WO2020155742A1 - 衍生醛基嘧啶的方法、检测5-醛基胞嘧啶的方法以及醛基嘧啶衍生物的应用 - Google Patents

衍生醛基嘧啶的方法、检测5-醛基胞嘧啶的方法以及醛基嘧啶衍生物的应用 Download PDF

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WO2020155742A1
WO2020155742A1 PCT/CN2019/117081 CN2019117081W WO2020155742A1 WO 2020155742 A1 WO2020155742 A1 WO 2020155742A1 CN 2019117081 W CN2019117081 W CN 2019117081W WO 2020155742 A1 WO2020155742 A1 WO 2020155742A1
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aldehyde
cytosine
uracil
structural unit
pyrimidine
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French (fr)
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李坤
周倩
余孝其
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四川大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/47—One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • G01N21/6428—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

Definitions

  • the present invention relates to the technical field of nucleic acid chemistry, in particular to a method for derivatizing aldehyde pyrimidine, a method for detecting 5-aldehyde cytosine, and the application of aldehyde pyrimidine derivatives.
  • DNA methylation means that DNA methyltransferase (DNMT) uses adenine methionine as a methyl donor to add a methyl group to the 5-carbon of cytosine to convert it to 5-methylcytosine (5mC) Process, and this process is dynamically reversible, that is, DNA demethylation.
  • DNMT DNA methyltransferase
  • 5mC 5-methylcytosine
  • the former is due to the inhibition of DNA methylation maintenance mechanisms, such as DNMT1 inactivation, and the DNA methylation level is gradually diluted during the continuous semi-reserved replication of DNA; the latter refers to the 5mC with the participation of various enzymes such as TET. Continuous oxidation and final conversion to cytosine through a series of intermediates.
  • 5-aldehyde cytosine As an intermediate in the active demethylation process, 5-aldehyde cytosine (5fC) is of great significance for maintaining the normal level of methylation in life.
  • 5fC 5-aldehyde cytosine
  • 5fC has more protein binding sites and more unique base distribution rules, enabling it to be used in special biological events such as gene regulation, changes in DNA structure, cell differentiation, and major diseases. Play a more important role in
  • Liquid chromatography-tandem mass spectrometry has significant advantages in this regard due to its own characteristics, but the abundance of 5fC in the body is extremely low, the ionization efficiency is also low, and other high-content classic bases or Rare bases and various impurity interferences in the sample matrix are still challenging to accurately determine 5fC using LC-MS/MS technology.
  • fluorescence sensing technology has the advantages of good selectivity, short response time, simple operation, and naked-eye observation. It has been widely used in living cells and even active oxygen and active sulfur in animals. , Enzyme, nucleic acid and other biological targets. Especially small organic molecule fluorescent probes are favored by many scientific researchers because of their easy modification and low cost. In recent years, focusing on the C-5 aldehyde group of 5fC, there have been reports that it can be fluorescently labeled with derivatives such as -NH 2 , -NHNH 2 , -ONH 2 , and indole.
  • 5-aldehyde uracil as the oxidation product of thymine (T), is very similar in structure to 5fC, but its aldehyde group is far more reactive than 5fC, so the above is based on Schiff base reaction or Aldol condensation
  • the fluorescent probe will preferentially react with 5fU. In other words, this kind of reagents cannot distinguish between the two aldehyde-based pyrimidines, and cannot realize the specific fluorescence recognition of 5fC.
  • the disclosed reagents also have a side chain activity that can undergo intramolecular cyclization with 5-NH 2 outside the ring of 5fC. Group.
  • both 5-CHO and 4-NH 2 are indispensable, which is also the key to achieving highly specific 5fC labeling.
  • Another similar report discloses an example of a small organic molecule containing the structure of -CH 2 CN, in which -CH 2 -is responsible for the condensation with 5-CHO of 5fC, and -CN is responsible for the cyclization with 4-NH 2 to select sexually lights up 5fC.
  • the purpose of the present invention is to provide a method for derivatizing aldehyde pyrimidine, a method for detecting 5-aldehyde cytosine and the application of aldehyde pyrimidine derivatives, so as to solve the time-consuming and condition existing in the existing 5fC detection and identification process Harsh questions.
  • the Wittig reaction (Yllid reaction) was discovered in 1953 by the German chemist G. Wittig, and is mainly used for the direct synthesis of olefins from aldehydes and ketones. This method has the advantages of high yield, mild reaction conditions, and good selectivity.
  • the applicant of the present invention also summed up previous experience, using Wittig reagent substituted by cyano group to achieve selective fluorescence of 5fC through Wittig olefination and photo-assisted intramolecular ring-closure reaction Identification and quantitative detection.
  • one aspect of the present invention provides a method for derivatizing aldopyrimidine, wherein the method comprises: mixing aldopyrimidine and cyano substituted Wittig reagent in an organic solvent, and then irradiating with ultraviolet light, The aldehyde pyrimidine derivative is prepared; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.
  • the 5-aldehyde cytosine is a substance containing 5-aldehyde cytosine structural unit
  • the substance containing 5-aldehyde cytosine structural unit is 5-aldehyde cytosine, 5-aldehyde cytosine At least one of pyrimidine nucleosides, 5-aldehyde cytosine nucleotides, 5-aldehyde cytosine-containing DNA, 5-aldehyde cytosine-containing RNA, and a mixture containing at least one compound;
  • the 5-aldehyde uracil is a substance containing 5-aldehyde uracil structural unit, and the substance containing 5-aldehyde uracil structural unit is 5-aldehyde uracil, 5-aldehyde uracil nucleoside At least one of 5-aldehyde uracil nucleotides and DNA containing 5-aldehyde uracil, RNA containing 5-aldehyde uracil, and a mixture containing at least one compound.
  • the aforementioned Wittig reagent has the following structural formula:
  • R 1 is one of hydrogen, cyano, halogen, hydrocarbon group, and hydrocarbon group containing O, N, halogen, P, S, or Si.
  • 5-aldehyde cytosine derivatives include derivatives having a structure represented by formula (1) and having a structure represented by formula (2):
  • R is each independently one of hydrogen, hydrocarbyl, ribose and deoxyribose
  • R 1 is independently hydrogen, cyano, halogen, hydrocarbyl and O, N, halogen, P, S, or Si-containing One of the hydrocarbon groups.
  • the 5-aldehyde uracil derivative has the structural formula shown in formula (3):
  • R is one of hydrogen, hydrocarbyl, ribose and deoxyribose
  • R 1 is one of hydrogen, cyano, halogen, hydrocarbyl, and hydrocarbyl containing O, N, halogen, P, S, or Si.
  • the above-mentioned organic solvent includes methanol, ethanol, isopropanol, ethylene glycol methyl ether, ethylene glycol dimethyl ether, acetonitrile, toluene, dichloromethane, N,N- One or more combinations of dimethylformamide, N,N-dimethylacetamide, dioxane and dimethylsulfoxide.
  • the mixing ratio may be any ratio.
  • the molar ratio of aldehyde pyrimidine and Wittig reagent is 1: (1-500), the reaction temperature is 15-80° C., and the reaction time is 5 min-48 h.
  • the molar ratio of aldopyrimidine and Wittig reagent is preferably 1:(20-250), more preferably 1:(20-50), and most preferably 1:20.
  • the reaction temperature is preferably 50-70°C, more preferably 55-65°C, and most preferably 60°C.
  • the reaction time is preferably 0.5h-6h, more preferably 0.5h-2h, and most preferably 1h.
  • the wavelength of the ultraviolet light is 200-400 nm, and the radiation time is 1 min-24h.
  • the wavelength of ultraviolet light is preferably 253-365 nm, more preferably 280-310 nm, and most preferably 280 nm.
  • the irradiation time of ultraviolet light is preferably 0.5h-12h, more preferably 0.5h-4h, and most preferably 1.5h.
  • the second aspect of the present invention provides aldopyrimidine derivatives obtained by the method of the present invention.
  • the third aspect of the present invention provides the application of the aldehyde-based pyrimidine derivative provided by the present invention in detecting 5-aldehyde-based cytosine and 5-aldehyde-based uracil, which is detected by the LC-MS/MS technology assisted by chemical derivatization.
  • the 5-aldehyde cytosine is a substance containing 5-aldehyde cytosine structural unit
  • the substance containing 5-aldehyde cytosine structural unit is 5-aldehyde cytosine, 5-aldehyde cytosine At least one of pyrimidine nucleosides, 5-aldehyde cytosine nucleotides, 5-aldehyde cytosine-containing DNA, 5-aldehyde cytosine-containing RNA, and a mixture containing at least one compound;
  • the 5-aldehyde uracil is a substance containing 5-aldehyde uracil structural unit, and the substance containing 5-aldehyde uracil structural unit is 5-aldehyde uracil, 5-aldehyde uracil nucleoside At least one of 5-aldehyde uracil nucleotides and DNA containing 5-aldehyde uracil, RNA containing 5-aldehyde uracil, and a mixture containing at least one compound.
  • the fourth aspect of the present invention provides the application of the aldehyde pyrimidine derivative with the structure represented by formula (1) and the structure represented by formula (2) prepared by the method of the present invention in the detection of 5-aldehyde cytosine, using fluorescence Identification technology for identification and detection.
  • the 5-aldehyde cytosine is a substance containing 5-aldehyde cytosine structural unit
  • the substance containing 5-aldehyde cytosine structural unit is 5-aldehyde cytosine, 5-aldehyde cytosine At least one of pyrimidine nucleosides, 5-aldehyde cytosine nucleotides, 5-aldehyde cytosine-containing DNA, 5-aldehyde cytosine-containing RNA, and a mixture containing at least one compound described above.
  • the fifth aspect of the present invention provides a method for detecting 5-aldehyde cytosine, wherein the method comprises: mixing 5-aldehyde cytosine and Wittig reagent in an organic solvent, and then irradiating with ultraviolet light to obtain 5 -Aldehyde cytosine derivatives, and then using fluorescence recognition technology to identify and detect 5-aldehyde cytosine derivatives.
  • the 5-aldehyde cytosine is a substance containing 5-aldehyde cytosine structural unit
  • the substance containing 5-aldehyde cytosine structural unit is 5-aldehyde cytosine, 5-aldehyde cytosine At least one of pyrimidine nucleosides, 5-aldehyde cytosine nucleotides, 5-aldehyde cytosine-containing DNA, 5-aldehyde cytosine-containing RNA, and a mixture containing at least one compound described above.
  • the present invention firstly uses Wittig reagent to achieve the chemical derivatization of 5fC and 5fU at the same time. Due to the introduction of the hydrophobic acrylonitrile structure, the retention time of the derivatized aldopyrimidine is prolonged, and the separation resolution with other bases is also improved. LC-MS/MS analysis assisted by chemical derivatization is more advantageous. Furthermore, after Wittig reagent olefination, combined with the photocatalytic "domino" reaction, the selective fluorescence recognition and quantitative detection of 5fC are realized.
  • the cyano-substituted Wittig reagent screened in the present invention can specifically target 5fC through a three-step continuous reaction, which is also not available in derivatives such as amine groups and indole.
  • the preferred high-reactivity phosphorous ylide and the light-assisted rapid ring-closure strategy of the present invention effectively compensate for the shortcomings of the time-consuming reaction of active methylene compounds.
  • the Wittig derivatization reaction provided by the present invention for 5-aldehyde cytosine has the advantages of mild conditions, high efficiency and rapidity, and economic and easy availability of raw materials.
  • Fig. 1 is a process diagram of the derivatization of aldehyde pyrimidine in an embodiment of the present invention.
  • Figure 2 is a comparison chart of HPLC traces of 5fC and 5fU before and after derivatization with other classic bases or rare bases.
  • Figure 3 shows the excitation spectrum and emission spectrum of the ring-closure product 5fC-CN-Close derived from an embodiment of the present invention, where the dotted line is the excitation spectrum, and the solid line is the emission spectrum.
  • Figure 4 is a graph showing the change in fluorescence intensity of trans-5fC-CN over time under ultraviolet radiation.
  • Figure 5 is a fluorescence selective detection spectrum of a 5fC-derived sample according to an embodiment of the present invention.
  • Figure 6 is a standard curve for the fluorescence quantification of 5fC with cyanomethylenetriphenylphosphine.
  • Fig. 7 is a graph showing changes in fluorescence intensity of 5fC-derived samples before and after ⁇ -ray irradiation according to an embodiment of the present invention.
  • the Wittig reagent used to label aldopyrimidine is cyanomethylene triphenylphosphine, and its structural formula is
  • the synthetic route of the embodiment of the present invention is shown in Figure 1.
  • the aldehyde pyrimidine and Wittig reagent are mixed in an organic solvent, and then irradiated with ultraviolet light to prepare the aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde Cytosine or 5-aldehyde uracil.
  • This embodiment is a method for derivatizing aldehyde pyrimidine, which includes:
  • the aldehyde pyrimidine and Wittig reagent were mixed in ethanol at a molar ratio of 1:1, reacted at 15°C for 48 hours, and then irradiated with ultraviolet light with a wavelength of 200 nm for 24 hours to prepare the aldehyde pyrimidine derivative; where,
  • the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.
  • This embodiment is a method for derivatizing aldehyde pyrimidine, which includes:
  • aldehyde pyrimidine and Wittig reagent were mixed in N,N-dimethylformamide at a molar ratio of 1:500, reacted at 80°C for 5 min, and then irradiated with ultraviolet light with a wavelength of 400 nm for 1 min.
  • This embodiment is a method for derivatizing aldehyde pyrimidine, which includes:
  • aldehyde pyrimidine and Wittig reagent were mixed in ethylene glycol dimethyl ether at a molar ratio of 1:20, reacted at 50°C for 6 hours, and then irradiated with ultraviolet light with a wavelength of 253 nm for 12 hours to prepare aldehyde pyrimidine Derivatives; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.
  • This embodiment is a method for derivatizing aldehyde pyrimidine, which includes:
  • aldehyde pyrimidine and Wittig reagent were mixed in ethylene glycol dimethyl ether at a molar ratio of 1:250, reacted at 70°C for 0.5 h, and then irradiated with ultraviolet light with a wavelength of 365 nm for 0.5 h to prepare aldehyde Pyrimidine derivatives; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.
  • This embodiment is a method for derivatizing aldehyde pyrimidine, which includes:
  • aldehyde pyrimidine and Wittig reagent were mixed in ethylene glycol dimethyl ether at a molar ratio of 1:50, reacted at 65°C for 1 h, and then irradiated with ultraviolet light with a wavelength of 300 nm for 45 minutes to prepare aldehyde pyrimidine Derivatives; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.
  • This embodiment is a method for derivatizing aldehyde pyrimidine, which includes:
  • aldehyde pyrimidine and Wittig reagent were mixed in ethylene glycol dimethyl ether at a molar ratio of 1:40, reacted at 65°C for 40 minutes, and then irradiated with ultraviolet light with a wavelength of 310 nm for 30 minutes to prepare aldehyde pyrimidine Derivatives; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.
  • This embodiment is a method for derivatizing aldehyde pyrimidine, which includes:
  • the aldehyde pyrimidine and Wittig reagent were mixed in ethylene glycol dimethyl ether at a molar ratio of 1:20, reacted at 60°C for 1 hour, and then irradiated with ultraviolet light with a wavelength of 280 nm for 1.5 hours to prepare the aldehyde group Pyrimidine derivatives; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.
  • the aldehyde-based pyrimidine derivatives 5-aldehyde-based cytosine derivatives and 5-aldehyde-based uracil derivatives are derived through the embodiment of the present invention, which can be used to detect aldehyde-based cytosine and aldehyde-based uracil.
  • the detection method can adopt LC-MS/MS analysis method.
  • 5-aldehyde cytosine can also be detected to determine the 5fC level in DNA.
  • 5-aldehyde cytosine reacts with Wittig reagent, its product trans-5fC-CN is transformed into its heterogeneous structure under ultraviolet radiation: cis-5fC-CN, cis-5fC-CN outside the ring 4-NH 2 and The closer -CN undergoes intramolecular condensation and cyclization reactions to obtain the final derivative that can emit fluorescence: 5fC-CN-Close.
  • the detection and quantitative analysis of 5fC can be realized by the fluorescent labeling method.
  • the retention time of other bases is concentrated in 4-6min, and the separation resolution between each other is low, especially the three bases of G, T, and 5mC are very close to 5fC and 5fU, which is extremely disadvantageous for LC-MS/MS analysis.
  • the retention time of aldehyde-based pyrimidines derivatized with Wittig reagent is directly extended to more than 10 minutes, which is obviously different from other bases. In this way, they can minimize their 5fC and 5fU in the LC-MS/MS analysis process. Ion suppression.
  • the derivatized acrylonitrile structure leads to increased hydrophobicity, which means that a less polar mobile phase (that is, a larger proportion of organic solvent) is required for elution, which is also conducive to improving the ionization efficiency of the target analyte.
  • the Wittig reagent can be used in the present invention to efficiently derive 5fC and 5fU at the same time, and the retention time of the generated nucleoside adducts is prolonged, and the separation from other bases is also greatly improved, which can improve the performance of 5fC and 5fU in LC-MS/ Sensitivity in MS detection.
  • the total reaction time of the present invention can be shortened to 2.5 h, which is a great improvement compared with the 10-24 h of the prior art, greatly saves time and cost, and improves detection efficiency.
  • the derived samples corresponding to only 5fC have obvious fluorescence enhancement at 410nm, A, G, C, T, U, 5mC, 5hmC, 5hmU, and even 5fU. Its fluorescence emission spectrum overlaps with the probe set, indicating that cyanomethylene triphenyl phosphine has a highly specific fluorescence recognition ability for 5fC.
  • Test Example 4 Fluorescence quantitative analysis of 5fC mutation caused by ⁇ -ray radiation by cyanomethylenetriphenylphosphine
  • 5mC can be mutated to 5fC by gamma radiation.
  • This test example intends to detect the mutation ability of gamma rays to 5mC.
  • the present invention utilizes the Wittig reagent substituted with cyano groups to simultaneously complete the efficient derivatization of 5-aldehyde cytosine (5fC) and 5-aldehyde uracil (5fU) using a one-pot method to obtain a core containing an acrylonitrile structure.
  • Glycoside adducts and mainly E-type configuration.
  • the hydrophobicity of the nucleoside adducts obtained in the present invention is increased, the retention time is prolonged during reversed-phase HPLC separation, and the resolution with other classic bases or rare bases is also greatly improved, which is useful for improving the performance of 5fC and 5fU in LC-MS.
  • the detection sensitivity during MS analysis is very advantageous.
  • the E-type nucleoside derivative corresponding to 5fC, the acrylonitrile structure can undergo cis-trans isomerization under ultraviolet light radiation, thereby shortening the space distance between the cyano group and the 4-amino group outside the ring, creating an intramolecular cycloaddition reaction Under favorable conditions, a new type of nucleoside with strong fluorescence is finally generated; however, 5fU lacks the extracyclic 4-amino group and cannot fully undergo the above process, which makes the strategy of selective fluorescence detection of 5fC possible.
  • the cyano-substituted Wittig reagent screened in the present invention has been commercialized.
  • the derivatization reaction conditions are mild, efficient and fast, effectively expanding the application of Wittig reagent in the field of epigenetics, and it is also a chemical marker and reactive type of aldopyrimidine.
  • the design of 5fC fluorescent probe provides new ideas.

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Abstract

本发明公开了一种衍生醛基嘧啶的方法、检测5-醛基胞嘧啶的方法以及醛基嘧啶衍生物的应用。本发明将醛基嘧啶与Wittig试剂于有机溶剂中混合，然后用紫外光进行辐射，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。本发明反应条件温和、高效快速，有效拓展了Wittig试剂在表观遗传学领域的应用，也为醛基嘧啶的化学标记和反应型5fC荧光探针的设计提供了新思路。

Description

衍生醛基嘧啶的方法、检测5-醛基胞嘧啶的方法以及醛基嘧啶衍生物的应用 技术领域
本发明涉及核酸化学技术领域，具体涉及一种衍生醛基嘧啶的方法、检测5-醛基胞嘧啶的方法以及醛基嘧啶衍生物的应用。
背景技术
哺乳动物细胞或组织DNA中，特别是CpG岛，都存在着不同程度的甲基化修饰，这与胚胎发育，肿瘤、精神、血液系统等重大疾病的发生发展密切相关。DNA甲基化是指DNA甲基转移酶(DNMT)以腺甲硫氨酸为甲基供体在胞嘧啶的5位碳上添加甲基使之转变为5-甲基胞嘧啶(5mC)的过程，并且此过程是动态可逆的，即DNA去甲基化。DNA去甲基化可分为主动和被动两种不同的形式。前者是由于DNA甲基化维持机制受到抑制，例如DNMT1失活，DNA甲基化水平在DNA的连续半保留复制过程中逐渐被稀释；后者是指在TET等多种酶的参与下，5mC持续氧化并经由一系列中间体最终转化为胞嘧啶的过程。
5-醛基胞嘧啶(5fC)作为主动去甲基化过程的中间体，对于维持生命体正常的甲基化水平具有重要意义。2015年，有文献报道称通过监测不同生长阶段小鼠组织DNA中所有胞嘧啶衍生物的含量变化，首次提出5fC是一种稳定的表观遗传学修饰，其丰度与其上游产物5-甲基胞嘧啶(5mC)、5-羟甲基胞嘧啶(5hmC)及下游产物5-羧基胞嘧啶(5caC)没有必然联系。另有报道表明，与5hmC、5caC相比，5fC拥有更多的蛋白结合位点及更为独特的碱基分布规律，使之在基因调控、改变DNA结构、细胞分化及重大疾病等特殊生物事件中扮演着更为重要的角色。
在复杂的生理过程中要想更加深入地研究5fC的生理学功能，关键在于开发高选择性、高灵敏度的检测技术来准确测定DNA中的5fC水平。
液相色谱-串联质谱(LC-MS/MS)因其自身特点在这方面具有显著优势，但是5fC在体内的丰度极低，离子化效率也低，再加上其他高含量经典碱基或稀有碱基以及样品基质中各种各样的杂质干扰，使用LC-MS/MS技术准确测定5fC仍然具有一定的挑战性。
鉴于此，研究者们开始尝试利用化学反应将一些更为疏水的或易于离子化的基团共价修饰到5fC的骨架上去，从而改善5fC在液相分离时与诸多干扰项的分离分辨率，同时提高在质谱检测过程中的离子化效率。但是目前，针对5fC的活泼醛基，仅有酰肼和 磺酰肼类衍生物涉及到此类应用。但是，这种基于席夫碱反应机理生成的C＝N双键不够稳定，易于水解，这对本身就比较耗时的LC-MS/MS来说极为不利。
另外，相比于传统的LC-MS/MS，荧光传感技术拥有选择性好、响应时间短、操作简单、可裸眼观察等优势，先后被广泛应用于活细胞乃至动物体内活性氧、活性硫、酶、核酸等生物靶标的检测。特别是有机小分子荧光探针因其具有易修饰、成本低等特点，受到了众多科研工作者的青睐。近年来，着眼于5fC的C-5位醛基，相继有报道称其能被-NH 2、-NHNH 2、-ONH 2、吲哚等衍生物荧光标记。然而，5-醛基尿嘧啶(5fU)作为胸腺嘧啶(T)的氧化产物，与5fC的结构极为类似，但其醛基的反应活性远胜于5fC，所以上述基于席夫碱反应或Aldol缩合的荧光探针将优先与5fU反应。也就是说，这类试剂并不能区分这两种醛基嘧啶，无法实现5fC的特异性荧光识别。
目前，有研究者利用含有侧链活性基团的活泼亚甲基化合物，例如丙二腈、1,3-茚满二酮、乙酰乙酸乙酯等，首次实现了5fC的特异性化学标记，并将此法进一步应用于5fC相关的测序、检测、成像及诊疗等方面。公开的这类试剂，除了含有与醛基缩合的活泼亚甲基之外，还在紧邻亚甲基的位置设置了能与5fC的环外4-NH 2发生分子内环化反应的侧链活性基团。如此一来，要想成功被这类试剂衍生，5-CHO和4-NH 2缺一不可，这也是实现5fC高度特异性标记的关键所在。另外一则类似的报道，公开了一例含有-CH 2CN结构的有机小分子，其中-CH 2-负责与5fC的5-CHO缩合，而-CN则负责与4-NH 2环化，从而选择性点亮5fC。
但是，上述鲜见的两例拥有高度特异性的醛基化试剂在标记5fC时均存在耗时较长，需10～24h，推测可能的原因是-CH 2-对-CHO的亲核性较弱导致反应活性低。
因此，发展更为高效、快速的5fC标记试剂及检测方法在本领域具有重大意义。
发明内容
本发明的目的在于提供一种衍生醛基嘧啶的方法、检测5-醛基胞嘧啶的方法以及醛基嘧啶衍生物的应用，以解决现有5fC的检测和识别工艺中存在的耗时长、条件苛刻的问题。
Wittig反应(叶立德反应)是德国化学家G.Wittig于1953年发现的，主要用于醛、酮直接合成烯烃。该方法具有收率高、反应条件温和、选择性好等优点。本发明的申请人鉴于5fC带有一个活泼的醛基，同时总结前人的经验，采用氰基取代的Wittig试剂，通过Wittig烯化及光辅助的分子内关环反应实现对5fC的选择性荧光识别和定量检测。 同时，由于5fU的反应活性高于5fC，Wittig烯化定能同时衍生这两种醛基嘧啶，这也为5fC/5fU的LC-MS/MS分析提供了一种新的衍生策略。
为了实现上述目的，本发明一方面提供一种衍生醛基嘧啶的方法，其中，所述方法包括：将醛基嘧啶与氰基取代的Wittig试剂于有机溶剂中混合，然后用紫外光进行辐射，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
进一步地，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种；
所述5-醛基尿嘧啶为含有5-醛基尿嘧啶结构单元的物质，所述含有5-醛基尿嘧啶结构单元的物质为5-醛基尿嘧啶、5-醛基尿嘧啶核苷、5-醛基尿嘧啶核苷酸以及含有5-醛基尿嘧啶的DNA、含有5-醛基尿嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种。
进一步地，在本发明较佳的实施例中，上述Wittig试剂具有如下结构式：
Figure PCTCN2019117081-appb-000001
其中，R 1为氢、氰基、卤素、烃基和含O、N、卤素、P、S或Si的烃基中的一种。
进一步地，在本发明较佳的实施例中，5-醛基胞嘧啶衍生物包括具有式(1)所示和具有式(2)所示结构的衍生物：
Figure PCTCN2019117081-appb-000002
其中，R各自独立地为氢、烃基、核糖基和脱氧核糖基中的一种，R 1各自独立地为氢、氰基、卤素、烃基和含O、N、卤素、P、S或Si的烃基中的一种。
进一步地，在本发明较佳的实施例中，5-醛基尿嘧啶衍生物具有式(3)所示结构式：
Figure PCTCN2019117081-appb-000003
其中，R为氢、烃基、核糖基和脱氧核糖基中的一种，R 1为氢、氰基、卤素、烃基和含O、N、卤素、P、S或Si的烃基中的一种。
进一步地，在本发明较佳的实施例中，上述有机溶剂包括甲醇、乙醇、异丙醇、乙二醇甲醚、乙二醇二甲醚、乙腈、甲苯、二氯甲烷、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、二氧六环和二甲基亚砜中的一种或多种组合。当为多种溶剂组合时，混合比例可以是任意比。
进一步地，在本发明较佳的实施例中，醛基嘧啶和Wittig试剂的摩尔比为1:(1-500)，反应温度为15-80℃，反应时间为5min-48h。
醛基嘧啶和Wittig试剂的摩尔比优选为1:(20-250)，更优选为1:(20-50)，最优选为1:20。
反应温度优选为50-70℃，更优选为55-65℃，最优选为60℃。
反应时间优选为0.5h-6h，更优选为0.5h-2h，最优选为1h。
进一步地，在本发明较佳的实施例中，紫外光的波长为200-400nm，辐射时间为1min-24h。
紫外光的波长优选为253-365nm，更优选为280-310nm，最优选为280nm。
紫外光的辐射时间优选为0.5h-12h，更优选为0.5h-4h，最优选为1.5h。
本发明第二方面提供本发明所述方法制得的醛基嘧啶衍生物。
本发明第三方面提供本发明提供的醛基嘧啶衍生物在检测5-醛基胞嘧啶和5-醛基尿嘧啶中的应用，采用化学衍生辅助的LC-MS/MS技术进行检测。
进一步地，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种；
所述5-醛基尿嘧啶为含有5-醛基尿嘧啶结构单元的物质，所述含有5-醛基尿嘧啶结构单元的物质为5-醛基尿嘧啶、5-醛基尿嘧啶核苷、5-醛基尿嘧啶核苷酸以及含有5-醛 基尿嘧啶的DNA、含有5-醛基尿嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种。
本发明第四方面提供本发明所述方法制得的具有式(1)所示和具有式(2)所示结构的醛基嘧啶衍生物在检测5-醛基胞嘧啶中的应用，采用荧光识别技术进行识别和检测。
进一步地，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种。
本发明第五方面提供一种检测5-醛基胞嘧啶的方法，其中，所述方法包括：将5-醛基胞嘧啶与Wittig试剂于有机溶剂中混合，然后用紫外光进行辐射，得到5-醛基胞嘧啶衍生物，然后，采用荧光识别技术进行识别和检测5-醛基胞嘧啶衍生物。
进一步地，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种。
本发明具有以下有益效果：
本发明先利用Wittig试剂同时实现5fC和5fU的化学衍生，由于疏水性丙烯腈结构的引入，使得衍生后的醛基嘧啶保留时间延长，与其他碱基的分离分辨率也得到提高，由此对化学衍生辅助的LC-MS/MS分析更为有利。进一步地，在Wittig试剂烯化之后，结合光催化的“多米诺”反应，实现5fC的选择性荧光识别和定量检测。与传统的胺基衍生物基于席夫碱反应构建的C＝N双键相比，本发明利用磷叶立德基于Wittig反应构建的C＝C双键要稳定得多。此外，本发明筛选所得的氰基取代的Wittig试剂可通过三步连续反应特异性靶向5fC，这也是胺基、吲哚等衍生物所不具备的。最后，本发明优选的高反应活性的磷叶立德及光辅助的快速关环策略有效弥补了活泼亚甲基类化合物反应耗时长的缺陷。本发明针对5-醛基胞嘧啶提供的Wittig衍生反应具有条件温和、高效快速、原料经济易得的优势。
附图说明
图1为本发明实施例衍生醛基嘧啶的工艺路线图。
图2为5fC与5fU在衍生前后与其他经典碱基或稀有碱基的HPLC轨迹对比图。
图3为本发明实施例衍生得到的关环产物5fC-CN-Close的激发光谱和发射光谱，其中，虚线为激发光谱，实线为发射光谱。
图4为trans-5fC-CN在紫外光辐射下荧光强度随时间的变化曲线图。
图5为本发明实施例的5fC衍生样品的荧光选择性检测图谱。
图6为氰亚甲基三苯基膦对5fC进行荧光定量的标准曲线。
图7为本发明实施例的5fC衍生样品在γ射线辐射前后的荧光强度变化图。
具体实施方式
以下结合附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下实施例中用于标记醛基嘧啶的Wittig试剂为氰亚甲基三苯基膦，其结构式为
Figure PCTCN2019117081-appb-000004
本发明实施例的合成路线如图1所示，将醛基嘧啶与Wittig试剂于有机溶剂中混合，然后用紫外光进行辐射，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
实施例1
本实施例为衍生醛基嘧啶的方法，包括：
将醛基嘧啶与Wittig试剂按照摩尔比为1：1的比例于乙醇中混合，在15℃下反应为48h，然后用波长为200nm的紫外光辐射24h，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
实施例2
本实施例为衍生醛基嘧啶的方法，包括：
将醛基嘧啶与Wittig试剂按照摩尔比为1：500的比例于N,N-二甲基甲酰胺中混合，在80℃下反应为5min，然后用波长为400nm的紫外光辐射1min，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
实施例3
本实施例为衍生醛基嘧啶的方法，包括：
将醛基嘧啶与Wittig试剂按照摩尔比为1：20的比例于乙二醇二甲醚中混合，在50℃下反应为6h，然后用波长为253nm的紫外光辐射12h，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
实施例4
本实施例为衍生醛基嘧啶的方法，包括：
将醛基嘧啶与Wittig试剂按照摩尔比为1：250的比例于乙二醇二甲醚中混合，在70℃下反应为0.5h，然后用波长为365nm的紫外光辐射0.5h，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
实施例5
本实施例为衍生醛基嘧啶的方法，包括：
将醛基嘧啶与Wittig试剂按照摩尔比为1：50的比例于乙二醇二甲醚中混合，在65℃下反应为1h，然后用波长为300nm的紫外光辐射45min，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
实施例6
本实施例为衍生醛基嘧啶的方法，包括：
将醛基嘧啶与Wittig试剂按照摩尔比为1：40的比例于乙二醇二甲醚中混合，在65℃下反应为40min，然后用波长为310nm的紫外光辐射30min，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
实施例7
本实施例为衍生醛基嘧啶的方法，包括：
将醛基嘧啶与Wittig试剂按照摩尔比为1：20的比例于乙二醇二甲醚中混合，在60℃下反应为1h，然后用波长为280nm的紫外光辐射1.5h，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
通过本发明实施例衍生得到醛基嘧啶衍生物：5-醛基胞嘧啶衍生物和5-醛基尿嘧啶衍生物，可用于检测醛基胞嘧啶和醛基尿嘧啶。检测的方法可采用LC-MS/MS分析法。
基于本发明实施例的衍生方法，还可以对5-醛基胞嘧啶进行检测，以确定DNA中的5fC水平。5-醛基胞嘧啶在与Wittig试剂反应后，其产物trans-5fC-CN在紫外光辐射转变为其异构结构：cis-5fC-CN，cis-5fC-CN的环外4-NH 2与距离较近的-CN进行分子内缩合环化反应，得到能够发射荧光的最终衍生物：5fC-CN-Close。以此，再通过荧光标记方法可实现对5fC的检测和定量分析。
试验例1 5fC和5fU经氰亚甲基三苯基膦衍生后的HPLC行为变化研究
取两个1.5mL离心管，分别加入0.5mg 5fC和0.5mg 5fU，然后分别加入11.8mg氰亚甲基三苯基膦(20eq.)以及0.75mL甲醇于两个离心管中，超声溶解并混匀，置于60℃摇床反应1h，然后加入等体积的水稀释一倍，进行HPLC分析。同时，将未经修饰的经典碱基或稀有碱基配成0.5mg/mL的水溶液，包括腺嘌呤(A)、鸟嘌呤(G)、尿嘧啶(U)、胞嘧啶(C)、5-羟甲基尿嘧啶(5hmU)、T、5mC、5hmC、5fC和5fU，并在同等条件下进行HPLC分析。
如图2所示，除A之外，其他碱基的保留时间均集中在4～6min，且相互之间的分离分辨率较低，尤其是G、T、5mC三种碱基非常逼近5fC和5fU，这对于LC-MS/MS分析来说是极为不利的。而经Wittig试剂衍生后的醛基嘧啶，保留时间直接延长到10min以上，明显与其他碱基区别开，如此一来可以尽可能地减小他们对5fC、5fU在LC-MS/MS分析过程中的离子抑制。另外，衍生引入的丙烯腈结构导致疏水性增加，意味着需要极性更小的流动相(也就是更大比例的有机溶剂)来洗脱，这也有利于提高目标分析物的离子化效率。
由此，本发明利用Wittig试剂可以同时高效地衍生5fC和5fU，生成的核苷加合物保留时间延长，与其他碱基的分离度也大大提升，这可以提高5fC和5fU在LC-MS/MS检测中的灵敏度。
试验例2 光辅助的分子内关环反应研究
Figure PCTCN2019117081-appb-000005
将750μL trans-5fC-CN的PBS水溶液(0.1mM)置于1cm石英比色皿中，搅拌条件下，用280nm的LED光源辐射石英比色皿的一侧，每隔一段时间取出50μL稀释至3mL PBS中测荧光(λ ex＝345nm)，记录410nm处的荧光发射强度(图3为关环产物5fC-CN-Close的激发光谱和发射光谱)，并绘制强度-时间变化曲线。
如图4所示，在开始辐射的1.5h内，荧光强度随辐射时间迅速增长，随后趋于平衡，并在很长一段时间内保持荧光强度基本不变，说明关环产物的光稳定性较好。由此可知，1.5h的紫外光辐射足以使trans-5fC-CN完全转化为关环产物。因此，先经Wittig试剂衍生，再经历1.5h的紫外光辐射，可以直接扫描样品的荧光光谱进行定量分析。加上1h的衍生反应时间，本发明的反应总时可缩短至2.5h，相比于现有技术的10～24h，有很大进步，大大节约了时间成本，提高了检测效率。
试验例3 氰亚甲基三苯基膦对5fC的荧光选择性研究
将核苷(10mM溶于DMSO,1μL)、氰亚甲基三苯基膦(100mM溶于DMSO,2μL)以及100μL甲醇加入1.5mL离心管中，混匀，60℃反应1h。反应结束后，加入900μL PBS，混匀后转移至1cm石英比色皿中，搅拌条件下，用280nm的LED光源辐射1.5h，随后不经额外纯化直接扫描荧光光谱(λ ex＝345nm)。
如图5所示，仅5fC对应得到的衍生样品在410nm处有明显的荧光增强，A、G、C、T、U、5mC、5hmC、5hmU，甚至5fU，与Wittig试剂共孵并光照后，其荧光发射光谱与探针组基本重叠，说明氰亚甲基三苯基膦对5fC具有高度特异性的荧光识别能力。
试验例4 氰亚甲基三苯基膦对γ射线辐射造成的5fC突变进行荧光定量分析
据文献报道(Madugundu,G.S.et al.Nucleic Acids Res.2014,42,7450)，5mC经γ射线辐射可突变为5fC。本试验例意欲检测γ射线对5mC的突变能力。
首先，将500μL 5mC的水溶液(10mM)暴露于 60Coγ射线源之下，并以16.7Gy/min的速率辐射90min。辐射结束后，冻干，继续加入氰亚甲基三苯基膦和100μL甲醇，混匀，60℃反应1h。反应结束后，加入900μL PBS，混匀后转移至1cm石英比色皿中，搅拌条件下，用280nm的LED光源辐射1.5h，随后不经额外纯化直接扫描荧光光谱(λ ex＝345nm)，如图7所示。记录410nm处的荧光强度，再根据如图6所示的标准曲线计算经γ射线辐射后样品中的5fC含量，进而求得γ射线对5mC的突变能力为(0.255fC/10 6碱基)/Gy。
综上所述，本发明利用氰基取代的Wittig试剂采用一锅法同时完成5-醛基胞嘧啶(5fC)和5-醛基尿嘧啶(5fU)的高效衍生，得到包含丙烯腈结构的核苷加合物，并以E式构型为主。本发明所得核苷加合物疏水性增加，在进行反相HPLC分离时保留时间延长，同时与其他经典碱基或稀有碱基的分离度也大大提高，这对于提高5fC和5fU在LC-MS/MS分析过程中的检测灵敏度十分有利。5fC对应的E式核苷衍生物，其丙烯腈结构在紫外光辐射下可发生顺反异构化，从而拉近氰基与环外4-氨基的空间距离，为分子内环加成反应创造了有利条件，最终生成发射强荧光的新型核苷；而5fU因缺乏环外4-氨基，不能完整地经历上述过程，这使得选择性荧光检测5fC的策略得以实现。
本发明筛选所得的氰基取代的Wittig试剂已经商业化，同时该衍生反应条件温和、高效快速，有效拓展了Wittig试剂在表观遗传学领域的应用，也为醛基嘧啶的化学标记和反应型5fC荧光探针的设计提供了新思路。
以上对本发明所提供的利用Wittig试剂高效衍生5-醛基胞嘧啶的方法及其在LC-MS/MS分析和荧光定量检测方面的应用进行了详细介绍。为了更清楚地阐述本发明的原理和技术方案，本文应用了部分具体实施例及相关附图辅助说明。应当指出，以上实施例及相关附图只是用于帮助理解本发明的方法及中心思想，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (14)

  1. 一种衍生醛基嘧啶的方法，其特征在于，所述方法包括：将醛基嘧啶与氰基取代的Wittig试剂于有机溶剂中混合，然后用紫外光进行辐射，制得醛基嘧啶衍生物；其中，醛基嘧啶为5-醛基胞嘧啶或5-醛基尿嘧啶。
  2. 根据权利要求1所述的方法，其中，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种；
    所述5-醛基尿嘧啶为含有5-醛基尿嘧啶结构单元的物质，所述含有5-醛基尿嘧啶结构单元的物质为5-醛基尿嘧啶、5-醛基尿嘧啶核苷、5-醛基尿嘧啶核苷酸以及含有5-醛基尿嘧啶的DNA、含有5-醛基尿嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种。
  3. 根据权利要求1所述的衍生醛基嘧啶的方法，其中，所述Wittig试剂具有如下结构式：
    Figure PCTCN2019117081-appb-100001
    其中，R 1为氢、氰基、卤素、烃基和含O、N、卤素、P、S或Si的烃基中的一种。
  4. 根据权利要求1或2所述的衍生醛基嘧啶的方法，其中，5-醛基胞嘧啶衍生物包括具有式(1)所示和具有式(2)所示结构的衍生物：
    Figure PCTCN2019117081-appb-100002
    其中，R各自独立地为氢、烃基、核糖基和脱氧核糖基中的一种，R 1各自独立地为氢、氰基，卤素，烃基和含O、N、卤素、P、S或Si的烃基中的一种。
  5. 根据权利要求1或2所述的衍生醛基嘧啶的方法，其中，5-醛基尿嘧啶衍生物具有式(3)所示结构式：
    Figure PCTCN2019117081-appb-100003
    其中，R为氢、烃基、核糖基和脱氧核糖基中的一种，R 1为氢、氰基、卤素、烃基和含O、N、卤素、P、S或Si的烃基中的一种。
  6. 根据权利要求1或2所述的衍生醛基嘧啶的方法，其中，所述有机溶剂选自甲醇、乙醇、异丙醇、乙二醇甲醚、乙二醇二甲醚、乙腈、甲苯、二氯甲烷、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、二氧六环和二甲基亚砜中的一种或多种组合。
  7. 根据权利要求1-6中任意一项所述的衍生醛基嘧啶的方法，其中，醛基嘧啶和Wittig试剂的摩尔比为1:(1-500)，反应温度为15-80℃，反应时间为5min-48h，紫外光的波长为200-400nm，辐射时间为1min-24h。
  8. 权利要求1-7中任一项所述的方法制得的醛基嘧啶衍生物。
  9. 权利要求1-7中任一项所述的方法制得的醛基嘧啶衍生物或者权利要求8所述的醛基嘧啶衍生物在检测5-醛基胞嘧啶和5-醛基尿嘧啶中的应用，采用化学衍生辅助的LC-MS/MS技术进行检测。
  10. 根据权利要求9所述的应用，其中，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种；
    所述5-醛基尿嘧啶为含有5-醛基尿嘧啶结构单元的物质，所述含有5-醛基尿嘧啶结构单元的物质为5-醛基尿嘧啶、5-醛基尿嘧啶核苷、5-醛基尿嘧啶核苷酸以及含有5-醛基尿嘧啶的DNA、含有5-醛基尿嘧啶的RNA，以及含有上述至少一种化合物的混合物 中的至少一种。
  11. 权利要求1-7中任一项所述的方法制得的具有式(1)所示和具有式(2)所示结构的醛基嘧啶衍生物在检测5-醛基胞嘧啶中的应用，采用荧光识别技术进行识别和检测。
  12. 根据权利要求11所述的应用，其中，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种。
  13. 一种检测5-醛基胞嘧啶的方法，其特征在于，所述方法包括：将5-醛基胞嘧啶与Wittig试剂于有机溶剂中混合，然后用紫外光进行辐射，得到5-醛基胞嘧啶衍生物，然后，采用荧光识别技术进行识别和检测5-醛基胞嘧啶衍生物。
  14. 根据权利要求13所述的方法，其中，所述5-醛基胞嘧啶为含有5-醛基胞嘧啶结构单元的物质，所述含有5-醛基胞嘧啶结构单元的物质为5-醛基胞嘧啶、5-醛基胞嘧啶核苷、5-醛基胞嘧啶核苷酸以及含有5-醛基胞嘧啶的DNA、含有5-醛基胞嘧啶的RNA，以及含有上述至少一种化合物的混合物中的至少一种。
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