CN102898464A - Photosensitive group-protected functional group phosphoramidite and its preparation method and use - Google Patents
Photosensitive group-protected functional group phosphoramidite and its preparation method and use Download PDFInfo
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Abstract
The invention discloses a photosensitive group-protected functional group phosphoramidite and its preparation method and use, and belongs to the field of nucleotide solid-phase synthesis. The photosensitive group-protected functional group phosphoramidite shown in the structural formula I or II has a nitrophenethyl alcohol matrix structure and a photo-induced cleavage activity, and can be used as a photoactivity protection group in oligonucleotide solid-phase synthesis. In the oligonucleotide solid-phase synthesis, the photosensitive group-protected functional group phosphoramidite can be used as the last monomer, is added into an end 5' of ODN and can protect a phosphate group, an amino group, a carboxyl group, a sulfydryl group or a carbonyl group. A test result shows that in oligonucleotide solid-phase synthesis, the photosensitive group-protected functional group phosphoramidite shown in the structural formula I or II can be used as the last monomer and is added into an end 5' of ODN so that an oligonucleotide product is obtained; and after the oligonucleotide product is radiated, the protection is eliminated and a corresponding functional molecule or marker is bonded to the end 5'.
Description
Technical field
The present invention relates to phosphoramidite compound; relate in particular to the phosphoramidite compound and preparation method thereof of the functional group of photaesthesia radical protection; the invention still further relates to the application of this phosphoramidite compound in oligonucleotide solid phase synthesis and 5 ' end functionalization, belong to solid phase synthesis and the functionalization field of oligonucleotide.
Background technology
Optical active group refers under photon excitation, can by forming free radical or heterolytic fission intermediate, the molecular structure of chemical transformation occur.It makes in complex system, especially under physiological environment, utilizes the variation of light guide small molecules or biomacromolecule to become possibility.Generally believe, compare with other the control measures such as temperature, potential of hydrogen, the light regulation and control have accurate positioning, regulation and control in time, on plurality of advantages such as the system impact are little.Usually the optical active group of indication can be divided into two classes, that is: photoisomerization group and light can excise group.The former photoprocess is reversible, and latter is irreversible chemical process.The concept that light can excise group (caged/caging moieties) proposed 1 by J.F.Hoffman in 1978 the earliest, and this class group can absorb the photon of this energy effectively to the photaesthesia in the 340-370nm scope.It is O-Nitrophenylfluorone and derivative thereof that modal light can excise group, also has in addition para hydroxybenzene acetoxyl group, coumarins, xanthone phenylacetic acid compound etc.Because the existence of ortho position nitro, after optical excitation, the o-nitrophenylethanol compounds forms the five-membered cyclic intermediate, and then obtains adjacent nitroso-group acetophenone compounds, with the other parts disconnection in the compound.Because the structure of o-nitrophenylethanol and derivative thereof is compared that other light can excise group in synthetic or is comparatively stable under the physiological environment, therefore in most of living things systems, be widely adopted, such as ATP, DNA, RNA, protein, aptamers etc.
Oligonucleotide can be used for doing the PCR primer, be used for probe, allele specific oligonucleotide oligonucleotide analysis (Allele-specificoligonucleotide that nucleotide sequence detects, ASO), single base diversity analysis (Single nucleotide polymorphism, SNP), antisense oligonucleotide, it is widely used in biology field.
In recent decades, oligonucleotide is being brought into play more and more important effect in molecular biology, becomes gradually a kind of very effective research tool.Particularly in recent years, how relevant research focuses mostly on utilizes oligonucleotide from the mechanism of production of gene level research various diseases and the expression network of genes involved thereof, and make its expression amount by regulatory gene prevent, diagnose and treat relative disease, as the another kind of thinking of medicament research and development.
The traditional method of oligonucleotide functionalization and application: usually, the functionalization of 5 ' end is the functional group that adds the small molecules band protecting group of phosphorus reagent after oligonucleotide is synthetic, connects other functional molecular via these functional groups again behind the deprotection.Which kind of protecting group is the module that pair methoxyl group triphenyl (MMTr) protection and trifluoroacetyl (TFA) protection are arranged such as 5 ' amino select determined by the aftertreatment of oligonucleotide.Connecting arm is generally 6 carbon, and the connecting arm of 12 carbon is used for some particular condition, as closely occuring in the molecule of fluorescent quenching.5 ' sulfydryl has triphenyl (Tr) protecting group and two kinds of modules of disulfide linkage protection; different from MMTr, Tr is stable to acid, needs with Silver Nitrate it to be sloughed carefully; centrifugal removing after excessive Silver Nitrate precipitates with TCEP, and excessive TCEP needs remove through desalination in this step.Not oxidized for guaranteeing thioether bond or disulfide key, final step need be used the iodine liquid of 0.2M in the synthesis cycle.And the module of disulfide linkage protection can disconnect with TCEP, directly uses after the desalination.End is that the ODN of sulfydryl can be combined with fluorescein, vitamin H or metallic nanoparticle in continuation, is used for the DNA chip analysis, fluorescent probe equimolecular Biological Detection.
After one section end is connected into the valeric acid NHS activator that contains disulfide in for amino oligonucleotide can with gold and silver nanoparticle surface bonding, form two sulphur-Jin Jian, in conjunction with efficient can reach 91%, single sulfydryl then only 48%.This strong DNA-gold surface binding pattern provides another kind of key tactics for the nanoparticle self-assembly that DNA controls.Initial introducing 5 ' need use the phosphorus reagent that contains sulfone when holding phosphate, if but synthesize last DMTr on ODN, then ammoniacal liquor is taken off the exposure phosphate in meeting with this protecting group, cause difficulty can for like this purifying of reversed-phase HPLC.
The amino of o-nitro benzyl alcohol structural defence is connected with the base of carboxyl with the thymus pyrimidine monomer, after oligonucleotide is synthetic and after the photo-irradiation treatment, can exposes amino and carboxyl.Add functional molecular and also can realize by the dna single body of modifying in chain, namely first optical active group being connected into thymus pyrimidine 2 ' position, to carry out DNA synthetic, is connected into functional molecular behind the light deprotection again.
Summary of the invention
One of purpose of the present invention provides the phosphoramidite compound of functional group of the photaesthesia radical protection of a kind of solid phase synthesis that can be applied to oligonucleotide and functionalization;
Two of purpose of the present invention provides a kind of method of synthetic described phosphoramidite compound;
Three of purpose of the present invention is solid phase synthesis and the functionalization that described phosphoramidite compound are applied to oligonucleotide.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Be applied to the phosphoramidite compound of functional group of the photaesthesia radical protection of oligonucleotide solid phase synthesis and functionalization, be the compound of structure shown in formula I or the formula II:
Wherein, R1 is selected from hydrogen, CH
3, COOCH
3R2 be selected from hydrogen or
R3 is selected from hydrogen or alkoxyl group (alkoxy); R4 is selected from hydrogen or alkoxyl group (alkoxy); Or R3-R4 is-OCH
2O; X is oxygen or nitrogen; Y is carbon, nitrogen or sulphur; Z is CH
2Or OCH
2CH
2N is the arbitrary integer of 1-6;
R2 be selected from hydrogen or
R3 is selected from hydrogen or alkoxyl group (alkoxy); R4 is selected from hydrogen or alkoxyl group (alkoxy); Or R3-R4 is-OCH
2O; R5 is hydrogen, CH
3Or phenyl; Z is CH
2Or OCH
2CH
2N is the arbitrary integer of 1-6.
Compound can prepare by the following method shown in formula I of the present invention or the formula II:
The method of compound shown in a kind of synthesis type I comprises: will obtain o-nitro benzyl alcohol after the reduction of ortho-nitrophenyl ethyl ketone; O-nitro benzyl alcohol is dissolved in methylene dichloride under nitrogen protection, ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts under room temperature (20-30 ℃) condition, and get final product.
The method of compound shown in a kind of synthesis type I comprises: (1) forms intermediate with o-nitro benzyl alcohol and DSC, and direct and 6-amino-hexanol effect gets 1-(2-nitrophenyl) ethyl 6-hydroxyl hexylamine base carbonic ether; (2) 1-(2-nitrophenyl) ethyl 6-hydroxyl hexylamine base carbonic ether is dissolved in methylene dichloride under nitrogen protection, ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts in room temperature (20-30 ℃), and get final product.
The method of compound shown in a kind of synthesis type I comprises: (1) o-nitro benzyl alcohol obtains 1-(1-bromotrifluoromethane)-2-oil of mirbane through bromo; Under acetone and mineral alkali effect, obtain 6-(6-(tertiary butyl dimethyl Si) caproic acid 1-(2-nitrophenyl) ethyl ester with 1-(1-bromotrifluoromethane)-2-oil of mirbane (2) with by the 6 hydroxycaproic acid of protected silane; (6-(tertiary butyl dimethyl Si) caproic acid 1-(2-nitrophenyl) ethyl ester is sloughed the protected silane base and is obtained 6 hydroxycaproic acid 1-(2-nitrophenyl) ethyl ester with 6-; (2) 6 hydroxycaproic acid 1-(2-nitrophenyl) ethyl ester is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts in room temperature (20-30 ℃), and get final product.
The method of compound shown in a kind of synthesis type I comprises: (1) is obtained 6-[1-(2-nitrophenyl) second sulfydryl with 1-(1-bromotrifluoromethane)-2-oil of mirbane by 6-sulfydryl hexanol nucleophilic substitution] the 1-hexanol; (2) 6-[1-(2-nitrophenyl) second sulfydryl] the 1-hexanol is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts in room temperature (20-30 ℃), and get final product.
The method of compound shown in a kind of synthesis type I comprises: after the hydroxyl of (1) o-nitro benzyl alcohol is activated by DSC, obtain S-6-hydroxyl hexyl O-1-(2-oil of mirbane) ethylenebis dithiocarbamate carbonic ether; (2) S-6-hydroxyl hexyl O-1-(2-oil of mirbane) ethylenebis dithiocarbamate carbonic ether is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts in room temperature (20-30 ℃), and get final product.
The method of compound shown in a kind of synthesis type II, comprise: (1) ortho-nitrophenyl ethyl glycol and 4-hydroxy-2-butanone are dissolved among the THF, under the dehydration of the catalysis of p-methyl benzenesulfonic acid and molecular sieve, obtain 4-hydroxyl fourth-2-ketone 1-(2-nitrophenyl)-1，2-ethandiol ketal; (2) 4-hydroxyl fourth-2-ketone 1-(2-nitrophenyl)-1，2-ethandiol ketal is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts under room temperature (20-30 ℃) condition, and get final product.
The temperature range of " room temperature " described in the inventive method is 20-30 ℃, is preferably 25 ℃.
The compound of structure all has the precursor structure of o-nitrophenylethanol shown in formula I of the present invention or the formula II, has the light nicking activity, in the solid phase synthesis of oligonucleotide, can be used as the photolytic activity protecting group it is joined 5 of ODN ' end as last monomer, can protect phosphate, amino, carboxyl, sulfydryl or carbonyl; Slough protecting group after the 365nm illumination and expose corresponding functional group; connect again corresponding functional molecular (such as steroidal, phenolphthalein, crown ether, hydrophilic alkyl chain) or marker (dyestuff small molecules; such as pyrene, rhodamine); realize the functionalization of oligonucleotide, also can form by the two products self of modifying in two ends the oligonucleotide of ring-type.
For amino, phosphate, can cut from solid phase first, remove the base protecting group, obtain the ODN with the photolytic activity protecting group behind the purifying, carry out the dynamic analysis of illumination reaction, obtain the ODN of exposed functional group behind the final purification.The two characterizes with high resolution mass spectrum.Further, amino is connected with dye molecule 5-TAMRA.
For carboxyl, as cutting with ammoniacal liquor first, then also may also can destroy the carboxylic acid ester bond of 5 ' protecting group, therefore should carry out illumination in solid phase first, remove the base protecting group with ammoniacal liquor again.Should connect dye molecule in solid phase after the illumination, to confirm being that illumination rather than ammoniacal liquor hydrolysis remove protecting group.
For sulfydryl, because the method for generally using is at AgNO
3Buffered soln in take off the triphenyl protecting group, add TCEP or DTT and remove excessive AgNO
3, prevent from simultaneously generating disulfide linkage.Then desalination and without the HPLC purifying.
Test-results shows; the compound of structure shown in formula I of the present invention or the formula II joins the resulting oligonucleotide product of 5 of ODN ' end as last monomer in the solid phase synthesis of oligonucleotide can slough protection through after the illumination, can connect corresponding functional molecular (such as steroidal, phenolphthalein, crown ether, hydrophilic alkyl chain etc.) or marker (such as fluorescence dye etc.) at 5 ' end.
Description of drawings
The structural formula of the phosphoramidite compound of the functional group of Fig. 1 photaesthesia radical protection of the present invention.
The synthetic route chart of the phosphoramidite compound of the functional group of Fig. 2 photaesthesia radical protection of the present invention.
What occur before Fig. 3 ODN1 illumination is bimodal, is the oligonucleotide of a pair of optical siomerism.
The curve that Fig. 4 ODN1/2 illumination deprotection efficient changes with light application time.
Fig. 5 ODN 2 is with the HPLC analytical results of light application time.
Change after the illumination of Fig. 6 ortho-nitrophenyl ethyl-sulfide compounds.
The HPLC analytical results of Fig. 7 ODN 5.
MALDI-TOF result before Fig. 8 ODN 6 illumination.
Fig. 9 ODN 2 5' amino labeled schematic diagram.
Ultraviolet-visible spectrogram before and after Figure 10 ODN 2 marks.
Figure 11 ODN 3 and pyrene connection diagram.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
Test materials
1, methylene dichloride, triethylamine, tetrahydrofuran (THF), acetonitrile equal solvent use hydrolith dry, and distillation before each the use.All reagent are buied from enterprises such as the chemical company limited of A Faaisha (Tianjin), Sigma's aldrich (China) company limited, the uncommon love of ladder (Shanghai) development of chemical industry company limited, the scientific and technological (Shanghai) Co., Ltd. of splendid chemistry far away.All reagent of buying from the commercial channel are before use all without being further purified.
2, all mixed solvents are with its volume ratio report.
3, all reaction process detect by thin-layer chromatography, and thin layer chromatography board is produced by Merck company, and the thick F of 0.24nm is arranged on it
254Silica gel.Column chromatography used silica gel (200-300 order) is produced by Haiyang Chemical Plant, Qingdao.
4,400MHz
1H nucleus magnetic resonance, 100MHz
13C nucleus magnetic resonance, 162MHz
31The P nucleus magnetic resonance by Bruker AVANCE II 400 nuclear magnetic resonance analyser at room temperature (298K) measure.Carbon spectrum and hydrogen spectrum chemical shift (δ, ppm) determine with respect to the generally acknowledged chemical shift of residual solvent, and the chemical shift that phosphorus is composed is take 85% phosphoric acid as external standard, and coupling constant (J) is rounded to 1Hz.Mass spectrum is recorded by ESI-Q-TOF.Described reaction and operation are all carried out in the darkroom.
5, the oligonucleotide solid phase synthesis with separate universal method
Oligonucleotide is synthetic by Applied Biosystems Incorporated 394 automatic DNA synthesizer DNAs.CPG:dG and dna single body are buied by AuGCT biotechnology company.Oligonucleotide carries out the synthetic of 1 μ mol scale by the standard DNA synthesis cycle at the LCAA-CPG of CPG:dG or modification.For 5 ' terminal reaction, the reaction times is extended for 120 seconds, and repeats once.
Half preparative HPLC Varian Prostar with Agilent C18 column (5 μ m, 9.4 * 250mm) carry out condition: solution A, 0.05M triethylamine-acetate buffer solution; Solution B, acetonitrile.Initial intensity 15%B, gradient 1%B/min, flow velocity 1mL/min, single passage UV detector detects wavelength 260nm.Moving phase is crossed 0.22 μ m water system/organic system filter membrane, the ultrasonic rear use of buffer salt solution before use.
(3.5 μ m, 4.6 * 75mm) carry out, initial intensity 0%B, gradient 1%B/min, flow velocity 1mL/min, PAD detector, detection wavelength 210-600nm with Symmetry C18 column at Watersalliance e2695 to analyze HPLC.
Mass spectrum is recorded by ESI-Q-TOF or MALDI-TOF.Uv-vis spectra is recorded by Beckmann DUSeries 800 spectrographs.
Photodissociation: carry out under the 365nm wavelength at UVP high intensity ultraviolet lamp, remain on below 30 ℃ for making system temperature, in case of necessity ice bag is placed under the quartz cell.If be the suspension of solid phase in the quartz cell, then add small stirrer, place and carry out illumination on the magnetic stirring apparatus.
The ammonia solution: add strong aqua (500 μ L) in the EP pipe that solid phase powder or dry oligonucleotide are arranged, at room temperature suspendible is 24 hours, gets the liquid evaporate to dryness.
The acetic acid solution: for sloughing 5 ' end DMTr protecting group, to the aqueous acetic acid (500 μ L) of the interior adding 80% of EP pipe that dry oligonucleotide is arranged, at room temperature suspendible is 30 minutes, gets the liquid evaporate to dryness.
The small molecules coupling agent that adds (BOP, HOBt etc.) is made into its DMF solution before use, and namely the coupling agent of dissolving 20 μ mol in the 1mL dry DMF is got 100 μ L and is added in solid phase or the dry oligonucleotide.
The preparation of embodiment 1 1-(2-oil of mirbane) ethanol (1-(2-nitrophenyl) ethanol) (compound 1a)
1-(2-oil of mirbane) ethyl ketone (3.51g, 21.3mmol) be dissolved in 1, the mixing solutions of 4-dioxy six alkane (15mL) and methyl alcohol (9mL), stirred 10 minutes at 0 ℃, slowly add sodium borohydride (1.60g, 42.3mmol), reaction system is edged up to room temperature, stir after 1 hour, with acetone (15mL) cancellation, solvent evaporated, ethyl acetate (70mL) dissolving residuum, washing organic phase (anhydrous sodium sulfate drying after 3 * 50mL), filtering and concentrating organic phase, evaporate to dryness get compound 1a, yellow oily liquid (3.52g, 21.1mmol, 99% productive rate);
1H NMR (400MHz, CDCl
3) δ=7.84 (dd, 2H, J=8,11Hz), 7.63 (t, 1H, J=7Hz), 7.40 (t, 1H, J=8Hz), 5.39 (q, 1H, J=6Hz), 1.54 (d, 3H, J=6Hz).
13C NMR (100MHz, CDCl
3) δ=147.8,141.0,133.5,128.0,127.5,124.2,65.5,24.2.
The preparation of embodiment 2 2-acetonitrile oxygen bases [1-(2-nitrophenyl) ethyl] di-isopropyl phosphoramide compound (1b)
Compound 1a (0.5mmol; 1.0eq) with behind tetrahydrofuran (THF) (THF) azeotropic water removing three times in reaction flask quantitatively; under nitrogen protection, be dissolved in methylene dichloride (10mL); ice bath; add successively 2-cyanoethyl diisopropylaminoethyl phosphine chlorine (1.5eq) and triethylamine (TEA) in it (3.0eq), edge up to room temperature the TLC(thin-layer chromatography) monitoring reaction; add the methylene dichloride dilution when reaction is finished, use 10%NaHCO
3(2 * 30mL) wash anhydrous sodium sulfate drying after the organic phase.Concentrated organic phase, silicagel column separates, and moving phase is sherwood oil/methylene dichloride and adds 3% TEA, obtains compound 1b, is yellow oily liquid.
1b, (R/S, 71% productive rate):
1H NMR (400MHz, CDCl
3) δ=7.80 (m, 2H), 7.59 (m, 1H), 7.35 (m, 1H), 5.46 (m, 1H), 3.81 (m, 1H), 3.60 (m, 2H), 3.45 (m, 1H), (2.62-2.46 m, 2H), 1.52 (m, 3H), 1.15-0.86 (m, 12H).
13C NMR (100MHz, CDCl
3) δ=146.9 (m), 140.0 (m), 133.2 (m), 128.2 (m), 127.6 (m), 123.6 (m), 117.2 (m), 67.0 (m), 58.2 (m), 42.9 (m), 24.9-24.0 (m), 23.8 (m), 19.9 (m)
31P NMR (162MHz, CDCl
3) δ=147.7 (s), 147.3 (s).
Compound 1a (425mg, 2.50mmol), triphenyl phosphorus (984mg, 3.75mmol) and carbon tetrabromide (1.25g, 3.75mmol) be dissolved in tetrahydrofuran (THF) (THF) (10mL) in, stirred under the room temperature 30 minutes.Filter, concentrated mother liquor, silicagel column separates, and moving phase petrol ether/ethyl acetate ratio is 4/1, gets compound 2: brown color oily liquids (558mg, 2.43mmol, 97% productive rate);
1H NMR (400MHz, CDCl
3) δ=7.88 (dd, 1H, J=1,4Hz), 7.81 (dd, 1H, J=1,4Hz), 7.65 (dd, 1H, J=1,4Hz), 7.42 (dd, 1H, J=1,7Hz), 5.80 (q, 1H, J=7Hz), 2.07 (d, 3H, J=7Hz).
13C NMR (100MHz, CDCl
3) δ=147.5,137.7,133.3,129.8,128.8,124.2,41.7,27.1.
The preparation of embodiment 3 1-(2-nitrophenyl) ethyl 6-hydroxyl hexylamine base carbonic ether (1-(2-nitrophenyl) ethyl 6-hydroxyhexylcarbamate) (compound 3a)
Compound 1a (1.03g, 6.17mmol) and N, N'-two succinimidyl carbonates (DSC) (2.37g, 9.26mmol) are dissolved in the acetonitrile (20mL), drip TEA (2.08mL, 15.0mmol) in mixed system.Stir under the room temperature and add 6-amino-hexanol (1.45g after 3 hours, 12.4mmol) dichloromethane solution (5mL), stir under the room temperature after 30 minutes, with methylene dichloride (50mL) dilution, wash organic phase (anhydrous sodium sulfate drying after 3 * 50mL) with water.Concentrated organic phase, silicagel column separates, and moving phase petrol ether/ethyl acetate ratio is 1/2, gets compound 3a: yellow oily liquid (1.78g, 5.74mmol, 93% productive rate);
1H NMR (400MHz, CDCl
3) δ=7.90 (d, 1H, J=8Hz), 7.60 (d, 2H, J=3Hz), 7.39 (d, 1H, J=4Hz), 6.20 (d, 1H, J=6Hz), 4.90 (s, 1H), 3.56 (t, 2H, J=6Hz), 3.09 (t, 2H, J=6Hz), 1.83 (s, 1H) 1.59 (d, 3H, J=6Hz), 1.57-1.24 (m, 8H, J=7Hz).
13CNMR (100MHz, CDCl
3) δ=155.3,147.6,138.7,133.4,128.1,127.0,124.3,68.4,62.5,40.7,32.4,29.8,26.2,25.2,22.2.m/z MS (ESI-TOF
+) measured value [M]
+333.1017, C
15H
22N
2O
5Theoretical value 333.1426.
Embodiment 4({6-{[(2-acetonitrile oxygen base) (diisopropylaminoethyl) phosphine] oxo } base } amino carbonic acid 1-(2-nitrophenyl) ethyl ester) preparation of (compound 3b)
Compound 3a (0.5mmol; 1.0eq) with behind the THF azeotropic water removing three times in reaction flask quantitatively; under nitrogen protection, be dissolved in methylene dichloride (10mL); ice bath; add successively 2-cyanoethyl diisopropylaminoethyl phosphine chlorine (1.5eq) and TEA (3.0eq) in it; edge up to room temperature the TLC monitoring reaction.Add the methylene dichloride dilution when reaction is finished, use 10%NaHCO
3(2 * 30mL) wash anhydrous sodium sulfate drying after the organic phase.Concentrated organic phase, silicagel column separates, and moving phase is sherwood oil/methylene dichloride and adds 3% TEA, obtains compound 3b, is yellow oily liquid.
3b, (59% productive rate):
1H NMR (400MHz, CDCl
3) δ=7.92 (d, 1H, J=8Hz), 7.61 (d, 2H, J=4Hz), (7.40 m, 1H), 6.22 (q, 1H, J=6Hz), 4.75 (br, 1H), 3.82 (m, 2H), 3.58 (m, 4H), 3.12 (m, 2H), 2.63 (t, 2H), 1.60 (d, 3H, J=6Hz), 1.58 (m, 2H), 1.47 (m, 2H), (1.34 m, 4H), 1.17 (t, 12H, J=4Hz).
13C NMR (100MHz, CDCl
3) δ=155.2,147.5,138.7,133.3,128.0,127.0,124.2,117.6,68.3,63.3 (d, J=17Hz), 58.1 (d, J=19Hz), 42.9 (d, J=12Hz), 40.8,30.9 (d, J=7Hz), 29.7,26.2,25.4,24.4 (d, J=8Hz), 22.1,20.2 (d, J=7Hz).
31P NMR (162MHz, CDCl
3) δ=147.2 (s).
The preparation of embodiment 5 6-(tertiary butyl dimethyl Si) caproic acid (6-(tert-butyldimethylsilyloxy) hexanoic acid) (compound 4a)
Under the anhydrous and oxygen-free environment, TERT-BUTYL DIMETHYL CHLORO SILANE (TBDMS-Cl) (1.84g, 12.2mmol), imidazoles (800mg, 11.8mmol) is dissolved in dimethyl formamide (DMF) (10mL).Add 6-hydroxyl acetic ester (650 μ L, 4.00mmol) in system, stirring at room adds ether (50mL) after 24 hours, washes organic phase (anhydrous sodium sulfate drying after 3 * 50mL) with water.Concentrated solvent gets yellow oil, and it is dissolved in Triton B(main component is benzyltrimethylammonium hydroxide) methanol solution (40%w/w, 10mL) at room temperature stirred 1 hour.Concentrate system adds water (20mL) dilution, and adjusts pH to 4 with 1M HCl.The water layer extracted with diethyl ether (5 * 20mL), the organic phase anhydrous sodium sulfate drying, solvent evaporated gets compound 4a: yellow oily liquid,
1H NMR (400MHz, CDCl
3) δ=3.60 (t, 2H, J=6Hz), 2.36 (t, 2H, J=7Hz), 1.65 (m, 2H, J=8Hz), 1.53 (m, 2H, J=7Hz), 1.40 (dd, 2H, J=4,6Hz), 0.89 (s, 9H), 0.04 (s, 6H).
13C NMR (100MHz, CDCl
3) δ=179.1,62.9,33.9,32.4,26.0,25.3,24.5,18.3 ,-5.3.
The embodiment 6 6-(preparation of 6-(tertiary butyl dimethyl Si) caproic acid 1-(2-nitrophenyl) ethyl ester (1-(2-nitrophenyl) ethyl 6-(tert-butyldimethylsilyloxy) hexanoate) (compound 4b)
The preparation of embodiment 76 hydroxycaproic acid 1-(2-nitrophenyl) ethyl ester (1-(2-nitrophenyl) ethyl6-hydroxyhexanoate) (compound 5a)
Embodiment 8(6-{[(2-acetonitrile oxygen base) (diisopropylaminoethyl) phosphine] oxo } caproic acid 1-(2-nitrophenyl) ethyl ester) preparation of (compound 5b)
Compound 5a (0.5mmol; 1.0eq) with behind the THF azeotropic water removing three times in reaction flask quantitatively; under nitrogen protection, be dissolved in methylene dichloride (10mL); ice bath; add successively 2-cyanoethyl diisopropylaminoethyl phosphine chlorine (1.5eq) and TEA (3.0eq) in it; edge up to room temperature the TLC(thin-layer chromatography) monitoring reaction.Add the methylene dichloride dilution when reaction is finished, use 10%NaHCO
3(2 * 30mL) wash anhydrous sodium sulfate drying after the organic phase.Concentrated organic phase, silicagel column separates, and moving phase is sherwood oil/methylene dichloride and adds 3% TEA, gets compound 5b, is yellow oily liquid.
5b, (63% productive rate):
1H NMR (400MHz, CDCl
3) δ=7.91 (d, 1H, J=8Hz), 7.61 (d, 2H, J=4Hz), 7.42 (m, 1H), (6.30 t, 1H, J=6Hz), 3.85-3.75 (m, 2H), 3.66-3.53 (m, 4H), 2.62 (t, 2H, J=6Hz), 2.32 (m, 2H), (1.64-1.34 m, 11H), 1.16 (m, 12H).
13C NMR (100MHz, CDCl
3) δ=172.3,147.8,138.0,133.4,128.3,127.1,117.6,67.8,63.4,63.2 (d, J=17Hz), 58.2 (d, J=19Hz), 43.0 (d, J=12Hz), 34.2, (30.8 d, J=8Hz), 25.4,24.5 (d, J=8Hz), 21.9,20.3 (d, J=7Hz).
31P NMR (162MHz, CDCl
3) δ=147.3 (s).
Embodiment 9 6-[1-(2-nitrophenyl) second sulfydryl] preparation of 1-hexanol (6-[1-(2-nitrophenyl) ethylthio] hexan-1-ol) (compound 6a)
Under nitrogen, cesium carbonate (438mg, 1.34mmol), Tetrabutyl amonium bromide (TBAB) (432mg, 1.34mmol) and sodium iodide (227mg, 1.51mmol) be dissolved in DMF (6mL), add 6-sulfydryl hexanol (183 μ L in the system, 2.07mmol), stir after 1 hour under the room temperature and be cooled to 0 ℃.With compound 2 (302mg, 1.31mmol) being added dropwise to system after being dissolved in DMF (5mL). mixture edges up to room temperature, stir after 12 hours and filter, concentrated filtrate, silicagel column separates, and moving phase petrol ether/ethyl acetate ratio is 2/1, get compound 6a, yellow oily liquid (297mg, 1.05mmol, 80% productive rate);
1H NMR (400MHz, CDCl
3) δ=7.81 (d, 1H, J=8Hz), 7.69 (d, 1H, J=8Hz), 7.56 (t, 1H, J=8Hz), 7.32 (t, 1H, J=8Hz), 4.55 (q, 1H, J=7Hz), (3.55 t, 2H, J=7Hz), 2.29 (m, 2H, J=5Hz), 1.56 (d, 3H, J=7Hz), (1.45 m, 4H), 1.26 (m, 4H).
13C NMR (100MHz, CDCl
3) δ=149.3,139.1,132.8,129.4,127.4,123.5,62.6,38.1,32.4,31.5,29.2,28.4,25.1,22.8.m/z MS (ESI-TOF
+) measured value [M]
+306.0832, C
14H
21NO
3S theoretical value 306.1140.
Embodiment 10(2-acetonitrile oxygen base 6-[1-(2-nitrophenyl) second sulfydryl] oxygen base di-isopropyl phosphoramide) preparation of (compound 6b)
Compound 6a (0.5mmol; 1.0eq) with behind the THF azeotropic water removing three times in reaction flask quantitatively; under nitrogen protection, be dissolved in methylene dichloride (10mL); ice bath; add successively 2-cyanoethyl diisopropylaminoethyl phosphine chlorine (1.5eq) and TEA (3.0eq) in it; edge up to room temperature the TLC monitoring reaction.Add the methylene dichloride dilution when reaction is finished, use 10%NaHCO
3(2 * 30mL) wash anhydrous sodium sulfate drying after the organic phase.Concentrated organic phase, silicagel column separates, and moving phase is sherwood oil/methylene dichloride and adds 3% TEA, obtains compound 6b, is yellow oily liquid.
6b, (54% productive rate):
1H NMR (400MHz, CDCl
3) δ=7.84 (d, 1H, J=8Hz), 7.71 (d, 1H, J=8Hz), (7.58 t, 1H, J=8Hz), 7.34 (t, 1H, J=8Hz), (4.56 q, 1H, J=7Hz), 3.80 (m, 2H), 3.58 (m, 4H), 2.62 (t, 2H, J=6Hz), 2.30 (m, 2H), (1.59 d, 3H, J=7Hz), 1.54 (m, 2Hz), 1.44 (m, 2Hz), 1.28 (m, 4Hz), 1.16 (t, 12H, J=7Hz).
13C NMR (100MHz, CDCl
3) δ=149.4,139.2,132.8,129.4,127.5,123.6,117.6,63.5 (d, J=17Hz), (58.3 d, J=18Hz), 43.0 (d, J=12Hz), 38.2,31.6,30.9 (d, J=7Hz), 29.1,28.4,25.4,24.5 (t, J=8Hz), 22.9,20.3 (d, J=7Hz).
31P NMR (162MHz, CDCl
3) δ=147.3 (s).
The preparation of embodiment 11 S-6-hydroxyl hexyl O-1-(2-oil of mirbane) ethylenebis dithiocarbamate carbonic ethers (S-6-hydroxyhexyl O-1-(2-nitrophenyl) ethyl carbonothioate) (compound 7a)
Compound 1a (1.28g; 7.66mmol), N, N'-two succinimidyl carbonates (DSC) (2.94g; 11.5mmol) under nitrogen protection, be dissolved in acetonitrile (20mL). in mixed system, drip TEA (1.59mL, 11.5mmol).Stir after 1 hour under the room temperature and add DMAP (DMAP) (1.40g, 11.5mmol), 6-sulfydryl hexanol (1.45mL, 11.5mmol), continue under the room temperature to stir after 15 minutes to add entry (80mL) termination reaction, with methylene dichloride (3 * 50mL) aqueous phase extracted, merge organic phase and use anhydrous sodium sulfate drying, concentrated organic phase, silicagel column separates, and moving phase petrol ether/ethyl acetate ratio is 2/1, gets compound 7a: yellow oily liquid (1.32g, 4.04mmol, 57% productive rate);
1H NMR (400MHz, CDCl
3) δ=7.94 (d, 1H, J=8Hz), 7.64 (d, 2H, J=4Hz), 7.42 (m, 1H), 6.41 (q, 1H, J=6Hz), 3.58 (t, 2H, J=6Hz), 2.78 (m, 2H), 1.78 (br, 1H), 1.65 (d, 3H, J=7Hz), (1.60-1.49 m, 4H), 1.33 (m, 4H).
13C NMR (100MHz, CDCl
3) δ=170.3,147.2,137.4,133.8,128.5,127.0,124.4,70.9,62.5,32.3,30.7,29.5,28.2,25.1,22.0.m/z MS (ESI-TOF
+) measured value [M]
+350.1514, C
14H
21NO
3S theoretical value 350.1038.
Embodiment 12(S-{6-{[(2-acetonitrile oxygen base) (diisopropylaminoethyl) phosphine] oxo } base } O-1-(2-nitrophenyl) ethylenebis dithiocarbamate carbonic ether) preparation of (compound 7b)
Compound 7a (0.5mmol; 1.0eq) with behind the THF azeotropic water removing three times in reaction flask quantitatively; under nitrogen protection, be dissolved in methylene dichloride (10mL); ice bath; add successively 2-cyanoethyl diisopropylaminoethyl phosphine chlorine (1.5eq) and TEA (3.0eq) in it; edge up to room temperature the TLC monitoring reaction.Add the methylene dichloride dilution when reaction is finished, use 10%NaHCO
3(2 * 30mL) wash anhydrous sodium sulfate drying after the organic phase.Concentrated organic phase, silicagel column separates, and moving phase is sherwood oil/methylene dichloride and adds 3% TEA, obtains compound 7b, is yellow oily liquid.
7b, (73% productive rate):
1H NMR (400MHz, CDCl
3) δ=7.95 (d, 1H, J=8Hz), 7.64 (d, 2H, J=4Hz), 7.43 (m, 1H), 6.40 (q, 1H, J=8Hz), 3.80 (m, 2H), 3.58 (m, 4H), (2.77 m, 2H), 2.61 (t, 2H, J=10Hz), (1.65 d, 3H, J=8Hz), 1.57 (m, 4H), (1.34 m, 2H), 1.15 (t, 12H, J=8Hz).
13C NMR (100MHz, CDCl
3) δ=170.3,147.3,137.5,133.8,128.5,127.1,124.4,117.6,70.9,63.4 (d, J=17Hz), 58.2 (d, J=18Hz), 42.9 (d, J=12Hz), (30.9 d, J=7Hz), 30.8,29.5,28.2,25.3,24.5 (t, J=8Hz), 24.4,22.0,20.3 (d, J=7Hz).
31P NMR (162MHz, CDCl
3) δ=147.2 (s).
Embodiment 13 4-hydroxyl fourths-2-ketone 1-(2-nitrophenyl)-1，2-ethandiol ketal (4-hydroxybutan-2-one 1-(2-nitrophenyl)-1,2-diyl ketal) preparation (8a)
1-(2-nitrophenyl) ethylene glycol (183mg, 1.00mmol), 4-hydroxy-2-butanone (86 μ L, 1.00mmol), p-methyl benzenesulfonic acid (3.8mg, 0.02mmol), a small amount of molecular sieve and THF (15mL), place built that the reaction vessel of water trap is arranged, back flow reaction 6 hours is filtered, concentrated organic phase, silicagel column separates, moving phase petrol ether/ethyl acetate ratio is 3/1, obtains compound 8a: yellow oily liquid (100mg, 0.40mmol, 40% productive rate contains enantiomer);
1H NMR (400MHz, CDCl
3) δ=8.07 (d, 1H, J=8Hz), 7.95 (d, 1H, J=8Hz), (7.69 t, 1H, J=8Hz), 7.47 (dt, 1H, J=1,8Hz), 5.66 (t, 1H, J=7Hz), 4.75 (dd, 0.1H, J=7,8Hz), 4.68 (dd, 0.9H, J=7,8Hz), (3.96 m, 1H), 3.86 (m, 1H), 3.76 (m, 1H), (2.18 m, 2H), 1.61 (s, 0.4H), 1.50 (s, 2.6H).
13C NMR (100MHz, CDCl
3) δ=147.3,136.5,134.2,128.6,127.6,127.4,124.8,111.4,73.7,71.2,59.0,41.1,23.0.m/z MS (ESI-TOF
+) measured value [M]
+276.0582, C
15H
22N
2O
5Theoretical value 276.0848.
Embodiment 14(2-second cyanato-{ 2-[2-methyl-4-(2-nitrophenyl)-1,3-oxa--2-ketal] ethyl } di-isopropyl phosphoramide) preparation of (compound 8b)
Compound 8a (0.5mmol; 1.0eq) with behind the THF azeotropic water removing three times in reaction flask quantitatively; under nitrogen protection, be dissolved in methylene dichloride (10mL); ice bath; add successively 2-cyanoethyl diisopropylaminoethyl phosphine chlorine (1.5eq) and TEA (3.0eq) in it; edge up to room temperature the TLC monitoring reaction.Add the methylene dichloride dilution when reaction is finished, use 10%NaHCO
3(2 * 30mL) wash anhydrous sodium sulfate drying after the organic phase.Concentrated organic phase, silicagel column separates, and moving phase is sherwood oil/methylene dichloride and adds 3% TEA, obtains compound 8b, is yellow oily liquid.
8b, (74% productive rate):
1H NMR (400MHz, CDCl
3) δ=8.04 (d, 1H, J=8Hz), 7.92 (d, 1H, J=8Hz), (7.67 t, 1H, J=8Hz), 7.45 (t, 1H, J=8Hz), (5.59 m, 1H), 4.60 (t, 1H, J=8Hz), 3.88-3.59 (m, 7H), 2.64 (t, 2H, J=3Hz), 2.24 (t, 0.9H, J=7Hz), 2.12 (t, 1.1H, J=7Hz), 1.60 (s, 1.7H), (1.48 s, 1.3H), 1.19 (dd, 12H, J=4,6Hz).
13C NMR (100MHz, CDCl
3) δ=147.3,137.0,136.6,134.0,128.4,127.6,124.6,117.5,110.5,110.2,74.3,71.3,59.6 (d, J=18Hz), 58.3 (d, J=19Hz), (43.1 d, J=12Hz), 40.9,39.5 (d, J=7Hz), 24.6,23.5,20.3.
31P NMR (162MHz, CDCl
3) δ=147.7 (s)
Embodiment 15 5 ' end contains the solid phase synthesis of the oligonucleotide of photolytic activity protecting group compound (1b, 3b, 5b, 6b, 7b or 8b)
Use automatic dna synthesizer, 3 ' hold synthetic 5 ' XTTT TTC TCT CTC TCT G 3 ' (table 1) into the solid phase CPG-G of guanylic acid.At this moment still have the DMTr protecting group on last base of 5 ' end.With the coupling of phosphorus reagent the time, itself and 5 ' hydroxyl are manually extended to 120 seconds in the reaction times of tetrazole activation, and repeat coupling once.
For protecting group 6b, the 7b of sulfydryl, normal oxygenant has been used in this experiment, and namely the THF/ of 0.02M iodine pyridine/water thinks that than the solution that is 7/2/1 it all has no adverse effects to 6b, 7b, can be not sulfoxide or sulfone with sulfide oxidation.
Table 15 ' end adds the oligonucleotide of photolytic activity protecting group (1b, 3b, 5b, 6b, 7b or 8b)
The light that test example 1 contains 5 ' end photolytic activity protecting group ODN removes test
Contain oligonucleotide ODN 1 and the ODN 2 of protecting group for embodiment 15 5 prepared ' ends, with the strong aqua hydrolysis, it is cut from solid phase first, remove simultaneously the protecting group on the base.The HPLC purifying contains the ODN of photolytic activity protecting group.Because this optical active group is responsive to uv-radiation, and the attached mass-spectrometric technique of matter assisted laser desorption (Matrix-assisted laser desorption, MALDI) the laser radiation sample of normal operation 336nm wavelength, therefore should avoid using the method for MALDI to measure mass spectrum, and use electron spray(ES) (ESI) method to measure.
Consider under the environment of different concns and solvent, the efficient of illumination can be different, before the removal efficiency of protecting group is measured in illumination, ODN need to be diluted to 20 μ M with pure water.When calculating absorbancy, only consider the specific absorbance of base, ignore the specific absorbance of ortho-nitrophenyl under 260nm.Absorbancy by measuring 260nm solution and, by the specific absorbance of calculating, can obtain the concentration of the ODN aqueous solution.
During 365nm illumination, consistent for guaranteeing experiment condition, the bias light environment is black, and the relative position of quartz container and UV light source is fixed.HPLC result shows, the retention time shortening of oligonucleotide on reverse-phase chromatographic column after the illumination, and the water-soluble change of ODN is large, and prompting has removed terminal non-polar group ortho-nitrophenyl after optical radiation.Simultaneously, mass spectral:mass spectrographic result has also confirmed this result's (table 2).
Mass spectrum result and the HPLC retention time of oligonucleotide before and after table 2 illumination
In addition, the result of HPLC also shows almost there is not other assorted peak in the system, shows that the process that this light removes is clean, can not destroy the structure of ODN itself.Under the 280nm UV-irradiation, cytosine(Cyt) can form dimer, and then destroys the primary structure of DNA, and used 365nm UV-light is very slowly to the destruction of DNA here.
For 5 ' hold the ODN 1 into the phosphate protecting group.HPLC analyzes and finds after the ammonia solution, approximate two peaks (Fig. 3) of integral area occur.After reclaiming respectively, the ESI of the two comes to the same thing, and consistent with expection (actual measurement 4983.7 and 4984.4, theoretical 4980.8), and this has confirmed that these two peaks are a pair of enantiomorphs.Calculate the ratio of the two peak area, obtain its percent enantiomeric excess (e.e. is 5%) in limit of error.ODN for corresponding which kind of optical property of different retention time can't learn.Consider coming to the same thing after the two illumination, only select one to carry out dynamic analysis.Concentration of aqueous solution at oligomer is under the condition of 20 μ M, and the research different light time is on the impact of protecting group removal effect, the i.e. kinetics of this process.At this, with the peak area of deprotection base with remove and not the ratio of deprotection base two peak area sums as the parameter of weighing this efficient.Two kinds of optical siomerisms of this light reaction procedure are respectively 98.7 ± 0.8% and 96.7 ± 0.4% (n=3) at 15 minutes transformation efficiency.Carry out significance analysis t check S
R=0.664, t=3.688, and t
0.02,4=3.747, t＜t
0.02,4, i.e. two photoresponse dynamic behavior and the results of optical isomer under this concentration, on 99% confidence level without significant difference.Simultaneously, this process meets first order reaction kinetics, and speed of reaction is directly proportional k=0.358min with the first power of oligonucleotide concentration
-1, r
2=0.975(Fig. 4).
For N-terminal oligomer ODN 2, be under the condition of 20 μ M at concentration of aqueous solution, the transformation efficiency of this light reaction procedure in the time of 15 minutes is 95.6 ± 2.2%.Think accordingly, this photoresponse is quite efficient under this concentration.Light removal efficiency under other concentration is further research not.This process also meets first order reaction kinetics, k=0.293min
-1, r
2=0.986(Fig. 5).
For 5 ' and end carboxyl ODN 3, because strong aqua is removing solid phase and base protecting group simultaneously, also can hydrolysising carboxy acid ester's key, therefore with (the acetonitrile/water ratio is 4/1,3mg/mL) carries out illumination in 30 minutes in the direct dispersion of solid phase and acetonitrile and the water mixed solvent.Ammoniacal liquor hydrolysis solid phase is also with behind the HPLC purifying; the mass spectrum result has the oligomer relative molecular mass of carboxyl consistent with 5 of expection ' end; but this can not prove protecting group owing to ultraviolet lighting removes, and will be proven by the linked reaction to 5 ' end carboxyl and pyrene methylamine in the test example 2.
For 5 ' sulfydryl ODN 4, the material behind the ammonia solution HPLC purifying turns out to be the protected oligomer of sulfydryl through mass spectrum, actual measurement relative molecular mass 5100.But through illumination, the relative molecular mass of the primary product behind the HPLC purifying is constant, still records 5099.Think that according to document bonding has further occured for the sulfydryl that exposes after the illumination and the nitrogen-atoms of nitrosobenzene, obtains 1-thioether-dihydrobenzo isoxzzole by product (Fig. 6).Under the concentration of 0.2M, the transformation efficiency after the illumination in 15 minutes reaches 94.3 ± 0.4%.With this illumination intermediate (20 μ M) and DTT reactant aqueous solution 12 hours, HPLC found that this intermediate still exists, and has generated simultaneously the oligonucleotide of sulfydryl modification seldom subsequently.
In order further to study this phenomenon, with the ODN 5 ammonia solutions that thiocarbonic ester is modified, behind the HPLC purifying, mass spectrum shows the adduct that has obtained sulfydryl and vinyl cyanide.Add dithiothreitol (DTT) (DTT) (0.2M) in the ammonia solution, the main peaks that then obtains is for exposing the oligonucleotide of sulfydryl.Be that ammoniacal liquor can make the thiocarbonic acid SOH ester-type hydrolysis, and DTT can compete with sulfydryl, and from the propylene cyanogen reaction that the phosphoric acid skeleton is taken off, sulfydryl is exposed.
If at first illumination ODN 5, then use the concentrated ammonia solution (0.2M) of DTT with its hydrolysis, the analytical results of HPLC and MALDI-TOF shows (Fig. 7), product is the oligonucleotide (actual measurement 4950 for exposing sulfydryl only, theoretical 4948) product (surveying 5002, theory 5001) and on the oligomer after sulfydryl and the addition of propylene cyanogen.This result confirms, can not generate 1-thioether-dihydrobenzo isoxzzole structure after the thiocarbonic ester illumination, otherwise the sulfydryl that can not occur exposing, and the adduct of sulfydryl and propylene cyanogen also can not occur.
For 5 ' and end carbonyl ODN 6, adopt the method identical with amino, be 21.85min with ketal protected oligomer retention time after the ammonia solution.And after the illumination, the main peaks of 11.75min appears, the oligonucleotide that the end of concentrated front mass spectrum result and expection contains carbonyl coincide.Yet we are surprised to find that, concentrate before the illumination and the ODN after the illumination, and the peak that HPLC analyzes 12min again all can disappear, and the peak of 15min is only arranged, and mass spectrum is the result show, 5 ' only have phosphate.Accordingly, think that the β elimination of phosphodiester bond has occured this part, take off 2-butanone, only keep phosphate group.
In addition, this experiment is also found, the ODN 6 usefulness MALDI before the illumination are surveyed molecular weight, the main peaks that obtains than the expection lack 16 dalton (actual measurement climax 5055, inferior peak 5071, the theory 5067, Fig. 8).Yet, obtain expected results (actual measurement 5070, theoretical 5067) with the ESI test.According to the people such as Zhang (Zhang, J.; Frankevich, V.; Knochenmuss, R.; Friess, S.D.; Zenobi, R.J.Am.Soc.Mass S sherwood oil ctrom.2003,14,42.; Frankevich, V.; Zhang, J.; Friess, S.D.; Deashtiev, M.; Zenobi, R.Anal.Chem.2003,75,6063.) the MALDI matrix hydrogenating reduction phenomenon reported infers, we believe this be since sample due to matrix (3-hydroxy-picolinic acid) the generation deoxidation and reduction of MALDI.
The mark of test example 25 ' end functional group ODN
1, ODN 2 is connected (Fig. 9) with 5-TAMRA-NHS:
5-TAMRA(5-carboxytetramethylrhodamine), namely 5-carboxyl tetramethyl-rhodamine is a kind of fluorescence dye, multiplex it is soluble in DMF in labeling polypeptide or oligonucleotide, DMSO and water, maximum absorption wavelength 548nm has maximum emission wavelength 574nm.Use the NHS activator of its carboxyl in this work.After illumination, direct and solid phase ODN 2 reactions are lower from the solid phase cutting with ODN with ammoniacal liquor again with 5-TAMRA-NHS, and with mass spectrum (table 3) and uv-vis spectra (Figure 10) sign, the result who obtains is consistent with expection behind the HPLC purifying.
Concrete test method is as follows: get the mixing solutions that 5 ' amino solid phase ODN 2 (3mg) after synthetic is suspended in acetonitrile/water (the v/v ratio is 4/1,0.5mL), 365nm UV illumination 30 minutes.Suspension is centrifugal, abandons supernatant liquor, washes after the solid phase dry with acetonitrile (1 * 500 μ L), ether (1 * 500 μ L).Add 10 equivalents on solid phase ODN 5 '-TAMRA-NHS, DMF (500 μ L), 0.1M NaHCO
3(500 μ L), 30 ℃ of suspendibles 24 hours.Use subsequently DMF (3 * 500 μ L), acetonitrile (3 * 500 μ L), ether (1 * 500 μ L) is washed solid phase, drying, HPLC analyzes after the ammonia solution, and calculating productive rate by the peak area ratio of HPLC is 44%.MALDI-TOF characterizes, measured value 5345, theoretical value 5345(table 3).
Because previous document is protecting group after the ammonia solution at the NaHCO of 0.1M
3React in the aqueous solution, but the aqueous solution can make the solid phase autohemagglutination.By attempting discovery, under the TAMRA of 10eq, when adding organic bases TEA when only making solvent with DMF, this coupling efficiency is very low, is using DMF and 0.1MNaHCO
3Productive rate is 44% during the aqueous solution system.
2, ODN 3 is by pyrene (1-pyrene methylamine hydrochloride) mark
Get the mixing solutions that 5 ' carboxyl solid phase ODN 3 (3mg) after synthetic is suspended in acetonitrile/water (the v/v ratio is 4/1,0.5mL), 365nm UV illumination 30 minutes.Suspension is centrifugal, abandons supernatant liquor, and (500 μ L) washes solid phase, drying with acetonitrile.Solid phase adds the BOP of 10 equivalents ODN on solid phase in the EP pipe, HOBt, DIpeA, and the DMSO of 1-pyrene methylamine hydrochloride (100 μ L), 30 ℃ of lower suspendibles 24 hours.Then use DMF (3 * 500 μ L), acetonitrile (3 * 500 μ L), ether (1 * 500 μ L) are washed solid phase and dry, and HPLC analyzes after the ammonia solution, and calculating productive rate by the peak area ratio of HPLC is 82%.MALDI-TOF characterizes, measured value 5163, theoretical value 5162.
5 ' carboxyl is connected (Figure 11) with the pyrene methylamine hydrochloride: pyrene is the tetranuclear aromatics compounds, is soluble in non-polar solvent, commonly uses and does dyestuff, and maximum absorption wavelength is 340nm, and excitation wavelength is 376nm and 395nm, uses the hydrochloride of 1-pyrene methylamine among the present invention.Carboxyl ODN is after illumination on the solid phase, and direct and this compound reacts.The productive rate that this step solid phase obtains in DMSO is better than DMF, may be because the solubleness of hydrochloride in DMF of pyrene methylamine is limited.Then, use ammoniacal liquor to cut the HPLC purifying from solid phase.The result of mass spectrum (table 3) and uv-vis spectra has confirmed it is that illumination rather than ammonia solution remove the photolytic activity protecting group.Peak area ratio by HPLC calculates, and the mark productive rate is 82%.
The oligonucleotide mass spectrum of table 3 mark and chromatogram result (
A)Be the analytical column result)
Claims (10)
1. the phosphoramidite compound of the functional group of photaesthesia radical protection is characterized in that, it is the compound of structure shown in formula I or the formula II:
Wherein, R1 is selected from hydrogen, CH
3, COOCH
3R2 be selected from hydrogen or
R3 is selected from hydrogen or alkoxyl group; R4 is selected from hydrogen or alkoxyl group; Or R3-R4 is-OCH
2O; X is oxygen or nitrogen; Y is carbon, nitrogen or sulphur; Z is CH
2Or OCH
2CH
2N is the arbitrary integer of 1-6;
R2 be selected from hydrogen or
R3 is selected from hydrogen or alkoxyl group; R4 is selected from hydrogen or alkoxyl group; Or R3-R4 is-OCH
2O; R5 is hydrogen, CH
3Or phenyl; Z is CH
2Or OCH
2CH
2N is the arbitrary integer of 1-6.
2. the method for the described formula I of synthetic claim 1 compound is characterized in that, comprising: will obtain o-nitro benzyl alcohol after the reduction of ortho-nitrophenyl ethyl ketone; O-nitro benzyl alcohol is dissolved in methylene dichloride under nitrogen protection, ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts under the room temperature condition, and get final product.
3. the method for the described formula I of synthetic claim 1 compound comprises:
(1) o-nitro benzyl alcohol and DSC are formed intermediate, direct and 6-amino-hexanol effect gets 1-(2-nitrophenyl) ethyl 6-hydroxyl hexylamine base carbonic ether;
(2) 1-(2-nitrophenyl) ethyl 6-hydroxyl hexylamine base carbonic ether is dissolved in methylene dichloride under nitrogen protection, ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, and room temperature is reacted, and get final product.
4. the method for compound shown in the synthetic claim 1 formula I comprises:
(1) o-nitro benzyl alcohol obtains 1-(1-bromotrifluoromethane)-2-oil of mirbane through bromo; Under acetone and mineral alkali effect, obtain 6-(6-(tertiary butyl dimethyl Si) caproic acid 1-(2-nitrophenyl) ethyl ester with 1-(1-bromotrifluoromethane)-2-oil of mirbane (2) with by the 6 hydroxycaproic acid of protected silane; (6-(tertiary butyl dimethyl Si) caproic acid 1-(2-nitrophenyl) ethyl ester is sloughed the protected silane base and is obtained 6 hydroxycaproic acid 1-(2-nitrophenyl) ethyl ester with 6-;
(2) 6 hydroxycaproic acid 1-(2-nitrophenyl) ethyl ester is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, and room temperature is reacted, and get final product.
5. the method for compound shown in the synthetic claim 1 formula I, comprising: (1) is obtained 6-[1-(2-nitrophenyl) second sulfydryl with 1-(1-bromotrifluoromethane)-2-oil of mirbane by 6-sulfydryl hexanol nucleophilic substitution] the 1-hexanol; (2) 6-[1-(2-nitrophenyl) second sulfydryl] the 1-hexanol is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, and room temperature is reacted, and get final product.
6. the method for compound shown in the synthetic claim 1 formula I comprises: after the hydroxyl of (1) o-nitro benzyl alcohol is activated by DSC, obtain S-6-hydroxyl hexyl O-1-(2-oil of mirbane) ethylenebis dithiocarbamate carbonic ether; (2) S-6-hydroxyl hexyl O-1-(2-oil of mirbane) ethylenebis dithiocarbamate carbonic ether is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, and room temperature is reacted, and get final product.
7. the method for compound shown in the synthetic claim 1 formula II, comprise: (1) ortho-nitrophenyl ethyl glycol and 4-hydroxy-2-butanone are dissolved among the THF, under the dehydration of the catalysis of p-methyl benzenesulfonic acid and molecular sieve, obtain 4-hydroxyl fourth-2-ketone 1-(2-nitrophenyl)-1，2-ethandiol ketal; (2) 4-hydroxyl fourth-2-ketone 1-(2-nitrophenyl)-1，2-ethandiol ketal is dissolved in methylene dichloride under nitrogen protection, and ice bath adds 2-cyanoethyl diisopropylaminoethyl phosphine chlorine and TEA more successively, reacts under the room temperature condition, and get final product.
8. according to any one described method of claim 2-7, it is characterized in that: the temperature range of described room temperature is 20-30 ℃, is preferably 25 ℃.
9. an oligonucleotide is characterized in that: contain phosphoramidite compound claimed in claim 1.
10. the application of phosphoramidite compound claimed in claim 1 in the solid phase synthesis and 5 of oligonucleotide ' end functionalization comprises: in the solid phase synthesis of oligonucleotide, it is joined 5 of oligonucleotide ' end as last monomer; Slough protection after the illumination, connect again corresponding functional molecular or marker.
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| CN103232511A (en) * | 2013-04-11 | 2013-08-07 | 北京大学 | Light-cleavable group protected deoxyuridine triphosphate derivatives and a synthesis method and applications thereof. |
| CN108264522A (en) * | 2016-12-30 | 2018-07-10 | 杭州韶法医药技术有限公司 | The phosphoramide of the ether group of silicon containing aryl |
| CN108484708A (en) * | 2018-04-22 | 2018-09-04 | 温州瑞昇生物技术有限公司 | Nucleic acid and preparation method thereof containing photosensitive unit |
| CN109810141A (en) * | 2019-01-30 | 2019-05-28 | 中国石油大学(华东) | Sulfur-bearing phosphorous imide ester, preparation method and its application |
| CN109824721A (en) * | 2019-01-30 | 2019-05-31 | 中国石油大学(华东) | Sulfur-bearing phosphorous imide ester, preparation method and its application |
| CN109879922A (en) * | 2019-01-30 | 2019-06-14 | 中国石油大学(华东) | Nucleic acid, preparation method and its application containing photosensitive unit |
| CN114671912A (en) * | 2022-03-03 | 2022-06-28 | 西北工业大学 | Method for homogeneous synthesis of blue copper tripeptide with assistance of photo-shearing carrier |
| CN114805432A (en) * | 2022-03-03 | 2022-07-29 | 西北工业大学 | Novel photocatalytic-cleavable carboxylic acid protecting group and preparation method of amino acid derivative thereof |
| WO2023282245A1 (en) * | 2021-07-05 | 2023-01-12 | 国立研究開発法人科学技術振興機構 | Method for purifying nucleotides, device for purifying nucleotides, hydrophobic reagent, and hydrophobic substrate |
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| CN103232511A (en) * | 2013-04-11 | 2013-08-07 | 北京大学 | Light-cleavable group protected deoxyuridine triphosphate derivatives and a synthesis method and applications thereof. |
| CN103232511B (en) * | 2013-04-11 | 2015-06-17 | 北京大学 | Light-cleavable group protected deoxyuridine triphosphate derivatives and a synthesis method and applications thereof. |
| CN108264522A (en) * | 2016-12-30 | 2018-07-10 | 杭州韶法医药技术有限公司 | The phosphoramide of the ether group of silicon containing aryl |
| CN108484708B (en) * | 2018-04-22 | 2022-07-01 | 北京超精核酸科技有限公司 | Nucleic acid containing photosensitive unit and preparation method thereof |
| CN108484708A (en) * | 2018-04-22 | 2018-09-04 | 温州瑞昇生物技术有限公司 | Nucleic acid and preparation method thereof containing photosensitive unit |
| CN109810141A (en) * | 2019-01-30 | 2019-05-28 | 中国石油大学(华东) | Sulfur-bearing phosphorous imide ester, preparation method and its application |
| CN109824721A (en) * | 2019-01-30 | 2019-05-31 | 中国石油大学(华东) | Sulfur-bearing phosphorous imide ester, preparation method and its application |
| CN109879922A (en) * | 2019-01-30 | 2019-06-14 | 中国石油大学(华东) | Nucleic acid, preparation method and its application containing photosensitive unit |
| CN109810141B (en) * | 2019-01-30 | 2021-08-24 | 中国石油大学(华东) | Sulfur-containing phosphoramidite ester, preparation method and application thereof |
| WO2023282245A1 (en) * | 2021-07-05 | 2023-01-12 | 国立研究開発法人科学技術振興機構 | Method for purifying nucleotides, device for purifying nucleotides, hydrophobic reagent, and hydrophobic substrate |
| CN114671912A (en) * | 2022-03-03 | 2022-06-28 | 西北工业大学 | Method for homogeneous synthesis of blue copper tripeptide with assistance of photo-shearing carrier |
| CN114805432A (en) * | 2022-03-03 | 2022-07-29 | 西北工业大学 | Novel photocatalytic-cleavable carboxylic acid protecting group and preparation method of amino acid derivative thereof |
| CN114671912B (en) * | 2022-03-03 | 2024-05-14 | 西北工业大学 | Method for homogeneously synthesizing blue copper tripeptide with assistance of light shearing carrier |
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