Methods for Producing Polynucleotides

Abstract

Provided herein is a method for producing a polynucleotide or an analog or derivative thereof, comprising providing a support-bound nucleoside, nucleotide, polynucleotide or derivative or analog thereof, the support-bound nucleoside, nucleotide, polynucleotide or derivative or analog thereof, the support-bound nucleoside, nucleotide, polynucleotide or derivative or analog thereof being a compound of formula (I) as defined herein and being (II) bound to a solid support, (ii) extending the chain length of the support-bound nucleoside, nucleotide, polynucleotide or derivative or analog thereof by introducing one or more additional nucleoside units, nucleotide units and / or analogs or derivatives thereof, and (iii) cleaving the resulting polynucleotide from the solid support by irradiating the compound of formula (I). Also provided are related compounds, uses, and polynucleotides obtained by the above methods. [Formula 1] TIFF2024542724000067.tif35114

Description

Detailed Description of the Invention

[0001] FIELD OF THEINVENTION The present invention relates to a method for producing a polynucleotide or an analogue or derivative thereof. The present invention also relates to compounds for use in said method and to a method of sequencing using said polynucleotide. The present invention further relates to a solid support column comprising said compounds for solid phase polynucleotides, to the use of said compounds for synthesizing a polynucleotide or a derivative or analogue thereof, and to a polynucleotide or a derivative or analogue thereof obtained by said method.

[0002] 2. Background of the Invention Polynucleotides are biopolymers that contain nucleotide monomer units and have applications in many different fields, including therapeutics, diagnostics, and research. Polynucleotides with customized nucleotide sequences can be synthesized in vitro, for example, using solution phase synthesis or solid phase synthesis.

[0003] Solid-phase chemical synthesis was developed in the 1960s and has many advantages over solution-phase synthesis: reactions can be driven to completion quickly using excess solution-phase reagents, impurities and excess reagents can be thoroughly washed away facilitating easy isolation of the growing polynucleotide without the need for purification between reaction steps, and the process is amenable to automation on computer-controlled solid-phase synthesizers.

[0004] In solid-phase polynucleotide synthesis, a polynucleotide or initial nucleoside / nucleotide monomer is typically attached to a solid support, and a series of reactions are carried out to extend the chain length of the support-attached polynucleotide / initial monomer, such that synthesis is carried out on the solid support. The solid support is typically immobilized between filters in a synthesis column. While solution phase reagents and solvents can flow freely through the column, the solid support and its attached compounds are fixed in place. The polynucleotide or initial nucleoside / nucleotide monomer is typically attached to the solid support using a linker.

[0005] Designing a suitable linker for use in solid-phase polynucleotide synthesis is a challenge. For example, the linker must be stable under the reaction conditions used during polynucleotide synthesis, but cleavable under specific conditions once polynucleotide synthesis is complete to obtain a free polynucleotide product. The linker must also be such that it attaches to the desired position of the polynucleotide to facilitate controlled synthesis.

[0006] There are several known chemical linkers that have been used to attach solid supports to polynucleotides or initial nucleoside / nucleotide monomers for solid phase polynucleotide synthesis.

[0007] Most commonly, succinyl-based linkers are used, examples of which are shown below:

[0008] [ka]

[0009] The succinyl linker can be hydrolytically cleaved to release a free nucleotide without any additional reaction steps. However, the reaction conditions for succinyl cleavage are relatively harsh, and usually require the use of concentrated ammonium hydroxide. The use of harsh reaction conditions can make the setup of the equipment required for the synthesis reaction more complicated and limit the range of polynucleotides that can be synthesized. For example, if the chemical group that constitutes the desired modification is susceptible to cleavage or modification by the reagents used throughout the polymer synthesis, it may not be possible to include said modification in the synthesized polynucleotide (e.g., specifically modified nucleotide monomers).

[0010] Moreover, in reality, when using succinyl linkers, the initial nucleoside / nucleotide monomer usually needs to be pre-bound to a solid support. This has the disadvantage that different synthesis columns are usually required depending on the identity of the nucleobase desired for the initial nucleotide monomer. Since a different column is usually required for each different monomer, the columns are usually available only for natural nucleobases (e.g. adenine (A), cytosine (C), guanine (G) or thymine (T)) and a narrow range of modified derivatives. However, if suitable columns are desired for non-natural nucleobases, they may not be readily available, limiting the range of polynucleotides that can be synthesized by the method.

[0011] To address this concern, attempts have been made to provide linkers that can be coupled to any desired nucleoside / nucleotide monomer after it has been synthesized on a solid support. In such "universal supports" for polynucleotide synthesis, the first monomer of the polynucleotide is usually added as the first coupling reaction and is not pre-coupled. Universal linkers usually function by coupling the first nucleotide to be incorporated into the polynucleotide chain to the support via a phosphate.

[0012] Examples of known linkers for universal support are shown below with the first nucleotide attached:

[0013] [ka]

[0014] The group bearing the DMT-protected OH functionality can be attached to a solid support, for example via an amide bond, as shown above, for reaction with the first nucleotide to be incorporated into the polymer chain. Once polymer synthesis is complete, the strained fused ring portion of the linker is released by ester hydrolysis, followed by intramolecular reaction at the phosphate group of the initial nucleotide to release the polynucleotide.

[0015] Even if the universal linker allows a range of nucleotides to be incorporated as initial nucleotides into the synthesized polynucleotide, significant problems remain. For example, many reaction steps under relatively harsh conditions are usually required to produce the free polynucleotide product. In particular, hydrolysis of the phosphate group usually leaves the phosphate moiety attached to the terminal sugar ring of the polynucleotide, which is usually not compatible with downstream enzymatic reactions and usually requires a terminal hydroxyl group at the corresponding position. This means that cleaving the phosphate group usually requires treatment of the synthesized polynucleotide with additional reagents, adding additional steps, additional costs, and the need for additional purification steps. Furthermore, if the cleaved linker is generated as a solution-phase by-product, the desired polynucleotide product must be isolated from this by-product. A linker that addresses these concerns is needed.

[0016] Photocleavable linkers, or "photolabile" linkers, that undergo cleavage under photoexcitation are known. Photocleavable linkers have the advantage that the cleavage conditions are relatively mild, do not usually alter the structure of the polynucleotide product, and are orthogonal to the usual reaction conditions used during polynucleotide synthesis.

[0017] Previous attempts have focused on photocleavable linkers containing a carboxyl group to attach the initial nucleotide to the support. Such linkers have typically focused on the use of ester or carbonate groups. Known photocleavable linkers include:

[0018] [ka]

[0019] While the above groups are photochemically cleavable, significant problems remain. For example, diester-containing linkers such as the succinate-based linker described above (the first of the two linkers depicted above) have the disadvantage that photocleavage of the linker does not usually produce free nucleotides in a single reaction step, but rather produces succinyl-functionalized nucleotides. Similarly, carbonate-containing linkers such as the second of the two linkers depicted above also produce carbonate-functionalized nucleotides. Further reaction steps are then required to obtain the free (poly)nucleotide, adding to the complexity, cost and time required in the synthesis process. Harsh reaction conditions are required to remove the succinyl or carbonate group from the succinyl- or carbonate-functionalized nucleotide. As a result, the photocleavable linkers depicted above have similar disadvantages to the succinyl-based linkers described above, since the use of harsh reaction conditions increases the complexity of the equipment setup required for the synthesis reaction and may also limit the range of polynucleotides that can be synthesized. For example, it may not be possible to generate (poly)nucleotides with protected nucleobases, since the protecting groups of the nucleobases may be at least partially removed during removal of the succinyl or carbonate group.

[0020] Furthermore, both the ester and carbonate motifs used in photocleavable linkers such as those depicted above are not stable under normal hydrolysis conditions, which is problematic as will be explained below.

[0021] More specifically, during polynucleotide synthesis, it is common to use protecting groups to protect various chemical groups present in the nucleosides, nucleotides and / or polynucleotides involved in the synthesis. In particular, protecting groups are often present on the nucleic acid bases and / or phosphorus-based bonds. These protecting groups are usually removed under hydrolysis conditions.

[0022] In many cases, it is desirable to remove the protecting groups present on the nucleobases and / or phosphodiester backbone of the synthesized polynucleotide before the polynucleotide is cleaved from its solid support, i.e. while the polynucleotide is retained on the support. This facilitates thorough washing of the by-products of the deprotection reaction, such as the removed protecting groups. However, as explained above, photocleavable linkers based on ester or carbonate bridges are not stable under the usual deprotection conditions used to remove protecting groups from synthesized polynucleotides. This creates problems during the synthesis process. If deprotection is performed before photocleavage, the photocleavable nature of the linker is largely unnecessary, since the polynucleotide product will necessarily be cleaved from the support, which would then require purification from the deprotected product. If photocleavage is performed before the deprotection step (indeed, if necessary), downstream processing of the cleaved polynucleotide is still required, and solution-based purification of the desired polynucleotide from the cleaved protecting groups is still required. Thus, there is a need for improvement in this area.

[0023] Another concern is that the saccharide ring of a nucleotide usually has multiple hydroxyl group substituents. In DNA nucleotides, hydroxyl groups are present at the 3' and 5' positions, whereas in RNA nucleotides, hydroxyl groups are present at the 2', 3' and 5' positions. In solid-state polynucleotide synthesis, it is usually preferred that the polynucleotide / initial monomer unit is attached to the solid support via its 3' hydroxyl group. This is because, although polynucleotide synthesis can be performed in either the 3' to 5' or 5' to 3' direction, it is often preferred to perform polynucleotide synthesis in the 3' to 5' direction. One reason that polynucleotide synthesis in the 3' to 5' direction is preferred in some cases is that the monomer units associated with 3' to 5' synthesis tend to be less expensive. Another reason is that the variety of readily available monomer units is higher in the 3' to 5' direction than in the 5' to 3' direction. For example, a wide variety of unnatural monomers, modified monomers, suitable for 3' to 5' synthesis are commercially available, which is not the case for 5' to 3' synthesis. A drawback of some known linkers is that during the synthesis of the linker between the nucleotide and the solid support, a significant amount of the linker may be bonded to the 2' hydroxyl group and / or the 5' hydroxyl group, instead of preferentially bonded to the 3' hydroxyl group. There is also a need for improvement in this area.

[0024] Photocleavable groups have also been used in RNA synthesis, e.g., to temporarily attach detectable labels to the terminal residues of synthesized strands to ensure strand synthesis. For example, photocleavable linkers based on acetal bridges with nitrobenzene groups conjugated with alkyl diamide linkers have been described to selectively link the terminal 2'-OH groups of RNA polymers with biotin for binding to streptavidin-functionalized beads to ensure successful strand synthesis.

[0025] The use of chemical linkers to connect a first (poly)nucleotide to a second (poly)nucleotide is also known. When the linker is attached to the 3' end of one (poly)nucleotide and the 5' end of the other (poly)nucleotide, the overall structure can be seen as a single polynucleotide chain with an interrupted linker unit. When the interrupted linker unit is photosensitive, it provides an approach to cleave the polynucleotide chain in situ into two separate (poly)nucleotide components using photoexcitation. Such linkers are also called "caged strand breakers".

[0026] One example of a known caged strand breaker is "caged strand breaker II CEP." The structure of "caged strand breaker II CEP" in its unbound form is shown below:

[0027] [ka]

[0028] When this linker is introduced into a polynucleotide sequence during polynucleotide synthesis, it is possible to produce a polynucleotide chain with a photolabile interrupted linker having the structure shown below:

[0029] [ka]

[0030] While linkers such as "caged strand breaker II CEP" allow for in situ photochemical cleavage of polynucleotide strands, significant problems remain.

[0031] For example, it may be impractical to use a phosphoramidite nucleotide monomer to introduce the first 3' nucleotide into the "caged strand breaker II CEP" because the resulting phosphodiester bond is anionic and therefore less susceptible to removal during photocleavage of the "caged strand breaker II CEP" linker. Therefore, it may be essential to introduce the first 3' nucleotide using a methyl nucleotide phosphonamidite monomer, which results in a neutrally charged methyl phosphonate bond.

[0032] Phosphoramidite monomers are much cheaper than methylphosphonamidite monomers, and a much wider range of phosphoramidite monomers, including non-natural nucleotide phosphoramidite monomers and modified nucleotide phosphoramidite monomers, are commercially available. Therefore, it would be desirable to be able to use phosphoramidite monomers to introduce the first 3' nucleotide into the linker, and there is a need for improvements in this area.

[0033] Thus, it is desirable to provide a linker for attaching a polynucleotide or monomeric unit thereof to a solid support that (i) can be cleaved under mild conditions orthogonal to those typically used in polynucleotide synthesis, (ii) is cleaved in a single reaction step to provide a free polynucleotide with minimal reaction by-products, (iii) is stable under reaction conditions typically used to deprotect synthesized polynucleotides, and (iv) preferentially binds to the 3' hydroxyl group of the polynucleotide / monomer unit.

[0034] It may also be desirable to provide a linker that joins the first polynucleotide to a second polynucleotide that is compatible with the phosphoramidite monomer.

[0035] The present invention aims to address some or all of these concerns. overview The present inventors have developed compounds of formula (I) as nucleosides, nucleotides, polynucleotides, and their derivatives and analogs having excellent photocleavability suitable for use in solid-phase polynucleotide synthesis. In particular, the linkers present in these compounds typically have each of the desirable advantages (i) to (iv) described above.

[0036] Accordingly, the present invention provides a method for producing a polynucleotide or an analog or derivative thereof, comprising the steps of: (i) (A) a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support.

[0037] [ka]

[0038] (In the formula, - (N) is a nucleoside, nucleotide, polynucleotide, or a derivative or analog thereof; Q is an oxygen atom or a sulfur atom, - R 2 are each independently selected from hydrogen, methyl, ethyl, C1-C2 haloalkyl, and halogen groups; - R 3 is methyl, ethyl or C1-C2 haloalkyl; - (A) is a 2-nitrobenzyl group, and the 2-nitrobenzyl group is selected from halogen, optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b and -NHC(O)R b wherein R aare each independently selected from hydrogen, optionally substituted C1-C2 alkyl, and optionally substituted C1-C2 alkoxyl; R b are each independently selected from hydrogen and an optionally substituted C1-C4 alkyl group; (ii) extending the length of the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, by introducing one or more additional nucleoside units, nucleotide units, and / or analogs or derivatives thereof; (iii) cleaving the resulting polynucleotide from the solid support by irradiating the compound of formula (I); The present invention provides a method comprising:

[0039] In some embodiments, Q is an oxygen atom, and / or R 2 are each independently selected from hydrogen, methyl, C1 fluoroalkyl and fluorine, preferably R 2 are each hydrogen, and / or R 3 is methyl, ethyl or C1-C2 fluoroalkyl, preferably R 3 is methyl.

[0040] In some embodiments, (A) is covalently attached to a linking group, which is covalently attached to a solid support.

[0041] In some embodiments, (A) is represented by formula (A-1) or formula (A-2):

[0042] [ka]

[0043] (In the formula, R 4 , R 5 , R 6 and R 7 each independently represents hydrogen, halogen, an optionally substituted C1-C4 alkyl, -ORa , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b and -NHC(O)R b where R a and R b is as defined herein, L is a linking group, and Z is a solid support.

[0044] In some embodiments, R 4 , R 5 , R 6 and R 7 each independently represents hydrogen, fluorine, an optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -OC(O)R b and -NHC(O)R b Selected from R a and R b is as defined herein, preferably R 4 , R 5 , R 6 and R 7 are each hydrogen or methoxy.

[0045] In some embodiments, the linking group is a C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 alkynylene groups and / or nucleotides or polynucleotides, wherein the alkylene, alkenylene or alkynylene groups are each independently selected from the group consisting of heteroatoms; phosphite groups; phosphate groups; carbonyl groups; C6-C 10 Aryl group; C5~C 10The linking group may be interrupted and / or terminated with one or more groups selected from a carbocyclyl group, a 5-10 membered heteroaryl group, and a 5-10 membered saturated or partially unsaturated heterocyclic group, and may be further substituted. In some embodiments, the linking group is attached to the solid support via a phosphoramidite group.

[0046] In some embodiments, the solid support comprises particles having a diameter of about 10 to about 1000 μm.

[0047] In some embodiments, the solid support comprises glass, silica, ceramic, or a polymeric resin.

[0048] In some embodiments, (N) is attached to adjacent oxygen atoms of formula (I) at the 3' or 5' position of the nucleoside, nucleotide, polynucleotide, or derivative or analog thereof.

[0049] In some embodiments, (N) is represented by formula (N-1) or formula (N-2):

[0050] [ka]

[0051] (wherein X is an oxygen atom, a nitrogen atom, a sulfur atom or -C(R a R a )-and;R 1 and R DR each independently represents hydrogen, halogen, an optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a and -C(O)R b where R a and R b is as defined herein, and R DR -ORa If R a Groups and R 1 One of the groups may be linked together to form a bridging motif, R P is hydrogen, a hydroxyl protecting group, a phosphoryl group or its salt or acid, a diphosphoryl group (-P(O2 - )-OP(O3 - )) or its salt or acid, a triphosphoryl group (-P(O2 - )-P(O2 - )-OP(O3 - )) or a salt or acid thereof, or a phosphorus-based bond to a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof, R B is an optionally substituted natural or non-natural nucleobase or a derivative or analogue thereof, and the wavy line indicates the point of attachment to the compound of formula (I).

[0052] In some embodiments, X is an oxygen atom and / or R 1 are each independently selected from hydrogen, halogen, C1-C2 alkyl, C1-C2 haloalkyl, and C1-C2 alkoxyl, preferably R 1 are each hydrogen, and / or R DR is hydrogen or halogen, preferably R DR is hydrogen, and / or R P is a hydrogen or hydroxyl protecting group.

[0053] In some embodiments, step (i) comprises: (a) A compound of formula (Ia)

[0054] [ka]

[0055] (In the formula, Q, R 2 , R 3 and (A) are as defined herein; R S is C1-C4 alkyl) To prepare the following: (b) reacting a compound of formula (Ia) with a nucleoside, nucleotide, polynucleotide or a derivative or analogue thereof, preferably a nucleoside, nucleotide, polynucleotide or a derivative or analogue thereof of formula (N-1) or formula (N-2) as defined herein, to form a compound of formula (I); Includes.

[0056] In some embodiments, step (i) comprises reacting a compound of formula (I) or formula (Ia)

[0057] [ka]

[0058] (In the formula, (N), Q, R 2 , R 3 , R S and (A) is as defined herein. to a solid support.

[0059] In some embodiments, extending the chain length in step (ii) comprises contacting the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof with a nucleoside or derivative thereof to form a phosphorus-based bond one or more times.

[0060] In some embodiments, the chain extending in step (ii) comprises: (a) providing a nucleoside having (i) a phosphoramidite group at the 3' position and a hydroxyl protecting group at the 5' position, or (ii) a phosphoramidite group at the 5' position and a hydroxyl protecting group at the 3' position; (b) activating the nucleoside phosphoramidite of step (a); (c) contacting the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof with an activated nucleoside phosphoramidite to form a phosphite triester bond; (d) oxidizing the phosphite triester bond to form a phosphotriester bond; (e) removing the hydroxyl protecting group from the terminal nucleoside of the resulting support-bound polynucleotide; The method includes carrying out the above steps (a) to (e) one or more times.

[0061] In some embodiments, after step (ii) and before step (iii), the method further comprises one or more steps of deprotecting the support-bound polynucleotide.

[0062] In some embodiments, step (iii) comprises photoirradiating the compound of Formula (I) with UV light having a wavelength of about 300 to about 500 nm.

[0063] Also provided herein is a support-bound nucleoside, nucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support at (A).

[0064] [ka]

[0065] (In the formula, Q, R 2 , R 3 and (A) is as defined herein, and (N) is a nucleoside, nucleotide or a derivative or analogue thereof, preferably a nucleoside, nucleotide or a derivative or analogue thereof represented by formula (N-1) or formula (N-2) as defined herein.

[0066] Also provided herein is a compound of formula (I)

[0067] [ka]

[0068] (In the formula, Q, R 2 , R 3 and (A) is as defined herein, (N) is represented by formula (N-1) or formula (N-2) as defined herein, and (A) is optionally bound to a solid support. Thus, the compound may or may not be in the presence of a solid support.

[0069] Also provided herein is a support-bound compound of formula (Ia), which is bound to a solid support at (A).

[0070] [ka]

[0071] (In the formula, Q, R 2 , R 3 and (A) are as defined herein). Also provided herein is a method for producing a support-bound nucleoside, nucleotide, or derivative or analogue thereof, comprising: (a) providing a compound of formula (Ia) as defined herein (A) that is bound to a solid support; and (b) reacting the compound of formula (Ia) with a nucleoside, nucleotide, or derivative or analogue thereof to produce the support-bound nucleoside, nucleotide, or derivative or analogue thereof.

[0072] Also provided herein is a solid support column for solid phase polynucleotide synthesis, comprising a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support at (A).

[0073] [ka]

[0074] (In the formula, (N), Q, R 2 , R 3 and (A) are as defined herein). Also provided is the use of a support-bound nucleoside, nucleotide or derivative or analogue thereof, a compound of formula (I) as defined herein, or a compound of formula (Ia) as defined herein, for the synthesis of a polynucleotide or a derivative or analogue thereof.

[0075] Further provided is a polynucleotide or a derivative or analogue thereof obtained by the method defined herein.

[0076] Detailed Description of the Invention definition The term "nucleosides, nucleotides, polynucleotides and derivatives and analogs thereof" includes nucleosides; derivatives of nucleosides; analogs of nucleosides; nucleotides; derivatives of nucleotides; analogs of nucleotides; polynucleotides; derivatives of polynucleotides; and analogs of polynucleotides.

[0077] As used herein, a "derivative" of a nucleoside, nucleotide, or polynucleotide is a modified form of the nucleoside, nucleotide, or polynucleotide that contains one or more chemical modifications to the phosphate backbone, sugar ring, or nucleobase of the DNA or RNA. A derivative of a nucleoside may thus be a chemically modified nucleoside, as described in more detail herein.

[0078] As used herein, a "nucleoside, nucleotide or polynucleotide" analog is a compound that is structurally similar to a nucleotide, such as DNA and RNA, but contains modifications at one or more positions, such as the phosphate backbone, sugar ring or nucleobase of the DNA or RNA. Nucleotide analogs include peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), and other synthetic polymers with nucleotide side chains. Preferably, the nucleotide analogs include peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and locked nucleic acid (LNA).

[0079] Unless otherwise specified, the term "nucleoside" includes protected nucleosides, i.e., nucleosides modified to include protecting groups in their chemical structure. Unless otherwise specified, the term "nucleotide" includes protected nucleotides, i.e., nucleotides modified to include protecting groups in their chemical structure. Unless otherwise specified, the term "polynucleotide" includes protected polynucleotides, i.e., polynucleotides modified to include protecting groups in their chemical structure. The above protecting groups are usually distinguished from modifications that can be made to nucleotide derivatives for functional reasons, e.g., to change the properties of the nucleotide or polynucleotide.

[0080] As used herein and unless otherwise specified, the term "protecting group" refers to any suitable protecting group known in the art. Various protecting groups are suitable for protecting various chemical groups. For example, as used herein and unless otherwise specified, the term "hydroxyl protecting group" refers to any suitable protecting group known in the art for protecting hydroxyl groups. Similarly, as used herein and unless otherwise specified, the term "amine protecting group" refers to any suitable protecting group known in the art for protecting amine groups. Further, as used herein and unless otherwise specified, the terms "phosphate protecting group" and "phosphite protecting group" refer to any suitable protecting group known in the art for protecting phosphate and phosphite groups, respectively.

[0081] Suitable hydroxyl protecting groups include acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), 4,4′-dimethoxytrityl (DMT), methoxymethyl ether (MOM), 4-monomethoxytrityl (MMT), levulinyl, 9-fluorenylmethyloxycarbonyl (Fmoc), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), silyl ethers such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS), methyl ether, ethoxyethyl ether, allyl ether and tert-butyl ether.

[0082] Suitable amine protecting groups include carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (Moz), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), trifluoroacetyl, benzoyl (Bz), benzyl (Bn), benzylidene, trityl (Tr), carbamate, phthalimide, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), trichloroethyl chloroformate (Troc), tosyl (Ts), nosyl, and 2-nitrophenylsulfenyl (Nps).

[0083] Suitable phosphate and phosphite protecting groups are alkyl groups, e.g. halogens, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b , -NHC(O)R b and -CN, in particular methyl, ethyl and 2-cyanoethyl, preferably 2-cyanoethyl; o-chlorophenyl; and p-chlorophenyl.

[0084] As used herein, an alkyl group may be linear, branched or cyclic, but is preferably linear. 20 The alkyl group is a linear or branched alkyl group containing 1 to 20 carbon atoms. 20 The alkyl group is usually C1-C 10It is an alkyl group, for example a C1-C6 alkyl group, a C1-C5 alkyl group or a C1-C4 alkyl group. Suitable C1-C4 alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl and tert-butyl. The C1-C4 alkyl group is preferably a C1-C3 alkyl group, more preferably ethyl or methyl. Unless otherwise specified, the alkyl group may be unsubstituted or substituted as described herein. For the avoidance of doubt, when two alkyl groups are present, the alkyl groups may be the same or different.

[0085] As used herein, an alkenyl group may be linear, branched or cyclic, but is preferably linear. An alkenyl group has one or more, for example one or two, usually one double bond. C2-C 20 The alkenyl group is a linear or branched alkenyl group containing 2 to 20 carbon atoms. 20 Alkenyl groups are usually C2-C 10 An alkenyl group, for example a C2-C6 alkenyl group, a C2-C5 alkenyl group or a C2-C4 alkenyl group. The C2-C4 alkenyl group is preferably a C2-C3 alkenyl group. C2-C4 alkenyl groups include ethenyl, propenyl and butenyl. Unless otherwise specified, an alkenyl group may be unsubstituted or substituted as described herein. For the avoidance of doubt, when two alkenyl groups are present, said alkenyl groups may be the same or different.

[0086] As used herein, an alkynyl group may be linear, branched or cyclic, but is preferably linear. An alkenyl group has one or more, for example one or two, usually one triple bond. C2-C 20 The alkynyl group is a linear or branched alkynyl group containing 2 to 20 carbon atoms. 20 Alkynyl groups are usually C2-C 10An alkynyl group, for example a C2-C6 alkynyl group, a C2-C5 alkynyl group or a C2-C4 alkynyl group. The C2-C4 alkynyl group is preferably a C2-C3 alkynyl group. C2-C4 alkynyl groups include ethynyl, propynyl and butynyl. Unless otherwise specified, an alkynyl group may be unsubstituted or substituted as described herein. For the avoidance of doubt, when two alkynyl groups are present, said alkynyl groups may be the same or different.

[0087] As used herein, an alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from an alkane. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms. As used herein, an alkylene group may be linear, branched or cyclic, but is preferably linear. C1-C 20 The alkylene group is a linear or branched alkylene group containing 1 to 20 carbon atoms. A C1-C2 alkylene group is usually a C1-C 10 It is an alkylene group, for example a C1-C6 alkylene group, a C1-C5 alkylene group or a C1-C4 alkylene group. Suitable C1-C4 alkylene groups include methylene, ethylene, n-propylene, i-propylene, n-butylene, sec-butylene and tert-butylene. The C1-C4 alkylene group is preferably a C1-C3 alkylene group, more preferably ethylene or methylene. Unless otherwise specified, an alkylene group may be unsubstituted or substituted as described herein. For the avoidance of doubt, when two alkylene groups are present, the alkylene groups may be the same or different.

[0088] As used herein, an alkenylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from an alkene. The two hydrogen atoms may be removed from the same carbon atom or from different carbon atoms. As used herein, an alkenylene group may be linear, branched or cyclic, but is preferably linear. An alkenylene group has one or more, for example one or two, typically one double bond. C2-C 20 The alkenylene group is a linear or branched alkenylene group containing 2 to 20 carbon atoms. 20 The alkenylene group is usually C2-C 10 An alkenylene group, for example a C2-C6 alkenylene group, a C2-C5 alkenylene group or a C2-C4 alkenylene group. The C2-C4 alkenylene group is preferably a C2-C3 alkenylene group. C2-C alkenylene groups include ethenylene, propenylene and butenylene. Unless otherwise specified, an alkenylene group may be unsubstituted or substituted as described herein. For the avoidance of doubt, when two alkenylene groups are present, the alkenylene groups may be the same or different.

[0089] As used herein, an alkynylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from an alkyne. As used herein, an alkynylene group may be linear, branched or cyclic, but is preferably linear. An alkynylene group has one or more, for example one or two, usually one triple bond. C2-C 20 The alkynylene group is a linear or branched alkynylene group containing 2 to 20 carbon atoms. 20 The alkynylene group is usually C2-C 10An alkynylene group, for example a C2-C6 alkynylene group, a C2-C5 alkynylene group or a C2-C4 alkynylene group. The C2-C4 alkynylene group is preferably a C2-C3 alkynylene group. C2-C4 alkynylene groups include ethynylene, propynylene and butynylene. Unless otherwise specified, an alkynylene group may be unsubstituted or substituted as described herein. For the avoidance of doubt, when two alkynylene groups are present, said alkynylene groups may be the same or different.

[0090] As used herein, a halogen is typically chlorine, fluorine, bromine or iodine, preferably chlorine, fluorine or bromine, more preferably chlorine or fluorine.

[0091] As used herein, C6-C 10 The aryl group is an aryl group having 6 to 10 carbon atoms, such as phenyl or naphthyl, preferably phenyl. The aryl group or moiety may be substituted or unsubstituted as described herein.

[0092] As used herein, C5-C 10 The carbocyclyl group is C5, C6, C7, C8, C9 or C 10 and preferably cyclopentyl or cyclohexyl. Typically, the cycloalkyl group is substituted with up to three substituents, for example one or two substituents, or unsubstituted.

[0093] As used herein, a 5-10 membered heteroaryl group is a 5-10 membered ring system in which the ring is fully unsaturated and aromatic and contains at least one heteroatom. Typically, the ring contains up to 3 or 4 heteroatoms, for example 1 or 2 heteroatoms, selected from O, N and S. Thus, a 5-10 membered heteroaryl group is typically a 5-10 membered aromatic ring containing 1, 2 or 3 heteroatoms selected from O, N and S. Preferably, the heteroatoms are selected from O and N. Suitable heteroaryl groups include, for example: Monocyclic 5- to 7-membered heteroaryl rings, such as furanyl, oxepinyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, azepinyl, thiophenyl, oxepinyl, and thiepinyl; and Bicyclic 8-10 membered heteroaryl rings, such as benzofuranyl, indolyl, isoindolyl, indolizinyl, indazolyl, benzimidazolyl, azaindolyl, azaidazolyl, purinyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, phthalazinyl, quinazolinyl, cinnolinyl, naphthyridinyl, pteridinyl and benzothiophenyl, preferably benzofuranyl, indolyl, isoindolyl, quinolinyl and isoquinolinyl. Examples include:

[0094] Preferably, the 5-10 membered heteroaryl group is a monocyclic 5-7 membered heteroaryl ring selected from furanyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, isoxazolyl, pyridinyl, pyridazinyl, pyrimidinyl and pyrazinyl.

[0095] As used herein, a 5-10 membered saturated heterocyclic group is a 5-10 membered saturated ring system, the ring containing at least one heteroatom. Typically, the ring contains up to 3 or 4 heteroatoms, such as 1 or 2 heteroatoms, selected from O, S and N. Thus, a 5-10 membered saturated heterocyclic group is a 5-10 membered ring, usually containing 1, 2 or 3 heteroatoms selected from O, S and N. Suitable saturated heterocyclic groups include, for example, monocyclic 5- to 8-membered saturated rings, more preferably 5- to 7-membered rings, such as tetrahydrofuranyl, piperidinyl, oxazolidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, dioxolanyl, piperidonyl, azepanyl, oxepanyl, piperazinyl, tetrahydropyranyl, and 1,4-diazepanyl, more preferably pyrrolidinyl, morpholinyl, piperazinyl, tetrahydropyranyl, piperidinyl, azepanyl, and 1,4-diazepanyl.

[0096] As used herein, a 5-10 membered partially unsaturated heterocyclic group is a 5-10 membered ring system in which the ring contains at least one unsaturated bond and at least one heteroatom, but the ring is not fully unsaturated or aromatic. Typically, the ring contains up to 3 or 4 heteroatoms, such as 1 or 2 heteroatoms, selected from O, N and S. Thus, a 5-10 membered partially unsaturated heterocyclic group is typically a 5-10 membered ring containing 1, 2 or 3 heteroatoms selected from O, N and S. Preferably, the heteroatoms are selected from O and N. Suitable partially unsaturated heterocyclic groups include, for example: monocyclic partially unsaturated 5- to 7-membered heterocyclic rings, such as dihydrofuranyl, pyranyl, dihydropyranyl, dioxinyl, dihydrooxepinyl, tetrahydrooxepinyl, pyrrolinyl, pyrazolinyl, imidazolinyl, dihydrooxazolyl, dihydroisoxazolyl, dihydrothiazolyl, dihydroisothiazolyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyridazinyl, tetrahydropyridazinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, dihydropyrazinyl, tetrahydropyrazinyl, oxazinyl, dihydrooxazinyl, thiazinyl, dihydrothiazinyl, dihydroazepinyl, tetrahydroazepinyl, dihydrothiophenyl, thiopyranyl, dihydrothiopyranyl, dihydrothiepinyl, and tetrahydrothiepinyl; and Bicyclic partially unsaturated 8-10 membered heterocyclic rings, such as dihydrobenzofuranyl, dihydroisobenzofuranyl, benzopyranyl, dihydrobenzopyranyl, benzodioxolyl, indolinyl, isoindolinyl, dihydroquinolinyl, tetrahydroquinolinyl, benzoxazinyl, dihydrobenzothiophenyl and benzodithiol; preferably dihydrobenzofuranyl, benzopyranyl, dihydrobenzopyranyl, benzodioxolyl, indolinyl, isoindolinyl, dihydroquinolinyl and tetrahydroquinolinyl. Examples include:

[0097] Preferably, the 5-10 membered partially unsaturated heterocyclic group is a monocyclic partially unsaturated 5-7 membered ring selected from dihydrofuranyl, pyranyl, pyrrolinyl and oxazinyl.

[0098] The heterocyclic and / or heteroaryl groups may be substituted or unsubstituted. Each ring atom may be unsubstituted or may have one or two substituents. The nitrogen atom may be disubstituted to be a positively charged heteroatom. The sulfur atom may be substituted to be a positively charged heteroatom. Typically, a heterocyclic or heteroaryl group has up to three substituents, for example one or two substituents. The heterocyclic or heteroaryl ring may be connected to the rest of the molecule by a bond at any available ring position of the heterocyclic or heteroaryl ring.

[0099] As used herein, an optionally substituted group, unless otherwise specified, includes halogens such as chlorine and / or fluorine, cyano groups, -OR x , -SR x , -NR x R x , -C(O)OR x , -C(O)NR x R x , -C(O)R x and may be substituted with suitable substituents, which may include C1-C4 alkyl groups such as methyl and / or ethyl, the C1-C4 alkyl substituents being themselves either unsubstituted or substituted with 1 to 3 halogen atoms. x are each independently selected from hydrogen and a C1-C4 alkyl group that is unsubstituted or substituted with one, two or three halogen groups. The optional substituents are preferably hydroxyl groups, halogens such as chlorine or fluorine, or C1-C4 alkyl groups such as methyl or ethyl.

[0100] Unless otherwise specified, the above also includes known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to a carboxylic acid, carboxyl group, or carboxyl group (-COOH) also includes its anionic (carboxylate) form (-COO - ), a salt or solvate, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N+ HR 1 R 2 ), a salt or solvate of an amino group, such as a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxy or hydroxyl group (-OH) also includes its anionic form (-O - ), salts or solvates, as well as conventional protected forms. Salts include salts formed with acids and bases. Suitable acids include both inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid, and organic acids, such as oxalic acid, citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid. Suitable bases include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides, as well as organic bases, such as alkylamines, aralkylamines, and heterocyclic amines. Hydrochloride and acetate salts, especially hydrochloride salts, are preferred. When intended for use in pharmaceutical applications, the salts of the compounds provided herein may preferably be pharma- ceutically acceptable salts.

[0101] A compound may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis and trans forms; E and Z forms; c, t, and r forms; endo and exo forms; R, S, and meso forms; D and L forms; d and l forms; (+) and (-) forms; keto, enol, and enolate forms; syn and anti forms; synclinal and anticlinal forms; α and β forms; axial and equatorial forms; boat, chair, twisted, envelope, and half-chair forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms"). Unless otherwise specified, a reference to a particular compound includes (wholly or partially) all such isomeric forms, including its racemic and other mixtures. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic methods) of such isomeric forms are either known in the art or readily obtainable by employing known methods in known procedures.

[0102] Unless otherwise specified, a reference to a particular compound or complex also includes ionic, salt, solvated and protected forms.

[0103] Photocleavable Linker In some embodiments, the present invention provides a method for producing a polynucleotide, the method comprising providing a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I) as defined herein and is bound to a solid support as described herein in group (A) of formula (I).

[0104] [ka]

[0105] (In the formula, (N), (A), Q, R 2 and R 3as specified above and below) Includes.

[0106] Also provided is the compound of formula (I) itself as well as derivatives thereof, such as the compound of formula (Ia) described herein. The compounds of formula (I) have utility in the preparation of support-bound compounds for use in the methods provided, as well as other uses as described herein.

[0107] The compound of formula (I) is a nucleoside, nucleotide, polynucleotide or derivative or analog thereof linked to a photocleavable linker. In formula (I) and related formulae shown herein, (N) is a nucleoside, nucleotide, polynucleotide or derivative or analog thereof, and the remaining portions of formula (I) and related formulae form a photocleavable linker that cleaves upon sufficient photoexcitation. Photocleavage is described in more detail herein. When this photocleavable linker cleaves upon photoexcitation, (N) is cleaved from the photocleavable linker such that the immediate product of cleavage is a nucleoside, nucleotide, polynucleotide or derivative or analog thereof that has a hydroxyl group instead of the photocleavable linker. Thus, using photoexcitation, the compound of formula (I) can be converted to a compound represented by (N)-OH in a single reaction step.

[0108] More specifically, (A) is a 2-nitrobenzyl group that facilitates cleavage of the linker during photoexcitation. Without being bound by theory, the inventors believe that the mechanism of this cleavage is illustrated by one specific example below.

[0109] [ka]

[0110] where HB is an acid and B is a base. However, the invention is not limited to this particular compound or to methods using this particular compound.

[0111] Those skilled in the art will appreciate that the compounds of formula (I) advantageously contain a photocleavable linker that can be cleaved using photoexcitation under mild conditions orthogonal to those typically used in polynucleotide synthesis.The photocleavable linker is advantageously cleaved in a single reaction step, usually a concerted reaction step, to provide a free nucleoside, nucleotide, polynucleotide, or derivative or analog thereof.Furthermore, the ketone by-product is volatile and therefore easily removed.

[0112] The photocleavable linker present in the compound of formula (I) does not contain hydrolyzable motifs such as ester or carbonate groups.Therefore, it is usually stable under the reaction conditions usually used to deprotect synthesized polynucleotides, for example, under the deprotection conditions used to remove hydrolyzable protecting groups from the protected nucleobases present in the synthesized polynucleotides.For example, the photocleavable linker is usually stable to concentrated ammonium hydroxide and / or ammonia.This allows the advantageous removal of various protecting groups from the synthesized polynucleotides before the synthesized polynucleotides are cleaved from their solid support, and allows the easy isolation of the deprotected polynucleotides.

[0113] The inventors have found that during the synthesis of the photocleavable linker present in the compound of formula (I), the linker advantageously preferentially binds to the nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof at the 3' hydroxyl group of said nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof. For example, when linked to a (poly)nucleotide having a free 3'-OH group and a free 5'-OH group, the linker usually binds more easily to the 3' hydroxyl group than to the 5'-OH group. When linked to a (poly)nucleotide having a free 2'-OH group, a free 3'-OH group, and a free 5'-OH group, the linker usually binds more easily to the 3' hydroxyl group than to the 2'-OH or 5'-OH group. Preferably, in the present invention, the linker does not bind to the 2'-hydroxyl group. In this way, it is easier to prepare the compound of formula (I) in which the nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof is bound to adjacent oxygen atoms at the 3' position. The above compounds are particularly useful for carrying out polynucleotide synthesis in the 3' to 5' direction. However, those skilled in the art should understand that, if required, the linker can still be effectively attached to, for example, the 5'-hydroxyl group. This is an operational parameter of the present invention. For example, when linking to a (poly)nucleotide that has a free 5'-OH group but no free 3'-OH group, the linker can be attached to the 5'-hydroxyl group.

[0114] The methods and compounds of the invention are described in more detail below. Formula (I) The present invention provides methods that include the use of a compound of formula (I). The present invention also provides a compound of formula (I) itself. In some embodiments provided herein, the compound of formula (I) is bound to a solid support at position (A).

[0115] [ka]

[0116] In the formula, Q is an oxygen atom or a sulfur atom. Preferably, Q is an oxygen atom. R 2 are each independently selected from hydrogen, methyl, ethyl, C1-C2 haloalkyl and halogen groups. 2 are each independently hydrogen, methyl, C1 fluoroalkyl or fluorine. More preferably, R 2 are each independently hydrogen or methyl. More preferably, R 2 are each hydrogen. This allows for easier synthesis of the compounds of formula (I).

[0117] R 2 The groups may be the same or different. Preferably, R 2 The groups are identical. Preferably, one R 2 The group is H or methyl, and the other R 2 The group is H or methyl. More preferably, one R 2 The group is H, and the other R 2 The groups are H or methyl. Even more preferably, both R 2 The group is H.

[0118] R 3 is methyl, ethyl or C1-C2 haloalkyl. Preferably, R 3 is methyl, ethyl or C1-C2 fluoroalkyl. More preferably, R 3 is methyl or ethyl; or R 3 may be methyl or CF. Most preferably, R 3 is methyl. 3 is one of these groups, the linker connecting (N) and (A) may be more easily cleaved using photoexcitation and / or the linker is preferentially attached at the 3' position of the nucleoside of the sugar ring.

[0119] (A) is a 2-nitrobenzyl group, and the 2-nitrobenzyl group is selected from halogen, optionally substituted C1-C4 alkyl, -OR a , -SR a, -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b and -NHC(O)R b may be substituted with one, two or three groups independently selected from

[0120] R a are each independently selected from hydrogen, optionally substituted C1-C2 alkyl, and optionally substituted C1-C2 alkoxyl. a are each independently selected from hydrogen or methyl, more preferably R a are hydrogen.

[0121] R b are each independently selected from hydrogen and an optionally substituted C1-C4 alkyl group. b are each independently selected from hydrogen or methyl, more preferably R b are hydrogen.

[0122] As used herein, the term "2-nitrobenzyl group" refers to a 2-nitrobenzyl group in which a nitro group is -C(R 3 )--Q- refers to nitrobenzyl groups attached to adjacent carbon atoms of formula (I) such that they are in the 2-position relative to the moiety.

[0123] [ka]

[0124] That is, in the 2-nitrobenzyl group represented by (A), the nitro group is in the ortho position relative to adjacent carbon atoms in formula (I); that is, the nitro group is in the -C(R 3 )-It is in the ortho position to the carbon atom.

[0125] Those skilled in the art will understand that the 2-nitrobenzyl group represented by (A) can also be referred to as a 2-nitrophenyl group.

[0126] The nitrobenzene group (A) is attached to a solid support in some embodiments provided herein. The nitrobenzene group (A) may be further substituted or otherwise unsubstituted. When (A) is further substituted, it may be substituted with one, two or three substituents independently selected from, for example, electron donating groups. (A) may be selected from halogen, optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b and -NHC(O)R b and R a and R b is as defined above. Preferably, when (A) is substituted, it is C1-C4 alkyl, -OR a , -SR a , -NR a R a , -OC(O)R b and -NHC(O)R b and preferably one or two groups selected from the group consisting of: a , and -OC(O)R b , preferably methoxy. Most preferably, (A) is unsubstituted apart from the nitro group of the nitrobenzene and apart from any linking group attached to the solid support.

[0127] In formula (I), (N) is a nucleoside, nucleotide, polynucleotide, or derivative or analog thereof. Those skilled in the art will understand that during the method of the present invention, the chain length of the nucleoside, nucleotide, polynucleotide, or derivative or analog thereof bound to the support is extended. Thus, when the method of the present invention or a step thereof is repeated multiple times, the composition of (N) may change between cycles because the chain length is extended from the previous step.

[0128] The nucleoside, nucleotide, polynucleotide or derivative or analogue thereof represented by (N) may be selected from any suitable natural, non-natural, functionalized and / or modified nucleosides, nucleotides and polynucleotides. The above nucleoside, nucleotide and polynucleotide derivatives and analogues are known in the art.

[0129] For example, (N) includes nucleosides, nucleotides and polynucleotides in which any group is modified with a protecting group. In particular, it is common for the nucleosides, nucleotides and polynucleotides to have protecting groups on their saccharide rings, nucleobases and / or phosphorus-based bonds. Commonly used protecting groups are known in the art.

[0130] For example, amino groups present in nucleobases (e.g., adenine, guanine, cytosine and their derivatives and analogs) may be protected with amine protecting groups known in the art, including acyl groups, such as acetyl, oxyacetyl (including phenoxyacetyl and t-butylphenoxyacetyl), propionyl, isobutyryl, benzoyl and benzylidene. Formamidine groups, such as dimethylformamidine and di-N-butylformamidine groups, may also be used.

[0131] The phosphate linkages may be protected with a phosphate protecting group, e.g., ethyl or 2-cyanoethyl, and the phosphite linkages may be protected with a phosphite protecting group, e.g., ethyl or 2-cyanoethyl. For example, a protected phosphodiester backbone linkage may be present as a phosphotriester, e.g., 2-cyanoethyl phosphotriester.

[0132] (N) also includes derivatives and analogs of nucleosides, nucleotides and polynucleotides in which the saccharide ring is a non-natural saccharide, such as arabinose or "locked ribose," or saccharide ring analogs in which the oxygen atoms normally present in the saccharide ring are replaced with nitrogen atoms, sulfur atoms or methylene motifs, respectively, such as imino, thio or carba sugar rings. "Locked ribose" is a modified ribose moiety having an additional bridge connecting the 2' oxygen and the 4' carbon. An example of a locked ribose moiety is shown below:

[0133] [ka]

[0134] Nucleic acids containing a "locked ribose" saccharide ring are known as "locked nucleic acids" or LNA. LNA has high stability against enzymatic degradation and is a commercially important nucleic acid analogue of RNA and DNA.

[0135] (N) is preferably a nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, in which the saccharide ring is a ribose ring or a deoxyribose ring. Thus, (N) is preferably a ribonucleoside, ribonucleotide, polyribonucleotide, deoxyribonucleoside, deoxyribonucleotide, polydeoxyribonucleotide, or derivative or analog thereof. More preferably, the saccharide ring is a deoxyribose ring. Thus, (N) is more preferably a deoxyribonucleoside, deoxyribonucleotide, polydeoxyribonucleotide, or derivative or analog thereof.

[0136] (N) includes derivatives and analogs of polynucleotides in which the phosphorus-based linkages connecting the nucleoside monomer units are phosphodiester linkages, phosphotriester linkages such as alkyl phosphotriester linkages, alkyl phosphotriester linkages, phosphorothioate linkages, phosphorodithioate linkages, and / or alkyl phosphonate linkages such as methyl phosphonate linkages. In particular, the phosphoramidite method is a common method of polynucleotide synthesis that initially produces polynucleotides in which the phosphorus-based linkages are phosphite-triester linkages. The phosphite-triester linkages can also be oxidized to produce phosphotriester linkages, which can be deprotected to produce phosphodiester linkages.

[0137] (N) is preferably an oxygen atom adjacent to the 3'-position or 5'-position of formula (I) of the nucleoside, nucleotide, polynucleotide, or derivative or analog thereof represented by (N) (i.e., -OC(R 2 ) is linked to the 2-moiety. This advantageously facilitates nucleotide chain growth in either the 3' to 5' or 5' to 3' direction. More preferably, (N) is linked to adjacent oxygen atoms at the 3' position of formula (I). This facilitates nucleotide chain growth in the 3' to 5' direction, which is usually cheaper and more versatile.

[0138] Preferably, (N) is a moiety represented by formula (N-1) or formula (N-2). More preferably, (N) is represented by formula (N-1).

[0139] [ka]

[0140] The wavy line indicates the point of attachment to the compound of formula (I). 2 )2-part) represents a bond.

[0141] X is an oxygen atom, a nitrogen atom, a sulfur atom, or -C(R a R a )-. Preferably, X is an oxygen atom. When X is an oxygen atom, the synthesis of the compound of formula (I) is easier.

[0142] R 1 and R DR each independently represents hydrogen, halogen, an optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a and -C(O)R b Selected from R DR -OR a If R a Groups and R 1 One of the groups may be linked together to form a bridging motif.

[0143] Preferably, R 1 are each independently selected from hydrogen, halogen, C1-C2 alkyl, C1-C2 haloalkyl, and C1-C2 alkoxyl. More preferably, R 1 are each selected from hydrogen, fluorine, methyl and methoxy. More preferably, R 1 are hydrogen.

[0144] Preferably, R DR is hydrogen, halogen, C1-C2 alkyl or -OR a More preferably, R DR is hydrogen, fluorine, methyl or -OR a More preferably, R DR is hydrogen, hydroxyl or methoxy. Even more preferably, R DR is hydrogen or hydroxyl. In some cases, R DR is not hydroxyl. Most preferably, R DR is hydrogen.

[0145] R P is a hydrogen, a hydroxyl protecting group, a phosphoryl group (-P(O3 - )) or its salt or acid, diphosphoryl group (-P(O2 - )-OP(O3 - )) or its salt or acid, a triphosphoryl group (-P(O2 - )-P(O2 - )-OP(O3 - )) or a salt or acid thereof, or a phosphorus-based bond to a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof.

[0146] Preferably, R P is hydrogen, a hydroxyl protecting group, or a phosphotriester bond to a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof. More preferably, R P is hydrogen or a hydroxyl protecting group. More preferably, R P is hydrogen or a hydroxyl protecting group selected from 4,4'-dimethoxytrityl, 4-monomethoxytrityl, levulinyl and Fmoc groups. Even more preferably, R P is hydrogen or a 4,4'-dimethoxytrityl group. Most preferably, R P is the 4,4'-dimethoxytrityl group.

[0147] RP is a phosphorus-based bond with a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof, the phosphorus-based bond may be a phosphodiester bond, a phosphotriester bond such as an alkyl phosphotriester bond, a phosphorous-triester bond such as an alkyl phosphorous-triester bond, a phosphorothioate bond, a phosphorodithioate bond or an alkyl phosphonate bond such as a methyl phosphonate. Preferably, the phosphorus-based bond is a phosphodiester bond, a phosphotriester bond or a phosphorous-triester bond. More preferably, the phosphorus-based bond is represented by formula (P-1) or formula (P-2):

[0148] [ka]

[0149] The leftmost connection point of each of formula (P-1) and formula (P-2) is marked as connection point A, and is a connection point with the adjacent oxygen atom of formula (N-1) or formula (N-2). The rightmost connection point of each of formula (P-1) and formula (P-2) is marked as connection point B, and is a connection point with a nucleoside, nucleotide, or polynucleotide, or a derivative or analog thereof. R PP is hydrogen, a lone pair of electrons resulting in a negative charge, a phosphate protecting group or a phosphite protecting group. PP is hydrogen, a lone pair that gives a negative charge, o-chlorophenyl, p-chlorophenyl, or a halogen, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b , -NHC(O)R b and -CN. More preferably, R PPis hydrogen, a lone pair of electrons resulting in a negative charge, o-chlorophenyl, p-chlorophenyl, a methyl group, or a 2-cyanoethyl group. More preferably, R PP is the 2-cyanoethyl group.

[0150] R P is a phosphorus-based bond to a nucleoside, nucleotide or polynucleotide, or a derivative or analog thereof, the nucleoside, nucleotide or polynucleotide, or a derivative or analog thereof, may be any nucleoside, nucleotide or polynucleotide, or a derivative or analog thereof, which may be represented by (N), such that (N) may include multiple nucleosides, nucleotides or polynucleotides, or derivatives or analogs thereof linked by phosphorus-based bonds.

[0151] R B is an optionally substituted natural or non-natural nucleobase or a derivative or analogue thereof. Natural nucleobases include adenine, cytosine, guanine, thymine and uracil. Non-natural nucleobases are known in the art and include, for example, analogues of natural nucleobases such as 2-thiothymine, natural nucleobases such as isoguanine, isocyanine and 5-aza-7-deazaguanine, and other isomers of groups used in the art as synthetic nucleobases such as xanthine. For example, unnatural nucleobases include 2-thiothymine, isoguanine, isocytosine, 1-methylcytosine, 5-aza-7-deazaguanine, 6-amino-5-nitropyridin-2-one, 1-methylcytosine, xanthine, hypoxanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, 2,6-diaminopurine, 6,8-diaminopurine, fluorocytosine, fluorouracil, mercaptopurine, thioguanine, 5-hydroxymethyluracil, 5-carboxycytosine, and fluorescent nucleobases such as 2-aminopurine and tricyclic cytosine tC, tCO, and tCnitro.

[0152] Derivatives or analogues of natural and non-natural nucleobases include protected natural and non-natural nucleobases, ie, nucleobases that have been modified to include protecting groups in their chemical structure.

[0153] Preferably, R B is a protected natural or non-natural nucleobase. More preferably, R B is a protected natural nucleobase.

[0154] R DR -OR a and R a Groups and R 1 When one of the groups is linked together to form a bridging motif, the moiety of formula (N-1) or formula (N-2) is preferably a moiety of formula (N-L1) or formula (N-L2):

[0155] [ka]

[0156] (In the formula, X, R 1 Each of R P and R B as specified above). Binding to the support As explained above, methods are provided herein that include the use of a support-bound nucleoside, nucleotide, polynucleotide, or derivative thereof, which is a compound of formula (I) bound to a solid support. In some embodiments, the present invention further provides a support-bound compound of formula (I) or related formula, such as formula (Ia), described herein. The compound is bound to the solid support at the group (A) of the compound.

[0157] In the above embodiment in which the compound is bound to the support, preferably, (A) is chemically bound to the support. Preferably, (A) is bound to the solid support by a covalent bond. More preferably, (A) is bound to the linking group by a covalent bond, and the linking group is bound to the solid support by a covalent bond. This provides a compound (e.g., a compound of formula (I)) that is stably bound to the solid support.

[0158] In some embodiments, (A) is a moiety of formula (A'):

[0159] [ka]

[0160] In formula (A'), R 4 , R 5 , R 6 and R 7 is substituted with the moiety -LZ, where L is a linking group and Z is a solid support, 4 , R 5 , R 6 and R 7 The remaining groups are as follows for Formula (A-1) and Formula (A-2):

[0161] More preferably, the linking group is attached to the 4- or 5-position of the 2-nitrobenzyl group represented by (A) (as shown above, the nitro group is -C(R 3 ) when numbered as being at the 2-position relative to the-Q-moiety), that is, the linking group is preferably at the para or meta position relative to the nitro group of the 2-nitrobenzyl group.

[0162] More preferably, (A) is a moiety of formula (A-1) or formula (A-2).

[0163] [ka]

[0164] Preferably, (A) is a moiety of formula (A-1). In formula (A-1) and formula (A-2), R 4 , R 5 , R 6 and R 7 are each independently selected from hydrogen and a substituent which may be an electron donating group. 4 , R 5 , R 6 and R 7 are usually independently hydrogen, halogen, optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b and -NHC(O)R b Selected from R a and R b is as set out above.

[0165] Preferably, R 4 , R 5 , R 6 and R 7 each independently represents hydrogen, fluorine, an optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -OC(O)R b and -NHC(O)R b More preferably, R 4 , R 5 , R 6 and R 7 are each independently hydrogen, -OR a , -SR a , -NR a R a , -OC(O)R b and -NHC(O)R b Even more preferably, R 4 , R5 , R 6 and R 7 are each independently hydrogen, -OR a , and -OC(O)R b More preferably, R 4 , R 5 , R 6 and R 7 are each independently selected from hydrogen and methoxy. Without being bound by theory, the inventors believe that R 4 , R 5 , R 6 and R 7 It is believed that the linker is more susceptible to cleavage upon photoexcitation when one or more of R 4 , R 5 , R 6 and / or R 7 When is hydrogen, the compounds of formula (I) are easier to synthesise.

[0166] Preferably, R 4 , R 5 , R 6 and / or R 7 At least one of R is hydrogen. For example, 4 , R 5 , R 6 and / or R 7 Preferably, one, two or three of R 4 and R 7 One of the groups is hydrogen, and R 4 and R 7 The other is hydrogen, -OR a , and -OC(O)R b Preferably, R 5 or R 6 is hydrogen. For example, in some embodiments, R 4 and R 7 One of the groups is hydrogen, and R 4 and R 7 The other is hydrogen, -OR a , and -OC(O)R b Selected from R 5 or R6 is hydrogen or methoxy.

[0167] When the compound is linked to the solid support (Z) by a linking group (L), as in the above formulas (A'), (A-1) and (A-2), L is the linking group and Z is the solid support.

[0168] The linking group L may be any linking group suitable for linking a polynucleotide to a solid support. 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 is or comprises an alkynylene group and / or a nucleotide or polynucleotide, said alkylene, alkenylene or alkynylene group being selected from the group consisting of a heteroatom or other hetero group as described herein; a phosphite group; a phosphate group; a carbonyl group; a C6-C 10 Aryl group; C5~C 10 The linking group may be interrupted and / or terminated by one or more groups selected from a carbocyclyl group, a 5- to 10-membered heteroaryl group, and a 5- to 10-membered saturated or partially unsaturated heterocyclic group, and may be further substituted. Suitable heteroatoms and hetero groups are -O-, -S-, -SO-, -SO2-, -NR z -, -C(O)NR z Including R z is H or methyl, preferably H. An example of an alkylene group interrupted and / or terminated with an -O- atom is an alkylene glycol group, so that the linking group may comprise or consist of a polyalkylene glycol, such as polyethylene glycol (PEG). Typically, the linking group is not linked to a biotin or streptavidin moiety.

[0169] More preferably, the linking group is a C1-C 10 Alkylene group, C2-C 10 alkenylene groups, and / or polynucleotides, wherein the alkylene or alkenylene groups are -O-, -S-, -SO-, -SO2- , -NR z -, -C(O)-, -C(O)NR z and R z is H or methyl; a phosphite group; a phosphate group; a C6 aryl group; a C5-C6 carbocyclyl group; a 5-6 membered heteroaryl group; and a 5-6 membered saturated or partially unsaturated heterocyclic group. Even more preferably, the linking group is 10 is or comprises an alkylene group and / or a polynucleotide, said alkylene group being -O-, -NR z -, -C(O)-, -C(O)NR z -, and R z is H or methyl; a C6 aryl group; a C5-C6 carbocyclyl group; a 5-6 membered heteroaryl group; and a 5-6 membered saturated or partially unsaturated heterocyclic group. Preferably, when the linking group is or contains a 5-6 membered heteroaryl group, the heteroaryl group is a triazole or imidazole, preferably a triazole.

[0170] The linking group may be or include a 1,2,3-triazole, a polyethylene glycol, a phosphonate group, a phosphoramidite group, and / or a succinyl group.

[0171] In some embodiments, the linking group is or includes a moiety of the form -ABC-, where A is an alkyl group that is unsubstituted or substituted as described herein, preferably an unsubstituted C1-C5 alkyl group, B is a cyclic group selected from a C6 aryl group; a C5-C6 carbocyclyl group; a 5-6 membered heteroaryl group; and a 5-6 membered saturated or partially unsaturated heterocyclic group, and C is an alkyl group that is unsubstituted or substituted as described herein, preferably an unsubstituted C1-C5 alkyl group.

[0172] Preferably, when the linking group is or contains a moiety of the form -ABC-, A is an unsubstituted C1-C4 alkyl group; B is a cyclic group selected from a C6 aryl group; a C5-C6 carbocyclyl group; a 5-6 membered heteroaryl group; and a 5-6 membered saturated or partially unsaturated heterocyclic group; and C is an unsubstituted C1-C4 alkyl group.

[0173] More preferably, when the linking group is or contains a moiety of the form -ABC-, A is an unsubstituted C1-C4 alkyl group; B is a cyclic group selected from triazole, benzene, cyclohexane, piperidine, pyridazine, pyridine, thiazole, imidazole, preferably triazole; and C is an unsubstituted C1-C4 alkyl group.

[0174] In some embodiments, the linking group is or includes a moiety of the form:

[0175] [ka]

[0176] (where the wavy line indicates the point of attachment to the rest of the molecule). Preferably, the linking group is attached to the solid support via a phosphoramidite ((RO)2PNR2) or phosphonate group, more preferably a phosphoramidite group. Examples of suitable phosphoramidite groups include -OP(OR q )(NR P )2 groups (in the formula, R q is a C1-C4 alkyl substituted with a substituent such as -CN, and R P where each is an optionally substituted C1-C6 alkyl group, preferably an unsubstituted C2-C4 alkyl group such as isopropyl. Thus, an example of a suitable phosphoramidite group is 2-cyanoethyldiisopropylphosphoramidite (OCEP). Reaction of the above group with a reactive functional group on the solid support bonds the linking group, thus linking the support to the compound.

[0177] SPECIFIC DISCLAIMED EMBODIMENTS Preferably, in formula (I), Q is an oxygen atom, - R 2 are each independently hydrogen, methyl, C1 fluoroalkyl or fluorine; - R 3 is methyl, ethyl or C1-C2 fluoroalkyl; - (A) is a 2-nitrobenzyl group, the 2-nitrobenzyl group being C1-C4 alkyl, -OR a , -SR a , -NR a R a , -OC(O)R b and -NHC(O)R b may be substituted with one, two or three, preferably one or two, groups selected from - R a are each independently selected from hydrogen, optionally substituted C1-C2 alkyl, and optionally substituted C1-C2 alkoxyl; - R b are each independently selected from hydrogen and an optionally substituted C1-C4 alkyl group; - (N) is a part of formula (N-1) or formula (N-2)

[0178] [ka]

[0179] (In the formula, The wavy line indicates the point of attachment to the compound of formula (I), X is an oxygen atom, a nitrogen atom, a sulfur atom or -C(R a R a )-and ·R 1 are each independently selected from hydrogen, halogen, C1-C2 alkyl, C1-C2 haloalkyl, and C1-C2 alkoxyl; ·R DRis hydrogen, halogen, C1-C2 alkyl or -OR a and ·R P is hydrogen, a hydroxyl protecting group, or a phosphodiester bond to a nucleoside, nucleotide or polynucleotide, or a derivative or analog thereof; ·R B is an optionally substituted natural or non-natural nucleobase, - when the compound of formula (I) is bound to a support as described herein, (A) is a moiety of formula (A')

[0180] [ka]

[0181] (In the formula, R 4 , R 5 , R 6 and R 7 is substituted with a moiety -LZ, where L is a linking group and Z is a solid support; 4 , R 5 , R 6 and R 7 The remaining groups are hydrogen, C1-C4 alkyl, -OR a , -SR a , -NR a R a , -OC(O)R b and -NHC(O)R b (In the formula, R a and R b are as defined above), L is C1~C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 is or comprises an alkynylene group and / or a nucleotide or polynucleotide, the alkylene, alkenylene or alkynylene group being selected from the group consisting of a heteroatom or other hetero group as described herein; a phosphite group; a phosphate group; a carbonyl group; a C6-C 10 Aryl group; C5~C10 a carbocyclyl group; a 5- to 10-membered heteroaryl group; and a 5- to 10-membered saturated or partially unsaturated heterocyclic group, the linking group being optionally interrupted and / or terminated by one or more groups selected from the group consisting of an optionally further substituted linking group; The linking group is attached to the solid support via a phosphoramidite ((RO)2PNR2) or phosphonate group).

[0182] More preferably, in formula (I), Q is an oxygen atom, - R 2 are each independently hydrogen or methyl, preferably hydrogen; - R 3 is methyl, ethyl or CF3, preferably methyl; - (A) is a 2-nitrobenzyl group, said 2-nitrobenzyl group being -OR a , and -OC(O)R b and more preferably, (A) is unsubstituted apart from the nitro group at position 2 and apart from any LZ moieties as defined herein; - R a are each independently selected from hydrogen and methyl; - R b are each independently selected from hydrogen and methyl; - (N) is a part of formula (N-1) or formula (N-2)

[0183] [ka]

[0184] (In the formula, The wavy line indicates the point of attachment to the compound of formula (I), X is an oxygen atom, ·R 1 are each independently selected from hydrogen, fluorine, methyl and methoxy, preferably hydrogen; ·RDR is hydrogen, hydroxy or methoxy, preferably hydrogen; ·R P is hydrogen, a hydroxyl protecting group, or a phosphodiester bond to a nucleoside, nucleotide or polynucleotide, ·R B is an optionally substituted natural or non-natural nucleobase, preferably a natural nucleobase), When a compound of formula (I) is support-bound as described herein, (A) is a moiety of formula (A-1) or formula (A-2):

[0185] [ka]

[0186] (In the formula, R 4 , R 5 , R 6 and R 7 are independently hydrogen, -OR a , and -OC(O)R b (In the formula, R a and R b is as specified above) are independently selected from L is C1~C 10 Alkylene group, C2-C 10 alkenylene groups, and / or polynucleotides, wherein the alkylene or alkenylene groups are -O-, -S-, -SO-, -SO2-, -NR z -, -C(O)-, -C(O)NR z and R z is H or methyl; a phosphite group; a phosphate group; a C6 aryl group; a C5-C6 carbocyclyl group; a 5-6 membered heteroaryl group; and a 5-6 membered saturated or partially unsaturated heterocyclic group, The linking group is attached to the solid support via a phosphoramidite group.

[0187] Method for Producing Polynucleotides or Their Analogs or Derivatives As discussed above, the present invention provides a method for producing a polynucleotide, comprising: (i) (A) attaching to a solid support a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I)

[0188] [ka]

[0189] (In the formula, (N), Q, R 2 , R 3 and (A) is as defined above). To prepare the following: (ii) extending the length of the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, by introducing one or more additional nucleoside units, nucleotide units, and / or analogs or derivatives thereof; (iii) cleaving the polynucleotide obtained from the solid support by irradiating the compound of formula (I) with light; The present invention provides a method comprising:

[0190] Step (i) of the method of the invention may comprise providing a pre-prepared support-bound nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof, which is a solid support-bound compound of formula (I) as described herein.

[0191] Alternatively, step (i) may comprise preparing a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof, which is a solid support-bound compound of formula (I). Thus, step (i) may comprise binding the compound of formula (I) to a solid support.

[0192] Alternatively, step (i) may comprise preparing a solid support-bound compound of formula (I) at (A) from a solid support-bound compound of formula (Ia), where formula (I) is

[0193] [ka]

[0194] (In the formula, Q, R 2 , R 3 and (A) are as defined above, and R S is C1-C4 alkyl).

[0195] Thus, step (i) may comprise attaching a compound of formula (Ia) to a solid support at (A). For example, step (i) of the method of the invention may comprise: (a) A compound of formula (Ia)

[0196] [ka]

[0197] (In the formula, Q, R 2 , R 3 and (A) are as defined above, and R S is C1-C4 alkyl) To prepare the following: (b) reacting a compound of formula (Ia) with a nucleoside, nucleotide, polynucleotide or a derivative or analogue thereof, preferably a nucleoside, nucleotide, polynucleotide or a derivative or analogue thereof of formula (N-1) or (N-2) as defined above, to form a compound of formula (I); may also include

[0198] Preferably, R S is methyl. Step (ii) of the method of the present invention preferably comprises performing one or more steps of forming a phosphorus-based bond by contacting a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof with a nucleoside or a derivative thereof.

[0199] The nucleoside or its derivative may be a nucleoside phosphoramidite, a nucleoside H-phosphonate or a nucleoside phosphotriester, preferably an activated nucleoside phosphoramidite, an activated nucleoside H-phosphonate or an activated nucleoside phosphotriester. The nucleoside phosphoramidite may be activated by reacting with tetrazole or its derivative as an activating agent, for example, the group of the phosphoramidite (e.g., the diisopropylamino group of the phosphoramidate) may be protonated by acidic tetrazole or its derivative. The nucleoside H-phosphonate may be activated by reacting with a chlorinating agent such as pivaloyl chloride. The nucleoside nucleoside phosphotriester may be activated by reacting with a coupling agent such as 1-(mesitylsulfonyl)-3-nitro-1H-1,2,4-triazole.

[0200] Preferably, in step (ii), the nucleoside or derivative thereof is an activated nucleoside phosphoramidite. Preferably, the activated nucleoside phosphoramidite has an activated phosphoramidite group at the 3' or 5' position. More preferably, the activated nucleoside phosphoramidite has an activated phosphoramidite group at the 3' position. Preferably, the activated nucleoside phosphoramidite having an activated phosphoramidite group at the 3' position has a hydroxyl protecting group at the 5' position. Preferably, the activated nucleoside phosphoramidite having an activated phosphoramidite group at the 5' position has a hydroxyl protecting group at the 3' position. Preferably, the activated phosphoramidite group is represented by the formula (PH-A):

[0201] [ka]

[0202] R PP and R B is as defined herein. The phosphorus-based bond may be a phosphodiester bond, a phosphotriester bond such as an alkyl phosphotriester bond, a phosphorous-triester bond such as an alkyl phosphorous-triester bond, a phosphorothioate bond, a phosphorodithioate bond, or an alkyl phosphonate bond such as a methyl phosphonate bond. Preferably, the phosphorous-based bond is a phosphorous-triester bond. More preferably, the phosphorous-based bond is a 2-cyanoethyl phosphorous-triester bond.

[0203] Preferably, step (ii) comprises performing one or more of the steps of providing a nucleoside phosphoramidite, activating the nucleoside phosphoramidite, and contacting a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof with the activated nucleoside phosphoramidite to form a phosphite triester bond.

[0204] The nucleoside phosphoramidite preferably has a phosphoramidite group at the 3' or 5' position. More preferably, the nucleoside phosphoramidite has a phosphoramidite group at the 3' position. Preferably, the nucleoside phosphoramidite having a phosphoramidite group at the 3' position has a hydroxyl protecting group at the 5' position. Preferably, the nucleoside phosphoramidite having a phosphoramidite group at the 5' position has a hydroxyl protecting group at the 3' position. Preferably, the phosphoramidite group is represented by the formula (PH):

[0205] [ka]

[0206] RPP and R a is as defined herein. Step (ii) may optionally further comprise oxidizing the phosphite triester bond to form a phosphotriester bond.

[0207] Step (ii) may optionally further comprise removing the hydroxyl protecting group.

[0208] More preferably, step (ii) comprises (a) providing a nucleoside having (i) a phosphoramidite group at the 3' position and a hydroxyl protecting group at the 5' position; or (ii) a phosphoramidite group at the 5' position and a hydroxyl protecting group at the 3' position; (b) activating the nucleoside phosphoramidite of step (a); (c) contacting the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof with an activated nucleoside phosphoramidite to form a phosphite triester bond; (d) oxidizing the phosphite triester bond to form a phosphotriester bond; (e) removing the hydroxyl protecting group from the terminal nucleoside of the resulting support-bound polynucleotide; The method includes carrying out the above steps (a) to (e) one or more times.

[0209] In step (a), the nucleoside preferably has a phosphoramidite group at the 3' position and a hydroxyl protecting group at the 5' position.

[0210] Step (b) typically involves contacting the nucleoside phosphoramidite with a suitable activating agent. Any suitable activating agent can be used. Typically, the activating agent is tetrazole or a derivative thereof. Typically, the activating agent and the nucleoside phosphoramidite are contacted in an organic solvent such as acetonitrile.

[0211] Step (c) may be carried out by flowing the activated nucleoside phosphoramidite of step (b) through a column containing a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof.

[0212] Step (d) typically involves oxidizing the phosphite triester using a suitable oxidizing agent. Any suitable oxidizing agent can be used. Typically, the oxidizing agent is iodine. Iodine may be used in the reaction together with a suitable solvent, for example, water and pyridine (for example, water / pyridine / THF 2 / 20 / 78, with iodine at a concentration of about 0.01-0.02M).

[0213] Step (e) may be carried out by contacting the support-bound polynucleotide obtained in step (d) with a suitable reagent. Typically, deprotection involves contacting the support-bound polynucleotide with a concentrated aqueous base, such as aqueous ammonia. The aqueous solution may be removed by evaporation.

[0214] Step (ii) may include repeating the reacting step at least once, at least twice, at least five times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 500 times, at least 1000 times, or more. Thus, step (ii) may include forming a polynucleotide that is at least 2 monomer units, at least 5 monomer units, at least 10 monomer units, at least 20 monomer units, at least 30 monomer units, at least 40 monomer units, at least 50 monomer units, at least 100 monomer units, at least 150 monomer units, at least 200 monomer units, at least 500 monomer units, at least 1000 monomer units, or more in length. One skilled in the art will appreciate that the nucleoside or derivative thereof bound to the support-bound nucleoside provided in step (i) may be the same or different in each repetition. Thus, the sequence of a polynucleotide synthesized by the method can be determined by a user of the method, for example, an operator of an instrument configured for synthesizing polynucleotides.

[0215] Preferably, the method of the invention comprises one or more steps of deprotecting the support-bound polynucleotide. Preferably, these steps are carried out after step (ii) and before step (iii). Preferably, deprotecting the support-bound polynucleotide comprises removing protecting groups from one or more nucleobases and / or one or more phosphorus-based bonds present in the support-bound polynucleotide. Deprotection can be achieved by treating the support-bound polynucleotide with a suitable reagent depending on the protecting group used.

[0216] For example, benzoyl, isobutyryl and dimethylformamidyl protecting groups commonly used to protect nucleobases can be removed using concentrated ammonium hydroxide. Ultra-mild protecting groups used to protect nucleobases, such as phenoxyacetyl, acetyl and isopropylphenoxyacetyl groups, can usually be removed using a methanolic solution of potassium carbonate or a mixture of aqueous ammonia and aqueous methylamine. Cyanoethyl phosphotriester groups can be used to protect phosphodiester backbones and can be removed using concentrated ammonium hydroxide. Phosphate groups can be protected as methyl triesters and removed using thiophenol.

[0217] In step (iii), the resulting polynucleotide is cleaved from the solid support by irradiating the compound of formula (I).

[0218] Step (iii) of the present invention preferably comprises irradiating the compound of formula (I) with UV light having a wavelength of about 300 to about 500 nm, preferably about 325 to about 475 nm, more preferably about 350 to about 450 nm. Irradiation can be carried out on any suitable time scale, depending on the power of the light source used. For example, a power of about 100 W to about 5000 W, for example about 200 W to about 1000 W, for example about 500 W, can be applied for about 1 minute to about 10 hours, for example about 10 minutes to about 1 hour, for example about 30 minutes. Suitable irradiation can be provided by a Hg lamp. The selection of suitable irradiation conditions is a routine procedure for those skilled in the art. Suitable irradiation conditions can be determined by monitoring the degree of cleavage and by selecting conditions that allow substantial or complete cleavage in the shortest time possible, according to the equipment used, without causing damage to the synthesized polynucleotide. The products of polynucleotide synthesis can be evaluated by monitoring the reaction using techniques such as NMR spectroscopy and mass spectroscopy.

[0219] The method of the present invention can be carried out at any suitable temperature and under conventional solvent conditions readily available to those skilled in the art. Usually, the reaction is carried out at about 10° C. to about 100° C., for example, about 20° C. to about 50° C., for example, about 25° C. to about 30° C. In many cases, the reaction can be carried out at room temperature.

[0220] The compound of formula (I) used in the method of the present invention is compatible with various well-known polynucleotide synthesis methods, such as phosphoramidite method, H-phosphonate method and phosphotriester method.Thus, the method of the present invention includes the phosphoramidite method of polynucleotide synthesis, the H-phosphonate method of polynucleotide synthesis and the phosphotriester method of polynucleotide synthesis.Preferably, the method of the present invention is the phosphoramidite method of polynucleotide synthesis.

[0221] Further compounds Also provided herein is a support-bound nucleoside, nucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support at (A).

[0222] [ka]

[0223] (In the formula, Q, R 2 , R 3 and (A) is as defined above, and (N) is a nucleoside, a nucleotide, or a derivative or analog thereof. Preferably, (N) is represented by formula (N-1) or formula (N-2) as defined above, and R P is hydrogen, a hydroxyl protecting group, a phosphoryl group or a salt or acid thereof, a diphosphoryl group or a salt or acid thereof, or a triphosphoryl group or a salt or acid thereof.

[0224] This compound comprises a single monomeric unit of a polynucleotide and is useful as a starting point for polynucleotide synthesis. In particular, this compound may be prepared in step (i) of the method of the invention.

[0225] The present invention also provides a compound of formula (I)

[0226] [ka]

[0227] (In the formula, Q, R 2 , R 3 and (A) is as defined above, (N) is represented by formula (N-1) or formula (N-2) as defined above, and (A) is optionally bound to a solid support). (N) is preferably represented by formula (N-1) as defined above.

[0228] The compounds include a nucleoside, nucleotide or polynucleotide of formula (I) or a derivative or analog thereof linked to a photocleavable linker at the 3' or 5' position, preferably the 3' position, of formula (I). As such, the compounds are ideal for use in polynucleotide synthesis in the 3' to 5' or 5' to 3' direction.

[0229] When (A) is bound to a solid support, this compound may be prepared by step (i) of the method of the invention.

[0230] When (A) is not bound to a solid support, this compound can be used to generate a compound of formula (I) bound to a solid support at (A). Alternatively, this compound is also useful as a blocked nucleotide compound. In particular, the photocleavable group of this compound can act as a protecting group that blocks the reactivity of the nucleoside hydroxyl group to which it is bound, but can be optionally removed using photoexcitation. Removal of the photocleavable group generates a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof with a free hydroxyl group, which can undergo a variety of reactions, such as coupling reactions with polynucleotide chains to extend the chain length.

[0231] Thus, the compound may exist in solution along with a mixture of other compounds, but is inactive and inert until a desired time point when the compound is activated using photoexcitation.

[0232] The present invention also provides a compound of formula (Ia) attached to a solid support at (A).

[0233] [ka]

[0234] (In the formula, Q, R 2 , R 3 and (A) is as defined above). This compound is useful for producing a compound of formula (I) which is attached to a solid support at (A).

[0235] solid support In the embodiments provided herein, the compounds provided herein may be bound to a solid support. For example, in some embodiments, the compounds of formula (I) or formula (Ia) are bound to a solid support at group (A) of the compounds.

[0236] Any suitable solid support can be used. Preferably, the solid support comprises glass, silica, ceramic, or polymer resin. More preferably, the solid support comprises controlled pore glass (CPG) or polystyrene. The CPG may be treated with a suitable surface treatment to introduce one or more functional groups. A suitable surface treatment includes silanes, such as (3-aminopropyl)triethoxysilane, to give aminopropyl CPG. The functionalized CPG can be derivatized by reacting the amino moiety of the aminopropyl group with a suitable moiety, such as an ester group. Suitable polystyrene materials are usually low-swelling and / or crosslinked. For example, polystyrene can be obtained by polymerizing divinylbenzene, styrene, and 4-chloromethylstyrene in the presence of a porosity agent. The resulting macroporous chloromethyl MPPS can be converted to aminomethyl MPPS.

[0237] Typically, the solid support is not functionalized with a protein such as streptavidin. Preferably, the solid support comprises particles having a diameter of about 1 μm to about 10,000 μm, more preferably about 5 μm to about 1,000 μm, even more preferably about 10 μm to about 500 μm, for example, about 50 to about 250 μm.

[0238] Preferably, the solid support material particles are porous. Preferably, when the solid support material is porous, the pore size is from about 1 to about 1000 nm, such as from about 10 to about 100 nm, for example about 50 nm.

[0239] Preferably, the support material is or comprises beads. The beads may be magnetic, e.g. comprise a magnetic material. The beads may be ferromagnetic or paramagnetic. The beads may have an iron oxide core.

[0240] The use of beads can be advantageous when the synthesized polynucleotides are used in sequencing techniques, such as droplet-based sequencing as described herein.

[0241] Preferably, the support material is contained within a column. The total volume of the column may be about 100 μL to about 100 L, for example, about 1 mL to about 1 L. Any suitable total volume can be used depending on the envisaged synthesis scale.

[0242] Thus, provided herein is a solid support column for solid phase polynucleotide synthesis comprising a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support in (A), comprising (N), Q, R 2 , R 3 and (A) is as defined in any one of the preceding claims.

[0243] Products of the disclosed methods The methods of the present disclosure can be used to generate polynucleotides or analogs or derivatives thereof. Accordingly, also provided herein are polynucleotides or derivatives or analogs thereof obtainable or obtainable by the methods provided herein.

[0244] The term "polynucleotide" refers to a single- or double-stranded covalently linked sequence of nucleotides in which the 3' and 5' ends of each nucleotide are joined by a phosphorus-based bond. The phosphorus-based bond present in the products of the disclosed method may be a phosphodiester bond, as in natural polynucleotides, or a non-natural phosphorus-based bond, such as a phosphotriester bond, e.g., an alkyl phosphotriester bond, such as a methyl phosphotriester bond and an ethyl phosphotriester bond; a phosphorothioate bond; a phosphorodithioate bond; and an alkyl phosphonate bond, e.g., a methyl phosphonate bond.

[0245] A polynucleotide may be composed of deoxyribonucleotide or ribonucleotide bases. A nucleic acid may further include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has been subjected to post-translational modifications, such as 5'-capping with 7-methylguanosine, 3'-processing such as cleavage and polyadenylation, and splicing. A nucleic acid may also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA). The size of a nucleic acid, also referred to herein as a "polynucleotide", is usually expressed in terms of the number of base pairs (bp) for double-stranded polynucleotides, or as the number of nucleotides (nt) for single-stranded polynucleotides. 1000 bp or 1000 nt is equal to 1 kilobase (kb). Polynucleotides less than about 40 nucleotides in length are commonly referred to as "oligonucleotides" and may include primers for use in DNA manipulation, for example, via the polymerase chain reaction (PCR).

[0246] A synthetic polynucleotide may have any combination of any nucleotides. The nucleotides may be naturally occurring or artificial. One or more nucleotides of the polynucleotide may be oxidized or methylated. One or more nucleotides of the polynucleotide may be damaged. For example, the polynucleotide may include pyrimidine dimers. The dimers are usually associated with ultraviolet light damage and are the main cause of skin melanoma. One or more nucleotides of the polynucleotide may be modified, for example with a label or tag, suitable examples of which are known by those skilled in the art. The polynucleotide may include one or more spacers. A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group, as described in more detail herein. The nucleobase and the sugar form a nucleoside. The nucleobase is usually heterocyclic. The nucleobase includes, but is not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is usually a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. The polynucleotide preferably includes the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC). The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides typically contain monophosphates, diphosphates, or triphosphates. The nucleotides may contain more than three phosphates, for example, four or five phosphates. The phosphates may be attached to the 5' or 3' positions of the nucleotide. The nucleotides of the polynucleotide may be attached to each other in any manner. The nucleotides are typically attached by their sugar and phosphate groups, similar to nucleic acids. The nucleotides may be linked via their nucleobases, such as in pyrimidine dimers.

[0247] The nucleotides contained in the polynucleotide include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP) and deoxymethylcytidine monophosphate.The nucleotide is preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP.The nucleotide may be abasic (i.e., lacking a nucleobase). A nucleotide may also lack a nucleobase and a sugar (ie, a C3 spacer).

[0248] Typically, a polynucleotide consists of or contains DNA and / or RNA, preferably DNA.

[0249] The length of a polynucleotide may be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotides or nucleotide pairs.

[0250] Uses of polynucleotides generated by the methods of the present disclosure The polynucleotides provided by the methods of the present disclosure and provided herein have many potential applications, and the applications are not particularly limited, and the polynucleotides provided herein and by the methods disclosed herein can be used for any application in which a polynucleotide is needed.

[0251] In some embodiments, the methods provided herein allow for the preparation of polynucleotides with very high purity.Therefore, the polynucleotides provided herein are particularly useful for applications that require high purity.Preferably, the purity of the polynucleotides obtained by the methods disclosed herein is at least 99%, such as at least 99.9%, such as at least 99.99%, more preferably at least 99.999% or more.

[0252] One application of the polynucleotides provided herein is in PCR (polymerase chain reaction). Methods for carrying out PCR are well known to those skilled in the art. For example, one embodiment of carrying out PCR includes contacting a primer that is complementary to the target polynucleotide under conditions that allow polymerase to catalyze chain extension with the target polynucleotide, thereby amplifying the target polynucleotide. The polynucleotides provided herein are particularly useful as primers for PCR due to their high purity.

[0253] Another use of the polynucleotides provided herein is in the preparation of therapeutic polynucleotides, which are being investigated for use in the treatment of a variety of disease states and have particular use as anti-cancer and anti-viral drugs.

[0254] Further applications of the polynucleotides provided herein include applications in next-generation sequencing technology.For example, the polynucleotides provided herein can be used as barcodes in single-cell sequencing technology, such as the drop sequence sequencing method described below.

[0255] Single-cell sequencing technology The methods of the invention may be advantageously used to generate support-bound polynucleotides for use in methods of sequencing the genome of a cell, in particular for use in the cell sequencing technique known as "drop sequencing" technology. Similarly, some compounds of the invention may be advantageously used in methods involving drop sequencing technology.

[0256] In standard drop sequencing techniques, a microfluidic device is used to generate an emulsion of water-in-oil droplets, each of which contains a maximum of a single biological cell (e.g., a mammalian cell, such as a human cell, or a bacterial cell) and a barcoded solid support, e.g., a barcoded bead, bound to a polynucleotide sequence that defines a polynucleotide "barcode." The support-bound polynucleotides of the present invention can be used in this approach.

[0257] More specifically, support-bound polynucleotides for use in drop sequence sequencing typically include a molecular beacon for identifying cellular target polynucleotides, a polynucleotide "barcode" and a universal sequence. Support-bound polynucleotides typically include a 3'-polyT end, i.e., there are several consecutive, e.g., about 5 to about 50, e.g., about 10 to about 40, e.g., about 30, nucleotide units having thymine as their nucleobase at the 3' end of the polynucleotide. The molecular beacon may include about 5 to about 15 nucleotides. The barcode may have about 4 to about 50, e.g., about 5 to about 30, e.g., about 6 to about 20, e.g., about 8 to about 15 monomer units. The universal sequence may be of any desired length.

[0258] Cells usually contain mRNAs with 5'-polyA regions; that is, there are several consecutive nucleotides at the 5' end of the mRNA with adenine as the nucleobase. Typically, in drop sequencing techniques, cells are lysed and their mRNAs are released into their respective droplets. The 5'-polyA regions of the mRNA strands hybridize to the 3'-polyT ends of the support-bound polynucleotides, thereby capturing the mRNA strands from a single cell onto a single solid support. The resulting support-bound polynucleotide-mRNA ensembles are often referred to as "STAMPs" - Single cell Transcriptomes Attached to MicroParticles.

[0259] The emulsion may then be broken, releasing the STAMPS, which can be used to create a transcriptome library. STAMPS can be identified by barcodes on the solid support and subjected to reverse transcription, PCR and sequencing to determine the mRNA strand sequence.

[0260] Traditionally, support-bound polynucleotides with 3'-polyT tails used in drop-sequencing technology were synthesized directly on the solid support using 5' to 3' solid-phase polynucleotide synthesis to provide a free 3' end available for hybridization with the mRNA strand.

[0261] However, the compounds and methods of the present invention can be used to generate support-bound polynucleotides for use in drop-sequencing technology that are barcoded at the 3' end and bound to solid supports. This has several advantages, including: (i) the reagents required to generate barcoded support-bound polynucleotides are much cheaper, in particular the so-called "standard" nucleotide phosphoramidites typically used in 3' to 5' solid-phase polynucleotide synthesis are approximately an order of magnitude cheaper than the so-called "reverse nucleotide phosphoramidites" typically used in 5' to 3' synthesis; (ii) the coupling efficiency of 3' to 5' solid-phase polynucleotide synthesis is usually higher than that of 5' to 3' synthesis; and (iii) the variety of commercially available "standard" nucleotide phosphoramidites is much greater than the commercially available "reverse nucleotide phosphoramidites", which allows for greater customization and therefore improvement of polynucleotides that capture mRNA strands.

[0262] These barcoded support-bound polynucleotides, synthesized in the 3' to 5' direction, can be used in a drop sequencing technique where photoexcitation is used to cleave a photocleavable linker, cleaving the polynucleotides from their barcoded supports prior to breaking the emulsion. Subsequent reverse transcription and PCR steps can then be performed "in the droplets" to sequence the mRNA strands present in each individual droplet.

[0263] Thus, provided herein is a method of sequencing a polynucleotide expressed by a cell, comprising: providing a polynucleotide bound to a support as described herein, wherein the polynucleotide is bound to the support by a photocleavable linker as described herein; contacting the support-bound polynucleotide or a photocleavage product of the support-bound polynucleotide with a target polynucleotide under conditions in which the support-bound polynucleotide or the photocleavage product hybridizes to the target polynucleotide; enzymatically synthesizing a complementary polynucleotide strand complementary to the target polynucleotide; and determining a sequence of the target polynucleotide by sequencing the synthesized complementary polynucleotide strand or the complementary polynucleotide strand.

[0264] Further embodiments of the present invention Also herein, - a solid support modified with a photocleavable linker, such as a compound of formula (Ia), as described herein; - preferably a reagent for synthesizing a polynucleotide comprising a plurality of nucleotide monomers; A kit for preparing a polynucleotide is provided, comprising:

[0265] Preferably, the solid support and the photocleavable linker are as defined herein. Further provided is an apparatus for performing polynucleotide synthesis, comprising: - a solid support modified with a photocleavable linker as described herein; - a light source for irradiating the solid support and cleaving the photocleavable linker; An apparatus is provided comprising:

[0266] Preferably, the solid support, the light source and the photocleavable linker are as defined herein.

[0267] method The compounds of the invention may be prepared by any suitable method. Details of typical synthetic routes for representative compounds of the invention are set out below and in the Examples.

[0268] In summary, the compounds of the present invention can generally be prepared by reactions according to the following schemes:

[0269] [ka]

[0270] The starting material SM is readily available. Reaction of A:SM with, for example, SOCl2 can be carried out by, for example, reacting R 3 B: Reduction of the carboxylic acid can be accomplished using, for example, NaBH4 to give the alcohol (when Q is O). When Q is other than O, standard transformations can be used to convert the alcohol to other functional groups. C: (D:) Sulfur-containing CR 2 NaN3 can be used to convert the bromine to an azide before reacting with 2 derivative to introduce a reactive group for reaction with a nucleotide, nucleoside or polynucleotide in step E. The azide can then be converted to a linking group using standard chemistry for attachment to a solid support, e.g., via one or more nucleophilic displacement steps of F.

[0271] Detailed synthetic routes for exemplary compounds of the invention are set forth below. Further embodiments The following are numbered aspects of the present invention.

[0272] [1] 1. A method for producing a polynucleotide or an analog or derivative thereof, comprising: (i) (A) a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support.

[0273] [ka]

[0274] (In the formula, - (N) is a nucleoside, nucleotide, polynucleotide, or a derivative or analog thereof; Q is an oxygen atom or a sulfur atom, - R 2 are each independently selected from hydrogen, methyl, ethyl, C1-C2 haloalkyl, and halogen groups; - R 3 is methyl, ethyl or C1-C2 haloalkyl; - (A) is a 2-nitrobenzyl group, and the 2-nitrobenzyl group is selected from halogen, optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b and -NHC(O)R b wherein R a are each independently selected from hydrogen, optionally substituted C1-C2 alkyl, and optionally substituted C1-C2 alkoxyl; R b are each independently selected from hydrogen and an optionally substituted C1-C4 alkyl group. To prepare the following: (ii) extending the length of the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, by introducing one or more additional nucleoside units, nucleotide units, and / or analogs or derivatives thereof; (iii) cleaving the polynucleotide obtained from the solid support by irradiating the compound of formula (I) with light; A method comprising:

[0275] [2] Q is an oxygen atom, and / or - R 2 are each independently selected from hydrogen, methyl, C1 fluoroalkyl and fluorine, preferably R 2 are each hydrogen, and / or - R 3 is methyl, ethyl or C1-C2 fluoroalkyl, preferably R 3 The method of embodiment 1, wherein is methyl.

[0276] [3] The method of embodiment 1 or embodiment 2, wherein (A) is covalently attached to a linking group, and the linking group is covalently attached to the solid support.

[0277] [4] The method of embodiment 3, wherein (A) is represented by formula (A-1) or formula (A-2).

[0278] [ka]

[0279] (In the formula, - R 4 , R 5 , R 6 and R 7 each independently represents hydrogen, halogen, an optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a, -C(O)OR a , -C(O)NR a R a , -C(O)R b , -OC(O)R b and -NHC(O)R b where R a and R b is as defined in aspect 1; L is a linking group; - Z is a solid support).

[0280] [5] R 4 , R 5 , R 6 and R 7 each independently represents hydrogen, fluorine, an optionally substituted C1-C4 alkyl, -OR a , -SR a , -NR a R a , -OC(O)R b and -NHC(O)R b Selected from R a and R b is as defined in aspect 1, preferably R 4 , R 5 , R 6 and R 7 The method of embodiment 4, wherein each is hydrogen or methoxy.

[0281] [6] The linking group is C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 alkynylene groups and / or nucleotides or polynucleotides, wherein the alkylene, alkenylene or alkynylene groups are each independently selected from the group consisting of heteroatoms, phosphite groups, phosphate groups, carbonyl groups, C6-C 10 Aryl group; C5~C 10The method of any one of embodiments 3 to 5, wherein the linking group is optionally interrupted and / or terminated with one or more groups selected from a carbocyclyl group; a 5-10 membered heteroaryl group; and a 5-10 membered saturated or partially unsaturated heterocyclic group, and the linking group is optionally further substituted.

[0282] [7] The method of any one of embodiments 2 to 6, wherein the linking group is attached to the solid support via a phosphoramidite group.

[0283] [8] The method of any one of the preceding embodiments, wherein the solid support contains particles having a diameter of about 10 to about 1000 μm.

[0284] [9] The method of any one of the preceding embodiments, wherein the solid support comprises glass, ceramic, or a polymeric resin.

[0285]

[10] The method of any one of the preceding embodiments, wherein (N) is attached to adjacent oxygen atoms of formula (I) at the 3' or 5' position of the nucleoside, nucleotide, polynucleotide, or derivative or analog thereof.

[0286]

[11] The method of any one of the preceding embodiments, wherein (N) is represented by formula (N-1) or formula (N-2).

[0287] [ka]

[0288] (In the formula, - X is an oxygen atom, a nitrogen atom, a sulfur atom or -C(R a R a )-and - R 1 and R DR each independently represents hydrogen, halogen, an optionally substituted C1-C4 alkyl, -OR a , -SRa , -NR a R a , -C(O)OR a , -C(O)NR a R a and -C(O)R b where R a and R b is as defined in aspect 1, and R DR -OR a If R a Groups and R 1 one of the groups may be linked together to form a bridging motif; - R P is hydrogen, a hydroxyl protecting group, a phosphoryl group or its salt or acid, a diphosphoryl group (-P(O2 - )-OP(O3 - )) or its salt or acid, a triphosphoryl group (-P(O2 - )-P(O2 - )-OP(O3 - )) or a salt or acid thereof, or a phosphorus-based bond to a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof, - R B is an optionally substituted natural or non-natural nucleobase or a derivative or analog thereof, - the wavy line indicates the point of attachment to the compound of formula (I).

[0289]

[12] - X is an oxygen atom, and / or - R 1 are each independently selected from hydrogen, halogen, C1-C2 alkyl, C1-C2 haloalkyl, and C1-C2 alkoxyl; preferably, R 1 are each hydrogen, and / or - R DR is hydrogen or halogen, preferably R DR is hydrogen, and / or - R P The method of embodiment 11, wherein is hydrogen or a hydroxyl protecting group.

[0290]

[13] Step (i) is (a) A compound of formula (Ia)

[0291] [ka]

[0292] (In the formula, Q, R 2 , R 3 and (A) are as defined in aspects 1-9, and R S is C1-C4 alkyl) To prepare the following: (b) reacting a compound of formula (Ia) with a nucleoside, nucleotide, polynucleotide or a derivative or analogue thereof, preferably a nucleoside, nucleotide, polynucleotide or a derivative or analogue thereof of formula (N-1) or formula (N-2) as defined in embodiment 11 or embodiment 12, to form a compound of formula (I);

[0026] The method of any one of the preceding aspects, comprising:

[0293]

[14] Step (i) comprises reacting a compound of formula (I) or formula (Ia)

[0294] [ka]

[0295] (In the formula, (N), Q, R 2 , R 3 , R S and (A) is as defined in any one of the preceding embodiments.

[0023] The method of any one of the preceding embodiments, comprising binding to a solid support.

[0296]

[15] The method of any one of the preceding embodiments, wherein extending the chain length in step (ii) comprises performing one or more steps of forming a phosphorus-based bond by contacting the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analogue thereof with a nucleoside or derivative thereof.

[0297]

[16] In step (ii), chain lengthening is carried out (a) providing a nucleoside having (i) a phosphoramidite group at the 3' position and a hydroxyl protecting group at the 5' position; or (ii) a phosphoramidite group at the 5' position and a hydroxyl protecting group at the 3' position; (b) activating the nucleoside phosphoramidite of step (a); (c) contacting the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof with an activated nucleoside phosphoramidite to form a phosphite triester bond; (d) oxidizing the phosphite triester bond to form a phosphotriester bond; (e) removing the hydroxyl protecting group from the terminal nucleoside of the resulting support-bound polynucleotide;

[0023] The method of any one of the preceding embodiments, comprising performing the above steps (a)-(e) one or more times.

[0298]

[17] The method of any one of the preceding embodiments, further comprising, after step (ii) and before step (iii), one or more steps of deprotecting the support-bound polynucleotide.

[0299]

[18] The method of any one of the preceding embodiments, wherein step (iii) comprises photoirradiating the compound of Formula (I) using UV light having a wavelength of about 300 to about 500 nm.

[0300]

[19] (A) A support-bound nucleoside, nucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support.

[0301] [ka]

[0302] (In the formula, Q, R 2 , R 3 and (A) is as defined in any one of aspects 1 to 9; (N) is a nucleoside, a nucleotide or a derivative or analog thereof, preferably a nucleoside, a nucleotide or a derivative or analog thereof represented by formula (N-1) or formula (N-2) as defined in embodiment 11 or 12).

[0303]

[20] Compounds of formula (I)

[0304] [ka]

[0305] (In the formula, Q, R 2 , R 3 and (A) is as defined in any one of aspects 1 to 9; (N) is represented by formula (N-1) or formula (N-2) as defined in embodiment 11 or 12).

[0306] In this embodiment, for the avoidance of doubt, (A) may optionally be associated with a solid support, i.e. a solid support may or may not be present.

[0307] [twenty one] (A) A support-bound compound of formula (Ia) bound to a solid support.

[0308] [ka]

[0309] (In the formula, Q, R 2 , R 3 and (A) is as defined in any one of the preceding embodiments.

[0310] [twenty two] 20. A method for producing a support-bound nucleoside, nucleotide, or derivative or analogue thereof as defined in embodiment 19, comprising the steps of: (a) providing a compound of formula (Ia) bound to a solid support as defined in embodiment 21 (A); (b) reacting a compound of formula (Ia) with a nucleoside, nucleotide, or a derivative or analog thereof to produce a support-bound nucleoside, nucleotide, or a derivative or analog thereof; A method comprising:

[0311] [twenty three] (A) a solid support column for solid phase polynucleotide synthesis comprising a support-bound nucleoside, nucleotide, polynucleotide or derivative or analogue thereof, the compound of formula (I) being bound to a solid support.

[0312] [ka]

[0313] (In the formula, (N), Q, R 2 , R 3 and (A) is as defined in any one of the preceding embodiments.

[0314] [twenty four] Use of a support bound nucleoside, nucleotide or derivative or analogue thereof as defined in embodiment 19, a compound of formula (I) as defined in embodiment 20, or a compound of formula (Ia) as defined in embodiment 21 for synthesizing a polynucleotide or a derivative or analogue thereof.

[0315] [twenty five] 19. A polynucleotide or a derivative or analogue thereof obtained by a method as defined in any one of embodiments 1 to 18.

[0316] Although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein for the method according to the present invention, it should be understood that various changes or modifications in form and details can be made without departing from the scope and spirit of the present invention. The following examples are provided to better illustrate specific embodiments, and should not be considered as limiting this application. This application is limited only by the claims. EXAMPLES

[0317] Working Example Synthesis Example 1 Synthesis of 1-(5-methyl-2-nitrophenyl)ethan-1-one

[0318] [ka]

[0319] To a solution of 5-methyl-2-nitrobenzoic acid (5 g, 27.6 mmol) in anhydrous toluene (30 mL) was added thionyl chloride (6 mL, 82.8 mmol) and the mixture was refluxed under argon at 80° C. for 3 h. The mixture was concentrated to give 5-methyl-2-nitrobenzoyl chloride as a yellow oil (5.5 g) which was carried on without further purification.

[0320] To a suspension of anhydrous magnesium chloride (2.36 g, 24.8 mmol) and diethyl malonate (5 mL, 33 mmol) in anhydrous ethyl acetate (80 mL) was added anhydrous triethylamine (16 mL) slowly at 0°C. After stirring for 45 min, a solution of 5-methyl-2-nitrobenzoyl chloride (5.5 g) in anhydrous ethyl acetate (10 mL) was added. The mixture was heated to 60°C and stirred for 30 min. Once complete, the reaction was extracted with ethyl acetate, washed with dilute hydrochloric acid (1 M), water, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. A solution of glacial acetic acid (30 mL) and concentrated sulfuric acid (6 mL) was added and the mixture was refluxed for 12 h. The reaction mixture was cooled in an ice bath, made alkaline with sodium hydroxide and the product was extracted with dichloromethane before purification by flash chromatography (0-50% ethyl acetate in hexane) to give the title compound (3.79 g, 21.2 mmol, 77%).

[0321] 1 H NMR (400 MHz, CDCl3) δ ppm 8.03 (d, 1H, J = 8.4 Hz, C H Ar), 7.38 (m, 1H, C H Ar), 7.18 (m, 1H, C H Ar), 2.54 (s, 1H, C H 3CO), 2.48 (s, 1H, C H 3). Synthesis Example 2 Synthesis of 1-(5-(bromomethyl)-2-nitrophenyl)ethan-1-one

[0322] [ka]

[0323] To a solution of 1-(5-methyl-2-nitrophenyl)ethan-1-one (3.79 g, 21.2 mmol) in anhydrous acetonitrile (25 mL) were added N-bromosuccinimide (4.14 g, 23.3 mmol) and 1,1'-azobis(cyclohexanecarbonitrile) (0.52 g, 2.12 mmol), and the mixture was refluxed for 12 h. After cooling to room temperature, the solvent was removed and the residue was dissolved in toluene (25 mL) and filtered. The filtrate was concentrated and purified by flash chromatography (0-50% ethyl acetate in hexane) to give the title compound (3.74 g, 14.5 mmol, 68%).

[0324] 1 H NMR (400 MHz, CDCl3) δ ppm 8.09 (d, 1H, J = 8.4 Hz, C H Ar), 7.62 (dd, 1H, J = 8.5, 2.0 Hz, C H Ar), 7.43 (d, 1H, J = 2.0 Hz, C H Ar), 4.50 (s, 2H, C H 2Br), 2.57 (s, 1H, C H 3CO). Synthesis Example 3 Synthesis of 1-(5-(bromomethyl)-2-nitrophenyl)ethan-1-ol

[0325] [ka]

[0326] 1-(5-(bromomethyl)-2-nitrophenyl)ethan-1-one (3.74 g, 14.5 mmol) was dissolved in a mixture of anhydrous methanol (38 mL) and anhydrous dioxane (25 mL) whereupon sodium borohydride (0.822 g, 21.7 mmol) was added slowly at 0° C. before it was allowed to warm to room temperature over 12 h. Water (60 mL) and 1 M hydrochloric acid (15 mL) were added and the suspension was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated and then purified by flash chromatography (0-50% ethyl acetate in hexanes) to give the title compound (3.38 g, 13.0 mmol, 90%).

[0327] 1 H NMR (400 MHz, CDCl3) δ ppm 7.91 (d, 1H, J = 8.4 Hz, C H Ar), 7.87 ppm (d, 1H, J = 2.0 Hz, C H Ar), 7.45 (m, 1H, C H Ar), 5.46 (q, 1H, J = 6.4 Hz, CH3C H ), 4.51 (s, 1H, C H 2Br), 2.27 (br. s, 1H, O H ), 1.58 (d, 3H, J = 6.4 Hz, C H 3CH). Synthesis Example 4 Synthesis of 1-(5-(azidomethyl)-2-nitrophenyl)ethan-1-ol

[0328] [ka]

[0329] To a solution of 1-(5-(bromomethyl)-2-nitrophenyl)ethan-1-ol (2.49 g, 9.57 mmol) in anhydrous dimethylformamide (30 mL) was added sodium azide (0.93 g, 14.4 mmol) and the mixture was stirred at room temperature for 3 h. The mixture was concentrated and then extracted from ethyl acetate with water, dried over anhydrous sodium sulfate, filtered, and concentrated before purification by flash chromatography (0-50% ethyl acetate in hexanes) to give the title compound (1.95 g, 8.77 mmol, 92%).

[0330] 1 H NMR (400 MHz, CDCl3) δ ppm 7.95 (d, 1H, J = 8.4 Hz, C H Ar), 7.81 (d, 1H, J = 2.0 Hz, C H Ar), 7.39 (dd, 1H, J = 8.4, 2.0 Hz, C H Ar), 5.48 (q, 1H, J = 6.4 Hz, CH3C H ), 4.49 (s, 2H, C H 2N3), 2.17 (br. s, 1H, O H ), 1.59 (d, 3H, J = 6.4 Hz, C H 3CH). Synthesis Example 5 Synthesis of ((1-(5-(azidomethyl)-2-nitrophenyl)ethoxy)methyl)(methyl)sulfane

[0331] [ka]

[0332] To a solution of 1-(5-(azidomethyl)-2-nitrophenyl)ethan-1-ol (1.65 g, 7.4 mmol) in DMSO (38 mL) were added acetic anhydride (57 mL) and glacial acetic acid (38 mL) and the reaction mixture was stirred at room temperature for 20 h. The reaction mixture was poured into ice-cold saturated aqueous sodium carbonate solution and stirred for a further 30 min before being extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium carbonate, water and brine before being dried over anhydrous sodium sulfate and concentrated. The crude material was purified by flash chromatography (0-30% ethyl acetate in hexanes) to give the title compound (1.7 g, 6 mmol, 81%).

[0333] 1 H NMR (400 MHz, CDCl3) δ ppm 7.96 (d, 1H, J = 8.4 Hz, C H Ar), 7.72 (d, 1H, J = 2.0 Hz, C H Ar), 7.39 (dd, 1H, J = 8.4, 2.0 Hz, C H Ar), 5.45 (q, 1H, J = 6.4 Hz, CH3C H ), 4.64 (d, 1H, J = 11.5 Hz, C H 2SCH3), 4.49 (s, 2H, C H 2N3), 4.35 (d, 1H, J = 11.5 Hz, C H 2SCH3), 2.13 (s, 3H, CH2SC H 3), 1.56 (d, 3H, J = 6.5 Hz, C H 3CH). Synthesis Example 6 Synthesis of 3'-O-((1-(5-(azidomethyl)-2-nitrophenyl)ethoxy)methyl)thymidine

[0334] [ka]

[0335] To a solution of ((1-(5-(azidomethyl)-2-nitrophenyl)ethoxy)methyl)(methyl)sulfane (1.7 g, 6 mmol) and 1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (2 g, 5.7 mmol) in anhydrous tetrahydrofuran (18 mL) at -40°C, N-iodosuccinimide (1.35 g, 6 mmol) and 4 Å molecular sieves (1.8 g) were added followed by triflic acid (0.53 mL, 6 mmol) and the mixture was stirred for 30 min. The reaction was quenched with triethylamine, filtered through Celite, and concentrated in vacuo. The residue was dissolved in ethyl acetate and washed with saturated aqueous sodium thiosulfate. The combined organic layers were washed with saturated sodium carbonate, water and brine, then dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was dissolved in anhydrous methanol (54 mL) and ammonium fluoride (2.1 g, 57 mmol) was added and the mixture was refluxed for 2 h and then cooled to room temperature for 12 h. The reaction mixture was concentrated in vacuo and purified by flash chromatography (0-100% ethyl acetate in hexanes) to give the title compound (1.2 g, 2.52 mmol, 42%).

[0336] 1 H NMR (400 MHz, CDCl3) δ ppm 8.66 (br. S., 1H, N H ), 7.96 (d, 0.5H, J = 2.6 Hz, C H Ar), 7.94 (d, 0.5H, J = 2.7 Hz, C H Ar), 7.73-7.70 (m, 1H, C H Ar), 7.42-7.32 (m, 2H, C H Ar, C H 6 ), 6.06 (t, 0.5H, J = 6.9 Hz, C H 1’ ), 6.01 (t, 0.5H, J = 6.9 Hz, C H 1’), 5.41-5.34 (m, 1H, C H CH3), 4.77 (d, 0.5H, J = 7.3 Hz, OC H 2O), 4.74 (d, 0.5H, J = 7.2 Hz, OC H 2O), 4.6 (d, 0.5H, J = 7.2 Hz, OC H 2O), 4.57 (d, 0.5H, J = 7.3 Hz, OC H 2O), 4.52-4.45 (m, 2.5H, C H 2N3, C H 3’ ), 4.38 (m, 0.5H, C H 3’ ), 4.09 (q, 0.5H, J = 2.9 Hz, C H 4’ ), 3.99-3.91 (m, 1H, C H 4’ , C H 5’ ), 3.86-3.78 (m, 1H, C H 5’ ), 3.56-3.5 (m, 0.5H, C H 5’ ), 2.69 (br. S., 0.5H, O H ), 2.59 (br. S., 1H, O H ), 2.44-2.38 (m, 1H, C H 2’ ), 2.34-2.25 (m, 0.5H, C H 2’ ), 2.18-2.12 (m, 0.5H, C H 2’ ), 1.93-1.90 (m, 3H, C H 3), 1.57 (d, 3H, J = 6.4 Hz, CHC H 3). Synthesis Example 7 Synthesis of 5'-O-(4,4'-dimethoxytrityl)-3'-O-((1-(5-(azidomethyl)-2-nitrophenyl)ethoxy)methyl)thymidine

[0337] [ka]

[0338] 3'-O-((1-(5-(azidomethyl)-2-nitrophenyl)ethoxy)methyl)thymidine (1.2 g, 2.52 mmol) was coevaporated three times with anhydrous pyridine and then dissolved in anhydrous pyridine (10 mL) under argon. 4,4'-Dimethoxytrityl chloride (1 g, 3 mmol) was added in small portions over 1 h and the reaction was stirred at room temperature for an additional 11 h. The reaction was quenched with methanol (5 mL) and triethylamine (1 mL) was added. The reaction was stirred for 10 min before the solvent was removed in vacuo. The crude product was purified via flash chromatography (0-100% ethyl acetate (dichloromethane with 0.1% triethylamine)) to give the title compound (1.85 g, 2.38 mmol, 94%).

[0339] 1 H NMR (400 MHz, DMSO d6) δ ppm 11.35-11.34 (m, 1H, N H ), 7.99-7.94 (m, 1H, C H Ar), 7.70-7.67 (m, 1H, C H Ar), 7.55-7.21 (m, 11H, C H Ar, C H 6 , 9 x C H DMTr), 6.92-6.86 (m, 4H, C H DMTr), 6.13 (t, 0.5H, J = 7.0 Hz, C H 1’ ), 6.10-6.04 (m, 0.5H, C H 1’ ), 5.23-5.10 (m, 1H, C H CH3), 4.72 (d, 1H, J = 7.1 Hz, OC H 2O), 4.63-4.53 (m, 2.5H, C H 2N3, OC H2O), 4.5 (d, 0.5H, J = 7.5 Hz, OC H 2O) 4.44-4.36 (m, 1H, C H 3’ ), 4.01-3.97 (m, 0.5H, C H 4’ ), 3.92-3.88 (m, 0.5H, C H 4’ ), 3.73 (s, 6H, 2 x OC H 3), 3.27-3.19 (m, 1H, C H 5’ ), 3.18-3.08 (m, 1H, C H 5’ ), 2.36-2.30 (m, 1H, C H 2’ ), 2.22-2.01 (m, 1H, C H 2’ ), 1.49-1.43 (m, 4.5H, C H 3. CHC H 3), 1.34 (d, 1.5HJ = 6.4 Hz, CHC H 3). Synthesis Example 8 Synthesis of 5'-O-(4,4'-dimethoxytrityl)-3'-O-((1-(5-((4-(4-hydroxybutyl)-1H-1,2,3-triazol-1-yl)methyl)-2-nitrophenyl)ethoxy)methyl)thymidine

[0340] [ka]

[0341] 5'-O-(4,4'-dimethoxytrityl)-3'-O-((1-(5-(azidomethyl)-2-nitrophenyl)ethoxy)methyl)thymidine (0.7 g, 0.9 mmol) and 5-hexyn-1-ol (0.15 mL, 1.35 mmol) were added to a 3:1:1 mixture of tetrahydrofuran / tert-butanol / water (13.5 mL / 4.5 mL / 4.5 mL). 7.5% aqueous copper(II) sulfate (2 mL) and 1 M aqueous (+)-sodium L-ascorbic acid (2.25 mL) were added and the reaction was stirred at room temperature for 2 h. The mixture was diluted with ethyl acetate and the organic phase was washed with saturated sodium bicarbonate and brine (diluted to 10% with water), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by flash chromatography (dichloromethane containing 0-10% methanol (0.1% triethylamine)) to give the title compound (0.7 g, 0.8 mmol, 59%).

[0342] 1 H NMR (400 MHz, DMSO d6) δ ppm 11.35-11.34 (m, 1H, N H ), 7.96-7.89 (m, 2H, C H Ar, C H triazole), 7.61-7.59 (m, 1H, C H 6 ), 7.5-7.47 (C H Ar), 7.42-7.20 (m, 10H, C H Ar, C H DMTr), 6.92-6.86 (m, 4H, C H DMTr), 6.16-6.04 (m, 1H, C H 1’ ), 5.67 (s, 1H, C H 2N), 5.63 (s, 1H, C H 2N), 5.17 (q, 0.5H, J = 6.4 Hz, C H CH3), 5.1 (q, 0.5H, J = 6.3 Hz, C H CH3), 4.67 (d, 1H, J = 7.2 Hz, OCH 2O), 4.51 (d, 0.5HJ = 7.2, OC H 2O), 4.45 (d, 0.5H, J = 7.3 Hz, OC H 2O), 4.43-4.33 (m, 2H, C H 3’ , O H ), 4.0-3.95 (m, 0.5H, C H 4’ ), 3.95-3.9 (m, 0.5H, C H 4’ ), 3.74-3.72 (m, 6H, 2 x OC H 3), 3.42-3.36 (m, 2H, C H 2OH), 3.28-3.20 (m, 1H, C H 5’ ), 3.19-3.09 (m, 1H, C H 5’ ), 2.59 (t, 2H, J = 7.6 Hz, C H 2CH2CH2), 2.38-2.29 (m, 1H, C H 2’ ), 2.24-2.03 (m, 1H, C H 2’ ), 1.64-1.54 (m, 2H, CH2C H 2CH2), 1.51-1.38 (m, 6.5H, CH2CH2C H 2, C H 3. C H 3), 1.3 (d, 1.5H, J = 6.5 Hz). Synthesis Example 9 Synthesis of 5'-O-(4,4'-dimethoxytrityl)-3'-O-((1-(5-((4-(4-[(2-cyanoethyl)(N,N-diisopropylamino)phosphino]oxybutyl)-1H-1,2,3-triazol-1-yl)methyl)-2-nitrophenyl)ethoxy)methyl)thymidine

[0343] [ka]

[0344] A solution of 5'-O-(4,4'-dimethoxytrityl)-3'-O-((1-(5-((4-(4-hydroxybutyl)-1H-1,2,3-triazol-1-yl)methyl)-2-nitrophenyl)ethoxy)methyl)thymidine (0.7 g, 0.8 mmol) in anhydrous dichloromethane (6 mL) was degassed under argon for 5 minutes before adding anhydrous diisopropylethylamine (0.42 mL, 2.4 mmol). 2-Cyanoethoxy-N,N-diisopropylaminochlorophosphine (0.214 mL, 0.96 mmol) was added dropwise and the reaction was stirred at room temperature for 1 hour. The reaction mixture was diluted with anhydrous dichloromethane (2 x 20 mL), washed with degassed saturated potassium chloride (30 mL), passed through anhydrous sodium sulfate, concentrated and dried under high vacuum. The crude product was purified by flash chromatography under argon (100% pyridine in ethyl acetate containing 0.2% ethyl acetate) to give the title compound (0.61 g, 0.57 mmol, 71%, mixture of diastereomers) as a colorless oil which was concentrated and aliquoted before being dissolved in degassed anhydrous acetonitrile and filtered through a 0.45 μm syringe filter.

[0345] 1 H NMR (400 MHz, CD3CN) δ ppm 8.96 (br. S., 1H, N H ), 7.87 (d, 0.5H, J = 8.4 Hz, C H Ar), 7.84 (d, 0.5H, J = 8.3 Hz, C H Ar), 7.57 (d, 0.5H, J = 6.7 Hz, C H Ar), 7.56 (d, 0.5H, J = 6.2 Hz, C H Ar), 7.53 (s, 0.5H, C H Triazole), 7.51 (s, 0.5H, C H triazole), 7.46-7.40 (m, 3H, C H 6 , 2 x C HDMTr), 7.35-7.22 (m, 8H, C H Ar, 7 x C H DMTr), 6.89-6.85 (m, 4H, 4 x C H DMTr), 6.14 (dd, 0.5H, J = 7.8 Hz, 6.1 Hz, C H 1’ ) 6.09 (dd, 0.5H, J = 7.6 Hz, 6.2 Hz, C H 1’ ), 5.55 (s, 1H, C H 2N), 5.51 (s, 1H, C H 2N), 5.24 (q, 0.5H, J = 6.5 Hz, C H CH3), 5.17 (q, 0.5H, J = 6.4 Hz, C H CH3), 4.65 (d, 0.5H, J = 7.2 Hz, OC H 2O), 4.64 (d, 0.5H, J = 7.3 Hz, OC H 2O), 4.5 (d, 0.5H, J = 7.4 Hz, OC H 2O), 4.46 (d, 0.5H, J = 7.3 Hz, OC H 2O), 4.42-4.36 (m, 1H, C H 3’ ), 4.00-3.98 (m, 0.5H, C H 4’ ), 3.91-3.89 (m, 0.5H, C H 4’ ) 3.79-3.52 (m, 6H, 2 x C H (CH3)2, C H 2CH2CN, C H 2OP), 3.76 (s, 6H, 2 x OC H 3), 3.29 (dd, 1H, J = 3.67 Hz, 1.96 Hz, C H 5’ ), 3.18 (d, 1H, J = 3.55 Hz, C H 5’), 2.66 (t, 2H, J = 7.3 Hz, C H 2CH2CH2), 2.69-2.59 (m, 2H, CH2C H 2CN), 2.4-2.24 (m, 1H, C H 2’ ), 2.1-2.0 (m, 1H, C H 2’ ), 1.72-1.56 (m, 4H, CH2CH2C H 2, CH2C H 2CH2), 1.51 (d, 1.5H, J = 1.1 Hz, C H 3), 1.47 (d, 1.5H, J = 1.2 Hz, C H 3), 1.45 (d, 1.5H, J = 6.4 Hz, CHC H 3), 1.35 (d, 1.5H, J = 6.5 Hz, CHC H 3) 1.13 (dd, 12 H, J = 10.3 Hz, 6.8 Hz, 2 x CH(C H 3)2). 31 P { 1 H} NMR (162 MHz, CD3CN) δ ppm 148.3. UV cutting consideration UV cleavage was performed using a handheld Analytik Jena UVP UVGL-25 4W UV lamp positioned approximately 2 cm from the sample. Irradiation of the samples in milli-Q water at 365 nm for 20 min resulted in nearly quantitative cleavage.

[0346] Standard DNA phosphoramidites, solid supports, and reagents were purchased from Link Technologies and Applied Biosystems. Automated solid-phase synthesis of polynucleotides was performed on a K&A H-8 SE DNA / RNA synthesiser. Synthesis was performed on a 0.2 or 1.0 micromole scale, with cycles of acid-catalyzed detritylation, coupling, capping, and iodine oxidation. 60 units of standard DNA phosphoramidites were coupled. Coupling efficiencies and overall synthesis yields were measured by a built-in automated trityl cation conductivity monitoring facility and were ≥98.0% in all cases. Polynucleotides were then cleaved from the solid support with concentrated ammonium hydroxide at room temperature for 60 min on an Applied Biosystems 394 synthesiser, followed by heating at 55°C for 5 h in a sealed tube to remove protecting groups from the nucleobases and backbone. Mass spectra of polynucleotides were obtained using a XEVO G2-QTOF MS instrument. - UV absorbance was performed on an Agilent Technologies Cary 60 UV-Vis at 260 nm in water.

[0347] Example 1 The 21mer polynucleotide sequence (TTTTTTTTXTTTTTTTTTT, where X is a photocleavable linker) was synthesized, cleaved from the solid support and used without further purification. The calculated mass was 6658.48 and the found mass was 6659.0.

[0348] A 1 mL milli-Q solution of 4 mM polynucleotide in water was irradiated at 365 nm for 20 min and the results analyzed by mass spectrometry. The major product was the desired TTTTTTTTT strand that had been detached by a photocleavage mechanism. The calculated mass was 2675.77, the found mass was 2675.6. The remaining material consisted of a series of polynucleotide strands containing remnants of the photocleavable linker. None of the 21mer starting sequence remained intact.

[0349] The experiment was repeated using the same sequence but without cleaving the polynucleotide from the solid support. Approximately 0.05 μmol equivalent of dried resin was placed in 1 mL of milli-Q water and irradiated at 365 nm for 20 min. The solution was passed through a 0.45 μm syringe filter and then analyzed by mass spectrometry. The only product found was the desired TTTTTTTTT strand with a calculated mass of 2675.77 and a found mass of 2674.6. UV-Vis absorbance of the resulting solution indicated that cleavage was nearly quantitative.

Claims

1. 1. A method for producing a polynucleotide or an analog or derivative thereof, comprising: (i) (A) A support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support. 【Chemical 1】 (In the formula, - (N) is a nucleoside, nucleotide, polynucleotide, or derivative or analog thereof; Q is an oxygen atom or a sulfur atom, -R ２ are each independently hydrogen, methyl, ethyl, C １ ~C ２ selected from haloalkyl and halogen groups; -R ３ is methyl, ethyl or C １ ~C ２ is haloalkyl, - (A) is a 2-nitrobenzyl group, and the 2-nitrobenzyl group is selected from the group consisting of halogen, optionally substituted C １ ~C ４ Alkyl, -OR ａ , -SR ａ , -NR ａ R ａ , -C(O)OR ａ , —C(O)NR ａ R ａ , -C(O)R ｂ , -OC(O)R ｂ and -NHC(O)R ｂ and optionally substituted with one, two, or three groups independently selected from ａ are each independently hydrogen, optionally substituted C １ ~C ２ Alkyl and optionally substituted C １ ~C ２ alkoxyl, R ｂ are each independently hydrogen and optionally substituted C １ ~C ４ alkyl groups) To prepare the following: (ii) extending the chain length of the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof by introducing one or more additional nucleoside units, nucleotide units, and / or analogs or derivatives thereof; (iii) cleaving the resulting polynucleotide from the solid support by irradiating the compound of formula (I) with light; A method comprising:

2. Q is an oxygen atom, and / or -R ２ are each independently hydrogen, methyl, C １ fluoroalkyl and fluorine, preferably R ２ are each hydrogen, and / or -R ３ is methyl, ethyl or C １ ~C ２ fluoroalkyl, preferably R ３ is methyl, and / or The method of claim 1, wherein (A) is covalently bound to a linking group, and said linking group is covalently bound to said solid support.

3. The method according to claim 2, wherein (A) is represented by formula (A-1) or formula (A-2). 【Chemistry 2】 (In the formula, -R ４ , R ５ , R ６ and R ７ are each independently hydrogen, halogen, or optionally substituted C １ ~C ４ Alkyl, -OR ａ , -SR ａ , -NR ａ R ａ , -C(O)OR ａ , —C(O)NR ａ R ａ , -C(O)R ｂ , -OC(O)R ｂ and -NHC(O)R ｂ is selected from Preferably, R ４ , R ５ , R ６ and R ７ are each independently hydrogen, fluorine, or optionally substituted C １ ~C ４ Alkyl, -OR ａ , -SR ａ , -NR ａ R ａ , -OC(O)R ｂ and -NHC(O)R ｂ More preferably, R ４ , R ５ , R ６ and R ７ are each hydrogen or methoxy, where R ａ and R ｂ is as defined in claim 1, L is the linking group; - Z is the solid support).

4. The linking group is C １ ~C ２０ Alkylene group, C ２ ~C ２０ Alkenylene group, C ２ ~C ２０ alkynylene groups and / or nucleotides or polynucleotides, wherein the alkylene group, the alkenylene group, or the alkynylene group is / are selected from the group consisting of heteroatoms; phosphite groups; phosphate groups; carbonyl groups; C ６ ~C １０ Aryl group; C ５ ~C １０ and optionally interrupted and / or terminated with one or more groups selected from a carbocyclyl group; a 5- to 10-membered heteroaryl group; and a 5- to 10-membered saturated or partially unsaturated heterocyclic group, and said linking group may be further substituted. The method of claim 2, wherein the linking group is preferably attached to the solid support via a phosphoramidite group.

5. 3. The method of claim 1 or claim 2, wherein (i) the solid support comprises glass, silica, ceramic, or polymeric resin, and / or (ii) the solid support comprises particles having a diameter of about 10 to about 1000 μm.

6. The method of claim 1 or claim 2, wherein (N) is connected to adjacent oxygen atoms of formula (I) at the 3' or 5' position of the nucleoside, nucleotide, polynucleotide, or derivative or analog thereof.

7. The method according to claim 1, wherein (N) is represented by formula (N-1) or formula (N-2). 【Chemistry 3】 (In the formula, X is an oxygen atom, a nitrogen atom, a sulfur atom, or —C(R ａ R ａ ) - and -R １ and R ＤＲ are each independently hydrogen, halogen, or optionally substituted C １ ~C ４ Alkyl, -OR ａ , -SR ａ , -NR ａ R ａ , -C(O)OR ａ , —C(O)NR ａ R ａ and -C(O)R ｂ where R ａ and R ｂ is as defined in claim 1, and R ＤＲ Ga-OR ａ When R ａ group, and the R １ one of the groups may be linked together to form a bridging motif; -R Ｐ is hydrogen, a hydroxyl protecting group, a phosphoryl group or a salt or acid thereof, a diphosphoryl group (-P(O ２ － )-O-P(O ３ － )) or its salt or acid, a triphosphoryl group (—P(O ２ － )-P(O ２ － )-O-P(O ３ － )) or a salt or acid thereof, or a phosphorus-based bond with a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof, -R Ｂ is an optionally substituted natural or unnatural nucleobase or a derivative or analog thereof, - the wavy line indicates the point of attachment to the compound of formula (I), More preferably, X is an oxygen atom, and / or -R １ are each independently hydrogen, halogen, or C １ ~C ２ Alkyl, C １ ~C ２ Haloalkyl and C １ ~C ２ alkoxyl; preferably, R １ are each hydrogen, and / or -R ＤＲ is hydrogen or halogen, preferably R ＤＲ is hydrogen, and / or -R Ｐ is a hydrogen or hydroxyl protecting group).

8. Step (i) is (a) a compound of formula (Ia) 【Chemistry 4】 (In the formula, Q, R ２ , R ３ and (A) are as defined in claim 1 or claim 2, and R Ｓ is C １ ~C ４ alkyl) To prepare the following: (b) reacting the compound of formula (Ia) with a nucleoside, nucleotide, polynucleotide, or a derivative or analog thereof to form a compound of formula (I).

9. Step (i) is a reaction of a compound of formula (I) or formula (Ia) 【Chemistry 5】 (In the formula, (N), Q, R ２ , R ３ , R Ｓ and (A) is as defined in claim 1 or claim 2). The method of claim 1 or claim 2, comprising binding to a solid support.

10. In step (ii), extending the chain length is contacting the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof with a nucleoside or derivative thereof to form a phosphorus-based bond. one or more times, In step (ii), extending the chain length is (a) providing a nucleoside having (i) a phosphoramidite group at the 3' position and a hydroxyl protecting group at the 5' position; or (ii) a phosphoramidite group at the 5' position and a hydroxyl protecting group at the 3' position; (b) activating the nucleoside phosphoramidite of step (a); (c) contacting the support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof with the activated nucleoside phosphoramidite to form a phosphite triester bond; (d) oxidizing the phosphite triester bond to form a phosphotriester bond; (e) removing the hydroxyl protecting group from the terminal nucleoside of the resulting support-bound polynucleotide; The method according to claim 1 or claim 2, preferably comprising carrying out steps (a) to (e) of the above once or a plurality of times.

11. 3. The method of claim 1 or claim 2, further comprising one or more steps of deprotecting the support-bound polynucleotide after step (ii) and before step (iii).

12. 3. The method of claim 1 or claim 2, wherein step (iii) comprises photoirradiating the compound of formula (I) using UV light having a wavelength of about 300 to about 500 nm.

13. (A) A support-bound nucleoside, nucleotide, or derivative or analog thereof, which is a compound of formula (I) bound to a solid support. 【Chemistry 6】 (In the formula, Q, R ２ , R ３ and (A) is as defined in claim 1; (N) is a nucleoside, nucleotide, or derivative or analog thereof).

14. Compounds of formula (I) 【Chemistry 7】 (In the formula, Q, R ２ , R ３ and (A) is as defined in claim 1; (N) is represented by formula (N-1) or formula (N-2), 【Chemistry 8】 (In the formula, X is an oxygen atom, a nitrogen atom, a sulfur atom or —C(R a R a )—; - R 1 and R DR are each independently selected from hydrogen, halogen, optionally substituted C 1 -C 4 alkyl, -OR a , -SR a , -NR a R a , -C(O)OR a , -C(O)NR a R a and -C(O)R b , where R a and R b are as defined in claim 1 and when R DR is -OR a , the R a group and one of the R 1 groups may be linked together to form a bridging motif; -R P is hydrogen, a hydroxyl protecting group, a phosphoryl group or a salt or acid thereof, a diphosphoryl group (-P(O 2 - )-O-P(O 3 - )) or a salt or acid thereof, a triphosphoryl group (-P(O 2 - )-P(O 2 - )-O-P(O 3 - )) or a salt or acid thereof, or a phosphorus-based bond to a nucleoside, nucleotide or polynucleotide or a derivative or analog thereof; R B is an optionally substituted natural or non-natural nucleobase or a derivative or analogue thereof; - the wavy line indicates the point of attachment to the compound of formula (I), More preferably, X is an oxygen atom, and / or each R 1 is independently selected from hydrogen, halogen, C 1 -C 2 alkyl, C 1 -C 2 haloalkyl and C 1 -C 2 alkoxyl; preferably, each R 1 is hydrogen, and / or R DR is hydrogen or halogen, preferably R DR is hydrogen, and / or - R P is hydrogen or a hydroxyl protecting group).

15. (A) a support-bound compound of formula (Ia) attached to a solid support; 【Chemistry 9】 (In the formula, Q, R ２ , R ３ and (A) is as defined in claim 1).

16. 14. A method for producing a support-bound nucleoside, nucleotide, or derivative or analog thereof as defined in claim 13, comprising: (a) providing a compound of formula (Ia) bound to a solid support as defined in claim 15 (A); (b) reacting said compound of formula (Ia) with a nucleoside, nucleotide, or derivative or analog thereof to produce a support-bound nucleoside, nucleotide, or derivative or analog thereof; A method comprising:

17. A solid support column for solid phase polynucleotide synthesis, comprising a support-bound nucleoside, nucleotide, polynucleotide, or derivative or analog thereof, which is a compound of formula (I) attached to a solid support in (A). 【Chemistry 10】 (In the formula, (N), Q, R ２ , R ３ and (A) is as defined in claim 1 or claim 2).

18. 16. Use of a support-bound nucleoside, nucleotide or derivative or analog thereof as defined in claim 13, a compound of formula (I) as defined in claim 14, or a compound of formula (Ia) as defined in claim 15, for synthesizing a polynucleotide or a derivative or analog thereof.

19. A polynucleotide or a derivative or analog thereof obtained by the method defined in claim 1 or claim 2.

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