CN109678802A - The application of the method for derived aldehydes yl pyrimidines, the method for detecting 5- aldehyde radical cytimidine and aldehyde radical pyrimidine derivatives - Google Patents
The application of the method for derived aldehydes yl pyrimidines, the method for detecting 5- aldehyde radical cytimidine and aldehyde radical pyrimidine derivatives Download PDFInfo
- Publication number
- CN109678802A CN109678802A CN201910079972.7A CN201910079972A CN109678802A CN 109678802 A CN109678802 A CN 109678802A CN 201910079972 A CN201910079972 A CN 201910079972A CN 109678802 A CN109678802 A CN 109678802A
- Authority
- CN
- China
- Prior art keywords
- aldehyde radical
- cytimidine
- pyrimidine
- alkyl
- pyrimidines
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
Abstract
The invention discloses a kind of method of derived aldehydes yl pyrimidines, detect the method for 5- aldehyde radical cytimidine and the application of aldehyde radical pyrimidine derivatives.The present invention mixes aldehyde radical pyrimidine with Wittig reagent in organic solvent, is then radiated with ultraviolet light, and aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical pyrimidine is 5- aldehyde radical cytimidine or 5- aldehyde radical uracil.Reaction condition of the present invention is mild, efficiently quick, has effectively expanded Wittig reagent in the application in epigenetics field, also the design for the chemical labeling of aldehyde radical pyrimidine and response type 5fC fluorescence probe provides new approaches.
Description
Technical field
The present invention relates to nucleic acid chemistry fields, and in particular to a kind of method of derived aldehydes yl pyrimidines, detection 5- aldehyde radical
The method of cytimidine and the application of aldehyde radical pyrimidine derivatives.
Background technique
In mammalian cell or tissue DNA, the especially island CpG, all the methylation modification of various degrees, this with
The occurrence and development of embryonic development, the major diseases such as tumour, spirit, hematological system are closely related.DNA methylation refers to DNA methyl
Transferase (DNMT) adds methyl using gland methionine as methyl donor on 5 carbon of cytimidine and is allowed to be changed into 5- methyl born of the same parents
The process of pyrimidine (5mC), and this process is dynamic reversible, i.e. DNA demethylation.DNA demethylation can be divided into actively and
Passive two different forms.The former be since DNA methylation support mechanism is suppressed, such as DNMT1 inactivation, DNA methyl
Change level is gradually diluted during the continuous semi-conservative replication of DNA；The latter refers in the presence of a variety of enzymes such as TET, 5mC
Continued oxidation and a series of process that cytimidine is eventually converted into via intermediates.
Intermediate of the 5- aldehyde radical cytimidine (5fC) as active demethylation process, first normal for the body that sustains life
Base level is of great significance.2015, there is document report to claim by owning in monitoring different stages of growth mouse tissue DNA
The changes of contents of cytosine derivative, being put forward for the first time 5fC is a kind of stable epigenetics modification, and abundance and its upstream produce
Object 5-methylcytosine (5mC), 5- hydroxymethyl cytosine (5hmC) and downstream product 5- carboxyl cytimidine (5caC) are without inevitable
Connection.Show compared with 5hmC, 5caC according to another report, 5fC possesses more protein binding sites and more unique base point
Cloth rule is allowed to play more in the special biological events such as gene regulation, change DNA structure, cell differentiation and major disease
For important role.
The physiologic function of 5fC is more in depth studied in complicated physiology course, key is to develop Gao Xuan
Selecting property, highly sensitive detection technique carry out the level of the 5fC in Accurate Determining DNA.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is because its own feature has significant advantage in this respect, but 5fC exists
Intracorporal abundance is extremely low, and Ionization Efficiency is also low, along with other high-content classics bases or rare bases and sample substrate
In the interference of various impurity, still there is certain challenge using LC-MS/MS technology Accurate Determining 5fC.
In consideration of it, researchers begin trying using chemical reaction by it is some more hydrophobic or be easy to ionize group
Gone on covalent modification to the skeleton of 5fC, so as to improve 5fC in liquid phase separation with the separation resolution ratio of many distracters, simultaneously
Improve Ionization Efficiency during Mass Spectrometer Method.But currently, it is directed to the active aldehyde of 5fC, only hydrazides and sulfohydrazide
Analog derivative is related to such application.But this C=N double bond generated based on schiff base reaction mechanism is not sufficiently stable, and is easy to
Hydrolysis, this to inherently than relatively time-consuming LC-MS/MS for it is totally unfavorable.
In addition, compared to traditional LC-MS/MS, fluorescence sense technology possesses that selectivity is good, the response time is short, operation is simple
It is single, can the advantages such as naked eye, be successively widely used in active oxygen, active sulfur, enzyme, nucleic acid etc. in living cells or even animal body
The detection of biological target.Especially small organic molecule fluorescence probe because its have the characteristics that easily modify, it is at low cost, receive numerous
The favor of researcher.In recent years, be conceived to the position the C-5 aldehyde radical of 5fC, in succession it has been reported that its can by-NH2 ,-NHNH2 ,-
The derivatives fluorescents such as ONH2, indoles label.However, oxidation product of the 5- aldehyde radical uracil (5fU) as thymidine (T), with
The structure of 5fC is extremely similar, but the reactivity of its aldehyde radical outclass 5fC, so above-mentioned contracted based on schiff base reaction or Aldol
The fluorescence probe of conjunction will preferentially be reacted with 5fU.That is, this kind of reagent can not distinguish both aldehyde radical pyrimidines, Wu Fashi
The specificity fluorescent identification of existing 5fC.
Currently, there is researcher using the active methylene compound containing pendant reactive group, such as malononitrile, 1,3- indenes
Full diketone, ethyl acetoacetate etc., realize the specific chemical label of 5fC for the first time, and this method is further applied 5fC phase
Sequencing, detection, imaging and diagnosis and treatment of pass etc..Disclosed this kind of reagent, in addition to containing the active methylene group being condensed with aldehyde radical
Except, the pendant reactive of intramolecular cyclization reaction can be occurred by 4-NH2 outside the ring with 5fC by being also provided in the position close to methylene
Group.In this way, be derived to success by this kind of reagent, 5-CHO and 4-NH2 are indispensable, this is also to realize 5fC height
The key point of specific marker.In addition first similar report, discloses organic small point that an example contains-CH2CN structure
Son, wherein-CH2- is responsible for being condensed with the 5-CHO of 5fC, and-CN is then responsible for being cyclized with 4-NH2, so that selectivity lights 5fC.
But above-mentioned two rarely seen aldehyde radical reagents for possessing high degree of specificity exist when marking 5fC it is time-consuming compared with
It grows, need 10~for 24 hours, thus it is speculated that cause reactivity low the possible reason is the nucleophilicity of the p- CHO of-CH2- is weaker.
Therefore, develop highly efficient, quick 5fC labelled reagent and detection method is of great significance in this field.
Summary of the invention
The purpose of the present invention is to provide a kind of method of derived aldehydes yl pyrimidines, detect 5- aldehyde radical cytimidine method and
The application of aldehyde radical pyrimidine derivatives, to solve the detection of existing 5fC and identify that time-consuming present in technique, condition is harsh to ask
Topic.
Wittig react (Ylide reaction) be that German chemist G.Wittig has found in nineteen fifty-three, be mainly used for aldehyde,
Ketone directly synthesizes alkene.This method has that high income, reaction condition be mild, good selective.Applicant of the present invention's mirror
An active aldehyde radical is had in 5fC, while summarizing the experience of forefathers, the Wittig reagent replaced using cyano is passed through
Wittig alkylene and the Intra-molecular condensation of light auxiliary realize selective fluorescence identifying and quantitative detection to 5fC.Meanwhile by
It is higher than 5fC in the reactivity of 5fU, Wittig alkylene surely derives both aldehyde radical pyrimidines simultaneously, this is also 5fC/5fU's
LC-MS/MS analysis provides a kind of new derivative strategy.
Technical scheme is as follows:
A kind of method of derived aldehydes yl pyrimidines, the Wittig reagent that aldehyde radical pyrimidine and cyano are replaced mix in organic solvent
It closes, is then radiated with ultraviolet light, aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical pyrimidine is 5- aldehyde radical cytimidine or 5- aldehyde
Base uracil.
Further, in preferred embodiments of the present invention, above-mentioned Wittig reagent has the following structure formula:
Wherein, R1For hydrogen, cyano, halogen, alkyl or containing O, N, halogen, P, S or Si alkyl.
Further, in preferred embodiments of the present invention, 5- aldehyde radical cytosine derivative includes having the following structure formula
Two kinds of derivatives:
Wherein, R is hydrogen, alkyl, ribosyl or deoxyribosyl, R1For hydrogen, cyano, halogen, alkyl or containing O, N, halogen
The alkyl of element, P, S or Si.
Further, in preferred embodiments of the present invention, 5- aldehyde radical uracil derivative has the following structure formula:
Wherein, R is hydrogen, alkyl, ribosyl or deoxyribosyl, R1For hydrogen, cyano, halogen, alkyl or containing O, N, halogen
The alkyl of element, P, S or Si.
Further, in preferred embodiments of the present invention, above-mentioned organic solvent includes methanol, ethyl alcohol, isopropanol, second two
Alcohol methyl ether, glycol dimethyl ether, acetonitrile, toluene, methylene chloride, N,N-dimethylformamide, DMAC N,N' dimethyl acetamide, dioxy
One of six rings and dimethyl sulfoxide or multiple combinations.When for multi-solvents combination, mixed proportion can be any ratio.
Further, in preferred embodiments of the present invention, the molar ratio of aldehyde radical pyrimidine and Wittig reagent is 1:(1-
500), reaction temperature is 15-80 DEG C, reaction time 5min-48h, and the wavelength of ultraviolet light is 200-400nm, and radiated time is
1min-24h。
The molar ratio of aldehyde radical pyrimidine and Wittig reagent is preferably 1:(20-250), more preferably 1:(20-50), most preferably
For 1:20.
Reaction temperature is preferably 50-70 DEG C, more preferably 55-65 DEG C, most preferably 60 DEG C.
Reaction time is preferably 0.5h-6h, more preferably 0.5h-2h, most preferably 1h.
The wavelength of ultraviolet light is preferably 253-365nm, more preferably 280-310nm, most preferably 280 nm.
The radiated time of ultraviolet light is preferably 0.5h-12h, more preferably 0.5h-4h, most preferably 1.5 h.
Aldehyde radical pyrimidine derivatives made from above-mentioned method.
Application of the above-mentioned aldehyde radical pyrimidine derivatives in detection 5- aldehyde radical cytimidine and 5- aldehyde radical uracil.
Further, in preferred embodiments of the present invention, 5- is detected using the LC-MS/MS technology of chemical derivatization auxiliary
Aldehyde radical cytimidine and 5- aldehyde radical uracil；5- aldehyde radical cytimidine is identified and detected using fluorescence identification technique.
Aldehyde radical pyrimidine derivatives are made according to the present invention: 5- aldehyde radical cytosine derivative and 5- aldehyde radical uracil derivative are logical
5- aldehyde radical cytimidine and 5- aldehyde radical uracil can be detected simultaneously by crossing LC-MS/MS technology；Further, pass through fluorescence identifying
Technology can identify 5- aldehyde radical cytimidine, and 5- aldehyde radical cytimidine and 5- aldehyde radical uracil are distinguished.
A method of detection 5- aldehyde radical cytimidine mixes 5- aldehyde radical cytimidine and Wittig reagent in organic solvent
It closes, is then radiated with ultraviolet light, obtain 5- aldehyde radical cytosine derivative and then identified using fluorescence identification technique
With detection 5- aldehyde radical cytosine derivative.
The invention has the following advantages:
The present invention realizes the chemical derivatization of 5fC and 5fU first with Wittig reagent simultaneously, due to hydrophobicity acrylonitrile structure
Introducing be also improved with the separation resolution ratio of other bases, thus so that the aldehyde radical pyrimidine retention time after derivative extends
The LC-MS/MS of chemical derivatization auxiliary is analyzed advantageously.Further, after Wittig reagent alkylene, in conjunction with photocatalysis
" domino " reaction, realize the selective fluorescence identifying and quantitative detection of 5fC.Schiff is based on traditional amido derivative
The C=N double bond of alkali reaction building is compared, and C=C double bond of the present invention using phosphorus ylide based on Wittig reaction building is steady
It is much fixed.In addition, the Wittig reagent that the present invention screens resulting cyano substitution can be selectively targeted by three step successive reactions
5fC, this is also not available for the derivatives such as amido, indoles.Finally, the phosphorus ylide of currently preferred high reaction activity and
The quick cyclization strategy of light auxiliary effectively compensates for the long defect of active methylene group class compound reaction time consumption.The present invention is directed to 5-
The advantage that there is the Wittig derivatization reaction that aldehyde radical cytimidine provides mild condition, efficiently quick, raw material economics to be easy to get.
Detailed description of the invention
Fig. 1 is the process route chart of derived aldehydes of embodiment of the present invention yl pyrimidines.
Fig. 2 is HPLC track comparison diagram of the 5fC and 5fU before and after derivative with other classical bases or rare bases.
Fig. 3 is the excitation spectrum and emission spectrum of the derivative obtained cyclization product 5fC-CN-Close of the embodiment of the present invention.
Fig. 4 is trans-5fC-CN fluorescence intensity versus time curve figure under ultraviolet radiation.
Fig. 5 is the fluorescence selectivity test map of the derivative sample of 5fC of the embodiment of the present invention.
Fig. 6 is the standard curve that cyanogen methylene triphenyl phosphine carries out fluorescent quantitation to 5fC.
Fig. 7 is that fluorescence intensity of the derivative sample of 5fC of the embodiment of the present invention before and after gamma Rays changes with time
Curve graph.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.The person that is not specified actual conditions in embodiment, according to normal conditions or the item suggested of manufacturer
Part carries out.Reagents or instruments used without specified manufacturer is the conventional products that can be obtained by commercially available purchase.
For marking the Wittig reagent of aldehyde radical pyrimidine for cyanogen methylene triphenyl phosphine in following embodiment, structural formula is
The synthetic route of the embodiment of the present invention is as shown in Figure 1, aldehyde radical pyrimidine and Wittig reagent are mixed in organic solvent
It closes, is then radiated with ultraviolet light, aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical pyrimidine is 5- aldehyde radical cytimidine or 5- aldehyde
Base uracil.
Embodiment 1
The method of the present embodiment derived aldehydes yl pyrimidines, comprising:
Aldehyde radical pyrimidine and Wittig reagent are mixed in ethyl alcohol according to the ratio that molar ratio is 1:1, reacted at 15 DEG C
For 48h, then with wavelength be 200nm ultraviolet radiation for 24 hours, aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical pyrimidine is 5- aldehyde
Base cytimidine or 5- aldehyde radical uracil.
Embodiment 2
The method of the present embodiment derived aldehydes yl pyrimidines, comprising:
Aldehyde radical pyrimidine and Wittig reagent are mixed in N,N-dimethylformamide according to the ratio that molar ratio is 1:500
It closes, reaction is 5min, the ultraviolet radiation 1min for being then 400nm with wavelength at 80 DEG C, and aldehyde radical pyrimidine derivatives are made；
Wherein, aldehyde radical pyrimidine is 5- aldehyde radical cytimidine or 5- aldehyde radical uracil.
Embodiment 3
The method of the present embodiment derived aldehydes yl pyrimidines, comprising:
Aldehyde radical pyrimidine and Wittig reagent are mixed in glycol dimethyl ether according to the ratio that molar ratio is 1:20,50
Reaction is 6h, the ultraviolet radiation 12h for being then 253nm with wavelength at DEG C, and aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical is phonetic
Pyridine is 5- aldehyde radical cytimidine or 5- aldehyde radical uracil.
Embodiment 4
The method of the present embodiment derived aldehydes yl pyrimidines, comprising:
Aldehyde radical pyrimidine and Wittig reagent are mixed in glycol dimethyl ether according to the ratio that molar ratio is 1:250,
Reaction is 0.5h, the ultraviolet radiation 0.5h for being then 365nm with wavelength at 70 DEG C, and aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde
Yl pyrimidines are 5- aldehyde radical cytimidine or 5- aldehyde radical uracil.
Embodiment 5
The method of the present embodiment derived aldehydes yl pyrimidines, comprising:
Aldehyde radical pyrimidine and Wittig reagent are mixed in glycol dimethyl ether according to the ratio that molar ratio is 1:50,65
Reaction is 1h, the ultraviolet radiation 45min for being then 300nm with wavelength at DEG C, and aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical
Pyrimidine is 5- aldehyde radical cytimidine or 5- aldehyde radical uracil.
Embodiment 6
The method of the present embodiment derived aldehydes yl pyrimidines, comprising:
Aldehyde radical pyrimidine and Wittig reagent are mixed in glycol dimethyl ether according to the ratio that molar ratio is 1:40,65
Reaction is 40min, the ultraviolet radiation 30min for being then 310nm with wavelength at DEG C, and aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde
Yl pyrimidines are 5- aldehyde radical cytimidine or 5- aldehyde radical uracil.
Embodiment 7
The method of the present embodiment derived aldehydes yl pyrimidines, comprising:
Aldehyde radical pyrimidine and Wittig reagent are mixed in glycol dimethyl ether according to the ratio that molar ratio is 1:20,60
Reaction is 1h, the ultraviolet radiation 1.5h for being then 280nm with wavelength at DEG C, and aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical is phonetic
Pyridine is 5- aldehyde radical cytimidine or 5- aldehyde radical uracil.
Derive through the embodiment of the present invention and obtain aldehyde radical pyrimidine derivatives: 5- aldehyde radical cytosine derivative and 5- aldehyde radical urine
Pyrimidine derivatives can be used for detecting aldehyde radical cytimidine and aldehyde radical uracil.LC-MS/MS analytic approach can be used in the method for detection.
Deriving method based on the embodiment of the present invention can also detect 5- aldehyde radical cytimidine, to determine in DNA
5fC is horizontal.After reacting with Wittig reagent, product trans-5fC-CN changes 5- aldehyde radical cytimidine in ultraviolet radiation
For its heterogeneous structure: the outer 4-NH of the ring of cls-5fC-CN, cis-5fC-CN2Intramolecular condensation ring is carried out with-the CN being closer
Change reaction, obtains the final derivative that can emit fluorescence: 5fC-CN-Close.With this, then can be real by fluorescence labeling method
Now to the detection of 5fC and quantitative analysis.
HPLC Behavioral change research of test example 1 5fC and 5fU after cyanogen methylene triphenyl phosphine is derivative
Two 1.5mL centrifuge tubes are taken, 0.5mg 5fC and 0.5mg 5fU is separately added into, it is sub- to be then respectively adding 11.8mg cyanogen
In two centrifuge tubes, ultrasonic dissolution is simultaneously mixed for methyl triphenyl phosphine (20eq.) and 0.75mL methanol, and it is anti-to be placed in 60 DEG C of shaking tables
1h is answered, isometric water is then added and dilutes one times, carries out HPLC analysis.Meanwhile by unmodified classical base or rare
Base is made into the aqueous solution of 0.5mg/mL, including adenine (A), guanine (G), uracil (U), cytimidine (C), 5- hydroxyl first
Base uracil (5hmU), T, 5mC, 5hmC, 5fC and 5fU, and under equal conditions carry out HPLC analysis.
As shown in Fig. 2, the retention time of other bases concentrates on 4~6min, and mutual separation in addition to A
Resolution ratio is lower, and especially tri- kinds of bases of G, T, 5mC approach 5fC and 5fU very much, this is extremely for LC-MS/MS analysis
Unfavorable.And the aldehyde radical pyrimidine after Wittig reagent is derivative, retention time directly extend to 10min or more, hence it is evident that with other
Base differentiates, and can thus reduce them as much as possible and press down to the ion of 5fC, 5fU in LC-MS/MS analytic process
System.In addition, the derivative acrylonitrile structure introduced causes hydrophobicity to increase, it is meant that need the smaller mobile phase of polarity (namely
The organic solvent of greater proportion) it elutes, this is also beneficial to the Ionization Efficiency for improving target analytes.
The present invention can efficiently derive 5fC and 5fU, the nucleosides adduct of generation using Wittig reagent simultaneously as a result,
Retention time extends, and also greatly promotes with the separating degree of other bases, and 5fC and 5fU can be improved in LC-MS/MS detection in this
Sensitivity.
Fig. 3 is the excitation spectrum and emission spectrum of cyclization product 5fC-CN-Close.
The Intra-molecular condensation research of 2 light of test example auxiliary
The PBS aqueous solution (0.1mM) of 750 μ L trans-5fC-CN is placed in 1cm quartz colorimetric utensil, under stirring condition,
With the side of the LED light source radiation quartz colorimetric utensil of 280nm, take out at regular intervals 50 μ L be diluted in 3mL PBS survey it is glimmering
Light (λex=345nm), the fluorescent emission intensity at 410nm is recorded, and draw strength versus time change curve.
As shown in figure 4, fluorescence intensity increases rapidly with radiated time in the 1.5h for starting radiation, then tend to balance,
And keep fluorescence intensity to be basically unchanged within a very long time, illustrate that the photostability of cyclization product is preferable.It follows that
The ultraviolet radiation of 1.5h is enough that trans-5fC-CN is made to be fully converted to cyclization product.It is therefore, first derivative through Wittig reagent,
The ultraviolet radiation of 1.5h is undergone again, and the fluorescence spectrum that can directly scan sample carries out quantitative analysis.In addition the derivative of 1h is anti-
Between seasonable, 2.5h can be foreshortened to when reaction of the invention is total, compared with the prior art 10~for 24 hours, have great progress, save significantly
About time cost, improves detection efficiency.
3 cyanogen methylene triphenyl phosphine of test example studies the fluorescence selectivity of 5fC
By nucleosides (10mM is dissolved in DMSO, 1 μ L), cyanogen methylene triphenyl phosphine (100mM is dissolved in DMSO, 2 μ L) and 100 μ
L methanol is added in 1.5mL centrifuge tube, mixes, 60 DEG C of reaction 1h.After reaction, 900 μ L PBS are added, are transferred to after mixing
In 1cm quartz colorimetric utensil, under stirring condition, with the LED light source radiation 1.5h of 280nm, then directly scanned without additional purification
Fluorescence spectrum (λex=345nm).
As shown in figure 5, only the corresponding obtained derivative sample of 5fC has an apparent fluorescence enhancement at 410nm, A, G, C, T,
U, 5mC, 5hmC, 5hmU or even 5fU, after incubating simultaneously illumination altogether with Wittig reagent, fluorescence emission spectrum and probe groups weigh substantially
It is folded, illustrate that cyanogen methylene triphenyl phosphine has the fluorescence identifying ability of high degree of specificity to 5fC.
4 cyanogen methylene triphenyl phosphine 5fC caused by gamma Rays of test example mutation carries out quantitative fluorescence analysis
According to the literature (Madugundu, G.S.et al.Nucleic Acids Res.2014,42,7450), 5mC warp
Gamma Rays can sport 5fC.This test example is intended to detect gamma-rays to the mutation ability of 5mC.
Firstly, the aqueous solution (10mM) of 500 μ L 5mC is exposed to60Under Co gamma ray projector, and with 16.7Gy/min's
Rate radiates 90min.After radiation, freeze-drying continuously adds cyanogen methylene triphenyl phosphine and 100 μ L methanol, mixes, and 60 DEG C anti-
Answer 1h.After reaction, 900 μ L PBS are added, is transferred to after mixing in 1cm quartz colorimetric utensil, under stirring condition, uses 280nm
LED light source radiation 1.5h, fluorescence spectrum (λ is then directly scanned without additional purificationex=345nm), as shown in Fig. 7.Record
Fluorescence intensity at 410nm calculates the 5fC content after gamma Rays in sample further according to standard curve as shown in FIG. 6,
And then acquiring gamma-rays is (0.25 5fC/10 to the mutation ability of 5mC6Base)/Gy.
In conclusion using one kettle way to be completed at the same time 5- aldehyde radical born of the same parents phonetic for the Wittig reagent that is replaced using cyano of the present invention
The efficient derivative of pyridine (5fC) and 5- aldehyde radical uracil (5fU) obtains the nucleosides adduct comprising acrylonitrile structure, and with E formula structure
Based on type.Gained nucleosides adduct hydrophobicity of the invention increases, and when carrying out reversed-phase HPLC separation, retention time extends, while with
Other classical bases or the separating degree of rare bases also greatly improve, this is for improving 5fC and 5fU in LC-MS/MS analytic process
In detection sensitivity it is highly beneficial.The corresponding E formula nucleoside derivates of 5fC, acrylonitrile structure can be sent out under ultraviolet radiation
Raw cis-trans isomerization, so that the space length of further cyano and the outer 4- amino of ring, creates favorably for Intramolecular cycloaddition
Condition ultimately generates the novel nucleoside of transmitting hyperfluorescence；And 5fU cannot completely undergo above-mentioned mistake due to a lack of 4- amino outside ring
Journey, this is achieved the strategy of selective fluorescence detection 5fC.
The present invention screens the Wittig reagent that resulting cyano replaces and has been commercialized, while the derivatization reaction condition temperature
With efficiently quickly, effectively expanded Wittig reagent in the application in epigenetics field, be also the chemical labeling of aldehyde radical pyrimidine
Design with response type 5fC fluorescence probe provides new approaches.
Above to provided by the present invention using the method for the efficiently derivative 5- aldehyde radical cytimidine of Wittig reagent and its in LC-
Application in terms of MS/MS analysis and fluorogenic quantitative detection is described in detail.In order to more clearly illustrate the principle of the present invention
And technical solution, some embodiments are applied herein and relevant drawings aid in illustrating.It should be pointed out that above embodiments and phase
It closes attached drawing and is merely used to help understand method and central idea of the invention, for those of ordinary skill in the art,
Without departing from the principles of the invention, can be with several improvements and modifications are made to the present invention, these improvement and modification are also fallen
Enter in the protection scope of the claims in the present invention.
Claims (10)
1. a kind of method of derived aldehydes yl pyrimidines, which is characterized in that by Wittig reagent that aldehyde radical pyrimidine replaces with cyano in having
It mixes in solvent, is then radiated with ultraviolet light, aldehyde radical pyrimidine derivatives are made；Wherein, aldehyde radical pyrimidine is 5- aldehyde radical born of the same parents
Pyrimidine or 5- aldehyde radical uracil.
2. the method for derived aldehydes yl pyrimidines according to claim 1, which is characterized in that Wittig reagent has the following structure
Formula:
Wherein, R1For hydrogen, cyano, halogen, alkyl or containing O, N, halogen, P, S or Si alkyl.
3. the method for derived aldehydes yl pyrimidines according to claim 1, which is characterized in that 5- aldehyde radical cytosine derivative includes
Have the following structure two kinds of derivatives of formula:
Wherein, R is hydrogen, alkyl, ribosyl or deoxyribosyl, R1For hydrogen, cyano, halogen, alkyl or containing O, N, halogen, P, S
Or the alkyl of Si.
4. the method for derived aldehydes yl pyrimidines according to claim 1, which is characterized in that 5- aldehyde radical uracil derivative has
Following structural formula:
Wherein, R is hydrogen, alkyl, ribosyl or deoxyribosyl, R1For hydrogen, cyano, halogen, alkyl or containing O, N, halogen, P, S
Or the alkyl of Si.
5. the method for derived aldehydes yl pyrimidines according to claim 1, which is characterized in that the organic solvent include methanol,
Ethyl alcohol, isopropanol, ethylene glycol monomethyl ether, glycol dimethyl ether, acetonitrile, toluene, methylene chloride, N,N-dimethylformamide, N, N-
One of dimethyl acetamide, dioxane and dimethyl sulfoxide or multiple combinations.
6. the method for derived aldehydes yl pyrimidines according to claim 1-5, which is characterized in that aldehyde radical pyrimidine and
The molar ratio of Wittig reagent is 1:(1-500), reaction temperature is 15-80 DEG C, reaction time 5min-48h, the wave of ultraviolet light
A length of 200-400nm, radiated time 1min-24h.
7. aldehyde radical pyrimidine derivatives made from method described in any one of claims 1-6.
8. application of the aldehyde radical pyrimidine derivatives as claimed in claim 7 in detection 5- aldehyde radical cytimidine and 5- aldehyde radical uracil.
9. application according to claim 8 detects 5- aldehyde radical cytimidine using the LC-MS/MS technology of chemical derivatization auxiliary
With 5- aldehyde radical uracil；5- aldehyde radical cytimidine is identified and detected using fluorescence identification technique.
10. a kind of method for detecting 5- aldehyde radical cytimidine, which is characterized in that by 5- aldehyde radical cytimidine with Wittig reagent in organic
It mixes in solvent, is then radiated with ultraviolet light, 5- aldehyde radical cytosine derivative is obtained, then, using fluorescence identification technique
Identified and detected 5- aldehyde radical cytosine derivative.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910079972.7A CN109678802B (en) | 2019-01-28 | 2019-01-28 | Method for deriving aldehyde pyrimidine, method for detecting 5-aldehyde cytosine and application of aldehyde pyrimidine derivative |
| PCT/CN2019/117081 WO2020155742A1 (en) | 2019-01-28 | 2019-11-11 | Method for deriving aldehyde-based pyrimidine, method for detecting 5-aldehyde cytosine, and use of aldehyde-based pyrimidine derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910079972.7A CN109678802B (en) | 2019-01-28 | 2019-01-28 | Method for deriving aldehyde pyrimidine, method for detecting 5-aldehyde cytosine and application of aldehyde pyrimidine derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109678802A true CN109678802A (en) | 2019-04-26 |
| CN109678802B CN109678802B (en) | 2020-12-29 |
Family
ID=66194975
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910079972.7A Active CN109678802B (en) | 2019-01-28 | 2019-01-28 | Method for deriving aldehyde pyrimidine, method for detecting 5-aldehyde cytosine and application of aldehyde pyrimidine derivative |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN109678802B (en) |
| WO (1) | WO2020155742A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110174451A (en) * | 2019-06-11 | 2019-08-27 | 山东农业大学 | A method of 5fC is detected based on tungsten sulfide-black titanium dioxide hetero-junctions photoelectrochemical assay |
| WO2020155742A1 (en) * | 2019-01-28 | 2020-08-06 | 四川大学 | Method for deriving aldehyde-based pyrimidine, method for detecting 5-aldehyde cytosine, and use of aldehyde-based pyrimidine derivative |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022171606A2 (en) | 2021-02-09 | 2022-08-18 | F. Hoffmann-La Roche Ag | Methods for base-level detection of methylation in nucleic acids |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0235301A1 (en) * | 1985-09-09 | 1987-09-09 | Teijin Limited | Pyridopyrimidine nucleotide derivatives |
| JPH09263584A (en) * | 1996-03-29 | 1997-10-07 | Ube Ind Ltd | 7-amino-2,3-dihydro-2-oxo-pyrido(2,3-d)pyrimidine and its production |
| CN103242242A (en) * | 2012-02-08 | 2013-08-14 | 河南师范大学 | 5-electron-defect alkenyl substituted pyrimidine carbocyclic ring nucleoside and preparation method |
| WO2014151682A1 (en) * | 2013-03-14 | 2014-09-25 | Icahn School Of Medicine At Mount Sinai | Pyrimidine compounds as kinase inhibitors |
| CN104311618A (en) * | 2013-09-27 | 2015-01-28 | 北京大学 | 5-aldehyde cytosine specific chemical labeling method and application thereof to sequencing, detection, imaging and treatment |
| CN106957350A (en) * | 2017-02-28 | 2017-07-18 | 北京大学 | The labeling method of 5 aldehyde radical cytimidines and its application in the sequencing of single base resolution ratio |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3753938B2 (en) * | 2000-12-15 | 2006-03-08 | 独立行政法人科学技術振興機構 | Method for point mutation of base using photoligating nucleoside-containing DNA |
| JP4777373B2 (en) * | 2008-02-14 | 2011-09-21 | 独立行政法人科学技術振興機構 | Method for producing photoresponsive nucleic acid |
| CN107573289B (en) * | 2017-09-13 | 2019-07-09 | 武汉大学 | The compound and labeling method of a kind of specific chemical label 5- aldehyde radical uracil and application |
| CN109678802B (en) * | 2019-01-28 | 2020-12-29 | 四川大学 | Method for deriving aldehyde pyrimidine, method for detecting 5-aldehyde cytosine and application of aldehyde pyrimidine derivative |
-
2019
- 2019-01-28 CN CN201910079972.7A patent/CN109678802B/en active Active
- 2019-11-11 WO PCT/CN2019/117081 patent/WO2020155742A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0235301A1 (en) * | 1985-09-09 | 1987-09-09 | Teijin Limited | Pyridopyrimidine nucleotide derivatives |
| JPH09263584A (en) * | 1996-03-29 | 1997-10-07 | Ube Ind Ltd | 7-amino-2,3-dihydro-2-oxo-pyrido(2,3-d)pyrimidine and its production |
| CN103242242A (en) * | 2012-02-08 | 2013-08-14 | 河南师范大学 | 5-electron-defect alkenyl substituted pyrimidine carbocyclic ring nucleoside and preparation method |
| WO2014151682A1 (en) * | 2013-03-14 | 2014-09-25 | Icahn School Of Medicine At Mount Sinai | Pyrimidine compounds as kinase inhibitors |
| CN104311618A (en) * | 2013-09-27 | 2015-01-28 | 北京大学 | 5-aldehyde cytosine specific chemical labeling method and application thereof to sequencing, detection, imaging and treatment |
| CN106957350A (en) * | 2017-02-28 | 2017-07-18 | 北京大学 | The labeling method of 5 aldehyde radical cytimidines and its application in the sequencing of single base resolution ratio |
Non-Patent Citations (2)
| Title |
|---|
| PAUL F. TORRENCE等: "Structurally diverse 5-substituted pyrimidine nucleosides as inhibitors of Leishmania donovani promastigotes in vitro", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| XUE SEN FAN,等: "Preparation of nucleoside–pyridine hybrids and pyridine attached acylureas from an unexpected uracil ring-opening and pyridine ring-forming sequence", 《CHINESE CHEMICAL LETTERS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020155742A1 (en) * | 2019-01-28 | 2020-08-06 | 四川大学 | Method for deriving aldehyde-based pyrimidine, method for detecting 5-aldehyde cytosine, and use of aldehyde-based pyrimidine derivative |
| CN110174451A (en) * | 2019-06-11 | 2019-08-27 | 山东农业大学 | A method of 5fC is detected based on tungsten sulfide-black titanium dioxide hetero-junctions photoelectrochemical assay |
| CN110174451B (en) * | 2019-06-11 | 2020-06-02 | 山东农业大学 | Method for detecting 5fC based on photoelectrochemical analysis of tungsten sulfide-black titanium dioxide heterojunction |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109678802B (en) | 2020-12-29 |
| WO2020155742A1 (en) | 2020-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109678802A (en) | The application of the method for derived aldehydes yl pyrimidines, the method for detecting 5- aldehyde radical cytimidine and aldehyde radical pyrimidine derivatives | |
| EP0185547B1 (en) | Polynucleotide hybridization assays employing catalyzed luminescence | |
| JP2619866B2 (en) | Competitive homogeneous test | |
| Kenten et al. | Rapid electrochemiluminescence assays of polymerase chain reaction products | |
| EP0131830B1 (en) | Labelled nucleic acid probes and adducts for their preparation | |
| US7855054B2 (en) | Multiplexed analyses of test samples | |
| US4950588A (en) | Prolonged enhanced chemiluminescence | |
| CN1612860A (en) | Compositions, methods and kits pertaining to luminescent compounds | |
| US20110136099A1 (en) | Multiplexed Analyses of Test Samples | |
| JPH02135100A (en) | Detecting method for specific nucleic acid configuration | |
| JP2006518587A (en) | Multiple analysis platform using molecular tags | |
| JPS62240864A (en) | Detection of material to be analyzed by energy transmission | |
| JP2002537858A (en) | Methods for direct sequencing of nucleic acids | |
| RU2007115421A (en) | GENOMIC ANALYSIS | |
| Hiratsuka | New fluorescent analogs of cAMP and cGMP available as substrates for cyclic nucleotide phosphodiesterase. | |
| JP2553519B2 (en) | Optical emission method | |
| JPH07270319A (en) | Method for measuring substance containing adenyl group using heteropoly acid | |
| CN1902490B (en) | Method of detecting biological molecules, and labeling dye and labeling kit used for the same | |
| Xu et al. | A genetically encoded fluorescent biosensor for detecting nitroreductase activity in living cancer cells | |
| CN110357865A (en) | A kind of near infrared fluorescent probe and its synthetic method and application for detecting hNQO1 enzyme | |
| Wang et al. | Visible‐Light Facilitated Fluorescence “Switch‐On” Labelling of 5‐Formylpyrimidine RNA | |
| Hashimoto et al. | Synthesis of tag introducible (3-trifluoromethyl) phenyldiazirine based photoreactive phenylalanine | |
| EP1390529B1 (en) | Method of analysing dna methylation using fluorescence polarisation | |
| CN108424402B (en) | Gamma-glutamyl transpeptidase biological probe and preparation method and application thereof | |
| CN115372331A (en) | Method for imaging magnesium ions in living cells based on chemically modified deoxyribozymes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |