WO2009117227A2

WO2009117227A2 - Deprotection of oligonucleotides that contain one or more ribonucleotides

Info

Publication number: WO2009117227A2
Application number: PCT/US2009/035240
Authority: WO
Inventors: Raymond Cvetovich; Daniel R. Sidler; Daniel Zewge
Original assignee: Merck & Co., Inc.
Priority date: 2008-03-18
Filing date: 2009-02-26
Publication date: 2009-09-24
Also published as: WO2009117227A3

Abstract

This invention concerns an efficient, friendly and safer (easy to handle and environmentally less hazardous) protocol for deprotection of silyl protecting groups during the synthesis of oligonucleotides containing one or more ribonucleotides. This protocol can be applied to a pure RNA molecule or a molecule that has combinations of RNA and DNA, composed of pure or mixtures of modified, unmodified and abasic oligonucleotides. Specifically, the present invention features a method for the removal of protecting groups from 2' -hydroxyl (2' -OH) allowing the subsequent purification and isolation of oligonucleotides comprising of one or more ribonucleotides.

Description

MRL-B1O-22511

TITLE OF THE INVENTION

DEPROTECTION OF OLIGONUCLEOTIDES THAT CONTAIN ONE OR MORE

RIBONUCLEOTIDES

5 BACKGROUND OF THE INVENTION

This invention relates to the deprotection of oligonucleotides comprising one or more ribonucleotides. Further the invention relates to deprotection steps in an overall synthesis of oligonucleotides that contain one or more ribonucleotides, including siRNA and raiRNA for therapeutic uses.

10 The following discussion relates to the synthesis, deprotection, and purification of oligonucleotides containing one or more ribonucleotides including modified, unmodified and abasic nucleotides. The discussion is not meant to be complete and is provided only for understanding the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

15 Research in the many roles of ribonucleic acids has, in the past, been hindered by limited means of producing such biologically relevant molecules (Cech, 1992, Nucleic Acids Research, 17, 7381-7393; Francklyn and Schirαmel, 1989, Nature, 337, 478-481; Cook et al, 1991, Nucleic Acids Research, 19, 1577-1583; Gold, 1988, Annu. Rev. Biochemistry, 57, 199- 233). Although enzymatic methods existed, protocols that allowed one to probe structure

20 function relationships were limited. Only uniform post-synthetic chemical modification (Karaoglu and Thurlow, 1991, Nucleic Acids Research, 19, 5293-5300) or site directed mutagenesis (Johnson and Benkovic, 1990, The Enzymes, Vol. 19, Sigman and Boyer, eds., 159- 211) were available. In the latter case, researchers were limited to usage of natural bases. Fortunately, adaptation of the phosphoramidite protocol for DNA synthesis to RNA synthesis

25 has greatly accelerated our understanding of RNA. Site-specific introduction of modified nucleotides to any position in a given RNA has now become routine. Furthermore, one is not confined to a single modification but can include many variations in each molecule.

It is seemingly out of proportion that one small structural modification could cause such a dilemma. However, the presence of a single hydroxyl at the 2'~position of the

30 ribofuranose ring, has been the major reason that research in the RNA field has lagged so far behind comparable DNA studies. Progress has been made in improving methods for DNA synthesis that have enabled the production of large amounts of antisense deoxyoϊigonucleotides for structural and therapeutic applications. Only recently have similar gains been achieved for ribonucleotides (Wincott et aL, 1995, Nucleic Acids Research, 23, 2677-2684; Sproat et ai, MRL-B1O-22511

1995, Nucleosides and Nucleotides, 14, 255-273; Vargeese et ah, 1998, Nucleic Acids Research, 26, 1046-1050).

The chasm between DNA and RNA synthesis is due to the difficulty of identifying orthogonal protecting groups for the 5'- and 2'-hydroxyls. Historically, two standard 5 approaches have been taken by scientists attempting to solve the RNA synthesis problem; developing a method that is compatible with state-of the-art DNA synthesis or designing an approach specifically suited for RNA. Although adaptation of the DNA process provides a more universal procedure in which non-RNA phosphoramidites can easily be incorporated into RNA oligomers, the advantage to the latter approach is that one can develop a process that is best for 0 RNA synthesis and as a result, better yields can be realized. However, in both cases similar issues are faced, for example, identifying protecting groups that are compatible with synthesis conditions, yet, can be removed at the appropriate juncture. This problem does not refer only to the T- and 5'-OH groups, but includes the base and phosphate protecting groups as well. Consequently, the accompanying deprotection steps, in addition to the choice of ancillary agents, 5 are impacted. Another shared issue is the need for efficient synthesis of the monomer building blocks.

Solid phase synthesis of oligoribonucleotides follows the same pathway as DNA synthesis. A universal support attached to the first nucleoside or a solid support with an attached nucleoside is subjected to removal of the protecting group on the 5'-hydroxyl. The incoming 0 phosphoramidite is coupled to the growing chain in the presence of an activator. Any unreacted 5'-hydroxyl is capped/washed and the phosphite triester is then oxidized to provide the desired phosphotriester linkage. The process is then repeated until an oligomer of the desired length results. The actual reagents used may vary according to the 5'- and 2 '-protecting groups. Other ancillary reagents may also differ. 5 Once the oligoribonucleotide has been synthesized, it must then be deprotected.

This is typically a two-step process that entails (1) cleavage of the oligomer from the support and deprotection of the base and phosphate blocking groups, followed by (2) removal of the 2'OH- protecting groups.

Occasionally, a different order of reactions or separate deprotection of the

30 phosphate groups is required. In all cases, it is imperative that indiscriminate removal of protecting groups not occur, this is particularly an issue in the classic situation wherein the first step is base mediated. In this case, if the 2'-hydroxyl is revealed under these conditions, strand scission will result due to attack of the vicinal hydroxyl group on the neighboring phosphate backbone. Two other concerns that are prevalent in RNA synthesis but play no part in DNA are MRL-B1O-22511

the propensity for 3'-2' phosphodiester migration to provide undesired 2'-5' linkages and the susceptibility of oligoribonucleotides to degradation by ribonucl eases. The latter fact has led many researchers to develop 2 '-protecting groups that can remain in place until the oligomer is required for the desired experiment.

5 The prospect of specifically suppressing the expression of disease-causing genes has generated a lot of enthusiasm for developing RNAi-based therapies (Dykxhoorn, D., Palliser, D., Lieberman, J. Gene Therapy, 2006, 13, 541 - 552). Small interfering RNAs (siRNAs) are widely exploited for sequence-specific gene knockdown, predominantly to investigate gene function in cultured vertebrate cells, and also hold promise as therapeutic agents (Pei, Y., Tuschl,0 T. Nature methods, 2006, 30, 670 - 676). With milestone discoveries of RJSfA silencing or

RNA interference (RNAi), the need for fast, efficient and environmentally benign RNA synthetic methodologies is in demand more than ever.

Researchers have been searching for alternative 2'-OH protecting groups (Manoharan, M. et al, Org Lett, 2003, 5, 403 - 406), (Ohgi, T. OrgLett, 2005, 7, 3477 - 3480). 5 However, the TBDMS protecting group remains the most widely utilized and reliable protecting group. TBDMS is usually preferred due to ease of preparation of 2'-TBDMS ribonucleotide derivatives and the facile removal of 2'-TBDMS protecting groups by the action of a fluoride ion sources, such as tetrabutylammonium fluoride (TBAF) and triethylamine trifmoride (TEA.3HF), (Reese,C. B. Org. Biomol Chem., 2005, 3, 3851 - 3868). 0 In addition to generating tetraalkylammonmm salts that are difficult to remove during purification, the use of TBAF is known to result in slow rate of conversion (deprotection takes over 12 h). As a result TEA.3HF has been the reagent of choice for TBDMS ether cleavage in oligonucleotide synthesis (Westman, E., Stronmberg, R. Nucleic Acids Res,, 1994, 22, 2430 - 2431). This reagent was also reported to result in degradation of some 5 oligonucleotides resulting in lower yields in RNA synthesis. (Usman et al. Nucleic Acids Res,, 1995, 2677 - 2684). Despite its high toxicity (contact with a highly concentrated solution of HF may lead to acute hypocalcemia, followed by cardiac arrest and death) large excess amounts of TEA.3HF are commonly used for deprotection of TBDMS ethers.

As such there exists an unmet need for safer (easy to handle and less hazardous), 0 fast and efficient protocols which allow for the complete deprotection of 2'-O-protected RNA molecules. Such a method will simplify and facilitate large scale synthesis of such molecules for use as therapeutic agents and the small scale synthesis of such molecules for combinatorial screening. MRL-B1O-22511

SUMMARY OF THE INVENTION

This invention concerns an efficient and safer (easy to handle and environmentally less hazardous) protocol for deprotection of silyl protecting groups during the synthesis of 5 oligonucleotides. Specifically, the present invention features a method for the removal of protecting groups from 2'-hydroxyl (2'-OH) allowing the deprotection and subsequent purification of oligonucleotides comprising of one or more ribonucleotides.

DETAILED DESCRIPTION OF THE INVENTION 0 This invention concerns an efficient and safer (easy to handle and environmentally less hazardous) protocol for deprotection of silyl protecting groups during the synthesis of oligonucleotides. Specifically, the present invention features a method for the removal of protecting groups from 2'-liydroxyl (2'-OH) allowing the deprotection and subsequent purification of oligonucleotides. 5 Synthesis of oligonucleotides that contain one or more ribonucleotides including modified, unmodified or abasic nucleotides, comprises the steps of: (a) solid phase, solution phase, and/or hybrid phase, ( e.g.; phosphoramidite-based or H-phosphonate-based) oligonucleotide synthesis comprising the steps of detritylation, activation, coupling, capping, and oxidation or the equivalent thereof, in any suitable order, followed by (b) deprotection 0 comprising contacting the oligonucleotide having one or more ribonucleotides with a base, followed by (c) contacting the partially deprotected oligonucleotide comprising of one or more ribonucleotides with fluoride salts in the absence or presence of a co-solvent at pH ranges of 4 - 13, and followed by aging of reaction mixture over 5 min to 240 min at 10 to 100⁰C (preferably at 55°C) to remove 2'-hydroxyl protecting groups (for example, t-butyldimethylsilyl). 5 In an embodiment, the instant invention features a process for the rapid deprotection of oligonucleotides comprising ribonucleotides which are protected with alkylsilyl based protecting groups. Additionally, the invention provides a process for the deprotection of oligonucleotides comprising both ribonucleotides and 2'-deoxy- ribofuranose moieties which are protected with t-butyldimethylsilyl (TBDMS) or other silyl or silyl derivative protecting groups.0 In another embodiment, the instant invention features the use of an aqueous methylamine solution or other basic reagents to partially deprotect oligonucleotides followed by treatment with fluoride salts (for example, potassium fluoride) in the absence or presence of a co-solvent (for example, DMSO, DMAC, DMF, NMP) for the complete removal of 2'-O- silyl protecting groups. MRL-B1O-22511

In an embodiment, the invention features a deprotection process, comprising (a) contacting the oligonucleotide containing a 2'-hydroxy silyl protecting group with a base (neat or aqueous) at about 10 to 100⁰C or 20⁰C to 80°C or 30⁰C to 65°C or preferably 35⁰C or 65⁰C to partially deprotect the oligonucleotide (b) contacting the partially deprotected oligonucleotide 5 with potassium fluoride (or other fluoride salts including but not limited to sodium fluoride, calcium fluoride, magnesium fluoride etc.) in the absence or presence of a co-solvent (for example DMSO, DMAC, DMF, HMPA, ethanol, methanol, isopropanol, N-methylpyrrolidinone and others) at a pH range of 4 - 13, and (c) heating at about 10 to 100⁰C, preferably at about 55 0C, for about 5 to 240 minutes, preferably about 60 minutes, to remove 2'-hydroxyl protecting0 groups (for example, t-butyldimethylsilyl).

In another embodiment steps (b) and (c) in the above embodiment can precede step (a) or steps (a) to (c) can be combined to a one pot protocol.

In another embodiment, the deprotection reaction can be directly filtered to obtain the desired product or product may be precipitated using an anti-solvent. If necessary product 5 may be purified and lyophilized.

In another embodiment, the partially deprotected oligonucleotide is filtered using a suitable filtering medium, such as sintered glass or a polymer, and washed with a polar solvent (for example, DMSO, DMAC, DMF, ethanol, methanol, isopropanol, and/or N- methylpyrrolidinone) prior to treatment with potassium fluoride (KF). In another embodiment,0 the filtrate may or may not be cooled prior to treatment with a potassium fluoride reagent.

In another embodiment, the partially deprotected oligonucleotide is treated with a fluoride source (for example, potassium fluoride) without prior filtration,

In another embodiment, the invention features a deprotection process where optimal pH is between 8 to 12, however pH ranges of 4 to 13 can be used. 5 In another embodiment, the pH adjustment could be achieved by adding neat or aqueous mineral acids (for example HCl or H₃PO4) or organic acids (for example, malonic acid, glycolic acid, citric acid and tartaric acid).

In another embodiment, the pH adjustment could be achieved by removing added volatile amine base (for example, methyl amine) via vacuum or gas purge (for example N₂, Ar, 0 air).

In another embodiment, potassium hydrogen difluoride (solid or aqueous solution) can be used to adjust pH to 4 to 13 and effect deprotection of silyl protecting groups during oligonucleotide synthesis. MRL-B1O-22511

In another embodiment,, the process for deprotection of oligonucleotides of the present invention is used to deprotect an oligonucleotide synthesized using a multi-well plate format. In another embodiment, the instant invention provides a high throughput deprotection of oligonucleotides in a multi-well plate format (for example, a 96-well plate or a 256, 384 well 5 plate). Additionally, rapid deprotection of enzymatically active oligonucleotides (siRNA, miRNA and other RNAs) in greater than microgram quantities with high biological activity is featured. It has been determined that the recovery of enzymatically active oligonucleotides in high yield and quantity is dependent upon certain critical steps used during its deprotection.

In additional embodiments, the process for deprotection of oligonucleotides of the0 present invention is used to deprotect an oligonucleotide synthesized in both a trityl-on and trityl-off manner in nanomolar to molar synthesis scale. It will be recognized by those skilled in the art that modifications concerning time and temperature parameters can be used to optimize deprotection conditions for reactions of differing scale and/or molecules of differing composition. The use of different time and temperature parameters for varying molecular5 content and/or different reaction scale applications is hence within the scope of the invention.

In an embodiment, the instant invention provides a deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl silyl protecting groups with abase (neat or aqueous) at a temperature about 10 to 100⁰C to partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with one or more0 fluoride salts in the absence or presence of a co-solvent at a pH range of about 6 - 13, and (c) heating at about 10 to 100⁰C, for about 5 to 240 minutes, to remove 2'-hydroxyl silyl protecting groups.

In another embodiment, the instant invention provides a deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl t-butyldimethyl silyl 5 (TBDMS) protecting groups with a base (neat or aqueous) at a temperature about 10 to 100⁰C to partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with organic or mineral acids to adjust pH to 6 - 13, at temperature range of 0 ⁰C to 65 ⁰C (c) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the absence or presence of a co-solvent, and (d) heating at about 10 to 100 ⁰C, for about 5 to 240 0 minutes, to remove 2'-hydroxyl silyl protecting groups.

In another embodiment, the instant invention provides a deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl t-butyldimethyl silyl (TBDMS) protecting groups with a base (neat or aqueous) at a temperature about 10 to 100⁰C to MRL-B1O-22511

partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the absence or presence of a co-solvent at a pH range of about 6 - 13, and (c) heating at about 10 to 100 ⁰C, for about 5 to 240 minutes, to remove 2'-hydroxyl silyl protecting groups. 5 In another embodiment, the step (a) base is aqueous methyl amine.

In another embodiment, the step (a) temperature is about 20⁰C to 8O⁰C.

In another embodiment, the step (a) temperature is about or 3O⁰C to 65°C.

In another embodiment, the step (a) partially deprotected oligonucleotide may be cooled prior to step (b). 0

In another embodiment, the step (a) partially deprotected oligonucleotide may be cooled to about -10 to 10 ⁰C prior to step (b).

In another embodiment, the step (b) fluoride salts are selected from: potassium fluoride, lithium fluoride, sodium fluoride, calcium fluoride, cesium fluoride and magnesium 5 fluoride.

In another embodiment, the step (V) fluoride salt is potassium fluoride.

In another embodiment, the step (b) solvent is selected from: DMSO, DMAC, DMF, HMPA, ethanol, methanol, isopropanol, N-methylpyrrolidinone and diglyme.

In another embodiment, the step (b) solvent is DMSO. 0 In another embodiment, the step (b) pH range is between 8-12.

In another embodiment, the step (c) temperature is about 55⁰C.

In another embodiment, the instant invention provides a deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl t-butyldimethyl silyl (TBDMS) protecting groups with aqueous methyl amine at a temperature about 30 to 65⁰C to5 partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with potassium fluoride salt in the presence of DMSO at a pH range of about 8-12, and (c) heating at about 55 ⁰C, for about 5 to 240 minutes, to remove 2'-hydroxyl protecting groups.

In another embodiment, the instant invention provides a deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl t-butyldimethyl silyl0 (TBDMS) protecting groups with a base (neat or aqueous) at a temperature about 10 to 100⁰C to partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with organic or mineral acids to adjust pH to 6 - 13 (c) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the absence or presence of a co-solvent, and MRL-B1O-22511

(d) heating at about 10 to 100 ⁰C, for about 5 to 240 minutes, to remove 2'-hydroxyl silyl protecting groups.

In another embodiment, the step (b) organic acids are selected from citric acid, malonic acid, glycolic acid and tartaric acid, and mineral acids are selected from hydrochloric 5 acid, phosphoric acid and perchloric acid

In another embodiment, the instant invention provides a deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl t-butyldimethyl silyl (TBDMS) protecting groups with a base (neat or aqueous) at a temperature about 10 to 100⁰C to partially deprotect the oligonucleotide, (b) adjusting the pH to 6 -13 by removing the base by 0 applying a vacuum or gas purge and (c) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the absence or presence of a co-solvent, and (d) heating at about 10 to 100 ⁰C, for about 5 to 240 minutes, to remove 2'-hydroxyl silyl protecting groups.

In another embodiment, the instant invention provides a process to remove one or more 2'-hydroxyl silyl protecting groups from oligonucleotides by contacting the 5 oligonucleotides that contain one or more silyl protecting groups at the 2'-0 position with a fluoride salt in the absence or presence of a cosolvent at a pH range of 4-13 followed by heating at about 10 to 100⁰C for about 5 to 240 minutes to effect desilylation.

In another embodiment, the process is performed in a one pot reaction along with the deprotection of oligonucleotides from a solid support. 0 hi another embodiment, the fluoride salt includes potassium fluoride or any other alkali or alkaline or transition metal fluoride salts or silica/alumina-supported fluoride salts.

In another embodiment, the process may require adjusting the pH of the reaction mixture to 4 - 13, or to an optimal pH using mineral acids, organic acids, or buffers, or by removal of volatile base via vacuum or gas purge (for example, N₂, Ar). 5 In another embodiment, optimal pH is between pH 8 and 12, however pH ranges of 4 to 13 effect clean deprotection of silyl protecting groups from 2'-0 position during the synthesis of oligonucleotides.

In another embodiment, the organic acid could be citric acid, malonic acid, tartaric acid glycolic acid or any other organic acid. 0 In another embodiment, the mineral acid could be hydrochloric acid, phosphoric acid or any other mineral acid.

In another embodiment, the organic or mineral acids could be added neat or as solutions (aqueous or organic) in various concentrations. MRL-B1O-22511

In another embodiment, pH adjustment could be done before or after adding the fluoride source (example; potassium fluoride, potassium hydrogen difluoride).

In another embodiment, pH adjustment could also be done by adding various buffering solutions.

5 In another embodiment, fluoride sources (including but not limited to potassium fluoride, potassium hydrogen difluoride) could be added as solid or solutions (aqueous or organic).

In another embodiment the amount of fluoride source added could be greater than or equal to 5 equivalents per silyl group to be deprotected. 0 In another embodiment, potassium hydrogen difluoride could be used by itself or after pH is adjusted with organic acids, mineral acids or with buffer solutions.

In another embodiment, 2'-OH protecting groups comprise t-butyldimethylsilyl (TBDMS), or any other silyl protecting group and derivatives thereof.

By "trityl-on" is meant, a molecule, for example an oligonucleotide, synthesized5 in a manner which leaves the 5'-terminal dimethoxytrityl protecting group or an equivalent protecting group intact.

By "trityl-off" is meant, a molecule, for example an oligonucleotide, synthesized in a manner which removes the5'-terminal dimethoxytrityl protecting group or an equivalent protecting group. 0 By "solid phase" is meant, synthesis comprising a solid support (for example, polystyrene or controlled pore glass) which is used as a scaffold for the sequential addition of subunits in the synthesis of a polymeric molecule such as an oligonucleotide. The solid support can be exposed sequentially to reagents in solution, thereby eliminating the need for repeated purification and isolation steps during synthesis. A linker molecule can be used as an interface5 between the solid support and the growing polymer. Solid phase synthesis can be used for both phosphoramidite and H-phosphonate methods of oligonucleotide synthesis.

By "solution phase" is meant, synthesis comprising the combining of reactants and reagents in solution, such as in a solvent which provides a homogeneous mixture. Solution phase synthesis can be a preferred method for the synthesis of oligonucleotides in large0 quantities in consideration of lower costs, more efficient reactivity of reagents, and engineering factors.

By "hybrid phase" is meant, synthesis comprising both solid phase and solution phase synthesis elements. MRL-B1O-22511

By "oligonucleotide" is meant a nucleic acid molecule comprising at least one ribonucleotide residue. The oligonucleotide can be single, double or multiple stranded and may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof, which are well known in the art. The oligonucleotide may be RNA 5 (modified or unmodified, enzymatic or nonenzymatic) DNA (modified or unmodified). The oligonucleotide may be an antisense or enzymatic RNA molecule including siRNA and miRNA. Oligonucleotides can be used for purposes including but not limited to use as therapeutic agents, diagnostic reagents, and research reagents.

By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position0 of a β-D-ribo-furanose moiety.

MODIFIED OLIGONUCLEOTIDES

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into oligonucleotides. For example, oligonucleotides may incorporate modified nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O~ 5 methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry , 35, 14090). Sugar modifications of oligonucleotides have been extensively described in the art (see Eckstein et al, International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991 , 253, 314-317; Usman and Cedergren, Trends0 in Biochem. ScI , 1992, 17, 334-339; Usman et al. International Publication PCTNo. WO 93/15187; Sproat, US Patent No. 5,334,711 and Beigelman et al, 1995, / Biol. Chem., 270, 25702; Beigelman et al, International PCT publication No, WO 97/26270; Beigelman et al, US Patent No. 5,716,824; Usman et al, US patent No. 5,627,053; Woolf et al, International PCT Publication No. WO 98/13526; Thompson et al., USSN 60/082,404 which was filed on April 20,5 1998; Karpeisky et al, 1998, Tetrahedron Lett. , 39, 1131 ; Eamshaw and Gait, 1998, Biopolymers (Nucleic acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al, 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated by reference herein in their totalities).

In another aspect the oligonucleotides comprise a 5' and/or a 3'- cap structure. 0 By "cap structure" is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Wincott et al, WO 97/26270, incorporated by reference herein). The cap may be present at the 5 '-terminus (5 '-cap) or at the 3 '-terminus (3'- cap) or may be present on both termini. In non-limiting examples the 5'-cap is selected from the group comprising inverted abasic residue (moiety), 4',5'-methylene nucleotide; l-(beta-D- MRL-B1O-22511

erythrofbranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-mαcleotides; modified base nucleotide; phosphorodithioate linkage; *Areopentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4- dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide 5 moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3 '-2 '-inverted abasic moiety; 1 ,4-butanediol phosphate; 3^!-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3^?-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein). 0 By "non-nucleotide" is meant any group or compound which can be incorporated into an oligonucleotide in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. 5 By "abasic" is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1 ' position, (for more details, see Wincott et al., International PCT publication No. WO 97/26270).

By "unmodified nucleoside" is meant one of the bases adenine, cytosine, guanine, thymine, uracil joined to the V carbon of β-D-ribo-furanose. 0 By "modified nucleoside" is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. In connection with 2'-raodified nucleotides as described for the present invention, by "amino" is meant 2'-NH₂ or T-O- NH₂, which may be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Patent 5,672,695 and Matulic-Adamic et al., WO5 98/28317, respectively, which are both incorporated by reference herein in their entireties.

Other features and advantages of the invention will be apparent from the following procedures, examples, and from the claims. Procedure- 1 Schematic representation of the deprotection of oligonucleotides

Q R=SiIyI protecting group MRL-B1O-22511

KP deprotection of TBDMS ethers in Oligonucleotides

Materials MW Amount Moles

Oligonucelotide 25 μmol

Methyl Amine (40 wt% in H2O) 31.06 4 mL

8NHC1 1 - 2 mL

ICF (IlM m H₂O) 58.10 1.4 mL 15 mmol (600 eq) 0

Process Description: To a 20 rnL reaction flask/bottle/vial was added solid CPG protected oligonucleotide, followed by methyl amine (4 mL), and the reaction mixture was aged at 35 ⁰C.

After methyl amine deprotection, the reaction mixture was filtered and washed with DMSO (2 x

4mL). The filtrate (pH 13 - 14) was sampled for HPLC. The reaction mixture was then treated 5 with HCl (8N₅ 1.5 - 2.OmL) to adjust pH to 8 - 9. Potassium fluoride (1 IM, 1.4 mL, 15 mmol,

600 eq) was then added, and the reaction mixture was aged at 55 ⁰C over Ih. Progress of

TBDMS deprotection was followed by HPLC.

Sample preparation: 20μL of reaction mixture is diluted to 600 μL with H₂O.

HPLC conditions: Eclipse XDB-Cδ, 4.6 X 150 mm Column. Flow rate - 1.5 mL/min. 0 Temperature = 65 ⁰C, injection volume = 10 μL_> UV detector at 260 ma. Gradient Method: eluent A. 200 raM TEAA in DI water. B = 200 HiM TEAA in ACN. Run time = 30 min; post time = 5 min; % B, 0 min = 5, 10 min = 20, 20 min = 80. Method may be optimized for various types of oligonucleotide sequences.

Notes: 5 1. pH could be adjusted to 8 - 12 with organic acids, including but not limited to: malonic acid, citric acid, glycolic acid, tartaric acid and oxalic acid.

2. pH could also be adjusted with mineral acids including but not limited to: phosphoric acid and perchloric acid.

3. Mineral or organic acids could be added neat or as solutions. 0 4. pH could also be adjusted using buffering agents.

5. Potassium fluoride could be added as a solid or a solution with various concentrations. MRL-B1O-22511

6. While optimal pH is between 8 and 12, deprotection can be effected at a wide pH range (4 to 14), however significant detritylation is detected at lower pH (< 7). This observation needs to be considered when deprotecting a trityl-on oligonucleotide.

7. Our studies indicate that minimal detritylation is detected when vising organic acids for pH adjustment. This is especially important for small scale synthesis of oligonucleotides, where the 5'trityl-on needs to be preserved for final purification.

8. This protocol has been effectively applied to molar scale reactions in oligonucleotide synthesis.

Procedure-2 0 KF deprotection of TBDMS ethers in Oligonucleotides

KF/H⁺

- Ohgo— {-oπ)_χ

^ ^Jx 1h @ 55 oC

Materials MW Amount Moles 5 Oligomicelotide 25 μmol

Methyl Amine (40 wt% in H20) 31.06 4 mL

Malonic acid (4.8M) 2 - 3 mL

KP (I lM m H₂O) 58.10 1.4 mL 15 mmol (600 eq) 0 Process Description: To a 20 mL reaction flask/bottle/vial was added solid CPG protected oligonucleotide, followed by methyl amine (4 mL), and the reaction mixture was aged at 35 ⁰C. After methyl amine deprotection, the reaction mixture was filtered and washed with DMSO (2 x 4mL). The filtrate (pH 13 - 14) was sampled for HPLC. The reaction mixture was then treated with Malonic acid (4.8M₅ 2 - 3 mL) to adjust pH to 8 - 9. Potassium fluoride (1 IM, 1.4 mL, 15 5 mmol, 600 eq) was then added, and the reaction mixture was aged at 55 ⁰C over Ih. Progress of TBDMS deprotection was followed by HPLC.

Sample preparation: 20μL of reaction mixture is diluted to 600 μL with H₂O, HPLC conditions: Eclipse XDB-C8, 4.6 X 150 mm Column. Flow rate = 1.5 mL/min. Temperature = 65 ⁰C, injection volume = 10 μL, UV detector at 260 nm. Gradient Method:

30 eluent A. 200 mM TEAA in DI water. B = 200 mM TEAA in ACN. Run time - 30 min, post time = 5 min. % B, 0 min = 5, 10 min = 20, 20 min = 80. Method may be optimized for various types of oligonucleotide sequences. Notes: MRL-B1O-22511

1. pH could also be adjusted with mineral acids including but not limited to: hydrochloric acid, phosphoric acid and perchloric acid.

2. Mineral or organic acids could be added neat or as solutions.

3. Addition of mineral or organic acids for pH adjustment could be done at temperature ranges of 5 -10 C to 65 ⁰C.

4. pH could also be adjusted using buffering agents.

5. Potassium fluoride could be added as a solid or a solution with various concentrations.

6. While optimal pH is between 8 and 12, deprotection can be effected at a wide pH range (4 to 14), however significant detritylation is detected at lower pH (< 7). This observation needs to be 0 considered when deprotecting a trityl-on oligonucleotide.

7. Our studies indicate that minimal detritylation is detected when using organic acids for pH adjustment. This is especially important for small scale synthesis of oligonucleotides, where the 5'trityl-on needs to be preserved for final purification.

8. This protocol has been effectively applied to molar scale reactions in oligonucleotide 5 synthesis.

Procedure-3

KHF? deprotection of TBDMS ethers in Oligonucleotides Materials MW Amount Moles

Oligonucleotide 25 μmol 0 Methyl Amine (40 wt% in H2O) 31.06 4 mL

KHF₂ (2.6M) 58.10 7 - 8 mL

Process Description: To a 20 mL reaction flask/bottle/vial was added solid CPG protected oligonucleotide, followed by methyl amine (4 mL)_> and the reaction mixture was aged at 35 ⁰C. 5 After methyl amine deprotection, the reaction mixture was then filtered and washed with DMSO (2 x 4mL). Filtrate (pH 13 - 14) was sampled for HPLC (20 μL diluted to SOO μL with H₂O). The pH was then adjusted (8 - 10) by drop wise addition of KHF₂ ( 7- 8mL). Reaction mixture was aged at 55 ⁰C over Ih. Progress of reaction was followed by HPLC. Caution: KHF₂ solution (2.6 M, pH = 5.0) or solid releases HF. Keep reagent cold and perform 0 reaction in a hood with appropriate ventilation and with proper personal protection equipment. Notes: KHF₂ could be added as a solid or as a solution (aqueous or organic) in various concentrations. KHF₂ can also be added after pH is adjusted with mineral or organic acids (neat or aqueous) or with buffer solutions.

Procedure-4 MRL-B1O-22511

Nanomolar-scale (5'-Trityl-0n) Library synthesis KF deprotection of TBDMS ethers irt Oligonucleotides FFlluuoorriiddee ssaalltt ( ( 'eexxaammpp ^ellee KKFF)) ' "~

R=SiIyI protecting group

5 Process Description: Methylamine (120 μL, 40% in H₂O) was added to each column in a plate (96-well plate column). The CPG bed was saturated by pulsing vacuum. After aging for 4 - 8 minutes DMSO (200 μL) was added to each synthesis column, and oligonucleotides were collected into a pure plate via vacuum. The collection plate was then aged at 35 ⁰C over 45 minutes. The collection plate was removed from oven and each well was treated with aqueous 0 malonic acid (20 μL, 4.8M) followed by aqueous potassium fluoride (100 μL, 11 M). Plate was aged at 65 ⁰C over Ih. The collection plate is removed from oven and each well was diluted with sodium acetate or sodium chloride solutions (aqueous) Final crude oligonucleotide was then purified on a 96-well plate C-18 column. Notes:

15 1. pH could also be adjusted with mineral acids including but not limited to: hydrochloric acid, phosphoric acid and perchloric acid.

2. pH could be adjusted to 8 - 12 with other organic acids, including but not limited to: citric acid, glycohc acid, tartaric acid and oxalic acid.

3. Mineral or organic acids could be added neat or as solutions, 0 4. pH could also be adjusted using buffering agents.

6. While optimal pH is between 8 and 12, deprotection can be effected at a wide pH range (4 to 14), however significant detritylation is detected at lower pH (< 7). This observation needs to be considered when deprotecting a trityl-on oligonucleotide. 5 7. Our studies indicate that minimal detritylation is detected when using organic acids for pH adjustment. This is especially important for small scale synthesis of oligonucleotides, where the

5 'trityl-on needs to be preserved for final purification.

8. This protocol has been effectively applied to molar scale reactions in oligonucleotide synthesis. 30 Procedure- 5

KF deprotection of TBDMS ethers in Oligonucleotides MRL-B1O-22511

803 μmol reaction

Materials MW Amount Moles

Oligonucelotide 10.3g 803 μmol

Methyl Amine (40 wt% in H2O) 31.06 16O mL 5 Malonic acid (4.SM) 173 mL

KF (I lM m H₂O) 58.10 7O mL 770 mmo

DMSO 25O mL

H₂O 150O mL

Sequence: 0 rU;rC;rC;fluU;fluU;fluU;fluC;fluU;fluC;flυU;fluU;omeA;fluU;fluU;omeG;fluU;fluC;omeA;ome A;omeU. The starting material is OmeU CPG (78 μmol/g) supported oligonucleotide with 3- TBDMS groups. Amount of Support = 10.3 g = 803 μmol

Process Description: After the synthesis was completed CPG was suspended in 40% aq MeNH₂ (160 ml) and stirred at 35°C (external water bath) for Ih. The slurry was cooled to < 10 ⁰C (ice 5 bath), and filtered. The cake was washed with DMSO (250 ml). The filtrate was transferred to a 3L flask fitted with over head stirrer and thermometer probe. Batch was cooled to 2 ⁰C and aqueous malonic acid (4.8M, 173 mL) was added dropwise over 25 min. Process was exothermic and temp was kept between 2-19 ⁰C employing cooling bath (ice/acetone). pH of 8.3 was attained after adding 173 mL of malonic acid. KF (11M, 70 mL) was then added to the0 reaction mixture (no exotherm was detected). Reaction mixture was heated to 65 ⁰C (over shot to 74 ⁰C for about 10 min) and aged at 60-65 ⁰C for 1.5h. Progress of reaction was followed by HPLC. Once conversion is complete (after 1.5h) batch was cooled to 14°C (ice bath) and aged over for 40 min. The batch was treated with IL of water (exothermic process, ~ + 8 ⁰C). pH of reaction mixture was 6.9 (desired range 6.5 to 7.5). Batch was filtered and diluted to a final 5 volume of 191I mL.

ODU, LC/MS and HPLC analysis: 200 uL sample to 10 mL, A260 = 1.094. ODU = 1.094 x 50 x 1911 - 104531.7 ODU/μmol = 104531.7/803 - 130 0 LCMS, M-I = 6577.88, cald m/z - 6578.82 Purification: purified by SAX Volume = 980 mL, A 260 (x50 dilution) = 0.879 Total ODUs = 43,084.8. co efficient = 32.9 μg/ODU amount = 1.42g

43084.8/803 = 54 ODU/μmol, HPLC = 79.96 LCAP MRL-B1O-22511

UPLC= LCAP

Yield after purification, extinction coefficients 32.9μg/ODU. amount of single strand =

32.9x10"⁶ x 43,084.8 = 1.42g, 803 x 10^~6 x 6578.82 = g % yield = 1.42/5.28 x 100 - 27% uncorrectedfor UPLC puny, 5 EXAMPLE l

Deprotection of an oligonucleotide, 19 mer, trityl-on [2¹FCp¹OMeG]P¹FC] [rU][2'FC][2'OMeG] [ 2'0MeA][2'FC][rU] [ 2OMeG] [2 ' OMeA] [rU] [2OMeG][2'OMeA][2'OMeG] [2'OMeG] [2'FC] [2'OMeG] { 2'0MeC] comprising three ribonucleotides with 2'-0-TBDMS group. Oligoribonucleotides with 2'-0-TBDMS group are0 highlighted.

Reverse phase HPLC results, wavelength = 260 run

MRL-B1O-22511

RP HPLC results of a 19-mer oligo indicate that when raethylamine is removed via vacuum to adjust pH to 11.4, reaction was clean and no detritylation was detected. However when pH is adjusted with HCl (8N), close to 20% detritylation was detected.

EXAMPLE 2

Deprotection of an oligonucleotide, 22 mer, trityl-on [2'FC][rU][rU] [2'0MeG]

[2'OMeA][2'OMeA] [2'OMeA] [rU] [2'0MeA] [2¹OMeG] [2¹FC] [2'0MeA]

[2'0MeA][rU][2'0MeA] [rϋ][rU][rU] [2'OMeA][rU][rU][2'OMeG] comprising nine ribonucleotides with 2'-O-TBDMS group. Oligoribonucleotides with 2'-0-TBDMS group are highlighted. 0 Reverse phase HPLC results, wavelength = 260 nm

RP HPLC results of a 22-mer oligo indicate minimal detritylation when pH is adjusted with organic acids like malonic acid. MRL-B1O-22511

EXAMPLE 3

Deprotection of an oligonucleotide, 22 rner, trityl-off, [2TC][rU][rU] [2¹OMeG]

[2OMeA] [2'0MeA] [2 'OMeA] [rU] [2 'OMeA] [2'0MeG] [2'FC] [2'0MeA]

[2'OMeA][rU][2^rOMeA] [rU][rU][rU] [2'0MeA] [rU][rU] [2¹OMeG] comprising nine ribonucleotides with 2'-0-TBDMS group. Oligoribonucleotides with 2'-0-TBDMS group are highlighted.

Reverse phase HPLC results, wavelength = 260 nm

Method of cleavage is indicated in diagram. Comparable to cleaner (to TEA.3HF reaction) 10 deprotection was recorded for potassium fluoride reaction. EXAMPLE 4

Deprotection of an oligonucleotide, 21 mer, trityl-on [rU][rU][rC] [rG] [rC][rU] [rC][rG][rA] [rC][rG][rU] [rU][rU][rA] [rA][rG][rC] [rA][rC] [2¹OMeU] comprising twenty MRL-B1O-22511

ribonucleotides with 2'-O-TBDMS group. Oligoribomicleotides with 2'-OTBDMS group are highlighted.

Reverse phase HPLC results, wavelength = 260 nm

Claims

MRL-B1O-22511WHAT IS CLAIMED IS-

1. A deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl silyl protecting groups with abase (neat or aqueous) at a temperature about 10 to 5 100⁰C to partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the absence or presence of a co-solvent at a pH range of about 6 - 13, and (c) heating at about 10 to 100 ⁰C, for about 5 to 240 minutes, to remove 2'-hydroxyl silyl protecting groups. 0 2. A deprotection process, comprising (a) contacting an oligonucleotide containing

2'~hydroxyl t-butyldimethyl silyl (TBDMS) protecting groups with a base (neat or aqueous) at a temperature about 10 to 100⁰C to partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the absence or presence of a co-solvent at a pH range of about 6 - 13, and (c) heating at about 10 to 100 ⁰C, for about 5 to 5 240 minutes, to remove 2'-hydroxyl silyl protecting groups.

3. The deprotection process of Claim 2, wherein the step (a) base is aqueous methyl amine. 0 4. The deprotection process of Claim 2, wherein the step (a) temperature is about

20⁰C to 8O⁰C.

5 The deprotection process of Claim 2, wherein the step (a) temperature is about or 3O⁰C to 65°C. 25

6. The deprotection process of Claim 2, wherein the step (b) fluoride salts are selected from: potassium fluoride, lithium fluoride, sodium fluoride, calcium fluoride, cesium fluoride and magnesium fluoride.

30 7. The deprotection process of Claim 2, wherein the step (b) fluoride salt is potassium fluoride. MRL-B1O-22511

8, The deprotection process of Claim 2, wherein the step (b) solvent is selected from: DMSO, DMAC, DMF, HMPA, ethanol, methanol, isopropanol, N-methylpyrrolidmone and diglyme.

5 9. The deprotection process of Claim 2, wherein the step (b) solvent is DMSO.

10. The deprotection process of Claim 2, wherein the step (b) pH range is between 8- 12.

10 11. The deprotection process of Claim 2, wherein the step (c) temperature is about

55°C.

12. A deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl t-butyldimethyl silyl (TBDMS) protecting groups with aqueous methyl amine at a 15 temperature about 30 to 65°C to partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with potassium fluoride salt in the presence of DMSO at a pH range of about 8-12, and (c) heating at about 55 ⁰C, for about 5 to 240 minutes, to remove 2'- hydroxyl protecting groups.

20 13. A deprotection process, comprising (a) contacting an oligonucleotide containing

2'-hydroxyl t-butyldimethyl silyl (TBDMS) protecting groups with a base (neat or aqueous) at a temperature about 10 to 100°C to partially deprotect the oligonucleotide, (b) contacting the partially deprotected oligonucleotide with organic or mineral acids to adjust pH to 6 - 13, (c) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the

25 absence or presence of a co-solvent, and (d) heating at about 10 to 100 ⁰C, for about 5 to 240 minutes, to remove 2'-hydroxyl silyl protecting groups.

14. The deprotection process of Claim 13, wherein the step (b) organic acids are selected from citric acid, malonic acid, glycolic acid and tartaric acid, and mineral acids are

30 selected from hydrochloric acid, phosphoric acid and perchloric acid.

15. A deprotection process, comprising (a) contacting an oligonucleotide containing 2'-hydroxyl t-butyldimethyl silyl (TBDMS) protecting groups with a base (neat or aqueous) at a MRL-B1O-22511

temperature about 10 to 100⁰C to partially deprotect the oligonucleotide, (b) adjusting the pH to 6 -13 by removing the base by applying a vacuum or gas purge and (c) contacting the partially deprotected oligonucleotide with one or more fluoride salts in the absence or presence of a co- solvent, and (d) heating at about 10 to 100 ⁰C , for about 5 to 240 minutes, to remove 2'-hydroxyl 5 silyl protecting groups.