WO2003070741A1

WO2003070741A1 - Phosphoramidites and use thereof in the preparation of oligonucleotides

Info

Publication number: WO2003070741A1
Application number: PCT/FR2003/000562
Authority: WO
Inventors: Pascale Clivio; Martial Thomas; Javier Ulises Ortiz Mayo
Original assignee: Centre National De Recherche Scientifique (Cnrs)
Priority date: 2002-02-20
Filing date: 2003-02-20
Publication date: 2003-08-28
Also published as: FR2836145B1; FR2836145A1; AU2003222938A1

Abstract

The invention relates to compound of formula (1) where A and B independently = a substituent group selected from cytosine, uracil or thymine, R1 = methyl, 2-cyanoethyl, 2-silylethyl or allyl and Lev = levulinyl.

Description

PHOSPHORAMIDITES AND THEIR USE IN THE PREPARATION OF OLIGONUCLEOTIDES

The subject of the present invention is phosphoramidite precursors, their process for preparation, the phosphoramidites obtained and their use in the synthesis of oligonucleotides specifically containing photochemical lesions in defined position. Exposure of cellular DNA to ultraviolet light causes lesions which form at the level of two adjacent pyrimidines and which are expressed at the molecular level by obtaining cyclobutanic dimers and pyrimidine-type adducts (6-4 pyrimidinone ) which can beomerized into Dewar-type adducts.

In the absence of repair and during the replication of damaged DNA, these lesions can cause errors in reading the polymerases and therefore be mutagenic. They can also induce a munosuppressive response which plays an important role in the development of the carcinogenesis process. These lesions are responsible in humans for most skin cancers. This represents a major public health problem in today's world.

Exacerbated by the depletion of the ozone layer, it can be expected that the increase in UVB radiation capable of crossing the atmosphere will also have uncontrollable repercussions on other living organisms which could dramatically alter the balance of many ecosystems. This is why the inventors are interested in synthetic compounds containing specifically this type of lesions in defined position and which make it possible to study this damage. Currently, there are three ways of synthesizing phosphoramidites which make it possible to obtain, on a solid support, oligonucleotides containing specifically and in a defined position the cis-syn cyclobutane adduct of thymine (Taylor JS, Brockie IR, O'Day CL., "A building block for the sequence-specific introduction of cis-syn thymine di ers into oligonucleotides", Solid-phase synthesis of TpT [c, s] pTpT. J. Am. Chem. Soc.

(1987), 109, 6735-6742; Murata T., Iwai S, Ohtsuka E,

"Synthesis and characterization of a substra te for T4 endonuclease V containing a phosphorodithioate linkage a t the thymine di er site", Nucleic Acids Res. (1990), 18, 7279-7286; Kosmoski JV, Smerdon. J., "Synthesis and nucleosome structure of DNA containing a UV photoproduct at a Specify Site", Biochemistry (1999), 38, 9485-9494; Tommasi S., Swiderski PM, Tu Y, Kaplan BE, Pfeifer GP, "Inhibition of transcription factor binding by ultraviolet-induced pyrimidine dimers" _r Biochemistry (1996), 35, 15693-15703).

The syntheses leading to these phopshora idites all follow the strategy initially developed by Taylor J.S and are based on the use, before the photochemical step, of a dinucleotide whose phosphodiester and hydroxyl functions in 3 'are protected.

The differences between these syntheses relate to the choice of protective groups, the grouping protector of the phosphate function which may be methyl or cyanoethyl, the 3 'hydroxyl function being protected either in the form of silyl ether or in the form of a levulinic acid ester.

Diagram 1 of these syntheses is as follows:

7a R ₂ = CH ₃ , R ₄ = P (OCH ₃ ) (NC ₄ H ₈ 0)

7a 'R ₂ = CH ₃ , R ₄ = P (OC ₂ H ₄ CN) N (CH (CH ₃ ) ₂ ) ₂

7b R ₂ = C ₂ H ₄ CN, R ₄ = P (OC ₂ H ₄ CN) N (CH (CH ₃ ) 2) ₂

Diagram 1

The photolysis of 8 leads to the thymine dimer Cis- Syn 9 with a yield of 41% for 9a and 42% for 9b. The 5 ′ hydroxyl function of compound 9 is protected by the dimethoxytrityl group (step i; yield: 60% and 76%), then the 3 ′ hydroxyl function is deprotected (step ii; yield: 73% and 78%) then functionalized in phosphoramidite (step iii; yield: 53% and 54%). The order of the 5 'protection and 3' deprotection steps can be reversed (step iv and i).

Compound 8 is obtained according to the routes illustrated in scheme 2. By the phosphoramidite route (8a-b) in the 3 ′ -> 5 ′ direction, a dimethoxytrityl derivative in 5 ′ and phosphoramidite in 3 ′ of thymidine is condensed (10a-b) and a 3 'protected derivative of thymidine (11a described by Taylor JS; 11b described by Murata T.); by the chlorophosphite route (8a) in the 5 '->3' direction, the 5 'tritylated derivative of thymidine (10c) is condensed with the 5' chlorophosphite derivative of thymidine protected at 3 '(lie described by Kosmoski JV). The phosphite triester is then oxidized to the phosphate triester and the hydroxyl function in 5 'detritylated to yield 8a.

8a R ^ CHa R ₃ = Silyl

8b R ₂ = C ₂ H4CN, R ₃ = Levulinyl

Diagram 2 The major drawback of this general synthetic route, linked to the fact that the dimethoxytrityl group 03 00562

is not compatible with the photochemical irradiation step, is to deprotect the hydroxyl function in 5 'of the precursor of 8 (Diagram 2, step iii and i') then to reprotect this function at level 9 to obtain the phosphoramidite 7 (Diagram 1, step i). These different stages cause a yield loss of at least 44%. In addition, the photochemical adduct 9 must be deprotected at 3 'to obtain phosphoramidite 7, which represents an additional loss of at least 22% of the photoproduct.

Patent application EP 0 787 740 describes a process for the preparation of a photoproduct (6-4) from a thymidine dimer of formula (II)

wherein R ₂ represents a methyl or cyanoethyl group and R ₇ represents a levulinyl group, or tert-butyldimethylsilyl.

This preparation process includes:

a step of irradiating the compound of formula II,

a step for protecting the 5 ′ OH group, a step for deprotecting the 3 ′ OH group, then the reaction of the product obtained with, for example, a phosphochlorinated derivative, to obtain the expected phosphoramidite of formula (I)

Although this method overcomes the drawbacks of the methods known from the prior art, it still comprises four steps, including several successive protection and deprotection steps which harm the good performance of the process.

However, the inventors have discovered that, if the hydroxyl in the 5 ′ position is protected by a levulinyl group, it was not necessary to remove this group before the photochemical irradiation step. This group was found to be both stable under irradiation conditions and compatible with the conditions for synthesis on a solid support.

The present invention therefore relates to derivatives corresponding to formula (1)

in which A and B each independently of one another represent a substituent chosen from the group consisting of cytosine, uracil and thymine,

Ri represents a methyl, 2-cyanoethyl group,

2-silylethyl or allyl, and

Lev represents a levulinyl group.

In a preferred embodiment of the invention,

A and B each represent a thymine and the derivative corresponds to the O-cyanoethyl phosphate of 5'-O-levulinylthymidilyl (3 '-5') thymidine.

The present invention also relates to a process for the preparation of the compounds of formula (2)

in which

C represents a substituent chosen from

and (B) in which Xi, X ₂ , R ₅ and R ₆ are defined as illustrated in the following table:

R 1 represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and - Lev represents a levulinyl group, by irradiation of a compound of formula (1).

The compounds of formula (2a), in which C represents a substituent (a) as defined above, - Ri represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and Lev represents a levulinyl group; and the compounds of formula (2b) in which

C represents a substituent (b) as defined above,

R 1 represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and Lev represents a levulinyl group, also form part of the invention. The compounds of formula (2b) can be in cis-syn, trans-syn 1 or trans-syn 2 form.

In a preferred embodiment of the invention, C represents a substituent (bl)

(bl) The present invention also relates to compounds of formula (3)

in which

C represents a substituent chosen from (a) and (b) as defined above,

Ri represents a methyl, 2-cyanoethyl group,

2-silylethyl or allyl, and

R ₂ represents a group (X ₃ ) or (X ₄ )

χ _{3 Λ} where R ₃ represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and R ₄ represents a substituent chosen from the groups -NR'R '', morpholino, N-pyrrolidinyl and 2, 2, 6, 6, -tetramethyl-N-piperidyl, R 'and R''each being a (Cι-C) alkyl group, and

Lev represents a levulinyl group, in particular the compound of formula

The compounds of formula (3) can be prepared by a process comprising the following steps: irradiation of the compound of formula (1) to obtain a compound of formula (2), reaction of the compound of formula (2) with a compound of formula ( 4)

(4a) (4b) in which

R ₃ represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and

R ₄ represents a group chosen from the groups -NR'R '', morpholino, N-pyrrolidinyl and 2,2,6,6, -tetramethyl-N-piperidyl, R 'and R "each being a group (Cι-C ₄ ) alkyl to obtain the compound of formula (3).

To obtain a compound of formula (2a), the compound of formula (1) can be irradiated with a UV radiation at 254 nm in an aqueous acetonitrile solution, at a temperature between 0 and 20 ° C.

For the preparation of the compounds of formula (2b), irradiation can be carried out under conventional conditions known to a person skilled in the art, in particular at a wavelength greater than 290 nm in an aqueous solution of acetonitrile in the presence of 'acetone.

The preparation of the compounds of formula (3) from the compounds of formula (2) and of formula (4) is carried out by methods known to those skilled in the art.

The compounds of formula (3) can be used for the preparation of DNA containing photochemical lesions of formula (a) and (b) using a DNA synthesizer according to conventional methods known to those skilled in the art.

The following examples illustrate the invention.

Example 1: Synthesis of O-cyanoethyl phosphate 5 '-O- levulinyl-P- (2-cyanoethyl) -T (cis-syn) T-3' - (2-cyanoethyl) - N, N-diisopropylphosphoramidite:

1.1. Synthesis of O-cyanoethyl phosphate of δ'-O-levulinylthymidilyl (3 '-5') thymidine.

To a solution of 3'-O _r (4,4'-dimethoxytrityl) thymidine prepared according to the method described by Wagner T. et al., Nucleosides and Nucleotides (1997), 16, 1657-1660 (168 mg; 0.31 mmol) in anhydrous acetonitrile (20 ml) are added by cannulation, a solution of tetrazole (71 mg; 1.023 mmol; 3.3 eq) in anhydrous acetonitrile (10 ml) then a solution of 5 '-O- levulinylthymidine-3 '- (2-cyanoethyl) -N, N-diisopropylphosphoramidite (Che.mGe.nes Corp., Ashland, MA, USA) (250 mg; 0.46 mmol; 1.5 eq) in anhydrous acetonitrile (20 ml). The reaction mixture, placed under an argon atmosphere, is stirred at room temperature for 30 minutes. A 0.36 M iodine solution (2.5 ml) in the tetrahydrofuran / water / lutidine mixture (50/25/25) is then added. After 20 minutes of stirring, the solution is concentrated under vacuum at room temperature and taken up in dichloromethane then a saturated solution of sodium thiosulfate is added dropwise until discoloration of the organic phase. After adding water until the two phases are equalized, the organic phase is washed and then dried over anhydrous sodium sulfate and concentrated to dryness. The residue obtained is taken up in 2.5 ml of a 3% solution of trifluoroacetic acid in anhydrous dichloromethane and the solution is stirred for 10 minutes. After three successive coevaporations with methanol, the residue is purified by chromatography on silica gel. Elution is carried out with an increasing gradient of methanol (0-10%) in dichloromethane. The expected compound is obtained in the form of a white foam with an overall yield of 87% (194 mg; 0.27 mmol).

¹ E NMR (δ, pp; J, Hz) (CD ₃ OD, 300 MHz)

7.53 and 7.51 (1H, si, H6 Tp ^a ); 7.47 (1H, s, H6 pT ^a ); 6.26 (1H, t, J = 6.8, H1 'pT); 6.23 (1H, t, J = 7.0, Hl 'Tp); 5.12 (1H, m, H3 'Tp); 4.49-4.25 (8H, m, H3 'pT, H4' Tp ^b , H5'H5 "pT, H5'H5" Tp, OCH ₂ CH ₂ CN); 4.08 (1H, s, H4 'pT ^b ); 2.93 (2H, m, OCH ₂ CH ₂ CN); 2.82 (2H, m, OCOCH ₂ CH ₂ COMe); 2.70-2.80 (3H, m, OCOCH ₂ CH ₂ COMe, H2'pT); 2.47 (1H, m, H2 "Tp ^c ); 2.29 (2H, m, H2'H2"pT); 2.16 (3H, s, OCOCH ₂ CH ₂ COMe); 1.89 (3H, s, Me pT ^d ); 1.88 (3H, s, Me Tp ^d ). ¹³ C NMR (δ, ppm) (CD ₃ OD, 75 MHz)

209.6 (OCOCH ₂ CH ₂ COMe); 174.3 (OCOCH ₂ CH ₂ COMe); 166.4 (C4 Tp, C4 pT); 152.4 (C2 Tp ^a ); 152.3 (C2 pT ^a ); 138.0 (C6 Tp *); 137.6 (C6 pT ^b ); 118.0 (CN); 112.2 (C5 Tp, C5 pT); 86.7 (Cl 'Tp, Cl'pT); 86.1 (C4 'pT ^c ); 84.2 (C4 'Tp ^c ); 80.1 (C3 'Tp); 71.8 (C3 'pT); 69.5 (C5 'pT); 64.7 (C5'Tp, OCH ₂ CH ₂ CN); 40.5 (C2 'pT); 39.1 (C2 'Tp); 38.8 (COCH ₂ CH ₂ COMe); 29.9 (OCOCH ₂ CH ₂ COMe) 28.9 (OCOCH ₂ CH ₂ COMe); 20.4 (OCH ₂ CH ₂ CN); 12.7 (CH ₃ pT, CH ₃ Tp). ³¹ P NMR (δ, ppm): -1.65 (CD ₃ OD, 121 MHz) SM HR (FAB): (M + H) ⁺ calculated for C ₂₈ H ₃₇ Oι ₄ N ₅ P 698,2074, found 698,2092.

1.2. Synthesis of

A 0.7 mM solution of the compound obtained in the previous step (400 ml) in a water / acetonitrile mixture (70:30) is placed in a 500 ml pyrex reactor. After bubbling argon for 30 minutes, 20 ml of acetone are added. Ultraviolet irradiation is carried out using a high-pressure mercury lamp (Hanau Q81; 80 watts) fitted with a refrigerated pyrex envelope, and positioned inside the reactor. The duration of irradiation is 4 hours. The photolysate is then concentrated and then purified by high performance liquid chromatography (HPLC) in reverse phase.

Preparatory column: Waters Preppak Cartrige, bondapak

C18, 125Â (15-20 μ), 47x300 mm.

Conditions: Detection: 230 nm, Flow rate: 50 ml / minutes Gradient table

The fraction of retention time 54 minutes, obtained with a yield of 30%, corresponds to the expected compound. It consists of an equimolecular mixture of the two diastereoisomers of the Cis-Syn isomer.

¹ H NMR (δ, ppm; J, Hz) (D ₂ 0, 400 MHz)

6.25 (0.5H, t, J = 6.5, H1 'pT); 6.06 (0.5H, m, H1 'pT); 5.74 (0.5H, dd, J ≈ 10.4; 4.2 Hl 'Tp); 5.38 (0.5H, dd, J = 10.2; 4.2, Hl 'Tp), 5.11; 5.07 (1H, si, H3 'Tp); 1.61 (3H, s, CH ₃ Tp *); 1.54 (3H, s, CH ₃ pT *).

31 P NMR (δ, ppm) -4.02, -4.52 (D ₂ 0, 243 MHz)

SM HR (FAB): (M + H) ⁺ calculated for C ₂₈ H ₃₇ 0ι ₄ N ₅ P 698,2074, found 698,2076.

The 54-minute retention time fraction is treated with a 32% aqueous ammonia solution overnight at room temperature. Analysis of the NMR of the crude reaction ^X H spectrum shows the presence of a single compound. This is identified with the Cis-Syn cyclobutane dimer of the thymine dinucleotide of formula

by comparison with the proton NMR data from the literature ((a) Kemmink J., Boelens R., Kaptein R., "Eur. Biophys. J" (1987), 14, 293-299. (b) Koning TMG , Van Soest JJG, Kaptein R., "Eur. J. Biochem." (1991), 195, 29-40). The compound obtained effectively corresponds to the diastereoisomeric mixture of the Cis-Syn isomer.

^X H NMR (δ, ppm; J, Hz) (D ₂ 0, 300 MHz)

5.98 (1H, dd, J = 5.7 and 8.8, Hl'pT); 5.66 (1H, dd, J = 5.3; 8.6, Hl'Tp); 4.65 (1H, m, H3 'Tp); 4.33 (2H, m, H6 pT, H3 'pT); 4.25 (1H, d, J = 5.9, H6 Tp); 4.19-3.89 (4H, m, H4 'Tp, H4' pT, H5'H5 "pT); 3.70 (2H, m, H5'H5" Tp); 2.61 (1H, ddd, J = 13.8; 8.6; 5.3, H2 'Tp); 2.43-2.25 (2H, m, H2 "Tp, H2 'pT); 2.09 (1H, ddd, J = 13.6; 5.7; 3.5 H2"

1.3. Synthesis of 5 '-O-levulinyl-P- (2-cyanoethyl) - (cis-syn) T-3' - (2-cyanoethyl) -N, N-diisopropylphosphoramidite.

The photoproduct obtained in the previous step (100 mg, 0.14 mmol), dried overnight under vacuum in a desiccator containing phosphorus pentoxide, is taken up in a mixture of tetrahydrofuran / acetonitrile

(1/1) anhydrous (2ml). After adding the diisopropylethylamine (100 μl, 0.57 mmol, 4 eq), the reaction is stirred for 30 minutes at room temperature, under an argon atmosphere. The chloro- (2-cyanoethyl) -bis- N, N'-diisopropylphosphoramidite (64 μl, 0.28 mmol, 2 eq) is added and the reaction stirred for 10 minutes. The reaction mixture is then concentrated and then taken up in dichloromethane and a saturated solution of sodium bicarbonate. After washing, the organic phase is dried over anhydrous sodium sulfate and then evaporated. The residue obtained is purified by chromatography on silica gel (particle size 6-35 microns) with a gradient of acetone in ethyl acetate (0-40%). The expected compound is obtained in the form of a translucent lacquer with a yield of 58% (75 mg). RM ³¹ P (δ, ppm): 149.78; 149.63; -4.06 (CD ₃ CΝ, 243 MHz)

Example 2: Synthesis of 10 and 32 mer oligonucleotides containing the cyclobutane cis-syn adduct of thymine

The compound of Example 1 (es) was used to synthesize two oligonucleotides 10 and 32 mothers of sequence:

10th mother: CGCAT [es] TACGC

32 mother: GATCTCGGCGACATCGGT [es] TCCGTCCTAACTCG

2.1. Synthesis on solid support.

The syntheses were carried out on an Applied Biosystems DNA synthesizer, 392 DNA / RNA Synthesizer on a micromolar scale.

Standard phosphoramidites were used. The phosphoramidite of Example 1 is used at a concentration of 0.1 M in a tetrahydrofuran / acetonitrile mixture (1/3).

A modified cycle was used during the coupling of this phosphoramidite. The modifications compared to the standard cycle include: an extended coupling time to 20 minutes, a double styling (capping sleeve), and an additional rinsing, - elimination of the detritylation step replaced by deprotection of the levulinyl group with a solution of 0.5 M hydrazine monohydrate in a pyridine / acetic acid mixture (3/2, v / v) in two passages of 5 minutes followed by five washes of 30 seconds with 1 acetonitrile.

The coupling yields of the phosphoramidite of Example 1 were evaluated by the conductimetric measurement of the Dmt ⁺ cation released for subsequent couplings compared to couplings preceding the incorporation of said phosphoramidite. In the case of the 10 mer, the coupling efficiency for the phosphoramidite used (position 5) and the following one (A6) compared to that preceding the modified phosphoramidite (A4) is of the order of 87%. In the case of the 32 mother, the coupling yield for the phosphoramidite used (position 14), the preceding one (C13) and the two following ones (G15 and G16) with respect to C12 is of the order of 91%.

2.2. Deprotection and purification. 2.2.1. Deprotection of nucleic bases, phosphate triester and release of the support The oligonucleotides are deprotected and "unhooked" from the support by the action of a 32% aqueous ammonia solution for 24 h at room temperature, protected from light. After evaporation to dryness, the tritylated oligonucleotides are purified by HPLC in reverse phase polarity.

Semi-preparative column: Aters Prepak Delta-Pak C18 25 x 100 mm (15 μm, 300 A).

Conditions: Detection at 260 nm, Flow rate 3 ml / minutes

Gradient table

32 mother retention time: 41 minutes.

2. 2.2. Detritylation of oligonucleotides.

The tritylated oligonucleotides are detritylated by treatment with an aqueous solution of 40% acetic acid for 2 hours then purified by HPLC in reverse phase polarity. Analytical column: Waters Delta-Pak C18 3.9 x 150 mm

(5μm, 300 Â).

Conditions: Detection at 260 nm, Flow rate 1 ml / minute

Gradient table

Eluent

Time TEA CH ₃ CN acetate (minutes) 0.1 M pH 7

Initial 95

30 60 40

Retention time of the 10th mother: 12.8 minutes. 32 mother retention time: 11.9 minutes. 2.2.3. Characterization .

MALDI mass spectrometry analysis (positive mode) of oligonucleotides 10 and 32 mothers is as follows: 10 mother Mass (M + H) ⁺ calculated 2988.005, measured

2988.7 +/- 0.5u

32 dam Mass (M + H) ⁺ calculated 9767.371, measured 9764.3 +/- 5u. Thus, the use of derivatives of formula (1) according to the invention makes it possible to quickly and economically obtain adducts of formula (3) which can be incorporated into oligonucleotides suitable for use by biophysicists and physicochemists for structural studies, by biochemists and biologists for studies in the field of DNA repair, transcription and its control, in the immune field and in the field of signal transduction following exposure UV radiation.

Claims

1. Derivative corresponding to formula (1)

in which

A and B each independently of one another represent a substituent chosen from the group consisting of cytosine, uracil and thymine,

Ri represents a methyl, 2-cyanoethyl group,

2-silylethyl or allyl, and

Lev represents a levulinyl group.

2. A derivative according to claim 1, for which A and B each represent a thymine and which corresponds to the O-cyanoethyl phosphate of 5 '-O-levulinylthymidilyl (3' - 5 ') thymidine.

3. Process for the preparation of the compounds of formula (2)

in which C represents a substituent chosen from

in which Xi, X ₂ , R ₅ and Rε are defined as illustrated in the following table:

R 1 represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and

Lev represents a levulinyl group by irradiation of a compound of formula (1) according to any one of claims 1 and 2.

4. Compounds of formula (2a) in which

C represents a substituent (a) as defined in claim 3,

Ri represents a methyl, 2-cyanoethyl group,

2-silylethyl or allyl, and - Lev represents a levulinyl group.

Compounds of formula (2b) in which C represents a substituent (b) as defined in claim 3,

R 1 represents a methyl, 2-cyanoethyl 2-silylethyl or allyl group, and Lev represents a levulinyl group in cis-syn, trans-syn 1 or trans-syn 2 form.

6. Compounds according to claim 5 in which C represents a substituent (bl)

7. Compounds of formula (3)

in which

C represents a substituent chosen from (a) and (b) as defined in claim 3,

Ri represents a methyl, 2-cyanoethyl group,

2-silylethyl or allyl, and

R ₂ represents a group (X ₃ ) or (X ₄ )

X4 where R ₃ represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and R ₄ represents a substituent chosen from the groups -NR'R '', morpholino, N-pyrrolidinyl and 2, 2, 6, 6, - tetramethyl-N-piperidyl, R 'and R''each being a group (Cι ~ C ₄ ) alkyl, and Lev represents a levulinyl group.

8. Compounds according to claim 7 corresponding to the formula

9. A method of preparing the compounds according to any one of claims 7 and 8, comprising the following steps: irradiation of the compound of formula (1) to obtain a compound of formula (2), and reaction of the compound of formula (2) with a compound of formula (4)

(4a) 4b) in which R ₃ represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and R ₄ represents a group chosen from the groups -NR 'R'', morpholino, N-pyrrolidinyl and 2, 2, 6, 6, -tetramethyl-N-piperidyl, R 'and R''each being a (Cι-C ₄ ) alkyl group to obtain the compound of formula (3).

10. Use of the compounds of formula (3) according to any one of claims 7 and 8 for the preparation of oligonucleotides.