JP2000505081A - Solid phase synthesis of oligonucleotides N3 '→ P5' phosphoramidate - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for synthesizing oligonucleotides N3 ′ → P5 ′ phosphoramidates using a phosphoramidite amine exchange reaction, wherein the deprotection 3′-amino acid of the solid support oligonucleotide chain is provided. The group is exchanged for the amino moiety of the 5'-phosphoramidite of the incoming monomer having a protected 3'-amino group. The resulting internucleoside phosphoramidite linkage is then oxidized to form a stable protected phosphoramidate linkage. The process of the present invention greatly improves product yields and reduces reagent usage over currently available synthetic methods for compounds of the above type.

Description

DETAILED DESCRIPTION OF THE INVENTION           Solid-phase synthesis of oligonucleotide N3 '→ P5' phosphoramidate                                 Field of the invention   The present invention relates generally to nucleic acid polymer chemistry, and more particularly to oligonucleotides. The present invention relates to a method for synthesizing otide N3 ′ → P5 ′ phosphoramidate.                                 Background of the Invention   Nucleic acid polymer chemistry has the pharmaceutical, diagnostic and analytical fields, and more specifically, Antisense diagnosis and anti-gene therapy, combinatorial chemistry, branched DN A signal amplification, and sequence-based DNA diagnostics and analysis (e.g., Important roles in many developing technologies .   Much of this chemistry is based on the binding strength, specificity, and specificity of natural nucleic acid polymers (eg, DNA). And to improve nuclease resistance. Unfortunately, one characteristic Improvements (e.g., nuclease resistance) often involve other properties (e.g., binding strength). Trade-off). Such a trade-off Many examples: Peptide nucleic acid (PNA) shows good nuclease resistance and binding strength However, cell uptake in test cultures is low (eg, Hanvey et al., Science, 258: 148). 1-1485 (1992)); phosphorothioate has good nuclease resistance and solubility But typically synthesized as a P-chiral mixture, with some sequence non-specific Exhibit significant biological effects (eg, Stein et al., Science, 261: 1004-1012 (1993)); Methylphosphonate shows good nuclease resistance and cell uptake, but Typically, they are also synthesized as P-chiral mixtures and have low duplex stability (e.g., Mesma eker et al. (above)).   Recently, a new class of oligonucleotide analogs has been developed, Having a so-called N3 ′ → P5 ′ phosphoramidate internucleoside bond, It exhibits very favorable binding properties, nuclease resistance and solubility (Gryazno v and Letsinger, Nucleic Acids Research, 20: 3403-3409 (1992); Chen et al., N ucleic Acids Research, 23: 2661-2668 (1995); Gryaznov et al., Proc. Natl. Acad .Sci., 92: 5798-5802 (1995); and Gryaznov et al. Am. Chem. Soc., 116: 31 43-3144 (1994)). Unfortunately, published compounds have The low synthesis yields have hindered their commercial use.   The utility of this new class of oligonucleotide analogs has been Modifications to improve the synthesis yield of any of the preferred properties without loss If unusual and novel synthetic approaches are found, they will be greatly enhanced.                                 Summary of the Invention   In view of the above, it is an important object of the present invention that the sequential coupling yield Significant increase in solid-phase synthesis of oligonucleotides N3 '→ P5' phosphoramidate It is to provide a new approach.   Another object of the present invention is to provide a novel 3′-protected amino-5 ′ for use in the method of the present invention. -To provide phosphoramidite monomers.   Still another object of the present invention is to provide an oligonucleotide N3 ′ → P5 ′ phosphoramidate (In particular, 2'-deoxyoligonucleotide N3 '→ P5' phosphoramidate) Another object of the present invention is to provide a practical large-scale synthesis method.   These and other objects of the present invention are directed to the use of oligonucleotides with amine exchange reactions. Achieved by providing a method for synthesizing leotide N3 '→ P5' phosphoramidate In this method, the deprotected 3'-amino group of the solid support oligonucleotide chain is 5'-Phosphoramido, an incoming monomer having a protected 3'-amino group Replaced with the amino part of the dye. The obtained internucleoside phosphoramid The diite linkage is then oxidized to form a stable protected phosphoramidate linkage I do. The general scheme of this reaction is depicted below.   Generally, the method of the invention comprises the following steps: (a) bound to a solid support Providing a first nucleoside, wherein the first nucleoside comprises a protected 3'- (B) deprotecting the protected 3'-amino group to form a free 3'-amino group (C) converting the free 3′-amino group to a 3′-protected aminonucleoside-5′-phosphoramido React with the dye monomer to form the internucleoside N3 '→ P5' phosphoramidite (D) oxidizing the bond; and (e) the desired oligonucleotide Steps (b) to (d) are repeated until leotide N3 ′ → P5 ′ phosphoramidate is synthesized. Process. Preferably, the 3'-protected aminonucleoside-5'-phosphoramidite The nitrogen moiety of the monomer 5'-phosphoramidite has a pK of at least 10_aStanding with It is a disabling amine.   The present invention further provides a 3'-protected aminonucleoside-5'-phosphoramidye of the following formula: , Which are particularly useful in the method of the invention: Wherein B is pyrimidine, purine, or an analog thereof; R¹Is the e Is a phosphate protecting group; W is -NHR^TwoOr -OR⁷Where R^Two Is an amino protecting group and R⁷Is a hydroxyl protecting group; R^ThreeIs hydrogen , Hydroxyl, fluoro or -OR ', wherein R' is one to three carbons Alkyl having an atom, or a 2'-hydroxyl protecting group (e.g., alkylsilyl And, for example, t-butyldimethylsilyl); and R^FourAnd R^FiveAre those Together with the attached nitrogen forms carbon atoms and / or oxygen, sulfur and nitrogen. Alkylamino leaving groups with up to 40 heteroatoms selected from the group consisting of Or forms an arylamino leaving group. -OR⁷A monomer having W as 3 '→ P5' phosphoramidate linkages and other linkages (e.g., phosphodiesters, Chimeric oligonucleotides containing both It is particularly useful for   The present invention relates to an orifice having either fully or partially amidated linkages. Significant drawbacks of conventional methods for synthesizing the oligonucleotide N3 '→ P5' phosphoramidate Overcoming and paving the way for commercial scale production of such compounds. In particular, the invention Has greatly improved coupling using much lower molar equivalents of monomer reactants. The oligonucleotide N3 ′ → P5 ′ phosphoramide Enables commercially feasible synthesis of the sheet. The present invention covers a wide range of fields (Science Widespread application of this compound in clinical and industrial research, Enable.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows N6-benzoyl-3′-tritylamino-2′-deoxyadenosine-5 ′-(2-cy Anoethyl-N, N-diisopropyl) -phosphoramidite and tetrazole Compound³¹It is a P-NMR spectrum.   FIG. 2 shows 3′-tritylaminothymidine-5 ′-(2-cyanoethyl- (2,2,6,6-tetra Methylpiperidinyl))-phosphoramidite and tetrazole³¹P-N It is an MR spectrum.   3a and 3b show two crude oligonucleotides synthesized by the transamination reaction of the present invention. It is an ion-exchange chromatogram of nucleotide N3 '→ P5' phosphoramidate.   FIG. 4 shows the oligo-2′-fluoronucleoside N3 ′ → P5 ′ phosphoramide of Example 19. 4 is an ion exchange HPLC chromatogram of a crude reaction mixture from the synthesis of a salt.                                   Definition   If the oligonucleotide is represented by a series of letters (e.g., `` ATGUCCTG '') Always, this nucleotide is in 5 'to 3' order from left to right and no other indication. If so, "A" represents deoxyadenosine and "C" represents deoxycytidine. Represents, "G" represents deoxyguanosine, "T" represents thymidine, And it is understood that "U" represents deoxyuridine.   As used herein, “N3 ′ → P5 ′ phosphoramidate” refers to the following forms of i. Stands for internucleosidic linkage:                       3'-NH-P (= X) (OR<sup>1</sup>) -O-5 ' Where 3 ′ and 5 ′ are the sugar moieties of the consecutive nucleosides connected by this bond , Where R<sup>1</sup>Is a hydrogen or phosphate protecting group, And X is a chalcogen (preferably oxygen or sulfur). Further identify And R<sup>1</sup>When is a phosphate protecting group, it is an atom containing up to 10 carbon atoms. Alkyl, alkenyl, aryl, aralkyl or cycloalkyl. Like Or R<sup>1</sup>When is a phosphate protecting group, it has from 1 to 6 carbon atoms Alkyl having an electron-withdrawing β-substituted ethyl (particularly, β-trihalomethyl-, β- Cyano-, β-sulfo- or β-nitro-substituted ethyl); electron-withdrawing substituted phenyl (Especially halo-, sulfo-, cyano- or nitro-substituted phenyl); It is a substituted phenylethyl atom withdrawing property. More preferably, R<sup>1</sup>Is phosphate protected When it is a group, it is methyl, β-cyanoethyl or 4-nitrophenylethyl is there. Most preferably, R<sup>1</sup>Is hydrogen, methyl or β-cyanoethyl. Electric The child-withdrawing substituent is typically a halo, cyano, nitro, sulfo, or mono- , Di- or trihalomethyl. Halogen atom substituents are usually fluoro Ro, chloro, bromo or iodo; preferably they are fluoro or chloro Is chloro. "Electron withdrawing" refers to the fact that a certain substituent is Indicates a tendency to attract children (ie, electronegative) (eg, March, Adva nced Organic Chemistry, pgs. 16-18 (John Wiley, New York, 1985)). Phosph Guidance on selecting a carbonate protecting group can be found in Beaucage and Iyer, Tetrahedro n 48: 2223-2311 (1992). For convenience, the nucleotide phosphoramide May sometimes be referred to as N3 ′ → P5 ′ phosphoramidate and P3 ′ → N5 ′ Phosphoramidates are indicated by subscripts “np” or “pn” respectively It is. Therefore, "U<sub>np</sub>U '' means that 3'-aminouridine and uridine are N3 '→ P5' Dinucleotides linked by a suformamidate linkage. Similarly, 2'-f Fluoro substituents are indicated by the superscript "f". Therefore, "U<sup>f</sup> <sub>np</sub>U "is 5'-mo Strike (5'-most) 3'-amino-2'-fluorouridine is N3 '→ P5' phosphoramidate A dinucleotide optionally linked to uridine. Lead alone g) The subscript "p" indicates 5'-monophosphate and follows alone (t ailing) The subscript "n" indicates a 3'-amino group.   "N3 '→ P5' phosphoramidite linkage" as used herein (with emphasis) The term means the phosphorus (III) intermediate of the N3 '→ P5' phosphoramidate linkage. According to the present invention, the N3 ′ → P5 ′ phosphoramidate linkage is an N3 ′ → P5 ′ phosphoramidate. It is formed by the oxidation of site bonds.   As used herein, `` nucleoside '' includes natural nucleosides, This includes the 2'-deoxy and 2'-hydroxyl forms (e.g., Kornberg and And Baker, DNA Replication, 2nd edition (Freeman, San Francisco, 1992) ) Is included. “Analogs” related to nucleosides include modified base moieties and And / or synthetic nucleosides having modified sugar moieties, such as, for example, And generally described in Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Have been. Such analogs include those disclosed in Uhlmann and Peyman (supra). Designed to enhance the binding properties (e.g., stability, specificity, etc.) And adult nucleosides.   As used herein, "pyrimidine" refers to a pyrimidine that occurs in a natural nucleoside. Gins (cytosine, thymine, and uracil, and their common analogs ( For example, oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio , And those containing substituents such as halo). Used herein The term further includes pyrimidines (e.g., N^Four-Ben Zoylcytosine). Common pyrimidine protecting groups further include Beaucage And Iyer (supra).   As used herein, “purine” refers to a purine (adequate) occurring in a natural nucleoside. Nin, guanine, and hypoxanthine, and their common analogs (eg, For example, oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, Including those containing substituents such as halo). As used herein The term further refers to purines (e.g., N^Two-Benzoylug Anin, N^Two-Isobutyrylguanine, N⁶-Benzoyl adenine) . More common purine protecting groups are disclosed in Beaucage and Iyer (supra) .   As used herein, “oligonucleotide N3 ′ → P5 ′ phosphoramidate” Is a nucleic acid linked by at least one N3 ′ → P5 ′ phosphoramidate linkage. Osido subunit oligomer (usually linear) is meant. This nucleoside A buunit usually contains a nucleoside or nucleoside analog, More common moieties with compatible chemistry (eg, non-basic sugars (abasi c sugars) and other hydrocarbon moieties, such as those described in the following references) May contain: Newton et al., Nucleic Acids Research, 21: 1155-1162 (199 3); Griffin et al. Am. Chem. Soc., 114: 7976-7982 (1992); Jaschke et al., Tetrahed. ron Letters, 34: 301-304 (1992); Ma et al., International Application PCT / CA92 / 00423; Zon et al., International Publication No. PCT / US90 / 06630; Durand et al., Nucleic Acids Research, 18: 6353-6359 (1990); Sa lunkhe et al. Am. Chem. Soc., 114: 8768-8772 (1992); More preferably The term refers to all internucleoside linkages as N3 '→ P5' phosphoramidate Means an oligonucleotide substituted with a bond, i.e., the term And fully "amidated" oligomers. Even more preferably, it is All internucleoside linkages are replaced with N3 '→ P5' phosphoramidate linkages The nucleoside subunit is a natural nucleoside or analog thereof It means a certain oligonucleotide. All bonds are N3 '→ P5' phosphoramidate An oligonucleotide N3 ′ → P5 ′ of the invention that is a bond (“fully amidated”). Phosphoramidate embedded in other oligonucleotides or polynucleotides To form larger oligomers that are "partially amidated" Can be achieved. For example, this fully amidated oligonucleotide N3 '→ P5' phosphoramid Dating A_npA_npG_npC_npC_nIs the larger nucleotide GGCCAAAA_npA_npG_npC_npC_np Oligonucleotide embedded in ACTAT (SEQ ID NO: 1) or larger: A_np A_npG_npC_npC_npIt is linked to "TTTATC" as TTTATC (SEQ ID NO: 2). PCR ply Such chimeric oligonucleotides that can be used as primers, capture probes, etc. Tides are included within the scope of the present invention.   As used herein, terms such as "oxidize" and "oxidation" refer to phosphorus-containing In connection with the nucleoside linkage, the phosphorus atom of this linkage is removed from the phosphorus (III) form to phosphorus ( V) means a method or process of converting to a form.   As used herein, the term "phosphoramidite amino group" refers to a phosphoramidite amino group. Amino group bonded to phosphorus atom of amidite group, -NR_FourR_FiveAnd "phospho The term "luamidite nitrogen" refers to the nitrogen atom of this phosphoramidite amino group. means.   As used herein, terms such as "sterically hindered," "sterically hindered," It refers to the effect of “bulky” groups on chemical reactivity (eg, Morris on and Boyd, Organic Chemistry, page 603 (Allyn and Bacon, Boston, 197 8)).                             Detailed description of the invention   The present invention provides a solid phase method for synthesizing oligonucleotide N3 '→ P5' phosphoramidate. Method, where the ability of the phosphoramidite monomer to attach to the free amino groups of the growing chain. The coupling is the free 3 'of this growing chain of the phosphoramidite amino group of the monomer. Proceeds via exchange with an amino group. Preferably, the ozone produced by the method of the present invention The lignonucleotide N3 '→ P5' phosphoramidate is described by the following formula:Where B is a purine or pyrimidine or an analog thereof; X is Lucogen (preferably oxygen or sulfur, most preferably oxygen);^ThreeIs , Hydrogen, fluoro or hydroxyl, preferably hydrogen; R⁶Is amino Or hydroxyl; and Z is hydrogen or a cationic counterion (eg, , Alkali metals), amine cations (e.g., ammonium, triethylammonium) Um). Preferably, n is in the range of one to several hundred; more preferably , N ranges from 1 to about 50; most preferably, n ranges from 1 to 30.   Preferably, the oligo-2-fluoronucleotide N3 ′ → P5 ′ phosphoramido of the present invention Dates are between 2 and 30 nucleotides in length. More preferably, this Are between 8 and 25 nucleotides in length, most preferably these are , Between 8 and 20 nucleotides in length.   As mentioned above, the general steps of the method include (a) a second step attached to a solid support. Providing one nucleoside, wherein the first nucleoside comprises a protected 3′-amino acid. (B) deprotecting the protected 3'-amino group to form a free 3'-amino group (C) converting the free 3'-amino group to a 3'-protected aminonucleoside-5'-phosphorua Reacts with the midite monomer to form the internucleoside N3'-P5 'phosphoramida (D) oxidizing the bond; and (e) the desired oligonucleotide. Steps (b) to (d) are repeated until nucleotide N3 ′ → P5 ′ phosphoramidate is synthesized. Returning.   The transamination reaction of the present invention can be performed even if a reversible equilibrium between reactants and products is obtained. (Shown below in reaction equations 1a and 1b). Most approaches to solid-phase oligonucleotide synthesis involving an inverse coupling step Coupling conditions, protecting groups, solid supports, binding groups, Protection reagents, reagents that cleave products from solid supports, product purification, etc. Important guidance for making choices can be found in, for example, the following literature: Literature.   In the present invention, a wide range of solid supports can be used, including controlled pore glass (CPG). ), Highly crosslinked polystyrene, acrylic copolymer, cellulose, nylon, dext Orchids, latex, polyacrolein, etc. It is shown: The support is further grafted with polystyrene beads; polyethylene glycol Polystyrene (e.g., TentaGel^TMRapp Polymere, Tubingen Germany) And so on. Properties of this support (e.g., material, porosity, size, shape, etc.) , And the type of binding moiety used will depend on various factors (e.g., Protecting groups, length of the final product, amount of the final product, etc.). Typical joint Minutes are disclosed below: Such.   Preferred solid supports for use in the present invention include CPG, and polyethylene glycol. Polystyrene having a terminal amino group (e.g., TentaGel −NH_Two ^TMRapp Polymere, Tubingen Germany). This aminopropyl group is , Are preferred spacers between CPG and nucleoside linkages. This first nucleus A preferred linkage to the 5'-hydroxyl of an osid is a succinyl group, which is Base labile ester linkages (typically after synthesis using aqueous ammonia) To be cleaved).   The monomers of the present invention include 2'-fluoro-3'-protected aminonucleoside-5'-phospho Luamidite, 2'-deoxy-3'-protected aminonucleoside-5-phosphoramidite 2'-protected-3'-protected aminoribonucleoside-5'-phosphoramidite; and And their 3'-protected-3'-hydroxyl counterparts. Preferred Alternatively, the monomers of the present invention are defined by the formula: Where B, W, R¹, R^Three, R^FourAnd R^FiveIs as defined above.   More preferably, -NR^FourR^FiveIs a sterically hindered amino group, which is preferably New alternatives: First, R^FourAnd R^FiveIs independently alkyl, Aralkyl, cycloalkyl or cycloalkylalkyl, wherein R^Four And R^FiveHas a total of from 6 to 20 carbon atoms. Even more preferably, R^Four And R^FiveIs independently alkyl having 1 to 8 carbon atoms. further Preferably, R^FourAnd R^FiveIs independently isopropyl, sec-butyl, isobutyl , T-butyl, cyclohexyl or 2-ethylhexyl. Most preferably, Independently, R^FourIs isopropyl while R^FiveIs t-butyl.   Second, R^FourAnd R^FiveTogether contain up to 12 carbon atoms in their main chain And form an alkylene chain containing a total of 4 to 20 carbon atoms, said chain Both terminal valence bonds of R^FourAnd R^FiveIs bonded to the bonded nitrogen atom. Sa More preferably, R^FourAnd R^FiveTogether form up to 6 carbon atoms in the main chain And may form an alkylene chain containing 4 to 12 carbon atoms in total. , Both terminal valence bonds of the chain are R^FourAnd R^FiveIs attached to the attached nitrogen atom You.   Third, R^FourAnd R^FiveTogether, together with the nitrogen to which they are attached, Chains of up to 10 carbon or heteroatoms and a total of 4 to 20 carbon atoms Form a saturated nitrogen heterocycle with an atom or heteroatom, such that R^FourAnd R^Five Together, together with the nitrogen atom to which they are attached, nitrogen, oxygen and sulfur And contains up to three heteroatoms selected from the group consisting of: Even more preferred Or R^FourAnd R^FiveTogether form, together with the nitrogen atom to which they are attached, 10 Up to carbon atoms and is selected from the group consisting of nitrogen, oxygen and sulfur A saturated nitrogen heterocycle having up to three additional heteroatoms is formed. Even better Preferably, R^FourAnd R^FiveTogether, together with the nitrogen atom to which they are attached, Loridino, morpholino, tetramethylguanidinyl or piperidino. Sa More preferably, R^FourAnd R^FiveAre together the nitrogen atom to which they are attached With dimethylpiperidinyl, pyrrolidinyl, dimethylmorpholino, tetrame Tylmorpholino, dimethylpyrrolidinyl, tetramethylpyrrolidinyl or tet It becomes lamethylpiperidinyl. Even more preferably, R^FourAnd R^FiveAre together Thus, together with the nitrogen atom to which they are attached, 2,2,6,6-tetramethylpiperidinyl, It becomes 2,6-dimethylpiperidinyl or 2,5-dimethylpyrrolidinyl. Most preferred H, R^FourAnd R^FiveTogether, together with the nitrogen atom to which they are attached, 2,2,6, It becomes 6-tetramethylpiperidinyl.   Preferably, the monomer used in this method is a 2'-deoxy-3'-protected amino It is a nucleoside-5'-phosphoramidite. More preferably, the phosphor Amidite amino groups have a PKa of at least 10.0. Even more preferably The phosphoramidite amino group is a tetrazole active as defined below. Equilibrium constant K₁(Measured in Example 9) is 10M^-1To be larger, Selected. Even more preferably, the equilibrium constant is 100 M^-1Bigger and most Preferably, this equilibrium constant is 1000M^-1Greater than.   Tetazole activation equilibrium constant K₁Is defined as: Here, [tetrazoridyl amidite] is the equilibrium of the tetrazodyl amidite intermediate [Tetrazole] is the equilibrium concentration of tetrazole [Midite] is the equilibrium concentration of the phosphoramidite monomer, and [R_TwoNH_Two ⁺ Tetrazolide^-] Is the tetrazole of the amino leaving group of this phosphoramidite monomer. This is the equilibrium concentration of the lido salt.   Preferably, the 3'-amino protecting group of this monomer, R^TwoIs an acid labile group (e.g., For example, triphenylmethyl (i.e., trityl), p-anisyldiphenylmethyl ( That is, monomethoxytrityl or MMT), di-p-anisylphenylmethyl ( That is, dimethoxytrityl or DMT) or acid-labile urethane). Most Also preferably, the 3'-amino protecting group is triphenylmethyl. these The protecting group may be an acidic solution (most preferably a 3% solution of dichloroacetic acid in methylene chloride). Removed by processing. Similarly, the 3'-hydroxyl protection of this monomer The group R7 is an acid labile group (e.g., trityl, MMT, DMT or urethane). You. Most preferably, the 3'-hydroxyl protecting group is DMT.   The term "free amino group" as used herein relates to the monomers of the present invention. Amino acids available to react with phosphoramidite groups of incoming monomers Means a group. Preferably, the free amino groups are primary amines. Detritylation After the step, the amino group is generally combined with the conjugate base of the acid used for detritylation. It exists in its salt form. After the detritylation step, if necessary, With a basic solution (eg, 2% triethylamine or pyridine in acetonitrile) Can be neutralized.   The coupling step of the present invention can be performed in a temperature range of -20 to 200C. Better Preferably, the reaction is performed at room temperature (about 15-30 ° C). This reaction is Mididite monomer solution and activator solution (or phosphoramidite monomer And a solution containing an activator) on a solid support. The reaction is carried out by adding to a reaction vessel containing the free amino group of otide. In general, The activator of the present invention is a nucleophilic catalyst, which is a more stable phosphoramidite. Replace the toamino group to provide a more reactive (and less stable) intermediate This intermediate then forms the solid supported oligonucleotide N3 '→ P5' Reacts with free 3 'amino groups of suformamidate. The mixture is then mechanically The mixture is mixed by a method such as gentle vortexing or sparging of an inert gas. Alternatively, The solution of theomer and activator was solid supported (e.g., Re (G) manufactured to flow through a reaction vessel (or column) containing nucleotides; Can be These monomers and activator may be pre-mixed or Mixed in a valve block or mixed in a pre-activated vessel Can be pre-equilibrated or added separately to the reaction vessel.   Examples of activators used in the present invention include tetrazole, 5- (ethylthio) tet Lazole, 5- (4-nitrophenyl) tetrazole, 5- (2-thienphen) tetrazole , Triazoles, pyridinium chlorides and the like (e.g. Beaucage and I yer (supra); Berner et al., Nucleic Acids Research, 17: 853-864 (1989); Benson, C hem. Rev. 41: l-61 (1947)). "Tetrazole activator" as used herein The term means an activator that is a tetrazole or tetrazole derivative . The most preferred activator is tetrazole. Suitable solvents include Examples include ril, tetrahydrofuran, and methylene chloride. Acetonitrile is preferred No. A very dry (water-free) monomer, activator, and Wash the solid support just before the coupling step and the solvent for the coupling step Great care should be taken in the solvent used to do so.   The choice of monomer (especially the choice of the phosphoramidite amino group) depends on its suitability. Depends on In general, experiments using a commercially available DNA based machine (for example, ABI Model 394) For laboratory-scale (0.01-10 μmol) phosphoramidate oligonucleotide synthesis, Relatively stable and relatively unreactive (this is due to the equilibrium constant K₁Defined by; above It is useful to use monomers, so that this solution can last for several weeks. Over time, it is left in the instrument for multiple synthesis. This use Other reasons why it is preferable to use a relatively less reactive monomer for Instruments of this type are typically designed to minimize the volume of reagents used. There is a fact that it is not measured. In practice, laboratory-scale synthesizers are typically Is controlled by flowing a monomer solution through this reaction vessel (column) to some extent. As some of the soluble product is produced from this column (and Et al.) Are removed, thus helping to drive this equilibrium towards completion. Also, The price of monomers used on this scale depends on other factors (eg, labor costs, convenience, etc.). A) will not be as important. Generally, less reactive monomers (e.g., , Jii (Sopropylamino phosphoramidite) In comparison, a relatively large excess of monomer (10-50 times) must be used, a reasonable Use a large excess of activator to achieve reaction rate and conversion (yield) There is a need.   Minimize the amount of monomer required (equivalent number) to obtain the desired coupling yield In particular, using relatively less reactive monomers, the small-scale When synthesizing the compound, it is very important to perform the coupling step and the oxidation step twice. It is useful in this case, in each of the two coupling steps, a lower concentration ( And low equivalent weight) of the monomer. This is because the coupling reaction is reversible (This is exemplified by the reaction formulas la and lb). Use this method The first coupling in this cycle uses significantly less monomer. And the equilibration of the desired phosphoramidite bonds formed under these conditions. The concentration is then determined by performing the first oxidation step of the cycle, It is "locked-in" as luamidate. Then, a relatively small amount of A second coupling step is carried out again using the nomer, followed by a second oxidation step. The process is performed. To achieve the desired yield using this method, all monomers must be Overall less monomer than when used in a single coupling Only needed.   The economics of large-scale production requires minimizing the amount (equivalent number) of monomers used There is. In this application, labor costs and some other prices are Since it does not rise linearly, the price of this monomer generally Mostly occupy. Large oligonucleotide synthesizers are also generally The model synthesizer is operated in a batch mode rather than a common flow mode, so that The reaction is driven to completion by removing soluble products from the vessel Operation is not practical. In this application, it is relatively reactive (this₁Defined by Higher; see above). Such monomers are Generally, relatively basic and / or sterically hindered phosphoramido It has a ditoamino group. The use of such reactive monomers reduces the use of lower concentrations. The use of an activator makes it possible to achieve a reasonable reaction rate. These Lower concentrations of activator prevent activation of the desired product with the activator and prevent activation. Helps to form an intermediate (Scheme lb). For these reasons, significantly lower amounts (1-5 equivalents) of monomer is required. Generally, use the highest purity monomer. It is very important to use   After this coupling step, the resulting phosphoramidite linkage is oxidized (Sulphided), stable protective phosphoramidate (phosphorothioamidate) bond Form a union. This oxidation step is performed immediately after the coupling solution is discharged from the reaction vessel. Or with a solvent wash in between. This phosphorua Since the midite bond can be hydrolyzed in the presence of tetrazole, the washing solution is Preferably, it is very dry and / or basic. Cleaning process used If not used, the oxidizing solution is basic and / or very dry Is preferred or is sufficiently reactive to be successfully completed by hydrolysis. The agent is selected.   Oxidizing agents useful in the method of the present invention include iodine, chlorine, bromine, peracids (e.g., Lorobenzoic acid), hydroperoxides (e.g., t-butyl hydroperoxide, Tyl hydroperoxide, methyl hydroperoxide, etc.), ozone, mixed reed Rusulfinic anhydride (e.g., 3H-2, l-benzoxathiolan-3-one-l-oxy ), Persulfate salts (eg, sodium persulfate, ammonium persulfate and persulfate) Acid tetrabutylammonium), monoperoxysulfate (e.g., oxone^TM), Sodium and / or other hypochlorites, peroxides (eg, Or bis (trimethylsilyl) peroxide, or hydrogen peroxide or non-aqueous peracid Hydride equivalents (eg, urea / hydrogen peroxide complexes). Phosphorus (111) Other useful oxidizing agents that can be used to convert to phosphorus (V) are Beaucage and Iye It is described in r (above). The sulfurizing agent used in the present invention includes elemental sulfur, thiura Mudisulfide (eg, tetraethylthiuram disulfide), acyl disulphide Sulfides (eg, phenacyl disulfide), phosphinothionyl disulfide (For example, S-Tetra^TM), And 1, l, -dioxo-3H-l, 2-benzodithiol-3-one Is mentioned. Hydrogen peroxide is a preferred oxidizing agent for use in the present invention. preferable In an embodiment, a solution of 1.5% hydrogen peroxide, 3.5% water, 20% pyridine and 75% THF (75%) was used. Use liquid I do.   In one embodiment of the invention, the residue is after the (last) oxidation step of the cycle. Unreacted 3'-amino groups are cleaned with a suitable capping agent before the next detritylation step. Can be encapsulated, they are inert to subsequent coupling steps . This capping step improves the HPLC profile and makes purification easier. And possibly removing unreacted 3 'amino groups from competition at equilibrium. This significantly improves the overall yield of the product. Caps useful in the method of the invention The electrophoretic reagents include electrophilic reagents such as acetic anhydride and isobutyric anhydride, acid chloride (For example, adamantyl carbonyl chloride, pivaloyl chloride, etc.) , Isothiocyanate, chloroformate and the like. Activate before oxidation Phosphoramidites, and acid chlorides (eg, pivaloy) in combination with an agent. Chloride or adamantyl carbonyl chloride). H-phosphonate salts (eg, triethylammonium isopropyl-H-pho Sulfonates) are also useful.   The 3'-amino protecting group in the monomer (eg, trityl) Lower reactivity of the monomer with the phosphoramidite group and the capping agent. However, this protection is not the same as the protected 5'-hydroxy of normal phosphodiester synthesis. Not as complete as in the case of droxyl groups. For this reason, phosphodiesters A slightly less reactive capping agent than acetic anhydride, which is most commonly used in synthesis For example, isobutyric anhydride) is preferred. Either isobutyric anhydride or acetic anhydride Used as a 1: 1: 8 (volume) anhydride: lutidine: tetrahydrofuran solution L-methylimid, supplied by PE Applied Biosystems (Foster City, CA). It can be used in equal parts to a solution of dazole in tetrahydrofuran.   The oligonucleotide is cleaved from the solid support and this strand assembly After completion, use aqueous ammonia or other means as described in the references cited above. And deprotected. The oligonucleotide has its terminal amino protecting group (or In some cases, the hydroxyl protecting group) can be cleaved from the support while remaining intact. This is because trityl or other protecting groups can be used, for example, in reverse phase or ion exchange HP. It is desirable in situations such as those used to aid purification by LC. However , Acid treatment after cleavage from support, deprotection, and purification using trityl protecting group The deprotected phosphoramidate linkage, when removed at At this stage, a great deal of care is needed because of the strong tendency to be subject to change.   Alternatively, a terminal amino protecting group (eg, trityl) is added prior to cleavage from the support. , Can be removed with an acid. In this case, the phosphoramidate linkage is still preserved. Protected and fragmentation is avoided. The oligonucleotide is then supported Cleaved from the body and deprotected as described above. This phosphoramidate oligo Nucleotides can be purified by ion exchange HPLC, reverse phase HPLC or other means .                                 Example 1                   2'- Deoxy-3'-tritylaminothymidine- Five'- Preparation of phosphoramidite monomer   3 '-(trityl) amino-3'-deoxythymidine-5'-(2-cyanoethyl N, N-diiso The synthesis of propyl) phosphoramidite (4t) is outlined in Scheme I. 3'-azide-5 '-0- (4-Methoxybenzoyl) -3'-dioxythymidine (lt) was converted to Czemecki and Vale ry, Synthesis, 1991: 239.                               Scheme I   3 '-(trityl) amino-5'-(4-methoxybenzoyl) -3'-deoxythymidine (2 t). 3'-azido-5'-0- (4-methoxybenzoyl) -3'-dioxythymidine (lt) (10.0 g, 24.9 mmol) in ethanol (500 mL) and in the presence of 10% Pd / C (1.0 g) Hydrogenation (4.2.10^Five Pa, 60 psi H_Two). pull Subsequently, the catalyst was removed by filtration and the solvent was evaporated under vacuum to give a 92% yield (8% (6 g, 22.9 mmol) of the corresponding 3'-amine, which was used directly in the next reaction. This 5 '-(4-methoxybenzoyl) -3'-amino-3'-deoxythymidine (8.6 g, 22.9 m mol) was azeotroped from pyridine (2 × 50 mL) and dissolved in anhydrous pyridine (50 mL). To this solution was added triethylamine (6.7 mL. 48. Mouth ride (7.0 g, 25.2 mmol) was added. The mixture was stirred at room temperature for 2 hours, Additional trityl chloride (1.9 g, 6.9 mmol) was added and the reaction was stirred for an additional 2 hours did. The solvent was removed under vacuum and the crude product was purified on silica (2-5% Me0H / CH_TwoCl_TwoOn) And 90% yield (12.7 g, 20.6 mmol) of 3 '-(trityl) amino-5'-(4-methoxy Benzoyl) -3′-deoxythymidine (2t) was obtained.   3 '-(Trityl) amino-3'-deoxythymidine (3t). 2t (30.lg, 48.7mmol) in 5 7:43 dissolved in l, 4-dioxane / Me0H (150 mL) followed by a 2M aqueous NaOH solution (73 .5 mL, 146.2 mmol) to remove the 5'-0-anisoyl protecting group. Was. After stirring at room temperature for 1.5 hours, the reaction mixture was subjected to Dowex 50W-X8 cation exchange Resin (about 150 g of dried pyridinium H⁺-1.6 meq / g). Once that p When H becomes neutral (about 10 minutes), the resin is filtered and CH_TwoCl_TwoAnd wash thoroughly with Me0H And the crude product was concentrated under vacuum. The residue was redissolved in Et0Ac (500 mL) and And saturated NaHC0_ThreeAqueous solution (2 x 250 mL), H_Two0 (250 mL) and saturated aqueous NaCl (250 mL) Extracted. Na_TwoSO_FourAfter drying and filtering, the solvent is removed in vacuo and the resulting foam 95: 5 CH_TwoCl_Two/ Me0H (300 mL). This solution is rapidly stirred. : 1Et_Two0 / hexane mixture (l250mL) and add pure 3 '-(trityl ) Amino-3'-deoxythymidine (3t) was precipitated in 90% yield (21.2 g, 43.8 mmol) . Conversion to phosphoramidite monomers and / or succinylated nucleosides The conversion is described in Examples 5-7 below.                                 Example 2                 2'- Deoxy-3'-tritylaminocytidine-5'- Preparation of phosphoramidite monomer   N^Four-Benzoyl-3 '-(trityl) amino-2', 3'-dideoxycytidine-5 '-(2-cy Anoethyl N, N-diisopropyl) phosphoramidite (4c) was synthesized according to Scheme II Outlined in                               Scheme II   3'-azido-5'-0- (tert-butyldimethylsilyl) -2 ', 3'-dideoxyuridine, 1du. 2′-Deoxyuridine (11.4 g, 50 mmol) was added under vacuum to anhydrous DMF (2 × 100 mL) And evaporated to dryness. Then, DMF (100 mL) Followed by triethylamine (8.36 mL, 60 mmol), 4-dimethylamino Pyridine (0.31 g, 2.5 mmol) and tert-butyldimethylsilyl chloride (8.29 g, 55.0 mmol). The reaction mixture was stirred at room temperature for 1 hour and dichloromethane ( 600 mL) and H_TwoExtract with O (3 × 200 mL) and saturated aqueous NaCl (200 mL) Was. The organic layer is Na_TwoSO_Four, Filtered, and concentrated in vacuo. Got Purify the residue on silica (2-10% MeOH / CH_TwoCl_Two) To yield 80% (13.7 g, 40.0 mmo 1) 5′-0- (tert-butyldimethylsilyl) -2′-deoxyuridine was obtained. Trihue Nylphosphine (16.8 g, 64.0 mmol) and DMF (100 mL) are added and the stirred mixture In a solution of diisopropyl azodicarboxylate (12.6 mL, 64.0 mmol) in DMF (20 mL) The liquid was added. After stirring at room temperature for 2 hours, the reaction mixture is concentrated under vacuum to about 30 mL And Et_TwoPoured into O (1200 mL). After 10 minutes of rapid stirring, the desired 2,3'-anne Hydro-5'-0- (tert-butyldimethylsilyl) -2'-deoxyuridine begins to precipitate Was. The resulting mixture was placed in the refrigerator overnight, and the precipitate was collected by filtration and additional cold Et_TwoWash with O (2 × 300 mL) and dry under vacuum to give 90% as a white solid. % Yield (11.7 g, 36.0 mmol) of 2,3′-anhydro-5′-0- (tert-butyldimethylsilyl) Lu) -2'-deoxyuridine, which was not further purified. Then, this 2 , 3'-Anhydro-5'-0- (tert-butyldimethylsilyl) -2'-deoxyuridine (33.8 g, 104.2 mmol) with LiN in DMF (300 mL) at 95-100 ° C. for 48 h._Three(7.65g , 156.3 mmol). The resulting brown, homogeneous mixture was then cooled to room temperature. And concentrated under vacuum to an oil, dissolved in EtOAc (800 mL), and_TwoO (200mL) Extracted. The aqueous layer was extracted twice more with EtOAc (75 mL) and the combined organics were extracted with H_TwoO ( 3 × 250 mL) and once with saturated aqueous NaCl (250 mL). This EtOAc Na solution_TwoSO_Four, Filtered and concentrated in vacuo to give a brownish foam As 87% yield (33.2 g, 90.3 mmol) of 3′-azido-5′-0- (tert-butyldimethyl (Silyl) -2 ', 3'-dideoxyuridine (1 du) was obtained and used directly for hydrogenation.   3 '-(trityl) amino-5'-0- (tert-butyldimethylsilyl) -2', 3'-dideoxy Uridine (2du). Crude 1 du (33.2 g, 90.3 mmol) was added to 2: 1 EtOH / CH_TwoCl_Two(300mL) Dissolve and hydrogenate (4.2.10.10) in the presence of 10% Pd / C (3.0 g) for 18 hours.^Five  Pa, 60 psi H_Two). Filtration and evaporation of the solvent under vacuum Depending on the rate, the catalyst is subsequently removed, corresponding to a quantitative yield (30.4 g, 89.8 mmol). To give the 3'-amine which was used directly in the next reaction. This 5'-0- (tert-butyl (Dimethylsilyl) -3'-amino-2 ', 3'-dideoxyuridine (30.4 g, 89.8 mmol) Azeotrope from lysine (2 × 300 mL) and CH_TwoCl_Two(600 mL) and anhydrous pyridine (70 mL) Was dissolved in the mixture. To this solution was added triethylamine (25.0 mL, 179.6 mmol) and And trityl chloride (35.0 g, 125.7 mmol) was added and the reaction mixture was allowed to stand at room temperature for 2 hours. While stirring. The solvent was removed under vacuum and the crude product was purified on silica (1- 1 5% MeOH / CH_TwoCl_Two) To give 85% yield (44.3 g, 75.9 mmol) of 3 '-(trityl) amino-5 '-0- (tert-Butyldimethylsilyl) -2', 3'-dideoxyuridine (2du) was obtained.   N^Four-Benzoyl-3 '-(trityl) amino-5'-0- (tert-butyldimethylsilyl) -2', 3'-dideoxycytidine (2c). 1,2,4- in anhydrous acetonitrile (125 mL) at 0 ° C Of triazole (11.1 g, 161.1 mmol) and phosphorus oxychloride (3.5 mL, 37.1 mmol) To the stirred mixture was added triethylamine (22.5 mL, 161.1 mmol) dropwise over 10 minutes. I dropped it. To this cold stirred mixture was added 2 du (9.4 g, 16 mL) as an acetonitrile (50 mL) solution. .1 mmol) was added. The mixture was stirred at room temperature for 2 hours and triethylamine (30 mL ) And H_TwoO (10 mL) is added to quench the reaction, facilitate dissolution, and evaporate the solvent. Removed under vacuum. The resulting brown solid is CH_TwoCl_Two(250 mL) and saturated NaH CO_Three(3 x 150 mL) aqueous solution, extracted with saturated aqueous NaCl,_TwoSO_Four, Filtered and filtered. And concentrated under vacuum to give a quantitative yield (10.2 g, 16.1 mmol) as an orange-yellow solid. l) 3 ′-(Trityl) amino-5′-0- (tert-butyldimethylsilyl) -2 ′, 3′-dideoxy Ci-4- (1,2,4-triazol-1-yl) uridine was obtained. This crude material is Dissolved in sun (200 mL) and cold concentrated NH_FourOH (50 mL) was added. Bring the reaction mixture to room temperature For 4 hours and concentrated in vacuo to a quantitative solid as a beige solid. Rate (9.4 g, 16.1 mmol) of 3 ′-(trityl) amino-5′-0- (tert-butyldimethylsilyl) ) -2 ', 3'-Dideoxycytidine was obtained. This crude material was then combined with anhydrous pyridine (2 × (200 mL) and redissolved in pyridine (200 mL). Benzoyl (2.2 mL, 19.3 mmol) was added. Then, the reaction system was stirred at room temperature for 16 hours. And cooled to 0 ° C from outside,_TwoO (40mL) After quenching and stirring for 5 minutes, concentrated aqueous ammonia (40 mL) was added and the reaction was mixed. The mixture was stirred at 0 ° C. for another 15 minutes. The solvent was removed under vacuum and the residue was_TwoCl_Two(12 (5 mL) and saturated NaHCO_ThreeExtract with aqueous solution (3 × 75 mL)_TwoSO_FourDry in , Filtered and concentrated under vacuum. This crude material was combined with silica (1-5% MeOH / CH_TwoC l_Two) To give a 92% yield (10.2 g, 14.8 mmol) of N^Four-Benzoyl-3 '-(trityl) Amino-5'-0- (tert-butyldimethylsilyl) -2 ', 3'-dideoxycytidine (2c) Obtained.   N^Four-Benzoyl-3 '-(trityl) amino-2', 3'-dideoxycytidine (3c). 2c (5 .8 g, 8.5 mmol) in 1: 1 CH_TwoCl_Two/ Pyridine (25 mL) and dissolve for 16 hours What, Et_ThreeBy reacting with N.3HF (6.9 mL, 42.6 mmol), the 5′-TBDMS protecting group was Removed. The reaction mixture is CH_TwoCl_Two(200 mL) and diluted with H_TwoO (2 x 50 mL) and Extracted with aqueous NaCl solution (50 mL). Organic layer with Na_TwoSO_Four, Filtered, and solvent removed Was removed under vacuum. The crude product is purified on silica (3% MeOH / CH_TwoCl_Two) Then 75 % Yield (3.7 g, 6.4 mmol) of N^Four-Benzoyl-3 '-(trityl) amino-2', 3'-dideoxy Cytidine (3c) was obtained. Phosphoramidite monomer and / or succini Conversion to fluorinated nucleosides is described in Examples 5-7 below.                                 Example 3                 2'- Deoxy-3'-lithylaminoguanosine- Five'- Preparation of phosphoramidite monomer   N^Two-Isobutyryl-3 '-(trityl) amino-2', 3-dideoxyguanosine-5 '-(2- Anoethyl N, N-diisopropyl) phosphoramidite (4 g) was synthesized according to Scheme I. Outlined in II. 5'-0-benzoyl-N^Two-Isobutyryl-2'-deoxyguanosine and And 3'-0-benzoyl-N^Two-Isobutyryl-2'-deoxyxyloganosine is available from Reese et al. J. Chem. Soc. Prepared as described previously in Perkin Trans. I, 1984: 1263 .                               Scheme III  5'-O- (tert-butyldimethylsilyl) -N^Two-Isobutyryl-3'-azide-2 ', 3'-dide Oxyguanosine (1 g). 3'-O-benzoyl-N^Two-Isobutyryl-2'-deoxygua To a stirred solution of nosin (4.86 g, 11.0 mmol) in DMF (20 mL) was added triethylamine (3.4 mL, 2 mL). 4.2 mmol), 4-dimethylaminopyridine (54 mg, 0.44 mmol) and tert-butyldim Tylsilyl chloride (3.31 g, 22.0 mmol) was added. The reaction was stirred at room temperature for 2 hours. After stirring, adding methanol (10 mL) and stirring for another 5 minutes, the reaction mixture Was concentrated under vacuum. CH residue_TwoCl_Two(150 mL), and_TwoO (3 × 40mL) and Washed with saturated aqueous NaCl (60 mL). Organic layer with Na_TwoSO_Four, Filtered and filtered. Concentration under air afforded 6.40 g of a reddish foam. 2M NaOH pre-cooled (about 5 ° C.) pyridine: MeOH: H_TwoO (65: 30: 5, 44.0 mL, 87.9 mmol) solution was added. The reaction mixture was stirred in an ice bath for 20 minutes and 1M HCl (97%). (mL) to pH7. The reaction mixture is concentrated under vacuum to about 50 mL and CH_TwoCl_Two (3 × 75 mL). Wash the combined organics with saturated aqueous NaCl (50 mL) and add Na_Two SO_Four, Filtered, and concentrated in vacuo to a 82% yield as a sandy foam. Rate (4.1 g, 9.1 mmol) of 5'-O- (tert-butyldimethylsilyl) -N^Two-Isobutyryl-2 ' -Deoxyxyloganosine was obtained, which was used in the next reaction without further purification. Used in. Crude 5'-O- (tert-butyldimethylsilyl) -N^Two-Isobutyryl-2'-deo To xyxiloganosine (47.3 g, 104.7 mmol), LiN_Three(15.4 g, 314.1 mmol), Phenylphosphine (41.2 g, 157.1 mmol) and anhydrous DMF (1000 mL) were added. The Ethyl azodicarboxylate (24.7 mL, 157.1 mmol) was added and the reaction mixed The material was stirred at room temperature under argon for 5 hours. H_TwoO (20 mL) is added and The reaction mixture was concentrated under vacuum. The residue was dissolved in EtOAc (1500 mL) and H_TwoO (3 × 1000mL ), Washed with a saturated aqueous NaCl solution (1000 mL),_TwoSO_Four, Filtered, and under vacuum And concentrated. Purify the residue on silica (1-5% MeOH / CH_TwoCl_Two) Yield of more than 100% (112.7 g) of impure 5'-O- (tert-butyldimethylsilyl) -N^Two- 1 g of isobutyryl-3′-azido-2 ′, 3′-dideoxyguanosine was obtained. This impurity The contained product was not further purified and was used directly for hydrogenation to afford the 3'-amine Purified.   5'-O- (tert-butyldimethylsilyl) -N^Two-Isobutyryl-3 '-(trityl) amino- 2 ', 3'-dideoxyguanosine (2g). Crude compound 1 g (49.9 g, 104.7 mmol) Dissolve in ethanol (1600 mL) and add 16% at room temperature in the presence of 10% Pd / C (2.5 g). Hydrogenated over time (4.2.10^FivePa, 60psi H_Two). The catalyst is removed by filtration and the The medium was evaporated under vacuum to give crude 5'-O- (tert-butyldimethylsilyl) -N^Two- Isobutyryl-3'-amino-2 ', 3'-dideoxyguanosine was obtained, which was (2-6% MeOH / CH_TwoCl_TwoNext to 1% Et_ThreeN / 6% MeOH / CH_TwoCl_Two) Then off As a white foam, 60% yield (28.5 g, 63.2 mmol) of pure 5′-O- (tert-butyl Dimethylsilyl) -N^Two-Isobutyryl-3'-amino-2 ', 3'-dideoxyguanosine Obtained. 3′-amine (28.5 g, 63.2 mmol) in pyridine (500 mL) at room temperature for 16 hours Over time, triethylamine (17.6 mL, 126.4 mmol) and trityl chloride (28 .2 g, 101.1 mmol). The solvent is removed under vacuum, The residue is purified on silica (1-5% MeOH / CH_TwoCl_Two) And quantitative yield (43.8 g, 63.2 mmol) of 5'-O- (tert-butyldimethylsilyl) -N^Two-Isobutyryl-3 '-(trityl ) Amino-2 ', 3'-dideoxyguanosine (2g) was obtained.   N^Two-Isobutyryl-3 '-(trityl) amino-2', 3'-dideoxyguanosine (3 g). 2 g (5.9 g, 8.5 mmol) in CH_TwoCl_Two/ Pyridine (25 mL) and added for 16 hours. And Et_ThreeThe 5'-TBDMS protecting group was removed by reacting with N3HF (6.9 mL, 42.6 mmol). I left. The reaction mixture is CH_TwoCl_Two(200 mL) and diluted with H_TwoO (2 x 50 mL) and saturated Extracted with aqueous NaCl (50 mL). Organic layer with Na_TwoSO_Four, Filter and remove the solvent Removed under vacuum. The crude product is purified on silica (2-5% MeOH / CH_TwoCl_Two) Then 7 3% yield (3.6 g, 6.2 mmol) of N^Two-Isobutyryl-3 '-(trityl) amino-2', 3'-dide 3 g of oxyguanosine was obtained. Phosphoramidite monomer and / or screen Conversion to a silylated nucleoside is described in Examples 5-7 below.                                 Example 4                 2'- Deoxy-3'-tritylaminoadenosine- Five'- Preparation of phosphoramidite monomer   N⁶-Benzoyl-3 '-(trityl) amino-2', 3'-dideoxyadenosine-5 '-(2- Synthesis of anoethyl N, N-diisopropyl) phosphoramidite (4a) Outlined in V. N⁶-Benzoyl-9- (5-O-benzoyl-2-deoxy-β-D-threo-pe (Nantafuranosyl) adenosine is described in Herdewijn, J .; Org. Chem., 53: 5050 (1988). Synthesized by the method.                                 Scheme IV  5'-O- (benzoyl) -N⁶-Benzoyl-3'-azido-2 ', 3'-dideoxyadenosine, 1a. N⁶-Benzoyl-9- (5-O-benzoyl-2-deoxy-β-D-threo-pentofurano (Sil) adenosine (7.0 g, 15.3 mmol), triphenylphosphine (6.0 g, 22.9 mmol) ) And LiN_Three(2.8 g, 56.4 mmol) was dissolved in DMF (100 mL). To this stirred mixture, Diethyl azodicarboxylate (3.6 mL, 22.9 mmol) was added all at once, The reaction was stirred at room temperature for 2.5 hours and the reaction was_TwoQuenched with O (10 mL). Solvent Removed under vacuum, redissolved the residue in EtOAc (300 mL) and_TwoO (2 x 200 mL) and Extracted with saturated aqueous NaCl (200 mL). Organic layer with Na_TwoSO_Four, Filtered, and Concentrated under vacuum. The obtained residual oil is SiO_TwoPurified on (2-5% MeOH / CH_TwoCl_Two) Purified And 5'-O-benzoyl-N as an amber foam⁶-Benzoyl-3'-azide-2 ', 3'- Dideoxyadenosine (1a) was obtained, which was used directly for hydrogenation.   N⁶-Benzoyl-3 '-(trityl) amino-2', 3'-dideoxyadenosine (3a). Crude 1a to 3: 1 EtOH / CH_TwoCl_Two(200 mL) and in the presence of 10% Pd / C (0.7 g) , 18 hours, hydrogenation (4.2.10^Five Pa, 60psi H_Two). Filtration and The catalyst was subsequently removed by evaporation of the solvent under vacuum and vacuum to give 57% (4.0%). g, 8.7 mmol) of the corresponding 3'-amine, which was used directly in the next reaction. Five'- O-benzoyl-N⁶-Benzoyl-3'-amino-2 ', 3'-dideoxyadenosine (3.9 g, 8 .5 mmol) in CH_TwoCl_Two(50 mL) and triethylamine (2.9 mL, 20.8 mmol) was added. This was followed by the addition of trityl chloride (2.9 g, 10.2 mmol). Stir at room temperature for 1.5 hours After stirring, the reaction mixture was_TwoCl_Two(50 mL) and H_TwoO (2 x 50 mL) and Extracted with aqueous NaCl solution (50 mL). Organic layer with Na_TwoSO_FourDry, filter, and vacuum Concentrate under the crude 5'-O-benzoyl-N⁶-Benzoyl-3 '-(trityl) amino-2', 3 '-Dideoxyadenosine, which is directly converted to the hydrolysis of the 5'-benzoyl protecting group. used. Crude 5'-O-benzoyl-N⁶-Benzoyl-3 '-(trityl) amino-2', 3'-di Deoxyadenosine (about 6.0 g, 8.5 mmol) was dissolved in THF / MeOH (1: 1, 100 mL), And it cooled to 0 degreeC. To this mixture was added a pre-cooled 2M aqueous NaOH solution (1 (3.7 ml, 27.4 mmol) was added and the reaction mixture was stirred at 0 ° C. for 20 minutes. at this point, The reaction appeared to be only about 50% complete, so additional 2 M aqueous NaOH (10.0 mL, 2 mL) was added. 0 mmol) was added. After stirring at 0 ° C. for a further 15 minutes, the reaction was washed with Dowex 50W-X8 Thion exchange resin (about 40 g of dry pyridinium H⁺-Form, 1.6meq / g) neutralized to pH 6 did. The resin is filtered and washed thoroughly with MeOH and THF, and the solvent is removed in vacuo. Removed below. CH residue_TwoCl_Two(300 mL) and H_TwoO (150 mL), saturated NaH CO_ThreeAqueous solution (2 x 150 mL), H_TwoExtracted with O (150 mL) and saturated aqueous NaCl (150 mL). N a_TwoSO_FourAfter drying with, the solution is filtered And concentrated under vacuum. Residue is SiO_TwoPurified on (2-3% MeOH / CH_TwoCl_Two) To give a 71% yield (3.6 g, 6.0 mmol) of N as a white foam.⁶-Benzoyl-3 '-(tri Cyl) amino-2 ', 3'-dideoxyadenosine (3a) was obtained. Phosphoramidite Conversion to nomers and / or succinylated nucleosides is described in Examples 5-5 below. Described in 7.                                 Example 5             3'- (Trityl) amino-2 ', 3'-dideoxynucleoside- 5 '-(2- Cyanoethyl N, N-diisopropyl) phosphoramidite, 4a , 4c, 4g and 4t preparation   CH_TwoCl_Two3 ′-(Trityl) amino-2 ′, 3′-dideoxynucleoside (3a, 3 c, 3g and 3t) (CH_Three8.4 mmol, previously azeotroped twice from CN) under argon 2.0 mL (11.8 mmol) of N, N-diisopropylethylamine and 2-cyanoethyl N, N 2.1 mL (9.4 mmol) of -diisopropylchlorophosphoramidite was added. 15 minutes After stirring, the reaction system was_TwoCl_TwoDiluted with and saturated NaHCO_ThreeAqueous solution and saturated NaCl Extracted with aqueous solution. Dry the organic layer (Na_TwoSO_Four), Filtered and concentrated in vacuo . N⁶-Benzoyl-3 '-(trityl) amino-2', 3'-dideoxyadenosine-5 '-(2- Anoethyl N, N-diisopropyl) phosphoramidite (4a)_TwoPurified on (5 % Et_ThreeN / 2% methanol / toluene yielded 5.82 g of pure phosphoramidite (87.1%). %)was gotten.³¹P NMR (CD_ThreeCN) δ 148.6, 149.2. N^Four-Benzoyl-3 '-(trityl) Amino-2 ', 3'-dideoxycytidine-5'-(2-cyanoethyl N, N-diisopropyl) Suformamidite (4c), SiO_TwoPurified on (3% MeOH / 5% Et_ThreeN / toluene) This gave 5.58 g (86.0%) of pure product.³¹P NMR (CD_ThreeCN) δ 149.3, 149.6. N^Two -Isobutyryl-3 '-(trityl) amino-2', 3'-dideoxyguanosine-5 '-(2-sia Noethyl N, N-diisopropyl) phosphoramidite (4 g) H_TwoCl_Two(10 mL) to ethyl ether (200 mL) and Precipitate in xane (200 mL) and remove the hydrogen phosphonamidate impurity. (Which in this case cannot be removed by column chromatography) Was. The solid is filtered, washed with hexane, And dried under vacuum. This precipitation step is repeated, and the resulting solid is treated with SiO 2_Two Further purification on (10% Et_ThreeN / CH_TwoCl_TwoThe pure phosphoramidite product 4.5 1 g (69%) was obtained.³¹P NMR (CD_ThreeCN) δ 148.7, 149.4. 3 '-(trityl) amino-3 '-Deoxythymidine-5'-(2-cyanoethyl N, N-diisopropyl) phosphoramidi (4t) SiO_TwoFurther purification (2% Et_ThreeN / CH_TwoCl_TwoThen, pure phosphoramid 4.06 g (70.7%) of daite and some mixed fractions were obtained.³¹P NMR (CD_ThreeCN) δ14 9.4, 149.5.                                 Example 6           3 '-( (Trityl) amino-2 ', 3'-dideoxynucleoside-5'- (2- Cyanoethyl-2,2,6,6-tetramethylpiperidine)               Preparation of phosphoramidites, 5a, 5c, 5g and 5t:   CH cooled to 4 ° C_TwoCl_Two3 '-(Trityl) amino-2', 3'-dideoxy in (25 mL) Nucleosides (3a, 3c, 3g and 3t) (this is CH_ThreePre-azeotrope twice from CN 8.4 mmol, under argon, DBU (1.9 mL, 12.6 mmol) and 2-cyanoethyl CH of 2,2,6,6-tetramethylpiperidine chlorophosphoramidite_TwoCl_TwoSolution 5.2 m L (8.4 mmol) (concentration = 1.626 mmol / mL) was added. Remove the ice bath and leave the solution for 30-60 minutes While stirring. To avoid decomposition, the crude solution was purified with SiO_TwoColumn (5a, 5 3% MeOH / 5% Et for c and 5t_Three10% Et for N / toluene and 5g_ThreeN / CH_Two Cl_Two). In each case, as indicated for each product, Further purification was required. N⁶-Benzoyl-3 '-(trityl) amino-2', 3'-dideo Xyadenosine-5 '-(2-cyanoethyl 2,2,6,6, -tetramethylpiperidine) phospho Luamidite (5a), SiO_Two(3% MeOH / 5% Et_ThreeN / toluene) 5.21 g (74.3%) of pure phosphoramidite were obtained.³¹P NMR (CD_ThreeCN) δ164.8,165 .Four. N^Four-Benzoyl-3 '-(trityl) amino-2', 3'-dideoxycytidine-5 '-(2-cy (Anoethyl 2,2,6,6-tetramethylpiperidine) phosphoramidite, 5c, SiO_Two Purified on (3% MeOH / 5% Et_ThreeN / toluene) yielded 5.07 g (74.2%) of pure product and And some mixed fractions were obtained.³¹P NMR (CD_ThreeCN) δ 164.8, 165.7. N^Two-Isobuti Lil-3 '-(trityl) amino-2', 3'-dideoxy Guanosine-5 '-(2-cyanoethyl 2,2,6,6, -tetramethylpiperidine) phosphorua Mididite (5 g) was added to CH under argon._TwoCl_Two(2 mL) and rapidly stirred at 4 ° C. Precipitated in ethyl ether (40 mL) and hexane (40 mL). Amidate impurities (which in this case could be removed by column chromatography) Was removed. Filter the solid, wash with hexane and dry under vacuum did. This precipitation step is repeated twice and the resulting solid is treated with SiO 2_TwoMore elaborate on (10% Et_ThreeN / CH_TwoCl_Two) Gives 0.89 g (12.8%) of pure phosphoramidite product was gotten.³¹P NMR (CD_ThreeCN) δ 165.2, 165.5. 3 '-(trityl) amino-3'-deoxy Cythymidine-5 '-(2-cyanoethyl 2,2,6,6, -tetramethylpiperidine) phosphorua Midite (5t) SiO_TwoPurified on (5% MeOH / 5% Et_Three(N / toluene) 3.33 g (54.8%) of suformamidite were obtained.³¹P NMR (CD_ThreeCN) δ 165.3, 166.1.                                 Example 7           3 '-( (Trityl) amino-2 ', 3'-dideoxynucleoside-5'- Preparation of succinilate, 6a, 6c and 6t:   CH_TwoCl_Two3 '-(Trityl) amino-2', 3'-dideoxynucleoside (3t, 3t) in 5 ml). c and 3a) To 1.5 mmol, 0.22 g (1.8 mmol) of N, N-dimethylaminopyridine is added, Then, 0.18 g (1.8 mmol) of succinic anhydride was added. After stirring at room temperature for 1 hour, The reaction was quenched by the addition of 0.6 mL of methanol and CH_TwoCl_TwoDiluted with, and cold 1 Extracted with 0% citric acid, water and saturated aqueous NaCl. Dilute the organic layer (Na_TwoSO_Four), Filtered and concentrated to a foam. N⁶-Benzoyl-3 '-(trityl) amino-2' , 3'-Dideoxyadenosine-5'-succinylate (6a). Yield 1.15 g (100%). N^Four- Benzoyl-3 '-(trityl) amino-2', 3'-dideoxycytidine-5'-succinylene G, 6c. Yield 0.77 g (76.3%). 3 '-(trityl) amino-3'-deoxythymidine-5'- Succinilate (6t). Yield 0.82 g (94.0%).                                 Example 8           3 '-( (Trityl) amino-2 ', 3'-dideoxynucleoside-5'- Preparation of succinyl-loaded CPG   3 ′-(Trityl) amino-2 ′, 3′-dide in 5 mL of N-methylpyrrolidine and 5 mL of DMSO 1 mmol of oxynucleoside-5'-succinylate (6a, 6c or 6t) and 1-hydro 0.13 g (0.95 mmol) of xybenzotriazole was added to N, N-diisopropylethylamine 0.35 mL (2.0 mmol) was added, followed by 2- (1H-benzotriazol-1-yl) -1,1,3, Add 0.36 g (0.95 mmol) of 3-tetramethyluronium hexafluorophosphate did. The solution was stirred for 5 minutes, added to aminopropyl-CPG (10.0 g) and shaken Placed in a bowl for 6 hours. CPG is filtered and DMF, methanol and ethyl ether And washed with water. Unreacted amino groups on the CPG are removed by standard ABI Acetylation was carried out using a solution for ripening. Nucleoside addition is 40.7umol^-1cm^-1No 20% TFA / CHCl using the extinction coefficient_ThreeIn a trityl assay at 432 nm It was measured. 38.6 umol / g for A, 33.6 umol / g for C and 29.0 u for T mol / g.                                 Example 9                   Tetazole activation equilibrium constant K ₁ Measurement   The equilibrium represented by the reaction formula 1 is expressed by N⁶-Benzoyl-3'-tritylamino-deoxyadeno Syn-5 '-(2-cyanoethyl N, N-diisopropyl) phosphoramidite, (4a, 56.1m g, 65.2 μmol) in 0.300 mL of anhydrous deutero acetonitrile. , And by adding 0.300 mL of a 0.5 M solution of tetrazole in acetonitrile , All were studied under an argon atmosphere. Transfer the solution under argon to a test tube for NMR. Moved to After 2 minutes, shown in FIG.³¹P-NMR spectra were recorded.   Spectra were phosphoramidite monomer (143.07, 143.72 ppm), tetrazo Lysyl-amidite intermediate (127.58 ppm), (via tetrazolidyl amidite intermediate Hydrogen phosphonate (10.24, 9.61 ppm) obtained from partial hydrolysis of the monomer) And minor resonances corresponding to side reactants. The total sum of these species is Estimated to be equal to the initial phosphoramidite concentration of 0.0993M, the concentration at equilibrium Was calculated from the relative integration of the individual resonances. It does not contain phosphorus and is therefore The concentration of species not appearing in torr was calculated as follows. Diisopropyl ammo The concentration at equilibrium (0.0824 M) of the potassium tetrazolide It was assumed to be equal to the initial concentration of the mer (0.0993M)-the equilibrium concentration (0.0169M). Tetrazo Concentration at equilibrium (0.0875 M) is the initial tetrazole concentration (0.2286 M) Zolidyl amidite intermediate concentration (0.0587M) and diisopropylammonium It was assumed to be equal to the sum of the trazolide concentrations (0.0824M). Activation equilibrium constant K₁To It was calculated as follows:   The above experiment was performed using 3'-tritylaminothymidine-5'-diisopropylaminophosphor Repeat with amidite monomer (4t) and K₁Is 56.2M^-1That it is Do you get it. Experiments were performed with 3'-tritylaminothymidine-5-tetramethylpiperidinyl Repeated again using the formamidite monomer (5t). The 31P-NMR spectrum This is shown in FIG.   Under the conditions of this experiment, tetramethylpiperidinyl pho Sformamidite monomer (expected 165.3, 166.1 ppm) was not detected Was. A monomer has a noise level in its spectrum if its concentration is at least 0.19 mM. It should be detectable above the bell. From this information, the K of this equilibrium₁Is at least 5260 M^-1That is, diisopropylamino phosphoramidite It can be calculated that it must be 93 times greater than the value of the monomer. Experiment, 3'- Tritylaminothymidine-5 '-(N-isopropyl-N-t-butyl) phosphoramidi Repeated one more time with trimonomer. Tetramethylpiperidinyl phosphor As with the amidite monomer, this monomer, at equilibrium,³¹P-NMR It could not be detected in the spectrum.                                 Example 10           Using diisopropylamino phosphoramidite monomer Oligo-2'-deoxynucleotide N3 '→ P5' phosphoramidate 1 μmol scale synthesis   On an ABI 392 DNA synthesizer, the oligonucleotide N3 '→ P5' Prepare foramidate and purify by preparative ion exchange chromatography did. The synthesis was carried out in a column with 3 ′-(trityl) amino-2 ′, 3′-dideoxynucleotide. Using Cid-5'-succinyl-added CPG, (3 'programmed on a commercial synthesizer Perform in the 5 '→ 3' direction (not in the 5 'direction). 3'-tritylamino-5'-diisopropylpho Sformamidite monomer was prepared as a 0.1 M solution in acetonitrile. Activity The sensitizing solution was 0.5 M tetrazole in acetonitrile (PE Applied Biosys tems, Foster City, CA). The detritylation solution is 3% in dichloromethane (DCM) Dichloroacetic acid (DCA) and the oxidation solution was tetrahydrofuran / pyridine / 0.1 M iodine in water (75/20/2, v / v / v) solution (PE Applied Biosystems, Fos ter City, CA).   Following detritylation, from coupling, oxidation, coupling and oxidation strategies Oligonucleotide N3 '→ P5' phosphoramide Was synthesized. Detritylation of the 3'-amine of the support-bound nucleoside is 3% D CA / DCM for 40 seconds for G and A nucleosides, and T and C nucleosides For 50 seconds. Then, the support-bound 3'-aminonu The creoside was delivered for 10 seconds with acetonitrile delivery / 5 seconds with argon flow. Washed 6 times with a brush combination. Amine and 5'-phosphoramidite-3'-trityl Coupling with aminonucleosides is achieved by combining monomer + torazole and tetrazo Delivery to the column alone, for about 10 seconds followed by a 5 minute wait More achieved. The monomer is flushed from the column with argon and then The iodine solution was immediately added, followed by a 2 minute wait. When oxidation is complete, Growing support-bound oligomers were treated with acetonitrile delivery for 20 seconds / 5 seconds Wash once with Lugon Flush combination and deliver acetonitrile for 10 seconds / 5 seconds Washed 5 times with a combination of argon flush during. No more coupling and oxidation Repeated once, followed by washing before detritylation. Using this procedure, For each coupling step, approximately 15 equivalents (initial addition of support-bound nucleoside and (Compared to) monomer, and therefore each synthesis cycle requires about 30 equivalents of monomer. Is used.   Upon completion of the synthesis, the support-bound 3'-detritylated oligonucleotide N3 '→ P5' Deliver phosphoramidate and place in concentrated aqueous ammonia at 55 ° for 12 hours. The base was deprotected. Cleaved and deprotected oligonucleotide N3 '→ P5' The phosphoramidate solution is removed from the CPG and the CPG is washed twice with 200 μl of ammonia Was cleaned. Combine all ammonia washes, buffer solution to 0.01 M NaOH, and Ammonia was removed under vacuum. Following filtration, the crude oligonucleotide was , Purified by a preparative anion exchange column (Pharmacia MonoQ 10/10), and separated by Sephadex G-25 (P desalted on harmacia NAP-5) and lyophilized.   The sequences listed below may be used as the phosphoramidite amine exchange method of the present invention, or Atherton-Todd oxidative coupling approach (e.g., Letsinger et al. U.S. Patent No. 5,476,925) was synthesized on an ABI 392 synthesizer. . The results are tabulated below and are shown in FIGS. 3a and 3b, which are the two listed in the table. Figure 3 shows a chromatogram of an ion exchange HPLC separation of mixed base sequences of species.   1 The sequence synthesized by the Atherton-Todd oxidative coupling method is 3'-terminal water While it has an acid group, the sequence synthesized by the amine exchange method has a 3'-terminal It has an amino group.                                 Example 11               Using diisopropylamino phosphoramidite Oligo-2'-deoxynucleoside N3 '→ P5' phosphoramidate 1 μmol scale synthesis Monomer: ratio of coupling / oxidation and coupling / oxidation / coupling / oxidation Comparison:   The effect of a single coupling / oxidation step on product yield is as follows: The double coupling / oxidation process (coupling / oxidation / coupling / oxidation) Compared to that: The sequence of 5'-AAC-ATG-GAG-AGC-GTC-3 '(SEQ ID NO: 7) was Synthesized using isopropylamino phosphoramidite monomer, and An example Compared to a second synthesis similar to the above except for the following: 1) per cycle (not twice) D) a single coupling / oxidation was used, and 2) the concentration of the monomer solution was (0. 0.2M (instead of 1M). Therefore, in a single coupling / oxidation experiment, In the synthesis cycle, about 30 equivalents of monomer were used, whereas coupling / acid For the coupling / coupling / oxidation experiments, approximately 15 equivalents were obtained in each of the two coupling steps. About 30 equivalents of monomer per synthesis cycle It was used. The results shown below show the efficiency using the 2x (coupling / oxidation) method Has been improved.                                 Example 12         Oligo-2'-deoxynucleoside N3 '→ P5' phosphoramidate 1 μmol scale synthesis: uncapped, acetic anhydride capped and Comparison of isobutyric anhydride capping   The sequence of 5′-AAC-ATG-GAG-AGC-GTC-3 ′ (SEQ ID NO: 7) was converted to 1 using the above procedure. The synthesis was performed three times on a μmol scale, but two of these synthesis were performed in each cycle. After the last oxidation step and before the detritylation step, cap for 60 seconds. A chemical conversion step was inserted. In one of these experiments, acetic anhydride was used as the capping reagent. / NMI (PE Applied Biosystems, Foster City, CA) and, on the other hand, no Water isobutyric acid (1/1/8 isobutyric anhydride / 2,6-lutidine / THF) / NMI (PE Applied Biosy stems, Foster City, CA). The results shown below are It demonstrates improved yield and purity.                                 Example 13Oligo-2'-deoxynucleoside N3 '→ P5' phosphoramidate 1 μmol scale synthesis: comparison of I ₂ and H ₂ O ₂ oxidation   The following sequences were synthesized on a 1 μmol scale using the above procedure. However, 1 In a pair of experiments, the iodine oxidizer was changed to 1.5% H_TwoO_Two/3.5%H_TwoO / 20% pyridine / 75% THF Oxidizing agent. No capping agent was used in these experiments .                                 Example 14         Oligo-2'-deoxynucleoside N3 '→ P5' phosphoramidate Ten μmol scale synthesis: diisopropyl and Comparison of tetramethylpiperidinyl phosphoramidite monomers   The following general procedure was followed for the synthesis on a 10 μmol scale: modified 390Z ABI DN A On a 10-umol scale, synthesizer N3 '→ P5' phosphoramide A plate was prepared and purified by preparative ion exchange chromatography. 3'- Tritylamino 5'-phosphoramidite monomer was dissolved in 0.1 M solution in acetonitrile. It was prepared as a liquid. The activator was 0.15M tetrazole in acetonitrile. Was. The detritylation solution is 3% dichloroacetic acid (DCA) in dichloromethane (DCM). And the oxidation solution is in tetrahydrofuran / pyridine / water (75/20/2, v / v / v) 0.1 M iodine (PE Applied Biosystems, Foster City, CA).   Batch type iterative synthesis cycle consisting of detritylation followed by coupling and oxidation. Oligonucleotides were synthesized using a loop. List the individual modules, Combined to form a complete synthesis cycle. More specifically, this The synthesis was performed using 10 μmol of 3 ′-(trityl) amino-2 ′, 3′-dideoxynucleoside-5′-s The reaction is performed in the 5 ′ → 3 ′ direction using a xyl-added CPG. Detritylation of 3'-amines , Followed by a repetitive flow of 3% DCA / DCM to the top of the reaction vessel, followed by 3 seconds of stirring and And emissions. The total acid exposure time was approximately 2 minutes. Then The support-bound 3'-aminonucleoside was added from the bottom and top of the reaction vessel. Were washed 10 times with a series of alternating washes with agitation and drainage. Free amine obtained (Probably its dichloroacetate) and 3 '-(trityl) aminonucleoside-5'-phos Coupling with foramidite monomer allows for initial delivery of monomer to the reactor This was followed using tetrazole delivery. The coupling mixture is then Stirred for 5 minutes. After draining the reaction vessel, add the iodine solution immediately. And stirred for 2 minutes. Drain the oxidizing solution and react the support A series of alternating acetonitriles from the bottom and from the top of the vessel with stirring and draining The plate was washed 10 times by the trill washing.   When synthesis is complete, the 3'-deprotected oligonucleotide is cleaved from the support and And deprotected in concentrated aqueous ammonia at 55 ° for 12 hours. 0.01M solution Buffered in NaOH and the ammonia was removed under vacuum. Following filtration, the crude The solution was purified on a preparative anion exchange column (Pharmacia MonoQ 10/10), desalted, And freeze-dried.   Using the above method, different concentrations (and thus different equivalent numbers) of tetrazole, Diisopropylaminophosphoramidite monomer or tetramethylpiperidi With any of the phosphoramidite monomers, 5'- TT-3 'dimer was synthesized. The result is a low potency diisopropylaminophosphor Tetramethylpiperidinyl phosphoramida is more potent than amidite monomer Phosphoramidite synthesis can be carried out using very small equivalents of site monomers This is clearly shown.                                Example 15         Oligo-2'-deoxynucleoside N3 '→ P5' phosphoramidate Ten μmol scale synthesis   Using the above procedure, and using the following amounts of tetramethylpiperidinyl phosphoramida The following sequence was synthesized on a 10 μmol scale using the site monomer.   1 This oligo is a support having a terminal trityl group remaining on the dimer. Disconnected.   2 This HPLC yield is the benzene formed from the deprotection of the benzoyl protecting group on the base. Corrected for amide.                                 Example 16         N ^4- Benzoyl-2'-fluoro-3 '-(4-methoxytrityl) amino- 2 ', 3'- Dideoxyuridine-5'- (2- Cyanoethyl N, N-diisopropyl) phosphoramidite   The 2'-fluoro nucleoside phosphoramidite monomer of the present invention is represented by the scheme Prepared as exemplified in V and VI. In summary, ribonucleosides are 5'- Converted to hydroxyl-protected-2 ', 3'-amhydroxyl nucleoside, followed by 2' The 3,3'-epoxy ring is opened by treatment with a reagent such as sodium azide. Forms a 5'hydroxyl-protected-3'-azide-3'-deoxyarabinonucleoside You. After purification, 5'-hydroxyl-protected-3'-azide-3'-deoxyarabinonucleoside Treated with a reagent such as diethylaminosulfatrifluoride (DAST). And fluorinates at the 2 ′ position, and then reduces the azide group to give 3′-amino obtain. After appropriate protection of the 3'-amino and release of the 5'-hydroxyl protecting group, Osido is phosphitylated with 5'-oxygen to give the crude phosphoramidite monomer Get.                               Scheme V   Referring to Scheme V, uridine 3 is mesylated and then described in the literature. Procedures (e.g., Codington, J.F .; Fecher, R .; Fox, JJJ Am. Chem. Soc. 19). 60, 82, 2794-2803) to give 2,2 ′ by treatment with sodium benzoate. -Selectively benzoylated, with formation of an anhydro ring. Of these reactions As a result, compound 7 was obtained with a total yield of 69 to 77%. Other literature methods (Codington, J. F .; Fecher, R .; Fox, J.J. J. Organic Chem. 1962, 27, 163-167), 2,3'- Anhydroarabinonucleoside 7 can be converted to 2 ', 3'-anhydrolyki in two steps. Converted to Souridine 8. For this, 7 is treated with hydrochloric acid to give 3'-mesyl-5'-benzo. It is involved in the formation of ilarabinouridine, which is Lyso-2 ′, 3′-epoxide in 63-77% overall yield upon treatment with 8 is formed. Then, also refer to the published procedures (Reichman, U .; Hollenberg, D.H .; Chu, C.K .; Watanabe, K.A .; Fox, J.J. J. Organic Chem. 1976, 41, 2042-204 According to 3), 2 ', 3'-anhydrolyxonucleoside 8 is converted to ammonium azide Heat together. Contrary to literature suggestions, this reaction is not completely stereoselective. Without the desired 5'-benzoyl-3'-azidoarabininonucleotide in a ratio of approximately 2.5: 1. Chromatography of Sid 9 and its 2'-azido-2'-deoxy regioisomer 9i A mixture was formed that could not be resolved by chromatography. Crude arabinonucleoside 9 was converted to DA Fluorination with ST to give 2′-fluoro-3′-azidonucleoside 10, followed by catalyst To give 2'-fluoro-3'-azidonucleoside 11, which is It could be separated from its regioisomer by Kagel chromatography. 3'- Protection of the amine with the monomethoxytrityl (MMT) group followed by 5 'monodebenzoylation Yields intermediate 13 which, by 5′-phosphitylation, yields anhydronucleoside The desired phosphoramidite building block 2u is formed in a total yield of 22% from Was. More specifically, these steps were performed as follows:   3'-O-methanesulfonyl-5'-O-benzoyl-2,2'-anhydroarabinouridine 7 was prepared according to the procedure of Codington et al. (Cited above, J. Organic Chem. Soc.). Prepared in 3 to 2 steps with 69-77% yield.   5'-O-benzoyl-2 ', 3'-anhydroxyuridine 8 is described in Codington et al. (Cited above). J. Am. Chem.), And prepared in 7 to 2 steps in 63-77% overall yield. Was made.   3'-Azido-5'-O-benzoyl-3'-deoxyarabinouuridine 9 was obtained from Reichman et al. 8 and anhydrous NH._FourN_Three(Obenland, C.O. Mangold, D.J .; Marino, M.P. Inorg. Synth. 1966, 8, 53-56 ), But could not be recrystallized successfully. The mass yield was 98% or more, but NMR was Is the regioisomer 2'-azido-5'-O-benzoyl-2'-deoxyxyluridine, 9i (which eluted with the desired product by silica gel TLC) at 25-35% There was a suggestion. Principal component¹H NMR 9:   2'-Fluoro-3'-azido-5'-O-benzoyl-2 ', 3'-dideoxyuridine 10 is Prepared as follows: anhydrous CH_TwoCl_TwoCrude 9 (containing 25% 9i) in (30 mL) To 5.0 g (13.4 mmol), 8.8 mL (66.6 mmol) of diethylaminosulfatrifluoride Was added. After stirring for 48 hours, the mixture was_TwoCl_Two(100 mL) and saturated N aHCO_ThreeThe solution was poured into 200 mL. When gas generation is over, CH_TwoCl_TwoLayer with new Na HCO_ThreeWashed with 100 mL of solution and then with water (2 × 100 mL). CH_TwoCl_TwoLayer under vacuum And chromatographed on flash chromatography. 3.5 g of a product containing 10i (20%), an isomer impurity which cannot be resolved %)was gotten.¹H NMR main component 10:  2'-Fluoro-3'-amino-5'-O-benzoyl-2 ', 3'-dideoxyuridine 11 is Prepared as follows: 3.5 g of crude 10 (20% of 10i) in 200 mL of 95% ethanol To (9.3 mmol) was added 600 mg of 10% palladium on carbon. 2.10 suspension^FivePa (40 (psi) overnight, then the catalyst was removed by filtration. Solvent under vacuum A light yellow solid consisting of two compounds which can be removed by TLC 3 g (90%) were obtained. Flash chromatography gives a pure white solid This gave 1.96 g (60% yield) of the desired product. Mass spectrum, FAB⁺, M + H⁺Is 350 .1152 (calculated value) and 350.1152 (actual value).   2'-fluoro-3 '-(4-methoxytrityl) amino-2', 3'-dideoxyuridine 12 is Was prepared as follows: 11 (1.0 g, 2.9 mmol) in 50 mL of anhydrous pyridine, 1.0 g (3.2 mmol) of 4-methoxytrityl chloride was added. Stir the mixture overnight , Saturated NaHCO_Three5 mL of an aqueous solution is added and the mixture is concentrated under vacuum to an oil Was. Dissolve the oil in 125 mL of ethyl acetate, wash it with water (3 × 100 mL), and Reconcentrate under vacuum to 2.05 g foam.   The foam was dissolved in a mixture of 40 mL of methanol, 40 mL of dioxane and 10 mL of water . NaOH (1 g, 25 mmol) was added and the mixture was stirred overnight. The solution under vacuum Concentrate to a syrup, dissolve in 100 mL of ethyl acetate, and add water (3 × 100 mL) And washed. Concentration of the organic layer under vacuum gave 1.11 g of a foam which was filtered. Lash chromatography provided 1.05 g (76%) of a white solid. Mass spectrum, FAB⁺, M + H⁺Are 518.2091 (calculated value) and 518.2076 (actual value). Was.   N^Four-Benzoyl-2'-fluoro-3 '-(4-methoxytrityl) amino-2', 3'-dideoxy Ciuridine 5 '-(2-cyanoethyl N, N-diisopropyl) phosphoramidite 2u Was prepared as follows: anhydrous CH_TwoCl_Two12 (700 mg, 1.35 mmol) in (20 mL) In addition, 200 mg (1.17 mmol) of diisopropylammonium tetrazolide and 2-cyano 0.5 mL (1.57 mmol) of ethyl N, N, N ', N'-tetraisopropyl phosphorodiamidite Was added. After stirring the mixture for 3 hours, the solvent was removed in vacuo and the residue was removed. Using a 4mm plate, and CH_TwoCl_Two0-3% methanol, 0.5% triethylamide Purified on Chromatotron eluting with Concentrate the product under vacuum to an oil , This, CH_TwoCl_Two(10 mL) and slowly stirred into 100 mL of rapidly stirring hexane. Precipitation was achieved by addition. After decanting the supernatant, the product is_TwoO_Fiveso Vacuum drying afforded 680 mg (70%) of a white powder. Mass spectrum, FAB⁺, M + H⁺Is 718.3170 (calculated value) and 718.3194 (actual value).                                 Example 17         N ^4- Benzoyl-2'-fluoro-3 '-(4-methoxytrityl) amino- 2 ', 3'- Dideoxycytidine 5 '-(2- (Cyanoethyl N, N-diisopropyl) phosphoramidite   Crude intermediate 10 was appropriately protected as shown in Scheme VI below. It was used for the preparation of suformamidite 2c. Ten uracil bases are Divakar and Re ese, J. Chem. Soc., Perkin. Trans. 1 Apply the method of 1982, 1171-1176. And was converted to cytosine. Subsequent 4-N benzoylation and 3'-azide Reduction to the amino group gave compound 13, which was chromatographed on silica gel. By the fee, it was separable from the positional isomer. For protection of 3'-amine with MMT group Subsequent selective 5'-O-debenzoylation yielded intermediate 15. Continue 5 '-Phosphitylation to give a total yield of 10% based on anhydronucleoside 8 This results in the desired phosphoramidite 2c.                                 Scheme VI More specifically, these steps were performed as follows: N^Four, 5'-O-jiben Zoyl-2'-fluoro-3'-amino-2 ', 3-dideoxycytidine 13 is as follows: Prepared: anhydrous CH_Three6.9 g of crude 10 (containing 35% of 10i) in CN (50 mL) (18. 11.7 g (169 mmol) of 1,2,4-triazole and P0Cl_Three(3.35 mL, 36.1 mmol) Anhydrous CH_ThreeAn ice-cold solution of CN (90 mL) was added. The mixture is cooled in an ice bath and Liethylamine (23 mL, 165 mmol) is added, then the reaction is stirred while stirring. Warmed to room temperature. 90 minutes later, add 15 mL (108 mmol) of triethylamine and 4 mL of water And the mixture was stirred for 10 minutes. The solvent is removed under vacuum, then the ethyl acetate When 250 mL of the reagent is added, the TLC of the solution indicates that the fluorescent intermediate has the same mobility as the starting material. showed that.   The mixture was concentrated under vacuum to 6.7 g foam. Dioxane (100 mL) and concentrated 20 ml of aqueous ammonia are added and after 3 hours the mixture is concentrated in vacuo to a yellow gel. And The gel was dissolved in 100 mL of ethyl acetate and washed with water (3 × 200 mL). true Concentration under air and P_TwoO_FiveVacuum drying at 5.4 g yielded 5.4 g of a solid which was Only one spot was provided by Kagel TLC.¹⁹Significant signal by F NMR Only two were observed. Main component: δ-192.8 (ddd, J = 22.8, 22.8, 53.1 Hz); Secondary component: δ-200.7 (ddd, J = 13.6, 19.9, 53.4 Hz).   Anhydrous pyridine (100 mL) was added and the solution was cooled to 4 ° C. While stirring, salt Benzoyl bromide (11.7 mL, 100 mmol) was added and the mixture was warmed to room temperature. 2 After time, 5 mL of water was added and the solvent was removed under vacuum to give a brown oil, This was dissolved in 200 mL of ethyl acetate, washed with water (3 × 200 mL), and then re- Concentrated to an oily foam.   Add ethanol (150 mL), and 2 g of 10% palladium on activated carbon, and 2.10 mixture^FivePa (40psi) H_TwoAnd hydrogenated overnight. By TLC, two new and new The formation of a compound that migrates in close proximity to the cavity was shown.   The catalyst is removed by filtration, and the filtrate is concentrated in vacuo to a yellow oily foam. Things. Silica gel flash chromatography (500 mL silica, CH_TwoCl_TwoIn 0-3% CH_ThreeEluting with OH) gave 1.85 g of semi-pure product, , CH_TwoCl_Two(10 mL). A solid precipitated rapidly and was collected by filtration. The new ch_TwoCl_TwoAnd washed. Vacuum drying gives 1.5 g of raw material as fine white crystals. The product 13 (11% yield from 9 and 9i) was obtained. Mass spectrum, FAB⁺, M + H⁺Is 4 53.1574 (calculated value) and 453.1574 (actual value).  N^Four, 5'-O-Dibenzoyl-2'-fluoro-3- (4-methoxytrityl) amino-2 ', 3'-di Deoxycytidine 14 was prepared as follows: 13 in 25 mL of anhydrous pyridine. (0.9 g, 2.0 mmol), 4-methoxytrityl chloride 0.86 g (2.8 mmol) Was added and the mixture was stirred overnight. Reaction system H_TwoQuench with O (0.5 mL) and And concentrated under vacuum. CH_TwoCl_Two(50 mL) and saturated NaHCO_Three50 mL of aqueous solution And washed with water (2 × 50 mL). The solvent was removed under vacuum and CH_TwoCl_Two(10mL), It was then pipetted into 80 mL of rapidly stirred hexane / ether (1/1). 2 more After stirring for hours, the product was collected by filtration and dried under vacuum to give a white powder. As a result, 1.3 g (yield: 88%) of a product was obtained. Mass spectrum, FAB⁺, M + H⁺Is 725 .2775 (calculated value) and 725.2761 (actual value).   N^Four-Benzoyl-2'-fluoro-3 '-(4-methoxytrityl) amino-2', 3'-dideoxy Cycitidine 15 was prepared as follows: pyridine / meta cooled in an ice bath To 14 (1.3 g, 1.75 mL) in 20 mL of methanol / water (65/30/5) was added pyridine / methanol / water (6 Cold 2M NaOH (5/30/5) (10 mL) was added. Cool the mixture and stir for 20 minutes did. After 5 minutes, the resin was removed by filtration and washed with methanol. Matching The filtrate and washings were concentrated in vacuo to an oil, which was taken up in 100 mL of ethyl acetate. Dissolved. NaHCO saturated mixture_ThreeWashed with aqueous solution (100 mL) and water (2 × 100 mL) . After concentrating in vacuo to a foam, the product was washed with CH 2_TwoCl_Two(10 mL) and Pets were placed in rapidly stirred hexane / ether (2/1). The product is filtered Collect and dry under vacuum to give 1.13 g of product as a white powder (102% yield) I got Mass spectrum, FAB⁺, M + H⁺Means 753.1489 (calculated value), 753.1499 (actual value )Met.   N^Four-Benzoyl-2'-fluoro-3 '-(4-methoxytrityl) amino-2', 3'-dideoxy Cytidine 5 '-(2-cyanoethyl N, N-diisopropyl) phosphoramidite 2c is Prepared as follows: anhydrous CH_TwoCl_TwoTo 15 (970 mg, 1.56 mmol) in (25 mL) 200 mg (1.17 mmol) of diisopropylammonium tetrazolide and 2-cyanoethyl 1.0 mL (3.15 mmol) of N, N, N ', N'-tetraisopropyl phosphorodiamidite did. After stirring the mixture for 3 hours, the solvent was removed in vacuo and the residue using mm plate and CH_TwoCl_Two0-1.5% methanol, 0.5% triethylamine Purified on Chromatotron eluting with. The product is concentrated to a foam under vacuum and , CH_TwoCl_Two(10 mL) and slowly added to 40 mL of rapidly stirring hexane Was precipitated. After decanting the supernatant, the product is_TwoO_FiveVacuum drying Drying yielded 880 mg (69%) of a white powder. Mass spectrum, FAB⁺, M + H⁺Is 953.25 68 (calculated value) and 953.2531 (actual value).                                 Example 18         N ^4- Benzoyl-2'-fluoro-3 '-(4-methoxytrityl) amino- 2 ', 3'- Dideoxycytidine-5'-succinyl-added CPG   Intermediate 15 is 5'-succinylated and standard procedures (For example, ) Added to the CPG solid support. More specifically, N4-benzoyl-2'-phenyl Luoro-3 '-(4-methoxytrityl) amino-2', 3'-dideoxycytidine 5'-succini An addition CPG was prepared as follows: anhydrous CH_TwoCl_Two15 (100 mg, 0 .16 mmol), 55 mg (0.55 mmol) of succinic anhydride and 65 mg (0. .53 mmol) was added. The mixture was stirred for 2 hours and then evaporated under vacuum Oil. CH oil_TwoCl_Two(20 mL) and saturated NaHCO_Three20 mL of aqueous solution and Washed with water (2 × 20 mL) and then re-concentrated under vacuum to a foam. In the foam, 1 mL of 0.4 M diisopropylethylamine in DMSO / N-methylpyrrolidine (1/1) and 0.2M 1-hydroxybenzotriazole in DMSO / N-methylpyrrolidine (1/1), 0 .2M 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hex 0.7 mL of safluorophosphate was added. After 3 minutes, the mixture is washed with a long chain alkyl It was taken up in a 10 mL syringe containing Mino-CPG (1.2 g). Additional DMSO (5 mL) wash It was also incorporated into a syringe. Mix the CPG-nucleoside mixture for 1.5 hours, then The CPG was washed with 5 volumes of anhydrous acetonitrile. Unreacted CPG amino group is 2 minutes Over time, use standard capping solutions (PE Applied Biosystems, Foster City , CA). CPG was combined with 5 volumes of acetonitrile and 5 volumes of CH_Two Cl_TwoWashes again. Nucleoside loading can be determined by standard trityl assays. , Approximately 5 μmole / g.                                 Example 19                           Oligo-2'-nucleotide N3 ' → P5 'phosphoramidate solid-phase synthesis   Oligo-2′-nucleotide N3 ′-> P5 ′ phosphoramidates were prepared according to Schemes V and VI Was synthesized on a solid support using the following phosphoramidite monomers. Compound 22 -25 (Table 1) were synthesized with phosphoramidite monomers. Free MMT-Click The average coupling efficiency determined by the on-assay is single About 94% for the coupling of and the double application of step 2 per synthesis cycle Was about 96%. A typical IE HPLC profile for crude oligomer synthesis is As shown in FIG.   The uniformly denatured oligo-2'-nucleotide N3 '→ P5' phosphoramidate has the following Prepared by amidite transfer reaction on ABI 380B synthesizer using protocol Was.   1) Detritylation, 5% dichloroacetic acid in dichloromethane, 1 minute.   2) Coupling, 0.1 M phosphoramidite 2u or 2c (Scheme V, respectively) Or VI) and 0.45M tetrazole in acetonitrile, 3 minutes.   3) Oxidation, 0.1M iodine in tetrahydrofuran / pyridine / water (10/10/1, v / v / v) 1 minute.   4) Capped, standard PE Applied Biosystems (Foster City, CA) cap Acetylation of unreacted 3'-amino group by chlorination solution, 30 seconds.   Following the chemical steps in the cycle, acetonitrile washes and dry argon were applied. The flushing used (0.2-0.4 minutes) is performed. Cutting from solid support and concentrated en After deprotection using Monia water (1-1.5 hours, 55 ° C.), the oligonucleotide was It was analyzed and purified by HPLC. Oligonucleotides were purified immediately after Pharmacia NA Desalte on a P-5 or NAP-10 gel filtration column and store frozen at -18 ° C or Lyophilized.   The preparation of 5'-phosphorylated oligonucleotides involves sulfone-derivatized CPG (e.g., Gryaznov, S.M .; Letsinger, R.L. Nucleic Acids Res. 1993, 21, 1403-1408 ).   Use the Dionex DX300 or DX500 system for IE analysis and oligonucleotide purification. Using. For analysis and purification of crude oligomers, use Pharmacia MonoQ 10/10 columns Was used to elute 1.5 M NaCl in 10 mM NaOH with a gradient of 2% per minute. other For all IE HPLC analyses, 1.5 M NaCl in 10 mM NaOH was applied at a 1.5% gradient per minute. Dionex NucleoPac PA100 column was used. 0.1M acetic acid for RP HPLC 1% gradient per minute of acetonitrile in triethylammonium (pH 7.0) Uses Hewlett Packard Hypersil ODS (5μ column) from Waters HPLC system did.   NMR spectra were recorded on a Bruker DRX-400 spectrometer. Chemical shift,¹H Vector,¹⁹F spectrum and³¹For the P spectrum, TMS and CCl, respectively_ThreeF And H_ThreePO_FourReport compared to   Thin layer chromatography (TLC) uses a methanol / dichloromethane eluent Made on Whatman polyester lined silica gel plate.   Dimer dU^f _np0.17 OD for T₂₆₀Acid hydrolysis at 55 ° C for 2 hours with 64% vinegar Performed in 25 μL of acid and the reaction mixture was analyzed by RP HPLC. Dimer Approximately 83% of the retention time (Rt) of 15.0 minutes is hydrolyzed to form 5′-thymidylic acid (R t 10.6 min) and hydrolyze to 2'-fluoro-3'-aminouridine (Rt 11.2 min). , Identified by co-injection with authentic standards. Also, about 7.5% of the reaction mixture Thymidine (Rt 12.1 min) was also found.                                   Table 1               T of oligonucleotides and their duplexes_mvalue   a all 2'-deoxy compounds; np, f, p and n are each 3'-N HP (O) (O-) 0-5 'internucleoside linkage, 2'-fluorine, 5'-phosphate, and 3'- Represents min.   b Tm was determined at 3 μM oligonucleotide; the first value was 10 mM Measured in sodium phosphate, 150 mM sodium chloride, pH 7.04; Further measurements were made in a similar buffer containing 10 mM magnesium chloride; T not determined_m ^SIt is about.   c complementary target; poly (dA) or poly (rA) for experiments 1-6, experiment 7, For 8 d (ATAAGGAAGAAGC) or r (AUAAGGAAGAAGC).

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventors Filon and Karen L.             United States California 94587,             Union City, Redlands Street             Tote 4339 (72) Inventors Griaznov, Sergey M.             United States California 94402,             San Mateo, West Bellevue             138 (72) Inventors McCardy, Sarah N.             United States California 94403,             San Mateo, Sylvan Avenue             347 (72) Inventors Nelson, Jeffrey S.             United States California 94536,             Fremont, Tres Avenue 4375 (72) Inventors Schulz, Ronald G.             United States California 94545,             Hayward, Bay Center Place             3832, Links Therapeutics,             Incorporated

Claims (1)

[Claims] 1. A method for synthesizing an oligonucleotide N3 ′ → P5 ′ phosphoramidate, A method comprising the following steps:   (a) providing a first nucleoside bound to a solid support, the first nucleoside comprising: The step wherein the nucleoside has a protected 3'-amino group;   (b) deprotecting the protected 3′-amino group to form a free 3′-amino group;   (c) the free 3'-amino group is 3'-protected aminonucleoside-5'-phosphoramidite React with monomer to form an internucleoside N3'-P5 'phosphoramidite linkage Forming a;   (d) oxidizing the bond. 2. Further comprising a step of repeating the reaction step and the oxidation step a plurality of times. Item 1. The method according to Item 1. 3. 3. The method according to claim 2, wherein the plurality of times is two or more and four or less. 4. Steps until the oligonucleotide N3 ′ → P5 ′ phosphoramidate is synthesized 4. The method of claim 3, further comprising repeating steps (b)-(d). 5. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 10M^-15. The method of claim 4, having a tetrazole activation equilibrium constant of 6. Reacting with the 3′-protected aminonucleoside-5′-phosphoramidite monomer 6. The method of claim 5, further comprising the step of capping said free 3'-amino group. The described method. 7. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 100M^-17. The method of claim 6, having a tetrazole activation equilibrium constant of: 8. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 10 pK_aThe phosphoramidite having a phosphoramidite amino group having the formula: the method of. 9. The 3′-protected aminonucleoside-5′-phosphoramidite monomer has the following formula: 9. The method according to claim 8, defined by:here,   B is pyrimidine, purine, or an analog thereof;   R¹Is a phosphate protecting group;   W is -NHR^Two-Or-OR⁷Where R^TwoIs an amino protecting group And then R⁷Is a hydroxyl protecting group;   R^ThreeIs hydrogen, hydroxyl, fluoro or -OR ', wherein R' is one An alkyl having ３3 carbon atoms, or a hydroxyl protecting group; and   R^FourAnd R^FiveTogether with the nitrogen to which they are bound to form carbon, oxygen, sulfur and With up to 40 atoms selected from the group consisting of nitrogen and nitrogen Forming a group or an arylamino leaving group. Ten. R^ThreeIs hydrogen, and R^FourAnd R^FiveBut independently, a total of 6-20 charcoal Alkyl, aralkyl, cycloalkyl or cycloalkyl 10. The method of claim 9, wherein the method is a quill. 11． Wherein the reaction step is the 3′-protected aminonucleoside-5′-phosphoramidite Further comprising treating the monomer with a nucleophilic catalyst to form a reactive intermediate. The method of claim 10, wherein 12． 12. The method according to claim 11, wherein said nucleophilic catalyst is a tetrazole activator. 13. The oxidation step selects the bond from the group consisting of hydrogen peroxide and iodine 13. The method of claim 12, further comprising treating with an oxidizing agent. 14. R^ThreeIs hydrogen, and R^FourAnd R^FiveBut together, up to 12 Form an alkylene chain containing from 4 to 20 carbon atoms in total And both terminal valence bonds of the chain are R^FourAnd R^FiveIs bonded to the bonded nitrogen atom The method of claim 9, wherein 15． Wherein the reaction step is the 3′-protected aminonucleoside-5′-phosphoramidite Further comprising treating the monomer with a nucleophilic catalyst to form a reactive intermediate. 15. The method according to claim 14, wherein 16． 16. The method according to claim 15, wherein said nucleophilic catalyst is a tetrazole activator. 17． The oxidation step selects the bond from the group consisting of hydrogen peroxide and iodine 17. The method of claim 16, further comprising treating with an oxidizing agent. 18． R^ThreeIs hydrogen, and R^FourAnd R^FiveBut together they joined Together with nitrogen, having up to 10 carbon or heteroatoms in its main chain, and Form a saturated nitrogen heterocycle having from 4 to 20 carbon or heteroatoms, As a result, R^FourAnd R^FiveBut, together with the nitrogen atom to which they are attached, Containing up to three heteroatoms selected from the group consisting of: oxygen and sulfur The method of claim 9. 19. Wherein the reaction step is the 3′-protected aminonucleoside-5′-phosphoramidite Further comprising treating the monomer with a nucleophilic catalyst to form a reactive intermediate. 19. The method of claim 18, wherein the method comprises: 20. 20. The method of claim 19, wherein said nucleophilic catalyst is a tetrazole activator. twenty one. The oxidation step selects the bond from the group consisting of hydrogen peroxide and iodine 21. The method of claim 20, further comprising treating with an oxidizing agent. twenty two. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 10 pK_aThe phosphoramidite having a phosphoramidite amino group having the formula: the method of. twenty three. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 10M^-123. The method of claim 22, having a tetrazole activation equilibrium constant of: twenty four. Reacting with the 3′-protected aminonucleoside-5′-phosphoramidite monomer 24.The method according to claim 23, further comprising capping the free 3'-amino group. The described method. twenty five. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 100M^-125. The method of claim 24, having a tetrazole activation equilibrium constant K of . 26. Wherein the reaction step is the 3′-protected aminonucleoside-5′-phosphoramidite Further comprising treating the monomer with a nucleophilic catalyst to form a reactive intermediate. 26. The method of claim 25, wherein 27. 27. The method of claim 26, wherein said nucleophilic catalyst is a tetrazole activator. 28. The oxidation step selects the bond from the group consisting of hydrogen peroxide and iodine 28. The method of claim 27, further comprising treating with an oxidizing agent. 29. Steps until the oligonucleotide N3 ′ → P5 ′ phosphoramidate is synthesized The method of claim 1, further comprising repeating steps (b)-(d). 30. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 10 pK_aHaving a sterically hindered phosphoramidite amino group having 9. The method according to 9. 31. Reacting with the 3′-protected aminonucleoside-5′-phosphoramidite monomer 31.The method of claim 30, further comprising capping the free 3'-amino group. The described method. 32. The sterically hindered phosphoramidite amino group has 6 to 20 carbon atoms Alkylamino, dialkylamino, cycloalkylamino, dicycloalkyl 32. The method of claim 31, which is ruamino or aralkylamino. 33. Wherein the reaction step is the 3′-protected aminonucleoside-5′-phosphoramidite Further comprising treating the monomer with a nucleophilic catalyst to form a reactive intermediate. 33. The method of claim 32, wherein 34. 34. The method of claim 33, wherein said nucleophilic catalyst is a tetrazole activator. 35. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 100M^-135. The method of claim 34, having a tetrazole activation equilibrium constant of 36. The oxidation step selects the bond from the group consisting of hydrogen peroxide and iodine 36. The method of claim 35, further comprising treating with an oxidizing agent. 37. A method for synthesizing an oligonucleotide of the formula:Wherein B is a purine or pyrimidine or an analog thereof; X is an acid Is sulfur or sulfur; R^ThreeIs hydrogen, fluoro or hydroxyl; R⁶Is , Amino or hydroxyl; and Z is hydrogen, an alkali metal or And n is at least 1; the method comprises the following steps: Including:   (a) providing a first nucleoside bound to a solid support, the first nucleoside comprising: The step wherein the nucleoside has a protected 3'-amino group;   (b) deprotecting the protected 3'-amino group to form a free 3'-amino group;   (c) converting the free 3'-amino group to a 3'-protected aminonucleotide in the presence of a nucleophilic catalyst; Reaction with the sid-5'-phosphoramidite monomer gives the internucleoside N3 ' -Forming a P5 'phosphoramidite bond;   (d) oxidizing the bond; and   (e) repeating steps (b) to (d) until the oligonucleotide is synthesized . 38. Reacting with the 3′-protected aminonucleoside-5′-phosphoramidite monomer 38.The method according to claim 37, further comprising capping the free 3'-amino group. The described method. 39. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 10 pK_aHaving a sterically hindered phosphoramidite amino group having 8. The method according to 8. 40. Steps (c) and (d) are repeated a plurality of times during each cycle of the steps (b) to (d). 40. The method of claim 39, further comprising the step of: 41. The steps (c) and (d) are repeated twice during each cycle of the steps (b) to (d). 41. The method of claim 40, wherein the method is performed. 42. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is as follows: 42. The method of claim 41, which is in the form of here,   B is pyrimidine, purine, or an analog thereof;   R¹Is a phosphate protecting group;   W is -NHR^Two-Or-OR⁷Where R^TwoIs an amino protecting group And then R⁷Is a hydroxyl protecting group;   R^ThreeIs hydrogen, hydroxyl, fluoro or -OR ', wherein R' is one An alkyl having 〜3 carbon atoms, or a hydroxyl protecting group: and   R^FourAnd R^FiveTogether with the nitrogen to which they are bound to form carbon, oxygen, sulfur and With up to 40 atoms selected from the group consisting of nitrogen and nitrogen Forming a group or an arylamino leaving group. 43. R^ThreeIs hydrogen, the nucleophilic catalyst is a tetrazole activator, and R^FourAnd R^FiveIndependently represents an alkyl, a, having 6 to 20 carbon atoms in total. Aralkyl, cycloalkyl or cycloalkylalkyl, according to claim 42. The described method. 44. The oxidation step converts the bond to iodine, chlorine, hydroperoxide, peracid, Mixed acylsulfinic anhydride, peroxide, ozone, elemental sulfur, thiuramdis Sulfide, acyl disulfide, phosphinothionyl disulfide and 1,1,- An oxidizing agent selected from the group consisting of dioxo-3H-1,2-benzodithiol-3-one 44. The method of claim 43, further comprising the step of treating. 45. R¹Is methyl, β-cyanoethyl or 4-nitrophenylethyl, and R^Two Is triphenylmethyl, and n ranges from 1 to 50. The described method. 46. R^ThreeIs hydrogen, and R^FourAnd R^FiveBut together, up to six And an alkylene chain having 4 to 12 carbon atoms in total. Form, both terminal valence bonds of the chain are R^FourAnd R^FiveBonds to the nitrogen atom 43. The method of claim 42, wherein 47. The nucleophilic catalyst is a tetrazole activator;¹But methyl, β-sia Noethyl or 4-nitrophenylethyl; R^TwoIs triphenylmethyl And n is in the range of 1 to 50; and the oxidizing step converts the bond to iodine. , Chlorine, hydroperoxide, peracid, mixed acylsulfinic anhydride, peroxide , Ozone, elemental sulfur, thiuram disulfide, acyl disulfide, phosphite Nothionyl disulfide and 1,1, -dioxo-3H-1,2-benzodithiol-3-one Further comprising the step of treating with an oxidizing agent selected from the group consisting of: The described method. 48. R^ThreeIs hydrogen, and R^FourAnd R^FiveBut together they joined Together with nitrogen, having up to 10 carbon or heteroatoms in its main chain, and Form a saturated nitrogen heterocycle having 4 to 20 carbon or heteroatoms with As a result, R^FourAnd R^FiveTogether, together with the nitrogen atom to which they are attached, Contains up to three heteroatoms selected from the group consisting of elemental, oxygen and sulfur 43. The method of claim 42. 49. The nucleophilic catalyst is a tetrazole activator, and the oxidation step comprises: The bond is iodine, chlorine, hydroperoxide, peracid, mixed acylsulfin Acid anhydride, peroxide, ozone, elemental sulfur, thiuram disulfide, acyldis Sulfide, phosphinothionyl disulfide and 1,1, -dioxo-3H-1,2-ben Treating with an oxidizing agent selected from the group consisting of zodithiol-3-ones. 49. The method of claim 48, comprising: 50. R¹Is methyl, β-cyanoethyl or 4-nitrophenylethyl, and R^Two Is triphenylmethyl, and n ranges from 1 to 50. The described method. 51. Compound of the formula: here,   B is pyrimidine, purine, or an analog thereof;   R¹Is a phosphate protecting group;   W is -NHR^Two-Or-OR⁷Where R^TwoIs an amino protecting group And then R⁷Is a hydroxyl protecting group;   R^ThreeIs hydrogen, hydroxyl, fluoro or -OR ', wherein R' is one Alkyl having up to 3 carbon atoms, or a hydroxyl protecting group; hand   R^FourAnd R^FiveTogether with the nitrogen to which they are bound to form carbon, oxygen, sulfur and With up to 40 atoms selected from the group consisting of nitrogen and nitrogen Form a group or an arylamino leaving group; However, the following compounds are excluded:   A compound having the above formula, wherein:     B is N⁶-Benzoyl adenine or thymine;     R¹Is trimethylsilyl;     W is 0-4,4'-dimethoxytrityl;     R^ThreeIs hydrogen; and     R^FourAnd R^FiveAre each isopropyl;   A compound of the above formula, wherein:     B is protected with dimethylformamide protected adenine, isobutyryl Cytosine, or thymine;   R¹Is β-cyanoethyl;   W is 0-dimethoxytrityl;   R^ThreeIs hydrogen; and   R^FourAnd R^FiveAre each isopropyl. 52. R^ThreeIs hydrogen; and R^FourAnd R^FiveBut independently, a total of 6-20 charcoal Alkyl, aralkyl, cycloalkyl or cycloalkyl 52. The compound of claim 51, which is alkyl. 53. R¹Is methyl, β-cyanoethyl or 4-nitrophenylethyl, and R^Two Is triphenylmethyl, and R⁷Is di-p-anisylphenylmethyl, And R^FourAnd R^FiveBut independently isopropyl, sec-butyl, isobutyl, t-butyl The compound according to claim 51, which is tyl, cyclohexyl or 2-ethylhexyl. . 54. R^ThreeIs hydrogen, and R^FourAnd R^FiveBut together, up to six And an alkylene chain having 4 to 12 carbon atoms in total. And both terminal valence bonds of the chain are R^FourAnd R^FiveBonds to the nitrogen atom 52. The compound of claim 51, wherein 55. R^ThreeIs hydrogen, and R^FourAnd R^FiveBut together they joined Together with nitrogen, having up to 10 carbon or heteroatoms in its main chain, and Form a saturated nitrogen heterocycle having from 4 to 20 carbon or heteroatoms, As a result, R^FourAnd R^FiveBut, together with the nitrogen atom to which they are attached, Containing up to three heteroatoms selected from the group consisting of: oxygen and sulfur 52. The compound of claim 51. 56. R¹Is methyl, β-cyanoethyl or 4-nitrophenylethyl, and R^Two Is triphenylmethyl, and R^FourAnd R^FiveBut together, they Along with the attached nitrogen atom, dimethylpipiridinyl, Loridinyl, dimethyl morpholino, tetramethyl morpholino, dimethyl pyrrolidi Nyl, tetramethylpyrrolidinyl or tetramethylpiperidinyl Item 55. The compound according to item 55. 57. Synthesis of oligonucleotide N3 '→ P5' phosphoramidate on solid support A method comprising the following steps:   3'-Protected aminonucleoside-5'-phospho with phosphoramidite amino group Providing a luamidite monomer; and   The phosphoramidite amino group of the monomer is replaced with the oligonucleotide N3 ′ → P5 ′ The 3′-protected amino is replaced by replacing the 3′-primary amino group of The nucleoside-5'-phosphoramidite monomer is converted to the oligonucleotide N3 '→ P Coupling to the 3'-primary amino group of 5 'phosphoramidate. 58. The above 3′-protected aminonucleoside-5′-phosphoramidite monomer The suformamidite amino group has a pK of at least 10_aThe method of claim 57, comprising: Method. 59. The coupling step further comprises forming a N3 ′ → P5 ′ phosphoramidite bond. 59. The method of claim 58, comprising. 60. The N3 ′ → P5 ′ phosphoramidite bond is oxidized to give N3 ′ → P5 ′ phosphoramidite. Further comprising the step of forming an N3 ′ → P5 ′ phosphorothioamidate bond. 60. The method of claim 59, comprising: 61. The 3′-protected aminonucleoside-5′-phosphoramidite monomer is less At least 100M^-161. The method of claim 60, having a tetrazole activation equilibrium constant of 62. The method further includes a step of repeating the coupling step and the oxidation step a plurality of times. 62. The method of claim 61, wherein 63. 63. The method of claim 62, wherein the plurality is two. 64. The 3′-protected aminonucleoside-5′-phosphoramidite monomer and cup Further comprising a step of capping the 3′-primary amino group that does not ring. 64. The method of claim 63.