CN107474091A - The synthesis and application of 5 aldehyde radical cytidine phosphoramidite monomers of photosensitive protective group protection and preparation method thereof and oligonucleotide - Google Patents
The synthesis and application of 5 aldehyde radical cytidine phosphoramidite monomers of photosensitive protective group protection and preparation method thereof and oligonucleotide Download PDFInfo
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- CN107474091A CN107474091A CN201710600724.3A CN201710600724A CN107474091A CN 107474091 A CN107474091 A CN 107474091A CN 201710600724 A CN201710600724 A CN 201710600724A CN 107474091 A CN107474091 A CN 107474091A
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- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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Abstract
The present invention relates to the field of chemical synthesis of oligonucleotide, discloses the synthesis and application of 5 aldehyde radical cytidine phosphoramidite monomers of photosensitive protective group protection and preparation method thereof and oligonucleotide.Shown in the chemical constitution such as formula (1) of 5 aldehyde radical cytidine phosphoramidite monomers of photosensitive protective group protection, wherein, R is methyl or phenyl.5 aldehyde radical cytidine phosphoramidite monomer of the present invention is used for the nucleotides for preparing 5 aldehyde radical cytimidine pointed decorations, and 5 aldehyde radical cytimidines are particularly with being had a wide range of applications in terms of protein interaction in the biochemical property for studying 5 aldehyde radical cytimidines.
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- nitrobenzophenones) propane-
The 5- of 5- aldehyde radical cytidine phosphoramidite monomers and 1- (2- nitrobenzophenones) propane -1,3- glycerol protections of 1,3- glycerol protections
The synthesis and application of the oligonucleotide of aldehyde radical cytimidine pointed decoration.
Background technology
5- aldehyde radical cytimidines are a kind of new base existence form [Science found recently in eukaryotic
2011,333,1300‐1303].5- aldehyde radical cytimidines are generally considered to be centre of the 5-methylcytosine during demethylation
Body [Chem.Rev.2015,115,2225-2239], but also evidence suggests 5- aldehyde radicals cytimidine can be long-term in vivo
Be stabilized, and the 26S Proteasome Structure and Function of genomic DNA is produced 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 cytimidines in DNA, it is necessary to prepare 5- aldehyde radical cytimidine pointed decorations
Oligonucleotide.Conventional means are first to synthesize 5- aldehyde radical cytidine phosphoramidite monomers, then pass through the plan of DNA synthesis in solid state
Slightly 5- aldehyde radical cytidine phosphoramidites monomer is pinpointed and introduces oligonucleotide, then ammoniacal liquor is deprotected and purifies to obtain pure widow
Polynucleotide is used for follow-up Biochemical Research.The aldehyde radical of 5- aldehyde radical cytidine phosphoramidite monomers be generally acknowledged that it is more stable,
All keep constant 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 phases
Into.If recently also it has been reported that not protecting the aldehyde radical of 5- aldehyde radical cytidine phosphoramidite monomers, DNA is synthesized and was deprotected
Journey has some side reactions, therefore has developed 1,3-PD and protected 5 aldehyde radicals, recovers 5- by acid treatment after ammoniacal liquor deprotection
Aldehyde radical [Angew.Chem.Int.Ed.Engl.2014,53,315-318.].
In some cases, such as during research 5- aldehyde radical cytidine phosphoramidite monomers and protein interaction, it is necessary to
One stable in DNA synthesis and purge process, but can efficient quantitative removes under conditions of more gentle 5- aldehyde radicals
Protection group.
Therefore, the DNA synthesis and deprotection procedure that Development of Novel can be with routine are compatible, efficiently fixed under mild conditions
5 aldehyde radical protection groups for measuring the 5- aldehyde radical cytidine phosphoramidite monomers of removing are necessary.
The content of the invention
The defects of the invention aims to overcome prior art to exist, there is provided a kind of to be related to what photosensitive protective group was protected
The synthesis and application of 5- aldehyde radical cytidine phosphoramidite monomers and preparation method thereof and oligonucleotide, and in particular to a kind of 1-
5- aldehyde radical cytidine phosphoramidite monomers of (2- nitrobenzophenones) propane -1,3- glycerol protections and preparation method thereof and 1- (2-
Nitrobenzophenone) propane -1,3- glycerol protections 5- aldehyde radical cytimidine pointed decorations oligonucleotide synthesis and application.
To achieve these goals, it is phonetic to provide a kind of 5- aldehyde radical born of the same parents of photosensitive protective group protection for first aspect present invention
Pyridine phosphoramidite monomer, wherein, the 5- aldehyde radical cytidine phosphoramidite monomer chemistries structural formulas are：
Wherein, R is methyl or phenyl.
Second aspect of the present invention provides a kind of system of the 5- aldehyde radical cytidine phosphoramidite monomers of photosensitive protective group protection
Preparation Method, wherein, this method comprises the following steps：
(1) by 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, 1- (2- nitrobenzophenones) third
Alkane -1,3- glycol, triethyl orthoformate and dichloromethane cool down after carrying out the first mixing, form the first mixed solution；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, post layer
Analysis obtains reaction product；
(2) reaction product and the tetrabutyl ammonium fluoride and the tetrahydrofuran are carried out into second mix, formation the
Two mixed solutions, then by second mixed solution rotate remove solvent, column chromatography obtain 5- [4- (2- nitrobenzophenones) -1,
3- dioxane -2- bases] -2 '-dideoxycytosine；
(3) by the 5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosines and trimethyl chlorine
Silane, pyridine carry out the 3rd mixing, form the 3rd mixed solution；The 3rd mixed solution is reacted with acetic anhydride again；
Then by the organic phase obtained after reaction washing, dry, filtering, column chromatography obtain 4-N- acyl groups -5- [4- (2- nitrobenzophenones) -
1,3- dioxane -2- bases] -2 '-dideoxycytosine；
(4) by the 4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosine
Reacted with 4,4'- dimethoxytriphenylmethyls chlorine, pyridine, then by the organic phase obtained after reaction washing, dry, mistake
Filter, column chromatography obtain 5 '-(dimethoxytrityl) -4-N- acetyl group -5- [4- (2- nitrobenzophenones) -1,3- dioxies six
Ring -2- bases] -2 '-dideoxycytosine；
(5) by described 5 '-(dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxies six
Ring -2- bases] -2 '-dideoxycytosine enters with double (diisopropylaminoethyl) (2- cyanoethoxies) phosphine, 1H-TETRAZOLE and dichloromethane
Row reaction, the solution obtained after reaction is rotated and removes solvent, then carries out column chromatography.
Preferably, in step (1), 1- (2- nitrobenzophenones) propane -1,3- glycol, 3 ', the 5 '-(tertiary butyl
Dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, the triethyl orthoformate, the dichloromethane and the titanium tetrachloride
The mol ratio of dosage be 1mmol:(3-5)mmol:(1.2-1.5)mmol: (5-7)mL：(0.4-0.5)mmol；
The condition of first mixing includes：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：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 a manner of being added dropwise in first mixed solution, and the reaction described in step (1)
Journey includes two stages, and the reaction condition of first stage includes：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 drops/minute；And the reaction condition bag 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 drops/minute.
Preferably, in step (2), described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine,
The ratio of the dosage of the tetrabutyl ammonium fluoride and the tetrahydrofuran is 1mmol:(3-4)mmol: (5-7)mL；And
The condition of second mixing includes：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- nitrobenzophenones) -1,3- dioxane -2- bases]
Cytimidine and the ratio of the dosage of the trim,ethylchlorosilane, the pyridine and the acetic anhydride are 1mmol:(5-7)mmol:(9-
11)mL：(1.1-1.3)mmol；And
The condition of 3rd mixing includes：Temperature is 18-25 DEG C, and the time is 1-2 hours, mixing speed 250-500
Rev/min；And the condition of the reaction includes：Temperature is 18-25 DEG C, and the time is 3-5 hours, mixing speed 250-500
Rev/min.
Preferably, in step (4), the 4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -
The ratio of 2 '-dideoxycytosine and 4,4'- dimethoxytriphenylmethyls chlorine, the dosage of pyridine is 1mmol:(1.5-2)mmol:
(9-11)mL；And
The condition of the reaction includes：Temperature is 18-25 DEG C, and the time be 12-18 hours, mixing speed is 250-500 turns/
Minute.
Preferably, in step (5), described 5 '-(dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitrobenzene
Base) -1,3- dioxane -2- bases] -2 '-dideoxycytosine and double (diisopropylaminoethyl) (2- cyanoethoxies) phosphine, 1H- tetra-
The ratio of the dosage of azoles and dichloromethane is 1mmol:(1.3-1.6)mmol:(1.3 -1.6)mmol:(10-30)mL；And
The condition of the reaction includes：Temperature is 18-25 DEG C, and the time be 2-4 hours, mixing speed is 250-500 turns/
Minute.
Third aspect present invention provides a kind of 5 aldehyde radical cytimidines of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections
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 monomers for the photosensitive protective group protection that method is prepared.
Preferably, the preparation method is carried out on ABI 394DNA/RNA synthesizers, wherein：
Preferably, the synthetic agent also includes Ac-dC, Bz-dA, dmf-dG and dT；
Preferably, trichloroacetic acid/dichloromethane is used to be deprotected；
Preferably, Cap A reagents are 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 reagents are the composition of N- methylimidazoles and tetrahydrofuran, and N- methylimidazoles 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
State
Preferably, the Coupling time of the 5- aldehyde radical cytidine phosphoramidite monomers of the photosensitive protective group protection is 4-6 points
Clock；
Preferably, the oligonucleotide for above-mentioned solid phase being synthesized to obtain handles 1-2h with 1 milliliter of AMA at 60-70 DEG C, so
Afterwards, after taking out supernatant centrifugal concentrating, the modacrylic using 20 weight % is gel purified；And
Preferably, the AMA is the mixed solution of 40% methylamine water solution and 28% ammoniacal liquor, and the methylamine water solution and
The ratio of the volume of the ammoniacal liquor is 1:(0.5-1.5).
Fourth aspect present invention provide 1- (2- nitrobenzophenones) propane for being prepared by preparation method described above-
The oligonucleotide of the 5- aldehyde radical cytimidine pointed decorations of 1,3- glycerol protection for the operational applications of DNA, wherein, should
The operational applications of DNA include carrying out 5 ' phosphorylations with PNK enzymes, and ligase is attached, and carry out DNA- albumen with albumen
The assembling of complex.
The aspect of present aspect the 5th provides a kind of preparation side of the oligonucleotide containing 5- aldehyde radical cytimidine pointed decorations
Method, wherein, 1- (2- nitrobenzophenones) propane -1,3- glycol that this method includes preparation method described above being prepared is protected
The oligonucleotide of the 5- aldehyde radical cytimidine pointed decorations of shield illumination 4-6 minute in-situ preparations under 340-360nm illumination obtain
Arrive.
It is an advantage of the invention that：(1) the 5- aldehyde radical cytimidine phosphorous acyls of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections
Amine monomers synthetic method is simple；(2) 1- (2- nitrobenzophenones) propane -1,3- glycol of 5- aldehyde radicals cytidine phosphoramidite monomer is protected
Base is protected all stable in oligonucleotide synthesis and purge process；(3) after obtaining pure oligonucleotide, 1- (2- nitrobenzene
Base) protection group can quantify removing by propane -1,3- glycerol protections base by illumination experiment.The removing mild condition, and almost
All biosystems are compatible.The synthetic method of the nucleotides of 5- aldehyde radicals cytimidine pointed decoration of the present invention, is being studied
Before the biochemical property of 5- aldehyde radical cytimidines particularly 5- aldehyde radicals cytimidine has a wide range of applications with protein interaction aspect
Scape.
Brief description of the drawings
Fig. 1 is the oligonucleotides of the 5- aldehyde radicals cytimidine modification containing 1- (2- nitrobenzophenones) propane -1,3- glycerol protections
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.
Embodiment
The end points of disclosed scope and any value are not limited to the accurate scope or value herein, these scopes or
Value should be understood to comprising the value close to these scopes or value.For number range, between the endpoint value of each scope, respectively
It can be combined with each other between the endpoint value of individual scope and single point value, and individually between point value and obtain one or more
New number range, these number ranges should be considered as specific open herein.
First aspect present invention provides a kind of 5- aldehyde radicals cytidine phosphoramidite monomer, wherein, its chemical structural formula is：
Wherein, R is methyl or phenyl.
Compound shown in formula (1) is properly termed as 3 '-O- (N, N- diisopropyl amido-O-beta- cyanoethoxyls-phosphine
Base) -5 '-(dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 ' -
Dideoxycytosine.
Wherein, in formula (1), " DMTrO- " is dimethoxytrityl, " N (iPr)2- " it is N, N- diisopropylamines
Base.
Second aspect of the present invention provides the preparation method of 5- aldehyde radical cytidine phosphoramidite monomers described above, its
In, this method comprises the following steps：
(1) by 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, 1- (2- nitrobenzophenones) third
Alkane -1,3- glycol, triethyl orthoformate and dichloromethane cool down after carrying out the first mixing, form the first mixed solution；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, post layer
Analysis obtains reaction product；
Will be after the first reaction wherein it is possible to be washed successively from saturated sodium bicarbonate aqueous solution, saturated nacl aqueous solution
Organic phase, then dried from anhydrous magnesium sulfate, then will be revolved after washing dried first mixed solution filtering
Dry filtrate, column chromatography obtain the reaction product of faint yellow solid.
(2) reaction product and the tetrabutyl ammonium fluoride and the tetrahydrofuran are carried out into second mix, formation the
Two mixed solutions, then by second mixed solution rotate remove solvent, column chromatography obtain 5- [4- (2- nitrobenzophenones) -1,
3- dioxane -2- bases] -2 '-dideoxycytosine；
Wherein, the tetrahydrofuran is dry tetrahydrofuran, and column chromatography obtains 5- [4- (2- nitrobenzophenones) -1,3- bis-
Ring -2- the bases of oxygen six] -2 '-dideoxycytosine is faint yellow solid.
(3) by the 5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosines and trimethyl chlorine
Silane, pyridine carry out the 3rd mixing, form the 3rd mixed solution；Again by the 3rd mixed solution and acetic anhydride (chlorobenzoyl chloride)
Reacted；Then the organic phase obtained after reaction washing, dry, filtering, column chromatography are obtained into 4-N- acyl groups -5- [4- (2- nitre
Base phenyl) -1,3- dioxane -2- bases] -2 '-dideoxycytosine；
Wherein, the pyridine is dry pyridine, can be successively from dichloromethane extraction, saturated sodium bicarbonate aqueous solution
Washing is dried through the 3rd reacted organic phase, then from anhydrous magnesium sulfate, is spin-dried for filtrate after then filtering, column chromatography obtains light
The reaction product of yellow solid.
(4) by the 4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosine
Reacted with 4,4'- dimethoxytriphenylmethyls chlorine, pyridine, then by the organic phase obtained after reaction washing, dry, mistake
Filter, column chromatography obtain 5 '-(dimethoxytrityl) -4-N- acetyl group -5- [4- (2- nitrobenzophenones) -1,3- dioxies six
Ring -2- bases] -2 '-dideoxycytosine；
Wherein, the pyridine is dry pyridine, can be molten from dichloromethane extraction, saturated sodium bicarbonate water successively
Liquid, saturated sodium-chloride water solution washing are dried through the 4th reacted organic phase, then from anhydrous magnesium sulfate, are revolved after then filtering
Dry filtrate, column chromatography obtain the reaction product of faint yellow solid.
(5) by described 5 '-(dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxies six
Ring -2- bases] -2 '-dideoxycytosine enters with double (diisopropylaminoethyl) (2- cyanoethoxies) phosphine, 1H-TETRAZOLE and dichloromethane
Row reaction, the solution obtained after reaction is rotated and removes solvent, then carries out column chromatography.
In the present invention, 3 '-O- (N, N- diisopropyl amido-O-beta- cyanogen can be obtained through above-mentioned preparation method
Ethyoxyl-phosphino-) -5 '-(dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine；
Wherein, the dichloromethane is dry dichloromethane.
According to the present invention, wherein, in step (1), 1- (2- nitrobenzophenones) propane -1,3- glycol, 3 ',
5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, the triethyl orthoformate, the dichloromethane and institutes
The mol 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, the 1- (2- nitrobenzophenones) propane -1,3- glycol equivalent to 1mmol, described 3 ', 5 '-(special
Butyldimethyl silicon substrate) dosage of -5- aldehyde radicals -2 '-dideoxycytosine is 3-5mmol, the dosage of the triethyl orthoformate is
1.2-1.5mmol, the dosage of the dichloromethane is 5-7mL, and the dosage of the titanium tetrachloride is 0.4-0.5mmol；Although institute
State 1- (2- nitrobenzophenones) propane -1,3- glycol, 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-deoxidation born of the same parents
Pyrimidine, the triethyl orthoformate, the mol ratio of dosage of the dichloromethane and the titanium tetrachloride meet above range i.e.
The present invention can be completed, it is, however, preferable that the 1- (2- nitrobenzophenones) propane -1,3- glycol, 3 ', the 5 '-(tertiary butyl two
Methylsilyl) -5- aldehyde radicals -2 '-dideoxycytosine, the triethyl orthoformate, the dichloromethane and the titanium tetrachloride
The mol ratio of dosage is 1mmol:4 mmol:1.2mmol:6mL：During 0.4mmol；Effect is more preferable；
The condition of first mixing can include：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 can include：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 a manner of being added dropwise in first mixed solution, and the reaction described in step (1)
Journey includes two stages, and the reaction condition of first stage includes：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 drops/minute, preferably 10 drops/minute；It is more preferable that effect is carried out under optimum condition；And
The condition of the second stage can include：Temperature is 18-25 DEG C, and preferably 20 DEG C, the time is 5-7 hours, excellent
Elect as 6 hours, mixing speed is 250-500 revs/min, and preferably 400 revs/min, the drop rate of the titanium tetrachloride is
10-15 drops/minute, preferably 15 drops/minute, it is more preferable that effect are carried out under the preferred conditions.
According to the present invention, in step (2), described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -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 the silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine equivalent to 1mmol, 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
The present invention can be completed by enclosing, it is, however, preferable that described 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-deoxidation born of the same parents are phonetic
The ratio of the dosage of pyridine, the tetrabutyl ammonium fluoride and the tetrahydrofuran is 1mmol:4mmol:During 6mL, effect is more preferable；And
The condition of second mixing can include：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- nitrobenzophenones) -1,3- dioxane -2- bases] -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, the 5- [4- (the 2- nitros equivalent to 1mmol
Phenyl) -1,3- dioxane -2- bases] -2 '-dideoxycytosine and the trim,ethylchlorosilane, the use of the trim,ethylchlorosilane
Measure as 5-7mmol, 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
Manage each dosage and meet that above range can complete the present invention, it is, however, preferable that the 5- [4- (2- nitrobenzophenones) -1,3-
Dioxane -2- bases] -2 '-dideoxycytosine and the trim,ethylchlorosilane, the pyridine and the acetic anhydride (benzoyl
Chlorine) the ratio of dosage be 1mmol:6mmol:10mL：During 1.2mmol, effect is more preferable；And
The condition of 3rd mixing can include：Temperature is 18-25 DEG C, and the time is 1-2 hours, mixing speed 250-
500 revs/min；And the condition of the reaction includes：Temperature is 18-25 DEG C, and the time is 3-5 hours, mixing speed 250-
500 revs/min；
Preferably, the condition of the 3rd mixing includes：Temperature is 20 DEG C, and the time is 1.5 hours, mixing speed 400
Rev/min；And the condition of the reaction includes：Temperature is 19-21 DEG C, and 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 groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine and 4,4'- dimethoxytriphenylmethyls chlorine, the ratio of dosage of pyridine can be 1mmol:(1.5-
2)mmol:(9-11)mL；That is, the 4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxies equivalent to 1mmol
Six ring -2- bases] -2 '-dideoxycytosine, the dosage of 4, the 4'- dimethoxytriphenylmethyls chlorine is 1.5-2mmol, described
The dosage of pyridine is 9-11mL；Although each dosage meets that above range can complete the present invention, it is, however, preferable that the 4-
N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosines and 4,4'- dimethoxy triphens
Ylmethyl chlorine, the ratio of dosage of pyridine can be 1mmol:2mmol:During 10mL, effect is more preferable；And
The condition of the reaction can include：Temperature is 18-25 DEG C, and the time is 12-18 hours, mixing speed 250-
500 revs/min；
Preferably, the condition of the reaction includes：Temperature is 20 DEG C, and the time is 16 hours, and mixing speed is 400 revs/min
Clock.
According to the present invention, in step (5), described 5 '-(dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitre
Base phenyl) -1,3- dioxane -2- bases] -2 '-dideoxycytosine and double (diisopropylaminoethyl) (2- cyanoethoxies) phosphines,
The ratio of the dosage of 1H-TETRAZOLE and dichloromethane is 1mmol:(1.3-1.6)mmol: (1.3-1.6)mmol:(10-30)mL；
That is described 5 '-(the dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- equivalent to 1mmol
Dioxane -2- bases] -2 '-dideoxycytosine, the dosage of described double (diisopropylaminoethyl) (2- cyanoethoxies) phosphines is 1.3-
1.6mmol, the dosage of the 1H- tetrazoliums is 1.3-1.6mmol, and the dosage of the dichloromethane is 10-30mL；It is although each
Dosage meets that above range can complete the present invention, it is, however, preferable that described 5 '-(dimethoxytrityl) -4-N- acyls
Base -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosines and double (diisopropylaminoethyl) (2- cyanogen
Base oxethyl) ratio of dosage of phosphine, 1H-TETRAZOLE and dichloromethane is 1mmol:1.5 mmol:1.5mmol:During 10mL, effect is more
It is good；And
The condition of the reaction can include：Temperature is 18-25 DEG C, and the time is 2-4 hours, mixing speed 250-500
Rev/min；
Preferably, the condition of the reaction includes：Temperature is 20 DEG C, 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 cytimidines of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections
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 monomers of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections that method is prepared.
According to the present invention, the preparation method can be entered on ABI 394DNA/RNA synthesizers by conventional program and reagent
The synthesis of row oligonucleotide is carried out, wherein：
Preferably, the synthetic agent can also include Ac-dC, Bz-dA, dmf-dG and dT；
Preferably, trichloroacetic acid/dichloromethane can be selected to be used to be deprotected；
Preferably, Cap A reagents can be the composition of acetic anhydride, 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,
Said composition contains 10 volume % acetic anhydride, 5-15 volumes % pyridine, 75-85 volumes % tetrahydrofuran；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 reagents can be the composition of N- methylimidazoles and tetrahydrofuran, and the N- methylimidazoles
Volume ratio with the tetrahydrofuran can be 10 volume %：(85-95 volume %)；That is, said composition contains 10 bodies
The product % N- methylimidazoles, 85-95 volumes % tetrahydrofuran；Preferably, the N- methylimidazoles and the tetrahydrochysene furan
The volume ratio muttered is 10 volume %：(88-92 volume %)；
Preferably, activator is the composition of ethlythiotetrazole and acetonitrile, the activator can be commercially available or
Person synthesizes to obtain using the method for prior art, and in the present invention, the activator is commercially available from, and the concentration of the activator
For 0.25M, i.e. 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 said composition contains 0.02M iodine, 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 bodies
Product %)：(15-25 volume %)：(76-80 volume %)；And
Preferably, the Coupling time of the 5- aldehyde radicals cytidine phosphoramidite monomer can be 4-6 minutes, preferably 4.5-
5.5 minute；
Preferably, the oligonucleotide for above-mentioned solid phase being synthesized to obtain can handle 1- with 1 milliliter of AMA at 60-70 DEG C
2h, then, after taking out supernatant centrifugal concentrating, the modacrylic using 20 weight % is gel purified；And
Preferably, the AMA is the mixed solution of 40% methylamine water solution and 28% ammoniacal liquor, and the methylamine water solution and
The ratio of the volume of the ammoniacal liquor 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- nitrobenzophenones) being prepared by preparation method described above
The oligonucleotide of the 5- aldehyde radical cytimidine pointed decorations of propane -1,3- glycerol protection for the operational applications of DNA, its
In, the operational applications of the DNA include carrying out 5 ' phosphorylations with PNK enzymes, and ligase is attached, and carry 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 radicals cytimidine pointed decoration, its
In, this method includes 1- (2- nitrobenzophenones) propane -1,3- glycerol protection that preparation method described above is prepared
The oligonucleotide of 5- aldehyde radical cytimidine pointed decorations illumination 4-6 minute in-situ preparations under 340-360nm illumination obtain.
Preferably, in the present invention, the illumination 4-6 minutes under 340-360nm illumination are passed through when any need, it is preferable that
Pass through the illumination 4.5-5.5 minutes under 345-355nm illumination, you can quantitatively remove 1- (2- nitrobenzophenones) propane -1,3- bis-
Alcohol protection group, recover the intact form of 5- aldehyde radical cytidine phosphoramidite monomers, play 5- aldehyde radical cytidine phosphoramidite monomers
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 radicals -2 '-dideoxycytosines (self-control), 1- (2- nitrobenzophenones) 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 acid carbonate (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)), tetrahydrochysene
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 cytimidines of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections of the present invention
The synthesis of phosphoramidite monomer.
Its synthesis step is as follows：
The preparation of compound (4)：Weigh monomer shown in 1.31g (2.71mmol, 1.0eq (equivalent)) formula (2) (wherein,
In formula (2), " TBS- " is tertiary butyl dimethyl silicon substrate), dissolved under argon gas protection with 20mL dry methylene chlorides；Then to
2.14g (10.84 mmol, 4eq) 1- (2- nitrobenzophenones) propane -1,3- glycol and 537uL are 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)
(1.08mmol, 0.4eq, 1M dichloromethane solution (that is, the titanium tetrachloride containing 1M in dichloromethane)) titanium tetrachloride is molten
Liquid, it is warmed to room temperature (20 DEG C).After stirring 6h, 542 μ L (0.54mmol, 0.2eq, 1M dichloromethane solution) four chlorinations are added dropwise
Titanium solution continues to react 6h.Reaction is quenched from saturated sodium bicarbonate aqueous solution, is extracted with dichloromethane (80mL), what is separated has
Machine is mutually washed with saturation NaCl solution (20 mL).Anhydrous magnesium sulfate is dried, and revolving removes solvent and obtained shown in crude product formula (3)
Monomer.The product of acquisition is dissolved in the tetrahydrofuran of 20mL dryings under argon gas protection, adds 4.34mL (4.34 mmol, 1M
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 solids (4) (1.36mmol, the weight % of yield 50).Production
Thing is a pair of diastereoisomers (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)：The monomer shown in 594mg (1.37mmol, 1.0eq) formula (4) is weighed, with dry pyridine
After corotation 2 times, argon gas protection is lower to use 15mL pyridinium dissolutions.1.27mL (3.5mmol, 6eq) trimethyl chlorine is added to reaction system
Silane simultaneously adds 150 μ L (1.51mmol, 1.1eq) acetic anhydrides under normal temperature condition after stirring 1.5h, continues under normal temperature (25 DEG C)
After reacting 4h, it is quenched with 5mL distilled water, revolving removes solvent.Add appropriate saturated sodium bicarbonate solution and use dichloromethane
(60mL) is extracted, and merges organic phase, and anhydrous magnesium sulfate is dried.Revolving remove solvent, with ethanol/methylene (v/v, 3%~
4%~6%) column chromatography is carried out, obtains monomer (0.73mmol, 54%) of the 350mg faint yellow solids as shown in formula (5).Product is
A pair of diastereoisomers (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 shown in 0.33g (0.95mmol, 1eq) formula (5) monomer (in formula (5),
" DMTr- " is dimethoxytrityl), the pyridinium dissolution dried under argon gas protection with 10mL.It is then slowly added into 470mg
(1.38mmol, 2eq) 4,4'- dimethoxytriphenylmethyl chlorine, is stirred at room temperature 16h.After TLC monitoring reactions completely, add
Reaction is quenched in 10mL saturated sodium bicarbonates, and is extracted with dichloromethane, and one is washed with the NaCl solution of saturation after merging organic phase
Secondary, anhydrous magnesium sulfate is dried.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 non-a pair
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 shown in 100mg (0.13mmol, 1eq) formula (6) monomer (in formula (6), N
(iPr)2- " it is N, N- diisopropylaminoethyls), 13.5mg (0.19mmol, 1.5eq) 1H-TETRAZOLEs in 25mL flasks, protect by argon gas
The lower 5mL dry methylene chlorides of shield dissolve.Then add double (diisopropylaminoethyl) (the 2- cyano group of 62 μ L (0.19mmol, 1.5eq)
Ethyoxyl) phosphine, stirs 3h under normal temperature.After reaction terminates, by system evaporated under reduced pressure under argon gas protection, then with a small amount of dichloromethane
Alkane dissolves, and rapid column chromatography ethanol/methylene (v/v, 0.5%~1% ,+2% triethylamine) purifying obtains light yellow solid
108mg (0.11mmol, the weight % of yield 85).Product has 4 isomers, and ratio is：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 radicals by implementing 1 1- (2- nitrobenzophenones) propane -1,3- glycerol protections prepared
Cytidine phosphoramidite monomer (7) carries out DNA synthesis in solid state DNA sequence dna 8：CGTTT7AGCGGTGCTAG (as shown in Figure 1).
1mol oligonucleotide synthesis is carried out by conventional program and reagent on the DNA/RNA synthesizers of ABI 394.Close
Include Ac-dC, Bz-dA, dmf-dG, dT and the 5- aldehyde radical cytidine phosphoramidite monomers for implementing 1 preparation into reagent；3 weight %
Trichloroacetic acid/dichloromethane is used to be deprotected；Cap A reagents：The volume % tetra- of volume % pyridines of 10 volume % acetic anhydrides/10/80
Hydrogen furans (v/v/v), Cap B reagents：The volume % tetrahydrofurans (v/v) of 10 volume %N- methylimidazoles/90；Activator：
0.25M ethlythiotetrazoles/acetonitrile；Oxidant：The volume % tetrahydrofurans of volume % pyridines of the volume % water of 0.02M iodine/2/20/78
(v/v/v).The Coupling time of 5- aldehyde radical cytidine phosphoramidite monomers is 5 minutes.
Above-mentioned solid phase synthesizes obtained carrier 1 milliliter of AMA (40 weight % methylamine water solutions：28 weight % ammoniacal liquor (volumes
Than 1:1) after 1h. taking-up supernatant centrifugal concentratings) are handled at 65 DEG C, 20 weight % modacrylics are gel purified to be contained
There is the oligonucleotide 8 of the 5- aldehyde radicals cytimidine modification of 1- (2- nitrobenzophenones) propane -1,3- glycerol protection, as shown in Figure 1.
It can be seen that implement the 5- aldehyde radical cytidine phosphoramidites of 1 1- (2- nitrobenzophenones) propane -1,3- glycerol protection prepared
Monomer (7) can be used for conventional DNA synthesis in solid state and purifying.
Embodiment 3
The present embodiment indicates that the 5- aldehyde radicals by implementing 2 1- (2- nitrobenzophenones) propane -1,3- glycerol protections prepared
5 ' phosphorylations of the oligonucleotide of cytimidine pointed decoration connect (as shown in Fig. 1) with DNA ligase.
200 μ L are included into 3nmol oligonucleotides 9 (as shown in Figure 1), 1 × CutSmart buffer (20 mM Tris-
Ac pH 7.9,5mM DTT, 1mM ATP) and 40U of T4 PNK reaction system be incubated 4h at 37 DEG C after, 95 DEG C of conditions
Reaction is quenched in lower heating 20min.Then to reaction system be separately added into 3.5nmol oligonucleotides 10 (as shown in Figure 1) and
3.5nmol oligonucleotides 11 (as shown in Figure 1), room temperature is naturally cooled to after heating 2min under the conditions of 95 DEG C.After add 1.5 μ
MM ATP and T4 the DNA ligase (1600U) of L 100,12h is incubated at 16 DEG C.DNA finally is isolated with ethanol precipitation,
And purified (as shown in Figure 2) with HPLC.
It can be seen that the 5- aldehyde radicals cytimidine fixed point obtained above containing 1- (2- nitrobenzophenones) propane -1,3- glycerol protection
The oligonucleotide of modification can be used for conventional DNA operations, including PNK enzymes carry out 5 ' phosphorylations and connected with DNA ligase.
Embodiment 4
Illumination take off 1- (2- nitrobenzophenones) propane on the 5- aldehyde radical cytidine phosphoramidite monomers of oligonucleotide-
1,3- glycerol protection bases.
It is 350nm fluorescent tubes that the aqueous solution (100 μM) of oligonucleotide 11, which is placed in equipped with 16 emission peaks,
In Rayonet Photoreactors (RPR-100), illumination 5min, obtain being stripped of 1- (2- nitrobenzophenones) propane -1,3- glycerol protection
The oligonucleotide 12 and 13 of the 5- aldehyde radical cytimidines Asia pointed decoration of base.By the oligomerization core of 5- aldehyde radical cytimidine pointed decorations
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 decorations of 1- (2- nitrobenzophenones) propane -1,3- glycerol protection bases
12 and 13, it can be seen that, pass through 350nm illumination 5 minutes, you can quantitatively remove 1- (2- nitrobenzophenones) propane -1,3- glycol
Protection group, the intact form of 5- aldehyde radical cytimidines can be recovered, whole biochemical functions of 5- aldehyde radical cytimidines can be played.
It is phonetic based on 1- (2- nitrobenzophenones) propane -1,3- glycerol protection 5- aldehyde radical born of the same parents from above-described embodiment, the present invention
The aldehyde radical of pyridine, the 5- aldehyde radicals cytimidine that 1- (2- nitrobenzophenones) propane -1,3- glycerol protection is prepared by DNA synthesis in solid state pinpoint
The oligonucleotide of modification.1- (2- nitrobenzophenones) propane -1,3- glycerol protection base is all stable in synthesis and purge process, obtains
To after pure oligonucleotide, 1- (2- nitrobenzophenones) propane -1,3- glycerol protections base can be divided by 350nm illumination 5
Clock quantitatively removes, and obtains the oligonucleotide chain of 5- aldehyde radical cytidine phosphoramidite monomer pointed decorations, can be used for biochemistry and grind
Study carefully.
The preferred embodiment of the present invention described in detail above, still, the present invention is not limited thereto.In the skill of the present invention
In art concept, technical scheme can be carried out a variety of simple variants, including each technical characteristic with it is any its
Its suitable method is combined, and these simple variants and combination should equally be considered as content disclosed in this invention, belong to
Protection scope of the present invention.
Claims (11)
1. the 5- aldehyde radical cytidine phosphoramidite monomers of a kind of photosensitive protective group protection, it is characterised in that its chemical structural formula is：
Wherein, R is methyl or phenyl.
A kind of 2. preparation method of the 5- aldehyde radical cytidine phosphoramidite monomers of photosensitive protective group protection, it is characterised in that the party
Method comprises the following steps：
(1) by 3 ', 5 '-(tertiary butyl dimethyl silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, 1- (2- nitrobenzophenones) propane -1,
3- glycol, triethyl orthoformate and dichloromethane cool down after carrying out the first mixing, form the first mixed solution；Again by described first
Mixed solution is reacted with titanium tetrachloride；Then the organic phase obtained after reaction washing, dry, filtering, column chromatography are obtained
Reaction product；
(2) reaction product is carried out into second with the tetrabutyl ammonium fluoride and the tetrahydrofuran to mix, it is mixed forms second
Solution is closed, then second mixed solution is rotated and removes solvent, column chromatography obtains 5- [4- (2- nitrobenzophenones) -1,3- bis-
Ring -2- the bases of oxygen six] -2 '-dideoxycytosine；
(3) by the 5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosines and trimethylchloro-silicane
Alkane, pyridine carry out the 3rd mixing, form the 3rd mixed solution；The 3rd mixed solution is reacted with acetic anhydride again；So
Afterwards by the organic phase obtained after reaction washing, dry, filtering, column chromatography obtain 4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,
3- dioxane -2- bases] -2 '-dideoxycytosine；
(4) by the 4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosine and 4,
4'- dimethoxytriphenylmethyls chlorine, pyridine are reacted, then the organic phase obtained after reaction is washed, dried, is filtered,
Column chromatography obtains 5 '-(dimethoxytrityl) -4-N- acetyl group -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine；
(5) by described 5 '-(dimethoxytrityl) -4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2-
Base] -2 '-dideoxycytosine carried out with double (diisopropylaminoethyl) (2- cyanoethoxies) phosphines, 1H-TETRAZOLE and dichloromethane it is anti-
Should, the solution obtained after reaction is rotated and removes solvent, then carries out column chromatography.
3. preparation method according to claim 2, wherein, in step (1), 1- (2- nitrobenzophenones) propane -1,
3- glycol, 3 ', 5 '-(tertiary butyl dimethyl the silicon substrate) -5- aldehyde radicals -2 '-dideoxycytosine, the triethyl orthoformate, institute
The mol ratio for stating the dosage of dichloromethane and the titanium tetrachloride is 1mmol：(3-5mmol)mmol:(1.2-1.5)mmol:(5-
7)mL：(0.4-0.5)mmol；
The condition of first mixing includes：Temperature is 18-25 DEG C, and the time be 0.5-1 hours, mixing speed is 250-500 turns/
Minute；
The condition of the cooling includes：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 a manner of being added dropwise in first mixed solution, and the course of reaction bag described in step (1)
Two stages are included, the reaction condition of first stage includes：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 10-15 drops/minute；And the reaction condition of second stage includes：Temperature
Spend for 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
10-15 drops/minute.
4. preparation method according to claim 2, wherein, in step (2), described 3 ', 5 '-(tertiary butyl dimethyl-silicon
Base) ratio of dosage of -5- aldehyde radicals -2 '-dideoxycytosine, the tetrabutyl ammonium fluoride and the tetrahydrofuran is 1mmol:(3-
4)mmol:(5-7)mL；And
The condition of second mixing includes：Temperature is 18-25 DEG C, and the time be 9-11 hours, mixing speed is 250-500 turns/
Minute.
5. preparation method according to claim 2, wherein, in step (3), the 5- [4- (2- nitrobenzophenones) -1,3-
Dioxane -2- bases] -2 '-dideoxycytosine and the trim,ethylchlorosilane, the pyridine and the acetic anhydride dosage ratio
For 1mmol:(5-7)mmol:(9-11)mL：(1.1-1.3)mmol；And
The condition of 3rd mixing includes：Temperature is 18-25 DEG C, and the time is 1-2 hours, and mixing speed is 250-500 revs/min
Clock；And the condition of the reaction includes：Temperature is 18-25 DEG C, and the time is 3-5 hours, and mixing speed is 250-500 revs/min
Clock.
6. preparation method according to claim 2, wherein, in step (4), 4-N- acyl groups -5- [4- (the 2- nitros
Phenyl) -1,3- dioxane -2- bases] -2 '-dideoxycytosine and 4,4'- dimethoxytriphenylmethyls chlorine, the dosage of pyridine
Ratio be 1mmol:(1.5-2)mmol:(9-11)mL；And
The condition of the reaction includes：Temperature is 18-25 DEG C, and the time is 12-18 hours, and mixing speed is 250-500 revs/min
Clock.
7. preparation method according to claim 2, wherein, in step (5), described 5 '-(dimethoxytrityl)-
4-N- acyl groups -5- [4- (2- nitrobenzophenones) -1,3- dioxane -2- bases] -2 '-dideoxycytosines and double (diisopropylaminoethyls)
The ratio of the dosage of (2- cyanoethoxies) phosphine, 1H-TETRAZOLE and dichloromethane is 1mmol:(1.3-1.6)mmol:(1.3-1.6)
mmol:(10-30)mL；And
The condition of the reaction includes：Temperature is 18-25 DEG C, and the time is 2-4 hours, and mixing speed is 250-500 revs/min.
8. a kind of oligonucleotide of the 5- aldehyde radical cytimidine pointed decorations of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections
Preparation method, it is characterised in that the synthetic agent in the preparation method includes claim 1 and claim 2-7 any one
The 5- aldehyde radical cytidine phosphoramidites of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections that described preparation method is prepared
Monomer.
9. preparation method according to claim 8, wherein, the preparation method is enterprising in ABI 394DNA/RNA synthesizers
OK, wherein：
The synthetic agent also includes Ac-dC, Bz-dA, dmf-dG and dT；
Trichloroacetic acid/dichloromethane is used to be deprotected；
Cap A reagents are the composition of acetic anhydride, pyridine and tetrahydrofuran, and the acetic anhydride, the pyridine and the tetrahydrochysene
The volume ratio of furans is 10 volume %：(5-15 volume %)：(75-85 volume %)；
Cap B reagents are the composition of N- methylimidazoles and tetrahydrofuran, and the N- methylimidazoles 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 tetrahydrochysene 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 radicals cytidine phosphoramidite monomer is 4-6 minutes；
The oligonucleotide that above-mentioned solid phase synthesizes to obtain 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% ammoniacal liquor, and the methylamine water solution and the ammoniacal liquor
The ratio of volume is 1：(0.5-1.5).
10. 1- (2- nitrobenzophenones) propane -1,3- glycerol protections that the preparation method described in claim 8 or 9 is prepared
The oligonucleotide of 5- aldehyde radical cytimidine pointed decorations is for the operational applications of DNA, it is characterised in that in the DNA operations
Application include with PNK enzymes carrying out 5 ' phosphorylations, ligase is attached, and the group with albumen progress DNA- protein complexes
Dress.
A kind of 11. preparation method of the oligonucleotide containing 5- aldehyde radical cytimidine pointed decorations, it is characterised in that this method bag
Include the 5- aldehyde of 1- (2- nitrobenzophenones) propane -1,3- glycerol protections that the preparation method described in claim 8 or 9 is prepared
The oligonucleotide of base cytimidine pointed decoration illumination 4-6 minute in-situ preparations under 340-360nm illumination obtain.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113993876A (en) * | 2019-06-11 | 2022-01-28 | 豪夫迈·罗氏有限公司 | Method for preparing oligonucleotides using improved oxidation protocols |
| CN114524855A (en) * | 2022-02-22 | 2022-05-24 | 武汉大学 | Synthesis method of 5-aldehyde cytosine phosphoramidite monomer |
| CN114685560A (en) * | 2020-12-31 | 2022-07-01 | 沈阳药科大学 | Synthesis and application of phosphoramidite monomer containing piperidine skeleton and oligonucleotide |
-
2017
- 2017-07-21 CN CN201710600724.3A patent/CN107474091B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| ARNE S.SCHRODER等: "Synthesis of a DNA Promoter Segment Containing All Four Epigenetic Nucleosides", 《ANGEW. CHEM. INT. ED.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113993876A (en) * | 2019-06-11 | 2022-01-28 | 豪夫迈·罗氏有限公司 | Method for preparing oligonucleotides using improved oxidation protocols |
| CN114685560A (en) * | 2020-12-31 | 2022-07-01 | 沈阳药科大学 | Synthesis and application of phosphoramidite monomer containing piperidine skeleton and oligonucleotide |
| CN114685560B (en) * | 2020-12-31 | 2024-05-14 | 沈阳药科大学 | Synthesis and application of phosphoramidite monomer containing piperidine skeleton and oligonucleotide |
| CN114524855A (en) * | 2022-02-22 | 2022-05-24 | 武汉大学 | Synthesis method of 5-aldehyde cytosine phosphoramidite monomer |
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