Reactions of phosphorous containing compounds

Abstract

The present invention relates to a method for the chalcogenation (e.g. thionation, oxygenation or selenation) of phosphorous (III) containing compounds.

Description

REACTIONS OF PHOSPHOROUS CONTAINING COMPOUNDS INTRODUCTION

[0001] The present invention relates to a method for the chalcogenation (e.g. oxygenation, sulfurization (also referred to as thionation) or selenation) of phosphorous (III) containing compounds.BACKGROUND OF THE INVENTION

[0002] Several phosphate backbone variants have been developed in an attempt to alter the chemical properties of native-state DNA and therefore overcome the two major challenges involved with using oligonucleotides in vivo, including: 1) delivery to the interior of the cell through the plasma membrane, a lipid bilayer that without transport proteins, is mostly impermeable to polar molecules, and 2) extension of the effective molecular lifetime by minimizing extra and intracellular nuclease degradation.

[0003] One of the original and still most widely-used backbone variants is phosphorothioate, commonly referred to as S-oligo when incorporated into an oligonucleotide. Phosphorothioate has been found to help alleviate the second major challenge associated with using oligonucleotides in vivo by reducing the activity of a variety of extra and intracellular nucleases.

[0004] Therefore, phosphorothioate containing oligonucleotides are backbone modified oligonucleotides which can provide therapeutics with higher metabolic stability, bioavailability (e.g. Inclisiran / Leqvio Novartis), bioactivity, selectivity, cellular uptake, nuclease resistance and half-life extension. For example, in serum and cellular environments, phosphorothioate-modified oligonucleotides exhibit prolonged half-lives.

[0005] Phosphorothioate containing oligonucleotides are produced by introducing a sulfur modification onto the phosphate linker group by thionation. Thionation of nucleotides is an essential method for the preparation of phosphorothioates, which have found application in both academic research and industrial production of siRNA drugs.

[0006] Existing thionation methods often utilise expensive and toxic reagents, and can result in chemical yields which are not ideal. Commonly, disulfide reagents are used for this purpose, such as phenylacetyl disulfide (PADS), 3-[(dimethylamino-methylidene)amino]-3H-1,2,4-dithiazole-3-thione (DDTT), 3-amino-1,2,3-dithiazole-5-thione (ADTT). These reagents are used in stoichiometric amounts, which implies atom and cost-inefficiency. Additionally, they are known to be moderately toxic.

[0007] US8058448B2 describes the use of 3-amino-1,2,4-dithiazolidine-5-one as a reagent for sulfurization of oligonucleotides. This reagent is described as superior to such reagents, such as PADS, Beaucage reagent, elemental sulfur, dibenzoyl tetra sulfide, tetraethylthiuram disulfide(TETD), and bis(O, O-diisopropoxyphosphinothioyl) disulfide. Beaucage reagent is not particularly stable and forms a secondary reaction product which is an oxidizing agent that can lead to undesired oxidation side-reaction and formation of unwanted oxidation side-products. Tetraethylthiuram disulfide can also have a slow reaction rate.

[0008] Krotz et al (Phosphorothioate Oligonucleotides with Low Phosphate Diester Content: Greater than 99.9% Sulfurization Efficiency with “Aged” Solutions of Phenylacetyl Disulfide (PADS). Organic Process Research & Development 8, 852-858 (2004)) describes that “aged” solution of PADS is much more efficient in sulfurization than the fresh reagent. They describe that over prolonged standing of PADS in a solution of MeCN / 3-picoline it forms some active S-transfer reagent which is responsible for sulfurization reaction. A disadvantage of PADS is that it may be hard to control the reaction since the actual sulfurizing reagent is not PADS itself.

[0009] The present invention was devised with the foregoing in mind.SUMMARY OF THE INVENTION

[0010] According to one aspect of the present invention, there is provided a method for photocatalytic chalcogenation of an organo phosphorous (III) compound, the method comprising reacting a phosphorus (III) compound (e.g. a nucleotide precursor) with a chalcogen source in the presence of a photocatalyst and light irradiation.

[0011] Suitably, the organo phosphorous (III) compound is selected from phosphorus (III) nucleotide precursors, phosphoramidites, phosphorothioates, phosphoramidothioates, phosphites, aminophosphites, phosphines and aminophosphines.

[0012] Suitably, the chalcogenation involves the oxygenation, sulfurization (also referred to as thionation) or selenation of the organo phosphorous (III) compound. More suitably, the chalcogenation involves the oxygenation or sulfurization of the organo phosphorous (III) compound. Most suitably, the chalcogenation involves the sulfurization of the organo phosphorous (III) compound.

[0013] Suitably therefore, the chalcogen source is an oxygen source, a sulfur source or a selenium source. More suitably, the chalcogen source is an oxygen source or a sulfur source. Most suitably, the chalcogen source is a sulfur source.Chalcogenation of a Phosphorus (III) Compounds

[0014] Suitably, the organo phosphorous (III) compound comprises a moiety according to the formula (I nt), below:XaR-P-Xb(Int)wherein:Xa and Xb are each independently selected from O or S; andR is any suitable functional group, which may be optionally protected.

[0015] The moiety according to the formula (Int) may be comprised within any suitable organo phosphorous (III) compound, examples of which are discussed herein. Non-limiting examples of organo phosphorous (III) compounds include phosphorus (III) nucleotide precursors, phosphoramidites, phosphorothioates, phosphoramidothioates, phosphites, aminophosphites, phosphines and aminophosphines.

[0016] The method described herein may be applied to any compound comprising the (Int) subunit, including but not limited to the compounds disclosed herein, such as phosphorus (III) nucleotide precursors, phosphoramidites, phosphorothioates, phosphoramidothioates, phosphites, aminophosphites, phosphines and aminophosphines.

[0017] In a preferred embodiment, the organo phosphorous (III) compound is a phosphorus (III) nucleotide precursor. Thus, the organo phosphorous (III) compound may comprise at least two nucleosides linked by a moiety according to formula (Int) above.

[0018] Preferably, Xaand Xb are each O.

[0019] Suitably, the nucleotide precursor comprises at least two nucleosides linked by a moiety according to the formula (Int-A), below:(Int-A)wherein R is any suitable functional group, which may be optionally protected.

[0020] Suitably, the nucleotide precursor comprises at least two nucleosides linked by a moiety according to the formula (Int-B), below:(Int-B)wherein R is any suitable functional group, which may be optionally protected.

[0021] Suitably, R is selected from a ORH, SRS, NRN1RN2or CH2Rc1;wherein:a) RHis hydrogen or a phosphorous hydroxyl protecting group (e.g. cyanoethyl, t-butyl or benzyl);b) Rsis hydrogen or a thiol protecting group (e.g. cyanoethyl, benzyl, 4- chlorobenzyl or 2,4 dichlorobenzyl)c) each of RN1and RN2are independently selected from:i) an amine protecting group, e.g. SO2(1-6C)alkyl or tosyl;ii) (1-6C)alkyl, (3-8C)cycloalkyl, 3- to 12 membered heterocyclyl or 5 to 10 membered heteroaryl, wherein any (1-6C)alkyl, (3- 8C)cycloalkyl, 3- to 12-membered heterocyclyl, 5 to 10 membered heteroaryl is optionally substituted by one or more substituents independently selected from halo, (1-2C)alkyl, (1-2C)alkoxy, (1- 2C)haloalkyl, (1-2C)haloalkoxy, cyano or NMe2;iii) RN1and RN2are linked such that, together with the nitrogen atom to which they are attached, form a 4- to 7-membered heterocyclic ring;d) RC1is selected from (1-6C)alkyl, (3-8C)cycloalkyl, 3- to 12 membered heterocyclyl, 5 to 10 membered heteroaryl or (1-6C)alkoxy, wherein any (1- 6C)alkyl, (3-8C)cycloalkyl, 3- to 12-membered heterocyclyl, 5 to 10 membered heteroaryl or (1-6C)alkoxy is optionally substituted by one or more substituents independently selected from halo, (1-2C)alkyl, (1-2C)alkoxy, (1- 2C)haloalkyl, (1-2C)haloalkoxy, cyano or NMe2.

[0022] Suitably, R is selected from a ORH, SRS, NRN1RN2or CH2RC1;wherein:RHis hydrogen or a phosphorous hydroxyl protecting group (e.g. cyanoethyl, t- butyl or benzyl);Rsis hydrogen or a thiol protecting group (e.g. cyanoethyl, benzyl or 2,4 dichlorobenzyl);each of RN1and RN2are independently selected from an amine protecting group, e.g. (1-6C)alkyl, SO2(1-6C)alkyl or tosyl); andRC1is selected from aryl, heteroaryl, (1-6C)alkyl and (1-6C)alkoxy.

[0023] Suitably, the nucleotide precursor comprises a moiety according to the formula (lnt-1), below:wherein:Xa and Xb are each independently selected from O or S;R is selected from a ORH, SRS, NRN1RN2or CH2RC1, wherein RH, RS, RN1, RN2and RC1are as defined anywhere herein;R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group;R4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl;R2bis selected from hydrogen, methoxy, F, OP2b, O(CH2)2OMe; wherein P2b is a hydroxyl protecting group; andR4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2b-X4b-, wherein one of X2b and X4b is (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3and Rb4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl; andB1and B2are independently selected optionally protected nucleobases.

[0024] Suitably, Xaand Xb are O, Thus, the nucleotide precursor comprises a moiety according to the formula (lnt-1 A), below:wherein:R is selected from a ORH, SRS, NRN1RN2or CH2RC1, wherein RH, RS, RN1, RN2and RC1are as defined anywhere herein;R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group;R4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl;R2bis selected from hydrogen, methoxy, F, OP2bor O(CH2)2OMe; wherein P2bis a hydroxyl protecting group; andR4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2b-X4b-, wherein one of X2b and X4b is (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3and Rb4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl; andB1and B2are independently selected optionally protected nucleobases.

[0025] Suitably, the nucleotide precursor has a structure according to Formula (lnt-2) below:P1 Bi1O Ri2bP2(lnt-2)wherein:Xa is selected from O or S;Xb is selected from O or S;P1 is a hydroxyl protecting group;P2 is a hydroxyl protecting group, a linker moiety attached to a solid support optionally via one or more further nucleotides, or a group with the formula:RRPO-P-N(iPr)2wherein RPis a phosphorous hydroxyl protecting group; andR is selected from a ORH, SRS, NRN1RN2or CH2Rc1, wherein RH, Rs, RN1, RN2and RC1are as defined anywhere herein;R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group;R4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl;R2bis selected from hydrogen, methoxy, F, OP2bor O(CH2)2OMe; wherein P2bis a hydroxyl protecting group; andR4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2b-X4b-, wherein one of X2b and X4b is (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3and Rb4areindependently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl; andB1and B2are independently selected optionally protected nucleobases;or P2 and R2b are linked such that, together with the atoms to which they are attached, they form a fused 5- or 6-membered heterocyclic ring, or a 5- or 6-membered silyl containing heterocyclic ring, which may be optionally substituted by one or more substituents selected from (1-4C)alkyl, (1-4C)alkoxy, benzyl or phenyl, any of which may in turn be optionally further substituted by one or more substituents selected from (1-4C)alkyl or (1-4C)alkoxy.

[0026] Suitably, Xaand Xb are both O. Thus, the nucleotide precursor has a structure according to Formula (lnt-2A) below:(lnt-2A)wherein R, B1, B2, R2a, R2b, R4a, R4b, P1 and P2 are as defined anywhere herein.

[0027] P2aand P2bare independently selected hydroxyl protecting groups. A number of appropriate hydroxyl protecting groups are available to those skilled in the art, such as those described in the definitions section herein. Non-limiting examples of suitable protecting groups are described herein and are not intended to unduly limit the scope of the present invention.

[0028] Suitably, the hydroxyl protecting groups P2aand P2bare each independently selected from acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy) methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

[0029] More suitably, the hydroxyl protecting groups P2a and P2b are each independently selected from benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMTr), 9-phenylxanthine-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthine-9-yl (MOX), tetrahydropyranyl (THP), 2-(trimethylsilyl)ethoxymethyl (SEM) and methoxymethyl (MOM),

[0030] As described herein, R is selected from ORH, SRS, NRN1RN2or CH2Rc1. Preferably, R is selected from ORH, SRSor NRN1RN2. More preferably, R is ORHor SRs. Most preferably, R is ORH

[0031] It will be appreciated that a wide variety of protecting groups may be used for each of RH, Rs, RN1, RN2and Rc. Such protecting groups may be easily identified by the skilled person. Non-limiting examples of suitable protecting groups are described herein and are not intended to unduly limit the scope of the present invention.

[0032] Suitably RHis selected from hydrogen, methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tertbutyl, 4-methoxybenzyl, 4-chlorobenzyl, 2-chlorophenyl and 2-cyanoethyl. More suitably, RHis selected from methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4-methoxybenzyl, 4-chlorobenzyl, 2-chlorophenyl and 2-cyanoethyl. Most suitably, RHis 2-cyanoethyl.

[0033] Suitably, Rsis selected from hydrogen, 2-cyanoethyl, benzyl, 4-chlorobenzyl and 2,4 dichlorobenzyl. More suitably, Rsis selected from 2-cyanoethyl, benzyl, 4-chlorobenzyl and 2,4 dichlorobenzyl.

[0034] Suitably, each of RN1and RN2are independently selected from (1-6C)alkyl, SO2(1-6C)alkyl or tosyl. More suitably, each of RN1and RN2are independently selected from isopropyl, SO2Me or tosyl.

[0035] Suitably, RC1is selected from phenyl, 5- or 6-membered heteroaryl, (1 -6C)alkyl and (1-6C)alkoxy. More suitably, RC1is selected from methyl or methoxy.

[0036] In certain embodiments, the nucleosides may comprise a locked nucleic acid.

[0037] In certain embodiments, R2a and R4a may be linked to form a locked nucleic acid of the formula -X2a-X4a- (where X2a is in the R2a position, and X4a is in the R4a position) for example:wherein one of X2a and X4a is (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1-2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl.

[0038] Suitably, if R2a and R4a are linked to form a locked nucleic acid, then it is of the formula -X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2) and the other is O; wherein each of Ra1and Ra2are independently selected from hydrogen or methyl. More suitably, if R2a and R4a are linked to form a locked nucleic acid, then it is of the formula -CH2-O- or -O-CH2-. Most suitably, the locked nucleic acid formed by R2a and R4a is of the formula -O-CH2, e.g.

[0039] In certain embodiments, R2b and R4b may be linked to form a locked nucleic acid of the formula -X2b-X4b-, (where X2bis in the R2b position, and X4b is in the R4b position), i.e.:wherein one of X2b and X4b is (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3and Rb4are independently selected from hydrogen, (1-2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRdis selected from hydrogen or a (1 -6C)alkyl.

[0040] The hydroxy and amino substituents in the groups described above may be protected with any of the protecting groups described herein.

[0041] Suitably, if R2b and R4b are linked to form a locked nucleic acid, then it is of the formula -X2b-X4b-, wherein one of X2b and X4b is (CRb1Rb2) and the other is O; wherein each of Rb1and Rb2are independently selected from hydrogen or methyl. More suitably, if R2b and R4b are linked to form a locked nucleic acid, then it is of the formula -CH2-O- or -O-CH2-. Most suitably, the locked nucleic acid formed by R2b and R4b is of the formula -O-CH2, e.g.:P2

[0042] Suitably:R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group; andR4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid of the formula -O-CH2-.

[0043] More suitably, R2ais selected from hydrogen, methoxy or OP2a; wherein P2ais a hydroxyl protecting group; and R4ais hydrogen. Most suitably, R2ais selected from hydrogen or OP2a; wherein P2ais a hydroxyl protecting group; and R4ais hydrogen.

[0044] Suitably:R2bis selected from hydrogen, methoxy, F, OP2bor O(CH2)2OMe; wherein P2bis a hydroxyl protecting group; andR4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid of the formula -O-CH2-.

[0045] More suitably, R2b is selected from hydrogen, methoxy or OP2a; wherein P2ais a hydroxyl protecting group; and R4b is hydrogen. Most suitably, R2b is selected from hydrogen or OP2a; wherein P2b is a hydroxyl protecting group; and R4b is hydrogen.

[0046] Suitably, the method comprises reacting a nucleotide precursor comprising a moiety according to the formula (I nt- 1 ), below:with a chalcogen source, in the presence of a photocatalyst and light irradiation;to form a nucleotide comprising a moiety according to formula (I) below:wherein Xaand Xb are each independently selected from O or S, X is a chalcogen atom, and R, R2a, R4a, B1, R2b, R4b and B2are as defined anywhere herein. Preferably, Xaand Xb are each O.

[0047] Suitably, the method comprises reacting a nucleotide precursor comprising a moiety according to the formula (I nt- 1 A), below:with a chalcogen source, in the presence of a photocatalyst and light irradiation;to form a nucleotide comprising a moiety according to formula (IA) below:wherein X is a chalcogen atom, and R, R2a, R4a, B1, R2b, R4b and B2are as defined anywhere herein.

[0048] Suitably, X is selected from O, S or Se. More suitably, X is selected from O or S.

[0049] P1 may be any suitable protecting group known in the art. Suitably, P1 is a hydroxyl protecting group selected from acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthine-9-yl (MOX) and methoxymethyl. More suitably, P1 is Dimethoxytrityl (DMTr) or acetyl.

[0050] P2 may be any suitable protecting group known in the art, or may be a linker moiety attached to a solid support optionally via one or more further nucleotides.

[0051] If P2 is a protecting group, it may be selected from a hydroxyl protecting group. Suitably, P2 is selected from acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthine-9-yl (MOX) and methoxymethyl. More suitably, P2 is acetyl.

[0052] In certain preferred embodiments, P2 may also be a linker moiety attached to a solid support optionally via one or more further nucleotides. Thus, the reaction may be performed using a “solid phase”. A number of appropriate linker moieties and solid supports are available to those skilled in the art, for example Controlled Pore Glass (CPG) and polystyrene resin beads. By way of example, the linker moiety and solid support may have the formula:wherein represents a solid support, such as Controlled Pore Glass (CPG) having a 500 to 2000 A pore size.

[0053] P2 may also be a group of the formula:RRpO-P-N(iPr)2wherein RP is a phosphorous hydroxyl protecting group. Suitably, RP is 2-cyanoethyl.

[0054] P2 and R2b may be linked such that, together with the atoms to which they are attached, they form a fused 5- or 6-membered heterocyclic ring, or a fused 5- or 6-membered silyl heterocyclic ring, which may be optionally substituted by one or more substituents selected from (1-4C)alkyl, (1-4C)alkoxy, benzyl or phenyl, any of which may in turn be optionally further substituted by one or more substituents selected from (1-4C)alkyl or (1-4C)alkoxy; and / or the ring formed by P2 and R2b may be attached to a solid support.

[0055] More suitably, P2 and R2b may be linked such that, together with the atoms to which they are attached form a fused 5-membered cyclic ether or cyclic silyl ether, which may be optionally substituted by one or more (1 -4C)alkyl groups.

[0056] Suitably, P2 and R2b, together with the atoms to which they are attached, may form a ring with the formula:wherein each of Rwand Rxis independently selected from hydrogen, (1-4C)alkyl, (1-4C)alkoxy, benzyl or phenyl, wherein any of which may in turn be optionally substituted by one or more substituents selected from ( 1 -4C)alkyl or methoxy; or one or Rwand Rxis hydrogen, and the other is linked to a solid support via a linker group; andeach of Ryand Rzis independently selected from hydrogen, (1 -6C)alkyl, (1-4C)alkoxy, benzyl or phenyl, wherein any of which may in turn be optionally substituted by one or more substituents selected from (1 -4C)alkyl or methoxy.

[0057] More suitably, P2 and R2b may form a ring with the formula:wherein each of Rwand Rxis independently selected from hydrogen, (1 -4C)alkyl or (1-4C)alkoxy. Most suitably, each of Rwand Rxare methyl, i.e. P2 and R2b may form a ring with the formula:(i.e. an isopropylidene protecting group).

[0058] The reaction may be performed at various temperatures. Suitably, the reaction is performed a temperature of from 0°C to 75°C. More suitably, the reaction is performed at a temperature of from 15 to 30°C.

[0059] Suitably, the method further comprises removing any protecting groups present and, if necessary cleavage from a solid support to form an oligonucleotide comprising a moiety of the formula:wherein:X is a chalcogen (e.g. oxygen, sulfur or selenium); andXa and Xb are each independently selected from O and S;R, R2a, R4a, B1, R2b, R4b and B2are as defined anywhere herein.

[0060] Preferably, Xaand Xb are O. Thus, the oligonucleotide formed may comprise a moiety of the formula:wherein X is a chalcogen (e.g. oxygen, sulfur or selenium); R, R2a, R4a, B1, R2b, R4b and B2are as defined anywhere herein.

[0061] Suitably, the chalcogen source is a sulfur source. Suitably the sulfur source is a thiosulfate, such as barium thiosulfate, magnesium thiosulfate, potassium thiosulfate, cesium thiosulfate or sodium thiosulfate. More suitably, the sulfur source is sodium thiosulfate.

[0062] The relative amount of the sulfur source to the starting material may be adjusted as appropriate. The sulfur source may be added in a stoichiometric equivalent ratio of from 1 to 10 (in relation to starting material, e.g. a compound comprising a moiety to the formula lnt-1), suitably from 2 to 8, more suitably 4 to 8, most suitably 7 to 8.

[0063] The relative amount of the sulfur source to the starting material may be adjusted as appropriate. The sulfur source may be added in a stoichiometric equivalent ratio of from 1 to 10 (in relation to starting material, e.g. a compound comprising a moiety to the formula lnt-1), suitably from 2 to 8, more suitably 4 to 8, most suitably 7 to 8.

[0064] Suitably, the chalcogen source is an oxygen source. The oxygen source may be oxygen gas or atmospheric oxygen.

[0065] Suitably, the chalcogen source is a selenium source. Suitably the selenium source is a selenosulfate. More suitably, the selenium source is sodium selenosulfate.

[0066] Suitably, the reaction is performed in a suitable solvent. The preferred solvents will depend on the nature of substituent groups such as P1 and P2, and can be readily determined by those skilled in the art.

[0067] Examples of suitable solvents include water and optionally one or more aprotic solvents, such as acetonitrile, THF, dioxane and acetone.

[0068] In embodiments where the reaction does not involve a solid phase (i.e. P2 is not a linker moiety attached to a solid support (optionally via one or more further nucleotides)) the solvent preferably comprises water and one or more aprotic solvents. The aprotic solvent may be selected from acetonitrile, THF, dioxane and acetone. Most suitably, the solvent comprises a mixture of water and acetonitrile.

[0069] In embodiments where the reaction does not involve a solid phase (i.e. P2 is not a linker moiety attached to a solid support (optionally via one or more further nucleotides)) the solvent may comprise a mixture of a water and an aprotic solvent in a ratio of from 1:2 to 1:4, more suitably from 1:2.5 to 1:3.5, for example 1:3.

[0070] In some embodiments, particularly embodiments where P2 is not a linker moiety attached to a solid support (optionally via one or more further nucleotides), the amount of water present in the solvent is the minimal amount needed to allow the sulfur source to dissolve in the mixture of water and aprotic solvent.

[0071] In certain embodiments, if P2 is a linker moiety attached to a solid support (optionally via one or more further nucleotides), the solvent may comprise a greater amount of water than other solvents (e.g. greater than 50 wt.% water, greater than 75 wt.% water or greater than 95wt.% water).

[0072] In preferred embodiments, if P2 is a linker moiety attached to a solid support (optionally via one or more further nucleotides), the solvent is water. The use of a solid phase can therefore remove the need for the presence of further solvents other than water.

[0073] The concentration of the starting material may be adjusted as appropriate. Suitably, the concentration of the starting material in the solvent is from 0.005M to 0.04 M.

[0074] A number of photocatalysts may be applied to the method of the present invention. However, it is known that DNA and RNA molecules can undergo photodegradation under certain wavelengths. It is therefore preferable that the photocatalyst is activated by light which has a wavelength which will not damage the DNA or RNA structure. Suitably, the photocatalyst is selected from a catalyst which is activated by light with a wavelength greater than or equal to 380 nm. More suitably, the photocatalyst is selected from a catalyst which is activated by light with a wavelength greater than or equal to 420 nm, i.e. light with a wavelength greater or equal than that of blue light.

[0075] In certain embodiments, the photocatalyst does not comprise a transition metal.

[0076] Suitably, the light irradiation will be adjusted depending on the wavelength at which the photoacatalyst is activated. Suitably, the light irradiation comprises visible light, such as blue, green or red light.

[0077] More suitably, the light irradiation does not comprise electromagnetic radiation which will damage DNA or RNA, such as UV light or in some cases violet, indigo or blue light.

[0078] The source of light irradiation may be one or more LEDs, e.g. one or more blue or green LEDs.

[0079] The photocatalysts which may be utilised in the methods of the invention include organophotocatalysts and metallophotocatalysts. Examples include flavin photocatalysts, iridium photocatalysts, ruthenium photocatalysts, cyanoarene-based donor-acceptor photocatalysts and acridinium based photocatalysts.

[0080] Particular examples of photocatalysts which may be utilised in the methods of the invention include any one or more of riboflavin, flavin adenine dinucleotide, flavin mononucleotide, flavin derivatives, Tris(bipyridine)ruthenium(ll) chloride (Ru-bpy), 2,4,6-triphenylpyrylium tetrafluoroborate (TPT), [4,4'-B / s(1,1-dimethylethyl)-2,2'-bipyridine-A / 1, A / 1 ']jb / s[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-A / ]phenyl-C]lridium(l 11) hexafluorophosphate ([Ir[dF(CF3)ppy]2(dtbbpy)]PF6), 9-Mesityl-10-methylacridinium tetrafluoroborate ([MesAcrMe]BF4), Erythrosin B, Eosin Y, Rose Bengal, Methylene blue, 9-Mesityl-1,3,6,8-tetramethoxy-10-phenylacridin-10-ium tetrafluoroborate([Mes(MeO)4AcrPh]BF4), 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzlPN), 2,4,6-Tri(9-carbazol-9-yl)-5-chloroisophthalonitrile (3CzClIPN), Iridium(lll) hexafluorophosphate ([lr[dF(CF3)ppy]2(dtbbpy)]PF6), Tris(2,2'-bipyridyl)ruthenium(l I) chloride hexahydrate ([Ru(bpy)3]Ch*6H2O) or Triphenyl phosphate (TPP).

[0081] In a another embodiment, the photocatalyst does not comprise a transition metal, for example it may be selected from riboflavin, flavin adenine dinucleotide, flavin mononucleotide, flavin derivatives, 2,4,6-triphenylpyrylium tetrafluoroborate (TPT), 9-Mesityl-10-methylacridinium tetrafluoroborate ([MesAcrMe]BF4), Erythrosin B, Eosin Y, Rose Bengal, Methylene blue, 9-Mesityl-1,3,6,8-tetramethoxy-10-phenylacridin-10-ium tetrafluoroborate ([Mes(MeO)4AcrPh]BF4), 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzlPN), 2,4,6-Tri(9-carbazol-9-yl)-5-chloroisophthalonitrile (3CzClIPN), or Triphenyl phosphate (TPP).

[0082] In a particular embodiment, the light irradiation comprises or is green light, and the photocatalyst is selected from 9-Mesityl-10-methylacridinium tetrafluoroborate, 9-Mesityl-1, 3,6,8-tetramethoxy-10-phenylacridin-10-ium tetrafluoroborate, 9-Mesityl-3,6-di-tert-butyl-10-phenylacridinium tetrafluoroborate, 4CzlPN, 3CzCIIPN, Erythrosin B, Eosin Y or Rose Bengal (referred to as C3, C4, C5, C6, C7, C8, C9 or C10, respectively in the examples section).

[0083] In a particular embodiment, the light irradiation comprises or is green light, and the photocatalyst is selected from Erythrosin B, Eosin Y, Rose bengal, Methylene blue, [MesAcrMe]BF4, [Mes(2,6-di-fBu)AcrPh]BF4, [Mes(MeO)4AcrPh]BF4, 4CzlPN and 3CzCIIPN.

[0084] In a particular embodiment, the light irradiation comprises or is green light, and the photocatalyst is a acridinium photocatalyst, such as [MesAcrMe]BF4, [Mes(2,6-di-fBu)AcrPh]BF4 or [Mes(MeO)4AcrPh]BF4.

[0085] In another embodiment, the light irradiation comprises or is blue light, and the photocatalyst is selected from catalyst tris(2,2'-bipyridyl)dichlororuthenium(l I) hexahydrate or (lr[dF(CF3)ppy]2(dtbpy))PF6.

[0086] In another embodiment, the light irradiation comprises or is blue light, and the photocatalyst is selected from riboflavin, flavin adenine dinucleotide, flavin mononucleotide, flavin derivatives, [Ir[dF(CF3)ppy]2(dtbbpy)]PF6, [Ru(bpy)3]Cl2*6H2O or TPP.

[0087] In a particular embodiment, the light irradiation comprises or is blue light, and the photocatalyst is riboflavin. Suitably, in this embodiment, the reaction may be performed using a solid support, e.g. P2 defined herein is a linker moiety attached to a solid support.

[0088] In another embodiment, the light irradiation comprises or is blue light, and the photocatalyst is selected from [Ir[dF(CF3)ppy]2(dtbbpy)]PF6, [Ru(bpy)3]Cl2*6H2O or TPP.

[0089] In another embodiment, the light irradiation comprises or is blue light, the photocatalyst is an iridium photocatalyst, such as [Ir[dF(CF3)ppy]2(dtbbpy)]PF6.

[0090] The catalyst loading may be adjusted as appropriate. Suitably, the catalyst loading is from 0.1 mol% to 10 mol% relative to the starting material, more suitably from 0.5 to 5 mol%, most suitably from 0.75 to 3 mol%.

[0091] Suitably, the method of the present invention further comprises coupling with one or more nucleotides (e.g. to form an oligonucleotide).

[0092] Suitably, if the photocatalytic reaction involves the sulfurization of the organo phosphorous (III) compound, the reaction is performed in an inert atmosphere, e.g. under a nitrogen or argon atmosphere.

[0093] In another aspect, there is provided a nucleotide obtained by, obtainable by, or directly obtained by the method of the present invention.

[0094] In another aspect, there is provided a method preparing a preparing a phosphorothioate containing oligonucleotide, the method comprising reacting a phosphorus (III) nucleotide precursor with a sulfur source in the presence of a photocatalyst and light irradiation, and subsequently coupling with one or more nucleotides.

[0095] In another aspect, there is provided a phosphorothioate containing oligonucleotide obtained by, obtainable by, or directly obtained by the method of the present invention.Chalcogenation of Further Phosphorus (III) Compounds

[0096] The photocatalytic chalcogenation (e.g. oxygenation and sulfurization) reaction demonstrated herein may also applied to a number of organo phosphorous (III) compounds including but not limited to phosphoramidites, phosphorothioates, phosphoramidothioates, phosphites, aminophosphites, phosphines and aminophosphines. Any of the embodiments of the invention relating to the photocatalytic chalcogenation of a phosphorus (III) nucleotide precursor may be applied equally to the photocatalytic thionation or oxidation of such compounds.

[0097] Suitably, the organo phosphorous (III) compound comprises a moiety according to formula (I nt), below:TR'P\Xb(Int)wherein:Xa and Xb are each independently selected from O or S; andR is any suitable functional group, which may be optionally protected.

[0098] Suitably, R is as defined anywhere herein.

[0099] Suitably, the organo phosphorous (III) compound comprises a moiety according to the formula (Int-A), below:(Int-A)wherein R is any suitable functional group, which may be optionally protected.

[0100] Suitably, the organo phosphorous (III) compound comprises a moiety according to the formula (Int-B), below:(Int-B)wherein R is any suitable functional group, which may be optionally protected.

[0101] In another embodiment, the organo phosphorous (III) compound is a phosphoramidite compound.

[0102] The phosphoramidite compound may have a formula (II), below:PziPZO'^N'RN1RN2(II)wherein:Pz is a protecting group or optionally substituted (1 -6Calkyl), (3-12C)cycloalkyl, heterocycyclyl, heteroaryl or aryl;Pzi is a nucleoside, a protecting group or optionally substituted (1 -6Calkyl), (3- 12C)cycloalkyl, heterocycyclyl, heteroaryl or aryl;or Pz and Pzi are joined to form a linked protecting group, e.g. a dinapthyl group; andRNI and RN2 are independently selected from (1-6C)alkyl, e.g. isopropyl ('Pr), or RNI and RN2 are linked such that, together with the nitrogen to which they are attached, they form a 4- to 7 membered heterocyclic ring (for example piperazinyl and morpholinyl).

[0103] The phosphoramidite compound may have a formula (Ila), below:wherein:R2cis selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein Pzais a hydroxyl protecting group;R4a is hydrogen;or R2aand R4aare linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-PY is a protecting group, e.g. DMTr;Pz is a protecting group, e.g. 2-cyanoethyl;RNI and RN2 are independently selected from (1 -6C)alkyl, e.g. isopropyl ('Pr), or RNI and RN2 are linked such that, together with the nitrogen to which they are attached, they form a 4- to 7 membered heterocyclic ring (for example piperazinyl and morpholinyl); and B3is a nucleobase which may be optionally protected.

[0104] Suitably, the phosphoramidite compound has the following formula (lib)wherein B3is a nucleobase which may be optionally protected.

[0105] In an embodiment, the phosphoramidite compound is:

[0106] An exemplary reaction involving the photocatalytic sulfurization of a phosphoramidite compound is as follows:Na2S2O3(2 eq.) [Mes(MeO)4AcrPh]BF4(5 mol%)Ar, Green LED, 3.5 h CD3CN / D2O (3:1)96% NMR yield20 μmol scale

[0107] In another embodiment, the organo phosphorous (III) compound is a phosphoramidothioate compound.

[0108] The phosphoramidothioate compound may have a formula (IIS), below:wherein:R2Cis selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group;R4a is hydrogen;or R2aand R4aare linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-PY is a protecting group, e.g. DMTr;Ps is selected from hydrogen or a thiol protecting group (e.g. cyanoethyl, benzyl, 4- chlorobenzyl or 2,4 dichlorobenzyl);RNI and RN2 are independently selected from (1 -6C)alkyl, e.g. isopropyl ('Pr), or RNI and RN2 are linked such that, together with the nitrogen to which they are attached, they form a 4- to 7 membered heterocyclic ring (for example piperazinyl and morpholinyl); and B3is a nucleobase which may be optionally protected.

[0109] Suitably, the phosphoramidothioate compound has the following formula (IISa)(IlSa)wherein B3is a nucleobase which may be optionally protected.

[0110] Suitably, the phosphoramidothioate compound is:

[0111] In another embodiment, the organo phosphorous (III) compound is a phosphite compound, for example triphenyl phosphite.

[0112] The phosphite compound may have a formula (III), below:R6XOXPX°'R7%8(HI)wherein Re, R? and Rs are independently selected from optionally substituted (1-6Calkyl), (3-12C)cycloalkyl, heterocycyclyl, heteroaryl or aryl, or Re and R? are linked to form a linked protecting group, e.g. a binapthyl group:

[0113] Specific examples of such organic phosphites include, but are not limited to diphenyl 2-ethylhexyl phosphite, triphenyl phosphite, tris(2,5-di-tert.-butyl-phenyl)phosphite, tris(2-tert.-butylphenyl)phosphite, tris(2-phenylphenyl)phosphite, tris{2-(1, 1 -dimethylpropyl)phenyl)phosphite, tris(2-cyclohexylphenyl)phosphite, tris(2-tert.-butyl-4-phenylphenyl)phosphite, tris)2-tert.-butyl-4-methylphenyl)phosphite, tris(2,4-di-tert.-amylphenyl)phosphite and tris(2,4-di-tert.-butylphenyl)phosphite.

[0114] An exemplary reaction involving the photocatalytic sulfurization of a phosphite compound is as follows:Na2S2O3(2 eq.)[Ir(dF(CF3)ppy]2(dtbbpy)]PF6(1 mol%) Ar, Blue LED, 18h MeCN / H2O (3:1) 71%

[0115] In another embodiment, the organo phosphorous (III) compound is a phosphine compound, for example triphenylphosphine.

[0116] The phosphine compound may have a formula (IV), below:RiopRiiiR9(IV)wherein Rg, Rwand Rn are independently selected from optionally substituted (1-6Calkyl), (3-12C)cycloalkyl, heterocycyclyl, heteroaryl or aryl.

[0117] Examples of phosphine compounds include, but are not limited to, dicyclohexylphosphine, tricyclohexylphosphine, triethylphosphine, tributylphosphine, diethylphenylphosphine, dicyclohexylphenylphosphine, tribenzylphosphine, orthotolyldiphenylphosphine, di(ortho-tolyl)phenylphosphine, triisopropylphosphine, triisobutylphosphine, triterbutylphosphine, phenylphosphine, diphenylphosphine, tris(4-methoxyphenyl)phoshine, 2-(diphenylphosphineyl)pyridine and triphenylphosphine.

[0118] An exemplary reaction involving the photocatalytic sulfurization of a phosphine compound is as follows:Na2S2O3(2 eq.)[Ir(dF(CF3)ppy]2(dtbbpy)]PF6(1 mol%) Ar, Blue LED, 18h MeCN / H2O (6:1)200 μmol scale 71%BRIEF DESCRIPTION OF THE FIGURE

[0119] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings:Figure 1: Application of the method to the solid-phase oligonucleotide synthesis.Conditions for 9: 5’-O-DMT-thymidine-3’-lcaa-CPG (25.0 pmol / g, 1.00 g, 25.0 pmol); detritylation: DCA (900 mg, 7.00 mmol) as a 3% solution in MeCN / toluene (8:2), r.t., 3 min; coupling: phosphoramidite (0.40 mmol), 5-(ethylthio)-1H-tetrazole (260 mg, 2.00 mmol) as a solution in 4 mL MeCN, Ar, r.t., 1 h; thionation: Na2S2O3(1.00 g, 7.00 mmol), riboflavin (5.00 mg, 13.0 pmol, 4 mol%), as a solution in 5 mL water, blue LEDs 456 nm (50W), 1000 rpm, Ar, r.t., 30 min; cleavage: methylamine (3 mL, 40% aq.), r.t., 2 h. Yield is given for the isolated product.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0120] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0121] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0122] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or examples of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or processso disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0123] In this specification the term “alkyl” includes both straight (i.e. linear) and branched chain alkyl groups. References to individual alkyl groups such as “propyl” are specific for the straight chain version only and references to individual branched chain alkyl groups such as “isopropyl” are specific for the branched chain version only. For example, “Ci-ealkyl” includes Ci-4alkyl, Ci-3alkyl, propyl, isopropyl and t-butyl. A similar convention applies to other radicals, for example “phenyl(Ci-6alkyl)” includes phenyl(Ci-4alkyl), benzyl, 1-phenylethyl and 2-phenylethyl.

[0124] The term "(m-nC)" or “Cm-n”, or "(m-nC) group" or “Cm-n” used alone or as a prefix, refers to any group having m to n carbon atoms.

[0125] The term "alkenyl", as used herein, refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyls" and "substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0126] The term "alkynyl", as used herein, refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyls" and "substituted alkynyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are included or not included in one or more triple bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0127] An “alkylene” group is an alkyl group that is positioned between and serves to connect two other chemical groups. Thus, “Ci-3alkylene” means a linear saturated divalent hydrocarbon radical of one to three carbon atoms or a branched saturated divalent hydrocarbon radical of three atoms, for example, methylene, ethylene, propylene, and the like.

[0128] The term “Cm-ncycloalkyl” means a hydrocarbon ring containing from m to n carbon atoms, for example “Cs-ecycloalkyl” means a hydrocarbon ring containing from 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The term “Cm-n-cycloalkyl” also encompasses non-aromatic saturated or partially saturated monocyclic, fused, bridged, or spiro bicyclic carbocyclic ring system(s). The term “Cm-ncycloalkyl” includes both monovalent species and divalent species. Monocyclic “Cm-ncycloalkyl” rings contain from about 3 to 12 (suitably from 3 to 8, most suitably from 5 to 6) ring carbon atoms. Bicyclic “Cm-ncycloalkyl” contain from 5 to 17 ring carbon atoms, suitably 5 to 12 ring carbon atoms. Bicyclic “Cm-n-cycloalkyl” rings may be fused, spiro, or bridged ring systems.

[0129] The term "cycloalkoxy" means a cycloalkyl-O-group in which the cycloalkyl group is as previously defined, for example C3-4cycloalkoxy (or-O-C3-4cycloalkyl) means a hydrocarbonring containing from 3 to 4 carbon atoms, linked to an O atom e.g.

[0130] The term “halo” or “halogeno” refers to fluoro, chloro, bromo and iodo.

[0131] The term “heterocyclyl”, “heterocyclic” or “heterocycle” means a non-aromatic saturated or partially saturated monocyclic, fused, bridged, or spiro bicyclic heterocyclic ring system(s). The term heterocyclyl includes both monovalent species and divalent species. Monocyclic heterocyclic rings contain from about 3 to 12 (suitably from 3 to 7, most suitably from 5 to 6) ring atoms, with from 1 to 5 (suitably 1, 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur in the ring. Bicyclic heterocycles contain from 5 to 17 member atoms, suitably 5 to 12 member atoms, in the ring. Bicyclic heterocycles contain from about 5 to about 17 ring atoms, suitably from 5 to 12 ring atoms. Bicyclic heterocyclic(s) rings may be fused, spiro, or bridged ring systems. Examples of heterocyclic groups include cyclic ethers such as oxiranyl, oxetanyl, tetrahydrofuranyl, dioxanyl, and substituted cyclic ethers. Heterocycles containing nitrogen include, for example, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrotriazinyl, tetrahydropyrazolyl, and the like. Typical sulfur containing heterocycles include tetrahydrothienyl, dihydro-1, 3-dithiol, tetrahydro-2 / 7-thiopyran, and hexahydrothiepine. Other heterocycles include dihydro-oxathiolyl, tetrahydro-oxazolyl, tetrahydro-oxadiazolyl, tetrahydrodioxazolyl, tetrahydro-oxathiazolyl, hexahydrotriazinyl, tetrahydro-oxazinyl, morpholinyl, thiomorpholinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl. For heterocycles containing sulfur, the oxidized sulfur heterocycles containing SO or SO2 groups are also included. Examples include the sulfoxide and sulfone formsof tetrahydrothienyl and thiomorpholinyl such as tetrahydrothiene 1,1 -dioxide and thiomorpholinyl 1,1 -dioxide. A suitable value for a heterocyclyl group which bears 1 or 2 oxo (=0) or thioxo (=S) substituents is, for example, 2-oxopyrrolidinyl, 2-thioxopyrrolidinyl, 2-oxoimidazolidinyl, 2-thioxoimidazolidinyl, 2-oxopiperidinyl, 2,5-dioxopyrrolidinyl, 2,5-dioxoimidazolidinyl or 2,6-dioxopiperidinyl. Particular heterocyclyl groups are saturated monocyclic 3 to 7 membered heterocyclyls containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen or sulfur, for example azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothienyl, tetrahydrothienyl 1,1-dioxide, thiomorpholinyl, thiomorpholinyl 1,1-dioxide, piperidinyl, homopiperidinyl, piperazinyl or homopiperazinyl. As the skilled person would appreciate, any heterocycle may be linked to another group via any suitable atom, such as via a carbon or nitrogen atom. However, reference herein to piperidino or morpholino refers to a piperidin-1-yl or morpholin-4-yl ring that is linked via the ring nitrogen.

[0132] A “carbon-linked heterocyclyl” means a heretocycle group as defined above that is connected via a carbon atom, rather than a heteroatom such as nitrogen.

[0133] By “spirocyclic ring systems” it is meant a compound which at least two rings which have only one atom in common and are not linked by a bridge.

[0134] By “fused ring systems” it is meant a compound in which two rings share two adjacent atoms. In other words, the rings share one covalent bond.

[0135] By “bridged ring systems” is meant ring systems in which two rings share more than two atoms, see for example Advanced Organic Chemistry, by Jerry March, 4thEdition, Wiley Interscience, pages 131-133, 1992. Examples of bridged heterocyclyl ring systems include, aza-bicyclo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza-bicyclo[3.2.1]octane and quinuclidine.

[0136] The term “heteroaryl” or “heteroaromatic” means an aromatic mono-, bi-, or polycyclic ring incorporating one or more (for example 1-4, particularly 1, 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur. The term heteroaryl includes both monovalent species and divalent species. Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10-membered bicyclic ring, for example a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to about four heteroatoms typically selected from nitrogen, sulfur and oxygen. Typically the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in thecase of an indole or pyrrole nitrogen. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.

[0137] Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-o-oxazinyl, 1 H-pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,1-b]thiazolyl, imidazo[1,2-b][1,2,4]triazinyl. “Heteroaryl” also covers partially aromatic bi- or polycyclic ring systems wherein at least one ring is an aromatic ring and one or more of the other ring(s) is a non-aromatic, saturated or partially saturated ring, provided at least one ring contains one or more heteroatoms selected from nitrogen, oxygen or sulfur. Examples of partially aromatic heteroaryl groups include for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-1,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro-benzo[1,4]dioxinyl, benzo[1,3]dioxolyl, 2,2-dioxo-1,3-dihydro-2-benzothienyl, 4,5,6,7-tetrahydrobenzofuranyl, indolinyl, 1.2.3.4-tetrahydro-1,8-naphthyridinyl, 1,2,3,4-tetrahydropyrido[2,3-b]pyrazinyl and 3.4-dihydro-2 / 7-pyrido[3,2-b][1,4]oxazinyl.

[0138] Examples of five membered heteroaryl groups include but are not limited to pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups.

[0139] Examples of six membered heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl.

[0140] A bicyclic heteroaryl group may be, for example, a group selected from:a benzene ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;a pyridine ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;a pyrimidine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;a pyrrole ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;a pyrazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;a pyrazine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;an imidazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;an oxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;an isoxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;a thiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;an isothiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;a thiophene ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;a furan ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;a cyclohexyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1, 2 or 3 ring heteroatoms; anda cyclopentyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1, 2 or 3 ring heteroatoms.

[0141] Particular examples of bicyclic heteroaryl groups containing a six membered ring fused to a five membered ring include but are not limited to benzfuranyl, benzthiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (e.g., adeninyl, guaninyl), indazolyl, benzodioxolyl and pyrazolopyridinyl groups.

[0142] Particular examples of bicyclic heteroaryl groups containing two fused six membered rings include but are not limited to quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodioxanyl, quinolizinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl and pteridinyl groups.

[0143] The term “aryl” means a cyclic or polycyclic aromatic ring having from 5 to 12 carbon atoms. The term aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like. In particular embodiment, an aryl is phenyl.

[0144] The term "optionally substituted" refers to either groups, structures, or molecules that are substituted and those that are not substituted.

[0145] Where optional substituents are chosen from “one or more” groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups.

[0146] The term “protecting group,” as used herein, refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for furtherreactions. Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.

[0147] Groups can be selectively incorporated into oligomeric compounds as provided herein as precursors. For example an amino group can be placed into a compound as provided herein as an azido group that can be chemically converted to the amino group at a desired point in the synthesis. Generally, groups are protected or present as precursors that will be inert to reactions that modify other areas of the parent molecule for conversion into their final groups at an appropriate time. Further representative protecting or precursor groups are discussed in Agrawal et al., Protocols for Oligonucleotide Conjugates, Humana Press; New Jersey, 1994, 26, 1-72.

[0148] The term “orthogonally protected” refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et al., J. Am. Chem. Soc., 1977, 99, 7363-7365; Barany et al., J. Am. Chem. Soc., 1980, 102, 3084-3095). Orthogonal protection is widely used in for example automated oligonucleotide synthesis. A functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired compound or oligomeric compound.

[0149] Examples of hydroxyl protecting groups which may be utilised in the present invention include without limitation, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy) methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl),9-(p-methoxyphenyl)xanthine-9-yl (MOX) and methoxymethyl (MOM). More preferred hydroxyl protecting groups include without limitation, benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMTr), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

[0150] Examples of protecting groups commonly used to protect phosphate and phosphorus hydroxyl groups (e.g. ORH) include without limitation, methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, allyl, cyclohexyl (cHex), 4-methoxybenzyl, 4-chlorobenzyl, 4-nitrobenzyl, 4-acyloxybenzyl, 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, diphenylmethyl, 4-methylthio-1 -butyl, 2-(S-Acetylthio)ethyl (SATE), 2-cyanoethyl, 2-cyano-1,1-dimethylethyl (CDM), 4-cyano-2-butenyl, 2-(trimethylsilyl)ethyl (TSE), 2-(phenylthio)ethyl, 2-(triphenylsilyl)ethyl, 2-(benzylsulfonyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,3-dibromopropyl, 2,2,2-trifluoroethyl, thiophenyl, 2-chloro-4-tritylphenyl, 2-bromophenyl, 2-[N-isopropyl-N-(4-methoxybenzoyl)amino]ethyl, 4-(N-trifluoroacetylamino)butyl, 4-oxopentyl, 4-tritylaminophenyl, 4-benzylaminophenyl and morpholino. More preferred phosphate and phosphorus hydroxyl protecting groups include without limitation, methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4-methoxybenzyl, 4-chlorobenzyl, 2-chlorophenyl and 2-cyanoethyl. Most preferred is 2-cyanoethyl.

[0151] Examples of amine protecting groups include without limitation, isopropyl, SO2-Me, tosyl, carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyl-oxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl.

[0152] The term "thiol-protecting group" refers to a readily cleavable group bonded to the sulfur of a thiol (-SH) group. Examples of thiol protecting groups include, without limitation, triphenylmethyl (trityl, Trt), acetamidomethyl (Acm), benzamidomethyl, 1 -ethoxyethyl, benzoyl, cyanoethyl, benzyl, 2,4-dichlorobenzyl, and the like. The related term "protected thiol group" refers to a thiol group that is bonded to a thiol-protecting group. General examples of protected thiol groups include, without limitation, -S-alkyl (alkylthio, e.g., C1-C10alkylthio), -S-aryl (thiophenyl), -S-acyl (acylthio), thioacetal, -S-aralkyl (aralkylthio, e.g., aryl(Ci-C4)alkylthio), where some specific protected thiols groups include methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio, isopentylthio, neopentylthio, hexylthio, heptylthio, nonylthio, cyclobutylthio, cyclopentylthio and cyclohexylthio, benzylthio, phenethylthio, propionylthio, n-butyrylthio and iso-butyrylthio. Thio protecting groups and protected thio groups are described in, e.g., C. B. Reese and E. Haslam, " Protective Groups in Organic Chemistry," J. G. W. McOmie, Ed., Plenum Press, New York, N. Y., 1973, Chapters 3 and 4, respectively, and T. W. Greene and P. G. M. Wuts, " Protective Groups in Organic Synthesis," Second Edition, John Wiley and Sons, New York, N. Y., 1991,.

[0153] Examples of thiol protecting groups include without limitation, triphenylmethyl (trityl), benzyl (Bn), and the like.

[0154] The term “oligonucleotide” refers to a polynucleotide strand. It will be appreciated by those skilled in the art that an oligonucleotide has a 5’ and a 3’ end and comprises a sequence of nucleosides linked together by inter-nucleoside linkages.

[0155] “Nucleobase” refers to a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring of a type that is commonly found in nucleic acids. Typically, but not necessarily, the nucleobase is capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with an appropriately complementary nucleobase. The nucleobases may be naturally occurring, such as the naturally-occurring encoding nucleobases A, G, C, T and II, or they may be modified or synthetic. The term “nucleobase” as defined herein therefore refers to both naturally occurring nucleobases which function as the fundamental units of genetic code (i.e. adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (II)) and also any modified or synthetic nucleobases which are known in the art. The skilled person will appreciate there to be numerous natural and synthetic nucleobase analogues available in the art which could be employed in the present invention. As such, the skilled person will readily be able to identify suitable nucleobase analogues for use in the present invention. Commonly available nucleobase analogues are commercially available from a number of sources (for example, see the Glen Research catalogue).

[0156] It will also be appreciated that the term “modified nucleobases” covers but is not limited to universal / degenerate bases (e.g. 3-nitropyrrole, 5-nitroindole and hypoxanthine); fluorescent bases (e.g. tricyclic cytosine analogues (tCO, tCS) and 2-aminopurine); base analogues bearing reactive groups selected from alkynes, thiols or amines; and base analogues that can crosslink oligonucleotides to DNA, RNA or proteins (e.g. 5-bromouracil or 3-cyanovinyl carbazole).

[0157] Common modified or synthetic nucleobases include 2-aminoadenine, 5-propynylcytosine, 5- propynyl uracil, 5-methylcytosine, 3-methyluracil, 5,6-dihydrouracil, 4-thiouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 6-dimethyl aminopurine, 6-methyl amino purine, 2-amino purine, 2,6-diamino purine, 6-amino-8-bromo purine, inosine, 5-methyl cytosine, 7-deazaadenine, 7-deazaguanosine, 3-cyanovinyl carbazole, 3-nitropyrrole, 5-nitroindole, hypoxanthine and G-clamp (a tricyclic aminoethyl-phenoxazine 2’-deoxyCytidine analogue whichhas the structure-L ).

[0158] Additional non-limiting examples of modified or synthetic nucleobases of which the target nucleic acid may be composed can be found in Fasman, CRC PRACTICAL HANDBOOK OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1985, pp. 385-392; Beilstein's Handbuch derOrganischen Chemie, Springer Verlag, Berlin and Chemical Abstracts, the entirety of which is incorporated herein by reference, and which provide references to publications describing the structures, properties and preparation of such nucleobases.

[0159] Further non-limiting examples of modified or synthetic nucleobases include fleximers, such as those described by Khandazhinskaya et al (Org. Biomol. Chem., 2021,19, 7379-7389), where a heterocyclic purine base is split into its two components, i.e. pyrimidine and imidazole. Other fleximers are described by Chen et al (ACS Infect. Dis. 2015, 1, 8, 357-366). Non-limiting examples of fleximer bases include:• 5-(1 / 7-imidazol-5-yl)-2,4-dimethoxypyrimidine;• 5-(1 / 7-imidazol-4-yl)-4-methoxypyrimidin-2-amine:5-(1 / 7-imidazol-4-yl)-4-methoxy- / \ / -methylpyrimidin-2-amine:• 1 / 7-imidazo[4',5':4,5]thieno[3,2-c / ]pyrimidin-5(6 / - / )-one:

[0160] It will be appreciated to the person skilled in the art that the present invention may be applied to compounds comprising a wide range of nucleobases. The particular examples described herein are included by way of example only, and are not intended to limit the potential nucleobases which may be utilised with the present invention.

[0161] As will be recognized by those of skill in the art, many of the above-described modified or synthetic nucleobases are capable of forming Watson-Crick base pairing interactions with the naturally occurring encoding nucleobases A, T, C, G and U. However, in certain embodiments of the invention, it may be desirable to include in a nucleobase polymer synthetic nucleobases which are not capable of forming Watson-Crick base pairs with either the naturally occurring encoding nucleobases A, T, C, G, and U and / or common analogs thereof, but that are capable of forming non-standard (i.e., non-Watson-Crick) base pairs with one another. Nucleobases having these properties are referred to herein as “non-standard synthetic” nucleobases. Examples of such non-standard synthetic nucleobases include, but are not limited to, iso-guanine (iso-G), iso-cytosine (iso-C), xanthine (X), kappa (K), nucleobase H, nucleobase J, nucleobase M and nucleobase N (see U. S. Pat. No. 6,001,983). These non-standard synthetic nucleobases base-pair with one another to form the following non-standard base pairs: iso-C*iso-G, K*X, H*J and M*N. Each of these non-standard base pairs has three hydrogen bonds. Additional non-standard synthetic nucleobases, as well as methods for their synthesis and methods for incorporating them into nucleobase polymers are found in U. S. Pat. Nos. 5,432,272, 5,965,364 and 6,001,983, the disclosures of which are incorporated herein by reference.

[0162] In the photocatalytic chalcogenation (e.g. sulfurization or oxygenation) method of the present invention, the nucleobases may be protected and comprise a protecting group which is removed following the chalcogenation reaction.

[0163] A protected nucleobase is a nucleobase in which reactive functional groups of the nucleobase are protected with nucleobase protecting groups. Typically, nucleobases have amine groups which can be protected with an amine protecting group. Amine protecting groups are known to those skilled in the art. For examples of amine protecting groups see Greene, et al, Protective Groups in Organic Synthesis (1991), John Wiley & Sons, Inc., pages 309-405, the teachings of which are incorporated herein by reference in their entirety. Preferably, amines are protected as amides. The amine groups of adenine and cytosine are typically protected with benzoyl protecting groups, and the amine groups of guanine is typically protected with an isobutyryl protecting group. However, other protection schemes maybe used. For example, for fast deprotection, the amine groups of adenine and guanine can be protected with phenoxyacetyl groups, and the amine group of cytosine can be protected with an acetyl or isobutyryl group. Conditions for removal of the nucleobase protecting group to generate the original functional group will depend on the protecting group used.

[0164] The nucleobase is attached to a sugar moiety (typically ribose or deoxyribose) or a ribose or deoxyribose mimic, for example a chemically modified sugar derivative (e.g. a chemically modified ribose or deoxyribose) or a cyclic group that functions as a synthetic mimic of a ribose or deoxyribose sugar moiety (e.g. the morpholino ring present in morpholino oligonucleotides). A chemically modified sugar derivative includes sugars modified at the 2’ position, for example to include 2'-O-methyl, 2'-O-methoxy-ethyl, 2’-NH2 and 2’-F modifications. Such sugars may be located in any of the nucleotides present in the oligonucleotides of the present invention.

[0165] The term “nucleoside” is used herein to refer to a moiety composed of a sugar / a ribose or deoxyribose mimic bound to a nucleobase / nucleobase analogue. The term nucleoside as used herein excludes the inter-nucleoside linkage that connects adjacent nucleosides together. An “inter-nucleoside linkage” is a linking group that connects the rings of the sugar / ribose or deoxyribose mimic of adjacent nucleosides. Thus, a “nucleotide” is a nucleoside with one or more inter-nucleoside linkage attached.

[0166] The terms “locked nucleic acid”, “LNA” or “locked nucleoside” are used herein to refer to nucleic acids or nucleosides comprising a ribose or deoxyribose moiety in which the conformation of the ribose or deoxyribose ring is fixed or locked in a specific conformation, typically by a bridging group. Typically the bridging group connects the 2’ and 4’ carbon atoms of the ribose or deoxyribose rings and locks the ribose or deoxyribose in the 3’-endo conformation(which is often found in A-form duplexes). Examples of locked nucleic acid / nucleoside structures are well known in the art and are commercially available.

[0167] A solid support for oligonucleotide synthesis is an organic or inorganic polymer that is insoluble in the reagents used for oligonucleotide synthesis. Typically, rigid polystyrene or controlled-pore glass silica is used as a solid support in oligonucleotide synthesis. Additionally, microporous or soft gel supports, especially poly(acrylamide) supports, such as those more commonly employed for the solid phase synthesis of peptides may be employed if desired. Preferred poly(acrylamide) supports are amine-functionalized supports, especially those derived from supports prepared by copolymerization of acryloyl-sarcosine methyl ester, N, N-dimethylacryamide and bis-acryloylethylenediamine, such as the commercially available (Polymer Laboratories) support sold under the catalogue name PL-DMA. The procedure for preparation of the supports has been described by Atherton, E. and Sheppard, R. C. in Solid Phase Peptide Synthesis: A Practical Approach, (1984) IRL Press at Oxford University Press, the microporous supports of which are incorporated herein by reference. The functional group on such supports is a methyl ester and this is initially converted to a primary amine functionality by reaction with an alkyl diamine, such as ethylene diamine.

[0168] A suitable pharmaceutically acceptable salt of an oligonucleotide of the invention is, for example, an acid-addition salt of an oligonucleotide of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoroacetic, formic, citric methane sulfonate or maleic acid. In addition, a suitable pharmaceutically acceptable salt of an oligonucleotide of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

[0169] It is also to be understood that certain oligonucleotides of the invention may exist in solvated as well as unsolvated forms such as, for example, hydrated forms.

[0170] By visible light it is meant wavelengths of around 380-750 nm. Visible light encompasses the colours violet, indigo, blue, green, yellow, orange and red. Violet light has a wavelength of from 380 to 450nm; indigo light has a wavelength of from 420 to 440 nm; blue light has a wavelength of from 450 to 495 nm; green light has a wavelength of from 495 to 570 nm; yellow light has a wavelength of from 570 to 590 nm; orange light has a wavelength of from 590 to 620 nm; and red light has a wavelength of from 620 to 750 nm.Numbered ParagraphsThe following paragraphs are not claims but serve to define particular aspects and embodiments of the invention.Paragraph 1. A method for the photocatalytic chalcogenation (e.g. oxygenation or sulfurization) of an organo phosphorous (III) compound, the method comprising reacting an organo phosphorous (III) compound with a chalcogen source (e.g. an oxygen source or a sulfur source) in the presence of a photocatalyst and light irradiation.Paragraph 2. The method according to paragraph 1, wherein the chalcogen source is an oxygen source, a sulfur source or a selenium source.Paragraph 3. The method according to paragraph 1 or paragraph 2, wherein the chalcogen source is an oxygen source or a sulfur source.Paragraph 4. The method according to any one the preceding paragraphs, wherein the organo phosphorous (III) compound is selected from phosphorus (III) nucleotide precursors, phosphoramidites, phosphorothioates, phosphoramidothioates, phosphites, aminophosphites, phosphines and aminophosphines.Paragraph 5. The method according to any one of the preceding paragraphs, wherein the organo phosphorous (III) compound is selected from phosphorus (III) nucleotide precursors, phosphoramidites, phosphites and phosphines.Paragraph 6. The method according to any one of the preceding paragraphs, wherein the organo phosphorous (III) compound is a phosphorus (III) nucleotide precursor.Paragraph 7. The method according to any one of paragraphs 1 to 6, wherein the organo phosphorous (III) compound comprises a moiety according to the formula (Int), below:T IP- R xb(Int)wherein:Xa and Xb are each independently selected from O or S (preferably Xaand Xb are O); R is any suitable functional group, which may be optionally protected.Paragraph 8. The method according to any one of the preceding paragraphs, wherein R is selected from a ORH, SRS, NRN1RN2or CH2Rc1;wherein:a) RHis a hydrogen or a phosphorous hydroxyl protecting group (e.g. cyanoethyl, t-butyl or benzyl);b) Rsis hydrogen or a thiol protecting group (e.g. cyanoethyl, benzyl, 4- chlorobenzyl and 2,4 dichlorobenzyl);c) each of RN1and RN2are independently selected from:i) an amine protecting group, e.g. SO2(1-6C)alkyl or tosyl; or ii) (1-6C)alkyl, (3-8C)cycloalkyl, 3- to 12 membered heterocyclyl or 5 to 10 membered heteroaryl, wherein any (1-6C)alkyl, (3- 8C)cycloalkyl, 3- to 12-membered heterocyclyl, 5 to 10 membered heteroaryl is optionally substituted by one or more substituents independently selected from halo, (1-2C)alkyl, (1-2C)alkoxy, (1- 2C)haloalkyl, (1-2C)haloalkoxy, cyano or NMe2;iii) RN1and RN2are linked such that, together with the nitrogen atom to which they are attached, form a 4 to 7 membered heterocyclic ring;d) RC1is selected from (1-6C)alkyl, (3-8C)cycloalkyl, 3- to 12 membered heterocyclyl, 5 to 10 membered heteroaryl or (1-6C)alkoxy, wherein any (1- 6C)alkyl, (3-8C)cycloalkyl, 3- to 12-membered heterocyclyl, 5 to 10 membered heteroaryl or (1-6C)alkoxy is optionally substituted by one or more substituents independently selected from halo, (1-2C)alkyl, (1-2C)alkoxy, (1- 2C)haloalkyl, (1-2C)haloalkoxy, cyano or NMe2.Paragraph 9. The method according to any one of the preceding paragraphs, wherein the organo phosphorous (III) compound is a phosphorus (III) nucleotide precursor, and wherein the method comprises reacting a nucleotide precursor comprising a moiety according to the formula (lnt-1), below:with a chalcogen source, in the presence of a photocatalyst and light irradiation; wherein:Xa and Xb are each independently selected from O or S (preferably Xaand Xb are O); R is as defined in paragraph 7;R2a is selected from hydrogen, methoxy, F, 0P2a or O(CH2)2OMe; wherein P2a is a hydroxyl protecting group;R4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl;R2bis selected from hydrogen, methoxy, F, OP2b or O(CH2)2OMe; wherein P2b is a hydroxyl protecting group;R4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2b and X4b is (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3and Rb4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl; andB1and B2are independently selected optionally protected nucleobases;to form a nucleotide comprising a moiety according to formula (I) below:(I)wherein X is a chalcogen atom (e.g. oxygen, sulfur or selenium, preferably oxygen or sulfur); wherein R, R2a, R4a, B1, R2b, R4b and B2are as defined in any one of the preceding paragraphs.Paragraph 10. The method according to any one of the preceding paragraphs, wherein the organo phosphorous (III) compound is a phosphorus (III) nucleotide precursor, and the nucleotide precursor has a structure according to Formula (lnt-2) below:(lnt-2)wherein:Xa is selected from O or S (preferably O);Xb is selected from O or S (preferably O);P1 is a hydroxyl protecting group;P2 is selected from a hydroxyl protecting group, or a group with the formula:RpOxPxN('Pr)2wherein Rp is a phosphorous hydroxyl protecting group;or P2 is a linker moiety attached to a solid support optionally via one or more further nucleotides;R is as defined in paragraph 7;R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group;R4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl;R2bis selected from hydrogen, methoxy, F, OP2b or O(CH2)2OMe; wherein P2b is a hydroxyl protecting group;R4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2b and X4b is (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3and Rb4are independently selected from hydrogen, (1 -2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; andRcis selected from hydrogen or a (1 -6C)alkyl; andor P2 and R2b may be linked such that, together with the atoms to which they are attached, they form a fused 5- or 6-membered heterocyclic ring, or a 5- or 6-membered silyl heterocyclic ring, which may be optionally substituted by one or more substituents selected from (1-4C)alkyl, (1-4C)alkoxy, benzyl or phenyl, any of which may in turn be optionally further substituted by one or more substituents selected from (1-4C)alkyl or (1-4C)alkoxy, and / or the ring formed by P2 and R2bmay be attached to a solid support; andB1and B2are independently selected optionally protected nucleobases.Paragraph 11. The method according to paragraph 10, wherein P1 is selected from acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl), 2-(trimethylsilyl)ethoxymethyl (SEM) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).Paragraph 12. The method according to paragraph 10 or 11, wherein P1 is dimethoxytrityl (DMTr).Paragraph 13. The method according to any one of paragraphs 10 to 12, wherein P2 is as defined in item i), ii), iii) or iv) below:i) P2 is a protecting group selected from acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1 -ethoxyethyl, 1-(2-chloroethoxy)ethyl, p- chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p- nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9- phenylxanthine-9-yl (Pixyl), 2-(trimethylsilyl)ethoxymethyl (SEM) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX); orii) P2 isRpO-N(iPr)2.wherein Rpis 2-cyanoethyl; oriii) P2 is a linker moiety attached to a solid support optionally via one or more further nucleotides;iv) or P2 and R2b may be linked such that, together with the atoms to which they are attached form a fused 5-membered cyclic ether or cyclic silyl ether, which may be optionally substituted by one or more (1-4C)alkyl groups; preferably P2 and R2b, together with the atoms to which they are attached, form a ring with the formula:Paragraph 14. The method according to any one of paragraphs 10 to 13, wherein P2 is acetyl or is a linker moiety attached to a solid support.Paragraph 15. The method according to paragraph 14, wherein P2 is a linker moiety attached to a solid support, and wherein the linker moiety and solid support have the formula:wherein:is a solid support, e.g. Controlled Pore Glass (CPG) with a 500- 2000 A pore size.Paragraph 16. The method according to any one of paragraphs 8 to 15, wherein:RHis selected from methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4- methoxybenzyl, 4-chlorobenzyl, 2,4 dichlorobenzyl, 2-chlorophenyl and 2-cyanoethyl, optionally wherein RHis 2-cyanoethyl;Rsis selected from 2-cyanoethyl, benzyl, 4-chlorobenzyl and 2,4 dichlorobenzyl;RN1and RN2are independently selected from isopropyl, SO2Me or tosyl; andRcis selected from methyl and methoxy.Paragraph 17. The method according to any one of paragraphs 8 to 16, wherein R is ORH, wherein RHis a hydrogen or a phosphorous hydroxyl protecting group.Paragraph 18. The method according to any one of paragraphs 8 to 17, wherein RHis selected from methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4-methoxybenzyl, 4-chlorobenzyl, 2-chlorophenyl and 2-cyanoethyl.Paragraph 19. The method according to any one of paragraphs 8 to 18, wherein RHis selected from cyanoethyl, t-butyl and benzyl.Paragraph 20. The method according to any one of paragraphs 8 to 19, wherein RHis 2-cyanoethyl.Paragraph 21. The method according to any one of paragraphs 8 to 20, wherein:R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group; andR4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2) and the other is O; wherein each of Ra1and Ra2are independently selected from hydrogen or methyl; e.g. R2a and R4a are linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-.Paragraph 22. The method according to any one of paragraphs 8 to 21, wherein:R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group; andR4a is hydrogen;or R2aand R4aare linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-.Paragraph 23. The method according to any one of paragraphs 9 to 22, wherein:R2a is selected from hydrogen, methoxy or OP2a; wherein P2a is a hydroxyl protecting group; andR4a is hydrogen.Paragraph 24. The method according to any one of paragraphs 9 to 23, wherein:R2a is selected from hydrogen or OP2a; wherein P2a is a hydroxyl protecting group; and R4a is hydrogen.Paragraph 25. The method according to any one of paragraphs 9 to 24, wherein:R2bis selected from hydrogen, methoxy, F, OP2bor O(CH2)2OMe; wherein P2bis a hydroxyl protecting group; andR4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid of the formula -X2b-X4b-, wherein one of X2b and X4b is (CRb1Rb2) and the other is O; wherein each of Rb1and Rb2are independently selected from hydrogen or methyl; e.g. R2b and R4b are linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-.Paragraph 26. The method according to any one of paragraphs 9 to 25, wherein:R2bis selected from hydrogen, methoxy, F, OP2bor O(CH2)2OMe; wherein P2bis a hydroxyl protecting group; andR4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-.Paragraph 27. The method according to any one of paragraphs 9 to 26, wherein:R2bis selected from hydrogen, methoxy or OP2b; wherein P2b is a hydroxyl protecting group; andR4bis hydrogen.Paragraph 28. The method according to any one of paragraphs 9 to 27, wherein:R2b is selected from hydrogen or OP2b; wherein P2b is a hydroxyl protecting group; and R4b is hydrogen.Paragraph 29. The method according to any one of the preceding paragraphs, wherein the reaction is performed at a temperature of from 0 to 75°C.Paragraph 30. The method according to any one of the preceding paragraphs, wherein the reaction is performed at a temperature of from 15 to 30°C.Paragraph 31. The method according to any one paragraphs 9 to 30, wherein the method further comprises removing any protecting groups present and, if necessary, cleavage from any solid support to form an oligonucleotide comprising a moiety of the formula:wherein:X is a chalcogen (e.g. oxygen, sulfur or selenium, preferably oxygen or sulfur); and R, B1, B2, R2a, R2b, R4aand R4b are as defined in any one of the preceding paragraphs.Paragraph 32. The method according to paragraph 31, wherein X is oxygen, sulfur or selenium.Paragraph 33. The method according to paragraph 31 or 32, wherein X is oxygen or sulfur.Paragraph 34. The method according to any one of the preceding paragraphs, wherein the reaction is a sulfurization reaction and the chalcogen source is a sulfur source.Paragraph 35. The method according to any one of the preceding paragraphs, wherein the reaction is a sulfurization reaction and the sulfur source is a thiosulfate.Paragraph 36. The method according to any one of the preceding paragraphs, wherein the reaction is a sulfurization reaction and the sulfur source is selected from barium thiosulfate, magnesium thiosulfate, potassium thiosulfate, cesium thiosulfate and sodium thiosulfate.Paragraph 37. The method according to any one of the preceding paragraphs, wherein the reaction is performed in a suitable solvent.Paragraph 38. The method according to paragraph 37, wherein if P2 is not a linker moiety attached to a solid support, the solvent comprises one or more of water and one or more aprotic solvents.Paragraph 39. The method according to paragraph 37 or 38, wherein if P2 is not a linker moiety attached to a solid support, the solvent comprises one or more of water and one or more aprotic solvents selected from acetonitrile, THF, dioxane or acetone.Paragraph 40. The method according to paragraph 37, 38 or 39, wherein if P2 is not a linker moiety attached to a solid support, the solvent comprises a mixture of water and acetonitrile.Paragraph 41. The method according to paragraph 37, wherein if P2 is a linker moiety attached to a solid support, the solvent is water.Paragraph 42. The method according to any one of the preceding paragraphs, wherein the photocatalyst is a catalyst which is activated by light with a wavelength greater than or equal to 380 nm.Paragraph 43. The method according to any one of paragraphs 1 to 42, wherein the photocatalyst is a catalyst which is activated by light with a wavelength greater than or equal to 420 nm.Paragraph 44. The method according to any one of paragraphs 1 to 41, wherein the photocatalyst is selected from one or more of riboflavin, flavin adenine dinucleotide, flavin mononucleotide, flavin derivatives, tris(bipyridine)ruthenium(ll) chloride (Ru-bpy), 2,4,6-triphenylpyrylium tetrafluoroborate (TPT), [4,4'-S / s(1, 1-dimethylethyl)-2,2'-bipyridine-A / 1, / 1 ']£> / s[3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-A / ]phenyl-C]lridium(l 11) hexafluorophosphate ([Ir[dF(CF3)ppy]2(dtbbpy)]PF6), 9-Mesityl-10-methylacridinium tetrafluoroborate ([MesAcrMe]BF4), Erythrosin B, Eosin Y, Rose Bengal, Methylene blue, 9-Mesityl-1, 3,6,8-tetramethoxy-10-phenylacridin-10-ium tetrafluoroborate ([Mes(MeO)4AcrPh]BF4), 1,2, 3, 5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzlPN), 2,4,6-Tri(9-carbazol-9-yl)-5-chloroisophthalonitrile (3CzClIPN), Iridium(lll) hexafluorophosphate ([Ir[dF(CF3)ppy]2(dtbbpy)]PF6), Tris(2,2'-bipyridyl)ruthenium(l I) chloride hexahydrate ([Ru(bpy)3]Ch*6H2O) or Triphenyl phosphate (TPP).Paragraph 45. The method according to any one of the preceding paragraphs, wherein the method further comprises coupling with one or more nucleotides.EXAMPLES

[0171] In the present work, a method for photocatalytic sulfurization (also known as thionation) of organo phosphorous (III) compounds, such as nucleotides, has been developed utilizing an inexpensive and non-toxic sulfur source. The method comprises the treatment of phosphorus (III) nucleotide precursor with a sulfur source and photocatalyst under light irradiation in an acetonitrile-water solvent mixture. Initially, the blue light source was used. However, it is known that DNA and RNA molecules can undergo photodegradation even under blue light. Therefore, a milder green light has been successfully employed with an appropriate photocatalyst. The method can also be applied to the synthesis of thionated phosphoramidites, phosphites, and phosphines under similar reaction conditions. The influence of light intensity on the reaction time has also been studied.

[0172] In summary, a mild method for photocatalytic sulfurization of nucleotides has been developed. The present methodology has improved atom and cost-efficiency and is moreenvironmentally friendly compared to the previously reported protocols. Hence, it can find an industrial application in the field of siRNA drugs.

[0173] A method for the photocatalytic oxygenation of organo phosphorous (III) compounds, such as nucleotides has also been developed utilizing air as an inexpensive oxygen gas source.

[0174] For sulfurization reactions, several reaction parameters have been tested, as described below:1) Na2S2O3 amount testNa2S2O3(n eq.) [Mes(MeO)4AcrPh]BF4(1 mol%) green LED (tube with LED strip) Ar MeCN / H2O (3:1), 0.01 Mr.t., 6 h Table 1: Na2S2O3 amount test resultsEntry Na2S2O3, eq. NMR yield, %1 2 652 4 713 6 754 8 77

[0175] From this experiment series it is concluded that the reaction works well with the sodium thiosulfate amounts in the range of 2 to 8 equivalents (in relation to starting material).2) Concentration testNa2S2O3(8 eq.)[Mes(MeO)4AcrPh]BF4(1 mol%) green LED (tube with LED strip) Ar MeCN / H2O (3:1)concentration (n M) r.t., 6 hTable 2: Concentration Test ResultsEntry Concentration, M NMR yield, %1 0.005 802 0.01 813 0.02 844 0.04 84

[0176] From this experiment series it is we concluded that the reaction works well in the concentration range of 0.005M to 0.04 M.3) Photocatalyst loading testNa2S2O3(8 eq.) [Mes(MeO)4AcrPh]BF4(n mol%) green LED (tube with LED strip) Ar MeCN / H2O (3:1), 0.02 Mr.t., 6 h Table 3: Photocatalyst loading test results:Entry Cat. loading, mol% NMR yield, %1 0.1 182 0.5 603 1 844 2.5 805 5 76

[0177] From this experiment series it is concluded that the reaction works well with the catalyst loading in range of 0.5 mol% to 5 mol%.4) Solvent testNMR yield,Entry Solvent%1 MeCN / H2O (3:1) 842 THF / H2O (3:1) 48Dioxane / H2O3 62(3:1)Acetone / H2O4 34(3:1)

[0178] From that experiment series it is concluded that the reaction works well in solvent mixtures of MeCN / H2O (3:1) and THF / H2O (3:1), preferably MeCN / H2O (3:1).5) Photocatalyst testNa2S2O3(8 eq.) photocatalyst (1 mol%) light Ar MeCN / H2O (3:1), 0.02 M r.t., 6 hTable 5: Photocatalyst testEntry Light source Photocatalyst NMR yield, %1 green LED Erythrosin B 362 green LED Eosin Y 253 green LED Rose bengal 644 green LED Methylene blue 185 green LED [MesAcrMe]BF4 846 green LED [Mes(2,3,6-di-tBu)AcrPh]BF4737 green LED [Mes(MeO)4AcrPh]BF4 848 green LED 4CzlPN 179 green LED 3CzCIIPN 1010 blue LED [Ir[dF(CF3)ppy]2(dtbbpy)]PF68611 blue LED [Ru(bpy)3]Cl2*6H2O 612 blue LED TPT 1013 blue LED Riboflavin 85

[0179] From this experiment series it is concluded that under green light the reaction works well with Acridinium photocatalysts (entries 5-7), it is less efficient with Erythrosin B, Eosin Y, and Rose Bengal (entries 1-3). The reaction is inefficient with Methylene blue, 4CzlPN, and 3CzCIIPN under green light (entries 4, 8-9). As for the blue light, the reaction works very well with Ir photocatalyst (entry 10) and Riboflavin (entry 13), but is less efficient with Ru and TPT photocatalysts (entries 11-12).

[0180] Further experiments and details of synthetic procedures is described below.General information

[0181] All chemicals were reagent grade (Acros Organics, Alfa Aesar, Apollo, Fluka, Fluorochem, Riedel-de Haen, Sigma-Aldrich, TCI) and used as supplied. All reactions were carried out in dried glassware under an Ar atmosphere unless stated otherwise. Toluene (99.5%, Extra Dry, over Molecular Sieves, Stabilized, AcroSeal®, Code: 364415000) and CH2CI2 (99.5%, Extra Dry, over Molecular Sieves, Stabilized, AcroSeal®, Code: 348465000) were purchased from Acros Organics. Petroleum ether (40-60 °C) was purchased from Biosolve. Extracts were dried over technical grade Na2SO4. Analytical thin layer chromatography (TLC) was performed on pre-coated Merck silica gel 60 F254 plates (0.25 mm) and visualised by UV. Flash column chromatography was carried out on Silicycle SiliaFlash P60 (230 - 400 mesh) and ZEOprep C18 silica gel (40 to 63 pm, 90 A). Concentration in vacuo was performed by rotary evaporation to ~ 10 mbar at 40 °C, drying at ~ 0.5 mbar and at r.t. Optical rotations of enantioenriched diastereomeric mixtures were obtained at r.t. on a Jasco P-2000 polarimeter at 589 nm using a 1.00 mL cell with a length of 100 mm; optical rotations are reported in (° mL) / (g dm), concentrations in g / 100 mL. IR spectra were measured on an ATR Varian Scimitar 800 FT-IR spectrometer and are reported in cm-1. The intensities of the bands are reported as: w = weak, m = medium, s = strong.1H NMR and13C NMR spectra were recorded on a Bruker Avance III 500 MHz or 400 MHz spectrometer at 298 K in CDCI3 supplied by Cambridge Isotope Laboratories (DLM-7TB-100S). Chemical shifts (5) are reported in ppm relative to tetramethylsilane (0.00 ppm). The multiplicities are reported in Hz as: s = singlet, br = broad singlet, d = doublet, t = triplet, q = quartet and m = multiplet. DEPT135 and 2-dimensional experiments (COSY, HMBC, HMQC, NOESY, TOCSY) were used to support assignments but are not included in this document. Both carbon and phosphorus were decoupled of the proton. High-resolution spectrometry (HR-ESI) was performed by Dr. Michael Pfeffer of the University of Basel on a Bruker maXis 4G QTOF ESI mass spectrometer. X-ray crystallography was performed by Dr. Alessandro Prescimone on a Bruker Kappa Apex 2 or a Stoe StadiVari diffractometer. Enantiomeric ratios were determined by HPLC on a chiral stationary phase using the indicated analytical columns and eluents with the retention times (Rt) reported in minutes. The phosphorane compounds were named according to previous literature1and the IUPAC rules (Nomenclature of Inorganic Chemistry IUPAC Recommendations 2005, sections 9.3.2, 9.3.3 and 9.3.4)2.Substrate synthesisDMAP, Et3N CH2CI2, r.t., 16 h 5-ETT2) TCA, CH2CI2, r t, 1 h CH2CI2 / CH3CNGP A r t, 16 hGP BCH2CI2 / CH3CN r t, 16 hGP BGeneral Procedure A: Synthesis of 3’-O-acetyl protected 2’-deoxynucleosides1) Acetylation

[0182] To a stirred solution of 5’-O-dimethoxytrityl-protected nucleoside (1.00 eq.) and / V, / V-dimethylpyridin-4-amine (0.20 eq.) in CH2CI2 (0.10 molL-1) was added acetic anhydride (1.50 eq.) and triethylamine (1.50 eq.) under Ar atmosphere at r.t. The progress of the reaction was monitored by TLC (CF^Ch / MeOH, 95:5 mixture). The mixture was diluted with ethyl acetate (200 mL) and subsequently washed with aqueous HCI (1.00 molL-1, 2 x 20 mL) and brine (20 mL). The organic phase was dried over Na2SC>4, and concentrated under reduced pressure. The product thus obtained was used directly in the next step without further purification.2) Detritylation

[0183] To a stirred solution of the product from the previous step in toluene (0.10 molL-1) was added 2,2,2-trichloroacetic acid (TCA, 2.00 eq.) at r.t. The mixture was stirred for 1 h. After completion of the reaction, MeOH (20 mL) was added, and the mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography on previously deactivated (EtaN) silica gel (CF^Ch / MeOH / EtaN, 100:0:0 to 90:9.99:0.01) to afford the desired product.General Procedure B: Phosphoramidite coupling

[0184] Phosphoramidite (1.00 eq.) and protected nucleoside (1.20 eq.) were suspended in CH2CI2 (0.10 molL-1) under Ar atmosphere. To this stirred mixture, a solution of 5-(ethylthio)-1 H-tetrazole (5-ETT, 0.36 molL-1in MeCN, 1.20 eq.) was added dropwise over 30 min at r.t. The reaction mixture was stirred for 16 h. The solvent was evaporated under reduced pressure. The residue was taken in CH2CI2 (30 mL) and subsequently washed with saturated aqueous NaHCO3(2 x 30 mL) and brine (30 mL). The organic phase was dried with Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography on previously deactivated (Et3N) silica gel (CH2CI2 / MeOH / Et3N, 100:0:0 to 90:9.99:0.01) to afford the desired product.

[0185] Compound 1a was prepared according to a reported procedure (characterization data in accordance with the literature Paul et al. (Oxidative Substitution of Boranephosphonate Diesters as a Route to Post-synthetically Modified DNA. J. Am. Chem. Soc. 137, 3253-3264 (2015)).5’-O-DMTr-dA(Bz)-3’-O-Ac-dT phosphite (1b)

[0186] Following the general procedure B, the reaction of 3’-O-Ac-dT (273 mg, 0.96 mmol, 1.20 eq.), 5’-O-DMTr-dA(Bz)-phosphoramidite (686 mg, 0.80 mmol, 1.00 eq.) and 5-(ethylthio)-1 H-tetrazole (125 mg, 0.96 mmol, 1.20 eq. in MeCN (3.20 mL)) in CH2CI2 (8.50 mL) provided the title compound as white solid (710 mg, 0.68 mmol, 97%); Rf0.45 (CH2CI2 / MeOH, 95:5); IR vmax(neat): 3646w, 3565w, 3031w, 2555w, 2361w, 1691s, 1609m, 1582m, 1510m, 1454m, 1371w, 1297w, 1247s, 1178m, 1072m, 1031s, 958w, 909s, 830m, 812w, 728s, 646m;1H NMR (500 MHz, CDCI3) 59.05 - 8.94 (m, 1H, NH), 8.77 - 8.68 (m, 1H, C2 H). 8.50 - 8.41 (m, 1H, NH), 8.22 - 8.11 (m, 1 H, C8AH). 8.06 - 7.99 (m, 2H, ArBz-o-H), 7.64 - 7.58 (m, 1 H, ArBz-p-H), 7.57 - 7.50 (m, 2H, ArBz-m-H), 7.42 - 7.35 (m, 3H, C6TH and DMTr-H), 7.32 - 7.13 (s, 7H overlapping with CDCI3, DMTr- / - / ), 6.85 - 6.76 (m, 4H, DMTr-H), 6.55 - 6.47 (m, 1H, CTH), 6.33 - 6.20 (m, 1H, CTH), 5.33 -5.23 (m, 1 H, ribose- / - / ), 5.20 - 5.02 (m, 1 H, ribose-H), 4.45 - 4.08 (m, 4H, ribose-H and C5’TH), 4.07 - 3.92 (m, 2H, OC / 72CH2CN), 3.82 - 3.72 (m, 6H, DMTr-OC / 73), 3.55 - 3.35 (m, 2H, C5’AH), 3.22 - 3.00 (m, 2H, C2’H2), 2.85 - 2.55 (m, 2H, OCH2C / 72CN), 2.47 - 2.16 (m, 2H, C2’H2), 2.13 -2.06 (m, 3H, OAc-CH3), 1.96 - 1.83 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.6, 165.0, 163.8, 158.7 (2C), 152.6, 151.7, 150.6, 149.9, 144.4, 141.8, 135.6 (2C), 135.2, 133.7,132.8, 130.1 (4C), 128.8 (2C), 128.1 (6C), 127.1, 123.8, 117.2, 113.3 (4C), 111.7, 86.8, 85.7, 84.9 (2C), 83.5, 74.2 (2C), 63.1, 62.4, 57.9, 55.4 (2C), 39.1, 37.4, 21.1, 20.4, 12.7;31P{1H} NMR (162 MHz, CDCI3) 6 139.4, 139.2. HRMS (ESI+): m / z calcd. 1041.3542 for ([M+H]+), found 1041.3542.5’-O-DMTr-dC(Ac)-3’-O-Ac-dT phosphite (1c)NHAcOAc

[0187] Following the general procedure B, the reaction of 3’-O-Ac-dT (239 mg, 0.84 mmol, 1.20 eq.), 5’-O-DMTr-dC(Ac)-phosphoramidite (540 mg, 0.70 mmol, 1.00 eq.) and 5-(ethylthio)-1 H-tetrazole (109 mg, 0.84 mmol, 1.20 eq. in MeCN (2.80 mL)) in CH2CI2 (7.40 mL) provided the title compound as white solid (240 mg, 0.25 mmol, 36%); Rf0.30 (C^Ch / MeOH, 95:5); IR vmax(neat): 3773w, 3691w, 3021w, 2357w, 1665s, 1611m, 1562m, 1494s, 1442w, 1393w, 1318m, 1241s, 1178m, 1112m, 1032s, 1001m, 961m, 909w, 829m, 747s, 667m, 641w, 616m;1H NMR (500 MHz, CDCI3) 6 9.43 - 9.21 (m, 1H, NH), 9.10 - 8.88 (m, 1H, NH), 8.24 - 8.10 (m, 1H, C6cH).7.39 - 7.23 (m, 10H, DMTr-H and C6TH overlapping with CDCI3), 7.22 - 7.14 (m, 1H, C5cH).6.89 - 6.79 (m, 4H, DMTr-H), 6.40 -6.26 (m, 1H, CTH), 6.26 - 6.18 (m, 1H, CTH), 5.32 - 5.14 (m, 1H, ribose- / - / ), 4.96 - 4.80 (m, 1H, ribose-H), 4.28 - 4.17 (m, 1H, ribose-H), 4.17 - 3.86 (m, 5H, OCH2CH2CN and C5'H2and ribose-H), 3.86 - 3.76 (m, 6H, OCW3), 3.59 - 3.47 (m, 1 H, C5'H), 3.45 - 3.34 (m, 1 H, C5'H), 2.84 - 2.67 (m, 1 H, C2'H), 2.65 - 2.53 (m, 2H, OCH2C / 72CN), 2.46 -2.27 (m, 2H, C2'H), 2.28- 2.21 (m, 3H, Ac-CH3), 2.19 -2.12 (m, 1H, C2'H), 2.15 -2.04 (m, 3H, AC-C / 73), 1.91 - 1.84 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.7, 170.6, 163.7, 162.7, 158.9 (2C), 155.1, 150.4, 144.2 (2C), 135.2 (3C), 130.2 (4C), 128.2 (4C), 127.4, 117.2, 113.5 (4C), 111.5, 96.5, 87.3, 86.9, 85.4, 84.8, 83.4, 74.2, 72.3, 62.2, 62.0, 57.6, 55.4 (2C), 41.0, 37.4, 25.1, 21.1, 20.3, 12.8;31P{1H} NMR (202 MHz, CDCI3) 5 139.2, 139.1. HRMS (ESI+): m / z calcd. 977.3093 for ([M+Na]+), found 977.3091.5’-O-DMTr-dG(lb)-3’-O-Ac-dT phosphite (1 d)oOAc

[0188] Following the general procedure B, the reaction of 3’-O-Ac-dT (199 mg, 0.70 mmol, 1.00 eq.), 5’-O-DMTr-dG(lb)-phosphoramidite (882 mg, 1.05 mmol, 1.50 eq.) and 5-(ethylthio)-1 H-tetrazole (137 mg, 1.05 mmol, 1.20 eq. in MeCN (2.80 mL)) in CH2CI2 (7.40 mL) provided the title compound as white solid (231 mg, 0.23 mmol, 32%); Rf0.31 (CH2CI2 / MeOH, 95:5); IR vmax(neat): 3625w, 3509w, 3063w, 2361s, 1682s, 1608m, 1559w, 1509w, 1469w, 1403w, 1327w, 1247m, 1222m, 1102m, 1067m, 1030m, 956w, 831w, 772s, 648w, 618w;1H NMR (500 MHz, CDCI3) 6 12.42 - 11.80 (br. s, 2H, N / 7), 9.75 - 9.47 (br. s, 1 H, N / 7), 7.88 - 7.64 (m, 1 H, C8GH), 7.58 - 7.45 (m, 1H, C6T / 7), 7.44 - 7.34 (m, 2H, DMTr-H), 7.33 - 7.12 (m, 7H, DMTr-H), 6.85 - 6.67 (m, 4H, DMTr-H), 6.33 - 6.21 (m, 1 H, ribose-H), 6.21 - 6.08 (m, 1 H, ribose-H), 5.41 - 5.27 (m, 1 H, ribose- / 7), 5.04 - 4.89 (m, 1H, ribose-H), 4.27 - 3.86 (m, 6H, ribose-H and C5’GH2and OC / 72CH2CN), 3.83 - 3.69 (m, 6H, DMTr-OC / 73), 3.41 - 3.20 (m, 2H, C5’TH2), 2.72 - 2.51 (m, 5H, C2’GH2and OCH2CH2CN and / Pr-CH(CH3)2), 2.51 - 2.29 (m, 2H, C2’TH2), 2.20 - 2.06 (m, 3H, Ac-CH3), 2.05 - 1.87 (m, 3H, C5TC / 73), 1.20 - 1.09 (m, 6H, 'Pr-CH(CH3)2);13C NMR (126 MHz, CDCI3) 5 179.4, 170.6, 163.9, 158.7 (2C), 155.5, 150.2, 147.9 (2C), 144.4, 136.9, 136.3, 135.4 (2C), 130.0 (4C), 128.0 (4C), 127.1, 121.7, 117.5, 113.2 (4C), 111.2, 86.8, 86.3, 85.9, 84.7, 84.0, 74.8, 74.1, 63.4, 62.6, 58.0, 55.3 (2C), 39.9, 37.3, 36.0, 21.2, 20.3, 19.0 (2C), 12.6;31P NMR (162 MHz, CDCI3) 5 142.6, 140.5. HRMS (ESI+): m / z calcd. 1023.3648 for ([M+H]+), found 1023.3666.5’-O-DMTr-dT-3’-O-Ac-dA(Bz) phosphite (1e)

[0189] Following the general procedure B, the reaction of 3’-O-Ac-dA(Bz) (100 mg, 0.25 mmol, 1.20 eq.), 5’-O-DMTr-dT-phosphoramidite (200 mg, 0.27 mmol, 1.00 eq.) and 5-(ethylthio)-1 H-tetrazole (35.0 mg, 0.27 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2CI2(5.00 mL) provided the title compound as white solid (121 mg, 0.12 mmol, 41%); Rr 0.32 (CH2CI2 / MeOH, 95:5); IR vmax(neat): 3050m, 2273m, 1971m, 1572s, 1435s, 1311w, 1106s, 988m, 710s;1H NMR (500 MHz, CDCI3) 69.06 - 8.93 (d, 1 H, NH), 8.71 - 8.64 (m, 1 H, C2AH), 8.43 - 8.35 (m, 1 H, C8AH), 7.75 -7.64 (m, 2H, ArBz-o-H), 7.59 - 7.53 (m, 1H, ArBz-p-H), 7.46 - 7.34 (m, 3H, ArBz-m-H and C6TH).7.33 - 7.19 (m, 9H, DMTr-H), 6.90 - 6.75 (m, 4H, DMTr-H), 6.54 - 6.45 (m, 1H, CTH), 6.43 -6.31 (m, 1H, CTH), 5.46- 5.36 (m, 1H, ribose-H), 4.99 -4.84 (m, 1H, ribose-H), 4.30 - 4.22 (m, 1H, ribose- / - / ), 4.19 - 4.03 (m, 3H, C5’H2and ribose-H), 3.89 - 3.80 (m, 2H, OC / 72CH2CN), 3.79 - 3.74 (m, 6H, DMTr-OC / 73), 3.54 - 3.46 (m, 1 H, C5’H), 3.37 - 3.30 (m, 1 H, C5’H), 2.97 - 2.85 (m, 1 H, C2’H), 2.66 - 2.58 (m, 1 H, C2’H), 2.55 - 2.27 (m, 4H, OCH2CH2CN and C2’H2), 2.11 (m, 3H, OAC-CH3), 1.46 (s, 3H, C5TC / 73);13C{1H} NMR (126 MHz, CDCI3) 6 172.0, 170.4, 163.8, 158.9 (2C), 152.9, 152.3, 151.0, 150.5, 144.3, 143.7, 135.5, 135.3 (2C), 134.3, 133.0, 130.2 (4C), 129.3 (2C), 129.0, 128.7 (2C), 128.2 (4C), 127.3, 117.3, 113.4 (4C), 111.5, 87.2, 85.2, 84.7, 84.6, 84.3, 74.5, 73.7, 63.0, 62.4, 57.6, 55.4 (2C), 39.9, 37.6, 21.0, 20.2, 11.9;31P{1H} NMR (202 MHz, CDCI3) 6 139.5, 139.0. HRMS (ESI+): m / z calcd. 1041.3542 for ([M+H]+), found 1041.3539.5’-O-DMTr-dT-3’-O-Ac-dC(Ac) phosphite (1f)oOAc

[0190] Following the general procedure B, the reaction of 3’-O-Ac-dC(Ac) (112 mg, 0.36 mmol, 1.20 eq.), 5’-O-DMTr-dT-phosphoramidite (223 mg, 0.30 mmol, 1.00 eq.) and 5-(ethylthio)-1 H-tetrazole (47.0 mg, 0.36 mmol, 1.20 eq. in MeCN (1.20 mL)) in CH2CI2(3.20 mL) provided the title compound as white solid (120 mg, 0.13 mmol, 42%); Rr 0.27 (CH2CI2 / MeOH, 95:5); IR vmax (neat): 3722w, 3625m, 2338w, 1680s, 1561m, 1494s, 1371w, 1307m, 1245s, 1178m, 1134m, 1032s, 909s, 830m, 728s, 598m;1H NMR (500 MHz, CDCI3) 6 9.79 - 9.66 (m, 1H, NH), 9.38 -9.13 (m, 1H, NH), 8.03 - 7.98 (m, 1H, C6cH). 7.48 - 7.41 (m, 1H, C6TH). 7.37 - 7.30 (m, 1H, C5cH). 7.31 - 7.25 (m, 2H, DMTr-H), 7.23 - 7.05 (m, 7H, DMTr-H), 6.84 - 6.64 (m, 4H, DMTr- / - / ), 6.29 - 6.19 (m, 1H, C1’H), 6.19 - 6.10 (m, 1H, CTH), 5.15 - 5.06 (m, 1H, ribose-H), 4.91 -4.74 (m, 1H, ribose- / - / ), 4.16 -4.08 (m, 1H, ribose-H), 4.07 - 4.02 (m, 1H, ribose-H), 4.02 - 3.92 (m, 2H, C5’ / 72), 3.91 - 3.75 (m, 2H, OCH2CH2CN), 3.72 - 3.63 (m, 6H, OCW3), 3.46 - 3.37 (m, 1H, C5’H), 3.26 - 3.20 (m, 1H, C5’H), 2.69 - 2.57 (m, 1H, C2’H), 2.52 - 2.43 (m, 2H, OCH2CH2CN), 2.40 - 2.32 (m, 1H, C2’H), 2.31 - 2.18 (m, 1H, C2’H), 2.18 - 2.13 (m, 3H, Ac-CW3), 2.04 - 1.92 (m, 4H, C2’H and Ac-CH3), 1.42 - 1.29 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCh) 6 171.1, 170.6, 164.1, 163.0, 158.9 (2C), 155.1, 150.4, 144.3, 143.9, 135.5 (3C),130.2 (4C), 128.3 (2C), 128.1 (2C), 127.3, 117.2, 113.4 (4C), 111.4, 96.8, 87.3, 87.2, 85.2, 84.8, 84.3, 74.1, 73.7, 63.0, 62.3, 57.7, 55.4 (2C), 40.0, 39.0, 25.0, 21.0, 20.4, 11.9;31P{1H} NMR (202 MHz, CDCI3) 6 139.6, 139.1. HRMS (ESI+): m / z calcd. 955.3274 for ([M+H+), found 955.3284.5’-O-DMTr-dT-3’-O-Ac-dG(lb) phosphite (1g)

[0191] Following the general procedure B, the reaction of 3’-O-Ac-dG(lb) (228 mg, 0.60 mmol, 1.20 eq.), 5’-O-DMTr-dT-phosphoramidite (372 mg, 0.50 mmol, 1.00 eq.) and 5-(ethylthio)-1 H-tetrazole (78.0 mg, 0.60 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2CI2 (10.0 mL) provided the title compound as white solid (289 mg, 0.25 mmol, 51%); Rr 0.22 (C^Ch / MeOH, 95:5); IR vmax (neat): 3673 m, 2973m, 2902m, 2361 s, 1682m, 1607w, 1560w, 1508w, 1465w, 1405w, 1248s 1055s, 908m, 790w, 728m, 632w;1H NMR (500 MHz, CDCI3) 6 12.04 (br. s, 1H, NH), 9.07 -8.91 (m, 1H, NH), 8.81 (br. s, 1H, NH), 7.82 - 7.73 (m, 1H, C8GH), 7.51 - 7.46 (m, 1H, C6TH).7.33 - 7.28 (m, 2H, DMTr-H), 7.26 - 7.10 (m, 7H, DMTr-H overlapping with CDCI3), 6.80 - 6.73 (m, 4H, DMTr-H), 6.36 - 6.24 (m, 1H, CTH), 6.15 - 6.09 (m, 1H, CTH), 5.38 - 5.29 (m, 1H, ribose- / - / ), 4.91 - 4.79 (m, 1H, ribose-H), 4.18 - 3.75 (m, 6H, ribose-H and OC / 72CH2CN and C5’G / 72), 3.74 - 3.70 (s, 6H, DMTr-OC / 73), 3.44 - 3.35 (m, 1 H, C5’TH), 3.31 - 3.23 (m, 1 H, C5’TH), 3.01 - 2.85 (m, 1H, C2’H), 2.66 - 2.19 (m, 6H, C2’H and OCH2C / 72CN and CH(CH3)2), 2.03 -1.98 (m, 3H, OAC-CH3), 1.43 - 1.34 (m, 3H, C5TCH3), 1.21 - 1.07 (m, 6H, CH(CH3)2);13C{1H} NMR (126 MHz, CDCI3) 6 179.0, 170.5, 163.8, 158.9 (2C), 155.6, 150.5, 148.2, 147.9, 144.3, 137.7, 135.6, 135.3 (2C), 130.3 (4C), 128.3 (2C), 128.2 (2C), 127.4, 122.1, 117.5, 113.5 (4C), 111.6, 87.2, 85.3, 84.7, 84.6, 83.9, 74.7, 73.9, 63.1, 62.5, 57.6, 55.4 (2C), 39.9, 36.7, 36.4, 21.1, 20.5, 19.1 (2C), 11.9;31P{1H} NMR (202 MHz, CDCI3) 6 139.7, 138.7. HRMS (ESI+): m / z calcd.1023.3648 for ([M+H]+), found 1023.3639.5’-O-DMTr-2’-F-dA(Bz)-3’-O-Ac-dT phosphite (1 h)BzHNOAc

[0192] Following the general procedure B, the reaction of 3’-O-Ac-dT (130 mg, 0.46 mmol, 1.00 eq.), 5’-O-DMTr-2’-F-dA(Bz)-phosphoramidite (481 mg, 0.55 mmol, 1.20 eq.) and 5-(ethylthio)-1H-tetrazole (71 mg, 0.55 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2CI2 (10.0 mL) provided the title compound as white solid (320 mg, 0.30 mmol, 66%); Rr 0.31 (CFhCh / MeOH, 95:5); IR vmax(neat): 3005w, 2362w, 2250w, 1690m, 1608m, 1509m, 1454m, 1246s, 1177m, 1030m, 907m, 828m, 727s, 645w;1H NMR (500 MHz, CDCI3) 69.72 - 9.55 (br. s, 1H, N / 7), 9.54 - 9.34 (m, 1H, N / 7), 8.81 - 8.74 (m, 1H, C2AH), 8.32 - 8.21 (m, 1H, C8AH), 8.10 - 8.01 (m, 2H, ArBz-o- / 7), 7.62 - 7.56 (m, 1H, ArBz-p-H), 7.55 - 7.47 (m, 2H, ArBz-m- / 7), 7.42 - 7.33 (m, 3H, DMTr-H and C6TH). 7.31 - 7.15 (m, 7H, DMTr-H overlapping with CDCI3), 6.82 - 6.77 (m, 4H, DMTr-H), 6.38 - 6.25 (m, 2H, C1’H), 5.90 - 5.72 (m, 1H, ribose-H), 5.39 - 5.18 (m, 2H, ribose- / 7), 4.41 - 4.35 (m, 1H, ribose-H), 4.27 - 3.88 (m, 5H, ribose-H and C5’H2and OC / 72CH2CN), 3.81 - 3.73 (m, 6H, DMTr-OC / 73), 3.68 - 3.59 (m, 1 H, C5’H), 3.48 - 3.34 (m, 1 H, C5’H), 2.61 -2.51 (m, 2H, OCH2CH2CN), 2.40 -2.30 (m, 1H, C2’TH), 2.25 -2.11 (m, 1H, C2’TH), 2.11 - 2.06 (m, 3H, OAC-CH3), 1.93- 1.80 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.6, 165.0, 163.8, 158.7 (2C), 152.9, 151.5, 150.5, 150.1, 144.3, 142.0, 135.4 (3C), 133.6, 132.9, 130.1 (4C), 128.8 (2C), 128.2 (4C), 128.0 (2C), 127.2, 123.8, 117.1, 113.3 (4C), 111.6, 91.8, 87.3, 86.8, 84.7, 83.4, 82.0, 74.2, 70.0, 62.6, 61.5, 57.8, 55.3 (2C), 37.3, 21.0, 20.3, 12.6;31P{1H} NMR (202 MHz, CDCI3) 5 139.3 (d,4JP-F= 13.0 Hz), 138.9 (d,4JP. F= 8.4 Hz);19F{1H} NMR (471 MHz, CDCI3) 5 -202.4 (d,4JF-P = 8.4 Hz), -202.6 (d,4JF-P = 12.9 Hz). HRMS (ESI+): m / z calcd. 1059.3448 for ([M+H]+), found 1059.3456.5’-O-DMTr-2’-O-Me-U-2’,3’-O-isopropylidene-U phosphite (1i)

[0193] Following the general procedure B, the reaction of 2’,3’-O-isopropylidene-U (255 mg, 0.90 mmol, 1.20 eq.), 5’-O-DMTr-2’-O-Me-U-phosphoramidite (570 mg, 0.75 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (120 mg, 0.90 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2CI2 (10.0 mL) provided the title compound as white solid (750 mg, 0.80 mmol, 88%); Rr 0.40 (CFhCh / MeOH, 95:5); IR vmax(neat): 3196m, 3058m, 2938m, 2249m, 1687s, 1509m, 1458m, 1380m, 1251m, 1069m, 1033m, 909m, 828w, 729s, 644w;1H NMR (500 MHz, CDCI3) 69.21 - 9.05 (m, 2H, N / 7), 7.95 - 7.87 (m, 1 H, C6uH). 7.39 - 7.18 (m, 10H DMTr-H and C6uH overlapping with CDCI3), 6.89 -6.82 (m, 4H, DMTr- / 7), 6.02 - 5.98 (m, 1H, CTuH), 5.68-5.66 (m, 1H, C1’uH), 5.66- 5.56 (m, 1 H, C5uH). 5.32 - 5.25 (m, 1 H, C5uH). 5.00 - 4.94 (m, 1 H, ribose-H), 4.88 - 4.72 (m, 2H, ribose- / 7), 4.29 - 4.20 (m, 2H, ribose-H), 4.14 - 3.82 (m, 5H, ribose-H and C5’uH2and OC / 72CH2CN), 3.82 - 3.78 (m, 6H, DMTr-OC / 73), 3.62 - 3.56 (m, 1 H, C5’uH), 3.55 - 3.53 (m, 3H, OCW3), 3.47 - 3.41 (m, 1H, C5’uH), 2.61 (app t, J = 6.3 Hz, 1H, OCH2C / 72CN), 2.57 - 2.52 (m, 1H, OCH2CH2CN), 1.56 - 1.54 (m, 3H, C(CW3)2), 1.34 - 1.30 (m, 3H, C(CW3)2);13C{1H} NMR (126 MHz, CDCI3) 6 163.3, 163.2, 159.0 (2C), 150.5, 150.1, 144.2, 142.6, 139.9, 135.0 (2C), 130.4 (2C), 130.3 (2C), 128.4 (2C), 128.2 (2C), 127.5, 117.4, 114.6, 113.5 (4C), 102.73, 102.66, 95.0, 87.4, 87.0, 86.7, 84.6, 83.1, 82.3, 81.2, 69.9, 62.7, 61.3, 58.7, 57.4, 55.4 (2C), 27.3, 25.4, 20.3;31P{1H} NMR (202 MHz, CDCI3) 6 139.6, 139.1. HRMS (ESI+): m / z calcd. 944.3114 for ([M+H]+), found 944.3126.5’-O-DMTr-2’-O-TBS-U-2’,3’-O-isopropylidene-U phosphite (1j)

[0194] Following the general procedure B, the reaction of 2’,3’-O-isopropylidene-U (275 mg, 0.96 mmol, 1.20 eq.), 5’-O-DMTr-2’-O-TBS-U-phosphoramidite (690 mg, 0.80 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (125 mg, 0.96 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2CI2 (10.0 mL) provided the title compound as white solid (470 mg, 0.450 mmol, 49%); Rr 0.38 (CH2CI2 / MeOH, 95:5); IR vmax(neat): 2855m, 2842m, 2382m, 2350m, 1677m, 1508m, 1480 m, 1394m, 1175m, 1107m, 1020m, 921m, 843 m, 702s, 645m;1H NMR (500 MHz, CDCI3) 59.29 -8.92 (m, 2H, N / 7), 7.93-7.84 (m, 1H, C6uH), 7.38-7.10 (m, 10H, DMTr- / 7 and C6u / 7 overlapping with CDCI3), 6.88 - 6.82 (m, 4H, DMTr-H), 5.97 - 5.92 (m, 1 H, C1’uH), 5.71 - 5.50 (m, 2H, C5uH).5.31 - 5.26 (m, 1 H, ribose-H), 5.02 - 4.95 (m, 1 H, ribose-H), 4.86 - 4.74 (m, 1 H, ribose-H), 4.64- 4.57 (m, 1 H, ribose- / - / ), 4.46 - 4.39 (m, 1 H, ribose-H), 4.28 - 4.19 (m, 2H, ribose-H), 4.08 -3.83 (m, 4H, C5’uH2and OCH2CH2CN), 3.82 - 3.78 (d, 6H, DMTr-OC / 73), 3.61 - 3.51 (m, 1 H, C5’uH), 3.48 - 3.38 (m, 1 H, C5’uH), 2.65 - 2.57 (m, 1 H, OCH2CH2CN), 2.54 - 2.49 (app t, 1 H, OCH2CH2CN), 1.57 - 1.51 (m, 3H, C(CW3)2), 1.35 - 1.28 (m, 3H, C(CW3)2), 0.91 - 0.85 (m, 9H, TBS-H), 0.13-0.08 (m, 6H, TBS-H);13C{1H} NMR (126 MHz, CDCI3) 6163.3, 163.2, 158.9 (2C), 150.7, 150.0, 144.3, 142.8, 140.1, 135.0 (2C), 130.4 (2C), 130.3 (2C), 128.3 (2C), 128.2 (2C), 127.5, 117.4, 114.6, 113.5 (4C), 102.6 (2C), 95.1, 88.3, 87.6, 86.7, 84.6, 82.5, 81.2, 75.5, 72.4, 62.6, 62.1, 57.2, 55.4 (2C), 27.2, 25.8 (3C), 25.4, 20.3, 18.2, -4.6 (2C);31P{1H} NMR (202 MHz, CDCI3) 6 138.8, 138.4. HRMS (ESI+): m / z calcd. 1044.3822 for ([M+H]+), found 1044.3822.5’-O-DMTr-2’-O-TBS-A(Bz)-2’,3’-O-isopropylidene-U phosphite (1 k)

[0195] Following the general procedure B, the reaction of 2’,3’-O-isopropylidene-U (102 mg, 0.36 mmol, 1.20 eq.), 5’-O-DMTr-2’-O-TBS-A(Bz)-phosphoramidite (296 mg, 0.30 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (47.0 mg, 0.36 mmol, 1.20 eq. in MeCN (1.20 mL)) in CH2Cl2(3.20 mL) provided the title compound as white solid (193 mg, 0.17 mmol, 55%); Rr 0.41 (CH2CI2 / MeOH, 95:5); IR vmax(neat): 3703w, 3619w, 3103w, 2932s, 2364s, 2341s, 1692s, 1609m, 1581m, 1510m, 1456m, 1381w, 1332w, 1296w, 1520s, 1219s, 1177m, 1071m, 1031s, 916w, 837m, 750s, 708m, 668m, 619m;1H NMR (500 MHz, CDCI3) 69.75 - 9.43 (m, 1H, NH), 9.37 - 9.23 (m, 1H, NH), 8.80 - 8.67 (m, 1H, C2AH), 8.32 - 8.20 (m, 1H, C8AH), 8.13 - 8.00 (m, 2H, ArBz-o- / 7), 7.63 - 7.57 (m, 1 H, ArBz-p-H), 7.56 - 7.48 (m, 2H, ArBz-m-H), 7.48 - 7.41 (m, 2H, DMTr-H), 7.38 - 7.17 (m, 8H, DMTr-H and C6uH overlapping with CDCI3), 6.91 - 6.78 (m, 4H, DMTr-H), 6.16-6.05 (m, 1H, C1’AW), 5.75 -5.53 (m, 2H, C5uH and C1’uH), 5.15 - 5.04 (m, 1H, ribose- / - / ), 5.06 - 4.91 (m, 1H, ribose-H), 4.89 - 4.71 (m, 1H, ribose-H), 4.71 - 4.63 (m, 1H, ribose- / - / ), 4.41 - 4.34 (m, 1H, ribose-H), 4.33 - 4.19 (m, 1H, ribose-H), 4.19 - 3.83 (m, 4H, OCH2CH2CN and C5’uH2), 3.83 - 3.74 (m, 6H, DMTr-OC / 73), 3.65 - 3.52 (m, 1H, C5’AH), 3.43 - 3.33 (m, 1H, C5’ H), 2.68 - 2.58 (m, 1H, OCH2CH2CN), 2.59 - 2.46 (app t, J = 6.3 Hz, 1H, OCH2C / 72CN), 1.64 - 1.49 (m, 3H, C(CW3)2), 1.41 - 1.26 (m, 3H, C(CW3)2), 0.89 - 0.67 (m, 9H, TBS-H), 0.00 - -0.11 (m, 3H, TBS-H), -0.18 - -0.24 (s, 3H, TBS-H);13C{1H} NMR (126 MHz, CDCI3) 5 164.8, 163.2, 158.8 (2C), 152.9, 151.9, 150.1, 149.9, 144.5, 142.5, 142.1, 135.6 (2C),133.8, 132.9, 130.2 (4C), 128.9 (2C), 128.3 (2C), 128.1 (4C), 127.2, 123.6, 117.3, 114.6, 113.4 (4C), 102.7, 95.0, 88.2, 87.0, 86.7, 84.6, 83.6, 81.2, 74.6, 73.6, 62.9, 62.5, 57.2, 55.4 (2C), 27.3, 25.7 (3C), 25.4, 20.3, 18.1, -4.6, -4.9;31P{1H} NMR (202 MHz, CDCI3) 6 139.04, 138.79. HRMS (ESP): m / z calcd. 1171.4356 for ([M+H]+), found 1171.4354.5’-O-DMTr-2’-O-MOE-5-Me-C(Bz)-2’,3’-O-isopropylidene-U phosphite (11)

[0196] Following the general procedure B, the reaction of 2’,3’-O-isopropylidene-U (102 mg, 0.36 mmol, 1.20 eq.), 5’-O-DMTr-2’-O-MOE-5-Me-C(Bz)-phosphoramidite (254 mg, 0.30 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (47.0 mg, 0.36 mmol, 1.20 eq. in MeCN (1.20 mL)) in CH2Cl2 (3.20 mL) provided the title compound as white solid (195 mg, 0.19 mmol, 63%); Rr 0.50 (CH2CI2 / MeOH, 95:5); IR vmax(neat): 3703w, 3630m, 3015w, 2361s, 2341s, 1694s, 1601m, 1565s, 1509m, 1454m, 1377m, 1311w, 1251s, 1176m, 1117m, 1069m, 1032s, 910m, 830m, 751s, 714m, 682m, 615m;1H NMR (500 MHz, CDCI3) 5 13.30 - 13.28 (m, 1H, N / 7), 8.76 - 8.66 (m, 1H, N / 7), 8.33 - 8.25 (m, 2H, ArBz-o-H), 7.84 - 7.80 (m, 1H, C65-Me-cH). 7.53 - 7.49 (m, 1H, ArBz-p- / 7), 7.45 - 7.38 (m, 4H, DMTr-H and ArBz-m- / 7), 7.35 - 7.19 (m, 8H, DMTr-H and C6uH overlapping with CDCI3), 6.90 - 6.82 (m, 4H, DMTr- / 7), 6.10 - 6.07 (m, 1H, CTs-Me-c / ), 5.72 -5.57 (m, 2H, C1’uH and C5uH). 4.97 - 4.92 (m, 1H, C4’uH), 4.90 - 4.74 (m, 2H, ribose-H), 4.37 - 4.29 (m, 1H, C4’5-Me-cH), 4.29 - 4.17 (m, 2H, ribose-H), 4.11 - 3.82 (m, 6H, OC / 72CH2CN and C5’UH2and MOE-CH2), 3.81 - 3.80 (m, 6H, DMTr-OC / 73), 3.65 - 3.52 (m, 3H, C5’5-Me-cH and MOE-CH2), 3.41 - 3.33 (m, 1H, C5’5-Me-cH). 3.32 - 3.30 (m, 3H, OCH2CH2OC / 73), 2.63 (app t, J = 6.3 Hz, 1H, OCH2CH2CN), 2.58 - 2.47 (m, 1H, OCH2C / 72CN), 1.55 - 1.55 (m, 3H, C(CW3)2), 1.51 - 1.49 (m, 3H, C55-Me-cCH3), 1.33 - 1.31 (m, 3H, C(CH3)2);13C{1H} NMR (126 MHz, CDCI3) 5 179.8, 162.9, 159.8, 159.0 (2C), 150.0, 148.4, 144.2, 142.3, 137.0, 136.8, 135.2 (2C), 132.6, 130.4 (4C), 130.1 (2C), 128.5 (2C), 128.2 (4C), 127.5, 117.5, 114.6, 113.5 (4C), 112.5, 102.7, 94.6, 87.4, 86.6, 84.6, 82.7, 82.1, 81.0, 72.5, 71.1, 70.5, 62.6, 62.0, 59.1, 57.2, 55.5 (2C), 27.3, 25.4, 20.4, 12.8;31P{1H} NMR (202 MHz, CDCI3) 6 139.2, 139.1. HRMS (ESP): m / z calcd.1105.3954 for ([M+H]+), found 1105.3965.5’-O-DMTr-LNA-A(Bz)-2’,3’-O-isopropylidene-U phosphite (1m)BzHNM °e'X M°e

[0197] Following the general procedure B, the reaction of 2’,3’-O-isopropylidene-U (94.6 mg, 0.33 mmol, 1.20 eq.), 5’-O-DMT r-LNA-A(Bz)-phosphoramidite (246 mg, 0.28 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (43.0 mg, 0.33 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2CI2 (5.00 mL) provided the title compound as white solid (129 mg, 0.121 mmol, 43%); Rr 0.37 (CFhCh / MeOH, 95:5); IR vmax (neat): 3198w, 3059w, 2953w, 2361w, 2252w, 1690s, 1609m, 1510m, 1456m, 1381w, 1250m, 1178w, 1034m, 909m, 831w, 731s, 630s;1H NMR (500 MHz, CDCI3) 6 9.75 -9.59 (m, 1 H, NH), 9.43 - 9.33 (m, 1 H, NH), 8.85 - 8.77 (m, 1 H, C2AH), 8.50 - 8.43 (m, 1 H, C8AH), 8.13 - 8.00 (m, 2H, ArBz-o-H), 7.65 - 7.58 (m, 1H, ArBz-p-H), 7.56 - 7.50 (m, 2H, ArBz-m- / 7), 7.49 - 7.43 (m, 2H, DMTr-H), 7.38 - 7.20 (m, 8H, DMTr-H and C6uH overlapping with CDCI3), 6.88 -6.82 (m, 4H, DMTr-H), 6.25 - 6.13 (m, 1H, CTH), 5.65 - 5.56 (m, 1H, C5uH). 5.49 - 5.44 (m, 1H, ribose- / - / ), 4.96-4.87 (m, 2H, ribose-H), 4.76-4.68 (m, 1H, ribose-H), 4.19-3.76 (m, 14H, ribose- / - / , OCH2CH2CN, C5’H2, LNA-C / 72and DMTr-OC / 73), 3.60 - 3.45 (m, 2H, C5’H2), 2.50 -2.42 (m, 2H, OCH2C / 72CN), 1.52 - 1.47 (m, 3H, C(CW3)2), 1.26 - 1.18 (m, 3H, C(CW3)2);13C{1H} NMR (126 MHz, CDCI3) 6 165.2, 163.2, 158.8 (2C), 154.1, 153.0, 151.3, 150.1, 144.4, 143.1, 140.8, 135.4 (2C), 133.7, 132.9, 130.2 (4C), 129.0 (2C), 128.2 (6C), 127.2, 123.7, 117.1, 114.5, 113.5 (4C), 102.7, 95.8, 87.7, 86.8, 86.5, 84.7, 84.4, 81.0, 79.3, 72.8, 71.7, 65.9, 62.9, 58.1, 55.4 (2C), 27.2, 25.3, 20.2;31P{1H} NMR (202 MHz, CDCI3) 6 138.5, 137.9. HRMS (ESI+): m / z calcd.1069.3492 for ([M+H]+), found 1069.3502.5’-O-DMTr-2’-F-dA(Bz)-2’-F-3’-O-Ac-U phosphite (1 n)

[0198] Following the general procedure B, the reaction of 2’-F-3’-O-Ac-U (79.0 mg, 0.27 mmol, 1.20 eq.), 5’-O-DMTr-2’-F-dA(Bz)-phosphoramidite (200 mg, 0.23 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (35.7 mg, 0.27 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2CI2 (5.00 mL) provided the title compound as white solid (125 mg, 0.118 mmol, 52%); Rr 0.45 (CF^Ch / MeOH, 95:5); IR vmax (neat): 3139w, 3050w, 2893w, 2341w, 2238w, 1690s, 1609m, 1510m, 1456m, 1381w, 1250m, 1178w, 1034m, 925m, 845w, 732s, 675s;1H NMR (500 MHz, CDCI3) 6 9.20 -9.06 (m, 2H, N / 7), 8.83 - 8.74 (m, 1H, C2AH), 8.30 - 8.20 (m, 1H, C8AH), 8.08 - 8.00 (m, 2H, ArBz-o- / 7), 7.65 - 7.58 (m, 1H, ArBz-p-H), 7.56 - 7.49 (m, 2H, ArBz-m- / 7), 7.42 - 7.18 (m, 10H, DMTr-H and C6uH overlapping with CDCI3), 6.83 - 6.76 (m, 4H, DMTr-H), 6.36 - 6.27 (m, 1H, C1’ / ), 5.83 - 5.70 (m, 2H, ribose-H), 5.68 - 5.58 (m, 1H, C5uH). 5.39 - 5.16 (m, 3H, ribose-H), 4.39 - 4.32 (m, 1H, ribose-H), 4.31 - 4.26 (m, 1H, ribose-H), 4.17 - 3.87 (m, 4H, OC / 72CH2CN and C5’ / 72), 3.81 - 3.73 (m, 6H, DMTr-OC / 73), 3.64 - 3.53 (m, 1H, C5’H), 3.47 - 3.32 (m, 1H, C5’ / 7), 2.58-2.52 (m, 2H, OCH2C / 72CN), 2.15-2.07 (m, 3H, OAc-CH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.0, 164.9, 162.7, 158.8 (2C), 153.0, 151.5, 150.1, 149.8, 144.4, 142.0, 140.9, 135.5 (2C), 133.6, 133.0, 130.2 (4C), 129.0 (2C), 128.2 (6C), 127.2, 123.8, 117.3, 113.4 (4C), 103.0, 94.1, 91.7, 91.6, 90.8, 90.3, 87.1, 81.9, 80.1, 69.4, 61.4 (2C), 57.8, 55.4 (2C), 20.6, 20.3;31P{1H} NMR (202 MHz, CDCI3) 5 139.6 (d, JP. F= 11.1 Hz), 139.3 (d, JP. F= 8.0 Hz);19F{1H} NMR (471 MHz, CDCh) 5 -199.3 (1stdiastereomer), -199.6 (2nddiastereomer), -202.2 - -202.3 (m, 1F, 1stand 2nddiastereomers). HRMS (ESI+): m / z calcd. 1063.3198 for ([M+H]+), found 1063.3207.5’-O-DMTr-2’-O-Me-U-2’-O-Me-3’-O-Ac-U phosphite (1o)

[0199] Following the general procedure B, the reaction of 2’-O-Me-3’-O-Ac-U (120 mg, 0.40 mmol, 1.20 eq.), 5’-O-DMTr-2’-O-Me-U-phosphoramidite (253 mg, 0.33 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (52.0 mg, 0.40 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2Cl2 (5.00 mL) provided the title compound as white solid (225 mg, 0.23 mmol, 70%); Rr 0.41 (C^Ch / MeOH, 95:5); IR vmax (neat): 3256w, 3105w, 2953w, 2428w, 2238w, 1728s, 1597m, 1510m, 1396m, 1361w, 1242m, 1159w, 1051m, 934m, 873w, 752s, 694s;1H NMR (500 MHz, CDCh) 6 8.89 -8.79 (m, 1H, N / 7), 8.77-8.66 (m, 1H, N / 7), 7.96-7.90 (m, 1H, C6uH). 7.61 -7.55 (m, 1H, C6uH), 7.39 - 7.21 (m, 9H, DMTr- / 7), 6.90 - 6.82 (m, 4H, DMTr- / 7), 6.02 - 5.88 (m, 2H, CTH), 5.75 -5.69 (m, 1 H, C5u / 7), 5.33 - 5.25 (m, 1 H, C5uH). 5.21 - 5.07 (m, 1 H, ribose-H), 4.85 - 4.72 (m, 1H, ribose-H), 4.28 - 3.83 (m, 8H, ribose-H and C5’H2and OC / 2CH2CN), 3.82 - 3.77 (m, 6H, DMTr-OC / 73), 3.68 - 3.62 (m, 1H, C5’H), 3.58 - 3.54 (m, 3H, OCW3), 3.46 - 3.40 (m, 4H, OCW3and C5’H), 2.63 (app t, J= 6.0 Hz, 1H, OCH2CH2CN), 2.57 -2.52 (m, 1H, OCH2CH2CN), 2.16 -2.10 (m, 3H, OAC-C / 73);13C{1H} NMR (126 MHz, CDCI3) 6 170.3, 163.0, 162.9, 159.0 (2C), 150.2 (2C), 144.1, 139.9 (2C), 135.0 (2C), 130.4 (4C), 128.4 (2C), 128.2 (2C), 127.5, 117.3, 113.5 (4C), 103.0, 102.7, 88.4, 87.9, 87.5, 87.1, 83.0, 81.6, 80.9, 70.2, 69.9, 61.1 (2C), 59.2, 58.7, 57.5, 55.5 (2C), 20.9, 20.4;31P{1H} NMR (202 MHz, CDCI3) 6 140.3, 139.7. HRMS (ESI+): m / z calcd.977.3328 for ([M+NH4]+), found 977.3345.5’-O-DMTr-2’-F-dA(Bz)-2’-O-Me-3’-O-Ac-U phosphite (1 p)

[0200] Following the general procedure B, the reaction of 2’-O-Me-3’-O-Ac-U (120 mg, 0.40 mmol, 1.20 eq.), 5’-O-DMTr-2’-F-dA(Bz)-phosphoramidite (292 mg, 0.33 mmol, 1.00 eq.) and 5-(ethylthio)-1H-tetrazole (52.0 mg, 0.40 mmol, 1.20 eq. in MeCN (2.00 mL)) in CH2Cl2(5.00 mL) provided the title compound as white solid (150 mg, 0.14 mmol, 42%); Rr 0.27 (CH2CI2 / MeOH, 95:5); IR vmax(neat): 2972w, 1768m, 1600w, 1518s, 1232m, 1067m, 1011s, 827w, 754w, 678w;1H NMR (500 MHz, CDCI3) 6 9.35 - 9.12 (m, 2H, NH), 8.81 - 8.75 (m, 1H, C2 H). 8.32 - 8.26 (m, 1H, C8AH). 8.08 - 8.01 (m, 2H, ArBz-o-H), 7.63 - 7.58 (m, 1H, ArBz-p-H), 7.56 - 7.48 (m, 3H, ArBz-m- / 7and C6uH). 7.41 -7.32 (m, 2H, DMTr-H), 7.31 -7.13 (m, 7H, DMTr-H overlapping with CDCI3), 6.84-6.76 (m, 4H, DMTr-H), 6.36-6.27 (m, 1H, C1’H), 5.93-5.88 (m, 1H, CTH), 5.87 - 5.71 (m, 1H, C2’AF / 7), 5.71 - 5.63 (m, 1H, C5uH). 5.40 - 5.20 (m, 1H, ribose-H), 5.18 - 5.09 (m, 1H, ribose- / - / ), 4.39 - 4.32 (m, 1H, ribose- / - / ), 4.28 - 3.87 (m, 6H, ribose- / - / and C5’ / 72and OCH2CH2CN), 3.79 - 3.75 (m, 6H, DMTr-OC / 73), 3.66 - 3.53 (m, 1 H, C5’H), 3.46 - 3.32 (m, 4H, C5’ / 7and OCW3), 2.59-2.51 (m, 2H, OCH2CH2CN), 2.15-2.09 (m, 3H, OAc-CH3);13C{1H} NMR (126 MHz, CDCI3) 6 170.3, 164.9, 162.9, 158.8 (2C), 153.0, 151.5, 150.2, 150.1, 144.3, 142.0, 139.8, 135.4 (2C), 133.6, 133.0, 130.2 (4C), 129.0 (2C), 128.2 (6C), 127.2, 123.9, 117.2, 113.4 (4C), 102.9, 91.7, 88.3, 87.4, 86.8, 82.0, 81.6, 80.8, 70.0, 69.8, 61.4 (2C), 59.1, 57.8, 55.4 (2C), 20.8, 20.4;31P{1H} NMR (202 MHz, CDCI3) 6 139.8 - 139.5 (m);19F{1H} NMR (471 MHz, CDCI3) 5-202.1 (d, J = 7.8 Hz), -202.4 (d, J= 12.5 Hz). HRMS (ESI+): m / z calcd. 1075.3415 for ([M+H]+), found 1075.3397.

[0201] DMTrOptimisation of the reaction conditions for photocatalytic sulfurization of oligonucleotidesGeneral procedure for the optimization of the reaction conditions for the photocatalytic sulfurization of oligonucleotides

[0202] In a 5 mL screw cap vial with a septum, compound 1a (23.2 mg, 25.0 pmol, 1.00 eq.), sodium thiosulfate (31.6 mg, 200 pmol, 8.00 eq.), triphenylphosphine oxide (as an internal standard, 6.60 mg, 25.0 pmol, 1.00 eq.), and photocatalyst (0.25 pmol, 1.00 mol%) were dissolved in the specified solvent under an Ar atmosphere. The mixture was evacuated and backfilled with Ar three times. The reaction vial was irradiated with a specified light with cooling by a fan at r.t. for the time indicated. The mixture was diluted with brine (3 mL), and extracted with ethyl acetate (3x5 mL). The organic phase was dried with Na2SO4 and concentrated under reduced pressure. The residue was analyzed by31P NMR to determine the yield and conversion.Table 6. Catalyst, concentration, and solvent evaluation for photocatalytic sulfurization of 1a on 25.0 pmol scaleLight loading, Cone., Solvent Time, Yield, %aEntry PCsource mol% molL-1(3:1) h P=SbP=OCpd Blue 1.00 0.02 MeCN / H2O 6 9 77 1 C1 6LEDBlue 1.00 0.02 MeCN / H2O 6 1 0 2 C2 86LEDGreen 1.00 0.02 MeCN / H2O 6 2 0 3 C3 84LEDGreen 1.00 0.02 MeCN / H2O 6 5 0 4 C4 84LEDGreen 1.00 0.02 MeCN / H2O 6 12 0 5 C5 73LEDGreen 1.00 0.02 MeCN / H2O 6 17 58 C6 17 LEDGreen 1.00 0.02 MeCN / H2O 6 19 62C7 10 LEDGreen 1.00 0.02 MeCN / H2O 6 19 32C8 36 LEDGreen 1.00 0.02 MeCN / H2O 6 12 48C9 25 LEDGreen 1.00 0.02 MeCN / H2O 6 9 1C10 64 LEDBlue 1.00 0.02 MeCN / H2O 6 6 82C11 10 LEDGreen C4 1.00 0.005 6 3 0MeCN / H2O 80LEDGreen C4 1.00 0.01 6 4 0MeCN / H2O 81LEDGreen C4 1.00 0.04 6 5 0MeCN / H2O 84LEDGreen C4 1.00 0.02 THF / H2O 5 296 48LEDGreen C4 1.00 0.02 Dioxane / H212 66 62LED 0Green C4 1.00 0.02 Acetone / H210 36 34LED 0Green C4 0.10 0.02 MeCN / H2O 6 14 5218LEDGreen C4 0.50 0.02 MeCN / H2O 6 18 760LEDGreen C4 2.50 0.02 MeCN / H2O 6 2 080LEDGreen C4 5.00 0.02 MeCN / H2O 6 5 0 22 76LED23 40 W C4 1.00 0.02 MeCN / H2O 4 93(84) <1 0 LED525 nm(green)aNMR yield with triphenylphosphine oxide as internal standard; isolated yield in parenthesis.bPhosphorothioate product.cPhosphate product from oxygenation side-reaction.dUnreacted starting material 1a.

[0203] The catalysts used in the examples are shown below:MeO Mes OMeC3 C4 E1 / 2(C7C’) = 2.08 V E1 / 2(C7C’) = 1.62 VC7 C8 E1 / 2(C7C’) = 2.08 V E1 / 2(C* / C-) = 1.79 V E1 / 2(C7C’) = 1 20 VPhBF4- C11E1 / 2(C7C’) = 2.55 v The catalysts are as follows:• C1 is Tris(2,2'-bipyridyl)dichlororuthenium(ll) hexahydrate;• C2 is (lr[dF(CF3)ppy]2(dtbpy))PF6;• C3 is 9-Mesityl-10-methylacridinium tetrafluoroborate;• C4 is 9-Mesityl-1,3,6,8-tetramethoxy-10-phenylacridin-10-ium tetrafluoroboratek• 05 is 9-Mesityl-3,6-di-tert-butyl-10-phenylacridinium tetrafluoroborate;• C6 is 4CzlPN;• 07 is 30zCIIPN;• 08 is Erythrosin B;• 09 is Eosin Y;• 010 is Rose Bengal; and• 011 is 2,4,6-triphenylpyrylium tetrafluoroborate.General procedure for photocatalytic sulfurization of oligonucleotidesNa2S2O3C4 (1.00 mol%) MeCN / H2O green light Ph BF4- Ar, r.t.General procedure C: photocatalytic sulfurization of dinucleotides

[0204] To a mixture of dinucleotide P(lll) precursor 1a-p (50.0 pmol, 1.00 eq.), sodium thiosulfate (61 mg, 400 pmol, 8.00 eq.), and photocatalyst C4 (0.29 mg, 0.50 pmol, 1.00 mol%) in a 20 mL crimp cap vial under an Ar atmosphere was added MeCN (1.9 mL) and H2O (0.6 mL). The mixture was evacuated and backfilled with Ar three times. The reaction vial was irradiated with green light (525 nm LED lamp, 40W, ~5 cm from the light source) with cooling by a fan at r.t. for 4 hours. The sulfurization / oxygenation ratio was determined by31P NMR of the reaction sample. The crude was purified without work-up using reverse-phase column chromatography (C18 silica, MeCN / H2O, 5:95 to 90:10) to obtain the desired product after evaporation under reduced pressure.

[0205] Groups R1to R5shown in the reaction scheme above vary as will be understood with reference to compounds 1a-k and 2a-k described below.5’-O-DMTr-dT-3’-O-Ac-dT phosphorothioate (2a)oOAc

[0206] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1a (46.6 mg, 50.0 pmol, 1.00 eq.). The title compound was obtained as a white solid (41.0 mg, 42.7 pmol, 84%, >99:1 sulfurization / oxygenation ratio); IR vmax(neat): 3629w, 3505w, 3062w, 2362s, 2335m, 1685s, 1608w, 1509m, 1467m, 1369w, 1248s, 1178m, 1107m, 1000s, 908s, 831m, 727s, 648m, 619m;1H NMR (500 MHz, CDCI3) 6 8.54 - 8.33 (m, 2H, N / 7), 7.59 - 7.53 (m, 1H, C6TH). 7.43 - 7.36 (m, 2H, DMTr-H), 7.33 - 7.22 (m, 8H, DMTr-H and C6TH). 6.92 - 6.78 (m, 4H, DMTr-H), 6.43 - 6.35 (m, 1 H, CTTH), 6.34 - 6.25 (m, 1 H, CTTH), 5.39 - 5.32 (m, 1 H, ribose- / 7), 5.32 - 5.18 (m, 1H, ribose-H), 4.44 - 4.07 (m, 6H, OCH2CH2CN and C5’TH2and ribose-H), 3.83 - 3.74 (m, 6H, OCW3), 3.53 - 3.37 (m, 2H, C5’TH2), 2.83 - 2.57 (m, 3H, OCH2CH2CN and C2’TH), 2.51 -2.17 (m, 3H, C2’TH), 2.15-2.08 (m, 3H, OAc-CH3), 1.99- 1.88 (m, 3H, C5TCH3), 1.49 - 1.43 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.7, 163.4 (2C), 159.0 (2C), 150.3 (2C), 144.2, 135.3 (4C), 130.2 (4C), 128.2 (4C), 127.5, 116.3, 113.6 (4C), 111.9 (2C), 87.5, 85.5, 85.0, 84.6, 82.7, 80.3, 74.0, 67.9, 63.4, 62.9, 55.5 (2C), 39.1, 37.0, 21.0, 19.6, 12.7, 11.9;31P{1H} NMR (202 MHz, CDCI3) 567.0, 66.9. HRMS (ESI+): m / z calcd. 977.3151 for ([M+NH4]+), found 977.3165.5’-O-DMTr-dA(Bz)-3’-O-Ac-dT phosphorothioate (2b)BzHNOAc

[0207] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1b (52.0 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (46.2 mg, 43.1 pmol, 86%, 98:2 sulfurization / oxygenation ratio); IR vmax (neat): 2982m, 2850w, 2377s, 2347s, 1750m, 1608m, 1589 m, 1421m, 1222s, 1104m, 790s;1H NMR (500 MHz, CDCI3) 59.23 - 9.00 (m, 2H, N / 7), 8.74 - 8.69 (m, 1H, C2AH), 8.21 – 8.15 (m, 1H, C8AH), 8.07 - 8.01 (m, 2H, ArBz-o- / - / ), 7.64 - 7.55 (m, 1 H, ArBz-p-H), 7.55 - 7.48 (m, 2H, ArBz-m-H), 7.41 - 7.31 (m, 3H, DMTr-H and C6TH). 7.31 - 7.16 (m, 7H, DMTr-H overlapping with CDCI3), 6.85 - 6.76 (m, 4H, DMTr-H), 6.53 -6.46 (m, 1 H, CTH), 6.39 -6.23 (m, 1 H, CTH), 5.48 - 5.41 (m, 1 H, ribose-H), 5.34 - 5.24 (m, 1 H, ribose- / - / ), 4.47 - 4.11 (m, 6H, C5’H2and OCH2CH2CN and ribose-H), 3.81 - 3.75 (m, 6H, DMTr-OC / 73), 3.51 - 3.38 (m, 2H, C5’H2), 3.20 - 3.00 (m, 1 H, C2’H), 2.84 - 2.58 (m, 3H, OCH2CH2CN and C2’H), 2.46 - 2.35 (m, 1H, C2’H), 2.34 - 2.19 (m, 1H, C2’H), 2.12 - 2.06 (m, 3H, AC-C / 73), 1.98- 1.91 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCI3) 5170.7, 164.8, 163.5, 158.8 (2C), 152.8, 151.7, 150.3, 149.9, 144.4, 141.6, 135.4 (3C), 133.7, 132.9, 130.2 (4C), 129.0 (4C), 128.2 (4C), 127.2, 123.7, 116.4, 113.4 (4C), 111.9, 87.1, 85.5, 84.9, 84.7, 82.7, 80.4, 74.1, 67.9, 63.2, 62.9, 55.4 (2C), 38.3, 37.0, 21.0, 19.7, 12.7;31P{1H} NMR (202 MHz, CDCI3) 566.9, 66.8. HRMS (ESI+): m / z calcd. 1073.3286 for ([M+H]+), found 1073.3263.5’-O-DMTr-dC(Ac)-3’-O-Ac-dT phosphorothioate (2c)NHAcOAc

[0208] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1c (47.7 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (45.5 mg, 46.1 pmol, 92%, >99:1 sulfurization / oxygenation ratio); IR vmax(neat): 2982m, 2345m, 2250m, 1690m, 1608m, 1509 m, 1454m, 1246s, 1139 m, 1040m, 902m, 815 m, 717 s, 645m;1H NMR (500 MHz, CDCI3) 5 8.75 - 8.71 (m, 1H, NH), 8.56 - 8.36 (m, 1H, NH), 8.12 - 8.05 (m, 1H, C6cH). 7.39 -7.20 (m, 10H, DMTr-Hand C6TH), 7.17-7.14 (m, 1H, C5cH). 6.89-6.81 (m, 4H, DMTr-H), 6.35 - 6.27 (m, 1H, CTH), 6.27 - 6.18 (m, 1H, CTH), 5.30 - 5.21 (m, 2H, ribose-H), 4.42 - 4.03 (m, 6H, ribose- / - / and C5’H2and OCH2CH2CN), 3.82 - 3.78 (m, 6H, DMTr-OC / 73), 3.53 - 3.40 (m, 2H, C5’ / 72), 2.96 - 2.83 (m, 1 H, C2’H), 2.81 - 2.75 (m, 1 H, OCH2CH2CN), 2.72 - 2.58 (m, 1 H, OCH2C / 72CN), 2.47 - 2.22 (m, 3H, C2’H), 2.23 - 2.20 (m, 3H, OAc-CH3), 2.11 - 2.08 (m, 3H, OAC-C / 73), 1.94 - 1.89 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCI3) 6 170.7 (2C), 163.5, 162.4, 158.9 (2C), 155.2, 150.3, 144.1 (2C), 135.2 (3C), 130.3 (4C), 128.2 (4C), 127.4, 116.4, 113.5 (4C), 111.9, 96.4, 87.2 (2C), 85.1 (2C), 82.6, 79.1, 74.0, 68.0, 62.8 (2C), 55.4 (2C), 40.3, 37.0, 25.1, 21.0, 19.6, 12.7;31P{1H} NMR (202 MHz, CDCI3) 6 67.1, 66.8. HRMS (ESI+): m / z calcd. 987.2994 for ([M+H]+), found 987.2993.5’-O-DMTr-dG(lb)-3’-O-Ac-dT phosphorothioate (2d)

[0209] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1d (51.3 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (37.0 mg, 33.3 pmol, 67%, >99:1 sulfurization / oxygenation ratio); IR vmax (neat): 3181w, 2936w, 1685s, 1607s, 1559m, 1508m, 1466m, 1404w, 1365w, 1248s, 1000s, 907s, 828m, 727s, 647m, 585w;1H NMR (500 MHz, CDCI3) 6 12.2 - 12.1 (d, 1H, NH), 10.1 - 9.0 (m, 2H, NH), 7.7 -7.7 (d, 1H, C8GH), 7.6 - 7.5 (dd, J = 17.4, 1.4 Hz, 1H, C6TH), 7.4 - 7.3 (m, 2H, DMTr-H), 7.3 - 7.1 (m, 7H, DMTr-H), 6.9 -6.7 (m, 4H, DMTr-H), 6.5 -6.0 (m, 2H, CTH), 5.5- 5.3 (m, 2H, ribose-H), 4.5 -4.1 (m, 6H, OCH2CH2CN and ribose- / - / and C5’H2), 3.8 - 3.7 (m, 6H, DMTr-OC / 73), 3.4 - 3.2 (m, 2H, C5’H2), 2.9 -2.3 (m, 7H, OCH2C / 72CN and CH(CH3)2and C2’H2), 2.2 -2.0 (m, 6H, OAc-CH3and C5TH).1.3 - 1.1 (m, 6H, CH(C / 73)2);13C{1H} NMR (126 MHz, CDCI3) 6 179.8, 170.7, 164.9, 158.8 (2C), 155.5, 150.4, 148.4 (2C), 144.4, 136.7 (2C), 135.4 (2C), 130.2 (4C), 129.3, 128.1 (4C), 127.2, 121.6, 116.6, 113.3 (4C), 111.4 (d), 87.1, 86.2, 85.1, 83.8, 83.0, 80.7, 74.0, 67.6, 63.3 (2C), 55.4 (2C), 39.2, 37.0, 36.0, 21.0, 19.7, 19.1, 12.7;31P{1H} NMR (202 MHz, CDCI3) 668.2, 67.7. HRMS (ESI+): m / z calcd. 1055.3369 for ([M+H]+), found 1055.3379.5’-O-DMTr-dT-3’-O-Ac-dA(Bz) phosphorothioate (2e)oOAc

[0210] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1e (52.0 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (47.0 mg, 43.8 pmol, 88%, >99:1 sulfurization / oxygenation ratio); IR vmax (neat): 3673m, 2973m, 2902m, 2361s, 1697m, 1603w, 1508w, 1450w, 1407w, 1248s, 1055s, 908m, 830w, 790w, 727m, 649.w;1H NMR (500 MHz, CDCh) 6 8.72 - 8.68 (m, 1H, C2 H). 8.68 – 8.61 (m, 1H, NH), 8.41 - 8.35 (m, 1H, C8AH). 7.73 - 7.67 (m, 2H, ArBz-o-H), 7.58 - 7.53 (m, 1H, ArBz-p-H), 7.44 - 7.35 (m, 3H, ArBz-m-H and C6TH). 7.33 - 7.19 (m, 9H, DMTr-H overlapping with CDCh), 6.88 - 6.80 (m, 4H, DMTr- / - / ), 6.55 - 6.46 (m, 1 H, CTH), 6.44 - 6.34 (m, 1H, CTH), 5.53 - 5.40 (m, 1 H, ribose-H), 5.36 -5.26 (m, 1 H, ribose- / - / ), 4.43 - 3.98 (m, 6H, ribose-H and C5’H2and OCH2CH2CN), 3.80 - 3.76 (m, 6H, DMTr-OC / 73), 3.49- 3.37 (m, 2H, C5’H2), 3.02 -2.92 (m, 1 H, C2’H), 2.73 -2.58 (m, 4H, C2’ / 7 and OCH2CH2CN), 2.47 - 2.33 (m, 1H, C2’H), 2.13 - 2.10 (m, 3H, OAc-CH3), 1.48 - 1.42 (m, 3H, C5TC / 73);13C{1H} NMR (126 MHz, CDCh) 6172.0, 170.5, 163.6, 158.9 (2C), 152.9, 152.4, 151.1, 150.4, 144.2, 143.7, 135.3 (3C), 134.3, 133.0, 130.2 (4C), 129.4 (2C), 129.1, 128.7 (2C), 128.2 (4C), 127.3, 116.5, 113.5 (4C), 111.7, 87.4, 84.9, 84.8, 84.6, 83.5, 80.1, 74.4, 67.7, 63.4, 62.8, 55.4 (2C), 39.1, 37.3, 21.0, 19.4, 11.9;31P{1H} NMR (202 MHz, CDCh) 567.2, 67.0. HRMS (ESP): m / z calcd. 1095.3083 for ([M+Na]+), found 1095.3103.5’-O-DMTr-dT-3’-O-Ac-dC(Ac) phosphorothioate (2f)OAc

[0211] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1f (47.7 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (46.0 mg, 46.6 pmol, 93%, >99:1 sulfurization / oxygenation ratio); IR vmax(neat): 3675w, 3630w, 3026w, 2919w, 2361m, 2341m, 1683s, 1610m, 1563m, 1493s, 1400w, 1372m, 1308m, 1245s, 1178m, 1111m, 999s, 910m, 829m, 728s, 648m;1H NMR (500 MHz, CDCh) 6 9.78 - 8.73 (m, 2H, NH), 8.13 -7.90 (m, 1 H, C6cH). 7.61 - 7.51 (m, 1 H, C6TH). 7.47 - 7.35 (m, 3H, DMTr-H and C5cH). 7.35 -7.18 (m, 7H, DMTr-H overlapping with CDCh), 6.91 - 6.75 (m, 4H, DMTr-H), 6.51 - 6.17 (m, 2H, C1’TH and CTcH), 5.39 - 5.17 (m, 2H, ribose-H), 4.47 - 4.00 (m, 6H, OCH2CH2CN and C5’TH2and ribose- / - / ), 3.83 - 3.76 (m, 6H, DMTr-OC / 73), 3.52 - 3.29 (m, 2H, C5’cH2), 2.91 - 2.12 (m, 6H, OCH2CH2CN and C2’cH2and C2’TH2), 2.26 -2.19 (m, 3H, Ac-CH3), 2.12 -2.05 (m, 3H, Ac-CW3), 1.52 - 1.38 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCh) 6 170.7 (2C), 163.9, 162.9, 158.9 (2C), 155.3, 150.5, 144.2 (2C), 135.4 (3C), 130.2 (4C), 128.2 (4C), 127.3, 116.4, 113.5 (4C), 111.7, 96.9, 94.2, 87.5, 86.3, 84.7, 83.6, 80.1, 74.1, 67.8, 63.4, 62.9, 55.4 (2C), 39.2, 38.8, 25.1, 21.0, 19.5, 11.9;31P{1H} NMR (202 MHz, CDCh) 6 67.6, 67.1. HRMS (ESP): m / z calcd.987.2994 for ([M+H]+), found 987.3010.5’-O-DMTr-dT-3’-O-Ac-dG(lb) phosphorothioate (2g)

[0212] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1g (51.1 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (46.0 mg, 43.6 pmol, 87%, 97:3 sulfurization / oxygenation ratio); IR v (neat): 3673m, 2973m, 2902m, 2361s, 1682m, 1607w, 1560w, 1508w, 1465w, 1405w, 1248s 1055s, 908m, 790w, 728m, 632w;1H NMR (500 MHz, CDCI3) 6 12.16 (br. s, 1H, NH), 9.57-9.27 (m, 1H, NH), 7.86 - 7.81 (m, 1H, C8GH), 7.57 - 7.51 (m, 1H, C6TH). 7.41 - 7.34 (m, 2H, DMTr-H), 7.32 - 7.17 (m, 7H, DMTr-H), 6.86 -6.79 (m, 4H, DMTr-H), 6.39 - 6.31 (m, 1H, CT / 7), 6.24 -6.17 (m, 1H, CT / 7), 5.49 - 5.38 (m, 1H, ribose- / - / ), 5.32 - 5.24 (m, 1H, ribose- / - / ), 4.42 - 4.05 (m, 6H, ribose- / - / and 05’ H2 and OCH2CH2CN), 3.79-3.75 (m, 6H, DMTr-OC / 73), 3.49 -3.34 (m, 2H, 05’ W2), 3.19-2.97 (m, 1H, 02’ / - / ), 2.76 - 2.33 (m, 6H, OCH2CH2CN and C2’H and CH(CH3)2), 2.10 - 2.01 (m, 3H, OAc-CW3), 1.48 - 1.41 (m, 3H, C5TCH3), 1.26 - 1.16 (m, 6H, CH(CH3)2);13C{1H} NMR (126 MHz, CDCl3) δ 179.3, 170.4, 163.8, 158.8 (2C), 155.6, 150.7, 148.0, 144.2 (2C), 138.1, 135.2 (3C), 130.1 (4C), 128.1 (4C), 127.3, 121.9, 116.7, 113.4 (4C), 111.7, 87.3, 85.1, 84.6, 84.4, 82.9, 80.1, 74.2, 67.4, 63.3, 62.8, 55.3 (2C), 38.9, 36.2, 35.9, 20.9, 19.4, 19.0 (20), 11.8.31P{1H}NMR (202 MHz, CDCI3) 666.9, 66.4. HRMS (ESI+): m / z calcd. 1077.3188 for ([M+Na]+), found 1077.3201.5’-O-DMTr-2’-F-dA(Bz)-3’-O-Ac-dT phosphorothioate (2h)

[0213] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1h (53.3 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (50.6 mg, 46.9 pmol, 93%, >99:1 sulfurization / oxidation ratio); IR v (neat): 3005m, 2362m, 2250m, 1690m, 1608m, 1509m, 1454m, 1246s, 1177m, 1030m, 907m, 828m, 727s, 645m;1H NMR (500 MHz,CDCI3) 69.22 - 9.15 (m, 1H, NH), 8.81 - 8.75 (m, 1H, C2AH), 8.31 - 8.22 (m, 1H, C8AH), 8.09 -8.03 (m, 2H, ArBz-o-H ), 7.67 - 7.60 (m, 1 H, ArBz-p-H), 7.59 - 7.51 (m, 2H, ArBz-m- / 7), 7.47 - 7.16 (m, 10H, DMTr-H and C6TH overlapping with CDCI3), 6.88 - 6.78 (m, 4H, DMTr-H), 6.39 - 6.29 (m, 2H, C1’ / 7 ), 6.08 - 5.62 (m, 2H, ribose-H and C2’FH), 5.35 - 5.22 (m, 1 H, ribose-H), 4.50 -3.92 (m, 6H, ribose-H and OCH2CH2CN),), 3.84 - 3.77 (m, 6H, DMTr-OC / 73), 3.72 - 3.56 (m, 1 H, C5’ / 7), 3.53 - 3.38 (m, 1H, C5’H), 2.85 - 2.73 (m, 1 H, OCH2CH2CN), 2.68 - 2.52 (m, 1H, OCH2CH2CN), 2.48 - 2.34 (m, 1 H, C2’H), 2.28 - 2.18 (m, 1 H, C2’H), 2.15 - 2.08 (m, 3H, OAc-CW3), 1.97- 1.90 (m, 3H, C5TC / 73);13C{1H} NMR (126 MHz, CDCI3) 5 170.6, 164.8, 163.4, 158.8 (2C), 153.1, 151.5, 150.3, 149.9, 144.3, 141.9, 135.3 (3C), 133.6, 133.0, 130.2 (4C), 129.3, 129.0 (2C), 128.3, 128.2 (4C), 127.3, 123.7, 116.4, 113.4 (4C), 111.8, 94.1, 87.1, 87.0, 86.9, 84.8, 82.6, 74.6, 74.2, 68.1, 63.0, 61.6, 55.4 (2C), 37.1, 21.0, 19.5, 12.6;19F{1H} NMR (471 MHz, CDCI3) 5 -202.9, -204.9 (d, J = 27.7 Hz);31P{1H} NMR (202 MHz, CDCI3) 567.5, 67.4. HRMS (ESI+): m / z calcd. 1091.3164 for ([M+H]+), found 1091.3169.5’-O-DMTr-2’-O-Me-U-2’,3’-O-isopropylidene-U phosphorothioate (2i)

[0214] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1i (47.2 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (43 mg, 44.0 pmol, 88%, 99:1 sulfurization / oxidation ratio); IR vmax (neat): 3189m, 3061m, 2959m, 2837m, 2253m, 1763m, 1686s, 1509m, 1458m, 1380m, 1251s, 1176m, 1120m, 1031s, 909m 829m, 729s, 648w;1H NMR (500 MHz, CDCI3) 5 9.24 (br. s, 2H, N / 7), 7.80 (m, 1H, C6 / 7), 7.46 - 7.07 (m, 10H, DMTr-H and C6 / 7), 6.89 - 6.82 (m, 4H, DMTr-H), 6.05 (s, 1H, CTH), 5.75 - 5.49 (m, 2H, C5 / 7), 5.32 - 5.26 (m, 1H, CTH), 5.24 - 5.16 (m, 1H, ribose-H), 5.04 - 4.99 (m, 1H, ribose- / 7), 4.92 - 4.82 (m, 1H, ribose-H), 4.43 - 3.90 (m, 7H, ribose-H and OCH2CH2CN), 3.85 - 3.73 (m, 6H, DMTr-OC / 73), 3.73 - 3.37 (m, 5H, OCW3and C5’H2), 2.79 (app t, J = 6.4 Hz, 1H, OCH2C / 72CN), 2.66 - 2.51 (m, 1H, OCH2CH2CN), 1.57 - 1.52 (m, 3H, C(CW3)2), 1.33 (m, 3H, C(C / 73)2);13C{1H} NMR (126 MHz, CDCI3) 5 163.4, 163.0, 159.0 (2C), 150.7, 150.2, 144.1, 142.8 (2C), 139.7, 134.8 (2C), 130.4 (4C), 128.3 (4C), 127.5, 116.5, 114.7, 113.5 (4C), 103.0, 102.9, 95.8, 95.4, 87.7, 86.5, 85.9, 84.5, 81.1, 74.9, 68.2, 62.6, 61.8, 58.9, 55.5 (2C), 27.2, 25.4, 19.4;31P{1H} NMR (202 MHz, CDCI3) 5 67.3, 66.9. HRMS (ESI+): m / z calcd. 976.2834 for ([M+H]+), found 976.2822.5’-O-DMTr-2’-O-TBS-U-2’,3’-O-isopropylidene-U phosphorothioate (2j)

[0215] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1j (53.0 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (49 mg, 45.5 pmol, 90%, 96:4 sulfurization / oxygenation ratio); IR vmax (neat): 3202w, 3060w, 2954w, 2363w, 1689s, 1509m, 1459m, 1381m, 1252m, 1158m, 1031s, 911m, 834m, 788w, 731m, 673w;1H NMR (500 MHz, CDCI3) 6 9.33 - 8.53 (m, 2H, N / 7), 7.88 - 7.74 (m, 1H, C6uH). 7.41 - 7.04 (m, 10H, DMTr-H and C6uH overlapping with CDCI3), 6.89 - 6.82 (m, 4H, DMTr-H), 6.04 - 5.91 (m, 1H, CTuH), 5.74-5.59 (m, 1H, C5uH). 5.58-5.44 (m, 1H, ribose-H), 5.35-5.26 (m, 1H, C5uH).5.08 - 4.97 (m, 2H, ribose-H), 4.88 - 4.78 (m, 1 H, ribose-H), 4.52 - 4.45 (m, 1 H, ribose-H), 4.40 - 3.96 (m, 6H, ribose-H and C5’uH2and OCH2CH2CN), 3.82 - 3.79 (m, 6H, DMTr-OC / 73), 3.62 -3.35 (m, 2H, C5’uH2), 2.76 (app t, J = 6.5 Hz, 1H, OCH2CH2CN), 2.66 - 2.49 (m, 1H, OCH2CH2CN), 1.58 - 1.48 (m, 3H, C(CW3)2), 1.36 - 1.29 (m, 3H, C(CW3)2), 0.90 - 0.86 (m, 9H, TBS-H), 0.14-0.06 (m, 6H, TBS- / 7);13C{1H} NMR (126 MHz, CDCI3) 5163.1, 162.7, 158.8 (2C), 150.6, 149.9, 144.0, 142.8, 139.7, 134.8 (2C), 130.2 (4C), 128.2 (4C), 127.4, 116.2, 114.5, 113.4 (4C), 103.0, 102.8, 96.0, 87.9, 87.6, 87.4, 86.3, 84.4, 82.0, 81.0, 74.6, 68.2, 62.7, 62.4, 55.3 (2C), 27.1, 25.6 (3C), 25.2, 19.3, 18.0, -4.8 (2C);31P{1H} NMR (202 MHz, CDCI3) 568.1, 67.2. HRMS (ESI+): m / z calcd. 1098.3362 for ([M+Na]+), found 1098.3362.5’-O-DMTr-2’-O-TBS-A(Bz)-2’,3’-O-isopropylidene-U phosphorothioate (2k)

[0216] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1k (58.6 mg, 50.0 pmol, 1.00 eq.). The title compound was obtained as a white solid (45.0 mg, 37.4 pmol, 75%, >99:1 sulfurization / oxygenation ratio); IR ymax(neat): 3660w, 3622w, 3068w, 2952m, 2243w, 1693s, 1609m, 1510m, 1456m, 1381m, 1252s, 1177m, 1072s, 1029s, 910mm, 837m, 730s, 647m;1H NMR (500 MHz, CDCI3) 5 9.16 - 9.05 (m, 2H, NH), 8.85 - 8.69 (m, 1H, C2AH), 8.26 - 8.18 (m, 1H, C8AH), 8.13 - 7.95 (m, 2H, ArBz-o-H), 7.65 - 7.58 (m, 1H, ArBz-p-H), 7.57 -7.50 (m, 2H, ArBz-m-H), 7.49 - 7.43 (m, 2H, DMTr-H), 7.39 - 7.33 (m, 4H, DMTr-H), 7.33 - 7.28 (m, 2H, DMTr-H), 7.27 - 7.16 (m, 2H, DMTr-H and C6uH). 6.95 - 6.77 (m, 4H, DMTr-H), 6.16 -6.08 (m, 1H, CTAH), 5.73 - 5.64 (m, 1H, C5uH). 5.59 - 5.53 (m, 1H, CTuH), 5.23 - 5.08 (m, 2H, ribose- / - / ), 5.08 - 5.00 (m, 1H, ribose-H), 4.91 - 4.78 (m, 1H, ribose-H), 4.56 - 4.44 (m, 1H, ribose- / - / ), 4.41 - 4.05 (m, 4H, OCH2CH2CN and C5’AH), 3.81 - 3.76 (m, 6H, OCW3), 3.62 - 3.51 (m, 1 H, C5’uH), 3.45 - 3.35 (m, 1 H, C5’uH), 2.79 - 2.73 (m, 1 H, OCH2CH2CN), 2.68 - 2.51 (m, 1H, OCH2CH2CN), 1.58 - 1.47 (m, 3H, C(CW3)2), 1.38 - 1.27 (m, 3H, C(CW3)2), 0.76 - 0.67 (s, 9H, TBS-H), -0.01 - -0.05 (d, 3H, TBS-H), -0.19 - -0.30 (d, 3H, TBS-H);13C NMR (126 MHz, CDCI3) 6 164.7, 162.9, 158.8 (2C), 153.0, 152.0, 150.0, 149.9, 144.5, 142.8, 141.7, 135.5 (2C), 133.8, 133.0, 130.3 (4C), 129.0 (2C), 128.2 (2C), 128.1 (4C), 127.3, 123.4, 116.4, 114.7, 113.5 (4C), 102.9, 96.2, 95.7, 87.8, 87.2, 86.3, 84.6, 83.0, 81.2, 74.3, 68.2, 63.1, 62.8, 55.4 (2C), 27.2, 25.6 (3C), 25.4, 19.5, 18.0, -4.6, -5.1;31P NMR (202 MHz, CDCI3) 6 68.2, 67.7. HRMS (ESI+): m / z calcd. 1203.4077 for ([M+H]+), found 1203.4078.05’-O-DMTr-2’-O-MOE-5-Me-C(Bz)-2’,3’-O-isopropylidene-U phosphorothioate (2I)

[0217] Prepared according to the general procedure C using dinucleotide P(lll) precursor 11 (55.3 mg, 50.0 pmol, 1.00 eq.). The title compound was obtained as a white solid (48.0 mg, 42.2 pmol, 84%, >99:1 sulfurization / oxygenation ratio); IR ymax(neat): 3677w, 3634w, 3067w, 2932w, 2361s, 2255w, 1694s, 1602m, 1565s, 1509m, 1452m, 1378m, 1309w, 1251s, 1176m, 1117m, 1070ms, 1028sm, 908s, 831m, 728s, 682w,648w, 615w;1H NMR (500 MHz, CDCI3) 5 13.30 -13.19 (m, 1H, NH), 9.26 - 8.69 (m, 1H, NH), 8.37 - 8.23 (m, 2H, Bz-o-H), 7.80 – 7.71 (m, 1H, C6cH). 7.59 - 7.47 (m, 1H, Bz-p-H), 7.47 - 7.38 (m, 4H, Bz-m-H and DMTr-H), 7.38 - 7.07 (m, 8H, DMTr-H and C6uH). 6.96 - 6.80 (m, 4H, DMTr-H), 6.21 - 6.05 (m, 1H, CTH), 5.76 - 5.47 (m, 2H, C5uH and CTH), 5.35 - 5.21 (m, 1H, ribose-H), 5.11 - 4.96 (m, 1H, ribose-H), 4.96 -4.78 (m, 1H, ribose- / - / ), 4.50 - 3.95 (m, 7H, OCH2CH2CN and C5’H2and ribose-H), 3.93 - 3.76 (m, 8H, DMTr-OC / 73and OCH2CH2OCH3), 3.72 - 3.37 (m, 4H, OCH2CH2OCH3and C5’H2), 3.37 - 3.25 (m, 3H, OCH2CH2OCH3), 2.88 - 2.74 (m, 1H, OCH2CH2CN), 2.71 - 2.49 (m, 1H, OCH2C / 72CN), 1.59 - 1.46 (m, 6H, C5cCH3and C(CW3)2), 1.42 - 1.26 (m, 3H, C(CW3)2);13C{1H} NMR (126 MHz, CDCI3) 5 179.8, 163.0, 159.6, 159.0 (2C), 150.1, 148.5, 144.2, 142.6, 137.2, 136.5, 135.2 (2C), 132.6, 130.4 (3C), 130.3, 130.1 (2C), 128.4 (2C), 128.3 (4C), 127.5, 116.6, 114.6, 113.5 (4C), 112.7, 102.9, 95.5, 87.6, 86.9, 86.1, 84.6, 82.3, 81.0 (2C), 76.2, 72.6, 70.7, 68.2, 62.7, 62.5, 59.1, 55.5 (2C), 27.2, 25.4, 19.4, 12.8;31P{1H} NMR (202 MHz, CDCI3) 567.2, 66.8. HRMS (ESI+): m / z calcd. 1137.3675 for ([M+H]+), found 1137.3675.5’-O-DMTr-LNA-A(Bz)-2’,3’-O-isopropylidene-U phosphorothioate (2m)

[0218] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1m (53.5 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (49 mg, 44.5 pmol, 93%, 96:4 sulfurization / oxygenation ratio); IR vmax (neat): 3197w, 3059w, 2958w, 2360m, 2250w, 1682s, 1698s; 1568m; 1509m, 1457m, 1249m, 1087w, 1034w, 908s, 829w, 720m, 624m;1H NMR (500 MHz, CDCI3) 6 10.32 (br. s, 1H, NH), 9.67 -9.35 (m, 1H, NH), 8.92 -8.68 (m, 1H, C2A / 7), 8.50 - 8.23 (m, 1H, C8AH), 8.08 - 8.00 (m, 2H, ArBz-o-H), 7.61 - 7.55 (m, 1H, ArBz-p-H), 7.53 - 7.46 (m, 2H, ArBz-m-H), 7.46 - 7.39 (m, 2H, DMTr-H), 7.36 - 7.14 (m, 8H, DMTr-H and C6uH). 6.85 - 6.82 (m, 4H, DMTr-H), 6.22 - 6.07 (m, 1 H, CTH), 5.64 - 5.56 (m, 1 H, C5uH). 5.53 - 5.50 (m, 1H, ribose- / - / ), 5.29 - 5.24 (m, 1H, ribose-H), 5.07 - 4.88 (m, 2H, ribose-H), 4.75 -4.67 (m, 1H, ribose- / - / ), 4.29 - 3.81 (m, 7H, ribose-H and OCH2CH2CN and CLNAH2), 3.81 - 3.75 (m, 6H, DMTr-OC / 73), 3.61 - 3.44 (m, 2H, C5’H2), 2.65 - 2.59 (app t, J = 6.2 Hz, 1H, OCH2CH2CN), 2.55 - 2.37 (m, 1H, OCH2CH2CN), 1.51 - 1.47 (d, 3H, C(CW3)2), 1.28 - 1.19 (m, 3H, C(C / 73)2);13C{1H} NMR (126 MHz, CDCI3) 6 165.0, 163.5, 158.6 (2C), 152.7, 151.2, 150.3, 149.8, 144.2, 142.7, 140.5, 135.3 (2C), 133.4, 132.8, 130.1 (4C), 128.8 (2C), 128.2 (2C), 128.0 (4C), 127.1, 123.6, 116.3, 114.5, 113.4 (4C), 102.7, 95.3, 87.7, 87.2, 86.5, 85.8, 84.3, 80.0, 78.3, 75.8, 72.5, 68.2, 62.7, 58.9, 55.3 (2C), 27.0, 25.1, 19.1;31P{1H} NMR (202 MHz, CDCI3) 666.9, 66.3. HRMS (ESI+): m / z calcd. 1101.3212 for ([M+H]+), found 1101.3229.5’-O-DMTr-2’-F-dA(Bz)-2’-F-3’-O-Ac-U (2n)BzHNOAc F

[0219] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1n (53.1 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (52 mg, 47.5 pmol, 95%, >99:1 sulfurization / oxygenation ratio); IR vmax(neat): 2959w, 2360m, 2250w, 2084w, 1980w, 1698s; 1610m; 1510m, 1457m, 1249m, 1034w, 908s, 730m, 631s;1H NMR (500 MHz, CDCI3) 69.50 - 8.97 (m, 2H, NH), 8.82 - 8.74 (m, 1 H, C2AH), 837 - 8.20 (m, 1 H, C8AH), 8.07 -8.01 (m, 2H, ArBz-o-H), 7.65 - 7.58 (m, 1H, ArBz-p-H), 7.57 - 7.49 (m, 2H, ArBz-m-H), 7.43 -7.14 (m, 10H, DMTr-H and C6uH overlapping with CDCI3), 6.86 - 6.76 (m, 4H, DMTr-H), 6.37 - 6.19 (m, 1H, ribose- / - / ), 5.96 - 5.60 (m, 4H, ribose-H and C5uH). 5.54 - 5.21 (m, 2H, ribose-H), 4.55 - 4.13 (m, 6H, ribose- / - / and OCH2CH2CN), 4.02 - 3.84 (m, 1H, ribose-H), 3.82 - 3.75 (m, 6H, DMTr-OC / 73), 3.66 - 3.34 (m, 1 H, ribose-H), 2.84 - 2.50 (m, 2H, OCH2CH2CN), 2.20 - 2.06 (m,3H, OAC-CH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.2, 169.9, 164.8, 162.8, 158.8 (2C), 153.1, 152.6, 150.7, 149.9, 147.5, 144.2, 143.3, 141.9, 141.4, 140.8, 139.6, 133.1, 130.3, 130.2, 129.3, 129.0 (2C), 128.2 (2C), 128.0 (2C), 127.2, 124.5, 116.5, 113.4 (4C), 103.3, 91.7, 90.1, 89.0, 87.8, 87.0, 86.1, 81.6, 79.2, 74.6, 69.2, 66.0, 63.0, 61.9, 55.4 (2C), 20.6, 19.4;31P{1H} NMR (202 MHz, CDCI3) 667.7, 67.4, 67.4, 66.9;19F{1H} NMR (471 MHz, CDCI3) 6 -197.1, -198.2, -198.5, -199.3, -203.5, -204.7, -209.4, -209.6. HRMS (ESI+): m / z calcd. 1095.2918 for ([M+H]+), found 1095.2925.5’-O-DMTr-2’-O-Me-U-2’-O-Me-3’-O-Ac-U (2o)

[0220] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1o (48 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (44.0 mg, 44.5 pmol, 89%, >99:1 sulfurization / oxygenation ratio); IR ymax(neat): 3197m, 3059m, 2939m, 2363m, 2253m, 1684s, 1509m, 1459m, 1380m, 1247s, 1178s, 1119s, 1247s, 908s, 829m, 728s, 631s;1H NMR (500 MHz, CDCI3) 67.92 - 7.81 (m, 1 H, C6uH). 7.63 - 7.43 (m, 1H, C6uH). 7.42 - 7.24 (m, 9H, DMTr-H), 6.92 - 6.84 (m, 4H, DMTr-H), 6.09 - 6.04 (m, 1H, CTH), 6.01 - 5.87 (m, 1H, CTH), 5.84-5.76 (m, 1H, C5uH). 5.33-5.27 (m, 1H, C5uH). 5.27-5.11 (m, 2H, ribose-H), 4.52 - 3.85 (m, 8H, ribose-H and OCH2CH2CN), 3.83 - 3.81 (m, 6H, DMTr-OC / 73), 3.72 - 3.53 (m, 4H, ribose- / - / and OCW3), 3.52 - 3.42 (m, 4H, ribose-H and OCW3), 2.84 - 2.78 (app t, J = 6.1 Hz, 1H, OCH2CH2CN), 2.68 - 2.51 (m, 1H, OCH2CH2CN), 2.19 - 2.15 (m, 3H, OAc-CH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.3, 163.1 (2C), 159.0 (2C), 150.4 (2C), 143.9, 139.8 (2C), 134.8 (2C), 130.4 (2C), 128.4 (2C), 128.2 (2C), 127.5, 116.5, 113.5 (4C), 103.1 (2C), 88.5, 87.7, 87.1, 86.7, 82.1, 81.8, 81.4, 81.3, 79.9, 75.0, 69.9, 66.6, 62.8, 61.4, 59.2, 58.9, 55.5 (2C), 20.8, 19.5;31P{1H} NMR (202 MHz, CDCI3) 667.6, 66.8. HRMS (ESI+): m / z calcd. 1009.3049 for ([M+NH4]+), found 1009.3043.5’-O-DMTr-2’-F-dA(Bz)-2’-O-Me-3’-O-Ac-U (2p)

[0221] Prepared according to the general procedure C using dinucleotide P(lll) precursor 1p (53.8 mg, 50 pmol, 1.00 eq.). The title compound was obtained as a white solid (49.5 mg, 44.7 pmol, 89%, >99:1 sulfurization / oxygenation ratio); IR v (neat): 3672w, 2972m, 2361m, 1691m, 1608m, 1509m, 1454m, 1390w, 1246s, 1177m, 1033s, 906s, 829w, 726s, 646w;1H NMR (500 MHz, CDCI3) 69.47 - 9.20 (m, 2H, NH), 8.82 - 8.73 (m, 1 H, C2AH), 8.28 - 8.21 (m, 1 H, C8AH), 8.08 - 8.00 (m, 2H, ArBz-o-H), 7.64 - 7.58 (m, 1H, ArBz-p-H), 7.57 - 7.46 (m, 3H, ArBz-m-H and C6uH). 7.42 -7.34 (m, 2H, DMTr-H), 7.32 - 7.13 (m, 7H, DMTr-H overlapping with CDCI3), 6.87 - 6.75 (m, 4H, DMTr-H), 6.37 - 6.29 (m, 1 H, CTH), 6.06 - 5.82 (m, 2H, CT / 7 and C2’AFH), 5.80 - 5.60 (m, 2H, C5uH and ribose-H), 5.20 - 5.10 (m, 1H, ribose-H), 4.46 - 3.88 (m, 7H, OCH2CH2CN and ribose- / - / and C5’H2), 3.82 - 3.74 (m, 6H, DMTr-OC / 73), 3.67 - 3.57 (m, 1H, C5’H), 3.48 - 3.34 (m, 4H, C5’H and OCW3), 2.84 -2.70 (m, 1H, OCH2C / 72CN), 2.67 -2.46 (m, 1H, OCH2CH2CN), 2.16 - 2.09 (m, 3H, OAc-CH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.3, 164.9, 163.0, 158.8 (20), 153.0, 152.5, 150.8, 150.3, 144.3, 141.9, 139.7, 135.3 (20), 133.5, 133.0, 130.2 (40), 129.3 (20), 129.0 (20), 128.1 (40), 127.2, 123.8, 116.4, 113.3 (40), 103.1, 91.1, 88.4, 88.2, 87.0, 81.6, 81.4, 80.0, 74.9, 69.7, 66.8, 63.1, 61.6, 59.2, 55.4 (20), 20.8, 19.6;31P{1H} NMR (202 MHz, CDCI3) 5 67.8, 67.7;19F{1H} NMR (471 MHz, CDCI3) 5 -203.3, -204.5. HRMS (ESI+): m / z calcd. 1107.3118 for ([M+H]+), found 1107.3132.General procedure for photocatalytic sulfurization of non-nucleotide phosphorus (III) compoundsNa2S2O3SC4 (1.00 mol%)MeCN / H2Ogreen light, Ar, rt3a-fGeneral procedure D: photocatalytic sulfurization of P (III) compounds

[0222] To a mixture of P(lll) precursor 1a-n (0.50 mmol, 1.00 eq.), sodium thiosulfate (632 mg, 4.00 mmol, 8.00 eq.), and photocatalyst C4 (2.91 mg, 5.00 pmol, 1.00 mol%) in a 20 mL crimp cap vial under an Ar atmosphere was added MeCN (3.75 mL) and H2O (1.25 mL). The mixture was evacuated and backfilled with Ar three times. The reaction vial was irradiated with green light (525 nm LED lamp, 40W ~5 cm from the light source) with cooling by a fan at r.t. for the time indicated. The sulfurization / oxygenation ratio was determined by31P NMR of the reaction sample. The mixture was directly purified by column chromatography (mesh) to obtain the desired product.Tris(4-methoxyphenyl)phosphine sulfide (3a)OMe

[0223] Prepared according to the general procedure D using tris(4-methoxyphenyl)phosphane (176 mg, 500 pmol, 1.00 eq.). Reaction time: 24 h. The title compound was obtained as a white solid (189 mg, 492 pmol, 98%, 96:4 sulfurization / oxygenation ratio); IR vmax (neat): 3708w, 2970m, 2867w, 2361m, 1422w, 1264m, 1204m, 1147w, 1054m, 1013m, 9744m, 896w, 828w, 732s;1H NMR (500 MHz, CDCI3) 67.70 - 7.57 (m, 6H, C2H), 7.01 - 6.88 (m, 6H, C3H), 3.83 (s, 9H, OCH3);13C{1H} NMR (126 MHz, CDCI3) 6 162.2 (d,4JC-p = 2.9 Hz, 3C, C4), 134.1 (d,2JC. P= 12.2 Hz, 6C, C2), 124.9 (d,1Jc-p = 91.8 Hz, 3C, C1), 114.1 (d,3JC-p = 13.7 Hz, 6C, C3), 55.5 (3C, OCH3);31P{1H} NMR (202 MHz, CDCI3) 640.9. HRMS (ESI+): m / z calcd. 385.1022 for ([M+H]+), found 385.1022.(4-(Dimethylamino)phenyl)diphenylphosphine sulfide (3b)

[0224] Prepared according to the general procedure D using 4-(diphenylphosphaneyl)- / V, / \ / -dimethylaniline (153 mg, 500 pmol, 1.00 eq.). Reaction time: 6 h. The title compound was obtained as a white solid (115 mg, 341 pmol, 80%, >99:1 sulfurization / oxygenation ratio).; IR: 3053 w, 2901w, 2361 m, 1659 w, 1593 m, 1513 m, 1435 m, 1365 m, 1307w, 1102 s, 999 w, 814 w, 814 m, 711 s, 659 s;1H NMR (500 MHz, CDCI3) 6 7.67 - 7.62 (m, 3H), 7.49 - 7.44 (m, 2H), 7.43 - 7.37 (m, 2H), 7.37 - 7.31 (m, 4H), 6.64 - 6.60 (m, 2H), 2.93 (s, 6H);13C NMR (126 MHz, CDCI3) 5152.2, 152.2, 134.5, 133.9, 133.7, 132.2, 132.1, 131.1, 131.1, 128.4, 128.3, 116.8,116.0, 111.4, 111.3, 77.3, 77.0, 76.8, 40.0;31P NMR (202 MHz, CDCh) 6 42.5. HRMS (ESI+): m / z calcd. 338.1127 for ([M+H]+), found 338.1132.Diphenyl(pyridin-2-yl)phosphine sulfide (3c)

[0225] Prepared according to the general procedure D using 2-(diphenylphosphaneyl)pyridine (132 mg, 500 pmol, 1.00 eq.). Reaction time: 24 h. The title compound was obtained as a white solid (123 mg, 416 pmol, 83%, >99:1 sulfurization / oxygenation ratio).; IR v (neat): 3708w, 2970m, 2867w, 2361m, 1422w, 1264m, 1204m, 1147w, 1054m, 1013m, 9744m, 896w, 828w, 732s;1H NMR (500 MHz, CDCh) 68.65 (ddt, J = 4.8, 1.6, 0.7 Hz, 1 H), 8.51 - 8.45 (m, 1 H), 7.86 - 7.80 (m, 4H), 7.77 (tdd, J = 7.8, 4.2, 1.8 Hz, 1 H), 7.45 - 7.38 (m, 2H), 7.38 - 7.31 (m, 4H), 7.29 (dddd, J= 7.7, 4.8, 2.9, 1.3 Hz, 1H);13C{1H} NMR (126 MHz, CDCI3) 5156.7, 155.8, 149.6, 136.4, 132.9, 132.4, 132.2, 131.5, 128.6, 128.3, 124.9, 77.3, 77.1, 76.8;31P{1H} NMR (202 MHz, CDCh) 5 37.5. HRMS (ESI+): m / z calcd. 296.0657 for ([M+H]+), found 296.0660.Tris(4-(trifluoromethyl)phenyl)phosphine sulfide (3d)

[0226] Prepared according to the general procedure D using tris(4-(trifluoromethyl)phenyl)phosphane (233 mg, 500 pmol, 1.00 eq.). Reaction time: 24 h. The title compound was obtained as a white solid (229 mg, 460 pmol, 92%, 98:2 sulfurization / oxygenation ratio); IR v (neat): 3708w, 2970m, 2867w, 2361m, 1422w, 1264m, 1204m, 1147w, 1054m, 1013m, 9744m, 896w, 828w, 732s;1H NMR (500 MHz, CDCh) 67.89 -7.82 (m, 6H, C2H), 7.80 - 7.71 (m, 6H, C3H);13C{1H} NMR (126 MHz, CDCh) 6 136.2 (d,1JC. P= 84.0 Hz, 3C, C1), 134.3 (qd,2JC-F = 33.1 Hz,4JC-p = 3.2 Hz, 3C, C4), 132.8 (d,2JC. P= 11.3 Hz, 6C, C2), 126.0 (dq,3JC. P= 12.9 Hz,3JC-F = 3.7 Hz, 6C, C3), 123.5 (q,1JC-F = 272.9 Hz, 3C, CF3);19F{1H} NMR (471 MHz, CDCh) 6 -63.3 (s, 9F, CF3);31P{1H} NMR (202 MHz, CDCh) 6 41.5. HRMS (ESI+): m / z calcd.499.0326 for ([M+H]+), found 499.0331.0,0,0-Triphenyl phosphorothioate (3e)

[0227] Prepared according to the general procedure D using triphenyl phosphite (155 mg, 500 pmol, 1.00 eq.). Reaction time: 24 h. The title compound was obtained as a white solid (167 mg, 488 pmol, 98%, >99:1 sulfurization / oxygenation ratio); IR v (neat): 3708w, 2970m, 2867w, 2361m, 1422w, 1264m, 1204m, 1147w, 1054m, 1013m, 9744m, 896w, 828w, 732s;1H NMR (500 MHz, CDCh) 67.34 - 7.19 (m, 6H, C3H), 7.18 - 7.04 (m, 9H, C2H and C4H);13C{1H} NMR (126 MHz, CDCh) 6 150.8 (d,2JC. P= 8.0 Hz, 3C, C1), 129.9 (d,4JC-p = 1.5 Hz, 6C, C3), 125.9 (d,5Jc-p = 2.0 Hz, 3C, C4), 121.3 (d,3JC-p = 4.8 Hz, 6C, C2);31P{1H} NMR (202 MHz, CDCh) 553.0. HRMS (ESI+): m / z calcd. 343.0552 for ([M+H]+), found 343.0550.(11 bS)-4-(Dimethylamino)dinaphtho[2,1 -c / :1',2'-f][1,3,2]dioxaphosphepine 4-sulfide (3f)

[0228] Prepared according to the general procedure D using (11bS)-A / , A / -dimethyldinaphtho[2,1-d:1',2'- / ][1,3,2]dioxaphosphepin-4-amine (180 mg, 500 pmol, 1.00 eq.). Reaction time: 24 h. The title compound was obtained as a white solid (182 mg, 465 pmol, 93%, >99:1 sulfurization / oxygenation ratio); IR v (neat): 3708w, 2970m, 2867w, 2361m, 1422w, 1264m, 1204m, 1147w, 1054m, 1013m, 9744m, 896w, 828w, 732s;1H NMR (500 MHz, CDCh) 5 8.04 (d,3JH-H = 8.8, 1H, C1H or C7H), 7.99 (d,3JH-H = 8.9 Hz, 1H, C1H or C7H), 7.95 (d,3JH-H = 8.3 Hz, 2H, C8H and C15H), 7.59 (dd,3JH-H = 8.8,4JH-P = 1.2 Hz, 1 H, C2H or C6H), 7.52 - 7.42 (m, 4H, C2H or C6H and Ar-CH), 7.36 - 7.25 (m, 3H, Ar-CH), 2.74 (d,3JH-P = 11.2 Hz, 6H, N(CH3)2);13C{1H} NMR (126 MHz, CDCh) 5 149.1 (d,2JC. P= 14.0 Hz, C2a or C5a), 147.0 (d,2JC. P = 8.6 Hz, C2a or C5a), 132.6 (d,4JC-p = 1.6 Hz, 2C, C11a and C11d), 132.0 (d, J= 1.7 Hz, C7a or C15a), 131.5 (d, J= 1.5 Hz, C7a or C15a), 131.0 (d,4JC-p = 1.5 Hz, C1 or C7), 130.9 (d,4JC-p = 1.5 Hz, C1 orC7), 128.7 (C8 orC15), 128.5 (C8 or C15), 127.4 (Ar-C), 127.2 (Ar-C), 126.9 (Ar-C), 126.6 (Ar-C), 125.8 (C11 or C12), 125.7 (C11 or C12), 122.1 (2C, C11b and C11c), 121.9 (d,3Jc-p = 2.9 Hz, C2 orC6), 121.0 (d,3JC. P= 2.9 Hz, C2 orC6), 38.7 (d,2JC. P= 3.9 Hz, 2C, N(CH3)2);31P{1H} NMR (202 MHz, CDCh) 583.85. HRMS (ESI+): m / z calcd. 392.0869 for ([M+H]+), found 392.0869.Synthesis of dinucleotide thiophosphoramidate

[0229] To a solution of 1-chloro- / V, / V, / \ / ', / \ / '-tetraisopropylphosphanediarnine (268 mg, 1.00 mmol, 1.00 eq.) in 10 mL CH2CI2 was added triethylamine (101 mg, 1.00 mmol, 1.00 eq.) and N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (658 mg, 1.00 mmol, 1.00 eq.). The reaction was stirred for 1 h at room temperature then 5-(ethylthio)-1H-tetrazole (250 mg, 1.92 mmol, 2.00 eq.) and 3'-O-acetylthymidine (284 mg, 1.00 mmol, 1.00 eq.) were added. After 2h, the crude was purified on silica pad using degassed ethyl acetate and methanol to yield compound (689 mg, 0.64 mmol, 64%, 70% purity by31P NMR) over the two steps. This compound was used in the next step without further purification.Na2S2O3, C4 (1.00 mol%) MeCN / H2O, green light

[0230] Prepared according to the general procedure C using dinucleotide P(lll) precursor described above (69.9mg, 50 mol, 1.00 eq.). The title compound (4) was obtained as a white solid (67%);1H NMR (500 MHz, CDCI3) 69.11 (d, J= 25.6 Hz, 1H), 8.76 (d, J= 3.9 Hz, 1H), 8.25 - 8.18 (m, 1H), 8.09 - 8.03 (m, 2H), 7.67 - 7.59 (m, 1H), 7.58 - 7.52 (m, 2H), 7.51 - 7.47 (m, 1H), 7.45 - 7.37 (m, 2H), 7.31 (m, 4H), 7.28 - 7.19 (m, 2H), 6.82 (dtd, J = 9.1, 4.2, 2.9 Hz, 4H), 6.55 (ddd, J = 12.6, 8.3, 5.7 Hz, 1H), 6.22 (ddd, J= 19.4, 8.8, 5.5 Hz, 1H), 5.45 - 5.39 (m, 1H,), 5.34 - 5.21 (m, 1H), 4.61 - 4.42 (m, 1H), 4.33 - 4.11 (m, 3H,), 3.81 - 3.79 (m, 6H), 3.78 - 3.71 (m, 2H), 3.54 - 3.41 (m, 2H,), 3.13 - 2.79 (m, 2H), 2.44 (m, 1H), 2.21 - 2.13 (m, 1 H,), 2.13 -2.07 (m, 3H,), 1.95 (s, 3H), 1.37 - 1.24 (m, 12H);13C NMR (126 MHz, CDCI3) 6 170.5, 163.3, 158.6, 152.6, 151.5, 150.0, 149.6, 144.3, 141.3, 135.5, 135.0, 134.9, 133.7, 132.8, 130.0, 128.8,, 128.0, 127.9, 127.0, 113.2, 111.5, 86.9, 86.8, 85.4, 85.1, 84.7, 77.3, 77.0, 76.8, 74.2, 74.0, 63.5, 55.2, 47.3, 37.3, 22.6, 20.9, 12.4;31P NMR (202 MHz, CDCI3) 670.98, 70.75. HRMS (ESI+): m / z calcd. 1103.4097 for ([M+H]+), found 1103.4093.Synthesis of nucleotide phosphoramidodithioateCl1) NaH, 0 °C, 4 h('Pr)2N'P^N('Pr)2Et2O, r t, 2 h

[0231] (2,4-dichlorophenyl)methanethiol (250 mg, 1.29 mmol, 1.00 eq.) was dissolved in dry Et20 (50.0 mL), treated with sodium hydride (dispersion in oil ~ 60%, 54.0 mg, 1.36 mmol, 1.05 eq), and cooled to 0°C and stirred for 4 h. A solution of 1 -chloro- N, N, N', N'-tetraisopropylphosphanediamine (363 mg, 1.36 mmol, 1.05 eq.) in dry Et20 (30.0 mL) was added dropwise, the mixture was allowed to warm to room temperature and stirred for 2 h. The white solid was filtered off, the solvent was removed in vacuo to give 1-((2,4-dichlorobenzyl)thio)- / V, / V, / V', / V-tetraisopropylphosphanediamine S3 as a white wax (391 mg, 1.29 mmol, 71%). The compound thus obtained was used directly in the next step without further purification.1H NMR (400 MHz, CDCh) 67.49 (d,3JH-H = 8.3 Hz, 1H, C6 / 7), 734 (d,4JH-H = 2.1 Hz, 1H, C3 / 7), 7.19 (dd,3JH-H = 8.3,4JH-H = 2.2 Hz, 1H, C5 / 7), 3.74 (d,3JH-p = 7.3 Hz, 2H, Bn-CH2), 3.68 - 3.55 (m, 4H, N(CH(CH3))2), 1.24 - 1.13 (m, 24H, N(CH(CH3)2)2);31P{1H} NMR (162 MHz, CDCh) 694.3.tetrazole, MeCN2) Na2S2O31C4 (1.00 mol%)MeCN / H2O, green lightAr, r t, 4 h

[0232] To a stirred solution of P(lll) precursor described above (27.5 mg, 100 pmol, 1.00 eq.) and 5’-O-DMTr-dA(Bz) (65.8 mg, 100 pmol, 1.00 eq.) in CH2CI2(10.0 mL) was added tetrazole (3-4% in MeCN, 234 pL, 100 pmol, 1.00 eq.) at r.t. After 30 min, the solution was diluted with CH2CI2(30 mL) and washed with H2O (30 mL). The organic phase was separated and dried with Na2SC>4. The solvent was removed in vacuo. The product thus obtained was used directly without further purification. To a mixture of this material and photocatalyst C4 (0.58 mg, 1.00 pmol, 1.00mol%) in a 10 mL crimp cap vial under an Ar atmosphere, MeCN (4.00 mL) was added. The mixture was evacuated and backfilled with Ar for three times. In a separate vial, a sodium thiosulfate solution (126 mg, 800 pmol, 8.00 eq.) in H2O (1.50 mL) was prepared and evacuated and backfilled with Ar for three times. This solution was added to the mixture of starting material and catalyst. The reaction vial was irradiated with green light (Kessil PR160L 525 nm lamp, 40W, ~5 cm from the light source) with cooling from the desk fan at r.t. for 4 h with stirring at 1000 rpm. The mixture was directly purified by reverse-phase column chromatography (ZEOprep 90 C18 15-25 pm, MeCN / H2O 20:80 to 100:0) to obtain 5’-O-DMT-dA(Bz)-S-(2,4-dichlorobenzyl) phosphoramidodithioate (5) as an off-white solid (72 mg, 71.0 pmol, 71%).1H NMR (500 MHz, CDCI3) 68.94 (br. s, 1H, NH), 8.75 (s, 1H, C2AH), 8.19-8.12 (m, 1H, C8AH), 8.05-7.99 (m, 2H, ArBz-o- / 7), 7.65 - 7.58 (m, 1 H, ArBz-p-H), 7.57 - 7.50 (m, 2H, ArBz-m-H), 7.49 - 7.46 (m, 1 H, ArBn- / - / ), 7.43 - 7.34 (m, 3H, DMT- / - / and ArBn-H), 7.33 - 7.12 (m, 8H, DMT- / - / and ArBn-H), 6.83 - 6.74 (m, 4H, DMT- / - / ), 6.50 -6.41 (m, 1H, C1’AW), 5.50 - 5.42 (m, 1H, ribose-H), 4.58 -4.48 (m, 1H, ribose- / - / ), 4.24-4.10 (m, 2H, Bn-CH2), 3.87-3.73 (m, 8H, DMT-OCH3 and N(CH(CH3)2)2), 3.52 -3.39 (m, 2H, C5’AH2), 3.09-2.69 (m, 2H, C2’AH2), 1.36- 1.23 (m, 12H, N(CH(CH3)2)2);13C{1H} NMR (126 MHz, CDCI3) 5 164.5, 158.6 (2C), 152.7, 151.5, 149.5, 144.4, 141.3, 135.5 (2C), 135.0, 134.1, 133.7, 132.8, 132.0, 130.1 (4C), 129.5, 129.0, 128.9 (2C), 128.2, 128.1, 127.9, 127.8 (2C), 127.3, 127.0, 125.3, 123.3, 113.2 (4C), 86.8, 85.4, 84.8, 78.6, 63.4, 55.2 (2C), 48.1 (2C), 37.9, 23.0, 22.4 (4C);31P{1H} NMR (202 MHz, CDCI3) 590.5, 89.8. HRMS (ESI+): m / z calcd.1011.2655 for ([M+H]+), found 1011.2659.Synthesis of 5'-O-monophosphorothioateN('Pr)2ABnO OBn, tetrazole

[0233] To a stirred solution of dibenzyl diisopropylphosphoramidite (569 mg (90% purity), 1.48 mmol, 1.20 eq.) and 2',3'-O-isopropylidene-U (351 mg, 1.23 mmol, 1.00 eq.) in CH2CI2(15.0 mL), tetrazole (1.08 mL, 0.62 mmol, 0.50 eq., 3-4% solution in MeCN) was added dropwise at r.t. After 3 h, volatiles were removed by rotary evaporation and the residue was purified by flash column chromatography (CH2CI2 / MeOH 100:0 to 95:5) to give the product as a clear oil (450 mg, 0.85 mmol, 69%); IR vmax(neat): 2990m, 1685s, 1455m, 1381m, 1264m, 1213m, 1067w, 1058s, 997s, 968s, 908m, 810w, 729s, 696s, 568w, 504w;1H NMR (500 MHz, CDCI3) 6 9.39 (s, 1H, NH), 7.42 (d,3JH-H = 8.1 Hz, 1H, C6uH). 7.38 -7.26 (m, 10H, ArBn-H), 5.86 (d,3JH-H = 2.6 Hz, 1H, CTuH), 5.59 (d,3JH-H = 8.1 Hz, 1H, C5uH). 4.87 (dd,2JH-H = 8.5,3JP. H= 1.9 Hz, 4H, Bn-CH2), 4.72- 4.65 (m, 2H, C2’uH and C3’uH), 4.32 - 4.27 (m, 1 H, C4’uH), 4.09 - 3.96 (m, 2H, C5’uH2), 1.56 (s, 3H, C(CW3)2), 1.32 (s, 3H, C(CW3)2);13C{1H} NMR (126 MHz, CDCI3) 5 162.9, 150.0, 141.1, 137.9 (t, J= 4.7 Hz, 2C), 128.7 (d, J= 1.4 Hz, 4C), 128.2 (d, J= 3.8 Hz, 2C), 127.6 (d, J= 12.2 Hz, 4C), 114.6, 102.5, 92.6, 85.5 (d, J = 5.8 Hz), 84.8, 80.7, 64.9 (dd, J = 18.0, 11.9 Hz, 2C), 62.3 (d, J= 9.0 Hz), 27.3, 25.5;31P{1H} NMR (202 MHz, CDCI3) 5140.4. HRMS (ESI+): m / z calcd.529.1734 for ([M+H]+), found 529.1738.Na2S2O3, C4 (1.00 mol%) MeCN / H2O, green lightAr, r.t., 4 hS3 6

[0234] To a mixture of dinucleotide P(lll) precursor described above (52.8 mg, 100 mol, 1.00 eq.) and photocatalyst C4 (0.58 mg, 1.00 pmol, 1.00 mol%) in a 10 mL crimp cap vial under an Ar atmosphere was added MeCN (4.00 mL). The mixture was evacuated and backfilled with Ar three times. In a separate vial, sodium thiosulfate solution (126 mg, 800 pmol, 8.00 eq.) in H2O (1.50 mL) was prepared and evacuated and backfilled with Ar three times. This solution was added to the mixture of starting material and catalyst. The reaction vial was irradiated with green light (Kessil PR160L 525 nm lamp, 40W, ~5 cm from the light source) with cooling from the desk fan at r.t. for 4 h with stirring at 1000 rpm. The sulfurization / oxygenation ratio was determined by31P NMR of the reaction sample. The mixture was directly purified by reverse-phase column chromatography (Biotage® Star C18 D - Duo 100 A 30 pm, MeCN / H2O 20:80 to 100:0) to obtain O, O-dibenzyl-O-2',3'-O-isopropylidene-U phosphorothioate (6) as a white solid (52.0 mg, 92.8 pmol, 93% >99:1 sulfurization / oxygenation ratio); IR vmax (neat): 2990w, 1685s, 1455m, 1380m, 1267m, 1214m, 1157w, 1067m, 1006s, 859m, 819m, 734s, 697s, 668w, 594w, 568w, 503w;1H NMR (500 MHz, CDCI3) 6 8.48 (s, 1H, N / 7), 7.41 - 7.28 (m, 11H, ArBn-H and C6uH).5.81 (d,3JH-H = 2.7 Hz, 1H, CTuH), 5.57 (d,3JH-H = 8.1 Hz, 1H, C5uH). 5.15 - 4.97 (m, 4H, Bn-CW2), 4.64 (dd,3JH-H = 6.4,3JH-H = 3.2 Hz, 1H, C3’uH or C2’uH), 4.60 (dd,3JH-H = 6.4,3JH-H = 2.7 Hz, 1H, C3’uH or C2’uH). 4.32 - 4.26 (m, 1H, C4’uH), 4.21 - 4.15 (m, 2H, C5’uH2), 1.55 (s, 3H, C(CW3)2), 1.31 (s, 3H, C(C / 73)2);13C{1H} NMR (126 MHz, CDCI3) 6 162.8, 150.0, 141.2, 135.7 (d,3Jc-p = 4.9 Hz), 135.6 (d,3JC-p = 5.3 Hz), 128.92, 128.89, 128.8 (4C), 128.4 (2C), 128.3 (2C), 114.7, 102.8, 92.9, 84.8 (d,3JC-p = 9.1 Hz), 84.6, 80.6, 70.3 (dd,2JC. P= 5.3 Hz,2JC. P= 5.3 Hz, 2C), 67.3 (d,2JC-p = 5.3 Hz), 27.3, 25.5;31P{1H} NMR (202 MHz, CDCI3) 5 69.1. HRMS (ESI+): m / z calcd. 583.1274 for ([M+Na]+), found 583.1275.Photocatalytic sulfurization for solid phase oligonucleotide synthesis

[0235] 5’-O-DMT-dT-3’-lcaa-controlled pore glass (CPG, 500 A) beads (200 mg, 25.0 pmol / g) were used for the synthesis. An initial deprotection step was carried out in a filtration funnel using 5% trifluoroacetic acid (TFA) solution in CH2CI2 (2.00 mL) for 1 h. Washing was performed with MeCN (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-15’-O-DMT-dC(Ac)-phosphoramidite and 0.50 molL-15-(ethylthio)-1H-tetrazole (1.50 mL) in MeCN under Ar for 1 h. Filtration was performed followed by washing with MeCN (2 x 8.00 mL). Beads were then placed in a closed vial along with 2.00 mL of an aqueous solution of 0.25 mmolL-1(4.00 mol%) riboflavin and 1.20 molL-1Na₂S₂O₃ and were stirred at 1000 rpm for 1 h under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x 8.00 mL). The beads were then treated with 5% TFA solution in CH2CI2 (2.00 mL) for 3 min. Washing with was performed using MeCN (8.00 mL) followed by deprotection and cleavage from the beads by treatment with methylamine aqueous solution (40%, 2.00 mL) for 18 h at r.t. Filtration provided the product in the aqueous solution which was purified directly by reverse phase chromatography (Biotage® Star C18 D - Duo 100 A 30 pm, MeCN / H₂O 20:80 to 100:0) to provide the product as a white solid (2.41 mg, 4.41 pmol, 88%).HRMS (ESI’): m / z calcd. 546.1065 for ([M] ), found 546.1073.

[0236] 5’O-DMT-thymidine-3’-lcaa-controlled pore glass (CPG) 1 g at 25 pmol / g was used for this synthesis. An initial deprotection step was carried out using 3% dichloro acetic acid (DCA) solution in acetonitrile:toluene 8:2 for 3 min. Washing was performed using 8 mL CH2CI2, then 2 x 8 mL toluene. The beads were then treated with 4 mL of a solution composed of 0.1 M phosphoramidite and 0.5 M 5-(ethylthio)-1H-tetrazole in acetonitrile under argon for 1 h. Filtration was performed followed by washing using 3 x 8 mL acetonitrile. Beads were then placed in a closed vial along with 5 mL of an aqueous solution of 0.25 mmolar (4 mol%) riboflavin and 1.20 M aqueous Na₂S₂O₃ and were stirred at 1000 rpm for 30 min under blue light irradiation (456 nm LED lamp, 40W). Filtration was performed followed by washing with 2 x 8 mL water and 2 x 8 mL acetonitrile. The beads were then treated with 3% DCA solution in acetonitrile for 3 min. Washing was performed using 8 mL CH2CI2 then 2x8 mL toluene. The beads were then treated with 4 mL of a solution composed of 0.1 M phosphoramidite and 0.5 M 5-(ethylthio)-1H-tetrazole in acetonitrile under argon for 1 h. Filtration was performed followed by washing using 3 x 8 mL acetonitrile. Beads were then placed in a closed vial along with 5 mL of an aqueous solution of 0.25 mmolar (4 mol%) riboflavin and 1.20 M aqueous Na₂S₂O₃ and were stirred at 1000 rpm for 30 min irradiated with blue light (456 nm LED lamp, 40W). Filtration was performed followed by washing with 2 x 8 mL water and 2 x 8 mL MeCN. The beads were then treated with 3% DCA solution in MeCN for 3 min. Washing with 8 mL acetonitrile was performed followed by deprotection and cleavage from the beads by treatment with 3 mL of 40 %. methylamine solution in water for 2 h at room temperature. Filtration provided the product in the aqueous solution (18 mg, 21 pmol, 86%) that was purified by reverse phase chromatography if high purity was needed.HRMS (ESI+): m / z calcd. 884.1392 for ([M+H]+), found 884.1400z31P NMR (202 MHz, D2O_salt) δ 56.83, 55.79, 55.71, 55.12.OH

[0237] 5’-O-DMT-dT-3’-lcaa-controlled pore glass (CPG, 500 A) beads (1.00 g, 25.0 pmol / g) were used for the synthesis. An initial deprotection step was carried out in a filtration funnel using 3% dichloroacetic acid (DCA) solution in MeCN:toluene (8:2, 5.00 mL) for 3 min. Washing was performed with CH2CI2 (8.00 mL) followed by toluene (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-1 5’-O-DMT-dC(Ac)-phosphoramidite and 0.50 molL-1 5-(ethylthio)-1H-tetrazole (4.00 mL) in MeCN under Ar for 1 h. Filtration was performed followed by washing with MeCN (3 x 8.00 mL). Beads were then placed in a closed vial along with 5.00 mL of an aqueous solution of 0.25 mmolL-1 (4.00 mol%) riboflavin and 1.20 molL-1 Na₂S₂O₃ and were stirred at 1000 rpm for 30 min under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x 8.00 mL). The beads were then treated with 3% DCA solution in MeCN (2.00 mL) for 3 min. Washing was performed using CH2CI2 (8.00 mL) followed by toluene (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-1 5’-O-DMT-2’-O-Me-U-phosphoramidite and 0.50 molL-1 5-(ethylthio)-1H-tetrazole in MeCN (4.00 mL) under Ar for 1 h. Filtration was performed followed by washing using MeCN (3 x 8.00 mL). Beads were then placed in a closed vial along with 5.00 mL of an aqueous solution of 0.25 mmolL-1 (4.00 mol%) riboflavin. An oxygen balloon was connected to the vial. The mixture was stirred at 1000 rpm for 30 min under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x 8.00 mL). The beads were then treated with 3% DCA solution in MeCN (2.00 mL) for 3 min. Washing with was performed using MeCN (8.00 mL) followed by deprotection and cleavage from the beads by treatment with methylamine aqueous solution (40%, 3.00 mL) for 2 h at r.t. Filtrationprovided the product in the aqueous solution which was purified directly by reverse phase chromatography (Biotage® Star C18 D- Duo 100 A 30 pm, MeCN / H2O 20:80 to 100:0) to provide the product as a white solid (11.4 mg, 13.2 pmol, 66%).HRMS (ESI+): m / z calcd. 432.5701 for ([M]2−), found 432.5704.OOH

[0238] 5’-O-DMT-dT-3’-lcaa-controlled pore glass (CPG, 500 A) beads (1.00 g, 25.0 pmol / g) were used for the synthesis. An initial deprotection step was carried out in a filtration funnel using 3% dichloroacetic acid (DCA) solution in MeCN:toluene (8:2, 5.00 mL) for 3 min. Washing was performed with CH2CI2 (8.00 mL) followed by toluene (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-15’-O-DMT-dC(Ac)-phosphoramidite and 0.50 molL-15-(ethylthio)-1H-tetrazole (4.00 mL) in MeCN under Ar for 1 h. Filtration was performed followed by washing with MeCN (3 x 8.00 mL). Beads were then placed in a closed vial along with 5.00 mL of an aqueous solution of 0.25 mmolL'1(4.00 mol%) riboflavin. An oxygen balloon was connected to the vial. The mixture was stirred at 1000 rpm for 30 min under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x 8.00 mL). The beads were then treated with 3% DCA solution in MeCN (2.00 mL) for 3 min. Washing was performed using CH2CI2 (8.00 mL) followed by toluene (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-15’-O-DMT-2’-O-Me-U-phosphoramidite and 0.50 molL'15-(ethylthio)-1H-tetrazole in MeCN (4.00 mL) under Ar for 1 h. Filtration was performed followed by washing using MeCN (3 x 8.00 mL). Beads were then placed in a closed vial along with 5.00 mL of an aqueous solution of 0.25 mmolL'1(4.00 mol%) riboflavin and 1.20 molL'1Na₂S₂O₃ and were stirred at 1000 rpm for 30 min under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x8.00 mL). The beads were then treated with 3% DCA solution in MeCN (2.00 mL) for 3 min. Washing with was performed using MeCN (8.00 mL) followed by deprotection and cleavage from the beads by treatment with methylamine aqueous solution (40%, 3.00 mL) for 2 h at r.t. Filtration provided the product in the aqueous solution which was purified directly by reverse phase chromatography (Biotage® Star C18 D- Duo 100 A 30 pm, MeCN / H₂O 20:80 to 100:0) to provide the product as a white solid (16.2 mg, 17.4 pmol, 87%).HRMS (ESI+): m / z calcd. 464.0868 for ([M]2−), found 464.0877.OH

[0239] 5’-O-DMT-dT-3’-lcaa-controlled pore glass (CPG, 500 A) beads (1.00 g, 25.0 pmol / g) were used for the synthesis. An initial deprotection step was carried out in a filtration funnel using 3% dichloroacetic acid (DCA) solution in MeCN:toluene (8:2, 5.00 mL) for 3 min. Washing was performed with CH2CI2 (8.00 mL) followed by toluene (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-15’-O-DMT-dC(Ac)-phosphoramidite and 0.50 molL-15-(ethylthio)-1H-tetrazole (4.00 mL) in MeCN under Ar for 1 h. Filtration was performed followed by washing with MeCN (3 x 8.00 mL). Beads were then placed in a closed vial along with 5.00 mL of an aqueous solution of 0.25 mmolL'1(4.00 mol%) riboflavin and 1.20 molL-1Na₂S₂O₃ and were stirred at 1000 rpm for 30 min under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x 8.00 mL). The beads were then treated with 3% DCA solution in MeCN (2.00 mL) for 3 min. Washing was performed using CH2CI2 (8.00 mL) followed by toluene (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-15’-O-DMT-2’-O-Me-C-phosphoramidite and 0.50 molL-15-(ethylthio)-1H-tetrazole in MeCN (4.00 mL) under Ar for 1 h. Filtration was performed followed by washing using MeCN (3 x 8.00 mL). Beads were then placed in a closed vial along with 5.00 mL of an aqueous solution of 0.25 mmolL'1(4.00 mol%) riboflavin. An oxygen balloon was connected to the vial. The mixture was stirred at 1000 rpm for 30 min under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x 8.00 mL). The beads were then treated with 3% DCA solution in MeCN (2.00 mL) for 3 min. Washing was performed using CH2CI2 (8.00 mL) followed by toluene (2 x 8.00 mL). The beads were then treated with a solution composed of 0.10 molL-15’-O-DMT-2’-O-Me-A-phosphoramidite and 0.50 molL-15-(ethylthio)-1H-tetrazole in MeCN (4.00 mL) under Ar for 1 h. Filtration was performed followed by washing using MeCN (3 x 8.00 mL). Beads were then placed in a closed vial along with 5.00 mL of an aqueous solution of 0.25 mmolL-1(4.00 mol%) riboflavin and 1.20 molL-1Na2S2C>3 and were stirred at 1000 rpm for 30 min under blue light irradiation (Kessil PR160L 456 nm lamp, 40W, ~ 4 cm from the light source). Filtration was performed followed by washing with H2O (2 x 8.00 mL) and MeCN (2 x 8.00 mL). The beads were then treated with 3% DCA solution in MeCN (2.00 mL) for 3 min. Washing with was performed using MeCN (8.00 mL) followed by deprotection and cleavage from the beads by treatment with methylamine aqueous solution (40%, 3.00 mL) for 2 h at r.t. Filtration provided the product in the aqueous solution which was purified directly by reverse phase chromatography (Biotage® Star C18 D - Duo 100 A 30 pm, MeCN / H₂O 20:80 to 100:0) to provide the product as a white solid (18.0 mg, 21.0 pmol, 86%). HRMS (ESI+): m / z calcd. 1227.2074 for ([M+H]+), found 1227.2089.Photocatalytic oxygenation of oligonucleotides

[0240] To a mixture of a dinucleotide P(lll) precursor 1c (47.7 mg, 50.0 pmol, 1.00 eq.) and photocatalyst C4 (0.29 mg, 0.50 pmol, 1.00 mol%) in a 10 mL crimp cap vial under an ambient atmosphere, MeCN (1.95 mL) and H2O (0.65 mL) were added. Three needles were inserted through the cap of the vial for gas exchange with the open atmosphere. The reaction vial was irradiated with green light (Kessil PR160L 525 nm lamp, 40W, ~5 cm from the light source) with cooling from the desk fan at r.t. for 16 h. The mixture was directly purified by reverse-phasecolumn chromatography (ZEOprep 90 C18 15-25 pm, MeCN / H2O, 5:95 to 90:10) to obtain 5’-O- DMT-dC(Ac)-3’-O-Ac-dT phosphate (7) as a white solid (38 mg, 39.1 pmol, 78%); IR vmax(neat): 3632w, 3549w, 2362s, 1666s, 1612m, 1563m, 1493s, 1443m, 1393m, 1307m, 1247s, 1177m, 1114m, 1030s, 910s, 830s, 728s, 648m;1H NMR (500 MHz, CDCI3) 69.32 - 8.60 (m, 2H, N / 7), 8.17 - 7.97 (m, 1H, C6cH). 7.40 - 7.22 (m, 10H, DMT- / - / and C6TH). 7.20- 7.12 (m, 1H, C5cH).6.93 -6.75 (m, 4H, DMT-H), 6.35 - 6.16 (m, 2H, C5’H), 5.34 - 5.19 (m, 1H, C3’TH), 5.18 - 5.06 (m, 1H, C3’cH), 4.47 - 4.03 (m, 6H, C5’TH2and OC / 72CH2CN and C4’H), 3.87 - 3.76 (m, 6H, DMT-OCH3), 3.57 - 3.36 (m, 2H, C5’cH2), 2.98 - 2.82 (m, 1H, C2’cH), 2.81 - 2.58 (m, 2H, OCH2CH2CN), 2.46 -2.24 (m, 3H, C2’cH and C2’TH2), 2.26 -2.11 (m, 3H, Ac-CH3), 2.11 -2.06 (s, 3H, AC-C / 73), 1.95 - 1.82 (m, 3H, C5TCH3);13C{1H} NMR (126 MHz, CDCI3) 5 170.7 (2C), 163.7, 162.6, 158.9 (2C), 155.2, 150.4, 144.1 (2C), 135.2 (3C), 130.2 (4C), 128.2 (4C), 127.4, 116.3, 113.5 (4C), 111.9, 96.5, 87.4, 86.9, 85.2 (2C), 82.6, 78.2, 73.8, 67.8, 62.6 (2C), 55.4 (2C), 40.3, 36.9, 25.1, 21.0, 19.8, 12.6;31P{1H} NMR (202 MHz, CDCI3) 5 -2.6, -2.7. HRMS (ESI+): m / z calcd. 993.3042 for ([M+Na]+), found 993.3045.

[0241] Sodium sulfite (12.6 g, 100 mmol, 1.00 eq.) was dissolved in distilled H2O (40.0 mL) until the appearance of a clear solution. The stirred solution is then warmed to 70°C. Elemental selenium (7.90 g, 100 mmol, 1.00 eq.) is slowly added at 70°C during a period of 2 hours. The mixture is then stirred at 70°C and 1000 rpm for 12 h. The solution is filtered to obtain a sodium selenosulfate solution. Upon filtration, the solution is sealed under nitrogen and stored at 70 °C. To a mixture of dinucleotide P(lll) precursor 1b (50.0 pmol, 1.00 eq.) and photocatalyst C4 (0.29 mg, 0.50 pmol, 1.00 mol%) in a 10 mL crimp cap vial under an Ar atmosphere was added MeCN (1.95 mL) and sodium selenosulfate solution obtained as described above (0.65 mL). The mixture was evacuated and backfilled with Ar three times. The reaction vial was irradiated with green light (Kessil PR160L 525 nm lamp, 40W, ~5 cm from the light source) with cooling by a desk fan at r.t. for 4 h with stirring at 1400 rpm. The mixture was directly purified by reverse-phase column chromatography (ZEOprep 90 C18 15-25 pm, MeCN / H2O 5:95 to 100:0) to obtain 5’-O-DMT-dA(Bz)-3’-0-Ac-dT phosphoroselenoate as a white solid (54 mg, 48.2 pmol, 96%); IR vmax(neat): 3735w, 3634w, 2945w, 2838w, 2341s, 2337s, 1700m, 1695m, 1684m, 1608m, 1507m, 1456m, 1250s, 1177m, 1073m, 1032m, 1000m, 905m, 827w, 727s, 720s, 701m, 668s, 649w;1H NMR (500 MHz, CD3CN) 5 9.53 - 9.12 (m, 2H, N / 7), 8.56 - 8.50 (m, 1H, C2 H). 8.25 - 8.18 (m, 1H, C8AH). 8.02 - 7.97 (m, 2H, ArBz-o-H), 7.67 - 7.60 (m, 1H, ArBz-p-H), 7.57 - 7.50 (m, 2H, ArBz-m- / 7), 7.42 - 7.34 (m, 3H, DMT- / - / and C6TH). 7.30 - 7.15 (m, 7H, DMT-H), 6.82 - 6.75 (m, 4H, DMT-H), 6.48 - 6.40 (m, 1H, CTH), 6.24 - 6.13 (m, 1H, CTH), 5.58 - 5.46 (m, 1H, ribose-H), 5.26 - 5.19 (m, 1H, ribose-H), 4.42 - 4.15 (m, 6H, C5’H2and OCH2CH2CN and ribose-H), 3.75 - 3.71 (m, 6H, DMT-OCH3), 3.42 - 3.31 (m, 2H, C5’H2), 3.30 - 3.15 (m, 1H, C2’H), 2.83 - 2.70 (m, 3H, OCH2CH2CN and C2’H), 2.36 - 2.22 (m, 2H, C2’H2), 2.06 - 2.00 (m, 3H, Ac-CH3), 1.85 - 1.79 (m, 3H, C5TC / 73);13C{1H} NMR (126 MHz, CD3CN) 5 171.0, 164.1, 164.1, 159.3 (2C), 152.4, 151.0, 150.9, 150.5, 145.4, 143.3, 136.2 (3C), 134.4, 133.2, 130.6 (4C), 129.2 (4C), 128.5 (4C), 127.5, 125.4, 113.6 (4C), 111.4, 86.9, 85.5, 85.3, 85.0, 82.6, 80.6, 74.3, 69.0, 64.4, 63.7, 55.5 (2C), 37.4, 36.9, 20.7, 19.5, 12.3, the carbon OCH2CH2CN was overlapping with CD3CN;31P{1H} NMR (202 MHz, CD3CN) 571.6, 71.3.HRMS (ESI+): m / z calcd. 1121.2715 for ([M+Na]+), found 1121.2726.

[0242] In oligonucleotide synthesis, solid-phase methods have become the gold standard due to their efficiency, ease of automation, and the ability to rapidly assemble long sequences with high purity. This technique involves attaching the growing oligonucleotide chain to an insoluble support, allowing for the selective removal of by-products and unreacted reagents. The photocatalytic thionation method provided by the inventors is fully compatible with traditional solid-phase oligonucleotide synthesis, as demonstrated by the synthesis of the 2-mer and 3-mer compounds 8 and 9, requiring minimal adjustments to the existing phosphoramidite method (Fig.1). The reaction is performed in water as a single solvent in combination with riboflavin (vitamin B2) as a benign and water-soluble photocatalyst. The loss in yield is, by a large degree, ascribable to the purification of an isolated product. This improvement not only makes the process more sustainable but also offers a safer, more cost-effective alternative without significantly altering the standard workflow, making it an ideal upgrade to established methods.

[0243] In conclusion, the inventors have developed a mild and general catalytic method for the thionation of oligonucleotides with inexpensive and non-toxic thiosulfate as sulfur source. The method provided phosphorothioates in good to excellent yield with minimal side-oxygenation. The protocol is easily switchable to oxygenation for the synthesis of phosphates, and to the synthesis of phosphoroselenoates, by modifying the reaction conditions. Furthermore, the utility of the methodology was demonstrated by its application to solid-phase synthesis. The present development offers a general, sustainable, and cost-efficient addition to existing methods of oligonucleotide synthesis, showing potential for future industrial applications.

Claims

CLAIMS1. A method for the photocatalytic chalcogenation (e.g. oxygenation or sulfurization) of an organo phosphorous (III) compound, the method comprising reacting an organo phosphorous (III) compound with a chalcogen source (e.g. an oxygen source or a sulfur source) in the presence of a photocatalyst and light irradiation.

2. The method according to claim 1, wherein the organo phosphorous (III) compound is selected from phosphorus (III) nucleotide precursors, phosphoramidites, phosphorothioates, phosphoramidothioates, phosphites, aminophosphites, phosphines and aminophosphines; optionally wherein the organo phosphorous (III) compound is selected from phosphorus (III) nucleotide precursors, phosphoramidites, phosphites and phosphines;further optionally wherein the organo phosphorous (III) compound is a phosphorus (III) nucleotide precursor.

3. The method according to claim 1 or claim 2, wherein the organo phosphorous (III) compound comprises a moiety according to the formula (Int), below:I IxP'R xby(Int)wherein:Xa and Xb are each independently selected from O or S (preferably Xaand Xb are O); R is any suitable functional group, which may be optionally protected;optionally wherein R is selected from a ORH, SRS, NRN1RN2or CH2Rc1;wherein:a) RHis a hydrogen or a phosphorous hydroxyl protecting group (e.g. cyanoethyl, t-butyl or benzyl);b) Rsis hydrogen or a thiol protecting group (e.g. cyanoethyl, benzyl, 4- chlorobenzyl or 2,4 dichlorobenzyl);c) each of RN1and RN2are independently selected from:i) an amine protecting group, e.g. SO2(1-6C)alkyl or tosyl; or ii) (1-6C)alkyl, (3-8C)cycloalkyl, 3- to 12 membered heterocyclyl or 5 to 10 membered heteroaryl, wherein any (1-6C)alkyl, (3- 8C)cycloalkyl, 3- to 12-membered heterocyclyl, 5 to 10 memberedheteroaryl is optionally substituted by one or more substituents independently selected from halo, (1-2C)alkyl, (1-2C)alkoxy, (1- 2C)haloalkyl, (1-2C)haloalkoxy, cyano or NMe2;iii) RN1and RN2are linked such that, together with the nitrogen atom to which they are attached, form a 4 to 7 membered heterocyclic ring;d) RC1is selected from (1-6C)alkyl, (3-8C)cycloalkyl, 3- to 12 membered heterocyclyl, 5 to 10 membered heteroaryl or (1-6C)alkoxy, wherein any (1- 6C)alkyl, (3-8C)cycloalkyl, 3- to 12-membered heterocyclyl, 5 to 10 membered heteroaryl or (1-6C)alkoxy is optionally substituted by one or more substituents independently selected from halo, (1-2C)alkyl, (1-2C)alkoxy, (1- 2C)haloalkyl, (1-2C)haloalkoxy, cyano or NMe2.

4. The method according to any one of claims 1 to 3, wherein the organo phosphorous (III) compound is a phosphorus (III) nucleotide precursor, and wherein the method comprises reacting a nucleotide precursor comprising a moiety according to the formula (lnt-1), below:B1CX^4aR“ B2R'P'xb^4b | IO R2b(lnt-1)with a chalcogen source, in the presence of a photocatalyst and light irradiation; wherein:Xa and Xb are each independently selected from O or S;R is as defined in claim 3;R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group;R4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1-2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; and Rcis selected from hydrogen or a (1 -6C)alkyl;R2bis selected from hydrogen, methoxy, F, OP2b or O(CH2)2OMe; wherein P2b is a hydroxyl protecting group; andR4bis hydrogen;or R2b and R4b are linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2b and X4b is (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3and Rb4are independently selected from hydrogen, (1-2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; and Rcis selected from hydrogen or a (1 -6C)alkyl; andB1and B2are independently selected optionally protected nucleobases;to form a nucleotide comprising a moiety according to formula (I) below:wherein X is a chalcogen atom (e.g. oxygen, sulfur or selenium, preferably oxygen or sulfur); wherein R, R2a, R4a, B1, R2b, R4b and B2are as defined in any one of the preceding claims; optionally wherein Xaand Xb are O.

5. The method according to any one of claims 1 to 3, wherein the organo phosphorous (III) compound is a phosphorus (III) nucleotide precursor, and the nucleotide precursor has a structure according to Formula (lnt-2) below:P1 Bi1O Ri2bP2(lnt-2)wherein:Xa is selected from O or S (preferably O);Xb is selected from O or S (preferably O);P1 is a hydroxyl protecting group;P2 is selected from a hydroxyl protecting group, or a group with the formula:RRPO-P-N(iPr)2wherein RP is a phosphorous hydroxyl protecting group;or P2 is a linker moiety attached to a solid support optionally via one or more further nucleotides;R is as defined in claim 3;R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group;R4ais hydrogen;or R2aand R4aare linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2aand X4ais (CRa1Ra2)x(where x is selected from 1 or 2); and the other is selected from CRa3Ra4, O, NRCor S; wherein each of Ra1, Ra2, Ra3and Ra4are independently selected from hydrogen, (1-2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; and Rcis selected from hydrogen or a (1 -6C)alkyl;R2b is selected from hydrogen, methoxy, F, OP2b or O(CH2)2OMe; wherein P2bis a hydroxyl protecting group; andR4bis hydrogen;or R2band R4bare linked to form a locked nucleic acid, e.g. a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2band X4bis (CRb1Rb2)x(where x is selected from 1 or 2); and the other is selected from CRb3Rb4, O, NRdor S; wherein each of Rb1, Rb2, Rb3andRb4are independently selected from hydrogen, (1-2C)alkyl, hydroxy, amino or halo, wherein the hydroxy and amino optionally comprise an appropriate protecting group; and Rcis selected from hydrogen or a (1 -6C)alkyl; andor P2 and R2b may be linked such that, together with the atoms to which they are attached, they form a fused 5- or 6-membered heterocyclic ring, or a 5- or 6-membered silyl heterocyclic ring, which may be optionally substituted by one or more substituents selected from (1-4C)alkyl, (1-4C)alkoxy, benzyl or phenyl, any of which may in turn be optionally further substituted by one or more substituents selected from (1 -4C)alkyl or (1- 4C)alkoxy, and / or the ring formed by P2 and R2b may be attached to a solid support; and B1and B2are independently selected optionally protected nucleobases;optionally wherein:P1 is selected from acetyl, t-butyl, t-butoxym ethyl, methoxym ethyl, tetrahydropyranyl, 1- ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6- dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2- trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9- phenylxanthine-9-yl (Pixyl), 2-(trimethylsilyl)ethoxymethyl (SEM) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX); and / orP2 is as defined in item i), ii), iii) or iv) below:i) P2 is a protecting group selected from acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1 -ethoxyethyl, 1-(2-chloroethoxy)ethyl, p- chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p- nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9- phenylxanthine-9-yl (Pixyl), 2-(trimethylsilyl)ethoxymethyl (SEM) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX); orii) P2 isRpO-N(iPr)2.wherein Rpis 2-cyanoethyl; oriii) P2 is a linker moiety attached to a solid support optionally via one or more further nucleotides;iv) or P2 and R2b may be linked such that, together with the atoms to which they are attached form a fused 5-membered cyclic ether or cyclic silyl ether, which may be optionally substituted by one or more (1-4C)alkyl groups; preferably P2 and R2b, together with the atoms to which they are attached, form a ring with the formula:further optionally wherein:P1 is dimethoxytrityl (DMTr); and / orP2 is acetyl or P2 is a linker moiety attached to a solid support, e.g. the linker moiety and solid support have the formula:wherein: ⬤ is a solid support, e.g. Controlled Pore Glass (CPG) with a 500-2000 Å pore size.

6. The method according to any one of claims 3, 4 or 5, wherein:RHis selected from methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, 4- methoxybenzyl, 4-chlorobenzyl, 2,4 dichlorobenzyl, 2-chlorophenyl and 2-cyanoethyl, optionally wherein RHis 2-cyanoethyl;Rsis selected from 2-cyanoethyl, benzyl, 4-chlorobenzyl and 2,4 dichlorobenzyl;RN1and RN2are independently selected from isopropyl, SO2Me or tosyl; andRcis selected from methyl and methoxy.

7. The method according to any one of claims 3 to 6, wherein R is ORHand RHis as defined in any one of the preceding claims;optionally wherein RHis selected from methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tertbutyl, 4-methoxybenzyl, 4-chlorobenzyl, 2-chlorophenyl and 2-cyanoethyl;further optionally wherein RHis 2-cyanoethyl.

8. The method according to any one of claims 4 to 7, wherein:R2ais selected from hydrogen, methoxy, F, OP2aor O(CH2)2OMe; wherein P2ais a hydroxyl protecting group; andR4a is hydrogen;or R2a and R4a are linked to form a locked nucleic acid of the formula -X2a-X4a-, wherein one of X2a and X4a is (CRa1Ra2) and the other is O; wherein each of Ra1and Ra2are independently selected from hydrogen or methyl; e.g. R2a and R4a are linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-;optionally wherein:R2a is selected from hydrogen, methoxy or OP2a; wherein P2a is a hydroxyl protecting group; andR4a is hydrogen;further optionally wherein:R2a is selected from hydrogen or OP2a; wherein P2a is a hydroxyl protecting group; and R4a is hydrogen.

9. The method according to any one of claims 4 to 8, wherein:R2bis selected from hydrogen, methoxy, F, OP2bor O(CH2)2OMe; wherein P2bis a hydroxyl protecting group; andR4bis hydrogen;or R2band R4b are linked to form a locked nucleic acid of the formula -X2b-X4b-, wherein one of X2b and X4b is (CRb1Rb2) and the other is O; wherein each of Rb1and Rb2are independently selected from hydrogen or methyl; e.g. R2b and R4b are linked to form a locked nucleic acid of the formula -O-CH2- or -CH2-O-;optionally wherein:R2bis selected from hydrogen, methoxy or OP2b; wherein P2b is a hydroxyl protecting group; andR4bis hydrogen;further optionally wherein:R2bis selected from hydrogen or OP2b; wherein P2b is a hydroxyl protecting group; and R4bis hydrogen.

10. The method according to any one of the preceding claims, wherein the reaction is performed at a temperature of from 0 to 75°C, optionally from 15 to 30°C.

11. The method according to any one of the preceding claims, wherein the method further comprises removing any protecting groups present and, if necessary, cleavage from any solid support to form an oligonucleotide comprising a moiety of the formula:wherein:X is a chalcogen (e.g. oxygen, sulfur or selenium, preferably oxygen or sulfur); and R, B1, B2, R2a, R2b, R4aand R4bare as defined in any one of the preceding claims.

12. The method according to any one of the preceding claims, wherein the reaction is a sulfurization reaction and the chalcogen source is a sulfur source;optionally wherein the sulfur source is a thiosulfate;further optionally wherein the sulfur source is selected from barium thiosulfate, magnesium thiosulfate, potassium thiosulfate, cesium thiosulfate and sodium thiosulfate.

13. The method according to any one of the preceding claims, wherein the reaction is performed in a suitable solvent; optionally wherein:a) if P2 is not a linker moiety attached to a solid support, the solvent comprises one or more of water and optionally one or more aprotic solvents e.g. acetonitrile, THF, dioxane or acetone;optionally wherein the solvent comprises a mixture of water and acetonitrile; or b) if P2 is a linker moiety attached to a solid support, the solvent is water.

14. The method according to any one of the preceding claims, wherein the photocatalyst is selected from:i) a catalyst which is activated by light with a wavelength greater than or equal to 380 nm;ii) a catalyst which is activated by light with a wavelength greater than or equal to 420 nm; oriii) one or more of riboflavin, flavin adenine dinucleotide, flavin mononucleotide, flavin derivatives, tris(bipyridine)ruthenium(ll) chloride (Ru-bpy), 2,4,6-triphenylpyrylium tetrafluoroborate (TPT), [4,4'-S / s(1, 1-dimethylethyl)-2,2'-bipyridine-A / 1, / 1 ']£> / s[3,5- difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-A / ]phenyl-C]lridium(l 11) hexafluorophosphate ([lr[dF(CF3)ppy]2(dtbbpy)]PF6), 9-Mesityl-10-methylacridinium tetrafluoroborate ([MesAcrMe]BF4), Erythrosin B, Eosin Y, Rose Bengal, Methylene blue, 9-Mesityl- 1,3,6,8-tetramethoxy-10-phenylacridin-10-ium tetrafluoroborate ([Mes(MeO)4AcrPh]BF4), 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzlPN), 2,4,6-Tri(9-carbazol-9-yl)-5-chloroisophthalonitrile (3CzClIPN), Iridium(lll) hexafluorophosphate ([Ir[dF(CF3)ppy]2(dtbbpy)]PF6), Tris(2,2'- bipyridyl)ruthenium(ll) chloride hexahydrate ([Ru(bpy)3]Cl2·6H2O) or Triphenyl phosphate (TPP).

15. The method according to any one of the preceding claims, wherein the method further comprises coupling with one or more nucleotides.

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