CN109678802B

CN109678802B - Method for deriving aldehyde pyrimidine, method for detecting 5-aldehyde cytosine and application of aldehyde pyrimidine derivative

Info

Publication number: CN109678802B
Application number: CN201910079972.7A
Authority: CN
Inventors: 李坤; 周倩; 余孝其
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2020-12-29
Anticipated expiration: 2039-01-28
Also published as: WO2020155742A1; CN109678802A

Abstract

The invention discloses a method for deriving aldehyde pyrimidine, a method for detecting 5-aldehyde cytosine and application of an aldehyde pyrimidine derivative. Mixing aldehyde pyrimidine and a Wittig reagent in an organic solvent, and then radiating with ultraviolet light to prepare an aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil. The invention has mild reaction conditions, high efficiency and high speed, effectively expands the application of the Wittig reagent in the field of epigenetics, and also provides a new idea for the chemical marking of aldehyde pyrimidine and the design of a reactive 5fC fluorescent probe.

Description

Method for deriving aldehyde pyrimidine, method for detecting 5-aldehyde cytosine and application of aldehyde pyrimidine derivative

Technical Field

The invention relates to the technical field of nucleic acid chemistry, in particular to a method for deriving aldehyde pyrimidine, a method for detecting 5-aldehyde cytosine and application of an aldehyde pyrimidine derivative.

Background

The DNA of mammalian cells or tissues, particularly CpG islands, have methylation modification in different degrees, which is closely related to the occurrence and development of serious diseases such as embryonic development, tumor, mental, blood system and the like. DNA methylation refers to the process by which DNA methyltransferase (DNMT) adds a methyl group to the 5-carbon of cytosine using ademetionine as the methyl donor to convert it to 5-methylcytosine (5mC), and is dynamically reversible, i.e., DNA demethylation. DNA demethylation can be divided into two distinct forms, active and passive. The former is due to inhibition of DNA methylation maintenance mechanisms, such as inactivation of DNMT1, DNA methylation levels are gradually diluted during the continuous semi-retentive replication of DNA; the latter refers to the process of sustained oxidation of 5mC in the presence of various enzymes such as TET and ultimately conversion to cytosine via a series of intermediates.

5-aldehyde cytosine (5fC) is used as an intermediate in an active demethylation process and has important significance for maintaining normal methylation level of a living body. In 2015, it was reported that by monitoring the content of all cytosine derivatives in mouse tissue DNA at different growth stages, 5fC was first proposed to be a stable epigenetic modification, whose abundance is not necessarily linked to its upstream product 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and downstream product 5-carboxycytosine (5 caC). In addition, it is reported that 5fC has more protein binding sites and more unique base distribution rules than 5hmC and 5caC, so that it plays a more important role in gene regulation, DNA structure modification, cell differentiation, and special biological events such as major diseases.

In order to study the physiological functions of 5fC more deeply in complex physiological processes, it is critical to develop highly selective, highly sensitive detection techniques to accurately determine the level of 5fC in DNA.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has significant advantages in this regard due to its own characteristics, but 5fC is very low in abundance in vivo, ionization efficiency is also low, and accurate determination of 5fC using LC-MS/MS techniques remains somewhat challenging, coupled with interference from other high content of classical or rare bases and various impurities in the sample matrix.

In view of this, researchers have attempted to covalently modify some groups that are more hydrophobic or easy to ionize onto the backbone of 5fC by chemical reaction, so as to improve the resolution of 5fC separation with many interfering items during liquid phase separation, and increase the ionization efficiency during mass spectrometric detection. However, currently, only hydrazide and sulfonyl hydrazide derivatives are involved in such applications for the reactive aldehyde group of 5 fC. However, the C ═ N double bonds formed by this schiff base reaction mechanism are not sufficiently stable and are susceptible to hydrolysis, which is extremely disadvantageous for LC-MS/MS which is relatively time-consuming in itself.

In addition, compared with the traditional LC-MS/MS, the fluorescence sensing technology has the advantages of good selectivity, short response time, simplicity in operation, capability of being observed by naked eyes and the like, and is sequentially and widely applied to detection of biological targets such as active oxygen, active sulfur, enzyme, nucleic acid and the like in living cells and even in animals. In particular, the organic small-molecule fluorescent probe is favored by a plurality of scientific researchers due to the characteristics of easy modification, low cost and the like. In recent years, attention has been paid to the C-5 aldehyde group of 5fC, and it has been reported that it is fluorescently labeled with derivatives such as-NH 2, -NHNH2, -ONH2, and indole. However, 5-aldehyde uracil (5fU) as an oxidation product of thymine (T) has a structure very similar to that of 5fC, but the reactivity of the aldehyde group is much better than that of 5fC, so the above-mentioned fluorescent probe based on Schiff base reaction or Aldol condensation will preferentially react with 5 fU. That is, such reagents do not distinguish between the two aldehyde pyrimidines and do not achieve specific fluorescent recognition of 5 fC.

Currently, researchers utilize active methylene compounds containing side chain active groups, such as malononitrile, 1, 3-indandione, ethyl acetoacetate and the like, to realize specific chemical labeling of 5fC for the first time, and the method is further applied to aspects such as sequencing, detection, imaging, diagnosis and treatment and the like related to 5 fC. In addition to containing an active methylene group condensed with an aldehyde group, the reagent disclosed therein is provided with a side chain active group capable of undergoing intramolecular cyclization reaction with the exocyclic 4-NH2 of 5fC at a position adjacent to the methylene group. As such, the lack of 5-CHO and 4-NH2, which are successfully derivatized by such reagents, is also critical to achieving highly specific labeling of 5 fC. Another similar report discloses a small organic molecule containing the structure-CH 2CN, in which-CH 2-is responsible for 5-CHO condensation with 5fC and-CN is responsible for cyclization with 4-NH2, thereby selectively illuminating 5 fC.

However, the two fresh cases of the aldehydizing reagents with high specificity are both long in time consumption and require 10-24 hours for marking 5fC, and the probable reason is that the reactivity is low due to the weak nucleophilicity of-CH 2-para-CHO.

Therefore, the development of more efficient and rapid 5fC labeling reagents and detection methods is of great significance in the field.

Disclosure of Invention

The invention aims to provide a method for deriving aldehyde pyrimidine, a method for detecting 5-aldehyde cytosine and application of an aldehyde pyrimidine derivative, so as to solve the problems of long time consumption and harsh conditions in the existing 5fC detection and identification process.

The Wittig reaction (ylide reaction) is discovered by German chemist G.Wittig in 1953, and is mainly used for directly synthesizing olefin from aldehyde and ketone. The method has the advantages of high yield, mild reaction conditions, good selectivity and the like. In view of the fact that 5fC has an active aldehyde group, the applicant of the invention summarizes the experience of the prior people, adopts a cyano-substituted Wittig reagent, and realizes selective fluorescence recognition and quantitative detection of 5fC through Wittig olefination and light-assisted intramolecular ring closure reaction. Meanwhile, as the reactivity of 5fU is higher than that of 5fC, the Wittig olefination can simultaneously derive the two aldehyde pyrimidines, and a new derivation strategy is provided for LC-MS/MS analysis of 5fC/5 fU.

The technical scheme of the invention is as follows:

a method for deriving aldehyde pyrimidine, mix aldehyde pyrimidine and cyano-substituted Wittig reagent in organic solvent, then radiate with the ultraviolet light, make aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Further, in a preferred embodiment of the present invention, the Wittig reagent has the following structural formula:

wherein R is¹Is hydrogen, cyano, halogen, hydrocarbyl or O, N-, halogen-, P, S-or Si-containing hydrocarbyl.

Further, in a preferred embodiment of the present invention, the 5-aldehyde cytosine derivative includes two derivatives having the following structural formula:

wherein R is hydrogen, hydrocarbyl, ribosyl or deoxyribosyl, R¹Is hydrogen, cyano, halogen, hydrocarbyl or O, N-, halogen-, P, S-or Si-containing hydrocarbyl.

Further, in a preferred embodiment of the present invention, the 5-carboxaldehyde uracil derivative has the following structural formula:

Further, in a preferred embodiment of the present invention, the organic solvent includes one or more of methanol, ethanol, isopropanol, ethylene glycol methyl ether, ethylene glycol dimethyl ether, acetonitrile, toluene, dichloromethane, N-dimethylformamide, N-dimethylacetamide, dioxane and dimethylsulfoxide. When a plurality of solvents are combined, the mixing ratio may be any ratio.

Further, in a preferred embodiment of the invention, the molar ratio of the aldehyde pyrimidine to the Wittig reagent is 1: (1-500), the reaction temperature is 15-80 ℃, the reaction time is 5min-48h, the wavelength of ultraviolet light is 200-400nm, and the radiation time is 1min-24 h.

The molar ratio of the aldehyde pyrimidine to the Wittig reagent is preferably 1: (20-250), more preferably 1: (20-50), most preferably 1: 20.

The reaction temperature is preferably 50 to 70 ℃, more preferably 55 to 65 ℃, and most preferably 60 ℃.

The reaction time is preferably 0.5h to 6h, more preferably 0.5h to 2h, most preferably 1 h.

The wavelength of the ultraviolet light is preferably 253 to 365nm, more preferably 280 to 310nm, most preferably 280 nm.

The irradiation time of the ultraviolet light is preferably 0.5h to 12h, more preferably 0.5h to 4h, and most preferably 1.5 h.

The aldehyde pyrimidine derivative prepared by the method.

The aldehyde pyrimidine derivative is applied to detection of 5-aldehyde cytosine and 5-aldehyde uracil.

Further, in the preferred embodiment of the present invention, the 5-aldehyde cytosine and 5-aldehyde uracil are detected by chemical derivatization assisted LC-MS/MS technology; and (3) identifying and detecting the 5-aldehyde cytosine by adopting a fluorescence identification technology.

Aldehyde pyrimidine derivatives prepared according to the invention: 5-aldehyde cytosine derivative and 5-aldehyde uracil derivative can be simultaneously detected by LC-MS/MS technology; further, 5-aldehyde cytosine can be identified by a fluorescence identification technology, and the 5-aldehyde cytosine is distinguished from the 5-aldehyde uracil.

A method for detecting 5-aldehyde cytosine comprises the steps of mixing 5-aldehyde cytosine with a Wittig reagent in an organic solvent, then radiating with ultraviolet light to obtain a 5-aldehyde cytosine derivative, and then identifying and detecting the 5-aldehyde cytosine derivative by adopting a fluorescence identification technology.

The invention has the following beneficial effects:

according to the invention, the Wittig reagent is firstly utilized to simultaneously realize chemical derivatization of 5fC and 5fU, due to the introduction of a hydrophobic acrylonitrile structure, the retention time of the aldehyde pyrimidine after derivatization is prolonged, and the separation resolution of the aldehyde pyrimidine from other basic groups is also improved, so that the method is more favorable for LC-MS/MS analysis assisted by chemical derivatization. Further, after Wittig reagent olefination, selective fluorescence recognition and quantitative detection of 5fC are realized by combining photocatalysis 'domino' reaction. Compared with the C ═ N double bond constructed by the traditional amino derivative based on Schiff base reaction, the C ═ C double bond constructed by the phosphorus ylide based on Wittig reaction is much more stable. In addition, the cyano-substituted Wittig reagent obtained by screening can specifically target 5fC through three-step continuous reaction, which is not possessed by derivatives such as amino, indole and the like. Finally, the optimized high-reactivity phosphorus ylide and photo-assisted rapid ring closing strategy effectively makes up for the defect of long reaction time consumption of the active methylene compounds. The invention aims at the Wittig derivatization reaction provided by 5-aldehyde cytosine and has the advantages of mild condition, high efficiency, rapidness, economy and easy obtainment of raw materials.

Drawings

FIG. 1 is a schematic diagram of a process for derivatizing an formylpyrimidine of the present invention.

FIG. 2 is a graph comparing HPLC traces of 5fC and 5fU before and after derivatization with other classical bases or rare bases.

FIG. 3 shows the excitation spectrum and emission spectrum of the ring-closed product 5fC-CN-Close derived from the example of the present invention.

FIG. 4 is a graph showing the change of fluorescence intensity of trans-5fC-CN with time under UV irradiation.

FIG. 5 is a fluorescence selectivity profile of a 5 fC-derived sample according to an embodiment of the present invention.

FIG. 6 is a standard curve for fluorescence quantification of cyanomethylenetriphenylphosphine on 5 fC.

FIG. 7 is a graph of fluorescence intensity before and after gamma irradiation with time for a 5 fC-derived sample of an example of the present invention.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the following examples, the Wittig reagent used for labeling formylpyrimidine is cyanomethylene triphenylphosphine, and the structural formula is

The synthetic route of the embodiment of the invention is shown in figure 1, aldehyde pyrimidine and Wittig reagent are mixed in an organic solvent, and then ultraviolet light is used for radiation to prepare the aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Example 1

The method for derivatizing aldehyde pyrimidine in the embodiment comprises the following steps:

mixing aldehyde pyrimidine and a Wittig reagent according to a molar ratio of 1: 1 in the proportion of ethanol, reacting for 48 hours at 15 ℃, and then radiating for 24 hours by using ultraviolet with the wavelength of 200nm to prepare an aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Example 2

mixing aldehyde pyrimidine and a Wittig reagent according to a molar ratio of 1: 500 in N, N-dimethylformamide, reacting at 80 deg.C for 5min, and irradiating with ultraviolet light with wavelength of 400nm for 1min to obtain formyl pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Example 3

mixing aldehyde pyrimidine and a Wittig reagent according to a molar ratio of 1:20 in proportion, mixing the mixture in ethylene glycol dimethyl ether, reacting for 6 hours at 50 ℃, and then radiating for 12 hours by using ultraviolet light with the wavelength of 253nm to prepare an aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Example 4

mixing aldehyde pyrimidine and a Wittig reagent according to a molar ratio of 1: 250 proportion in glycol dimethyl ether, reacting for 0.5h at 70 ℃, and then radiating for 0.5h by using ultraviolet light with the wavelength of 365nm to prepare aldehyde pyrimidine derivatives; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Example 5

mixing aldehyde pyrimidine and a Wittig reagent according to a molar ratio of 1: mixing 50 parts of the aldehyde pyrimidine derivative in ethylene glycol dimethyl ether, reacting for 1 hour at 65 ℃, and then radiating for 45min by using ultraviolet light with the wavelength of 300nm to prepare the aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Example 6

mixing aldehyde pyrimidine and a Wittig reagent according to a molar ratio of 1: 40 in proportion in ethylene glycol dimethyl ether, reacting for 40min at 65 ℃, and then radiating for 30min by ultraviolet light with the wavelength of 310nm to prepare an aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

Example 7

mixing aldehyde pyrimidine and a Wittig reagent according to a molar ratio of 1:20 in proportion, mixing the mixture in ethylene glycol dimethyl ether, reacting for 1h at 60 ℃, and then radiating for 1.5h by using ultraviolet light with the wavelength of 280nm to prepare an aldehyde pyrimidine derivative; wherein the aldehyde pyrimidine is 5-aldehyde cytosine or 5-aldehyde uracil.

The aldehyde pyrimidine derivative is obtained by derivation of the embodiment of the invention: the 5-aldehyde cytosine derivative and the 5-aldehyde uracil derivative can be used for detecting aldehyde cytosine and aldehyde uracil. The detection method can adopt an LC-MS/MS analysis method.

Based on the derivatization method of the embodiment of the invention, 5-aldehyde cytosine can also be detected to determine the level of 5fC in DNA. After the 5-aldehyde cytosine reacts with a Wittig reagent, the product trans-5fC-CN is converted into an isomeric structure thereof by ultraviolet radiation: cls-5fC-CN, exocyclic 4-NH of cis-5fC-CN₂And carrying out intramolecular condensation cyclization reaction with-CN at a close distance to obtain a final derivative capable of emitting fluorescence: 5 fC-CN-Close. Therefore, the detection and quantitative analysis of 5fC can be realized by a fluorescence labeling method.

Experimental example 15 fC and 5fU HPLC behavior Change study after derivatization with cyanomethylenetriphenylphosphine

Two 1.5mL centrifuge tubes were taken, 0.5mg of 5fC and 0.5mg of 5fU were added, respectively, then 11.8mg of cyanomethylenetriphenylphosphine (20eq.) and 0.75mL of methanol were added to the two centrifuge tubes, respectively, ultrasonically dissolved and mixed, placed in a 60 ℃ shaker for reaction for 1h, then diluted one time by adding an equal volume of water, and subjected to HPLC analysis. Meanwhile, unmodified classical bases or rare bases were prepared as an aqueous solution of 0.5mg/mL including adenine (A), guanine (G), uracil (U), cytosine (C), 5-hydroxymethyluracil (5hmU), T, 5mC, 5hmC, 5fC and 5fU, and subjected to HPLC analysis under the same conditions.

As shown in FIG. 2, the retention times of other bases except A are all concentrated at 4-6 min, and the separation resolution between the bases is low, especially the G, T and 5mC three bases are very close to 5fC and 5fU, which is very unfavorable for LC-MS/MS analysis. The retention time of the aldehyde pyrimidine derived by the Wittig reagent is directly prolonged to more than 10min, and the aldehyde pyrimidine is obviously distinguished from other bases, so that the ion inhibition of the aldehyde pyrimidine in the LC-MS/MS analysis process of 5fC and 5fU can be reduced as much as possible. In addition, derivatizing the incorporated acrylonitrile structure results in increased hydrophobicity, meaning that a less polar mobile phase (i.e., a greater proportion of organic solvent) is required for elution, which also contributes to increased ionization efficiency of the target analyte.

Therefore, the invention utilizes Wittig reagent to simultaneously and efficiently derive 5fC and 5fU, the retention time of the generated nucleoside adduct is prolonged, the separation degree with other bases is greatly improved, and the sensitivity of the 5fC and 5fU in LC-MS/MS detection can be improved.

FIG. 3 shows the excitation spectrum and emission spectrum of the ring-closed product 5 fC-CN-Close.

Experimental example 2 photo-assisted intramolecular cyclization reaction study

750 μ L of trans-5fC-CN in PBS (0.1mM) was placed in a 1cm quartz cuvette, one side of the cuvette was irradiated with a 280nm LED light source with stirring, and 50 μ L of the cuvette was removed at intervals and diluted to 3mL PBS for fluorescence (λ `) measurement_ex345nm), the fluorescence emission intensity at 410nm is recorded and an intensity-time curve is plotted.

As shown in FIG. 4, the fluorescence intensity rapidly increased with the irradiation time within 1.5h from the start of irradiation, then reached equilibrium, and remained substantially constant for a long period of time, indicating that the light stability of the product of the ring closure was better. It can be seen that 1.5h of UV irradiation is sufficient to completely convert trans-5fC-CN into the ring-closed product. Therefore, the fluorescence spectrum of the sample can be directly scanned for quantitative analysis by first deriving with a Wittig reagent and then irradiating with ultraviolet light for 1.5 h. In addition, the derivatization reaction time is 1h, the total reaction time of the method can be shortened to 2.5h, compared with the reaction time of 10-24 h in the prior art, the method has great progress, the time cost is greatly saved, and the detection efficiency is improved.

Experimental example 3 fluorescence selectivity study of cyanomethylenetriphenylphosphine on 5fC

Nucleoside (10mM in DMSO, 1. mu.L), cyanomethylene triphenylphosphine (100mM in DMSO, 2. mu.L) and 100. mu.L of methanol were added to a 1.5mL centrifuge tube, mixed well and reacted at 60 ℃ for 1 h. Inverse directionAfter the reaction, 900 μ L PBS was added, mixed well and transferred to a 1cm quartz cuvette, irradiated with a 280nm LED light source for 1.5h with stirring, and then the fluorescence spectrum (λ) was directly scanned without additional purification_ex＝345nm)。

As shown in FIG. 5, the fluorescence enhancement of the derived samples obtained corresponding to only 5fC at 410nm is significant, and A, G, C, T, U, 5mC, 5hmC, 5hmU, and even 5fU, after being co-incubated with Wittig reagent and illuminated, the fluorescence emission spectra of the derived samples are basically overlapped with the probe set, which indicates that cyanomethylenetriphenylphosphine has highly specific fluorescence recognition capability on 5 fC.

Test example 4 quantitative fluorescence analysis of 5fC mutation by gamma-ray radiation with cyanomethylenetriphenylphosphine

It is reported in the literature (Madugundu, G.S.et al.nucleic Acids Res.2014,42,7450) that 5mC is mutable to 5fC by gamma irradiation. This test example is intended to detect the mutational ability of gamma rays to 5 mC.

First, 500. mu.L of 5mC in water (10mM) was exposed to⁶⁰Co gamma ray source, and irradiating at 16.7Gy/min rate for 90 min. After the radiation is finished, freeze-drying, continuously adding cyanomethylene triphenylphosphine and 100 mu L methanol, uniformly mixing, and reacting for 1h at 60 ℃. After the reaction, 900 μ L PBS was added, mixed well and transferred to a 1cm quartz cuvette, irradiated with a 280nm LED light source for 1.5h with stirring, and then the fluorescence spectrum (λ) was directly scanned without additional purification_ex345nm) as shown in fig. 7. Recording the fluorescence intensity at 410nm, calculating the content of 5fC in the sample after gamma-ray radiation according to the standard curve shown in FIG. 6, and further obtaining the mutation capability of the gamma-ray to 5mC (0.255 fC/10)⁶base)/Gy.

In conclusion, the invention utilizes a cyano-substituted Wittig reagent to simultaneously complete the efficient derivatization of 5-aldehyde cytosine (5fC) and 5-aldehyde uracil (5fU) by a one-pot method to obtain a nucleoside adduct containing an acrylonitrile structure, and the nucleoside adduct is mainly in an E-type configuration. The nucleoside adduct obtained by the invention has increased hydrophobicity, prolonged retention time when reversed-phase HPLC separation is carried out, and greatly improved separation degree with other classical bases or rare bases, which is very beneficial to improving the detection sensitivity of 5fC and 5fU in the LC-MS/MS analysis process. The acrylonitrile structure of the E-type nucleoside derivative corresponding to 5fC can generate cis-trans isomerization under ultraviolet radiation, so that the space distance between cyano and exocyclic 4-amino is shortened, favorable conditions are created for intramolecular cycloaddition reaction, and finally, a novel nucleoside emitting strong fluorescence is generated; whereas 5fU, lacking the exocyclic 4-amino group, could not undergo the above process in its entirety, which enabled a strategy for selective fluorescent detection of 5 fC.

The cyano-substituted Wittig reagent obtained by screening is commercialized, and meanwhile, the derivatization reaction condition is mild, efficient and rapid, so that the application of the Wittig reagent in the field of epigenetics is effectively expanded, and a new thought is provided for the chemical labeling of aldehyde pyrimidine and the design of a reactive 5fC fluorescent probe.

The method for efficiently deriving the 5-aldehyde cytosine by utilizing the Wittig reagent and the application thereof in LC-MS/MS analysis and fluorescent quantitative detection are described in detail above. In order to clearly illustrate the principle and technical solution of the present invention, some specific embodiments and related drawings are used for assisting the description. It should be noted that the above embodiments and the related drawings are only used for assisting understanding of the method and the central idea of the present invention, and it is obvious to those skilled in the art that a plurality of improvements and modifications can be made to the present invention without departing from the principle of the present invention, and the improvements and modifications also fall into the protection scope of the claims of the present invention.

Claims

1. A method for detecting 5-aldehyde cytosine is characterized in that 5-aldehyde cytosine and a Wittig reagent are mixed in an organic solvent, then ultraviolet light is used for radiation to obtain a 5-aldehyde cytosine derivative, and then a fluorescence recognition technology is adopted for recognition and detection of the 5-aldehyde cytosine derivative; the molar ratio of the 5-aldehyde cytosine to the Wittig reagent is 1: (1-500), the reaction temperature is 15-80 ℃, the reaction time is 5min-48h, the wavelength of ultraviolet light is 200-400nm, and the radiation time is 1min-24 h;

the Wittig reagent has the following structural formula:

wherein R is¹Is hydrogen, cyano, halogen, hydrocarbyl or O, N-, halogen-, P, S-or Si-containing hydrocarbyl;

the 5-aldehyde cytosine derivative is two derivatives with the following structural formula:

wherein R is ribosyl or deoxyribosyl, R¹Is hydrogen, cyano, halogen, hydrocarbyl or O, N-, halogen-, P, S-or Si-containing hydrocarbyl.