CN107474091B - The synthesis and application of 5- aldehyde radical cytidine phosphoramidite monomer of photosensitive protective group protection and preparation method thereof and oligonucleotide - Google Patents
The synthesis and application of 5- aldehyde radical cytidine phosphoramidite monomer of photosensitive protective group protection and preparation method thereof and oligonucleotide Download PDFInfo
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Abstract
The present invention relates to the field of chemical synthesis of oligonucleotide, disclose the synthesis and application of 5- aldehyde radical cytidine phosphoramidite monomer of photosensitive protective group protection and preparation method thereof and oligonucleotide.Shown in the chemical structure such as formula (1) of the 5- aldehyde radical cytidine phosphoramidite monomer of photosensitive protective group protection, wherein R is methyl or phenyl.5- aldehyde radical cytidine phosphoramidite monomer of the present invention is used to prepare the nucleotide of 5- aldehyde radical cytimidine pointed decoration, has a wide range of applications in terms of the biochemical property especially 5- aldehyde radical cytimidine of research 5- aldehyde radical cytimidine and protein interaction.
Description
Technical field
The present invention relates to the field of chemical synthesis of oligonucleotide, and in particular to the 5- aldehyde radical born of the same parents of photosensitive protective group protection
The synthesis and application of pyrimidine phosphoramidite monomer and oligonucleotide, more particularly, to 1- (2- nitrobenzophenone) propane-
The 5- of 5- aldehyde radical cytidine phosphoramidite monomer and 1- (2- nitrobenzophenone) propane -1,3- glycerol protection of 1,3- glycerol protection
The synthesis and application of the oligonucleotide of aldehyde radical cytimidine pointed decoration.
Background technique
5- aldehyde radical cytimidine is a kind of new base existence form [Science found in eukaryocyte recently
2011,333,1300‐1303].5- aldehyde radical cytimidine is generally considered to be centre of 5-methylcytosine during demethylation
Body [Chem.Rev.2015,115,2225-2239], can be long-term in vivo but also evidence suggests 5- aldehyde radical cytimidines
Be stabilized, and to the structure and function of genomic DNA generate certain influence [Nat.Struct.Mol.Biol.2015,22,
44‐49；Nat.Struct.Mol. Biol.2012,19,831‐833；Genome Biol.2013,14,R119.].
In order to study biochemical property of the 5- aldehyde radical cytimidine in DNA, need to prepare 5- aldehyde radical cytimidine pointed decoration
Oligonucleotide.Conventional means are first to synthesize 5- aldehyde radical cytidine phosphoramidite monomer, then pass through the plan of DNA synthesis in solid state
Slightly 5- aldehyde radical cytidine phosphoramidite monomer is pinpointed and introduces oligonucleotide, then ammonium hydroxide is deprotected and purifies to obtain pure widow
Polynucleotide is used for subsequent Biochemical Research.The aldehyde radical of 5- aldehyde radical cytidine phosphoramidite monomer be generally acknowledged that it is more stable,
It is all remained unchanged in conventional DNA synthesis in solid state and deprotection steps, therefore protection can not had to, be directly used in the conjunction of DNA solid phase
At.Recently also it has been reported that DNA is synthesized and was deprotected if not protecting the aldehyde radical of 5- aldehyde radical cytidine phosphoramidite monomer
Journey has some side reactions, therefore has developed 1,3-PD and protected 5 aldehyde radicals, leads to peracid treatment after ammonium hydroxide deprotection and restores 5-
Aldehyde radical [Angew.Chem.Int.Ed.Engl.2014,53,315-318.].
In some cases, it when for example research 5- aldehyde radical cytidine phosphoramidite monomer interacts with protein, needs
One is stablized in DNA synthesis and purification process, but can under conditions of more mild the 5- aldehyde radical of efficient quantitative removing
Protecting group.
Therefore, Development of Novel can be compatible with conventional DNA synthesis and deprotection procedure, efficiently fixed under mild conditions
5 aldehyde radical protecting groups for measuring the 5- aldehyde radical cytidine phosphoramidite monomer of removing are necessary.
Summary of the invention
The purpose of the invention is to overcome defect of the existing technology, provide it is a kind of be related to photosensitive protective group protection
The synthesis and application of 5- aldehyde radical cytidine phosphoramidite monomer and preparation method thereof and oligonucleotide, and in particular to a kind of 1-
5- aldehyde radical cytidine phosphoramidite monomer of (2- nitrobenzophenone) propane -1,3- glycerol protection and preparation method thereof and 1- (2-
Nitrobenzophenone) propane -1,3- glycerol protection 5- aldehyde radical cytimidine pointed decoration oligonucleotide synthesis and application.
To achieve the goals above, it is phonetic to provide a kind of 5- aldehyde radical born of the same parents that photosensitive protective group is protected for first aspect present invention
Pyridine phosphoramidite monomer, wherein the 5- aldehyde radical cytidine phosphoramidite monomer chemistries structural formula are as follows:
Wherein, R is methyl or phenyl.
Second aspect of the present invention provides a kind of system of the 5- aldehyde radical cytidine phosphoramidite monomer of photosensitive protective group protection
Preparation Method, wherein method includes the following steps:
(1) by 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, 1- (2- nitrobenzophenone) third
Alkane -1,3- glycol, triethyl orthoformate and methylene chloride carry out cooling, the first mixed solution of formation after the first mixing；Again by institute
The first mixed solution is stated to be reacted with titanium tetrachloride；Then by the organic phase obtained after reaction washing, dry, filtering, column layer
Analysis obtains reaction product；
(2) reaction product and the tetrabutyl ammonium fluoride and the tetrahydrofuran are carried out second mix, formation the
Then second mixed solution is rotated and removes solvent by two mixed solutions, column chromatograph to obtain 5- [4- (2- nitrobenzophenone) -1,
3- dioxane -2- base] -2 '-dideoxycytosine；
(3) by the 5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine and trimethyl chlorine
Silane, pyridine carry out third mixing, form third mixed solution；The third mixed solution is reacted with acetic anhydride again；
Then it chromatographs the organic phase obtained after reaction washing, dry, filtering, column to obtain 4-N- acyl group -5- [4- (2- nitrobenzophenone) -
1,3- dioxane -2- base] -2 '-dideoxycytosine；
(4) by the 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine
It is reacted with 4,4'- dimethoxytriphenylmethyl chlorine, pyridine, then by the organic phase obtained after reaction washing, dry, mistake
Filter, column chromatograph to obtain 5 '-(dimethoxytrityl) -4-N- acetyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxy six
Ring -2- base] -2 '-dideoxycytosine；
(5) by described 5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxy six
Ring -2- base] -2 '-dideoxycytosine and bis- (diisopropylaminoethyls) (2- cyanoethoxy) phosphine, 1H-TETRAZOLE and methylene chloride into
Row reaction, the solution obtained after reaction is rotated and removes solvent, then carries out column chromatography.
Preferably, in step (1), 1- (2- nitrobenzophenone) propane -1,3- glycol, 3 ', 5 '-(tertiary butyl
Dimethyl silicon substrate) -5- aldehyde radical -2 '-dideoxycytosine, the triethyl orthoformate, the methylene chloride and the titanium tetrachloride
Dosage molar ratio be 1mmol:(3-5) mmol:(1.2-1.5) mmol:(5-7) mL:(0.4-0.5) mmol；
Described first mixed condition includes: that temperature is 18-25 DEG C, and the time is 0.5-1 hours, mixing speed 250-
500 revs/min；
The condition of the cooling includes: that temperature is 0-4 DEG C, and the time is 0.1-0.5 hours, mixing speed 250-500
Rev/min；And
The titanium tetrachloride is added in first mixed solution in a manner of being added dropwise, and reaction described in step (1)
Journey includes two stages, and the reaction condition of first stage includes: that temperature is 18-25 DEG C, and the time is 5-7 hours, and mixing speed is
250-500 revs/min, the drop rate of the titanium tetrachloride is 10-15 drop/minute；And the reaction condition packet of second stage
Include: temperature is 18-25 DEG C, and the time is 5-7 hours, and mixing speed is 250-500 revs/min, the dropwise addition speed of the titanium tetrachloride
Rate is 10-15 drop/minute.
Preferably, in step (2), described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radical -2 '-dideoxycytosine,
The ratio of the dosage of the tetrabutyl ammonium fluoride and the tetrahydrofuran is 1mmol:(3-4) mmol:(5-7) mL；And
Described second mixed condition includes: that temperature is 18-25 DEG C, and the time is 9-11 hours, mixing speed 250-500
Rev/min.
Preferably, in step (3), -2 '-deoxidation of the 5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base]
The ratio of the dosage of cytimidine and the trim,ethylchlorosilane, the pyridine and the acetic anhydride is 1mmol:(5-7) mmol:(9-
11) mL:(1.1-1.3) mmol；And
It is 18-25 DEG C that the condition of the third mixing, which includes: temperature, and the time is 1-2 hours, mixing speed 250-500
Rev/min；And it is 18-25 DEG C that the condition of the reaction, which includes: temperature, the time is 3-5 hours, mixing speed 250-500
Rev/min.
Preferably, in step (4), the 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -
The ratio of the dosage of 2 '-dideoxycytosines and 4,4'- dimethoxytriphenylmethyl chlorine, pyridine is 1mmol:(1.5-2) mmol:
(9-11)mL；And
The condition of the reaction includes: that temperature is 18-25 DEG C, and the time is 12-18 hour, mixing speed be 250-500 turn/
Minute.
Preferably, in step (5), 5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzene
Base) -1,3- dioxane -2- base] -2 '-dideoxycytosine and bis- (diisopropylaminoethyls) (2- cyanoethoxy) phosphine, 1H- tetra-
The ratio of the dosage of azoles and methylene chloride is 1mmol:(1.3-1.6) mmol:(1.3-1.6) mmol:(10-30) mL；And
The condition of the reaction includes: that temperature is 18-25 DEG C, and the time is 2-4 hour, mixing speed be 250-500 turn/
Minute.
Third aspect present invention provides a kind of 5 aldehyde radical cytimidines of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
The preparation method of the oligonucleotide of pointed decoration, wherein the synthetic agent in the preparation method includes preparation described above
The 5- aldehyde radical cytidine phosphoramidite monomer for the photosensitive protective group protection that method is prepared.
Preferably, which carries out on ABI 394DNA/RNA synthesizer, in which:
Preferably, the synthetic agent further includes Ac-dC, Bz-dA, dmf-dG and dT；
Preferably, trichloroacetic acid/methylene chloride is for being deprotected；
Preferably, Cap A reagent is the composition of acetic anhydride, pyridine and tetrahydrofuran, and the acetic anhydride, the pyridine
Volume ratio with the tetrahydrofuran is 10 volume %:(5-15 volume %): (75-85 volume %)；
Preferably, Cap B reagent is the composition of N- methylimidazole and tetrahydrofuran, and the N- methylimidazole and described
The volume ratio of tetrahydrofuran is 10 volume %:(85-95 volume %)；
Preferably, activator is the composition of ethlythiotetrazole and acetonitrile；
Preferably, oxidant is the composition of iodine, water, pyridine and tetrahydrofuran, and the iodine, the water, the pyridine
Volume ratio with the tetrahydrofuran is 0.02M:(1-2 volume %): (10-30 volume %): (75-85 volume %)；And institute
It states
Preferably, the Coupling time of the 5- aldehyde radical cytidine phosphoramidite monomer of the photosensitive protective group protection is 4-6 points
Clock；
Preferably, the oligonucleotide that above-mentioned solid phase synthesizes is handled into 1-2h at 60-70 DEG C with 1 milliliter of AMA, so
Afterwards, after taking out supernatant centrifugal concentrating, the modacrylic using 20 weight % is gel purified；And
Preferably, the AMA be 40% methylamine water solution and 28% ammonium hydroxide mixed solution, and the methylamine water solution and
The ratio of the volume of the ammonium hydroxide is 1:(0.5-1.5).
Fourth aspect present invention provides 1- (2- nitrobenzophenone) propane-being prepared by preparation method described above
The oligonucleotide of the 5- aldehyde radical cytimidine pointed decoration of 1,3- glycerol protection is being used for the operational application of DNA, wherein should
The operational application of DNA includes carrying out 5 ' phosphorylations with PNK enzyme, and ligase is attached, and carries out DNA- albumen with albumen
The assembling of complex.
The 5th aspect of present aspect provides a kind of preparation side of oligonucleotide containing 5- aldehyde radical cytimidine pointed decoration
Method, wherein this method includes that 1- (2- nitrobenzophenone) propane -1,3- glycol that preparation method described above is prepared is protected
The oligonucleotide of the 5- aldehyde radical cytimidine pointed decoration of shield illumination 4-6 minutes in-situ preparation under the illumination of 340-360nm obtains
It arrives.
The invention has the advantages that the 5- aldehyde radical cytimidine phosphorous acyl of (1) 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
Amine monomers synthetic method is simple；(2) 1- (2- nitrobenzophenone) propane -1,3- glycol of 5- aldehyde radical cytidine phosphoramidite monomer is protected
Base is protected all stable in oligonucleotide synthesis and purification process；(3) after obtaining pure oligonucleotide, 1- (2- nitrobenzene
Base) protecting group can quantify removing by illumination experiment by propane -1,3- glycerol protection base.The removing mild condition, and almost
All biosystems are compatible.The synthetic method of the nucleotide of 5- aldehyde radical cytimidine pointed decoration of the present invention, is being studied
Before the biochemical property of 5- aldehyde radical cytimidine especially 5- aldehyde radical cytimidine and protein interaction aspect have a wide range of applications
Scape.
Detailed description of the invention
Fig. 1 is the oligonucleotides of the 5- aldehyde radical cytimidine modification containing 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
5 ' the phosphorylations and DNA ligase connection strategy of acid.
Fig. 2 is the ultra high efficiency liquid phase analysis figure of oligonucleotide 12 and 13.
Fig. 3 is the mass spectrogram of oligonucleotide 12 and 13.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
First aspect present invention provides a kind of 5- aldehyde radical cytidine phosphoramidite monomer, wherein its chemical structural formula are as follows:
Wherein, R is methyl or phenyl.
Formula (1) compound represented is properly termed as 3 '-O- (N, N- diisopropyl amido-O-beta- cyanoethoxyl-phosphine
Base) -5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 ' -
Dideoxycytosine.
Wherein, in formula (1), " DMTrO- " be dimethoxytrityl, " N (iPr)2" it is N, N- diisopropylamine
Base.
Second aspect of the present invention provides the preparation method of 5- aldehyde radical cytidine phosphoramidite monomer described above,
In, method includes the following steps:
(1) by 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, 1- (2- nitrobenzophenone) third
Alkane -1,3- glycol, triethyl orthoformate and methylene chloride carry out cooling, the first mixed solution of formation after the first mixing；Again by institute
The first mixed solution is stated to be reacted with titanium tetrachloride；Then by the organic phase obtained after reaction washing, dry, filtering, column layer
Analysis obtains reaction product；
Wherein it is possible to successively select saturated sodium bicarbonate aqueous solution, saturated sodium chloride solution washing will be after the first reaction
Organic phase, then select anhydrous magnesium sulfate dry, first mixed solution after washing is dry then filtered into back spin
Dry filtrate, column chromatograph to obtain the reaction product of faint yellow solid.
(2) reaction product and the tetrabutyl ammonium fluoride and the tetrahydrofuran are carried out second mix, formation the
Then second mixed solution is rotated and removes solvent by two mixed solutions, column chromatograph to obtain 5- [4- (2- nitrobenzophenone) -1,
3- dioxane -2- base] -2 '-dideoxycytosine；
Wherein, the tetrahydrofuran is dry tetrahydrofuran, and column chromatographs to obtain 5- [4- (2- nitrobenzophenone) -1,3- bis-
Six ring -2- base of oxygen] -2 '-dideoxycytosine be faint yellow solid.
(3) by the 5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine and trimethyl chlorine
Silane, pyridine carry out third mixing, form third mixed solution；Again by the third mixed solution and acetic anhydride (chlorobenzoyl chloride)
It is reacted；Then it chromatographs the organic phase obtained after reaction washing, dry, filtering, column to obtain 4-N- acyl group -5- [4- (2- nitre
Base phenyl) -1,3- dioxane -2- base] -2 '-dideoxycytosine；
Wherein, the pyridine is dry pyridine, can successively select be extracted with dichloromethane, saturated sodium bicarbonate aqueous solution
The organic phase after third is reacted is washed, then selects anhydrous magnesium sulfate dry, is spin-dried for filtrate after then filtering, column chromatographs to obtain light
The reaction product of yellow solid.
(4) by the 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine
It is reacted with 4,4'- dimethoxytriphenylmethyl chlorine, pyridine, then by the organic phase obtained after reaction washing, dry, mistake
Filter, column chromatograph to obtain 5 '-(dimethoxytrityl) -4-N- acetyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxy six
Ring -2- base] -2 '-dideoxycytosine；
Wherein, the pyridine is dry pyridine, can successively select be extracted with dichloromethane, saturated sodium bicarbonate water it is molten
The organic phase of liquid, saturated sodium-chloride water solution washing after the 4th reaction, then select anhydrous magnesium sulfate dry, then filter back spin
Dry filtrate, column chromatograph to obtain the reaction product of faint yellow solid.
(5) by described 5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxy six
Ring -2- base] -2 '-dideoxycytosine and bis- (diisopropylaminoethyls) (2- cyanoethoxy) phosphine, 1H-TETRAZOLE and methylene chloride into
Row reaction, the solution obtained after reaction is rotated and removes solvent, then carries out column chromatography.
In the present invention, through the above-mentioned available 3 '-O- of preparation method (N, N- diisopropyl amido-O-beta- cyanogen
Ethyoxyl-phosphino-) -5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine；
Wherein, the methylene chloride is dry methylene chloride.
According to the present invention, wherein in step (1), 1- (2- nitrobenzophenone) propane -1,3- glycol, 3 ',
5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radical -2 '-dideoxycytosine, the triethyl orthoformate, the methylene chloride and institutes
The molar ratio for stating the dosage of titanium tetrachloride can be 1mmol:(3-5) mmol:(1.2-1.5) mmol:(5-7) mL:(0.4-
0.5)mmol；That is, being equivalent to the 1- (2- nitrobenzophenone) propane -1,3- glycol of 1mmol, described 3 ', 5 '-is (special
Butyldimethyl silicon substrate) -5- aldehyde radical -2 '-dideoxycytosine dosage is 3-5mmol, the dosage of the triethyl orthoformate is
1.2-1.5mmol, the dosage of the methylene chloride are 5-7mL, and the dosage of the titanium tetrachloride is 0.4-0.5mmol；Although institute
State 1- (2- nitrobenzophenone) propane -1,3- glycol, described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radical -2 '-deoxidation born of the same parents
The molar ratio of the dosage of pyrimidine, the triethyl orthoformate, the methylene chloride and the titanium tetrachloride is met the above range i.e.
The achievable present invention, it is, however, preferable that the 1- (2- nitrobenzophenone) propane -1,3- glycol, 3 ', 5 '-(tertiary butyl two
Methylsilyl) -5- aldehyde radical -2 '-dideoxycytosine, the triethyl orthoformate, the methylene chloride and the titanium tetrachloride
When the molar ratio of dosage is 1mmol:4 mmol:1.2mmol:6mL:0.4mmol；Effect is more preferable；
Described first mixed condition may include: that temperature is 18-25 DEG C, and the time is 0.5-1 hours, and mixing speed is
250-500 revs/min；Preferably, temperature is 20 DEG C, and the time is 0.5 hour, and mixing speed is 400 revs/min；
The condition of the cooling may include: that temperature is 0-4 DEG C, and the time is 0.1-0.5 hours, mixing speed 250-
500 revs/min；Preferably, temperature is 0 DEG C, and the time is 0.5 hour, and mixing speed is 400 revs/min；And
The titanium tetrachloride is added in first mixed solution in a manner of being added dropwise, and reaction described in step (1)
Journey includes two stages, and the reaction condition of first stage includes: that temperature is 18-25 DEG C, and preferably 20 DEG C, the time is 5-7 hours,
Preferably 6 hours, mixing speed be 250-500 revs/min, preferably 400 revs/min, the drop rate of the titanium tetrachloride
For 10-15 drop/minute, preferably 10 drops/minute；It is more preferable that effect is carried out under optimum condition；And
The condition of the second stage may include: that temperature is 18-25 DEG C, and preferably 20 DEG C, the time is 5-7 hours, excellent
It is selected as 6 hours, mixing speed is 250-500 revs/min, and preferably 400 revs/min, the drop rate of the titanium tetrachloride is
It is more preferable to carry out effect under the preferred conditions for 10-15 drop/minute, preferably 15 drops/minute.
According to the present invention, in step (2), described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radical -2 '-deoxidation born of the same parents
The ratio of the dosage of pyrimidine, the tetrabutyl ammonium fluoride and the tetrahydrofuran can be 1mmol:(3-4) mmol:(5-7) mL；
That is, described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine of 1mmol are equivalent to, it is described
The dosage of tetrabutyl ammonium fluoride is 3-4mmol, and the dosage of the tetrahydrofuran is 5-7mL；Although each dosage meets above-mentioned model
It encloses and the present invention can be completed, it is, however, preferable that described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radical -2 '-deoxidation born of the same parents are phonetic
Pyridine, the tetrabutyl ammonium fluoride and the tetrahydrofuran dosage ratio be 1mmol:4mmol:6mL when, effect is more preferable；And
Described second mixed condition may include: that temperature is 18-25 DEG C, and the time is 9-11 hours, and mixing speed is
250-500 revs/min；Preferably, temperature is 20 DEG C, and the time is 10 hours, and mixing speed is 400 revs/min.
According to the present invention, in step (3), the 5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 ' -
Dideoxycytosine and the ratio of the dosage of the trim,ethylchlorosilane, the pyridine and the acetic anhydride (chlorobenzoyl chloride) are
1mmol:(5-7) mmol:(9-11) mL:(1.1-1.3) mmol；That is, being equivalent to the 5- [4- (the 2- nitro of 1mmol
Phenyl) -1,3- dioxane -2- base] -2 '-dideoxycytosine and the trim,ethylchlorosilane, the use of the trim,ethylchlorosilane
Amount is 5-7mmol, and the dosage of the pyridine is 9-11mL, and the dosage of the acetic anhydride (chlorobenzoyl chloride) is 1.1-1.3mmol；To the greatest extent
It manages each dosage and meets the above range and the present invention can be completed, it is, however, preferable that the 5- [4- (2- nitrobenzophenone) -1,3-
Dioxane -2- base] -2 '-dideoxycytosine and the trim,ethylchlorosilane, the pyridine and the acetic anhydride (benzoyl
Chlorine) dosage ratio be 1mmol:6mmol:10mL:1.2mmol when, effect is more preferable；And
It is 18-25 DEG C that the condition of the third mixing, which may include: temperature, and the time is 1-2 hours, mixing speed 250-
500 revs/min；And it is 18-25 DEG C that the condition of the reaction, which includes: temperature, the time is 3-5 hours, mixing speed 250-
500 revs/min；
Preferably, it is 20 DEG C that the condition of the third mixing, which includes: temperature, and the time is 1.5 hours, mixing speed 400
Rev/min；And it is 19-21 DEG C that the condition of the reaction, which includes: temperature, the time is 3.5-4.5 hours, and mixing speed is
350-450 revs/min.
According to the present invention, in step (4), the 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine and 4,4'- dimethoxytriphenylmethyl chlorine, pyridine the ratio of dosage can be 1mmol:(1.5-
2)mmol:(9-11)mL；That is, being equivalent to the 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxy of 1mmol
Six ring -2- bases] -2 '-dideoxycytosine, the dosage of 4, the 4'- dimethoxytriphenylmethyl chlorine is 1.5-2mmol, described
The dosage of pyridine is 9-11mL；Although each dosage, which is met the above range, can be completed the present invention, it is, however, preferable that the 4-
N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine and 4,4'- dimethoxy triphen
When the ratio of the dosage of ylmethyl chlorine, pyridine can be 1mmol:2mmol:10mL, effect is more preferable；And
The condition of the reaction may include: that temperature is 18-25 DEG C, and the time is 12-18 hours, mixing speed 250-
500 revs/min；
Preferably, it is 20 DEG C that the condition of the reaction, which includes: temperature, and the time is 16 hours, and mixing speed is 400 revs/min
Clock.
According to the present invention, in step (5), 5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitre
Base phenyl) -1,3- dioxane -2- base] -2 '-dideoxycytosine and bis- (diisopropylaminoethyls) (2- cyanoethoxy) phosphine,
The ratio of the dosage of 1H-TETRAZOLE and methylene chloride is 1mmol:(1.3-1.6) mmol:(1.3-1.6) mmol:(10-30) mL；?
That is being equivalent to described 5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- of 1mmol
Dioxane -2- base] -2 '-dideoxycytosine, the dosage of bis- (diisopropylaminoethyls) (2- cyanoethoxy) phosphine is 1.3-
1.6mmol, the dosage of the 1H- tetrazolium are 1.3-1.6mmol, and the dosage of the methylene chloride is 10-30mL；Although each
Dosage, which is met the above range, can be completed the present invention, it is, however, preferable that described 5 '-(dimethoxytrityl) -4-N- acyl
Base -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine and bis- (diisopropylaminoethyl) (2- cyanogen
Base oxethyl) phosphine, 1H-TETRAZOLE and methylene chloride dosage ratio be 1mmol:1.5 mmol:1.5mmol:10mL when, effect is more
It is good；And
The condition of the reaction may include: that temperature is 18-25 DEG C, and the time is 2-4 hours, mixing speed 250-500
Rev/min；
Preferably, it is 20 DEG C that the condition of the reaction, which includes: temperature, and the time is 3 hours, and mixing speed is 400 revs/min
Clock.
Third aspect present invention provides a kind of 5- aldehyde radical cytimidine of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
The preparation method of the oligonucleotide of pointed decoration, wherein the synthetic agent in the preparation method includes preparation described above
The 5- aldehyde radical cytidine phosphoramidite monomer of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection that method is prepared.
According to the present invention, the preparation method can on ABI 394DNA/RNA synthesizer by conventional program and reagent into
The synthesis of row oligonucleotide carries out, in which:
Preferably, the synthetic agent can also include Ac-dC, Bz-dA, dmf-dG and dT；
Preferably, trichloroacetic acid/methylene chloride can be selected for being deprotected；
Preferably, Cap A reagent can be acetic anhydride, the composition of pyridine and tetrahydrofuran, and the acetic anhydride, described
The volume ratio of pyridine and the tetrahydrofuran can be 10 volume %:(5-15 volume %): (75-85 volume %)；That is,
The composition contains the acetic anhydride of 10 volume %, the pyridine of 5-15 volume %, the tetrahydrofuran of 75-85 volume %；Preferably, institute
The volume ratio for stating acetic anhydride, the pyridine and the tetrahydrofuran is 10 volume %:(8-12 volume %): (78-82 volume %)；
Preferably, Cap B reagent can be the composition of N- methylimidazole and tetrahydrofuran, and the N- methylimidazole
Volume ratio with the tetrahydrofuran can be 10 volume %:(85-95 volume %)；That is, the composition contains 10 bodies
The N- methylimidazole of product %, the tetrahydrofuran of 85-95 volume %；Preferably, the N- methylimidazole and the tetrahydro furan
The volume ratio muttered is 10 volume %:(88-92 volume %)；
Preferably, activator be ethlythiotetrazole and acetonitrile composition, the activator can be commercially available or
Person synthesizes to obtain using the method for the prior art, and in the present invention, the activator is commercially available from, and the concentration of the activator
For 0.25M, that is, the equivalent concentration of ethlythiotetrazole is 0.25M in the activator.
Preferably, oxidant is the composition of iodine, water, pyridine and tetrahydrofuran, and the iodine, the water, the pyridine
Volume ratio with the tetrahydrofuran can be 0.02M:(1-2 volume %): (10-30 volume %): (75-85 volume %)；?
That is the composition contains the iodine of 0.02M, water is 1-2 volume %, and pyridine is 10-30 volume %, tetrahydrofuran 75-85
Volume %；Preferably, the volume ratio of the iodine, the water, the pyridine and the tetrahydrofuran is 0.02M:(1.5-2 body
Product %): (15-25 volume %): (76-80 volume %)；And
Preferably, the Coupling time of the 5- aldehyde radical cytidine phosphoramidite monomer can be 4-6 minutes, preferably 4.5-
5.5 minute；
Preferably, the oligonucleotide that above-mentioned solid phase synthesizes can be handled into 1- at 60-70 DEG C with 1 milliliter of AMA
2h, then, after taking out supernatant centrifugal concentrating, the modacrylic using 20 weight % is gel purified；And
Preferably, the AMA be 40% methylamine water solution and 28% ammonium hydroxide mixed solution, and the methylamine water solution and
The ratio of the volume of the ammonium hydroxide is 1:(0.5-1.5), preferably 1:(0.8-1.2), more preferably 1:1.
Fourth aspect present invention provides a kind of 1- (2- nitrobenzophenone) being prepared by preparation method described above
The oligonucleotide of the 5- aldehyde radical cytimidine pointed decoration of propane -1,3- glycerol protection is being used for the operational application of DNA,
In, the operational application of the DNA includes carrying out 5 ' phosphorylations with PNK enzyme, and ligase is attached, and carries out DNA- with albumen
The assembling of protein complexes.
Fifth aspect present invention provides a kind of preparation method of the oligonucleotide of 5- aldehyde radical cytimidine pointed decoration,
In, this method includes 1- (2- nitrobenzophenone) propane -1,3- glycerol protection that preparation method described above is prepared
The oligonucleotide of 5- aldehyde radical cytimidine pointed decoration illumination 4-6 minutes in-situ preparation under the illumination of 340-360nm obtains.
Preferably, in the present invention, by illumination 4-6 minutes under the illumination of 340-360nm when any need, it is preferable that
By illumination 4.5-5.5 minutes under the illumination of 345-355nm, 1- (2- nitrobenzophenone) propane -1,3- bis- can be quantitatively removed
Alcohol protection group restores the intact form of 5- aldehyde radical cytidine phosphoramidite monomer, plays 5- aldehyde radical cytidine phosphoramidite monomer
Whole biochemical functions.
The present invention will be described in detail by way of examples below.
3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radical -2 '-dideoxycytosines (self-control), 1- (2- nitrobenzophenone) third
Alkane -1,3- glycol (self-control), triethyl orthoformate (Tianjin chemical reagent supply and marketing company, article No. HWMT818864) and dichloro
Methane (Tianjin chemical reagent supply and marketing company, article No. 3975), sodium bicarbonate (Tianjin chemical reagent supply and marketing company, article No.
166), sodium chloride (Tianjin chemical reagent supply and marketing company, article No. 017), tetrabutyl ammonium fluoride (TCI, article No. T1338)), tetrahydro
Furans (Tianjin chemical reagent supply and marketing company, article No. 2287), trim,ethylchlorosilane (Tianjin chemical reagent supply and marketing company, goods
Number 110121000), pyridine (Aladdin, article No. P111511), acetic anhydride (Tianjin chemical reagent supply and marketing company, article No. 282),
The raw materials such as 4,4'- dimethoxytriphenylmethyl chlorine (Adamas, article No. 24127C), 1H-TETRAZOLE (TCI, article No. S014625)
For commercially available product.
Embodiment 1
The present embodiment indicates that the 5- aldehyde radical cytimidine of 1- of the invention (2- nitrobenzophenone) propane -1,3- glycerol protection
The synthesis of phosphoramidite monomer.
Its synthesis step is as follows:
The preparation of compound (4): monomer shown in 1.31g (2.71mmol, 1.0eq (equivalent)) formula (2) is weighed (wherein,
In formula (2), " TBS- " is tertiary butyl dimethyl silicon substrate), it is dissolved under protection of argon gas with 20mL dry methylene chloride；Then to
The pure and mild 537uL of 2.14g (10.84 mmol, 4eq) 1- (2- nitrobenzophenone) propane -1,3- bis- is added in reaction system
(3.25mmol, 1.2eq) triethyl orthoformate, and 1.08mL is added dropwise under condition of ice bath (temperature is 0 DEG C)
(dichloromethane solution (that is, containing the titanium tetrachloride of 1M in methylene chloride) of 1.08mmol, 0.4eq, 1M) titanium tetrachloride is molten
Liquid is warmed to room temperature (20 DEG C).After stirring 6h, 542 μ L (dichloromethane solution of 0.54mmol, 0.2eq, 1M) four chlorinations are added dropwise
Titanium solution the reaction was continued 6h.Saturated sodium bicarbonate aqueous solution quenching reaction is selected, is extracted with methylene chloride (80mL), what is separated has
Machine is mutually with saturation NaCl solution (20 mL) washing.Anhydrous magnesium sulfate is dry, and revolving removes solvent and obtains shown in crude product formula (3)
Monomer.The product of acquisition is dissolved in the dry tetrahydrofuran of 20mL under protection of argon gas, 4.34mL (4.34 mmol, 1M are added
Tetrahydrofuran solution) tetrabutyl ammonium fluoride, stir 10h at room temperature.Revolving removes solvent, and with ethanol/methylene, (gradient is washed
It is de-, v/v, 3%~6%~10%) column chromatography, obtain 590mg faint yellow solid (4) (1.36mmol, 50 weight % of yield).It produces
Object is a pair of of diastereoisomer (1:1).
1H NMR(400MHz,d4-CD3OD): δ (ppm) 8.22 (s, 1H), 8.18 (s, 1H), 7.95 (d, J=8.2Hz,
2H), 7.87 (d, J=4.2Hz, 1H), 7.85 (d, J=3.9Hz, 1H), 7.75-7.71 (m, 2H), 7.54-7.50 (m, 2H),
6.26-6.21(m,2H),5.62(s,2H),5.48-5.45(m,2H), 4.37-4.30(m,4H),4.18-4.11(m,2H),
3.96-3.92(m,2H),3.82-3.70(m,4H), 2.41-2.34(m,2H),2.17-2.10(m,12H),2.07-2.03
(m,4H).13C NMR(101 MHz,d4-CD3OD):δ(ppm)164.9,157.6,148.9,142.2,142.2,137.4,
134.9,129.9, 129.5,125.3,106.1,106.0,99.9,99.8,89.0,88.9,87.9,87.7,76.6,76.5,
72.0, 71.9,68.3,62.8,42.2,42.1,33.8.HRMS(ESI):C19H22N4O8,[M+H]+cal. 435.1516,
found 435.1489.
The preparation of compound (5): weighing monomer shown in 594mg (1.37mmol, 1.0eq) formula (4), with dry pyridine
After corotation 2 times, argon gas protection is lower to use 15mL pyridinium dissolution.1.27mL (3.5mmol, 6eq) trimethyl chlorine is added to reaction system
150 μ L (1.51mmol, 1.1eq) acetic anhydrides are added after silane and under normal temperature conditions stirring 1.5h, continue under room temperature (25 DEG C)
It after reacting 4h, is quenched with 5mL distilled water, revolving removes solvent.Appropriate saturated sodium bicarbonate solution is added and uses methylene chloride
(60mL) extraction, merges organic phase, and anhydrous magnesium sulfate is dry.Revolving remove solvent, with ethanol/methylene (v/v, 3%~
4%~6%) column chromatography is carried out, 350mg faint yellow solid monomer as shown in formula (5) (0.73mmol, 54%) is obtained.Product is
A pair of of diastereoisomer (1:1).
1H NMR(400MHz,CDCl3):δ(ppm)9.03(br,1H),8.43-8.42(m,1H), 8.95-7.93(m,
1H), 7.77-7.66 (m, 2H), 7.48-7.43 (m, 1H), 6.10 (t, J=5.4Hz, 1H), 5.61 (m, 1H), 5.50-5.45
(m,1H),4.55-4.48(m,1H),4.37-4.34(m,1H), 4.18-4.12(m,1H),3.99-3.95(m,1H),3.88
(m,2H),2.61(m,3H),2.54-2.47(m, 1H),2.31-2.22(m,1H),2.15-2.12(m,1H),2.09-2.02
(m,1H).13C NMR(101 MHz,d6-DMSO):δ(ppm)170.7,158.8,153.3,147.3,142.3,143.6,
135.3,135.2, 134.0,129.1,128.0,124.2,105.2,97.5,88.1,86.5,86.5,74.5,74.4,
70.2,70.0, 66.5,61.1,40.8,32.4,25.6.HRMS(ESI):C21H24N4O9,[M+H]+cal.477.1622,
found 477.1673.
The preparation of compound (6): weigh monomer shown in 0.33g (0.95mmol, 1eq) formula (5) (in formula (5),
" DMTr- " is dimethoxytrityl), the pyridinium dissolution dry with 10mL under argon gas protection.It is then slowly added into 470mg
(1.38mmol, 2eq) 4,4'- dimethoxytriphenylmethyl chlorine, is stirred at room temperature 16h.After TLC monitors fully reacting, it is added
10mL saturated sodium bicarbonate quenching reaction, and be extracted with dichloromethane, one is washed with the NaCl solution of saturation after merging organic phase
Secondary, anhydrous magnesium sulfate is dry.Revolving remove solvent, with ethanol/methylene (gradient elution, v/v, 1%~2%~3% ,+
0.5%, v/v triethylamine) column chromatography is carried out, obtain faint yellow solid (6) 350mg (0.45 mmol, 65%).Product is a pair of non-
Enantiomter (1:1).
1H NMR(400MHz,CDCl3):δ(ppm)9.12-9.11(m,2H),8.53(s,1H),8.46(s, 1H),8.00
(d, J=8.4Hz, 2H), 7.73-7.65 (m, 4H), 7.49-7.44 (m, 2H), 7.37-7.35 (m, 4H), 7.25-7.21 (s,
12H), 7.15-7.11 (m, 2H), 6.78-6.74 (m, 8H), 6.54 (t, J=6.4Hz, 1H), 6.45 (t, J=6.4Hz,
1H),4.67-4.64(m,1H),4.62-4.60(m,1H),4.55-4.52(m, 1H),4.52-4.49(m,1H),4.41(s,
1H), 4.25 (s, 1H), 4.23-4.22 (m, 1H), 4.12-4.11 (m, 1H), 4.03 (dd, J=4.3,11.6,2H), 3.69
(s, 3H), 3.67 (s, 3H), 3.66 (s, 3H), 3.65 (s, 3H), 3.58 (dd, J=2.8,10.7,1H), 3.53 (dd, J=
), 2.5,10.7,1H 3.39-3.32 (m, 2H), 3.26 (dd, J=2.4,10.8,1H), 3.19 (dd, J=3.5,10.7,
1H),2.74-2.63(m,8H), 2.40-2.33(m,1H),2.29-2.22(m,1H),1.97-1.94(m,1H),1.88-
1.85(m,1H), 1.82-1.73(m,2H).13C NMR(101MHz,CDCl3):δ(ppm)172.71,159.24,159.20,
158.73,154.35,146.82,146.53,144.57,144.10,143.72,143.47,136.44,136.26,
135.57,135.36,135.34,135.29,134.36,134.32,130.10,130.04,130.01,128.62,
128.54,128.27,128.18,128.14,128.04,128.00,127.26,127.18,124.55,113.39,
105.42,105.21,99.83,87.26,87.01,86.92,86.90,86.85,86.39,77.24,75.49, 75.24,
72.11,71.87,67.07,66.90,63.70,63.26,55.14,55.11,42.35,42.27,32.85, 26.90,
26.83.ESI-Q-TOF:C42H42N4O11,[MH]+cal.779.2928；found 779.2969.
The preparation of compound (7): weigh monomer shown in 100mg (0.13mmol, 1eq) formula (6) (in formula (6), N
(iPr)2" be N, N- diisopropylaminoethyl), 13.5mg (0.19mmol, 1.5eq) 1H-TETRAZOLE in 25mL flask, protect by argon gas
The lower 5mL dry methylene chloride of shield dissolves.Bis- (diisopropylaminoethyl) (the 2- cyano of 62 μ L (0.19mmol, 1.5eq) are then added
Ethyoxyl) phosphine, stirs 3h under room temperature.After reaction, under protection of argon gas by system evaporated under reduced pressure, then with a small amount of dichloromethane
Alkane dissolution, rapid column chromatography ethanol/methylene (v/v, 0.5%~1% ,+2% triethylamine) purifying obtain light yellow solid
108mg (0.11mmol, 85 weight % of yield).Product has 4 isomers, ratio are as follows: 3:3:1:1.
31P NMR(162MHz,CDCl3)δ(ppm)149.4(s,3P),149.0(s,3P)148.7(s,1P), 148.5
(s,1P)；ESI-Q-TOF:C51H59N6O12P,[MH]+cal.979.4007；found 979.3730.
Embodiment 2
The present embodiment indicates that the 5- aldehyde radical of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection by implementing 1 preparation
Cytidine phosphoramidite monomer (7) carries out DNA synthesis in solid state DNA sequence dna 8:CGTTT7AGCGGTGCTAG (as shown in Figure 1).
The synthesis of 1mol oligonucleotide is carried out by conventional program and reagent on 394 DNA/RNA synthesizer of ABI.It closes
It include Ac-dC, Bz-dA, dmf-dG, the 5- aldehyde radical cytidine phosphoramidite monomer of 1 preparation of dT and implementation at reagent；3 weight %
Trichloroacetic acid/methylene chloride is for being deprotected；Cap A reagent: 10 volume % acetic anhydrides/10 volume % pyridines/80 volume % tetra-
Hydrogen furans (v/v/v), Cap B reagent: 10 volume %N- methylimidazoles/90 volume % tetrahydrofurans (v/v)；Activator:
0.25M ethlythiotetrazole/acetonitrile；Oxidant: 0.02M iodine/2 volume % water/20 volume % pyridines/78 volume % tetrahydrofurans
(v/v/v).The Coupling time of 5- aldehyde radical cytidine phosphoramidite monomer is 5 minutes.
The carrier that above-mentioned solid phase synthesizes 1 milliliter of AMA (40 weight % methylamine water solutions: 28 weight % ammonium hydroxide (volumes
Than 1:1)) at 65 DEG C handle 1h. take out supernatant centrifugal concentrating after, 20 weight % modacrylics are gel purified to be contained
There is the oligonucleotide 8 of the 5- aldehyde radical cytimidine modification of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection, as shown in Figure 1.
As it can be seen that implementing the 5- aldehyde radical cytidine phosphoramidite of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection of 1 preparation
Monomer (7) can be used for conventional DNA synthesis in solid state and purifying.
Embodiment 3
The present embodiment indicates that the 5- aldehyde radical of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection by implementing 2 preparations
5 ' phosphorylations of the oligonucleotide of cytimidine pointed decoration are connected with DNA ligase (as shown in Fig. 1).
It include 3nmol oligonucleotide 9 (as shown in Figure 1), 1 × CutSmart buffer (20 mM Tris- by 200 μ L
Ac pH 7.9,5mM DTT, 1mM ATP) and 40U of T4 PNK reaction system be incubated for 4h at 37 DEG C after, 95 DEG C of conditions
Lower heating 20min quenching reaction.Then to reaction system be separately added into 3.5nmol oligonucleotide 10 (as shown in Figure 1) and
3.5nmol oligonucleotide 11 (as shown in Figure 1) heats cooled to room temperature after 2min under the conditions of 95 DEG C.After 1.5 μ are added
100 mM ATP and T4 DNA ligase (1600U) of L, is incubated for 12h at 16 DEG C.DNA finally is isolated with ethanol precipitation,
And purify (as shown in Figure 2) with HPLC.
As it can be seen that the 5- aldehyde radical cytimidine fixed point obtained above containing 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
The oligonucleotide of modification can be used for conventional DNA operation, including PNK enzyme carries out 5 ' phosphorylations and connects with DNA ligase.
Embodiment 4
1- (2- nitrobenzophenone) propane-on the 5- aldehyde radical cytidine phosphoramidite monomer of oligonucleotide is taken off in illumination
1,3- glycerol protection base.
It is 350nm fluorescent tube that the aqueous solution (100 μM) of oligonucleotide 11, which is placed in equipped with 16 emission peaks,
In Rayonet Photoreactor (RPR-100), illumination 5min obtains being stripped of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
The oligonucleotide 12 and 13 of the 5- aldehyde radical cytimidine Asia pointed decoration of base.By the oligomerization core of 5- aldehyde radical cytimidine pointed decoration
Thuja acid 12 and 13 carries out ultra high efficiency liquid phase and mass spectral analysis, as shown in Figures 2 and 3, it is shown that after illumination in 5 minutes, it is fixed
Amount has taken off the oligonucleotide of the 5- aldehyde radical cytimidine pointed decoration of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection base
12 and 13, it can be seen that, by illumination 5 minutes of 350nm, it can quantitatively remove 1- (2- nitrobenzophenone) propane -1,3- glycol
Protecting group can restore the intact form of 5- aldehyde radical cytimidine, can play whole biochemical functions of 5- aldehyde radical cytimidine.
As can be seen from the above embodiments, the present invention is based on 1- (2- nitrobenzophenone) propane -1,3- glycerol protection 5- aldehyde radical born of the same parents are phonetic
The aldehyde radical of pyridine is pinpointed by the 5- aldehyde radical cytimidine that DNA synthesis in solid state prepares 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
The oligonucleotide of modification.1- (2- nitrobenzophenone) propane -1,3- glycerol protection base is all stable in synthesis and purification process, obtains
To after pure oligonucleotide, which can be divided by the illumination 5 of 350nm
Clock quantitatively removes, and obtains the oligonucleotide chain of 5- aldehyde radical cytidine phosphoramidite monomer pointed decoration, can be used for biochemistry and grind
Study carefully.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention
In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to
Protection scope of the present invention.
Claims (6)
1. a kind of 5- aldehyde radical cytidine phosphoramidite monomer of photosensitive protective group protection, which is characterized in that its chemical structural formula are as follows:
Wherein, R is methyl or phenyl, and " DMTrO- " is dimethoxytrityl, " N (iPr)2" it is N, N- diisopropylamine
Base；
Wherein, the photosensitive protective group is 1- (2- nitrobenzophenone) propane -1,3- glycol.
2. the preparation method of the 5- aldehyde radical cytidine phosphoramidite monomer of photosensitive protective group protection described in claim 1, special
Sign is, method includes the following steps:
(1) by 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, 1- (2- nitrobenzophenone) propane -1,
3- glycol, triethyl orthoformate and methylene chloride carry out cooling, the first mixed solution of formation after the first mixing；Again by described first
Mixed solution is reacted with titanium tetrachloride；Then the organic phase obtained after reaction washing, dry, filtering, column are chromatographed to obtain
Reaction product；
(2) reaction product is carried out second with tetrabutyl ammonium fluoride and tetrahydrofuran to mix, forms the second mixed solution, so
Second mixed solution is rotated afterwards and removes solvent, column chromatographs to obtain 5- [4- (2- nitrobenzophenone) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine；
(3) by the 5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine and trimethylchloro-silicane
Alkane, pyridine carry out third mixing, form third mixed solution；The third mixed solution is reacted with acetic anhydride again；So
Afterwards by the organic phase obtained after reaction washing, dry, filtering, column chromatograph to obtain 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,
3- dioxane -2- base] -2 '-dideoxycytosine；
(4) by the 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine and 4,
4'- dimethoxytriphenylmethyl chlorine, pyridine are reacted, then the organic phase obtained after reaction is washed, is dry, is filtered,
Column chromatographs to obtain 5 '-(dimethoxytrityl) -4-N- acetyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine；
(5) by described 5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine and bis- (diisopropylaminoethyls) (2- cyanoethoxy) phosphine, 1H-TETRAZOLE and methylene chloride carry out instead
It answers, the solution obtained after reaction is rotated and removes solvent, then carry out column chromatography；
Wherein, in step (1), 1- (2- nitrobenzophenone) propane -1,3- glycol, 3 ', 5 '-(the tertiary butyl dimethyl
Silicon substrate) -5- aldehyde radical -2 '-dideoxycytosine, the triethyl orthoformate, the methylene chloride and the titanium tetrachloride dosage
Molar ratio be 1mmol:(3-5) mmol:(1.2-1.5) mmol:(5-7) mL:(0.4-0.5) mmol；
Described first mixed condition includes: that temperature is 18-25 DEG C, and the time is 0.5-1 hour, mixing speed be 250-500 turn/
Minute；
The condition of the cooling includes: that temperature is 0-4 DEG C, and the time is 0.1-0.5 hours, and mixing speed is 250-500 revs/min
Clock；And
The titanium tetrachloride is added in first mixed solution in a manner of being added dropwise, and reaction process packet described in step (1)
Two stages are included, the reaction condition of first stage includes: that temperature is 18-25 DEG C, and the time is 5-7 hours, mixing speed 250-
500 revs/min, the drop rate of the titanium tetrachloride is 10 drops/minute；And the reaction condition of second stage includes: temperature
It is 18-25 DEG C, the time is 5-7 hours, and mixing speed is 250-500 revs/min, and the drop rate of the titanium tetrachloride is 15
Drop/minute；
Wherein, in step (2), described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radical -2 '-dideoxycytosine, described four
The ratio of the dosage of butyl ammonium fluoride and the tetrahydrofuran is 1mmol:(3-4) mmol:(5-7) mL；And
Described second mixed condition includes: that temperature is 18-25 DEG C, and the time is 9-11 hour, mixing speed be 250-500 turn/
Minute；
Wherein, in step (3), 5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-dideoxycytosine with
The ratio of the dosage of the trim,ethylchlorosilane, the pyridine and the acetic anhydride is 1mmol:(5-7) mmol:(9-11) mL:
(1.1-1.3)mmol；And
It is 18-25 DEG C that the condition of the third mixing, which includes: temperature, and the time is 1-2 hours, and mixing speed is 250-500 revs/min
Clock；And it is 18-25 DEG C that the condition of the reaction, which includes: temperature, the time is 3-5 hours, and mixing speed is 250-500 revs/min
Clock；
Wherein, in step (4), the 4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,3- dioxane -2- base] -2 '-is de-
The ratio of the dosage of oxygen cytimidine and 4,4'- dimethoxytriphenylmethyl chlorine, pyridine is 1mmol:(1.5-2) mmol:(9-11)
mL；And
The condition of the reaction includes: that temperature is 18-25 DEG C, and the time is 12-18 hours, and mixing speed is 250-500 revs/min
Clock；
Wherein, in step (5), 5 '-(dimethoxytrityl) -4-N- acyl group -5- [4- (2- nitrobenzophenone) -1,
3- dioxane -2- base] -2 '-dideoxycytosine and bis- (diisopropylaminoethyls) (2- cyanoethoxy) phosphine, 1H-TETRAZOLE and two
The ratio of the dosage of chloromethanes is 1mmol:(1.3-1.6) mmol:(1.3-1.6) mmol:(10-30) mL；And
The condition of the reaction includes: that temperature is 18-25 DEG C, and the time is 2-4 hours, and mixing speed is 250-500 revs/min.
3. a kind of oligonucleotide of the 5- aldehyde radical cytimidine pointed decoration of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection
Preparation method, which is characterized in that the synthetic agent in the preparation method includes 1- described in claim 1 (2- nitrobenzophenone) third
The 5- aldehyde radical cytidine phosphoramidite monomer of alkane -1,3- glycerol protection.
4. preparation method according to claim 3, wherein the preparation method is enterprising in ABI 394DNA/RNA synthesizer
Row, in which:
The synthetic agent further includes Ac-dC, Bz-dA, dmf-dG and dT；
Trichloroacetic acid/methylene chloride is for being deprotected；
Cap A reagent is the composition of acetic anhydride, pyridine and tetrahydrofuran, and the acetic anhydride, the pyridine and the tetrahydro
The volume ratio of furans is 10 volume %:(5-15 volume %): (75-85 volume %)；
Cap B reagent is the composition of N- methylimidazole and tetrahydrofuran, and the N- methylimidazole and the tetrahydrofuran
Volume ratio is 10 volume %:(85-95 volume %)；
Activator is the composition of ethlythiotetrazole and acetonitrile；
Oxidant is the composition of iodine, water, pyridine and tetrahydrofuran, and the iodine, the water, the pyridine and the tetrahydro furan
The volume ratio muttered is 0.02M:(1-2 volume %): (10-30 volume %): (75-85 volume %)；And
The Coupling time of the 5- aldehyde radical cytidine phosphoramidite monomer is 4-6 minutes；
The oligonucleotide that above-mentioned solid phase synthesizes is handled into 1-2h with 1 milliliter of AMA at 60-70 DEG C, then, takes out supernatant
After liquid centrifugal concentrating, the modacrylic using 20 weight % is gel purified；And
The AMA is the mixed solution of 40% methylamine water solution and 28% ammonium hydroxide, and the methylamine water solution and the ammonium hydroxide
The ratio of volume is 1:(0.5-1.5).
5. the 5- of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection that preparation method described in claim 3 or 4 is prepared
The oligonucleotide of aldehyde radical cytimidine pointed decoration is being used for the operational application of DNA, which is characterized in that the DNA is operational
Using including carrying out 5 ' phosphorylations with PNK enzyme, ligase is attached, and the group of DNA- protein complexes is carried out with albumen
Dress.
6. a kind of preparation method of the oligonucleotide containing 5- aldehyde radical cytimidine pointed decoration, which is characterized in that this method packet
Include the 5- aldehyde of 1- (2- nitrobenzophenone) propane -1,3- glycerol protection that preparation method described in claim 3 or 4 is prepared
The oligonucleotide of base cytimidine pointed decoration illumination 4-6 minutes in-situ preparation under the illumination of 340-360nm obtains.
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