JPH05501418A - Oligonucleotide functionalization methods - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Oligonucleotide functionalization methods The present invention uses amine-containing reagents to functionalize oligonucleotides at their 5'OH ends. related to the process of enabling.

The present invention describes dideoxynucleoside phosphoramidite derivatives useful in the process. It also relates to a synthon consisting of a body.

Synthesis of oligonucleotides covalently linked to various reactants is a field of biochemistry and molecular biology. Due to the extensive use of these molecules in medicine as well as as potential therapeutic agents, Very interesting for their use.

In particular, the attachment of intercalating agents to oligonucleotides involves the complementary sequence of these oligomers. (U, As5eline et al. ,,Proc, Natl, Acad, Sci.

USA, 81.3297-3301.1984), improves cell membrane permeation of the latter. (P, Verspleren et al, Gene, 61.307-315 ．． 1987).

Additionally, oligonucleotides (S, Agrav al, C, Christodoulou and M, J, Galt.

Nuclelc Ac1ds Res, 14.6227-8245.1986 ;P, Richterich.

Nucleic Ac1ds Res, 17.2181-2185.1989 ) are easily detected using highly sensitive fluorescent or enzymatic and colorimetric methods, respectively. be done. These compounds are used as cold molecular probes and also as radioactive probes. can be substituted advantageously.

Finally, chemically activated vines (C, B, Chen and DJ).

Sigman, Proc, NaL, Acad, Sc1, USA, 83, 7147-7151; T. LeDoan et al., Nucl eic Ac1ds Res, 15.8843-8659.1987; J, C ,Franqols et al., ,Biochemistry, 27.227 2-2278, 1988; S, A, 5trobe1. H, E, Mo5er a nd P, B, Dervan, J, As, Chem.

Soc,, 110.7927-7929.1988; S, B, Lin et a. l.

Biochemistry, 28.1054-10B1.1989) or photochemistry (T, Le Doan et al, Nucleic Ac 1ds Res,, 15°7749-7760.1987) bonded to the reactive group Oligonucleotides bind to nucleic acids at the level of complementary sequences, causing irreversible damage. can be induced locally. This group of compounds modulates the biological function of a given gene. It may also be used for selective inhibition. The role of oligomer sequences is determined by the genes involved. The goal is to ensure the specificity of recognition at the level of the complementary sequences carried; Scientific activator deals damage to this target.

In this patent application, "oligonucleotide" refers to an oligodeoxynucleotide in a DNA system. It is understood that in the leotide and RNA systems oligoribonucleotides are meant.

Additionally, an "oligonucleotide" may have a β-anomeric configuration or a non-natural α-anomeric configuration. It is also understood to mean an oligonucleotide.

Finally, the nucleotides that make up the oligonucleotide are true nucleotides, i.e. Nucleotides with a phosphate at the 5' end of the corresponding nucleoside or more commonly found In particular, derivatives of the phosphorothiolic acid or methylphosphonic acid type.

In other patent applications, the applicant has used amine-containing reagents to synthesize oligonucleotides. A process for functionalization with 5'OH has also been described. For this reason, the present invention For a better understanding, please refer to French patent application FR No. 83.01°223 (2,5 No. 40,122) and No. 84.117゜955 (2,568,254) It is useful to refer to the literature, where at least one insertion group is strands of optionally modified β-anomeric natural nucleotides linked together Compounds consisting of oligonucleotides were described.

Synthesis and sequence specificity of oligonucleotide compounds attached to activatable chemical groups Their use as synthetic artificial nucleases is described in French Patent Application No. 87.03.3. No. 66 (2,586,707).

The method by which oligonucleotides are stabilized in the form of phosphotriesters has been published in international patents. It was described in the application PCT WO 83101451.

Oligos stabilized by replacing natural phosphodiester bonds with phosphonic acid bonds Nucleotides are described in US Pat. No. 4,469,863.

Production of α-anomeric oligonucleotides and intercalation agents or chemical or photochemical The coupling of those with active "H" groups is described in International Application PCT WO 881. It was described in the specification of No. 04301.

In this application, "reactant" refers to a group corresponding to an intercalating agent, also referred to as an intercalating agent; chemical groups, such as groups with functional groups that react directly or indirectly with the leotide chain. or a photoactivatable group or a group whose presence allows for easy and selective detection. Then it is understood.

Photoforms on solid supports are used to produce α-oligodeoxyribonucleotide compounds. The synthesis using the sphoramidite method is described in French patent application no. 87.04,339. phosphorylation on a solid support of α-oligoribonucleotide compounds. Synthesis using the amidite method is described in French Patent Application No. 88.12.264. Are listed.

To date, in the process of functionalizing oligonucleotides with reactants, linkers - derivatives of the above reactants functionalized with - e.g. phosphoramidites - solid supports In the synthesis case above, the phosphoramidite is the final stage of oligonucleotide extension. used in stages. For this reason, 5tein et al, Gene 72 (1988) 333-341, oligonucleotides, in this case oligonucleotides, A conventional method of functionalizing crizine (ACr).

This method can be illustrated as follows: a) by linker (m-3 or 5); Functionalization of acridine b) Formation of the corresponding phosphoramidite C) Synthesis on a solid support using 1 in the final step of mechanical elongation d) Removal of the solid support and deprotection of the oligomer using C6H55H. these Under the conditions, side reactions were observed with acridine in the final oligomer upon deprotection. (CI is replaced with C6Hs　S).

The approach described in this paper requires the introduction of different reactants each time it is desired to introduce different reactants. requires that a corresponding phosphoramidite be synthesized: To overcome this drawback, the 3'OH end of the oligodeoxyribonucleotide Covalent bonding with the amino reagent (R-NH2) at the end of the oligonucleate follows the following route. Aldehyde functional group and amino derivative of the abasic site (AP) formed in leotide (J, -J, Vasseur, C, Ga Uthier, B. Rayner.

J, Paoletti and J,-L, Imbach, Biochea, B iophys, f? es.

Cowiun, 152, 56-61, 1988; J, -R, Be rtrand, J, -J, Vasseur.

A., Gouyette, B., Rayner, J.-L., Imbach, C., Pa. oletti and C.

Malvy, J., Biol, Che+g, 264.14172-14178. 1989): this approach, which is nevertheless very general in itself. Since a large number of R-NH2 reagents are possible, the AP site (2'-deo xyribose) formation.

In fact, the latter is produced by acid hydrolysis of the glycosidic bond of deoxyadenosine (dA). can be obtained.

At that time, the oligonucleotide chain is acid-hydrolysed so that dC and dT are more susceptible to acid hydrolysis than dA or dG. It would contain only pyrimidine nucleosides.

Thus, in other words, pyrimidine nucleosides can be prepared using amine-containing reagents. This method can be used to functionalize oligodeoxyribonucleotides consisting of It is impossible.

The present invention can be synthesized on a solid support, where appropriate any conventional DN A and RNA bases (A.

C, G, T or U) and modified bases (DNA-based and RNA-based A general rule for forming a non-basic site at the 5'OH end of an oligonucleotide We are proposing a process. Therefore, this original process Oligonucleotides of any sequence can be functionalized using reactive agents containing Wear.

The main feature of the functionalization method according to the invention is the use of dideoxyribonucleosides. , it is possible to generate oligonucleotides of any sequence by automated synthesis on a solid support. The molecules can also be functionalized at their 5'OH ends.

The process according to the invention can be applied to oligomers (natural systems in the β-configuration, non-natural systems in the α-configuration, D regardless of structural modifications carried out with phosphorothioic acid, methylphosphonic acid, etc.) Applicable to NA or RNA systems.

More particularly, the subject matter of the present invention provides oligonucleation using amine-containing reagents of formula RNH2. A process for functionalizing cleotide at its 5' OH end; It is characterized by the following stages: a) said oligonucleotide is synthesized; b) said dideoxynucleotide is synthesized; attached at its 5' end to the 5' OH terminus of the oligonucleotide; C) covalently linked; the dideoxynucleotide at the 5'OH end of the oligonucleotide. 5 of the oligonucleotide derived from the opening by acid hydrolysis of the glycoside ring of 'Reductive amination reaction between the aldehyde functional group at the OH terminal and the above amino reagent. using the above oligonucleotide and the above RNH2 amino reagent.

Therefore, the present invention relates to oligonucleotide systems considered (natural or modified). At the 5'OH end of the oligomer, which can contain any base regardless of A novel approach to forming non-basic dideoxyribose moieties is provided.

These abasic oligonucleotides are characterized by the fact that they contain amines. After reductive amination with reactants, they can be easily and quantitatively derived into their derivatized forms. It is not isolated from.

In an embodiment of the process according to the invention with respect to steps a) and b), the oligonucleotide Otide synthesis is a phosphoramidite bond synthesis on a solid support. (phosphoramidte-auaching syr+thesis ), and in the final step, the phosphoramidite derivative is dideoxynucleotide attached to the 5' end of the compound using an addition synthesis cycle It is used at the 5' end of the dideoxy nucleoside corresponding to

The dideoxynucleoside phosphoramidite derivatives according to the invention much more sensitive to acid hydrolysis than silibonucleosides or ribonucleosides. Rula I7. Among the latter, as can be seen further, the dideoxynuclides of nebularine Rheoside derivatives are more easily hydrolyzed.

However, the same holds true for other dideoxynucleoside derivatives, especially those of the formula Corresponding hydrogenophosphonic acid derivatives: This applies from synthesis using phosphoramidites to other types of synthesis, In particular, there is a possibility of transitioning to synthesis using hydrogen phosphonate derivatives (Nucle ic Aclds Research, Vol. R4, No. 13.1988).

The solid-phase phosphoramidite process consists of two main steps: -5' protected by one of its 2' or 3' hydroxyl functions. a creoside derivative is immobilized on a solid support; Protected by condensation of monomers consisting of -5' and 2'-protected nucleosides An oligonucleotide chain substituted with a phosphoramidite group at the 3' position is attached to this support in a manual or automated synthesis reactor; - the oligonucleotide is It is obtained after removing the obtained oligomer from the support and removing the protecting groups.

Suitably, the attachment of the oligonucleotide is in the case of the first monomer, a fixed nucleotide. Fixed nucleoside compound or in the case of subsequent monomers of the above fixed nucleoside compound The 5' hydroxyl functional group of the protected intermediate polynucleotide compound immobilized on the It is carried out by condensation between the 3' functional group of the above monomer in the presence of an active agent. .

In particular, activators for the condensation of the monomers mentioned above are tetrazole and p-nitrophe. selected from its derivatives such as niltetrazole.

In the process according to the invention, preferably after steps a) and b) and after step C) before the above-mentioned dideoxynucleotide linked at the 5' end. Compounds consisting of oligonucleotides are removed from the solid support and the protecting groups are removed. be removed.

The reductive amination reaction in step C) can be carried out, for example, with sodium cyanoborohydride in an acid medium. This is done using thorium treatment.

In this embodiment of the process, the phosphoramidite synthesis involves the dideoxynuclease of the following formula: Using creoside phosphoramidite derivatives: In the above formula, -R and R are 1 2 3' like CHCH(CH3)2 represents optionally substituted C-C alkyl or R and R2 taken together forming a morpholino group; -R is optionally substituted C-C alkyl, especially CH or CH2CH2CN -B represents a nucleobase, in particular adenine, guanine or nebularine.

In the case of nucleobase B, adenine, guanine or nebularine are other nucleobases. In practice, it is preferable to select them if their acid hydrolysis is easy compared to the above.

More preferably, a phosphoramidite derivative of the formula (■) in which B represents nebularin is used. It will be done.

The originality and advantage of the approach according to the present invention is that oligonucleotides on a solid support are During the final step of the synthesis of leotide, a similar dielectric It consists in the use of deoxynucleoside phosphoramidite derivatives. deoxy sugar ring After ring opening, dideoxynucleoside phosphoramidite derivatives become real oligonucleotides. Provides a "linker" between the leotide and the reactant. Furthermore, according to the present invention, deprotection is No longer carried out with functionalized oligomers due to the risk of reaction; - is deprotected before introducing the reactants. Therefore, the reactants, i.e. generally very The consumption of a substance that is expensive due to the present invention is avoided because it is introduced at the final stage of the synthesis. It is less in the process of

In previous publications, such as the paper by 5tefn et al., in the production of phosphoramidites, requires the prior introduction of a linker, and in solid-phase synthesis an excess amount (20 times the stoichiometric linker) is required. There was a need to use sphoramidite derivatives 1).

In a specific embodiment, according to the process according to the invention, compounds of formula RNH is the monovalent residue of the amine-containing reactant RNH2; -J-H or OH.

The single residues B may be the same or different from each other, each in the α or β-anomeric configuration. represents an optionally modified base of a nucleic acid attached to a glycoside ring; may be the same or different from each other, oxo anion O-, thio anion s -, alkyl group, alkoxy or -ruoxy group or aminoalkyl, alkyl group, Represents a minoalkoxy or thioalkyl group; -n is 1-50.

More specifically, mention may be made of the compound of formula 1, which is x-o-1s- or cH3.

In the case of X-8-, the usual oxidation step after detritylation, addition and capping may be replaced by a sulfidation cycle (Gene, 72.343-347) (W, J , 5tec, G., Zon, V., Egan and B.

5tec, J, Amer, CheLSoc,, 108, 6077- 6079, 1984: R, P, Iyer.

W., Egan, J., B. Regan and S. L., Beaucage, J. AIIer, Chem, Soe,.

112.1253-1254.1990). For CH3, 5'- (dimethoxytrityl) nucleoside 3’-(N,N-diisopropylmethytyl) phosphoramidite) synthon replaces the phosphoramidite synthon in the addition step. May be used instead (M, A, Dorsan, S, A, Noble, L, J, M cBride and M, H.

Caruthers, Tetrahedron, 40.95-102, 1984 ;A, Jiger and J, Engels, Tetrahedron Let ters, 25.1437-1440.1984).

As already mentioned, the reactive group of formula RNH- according to the invention is well known in the art involving nucleic acids. It corresponds to the reactant, which is a known compound. They are found in the structure of DNA or RNA, for example. It is a compound that can be “inserted” into.

These intercalating agents are generally acridine, furocoumarin, daunomycin, 1.10 -Phenanthroline, phenanthridinium, profilins, (1*2- a: 3’.

2'-d) Derivatives of dipyridoimidazole, eributicine, eributidinium or are polycyclic compounds with planar configuration such as their derivatives with NH2 functionality. Consisting of

These reactants may be reactive chemical groups that cleave chemical groups, i.e. These groups can react directly or indirectly to split the nucleotide chain. Can be done. These reactive chemical groups are e.g. chemically or photochemically activated scales. is preferable. Activated vine cleavage reactive groups are for example: ethylenediaminetetraacetic acid, di Ethylenetriaminepentaacetic acid, porphyrins, 1.10-phenanthroline, but Induction of compounds such as psoralen, U, V, and other aromatic groups that absorb near visible light. It is a conductor.

These chemical groups, which can be activated in the presence of metal ions, oxygen and reducing agents, give them Inducing cleavage at adjacently located nucleic acid sequences.

More specifically, according to the invention, the RNH residues of the reactants are: - Ethylenediaminetetraacetic acid derivative group of the following formula base -Methylpyroporphyrin derivative group of the following formula -Phenanthroline derivative group of the following formula D -Acridine derivative group: -Proflavin-derived group: l [R represents an amino (NH) or azido (N3) group] -9-aminoeributicine : Finally, the subject of the invention is a synthon useful in the process according to the invention, which It is characterized by corresponding to the following general formula: In the above formula, R1, R2, R3 and B have the meanings given in claim 4.

As already mentioned, preferably in the synthon according to the invention B is preferably represents adenine, guanine or nebularine.

Other advantages and features of the invention will become apparent from the examples below. dideoxyribose residue) to form a 2',3'-dideoxyribose residue Uses of synucleoside phosphoramidites Produce a phosphoramidite of the following formula: Desired oligonucleotide in a synthesizer Additions that allow the incorporation of phosphoramidite 1 after complete assembly of leotide Do the cycle.

The corresponding oligomer 2 is obtained after deprotection by conventional methods and then optionally analyzed by HPLC. refine. Treatment in acid medium (30 g MHC1, 37°, 12-15 iin ) and thus selectively hydrolyze dideoxyribose residues from ' with the dideoxyribose end, i.e. 3, and the latter with cyanoborohydride in buffer. React with the desired amine in the presence of lium. The experimental conditions at this stage were not used. depending on the properties of the amine (solubility, reactivity, etc.) 4 is isolated by HPLC.

According to another embodiment, the reductive amination reaction in step C) of the process according to the invention is carried out using sodium cyanoborohydride treatment in acid medium.

Dideoxynucleoside phosphoramidite derivatives of nebulalin are more tolerant than pyrimidine or purine nucleosides, especially than adenine nucleosides. It seems to be easily hydrolyzed.

The following example describes the synthesis and synthesis of the following types of ddA and ddN 5' phosphoramidites. Characteristics of 2B cotton nebulaline described However, various other phosphorus substituents: CNCH2CH2 instead of CH3 Other phosphoramidite derivatives protected with can also be used.

This for deriving various types of oligodeoxynucleotides (β or α) Reduction of the product obtained after the use of phosphoramidites with amine-containing reactants Amination is possible as long as the steps in this sequence are performed without isolating the intermediate oligomer. It is listed as follows.

f412:ddA*sphoramidite:5'-diisofuropylaminomethoxy Synthesis of posphinyl-2',3'-dideoxyadenosine N,N,N-diisopropylethylamine (1,39μL 8ml1ol) and Chloro-N,N-diisopropylaminomethoxyphosphine (0.98 ml, 5 mmol) in anhydrous dichloromethane (61). (470.5 mg 12 smol). Mixed liquid under argon atmosphere Stir at room temperature for 30 minutes, then pour into a separatory funnel containing ethyl acetate (80 ml). . The resulting solution is washed with brine (4×50 ml). Sodium sulfate organic phase dry, filter and evaporate to dryness under reduced pressure. The resulting residue was applied to a silica gel column. The elution was carried out using cyclohexane, dichloromethane and trichloromethane. Ethylamine mixture i! (1990/9/1 to 30/69/1゜v/v/v) . The fractions containing the desired pure product were combined, evaporated to dryness, and the residue reconstituted in dioxane. Dissolve and lyophilize. A colorless powder (345.31 g/44% yield) is obtained.

-31pN Month R (CD3ON) 149-1i9pp■-'HNMI? (CD3 C13) δ 11.33.1! , 32 and 8.28<3s, 2H.

H2 and Ha); 6.32 (m, LH, o, ); 4.34 (i, 11.) +4); 3.83 (Il, 2H1H5, and H5, ); 3.5g (s, 2H, OH(IPr)2); 3.44 and 3.43 (2d, 3)1. C) 130) :2-50(1,2H,H2,and H2,):2.14(11,2 old H3, and and Hal, ):1. +6 (m, 12L CHa (lPr) 2L Note: S- Singlet, d-doublet, m-multiplet. 6-7MS signal and ratio Chemical shift expressed in pp■ compared to One mass spectrum: FAB<0, polyethylene glycol matrix: (MH)--395; B--134; CH30-P-(0-)N-(IPr)2-178゜FAB) 0, polyethylene glycol matrix: (M”H)”-397: (M "H-)IN(IPr)2)"-298;(M1H-BH)"-26 2; (M+H-CH3O-P(OH)N-<IPr)2)"-218:C H30-P-N (IPr) 2 and B-C”-0-182; (B”H2)” -138゜(lIJ3: ddN phosphoramidite: 5'-diisopropyl Synthesis of aminomethoxyphosphinyl-2',3'-dideoxynebularin N,N,N-diisopropylethylamine (0,671,3,844B+mol ) and chloro-N,N-diisopropylaminomethoxyphosphine (0,35m 1.8 mmol) of 2',3'-dideoxy in anhydrous dichloromethane (51) Add to suspension of cinebraline (211.6 mg, 0.961a + 1 ol) . The mixture was stirred at room temperature under an argon atmosphere for 30 minutes and then diluted with ethyl acetate (40 1) Pour into the containing separatory funnel. Wash the resulting solution with brine (4X 25ml) . The organic phase is dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. obtained The residue was dissolved in dichloromethane and triethylamine (90/9/1 to 50/49/1 , v/v/v> and subjected to silica gel column chromatography. desirable purity The product-containing fractions were combined and evaporated to dryness, and the residue, redissolved in benzene, was lyophilized. dry A colorless powder (165.3 mg, yield 45%) is obtained.

-31P NMR (CD3 CN) 150.09pp -'HIJMR<CD CI) 69.03 (s, 1N, H, H or 33 2 B H): 8.88 (s, IH,)l SH or H8); 11.59 and 8.55 ( 2s, IH? , H5) le or Hg); 6.40 (+g, IH, Hr); 4 ．． 32 (m.

1H1H4 to 3.93-3.67 (m, 2H2H5, and) +5. , ):3. 81-3.51 (m, 2H5CH (IPr) 2); 3.38 and 3.35 ( 2d, 3)1. CH30, JHP-12, 9 and 13.5Hz); 2L, 54 (i, 2H, H, and H2,,); 2.18 (m.

28, H3, and H3, ); 1.13(m, 12H,0H3(jPr)).

−Mass vector: FAB (0; polyethylene glycol matrix: (M-11)-380; B--119; CH30-P-(0-)N-(iPr)2 = 178°FAB >O, polyethylene glycol matrix: (M"H)" = 382; (M +H-HN(iPr)2)"-281; (M+H-BH)"=262 ;(M+H-Cl 30-P(OH)N-(iPr)2)"-203: CH30-P-N (iPr) 2 and B-C”!0-182; B-Ci=( 1147; (B+)12)” -121° Biliary: dideoxy nebula on a solid support using the phosphoramidite method Synthesis of oligonucleotides containing phosphorus at the 5' end Dideoxynebularine was prepared using methods described in the literature (e.g., β-configured oligodeoxy In the case of xyribonucleotides, the cyanoethyl phosphoramidite method and the α = In the case of oligodeoxyribonucleotides, the methylphosphoramidite method: F., Marvan, B., Rayner, J., P. Leonettfand, J. , L, 1sbach, Nuclelc Ac1ds Res, , 18.833- 847.1988) in an amount of μ-01 by a conventional method. Incorporated in the final step of the synthesis of the compound.

The coupling cycle of the ddN methylphosphoramidite synthon is This is the cycle used in the foramidite method (see above reference). Detritylation The step is omitted at the end of this cycle.

Deprotection of oligonucleotides: This deprotection is quite conventional and depends on the nature of the phosphate protecting group used. .

Thus, in the case of α-configured oligonucleotides, phosphate deprotection at room temperature using a solution of Et3N/dioxane (1/l/2.V/V/V). Oligonucleotide removal from the support was carried out for 30 minutes at room temperature. treatment with 32% NH4OH three times to remove the acyl group (by capping). Deprotection of protected functional groups was carried out using 32% NH4OH at 55'' for 16 hours. Ru.

Only NH4OH treatment is used in the case of oligodeoxyribonucleotides of the configuration. Ru.

Example 5: Adding an oligonucleotide to the “cap” end of the rabbit globin gene Functionalized dideoxynebularin with 9-aminoeributicine at the 5′OH end Oligonucleotide contained at the 5′ end (crude reaction mixture obtained after deprotection, 26 72 absorbance units at 0 nm) is dissolved in a 1005 M solution of hydrochloric acid (300 μn).

The mixture is left at 37°C for 14 minutes. The progress of the reaction was monitored by HPLC under the conditions described below. monitor.

Starting oligonucleotide (RT -17,20sin, 5ax=259.2ns ) and the disappearance of the chain 0CH-(CH2)2-CHO)I-CH20-P-(0″″ )0° (RT-15,0311in, λIIax-259,2nm) at the 5' end Formation of oligonucleotides containing (λl1ax - 263.4 nm) is observed. .

At the end of these 14 minutes of acid treatment, add 1M sodium acetate buffer, pH 5 (400 μg) of sodium cyanoborohydride (6.2 mg, 100 μmol) solution of 9-aminoeributicine in water (500 μg) and then 205 M hydrochloric acid. Add nM solution continuously. The reaction mixture is left at room temperature for 30 minutes. At this stage In HPLC analysis of the reaction mixture, the intermediate oligonucleotide (R knee 15. 03iin) and the oligonucleotide conjugated to aminoeributicine ( R-r-17-80iin, λmax-259,2nm and 307.2nm) Formation is shown. This latter compound is then purified by HPLC.

The amount of functionalized oligonucleotide obtained is 29 absorbance units at 260 tv.

The spectrometric purity of this compound is 99% at this same wavelength.

HPLC conditions used Injection: 10μg 3 u Xl-0DS Cta Beckman column flow rate 1 ml/win for 30 minutes pH ranging from 5,9 to 0. of acetonitrile in 1M ammonium acetate buffer Linear gradient from 5% solution to 15% solution of acetonitrile in the same buffer Example 6: β-oligin against the splicing site of the tat gene of the HIV1 virus Functionalization with 9-aminoeributicine at the 5'OH end of the oligonucleotide Oligonucleotide containing dideoxynebularine at the 5' end (obtained after deprotection) The crude reaction mixture (80.5 absorbance units at 260 nm) was dissolved in water (700 μII). Understand.

A 100 mM solution of hydrochloric acid (300 μg) is then added. Heat the mixture at 37℃ for 12 minutes. Leave it for a while. The progress of the reaction is monitored by HPLC under the conditions described below. Departure o of oligonucleotide (RT=16.78m1n, λwax-262.4nw) Disappearance and Nebularin base (RT-8, 91m1n, λ5ax-263.4nw) ) and the chain 0CH-(CH)-CHoH-CH20-P-(0-)O□- Oligonucleotide containing one terminal (RT=16.18 sin, λmax-28 Simultaneous formation of 2,4nII) is observed.

At the end of these 12 minutes of acid treatment, add 1M sodium acetate buffer, pH 5 (400 A solution of sodium cyanoborohydride (6,21,100 μmol) in μg), 10 nM of 9-aminoeributicine in 20 IIM hydrochloric acid after 500 μN of water. Add solution continuously. The reaction mixture is left at room temperature for 30 minutes. The smell at this stage Therefore, HPLC analysis of the reaction mixture revealed that the intermediate oligonucleotide CRT-11i,1 6m1n) and the disappearance of the oligonucleotide (R T-17,88m1n, λwax-282,4nII and 307.2ni) The structure is shown below. This latter compound is then purified by HPLC.

The amount of functionalized oligonucleotide obtained is 33 absorbance units at 260 nm.

The spectrometric purity of this compound is 94% at this same wavelength.

HPLC conditions used Injection: 10μg 3μ x LODS018 Beckman column flow rate 1ml/1n for 20 minutes From a 0.1 M solution of ammonium acetate buffer at pH 5.9 to Linear gradient to 20% solution of tolyl Summary book The subject matter of the present invention is to prepare oligonucleotides using amine-containing reagents of the formula RNH2. A process for functionalizing at the 5'OH end of The following stages: a) said oligonucleotide is synthesized; b) said dideoxynucleotide is synthesized; attached at its 5' end to the 5'OH terminus of the oligonucleotide; C) covalently attached; However, at the 5'OH end of the above oligonucleotide, there is a dideoxynucleotide at the 5'OH end. 5' of oligonucleotides derived from acid hydrolytic opening of the glycoside ring Using a reductive amination reaction between the aldehyde functional group at the OH terminal and the above amino reagent, carried out with the above oligonucleotide and the above RNH2 amino reagent; It is characterized by

international search report international search report FR9100503 S^　48788

Claims (10)

[Claims] 1. An amine-containing reagent of formula RNH2 is used to react an oligonucleotide with its 5'OH A method for terminally functionalizing, comprising: The following stages: a) said oligonucleotide is synthesized; b) said dideoxynucleotide is synthesized; attached at its 5' end to the 5'OH end of the oligonucleotide; c) covalently linked; However, at the 5'OH end of the above oligonucleotide, there is a dideoxynucleotide at the 5'OH end. 5' of oligonucleotides derived from acid hydrolytic opening of the glycoside ring Using a reductive amination reaction between the aldehyde functional group at the OH terminal and the above amino reagent, carried out with the above oligonucleotide and the above RNH2 amino reagent; A method characterized by: 2. In steps a) and b), the synthesis of oligonucleotides is carried out on a solid support. phosphoramidite bond synthesis in which the final step is to A derivative is added to the 5' end of the oligonucleotide using an addition synthesis cycle. at the 5' of the dideoxynucleoside corresponding to the dideoxynucleotide to be bound. The method according to claim 1, wherein the method is used. 3. After steps a) and b) and before step c), at the 5' end A compound consisting of an oligonucleotide to which a oxynucleotide is attached is a solid support. 3. The method according to claim 1 or 2, wherein the protecting group is removed from the molecule and the protecting group is removed. 4. The dideoxynucleoside phosphoramidite derivative has the following formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the above formula: -R1 and R2 are optionally substituted C1 such as CH3, CH(CH3)2 -C7 alkyl or R1 and R2 together represent a morpholino group: ▲ Formula , chemical formulas, tables, etc.▼ form; -R3 is optionally substituted C1-C7 alkyl, especially CH3 or CH2CH 2CN; -B represents a nucleobase, in particular adenine, guanine or nebulaline ] The method according to any one of claims 1 to 3. 5. The functionalized oligonucleotide has the following formula: ▲ There are mathematical formulas, chemical formulas, tables, etc. S▼ [In the above formula: -RNH is the monovalent residue of the amine-containing reactant RNH2; -J=H or OH; - the residues B may be the same or different from each other, each in the α or β-anomeric configuration; represents an optionally modified base of a nucleic acid attached to a glycosidic ring at the - group X may be the same or different from each other, oxo anion O-, thio anion S-, alkyl group, alkoxy or aryloxy group or aminoalkyl , represents an aminoalkoxy or thioalkyl group; -n is 1 to 50] The method according to any one of claims 1 to 4. 6. The person according to any one of claims 1 to 5, wherein X = O-, S- or CH3. Law. 7. The RNH residue of the reactant is: -Ethylenediaminetetraacetic acid derivative group of the following formula: ▲Mathical formula, chemical formula, table, etc. are available▼ -diethylenetriaminepentaacetic acid derivative group -Methylpyroporphyrin derivative group of the following formula: ▲Mathical formula, chemical formula, table, etc. are available▼ - A phenanthroline derivative group of the following formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ -Acridine derivative group: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ -proflapine-derived group: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [R1 represents an amino (NH2) or azide (N3) group]-9-aminoellipti Shin: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 7. A method according to any one of claims 1 to 6. 8. The reductive amination reaction in step c) involves sodium cyanoborohydride in an acidic medium. 8. The method according to any one of claims 1 to 7, which is carried out using a treatment. 9. General formula below: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (In the above formula, R1, R2, R3 and B have the meanings indicated in claim 4) Useful in the method according to any one of claims 1 to 8, characterized in that Sinton. 10. Synthesis according to claim 9, wherein B represents adenine, guanine or nebularine. hmm.