WO2000075157A1

WO2000075157A1 - Oligonucleotide synthesis with lewis acids as activators

Info

Publication number: WO2000075157A1
Application number: PCT/US2000/012530
Authority: WO
Inventors: Xiu C. Wang
Original assignee: Abbott Laboratories
Priority date: 1999-06-03
Filing date: 2000-05-08
Publication date: 2000-12-14
Also published as: EP1181301A1; CA2376016A1; MXPA01012444A; JP2003514766A

Abstract

A process for synthesizing oligonucleotides by phosphoramidite chemistry wherein the improvement is the use of Lewis acids as activators for formation of the phosphorous-oxygen bond.

Description

OLIGONUCLEOTIDE SYNTHESIS WITH LEWIS ACIDS AS ACTIVATORS

Technical Field

The present invention relates to a process utilizing Lewis acids as activators in the preparation of oligonucleotides by phosphoramidite chemistry.

Background of the Invention

The present invention relates generally to the fields of organic chemistry and biology. In particular, the present invention is directed to compositions and methods for use in oligonucleotide synthesis.

Phosphoramidite chemistry (Beaucage, S. L., and Lyer, R. P. Tetrahedron (1992), 48, 2223-2311) has become by far the most widely used coupling chemistry for the synthesis of oligonucleotides. As is well known to those skilled in the art, phosphoramidite synthesis of oligonucleotides involves activation of nucleoside phosphoramidite monomer precursors by reaction with an activating agent to form activated intermediates, followed by sequential addition of the activated intermediates to the growing oligonucleotide chain (generally anchored at one end to a suitable solid support) to form the oligonucleotide product. Tetrazole is commonly used for the activation of the nucleoside phosphoramidite monomers; the activation occurs by the mechanism depicted in Scheme I. Tetrazole has an acidic proton which presumably protonates the basic nitrogen of the diisopropylamino phosphine group, thus making the diisopropylamino group a leaving group. The negatively charged tetrazolium ion then makes an attack on the trivalent phosphorous, forming a transient phosphorous tetrazolide species. The 5'-OH group of the solid support bound nucleoside then attacks the active trivalent phosphorous species, resulting in the formation of the internucleotide linkage.

The trivalent phosphorous is finally oxidized to the pentavalent phosphorous. Scheme 1

1 ) Repetition of cycles

2) Cleave and deprotect with NH₄OH or CH₃NH2/NH4OH

Biologically active oligonucleotides

Principal drawbacks of tetrazole are its cost and instability which includes its potential to explode (Material Safety Data Sheets or MSDS lists IH-tetrazole as a severe explosion hazard). Because of its inherent instability, sublimed tetrazole is generally required to ensure desired coupling yields. Further, tetrazole (which is typically used near its saturated solubility of 0.5M) tends to precipitate out of acetonitrile solution at cold temperatures; this can lead to valve blockage on some automated DNA synthesizers. Other activators which work almost as efficiently as tetrazole have similar drawbacks to those of tetrazole as discussed above. These activators, which are all proton donors, include the following members of the tetrazole class of activators: 5-(p- nitrophenyl) tetrazole (Froehler, B. C. and Mattcucci, M. D., Tetrahedron Letters (1983), 24, 3171-3174); 5-(p-nitrophenyl) tetrazole and DMAP (Pon, R.T., Tetrahedron Letters (1987), 28, 3643-3646); and 5-(ethylthio)- IH-tetrazole (Wright, P. et al., Tetrahedron

Letters (1993), 34, 3373-3376). In addition to the tetrazole class of activators, the following activators have been employed: N-methylaniline trifluoroacetate (Fourrey, J. L. and Varenne, J., Tetrahedron Letters (1984), 25, 4511-4514); N-methyl anilinium trichloroacetate (Fourrey, J. L. et al., Tetrahedron Letters (1987), 28, 1769-1772); 1-methylimidazoletrifluoromethane sulfonate (Arnold, L. et al., Collect. Czech. Chem. Commun. (1989), 54, 523-532); octanoic acid or triethylamine (Stec, W. J. and Zon, G.,

Tetrahedron Letters (1984), 25, 5279-5282); 1-methylimidazole HC1, 5-trifluoromethyl- lH-tetrazole, N,N-dimethylaniline HC1, and N,N-dimethylaminopyridine HC1 (Hering, G. et al., Nucleosides and Nucleotides (1985), 4, 169-171). Overall, these activators gave inferior performance relative to tetrazole. JP 08301878 discloses the use of another proton activator. A 1 :1 mixture of benzimidazole and BF₃ etherate is disclosed wherein the BF₃ component acts to increase the acidity of the benzimidazole proton necessary for activation of the phosphoramidite (intermediates). The benzimidazole BF₃ complex acts in a manner similar to tetrazole described in Scheme 1. The present invention does not activate phosphoramidite intermediates with a proton donor but instead utilizes Lewis acids for activation. In particular, BF₃ etherate is used in the present invention. The advantages of BF₃ etherate over the benzimidazole BF₃ complex include commercial availability and ease of removal of diethyl ether versus removal of benzimidazole.

Furthermore, the activated phosphoramidite intermediates are highly sensitive to moisture. An excess of 50% to 100% of the highly valuable phosphoramidites are required for sequencing even with anhydrous solvents (<20 ppm moisture content). The presence of trace amounts of moisture results in considerable loss of yield and an increase in deleted sequencing impurities. In addition to activating phosphoramidite intermediates, Lewis acids can act as moisture scavengers minimizing decomposition of the phosphoramidite. Therefore, the use of Lewis acids for activation of phosphoramidite intermediates leads to improved coupling efficiency, lower cost, and convenient material handling and operation.

Addition of a Lewis acid, however, can lower the pH of the reaction mixture. The increased acidity in the reaction mixture may cause the removal of N- and O-protecting groups on the phosphoramidite intermediate leading to undesirable products and reduced yields. The addition of pyridine may therefore be used to increase the pH to a level that the phosphoramidite protecting groups can tolerate.

It is an object of the present invention to provide Lewis acid activated nucleosides for use in synthesis, including solid phase synthesis, which do not exhibit all of the drawbacks of the prior art.

It is a further object of the present invention to provide methods for the preparation and use of Lewis acid activated nucleosides as hereinafter described.

Summary of the Invention In its principle embodiment, the present invention discloses a process for activating phophoramidite monomers of formula I:

I, wherein B¹ is selected from the group consisting of a purine base and a pyrimidine base; R¹ is a secondary amine, a preferred amine is diisopropylamine;

R² is selected from the group consisting of alkoxy, alkyl, alkylsulfonylalkoxy arylsulfonylalkoxy, cyanoalkoxy, and haloalkoxy;

R³ is a hydroxy-protecting group, a preferred group is 4-4'-dimethoxytrityl; and

R⁴ is selected from the group consisting of hydrogen and -OR⁷ wherein, R⁷ is a hydroxy- protecting group; comprising treating the phosphoramidite monomers of formula I with an optional amount of pyridine and a Lewis acid, preferred Lewis acids are selected from aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride and zirconium(IN) chloride. Detailed Description of the Invention

All patents, patent applications, and literature references cited in the specification are hereby incorporated by reference in their entirety. In the case of inconsistencies, the present disclosure, including definitions, will prevail.

Definition of Terms

As used in the specification and the appended claims, the following terms have the meanings specified.

The term "alkoxy," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein.

Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and the like.

The term "alkyl," as used herein, refers to a straight or branched chain hydrocarbon containing from 1 to 5 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, and the like.

The term "alkylcarbonyl," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein. Representative examples of alkylcarbonyl include, but are not limited to, acetyl, ethylcarbonyl, and the like.

The term "alkylcarbonyloxy," as used herein, refers to an alkylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein. Representative examples of alkylcarbonyloxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, t-butylcarbonyloxy, and the like. The term "alkylsulfonyl," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein. Representative examples of alkylsulfonyl include, but are not limited to, methylsulfonyl, ethylsulfonyl, and the like.

The term "alkylsulfonylalkoxy," as used herein, refers to an alkylsulfonyl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of alkylsulfonylalkoxy include, but are not limited to, 2-methylsulfonylethoxy, 2-ethylsulfonylethoxy, and the like. The term "amino," as used herein, refers to a -NH₂ group. The term "amino-protecting group" or "N-protecting group,"refers to groups intended to protect an amino group against undersirable reactions during synthetic procedures. Commonly used nitrogen-protecting groups are disclosed in Greene, T. W., &

Wuts, P. G. M. (1991). Protectective Groups In Organic Synthesis (2nd ed.). New York:

John Wiley & Sons. Preferred nitrogen-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

The term "aryl," as used herein, refers to an aromatic monocyclic ring system, or a bicyclic-fused ring system wherein one or both of the fused rings are aromatic.

Representative examples of aryl include, but are not limited to, azulene, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The aryl groups of this invention can be substituted with 1, 2, or 3 substituents independently selected from alkyl, cyano, halogen, haloalkyl, and nitro.

The term "arylalkoxy," as used herein, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.

Representative examples of arylalkoxy include, but are not limited to, 2-phenylethoxy, 2-naphthylethoxy, 2-(4-nitrophenyl)ethoxy, and the like.

The term "arylsulfonyl," as used herein, refers to an aryl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein.

Representative examples of arylsulfonyl include, but are not limited to, phenylsulfonyl, naphthylsulfonyl, and the like. The term "arylsulfonylalkoxy," as used herein, refers to an arylsulfonyl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of arylsulfonylalkoxy include, but are not limited to, 2-phenylsulfonylethoxy, 3-phenylsulfonylpropoxy, and the like. The term "carbonyl," as used herein, refers to a -C(O)- group. The term "catechol," as used herein, refers to a C₆H₄- 1 ,2-(O-)₂ group, wherein both oxygen atoms are attached to M, as defined herein. The term "cyano," as used herein, refers to a -CN group.

The term "cyanoalkoxy," as used herein, refers to a cyano group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of cyanoalkoxy include, but are not limited to, 2-cyanoethoxy, 3-cyanopropoxy, 1 -methyl-2-cyanoethoxy, l,l-dimethyl-2-cyanoethoxy, and the like.

The term "halo" or "halogen," as used herein, refers to -Cl, -Br, -I or -F. The term "haloalkoxy," as used herein, refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of haloalkoxy include, but are not limited to, 2,2,2- trichloroethoxy, l,l-dimethyl-2,2,2-trichloroethoxy, trifluoromethoxy, and the like.

The term "haloalkyl," as used herein, refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2- fluoroethyl, trifiuoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.

The term "hydroxy-protecting group" or "O-protecting group" refers to groups intended to protect a hydroxy group against undesirable reactions during synthetic procedures. Commonly used hydroxy-protecting groups are disclosed in T.H. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis. 2nd edition, John Wiley & Sons, New York (1991), which is hereby incorporated by reference. Examples of hydroxy- protecting groups include, but are not limited to, substituted methyl ethers, for example, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl)- ethoxymethyl, benzyl, and triphenylmethyl; tetrahydropyranyl ethers; substituted ethyl ethers, for example, 2,2,2-trichloroethyl and t-butyl; silyl ethers, for example, trimethylsilyl, t-butyldimethylsilyl and t-butyldiphenylsilyl; cyclic acetals and ketals, for example, methylene acetal, acetonide and benzylidene acetal; cyclic ortho esters, for example, methoxymethylene; cyclic carbonates; cyclic boronates; carbonyl derivatives, for example, acetyl, p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,4- dinitrophenylethoxylcarbonyl, 2-(methylthiomethoxymethyl)benzoyl, 2- (isopropylthiomethoxymethyl)benzoyl, 2-(2,4- dinitrobenzenesulphenyloxymethyl)benzoyl, 4-(methylthiomethoxy)butyryl, and levulinyl; trityl derivatives, for example, 4,4'-dimethoxytrityl, 4,4',4"-tris-(benzyloxy)trityl, 4,4',4"- tris-(4,5-dichlorophthalimido)trityl, 4,4',4"-tris-(levulinyloxy)trityl, 3-(imidazolylmethyl)- 4,4'-dimethoxytrityl, 4-decyloxytrityl, 4-hexadecyloxytrityl and l,l-bis-(4-methoxyhenyl)- l'-pyrenylmethyl; substituted xanthenyl groups, for example, pixyl(9-phenylxanthen-9-yl), 9-(p-methoxyphenyl)xanthen-9-yl), and 9-(4-octadecyloxyphenyl)xanthene-9-yl.

The term "Lewis acid," as used herein, refers to a chemical species, other than a proton, that has a vacant orbital or accepts an electron pair. It is to be understood that Lewis acids can be purchased or prepared as complexes including but not limited to, etherates, hydrates, and thioetherates. It is to be further understood that complexes purchased or prepared for the present invention do not contain an acidic proton.

Representative examples of Lewis acid include, but are not limited to, aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride, zirconium(IV) chloride, and the like. The term "methylenedioxy," as used herein, refers to a -OC(R⁸⁰)(R⁸¹)O- group wherein R⁸⁰ and R⁸¹ are independently selected from hydrogen and alkyl. The oxygen atoms of the methylenedioxy group are attached to the parent molecular moiety through two adjacent carbon atoms. Representative examples of a methylenedioxy group include, but are not limited to, 1,3-dioxolanyl, 2,2-dimethyl-l,3-dioxolanyl, 2-methyl-l,3- dioxolanyl, and the like.

The term "oxy," as used herein, refers to (-O-).

The term "purine base," as used herein, refers to an organic base selected from 9H-purin-6-ylamine (adenine) and 2-amino-l,9-dihydro-6H-purin-6-one (guanine). The amino group attached to adenine can be protected with a nitrogen-protecting group. The term "proton," as used herein, refers to H⁺.

The term "pyrimidine base," as used herein, refers to an organic base selected from 2,4(lH,3H)-pyrimidinedione (uracil), 5-methyl-2,4(lH,3H)-pyrimidinedione (thymine), and 4-amino-2(lH)-pyrimidinone (cytosine). The amino group attached to cytosine can be protected with a nitrogen-protecting group. The term "sulfonyl," as used herein, refers to a -SO₂- group.

The term "trifluoromethane," as used herein, refers to a -CF₃ group. The term "trifluoromethanesulfonyl," as used herein, refers to a trifluoromethane group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein.

The term "trifluoromethanesulfonyloxy," as used herein, refers to a trifluoromethanesulfonyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein.

Abbreviations

The following abbreviations are used: DMA for dimethylacetamide, DMT for 4,4'- dimethoxytrityl, EtOAc for ethyl acetate, eq for equivalents, MSDS for Material Safety

Data Sheets, NaHCO₃ for sodium bicarbonate, Na₂SO₄ for sodium sulfate, and TLC for thin layer chromatography.

Synthetic Methods The compounds and processes of the present invention will be better understood in connection with the following synthetic scheme which illustrates the method by which the compounds of the invention may be prepared.

Scheme 1

^{π -}2 equivalents Lewis acid 0-2 equivalents

pyridine

(i) (ϋ)

(iii)

General procedure using Lewis acids as activators:

A solution of phosphoramidite (i) (1.0 eq) and nucleoside (ii) (1.0 eq) in acetonitrile or DMA (0.1 M) was treated with a Lewis acid (Table 1). The solution was mixed at room temperature and was monitored by TLC using EtOAc :triethylamine (95:5) as the developing solvent. After a range of less than 5 minutes to 60 minutes (Table 1), the reaction mixture was quenched with aqueous NaHCO₃ and extracted with EtOAc. The organic layer was washed with aqueous NaHCO₃, dried (Na₂SO₄), and evaporated under vacuum to provide the dimer product (iii) in approximately quantitative yield. ^3,P NMR (500 MHz, CD₂C1₂) δ 139.5, 139.7;

MS (ESr) m/z: 1041.4 (M⁺). General procedure using Lewis acids and pyridine as activators: A solution of phosphoramidite (i) (1.0 eq) and nucleoside (ii) (1.0 eq) in acetonitrile or DMA (0.1 M) was treated with pyridine (2 eq) followed by the addition of a Lewis acid (2 eq). The solution was mixed at room temperature and was monitored by TLC using EtOAc :triethylamine (95:5) as the developing solvent. After less than 5 minutes, the reaction mixture was quenched with aqueous NaHCO₃ and extracted with EtOAc. The organic layer was washed with aqueous NaHCO₃, dried (Na₂SO₄), and evaporated under vacuum to provide the dimer product (iii) in approximately quantitative yield.

³¹P NMR (500 MHz, CD₂C1₂) δ 139.5, 139.7; MS (ESL) m/z: 1041.4 (M⁺).

Table 1

It is to be understood that dimer (iii) can be oxidizied using standard conditions know to those of ordinary skill in the art to give the phosphate, (J. Am. Chem. Soc, (1976), 98, 3655-3661). The 5'-OH of the oxidized dimer can be deprotected and treated with a Lewis acid activated phophoramidite monomer to form a trimer. This sequence of steps can be repeated until an oligonucleotide of desired length has been synthesized such that the process of the present invention can be used for preparing oligonucleotides, including solid phase synthesis thereof.

Claims

WE CLAIM:

1. A composition comprising a Lewis acid, an optional amount of pyridine, and a compound of formula I:

R²^^P _Rι I, wherein,

B¹ is selected from the group consisting of a purine base and a pyrimidine base; R¹ is -NR⁵R⁶, wherein, R⁵ and R⁶ are independently selected from the group consisting of alkyl and arylalkyl; R² is selected from the group consisting of alkoxy, alkyl, alkylsulfonylalkoxy arylsulfonylalkoxy, cyanoalkoxy, and haloalkoxy; R³ is a hydroxy-protecting group;

R⁴ is selected from the group consisting of hydrogen and -OR⁷, wherein R⁷ is a hydroxy-protecting group.

A composition according to claim 1 comprising said Lewis acid of formula II:

π, wherein, M is selected from the group consisting of aluminum, antimony, bismuth, boron, cadmium, cobalt, copper, chromium, gold, hafnium, iridium, iron, lanthanum, magnesium, manganese, mercury, molybdenum, nickel, niobium, osmium, palladium, platinum, phosphorous, rhenium, ruthenium, scandium, silver, tantalum, tellurium, tin, tungsten, titanium, vanadium, zinc, zirconium, and yttrium; R¹⁰ is selected from the group consisting of alkoxy, alkylcarbonyloxy, trifluoromethanesulfonyloxy, cyano, and halogen; R" is absent or selected from the group consisting of alkoxy, alkylcarbonyloxy, trifluoromethanesulfonyloxy, cyano, and halogen; or R¹⁰ and R¹¹ taken together form a catechol wherein both oxygen atoms are attached to M;

R¹² is absent or selected from the group consisting of alkoxy, alkylcarbonyloxy, trifluoromethanesulfonyloxy, cyano, and halogen;

R¹³ is absent or selected from the group consisting of alkoxy, alkylcarbonyloxy, trifluoromethanesulfonyloxy, cyano, and halogen; and

R¹⁴ is absent or selected from the group consisting of halogen.

3. A composition according to claim 2 comprising said Lewis acid of formula II wherein,

M is selected from the group consisting of aluminum, bismuth, boron, iron, magnesium, manganese, titanium, zinc, and zirconium;

R¹⁰ is selected from the group consisting of alkoxy, cyano, halogen and trifluoromethanesulfonyloxy;

R^H is selected from the group consisting of alkoxy, cyano, halogen and trifluoromethanesulfonyloxy; or

R¹⁰ and R" taken together form a catechol wherein both oxygen atoms are attached to M;

R¹² is absent or selected from the group consisting of alkoxy and halogen;

R¹³ is absent or selected from the group consisting of alkoxy and halogen; and

R¹⁴ is absent. 4. A composition according to claiml comprising, said Lewis acid selected from the group consisting of aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride and zirconium(IV) chloride.

5. A composition according to claiml , wherein R⁵ and R⁶ are independently selected from the group consisting of alkyl.

6. A composition according to claim 1, wherein R⁵ is isopropyl; and

R⁶ is isopropyl.

7. A composition according to claim 1, wherein R² is selected from the group consisting of cyanoalkoxy.

8. A composition according to claim 1, wherein R² is 2-cyanoethoxy.

9. A composition according to claim 1, wherein R¹ is diisopropylamino; and R² is 2-cyanoethoxy.

10. A composition according to claim 1, wherein R³ is selected from the group consisting of 4,4'-dimethoxytrityl, 4,4',4"-tris-(benzyloxy)trityl, 4,4',4"-tris-(4,5- dichlorophthalimido)trityl, 4,4',4"-tris-(levulinyloxy)trityl, 3-(imidazolylmethyl)- 4,4',-dimethoxytrityl, pixyl(9-phenylxanthen-9-yl), 9-(p- methoxyphenyl)xanthen-9-yl), 4-decyloxytrityl, 4-hexadecyloxytrityl, 9-(4- octadecyloxyphenyl)xanthene-9-yl, 1 , 1 -bis-(4-methoxyhenyl)-l'-pyrenylmethyl, p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,4- dinitrophenylethoxylcarbonyl, 4-(methylthiomethoxy)butyryl, 2- (methylthiomethoxymethyl)benzoyl, 2-(isopropylthiomethoxymethyl)benzoyl, 2-

(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl, and levulinyl.

11. A composition according to claim 1, wherein R³ is 4,4'-dimethoxytrityl.

12. A composition according to claim 1 comprising 1-4 molar equivalents of pyridine.

3. A composition according to claim 1 comprising, said Lewis acid selected from the group consisting of aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride and zirconium(IV) chloride;

0-4 molar equivalents of pyridine; and said compound of formula I wherein,

R¹ is diisopropylamino;

R² is 2-cyanoethoxy; and R³ is 4,4'-dimethoxytrityl.

14. A process for the preparation of a compound of formula III:

III, wherein B¹ and B² are independently selected from the group consisting of a purine base and a pyrimidine base;

R³ is a hydroxy-protecting group; R⁴ is selected from the group consisting of hydrogen and -OR⁷;

R⁸ is -OR⁷; and

R⁹ is selected from the group consisting of hydrogen and -OR⁷; or

R⁸ and R⁹ taken together form a methylenedioxy group; said process comprising treating a composition according to claim 1 with a compound of formula IV,

IV.

15. A process according to claim 14, wherein R² is alkyl.

16. A process according to claim 14, wherein R² is cyanoalkoxy.

17. A process according to claim 14, wherein

R² is 2-cyanoethoxy; and R⁸ and R⁹ taken together form a methylenedioxy group.

18. A process according to claim 14, wherein R² is 2-cyanoethoxy;

R³ is 4,4'-dimethoxytrityl;

R⁴ is hydrogen;

R⁸ and R⁹ taken together form a methylenedioxy group.

19. A process of oligonucleotide synthesis in which nucleoside phosphoramidite monomer precursors are activated by treatment with a Lewis acid to form activated intermediates and the activated intermediates are sequentially added to form an oligonucleotide product, wherein the improvement comprises using said Lewis acid as a phosphoramidite monomer activator.

0. A process according to claim 19 wherein, said Lewis acid is selected from the group consisting of aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride and zirconium(IV) chloride.