CN103232507B - Modified nucleoside monomer and synthetic method thereof and application - Google Patents
Modified nucleoside monomer and synthetic method thereof and application Download PDFInfo
- Publication number
- CN103232507B CN103232507B CN201210130023.5A CN201210130023A CN103232507B CN 103232507 B CN103232507 B CN 103232507B CN 201210130023 A CN201210130023 A CN 201210130023A CN 103232507 B CN103232507 B CN 103232507B
- Authority
- CN
- China
- Prior art keywords
- compound
- modified nucleoside
- dissolved
- dimethoxytrityl
- oxygen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 0 CC([*@]([C@@]1NC(C*)*C1)C=CC(C)=Cl)=N Chemical compound CC([*@]([C@@]1NC(C*)*C1)C=CC(C)=Cl)=N 0.000 description 5
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses two class modified nucleoside monomers and synthetic method thereof and application, the modified nucleoside monomer that two classes provided by the invention are new, there is the structure such as Formulas I or Formula II, the present invention also provides for its synthetic method, and this monomer can be advantageously used in the solid phase synthesis of oligonucleotide puted together with polypeptid covalence, and between polypeptide and oligonucleotide, build two sulfur connect bases or triazole connects base.Adopt modified nucleoside monomer provided by the present invention to carry out the synthesis of the oligonucleotide puted together with polypeptid covalence, have that atopic is strong, carrying capacity is relatively high and the advantage such as easy and simple to handle.
Description
Technical field
The present invention relates to a class modified nucleoside monomer, further relate to synthetic method and the application in oligonucleotide conjugates synthesizes thereof.The invention belongs to synthesis chemical field.
Background technology
Wanting to develop into useful medicine based on some medicines such as antisense oligonucleotide, peptide nucleic acid(PNA) of nucleic acid and the siRNA (siRNA) that gets a good chance of, improving its cell permeability is one of the key issue that must solve.A lot of technology are already used to improve the cellular uptake of this compounds, for instance, high pressure intravenous injection, liposome and nanometer polymer etc., but all there is side effect in various degree and limit its development in these methods.
Cell-permeant peptide is the emerging transport vehicle grown up in the last few years, is widely used for the intracellular transport of some acellular membrane permeability macromole.And use it for nucleic acid drug intracellular transport, mainly through permeable membrane peptide and oligonucleotide covalency are puted together realization.Chemically realize conjugate generation and mainly have three kinds of methods: solid phase Stepwise synthesis, solid phase fragment condensation and liquid-phase fragment condensation method.The advantages such as in these three method, solid phase Stepwise synthesis has easy and simple to handle, and substrate consumption is few.
Summary of the invention
In order to solve the problems of the prior art, it is an object of the invention to provide the new nucleoside of two classes and modify monomer, this monomer can be advantageously used in the synthesis that polypeptid covalence puts together oligonucleotide, and builds two sulfur connection bases or triazole connection base between polypeptide and oligonucleotide.
In order to achieve the above object, the present invention adopts the following technical scheme that
One aspect of the present invention, it is provided that modified nucleoside monomer there is the structure such as Formulas I or Formula II:
Sugar therein is L-configuration or D-form；Described X is O or S-S；Described R1For hydroxyl, hydrogen, substituted hydroxy, amino, substituted-amino, sulfydryl and or substituted sulfhydryl, R2For having the protection base of uv absorption, R3For hydrogen, natural purine, pyrimidine bases, non-natural or the purine of manually modified synthesis, pyrimidine bases；
R4For-R-NH2,-R-COOH、Wherein R is C1-C8Low alkyl group, alkenyl, alkynyl group, aryl, aralkyl or containing heteroatomic above-mentioned group.
In the present invention, it is preferred to R1For hydrogen, hydroxyl or amino, R2For benzoyl, 4,4 '-dimethoxytrityl or monomethoxytrityl；
R3ForWherein R5For methyl, hydrogen, halogen or replacement halogen, R6For hydroxyl, amino, hydrogen, methyl, halogen or replacement halogen；R4For-R-NH2OrWherein R is C1-C8Low alkyl group.
It is furthermore preferred that R3For pyrimidine bases, R is methylene or ethylidene.
Present invention also offers a kind of method synthesizing described modified nucleoside monomer, it is characterised in that when the X in Formulas I or Formula II is S-S, comprise the steps:
1) 2 '-deoxyuridine is dissolved in anhydrous pyridine, adds DMTrCl, reaction 3 hour is stirred at room temperature, obtains 5'-oxygen-(4,4'-dimethoxytrityl)-2'-deoxyuridine, be compound 1 under condition of ice bath；
2) compound 1 is dissolved in anhydrous pyridine, MsCl, room temperature reaction 6 hours, solvent evaporated it is slowly added dropwise under condition of ice bath, dichloromethane extraction, remnants after being evaporated being dissolved in anhydrous acetonitrile, adds potassium carbonate, 85 DEG C are refluxed 2 hours, obtain 5'-oxygen-(4,4'-dimethoxytrityl)-2,3'-dehydration-2'-deoxyuridines, it is compound 2；
3) being dissolved in dry DMF by compound 2, added thiobenzoate sodium every 30 minutes, 110 DEG C are refluxed 3 hours, obtain 5'-oxygen-(4,4'-dimethoxytrityl)-3'-S-benzoyl-2'-deoxyuridine, be compound 3；
4) compound 3 and 2-(2-pyridine radicals dimercapto)-ethylamine are dissolved in the sodium hydroxide alcoholic solution of 0.4M; under argon shield; room temperature reaction 6 hours; obtain 5'-oxygen-(4; 4'-dimethoxytrityl)-3'-(2-amino-ethyl disulfide group)-2'-deoxyuridine, it is compound 4；
5) compound 3 and 3-(2-pyridine radicals dimercapto)-propanoic acid are dissolved in 0.4M sodium hydroxide alcoholic solution; under argon shield; room temperature reaction 3 hours; obtain 5'-oxygen-(4; 4'-dimethoxytrityl)-3'-(1-propanoic acid disulfide group)-2'-deoxyuridine, it is compound 5.
When the X in Formulas I or Formula II is O, comprise the steps:
1) 2 '-deoxyuridine is dissolved in anhydrous pyridine, adds DMTrCl, reaction 3 hour is stirred at room temperature, obtains 5'-oxygen-(4,4'-dimethoxytrityl)-2'-deoxyuridine, be compound 1 under condition of ice bath；
2) compound 1 and Anhydrous potassium carbonate are blended in anhydrous acetonitrile, add 3-propargyl bromide, 50 DEG C of stirring reactions 10 hours, obtain 5'-oxygen-(4,4'-dimethoxytrityl)-3'-oxygen-(2-propynyl)-2'-deoxyuridine.
The present invention also provides for the application in oligonucleotide conjugates synthesizes of the described modified nucleoside monomer.Described application is the liquid-phase synthesis process utilizing described modified nucleoside monomer sequentially to be synthesized the oligonucleotide conjugates being made up of two macromole conjugates on solid phase carrier by solid phase.
Described solid phase carrier mainly but is not limited to use in the resin of solid phase synthesis.
Described macromole conjugate is mainly but not limited to the molecular weight molecule more than 500.
Described a kind of macromole conjugate be mainly but not limited to oligonucleotide with and the like.Described oligonucleotide and the like be mainly but not limited to DNA, RNA, PNA and their own and formed each other two, three, the tetramer.
Wherein another kind of macromole conjugate is mainly but not limited to peptide, protein, polysaccharide, antibody, polycation, liposome, nanoparticle.
The present invention also provides for the application in relevant biological activity research of the above-mentioned oligonucleotide conjugates.Described research and application mainly include but not limited to carry out on mammal.
The present invention is by providing the nucleoside that two classes are new to modify monomer, and this monomer can be advantageously used in the solid phase synthesis that polypeptid covalence puts together oligonucleotide, and builds two sulfur connection bases or triazole link base between polypeptide and oligonucleotide.It is short that the nucleoside of the present invention modifies monomer synthetic route, and synthesis is convenient, and it can point out carrying capacity information in solid phase synthesis.Modify monomer by nucleoside provided by the present invention to carry out polypeptid covalence and put together the synthesis of oligonucleotide there is the advantages such as atopic is strong, carrying capacity is relatively high, easy and simple to handle.
Detailed description of the invention
Provide defined below firstly, for the term occurred in this specification: term " nucleoside " and " nucleoside analog " can exchange use, refer to the compound comprising the sugar moieties with heterocycle covalent coupling.Particularly preferred heterocycle includes aromatic heterocycle, even more preferably from heterocycle include purine, pyrimidine or purine/pyrimidine analogue.Term " oligonucleotide " refers to that at least two and two or more nucleoside are coupled by phosphoric acid ester bond.
Term " protection base " refers to covalently bound with the oxygen atom of the compound considered or nitrogen-atoms, it is prevented that the chemical group that described oxygen atom or nitrogen-atoms react further in the derivatization process of other functional groups in considered compound.The protection base of miscellaneous oxygen and nitrogen is that the known to the skilled of organic synthesis field (is write referring to such as ProtectingGroupsinOrganicSynthesis, JamesR.Hanson, BlackwellScienceInc；ISBN:063204506X, or ActivatingAgentsandProtectingGroups, HandbookofReagentsforOrganicSynthesis, WilliamR.Roush and AnthonyJ.Pearson write；JohnWiley&SonLtd；ISBN:0471979279, the two list of references is all incorporated herein by reference).
The particularly preferred blocking group containing uv absorption of hydroxyl mainly includes but not limited to:
Term " low alkyl group " refers to methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, sec-butyl or n-hexyl, also includes having the ring-type of 1-4 carbon atom, branched-chain or straight-chain alkyl.Term " alkenyl " refers to and comprises at least one double bond structure in above-mentioned low alkyl group.Term " alkynyl group " refers to and comprises at least one three bond structure in above-mentioned low alkyl group.Term " aryl " refers to the unsaturated aromatic carbocyclic radical with a monocycle (such as phenyl) or two condensed ring (such as naphthyl), and described aryl can replace by functional group or without functional group.
Term " containing hetero atom " refers to that in substituent group, at least one carbon atom is replaced by non-carbon such as oxygen, sulfur, nitrogen, phosphorus.Term " substituted hydroxy " refers to that the hydrogen of hydroxyl is replaced by other groups outside hydrogen atom.Term " substituted-amino " refers to that in ammonia molecule, at least one hydrogen is replaced by other groups outside hydrogen atom.Term " substituted sulfhydryl " refers to that the hydrogen of sulfydryl is replaced by other groups outside hydrogen atom.Term " replacement halogen " refers to and at least contains a halogen atom in substituent group.
Term " L-nucleoside " refers to the nucleoside compound with L-configuration sugar moieties.Equally, term " D-nucleoside " refers to the nucleoside compound with D-form sugar moieties.Term " halogen " refers to that halogen includes fluorine, chlorine, bromine, iodine etc..
According to the present invention, it is provided that new modified nucleoside monomer as follows:
Sugar therein is L-configuration or D-form；Described R1Mainly it is but not limited to hydroxyl, hydrogen, substituted hydroxy, amino, substituted-amino, sulfydryl and substituted sulfhydryl, R2Mainly it is but not limited to the protection base with uv absorption, R3Mainly it is but not limited to hydrogen, natural purine, pyrimidine bases, non-natural and the purine of manually modified synthesis, pyrimidine bases；
Wherein R5Mainly it is but not limited to methyl, hydrogen, halogen and replacement halogen, R6Mainly it is but not limited to hydroxyl, amino, hydrogen, methyl, halogen and replacement halogen.
Wherein R is mainly but not limited to C1-C4Low alkyl group, alkenyl, alkynyl group, aryl or aralkyl and containing heteroatomic above-mentioned group.
According to the present invention, utilizing the modified nucleoside monomer in the present invention to prepare oligonucleotide covalent conjugates, its feature mainly comprises the steps that
A. synthetic peptide sequence on solid phase carrier, this peptide sequence terminal amino group is not necessary to functional group's conversion (formula III) or carries out functional group's conversion (Formulas I V) by specific reagent (such as succinic anhydride).Carry out being converted to (Formula V) also by nitrine caproic acid.
B. by immobilized to formula (III) and modified nucleoside formula (3), (4), (5), (6) condensation or by immobilized to formula (IV) and modified nucleoside formula (1), (2), (7), (8) condensation, it is also possible to formula (V) and formula (9) are reacted immobilized by Click.Formula (VI), (VII), (VIII) is respectively obtained for formula (1), (5), (9).
C. formula (VI), (VII) and (VIII) can direct sequence synthetic oligonucleotide on DNA synthesizer.
D. conjugate is obtained target conjugate after cutting, purification solid phase carrier.
Further describe preparation method and the beneficial effect of the present invention by the following examples, it should be understood that these embodiments only for illustration purposes, are never limited in protection scope of the present invention.
A. synthetic modification nucleoside
Modified nucleoside shown in embodiment 1 synthesis type (1) (5)
5'-oxygen-4,4'-dimethoxytrityl-2'-Brdurd (compound 1)
2 '-deoxyuridine (1.5g, 6.57mmol) is dissolved in 20mL anhydrous pyridine, adds DMTrCl (2.67g, 7.89mmol) under condition of ice bath.Reaction 3 hour is stirred at room temperature, reacts with a small amount of methanol cancellation, evaporated under reduced pressure solvent.Reduced pressure chromatography purification, dichloromethane: methanol=9:1 affords white blister solid 3.31g, is the compound described in title, yield 95%.ESI-MS:553.2(M+Na+)。
5'-oxygen-4,4'-dimethoxytrityl-2,3'-dehydration-2'-Brdurd (compound 2)
Compound 1 (3.0g, 5.65mmol) is dissolved in 10mL anhydrous pyridine, is slowly added dropwise MsCl (1.4mL, 16.95mmol) under condition of ice bath, and room temperature reaction, after 6 hours, reacts with a small amount of methanol cancellation.Evaporated under reduced pressure solvent, saturated sodium carbonate solution washs, dichloromethane extraction (3 × 30mL), merges organic facies, and anhydrous sodium sulfate dries.Remnants after being evaporated are dissolved in 50mL anhydrous acetonitrile, add K2CO3(2.4g, 16.95mmol), 85 DEG C are refluxed 2 hours.Filter, evaporated under reduced pressure solvent.Reduced pressure chromatography purification, dichloromethane: methanol=95:5 affords light yellow blister solid 2.7g, is the compound described in title, two step total recoverys 81%.
1HNMR(400MHz,CDCl3) δ (ppm) 7.42-7.17 (m, 9H, ArH), 7.04 (d, J=7,36Hz, 1H, 6H), 6.83-6.79 (m, 4H, ArH), 5.93 (d, J=7.2Hz, 1H, 5H), 5.47 (m, 1H, 4'H), 5.17 (m, 1H, 1'H), 4.27 (m, 1H, 3'H), 3.79 (s, 6H, CH3O×2),3.40-3.31(m,2H,5'H×2),2.61-2.38(m,2H,2'-H×2).
5'-oxygen-4,4'-dimethoxytrityl-3'-S-benzoyl-2'-Brdurd (compound 3)
Compound 2 (417mg, 0.813mmol) is dissolved in 10mL dry DMF, adds BzSNa (232mg, 2.04mmol) every 30 minutes, and 110 DEG C are refluxed 3 hours, add AcSK1.39g altogether.After having reacted, washing with saturated sodium bicarbonate solution, extraction into ethyl acetate (3 × 10mL), merge organic facies, anhydrous sodium sulfate dries.Reduced pressure chromatography purification, petroleum ether: ethyl acetate=2:1 affords faint yellow blister solid 334mg, is the compound described in title, yield 70%.
1HNMR(400MHz,CDCl3) δ (ppm) 8.83 (s, 1H, NH), 8.03 (d, J=7.8,1H, 6H), 7.42-7.16 (m, 9H, ArH), 6.84 (d, J=7.8Hz, 4H, ArH), 6.15 (d, J=7.8,1H, 5H), 5.32 (m, 1'H), 4.26 (m, 1H, 4'H), 3.80 (s, 6H, CH3O×2),3.53-3.40(m,2H,5'H×2),2.95-2.83(m,1H,3'H),2.60-2.44(m,2H,2'-H×2),2.39(s,3H,COCH3).
5'-oxygen-(4,4'-dimethoxytrityl)-3'-(2-amino-ethyl disulfide group)-2'-Brdurd (compound 4)
Compound 3 (170mg, 0.20mmol) and 2-(2-pyridine radicals dimercapto)-ethylamine are dissolved in the sodium hydroxide alcoholic solution of 10mL0.4M, under argon shield, and room temperature reaction 6 hours.Concentration, reduced pressure chromatography purification, dichloromethane: methanol=40:1 affords faint yellow blister solid 100mg, is the compound described in title, yield 80%.
1HNMR(300MHz,DMSO-d6) δ (ppm) 11.37 (s, 1H, 3NH), 7.76 (d, J=8.1Hz, 1H, 6H)；7.37-7.21 (m, 9H, ArH), 6.86 (m, 4H, ArH), 6.03 (t, J=6.0Hz, 1H, 1'H), 5.35 (d, J=7.8Hz, 1H, 5H), 4.03 (m, 1H, 4'H), 3.90-3.71 (m, 2H, 5'H × 2), 3.78 (s, 6H, CH3O×2),3.40-3.28(m,5H,3'H,SSCH2CH2),2.50-2.40(m,2H,2'H×2),2.00(m,2H,CH2NH2).
5'-oxygen-(4,4'-dimethoxytrityl)-3'-(1-propanoic acid disulfide group)-2'-Brdurd (compound 5)
Compound 3 (200mg, 0.22mmol) and 3-(2-pyridine radicals dimercapto)-propanoic acid are dissolved in the sodium hydroxide alcoholic solution of 10mL0.4M, under argon shield, and room temperature.React concentration in 6 hours, reduced pressure chromatography purification, dichloromethane: methanol=15:1 affords faint yellow blister solid 120mg, is the compound described in title, yield 75%.ESI-MS:663.18(M-H-)。
Modified nucleoside shown in the 2-in-1 accepted way of doing sth of embodiment (9)
5'-oxygen-(4,4'-dimethoxytrityl)-2'-deoxyuridine (compound 1)
2'-deoxyuridine (1.5g, 6.57mmol) is dissolved in anhydrous pyridine (20mL), adds DMTrCl (2.67g, 7.89mmol, 1.2eq) under condition of ice bath.Reaction 3 hour is stirred at room temperature, reacts with a small amount of methanol cancellation, evaporated under reduced pressure solvent.Reduced pressure chromatography dichloromethane: absolute methanol=9:1, obtains white blister solid 3.31g, is the compound described in title, yield 95%.ESI-MS:553.2(M+Na+)。
5'-oxygen-(4,4'-dimethoxytrityl)-3'-oxygen-(2-propynyl)-2'-deoxyuridine (compound 2)
Compound 1 (530mg) and Anhydrous potassium carbonate (150mg) are blended in anhydrous acetonitrile (10ml), add 3-propargyl bromide (100 μ l), 50 DEG C of stirring reactions 10 hours.Leaching insoluble matter, filter vacuum concentrates, residue normal pressure column chromatography for separation, and dichloromethane: ethyl acetate=3:1 obtains faint yellow blister solid 503mg, is the compound described in title, yield 88.1%.ESI-MS:569.3(M+H+), 591.2 (M+Na+)。
1HNMR(300MHz,CDCl3) 7.808 (d, J=8.1Hz, 1H, H-6), 7.39-7.26 (m, 9H, Ar-H), 6.85 (d, J=8.7Hz, 4H, Ar-H), 6.33 (t, J=6.3Hz, 1H, H-5), 5.50 (d, J=8.1Hz, 1H, H-1 '), 4.69 (d, J=2.4Hz, 2H, 3 '-O-CH2), 4.56 (s, 1H, NH), 4.02 (d, J=3.3Hz, 1H, H-4 '), 3.80 (s, 6H, 2OCH3),3.49-3.45(m,2H,5’-CH2),2.46-2.26(m,2H,2’-CH2), 2.17 (s, 1H, C ≡ CH), 1.84 (d, J=3.9Hz, 1H, H-3 ').
B. peptide sequence synthesis
On the CPG that hydroxylating is modified, for sequence KALLAL, synthesize target sequence according to Fmoc or Boc method, and remove the protection base on sequence terminal amino group with suitable reagent.For Boc method: Boc-Lys (Fmoc)-OH, HOBt and the HBTU that the side-chain amino group Fmoc of 2-8 equivalent is protected; it is dissolved in 4mLDMF, adds the DIPEA of 2-8 equivalent, after activating 15 minutes; joining in the CPG that certain hydroxylization is modified, shaken at room temperature reacts 1 hour.Filter reactant liquor, with each 5mL of DMF, methanol and DCM successively washing resin three times, fully dry.Taking 1-3mgCPG, add appropriate 20% piperidines/DMF solution, the light absorption value measuring 304nm after constant volume is calculated as 80 μm of ol/g (i.e. the amount of Lys in every gram of resin).After reaching desired value, remove Boc with the dichloromethane solution of 33%TFA and protect base, with DMF, methanol and DCM washing resin successively, fully dry.Carry out next amino acid couplings, detect reaction with 1,2,3-indantrione monohydrate and complete, close unreacted amino with acetic anhydride, with each 5mL of DMF, methanol and DCM successively washing resin three times, fully dry.Repetitive operation is until aminoacid sequence synthesis is complete.Remove Boc with the dichloromethane solution of 33%TFA and protect base, with DMF, methanol and DCM washing resin successively, fully dry.
C. functional group's conversion
Taking the succinic anhydride (being equivalent to the amount of hydroxyl in hydroxylating resin) of 1-5 equivalent, the DMAP of 1-5 equivalent is dissolved in appropriate anhydrous pyridine, joins in the peptide resin of above-mentioned synthesis, and shaken at room temperature reacts 6 hours.It is filtered to remove reactant liquor, with dichloromethane (3 × 5ml), absolute methanol (3 × 5ml) and dichloromethane (3 × 5ml) washing, dry.1,2,3-indantrione monohydrate detection extent of reaction, if the display positive, repeats derivatization operation, until display negative findings, it is achieved end function base is become carboxyl from amino.Take the nitrine caproic acid (be equivalent to hydroxyl in hydroxylating resin amount) of 1-5 equivalent, take 2-5 equivalent 6-nitrine caproic acid (be equivalent to hydroxyl in hydroxylating resin amount), 2-5 eq. HOBt per, 2-5 equivalent HBTU, it is dissolved in appropriate anhydrous DMF without amine, after adding 2-5 equivalent DIPEA activation 5-20 minute, join in the peptide resin of above-mentioned synthesis, room temperature concussion reaction 6 hours.It is filtered to remove reactant liquor, with dichloromethane (3 × 5ml), absolute methanol (3 × 5ml) and dichloromethane (3 × 5ml) washing, dry.1,2,3-indantrione monohydrate detection extent of reaction, if the display positive, repeats derivatization operation, until display negative findings, it is achieved end function base is become azido from amino.
D. monomer is modified immobilized
By immobilized for the modified nucleoside shown in formula (1) in formula (IV)
Take the modified nucleoside shown in 5-8 equivalent formula (1) (being equivalent to the amount of hydroxyl in hydroxylating resin), 5-8 equivalent HBTU, the DMAP of 1-3 equivalent adds in the CPG of above-mentioned succinic anhydride derivatization, adds appropriate anhydrous acetonitrile, and shaken at room temperature reacts 12 hours.With dichloromethane (3 × 5ml), absolute methanol (3 × 5ml) and dichloromethane (3 × 5ml) washing, dry.Repeat to upload once.Taking a small amount of CPG in 5mL volumetric flask, with 3% dichloroacetic acid/dichloromethane solution constant volume, 507nm measures absorption value, calculates carrying capacity.
By immobilized for the modified nucleoside shown in formula (9) in formula (V)
Take the modified nucleoside shown in 5-8 equivalent formula (9) (being equivalent to the amount of hydroxyl in hydroxylating resin), 5-8 equivalent DIPEA, the CuI of 1-3 equivalent adds in the CPG of above-mentioned nitrine caproic acid derivatization, adds appropriate anhydrous acetonitrile, and shaken at room temperature reacts 12 hours.With dichloromethane (3 × 5ml), absolute methanol (3 × 5ml) and dichloromethane (3 × 5ml) washing, dry.Repeat to upload once.Taking a small amount of CPG in 5mL volumetric flask, with 3% dichloroacetic acid/dichloromethane solution constant volume, 507nm measures absorption value, calculates carrying capacity.
E. synthetic peptide puts together oligonucleotide
By the CPG of above-mentioned immobilized modified nucleoside, ABI392 type DNA/RNA synthesizer synthesizes by standard phosphoramidite method the oligonucleotide sequences (3'-UUAGCCCUUUAAAGAGAUAAU-5') specified.Synthesis scale is 1 μM, obtains conjugate sequence (KALLAL-3'-UUAGCCCUUUAAAGAGAUAAU-5').Detect DMT content through spectrophotometer, calculate and often walk Coupling efficiency more than 99%.The quality HPLC of conjugate is controlled, and purity is more than 95%.The correctness of conjugate is detected with MALDI-TOF-MS.Wherein, the molecular weight of the conjugate formed based on the modified nucleoside shown in formula (1) is for 7451.2, and value of calculation is 7450.4.The molecular weight of the conjugate formed based on the modified nucleoside shown in formula (9) is for 7448.2, and value of calculation is 7442.4.
The information shown herein and be described in detail is enough to realize the above-mentioned purpose of the present invention, and therefore the preferred embodiments of the invention represent the theme of the present invention, and this theme is that the present invention extensively contains.The scope of the present invention is fully contemplated by other embodiment that will be apparent to persons skilled in the art, therefore, the scope of the present invention is not limited by any content except claims, wherein except clearly stating, the singulative of element used does not imply that " one with unique ", and refers to " one or more ".For persons skilled in the art, therefore all known above-mentioned preferred embodiments and the structure of additional embodiment part, composition and equivalent functionally are incorporated herein by, and attempt to be contained by the claim of the present invention.
Furthermore, it is not necessary that certain equipment or method express each problem solved by the invention, because they include within the claim of the present invention all.It addition, no matter all parts in the disclosure fact, composition, or whether method step clearly described in the claims, and they are all without contributing to the public.But, for those of ordinary skills, it is evident that under the premise without departing substantially from the spirit and scope of the invention as illustrated in claims, it is possible to make various change and modification in form, reagent and synthesis details.The foregoing is only presently preferred embodiments of the present invention, be merely illustrative for the purpose of the present invention, and nonrestrictive.Those skilled in the art is understood, and it can be carried out many changes in the spirit and scope that the claims in the present invention limit, amendment, even equivalence, but falls within protection scope of the present invention.
Claims (3)
1. modified nucleoside monomer, has the structure such as Formulas I
Wherein said X is S-S；Described R1For hydrogen, R2It is 4,4 '-dimethoxytrityl, R3For uracil, R4For-R-NH2, wherein R is methylene or ethylidene.
2. the method for the modified nucleoside monomer that a kind synthesizes described in claim 1, it is characterised in that when the R in Formulas I is ethylidene, comprise the steps:
1) 2'-deoxyuridine is dissolved in anhydrous pyridine, adds DMTrCl, reaction 3 hour is stirred at room temperature, obtains 5'-oxygen-(4,4'-dimethoxytrityl)-2'-deoxyuridine, be compound 1 under condition of ice bath；
2) compound 1 is dissolved in anhydrous pyridine, MsCl, room temperature reaction 6 hours, solvent evaporated it is slowly added dropwise under condition of ice bath, dichloromethane extraction, remnants after being evaporated being dissolved in anhydrous acetonitrile, adds potassium carbonate, 85 DEG C are refluxed 2 hours, obtain 5'-oxygen-(4,4'-dimethoxytrityl)-2,3'-dehydration-2'-deoxyuridines, it is compound 2；
3) being dissolved in dry DMF by compound 2, added thiobenzoate sodium every 30 minutes, 110 DEG C are refluxed 3 hours, obtain 5'-oxygen-(4,4'-dimethoxytrityl)-3'-S-benzoyl-2'-deoxyuridine, be compound 3；
4) compound 3 and 2-(2-pyridine radicals dimercapto)-ethylamine are dissolved in the sodium hydroxide alcoholic solution of 0.4M; under argon shield; room temperature reaction 6 hours; obtain 5'-oxygen-(4; 4'-dimethoxytrityl)-3'-(2-amino-ethyl disulfide group)-2'-deoxyuridine, it is compound 4.
3. the application in oligonucleotide conjugates synthesizes of the modified nucleoside monomer described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210130023.5A CN103232507B (en) | 2012-04-27 | 2012-04-27 | Modified nucleoside monomer and synthetic method thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210130023.5A CN103232507B (en) | 2012-04-27 | 2012-04-27 | Modified nucleoside monomer and synthetic method thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103232507A CN103232507A (en) | 2013-08-07 |
| CN103232507B true CN103232507B (en) | 2016-07-06 |
Family
ID=48880586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210130023.5A Expired - Fee Related CN103232507B (en) | 2012-04-27 | 2012-04-27 | Modified nucleoside monomer and synthetic method thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103232507B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106215821B (en) * | 2016-08-12 | 2018-08-17 | 江苏泽成生物技术有限公司 | A kind of preparation method of magnetic microparticle chemiluminescence immune assay reagent carboxyl magnetic bead |
| CN113549121B (en) * | 2021-07-21 | 2023-05-02 | 上海兆维科技发展有限公司 | Preparation method of nucleoside modifier |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007122333A2 (en) * | 2006-04-24 | 2007-11-01 | Universite Joseph Fourier (Grenoble 1) | Process for preparing disulphides and thiosulphinates and compounds prepared |
| CN101899092A (en) * | 2009-06-01 | 2010-12-01 | 北京大学 | Novel peptide-link base-conjugate and solid phase synthesis method thereof |
| WO2012012776A1 (en) * | 2010-07-22 | 2012-01-26 | Gilead Sciences, Inc. | Methods and compounds for treating paramyxoviridae virus infections |
-
2012
- 2012-04-27 CN CN201210130023.5A patent/CN103232507B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007122333A2 (en) * | 2006-04-24 | 2007-11-01 | Universite Joseph Fourier (Grenoble 1) | Process for preparing disulphides and thiosulphinates and compounds prepared |
| CN101899092A (en) * | 2009-06-01 | 2010-12-01 | 北京大学 | Novel peptide-link base-conjugate and solid phase synthesis method thereof |
| WO2012012776A1 (en) * | 2010-07-22 | 2012-01-26 | Gilead Sciences, Inc. | Methods and compounds for treating paramyxoviridae virus infections |
Non-Patent Citations (2)
| Title |
|---|
| A solid-phase method for peptide–siRNA covalent conjugates based on click chemistry;Yang Liu，等;《Med. Chem. Commun.》;20120229;第3卷;第506-511页 * |
| Synthesis of oligodeoxyribonucleotide N3"→P5"phosphoramidates;Jer-Kang Chen，等;《Nucleic Acids Research》;19951231;第23卷(第14期);第2661-2668页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103232507A (en) | 2013-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11279736B2 (en) | Conformationally-preorganized, MiniPEG-containing gamma-peptide nucleic acids | |
| US7795423B2 (en) | Polynucleotide labeling reagent | |
| CA2452474C (en) | Nucleotide compositions comprising photocleavable markers and methods of preparation thereof | |
| JPH05504564A (en) | Uncharged morpholino-based polymerases with achiral internal subunit linkages | |
| JP2000502341A (en) | Reusable solid support for oligonucleotide synthesis, its preparation and its use | |
| US8889843B2 (en) | Nucleic acid synthesizing dimer amidite and nucleic acid synthesizing method | |
| JP2005514505A (en) | Preparation and use of multi-arm dendritic and functional PEG | |
| CN103232507B (en) | Modified nucleoside monomer and synthetic method thereof and application | |
| US9441002B2 (en) | Dithiolane based thiol modifier for labeling and stronger immobilization of bio-molecules on solid surfaces | |
| JP6153116B2 (en) | Triazole-linked cyclic dinucleotide analogs | |
| AU2006316903B2 (en) | Polynucleotide labelling reagent | |
| WO2003076412A1 (en) | Novel hairpin polyamide | |
| EP4083054A1 (en) | Method for producing pna oligomer in solution process | |
| CN103073606A (en) | Method for synthesizing and preparing 5'-S-(4, 4'-dimethoxytriphenylmethyl)-2'-deoxyinosine | |
| CN114920684B (en) | Selenium-containing benzamide compound and synthetic method and application thereof | |
| Kore | Solid-phase synthesis of new ribo and deoxyribo BrdU probes for labeling and detection of nucleic acids | |
| CN114773412A (en) | Non-basal-protection single-arm carbohydrate-containing compound and preparation method thereof | |
| CN117624266A (en) | Liver targeting compound, oligonucleotide conjugate thereof, coupling method and application | |
| CN114736150A (en) | off-DNA and on-DNA synthesis method of arylselenoindole compound | |
| JP3837633B2 (en) | Novel functional peptide nucleic acid and production method thereof | |
| CN116003306A (en) | Synthesis method of linker compound of maleimidocaprooic acid-PEGn analogue | |
| US20030208050A1 (en) | Nucleoside derivatives | |
| KR20120058699A (en) | Oligomer and the synthesis of the oligomer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160706 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |