Technical field
[0001]
　The present invention is a manufacturing method morpholino oligonucleotides and to morpholinoethyl nucleotides used as the starting material for the production method.
Background technique
[0002]
　Morpholinophenyl oligonucleotides, DNA, high affinity for RNA, is resistant to various nucleases are stable in vivo, for toxicity is low, is the compound use as antisense oligonucleotides are noted (see non-Patent Document 1).
[0003]
　As a method for producing morpholinoethyl oligonucleotides are solid phase synthesis and liquid phase synthesis methods are reported (Non-Patent Document 2, see Patent Documents 1-5).
　Solid phase synthesis methods are advantageous in and speed surface can be automated synthesis of, there are facilities constraints on the scale-up is limited, also because of its low reactivity, a reagent used in nucleotide extension reactions monomer it is necessary to use an excess, not suitable for industrial large-scale synthesis. Further, confirmation of the progress of the reaction at an intermediate stage, there is also a disadvantage that the intermediate structure analysis and the like is also difficult.
　On the other hand, the liquid phase synthesis method, there are issues such as solubility and complexity of the post-processing operation, a large amount of chain length morpholino oligonucleotides may be used as antisense pharmaceuticals be rapidly synthesized is difficult .
CITATION
Patent Document
[0004]
Patent Document 1: WO 91/09033 Patent
Patent Document 2: WO 2008/008113
Patent Document 3: U.S. Patent Application Publication No. 2009/0131632 Pat
Patent Document 4: WO 2009/064471 Patent
JP 5: International Publication No. WO 2012/043730
Non-patent literature
[0005]
非特許文献1 : Ｓｕｍｍｅｒｔｏｎ，Ｊ．等，Ａｎｔｉｓｅｎｓｅ　ａｎｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｄｒｕｇ　Ｄｅｖｅｌｏｐｍｅｎｔ，１９９７年，Ｖｏｌ．７，ｐ．１８７．
非特許文献2 : Ｈａｒａｋａｗａ等，Ｂｉｏｏｒｇａｎｉｃ　＆　Ｍｅｄｉｃｉｎａｌ　Ｃｈｅｍｉｓｔｒｙ　Ｌｅｔｔｅｒｓ，２０１２年，Ｖｏｌ．２２，ｐ．１４４５－１４４７
Summary of the Invention
Problems that the Invention is to Solve
[0006]
　An object of the present invention is to provide a method, and a new morpholinophenyl nucleotide which is a raw material of the method of manufacturing morpholino oligonucleotides efficiently and in high yield by the liquid phase method.
Means for Solving the Problems
[0007]
　The present inventors have found that in the process of manufacturing morpholino oligonucleotide by a condensation reaction in the liquid phase, for a method for isolating the desired product by precipitation after deprotection reaction and condensation reaction, the operation is complicated, the operation time paying attention to the point that becomes huge, Work-up by extraction instead of precipitation, by a method to remove impurities in the aqueous layer side, it found efficiently to the target compound can be isolated. Furthermore a post-treatment with extraction operation after the deprotection reaction, by combining a post-treatment by extraction after condensation reaction can be carried out extension reaction morpholino oligonucleotides in one reaction vessel, it can be called one-pot synthesis It was found to be a.
　The present inventors have found that in the process of manufacturing morpholino oligonucleotide by a condensation reaction in the liquid phase, morpholinoethyl nucleoside monomer material remaining after the condensation reaction causes a double additional involved in the reaction in the next step, to produce by-products, purified as chain length increases it becomes difficult, even extension morpholino oligonucleotides focused on the point to be difficult, employing an extraction operation in post-treatment after the condensation reaction, for example, the particular compound ( by treating the system with quenching agent) was found to be able to efficiently remove the water layer side extraction raw material monomers derived from impurities remaining.
　The present inventors have found that in the process of manufacturing morpholino oligonucleotide by a condensation reaction in the liquid phase, from protecting groups resulting from deprotection of the protecting group such as trityl group protecting the nitrogen of the morpholine ring before condensation compounds, since involved in the reaction in the next condensation step produces a byproduct, which also focuses on the point that a difficult manufacturing morpholino oligonucleotide of long chain in the liquid phase synthesis, after deprotection reaction employing an extraction operation to a post-treatment, for example, by treating the system with specific compound (a cation scavenger), to be able to efficiently remove the aqueous layer side extraction of impurities from protecting groups such as trityl group heading was.
　The present inventors have found that in the process of manufacturing morpholino oligonucleotide by a condensation reaction in the liquid phase, 5 'and / or nucleobase alkyl and / or 10 or more carbon atoms having 10 to 300 carbon 300 the following protective groups having an alkenyl group (hereinafter, also referred to as "anchor".) be protected with a (hereinafter, also referred to as "anchoring".) Accordingly, the condensation reaction and deprotection reaction proceeds satisfactorily, further extracted operated to cull impurities to the aqueous layer side, improved solubility or partial layer of the non-polar solvent morpholino nucleotides, can be efficiently removed aqueous layer side extraction of impurities, more efficient morpholino oligonucleotides were found to be extended synthesized.
　The present inventors have found that in the process of manufacturing morpholino oligonucleotide by a condensation reaction in the liquid phase, nucleobase having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms by using morpholino nucleoside monomers that are protected by a protecting group, the condensation reaction and deprotection reaction proceeds satisfactorily, when cull further extraction with impurities to the aqueous layer side, a non-polar solvent morpholino nucleotides solubility and improved partial layer resistance to found that impurities can be efficiently removed to the aqueous layer side to extraction.
　The present inventors have found that the condensation reaction in the liquid phase in the process of manufacturing morpholino oligonucleotides, alkyl and / or 300 or less 10 or more carbon atoms having 10 to 300 carbon atoms as a protecting group as described above when using a protecting group having an alkenyl group, by employing a protecting group with an alkyl group and / or an alkenyl group having 300 or less branched having 10 or more carbon atoms of 300 or less branched having 10 or more carbon atoms, wherein the condensation reaction and the deprotection reaction proceeds satisfactorily, further when culling impurities to the aqueous layer side extraction to improve solubility and partial layer of the non-polar solvent morpholino nucleotides, extraction of impurities in efficiently be removed to the aqueous layer side, it was found to be able to stretch synthesized more efficiently morpholinophenyl oligonucleotide.
　The inventors have by these methods, found that can be produced efficiently and in high yield morpholino oligonucleotide by a liquid phase method, and completed the present invention.
　That is, the present invention includes the following.
[1] 5 'position hydroxyl group is activated phosphoramidate reduction, and morpholine ring nitrogen atom p pieces protected with a removable temporary protective group under acidic conditions polymerization morpholinoethyl oligonucleotides (p is 1 represents any integer greater than or equal to. the), 5 'end and / or nucleic acid bases, each independently, a protecting group having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms or the protected by morpholine ring nitrogen temporary custody group and different conditions can be removed by a protecting group of atoms, and n pieces polymerization morpholinoethyl oligonucleotides (n that morpholine ring nitrogen atom is not protected, one or more indicates an arbitrary integer.) and, after condensation by phosphoramidate bond or phosphorodiamidate bonds through the morpholine ring nitrogen atom, the resulting reaction Subjected compounds in extraction, separating the n + p pieces polymerization morpholinophenyl oligonucleotide is the product in an organic layer side (hereinafter, "step (2)" hereinafter) containing, for n + p pieces polymerization morpholinophenyl oligonucleotide Production method.
[2] n pieces polymerization morpholino oligonucleotide 5 'terminus and the nucleic acid bases, as well as among the p number polymerization morpholinophenyl oligonucleotide nucleobases, at least one of a branched alkyl group having 10 to 300 carbon atoms and / or It is protected with a protecting group having a branched alkenyl group having 10 to 300 carbon atoms, [1] the method according.
[3] 5 'end of the n polymer morpholinophenyl oligonucleotide is protected with a protecting group having a branched chain alkyl groups and / or branched alkenyl group having 10 to 300 carbon atoms 10 to 300 carbon atoms [1] the method according.
[4] p pieces polymerization morpholinophenyl oligonucleotide nucleobases, each independently, a protecting group or the morpholine ring nitrogen with an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms a protecting group of atoms are protected under different conditions can be removed by a protecting group, [1] the method according.
[5] Prior to extraction, the reaction mixture is treated with a quenching agent, [1] The method according.
[6] quenching agent is a compound having a secondary amino group and carboxy group, or a quenching agent comprising a compound having a phosphono group, [5] The method according.
[7] quenching agent is a quenching agent consisting of a compound having one secondary amino group and one or two carboxyl groups, [5] The method according.
[8] quenching agent is a prolyl glutamic acid, [5] The method according.
[9] quenching agent is a prolyl proline, [5] The method according.
[10] quenching agent is a quenching agent consisting of a compound having a phosphono group, [5] The method according.
[11] quenching agent is a phenylphosphonic acid, [5] The method according.
[12] further comprises the steps of, [1] the production method according to any one of to [11]; before step according to [1] (Step (2)), in a non-polar solvent , morpholine ring nitrogen atom is protected with a temporary protecting group which can be removed under acidic conditions, and the 5 'end and / or nucleic acid bases, each independently, an alkyl group having 10 to 300 carbon atoms and / or carbon atoms of n polymer morpholinophenyl oligonucleotide protected by removable protecting group with a protecting group, or under conditions different from the protecting group of the morpholine ring nitrogen atom having 10 or more 300 or less alkenyl group, a morpholine ring nitrogen atom removing temporary protective group, subjected to extraction the reaction mixture obtained, separating the n number polymerization morpholinophenyl oligonucleotide is the product in an organic layer side (hereinafter, referred to as "step (1)") .
[13] is reacted with an acid in the presence of a cation scavenger, to remove temporary protective group for morpholine ring nitrogen atom, [12] The method according.
[14] a cation scavenger is a compound having a mercapto group and a carboxy group, or a cation scavenger consisting of indole compound having a carboxy group, [13] The method according.
[15] a cation scavenger is a cation scavenger comprising a compound having one mercapto group and one or two carboxyl groups, [13] The method according.
[16] cation trapping agent is thiomalate or 3-mercaptopropionic acid, [13] The method according.
[17] morpholine ring nitrogen temporary custody protecting group which can be removed under different conditions and groups of atoms, a silyl protecting group, the production method according to [1].
[18] morpholine ring nitrogen temporary custody protecting group which can be removed under different conditions and groups of atoms, tert- butyldimethylsilyl group, diisopropylphenyl silyl group, is triphenylsilyl group, or a diphenyl tert- butoxysilyl group [1] the method according.
[19] is p is 1, The process according to any one of [1] to [18].
[20] a protecting group having an alkyl group and / or alkenyl group having 10 to 300 carbon atoms having 10 to 300 carbon atoms, by the following formula (II):
[0008]
[Formula 1]

[0009]
[L represents a single bond or the formula (a1):
[0010]
[Formula 2]

[0011]
Wherein * indicates the bonding position to Y;
** is protected, indicate the bonding position to an oxygen atom or a nitrogen atom;
L 1 is a divalent optionally substituted C 1 -22 represents a hydrocarbon group; and
L 2 is, C (= O) or show, or *** N (R 3 ) -R 1 -N (R 2 ) C (= O) ** (wherein , ** is L 1 represents a bonding position with, *** indicates the bonding position to Y, R 1 represents a C substituted 1-22 represents an alkylene group, R 2 and R 3 are each independently hydrogen atom or an optionally substituted C 1-22 represents an alkyl group, or R 2 and R 3 together are a C substituted 1-22It may form a alkylene linkage. ) A group represented by. Represents a group represented by],
Y represents a single bond, an oxygen atom, or NR (. R may represent a hydrogen atom, an alkyl group or an aralkyl group), as well as
Z has the formula (a2):
[0012]
[Formula 3]

[0013]
Wherein * indicates the bonding position to Y;
ring A represents a benzene ring or a cyclohexane ring;
R 4 is either a hydrogen atom, or R b is represented by the following formula (a3) that a group, and when ring a and ring B are both benzene rings, R 6 represents a single bond or O- together with, form a fluorenyl group or a xanthenyl group together with the ring B It is good;
is the k Q, or each independently represents a single bond or -O -, - S -, - OC (= O) -, - NHC (= O) - or -NH- are shown;
the k R 5 is independently each represents an organic group having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms;
k represents an integer of 1-4 ;
ring a, k-number of QR 5 in addition to further halogen atom, a halogen atom In an optionally substituted C 1-6 alkyl group, and even a C substituted by a halogen atom 1-6 may have a substituent group selected from the group consisting of an alkoxy group;
R a is It represents a hydrogen
atom; R b represents a hydrogen atom, or the formula (a3):
[0014]
[Chemical Formula 4]

[0015]
(Wherein, * represents a bonding position;
ring B represents a benzene ring or a cyclohexane ring;
j is 0 to an integer of 4;
j-number of Q represents the same meanings as defined above;
j-number of R 7 each independently represents an organic group having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon
atoms; R 6 is either a hydrogen atom, or R 4 shows a together a single bond or a O- and may form a fluorenyl group or a xanthenyl group together with ring a; and
ring B, j-number of QR 7 in addition to further halogen atom, a halogen atom in an optionally substituted C 1-6 alkyl group, and even a C substituted by a halogen atom 1-6 tables in may have a substituent group selected from the group consisting of an alkoxy group.) It represents a group that is, or R a and b are taken together to form a carbonyl group. Is a group represented by the process according to any one of [1] to [19].
[21] further comprises the following steps [1] to [20] the production method according to any one
of; [1], wherein step (step (2)) obtained in n + p pieces polymerization morpholinophenyl oligo removing any protecting groups of a nucleotide (hereinafter referred to as "step (4)").
[22] temporary protection which can be removed under acidic conditions group, trityl group, dimethoxytrityl group, or a mono-methoxytrityl group, The process according to any one of [1] to [21].
[23] Non-polar solvent is a halogenated solvents, aromatic solvents, ester solvents, aliphatic solvents, non-polar ether solvent, and a solvent selected from the group consisting of, [1] the process according to any one of - [22].
As used [24] according to [12] Step (Step (1)) in the obtained reaction mixture according to it [1] morpholino oligonucleotide without isolation step (step (2)), [12] the method according to any one of - [23].
[25] In the non-polar solvent, morpholine ring nitrogen atom is protected with a temporary protecting group which can be removed under acidic conditions, and the 5 'end of several 10 to 300 of the alkyl group and / or having 10 or more carbon-carbon 300 the following protected groups or the morpholine ring nitrogen n protected with a protecting group different conditions can be removed by a protecting group of atoms pieces polymerization morpholinophenyl oligonucleotide having an alkenyl group, in the presence of a cation scavenger is reacted with an acid, removing temporary protecting group of morpholine ring nitrogen atom (step (1)), including, n pieces polymerization morpholinoethyl oligonucleotides (n that morpholine ring nitrogen atom is not protected, It indicates one or more optional integer.) the method of producing.
[26] a cation scavenger is a compound having a mercapto group and a carboxy group, or a cation scavenger consisting of indole compound having a carboxy group, [25] The method according.
[27] a cation scavenger is a cation scavenger comprising a compound having one mercapto group and one or two carboxyl groups, [25] The method according.
[28] cation trapping agent is thiomalate or 3-mercaptopropionic acid, [25] The method according.
[29] Among the p number polymerization morpholinophenyl oligonucleotide and the n polymerization morpholinophenyl nucleobase oligonucleotide has at least one is an alkyl group and / or the number 10 to 300. alkenyl group of carbon atoms of 10 to 300 carbon atoms It is protected with a protecting group having the [1] the method according.
[30] a protecting group having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms, 10 to 300. branched alkyl groups and / or having 10 to 300 carbon atoms is a protecting group having a branched chain alkenyl group, [29] the method according.
[31] the general formula (I):
[0016]
[Formula 5]

[0017]
Wherein,
m represents an arbitrary integer of 0 or more,
P 1 is a hydrogen atom or a temporary protecting group which can be removed under acidic conditions,
P 2 is and alkyl group of 10 to 300 carbon atoms / or a protecting group, or P having an alkenyl group having 10 to 300 carbon atoms 1 shows a possible removal under different conditions protecting group,
base 1 represents a nucleic acid base which may be protected by a protecting group,
m number of base 2 are independently represents a nucleic acid base which may be protected by a respective protecting group,
m-number of X is independently each di C 1-6 alkylamino group or 4-position nitrogen atom is protected, protected by groups further show optionally substituted 1-piperazinyl group,
m pieces of W represents an oxygen atom.
However, 1) Base 1 when is protected with a protecting group protecting group, m pieces of any Base 2 any protecting groups which may have been protected by a protecting group, and P 2 protecting groups represented by, at least one is a protecting group having an alkyl group and / or 300 an alkenyl group having 10 or more carbon atoms having 10 to 300 carbon atoms, and
2) P 2If There is a protecting group having a straight-chain alkyl groups and / or 300 the following linear alkenyl group having 10 or more carbon atoms having 10 to 300 carbon atoms, Base 1 and m any Base 2 at least one of, is a nucleobase that is protected with a protecting group having an alkyl group and / or alkenyl group having 10 to 300 carbon atoms having 10 to 300 carbon atoms. ]
Morpholinophenyl nucleotides denoted by.
[32] m is an integer of 0 to 19, [31], wherein morpholino nucleotide.
[33] a protecting group having an alkyl group and / or alkenyl group having 10 to 300 carbon atoms having 10 to 300 carbon atoms, by the following formula (II):
[0018]
[Formula 6]

[0019]
[L represents a single bond or the formula (a1):
[0020]
[Chemical Formula 7]

[0021]
Wherein * indicates the bonding position to Y;
** is protected, indicate the bonding position to an oxygen atom or a nitrogen atom;
L 1 is a divalent optionally substituted C 1 -22 represents a hydrocarbon group; and
L 2 is, C (= O) or show, or *** N (R 3 ) -R 1 -N (R 2 ) C (= O) ** (wherein , ** is L 1 represents a bonding position with, *** indicates the bonding position to Y, R 1 represents a C substituted 1-22 represents an alkylene group, R 2 and R 3 are each independently hydrogen atom or an optionally substituted C 1-22 represents an alkyl group, or R 2 and R 3 together are a C substituted 1-22It may form a alkylene linkage. ) A group represented by. Represents a group represented by],
Y represents a single bond, an oxygen atom, or NR (. R may represent a hydrogen atom, an alkyl group or an aralkyl group), as well as
Z has the formula (a2):
[0022]
[Formula 8]

[0023]
Wherein * indicates the bonding position to Y;
ring A represents a benzene ring or a cyclohexane ring;
R 4 is either a hydrogen atom, or R b is represented by the following formula (a3) that a group, and when ring a and ring B are both benzene rings, R 6 represents a single bond or O- together with, form a fluorenyl group or a xanthenyl group together with the ring B It is good;
is the k Q, or each independently represents a single bond or -O -, - S -, - OC (= O) -, - NHC (= O) - or -NH- are shown;
the k R 5 is independently each represents an organic group having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms;
k represents an integer of 1-4 ;
ring a, k-number of QR 5 in addition to further halogen atom, a halogen atom In an optionally substituted C 1-6 alkyl group, and even a C substituted by a halogen atom 1-6 may have a substituent group selected from the group consisting of an alkoxy group;
R a is It represents a hydrogen
atom; R b represents a hydrogen atom, or the formula (a3):
[0024]
[Formula 9]

[0025]
(Wherein, * represents a bonding position;
ring B represents a benzene ring or a cyclohexane ring;
j is 0 to an integer of 4;
j-number of Q represents the same meanings as defined above;
j-number of R 7 each independently represents an organic group having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon
atoms; R 6 is either a hydrogen atom, or R 4 shows a together a single bond or a O- and may form a fluorenyl group or a xanthenyl group together with ring a; and
ring B, j-number of QR 7 in addition to further halogen atom, a halogen atom in an optionally substituted C 1-6 alkyl group, and even a C substituted by a halogen atom 1-6 tables in may have a substituent group selected from the group consisting of an alkoxy group.) It represents a group that is, or R a and b are taken together to form a carbonyl group. Is a group represented by, morpholinoethyl nucleotide according to [31] or [32].
[34] a protecting group having an alkyl group and / or 300 an alkenyl group having 10 or more carbon atoms having 10 to 300 carbon atoms,
3,4,5-tri (octadecyl) benzoyl group, and 3,4,5-tri (2 ', 3'-dihydro phytyl oxy) is selected from the group consisting of a benzoyl group, [31] or [32 morpholinophenyl nucleotides described.
[35] P 2 is a silyl protecting group, [31] morpholinophenyl nucleotides of any one of - [34].
[36] P 2 is, tert- butyldimethylsilyl group, diisopropylphenyl silyl group, a triphenylsilyl group, or a diphenyl tert- butoxysilyl group, [31] according to any one of - [35] morpholinophenyl nucleotides .
[37] P 1 is trityl group, monomethoxytrityl group or a dimethoxytrityl group, [31] - morpholinophenyl nucleotides according to any one of [36].
Effect of the Invention
[0026]
　According to the present invention, in the liquid phase synthesis morpholino oligonucleotides, conveniently aqueous layer side by extraction of impurities from the protecting group occurring by deprotection prior morpholinoethyl nucleoside monomers and the condensation reaction of the starting materials remaining after the condensation reaction be removed, since the side reaction of these impurities is suppressed, it becomes possible to manufacture large quantities and efficiently high quality morpholino oligonucleotide.
　The protection with a particular 5 'hydroxyl group or an amino group present at the end, and / or nucleobase an alkyl and / or alkenyl group having 10 or more carbon atoms of 300 or less branched branched having 10 to 300 carbon atoms by protected with groups, and preferably by a protecting group having such protected by protecting a group, and preferably branched-chain alkyl groups and / or branched-chain alkenyl groups of the nucleobases, also condensation reaction and deprotection reaction proceeded satisfactorily, further improves the solubility and partial layer of morpholinoethyl oligonucleotides in extraction, it becomes possible to perform the post-processing more efficiently.
　Morpho That is, according to the present invention, the condensation reaction was remarkably improved compared to the phase the solid reactive by performing in a liquid phase, a monomer equivalent of using can be reduced remarkably, after further reaction by extraction it is possible to easily isolated and purified Reno oligonucleotide, it becomes possible to produce efficiently and in high yield a chain length morpholino oligonucleotides that can be utilized in pharmaceutical in the liquid phase synthesis method.
DESCRIPTION OF THE INVENTION
[0027]
[Explanation of Terms]
　Unless defined otherwise, all technical and scientific terms used herein have the same meaning as the present invention is commonly understood by one of ordinary skill in the belonging art. Although any methods and materials similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned herein, for example, are described in publications can be used in connection with the described invention, constructs and for the purpose of describing and disclosing the methodologies, hereby by reference It is incorporated in the book.
[0028]
　In the present specification, the structural units morpholino oligonucleotides "morpholinoethyl nucleoside" is a compound represented by the following formula (1).
[0029]
[Formula 10]

[0030]
(Wherein, Base shows a nucleic acid base which may be protected)
　morpholinoethyl nucleoside (1) are prepared by methods known per se (e.g., methods described in WO91 / 09033A1) prepared according to a method analogous, or in be able to. Specifically, as shown in the following scheme, the corresponding ribonucleoside (2) 2 corresponding oxidized opened with sodium periodate or the like ', and 3'-dialdehyde (3), 2', 3 '- dialdehyde (3) and ring closure with ammonia 2', 3'-dihydroxy-morpholinophenyl nucleoside (4), and 2 ', 3'-dihydroxy-morpholinophenyl nucleoside (4) a reducing agent (e.g., cyanoborohydride sodium borohydride, by reduction with sodium triacetoxyborohydride, etc.), morpholinoethyl nucleoside (1) can be obtained.
[0031]
[Of 11]

[0032]
　In this specification, the position numbers (1 ', 2', etc.) morpholino nucleoside shall be used those corresponding to the position number of the carbon atoms of the ribose of the ribonucleoside which is a raw material (2).
[0033]
　In the present specification, the morpholinoethyl oligonucleotides, means a compound that 2 or more morpholino nucleoside 5 'position through a hydroxyl group and the nitrogen atom of the morpholine ring polymerized by phosphoramidate bond or phosphorodiamidate bond and, for example, the m '+ 1 or polymerized morpholinophenyl oligonucleotide, include compounds represented by the following formula (5).
[0034]
[Chem. 12]

[0035]
(Wherein,
m 'is 1 or more represents any
integer, Base 1 and m' number of Base 2 are independently represents a nucleic acid base which may be protected respectively,
m 'number of X is, independently each di C 1-6 alkylamino group, the 4-position nitrogen atom is protected with a protecting group, it may be additionally substituted 1 shows a piperazinyl group, etc.,
m 'number of W represents an oxygen atom .)
[0036]
　As used herein, "4-position nitrogen atom is protected by a protecting group, it may be additionally substituted 1-piperazinyl group" means that the 4-position nitrogen atom of the piperazinyl group is protected with a protecting group and it is protected by a protective group capable of withstanding the deprotection conditions morpholine ring nitrogen atom morpholino nucleotide 1-piperazinyl group. Preferably an acyl group as such "protecting group for piperazinyl group 4-position nitrogen atom", for example, monofluoromethyl acetyl group, difluoroacetyl group, trifluoroacetyl group, 2-fluoro-propionyl group, 2,2-difluoro-propionyl group, 3 , 3,3-trifluoropropyne propionyl group, 2,3,3,3-tetrafluoro-propionyl group, pentafluoropropionyl more preferably an acyl group having a fluoroalkyl group on the carbon chain, such groups (WO 2008/008113 pamphlet reference). Piperazinyl group, which may be a hydrogen atom bonded to a carbon atom of the piperazinyl group is substituted, the substituent, an alkyl group such as a methyl group (preferably having from 1 to 3 carbon atoms) and the like.
[0037]
　In this specification, copying the practice of nucleic acid chemistry, "5'-end" morpholino nucleoside end on the side having a free hydroxyl group of the 5'-position morpholino oligonucleotides (lower left side of the equation (5)), morpholino nucleoside of opposite end (the upper right side of the equation (5)) shall be referred to as "3'-end".
[0038]
　As used herein, "nucleobase" includes not particularly limited as long as it is used in the synthesis of nucleic acid, for example, Shitoshiru group, uracil group, a pyrimidine base, such as thyminyl group, adenyl group, such as a guanyl group mention may be made of the purine base. More nucleic acid bases present morpholinophenyl nucleotides may be a nucleobase heterologous, it may be homologous nucleobases. Furthermore, "it is protected nucleic acid base which may", for example, nucleic acid bases can be mentioned an amino group and a hydroxyl group present on the nucleobases are protected, for example, adenyl group is a nucleobase having an amino group , guanyl group or at Shitoshiru group, it may be an amino group is protected, the nucleic acid is protected by a protective group which amino group of the nucleobase can withstand deprotection conditions morpholine ring nitrogen atom morpholino nucleotide bases It is preferred. Examples of the "protecting group of amino group" is not particularly limited, for example, Greens Protective Groups in Organic Synthesis (Greene's PROTECTIVE GROUPS in ORGANIC SYNTHESIS), 4th edition, Wiley-Interscience ( Wiley-Interscience) publication (can be exemplified protecting group described in 2006) and the like. Specific examples of the "protecting group of amino group", for example, a pivaloyl group, pivaloyloxymethyl group, trifluoroacetyl group, phenoxyacetyl group, 4-isopropyl phenoxyacetyl, 4-tert-butyl phenoxyacetyl include an acetyl group, a benzoyl group, an isobutyryl group, dimethylformamidine isoxazolidinyl group, a 9-fluorenylmethyloxycarbonyl group. Among these, phenoxyacetyl, 4-isopropyl phenoxyacetyl group, an acetyl group, a benzoyl group, an isobutyryl group and dimethylformamidine isoxazolidinyl group. Also, may be the carbonyl group of the nucleobase is protected, for example, phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5 Jikurorofe Nord, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylamino-phenol, pentafluorophenol, 4-pivaloyloxymethyl benzyl alcohol, 4-nitro phenethyl alcohol, 2- (methylsulfonyl) ethanol, 2- (phenylsulfonyl) ethanol, 2-cyano ethanol, 2- (trimethylsilyl) ethanol, dimethylcarbamoyl chloride, diethyl carbamic acid chloride, ethyl phenyl carbamic acid chloride, 1-pyrrolidinecarboxylic acid chloride, 4-morpholine carboxylic acid chloride, by reacting diphenyl carbamic acid chloride and the like, can be protected carbonyl group. Here, the protecting group of the carbonyl group, it may not be necessary particularly introduced. Further, in the "nucleobase" includes, in addition to the foregoing groups, the nucleobase is any substituent (e.g., a halogen atom, an alkyl group, an aralkyl group, an alkoxy group, an acyl group, an alkoxyalkyl group, hydroxy group, amino group, monoalkylamino group, a dialkylamino group, a carboxy group, a cyano group, modified nucleic acid bases which is substituted 1-3 at an arbitrary position by nitro group) (eg, 8-Buromoadeniru group, 8-bromo guanyl group, 5-Buromoshitoshiru group, 5-Yodoshitoshiru group, 5-bromouracil group, 5-iodo uracil group, 5-fluorouracil group, 5-Mechirushitoshiru group, 8-oxo-guanyl group, Hipokisanchiniru group) are also encompassed. Chloride, diethyl carbamic acid chloride, ethyl phenyl carbamic acid chloride, 1-pyrrolidine carbonyl chloride, 4-morpholine carboxylic acid chloride, by reacting diphenyl carbamic acid chloride and the like, can be protected carbonyl group. Here, the protecting group of the carbonyl group, it may not be necessary particularly introduced. Further, in the "nucleobase" includes, in addition to the foregoing groups, the nucleobase is any substituent (e.g., a halogen atom, an alkyl group, an aralkyl group, an alkoxy group, an acyl group, an alkoxyalkyl group, hydroxy group, amino group, monoalkylamino group, a dialkylamino group, a carboxy group, a cyano group, modified nucleic acid bases which is substituted 1-3 at an arbitrary position by nitro group) (eg, 8-Buromoadeniru group, 8-bromo guanyl group, 5-Buromoshitoshiru group, 5-Yodoshitoshiru group, 5-bromouracil group, 5-iodo uracil group, 5-fluorouracil group, 5-Mechirushitoshiru group, 8-oxo-guanyl group, Hipokisanchiniru group) are also encompassed. Chloride, diethyl carbamic acid chloride, ethyl phenyl carbamic acid chloride, 1-pyrrolidine carbonyl chloride, 4-morpholine carboxylic acid chloride, by reacting diphenyl carbamic acid chloride and the like, can be protected carbonyl group. Here, the protecting group of the carbonyl group, it may not be necessary particularly introduced. Further, in the "nucleobase" includes, in addition to the foregoing groups, the nucleobase is any substituent (e.g., a halogen atom, an alkyl group, an aralkyl group, an alkoxy group, an acyl group, an alkoxyalkyl group, hydroxy group, amino group, monoalkylamino group, a dialkylamino group, a carboxy group, a cyano group, modified nucleic acid bases which is substituted 1-3 at an arbitrary position by nitro group) (eg, 8-Buromoadeniru group, 8-bromo guanyl group, 5-Buromoshitoshiru group, 5-Yodoshitoshiru group, 5-bromouracil group, 5-iodo uracil group, 5-fluorouracil group, 5-Mechirushitoshiru group, 8-oxo-guanyl group, Hipokisanchiniru group) are also encompassed.
　Moreover, the "protecting group of amino group" includes protecting groups having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms, a method known per se or a method analogous thereto it is possible to protect an amino group according to. For example, it can be protected by reacting the nitrogen atom on the pyrimidine ring of thyminyl group, a 3,4,5-tri (octadecyloxy) benzoic acid chloride in the presence of a base.

The scope of the claims
[Claim 1]
　5 'position hydroxyl group is activated phosphoramidate reduction, and p number polymerization morpholinoethyl oligonucleotides (p protected by the morpholine ring nitrogen atom temporary protecting group which can be removed under acidic conditions, one or more optional of an integer a.), 5 'end and / or nucleobases each independently a protecting group or the Morpho having an alkyl group and / or the number 10 to 300. alkenyl group of carbon atoms of 10 to 300 carbon atoms protected by phosphorus ring nitrogen atom of the temporary protecting groups and removable under different conditions protecting group, and n pieces polymerization morpholinoethyl oligonucleotides (n that morpholine ring nitrogen atom is not protected, an arbitrary integer of 1 or more It is shown.) and, after condensation by phosphoramidate bond or phosphorodiamidate bonds through the morpholine ring nitrogen atom, the reaction mixture obtained Subjected to extraction, comprising the step of separating the n + p pieces polymerization morpholinophenyl oligonucleotide is the product in an organic layer side, n + p pieces polymerization method for producing morpholino oligonucleotide.
[Claim 2]
　n number polymerization morpholinophenyl oligonucleotide 5'-end and nucleic acid bases, as well as among the p number polymerization morpholinophenyl oligonucleotide nucleobases, at least one is branched chain alkyl groups and / or 10 carbon atoms having 10 to 300 carbon atoms It is protected with a protecting group having 300 or less branched alkenyl group or process according to claim 1, wherein.
[Claim 3]
　n pieces 5 'end of the polymerization morpholinoethyl oligonucleotide is protected with a protecting group having a branched chain alkyl groups and / or branched alkenyl group having 10 to 300 carbon atoms 10 to 300 carbon atoms, claims production method of 1, wherein the.
[Claim 4]
　p number polymerization morpholinophenyl oligonucleotide nucleobases, each independently, of protecting groups or the morpholine ring nitrogen atom with an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms It is protected with a removable protecting group under conditions different from the group, the process according to claim 1, wherein.
[Claim 5]
　Before extraction, the reaction mixture is treated with a quenching agent, production method according to claim 1.
[Claim 6]
　Quenching agent is a compound having a secondary amino group and carboxy group, or a quenching agent comprising a compound having a phosphono group, a manufacturing method of claim 5.
[Claim 7]
　Quenching agent is a quenching agent consisting of a compound having one secondary amino group and one or two carboxyl groups, a manufacturing method of claim 5, wherein.
[8.]
　Quenching agent is a prolyl glutamic acid The process of claim 5 wherein.
[Claim 9]
　Quenching agent is a prolyl proline, a manufacturing method of claim 5, wherein.
[Claim 10]
　Quenching agent is a quenching agent consisting of a compound having a phosphono group, a manufacturing method of claim 5, wherein.
[Claim 11]
　Quenching agent is a phenylphosphonic acid, the production method of claim 5, wherein.
[Claim 12]
　Further comprises the steps, the production method according to any one of claims 1 to 11; before described process in claim 1, in a non-polar solvent, morpholine ring nitrogen atom acidic conditions in protected with a removable temporary protective group, and the 5 'end and / or nucleic acid bases, each independently, protection having an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms from group or the morpholine ring of n are protected by removable protective group under conditions different from the protecting group for the nitrogen atom polymerization morpholinophenyl oligonucleotide, was removed temporary protecting group of morpholine ring nitrogen atom, the resulting the reaction mixture was subjected to extraction, separating the n polymerization morpholinophenyl oligonucleotide is the product in an organic layer side.
[Claim 13]
　It is reacted with an acid in the presence of a cation scavenger, to remove temporary protective group for morpholine ring nitrogen atom, the production method of claim 12, wherein.
[Claim 14]
　Cation scavengers, compounds having a mercapto group and a carboxy group, or a cation scavenger consisting of indole compound having a carboxyl group, a manufacturing method of claim 13.
[Claim 15]
　A cation scavenger is a cation scavenger comprising a compound having one mercapto group and one or two carboxyl groups, a manufacturing method of claim 13, wherein.
[Claim 16]
　Cation trapping agent is thiomalate or 3-mercaptopropionic acid, The process of claim 13 wherein.
[Claim 17]
　Morpholine ring nitrogen temporary custody protecting group which can be removed under different conditions and groups of atoms, a silyl-based protective group, The process according to claim 1, wherein.
[Claim 18]
　Morpholine ring nitrogen temporary custody protecting group which can be removed under different conditions and groups of atoms, a tert- butyldimethylsilyl group, diisopropylphenyl silyl group, a triphenylsilyl group, or a diphenyl tert- butoxysilyl group, claims production method of 1, wherein the.
[Claim 19]
　p is 1, The method according to any one of claims 1 to 18.
[Claim 20]
　Protecting group with an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms, following formula (II):
[of

1] [L represents a single bond or the formula (a1):
[formula 2]

wherein, * represents a bonding position to Y;
** is protected, it indicates the bonding position to an oxygen atom or a nitrogen atom;
L 1 may be substituted 2 valent C 1-22 represents a hydrocarbon group; and
L 2 is, C (= O) or show, or *** N (R 3 ) -R 1 -N (R 2 ) C (= O) * * (where ** is L 1 represents a bonding position with, *** indicates the bonding position to Y, R 1 represents a C substituted 1-22 represents an alkylene group, R 2 and R 3 are optionally independently be hydrogen or optionally substituted C1-22 represents an alkyl group, or R 2 and R 3 together are a C substituted 1-22 may form an alkylene bond. ) A group represented by. Represents a group represented by],
Y represents a single bond, an oxygen atom, or NR (R represents a hydrogen atom, an alkyl group or an aralkyl group.) Indicates, and
Z has the formula (a2): [Chemical
formula 3 ]

wherein * represents the bonding position to Y;
ring a represents a benzene ring or a cyclohexane
ring; R 4 is either a hydrogen atom, or R b is, the table the following formula (a3) a group is, and when ring a and ring B are both benzene rings, R 6 represents a single bond or O- together with, to form a fluorenyl group or a xanthenyl group together with the ring B at best;
k number of Q are either each independently a single bond, or -O -, - S -, - OC (= O) -, - NHC (= O) - or -NH- are shown ;
k-number of R 5 is independently an alkyl group having 10 to 300 carbon atoms and / or Represents an organic group having an alkenyl group having 10 to 300 carbon atoms;
k represents an integer of 1-4;
ring A, k pieces of QR 5In addition to further halogen atom, a C substituted with halogen atom 1-6 alkyl group, and even a C substituted by a halogen atom 1-6 substituents selected from the group consisting of an alkoxy group
; it may have R a represents a hydrogen
atom; R b represents a hydrogen atom, or the formula (a3):
[Chemical formula 4]

(wherein, * represents a bonding position;
ring B is a benzene ring or it shows a cyclohexane ring;
j represents an integer of 0 ~ 4;
j-number of Q represents the same meanings as defined above;
j-number of R 7 is independently each alkyl of 10 to 300 carbon atoms It represents an organic group having a group and / or the number 10 to 300. alkenyl group having a carbon;
R 6 is either a hydrogen atom, or R 4 together with by a single bond or a O-, with ring a It may form a fluorenyl group or a xanthenyl group ; And
ring B, j-number of QR 7 in addition to further halogen atom, a C substituted with halogen atom 1-6 alkyl group, and may be substituted by a halogen atom C 1-6It may have a substituent group selected from the group consisting of an alkoxy group. ) Represents a group represented by, or R a and R b are taken together to form a carbonyl group. Is a group represented by the process according to any one of claims 1 to 19.
[Claim 21]
　Further comprises the steps of, claims 1 to 20 The process according to any one of; a step of removing all the n + p-number obtained in step of claim 1 polymerized morpholinophenyl oligonucleotide protecting groups .
[Claim 22]
　Temporary protection which can be removed under acidic conditions group, trityl group, dimethoxytrityl group, or a mono-methoxytrityl group, The process according to any one of claims 1 to 21.
[Claim 23]
　Nonpolar solvents, halogenated solvents, aromatic solvents, ester solvents, aliphatic solvents, non-polar ether solvent, and a solvent selected from the group consisting of, according to claim 1 to 22 the process according to any one.
[Claim 24]
　As used according the claim 12 the reaction mixture obtained in the process described in described as in claim 1 without isolating morpholino oligonucleotides manufacturing method as set forth in any one of claims 12-23 .
[Claim 25]
　In nonpolar solvents, morpholine ring nitrogen atom is protected with a removable temporary protective group under acidic conditions, and the 5 'end with an alkyl group and / or 300 or less 10 or more carbon atoms having 10 to 300 carbon atoms the protecting group or the morpholine ring of n are protected by removable protective group under conditions different from the protecting group for the nitrogen atom polymerization morpholinophenyl oligonucleotide having an alkenyl group, an acid in the presence of a cation scavenger reaction by a step of removing temporary protective group for morpholine ring nitrogen atoms
including, n pieces polymerization morpholinophenyl oligonucleotides morpholine ring nitrogen atom is not protected (n represents one or more optional integer.) the method of production.
[Claim 26]
　Cation scavengers, compounds having a mercapto group and a carboxy group, or a cation scavenger consisting of indole compound having a carboxy group, The process of claim 25 wherein.
[Claim 27]
　Manufacturing method of a cation scavenger is a cation scavenger comprising a compound having one mercapto group and one or two carboxyl groups, according to claim 25, wherein.
[Claim 28]
　Cation trapping agent is thiomalate or 3-mercaptopropionic acid, The process of claim 25 wherein.
[Claim 29]
　Of the nucleic acid bases included in the p number polymerization morpholinophenyl oligonucleotide and the n polymerization morpholinophenyl oligonucleotide, at least one is protected with an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms It is protected by a group, the method according to claim 1, wherein.
[Claim 30]
　Protecting group with an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms, branched chain alkyl groups and / or 300 or less branched having 10 or more carbon atoms having 10 to 300 carbon atoms is a protecting group having an alkenyl group, the method of claim 29, wherein.
[Claim 31]
　General formula (I):
[Chemical Formula 5]

[wherein,
m represents an arbitrary integer of 0 or more,
P 1 is a hydrogen atom or a temporary protecting group which can be removed under acidic conditions,
P 2 is protecting group or P having an alkyl group and / or alkenyl group having 10 to 300 carbon atoms having 10 to 300 carbon atoms 1 shows a removable protective group under conditions different from,
base 1 is protected with a protecting group shows the nucleic acid base which may have,
m-number of base 2 are independently represents a nucleic acid base which may be protected by a respective protecting group,
m-number of X is independently each di C 1-6 alkyl amino or 4-position nitrogen atom, is protected by a protecting group, further show optionally substituted 1-piperazinyl group,
m pieces of W represents an oxygen atom.
However, 1) Base 1 when is protected with a protecting group protecting group, m pieces of any Base 2 any protecting groups which may have been protected by a protecting group, and P 2 protecting groups represented by, at least one is a protecting group having an alkyl group and / or 300 an alkenyl group having 10 or more carbon atoms having 10 to 300 carbon atoms, and
2) P 2If There is a protecting group having a straight-chain alkyl groups and / or 300 the following linear alkenyl group having 10 or more carbon atoms having 10 to 300 carbon atoms, Base 1 and m any Base 2 at least one of, is a nucleobase that is protected with a protecting group having an alkyl group and / or alkenyl group having 10 to 300 carbon atoms having 10 to 300 carbon atoms. ]
Morpholinophenyl nucleotides denoted by.
[Claim 32]
　m is an integer of 0 to 19, claim 31 morpholino nucleotide.
[Claim 33]
　Protecting group with an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms, following formula (II):
[of

6] [L represents a single bond or the formula (a1):
[Chemical formula 7]

wherein, * represents a bonding position to Y;
** is protected, indicate the bonding position to an oxygen atom or a nitrogen atom;
L 1 may be substituted 2 valent C 1-22 represents a hydrocarbon group; and
L 2 is, C (= O) or show, or *** N (R 3 ) -R 1 -N (R 2 ) C (= O) * * (where ** is L 1 represents a bonding position with, *** indicates the bonding position to Y, R 1 represents a C substituted 1-22 represents an alkylene group, R 2 and R 3 are optionally independently be hydrogen or optionally substituted C1-22 represents an alkyl group, or R 2 and R 3 together are a C substituted 1-22 may form an alkylene bond. ) A group represented by. Represents a group represented by],
Y represents a single bond, an oxygen atom, or NR (R represents a hydrogen atom, an alkyl group or an aralkyl group.) Indicates, and
Z has the formula (a2): [Formula
8 ]

wherein * represents the bonding position to Y;
ring a represents a benzene ring or a cyclohexane
ring; R 4 is either a hydrogen atom, or R b is, the table the following formula (a3) a group is, and when ring a and ring B are both benzene rings, R 6 represents a single bond or O- together with, to form a fluorenyl group or a xanthenyl group together with the ring B at best;
k number of Q are either each independently a single bond, or -O -, - S -, - OC (= O) -, - NHC (= O) - or -NH- are shown ;
k-number of R 5 is independently an alkyl group having 10 to 300 carbon atoms and / or Represents an organic group having an alkenyl group having 10 to 300 carbon atoms;
k represents an integer of 1-4;
ring A, k pieces of QR 5In addition to further halogen atom, a C substituted with halogen atom 1-6 alkyl group, and even a C substituted by a halogen atom 1-6 substituents selected from the group consisting of an alkoxy group
; it may have R a represents a hydrogen
atom; R b represents a hydrogen atom, or the formula (a3):
[Chemical formula 9]

(wherein, * represents a bonding position;
ring B is a benzene ring or it shows a cyclohexane ring;
j represents an integer of 0 ~ 4;
j-number of Q represents the same meanings as defined above;
j-number of R 7 is independently each alkyl of 10 to 300 carbon atoms It represents an organic group having a group and / or the number 10 to 300. alkenyl group having a carbon;
R 6 is either a hydrogen atom, or R 4 together with by a single bond or a O-, with ring a It may form a fluorenyl group or a xanthenyl group ; And
ring B, j-number of QR 7 in addition to further halogen atom, a C substituted with halogen atom 1-6 alkyl group, and may be substituted by a halogen atom C 1-6It may have a substituent group selected from the group consisting of an alkoxy group. ) Represents a group represented by, or R a and R b are taken together to form a carbonyl group. It is a group represented by, morpholinoethyl nucleotide of claim 31 or 32.
[Claim 34]

Protecting　group with an alkyl group and / or the number 10 to 300. alkenyl group having a carbon number of 10 to 300 carbon atoms, 3,4,5-tri (octadecyl) benzoyl group, and 3,4,5-tri ( 2 ', 3'-dihydro phytyl oxy) is selected from the group consisting of a benzoyl group, morpholinoethyl nucleotide of claim 31 or 32.
[Claim 35]
　P 2 is a silyl protecting group, morpholinoethyl nucleotides according to any one of claims 31 to 34.
[Claim 36]
　P 2 is, tert- butyldimethylsilyl group, diisopropylphenyl silyl group, a triphenylsilyl group, or a diphenyl tert- butoxysilyl group, morpholinoethyl nucleotides according to any one of claims 31-35.
[Claim 37]
　P 1 is trityl group, monomethoxytrityl group or a dimethoxytrityl group, morpholinoethyl nucleotides according to any one of claims 31-36.