CN102702010B - Photosensitive functionalized solid-supported phase, preparation method and application thereof - Google Patents
Photosensitive functionalized solid-supported phase, preparation method and application thereof Download PDFInfo
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- CN102702010B CN102702010B CN201210162973.6A CN201210162973A CN102702010B CN 102702010 B CN102702010 B CN 102702010B CN 201210162973 A CN201210162973 A CN 201210162973A CN 102702010 B CN102702010 B CN 102702010B
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Abstract
The invention discloses a photosensitive functionalized solid-supported phase, a preparation method and application thereof. The invention first provides photosensitive functionalized compounds with a structural formula of I or II. Further, the compound with the structural formula of I or II provided by the invention is connected onto amino solid-phase to obtain the photosensitive functionalized solid-supported phase with a structural formula of III or IV. In the solid-phase synthesis of oligonucleotides, the photosensitive functionalized solid-supported phase can be used as 3' end of ODN of the oligonucleotide. After the synthesis process, the oligonucleotides can be cleaved from the solid-supported phase and removed protective groups effectively under illumination to expose the corresponding functional groups, which can be connected with small molecule dyes, or cyclic oligonucleotides are formed through products with double-modified two ends themselves. Results of loading rate and photo-cleavage efficiency experiments show that the compounds with the structural formula of III or IV have higher loading rates and excellent photo-cleavage cutting efficiencies and can be used for solid-phase synthesis and functionalization of the oligonucleotides.
Description
Technical field
The present invention relates to the immobilized phase of functionalization of oligonucleotide, immobilized phase of light activated functionalization that relates in particular to light activated functionalized compounds and prepare with this compound and preparation method thereof, the invention still further relates to the immobilized application in oligonucleotide solid phase synthesis and functionalization of described light activated 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, light regulation and control have accurate positioning, regulation and control in time, on plurality of advantages such as system impact are little.Conventionally 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) is proposed in 1978 by J.F.Hoffman the earliest
1, this class group, to the photaesthesia within the scope of 340-370nm, can absorb the photon of this energy effectively.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.Due to the existence of ortho position nitro, after optical excitation, o-nitrophenylethanol compounds forms five-membered cyclic intermediate, and then obtains adjacent nitroso-group acetophenone compounds, disconnects with the other parts in compound.Due to the structure of o-nitrophenylethanol and derivative thereof, compare that other light can excise group in synthetic or comparatively stable under physiological environment, therefore in most of living things systems, be widely adopted, as ATP, DNA, RNA, protein, aptamers etc.
Oligonucleotide can be used for doing PCR primer, the probe, allele specific oligonucleotide oligonucleotide analysis (the Allele-specific oligonucleotide that for nucleotide sequence, detect, 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 to study the mechanism of production of various diseases and the expression network of genes involved thereof from gene level, 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.
When oligomer needs double-tagging maybe to need 3 ' restriction endonuclease to stablize, just need modify at 3 ' end.For introducing 3 ' amino solid phase, its advantage is that Fmoc can optionally remove in solid phase, thereby the functionalization that continuation is carried out ODN in solid phase is puted together.Meanwhile, this solid phase has been introduced a chiral carbon atom, may find the oligomer of its a pair of optical siomerism when HPLC purifying.Its deficiency is, although comparatively stable in oligomer building-up process, if misfeasance, some Fmoc protecting group can be sloughed, so freely amino can with synthesis cycle in seal step acetic anhydride used and react, the productive rate of expection oligomer reduces.The amino solid phase of another kind of introducing is to utilize phthalic imidine molecule, uses strong aqua oligomer can be cut and exposes amino from solid phase.This connecting arm is to synthetic stable circulation and do not have chiral centre.In addition also having first base in solid phase is the amination method of cytosine(Cyt) or the thymus pyrimidine of tool amino side-chain.
CPG for the sulfydryl modification that is modified with disulfide linkage protection of 3 ' sulfydryl; not oxidized for guaranteeing disulfide linkage in synthetic; all oxidation steps need be used the iodine liquid of 0.2M; after synthetic, with strong aqua, from solid phase, cut (55 ℃; 4 hours); after HPLC purifying, with the TCEP aqueous solution, disconnect disulfide linkage, do not need can use through HPLC purifying.Shankar study group once reported through the solid phase of its modification first through mercuric perchlorate processing, and then in the mixed system of THF and methyl alcohol, 350nm illumination 2 hours can be higher than small molecules such as the Fmoc-alanine being connected in 70% yield release solid phase and cholesterol.And solid phase is directly exposed under daylight, within 7 days, only discharge 5%.This method can reduce the susceptibility of solid phase to daylight, convenient operation.The people such as Greenberg report claim 3 ' hold the ODN for phosphate can from solid phase, do not cut by strong aqua; and the ODN of deprotection can be used as hybridization probe in solid phase; duplex oligomer after illumination after hybridization cuts from solid phase, has the antisense strand side of base mismatch not to be combined in solid phase.
The solid phase Pd of 3 ' hydroxyl
0under the environment that is 5.5 at pH, remove base and phosphoric protecting group, then use up from solid phase cutting.This method can be introduced non-natural 5-hydroxyl cytosine(Cyt) responsive to strong aqua or that can not obtain through synthetic conversion in ODN.With 3 ' amino solid phase synthesis ODN, then photodissociation, in liquid phase, 3 ' end is connected into the small molecules such as pyrene, steroidal, vitamin H, tripeptides, then sloughs base protecting group and 5 '-DMTr.Or after solid phase synthesis, de-DMTr protecting group, 5 ' end is connected into functional molecular, in liquid phase, after photodissociation, at 3 ' end, is connected into other molecule, last strong aqua dealkalize base protecting group and acetonitrile oxygen base
39.This has enriched the synthetic method of oligonucleotide conjugate to a great extent.The functionalization of 3 ' carboxyl also has report.
The amino of o-nitro benzyl alcohol structural defence is connected with the base of thymus pyrimidine monomer with carboxyl, after oligonucleotide is synthetic and after photo-irradiation treatment, can exposes amino and carboxyl.In chain, add functional molecular also can realize by the DNA single body of modifying, first optical active group being connected into thymus pyrimidine 2 ' position, to carry out DNA synthetic, is connected into functional molecular after light deprotection again.
Summary of the invention
One of the object of the invention is to provide light activated functionalization small molecules;
Two of the object of the invention is to provide the immobilized phase of light activated functionalization;
Three of the object of the invention is to provide the method for synthetic described light activated functionalization small molecules and the immobilized phase of light activated functionalization;
Four of object of the present invention is by described light activated functionalization small molecules and immobilized solid phase synthesis or the functional modification that is applied to mutually oligonucleotide of light activated functionalization.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Light activated functionalized compounds, its structural formula is with shown in following formula I or formula II:
Wherein, R1 is hydrogen or two (4-p-methoxy-phenyl) (phenyl) methoxyl group (DMTr); R2 is hydrogen or alkoxyl group (alkoxy); R3 is selected from hydrogen or alkoxyl group (alkoxy); Or R2-R3 is-OCH
2o; X is oxygen or nitrogen; Y is carbon, nitrogen or sulphur; Z is CH
2or OCH
2cH
2; M or n are the arbitrary integer of 1-6;
Wherein, R1 is hydrogen or DMTr; R2 is hydrogen or alkoxyl group (alkoxy); R3 is selected from hydrogen or alkoxyl group (alkoxy); Or R2-R3 is-OCH
2o; M is the arbitrary integer of 1-6.
Another object of the present invention has been to provide the immobilized phase of light activated functionalization, and its structural formula is the compound shown in following formula III or formula IV:
Wherein, R1 is hydrogen or DMTr; R2 is hydrogen or alkoxyl group (alkoxy); R 3 is selected from hydrogen or alkoxyl group (alkoxy); Or R2-R3 is-OCH
2o; X is oxygen or nitrogen; Y is carbon, nitrogen or sulphur; Z is CH
2or OCH
2cH
2; M or n are the arbitrary integer of 1-6; CPG is Bio-Glas;
Wherein, R1 is hydrogen or DMTr; R2 is hydrogen or alkoxy; R3 is selected from hydrogen or alkoxy; Or R2-R3 is-OCH
2o; M is the arbitrary integer of 1-6; CPG is Bio-Glas.
Another object of the present invention has been to provide the method for the immobilized phase compound of light activated functionalization shown in light activated functionalization small molecules described in synthesis type I or formula II and formula III or formula IV.
A kind of method of compound shown in synthesis type II, comprise: (1) by 2-amino-1-(2-nitrophenyl) ethanol and butynoic acid in dimethyl formamide (DMF), by become amido linkage to obtain N-[2-hydroxyl-2-(2-nitrophenyl) ethyl with I-hydroxybenzotriazole (HOBt) with ethylene dichloride (EDC)] 4-alkynes butyramide (compound 11a); (2) by N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] 4-alkynes butyramide and DMTr-Cl be dissolved in pyridine, and at room temperature reaction, obtains formula II compound (compound 11b).
A method for compound shown in synthesis type I, comprising: in water-less environment, by N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] 4-alkynes butyramide (compound 11a), DSC and DMAP (DMAP) are dissolved in and in acetonitrile, obtain mixed system; The dichloromethane solution that this mixed system is at room temperature stirred to rear dropping 6-amino-hexanol continues at room temperature stirring reaction, obtains 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl 6-hydroxyl hexyl amino-carbon acid esters (compound 12a); (2), under water-less environment, 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl 6-hydroxyl hexyl amino-carbon acid esters (12a) and DMTr-Cl are dissolved in methylene dichloride; Drip triethylamine, under room temperature, react, obtain compound 12b.
A kind of method of compound shown in synthesis type I, comprise: under water-less environment, 6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] caproic acid triethylamine (compound 10), 1,3-dicyclohexylcarbodiimide (DCC) and DMAP are dissolved in dimethyl formamide (DMF), at room temperature after stirring reaction, add N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] 4-alkynes butyramide (compound 11a), at 70 ℃, continue stirring reaction, obtain compound 13.
A kind of method of compound shown in synthesis type I, comprise: under water-less environment, by N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] 4-alkynes butyramide (11a), DSC and DMAP be dissolved in acetonitrile, at room temperature after stirring reaction, add 6-sulfydryl hexanol, continue stirring reaction, obtain S-6-hydroxyl hexyl O-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ether (compound 14a); Under water-less environment, S-6-hydroxyl hexyl O-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ether and DMTr-Cl are dissolved in methylene dichloride; Drip triethylamine, under room temperature, react, obtain compound 14b.
A method for compound shown in synthesis type III or formula IV, comprises the following steps:
(1) 4-bromo-butyric acid methyl esters is carried out to azide then with the methanol solution hydrolysis of LiOH, obtain 4-nitrine butyric acid (compound 15); 4-nitrine butyric acid and LCAA-CPG are formed to amido linkage, and azido-is modified in amino solid phase; (2) with xitix, copper sulfate, the DIPEA of 10 equivalents, the alkynyl in the nitrine in amino solid phase and formula I compound is carried out to Huisgen trinitride-alkynes cycloaddition reaction, in sealing solid phase, the amino of unmodified, obtains.
Room temperature described in the present invention is 20-30 ℃, is preferably 25 ℃.
The immobilized precursor structure that all has mutually o-nitrophenylethanol of the light activated functionalization of structure shown in formula III of the present invention or formula IV, there is light nicking activity, in the solid phase synthesis of oligonucleotide, can be used as 3 of the ODN of photolytic activity protecting group using it as oligonucleotide ' end, can protect phosphate, amino, carboxyl or sulfydryl; After oligonucleotide end of synthesis, under 365nm illumination, can effectively oligonucleotide be excised and slough protection from solid phase, expose corresponding function group; Oligonucleotide can connect dyestuff small molecules (as pyrene, rhodamine) by these functional groups (as amino, carboxyl), also can by the two products self of modifying in two ends, form the oligonucleotide of ring-type, reaches the object of oligonucleotide functionalization.Immobilized rate and light cutting efficiency experimental result show, the compound of structure shown in formula III of the present invention or formula IV has higher immobilized rate and excellent light cutting efficiency, can be applied to solid phase synthesis and the functionalization of oligonucleotide.
Accompanying drawing explanation
The compound of structure shown in Fig. 1 formula I of the present invention or formula II.
The immobilized phase of light activated functionalization shown in Fig. 2 formula III of the present invention or formula IV.
The synthetic route chart of Fig. 3 formula I of the present invention or formula II compound.
The synthetic method of the immobilized phase of light activated functionalization shown in Fig. 4 formula III of the present invention or formula IV.
Embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to 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 the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
Test materials and method
1, methylene dichloride, triethylamine, tetrahydrofuran (THF), acetonitrile equal solvent are dried with hydrolith, and distillation before each use.All reagent changes into the enterprises such as industrial development company limited, the scientific and technological (Shanghai) Co., Ltd. of splendid chemistry far away and buys from A Faaisha (Tianjin) Chemical Co., Ltd., Sigma's aldrich (China) company limited, the uncommon love of ladder (Shanghai).
2, all mixed solvents are with its volume ratio report.All reaction process detect by thin-layer chromatography, and thin layer chromatography board is produced by Merck company, have the F that 0.24nm is thick on it
254silica gel.Column chromatography used silica gel (200-300 order) is produced by Haiyang Chemical Plant, Qingdao.400MHz
1h nucleus magnetic resonance, 100MHz
13c nucleus magnetic resonance, 162MHz
31p 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 be 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 darkroom.
3, oligonucleotide solid phase synthesis and separated universal method
Oligonucleotide is synthetic by Applied Biosystems Incorporated 394 automatic DNA synthesizer DNAs.Amino solid phase LCAA-CPG buys from Millipore Corporation.CPG:dG and DNA single body are buied by AuGCT biotechnology company.Oligonucleotide is carried out the synthetic of 1 μ mol scale on the LCAA-CPG of CPG:dG or modification by standard DNA synthesis cycle.For the reaction of 5 ' end, the reaction times is extended for 120 seconds, and repeats once.
Half preparative HPLC carries out condition with Agilent C18 column (5 μ m, 9.4 * 250mm) on Varian Prostar: 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.
Analyze HPLC and with Symmetry C18 column (3.5 μ m, 4.6 * 75mm), carry out on Waters alliance e2695, initial intensity 0%B, gradient 1%B/min, flow velocity 1mL/min, PAD detector, detects wavelength 210-600nm.
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 365nm wavelength at UVP high intensity ultraviolet lamp, for system temperature is remained on below 30 ℃, if desired ice bag is placed under quartz cell.If be the suspension of solid phase in quartz cell, add small stirrer, be placed in and on magnetic stirring apparatus, carry out illumination.
Ammonia solution: add strong aqua (500 μ L) to having in the EP pipe of solid phase powder or dry oligonucleotide, at room temperature suspendible is 24 hours, gets liquid evaporate to dryness.
Acetic acid solution: for sloughing 5 ' end DMTr protecting group, to there being the aqueous acetic acid (500 μ L) that adds 80% in the EP pipe of dry oligonucleotide, at room temperature suspendible is 30 minutes, gets liquid evaporate to dryness.
The small molecules coupling agent adding (BOP, HOBt etc.) is made into its DMF solution before use, in 1mL dry DMF, dissolves the coupling agent of 20 μ mol, gets 100 μ L and is added in solid phase or dry oligonucleotide.
The preparation of embodiment 1 2-amino-1-(2-nitrophenyl) ethanol (compound 9)
Ortho-nitrophenyl ethyl ketone (5.00g, 30.3mmol), CuBr
2(12.8g, 62.0mmol) is dissolved in the mixed solvent of chloroform (15mL) and ethyl acetate (10mL), is warming up to 80 ℃ of backflows, adds 2 Br
2continue reaction 2 hours, add ethyl acetate (50mL) diluted system, washing (3 * 80mL), concentrated organic phase after anhydrous sodium sulfate drying, residuum is dissolved in acetone (15mL), and ice bath slowly adds NaN
3(2.36g, 36.4mmol), finishes, edge up to room temperature, continue reaction and after 1 hour, add ethyl acetate dilution, filter and concentrated mother liquor, resistates is dissolved in the mixed solvent of methyl alcohol (10mL) and dioxy six alkane (5mL), ice bath also stirs, by NaBH
4(1.14g, 30.0mmol) is dissolved in dioxy six alkane (5mL) and is added dropwise to above-mentioned system, after within 10 minutes, finishing, is stopped reaction, acetone cancellation, concentrate system silicagel column are separated, and moving phase petrol ether/ethyl acetate ratio is 2/1, obtains 2-nitrine-1-(2-nitrophenyl) ethanol.Be dissolved in tetrahydrofuran (THF) (THF) (15mL) in, ice bath also stirs, by PPh
3(6.50g, 24.8mmol) slowly add in this system, finish and edge up to room temperature, continue reaction concentrated after 3 hours, silicagel column is separated, and mobile phase dichloromethane/MeOH/ triethylamine (TEA) is than being 40/1/1, obtain compound 9: faint yellow solid (2.18g, 12.0mmol, 40%, totally 3 step productive rates).
1h NMR (400MHz, MeOH-d4) δ=7.80 (dd, 2H, J=8,11Hz), 7.62 (t, 1H, J=7Hz), 7.40 (t, 1H, J=8Hz), 5.06 (d, 1H, J=3,8Hz), 2.90 (dd, 1H, J=2,13Hz), 2.64 (dd, 1H, J=8,13Hz).
13c NMR (100MHz, MeOH-d4) δ=149.4,139.7,134.3,129.5,129.4,125.1,49.6.m/z MS (ESI-TOF
+) measured value [M]
+205.0586, C
8h
10n
2o
3theoretical value 205.0589.
Embodiment 2 6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] preparation of caproic acid triethylamine (compound 10)
In water-less environment, to DMTr-Cl(two (4-p-methoxy-phenyl) (phenyl) methyl chloride; Have another name called: 4,4-dimethoxytrityl chloride, 4,4-dimethoxytrityl chloride) (500mg, in methylene dichloride 1.48mmol) (10mL) solution, add 6 hydroxycaproic acid ethyl ester (200 μ L, 1.23mmol) and triethylamine (TEA) (430 μ L, 3.08mmol).System at room temperature stirs after 1 hour and uses NaHCO
3aqueous solution extraction (0.1M, 2 * 30mL), anhydrous sodium sulfate drying.Concentrated organic phase, residue is dissolved in the NaOH aqueous solution (6M, 25mL) and ethanol (4mL), at room temperature stirs, until TLC(thin-layer chromatography) disappearance of monitoring ester class.By chloroform (50mL) diluted system, washing (3 * 50mL).Concentrated organic phase, silicagel column is separated, and mobile phase methanol/methylene dichloride/TEA ratio is 3/7/1, obtains compound 10: white solid (550mg, 1.08mmol, 87% productive rate);
1h NMR (400MHz, CDCl
3) δ=7.42-7.18 (m, 10H), 6.80 (d, 4H, J=9Hz), 3.78 (s, 6H), 3.47 (s, 1H), 3.01 (t, 2H, J=7Hz), 2.84 (q, 6H, J=7Hz), 2.21 (t, 2H, J=8Hz), 1.60 (m, 4H, J=8Hz), 1.38 (dd, 2H, J=7,12Hz), 1.16 (t, 3H, J=7Hz).
13c NMR (100MHz, CDCl
3) δ=179.2,158.3,145.5,136.8,130.2,128.2,127.6,126.5,113.0,85.6,63.6,55.2,45.1,36.5,30.1,26.4,26.0,9.5.
Embodiment 3 N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] preparation of 4-alkynes butyramide (compound 11a)
4-alkynes butyric acid (640mg, 6.51mmol), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC-HCl) (1.24g, 6.47mmol) and I-hydroxybenzotriazole (HOBt) (996mg, 6.50mmol) be dissolved in dimethyl formamide (DMF) (5mL), at room temperature stir 1 hour backward its and drip DMF (3mL) solution of compound 9 (988mg, 5.40mmol).System at room temperature continues to stir 3 hours, with ethyl acetate (50mL) dilution, and washing (3 * 50mL) anhydrous sodium sulfate drying.Concentrated organic phase, silicagel column is separated, and moving phase petrol ether/ethyl acetate ratio is 1/1, obtains compound 11a: pale yellow oily liquid body (1.13g, 4.31mmol, 80% productive rate).
1h NMR (400MHz, CDCl
3) δ=7.95 (m, 2H), 7.67 (m, 1H, J=8Hz), 7.45 (m, 1H, J=1,8Hz), 6.22 (s, 1H), 5.38 (t, 1H, J=4Hz), 3.70 (dd, 2H, J=5,6Hz) .2.55 (m, 2H, J=2Hz), 2.46 (m, 2H, J=2Hz), 2.02 (m, 1H, J=3Hz).
13c NMR (100MHz, CDCl
3) δ=173.8,147.7,137.5,133.6,128.8,128.5,124.5,82.6,70.8,69.7,47.8,35.1,14.9.m/z MS (ESI-TOF
+) measured value [M]
+285.0812, C
13h
14n
2o
4theoretical value 285.0852.
Embodiment 4 N-{2-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group]-2-(2-nitrophenyl) ethyl } preparation of 4-alkynes butyramide (compound 11b)
In water-less environment, compound 11a (92mg, 351 μ mol) and DMTr-Cl (150mg, 443 μ mol) are dissolved in pyridine (5mL) and at room temperature stir and spend the night.Concentrate system, silicagel column is separated, and moving phase petrol ether/ethyl acetate ratio is 1/1 to add 1%TEA, obtains compound 11b: colourless oil liquid (154mg, 273 μ mol, 78% productive rate).
1h NMR (400MHz, CDCl
3) δ=7.77 (d, 1H, J=8Hz), 7.66 (d, 1H, J=8Hz), 7.49-7.18 (m, 11H), 6.68 (dd, 4H, J=9,22Hz), 5.38 (t, 1H, J=5Hz), 3.78 (d, 6H, J=8Hz), 2.43 (m, 2H), 2.32 (m, 2H), 1.96 (t, 1H, J=2Hz).
13c NMR (100MHz, CDCl
3) δ=170.5,158.6,158.5,146.8,145.1,138.1,135.6,135.3,132.7,130.2,130.1,130.0,127.9,127.8,127.3,126.9,123.7,113.2,113.0,87.6,82.9,69.8,69.2,46.2,45.1,35.3,14.6.m/z MS (ESI-TOF
+) measured value [M]
+587.2598, C
34h
32n
2o
6theoretical value 587.2158.
The preparation of embodiment 5 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl 6-hydroxyl hexyl amino-carbon acid esters (12a)
In water-less environment, compound 11a (131mg, 500 μ mol), N, N '-bis-succinimidyl carbonate (DSC) (154mg, 601 μ mol), with DMAP (DMAP) (74mg, 606 μ mol) is dissolved in acetonitrile (7mL), this system at room temperature stirs and after 30 minutes, drips 6-amino-hexanol (70mg, 597 μ mol) methylene dichloride (5mL) solution, continues at room temperature to stir 10 minutes.Concentrated this system, the separated resistates of silicagel column, moving phase is that methylene dichloride/TEA ratio is 10/1, obtains compound 12a: yellow solid (150mg, 370 μ mol, 74% productive rate).
1h NMR (400MHz, CDCl
3) δ=7.93 (d, 1H, J=8Hz), 7.63 (m, 2H), 7.42 (m, 1H), 6.65 (br, 1H), 6.19 (m, 1H), 5.50 (br, 1H), 3.85 (m, 1H, J=7Hz), 3.55 (m, 3H), 3.05 (dd, 2H, J=6,12Hz), 2.49 (s, 1H), 2.43 (m, 2H, J=2Hz), 2.35 (d, 2H, J=14Hz), 1.96 (s, 1H), 1.51-1.41 (m, 4H), 1.31-1.28 (m, 4H).
13c NMR (100MHz, CDCl
3) δ=171.4,155.4,134.4,133.5,128.8,128.0,124.6,82.6,71.0,69.2,62.3,43.8,40.8,35.1,32.3,29.5,26.2,25.2,14.7.m/zMS (ESI-TOF
+) measured value [M]
+428.1340, C
20h
27n
3o
6theoretical value 428.1798.
Embodiment 6 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl-6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] preparation of hexyl amino-carbon acid esters (compound 12b)
Under water-less environment, compound 12a (97mg, 240 μ mol) and DMTr-Cl (100mg, 261 μ mol) are dissolved in methylene dichloride (5mL) and obtain a purplish red solution.To this system, drip after TEA (82 μ L, 590 μ mol), under room temperature, stir after 30 minutes with methylene dichloride (50mL) dilution, saturated NaHCO
3the aqueous solution (2 * 50mL) is washed, anhydrous sodium sulfate drying.Concentrated organic phase, silicagel column is separated, and moving phase petrol ether/ethyl acetate ratio is 1/1 to add 1%TEA, obtains compound 12b: colourless foam (127mg, 187 μ mol, 78% productive rate).
1h NMR (400MHz, CDCl
3) δ=7.97-7.17 (m, 13H), 6.81 (d, 4H, J=9Hz), 6.26 (br, 1H), 6.16 (m, 1H), 4.92 (br, 1H), 3.89 (m, 1H, J=7Hz), 3.78 (s, 6H), 3.70 (m, 1H), 3.09 (m, 2H, J=6Hz), 3.02 (m, 2H, J=6Hz), 2.47 (t, 2H, J=6Hz), 2.37 (t, 2H, J=7Hz), 1.96 (d, 1H, J=2Hz), 1.72 (s, 1H), 1.57 (t, 2H, J=7Hz), 1.44 (t, 2H, J=7Hz), 1.35 (t, 2H, J=7Hz), 1.25 (t, 3H, J=7Hz).
13c NMR (100MHz, CDCl
3) δ=171.0,158.3,155.2,145.4,136.7,133.6,130.0,129.0,128.2,128.0,127.7,126.6,124.7,113.0,85.6,82.8,71.1,69.3,63.2,55.2,44.0,41.1,35.3,29.9,29.7,26.6,25.9,14.8,14.2.m/z MS (ESI-TOF
+) measured value [M]
+730.2100, C
41h
45n
3o
8theoretical value 730.3105.
Embodiment 7 6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] preparation of caproic acid 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl ester (compound 13)
Under water-less environment, compound 10 (270mg, 504 μ mol), 1,3-dicyclohexylcarbodiimide (DCC) (210mg, 1.02mmol) and DMAP (DMAP) (18mg, 80 μ mol) be dissolved in DMF (5mL), at room temperature stir and after 1 hour, add compound 11a (100mg, 382 μ mol), at 70 ℃, continue to stir 3 hours, with ethyl acetate (50mL) dilution, washing (3 * 50mL) anhydrous sodium sulfate drying.Concentrated organic phase, silicagel column is separated, and moving phase petrol ether/ethyl acetate ratio is 1/1 to add 1%TEA, obtains compound 13: yellow oily liquid (121mg, 185 μ mol, 48% productive rate).
1h NMR (400MHz, CDCl
3) δ=7.90-7.08 (m, 13H), 6.73 (d, 4H, J=8Hz), 6.28 (m, 1H, J=4Hz), 6.00 (br, 1H), 4.03 (q, 1H, J=7Hz), 3.82 (m, 1H, J=7Hz), 3.70 (s, 6H), 2.39-2.23 (m, 6H), 1.96 (s, 2H), 1.88 (s, 1H), 1.53-1.25 (m, 6H).
13c NMR (100MHz, CDCl
3) δ=172.6,170.9,158.3,148.0,145.3,136.5,133.6,133.5,129.9,129.1,129.0,128.1,128.0,127.8,127.7,127.6,126.5,124.6,113.1,112.9,85.6,82.8,70.2,69.3,63.0,60.3,55.1,43.5,35.1,34.0,25.8,25.1,24.6,24.3,14.7,14.1.m/z MS (ESI-TOF
+) measured value [M]
+701.2128, C
40h
42n
2o
8theoretical value 701.2839.
The preparation of embodiment 8 S-6-hydroxyl hexyl O-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ethers (compound 14a)
Under water-less environment, compound 11a (199mg, 400 μ mol), DSC (123mg, 480 μ mol), DMAP (60mg, 491 μ mol) be dissolved in acetonitrile (10mL), system at room temperature stirs and after 30 minutes, adds 6-sulfydryl hexanol (65 μ L, 515 μ mol).Continue to stir after 1.5 hours, concentrate system, silicagel column is separated, and moving phase petrol ether/ethyl acetate ratio is 1/3, obtains compound 14a: yellow oily liquid (123mg, 292 μ mol, 77% productive rate).
1h NMR (400MHz, CDCl
3) δ=8.01 (d, 1H, J=8Hz), 7.65 (m, 2H), 7.48 (dd, 1H, d=2,4Hz), 6.45 (dd, 1H, J=4,7Hz), 6.18 (br, 1H), 3.90-3.79 (m, 2H), 3.62 (dd, 2H, J=2,7Hz), 2.81 (t, 2H, J=7Hz), 2.44 (m, 2H), 2.37 (m, 2H), 1.72-1.45 (m, 8H).
13c NMR (100MHz, CDCl
3) δ=171.0,170.7,147.7,133.8,133.1,129.2,127.9,124.9,82.8,73.2,69.4,62.7,43.3,39.0,35.2,32.5,31.0,29.4,28.2,29.2,25.5,14.7.m/z MS (ESI-TOF
+) measured value [M]
+445.1453, C
20h
26n
2o
6s theoretical value 445.1409.
Embodiment 9 S-6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] preparation of hexyl-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ether (compound 14b)
Under water-less environment, compound 14a (123mg, 292 μ mol) and DMTr-Cl (108mg, 320 μ mol) are dissolved in methylene dichloride (5mL) and obtain a purplish red solution.To this system, drip after TEA (89 μ L, 640 μ mol), under room temperature, stir after 30 minutes with methylene dichloride (40mL) dilution, saturated NaHCO
3the aqueous solution (2 * 40mL) is washed, anhydrous sodium sulfate drying.Concentrated organic phase, silicagel column is separated, and moving phase petrol ether/ethyl acetate ratio is 1/1 to add 1%TEA, obtains compound 14b, colourless foam (160mg, 221 μ mol, 78% productive rate).
Compound 14b:
1h NMR (400MHz, CDCl
3) δ=8.03-7.25 (m, 13H), 6.81 (d, 4H, J=9Hz), 6.47 (dd, 1H, J=4,7Hz), 6.08 (br, 1H), 3.88-3.81 (m, 2H), 3.77 (s, 6H), 3.02 (t, 2H, J=7Hz), 2.78 (t, 2H, J=7Hz), 2.45 (t, 2H, J=2Hz), 2.44 (m, 2H), 1.99 (t, 2H, J=3Hz), 1.59-1.55 (m, 4H), 1.35-1.30 (m, 4H).
13cNMR (100MHz, CDCl
3) δ=170.8,170.6,158.3,147.7,145.3,136.6,133.7,133.2,130.0,129.2,128.1,127.9,127.6,126.5,124.9,112.9,85.6,82.8,73.1,69.5,63.1,55.1,43.3,35.2,31.1,29.8,29.5,28.5,25.7,14.7.m/z MS (ESI-TOF
+) measured value [M]
+747.2657, C
41h
44n
2o
8s theoretical value 747.2716.
The preparation of embodiment 10 4-nitrine butyric acid (compound 15)
Bromo-butyric acid methyl esters (600 μ L, 4.77mmol), sodium azide (372mg, 5.72mmol) is dissolved in DMF (5mL), stirs after 2 hours with methylene dichloride (50mL) dilution, washing (4 * 50mL), anhydrous sodium sulfate drying under room temperature.Concentrate organic phase, add MeOH (7mL) solution of LiOH (228mg, 9.54mmol), under room temperature, stir and spend the night.Concentrate system, adds water (10mL), uses NH
4in Cl saturated aqueous solution and system to pH=5.Methylene dichloride (3 * 30mL) extraction, anhydrous sodium sulfate drying organic phase, evaporate to dryness, obtains compound 15: deep yellow oily liquid (395mg, 3.06mmol, 64% liang of step productive rate).
1h NMR (400MHz, CDCl
3) δ=3.36 (t, 2H, J=7Hz), 2.45 (t, 2H, J=7Hz), 1.90 (m, 2H, J=7Hz).
13c NMR (100MHz, CDCl
3) δ=177.7,50.5,30.8,24.0.
The preparation of the amino solid phase of embodiment 11 (CPG 11)
Benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate (HBTU) (38mg, 100 μ mol), DMAP (12mg, 100 μ mol) and 4-nitrine butyric acid (43mg, 330 μ mol) be dissolved in dry DMF (1mL), under room temperature, suspendible adds LCAA-CPG (100mg after 30 minutes, 99.2 μ mol/g, 9.9 μ mol).At room temperature continue suspendible after 24 hours, use successively methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (3 * 1mL) to wash solid phase.For detecting the first step coupling effect, get few solid phase, drip the DMF solution of 10% diisopropyl ethyl amine (DIPEA) and the DMF solution of 1%2,4,6-trinitro-benzene-sulfonic acid (TNBS).Solid phase not yet white shows that on it, amino substantially reacts.Solid phase dispersion is in (1mL) solution of the dimethyl sulfoxide (DMSO) (DMSO) that is dissolved with compound 11b (200 μ mol), add the anti-hematic acid sodium of L-(20mg, 100 μ mol), anhydrous cupric sulfate (8mg, 50 μ mol) and diisopropyl ethyl amine (DIPEA) (8 μ L, 50 μ mol).Reaction system suspendible at 25 ℃ is used DMSO (1 * 1mL), 0.1MNaHCO successively after 24 hours
3the aqueous solution (1 * 1mL), methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (1 * 1mL).After this, to the acetonitrile solution (500 μ L) that adds the pyridine of N-Methylimidazole and acetonitrile solution (500 μ L), acetic anhydride in solid phase, i.e. Cap A, CapB, under room temperature, suspendible is 30 minutes, with acetonitrile (3 * 1mL), washes solid phase, obtains.
The preparation of the amino solid phase of embodiment 12 (CPG 12)
HBTU (38mg, 100 μ mol), DMAP (12mg, 100 μ mol) and 4-nitrine butyric acid (43mg, 330 μ mol) be dissolved in dry DMF (1mL), under room temperature, suspendible adds LCAA-CPG (100mg after 30 minutes, 99.2 μ mol/g, 9.9 μ mol).At room temperature continue suspendible after 24 hours, use successively methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (3 * 1mL) to wash solid phase.For detecting the first step coupling effect, get few solid phase, drip the DMF solution of 10%DIPEA and the DMF solution of 1%TNBS.Solid phase not yet white shows that on it, amino substantially reacts.Solid phase dispersion, in DMSO (1mL) solution that is dissolved with compound 12b (200 μ mol), adds the anti-hematic acid sodium of L-(20mg, 100 μ mol), anhydrous cupric sulfate (8mg, 50 μ mol) and DIpeA (8 μ L, 50 μ mol).Reaction system suspendible at 25 ℃ is used DMSO (1 * 1mL), 0.1M NaHCO successively after 24 hours
3the aqueous solution (1 * 1mL), methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (1 * 1mL).After this, to the acetonitrile solution (500 μ L) that adds the pyridine of N-Methylimidazole and acetonitrile solution (500 μ L), acetic anhydride in solid phase, i.e. CapA, CapB, under room temperature, suspendible is 30 minutes, with acetonitrile (3 * 1mL), washes solid phase, obtains.
The preparation of the amino solid phase of embodiment 13 (CPG 13)
HBTU (38mg, 100 μ mol), DMAP (12mg, 100 μ mol) and 4-nitrine butyric acid (43mg, 330 μ mol) be dissolved in dry DMF (1mL), under room temperature, suspendible adds LCAA-CPG (100mg after 30 minutes, 99.2 μ mol/g, 9.9 μ mol).At room temperature continue suspendible after 24 hours, use successively methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (3 * 1mL) to wash solid phase.For detecting the first step coupling effect, get few solid phase, drip the DMF solution of 10%DIPEA and the DMF solution of 1%TNBS.Solid phase not yet white shows that on it, amino substantially reacts.Solid phase dispersion, in DMSO (1mL) solution that is dissolved with compound 13 (200 μ mol), adds the anti-hematic acid sodium of L-(20mg, 100 μ mol), anhydrous cupric sulfate (8mg, 50 μ mol) and DIpeA (8 μ L, 50 μ mol).Reaction system suspendible at 25 ℃ is used DMSO (1 * 1mL), 0.1M NaHCO successively after 24 hours
3the aqueous solution (1 * 1mL), methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (1 * 1mL).After this, to the acetonitrile solution (500 μ L) that adds the pyridine of N-Methylimidazole and acetonitrile solution (500 μ L), acetic anhydride in solid phase, i.e. CapA, CapB, under room temperature, suspendible is 30 minutes, with acetonitrile (3 * 1mL), washes solid phase, obtains.
The preparation of the amino solid phase of embodiment 14 (CPG 14)
HBTU (38mg, 100 μ mol), DMAP (12mg, 100 μ mol) and 4-nitrine butyric acid (43mg, 330 μ mol) be dissolved in dry DMF (1mL), under room temperature, suspendible adds LCAA-CPG (100mg after 30 minutes, 99.2 μ mol/g, 9.9 μ mol).At room temperature continue suspendible after 24 hours, use successively methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (3 * 1mL) to wash solid phase.For detecting the first step coupling effect, get few solid phase, drip the DMF solution of 10%DIPEA and the DMF solution of 1%TNBS.Solid phase not yet white shows that on it, amino substantially reacts.Solid phase dispersion, in DMSO (1mL) solution that is dissolved with compound 14b (200 μ mol), adds the anti-hematic acid sodium of L-(20mg, 100 μ mol), anhydrous cupric sulfate (8mg, 50 μ mol) and DIpeA (8 μ L, 50 μ mol).Reaction system suspendible at 25 ℃ is used DMSO (1 * 1mL), 0.1M NaHCO successively after 24 hours
3the aqueous solution (1 * 1mL), methyl alcohol (3 * 1mL), methylene dichloride (3 * 1mL), ether (1 * 1mL).After this, to the acetonitrile solution (500 μ L) that adds the pyridine of N-Methylimidazole and acetonitrile solution (500 μ L), acetic anhydride in solid phase, i.e. CapA, CapB, under room temperature, suspendible is 30 minutes, with acetonitrile (3 * 1mL), washes solid phase, obtains.
Synthesizing of embodiment 15 oligonucleotides
The solid phase synthesis that carries out oligonucleotide by four kinds of prepared solid phases of embodiment 11-14, DNA synthesizes ordinary method automatically, has finally synthesized 5 ' TTT TTC TCT CTC TCT tetra-chains of Y3 ' (table 1).
In order to apply this strategy synthesis of cyclic oligonucleotide, at oligomerization two ends, use respectively amino solid phase and carboxyl-protecting group molecule, as ODN 11.
Table 13 ' hold as modifying the oligonucleotide of solid phase and the oligomer of difunctionalization
The immobilized efficiency of four kinds of amino solid phases that test example 1 is prepared and the determination test of light cutting efficiency
For the immobilized efficiency of four kinds of prepared solid phases of mensuration embodiment 11-14, the solid phase taking a morsel after sealing in this step, add the dichloromethane solution of 3%TFA, take off DMTr protecting group.With anhydrous methylene chloride, make blank, the absorbance of the different weaker concns of measuring this solution under 504nm, and linear regression is extrapolated to the absorbancy (table 2) of this solution.The specific absorbance 78000M of the DMTr positive ion by reported literature
-1cm
-1, calculate the amount of substance concentration of DMTr positive ion, and then calculate the supported quantity (table 3) after modification.From result, we can find out, in the group of four kinds of solid phases, between parallelism and group, parallelism is all good, and the efficiency of this two single-step solid phase reaction is about 60%.Result shows, this modifying method is feasible, effective, reproducible.
For measuring the light cutting efficiency of immobilized phase, the solid phase that again takes a morsel (embodiment 11-14 is prepared), be suspended in acetonitrile/water (4/1,6mg/1mL), 365nm illumination after 30 minutes, measure as stated above respectively in its solution and solid phase on the amount of substance of residual DMTr positive ion, the amount in solution is the light cutting efficiency (table 3) of these four kinds of solid phases under this condition with the ratio of total amount of substance.
The immobilized rate measurement result of table 2 (showing DMTr absorbancy)
The immobilized rate of four kinds of solid phases of table 3 and light cutting efficiency
Test example 2 is containing the light cutting of 3 ' terminal modified solid phase ODN
Four kinds of solid phase oligonucleotides of embodiment 15 synthesizeds are used up and carried out cutting test.
Mass spectrum and the stratographic analysis result of the oligonucleotide of table 43 ' end functionalization
Prepared ODN 7, ODN 8, ODN 9 and ODN 10 illumination (365nm) of embodiment 15, after 30 minutes, are hydrolyzed the ODN cutting, and carry out HPLC purifying with strong aqua.If complete error-free ODN, its 5 ' end has DMTr, and it is than ethanoyl sealing for synthetic ODN(5 ' end of makeing mistakes, and there is no DMTr) have longer retention time, be easy to reversed-phase column separation.Again the ODN obtaining is removed to DMTr protecting group with 80% aqueous acetic acid, after HPLC purifying, obtain the ODN that target is modified.Mass spectral characteristi result (table 4) has confirmed that these ODN are desired.
After above-mentioned off-test; by reference to document, can find out that the phosphate solid phase of above-mentioned modification is stable for strong aqua because there is no carboxylic acid ester bond; be the acetonitrile oxygen base that strong aqua only can be removed base protecting group and tricresyl phosphate ester bond, and ODN can not cut down from solid phase.Therefore for 3 ' phosphate of end also can first ammonia solution, then it is modified in solid phase, last photodissociation obtains 3 ' hold the oligomer into phosphate.
Synthesizing of the mark of test example 33 ' end functional group ODN and cyclic oligomeric thing
ODN 8(embodiment 15 is prepared) after solid phase illumination, with the oligonucleotide of functional group in liquid phase.Before ammonia solution, it is reacted with 1-pyrene butyric acid.By attempting thinking that DMF does comparatively ideal of solvent effect after acetonitrile, ethylene dichloride, DMSO and DMF equal solvent, the productive rate being obtained by HPLC calculated by peak area is 85%.And then removing 5 ' end DMTr with acetic acid, after HPLC purifying, with mass spectral characteristi, its molecular weight is confirmed (table 5).
The oligonucleotide mass spectrum of table 5 mark and chromatogram result (a) are analytical column result)
Claims (11)
1. light activated functionalized compounds, its structural formula is with shown in following formula I or formula II:
Wherein, R1 is hydrogen or two (4-p-methoxy-phenyl) (phenyl) methyl; R2 is hydrogen; R3 is hydrogen; Or R2-R3 is-OCH
2o; X is oxygen or nitrogen; Y is carbon, nitrogen or sulphur; Z is CH
2or OCH
2cH
2; M or n are the arbitrary integer of 1-6;
Wherein, R1 is hydrogen or two (4-p-methoxy-phenyl) (phenyl) methyl; R2 is hydrogen; R3 is hydrogen; Or R2-R3 is-OCH
2o; M is the arbitrary integer of 1-6.
2. the immobilized phase of light activated functionalization, it is compound shown below:
R be selected from DMTr,
wherein, DMTr is two (4-p-methoxy-phenyl) (phenyl) methyl; CPG is Bio-Glas.
3. N-{2-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group in compound shown in formula II described in a synthetic claim 1]-2-(2-nitrophenyl) ethyl } method of 4-alkynes butyramide, comprising: by N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] 4-alkynes butyramide and two (4-p-methoxy-phenyl) (phenyl) methyl chloride is dissolved in pyridine, at room temperature reaction, obtains.
4. 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl-6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group in compound shown in formula I described in a synthetic claim 1] method of hexyl amino-carbon acid esters, comprise: under water-less environment, 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl 6-hydroxyl hexyl amino-carbon acid esters and two (4-p-methoxy-phenyl) (phenyl) methyl chloride are dissolved in methylene dichloride, drip triethylamine, under room temperature, react, obtain.
5. 6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group in compound shown in formula I described in a synthetic claim 1] method of caproic acid 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl ester, comprise: under water-less environment, 6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] caproic acid triethylamine, 1,3-dicyclohexylcarbodiimide and DMAP are dissolved in dimethyl formamide, at room temperature after stirring reaction, add N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] 4-alkynes butyramide, at 70 ℃, continue stirring reaction, obtain.
6. the methoxyl group of compound S-6-[two (4-p-methoxy-phenyl) (phenyl) shown in formula I described in a synthetic claim 1] method of hexyl-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ether, comprise: under water-less environment, by N-[2-hydroxyl-2-(2-nitrophenyl) ethyl] 4-alkynes butyramide, N, N'-bis-succinimidyl carbonates and DMAP are dissolved in acetonitrile, at room temperature after stirring reaction, add 6-sulfydryl hexanol, continue stirring reaction, obtain S-6-hydroxyl hexyl O-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ether, under water-less environment, S-6-hydroxyl hexyl O-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ether and two (4-p-methoxy-phenyl) (phenyl) methyl chloride is dissolved in methylene dichloride, drip triethylamine, under room temperature, react, obtain.
7. a method for compound shown in synthetic claim 2, comprises the following steps:
(1) 4-bromo-butyric acid methyl esters is carried out to azide then with the methanol solution hydrolysis of LiOH, obtain 4-nitrine butyric acid, 4-nitrine butyric acid and amino solid phase LCAA-CPG are formed to amido linkage, and azido-is modified on amino solid phase LCAA-CPG, (2) with xitix, copper sulfate, diisopropyl ethyl amine is by the nitrine on amino solid phase LCAA-CPG and 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl-6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] hexyl amino-carbon acid esters, 6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] caproic acid 1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethyl ester or S-6-[two (4-p-methoxy-phenyl) (phenyl) methoxyl group] alkynyl in hexyl-1-(2-nitrophenyl)-2-(4-alkynes amide-based small) ethylenebis dithiocarbamate carbonic ether carries out Huisgen trinitride-alkynes cycloaddition reaction, seal the amino of the upper unmodified of amino solid phase LCAA-CPG, obtain.
8. the method described in any one according to claim 3-6, is characterized in that: described room temperature is 20-30 ℃.
9. it is characterized in that in accordance with the method for claim 8: described room temperature is 25 ℃.
10. an oligonucleotide, is characterized in that: contain light activated functionalized compounds claimed in claim 1 or the immobilized phase of light activated functionalization claimed in claim 2.
11. light activated functionalized compounds claimed in claim 1 or the immobilized application in oligonucleotide solid phase synthesis of light activated functionalization claimed in claim 2.
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