CN1900103A - Nucleoside phosphoramidite used in RNA oligo-nucleotide synthesis and its synthesizing method - Google Patents
Nucleoside phosphoramidite used in RNA oligo-nucleotide synthesis and its synthesizing method Download PDFInfo
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Abstract
The present invention discloses a kind of nucleoside phosphoramidite for synthesizing RNA oligonucleotide and its synthesis. The present invention adopts [2, 2-dimethyl-2-(o-nitrophenyl) acetylmethoxy] ether (DNPA) as the 2'-hydroxy protecting radical. The protecting radical DNPA is compatible with convenient 5'-hydroxy protecting radical, and may be eliminated under mild condition, so that it can ensure the RNA integrality and is suitable for synthesis of RNA oligonucleotide. The present invention provides one new path for synthesizing RNA oligonucleotide and may be used for the RNA molecule synthesis of various kinds of natural nucleoside, non-natural nucleoside, labeling nucleoside or their derivatives.
Description
Technical field
The present invention relates to a kind of chemical synthesis process of oligonucleotide, more particularly, the present invention relates to synthetic nucleoside phosphoramidites of a kind of RNA of being used for oligonucleotide (or claiming the RNA oligonucleotide derivative) and synthetic method thereof.
Background technology
Disturb the research of (RNAi) at an unprecedented rate to develop about RNA, its range of application also more and more widely, for example, carried out big quantity research at aspects such as functional genome, drug target affirmation and biological functions, this has caused being badly in need of siRNA molecule derivant low-cost, that high efficiency siRNA is synthetic and chemical process improves.At present, the successful strategies of the synthetic RNA of great majority is to be based upon on the basis of DNA synthetic method before, must be protected except 2 '-hydroxyl, and other chemical process and DNA building-up process are very similar.It is 2 '-hydroxyl that most strategies are to use tertiary butyl dimethyl-silicon ether (TBDMS) protection ribose.This guard method makes needs 10 minutes by each round-robin coupling time lengthening usually, because the spatial volume of the TBDMS group that closes on has hindered the reaction with 3 '-phosphoramidite.
Though it is very general work that DNA synthesizes in countless research laboratories, RNA's is synthetic so not successful.Although a lot of research groups used this TBDMS guard method to synthesize oligo rna, this method has run into a lot of difficulties in actual applications.Up to now, expect that high-quality nucleoside phosphoramidites monomer is all very difficult.Difficult problem is that 2 '-O-TBDMS group can easily be moved on 3 '-OH position and forms isomer; Another difficult problem is that the coupling efficiency of nucleoside phosphoramidites always is not very high.As a rule, the coupling efficiency of whole each step of synthetic should be all greater than 98% efficient, actual application value is so just arranged.At last, the impurity that the finished product contained is because lower coupling and the byproduct of efficient causes.The reason that produces about these impurity has a lot of arguements, can not reach usually but last result is the synthetic of RNA.
The silyl guard method of improvement is the blocking group of triisopropyl silica methyl (TOM) ether as 2 '-hydroxyl.The TOM blocking group has been avoided the formation of isomer and has been improved coupling efficiency, and available same quadrat method of removing TBDMS is removed the TOM blocking group.The TOM blocking group than the steric hindrance of 2 '-O-TBDMS group still less, so the TOM blocking group can reach higher coupling efficiency.But the monomeric synthetic cost of 2 '-TOM nucleoside phosphoramidites is very high.
Carry out RNA synthetic difficulty at the DNA synthetic method of application standard, people have developed multiple other the blocking group of 2 '-hydroxyl.Searge Beaucage etc. sums up various method.The blocking group that these methods concentrate on simply mostly with other types substitutes 2 '-O-TBDMS, also eliminates it when removing blocking group under different condition to shift to 3 '-hydroxyl.For example, to the group of photo-labile,, once be used as the blocking group of 2 '-hydroxyl, but do not used widely by other investigators as ortho position nitrobenzyl group.The difficulty of this method is that nucleic acid can absorb UV consumingly, and the group of photo-labile is difficult to do not had by product by a large amount of removing.Another kind method is then utilized sour unsettled acetal group, and this method is the Reese discovery and is utilized by other people after 25 again.Utilize the difficult point of the synthetic route of this blocking group to be, find one at DMTr (4,4 '-dimethoxytrityl) when the group acidic conditions goes to protect, at all stable acetal of each nucleosides addition step.Simultaneously, this acetal goes last acid that to protect under the condition of step must be unsettled, the RNA product that can not degrade natural.Recently, find and reported that a kind of new acetal (ACE) is that bis (2-acetoxyethoxy) methyl ortho ester group is protected 2 '-oh group, and be used for silylation protection 5 '-oh group, can meet these standards.Although it is promising that these methods look like, when big specification is synthetic, still have some obstacles, and cost is very high.
Though there are some defectives in the acetal method, it seems be the protection 2 '-hydroxyl method in very attractive group, reason is as follows: acetal is low, the very easy introducing 2 ' of a kind of cost-position and high again method of output.Except 5 '-O-DMTr acid go to protect in the step, they seem all very stable under all oligonucleotide synthetic conditions.At last, this compounds has been contained the unstable (labilities) of wide range.Therefore, acetal is can be in oligonucleotide is synthetic highly stable and be easy to again remove in last protective reaction.Last to go to protect be fully under the condition of the acidic solution of gentleness, and not with an organic solvent, and this is its another attracting feature.RNA is very soluble in water, but the common less organic solvent that is dissolved in, and the structure of RNA may complicated folding up in organic solvent.
The obstacle that uses unsettled acid acetal is 5 '-O-DMTr group, and it has also been removed by acid together.In in the past a lot of years, it is slow to discover that further RNA degrades when pH 2, thereby must seek a kind of special unsettled acid 2 '-blocking group.Therefore, in the end tart goes to protect step, must remain on minimum acid concentration and remove guard time.As a result, the optional group of acetal and the 5 '-end of 2 '-end combined and can be removed under non-acid condition during RNA synthesized.For example, FMOC group and levulinoyl group are used.Yet they are not applied to 2 '-TBDMS method practice.
DNA and RNA oligonucleotide synthetic methodology all are according to protecting 5 '-hydroxyl with DMTr group and silyl-group now.By years of researches, chemical coupling and former most blocking group be by accuracy or replaced, yet still be utilized at 5 '-hydroxyl terminal DMTr.People attempt to seek a suitable surrogate, but not success.Perhaps be the reason of the characteristic of 5 '-hydroxy-protective group when synthetic oligonucleotide.DMTr has the necessary main characteristic of a lot of protections 5 '-hydroxyl: (1), stability when storing synthetic at phosphoramidite; (2) rapid and quantitative removing under the condition of gentleness; (3) remove the non-degradable polymer of condition; (4) stability under the oligonucleotide synthesis condition; (5) in monomeric building-up process; to the regioselectivity of 5 ' main-hydroxyl with respect to accessory 2 ' and 3 '-hydroxyl; visible properties when (6) going to protect helps the synthetic of monitoring oligonucleotide, (7) cost may command.1,2,3,4 pairs of efficient synthetic oligonucleotides of condition are necessary.Condition 5,6,7 is not synthetic necessary, but can make chemosynthesis be used for practice.The DMTr group is very suitable to these prerequisites.It is extremely important that DMTr becomes in the oligonucleotide building-up process.The site of other protection is at A, C, the outer amido of G ring, phosphate backbone and 2 '-hydroxyl.The site of protection must be stable under the oligonucleotide synthesis condition, when particularly 5 '-O-DMTr group acidity goes to protect in the process that each base adds.This is limited in when DNA synthesizes and does not exist, but can influence the synthetic of RNA.
Because the restriction in site when selecting 2 '-hydroxyl protection, the effect of DMTr group is unsatisfactory in RNA is synthetic.When synthetic RNA, additional 2 '-hydroxy-protective group must be easy to introduce, and stable in the oligonucleotide building-up process, it is synthetic to finish RNA at last, must high-quality a large amount remove be last 2 '-blocking group.Natural not protected RNA is easy to by chemical process and enzyme liberating.Aspect chemical, RNA is unstable under high or low pH, but just stable many when neutral PH.Thereby, remove 2 '-hydroxyl at last and must under the condition of gentleness, carry out.The constraint of these RNA 2 '-hydroxyl protection and the constrained of additional 5 '-O-DMTr the selection of 2 '-group.Though it is possible that the result causes RNA synthetic, it is synthetic successfully not resemble DNA.
Acetals normally 2 '-blocking group is selected at most.The main shortcoming of acetals is compatible relatively poor with the DMTr of acid instability.
Summary of the invention
The purpose of this invention is to provide a kind of new blocking group that can satisfy above every kind of demand, can obtain the very low nucleoside phosphoramidites monomer of foreign matter content by this blocking group, this monomer can be used for the synthetic of RNA oligonucleotide.
On the one hand, the invention provides the purposes of hydroxyl in a kind of utilization-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group protection chemosynthesis process or the chemical reaction.
More preferably; above-mentioned-[2; 2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group is the 2 '-hydroxyl that is used to protect on nucleosides or the nucleoside derivates; nucleosides comprises adenosine, guanosine, cytidine or uridine, and the derivative of nucleosides comprises adenosine derivative, guanosine derivative, cytidine derivatives or uridine derivatives.The blocking group of blocking group of the present invention and conventional 5 '-hydroxyl is as 4,4 '-DMTr, 4,4 ', 4 "-TMTr etc. has compatibility.
On the other hand, the invention provides a kind of nucleoside phosphoramidites with structure shown in the chemical general formula (I), this nucleoside phosphoramidites can be used for the synthetic of RNA oligonucleotide:
Derivative;
R
2Be 4,4 '-dimethoxytrityl or other hydroxy-protective groups;
R
3Be phosphoramidate or phosphate group;
R
4Be hydrogen, methyl or other groups;
R
5Be 2,2-dimethyl-2-(ortho-nitrophenyl base) ethanoyl or 2,2-dimethyl-2-(the ortho-nitrophenyl base of replacement) ethanoyl.
In above-mentioned chemical general formula (I), the blocking group R on 2 '-hydroxyl
4-CH-OR
5Be preferably-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether or-[2,2-dimethyl-2-(the ortho-nitrophenyl base of replacement) acetyl yloxymethyl] ether.
Preferably, above-mentioned nucleoside phosphoramidites has the structure shown in the chemical general formula (II):
In the formula, B
PVITAMIN B4, guanine, cytosine(Cyt), uridylic or its corresponding derivative of expression N protection; Preferably, B
PVITAMIN B4, guanine, cytosine(Cyt) or the uridylic of expression N protection; Further preferably, B
PThe VITAMIN B4 or the cytosine(Cyt) of expression N protection;
DNPA represents-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether.
More preferably, above-mentioned nucleoside phosphoramidites is the adenosine phosphoramidite with structure shown in the chemical general formula (III):
Again on the one hand; the invention provides the synthetic method of the synthetic nucleoside phosphoramidites of a kind of RNA of being used for oligonucleotide; the method is characterized in that; it adopts-and [2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group protects the step of 2 '-hydroxyl on nucleosides or the nucleoside derivates.Wherein, the monomeric synthetic route of nucleoside phosphoramidites of a kind of employing 2 '-DNPA protection is as follows:
(synthetic route I:2 '-the monomeric synthetic route of nucleoside phosphoramidites of DNPA protection)
Be that above-mentioned method can in turn include the following steps:
(1) base on the nucleosides is protected, described base is VITAMIN B4, guanine, cytosine(Cyt) or uridylic;
(2) usefulness-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group of 2 '-hydroxyl on the nucleosides is protected;
(3) 2 '-hydroxyl is protected by the 5 '-hydroxyl protection of-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group, on the nucleosides;
(4) 3 '-hydroxyl to resulting intermediate product in the step (3) carries out phosphorylation reaction, obtains required nucleoside phosphoramidites;
Wherein, in step (2) before, the preferred earlier 3 '-hydroxyl and 5 ' on the nucleosides-hydroxyl the protection, then step (2) afterwards, step (3) before, remove the blocking group on 3 '-hydroxyl and 5 '-hydroxyl.
With the adenosine is example, and the method for synthetic nucleosides phosphoramidite monomer of the present invention is described in further detail:
Adenosine is at first followed the reaction of three silicomethane chlorine in pyridine, then with the Benzoyl chloride reaction, add ammonium hydroxide at last and remove front three silicon generation 6-N-benzoyl-adenosine in same reactor, and productive rate reaches 94%.6-N-benzoyl-adenosine and 1,3-two chloro-1,1,3,3-tetra isopropyl-1,3-sily oxide (TIPDSCl
2) reaction generation 6-N-benzoyl-3 ' in pyridine, 5 '-O-(tetra isopropyl disiloxane-1,3-diether) adenosine, productive rate is 90%.In DMF, handle through NaH earlier, with the DNPA-Cl reaction, generate 6-N-benzoyl-2 '-DNPA-3 ', 5 '-O-(tetra isopropyl-1,3-two silica) adenosine then.After the DNPA group is introduced in 2 ' position, handle with tetrabutyl ammonium fluoride, remove 3 '; 5 '-tetra isopropyl disiloxane-1,3-two ether groups generate 6-N-benzoyl-2 '-O-[2; 2-dimethyl-2-(ortho-nitrophenyl base) ethanoyl oxygen methyl ether-adenosine, productive rate are 90%.Earlier with 4; the diffusing phenmethyl chlorine of 4 '-dimethoxy is optionally protected 5 '-hydroxyl; under standard conditions; again with O-2-second cyano group-N; the inferior phosphoryl chloride reaction of N-diisopropylamine generates 6-N-benzoyl-2 '-O-DNPA-5 '-O-(4; 4 '-dimethoxytrityl) (2-second cyano group-N, N-diisopropylphosphoramidite, productive rate are 65-86% to adenosine-3 '-O-.
Other nucleoside phosphoramidites all can be synthesized by above-mentioned same quadrat method or similar approach.
Utilize the above-mentioned nucleoside phosphoramidites monomer that obtains, can synthesize the RNA oligonucleotide chain, for example the oligonucleotide of 20-25 Nucleotide.For example, utilize DNPA blocking group recited above, prepare 2 '-O-DNPA-5 '-O-(4; 4 '-dimethoxytrityl) nucleosides-3 '-O-(2-second cyano group-N; the N-diisopropylphosphoramidite, as synthetic RNA phosphoramidite monomer, press route synthetic oligonucleotide as follows:
(synthetic route II: the solid phase synthesis route of oligonucleotide)
Utilize method of the present invention, can synthesis of natural or non-natural RNA oligonucleotide.2 '-O-[2 described in the present invention; 2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] building-up process of ether nucleosides (G, A, C and U) phosphoramidite, and can be used for any natural nucleus glycoside, non-natural nucleoside, mark nucleosides and signal group or synthetic at other derivatives of RNA oligonucleotide molecules.
When production nucleoside phosphoramidites monomer; adopt of the present invention 2; 2-dimethyl-2-(ortho-nitrophenyl base) ethanoyl oxygen methyl ether (DNPA) is as the blocking group of stable nucleosides 2 '-position hydroxyl; the unexistent advantage of other all protecting groups before having can obtain highly purified nucleoside phosphoramidites monomer.In addition, the condition of removal DNPA group does not influence oligonucleotide product and quality as mild as a dove.
Below, describe the present invention in detail in conjunction with embodiment.But these specific embodiments are not construed as limiting the scope of the present invention; Persons of ordinary skill in the art may appreciate that on basis of the present invention, need not any creative work, can suitably improve or change the present invention, thereby all these improvement or change still in protection scope of the present invention.
Embodiment
The condition that is adopted in the experiment is common synthetic general condition.Wherein, solvent must guarantee drying, and methylene dichloride, second are fine, pyrimidine is by hydrolith distillation preparation; Be reflected under the argon gas stream protection and carry out; TLC: filtering layer silica gel thin-layer 60 F254 from EM, Flash chromatogram (FC): silica gel 60 , EM mesh 180-240.
1Carry out in H NMR spectrum Varian VNMR 400 spectrometers.With aforesaid method steam CDCl
3Or DMSO-d
6As solvent, data use ppm as record.All solvents, organic chemical, inorganic chemistry medicine are bought from ACROS or Aldrich.
Synthesizing of embodiment 1 adenosine phosphoramidite
Synthetic 6-N-benzoyl adenosine (compound 1): with adenosine (10.68g; 39.96mmol) be dissolved in the 200mL pyridine and form suspension; add trimethylchlorosilane (38.1mL; 300mmol) in wherein; at room temperature; the argon gas stream protection is thorough mixing reaction 45min down; add benzoyl chlorine (23.1mL then; 200mmol) thorough mixing reaction 4h; mixture adds 40mL water after cooling off in ice bath; (96mL 713.3mmol), at room temperature mixes 45min slowly to add the aqueous solution of 29% ammonium hydroxide behind the 20min; the centrifugal dry powder that is evacuated to adds the 320mL water dissolution.Solution once forms crystallization at once with ethyl acetate (100mL) flushing.Use 30mL EtOAc, 40mL frozen water, 40mL acetone (twice) flushing to obtain white solid 6-N-benzoyl adenosine (compound 1) (14.17g, 95.6%): R after the filtration successively
f=0.24 (13.3% methanol mixed chloroform);
1H NMR (400MHz, DMSO-d
6) δ 3.55-3.70 (m, 2H), 3.96-3.99 (m, 1H), 4.17-4.19 (m, 1H), and 4.63-4.67 (m, 1H), 5.14 (t, J=5.50Hz, 1H), 5.26 (d, J=4.77Hz, 1H), 5.58 (d, J=5.87Hz, 1H), 6.04 (d, J=5.51Hz, 1H), 7.53-7.64 (m, 3H), 8.03-8.04 (m, 2H), 8.72 (s, 1H), 8.75 (s, 1H), 11.22 (br s, 1H);
13C NMR (100MHz, DMSO-d
6) δ 62.00,71.05,74.33,86.40,88.24,126.58,129.18,133.17,133.99,143.86,151.09,152.33,152.91,166.31.
Synthetic 6-N-benzoyl-3 '; 5 '-O-(tetra isopropyl disiloxane-1; the 3-diether) adenosine (compound 6): under 0 ℃, be that 6-N-benzoyl adenosine (14.45mmol) is dissolved in 200mL anhydrous pyridine formation suspension, slowly add 1 again with compound 1; 3-two chloro-1; 1,3,3-tetra isopropyl-1; the 3-sily oxide (5.5mL, 17.34mmol).At room temperature, argon gas stream is protected thorough mixing reaction 6h down, adding 4mL water end reaction under 0 ℃ then.Except that after desolvating, add 600mL CH in the residue
2Cl
2Dissolving adds the saturated NaCl flushing of 100mL water and 90mL then.Organic layer can be by the dry .Na of anh
2SO
4Under low pressure remove with solvent.Residue is by flash column chromatography (SiO
2) to obtain white solid compound 2 be 6-N-benzoyl-3 ' to purifying, 5 '-O-(tetra isopropyl disiloxane-1,3-diether) adenosine (94.0%): R
f=0.57 (9.1% methanol mixed chloroform).Product passes through
1H NMR (400MHz, CDCl
3),
13CNMR (100MHz, CDCl
3) and ESI-MS be confirmed.
Synthetic 6-N-benzoyl-2 '-O-DNPA-3 ', 5 '-O-(tetra isopropyl disiloxane-1, the 3-diether) adenosine (compound 3): with compound 2 is 6-N-benzoyl-3 ', 5 '-O-(tetra isopropyl disiloxane-1, the 3-diether) adenosine (21.87mmol) is drained dry 5h by oil pump, is dissolved in 250mLDMF. then at 0 ℃, and NaH (64.5mmol) adds in the above solution, stir 5h, slowly add DNPA-Cl (64.5mmol) with syringe again.At thorough mixing reaction 3h under 0 ℃, under the argon gas stream protection, at 80 ℃ of heating 2h, under low-temp low-pressure, remove and desolvate then, residue is dissolved in 800mL CHCl
3, use saturated NaHCO
3(100mL) and water (100mL) flushing.Water layer is by CHCl
3Extraction, the bonded organic layer passes through Na
2SO
4Drying, solvent is removed by low pressure.Residue is by flash column chromatography (SiO
2) purifying obtains white solid 6-N-benzoyl-2 '-O-DNPA-3 ', 5 '-O-(tetra isopropyl disiloxane-1,3-diether) adenosine (compound 3) (97.0%): R
f=0.67 (75%EtOAc in hexane); Product passes through
1H NMR (400MHz, CDCl
3),
13C NMR (100MHz, CDCl
3) and ESI-MS be confirmed.Synthetic 6-N-benzoyl-2 '-O-DNPA-adenosine (compound 4): under 0 ℃, argon gas stream protection; with compound 3 is 6-N-benzoyl-2 '-O-DNPA-3 '; 5 '-O-(tetra isopropyl disiloxane-1; the 3-diether) adenosine (5.24mmol) is dissolved in THF (200mL); add tetrabutyl ammonium fluoride (5.3mL again; 5.24mmol), thorough mixing reaction 6h adds 5%NH
4HCO
3(6mL) termination reaction.After solvent was removed, residue added CH
2Cl
2And pyridine (600mL, mixed solution dissolving v/v=3/1), the saturated NaHCO of usefulness 50mL
3Clean (2 times).The bonded organic layer passes through Na
2SO
4Drying, solvent is removed by low pressure.Residue is by flash column chromatography (SiO
2) purifying obtains white solid 6-N-benzoyl-2 '-O-DNPA-adenosine (90%) product and passes through:
1HNMR (400MHz, CDCl
3),
13C NMR (100MHz, DMSO-d
6) and ESI-MS be confirmed.
Synthetic 6-N-benzoyl-2 '-O-DNPA-5 '-O-(4; 4 '-dimethoxytrityl) adenosine (compound 5). with the solution (1.13mmol) and 4 of compound 4; 4 '-dimethoxytrityl chlorine (0.763g; 2.25mmol) (using the vacuum pump dried overnight) in pyridine (40mL); add diisopropyl ethyl amine (0.98mL, 5.63mmol). these reaction mixtures stirring at room 12 hours under argon shield.After adding ethanol (2mL), remove and desolvate.Resistates is dissolved in CHCl
3(300mL), with saturated NaHCO
3(30mL) solution and saturated NaCl (30mL) solution washing. organic layer passes through anhydrous Na
2SO
4Drying, and solvent is under low pressure removed.Resistates flash column chromatography (SiO
2) purifying, obtain the faint yellow solid of compound 5 (90.1%): R
f=0.36 (4.8% methanol mixed chloroform). product is used
1H NMR (400MHz, CDCl
3),
13C-NMR (100MHz, CDCl
3) and the ESI-MS evaluation.
Synthetic 6-N-benzoyl-2 '-O-DNPA-5 '-O-(4,4 '-dimethoxytrityl) adenosine-3 '-O-(2-second cyano group-N, N-diisopropylphosphoramidite (compound 6): compound 5 (1.11mmol) is used the oil pump dried overnight, is dissolved in anhydrous CH under the ice bath cooling
2Cl
2(25mL).(0.794mL, 4.56mmol) with 2-second cyano group-N, the inferior phosphoryl chloride of N-diisopropylamine adds above solution to diisopropylethylamine under argon gas stream.After spending 20 minutes,, add 250mL EtOAc diluted reaction mixture, the saturated NaHCO of organic layer as for hybrid reaction 4.5h under the room temperature
3(25mL) with saturated NaCl (25mL) washing.Organic layer Na
2SO
4Drying, solvent is under low pressure removed.Residue column chromatography (SiO
2) purifying obtains the yellow solid (75.5%) of compound 6: R
f=0.59,0.70 (4.8%EtOAc in chloroform).Product is used
1H NMR (400MHz, CDCl
3),
13C-NMR (100MHz, CDCl
3) and the ESI-MS evaluation.
Synthesizing of embodiment 2 cytidine phosphoramidites
Synthetic 4-N-benzoyl cytidine (compound 7): (9.72g 39.96mmol) is added to the 200mL pyrimidine and forms suspension cytidine, and (38.1mL 300mmol) adds wherein with trimethylchlorosilane then.Thorough mixing reaction 1h under the argon gas stream protection at room temperature; at room temperature add Benzoyl chloride (23.0mL then; 200mmol) hybrid reaction 4h; mixture adds 40mL water after cooling off in ice bath; (60mL 445.8mmol), at room temperature mixes 25min to add 29% solution of ammonium hydroxide behind the 20min; the centrifugal dry powder that is evacuated to adds the 400mL water dissolution.After adding 150mLEtOAc more just crystallization can appear.Use 30mL acetyl triethyl, 30mL frozen water, 30mL acetone (twice) flushing to obtain the 4-N-benzoyl cytidine (compound 7) (13.1g, 94.4%) of white solid after the filtration successively.R
f=0.55 (20% methyl alcohol-acetyl triethyl);
1H NMR (DMSO-d
6, 400MHz) δ 3.57-3.77 (m, 2H), 3.89-4.01 (m, 3H), 5.06 (d, J=5.47Hz, 1H), 5.18 (t, J=5.08Hz, 1H), 5.52 (d, J=5.09Hz, 1H), 5.79 (d, J=2.74Hz, 1H), 7.31 (m, 1H), 7.48-7.63 (m, 3H), and 7.97-7.99 (m, 2H), 8.47 (d, J=7.42Hz, 1H), 11.16 (br s, 1H);
13C NMR (DMSO-d
6, 100MHz) δ 60.57,69.32, and 75.23,84.90,90.88,96.80,129.14,133.42,133.86,145.94,155.22,163.70,168.03.
Synthetic 4-N-benzoyl-3 ', 5 '-O-(tetra isopropyl disiloxane-1, the 3-diether) cytidine (compound 8): (5.02g 14.45mmol) is added in the 200mL anhydrous pyridine and forms suspension, slowly adds 1 again with 4-N-benzoyl cytidine (compound 7) under 0 ℃, 3-two chloro-1,1,3,3-tetra isopropyl-1, the 3-sily oxide (5.5mL, 17.34mmol).At room temperature, argon gas stream is protected thorough mixing reaction 6h down, adding 4mL water end reaction under 0 ℃ then.Except that after desolvating, add 600mL CH in the residue
2Cl
2Dissolving adds the saturated NaCl flushing of 100mL water and 90mL then.Organic layer Na
2SO
4Desolvate with under low pressure removing in dry back.Residue is by column chromatography (SiO
2) purifying obtains white solid 4-N-benzoyl-3 ', 5 '-O-(tetra isopropyl disiloxane-1,3-diether) cytidine (compound 8) (8g, 94.0%), R
f=0.57 (9: 1=methyl alcohol: chloroform);
1H NMR (400MHz, CDCl
3) δ 0.94-1.11 (m, 28H), 2.88 (br s, 1H), 4.01-4.05 (dd, J=13.44,2.69Hz, 1H) 4.20-4.35 (m, 4H), 5.86 (s, 1H), 7.50-7.63 (m, 4H), 7.87-7.89 (d, J=6.59Hz, 2H), 8.22 (d, J=6.60Hz, 1H), 8.67 (br s, 1H);
13C NMR (100MHz, CDCl
3) δ 12.70,13.13,13.15,13.60,17.05,17.12,17.21,17.52,17.63,17.68,60.23,68.82,75.46,82.26,91.80,96.24,127.70,129.34,133.49,144.92,162.56; ESI-MS is calculated as M 589.2640, detects to be (M+H) 590.
All the other steps are identical with corresponding steps among the embodiment 1, make 6-N-benzoyl-2 '-O-DNPA-5 '-O-(4,4 '-dimethoxytrityl) cytidine-3 '-O-(2-second cyano group-N, N-diisopropylphosphoramidite at last.
Synthesizing of embodiment 3 guanosine phosphoramidites
By with embodiment 1 in corresponding method make 6-N-benzoyl-2 '-O-DNPA-5 '-O-(4,4 '-dimethoxytrityl) guanosine-3 '-O-(2-second cyano group-N, N-diisopropylphosphoramidite.
Synthesizing of embodiment 4 uridine phosphoramidites
By with embodiment 1 in corresponding method make 6-N-benzoyl-2 '-O-DNPA-5 '-O-(4,4 '-dimethoxytrityl) uridine-3 '-O-(2-second cyano group-N, N-diisopropylphosphoramidite.
Synthesizing of embodiment 5 oligonucleotide
2 '-O-DNPA-5 '-O-(4 of embodiment 1-4 preparation above utilizing respectively, 4 '-dimethoxytrityl) adenosine-3 '-O-(2-second cyano group-N, the N-diisopropylphosphoramidite, 2 '-O-DNPA-5 '-O-(4,4 '-dimethoxytrityl) cytidine-3 '-O-(2-second cyano group-N, the N-diisopropylphosphoramidite, 2 '-O-DNPA-5 '-O-(4,4 '-dimethoxytrityl) guanosine-3 '-O-(2-second cyano group-N, the N-diisopropylphosphoramidite, 2 '-O-DNPA-5 '-O-(4,4 '-dimethoxytrityl) uridine-3 '-O-(2-second cyano group-N, the N-diisopropylphosphoramidite is the RNA phosphoramidite monomer, with the synthetic RNA oligonucleotide of Applied Biosystems DNA/RNA synthesizer (Model394) automatization.Synthetic good RNA oligonucleotide, the complete first-class blocking group of lixiviating base of reaction 24h energy in ammonium hydroxide; SnCl with 1.0M
2DMF solution, 5h removes the DNPA blocking group under 50 ℃ of temperature, desalts by the oligonucleotide purifying afterwards.
Claims (10)
1, a kind of nucleoside phosphoramidites with structure shown in the chemical general formula (I):
In the formula, R
1Be VITAMIN B4, guanine, cytosine(Cyt), uridylic or its corresponding derivative of N protection;
R
2Be 4,4 '-dimethoxytrityl or other hydroxy-protective groups;
R
3Be phosphoramidate or phosphate group;
R
4Be hydrogen, methyl or other groups;
R
5Be 2,2-dimethyl-2-(ortho-nitrophenyl base) ethanoyl or 2,2-dimethyl-2-(the ortho-nitrophenyl base of replacement) ethanoyl.
2, nucleoside phosphoramidites as claimed in claim 1 is characterized in that, the blocking group R in the chemical general formula (I) on 2 '-hydroxyl
4-CH-OR
5For-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether or-[2,2-dimethyl-2-(the ortho-nitrophenyl base of replacement) acetyl yloxymethyl] ether.
3, nucleoside phosphoramidites as claimed in claim 2 is characterized in that, described nucleoside phosphoramidites has the structure shown in the chemical general formula (II):
In the formula, B
PVITAMIN B4, guanine, cytosine(Cyt), uridylic or its corresponding derivative of expression N protection;
DNPA represents-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether.
4, nucleoside phosphoramidites as claimed in claim 3 is characterized in that, described B
PVITAMIN B4, guanine, cytosine(Cyt) or the uridylic of expression N protection.
5, as claimed in claim 4, it is characterized in that described nucleoside phosphoramidites is the adenosine phosphoramidite with structure shown in the chemical general formula (III):
6, nucleoside phosphoramidites as claimed in claim 4 is characterized in that, described B
PThe cytosine(Cyt) of expression N protection.
7 ,-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group is used for the purposes of chemosynthesis process or chemical reaction protection hydroxyl.
8, purposes as claimed in claim 7; it is characterized in that; described-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group is the 2 '-hydroxyl that is used to protect on nucleosides or the nucleoside derivates, and described nucleosides is adenosine, guanosine, cytidine or uridine.
9, a kind of synthetic method that is used for the synthetic nucleoside phosphoramidites of RNA oligonucleotide; it is characterized in that; this method comprises that employing-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group protects the step of 2 '-hydroxyl on nucleosides or the nucleoside derivates.
10, method as claimed in claim 9 is characterized in that, this method in turn includes the following steps:
(1) base on the nucleosides is protected, described base is VITAMIN B4, guanine, cytosine(Cyt) or uridylic;
(2) usefulness-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group of 2 '-hydroxyl on the nucleosides is protected;
(3) 2 '-hydroxyl is protected by the 5 '-hydroxyl protection of-[2,2-dimethyl-2-(ortho-nitrophenyl base) acetyl yloxymethyl] ether group, on the nucleosides;
(4) 3 '-hydroxyl to resulting intermediate product in the step (3) carries out phosphorylation reaction, obtains required nucleoside phosphoramidites;
Preferably, in step (2) before, earlier the 3 '-hydroxyl and 5 ' on the nucleosides-hydroxyl is protected, then step (2) afterwards, step (3) before, remove the blocking group on 3 '-hydroxyl and 5 '-hydroxyl.
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