Patent Application
20250154191
The present invention relates to a solid phase synthesis method of an oligonucleotide that enables cleavage of a synthesized oligonucleotide even under mild conditions by using a nucleoside phosphoramidite with a specific structure, and the like.
Solid phase synthesis methods using the phosphoramidite method have been widely used for chemical synthesis of oligonucleotides (nucleic acids) such as DNA and RNA. At the first stage of the synthesis cycle, however, the 3′-terminal nucleoside of the oligonucleotide (nucleic acid) sequence to be synthesized needs to be carried on a solid phase support.
In this case, there is a method including preparing, depending on the nucleoside that becomes the 3′-terminus of the oligonucleotide to be synthesized, a solid phase support on which the nucleoside is carried in advance and performing the subsequent nucleotide elongation reaction. However, it is complicated to prepare each time a solid phase support on which the 3′-terminal nucleoside of the oligonucleotide to be synthesized is carried in advance. On the other hand, when a solid phase support carrying a universal linker capable of coupling with any nucleoside (e.g., Patent Literature 1, Patent Literature 2) is used, the nucleoside at the 3′-terminal of oligonucleotide can be freely introduced as the first nucleoside in the step of elongating the oligonucleotide onto the universal linker, regardless of the sequence of the oligonucleotide to be synthesized, and an oligonucleotides with any sequence can be effectively synthesized regardless of the nucleoside at the 3′-terminal. Therefore, such universal linkers are widely used in solid phase synthesis.
More specifically, an universal linker is carried in advance on a solid phase support via a cleavable linker (spacer) such as succinyl group and the like, and any nucleoside at the 3′-terminus is coupled to the universal linker, after which an oligonucleotide elongation reaction generally including the following steps:
By repeating the above synthesis cycle and proceeding with the elongation reaction of the oligonucleotide from the 3′-terminus to the 5′-terminus, an oligonucleotide (nucleic acid) having the desired sequence is synthesized.
Finally, the cleavable linker is hydrolyzed with aqueous ammonia, methylamine solution, and the like, and the synthesized oligonucleotide (nucleic acid) is cleaved from the solid phase support and the universal linker.
The nucleoside phosphoramidites (nucleic acid monomers) widely used in the above-mentioned conventional solid phase phosphoramidite methods are a group of nucleoside phosphoramidites represented by a compound shown by the following formula (A):
However, when a widely used nucleoside phosphoramidite is used for coupling with a universal linker, in the final step of solid phase synthesis in which the cleavable linker is hydrolyzed with aqueous ammonia or methylamine solution and the synthesized oligonucleotide is separated from the solid phase support and the universal linker, strongly basic conditions such as “treatment with concentrated aqueous ammonia, 55° C., 8 hr” are often required. It has thus been difficult to use modified nucleic acids and functional molecules, that are vulnerable to basic conditions, in oligonucleotide (nucleic acid) synthesis. For this reason, the development of a solid phase synthesis method of oligonucleotides (nucleic acids) that enables cleavage of synthesized oligonucleotides (nucleic acids) even under milder conditions is awaited.
The present inventors have conducted intensive studies in an attempt to solve the aforementioned problems and found that the above-mentioned problems can be solved by converting the “cyanoethyl group” used as a hydroxy-protecting group on the phosphorus atom for the widely-used nucleoside phosphoramidite to an alkoxy group and the like, and created a solid phase oligonucleotide (nucleic acid) synthesis method that enables cleavage of synthesized nucleic acid even under mild conditions.
Furthermore, they have also found that, in the solid phase oligonucleotide (nucleic acid) synthesis method of the present invention, a linker with a simpler structure can also be used as a universal linker in place of the linkers widely used at present.
That is, one embodiment of the present invention is, in a solid phase synthesis of an oligonucleotide by using a universal linker, a solid phase synthesis method of an oligonucleotide, including coupling, as a nucleoside phosphoramidite for introducing a 3′-terminal nucleoside, a nucleotide derivative represented by the following formula (I):
More specific embodiments of the present invention are shown in the following.
[1]A solid phase synthesis method of an oligonucleotide, comprising a step of subjecting an oligonucleotide having, at the 3′-terminus of an oligonucleotide sequence thereof, a nucleotide derivative represented by the formula (II):
Y1—O—C(X1)(X2)C(X3)(X4)—O—Y2 (III)
According to the present invention, a solid phase synthesis method of an oligonucleotide that enables cleavage of a synthesized oligonucleotide (nucleic acid) even under mild conditions, a novel nucleoside phosphoramidite useful for practicing the solid phase synthesis method of an oligonucleotide of the present invention, and a universal linker with a simple structure useful for practicing the solid phase synthesis method of an oligonucleotide of the present invention are disclosed.
A solid phase synthesis method of an oligonucleotide as one embodiment of the present invention is
[1] a solid phase synthesis method of an oligonucleotide, comprising a step of subjecting an oligonucleotide having, at the 3′-terminus of an oligonucleotide sequence thereof, a nucleotide derivative represented by the formula (II):
wherein * is a bonding position to the universal linker;
More specifically,
[2] a solid phase synthesis method of an oligonucleotide, including
As used herein, since the “oligonucleotide having the desired sequence” obtained in step (2) corresponds to “the oligonucleotide having, at the 3′-terminus of an oligonucleotide sequence thereof, a nucleotide derivative represented by the formula (II):
The “oligonucleotide (nucleic acid)” of interest in the present invention means a chain compound in which nucleosides are linked by phosphodiester bonds, and includes DNA, RNA, and the like. The oligonucleotide (nucleic acid) may be either single-stranded or double-stranded, but is preferably single-stranded because it can be efficiently synthesized using an oligonucleotide (nucleic acid) synthesizer. In addition, the “oligonucleotide (nucleic acid)” includes not only oligonucleotides containing purine bases such as adenine (A) and guanine (G) and pyrimidine bases such as thymine (T), cytosine (C), and uracil (U), but also modified oligonucleotides (nucleic acids) containing other modified heterocyclic bases. In the present invention, nucleosides linked by phosphorothioate bonds are also included in the “modified oligonucleotide (nucleic acid)”. In addition, cases where the sugar moiety of nucleoside is modified or where a crosslinked structure is formed between the atoms constituting the sugar moiety (bridged nucleotide, BNA/LNA) are also included in the “modified oligonucleotide (nucleic acid)”.
As described above, the present invention is characterized in that the desired oligonucleotide is synthesized by carrying a nucleotide derivative represented by the formula (I):
Each group in the nucleotide derivative (I) is described below.
In the present specification, examples of the “halogen atom” include fluorine, chlorine, bromine, and iodine.
In the present specification, the “alkyl group” (including “alkyl moiety” constituting the group) refers to a linear or branched chain saturated hydrocarbon group, where one having 1 to 6 carbon atoms is preferred. Examples of the “C1-6 alkyl group” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl.
In the present specification, the “alkenyl group” (including “alkenyl moiety” constituting the group) refers to a linear or branched chain hydrocarbon group having one or more double bonds in a molecule, where one having 2 to 6 carbon atoms is preferred. Examples of the “C2-6 alkenyl group” include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, and 5-hexenyl.
In the present specification, the “alkynyl group” (including “alkynyl moiety” constituting the group) refers to a linear or branched chain hydrocarbon group having one or more triple bonds in a molecule, where one having 2 to 6 carbon atoms is preferred. Examples of the “C2-6 alkynyl group” include ethynyl, 1-propynyl, 2-propynyl(propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, and 4-methyl-2-pentynyl.
In the present specification, the “alkoxy group” (including “alkoxy moiety” constituting the group) refers to a group having a structure in which a hydrogen atom of the hydroxy group is substituted by the aforementioned “alkyl group” where one having 1 to 6 carbon atoms is preferred. Examples of the “C1-6 alkoxy group” include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy group, sec-pentyloxy group, tert-pentyloxy group, hexyloxy group, and the like.
In the present specification, the “alkylthio group” (including “alkylthio moiety” constituting the group) refers to a group having a structure in which a hydrogen atom of the thiol group is substituted by the aforementioned “alkyl group” where one having 1 to 6 carbon atoms is preferred. Examples of the “C1-6 alkylthio group” include methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio, isopentylthio, neopentylthio, 1-ethylpropylthio, hexylthio, isohexylthio, 1,1-dimethylbutylthio, 2,2-dimethylbutylthio, 3,3-dimethylbutylthio, and 2-ethylbutylthio.
In the present specification, the “aryl group” (including “aryl moiety” constituting the group) refers to an aromatic hydrocarbon ring group where one having 6 to 14 carbon atoms is preferred. Examples of the C6-14 aromatic hydrocarbon ring group include phenyl, naphthyl, and the like.
In the present specification, the “cyclic group” (including “cyclic group moiety” constituting the group) refers to “hydrocarbon ring group” and “heterocyclic group”.
In the present specification, examples of the “hydrocarbon ring group” include aromatic hydrocarbon ring group (preferably, C6-14 aromatic hydrocarbon ring group having 6 to 14 carbon atoms), cycloalkyl (preferably, C3-10 cycloalkyl having 3 to 10 carbon atoms), and cycloalkenyl (preferably, C3-10 cycloalkenyl having 3 to 10 carbon atoms).
In the present specification, examples of the “C6-14 aromatic hydrocarbon ring group” include phenyl and naphthyl.
In the present specification, examples of the “C3-10 cycloalkyl” include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
In the present specification, examples of the “C3-10 cycloalkenyl” include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.
In the present specification, examples of the “heterocyclic group” include (i) an aromatic heterocyclic group, (ii) a non-aromatic heterocyclic group, and (iii) a 7- to 10-membered crosslinked heterocyclic group, each containing, as a ring-constituting atom besides carbon atom, 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom.
In the present specification, examples of the “aromatic heterocyclic group” (including “5- to 14-membered aromatic heterocyclic group”) include a 5- to 14-membered (preferably, 5- to 10-membered) aromatic heterocyclic group containing, as a ring-constituting atom besides carbon atom, 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.
Preferred examples of the “aromatic heterocyclic group” include 5- or 6-membered monocyclic aromatic heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and the like; and 8- to 14-membered fused polycyclic (preferably, bi or tricyclic) aromatic heterocyclic groups such as benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl, phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like.
In the present specification, examples of the “non-aromatic heterocyclic group” (including “3- to 14-membered non-aromatic heterocyclic group”) include a 3- to 14-membered (preferably, 4- to 10-membered) non-aromatic heterocyclic group containing, as a ring-constituting atom besides carbon atom, 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.
Preferred examples of the “non-aromatic heterocyclic group” include 3- to 8-membered monocyclic non-aromatic heterocyclic groups such as aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanyl, azocanyl, diazocanyl, and the like; and 9- to 14-membered fused polycyclic (preferably, bi or tricyclic) non-aromatic heterocyclic groups such as dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzisothiazolyl, dihydronaphtho[2,3-b]thienyl, tetrahydroisoquinolyl, tetrahydroquinolyl, 4H-quinolizinyl, indolinyl, isoindolinyl, tetrahydrothieno[2,3-c]pyridinyl, tetrahydrobenzazepinyl, tetrahydroquinoxalinyl, tetrahydrophenanthridinyl, hexahydrophenothiazinyl, hexahydrophenoxazinyl, tetrahydrophthalazinyl, tetrahydronaphthyridinyl, tetrahydroquinazolinyl, tetrahydrocinnolinyl, tetrahydrocarbazolyl, tetrahydro-β-carbolinyl, tetrahydroacrydinyl, tetrahydrophenazinyl, tetrahydrothioxanthenyl, octahydroisoquinolyl, and the like.
In the present specification, preferred examples of the “7- to 10-membered crosslinked heterocyclic group” include quinuclidinyl and 7-azabicyclo[2.2.1]heptanyl.
Examples of the “heterocycle” when Z1 and Z2 are bonded to each other to form, together with a nitrogen atom bonded thereto, an optionally substituted heterocycle include a nitrogen-containing aromatic heterocycle and a nitrogen-containing non-aromatic heterocycle, each containing, as a ring-constituting atom, 1 to 4 nitrogen atoms besides carbon atom, and further, 1 to 4 hetero atoms selected from a sulfur atom and an oxygen atom.
In the present specification, preferred examples of the “nitrogen-containing aromatic heterocycle” include 5- or 6-membered monocyclic nitrogen-containing aromatic heterocycles such as imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, 1,2,4-oxadiazole, 1,3,4-oxadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, triazole, tetrazole, triazine, and the like; and
In the present specification, preferred examples of the “nitrogen-containing non-aromatic heterocycle” include 3- to 8-membered (more preferably, 5- or 6-membered) monocyclic nitrogen-containing non-aromatic heterocycles such as aziridine, pyrroline, pyrrolidine, imidazoline, imidazolidine, oxazoline, oxazolidine, pyrazoline, pyrazolidine, thiazoline, thiazolidine, tetrahydroisothiazole, tetrahydroxazole, tetrahydroisoxazole, piperidine, piperazine, tetrahydropyridine, dihydropyridine, tetrahydropyrimidine, tetrahydropyridazine, morpholine, thiomorpholine, and the like; and 9- to 14-membered fused polycyclic (preferably, bi or tricyclic) nitrogen-containing non-aromatic heterocycles such as dihydrobenzimidazole, dihydrobenzoxazole, dihydrobenzothiazole, dihydrobenzisothiazole, tetrahydroisoquinoline, tetrahydroquinoline, 4H-quinolizine, indoline, isoindoline, tetrahydrothieno[2,3-c]pyridine, tetrahydrobenzazepine, and the like.
In the present specification, the “hydroxy-protecting group” for Y is not particularly limited, and those generally used for protecting the 5′-hydroxy group in the solid phase synthesis of oligonucleotides (nucleic acids) can be used. For example, trityl-based protecting groups, silyl-based protecting groups, and acyl-based protecting groups can be mentioned.
Examples of the “trityl-based protecting group” include trityl groups optionally substituted by any substituents (e.g., substituents selected from C1-6 alkoxy group, C1-6 alkyl group, halogen atom, and the like (two or more substituents may be joined to form a ring)). Specifically, a trityl group (Tr), a mono-methoxytrityl group (e.g., 4-methoxytrityl group (MMTr)), a dimethoxytrityl group (e.g., 4,4′-dimethoxytrityl group (DMTr)), a 9-phenylxanthen-9-yl group (pixyl group), and the like, preferably, a 4,4′-dimethoxytrityl group (DMTr), can be mentioned.
Examples of the “silyl-based protecting group” include silyl groups tri-substituted by any substituents (e.g., substituents selected from C1-6 alkoxy group, C1-6 alkyl group, phenyl group, and the like). Specifically, a trimethylsilyl group, a triethylsilyl group, an isopropyldimethylsilyl group, a tert-butyldimethylsilyl group, a dimethylmethoxysilyl group, a methyldimethoxysilyl group, a tert-butyldiphenylsilyl group, and the like, preferably, a trimethylsilyl group, can be mentioned.
Examples of the “acyl-based protecting group” include an acetyl group, a t-butoxycarbonyl group, a benzoyl group, a levulinoyl group, and the like.
Among the above-mentioned hydroxy-protecting groups, a trityl-based protecting group is preferred and a 4,4′-dimethoxytrityl group (DMTr) is more preferred, because deprotection is easily performed using an acid in the case of hydroxy-protecting groups that are removed by acids.
In the present specification, “optionally substituted” means an embodiment without substitution or with substitution with 1 to 3 substituents at substitutable positions. In the case of 2 or 3 substitutions, each substituent may be the same or different.
In the present specification, “substituted” means an embodiment with substitution with 1 to 3 substituents at substitutable positions. In the case of 2 or 3 substitutions, each substituent may be the same or different.
Examples of the substituent that each group of the nucleotide derivative (I), such as “an optionally substituted alkoxy group”, “an optionally substituted cyclic group-oxy group”, “an optionally substituted alkyl group”, “an optionally substituted alkenyl group”, “an optionally substituted alkynyl group”, “an optionally substituted aryl group”, and the like, can have include
In the “optionally substituted alkoxy group”, “optionally substituted alkyl group”, “optionally substituted alkenyloxy group”, and “optionally substituted alkynyloxy group” of the nucleotide derivative (I), when the “alkoxy group”, “alkyl group”, “alkenyloxy group”, and “alkynyloxy group” are each substituted by a “substituent” selected from a formylamino group, a C1-10 alkyl-carbonylamino group, a carbamoyl group, and a C1-10 alkyl-carbamoyl group, the total carbon number of the constituent moieties other than —CONH— (or —NHCO—) is preferably 1 to 10.
In the present specification, the “optionally modified nucleobase” is not particularly limited as long as it is used for the synthesis of oligonucleotides (nucleic acids). For example, cytosyl group, uracil group, thyminyl group, adenyl group, guanyl group, and modified nucleobases of these such as 8-bromoadenyl group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, 5-methylcytosyl group, 8-oxoguanyl group, hypoxanthinyl group, and the like can be mentioned.
Here, in the “nucleobase”, for example, an amino group of the adenyl group, guanyl group and cytosyl group or modified nucleobase may be protected. The amino-protecting group is not particularly limited as long as it is used as a nucleic acid-protecting group, and specifically, for example, benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl and the like are optionally used for protection.
Preferred embodiments of each symbol in the nucleotide derivative (I) are described below.
Y is as defined above, and is preferably
Z1 and Z2 are each independently as defined above, and preferably
R1 is as defined above, and is preferably
R2 is as defined above, and is preferably
R3 is as defined above, and is preferably
R2 and R3 are bonded to each other to form a group represented by the following formula (shown in the order of -R2-R3-):
—O—C(Ra)2—,—O—C(Rb)2—C(Rc)2—,—O—C(Rd)2—O—,—N(Re)—C(Rf)2—,—N(Rg)—CO—,—S—C(Rh)2—, or —C(Ri)2—C(Rj)2—
—O—C(Ra)2—, —O—C(Rb)2—C(Rc)2—, —O—C(Rd)2—O—,—N(Re)—C(Rf)2—, —N(Rg)—CO—,—S—C(Rh)2-, or —C(Ri)2-C(Rj)2-
—O—C(Ra)2—, —O—C(Rb)2—C(Rc)2—, —O—C(Rd)2—O—, —N(Re)—C(Rf)2—, —N(Rg)—CO—, —S—C(Rh)2—, or —C(Ri)2—C(Rj)2—
Base is an optionally modified nucleobase.
In the nucleotide derivative (I) of the present invention, when X is an optionally substituted (C4-C5)alkoxy group, the nucleotide derivative (I) is a novel compound.
The nucleotide derivative (I) in the present invention can be produced by appropriately applying the specific methods described in Examples/Production Examples below or methods known in the pertinent technique field. When the nucleotide derivative (I) is commercially available, commercially available products may also be used.
Preferred embodiments of each symbol of the nucleotide derivative residue (II) are the same as those described above for nucleotide derivative (I).
Preferred nucleotide derivatives (I) are described below.
[Nucleotide derivative (I-A)]
A nucleotide derivative (I), wherein
—O—C(Ra)2—, —O—C(Rb)2—C(Rc)2—, —O—C(Rd)2—O—,—N(Re)—C(Rf)2—, —N(Rg)—CO—, —S—C(Rh)2—, or —C(Ri)2—C(Rj)2—
A nucleotide derivative (I), wherein
—O—C(Ra)2—, —O—C(Rb)2—C(Rc)2—, —O—C(Rd)2—O—,—N(Re)—C(Rf)2—, —N(Rg)—CO—,—S—C(Rh)2—, or —C(Ri)2—C(Rj)2—
A nucleotide derivative (I-A) or (I-B), wherein
Y is a dimethoxytrityl group.
A nucleotide derivative (I-A), (I-B) or (I-C), wherein R1 is a hydrogen atom, a halogen atom, a hydroxy group,
As preferred nucleotide derivative residue (II), reference is made to those mentioned above for nucleotide derivative (I).
In practicing the present invention, the above-mentioned nucleotide derivative (I) is carried on a solid phase support via a universal linker, more specifically, the nucleotide derivative (I) is linked onto a solid phase support via a covalent bond in an embodiment shown by (solid phase support (SM))-(spacer (L))-(universal linker (UL))-(nucleotide derivative (I)) (hereinafter to be also denoted as “solid phase support linked-nucleotide derivative (I)”).
When the universal linker can be directly linked onto the solid phase support, the spacer is not essential.
In the following, the conjugate of (solid phase support (SM))-(spacer (L))-(universal linker (UL)) is sometimes simply denoted as a “solid phase support”.
In the present specification, “solid phase support” (to be also denoted as “SM”) is not particularly limited as long as it is a carrier for solid phase synthesis from which excess reagents can be easily removed by washing. Examples include a glass-based porous support, a porous polymer support such as polystyrene-based porous support, acrylamide-based porous support, and the like, and the like, and a polystyrene-based porous support and a glass-based porous support are preferred.
In the present specification, the “glass-based porous support” refers to a porous.support containing glass as a constituent component. For example, porous glass particles (CPG) in a particle form and the like can be mentioned, but it is not limited thereto. More specifically, as the aforementioned CPG, a CPG solid phase support having a long chain amino alkyl spacer (LCAA-CPG solid phase support) is preferably used. In the case of synthesis of a long chain nucleotide, CPG with pores of 20 to 400 nm, more preferably 50 to 200 nm, further preferably 100 nm, is used.
In the present specification, the “polystyrene-based porous support” is a porous support made of a resin mainly composed of a structural unit of styrene or a derivative thereof, and in particular, a polystyrene-based porous support having amino group and/or hydroxy group is preferred.
Examples of the polystyrene-based porous support include a porous support composed of styrene-hydroxystyrene-divinylbenzene-based copolymer particles (JP-A-2005-097545, JP-A-2005-325272 and JP-A-2006-342245), a porous support composed of a styrene-(meth)acrylonitrile-hydroxystyrene-divinylbenzene-based copolymer (JP-A-2008-074979), and the like.
In the present specification, the “acrylamide-based porous support” is a porous support made of a resin mainly composed of a structural unit of acrylamide or a derivative thereof, and in particular, an acrylamide-based porous support having amino group and/or hydroxy group is preferred and an acrylamide-based porous support having a hydroxy group is preferred.
Examples of the acrylamide-based porous support include a porous support composed of an aromatic mono-vinyl compound-divinyl compound-(meth)acrylamide derivative-based copolymer and the like. When the support is an acrylamide-based solid phase support and the content of the structural unit derived from (meth)acryl amide derivative monomer is too low, the effect of preventing a decrease in the amount of nucleic acid synthesized and a decrease in the synthesis purity cannot be obtained, whereas when the content is too high, the formation of porous resin beads becomes difficult. Accordingly, the content is preferably 0.3 to 4 mmol/g, more preferably 0.4 to 3.5 mmol/g, further preferably 0.6 to 3 mmol/g.
The above-mentioned solid phase support may be any solid phase support having a functional group capable of forming a covalent bond with a spacer (cleavable linker). In particular, a solid phase support having an amino group and/or a hydroxy group is preferred. In this case, the bond between the solid phase support (SM) and the spacer (L) is, for example, an amide bond or an ester bond.
In the present invention, while the content of the functional group in the solid phase support is not particularly limited, when the content of the functional group is too low, the yield of the oligonucleotide (nucleic acid) decreases, whereas when the content of the functional group is too high, the purity of the obtained oligonucleotide (nucleic acid) decreases. Those of ordinary skill in the art can appropriately select preferred contents.
In the present specification, the “spacer” (to be also denoted as “L”) refers to a molecule that connects the universal linker and the solid phase support via a covalent bond. In order to efficiently cleave the resultant product from the solid phase support in the final stage of solid phase synthesis, it is preferred that the spacer be cleavable, and more preferably, can be hydrolyzed by an alkaline solution such as aqueous ammonia or a methylamine solution. Known spacers used in the pertinent technique field for solid phase synthesis of oligonucleotides can also be used in practicing the present invention.
The “spacer” is as defined above, and is preferably a divalent group represented by the formula (III):
When the structure of the spacer is an asymmetric structure, either the left or right bonding site may be bonded to the nucleotide derivative (I) of the present invention.
The “inactive” in the “inactive divalent group” refers to the absence of a functional group that inhibits solid phase synthesis reactions, such as a hydroxy group, an amino group, a carboxy group, a sulfanyl group, a sulfo group, and the like.
La as the “inactive divalent group” is more preferably an inactive divalent group whose main chain is composed of atoms selected from a carbon atom, an oxygen atom, a sulfur atom, and a nitrogen atom (e.g., 1 to 200 atoms, preferably, 1 to 150 atoms, more preferably, 1 to 100 atoms, further preferably, 1 to 50 atoms, further more preferably, 1 to 10 atoms, and particularly preferably, 1 to 5 atoms).
La is preferably —CH2OC6H4OCH2—, —CH2CH2—, or —CH2CH2CH2—, more preferably —CH2OC6H4OCH2— or —CH2CH2—.
In the present invention, the length of the spacer is not particularly limited. It can be appropriately set according to the chain length of the desired oligonucleotide (nucleic acid) sequence.
In the present invention, the “universal linker” (to be also denoted as “UL”) is a linker that is linked to a solid phase support via a covalent bond or a spacer, and is used as a starting point for synthesizing the desired oligonucleotide on the solid phase support. By using this universal linker, the desired oligonucleotide can be obtained, regardless of the type of nucleoside at the 3′-terminus of the desired oligonucleotide, by starting the synthesis by reacting the nucleoside phosphoramidite to be the 3′-terminal in the same step as in a general automated nucleic acid synthesis, synthesizing the desired oligonucleotide, and cleaving same from the solid support by a method similar to the general method.
The universal linker used in the present invention is not particularly limited, and may be the universal linker disclosed or cited in JP-A-2011-088843, the universal linker disclosed in JP-A-2016-204316, or the like. Alternatively, it may be a commercially available universal support in which a universal linker is linked to a solid phase support. More specifically, for example, UnyLinker (trade name) universal support for oligonucleotide synthesis (Chem Genes), Universal Syn Base (trade name) and Universal Q Syn Base (trade name) (Biosearch Technologies), and Universal Support I, II, and III (Glen Research), and the like are used.
In this step (1), the aforementioned “solid phase support linked-nucleotide derivative (I)” can be produced by those of ordinary skill in the art by appropriately applying the specific methods described in Examples/Production Examples below or methods known in the pertinent technique field.
For example, a group represented by HO-L- is reacted with the corresponding acid anhydride to introduce a spacer into a universal linker (UL), and then a reaction with a solid phase support is performed in a solvent in the presence of a condensing agent and a base to obtain a “(solid phase support (SM))-(spacer (L))-(universal linker (UL)) conjugate”. The conjugate is then loaded into a reaction column of an oligonucleotide automatic synthesizer and the nucleotide derivative (I) of the present invention, which serves as the starting point for the subsequent nucleotide elongation reaction, is bonded as the first (3′-terminal) nucleotide to the universal linker (UL) by appropriately applying the specific methods described in Examples/Production Examples below or methods known in the pertinent technique field, whereby the “solid phase support linked-nucleotide derivative (I)” can be produced.
When the “(solid phase support (SM))-(spacer (L))-(universal linker (UL)) conjugate” is commercially available, commercially available products may also be used.
Step (2) is a step of sequentially condensing nucleic acid monomers according to the desired oligonucleotide sequence, by using the nucleotide derivative (I) of the “solid phase support linked-nucleotide derivative (I)” obtained in step (1) as a starting point, to elongate the nucleotide.
In the “solid phase support linked-nucleotide derivative (I)”, the nucleotide derivative (I) specifically takes the form of “nucleotide derivative residue (II)”.
The “nucleic acid monomer” here is a nucleoside phosphoramidite appropriately selected according to the oligonucleotide sequence and can be produced by appropriately applying the specific methods described in Examples/Production Examples below or methods known in the pertinent technique field. When the “nucleic acid monomer” is commercially available, commercially available products may also be used. For example, a group of nucleoside phosphoramidites represented by a compound of the following formula (A):
As the nucleic acid monomer, one or two or more “nucleotide derivatives (I)” of the present invention may also be used.
In the nucleotide elongation reaction in step (2), step A: a step of deprotecting 5′ hydroxy group of nucleotide (including nucleotide derivative (I) at the 3′-terminus)—step B: a step of condensing with nucleic acid monomer (phosphoramidite)—step C: a step of oxidizing/sulfurizing nucleotide linked moiety, are successively repeated once or twice or more according to the desired nucleic acid sequence, whereby an oligonucleotide having the desired sequence can be obtained. The respective steps A to C can be performed by those of ordinary skill in the art by appropriately applying the specific methods described in Examples/Production Examples below or methods known in the pertinent technique field.
Step (3) is a step of subjecting the obtained oligonucleotide having the desired sequence to a reaction for cleaving from the solid phase support.
The reaction for cleaving in this step can be performed by those of ordinary skill in the art by appropriately applying the specific methods described in Examples/Production Examples below or methods known in the pertinent technique field. For example, it is performed by treating the solid phase support on which the oligonucleotide has been bonded with ammonia, amines and/or base and recovering the oligonucleotide with the desired sequence.
Examples of ammonia include concentrated aqueous ammonia. Examples of amines include alkylamine (methylamine, ethylamine, isopropylamine, ethylenediamine, diethylamine, triethylamine, etc.). Examples of base include metal carbonates such as potassium carbonate and the like. One or more kinds of ammonia, amines, and/or base can be used in combination.
It is desirable to use ammonia, amines, and/or base in a mixture with a solvent. Examples of the solvent include water, alcohols (e.g., methanol, ethanol, etc.) and the like. Two or more kinds of these solvents may be used in a mixture in an appropriate ratio.
More specifically, step (3) is performed by pouring a solution of ammonia, amines, and/or base into a column containing a solid phase support to which an oligonucleotide has been bonded, thereby bringing the solution into contact with the solid phase support to which the oligonucleotide is bonded.
In the reaction for cleaving in step (3), the reaction temperature thereof is appropriately selected according to the reaction conditions and is not particularly limited. The reaction is generally performed in the range of from room temperature to under warming. The reaction time is appropriately selected according to the reaction conditions and is not particularly limited, but the reaction is generally performed within the range of 1 minute to 10 hours.
The reaction for cleaving in step (3) of the present invention is preferably performed by contacting the solid phase support, on which the oligonucleotide has been carried via a universal linker, with
Further preferably, it is performed by contacting the solid phase support, on which the oligonucleotide has been carried via a universal linker, with
In the present invention, the nucleotide derivative (I) is used as the 3′-terminal nucleotide in oligonucleotide synthesis. Because of this, the present invention is characterized in that the desired oligonucleotide can be efficiently cleaved not only under general cleavage conditions, but also under cleavage conditions with extremely mild basic conditions. This point is specifically described in the Examples below.
As described above, in oligonucleotide synthesis using the nucleotide derivative (I) of the present invention, the desired oligonucleotide can be efficiently cleaved even under extremely mild cleavage conditions. The present inventors have also found that a simple 1,2-diol derivative having a 1,2-diol structure can be used as a universal linker when the nucleotide derivative (I) of the present invention is used.
More specifically, they have found that a 1,2-diol derivative represented by the following formula (III):
Y1-O—C(X1)(X2)C(X3)(X4)-O-Y2 (III)
Each group in the 1,2-diol derivative (III) is described below.
As the “chain or cyclic substituent”, the “alkyl group”, “alkenyl group”, “alkynyl group”, and “alkoxy group” described above for the nucleotide derivative (I) can be mentioned as the “chain substituent”, and the aforementioned “cyclic group” can be mentioned as the “cyclic substituent”. As used herein, each group may be substituted.
As the ring in the “optionally substituted ring”, “heterocycle” can be mentioned.
Examples of the “heterocycle” include an aromatic heterocycle and a non-aromatic heterocycle, each containing, as a ring-constituting atom besides carbon atom, 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom.
Examples of the “aromatic heterocycle” include a 5- to 14-membered (preferably, 5- to 10-membered, more preferably, 5- or 6-membered) aromatic heterocycle containing, as a ring-constituting atom besides carbon atom, 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom. Preferred examples of the “aromatic heterocycle” include 5- or 6-membered monocyclic aromatic heterocycles such as thiophene, furan, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, 1,2,4-oxadiazole, 1,3,4-oxadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, triazole, tetrazole, triazine, and the like.
In addition, examples of the “non-aromatic heterocycle” include a 3- to 14-membered (preferably, 4- to 10-membered, more preferably, 5- or 6-membered) non-aromatic heterocycle containing, as a ring-constituting atom besides carbon atom, 1 to 4 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom. Preferred examples of the “non-aromatic heterocycle” include 3- to 8-membered (more preferably, 5- or 6-membered) monocyclic non-aromatic heterocycles such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, imidazoline, imidazolidine, oxazoline, oxazolidine, pyrazoline, pyrazolidine, thiazoline, thiazolidine, tetrahydroisothiazole, tetrahydroxazole, tetrahydroisoxazole, piperidine, piperazine, tetrahydropyridine, dihydropyridine, dihydrothiopyran, tetrahydropyrimidine, tetrahydropyridazine, dihydropyran, tetrahydropyran, tetrahydrothiopyran, morpholine, thiomorpholine, azepane, diazepane, azepine, azocane, diazocane, oxepane, and the like.
Examples of the “hydroxy-protecting group” include the “hydroxy-protecting group” described above for nucleotide derivative (I).
Specific preferred examples of each group above and the substituent with which each group is optionally substituted include those described for the nucleotide derivative (I).
Preferred embodiments of each group in the 1,2-diol derivative (III) are as follows.
The usefulness of the 1,2-diol derivative (II) as a universal linker is specifically described in the Examples below.
Another preferred embodiment of the present invention is as follows.
[1-A]A solid phase synthesis method of an oligonucleotide, comprising a step of subjecting an oligonucleotide having, at the 3′-terminus of an oligonucleotide sequence thereof, a nucleotide derivative represented by the formula (II):
—O—C(Ra)2—, —O—C(Rb)2—C(Rc)2—, —O—C(Rd)2—O—, —N(Re)—C(Rf)2—, —N(Rg)—CO—,—S—C(Rh)2—, or —C(Ri)2—C(Rj)2—
—O—C(Ra)2—, —O—C(Rb)2—C(Rc)2—, —O—C(Rd)2—O—, —N(Re)—C(Rf)2—, —N(Rg)—CO—,—S—C(Rh)2—, or —C(Ri)2—C(Rj)2—
The present invention is described in detail in the following by referring to specific Examples. However, the present invention is not limited thereto.
In the above-mentioned formulas, DMTr is a 4,4′-dimethoxytrityl group, Me is a methyl group, Et is an ethyl group, iPr is an isopropyl group, DIPEA is diisopropyl ethylamine, and ETT is ethylthiotetrazole.
In the following, phosphoramidites represented by the above-mentioned formulas 3a to 3f, and 3h are also denoted as nucleotide derivatives 3a to 3f, and 3h, and phosphoramidite represented by the formula 3g is also denoted as compound 3g. Compound 3g is a known phosphoramidite and used as a control for the nucleotide derivative (I) of the present invention.
It was synthesized according to a reported literature (Bioconjugate Chem., 2008, 19, 1696-1706). Under an argon stream, to a solution of commercially available compound 1 (3.00 g, 5.51 mmol) in anhydrous dichloromethane (20 mL) was added diisopropyl ethylamine (2.40 mL, 13.2 mmol). At 0° C., bis(diisopropylamino)chlorophosphine (1.56 g, 6.61 mmol) was added, and the mixture was stirred at room temperature for 1.5 hr. After completion of the reaction, the reaction mixture was evaporated under reduced pressure, and a crude product was purified by silica gel column chromatography (hexane/ethyl acetate=2:1) to give compound 2 (2.62 g, yield 61%) as a white solid.
It was synthesized according to a reported literature (Bioorg. Med. Chem. Lett., 2015, 25, 3610-3615.). Under an argon stream, to a solution of compound 2 (150 mg, 0.194 mmol) in anhydrous acetonitrile (2 mL) were added diisopropyl ethylamine (40.5 μL, 0.232 mmol) and ethanol (13.5 μL, 0.232 mmol). Ethylthiotetrazole (30.2 mg, 0.232 mmol) was added, and the mixture was stirred at room temperature for 1 hr. After completion of the reaction, the mixture was diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure. A crude product was purified by silica gel column chromatography (hexane/ethyl acetate=3:1) to give nucleotide derivative 3b (78.0 mg, yield 56%) as a white solid.
Under an argon stream, to a solution of compound 2 (150 mg, 0.194 mmol) in anhydrous acetonitrile (2 mL) were added diisopropyl ethylamine (40.5 μL, 0.232 mmol) and isopropanol (17.9 μL, 0.232 mmol). Ethylthiotetrazole (30.2 mg, 0.232 mmol) was added, and the mixture was stirred at room temperature for 2 hr. The mixture was diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure. A crude product was purified by silica gel column chromatography (hexane/ethyl acetate=3:1) to give nucleotide derivative 3c (64.0 mg, yield 45%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 7.99 (brs, 1H), 7.66,7.63(d×2,1H,J=1.0 Hz), 7.42-7.39 (m, 2H), 7.31-7.22 (m, 6H), 6.84-6.81 (m, 4H), 6.44-6.39 (m, 1H), 4.65-4.59 (m, 1H), 4.21,4.18(d×2,1H,J=2.0 Hz), 4.07-3.89 (m, 1H), 3.79 (s, 6H), 3.59-3.46 (m, 3H), 3.36-3.30 (m, 1H), 2.57-2.45 (m, 1H), 2.33-2.26 (m, 1H), 1.39-1.36 (m, 3H), 1.26-1.04 (m, 18H) 31P-NMR (202 MHz, CDCl3) δ 145.2,144.6
IR(ATR): 2966.0,2928,1686,1607,1508 cm−1
HRMS (ESI-TOF): calcd. for C40H52N3aO8P
[M+Na]+756.3390, found 756.3391
Under an argon stream, to a solution of compound 2 (150 mg, 0.194 mmol) in anhydrous acetonitrile (2 mL) were added diisopropyl ethylamine (40.5 μL, 0.232 mmol) and 2-methylpropanol (21.5 μL, 0.232 mmol). Ethylthiotetrazole (30.2 mg, 0.232 mmol) was added, and the mixture was stirred at room temperature for 1 hr. The mixture was diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure. A crude product was purified by silica gel column chromatography (hexane/ethyl acetate=3:1) to give nucleotide derivative 3d (68.0 mg, yield 44%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 7.96 (brs, 1H), 7.66,7.62(brs×2,1H), 7.41-7.39 (m, 2H), 7.31-7.22 (m, 6H), 6.83-6.82 (m, 4H), 6.43-6.39 (m, 1H), 4.67-4.62 (m, 1H), 4.22,4.15(d×2,1H; J=2.0 Hz), 3.79 (s, 6H), 3.61-3.46 (m, 3H), 3.42-3.16 (m, 3H), 2.56-2.45 (m, 1H), 2.33-2.28 (m, 1H), 1.88-1.66 (m, 1H), 1.40,1.39(s×2,3H), 1.16-1.04 (m, 12H), 0.92-0.81 (m, 6H) 31P-NMR (202 MHz, CDCl3) δ 147.2,146.9
IR(ATR): 2963.0,2929,1686,1607,1508 cm−1
HRMS (ESI-TOF): calcd. for C41H54N3NaO8P
[M+Na]+770.3546, found 770.3539
Under an argon stream, to a solution of compound 2 (200 mg, 0.258 mmol) in anhydrous acetonitrile (2 mL) were added diisopropyl ethylamine (54.1 μL, 0.310 mmol) and 2,2-dimethyl-1-propanol (27.3 mg, 0.310 mmol). Ethylthiotetrazole (40.4 mg, 0.310 mmol) was added, and the mixture was stirred at room temperature for 2 hr. After completion of the reaction, the mixture was diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure. A crude product was purified by silica gel column chromatography (hexane/ethyl acetate=5:1) to give nucleotide derivative 3e (94.8 mg, yield 47%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 8.03 (brs, 1H), 7.66,7.61(d×2,1H,J=1.0 Hz), 7.40(dd,2H,J=2.0 Hz,J=8.0 Hz), 7.30-7.22 (m, 6H), 6.84-6.81 (m, 4H), 6.43-6.40 (m, 1H), 4.68-4.62 (m, 1H), 4.20,4.14(d×2,1H,J=2.0 Hz), 3.79 (s, 6H), 3.60-3.45 (m, 3H), 3.39-3.26 (m, 2H), 3.19-3.06 (m, 1H)2.55-2.45 (m, 1H), 2.33-2.32 (m, 1H), 1.40-1.38.(m,3H), 1.27-1.25 (m, 1H), 1.17-1.04 (m, 12H), 0.91,0.82(sx2,9H) 31P-NMR (202 MHz, CDCl3) δ 147.6,147.4
IR(ATR)cm−1:2962,1684,1607,1508
HRMS(ESI-TOF):calcd. for C42H56N3NaO8P [M+Na]+784.3703, found 784.3701
It was synthesized according to a reported literature (J. Org. Chem., 1995, 60, 925-930.). Under an argon stream, to a solution of compound 2 (500 mg, 0.645 mmol) in anhydrous acetonitrile (6 mL) were added diisopropyl ethylamine (270 μL, 1.55 mmol) and phenol (70.0 mg, 0.774 mmol). Ethylthiotetrazole (101 mg, 0.774 mmol) was added, and the mixture was stirred at room temperature for 3 hr. After completion of the reaction, a 5% sodium hydrogen carbonate aqueous solution was added to quench the reaction, and the mixture was diluted with ethyl acetate. The organic layer was washed twice with water and once with saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure. A crude product was purified by silica gel column chromatography (hexane/ethyl acetate=2:1) to give nucleotide derivative 3f (328 mg, yield 63%) as a white solid.
Under an argon stream, to a solution of commercially available compound 4 (1.0 g, 1.88 mmol) in anhydrous dichloromethane (10 mL) was added diisopropyl ethylamine (0.788 mL, 4.51 mmol). At 0° C., bis(diisopropylamino)chlorophosphine (534 mg, 2.26 mmol) was added, and the mixture was stirred at room temperature for 1.5 hr. After completion of the reaction, the reaction mixture was evaporated under reduced pressure and a crude product was purified by silica gel column chromatography (hexane/ethyl acetate=2:1) to give compound 5 (1.09 g, yield 73%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 8.34 (brs, 1H), 8.01(d,2H,J=8.0 Hz), 7.40 (d, 2H, J=7.5 Hz), 7.30-7.22 (m, 7H), 6.84-6.82 (m, 4H), 6.02(d,J=8.0 Hz,1H), 5.25(d,J=8.0 Hz,1H), 4.37-4.33 (m, 1H), 4.26-4.25 (m, 1H), 3.88 (dd, J=4.0, 3.5 Hz, 1H), 3.79 (s, 6H), 3.59-3.42 (m, 6H), 3.56 (s, 3H), 1.17 (d, J=2 Hz, 6H), 1.16 (d, J=2 Hz, 6H), 1.14(d,J=7.0 Hz,6H), 1.04 (d, J=6.5 Hz, 6H).
13C-NMR (125 MHz, CDCl3) δ 162.9, 158.7, 149.9, 144.3, 140.2, 135.3, 135.1, 130.3, 128.3, 127.9, 127.1, 113.2, 101.9, 87.4, 87.2, 84.0, 83.9, 82.9, 82.8, 70.4, 70.3, 62.1, 58.6, 55.2, 44.7, 44.6, 24.6, 24.5, 24.4, 24.2, 24.1, 24.0. 31P-NMR (202 MHz, CDCl3) δ 118.3.
IR(ATR)cm−1: 2968, 1684, 1508.
HRMS (FAB): calcd for C43H60N4O8P [M+H]+, 791.4149, found 791.4147.
Under an argon stream, to a solution of commercially available compound 6 (310 mg, 0.543 mmol) in anhydrous dichloromethane (5 mL) was added diisopropyl ethylamine (0.227 mL, 1.30 mmol). At 0° C., bis(diisopropylamino)chlorophosphine (154 mg, 0.652 mmol) was added, and the mixture was stirred at room temperature for 4 hr. After completion of the reaction, the reaction mixture was evaporated under reduced pressure, and a crude product was purified by silica gel column chromatography (hexane/ethyl acetate=2:1) to give compound 7 (210 mg, yield 48%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 8.30 (brs, 1H), 7.69(d,1H,J=1.0 Hz), 7.49-7.47 (m, 2H), 7.37-7.34 (m, 4H), 7.30-7.22 (m, 5H), 6.84-6.82 (m, 4H), 4.59 (s, 1H), 4.08 (d, 1H, J=9.0 Hz), 3.89, 3.85(ABq,2H,J=18.0,7.5 Hz), 3.79 (s, 6H), 3.58 (d,1H,J=11.0 Hz), 3.43-3.36 (m, 5H), 1.73 (s, 3H), 1.13 (d, J=6.5 Hz, 6H), 1.10(d,J=7.0 Hz,6H), 1.03 (d, J=6.5 Hz, 6H), 0.91 (d, J=6.5 Hz, 6H).
31P-NMR (202 MHz, CDCl3) δ 117.6.
IR(ATR)cm−1:2966,1687,1607,1508.
HRMS (ESI-TOF): calcd for C44H60N4O8P [M+H]+ 803.4149, found 803.4148.
Under an argon stream, to a solution of compound 5 (600 mg, 0.759 mmol) in anhydrous acetonitrile (5 mL) were added diisopropyl ethylamine (0.16 mL, 0.911 mmol) and ethanol (53 μL, 0.911 mmol). Ethylthiotetrazole (119 mg, 0.911 mmol) was added, and the mixture was stirred at room temperature for 2.5 hr. The mixture was diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure. A crude product was purified by silica gel column chromatography (hexane/ethyl acetate=4:1) to give compound 3bOMe (219 mg, yield 39%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 8.73 (brs, 1H), 8.08(d,0.3H,J=8.0 Hz,1H), 8.04(d,0.7H,J=8.0 Hz), 7.86 (brs, 1H), 7.43-7.36 (m, 2H), 7.37-7.22 (m, 7H), 6.85-6.82 (m, 4H), 5.99(d,0.7H,J=8.0 Hz), 5.97(d,0.3H,J=8.0 Hz), 5.18-5.15 (m, 1H), 4.59-4.55(m,0.3H), 4.48-4.44(m,0.7H), 4.24-4.21 (m, 1H), 3.84-3.82 (m, 1H), 3.80 (s, 6H), 3.67-3.47 (m, 9H), 1.26-1.03 (m, 16H). 31P-NMR (202 MHz, CDCl3) δ 148.3,147.9.
IR(ATR)cm−1:2966,1607,1508.
HRMS(FAB):calcd for C39H51N3O9P [M+H]+ 736.3363, found 736.3375.
Under an argon stream, to a solution of compound 7 (110 mg, 0.134 mmol) in anhydrous acetonitrile (2 mL) were added diisopropyl ethylamine (28.6 μL, 0.164 mmol) and ethanol (9.6 μL, 0.164 mmol). Ethylthiotetrazole (21.3 mg, 0.164 mmol) was added, and the mixture was stirred at room temperature for 4 hr. The mixture was diluted with ethyl acetate, and the organic layer was washed twice with water and once with saturated brine, and dried over sodium sulfate. The solvent was evaporated under reduced pressure. A crude product was purified by silica gel column chromatography (hexane/ethyl acetate=3:1) to give nucleotide derivative 3bLNA (33.0 mg, yield 32%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 8.21 (brs, 1H), 7.70 (brs, 1H), 7.49-7.46 (m, 2H), 7.37-7.22 (m, 7H), 6.85-6.82 (m, 4H), 5.65(s,0.7H), 5.64(s,0.3H), 4.57(s,0.7H), 4.51(s,0.3H), 4.35(d,0.3H,J=9.0 Hz), 4.26(d,0.7H,J=6.5 Hz), 3.87 (d, 1H, J=7.5 Hz), 3.80-3.79 (m, 7H), 3.71-3.47 (m, 5H), 3.45-3.41 (m, 1H), 1.61 (s, 3H), 1.22-0.99 (m, 15H).
31P-NMR (202 MHz, CDCl3) δ 147.2.
IR(ATR)cm−1:2966,1687,1607,1508.
HRMS (ESI-TOF): calcd for C40HsoN3NaO9P
[M+Na]+770.3182, found 770.3182.
Under an argon stream, to a suspension of solid phase support lcaa-CPG (Amino-SynBase™ CPG 1000/110 (LCAA), manufactured by Biosearch Technologies) (200 mg, amino group content: 11.2 μmol) in anhydrous acetonitrile (1 mL) were added diisopropyl ethylamine (3.80 μL, 22.4 μmol), HBTU (4.25 mg, 11.2 μmol) and compound 8 (5.20 mg, 11.2 μmol) synthesized according to a literature (Bioorganic & Med. Chem., 1997, 5, 2235-2243.). After stirring at room temperature for 1 hr, the reaction mixture was filtered, and a solid phase support was washed with acetonitrile and further vacuum dried overnight. The obtained solid phase support was suspended in CapA solution (acetonitrile solution of 1-methylimidazole, 0.5 mL) and CapB solution (acetonitrile solution of acetic anhydride, 0.5 mL) and the suspension was stirred for 30 min. The reaction mixture was filtered again, and the solid phase support was washed with acetonitrile and vacuum dried overnight to give CPG1 solid phase support. The amount of compound 8 supported by CPG1 was quantified by the DMTr assay method (*) and found to be 39±2.8 mol/g.
By a method similar to that used for CPG1 and using compound 9 (5.36 mg, 11.2 μmol) synthesized according to a literature (Nucleic Acids Res., 1987, 15, 3113-3129.), CPG2 solid phase support was synthesized. The amount of compound 9 supported by CPG2 was quantified by the DMTr assay method and found to be 33±0.9 mol/g.
(*) DMTr assay method: a method for indirectly quantifying the supported amount by treating solid phase support with a deblocking solution (3 w/v % dichloromethane solution of trichloroacetic acid) and performing absorbance measurement (504 nm) of the amount of deprotected DMTr (dimethoxytrityl) group.
Oligonucleotide was synthesized using a GeneDesign nS-811 Oligonucleotides Synthesizer (manufactured by GeneDesign, Inc.) and according to the general phosphoramidite method. The synthesis scale was set to 0.2 μmol, and the synthesis was performed under trityl OFF conditions. Commercially available phosphoramidite of T (same as compound 3g), commercially available phosphoramidite of AcC, commercially available phosphoramidite of PacA, and commercially available phosphoramidite of iPrPacG were prepared as 0.1 M solutions in anhydrous acetonitrile and used. As the activator, 5-ethylthio-1H-tetrazole (0.25 M anhydrous acetonitrile solution) was used, and the condensation time was 10 minutes for nucleotide derivatives 3a-3f, 3h, and compound 3g (all in the case of condensation with universal linker), and 25 seconds for commercially available phosphoramidite of T, commercially available phosphoramidite of AcC, commercially available phosphoramidite of PacA, and commercially available phosphoramidite of iPrPacG (all in the case of condensation with nucleotide other than universal linker).
The outline of the synthesis scheme and the structures of the synthesized oligonucleotides are shown in the following. In the present specification, AcC, PacA, and iPrPacG are respectively also denoted as CAc, APac, and GiPrPac.
In the above-mentioned formulas, DMTr, Me and Et are each as defined above, CE is a 2-cyanoethyl group, T is a thyminyl group, U is a uracil group, CAc is a N-acetylcytosyl group, APac is a N-phenoxyacetyladenyl group, and GiPrPac is a N-(4-isopropylphenoxyacetyl)guanyl group (here, thyminyl group, uracil group, cytosyl group, adenyl group, and guanyl group respectively show thymine monovalent group, uracil monovalent group, cytosine monovalent group, adenine monovalent group, and guanine monovalent group).
When T, CAc, APac, and GiPrPac are described as nucleic acid monomers constituting oligonucleotide sequences, each nucleobase means a deoxyribonucleotide bonded to deoxy-D-ribose. When U is described as a nucleic acid monomer constituting an oligonucleotide sequence, the nucleobase means a ribonucleotide bonded to D-ribose.
Using commercially available CPG3 (Universal Support manufactured by Glen Research) as a solid phase support and a DNA automatic synthesizer, T-10 mer oligonucleotides (CPG3a-h) into which the aforementioned nucleotide derivatives 3a-3f, 3h, 3bOMe, 3bLNA, compound 3g were introduced at the 3′-terminus, T-9 mer-UOMe oligonucleotide (CPG3bOMe), T-9 mer-TLNA oligonucleotide (CPG3bLNA), and (11 mer constituted of plural kinds of nucleic acid monomers)-T oligonucleotide (CPG3e-mix) were synthesized.
The outline of the synthesis scheme and the structures of the synthesized oligonucleotides are shown in the following.
In the above-mentioned formulas, DMTr, CE, and T are each as defined above.
Using CPG1 synthesized according to Example B1 as a solid phase support and a DNA automatic synthesizer, T-10 mer oligonucleotides (CPG1a and CPG1g) into which nucleotide derivative 3a or compound 3g was introduced at the 3′-terminus were synthesized.
Using CPG2 synthesized according to Example B2 as a solid phase support, and according to a method similar to that for CPG1a, T-10 mer oligonucleotides (CPG2a and CPG2g) were synthesized.
D. Evaluation of Cleavage Efficiency of Oligonucleotide from Solid Phase (CPG)
The outline of the cleavage of oligonucleotide is shown in the following.
In the above-mentioned formulas, CE, T, Me, Et, U, CAc, APac, and GiPrPac are each as defined above,
Oligonucleotides were cleaved from CPG3a-h, CPG3bOMe, and CPG3bLNA under the following conditions 1) to 3).
In addition, oligonucleotide was cleaved from CPG3e-mix under the above-mentioned conditions 1) and 3).
After treatment under the above-mentioned respective conditions, the cleavage results were analyzed by reversed-phase HPLC. The HPLC analysis conditions are shown in the so following Table 1.
HPLC charts showing the cleavage results of oligonucleotides from CPG3a, CPG3e, CPG3g, CPG3bOMe, and CPG3bLNA are shown in
HPLC charts showing the cleavage results of oligonucleotides from CPG3e-mix are shown in
A table summarizing the production ratio of the oligonucleotide (T10) of interest and the by-produced oligonucleotide (T10-adduct) which are released upon cleavage of each CPG3a-h is also shown (Table 2). Here, the structure of the T10-adduct was identified by comparison with the description in a literature (Tetrahedron, 2021, 92, 132261.)
Similarly, tables summarizing the production ratio of the oligonucleotides (T9UOMe and T9TLNA) of interest and the by-produced oligonucleotides (T9UOMe-adduct and T9TLNA-adduct) which are released upon cleavage of CPG3bOMe and CPG3bLNA are also shown (Table 3 and Table 4).
Furthermore, CPG3e and CPG3g were subjected to cleavage by 4) treatment with 28% aqueous ammonia at 80° C. or 5) AMA solution at 55° C., and monitored over time by HPLC (HPLC gradient; SOLUTION B: 8-18% (30 min)).
HPLC charts showing the cleavage results of oligonucleotides from CPG3e and CPG3g are shown in
Tables summarizing the production ratio of the oligonucleotide (T10) of interest and the by-produced oligonucleotide (T10-adduct) (T10:T10-adduct) which are released under conditions 4) and conditions 5) are shown in Table 5 and Table 6.
In oligonucleotide synthesis, phosphoramidites (compound 3g) having a cyanoethoxy group as a protecting group for the phosphoric acid moiety have been widely used. However, when synthesizing oligonucleotides by using a universal support conventionally used widely, the use of the nucleotide derivatives (I) of the present invention (corresponding to nucleotide derivatives 3a-3f, 3h), having a more stable substituent such as an alkoxy group as a protecting group for the phosphoric acid moiety, as a phosphoramidite strikingly increased the production of T10 oligonucleotides, and it was confirmed that the oligonucleotides can be released from the solid phase support under mild base conditions. This superior effect was also confirmed when OMe or LNA having modified sugar moieties was used.
The outline of the cleavage of oligonucleotide is shown in the following.
In the above-mentioned formulas, CE, T, T10, and T10-adduct are each as defined above.
Cleavage of oligonucleotides from CPG1a and CPG1g/CPG2a and CPG2g was performed by a treatment with a methanol solution of 50 mM potassium carbonate at room temperature for 4 hr. The results thereof were analyzed by reversed-phase HPLC. HPLC analysis conditions are shown in the following Table 7.
HPLC charts showing the cleavage results of oligonucleotides from CPG1a and CPGlg/CPG2a and CPG2g are shown in
The production ratio of the oligonucleotide (T10) (SEQ ID NO: 1) of interest and the by-produced oligonucleotide (T10-adduct) which are released upon cleavage of each CPG is shown in Table 8 as values calculated from peak area ratio on HPLC chart.
In the Table, * is a Triester form represented by the following formula.
T10-adduct (n=1 or 2) and Triester form as by products were each identified by ESI-MS analysis. The results thereof are shown in the following Table 9.
It was clarified that, by using the nucleotide derivative (I) of the present invention that is a phosphoramidite having stabler substituents, oligonucleotides can be cleaved sufficiently rapidly even when an ethyleneglycol resin (CPG1) with a “1,2-diol structure” which is the same as a universal support with a simpler structure than the universal support is used as a solid phase support.
In this way, one of the features of the production method of oligonucleotides of the present invention is that a 1,2-diol derivative having a simple structure can be used as a universal linker.
In each formula below, DMTr is a 4,4′-dimethoxytrityl group, Me is a methyl group, iPr is an isopropyl group, tBu is a t-butyl group, and Ac is an acetyl group.
According to the procedures in a known literature (Nucleosides, Nucleotides, and Nucleic Acids, 2005, 24, 815-818.), compound 10 was synthesized.
Compound 10 (10.0 g, 31.6 mmol) was subjected 3 times to azeotropic distillation with dehydration with pyridine. The Compound was successively dissolved in pyridine (80 mL), 4,4′-dimethoxytritylchloride (13.9 g, 41.1 mmol) was added and the mixture was stirred for 1.5 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=3/7→7/3) to give compound 11 (19.6 g, quant.).
1H-NMR (CDCl3, 400 MHz); δ 7.94 (1H, brs), 7.65 (1H, d, J=0.8 Hz), 7.42-7.39 (2H, m), 7.31-7.24 (7H, m), 6.85-6.82 (4H, m), 6.01(1H,d,J=4.0 Hz), 4.43 (1H, dd, J=10.8, 5.6 Hz), 4.15-4.11 (2H, m), 4.08-4.03 (1H, m), 3.79 (6H, s), 3.78-3.73 (1H, m), 3.65-3.60 (1H, m), 3.56-3.51 (3H, m), 3.42-3.39 (1H, m), 3.39 (3H, s), 1.37 (3H, s).
LRMS(ESI-QTOF):m/z 641.2360[M+Na]+
Compound 11 (4.00 g, 7.50 mmol) was dissolved in dichloromethane (33.0 mL), N,N-diisopropyl ethylamine (3.40 mL, 19.0 mmol) and bis(diisopropylamino)chlorophosphine (2.60 g, 9.70 mmol) were successively added, and the mixture was stirred for 2 hr. Then, 2-propanol (1.50 mL, 19.0 mmol) and 4,5-dicyanoimidazole (150 mg, 1.30 mmol) were successively added, and the mixture was stirred for 22 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane-→ethyl acetate/n-hexane=1/1) to give nucleotide derivative 12c (3.37 g, 65%).
1H-NMR (CD3CN, 400 MHz); δ 9.13 (1H, brs), 7.53-7.44 (3H, m), 7.36-7.23 (7H, m), 6.89-6.86 (4H, m), 5.92-5.90 (1H, m), 4.46-4.38 (1H, m), 4.21-3.91 (3H, m), 3.84-3.72 (8H, m), 3.64-3.49 (4H, m), 3.42-3.27 (5H, m), 1.39-1.35 (3H, m), 1.22-1.13 (12H, m), 1.09-1.03 (6H, m).
31P-NMR(CD3CN, 162 MHz); δ 147.6,147.1.
LRMS(ESI-QTOF):m/z 808.4109[M+H]+
Compound 11 (8.00 g, 13.0 mmol) was dissolved in dichloromethane (65.0 mL), N,N-diisopropyl ethylamine (6.80 mL, 39.0 mmol) and bis(diisopropylamino)chlorophosphine (5.20 g, 19.0 mmol) were successively added, and the mixture was stirred for 2 hr. Then, neopentyl alcohol (5.70 g, 65.0 mmol) and 4,5-dicyanoimidazole (310 mg, 2.60 mmol) were successively added, and the mixture was stirred for 6 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=3/7→1/1) to give nucleotide derivative 12e (5.69 g, 53%).
1H-NMR (CD3CN, 400 MHz); δ 9.11 (1H, brs), 7.53-7.44 (3H, m), 7.35-7.23 (7H, m), 6.88-6.86 (4H, m), 5.93-5.90 (1H, m), 4.50-4.42 (1H, m), 4.22-4.13 (2H, m), 3.86-3.72 (8H, m), 3.65-3.24 (10H, m), 3.20-3.06 (1H, m), 1.38-1.35 (3H, m), 1.24-1.14 (9H, m), 1.04-1.03 (3H, m), 0.90-0.80 (9H, m).
31P-NMR(CD3CN, 162 MHz); δ 149.8,149.5.
LRMS(ESI-QTOF):m/z 836.4615[M+H]+
According to the procedures in a known literature (J. Org. Chem. 2005, 70, 10453-10460.), compound 13 was synthesized.
According to the procedures in a known literature (J. Org. Chem. 2005, 70, 10453-10460.), nucleotide derivative 14g was synthesized.
Compound 13 (4.5 g, 7.50 mmol) was dissolved in dichloromethane (37.5 mL), N,N-diisopropyl ethylamine (3.92 mL, 22.5 mmol) and bis(diisopropylamino)chlorophosphine (3.0 g, 11.3 mmol) were successively added, and the mixture was stirred for 3 hr. Then, 2-propanol (1.72 mL, 22.5 mmol) and 4,5-dicyanoimidazole (177 mg, 1.50 mmol) were successively added, and the mixture was stirred for 21 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=1/1) to give nucleotide derivative 14c (3.57 g, 60%).
1H-NMR (CD3CN, 400 MHz); δ 9.01 (1H, brs), 7.86-7.75 (1H, m), 7.48-7.43 (2H, m), 7.36-7.24 (7H, m), 6.89-6.86 (4H, m), 5.84-5.83 (1H, m), 5.22-5.17 (1H, m), 4.47-4.32 (1H, m), 4.17-3.86 (5H, m), 3.77 (6H, m), 3.67-3.54 (2H, m), 3.49-3.36 (2H, m), 2.74-2.60 (2H, m), 1.23-1.10 (15H, m), 1.06-1.05 (3H, m)
31P-NMR(CD3CN, 162 MHz); δ 147.9, 146.9.
LRMS(ESI-QTOF):m/z 789.3420[M+H]+
According to the procedures in a known literature (Nucleosides and Nucleotides, 1992, 11, 1263-1273.), compound was synthesized.
According to the procedures in a known literature (Nucleosides and Nucleotides, 1992, 11, 1263-1273.), nucleotide derivative 16g was synthesized.
Compound 15 (4.5 g, 7.67 mmol) was dissolved in dichloromethane (38.4 mL), N,N-diisopropylethylamine (4.01 mL, 23.0 mmol) and bis(diisopropylamino)chlorophosphine (3.07 g, 11.5 mmol) were successively added, and the mixture was stirred for 2 hr. Then, 2-propanol (1.76 mL, 23.0 mmol) and 4,5-dicyanoimidazole (181 mg, 1.53 mmol) were successively added, and the mixture was stirred for 21 hr. After completion of the reaction, saturated aqueous sodium hydrogen carbonate the reaction was quenched with and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=1/4) to give nucleotide derivative 16c (3.23 g, 54%).
1H-NMR (CD3CN, 400 MHz); δ 9.03 (1H, brs), 7.84-7.73 (1H, m), 7.47-7.42 (2H, m), 7.36-7.23 (7H, m), 6.90-6.85 (4H, m), 5.99-5.87 (2H, m), 5.33-5.27 (1H, m), 5.24-5.14 (2H, m), 4.47-4.35 (1H, m), 4.28-3.94 (5H, m), 3.77 (6H, m), 3.65-3.53 (2H, m), 3.44-3.34 (2H, m), 1.22-1.13 (12H, m), 1.11-1.09 (3H, m), 1.06-1.04 (3H, m).
31P-NMR(CD3CN, 162 MHz); δ 147.5,147.2.
LRMS(ESI-QTOF):m/z 776.3499[M+H]+
According to the procedures in a known literature (J. Med. Chem. 2008, 51, 4957-4967.), compound 17 was synthesized.
(1) Compound 17 (14.1 g, 27.0 mmol) was dissolved in tetrahydrofuran (135 mL), triethylamine 3 hydrofluoric acid salt (6.59 mL, 40.5 mmol) was added, and the mixture was stirred for 3.5 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate, sodium chloride (10 g) was added, and the mixture was extracted with tetrahydrofuran. Then, the organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure to give crudely purified compound 18 (7.62 g, <27.0 mmol).
(2) The crudely purified compound 18 (7.62 g, <27.0 mmol) was azeotropically distilled three times with pyridine. The obtained residue was dissolved in pyridine (90 mL), 4,4′-dimethoxytritylchloride (11.0 g, 32.4 mmol) was added and the mixture was stirred for 15 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the mixture was azeotropically distilled three times with toluene. The residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=1/1) to give compound 19 (12.8 g, 81%).
1H-NMR (CDCl3, 400 MHz); δ 8.00-7.98 (2H, m), 7.41-7.37 (2H, m), 7.32-7.25 (7H, m), 6.87-6.83 (4H, m), 5.97 (1H, d, J=2.4 Hz), 5.28 (1H, dd, J=8.0, 2.4 Hz), 4.55-4.44 (3H, m), 4.24 (1H, dd, J=4.0, 2.8 Hz), 4.06(1H,dt,J=6.8,2.0 Hz), 3.80 (6H, s), 3.57-3.50 (2H, m), 2.53(1H,d,J=8.0 Hz), 2.51(1H,t,J=2.4).
LRMS(ESI-QTOF):m/z 607.1871[M+Na]+
Compound 19 (3.50 g, 5.99 mmol) was dissolved in dichloromethane (30 mL), 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (2.80 mL, 8.98 mmol) and 4,5-dicyanoimidazole (70.7 mg, 0.599 mmol) were successively added, and the mixture was stirred for 15.5 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. Then, the organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=7/10→ethyl acetate) to give compound 20g (4.33 g, 92%).
1H-NMR (400 MHz,CD3CN) δ 9.07 (1H, brs), 7.79-7.69 (1H, m), 7.44-7.39 (m, 2H), 7.33-7.20 (7H, m), 6.80-6.93 (4H, m), 5.86-5.84 (1H, m), 5.24-5.22 (1H, m), 4.51-4.41 (1H, m), 4.37-4.32 (2H, m), 4.28-4.23 (1H, m), 4.17-4.10 (1H, m), 4.06-4.00 (1H, m), 3.92-3.53 (8H, m), 3.43-3.31 (2H, m), 2.72-2.63 (2H, m), 2.50-2.47 (1H, m), 1.92-1.89 (1H, m), 0.97-1.27 (12H, m).
31P-NMR (162 MHz, CD3CN) δ 150.7,150.3.
LRMS(ESI-QTOF):m/z 807.3075[M+Na]+
Compound 19 (4.50 g, 7.70 mmol) was dissolved in dichloromethane (39 mL), N,N-diisopropyl ethylamine (4.02 mL, 23.1 mmol) and bis(diisopropylamino)chlorophosphine (3.08 g, 11.5 mmol) were successively added, and the mixture was stirred for 2 hr. Then, 2-propanol (3.53 mL, 46.2 mmol) and 4,5-dicyanoimidazole (182 mg, 1.54 mmol) were successively added, and the mixture was stirred for 5 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate and the mixture was extracted with ethyl acetate. Then, the organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=9/13) to give nucleotide derivative 20c (3.53 g, 59%).
1H-NMR (400 MHz,CD3CN) δ 9.05 (brs, 1H), 7.79-7.69 (1H, m), 7.48-7.40 (2H, m), 7.32-7.20 (7H, m), 6.86-6.82 (4H, m), 5.86 (1H, m), 5.26-5.22 (1H, m), 4.44-4.32 (2H, m), 4.24-4.18 (1H, m), 4.15-3.88 (2H, m), 3.73 (6H, s), 3.64-3.51 (2H, m), 3.41-3.29 (2H, m), 2.70-2.68 (1H, m), 1.92-1.89 (1H, m), 1.19-1.01 (18H, m).
31P-NMR (162 MHz, CD3CN) δ 147.7,147.6.
LRMS(ESI-QTOF):m/z 774.3848[M+H]+
According to the procedures in a known literature (Tetrahedron. 2021, 72, 153066.), compound 21 was synthesized.
Compound 21 (10.6 g, 21.7 mmol) was dissolved in tetrahydrofuran (80 mL), under ice-cooling, sodium hydride (60 wt %, oily) (2.60 g, 65.0 mmol) was added over 10 min, and the mixture was stirred for 15 min. Then, N-(3-bromopropyl)acetamide (5.85 g, 32.5 mmol) was dissolved in 6 mL of tetrahydrofuran. The mixture was stirred at room temperature for 15 hr. After completion of the reaction, the reaction was quenched with saturated ammonium chloride aqueous solution, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate. Then, the organic layer was dried over magnesium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/n-hexane=1/4→ethyl acetate→methanol/chloroform=1/19) to give compound 22 (3.25 g, 26%).
1H-NMR (400 MHz, DMSO-D6) δ 7.96 (1H, brs), 7.76 (1H, t, J=5.3 Hz), 7.65 (1H, d, J=8.2 Hz), 5.58 (1H, s), 5.53 (1H, d, J=8.2 Hz), 4.23 (1H, dd, J=8.0, 4.4 Hz), 4.13 (1H, d, J=12.3 Hz), 4.03 (1H,d,J=4.6 Hz), 3.99-3.90 (2H, m), 3.78-3.65 (2H, m), 3.14-3.07 (2H, m), 1.77 (3H, s), 1.69-1.63 (2H, m), 1.06-0.98 (28H, m).
LRMS(ESI-QTOF):m/z 608.3365[M+Na]+
Compound 22 (3.25 g, 5.55 mmol) was dissolved in tetrahydrofuran (28 mL), triethylamine 3 hydrofluoric acid salt (1.36 mL, 8.32 mmol) was added, and the mixture was stirred for 2.5 hr. After completion of the reaction, under ice-cooling, potassium carbonate (1.53 g, 11.1 mmol) and silica gel (6.0 g) were successively added, and tetrahydrofuran was evaporated. Purification by silica gel column chromatography (methanol/chloroform=3/22→1/4) gave compound 23 (1.46 g, 77%).
1H-NMR (400 MHz, DMSO-D6) δ 11.32 (1H, brs), 7.93 (1H, d, J=8.2 Hz), 7.77 (1H, t, J=5.7 Hz), 5.83 (1H, d, J=5.0 Hz), 5.64 (1H, d, J=7.8 Hz), 5.15-5.13 (2H, m), 4.09(1H,qd,J=5.2,1.8 Hz), 3.88-3.84 (2H, m), 3.65(1H,dq,J=12.3,2.7 Hz), 3.58-3.47 (2H, m), 3.17 (1H, d, J=5.0 Hz), 3.12-3.02 (2H, m), 1.78 (3H, s), 1.65-1.58 (2H, m).
LRMS(ESI-QTOF):m/z 366.1188 [M+Na]+
Compound 23 (1.46 g, 4.25 mmol) was azeotropically distilled three times with pyridine. The obtained residue was dissolved in pyridine (14 mL), 4,4′-dimethoxytritylchloride (1.73 g, 5.10 mmol) was added, and the mixture was stirred for 15 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate, and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and the mixture was azeotropically distilled twice with toluene. The residue was purified by silica gel column chromatography (methanol/chloroform=1/49→1/19) to give compound 24 (2.53 g, 92%). 1H-NMR (400 MHz, DMSO-D6) δ 11.38 (1H, brs), 7.79 (1H, t, J=5.3 Hz), 7.72 (1H, d, J=8.2 Hz), 7.39-7.23 (9H, m), 6.90 (4H, d, J=8.5 Hz), 5.79 (1H, d, J=3.7 Hz), 5.28 (1H, d, J=8.2 Hz), 5.20 (1H, d, J=6.4 Hz), 4.21-4.16 (1H, m), 3.98-3.94 (1H, m), 3.90 (1H, t, J=4.1 Hz), 3.74 (6H, s), 3.59 (2H, t, J=6.2 Hz), 3.25-3.20 (1H, m), 3.15-3.05 (2H, m), 1.78 (3H, s), 1.67-1.61 (2H, m).
LRMS(ESI-QTOF):m/z 668.2421[M+Na]+
Compound 24 (1.10 g, 1.70 mmol) was dissolved in dichloromethane (8.5 mL), 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.810 mL, 2.56 mmol) and 4,5-dicyanoimidazole (20.1 mg, 0.170 mmol) were successively added, and the mixture was stirred for 18 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol/(1% triethylamine-containing ethyl acetate)=1/99→1/19) to give nucleotide derivative 25g (1.05 g, 73%).
1H-NMR (400 MHz,CD3CN) δ 9.09 (1H, brs), 7.82-7.70 (1H, m), 7.41 (2H, m), 7.33-7.20 (7H, m), 6.85 (4H, m), 6.44 (1H, brs), 5.84-5.82 (1H, m), 5.21-5.17 (1H, m), 4.50-4.36 (1H, m), 4.15-4.09 (1H, m), 4.05-3.97 (2H, m), 3.87-3.52 (11H, m), 3.44-3.32 (2H, m), 3.21-3.14 (2H, m), 2.67-2.45 (2H, m), 1.80-1.79 (3H, m), 1.61-1.75 (2H, m), 0.99-1.23 (12H, m).
31P-NMR (162 MHz, CD3CN) δ 150.2,149.8.
LRMS(ESI-QTOF):m/z 868.3463[M+Na]+
Compound 24 (2.50 g, 3.87 mmol) was dissolved in dichloromethane (19 mL), N,N-diisopropyl ethylamine (2.02 mL, 11.6 mmol) and bis(diisopropylamino)chlorophosphine (1.55 g, 5.81 mmol) were successively added, and the mixture was stirred for 2 hr. Then, 2-propanol (1.78 mL, 23.2 mmol) and 4,5-dicyanoimidazole (91.4 mg, 0.774 mmol) were successively added, and the mixture was stirred for 6 hr. After completion of the reaction, the reaction was quenched with saturated aqueous sodium hydrogen carbonate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. Then, the organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol/(1% triethylamine-containing ethyl acetate)=1/99→1/19) to give nucleotide derivative 25c (2.27 g, 70%).
1H-NMR (400 MHz,CD3CN) δ 9.03 (1H, brs), 7.82-7.69 (1H, m), 7.48-7.39 (2H, m), 7.32-7.21 (7H, m), 6.89-6.83 (4H, m), 6.45 (1H, brs), 5.84-5.82 (1H, m), 5.23-5.16 (1H, m), 4.43-4.27 (1H, m), 4.13-3.89 (3H, m), 3.77-3.47 (10H, m), 3.44-3.28 (2H, m), 3.25-3.07 (2H, m), 1.80-1.79 (3H, m), 1.73-1.63 (2H, m), 1.23-1.01 (18H, m). 31P-NMR (162 MHz, CD3CN) δ 147.3,147.1.
LRMS(ESI-QTOF):m/z 835.3854[M+H]+
Oligonucleotide was synthesized using Oligpilot 100 (Cytiva) and according to the general phosphoramidite method. As a support, the following Primer Support 5G Unylinker 350(PS1) was used and the synthesis scale was set to 50 μmol, and the synthesis was performed under trityl OFF conditions.
In each formula below, DMTr is a 4,4′-dimethoxytrityl group, Ph is a phenyl group, Me is a methyl group, iPr is an isopropyl group, Ac is an acetyl group, and CE is a 2-cyanoethyl group.
Commercially available phosphoramidite of T (same as compound 3g) and commercially available MOE-T amidite (nucleotide derivative 12g shown by the following formula, manufactured by Hongene) were prepared as 0.15 mol/L solutions in anhydrous acetonitrile and used.
As the activator, 5-benzylthio-1H-tetrazole was used and the condensation time was 3 min for coupling of natural thymidine amidite and 12 min for coupling of 2′-modified nucleoside amidite.
The outline of the synthesis scheme and the structures of the synthesized oligonucleotides are shown in the following.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-9 mer-(MOE)T oligonucleotides (PS1cA (n=9), PS1eA (n=9), PS1gA (n=9)) into which the aforementioned nucleotide derivatives 12c, 12e, 12g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-1 mer-(2′-OCE)U oligonucleotides (PS1cB (n=1), PS1gB (n=1)) into which the aforementioned nucleotide derivatives 14c, 14g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-9 mer-(2′-OCE)U oligonucleotides (PSlcB (n=9), PS1gB (n=9)) into which the aforementioned nucleotide derivatives 14c, 14g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-1 mer-(2′-O-allyl)U oligonucleotides (PS1cC (n=1), PS1gC (n=1)) into which the aforementioned nucleotide derivatives 16c, 16g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-9 mer-(2′-O-allyl)U oligonucleotides (PS1cC (n=9), PSlgC (n=9)) into which the aforementioned nucleotide derivatives 16c, 16g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-1 mer-(2′-O-propargyl)U oligonucleotides (PS1cD (n=1), PS1gD (n=1)) into which the aforementioned nucleotide derivatives 20c, 20g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-9 mer-(2′-O-propargyl)U oligonucleotides (PS1cD (n=9), PS1gD (n=9)) into which the aforementioned nucleotide derivatives 20c, 20g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-1 mer-(2′-O-(3-acetamide)propyl)U oligonucleotides (PS1cE (n=1), PS1gE (n=1)) into which the aforementioned nucleotide derivatives 25c, 25g were introduced at the 3′-terminus were synthesized.
Using commercially available PS1 (Primer Support 5G Unylinker 350 manufactured by Cytiva) as a solid phase support and a DNA automatic synthesizer, T-9 mer-(2′-O-(3-acetamide)propyl)U oligonucleotides (PS1cE (n=9), PS1gE (n=9)) into which the aforementioned nucleotide derivatives 25c, 25g were introduced at the 3′-terminus were synthesized.
G. Evaluation of Oligonucleotide Cleavage Efficiency from Solid Phase (PS)
In each formula below, Ph is a phenyl group, Me is a methyl group, iPr is an isopropyl group, Ac is an acetyl group, and CE is a 2-cyanoethyl group.
T is a thyminyl group and U is a uracil group (wherein thyminyl group and uracil group respectively indicate thymine monovalent group and uracil monovalent group).
When T is described as a nucleic acid monomer constituting an oligonucleotide sequence, the nucleobase means a deoxyribonucleotide bonded to deoxy-D-ribose. When U is described as a nucleic acid monomer constituting an oligonucleotide sequence, the nucleobase means a ribonucleotide bonded to D-ribose.
The outline of the cleavage of oligonucleotide is shown in the following.
In the above-mentioned formulas, TnA (n=9) is T9-(MOE)T oligonucleotide (SEQ ID NO: 5), TnA-adduct (n=9) is an oligonucleotide in which a universal linker is bonded to the aforementioned each oligonucleotide without being cleaved.
Cleavage from PSlcA (n=9), PS1eA (n=9) and PSlgA (n=9) was performed under the following conditions.
After treatment under the above-mentioned respective conditions, the cleavage results were analyzed by reversed-phase HPLC. The HPLC analysis conditions are shown in the following Table 10.
HPLC charts showing the cleavage results of oligonucleotides from PS1cA (n=9), PS1eA (n=9) and PS1gA (n=9) are shown in
The structure of the oligonucleotide (TnA) of interest which is released upon cleavage from each solid phase was confirmed by LC-MS analysis. The results of the MS analysis are shown in the following Table 11.
In addition, the production ratio (HPLC area ratio; TnA:TnA-adducts) of the oligonucleotide (TnA) of interest and the by-produced oligonucleotide (TnA-adducts) which are released upon cleavage from each solid phase is shown in the following Table 12.
In oligonucleotide synthesis, phosphoramidites (compound: MOE-T amidite commercially available product, nucleotide derivative 12g) having a cyanoethoxy group as a protecting group for the phosphoric acid moiety have been widely used. However, when synthesizing oligonucleotides by using a universal support conventionally used widely, the use of the nucleotide derivatives (I) of the present invention (corresponding to nucleotide derivatives 12c, 12e), having a more stable substituent such as an alkoxy group as a protecting group for the phosphoric acid moiety, as a phosphoramidite strikingly increased the production of TnA oligonucleotides, and a superior effect was also confirmed that the oligonucleotides can be released from the solid phase support under mild base conditions even when MOE having modified 2′ moiety of sugar moiety was used.
The outline of the cleavage of oligonucleotide is shown in the following.
In the above-mentioned formulas, TnB—E (n=1) is T1-(2′-alkoxy)U oligonucleotide, TnB—E (n=9) is T9-(2′-alkoxy)U oligonucleotide (SEQ ID NO: 6-9), and
TnB—E-adduct (n=1, or n=9) is an oligonucleotide in which a universal linker is bonded to the aforementioned each oligonucleotide without being cleaved.
Cleavage from PS1cB—E (n=1), PS1cB—E (n=9), PS1gB—E (n=1), and PS1gB—E (n=9) was performed under the following conditions.
After treatment under the above-mentioned respective conditions, the cleavage results were analyzed by reversed-phase HPLC. The HPLC analysis conditions are shown in the following Table 13 (HPLC analysis conditions for TnB—D (n=1)), Table 14 (HPLC analysis conditions for TnE (n=1)), and Table 15 (HPLC analysis conditions for TnB—E (n=9)).
HPLC charts showing the cleavage results from 1) PS1cB (n=1) and PS1gB (n=1), 2) PS1cC (n=1) and PSlgC (n=1), 3) PS1cD (n=1) and PS1gD (n=1), 4) PS1cE (n=1), and PS1gE (n=1), 5) PS1cB (n=9) and PS1gB (n=9), 6) PS1cC (n=9) and PSlgC (n=9), 7) PS1cD (n=9) and PS1gD (n=9), and 8) PS1cE (n=9) and PS1gE (n=9) are respectively shown in 1)
The structures of the oligonucleotides (TnB—E) of interest which are released upon cleavage from respective solid phases were confirmed by LC-MS analysis. The results of the MS analysis are shown in Table 16.
Tn addition, the production ratio (HPLC area ratio; TnB—E:TnB—E-adduct) of the oligonucleotide (TnB—E) of interest and the by-produced oligonucleotide (TnB—E-adduct) which are released upon cleavage from each solid phase is shown in the following Table 17.
In oligonucleotide synthesis, phosphoramidites (compound: corresponding to nucleotide derivatives 14g, 16g, 20g, 25g) having a cyanoethoxy group as a protecting group for the phosphoric acid moiety have been widely used. However, when synthesizing oligonucleotides by using a universal support conventionally used widely, the use of the nucleotide derivatives (I) of the present invention (corresponding to nucleotide derivatives 14c, 16c, 20c, 25c), having a more stable substituent such as an alkoxy group as a protecting group for the phosphoric acid moiety, as a phosphoramidite strikingly increased the production of TnB—E oligonucleotides, and a superior effect was also confirmed that the oligonucleotides can be released from the solid phase support under mild base conditions even when nucleotides having 2′ moiety of sugar moiety modified with various functional groups were used.
The corresponding Table between the sequence Nos and the base sequences described in the above-mentioned Examples is shown below.
The present invention relates to a solid phase synthesis method of an oligonucleotide that enables cleavage of a synthesized oligonucleotide even under mild conditions by using a nucleoside phosphoramidite with a specific structure, and the like, and is useful in the technique fields requiring oligonucleotide synthesis (e.g., in the field of research and development of nucleic acid drugs).
This application is based on a patent application No. 2021-205491 filed in Japan (filing date: Dec. 17, 2021), the contents of which are incorporated in full herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-205491 | Dec 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/046549 | 12/16/2022 | WO |
