CN111153952B - N-azido acetyl-D-mannosamine derivative, preparation method thereof and application thereof in esterase detection - Google Patents
N-azido acetyl-D-mannosamine derivative, preparation method thereof and application thereof in esterase detection Download PDFInfo
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- 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
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- 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"
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- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention relates to application of N-azidoacetyl-D-mannosamine derivatives in detecting esterase. The invention mainly relates to an N-azido acetyl-D-mannosamine derivative shown as the following formula (I), a preparation method and application thereof in esterase detection:
wherein R is1Selected from H, F, Cl, Br, I, -OH, -NH2、‑NO2、‑N3、C1‑6Alkyl radical, C1‑6Alkoxy, -CO-C1‑6Alkyl, -CO-NH-C1‑6Alkyl, -COOC1‑6Alkyl, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy; said aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy optionally substituted with: F. cl, Br, I, -OH, -NH2、‑NO2、‑N3、C1‑6Alkyl radical, C1‑6An alkoxy group; r2、R3Identical or different, independently of one another, from the group consisting of O, S, NH, NMe; n is1、n2Identical or different, independently of one another, from the group consisting of integers from 1 to 4;
represents an a-key or an e-key.
Description
Technical Field
The invention relates to an N-azido acetyl-D-mannosamine derivative, a preparation method and application thereof, in particular to application of the N-azido acetyl-D-mannosamine derivative in esterase detection.
Background
In eukaryotic cells, polysaccharides are involved in the construction of cell membranes, and play an important role in information exchange, interaction and the like of cells. However, the molecular structure of polysaccharides in cells varies, and there is a certain difficulty in labeling and functional research. Some non-natural saccharides that are chemically modified can express polysaccharides on cell membranes through sugar metabolism processes of cells, thereby providing a new method for the research of polysaccharide functions. The bioorthogonal click reaction without catalyst catalysis is a safe and effective cell detection and labeling method due to the advantages of high specificity, high reaction speed, good biocompatibility and the like, has potential application in the aspects of labeling cancer cells, targeting drug delivery in vivo of nanoparticles and the like, and is increasingly concerned by people.
However, the influence of bioorthogonal click reaction without catalysis by a catalyst on cell functions and the therapeutic efficacy of the bioorthogonal click reaction on diseases still need to be further researched, and the work of detecting compounds in organisms based on sugar metabolism processes and by using the bioorthogonal click reaction is rarely reported, so that the research on the bioorthogonal click reaction and the sugar metabolism processes has important significance on the aspects of detection and marking of polysaccharides, recognition and treatment of cancer cells, drug release, detection of biomolecules and the like. In addition, esterases are important drug targets and prodrug activators, and esterase activity is involved in the metabolism of many drugs and prodrugs. The esterase probe has important application in the aspect of evaluating the treatment efficacy of various medicaments with ester bonds, but a plurality of fluorescence probes for detecting the esterase at present are far from reaching a satisfactory degree, so that a method for detecting the esterase by utilizing bio-orthogonal click reaction and a sugar metabolism process provides a new way for researching the function of the esterase.
Disclosure of Invention
The invention aims to provide an N-azidoacetyl-D-mannosamine derivative.
Another object of the present invention is to provide a process for producing the above N-azidoacetyl-D-mannosamine derivative.
The invention also aims to provide the application of the N-azidoacetyl-D-mannosamine derivative in detecting esterase.
The invention is realized by the following technical scheme:
an N-azidoacetyl-D-mannosamine derivative represented by the following formula (I):
wherein R is1Selected from H, F, Cl, Br, I, -OH, -NH2、-NO2、-N3、C1-6Alkyl radical, C1-6Alkoxy, -CO-C1-6Alkyl, -CO-NH-C1-6Alkyl, -COOC1-6Alkyl, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy; said aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy optionally substituted with: F. cl, Br, I, -OH, -NH2、-NO2、-N3、C1-6Alkyl radical, C1-6An alkoxy group;
R2、R3identical or different, independently of one another, from the group consisting of O, S, NH, NMe;
n1、n2identical or different, independently of one another, from the group consisting of integers from 1 to 4;
According to an embodiment of the invention, said R1Selected from H, F, Cl, Br, I, -OH, -NH2、-NO2、-N3、C1-6Alkyl radical, C1-6Alkoxy, -CO-C1-6Alkyl, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy; said aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy optionally substituted with: F. cl, Br, I, -OH, -NH2、-NO2。
According to an embodiment of the invention, R2Selected from O, S or NH, R3Selected from O or S.
According to an embodiment of the present invention, n1、n2Identical or different, independently of one another, are selected from 1 or 2.
As an example, the N-azidoacetyl-D-mannosamine derivative represented by said formula (I) is selected from the group consisting of, but not limited to, the following compounds:
the invention also provides a preparation method of the N-azidoacetyl-D-mannosamine derivative shown in the formula (I), which comprises the following steps:
wherein R is1、R2、R3、n1、n2、
As defined above;
reacting the compound (III) with the compound (II) to obtain the N-azidoacetyl-D-mannosamine derivative shown in the formula (I).
According to the present invention, there is provided,
the molar ratio of the compound (III) to the compound (II) is 1 (1-6), for example 1 (2-5).
The reaction is preferably carried out in the presence of an acid-binding agent; the acid scavenger is selected from organic bases such as triethylamine, N-diisopropylethylamine or mixtures thereof.
The reaction is carried out in a solvent environment, and the solvent is preferably a mixed solvent composed of dichloromethane and tetrahydrofuran.
According to the invention, the preparation method also comprises the step of separating and purifying the obtained N-azidoacetyl-D-mannosamine derivative shown in the formula (I) by using silica gel column chromatography.
According to the invention, the process also comprises the preparation of compound (III) comprising:
wherein R is3、n2、
As defined above;
and (3) reacting the compound (IV) with tetrabutylammonium fluoride to obtain a compound (III).
According to the present invention, there is provided,
the reaction is carried out in a solvent environment, preferably tetrahydrofuran.
The molar ratio of the compound (IV) to tetrabutylammonium fluoride is 1 (0.5-6), for example 1 (1-3).
According to the present invention, the compound (IV) can be prepared by the methods described in the references.
According to the invention, the compound of the formula (I) can react with near-infrared fluorescent dyes with alkynyl, particularly dyes with both diphenyl cyclooctyne structure and near-infrared luminescent property, such as cyanine Cy5.5 diphenyl cyclooctyne (DBCO-Cy5.5) in a click reaction without catalysis of a catalyst.
The invention also provides application of the N-azidoacetyl-D-mannosamine derivative shown as the formula (I) in detecting esterase.
According to the invention, the detection can be carried out in vitro and can also be used for detecting esterases in cells. According to the invention, the detection is carried out in the presence of a dye having both a diphenylcyclooctyne structure and a near-infrared fluorescent light-emitting structure, for example in the presence of the cyanine Cy5.5 diphenylcyclooctyne (DBCO-Cy5.5).
Advantageous effects
1) The compound of the formula (I) can generate a click reaction with an alkynyl near-infrared fluorescent dye DBCO-Cy5.5 without catalysis of a catalyst.
2) The reaction product of the compound of the formula (I) and DBCO-Cy5.5 can be subjected to enzymolysis reaction with esterase to break ester bonds. Furthermore, the method can be used for detecting by methods such as mass spectrometry, high performance liquid chromatography, absorption spectrometry and the like, thereby realizing the aim of detecting the esterase in vitro.
3) The compound of the formula (I) can be subjected to hydrolysis reaction with esterase, but not react with other compounds in organisms, so that the compound of the formula (I) has selective response to esterase and shows higher specificity.
4) The compounds of formula (I) according to the invention can be used for detecting esterases in cells.
5) After the compound of the formula (I) is hydrolyzed by intracellular esterase, the compound of the formula (III) which can directly participate in the sugar metabolism process of cells is generated, the compound of the formula (III) finally expresses polysaccharide with azide groups on the surface of cell membranes through a series of sugar metabolism processes, and the polysaccharide can generate a click reaction with near-infrared fluorescent dye DBCO-Cy5.5 with alkynyl groups added into a cell culture medium without catalysis of a catalyst, so that the polysaccharide with the azide groups expressed on the cell membranes is marked with the near-infrared fluorescence of Cy5.5, and the purpose of detecting the intracellular esterase is realized.
Definition and description of terms:
unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter of the application. In this application, unless otherwise indicated, the terms "include" and other forms, such as "comprises," "comprising," and "includes" are not limiting.
The term "alkyl" refers to a straight or branched alkyl group having 1 to 6, preferably 1 to 3, carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, neopentyl.
The term "aryl" is understood to mean preferably a mono-, bi-or tricyclic hydrocarbon ring having a monovalent aromatic or partially aromatic character of 6 to 20 carbon atoms, preferably "C6-14Aryl ". The term "C6-14Aryl "is to be understood as preferably meaning a mono-, bi-or tricyclic hydrocarbon ring having a monovalent or partially aromatic character with 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (" C6-14Aryl group "), in particular a ring having 6 carbon atoms (" C6Aryl "), such as phenyl; or biphenyl, or is a ring having 9 carbon atoms ("C9Aryl), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C10Aryl radicals), such as tetralinyl, dihydronaphthyl or naphthyl, or rings having 13 carbon atoms ("C13Aryl radicals), such as the fluorenyl radical, or a ring having 14 carbon atoms ("C)14Aryl), such as anthracenyl.
The term "heteroaryl" is understood to mean monocyclic, bicyclic and tricyclic ring systems containing 5 to 20 ring atoms, 5 to 14 ring atoms, or 5 to 12 ring atoms, or 5 to 10 ring atoms, or 5 to 6 ring atoms, at least one of which is aromatic, and at least one of which contains one or more heteroatoms (e.g., N, O, S, Se, etc.), wherein each ring system contains a ring of 5 to 7 atoms with one or more attachment points to the rest of the molecule. The heteroaryl group is optionally substituted with one or more substituents described herein. In some embodiments, a heteroaryl group of 5-10 atoms contains 1,2,3, or 4 heteroatoms independently selected from O, S, Se and N. In other embodiments, a 5-6 atom heteroaryl group contains 1,2,3, or 4 heteroatoms independently selected from O, S, Se and N.
Examples of monocyclic rings of heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and their benzo derivatives, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, and the like; or azocinyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like.
The term "heterocyclyl" means a monocyclic, bicyclic or tricyclic ring system in which one or more atoms in the ring are independently optionally substituted with a heteroatom, the ring may be fully saturated or contain one or more unsaturations, but is not aromatic, having one or more points of attachment to other molecules. One or more ring hydrogen atoms may be independently unsubstituted or substituted with one or more substituents described herein. Some of these embodiments are "heterocyclyl" groups which are monocyclic of 3 to 7 atoms, or bicyclic of 7 to 10 atoms, containing 1 to 5, preferably 1 to 3 heteroatoms selected from N, O, S and Se. In particular, the heterocyclic group may include, but is not limited to: 4-membered rings such as azetidinyl, oxetanyl; 5-membered rings such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclic group may be benzo-fused. The heterocyclyl group may be bicyclic, for example but not limited to a5, 5 membered ring, such as a hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl ring, or a5, 6 membered bicyclic ring, such as a hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl ring. The nitrogen atom containing ring may be partially unsaturated, i.e. it may contain one or more double bonds, such as but not limited to 2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4] thiadiazinyl, 4, 5-dihydrooxazolyl or 4H- [1,4] thiazinyl, or it may be benzo-fused, such as but not limited to dihydroisoquinolyl, 1, 3-benzoxazolyl, 1, 3-benzodioxolyl.
Unless otherwise indicated, heterocyclyl, heteroaryl include all possible isomeric forms thereof, e.g. positional isomers thereof. Thus, for some illustrative, non-limiting examples, pyridyl or pyridinylene includes pyridin-2-yl, pyridinylene-2-yl, pyridin-3-yl, pyridinylene-3-yl, pyridin-4-yl, and pyridinylene-4-yl; thienyl or thienylene includes thien-2-yl, thien-3-yl and thien-3-yl.
Drawings
FIG. 1 shows the use of compound (2) prepared in example 2 for detecting esterase in A549 cells.
FIG. 2 shows the compound (2) prepared in example 2 for detecting esterase in HEK293 cells.
FIG. 3 is a diagram showing the reaction mechanism of the compound of formula (I) for detecting esterase.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Preparation example 1
Preparation of Compound (III-1)
Compound (IV-1) (0.75g,1.5mM) was dissolved in anhydrous tetrahydrofuran in an ice-water bath, and a 1M solution of tetrabutylammonium fluoride (1.5eq.,2.25mM) in tetrahydrofuran was added, followed by stirring at room temperature for 3 hours. After completion of the reaction, the reaction was quenched with 10mL of a saturated aqueous solution of ammonium chloride, extracted with ethyl acetate (50mL × 3), and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was removed. The crude product was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (v/v ═ 1:2 to 1:3) to give a white solid (541mg, 93%).
Example 1
Preparation of Compound (1)
A compound of formula (III-1) (388mg, 1mM) was dissolved in 15mL of a dry mixed solution of tetrahydrofuran/dichloromethane (THF/DCM, 1:1v/v) under protection of argon in an ice-water bath, and triethylamine (0.809g, 8mM) and N, N-diisopropylethylamine (1.034g, 8mM) were further added, and after 10 minutes of reaction, 2- (furan-2-yl) ethyl chloroformate (696mg,4mM) dissolved in 10mL of a dry solution of THF/DCM (1:1v/v) was added dropwise. The reaction was continued at 0 ℃ for 1 hour, the ice-water bath was removed and the temperature was returned to room temperature for 3 hours. After completion of the reaction, the solvent was removed and the crude product was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (v/v ═ 1:1) to give a white solid (189mg, 36%).1H NMR(400MHz,CD3OD):δ7.511(d,1H),6.362(m,1H),6.125(d,1H),5.983(s,1H),5.328-5.098(m,4H) 4.733(s,1H),4.359-4.280(m,1H),4.150-4.070(m,2H),3.935(s,2H),2.98(t,2H),2.170-1.911(m,9H). ESI-HRMS calculated C21H26N4O12Na+(M+Na)+549.1, Experimental value 549.1.
Example 2
Preparation of Compound (2)
A compound of formula (III-1) (388mg, 1mM) was dissolved in 15mL of a dry mixed solution of tetrahydrofuran/dichloromethane (THF/DCM, 1:1v/v) under protection of argon in an ice-water bath, and triethylamine (0.809g, 8mM) and N, N-diisopropylethylamine (1.034g, 8mM) were further added, and after 10 minutes of reaction, benzyl chloroformate (680mg, 4mM) dissolved in 10mL of a dry solution of THF/DCM (1:1v/v) was added dropwise. The reaction was continued at 0 ℃ for 1 hour, the ice-water bath was removed and the temperature was returned to room temperature for 3 hours. After completion of the reaction, the solvent was removed and the crude product was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (v/v ═ 1:1) to give a white solid (198mg, 38%).1H NMR(400MHz,CD3OD) delta 7.32-7.35(d,5H),5.997(s,1H),5.330-5.090(m,4H),4.725(s,1H),4.356-4.281(m,1H),4.150-4.073(m,2H),3.936(s,2H),2.173-1.910(m,9H). ESI-MS calculated C22H26N4O11Na+(M+Na)+545.1, Experimental value 545.1.
Example 3
Preparation of Compound (3)
A compound of formula (III-1) (388mg, 1mM) was dissolved in 15mL of a dry mixed solution of tetrahydrofuran/dichloromethane (THF/DCM, 1:1v/v) under protection of argon in an ice-water bath, triethylamine (0.809g, 8mM) and N, N-diisopropylethylamine (1.034g, 8mM) were further added, and after 10 minutes of reaction, chloroformic acid dissolved in 10mL of a dry solution of THF/DCM (1:1v/v) was added dropwiseEthyl ester (432mg,4 mM). The reaction was continued at 0 ℃ for 1 hour, the ice-water bath was removed and the temperature was returned to room temperature for 3 hours. After completion of the reaction, the solvent was removed and the crude product was purified by silica gel column chromatography eluting with petroleum ether/ethyl acetate (v/v ═ 1:1) to give a white solid (184mg, 40%).1H NMR(400MHz,CD3OD) 6.001(s,1H),5.328-5.097(m,2H),4.726(s,1H),4.360-4.280(m,1H),4.24(m,2H),4.156-4.070(m,2H),3.942(s,2H),2.171-1.905(m,9H),1.35(t,3H), MALDI-TOF-MS17H25N4O11 +(M+H)+461.1, Experimental value 461.1.
Example 4
The compound (2) obtained in example 2 was applied to the detection of esterase in a549 cells: 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) was used as an inhibitor of intracellular esterases. Culturing group A cells with 0.2mM AEBSF for 24h, and adding 25 μ M compound (2) for 24 h; group B cells were cultured for 24h without AEBSF by directly adding 25. mu.M of Compound (2). Then both groups of cells were washed off with Compound (2), twice with PBS, and stained for 2h with 1. mu.M DBCO-Cy5.5. And washing DBCO-Cy5.5, adding Hoechst 33342 with the concentration of 10 mu g/mL for staining for 20min, finally washing the Hoechst 33342, and carrying out confocal microscope imaging on the cells. As can be seen from FIG. 1, in the case of group B cells which were not treated with AEBSF, the cell membrane surface exhibited significant near-infrared fluorescence, indicating that polysaccharides having azide groups were produced on the cell membrane. The near-infrared fluorescence intensity of the cell membrane surface of the group A cells treated with AEBSF is greatly reduced, which indicates that the polysaccharides with azide groups generated on the cell membrane are greatly reduced. Therefore, the polysaccharide with azido groups on the cell membrane is actually generated by the fact that the compound (2) is expressed on the cell membrane of A549 cells through a series of sugar metabolism processes after undergoing a hydrolysis reaction with intracellular esterase, and the fact indicates that the compound (2) obtained in example 2 can be used for detecting esterase in A549 cells.
Example 5
The compound (2) obtained in example 2 was applied to the detection of esterases in HEK293 cells: 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) was used as an inhibitor of intracellular esterases. Culturing group A cells with 0.2mM AEBSF for 24h, and adding 50 μ M compound (2) for 24 h; group B cells were cultured for 24h without AEBSF by directly adding 50. mu.M of Compound (2). Then both groups of cells were washed off with Compound (2), twice with PBS, and stained for 2h with 2. mu.M DBCO-Cy5.5. And washing DBCO-Cy5.5, adding Hoechst 33342 with the concentration of 10 mu g/mL for staining for 20min, finally washing the Hoechst 33342, and carrying out confocal microscope imaging on the cells. As can be seen from FIG. 2, the cell membrane surface exhibited significant near-infrared fluorescence for group B cells that were not pretreated with AEBSF, indicating that polysaccharides with azido groups were produced on the cell membrane. The near infrared fluorescence intensity of the cell membrane of the group A cells pretreated by AEBSF is greatly weakened, which indicates that the polysaccharide with azide groups generated on the cell membrane is greatly reduced. Therefore, the polysaccharide with azide groups on the cell membrane is actually generated by the fact that the compound (2) is expressed on the cell membrane of the HEK293 cell through a series of sugar metabolism processes after undergoing a hydrolysis reaction with the esterase in the cell, and the fact shows that the compound (2) obtained in the example 2 can be applied to detection of the esterase in the HEK293 cell.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (11)
1. An N-azidoacetyl-D-mannosamine derivative represented by the following formula (I):
（I）
wherein R is1Is selected from C1-6Alkyl radical, C6-14Aryl, 5-14 membered heteroaryl; said C is6-14Aryl, 5-14 membered heteroaryl optionally substituted with: F. cl, Br, I, -OH, -NH2、-NO2、-N3、C1-6Alkyl radical, C1-6An alkoxy group;
R2selected from O, R3Is selected from O;
n1、n2identical or different, independently of one another, from the group consisting of integers from 1 to 4;
“
"represents an a bond or an e bond.
2. The derivative of claim 1, wherein R is1Is selected from C1-6Alkyl radical, C6-14Aryl, 5-14 membered heteroaryl; said C is6-14Aryl, 5-14 membered heteroaryl optionally substituted with: F. cl, Br, I, -OH, -NH2、-NO2；
R2Selected from O, R3Is selected from O;
n1、n2identical or different, independently of one another, are selected from 1 or 2.
3. The derivative according to claim 1 or 2, wherein the derivative is selected from the following compounds:
4. a process for the preparation of the derivatives of any of claims 1 to 3, comprising the steps of:
wherein R is1、R2、R3、n1、n2As defined in any one of claims 1 to 3;
reacting the compound (III) with the compound (II) to obtain the N-azidoacetyl-D-mannosamine derivative shown in the formula (I).
5. The process according to claim 4, wherein the reaction is carried out in the presence of an acid-binding agent.
6. The method of claim 5, wherein the acid scavenger is selected from organic bases.
7. The method according to claim 4, wherein the reaction is carried out in a solvent environment, and the solvent is selected from a mixed solvent of dichloromethane and tetrahydrofuran.
8. The process according to claim 4, further comprising the preparation of compound (III) comprising the steps of:
wherein R is3、n2As defined in claim 1;
and (3) reacting the compound (IV) with tetrabutylammonium fluoride to obtain a compound (III).
9. Use of a derivative according to any one of claims 1 to 3 for the detection of an esterase.
10. Use according to claim 9, characterized in that the detection is carried out in the presence of a dye having both a diphenylcyclooctyne structure and a near-infrared fluorescent light-emitting structure.
11. Use according to claim 9, wherein the detection is carried out in vitro or for detecting an esterase in a cell.
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| WO2015112016A1 (en) * | 2014-01-24 | 2015-07-30 | Synaffix B.V. | Process for the cycloaddition of a halogenated 1,3-dipole compound with a (hetero)cycloalkyne |
| CN108699095A (en) * | 2015-10-07 | 2018-10-23 | 伊利诺伊大学评议会 | Triggering for marking and targeting for cancer selective can activating metabolism sugar precursor |
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| WO2015112016A1 (en) * | 2014-01-24 | 2015-07-30 | Synaffix B.V. | Process for the cycloaddition of a halogenated 1,3-dipole compound with a (hetero)cycloalkyne |
| CN108699095A (en) * | 2015-10-07 | 2018-10-23 | 伊利诺伊大学评议会 | Triggering for marking and targeting for cancer selective can activating metabolism sugar precursor |
Non-Patent Citations (3)
| Title |
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| Extracellular and intracellular esterase processing of SCFA–hexosamine analogs:Implications for metabolic glycoengineering and drug delivery;Mohit P. Mathew,et al.;《Bioorganic & Medicinal Chemistry Letters》;20120913;第22卷;第6929-6933页 * |
| Imaging Cell Surface Glycans with Bioorthogonal Chemical Reporters;Pamela V. Chang,et al.;《J. AM. CHEM. SOC.》;20070619;第129卷;第8400-8401页 * |
| In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy;Ran Xie,et al.;《PNAS》;20160510;第113卷(第19期);第5173-5178页 * |
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