CN101735298A - Phosphoric acid diester and preparation method thereof - Google Patents
Phosphoric acid diester and preparation method thereof Download PDFInfo
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- CN101735298A CN101735298A CN200910185839A CN200910185839A CN101735298A CN 101735298 A CN101735298 A CN 101735298A CN 200910185839 A CN200910185839 A CN 200910185839A CN 200910185839 A CN200910185839 A CN 200910185839A CN 101735298 A CN101735298 A CN 101735298A
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- acid ester
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Abstract
The invention discloses a phosphoric acid diester and a preparation method thereof, wherein the phosphoric acid diester is cyanogroup boron hydride organic phosphate. The preparation method comprises the following processes: a. nucleoside coupling, b. cyanogroup boron hydride replacement, c. deprotection, d. purification. Compared with the prior art, the invention provides a brand-new phosphoric acid diester- cyanogroup boron hydride organic phosphate, wherein one non- bridging oxygen atom of the phosphoric acid diester group is replaced by cyanogroup boron hydride. Compared with natural DNA, the phosphoric acid diester can stably and well resist nuclease hydrolysis in a wide rang of pH value; meanwhile, based on capryl alcohol partition experiment (namely lipotropism measurement), cyanogroup boron hydride organic phosphate can demonstrate greater membrane permeability than conventional oligonucleotides.
Description
Technical field
The invention belongs to phosphodiester and preparation method thereof, belong to phosphodiester that is used for antisense nucleic acid and gene probe field and preparation method thereof especially.
Background technology
Nucleotide is the compound that is formed by connecting by base (mainly being the derivative of purine, pyrimidine base), pentose (ribose or ribodesose) and phosphoric acid, oligonucleotide is the general name (Nucleotide that comprises thymus nucleic acid DNA or RNA (ribonucleic acid)) that a class has only the short chain Nucleotide of 20 left and right sides base pairs, oligonucleotide can be at an easy rate and their complementary pair link, so be commonly used to determine the structure of DNA or RNA, in being usually used in processes such as gene chip, electrophoresis, fluorescence in situ hybridization as probe.
In recent years, the research that is used for multiple treatment of diseases of oligonucleotide, particularly antisense oligonucleotide has been subjected to paying attention to widely.
Antisense nucleic acid is meant and target DNA or RNA base complementrity, and bonded section of DNA or RNA with it.
Antisense technology is meant and utilizes antisense nucleic acid to suppress some expression of gene specifically.1967, Belikova etc. proposed to utilize one section antisense oligonucleotide to come the imagination of inhibition of gene expression specifically.1978, Paul etc. utilized one section antisense DNA oligonucleotide chain successfully to suppress sarcoma viral the duplicating in Louth (Rous), cause that people pay close attention to greatly.The eighties in 20th century, the success of oligonucleotide synthetic technology, the research fast development of antisense nucleic acid is got up, and makes antisense technology obtain certain progress at aspects such as antitumor, antiviral and apoptosis mechanism, signal pass through mechanism.
The natural antisense oligonucleotide is the less poly oligonucleotide of molecule quality, they and the positive-sense strand of organism double-stranded DNA or one section sequence complementation of mRNA.Find by the regulation and control of plasmid replication in the E.Coli body the earliest.
Sense-rna can be obtained by synthetic and antisense expression vector dual mode.The latter utilizes gene recombination technology, and it is resulting oppositely to insert one section target gene between suitable promotor and transcription terminator, and the RNA of antisense expression can suppress homogenic expression, effect stability, lasting.The antisense oligonucleotide of synthetic comprises sense-rna and antisense DNA, is made up of 15～20 Nucleotide usually, for preventing the Decomposition of intracellular nucleic acid enzyme, often it is carried out chemically modified.They are transported to target cell, only produce fugitive answering, can not stay long lasting hereditary effect, this does not verify under the prerequisite of its side effect as yet for early-stage Study, and security is preferably arranged; The target sequence of its consumption and effect can artificially be controlled, and can not disturb the normal control methods of other gene structure and they self in principle; Especially antisense oligonucleotide can not produce resistance, also can adopt the multiple spot antisense to handle and strengthen the inhibition effect.The gene silencing function of sense-rna not only can be used for studying the unknown gene function, and can be used for multiple treatment of diseases.After reported first such as Zammeenik were duplicated with antisense oligonucleotide inhibition Rous sarcoma virus, antisense oligonucleotide just was applied to the research of tumour, virus infection, parasitosis rapidly.Liu song-kai etc. utilizes sense-rna and siRNA to suppress the synthetic of required cell protein CyPA in the HIV-1 reproduction process respectively, and the result has all obviously suppressed HIV-1 duplicating in the human T lymphocyte; They think two kinds of method mechanism differences, and the targeting moiety difference is united use and can be strengthened the inhibition effect.Ji Yinduo etc. find to suppress the expression of encoding gene by making up the antisense expression vector at the streptococcus aureus fatty acid synthetase behind this bacterium of transfection, make its growth be subjected to obvious inhibition, even stop.Antisense technology is applied to clinical treatment also report repeatly: as utilize the antisense oligonucleotide of synthetic to suppress the expression etc. of oncogene such as C-myc or tumor growth factor.
Sense-rna not only can be used as the negative regulatory factor of genetic expression, also can be used as positive regulating factor.For example: the formation of mRNA II level structure suppresses its translation sometimes, and the sense-rna that design stops mRNA II level structure to form can promote expression of gene.
We know that interior certain genetic expression deficiency of organism or over-expression all can cause the disease of organism.Be subjected to short apoptogene to reach the control that presses down apoptogene as apoptosis gene; During normal expression, in fetal development, can remove useless even deleterious cell, make normal, the complete form of histoorgan structure body; Remove the cell of aging, damage, canceration at the body of growing up, and replace newborn cell, stable complete to keep in the organ cell quantity with body defending system.If apoptogene is expressed not enough, cause apoptosis to be obstructed, can progressively develop into tumour; If over-expression then causes histoorgan developmental defect or function to reduce, and then causes body to produce a series of pathology.The sense-rna apoptosis capable of inhibiting cell of the short apoptogene of target; Otherwise, then promote apoptosis.
Sense-rna also has the possibility as the novel antibacterial medicine: infectious diseases is owing to the pathogeny bacterium grows in vivo, the imbalance of breeding or body normal microflora causes.Some material that produces in some composition of bacterium itself and/or the growth metabolism process has toxic action to particular organization's organ, and these toxicants are exactly the virulence factor of pathogenic bacterium.The fast development of nucleic acid sequence analysis technology is out of question the gene order of verifying the coding virulence factor.Design corresponding sense-rna according to gene order, change in the bacterial cell, suppress the expression of Disease-causing gene or the necessary protein gene of coding bacterial growth, can reach the purpose of attenuation, inhibition or kill bacteria by transporting carrier.Antisense technology is becoming indispensable means of human research and treatment virus disease, tumour and even each biomedical sector.External existing antisense drug is applied to clinical, in July, 1998 reported first antisense drug by FDA (Food and Drug Administration) authentication by the Science magazine, promptly is used for the treatment of the Virtravene of the retinitis of AIDS (AIDS) patient's cytomegalovirus infection.Virtravene is a kind of G 3139, be in the phosphodiester group, non-bridging oxygen atom is by oligonucleotide that sulphur atom replaced, this medicine is to be developed based on the oligonucleotide of the phosphodiester bond that contains modification by American I sis company, also has multiple antisense drug to be used for clinical trial in addition or has gone on the market.
But, antisense oligonucleotide is used for the method as the treatment disease, still there are several problem demanding prompt solutions at present: (1) transfection efficiency: no matter be the sense-rna or the external synthetic antisense oligonucleotide of vector expression, only enter target cell, just can combine with homologous gene, the performance regulating effect, and the sense-rna adjusting has tangible dose-dependently.It is an internalization process that exposed antisense oligonucleotide enters cell, and efficient is very low; Having the polar antisense oligonucleotide then is an active absorption process, and efficient is higher relatively.Yet this problem is still the maximum bottleneck in the sense-rna Application and Development so far.(2) selection of target sequence: many results of study show: the reticent effect of the antisense of some genes is better than other genes; Same gene, the reticent effect difference of different target sequences.The selection of target sequence relies on experience at present to a certain extent except that the mechanism of action according to above-mentioned antisense oligonucleotide, can not be determined by the analyzing gene group fully.(3) route of administration: medicine must reach certain amount and keep its pharmacological action of regular hour competence exertion in body.That is to say that medicine at first should be able to tolerate the effect of some enzyme of body and not be decomposed, and arrives safely target cell, simultaneously non-destination organization organ is had no side effect.In fact, still there is not the medicine that reaches these two requirements simultaneously at present.
In addition, the oligonucleotide with adjusting function can be used for suppressing the RNA fragment, prevents that it from translating into albumen, prevent can play a part aspect the cancer cells activity certain.Have the novel ligonucleotides of modifying phosphodiester bond,, might become anticancer disease and antiviral medical preparation, and receive much concern because of it can be used as the probe of biological chemistry and biology field.Up to now, many nucleotide analogs have been developed to the potential medical preparation, can be used for regulating the anti-gene of specific gene and the expression of anti-gene target.These researchs are faced with some significant challenges, need further exploring aspect biology field and the clinical application.
Summary of the invention
The 1st technical problem to be solved by this invention provides the strong phosphodiester of a kind of nuclease-resistant hydrolysis ability.
The 2nd technical problem to be solved by this invention is the preparation method of above-mentioned phosphodiester.
The technical scheme of technical solution problem of the present invention is: a kind of phosphodiester, described phosphodiester are cyano group borine phosphoric acid ester, and its molecular formula is: [(R
1O) (R
2O) P (O) (BH
2CN)]
-, its structural formula is:
Described R
1, R
2For straight chained alkyl, branched-chain alkyl, ribonucleoside, dezyribonucleoside with and corresponding derivative.
The structural formula of preferred phosphodiester is:
Described B
1And B
2Be VITAMIN B4, thymus pyrimidine, guanine, cytosine(Cyt) and corresponding derivative thereof;
Described R
3And R
4Oligodeoxynucleotide for hydrogen, alkyl, oligodeoxynucleotide, modification.
The structural formula of preferred phosphodiester is:
Described B
3And B
4Be VITAMIN B4, uridylic, GC and corresponding derivative thereof;
Described R
5And R
6Oligodeoxynucleotide for hydrogen, alkyl, oligodeoxynucleotide, modification.
Most preferred R
3, R
4Be hydrogen, B
1, B
2Be thymus pyrimidine;
Most preferred R
5, R
6Be hydrogen, B
3Be uridylic, B
4Be VITAMIN B4;
Preparation method of the present invention is: a, nucleoside coupling, b, cyanogroup boron hydride replacement, c, deprotection, d, purifying.
Described a, nucleoside coupling are:
The nucleosides or the deoxynucleoside phosphoramidite that will contain protecting group reacted in dimethyl formamide 1-3 hour with the acetyl nucleosides under the catalysis of tetrazole, obtained phosphorous acid ester; The nucleosides of protection or the mol ratio of deoxynucleoside phosphoramidite and acetyl nucleosides or acetyl deoxynucleoside are 1: 1.
Described b, cyanogroup boron hydride replacement are: under 50-80 ℃, a, adenosine are replaced resulting phosphorous acid ester and aniline cyano group borine reacted 2-3 hour in the tetrahydrochysene skin is muttered; The mol ratio of phosphorous acid ester and aniline cyano group borine is 1: 4-5.
Preferred phosphodiester is as the molecular probe of enzymatic and the research of non-enzymatic reaction stereochemistry; As B in the cancer boron neutron capture therapy
10The application of carrier.
In oligomerization picodna (ODN), use S
-, CH
3Or BH
3 -Replacing non-bridging oxygen atom in the natural phosphodiester can provide the ability of nuclease-resistant.For example, non-ionic methyl phosphorodithioate (CH
3-ODN) phosphodiesterase there is very strong resistibility; Anionic thiophosphatephosphorothioate (S
--ODN) can be by phosphodiesterase I hydrolysis, but far below the hydrolysis rate of natural phosphodiester.Borine phosphoric acid ester (BH
3 --ODN) compare S
--ODN more can resist some phosphodiesterase.With one in three hydrogen atoms of cyano group replacement borine, in view of BH
2CN
-The comparatively large vol of group and the shortage of lone-pair electron, [O=P-BH
2CN]
-Oligonucleotide is than [O=P-S]
-Oligonucleotide is lipophilic oil more.Studies show that the BH of electron deficiency
3Structure easily and Lewis base (for example tris phosphite) coordination form the borine tris phosphite.
Because cyano group also is a very strong drawing electron group ,-BH
2CN is with respect to-BH
3It is a stronger Lewis acid.
Because BH
2The comparatively large vol of CN, the BH of tris phosphite
2CN boronation velocity ratio BH
3Boronation speed is slow and to sterically hindered responsive more, but P-BH
2In a single day the coordinate bond of CN or covalent linkage form, and it will be than corresponding P-BH
3Coordinate bond and covalent linkage are stable.For example, N, the cyano group boronation speed of N-diisopropylaminoethyl tris phosphite is more a lot of slowly than methoxyl group tris phosphite.
In addition, the sterically hindered seemingly soluble phosphorous acid ester that large volume caused of two sec.-propyl amino and aniline-cyano group borane complex phenomenon of cyano group borine exchange slowly in the time of 68 ℃.On the other hand, although DMT (4,4 '-dimethoxytrityl) positively charged ion can cause the boronation effect of taking off of borine phosphoric acid ester, experimental result shows that cyano group borine phosphoric acid ester is stable under the situation that the DMT positively charged ion exists.
Be different from [O=P-O]
-, [O=P-BH
2CN]
-Key links structure can make that compound is easier to be passed cytolemma and enter cell.Contain [O=P-BH
2CN]
-Synthesizing of the DNA of phosphodiester bond and RNA analogue, provide possibility for preparing brand-new species compound (Nucleotide of modification and nucleic acid).They and natural acid have similarity, and the peculiar property of strong lipotropy and nuclease-resistant hydrolysis is also arranged.This strong lipotropy, ideal is water-soluble and novel combination nuclease-resistant is exceedingly useful in medicinal design.
The present invention compared with prior art provides a kind of brand-new phosphodiester---and cyano group borine phosphoric acid ester, wherein phosphodiester group non-bridging oxygen atom is by cyanogroup boron hydride replacement.With respect to n DNA, the phosphodiester that is constituted can both be stablized in very wide pH scope and can resist the nuclease hydrolysis well, simultaneously, based on the partition experiment (being that lipotropy is measured) of octanol, cyano group borine phosphoric acid ester may represent bigger membrane permeability than the oligonucleotide of routine.
Description of drawings
Fig. 1 is the process flow sheet of the embodiment of the invention 1.
Fig. 2 is the process flow sheet of the embodiment of the invention 2.
In Fig. 1,2, DMT is 4, and 4 '-dimethoxytrityl, Ac are that ethanoyl, T are that thymus pyrimidine, U are that uridylic, A are that VITAMIN B4, TBDMS are that tertiary butyl dimethyl is silica-based.
Embodiment
Embodiment 1:
DNA cyano group borine phosphoric acid ester analogue: two thymus nucleoside cyano group borine phosphoric acid ester (Tp
BH2CNT or Tp
CBSynthesizing T):
Double-core glycosides cyano group borine phosphoric acid ester (Tp
BH2CNT or Tp
CBT) synthetic route as shown in Figure 1.
A, nucleoside coupling:
Under the room temperature, the thymus nucleoside phosphoramidite of protection (
1) (0.5mmol buys from U.S. Chemgenes company) reacting 1 hour among dimethyl formamide DMF (3ml) with acetyl thymus nucleoside (0.5mmol) under the catalysis of tetrazole (0.5mmol), obtain phosphorous acid ester (
2).
B, cyanogroup boron hydride replacement:
Under 68 ℃, phosphorous acid ester (
2) same aniline-cyano group borane complex (4mmol) in the tetrahydrochysene skin is muttered (12ml), react obtained in 2 hours phosphorous acid ester-cyano group borine (
3).
C, deprotection:
With 3 normal remove DMT reagent (3% dichloro acetic acid is dissolved in methylene dichloride) handle compound (
3) be protected after half an hour double-core glycosides cyano group borine phosphoric acid ester (
4). after the decompression rotary evaporation is removed lower boiling solvent, compound (
4) with volume ratio be 1: 1 ammonia hydroxide/methanol at room temperature handle be converted into after three hours double-core glycosides cyano group borine phosphoric acid ester (
5) thick product.
D, purifying:
Double-core glycosides cyano group borine phosphoric acid ester (
5) thick product with QA-Cellulose (HCO
3-) ion exchange column (U.S. Amershan company) purifying, with 0.1M Ammonium bicarbonate food grade eluant solution, collect respective components, obtain the ammonia salt form double-core glycosides cyano group borine phosphoric acid ester (
5) (0.18mmol), from compound (
1) to double-core glycosides cyano group borine phosphoric acid ester (
5) (Tp
BH2CNT or Tp
CBT), overall yield is about 36%.
Double-core glycosides cyano group borine phosphoric acid ester (
5) structure pass through UV;
31P NMR;
1H NMR; MS (FAB
-); HRMS (FAB) (M
-) measure.
Double-core glycosides cyano group borine phosphoric acid ester (
5) (Tp
BH2CNT or Tp
CBT):
UV λ
Max=267nm;
31P NMR (D
2O, 161.9MHz). (ppm) 75 (br);
1H NMR (D
2O, 400MHz). (ppm) 7.52,7.48,7.47,7.46 (4s, 2H, H6), 6.15-6.08 (m, 2H, H1 '), (4.77-4.71 m, 1H, H3 '), 4.44-4.41 (m, 1H, H3 '), 4.04-3.99 (m, 4H, H5 '), 3.67-3.61 (m, 2H, H4 '), 2.38-2.19 (2m, 4H, H2 '), 1.75,1.71 (2s, 6H, 5-CH
3), 0.98-0.52 (br, 2H, BH
2CN); MS (FAB
-): m/z for M
-568.1; HRMS (FAB
-) calculated value C
21H
28O
11N
5PB (M
-) 568.1614, measured value 568.1626.
Embodiment 2:
RNA cyano group borine phosphoric acid ester analogue: uridine VITAMIN B4 cyano group borine phosphoric acid ester (Up
BH2CNA or Up
CBSynthesizing A).
Uridine VITAMIN B4 cyano group borine phosphoric acid ester (Up
BH2CNA or Up
CBA) synthetic route is shown in figure two.
A, nucleoside coupling:
Under the room temperature, the uridine phosphoramidite of protection (
6) (0.5mmol buys from U.S. Chemgenes company) reacting 2 hours among dimethyl formamide DMF (6ml) with diacetyl adenosine (0.5mmol) under the catalysis of tetrazole (0.5mmol), obtain phosphorous acid ester (
7).
B, cyanogroup boron hydride replacement:
Under 68 ℃, phosphorous acid ester (
7) same aniline-cyano group borine (4mmol) in tetrahydrofuran (THF) (16ml), react obtained in 3 hours phosphorous acid ester-cyano group borine (
8).
C, deprotection:
With 4 normal remove DMT reagent (3% dichloro acetic acid is dissolved in methylene dichloride) handle phosphorous acid ester-cyano group borine (
8) after 1 hour protected double-core glycosides cyano group borine phosphoric acid ester (
9). after the decompression rotary evaporation is removed lower boiling solvent, protected double-core glycosides cyano group borine phosphoric acid ester (
9) with 1: 1 ammonia hydroxide/methanol of volume ratio at room temperature handle be converted into after three hours 2 '-O-tertiary butyl dimethyl-silicon generation-3 '-O-uridine-5 '-O-VITAMIN B4 cyano group borine phosphoric acid ester (
10).Its character is:
31P NMR (D
2O, 161.9MHz) δ (ppm) 75 (br); Fast atom bombardment mass spectroscopy(FABMS): M
-(m/z, 709.2) and C
26H
39O
11N
8PBSi (M
-) conform to.
With 2 '-O-tertiary butyl dimethyl-silicon generation-3 '-O-uridine-5 '-O-VITAMIN B4 cyano group borine phosphoric acid ester (
10) (0.25mmol) be dissolved in 2ml tetra-tert fluoride amine t-Bu
4Stir in the F solution (the tetrahydrochysene skin of 1M mutter solution) generated in 2 hours uridine VITAMIN B4 cyano group borine phosphoric acid ester (
11) (Up
BH2CNA or Up
CBA) thick product.
D, purifying:
After the decompression rotary evaporation is removed lower boiling solvent, uridine VITAMIN B4 cyano group borine phosphoric acid ester (
11) thick product with QA-Cellulose (HCO
3-) the ion-exchange column purification, with 0.1M Ammonium bicarbonate food grade eluant solution, collect respective components, obtain the ammonia salt form uridine VITAMIN B4 cyano group borine phosphoric acid ester (
11) (0.09mmol).Overall yield be about 18% (from
6Extremely
11).
Uridine VITAMIN B4 cyano group borine phosphoric acid ester (
11) structure pass through UV;
31P NMR;
1H NMR; MS (FAB
-); HRMS (FAB) (M
-) measure.
UV λ
Max.258nm;
31P NMR (D
2O, 161.9MHz). (ppm) 74-78 (br);
1H NMR (D
2O, 400MHz). (ppm) 8.25,8.21 (2s, 1H, H-8), 8.06 (s, 1H, H-2), 7.62-7.58 (m, 1H, H-6), 5.94 (1H, H-1 '), 5.64 (m, 4H), 0.8-0.2 (br, 2H, BH
2CN) .MS (FAB
-): m/z for M
-595.1; HRMS (FAB
-) calculated value C
20H
25O
11N
8PB (M
-) 595.1471, measured value 595.1464.
Embodiment 3:
The essential property of cyano group borine phosphoric acid ester.
1, acid and alkali hydrolysis stability.
Cyano group borine phosphoric acid ester is highly stable in acid and alkali hydrolysis.At 37 ℃, under pH=3 or 11 the condition, with 100mM acetic acid/ammoniacal liquor handle two thymus nucleoside cyano group borine phosphoric acid ester (
5) 24 hours, utilize high performance liquid chromatography not detect the product of hydrolysis or degraded.High-efficient liquid phase chromatogram condition: eluent is that (Triethylammonium Acetate is TEAA) with 20% second cyanogen for 80%100mM acetate triethyl amine salt; Elution flow rate is 1 ml/min.
2, nuclease-resistant is water-disintegrable.
Cyano group borine phosphoric acid ester bond is very stable for the hydrolysis of snake venom phosphodiesterase (SVPDE) and bovine spleen phosphodiesterase (BSPDE).The nuclease-resistant hydrolytic process is monitored with high performance liquid chromatography.High-efficient liquid phase chromatogram condition: eluent is 80%100mM acetate triethyl amine salt (TEAA) and 20% second cyanogen; Elution flow rate is 1 ml/min.Surpass 99% during in natural pair of thymus nucleoside phosphoric acid ester (TpT) by the SVPDE hydrolysis, the double-core glycosides cyano group borine phosphoric acid ester among the embodiment 1 (
5) be stable greater than 99%.Surpass 99% during in natural pair of thymus nucleoside phosphoric acid ester (TpT) by the BSPDE hydrolysis, double-core glycosides cyano group borine phosphoric acid ester (
5) be stable greater than 99%.
3, strong lipotropy.
Water miscible double-core glycosides cyano group borine phosphoric acid ester (
5) dimer has a negative charge, its lipotropy is between common phosphoric acid ester and methyl phosphorodithioate.The lipotropy of compound can be come quantitatively with partition coefficient.Partition coefficient is defined as a certain compound when hot alcohol and water biphase-equilibrium, and this compound is at the concentration of octanol in mutually and the concentration ratio at aqueous phase.Partition measuring by octanol.Water miscible double-core glycosides cyano group borine phosphoric acid ester (
5) partition coefficient be 0.11.The partition coefficient of TpT is 8.7x10
-5This experiment show water miscible double-core glycosides cyano group borine phosphoric acid ester (
5) lipotropy be lipophilic 1260 times of natural TpT.
Claims (10)
1. phosphodiester, it is characterized in that: described phosphodiester is a cyano group borine phosphoric acid ester.
2. according to the described a kind of phosphodiester of claim 1, it is characterized in that: its molecular formula of described phosphodiester is: [(R
1O) (R
2O) P (O) (BH
2CN)]
-, its structural formula is:
Described R
1, R
2For straight chained alkyl, branched-chain alkyl, ribonucleoside, dezyribonucleoside with and corresponding derivative.
3. according to the described a kind of phosphodiester of claim 2, it is characterized in that: the structural formula of described phosphodiester is:
Described B
1And B
2Be VITAMIN B4, thymus pyrimidine, guanine, cytosine(Cyt) and derivative thereof;
Described R
3And R
4Oligodeoxynucleotide for hydrogen, alkyl, oligodeoxynucleotide, modification.
4. according to the described a kind of phosphodiester of claim 2, it is characterized in that: the structural formula of described phosphodiester is:
Described B
3And B
4Be VITAMIN B4, uridylic, guanine, cytosine(Cyt) and derivative thereof;
Described R
5And R
6Oligodeoxynucleotide for hydrogen, alkyl, oligodeoxynucleotide, modification.
5. according to the described a kind of phosphodiester of claim 3, it is characterized in that: described R
3, R
4Be hydrogen, B
1, B
2Be thymus pyrimidine.
6. according to the described a kind of phosphodiester of claim 4: it is characterized in that: described R
5, R
6Be hydrogen, B
3Be uridylic, B
4Be VITAMIN B4.
7. the preparation method of the described a kind of phosphodiester of claim 1 comprises following operation: a, nucleoside coupling, b, cyanogroup boron hydride replacement, c, deprotection, d, purifying.
8. the preparation method of a kind of phosphodiester according to claim 7, it is characterized in that: described a, nucleoside coupling are:
The nucleosides or the deoxynucleoside phosphoramidite that will contain protecting group reacted in dimethyl formamide 1-3 hour with acetyl nucleosides or acetyl deoxynucleoside under the catalysis of tetrazole, obtained phosphorous acid ester; The mol ratio of nucleosides or deoxynucleoside phosphoramidite and acetyl nucleosides or acetyl deoxynucleoside is 1: 1.
9. the preparation method of a kind of phosphodiester according to claim 7, it is characterized in that: described b, cyanogroup boron hydride replacement are: under 50-80 ℃, a, adenosine are replaced resulting phosphorous acid ester and aniline cyano group borine reacted 2-3 hour in the tetrahydrochysene skin is muttered; The mol ratio of phosphorous acid ester and aniline cyano group borine is 1: 4-5.
10. claim 3,4 described phosphodiesters are as the molecular probe of enzymatic and the research of non-enzymatic reaction stereochemistry; As B in the cancer boron neutron capture therapy
10The application of carrier.
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| CN103936805A (en) * | 2013-01-18 | 2014-07-23 | 昆山市工业技术研究院小核酸生物技术研究所有限责任公司 | Nucleotide and/or oligonucleotide and preparation method thereof |
-
2009
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103936805A (en) * | 2013-01-18 | 2014-07-23 | 昆山市工业技术研究院小核酸生物技术研究所有限责任公司 | Nucleotide and/or oligonucleotide and preparation method thereof |
| CN103936805B (en) * | 2013-01-18 | 2016-12-28 | 昆山市工业技术研究院小核酸生物技术研究所有限责任公司 | A kind of nucleotide and/or oligonucleotide and preparation method thereof |
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