CN104844674B - Novel polymeric zymolyte: fluorescence can produce polyphosphoric acids end mark nucleotide and its application - Google Patents
Novel polymeric zymolyte: fluorescence can produce polyphosphoric acids end mark nucleotide and its application Download PDFInfo
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Abstract
The present invention relates to the nucleotide of a kind of 5 '-terminal phosphate fluorescent markers, provide molecular structure and synthetic method.Such molecule can be identified preferably by archaeal dna polymerase, and fast transition of the fluorescence signal from closing to open state is realized under alkaline phosphatase assistance after being identified and correctly being combined by polymerase, to can be applied in the fields such as determining nucleic acid sequence as polymerase substrate.
Description
Technical field
The present invention relates to the nucleotide of a kind of 5 '-terminal phosphate fluorescent markers, and its in nucleic acid base sequence measuring method
In application.
Background technique
Measure etc. in applying, is directed to when DNA replication dna amplification that archaeal dna polymerase participates in, nucleotide sequence are in synthesis
The labelling technique of nucleic acid molecule, in recent years, the nucleotide of fluorescent marker are sufficiently applied in DNA high throughput sequencing technologies,
Wherein, labelling technique first is that different fluorophors is connected to the specific position of nucleotide base by the way that long-chain can be cut off
On, complete to identify and record removing the labelling groups by additional chemical means after fluorescence signal in DNA, and then be next
The nucleotide importing of wheel is prepared.The technology for commercial undertaking's mature of representative and occupies master by illumina company
Want market using status (US7057026；7566537).But it is this to carry out the mode of fluorescent marker still on nucleotide base
There are certain limitations --- and ' the molecule scar ' that each chemical ablation mark molecule is retained all causes newly synthesized DNA molecular
The inevitable morphologic change after to a certain degree, prevents effective combination of polymerase molecule, so that influencing sequencing reads length.
Thus, develop new nucleotide marker means, and then developing new DNA high throughput sequencing technologies is all existing market demand and section
Learn the hot spot that research is chased.Relative to the technology of the label carried out on nucleotide base, to 5 '-terminal phosphates of nucleotide into
The technology of line flag is also applied in biochemistry and related biotechnology research very early.By on terminal phosphate position
The marker for introducing different function can help people to understand the physiology course that various nucleotide participate in.And in nucleotide end
Fluorescence marker groups are introduced on phosphoric acid, nucleic acid polymerase is after identifying and correctly introducing nucleotide by automatic excision remainder
The fluorescent marker that phosphate group and its end have is conducive to polymerase to guarantee the natural structure of newly synthesized DNA
The rapidity and accuracy of base are continually introduced into long section.These potential features drive people by 5 '-terminal phosphate marks
Note nucleotide is applied in the practical application of such as high-throughput DNA sequencing, single nucleotide polymorphism screening etc..However, protecting
Demonstrate,prove that these are applied it is critical that this is labeled nucleotide after the 5 ' of nucleotide-phosphate terminal introducing fluorescence marker groups
It can smoothly be identified by archaeal dna polymerase.The research of Sood in 2005 et al. verified (Sood, A. etc.
J.Am.Chem.Soc.2005,127,2394), fluorescent tag molecule is connected directly between triphosphate deoxyribose nucleotide
It (dNTP) is not ideal mark mode on terminal phosphate, because on the one hand the fluorophor on label occupies phosphoric acid chain
On a negative electrical charge, reduce the binding ability of phosphoric acid Yu archaeal dna polymerase catalytic center metal ion；On the other hand, fluorescence
Steric effect caused by group is not easy to access nucleotide and enters the active catalytic center of archaeal dna polymerase, to subtract
Archaeal dna polymerase is delayed to the correct identification of labeled nucleic acid molecule and reaction rate.Thus, research synthesis has longer phosphoric acid
The terminal phosphate fluorescence-labeled nucleotides technology of chain is the target of people's research.
In the method for research long-chain terminal phosphate fluorescent marker, there are two different directions, one of direction is to make
It is remained unchanged for various stage photoluminescent properties of the fluorescent molecule before and after archaeal dna polymerase effect of label, thus length can be used
Chemical inertness link molecule by fluorescent marker ' anchoring ' on the terminal phosphate of nucleotide, this technology is with Stephen
The terminal phosphate labelling technique of W.Turner et al. development is representative (Nucleosides, Nucleotides&Nucleic
Acids, 2008,27,1072-1083)；Although mark molecule may be implemented far from polymerase catalysed site in this technology, by
In polymerase cannot be embodied by nucleotide and be integrated to the change in fluorescence on new synthetic DNA before and after this chemical process, thus can only
Applied to specific occasion.Second of technology, that is, the labelling technique that the present invention is discussed, the fluorescent molecule marked is with altogether
Valence link is directly in conjunction with phosphoric acid molecules, and ' closing ' is presented in the property of fluorescent molecule under ' in conjunction with ' and ' dissociation ' two states
' unlatching ' two different conditions, in other words its photoluminescent property when fluorescent tag molecule is in bonding state with terminal phosphate
In cancellation state, cannot be excited by the exciting light of specific wavelength；And immediately once discharged by polymerase and phosphatase
Return to can excited state, to provide fluorescence signal.This specific character develop for us generated based on fluorescence signal it is all
Such as DNA base sequencing technology application provides premise.Then in terms of polymeric enzyme reaction dynamics, nucleotide 5 '-phosphorus
The introducing of sour Terminal fluorescent group increases the steric effect that nucleotide enters polymerase catalysed center to a certain extent, thus
The kinetics property of polymerase is affected, so that reaction is fast compared with nature nucleotide (unmodified nucleotide)
It spends slack-off.In addition be labeled nucleotide background fluorescence signal, i.e., fluorophor be released before fluorescent quenching degree, and
Fluorescence intensity (product of extinction coefficient and quantum yield) after fluorophor release, photoluminescent property (absorption/launch wavelength),
All directly affected by selected fluorophor.
These there are aiming at the problem that, the present invention solves the fluorescence of labeled nucleotide using novel fluorescent dye
The fluorescence intensity and photoluminescent property problem of fluorophor after background and release.On the other hand, the present invention is in different length poly
New method is used in terms of the synthesis of phosphoric acid chain, is asked with solving the steric hindrance between mark molecule and polymerase catalytic activity center
Topic.In terms of the synthesis of phosphoric acid chain, Shiv Kumar etc. has made beneficial exploration (Nucleosides, Nucleotides&
Nucleic Acids, 2005,24,401；), but still there are synthetic method not directly, to be difficult to synthesize long phosphoric acid chain (such as more
In five phosphoric acid).Thus, the invention discloses the end mark nucleotide point that new synthesis has different length polyphosphoric acids chain
The method of son, and it is high by using absorptivity and quantum yield, and quenching effects are good, and excitation wavelength is suitble to nucleotide sequencing
The fluorescent molecule of application realizes the practical applications of this kind of terminal phosphate labeled nucleotides that there is fluorescence can produce property.
Summary of the invention
The present invention relates to the nucleotide of a kind of 5 '-terminal phosphate fluorescent markers, provide molecular structure and synthetic method.It should
Class molecule can be identified preferably by archaeal dna polymerase, and identified and correctly combined later in alkaline phosphatase by polymerase
Enzyme assists fast transition of the lower realization fluorescence signal from closing to open state, to can be applied to as polymerase substrate
In the fields such as determining nucleic acid sequence.The patent CN that the synthetic method of specific fluorescent molecule is applied in applicant in this case
It is described in more detail in 201510155218.9.
The present invention provides a kind of nucleotide structure of terminal phosphate fluorescent marker, it is characterised in that: has following general formula (1)
Shown structure,
Wherein Ra, RbIndependent it can be selected from-H ,-OH；N is the integer more than or equal to 1, and less than or equal to 5；
Wherein, the fluorophor in general formula (1) has following general formula (2) described structure,
Wherein, R in general formula (2)1, R5It is independent to be selected from-H, fluorine, chlorine, bromine, aryl, substituted aryl, C1-C6 alkyl, take
C1-C6 alkyl, C1-C6 alkoxy, the substituted C1-C6 alkoxy in generation；
R2-4, R6-8, R11-13Independent it can be selected from-H, fluorine, chlorine, bromine, aryl, substituted aryl, heteroaryl ,-CO2H、-
CO2R、-SO3H、-SO3R、-CH2CO2H、-CH2CO2R、-CH2SO3H、-CH2SO3R、-CH2NH2、-CH2NHR、-NO2, C1-C6 alkane
Base, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C6 alkoxy aryl, substituted C1-C6
Alkoxy aryl, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzene formyl
Base, wherein R is selected from C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C6 alcoxyl
Base aryl, substituted C1-C6 alkoxy aryl, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzene
Formoxyl, substituted benzoyl；
R9、R10Independent it can be selected from C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6
Alkoxy, substituted C1-C6 alkoxy aryl, benzyl, replaces benzyl at C1-C6 alkoxy aryl.
The present invention provide it is a kind of synthesize terminal phosphate fluorescent marker nucleotide structure method, it is characterised in that including with
Lower step:
(a) trimetaphosphate is reacted with the hydroxyl of fluorescent dye groups, among three metaphosphoric acids for generating fluorophor modification
Body；
(b) nucleosides with 5 '-phosphoric acid chains and three metaphosphoric acid intermediate reactions of fluorophor modification are selected, production is converted to
Object 5 '-polyphosphoric acids Terminal fluorescent labels nucleotide；
Wherein, the nucleotide structure of the terminal phosphate fluorescent marker has structure shown in following general formula (1):
Wherein Ra, RbIndependent it can be selected from-H ,-OH；N is the integer more than or equal to 1, and less than or equal to 5.The present invention
The method of the nucleotide structure of the synthesis terminal phosphate fluorescent marker of offer, for fluorescent molecule there is no special requirement,
Such as the oxa anthracenes containing hydroxyl, cumarin, resorufin class fluorescent molecule.
Preferred embodiment in accordance with the present invention, structure described in the fluorophor preferred formula (2).
Another preferred embodiment according to the present invention, n value are 3,4 or 5.
Wherein, in general formula (1) base can selected from adenine (A), guanine (G), cytimidine (C), thymidine (T),
Uracil (U), the nucleoside base or non-natural nucleoside base modified.
The step of preferred embodiment in accordance with the present invention, the nucleotide structure method of synthesis terminal phosphate fluorescent marker
(a) in, trimetaphosphate is activated in the presence of activating reagent, then is reacted with the hydroxyl of fluorescent dye groups, and intermediate is generated
Three metaphosphoric acids of fluorophor modification.Activating reagent can be acyl chlorides, sulfonic acid chloride, chlorination sulfone, thionyl chloride, the such as thionyl chloride
Preferred embodiment in accordance with the present invention, synthesize terminal phosphate fluorescent marker nucleotide structure method in, institute
Stating nucleotide structure is that 2 ' -5 '-monophosphates of deoxyribonucleoside (dNMP), 2 ' -5 '-diphosphonic acid of deoxyribonucleoside (dNDP) or 2 '-are de-
5 '-triphosphoric acid of oxygen nucleosides (dNTP).
Another preferred embodiment according to the present invention, the corresponding fluorescent molecule of the fluorophor be with flowering structure,
Preferred embodiment in accordance with the present invention, it is first in the nucleotide structure method for synthesizing terminal phosphate fluorescent marker
First by fluorophor first with activation trimetaphosphate react, generation three metaphosphoric acid compound intermediate of fluorophor, then with core
Glycosides 5 '-monophosphate or the reaction of nucleosides 5 '-diphosphonic acid or nucleosides 5 '-triphosphoric acid or 5 '-polyphosphoric acid.
Preferred embodiment in accordance with the present invention, the method are cooking-pot type reaction, and centre does not need to isolate and purify institute
State intermediate.The method does not include the protection and/or deprotection of the amino and/or hydroxyl of the nucleosides.
The nucleotide structure of terminal phosphate fluorescent marker provided by the present invention can be used as DNA or RNA polymerase bottom
Object is identified and be incorporated into DNA or RNA chain by DNA or RNA polymerase, and releases the polyphosphoric acids of fluorophor label
Molecule.The substrate that the nucleotide structure of the terminal phosphate fluorescent marker can be used as alkaline phosphatase continues to decompose, until
Whole phosphate groups are detached from from fluorescent molecule.
The nucleotide structure of the terminal phosphate fluorescent marker closes before being decomposed and releasing fluorophor for fluorescence
Closed state does not issue fluorescence signal significantly when the light that is excited irradiates.The nucleotide structure of the terminal phosphate fluorescent marker is gathered
Synthase and alkaline phosphatase release fluorophor after decomposing, and significantly issue fluorescence signal in exciting light irradiation.The fluorescence
The excitation wavelength range of group is 530-590nm, and wavelength of transmitted light range is 550-650nm.
Preferred embodiment according to the present invention, the nucleotide structure of formula (1) the terminal phosphate fluorescent marker are 5 '-ends
Six phosphate of end-ζ-phosphoric acid fluorescent marker nucleotide, 5 '-end-η-phosphoric acid fluorescent marker nucleotide, seven phosphate or
5 '-end-θ-phosphoric acid fluorescent marker, eight phosphate of nucleotide.
The present invention also provides the nucleic acid molecule and its synthetic method of a kind of terminal phosphate fluorescent marker, structure is fluorescence
Group connects 5-8 phosphoric acid molecules, is then consecutively connected to ribose and base；The wherein nucleic acid molecule of the fluorescent marker
It is to be reacted by trimetaphosphate with the hydroxyl of fluorescent dye groups, then reacts again with the nucleosides with 5 '-phosphoric acid chains
It obtains；The nucleic acid molecule of the terminal phosphate fluorescent marker can identify by archaeal dna polymerase, and reaction bonded it
Fluorescence signal may be implemented under alkaline phosphatase assistance afterwards to be changed from closing to open state.
One of the advantages of the present invention is that fluorescence marker groups are only in 5 '-poly phosphorus in invented nucleic acid molecule structure
Sour terminal position, thus as archaeal dna polymerase substrate for DNA synthesis etc. in related applications when do not generate molecule scar, so
Newly generated DNA molecular structure is not influenced.The two of advantage are that fluorescence-labeled nucleotides molecule provided by the invention polymerize in DNA
It is fluorescence closed state before enzyme association reaction, is changed into fluorescence open state with the hydrolysis of phosphatase after reacting.Advantage
Three for the present invention provides a kind of reaction method for easily preparing long-chain fluorescent marker polyphosphoric acids nucleotide, this method benefits
It is one of initial feed with the sodium trimetaphosphate that commercialization is easy to get, first by it in conjunction with fluorescent molecule, is conveniently formed
The fluorescent marker of the state of activation is cyclized phosphate, prepares target molecule, polyphosphoric acid chain length then at the polyphosphoric acid nucleosides reaction being easy to get
Longer advantage.The reaction method provided by the invention for preparing fluorescent marker polyphosphoric acids nucleotide, than passing through nucleotide cyclization
Reaction prepares the method for fluorescent marker polyphosphoric acids nucleotide, and not only method is simple, also avoids the generation of by-product after cyclization,
Preparation efficiency is improved, so that product yield significantly improves, there is the longer advantage of polyphosphoric acid chain length.
Detailed description of the invention
Fig. 1 .a) PO-dA6P itself absorption spectrum (shown in half dotted line)；B) phosphatase (CIP) does not divide PO-dA6P
Solution reaction (shown in dotted line)；C) phosphodiesterase (PDE) disconnects phosphoric acid chain, and phosphatase hydrolyzes rapidly and releases fluorescent base
Group, i.e. PO-dA6P+CIP+PDE (shown in solid).
Fig. 2 .a) PO-dA6P autofluorescent background (fluorescent quenching state, shown in dotted line)；B) it is passed through in the presence of DNA profiling
Polymerase (BST) is crossed to combine and fluorescence intensity (fluorescence open state, the solid line institute after phosphatase (CIP) hydrolysis removal phosphoric acid
Show).
Fig. 3 .PO-dA4P and PO-dA6P prolongs single base template under polymerase (BST) and phosphatase (CIP) effect
Stretch reaction rate comparison diagram.
Specific embodiment
Before open description the compound of the present invention, compound, reaction system, device, system/or method, it should manage
Solve specific synthetic method, the article used, device that these are mentioned in specific embodiment, special reagent (unless in addition providing) etc.
It is only for further explanation of the invention, used term is also used only for description particular form, does not constitute the present invention
Limiting factor.
Embodiment 1:
Providing a kind of fluorescein analog --- the fluorescein or anthracene class fluorescein, the substance that carbon bridge replaces have
The characteristics such as long wavelength, the fluorescence property of can produce, derivatization be simple and easy, extinction coefficient and quantum yield height, it is logical with having structure
Formula (1):
It is a kind of using anthracene structure as the fluorescent dye of parent, it is characterised in that: there is the structure as shown in general formula (1), wherein R0
It can be selected from-H, phosphate, substituted phosphate；R1, R5It is independent to be selected from-H, fluorine, chlorine, aryl, substituted aryl, C1-C6 alkane
Base, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy；R2-4, R6-8, R11-13Independent can be selected from-
H, fluorine, chlorine, bromine, aryl, substituted aryl, heteroaryl ,-CO2H、-CO2R、-SO3H、-SO3R、-CH2CO2H、-CH2CO2R、-
CH2SO3H、-CH2SO3R、-CH2NH2、-CH2NHR、-NO2, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, replace
C1-C6 alkoxy, C1-C6 alkoxy aryl, substituted C1-C6 alkoxy aryl, phenyl, substituted-phenyl, xenyl, substitution
Xenyl, benzyl, substituted benzyl, benzoyl, substituted benzoyl, wherein R is selected from C1-C6 alkyl, substituted C1-C6 alkane
Base, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C6 alkoxy aryl, substituted C1-C6 alkoxy aryl, phenyl,
Substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzoyl；R9、R10It can be independent
Be selected from C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C6 alkoxy aromatic
Base, substituted C1-C6 alkoxy aryl, benzyl, substituted benzyl.
The synthetic method of compound is as follows:
(1) by the halogenated m-hydroxybenzaldehyde of neighbour with hydroxy-protective group or the congener and band with other substituent groups
There is hydroxy phenyl metal reagent between hydroxy-protective group or reacted with hydroxy phenyl metal reagent between substituent group, is added
At product, that is, benzhydrol derivative.
(2) benzophenone derivates are converted through peroxidization by above-mentioned benzhydrol derivative.
(3) above-mentioned benzophenone derivates are reacted with metal reagent, obtains the diphenylmethane derivatives of dialkyl group substitution.
(4) above-mentioned dialkyl group diphenylmethane derivatives are passed through and is reacted at low temperature with metal reagent, generate metal aryl
Compound.
(5) above-mentioned aryle is reacted with benzaldehyde derivative, generates addition product, that is, benzyl alcohol derivative.
(6) above-mentioned benzyl alcohol derivative is generated into methanone derivatives through peroxidating
(7) above-mentioned methanone derivatives are obtained bisphenol compound, passes through dehydration by removing phenolic hydroxyl protecting group group
Ring closure reaction obtains target fluorescent object.
Embodiment 2.
According to compound described in embodiment 1, it is specifically as follows:
Embodiment 3.
According to compound as described in example 2, synthetic method specifically be can be such that
Reagent and reaction condition: i) THF, -40 DEG C；Ii) DCM, PCC, diatomite；iii)ZnMe2, TiCl4, DCM-40 DEG C；
Iv) t-Buli, THF；O-tolualdehyde；V) DCM, PCC；vi)BBr3；MeSO3H
1) synthesizes (the bromo- 5- methoxyphenyl of 2-) -3- methoxy benzyl alcohol 1a
In the dry round-bottomed bottle of the 250ml equipped with constant pressure funnel, the 80ml of m-methoxyphenyl magnesium chloride is done
Dry tetrahydrofuran solution is cooled to -40 DEG C, under protection of argon gas by the dry tetrahydro of the 20ml of 3- methoxyl group o-bromobenzaldehye (10.8g)
Tetrahydrofuran solution is added dropwise in reaction flask by constant pressure funnel, is kept for -40 DEG C stirring 2-6 hours, is monitored by TLC anti-
It answers, stops reaction after the disappearance of raw material 3- methoxyl group o-bromobenzaldehye, 20ml saturated aqueous ammonium chloride quenching reaction is added.Instead
It answers mixture that 100ml methylene chloride is added in two portions after Rotary Evaporators remove most tetrahydrofurans, is closed after liquid separation
And organic phase, organic phase pass through saturated common salt water washing, Na2SO4Silica gel column chromatography, expansion are used after dry, Rotary Evaporators concentration
Agent petrol ether/ethyl acetate~10/1 collects the product point of Rf~0.3, obtains 15g colourless liquid, yield 93%.
2) synthesizes (the bromo- 5- methoxyphenyl of 2-) -3- methoxy benzophenone 1b
(the bromo- 5- methoxyphenyl of 2-) -3- methoxy benzyl alcohol 1a (6.5g) is taken to be dissolved in 80ml methylene chloride, quickly stir
It mixes down and adds 10g pyridinium chloro-chromate (PCC), react 2-5h at room temperature, TLC monitors the reaction after raw material disappears substantially
Stop, reaction solution is filtered by diatomite drainage short column, filtrate is collected after eluent methylene chloride, through silicon after Rotary Evaporators concentration
The product point of Rf~0.6 is collected in rubber column gel column column chromatography, solvent petrol ether/ethyl acetate~10/1, and yellowish solid is obtained after concentration
6.1g, yield 95%.
3) synthesizes the bromo- 4- methoxyl group -2- of 1- (2- (3- methoxyphenyl isopropyl)) benzene 1c
70ml methylene chloride is added in dry 250ml round-bottomed bottle, is cooled to -40 DEG C, TiCl is added4(9ml), under stirring
It is slowly added dropwise to zinc methide solution (the toluene solution 80ml of 1M), is stirred 15 minutes under the conditions of -40 DEG C under argon gas protection.It takes
(the bromo- 5- methoxyphenyl of 2-) -3- methoxy benzophenone 1b (6.1g) is dissolved in 30ml methylene chloride, this solution is added dropwise to
It states in solution, is kept for -40 DEG C and be stirred to react 3 hours, be to slowly warm up to 0 DEG C later and be further continued for reaction 5~10 hours, TLC monitoring
The reaction is until raw material stops after disappearing substantially.Quenching reaction in trash ice, mixture warp will be poured under brown reaction solution stirring
Methylene chloride extraction, Na2SO4It is dry, use silica gel column chromatography after Rotary Evaporators concentration, solvent petrol ether/ethyl acetate~
15/1, the product point of Rf~0.6 is collected, 5.5g colourless liquid, yield 87% are concentrated to give.
4) synthesizes (4- methoxyl group -2- (2- (3- methoxyphenyl) isopropyl) phenyl) (2- tolyl) methanol 1d
Compound 1c (3g) is added in dry 100ml round-bottomed bottle, adds 30ml anhydrous tetrahydro furan and is dissolved,
It is cooled to -78 DEG C under argon gas protection, the hexane solution (9~10mmol) of tert-butyl lithium is added dropwise under stirring, is protected after being added dropwise
It holds temperature to react 1 hour within the scope of -78 DEG C~-60 DEG C, then o-tolualdehyde (11.5mmol) is dissolved in 10ml dry four
Hydrogen furans is slowly added to syringe into the reaction solution of -78 DEG C of compound 1c, it is enabled slowly freely to heat up after being added dropwise
Reaction 2~6 hours.10ml saturated ammonium chloride solution quenching reaction is added.Most tetrahydrofurans are removed through Rotary Evaporators
After add 100ml methylene chloride extraction three times, organic phase obtains crude Compound 1d, is directly used in down through drying after concentration
Single step reaction.
5) synthesizes (4- hydroxyl -2- (2- (3- hydroxy phenyl) isopropyl) phenyl) (2- tolyl) ketone 1e
Previous step compound 1d is dissolved in 40ml methylene chloride, quickly adds 4g pyridinium chloro-chromate under stirring
(PCC), 2-4h is reacted at room temperature, and TLC monitors the reaction until raw material stops after disappearing substantially, and reaction solution passes through diatomite drainage
Short column filters, and filtrate is collected after eluent methylene chloride, through silicagel column column chromatographic purifying, solvent petroleum after Rotary Evaporators concentration
Ether/ethyl acetate~7/1 collects the product point of Rf~0.5, and yellowish solid 2.9g is obtained after concentration.
6) synthesising target compound 1 " Beijing orange " (Peking Orange), is labeled as PO.
It takes compound 1e (2.9g) to be dissolved in 40ml dry methylene chloride, Boron tribromide (2-5eq) is added dropwise under ice-water bath is cooling,
It is added dropwise that the reaction was continued 2~5 hours.20ml ice water is carefully added into after completion of the reaction to be quenched, mixture continues stirring 30 minutes,
With saturation NaHCO3Aqueous solution adjusts PH to~7, and the extraction of 150ml methylene chloride is added, and organic phase is washed through washing, saturated common salt
It washs, anhydrous Na2SO4It after drying, is concentrated through Rotary Evaporators, using vacuum oil pump decompressing and extracting, obtains yellowish grease.
5ml methane sulfonic acid is added into the grease, 80~100 DEG C are heated under stirring, keeps stopping for heating lower reaction 1 hour.It will
It is poured into trash ice under reactant stirring, the solid of precipitation is collected by filtration, and using washing, vacuum drying obtains target production
1 crude product of object about 2.2g.Crude product passes through silica gel column chromatography separating-purifying, obtains 1.8g Orange red solid " Beijing orange " (Peking
Orange), yield 72%.
On this basis, the compound for obtaining the general formula (1) of other substituent groups belongs to the routine techniques hand of chemical field
Section.
Embodiment 4.
Synthesize 2 '-deoxyribonucleoside, six phosphate of 5 '-ζ-terminal phosphate fluorescent marker
1) three metaphosphate reactive intermediate of synthetic dyestuffs
Sodium trimetaphosphate (355mg, 1.16mmol) is taken to be dissolved in appropriate amount of deionized water, this solution passes through ion exchange column
(BioRad AG-50W-XB cation exchange resin) processing is replaced into three metaphosphoric acid tributyl amine salt, by the aqueous solution of this amine salt
It is freeze-dried, then 12h is drained through oil pump vacuum, obtain three dry metaphosphoric acid tributyl amine salt solids.Under argon atmosphere,
This solid is put into dry 25mL round bottom reaction flask, the 10mL acetonitrile solution of dried over anhydrous is added into this reaction flask, it will
This solution is cooling under agitation (10~-10 DEG C cooling, to select 0 DEG C here), be added dropwise into solution thionyl chloride (or other
Activating reagent) (109uL) and DMAP (to dimethylamino naphthyridine) (8mg), 430mg tri-butylamine is then added.Then this is molten
Liquid stirs 1~30 minute at 0 DEG C, is then added to fluorescent marker dyes ' Beijing orange ' 1 (164mg, 0.5mmol) above-mentioned anti-
Answer in liquid, which is stirred to react 1~5 hour under conditions of 0 DEG C to room temperature, until HPLC detect it is most glimmering
The disappearance of photoinitiator dye raw material can stop reacting, this solution includes midbody compound 2.The solution can be reserved in -20 DEG C of refrigerators
For use.
2) 2 '-deoxyribonucleoside, six phosphate of 5 '-ζ-terminal phosphate fluorescent marker is synthesized
Take 2 '--5 '-triphosphoric acid sodium salts (50umol) of desoxyadenossine to be dissolved in 1ml deionized water solution, by this solution pass through from
Sub- exchange column (BioRad AG-50W-XB cation exchange resin) processing, is converted to 2 '-desoxyadenossine three fourths of -5 '-triphosphoric acid
The aqueous solution of amine salt, this aqueous solution pass through low-temperature rotary evaporation under high vacuum and remove most aqueous solutions, and remaining solution adds
Enter the DMF of 1mL dried over anhydrous, low-temperature rotary evaporation of solvent, this process are repeated two more times under a high vacuum again, then will
This 2 '--5 '-triphosphoric acid tri-n-butylamine salt of desoxyadenossine for removing water is dissolved in the dried over anhydrous DMF of 2mL, at room temperature with stirring,
The acetonitrile solution 2mL of three metaphosphoric acid intermediates 2 of fluorescent marker obtained in step 1) is extracted and is added to by syringe
In this reaction solution, anhydrous MgBr is then added2.2Et2Reaction is stirred at room temperature in the DMF solution of O (0.4umol), this mixture
8-24 hours, reaction process used HPLC tracing detection, is not further added by product peak, can stop reacting.This reaction solution is existed
Low-temperature rotary evaporation removes the DMF solvent of the overwhelming majority under high vacuum, then dissolves the residue in the vinegar triethylenetetraminehexaacetic acid of the 100mM of 3mL
Amine buffer (PH 7.6), the inverted separation chromatography separating-purifying of this solution, using pressing preparative chromatograph, AQ C- in Agela
18-60g prepares post separation.Gradient elution: mobile phase A is 50mM acetic acid triethylamine buffer solution, PH 7.5；Mobile phase B is chromatography
Pure acetonitrile, elution program are as follows: 0-15min increases to 20%B by A, and 15-35min B component increases to 40%, flow velocity 15mL/
Min is collected and is contained target component fraction, is concentrated under reduced pressure, and through efficient liquid phase chromatographic analysis purity about 90%, fine is isolated and purified
Using highly effective liquid phase chromatographic system, Inertsil C18 semi-preparative column is isolated and purified, flow velocity 3mL/min, mobile phase A 50mM
Acetic acid triethylamine buffer solution, PH 7.5；Mobile phase B is trifluoroacetic acid aqueous solution, gradient elution program are as follows: 0-15min is increased to by A
20%B, 15-25min, which increase to 30%, 25-35min by B component, increases to 50%.Pure product fractions are collected, low temperature concentration is surveyed
Determine to be placed in -20 DEG C of refrigerators after concentration to save backup.Sterling is through MALDI-TOF mass spectral analysis, target product ' Beijing orange-
DA6P ' (or abbreviation PO-dA6P) molecular composition and calculated value C33H37N5O22P6, m/z 1041.0375. measured value 1040.1097
(M-H).Its yield is about 70%.
Other three kinds of 5 '-ζ-terminal phosphate fluorescent markers 2 '-deoxyribonucleoside, six phosphate (PO-dG6P, PO-dC6P,
PO-dT6P it) is respectively synthesized according to 4 the method for the present embodiment.Its structure is as follows:
Embodiment 5
The synthesis of 2 '-deoxyribonucleoside tetraphosphate esters of 5 '-δ-terminal phosphate fluorescent marker is according to 4 the method for the present embodiment
It carries out, only 2 '--5 '-triphosphoric acid sodium salts of deoxyribonucleoside need to be replaced with 2 '--5 '-monophosphate sodium salts of desoxyadenossine.
Embodiment 6
2 '-deoxyribonucleoside, five phosphate synthesis of 5 '-ε-terminal phosphate fluorescent marker is according to 4 the method for the present embodiment
It carries out, only 2 '--5 '-triphosphoric acid sodium salts of deoxyribonucleoside need to be replaced with 2 '--5 '-diphosphonic acid sodium salts of desoxyadenossine.
Embodiment 7
Six phosphate of nucleosides (PO-dN6P) of four kinds of 5 '-ζ-terminal phosphate fluorescent markers is in DNA according to embodiment 4
Application in terms of sequencing.
It is first that the ssDNA template for being used for sequential test is immobilized to glass-chip table by way of asymmetric Solid phase PCR
On face: chip surface having been planted to injection PCR mixed liquor in the channel for be implanted with primer, including the primer 1 of 1uM；125nM's
Primer 2；The DNA profiling of 100nM；The archaeal dna polymerase (Platinum Taq (Life technology)) of 0.3U/uL, 3mM's
Magnesium ion, 1x standard Taq buffer.The pre- amplification (95 DEG C of 30s, 65 DEG C of 15s, 72 DEG C of 45s) of 15~20 circulations is done, first to produce
Then raw enough single-stranded DNA templates make the amplification (95 DEG C of 30s, 65 DEG C, 150s) of 20~30 circulations so as to what is expanded in advance
SsDNA and chip surface primer annealing simultaneously effectively extend.After PCR, pure water cleans chip surface 10min, it is final obtain containing about
There is the chip surface of 5fmol/mm2 sequence concentration to be measured.
Before sequencing starts, by 2 '-deoxyribonucleoside, the six phosphate PO-dN6P of four kinds of 5 '-ζ-terminal phosphate fluorescent markers points
Be not individually placed in 4 DEG C of cooling sample bottles and be diluted to 1uM (using buffer be 50mM Tris-HCl pH 7.9,
50mM NaCl, 0.1%tween-20,1mM MnCl2, 1mM DTT), while also including alkaline phosphatase (CIP) in sample bottle,
Archaeal dna polymerase (Bst).When sequencing, a kind of PO-dN6P is added into chip channel each time, it is poly- to then heat to 65 DEG C of initiations
Synthase work, releases the polyphosphoric acids segment chain with fluorescent marker, and alkaline phosphatase fast decoupled this polyphosphoric acids chain is simultaneously
Fluorescent dye core element is released, temperature is reduced after the completion of polymerization reaction to room temperature, fluorescence signal is generated under excitation light, is somebody's turn to do
The base quantity that signal is correctly identified and is integrated in template with polymerase is in a linear relationship.Complete signal take pictures acquisition after rinse
Chip channel (50mM Tris-HCl pH 7.9,50mM NaCl, 0.1%tween-20,1mM EDTA, 1mM DTT) carries out
The addition of next labeled substrate PO-dN6P so circuits sequentially progress, until sequencing is completed.This sequencing approach does not limit to
Specific sequence, it is only necessary to be carried out building library processing accordingly according to actual sample source, and be carried out on specific reaction chip
Surface sample amplification.This method is short (every wheel reaction and signal acquisition are no more than 3 minutes) with the sequencing reaction period, noise
It is quasi- than high (the long excitation/emission wavelength with 540nm or more of fluorescent molecule avoids the pollution of the background fluorescences such as chip material)
Exactness is high, reads the features such as long.
Embodiment 8
2 '-deoxyribonucleoside tetraphosphate the ester according to embodiment 5, the identical sequencing described in embodiment 7 under the conditions of, are used
Make gene sequencing, sequencing reaction speed (time required for each round sequencing reaction is completed) will be slower than PO-dN6P, in entirety
As a result show as obtaining on it is identical to read the long required sequencing time longer, the reason for this is that polymerase is to having different length phosphoric acid chain
Nucleotide identification and combine speed different, and thus influence sequencing accuracy and mispairing caused by the reaction time extends
Equal error increases.
Embodiment 9
On the basis of embodiment 4, if the active the link group of fluorescent molecule is changed to amino or other bases by hydroxyl
Group, then the nucleotide structure for being formed by terminal phosphate fluorescent marker cannot be cut off by phosphatase, thus can not discharge fluorescence
Molecule leads to that fluorescence signal cannot be generated.
A specific embodiment of the invention is further describing for technical solution of the present invention, is to preferably describe
The effect of claimed content and select, be not the limitation for summary of the invention.Its step, parameter etc., such as
Design parameter is that those skilled in the art's design according to the present invention can choose in embodiment 4.
Claims (15)
1. a kind of nucleotide structure of terminal phosphate fluorescent marker, it is characterised in that: there is structure shown in following general formula (1),
Wherein Ra, RbIndependent it can be selected from-H ,-OH；N is the integer more than or equal to 3, and less than or equal to 5；
Wherein, the fluorophor in general formula (1) has following general formula (2) described structure,
Wherein, R in general formula (2)1, R5It is independent to be selected from-H, C1-C6 alkyl, C1-C6 alkoxy；
R2-4It is independent to be selected from-H, fluorine, chlorine ,-CO2H、-SO3H、-CH2CO2H、-CH2SO3H, C1-C6 alkyl, C1-C6 alcoxyl
Base；
R6、R13Selection-H；
R7,8,11,12Independent it can be selected from-H, fluorine, chlorine, C1-C6 alkyl, C1-C6 alkoxy ,-CO2H、-CH2CO2H；
R9、R10Selected from C1-C6 alkyl.
2. structure according to claim 1, which is characterized in that base is phonetic selected from adenine, guanine, born of the same parents in general formula (1)
Pyridine, thymidine, uracil, modification nucleoside base.
3. structure according to claim 1, which is characterized in that the corresponding fluorescent molecule of the fluorophor is following knot
Structure:
4. structure according to claim 1, which is characterized in that the nucleotide structure of the terminal phosphate fluorescent marker can be with
As DNA or RNA polymerase substrate, it can be identified and be incorporated into DNA or RNA chain by DNA or RNA polymerase, and release
The polyphosphoric acids molecule of fluorophor label.
5. structure according to claim 4, which is characterized in that the nucleotide structure of the terminal phosphate fluorescent marker can be with
Substrate as alkaline phosphatase continues to decompose, until whole phosphate groups are detached from from fluorescent molecule.
6. structure according to claim 1, it is characterised in that the nucleotide structure of the terminal phosphate fluorescent marker is in quilt
It decomposes and is fluorescence closed state before releasing fluorophor, do not issue fluorescence signal when the light that is excited irradiates significantly.
7. structure according to claim 1, which is characterized in that the nucleotide structure of the terminal phosphate fluorescent marker is gathered
Synthase and alkaline phosphatase release fluorophor after decomposing, and significantly issue fluorescence signal in exciting light irradiation.
8. structure according to claim 3, which is characterized in that the excitation wavelength range of the fluorophor is 530-
590nm, wavelength of transmitted light range are 550-650nm.
9. structure according to claim 1, which is characterized in that formula (1) structure is 5 '-end-ζ-phosphoric acid fluorescence mark
Six phosphate of nucleotide of note perhaps 5 '-end-η-phosphoric acid fluorescent marker nucleotide, seven phosphate or 5 '-end-θ-
Eight phosphate of nucleotide of phosphoric acid fluorescent marker.
10. a kind of method for the nucleotide structure for synthesizing terminal phosphate fluorescent marker, it is characterised in that the following steps are included:
(a) trimetaphosphate is reacted with the hydroxyl of fluorescent dye groups, generates three metaphosphoric acid intermediates of fluorophor modification；
(b) nucleosides with 5 '-phosphoric acid chains and three metaphosphoric acid intermediate reactions of fluorophor modification are selected, product is converted to
5 '-polyphosphoric acids Terminal fluorescent labels nucleotide；
Wherein, the nucleotide structure of the terminal phosphate fluorescent marker has structure shown in following general formula (1):
Wherein Ra, RbIndependent it can be selected from-H ,-OH；N is the integer more than or equal to 3, and less than or equal to 5；
Fluorophor in the general formula (1) has following general formula (2) described structure,
Wherein, R in general formula (2)1, R5It is independent to be selected from-H, C1-C6 alkyl, C1-C6 alkoxy；
R2-4It is independent to be selected from-H, fluorine, chlorine ,-CO2H、-SO3H、-CH2CO2H、-CH2SO3H, C1-C6 alkyl, C1-C6 alcoxyl
Base；
R6、R13Selection-H；
R7,8,11,12Independent it can be selected from-H, fluorine, chlorine, C1-C6 alkyl, C1-C6 alkoxy ,-CO2H、-CH2CO2H；
R9、R10Selected from C1-C6 alkyl.
11. according to the method described in claim 10, it is characterized in that, trimetaphosphate is in activating reagent in the step (a)
In the presence of be activated, then reacted with the hydroxyl of fluorescent dye groups, generate three metaphosphoric acids of intermediate fluorophor modification.
12. according to the method described in claim 10, it is characterized in that, first that fluorophor and three metaphosphoric acids of activation is anti-
Answer, generate three metaphosphoric acid compound intermediate of fluorophor, then with nucleosides 5 '-monophosphate or nucleosides 5 '-diphosphonic acid or core
Glycosides 5 '-triphosphoric acid or the reaction of 5 '-polyphosphoric acid.
13. according to the method described in claim 10, it is characterized in that, the method do not include the nucleosides amino and/or
The protection and/or deprotection step of hydroxyl.
14. synthetic method according to claim 10, which is characterized in that the method is cooking-pot type reaction, and centre is not required to
The purifying intermediate to be separated.
15. a method of gene sequencing is carried out using the nucleotide structure of terminal phosphate fluorescent marker, it is characterised in that described
Nucleotide structure is the nucleotide structure limited in claim 1.
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