrate, and marking the derivative as the carbonate compound;
s2, synthesizing the glucuronic acid/glucuronic acid derivative into a glucuronic acid derivative with a cleavable connecting arm;
s3, coupling the carbonate compound to the glucuronic acid derivative with the cleavable connecting arm to prepare the sugar derivative with the response of the beta-glucuronidase.
In one embodiment of the present invention, the reaction equation of the preparation method of the saccharide derivative of the present invention is shown in FIG. 2 below:
Figure BDA0003700763930000041
in one embodiment of the present invention, in step S1, the preparation of the activated ester derivative of the 4-nitrophenyl chloroformate activated sugar precursor substrate carbonate is performed according to the following equation, as shown in fig. 3:
Figure BDA0003700763930000042
in one embodiment of the present invention, in step S1, the sugar precursor substrate is activated by 4-nitrophenyl chloroformate to form said carbonate compound.
In one embodiment of the invention, in step S1, the sugar precursor substrate comprises Ac 3 ManNAz, etc.
In one embodiment of the present invention, the ratio of reactants in step S1 is 4-nitrophenylcarbonyl chlorate (1.2eq), sugar precursor substrate (1eq), TEA (2eq), r.t, 12 h.
In one embodiment of the present invention, in step S1, the sugar precursor substrate is prepared by the method of the literature (J Am Chem Soc,2010,132,9516-9518), and the reaction equation is shown in FIG. 4 below:
Figure BDA0003700763930000043
in one embodiment of the present invention, in step S2, the glucuronic acid/glucuronic acid derivative is subjected to ring opening by sodium methoxide, acetylation, followed by bromination to obtain a sugar donor for glycosylation, followed by reduction, and a carbonate intermediate is prepared by using 4-nitrophenyl chloroformate to obtain a carbamate intermediate, which is synthesized into the glucuronic acid derivative with a cleavable linking arm.
In one embodiment of the present invention, in step S2, the glucuronic acid/glucuronic acid derivative is synthesized into a glucuronic acid derivative with a cleavable linker arm by the methods of (J Am Chem Soc,2012,134, 3103-.
In one embodiment of the present invention, the coupling reaction is carried out in the presence of DMAP, DCM in step S3.
In one embodiment of the present invention, in step S3, the ratio of each reactant is: carbonate compound (1eq), compound 15(1.2eq), DCM, DMAP (1eq), DIPEA (2eq), r.t,4h.
The third purpose of the invention is to provide the application of the beta-glucuronidase response carbohydrate derivative in preparing tumor diagnosis and markers.
The fourth purpose of the invention is to provide a beta-glucuronidase response carbohydrate derivative or a preparation method of the beta-glucuronidase response carbohydrate derivative, and application of the beta-glucuronidase response carbohydrate derivative in tumor imaging.
The saccharide derivative is modified with a chemical reporter group, and the chemical reporter group is used for labeling or treatment. The beta-glucuronidase response carbohydrate derivative can be taken up by tumor cells in a large amount, and expresses non-natural sugar on the surface of the tumor cells through a carbohydrate metabolism path, so that the beta-glucuronidase response carbohydrate derivative is used for marking tumors and treating the tumors.
Has the advantages that: compared with the traditional carbohydrate derivative precursor with a chemical report group, the invention has the following advantages:
(1) the beta-glucuronidase-responsive carbohydrate derivative can selectively mark tumor cells through catalysis of the beta-glucuronidase, can be accumulated in the tumor cells in a large amount, avoids side effects on normal cells, and promotes the tumor marking efficiency and the tumor treatment efficiency. This strategy provides a simple and effective method to enhance the selective labeling of tumor cells, and can be further used for tumor imaging and tumor therapy, and can be widely applied in the field of tumor diagnosis and therapy.
(2) The saccharide derivative provided by the invention has the characteristic of response of beta-glucuronidase, can improve the selectivity of tumor cell markers, and can be accumulated in a large amount in tumor cells with high beta-glucuronidase activity, so that the tumor marking efficiency and the tumor treatment efficiency are promoted. The sugar metabolism labeled precursor compound with tumor cell selectivity is a sugar precursor responding to beta-glucuronidase, the existing sugar metabolism engineering is reasonably modified, and the sugar precursor derivative has high labeling efficiency, reduces the labeling of normal cells, and further reduces side effects. The invention can be used for marking, imaging, diagnosing and treating tumors.
(3) The preparation method of the beta-glucuronidase response carbohydrate derivative has high preparation efficiency, is easy to purify, and is easy to obtain the precursor of the compound.
Drawings
FIG. 1 is a schematic diagram of the reaction principle of the present invention.
FIG. 2 is a reaction scheme of the process for the preparation of the saccharide derivatives according to the present invention.
FIG. 3 is a reaction scheme for the preparation of 4-nitrophenyl chloroformate-activated monosaccharide carbonate activated ester derivatives in accordance with the present invention.
FIG. 4 is a reaction scheme for preparing a sugar precursor substrate according to the present invention.
FIG. 5 is a schematic diagram of β -glucuronidase-responsive acetylmannosamine (Glu-AAM) and its synthetic scheme in accordance with an embodiment of the present invention. The reaction conditions are as follows:
(a) (i) MeOH, MeONa; (ii) TEA, chloroacetic anhydride, r.t, 12 h;
(b)DMF，NaN 3 ，12h；
(c)TBDPSCl，Py，DMAP，50℃，24h；
(d)Py，AC 2 O，r.t，12h；
(e)TBAF，THF，r.t，12h；
(f) 4-nitrophenylcarbonyl chlorate, DCM, TEA, r.t, 12 h;
(g)(i)MeOH,MeONa；(ii)HClO 4 ,Ac 2 O；
(h)DCM,HBr/AcOH,0℃,4h；
(i)Ag 2 O,CH 3 CN, 4-hydroxy-3-nitrobenzaldehyde, r.t,3 h;
(j)NaBH 4 ,i-PrOH/CHCl 3 ,r.t,3h；
(k) 4-nitrophenylcarbonyl chlorate, DCM, TEA, r.t,3 h;
(l) Methyl (tert-butyl 2- (methylamino) ethyl) carbamate), DCM, DMAP, DIPEA, r.t,1 h;
(m)25％TFA/DCM,0℃,2h；
(n) Compound 7, DCM, DMAP, DIPEA, r.t,4h.
FIG. 6 is the NMR spectrum of Glu-AAM according to the example of the present invention.
FIG. 7 is the NMR carbon spectrum of Glu-AAM according to an example of the present invention.
FIG. 8 shows the observation of Ac labeled with DAPI, DBCO-Cy5 by confocal microscope 4 ManNAc、Ac 3 MANAz or Glu-AAM treated SKBR3 cells and NCM-460 cell map.
FIG. 9 shows the flow cytometry detection of DBCO-Cy 5-labeled Ac of the present invention 4 ManNAc、AC 3 Corresponding mean fluorescence intensity profile of MANAz or Glu-AAM treated cells: p<0.0001。
Detailed Description
The invention is further described with reference to the following figures and examples. The present invention will be better understood from the following examples. However, it is readily understood by those skilled in the art that the specific material proportions, process conditions and results thereof described in the examples are merely illustrative of the invention and should not, nor should they limit the invention as detailed in the claims.
Example 1
Synthesis of beta-glucuronidase-responsive carbohydrate derivatives
The embodiment of the invention designs acetyl-N-azidoacetyl-mannosamine responding to beta-glucuronidase, which modifies acetylated beta-glucuronic acid at the C6 position thereof through self-breaking connecting arms, namely a compound Glu-AAM, and has a structure shown as a formula (I-1):
Figure BDA0003700763930000071
the synthetic route is shown in figure 5, and the synthetic route of the beta-glucuronidase-responsive acetylmannosamine (Glu-AAM) and the reaction conditions are as follows:
s1, preparation of carbonate compound (compound 7):
first, compound 6 (Ac) 3 ManNAz) according to the literature (J Am Chem Soc,2010,132,9516-9518.) it was reported that the D-mannosamine hydrochloride from the sugar precursor substrate, compound 1, can be obtained in only five steps, specifically:
(a) (i) MeOH, MeONa; (ii) TEA, chloroacetic anhydride, r.t, 12 h;
(b)DMF，NaN 3 ，12h；
(c)TBDPSCl，Py，DMAP，50℃，24h；
(d)Py，AC 2 O，r.t，12h；
(e)TBAF，THF，r.t，12h；
then, Compound 6 (Ac) 3 ManNAz) was activated with 4-nitrophenyl chloroformate to give carbonate compound 7:
(f) compound 6(1eq), 4-nitrophenylcarbonyl chlorate (1.2eq), DCM, TEA (2eq), r.t, 12 h.
S2, glucuronic acid/glucuronic acid derivatives (compound 8) were synthesized as glucuronic acid derivatives with cleavable linker arms (compound 15):
according to the literature reports (J Am Chem Soc,2012,134, 3103-:
(g)(i)MeOH,MeONa；(ii)HClO 4 ,Ac 2 O；
(h)DCM,HBr/AcOH,0℃,4h；
(i)Ag 2 O,CH 3 CN, 4-hydroxy-3-nitrobenzaldehyde, r.t,3 h;
(j)NaBH 4 ,i-PrOH/CHCl 3 ,r.t,3h；
(k) 4-nitrophenylcarbonyl chlorate, DCM, TEA, r.t,3 h;
(l) Methyl (tert-butyl 2- (methylamino) ethyl) carbamate), DCM, DMAP, DIPEA, r.t,1 h;
(m)25％TFA/DCM,0℃,2h；
s3, coupling a carbonate compound (compound 7) to a glucuronic acid derivative (compound 15) with a cleavable linking arm to prepare a sugar derivative (Glu-AAM) with a beta-glucuronidase response:
the sugar precursor Glu-AAM was prepared by coupling compound 7 to compound 15 in DMAP, DCM:
(n) compound 7(1eq), compound 15(1.2eq), DCM, DMAP (1eq), DIPEA (2eq), r.t,4h.
The sugar precursor Glu-AAM obtained after the reaction was purified by silica gel column chromatography, and the yield was 66%. The structure of Glu-AAM is identified, and the NMR hydrogen spectrum and the NMR carbon spectrum of Glu-AAM are shown in figures 6 and 7, and the NMR spectra prove that the synthesized compound is correct, and the linking mode of glucuronic acid is beta-glucosyl glycosidic bond.
Example 2
Metabolic labeling of the sugar precursor Glu-AAM obtained in example 1 in different cell lines
Breast cancer cell line SKBR3 with high β -glucuronidase Activity and human Normal Colon epithelial NCM-460 cells with Low β -glucuronidase Activity, each incubated with 100 μ M Glu-AAM for 24 hours and labeled with DBCO-Cy5(50 μ M) at 37 ℃ for 1 hour, while using Ac 4 ManAc and Ac 3 ManAz served as negative and positive controls.
Cell images were collected by laser scanning confocal microscopy. As shown in FIG. 8, SKBR3 cells and NCM-460 cells were subjected to Ac 4 ManNAc、Ac 3 MANA Z Or Glu-AAM treatment and labeling by DAPI and DBCO-Cy5, wherein the merge group is the superposition of two fluorescence signals of DAPI and DBCO-Cy5, and SKBR3 cells can be successfully labeled by Glu-AAM metabolism and show stronger fluorescence signals. Glu-AAM was not labeled for NCM-460 cells with low β -glucuronidase activity. And Ac 3 MANA Z In contrast to the above-mentioned results,Glu-AAM has better selectivity for tumor cells and has similar metabolic efficiency. Further, as shown in fig. 9, the flow cytometry results demonstrate that Glu-AAM can selectively label tumor cells with high β -glucuronidase activity.
It should be noted that, the preparation method of the β -glucuronidase-responsive saccharide derivative shown in the structural formula (II) is the same as that in example 1, and in the same way, other β -glucuronidase-responsive saccharide derivatives can be prepared by the method similar to that in example 1, and the corresponding product similar to example 1 is also obtained, and meanwhile, the experimental determination result similar to example 2 is also obtained, and the details are not repeated here.
With the conventional compound Ac for tumor cell marking 4 Manaz or Ac 3 Compared with ManAz and the like, the compound Glu-AAM disclosed by the invention can be efficiently metabolized in tumor cells SKBR3 with higher beta-glucuronidase activity, has higher metabolic efficiency, and can not be metabolized in normal cells, so that the Glu-AAM compound disclosed by the invention not only maintains the efficient metabolic efficiency, but also has the selectivity of the tumor cells, and is more effectively used for diagnosis and marking of the tumor cells, and is used for related development and application of tumor diagnosis reagents and the like.
The above description is only of the preferred embodiments of the present invention, and it should be pointed out that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims (10)

1. A β -glucuronidase-responsive carbohydrate derivative, comprising:
a. the chemical structure of the response of the beta-glucuronidase comprises glucuronic acid/glucuronic acid derivatives and a self-breaking connecting arm,
b. the sugar precursor substrate comprises monosaccharide and any one of monosaccharide derivatives, disaccharide and disaccharide derivatives,
c. chemical reporter groups, including chemical groups that can be used in click chemistry reactions;
the chemical structure of the a, beta-glucuronidase response is modified on the hydroxyl group of the b.
2. The β -glucuronidase-responsive saccharide derivative according to claim 1, wherein the monosaccharide and monosaccharide derivative comprise any one of mannose, mannosamine, acetamidomannose, sialic acid, glucose, glucosamine, galactose, galactosamine, or fucose, and the disaccharide and disaccharide derivative comprise any one of trehalose, lactose, and maltose.
3. The β -glucuronidase-responsive carbohydrate derivative of claim 1, wherein the self-cleaving linker arm comprises a β -glycosidic bond,
when the self-breaking linker arm is a beta-glycosidic bond, the beta-glucuronidase-responsive chemical structure is a beta-glycosidic-linked glucuronic acid/glucuronic acid derivative.
4. The β -glucuronidase-responsive carbohydrate derivative of claim 1, characterized in that: the chemical reporter group is a group containing a bio-orthogonal functional group, and the bio-orthogonal functional group comprises any one structure of azide-N3, alkynyl, ketone and thiol.
5. The β -glucuronidase-responsive carbohydrate derivative of claim 1, characterized in that: the beta-glucuronidase-responsive chemical structure is modified at the hydroxyl group at the 6-position of the sugar precursor substrate.
6. The β -glucuronidase-responsive carbohydrate derivative of claim 1, characterized in that: the chemical reporter group is modified on the 2-position amino or hydroxyl of the monosaccharide or the 2-position hydroxyl or amino of the disaccharide.
7. The β -glucuronidase-responsive carbohydrate derivative of claim 1, wherein the β -glucuronidase-responsive carbohydrate derivative has a structure represented by formula (I) or (II):
Figure FDA0003700763920000021
8. a method for preparing a β -glucuronidase-responsive carbohydrate derivative, comprising the steps of:
s1, preparing a sugar precursor substrate carbonate activated ester derivative activated by 4-nitrophenyl chloroformate by using the sugar precursor substrate, and marking the derivative as a carbonate compound;
s2, synthesizing the glucuronic acid/glucuronic acid derivative into a glucuronic acid derivative with a cleavable connecting arm;
s3, coupling the carbonate compound to the glucuronic acid derivative with the cleavable connecting arm to prepare the sugar derivative with the response of the beta-glucuronidase.
9. The method of claim 8, wherein in step S1, the sugar precursor substrate is activated by 4-nitrophenyl chloroformate to form said carbonate compound;
in step S2, the glucuronic acid/glucuronic acid derivative is subjected to ring opening by sodium methoxide, acetylation, followed by bromination to obtain a sugar donor, which is then subjected to glycosylation, reduction, and preparation of a carbonate intermediate by using 4-nitrophenyl chloroformate, and finally a carbamate intermediate is obtained, and the glucuronic acid derivative with a cleavable connecting arm is synthesized;
in step S3, the coupling reaction was carried out in the presence of DCM, DMAP, DIPEA.
10. Use of the β -glucuronidase-responsive carbohydrate derivative as defined in any one of claims 1 to 7, or the β -glucuronidase-responsive carbohydrate derivative as defined in any one of claims 8 to 9, in a method for preparing a tumor imaging or a reagent for preparing a tumor marker.
CN202210721691.9A 2022-06-17 2022-06-17 Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof Pending CN114957356A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210721691.9A CN114957356A (en) 2022-06-17 2022-06-17 Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210721691.9A CN114957356A (en) 2022-06-17 2022-06-17 Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN114957356A true CN114957356A (en) 2022-08-30

Family

ID=82965412

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210721691.9A Pending CN114957356A (en) 2022-06-17 2022-06-17 Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN114957356A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112004556A (en) * 2018-04-03 2020-11-27 辛迪维亚公司 Conjugates of cytotoxic drugs and prodrug forms of said conjugates
CN114085256A (en) * 2021-11-23 2022-02-25 江南大学 Beta-galactosidase response carbohydrate derivative and application thereof
CN114341148A (en) * 2019-09-25 2022-04-12 北京大学 Anti-aging and anti-inflammatory prodrugs and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112004556A (en) * 2018-04-03 2020-11-27 辛迪维亚公司 Conjugates of cytotoxic drugs and prodrug forms of said conjugates
CN114341148A (en) * 2019-09-25 2022-04-12 北京大学 Anti-aging and anti-inflammatory prodrugs and methods of use thereof
CN114085256A (en) * 2021-11-23 2022-02-25 江南大学 Beta-galactosidase response carbohydrate derivative and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JONATHAN D. BARGH, 等: "A dual-enzyme cleavable linker for antibody–drug conjugates", 《CHEM. COMMUN.》, vol. 57, pages 3457, XP055976742, DOI: 10.1039/D1CC00957E *
YANCHUN LI, 等: "b-Galactosidase-dependent metabolic glycoengineering of tumor cells for imaging and immunotherapy", 《CHEM. COMMUN.》, vol. 58, pages 2568 *

Similar Documents

Publication Publication Date Title
CN114085256B (en) Beta-galactosidase responsive saccharide derivative and application thereof
JPS6345293A (en) Sialosylceramide compound and production thereof
EP0728763B1 (en) Ganglioside gm3 analog having sialic acid residue fluorinated at the 9-position and intermediate therefor
CN116217638B (en) Seleno-non-natural sugar and preparation method and application thereof
JP5150896B2 (en) Production method of sugar chain polymer
CN114957356A (en) Beta-glucuronidase response carbohydrate derivative and preparation method and application thereof
Han et al. Chemoenzymatic syntheses of sialyl Lewis X–chitosan conjugate as potential anti-inflammatory agent
EP0342599A2 (en) Polyacetyl oligosaccharide derivatives
CN106008617B (en) Tn antigen and its synthesis technology
Hanashima et al. Synthesis of the starfish ganglioside LLG-3 tetrasaccharide
CN105859807A (en) Beta-1,4-galactosyltransferase I substrate fluorescent molecular probe, and intermediate and preparation method thereof
JPH093088A (en) Production of aminodisaccharide and chitin or its analogue polysaccharides
CN109810157A (en) A kind of beta-glucuronidase enzyme sedimentation type fluorogenic substrate synthetic method
JP4813838B2 (en) O-linked sugar amino acid derivative having core 6 type structure and method for producing the same
JP5327945B2 (en) Glycoside and method for producing the same
US5972907A (en) Synthetic core 2-like branched structures containing GalNAc-lewisx and Neu5Acα2-3Galβ1-3GalNAc sequences as novel ligands for selectins
KR100550160B1 (en) METHOD FOR PREPARING INTERMEDIATES OF ARBUTIN AND alpha-TYPE ARBUTIN
CN116284165A (en) Integrated synthesis method of mucin-related tumor-related carbohydrate antigen
JP4781597B2 (en) N-acetylglucosaminyl tyrosine derivative
JP2007022987A (en) O-linked type sugar amino acid derivative having core 3 type structure and method for producing the same
CN109776625A (en) A kind of synthetic method of D-MANNOSE
JPH0656901A (en) Polymeric lewis x saccharide and its production
JP4599505B2 (en) Method for producing N-acetyl-β-hexosaminide derivative
JP2005036163A (en) Polymer material combined with sugar
Wang et al. A new and practical procedure for the preparation of the glucohexatose phytoalexin elicitor

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination