MXPA98000339A - In situ preparation of nucleosid fosforamiditas and synthesis of oligonucleot - Google Patents

Abstract

The invention provides novel bifunctional phosphorylating reagents and their in situ application of 5'-protected nucleoside phosphoro-mids and oligonucleotide synthesis, the bifunctional phosphorylating reagents according to the invention react rapidly with nucleosides under neutral or slightly basic conditions, without an additional activation step; moreover, the bifunctional phosphorylating reagents according to the invention chemoselectively generate the corresponding phosphoramidite nucleosides in situ without the need to purify the nucleoside phosphoramidites before using them in the oligonucleotide synthesis, finally, the bifunctional phosphorylating reagents according to the invention are relatively stable and easy to manage

Description

IN SITU PREPARATION OF NUCLEOSID PHOSPHORAMIDITES AND SYNTHESIS OF OLIGONUCLEOTIDE The present is a continuation in part of the US application Serial No. 08 / 539,939, filed on October 6, 1995.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in said synthesis.

BRIEF DESCRIPTION OF THE RELATED ART Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR for inhibiting the opposite direction of gene expression. This widespread use of oligonucleotides has led to an increasing demand for fast, low cost and efficient methods for synthesizing oligonucleotides. The synthesis of oligonucleotides for applications of opposite direction and diagnosis can now be achieved routinely. See, for example, Methods in Molecular Biology, Volume 20: Protocols for Oligonucleotides and Analogs, pages 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: ft Practical Approach, pages 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra, Agrawal and Iyer, Curr. Op. In Biotech .. 6: 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). The first synthetic approaches included phosphodiester chemistry and phosphotriester chemistry. Khorana and coauthors, J. Molec. Biol. 72: 209 (1972) describes the chemistry of phosphodiester for the synthesis of oligonucleotides. Reese, Tetrahedron Lett .. 34: 3143-3179 (1978), describes the chemistry of the phosphotriester for the synthesis of oligonucleotides and polynucleotides. These primitive approaches have yielded, to a large extent, to the more efficient phosphoramidite and H-phosphonate approaches for synthesis. Of them the phosphoramidite approach has become the most popular for most applications. Beaucage and Cruthers, Tetrahedron Lett. 22: 1859-1862 (1981), describe the use of deoxynucleoside phosphoramidites in the synthesis of the polynucleotide. The phosphoramidite approach has been used to synthesize oligonucleotides having a variety of modified internucleoside linkages. Agra al and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987) teach the synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly and co-authors, Biochemistry 23: 3443 (1984), describe the synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager and co-authors, Biochemistry. 27: 7237 (1988) describe the synthesis of oligonucleotide phosphoramidates, using the chemistry of phosphoramidite. The solid phase synthesis of oligonucleotides by the phosphoramidite approach can vary for different applications; but ordinarily it implies the same generalized protocol. Briefly, this approach comprises anchoring the nucleoside more 3 'to a solid support, functionalized with amino and / or hydroxyl portions, and subsequently adding the additional nucleosides step by step. The desired internucleoside ligations are formed between the 3'-phosphoramidite group of the arriving nucleoside and the 5'-hydroxyl group of the nucleoside more 5 'of the nascent oligonucleotide, attached to the support. It is still necessary to refine the methodologies; Nevertheless; particularly when a transition to large-scale synthesis is formed (10 μmol to 1 mmol and more). See Padmapriya and coauthors, Antisense Res. Dev., 4: 185 (1994). Several modifications of normal phosphoramidite methods have already been reported to facilitate the synthesis and isolation of oligonucleotides. See, for example, Padmapriya and coauthors, supra; Raviku ar and coauthors, Tetrahedron 50: 9255 (1994); Theisen and co-authors, Nucleosides and Nucleotides, 12: 43 (1994); and Iyer and co-authors, Nucleosides &; Nucleotides 14: 1349 (1995) (Kuijpers and co-authors, Nucí Acids Res. 18: 5197 (1990); and Reddy and co-authors, Tetrahedron Lett .. 35: 4311 (1994) .A major limiting factor for the synthesis of oligonucleotides a Efficient cost is the time and cost necessary to form and purify the monomeric nucleoside phosphoramidites Bodepudi and coauthors, Chem., Res. Toxicol .. 5: 608-617, describe that the preparation of phosphoramidites from 2'-deoxy -7,8-dihydro-8-oxoguanosine and 2'-deoxy-7,8-dihydro-8-oxoadenosine, according to the normal procedure, results in the extensive decomposition of the phosphoramidites during purification, due to their instability and its sensitivity to water.A potential approach to solving these problems is to generate the phosphoramidite in situ, as the oligonucleotide synthesis procedure is carried out.

Unfortunately, the numerous attempts to achieve this approach have been reasoned desire res. Moore and Beaucage, J. or rg. Chem. 50: 2019-2025 (1985) teaches the in situ preparation of phosphoramidites by reacting deoxy rribonucleosides with bis- (pyrrolidino) methoxyphosphine, activated with 4,5-dichloroimidazole in l-methyl-2-pi rrolidinone. However, this method was limited by deficient chemoselectivity, forming about 8-10% of the methylphosphite triester of the (3 ', 3'-dinuleoside), as a by-product. Barone and coauthors, Nucleic Acids Res., 12: 4051-4061 (1984) and Lee and Moon, Chem. Lett. , 1229-1232 (1984) describe better chemoselectivity in the preparation of phosphoramidites in situ, by reacting deoxyribonucleosides with bis- (N-dialkylamino) -alkoxyphosphines and lH "tetrazole or its N, N-diisopropylammonium salt. Unfortunately, the tetrazole-N, N-diisopropylammonium salt, whether added or generated in situ, can form precipitates within the synthesizer. Helinski and coauthors, Te rahedron Lett .. 32: 4981-4984 (1991 (and 34: 6451-6454 (1993)) describe the selective activation of bifunctional phosphitylation reagents containing a p-nitrophenoxy group, however, this methodology is not adaptable in current phosphoramidite approaches, because the p-nitrophenoxy group has to be activated using a strong base Finally, Fourrey and co-authors, Tetrahedron Lett .. 22: 729-732 (1981) and Cao and co-authors, Tetrahedron Lett ., 24: 1019-1020 (1983) describe phosphorus dichlorite and corresponding ditetrazolite and dithriazolite as reactive bifunctional phosphorylating agents Unfortunately, the application of these agents to the synthesis of oligonucleotides is generally problematic due to their extremely high reactivity and poor chemoselectivity Therefore, there is a need for new functional phosphorylating reagents and their application in the in situ preparation of nucleoside phosphoramid 5'-protected proteins and the synthesis of oligonucleotides without prior purification of nucleoside-phosphorates. Ideally, such reagents should react rapidly with nucleosides under neutral or weakly basic conditions, without an additional activation step; chemoselectively the corresponding nucleoside phosphoramidites in situ must be relatively stable and easy to handle.

BRIEF DESCRIPTION OF THE INVENTION This invention provides novel bifunctional phosphorylating reagents and their application in the in situ preparation of 5'-protected nucleoside-phosphoramidites and the synthesis of oligonucleotides. The bifunctional phosphorylating reagents according to the invention rapidly react with nucleosides under neutral or weakly basic conditions, without additional activation step. In addition, the bifunctional phosphorylating reagents according to the invention chemoselectively generate the corresponding nucleoside phosphoramidites in situ, without the need to purify the nucleoside phosphoramidites before using them in the synthesis of oligonucleotides. Finally, the bifunctional phosphorylating reagents according to the invention are relatively stable and easy to handle. In a first aspect, the invention provides bifunctional phosphorylating reagents which are useful for the in situ preparation of 5'-protected nucleoside phosphoramidites and the synthesis of oligonucleotides. The bifunctional phosphorylating reagents according to the invention have the general structure (I): > where 0, C or S more towards the rejection is the point of union where the N further to the left is the junction point to the fos where the N furthest to the left is the junction point to the phosphor ro.

The bifunctional phosphorylating reagents according to the invention react in the presence of a secondary or tertiary amine with 5-protected nucleosides to chemoselectively produce 5'-protected nucleoside-3 '-phosphoramidites. In a second aspect, the invention provides a method for generating 5'-protected nucleoside phosphoramidites in situ, without producing a precipitate and without the need to purify the nucleoside phosphoramidites prior to their use in the synthesis of oligonucleotides. In the process according to this aspect of the invention, bifunctional phosphorylating reagents according to the invention are reacted with the 5'-protected nucleosides, in the presence of a secondary or tertiary amine, to produce a 5'-protected phosphoramidite nucleoside. . In a third aspect, the invention provides an improved method for synthesizing an oligonucleotide. In the method according to this aspect of the invention, the improvement comprises the step of generating the nucleoside phosphoramidite in situ, instead of adding purified nucleoside phosphoramidites at the appropriate point, in a conventional procedure for the synthesis of the oligonucleotide. The reagents and methods according to the invention are useful for producing a wide variety of unmodified or chemically modified oligonucleotide compounds, or radiolabelled oligonucleotide compounds, all of which are generally referred to herein as "oligonucleotides". The reagents and methods according to the invention can be used or implemented on a scale ranging from a small laboratory scale to a large commercial scale.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows eight particularly preferred embodiments of bifunctional phosphorylating reagents according to the invention. Figure 2 shows a scheme for the in situ preparation of a 5'-protected nucleoside phosphoramidite and its incorporation into an increasing chain of oligonucleotide. Figure 3 shows two schemes for synthesizing bifunctional phosphorylating reagents according to the invention. Figure 4 shows a scheme for the in situ preparation of a 5'-DMT-nucleoside-phosphoramidite using a particularly preferred bifunctional phosphorylating reagent according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in said synthesis. The patents and publications identified in this specification are within the knowledge of those skilled in the art and are hereby incorporated by reference in their entirety. The invention provides novel bifunctional phosphorylating reagents and their application in the in situ preparation of 5'-protected nucleoside-phosphoramidites and the synthesis of oligonucleotides. The bifunctional phosphorylating reagents according to the invention react rapidly with the nucleosides under neutral or weakly basic conditions, without an additional activation step. The bifunctional phosphorylating reagents according to the invention chemoselectively generate the corresponding nucleoside phosphoramidites in situ, without the need to purify the nucleoside phosphoramidites before using them in the synthesis of oligonucleotides. Finally, the bifunctional phosphorylating reagents according to the invention are relatively stable and easy to handle. In a first aspect, the invention provides bifunctional phosphorylating reagents which are useful for the in situ preparation of 5'-protected nucleoside phosphoramidites and the synthesis of oligonucleotides. The bifunctional phosphorylating reagents according to the invention have the general structure (I): where 0, C or S more towards the rejection is the point of union where the N further to the left is the point of union where the N further to the left is the point of attachment to phosphorus. The bifunctional foefitilant reagents according to the invention react in the presence of a secondary or tertiary amine with the 5'-protected nucleosides to chemoselectively produce 5'-protected nucleoside-3'-phosphoramidites. Particularly preferred embodiments of bifunctional phosphorylating reagents according to the invention are shown in Figure 1. Each of these particularly preferred embodiments has a high reactivity to the 5'-protected nucleosides and can be selectively activated in the presence of a secondary amine or tertiary to form the 5'-protected nucleoside phosphoramidites. A particularly preferred bifunctional phosphorylating reagent according to the invention is 2-cyanoethoxy (N, N-diisopropylamino) -3-nitro-l, 2,4-triazolylphosphine, which is shown as compound 1 in Figure 1. It is a solid resembling wax, pale yellow, and it is quite stable both at -20 ° C and at room temperature. The bifunctional phosphorylating reagents according to the invention can be synthesized according to any of the schemes shown in Figure 3. Compounds having a diisopropylamino group in the Y-position (for example, compounds 1 and 2 of Figure 1) they can be prepared from alkoxy (chloro-N, N-diisopropylamino-phosphine and the appropriate heterocycle in the presence of triethylamine (upper scheme) Alternatively, those compounds can be prepared from alkoxy (chloro-N, N-diisopropyl Inofosphine and the t-rimethylsilyl derivative (TMS) of the appropriate heterocycle (lower scheme) Compounds having a dimethylamino, morpholino or pyrrolidino group in the Y-position (e.g., compounds 3-8 of Figure 1) can be synthesized by reacting alkoxydichlorophosphine with an appropriate (dialkylamino) trimethylsilane, followed by reaction with an appropriate heterocycle, either in the presence of triethylamine or using the TMS derivative of the heterocycle. In a second aspect, the invention provides methods for generating in situ 5'-protected nucleoside phosphoramidites, without producing a precipitate and without the need for purification of the nucleoside phosphoramidites before their use in the synthesis of oligonucleotides. In the process according to this aspect of the invention, the bifunctional phosphorylating reagents according to the invention are reacted with the 5'-protected nucleosides, in the presence of a secondary or tertiary amine to produce a 5'-protected phosphoramidite nucleoside. . Appropriate 5'-protected nucleosides include: adenosine, guanosine, cytosine, uridine, inosine and thymidine, as well as modified nucleosides (see, for example, Sanghvi in Antisense Research and Applications, pages 273-288 (Crook and Lebrleu, Eds. CRC Poress (1993) and the references cited there). The 5 'position of the nucleoside can be protected by any common protecting groups (see, for example, Sonveaux in Protocols for Oligonucleotide Conjugates, pages 1-72 (S. Agra al, Ed.) Humana Press (1994)) or with any suitable protecting group for the synthesis of oligonucleotides. In certain preferred embodiments, the 5 'position of the nucleotide is protected by a dimethoxytrityl group (DMT). For the purposes of the invention, the term in situ is intended to mean "without purification by means". Thus, the in situ generation of the 5'-protected nucleoside-phosphoramidites takes place provided that at least one of the 5'-protected nucleoside-phosphoramidites is generated and then used for the synthesis of oligonucleotide without purification by means of the nucleosides -5'-protected phosphoramidites. Said generation of the 5'-protected nucleoside phosphoramidites and the synthesis of the oligonucleotides can be carried out in the same reaction vessel or in different reaction vessels. In addition, the generation of the 5'-protected nucleoside phosphoramidites can take place before or contemporaneously with the synthesis of the oligonucleotide. The reaction between the bifunctional phosphtylation reagents and the 5'-protected nucleoside can be monitored by conventional 31 P NMR spectroscopy. The most preferred bifunctional phosphtylation reagents according to the invention will react to completion with the 5'-protected nucleoside within about 10 minutes. The exact rate of reaction will vary with the nature of N, N-dialkylamino and / or alkyl, 0-alkyl, 0- or S-cyanoethyl groups or other R groups.

When the bifunctional phosphorylating reagents according to the invention are used in the process according to this aspect of the invention, to obtain the chemoselectivity of the reaction for the desired 5'-protected nucleoside phosphoramidite, the reaction is preferably carried out in presence of a secondary or tertiary amine. Otherwise, it is possible to obtain, under certain circumstances, the dimer 3 ', 3' -nucleoeide, as a by-product. This is because the reaction between the phosphorylating reagent and the 5'-protected nucleoside produces both the 5'-protected nucleoside-phosphoramidite and a released heterocycle molecule. In cases where the released heterocycle is a weak acid, the dialkyla group can be activated in the 5'-protected nucleoside phosphoramidite, which can then be reacted with a 5-protected nucleoside to produce the dimer. Secondary or tertiary amines can avoid this side reaction by trapping the released heterocycle. Preferably this can be achieved by carrying out the reaction between the phosphtylation reagent and the 5'-nucleoside -protected in the presence of diisoprothylamine or diisopromine, most preferably in the presence of about one equivalent of either or both of them. In a third aspect, the invention provides an improved method for the synthesis of an oligonucleotide. In the method according to this aspect of the invention, the improvement comprises the step of generating the nucleoside phosphoramidite in situ, instead of adding purified nucleoside phosphoramidites, in the coupling step, in a conventional procedure for the synthesis of oligonucleotide. . The improvement according to this aspect of the invention can be incorporated into any common phosphoramidite synthesis protocol, using any automatic synthesizer. For example, for small-scale oligonucleotide synthesis, a common and current protocol for oligonucleotide synthesis at a scale of 0.2 to 1.0 micromoles can be followed, using an automated Millipore 8909 Expedite ™ synthesizer (Millipore, Bedford, MA, USA) , except that at the points where the 5'-protected nucleoside-phosphoramidites are normally added, instead, a 0.1M solution of the 5'-protected nucleoside phosphoramidite is generated in situ, adding a bifunctional phosphorylating reagent according to the invention to the reaction, and a 5-protected nucleoside in the presence of an equivalent of a secondary or tertiary amine, such as diisopropylethylamine or diisopropylamine. This procedure produces oligonucleotides in an average step yield of more than 98%. For larger scale synthesis, similar modifications can be made to a large-scale synthesis procedure, using a proportionally larger amount of bifunctional phosphorylating reagent and 5'-protected nucleoside.

The versatility of the improvement in accordance with this aspect of the invention allows it to be used for the synthesis of a large variety of different oligonucleotides. For the purposes of the invention, the term "oligonucleotide" includes polymers of two or more deoxyribonucleotide or 2'-O-substituted ribonucleotide monomers, or any combination thereof. Said monomers can be coupled together by any of the numerous known internucleoside linkages. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, phosphorodithioate, ethylphosphonate or phosphoramidate ligands, or combinations thereof. The term "oligonucleotide" also encompasses those polymers having bases or sugars chemically modified or radioisotopically labeled, and / or having additional substituents including, without limitation, lipophilic groups, intercalators, diamines and adamantane. For the purposes of the invention, the term "2'-O-substituted" means the substitution at the 2 'position of the pentose portion with a.-0-alkyl group containing from 1 to 6 saturated or unsaturated carbon atoms, or with an -0-aryl or allyl group having from 2 to 6 carbon atoms; wherein said alkyl, aryl or allyl group may be unsubstituted or may be substituted, for example, with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxy or amino groups; or said 2 'substitution can be with a hydroxy group (to produce a ribonucleoside), with an amino group or a halo group, but not with a 2'-H group. The following examples are intended to further illustrate certain preferred embodiments of the invention, and are not intended to have a limiting nature. Except as otherwise indicated, in each of the following examples, anhydrous acetonitrile, tetrahydrofuran and dichloromethane from Aldrich (Milwaukee, Wl, E.U.A.) were purchased. Triethylamine, diisopropylamine and diisopropylethylamine were also purchased from Aldrich, and distilled into calcium hydride before use. The anhydrous acetonitrile was purchased from J.T. Baker Inc. (Phillpsburg, NJ, E.U.A.). DT-CPG, 5'-DMT-deoxyadenosine- (Bz) cyanoethyl-phosphoramidite, 5'-DMT-deoxycytidine (Bz) -cianoethyl-phosphoramidite, 5'-DTM-deoxyguanosine (IBU) cyanoethyl-phosphoramidite, 5'- DMT-thymidine-dianoethyl-phosphoramidite, Cap A solutions, Cap B activator, oxidant and unblocked ra, from PerSeptive Biosystems (Fra ingham, MA, E: UA). The Beaucage reagent (3 H-l, 2-benzodithiol-3-one 1,1-dioxide) was purchased from R. I. Chemical (0 range, CA, E. U. A.). All other chemicals were purchased from Aldrich. The NMR spectra were recorded with 3 P (121.65 MHz) and the NMR spectra with 1 »(300 MHz) in a unit Vary 300 (the chemical shift was correlated with 85% of H3PO4 and tetramethylsilane, respectively). The synthesis of the oligonucleotide was carried out on an Expedite ™ 8909 DNA synthesizer (Millipore). The compound numbers, shown in bold, refer to the compounds shown in Figure 1.

EXAMPLE 1 SYNTHESIS OF CL0R0CN.N-DIMETHYLAMIN0) MET0XIF0SFINA To a solution of 10.0 g, 75.24 mmol, 7.1 mL, of methyl dichlorophosphite in 50 mL of CH 2 Cl 2, 12.1 mL, 8.8 g, 75.2 mmol, of N, N-dimethyltrimethylsilyl-amine at 0 ° C was added dropwise. The resulting mixture was stirred for two hours at room temperature. The solvent was removed under reduced pressure to give a colorless oil (10.33 g, 97%) as a product. NMR with iH (CDCI3) m 3.64 (d, J = 13.5 Hz, 3H), 2.66 (s, 3H), 2.62 (s, 3H); NMR with 3ip (CDCl 3) _ 179.4.

EXAMPLE 2 SYNTHESIS OF CL0R0 (MET0XI) PIRR0LIDIN0F0SFINA It was added dropwise to a solution of 10.0 g of 75.24 mmol, 7.1 ml of methyl dichlorophosphite in CH 2 Cl 2 (50 ml), 13.1 ml, 10.8 g, 75.2 mmol, of l-trimethylsilyl pi rrolidine at 0 ° C. The resulting mixture was stirred for two hours at room temperature. The solvent was removed under reduced pressure to give 11.97 g, 95% of a colorless oil, as a product. NMR with 3ip (CDCI3) .... 182.3.

EXAMPLE 3 SYNTHESIS OF CL0R0 (2-CIAN0ET0XnM0RF0LIN0F0SFINA To a solution of 3.0 g, 17.5 mmol, 2.2 ml of 2-cyanoethyl 2-dichlorophosphite in 45 ml of CH 2 Cl 2 was added dropwise 3.1 ml, 2.8 g, 17.5 mmol, of 4- (trimethylsilyl) -morpholine. The resulting mixture was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure to give 3.87 g, 86% pale yellow oil, as a product.

EXAMPLE 4 SYNTHESIS OF CHLORINE C2-CIAN0ET0XI) PIRR0LIDIN0F0SFI NA A solution of 2.0 g, 11.6 mmol, 1.5 ml of 2-cyanoethyl dichlorophosphite, 2.0 ml, 1.7 g, 11.6 mmol, of 4- (trimethylsilyl) pi rrolidine at 0 ° C was added dropwise. The resulting mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure to give 2.48 g, 88% of a colorless oil, as a product.

EXAMPLE 5 SYNTHESIS OF 2-CIAN0ET0XI (N.N-DIIS0PR0PILAMIN0 3NITR0-1.2.4- TRIAZOLIDPHOSFINA l) To a stirred solution of 9.64 g, 84.50 mmol, of 3-nitro-l, 2,4-triazole and 14.0 ml, 10.26 g, 101.4 mmol, of triethylamine in 200 ml of THF, 20.0 g, 84.50 mmol was added dropwise. , of chloro-2-cyanotoxy-N, N-diisopropylaminophosphine at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 24.9 g, 95%, of the crude product as a pale brown oil. After standing at room temperature, the oil becomes a pale yellow, wax-like solid. NMR with H (CDCl 3) "8.39 (s, 1H), 4.09 (m, 2H), 3.51 (, 2H), 2.81 (m, 2H), 1.19 (d, J = 6.0 Hz, 6H), 1.07 (d, J = 9.0 Hz, 6H). NMR with 3ip (CDCl 3). ". 133.9.

EXAMPLE 6 SYNTHESIS OF MET0XI-N.N-DIIS0PR0PILAMIN0 (3-NITR0-1.2.4- TRIAZOLIDPHOSPHINE (2) To a stirred solution of 0.69 g, 6.07 mmol, of 3-nitro-l, 2,4-triazole and 2.82 mL, 2.05 g, 20.2 mmol, of triethylamine in 10 mL of THF and 20 mL of CH2Cl, was added dropwise. 1.0 g, 5.06 mmoles, of chlorine (N, N-diisopropylamino) methoxyphosphine, at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 1.24 g, 89%, of the crude product as a pale brown oil. NMR with31P (CDC13) _ 135.9.

EXAMPLE 7 SYNTHESIS OF MET0XI (3-NITR0-1.2.4-TRIAZ0LIL) PIRR0LIDIN0F0SFINA Í31 To a stirred solution of 2.70 g, 23.71 mmol, of 3-nitro-l, 2,4-triazole and 11.0 ml, 8.0 g, 79.0 mmol, of triethylamine in 40 ml of THF and 20 ml of CH2Cl2, was added dropwise. 3.31 g, 19.76 mmoles, of chloro (methoxy) pyrrolidino-phosphine, at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 3.97 g, 82%, of the crude product, as a pale yellow oil. NMR with 31P (CDCI3) 132. 9.

EXAMPLE 8 SYNTHESIS OF N. N-DIMETHYLAMIN (MET0XIK3-NITR0-1.2.4- TRIAZOLIDPHOSPHINE (4) To a stirred solution of 2.86 g, 25.1 mmoles, of 3-nitro-l, 2,4-triazole and 14.0 ml, 10.2 g, 101 mmoles, of triethylamine in 40 ml of THF, 3.55 g, 25.1 mmoles was added dropwise. , of chloro (N, N-dimethylamino) methoxyphosphine in 10 ml of CH2Cl2, at room temperature. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 4.56 g, 83% of the crude product, as a yellow oil. NMR with 31P (CDCl 3). 134.9.

EXAMPLE 9 SYNTHESIS OF 2-CIAN0ET0XIC3-NITR0-1.2.4- TRIAZOLIDMORFOLINOFOSFINA (5) To a stirred solution of 2.57 g, 22.57 mmol, of 3-nitro-l, 2,4-triazole and 12.58 mL, 9.14 g, 90.28 mmol, of triethylamine in 40 mL of CH 2 Cl 2, 5.82 g was added dropwise, 22. 57 mmoles, of chlorine (2-cyanotetoxy) morpholinophosphine, at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 5.98 g, 79%, of the crude product, as a yellow oil. NMR with 3iP (CDCI3) .... 126.9.

EXAMPLE 10 SYNTHESIS OF 4.5-DICL0R0IMIDAZ0LIL (MET0XI) PIRR0LIDIN0F0SFINA (6) To a stirred solution of 4.62 g, 33.7 mmol, of 4,5-dichloroimidazole and 18.8 ml, 13.65 g, 134.9 mmoles, of triethylamine in 40 ml of THF, 3.31 g, 19.76 mmoles, of chlorine (methoxy) pi rrolidinophosphine in 13.4 ml of CH 2 Cl 2 were added dropwise to the temperature ambient. The mixture was stirred for 3 hours at room temperature. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 7.76 g, 86%, of the crude product as a pale yellow oil. NMR with 3 * P (CDCl 3) 125.9.

EXAMPLE 11 SYNTHESIS OF 4.5-DICL0R0IMIDAZ0LIL-1.4-DIMETHYLAMIN (MET0Xn- FOSFI A (7) To a stirred solution of 3.44 g, 25.1 mmol, of 4,5-dichloroimidazole and 14.0 ml, 10.16 g, 100.4 mmoles, of triethylamine in 30 ml of THF, 3.55 g, 25.1 mmoles, of chlorine (N, N-dimethylamino) methoxyphosphine were added dropwise in 10 ml of CH2CI2, at room temperature. The mixture was stirred at room temperature for two hours. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 2.81 g, 79%, of the crude product, as a yellow oil. NMR with 31P (CDCI3) ._. 129.3.

EXAMPLE 12 SYNTHESIS OF 2-CIAN0ET0XI (4.5-DICL0R0IMIDAZ0LIL) PIRR0LIDINQ- FOSFINA (8) To a stirred solution of 1.59 g, 11.63 mmol, of 4,5-dichloroimidazole and 1.95 mL, 1.41 g, 13.96 mmol, of triethylamine in 30 mL of CH 2 Cl 2, 2.40 g was added dropwise, 11. 63 mmoles, of chloro (2-cyanoethoxy) pyrrolidinophosphine in 10 ml of CH2Cl2, at room temperature. The mixture was stirred for 4 hours at room temperature. The reaction mixture was filtered to remove the resulting salt and the solvent was removed under reduced pressure to give 2.93 g, 82%, of the crude product, as a pale yellow oil. NMR with 31P (CDCI3 __ 123.2.

EXAMPLE 13 IN SITU PREPARATION OF 5 '-DMT-TIMIDINE-CED-FOSFORAMIDITAS To 1.65 mmol (0.90 g) of 5 '-DMT-thymidine in 8.3 ml of THF, a solution of 0.54 g, 1.73 mmol, of 1 and 0. 31 ml, 0.23 g, 1.78 mmoles of N, N-diisopropylethylamine in 89.3 ml of CH3CN, at room temperature. The mixture was stirred for 5 minutes and the solution (0.1 M) of phosphoramidite was ready for use.

EXAMPLE 14 IN SITU PREPARATION OF hP -ÍBU-5-DMT-2-DES0XIGUAN0SINA-CED- FOSFORAMIDITAS At 2.0 mmol (1.28 g) of 5'-DMT-dGiB "in 10.0 ml of THF, 0.66 g, 2.1 mmol, of 1 and 0.38 mL, 0.28 g, 2.2 mmol, of N, N-diisopropylethylamine in 10.0 mL of CH 3 CN were added at room temperature. The mixture was stirred for 5 minutes and the solution (0.1 M) of phosphoramidite was ready for use.

EXAMPLE 15 IN SITU PREPARATION OF N6-BZ-5-DMT-2-DES0XIADEN0SINA-CED-FOSFORAMIDITAS At 2.0 mmoles (1.32 g) of 5'-DMT-dAB * in 10.0 ml of THF, 0.66 g, 2.1 mmol, of a solution of 1 and 0.38 ml, 0.28 g, 2.2 mmol, of N, N-diisopropylethylamine was added. in 10.0 ml of CH3CN, at room temperature. The mixture was stirred for 5 minutes and the solution (0.1 M) of phosphoramidite was ready for use.

EXAMPLE 16 IN SITU PREPARATION OF N * -Bz-5 '-DMT-2' -DESOXICITIDINE-CED- PHOSPHORAMIDITES At 2.0 mmole (1.27 g) of 5'-DMT-dCB * in 10.0 ml of CH3CN, a solution of 0.66 g, 2.1 mmol of 1 and 0.38 mL, 0.28 g, 2.2 mmol, of N, N-diisopropylethylamine in 10.0 mL of CH3CN was added at room temperature. The mixture was stirred for 5 minutes and the solution (0.1 M) of phosphoramidite was ready for use.

EXAMPLE 17 ANALYSIS OF NMR SPECTROSCOPY WITH 3 * P OF NUCLEOSIDE-PHOSPHORAMIDITE MONOMERS To 0.20 mmol (62.79 mg) of 1, a solution of 92.4 mg, 0.17 mmol, of 5 '-DMT-thymidine and 0.035 mL, 25.9 mg, 0.20 mmol, of N, N-diisopropylethylamine in 0.7 mL of CDCI3 was added, at room temperature. After stirring for 10 minutes at room temperature, the solution was transferred to an NMR tube and examined by conventional NMR spectroscopy. The results showed that 98 percent of the expected product was the product of nucleoside phosphoramidite. Similar results were obtained using each of the reagents 2-8.

EXAMPLE 18 SYNTHESIS OF OLIGONUCLETICIDES The phosphorothioate and the phosphodiester of the oligonucleotide were synthesized, at a scale of 0.2 and 1 micromole, following the common and current protocol, using an automatic synthesizer (Millipore 8909 Expedite < MR >, Bedford, MA, USA), except that it replaced the step of adding the nucleoside phosphoramidite by the in situ generation of the nucleoside phosphoramidite, as described in example 17. For the phosphorothioate of the oligonucleotide, the oxidation step with iodine was replaced by sulfurization with 1,1- 3H-l, 2-benzodithiol-3-one dioxide (Beaucage reagent). The treatment was carried out for eight hours with ammonium hydroxide at 65 * C to remove the oligomer from the support and to deprotect the nucleoside bases. The mixture was filtered to remove the CPG. After the ammonium hydroxide solution was removed by Speed Vac, the remaining crude products were subjected to CE and IE-HPLC analysis.

Claims (6)

NOVELTY OF THE INVENTION CLAIMS

1. - A bifunctional phosphorylating reagent characterized because it has the general structure: R p where the 0, C or S more to the right is the point of union

2. - The bifunctional phosphorylating reagent according to claim 1, further characterized in that the reagent is selected from the group consisting of the compounds shown in Figure 1. 3.- A method for generating in situ 5'-protected nucleoside phosphoramidites, characterized said process because it comprises reacting a bifunctional phosphorylating reagent according to claim 1, with 5'-protected nucleosides, in the presence of a secondary or tertiary amine, to produce a 5'-protected nucleoside-phosphoramidite. 4. The method according to claim 3, further characterized in that the bifunctional phosphorylating reagent is selected from the group of compounds shown in figure 1. 5. An improved method for synthesizing an oligonucleotide, characterized by the improvement because it comprises generating in situ the nucleoside phosphoramidite according to the method of claim

3. 6. An improved method for synthesizing an oligonucleotide, characterized in that it comprises improving in situ generating the nucleoside phosphoramidite according to the method of claim 4.