AU703509C - Solid phase synthesis of oligonucleotide N3'-P5' phosphoramidates - Google Patents

Description

SOLID PHASE SYNTHESIS OF OLIGONUCLEOTIDE

N3'→P5' PHOSPHORAMIDATES

Field of the Invention

The invention relates generally to nucleic acid polymer chemistry, and more particularly, to methods of synthesizing oligonucleotide N3'→P5' phosphorami dates.

Background

Nucleic acid polymer chemistry has played a crucial role in many developing technologies in the pharmaceutical, diagnostic, and analytical fields, and more particularly in the subfields of antisense and anti-gene therapeutics, combinatorial chemistry, branched DNA signal amplification, and array-based DNA diagnostics and analysis e.g. Uhlmann and Peyman, Chemical Reviews, 90: 543-584 (1990); Milligan et al, J. Med. Chem. 36: 1923-1937 (1993); Mesmaeker et al, Current Opinion in

Structural Biology, 5: 343-355 (1995); Thuong et al, Angew. Chem. Int. Ed. Engl., 32: 666-690 (1993); Brenner et al, Proc. Natl. Acad. Sci., 89: 5381 -5383 (1992); Gold et al, Ann. Rev. Biochem., 64: 763-797 (1995); Gallop et al, J. Med. Chem. 37: 1233-1258 (1994); Gordon et al, J. Med. Chem. 37: 1385-1401 (1994); Gryaznov, International application PCT/US94/07557; Urdea et al, U.S. patent 5,124,246; Southern et al, Genomics, 13: 1008-1017 (1992); McGall et al, U.S. patent 5,412,087; Fodor et al, U.S. patent 5,424,186; Pirrung et al, U.S. patent 5,405,783; and the like.

Much of this chemistry has been directed to improving the binding strength, specificity, and nuclease resistance of natural nucleic acid polymers, such as DNA. Unfortunately, improvements in one property, such as nuclease resistance, often involve trade-offs against other properties, such as binding strength. Examples of such tradeoffs abound: peptide nucleic acids (PNAs) display good nuclease resistance and binding strength, but have reduced cellular uptake in test cultures, e.g. Hanvey et al, Science, 258: 1481-1485 (1992); phosphorothioates display good nuclease resistance and solubility, but are typically synthesized as P-chiral mixtures and display several sequence-non-specific biological effects, e.g. Stein et al, Science, 261 : 1004-1012

(1993); methylphosphonates display good nuclease resistance and cellular uptake, but are also typically synthesized as P-chiral mixtures and have reduce duplex stability, e.g. Mesmaeker et al (cited above); and so on.

Recently, a new class of oligonucleotide analog has been developed having so- called N3'→P5' phosphoramidate intemucleoside linkages which display very favorable binding properties, nuclease resistance, and solubility, Gryaznov and Letsinger, Nucleic Acids Research, 20: 3403-3409 (1992); Chen et al, Nucleic Acids Research, 23: 2661 - 2668 (1995); Gryaznov et al, Proc. Natl. Acad. Sci., 92: 5798-5802 (1995); and Gryaznov et al, J. Am. Chem. Soc, 116: 3143-3144 (1994). Unfortunately, low synthesis yields of these compounds with published protocols has inhibited their commercial application.

The utility of this new class of oligonucleotide analog would be significantly increased if modifications and new synthesis approaches could be found that would improve synthesis yields without a concomitant loss in any other of its other favorable properties outlined above.

Summary of the Invention

In view of the above, an important objective of our invention is to provide a new approach to solid phase synthesis of oligonucleotide N3'->P5' phosphoramidates in which stepwise coupling yields are significantly increased.

Another objective of our invention is to provide novel 3'-protected amino-5'- phosphoramidite monomers for use in the method of the invention.

A further objective of the invention is to provide a practical large-scale synthesis method for making oligonucleotide N3' →P5' phosphoramidates, particularly 2'- deoxyoligonucleotide N3'→P5' phosphoramidates.

These and other objects of our invention are accomplished by providing a method of synthesizing oligonucleotide N3'→P5' phosphoramidates using an amine- exchange reaction in which a deprotected 3 '-amino group of a solid phase supported oligonucleotide chain is exchanged for the amino portion of a 5 '-phosphoramidite of an incoming monomer which has a protected 3 '-amino group. The resulting

intemucleotide phosphoramidite linkage is then oxidized to form a stable protected phosphoramidate linkage. The general scheme of the reaction is depicted below.

Generally, the method of the invention includes the following steps: (a) providing a first nucleoside attached to a solid phase support, the first nucleoside having a protected 3' amino group; (b) deprotecting the protected 3' amino group to form a free 3' amino group; (c) reacting the free 3' amino group with a 3'-protected aminonucleoside-5'-phosphoramidite monomer to form an intemucleoside N3'→P5' phosphoramidite linkage; (d) oxidizing said linkage; and (e) repeating steps (b) through (d) until the desired oligonucleotide N3'→P5' phosphoramidate is synthesized.

Preferably, the nitrogen moiety of the 5'-phosphoramidite of the 3'-protected aminonucleoside-5'-phosphoramidite monomer is a sterically hindered amine having a pK_a of at least 10.

The invention further includes 3'-protected-aminonucleoside-5'- phosphoramidite monomers of the following formula, which monomers are particularly useful in the method of the invention:

wherein: B is pyrimidine, purine, or an analog thereof; R¹ is a phosphate protecting group; W is either -NHR² or -OR⁷, where R² is an amino protecting group and R⁷ is a hydroxyl protecting group; R³ is hydrogen, hydroxyl, fluoro or -OR', wherein R' is alkyl having from 1 to 3 carbon atoms or a 2'-hydroxyl protecting group, such as alkylsilyl, e.g. t-butyldimethylsilyl, or the like; and R⁴ and R⁵ together with the nitrogen to which they are attached form an alkylamino- or arylamino leaving group having up to 40 carbon atoms and/or heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen.

Monomers with W as -OR⁷ are particularly useful in synthesizing chimeric

oligonucleotides containing both N3'→P5' phosphoramidate linkages and other linkages, such as phosphodiester, phosphorothioate, and the like.

The invention overcomes critical shortcomings of prior art methods of synthesizing oligonucleotide N3'→P5' phosphoramidates having either fully amidated or partially amidated linkages and opens the way for commercial scale production of such compounds. In particular, the invention provides greatly increased coupling yields using much lower molar equivalents of monomer reactants which, in turn, allows commercially feasible synthesis of the oligonucleotide N3'→P5' phosphoramidates. The invention will permit the widespread application of the compounds in a wide range of fields including scientific and industrial research, therapeutics, and diagnostics. Brief Description of the Figures

Figure 1 is a ³¹P-NMR spectrum of a mixture of N6-benzoyl-3'-tritylamino-2'- deoxyadenosine-5'-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite and tetrazole.

Figure 2 is a ³¹P-NMR spectrum of a mixture of 3'-tritylaminothymidine-5'-(2- cyanoethyl-(2,2,6,6-tetramethylpiperidinyl))-phosphoramidite and tetrazole.

Figures 3a and 3b are ion exchange chromatograms of two crude

oligonucleotide N3'→P5' phosphoramidates synthesized by the amine-exchange reaction of the invention.

Figure 4 is an ion exchange HPLC chromatogram of the crude reaction mixture from synthesis of an oligo-2'-fluoronucleoside N3'→P5' phosphoramidate of Example 19.

Definitions

Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGUCCTG," it will be understood that the nucleotides are in 5'→3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T" denotes thymidine, and "U" denotes deoxyuridine, unless otherwise noted.

As used herein, "N3'→P5' phosphoramidate" refers to an intemucleosidic linkage of the form:

3'-NH-P(=X)(OR¹)-O- 5' wherein the 3' and 5' refer to the carbon atoms of the sugar moieties of consecutive nucleosides which are connected by way of the linkage, and wherein R¹ is hydrogen or a phosphate protecting group, and X is a chalcogen, preferably oxygen or sulfur. More particularly, when R¹ is a phosphate protecting group it may be alkyl, alkenyl, aryl, aralkyl, or cycloalkyl containing up to 10 carbon atoms. Preferably, when R¹ is a phosphate protecting group it is alkyl having from 1 to 6 carbon atoms; electron- withdrawing β-substituted ethyl, particularly β-trihalomethyl-, β-cyano-, β-sulfo-, or β- nitro- substituted ethyl; electron- withdrawing substituted phenyi, particularly halo-, sulfo-, cyano-, or nitro-, substituted phenyi; or electron- withdrawing substituted phenylethyl. More preferably, when R¹ is a phosphate protecting group it is methyl, β- cyanoethyl, or 4- nitrophenylethyl. Most preferably, R¹ is hydrogen, methyl, or β- cyanoethyl. Electron- withdrawing substituents are typically halo, cyano, nitro, sulfo, or mono-, di-, or trihalomethyl, and the like. Halogen atom substituents are usually fluoro, chloro, bromo, or iodo; and preferably, they are fluoro or chloro. "Electron- withdrawing" denotes the tendency of a substituent to attract valence electrons of the molecule of which it is a part, i.e. it is electronegative, e.g. March, Advanced Organic Chemistry, pgs. 16-18 (John Wiley, New York, 1985). Guidance for selecting a phosphate protecting group is provided in Beaucage and Iyer, Tetrahedron 48: 2223- 231 1 (1992). For convenience, nucleotide phosphoramidates are sometimes indicated herein by a subscripted "np" or "pn" for N3'→P5' phosphoramidates and P3'→N5' phosphoramidates, respectively. Thus, "U_npU" is a dinucleotide in which a 3'- aminouridine and a uridine are linked by an N3'→P5' phosphoramidate linkage.

Similarly, 2'-fluoro substituents are indicated by a superscripted "f '. Thus, "U^f _npU" is a dinucleotide in which the 5'-most 3'-amino-2'-fluorouridine is linked to a uridine by an N3'→P5' phosphoramidate linkage. A single leading subscripted "p" indicates a 5' monophosphate, and a single trailing subscripted "n" indicates a 3'-amino group.

As used herein, the term "N3'→P5' phosphoramidite linkage" (emphasis added) refers to the phosphorus (III) intermediate of the N3'→P5' phosphoramidate linkage. In accordance with the invention, an N3'→P5' phosphoramidate linkage is formed by oxidizing an N3'→P5' phosphoramidite linkage.

As used herein, "nucleoside" includes the natural nucleosides, including 2'- deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA

Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g. stability, specificity, or the like, such as disclosed by Uhlmann and Peyman (cited above).

As used herein, "pyrimidine" means the pyrimidines occurring in natural nucleosides, including cytosine, thymine, and uracil, and common analogs thereof, such as those containing oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, halo, and like, substituents. The term as used herein further includes pyrimidines with common protection groups attached, such as N⁴-benzoylcytosine. Further common pyrimidine protection groups are disclosed by Beaucage and Iyer (cited above).

As used herein, "purine" means the purines occurring in natural nucleosides, including adenine, guanine, and hypoxanthine, and common analogs thereof, such as those containing oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, halo, and like, substituents. The term as used herein further includes purines with common protection groups attached, such as N -benzoylguanine, N -isobutyrylguanine, N - benzoyladenine, and the like. Further common purine protection groups are disclosed by Beaucage and Iyer (cited above).

As used herein, "oligonucleotide N3'→P5' phosphoramidate" means an oligomer, usually linear, of nucleoside subunits linked by at least one N3'→P5' phosphoramidate linkage. The nucleoside subunits usually comprise nucleosides or nucleoside analogs, but may also comprise more general moieties having compatible chemistry, such as abasic sugars and other hydrocarbon moieties, such as described in the following references: Newton et al, Nucleic Acids Research, 21 : 1 155-1 162 (1993); Griffin et al, J. Am. Chem. Soc, 114: 7976-7982 (1992); Jaschke et al, Tetrahedron Letters, 34: 301-304 (1992); Ma et al, International application PCT/CA92/00423; Zon et al, International application PCT/US90/06630; Durand et al, Nucleic Acids Research, 18: 6353-6359 (1990); Salunkhe et al, J. Am. Chem. Soc, 1 14: 8768-8772 (1992); and the like. More preferably, the term means an oligonucleotide wherein all

intemucleosidic linkages are replaced by N3'→P5' phosphoramidate linkages, i.e. the term comprehends partially as well as fully "amidated" oligomers. Still more preferably, it means an oligonucleotide wherein all the intemucleosidic linkages are replaced by N3'→P5' phosphoramidate linkages and wherein the nucleoside subunits are the natural nucleosides or analogs thereof. An oligonucleotide N3'→P5'

phosphoramidate of the invention in which every linkage is an N3'→P5'

phosphoramidate linkage ("fully amidated") may be imbedded in or attached to other oligonucleotides or polynucleotides to form a larger oligomer which is "partially amidated." For example, the fully amidated oligonucleotide N3'-»P5' phosphoramidate A_npA_npG_npC_npC_n is embedded in the larger oligonucleotide

GGCCAAAA_npA_npG_npC_npC_npACTAT (SEQ ID NO: 1 ), or is attached to "TTTATC" as a larger oligonucleotide: A_npA_npG_npC_npC_npTTTATC (SEQ ID NO: 2). Such chimeric oligonucleotides which may be employed as PCR primers, capture probes, and the like, are included within the scope of the invention.

As used herein, the term "oxidize," "oxidation," or like terms, in reference to a phosphorus-containing intemucleosidic linkage means a process or treatment for converting the phosphorus atom of the linkage from a phosphorus (III) form to a phosphorus (V) form.

As used herein, the term "phosphoramidite amino group" refers to the amino group, -NR⁴R⁵, attached to the phosphorus atom of a phosphoramidite group, and the term "phosphoramidite nitrogen" refers to the nitrogen atom of the phosphoramidite amino group.

As used herein, the terms "steric hinderance," "sterically hindered," and the like, refer to the effect on chemical reactivity of "bulky" groups, e.g. Morrison and Boyd, Organic Chemistry, page 603 (Allyn and Bacon, Boston, 1978).

Detailed Description of the Invention

The invention is directed to a solid phase method of synthesizing

oligonucleotide N3'→P5' phosphoramidates in which coupling of phosphoramidite monomers to a free amino group of a growing chain proceeds through the exchange of the phosphoramidite amino group of the monomer with the free 3' amino group of the growing chain. Preferably, the oligonucleotide N3'→P5' phosphoramidates produced by the method of the invention are described by the formula:

wherein: B is a purine or pyrimidine or an analog thereof; X is a chalcogen, preferably, oxygen or sulfur, and most preferably, oxygen; R³ is hydrogen, fluoro, or hydroxyl, preferably, hydrogen; R⁶ is amino or hydroxyl; and Z is hydrogen, or a cationic counter-ion such as alkali metal, amine cation such as ammonium, triethylammonium, or the like. Preferably, n is in the range of from 1 to several hundred; more preferably, n is in the range of from 1 to about 50; and most preferably, n is in the range of from 1 to 30.

Preferably, oligo-2'-fluoronucleotide N3'→P5' phosphoramidates of the invention are between 2 and 30 nucleotides in length. More preferably, they are between 8 and 25 nucleotides in length; and most preferably, they are between 8 and 20 nucleotides in length.

As mentioned above the general steps of the method include (a) providing a first nucleoside attached to a solid phase support, the first nucleoside having a protected 3' amino group; (b) deprotecting the protected 3' amino group to form a free 3' amino group; (c) reacting the free 3' amino group with a 3'-protected aminonucleoside-5'- phosphoramidite monomer to form an intemucleoside N3'→P5' phosphoramidite linkage; (d) oxidizing said linkage; and (e) repeating steps (b) through (d) until the desired oligonucleotide N3'→P5' phosphoramidate is synthesized.

Even though the amine exchange reaction of the invention depends on a reversible equilibrium between reactants and products (shown below in equations 1 a and 1b)-- in contrast to most approaches to solid phase oligonucleotide synthesis which involve irreversible coupling steps, considerable guidance in making selections concerning coupling conditions, protecting groups, solid phase supports, linking groups, deprotection reagents, reagents to cleave products from solid phase supports, purification of product, and the like, in the context of the present invention can be found in literature, e.g. Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Amarnath and Broom, Chemical Reviews, Vol. 77, pgs. 183-217 (1977); Pon et al, Biotechniques, Vol. 6, pgs. 768-775 (1988); Ohtsuka et al, Nucleic Acids Research, Vol. 10, pgs. 6553-6570 (1982); Eckstein, editor (cited above), Greene and Wuts (cited above), Narang, editor, Synthesis and Applications of DNA and RNA (Academic Press, New York, 1987), Beaucage and Iyer (cited above), and like references.

A wide variety of solid phase supports may be used with the invention, including microparticles made of controlled pore glass (CPG), highly cross-linked polystyrene, acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like, disclosed in the following exemplary references: Meth. Enzymol., Section A, pages 1 1-147, vol. 44 (Academic Press, New York, 1976); U.S. patents 4,678,814; 4,413,070; and 4,046;720; and Pon, Chapter 19, in Agrawal, editor, Methods in

Molecular Biology, Vol. 20, (Humana Press, Totowa, NJ, 1993). Supports further include polystyrene beads; polystyrene grafted with polyethylene glycol (e.g.,

TentaGel^TM, R_app Polymere, Tubingen Germany); and the like. Selection of the support characteristics, such as material, porosity, size, shape, and the like, and the type of linking moiety employed depends on a variety of factors, such as protection groups employed, length of final product, quantity of final product, and the like. Exemplary linking moieties are disclosed in Pon et al, Biotechniques, 6:768-775 (1988); Webb, U.S. patent 4,659,774; Barany et al, International patent application PCT/US91/06103; Brown et al, J. Chem. Soc. Commun., 1989: 891 -893; Damha et al, Nucleic Acids Research, 18: 3813-3821 (1990); Beattie et al, Clinical Chemistry, 39: 719-722 (1993); Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992); and the like. Preferred solid supports for use in the invention are CPG and polystyrene grafted with polyethylene glycol and possessing a terminal amino group (e.g., TentaGel- NH₂ ^TM, Rapp Polymere, Tubingen Germany). The aminopropyl group is a preferred spacer between CPG and the nucleoside linkage. The preferred linkage to the 5'- hydroxyl of the first nucleoside is a succinyl group which provides a base-labile ester linkage that is typically cleaved after synthesis with aqueous ammonia.

Monomers of the invention include 2'-fluoro-3'-protected aminonucleoside-5'- phosphoramidites, 2'-deoxy-3 '-protected aminonucleoside-5'-phosphoramidites, 2'- protected-3'-protected aminoribonucleoside-5'-phosphoramidites, and their 3'-protected- 3'-hydroxyl counterparts. Preferably, monomers of the invention are defined by the formula:

wherein B, W, R¹ , R³, R⁴, and R⁵ are as defined above.

More preferably, -NR⁴R⁵ is a sterically hindered amino group which may consist of the following preferred alternatives: First, R⁴ and R⁵ taken separately are alkyl, aralkyl, cycloalkyl, or cycloalkylalkyl, wherein R⁴ and R⁵ have a combined total of from 6 to 20 carbon atoms. Still more preferably, R⁴ and R⁵ taken separately are alkyl having from 1 to 8 carbon atoms. In further preference, R⁴ and R⁵ when taken separately are isopropyl, sec-butyl, isobutyl, t-butyl, cyclohexyl, or 2-ethylhexyl. Most preferably, when taken separately R⁴ is isopropyl while R⁵ is t-butyl.

Second, R⁴ and R⁵ taken together may form an alkylene chain containing up to 12 carbon atoms in the principal chain and a total of from 4 to 20 carbon atoms with both terminal valence bonds of said chain being attached to the nitrogen atom to which R⁴ and R⁵ are attached. In further preference, R⁴ and R⁵ taken together form an alkylene chain containing up to 6 carbon atoms in the principal chain and a total of from 4 to 12 carbon atoms with both terminal valence bonds of said chain being attached to the nitrogen atom to which R⁴ and R⁵ are attached.

Third, R⁴ and R⁵ taken together and with the nitrogen to which they are attached form a saturated nitrogen heterocycle having up to 10 carbon atoms or heteroatoms in the principal chain and a total of from 4 to 20 carbon atoms or heteroatoms altogether, such that R⁴ and R⁵ taken together and with the nitrogen to which they are attached contain up to three heteroatoms selected form the group consisting of nitrogen, oxygen, and sulfur. In further preference, R⁴ and R⁵ taken together and with the nitrogen to which they are attached form a saturated nitrogen heterocycle having up to 10 carbon atoms and up to three additional heteroatoms selected form the group consisting of nitrogen, oxygen, and sulfur. Still more preferably, R⁴ and R⁵ taken together and with the nitrogen to which they are attached are pyrrolidino, morpholino,

tetramethylguanidinyl or piperidino. Still more preferably, R⁴ and R⁵ taken together and with the nitrogen to which they are attached are dimethylpiperidinyl, pyrrolidinyl, dimethylmorpholino, tetrarnethylmorpholino, dimethylpyrrolidinyl,

tetramethylpyrrolidinyl, or tetramethylpiperidinyl. Still more preferably, R⁴ and R⁵ taken together and with the nitrogen to which they are attached are 2,2,6,6- tetramethylpiperidinyl, 2,6-dimethylpiperidinyl, or 2,5-dimethylpyrrolidinyl. Most preferably, R⁴ and R⁵ taken together and with the nitrogen to which they are attached is 2,2,6,6-tetramethylpiperidinyl.

Preferably, monomers for use in the method are 2'-deoxy-3 '-protected aminonucleoside-5'-phosphoramidites. In further preference, the phosphoramidite amino group has a pKa of at least 10.0. Still more preferably, the phosphoramidite amino group is selected such that the tetrazole activation equilibrium constant K₁ as defined below, and as measured in Example 9, is greater than 10 M^-1. Still more preferably, the equilibrium constant is greater than 100 M^-1; and most preferably, the equilibrium constant is greater than 1000 M^-1 .

The tetrazole activation equilibrium constant K| is defined as follows:

where [Tetrazolidyl amidite] is the equilibrium concentration of the tetrazolidyl amidite intermediate, [Tetrazole] is the equilibrium concentration of tetrazole, [monomer amidite] is the equilibrium concentration of the phosphoramidite monomer, and

[R₂NH₂ ⁺ tetrazolide-] is the equilibrium concentration of the tetrazolide salt of the amino leaving group of the phosphoramidite monomer.

Preferably, the 3 '-amino protecting group of the monomer, R², is an acid-labile group, such as triphenylmethyl (i.e., trityl), p-anisyldiphenylmethyl (i.e.,

monomethoxytrityl or MMT), di-p-anisylphenylmethyl (i.e., dimethoxytrityl or DMT), or acid-labile urethane. Most preferably, the 3'-amino protecting group is

triphenylmethyl. These protecting groups are removed by treatment with acidic solutions, most preferably with a solution of 3% dichloroacetic acid in methylene chloride. Likewise, the 3'-hydroxyl protecting group of the monomer, R⁷, is an acid- labile group, such as trityl, MMT, DMT, or urethane. Most preferably, the 3'-hydroxyl protecting group is DMT.

As used herein, the term "free amino group" in reference to the monomers of the invention means an amino group available for reacting with the phosphoramidite group of an incoming monomer. Preferably, a free amino group is a primary amine. After the detritylation step, the amino group will generally be in the form of its salt with the conjugate base of the acid used for detritylation. This salt optionally may be neutralized with a basic solution such as 2% triethylamine or pyridine in acetonitrile after the detritylation step.

The coupling step of the invention may be carried out in the temperature range of -20 to 200 degrees Centigrade. More preferably, the reaction is carried out at ambient temperature (about 15-30 degrees Centigrade). The reaction is performed by adding a solution of the phosphoramidite monomer and a solution of an activator (or a solution containing the phosphoramidite monomer and the activator) to the reaction vessel containing the free amino group of an (oligo)nucleotide covalently attached to a solid support. Generally, activators of the invention are nucleophilic catalysts that displace the more stable phosphoramidite amino group to form a highly reactive (and much less stable) intermediate which, in turn, reacts with the free 3' amino group of a solid supported oligonucleotide N3'→P5' phosphoramidate. The mixture is then mixed by such methods as mechanically vortexing, sparging with an inert gas, etc.

Alternately, the solution(s) of monomer and activator can be made to flow through a reaction vessel (or column) containing the solid supported (oligo)nucleotide with a free 3'-amino group. The monomer and the activator either can be premixed, mixed in the valve-block of a suitable synthesizer , mixed in a pre-activation vessel and pre- equilibrated if desired, or they can be added separately to the reaction vessel.

Examples of activators for use in the invention are tetrazole, 5- (ethylthio)tetrazole, 5-(4-nitrophenyl)tetrazoIe, 5-(2-thiophene) tetrazole, triazole, pyridinium chloride, and the like, e.g. Beaucage and Iyer (cited above); Bemer et al, Nucleic Acids Research, 17: 853-864 (1989); Benson, Chem. Rev. 41 : 1-61 (1947). As used herein, the term "tetrazole activator" refers to activators which are tetrazole or derivatives of tetrazole. The most preferred activator is tetrazole. Suitable solvents are acetonitrile, tetrahydrofuran, methylene chloride, and the like. Acetonitrile is preferred. A great amount of care should be exercised to use very dry (free from water) monomer, activator , and solvent for the coupling step and for the solvent used to wash the solid support immediately before the coupling step.

The choice of monomer (particularly the choice of the phosphoramidite amino group) depends on the application. Generally, for research-scale (0.01-10 μmol) phosphoramidate oligonucleotide synthesis using commercially available DNA synthesizers such as the ABI Model 394 it is useful to use a monomer which is relatively stable and relatively less reactive (as defined by the equilibrium constant K_{1 ,} vide supra) so the solution can be left on the instrument for multiple syntheses over a period of weeks. Another reason favoring the use of a relatively less reactive monomer for this application is the fact that instruments of this type are typically not designed to minimize the volume of reagent used. In fact, since research-scale synthesizers typically operate to some extent by flowing monomer solution through the reaction vessel (column), some of the soluble products are removed from the column (and therefore from the equilibrium), thus helping to drive the equilibrium towards completion. Also, the cost of the monomer used at this scale may be relatively less important than other factors (such as labor cost, convenience, etc). Generally, when less reactive monomers (such as diisopropylaminophosphoramidites) are used, one needs to use relatively large excesses of monomer (10-50) compared to the free amino group and one needs to use large excesses of activator to achieve reasonable reaction rates and conversions (yield) .

To minimize the amount (number of equivalents) of monomer required to effect a desired coupling yield, especially when using a relatively less reactive monomer and performing a small-scale synthesis as outlined above, it is very useful to perform the couple and oxidation steps twice, using a lower concentration (and less equivalents) of monomer in each of the two coupling steps. This is because the coupling reaction is a reversible equilibration (illustrated in equations la and lb). Using this method the first coupling of the cycle is performed using fairly low amounts of monomer and the equilibrium concentration of desired phosphoramidite linkage which forms under these conditions is then "locked-in" as the phosphoramidate by performing the first oxidation step of the cycle. A second coupling step is then performed, again with relatively low amounts of monomer, followed by a second oxidation step. Less monomer overall is required to achieve a desired yield using this method than if all the monomer is used in a single coupling.

The economics of large-scale production require that the amount (number of equivalents) of monomer used be minimized. In this application the cost of monomer generally becomes a larger percentage of the overall cost since the labor and some other costs do not increase linearly with the scale of the synthesis. Also, large-scale oligonucleotide synthesizers generally operate in a batch-mode rather than the flow- through mode commonly found in research-scale synthesizers, such that the technique of driving the reaction towards completion by removing soluble products from the vessel is not practical. In this application it is necessary to select a monomer which is relatively more reactive (as defined by K₁ , vide supra). Such monomers generally possess phosphoramidite amino groups which are relatively more basic and/or more sterically hindered. The use of such reactive monomers allows the use of lower concentrations of activator to achieve reasonable reaction rates. These lower concentrations of activator help to prevent the reverse reaction of the desired product with activator to form activated intermediate (equation 1 b). For these reasons significantly lower amounts of monomer (1-5 equivalents) are required. Generally, it is very important to empoly monomer of the highest purity.

After the coupling step the resulting phosphoramidite linkage is oxidized (sulfurized) to form a stable protected phosphoramidate (phosphorothioamidate) linkage. The oxidation step can be performed immediately after the coupling solution is drained from the reaction vessel, or with a solvent wash in between. Since the phosphoramidite linkage could hydrolyze in the presence of tetrazole, the wash solution is preferably very dry and/or basic. If a wash step is not used, the oxidizing solution is preferably basic and/or very dry, or an oxidizing agent is selected that is sufficiently reactive to compete favorably with hydrolysis.

Oxidizing agents which are useful in the method of this invention include iodine, chlorine, bromine, peracids such as m-chlorobenzoic acid, hydroperoxides such as t-butylhydroperoxide, ethyl hydroperoxide, methyl hydroperoxide and the like, ozone, mixed acyl-sulfinic anhydrides such as 3H-2,1-benzoxathiolan-3-one-1-oxide, salts of persulfates such as sodium, ammonium, and tetrabutylammonium persulfate and the like, monoperoxysulfates such as oxone™, sodium and/or other hypochlorites, peroxides such as diethyl peroxide or bis(trimethylsiIyl)peroxide, or hydrogen peroxide or non aqueous hydrogen peroxide equivalents such as urea/hydrogen peroxide complex, etc. Other useful oxidizing agents which may be used to convert phosphorus (III) to phosphoms (V) are described in Beaucage and Iyer (cited above). Sulfurzing agents for use in the invention include elemental sulfur, thiuram disulfides such as tetraethyl thiuram disulfide, acyl disulfides such as phenacyldisulfide, phosphinothioyl disulfides such as S-Tetra™, and 1,1-dioxo-3H-1,2-benzodithiol-3-one. Hydrogen peroxide is a preferred oxidizing agent for use in this invention. A preferred

embodiment is to use a solution of 1.5% hydrogen peroxide, 3.5% water, 20% pyridine, and 75% THF.

In one embodiment of the invention the unreacted 3 '-amino groups remaining after the (last) oxidation step of a cycle may be capped with a suitable capping agent before the next detritylation step to render them inert to subsequent coupling steps. This capping step not only improves the HPLC profile to make purification more facile, but also significantly improves the overall yield of product, perhaps by eliminating the unreacted 3' amino groups from competing in the equilibrium. Capping reagents useful in the method of this invention include electrophilic reagents such as such as acetic anhydride and isobutyric anhydride, acid chlorides such as adamantyl carbonyl chloride, pivaoyl chloride, and the like, isothiocyanates, chloroformates, etc. Also useful are phosphoramidites in conjunction with an activator and followed by oxidation, and H- phosphonate salts such as triethylammonium isopropyl-H-phosphonate used in conjunction with an acid chloride such as pivaoyl chloride or adamantyl carbonyl chloride.

The 3'-amino protecting groups in the monomer, e.g. trityl, make the amino group less reactive with the phosphoramidite group of the monomers and with capping agents. However, this protection is not as complete as is the case with the similarly protected 5'-hydroxyl group of conventional phosphodiester synthesis. For this reason a slightly less reactive capping agent such as isobutyric anhydride is preferred over the acetic anhydride most often used in phosphodiester synthesis. Either the isobutyric anhydride or acetic anhydride can be used as a 1 : 1 :8 anhydride:lutidine:

tetrahydrofuran (by volume) solution and used in equal parts with a solution of 1 - methylimidizole in tetrahydrofuran as supplied by PE Applied Biosystems (Foster City, CA).

The oligonucleotide is cleaved from the solid support and deprotected after completion of the chain assembly using aqueous ammonia or other means as described in references cited above. The oligonucleotide may be cleaved from the support with its terminal amino-protecting group (or hydroxyl protecting group in some cases) intact. This is desirable in some situations in which the trityl or other protecting group is used as an aid in purification, such as by reverse phase or ion-exchange HPLC. However, when trityl protecting groups are used and are removed with acid treatment after cleavage from the support, deprotection, and purification there is a strong tendency for the deprotected phosphoramidate linkages to undergo undesired fragmentation, therefore great care is required during this step.

Alternately, the terminal amino protecting group (such as trityl) may be removed with acid before cleavage from the support. In this case the phosphoramidate linkages are still protected and fragmentation is avoided. The oligonucleotide is then cleaved from the support and deprotected as above. The phosphoramidate oligonucleotide may be purified by ion-exchange HPLC, reversed-phase HPLC, or other means.

Example 1

Preparation of 2'-Deoxy-3 '-tritylaminothymidine-5'-phosphoramidite Monomers

The synthesis of 3'-(Trityl)amino-3'-deoxythymidine-5'-(2-cyanoethyl N,N- diisopropyl) phosphoramidite, 4t is outlined in Scheme I. 3'-Azido-5'-O-(4- methoxybenzoyl)-3'-deoxythymidine, 1t, was synthesized by the method of Czemecki and Valery, Synthesis, 1991 : 239.

3'-(Trityl)amino-5'-(4-methoxybenzoyl)-3'-deoxythymidine, 2t. 3'-Azido-5'-(9- (4-methoxybenzoyl)-3'-deoxythymidine, It (10.0 g, 24.9 mmol), was dissolved in ethanol (500 mL) and reduced via hydrogenation (60 psi H₂) in the presence of 10% Pd/C (1.0 g) for 16 h. Subsequent removal of the catalyst via filtration and evaporation of solvent in vacuo, afforded 92% yield (8.6 g, 22.9 mmol) of the corresponding 3'- amine which was taken on directly to the next reaction. The 5'-(4-methoxybenzoyl)-3'- amino-3'deoxythymidine (8.6 g, 22.9 mmol) was azeotroped from pyridine (2 x 50 mL), and dissolved in anhydrous pyridine (50 mL). To this solution was added triethylamine (6.71 mL, 48.1 mmol) and trityl chloride (7.0 g, 25.2 mmol). This mixture was stirred for 2 h at ambient temperature, an additional portion of trityl chloride was added (1.9 g, 6.9 mmol), and the reaction was stirred an additional 2 h. Solvents were removed in vαcuo, and the cmde product was purified on silica (2 - 5% MeOH/CH₂Cl₂) to afford 90% yield (12.7 g, 20.6 mmol) of 3'-(Trityl)amino-5'-(4-methoxybenzoyl)-3'- deoxythymidine, 2t.

3'-(Trityl)amino-3'-deoxythymidine, 3t. The 5'-O-anisoyl protecting group was removed by dissolving 2t (30.1 g, 48.7 mmol) in 57:43 1 ,4-dioxane/MeOH (150 mL), followed by the addition of 2 M aq. NaOH (73.1 mL, 146.2 mmol). After stirring for 1.5 h at ambient temperature, the reaction mixture was neutralized with Dowex 50W- X8 cation exchange resin (ca. 150 g of dry pyridinium H -form, 1.6 meq/g). Once the pH was neutral (ca. 10 min), the resin was filtered, washed extensively with CH₂Cl₂ and MeOH, and the cmde product was concentrated in vacuo. The residue was redissolved in EtOAc (500 mL) and extracted with saturated, aq. NaHCO₃ (2 x 250 mL), H₂O (250 mL), and saturated, aq. NaCl (250 mL). After drying over Na₂SO₄ and filtration, the solvents were removed in vacuo, and the resulting foam was redissolved in 95:5 CH₂Cl₂/MeOH (300 mL). This solution was added slowly to a rapidly stirring mixture of 1 : 1 Et₂O/hexane (1250 mL) to precipitate the pure 3'-(Trityl)amino-3'- deoxythymidine, 3t, in 90% yield (21.2 g, 43.8 mmol). Conversion to phosphoramidite monomers and/or succinylated nucleoside is described in Examples 5-7 below.

Example 2

Preparation of 2'-Deoxy-3'-tritylaminocytidine-5'-phosphoramidite Monomers The synthesis of N -Benzoyl-3'-(trityl)amino-2',3'-dideoxycytidine-5'-(2- cyanoethyl N,N-diisopropyl) phosphoramidite, 4c is outlined in Scheme II.

- 3'-azido-5'- O-(tert-butyldimethylsilyl)-2',3'-dideoxyuridine, 1du. 2'- Deoxyuridine (1 1.4 g, 50 mmol) was thoroughly dried by coevaporating in vacuo with anhydrous DMF (2 x 100 mL). DMF (100 mL) was then added, followed by triethylamine (8.36 mL, 60 mmol), 4-dimethylaminopyridine (0.31 g, 2.5 mmol), and tert-butyldimethylsilyl chloride (8.29 g, 55.0 mmol). The reaction mixture was stirred for 1 h at RT, diluted with dichloromethane (600 mL) and extracted with H₂O (3 x 200 mL), and saturated aq. NaCl (200 mL). The organic layer was dried over Na₂SO₄, filtered and concentrated in vacuo. The resulting residue was purified on silica (2 - 10% MeOH/CH₂Cl₂) to afford 80% yield (13.7 g, 40.0 mmol) of 5'-O-(tert- butyldimethylsilyl)-2'-deoxyuridine. Triphenylphosphine (16.8 g, 64.0 mmol) and DMF ( 100 mL) were added, and to this stirring mixture was added a solution of diisopropylazodicarboxylate (12.6 mL, 64.0 mmol) in DMF (20 mL). After stirring 2 h at RT, the reaction mixture was concentrated in vacuo to ca. 30 mL, and poured into Et₂O (1200 mL). The desired 2,3'-anhydro-5'-O-(tert-butyldimethylsilyl)-2'- deoxyuridine began precipitating out after 10 min of rapid stirring. The resulting mixture was placed in the refrigerator overnight, the precipitate was collected by filtration, washed with additional cold Et₂O (2 x 300 mL), and dried in vacuo to afford 90% yield (11.7 g, 36.0 mmol) of 2,3'-anhydro-5^,-O-(tert-butyldimethylsilyl)-2'- deoxyuridine as a white solid, which was not purified further. The 2,3'-anhydro-5'-O- (tert-butyldimethylsilyl)-2'-deoxyuridine (33.8 g, 104.2 mmol) was then reacted with LiN₃ (7.65 g, 156.3 mmol) in DMF (300 mL) at 95-100°C for 48 h. The resulting brown, homogeneous mixture was then cooled to RT, concentrated in vacuo to an oil, dissolved in EtOAc (800 mL), and extracted with H₂O (200 mL). The aqueous layer was extracted twice more with EtOAc (75 mL), and the combined organics were washed with H₂O (3 x 250 mL) and once with saturated, aq. NaCl (250 mL). The

EtOAc solution was dried over Na₂SO₄, filtered, and concentrated in vacuo, to afford 87% yield (33.2 g, 90.3 mmol) of 3^,-azido-5^,-O-(tert-butyldimethylsilyl)-2',3'- dideoxyuridine, 1du, as a brownish foam, which was taken on directly to

hydrogenation.

3'-(Trityl)amino-5'-O-(tert-butyldimethylsilyl)-2',3'-dideoxyuridine, 2du. Crude ldu (33.2 g, 90.3 mmol) was dissolved in 2:1 EtOH/ CH₂Cl₂ (300 mL) and reduced via hydrogenation (60 psi H₂) in the presence of 10% Pd/C (3.0 g) for 18 h. Subsequent removal of the catalyst via filtration and evaporation of solvent in vacuo, afforded quantitative yield (30.4 g, 89.8 mmol) of the corresponding 3'-amine which was taken on directly to the next reaction. The 5'- O-(tert-butyldimethylsilyl)-3'-amino-2',3'- dideoxyuridine (30.4 g, 89.8 mmol) was azeotroped from pyridine (2 x 300 mL), and dissolved in a mixture of CH₂Cl₂ (600 mL) and anhydrous pyridine (70 mL). To this solution was added triethylamine (25.0 mL, 179.6 mmol) and trityl chloride (35.0 g, 125.7 mmol), and the reaction mixture was stirred for 2 h at ambient temperature. Solvents were removed in vacuo, and the crude product was purified on silica (1 - 5% MeOH/CH₂Cl₂) to afford 85% yield (44.3 g, 75.9 mmol) of 3'-(Trityl)amino-5'- O-(tert- butyldimethylsilyl)-2',3'-dideoxyuridine, 2du.

N⁴-Benzoyl-3'-(trityl)amino-5'-O-(tert-butyldimethylsilyl)-2',3'-dideoxycytidine,

2c. Triethylamine (22.5 mL, 161.1 mmol) was added dropwise over a period of 10 min to a stirring mixture of 1 ,2,4-triazole (11.1 g, 161.1 mmol) and phosphorus oxychloride (3.5 mL, 37.1 mmol) in anhydrous acetonitrile (125 mL) at 0° C. To this cold, stirring mixture was added 2du (9.4 g, 16.1 mmol) as a solution in acetonitrile (50 mL). This mixture was stirred at RT for 2 h, triethylamine (30 mL) and H₂O (10 mL) were added to quench the reaction and promote dissolution, and solvents were removed in vacuo. The resulting brown solid was redissolved in CH₂Cl₂ (250 mL), extracted with saturated, aq. NaHCO₃ (3 x 150 mL), saturated, aq. NaCl, dried over Na₂SO₄, filtered, and concentrated in vacuo to afford quantitative yield (10.2 g, 16.1 mmol) of 3'- (Trityl)amino-5'-O-(tert-butyldimethylsilyl)-2',3'-dideoxy-4-(l,2,4-triazol-1-yl)uridine as an orangish solid. This cmde material was dissolved in 1 ,4-dioxane (200 mL) and cold, concentrated NH₄OH (50 mL) was added. The reaction mixture was stirred at RT for 4 h, and concentrated in vacuo to afford quantitative yield (9.4 g, 16.1 mmol) of 3'- (Trityl)amino-5'-O-(tert-butyldimethylsilyl)-2',3'-dideoxycytidine as a beige solid. This cmde material was then azeotroped from anhydrous pyridine (2 x 200 mL), redissolved in pyridine (200 mL), and to this stirring solution was added benzoyl chloride (2.2 mL, 19.3 mmol) at 0°C. The reaction was then stirred at RT for 16 h, externally cooled to 0°C, quenched with H₂O (40 mL), and after stirring 5 min, cone. aq. ammonia (40 mL) was added and the reaction mixture was stirred for an additional 15 min at 0°C.

Solvents were removed in vacuo, the residue was redissolved in CH₂CI₂ (125 mL), and extracted with saturated, aq. NaHCO₃ (3 x 75 mL), dried over Na₂SO₄, filtered and concentrated in vacuo. This crude material was purified on silica (1 - 5% MeOH CH₂Cl₂) to afford 92% yield (10.2 g, 14.8 mmol) of N⁴-Benzoyl-3'-(trityl)amino-5'-O- (tert-butyldimethylsilyl)-2',3'-dideoxycytidine, 2c.

N⁴-Benzoyl-3'-(trityl)amino-2',3'-dideoxycytidine, 3c. The 5'-TBDMS protecting group was removed by dissolving 2c (5.8 g, 8.5 mmol) in 1 :1

CH₂Cl₂/pyridine (25 mL) and reacting with Et₃N 3HF (6.9 mL, 42.6 mmol) for 16 h. The reaction mixture was diluted with CH₂CI₂ (200 mL) and extracted with H₂O (2 x 50 mL), and saturated, aq. NaCl (50 mL). The organic layer was dried over Na₂SO₄, filtered, and the solvent was removed in vacuo. The crude product was purified on silica (3% MeOH/CH₂Cl₂) to afford 75% yield (3.7 g, 6.4 mmol) of N⁴-Benzoyl-3'- (trityl)amino-2',3'-dideoxycytidine, 3c. Conversion to phosphoramidite monomers and/or succinylated nucleoside is described in Examples 5-7 below. Example 3

Preparation of 2'-Deoxy-3'-tritylaminoguanosine-5'-phosphoramidite Monomers The synthesis of N²-Isobutyryl-3'-(trityl)amino-2',3'-dideoxyguanosine-5'-(2- cyanoethyl N,N-diisopropyl) phosphoramidite, 4g is outlined in Scheme III. 5'-O-

Benzoyl-Ν²-isobutyryl-2'-deoxyguanosine and 3'-O-Benzoyl-Ν² -isobutyryl-2'- deoxyxyloguanosine were prepared as described previously, Reese et al, J. Chem. Soc. Perkin Trans. I, 1984: 1263.

5'-O-(tert-Butyldimethylsilyl)-N -isobutyryl-3'-azido-2',3'-dideoxyguanosine, lg. To a stirring solution of 3'-O-Benzoyl-N -isobutyryl-2'-deoxyxyloguanosine (4.86 g, 11.0 mmol) in DMF (20 mL), was added triethylamine (3.4 mL, 24.2 mmol), 4- dimethylaminopyridine (54 mg, 0.44 mmol), and tert-butyldimethylsilyl chloride 33.31 g, 22.0 mmol). The reaction was stirred for 2 h at RT, methanol (10 mL) was added and after stirring an additional 5 min, the reaction mixture was concentrated in vacuo. The residue was redissolved in CH₂Cl₂ (150 mL), washed with H₂O (3 x 40 mL), and saturated, aq. NaCl (60 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo to afford 6.40 g of a reddish-colored foam. To this cmde material was added, a pre-chilled (ca. 5°C) solution of 2 M NaOH in 65:30:5 pyridine:MeOH:H₂O (44.0 mL, 87.9 mmol). The reaction mixture was stirred in an ice bath for 20 min and neutralized with 1 M HCI (97 mL) to pH 7. The reaction mixture was concentrated in vacuo to ca. 50 mL, and extracted with CH₂Cl₂ (3 x 75 mL). The combined organics were washed with saturated, aq. NaCl (50 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo to afford 82% yield (4.1 g, 9.1 mmol) of 5'-O-(tert- butyldimethylsilyl)-N -isobutyryl-2'-deoxyxyloguanosine as a sandy-colored foam, which was taken on to the next reaction without further purification. To cmde 5'-O- (tert-butyldimethylsilyl)-N -isobutyτyl-2'-deoxyxyloguanosine (47.3 g, 104.7 mmol), was added LiN₃ (15.4 g, 314.1 mmol), triphenylphosphine (41.2 g, 157.1 mmol), and anhydrous DMF (1000 mL). Diethylazodicarboxylate (24.7 mL, 157.1 mmol) was added and the reaction mixture was stirred for 5 h at RT under argon. H₂O (20 mL) was added and the reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc (1500 mL), washed with H₂O (3 x 1000 mL), saturated, aq. NaCl (1000 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified on silica (1 - 5% MeOH/CH₂Cl₂), although this afforded >100% yield (1 12.7 g) of impure 5'-O-(tert-Butyldimethylsilyl)-N²-isobutyryl-3'-azido-2',3'-dideoxy- guanosine, 1g. This contaminated product was not purified further, and was taken on directly to hydrogenation and purified as the 3'-amine.

5'-O-(tert-Butyldimethylsilyl)-N²-isobutyryl-3'-(trityl)amino-2',3'- dideoxyguanosine, 2g. Crude compound 1g (49.9 g„ 104.7 mmol) was dissolved in (warm) ethanol (1600 mL) and hydrogenated (60 psi H₂) in the presence of 10% Pd/C (2.5 g) for 16 h at RT. The catalyst was removed via filtration, and the solvent was evaporated in vacuo to afford the cmde 5'-O-(tert-butyldimethylsilyl)-N -isobutyryl-3'- amino-2',3'-dideoxyguanosine, which was purified on silica (2 - 6% MeOH/CH₂Cl₂ and then 1% Et₃N/6% MeOH/CH₂Cl₂) to afford 60% yield (28.5 g, 63.2 mmol) of pure 5'- O-(tert-butyldimethylsilyl)-N -isobutyryl-3'-amino-2',3'-dideoxyguanosine as an off- white foam. The 3'-amine (28.5 g, 63.2 mmol) was protected by reacting with triethylamine (17.6 mL, 126.4 mmol) and trityl chloride (28.2 g, 101.1 mmol) in pyridine (500 mL) for 16 h at RT. Solvents were removed in vacuo and the residue was purified on silica (1 - 5% MeOH/CH₂Cl₂) to afford quantitative yield (43.8 g, 63.2 mmol) of 5'- O-(tert-butyIdimethylsilyl)-N²-isobutyryl-3'-(trityl)amino-2',3'- dideoxyguanosine, 2g. N -Isobutyryl-3'-(trityl)amino-2',3'-dideoxyguanosine, 3g. The 5'-TBDMS protecting group was removed by dissolving 2g (5.9 g, 8.5 mmol) in 1 : 1

CH₂Cl₂/ρyridine (25 mL) and reacting with Et₃N 3HF (6.9 mL, 42.6 mmol) for 16 h. The reaction mixture was diluted with CH₂CI₂ (200 mL) and extracted with H₂O (2 x 50 mL), and saturated, aq. NaCl (50 mL). The organic layer was dried over Na₂SO₄, filtered, and the solvent was removed in vacuo. The crude product was purified on silica (2-5% MeOH/CH₂Cl₂) to afford 73% yield (3.6 g, 6.2 mmol) of N²-Isobutyryl-3'- (trityl)amino-2',3'-dideoxyguanosine, 3g. Conversion to phosphoramidite monomers and/or succinylated nucleoside is described in Examples 5-7 below.

Example 4

Preparation of 2'-Deoxy-3'-tritylaminoadenosine-5'-phosphoramidite Monomers The synthesis of N -Benzoyl-3'-(trityl)amino-2',3'-dideoxyadenosine-5'-(2- cyanoethyl N,N-diisoρropyl) phosphoramidite, 4a is outlined in Scheme IV. Ν - Benzoyl-9-(5-O-benzoyl-2-deoxy-β-D-threo-pentofuranosyl)adenine was synthesized by the method of Herdewijn, J. Org. Chem., 53: 5050 (1988).

5'-O-(Benzoyl)-N -benzoyl-3'-azido-2',3'-dideoxyadenosine, la. N⁶-Benzoyl-9- (5-O-benzoyl-2-deoxy-β-D-threo-pentofuranosyl)adenine (7.0 g, 15.3 mmol), triphenylphosphine (6.0 g, 22.9 mmol), and LiN₃ (2.8 g, 56.4 mmol) were dissolved in DMF (100 mL). To this stirring mixture was added diethylazodicarboxylate (3.6 mL, 22.9 mmol) in one portion, the reaction was stirred for 2.5 h at RT, and the reaction was quenched with H₂O (10 mL). The solvents were removed in vacuo, the residual oil was redissolved in EtOAc (300 mL) and extracted with H₂O (2 X 200 mL) and saturated. aq. NaCl (200 mL). The organic layer was dried over Na₂SO₄, filtered, and

concentrated in vacuo. The resulting residual oil was purified on SiO₂ (2 - 5%

MeOH/CH₂Cl₂) to afford 5'-O-Benzoyl-N⁶-benzoyl-3'-azido-2',3'-dideoxyadenosine, 1a, as an amber foam, which was taken on directly to hydrogenation.

N -Benzoyl-3'-(trityl)amino-2',3'-dideoxyadenosine, 3a. Crude 1a was dissolved in 3: 1 EtOH/ CH₂C1₂ (200 mL) and reduced via hydrogenation (60 psi H₂) in the presence of 10% Pd/C (0.7 g) for 18 h. Subsequent removal of the catalyst via filtration and evaporation of solvent in vacuo, afforded 57% (4.0 g, 8.7 mmol) of the corresponding 3'-amine which was taken on directly to the next reaction. 5'-OBenzoyl- N -benzoyl-3'-amino-2',3'-dideoxyadenosine (3.9 g, 8.5 mmol) was dissolved in CH₂Cl₂ (50 mL), triethylamine (2.9 mL, 20.8 mmol) was added, followed by the addition of trityl chloride (2.9 g, 10.2 mmol). After stirring for 1.5 h at RT the reaction mixture was diluted with CH₂CI₂ (50 mL), and extracted with H₂O (2 x 50 mL) and saturated, aq. NaCl (50 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo to afford cmde 5'-O-Benzoyl-N -benzoyl-3'-(trityl)amino-2',3'- dideoxyadenosine, which was taken on directly to hydrolysis of the 5'-benzoyl protecting group. The crude 5'-O-Benzoyl-N -benzoyl-3'-(trityl)amino-2',3'- dideoxyadenosine (ca. 6.0 g, 8.5 mmol) was dissolved in 1 :1 THF/MeOH (100 mL) and cooled to 0°C. To this mixture was added pre-chilled 2 M aq. NaOH (13.7 mL, 27.4 mmol), and the reaction mixture was stirred at 0°C for 20 min. At this time the reaction appeared to be only about 50% complete, so additional 2M aq. NaOH (10.0 mL, 20 mmol) was added. After stirring an additional 15 min at 0°C, the reaction was neutralized with Dowex 50W-X8 cation exchange resin (ca. 40 g of dry pyridinium H⁺- form, 1.6 meq/g) to pH 6. The resin was filtered and washed extensively with MeOH and THF, and solvents were removed in vacuo. The residue was redissolved in CH₂Cl₂ (300 mL) and extracted with H₂O (150 mL), saturated, aq. NaHCO₃ (2 x 150 mL), H₂O (150 mL), and saturated, aq. NaCl (150 mL). After drying over Na₂SO₄, the solution was filtered and concentrated in vacuo. The residue was purified on SiO₂ (2 - 3% MeOH/ CH₂Cl₂) to afford 71% yield (3.6 g, 6.0 mmol) of N⁶-Benzoyl-3'-(trityl)amino- 2',3'-dideoxyadenosine, 3a, as a white foam. Conversion to phosphoramidite monomers and/or succinylated nucleoside is described in Examples 5-7 below. Example 5

Preparation of 3'-(trityl)amino-2',3'-dideoxynucleoside-5'-(2-cyanoethyl N,N- diisopropyl) phosphoramidites, 4a, 4c, 4g, and 4t:

To 8.4 mmol 3'-(trityl)amino-2,'3'-dideoxynucleoside (3a, 3c, 3g, or 3t) (previously azeotroped 2 times from CH₃CΝ) in 25 mL of CH₂CI₂ under argon was added 2.0 mL (1 1.8 mmol) of N,N-diisopropylethylamine and 2.1 mL (9.4 mmol) of 2- cyanoethyl N,N-diisopropylchlorophosphoramidite. After stirring for 15 min the reaction was diluted with CH₂CI₂ and extracted with saturated, aq. ΝaHCO₃ and saturated, aq. NaCl. The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. N⁶-Benzoyl-3'-(trityl)amino-2',3'-dideoxyadenosine-5'-(2-cyanoethyl N,N- diisopropyl) phosphoramidite, 4a, was purified on SiO₂ (5% Et₃Ν/2%

methanol/toluene) which gave 5.82 g (87.1 %) of pure phosphoramidite. ³¹P NMR (CD₃CN) δ 148.6, 149.2. N⁴-Ben zoyl-3'-(trityl)amino-2',3'-dideoxycytidine-5'-(2- cyanoethyl N,N-diisopropyl) phosphoramidite, 4c, was purified on SiO₂ (3% MeOH/5% Et₃Ν/toluene) and gave 5.58 g (86.0%) pure product. ^{3 1}P NMR (CD₃CN) δ 149.3, 149.6. N²-Isobutyryl-3'-(trityl)amino-2',3'-dideoxyguanosine-5'-(2-cyanoethyl N,N- diisopropyl) phosphoramidite, 4g, was precipitated under argon from 10 mL CH₂CI₂ into rapidly stirring ethyl ether (200 mL) and hexane (200 mL) at 4 °C to remove the hydrogen phosphonamidate impurity which cannot be removed by column

chromatography in this case. The solid was filtered, washed with hexane, and dried in vacuo. This precipitation step was repeated and the resulting solid was purified further on Siθ2 (10% Et₃Ν/CH₂Cl₂) and gave 4.51 g (69%) pure phosphoramidite product. ³¹P NMR (CD₃CN) δ 148.7, 149.4. 3'-(Trityl)amino-3'-deoxythymidine-5'-(2- cyanoethyl N,N-diisopropyl) phosphoramidite, 4t, was purified on SiO₂ (2%

Et₃Ν/CH₂Cl₂) and afforded 4.06g (70.7%) pure phosphoramidite and some mixed fractions. ³¹P NMR (CD₃CN) δ 149.4, 149.5.

Example 6

Preparation of 3'-(trityl)amino-2',3'-dideoxynucleoside-5'-

(2-cyanoethyl-2,2,6,6-tetramethylpiperidine) phosphoramidites,

5a, 5c, 5g, and 5t:

To 8.4 mmol 3'-(trityl)amino-2,'3'-dideoxynucleoside (3a, 3c, 3g, or 3t) (previously azeotroped 2 times from CH₃CN) in 25 mL of CH₂CI₂ cooled to 4 °C under argon was added 1.9 mL (12.6 mmol) of DBU and 5.2 mL (8.4 mmol) of a solution of 2-cyanoethyl 2,2,6,6-tetramethylpiperidinechlorophosphoramidite in CH₂CI₂

(conc.= 1.626 mmol/mL). The ice bath was removed and the solution was stirred for 30-60 min. To avoid decomposition, the crude reaction was loaded directly onto a SiO₂ column (3% MeOH/5% Et₃N/toluene for 5a, 5c, and 5t, and 10% Et₃N/CH₂Cl₂ for 5g) for purification. In all cases further purification was necessary as indicated for each product. N⁶-Benzoyl-3'-(trityl)amino-2',3'-dideoxyadenosine-5^,-(2-cyanoethyl 2,2,6,6- tetramethylpiperidine) phosphoramidite, 5a, was purified on SiO₂ (3%

MeOH/5%Et₃Ν/toluene) which gave 5.21 g (74.3 %) of pure phosphoramidite. ³¹P NMR (CD₃CN) δ 164.8, 165.4. N⁴-Benzoyl-3'-(trityl)amino-2',3'-dideoxycytidine-5'- (2-cyanoethyl 2,2,6,6-tetramethylpiperidine) phosphoramidite, 5c, was purified on SiO₂ (3% MeOH/5% Et₃Ν/toluene) and gave 5.07 g (74.2%) pure product and some mixed fractions. ^{3 1}P NMR (CD₃CN) δ 164.8, 165.7. N²-Isobutyryl-3'-(trityl)amino-2',3'- dideoxyguanosine-5'-(2-cyanoethyl 2,2,6,6-tetramethylpiperidine) phosphoramidite, 5g, was precipitated from 2 mL of CH₂CI₂ into rapidly stirring ethyl ether (40 mL) and hexane (40 mL) at 4 °C under argon to remove the hydrogen phosphonamidate impurity which cannot be removed by column chromatography in this case. The solid was filtered, washed with hexane, and dried in vacuo. This precipitation step was repeated two times and the resulting solid was purified further on SiO₂ (10% Et₃Ν/CH₂CI₂) and gave 0.89 g (12.8%) pure phosphoramidite product. ³¹P NMR (CD₃CN) δ 165.2, 165.5. 3^,-(Trityl)amino-3'-deoxythymidine-5'-(2-cyanoethyl 2,2,6,6- tetramethylpiperidine) phosphoramidite, 5t, was purified on Siθ2 (5% MeOH/5% Et₃N/toluene) and afforded 3.33 g (54.8%) pure phosphoramidite. ^{3 1}P NMR (CDCI₃) δ 165.3, 166.1.

Example 7

Preparation of 3'-(trityl)amino-2',3'- dideoxynucleoside-5'-succinylates, 6a, 6c, and 6t:

To 1.5 mmol 3'-(trityl)amino-2',3'-dideoxynucleoside (3t, 3c, or 3a) in 5 mL

CH₂CI₂ was added 0.22 g (1.8 mmol) of N,N-dimethylaminopyridine and then 0.18 g (1.8 mmol) of succinic anhydride. After stirring at room temperature for 1 hour the reaction was quenched by addition of 0.6 mL of methanol, diluted with CH₂CI₂ and extracted with cold 10% citric acid, water, and saturated, aq. ΝaCl. The organic layer was dried (Νa₂SO₄), filtered, and concentrated to a foam. N⁶-Benzoyl-3'-(trityl)amino- 2'-3'-dideoxyadenosine-5'-succinylate, 6a. Yield 1.15 g (100 %). N⁴-Benzoyl-3'- (trityl)amino-2', 3'-dideoxycytidine-5'- succinylate, 6c. Yield 0.77 g (76.3 %). 3'- (Trityl)amino-3'-deoxythymidine-5'- succinylate, 6t. Yield 0.82 g (94.0 %). Example 8

Preparation of 3'-(trityl)amino-2',3'- dideoxynucleoside-5'-succinyl-loaded CPG

To 1 mmol 3'-(trityl)amino-2',3'-dideoxynucleoside-5'-succinylate (6a, 6c or 6t) and 0.13 g (0.95 mmol) of 1 -hydroxybenzotriazole in 5 mL N-methylpyrrolidine and 5 mL DMSO was added 0.35 mL (2.0 mmol) of N,N-diisopropylethylamine and then 0.36 g (0.95 mmol) of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate. The solution was stirred 5 min, added to 10.0 g aminopropyl- CPG, and put on the shaker for 6 hours. The CPG was filtered and washed with DMF, methanol, and ethyl ether. Unreacted amino groups on the CPG were acetylated using the standard ABI capping solutions for 30 min. The nucleoside loadings, determined by trityl assay at 432 nm in 20 % TFA/CΗCI3 using a molar extinction coefficient of 40.7 umol^{- 1} cm ^{- 1}, were 38.6 wmol/g for A, 33.6 «mol/g for C and 29.0 umol/g for T. Example 9

Measurement of the Tetrazole Activation Equilibrium Constant K₁ The equiiiorium represented by Equation 1 was studied by dissolving Ν⁶- benzoyl -3'-tritylamino-deoxyadenosine- 5'-(2-cyanoethyl N,N-di isopropyl)

phosphoramidite, (4a, 56.1 mg, 65.2 μmol) in 0.300 mL of dry deuteroacetonitrile and adding 0.300 mL of a solution of 0.5 M tetrazole in acetonitrile, all under an argon atmosphere. The solution was transferred to an ΝMR tube under argon. After 2 min, the P-ΝMR spectrum shown in Figure 1 was recorded.

The spectmm consists of resonances corresponding to the phosphoramidite monomer (143.07,143.72 ppm) the tetrazolidyl-amidite intermediate (127.58 ppm), the hydrogen phosphonate (10.24, 9.61 ppm) resulting from hydrolysis of some of the monomer (through the tetrazolidyl amidite intermediate), and minor side-reactions. The total of the integrations of these species was assumed to be equal to the initial phosphoramidite concentration of 0.0993 M, and the concentrations at equilibrium were calculated from the relative integrations of the individual resonances. The

concentrations of the species which contain no phosphorus, and therefore do not appear in the spectrum, were calculated as follows. The concentration of the

diisopropylammonium tetrazolide at equilibrium (0.0824 M) was assumed to be equal to the initial concentration (0.0993 M) minus the equilibrium concentration (0.0169 M) of the phosphoramidite monomer. The tetrazole concentration at equilibrium (0.0875 M) was assumed to be equal to the initial tetrazole concentration (0.2286 M) minus the sum of the tetrazolidyl amidite intermediate concentration (0.0587 M) and the diisopropylammonium tetrazolide concentration (0.0824 M). The activation

equilibrium constant K] was calculated as follows:

The above experiment was repeated using the 3'-tritylaminothymidine-5'- diisopropylaminophosphoramidite monomer (4t) and the K₁ was found to be 56.2 M^{- 1}. The experiment was repeated again using the 3'-tritylaminothymidine-5'- tetramethylpiperidinyl phosphoramidite monomer (5t). The ³¹P-nmr spectmm appears in Figure 2.

Under the conditions of the experiment, no tetramethylpiperidinyl

phosphoramidite monomer remaining at equilibrium (expected at 165.3,166.1 ppm) was detected . The monomer should be detectable above the noise level of the spectrum if its concentration were at least 0.19 mM. From this information, it may be calculated that the K₁ for this equilibrium must be at least 5260 M^-1, or 93 times greater than that for the diisopropylaminophosphoramidite monomer. The experiment was repeated once more using the 3'-tritylaminothymidine-5'-(N-isopropyl-N-t-butyl)phosphoramidite monomer. As in the case of the tetramethylpiperidinyl phosphoramidite monomer, this monomer was not detectable at equilibrium in the ³¹P-ΝMR spectra.

Example 10

1-μmol Scale Synthesis of Oligo-2'-deoxynucleoside N3'→P5' phosphoramidates using Diisopropylaminophosphoramidite Monomers:

Oligonucleotide N3'→P5' phosphoramidates were prepared on an ABI 392

DNA synthesizer at the 1 -umol scale and purified by preparative ion exchange chromatography. The synthesis is performed in the 5' to 3' direction (instead of the 3' to 5' direction which commercially available synthesizers are programmed for) using 1 μ mol of 3'-(trityl)amino-2',3'-dideoxynucleoside-5^,-succinyl-loaded CPG in the column. 3'-Tritylamino -5'-diisopropylphosphoramidite monomers were prepared as 0.1 M solutions in acetonitrile; the activation solution was 0.5 M tetrazole in acetonitrile (PE Applied Biosystems, Foster City, CA); the detritylation solution was 3% dichloroacetic acid (DC A) in dichloromethane (DCM), and the oxidation solution was 0.1 M iodine in tetrahydrofuran/pyridine/water, 75/20/2, v/v/v solution (PE Applied Biosystems, Foster City. CA).

Oligonucleotide N3'→P5' phosphoramidates were synthesized utilizing a repetitive synthesis cycle consisting of detritylation followed by a coupling, oxidation, coupling, oxidation strategy. Detritylation of the 3' amine of the support-bound nucleoside was achieved using a 40 second flow of 3% DCA/DCM for G and A nucleosides and 50 seconds for T and C nucleosides. The support-bound 3'- aminonucleoside was then washed six times with a 10 second acetonitrile delivery/5 second argon flush combination. Coupling of the amine with the 5'-phosphoramidite- 3'-tritylamino nucleoside was achieved using an alternating delivery to the column of monomer plus tetrazole and tetrazole alone for ~ 10 seconds followed by a 5 minute wait. The monomer was flushed from the column with argon and the iodine solution was immediately added, followed by a 2 minute wait. Upon completion of oxidation the growing support bound oligomer was washed one time with a 20 second acetonitrile delivery/5 second argon flush combination and five times with a 10 second acetonitrile delivery/5 second argon flush combination. Coupling and oxidation were repeated one additional time followed by washing prior to detritylation. Using this procedure ~ 15 equivalents (compared to initial loading of support-bound nucleoside) of monomer is used for each coupling step; therefore ~ 30 equivalents monomer is used for each synthesis cycle.

Upon synthesis completion, the support-bound 3'-detritylated oligonucleotide N3'→P5' phosphoramidate was cleaved and base-deprotected in concentrated aqueous ammonia at 55° for 12 hours. The cleaved and deprotected oligonucleotide N3'→P5' phosphoramidate solution was removed from the CPG and the CPG was washed 2 times with 200 μl of ammonia . All the ammonia washes were combined, the solution was buffered to 0.01M NaOH and the ammonia was removed under vacuum.

Following filtration, the cmde oligonucleotide was purified on a preparative anion exchange column (Pharmacia MonoQ 10/10), desalted on Sephadex G-25 (Pharmacia NAP-5), and lyophilized.

The sequences listed below were synthesized on an ABI 392 synthesizer using either the phosphoramidite amine exchange method of the invention or the Atherton- Todd oxidative coupling approach, e.g as described in Letsinger et al, U.S. patent 5,476,925. Results are tabulated below and illustrated in Figures 3a and 3b, which show chromatograms of ion-exchange HPLC separations of the two mixed-base sequences listed in the table.

Example 11

1-μmol Scale Synthesis of Oligo-2'-deoxynucleoside N3'→P5

phosphoramidates using Diisopropylaminophosphoramidite

Monomers: Comparison of Couple/Oxidation to CoupIe/Oxidation/couple/oxidation:

The effect on product yield of a single couple/oxidation step was compared with that of a double couple/oxidation step (couple/oxidation/couple oxidation) as follows: The sequence 5'-AAC-ATG-GAG-AGC-GTC-3' (SEQ ID NO: 7) was synthesized using diisopropylamino phosphoramidite monomers using the procedure described above and compared to a second synthesis which was like that above, but with the following exceptions: 1) a single couple / oxidation was used per cycle (instead of two), and 2) the concentration of monomer solution was 0.2 M (instead of 0.1 M). Therefore in the single couple / oxidation experiment ~30 equivalents of monomer were used in each synthesis cycle, whereas in the couple / oxidation / couple / oxidation experiment ~15 equivalents of monomer were used in each of the two coupling steps, for the same total of ~ 30 equivalents of monomer per synthesis cycle. The results, which appear below, demonstrate the improved efficiency using the 2X (couple / oxidation) method.

Example 12

1-μmol Scale Synthesis of oligo-2'-deoxynucleoside N3'→P5'

phosphoramidates: Comparison of no capping, acetic anhydride capping, and isobutyric anhydride capping

The sequence 5'-AAC-ATG-GAG-AGC-GTC-3' (SEQ ID NO: 7) was synthesized three times on a 1-μmol scale using the above procedure, but in two of the syntheses a 60 second capping step was inserted after the last oxidation step and before detritylation in each cycle. Acetic anhydride/NMI (PE Applied Biosystems, Foster City, CA) were used as capping reagents in one of the experiments and isobutyric anhydride (1/1/8 isobutyric anhydride/2,6-lutidine/THF) /NMI (PE Applied Biosystems, Foster City, CA) was used in the other. The results which appear below demonstrate the improved yield and purity with capping.

Example 13

1-μmol Scale Synthesis of oligo-2'-deoxynucleoside N3'→P5' phosphoramidates: Comparison of I₂and H₂O₂oxidation

The following sequences were synthesized on a 1-μmole scale using the above procedure, however in one set of experiments the iodine oxidation reagent was replaced with an oxidizing agent consisting of 1.5 % H ₂O₂/3.5 % H₂O / 20 % pyridine/ 75 % THF. No capping was employed in these experiments.

Example 14

10-μmol Scale Synthesis of oligo-2'-deoxynucleoside N3'→P5' phosphoramidates: Comparison of Diisopropyl and Tetramethylpiperidinyl Phosphoramidite Monomers The following general procedure was followed for 10-μmol-scale synthesis:

Oligonucleotide N3'→P5' phosphoramidates were prepared on a modified 390Z ABI DNA synthesizer at the 10-umol scale and purified by preparative ion exchange chromatography. 3'-Tritylamino 5 '-phosphoramidite monomers were prepared as 0.1 M solutions in acetonitrile; the activator was 0.15 M tetrazole in acetonitrile; the detritylation solution was 3% dichloroacetic acid (DCA) in dichloromethane (DCM), and the oxidation solution was 0.1 M iodine in tetrahydrofuran/pyridine/water, 75/20/2 (v/v/v) (PE Applied Biosystems, Foster City, CA).

Oligonucleotides were synthesized utilizing a batch-mode repetitive synthesis cycle consisting of detritylation followed by coupling and oxidation. Individual modules were written and combined to form the complete synthesis cycle. More specifically, the synthesis is performed in the 5' to 3' direction using 10 μmol of 3'- (trityl)amino-2',3'-dideoxyncleoside-5'-succinyl-loaded CPG. Detritylation of the 3'- amine was achieved using a repetitive flow of 3% DCA/DCM to the top of the reaction vessel followed by a 3 second vortex and drain. The total acid exposure time is approximately 2 minutes. The support-bound 3 '-aminonucleoside was then washed 10 times with a series of alternating washes from the bottom of the reaction vessel as well as from the top with vortexing and draining. Coupling of the resulting free amine (presumably as its dichloroacetate salt) with the 3'-(trityl)aminonucleoside-5'- phosphoramidite monomer was performed using an initial delivery of monomer to the reaction vessel followed by the delivery of tetrazole. The coupling mixture was then vortexed for 5 minutes. After draining the reaction vessel, the iodine solution was immediately added, and vortexed for 2 minutes. The oxidation solution was drained and the support was washed 10 times with a series of alternating acetonitrile washes from the bottom of the reaction vessel as well as from the top with vortexing and draining.

Upon completion of the synthesis, the 3 '-deprotected oligonucleotide was cleaved from the support and deprotected with concentrated aqueous ammonia at 55° for 12 hours. The solution was buffered to 0.01 M NaOH and the ammonia removed under vacuum. Following filtration, the cmde solution was purified on a preparative anion-exchange column (Pharmacia MonoQ 10/10), desalted, and lyophilized.

Using the above method, 5'-TT-3' dimers were synthesized on the 10μmole scale using varying concentrations (and therefore varying number of equivalents ) of tetrazole and of either the diisopropylamino phosphoramidite monomer or the tetramethylpiperidine phosphoramidite monomer. The results clearly indicate that phosphoramidate synthesis can be performed using significantly lower equivalents of the more potent tetramethylpiperidinyl phosphoramidite monomer than the less potent diisopropylamino phosphoramidite monomer.

Example 15

10-μmol Scale Synthesis of oIigo-2'-deoxynucleoside N3'→P5' phosphoramidates The following sequences were synthesized on the 10-μmole scale using the above procedure and using the following quantities of tetramethylpiperidinyl phosphoramidite monomers.

Example 16

N⁴-benzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxyuridine 5'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite

2'-Fluoronucleoside phosphoramidite monomers of the invention are prepared as illustrated in Schemes V and VI. Briefly, a ribonucleoside is transformed into a 5'-hydroxyl-protected-2',3'-anhydroxylnucleoside, after which the 2',3'-epoxy ring is opened by treatment with sodium azide, or like reagent, to form a 5'-hydroxyl- protected-3'-azido-3'-deoxyarabinonucleoside. After purification, the 5'-hydroxyl- protected-3'-azido-3'-deoxyarabinonucleoside is fluorinated at the 2' position by treatment with diethylaminosulfur trifluoride (DAST), or like reagent, after which the azido group is reduced to give a 3'-amino. After suitably protecting the 3'-amino and releasing the 5'-hydroxyl protecting group, the nucleoside is phosphitylated at the 5' oxygen to give the crude phorphoramidite monomer.

In reference to Scheme V, uridine 3 was mesylated and then selectively benzoylated with accompanying formation of the 2,2'-anhydrocycle by treatment with sodium benzoate according to literature procedure, e.g. Codington, J.F.; Fecher, R.; Fox, J.J. J. Am. Chem. Soc. 1960, 82, 2794-2803. These reactions resulted in compound 7 with 69-77% overall yields. By another literature method (Codington, J.F.; Fecher, R.; Fox, J.J. J. Organic Chem. 1962, 27, 163-167), 2,3'- anhydroarabinonucleoside 7 was transformed into 2',3'-anhydrolyxouridine 8 in two steps. This involved treatment of 7 with hydrochloric acid to form 3'-mesyl-5'- benzoylarabinouridine, which upon treatment with ammonium hydroxide closed to form the lyxo-2',3'-epoxide 8 in 63-77% overall yields. Then, also according to published procedure (Reichman, U.; Hollenberg, D.H.; Chu, C.K.; Watanabe, K.A.; Fox, J.J. J. Organic Chem. 1976, 41, 2042-2043), 2',3'-anhydrolyxonucleoside 8 was heated with ammonium azide. Contrary to literature suggestion, this reaction was not completely stereoselective, but produced a chromatographically unresolvable mixture of the desired 5'-benzoyl-3'-azidoarabinonucleoside 9 and it's 2'-azido-2'- deoxyregioisomer 9i in approximately a 2.5: 1 ratio. Crude arabinonucleoside 9 was fluorinated with DAST to give 2'-fluoro-3'-azidonucleoside 10, then catalytically hydrogenated to give 2'-fluoro-3'-aminonucleoside 11, which was separable from its regioisomer by silica gel chromatography. Protection of the 3'-amine with a

monomethoxytrityl (MMT) group, followed by 5'- debenzoylation produced

intermediate 13, with 5'-phosphitylation producing the desired phosphoramidite building block 2u in a 22% overall yield from anhydronucleoside 8. More particularly, the steps were carried out as follows:

3'-O-Methanesulfonyl-5'-O-benzoyl-2,2'-anhydroarabinouridine 7 was prepared in two steps from 3 according to the procedure of Codington et al. (cited above, J. Am. Chem. Soc.) in 69-77% overall yields.

5'-O-benzoyl-2',3'-anhydrolyxouridine 8 was prepared in two steps from 7 according to the procedure of Codington et al. (cited above, J. Organic Chem.) in 63- 77% overall yields.

3'-azido-5'-O-benzoyl-3'-deoxyarabinouridine 9 was prepared from 8 and anhydrous NH₄N₃ (described in Obenland, C.O.;Mangold, D.J.; Marino, M.P. Inorg. Synth. 1966, 8, 53-56) according to the procedure of Reichman et al., (cited above) but without successful recrystalhzation. Mass yields were 98% or greater, but NMR suggested 25-35% of the regioisomer, 2'-azido-5'-O-benzoyl-2'-deoxyxylouridine, 9i, which co-eluted with the desired product by silica gel TLC. ¹H NMR, Major component, 9: δ 10.8 (br s, 1H), 8.1 1 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.5 (m, 2H), 6.19 (d, J = 3.6 Hz, 1H), 5.40, (d, J = 8.0 Hz, 1H), 4.84 (m, 1H), 4.73 (d, J = 5.7 Hz, 1H), 4.63 (br d, J = 4.2 Hz, 1H), 4.2 (mm, 2H); Minor component, 9i: δ 10.6 (br s, 1H), 8.1 1 (d, J = 7.5 Hz, 2H), 7.81 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.5 (m, 2H), 5.85 (s, 1H), 5.47, (d, J = 8.1 Hz, 1H), 4.86 (m, 1H), 4.76 (d, J = 5.4 Hz, 1H), 4.62 (br d, J = 4.0 Hz, 1H), 4.3-4.2 (mm, 2H).

2^,-fluoro-3'-azido-5'-O-benzoyI-2',3^,-dideoxyuridine 10 was prepared as follows: To 5.0 g (13.4 mmol) of cmde 9 (containing 25% 9i) in 30 mL anhydrous CH₂CI₂ was added 8.8 mL (66.6 mmol) of diethylaminosulfur trifluoride. After stirring for 48 h, the mixture was diluted with 100 mL CH₂CI₂ and poured into 200 mL saturated aqueous NaHCO₃. When evolution of gas ceased, the CH₂CI₂ layer was washed with 100 mL fresh NaHCO₃ solution and then with water (2x100 mL). Concentration of the CH₂CI₂ layer in vacuo and flash chromatography gave 3.5 g (70%) of product containing 20% of the largely chromatographically unresolvable isomeric impurity, 10i. 1H NMR, Major component, 10: δ 8.7 (br s, 1H), 8.07 (d, J = 7.4 Hz, 2H), 7.62 (d, J = 7.5 Hz, 1H), 7.49 (dd, J = 7.6, 7.6 Hz, 2H), 7.39 (d, J = 8.1 Hz, 1H), 5.70 (d, J = 21.1 Hz, 1H), 5.65 (d, J = 8.2 Hz, 1H), 5.48 (dd, J = 4.7, 52.9 Hz, 1H), 4.7-4.4 (unresolved), 4.32 (dd, J = 4.7, 9.5 Hz, 1H), 4.27 (dd, J = 4.7, 9.5 Hz, 1H); Minor component, 10i: δ 8.7 (br s, 1H), 8.03 (d, J = 7.2 Hz, 2H), 7.64 (d, J = 7.6 Hz, 1H), 7.51 (dd, J = 7.4, 7.7 Hz, 2H), 7.33 (d, J = 8.2 Hz, 1H), 5.99 (d, J = 6.4 Hz, 1H), 5.67 (d, J = 9 Hz, 1H), 5.40 (ddd, J = 2.8, 5.0, 53.4 Hz, 1H), 4.8-4.4 (unresolved), 4.10 (mm, 2H).

2'-fluoro-3'-amino-5'-O-benzoyl-2',3'-dideoxyuridine 11 was prepared as follows: To 3.5 g (9.3 mmol) crude 10 (20% lOi) in 200 mL 95% ethanol was added 600 mg of 10% palladium on carbon. The suspension was hydrogenated at 40 psi overnight and then the catalyst removed by filtration. The solvent was removed in vacuo, giving 2.93 g (90%) of a light yellow solid consisting of two compounds which were resolvable by TLC. Flash chromatography provided 1.96 g (60% yield) of the desired product as a pure white solid. Mass-spectrometry, FAB⁺, M+H⁺, calculated: 350.1152, observed: 350.1152. ¹H NMR δ 8.14 (br s, 1H), 8.06 (d, J - 7.1 Hz, 1H), 7.64 (dd, J = 7.4, 7.4 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.50 (dd 7.7, 7.8 Hz, 1H), 5.86 (d, J = 18.5 Hz, 1H), 5.51 (d, J = 8.2 Hz, 1H) 5.00 (dd, J = 4.3, 52.4 Hz, 1H), 4.81 (dd J = 2.2, 12.8 Hz, 1H), 4.73 (dd, J = 3.5, 12.7 Hz, 1H), 4.14 (ddd, J = 2, 3, 10.2 Hz, 1H), 3.57 (ddd, J = 4, 10.5, 26.6 Hz, 1H); ¹⁹F NMR δ -198.3 (ddd, J = 18.5, 26.4, 52.2 Hz).

2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxyuridine 12 was prepared as follows: To 1.0 g (2.9 mmol) of 11 in 50 mL anhydrous pyridine was added 1.0 g (3.2 mmol) 4-methoxytrityl chloride. The mixture was stirred overnight, 5 mL saturated aqueous NaHCO₃ was added, and the mixture concentrated in vacuo to an oil. The oil was dissolved in 125 mL ethyl acetate, which was washed with water (3x100 mL) and reconcentrated in vacuo to 2.05 g of foam.

The foam was dissolved in a mixture of 40 mL methanol, 40 mL dioxane, and 10 mL water. NaOH (1 g, 25 mmol) was added and the mixture stirred overnight. The solution was concentrated in vacuo to a syrup, which was dissolved in 100 mL ethyl acetate and washed with water (3xl00mL). Concentration in vacuo of the organic layer gave 1.1 1 g of a foam, which upon flash chromatography gave 1.05 g (76%) of a white solid. Mass-spectrometry, FAB⁺, M+H⁺, calculated: 518.2091, observed:

518.2076. Η NMR δ 8.64 (br d J = 4.2 Hz, 1H), 8.14 (br s, 1H), 7.57 (mm, 5H), 7.48 (d J = 8.7 Hz, 1H), 7.3 (mm, 8H), 6.83 (d J = 8.8 Hz, 2H), 5.67 (d, J = 17.7 Hz, 1H), 5.62 (d, J = 8.1 Hz, 1H), 4.23 (m, 2H), 4.03 (br d, J = 10.2 Hz, 1H), 3.80 (s, 3H), 3.31 (dddd, J = 3.6, 10.3, 10.9, 25.8 Hz, 1H), 2.80 (dd. J = 3.6, 50.9 Hz, 1H), 2.51 (dd, J = 3.0, 11.2 Hz, 1H); ¹⁹F NMR δ -192.5 (dddd, J = 2.9, 17.7, 26.1 , 50.9 Hz).

N⁴-benzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxyuridine 5'-(2- cyanoethyl N,N-diisopropyl)phosphoramidite 2u was prepared as follows: To 700 mg (1.35 mmol) of 12 in 20 mL anhydrous CH₂CI₂ was added 200 mg (1.17 mmol) of diisopropylammonium tetrazolide and 0.5 mL (1.57 mmol) of 2-cyanoethyl N,N,N',N'- tetraisopropylphosphorodiamidite. After stirring the mixture for 3h, the solvent was removed in vacuo and the residue purified on a Chromatotron, using 4 mm plates and eluting with 0-3% methanol, 0.5% triethylamine in CH₂CI₂. The product was concentrated in vacuo to an oil, which was dissolved in 10 mL CH₂CI₂ and precipitated by slow addition into 100 mL of rapidly stirred hexane. After decanting the

supernatant, the product was vacuum desiccated over P₂O₅, giving 680 mg (70%) of white powder. Mass-spectrometry, FAB⁺, M+H⁺, calculated: 718.3170, observed: 718.3194. ¹⁹F NMR δ -190.9 (ddd, J = 21.7, 21.8, 51.3 Hz); ³¹P NMR δ 150.5, 149.5.

Example 17

N⁴-benzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxycytidine

5'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite

Crude intermediate 10 was used for preparation of the appropriately protected cytidine phosphoramidite 2c as shown in Scheme VI below. The uracil base of 10 was converted to cytosine by adaptation of the method of Divakar and Reese, J. Chem. Soc, Perkin. Trans. 1 1982, 1171-1 176. Subsequent 4-N benzoylation and reduction of the 3'-azido to an amino group gave compound 13, which was separable from its regioisomer by silica gel chromatography. Protection of the 3'-amine with an MMT group, followed by selective 5'-O-debenzoylation produced intermediate 15.

Subsequent 5'-phosphitylation lead to desired phosphoramidite 2c in a 10% overall yield based on anhydronucleoside 8.

More particularly, the steps were carried out as follows: N⁴,5'-O-dibenzoyl-2'-fluoro-3'- amino-2',3'-dideoxycytidine 13 was prepared as follows: To 6.9 g (18.4 mmol) of crude 10 (containing 35% 10i) in 50 mL anhydrous CH₃CN was added an ice-cold solution of 1 1.7 g (169 mmol) 1,2,4-triazole and 3.35 mL (36.1 mmol) POCl₃ in 90 mL anhydrous CH₃CN. The mixture was cooled in an ice bath and anhydrous triethylamine (23 mL, 165 mmol) was added, then the reaction allowed to warm to room temperature with stirring. After 90 min, 15 mL (108 mmol) triethylamine and 4 mL water were added and the mixture stirred for 10 min. The solvent was removed in vacuo, then 250 mL ethyl acetate was added, and the solution was TLC indicated a fluorescent intermediate with the same mobility as the starting material.

The mixture was concentrated in vacuo to 6.7 g of a foam. Dioxane (100 mL) and 20 mL concentrated aqueous ammonia were added, and after 3 h, the mixture was concentrated in vacuo to a yellow gel. The gel was dissolved in 100 mL ethyl acetate and washed with water (3x200 mL). Concentration in vacuo and vacuum desiccation over P₂O₅ yielded 5.4 g of a solid which gave only one spot by silica gel TLC. Only two significant signals were observed by ¹⁹F NMR, Major component: δ - 192.8 (ddd, J = 22.8, 22.8, 53.1 Hz); Minor component: δ -200.7 (ddd, J = 13.6, 19.9, 53.4 Hz).

Anhydrous pyridine (100 mL) was added and the solution cooled to 4 °C.

Benzoyl chloride (11.7 mL 100 mmol) was added with stirring and the mixture allowed to warm to room temperature. After 2 h, 5 mL water was added and the solvent removed in vacuo, giving a brown oil, which was dissolved in 200 mL ethyl acetate, washed with water (3x200 mL), and then reconcentrated in vacuo to an oily foam.

Ethanol (150 mL) and 2 g of 10% palladium on activated carbon were added and the mixture was hydrogenated at 40 psi H₂ overnight. TLC indicated formation of two new slower, closely-migrating compounds.

The catalyst was removed by filtration, and the filtrate concentrated in vacuo to an oily yellow foam. Silica gel flash chromatography (500 mL silica, eluted with 0-3% CH₃OH in CH₂CI₂) provided 1.85 g of semi-pure product, which was dissolved in 10 mL CH₂CI₂. A solid quickly precipitated, which was collected by filtration and washed with fresh CH₂CI₂. Vacuum desiccation yielded 1.5 g of product 13 (1 1% yield from 9 and 9i) as fine white crystals. Mass-spectrometry, FAB⁺, M+H⁺, calculated:

453.1574, observed: 453.1574. 1H NMR δ 8.21 (d, J = 7.5 Hz, 1H), 8.08-8.13 (mm, 3H), 7.94 (d, J = 7.4 Hz, 2H), 7.46-7.7 (mm, 8H), 6.04 (d, J = 16.9 Hz, 1H), 5.08 (dd, J = 3.6, 51.5 Hz, 1H), 4.85 (dd, J = 3.3, 12.8 Hz, 1H), 4.80 (dd, J = 2.1 , 12.8 Hz, 1H), 4.26 (m, 1H), 3.48 (dm, J = 27 Hz, 1H); ¹⁹F NMR δ -200.1 (m).

N⁴,5'-O-dibenzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxycytidine 14 was prepared as follows: To 0.9 g (2.0 mmol) of 13 in 25 mL anhydrous pyridine was added 0.86 g (2.8 mmol) 4-methoxytrityl chloride, and the mixture stirred overnight. The reaction was quenched with 0.5 mL H₂O and concentrated in vacuo. CH₂CI₂ (50 mL) was added and washed with 50 mL saturated aqueous NaHCO₃ and with water (2x50 mL). The solvent was removed in vacuo, replaced with 10 mL CH₂CI₂, and pipetted into 80 mL rapidly stirred 1/1 hexane/ether. After further stirring for 2 h, the product was collected by filtration and dried under vacuum, giving 1.3 g (88% yield) of product as a white powder. Mass-spectrometry, FAB⁺, M+H⁺, calculated: 725.2775, observed: 725.2761. 1H NMR δ 8.59 (br s, 1H), 8.07 (br d, J = 5.7 Hz, 1H), 7.89 (br d, J = 7 Hz, 2H), 7.83 (dd, J = 1.3, 6.7 Hz, 2H), 7.68 (dd, J = 7.4, 7.4 Hz, 2H), 7.5-7.6 (m, 8H), 7.43 (dd, J = 2.1, 6.9 Hz, 2H), 7.1-7.3 (mm, 7H), 6.71 (d, J = 8.9 Hz, 2H), 5.80 (d, J = 15.4 Hz, 1H), 5.03 (dd, J = 2.0, 13.0 Hz, 1H), 4.98 (dd, J = 2.3;, 13.1 Hz, 1H), 4.41 (br d, J = 10.5 Hz, 1H), 3.63 (s, 3H), 3.36 (dddd, J = 3.1, 1 1.1, 1 1.1, 25.7 Hz, 1H), 2.84 (dd, J = 3.1 , 49.9 Hz, 1H), 2.52 (dd, J = 2.7, 1 1.5 Hz, 1H); ¹⁹F NMR δ -196.3 (m).

N -benzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxycytidine 15 was prepared as follows: To 1.3 g ( 1.75 mmol) of 14 in 20 mL of 65/30/5

pyridine/methanol/water, cooled in an ice bath, was added 10 mL of cold 2 M NaOH in 65/30/5 pyridine/methanol/water. The mixture was stirred cold for 20 min, then neutralized with pyridinium-H⁺ form Bio-Rad AG^® 50W-X8 cation exchange resin. After 5 min, the resin was removed by filtration and washed with methanol. The combined filtrate and wash were concentrated in vacuo to an oil, which was dissolved in 100 mL ethyl acetate. The mixture was washed with 100 mL saturated aqueous NaHCO₃ and with water (2x100mL). After concentration in vacuo to a foam, the product was dissolved in 10 mL CH₂CI₂ and pipetted into 75 mL rapidly stirred hexane/ether, 2/1. The product was collected by filtration and dried under vacuum, giving 1.13 g (102%) yield) of product as a white powder. Mass-spectrometry, FAB⁺, M+Cs⁺, calculated: 753.1489, observed: 753.1499. ¹H NMR δ 8.30 (br d, J = 6.8 Hz, 1H), 7.89 (br d, J = 6.7 Hz, 2H), 7.64 (dd, J = 7.4, 7.4 Hz, 1H), 7.44-7.56 (mm, 9H), 7.22-7.32 (mm, 9H), 6.82 (d, J = 8.8 Hz, 2H), 5.80 (d, J = 15.7 Hz, 1H), 4.26 (mm, 2H), 4.13 (d, J = 10.2 Hz, 1H), 3.81 (s, 3H), 3.26 (dddd, J = 3.4, 10.7, 10.8, 26.5 Hz, 1H), 2.93 (dd, J = 3.3, 50.5 Hz, 1H), 2.50 (dd, J = 2.8, 1 1.0 Hz, 1H); ¹⁹F NMR δ -195.3 (m).

N -benzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxycytidine 5'-(2- cyanoethyl N,N-diisopropyl)phosphoramidite 2c was prepared as follows: To 970 mg (1.56 mmol) of 15 in 25 mL anhydrous CH₂CI₂ was added 200 mg (1.17 mmol) of diisopropylammonium tetrazolide and 1.0 mL (3.15 mmol) of 2-cyanoethyl N,N,N',N'- tetraisopropylphosphorodiamidite. After stirring the mixture for 3h, the solvent was removed in vacuo and the residue purified on a Chromatotron, using 4 mm plates and eluting with 0-1.5% methanol in 0.5%) triethylamine in CH₂CI₂. The product was concentrated in vacuo to a foam, which was dissolved in 10 mL CH₂CI₂, and

precipitated by slow addition to 40 mL of rapidly stirred hexane. After decanting the supernatant, the product was vacuum desiccated over P₂O₅, giving 880 mg (69%) of white powder. Mass-spectrometry, FAB⁺, M+Cs⁺, calculated: 953.2568, observed: 953.2531. ¹⁹F NMR δ -193.6 (m); ³¹P NMR δ 150.4, 149.4.

Example 18

N⁴-benzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2',3'-dideoxycytidine

5'-succinyl-loaded CPG

Intermediate 15 was 5'-succinylated and loaded upon CPG solid support by standard procedures, e.g. Atkinson, T.; Smith, M. In Oligonucleotide Synthesis. A Practical Approach, Gait, M.J. Ed., IRL Press, 1984, 35-81 ; and Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30, 1927-1930. More, particularly, N⁴-benzoyl-2'-fluoro-3'-(4-methoxytrityl)amino-2^,,3'-dideoxycytidine 5'- succinyl-loaded CPG was prepared as follows: To 100 mg (0.16 mmol) of 15 in 2 mL anhydrous CH₂CI₂ was added 55 mg (0.55 mmol) of succinic anhydride and 65 mg

(0.53 mmol) of dimethylaminopyridine. The mixture was stirred for 2 h, the evaporated in vacuo to an oil. The oil was dissolved in 20 mL CH₂CI₂, washed with 20 mL of saturated aqueous NaHCO₃ and with water (2x20mL), and then reconcentrated in vacuo to a foam. To the foam was added 1 mL 0.4 M diisopropylethylamine in DMSO/N- methylpyrrolidine, 1/1, and 0.7 mL 0.2 M 1-hydroxybenzotriazole, 0.2 M 2-(1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in DMSO/N- methylpyrrolidine, 1/1. After 3 min, the mixture was drawn into a 10 mL syringe containing 1.2 g of long-chain alkylamino-CPG. An additional 5 mL DMSO wash was also drawn into the syringe. The CPG-nucleoside mixture was mixed for 1.5 h, then the CPG washed with 5 volumes of anhydrous acetonitrile. Unreacted CPG amino groups were acetylated by standard capping solutions (PE Applied Biosystems, Foster City, CA) for 2 min. The CPG was again washed with 5 volumes of acetonitrile and 5 volumes of CH₂CI₂. Nucleoside loading was determined to be approximately 5 μmole/g by standard trityl assay.

Example 19

Solid Phase Synthesis of Oligo-2'-fluoronucleotide

N3'→P5' Phosphoramidates

Oligo-2'-fluoronucleotide N3'->P5' phosphoramidates were synthesized on solid phase supports using phosphoramidite monomers of Schemes V and VI.

Compounds 22-25 (Table 1) were synthesized by way of phosphoramidite monomers. The average coupling efficiency as determined by released MMT-cation assay was -94% with single coupling per cycle and -96% with double application of Step 2 per synthetic cycle. A representative IE HPLC profile of a cmde oligomer synthesis is shown in Figure 4.

Uniformly modified oligo-2'-fluoronucleotide N3'-»P5' phosphoramidates were prepared by amidite transfer reaction on an ABI 380B synthesizer using the following protocol:

1) detritylation, 5% dichloroacetic acid in dichloromethane, 1 min.

2) coupling, 0.1 M phosphoramidite 2u or 2c (Scheme V or VI, respectively) and 0.45 M tetrazole in acetonitrile, 3 min.

3) oxidation, 0.1 M iodine in tetrahydrofuran/pyridine/water, 10/10/1, v/v/v, 1 min. 4) capping, acetylation of unreacted 3'-amino groups by standard PE Applied

Biosystems (Foster City, CA) capping solutions, 30 sec.

Chemical steps within the cycle were followed by acetonitrile washings and flushings with dry argon for 0.2-0.4 min. After cleavage from the solid support and deprotection with concentrated aqueous ammonia, 1-1.5 h, 55 °C, oligonucleotides were analyzed and purified by IE HPLC. Oligonucleotides were desalted on Pharmacia

NAP-5 or NAP- 10 gel filtration columns immediately after purification and stored frozen or lyophilized at -18 °C.

Preparation of the 5'-phosphorylated oligonucleotides was done upon sulfone- derivatized CPG, e.g. Gryaznov, S.M.; Letsinger, R.L. Nucleic Acids Res. 1993, 21,

1403-1408.

Dionex DX300 or DX500 systems were used for IE analysis and purification of oligonucleotides. A Pharmacia MonoQ 10/10 column was used for analysis and purification of crude oligomers, eluted with a 2% per minute gradient of 1.5 M NaCl in 10 mM NaOH. A Dionex NucleoPac PA 100 column, eluted with a 1.5% per minute gradient of 1.5 M NaCl in 10 mM NaOH was used for all other IE HPLC analysis. A

Hewlett Packard Hypersil ODS, 5μ column on a Waters HPLC system was used for RP

HPLC, with a 1% per minute gradient of acetonitrile in 0.1 M triethylammonium acetate, pH 7.0.

NMR spectra were recorded on a Bruker DRX-400 spectrometer. Chemical shifts are reported relative to TMS, CCl₃F, and H₃PO₄, for ¹H, ¹⁹F, and ³¹P spectra, respectively.

Thin layer chromatography (TLC) was performed on Whatman polyester-backed silica gel plates with methanol/dichloromethane eluents.

Acid hydrolysis of 0.17 OD₂₆₀ of the dimer dU^f _npT wT as done in 25 μL of 64% acetic acid, 2 h at 55 °C, and the reaction mixture was analyzed by RP HPLC.

Approximately 83% of the dimer, retention time (Rt) 15.0 min, was hydrolyzed to mainly 5'-thymidylic acid, Rt 10.6 min, and 2'-fluor-3'-aminouridine, Rt 11.2 min, as were identified by co-injection with authentic standards. Also, -7.5% of thymidine, Rt 12.1 min, was found in the reaction mixture.

^a

Claims (64)

We claim:

1. A method of synthesizing an oligonucleotide N3'→P5' phosphoramidate, the method comprising the steps of:

(a) providing a first nucleoside attached to a solid phase support, the first nucleoside having a protected 3' amino group;

(b) deprotecting the protected 3' amino group to form a free 3' amino group;

(c) reacting the free 3' amino group with a 3'-protected aminonucleoside-5'- phosphoramidite monomer to form an intemucleoside N3'-»P5' phosphoramidite linkage; and

(d) oxidizing said linkage.

2. The method of claim 1 further including the step of repeating a plurality of times said steps of reacting and oxidizing.

3. The method of claim 2 wherein said plurality is between 2 and 4, inclusive.

4. The method of claim 3 further including a step of repeating steps (b) through (d) until said oligonucleotide N3'→P5' phosphoramidate is synthesized.

5. The method of claim 4 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a tetrazole activation equilibrium constant of at least 10 M^-1.

6. The method of claim 5 further including a step of capping said free 3' amino groups that fail to react with said 3 '-protected aminonucleoside-5 '-phosphoramidite monomer.

7. The method of claim 6 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a tetrazole activation equilibrium constant of at least 100 M^-1.

8. The method of claim 7 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a phosphoramidite amino group with a pK_a of at least 10.

9. The method of claim 8 wherein 3 '-protected aminonucleoside-5'- phosphoramidite monomer is defined by the formula: wherein:

B is pyrimidine, purine, or an analog thereof;

R¹ is a phosphate protecting group;

W is either -NHR² or -OR⁷, where R is an amino protecting group and where R⁷ is a hydroxyl protecting group;

R is hydrogen, hydroxyl, fluoro or -OR', where R' is alkyl having from 1 to 3 carbon atoms or a hydroxyl protecting group; and

R⁴ and R⁵ together with the nitrogen to which they are attached form an alkylamino- or arylamino leaving group having up to 40 atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen.

10. The method of claim 9 wherein R³ is hydrogen and wherein R⁴ and R⁵ taken separately are alkyl, aralkyl, cycloalkyl, or cycloalkylalkyl having a combined total of from 6 to 20 carbon atoms.

1 1. The method of claim 10 wherein said step of reacting further includes treating said 3'-protected aminonucleoside-5'-phosphoramidite monomer with a nucleophilic catalyst to form a reactive intermediate.

12. The method of claim 1 1 wherein said nucleophilic catalyst is a tetrazole activator.

13. The method of claim 12 wherein said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of hydrogen peroxide and iodine.

14. The method of claim 9 wherein R³ is hydrogen and wherein R⁴ and R⁵ taken together form an alkylene chain containing up to 12 carbon atoms in the principal chain and a total of from 4 to 20 carbon atoms with both terminal valence bonds of said chain being attached to the nitrogen atom to which R⁴ and R⁵ are attached.

15. The method of claim 14 wherein said step of reacting further includes treating said 3'-protected aminonucleoside-5'-phosphoramidite monomer with a nucleophilic catalyst to form a reactive intermediate.

16. The method of claim 15 wherein said nucleophilic catalyst is a tetrazole activator.

17. The method of claim 16 wherein said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of hydrogen peroxide and iodine.

18. The method of claim 9 wherein R is hydrogen and wherein R⁴ and R⁵ taken together and with the nitrogen to which they are attached form a saturated nitrogen heterocycle having up to 10 carbon atoms or heteroatoms in the principal chain and a total of from 4 to 20 carbon atoms or heteroatoms altogether, such that R⁴ and R⁵ taken together and with the nitrogen to which they are attached contain up to three heteroatoms selected form the group consisting of nitrogen, oxygen, and sulfur.

19. The method of claim 18 wherein said step of reacting further includes treating said 3'-protected aminonucleoside-5'-phosphoramidite monomer with a nucleophilic catalyst to form a reactive intermediate.

20. The method of claim 19 wherein said nucleophilic catalyst is a tetrazole activator.

21. The method of claim 20 wherein said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of hydrogen peroxide and iodine.

22. The method of claim 4 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a phosphoramidite amino group with a pK_a of at least 10.

23. The method of claim 22 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a tetrazole activation equilibrium constant of at least 10

M^-1.

24. The method of claim 23 further including a step of capping said free 3' amino groups that fail to react with said 3'-protected aminonucleoside-5'-phosphoramidite monomer.

25. The method of claim 24 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a tetrazole activation equilibrium constant, K₁ , of at least 100 M^-1.

26. The method of claim 25 wherein said step of reacting further includes treating said 3'-protected aminonucleoside-5'-phosphoramidite monomer with a nucleophilic catalyst to form a reactive intermediate.

27. The method of claim 26 wherein said nucleophilic catalyst is a tetrazole activator.

28. The method of claim 27 wherein said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of hydrogen peroxide and iodine.

29. The method of claim 1 further including the step of repeating steps (b) through (d) until said oligonucleotide N3'→P5' phosphoramidate is synthesized.

30. The method of claim 29 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a sterically hindered phosphoramidite amino group having a pK_a of at least 10.

31. The method of claim 30 further including a step of capping said free 3' amino groups that fail to react with said 3 '-protected aminonucleoside-5'-phosphoramidite monomer.

32. The method of claim 31 wherein said sterically hindered phosphoramidite amino group is alkylamino, dialkylamino, cycloalkylamino, dicycloalkylamino, or aralkylamino having from 6 to 20 carbon atoms.

33. The method of claim 32 wherein said step of reacting further includes treating said 3'-protected aminonucleoside-5'-phosphoramidite monomer with a nucleophilic catalyst to form a reactive intermediate.

34. The method of claim 33 wherein said nucleophilic catalyst is a tetrazole activator.

35. The method of claim 34 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a tetrazole activation equilibrium constant of at least 100 M^-1.

36. The method of claim 35 wherein said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of hydrogen peroxide and iodine.

37. A method of synthesizing an oligonucleotide of the formula:

wherein: B is a purine or pyrimidine or an analog thereof; X is oxygen or sulfur; R³ is hydrogen, fluoro, or hydroxyl; R⁶ is amino or hydroxyl; Z is hydrogen, alkali metal, or an amine cation; and n is at least one; the method comprising the steps of:

(a) providing a first nucleoside attached to a solid phase support, the first nucleoside having a protected 3' amino group;

(b) deprotecting the protected 3' amino group to form a free 3' amino group; (c) reacting the free 3' amino group with a 3'-protected aminonucleoside-5'- phosphoramidite monomer in the presence of a nucleophilic catalyst to form an intemucleoside N3'→P5' phosphoramidite linkage;

(d) oxidizing said linkage; and

(e) repeating steps (b) through (d) until the oligonucleotide is synthesized.

38. The method of claim 37 further including a step of capping said free 3' amino groups that fail to react with said 3'-protected aminonucleoside-5'-phosphoramidite monomer.

39. The method of claim 38 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer has a sterically hindered phosphoramidite amino group having a pK_a of at least 10.

40. The method of claim 39 further including a step of repeating said steps (c) and (d) a plurality of times during each cycle of said steps (b) through (d).

41. The method of claim 40 wherein said steps (c) and (d) are repeated twice during each cycle of said steps (b) through (d).

42. The method of claim 41 wherein said 3'-protected aminonucleoside-5'- phosphoramidite monomer of the form:

wherein:

B is pyrimidine, purine, or an analog thereof;

R¹ is a phosphate protecting group;

W is either -NHR² or -OR⁷, where R² is an amino protecting group and where

R⁷ is a hydroxyl protecting group;

R³ is hydrogen, hydroxyl, fluoro or -OR', where R' is alkyl having from 1 to 3 carbon atoms or a hydroxyl protecting group; and

R⁴ and R⁵ together with the nitrogen to which they are attached form an alkylamino- or arylamino leaving group having up to 40 atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen.

43. The method of claim 42 wherein R³ is hydrogen; said nucleophilic catalyst is a tetrazole activator; and R⁴ and R⁵ taken separately are alkyl, aralkyl, cycloalkyl, or cycloalkylalkyl having a combined total of from 6 to 20 carbon atoms.

44. The method of claim 43 wherein said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of iodine, chlorine, hydroperoxide, peracids, mixed acyl-sulfinic anhydrides, peroxides, ozone, elemental sulfur, thiuram disulfides, acyl disulfides, phosphinothioyl disulfides, and 1 ,1-dioxo-3H-1,2-benzodithiol-3-one.

45. The method of claim 44 wherein R¹ is methyl, β-cyanoethyl, or 4- nitrophenylethyl; R² is triphenylmethyl; and n is in the range of from 1 to 50.

46. The method of claim 42 wherein R³ is hydrogen and wherein R⁴ and R⁵ taken together form an alkylene chain containing up to 6 carbon atoms in the principal chain and a total of from 4 to 12 carbon atoms with both terminal valence bonds of said chain being attached to the nitrogen atom to which R⁴ and R⁵ are attached.

47. The method of claim 46 wherein: said nucleophilic catalyst is a tetrazole activator; R¹ is methyl, β-cyanoethyl, or 4-nitrophenyl ethyl; R² is triphenylmethyl; n is in the range of from 1 to 50; and said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of iodine, chlorine, hydroperoxide, peracids, mixed acyl-sulfinic anhydrides, peroxides, ozone, elemental sulfur, thiuram disulfides, acyl disulfides, phosphinothioyl disulfides, and 1,1-dioxo- 3H-1,2-benzodithiol-3-one.

48. The method of claim 42 wherein R³ is hydrogen and wherein R⁴ and R⁵ taken together and with the nitrogen to which they are attached form a saturated nitrogen heterocycle having up to 10 carbon atoms or heteroatoms in the principal chain and a total of from 4 to 20 carbon atoms or heteroatoms altogether, such that R⁴ and R⁵ taken together and with the nitrogen to which they are attached contain up to three heteroatoms selected form the group consisting of nitrogen, oxygen, and sulfur.

49. The method of claim 48 wherein said nucleophilic catalyst is a tetrazole activator and wherein said step of oxidizing further includes treating said linkage with an oxidizing agent selected from the group consisting of iodine, chlorine,

hydroperoxide, peracids, mixed acyl-sulfinic anhydrides, peroxides, ozone, elemental sulfur, thiuram disulfides, acyl disulfides, phosphinothioyl disulfides, and 1,1-dioxo- 3H-1,2-benzodithiol-3-one.

50. The method of claim 49 wherein R¹ is methyl, β-cyanoethyl, or 4- nitrophenylethyl; R² is triphenylmethyl; and n is in the range of from 1 to 50.

51. A compound of the formula: wherein:

B is pyrimidine, purine, or an analog thereof;

R¹ is a phosphate protecting group;

W is either -NHR² or -OR⁷, where R² is an amino protecting group and where R⁷ is a hydroxyl protecting group;

R³ is hydrogen, hydroxyl, fluoro or -OR', where R' is alkyl having from 1 to 3 carbon atoms or a hydroxyl protecting group; and

R⁴ and R⁵ together with the nitrogen to which they are attached form an alkylamino- or arylamino leaving group having up to 40 atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen.

52. The compound of claim 51 wherein R¹ is hydrogen; and R⁴ and R⁵ taken separately are alkyl, aralkyl, cycloalkyl, or cycloalkylalkyl having a combined total of from 6 to 20 carbon atoms.

53. The compound of claim 44 wherein R¹ is methyl, β-cyanoethyl, or 4- nitrophenylethyl; R² is triphenylmethyl; R⁷ is di-p-anisylphenylmethyl; and R⁴ and R⁵ taken separately are isopropyl, sec-butyl, isobutyl, t-butyl, cyclohexyl, or 2-ethylhexyl..

54. The compound of claim 51 wherein R³ is hydrogen and wherein R⁴ and R⁵ taken together form an alkylene chain containing up to 6 carbon atoms in the principal chain and a total of from 4 to 12 carbon atoms with both terminal valence bonds of said chain being attached to the nitrogen atom to which R⁴ and R⁵ are attached.

55. The method of claim 42 wherein R³ is hydrogen and wherein R⁴ and R⁵ taken together and with the nitrogen to which they are attached form a saturated nitrogen heterocycle having up to 10 carbon atoms or heteroatoms in the principal chain and a total of from 4 to 20 carbon atoms or heteroatoms altogether, such that R⁴ and R⁵ taken together and with the nitrogen to which they are attached contain up to three

heteroatoms selected form the group consisting of nitrogen, oxygen, and sulfur.

56. The compound of claim 55 wherein R¹ is methyl, β-cyanoethyl, or 4- nitrophenylethyl; R² is triphenylmethyl; and R⁴ and R⁵ taken together and with the nitrogen to which they are attached are dimethylpipiridinyl, pyrrolidinyl,

dimethylmorpholino, tetramethylmoφholino, dimethylpyrrolidinyl,

tetramethylpyrrolidinyl, or tetramethylpiperidinyl.

57. A method of synthesizing an oligonucleotide N3'-»P5' phosphoramidate on a solid phase support, the method comprising the steps of:

providing a 3'-protected aminonucleoside-5'-phosphoramidite monomer having a phosphoramidite amino group; and

coupling the 3'-protected aminonucleoside-5'-phosphoramidite monomer to a 3' primary amino group of the oligonucleotide N3'→P5' phosphoramidate by exchange of the phosphoramidite amino group of said monomer with the 3' primary amino group of the oligonucleotide N3'→P5' phosphoramidate.

58. The method of claim 57 wherein said phosphoramidite amino group of said 3'- protected aminonucleoside-5'-phosphoramidite monomer has a pKa of at least 8.

59. The method of claim 58 wherein said step of coupling further includes formation of an N3'→P5' phosphoramidite linkage.

60. The method of claim 59 further including the step of oxidizing said N3'→P5' phosphoramidite linkage to form an N3'→P5' phosphoramidate or an N3'→P5' phosphorothioamidate linkage.

61. The method of claim 60 wherein said 3 '-protected aminonucleoside-5'- phosphoramidite monomer a tetrazole activation equilibrium constant of at least 100 M-

62. The method of claim 61 further including a step of repeating said steps of coupling and oxidizing a plurality of times.

63. The method of claim 62 wherein said plurality is twice.

64. The method of claim 63 further including a step of capping said 3' primary amino groups that fail to couple with said 3 '-protected aminonucleoside-5'- phosphoramidite monomer.