Long-chain alkenyloxy-substituted benzoyl derivative and oligonucleotide synthesis method using same

A compound represented by the following formula (1) or a salt thereof:

• wherein,
• R each independently represents an optionally substituted alkenyl group having 14 to 60 carbon atoms;
• n represents 2 or 3, and the plurality of OR groups may be the same or different;
• m represents 0 or 1;
• p represents 1 or 2;
• X is C, N, O or S, and when X is C, it represents CH2 or may form, together with Y, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent, and when X is N, it represents NR1 or may form, together with Y, a 5- to 7-membered heterocyclic ring which may have a substituent, wherein R1 is H or a C1-6 alkyl group;
• Y is a single bond or B(CH2)t* (wherein B is a 5- to 7-membered carbocyclic ring which may have a substituent, t is 0, 1, or 2, and * indicates a bond to X), or may form, together with X, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent;
• Z is a single bond or (CH2)s (wherein s is an integer of 1 to 5), provided that when Y is a single bond, Z is not a single bond.

A nucleoside or nucleotide represented by any one of the following formulae (I), (II), (V), (VI), (VII), or (VIII):

• wherein,
• o represents any integer of 0 or more;
• each Base independently represents an optionally modified nucleic acid base;
• each d independently represents a hydrogen atom, a halogen atom, or a hydroxyl group which may be protected, or may form a crosslink with the 4′-position carbon;
• each Q1 independently represents a C1-4 alkyl group having an electron-withdrawing group, an allyl group, a C2-6 alkenyl group or a C6-10 aryl group having a cyano group, a nitro group or a halogen group, or a hydrogen atom;
• W/Tg or Tg/W represents W or Tg, and when the 5′ side is W, the 3′ side is Tg, whereas when the 5′ side is Tg, the 3′ side is W, wherein
• W represents a hydrogen atom or a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion;
• each R2 independently represents O or S;
• and
• Tg represents a group represented by the following formula (5):
• wherein,
• R each independently represents an optionally substituted alkenyl group having 14 to 60 carbon atoms;
• n represents 2 or 3, and the plurality of OR groups may be the same or different;
• m represents 0 or 1;
• p represents 1 or 2;
• X is C, N, O or S, and when X is C, it represents CH2 or may be combined with Y to form a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent, and when X is N, it represents NR1 or may form, together with Y, a 5- to 7-membered heterocyclic ring which may have a substituent, wherein R1 is H or a C1-6 alkyl group;
• Y is a single bond or B(CH2)t* (wherein B is a 5- to 7-membered carbocyclic ring which may have a substituent, t is 0, 1, or 2, and * indicates a bond to X), or may form, together with X, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent;
• Z is a single bond or (CH2)s, wherein s is an integer of 1 to 5, provided that when Y is a single bond, Z is not a single bond,
• ** indicates a bond to O;
• wherein,
• o represents any integer of 0 or more;
• each Base independently represents an optionally modified nucleic acid base;
• each V independently represents a C1-6 alkoxy group, a di(C1-6 alkyl)amino group, or a piperazino group in which the 4-position nitrogen is protected with a protecting group and which may be further substituted;
• W′ represents a hydrogen atom or a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion;
• each R2 independently represents O or S;
• Tg represents a group represented by the following formula (5):
• wherein,
• R each independently represents an optionally substituted alkenyl group having 14 to 60 carbon atoms;
• n represents 2 or 3, and the plurality of OR groups may be the same or different;
• m represents 0 or 1;
• p represents 1 or 2;
• X is C, N, O or S, and when X is C, it represents CH2 or may form, together with Y, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent, and when X is N, it represents NR1 or may form, together with Y, a 5- to 7-membered heterocyclic ring which may have a substituent, wherein R1 is H or a C1-6 alkyl group;
• Y is be a single bond or B(CH2)t* (wherein B is a 5- to 7-membered carbocyclic ring which may have a substituent, t is 0, 1, or 2, and * indicates a bond to X), or may form, together with X, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent;
• Z is a single bond or (CH2)s, wherein s is an integer of 1 to 5, provided that when Y is a single bond, Z is not a single bond,
• ** indicates a bond to O;
• wherein,
• o represents any integer of 0 or more;
• each Base independently represents an optionally modified nucleic acid base;
• each D independently represents a hydrogen atom, a halogen atom, or a hydroxyl group which may be protected, or may form a crosslink with the 4′-position carbon;
• each Q1 independently represents a C1-4 alkyl group having an electron-withdrawing group, an allyl group, a C2-6 alkenyl group or a C6-10 aryl group having a cyano group, a nitro group, or a halogen group, or a hydrogen atom;
• W each independently represents a hydrogen atom or a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion;
• each R2 independently represents O or S;
• Tg represents a group represented by the following formula (5):
• wherein,
• R each independently represents an optionally substituted alkenyl group having 14 to 60 carbon atoms;
• n represents 2 or 3, and the plurality of OR groups may be the same or different;
• m represents 0 or 1;
• p represents 1 or 2;
• X is C, N, O or S, and when X is C, it represents CH2 or may form, together with Y, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent, and when X is N, it represents NR1 or may form, together with Y, a 5- to 7-membered heterocyclic ring which may have a substituent, wherein R1 is H or a C1-6 alkyl group;
• Y is a single bond or B(CH2)t* (wherein B is a 5- to 7-membered carbocyclic ring which may have a substituent, t is 0, 1, or 2, and * indicates a bond to X), or may form, together with X, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent;
• Z is a single bond or (CH2)s, wherein s is an integer of 1 to 5, provided that when Y is a single bond, Z is not a single bond,
• ** indicates a bond to Base;
• wherein,
• o represents any integer of 0 or more;
• each Base independently represents an optionally modified nucleic acid base;
• each V independently represents a C1-6 alkoxy group, a di(C1-6 alkyl)amino group, or a piperazino group in which the 4-position nitrogen is protected with a protecting group and which may be further substituted;
• W′ each independently represents a hydrogen atom or a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion;
• each R2 independently represents O or S;
• Tg represents a group represented by the following formula (5):
• wherein,
• R each independently represents an optionally substituted alkenyl group having 14 to 60 carbon atoms;
• n represents 2 or 3, and the plurality of OR groups may be the same or different;
• m represents 0 or 1;
• p represents 1 or 2;
• X is C, N, O or S, and when X is C, it represents CH2 or may form, together with Y, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent, and when X is N, it represents NR1 or may form, together with Y, a 5- to 7-membered heterocyclic ring which may have a substituent, wherein R1 is H or a C1-6 alkyl group;
• Y is a single bond or B(CH2)t* (wherein B is a 5- to 7-membered carbocyclic ring which may have a substituent, t is 0, 1, or 2, and * indicates a bond to X), or may form, together with X, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent;
• Z is a single bond or (CH2)s, wherein s is an integer of 1 to 5, provided that when Y is a single bond, Z is not a single bond,
• ** indicates a bond to Base.

A method for producing an oligonucleotide by a liquid phase method using a carrier protecting group, comprising any one of step A, step B, or step C:

• (i) step A comprises the following steps (A1)-(A3):
• (A1) a step of reacting, in a nonpolar or polar solvent, an n-mer nucleotide (wherein n is any integer of 1 or more) in which the 3′- or 5′-position hydroxyl group is protected with a support protecting group and the 5′- or 3′-position hydroxyl group is protected with a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion, with an acid and a cation scavenger, or with a reagent containing a fluorine anion to remove the temporary protecting group at the 5′- or 3′-position hydroxyl group, and then separating the n-mer nucleotide from impurities in the reaction solution;
• (A2) a step of adding a p-mer nucleotide (p is any integer of 1 or more) in which the 3′-position hydroxyl group has been phosphoramiditized and the 5′-position hydroxyl group has been protected with a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion, or in which the 5′-position hydroxyl group has been phosphoramiditized and the 3′-position hydroxyl group has been protected with a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion, to a solution of the n-mer nucleotide obtained in step (A1) from which the temporary protecting group at the 5′-position hydroxyl group or the 3′-position hydroxyl group has been removed, thereby condensing the n-mer nucleotide and the p-mer nucleotide via the 5′-position hydroxyl group or the 3′-position hydroxyl group through a phosphite triester bond;
• and
• (A3) a step of converting the phosphite triester bond of the n+p-mer oligonucleotide obtained in step (A2) into a phosphate triester bond or a thiophosphate triester bond using an oxidizing agent or a sulfurizing agent;
• (ii) step B comprises the following steps (B1)-(B3):
• (B1) a step of reacting, in a non-polar or polar solvent, an n-mer nucleotide (n is any integer of 1 or more) in which a support protecting group is bound to a nucleic acid base at the 3′-terminal or a nucleic acid base at the 5′-terminal and the 3′-position hydroxyl group and the 5′-position hydroxyl group are protected with a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion, with an acid and a cation scavenger, or with a reagent containing a fluorine anion to remove the temporary protecting group at only one of the 5′-position hydroxyl group or the 3′-position hydroxyl group, and then separating the n-mer nucleotide from impurities in the reaction solution;
• (B2) a step of adding a p-mer nucleotide (p is any integer of 1 or more) in which the 3′-position hydroxyl group has been phosphoramiditized and the 5′-position hydroxyl group has been protected with a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion, or in which the 5′-position hydroxyl group has been phosphoramiditized and the 3′-position hydroxyl group has been protected with a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion, to a solution of the n-mer nucleotide obtained in step (B1) from which the temporary protecting group at the 5′-position hydroxyl group or the 3′-position hydroxyl group has been removed, thereby condensing the n-mer nucleotide and the p-mer nucleotide via the 5′-position hydroxyl group or the 3′-position hydroxyl group through a phosphite triester bond;
• and
• (B3) a step of converting the phosphite triester bond of the n+p-mer oligonucleotide obtained in step (B2) into a phosphate triester bond or a thiophosphate triester bond using an oxidizing agent or a sulfurizing agent;
• or
• (iii) step C comprises the following steps (C1)-(C4):
• (C1) a step of obtaining an n-mer nucleotide from which the temporary protecting group at the 5′-position hydroxyl group has been removed and a p-mer nucleotide from which the temporary protecting group at the 3′-position hydroxyl group has been removed by carrying out the following steps (a) and (b), respectively:
• (a) a step of reacting, in a nonpolar or polar solvent, an n-mer nucleotide (n is any integer of 1 or more) in which the 3′-position hydroxyl group is protected with a support protecting group and the 5′-position hydroxyl group is protected with a temporary protecting group removable under acidic conditions or with a reagent containing a fluorine anion, with an acid and a cation scavenger, or with a reagent containing a fluorine anion to remove the temporary protecting group at the 5′-position hydroxyl group, and then separating the n-mer nucleotide from impurities in the reaction solution;
• and
• (b) a step of reacting, in a non-polar or polar solvent, a p-mer nucleotide (p is any integer of 1 or more) in which a support protecting group is bound to the 3′-terminal nucleic acid base and the 3′-position hydroxyl group and the 5′-position hydroxyl group are protected with temporary protecting groups removable under acidic conditions or with a reagent containing a fluorine anion, with an acid and a cation scavenger, or with a reagent containing a fluorine anion to remove only the temporary protecting group at the 3′-position hydroxyl group, and then separating the p-mer nucleotide from impurities in the reaction solution;
• wherein the support protecting groups of (a) and (b) may be the same or different;
• (C2) a step of phosphoramiditizing the 5′-position hydroxyl group of the n-mer nucleotide from which the temporary protecting group at the 5′-position hydroxyl group has been removed obtained in step (C1), or a step of phosphoramiditizing the 3′-position hydroxyl group of the p-mer nucleotide from which the temporary protecting group at the 3′-position hydroxyl group has been removed obtained in step (C1),
• (C3) a step of adding the n-mer nucleotide whose 5′-position hydroxyl group has been phosphoramiditized obtained in step (C2) to a solution of the p-mer nucleotide from which the temporary protecting group at the 3′-position hydroxyl group has been removed obtained in step (C1), or adding the p-mer nucleotide whose 3′-position hydroxyl group has been phosphoramiditized obtained in step (C2) to a solution of the n-mer nucleotide from which the temporary protecting group at the 5′-position hydroxyl group has been removed obtained in step (C1), thereby condensing the n-mer nucleotide and the p-mer nucleotide through a phosphite triester bond;
• and
• (C4) a step of converting the phosphite triester bond of the n+p-mer oligonucleotide obtained in step (C3) into a phosphate triester bond or a thiophosphate triester bond using an oxidizing agent or a sulfurizing agent;
• wherein, the support protecting group is represented by the following formula (5):
• wherein,
• R each independently represents an optionally substituted alkenyl group having 14 to 60 carbon atoms;
• n represents 2 or 3, and the plurality of OR groups may be the same or different;
• m represents 0 or 1;
• p represents 1 or 2;
• X is C, N, O or S, and when X is C, it represents CH2 or may form, together with Y, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent, and when X is N, it represents NR1 or may form, together with Y, a 5- to 7-membered heterocyclic ring which may have a substituent, wherein R1 is H or a C1-6 alkyl group;
• Y is a single bond or B(CH2)t* (wherein B is a 5- to 7-membered carbocyclic ring which may have a substituent, t is 0, 1, or 2, and * indicates a bond to X), or may form, together with X, a 5- to 7-membered carbocyclic or heterocyclic ring which may have a substituent;
• Z is a single bond or (CH2)s, where s is an integer of 1 to 5, provided that when Y is a single bond, Z is not a single bond;
• ** indicates, in step A, a bond to the 3′- or 5′-position hydroxyl group, in step B, a bond to a nucleic acid base, and in step C, a bond to the 3′-position hydroxyl group or the 3′-terminal nucleic acid base.