HK40003927A - Enhanced coupling of stereodefined oxazaphospholidine phosphoramidite monomers to nucleoside or oligonucleotide - Google Patents

Description

Enhanced coupling of sterically defined oxaazaphospholane phosphoramidite monomers to nucleosides or oligonucleotides

Technical Field

The present invention relates to the field of sterically defined (steredofined) phosphorothioate oligonucleotides and to sterically defined nucleoside monomers and methods of using the monomers to synthesize sterically defined oligonucleotides. Disclosed herein are solvent compositions that provide enhanced solubility and stability to sterically defined nucleoside monomers and can be used to improve the coupling efficiency of these monomers in oligonucleotide synthesis.

Background

It has recently become apparent that the use of sterically defined phosphorothioate internucleoside linkages in oligonucleotides can optimise the pharmacological profile of therapeutic oligonucleotides. However, current preparation of stereodefined phosphorothioate oligonucleotides is relatively inefficient compared to non-stereodefined phosphorothioate oligonucleotides. There is therefore a need to improve the efficiency of synthesis of sterically defined oligonucleotides.

Wan et al, Nucleic Acids Research (advanced Access, published 11/14/2014) discloses a method for the synthesis of (S) cET Gapmer antisense oligonucleotides containing a chiral phosphorothioate linkage within the DNA gap region. The oligonucleotide prepared by Wan et al incorporates oxaazaphospholidine (oxazaphosphoridine) DNA monomers into an (S) cET Gapmer. The DNA amidate was prepared as a 0.2M solution in acetonitrile/toluene (1:1v/v) and coupled using a double coupling step. The (S) cET monomer is a standard (non-stereo defined) amidate.

WO2014/010250 discloses nucleoside monomers which, when incorporated into an oligonucleotide, provide chirally oriented stereocenters at the corresponding phosphorothioate internucleoside linkage positions. The coupling step reported in WO2014/010250 was carried out in acetonitrile.

In some embodiments, the invention is based on the following observations: oxaazaphospholane phosphoramidite monomers may be difficult to dissolve in many solvents and may be unstable even when dissolved so as to limit the ability to prepare sterically defined oligonucleotides to commercially relevant scales.

In addition to being able to provide a suitable stable solution of the oxaazaphospholane phosphoramidite monomer, the present invention is based on the following findings: the oxaazaphospholane phosphoramidite monomer solution can lead to relatively inefficient coupling during oligonucleotide synthesis.

By using an aromatic heterocyclic solvent in acetonitrile, the inventors have found that the solubility, stability and/or reactivity of the oxaazaphospholane phosphoramidite monomer can be improved.

Description of the invention

The present invention provides a method of coupling an oxaazaphospholane phosphoramidite monomer to the 5' -end of a nucleoside or oligonucleotide comprising the step of reacting the nucleoside or oligonucleotide with the oxaazaphospholane phosphoramidite monomer, wherein the reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent. The coupling method of the invention may be added to the oligonucleotide synthesis method.

The invention provides a method for synthesizing a stereospecifically defined phosphorothioate oligonucleotide, comprising the following steps:

a) deprotecting the protected 5' -hydroxy terminus of a nucleoside or oligonucleotide attached to a solid support,

b) coupling an oxaazaphospholane phosphoramidite monomer to the deprotected 5' -hydroxy terminus of a nucleoside or oligonucleotide, wherein the coupling reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent to form a phosphite triester intermediate, and

c) oxidizing the phosphite triester intermediate with a vulcanizing agent,

d) Optionally repeating steps a) -c) for one or more further extension cycles,

e) the oligonucleotide is deprotected from the solid support and cleaved.

The method of the invention may comprise a plurality of further extension cycles of step d), for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more further extension cycles.

The present invention provides a method of coupling an oxaazaphospholane phosphoramidite monomer to the 5' -end of a nucleoside or oligonucleotide or to a hydroxyl group (e.g., a linker) attached to a solid support, comprising the step of reacting the nucleoside, oligonucleotide or solid support with the oxaazaphospholane phosphoramidite monomer, wherein the reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent.

The present invention provides a method of coupling an oxaazaphospholane phosphoramidite monomer to the 5' -end of a nucleoside or oligonucleotide or to a hydroxyl group (e.g., linker) attached to a solid support, comprising the step of reacting the nucleoside, oligonucleotide or solid support with the oxaazaphospholane phosphoramidite monomer, wherein the reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent and an activator.

The invention provides a method for synthesizing oligonucleotides, which comprises a method for coupling oxaazaphospholane phosphoramidite monomers to the 5' -end of the nucleosides or oligonucleotides.

The present invention provides an acetonitrile solution composition comprising an oxaazaphospholane phosphoramidite monomer, acetonitrile and an aromatic heterocyclic solvent.

The present invention provides a method of dissolving an oxaazaphospholane phosphoramidite monomer comprising adding said monomer to a solvent composition comprising acetonitrile and an aromatic heterocyclic solvent, and optionally an activator.

The present invention provides the use of an aromatic heterocyclic solvent to enhance the stability and/or solubility of oxaazaphospholane phosphoramidites in acetonitrile.

The present invention provides the use of aromatic heterocyclic solvents to enhance reactivity, for example, the reactivity of oxaazaphospholane phosphoramidites in acetonitrile in an oligonucleotide synthesis coupling step.

As shown in the examples, the use of the solvent composition of the present invention (also referred to as acetonitrile and aromatic heterocyclic solvent composition) enhances the solubility and stability of oxaazaphospholane phosphoramidite monomers, which may lead to improved utility in oligonucleotide synthesis. In some embodiments, the oxaazaphospholane phosphoramidite monomer is soluble in the solvent composition for a period of at least 24 hours. The invention further provides a solution of an oxaazaphospholane phosphoramidite monomer comprising the monomer and the acetonitrile solvent composition of the invention (acetonitrile and aromatic heterocyclic solvent composition). In some embodiments, the solution of the oxaazaphospholane phosphoramidite monomer is stable for at least 24 hours.

Drawings

FIG. 1: stability of various L and D nucleoside monomers in solvent selection. 3 is quite unstable, 2 is moderately stable, and 1 is quite stable.

FIG. 2: solubility of various L and D nucleoside monomers in solvent selection.

FIG. 3: the stability of the L-LNA-G-iBu monomer (3a) and the L-LNA-G-DMF monomer measured after 24 hours in various solvents (see example 6).

FIG. 4: the addition of 5% pyridine to acetonitrile solvent reduced the coupling efficiency of conventional phosphoramidites.

FIG. 5: stability of L-LNA-A with and without triethylamine. Triethylamine stabilizes the L-LNA a monomer.

FIG. 6: relative coupling efficiencies in model systems using stereospecified L-LNA-a oxaazaphospholane phosphoramidite monomers and various different amine bases.

FIG. 7: relative coupling efficiency in a model system using different oxaazaphospholane phosphoramidite monomers in various solvents. Further testing of additional monomers showed that the solubility enhancing effect of the addition of pyridine was universal across a range of monomers. As in the case of D-LNA A, D-DNA A and L-DNA A, these monomers are insoluble after 24 hours in MeCN. However, the solubility of the monomer is maintained after the addition of pyridine. An increase in the reactivity of D-DNA A and L-LNA T is also observed when L-DNA A and D-LNA A react in a similar manner.

FIG. 8: conversion of full length product with and without 2.5% pyridine.

FIG. 9: theoretical yield (%) of-13 mer with and without pyridine.

FIG. 10: theoretical yield (%) of-16 mer with and without pyridine.

FIG. 11: conversion to full length product in the absence of pyridine, in the presence of 100% pyridine solvent and 2.5% pyridine, indicates that 2.5% pyridine results in conversion rates close to those achieved with non-stereospecific phosphoramide couplings.

FIG. 12: exemplary oxaazaphospholane phosphoramidite DNA monomers M1-M8. Ac ═ acetyl protecting group, Bz ═ benzoyl protecting group.

FIG. 13: exemplary oxaazaphospholane phosphoramidite DNA monomers M9-M16, wherein R¹Methyl group; ac ═ acetyl protecting group, Bz ═ benzoyl protecting group.

FIG. 14: exemplary oxaazaphospholane phosphoramidite LNA monomers M17-M24. Ac ═ acetyl protecting group, Bz ═ benzoyl protecting group.

FIG. 15: exemplary oxaazaphospholane phosphoramidite LNA monomers M25-M32; wherein R is¹(ii) methyl; ac ═ acetyl protecting group, Bz ═ benzoyl protecting group.

FIG. 16: exemplary oxaazaphospholane phosphoramidite LNA monomers M32-M40, where R ¹Selected from hydrogen and methyl; r_eIs methyl, which may be in S or R configuration, preferably in S configuration ((S) Cet), Ac ═ acetyl protecting group, Bz ═ benzoyl protecting group.

FIG. 17: exemplary oxaazaphospholane phosphoramidite DNA monomers (formulas 33-40). A ═ adenine, which may optionally be protected, for example, by acetyl or benzoyl; t is thymine; c ═ cytosine, optionally 5-methylcytosine, cytosine or 5-methylcytosine optionally protected, for example, by benzoyl or acetyl; g ═ guanine, which may optionally be protected, for example, with an acyl group, such as iBu or DMF; r³Selected from the following groups: CH (CH)₂ODMTr、CH₂alkyl-O-DMTr, CH-Me-O-DMTr, CH₂OMMTr、CH₂alkyl-O-MMTr, CH (Me) -O-MMTr, CH-R^a-O-DMTrR^bAnd CH-R^a-O-MMTrR^bIs preferably-CH₂-O-DMTr; r is aryl, preferably phenyl; r¹Is hydrogen or methyl; r⁹Is hydrogen.

FIG. 18: exemplary oxaazaphospholane phosphoramidite LNA monomers (formulas 41-48). A ═ adenine, which may optionally be protected, for example, by acetyl or benzoyl; t is thymine; c ═ cytosine, optionally 5-methylCytosine, cytosine or 5-methylcytosine may optionally be protected, for example, by benzoyl or acetyl; g-guanine, which may optionally be protected, e.g., with an acyl group, e.g., iBu for L-LNA-G monomers or an acyl group (e.g., iBu) for D-LNA-G monomers or DMF; r ³Selected from the following groups: CH (CH)₂ODMTr、CH₂alkyl-O-DMTr, CH-Me-O-DMTr, CH₂OMMTr、CH₂alkyl-O-MMTr, CH (Me) -O-MMTr, CH-R^a-O-DMTrR^bAnd CH-R^a-O-MMTrR^bIs preferably-CH₂-O-DMTr; r is aryl, preferably phenyl; r¹Is hydrogen or methyl; r⁹Is hydrogen.

FIG. 19: relative coupling efficiency in a model system using various oxaazaphospholane phosphoramidite monomers in acetonitrile with or without 2.5% pyridine. The figure illustrates that the coupling efficiency of L-LNA-G, L-LNA-C, D-DNA-C is significantly improved by the presence of 2.5% pyridine in the coupling solvent, that for the remaining monomers tested, the addition of pyridine increases the coupling efficiency (e.g., L-DNA-T or L-DNA-C) or has no adverse effect on the coupling efficiency, and that the results demonstrate that benefits can be seen for all monomers using a coupling solvent comprising a heterocyclic base solvent (e.g., pyridine) in view of the solubility and stability benefits of pyridine to these monomers.

Detailed Description

The term "aryl" as used herein refers to an aromatic ring in which each ring-forming atom is a carbon atom. Aryl rings are formed from five, six, seven, eight, nine, or more than nine carbon atoms. Aryl is substituted or unsubstituted. In one aspect, aryl is phenyl or naphthyl. Depending on the structure, the aryl group can be a monovalent group or a divalent group (i.e., arylene). In one aspect, aryl is C _6-10And (4) an aryl group. In some embodiments, aryl is phenyl. When substituted, the aryl group may be substituted with a group selected from the group consisting of: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl or C_6-14Aryl radical C_1-4An alkyl group. Multiple fetchingThe generations may be selected, non-independently or independently, from: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl or C_6-14Aryl radical C_1-4An alkyl group; or a group selected from halogen, such as iodine, fluorine, bromine or chlorine, such as phenyl substituted by halogen, such as iodine, fluorine, bromine or chlorine.

An "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl moiety may be a saturated alkyl (meaning that it does not contain any unsaturated units, such as carbon-carbon double bonds or carbon-carbon triple bonds), or the alkyl moiety may be an unsaturated alkyl (meaning that it contains at least one unsaturated unit). The alkyl moiety, whether saturated or unsaturated, may be branched, straight-chain or include cyclic moieties. The point of attachment of the alkyl group is on a carbon atom that is not part of the ring. An "alkyl" moiety may have from 1 to 10 carbon atoms (whenever appearing herein, a numerical range such as "1 to 10" refers to each integer in the given range; for example, "1 to 10 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the definition of the invention also includes the term "alkyl" appearing without the specified numerical range). Alkyl includes branched and straight chain alkyl groups. The alkyl group of the compounds described herein may be referred to as "C _1-6Alkyl "or similar names. By way of example only, "C_1-6Alkyl "means that there are one, two, three, four, five or six carbon atoms in the alkyl chain, i.e. the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, allyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, and the like. In one aspect, alkyl is C_1-6Or C_1-4Alkyl or C_1-3An alkyl group. C_1-3Alkyl refers to straight or branched chain alkyl groups having 1 to 3 carbon atoms. C_1-4Examples of alkyl are methyl, ethyl, propylAnd isopropyl. C_1-3Alkyl refers to straight or branched chain alkyl groups having 1 to 4 carbon atoms. C_1-3Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl.

"alkenyl" groups are straight, branched, and cyclic hydrocarbon groups containing at least one carbon-carbon double bond. The alkenyl group may be substituted.

"alkynyl" groups are straight, branched, and cyclic hydrocarbon groups containing at least one carbon-carbon triple bond. Alkynyl groups may be substituted.

An "alkoxy" group refers to an alkyl group attached to an oxygen, i.e., (alkyl) -O-group, wherein alkyl is as defined herein. Examples thereof include methoxy (-OCH)₃) Or ethoxy (-OCH)₂CH₃)。

An "alkenyloxy" group refers to an alkenyl group attached to an oxygen, i.e., a (alkenyl) -O-group, where alkenyl is as defined herein.

An "alkynyloxy" group refers to an alkynyl group attached to an oxygen, i.e., (alkynyl) -O-group, wherein alkynyl is as defined herein.

An "aryloxy" group refers to an aryl group attached to an oxygen, i.e., (aryl) -O-group, wherein aryl is as defined herein. Examples thereof include phenoxy (-OC)₆H₅)。

"silyl" refers to H₃Si-. As used herein, "substituted silyl" refers to a moiety having one or more silyl groups with hydrogen substituted. Examples include, but are not limited to, TBDMS (t-butyldimethylsilyl), TBDPS (t-butyldiphenylsilyl), or TMS (trimethylsilyl) groups.

The term "halogen" is intended to include fluorine, chlorine, bromine and iodine. The term "halide" includes fluoride, bromide, iodide and chloride.

"acyl protecting group" includes acyl-C (═ O) -R⁷Wherein R is⁷Are terminal groups, for example selected from alkyl-, alkenyl-, alkynyl-, cycloalkyl-and aryl-groups; or from unsubstituted alkyl-, unsubstituted alkenyl-, unsubstituted alkynyl-, unsubstituted cycloalkyl-or unsubstituted aryl A group of a radical-group; or a group selected from substituted alkyl-, substituted alkenyl-, substituted alkynyl-, substituted cycloalkyl-or substituted aryl-groups. In some embodiments, R⁷May be selected from unsubstituted C_1-6-alkyl-, unsubstituted C_2-6-alkenyl-, unsubstituted C_2-6-alkynyl-, unsubstituted C_3-7Cycloalkyl-or unsubstituted phenyl-radicals or substituted C_1-6-alkyl-, substituted C_2-6-alkenyl-, substituted C_2-6-alkynyl-, substituted C_3-7-a cycloalkyl-or substituted phenyl-group; wherein when substituted, the substituents may be mono-or poly-substituted, for example with one or more substituents selected from: halogen element, C_1-6Alkyl radical, C_2-6-alkenyl, C_2-6-alkynyl, C_1-6-alkoxy, optionally substituted aryloxy or optionally substituted aryl. In some embodiments, the acyl protecting group is isobutyryl (-C (O ═) CH (CH)₃)₂) (also referred to herein as iBu). The term isobutyryl group may also be a spelled isobutyryl group.

Oxaazaphospholane phosphoramidites

The present invention provides an acetonitrile solution of an oxaazaphospholane phosphoramidite (also referred to herein as a nucleoside monomer, or imide), such as a nucleoside monomer of formula 1, comprising acetonitrile, the nucleoside monomer, and an aromatic heterocyclic solvent.

In some embodiments, the nucleoside monomer is of formula 1:

wherein Z is a nucleoside, and wherein,

R⁵and R⁶Independently selected from the group consisting of: hydrogen, alkyl, cyclo-alkyl, aryl, heteroaryl, substituted alkyl, substituted cyclo-alkyl, substituted aryl and substituted heteroaryl, or R⁵And R⁶Together form a heterocyclic ring containing 3 to 16 carbon atoms and the N atom of formula 1;

R⁹is hydrogen;

R¹is selected from the following groups: hydrogen and C_1-3An alkyl group; and is

R is selected from the following groups: aryl, heteroaryl, substituted aryl, substituted heteroaryl, nitro, halogen, cyano, silyl, substituted silyl, sulfone, substituted sulfone (aryl substituted sulfone), fluorene and substituted fluorine;

wherein, when substituted, R may be substituted with a group selected from: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl radicals or C_6-14Aryl radical C_1-4An alkyl group. The multiple substitutions may be selected from the following groups, either independently or both: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl radicals or C _6-14Aryl radical C_1-4An alkyl group.

R and R of nucleosides of formula 1¹(R/R¹) The group provides a stereocenter that, when incorporated into an oligonucleotide, results in the formation of a phosphorothioate group that is sterically defined with the Sp 3' of the nucleoside.

In some embodiments, the stereogenic center is in the L position, as shown in formula 1 a. In some embodiments, the stereocenter is in the D position, as shown in formula 1 b.

Comprising R and R as shown in formula 1a¹The monomer of the group-generated stereocenter is referred to herein as the L monomer, which results in the formation of the Sp stereocenter. Comprising R and R as shown in formula 1b¹The monomer of the stereocenter generated by the group is herein referred to as the D monomer, whichResulting in the formation of an Rp stereocenter.

When substituted, R may be substituted by a group selected from C_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl or C_6-14Aryl radical C_1-4An alkyl group. Multiple substitutions may be selected from C independently or independently_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl or C_6-14Aryl radical C_1-4An alkyl group.

In some embodiments, R is selected from the group consisting of aryl, heteroaryl, substituted aryl, substituted heteroaryl, nitro, halogen, cyano, silyl, substituted silyl, sulfone, substituted sulfone (aryl-substituted sulfone), fluorene, and substituted fluorene.

In some embodiments, R is selected from aryl, heteroaryl, substituted aryl, and substituted heteroaryl.

In some embodiments, R is aryl, e.g., phenyl.

In some embodiments, when R is substituted aryl, R may be substituted with a halogen such as iodo, fluoro, bromo, or chloro, for example phenyl substituted with a halogen such as iodo, fluoro, bromo, or chloro.

In some embodiments, R¹Is hydrogen. In some embodiments, R¹Is C_1-3Alkyl radicals, such as methyl, ethyl or propyl. In some embodiments, R¹Is methyl.

In some embodiments, R is aryl such as phenyl and R is¹Is hydrogen.

In some embodiments, R is aryl, e.g., phenyl, and R is¹Is C_1-3Alkyl groups such as methyl, ethyl or propyl.

In some embodiments, R is

Wherein G is³¹、G³²And G³³Independently selected from C_1-4Alkyl radical, C_6-14Aryl radical C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl and C_6-14Aryl radical C_1-4An alkyl group.

In some embodiments, R is

Wherein G is²¹、G²²And G²³Independently hydrogen, nitro, halogen, cyano or C_1-3An alkyl group.

In some embodiments, R is

Wherein G is⁵¹、G⁵²And G⁵³Independently hydrogen, nitro, halogen, cyano or C_1-3Alkyl or C _1-3An alkoxy group.

In some embodiments, R⁵And R⁶Together form a heterocyclic ring (together with the ring nitrogen shown in formula 1) -a nucleoside monomer, known as a bicyclic oxaazaphospholane phosphoramidite. The heterocyclic ring may comprise, for example, 3 to 16 carbon atoms, for example 4 carbon atoms.

Bicyclic oxaazaphospholane phosphoramidite monomers

In some embodiments, the monomer is a bicyclic oxaazaphospholane phosphoramidite monomer, e.g., in some embodiments, R⁵And R⁶Together form a heterocyclic ring. In some embodiments, R⁵And R⁶Together form a heterocyclic ring (together with the ring nitrogen shown in formula 1) that contains 4 carbon atoms such that there are a total of 5 atoms (4 atoms) in the heterocyclic ringCarbon and one nitrogen shown in formula 1). For example, the compound of the invention may be a compound of formula 2a or 2 b:

r, R therein¹、R⁹And Z is as described for compounds of formula 1.

In some embodiments, R⁵And R⁶Together form a heterocyclic ring (together with the ring nitrogen shown in formula I) containing 4 carbon atoms such that there are a total of 5 atoms in the heterocyclic ring (4 carbons and one nitrogen shown in formula 1), and R is an aryl group, e.g., phenyl, R¹Is hydrogen or methyl. R⁹Is hydrogen.

The above Z group is a nucleoside wherein the 3' oxygen of the nucleoside is the exocyclic oxygen shown in formula 1, 1a, 1b, 2a or 2 b. In some embodiments, the Z group is a LNA nucleoside moiety. In some embodiments, the Z group is a DNA nucleoside moiety. Thus, in some embodiments, the compounds of the present invention may be represented by formula 3a or 3 b:

Wherein, R, R¹、R⁵、R⁶And R⁹As defined in the compounds of the invention;

b is a nucleobase group which is,

in some embodiments, B is a nucleobase selected from: adenine, guanine, cytosine, thymine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

In some embodiments, B is a purine nucleobase. In some embodiments, B is a pyrimidine nucleobase. In some embodiments, B is adenine. In some embodiments, B is thymine. In some embodiments, B is guanine. In some embodiments, B is cytosine. In some embodiments, when B is cytosine, B is 5-methyl-cytosine.

In some embodiments, B is not cytosine, for example, when the monomer is a D-DNA monomer of, for example, formula 20 or 22. In some embodiments, when the monomer is D-DNA-C, B is not an acetyl (Ac) -protected cytosine.

It will be appreciated that for use in oligonucleotide synthesis, the nucleobase B may be protected in an imide monomer (thymine is typically used without a protecting group). Suitable protecting groups include Dimethylformamide (DMF), Dimethoxytrityl (DMT) or acyl protecting groups such as isobutyryl (iBu), or acetyl (Ac) or benzoyl (Bz) protecting groups.

In some embodiments, for example when the monomer is L-LNA-G, B is not DMF protected guanine (G). R³Selected from the following groups: CH (CH)₂ODMTr、CH₂alkyl-O-DMTr, CH-Me-O-DMTr, CH₂OMMTr、CH₂alkyl-O-MMTr, CH (Me) -O-MMTr, CH-R^a-O-DMTrR^bAnd CH-R^a-O-MMTrR^b；

R²Selected from halogens such as-F, amino, azido, -SH, -CN, -OCN, -CF₃、-OCF₃、 -O(R^m) -alkyl, -S (R)^m) -alkyl, -N (R)^m) -alkyl, -O (R)^m) -alkenyl, -S (R)^m) -alkenyl, -N (R)^m) -an alkenyl group; -O (R)^m) -alkynyl, -S (R)^m) -alkynyl or-N (R)^m) -an alkynyl group; O-alkylene-O-alkyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl, O (CH)₂)₂SCH₃、 O-(CH₂)₂-O-N(R^m)(Rⁿ) Or O-CH₂C(＝O)-N(R^m)(Rⁿ)、-O-(CH₂)₂OCH₃and-O-CH₃Wherein R is^mAnd RⁿEach independently is H, an amino protecting group or a substituted or unsubstituted C_1-10An alkyl group;

R⁴selected from the group consisting of alkyl, cycloalkyl, heterocycloalkyl, O-alkyl, S-alkyl, NH-alkyl and hydrogen; in some embodiments, R⁴Is hydrogen. In some embodiments, R⁴Is hydrogen, and R²Is selected from the following groups: -O-CH₃and-O- (CH)₂)₂OCH₃。

Or in some embodiments, R²And R⁴Together represent a divalent bridge, e.g. consisting of 1, 2, 3 members selected from-C (R)^aR^b)-、-C(R^a)＝C(R^b)、-C(R^a)＝N、O、-Si(R^a)₂-、S-、-SO₂-、 -N(R^a) -and>c ═ Z groups/atom composition;

wherein R is^aAnd R^bEach (when present) is independently selected from hydrogen, optionally substituted C _1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkynyl, hydroxy, optionally substituted C_1-6-alkoxy, C_2-6Alkoxyalkyl group, C_2-6-alkenyloxy, carboxy, C_1-6Alkoxycarbonyl, C_1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di- (C)_1-6-alkyl) amino, carbamoyl, mono-and di- (C)_1-6-alkyl) -amino-carbonyl, amino-C_1-6-alkyl-aminocarbonyl, mono-and di- (C)_1-6-alkyl) amino-C_1-6-alkyl-aminocarbonyl, C_1-6-alkyl-carbonylamino, ureido, C_1-6Alkanoyloxy, sulfonyl (sulphono), C_1-6Alkylsulfonyloxy, nitro, azido, sulfanyl, C_1-6Alkylthio, halogen, wherein aryl and heteroaryl may be optionally substituted, and wherein two geminal substituents R^aAnd R^bTaken together may represent optionally substituted methylene (═ CH)₂) Wherein the asymmetric group may be either R or S oriented for all chiral centers.

In some embodiments, when incorporated into an oligoAmong nucleotides, nucleoside (Z) confers higher binding affinity to a complementary RNA target than an equivalent DNA nucleoside. Such nucleosides are known as high affinity nucleosides. Examples of high affinity nucleosides include 2' -O-MOE, 2' -fluoro, 2' -O-methyl, and LNA nucleosides. In embodiments where the nucleoside is a high affinity nucleoside, R ³May be, for example, CH₂-O-DMTr or CH₂-O-MMTr。

In some embodiments, R²Selected from fluorine (-F), -O- (CH)₂)₂OCH₃and-O-C_1-3Alkyl radicals, e.g. O-CH₃. In this embodiment, R⁴Optionally hydrogen.

In some embodiments, the nucleoside is a LNA nucleoside (also referred to as a bicyclic nucleoside) comprising a 2 '-4' bridge (divalent group).

In some embodiments, R²And R⁴Together represent a divalent bridge selected from the group consisting of bridge-C (R)^aR^b)-O-、–C(R^aR^b)C(R^aR^b)-O-、-CH₂-O-、-CH₂CH₂-O-、-CH(CH₃) -O-. In some embodiments, R²And R⁴Represents a divalent bridged-CH₂-O- (methylene-oxy, also known as oxy-LNA) or-CH (CH)₃) -O- (methyl-methylene-oxy). -CH (CH)₃) The O-bridge introduces a chiral center at the carbon atom within the bridge, which in some embodiments is located at the S position (e.g. a nucleoside known in the art as (S) cET-see EP 1984381)). In some embodiments, R²And R⁴Represents a divalent bridged-CH₂-O-, wherein the bridge is located at the β -D position (β -D-oxy LNA). In some embodiments, R²And R⁴Represents a divalent bridged-CH₂-O-, wherein the bridge is located at the alpha-L position (alpha-L-D-oxy LNA). In some embodiments, R²And R⁴Represents a divalent bridged-CH₂-S- (Sulfur LNA) or-CH₂-NH₂- (amino LNA). At R²And R ⁴In embodiments which together represent a divalent bridge, R³May be, for example, CH₂-O-DMTr or CH₂-O-MMTr。

In some embodiments where the nucleoside (Z) is a bicyclic nucleoside (LNA), e.g., β -D-O LNA, R is aryl, e.g., phenyl, and R is¹Is hydrogen or C_1-3An alkyl group. In said embodiment, R⁵And R⁶May together form a heterocyclic ring, such as a 5-membered heterocyclic ring as described herein (see, e.g., formulas 2a and 2 b).

In some embodiments, the oxaazaphospholane phosphoramidite monomer is selected from formulas 4a, 4b, 5a, 5b, 6a, 6b, 7a, and 7 b.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is selected from formula 8a, 8b, 8c or 8 d; or 9a, 9b, 9c or 9 d:

in some embodiments, the nucleobase B is an adenine, e.g., a Bz-protected adenine. In some embodiments, the nucleobase B is thymine. In some embodiments, the monomer is a D-DNA-A monomer (e.g., a monomer of formula 9c, and the nucleobase B is an adenine, e.g., a Bz-protected adenine). The examples illustrate that D-DNA-A monomers (e.g.formula 9c), L-LNA-A monomers and L-LNA-T monomers (e.g.formula 8a or 8b) show improved coupling when used in acetonitrile/aromatic heterocyclic solvents as described in the present invention.

DMF protected L-LNA-G

As shown in the examples, DMF protected L-LNA-G monomer is difficult to dissolve in acetonitrile solvent.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is not an L-LNA monomer comprising a DMF protected guanine nucleobase.

In some embodiments, the DMF protected guanine group (B) has the following structure:

in some embodiments, the oxaazaphospholane phosphoramidite monomer is not a monomer of formula 11 or 12:

r, R therein¹、R³、R⁵、R⁶And R⁹Is as described for the monomer of formula 1, and wherein for the monomer of formula 11, X and Y together represent a divalent bridge (e.g., according to R herein²And R⁴For example a bridged bridge selected from: -C (R)^aR^b)-O-、–C(R^aR^b)C(R^aR^b)-O-、-CH₂-O-、-CH₂ CH₂-O-、-CH(CH₃) -O-. In some embodiments, X and Y represent a divalent bridge-CH₂-O- (methylene-oxy, also known as oxy-LNA) or-CH (CH)₃) -O- (methyl-methylene-oxy). -CH (CH)₃) the-O-bridge introduces a chiral center at a carbon atom within the bridge, which in some embodiments is in the S position (e.g., a nucleoside known in the art as (S) cET-see EP 1984381)). In some embodiments, X and Y represent a divalent bridge-CH₂-O-, wherein the bridge is in the β -D position (β -D-oxy LNA). In some embodiments, X and Y represent a divalent bridge-CH ₂-O-, wherein the bridge is located in the α -L position (α -L-D-oxolna). In some embodiments, X and Y represent a divalent bridge-CH₂-S- (thio LNA) or-CH₂-NH₂- (amino LNA). In embodiments where X and Y together represent a divalent bridge, R³May be, for example, CH₂-O-DMTr or CH₂-O-MMTr。

In some embodiments, the oxaazaphospholane phosphoramidite monomer is not a monomer of formula 13 or 14:

x, Y, R, R therein¹、R⁹And R³As described in equations 11 and 12. The exocyclic oxygen of the guanine base may optionally be protected, for example, with a cyano group.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is not a monomer of formula 15 or 16:

x, Y, R therein¹And R³As described in equations 11 and 12. The exocyclic oxygen of the guanine base may optionally be protected, for example with a cyano group. In some embodiments of formula 15 or 16, R1 is hydrogen. In some embodiments of formula 15 or 16, R³Is CH₂-O-DMTr or CH₂-O-MMTr. In some embodiments, the oxaazaphospholanemamide monomers of the invention comprise an acyl protected nucleoside (Z).

Acyl protected L-LNA-G

As shown in the examples, DMF protected L-LNA-G monomer is poorly soluble in acetonitrile solvents. However, the present inventors have determined that the use of an acyl protecting group on guanosine of the L-LNA-G monomer circumvents the solubility problem.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is an L-LNA monomer comprising an acyl protected guanine nucleobase, such as an isobutyryl protected guanine.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is an L-LNA-G monomer of formula 23, 24, 25, 26, 27, 28, 29 or 30:

wherein, R, R¹、R²、R³、R⁴、R⁵、R⁹And R⁶As defined in the compounds of the invention, and-C (═ O) -R⁷Is an acyl protecting group on the exocyclic nitrogen of a guanine base, R⁸(when present) is a protecting group on the guanine exocyclic oxygen. In some embodiments, R⁸Is cyanoethyl. In some embodiments, R is phenyl, R is¹Is hydrogen or methyl, and R³Is optionally CH₂-O-DMTr or CH₂-O-MMTr. In some embodiments, R⁷Is isobutyryl. In formulas 31 and 32, Y and X are as described for formula 11.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is selected from: L-LNA-T, D-DNA-A, D-DNA-C, L-LNA-C and L-LNA-G (not DMF protected L-LNA-G) or L-DNA-C and L-DNA-T oxaazaphospholane phosphoramidite monomers. As shown in the examples, these monomers show improved coupling efficiency when used in the coupling solvent composition of the invention, and in addition have general solubility and stability benefits for oxaazaphospholane phosphoramidite monomers.

Solvent composition (solution)

The present invention provides an acetonitrile solution comprising an oxaazaphospholane phosphoramidite monomer, acetonitrile and an aromatic heterocyclic solvent.

In some embodiments, the acetonitrile solution further comprises an activator. Many activators for phosphoramidite oligonucleotide synthesis are known and typically comprise acidic azole catalysts such as 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthio-tetrazole, and 4, 5-dicyanoimidazole.

In some embodiments, the aromatic heterocyclic solvent has a pKa of about 4 to about 7. In some embodiments, the aromatic heterocyclic solvent has a pKa in water at 20 ℃ of about 7 to about 17.

In some embodiments, the aromatic heterocyclic solvent is an aromatic heterocyclic base.

In some embodiments, the aromatic heterocyclic solvent is an aromatic heterocyclic acid.

In some embodiments, the aromatic heterocyclic solvent is selected from pyridine, 2-methylpyridine, 4-methylpyridine, 3-methylpyridine, lutidine, and pyrrole.

In some embodiments, the aromatic heterocyclic solvent is pyridine.

In some embodiments, the aromatic heterocyclic solvent is pyrrole.

In some embodiments, the aromatic heterocyclic solvent is 3-methylpyridine.

In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 0.1% to about 50% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 40% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 30% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 25% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 10% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 5% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 1% to about 5% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is from about 1% to about 4% (v/v). In some embodiments, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 10% (v/v), for example about 1% to about 5% (v/v), such as about 2-3% (v/v), for example about 2.5% (v/v). In some embodiments, optionally the aromatic heterocyclic base solvent is pyridine.

In some embodiments, wherein the aromatic heterocyclic solvent is pyridine, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is from about 0.5% to about 10%, such as from about 1% to about 5%, for example from about 2-3%, such as about 2.5% or about 3.5%, or about 2-4%.

In some embodiments, wherein the aromatic heterocyclic solvent is pyrrole, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is from about 0.5% to about 10%, such as from about 1% to about 5%, for example 2-4% or about 2-3%, for example about 2.5%.

In some embodiments, wherein the aromatic heterocyclic solvent is 3-methylpyridine, the concentration (v/v) of the aromatic heterocyclic solvent in acetonitrile is from about 0.5% to about 10%, such as from about 1% to about 5%, for example 2-4%, or about 2-3%, for example about 2.5%.

Activating agent

An activator is a reagent used before or during the coupling step of oligonucleotide synthesis that activates the phosphoramidite monomer to allow the monomer to couple to the 5' terminal group attached to the solid support or oligonucleotide chain.

In some embodiments, the acetonitrile solvent composition further comprises an activating agent.

In some embodiments, the activator is selected from the group consisting of CMPT (N- (cyanomethyl) pyrrolidinium trifluoromethane sulfonate (CMPT), N- (phenyl) imidazolium triflate (PhIMT), benzimidazolium triflate (BIT), 4, 5-Dicyanoimidazole (DCI), tetrazole, and 5- (benzylthio) -1H-tetrazole.

In some embodiments, the activator is 4, 5-Dicyanoimidazole (DCI).

In some embodiments, the solvent composition comprises about 0.5 to about 2M DCI (or other activating agent), for example about 1M DCI (or other activating agent).

In some embodiments, the solvent composition further comprises N-methylimidazole, e.g., N-methylimidazole at a concentration of 0.01 to about 1M of N-methylimidazole, e.g., about 0.1M of N-methylimidazole.

In some embodiments, the activator comprises N-methylimidazole. In some embodiments, the activator comprises 4, 5-Dicyanoimidazole (DCI), tetrazole, or 5- (benzylthio) -1H-tetrazole. In some embodiments, the activator comprises 4, 5-Dicyanoimidazole (DCI), tetrazole, or 5- (benzylthio) -1H-tetrazole and N-methylimidazole.

In some embodiments, N-methylimidazole is used at a concentration of 0.01M to about 1M N-methylimidazole, for example about 0.1M N-methylimidazole. In some embodiments, the acetonitrile solution comprises N-methylimidazole at a concentration of 0.01M to about 1M of N-methylimidazole, for example about 0.1M of N-methylimidazole.

In some embodiments, the activator is DCI or tetrazole, or 5- (benzylthio) -1H-tetrazole, which can be used at a concentration of about 0.5 to about 2M, for example about 1M (e.g., in acetonitrile solutions of the invention).

In some embodiments, the activator is 4, 5-Dicyanoimidazole (DCI). In some embodiments, the solvent composition comprises about 0.5 to about 2M DCI, for example about 1 MDCI. It will be appreciated that in order to optimise coupling efficiency, it may be necessary to optimise the amount of activator used, as shown in the examples. In some embodiments, the DCI activator is used at a concentration of 0.5M to 1M DCI. In some embodiments, when the activator is DCI, the solvent composition further comprises N-methylimidazole (NMI), e.g., an N-methylimidazole of N-methylimidazole at a concentration of 0.01 to about 1M, e.g., about 0.1M N-methylimidazole. NMI is an agent that can enhance the solubility of other activating agents such as DCI.

Method for synthesizing oligonucleotides

The invention provides a method of synthesizing an oligonucleotide comprising a method of coupling an oxaazaphospholane phosphoramidite monomer to the 5' end of a solid support, a nucleoside or an oligonucleotide of the invention.

The invention provides a method for synthesizing a stereospecifically defined phosphorothioate oligonucleotide, comprising the following steps:

a) deprotecting the protected 5' -hydroxy terminus of a nucleoside or oligonucleotide attached to a solid support,

b) coupling an oxaazaphospholane phosphoramidite monomer to the deprotected 5' -hydroxy terminus of a nucleoside or oligonucleotide, wherein the coupling reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent to form a phosphite triester intermediate, and

c) Oxidizing the phosphite triester intermediate with a vulcanizing agent,

d) optionally repeating steps a) -c) for one or more further extension cycles,

e) the oligonucleotide is deprotected from the solid support and cleaved.

The method of the invention may comprise a plurality of further extension cycles of step d), for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more further extension cycles.

In some embodiments, after step c) or after step d), an optional amine wash step is performed. The amine washing step refers to an optional method for oligonucleotide synthesis, wherein the oligonucleotide is treated with a solution of a weak base in an organic solvent, such as 20% diethylamine in acetonitrile, or 1: and (3) treating triethylamine/acetonitrile. Amine washing results in removal of the cyanoethyl phosphate protecting group without cleaving the oligonucleotide from the solid support. The benefits of including an amine wash allow the avoidance of undesirable cyanothiol adducts, such as acrylonitrile, formed by side reactions of cyanoethyl phosphate protecting groups and heterocyclic bases, particularly thymine.

Typically, the chiral auxiliary is cleaved from the oligonucleotide during deprotection and cleavage from the solid support. For example, suitable deprotection/cleavage may be carried out in concentrated ammonium hydroxide at a temperature of about 55 ℃.

In some embodiments, after step e), the oligonucleotide may be purified. The purification step may be carried out using any suitable method for oligonucleotide purification, such as ion exchange purification or reverse phase chromatography, or both. In some embodiments, the purification comprises the sequential steps of: a) ion exchange purification, b) desalting, e.g., by diafiltration. Followed by c) lyophilization and d) reverse phase chromatography. Prior to purification, the ammonium hydroxide is typically removed or at least diluted. Alternatively, DMT-ON reverse phase purification followed by detritylation is also the choice for purifying oligonucleotides (see Capaldi and Scozzari, Chapter 14, Antisense Drug Technology: Principles, Strategies, and Applications, CRC Press 2008.

In some embodiments, the oligonucleotide may be conjugated after step e) or after an optional purification step. Alternatively, conjugation may be performed during oligonucleotide synthesis.

In some embodiments, the oligonucleotide produced by the methods of the invention, a sterically defined phosphorothioate oligonucleotide, is an antisense oligonucleotide or a mixed sequence oligonucleotide. In some embodiments, the sterically defined phosphorothioate oligonucleotide comprises sterically defined phosphorothioate internucleoside linkages and sterically random phosphorothioate internucleoside linkages.

Since the oxaazaphospholane phosphoramidite monomer introduces an Sp or Rp phosphorothioate internucleoside linkage, the methods of the invention can be used to synthesize sterically defined oligonucleotides. Accordingly, the present invention provides improved methods for synthesizing sterically defined phosphorothioate oligonucleotides.

These improvements include providing solutions of oxaazaphospholane phosphoramidite monomers, such as those described herein, with enhanced monomer solubility as compared to acetonitrile solutions of monomers without aromatic heterocyclic solvents. Alternatively, providing a more stable solution of oxaazaphospholane phosphoramidite monomers, such as those described herein, with enhanced monomer solution stability as compared to acetonitrile solutions of monomers without aromatic heterocyclic solvents; alternatively, providing a more reactive oxaazaphospholane phosphoramidite monomer, such as those described herein, has enhanced monomer reactivity compared to an acetonitrile solution of the monomer without an aromatic heterocyclic solvent. The skilled artisan will appreciate that one of the combined benefits of having a higher solubility, a more stable solution, and a higher reactivity will result in more efficient synthesis and more reliable and enhanced yields of oligonucleotide product. Benefits may also include avoiding or reducing undesirable side reactions, thereby achieving higher product purity.

In some embodiments, the 5' terminus is an-OH group attached to the solid support. the-OH group may be directly attached to the solid support, e.g. via a linker, e.g. a universal linker, or may be part of a nucleoside or oligonucleotide attached to the linker or the solid support.

In some embodiments, the oligonucleotide synthesis method is a solid phase phosphoramidite synthesis, wherein at least one coupling step is as a coupling method according to the invention.

The oligonucleotide synthesis method of the present invention may comprise the steps of:

a) providing a solid support having free 5' -OH groups;

b) activation of the oxaazaphospholane phosphoramidite monomer;

c) coupling an activated oxaazaphospholane phosphoramidite monomer to a free' 5-OH according to the method of the present invention to form a phosphite triester intermediate,

d) the phosphite triester intermediate is oxidized with a sulfurizing reagent such as xanthogen hydride (xanthogen hydride),

e) blocking any free-OH groups, e.g. using acetic anhydride,

f) r on the oxaazaphospholane phosphoramidite monomer³The group is deprotected, and the reaction is carried out,

g) optionally repeating steps b) -f),

h) deprotecting any remaining protecting groups (global deprotection) and cleaving the oligonucleotide from the solid support, e.g.by treatment with ammonium hydroxide at 60 ℃,

Wherein the free-OH group of the solid support can optionally be attached to a nucleotide or oligonucleotide chain attached to the solid support.

The solid support may be provided in a protected form, wherein the 5' OH group is protected, for example, by a DMT group. Prior to step a), the solid support (or the terminal nucleoside attached thereto) may be deblocked (detritylated) to provide a free 5' -OH group.

In some embodiments, steps b) to f) are repeated 7-25 times, for example 7-16 times, in the oligonucleotide synthesis. In some embodiments, the repetition of steps b) -f) is a continuous cycle in the oligonucleotide synthesis.

An exemplary protocol for the synthesis of phosphoramidite oligonucleotides using oxaazaphospholane phosphoramidite monomers:

in some embodiments, in addition to incorporating sterically defined phosphorothioate internucleoside linkages, synthetic methods can incorporate sterically random internucleoside linkages by using standard phosphoramidite monomers.

Sterically defined phosphorothioate oligonucleotides

Typically, the synthetic oligonucleotide phosphorothioates are random mixtures of Rp and Sp phosphorothioate linkages (also referred to as mixtures of diastereomers). In the methods of the invention, phosphorothioate oligonucleotides are provided wherein at least one phosphorothioate bond of the oligonucleotide is sterically defined, i.e. is Rp or Sp, in at least 75%, such as at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, such as at least 98%, such as at least 99%, or (substantially) all oligonucleotide molecules present in the oligonucleotide sample. The sterically defined oligonucleotide comprises at least one sterically defined phosphorothioate linkage. The term stereospecifically may be used to describe the specific chirality of one or more phosphorothioate internucleoside linkages as Rp or Sp, or may be used to describe an oligonucleotide comprising one (or more) such phosphorothioate internucleoside linkages. It has been recognized that a stereodefined oligonucleotide may contain a small amount of another stereoisomer at any one position, for example, Wan et al reported 98% stereoselectivity of Gapmer reported in NAR at 11 months 2014.

LNA oligonucleotides

An LNA oligonucleotide is an oligonucleotide comprising at least one LNA nucleoside. The LNA oligonucleotide may be an antisense oligonucleotide.

The term oligonucleotide as used herein is defined as a molecule comprising two or more covalently linked nucleosides as is commonly understood by those skilled in the art. For use as antisense oligonucleotides, oligonucleotides of 7-30 nucleotides in length are typically synthesized.

The term "antisense oligonucleotide" as used herein refers to an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on the target nucleic acid. An antisense oligonucleotide can also be defined by its complementarity to a target nucleic acid. The antisense oligonucleotide is single-stranded. Antisense oligonucleotides are not substantially double stranded and are therefore not sirnas. The antisense oligonucleotide comprises contiguous nucleotides complementary to the target nucleic acid. Antisense oligonucleotides typically contain one or more modified internucleoside linkages, and may be in the form of LNA gapmers or mixed wing (mixed wing) gapmers, as non-limiting examples. In other embodiments, the oligonucleotide may be a mixture of LNAs (LNA and non-LNA nucleotides, such as LNA and DNA (see, e.g., WO2007/112754, incorporated herein by reference), or LNA and 2'-O-MOE nucleotides, or LNA, DNA and 2' O-MOE nucleotides), or LNA monoliths (LNA nucleotides only-see, e.g., WO2009/043353, incorporated herein by reference).

The term "modified internucleoside linkage" is defined as a linkage other than a Phosphodiester (PO) linkage, as commonly understood by those skilled in the art, which covalently couples two nucleosides together. Modified internucleoside linkages are particularly useful for stabilizing oligonucleotides for in vivo applications, and may be useful for protecting against nuclease cleavage. Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments, at least 70%, such as at least 80% or such as at least 90% of the internucleoside linkages in the oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate. In some embodiments, all internucleoside linkages in the oligonucleotide or a contiguous nucleotide sequence thereof are phosphorothioate, wherein at least one phosphorothioate internucleoside linkage is a sterically defined phosphorothioate internucleoside linkage (resulting from incorporation of an oxaazaphospholane phosphoramidite monomer into the oligonucleotide during oligonucleotide synthesis). Other internucleoside linkers are disclosed in WO2009/124238 (incorporated herein by reference).

The term nucleobase includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term nucleobase also includes modified nucleobases, which may be different from naturally occurring nucleobases, but which are functional during nucleic acid hybridization. In some embodiments, the nucleobase moiety is modified by modifying or replacing the nucleobase. As used herein, "nucleobase" refers to naturally occurring nucleobases, such as adenine, guanine, cytosine, thymine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. These variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research vol 45,2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry suppl.371.4.1.

Nucleotides are building blocks for oligonucleotides and polynucleotides, and for the purposes of the present invention, nucleotides include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides, comprise a ribose moiety, a nucleobase moiety, and one or more phosphate groups (which are not present in nucleosides). Modified nucleosides and nucleotides are modified by introducing modifications to the ribose moiety, the nucleobase moiety, or, in the case of modified nucleotides, to the internucleoside linkage, as compared to the equivalent DNA or RNA nucleosides/nucleotides. Nucleosides and nucleotides can also be referred to interchangeably as "units" or "monomers".

The term "modified nucleoside" or "nucleoside modification" as used herein refers to a nucleoside that is modified by the introduction of one or more modifications of a sugar moiety or a (nucleobase) moiety, as compared to an equivalent DNA or RNA nucleoside. The term "modified nucleoside" is also used interchangeably herein with the term "nucleoside analog" or modified "unit" or modified "monomer". Examples of modified nucleosides are described in the "oligomer modification" section alone and in sub-sections thereof.

Acyl protected exocyclic nitrogen

The exocyclic nitrogen group of guanine is shown below (circled). In the monomers used in the present invention, this group is protected by an acyl group. Oxygen groups may also be optionally protected, for example with cyano groups.

Locked Nucleotide (LNA)

LNA nucleosides are modified nucleosides comprising a linking group (referred to as a divalent group or bridge) between C2 'and C4' of the ribose sugar ring of the nucleotide (i.e., wherein R is²And R⁴Together represent embodiments of divalent bridging).

These nucleosides are also referred to in the literature as bridge nucleic acids or Bicyclic Nucleic Acids (BNA).

In some embodiments, the oxaazaphospholane phosphoramidite monomer is or comprises an LNA nucleoside, for example, the monomer can be a compound of formula 17 or 18:

wherein B represents a nucleobase; r, R¹、R⁶、R³、R⁹、R⁵As shown in equation 1.

In some embodiments of formula 17, B is not DMF protected guanine. In some embodiments, B is adenine or thymine. In some embodiments, B is DMF protected adenine.

X represents a group selected from: -C (R)^aR^b)-、-C(R^a)＝C(R^b)-、-C(R^a)＝N-、-O-、 -Si(R^a)₂-、-S-、-SO₂-、-N(R^a) -and>C＝Z，

in some embodiments, X is selected from the group consisting of-O-, -S-, NH-, and NR^aR^b、-CH₂-、CR^aR^b、 -C(＝CH₂) -and-C (═ CR)^aR^b)-，

In some embodiments, X is-O-,

y represents a group selected from: -C (R)^aR^b)-、-C(R^a)＝C(R^b)-、-C(R^a)＝N-、-O-、 -Si(R^a)₂-、-S-、-SO₂-、-N(R^a) -and>C＝Z，

in some embodiments, Y is selected from the group consisting of-CH ₂-、-C(R^aR^b)-、–CH₂CH₂-、 -C(R^aR^b)-C(R^aR^b)-、–CH₂CH₂CH₂-、-C(R^aR^b)C(R^aR^b)C(R^aR^b)-、 -C(R^a)＝C(R^b) -and-C (R)^a)＝N-，

In some embodiments, Y is selected from the group consisting of-CH₂-、-CHR^a-、-CHCH₃-、CR^aR^b-，

or-X-Y-together represent a divalent linking group (also referred to as a radical) together represent a divalent linking group consisting of 1, 2 or 3 groups/atom selected from: -C (R)^aR^b)-、-C(R^a)＝C(R^b)-、 -C(R^a)＝N-、-O-、-Si(R^a)₂-、-S-、-SO₂-、-N(R^a) -and>C＝Z，

in some embodiments, -X-Y-represents a divalent group selected from-X-CH₂-、 -X-CR^aR^b-、-X-CHR^a-、-X-C(HCH₃)^-、-O-Y-、-O-CH₂-、-S-CH₂-、-NH-CH₂-、 -O-CHCH₃-、-CH₂-O-CH₂、-O-CH(CH₃CH₃)-、-O-CH₂-CH₂-、 OCH₂-CH₂-CH₂-、-O-CH₂OCH₂-、-O-NCH₂-、-C(＝CH₂)-CH₂-、-NR^a-CH₂-、 N-O-CH₂、-S-CR^aR^b-and-S-CHR^a-。

In some embodiments, -X-Y-represents-O-CH₂-or-O-CH (CH)₃) -, andR^aand R^bEach (when present) is independently selected from hydrogen, optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkynyl, hydroxy, optionally substituted C_1-6-alkoxy, C_2-6Alkoxyalkyl group, C_2-6-alkenyloxy, carboxy, C_1-6Alkoxycarbonyl, C_1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di- (C)_1-6-alkyl) amino, carbamoyl, mono-and di- (C)_1-6-alkyl) -amino-carbonyl, amino-C_1-6-alkyl-aminocarbonyl, mono-and di- (C)_1-6-alkyl) amino-C_1-6-alkyl-aminocarbonyl, C_1-6-alkyl-carbonylamino, ureido, C_1-6Alkanoyloxy, sulfonyl, C_1-6Alkylsulfonyloxy, nitro, azido, sulfanyl, C _1-6Alkylthio, halogen, wherein aryl and heteroaryl may be optionally substituted, and wherein two geminal substituents R^aAnd R^bTaken together may represent optionally substituted methylene (═ CH)₂) Wherein the asymmetric group may be either R or S oriented for all chiral centers.

R¹⁰May be hydrogen, or in some embodiments may be selected from optionally substituted C_1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkynyl, hydroxy, C_1-6-alkoxy, C_2-6Alkoxyalkyl group, C_2-6-alkenyloxy, carboxy, C_1-6Alkoxycarbonyl, C_1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di- (C)_1-6-alkyl) amino, carbamoyl, mono-and di- (C)_1-6-alkyl) -amino-carbonyl, amino-C_1-6-alkyl-aminocarbonyl, mono-and di- (C)_1-6-alkyl) amino-C_1-6-alkyl-aminocarbonyl, C_1-6-alkyl-carbonylamino, ureido, C_1-6Alkanoyloxy, sulfonyl, C_1-6Alkylsulfonyloxy, nitro, azido, sulfanyl, C_1-6Alkylthio, halogen, wherein aryl and heteroaryl may be optionally substituted, and wherein two geminal substituents taken together may represent oxo, thio, imino or optionally substituted methylene.

In some embodiments, R¹⁰Is selected from C_1-6Alkyl groups such as methyl and hydrogen.

In some embodiments, R¹⁰Is hydrogen.

In some embodiments, R^aIs hydrogen or methyl. In some embodiments, when present, R^bIs hydrogen or methyl.

In some embodiments, R^aAnd R^bOne or both of which are hydrogen.

In some embodiments, R^aAnd R^bOne is hydrogen and the other is not hydrogen.

In some embodiments, R^aAnd R^bOne is methyl and the other is hydrogen.

In some embodiments, R^aAnd R^bAre both methyl groups.

In some embodiments, the divalent group-X-Y-is-O-CH₂-and R¹⁰Is hydrogen. In some embodiments, the divalent group-X-Y-is-S-CH₂-and R¹⁰Is hydrogen.

In some embodiments, the divalent group-X-Y-is-NH-CH₂-and R¹⁰Is hydrogen.

In some embodiments, the divalent group-X-Y-is-O-CH₂-CH₂-or-O-CH₂-CH₂- CH₂-and R¹⁰Is hydrogen.

In some embodiments, the divalent group-X-Y-is-O-CH₂-and R¹⁰Is C_1-6Alkyl groups, such as methyl.

In some embodiments, the divalent group-X-Y-is-O-CR^aR^b-, wherein R^aAnd R^bOne or both of which are not hydrogen, e.g. methyl, and R¹⁰Is C_1-6Alkyl groups, such as methyl.

In some embodiments, the divalent group-X-Y-represents a divalent linking group-O-CH (CH)₂OCH₃) - (2' O-methoxyethyl bicyclic nucleic acid-Seth et al, 2010, J.org. chem.,2010,75(5), pp 1569-containing 1581). In some embodiments, the divalent group-X-Y-represents a divalent linking group-O-CH (CH)₂CH₃) - (2' O-ethylbicyclic nucleic acid-Seth et al, 2010, j. In some embodiments, the divalent group-X-Y-is-O-CHR^a-and R¹⁰Is hydrogen.

In some embodiments, the divalent group-X-Y-is-O-CH (CH)₂OCH₃) -and R¹⁰Is hydrogen. This LNA nucleoside is also known in the art as ring moe (cmoe) and is disclosed in WO 07090071.

In some embodiments, the divalent group-X-Y-represents a divalent linking group of R-or S-configuration-O-CH (CH)₃) -. In some embodiments, the divalent groups-X-Y-taken together represent a divalent linking group-O-CH₂-O-CH₂- (Seth et al, 2010, j. In some embodiments, the divalent group-X-Y-is-O-CH (CH)₃) -and R¹⁰Is hydrogen. This 6' methyl LNA nucleoside is also known in the art as the cET nucleoside and may be the (S) cET or (R) cET stereoisomer, which is disclosed in WO07090071(β -D) and WO2010/036698(α -L).

In some embodiments, the divalent group-X-Y-is-O-CR ^aR^b-, wherein R^aOr R^bAre not both hydrogen and R¹⁰Is hydrogen. In some embodiments, R^aAnd R^bAre both methyl groups.

In some embodiments, the divalent group-X-Y-is-S-CHR^a-and R¹⁰Is hydrogen.

In some embodiments, the divalent group-X-Y-is-C (═ CH)₂)-C(R^aR^b) -, for example-C (═ CH)₂)-CH₂-or-C (═ CH)₂)-CH(CH₃) -and R¹⁰Is hydrogen.

In some embodiments, the divalent group-X-Y-is-N (-OR)^a) -and R¹⁰Is hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl. In some embodiments, the divalent groups-X-Y-taken together represent a divalent linking group-O-NR^a-CH₃- (Seth et al, 2010, j. In some embodiments, the divalent group-X-Y-is-N (R)^a) -and R¹⁰Is hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl.

In some embodiments, R¹⁰Is C_1-6Alkyl groups such as methyl. In this embodiment, the divalent group-X-Y-may be selected from-O-CH₂-or-O-C (HCR)^a) -, e.g. -O-C (HCH)₃)-。

In some embodiments, the divalent group is-CR^aR^b-O-CR^aR^b-, e.g. CH₂-O-CH₂-and R¹⁰Is hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl.

In some embodiments, the divalent group is-O-CR^aR^b-O-CR^aR^b-, e.g. O-CH ₂-O-CH₂And R is¹⁰Is hydrogen. In some embodiments, R^aIs C_1-6Alkyl groups such as methyl.

It is understood that LNA nucleosides can be in the beta-D or alpha-L stereoisomeric form unless specifically indicated.

As shown in the examples, in some embodiments of the invention, an LNA nucleoside is or comprises a β -D-oxy-LNA nucleoside, e.g., where the 2 '-4' bridge is as described in formula I and where X is oxygen and Y is CH₂And R is¹⁰Is hydrogen.

DNA nucleosides

In some embodiments, the oxaazaphospholane phosphoramidite monomer is or comprises a DNA nucleoside, for example the monomer may be of formula 19 or formula 20:

wherein B represents a nucleobase; r, R¹、R⁶、R³、R⁹、R⁵As shown in equation 1. In some embodiments of formula 20, B is adenine, e.g., a protected adenine, e.g., a Bz protected adenine.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is represented by formulas 21 and 22:

wherein B represents a nucleobase; r, R¹、R³、R⁹As shown in equation 1. In some embodiments of formula 20 or 22, B is adenine, e.g., a protected adenine, e.g., a Bz protected adenine. In some embodiments of the monomer of formula 19, 20, 21 or 22, R is phenyl, and R is¹Is hydrogen or methyl. In some embodiments of monomers of formula 19, 20, 21, or 22, R ³Is CH₂-O-DMTr or CH₂-O-MMTr。

Oligonucleotides comprising DNA and/or affinity-enhancing nucleosides

In some embodiments, the oligonucleotide is a DNA phosphorothioate oligonucleotide. The DNA phosphorothioate oligonucleotide comprises only DNA nucleosides and, in some embodiments, may comprise only sterically defined phosphorothioate internucleoside linkages. The DNA phosphorothioate may be, for example, 18 to 25 nucleotides in length.

In some embodiments, the oligonucleotide comprises one or more affinity enhancing nucleosides, such as a LNA or a 2' substituted nucleoside as described herein. Affinity enhancing nucleosides, such as 2'-O-MOE or 2' -O methyl, are commonly used in antisense oligonucleotides, or in combination with other nucleosides, such as DNA nucleosides, in a format such as a mixture or Gapmer, or can be used in fully sugar-modified oligonucleotides, where all nucleosides are not DNA or RNA.

In some embodiments, the oligonucleotides synthesized by the methods of the invention may be gapmers, and LNA gapmers or mixed-wing gapmers.

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 33 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 34 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 35 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 36 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 37 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 38 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 39 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 40 (FIG. 17).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 41 (FIG. 18).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 42 (FIG. 18).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 43 (FIG. 18).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 44 (FIG. 18).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 45 (FIG. 18).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 46 (FIG. 18).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 47 (FIG. 18).

In some embodiments of the methods of the present invention, the oxaazaphospholane phosphoramidite monomer is formula 48 (FIG. 18).

In some embodiments, the oxaazaphospholane phosphoramidite monomer is a DNA monomer.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is an LNA monomer. In some embodiments, the oxaazaphospholane phosphoramidite monomer is an LNA-A (D-LNA-A or L-LNA-A) monomer.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is an LNA-C (D-LNA-A or L-LNA-A) monomer.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is an L-LNA-G (D-LNA-A or L-LNA-A) monomer, for example L-LNA-G, wherein the exocyclic nitrogen of the guanine residue is protected with an acyl protecting group, such as isobutyryl.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is not an L-LNA-G monomer, wherein the exocyclic nitrogen on the guanine residue is protected with a DMF protecting group. In some embodiments, the oxaazaphospholane phosphoramidite monomer is not a D-LNA-G monomer.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is not an LNA-T monomer, such as D-LNA-T or L-LNA-T.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is not an LNA-T monomer, for example a D-LNA-T or L-LNA-T or D-LNA-G monomer.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is a DNA monomer or an LNA monomer selected from the group consisting of an LNA-A monomer, an LNA-C monomer, and an acyl protected L-LNA-G monomer.

In some embodiments, the oxaazaphospholane phosphoramidite monomer is not an LNA-T monomer, a D-LNA-G monomer, or a DMF protected L-LNA-G monomer.

Gapmer

The term Gapmer as used herein refers to an antisense oligonucleotide comprising an RNase H recruiting oligonucleotide region (gap) flanked 5 'and 3' by one or more affinity enhancing modified nucleosides (flanking). Various Gapmer designs are described herein. The head (headers) and tail (tails) are oligonucleotides capable of recruiting RNase H in which one of the flanks is deleted, i.e. nucleosides in which only one of the ends of the oligonucleotide comprises an affinity enhancing modification. For the head, the 3 'flank is deleted (i.e., the 5' flank comprises an affinity enhancing modified nucleoside) and for the tail, the 5 'flank is deleted (i.e., the 3' flank comprises an affinity enhancing modified nucleoside). In some embodiments, the sterically defined phosphorothioate oligonucleotide is a Gapmer oligonucleotide, e.g., a LNA Gapmer oligonucleotide.

LNA Gapmer

The term LNA Gapmer is a Gapmer oligonucleotide in which at least one affinity enhancing modified nucleoside is a LNA nucleoside.

Hybrid wing Gapmer

The term mixed-wing Gapmer refers to an LNA Gapmer in which the flanking region comprises at least one LNA nucleoside and at least one non-LNA modified nucleoside, e.g. at least one 2' substituted modified nucleoside, e.g. 2' -O-alkyl-RNA, 2' -O-methyl-RNA, 2' -alkoxy-RNA, 2' -O-methoxyethyl-RNA, (moe), 2' -amino-DNA, 2' -fluoro-DNA, arabinonucleic acids (ANA), 2' -fluoro-ANA and 2' -F-ANA riboside. In some embodiments, one flank of the mixed-wing Gapmer comprises an LNA nucleoside (e.g., 5' or 3'), and the other flank (3 ' or 5', respectively) comprises a 2' substituted modified nucleoside.

Length of

When referring to the length of a nucleotide molecule as referred to herein, the length corresponds to the number of monomeric units, i.e. nucleotides, whether these monomeric units are nucleotides or nucleotide analogues. With respect to nucleotides, the terms monomer and unit are used interchangeably herein.

The method of the invention is particularly suitable for purifying short oligonucleotides, e.g., oligonucleotides consisting of 7 to 30 nucleotides, e.g., 7-10 nucleotides, e.g., 7, 8, 9, 10 or 10 to 20 nucleotides, e.g., 12 to 18 nucleotides, e.g., 12, 13, 14, 15, 16, 17 or 18 nucleotides.

Mixed sequence oligonucleotides

The oligonucleotides synthesized using the methods of the invention may be mixed sequence oligonucleotides. The invention provides a preparation method of synthetic mixed sequence oligonucleotide. The mixed sequence oligonucleotide comprises at least two, such as at least three (e.g., selected from A, T, C or G, wherein C is optionally 5-methyl-cytosine) of at least four different base moieties. Antisense oligonucleotides are typically mixed sequence oligonucleotides.

Other embodiments of the invention

Embodiment A:

1. a method of coupling an oxaazaphospholane phosphoramidite monomer to the 5' end of a nucleoside or oligonucleotide or to a hydroxyl group attached to a solid support comprising the step of reacting a nucleoside, oligonucleotide or solid support with an oxaazaphospholane phosphoramidite monomer wherein the reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent, and optionally an activator.

2. The method of embodiment 1 wherein the oxaazaphospholane phosphoramidite monomer is a compound of formula I

Wherein Z is a nucleoside, and wherein,

R⁵and R⁶Independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, substituted alkyl, substituted cycloalkyl, substituted aryl and substituted heteroaryl, or R ⁵And R⁶Together with the N atom of formula I form a packageA heterocycle having 3 to 16 carbon atoms;

R⁹is hydrogen;

R¹is selected from the following groups: hydrogen and C_1-3An alkyl group; and is

R is selected from the following groups: aryl, heteroaryl, substituted aryl, substituted heteroaryl, nitro, halogen, cyano, silyl, substituted silyl, sulfone, substituted sulfone (aryl substituted sulfone), fluorene and substituted fluorine;

wherein, when substituted, R may be substituted with a group selected from: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl radicals or C_6-14Aryl radical C_1-4An alkyl group. The multiple substitutions may be selected from the following groups, either independently or both: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl radicals or C_6-14Aryl radical C_1-4An alkyl group.

3. The method according to embodiment 1 or 2 a, wherein the aromatic heterocyclic solvent has a pKa in water of 4 to 7 or 7 to 17 at 20 ℃.

4. The method of any of embodiments a 1-3 wherein the aromatic heterocyclic solvent is an aromatic heterocyclic base.

5. The method of any one of embodiments a 1-3 wherein the aromatic heterocyclic solvent is an aromatic heterocyclic acid.

6. The process according to any one of embodiments a 1-3, wherein the aromatic heterocyclic solvent is selected from the group consisting of pyridine, 2-picoline, 4-picoline, 3-picoline, lutidine, and pyrrole.

7. The method of any one of embodiments a 1-6, wherein the concentration (v/v) of aromatic heterocyclic solvent in acetonitrile is about 0.1% to about 50% (v/v).

8. The method according to any of embodiments a 1-6, wherein the concentration of the aromatic heterocyclic solvent in acetonitrile (v/v) is from about 0.5% to about 10%, such as from about 1% to about 5%, for example from about 2-3%, such as about 2.5%.

9. The method of any one of embodiments 1-8, wherein the active agent comprises N-methylimidazole.

10. The process of any one of embodiments a 1-9, wherein the solvent mixture comprises N-methylimidazole at a concentration of 0.01 to about 1M N-methylimidazole, for example about 0.1M N-methylimidazole.

11. The method of any one of embodiments a 1-10, wherein the active agent comprises 4, 5-Dicyanoimidazole (DCI), tetrazole, or 5- (benzylthio) -1H-tetrazole.

12. The method according to any of a embodiments 1-11, wherein the solvent composition comprises about 0.5 to about 2M DCI (or other activator of a embodiment 11), for example about 1M DCI (or other activator of a embodiment 11).

13. The method according to any of embodiments a 1-12, wherein the oxaazaphospholane phosphoramidite monomer is a compound

Z, R, R therein¹、R⁶、R⁹And R⁵All as described in embodiment 2.

14. The method according to any one of embodiments a-11, a-1, wherein R is selected from the group consisting of aryl, heteroaryl, substituted aryl, and substituted heteroaryl.

15. The method according to any one of embodiments 1-11, wherein R is an aryl group, such as phenyl.

16. The method according to any one of embodiments 1-13, wherein R¹Is hydrogen.

17. The method according to any one of embodiments 1-13, wherein R¹Is C_1-3Alkyl groups, such as methyl.

18. The method according to any one of embodiments 1-15, wherein R⁵And R⁶Together form a heterocyclic ring containing from 3 to 16 (e.g. 4) carbon atoms, together with the N atom of formula (I), (Ia) or (1 b).

19. The method according to any one of embodiments 1-15, wherein R⁵And R⁶Together form a heterocyclic ring containing 4 carbon atoms, together with the N atom of formula (I), (Ia) or (1 b).

20. The method of any of embodiments a 1-19, wherein the phosphoramidite monomer compound is formula 2a or 2b

Wherein Z, R and R ¹As in any one of embodiments a 2-17.

21. The method according to any one of embodiments A1-20, wherein the oxaazaphospholane phosphoramidite monomer compound is of formula 3a or 3b

Wherein the content of the first and second substances,

R、R¹、R⁵、R⁶and R⁹As described in any one of embodiments a 2-18;

b is a nucleobase group which is,

R³is selected from CH₂ODMTr、CH₂alkyl-O-DMTr, CH-Me-O-DMTr, CH₂OMMTr、CH₂alkyl-O-MMTr, CH (Me) -O-MMTr, CH-R^a-O-DMTrR^bAnd CH-R^a-O-MMTrR^b；

R²Is selected from the following groups: halogen such as-F, amino, azido, -SH, -CN, -OCN, -CF₃、-OCF₃、-O(R^m) -alkyl, -S (R)^m) -alkyl, -N (R)^m) -alkyl, -O (R)^m) -alkenyl, -S (R)^m) -alkenyl, -N (R)^m) -alkenyl；-O(R^m) -alkynyl, -S (R)^m) -alkynyl or-N (R)^m) -an alkynyl group; O-alkylene-O-alkyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl, O (CH)₂)₂SCH₃、O-(CH₂)₂-O-N(R^m)(Rⁿ) Or O-CH₂C(＝O)-N(R^m)(Rⁿ)、 -O-(CH₂)₂OCH₃and-O-CH₃Wherein R is^mAnd RⁿEach independently is H, an amino protecting group or a substituted or unsubstituted C_1-10An alkyl group;

R⁴selected from alkyl, cycloalkyl, heterocycloalkyl, O-alkyl, S-alkyl, NH-alkyl and hydrogen;

or R²And R⁴Together represent a divalent bridge consisting of 1, 2, 3 members selected from the group consisting of-C (R)^aR^b)-、 -C(R^a)＝C(R^b)、-C(R^a)＝N、O、-Si(R^a)₂-、S-、-SO₂-、-N(R^a) -and>c ═ Z groups/atom composition;

wherein R is^aAnd R^bEach (when present) is independently selected from hydrogen, optionally substituted C _1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkynyl, hydroxy, optionally substituted C_1-6-alkoxy, C_2-6Alkoxyalkyl group, C_2-6-alkenyloxy, carboxy, C_1-6Alkoxycarbonyl, C_1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di- (C)_1-6-alkyl) amino, carbamoyl, mono-and di- (C)_1-6-alkyl) -amino-carbonyl, amino-C_1-6-alkyl-aminocarbonyl, mono-and di- (C)_1-6-alkyl) amino-C_1-6-alkyl-aminocarbonyl, C_1-6-alkyl-carbonylamino, ureido, C_1-6Alkanoyloxy, sulfonyl, C_1-6Alkylsulfonyloxy, nitro, azido, sulfanyl, C_1-6Alkylthio, halogen, wherein aryl andheteroaryl may be optionally substituted, and wherein two geminal substituents R^aAnd R^bTaken together may represent optionally substituted methylene (═ CH)₂) Wherein the asymmetric group can be either R or S oriented for all chiral centers.

22. The method according to any one of embodiments a 1-21, wherein the oxaazaphospholane phosphoramidite monomer is selected from the group consisting of formulas 4a, 4b, 5a, 5b, 6a, 6b, 7a and 7 b.

23. The method according to any of embodiments a-23, wherein the oxaazaphospholane phosphoramidite monomer comprises a nucleobase moiety which is a purine or pyrimidine, e.g. a nucleobase selected from the group consisting of adenine, guanine, uracil, thymine and cytosine, isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thiouracil, 2' -thiothymine, inosine, diaminopurine, 6-aminopurine, 2, 6-diaminopurine and 2-chloro-6-aminopurine.

24. The method according to any one of embodiments a 1-23, wherein B in the oxaazaphospholane phosphoramidite monomer is adenine or thymine.

25. The method according to any of embodiments 1-24, wherein the oxaazaphospholane phosphoramidite monomer is selected from the group consisting of formula 8a or formula 8b

Wherein B is adenine or thymine, and wherein R, R¹、R³And R⁹The method of any one of embodiments 1-24, wherein when B is adenine, it may be protected, for example with benzoyl).

26. A process according to any one of embodiments A1 to 24, wherein the oxaazaphospholane phosphoramidite monomer is as described in formula 9c

Wherein B is adenine, and wherein R, R¹、R³And R⁹As described in any of embodiments 1-24 of a, wherein when B is adenine, it may be protected, for example with benzoyl.

27. The method of any one of embodiments 1-26, wherein R is phenyl, R is¹Is hydrogen or methyl, R⁹Is hydrogen, and R³Is selected from CH₂ODMTr、CH₂alkyl-O-DMTr, CH-Me-O-DMTr, CH₂OMMTr、CH₂alkyl-O-MMTr, CH (Me) -O-MMTr, CH-R^a-O-DMTrR^bAnd CH-R^a-O-MMTrR^bE.g. CH₂-O-DMTr or CH₂-O-MMTr。

28. An acetonitrile solution comprising an oxaazaphospholane phosphoramidite monomer as described in any of embodiments 1-27 of a, acetonitrile and an aromatic heterocyclic solvent.

29. The acetonitrile solution of embodiment a 28 wherein the concentration of the oxaazaphospholane phosphoramidite monomer is from about 0.05M to about 2M, for example from about 0.1M to about 1M, for example from about 0.1M to about 0.2M, for example about 0.15M, or about 0.175M, or about 0.2M.

30. The acetonitrile solution of embodiment a 28 or 29 wherein the aromatic heterocyclic solvent is as described in any one of embodiments a 1-28.

31. The acetonitrile solution according to any one of embodiments a-28-30 wherein the oxaazaphospholane phosphoramidite monomer is according to any one of embodiments a-1-28.

32. The acetonitrile solution of any one of embodiments a-31, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is about 0.1% to about 50% (v/v).

33. The acetonitrile solution of any one of embodiments a-32, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is from about 0.5% to about 10%, e.g., from about 1% to about 5% (v/v), e.g., about 2-3%, e.g., about 2.5%.

34. The acetonitrile solution of any one of embodiments a-33 wherein the acetonitrile solution further comprises an activator, for example an activator as described in any one of embodiments a-9-12.

35. The acetonitrile solution of embodiment a 34, wherein the acetonitrile solution comprises about 0.5 to about 2M DCI, e.g., about 1M DCI.

36. The acetonitrile solution of any one of embodiments a to 35 wherein the acetonitrile solution comprises from about 0.01 to about 1M N-methylimidazole, for example about 0.1M N-methylimidazole.

37. A method of oligonucleotide synthesis comprising the method of any one of embodiments 1-27 of a.

38. A method of synthesizing an oligonucleotide as described in embodiment a 37, said method comprising the steps of:

a) providing a solid support having free 5' -OH groups;

b) activating an oxaazaphospholanepolylamine monomer according to any of embodiments A from 21 to 27, for example in a solution according to any of embodiments A from 1 to 36,

c) Coupling an activated oxaazaphospholane phosphoramidite monomer to the free' 5-OH according to the method of any of embodiments 1-27 of A to form a phosphite triester intermediate,

d) the phosphite triester intermediate is oxidized with a sulfurizing reagent such as hydrogenated xanthane,

e) blocking any free-OH groups, e.g. using acetic anhydride,

f) r on the oxaazaphospholane phosphoramidite monomer³The group is deprotected, and the reaction is carried out,

g) optionally repeating steps b) -f),

h) deprotecting any remaining protecting groups (global deprotection) and cleaving the oligonucleotide from the solid support, e.g.by treatment with ammonium hydroxide at 60 ℃,

wherein the free-OH group of the solid support can optionally be attached to a nucleoside or oligonucleotide chain attached to the solid support.

39. A method of dissolving an oxaazaphospholane phosphoramidite monomer, for example a monomer according to any of embodiments 1-27, comprising adding the monomer to a solvent composition comprising acetonitrile and an aromatic heterocyclic solvent, and optionally an activator.

40. Use of an aromatic heterocyclic solvent to enhance the stability and/or solubility and/or reactivity of an oxaazaphospholane phosphoramidite monomer, for example a monomer according to any of embodiments 1-27 in acetonitrile.

41. The process, method, acetonitrile solution or use according to any one of the preceding embodiments a, wherein the oxaazaphospholane phosphoramidite monomer is not an L-LNA-guanine monomer wherein the guanine is protected by DMF.

Embodiment B:

1. a method of synthesizing a sterically defined phosphorothioate oligonucleotide comprising the steps of:

a) deprotecting the protected 5' -hydroxy terminus of a nucleoside or oligonucleotide attached to a solid support,

b) coupling an oxaazaphospholane phosphoramidite monomer to the deprotected 5' -hydroxy terminus of a nucleoside or oligonucleotide, wherein the coupling reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent to form a phosphite triester intermediate, and

c) oxidizing the phosphite triester intermediate with a vulcanizing agent,

d) optionally repeating steps a) -c) for one or more further elongation cycles,

e) the oligonucleotide is deprotected from the solid support and cleaved.

2. The method of embodiment 2 wherein the method comprises a plurality of further extension cycles.

3. The method of embodiment 3 wherein the sterically defined phosphorothioate oligonucleotide is an antisense oligonucleotide.

4. A method of coupling an oxaazaphospholane phosphoramidite monomer to the 5' end of a nucleoside or oligonucleotide comprising the step of reacting the nucleoside or oligonucleotide with the oxaazaphospholane phosphoramidite monomer wherein the reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent.

5. The method of any of embodiments 1-4 wherein the aromatic heterocyclic solvent has a pKa in water of 4 to 7 or 7 to 17 at 20 ℃.

6. The method of any one of embodiments 1-5 wherein the aromatic heterocyclic solvent is an aromatic heterocyclic base.

7. The method of any one of embodiments 1-5 wherein the aromatic heterocyclic solvent is an aromatic heterocyclic acid.

8. The method according to any one of embodiments 1 to 5, wherein the aromatic heterocyclic solvent is selected from the group consisting of pyridine, 2-methylpyridine, 4-methylpyridine, 3-methylpyridine, lutidine, and pyrrole.

9. The method of any one of embodiments 1-8 wherein the aromatic heterocyclic solvent is pyridine.

10. The method of any one of embodiments 1-9 wherein the concentration (v/v) of aromatic heterocyclic solvent in acetonitrile is about 0.1% to about 50% (v/v), for example about 0.5% to about 25%.

11. The method of any one of embodiments 1-9 wherein the concentration (v/v) of aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 10%, such as about 1% to about 5%, for example about 2-4%, such as about 2.5%, or about 3.5%.

12. The method of any one of embodiments 1-11 wherein the acetonitrile solvent composition further comprises an activator.

13. The method of embodiment 12 wherein the activator is selected from the group consisting of CMPT (N- (cyanomethyl) pyrrolidinium triflate (CMPT), N- (phenyl) imidazolium triflate (PhIMT), benzimidazolium triflate (BIT), 4, 5-Dicyanoimidazole (DCI), tetrazole and 5- (benzylthio) -1H-tetrazole.

14. The method of embodiment 13 wherein the activator is 4, 5-Dicyanoimidazole (DCI).

15. The method of any of B embodiments 1-14, wherein the solvent composition comprises from about 0.5 to about 2M DCI (or other activating agent of B embodiment 13), for example about 1M DCI (or other activating agent of B embodiment 13).

16. The method of any of B embodiments 12-15 wherein the solvent composition further comprises N-methylimidazole, e.g., N-methylimidazole at a concentration of 0.01 to about 1M N-methylimidazole, e.g., about 0.1M N-methylimidazole.

17. A process according to any one of embodiments 1 to 16, wherein the oxaazaphospholane phosphoramidite monomer is a compound of formula I

Wherein Z is a nucleoside, and wherein,

R⁵and R⁶Independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, substituted alkyl, substituted cycloalkyl, substituted aryl and substituted heteroaryl, or R⁵And R⁶Together with the N atom of formula I form a heterocyclic ring containing from 3 to 16 carbon atoms;

R⁹is hydrogen;

R¹is selected from the following groups: hydrogen and C_1-3An alkyl group; and is provided with

R is selected from the following groups: aryl, heteroaryl, substituted aryl, substituted heteroaryl, nitro, halogen, cyano, silyl, substituted silyl, sulfone, substituted sulfone (aryl substituted sulfone), fluorene and substituted fluorine;

wherein, when substituted, R may be substituted with a group selected from: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C_1-4Alkoxy radical C_6-14Aryl radicals or C_6-14Aryl radical C_1-4An alkyl group.The multiple substitutions may be selected from the following groups, either independently or both: c_1-4Alkyl radical, C_6-14Aryl radical, C_1-4Alkoxy radical, C_7-14Aralkyl radical, C_1-4Alkyl radical C_6-14Aryl radical, C _1-4Alkoxy radical C_6-14Aryl radicals or C_6-14Aryl radical C_1-4An alkyl group.

18. The method according to any one of embodiments 1-17 of B, wherein the oxaazaphospholane phosphoramidite monomer is a compound

Z, R, R therein¹、R⁶、R⁹And R⁵All as described in embodiment B17.

19. A method as set forth in B embodiment 17 or 18 wherein R is selected from the group of aryl, heteroaryl, substituted aryl, and substituted heteroaryl.

20. The method of any one of embodiments 17-19 of B wherein R is an aryl group, such as phenyl.

21. The method of any one of embodiments 17-20 of B, wherein R¹Is hydrogen.

22. The method of any one of embodiments 17-21 of B, wherein R¹Is C_1-3Alkyl groups, such as methyl.

23. The method of any one of embodiments 17-22 of B, wherein R⁵And R⁶Together form a heterocyclic ring containing from 3 to 16 (e.g. 4) carbon atoms, together with the N atom of formula (I), (Ia) or (1 b).

24. The method of any one of embodiments 17-22 of B, wherein R⁵And R⁶Together form a heterocyclic ring containing 4 carbon atoms, together with the N atom of formula (I), (Ia) or (1 b).

25. The method of any of embodiments 1-24 of B wherein the phosphoramidite monomer compound is of formula 2a or 2B

Wherein Z, R and R¹The method according to any one of embodiments 17 to 24.

A process as in any one of embodiments 1-25 wherein the oxaazaphospholane phosphoramidite monomer compound is of formula 3a or 3B

Wherein the content of the first and second substances,

R、R¹、R⁵、R⁶and R⁹As described in any one of embodiments 2-18 of B;

b is a nucleobase group which is,

R³is selected from CH₂ODMTr、CH₂alkyl-O-DMTr, CH-Me-O-DMTr, CH₂OMMTr、CH₂alkyl-O-MMTr, CH (Me) -O-MMTr, CH-R^a-O-DMTrR^bAnd CH-R^a-O-MMTrR ^b；

R²Is selected from the following groups: halogen such as-F, amino, azido, -SH, -CN, -OCN, -CF₃、-OCF₃、-O(R^m) -alkyl, -S (R)^m) -alkyl, -N (R)^m) -alkyl, -O (R)^m) -alkenyl, -S (R)^m) -alkenyl, -N (R)^m) -an alkenyl group; -O (R)^m) -alkynyl, -S (R)^m) -alkynyl or-N (R)^m) -an alkynyl group; O-alkylene-O-alkyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl, O (CH)₂)₂SCH₃、O-(CH₂)₂-O-N(R^m)(Rⁿ) Or O-CH₂C(＝O)-N(R^m)(Rⁿ)、 -O-(CH₂)₂OCH₃and-O-CH₃Wherein R is^mAnd RⁿEach independently is H, an amino protecting group or a substituted or unsubstituted C_1-10An alkyl group;

R⁴selected from alkyl, cycloalkyl, heterocycloalkyl,O-alkyl, S-alkyl, NH-alkyl and hydrogen;

or R²And R⁴Together represent a divalent bridge consisting of 1, 2, 3 members selected from the group consisting of-C (R)^aR^b)-、 -C(R^a)＝C(R^b)、-C(R^a)＝N、O、-Si(R^a)₂-、S-、-SO₂-、-N(R^a) -and>c ═ Z groups/atom composition;

wherein R is^aAnd R^bEach (when present) is independently selected from hydrogen, optionally substituted C _1-6-alkyl, optionally substituted C_2-6-alkenyl, optionally substituted C_2-6-alkynyl, hydroxy, optionally substituted C_1-6-alkoxy, C_2-6Alkoxyalkyl group, C_2-6-alkenyloxy, carboxy, C_1-6Alkoxycarbonyl, C_1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di- (C)_1-6-alkyl) amino, carbamoyl, mono-and di- (C)_1-6-alkyl) -amino-carbonyl, amino-C_1-6-alkyl-aminocarbonyl, mono-and di- (C)_1-6-alkyl) amino-C_1-6-alkyl-aminocarbonyl, C_1-6-alkyl-carbonylamino, ureido, C_1-6Alkanoyloxy, sulfonyl, C_1-6Alkylsulfonyloxy, nitro, azido, sulfanyl, C_1-6Alkylthio, halogen, wherein aryl and heteroaryl may be optionally substituted, and wherein two geminal substituents R^aAnd R^bTaken together may represent optionally substituted methylene (═ CH)₂) Wherein the asymmetric group may be either R or S oriented for all chiral centers.

A method as in any one of embodiments 1-26 wherein the oxaazaphospholane phosphoramidite monomer is selected from the group consisting of formulas 4a, 4B, 5a, 5B, 6a, 6B, 7a and 7B.

R, R therein¹、R³、R⁹、R⁵、R⁶And B is as in embodiment 26 of B.

28. The method of any of embodiments 1-27 wherein the oxaazaphospholane phosphoramidite monomer comprises a nucleobase moiety which is a purine or pyrimidine, e.g., a nucleobase selected from the group consisting of adenine, guanine, uracil, thymine and cytosine, isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thiouracil, 2' -thiothymine, inosine, diaminopurine, 6-aminopurine, 2, 6-diaminopurine, and 2-chloro-6-aminopurine.

29. The method of any of B embodiments 1-28 wherein the oxaazaphospholane phosphoramidite monomer is selected from M1 to M40.

30. The method of any of B embodiments 1-29 wherein the base moiety (B) of the oxaazaphospholane phosphoramidite monomer comprises an adenine base.

31. The method of any of B embodiments 1-30 wherein the base moiety (B) of the oxaazaphospholane phosphoramidite monomer comprises a thymine base.

32. The method of any of B embodiments 1-30 wherein the base moiety (B) of the oxaazaphospholane phosphoramidite monomer comprises a guanine base.

33. The method of any of B embodiments 1-30 wherein the base moiety (B) of the oxaazaphospholane phosphoramidite monomer comprises a cytosine base.

34. The method of any of B embodiments 1-33 wherein the oxaazaphospholane phosphoramidite monomer is an L monomer.

35. The method of any of embodiments 1-33 of B wherein the oxaazaphospholane phosphoramidite monomer is a D monomer.

36. A method as in any one of embodiments 1-35 wherein the oxaazaphospholane phosphoramidite monomer is an LNA monomer, for example a β -D-oxolna monomer.

37. The method of any of embodiments 1-36 wherein the oxaazaphospholane phosphoramidite monomer is a DNA monomer.

38. The method of any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from formula 8a or formula 8B

Wherein B is thymine, and wherein R, R¹、R³And R⁹The method according to any one of embodiments 17 to 24.

39. The method of any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from formula 8a or formula 8B

Wherein B is adenine, and wherein R, R¹、R³And R⁹The compound of any one of embodiments 17 to 24, wherein adenine may be protected, for example with benzoyl).

40. A method as in any of B embodiments 1-28 wherein the oxaazaphospholane phosphoramidite monomer is selected from D-DNA-A or L-DNA-A monomers, for example of the formula

Wherein A is adenine, and wherein R, R¹、R³And R⁹As described in any of embodiments 1-24 of B, wherein the base adenine may be protected, for example with benzoyl.

41. A method as described in any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from a D-DNA-T or L-DNA-T monomer, for example an oxaazaphospholane phosphoramidite monomer of the formula

Wherein T is thymine, and wherein R, R¹、R³And R⁹As in any one of embodiments 1-24 of B.

42. A method as described in any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from a D-DNA-C or L-DNA-C monomer, for example an oxaazaphospholane phosphoramidite monomer of the formula

Wherein C is cytosine, and wherein R, R ¹、R³And R⁹The method of any one of embodiments 1 to 24 wherein the base cytosine may be protected, for example with acetyl or benzoyl, and wherein optionally the cytosine is a 5-methylcytosine.

43. The method of any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from a D-DNA-G or L-DNA-G monomer, for example an oxaazaphospholane phosphoramidite monomer of the formula

Wherein G is guanine, and wherein R, R¹、R³And R⁹As described in any of embodiments 1-24 of B, wherein the base guanine may be protected, for example with DMF or an acyl group such as iBu.

44. A method as described in any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from a D-LNA-A or L-LNA-A monomer, for example an oxaazaphospholane phosphoramidite monomer of the formula

Wherein A is adenine and wherein R, R¹、R³And R⁹As described in any of B embodiments 1-24, wherein the base adenine may be protected, for example with benzoyl.

45. A method as described in any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from a D-LNA-T or L-LNA-T monomer, for example an oxaazaphospholane phosphoramidite monomer of the formula

Wherein T is thymine, and wherein R, R¹、R³And R⁹As in any one of embodiments 1-24 of B.

46. A process as claimed in any one of embodiments 1 to 28, wherein the oxaazaphospholane phosphoramidite monomer is selected from D-LNA-C or L-LNA-C monomers, for example of the formula

Wherein C is cytosine, and wherein R, R¹、R³And R⁹As implemented in BThe method of any one of schemes 1-24, wherein the base cytosine is protected, e.g., with acetyl or benzoyl, and wherein optionally the cytosine is a 5-methylcytosine.

47. A method as described in any of embodiments 1-28 of B wherein the oxaazaphospholane phosphoramidite monomer is selected from a D-LNA-G or L-LNA-G monomer, for example an oxaazaphospholane phosphoramidite monomer of the formula

Wherein G is guanine, and wherein R, R¹、R³And R⁹The process according to any one of embodiments 1 to 24, wherein the base guanine may be protected by an acyl group such as iBu for L-LNA-G monomers or by an acyl group (such as iBu) or DMF for D-LNA-G monomers.

48. The method of any of embodiments 1-47 wherein the oxaazaphospholane phosphoramidite monomer is a DNA monomer or is an LNA monomer selected from the group consisting of: LNA-A monomers, LNA-C monomers and acyl protected L-LNA-G monomers.

49. The method of any one of embodiments 1-47 wherein the oxaazaphospholane phosphoramidite monomer is not an LNA-T monomer, a D-LNA-G monomer or a DMF protected L-LNA-G monomer.

50. The method of any one of embodiments 17-49 of B wherein R is phenyl and R is¹Is hydrogen or methyl, R⁹Is hydrogen, and R³Is selected from CH₂ODMTr、CH₂alkyl-O-DMTr, CH-Me-O-DMTr, CH₂OMMTr、CH₂alkyl-O-MMTr, CH (Me) -O-MMTr, CH-R^a-O-DMTrR^bAnd CH-R^a-O-MMTrR^bE.g. CH₂-O-DMTr or CH₂-O-MMTr。

51. The method of any one of embodiments 17-49 of B wherein R is phenyl and R is¹Is hydrogen or methyl, R⁹Is hydrogen, and R³is-CH₂-O-DMTr。

52. An acetonitrile solution comprising an oxaazaphospholane phosphoramidite monomer as described in any of embodiments B17-51, acetonitrile and an aromatic heterocyclic solvent.

53. The acetonitrile solution of embodiment B52 wherein the concentration of the oxaazaphospholane phosphoramidite monomer is from about 0.05M to about 2M, for example from about 0.1M to about 1M, for example from about 0.1M to about 0.2M, for example about 0.15M, or about 0.175M, or about 0.2M.

54. The acetonitrile solution of B embodiment 52 or 53 wherein the aromatic heterocyclic solvent is as described in any one of B embodiments 1-16.

55. The acetonitrile solution of any one of B embodiments 52-54, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is about 0.1% to about 50% (v/v), e.g., about 0.5% to about 25% (v/v).

56. The acetonitrile solution of any one of B embodiments 52-55, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is about 0.5% to about 10%, for example about 1% to about 5% (v/v), for example about 2-4%, for example about 2.5%, such as about 3.5%.

Examples

Example 1: general synthetic methods:

PCl was added to a solution of N-methylmorpholine in toluene (50mL) at-70 ℃ over a period of 10 minutes₃(2.93mL 33.4 mmol). Thereafter, a solution of proline (P5-D or P5-L) adjuvant (6.24g,35.2mmol) in toluene (50mL) was added over 30 minutes (see J.Am.chem.Soc.,2008,130, 16031-16037 for synthesis of P5-D and P5-L). The resulting mixture was stirred at room temperature for 1.5 hours, then the solvent and volatiles were removed in vacuo (40 ℃,15 mbar). The remaining residue was then dissolved in THF (50mL), cooled to-70 ℃ and NEt was added first₃(17.8mL,128 mmol) and then a solution of 5' -ODMT-DNA-nucleoside (16mmol) in THF (50mL) was added over 30 minutes. The reaction mixture was stirred at-77 ℃ for 30 minutes and then at room temperature for 2 hours. Thereafter, cold EtOAc (200mL) was added and the mixture was quenched with cold NaHCO₃Washed (150mL), brine (150mL), and dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography under argon, washed The stripping solution contained 7% NEt₃To avoid degradation on silica gel.

The product obtained is a solid, possibly containing small amounts of residual solvents, such as EtOAc, THF and NEt₃。

Using the above method, the following monomers were synthesized:

D-DNA A:³¹P NMR(160MHz,DMSO-d₆):δ150.3

L-DNA A:³¹P NMR(160MHz,DMSO-d₆):δ148.5

D-DNA T:³¹P NMR(160MHz,DMSO-d₆):δ151.0

L-DNA T:³¹P NMR(160MHz,DMSO-d₆):δ149.1

D-DNA C:³¹P NMR(160MHz,DMSO-d₆):δ151.7

L-DNA C:³¹P NMR(160MHz,DMSO-d₆):δ149.8

D-DNA G-i-Bu:³¹P NMR(160MHz,DMSO-d₆):δ151.7

L-DNA G-DMF:³¹P NMR(160MHz,DMSO-d₆):δ150.3

example 2

Synthesis of D-LNA-G-DMF

5' -ODMT-LNA-G (3.51G,5.00mmol) was co-evaporated with pyridine and then toluene to remove any residual water or other solvent. The residue was then dissolved in pyridine (10mL) and THF (10 mL). This solution was added to D-oxaazaphospholane (3.51g,5.00mmol), PCl at-77 deg.C₃(0.88mL,10.0mmol) and NEt₃(3.50mL,25.0 mmol). The resulting reaction mixture was then stirred at-77 ℃ for 15 minutes and then at room temperature for 1.5 hours. Thereafter, EtOAc (150mL) was added and the mixture was quenched with cold NaHCO₃Washed (100mL) with brine (100mL) and Na₂SO₄Dried, filtered and finally evaporated together with toluene.

By column chromatography (eluent: EtOAc solution of THF, from 10% to 30% + 7% NEt₃) The resulting residue was purified to give D-LNA-G-DMF (3.91G, estimated 84% yield).

¹H NMR(400MHz,DMSO-d₆):δ11.42(1H,s),8.56(1H,s),7.95(1H, s),7.49-7.16(14H,m),6.90-6.83(4H,m),5.96(1H,s),5.58(1H,d,J＝6.7 Hz),3.87(1H,d,J＝8.1Hz),3.72(6H,s),3.62-3.54(1H,m),3.45(2H,s), 3.40-3.33(1H,m),3.08(3H,s),2.99(3H,s),2.93-2.84(1H,m),1.53-1.39(2H, m),1.06-0.97(1H,m),0.79-0.63(1H,m)。

³¹P NMR(160MHz,DMSO-d₆):δ151.6

LRMS(ESI)m/z[M+H]⁺C₄₆H₄₉N₇O₈Calculated value of P858.3. Found 858.7.

Example 3

Synthesis of L-LNA-G-DMF

5' -ODMT-LNA-G (4.91G,7.00mmol) was co-evaporated with pyridine and then toluene to remove any residual water or other solvent. The residue was then dissolved in pyridine (10mL) and THF (15 mL). This solution was added to L-oxaazaphospholane (2.48g, 14.0mmol), PCl at-77 deg.C ₃(1.22mL,14.0mmol) and NEt₃(4.90mL,35.0 mmol). The resulting reaction mixture was then stirred at-77 ℃ for 15 minutes and then at room temperature for 1.5 hours. Thereafter, EtOAc (150mL) was added and the mixture was quenched with cold NaHCO₃Washed (100mL) with brine (100mL) and Na₂SO₄Dried, filtered and finally evaporated together with toluene.

Column chromatography (eluent: THF in EtOAc/DCM 1: 1, using 15% to 25% + 7% NEt₃Gradient of (D) purified the resulting residue to give D-LNA-G-DMF (3.41G, estimated 84% yield). The product was purified by column chromatography as described above.

¹H NMR(400MHz,DMSO-d₆):δ12.3-11.9(1H,br s),11.8-11.5(1H, br s),8.05(1H,s),7.45-7.40(2H,m),7.35-7.21(10H,m),7.02-6.97(2H,m), 6.92-6.86(4H,m),5.94(1H,s),5.09(1H,d,J＝6.5Hz),4.88(1H,d,J＝7.5 Hz),4.69(1H,s),3.89-3.81(2H.m),3.74(3H,s),3.73(3H,s),3.71-3.64(1H, m),3.48-3.38(3H,m),2.83-2.73(1H,m),2.71-2.64(1H,m),1.55-1.45(2H, m),1.14-1.05(1H,m),1.08(3H,d,J＝6.9Hz),1.05(3H,d,J＝6.9Hz), 0.76-0.66(1H,m)。

³¹P NMR(160MHz,DMSO-d₆):δ148.7

LRMS(ESI)m/z[M+H]⁺C₄₇H₅₀N₆O₉Calculated value of P873.3. Found 873.7.

Example 4

Synthesis of D-DNA G-DMF

PCl was added to a solution of N-methylmorpholine in toluene (50mL) at-70 ℃ over a period of 10 minutes₃(2.93mL,33.4 mmol). Thereafter, a solution of P5-D (6.24g, 35.2mmol) in toluene (50mL) was added over 30 minutes. The resulting reaction mixture was stirred at room temperature for 1.5 hours, then the solvent and volatiles were removed in vacuo (40 ℃,15 mbar). The remaining residue was then dissolved in THF (50mL), cooled to-70 ℃ and NEt was added first₃(17.8mL,128mmol) and then a solution of 5' -ODMT-DNA-G (9.99G,16.0mmol) in THF (50mL) was added over 30 minutes. The reaction mixture was stirred at-77 ℃ for 30 minutes and then at room temperature for 2 hours. After this time, cold EtOAc (200mL) was added and the mixture was quenched with cold NaHCO ₃Washed (150mL), brine (150mL), and dried (Na)₂SO₄) Filtered and evaporated to dryness. The crude product was purified by flash column chromatography under argon (eluent: DCM/EtOAc: 2/1+ 7% NEt)₃). The D-DNA-G-DMF was isolated as a white foam (10.6G, 72%) with traces of solvent impurities (EtOAc, toluene and NEt)₃)。

¹H NMR(400MHz,DMSO-d₆):δ11.36(1H,s),8.52(1H,s),7.96(1H, s),7.40-7.16(14H,m),6.83-6.77(4H,m),6.27(1H,t,J＝6.4Hz),5.65(1H, d,j＝6.5Hz),5.08-5.01(1H,m),4.02-3.98(1H,m),3.91-3.83(1H,m),3.71 (6H,s),3.45-3.35(1H,m),3.27-3.18(2H,m),3.07(3H,s),3.00(3H,s), 2.97-2.88(2H,m),2.49-2.40(1H,m),1.58-1.48(1H,m),1.47-1.38(1H,m), 1.16-1.09(1H,m),0.86-0.76(1H,m)。

³¹P NMR(160MHz,DMSO-d₆):δ151.7

LRMS(ESI)m/z[M-H]^-C₄₅H₄₇N₇O₇Calculated value of P828.3. Measured in factValue 828.6.

Example 5

Synthesis of L-DNA G-DMF

PCl was added to a solution of N-methylmorpholine in toluene (25mL) over a period of 5 minutes at-55 deg.C₃(1.33mL,15.2mmol) and then a solution of P5-L (2.84g,16.00mmol) in toluene (25mL) was added over 15 minutes. The resulting reaction mixture was stirred at-55-45 ℃ for 10 minutes and then at room temperature for 1.5 hours. The solvent and other volatiles were then removed in vacuo (40 ℃,6 mbar). The remaining residue was then dissolved in THF (25mL) and cooled to-77 ℃. Thereafter, NEt is added₃(8.92mL,64mmol) and then a solution of 5' -ODMT-DNA-G-DMF (4.5G, 7.2mmol) in THF (25mL) was added over 15 minutes. The reaction mixture was stirred at-77 ℃ for 15 minutes and then at room temperature for 3 hours. Thereafter, EtOAc (150mL) was added and the mixture was quenched with cold NaHCO₃(100mL), brine (50mL), and dried (Na) ₂SO₄) Filtered and evaporated.

Flash column chromatography under argon (eluent: EtOAc/DCM ═ 1/2+ 7% NEt₃) The product was isolated as a white foam (3.77g, 63%) with traces of EtOAc.

¹H NMR(400MHz,DMSO-d₆):δ11.36(1H,s),8.51(1H,s),7.96(1H, s),7.39-7.11(14H,m),6.80-6.73(4H,m),6.28(1H,t,J＝6.5Hz),5.72(1H, d,j＝6.5Hz),5.06-4.96(1H,m),4.02-3.95(1H,m),3.84-3.76(1H,m),3.70 (3H,s),3.69(3H,s),3.50-3.39(1H,m),3.27-3.18(2H,m),3.08(3H,s),3.02 (3H,s),2.98-2.83(2H.m),2.48-2.39(1H,m),1.58-1.40(2H,m),1.12-1.02 (1H,m),0.83-0.71(1H,m)。

³¹P NMR(160MHz,DMSO-d₆):δ150.3

LRMS(ESI)m/z[M+H]⁺C₄₅H₄₉N₇O₇Calculated value of P830.3. Found 830.6.

Example 6

Synthesis of L-LNA-G-Ibu monomer

Method for synthesizing 5' -OAP-LNA-G-iBu derivative

Step A: to a solution of N-methylmorpholine (1.76mL,16.0mmol) in toluene (15mL) at-55 deg.C was added PCl over a period of 5 minutes₃(0.66mL,7.6 mmol). Thereafter, a solution of (S) -phenyl- (R) -pyrrolidin-2-yl) methanol (P5-D) (1.42g,8.00mmol) in toluene (12mL) was added over the next 15 minutes. The reaction mixture was then stirred between-55 and-45 ℃ for 10 minutes, then at room temperature for 1.5 hours. The solvent and other volatile compounds were removed in vacuo at 40 ℃ and 6 mbar, followed by addition of THF (13 mL).

And B: the reaction mixture was then cooled to-77 deg.C, then triethylamine (5.54mL, 40mmol) was added, followed by a solution of 5' -ODMT-LNA-G-iBu (2.67G,4mmol) in THF (13mL) over 15 minutes. The resulting mixture was stirred at-77 ℃ for 15 minutes and then at room temperature for 3 hours. Thereafter, EtOAc (75mL) was added and the mixture was quenched with cold NaHCO ₃Washed (50mL) with brine (50mL) and Na₂SO₄Dried, filtered and evaporated in vacuo. Flash column chromatography (EtOAc: Hexane 1:4+ 7% NEt)₃) The crude product was purified under argon.

The product was obtained as a white foam (1.95g, estimated 55% yield).

In DMSO³¹P-NMR 148.8ppm+1％28.8ppm。

Additional optimization of D-LNA G-iBu and L-LNA G-iBu synthesis

A slight excess of PCl relative to the precursor (e.g., P5) was found₃Resulting in the formation of by-products which significantly reduce the yield of the product (e.g., OAP-LNA-GiBu). Therefore, it is preferred to use at least molar equivalent amounts of precursor and PCl₃. In some embodiments, step 1 the precursor is reacted with PCl₃Is greater than about 1, for example, greater than 1.05. In some embodiments, step 1 the precursor is reacted with PCl₃Is not more than 1.5.

It was found that using more than 2-fold molar equivalents of intermediate in step 2 resulted in the highest product yield (see table, entries 3 and 5). In some embodiments, an intermediate (e.g., 5' -ODMT-G/iBu) is combined with a precursor and PCl₃Is greater than 2.

Purity of the product is determined by³¹And (4) determining a P-NMR spectrum.

Example 7

Determination of product stability and solubility

To investigate the stability and solubility of L-LNA G-DMF and L-LNA G-i-Bu, the following experimental procedure was followed:

a1.5 mL vial was charged with 0.013mmol of phosphoramidite, and the solid material was dissolved in 0.13mL of solvent. Thereafter, the vial was capped, vortexed, and finally left at room temperature for 24 hours. Then, the solubility of the dissolved substance was visually checked (fig. 1). The solubility was set to "no" if the solution appeared cloudy or uneven. If the solution appeared to be completely homogeneous, the solubility was set to "yes" (repeat examination after 24 hours).

The stability determination method comprises the following steps: to complete the analysis, the stability of phosphoramidites was studied using an Agilent 1100 series HPLC-MS, using a gradient elution from 80% a (1% NH4OH in H2O) to 100% B (20% a in MeCN) and a Waters Xterra MS c182.1x100mm column. The mass and UV peak of the parent compound were identified at 0 and 24 hours. Thereafter, the relative stability compared to other by-products was reported by integrating the UV chromatogram (254nm) and normalizing the area to the chromatogram recorded at 0 hours (fig. 2).

The solubility data for the three monomers at 0 hours and 24 hours post synthesis are shown in figure 1. The stability data measured after 24 hours in various solvents are shown in FIG. 2 and in FIGS. 3a (L-LNA-G-iBu) and 3b (L-LNA-G-DMF).

The monomer L-LNA G-DMF is insoluble in most solvents (MeCN, MeCN: DCE, MeCN: toluene, MeCN: acetone, dioxane and THF). Solvents in which the monomers are soluble (MeCN: DCM, DMF, DMSO, NMP, DCM, DCE and toluene) show great instability. The best solvent is DCM, which leaves 10% phosphoramidite after 24 hours.

The monomer L-LNA G-i-Bu is soluble in all solvents studied (12 different solvents), and the best performing ones are MeCN, MeCN: acetone, DCM and DCE. All solvents investigated for the L-LNA G-i-Bu monomer showed significant improvement in solubility and stability.

Example 8

Relative coupling efficiency in model systems:

model system: 5 '-gcattggtatt(LNA A) cattgttgtttt-3'

To delay the coupling efficiency of conventional LNA phosphoramidites, LNA a was diluted to 0.025M in MeCN (with and without 5% pyridine). Thereafter, the amidate was used in the model system (5 '-gcattggtatt(LNA A) cattgttgtttt-3'). Here the 3' flank was identified in the crude mixture after deprotection and compared to the full length product to obtain the relative coupling efficiency of the monomers in question, i.e. LNA a 0.025M and LNA a 0.025M + 5% pyridine.

The results show that by reducing the concentration of monomer in solution, it is indeed possible to restart the coupling. However, it also shows that in the case of LNA a, the addition of pyridine decreases the reactivity (fig. 4).

Example 9 Triethylamine stabilizes the oxaazaphospholane phosphoramidite monomer solution but does not improve the coupling efficiency.

Here L-LNA A is monitored for Et₃Stability in the presence of N (5-10 equivalents compared to phosphoramidite).

To investigate the stability and solubility of L-LNA a, the following experimental procedure was followed.

A1.5 mL vial was charged with 0.013mmol of the phosphoramidite and the solid material was dissolved in 0.13mL of solvent (about 5-10 equivalents with and without Et 3N). Thereafter, the vial was capped, vortexed, and finally left at room temperature for 24 hours. To investigate the stability of the phosphoramidite, an Agilent 1100 series HPLC-MS was used, using 80% A (1% NH) ₄OH in H₂O) gradient elution to 100% B (20% a in MeCN) and Waters Xterra MS c182.1x 100mm column. The mass and UV peak of the parent compound were identified at 0 and 24 hours. Thereafter, other minor matters were reportedRelative stability of the product phase ratio. This process was repeated again after 48 hours.

The results (fig. 5) show that the stability of L-LNA a is very unstable over time in the presence of MeCN only. After 24 hours, most of the L-LNA A was degraded. After 48 hours, the L-LNA A monomer was completely degraded. In the presence of Et3N (about 5-10 equivalents compared to monomer) L-LNA A in MeCN was completely stable after 24 hours. After 48 hours, L-LNA A was partially present, but still most of the L-LNA A remained in solution.

Therefore, Et3N stabilized the phosphoramidite in solution. However, the use of these conditions in oligonucleotide synthesis only results in trace amounts of full-length product.

Example 10: relative coupling efficiency in model systems using L-LNA a oxaazaphospholane phosphoramidite monomers and various amine bases:

in order to find a suitable base which is tolerated during the coupling step, several different additives of the relevant nitrogenous bases were investigated in the model system (5 '-gcattggtatt(LNA A) cattgttgtttt-3').

Global deprotection (NH) of oligonucleotides₄OH, overnight at 60 ℃), 3' DNA flanks were identified and compared to the full-length product in the crude mixture to obtain a study of the values of relative coupling efficiency under this condition (solvent +/-base). The results are shown in FIG. 6.

Interestingly, the conventional oligonucleotide synthesis solvent MeCN itself was found to result in a moderate relative coupling efficiency of 59%. However, in the presence of pyridine, coupling is possible and in some cases results in improved relative coupling efficiency.

The amount of pyridine required to obtain maximum coupling efficiency was titrated and found to be optimal in MeCN between 5 and 1% v/v pyridine.

In addition, pyridine derivatives such as 3-methylpyridine also improve coupling efficiency.

Example 11: relative coupling efficiencies using various oxaazaphospholane phosphoramidite monomers and a variety of different solvents in a model system:

to investigate the effect of the added pyridine on the solvent of the monomers, a set of 5 additional monomers was investigated using a model system (5 '-gcattggtatt (stereospecified said phosphoramidite) cattgttgtttt-3').

Global deprotection (NH) of oligonucleotides₄OH, overnight at 60 ℃), 3' DNA flanks were identified and compared to the full-length product in the crude mixture to obtain a study of the values of relative coupling efficiency under this condition (solvent +/-base). The results are shown in FIG. 7.

It can be seen that the effect of adding pyridine to increase reactivity is not common in all monomers. Interestingly, certain monomers such as D-DNA A benefit from pyridine in terms of increased relative coupling yields.

In other cases, the results were comparable with or without pyridine, as in the case of L-DNA A. However, MeCN by itself is not sufficient to keep the monomer in solution over a 24 hour period, given the nature of solubility. The monomers will be kept in solution over a period of 24 hours by the addition of 2.5% pyridine.

Example 12: solubility of various oxaazaphospholane phosphoramidite monomers in MeCN +/-2.5% pyridine and stability of the solution:

the solubility of the following monomers was determined as in example 7.

DNA A is Bz-protected, DNA C is acetyl (Ac) -protected, DNA T is free of protecting groups, DNA G is DMF, LNA A is Bz-protected, LNA C is Bz, LNA T is free of protecting groups, LNA G is DMF (D-LNA) and Ibu (L-LNA) protected. Bz ═ benzoyl.

Unless otherwise indicated, all monomers have DMF protected nucleobases, except L-LNA-G-iBu, which has isobutyryl protecting groups.

Further testing of additional monomers showed that the solubility enhancing effect of the addition of pyridine is universal in the monomer series. As in the case of D-LNA A, D-DNA A and L-DNA A, these monomers are insoluble after 24 hours in MeCN. However, by adding pyridine, the solubility of the monomer is maintained. An increase in reactivity was also observed for D-DNA A and L-LNA T, but L-DNA A and D-LNA A reacted in a comparable manner.

Example 13: conversion of full length product with and without 2.5% pyridine and different activation concentrations.

Relative coupling transformations were obtained in model system 5 '-xtttttttttttttttt-3' (where X ═ L-LNA a). The unreacted fragment (5'-ttttttttttttttt-3') and full length product (5 '- (L-LNA-A) ttttttttttttttt-3') were integrated and compared to each other to obtain the relative coupling efficiencies in the system. Different concentrations of the activating agent were used to determine the optimum concentration. The addition of pyridine significantly improves the coupling efficiency relative to coupling without pyridine. As can be seen from the results (fig. 8), the addition of pyridine generally has benefits in terms of increased conversion rate regardless of the activator concentration. As is conventional in the art, it is evident that the concentration of the activating agent should be optimized, and in the case of DCI, activating agents at concentrations of 1M DCI and 0.1M NMI are typically used. Using the obtained conversion to full length product, a number of theoretical yields were calculated. It is apparent here that the addition of pyridine is crucial to obtaining useful yields for drug discovery. In view of the coupling efficiency data obtained experimentally, the theoretical yield of possible 13mer oligonucleotides is shown in fig. 9, for 16mer oligonucleotides, see fig. 10. The data are provided in the following table:

Table of actual conversion to full-length product and theoretical yields of 13-and 16-mer oligonucleotides.

This data shows the significant benefit of using the coupling solvents of the present invention to synthesize stereodefined oligonucleotides.

Example 14 improvement of stereospecific oligonucleotide Synthesis

In this example, the synthesis of stereochemical variants of LNA oligonucleotides as shown below was performed using standard conditions (acetonitrile coupling solvent) and conditions according to the invention:

5’-GS_pCS_paS_ptS_ptS_pgS_pgS_ptS_paS_ptS_pTS_pCS_pA-3’(SEQ ID NO 1)

x represents an LNA nucleotide

Lower case letters for DNA nucleotides

Footnote Sp ═ stereorandom phosphorothioate internucleoside linkages.

The prior art conditions are as follows: 49 compounds were synthesized on a 1 μmol scale using acetonitrile as the solvent for the stereospecific phosphoramidite and 0.25M DCI as the activator. By using acetonitrile, significant problems are observed with regard to the instability and solubility of the phosphoramidite, which leads to line plugging on the synthesis equipment and low lifetime of the phosphoramidite solution. All syntheses were performed ON DMT-ON, which means that no final acid treatment was performed ON the synthesis instrument. After synthesis, the oligonucleotides were cleaved from the solid support using concentrated ammonium hydroxide at room temperature. The oligonucleotide was then deprotected by allowing the resulting solution to stand at 60 ℃ for 24 hours. The oligonucleotides were then purified by purification using a DMTr-based reverse phase column. After concentration of the oligonucleotide in vacuo, the oligonucleotide was dissolved in 200. mu.L of PBS, the concentration was determined by absorbance at 260nm, and back-calculated to concentration using the theoretically calculated extinction coefficient. The mean concentration of the 49 oligonucleotide solutions was thus measured in 200. mu.L PBS at 391. mu.M.

New and improved conditions: 192 compounds were synthesized on a 1 μmol scale using 3, 5% pyridine in acetonitrile as solvent for the sterically defined phosphoramidite and 1M DCI +0.1M NMI as activator. By using this solvent for the sterically defined phosphoramidite, no problems with solubility were observed, and a longer lifetime of the phosphoramidite solution was seen. All syntheses were performed ON DMT-ON, which means that no final acid treatment was performed ON the synthesis instrument. After synthesis, the oligonucleotides were cleaved from the solid support using concentrated ammonium hydroxide at room temperature. The oligonucleotide was then deprotected by allowing the resulting solution to stand at 60 ℃ for 24 hours. The oligonucleotides were then purified by purification using a DMTr-based reverse phase column. After concentration of the oligonucleotide in vacuo, the oligonucleotide was dissolved in 200. mu.L PBS and the concentration was determined by absorbance at 260nm and back-calculated to concentration using the theoretically calculated extinction coefficient. The average concentration of 192 oligonucleotide solutions thus measured in 200. mu.L PBS was 1071. mu.M

Thus, comparing the solubility and reactivity enhancements throughout the series, we found a 2.7-fold increase in yield using pyridine compared to the pyridine-free condition.

Example 15 relative coupling efficiency in model systems with and without pyridine using various oxaazaphospholane phosphoramidite monomers:

to investigate the effect of the added pyridine on the solvent of the monomers, a set of 7 additional monomers was investigated using the model system (5 '-gcattggtatt (stereospecific imide) cattgttgtttt-3').

Global deprotection (NH) of oligonucleotides₄OH overnight at 60 ℃), 3' DNA flanking was identified and compared to the full length product in the crude mixture to obtain a study of the values of relative coupling efficiency under this condition (solvent +/-base). The results are shown in FIG. 19. The results show that, in addition to the benefits of improving the solubility and stability of all monomers, the use of a coupling solvent comprising a heterocyclic base solvent (e.g., pyridine) provides a significant improvement in the coupling efficiency of D-DNA-C, L-LNA-C and L-LNA-G monomers in addition to L-LNA-T and D-DNA-a monomers (see fig. 7). Furthermore, these results indicate that the presence of pyridine does not adversely affect the coupling efficiency of the other monomers.

Claims (23)

1. A method of synthesizing a sterically defined phosphorothioate oligonucleotide comprising the steps of:

a) deprotecting the protected 5' -hydroxyl terminus of the nucleoside or oligonucleotide attached to the solid support,

b) Coupling an oxaazaphospholane phosphoramidite monomer to the deprotected 5' -hydroxy terminus of a nucleoside or oligonucleotide, wherein the coupling reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent selected from pyridine, 2-methylpyridine, 4-methylpyridine, 3-methylpyridine and lutidine, to form a phosphite triester intermediate

c) Oxidizing the phosphite triester intermediate with a vulcanizing agent,

d) optionally repeating steps a) -c) for one or more further extension cycles,

e) the oligonucleotide is deprotected from the solid support and cleaved.

2. The method of claim 1, wherein the method comprises step d) of a plurality of further extension cycles.

3. The method of claim 2, wherein the sterically defined phosphorothioate oligonucleotide is an antisense oligonucleotide.

4. A method of coupling an oxaazaphospholane phosphoramidite monomer to the 5' -end of a nucleoside or oligonucleotide comprising the step of reacting the nucleoside or oligonucleotide with the oxaazaphospholane phosphoramidite monomer wherein the reaction is carried out in an acetonitrile solvent composition comprising acetonitrile and an aromatic heterocyclic solvent selected from pyridine, 2-methylpyridine, 4-methylpyridine, 3-methylpyridine and lutidine.

5. The method of any one of claims 1-4, wherein the aromatic heterocyclic solvent has a pKa in water of 4-7 or 7-17 at 20 ℃.

6. The method of any one of claims 1-4, wherein the aromatic heterocyclic solvent is pyridine.

7. The method of any one of claims 1-4, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 0.1% to 50% v/v.

8. The process of claim 7, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 0.5% to 25% v/v.

9. The method of any one of claims 1-4, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 0.5% to 10% v/v.

10. The method of claim 9, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 1% to 5% v/v.

11. The process of claim 9, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 2-4% v/v.

12. The process of claim 9, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 2.5% v/v.

13. The process of claim 9, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 3.5% v/v.

14. An acetonitrile solution comprising an oxaazaphospholane phosphoramidite monomer, acetonitrile, and an aromatic heterocyclic solvent selected from pyridine, 2-methylpyridine, 4-methylpyridine, 3-methylpyridine, and lutidine.

15. The acetonitrile solution of claim 14 wherein the concentration of oxaazaphospholane phosphoramidite monomer is 0.05M to 2M.

16. The acetonitrile solution of claim 14 wherein the concentration of the oxaazaphospholane phosphoramidite monomer is 0.1M to 1M.

17. The acetonitrile solution of claim 14 wherein the concentration of the oxaazaphospholane phosphoramidite monomer is from 0.1M to 0.2M.

18. The acetonitrile solution of claim 14 wherein the concentration of the oxaazaphospholane phosphoramidite monomer is 0.15M.

19. The acetonitrile solution of claim 14 wherein the concentration of the oxaazaphospholane phosphoramidite monomer is 0.175M.

20. The acetonitrile solution of claim 14 wherein the concentration of the oxaazaphospholane phosphoramidite monomer is 0.2M.

21. The acetonitrile solution of any one of claims 14-20 wherein the aromatic heterocyclic solvent is as described in any one of claims 1-13.

22. The acetonitrile solution of any one of claims 14-20, wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 0.1% to 50% v/v.

23. The acetonitrile solution of claim 22 wherein the concentration of aromatic heterocyclic solvent in acetonitrile is 0.5% to 25% v/v.