WO2012059510A1

WO2012059510A1 - Back pressure control during solid-phase synthesis on polymeric supports

Info

Publication number: WO2012059510A1
Application number: PCT/EP2011/069230
Authority: WO
Inventors: Hagen Cramer; Kevin James Finn; Vincent Ochieng Aduda; Thomas M. Rupp
Original assignee: Girindus America, Inc.
Priority date: 2010-11-02
Filing date: 2011-11-02
Publication date: 2012-05-10

Abstract

Described herein are methods for reducing the pressure in a solid-support column caused by swelling of the solid support in various solvents used in the detritylation and/or thiolation processes during oligonucleotide synthesis. Also described herein are methods for synthesizing oligonucleotides comprising contacting the solid-phase support with a washing fluid prior to and/or after thiolation and/or detritylation, wherein the washing fluid reduces pressure build up during oligonucleotide synthesis.

Description

Back pressure control during solid-phase synthesis on polymeric supports

This application claims priority to U.S. provisional application

No. 61/409256 filed November 2, 2010, the whole content of this application being incorporated herein by reference for all purposes.

FIELD

Described herein are methods for controlling back pressure build-up during the solid-phase synthesis of oligonucleotides using polymeric supports, wherein the methods comprise contacting the solid-phase support with a washing fluid prior to and/or after thiolation and/or prior to and/or after detritylation.

BACKGROUND

The manufacturing of non-swelling polymeric supports for solid-phase synthesis of oligonucleotides has been elusive. Some polymeric supports such as the macroporous polystyrene supports (e.g., PS-200 commercially available from GE Healthcare) have consistent swelling properties in a variety of solvents. Accordingly, during oligonucleotide synthesis, which often employs different solvent systems in the various steps of each nucleotide addition cycle, these macroporous polystyrene supports are advantageous because of the predictability of their swelling properties. However, these supports are unable to support a high load, and therefore, oligonucleotide syntheses employing macroporous polystyrene supports are limited in their overall yield of the desired

oligonucleotide.

High-load polystyrene supports have been developed and are commercially available. These solid-phase supports have the advantage of greatly increasing the oligonucleotide load in the support column. As such, these high-load polystyrene supports have increased the overall yields of oligonucleotides in a single synthesis compared to the macroporous polystyrene supports. However, unlike the macroporous polystyrene supports, the high-load polystyrene supports exhibit different swelling characteristics in different solvents commonly used for oligonucleotide synthesis. In certain solvents, the high-load polystyrene supports may swell to such an extent that the reagent flow in the support column is completely stopped, leading to costly and inefficient syntheses as well as significant reduction in the life of pumps and seals. Consequently, it is desirable to minimize or reduce the support column pressure caused by swelling of the polystyrene supports upon solvent change during each nucleotide addition cycle of an oligonucleotide synthesis.

SUMMARY

Described herein are methods for reducing or controlling the pressure in a solid-support column caused by swelling of the solid support in various solvents used in the detritylation and/or thiolation processes during oligonucleotide synthesis. Also described herein are methods for synthesizing oligonucleotides comprising contacting the solid-phase support with a washing fluid prior to and/or after thiolation and/or prior to and/or after detritylation, wherein use of the washing fluid reduces pressure build up during oligonucleotide synthesis.

Other embodiments, objects, features and advantages will be set forth in the detailed description of the embodiments that follows, and in part will be apparent from the description, or may be learned by practice, of the claimed invention. These objects and advantages will be realized and attained by the processes and compositions described and claimed herein. The foregoing Summary has been made with the understanding that it is to be considered as a brief and general synopsis of some of the embodiments disclosed herein, is provided solely for the benefit and convenience of the reader, and is not intended to limit in any manner the scope, or range of equivalents, to which the appended claims are lawfully entitled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 illustrates pressure changes in a solid support column during the detritylation and thiolation processes of each nucleotide addition cycle during the synthesis of a \6-mer oligonucleotide.

DESCRIPTION

While the present invention is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the claimed subject matter, and is not intended to limit the appended claims to the specific embodiments illustrated. The headings used throughout this disclosure are provided for convenience only and are not to be construed to limit the claims in any way. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

As used herein, the term "oligonucleotide" refers to an oligomer of nucleoside monomeric units comprising sugar units connected to nucleobases, wherein the nucleoside monomeric units are connected by internucleotide bonds. As used herein, the term "internucleotide bond" refers to a chemical linkage between two nucleoside moieties, such as the phosphodiester linkage typically present in nucleic acids found in nature, or other linkages typically present in synthetic nucleic acids and nucleic acid analogues. By way of example and without limitation, such internucleotide bond may include a phospho or phosphite group, and may include linkages where one or more oxygen atoms of the phospho or phosphite group are either modified with a substituent or replaced with another atom, e.g., a sulfur atom, or the nitrogen atom of a mono- or di-alkyl amino group. Typical internucleotide bonds are diesters of phosphoric acid or its derivatives, including but not limited to phosphates, thiophosphates, dithiophosphate, phosphoramidates, thio phosphoramidates.

As used herein, the term "nucleoside" refers to a compound consisting of a nucleobase connected to a sugar. The sugars may include, but are not limited to, a furanose ring such as ribose, 2'-deoxyribose and/or a non-furanose ring such as cyclohexenyl, anhydrohexitol, and morpholino. The modifications, substitutions and positions indicated hereinafter of the sugar included in the nucleoside are discussed with reference to a furanose ring, but the same modifications and positions are understood to also apply to analogous positions of other sugar rings. The sugar may be additionally modified. As non-limiting examples of the modifications thereof, the sugar may be modified at the 2'-, 3'-, and/or

4' -positions. In one embodiment, the 2'-position of a furanosyl sugar ring may be optionally modified to include for instance hydrogen ; hydroxyl ; C₁-C₂₀ alkoxy such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, and phenoxy ; azido ; amino ; alkylamino ; fluoro ; chloro and bromo. In another embodiment, 2'-4'- and/or 3'-4'-linked furanosyl sugar ring modifications may be made. In another embodiment, modifications to the furanosyl sugar ring may be made, including but not limited to substitutions for the ring 4'-0 by S, CH₂, NR, CHF or CF₂.

As used herein, the term "nucleobase" refers to a nitrogen-containing heterocyclic moiety capable of pairing with a complementary nucleobase or nucleobase analog. Typical nucleobases are the naturally occurring nucleobases including the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U), and modified nucleobases including other synthetic and natural nucleobases such as 5-methylcytosine

(5-me-C), 5 -hydroxy methyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,

8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine,

3-deazaguanine and 3-deazaadenine, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine

cytidine( lH-pyrimido[5,4-b] [ 1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H- pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Other potentially suitable bases include universal bases, hydrophobic bases, promiscuous bases and size- expanded bases.

As used herein, the term "solid support" refers to any particle, bead, or surface upon which synthesis of an oligonucleotide occurs. In one embodiment, the solid support may be an inorganic support. By way of example and without limitation, the inorganic support may be silica gel and/or controlled pore glass (CPG). In another embodiment, the solid support may be an organic support. By way of example and without limitation, the organic support may be highly crosslinked polystyrene, grafted copolymers consisting of a low crosslinked polystyrene matrix on which polyethylene glycol (PEG or POE) is grafted (e.g., Tentagel), polyvinylacetate (PVA), a copolymer of

polystyrene/divinyl benzene (e.g., Poros), aminopolyethyleneglycol and/or cellulose. In one embodiment, the solid support is highly crosslinked

polystyrene. In a further embodiment, the highly crosslinked polystyrene solid supports may be NittoPhase^®, NittoPhase^® HL, or UnyLinker™ NittoPhase^®, all of which are commercially available from Nitto Denko Corporation.

In one embodiment, the protected oligonucleotide may be attached to the solid support by means of a linkage. Linkages are known in the art as chemical moieties comprising a covalent bond or a chain of atoms that covalently attach a solid support to a nucleoside, nucleotide or oligonucleotide. So called "standard solid supports" carrying a nucleoside that has been pre-attached via a linker are commercially available. This nucleoside may become the 3' - or 5'- terminal residue of the final oligonucleotide after the cleavage and deprotection steps. Suitable linkers which can be used in this embodiment include, without limitation, succinyl, carbonate, or carbamate. In one embodiment, the linker is succinyl. In one embodiment, the standard solid support carries the 3' - or 5'-terminal nucleoside.

Solid supports without the 3' - or 5' - nucleoside pre-attached, namely the "universal" solid supports, are also known in the art and commercially available. Those supports do not have the intended 3' - or 5'- terminal nucleoside attached. Instead, the corresponding terminal nucleoside or residue is added in the first cycle, generating an undesired phosphate or thiophosphate linkage between this nucleoside and the universal support. This approach requires that the undesired phosphate or thiophosphate linkage be removed during the cleavage and/or deprotection step. Typical examples of the "universal" solid support are shown in scheme 1.

Scheme 1

Universal Support Type 1

Universal Support Type 2

Other types of commercially available solid supports may carry protected functional groups, which can be used for post-synthesis conjugation, or may carry the conjugated load directly, such as carbohydrates, lipophilic molecules, peptides, antibiotics, pharmaceuticals, vitamins, fluorescent labels, lipids, folate, cholesterol and dyes. Conjugation allows a desired oligonucleotide to be covalently linked to a reporter group with biologically relevant properties. In one embodiment, the solid support may be TAMRA functionalized succinyl resin. In another embodiment, supports containing bis-amino branched linkers may be employed. By way of example and without limitation, bis-amino branched linkers may be utilized to assemble conjugates of any desired oligonucleotide and peptides. In yet another embodiment, the solid support may contain a linker functionalized with a modified glyceryl group. In yet another embodiment, the solid support may contain a linker functionalized with a fatty acyl group having from 6 carbons to 30 carbons. In still another embodiment, the solid support may contain a linker functionalized with a fatty acyl group having from 10 to 25 carbons. In a further embodiment, the solid support may contain a linker functionalized with a fatty acyl group having from 15 carbons to 20 carbons. In yet a further embodiment, the solid support may contain a linker functionalized with a palmitoyl group.

In one embodiment described herein, the oligonucleotide synthesis yields an oligonucleotide of any desired length. In one embodiment, the

oligonucleotide may be from about 2 to about 200 nucleotides long. In another embodiment, the oligonucleotide may be from about 10 to about 150 base monomers long. In another embodiment, the oligonucleotide may be from about 10 to about 100 base monomers long. In yet another embodiment, the oligonucleotide may be from about 10 to about 75 monomers long. In yet another embodiment, the oligonucleotide may be from about 15 to about 25 base monomers long. In yet another embodiment, the oligonucleotide may be from about 25 to about 50 base monomers long. In yet another embodiment, the oligonucleotide may be up to about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195 and about 200 base monomers long.

In further embodiments, the methods described herein may produce oligonucleotides of DNA, RNA, BNA, UNA, any derivatives thereof, and in any combination thereof. In one embodiment, the BNA may be LNA or EN A. In another embodiment, the oligonucleotide synthesis may produce

oligonucleotides having the formula as described in U.S. Patent No. 6,465,628 at column 2, line 1 through column 3, line 15. U.S. Patent No. 6,465,628 is hereby incorporated by reference to the same extent as if it were set forth in its entirety herein.

As used herein, "DNA" refers to a polymer of deoxyribonucleic acid units.

As used herein, "RNA" refers to a polymer of ribonucleic acid units. As used herein, "BNA" refers to a polymer of bicyclic nucleic acids. As used herein, "LNA" refers to a polymer of locked nucleic acid units. As used herein, "EN A" refers to a polymer of 2'-0,4'-C-ethylene bridged nucleic acid. As used herein, "UNA" refers to a polymer of unlocked nucleic acids.

In another embodiment described herein, non limiting examples of naturally occurring nucleobases may be adenine, guanine, cytosine, uracil, and thymine. In another embodiment, non limiting examples of non-naturally occurring and rare naturally occurring nucleobases may be xanthine,

hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine.

In a further embodiment described herein, the oligonucleotide synthesis may produce an oligonucleotide comprising any desired nucleotide sequence. In another embodiment, the oligonucleotide sequence may be 5'-TCG TCG TTT TGT CGT TTT GTC GTT-3', which is a DNA sequence known commercially as CPG7909 (SEQ ID NO: l). In yet another embodiment, the oligonucleotide sequence may be an oligonucleotide comprising both LNA and DNA bases (SEQ ID NO:2).

In an additional embodiment, the oligonucleotide may be synthesized from the 3 '-terminus to the 5 '-terminus. In another embodiment, the oligonucleotide may be synthesized from the 5'-terminus to the 3'-terminus.

In an embodiment described herein, the oligonucleotide synthesis may comprise the following steps : (a) detritylation to remove the acid labile protecting group from the 5' or 3 '-terminal nucleoside of the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle), (b) coupling the unprotected 5' or 3 '-terminal nucleoside phosphoramidite of the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) to a protected nucleoside to form a phosphite triester linkage between the incoming nucleotide synthon and the support-bound oligonucleotide chain, and (c) the combined thiolation of the support-bound oligonucleotide to oxidize the phosphite triesters formed in the coupling step to phosphothiolates and capping to cap all of the 5'-hydroxy groups that failed to react in the coupling step. In one embodiment, these three cycles may be repeated as desired using the appropriate protected nucleoside phosphoramidite for assembly of any desired oligonucleotide sequences with a protecting group at the 5' terminus.

In another embodiment described herein, the oligonucleotide synthesis may comprise the following steps : (a) detritylation to remove the acid labile protecting group from the 5' or 3 '-terminal nucleoside of the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle), (b) coupling the unprotected 5' or 3 '-terminal nucleoside phosphoramidite of the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) to a protected nucleoside to form a phosphite triester linkage between the incoming nucleotide synthon and the support-bound oligonucleotide chain, (c) thiolation of the support-bound oligonucleotide to oxidize the phosphite triesters formed in the coupling step to phosphothiolates, and (d) capping the 5 '-hydroxy groups that failed to react in the coupling step. In a further embodiment these four cycles may be repeated as desired using the appropriate protected nucleoside phosphoramidite for assembly of any desired oligonucleotide sequences with a protecting group at the

5 '-terminus.

In an additional embodiment described herein, the oligonucleotide synthesis may comprise the following steps : (a) detritylation to remove the acid labile protecting group from the 5' or 3 '-terminal nucleoside of the support- bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle), (b) coupling the unprotected 5' or 3 '-terminal nucleoside phosphoramidite of the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) to a protected nucleoside to form a phosphate triester linkage between the incoming nucleotide synthon and the support-bound oligonucleotide chain, (c) oxidation of the support-bound oligonucleotide to oxidize the phosphite triesters formed in the coupling step to phosphate triesters, and (d) capping the 5'-hydroxy groups that failed to react in the coupling step. In a further embodiment these four cycles may be repeated as desired using the appropriate protected nucleoside

phosphoramidite for assembly of any desired oligonucleotide sequences with a protecting group at the 5 '-terminus.

Detritylation

In another embodiment described herein, each cycle of the solid-phase synthesis commences with removal of the acid labile protecting group of the 5' or 3 '-terminal nucleoside of the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle). Suitable nucleobase protecting groups are known to persons of ordinary skill in the art, including but not limited to benzoyl, isobutyryl, acetyl, phenoxyacetyl, aryloxyacetyl, phthaloyl, 2-(4-nitro-phenyl)ethyl, pent-4-enoyl,

dimethylformamidine (dmf), dialkylformamidine, and dialkylacetamidine. In one embodiment, suitable 5 '-hydroxyl protecting groups may include, but are not limited to, trityl groups. In one embodiment, the 5 '-hydroxyl protecting group may be a dimethoxy trityl group (DMTr) or a monomethoxy trityl group (MMTr). In another embodiment, the 5'-protecting group may be, but is not limited to, tert-butyl dimethylsilyl (TBDMS), levulinyl, benzoyl, fluorenemethoxycarbonyl (FMOC), 9-phenylthioxanthen-9-yl (S-pixyl).

Suitable 2' -protecting groups used in RNA synthesis include, but are not limited to 2'-0-protecting groups : tert-butyl dimethylsilyl (TBDMS),

9-phenylxanthen-9-yl (Px), 9-phenylthioxanthen-9-yl (SPx),l- [(2-chloro- 4methyl)pheny]-4-methoxypiperidin-4-yl (Ctmp), 1- (2-fluorophenyl)-4- methoxypiperidin-4-yl (Fpmp), [2-(methylthio)phenyl]thiomethyl (MTPM), bis-(Acetoxyethyloxy)methylester (ACE), (1-methyl-l- methoxyethyl)(MME),methoxy(ethoxymethyl( MEM), p- nitrophenylethylsylfonyl (NPES), p-cyanophenylethylsylfonyl (CPES), carbomethoxyethylsulfonyl (CEMS), TriisopropylsilylOxy Methyl (TOM) and 2' silyl-containing thiocarbonate protecting group.

In one embodiment described herein, the protecting group is removed from the 5 '-terminus by treatment with an acidic solution. In another embodiment, the acidic solution may comprise an organic acid dissolved in an organic solvent. In yet another embodiment, the organic acid may be a haloacetic acetic acid. By way of example and without limitation, the organic acid may be trifluoroacetic acid ("TFA"), fluoroacetic acid, trichloroacetic acid ("TCA"), dichloroacetic acid ("DCA"), chloroacetic acid, and any combinations thereof. In a further embodiment, the acidic solution may comprise a sulfonic acid. In yet a further embodiment, the sulfonic acid may be an alkyl sulfonic acid or an aryl sulfonic acid.

In another embodiment described herein, the acid may be dissolved in a solution comprising methylene chloride, an arene solvent, or substituted arene solvents, in any combination. In one embodiment, the substituted arene solvent may be an alkylbenzene and any combinations thereof. By way of example and without limitation, the alkylbenzene solvents may be toluene, xylene, hemimellitene, pseudodocumeme, mesitylene, prehnitene, isodurene, durene pentamethylbenzene, hexamethylbenzene, ethylbenzene, ethyltoluene, propylbenzene, propyltoluene, butylbenzene, pentanylbenzene, pentanyl toluene, hexanyl benzene, hexanyl toluene and any combinations thereof. The acid may also be dissolved in a solution of diphenylmethane, triphenylmethane, tetraphenylmethane, 1,2-diphenylethane and any combinations thereof. The acid may further be dissolved in a solution of styrene, stilbene, diphenylethylene, triphenylethylene tetraphenylethylene and any combination thereof. In yet a further embodiment, the acid may be dissolved in a solution of phenylacetylene, diphenylacetylene and any any combination thereof. The detritylation solution may further comprise acetonitrile in any combination with any of the foregoing solvents in any combination.

In a further embodiment, the acidic solution used for detritylation may contain from about 0.5 % to about 15 % (v/v) of acid. In another embodiment, the acidic solution used for detritylation may contain from about 1 % to about 10 % (v/v) of acid. In yet another embodiment, the acidic solution used for detritylation may contain about 2 % to about 5 % (v/v) of acid. In another embodiment, the acidic solution used for detritylation may contain

about 3 % (v/v) of acid. In still another embodiment, the acidic solution used for detritylation may contain about 0.5 %, about 1 %, about 1.5 %, about 2 %, about 2.5 %, about 3 %, about 3.5 %, about 4 %, about 4.5 %, about 5 %, about 5.5 %, about 6 %, about 6.5 %, about 7 %, about 7.5 %, about 8 %, about 8.5 %, about 9 %, about 9.5 % and about 10 % (v/v) of acid. In one another embodiment, the acidic solution used for detritylation may contain about 3 % DCA dissolved in toluene.

In yet a further embodiment, the acid may be present in an amount of from about 10 to about 120 molar equivalents. In another embodiment, the acid may be present in an amount of from about 30 to about 105 molar equivalents. In yet another embodiment, the acid may be present in an amount of from about 50 to about 90 molar equivalents. In still another embodiment, the acid may be present in an amount of about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, and about 120 molar equivalents. In still another embodiment described herein, the acidic solution may be contacted with the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 30 seconds to about 30 minutes. In another embodiment, the acidic solution may be contacted with the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 2 minute to about 15 minutes. In yet another embodiment, the acidic solution may be contacted with the support-bound oligonucleotide (or nucleoside, linker or other type of

functionalized support for the first cycle) for about 3 minutes to about 8 minutes. In a further another embodiment, the acidic solution may be contacted with the support-bound oligonucleotide (or nucleoside, linker or other type of

functionalized support for the first cycle) for about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 and about 30 minutes.

Detritylation plays a role in each synthesis cycle to produce high yields of quality nucleotides. A common side reaction during detritylation is depurination due to the acid lability of purine nucleotides and nucleosides under the acidic conditions required for deprotection of the 5'-terminus. Increasing the contact time between the acid and the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) may lead to depurination to an extent that could compromise the quality of the

oligonucleotide. However, incomplete detritylation may generate N-l mers and shorter sequence impurities. Accordingly, optimizing the detritylation conditions may be necessary to yield oligonucleotides of desired quality.

In an embodiment described herein, the solid support may be washed with a detritrylation washing fluid prior to detritylation of the protecting group at the 5 '-terminus of the support-bound oligonucleotide. In another embodiment, the solid support may be washed with a detritrylation washing fluid after detritylation of the protecting group at the 5 '-terminus of the support-bound oligonucleotide. In yet another embodiment, the solid support may be washed with a detritrylation washing fluid both prior to and after detritylation of the protecting group at the 5 '-terminus of the support-bound oligonucleotide.

Although not required, the detritrylation washing fluid may be the same solvent system used to prepare the acidic solution for detritylation. By way of example and without limitation, if detritylation employs an acidic solution of DCA in toluene, the column may be washed with toluene prior to and/or after detritylation. In one embodiment, the detritylation washing fluid may comprise methylene chloride, an arene solvent, or substituted arene solvents, in any combination. In one embodiment, the substituted arene solvent may be an alkylbenzene and any combinations thereof. By way of example and without limitation, the alkylbenzene solvents may be toluene, xylene, hemimellitene, pseudodocumeme, mesitylene, prehnitene, isodurene, durene

pentamethylbenzene, hexamethylbenzene, ethylbenzene, ethyltoluene, propylbenzene, propyltoluene, butylbenzene, pentanylbenzene, pentanyl toluene, hexanyl benzene, hexanyl toluene and any combinations thereof. In another embodiment, the detritylation washing fluid may comprise diphenylmethane, triphenylmethane, tetraphenylmethane, 1,2-diphenylethane and any combinations thereof. In another embodiment, the detritylation washing fluid may comprise styrene, stilbene, diphenylethylene, triphenylethylene tetraphenylethylene and any combination thereof. In another embodiment, the detritylation washing fluid may comprise phenylacetylene, diphenylacetylene and any any combination thereof. The detritylation washing fluid may further comprise acetonitrile in any combination with any of the foregoing solvents in any combination.

In an additional embodiment, each wash prior to and/or after detritylation may deliver from about 0.5-column volume to about 10-column volume of the detritylation washing fluid. In another embodiment, each wash prior to and/or after detritylation may deliver from about 1 -column volume to about 8-column volume of the detritylation washing fluid. In yet another embodiment, each wash prior to and/or after detritylation may deliver from about 3 -column volume to about 7-column volume of the detritylation washing fluid. In yet another embodiment, each wash prior to and/or after detritylation may deliver from about 4-column volume to about 7-column volume of the detritylation washing fluid. In a further embodiment, each wash prior to and/or after detritylation may deliver from about 6-column volume to about 7-column volume of the detritylation washing fluid. In still another embodiment, each wash prior to and or after detritylation may deliver about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 and about 10-column volume of the detritylation washing fluid. In yet a further embodiment described herein, washing the support column prior to and/or after detritylation may decrease undesired pressure increases in the support column. An increased pressure in the column support may block the flow of solvents and reagents through the support column, thereby decreasing the efficiency of the oligonucleotide synthesis. In one embodiment, detritylation washes may be employed as desired. In one embodiment, detritylation washes may be employed during each nucleotide addition cycle of the oligonucleotide synthesis. In another embodiment, detritylation washes may be employed only during nucleotide addition cycles in which an undesired increase in column pressure is observed. In yet another embodiment, detritylation washes may not be employed at all.

Coupling

In another embodiment described herein, the chain elongation step may be achieved by using standard phosphoramidite-coupling chemistry. During this step, the 5 '-hydroxy groups of the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) may be reacted with a solution of protected nucleoside phosphor amidite in the presence of an activator such as tetrazoles in an organic solvent. This reaction results in the formation of a phosphite triester linkage (three-coordinate phosphorus) between the incoming nucleotide synthon and the support-bound oligonucleotide chain. Excess reagents may be washed from the column reactor with solvent in an amount of from about 0.25-column volume to about 5-column volume. In one embodiment, the solvent may be acetonitrile. In a further embodiment, the excess reagents may be washed from the column reactor with solvent in an amount of about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.25, about 2.5, about 2.75, about 3, about 3.25, about 3.5, about 3.75, about 4, about 4.25, about 4.5, about 4.75, and

about 5-column volume. In another embodiment, the support column may be washed with acetonitrile prior to and after the coupling reaction.

In another embodiment, the support column may be washed with a coupling washing fluid. In yet another embodiment, the coupling washing fluid may comprise methylene chloride, an arene solvent, or substituted arene solvents, in any combination. In an additional embodiment, the substituted arene solvent may be an alkylbenzene and any combinations thereof. By way of example and without limitation, the alkylbenzene solvents may be toluene, xylene, hemimellitene, pseudodocumeme, mesitylene, prehnitene, isodurene, durene pentamethylbenzene, hexamethylbenzene, ethylbenzene, ethyltoluene, propylbenzene, propyltoluene, butylbenzene, pentanylbenzene, pentanyl toluene, hexanyl benzene, hexanyl toluene and any combinations thereof. In another embodiment, the coupling washing fluid may comprise diphenylmethane, triphenylmethane, tetraphenylmethane, 1,2-diphenylethane and any combinations thereof. In another embodiment, the coupling washing fluid may comprise styrene, stilbene, diphenylethylene, triphenylethylene tetraphenylethylene and any combination thereof. In another embodiment, the coupling washing fluid may comprise phenylacetylene, diphenylacetylene and any combination thereof. In yet another embodiment, the coupling washing fluid may be dimethyl formamide ("DMF"). In an additional embodiment, the coupling washing fluid may be dimethyl sulfoxide ("DMSO"). In a further embodiment, the coupling washing fluid may further comprise acetonitrile in any combination with any of the foregoing solvents in any combination.

In a further embodiment described herein, the concentration of the protected nucleoside phosphoramidite in solution may be from about 0.01 M up to its maximum concentration in the respective solvent. In another embodiment, the concentration of the protected nucleoside phosphoramidite in solution may be from about 0.1 M to about 0.7 M. In yet another embodiment, the concentration of the protected nucleoside phosphoramidite in solution may be from

about 0.15 M to about 0.4 M. In yet another embodiment, the concentration of the protected nucleoside phosphoramidite in solution may be from about 0.15 M to about 0.2 M. In still another embodiment, the concentration of the protected nucleoside phosphoramidite in solution may be about 0.2 M. In another embodiment, the concentration of the protected phosphoramidite in solution may be about 0.01, about 0.05, about 0.1, about 0.15, about 0.2, about 0.25., about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, and about 1.0 M. As used herein, "M" refers to molar concentration.

In another embodiment, the protected nucleoside phosphoramidite may be present in an amount of from about 1 to about 4 molar equivalents. In yet another embodiment, the protected nucleoside phosphoramidite may be present in an amount of from about 1.5 to about 2.5 molar equivalents. In yet another embodiment, the protected nucleoside phosphoramidite may be present in an amount of from about 1.7 to about 2.0 molar equivalents. In yet another embodiment, the protected nucleoside phosphoramidite may be present in an amount of from about 1, about 1.3, about 1.6, about 1.9, about 2.2, about 2.5, about 2.8, about 3.1, about 3.4, about 3.7 and about 4 molar equivalents.

In an additional embodiment, a tetrazole may be present during the coupling reaction. Suitable tetrazoles include without limitation, IH-tetrazole, 5-(ethylthio)-lH-tetrazole ("ETT"), 5-(benzylthio)-lH-tetrazole ("BTT"), and 4,5-dicyanoimidazole ("DCI"), saccharine 1-methylimidazole ("SMI"),

5-(bis-3.5-trifluoromethylphenyl)-lH-tetrazole ("Activator 42"), and any activator in combination with N-methylimidazole. In one embodiment, the tetrazole may be present in a concentration of about 0.1 M to about 1 M. In another embodiment, the tetrazole may be present in a concentration of about 0.2 M to about 0.3 M. In yet another embodiment, the tetrazole may be present in a concentration of about 0.5 M to about 0.6 M. In a further embodiment, the tetrazole may be present in a concentration of about 0.1, about 0.2, about 0.25, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9 and about 1 M.

In one embodiment, the solution of protected nucleoside phosphor amidite and IH-tetrazole may be contacted with the support column for about 1 minute to about 20 minutes. In another embodiment, the solution of protected nucleoside phosphor amidite and IH-tetrazole may be contacted with the support column for about 1.5 minutes to about 7 minutes. In yet another embodiment, the solution of protected nucleoside phosphoramidite and IH-tetrazole may be contacted with the support column for about 2 minutes to about 7 minutes. In yet another embodiment, the solution of protected nucleoside phosphoramidite and IH-tetrazole may be contacted with the support column for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 and about 20 minutes.

Combined Thiolation and Capping

In a further embodiment described herein, in each cycle, some fraction of the 5 '-hydroxy groups of the support-bound oligonucleotide may not react with the nucleoside phosphoramidite. To prevent these oligonucleotides from reacting with a nucleoside phosphoramidite in a subsequent cycle, thereby producing, by way of example and without limitation, an N-l mer, it may be desirable to cap these oligonucleotides to prevent any further reactivity. This may be accomplished by any suitable capping reaction using any suitable capping reagent known to those of ordinary skill in the art. In another embodiment, in each cycle, it may be desirable to thiolate the three-coordinate phosphate triesters formed in the coupling reaction to the more stable five- coordinate phosphorothiolates. This may be accomplished by any suitable thiolation reaction using any suitable thiolation reagent. In one embodiment, the thiolation and capping reaction may be combined into a single reaction step. In another embodiment, the thiolation and capping reactions may be performed as discrete reaction steps.

Standard thiolation reagents for use in oligonucleotide synthesis are common and well known in the art, and their use is contemplated herein. In one embodiment, the thiolation reagent may be dimethylthiuram disulfide ("DTD"). In another embodiment, the thiolation reagent may be phenylacetyl

disulfide ("PADS"). In another embodiment, the thiolation reagent may be xanthane hydride. In yet another embodiment, the thiolation reagent may be 3- ((dimethylamino-methylidene)amino)-3H-l,2,4-dithiazole-3-thione ("DDTT"). In still another embodiment, the thiolation reagent may be 3-ethoxy-l,2,4- dithiazoline-5-one ("EDITH"). In an additional embodiment, the thiolation reagent may be dibenzoyl tetrasulfide. In a further embodiment, the thiolation reagent may be 3-H-l,2-benzodithiol-3-one 1,1-dioxide ("Beaucage Reagent"), see Iyer et al., J. Org. Chem. 55, 4693-99 (1990). In yet a further embodiment, the thiolation reagent may be tetraethylthiuram disulfide ("TETD"). In yet another embodiment, the thiolation reagent may be 3 -phenyl- 1,2,4-dithiazoline- 5-one ("PolyOrg Sulfa" or "POS"). In still another embodiment, the thiolation reagent may be bis(0,0-diisopropoxy phosphinothioyl) disulfide

("Stec's Reagent").

Standard capping solutions for use in oligonucleotide synthesis are common and well known in the art, and their use is contemplated herein. In one embodiment, the capping solution may be a combination of a first capping solution and a second capping solution. In an additional embodiment, the first capping solution may be a solution comprising N-methylimidazole. In another embodiment, the first capping solution may further comprise pyridine and acetonitrile. By way of example and without limitation, the first capping solution may be a solution comprising N-methylimidazole, pyridine and acetonitrile in a ratio of about 2:3:5 (v:v). In another embodiment, the second capping solution may be a solution comprising an organic acid anhydride.

Suitable organic acid anhydrides include, without limitation, acetic anhydride, isobutyric anhydride, phenoxyacetic anhydride, and any combinations thereof. In another embodiment, the second capping solution may further comprising acetonitrile and/or tetrahydrofuran ("THF"). In yet another embodiment, the second capping solution may further comprise the thiolation reagent. In still another embodiment, the second capping solution in a solution of 20 % acetic anhydride, acetonitrile, and THF in a ratio of about 1:2:2 (v/v).

In an additional embodiment described herein, the thiolation reagent may be present in the second capping solution in a concentration of from

about 0.05 M up to the thiolation reagent's maximum concentration in the respective solvent. In another embodiment, the thiolation reagent may be present in the second capping solution in a concentration of from about 0.25 M to about 0.65 M. In yet another embodiment, the thiolation reagent may be present in the second capping solution in a concentration of from about 0.15 M to about 0.35 M. In still another embodiment, the thiolation reagent may be present in the second capping solution in a concentration of about 0.2 M. In a further embodiment, the thiolation reagent may be present in the second capping solution in a concentration of about 0.5, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9 and about 2 M.

In one embodiment described herein, thiolation and capping may be accomplished by contacting a solution comprising a combination of the first capping solution and the second capping solution with the support-bound oligonucleotide. In one embodiment, the solution comprising a combination of the first capping solution and the second capping solution may be prepared by combining about 85 g DTD, about 200 mL acetic anhydride, about 400 mL acetonitrile, and about 400 mL THF.

In a further embodiment described herein, the combination of the first capping solution and the second capping solution is contacted with the support column for about 0.1 minute to about 10 minutes. In another embodiment, the combination of the first capping solution and the second capping solution is contacted with the support column for about 2 minutes to about 8 minutes. In yet another embodiment, the combination of the first capping solution and the second capping solution is contacted with the support column for

about 3 minutes to about 6 minutes. In still another embodiment, the

combination of the first capping solution and the second capping solution is contacted with the support column for about 4 minutes. In an additional embodiment, the combination of the first capping solution and second capping solution is contacted with the support column for about 0.1, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 and about 10 minutes.

In another embodiment, the first and second capping solutions may, either individually or in combination, be delivered in an amount of from about

0.25-column volume to about 4-column volume. In another embodiment, the first and second capping solutions may, either individually or in combination, be delivered in an amount of from about 1.5-column volume to about 3.5-column volume. In yet another embodiment, the first and second capping solutions may, either individually or in combination, be delivered in an amount of from about 2-column volume to about 3-column volume. In still another embodiment, the first and second capping solutions may, either individually or in combination, be delivered in an amount of from about 0.25, about 0.5, about 0.75, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5 and 4-column volume.

In yet another embodiment described herein, the solid support may be washed with a washing fluid prior to the combined thiolation and capping of the support-bound oligonucleotide. In another embodiment, the solid support may be washed with a washing fluid after the combined thiolation and capping of the support-bound oligonucleotide. In yet another embodiment, the solid support may be washed with a washing fluid both prior to and after the combined thiolation and capping of the support-bound oligonucleotide. Although not required, the washing fluid used for washing prior to and/or after the combined thiolation and capping step may be the same organic solvent system used to prepare the overall reaction solutions. By way of example and without limitation, if thiolation and capping employs a combination of the first and second capping solutions, the washing fluid may contain pyridine, acetonitrile, or THF or any combinations thereof. In one embodiment, the solid support may be washed with a washing fluid comprising pyridine, acetontrile, THF and any combination thereof.

In an additional embodiment, each wash prior to and/or after the combined thiolation and capping may deliver from about 0.5-column volume to about 5-column volume of the washing fluid. In another embodiment, each wash prior to and/or after the combined thiolation and capping may deliver from about

1 -column volume to about 4-column volume of the washing fluid. In yet another embodiment, each wash prior to and/or after the combined thiolation and capping may deliver from about 1.5-column volume to about 3-column volume of the washing fluid. In a further embodiment, each wash prior to and/or after the combined thiolation and capping may deliver about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5 and about 5-column volume.

Sequential Thiolation and Capping

In one embodiment described herein, in each cycle, it may be desired to employ separate thiolation and capping steps. In another embodiment, the support-bound oligonucleotide is subjected to thiolation prior to capping.

disulfide ("PADS"). In another embodiment, the thiolation reagent may be xanthane hydride. In yet another embodiment, the thiolation reagent may be 3-((dimethylamino-methylidene)amino)-3H-l,2,4-dithiazole-3-thione ("DDTT"). In still another embodiment, the thiolation reagent may be 3-ethoxy-l,2,4- dithiazoline-5-one ("EDITH"). In an additional embodiment, the thiolation reagent may be dibenzoyl tetrasulfide. In a further embodiment, the thiolation reagent may be 3-H-l,2-benzodithiol-3-one 1,1-dioxide ("Beaucage Reagent"), see Iyer et al., J. Org. Chem. 55, 4693-99 (1990). In yet a further embodiment, the thiolation reagent may be tetraethylthiuram disulfide ("TETD"). In yet another embodiment, the thiolation reagent may be 3 -phenyl- 1,2,4-dithiazoline- 5-one ("PolyOrg Sulfa" or "POS"). In still another embodiment, the thiolation reagent may be bis(0,0-diisopropoxy phosphinothioyl) disulfide

("Stec's Reagent").

In another embodiment described herein, the phosphate triester formed in the coupling reaction may be converted to the corresponding phosphorothiolate triester due to the greater stability of the phosphorothiolate triester in vivo relative to the phosphate triester. In one embodiment, the phosphate triester may be treated with any thiolation reagent in any organic solvent. In another embodiment, the thiolation reaction may be carried out in any polar organic solvent. Suitable solvents may be but are not limited to nitrogen-containing solvents, including N-heterocycles, acetonitrile, dichloromethane,

dichloroethane, and furans, including tetrahydrofuran. In one embodiment, the solvent may be pyridine. In another embodiment, the solvent may be any substituted pyridine, such as picoline, lutidine, and collidine, and any

combinations thereof. In yet another embodiment, the thiolation reaction may be carried out in a mixture of an aprotic solvent and a protic or basic solvent. The solvent mixtures may contain suitable solvents in any desired. In one embodiment, the colvent mixture may contain suitable solvents in a ratio of about 1: 1 (v/v). Suitable solvent mixtures include but are not limited to acetontrile/pyridine, acetonitrile/picoline and acetonitrile/lutidine. Suitable aprotic solvents include but are not limited to pyridine and substituted pyridines such as picoline, lutidine, and collidine. In yet another embodiment, the thiolation reaction may be carried out in a mixture of acetonitrile and pyridine. In a further embodiment, the thiolation reaction may be carried out in a mixture of acetonitrile and picoline. In one embodiment, the thiolation reaction is carried out in a 1: 1 (v/v) mixture of acetontrile/pyridine, acetonitrile/picoline or acetonitrile/lutidine .

In an additional embodiment described herein, the thiolation reagent is present in a concentration of from about 0.05 M to about 1 M. In another embodiment, the thiolation reagent is present in a concentration of from about 0.15 M to about 0.8 M. In yet another embodiment, the thiolation reagent is present in a concentration of from about 0.2 M to about 0.6 M. In yet another embodiment, the thiolation reagent is present in a concentration of about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95 and about 1M. In still another embodiment, the thiolation reaction may employ a solution of about

0.2 M PADS.

In one embodiment, the solution containing the thiolation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 0.5 minutes to about 10 minutes. In another embodiment, the solution containing the thiolation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 1 minute to about 8 minutes. In yet another embodiment, the solution containing the thiolation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 2 minutes to about 6 minutes. In still another embodiment, the solution containing the thiolation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 3 minutes. In still another embodiment, the solution containing the thiolation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of

functionalized support for the first cycle) for about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 and about 10 minutes.

In a further embodiment, the thiolation reagent may be present in the column in an amount of about 1 to about 12 molar equivalents. In another embodiment, the thiolation reagent may be present in the column in an amount of about 2 to about 9 molar equivalents. In yet another embodiment, the thiolation reagent may be present in the column in an amount of about 3 to about 6 molar equivalents. In yet another embodiment, the thiolation reagent may be present in the column in an amount of about 6 to about 10 molar equivalents. In still another embodiment, the thiolation reagent may be present in an amount of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, and about 12 molar equivalents. As used herein, "molar equivalents" is relative to the load of the solid support.

In another embodiment described herein, the solid support may be washed with a thiolation washing fluid prior to thiolation of the support-bound oligonucleotide. In another embodiment, the solid support may be washed with a thiolation washing fluid after thiolation of the support-bound oligonucleotide. In yet another embodiment, the solid support may be washed with a thiolation washing fluid both prior to and after thiolation of the support-bound

oligonucleotide. Although not required, the thiolation washing fluid used for washing prior to and/or after thiolation may be the same organic solvent system used to prepare the thiolation solution. By way of example and without limitation, if thiolation employs PADS in a solution of acetonitrile and picoline, the column may be washed with an acetonitrile/picoline solution prior to and/or after thiolation. In one embodiment, the solid support may be washed with a solution comprising acetonitrile and picoline in any relative amount. In another embodiment, the solid support may be washed with solution of acetonitrile and pyridine in any relative amount. In another embodiment, the solid support may be washed with a solution of acetonitrile and lutidine in any relative amount. In another embodiment, the solid support may be washed with picoline, lutidine, pyridine and any combinations thereof.

In yet another embodiment, each wash prior to and/or after thiolation may deliver from about 0.5-column volume to about 10-column volume of the thiolation washing fluid. In another embodiment, each wash prior to and/or after thiolation may deliver from about 1 -column volume to about 8-column volume of the thiolation washing fluid. In yet another embodiment, each wash prior to and/or after thiolation may deliver from about 3 -column volume to about

7-column volume of the thiolation washing fluid. In yet another embodiment, each wash prior to and/or after thiolation may deliver from about 4-column volume to about 7-column volume of the thiolation washing fluid. In yet another embodiment, each wash prior to and/or after thiolation may deliver from about 6-column volume to about 7-column volume of the thiolation washing fluid. In still another embodiment, each wash prior to and or after thiolation may deliver about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 and about 10-column volume of the thiolation washing fluid.

In still another embodiment described herein, washing the support column prior to and/or after thiolation may decrease undesired pressure increases in the support column. An increased pressure in the column support may block the flow of solvents and reagents through the support column, thereby decreasing the efficiency of the oligonucleotide synthesis. In one embodiment, thiolation washes may be employed as desired. By way of example and without limitation, thiolation washes may be employed during each cycle of the oligonucleotide synthesis. In another embodiment, thiolation washes may be employed before, during, and/or after cycles in which an undesired increase in column pressure is observed. In another embodiment, thiolation washes may not be employed at all.

In another embodiment described herein, the capping reaction may employ, either alone or in combination, the first capping solution and the second capping solution, as previously set forth. In one embodiment, the first capping solution may comprise N-methylimidazole, pyridine, and acetonitrile in a ratio of about 2:3:5 volume by volume. In another embodiment, the second capping solution may contain 20 % acetic anhydride in acetonitrile. In yet another embodiment, any common capping reagents may be utilized.

In a further embodiment described herein, the first and second capping solutions may be combined and pumped through the support column. In one embodiment, the capping solutions, either alone or in combination, may be contacted with the support column containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 0.1 minutes to about 10 minutes. In another embodiment, the capping solutions, either alone or in combination, may be contacted with the support column containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 2 minute to about 8 minutes. In yet another embodiment, the capping solutions, either alone or in combination, may be contacted with the support column containing the support-bound oligonucleotide (or nucleoside, linker or other type of

functionalized support for the first cycle) for about 3 minutes to about 6 minutes. In still another embodiment, the capping solutions, either alone or in

combination, may be contacted with the support column containing the support- bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 4 minutes. In still another embodiment, the capping solutions, either alone or in combination, may be contacted with the support column containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 0.1, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 and about 10 minutes.

In one embodiment, the capping solutions, either alone or in combination, may be used in an amount of from about 0.5-column volume to about 10-column volume. In another embodiment, the capping solutions, either alone or in combination, may be used in an amount of from about 1 -column volume to about 8-column volume. In yet another embodiment, the capping solutions, either alone or in combination, may be used in an amount of from about 3-column volume to about 7-column volume. In yet another embodiment, the capping solutions, either alone or in combination, may be used in an amount of from about 4-column volume to about 6-column volume. In yet another embodiment, the capping solutions, either alone or in combination, may be used in an amount of about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 and about 10-column volume.

Oxidation

Due to the relative instability of phosphite triesters in vivo, it may be desirable to oxidize the phosphite triesters formed during the coupling reaction into the more stable phosphate triesters. In one embodiment, pressure increases in the solid- support column may be observed upon oxidation of phosphite triesters to phosphate triesters.

In one embodiment, the oxidation reagent may be any desired oxidation reagent. By way of example and without limitation, the oxidation reagent may be iodine, iodobenzene diacetate, tetrabutylammonium periodate, TMSOOTMS, hydrogen peroxide, tert-butyl hydroperoxide, cumene peroxide, di-tert-butyl peroxide, trimethylamine N-oxide, N-methylmorpholine-N-oxide, pyridine N-oxide, and dimethylsulfoxide.

In a further embodiment, the oxidation reagent may be present in the column in an amount of about 0.1 to about 12 molar equivalents. In another embodiment, the oxidation reagent may be present in the column in an amount of about 0.5 to about 9 molar equivalents. In yet another embodiment, the oxidation reagent may be present in the column in an amount of about 3 to about 6 molar equivalents. In yet another embodiment, the oxidation reagent may be present in the column in an amount of about 6 to about 10 molar equivalents. In still another embodiment, the oxidation reagent may be present in an amount of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, and about 12 molar equivalents.

In a further embodiment, the oxidation reaction may be carried out in any suitable solvent. In one embodiment, the solvent may be aqueous. In another embodiment, the solvent may be non- aqueous. Suitable solvents may be but are not limited to any nitrogen-containing compound, including nitrogen

heterocycles, any oxygen-containing compound, including oxygen heterocycles. In one embodiment, the oxidation reaction may be carried out in a mixture of pyridine and water in any ratio. In another embodiment, the oxidation reaction may be carried out in a mixture of pyridine, THF and water in any ratio. In yet another embodiment, the oxidation reaction may be carried out picoline and water in any ratio. In a further embodiment, the oxidation reaction may be carried out in lutidine and water in any ratio. In still a further embodiment, the oxidation reaction may be carried out in collidine and water in any ratio. In an additional embodiment, the oxidation may be carried out in pyridine, picoline, lutidine, collidine and any combination thereof. In one embodiment, the oxidation reaction may be carried out in a solution comprising acetonitrile and any of the foregoing solvents.

In an additional embodiment described herein, the oxidation reagent is present in a concentration of from about 0.01 M to about 1 M. In another embodiment, the oxidation reagent is present in a concentration of from about 0.15 M to about 0.8 M. In yet another embodiment, the oxidation reagent is present in a concentration of from about 0.2 M to about 0.6 M. In yet another embodiment, the oxidation reagent is present in a concentration of about 0.01, about, 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95 and about 1M.

In a further embodiment, the oxidation reaction may employ a solution of about 0.05 M iodine in a 10/90 (v/v) solution of water in pyridine. In a further embodiment, the oxidation reaction may employ a solution of from about 0.01 M to about 0.1 M iodine in a 10/90 (v/v) solution of water in pyridine.

In one embodiment, the solution containing the oxidation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 0.5 minutes to about 10 minutes. In another embodiment, the solution containing the oxidation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 1 minute to about

8 minutes. In yet another embodiment, the oxidation containing the thiolation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 2 minutes to about 6 minutes. In still another embodiment, the solution containing the oxidation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of functionalized support for the first cycle) for about 3 minutes. In still another embodiment, the solution containing the oxidation reagent may be contacted with the column support containing the support-bound oligonucleotide (or nucleoside, linker or other type of

In another embodiment described herein, the solid support may be washed with an oxidation washing fluid prior to oxidation of the support-bound oligonucleotide. In another embodiment, the solid support may be washed with an oxidation washing fluid after oxidation of the support-bound oligonucleotide. In yet another embodiment, the solid support may be washed with an oxidation washing fluid both prior to and after oxidation of the support-bound

oligonucleotide. Although not required, the oxidation washing fluid used for washing prior to and/or after oxidation may be the same solvent system used to prepare the oxidation solution. By way of example and without limitation, if oxidation employs 12 in a 10 % solution of water in pyridine, the column may be washed with an aqueous solution of pyridine prior to and/or after thiolation. In one embodiment, the solid support may be washed with a solution comprising water and pyridine in any relative amount. In another embodiment, the solid support may be washed with solution of pyridine, picoline, lutidine, collidine, and any combinations thereof.

In yet another embodiment, each wash prior to and/or after oxidation may deliver from about 0.5-column volume to about 10-column volume of the oxidation washing fluid. In another embodiment, each wash prior to and/or after oxidation may deliver from about 1 -column volume to about 8-column volume of the oxidation washing fluid. In yet another embodiment, each wash prior to and/or after oxidation may deliver from about 3 -column volume to about 7-column volume of the oxidation washing fluid. In yet another embodiment, each wash prior to and/or after oxidation may deliver from about 4-column volume to about 7-column volume of the oxidation washing fluid. In yet another embodiment, each wash prior to and/or after oxidation may deliver from about 6-column volume to about 7-column volume of the oxidation washing fluid. In still another embodiment, each wash prior to and or after oxidation may deliver about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 and about 10-column volume of the oxidation washing fluid.

In still another embodiment described herein, washing the support column prior to and/or after oxidation may decrease undesired pressure increases in the support column. An increased pressure in the column support may block the flow of solvents and reagents through the support column, thereby decreasing the efficiency of the oligonucleotide synthesis. In one embodiment, oxidation washes may be employed as desired. By way of example and without limitation, oxidation washes may be employed during each cycle of the oligonucleotide synthesis. In another embodiment, oxidation washes may be employed before, during, and/or after cycles in which an undesired increase in column pressure is observed. In another embodiment, oxidation washes may not be employed at all. Cleavage from Support Column

In an additional embodiment described herein, once a desired

oligonucleotide has been synthesized by any of the above processes, the oligonucleotide may be cleaved from the solid support and deprotection of the oligonucleotide backbone and nucleobases may be achieved by any desired means, as is commonly known in the art. In one embodiment, cleavage from the solid support and deprotection of the oligonucleotide backbone and nucleobases may be achieved by incubation with ammonium hydroxide at from about 40°C to about 65°C for up to 48 hours. In another embodiment, cleavage from the solid support and deprotection of the oligonucleotide backbone and nucleobases may be achieved by incubation with ammonium hydroxide at from about 45 °C to about 60°C for up to 48 hours. In yet another embodiment, cleavage from the solid support and deprotection of the oligonucleotide backbone and nucleobases may be achieved by incubation with ammonium hydroxide at from about 50°C to about 55°C for up to 48 hours.

In another embodiment described herein, once the desired oligonucleotide has been cleaved from the support column, the support may then be filtered and washed with any suitable solvent. In one embodiment, the support may be filtered with a solution of ethanol in water (1: 1 v/v). The combined filtrate and washings may then be concentrated to yield a crude 5 '-protected oligonucleotide solution, which may then be purified and characterized by any desired means, including without limitation reverse-phase high performance liquid

chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS). In yet another embodiment, the 5 '-terminus of the support-bound oligonucleotide may be deprotected prior to cleavage from the support column.

In a further embodiment described herein, the oligonucleotide may be used for therapeutic applications. In another embodiment, the oligonucleotide may be used for diagnostic applications. In yet another embodiment, the oligonucleotide may be used for research applications.

Should the disclosure of any patents, patent applications, and publications which are incorporated herein by reference conflict with the description of the present application to the extent that it may render a term unclear, the present description shall take precedence.

EXAMPLES

Example 1 - Synthesis of CPG7909 (SEQ ID NO:l)

General procedure. The synthesis of the phosphorothioate of

SEQ ID NO: l was performed on an Akta 100 synthesizer using a FL35 column having a 35 mm diameter FineLine 35 fixed bed design. The synthesis was carried out at a 4 mmol scale using 1.85 equivalents of commercially available phosphoramidites at a concentration of 0.170 M and a NittoPhase^® HL support having a loading of 350 μιηοΐ/g. Separate detritylation and thiolation steps were employed to synthesize SEQ ID NO: l. During detritylation, the 5'-terminus protecting groups were removed by treatment with 20 % DC A in toluene.

During thiolation, the phosphate triesters were thiolated to phosphorothiolate triesters by treating with a 0.2 M solution of PADS in a 1: 1 (v/v) acetonitrile and picoline. Upon completion of the synthesis on the solid support, the terminal 5'-0-4,4-dimethoxytrityl (DMTr) protecting group was removed from the support-bound oligonucleotide by treatment with DC A. The solid support containing the deprotected oligonucleotide was then treated with concentrated aqueous ammonium hydroxide (-30 % by weight in water) at about 55°C for 16 hours to cleave the oligonucleotide from the solid support. The oligonucleotide was then analyzed and characterized by RP-HPLC and LC/MS.

Three batches of oligonucleotides were synthesized and analyzed. The results are described below and summarized in Table 1.

Batch 1. Synthesis of the first batch of oligonucleotides of SEQ ID NO: 1 did not employ any washes prior to and/or after the detritylation and/or thiolation steps. Spikes of high pressures in the support column were observed throughout both the detritylation and thiolation reaction steps. This undesirable increase in pressure resulted in a higher than expected phosphodiester content and lower than expected full-length product content.

Batch 2. Synthesis of the second batch of oligonucleotides began with a large increase in pressure during the first detritylation cycle. Accordingly, the method was modified to reduce the P-Flow of the detritylation reagent during each detritylation step until about 1.2-column volume had passed through the support column, after which the P-Flow was increased to 400 cm/h. This reduction in P-Flow during the detritylation step increased contact time between the detritylation reagent and the support-bound oligonucleotide. Although a uniform contact time was not achieved for each detritylation step, this modification initially eliminated undesired pressure increases. However, large pressure increases were again observed during the detritylation step of the 20^th cycle. Slowing the flow rate of detritylation reagent increased the content of the full length product and decreased the phosphodiester content.

Additionally, high pressure spikes were observed beginning during the thiolation step of the 17th cycle. To compensate for this undesired increase in pressure, the height of the column bed was adjusted to 11.2 cm during the 20^th cycle to allow continuation of the flow of thiolation reagent. Reversing the direction of flow of the thiolation reagent did not decrease the undesired pressure.

Batch 3. The support column was packed with toluene. A spike of high pressure was observed during the first cycle. Consequently, the synthesis was modified to condition the support column with about 1 -column volume of toluene prior to delivery of the detritylation reagent and a second about 1 -column volume of toluene after delivery of the detritylation reagent. These toluene washings alleviated the occurrence of high pressure spikes during the

detritrylation process until the 23rd cycle. The method was similarly modified to condition the support column with about 1 -column volume of picoline at a flow of 227 cm/hr prior to delivery of the PADS reagent and about 1 -column volume after thiolation. In view of this modification, high pressure spikes were not observed during the thiolation process. Consequently, it has been shown that washing the support column with (i) toluene prior to and/or after detritylation and/or (ii) picoline prior to and after thiolation alleviates undesirable pressure build up in the support column during the synthesis of oligonucleotides.

Although these modifications alleviated the problems associated with the observed pressure increases in the absence of the washings prior to and/or after detritylation and/or thiolation, the overall yield of the full-length product decreased. For example, while about 77 % and 82 % of the content of oligonucleotides synthesized in the first and second batches were full length, respectively, only about 75 % of the synthesized oligonucleotides synthesized in the third batch were full length. This decrease in yield of the desired full-length oligonucleotide was likely a result of incomplete detritylation. Accordingly, the reaction parameters of the detritylation process may be adjusted in order to optimize the yield of the desired full length oligonucleotide.

TABLE 1

Example 2 - Synthesis of \6-mer LNA/DNA gapmer (SEQ ID NO:2)

General procedure. The synthesis of the phosphorothioate of

SEQ ID NO:2 was performed on an Akta 100 synthesizer using a FL35 column having a 35 mm diameter FineLine 35 fixed bed design. The synthesis was carried out at a 2.75 mmol scale using commercially available phosphoramidites and a UnyLinker™ NittoPhase^® support having a loading of about 200 μιηοΐ/g. Separate detritylation and thiolation steps were employed to synthesize

SEQ ID NO:2. During detritylation, the 5 '-terminus protecting groups were removed by treatment of 3 % DCA in toluene. During thiolation, the phosphate triesters were thiolated to phosphorothiolate triesters by treating with a 0.2 M solution of xanthane hydride in a solution of pyridine. Upon completion of the synthesis on the solid support, the terminal 5'-0-4,4-dimethoxytrityl (DMTr) protecting group was removed from the support-bound oligonucleotide by treatment with dichloroacetic acid. The solid support containing the deprotected oligonucleotide was then treated with concentrated aqueous ammonium hydroxide (-30 % by weight in water) at about 55°C for 16 hours to cleave the oligonucleotide from the solid support. The oligonucleotide was then analyzed and characterized by RP-HPLC and LC/MS.

Initially, the synthesis of the oligonucleotides of SEQ ID NO:2 employed only acetonitrile washes prior to or after the detritylation and thiolation reactions. While detritylation did not cause any undesired increases in pressure, pressure build up was observed during the thiolation reaction after about 10 cycles. For example, after addition of the 12^th base, the pressure during thiolation rose rapidly to about 18 bar. Upon thiolation of the 13^th base, the pressure limit was exceeded. To compensate for this increased pressure, the flow rate was reduced during the thiolation reaction to about 25 mL/min. However, the pressure continued to increase during thiolation of the 14^th cycle, wherein the flow rate of the thiolation solution was further decreased to about 20 mL/min. For the last two cycles, the method was modified to include washing the support column with a mixture of pyridine and acetonitrile (1: 1 v/v) prior to and after thiolation with xanthane hydride. These washings alleviated the pressure build up, and no further pressure issues were observed during the last two cycles. These results are summarized in Table 2 and Figure 1. Consequently, it has been shown that washing the column with a solution of pyridine and acetonitrile prior to and/or after thiolation alleviates undesirable pressure build up in the support column during the synthesis of oligonucleotides.

TABLE 2

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The use of the terms "a," "an" and "the" and similar references in the context of this disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as, preferred, preferably) provided herein, is intended merely to further illustrate the content of the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

Alternative embodiments of the claimed disclosure are described herein, including the best mode known to the inventors for practicing the claimed invention. Of these, variations of the disclosed embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing disclosure. The inventors expect skilled artisans to employ such variations as appropriate (e.g., altering or combining features or embodiments), and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of individual numerical values are stated as approximations as though the values were preceded by the word "about" or "approximately." Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about" or "approximately." In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms "about" and "approximately" when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered include the criticality of the element and/or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numerical values is not meant to limit how the use of the words "about" or "approximately" will serve to broaden a particular numerical value or range. Thus, as a general matter, "about" or "approximately" broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term "about" or "approximately." Thus, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

It is to be understood that any ranges, ratios and ranges of ratios that can be formed by, or derived from, any of the data disclosed herein represent further embodiments of the present disclosure and are included as part of the disclosure as though they were explicitly set forth. This includes ranges that can be formed that do or do not include a finite upper and/or lower boundary. Accordingly, a person of ordinary skill in the art most closely related to a particular range, ratio or range of ratios will appreciate that such values are unambiguously derivable from the data presented herein.

REFERENCES

NittoPhase^® Solid Support for Small to Large Scale Oligonucleotide Synthesis, Product Brochure from Kinovate Life Sciences.

UnyLinker™ NittoPhase^® Solid Support, Product Brochure from Kinovate Life Sciences.

Custom Oligonucleotide Synthesis, available at

www.bio.da.vidson.edii/Courses/Molbio/Mol.Students/Spring2003/

Holmberg/oligonucleotide_synthesis.htm.

Moore, M.N., et al., Overcoming Backpressure Problems during Solid-Phase Synthesis of Oligonucleotides, Organic Process Research & Development, Vol. 8, p. 271-74 (1994).

Wang, et al., Three-Step Cycle Automated Synthesis Using Phosphoramidite Chemistry and DTD, p. 52-55. Wang, et al., Four-Step Cycle Automated Synthesis Using Phosphoramidite Chemistry and PADS, p. 55-57.

U.S. Publication No. 2008/139797A

JP Publication No. 2009/280753A

U.S. Patent No. 6,538,128

U.S. Patent No. 6,040,438

U.S. Publication No. 2005/0165226

WO 01/51502

U.S. Patent No. 7,227,015

Kataoka, M., et al., Ethyl(methyl)dioxirane as as Efficient Reagent for the

Oxidation of Nucleoside Phosphites into Phosphates Under Nonbasic Anhydrous Conditions, Org. Lett., Vol. 3, p.815-18 (2001).

Uzagare, M.C., et al., NBS-DMSO as a Nonaqueous Nonbasic Oxidation Reagent for the Synthesis of Oligonucleotides, Bioorganic & Medicinal Chem. Lett., Vol. 13, p.3537-40 (2003).

Cvetovich, R., Hydrogen Peroxide Oxidation of Phosphite Triesters in

Oligonucleotide Syntheses, Organic Process Research & Development, Vol. 14, p.295-97 (2010).

Iyer et al., J. Org. Chem. 55, 4693-99 (1990).

U.S. Patent No. 6,399,765

U.S. Patent No. 6,795,402

U.S. Patent No. 7,273,933

Singh, Y., et al., Recent Developments in Oligonucleotide Conjugation, Chemical Society Reviews, Vol. 39, p. 2054-70 (2010).

U.S. Patent No. 6,465,628

SEQUENCE LISTING

SEQ ID NO: l 5'-TCG TCG TTT TGT CGT TTT GTC GTT-3' (DNA bases) SEQ ID NO:2 Ιβ-mer LNA/DNA gapmer

Claims

C L A I M S

1. A method of reducing pressure in a solid- support column during synthesis, the method comprising : a. providing a solid support in a column, wherein the solid support comprises a linker, a nucleoside, an oligonucleotide or other type of functional group attached to the solid support ; b. contacting the solid support with a first volume of a washing fluid prior to a modification of the nucleoside or oligonucleotide ; c. contacting the solid support with a volume of solution comprising a reagent to effect the modification of the nucleoside or oligonucleotide for an amount of time sufficient to achieve the modification ; and d. contacting the solid support with a second volume of the washing fluid after the modification of the nucleoside or oligonucleotide.

2. The method of claim 1, wherein the solid support swells upon a change in solvent in the solid- support column.

3. The method of claim 1 or 2, wherein the washing fluid comprises an alkyl-substituted arene solvent.

4. The method of claim 3, wherein the washing fluid comprises toluene.

5. The method of anyone of claims 1 to 4, wherein the amount of the first and second volume of the washing fluid is individually about 0.25-column volume to about 4-column volume.

6. The method of anyone of claims 1 to 5, wherein the washing fluid is a thiolation washing fluid used prior to and after thiolation of the nucleoside or oligonucleotide.

7. The method of claims 1 to 5, wherein the washing fluid is a detritylation washing fluid used prior to and after detritylation of the nucleoside or oligonucleotide.

8. A method of reducing pressure in a solid-support column during thiolation of a phosphotriester linkage of a support-bound nucleoside of oligonucleotide, the method comprising : a. providing a solid support in a column, wherein the solid support comprises a linker, a nucleoside, an oligonucleotide or other type of functional group attached to the solid support ; b. contacting the solid support with a first volume of a thiolation washing fluid prior to thiolation ; c. contacting the solid support with a volume of thiolation solution comprising a thiolation reagent for an amount of time sufficient to achieve thiolation ; and d. contacting the solid support with a second volume of the thiolation washing fluid after thiolation.

9. The method of claim 8, wherein the thiolation washing fluid comprises an alky 1- substituted arene solvent.

10. The method of claim 9, wherein the alky 1- substituted arene solvent is selected from the group consisting of picoline and lutidine.

11. The method of claim 9, wherein the thiolation washing fluid further comprises acetonitrile.

12. The method of anyone of claims 8 to 11, wherein the thiolation reagent comprises PADS.

13. The method of anyone of claims 8 to 12, wherein the amount of the first and second volume of the thiolation washing fluid is individually about 0.25-column volume to about 4-column volume.

14. The method of anyone of claims 8 to 13, wherein the solid support swells upon a change in solvent in the solid-support column.

15. A method of reducing pressure in a solid-support column during detritylation of a 5 '-terminus of a support-bound nucleoside of oligonucleotide, the method comprising : a. providing a solid support in a column, wherein the solid support comprises a linker, a nucleoside, an oligonucleotide or other type of functional group attached to the solid support ; b. contacting the solid support with a first volume of a detritylation washing fluid prior to detritylation ; c. contacting the solid support with a volume of detritylation solution

comprising a detritylation reagent for an amount of time sufficient to achieve detritylation ; and d. contacting the solid support with a second volume of the detritylation

washing fluid after detritylation.

16. The method of claim 15, wherein the detritylation washing fluid comprises an alkyl-substituted arene solvent.

17. The method of claim 16, wherein the alkyl-substituted arene solvent comprises toluene.

18. The method of anyone of claims 15 to 17, wherein the detritylation reagent comprises a haloacetic acid.

19. The method of claim 18, wherein the haloacetic acid comprises dichloroacetic acid.

20. The method of anyone of claims 15 to 19, wherein the amount of the first and second volume of the detritylation washing fluid is individually about

0.25-column volume to about 4-column volume.

21. The method of anyone of claims 15 to 20, wherein the solid support swells upon a change in solvent in the solid-support column.

22. A process for manufacturing an oligonucleotide which comprises reducing a solid-support column pressure, wherein said reducing comprises : a. providing a solid support in a column, wherein the solid support comprises a linker, a nucleoside, an oligonucleotide or other type of functional group attached to the solid support ; b. contacting the solid support with a first volume of a thiolation washing fluid prior to thiolation ; c. contacting the solid support with a volume of thiolation solution comprising a thiolation reagent for an amount of time sufficient to achieve thiolation ; and d. contacting the solid support with a second volume of the thiolation washing fluid after thiolation.

23. The method of claim 22, wherein the thiolation washing fluid comprises an alkyl-substituted arene solvent.

24. The method of claim 23, wherein the alkyl-substituted arene solvent is selected from the group consisting of picoline and lutidine.

25. The method of claim 23 or 24, wherein the thiolation washing fluid further comprises acetonitrile.

26. The method of anyone of claims 22 to 25, wherein the thiolation reagent comprises PADS.

27. The method of anyone of claims 22 to 26, wherein the amount of the first and second volume of the thiolation washing fluid is individually about 0.25-column volume to about 4-column volume.

28. The method of anyone of claims 22 to 27, wherein the solid support swells upon a change in solvent in the solid-support column.

29. The method of anyone of claims 22 to 28, wherein the column pressure increases above about 10 bar during thiolation.

30. The method of anyone of claims 22 to 29, wherein the column pressure increases above about 10 bar during detritylation.

31. A process for manufacturing an oligonucleotide which comprises reducing a solid-support column pressure, wherein said reducing comprises : a. providing a solid support in a column, wherein the solid support comprises a linker, a nucleoside, an oligonucleotide or other type of functional group attached to the solid support reto ; b. contacting the solid support with a first volume of a detritylation washing fluid prior to detritylation ; c. contacting the solid support with a volume of detritylation solution

washing fluid after detritylation.

32. The method of claim 31, wherein the detritylation washing fluid comprises an alkyl-substituted arene solvent.

33. The method of claim 32, wherein the alkyl-substituted arene solvent comprises toluene.

34. The method of anyone of claims 31 to 33, wherein the detritylation reagent comprises a haloacetic acid.

35. The method of claim 34, wherein the haloacetic acid comprises dichloroacetic acid.

36. The method of anyone of claims 31 to 35, wherein the amount of the first and second volume of the detritylation washing fluid is individually about

0.25-column volume to about 4-column volume.

37. The method of anyone of claims 31 to 36, wherein the solid support swells upon a change in solvent in the solid-support column.

38. The method of anyone of claims 31 to 37, wherein the column pressure increases above about 10 bar during thiolation.

39. The method of anyone of claims 31 to 38, wherein the column pressure increases above about 10 bar during detritylation.