FR2836145A1 - New levulinyl phosphoramidite derivatives used for synthesis of oligonucleotides - Google Patents

Abstract

Levulinyl phosphoramidite derivatives (I) are new. Levulinyl phosphoramidite derivatives of formula (I) are new. A, B' = cytosine, uracil or thymine; R1 = methyl, 2-cyanoethyl, 2-silylethyl or allyl, and Lev = levulinyl. Independent claims are also are included for: (1) preparation of compounds of formula (II) by irradiating (I), and (2) new compounds of formula (III), and (3) preparation of (III). Q = a group of formula (i) or (ii); X1, X2 = O, and R5, R6 = H and/or Me, or one of X1 and X2 = NH and the other is O, and R5, R6 = H, or one of them is H and the other is Me, or X1, X2 = O, and R5, R6 = Me; R2 = P(=O)(H)OH or P(OR3)R4; R3 = methyl, 2-cyanoethyl, 2-silylethyl or allyl; R4 = NR'R, morpholino, N-pyrrolidinyl or 2,2,6,6-tetramethyl-N-piperidinyl, and R', R = 1-4C alkyl.

Description

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 The present invention relates to phosphoramidite precursors, to a process for their preparation, to the phosphoramidites obtained and to their use in the synthesis of oligonucleotides that specifically contain photochemical lesions in a defined position.

 The exposure of cellular DNA to ultraviolet light causes lesions that form at the level of two adjacent pyrimidines and which are translated at the molecular level by obtaining cyclobutanic dimers and pyrimidine-type adducts (6-4 pyrimidinone). ) that can be isomerized to Dewar adducts.

 In the absence of repair and during the replication of the damaged DNA, these lesions can lead to errors in reading the polymerases and therefore be mutagenic.

 They can also induce an immunosuppressive response that plays an important role in the development of the carcinogenesis process. These lesions are responsible in humans for most skin cancers. This represents in the world today a major problem of public health.

 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 other uncontrollable impacts on other living organisms that could dramatically alter the balance of the ozone layer. many ecosystems.

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 This is why the inventors are interested in synthetic compounds specifically containing this type of lesions in a defined position and which make it possible to study this damage.

 Currently, there are three phosphoramidite synthesis routes which make it possible to obtain, on a solid support, oligonucleotides containing specifically and in a defined position the cis-syn cyclobutanic adduct of thymine (Taylor J. S, Brockie IR, O). A CLASS BLOCK FOR THE SEQUENCE-SPECIFIC INTRODUCTION OF cis-syn thymine various into oligonucleotides, Solidphase 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 substrate for T4 endonuclease V containing a phosphorodithioate linkage at the thymine dimer site", Nucleic Acids Res. (1990), 18, 7279-7286; Kosmoski, J.V., Smerdon M.J., "Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site", Biochemistry (1999), 38, 9485-9494; Tommasi S., Swiderski P.M., Tu Y, Kaplan B.E., Pfeifer G.P., "Inhibition of various transcription factor binding by ultraviolet-induced pyrimidine", Biochemistry (1996), 35, 15693-15703).

 The syntheses leading to these phopshoramidites all follow the strategy initially developed by Taylor J. S and is 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

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 Phosphate protecting agent which may be methyl or cyanoethyl, the 3 'hydroxyl function being protected either in the form of silyl ether or in the form of levulinic acid ester.

Figure 1 of these summaries is as follows:

Diagram 1
Photolysis of 8 leads to CisSyn 9 thymine dimer with a yield of 41% for 9a and 42% for 9b. The hydroxyl function at 5 'of compound 9 is protected by the dimethoxytrityl group (step i, yield: 60% and 76%), then the hydroxyl function at 3 is deprotected (step ii, yield: 73% and 78%) and functionalized to phosphoramidite (step iii, yield: 53% and 54%).

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 The order of protection steps in 5 'and deprotection in 3' can be reversed (step iv and i).

Compound 8 is obtained according to the routes illustrated in Scheme 2. By means of the phosphoramidites (8a-b) in the 3'-> 5 'direction, a dimethoxytrityl derivative is condensed in 5' and phosphoramidite in 3 'of thymidine (10a-b) and a 3'-protected derivative of thymidine (11a described by Taylor J. S, 11b described by Murata T.); Chlorophosphites (8a) in the 5'-> 3 'direction are condensed to the 5'-trityl derivative of thymidine (10c) with the 5'-chlorophosphite derivative of the 3' protected thymidine (IIc described by Kosmoski JV). The phosphite triester is then oxidized to phosphate triester and the 5 'hydroxyl function detritylated to yield 8a.

Figure 2
The major disadvantage of this general route of synthesis, related to the fact that the dimethoxytrityl group

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 is not compatible with the photochemical irradiation step, is to deprotect the 5 'hydroxyl function of the precursor of 8 (Scheme 2, step iii and II) and then to reprotect this function at 9 to obtain phosphoramidite 7 (Diagram 1, step i). These different stages result in 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.

dans laquelle Rs représente un groupe méthyle ou cyanoéthyle et R7 représente un groupe lévulinyle, ou tert-butyldiméthylsilyle. Patent Application EP 0 787 740 describes a process for preparing a photoproduct (6-4) from a thymidine dimer of formula (II)

wherein Rs is methyl or cyanoethyl and R7 is levulinyl, or tert-butyldimethylsilyl.

une étape d'irradiation du composé de formule II, une étape de protection du groupe 5'OH, - une étape de déprotection du groupe 3'OH, puis - la réaction du produit obtenu avec, par exemple, un dérivé phosphochloré, pour obtenir le phosphoramidite attendu de formule (I) This preparation process comprises:

a step of irradiation of the compound of formula II, a step of protecting the 5'OH group, a step of deprotection of the 3'OH group, then the reaction of the product obtained with, for example, a phosphochlorine derivative, to obtain the expected phosphoramidite of formula (I)

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Although this method overcomes the disadvantages of the known methods of the prior art, it still comprises four steps, including several successive protection and deprotection steps that affect the good performance of the process.

 However, the inventors have discovered that, if the hydroxyl at the 5 'position was 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 at the irradiation conditions and compatible with the solid support synthesis conditions.

dans laquelle The subject of the present invention is therefore derivatives having the formula (1)

in which

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A and B are each independently of one another a substituent selected from the group consisting of cytosine, uracil and thymine,
R 1 represents a methyl, 2-cyanoethyl group,
2-silylethyl or allyl, and
Lev represents a levulinyl group.

correspond au O-cyanoéthyle phosphate de la 5'-0lévulinylthymidilyl (3'-5') thymidine.
La présente invention a également pour objet un procédé de préparation des composés de formule (2)

dans laquelle - C représente un substituant choisi parmi

et

In a preferred embodiment of the invention, A and B each represent a thymine and the derivative

corresponds to O-cyanoethyl phosphate of 5'-levulinylthymidilyl (3'-5 ') thymidine.
The subject of the present invention is also a process for the preparation of the compounds of formula (2)

in which - C represents a substituent chosen from

and

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in which X 1, X 5 R 5 and R 6 are defined as illustrated in the following table:

<Tb>
<tb> X1 <SEP> X2 <SEP> R5 <SEP> R6
<tb> O <SEP> O <SEP> H <SEP> H
<tb> 0 <SEP> 0 <SEP> CH3 <SEP> H
<tb> 0 <SEP> 0 <SEP> H <SEP> CH3
<tb> O <SEP> NH <SEP> H <SEP> H
<tb> NH <SEP> 0 <SEP> H <SEP> H
<tb> 0 <SEP> 0 <SEP> CH3 <SEP> CH3
<tb> 0 <SEP> NH <SEP> CH3 <SEP> H
<tb> NH <SEP> 0 <SEP> H <SEP> CH3
<tb> NH <SEP> NH <SEP> H <SEP> H
<Tb>

R 1 represents a methyl, 2-cyanoethyl group,
2-silylethyl or allyl, 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,
R 1 represents a methyl, 2-cyanoethyl group,
2-silylethyl or allyl, and
Lev is a levulinyl group; and compounds of formula (2b) in which - C represents a substituent (b) as defined above,
R 1 represents a methyl, 2-cyanoethyl group,
2-silylethyl or allyl, and
Lev represents a levulinyl group, are also 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 (b1)

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dans laquelle - C représente un substituant choisi parmi (a) et (b) tels que définis précédemment, Ri représente un groupe méthyle, 2-cyanoéthyle,
2-silyléthyle ou allyle, et - R2 représente un groupe (X3) ou (X4)'

où R3 représente un groupe méthyle, 2-cyanoéthyle, 2-silyléthyle ou allyle, et R4 représente un substituant

choisi parmi les groupes-NR'R, morpholino, N-pyrrolidinyle et 2, 2, 6, 6, -tétraméthyl-N-pipéridyle, R' et R" étant chacun un groupe (CI-C4) alkyle, et
Lev représente un groupe lévulinyle, notamment le composé de formule The subject of the present invention is also compounds of formula (3)

in which - C represents a substituent chosen from (a) and (b) as defined above, R 1 represents a methyl, 2-cyanoethyl group,
2-silylethyl or allyl, and - R2 represents a group (X3) or (X4)

where R3 is methyl, 2-cyanoethyl, 2-silylethyl or allyl, and R4 is a substituent

selected from the groups -NR'R, morpholino, N-pyrrolidinyl and 2,2,6,6-tetramethyl-N-piperidyl, R 'and R "each being a (C 1 -C 4) alkyl group, and
Lev represents a levulinyl group, especially the compound of formula

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dans laquelle - R3 représente un groupe méthyle, 2-cyanoéthyle,
2-silyléthyle ou allyle, et - R4 représente un groupe choisi parmi les groupes

- NR'R", morpholino, N-pyrrolidinyle et 2, 2, 6, 6,-tétraméthyl-N-pipéridyle, R'et R"étant chacun un groupe (CI-C4) alkyle pour obtenir le composé de formule (3). The compounds of formula (3) may 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)

in which - R3 represents a methyl, 2-cyanoethyl group,
2-silylethyl or allyl, and - R4 represents a group selected from groups

- NR'R ", morpholino, N-pyrrolidinyl and 2,2,6,6-tetramethyl-N-piperidyl, R 'and R" each being a (C 1 -C 4) alkyl group to obtain the compound of formula (3 ).

 To obtain a compound of formula (2a), it is possible to irradiate the compound of formula (1) with a

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 UV radiation at 254 nm in an aqueous solution of acetonitrile, at a temperature between 0 and 20 C.

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

 The preparation of the compounds of formula (3) from the compounds of formula (2) and 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 5'-O-levulinylthymidilyl (3'-5 ') thymidine.

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 To a solution of 3'-O- (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) and then a solution of 5'-O-levulinylthymidine -3'- (2-cyanoethyl) -N, N-diisopropylphosphoramidite (ChemGenes 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 solution of 0.36 M iodine (2.5 ml) in tetrahydrofuran / water / lutidine (50/25/25) is then added. After stirring for 20 minutes, the solution is concentrated under vacuum at room temperature and taken up in dichloromethane and then a saturated solution of sodium thiosulfate is added dropwise until the organic phase is decolorized. After addition of water until the two phases are equalized, the organic phase

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is washed and then dried over anhydrous sodium sulphate 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 by 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).

209, 6 (OCOCH2CH2COMe) ; 174, 3 (OCOCHCOMe) ; 166, 4 (C4 Tp, C4 pT) ; 152, 4 (C2 Tpa) ; 152, 3 (C2 pTa) 138, 0 (C6 Tpb) ; 137, 6 (C6 pTb) ; 118, 0 (CN) ; 112, 2 (C5 Tp, C5 pT) ; 86, 7 (C1'Tp, C1'pT) ; 86, 1 (C4'pTc) ; 84, 2 (C4'Tpc) ; 80,1 (C3'Tp) ; 71,8 (C3'pT) ; 69,5 (C5'pT) ; 64,7 (C5'Tp, OCH2CH2CN) ; 40,5 (C2'pT) ; 39,1 (C2'Tp) ; 38,8 (COCH2CH2COMe) ; 29,9 (OCOCH2CH2COMe) 28,9 (OCOCH2CH2COMe) ; 20,4 (OCH2CH2CN) ; 12,7 (CH3 pT, CH3 Tp). 1 H NMR (0, ppm, J, Hz) (CD 30 D, 300 MHz) δ, 53 and 7.11 (1H, ss, H 6 Tpa); 7, 47 (1H, s, H6 pTa); 6, 26 (1H, t, J = 6.8, H1'pT); 6, 23 (1H, t, J = 7.0, H1'Tp); 5, 12 (1H, m, H3'Tp); 4.49-4.4 (8H, m, H3'pT, H4'Tpb, H5'H5 "pT, H5'H5" Tp, OCH2 CH2CN); 4. 08 (1H, s, H4'pTb); 2.93 (2H, m, OCH2CH2CN); 2.82 (2H, m, OCOCHsCHsCOMe); 2.70-2.80 (3H, m, OCOCH2CH2COMe, H2'pTc); 2.47 (1H, m, H2 "Top"); 2.29 (2H, m, H2'H2 "pT); 2.16 (3H, s, OCOCH2CH2COMe); 1.89 (3H, s, Me pTd); 1.88 (3H, s, Me Tpd). 13C NMR (#, ppm) (CD30D, 75 MHz)

209, 6 (OCOCH2CH2COMe); 174, 3 (OCOCHCOMe); 166.4 (C4 Tp, C4 pT); 152, 4 (C2 Tpa); 152, 3 (C2 pTa) 138.0 (C6 Tbp); 137.6 (C6 pTb); 118.0 (CN); 112, 2 (C5 Tp, C5 pT); 86, 7 (C1'Tp, C1'pT); 86.1 (C4'pTc); 84.2 (C4'Tpc); 80.1 (C3'Tp); 71.8 (C3'pT); 69.5 (C5'pT); 64.7 (C5'Tp, OCH2CH2CN); 40.5 (C2'pT); 39.1 (C2'Tp); 38.8 (COCH2CH2COMe); 29.9 (OCOCH2CH2COMe) 28.9 (OCOCH2CH2COMe); 20.4 (OCH 2 CH 2 CN); 12.7 (CH3 pT, CH3 Tp).

NMR 31p (0, ppm): -1.65 (CD30D, 121 MHz)

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 MS HR (FAB): (M + H) + calcd for C28H17014NsP 698.2074, found 698.2092.

1.2. Synthesis of

A 0.7 mM solution of the compound obtained in the preceding 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. The ultraviolet irradiation is carried out using a high-pressure mercury lamp (Hanau Q81, 80 watts) equipped with a refrigerated Pyrex envelope, and positioned inside the reactor. The duration of the irradiation is 4 hours. The photolysate is then concentrated and then purified by reverse phase high performance liquid chromatography (HPLC).

- Preparative column: Waters Preppak Cartrige, bondapak
C18, 125 (15-20 μm), 47x300 mm.

 - Conditions: Detection: 230 nm, Flow: 50 ml / minutes

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Gradient table

<Tb>
<tb> Eluent
<tb> Time <SEP> H2O / CH3CN <SEP> H2O / CH3CN
<tb> (minutes) <SEP> 90 <SEP>: <SEP> 10 <SEP> 50 <SEP>: <SEP> 50
<tb> Initial <SEP> 100 <SEP> 0
<tb> 65 <SEP> 87 <SEP> 13
<tb> 90 <SEP> 0 <SEP> 100
<Tb>

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 (O, ppm, J, Hz) (D 2 O, 400 MHz) 6.25 (0.5H, t, J = 6.5, HCl); 6.06 (0.5H, m, H, Pt); 5.74 (0.5H, dd, J = 10.4; 4.2H! Tp); 5.38 (0.5H, dd, J = 10.2, 4.2, H1'Tp), 5, 11; 5.07 (1H, ss, H3'Tp); 1.61 (3H, s, CH3 Tp *); 1, 54 (3H, s, CH3 pT *).

31 P NMR (8 ppm): -4.02; -4.52 (D2O, 243 MHz) MS HR (FAB): (M + H) + calcd for C28H37014NsP 698.2074, found 698, 2076.

 The fraction of retention time 54 minutes is treated with a 32% aqueous ammonia solution overnight at room temperature. Analysis of the 1 H NMR spectrum of the reaction crude shows the presence of a single compound. This is identified with the cyclobutanic dimer Cis-Syn of the thymine dinucleotide of formula

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par comparaison avec les données de RMN du proton de la littérature ( (a) Kemmink J., Boelens R., Kaptein R., "Eur. Biophys. J" (1987), 14,293-299. (b) Koning T. M. G., Van Soest J. J. G., Kaptein R.,"Eur. J. Biochem." (1991), 195,29-40). Le composé obtenu correspond effectivement au mélange diastéréoisomérique de l'isomère Cis-Syn.

by comparison with 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 corresponds effectively to the diastereoisomeric mixture of the Cis-Syn isomer.

1. 3. Synthèse de 5'-O-lévulinyl-P- (2-cyanoéthyl) -T (cissyn) T-3'- (2-cyanoéthyl) -N, N-diisopropylphosphoramidite. 1 H NMR (8 ppm, J, Hz) (D 2 O, 300 MHz) 5.98 (1H, dd, J = 5.7 and 8.8, HCl); 5.66 (1H, dd, J = 5.3, 8.6, H1'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); 3.10 (2H, m, H5'H5"Tp); 2.61 (1H, ddd, J = 13.8, 8.6, 5.3, H2'Tp); 2.43-2.25 (2H, m, H 2 Tp, H 2 Pt), 2.09 (1H, ddd, J = 13.6, 5.7, 3.5H 2 O.

1. 3. Synthesis of 5'-O-levulinyl-P- (2-cyanoethyl) -T (cissyn) 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 anhydrous tetrahydrofuran / acetonitrile (1/1) (2 ml). ). After adding the

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 diisopropylethylamine (100 μl, 0.57 mmol, 4 eq), the reaction is stirred for 30 minutes at room temperature under an argon atmosphere. Chloro- (2-cyanoethyl) -bis-N, N'-diisopropylphosphoramidite (64 g, 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 sulphate 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).

31 P NMR (8 ppm): 149.78; 149.63; -4.06 (CD3CN, 243 MHz) Example 2 Synthesis of 10 and 32-mer oligonucleotides containing the cis-syn cyclobutanic adduct of thymine
The compound of Example 1 (cs) was used to synthesize two oligonucleotides 10 and 32 mothers of sequence:
10 mother: CGCAT [cs] TACGC
32 mother: GATCTCGGCGACATCGGT [cs] TCCGTCCTAACTCG 2.1. Synthesis on solid support.

 The syntheses were performed on an Applied Biosystems DNA synthesizer, 392 DNA / RNA Synthesizer at the micromolar scale.

 Standard phosphoramidites have been employed.

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 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 when coupling this phosphoramidite. The modifications with respect to the standard cycle include: a coupling time extended to 20 minutes, a double capping, and an additional rinse, a suppression of the detritylation step replaced by a 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 washings of 30 seconds with acetonitrile.

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

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The oligonucleotides are deprotected and "unhooked" from the support by the action of a 32% aqueous ammonia solution for 24 hours at room temperature, protected from light. After evaporation to dryness, the tritylated oligonucleotides are purified by HPLC in reverse phase polarity.

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

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

<Tb>
<tb> Eluent
<tb> Time <SEP> Acetate <SEP> of <SEP> TEA <SEP> CH3CN
<tb> (minutes) <SEP> 0.1 <SEP> M <SEP> pH <SEP> 7
<tb> Initial <SEP> 95 <SEP> 5
<tb> 55 <SEP> 60 <SEP> 40
<Tb>
Retention time in mother: 41 minutes.

Retention time of 32 mother: 41 minutes.

 2.2. 2. Detritylation of oligonucleotides.

 The tritylated oligonucleotides are detitylated by treatment with a 40% aqueous acetic acid solution for 2 hours and then purified by HPLC in reverse phase polarity.

Analytical column: Waters Delta-Pak C18 3.9 x 150 mm (5Mm, 300 A).

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

<Tb>
<tb> Eluent
<tb> Time <SEP> Acetate <SEP> of <SEP> TEA <SEP> CH3CN
<tb> (minutes) <SEP> 0.1 <SEP> M <SEP> pH <SEP> 7
<tb> Initial <SEP> 95 <SEP> 5
<tb> 30 <SEP> 60 <SEP> 40
<Tb>
Maternal retention time: 12.8 minutes. Retention time of 32 mother: 11.9 minutes.

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2. 2. 3. Caractérisation.

2. 2. 3. Characterization.

The MALDI (positive mode) mass spectrometry analysis of the 10 and 32-mer oligonucleotides is as follows:
10 mother Mass (M + H) + calcd 2988.005, measured 2988.7 +/- 0.5u
32 dam Mass (M + H) + calcd. 9767.371, measured
9764.3 +/- 5u.

 Thus, the use of derivatives of formula (1) according to the invention makes it possible to obtain quickly and economically adducts of formula (3) which can be incorporated in 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 post-exposure signal transduction cellular UV radiation.

Claims (2)

A and B are each independently of one another a substituent selected from the group consisting of cytosine, uracil and thymine; R 1 is methyl, 2-cyanoethyl,  in which

 CLAIMS 1. Derivative corresponding to the formula (1) Lev represents a levulinyl group. 2-silylethyl or allyl, and 2. Derivative according to claim 1, wherein A and B each represents a thymine and which corresponds to the ocyanoethyl phosphate of 5'-O-levulinylthymidilyl (3'-5 ') thymidine.  wherein C represents a substituent selected from

3. Process for preparing the compounds of formula (2) <Desc / Clms Page number 22>

 in which X 1, X 5, R 5 and R 6 are defined as illustrated in the following table:

<Tb> <tb> NH <SEP> NH <SEP> H <SEP> H <tb> NH <SEP> 0 <SEP> H <SEP> CH3 <tb> 0 <SEP> NH <SEP> CH3 <SEP> H <tb> 0 <SEP> 0 <SEP> CH3 <SEP> CH3 <tb> NH <SEP> 0 <SEP> H <SEP> H <tb> 0 <SEP> NH <SEP> H <SEP> H <tb> 0 <SEP> 0 <SEP> H <SEP> CH3 <tb> 0 <SEP> 0 <SEP> CH3 <SEP> H <tb> 0 <SEP> 0 <SEP> H <SEP> H <tb> Xi <SEP> X2 <SEP> Rs <SEP> R6 <Tb>  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) wherein - C represents a substituent (a) as defined in claim 3, R1 represents a methyl, 2-cyanoethyl, 2-silylethyl or allyl group, and Lev represents a levulinyl group. 5. Compounds of formula (2b) in which <Desc / Clms Page number 23>  C represents a substituent (b) as defined in claim 3, R 1 represents a methyl, 2-cyanoethyl group; Lev represents a levulinyl group in cis-syn, trans-syn 1 or trans-syn 2 form. 2-silylethyl or allyl, and R 1 represents a methyl, 2-cyanoethyl group,  in which - C represents a substituent chosen from (a) and (b) as defined in claim 3,

 7. Compounds of formula (3)

Compounds according to claim 5 wherein C represents a substituent (b1) 2-silylethyl or allyl, and - ruz represents a group (X3) or (X4)

<Desc / Clms Page number 24>  where R3 is methyl, 2-cyanoethyl, 2silylethyl or allyl, and R4 is a substituent selected from the groups -NR'R ', morpholino, Npyrrolidinyl and 2,2,6,6-tetramethyl-N-piperidyl, R and R ', each being a (C 1 -C 4) alkyl group, and Lev is a levulinyl group.

 9. Process for the preparation of 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)

Compounds according to claim 7 having the formula <Desc / Clms Page number 25>  a group selected from the groups-NR'R ', morpholino, N-pyrrolidinyl and 2,2,6,6-tetramethyl-N-piperidyl, R' and R "each being a (C1-C4) alkyl group to obtain the compound of formula (3).

 (4a) (4b) wherein R3 is methyl, 2-cyanoethyl, 2-silylethyl or allyl, and R4 is 10. Use of the compounds of formula (3) according to any one of claims 7 and 8 for the preparation of oligonucleotides.