CN101575332A - Diazacyclo propenyl-contained non-natural nucleoside, preparation method and applications thereof - Google Patents
Diazacyclo propenyl-contained non-natural nucleoside, preparation method and applications thereof Download PDFInfo
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- CN101575332A CN101575332A CNA2009100857790A CN200910085779A CN101575332A CN 101575332 A CN101575332 A CN 101575332A CN A2009100857790 A CNA2009100857790 A CN A2009100857790A CN 200910085779 A CN200910085779 A CN 200910085779A CN 101575332 A CN101575332 A CN 101575332A
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Abstract
The invention provides a diazacyclo propenyl-contained non-natural nucleoside with a photosensitive group, and also discloses a preparation method and applications of the compound. The provided nucleotide can be photolyzed with higher wavelength (about 360nm), generates a high-activity carbene and can enter the cell to participate in the synthesis of new biomacromolecule in the cell; and the biomacromolecule mixed with an optical crosslinking reagent can be used as a photochemical treatment reagent, thus inducing crosslinking inside or between molecules.
Description
Technical field
The present invention relates to a kind of non-natural nucleoside, specifically, relate to a kind of non-natural nucleoside, its preparation method and application thereof that contains the diazacyclo propenyl.
Background technology
Non-natural nucleoside is incorporated in the oligonucleotide; for gene sequencing and identification aberrant gene, by antisense oligonucleotide regulatory gene and proteic expression, survey interaction between DNA-NDA, DNA-RNA and the DNA-albumen, describe the character of charge transfer reaction in the dna molecular and estimate hydrogen bond and interact effect in the stabilized DNA molecule of π～π, be very effective.
As everyone knows, the non-natural nucleoside that comprises photosensitive group has been used to discern nucleic acid-nucleic acid, nucleic acid-protein-interacting.For example, Koudan has developed 2 '-deoxyuridine derivative that light lives (J.Biomol.Structure Dynamics 2002,20 (3), 421-428); Taranenko developed band trifluoromethyl diazacyclo propenyl aryl non-natural nucleoside (Eur.J.Biochem.2003,270,2945-2949); Dezhurov has developed and has contained 4-nitrine-2, and the non-natural nucleoside of 5-two fluoro-3-chloropyridine bases (Russ.J.Bioorg.Chem.2003,29 (1), 66-72).At present, the non-natural nucleoside that photosensitive group particularly contains the diazacyclo propenyl that contains of novel structure has the market requirement widely.
The diazacyclo propenyl derivatives has been widely used in studying the 26S Proteasome Structure and Function of nucleic acid, albumen and other various macromolecular complexs.Compare other light affinity groups, the diazacyclo propylene has following several remarkable advantage: the first, and it is the photodissociation rapidly of energy quilt in the 350-360 nanometer wavelength range, and for most of biomacromolecules, this wavelength is outside their absorbing wavelength; The second, the diazacyclo propylene can be formed the Cabbeen intermediate of high reaction activity after the photodissociation, and Cabbeen can linked reaction take place with the various functional groups that comprise inert aliphatic carbon hydrogen bond; The 3rd, the carbon bond that produces with the Cabbeen coupling is stable under common experiment condition; The 4th, compare other light group of living, diazacyclo propenyl derivatives sterically hindered less has good chemical stability under the situation of the laboratory of medium tenacity illumination.
Intramolecularly and intermolecular linked reaction can take place in nucleoside molecule, and reaction will form a new covalent linkage after finishing.When linked reaction during intermolecular carrying out, linked reaction can take place with another biomolecules (for example nucleosides, peptide and antibody etc.) in nucleoside molecule.This research work can be applied to the interaction that (i) research albumen/DNA or albumen/RNA take place through photic coupling; DNA/RNA interaction, DNA reparation and the modified protein of (ii) catching and describe different cells are with the interaction between the accounting probe that contains analogue; (iii) these probe molecules are incorporated in siRNA or any and these molecule link coupled RNA fragment, thereby confirm the target cell (for example, little " tail " is incorporated into to help in the rna probe molecule to find out result from the body or external RNA/ albumen coupling mixture) of RNA molecule; (iv) these probe molecules are incorporated in DNA or the PNA antisense molecule, be illustrated in the genomic dna binding site or in gene therapy other potential target DNA/PNA.The linked reaction of DNA chain inside is a kind of means of the most effective cell killing, also is the mode of action of for example treating several chemicalses such as medicine-cis-platinum of cancer simultaneously.
Common strategy of cancer treatment is to cause that thereby dna damage causes the apoptosis of cancer cells.Some the most effectively anticarcinogens (for example endoxan, ametycin and cis-platinum) that use clinically are exactly by causing the crosslinked anticancer function of realizing of DNA chain.(Nat.rev.Cancer 2002 at Hurley for these, 2,188-200), Wang (Nat.Rev.Drug Discov.2005,4,307-320), Wang (J.Am.Chem.Soc.2003,125,1116-1117) and Yoshimura (Nucleic Acids Symp.Ser.2004 has argumentation in article 81-82).But present therapeutic strategy can only work in virus replication, and mechanism does not have selectivity, can kill normal cell, and toxicity is very big.
Therefore, for design treatment cancer and medicine for treating viral infections, can cause that the crosslinked nucleoside analog of DNA chain is very attractive molecule.
Summary of the invention
The purpose of this invention is to provide a kind of non-natural nucleoside that contains the diazacyclo propenyl with photosensitive group.
Another object of the present invention provides a kind of preparation method who contains the non-natural nucleoside of diazacyclo propenyl.
A further object of the present invention provides a kind of non-natural nucleoside that contains the diazacyclo propenyl as the application in the photosensitive crosslinker.
In order to realize the object of the invention, the present invention has the non-natural nucleoside compound that contains the diazacyclo propenyl of structural formula (I):
Wherein, R in the formula
1Be hydrogen, replacement or unsubstituted C
1～C
8Alkyl, n is 0,1,2 or 3, but preferably 0.
Ar is 5 yuan to 10 yuan hetero-aromatic ring or 5 yuan to 10 yuan aromatic ring, and these aromatic rings need possess two conditions: (a) have a substituting group that contains sugar, sugar analogue, nucleosides or nucleoside analog at least; (b) 0 to 3 comprises replacement or unsubstituted C
1～C
8Alkyl, hydroxyl and amino.Ar also can be with substituent 6 yuan to 10 yuan aromatic ring, for example phenyl or naphthyl.Described Ar can be with substituting groups such as ribose or ribodesose.Perhaps, Ar can be with nucleoside phosphoramidites monomer or the monomeric sugar analogue substituting group of deoxynucleoside phosphoramidite.Perhaps, Ar can be with nucleosides substituting groups such as modified sugar-phosphoric acid structural unit of multiple sugar-phosphoric acid structural unit, multiple or nucleoside analogs.Perhaps, Ar can be with substituting groups such as nucleoside analog such as peptide nucleic acid(PNA).
In general, Ar has a substituting group shown in molecular formula (II):
R
2, R
3, R
4Or R
5Be hydrogen or hydroxyl; R
6Be through protection or without the single, double or triphosphoric acid base of protecting; Perhaps R
3And R
6Energy in two groups and poly-nucleotide chain reaction coupling.Usually, R
3And R
6One in two groups is phosphoramidite, R
2, R
4Or R
5Be hydrogen or hydroxyl.
The art of phosphoramidite monomer chemistry is widely known by the people.For example, DNA can be with Matteucci at J.Am.Chem.Soc.1981, described in 103,3185 or with Yoo at J.Biol.Chem.1989, method or other well-known methods of the use phosphoramidite monomer solid phase synthesis described in 764,17078 are synthesized.
In addition, Ar can be with the substituting group shown in the formula (III):
R
7And R
9It is the group that can carry out linked reaction with poly-nucleotide chain; R
8Be hydrogen or hydroxyl.Usually, R
7And R
9Be hydroxyl or phosphoramidite.
Usually, the R in the formula (I)
1Be CX
3, X is a halogen.X generally is the F element.
Preferably, the compound that meets structural formula (I) is a formula (IV) in the formula (VII) one:
R shown in the formula
10Be H, replacement or unsubstituted C
1～C
8Alkyl, hydroxyl or amino in a kind of; R is glycosyl, glycosyl analogue, nucleic acid base or analogue with nucleic acid backbone; N and R
1Described in (I).
Further preferably, the compound that meets structural formula (I) is a formula (VIII) in the formula (XIII) one:
R is suc as formula described in (IV).
More preferably, the compound that meets structural formula (I) is a formula (XIV) in the formula (XVII) one:
The preparation method of structural formula of the present invention (I) compound, undertaken by following chemical reaction process:
Wherein, compound 6 is through synthetic obtaining of five steps.But reference (A N Topin, Nucleosides ﹠amp; Nucleotides 1998,17,1163-1175) preparation.
Compound 6 is used to prepare final phosphorus acylated reagent XIV (14 in the route 2).The concrete chemical reaction process of face as follows.
(but MA Cameron, Journal of OrganicChemistry 1997,62 9065-9069) prepare from the 2-deoxythymidine preparation reference of compound 9.
The preparation method of structural formula XIV comprises the steps: among the present invention
1) with 3-(4-iodine substituted phenyl)-3-(trifluoromethyl)-3H-diazacyclo propylene (6), 1,4-dehydration-3,5-two-oxygen (tertiary butyl dimethyl is silica-based)-2-deoxidation-D-erythro form-penta-1-glycal (9), nitrogen, nitrogen-dicyclohexyl methylamine, palladium, tetrabutylammonium chloride, the 4A molecular sieve of porphyrize and heavily steam nitrogen, nitrogen-dimethyl formamide mixes, to form suspension, under nitrogen atmosphere, be heated to 60 ℃, and be incubated 40 hours, use ethyl acetate extraction, organic phase wash dry after-filtration concentrate obtain oily matter (2R, 5R)-2-((tertiary butyl and methyl siloxy) methyl)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone (10);
2) then to the (2R of ice bath to 0 ℃, 5R)-2-((tertiary butyl and methyl siloxy) methyl)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) in the tetrahydrofuran solution of dihydrofuran-3 (2 hydrogen)-ketone (10), the tetrahydrofuran solution that adds Glacial acetic acid and tetrabutyl ammonium fluoride successively, stirring is concentrated after 1.5 hours down at 0 ℃ for reaction mixture, separation and purification obtains oily matter (2R, 5R)-2-(methylol)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone (11);
3) bathe (2R that is cooled to-10 ℃ to cryosel, 5R)-2-(methylol)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) in dihydrofuran-3 (2 the hydrogen)-acetonitrile of ketone (11) and the solution of Glacial acetic acid, add the triethoxy sodium borohydride, under nitrogen atmosphere, concentrate after 30 minutes in-10 ℃ of stirrings, separation and purification, obtain white powder (1R)-1,4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (12)
4) under 0 ℃, (1R)-1, the heavily steaming pyridine solution of 4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (12) stirred after 10 minutes, to wherein adding 4 successively, 4 '-dimethoxytrityl methyl chloride and 4-(dimethylamino) pyridine, under nitrogen atmosphere, reaction solution extracts with ether (2 * 50 milliliters) after stirring 6 hours under 0 ℃, merge organic phase, dry, filter, concentrated filtrate, separation and purification obtains (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (13);
5) (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (13), nitrogen, nitrogen-diisopropyl ethyl amine and 2-cyanogen oxyethyl group-nitrogen, the heavily steaming dichloromethane solution of nitrogen-di-isopropyl protochloride phosphamide stirred 1.5 hours down in 0 ℃ under nitrogen atmosphere, separation and purification, obtain white foam shape thing (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose-2-cyanogen oxyethyl group diisopropylphosphoramidite (14).
Nucleosides of the present invention can change into corresponding ribonucleotide triphosphate monomer with the method for knowing, ribonucleoside phosphoramidite monomer, deoxyribonucleoside triphosphate monomer or deoxyribonucleoside phosphoramidite monomer.
Compound for structural formula XV can adopt 3-(3-iodine substituted phenyl)-3-(trifluoromethyl)-3H-diazacyclo propylene, forms according to the preparation process of structural formula XIV is synthetic.
Can be for the compound that meets structural formula XVI by compound (1R)-1,4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (12) adopts the triphosphoric acid prepared in reaction.Can be for the compound that meets structural formula XVII by similar prepared in reaction.
The non-natural nucleoside compound that contains the diazacyclo propenyl of the present invention has following advantage:
1. nucleosides of the present invention can produce a highly active Cabbeen, this Cabbeen even can break aliphatic c h bond, and in nanosecond, finish reaction;
Nucleosides 2. of the present invention can photodissociation under higher wavelength (～360nm);
3. under medium laboratory lighting condition, just can operate;
4. nucleosides of the present invention and natural nucleosides space structure are similar, can directly be doped in the natural nucleotide.
Ribonucleoside triphosphote monomer of the present invention can be discerned by enzyme, and end of the chain transferring enzyme for example extends to 3 ' end of nucleic acid chains with the direct mode of non-template.The ribonucleoside triphosphote tail that this kind mode produces can be with the antibody Direct Recognition and without any need for the reporter group mark.Still can be after nucleosides mixes in oligonucleotide or the nucleic acid chains by for example restriction enzyme and the ligase enzyme identification of nucleic acid modifying enzyme.
Nucleosides of the present invention also comprises other non-natural nucleotide, for example modifies reporter group on this basis.The compound of invention has increased the molecular diversity of existing nucleoside analog.The oligonucleotide of the nucleosides preparation among application the present invention can increase the avidity to target molecule.
Oligonucleotide of the present invention can prepare with the phosphorus acylated method of standard (for example M.D.Matteucci is at J.Am.Chem.Soc.1981,103, the method for describing among the 3185-3191).
Nucleic acid base analogue XIV and XV can be doped in DNA, RNA or other nucleic acid superpolymer by solid phase synthesis as photocrosslinking reaction probe.These have the segmental base of diazacyclo propylene molecules for the interaction between nucleic acid-albumen, have very little space steric effect, can discharge nitrogen under UV-irradiation, generate the Cabbeen active intermediate.The Cabbeen intermediate is effective photocrosslinking species, can carry out crosslinked with contiguous albumen and nucleic acid residue.
Synthetic by solid phase DNA, phosphoramidite monomer 14 is introduced in the dna molecular.1) and 5 '-ATGAACCNGGAAAAC-3 ' (sequence number: 3) be synthesized and purifying (N=B in sequence number 1 and sequence number 3, B are the base analogues 14 that new synthetic comprises the diazacyclo propenyl) trimer TBT and 15 yuan of 5 '-ATCAAGNGCAAGAC-3 ' (sequence number:.
Be the stability under the solid phase oligomerization nucleosides synthesis condition of check diazacyclo propylene base standard, trimer TBT at room temperature spends the night with deprotection with the strong aqua reaction.In the auxiliary parsing of matrix flight time mass spectrum; the compound of deprotection provides three peaks, and these three peaks match with the molecular weight that trimeric molecular weight, trimer are sloughed the product that the molecular weight (the diazacyclo propenyl can be sloughed nitrogen under the auxiliary condition of resolving of matrix) of nitrogen and trimer obtain with substrate reaction respectively.When the compound of deprotection at aqueous phase during with near-ultraviolet light (hv＞300 nanometers) irradiation, its matrix is auxiliary resolves the peak that flight time mass spectrum provides a new product that reacts corresponding to Cabbeen and water, the disappearance at aforesaid three peaks of simultaneous.This observes the clear and definite existence of diazacyclo propylene group in the trimer that shown.
Can synthesize by similar scheme with the similar compound of dNTP and NTP (for example as molecular formula XVI and XVII compound).These compounds can be used as the analogue of dATP, dGTP, dTTP and dCTP, have potential biomarker and coupled action.They also can be introduced among DNA and the RNA by the catalytic nucleic acid of polysaccharase is synthetic.
Confirm among the present invention to contain the diazacyclo propenyl photo-crosslinking DNA base analogue can with the carrying out of contiguous nucleic acid or protein residues photo-crosslinking efficiently.
Nucleosides among the present invention can enter cell, and is just as known nucleoside analog, macromolecular synthetic as true tumor in 5-floxuridine and the Zidovodine participation cell.The biomacromolecule of mixing photo-crosslinking reagent can be used as photochemical therapy reagent, the interior or intermolecular cross-linking of inducing molecule.
After nucleosides among the present invention is incorporated into nucleic acid, utilize the hybridization characteristic of nucleic acid can be used for the antisense nucleic acid field.
Nucleosides character of the present invention is different with natural nucleus glycoside, thereby is fit to other purposes.For example, the interaction in vivo of nucleosides of the present invention and enzyme has important biological significance, and new antiviral therapy method is had material impact.
Oligonucleotide among the present invention (having an affinity groups or fluorophor) can enter into viable cell as siRNAs.Two photons stimulate in can inductor crosslinked.As seen cross-linked composite can become by fluorophor, also can separate obtaining by affinity groups.
(sequence number: 1) 4 dsDNA probes 15 of (N=B, B are the base analogues 14 that new synthetic comprises the diazacyclo propenyl) preparation, modified base B are corresponding with A, T, G, C respectively with the poly-5`ATCAAAGNGCAAGAC-3` of 15-.The diazacyclo propenyl is entrained in the centre of the poly-single stranded DNA of 15-.Obtain double chain DNA probe by single stranded DNA and complementary strand annealing, A, T, G, C match with B respectively.
DsDNA probe 15 is through the photo-crosslinking activity of UV irradiating and detecting B and antisense DNA chain.The denatured DNA gel is used to analyze crosslinked.The appearance of low migration band shows the formation of crosslinked dsDNA.In contrast, T replaces B to be mixed in the antisense DNA chain.Through the uv irradiating of different time, B and A, T, G, C paired sample are detected.Experiment shows that photoresponse can finish in 5 minutes, prolongs light application time cross-linking efficiency is not significantly improved.Yet as what expect, the diazacyclo propylene can be by photodissociation fast, and the life-span of photolytic product-Cabbeen has only the time of nanosecond.UV-crosslinked experiment shows that clearly diazacyclo propylene base is crosslinked with different efficient with antisense strand.G and B 5 minutes the irradiation after have 50% crosslinked, T and B crosslinked next, the crosslinked productive rate of A or C and B is also very considerable.
Table 1
In order to determine these results, four DNAs (dsDNA 8-11) in the table 1 are 0 ℃ of uv irradiating through different time (30uM dsDNA is dissolved in 10mM three (methylol) aminomethane hydrochloride, and pH 7.4,100mM NaCl), sex change then is with 20% poly-propionic acid amide gel analysis.The formation of crosslinked DNA product in the appearance explanation chain of low migration band, however do not shine through UV with, even the product of trace also detect less than.Different with B paired base, cross-linking efficiency is also different in the DNA chain.B and A pairing productive rate have only 5-10%, and B and G paired productive rate are up to 30-40%.Experiment shows that also the crosslinking reaction of diazacyclo propylene can finish in 10 minutes.Prolonging irradiation time does not improve cross-linking efficiency is significant.This meets expection, because the diazacyclo propylene can be by photodissociation fast, the life-span of photolytic product-Cabbeen has only the time of nanosecond.
(sequence number: 3) (N=B, B are the base analogues 14 that new synthetic comprises the diazacyclo propenyl) is annealed into two strands with the complementary strand that contains energy and B complementary A, T, G or C to 15 yuan of purified oligomerization nucleoside 5 '-ATGAACCNGGAAAAC-3 '.Measure the melting temperature(Tm) (Tm) (seeing Table 1) of double-stranded DNA s (10mM three (methylol) aminomethane hydrochloride, PH7.4,100Mm NaCl) with dsc.Remove B:A pairing (sequence number 1, table 1) and do not detect the behavior of unwinding, its excess-three kind pairing (sequence number 2,3 and 4, table 1) all is lower than normal DNA (sequence number 5,6, table 1) paired Tm value 18～21% (10～12 ℃).This gentle relatively Tm value reduces somewhat astonishing, in fact, have in dsDNA chain a simple phenyl modified base analogue and A, T, all can be violent when G, C are complementary the stability (reducing 31～41%) of minimizing two strands with respect to normal DNA paired Tm.The potential role of diazacyclo propenyl in the base pairing further structural research that awaits, but performance and the single phenyl of phenyl (trifluoromethyl) diazacyclo propenyl in the DNA pairing is not both very clearly.
In order to test nucleic acid molecule and proteic crosslinked ability, detected four dsDNA probes 15 and DNA and repaired, have the photo-crosslinking of the albumin A lkA of superhelix base structure.The poly-propionic acid amide gel of albumen is used to analyze crosslinking reaction, detects protein sample itself and albumin A lkA and each dsDNA probe shine or do not have the UV irradiation through UV sample.All four probes can connect albumen and DNA as effective photo-crosslinking reagent.Contained B in the probe is if can match with A, and cross-linking efficiency is the highest.
Above-mentioned experiment illustrated contain the diazacyclo propenyl photo-crosslinking DNA base analogue can with the carrying out of contiguous nucleic acid or protein residues photo-crosslinking efficiently, for biological chemistry and biomedical research provide strong tool.
The further purposes of the nucleoside molecule described in the present invention is that these molecules are combined as photochemical medicine with DNA or pna molecule.In case these medicines are present in the individual cells, optical radiation will cause the linked reaction of DNA chain inside or by photochemical medicine and intracellular double-stranded DNA generation covalent effect to form triple strand dna.The DNA linked reaction that this original position forms will make the target cell apoptosis effectively.Optical radiation can be controlled at some zones effectively, will have only cell apoptosis by this way seldom like this.These photochemical medicines may be target with telomere or relevant mixture, perhaps are designed to be associated with gene order in the known cancer cells and act on.
And the fragment gene sequence in these photochemical medicines possibility pair cells works.This will be incorporated on the cell chromosome particularly useful to the elimination retrovirus, for example the HIV cells infected.The photochemical therapy reagent of target cells infected has been represented the therapeutic strategy of a class novelty, even because the potential cells infected also can be killed by the photo-crosslinking strategy of target retrovirus.Can destroy these metabolism inactivations, the potential cells infected needs to want for the patient of treatment infected by HIV very much, and therapeutic strategy can only work in virus replication at present.
Nucleoside analog among the present invention can be crosslinked with the dna double chain under the irradiation of light, participates in forming three chain oligonucleotide sequences, makes its loss of function.Downward modulation particular target expression of gene is a very promising strategy of treatment human diseases, and Da Ros, Besch, Buchini and Leumann are described (Curr.Med.Chem.2005,12:71-88 to biological experiment; Curr.Drug Targets.2004,5:691-703; Curr.Opin.Chem.Biol.2003,7:717-726) DNA three chains are based on an application of this strategy.
Another application of the invention is the DNA of the nucleosides described in the present invention and short chain or PNA to be combined and then photic linked reaction takes place, thereby obtains the sequence of a specific dna molecular.
Nucleosides among the present invention can enter cell, and just as known nucleoside analog, it is macromolecular synthetic to participate in the cell true tumor as 5-floxuridine and Zidovodine.The biomacromolecule of mixing photo-crosslinking reagent can be used as photochemical therapy reagent, the interior or intermolecular cross-linking of inducing molecule.
The secondary structure of nucleic acid can influence the function of ribozyme.Nucleosides of the present invention can influence the formation of nucleic acid secondary structure, and natural nucleus glycoside or other modified nucleoside derivative do not possess this effect.
After nucleosides among the present invention is incorporated into nucleic acid, utilize the hybridization characteristic of nucleic acid can be used for the antisense nucleic acid field.
Nucleosides character among the present invention is different with natural nucleus glycoside, thereby is fit to other purposes.For example, the interaction in vivo of nucleosides among the present invention and enzyme has important biological significance, and new antiviral therapy method is had material impact.
Oligonucleotide among the present invention (having an affinity groups or fluorophor) can enter into viable cell as siRNAs.Two photons stimulate in can inductor crosslinked.As seen cross-linked composite can become by fluorophor, also can separate obtaining by affinity groups.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Synthesizing of embodiment 1 nucleosides
3-(4-iodine substituted phenyl)-3-(trifluoromethyl)-3H-diazacyclo propylene (6)
Compound (6) reference (A N Topin, Nucleosides ﹠amp; Nucleotides 1998,17,1163-1175) from 1, and 4-diiodo-benzene preparation (1).
1,4-dehydration-3,5-two-oxygen (tertiary butyl dimethyl is silica-based)-2-deoxidation-D-erythro form-penta-1-glycal 1,4-dehydration-3,5-two-oxygen (tertiary butyl dimethyl is silica-based)-2-deoxidation-D-erythro form-penta-1-glycal (9)
Compound (9) reference (MA Cameron, Journal of Organic Chemistry1997,62,9065-9069) prepare from the 2-deoxythymidine.
(2R, 5R)-2-((tertiary butyl and methyl siloxy) methyl)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone (10)
In a dry round-bottomed flask of crossing, add 3-(4-iodine substituted phenyl)-3-(trifluoromethyl)-3H-diazacyclo propylene (6) (0.368 gram successively, 1.18 mmole), 1,4-dehydration-3,5-two-oxygen (tertiary butyl dimethyl is silica-based)-2-deoxidation-D-erythro form-penta-1-glycal (9) (0.488 gram, 1.42 mmole), nitrogen, (0.30 milliliter in nitrogen-dicyclohexyl methylamine, 1.4 mmole), palladium (0.026 gram, 0.12 mmole), tetrabutylammonium chloride (0.328 gram, 1.18 mmole), the 4A molecular sieve of porphyrize and 3.0 milliliters heavily steam nitrogen, nitrogen-dimethyl formamide is to form suspension.Reaction mixture at room temperature stirs, and feeds nitrogen after about 30 minutes, is heated to 60 ℃ under nitrogen atmosphere, and is incubated 40 hours.After this, reaction mixture water (50 milliliters) dilution is extracted with ethyl acetate (1 * 50 milliliter).Organic phase is washed successively with saturated sodium bicarbonate solution (50 milliliters), saturated aqueous common salt (50 milliliters), concentrates with the anhydrous magnesium sulfate drying after-filtration.The enriched material silica gel column chromatogram separating purification, obtain oily matter (2R, 5R)-2-((tertiary butyl and methyl siloxy) methyl)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone (10) (0.225 gram, productive rate 46%).Proton nmr spectra (500 megahertzes, deuterochloroform) δ 7.54 (d, J=8.4Hz, 2H), 7.21 (d, J=8.4Hz, 2H), 5.19 (dd, J=11.0,5.9Hz, 1H), 4.04 (t, J=2.5Hz, 1H), 3.97 (m, 2H), 2.82 (dd, J=17.6,5.9Hz, 1H), 2.38 (dd, J=17.6,11.0Hz, 1H), 0.87 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).
(2R, 5R)-2-(methylol)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone (11)
(2R to ice bath to 0 ℃, 5R)-2-((tertiary butyl and methyl siloxy) methyl)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone (10) (0.460 gram, 1.11 in tetrahydrofuran solution mmole) (5 milliliters), add the tetrahydrofuran solution (3.0 milliliters) of the tetrabutyl ammonium fluoride of Glacial acetic acid (0.2 milliliter) and 1.0 mol successively.Stirring is concentrated after 1.5 hours down at 0 ℃ for reaction mixture.The enriched material silica gel column chromatogram separating purification obtains oily matter (2R, 5R)-2-(methylol)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone (11) (0.308 gram, productive rate 92%).Proton nmr spectra (500 megahertzes, deuterochloroform) δ 7.49 (d, J=8.1Hz, 2H), 7.24 (d, J=8.1Hz, 2H), 5.24 (dd, J=11.0,5.9Hz, 1H), 4.06 (t, J=3.3Hz, 1H), 3.96 (m, 2H), 2.90 (dd, J=18.0,5.9Hz, 1H), 2.48 (dd, J=18.0,11.0Hz, 1H).
(1R)-1,4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (12)
Bathe (the 2R that is cooled to-10 ℃ to cryosel, 5R)-2-(methylol)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) in the acetonitrile (10 milliliters) of dihydrofuran-3 (2 hydrogen)-ketone (11) and the solution of Glacial acetic acid (2 milliliters), disposable adding triethoxy sodium borohydride (0.529 gram, 2.50 mmoles).Reaction mixture concentrates after 30 minutes in-10 ℃ of stirrings under nitrogen atmosphere.The enriched material silica gel column chromatography separating purification obtains white powder (1R)-1,4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (12) (0.170 gram, productive rate 56%).Proton nmr spectra (500 megahertzes, deuterochloroform .) δ 7.37 (d, J=8.4Hz, 2H), 7.17 (d, J=8.4Hz, 2H), 5.16 (dd, J=9.9,5.5Hz, 1H), 4.40 (m, 1H), 4.02 (m, 1H), 3.75 (m, 2H), 2.24 (m, 1H), 1.95 (m, 1H).
(1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (13)
Under 0 ℃, (1R)-1,4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (12) (0.170 gram, 0.562 heavily steaming pyridine (5 milliliters) solution stirring mmole) is after 10 minutes, to wherein adding 4 successively, 4 '-dimethoxytrityl methyl chloride (0.286 gram, 0.844 mmole) and 4-(dimethylamino) pyridine (0.004 gram, 0.03 mmole).Under nitrogen atmosphere, reaction solution is used ether (2 * 50 milliliters) extraction then in 0 ℃ of stirring water (50 milliliters) dilution after 6 hours down.Merge organic phase, use anhydrous magnesium sulfate drying.Filter concentrated filtrate.The enriched material silica gel column chromatography separating purification, obtain white powder (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (13) (0.242 gram, productive rate 71%).Proton nmr spectra (500 megahertzes, deuterochloroform) δ 7.44 (m, 2H), 7.39 (d, J=8.4Hz, 2H), 7.33 (m, 4H), 7.26 (m, 2H), 7.21 (m, 1H), 7.14 (d, J=8.4Hz, 2H), 6.81 (m, 4H), 5.17 (dd, J=9.9,5.5Hz, 1H), 4.40 (m, 1H), 4.06 (m, 1H), 3.78 (s, 6H), 3.33 (dd, J=9.9,4.4Hz, 1H), 2.24 (ddd, J=13.2,5.5,1.8Hz, 1H), 1.97 (ddd, J=13.2,9.9,6.0Hz, 1H).Carbon-13 nmr spectra (126 megahertzes, deuterochloroform) δ 158.6,144.9,144.1,136.1,130.2,128.3,128.2,128.0,127.0,126.6,126.5,122.3 (q, J=274.8Hz), 113.3,86.6,86.4,79.4,74.6,64.5,55.3,43.9,28.5 (q, J=40.1Hz).
(1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose 2-cyanogen oxyethyl group diisopropylphosphoramidite monomer (14)
(1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose (13) (0.121 gram, 0.200 mmole), nitrogen, nitrogen-diisopropyl ethyl amine (0.10 milliliter, 0.57 mmole) and 2-cyanogen oxyethyl group-nitrogen, the heavily steaming dichloromethane solution (2 milliliters) of nitrogen-di-isopropyl protochloride phosphamide (0.07 milliliter, 0.3 mmole) stirred 1.5 hours down in 0 ℃ under nitrogen atmosphere.Reaction soln is directly used silica gel column chromatography separating purification, obtain white foam shape thing (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose 2-cyanogen oxyethyl group diisopropylphosphoramidite monomer (14) (0.136 gram, productive rate 84%).Proton nmr spectra (500 megahertzes, deuterochloroform) δ 7.46-7.43 (m, 4H), 7.35-7.25 (m, 9H), and 6.83-6.79 (m, 4H), 5.25-5.15 (m, 1H), 4.55-4.45 (m, 1H), and 4.25-4.15 (m, 1H), 3.90-3.20 (m, 12H), 2.70-1.90 (m, 4H), and 1.35-1.05 (m, 12H).Nucleus magnetic resonance phosphorus spectrum (202 megahertzes, deuterochloroform) δ 148.7,148.6.
Embodiment 2
1. the synthetic and evaluation of oligonucleotide
Oligonucleotide adopts the β-nitrile ethyl phosphoramidite chemical process of standard to synthesize on AppliedBiosystems Expedite nucleic acid synthesizer.All oligonucleotide synthesize with the pattern that the 5`-end takes off DMT, and ambient temperature overnight in strong aqua is downcut from CPG (alkylcontrolled pore glass) solid phase carrier at last.Oligonucleotide gathers propionic acid amide gel electrophoresis separation and purification with 20% sex change.The molar extinction coefficient of supposing the base that the diazacyclo propenyl is modified is zero under 260nm, carries out quantitatively with the uv-absorbing of this wavelength.Determine the exactness of all synthetic oligonucleotide chains with the MALDI mass spectrum.
2.ds photo-crosslinking in the chain of DNA
The DNA sample is dissolved in 10mM three (methylol) aminomethane hydrochloride, and (pH 7.4), in the damping fluid of 100mM NaCl, 0 ℃ of irradiation different time sections, 450 watts of used mercury lamps will be used the light wave of glass filter disc filtering less than 300nm.With the poly-propionic acid amide gel analysis sample of denatured DNA.
The expression and the purifying of (3.AlkA 3-methyladenine DNA Glycosylase II)
Conversion has the E.coli cell of AlkA expression plasmid, inserts to contain in the LB substratum of ammonia benzyl, and 37 ℃ of cultivations are up to cell density OD
600Reach 0.7, in nutrient solution, add IPTG (1Mm), induce 4h for 30 ℃.Centrifugal collection thalline is stored in-80 ℃.Following step is all carried out under 4 ℃.Thalline is resuspended in (10mM three (methylol) aminomethane hydrochloride, (pH 7.4), 100mM NaCl, 5% glycerine, 2mM CaCl in the 20ml lysate
2, 10mM MgCl
2, the 10mM 2 mercapto ethanol), ultrasonication, 12,000 leave heart 20min.Supernatant liquor is with the quick post of sepharose (amersham biosciences) purifying, pillar will be used buffer A (10mM three (methylol) aminomethane hydrochloride earlier, (pH 7.4), the 10mM2-mercaptoethanol) pre-equilibration, carry out gradient elution with the buffer A that contains 0 to 1.0M NaCl.Contain proteic component ultrafiltration and concentration, (amershambiosciences) is further purified with the Mono-S ion exchange column, and elutriant is to contain 0 to 1.0M NaCl buffer A.
4.ds DNA-AlkA is crosslinked
Albumen behind the purifying and dna probe are dissolved in and contain 10mM three (methylol) aminomethane hydrochloride, in the damping fluid of (PH 7.4) and 100mM NaCl, pay different time sections under 0 ℃, rayed 5min then, 450 watts of used mercury lamps will be with the light wave of glass filter disc filtering less than 300nm.Sample gathers the propionic acid amide gel analysis with sex change.
Above experimental result shows: utilize the nucleoside compound of preparation of the present invention can photodissociation under higher wavelength (～360nm) produce a highly active Cabbeen, thereby in the inducing molecule or intermolecular cross-linking reaction.Nucleosides of the present invention can enter cell, and it is macromolecular synthetic to participate in the interior true tumor of cell, and the biomacromolecule of mixing photo-crosslinking reagent can be used as photochemical therapy reagent.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
＜110〉Beijing Okeanos Tech Co., Ltd.
＜120〉contain non-natural nucleoside, its preparation method and the application thereof of diazacyclo propenyl
<130>KHP09112512.1
<160>9
<170>PatentIn?version?3.5
<210>1
<211>15
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉" n " is for comprises the base analogue 14 of diazacyclo propenyl
<400>1
atcaaagngc?aagac 15
<210>2
<211>15
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉" n " is for comprises the base analogue 14 of diazacyclo propenyl
<400>2
tagtttcncg?ttctg 15
<210>3
<211>15
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉" n " is for comprises the base analogue 14 of diazacyclo propenyl
<220>
<221>misc_feature
<222>(8)..(8)
<223>n?is?a，c，g，or?t
<400>3
atgaaccngg?aaaac 15
<210>4
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＜213〉artificial sequence
<400>4
tacttggacc?ttttg 15
<210>5
<211>15
<212>DNA
＜213〉artificial sequence
<400>5
tacttggtcc?ttttg 15
<210>6
<211>15
<212>DNA
＜213〉artificial sequence
<400>6
tacttgggcc?ttttg 15
<210>7
<211>15
<212>DNA
＜213〉artificial sequence
<400>7
tacttggccc?ttttg 15
<210>8
<211>15
<212>DNA
＜213〉artificial sequence
<400>8
atgaacctgg?aaaac 15
<210>9
<211>15
<212>DNA
＜213〉artificial sequence
<400>9
atgaacccgg?aaaac 15
Claims (10)
1. the non-natural nucleoside compound that contains the diazacyclo propenyl that has structural formula (I):
Wherein, R in the formula
1Be hydrogen, replacement or unsubstituted C
1～C
8Alkyl, n is 0,1,2 or 3, but preferably 0;
Ar is 5 yuan to 10 yuan hetero-aromatic ring or 5 yuan to 10 yuan aromatic ring, and these aromatic rings need possess two conditions: (a) have a substituting group that contains sugar, sugar analogue, nucleosides or nucleoside analog at least; (b) 0 to 3 comprises replacement or unsubstituted C
1～C
8Alkyl, hydroxyl and amino; Ar or with substituent 6 yuan to 10 yuan aromatic ring, or band ribose or ribodesose substituting group, or band nucleoside phosphoramidites monomer or the monomeric sugar analogue substituting group of deoxynucleoside phosphoramidite; Perhaps, Ar is the modified sugar-phosphoric acid structural unit of band multiple sugar-phosphoric acid structural unit, multiple or the nucleosides substituting group of nucleoside analog; Perhaps, Ar is the substituting group of the nucleoside analog of band peptide nucleic acid(PNA).
2. compound according to claim 1 is characterized in that, Ar has a substituting group shown in molecular formula (II):
R
2, R
3, R
4Or R
5Be hydrogen or hydroxyl; R
6Be through protection or without the single, double or triphosphoric acid base of protecting; Perhaps R
3And R
6Energy in two groups and poly-nucleotide chain reaction coupling.
3. compound according to claim 2 is characterized in that R
3And R
6One in two groups is phosphoramidite.
4. according to any described compound of claim 1-3, it is characterized in that Ar is with the substituting group shown in the formula (III):
R
7And R
9It is the group that can carry out linked reaction with poly-nucleotide chain; R
8Be hydrogen or hydroxyl.
5. compound according to claim 4 is characterized in that, the compound of structural formula (I) is that formula (IV) arrives in the formula (VII):
R shown in the formula
10Be H, replacement or unsubstituted C
1～C
8Alkyl, hydroxyl or amino in a kind of; R is glycosyl, glycosyl analogue, nucleic acid base or analogue with nucleic acid backbone; N and R
1Described in (I).
6. compound according to claim 5 is characterized in that, the compound of structural formula (I) is that formula (VIII) arrives in the formula (XIII):
R is suc as formula described in (IV).
7. compound according to claim 6 is characterized in that, the compound of structural formula (I) is that formula (XIV) arrives in the formula (XVII):
8. prepare the method for the described compound of claim 7, it is characterized in that, comprise the steps:
1) with 3-(4-iodine substituted phenyl)-3-(trifluoromethyl)-3H-diazacyclo propylene, 1,4-dehydration-3,5-two-oxygen (tertiary butyl dimethyl is silica-based)-2-deoxidation-D-erythro form-penta-1-glycal, nitrogen, nitrogen-dicyclohexyl methylamine, palladium, tetrabutylammonium chloride, the 4A molecular sieve of porphyrize and heavily steam nitrogen, nitrogen-dimethyl formamide mixes, to form suspension, under nitrogen atmosphere, be heated to 60 ℃, and be incubated 40 hours, use ethyl acetate extraction, organic phase wash dry after-filtration concentrate obtain oily matter (2R, 5R)-2-((tertiary butyl and methyl siloxy) methyl)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone;
2) then to the (2R of ice bath to 0 ℃, 5R)-2-((tertiary butyl and methyl siloxy) methyl)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) in the tetrahydrofuran solution of dihydrofuran-3 (2 hydrogen)-ketone, the tetrahydrofuran solution that adds Glacial acetic acid and tetrabutyl ammonium fluoride successively, stirring is concentrated after 1.5 hours down at 0 ℃ for reaction mixture, separation and purification obtains oily matter (2R, 5R)-2-(methylol)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) dihydrofuran-3 (2 hydrogen)-ketone;
3) bathe (2R that is cooled to-10 ℃ to cryosel, 5R)-2-(methylol)-5-(4-(3-trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl) in dihydrofuran-3 (2 the hydrogen)-acetonitrile of ketone and the solution of Glacial acetic acid, add the triethoxy sodium borohydride, under nitrogen atmosphere, concentrate after 30 minutes in-10 ℃ of stirrings, separation and purification obtains white powder (1R)-1,4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose;
4) under 0 ℃, (1R)-1, the heavily steaming pyridine solution of 4-dehydration-2-deoxidation-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose stirred after 10 minutes, to wherein adding 4 successively, 4 '-dimethoxytrityl methyl chloride and 4-(dimethylamino) pyridine, under nitrogen atmosphere, reaction solution extracts with ether (2 * 50 milliliters) after stirring 6 hours under 0 ℃, merge organic phase, drying is filtered, concentrated filtrate, separation and purification obtains (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose;
5) (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose, nitrogen, nitrogen-diisopropyl ethyl amine and 2-cyanogen oxyethyl group-nitrogen, the heavily steaming dichloromethane solution of nitrogen-di-isopropyl protochloride phosphamide stirred 1.5 hours down in 0 ℃ under nitrogen atmosphere, separation and purification, obtain white foam shape thing (1R)-1,4-dehydration-2-deoxidation-5-oxygen-(4,4 '-dimethoxy) triphenyl-1-C-(4-(3-(trifluoromethyl)-3 hydrogen-diazacyclo propenyl) phenyl)-D-erythro form-pentose-2-cyanogen oxyethyl group diisopropylphosphoramidite monomer.
9. the described compound of claim 1-8 is in the application that is used for preparing photocrosslinking reaction probe.
10. the described compound of claim 1-8 is in the application that is used for preparing photochemical medicine.
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|---|---|---|---|---|
| CN110141665A (en) * | 2019-05-23 | 2019-08-20 | 南京工业大学 | A kind of method of aziridine in the application modification of antibody coupling drug-linker |
| CN110237253A (en) * | 2016-03-09 | 2019-09-17 | 苏州大学 | The nano-particles self assemble aggregation and application that ultraviolet light mediates |
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2009
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110237253A (en) * | 2016-03-09 | 2019-09-17 | 苏州大学 | The nano-particles self assemble aggregation and application that ultraviolet light mediates |
| CN110237253B (en) * | 2016-03-09 | 2021-06-18 | 苏州大学 | Ultraviolet light mediated nanoparticle self-assembly aggregate and application |
| CN110141665A (en) * | 2019-05-23 | 2019-08-20 | 南京工业大学 | A kind of method of aziridine in the application modification of antibody coupling drug-linker |
| CN110141665B (en) * | 2019-05-23 | 2021-09-17 | 南京工业大学 | Application modification method of aziridine in antibody-coupled drug connector |
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